in vivo biotherapy of hl-60 myeloid leukemia with a genetically · is a stohlman scholar of the...

Vol. 3. 22/7-2227. Dece,nher /997 Clinical Cancer Research 2217

In Vivo Biotherapy of HL-60 Myeloid Leukemia with a Genetically

Engineered Recombinant Fusion Toxin Directed against the

Human Granulocyte Macrophage Colony-stimulating

Factor Receptor’

John P. Perentesis,2 Roland Gunther,Barbara Waurzyniak, Yuri Yanishevski,

Dorothea E. Myers, Onur Ek, Yoav Messinger,

Yu Shao, Lisa M. Chelstrom, Elizabeth Schneider,

William E. Evans, and Fatih M. UckunDepartments of Pediatrics Ii. P. P.. Y. S.. R. G.] and Biochemistry

Ii. P. P.], University of Minnesota Medical School. Minneapolis.

Minnesota 55455: Wayne Hughes Institute, St. Paul, Minnesota

55113 [B. W., D. E. M.. 0. E.. Y. M.. L. M. C.. E. S.. F. M. UI: and

Pharmaceutical Department. St. Jude Childrens Research Hospital.

Memphis. Tennessee 38101 lY. Y.. W. E. E.]

ABSTRACT

Acute myeloid leukemia (AML) is the most common

form of acute leukemia. Contemporary chemotherapy reg-

imens fail to cure most patients with AML. We have genet-

ically engineered a recombinant diphtheria toxin (DT)-

human granulocyte macrophage colony-stimulating factor

(GMCSF) chimeric fusion protein (DTCIGMCSF) that spe-

cifically targets the GMCSF receptor on fresh human AML

cells and myeloid leukemia cell lines. At a nontoxic dose

level, DTC�GMCSF therapy was superior to the standard

chemotherapeutic agents 1-�-D-arabinofuranosylcytosine

and Adriamycin, resulting in 60% long-term event-free sur-

vival of severe combined immunodeficient mice challenged

with an otherwise invariably fatal cell dose of the human

HL-60 myeloid leukemia. Notably, systemic exposure levels

of DTC�GMCSF, which were found to be therapeutic in the

severe combined immunodeficient mouse xenograft model of

Received 3/3/97: revised 8/29/97: accepted 9/4/97.

The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.I This study was supported by NIH Grants CA-53586 (to J. P. P.),

AI-00793 (to J. P. P.). and CA-42633 (to F. M. U.) and a Translational

Research Award from the Leukemia Society of America (to J. P. P.).

Additional funding included special grants from the Variety Childrens

Association, the University of Minnesota Childre&s Cancer Research

Fund, the Minnesota Medical Foundation, the Leukemia Task Force. theSkogquist Memorial Fund. the Midwest Athletes Against Childhood

Cancer Fund. and the National Childhood Cancer Foundation. F. M. U.is a Stohlman Scholar of the Leukemia Society of America. J. P. P. is a

Scholar of the University Children’s Foundation and recipient of a NIH

Physician Scientist Award.

2 To whom requests for reprints should be addressed, at University ofMinnesota Medical School. Box 422 UMHC, 420 Delaware Street SE.

Minneapolis. MN 55455. Phone: (612) 626-3297: Fax: (612) 626-4842.

human HL-60 myeloid leukemia, could be achieved in cyno-

molgus monkeys without any significant nonhematological

toxicities. The recombinant DTCIGMCSF fusion toxin might

be useful in the treatment of AML patients whose leukemias

have recurred and developed resistance to contemporary

chemotherapy programs.

INTRODUCTION

Therapy for AML.3 the most common form of acute leu-

kemia in adults and the second most frequent leukemia in

children ( 1 . 2). remains problematic. Despite the use of intensive

multiagent chemotherapy regimens. over half of all patients with

AML will succumb to their disease because of of the emergence

of dominant multidrug-resistant subclones of leukemia cells

(3-8). Increasing the dose intensity of AML therapy by the use

of myeloablative chemotherapy and supralethal radiochemo-

therapy followed by allogeneic or autologous hone marrow

transplantation has effected only modest improvements in the

overall survival of AML patients and is associated with consid-

erable morbidity and mortality (9-12). These observations un-

derscore the need for rational drug design-based therapies for

AML and the identification of novel AML-specific therapeuticagents with unique mechanisms of action and nonoverlapping

mechanisms of resistance.

One of the most toxic substances found in nature. DT is a

535-residue protei n secreted by Corvnehacterium thjthiheriae.

Native DT binds to human cells through a specific receptor.

which has recently been identified as a heparin-binding epider-

mal growth factor-like precursor and which is widely expressed

on human cells (13). The subsequent entry of a single DT

molecule into the cytoplasm is sufficient to result in the corn-

plete inactivation of cellular protein synthesis. leading to cell

death ( 14). DT inhibits protein synthesis by catalyzing the

ADP-ribosylation of EF-2, an essential protein synthesis cofac-

tor, at a highly conserved posttranslationally modified histidine

residue known as diphthamide ( 15). Biochemical. genetic. and

recent X-ray crystallographic analyses of DT have identified

three functionally distinct structural domains, including an NH2-

terminal domain containing the ADP-ribosyltransferase cata-

lytic site, a domain in the middle of the protein that is involved

in facilitating toxin translocation across membranes, and a

3 The abbreviations used are: AML. acute myeloid leukemia: DT. diph-theria toxin: EF-2, elongation factor 2: GMCSF, granulocyte macro-

phage colony-stimulating factor: DLGMCSF. DT-GMCSF chimeric

fusion protein: GMCSF-R. GMCSF receptor: ARA-C. l-�-o-arabino-

furanosylcytosine; SCID. severe combined immunodeficient: AUC. area

under the concentration-time curve.

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INHIBITION OF PROTEINSYNTHESIS ELONGATION

2218 in Vivo Therapy of HL-60 Human Myeloid Leukemia

DT,�,GMCSF Fusion Toxin

GMCSFY

Fig. 1 Mechanism of action of recombinant DTC,GMCSF fusion toxin. In DTCIGMCSF, both the catalytic (C) and translocation (T) domains of nativeDT are preserved, but the native receptor-binding domain of DT, which mediates its indiscriminate binding to human cells, is genetically replacedwith human GMCSF (32). DTCIGMCSF binds to high-affinity GMCSF-Rs on AML cells, is internalized by receptor-mediated endocytosis, andundergoes cleavage by lysosomal proteases to release the C domain (i.e. . DTCJ. The C domain is a specific ADP-ribosyltransferase, catalyzing thetransfer of ADP-ribose from NAD� to EF-2 at its diphthamide residue (32-34). EF-2 is an essential cofactor that is required for the elongation phaseof protein synthesis. ADP-ribosylated EF-2 is unable to productively interact with the ribosome, resulting in irreversible shutdown of protein synthesis.Inhbition of protein synthesis is followed by apoptotic death of leukemic cells, which is independent of the actions of p513 or the bcl-2 oncoprotein

(33, 34).

COOH-terminal domain that mediates binding to target cells

(16, 17). The profound toxicity of DT is a result of the catalytic

nature of its mechanism of action and the ubiquitous expression

of its receptor on human cells.

Although unmodified native DT has previously beenused for the direct in vivo treatment of human solid tumors

(18), recent preclinical and clinical investigations of novel

DT-based therapeutics have used genetic engineering to re-place the portion of DT that mediates indisciminate binding

to human cells with cytokines or growth factors that target

receptors on malignant cells (19-27). Because leukemic cellsfrom the vast majority of patients with AML express high-

affinity receptors for GMCSF (28-31), we have built upon

the investigations of the molecular and structural biology of

DT and GMCSF to create a novel recombinant fusion toxin,

DTCIGMCSF, that redirects the lethal action of DT to high-

affinity GMCSF-Rs on AML cells (Fig. 1; Ref. 32). This

fusion toxin preserves the portions of DT that include the

lethal catalytic ADP-ribosyltransferase domain (C domain)

and the contiguous proximal portion of the toxin that is

associated with translocation across cellular membranes (T

domain). The native receptor-binding domain (R domain) of

DT was genetically replaced with human GMCSF in the

construction of the DTC,GMCSF fusion toxin. DTC�GMCSF is

selectively cytotoxic to a wide range of GMCSF-R-positive

AML cell lines (32), including cell lines displaying high level

resistance to conventional chemotherapeutic agents because

of expression multidrug resistance associated with P-glyco-

protein or multidrug resistance-associated protein (33, 34).

Moreover, DTC�GMCSF efficiently induces rapid apoptotic

death in chemotherapy-resistant AML cell lines and primaryleukemic cells from therapy-refractory AML patients (34).

We now report the in vivo pharmacodynamic features,

toxicity, and biological activity of DTC�GMCSF in SCID mice

that were xenografted with the human HL-60 myeloid leuke-mia cell line, as well as in normal cynomolgus monkeys.

DTC�GMCSF therapy was superior to the standard chemothera-

peutic agents ARA-C and Adriamycin, resulting in 60% long-

term event-free survival of SCID mice that were challenged

with an otherwise invariably fatal dose of the human HL-60

myeloid leukemia cell line. Notably, systemic exposure levels of

DTC�GMCSF, which were found to be therapeutic in the SCID

mouse xenograft model of human AML, caused reversible dose-

limiting neutropenia and thrombocytopenia in cynomolgus

monkeys, but they were not associated with any significant

nonhematological toxicities. Thus, the DTC,GMCSF fusion



Clinical Cancer Research 2219

toxin might be useful in the treatment of AML patients whose

leukemias have recurred and developed resistance to contempo-

rary chemotherapy programs.

MATERIALS AND METHODS

DTC�GMCSF Fusion Toxin The DTCIGMCSF fusion

toxin is a 521-amino acid residue chimeric protein that contains

a predicted NH2-terminal methionine residue, followed by

amino acid residues 1-385 of DT (including the C and T

domains), a Ser-(Gly)4-Ser-Met linker peptide, and mature hu-

man GMCSF (32). The native receptor binding domain (R

domain) of DT was completely deleted in the construction ofDTCIGMCSF. The rationale for the construction of DTC,GMCSFwas based, in part, upon X-ray crystallographic structural stud-

ies of DT, which identified a small peptide loop located at amino

acid residues 380-386 that separates the R domain from the C

and T domains and allows the entire Mr 15,000 R domain to

flexibly rotate, as a unit, by 180#{176},with atomic movement of up

to 65 A (16, 17). In DTC�GMCSF, the Ser-(Gly)4-Ser-Met linker

peptide and mature human GMCSF segment were inserted in

the fusion toxin at the site of this small flexible peptide loop.

This construction provided a natural separation of the DT and

GMCSF moieties and ensured that the NH2-terminal helices of

GMCSF would be accessible for high-affinity receptor binding

(34). DTC�GMCSF was expressed with high-efficiency fermen-

tation methods in Escherichia coli and purified through sequen-

tial anti-DT immunoaffinity and Mono-Q (Pharmacia, Piscat-

away, NJ) high-pressure liquid chromatographic methods,

followed by exhaustive endotoxin removal (32).

SCID Mouse Xenograft Model of Human HL-60 Mye-

bid Leukemia. We used the p53-deficient human acute pro-

myelocytic leukemia cell line HL-60 (35) to establish a SCID

mouse xenograft model of human AML (36). HL-60 cells were

maintained in Iscove’s modified Dulbecco’s medium, 10% fetal

bovine serum, 50 units/ml penicillin, and 50 p.g/ml streptomy-

cm. One day following sublethal total body irradiation with 2

Gy, 6-week-old CB.17 SCID mice, obtained from Charles River

Laboratories (Wilmington, MA), were injected i.p. with 4 X 106

HL-60 cells. Beginning 24 h later, mice were treated according

to one of the following regimens: PBS for 5 days: 2 p.g/day

human GMCSF (Immunex, Seattle, WA) for 5 days; 1 p.g/day

DTCOGMCSF for 5 days; 200 p.g/day ARA-C (Shein Pharma-

ceuticals Steris Laboratories, Phoenix, AZ) for 10 days; or 4

p.g/day Adriamycin (Chiron Therapeutics. Emeryville, CA) for

10 days. All drugs and controls were administered as i.p. 0.2-mi

bolus injections. SCID mice were maintained in Micro-Isolator

cages (Lab Products, Inc., Maywood, NY) within the

AAALAC-approved specific pathogen-free facilities of the Bio-

therapy Institute, as described previously (37-39). Mice were

evaluated daily for signs of toxicity and survival, and all healthy

mice were euthanized 8 months after injection of leukemia cells

or earlier, if they became moribund. For histopathological stud-

ies, tissues were fixed in 10% neutral buffered formalin, dehy-

drated, and embedded in paraffin by routine methods. Glass

slides with affixed 6-p.m tissue sections were prepared and

stained with H&E. Toxicity and pharmacology studies included

mice that had not been inoculated with any leukemia cells.

Event-free survival was assessed by life-table methods using the

Kaplan-Meier method, and the logarithmic rank test was usedfor comparisons of outcome between groups, as previously

reported (37-39).

Cynomolgus Monkey Experiments. Three femalecynomolgus monkeys, Macaca fasciularis, were obtained from

the Biomedical Resources Foundation (Houston, TX). Prior to

entering the study, the monkeys were housed in a quarantine

room for 6 weeks. During this time, they were tested for tuber-

culosis three times, serologically screened for herpes virus

simiae, and screened for enteric bacterial, protozoal, and hel-

minth pathogens. Animal housing was located in a AAALAC-

approved secure indoor primate research facility with controlled

temperature, humidity, and noise levels. Ventilation consisted of

15-20 changes/h of unrecirculated air, and lighting was pro-

vided by fluorescent lights on a 12-h cycle. The monkeys were

singly housed in stainless steel cages and provided with toys and

treats for enrichment. Monkeys were fed commercial monkey

chow with fresh fruit and fresh water ad libidum. Animal care

and veterinary oversight was provided by trained veterinarians.

Monkeys were fasted overnight prior to anesthesia and first

treatment. After induction of anesthesia (10-15 mg/kg ketamine

hydrochloride), a catheter was placed percutaneously either into

the right or left cephalic vein using a sterile disposable kit. This

catheter was taped in place for administration of DTC�GMCSF or

maintenence fluids (normal saline at 4 mI/kg/hr via an infusion

pump) and for drawing of blood samples for toxicity and phar-

macokinetic studies. A Harvard infusion pump was used to

administer DTC�GMCSF as a constant iv. infusion over an 1-h

period. For toxicity analysis, animals were examined by two

veterinarians twice daily, and blood chemistry and hematologystudies were conducted three times a week for the week 1

postinfusion and weekly thereafter. Toxicity grades were as-

signed based on established toxicity grading criteria of the

Children’s Cancer Group, with slight modifications. Monkeys

were electively sacrificed for histological examination at 30

days posttreatment.

Pharmacokinetic Studies. The systemic disposition

studies in SCID mice and cynomolgus monkeys were performed

using unlabeled DTC,GMCSF, and the drug concentrations were

measured in the serum samples using the GMCSF Quantikine

ELISA kit from R&D Systems, a quantitative sandwich enzyme

immunoassay. In these studies, DTC�GMCSF was administered

by i.p. injection to SCID mice at doses of 1, 2, 5, and 10 p.g.

Four mice were used at each dose level, and six nonoverlapping

time samples were obtained from each pair of mice. Mice were

serially bled by retroorbital puncture at 10 and 30 mm and 1, 2,

4. 8. 24. and 48 h following the administration of the fusion

toxin. In cynomolgus monkeys, serum samples were obtained at

the time points 30 mm and 1, 2, 4, and 8 h postinfusion. A

two-compartment, first-order pharmacokinetic model was fit to

the plasma concentration versus time data for DTCIGMCSF.

Maximum likelihood estimation, as implemented in ADAPT II

software (40), was used to estimate the central compartment

volume of distribution, elimination rate constant, and distribu-

tion rate constants for DTC�GMCSF, as reported previously (37).

Tissue distribution studies in SCID mice were performed

using radioiodinated DTC�GMCSF, as described in detail in

previous publications from our laboratory (37). A flow-limited

physiological pharmacokinetic model was used to characterize




Table I Pharmacokinetic features of DT�S,GMCSF in SCID mice

The pharmacokinetic features of DTCGMCSF were examined in aSCID mouse model of human AML, as described in detail in Materialsand Methods.” Studies were performed in SCID mice with minimalleukemic burden at 24 h after inoculation of 4 X 106 HL-60 human

AML cells.

Dose (i.p.)

10Parameters (units) I p.g 2 p.g 5 jig p.g

Vc (ml) 346 419 65.7 49.7Ke (lTh) 1.3 0.98 1.56 1.6Kabs (p.g/h) 0.57 2.3 6.1 19.5

T,,,� (h) 0.53 0.71 0.44 0.43Clearance (ml/h) 450 41 1 103 80

AUC (ng/ml h/day) 2.2 4.9 48.5 125

the tissue disposition of DTC,GMCSF in healthy control SCIDmice, as well as in SCID mice xenografted with human AML

cells (37, 41). The model consisted of the following compart-

ments: plasma, heart, lungs, brain, liver, spleen, kidneys, skin,

muscle, and bone marrow. Each organ was configured as a

three-compartment structure corresponding to the vascular, in-

terstitial, and intracellular spaces. Physiological parameters of

the mice were those previously reported (41). A set of lineardifferential equations describing the mass balances of each

model compartment was used to estimate tissue partition coef-

ficients (i.e. , the ratio of the drug concentration in the tissue ofinterest to the drug concentration in the plasma at equilibrium)for each organ. These differential equations were simulta-

neously solved using the ADAPT II software and a maximum

likelihood algorithm was used to estimate model parameters

(40). Physiological parameters of the tissues and estimated rate

constants were used to derive tissue partition coefficients.

RESULTS

Pharmacodynamic Features, Toxicity, and Biological

Activity of DTC�GMCSF in SCID Mice. The pharmacoki-netic features of DTCIGMCSF were characterized in SCID mice

inoculated with 4 X 106 HL-60 human AML cells. A one-

compartment, first-order pharmacokinetic model with zero-

order absorption, as implemented in the ADAPT II software,

was fit to the data for plasma concentration versus time.

DTCIGMCSF was cleared rapidly from plasma, with an elimi-

nation half-life of 0.43-0.71 h (Table 1). Treatment of SCIDmice with a single dose of 1 p.g (50 p.glkg) of DTCIGMCSF

resulted in a systemic exposure level (i.e., AUC) of 2.2 ng/ml1i/

day. Both the volume of distribution and clearance of

DTC,GMCSF decreased, and the AUC increased as the dose

exceeded 2 p.g, consistent with a saturable receptor-dependentbinding of DTC�GMCSF to leukemia cells.

DTC�GMCSF demonstrated excellent distribution into the

parenchymal space of most tissues. Tissue partition coefficients

are shown in Table 2. Parenchymal concentrations were close to

total tissue concentrations, which is consistent with the good

capillary permeability of DTC�GMCSF. In SCID mice with

advanced human AML, the tissue partition coefficients were

Table 2 Tissue disposition of DTCSGMCSF in SCID mice

The tissue partition coefficients of DTC,GMCSF were examined ina SCID mouse model of human AML, as described in detail in “Mate-rials and Methods.” Studies were performed in mice with minimal

leukemia as in Table I. as well as in mice with advanced human AML,

at 4 weeks after inoculation of 4 X 106 HL-60 cells.

Tissue

Partition coefficient (ml/g)

Minimal leukemia_burdenLarge leukem ia burden

Bone marrow 4.3 4.66 X l0�Heart 0.5 2.31 X l0�

Liver 2.3 0.7

Kidney 3.4 5.13 X l0�

Lungs 6.1 0.43Muscle 0.5 2.43 X l0�

Skin 2.8 2.94 X l0�

Spleen 7.0 1.64 X l0�

substantially (i.e. , 1000-fold) higher in involved organs and

proportional to tissue leukemia burden, consistent with a selec-

tive binding of DTCIGMCSF to GMCSF-R-positive human leu-

kemia cells that had infiltrated the tissues.

DTC,GMCSF was not toxic to SCID mice at six different

dose levels, ranging from 5 p.g (250 p.glkg) to 30 p.g (1500

p.gfkg). None of the 35 mice treated with i.p. bolus injections of

DTCOGMCSF in this dose range experienced side effects or died

of toxicity during the 1-month observation period. No his-

topathological lesions were found in the organs of DT�5GMCSF-

treated mice that were electively killed at 35 days. At higher

doses, we encountered a dose-limiting severe renal toxicity due

to DT�,GMCSF-induced acute tubular necrosis (LD50 = 50

jig = 2500 p.g/kg). The necrotic tubular epithelial cells were

hypereosinophilic, with pyknotic or lysed nuclei. Sloughing of

the epithelium of the proximal renal tubules, dilation of affectedtubules, and numerous intratubular granular and hyaline casts

were prominent in the kidneys of affected mice.

Previously, we showed that DTCIGMCSF is selectively

cytotoxic to a wide range of GMCSF-R-positive AML cell lines

and primary AML cells from therapy-refractory patients

(32-34). We next evaluated the antileukemic efficacy of

DT�1GMCSF against the prototypic HL-60 human myeloid leu-

kemia cell line in SCID mice. All 5 1 control mice that were

treated with PBS died of disseminated human HL-60 leukemia,

with a median survival of 42 days (Table 3). Histopathological

examination of the bone marrows from these mice showed

multifocal and partially effacing leukemic cell infiltrates, which,

in some areas, extended through the cortical bone into the

surrounding soft tissues (Fig. 2, A and B). Involvement of thespleen consisted of patchy infiltration of the red and white pulp

by leukemic cells (Fig. 2, C and D). Involvement of the thymus

was diffuse with replacement of the normal tissue architecture

by sheets of closely packed leukemic cells, resulting in nearly

total effacement of the normal architecture (Fig. 2, E and F).

The kidneys had large, irregularly shaped, interstitial infiltrates

in the pelvis and peripelvic fat (Fig. 2, G and H). Infiltrates of

leukemic cells were present in the epicardium of the atrium in

the hearts; the pancreas had extensive interstitial infiltrates, with

massive replacement of the normal tissue architecture by sheets




Table 3 Antileukemic activity of DTCGMCSF fusion toxin against human AML in SCID mice

SCID mice were inoculated i.p. with 4 X 106 HL6O cells. Antileukemic therapy according to the indicated protocols was initiated 24 h later. EFS”was assessed by life-table methods using the Kaplan-Meier method, and the logarithmic rank test was used for comparison of outcome between

groups.

Treatment group Schedule No. of mice

EFS (%)

Median EFS (days) P (versus DTCGMCSF)60 days 120 days 180 days

ControlGMCSFARA-CAdriamycin

DTCGMCSF

‘I EFS, event-fr

PBS x 5 days 51

2 p.g/d X 5 days 13

200 p.g/d X 10 days 7

4 p.g/d X 10 days 7

1 p.g/d X 5 days 10

ee survival: NA. not applicable.

20 ± 68 ± 7

57 ± 29

86 ± 13

100 ± 0

2 ± 20 ± 0

14 ± 13

43 ± 1970 ± 15

0 ± 00 ± 0

NA

NA60 ± 16

424163

94>210

<0.0001<0.0001

0.01

0.1

ofdensely packed leukemic cells; and leukemic cell infiltrates in

the gastrointestinal tract were found in the serosa, smooth mus-

cle tunica, and submucosa of the stomach and large intestine.

Central nervous system leukemia was evidenced by leukemic

cell infiltrates in the subarachnoid space and on the meninges

(data not shown).

Similarly. all of the 13 control mice treated with native re-

combinant human GMCSF died of disseminated human HL-60

myeloid leukemia, with a median survival of 41 days (Fig. 3 and

Table 3). In contrast, 60 ± 16% ofthe SCID mice treated with 1 p.g

of DTC,GMCSF (= 10% of the LD30 dose level) daily, for a total

of 5 days, remained alive without clinical evidence of leukemia for

>2 10 days (Fig. 3 and Table 3). When these mice were electively

euthanized at 8 months, no leukemic infiltrates were found in any

of the organs. Taken together, these experiments demonstrated that

an AUC of 2.2 ng/mlh/day, which was achieved on the 1 p.glday

(= 50 p.g/kg/day) for 5 days treatment schedule, is a highly

effective and nontoxic systemic exposure level for DTC,GMCSF in

this HL-60 SCID mouse xenograft model of human AML. Treat-

ment of SCID mice with 2 mg (200 p.g for 10 days) of ARA-C or

40 p.g (4 p�g for 10 days) of Adriamycin also improved survival,

with median survival times of 63 days and 94 days, respectively.

However, these treatment regimens were not as effective as

DTC,GMCSF therapy (Fig. 3 and Table 3).

Toxicity, Pharmacodynamic Features, and Biological

Activity of DTC�GMCSF in Cynomolgus Monkeys. We

next evaluated the toxicity and pharmacokinetic features of

DTC,GMCSF in cynomolgus monkeys. The primary goal of

our toxicity study was to determine whether we could safely

achieve, in cynomolgus monkeys, the systemic exposure

level of DTC,GMCSF that was effective against human AML

in SCID mice. Three cynomolgus monkeys received daily 1-h

iv. infusions of DTC,GMCSF for 5 consecutive treatment

days, at dose levels of 7, 15, and 50 p.gfkg/day, respectively.

At the 7- and 15-p.g/kg dose levels, plasma concentrations of

DTC,GMCSF were too low to accurately determine its phar-

macokinetic parameters. A two-compartment, first-order

pharmacokinetic model was fit to the plasma concentration

versus time data for the 50-p.g/kg dose level of DTCIGMCSF.

As shown in Fig. 4, DTC�GMCSF was cleared from monkey

plasma at 439 ml/h/kg, with an elimination half-life (t112�) of

5.4 h. Thus, DTC,GMCSF had a substantially slower plasma

clearance and longer elimination half-life in the cynomolgus

monkey receiving 50 p.g/kg of the fusion toxin than it did in

mice treated at the same dose level. Consequently, the sys-

temic exposure level (AUC) of DTC,GMCSF at this dose level

was 1 14 ng/mlh/day, which exceeded, by >50-fold, the

target therapeutic systemic exposure levels (i.e. , 2.2 ng/mlh/

day) that was observed in SCID mice receiving the same dose

of DTC�GMCSF. Notably, the pharmacokinetic features of

DTC�GMCSF in monkeys were very similar to the pharma-

cokinetics of s.c. administered GMCSF in children with solid

tumors (42). The AUC values, normalized to the 50-p.glkg

dose level, were 1 14 ng/mlth/day for DTC,GMCSF in the

monkey and 178 (range, 68-469) ng/mlh/day for GMCSF in

children.

No clinical or laboratory evidence of significant toxicity

was observed in cynomolgus monkeys treated at the 7- and

l5-p.g/kg/day dose levels of DTC�GMCSF (Table 4). In con-

trast, treatment with 50 jig/kg/day DTC�GMCSF caused sig-

nificant toxicity. As shown in Fig. 4, the monkey treated at

this dose level showed evidence of severe myelosuppression,

with a nadir absolute neutrophil count of 200 on day 4. A

bone marrow aspirate sample from left posterior iliac crest

was obtained on day 5 and showed markedly decreased

erythroid and neutrophil precursors. The differential cell

count in the bone marrow concentrate showed 59% mono-

cytes, 38.6% lymphocytes, 1% erythroblasts, 0.2% neutro-

phils and precursors, 0.4% eosinophils and precursors, 0.4%

basophils and precursors, and 0.4% plasma cells. Megakaryo-

cytes were essentially absent in the marrow aspirate sample;

however, in the face of only a modest thrombocytopenia, this

finding may reflect a sampling error or an artifact related to

marrow specimen processing. This monkey developed severe

anemia, with a nadir hemoglobin of 3.0 g/dl on day 8. A

component of the anemia may have been associated with the

repeated phlebotomies in this study. The absolute lympho-

cyte count did not decrease after DTC�GMCSF therapy. The

myelosuppression was associated with an episode of staphy-

lococcal sepsis, which was accompanied by hypotension with

tachycardia and consumptive coagulopathy with thrombocy-

topenia (nadir platelet count = 109,000/p.l on day 9; see Fig.

4), epistaxis and hematochezia. During the bacterial sepsis,

serum alanine aminotransferase levels were transiently ele-

vated. These complications responded to parenteral antibiotic

therapy. Myelosuppression was transient, and peripheral



[V�#{149}

� � ;:

2222 In Viva Therapy of HL-60 Human Myeloid Leukemia

Fig. 2 SCII) 11OU5� xenograftmodel of human HL-60 mye-bid leukemia. HL-60 cells

cause disseminated humanAML in SCID mice when they

are injected I.p. A and B, bonemarrow involvement consistsof multifocal to partially effac-

ing infiltration of the marrow

cavity by human AML cells,which, in some areas, extend

through the cortical bone into

the surrounding soft tissue. Cand D. patchy infiltration by

leukemic cells is found in thesplenic red and white pulp. E

and F. the thymic architectureis nearly effaced by sheets of

closely packed leukemic cells.G and H, large. irregularlyshaped. interstitial infiltrates of

leukemic cells are present in therenal pelvis and peripelvic fat.

Original magnifications, X25(A. C. E. and G: X100 (B, D,

and F): and >:5o (if).

blood cell counts returned to their baseline levels by day 10. monocytes, 2.4C/c eosinophils and precursors. 0.8% basophils

A follow-up bone marrow aspirate sample from left posterior and precursors, l6.49�- lymphocytes. and 0.89� plasma cells.

iliac crest on day 30 revealed trilineage hematopoiesis. with Megakaryocytes appeared normal in number and morphol-

3 1 % erythroblasts. 40.6% neutrophils and precursors. 8% ogy. Extensive clinical monitoring and histopathological



1.0

a) 0.8

L�.

C0>

w0)C>

Cl) 0.4C0

00.0

�: 0.2

0 30 60 90 120 150 180 210


Fig. 3 In vivo antileukemic efficacy of

DTCGMCSF in a SCID mouse xenograft model ofhuman HL-60 myeloid leukemia. Treatment pro-tocols were initiated 1 day after the inoculation of4 X 106 HL-60 cells. SCID mice received i.p.injections of DTC,GMCSF (I p.g/day for 5 days,total dose = 5 �J.g; n = 10), PBS (control for 5days; n = 51), native human GMCSF (2 pg/day X

5 days, total dose = 10 p.g: n 13). Adriamycin

(topoisomerase II inhibitor; 4 p.g/day for 10 days,total dose - 40 p.g; n = 7), and ARA-C (antime-

tabolite; 200 p.g/day for 10 days, total dose 2

mg; n = 7). Cumulative proportions of mice sur-viving event-free are shown according to the num-

ber of days after inoculation of HL-60 cells.

study of the monkeys did not reveal evidence of any other

fusion toxin-related toxicity. In particular, renal toxicity,

which was dose limiting in mice. was not observed.

DISCUSSION

Murphy and coworkers ( 19) used genetic engineering to

redirect the lethal action of DT to cancer cells expressing

interleukin 2, interleukin 4 (20), interleukin 6 (21), epidermal

growth factor receptor (22), and the melanocyte-stimulating

hormone receptor (23). Early clinical trials of their DT-interleu-

kin 2 fusion toxins (i.e. , DAB486IL-2 and DAB389IL-2) have

generated very promising results, with complete and partial

remissions observed in relapsed patients with IL-2 receptor-

bearing malignancies (24-27). Other investigators have targeted

Pseudomonas exotoxin A to cytokine receptors (43-45). We

have focused our efforts on designing an effective anti-AML

fusion toxin (32-34).

Hematopoietic cytokines appear to play a prominent role in

the etiology and maintenance of AML, and large subsets of

AML patients exhibit direct or indirect (i.e. , by other cytokines)

activation of GMCSF-related growth loops in leukemic blasts

(3 1, 46-50). The autonomous growth of leukemic blasts related

to activation of hematopoietic growth factor-related prolifera-

tive pathways is associated with a markedly reduced survival in

AML (5 1, 52). Significantly, the autonomous proliferation of

AML blasts in culture can be abrogated in over 80% of cases by

use of a neutralizing anti-GMCSF antibody or an antisense

oligonucleotide directed against the GMCSF transcript (53).

Complementary studies have revealed that the majority of pa-

tients with AML possess leukemic blasts that express

GMCSF-Rs (28-30, 54). These observations indicate that

the GMCSF-R is a suitable leukemic cell surface target for

the directed biotherapy of AML. We have developed the

DTC�GMCSF fusion toxin to specifically target the GMCSF-R

on AML cells (32-34).

In previous studies, we have observed that DTC�GMCSF is

Time Following Inoculation of HL6O Cells (Days)

selectively cytotoxic to GMCSF-R-positive human AML cells,

including those expressing multidrug resistant phenotypes that

are associated with P-glycoprotein or multidrug resistance-

associated protein (33). DTCIGMCSF displays potent antileuke-

mic activity against AML cells that are deficient in p53 expres-

sion and radiation-resistant AML cells, as well as mixed lineage

leukemia cells expressing high levels of the antiapoptotic bcl-2

oncoprotein (33). Most recently. we found that DTC,GMCSF

induces rapid apoptotic cell death in multidrug-resistant AML

cell lines, as well as primary leukemic cells from therapy-

refractory, multiple-relapse AML patients (34). Here. we exam-

med the in vivo pharmacodynamic features and antileukemic

efficacy of DTC�GMCSF in a SCID mouse xenograft model of

human AML and found that DTCIGMCSF therapy, at a nontoxic

dose level yielding a systemic exposure level of 2.2 ng/ml1i/day,

was superior to the standard chemotherapeutic agents ARA-C

and Adriamycin, resulting in 60% long-term event-free survival

of SCID mice that were challenged with an otherwise invariably

fatal human AML. Notably. we found that systemic exposure

levels of DTC,GMCSF, which were 50-fold higher than those

found to be therapeutic in the SCID mouse xenograft model of

human AML, were achievable in cynomolgus monkeys without

causing any significant nonhematological toxicity.

Multiple previous studies have characterized the action of

human GMCSF in cynomolgus and rhesus monkeys and mdi-cate that they are appropriate preclinical models for studying the

toxicity of GMCSF-R-directed therapies. These studies demon-

strated that human recombinant GMCSF is a potent stimulator

of hematopoiesis in these animals, and they allowed for the

development of relevant primate models for human GMCSF

administration. pharmacology, and toxicity (55-65). In this

study of DTCIGMCSF administration to normal cynomolgus

monkeys, the fusion toxin produced significant but reversible

neutropenia, an expected finding in view of the ability of

GMCSF to stimulate the proliferation and differentiation of a

broad range of early-stage hematopoietic myeloid lineage pro-



E0)

0.C

0

Ca,0C0

0

PharmacokineticParameters Monkey 52H

Vc (mI/kg) 97.6

Ke (1/hr) 4.5

Kcp (1/hr) 0.11Kpc (1/hr) 0.13

T,,�cx (hr) 0.15

T,,�j1 (hr) 5.4

CI (mI/hr/kg) 439

AUC (ng/mrhr/day) 114

1000

A100

0 1 2 3 4 5

Hours

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

DTc, GMC5F Days


Fig. 4 Pharmacokinetic features and biological

activity of DTCGMCSF in cynomolgus monkeys.

A, after iv. administration of 50 p.g/kg/day

DTCGMCSF over 1 h. serial plasma samples were

examined for drug levels. A two-compartment,first-order pharmacokinetic model was fit to the

data for plasma concentration versus time. V�.

central volume of distribution; K�, elimination

rate constant: � and K,..�, peripheral distribution

rate constants: T,,2,,, a elimination half-life; T,126,

I� elimination half-life: Cl. systemic clearancefrom plasma. B. hematological effects of treat-

ment with 50 p.glkg/day DTCGMCSF adminis-

tration. Peripheral blood hematology profiles

were obtained before, during, and after theDTCGMCSF treatment course, as described in

‘Materials and Methods.”

genitor cells (66, 67). A Grade 4 neutropenia was observed to

begin approximately 48-72 h after the initiation of therapy,

consistent with the kinetics of DTC�GMCSF cytotoxic activity

against early marrow precursors (32).

A transient early paradoxical increase in WBC count and

absolute neutrophil count was observed 24 h after DTCIGMCSF

administration and may reflect a partial agonist effect on the

distribution of mature neutrophils. Other investigators have also

suggested that fusion toxins may produce early agonist effects in

target cells prior to the onset of cytotoxicity. For example, it has

been demonstrated that the early interaction of the interleukin

2-DT fusion (DAB486IL-2) with interleukin 2 receptor-bearing

T cells initially produces effects upon c-myc, IFN-�y, and inter-

leukin 2 receptor mRNA expression that are identical to those

mediated by interleukin 2 and that the cytotoxic effects of

protein synthesis inhibition mediated by the DAB486IL-2 DT

moiety are not manifest until several hours later (68). Transient

early neutrophilia preceding a period of marrow suppression has

also been observed in primates receiving GMCSF immediately

after treatment with myeloablative total body irradiation and

autologous bone marrow transplantation (65).

Nonhematological toxicities were observed in monkeys

treated with DTCIGMCSF. A reversible increase in serum

alanine aminotransferase was observed on days 5-8 after

DTC,GMCSF administration. Although this abnormality was

coincident with an episode of bacterial septicemia and resolved

with the successful treatment of the infection, it is notable that

the maximum tolerated dose of the DAB486IL2 fusion toxin in

humans has generally been defined by asymptomatic transient

hepatic transaminase elevation (24-27). The hepatic trans-

aminase elevations observed in human clinical trials of

DAB486IL-2 may be associated with the toxin moiety, and they

appear to be noncumulative and have decreasing intensity upon

repeat courses of the fusion toxin.




noteworthy that rare mutant tissue culture cell lines have been

Table 4 Toxicity of DTCIGMCSF in cynomolgus monkeys

Maximum toxicity grade”

52J 52C 52H

7 p.g/kg/day. I 5 p.g/kg/day 50 p.g/kglday

Toxicity X5days X5 days X 5 days

Decreased activity/poor 1 1 4 (days 8-9

p.o. intake

Fever 0 1) 0

Weight loss 0 0 1

Cardiac

Hypotension 0 0 2 (day 8)

Tachycardia 1 1 2 (day 10)

Pulmonary 0 0 1

Renal 0 (1 0

Liver”

ALT 0 0 3 (days 5-8)Bili 0 0 0

GastrointestinalVomiting 0 0 0

Diarrhea 0 2 (day 5) 4 (days 8-9)

Hematological

Leukopenia 0 0 4 (days 4-5)

Anemia I 2 4 (days 8-10)

Thrombocytopenia 0 0 3 (days 8-9)

Coagulopathy 0 0 1

Infection 0 0 3 (days 5-9)

Neurological 0 0 0

(S For each toxicity ofgrade >1, the onset and duration of toxicityare indicated in parentheses.

I’ ALT, alanine aminotransferase: Bili, bilirubin.

It has been observed that GMCSF may play a significant

role in pulmonary homeostasis. GMCSF-deficient mice de-

velop significant but generally asymptomatic pulmonary

findings that are characterized by a decreased clearance of

surfactant lipids and proteins (69), and cynomolgus monkeys

appear to demonstrate a significant increase in the number

and function of lung phagocytic cells upon GMCSF admin-

istration (70). We did not observe clinical or histopatholog-

ical evidence of pulmonary toxicity in the DT�1GMCSF-

treated cynomolgus monkeys . Nevertheless, patients treated

with DTC�GMCSF in future clinical trials will need to be

closely monitored for pulmonary toxicity.

Our current findings of minor and tolerable nonhemato-

logical toxicities of DTC,GMCSF in cynomolgus monkeys are

consistent with our previous in vitro studies indicating that the

cytotoxicity of DTC�GMCSF is dependent upon the expression

of high affinity GMCSF-Rs (32-34). Overall, these results in-

dicate that the targeted inhibition of protein synthesis is a

feasible mechanism to activate apoptotic death mechanisms in

myeloid leukemias that are highly resistant to contemporary

chemotherapy regimens (34). The recombinant fusion toxin

DTCIGMCSF may, thus, serve as an effective treatment for

AML, and it has exhibited superior cytotoxicity against AML in

in vitro (32-34) and in vivo (the present study) therapy models.

These observations carry the caveat that the pharmacological

distribution and toxicities associated with DTCIGMCSF admin-

istration to patients with a large tumor burden of refractory

AML may be significantly different from those observed in our

study of drug administration to normal primates. It is also

identified as resistant to the cytotoxicity of DT because they

possess an EF-2 that is defective in the posttranslational bio-

synthesis of diphthamide. which serves as the recognition site

for DT (7 1 ). It is possible that AML cells that are DT resistant

because of EF-2 diphthamide mutations or decreased expression

level of GMCSF-Rs may be encountered during the clinical

evaluation of DTCIGMCSF. Therefore, it will be important to

determine whether this new biotherapeutic agent might act in

synergy with other standard chemotherapies or conventional

immunotoxins containing toxins, radioisotopes, or cytotoxic

drugs (72-78).

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1997;3:2217-2227. Clin Cancer Res J P Perentesis, R Gunther, B Waurzyniak, et al. receptor.human granulocyte macrophage colony-stimulating factorengineered recombinant fusion toxin directed against the In vivo biotherapy of HL-60 myeloid leukemia with a genetically

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in vivo biotherapy of hl-60 myeloid leukemia with a genetically · is a stohlman scholar of the...

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