[CANCER RESEARCH57, 21872192,

June I, 1997)

Immunotherapy

of BALB/c Mice Bearing Ehrlich Ascites Tumor with Vitamin

D-binding Protein-derived
Nobuto

Yamamoto,2

Macrophage Activating Factor'

and Venkateswara

R. Naraparaju

Laboratory of Cancer Immunology and Molecular Biology, Albert Einstein Cancer Center, Philadelphia, Pennsylvania

19141

ABSTRACT

subsequent antigen presentation is the first step of the immune devel

opment cascade, the capacity of the hosts to activate their own
Vitamin D3-binding protein (DBP; human DBP is known as Gc protein)
macrophages with their serum provides an index for their immune
is the precursor of macrophage activating factor (MAF). Treatment of
potential (10).
mouse DBP with hnmobffized @3-galactosidase
or treatment of human Gc
Our accumulated evidence indicates that inflammation-primed
protein with immobifized @-galactosidaseand sialidase generated a re
macrophage activation requires the participation of B and T lympho
markably potent MAF, termed DBPMAF or GCMAF, respectively. The
cytes (3, 8, 1114)and serum DBP3 (human DBP is known as Gc
domain of Ge protein responsible for macrophage activation was cloned
and enzymatically converted to the cloned MAF, designated CdMAF. In protein) (1215).Gd protein (the major subgroup of human Gc
Ehrlich asates tumor-bearing mice, tumor-specific serum a-N-acetyl
protein) carries a trisaccharide composed of N-acetylgalactosamine
galactosaminidase
(NaGalase) activity increased linearly with time as the
with dibranched galactose and sialic acid termini ( 1517).As shown
transplanted
tumor cells grew in the peritoneal cavity. Therapeutic effects
in Fig. la, this oligosaccharide is hydrolyzed by membranous f3ofDBPMAF, GCMAF, and CdMAF on mice bearing Ehrlich ascites tumor
galactosidase of inflammation-primed B cells to yield a macrophage
were assessed by survival time, the total tumor cell count in the peritoneal
proactivating factor, which is in turn hydrolyzed by sialidase of
cavity, and serum NaGalase activity. Mice that received a single admin
T-cells
to yield a MAF (1517).Mouse DBP carries a disaccharide
istration of DBPMAF or GcMAF (100 pg/mouse) on the same day after
transplantation of tumor (1 x 1O@cells) showed a mean survival time of composed of N-acetylgalactosamine with a galactose terminal. Hy
35 4 days,whereastumor-bearing controls had a meansurvival time of drolysis of this disaccharide by /3-galactosidase of B cells alone
16 2 days. When mice received the second DBPMAF or GcMAF generates a potent MAF (Refs. 1820;Fig. lb). Thus, DBP and Gc
protein are precursors for MAFs (15, 18). Incubation of mouse DBP
administration
at day 4, they survived more than 50 days. Mice that
received two DBPMAF administrations, at days 4 and 8 after transplan
with immobilized (3-galactosidase or incubation of Gc protein with
teflon of 1 x
tumor cells, survived up to 32 4 days. At day 4 immobilized f3-galactosidase and sialidase generates a remarkably
posttransplantation,
the total tumor cell count in the peritoneal cavity was
high-titered MAF, termed DBPMAF or GcMAF, respectively (1618,
approximately 5 x 10@cells. Mice that received two DJ3PMAF adminis
21), whose potency and time course of effect suggest that these
trations, at days 0 and 4 after transplantation of 5 x 1O@
twnor cells, also
proteins act directly on macrophages and are the most active factors
survived up to 32 4 days, while control mice that received the 5 x 10@
known for this process.
ascites tumor cells only survived for 14 2 days. Four DBPMAF,
A recent important observation was that cancer patients had de
GeMAF, or CdMAF administrations to mice transplanted with 5 X 10@
creased
capability of macrophage activation with their own serum
Ehrlich ascites tumor cells with 4-day intervals showed an extended
survival of at least 90 days and an insignificantly low serum NaGalase because of decreased precursor activity of the serum Gc protein (10).
Loss of the precursor activity of the Gc protein was found to be due
level between days 30 and 90
to the deglycosylation of Gc protein by serum NaGalase derived from
cancerous cells (Fig. lc; Refs. 10 and 22). Once Gc protein is
INTRODUCTION
deglycosylated, lymphocyte glycosidases (i.e., j3-galactosidase and
sialidase of lymphocytes) can no longer convert the deglycosylated
Inflammation results in macrophage activation, leading to immune
Gc protein to the MAF (10). Thus, macrophages cannot be activated
development. Inflamed tissues release membranous lipid metabolites,
under such a circumstance. However, GcMAF and DBPMAF can
lysophosphatidylcholine and other lysophospholipids, that efficiently
bypass the decapitated macrophage activation cascade and efficiently
activate macrophages (13).Inflammation of cancerous tissues (e.g.,
activate macrophages (10, 2 1). Because macrophages have a potential
melanoma and bladder cancer) induced by intratumor administration
to kill and eliminate cancerous cells, we studied the efficacy of
of BCG (Bacille Calmette Guerin) or other bacterial cells can result in
DBPMAF, GcMAF, and its cloned derivative (CdMAF) on mice
the regression of local as well as metastasized tumors, suggesting the
bearing Ehrlich ascites tumor. The fate of the transplanted tumor cells
development of specific immunity against the cancerous cells (4, 5).
and tumor-specific serum NaGalase as a prognostic index was mon
Inflamed cancerous tissues release alkylglycerols as well as lysophos
itored as the therapy progressed.
pholipids, because cancerous tissues contain alkylphospholipids (6
8). Alkylglycerols are approximately 400 times more active macroph
age-activating agents than lysophospholipids in terms of the minimal
MATERIALS AND METHODS
dosages required for optimal macrophage activation (8). This may
Animals and Tumor
explain why inflammation in cancerous tissues is curative. In fact,
macrophages have a potential to eliminate cancerous cells when
Female BALB/c mice, 71
2 weeks of age, were obtained from The Jackson
activated (9). Because macrophage activation for phagocytosis and
Laboratory (Bar Harbor, ME). Mice were fed Purina Mouse Chow and water
ad libitum.

Received 11/26/96; accepted 4/2/97.

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 to indicate this fact.
1 Supported

in part

by USPHS

Grant

RO1

AI-32140

from

the

National

Institute

for

Allergy and Infectious Diseases.

2 To

whom

requests

for

reprints

Ehrlich

ascites

tumor

was obtained

passage

through

3 The

abbreviations

the BALB/c

used

are:

mouse

DBP,

peritoneal

vitamin

activating factor; DBPMAF, enzymatically

should

be addressed,

at the

Laboratory

of

from

the Division

of Cancer

Treatment, National Cancer Institute (Bethesda, MD) and maintained by serial

D3-binding

cavity.

protein;

generated DBP-derived

MAF,

macrophage

MAF: Ge, human

DBP; GcMAF, enzymaticallygeneratedGe protein-derivedMAF; CdMAF, cloned do

main of GcMAF responsible for macrophage activation; NaGalase, a-N-acetylgalac

Cancer

Immunology and Molecular Biology, Albert Einstein Cancer Center, Korman Research
Pavilion B-31, 5501 Old York Road, Philadelphia, PA 19141.

tosaminidase.
2187

Downloaded from cancerres.aacrjournals.org on December 12, 2015. 1997 American Association for Cancer
Research.

ANTITUMOR ACTIVITY OF MAP

4@tosase
@ofBc&@@IIs*

f@ase

GaIN

@ofTceIIs@IIs

Ac

aLaJ@Ach

@!ch@

hMacrophage
@..

activatingfactor

Gd protoin
(MAF)I

Gal-

@ofBc&@*@IIs*

Fig. I . Schematic illustration of the formation of

Ib.

the MAF derived from Ge protein (a) and mouse DBP

(b) and the deglycosylation of Ge protein and mouse

..

VMacrophage

Mouse DBP

DBP (c). a, lysophosphatidylcholine-treated

B cells.

activating

/@cetyl
C.

@@c@Ser

4,

factor

(MAF)

@a9
@..

Deglycosylated
Gc protein

Deglycosylated

Mouse DB

(Gc 1)

/3-galactosidaseand sialidase to yield the cloned MAF, CdMAF. As shown in

Fig. 2, the size of CdMAF is 18.5% of the length of the GcMAF peptide.
HumanGcproteinconsistsof threegeneticgroups(proteinpolymorphism): Incubation of mouse peritoneal macrophages with 10 pg Cd.MAF/ml was
Gclf, Gels, and Gc2. Both Gclf and Gels subtypes of Gel carry sialic acid, required for 7-fold-increased phagocytic capacities and 15-fold-increased
Proteins, Enzymes, and Reagents

whereas

0c2

does

not (15). Gel

protein

(a mixture

of Gclf

and Gels)

and

superoxide-generating

mouse DBP were purified by vitamin D-affinity chromatography (18, 21, 23).
Glycosidases were purchased from Boehringer Mannheim (Indianapolis, IN)

and immobilized on Sepharose (21). p-Nitrophenyl N-acetyl-a-D-galac

tosaminide

was purchased

from Sigma

Chemical

Co. (St. Louis,

contamination

in the concentrated

zymes, human Ge protein, mouse DBP, and culture media.

posttransplantation

and galactose (18

@3-galactosidase (0.1 unit/ml)

DBPMAF. A small amount (100 pg/mI) of this product activates mouse

peritoneal macrophages for 7-fold-increased phagocytic capacities and 15Treatment
protein

carries

superoxide-generating

of Gd

Protein

a trisaccharide

capacities

by Immobilized
composed

(18).

Glycosidases.

Human Gel

of N-acetylgalactosamine

MAF, GcMAF. A small amount (10 pg/ml) of this product was required to

Treatment

macrophages
for 7-fold-increased
phagocytic
superoxide-generating
capacities.

of the Cloned Ge Domain by Immobilized

capac

Glycosidases

to

Yield the MAF, CdMAF. Chemical (i.e., cyanogen bromide) and proteolytic
fragmentations

of Ge peptide

(Mr 52,000;

458 amino

acid residues)

revealed

that a domain containing 85 amino acid residues at the COOH-terminal carries

the glycosylation site and is responsible for macrophage activation. Thus, this
domain was cloned via baculovirus
4 Unpublished

peritoneal cavity, DBPMAF, GcMAF, and CdMAF were administered i.m. for

the activation of macrophages. Approximately 3.5 h after the administration of

MAF, systemic macrophages (including monocytes) were activated and re
cruited to inflamed lesions.

The efficacy of DBPMAF, GcMAF, and CdMAF was assessed by time

course analyses that determinedthe total ascites tumor cell counts in the
peritoneal cavity and the serum NaGalase activity after administrations of
GcMAF, CdMAF, or DBPMAF (100 pg/mouse) four times (days 0, 4, 8, and
12) at 4-day intervals. At each data point, six mice were anesthetized, and sera

with di

branched galactose and sialic acid termini (1517,

21). Stepwise digestion of
Gel glycoprotein by immobilized f3-galactosidase and sialidase yields the
activate mouse peritoneal
ities and 15-fold-increased

but before the increase in cell counts.

To avoid deglycosylation of MAF by highly concentrated NaGalase in the

in 1 mM PBS by tumbling motion at pH 7.4 for 1 h to yield the MAF,

fold-increased

by serial passage

soon as they were transplanted. To assess the fate of the known cell counts of

Treatment of Mouse DBP by Immobilized Glycosidase. Mouse DBP

with immobilized

Ehrlich ascites tumor had been maintained

the transplanted tumor, the first administration of MAF should be given 6 h

In Vitro Treatment of DBP and Its Cloned Derivative with Immobilized

fi-Galactosidase and Sialidase

20). DBP (2 I.Lg) was incubated

Because

throughthe BALB/cmouseperitonealcavity,the ascitestumorcells grew as

stock solutions of en

carries a disaccharide composed of N-acetylgalactosamine

Treatment of Ehrlich Ascites Tumor-bearing Mice with Enzymatically

Generated MAF

MO). Using

the Limulus amebocyte lysate assay (2), we routinely tested for freedom of
lipopolysaccharide

capacities.

vector4 and treated with immobilized

observations.

were collected

from the vena cava inferior for NaGalase

assays.

For ascites

tumor cell counts in the peritoneal cavity, 57

ml of PBS were injected into the
peritoneal cavity and aspirated with a syringe, and the total number of ascites
tumor cells in the peritoneal lavage was determined.
Assay for NaGalase in the Sera of Ehrlich Ascites Tumor-bearing Mice
Proteins in mouse sera (100 s.d)were precipitated by 70% saturated ammo
nium sulfate. This high-salt precipitation

procedure also dissociates

the product

inhibitor from the enzyme. The precipitates were dissolved in 50 mxi citrate
phosphate buffer (pH 6.0) and dialyzed against the same buffer at 4C
overnight. The dialysates were made up to 1 ml in volume and assayed for
enzyme activity (10, 22, 24). Substrate solution (250 pi) contained 5 @tmol
of
p-nitrophenyl

N-acetyl-a-D-galactosaminide

in 50 mM citrate

buffer

2188

Downloaded from cancerres.aacrjournals.org on December 12, 2015. 1997 American Association for Cancer
Research.

(pH 6.0).

ANTITUMOR AcrIvrI-Y OF MAP

-16 14K R V L V L L L A

14

-12

LVLLLALAFGHALERGRDYEKDKVCNE

I6FSHLGKEDFISLSLVLYSRKFPSGTFEQVSQLVKEVVSLTEACCA

14 I6LAMLGKEDFRSLSLI

H
M

+1 -->DomainI
@AF G H A L E R G R D Y E K N K V C K E

LYSRKFSSSTFEQVNQLVKEVVSLTEECCA

61EGADPDCYDTRTSALSAKSCESNSPFPVHPGTAECCTKEGLERKL
61 E G A D P 1 C Y D I R I S E I S V K S C E S D A P F P V H P G I P E C C I K E G L E R K I

H 106 C N A L I

H Q P Q E F P 1 Y V E P 1 N D E I C E A F R

D P K E Y A P4Q F 14W E V S I N

14106 C N A L I S H Q P Q E F P 1 Y V E P 1 N D E I C E A F R R D P K G P A D Q F L V E Y S S N
Fig. 2. Homology ofthe sequences ofhuman Ge
protein (11)and mouse (M) DBP. Underlined resi
dues of human Ge protein are not identical to the

residues of mouse DBP. Numbers to the left, amino

acid residue positions. Domain divisions are

marked on the top of the sequences. Bold letters,
amino acid residues ofthe cloned CdMAF that span

residues 374458of the Ge protein.

Domain

II

H151YGQAPLSILVSYTKSYLSMVGSCCTSASPTVCFLKERLQLKH
14 151 Y G Q A P L P L I VA

-->
LSL

Y I K N Y I S 14 V G S C C I S A N P 1 V C F V K E R I Q 14 K H L SI

H 196 1 1 1 L S N R V C S Q Y A A Y G E K K S R I S N I I K L A Q K V P 1 A D I E D V L P L A E
14 196 1 1 1 14 S N R V C S Q V A A V G K E K S R I S H I I K I A Q K V P 1 A N I E N V I P I A E
H 241 D ! I t( I I S
C C E S A S E D C 14A K E I P E H I
K @,,
C
N I S I K N S K F E D C C Q
14 241 D F I E I I S R C C E S I S E D C M A S E L P E H I I K I C Q N L S K K N S K F E E C C Q
H 286 E K I A N D V F V C T V F 14P A A Q I P E I P 0 V E I P 1 N K D V C D P G N T K V H D K V
M 286 E N I P 14N I F M C I V F 14P A A E P L 0 1 P A I K I P 1 G K D L C G 0 S T I Q A N D 0 V
Cck4AF - ->

H 331 @1F E I S R R I H I P E V F I S K V I E P 1 L K S I G E C C D V E D S I T C F N A K G P 1
14 331 1 F E I S R R I Q V P E V F I S K V I E P 1 I K T L R E C C 0 1 0 D S V A C F S I Q S P I
-->

Domain

III

H 376 1 K K E I S S F I D K G Q E I C A D V S E N I F T E V K K K I A E R I K A K I P D A I P K
M 376 1 K R 0 1 1 5 F I E K G Q E 14C A D V S E N T F I E V K K K I A E R I R I K I P N I S P A
H 421 E I A K I V N K R S D F A S N C C S I N S P P 1 V C D S E I D A E I K N I I
14 421 E I K D 14 V E K H S D F A S K C C S I N S P P I V C S S Q I D A E 14 I D T I 0 5

The reaction was initiated by the addition of 250 pi of the dialysates, kept at
37Cfor 60 mm, and terminated by adding 200 j.d of 10% trichloroacetic acid.
After centrifugation, 300 @&l
of 0.5 M Na2CO3 solution was added to the

enzymatically generated MAF (DBPMAF or GCMAF), they survived for

approximately 35 4 days, as shown in Table 1. The control mice that
received ascites tumor only died in 16 2 days. When these mice
supernatant. The amount of p-nitrophenol released was determined spectro
received an additional administration of MAF at 4 days posttransplanta
photometrically at 420 nm with a Beckman DU600 spectrophotometer, and
tion, all 6 mice survived at least 50 days.
NaGalase activity was expressed in nanomoles/min/mg protein.
Mice that received 2 DBPMAF administrations, at days 4 and 8
after transplantation of 1 X l0@ ascites tumor cells, survived for
RESULTS
approximately 32 4 days. When the total tumor cell count in the
peritoneal
cavity was examined at day 4 after transplantation of
Antitumor
Effect of the Enzymatically
Generated
MAFs
1
X
l0@ascites
tumor cells, approximately S X l0@tumor cells were
(DBPMAF and GCMAF) on Mice Transplanted
with Ehrlich
detected
in
the
peritoneal
lavage. When mice received two adminis
Ascites Tumor. When 6 BALB/c mice were transplanted with 1 X 10@
trations of DBPMAF or GcMAF at days 0 and 4 after transplantation
Ehrlich ascites tumor cells and treated 6 h later with 100 pg/mouse of the
of 5 X iO@tumor cells, they survived up to 32 4 days. The control
mice that received 5 X l0@ ascites tumor cells only died in 14 2
Table 1 Therapy of mice bearing Ehrlich ascites tumor with the enzymatically
generated MAF (DBPMAF and GcMAF)

No. of mice

Posttransplantation treatment

No. of mice survived/period

A. Experiment1 (1 X i0@tumorcells/mouse; 100 pg of DBPMAF/mouse)

6 mice
6mice
6 mice
6daysB.
mice

Untreatedcontrol
DayO
Days 0 and 4
Days 4 and 8

6 micell6 2 days
6mice/35 4days
6 mice/>5O days
6 mice/32 4

GcMAF/mouse)6
Experiment2 (1 X lO@tumorcells/mouse; 100 pg of
mice
6mice
daysC.
6 mice

Untreated control
DayO
Days 0 and 4

6 mice/16 2 days
6mice/35 Sdays
6 mice/>50

DBPMAF/mouse)6
Experiment3 (5 x 1O@
tumorcells/mouse; 100 pg of
mice
6mice
6 mice
daysD.

Untreatedcontrol
DaysOand4
Days 0, 4, and 8

6 mice/14 2 days
6mice/32 4days
2 mice/35 and 38 days
4 mice/>53

GcMAF/mouse)6
Experiment4 (5 X lO@tumorcells/mouse; 100 pg of
mice
6 mice

Untreated control
Days 0 and 4

6 mice/14 2 days
6 mice/32 5 days

days.
Therefore, 2 administrations of DBPMAF or GcMAF to mice, at
days 0 and 4 after transplantation with 5 X l0@ ascites tumor cells,
allowed the mice to survive no more than 32 4 days. When the
number of ascites tumor cells in the peritoneal cavity was counted on
the 8th day, approximately 1 X l0@ or 1 X l0@ tumor cells were
detected in the peritoneal lavage from the mice treated with DBPMAF
or GcMAF, respectively. As shown in Table 2, at day 12, cell counts
in all treated mice increased to approximately 5 X 10@and 5 X l0@
cells, respectively. At day 24, the cell count in the peritoneal cavity
increased to more than 2 X l0@ cells in all treated mice. The total
tumor burden of more than 108 ascites cells in the peritoneal cavity
killed mice at 32 4 days (Table 1). Thus, more than three MAF
administrations were required for eradication of the ascites tumor. To
assess MAF efficacy and the curative rate of Ehrlich ascites tumor
bearing mice, measurement of the fate of the transplanted ascites
tumor cells in the peritoneal cavity was required as a prognostic index.
Correlation of Serum NaGalase Activity with Tumor Burden in
Mice Transplanted
with Ehrlich Ascites Tumor CeHs. NaGalase
activity can be detected in the sera of all cancer patients, but not in
healthy humans (10, 22). When mice were transplanted with S X I0@
Ehrlich ascites tumor cells and assayed for serum NaGalase activity,
2189

aSurvival
period
values
represent
mean

SE
osix
f mice.
The
results
were
reproduced

three times with a BALB/e or Swiss mouse.

Downloaded from cancerres.aacrjournals.org on December 12, 2015. 1997 American Association for Cancer
Research.

ANTITUMOR ACFIVITY OF MAP

Table 2 Thefate of Ehrlich ascites tumor cells in the peritoneal cavity of mice after
two administrations
No. of mice

ofDBPMAF,

was more effective in eliminating tumor cells than the equivalent

GcMAF (100 pg/mouse) therapy. As can be seen in Fig. 4, 4 DBP

GcMAF, or CdMAF at days 0 and 4

Days posttransplantation

assay

Survived tumor cell counts

MAF

Untreated control
6 mice
6 mice
Treated
6 mice
6 mice
6 mice
cells/mouseB.
6 mice

Day 8
Day 12

1 X l0@cells/mouse
1 X l0@cells/mouse

Day 8
Day 14
Day 24
Day 30

t X lO@cells/mouse
5 X l0@cells/mouse
2 X l0@cells/mouse
>l0@

GeMAF/mouse)Untreated
Experiment2 (5 x l0@tumorcells/mouse; 100 pg of
control
6 mice
6 mice
Treated
6 mice
6 mice
6 mice
cells/mouseC.
6 mice

Day 8
Day 12

I X l0@cells/mouse
1 X 108cells/mouse

Day 8
Day 14
Day 24
Day 30

1X
5 X
6 X
>

lO@cells/mouse
l0@cells/mouse
iO@cells/mouse
l0@

CdMAF/mouse)Untreated
Experiment3 (5 x l0@tumorcells/mouse; 100 pg of
control
6 mice
6 mice

Day 8
Day 12

1 X l0@cells/mouse
1 X l0@cells/mouse

Day 8
Day 14
Day 24

3 x l0@cells/mouse
2 X l0@cells/mouse
3 X 1ff cells/mouse

Treated

6 mice
6 mice
6 mice
cells/mouseLi
6 mice

Day 30

Results were reproduced three times with BALB/e

administrations

with 4-day

intervals

resulted

in an extended

survival of over 90 days and an insignificantly low serum NaGalase

level (1.81 0.15 nmol/min/mg) equivalent to that of control mice
(that received no transplantation) at 3090days after tumor trans
plantation, indicating complete eradication of the tumor. After day 30,
no ascites tumor cells were detectable in the peritoneal lavage.
Therapeutic Efficacy of CdMAF for Mice Bearing Ascites Tu
mor Cells Was Assessed by Serum NaGalase Activity. As shown
in Fig. 2, the cloned MAF, CdMAF, contained 18.5% of GcMAF (85
amino acid residues at the COOH-terminal). Thus, the amount of
the immunogenic epitopes nonhomologous to DBPMAF (mouse
DBPMAF) in CdMAF would likely be reduced more than 5-fold as
compared with GcMAF. When BALB/c mice received 5 X 10@
Ehrlich ascites tumor cells and were treated with 100 pg CdMAF/
mouse on days 0 and 4, they survived for 32 4 days. The control
mice that received the ascites tumor only died within 14 2 days.
When these mice were injected with 2 additional administrations on
days 8 and 12 (total of 4 administrations) of CdMAF, the mice
survived for at least 90 days. As in DBPMAF and GcMAF therapy,
the most beneficial CdMAF (100 pg/mouse) therapy regimen con
sisted of 4 treatments given 4 days apart, starting 6 h after transplan
tation (i.e., on days 0, 4, 8, and 12; Fig. 4). When the total cell counts
in the peritoneal cavity and serum NaGalase activity were examined
on days 30, 60, and 90, a low level of serum NaGalase activity
(2.31 0.19 nmollmin/mg) and approximately 20 Ehrlich ascites
tumor cells in the peritoneal lavage were persistently detected.

A. ExperimentI (5 x l0@tumorcells/mouse: 100 pg of DBPMAF/mouse)

> l0@
or Swiss mouse.

they all showed serum NaGalase that deglycosylates mouse DBP (Fig.
lc). This serum enzyme activity increased as the ascites tumor cells
grew in the peritoneal cavity. As shown in Fig. 3, the serum NaGalase
enzyme activity increased linearly with time as the Ehrlich ascites
tumor cells grew. Healthy control mice had 1.98 0.15 nmoL/min/mg
of serum enzyme activity, which is a-galactosidase capable of hy
drolysis of the same substrate as that for NaGalase (10). The enzyme
activities beyond that of control mice are considered to be NaGalase
activity derived from cancerous cells. Thus, serum NaGalase activity
is proportional to tumor burden (Fig. 3). In support of this observation,
the serum NaGalase activity in nude mice transplanted with human
oral squamous cell carcinoma is proportional to the tumor size (by
weight) (22). Thus, the proportionality of the serum NaGalase activity
to tumor burden allowed us to use this serum enzyme for prognostic
and diagnostic purposes.
Therapeutic Effect of GcMAF on Mice Bearing Ascites Tumor
Cells Was Monitored by Serum NaGalase Activity. When BALB/c
mice were transplanted with Ehrlich ascites tumor (5 X l0@ cells/
mouse) and treated with 4 administrations of GcMAF (100 pg/mouse)
given 4 days apart, starting 6 h after transplantation (i.e., on days 0, 4,
8 and 12), they survived for at least 90 days. After 30 days, a very low
level of serum NaGalase (2.81 0.1 1 nmol/min/mg) was detected
(Fig. 4). Although all mice were healthy up to 90 days, approximately
90 ascites tumor cells were persistently detected in the peritoneal
lavage at days 30, 60, and 90.
Antitumor Effect of DBPMAF on Mice Bearing Ascites Tumor
Was Assessed by Tumor-specific
Serum NaGalase Activity.
Mouse DBP and human Ge protein are highly conserved and share a
78% identical amino acid sequence (Ref. 25; Fig. 2). It is possible that
mice can develop antibodies against nonhomologous epitopes in hu
man protein (GcMAF), which would reduce its effectiveness with
regimens involving multiple administration of this agent during pro
tracted time intervals. Indeed, DBPMAF (100 pg/mouse) treatment

DISCUSSION
The results of the present study indicate that Ehrlich ascites tumor
in BALB/c mice can be efficiently eradicated by macrophage-directed
immunotherapy using DBPMAF. MAFs (i.e., DBPMAF, GcMAF,
and CdMAF) can activate macrophages, monocytes, and other phago
cytes such as osteoclasts (19, 20) but cannot stimulate B and T cells
(17). Moreover, MAFs stimulate the progenitor cells, resulting in a
dramatic increase in macrophage counts (nearly 30-fold) in the 4 days
after MAF treatment (19, 20, 26). Thus, the most beneficial therapeu
tic regimen consisted of four treatments given 4 days apart, starting
immediately after transplantation (i.e., on days 0, 4, 8, and 12).
Although 1 or 2 administrations of MAFs reduced the ascites tumor
cell counts by more than 1000-fold in the mouse peritoneal cavity,
tumor cells began to grow back several days after MAF administra

0
Ca
U

CE

E?
1U
Ca

o'al
U
U
01@.@@

Z'U

12

16

Time(Days)
Fig. 3. Time course study of serum NaGalase activity in a BALB/c mouse transplanted
with 5 X lO@Ehrlich ascites tumor cells. The results were reproduced three times, with
slightly different time intervals.

2190

Downloaded from cancerres.aacrjournals.org on December 12, 2015. 1997 American Association for Cancer
Research.

ANTITUMOR ACTIVITY OF MAF

16

0
0
Ca
U

Ea@
CE

Ca

0 Control
. GcMAF
U CdMAF

DBPMAF

00
8

Fig. 4. Time course analysis of the therapeutic

efficacy of DBPMAF, GeMAF, and CdMAF as
monitored by serum NaGalase activity in a BALB/c
mouse transplanted

with 5 X lO@Ehrlich ascites

tumor cells. The results were reproduced three

times, with slightly different time intervals.

ZCU

11k

6@l 90

Time (Days)

tion. If the ascites tumor growth is repeatedly suppressed by 4 ad

ministrations of MAF over 12 days, the ascites tumor did not grow
back for at least 90 days. In a separate study, we observed that the
GcMAF-treated Ehrlich tumor-bearing mice survived for at least 150
days. However, very small numbers (< 100) of ascites tumor cells

became dormant cells (2729)

in the peritoneal cavity of the GcMAF
or CdMAF-treated mice. The effectiveness of multiple administration
of MAFs during protracted time intervals suggests that immunity
developed against the tumor. The protracted MAF treatment under
suppression of tumor growth for more than 12 days seemed to be
required for immune development against the cancerous cells. In
support

of this concept,

the transplanted

Ehrlich

ascites

tumor

cells

weekly administration of GcMAF (100 ng) to cancer patients shows

promising results with a variety of human cancers (32, 33). Because
mammalian DBPs are highly conserved, GcMAF can be used for
macrophage activation and cancer therapy in various mammals.
Like human Ge protein in cancer patients (10), the precursor
activity of mouse DBP in cancer-bearing mice was lost or reduced,
because mouse DBP was deglycosylated by tumor-derived serum
NaGalase (see Fig. lc). GcMAF can bypass inactivated DBP and act
directly on macrophages for efficient activation. Macrophages of
cancer-bearing mice are capable of being activated, as demonstrated
by the activity of macrophages 3.5 h after GcMAF treatment. In the
current paper, we showed that a minute amount (100 pg/mouse) of
GcMAF has a potent curative activity for mice bearing Ehrlich ascites
tumor. When DBPMAF (100 pg/mouse) was administered to Ehrlich
ascites tumor-bearing mice, DBPMAF was found to have more cura
tive activity than GcMAF. Four administrations of DBPMAF to
Ehrlich ascites tumor-bearing mice rendered excellent curative re
sponses.
DBPMAF and GcMAF can be generated only from the glycosy
lated DBP and Ge protein. The amino acid sequence of mouse DBP
is 78% identical to that of the human Ge protein (25). The DBP of all
mammalian species carries only one oligosaccharide near the COOH
terminal (18). Gel (one of the major Ge isoforms) is 100% glycosy
lated, whereas only 10% of mouse DBP molecules are glycosylated

cannot grow in mice previously immunized with killed Ehrlich ascites

tumor cells.4 In fact, the tumoricidal capacity of macrophages is
observed preferentially via the IgG (Fc-receptor)-mediated pathway
(9, 30, 31). Moreover, the MAFs are potent adjuvants in immunization
for antibody production (21).
All cancer patients carry serum NaGalase that deglycosylates Gc
protein (Fig. lc), resulting in inactivation of the precursor activity of
Ge protein (10). GcMAF can bypass the inactivated Ge protein and
act directly on macrophages for efficient activation. Administered
GcMAF in cancer patients circulates rapidly and interacts with sys
temic macrophages within approximately 30 mm.4 Although GcMAF
administered in cancer patients could be theoretically deglycosylated
DBP cannot be converted
to
by NaGalase, the activity of serum NaGalase complexed with a (15, 18). Because nonglycosylated
DBPMAF, f3-galactosidase-treated mouse DBP as DBPMAF prepa
product inhibitor in the blood stream deglycosylates only a very small
ration contained the active DBPMAF form in only 10% of the total
fraction of GcMAF during the 30-mm circulation period. The
protein. Although 100 pg/mouse of both GeMAF and DBPMAF were
MAF-primed macrophages require 3 h for the development of full
used for each injection, the latter (DBPMAF) has 10 pg of active
phagocytic/cytocidal
capacity. The half-life of activated human
MAF/mouse. In spite of the 10-fold difference in MAF activity, the
macrophages is 56days (8). Preliminary studies revealed that
2191

Downloaded from cancerres.aacrjournals.org on December 12, 2015. 1997 American Association for Cancer
Research.

ANTITUMOR ACI1VITY OF MAP

DBPMAF treatment in the present study was more effective in dim

mating Ehrlich aseites tumor cells than GeMAF therapy with the
equivalent dosage. Such a finding may be explained by the possibility
that mice can develop antibodies against human protein GcMAF,
which would reduce its effectiveness with regimens involving multi
plc administration of this agent during protracted time intervals.
Commercial availability of interspecies antibodies against Ge protein

supports the hypothesis that interspecies antibodies are raised against

nonhomologous

amino acid sequences of DBP. Because GcMAF is a

potent

for the antibody-producing

adjuvant

capacity

of mice

(21),

administration of this heterologous protein to mice may produce at

least a low level of antibodies against GcMAF, in spite of highly
conserved DBPs. Because DBPMAF is of mouse origin, repeated
administration of various doses of DBPMAF to Ehrlich aseites tumor
bearing mice showed an excellent curative effect to the tumor, without
adverse immunological effects.
The peptide of CdMAF contains 85 amino acid residues that
span Ge protein amino acid residues 374458 (COOH-terminal),
as shown in Fig. 2. Because CdMAF is only 18.5% segment of the
GcMAF peptide at the COOH-terminal, the possibility of devel
oping antibodies against nonhomologous epitopes within the do
main in mouse would be reduced more than 5-fold as compared
with that of Ge protein. In fact, the data presented in this paper
support this hypothesis (see Fig. 4). Like GcMAF, CdMAF pro
duces no side effects in humans. Of clinical significance is the fact
that CdMAF is a nonhuman blood product.
In the present communication, our accumulated evidence enabled
us to design the appropriate frequency (four times) of administration
of MAF (100 pg/mouse) to demonstrate differential efficacy of
DBPMAF, GeMAF, and CdMAF (Fig. 4). Because serum
NaGalase activity is proportional to tumor burden in the hosts (22,
34), serum NaGalase activity along with the total tumor cell counts
in the peritoneal cavity seemed to be the most precise index for
the prognosis and curative state of the disease.

capacities of macrophages photodynamically activated with hematoporphyrin deny

ative. Photochem. Photobiol., 56: 245250,1992.
10. Yamamoto, N., Naraparaju, V. R., and Asbell, S. 0. Deglycosylation of senun
vitamin D-binding protein and immunosuppression in cancer patients. Cancer Res.,
56: 28272831,1996.
I 1. Homma, S., and Yamamoto, N. Activation process of macrophages after in vitro
treatment of mouse lymphocytes with dodecylglycerol. CIin. Exp. Immunol., 79:

307313,1990.
12. Yamamoto, N., Homma, S., and Miliman, I. Identification of the serum factor
required for in vitro activation of macrophages: role of yitamin D-binding protein
(group-specific component, Ge) in lysophospholipid activation of mouse penitoneal
macrophages. J. Immunol., 147: 273280,1991.
13. Yamamoto, N., Homma, S., Haddad, J. G., and Kowalski, M. N. Vitamin D3-binding

protein required for in vitro activation of macnophages after dodecyiglycerol treat

ment of mouse penitoneal cells. Immunology, 74: 420424,1991.
14. Homma, S., Yamamoto, M., and Yamamoto, N. Vitamin D-binding protein (group
specific component, Ge) is the sole serum protein required for macrophage activation
after treatment of peritoneal cells with lysophosphatidylcholine. Immunol. Cell Biol..
71: 249257,1993.

15. Yamamoto, N., and Homma, S. Vitamin D3-binding protein (group-specific compo
nent) is a precursor for the macrophage activating signal from lysophosphatidylcho
line-treated lymphocytes. Proc. Nail. Acad. Sci. USA, 88: 85398543,1991.
16. Yamamoto, N., and Kumashiro, R. Conversion of vitamin D3-binding protein (group

specific component) to a macrophage-activatingfactor by the stepwise action of a-ga

lactosidase of B cells and sialidase of T cells. J. ImmunoL, 151: 2794-2902, 1993.
17. Naraparaju, V. R., and Yamamoto, N. Roles of@-galactosidase ofB lymphocytes and

sialidase of T lymphocytes in inflammation-primed activation of macrophages. hn

munol. Lets., 43: 143148,1994.
18. Yamamoto, N., and Naraparaju, V. R. Role of mouse vitamin D3-binding protein in

activation of macrophages. J. Immunol., 157: 17441751,1996.

19. Yamamoto, N., and Naraparaju, V. R. A defect in @-galactosidase
of B lymphocytes
in the osteopetrotic (op/op) mouse. Immunol. Lett., 50: 3540,1996.
20. Yamamoto, N., and Naraparaju, V. R. A defect in inducible
@-galactosidase of B
lymphocytes in the osteopetrotic (mi/mi) mouse. Immunology. 88: 604610, 1996.
21. Yamamoto, N. Structural definition of a potent macrophage activating factor derived

from vitamin D3-binding protein with adjuvant activity for antibody production. Mol.
Immunol.,33: 11571164,
1996.
22. Yamamoto, N., Naraparaju, V. R., and Urade, M. Prognostic utility of serum a-N-

acetylgalactosaminidase and immunosuppression resulted from deglycosylation of

serum Ge protein in oral cancer patients. Cancer Res., 57: 295299,1997.
23. Link, R. P., Perlman, K. L, Pierce, E. A., Schnoes, H. K., and DeLuca, H. F.

Purification of human serum vitamin D-binding protein by 25-hydroxyvitamin D3Sepharose chromatography. Anal. Biochem., 157: 262269,1986.
24. Yamamoto, N., Naraparaju, V. R., and Snnivasula, S. M. Structural modification of

serum vitamin D3-binding protein and immunosuppression in fflV-infected patients.

AIDS Res. Hum. Retroviruses, 11: 13731378,1995.
25. Yang, F., Bergeron, J. M.. Linehan, L. A., Lalley, P. A., Sakaguchi, A. Y., and

Bowman. B. H. Mapping and conservation of the group-specific component gene in

mouse. Genomics, 7: 509516,1990.

ACKNOWLEDGMENTS
We thank Dr. Sidney Weinhouse for his critical advice, guidance, and
interest in our work.

26. Yamamoto, N., Lindsay, D. D., Naraparaju, V. R., Ireland, R. A., and Popoff, S. N.
A defect in the inflammation-primed macrophage activation cascade in osteopetrotic
(op) rats. J. Immunol., 152: 51005107, 1994.
27. Weinhold, K. J., Goldstein, L. T., and Wheelock, E. F. The tumor-dormant state:

quantitation of L5178Y cells and host immune responses during the establishment and
course of dormancy in syngeneic DBA/2 mice. J. Exp. Med., 149: 732744,1979.

REFERENCES

28. Weinhold, K. J., Goldstein, L. T., and Wheelock, E. F. The tumor-dormant

state

established with L5178Y lymphoma cells in immunized syngeneic munine hosts.

Nature (Lond.), 270: 5961,1977.

1. Ngwenya, B. Z., and Yamamoto, N. Activation of peritoneal maerophages by lyso

phosphatidylcholine. Biochim. Biophys. Acts, 839: 915,1985.

29. Robinson, M. K., and Wheelock, E. F. Identification of macrophage-mediated cyto

lytic activity as a tumor-suppressive mechanism during maintenance of the L5178Y

2. Ngwenya, B. Z., and Yasnamoto, N. Effects of inflammation products on immune

systems: lysophosphatidyleholine stimulates macrophages. Cancer Immunol. Immu

tumor-dormant state in DBA/2 mice. J. Immunol., 126: 673679,1981.

nother.,
21: 174182,
1986.
3. Ngwenya,B. Z., andYamamoto,N. Contributionof lysophosphatidyleholine-treated 30. Weiner, L. M., Moldofsky, P. J., Gatenby, R. A., O'Dwyer, J., O'Brien, J., Litwin, S.,
and Comis, R. L. Antibody delivery and effector cell activation in Phase II trial of
nonadherent cells to mechanism of macrophage activation. Proc. Soc. Exp. Biol.
recombinant

Med., 193: 118124,1990.

4. Morton, D., Eibler, F. R., Malmgren, R. A., and Wood, W. C. Immunological factors
which influence response to immunotherapy in malignant melanoma. Surgery (St.
Louis), 68: 158164,1970.
5. Thar, B., and Tanaka, T. Immunotherapy of cancer: regression of tumors after
intralesional injection of living Mycobacterium bovis. Science (Washington DC),
172:271273,
1971.
6. Yamamoto, N., and Ngwenya, B. Z. Activation ofmacrophages by lysophospholipids

and ether derivatives of neutral lipids and phospholipids. Cancer Res., 47: 2008
2013,1987.
7. Yamamoto, N., Ngwenya, B. Z., Sery, T. W., and Pieringer, P. A. Activation of

macrophages by ether analogues of lysophospholipids. Cancer Immunol. Immu

nother.,
25:185192,
1987.
8. Yamamoto, N., St. Claire, D. A., Homma, S., and Ngwenya, B. Z. Activation of
mouse macrophages by alkylglycerols, inflammation products of cancerous tissues.
Cancer Res., 48: 60446049,1988.
9. Yamamoto, N., Hoober, J. K., Yamamoto, S., and Yamamoto, N. Tumoricidal

y-interferon and munine monoclonal antibody CO17lA in advanced

colorectal carcinoma. Cancer Res., 48: 25682573,1988.

31. Houghton, A. L., Mintzer, D., Cordon-Cardo,

C., Welt, S., Fliegel, S., Vadhan, S.,

Carswell, E., Melamed, M., Oettgen, H. F., and Old, L. J. Mouse monoclonal 1g03
antibody detecting GD3 ganglioside: a Phase I trial in patient with malignant mela
noma. Proc. Nail. Acad. Sci. USA, 82: 12421246,
1985.
32. Naraparaju, V. R., Wimmers, R. S., Neil, R. N., Orchard, P. J., and Yamamoto, N.

Originof immunosuppression
injuvenileleukemiaandtherapeuticefficacyof vita
mm D3-binding protein-derived macrophage-activating factor. Proc. Am. Assoc.
Cancer Res., 37: 213, 1996.
33. Yamamoto, N., Naraparaju, V. R., Neil, R. N., Suyama, H., and Nakazato, H.
Therapeutic efficacy of vitamin D3-binding protein-derived macnophage-activating

factor for prostate, breast, and colon cancers. Proc. Am. Assoc. Cancer Res., 38: 31,
1997.
34. Koga, Y.. Naraparaju, V. R., and Yamamoto, N. Antitumor effects of vitamin
D@-binding protein-derived macrophage-activating factor on Ehrlich tumor-bearing

mice. Proc. Am. Assoc. Cancer Res., 37: 481, 1996.

2192

Downloaded from cancerres.aacrjournals.org on December 12, 2015. 1997 American Association for Cancer
Research.

Immunotherapy of BALB/c Mice Bearing Ehrlich Ascites Tumor

with Vitamin D-binding Protein-derived Macrophage Activating
Factor
Nobuto Yamamoto and Venkateswara R. Naraparaju
Cancer Res 1997;57:2187-2192.

Updated version

E-mail alerts
Reprints and
Subscriptions
Permissions

Access the most recent version of this article at:

http://cancerres.aacrjournals.org/content/57/11/2187

Sign up to receive free email-alerts related to this article or journal.

To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at pubs@aacr.org.
To request permission to re-use all or part of this article, contact the AACR Publications
Department at permissions@aacr.org.

Downloaded from cancerres.aacrjournals.org on December 12, 2015. 1997 American Association for Cancer
Research.