In vitro enzymatic conversion of glycosylated mammalian vitamin D-binding protein to a potent macrophage activating factor

A novel macrophage activating factor is prepared in vitro by treating glycosylated mammalian vitamin D-binding protein with glycosidases. Vitamin D-binding protein, which is isolated from blood or plasma of animals by known procedures, is thus readily converted to a highly potent macrophage activating factor.

FIELD OF THE INVENTION 
The invention relates to macrophage activation, in particular to the in 
vitro enzymatic production of a potent macrophage activating factor. 
BACKGROUND OF THE INVENTION 
A. Inflammatory Response Results in Activation of Macrophages 
Microbial infections of various tissues cause inflammation which results in 
chemotaxis and activation of phagocytes. Inflamed tissues release 
lysophospholipids due to activation of phospholipase A. Inflamed cancerous 
tissues produce alkyl-lysophospholipids and alkylglycerols as well as 
lysophospholipids, because cancerous cells contain alkylphospholipids and 
monoalkyldiacylglyercols. These lysophospholipids and alkylglycerols, 
degradation products of membranous lipids in the inflamed normal and 
cancerous tissues, are potent macrophage activating agents (Yamamoto et 
al., Cancer Res. 7:2008, 1987; Yamamoto et al., Cancer Immunol. 
Immunother. 25:185, 1987; Yamamoto et al., Cancer Res. 24:6044, 1988). 
Administration of lysophospholipids (5-20 .mu.g/mouse) and alkylglycerols 
(10-100 ng/mouse) to mice activates macrophages to phagocytize 
immunoglobulin G-coated sheep red blood cells. The macrophages phagocytize 
the target red blood cells via their receptors recognizing the Fc portion 
of the immunoglobulin G but not the C3b portion of the complement 
(Yamamoto et al., Cancer Res. 47:2008, 1987). 
In vitro treatment of mouse peritoneal macrophages alone with 
lysophospholipids or alkylglycerols results in no enhanced ingestion 
activity (Yamamoto et al., Cancer Res. 48:6044, 1988). However, incubation 
of peritoneal cells (mixture of macrophages and B and T lymphocytes) with 
lysophospholipids or alkylglycerols for 2-3 hours produces markedly 
enhanced Fc-receptor-mediated phagocytic activity of macrophages (Yamamoto 
et al., Cancer Res. 47:2008, 1987; Yamamoto et al., Cancer Res. 48:6044, 
1988). 
Incubation of macrophages with lysophospholipid- or alkylglycerol-treated B 
and T lymphocytes in a medium containing 10% fetal calf serum developed a 
greatly enhanced phagocytic activity of macrophages (Yamamoto et al., 
Cancer Res. 48:6044, 1988; Homma and Yamamoto, Clin. Exp. Immunol. 79:307, 
1990). Analysis of macrophage activating signal transmission among the 
nonadherent (B and T) lymphocytes has revealed that lysophospholipid- or 
alkylglycerol-treated B-cells can transmit a signalling factor to T-cells; 
in turn, the T-cells modify the factor to yield a new factor, which is 
capable of the ultimate activation of macrophages for ingestion capability 
(Yamamoto et al., Cancer Res. 48:6044, 1988). 
B. Vitamin D-Binding Protein 
Vitamin D-binding protein, also known as DBP, is an evolutionary conserved 
glycoprotein among animals (Cooke and Haddad, Endocrine Rev. 10:294 1989). 
DBP from animals serologically cross-reacts with human DBP (Ogata et al., 
Comp. Bioch. Physiol. 90B:193, 1988). Animal DBP is a genetically 
polymorphic plasma protein in some species and has a relative molecular 
weight of about 52,000. It normally constitutes about 0.5% of the plasma 
proteins in animals. The plasma concentration is generally about 260 
.mu.g/ml. Polymorphism of the human DBP, known as "group specific 
component" or "Gc protein" is demonstrable by gel electrophoretic 
analysis, which reveals two major phenotypes: Gc1 and Gc2 (Hirschfeld et 
al., Nature 185:931, 1960). The entire nucleotide coding sequences of the 
Gc1 and Gc2 genes, and the predicted amino acid sequences, have been 
reported (Cooke, et al., J. Clin. Invest. 76:2420, 1985; Yang et al., 
Proc. Natl. Acad. Sci. USA 82:7994, 1985). Gc1 is further divided into 
Gc1f and Gc1s subtypes which migrate electrophoretically as two bands, 
"fast" and "slow", (Svasti et al., Biochem. 18:1611, 1979). 
Coopenhaver et al., Arch. Biochem. Biophys. 226, 218-223 (1983) reported 
that a post-translational glycosylation difference occurs at a threonine 
residue, which appeared in a region of the protein having an amino acid 
difference between Gc1 and Gc2. 
Viau et al., Biochem. Biophys. Res. Commun. 117, 324-331 (1983), reported a 
predicted structure for the O-glucosidically linked glycan of Gc1, 
containing a linear arrangement of sialic acid, galactose and 
N-acetylgalactosamine linked to a serine or threonine residue. 
Polymorphism of mammalian DBP can be demonstrated by isoelectric focusing 
(Gahne and Juneja, Anim. Blood Grps. Biochem. Genet. 9:37, 1978; Van de 
Weghe et al., Comp. Biochem. Physiol. 73B:977, 1982; Ogata et al., Comp. 
Biochem. Physiol. 90B:193, 1988). 
The animal DBP may be purified by a variety of means, which have been 
reported in the literature. For example, DBP may be purified by 
25-hydroxyvitamin D.sub.3 -Sepharose.RTM. affinity chromatography from 
plasma of various animal species (Link, et al., Anal. Biochem. 157:262, 
1986). DBP can also be purified by actin-agarose affinity chromatography 
due to its specific binding capacity to actin (Haddad et al., Biochem. J. 
218:805, 1984). 
Despite the characterization and intensive study of the human and animal 
vitamin D-binding protein, and the existence of ready methods for their 
purification, the conversion of these proteins to a potent macrophage 
activity factor has not been demonstrated until the present invention. 
SUMMARY OF THE INVENTION 
A process for the production of a potent macrophage activating factor is 
provided. Animal vitamin D-binding protein, which is an evolutionary 
conserved animal protein which is serologically cross-reactive with 
group-specific component in human serum, is a precursor of the macrophage 
activating factor. Animal DBP is converted to the macrophage activating 
factor by the action of glycosidases of B and T cells. 
According to a process for preparing macrophage activating factor, animal 
DBP is contacted in vitro (i) with .beta.-galactosidase, or (ii) with 
.beta.-galactosidase in combination with sialidase, .alpha.-mannosidase or 
a mixture thereof. A potent macrophage activating factor is obtained in 
large quantities. 
According to one embodiment of the invention, animal DBP, which is believed 
to possess an oligosaccharide moiety which includes galactose and sialic 
acid residues (hereinafter "DBPgs"), is contacted with 
.beta.-galactosidase and sialidase to provide the macrophage activating 
factor. According to another embodiment, DBP which is believed to possess 
an oligosaccharide moiety which includes galactose and .alpha.-mannose 
residues (hereinafter "DBPgm") is contacted with .beta.-galactosidase and 
.alpha.-mannosidase. In yet another embodiment, DBP which is believed to 
possess an oligosaccharide moiety which includes a galactose residue 
without sialic acid or .alpha.-mannose (hereinafter "DBPg") is contacted 
with .beta.-galactosidase alone to form the macrophage activating factor. 
Because of DBP genetic polymorphism, the macrophage activating factor is 
preferably prepared by contacting animal DBP with all three enzymes to 
obtain the macrophage activating factor, particularly when DBP purified 
from pooled plasma of different individuals is utilized. 
The invention also relates to a macrophage activating factor prepared 
according to the above process or any embodiment thereof, and compositions 
comprising the macrophage activating factor in combination with a 
pharmaceutically acceptable carrier, for veterinary use. 
The invention further relates to a method for inducing macrophage 
activation in an animal in need thereof by administering to such animal a 
macrophage activating effective amount of the novel macrophage activating 
factor. 
"Animal DBP" as used herein means the genetically polymorphic animal 
(exclusive of human) glycoprotein, also known as "vitamin D-binding 
protein", including all genetic variations thereof, such as DBPg, DBPgs 
and DBPgm. The singular expression "DBP" is thus understood to encompass 
all such variants, unless stated otherwise. 
By "macrophage activation" is meant the stimulation of macrophages to an 
increased level of phagocytic activity.

DETAILED DESCRIPTION OF THE INVENTION 
A serum factor, which has been identified as animal DBP, is converted to a 
macrophage activating factor by the action of B and T cell glycosidases. 
DBP exists as a polypeptide having attached thereto a specific 
oligosaccharide, portions of which are readily removable by treatment with 
readily available glycosidases. These glycosidases are equivalent to the 
functions of B and T cells upon the DBP. Upon treatment with specific 
glycosidases, DBP is unexpectedly converted to a highly potent macrophage 
activating factor. Thus, efficient conversion of DBP to the macrophage 
activating factor is achieved in vitro, in the absence of B- and T-cells. 
The novel macrophage activating factor formed by the enzymatic treatment 
of DBP is substantially pure and of such high potency that administration 
to a host of even a trace amount (500 picogram/kg of body weight) results 
in greatly enhanced phagocytic macrophage activity. Since the enzymatic 
generation of the novel factor bypasses the functions of B- and T-cells in 
macrophage activation, it has utility as a potent adjuvant for vaccination 
and as a post-infection therapeutic agent for serious infectious diseases. 
T-cell lymphokine macrophage activating factor, also known as 
.gamma.-interferon, is generated by lymphokine-producing T-cells in small 
amounts, or is obtained by genetic engineering. The novel macrophage 
activating factor of the invention, on the other hand, may be readily 
obtained from DBP which can be readily purified from the plasma of animal 
blood according to known purification procedures. 
The polymorphic DBP phenotypes are expressed inter alia as differences in 
the oligosaccharide attached to the polypeptide portion of the DBP 
molecule. The novel macrophage activating factor of the invention may be 
efficiently produced from animal DBP by incubation with a combination of 
.beta.-galactosidase and sialidase, or a combination of 
.beta.-galactosidase and .alpha.-mannosidase. In some instances, treatment 
of DBP with .beta.-galactosidase alone efficiently yields the macrophage 
activating factor. The in vitro conversion of DBP to macrophage activating 
factor by the action of commercially available enzymes is so efficient 
that an extremely high activity of macrophage activating factor is 
obtained. 
Due to its genetic polymorphism in many animal species, DBP is preferably 
treated with all three enzymes, as an enzyme mixture. In particular, DBP 
obtained from pooled blood from several individuals of the species may 
contain more than one DBP type. Complete conversion of DBP to macrophage 
activating factor may thus most expeditiously be achieved by treatment 
with all three enzymes, as an enzyme mixture. 
DBPg treated with .beta.-galactosidase alone efficiently activates 
macrophages. Therefore, removal of galactose from DPBg results in the 
formation of the macrophage activating factor. On the other hand, two 
glycosidases are required to convert DBP from DBPgs and DBPgm animals. 
Conversion of DBPgs to macrophage activity factor requires incubation with 
the combination of .beta.-galactosidase and sialidase. DBPgm conversion 
requires .beta.-galactosidase and .alpha.-mannosidase. 
It is believed that animal DBP phenotypes and subtypes are characterized as 
glycoproteins having the following oligosaccharide structures linked to an 
amino acid residue of the protein portion of the molecule: 
______________________________________ 
Representative 
DBP Type 
Oligosaccharide Animal Species 
______________________________________ 
DBPgs 
##STR1## monkey, bovine, sheep, goat, pig, horse 
DBPgm 
##STR2## bovine 
DBPg GalGalNAc dog, cat, rat, mouse 
______________________________________ 
Without wishing to be bound by any theory, it is believed that the above 
glycosylation occurs on the protein portion of DBP through a threonine or 
serine residue occurring at an amino acid position corresponding to about 
position 420 of human DBP, or through a threonine or serine residue in the 
same vicinity, thus forming the O-glycosidic linkage 
GalNAc.alpha.(1.fwdarw.0)-Thr or GalNAc.alpha.(1.fwdarw.0)-Ser. Thus, 
without wishing to be bound by any theory, the novel macrophage activating 
factor is believed to comprise a protein in substantially pure form having 
substantially the amino acid sequence of DBP and a terminal 
N-acetylgalactosamine group linked to an amino acid residue. 
Animal DBP of high purity for use in the process of the invention is most 
readily prepared by 25-hydroxyvitamin D.sub.3 -Sepharose.RTM. affinity 
chromatography of animal blood according to the procedure of Link et al., 
Anal. Biochem. 157, 262 (1986), the entire disclosure of which is 
incorporated herein by reference. DBP may also be purified by 
actin-agarose affinity chromatography according to the procedure of Haddad 
et al., Biochem. J. 218, 805 (1984), which takes advantage of the binding 
specificity of DBP for actin. The entire disclosure of Haddad et al., is 
incorporated herein by reference. Other methods of obtaining DBP in high 
purity are reported in the literature The known procedures utilized for 
purifying the corresponding human protein, Gc protein, are directly 
applicable to the purification of animal DBP. 
The glycosidases utilized in the practice of the invention are well known 
and commercially available. .beta.-Galactosidase, (.beta.-D-galactosidase 
galactohydrolase, EC 3.2.1.23) is obtained from Escherichia coli. 
.beta.-Galactosidase is available, for example, from Boehringer Mannheim 
Biochemicals, Indianapolis, Ind., cat. no. 634395. 
.alpha.-Mannosidase (.alpha.-D-mannoside mannohydrolase, EC 3.2.1.24) is 
obtained from the jack bean (Canavalia ensiformis). It is available, for 
example, from Boehringer Mannheim Biochemicals, cat. no. 269611. 
Sialidase, also known as "neuraminidase" (acylneuraminyl hydrolase EC 
3.2.1.18), is obtained from Clostridium perfringens, Vibrio cholerae or 
Arthrobacter ureafaciens. All three forms of sialidase are available from 
Boehringer Mannheim Biochemicals, cat. nos. 107590, 1080725 and 269611. 
DBP is readily converted to the macrophage activating factor by contact 
with a hydrolytic-effective amount of one or more of the above 
glycosidases. Any amount of enzyme sufficient to achieve substantially 
complete conversion of DBP to macrophage activating factor may be 
utilized. About 0.1 units (1 unit being the amount of enzyme which 
catalyzes 1 .mu.mole of substrate in 1 minute) of each enzyme per 1 .mu.g 
of DBP is more than sufficient for this purpose. Preferably, an excess of 
the amount of enzyme actually necessary to convert the glycoprotein to 
macrophage activating factor is utilized to insure complete conversion. 
The DBP and enzymes may be contacted in, for example, phosphate buffer or 
acetate buffer. A phosphate buffer is preferred (pH 5.5). Other media 
known to those skilled in the art for conducting enzymatic reactions may 
be substituted. 
The reaction may be carried out at any temperature suitable for conducting 
enzymatic reactions. Typically, the temperature may vary from 25.degree. 
C. to 37.degree. C., with about 37.degree. C. being preferred. The 
substrate and enzyme(s) are allowed to incubate in the reaction media 
until substantial conversion of DBP to macrophage activating factor is 
achieved. While it may be appreciated that the actual incubation times 
employed may depend upon several factors such as the concentration of the 
reactants, the reaction temperature, and the like, a reaction time of 
about 30 minutes at 37.degree. C. is generally sufficient to obtain 
complete conversion of DBP to macrophage activating factor. 
Conversion of DBP to macrophage activating factor may be conducted in any 
vessel suitable for enzymatic reactions. It is preferred that sialidase is 
utilized in insoluble form, e.g., attached to beaded agarose (Sigma 
Chemical Co., cat. no. N-4483), to avoid contamination of the resulting 
macrophage activating factor with sialidase fragments of similar molecular 
weight. The macrophage activating factor may be produced by adding the 
appropriate enzyme(s) to DBP in a liquid medium, followed by subsequent 
filtration of the liquid to recover the macrophage activating factor. For 
example, the enzyme-DBP reaction mixture may be passed through a 
sterilized 100 kDa cut off filter (e.g. Amicon YM 100) to remove the 
immobilized sialidase, .beta.-galactosidase (MW=540 kDa) and 
.alpha.-mannosidase (MW=190 kDa). The filtrate contains substantially pure 
macrophage activating factor of high activity. Where the conversion of 
large quantities of DBP to macrophage activating factor is desired, all 
enzymes are most advantageously contained in the solid phase. 
.beta.-Galactosidase, and sialidase or .alpha.-mannosidase, most 
preferably a mixture of all three enzymes, is affixed to, e.g., agarose 
beads with a suitable coupling agent such as cyanogen bromide. Methods for 
attaching enzymes to solid supports are known to those skilled in the art. 
Conversion of DBP to macrophage activating factor by means of incubation 
with immobilized enzymes is preferred, as the subsequent step of 
separating the macrophage activating factor from the enzyme mixture is 
obviated. 
Regardless of whether immobilized or liquid phase enzyme is utilized, it is 
desired to pass the product mixture through an ultrafilter, preferably a 
filter having a pore size no larger than about 0.45.mu., to provide an 
aseptic preparation of macrophage activating factor. 
B-cells possess the function corresponding to .beta.-galactosidase. T-cells 
carry the functions corresponding to sialidase and .alpha.-mannosidase. 
Without wishing to be bound by any theory, it is believed that DBP is 
modified in vivo in an ordered sequence by the membranous enzymes of B and 
T lymphocytes to yield macrophage activating factor. 
Activation of macrophages, which is characterized by their consequent 
enhanced phagocytic activity, is the first major step in a host's immune 
defense mechanism. Macrophage activation requires B and T lymphocyte 
functions, which modify DBP in a step-wise fashion, to yield the novel 
macrophage activating factor. Since the glycosidases used for in vitro 
conversion of DBP to macrophage activating factor according to the present 
invention correspond to the B- and the T-cell function required for 
production of macrophage activating factor, the in vitro enzymatic 
generation of the macrophage activating factor bypasses the functions of 
B- and T-cells. Moreover, since the herein described macrophage activating 
factor may be generated from blood of the same animal species undergoing 
treatment, side effects, such as immunogenicity, are believed to be 
minimal. 
Following infection, microbial antigens are bound by macrophages. Most of 
this surface-bound antigen is internalized (i.e., phagocytized), and 
processed by digestion. The macrophages return some processed antigens to 
their surfaces so that antigenic determinants can be "presented" 
efficiently to antigen-specific lymphocytes. However, the binding, 
phagocytosis, processing and presentation of antigens requires that the 
macrophage first be activated. Development of the immune response 
following infection is thus typically delayed for 1-2 weeks, pending 
complete macrophage activation. This is the period during which B- and 
T-cells participate in generating the macrophage activating factor. During 
this lag period, the infection may become well-established. 
I have observed the occurrence of macrophage activation in mice in less 
than six hours following administration of the macrophage activating 
factor prepared from DBP. Substantial antibody production is observed in 
mice in as little as 48 hours after coinjection of the macrophage 
activating factor and antigen. A large amount of antigen-specific antibody 
is produced within 96 hours. It is thus contemplated that the macrophage 
activating factor of the present invention, which is capable of inducing 
extemely rapid activation of macrophages, will be useful as an adjuvant 
for vaccination to enhance and accelerate the development of the immune 
response and to generate a large amount of antigen-specific antibodies. 
For the same reason, it is further contemplated that the macrophage 
activating factor will find utility as a post-infection therapeutic agent 
to accelerate antibody production, either alone or in combination with 
other therapeutic agents. This therapy should be particularly effective in 
treating infectious diseases with long incubating periods, such as rabies. 
To minimize any possible immunologic reaction from administration of the 
macrophage activating factor, it is preferred that each animal species 
would receive only macrophage activating factor derived from the blood of 
the same species. Similarly, the risk of immunologic reaction in 
individual animals would be minimized by administering only the same 
variant of DBP-derived macrophage activating factor, in situations wherein 
there is intraspecies DBP polymorphism. 
The macrophage activating factor may be administered to an animal to induce 
macrophage activation, either alone or in combination with other 
therapies. The amount of macrophage activating factor administered depends 
on a variety of factors, including the potency of the agent, the duration 
and degree of macrophage activation sought, the size and weight of the 
subject, the nature of the underlying affliction, and the like. Generally, 
administration of as little as about 0.5 ng of factor per kg of the 
subject's body weight will result in substantial macrophage activation. 
According to one treatment, an animal may receive about 2 ng of macrophage 
activating factor per kilogram of body weight every three to five days to 
maintain a significant level of macrophage activation. 
The macrophage activating factor may be administered by any convenient 
means which will result in delivery to the circulation of an amount of the 
factor sufficient to induce substantial macrophage activation. For 
example, it may be delivered by intravenous or intramuscular injection. 
Intramuscular administration is presently preferred as the route of 
administration. 
The macrophage activating factor may be taken up in pharmaceutically 
acceptable carriers, particularly those carriers suitable for delivery of 
proteinaceous pharmaceuticals. The factor is soluble in water or saline 
solution. Thus, the preferred formulation for veterinary pharmacological 
use comprises a saline solution of the agent. The formulation may 
optionally contain other agents, such as agents to maintain osmotic 
balance. For example, a typical carrier for injection may comprise an 
aqueous solution of 0.9% NaCl or phosphate buffered saline (a 0.9% NaCl 
aqueous solution containing 0.01M sodium phosphate, .apprxeq.pH 7.0). 
The invention is illustrated by the following non-limiting examples. 
EXAMPLE 1 
A. Conversion of DBP to Macrophage Activating Factor 
Purified DBP (1.0 .mu.g) obtained from (A) cow, (B) pooled blood of seven 
cows, (C) cat or (D) dog was combined with 1 ml of phosphate-buffered 
saline (PBS-Mg) containing 0.01M sodium phosphate, 0.9% NaCl and 1 mM 
MgSC.sub.4 and treated with 2 .mu.l of PBS-Mg containing 0.1 U of the 
enzyme combinations indicated in Table 1. The enzymes utilized were as 
follows: 
Sialidase (Boehringer Mannheim Biochemicals, cat. no. 107590). 
.alpha.-Mannosidase (Boehringer, cat. no. 107379). 
.beta.-Galactosidase (Boehringer, cat. no. 634395). 
The respective enzyme-DBP mixtures were incubated in microcentrifuge tubes 
for sixty minutes at 37.degree. C. The reaction mixture containing the 
enzyme-treated DBP was then diluted 10.sup.-4 in 0.1% egg albumin (EA) 
supplemented medium, for the following assay. 
B. In Vitro Assay of Macrophage Activating-Factor 
1. Preparation of Macrophage Tissue Culture 
Peritoneal cells were collected by injecting 5 ml of phosphate buffered 
saline, containing 0.01M sodium phosphate, 0.9% NaCl and 5 units/ml 
heparin into the peritoneal cavity of BALB/c mice. Peritoneal cells were 
removed and washed by low speed centrifugation and suspended in a tissue 
culture medium RPMI 1640 supplemented with 0.1% egg albumin (EA medium) at 
a concentration of 1-2.times.10.sup.6 cells/ml. 1 ml aliquots of the cell 
suspension were layered onto 12 mm coverglasses which had been placed in 
the 16 mm diameter wells of tissue culture plates (Costar, Cambridge, 
Mass.). The plates were incubated at 37.degree. C. in a 5% CO.sub.2 
incubator for 30 minutes to allow macrophage adherence to the coverglass. 
The coverglasses were removed, immersed with gentle agitation in RPMI 
medium to dislodge non-adherent B and T cells, and placed in fresh tissue 
culture wells containing EA-medium. 
2. Preparation of Sheep Erythrocyte/Rabbit Anti-erythrocyte IgG Conjugates 
Washed sheep erythrocytes were coated with subagglutinating dilutions of 
the purified IgG fraction of rabbit anti-sheep erythrocyte antibodies. A 
0.5% suspension of rabbit IgG-coated sheep erythrocytes in RPMI 1640 
medium was prepared for use in the following phagocytosis assay. 
3. Phagocytosis Assay. 
1 ml aliquots of the diluted reaction mixture from A., above, were layered 
onto the macrophage-coated cover-glasses from B.1., above, and incubated 
for 2 hours in a 5% CO.sub.2 incubator at 37.degree. C. The culture media 
was then removed and 0.5 ml of the 0.5% erythrocyte-IgG conjugate 
suspension were added to the macrophage-coated cover-glasses and incubated 
for 1 hour at 37.degree. C. The coverglasses were then washed in a 
hypotonic solution (1/5 diluted phosphate buffered saline in water) to 
lyse non-ingested erythrocytes. The macrophages with ingested erythrocytes 
were counted. The average number of erythrocytes ingested per macrophage 
was also determined. Macrophage phagocytic activity was calculated as an 
"Ingestion index" (the percentage of macrophages which ingested 
erythrocytes times the average number of erythrocytes ingested per 
macrophage). The data are set forth in Table 1. 
TABLE 1 
______________________________________ 
Macrophage Activation by 
Glycosidase-treated DBP 
Ingestion Index 
B 
Glycosidases for 
A .sup. pooled.sup. 
C D 
treatment of DBP 
bovine.sup. 
bovine.sup. 
cat dog 
______________________________________ 
-- 55 .+-. 10 
67 .+-. 15 
77 .+-. 12 
73 .+-. 19 
Sialidase 59 .+-. 15 
71 .+-. 19 
80 .+-. 21 
59 .+-. 10 
.beta.-galactosidase 
63 .+-. 18 
76 .+-. 15 
278 .+-. 35 
284 .+-. 41 
Mannosidase 
61 .+-. 13 
73 .+-. 28 
69 .+-. 15 
62 .+-. 26 
.beta.-galactosidase + 
295 .+-. 34 
335 .+-. 32 
269 .+-. 31 
265 .+-. 37 
sialidase 
.alpha.-mannosidase + 
67 .+-. 22 
54 .+-. 12 
73 .+-. 20 
67 .+-. 26 
sialidase 
.beta.-galactosidase + 
72 .+-. 15 
188 .+-. 38 
266 .+-. 38 
252 .+-. 33 
mannosidase 
______________________________________ 
It is apparent from Table 1 that bovine species display polymorphism with 
respect to DBP type. While the purified DBP from a single bovine 
individual (column A) was converted to macrophage activating factor by 
treatment with a combination of sialidase and .beta.-galactosidase, 
treatment with .beta.-galactosidase and either sialidase or 
.alpha.-mannosidase resulted in generation of macrophage activator from 
DBP purified from pooled bovine plasma of seven cows. It is thus apparent 
that the single bovine individual was of DBP type "gs" and that the pooled 
material was composed of DBP from both DBPgs and DBPgm individuals. 
Similarly, it is apparent from Table 1 that the cat and dog DBP donors 
were type DBPg, since treatment with galactosidase alone was sufficient 
for generation of macrophage activating factor. 
The effect of macrophage activating factor concentration on activity was 
investigated by treating the same bovine DBPgs, pooled bovine DBP, and cat 
DBPg according to Example 1, at glycosidase-treated DBP dilutions of 
10.sup.-4, 10.sup.-5 and 10.sup.-6 of the original 1.0 .mu.g/ml solution. 
The results are set forth in Table 2 (bovine DBPgs), Table 3 (pooled 
bovine DBP) and Table 4 (cat DBPg). 
TABLE 2 
______________________________________ 
Macrophage activation by 
Glycosidase-treated Bovine DBPgs 
Ingestion Index 
Dilution of Bovine DBP 
Glycosidase- Bovine DBP treated with 
Treated untreated .beta.-galactosidase 
Bovine DBPgs control and sialidase 
______________________________________ 
10.sup.-4 63 .+-. 12 289 .+-. 11 
10.sup.-5 59 .+-. 15 322 .+-. 35 
10.sup.-6 55 .+-. 18 116 .+-. 22 
______________________________________ 
TABLE 3 
______________________________________ 
Macrophage Activation by 
Glycosidase-treated pooled bovine DBP 
Dilution of 
Ingestion Index 
Glycosidase- 
Bovine Bovine DBP Bovine DBP 
Treated pooled 
DBP treated with 
treated with 
Bovine DBPgs 
untreated 
.beta.-galactosidase 
.beta.-galactosidase 
and DBPgm control and sialidase 
and .alpha.-mannosidase 
______________________________________ 
10.sup.-4 72 .+-. 25 
312 .+-. 38 285 .+-. 38 
10.sup.-5 83 .+-. 20 
297 .+-. 45 203 .+-. 36 
10.sup.-6 76 .+-. 18 
145 .+-. 34 122 .+-. 23 
______________________________________ 
TABLE 4 
______________________________________ 
Macrophage Activation by 
Glycosidase-treated Cat DBPg 
Ingestion Index 
Dilution of Cat DBP 
Glycosidase- untreated 
Cat DBP treated with 
Cat DBPg control .beta.-galactosidase 
______________________________________ 
10.sup.-4 68 .+-. 26 
320 .+-. 29 
10.sup.-5 65 .+-. 23 
275 .+-. 23 
10.sup.-6 76 .+-. 20 
108 .+-. 34 
______________________________________ 
EXAMPLE 3 
Purified DBP (1.0 .mu.g from each of the species identified in Table 5, 
below, was treated according to Example 1 with a mixture of 
.beta.-galactosidase, sialidase and .alpha.-manosidase (0.5 U each) in 1 
ml of PBS-Mg containing 0.01M sodium phosphate, 0.9% NaCl and 1 mM 
MgSO.sub.4 for sixty minutes at 37.degree. C. The reaction mixture 
containing each treated DBP was then diluted 10.sup.-4 in 0.1% 
supplemented EA medium and assayed for macrophage activation activity 
according to the in vitro assay of Example 1B. The results are set forth 
in Table 5. It may be observed that treatment with a mixture containing 
all three enzymes resulted in conversion of DBP to a potent macrophage 
activating factor, regardless of DBP polymorphism. 
TABLE 5 
______________________________________ 
Ingestion Index 
Treated with 
Glycosidase- Untreated .beta.-galactosidase + 
mannosidase control sialidase + .alpha. 
______________________________________ 
Monkey (Macaca fucata) 
72 .+-. 26 
295 .+-. 38 
Bovine (Bos taurus) 
52 .+-. 19 
320 .+-. 52 
Sheep (Ovis aries) 
48 .+-. 17 
313 .+-. 48 
Goat (Capra hircus) 
56 .+-. 24 
289 .+-. 32 
Pig (Sus scrofa) 
47 .+-. 12 
332 .+-. 27 
Horse (Equus caballus) 
69 .+-. 23 
266 .+-. 38 
Cat (Felis catus) 
58 .+-. 15 
328 .+-. 43 
Dog (Canis familigris) 
60 .+-. 17 
337 .+-. 18 
Rat (Fisher) 65 .+-. 25 
284 .+-. 37 
Mouse (BALB/C) 
71 .+-. 28 
276 .+-. 34 
______________________________________ 
EXAMPLE 4 
A. Conversion of DBP to Macrophage Activating Factor with Immobilized 
Enzyme 
1. Preparation of Immobilized Enzymes 
100 mg of CNBr-activated agarose (Sepharose 4B) was washed with 1 mM HCl 
and suspended in coupling buffer (300 .mu.l) containing NaHCO.sub.3 buffer 
(0.1M, pH 8.3) and NaCl (0.5M). .beta.-Galactosidase, .alpha.-mannosidase 
or sialidase, or a combination of all three enzymes (2 U each enzyme), 
were mixed in 600 .mu.l of the coupling buffer and incubated at room 
temperature for 2 hours in an end-over-end mixer. Remaining active groups 
in the agarose were blocked by incubation with 0.2M glycine in coupling 
buffer for 2 hours at room temperature. The agarose-immobilized enzyme was 
washed with coupling buffer to remove unabsorbed protein and glycine, 
followed by washing with acetate buffer (0.1M, pH 4) containing NaCl 
(0.5M), and additional coupling buffer. The agarose-immobilized enzyme 
preparations were stored at 4.degree. C. 
2. Conversion of DBP to Macrophage Activating Factor 
DBP in 1 ml of PBS-Mg (pH 5.5) was combined with a mixture of the 
above-prepared agarose-immobilized enzymes (2 units each enzyme) in 1 ml 
of PBS-Mg (pH 5.5). The reaction mixtures were incubated in 5 ml plastic 
tubes at 37.degree. C. in an end-over-end mixer for 30 minutes. The 
reaction mixtures were thereafter spun with a table-top centrifuge at 
2,000 rpm for 15 minutes. The supernatant of each reaction mixture was 
collected, filtered through a sterilized 0.45.mu. pore size filter (type 
HA, Millipore Company, Bedford, Mass.), and diluted. 
B. In Vivo Assay of Macrophage Activating Factor 
The enzymatically-modified DBP (100, 30, 10, 3 and 1 picogram samples) were 
administered intramuscularly to BALB/c mice weighing .about.20 grams. At 
18 hours post-administration, peritoneal cells were collected and placed 
on 12 mm coverglasses in the 16 mm wells of tissue culture plates. The 
plates were incubated at 37.degree. C. for 30 minutes to allow adherence 
of macrophages. The coverglasses were washed in RPMI 1640 medium to 
dislodge non-adherent cells, and then placed in new wells. Rabbit 
IgG-coated sheep erythrocytes as prepared in Example 1B.2. were layered 
onto the coverglass, and a phagocytosis assay was performed as in Example 
1B.3. The results are set forth in Table 6: 
TABLE 6 
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In Vivo Assay of Macrophage Activation 
by Glycosidase-treated Bovine DBPgs 
Dosage of 
Ingestion Index 
enzymatically 
Bovine DBP Dog DBP 
modified treated with treated with 
DBP .beta.-galacto- .beta.-galacto- 
(picogram/ 
untreated 
sidase and untreated 
sidase and 
mouse) control sialidase control 
sialidase 
______________________________________ 
100 63 .+-. 18 
283 .+-. 42 
55 .+-. 22 
272 .+-. 29 
30 56 .+-. 17 
341 .+-. 38 
43 .+-. 12 
295 .+-. 35 
10 52 .+-. 18 
315 .+-. 44 
63 .+-. 17 
277 .+-. 41 
3 51 .+-. 12 
141 .+-. 27 
51 .+-. 15 
128 .+-. 27 
1 65 .+-. 15 
86 .+-. 12 
60 .+-. 18 
89 .+-. 26 
______________________________________ 
The present invention may be embodied in other specific forms without 
departing from the spirit or essential attributes thereof and, 
accordingly, reference should be made to the appended claims, rather than 
to the foregoing specification, as indicating the scope of the invention.