Note: Descriptions are shown in the official language in which they were submitted.
1307737
USE OF ANTIBODY/ANTIGEN INTERACTIONS TO PROTECT OR
MODULATE BIOLOGICAL ACTIVITY
Backaround of the Invention
This invention relates to the use of
antibody/antigen interactions to protect biologically
active entities against in vivo and in vitro
inactivation.
Biologically active entities, such as enzymes,
hormones, growth factors, antibodies and drugs, are
used in a variety of medical and industrial
applications in which their useful life may be
shortened by inactivation. Such inactivation may
result from physical, chemical or biological
processes or conditions, or by self-destruction in
the case of certain enzymes, occurring concurrently
with the processes of the desired activity in a
particular application: the inactivation may result
from a combination of such inactivating processes.
In some cases the inactivation occurs rapidly,
necessitating frequent replacement of the active
entity.
V. V. Mozhaev et al, Enzyme Microb. Technol.,
1984, Vol. 6, page 50 et seq., review structure-
~3()7737
stability relationships in proteins and existingapproaches to stabilizing proteins. In Chemical &
Ena'r News, September 30, 1985, page 19 et seq., there
is a description of albumin/enzyme complexes that are
resistant to proteolytic and heat inactivation, while
U.S. patent No. 4,179,337 refers to polyethylene
glycol/enzyme complexes which are also inactivation-
resistant.
Biologically active entities are employed in a
labelled form in different environments, for example,
in immunological detection and diagnostic processes.
The label may typically be a radioactive isotope
label, enzyme label, fluorescent label or a label
which can be determined photometrically. One
limitation on the selection of the label is that it
should not react with, and potentially inactivate, a
site of desired biological activity of the
biologically active entity.
Several solutions have been proposed to 910w
deterioration of activity in specific cases where a
biologically active entity is subjected to
inactivating conditions. However, no general approach
has previously been formulated to counter the
different inactivation processes with one type of
agent.
Summar~ of the Invention
It is therefore an object of this invention to
employ interactions between antibodies and
biologically active antigens to effect a modulation,
and particularly a prolongation, of their activity.
.- ~ . . , , .. . . .. ; . ,,. ~ . . . . . ~ -, . . . . . . .. .
1307737
It is a further object of this invention to
provide a biologically active entity stabilized
against inactivation of a desired biological
activity.
It is yet another object of the present
invention to provide a mechanism for slow-release of
a biologically active entity in order to provide
sustained activity.
In accomplishing the foregoing objects, there
has been provided, in accordance with one aspect of
the present invention, a method of producing a
biologically active complex characterized by an
enhanced resistance to inactivation, comprising the
steps of (A) providing a complex comprised of a
molecule which is biologically active and an antibody
entity that recognizes that molecule, said complex
being biologically active; and (B) measuring a
prolongation of biological activity, when the
complex is subjected to a condition that inactivates
the uncomplexed molecule, relative to inactivation of
the molecule by the condition in question. In
preferred embodiments, step (A) comprises exposing
the biologically active molecule to either polyclonal
antibody or a monoclonal antibody that recognizes the
molecule. In another preferred embodiment, the
antibody entity is an antibody fragment or an
antigen-binding protein.
In accordance with another aspect of the
present invention, a complex has been provided that
comprises (i) a molecule having a biological activity
and (ii) an antibody entity recognizing that
molecule, which complex displays a biological
activity that is inactivation-resistant, relative to
1307737
that of the free molecule. In a preferred
embodiment, the molecule is an enzyme.
A method has also been provided, in accordance
with still another aspect of the present invention,
that comprises the steps of (l) providing a complex
comprised of a molecule which i8 biologically active
and an antibody entity that recognizes the molecule,
said complex presenting at least one binding site for
a labelling agent; (2) exposing the complex to the
labelling agent such that the labelling agent is
bound to the binding site; and then (3) effecting a
disassociation of the complex to release the molecule
carrying said labelling agent. In a preferred
embodiment, the biologically active molecule is
itself an antibody.
Other objects, features and advantages of the
present invention will become apparent from the
following detailed description. It should be
understood, however, that the detailed description
and the specific examples, while indicating preferred
embodiments of the invention, are given by way of
illustration only, since various changes and
modifications within the spirit and scope of the
invention will become apparent to those skilled in
the art from this detailed description.
Brief Description of the Drawinas
The present invention is illustrated in the
accompanying drawings, in which:
FIGURE 1 is a graph depicting the loss in
activity over time at 70C of ~-amylase compared with
the same enzyme stabilized in accordance with the
present invention.
1307737
FIGURE 2 is a graph showing the loss, with
increasing temperature, in activity of the
biologically active entity in Figure 1, compared with
the same entity stabilized in accordance with the
present invention.
FIGURE 3 i8 a graph wherein the residual
activity (in the presence of trypsin) of the
biologically active entity, asparaginase, is plotted
as a function of the increase in concentration of
different antibody entities, or other proteins,
employed to protect biological activity in accordance
with the present invention;
FIGURE 4 is a graph showing the residual
activity of asparaginase in the presence of trypsin,
when the enzyme is protected with different
combinations of monoclonal antibody in accordance
with the present invention with time.
FIGURE 5 is a graph depicting the loss of
activity of asparaginase when that biologically
active entity is subjected to pH 3.0 at 37C compared
with the prolongation of activity displayed by
protected enzyme, in accordance with this invention,
under the same conditions.
FIGURE 6 demonstrates graphically the loss of
activity due to self-destruction over time of a
biologically-active entity (trypsin) compared with
trypsin protected in accordance with the present
invention.
FIGURE 7 is a graph showing the loss of
activity by another biologically active entity,
subtilisin, in the presence of 0.05% NaOCl, compared
with the same entity protected in accordance with
this invention.
1307737
FIGURE 8 is a graph demonstration of the loss
of activity of glucoamylase when that biologically
active entity is exposed to increases in alcohol
concentration, compared with the same entity
protected in accordance with the invention.
Detailed Description of the Preferred Embodiments
It has been discovered that a wide variety of
biologically active molecules can be rendered
inactivation-resistant by exploiting antibody/antigen
interactions to protect vulnerable sites on such
molecules from the harmful effects of any
inactivation process, thereby dramatically slowing
loss of biological activity. Since the interaction
of an antibody with its antigen has been viewed
heretofore as the first step in the ultimate
destruction of the antigen, the use of an
antibody/antigen interaction, pursuant to the present
invention, to prolong the biological activity of an
antigen represents an original approach which would
not have been considered heretofore. More generally,
the innovative use of an antibody/antigen interaction
for protective purposes, rather than for destroying
biologically active entities, as defined below, is a
departure from the conventionally recognized use of
antibody/antigen interactions.
To achieve a prolongation of biological
activity in accordance with the present invention, a
binding entity (as further defined below) is prepared
that recognizes at least one site on a molecule
("biologically active entity"), that site being
necessary to activity and normally subject to
inactivation under certain conditions, such as
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1307737
disrupting temperature or pH or the presence of some
agent like a proteolytic enzyme, an alcohol or an
oxidant. A suitable binding entity in this context
can be an antibody which is raised by challenging the
immune system of a standard laboratory animal with
all or a portion of the biologically active entity.
Alternatively, the binding entity can be an antigen-
binding protein, as described below, which recognizes
the biologically active entity. In any event, it i6
a routine matter to screen putative binding entities,
pursuant to the present invention, to identify those
having the requisite specificity, i.e., those that
bind the biologically active entity in an
inactivation-inhibitive manner without unduly
lessening biological activity.
(i) "Biologically Active Entity~
More specifically a biologically active entity
suitable for the present invention can be any
molecule that promotes or actively participates in a
desired biological reaction and that has at least one
first site responsible for, contributing to or
participating in the desired biological reaction.
Generally, a suitable biologically active entity will
additionally have at least one second site which is
essentially noncontributing to the desired biological
reaction.
By "essentially noncontributing," it is
intended that the at least one second site is not
essential to the performance of the first site in the
desired biological reaction. In particular, the
second site may play a role in processes that result
in inactivation of the biologically active entity,
with respect to the desired biological activity.
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1307737
This inactivation may result from a physical,
chemical or biological process or from a combination
of such processes.
Biologically active entities which are used in
the present invention can be enzymes, hormones,
growth factors and antibodies, for example, as well
as chemical species which effect biological change,
such as drugs and medicines. Suitable biologically
active entities include such enzymes as amylases,
like ~-amylase and ~-amylase; glucoamylase, glucose
isomerase, invertase; proteases like trypsin and
subtilisin; pectinase, L-asparaginase, ~-1,4-
glucosidase, cholesteryl esterase, uricase, catalase,
superoxide dismutase and glucose-6-phosphatase.
Other biologically active entities are interferon,
tissue plasminogen activator and muteins thereof, and
such antibodies as CEA (carcinogen embryonic
antigen), antibodies to HTLV, and antibodies to mouse
IgG.
For enzymes, hormones and the like, there will
typically be a plurality of the active first sites
and, generallyl a plurality of the second sites.
Depending on the particular application, either the
first or second sites, but not both, will participate
in the antibody/antigen interaction. When the
binding entity (see below) is an antibody entity,
such sites will be epitopes to which the antibody
entity will bind. In the case of drugs and
medicines, the first site may be a chemical
configuration or ligand responsible for the desired
biological activity, and the second site may likewise
be a chemical configuration or ligand.
~307737
Preferably, the first and second sites are
spaced apart so that there is no stearic or other
interference of the first site by the second site,
after binding with the binding entity.
S (ii) "Binding Entity"
The binding entity is, in particular, an
antibody entity that "recognizes" (binds to) a
specific portion or site of the biologically active
entity. An antibody entity suitable for the present
invention can be polyclonal antibody, one or more
monoclonal antibodies, or an amino-acid sequence which
contains the variable region of an antibody. An
antibody entity can also comprise hypervariable
regions derived, respectively, from the heavy and
light chains of an antibody, which chains could be
linked:
-in their natural (in vivo) configuration, e.g.,
as in a Fab fragment,
-via chemical modification, using bifunctional
linkers, to effect a crosslinking in vitro, or
-through a linking entity comprised of a
variable-length peptide chain, thereby to
provide a single-chain antibody or so-called
"antigen binding protein," as disclosed, for
example, in U.S. patent No. 4,704,692.
In another embodiment of the present invention,
a protein which is a single-stranded polypeptide chain
comprising three distinct regions, wherein:
a region (A) of said protein includes a first
domain which is capable of biological activity,
and a second domain which contains an epitope
for binding to another portion of said protein;
a region (B) of said protein includes an anti-
body-like domain comprising polypeptide por-
~'
C
. .
1307737
tions corresponding to the liqht or heavy chain
portions of the hypervariable region of an
antibody that is capable of binding to said
epitope of region (A), said light or heavy
S chain variable portions being linked by a first
linker portion of said polypeptidei and
a region (C) of said protein includes a second
polypeptide linker portion linking region (A)
and region (B); and wherein said linear
polypeptide is capable of assuming a conforma-
tion such that said region (A) is functionally
active and able to effect said biological
activity, and said antibody-like domain of said
region (B) is bound in an antibody-antigen-like
fashion to said epitope of region (A); and
wherein said antibody-antigen-like binding con-
fers upon said protein resistance to a deacti-
vating condition with respect to said biologi-
cal activity.
In yet another embodiment of the present
invention, DNA encoding both the biologically active
entity and the antibody entity could be integrated
into a microorganism or animal cell, via recombinant
DNA techniques, to effect a simultaneous synthesis of
- 9a -
C
1307737
both entities, resulting in a complex of the two.
Alternatively, the same approach could be employed to
synthesize a new, covalently-integrated entity
linking the biologically active molecule and antibody
entity initially selected, either directly or via the
linking entity, where complimentary sites on the two
selected entities will form a complex.
An antibody entity within the present
invention thus should be capable of binding to the
second site(s) of the biologically active entity so
as to prevent substantially or delay participation by
the second site(s) in a process that leads to a
deterioration of the desired biological activity to
which at least one first site relates. The antibody
entity may be formed in an immunological response,
wherein the second site acts as an antigenic or
antibody recognition site; this is not essential,
however, as the antibody entity need only bind the
second site so that it does not participate in an
inactivation process. In addition, the antibody
entity should not bind or interfere with the sites
responsible for the desired biological activity to
such a degree that the desired biological activity
cann~t serve a useful purpose.
Procedures are well-known for producing
polyclonal antibodies and monoclonal antibodies, as
well as fragments of antibodies, that will bind to a
biologically active molecule. A suitable procedure
can involve immunizing a rabbit or other standard
laboratory animal with the biologically active
entity, which may have been modified beforehand so as
to "mask" the desired first sites and prevent the
generation of inhibitory antibodies which recognize
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1307737
the first sites. For example, an antibody could be
produced initially that binds a first site; then the
complex of that antibody and the biologically active
molecule would be used as the antigen to raise
noninhibitory polyclonal sera. By means of
conventional techniques, antisera taken from the
animal can then be used as a source for antibody that
binds the biologically active entity without
destroying its activity, i.e., to produce antibody
recognizing the second site(s), but not the active
site, of the biologically active entity.
If this technique is employed without
protection of the desired first site, e.g., if such
sites have not been identified, routine testing of
different polyclonal antibody samples thereby
developed can be carried out to identify those which
will recognize the biologically active entity and
form an active complex that is inactivation-
resistant.
By the same token, conventional somatic-fusion
procedures, as outlined, for example, by Kennett et
al, Curr. Top. Microbiol. Immunol. 81: 77-91 (1978),
can be employed to produce monoclonal antibodies
suitable for use in the present invention. For
example, mice can be immunized in a known manner with
the biologically active entity, or a part of it.
Spleen cells of the immunized mice are then removed
and fused with an immortalizing cell line, such as
murine myeloma line BALB/C NS-lAg4-1 (ATCC No.
TIB18), according to the method of Kohler and
Milstein, see, e.g., Nature 256: 495-97 (1975), and
the resulting fusion products are screened for those
that survive in culture and produce monoclonal
13()7737
antibody of the desired specificity. In this way,
different hybridomas can be tested for production of
monoclonal antibody that form, with the biologically
active entity, a complex which displays prolonged
activity (relative to the free molecule) under
inactivating conditions.
In accordance with the present invention, a
biologically active entity can be analyzed to
identify specific sites which are to be bound by the
binding entity. For example, fragments of the
biologically active entity that do not include any
active sites can be used as antigen to produce
antibody which binds the biologically active entity
without interfering with its activity.
When the biologically active entity is itself
an antibody, its antigen binding site can be
protected, pursuant to the present, using either
antigen normally bound by the antibody or an anti-
idiotypic antibody, i.e., an antibody that recognizes
the antigen binding site.
(iii) "Inactivation"
The biologically active entities employed in
this invention may be inactivated as a result of
physical, chemical or biological processes which may
occur in the working environment of the entity. For
example, disruptive increases or decreases in
temperature, as well as oxidation may, result in
inactivation. In the case in which the biologically
active entity is an enzyme, inactivation may also
result from enzymatic self-destruction.
A biologically active entity within the
present invention can be employed in any environment
in when the entity has previously been employed, as
1307737
well as in environments where it has not been
heretofore practical to use it because of short-
lived activity. For example, use of enzymes as drugs
or therapeutic agents is limited by their
biodegradation or inactivation in the body; the
present invention provides a means for overcoming
this problem.
By the same token, an antibody which is used,
according to the present invention, as a biologically
active entity can be labelled or complexed with a
desired drug, agent or other species without
interfering with an active site of the antibody which
is needed in the immunogenic or diagnostic process.
Thus, antibodies are often chemically modified for
the purpose of labelling. Most of these reactions
are random and leave a certain percentage of the
antibody inactive, due to the chemical modification
of binding sites. This can be prevented, pursuant to
the present invention, if before the chemical
modification (labelling) the antibody is reacted with
its antigen (preferably, with antigen immobilized on
a solid surface) so that the binding site is occupied
and protected. Thereafter, when the labelling
procedure is completed, the antibody/antigen complex
can be dissociated, for example, with low pH buffer,
and the labelled antibody collected and used.
Conversely, if an antigen is to be labelled,
its antibody can immobilized, the antigen added, and
labelling (and subsequent elution) carried out to
remove the antibody.
Growth factors like interferon and
erythropoietin have a very short half-life in serum,
mainly due to enzymatic degradation. This problem i5
1307~37
compounded when the drug is produced by engineered
organisms within normal glycosylation is not effected
and, as a consequence, carbohydrate residues found in
the naturally-occurring molecule are missing. In the
case of erythropoietin, these residues provide
protection against enzymatic degradation. Specific
antibodies can provide similar protection, allowing
for the use of the less expensive, genetically-
engineered erythropoietin. Growth factors like
epidermal growth factor (EGF), which are used to
affect growth and proliferation of certain cell types,
have an effectiveness that is diminished by degrading
enzymes secreted by growing cells. Their efficacy can
therefore be improved, in accordance with the present
invention, by binding with an antibody entity.
Animal growth hormone can be used to increase
body weight or milk production in farm animals. It is
believed, however, that rapid inactivation of the
injected hormone by proteolytic and other enzymes in
vitro has made it necessary to use daily injections of
the hormone, which is cumbersome. But protection of
the growth hormone by binding the hormone with
specific antibodies, in accordance with the invention,
can protect the hormone from enzyme degradation
without unduly reducing its potency. As a result, the
effective hormone level can be maintained with lower
doses and fewer injections.
In addition to protecting enzymes against
inactivation by other enzymes, the present invention
can be used to protect an enzyme against self-
degradation. For example, proteolytic enzyme, such as
the subtilisin used in many detergents, çan be
.~ .
1307737
protected against self-degradation by specific
antibody entities pursuant to the present invention.
In accordance with the present invention, it
is also possible to use a labelling entity which
might otherwise be unsuitable as a result of side
reactions affecting the desired site of activity.
Thus, the desired site is initially bound to the
binding entity to form a complex, thereafter the
complex is labelled with the labelling agent by
conventional procedures, the labelling entity being
bound by the at least one second site. After the
labelling entity is bound to the a second site, the
biding entity is removed, by conventional procedures,
from the labelled complex to provide the labelled
biologically active entity in which the first site is
free, the labelling agent being bound so that it is
no longer available for reaction with the first site.
By means of the present invention, a
biologically active species can be complexed with the
binding entity so as to assure the slow release over
time of the species. In this way a single high
dosage of the (complexed) species, which might
otherwise be unacceptable because of toxicity or side
effects, may be employed and frequent administrations
(or higher dosages of a rapidly degraded species)
consequently avoided.
The present invention is further described
below by reference to the following, illustrative
examples.
1307737
Example 1. The Effect of Temperature on Antibody
Protected and Unprotected ~-Amylase
As described in greater detail below,
comparison tests were carried out on~-amylase and ~-
amylase stabilized in accordance with the invention.
In Figure 1, Plot A shows that the stabilized ~-
amylase in accordance with the invention still had
100% activity after three hours, and 50% activity
after 16 hours, at 70DC, while ~-amylase not
stabilized in accordance with the invention (Plot B)
was completely inactivated (0% activity) after only
15 minutes at the same temperature. A similar,
relative resistance to heat-inactivation is evident
from a comparison of Plot C (stabilized ~-amylase)
with Plot D (free enzyme) in Figure 2.
A human salivary ~-amylase (EC 3.2.1.1; Sigma
catalog No. A052) stock solution (100 units/ml, or
0.1 mg protein/ml) was made up in 5 mM CaC12 and 0.9%
NaCl. To obtain a "protected" form of the enzyme,
the volume equivalent of 35 units of ~-amylase
solution was added 245yl of a 5 mM CaC12/0.9% NaCl
solution containing rabbit polyclonal (IgG) anti-
buman salivary ~-amylase antibody purchased from
Sigma Chemical Company (catalog No. A8273: protein
content: 2.85mg/ml; estimated specific antibody
content: 0.1425mg/ml). The mixture was then
incubated overnight at 4C.
The molar ratio of ~-amylase to specific IgG
of the resulting test composition was nominally 2:1,
and an enzyme activity of 58.8 units/ml was measured
usinq a commercial kit (No. 575- W ; product of Sigma
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1~07737
Chemical Co., St. Louis, M0) which monitors enzyme-
mediated maltose production as a function of
increased absorbance at 340nm. A control
(unprotected) composition with the same activity was
produced, pursuant to the same basic protocol, by
mixing ~-amylase with normal mouse IgG, i.e., IgG
from a mouse that was not exposed to human u-amylase.
Sample dilutions (100~1; 2.94 units/ml) with
CaC12/NaCl solution of the test and comparison
compositions, respectively, were placed in a Gilford
"Response" W -VIS spectrophotometer which had been
previously temperature-adjusted to a particular
temperature. After a 5-minute incubation, each
sample was removed and cooled in ice water. After
the spectrophotometer had been readjusted to 30C,
the samples were reintroduced, respectively,
equilibrated to 30UC, and tested (using the above-
mentioned Sigma kit) for enzyme activity, as measured
via an increase in absorbance at 340 nm.
Both the protected and unprotected samples
were subjected to the following temperatures: room
temperature (RT; about 22), 65-, 68, 70, 72, 75,
80, 85 and 90C. The linear rate constant at each
temperature was determined and percentage activity
calculated as:
Rate at T
X 100.
Rate at RT
The plot thus obtained of percent activity versus
temperature is shown in Figure 2.
In a separate experiment, test and control
dilutions were prepared as described above, and a
100~1 sample of unprotected or protected enzyme was
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added to each of five cuvettes of the Gilford
spectrophotometer, and the temperature was adjusted
to 70C. At the end of 0, 5, 10, 15 and 30 minutes,
respectively, one cuvette was taken out and cooled
immediately in ice water. After the final
incubation, the spectrophotometer was readjusted to
30C, the cuvettes were reinserted and, after 5
minutes to allow for equilibration of the samples,
each sample was tested for enzyme activity, as
previously described.
For long-term incubations, a water bath set at
70C was used. Two tubes containing 1.5 ml aliquots
of the protected and unprotected ~-amylase dilutions,
respectively, were incubated in the water bath for 1,
2, 3, 4, 5, 6, 16, 18 and 20 hours. At the end of
each time, 100 ~1 of each sample were pipetted out
into a cuvette and cooled in ice water. After five
minutes, the two samples were then placed in the
spectrophotometer, which had been adjusted to 30C,
and after five more minutes the enzyme activity of
each sample was measured.
The linear rate constant for each incubation
time was determined and % activity for a given
incubation time calculated as follows:
Rate at 70C for time T
-------------------------------------- X 100
Rate at RT (0 incubation
time at 70C)
The plot of percentage activity versus incubation
time at 70C is shown in Figure 1.
Tests substantially similar to those described
above were carried out with the enzymes subtilisin
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13(~'7737
and glucoamylase. For both biologically active
molecules, the use of polyclonal antibody pursuant to
the present invention resulted in a prolongation of
activity, under conditions of disruptively high
temperature, relative to the unprotected enzyme.
Thus, free subtilisin lost 50% of original activity
in leæs than five minutes at 65C, while the
subtilisin-antibody complex retained over 50% of its
activity for at least three hours at the same
temperature. By the same token, unprotected
glucoamylase lost 95% of its activity at 66~C in only
five minutes (half-life: about two minutes), whereas
the protected enzyme was still over 50% active after
three hours (half-life: >three hours).
Example 2. The Effect of Trypsin on Antibody-
Protected and Unprotected Asparaginase
A. Use of Polyclonal Antibody: Mouse
polyclonal anti-asparaginase sera was purified on a
protein-A column and dialyzed for 17 hours against
water. The dialyzed IgG fraction was then
concentrated by vacuum dialysis to a protein
concentration of 100 ~g per ml, the resulting
concentrate ("antibody solution") having an assumed
specific-IgG content of 5% Thereafter, 1.2 units of
L-asparaginase (EC 3.5.1.1) dissolved in a O.lM
borate-HCL/0.1 mM EDTA buffer (pH 9.0) were mixed
with 1.12 ml of antibody solution, giving a 1:1 molar
ratio of enzyme to specific antibody, and the mixture
was incubated overnight at 4-C. Water (0.82ml at pH
9.2) was then added to give a final concentration of
0.6 units of protected asparaginase/ml.
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13~7737
The antibody-protected and unprotected samples
of asparaginase (0.15 units per ml of each) were
preincubated with 5 units per ml of trypsin (Sigma
catalog No. T1005) in water pH 9.2 for 5 minutes at
37C. The trypsin-treated samples were then
transferred to two cuvettes in the thermal holder of a
Gilford spectrophotometer previously set at 37C. An
equal volume of substrate (2 mM L-asparagine in water;
pH 9.2) was then added and the conversion of L-
asparagine (1 mM final) to L-aspartic acid was
monitored at 197 nm at 37C.
The results, as shown in Table 1 below,
demonstrate the extremely low convercion rate of L-
asparagine by the unprotected asparaginase in the
lS presence of trypsin, as compared with asparaginase
protected in accordance with the present invention.
TABLE 1
Time for 50% con-
version of 1 mM
L-Asparagine to % Conversion
ExperimentL-Asparactic acid per minute
1 *AB Protected 20.5 mins. 2.5%
*Unprotected**7.15 hrs. 0.116%
25 2 *AB Protected 20.5 mins. 2.5%
*Unprotected***11.5 hrs. 0.07%
* Both protected and unprotected asparaginase were
treated with 5 units/ml of trypsin at an
asparaginase concentration of 0.15 units/ml.
** Obtained by extrapolation based on conversion
per minute.
3~
*** Obtained by interpolation based on endpoint
determination of conversion after 20 hours of
incubation with the substrate.
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B. Use of Vari~us Monoclonal Antibodies:
Monoclonal antibodies (MAbs) to L-asparaginase were
prepared according to the method of Kohler and
Milstein. Fifty micrograms of L-asparaginase
suspended in phosphate buffer and Complete Freund'~
Adjuvant (1:1 volume ratio) were injected
intraperitoneally into each of four BALB-C mice. A
day 12 post-injection, a first boost was given i.p.
in the form of 50 micrograms of the enzyme in
phosphate buffer. On day 15, the mice were bled and
their antibody titer was determined by the ELISA
method. Those with the highest titer were given a
second boost of the same constituency and, three days
later, were sacrificed and their spleen cells were
removed for use in a somatic fusion.
After 7 days, the supernatants from growing
hybridomas were tested by the ELISA method for
positive reaction against L-asparaginase. The most
promising hybrids were cloned by the "limiting
dilution method," as disclosed by Lefkovits and
Waldmann, LIMITING DILUTION ANALYSIS OF CELLS IN THE
IMMUNE SYSTEM (Cambridge Univ. Press 1979). Six
clones of producing monoclonal antibodies were
obtained and were labelled No. 12, No. 19, No. 29,
No. 33, No. 34 and No. 35, respectively.
Five samples of enzyme-antibody complex were
prepared as follows. To samples containing 25~1 (0.05
units) of L-asparaginase were added antibody in an
amount to provide ratios of 1, 2, 6, 10 and 20~g
protein per unit of enzyme, respectively. Water was
added to give a final sample volume of 500~1.
Samples were prepared in this way with monoclonals
No. 12, No. 29, No. 34 and No. 35, and with a bovine
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serum albumin (BSA), and were stored at 4C overnight
before testing, as described below.
In addition, 1.5 equivalents t3.75~g~ of each
enzyme-specific MAb were added to 0.5 unit (2.33~g)
of L-asparaginase. In like fashion, samples of
enzyme-antibody complex were prepared using four MAbs
(Nos. 12, 29, 34 and 35) and three MAbs (Nos. 12, 29
and 34), respectively, in combination.
A Gilford "Response" spectrophotometer with
temperature-controlled, 6-position, 10 mm-cuvette
holder was set at 37C, and 50~1 of each of the five
samples prepared using one monoclonal antibody was
added to separate cuvettes. Water (35~1) and trypsin
(15 ~1: 1.5 units) were added and the solutions
incubated at 37C for five minutes. At the end of
this time the cuvettes were taken out and cooled in
ice water. After spectrometer was readjusted to
25C, the cuvettes were reinserted and allowed five
minutes to equilibrate. Water (100~1; pH 9.0) and
substrate (200~1) were added and mixed with a pasteur
pipette. The conversion rate (decrease in
absorbance/minute at 197nm) of L-asparagine to L-
aspartic acid was determined for each sample.
In a separate experiment, 65~1 (0.065 units)
of each of the four multi-MAb samples were added to
separate cuvettes of the spectrophotometer. Water
(15~1), trypsin (20~1, 2 units) and substrate (200~1)
were added, and the conversion rate of L-asparagine
to L-aspartic acid was determined for each sample. A
control sample, prepared by diluting 25~1 of enzyme
with water to a final volume of 650~1, was also run
with and without the addition of trypsin.
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A plot of L-asparaginase residual activity
versus antibody concentration (~g protein added per
unit of L-asparaginase) for each of four single-MAb
samples and for BSA-MAb sample is shown in Figure 3.
5 The percent c:onversion of 1 mM of L-asparagine to L-
aspartic acid versus time is plotted in Figure 4 for
the multi-MAb samples. These results demonstrate
that (1) the degree of protection varied from MAb to
MAb, with No. 12 the most effective, and (2) an
10 unrelated protein (BSA) provided virtually no
protection. A significant level of protection was
achieved with MAb No. 12 alone -- unprotected,
trypsin-challenged enzyme lost over 90% of its
activity under the same conditions -- but protection
15 approximating that of the unchallenged control
required the use of four or five monoclonal
antibodies.
Example 3. Protection of L-Asparaginase Against the
Effect of Disrupting pH
Three equivalents (10.261~g) of each of four
MAbs from Example 2 (Nos. 12, 29, 34 and 35) were
added to 45 ~1 of L-asparaginase (0.9 units,
3.2141~g) to give a final volume was 2.lml. The
sample was incubated overnight at 4C. A control
sample was prepared by diluting 100~1 of L-
asparaginase to 2ml with water.
Both samples from were kept in ice and were
adjusted to pH 3 with dilute HCl. A 50-~1 aliquot of
each was added to a cuvette, 150~11 of water
(pH 9.2) with 2001.1 of substrate were added, and the
solutions were mixed. The activity of each sample,
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in terms of conversion rate of L-asparagine to L-
aspartic acid, was determined (at zero time, To) in a
Gilford "Response" spectrophotometer which had been
set to 25C.
The two samples were then placed in a water
bath set at 37C. Aliquots (50 ~1) of each were
taken at intervals of 5, 15, 45 and 65 minutes, and
after 3 and 18 hours. The activity of each of these
samples was determined as above. The percentage
activity for a given incubation time (Tx) was
calculated as follows:
Rate of conversion at Tx
x 100 .
Rate of conversion at To
The plot of L-asparaginase residual activity versus
incubation time at pH 3 is shown in Figure 5. Enzyme
protected with a mixture of four NAbs retained over
30% of its activity after two hours, whereas the
unprotected enzyme had less than 2% of its activity
after only 45 minutes.
Example 4. Protection of Trypsin Against Self-
Digestion
Rabbit polyclonal anti-trypsin serum was
obtained from Yentrex Laboratories (Portland, ME).
An IgG fraction was purified from the serum by the
use of a MAPS II protein A kit (product of Bio-Rad
Laboratories, Richmond, CA). The purified IgG
fraction was dialyzed against O.lN Tris-HCl (pH 8.0),
with several changes of buffer. The final protein
concentration of the resulting antibody solution was
800~g/ml and, with an assumed content of 5% for IgG
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specific for trypsin, the concentration of specific
antibody was 40 ~g/ml.
To 20 ~1 of trypsin solution (20 units: 2~g)
in the same Tris-HCL buffer were added 315~1
(12.6~g of specific IgG; 252~g of total IgG) of
antibody solution to give a 1:1 molar ratio of
trypsin to specific IgG. Water (65~1) was added to
give a final trypsin concentration of 50 units/ml.
Two controls were prepared, one containing
252~g of bovine serum albumin and the other
containing no protein. Final trypsin concentration
were also 50 units per ml.
All samples were incubated at 4C, and 50~1 of
each were assayed for trypsin activity at 0, 1, 3, 5
and 6 days by means of the above-mentioned Gilford
spectrophotometer (temperature: 25C; absorbance at
247nm). The linear rate constant for each sample
were then determined and the residual activity
calculated as described in Example 3.
Trypsin protected with a rabbit anti-trypsin
polyclonal antibody maintained 100% of its activity
for up to three days, whereas unprotected trypsin
lost 75% of its activity after one day, at 4C. As
shown in Figure 6, where percentage activity is
plotted against time (days), when nonspecific protein
(BSA) was added to trypsin, there was a 50%
protection of its activity after 3 days, but this
protection was significantly lower than that
associated with the antibody. Moreover, the
protection achieved with BSA went down to near-zero
after five days, while 30% protection was maintained
after six days when antibody was used.
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Example 5. Protection of Subtilisin Against
Inactivation by an Oxidizing Agent
Subtilisin-antibody complex was prepared as in
Example 3. An unprotected control was prepared by
adding 1.25~g of subtilisin (EC 3.4.21.14; Boehringer
Mannheim catalog No. 165905) to 136~g of BSA in 1.5ml
of 50 mM KCl and 50 mM Tris-HCl buffer (pH 8.0). A
O.5 mM solution of the enzyme substrate, N-succinyl-
ala-ala-pro-phe p-nitroanilide, was prepared in 0.1 M
Tris-HCl buffer (pH ~7.8). A commercial bleach
formulation (JAVEX~ , containing 6% sodium
hypochlorite, was used as the oxidizing agent.
Samples of subtilisin protected with mouse
anti-subtilisin polyclonal antibody and of
unprotected subtilisin, respectively, were subjected
to increasing concentrations of sodium hypochlorite
at 37-C for 15 minutes. Substrate was then added to
each sample and the activity of the enzyme
determined, at 37C, by monitoring an increase in
absorbance at 410nm which was correlated with the
rate of hydrolysis of the substrate.
In the oxidant-concentration range of 0.04% to
0.15%, the protected enzyme was at least twice as
active as the unprotected enzyme. By the same token,
it was found that the protected subtilisin, when
exposed to 0.05% of sodium hypochlorite for various
times, retained its activity longer than unprotected
enzyme subjected to the same conditions (see Figure
7). After 30 minutes preincubation with 0.05% sodium
hypochlorite, for example, the protected subtilisin
retained over 75% of original activity, whereas the
unprotected subtilisin displayed less than 25% of
original activity.
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Example 6. Protection of Glucoamylase Against the
Effect of Alcohol ~
A Glucoamylase (DIAZYME L-200; product of Miles
Laboratories) protected with rabbit anti-
glucoamylase polyclonal antibody was prepared in
accordance with Example 5. Sets of three samples
each of the enzyme-antibody complex and unprotected
glucoamylase plus nonimmune human IgG were exposed,
respectively, to no alcohol, 2.5~ ethanol and 5%
ethanol (v/v3. The samples were incubated for
various times at 37C before being assayed for enzyme
activity.
The results are illustrated in Figure 8.
Unprotected enzyme samples exposed to 2.5% and 5%
ethanol lost 50% of activity in 8 and 10 hours,
respectively. Protected samples, by contrast,
suffered an average loss of less than 5% of original
activities after 10 hours.
