CA1307737C - Use of antibody/antigen interactions entities to protect or modulatebiological activity - Google Patents
Use of antibody/antigen interactions entities to protect or modulatebiological activityInfo
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- CA1307737C CA1307737C CA000569368A CA569368A CA1307737C CA 1307737 C CA1307737 C CA 1307737C CA 000569368 A CA000569368 A CA 000569368A CA 569368 A CA569368 A CA 569368A CA 1307737 C CA1307737 C CA 1307737C
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Abstract
Abstract of the Disclosure A complex can be produced, comprising (i) an molecule having a biological activity and (ii) an antibody entity recognizing the molecule, that displays biological activity which is inactivation-resistant, relative to that of the molecule. The biologically active molecule can be an enzyme, for example, and can be used to produce a complex with an antibody or antigen-binding protein that resists inactivation resulting from a physical, chemical or biological process. Thus, the inactivation-resistant complex displays prolonged activity under conditions characterized by disrupting temperature, the presence of a proteolytic enzyme, disrupting pH, the presence of an oxidizing agent and/or the presence of an alcohol, among others.
Description
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.
.- ~ . . , , .. . . .. ; . ,,. ~ . . . . . ~ -, . . . . . . .. .
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 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.
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.
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 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.
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
. .
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
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 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 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 .~ .
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.
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 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 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
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 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.
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.
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.
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.
.- ~ . . , , .. . . .. ; . ,,. ~ . . . . . ~ -, . . . . . . .. .
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 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.
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.
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 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.
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
. .
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
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 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 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 .~ .
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.
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 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 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
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 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.
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.
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.
~307737 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 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.
13~)7737 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, 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
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.
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.
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.
*** Obtained by interpolation based on endpoint determination of conversion after 20 hours of incubation with the substrate.
~307737 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 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.
13~)7737 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, 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
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.
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.
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.
Claims (34)
1. A method of producing a biologically active complex characterized by an enhanced resistance to inactivation, comprising the steps of i) providing a complex comprised of a molecule which is biologically active and a mono-clonal antibody or a single-chain antibody that recognizes said molecule, said complex being biologically active; and ii) measuring a preservation of biological activity, for said complex, when said complex is subjected to a condition that inactivates said molecule alone.
2. A method according to claim 1, wherein step i) comprises exposing said molecule to a monoclonal antibody that recognizes said molecule, such that a molecule-monoclonal antibody complex is formed.
3. A method according to claim 1, wherein step i) comprises exposing said molecule to a single-chain antibody that recognizes said molecule, such that a molecule-single-chain antibody complex is formed.
4. A method according to claim 1, 2 or 3, wherein said molecule is an enzyme.
5. A method according to claim 1, wherein said condition is selected from the group consisting of denaturing temperature, the presence of a proteolytic enzyme, denaturing pH, the presence of an oxidizing agent, and the presence of an alcohol.
6. A method of producing a biologically active complex characterized by an enhanced resistance to inactivation, comprising the steps of:
(A) binding a molecule which is biologically active with a monoclonal antibody or a single-chain antibody that recognizes said molecule to produce a complex which retains the biological activity of said molecule; and (B) measuring a preservation of biological activity for said complex, wherein said biological activity is substantially the same as the activity of said molecule alone, when said complex is subjected to a condition that inactivates said molecule alone.
(A) binding a molecule which is biologically active with a monoclonal antibody or a single-chain antibody that recognizes said molecule to produce a complex which retains the biological activity of said molecule; and (B) measuring a preservation of biological activity for said complex, wherein said biological activity is substantially the same as the activity of said molecule alone, when said complex is subjected to a condition that inactivates said molecule alone.
7. A method according to claim 6, wherein said molecule is an enzyme.
8. A method according to claim 6 or 7, wherein said condition is selected from the group consisting of denaturing temperature, the presence of a proteolytic enzyme, denaturing pH, the presence of an oxidizing agent, and the presence of an alcohol.
9. A method according to claim 8, wherein said condition is the presence of a proteolytic enzyme.
10. A method according to claim 8, wherein said condition is selected from the group consisting of denaturing pH, the presence of an oxidizing agent, and the presence of an alcohol.
11. A method according to claim 6, 7, 9 or 10, wherein said molecule which is biologically active has subtilisin activity.
12. A method according to claim 6, 7, 9 or 10, wherein said molecule which is biologically active has .alpha.-amylase activity.
13. A method according to claim 6, 7, 9 or 10, wherein said molecule which is biologically active has L-asparaginase activity.
14. A method according to claim 6, 7, 9 or 10, wherein said molecule which is biologically active has trypsin activity.
15. A method according to claim 6, 7, 9 or 10, wherein said molecule which is biologically active has glucoamylase activity.
16. A method according to claim 6, 7, 9 or 10, wherein step (A) comprises binding a molecule which is biologically active with a monoclonal antibody that recognizes said molecule to produce a complex which retains the biological activity of said molecule.
17. A complex comprised of (i) a molecule having a biological activity, and (ii) an antibody entity selected from the group consisting of a monoclonal antibody and a single-chain antibody recognizing said molecule, said complex displaying a biological activity which is inactivation-resistant relative to, and which is substantially the same as, that of said molecule alone.
18. A complex according to claim 17, wherein said molecule is an enzyme.
19. A complex according to claim 18, wherein said molecule having a biological activity has subtilisin activity.
20. A complex according to claim 18, wherein said molecule having a biological activity has .alpha.-amylase activity.
21. A complex according to claim 18, wherein said molecule having a biological activity has L-asparaginase activity.
22. A complex according to claim 18, wherein said molecule having a biological activity has trypsin activity.
23. A complex according to claim 18, wherein said molecule having a biological activity has glucoamylase activity.
24. A complex according to claim 17, 18, 19, 20, 21, 22 or 23, wherein said complex is comprised of (i) a molecule having a biological activity, and (ii) a monoclonal antibody recognizing said molecule.
25. A complex according to claim 17, said complex being the product of a process comprising the steps of:
(A) binding said molecule and said antibody entity such that a molecule-antibody entity complex is formed; and (B) measuring a preservation of said biological activity by said complex, such that said biological activity is substantially the same as that of said molecule alone, when said complex is subjected to a condition that inactivates said molecule alone.
(A) binding said molecule and said antibody entity such that a molecule-antibody entity complex is formed; and (B) measuring a preservation of said biological activity by said complex, such that said biological activity is substantially the same as that of said molecule alone, when said complex is subjected to a condition that inactivates said molecule alone.
26. A complex according to claim 25, wherein said condition is the presence of a proteolytic enzyme.
27. A complex according to claim 25, wherein more than one monoclonal antibody or single-chain antibody is bound to said molecule.
28. A protein which is a single-stranded polypep-tide 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-tions corresponding to the light 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 chain variable portions being linked by a first linker portion of said polypeptide; 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.
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-tions corresponding to the light 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 chain variable portions being linked by a first linker portion of said polypeptide; 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.
29. A protein according to claim 28, wherein region (A) corresponds to an enzyme.
30. A protein according to claim 28, wherein region (A) corresponds to a hormone.
31. A protein according to claim 28, wherein region (A) corresponds to a growth factor.
32. A protein according to claim 28, wherein region (A) corresponds to a drug.
33. A protein according to claim 28, wherein region (A) corresponds to an antibody.
34. A protein according to claim 28, wherein said deactivating condition is selected from the group con-sisting of denaturing temperature, denaturing pH, the presence of a proteolytic enzyme, the presence of an oxidizing agent, and the presence of an alcohol.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US7186187A | 1987-07-07 | 1987-07-07 | |
| US071,861 | 1987-07-07 | ||
| US18253088A | 1988-04-18 | 1988-04-18 | |
| US182,530 | 1988-04-18 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1307737C true CA1307737C (en) | 1992-09-22 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000569368A Expired - Fee Related CA1307737C (en) | 1987-07-07 | 1988-06-13 | Use of antibody/antigen interactions entities to protect or modulatebiological activity |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1307737C (en) |
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1988
- 1988-06-13 CA CA000569368A patent/CA1307737C/en not_active Expired - Fee Related
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