Patent Grant
7989596
The present invention relates to a method of reversibly altering the binding specificity of monoclonal antibodies.
The present inventor has previously reported the discovery that blood and other bodily fluids from normal individuals contain a significant number of autoantibodies, that, when treated with an oxidizing agent, become capable of binding self antigens. See, for example, the following publications:
In these publications, it was reported that blood from normal individuals contains a significant number of autoantibodies, in a wide variety of isotypes and specificities, but that these autoantibodies become detectible only when certain body fluids or blood are exposed to oxidation, by, for example an oxidizing agent or electric current, according to a method described therein. It was reported that samples such as blood, plasma, serum, breast milk, cerebrospinal fluid, and purified immunoglobulin fractions can be treated by oxidation and then assayed with a variety of self antigens and other types of antigens to identify masked autoantibodies that can be unmasked by oxidation. Autoantibodies that have been unmasked by oxidation include the following in Table 2:
It has now been discovered that the binding specificity of monoclonal antibodies can be altered by similar treatments with an oxidizing agent or a direct electric current. This finding is significant, since monoclonal antibodies are typically intended to bind only a specific antigen. However, according to the method described herein, the spectrum of activity of a monoclonal antibody can be broadened to include antigens other than the specific antigen that the monoclonal antibody is intended to bind.
According to an aspect of the present invention, there is provided a method comprising providing a composition containing a monoclonal antibody, the monoclonal antibody having a binding specificity toward a specific antigen, and exposing the composition to an oxidizing agent or an electric potential sufficient to effect an alteration of the binding specificity of the monoclonal antibody.
According to another aspect of the present invention there is provided a composition comprising a monoclonal antibody having a binding specificity that has been altered by exposure of the monoclonal antibody to an oxidizing agent or an electric potential.
a) and (b) are graphs showing the effect of EMF exposure on a monoclonal antibody produced to a plasma membrane antigen found on the murine tumor cell line SP2/0. This monoclonal antibody was tailor made for the express purpose of controlling for the proprietary variables that may exist in commercially prepared monoclonal antibody preparations. For example, might the suspension solutions used by commercial monoclonal antibody producers be contaminated with animal serum as residual from the monoclonal culture growth media? Such contamination could interfere with masking and/or unmasking observations of the mouse monoclonals.
As shown in
The two graphs comprising
The present invention relates to a method of altering the binding specificity of a monoclonal antibody.
The term “altering the binding specificity” of a monoclonal antibody refers to a process whereby a monoclonal antibody is changed or altered, such as by oxidation and reduction, so that it becomes capable of specific binding of an antigen or ligand that it had not previously been capable of specifically binding. For example, the spectrum of antibody activity may be broadened so that the monoclonal antibody binds to other antigens. The term “altering the binding specificity” may also apply to a process whereby a monoclonal antibody is changed or altered, such as by oxidation or reduction so that it becomes incapable of specific binding of an antigen or ligand that it had previously been capable of specifically binding, but it is to be understood that in this context, the term refers to a reversible change and not a permanent, irreversible change such as denaturing of the protein.
The term “monoclonal antibody” refers generally to an antibody that has been selected on the basis of binding specificity towards a particular antigen and then cloned or otherwise manufactured to produce a set of antibody molecules each having an identical molecular structure. Monoclonal antibodies for many antigens are commercially available.
In the method of the present invention, the binding specificity of a monoclonal antibody is altered by exposing the monoclonal antibody to an oxidant or to an electric current. For example, the binding specificity of a monoclonal antibody can by altered so that the monoclonal antibody is able to bind an antigen that it was not able to bind before the method was carried out.
If an oxidizing agent is used to carry out the method of the invention, the oxidizing agent can be any compound that is capable of altering the redox state of a biological molecule. More specifically, the oxidizing agent is a molecule that has the ability to be reduced by acting as an electron acceptor for other molecules that act as electron donors. Suitable oxidizing agents include many compounds that contain a coordinated (transition) metal that can participate in oxidation-reduction reactions, for example a redox capable metal such as iron that can alternate between its ferric and ferrous states. Other examples of oxidizing agents include, but are not limited to hemin and the coordinated magnesium metal in chlorophyll molecules, sodium periodate (NaIO4) and potassium permanganate (KMnO4). Typically, when a transitional metal oxidizing agent is used, a mixture of the monoclonal antibody and the oxidizing agent must be incubated for a period of time, typically for 12-24 hours. The oxidizing agent should be used at a concentration sufficient to alter the binding specificity of the monoclonal antibody, but not at a concentration that might destroy or denature the monoclonal antibody. It has been found that different types of monoclonal antibodies can interact differently with different antioxidants.
If a DC electric current is used to carry out the method of the invention, the method may be carried out by any means of delivering an electric current, such as by immersing positive and negative electrodes into a conductive solution containing the sample to be treated. A solution containing a monoclonal antibody is exposed to an electric potential of a sufficient magnitude and of a sufficient duration to alter the binding specificity of the monoclonal antibody. It has been found that positive results may be obtained by exposing a solution to an electric potential of 6-24 volts for a few seconds to a few minutes. An extended exposure to an electric current may result in reversal of the alteration of the binding specificity. This has been seen by using a glycophorin A monoclonal antibody and flow cytometry and measuring the antibody's reactivity to red blood cells overtime of EMF exposure.
Without being bound to a specific theory, it is believed that exposure of a monoclonal antibody to the oxidizing agent or electric current can oxidize and/or reduce an antigen binding site in the Fab portion of the monoclonal antibody. It has been found in oxidation experiments conducted with IVIg and using detection by monoclonal anti-nitrotyrosine antibodies that IgG that has been exposed to oxidation by hemin has a greater degree of nitrosylation than non-treated IgG. Accordingly, it can be theorized that a similar mechanism might be operative with monoclonal antibodies and that the alteration of the antigen binding site of the monoclonal antibody is effected by reversible nitrosylation of aromatic ring containing amino acids (e.g., tyrosine and tryptophan) in and around the antibody hypervariable region, which may produce conformational changes in the antigen binding site.
Whether a particular monoclonal antibody of interest is one that has a binding specificity that can be altered by changing its redox state and the effectiveness of any set of conditions for altering the binding specificity of the monoclonal antibody of interest may be readily determined by subjecting the monoclonal antibody to a change in redox state by, for example, exposing the monoclonal antibody to an oxidizing agent or electric current and then using ELISA or other ligand-receptor assays to determine whether the binding specificity of the monoclonal antibody has been altered. In other words, an assay of a monoclonal antibody can be carried out before and after the monoclonal antibody is subjected to a change in redox conditions to see whether the process has altered the binding specificity of the monoclonal antibody. For example, the best oxidizing agent or method to alter a specific monoclonal antibody can be readily determined by simple experimentation. Experimental data have shown that different oxidizing agents can unmask different autoantibody specificities.
A further aspect of the present invention is a monoclonal antibody that has been altered by exposure to an oxidizing agent or electric current. As explained more fully in the examples below, it has been found to date that the binding specificity of the following monoclonal antibodies can be altered to change their binding profile with respect to at least one of cardiolipin, phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine: an anti-glycophorin A monoclonal antibody; an anti-CXCR4 monoclonal antibody; a CD 63 monoclonal antibody; a B2GP-1 monoclonal antibody; an anti-platelet IIb-IIIa monoclonal antibody; and a gamma 1 mouse isotype control monoclonal antibody. Alterations in binding properties of a monoclonal antibody to Factor VII and a monoclonal antibody to CD 44 as well as a monoclonal antibody to Factor IX were dependent upon the dilution buffers used for the ELISA testing.
Having described the invention, the following examples are given to illustrate specific applications of the invention, including the best mode now known to perform the invention. These specific examples are not intended to limit the scope of the invention described in this application.
Regarding each of the described herein, unless otherwise noted, the following procedure was typically used: 250 μl of monoclonal antibody was used directly from the vial or bottle supplied by the commercial vendor. It was placed as a bubble on a parafilm sheet. Graphite electrodes were connected to the positive and negative terminals of 6-9 volt battery or a power source (BK Precision) set at 8-volts and were submersed into the bubble solution containing monoclonal antibodies for 10 seconds. (Where noted herein, an alternative treatment was used in which the monoclonal antibody was combined with hemin and the mixture was incubated, with rocking or shaking, at 36° C. for a period of 12-24 hours.) Following the treatment with electric current or with hemin, a sample of the monoclonal antibody was tested for the presence of antiphospholipid antibodies (aPL) using a comprehensive in-house ELISA aPL format that provides separate aPL test results. The testing procedure is described in greater detail in the following publications, incorporated herein by reference: Wagenknecht, D R, et al., The Evolution, Evaluation and Interpretation of Antiphospholipid Antibody Assays, Clinical Immunology Newsletter, Vol. 15, No. 2/3 (1995) pp. 28-38 and McIntyre, J A, et al., Frequency and Specificities of Antiphospholipid Antibodies (aPL) in Volunteer Blood Donors, Immunobiology 207(1): 59-63, 2003.
Four aPL specificities were assessed, 1) aPS=antiphosphatidylserine, 2) aCL=anticardiolipin, 3) aPE=antiphosphatidylethanolamine, and 4) aPC=antiphosphatidylcholine. Each monoclonal antibody sample, before and after oxidation was diluted 1/10 into and assessed in the presence (dependent) and absence (independent) of a TRIS buffer diluents supplemented with either 10% adult bovine plasma (ABP), which contains the phospholipid-binding plasma proteins or 1% bovine serum albumin, (BSA, which is devoid of phospholipid-binding plasma proteins), respectively.
With the exception of
The results in the aPL specificities obtained for the various experiments described herein are given in the accompanying figures. The positive/negative findings are expressed in terms of optical density (OD). In describing the results herein, the term “unmasking” refers generally to the condition in which an alteration of binding specificity of a monoclonal antibody is observed, such as, for example, where an enhanced binding to a phospholipid antigen is observed.
A 250 μl bubble of the manufacturer's solution containing a mouse monoclonal IgG2b antibody to human glycophorin A was placed on a parafilm platform. The solution was exposed to 10 seconds of 8-volt EMF by immersing two electrodes (anode and cathode) for 10 seconds with a power source set at 8 volts. Each monoclonal antibody solution was assayed before and after oxidation for the following binding specificities: antiphosphatidylserine (aPS), anticardiolipin (aCL), antiphosphatidylethanolamine (aPE), and antiphosphatidylcholine (aPC) The control and redox exposed solutions were then assayed for aPS, aCL, aPE and aPC binding specificities each diluted 1/10 into separate TRIS diluent buffers, one supplemented with 10% adult bovine plasma (ABP) and the other supplemented with 1% bovine serum albumin (BSA). The binding profiles for the diluents containing ABP and BSA and the anti-glycophorin A monoclonal antibody before EMF treatment (“control”) and after EMF treatment (“redox”) are shown in
Example 1 was repeated, except that a mouse anti-CXCR4 monoclonal antibody, a co-receptor for the HIV infection of CD4 positive cells, was used as the monoclonal antibody instead of an anti-glycophorin A monoclonal antibody. The treatment and testing format was the same as for
Example 1 was repeated, except that a CD 63 monoclonal antibody was used as the monoclonal antibody instead of an anti-glycophorin A monoclonal antibody. The format of the ELISA testing of an IgG1 monoclonal antibody to CD 63 was identical to that described in
Example 1 was repeated, except that a monoclonal antibody to a plasma protein, beta2 glycoprotein I (β2GP-I) was used as the monoclonal antibody instead of an anti-glycophorin A monoclonal antibody. The binding profiles for the diluents containing ABP and BSA and the β2GP-I monoclonal antibody before EMF treatment (“control”) and after EMF treatment (“redox”) are shown in
Example 1 was repeated, except that an anti-factor VII monoclonal antibody was used as the monoclonal antibody instead of an anti-glycophorin A monoclonal antibody. The binding profiles for the diluents containing ABP and BSA and the anti-factor VII monoclonal antibody before EMF treatment (“control”) and after EMF treatment (“redox”) are shown in
Example 1 was repeated, except that a factor IX monoclonal antibody was used as the monoclonal antibody instead of an anti-glycophorin A monoclonal antibody. The binding profiles for the diluents containing ABP and BSA and the factor XI monoclonal antibody before EMF treatment (“control”) and after EMF treatment (“redox”) are shown in
Example 1 was repeated, except that gamma 1 mouse control monoclonal antibody was used as the monoclonal antibody instead of an anti-glycophorin A monoclonal antibody. Additionally, an alternative oxidative treatment was carried out using hemin under the conditions described above. In particular, hemin, 2.5 μl (15.15 mg/ml) was added to 0.5 ml of the monoclonal antibody solution and incubated overnight at 36° C. on a rocking platform. The binding profiles for the diluents containing ABP and BSA and the gamma 1 mouse control monoclonal antibody before either hemin or EMF treatment (“control”) and after “EMF” and “Hemin” treatment are shown in
Example 1 was repeated, except that a CD 44 monoclonal antibody was used as the monoclonal antibody instead of an anti-glycophorin A monoclonal antibody. The binding profiles for the diluents containing ABP and BSA and the CD 44 monoclonal antibody before treatment (“control”) and after “Hemin” or “EMF” treatment are shown in
Example 1 was repeated, except that an anti-platelet IIb-IIIa mouse monoclonal antibody was used as the monoclonal antibody instead of an anti-glycophorin A monoclonal antibody. The binding profiles for the diluents containing ABP and BSA and the CD 63 mouse monoclonal antibody before EMF treatment (“control”) and after EMF treatment (“redox”) are shown in
Example 1 was repeated, except that a tailor made monoclonal antibody to a murine tumor cell line SP2/0 was used as the monoclonal antibody instead of an anti-glycophorin A monoclonal antibody. This monoclonal antibody (Mab) was produced in culture media wherein all the components were known and samples of the culture media were obtained before and after growing the monoclonal to assure that all possible ELISA controls were performed. The binding profiles for the ABP (
The identical anti-glycophorin A monoclonal antibody used in example 1 was used to assess the effects of EMF oxidation on the monoclonals recognition of its red blood cell (RBC) membrane target antigen. The experimental design also used a 250 μl bubble of the monoclonal antibody solution on parafilm, but a 3 μl sample was withdrawn at each 5 second interval for the first minute to test by flow cytometry for RBC binding. After 60 seconds, an additional EMF treatment was done for another 60 seconds, uninterrupted (total EMF time 2 minutes). The results of this experiment are provided in
The reversibility of altering the binding properties of monoclonal antibodies is shown in
Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.
| Number | Name | Date | Kind |
|---|---|---|---|
| 20050054051 | Rosen | Mar 2005 | A1 |
| 20050101016 | McIntyre | May 2005 | A1 |
| 20050260681 | McIntyre | Nov 2005 | A1 |
| Number | Date | Country | |
|---|---|---|---|
| 20090071842 A1 | Mar 2009 | US |
