WO1987007147A1

WO1987007147A1 - Method for producing monoclonal antibodies to an immunogenic antibody-antigen conjunction

Info

Publication number: WO1987007147A1
Application number: PCT/US1987/001295
Authority: WO
Inventors: William Jackson Payne, Jr.
Original assignee: Murex Corporation
Priority date: 1986-05-28
Filing date: 1987-05-28
Publication date: 1987-12-03
Also published as: AU7485987A

Abstract

A method for the preparation of a monoclonal antibody capable of recognizing the juncture of an antigenic determinant and a corresponding antibody binding site when complexed to each other. Antigen may be subjected to limited proteolytic digest to obtain an immunochemically active fragment. Similarly, a monoclonal antibody directed against the antigenic determinant of interest is enzymatically cleaved to obtain the Fab or F(ab')2 fragment. The two fragments are combined so as to form a complex. This immunogenic complex is subsequently injected into recipient animal and the antibodies produced recognize specifically the juncture of the immunogenic complex.

Description

METHOD FOR PRODUCING MONOCLONAL ANTIBODIES TO AN IMMUNOGENIC ANTIBODY-ANTIGEN CONJUNCTION

FIELD OF THE INVENTION

The present invention relates to antibodies and methods for their production. More particularly, the present invention comprises a method for the production of monoclonal antibodies designed to recognize the juncture between an immunogenic antibody:antigen complex.

BACKGROUND OF THE INVENTION

In U. S. Application No. 6,790,077, which is incorporated by reference in its entirety herein, an assay method is described in which a first antibody recognizes and binds to an analyte, typically an antigen, suspected of being in a solution. A labeled second antibody, a monoclonal, recognizes and binds preferentially to the juncture between the first antibody, and any of the analyte present, yet will not bind to analyte alone or antibody alone. The presence or amount of labeled material present is measured and the quantity of analyte present determined. The assay is unique in that it is highly selective: only material bound to the first antibody will be recognized, and only the juncture will be bound by the second antibody. This selectivity eliminates a substantial amount of false or spurious signal resulting from the cross-reactive binding of labeled antibody to materials other than the analyte or to analyte not bound by the first antibody. SUMMARY OF THE INVENTION

The present invention relates to a method for the production of monoclonal antibodies to a unique antigen. More particularly, disclosed is a method for producing a monoclonal antibody directed against the juncture of the idiotypic determinant of a first monoclonal antibody and the antigenie determinant of an antigen when the monoclonal antibody and the antigen are bound together, comprising: (a) providing an antigen of interest; (b) subjecting the antigen to limited proteolytic digestion to produce one antigenic fragment capable of binding to an antibody directed against the antigen; (c) providing a first monoclonal antibody directed against the antigen and/or antigenic fragment; (d) separating the Fab or

F(ab') portion from the Fc portion of the first monoclonal antibody; 2

(e) combining the antigen or antigenic fragment and the Fab or F(ab'L portion in a manner favoring the formation of an immunogenic complex;

(f) injecting the immunogenic complex of step (e) into a host animal; and (g) obtaining the monoclonal antibodies directed against the immunogenic complex capable of recognizing the juncture between the antigen and the first antibody when complexed together.

DESCRIPTION OF THE INVENTION

The method of the present invention is useful for the production of highly selective monoclonal antibodies to a unique antigenic entity. Briefly, an antigen of interest is subjected to limited proteolysis so as to provide, among other components, a fragment that contains an active antigenic determinant. A monoclonal antibody directed against the antigenic determinant is cleaved enzymatically and Fab or F(ab') and Fc fragments are separated. The antigen fragment and the Fab or F(ab') fragment are then combined so as to produce a complex possessing immunogenic properties. Animal recipients are immunized with this immunαgen. Specific antibody-producing lymphocytes are fused with myeloma cells and monoclonal antibodies are selected. The resulting monoclonal antibody will selectively bind to the juncture of the antigen and the antibody, but not the individual components alone. Analyte selection will dictate the extent of proteolytic digestion. Typically, a small antigen, such as a hormone or drug, has one antigenic determinant, i.e., a sequence of amino acids or a molecular structure which is recognized by an antibody. The desired epitope structure on a small antigen or antigen fragment has a higher probability of being recognized than if present on a large antigen with multiple antigenic determinants. Therefore, a large antigen may be degraded into a smaller fragment, which under optimum circumstances possesses only the antigenic determinant desired.

Limited proteolysis of antigens is an established procedure, and discussed in greater detail in the Examples. Briefly, the antigen is subjected to the action of a proteolytic substance under conditions that allow the extent of degradation to be controlled. The products of this proteolytic cleavage are subjected to sodium dodecyl sulfate

("SOS") polyacrylamide gel electrophoresis ("PAGE") to separate the protein fragments by size. Protein fragments are transferred to nitrocellulose paper by the Western blot technique, which is well known in the art. Blots are developed using a specific monoclonal antibody in an indirect enzyme-linked im unosorbent assay ("ELISA") to identify the polypeptide fragment that contains the antigenic determinant. The identified fragment with the antigenic determinant is purified by molecular exclusion chromatography, or alternatively, 5 by SDS-PAGE followed by electroelutioπ.

A monoclonal antibody is used that is capable of binding to the antigenic fragment. In order to reduce the size of the antigenic fragment-antibody complex and to again make recognition of the juncture easier, the Fc fragment is removed from the antibody

10 molecule. In a syngeneic system, i.e., monoclonal antibody and recipient animal, the immune complex consisting of a whole antibody molecule bound to an antigen would be cleared by normal mechanisms without generating the desired unique antibody(ies). In an allogeneic or xenogeneic system, removal of the Fc portion would eliminate l ~ numerous potential antigenic determinants. Cleavage can be accomplished by one of several established techniques. The most common technique uses the enzyme pepsin which cleaves the IgG molecule just below the hinge region, yielding a Fc fragment and a single F(ab' )₂fragment possessing a molecular weight of approximately 100,000 0 daltons and having two antigen binding sites, also called paratopes. Alternatively, the antibody can be cleaved just above the hinge region by using the enzyme papain, yielding a larger Fc fragment and two Fab fragments, each with a molecular weight of 50,000 daltons and each having a single antigen binding site. For the purposes of 5 illustration only, the Fab fragment will be discussed in this application. The F(ab') or Fab fragment can be purified by using either molecular exclusion or affinity (Protein A) chromatography; both methods are discussed in detail in the Examples set forth below.

In order to create the im unogen necessary for immunization an 5 antibody:antigen complex must first be formed. The purified antigen preparation possessing the desired antigenic determinant is combined with the Fab fragment of the antibody under appropriate conditions, i.e., pH, molarity and temperature. The antigenic determinant is recognized and bound by the binding site of the Fab creating a more

20 exposed conjunction site with an enhanced probability of im unogenicity. It is this conjunction that will serve as the new immunogen for the production of the conjunction binding antibody. The complex is purified by a means gentle enough to allow the complex to remain intact; for example, one appropriate method is molecular l ~ exclusion chromatography, which will separate the complex from the reactants based upon molecular size.

Immunogenicity, the characteristic of a molecule or substance to evoke a hpst immune response can be enhanced by various means. Existing methods include chemical cross-linking (glutaraldehyde) and

20 heat-induced aggregation which might create a repeating polymeric structure of the complex. The use of adjuvants and multiple inoculations may also be used to enhance immunogenicity. These methods are all well known in the art.

In order to produce the desired unique antibodies necessary for

25 the invention, it is requisite to use monoclonal antibodies for production of the conjunction site. Moreover, in order to reduce the likelihood that the recipient animal will immunologically react with epitopes present on the Fab antibody fragment, it is preferable to use a syngeneic system, i.e., any antibodies used to create the conjunction site in the method should be produced from genetically identical animals as those used to make the conjunction binding antibodies. This reduces the possibility that a recipient will process an antibody as a foreign antigen, such as a Balb/c mouse recognizing a C57BL mouse antibody as foreign and producing antibodies to the antibody, rather than to the antigen-antibody conjunction as desired. Monoclonal antibodies are prepared essentially as described by Milstein and Kohler (Nature, 256:495-497, 1975). The process is well known and is reviewed in the Examples set forth below. Briefly, an appropriate animal, such as a mouse, is injected with an immunogen, such as the complex of antigen and antibody fragments. Subsequently, the mouse is sacrificed and cells removed from its spleen are fused with myeloma cells. Fusion results in the formation of immortal hybrid cells, known as "hybridomas," which synthesize antibodies. The hybridoma population produced is screened to identify the desired antibodies. The hybridoma populations thus identified are cloned by limiting dilution to isolate hybridomas derived from a single cell and thereafter are designated as monoclonal hybridoma. The antibody obtained via this procedure is the product of a single B-cell from the immunized recipient animal that is generated in response to a specific antigenic ^" determinant identified on the immunogen. In the case described it is the juncture which is recognized. In accordance with the process, the conjunction immunogen is injected into a suitable animal, such as a mouse. The resulting hybridoma cells are screened, such as by^' Microtiter ELISA technique, for the production of a monoclonal antibody that binds to an intact (non-digested) antigen-antibody complex, but not the- antibody or the intact antigen alone.

The monoclonal antibody thus produced can be utilized in an assay procedure to bind to any co plexed antigen:antibody present in the reaction mixture. Either this monoclonal antibody or the complexed monoclonal antibody can be tagged with one of manypossible labeling materials, such as an enzyme, fluorescent, chemiluminescent, bioluminescent, isotopic, ferromagnetic, or other common signal generating material. As a result, the sensitivity of the assay procedure will be high because of the unique selectivity of the conjunction binding monoclonal antibody.

An alternative embodiment involves first forming a complex of intact antigen and intact first monoclonal antibody. Then the Fc fragment is cleaved from the antibody using pepsin or papain, followed by limited protolytic digestion of the antigen under conditions that will not result in digestion of the bound antibody. The purified product is then used as the new immunogen to be injected into the recipient animal. Care must be taken to create reaction conditions conducive to the desired reaction. If the pH is too low dissociation of the antigen:antibody complex is possible. Furthermore, because papain and pepsin are relatively non-specific enzymes (other than the action on sulfhydryl groups) there is the possibility of degradation of the antigen. While the invention has been described in connection with a preferred embodiment, it is not intended to limit the scope of the invention to the particular form set forth, but, on the contrary, it is intended to cover such alternatives, modifications and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims.

y EXAMPLES Example 1

Using Chlamydia trachomi is major outer membrane protein (MOMP) as the antigen: A. Preparation of antigen fragment containing active antigenic determinant:

1. Procedure for limited proteolytic digestion of antigen: Purified (MOMP) eluted electrophoretically from gels is dissolved at approximately 0.5 mg/ml in sample buffer which contains 0.125 M Tris/HCl at pH 6.8, 0.5% SDS,

10% glycerol , and 0.0001% bromphenol blue. The samples are heated to 100°C for 2 minutes. Proteolytic digestion is carried out at 37°C for 30 minutes by addition of appropriate amounts of a particular protease. Selection of protease and optimal conditions are determined empirically for each antigen to provide the desired digestion products. Among the preferred proteases include, but are not limited to, endoproteinase Glu-C from Staphylococcus aureus V8, endoproteinase Arg-C from mouse submaxillary gland, endoproteinase Tys-C from Lysobacter enzymogenes, and the like. Following addition of 2-mercaptoethanol and SDS to final concentrations of 10% and 2%, respectively, proteolysis is stopped by boiling the samples for 2 minutes. 20 to 30ul (10 to 15ug) of each sample is loaded into a sample well of 15% acrylamide gel and the gel is run according to accepted procedures.

2. Identification of epitope-bearing portion of the proteolytic digest preparation:

SDS-polyacrylamide gel electrophoresis of the digest is performed. The preparations are transferred electrophoretically to nitrocellulose paper using the method of Towbin, et al , commonly referred .to as Western blot. To identify the polypeptide fragment of interest an ELISA is performed using a selected monoclonal antibody.

3. Purificati on of chl amydia MOMP fragment:

The MOMP fragment i s puri fied by SDS-PAGE and el ectroel uti on .

B . Preparation of Fab antibody fragment

1. Procedure for cl eavage of antibody mol ecul e :

(a) 0.5 ml Immobi l i zed papai n (pre-washed i n buffer) i s added to 10 mg IgG i n 1.0 ml freshly made 20 mM NaH PO + 20 mM cysteine HC1 + 10 mM EDTA-Na , pH

6.2.

(b) Incubated 5' hours at 37°C with rocking.

(c) 30 ml 10 mM Tris-HCl, pH 7.5 is added. The reactants are mixed and centrifuged. 2. Procedure for purification of Fab antibody fragment: (a) The supernatant is applied to a 5 ml Immobilized Protein A column equilibrated with 10 mM Tris-HCl, pH 7.5. (b) The column is washed with 15 ml 10 mM Tris buffer, pH 7.5. The protein eluded is Fab. (c) To recover Fc and undigested IgG, the gel is washed with 0.1 M glycine-HCl, pH 2.8.

C. Preparation and purification of complex consisting of Chlamydia antigen fragment and Fab fragment:

Purified antigen fragment and Fab preparations are mixed in a 20 mM phosphate buffer pH 7.4, with 1,50 mM NaCl. The mixture is allowed to incubate for a period of time until equilibrium is reached for the binding of the components. The incubation time- is determined by empirical observation. When equilibrium has been reached the mixture is subjected to molecular exclusion chromatography using, for example, Sephadex G-75 superfine gel filtration beads (Sigma, St. Louis, MO). Since the molecular weights of both the antigen fragments and the Fab fragments are known, fractions from a calibrated molecular exclusion column containing the complex are easily located by determining protein content using a spectrophotometric reading at 280 nm. The appropriate fractions are pooled and may be concentrated for use as an immunogen.

D. Preparation of monoclonal antibodies to purified conjunction immunogen: This antibody is prepared according to the accepted procedure described by Milstein & ohler (Nature 256:495-497, 1975).

The monoclonal antibodies are prepared by fusing spleen cells, from a mouse immunized against- the chlamydia antigen:antibody complex, with an appropriate murine myeloma cell line (P3X63-Ag8.653). The resultant product is then cultured in a standard hypoxanthiπe, aminopterin, and thymidine (HAT) medium. Immunoassays are utilized to screen the specific antibodies produced by the growing hybridoma cultures. Typical immunoassays that may be used include microtiter ELISA and dot-blot ELISA. The first is done in 96 well microtiter plates and the second is done on strips of nitrocellulose paper. The antibody alone, the antigen alone and the antibod :antigen complex are applied to the plates or paper as the antigen source for the conjunction binding antibody. After the antibody of interest has been allowed to react with the three potential antigen sources a second antibody, i.e., enzyme conjugated anti-mouse IgG, is added to indicate where the desired first antibody-.antigen complex and not the antibody or antigen alone will be selected.

The immunized spleen cells may be derived from any mammal, such as primates, humans, rodents (i.e., mice, rats, and rabbits), bovine, ovine, canine, or the like, but the present invention will be described in connection with mice. The mouse is immunized by several injections of the immunogenic complex generally over a period of several weeks. When the mouse shows sufficient antibody titre against the immunogenic complex, as determined by conventional assay methods, it is given a booster injection of the immunogenic complex, then sacrificed- and the spleen is removed. The fusion is then initiated utilizing spleen cells from immunized mice and an appropriate myeloma cell line (For Example, P3X63-Ag8.653).

The hybridomas yielding antibodies specific for the immunogenic complex are selected and cloned utilizing standard methods. The monoclonal antibodies from the clones are then tested to determine their specificity for the particular immunogenic complex. Selected monoclonal antibodies specific for the immunogenic complex are then conjugated with an appropriate label. Amounts of antibody sufficient for labeling and subsequent commercial production are obtained from monoclonal hybridomas by the accepted techniques, such as a batch or continuous tissue culture or culture in vivo in mammals, such as mice. The monoclonal antibodies may be labeled with signal generating material, such as an enzyme, fluorescent compound, luminescent compound, radioactive compound, ferromagnetic label, and the like. Examples of enzymes utilized as labels are alkaline phosphatase, glucose oxidase, galactosidase, peroxidase, urease, and the like. Conjugation with enzymes can be accomplished by any one of the conventional and accepted methods, such as the Staphylococcal Protein A method, the glutaraldehyde method, the benzoquinone method, the periodate method, or a biotin-a^'vidin bridge.

Example 2 The procedures according to Example 1 are followed except that in Step B the F(ab') antibody fragment is prepared rather than the Fab fragment using the following procedure: F(ab')„preparation and purification:

(a) 0.25 ml Immobilized Pepsin (pre-washed in buffer) is added to 10 mg IgG in 1.0 ml 20 M sodium acetate buffer, pH 4.5.

(b) Incubate 2 hours at 37°C with rocking.

(c) 3.0 ml 10 mM Tris HC1, pH 7.5 is added. Mix and centrifuge. (d) Supernatant is applied to 5 ml Immobilized Protein A column equilibrated with 10 mM Tris and HC1, pH 7.5.

(e) The column is washed with 15 ml 10 mM Tris buffer, pH 7.5. The protein eluted is F(ab'

(f) To recover Fc and undigested IgG, the gel is eluted with 0.1 M glycine HC1, pH 2.8.

Example 3

The procedure according to Examples 1 or 2 above is followed wherein substituted for the antigen is thyroxine and step A is eliminated, thereby permitting the use of the undigested thyroxine molecule as the antigen with which it will form the complex with the first monoclonal antibody, which itself is directed against thyroxine.

Examples 4-7 The procedure according to Examples 1 or 2 is followed wherein substituted for Chlamydia trachomatis is one of the following antigens and substituted for the first monoclonal antibody directed against Chlamydia trachomatis is a monoclonal antibody directed against the selected antigen: Candida albicans, Human T-Lymphotropic Virus - III, Giardia Iambiia, or glycosylated hemoglobin.

Claims

WHAT IS CLAIMED:

1. A method for producing a monoclonal antibody directed against the juncture of the idiotypic determinant of a first monoclonal antibody and the antigenic determinant of an antigen when said monoclonal antibody and said antigen are bound together, comprising:

(a) providing an antigen of interest;

(b) providing a first monoclonal antibody directed against said antigen and/or antigenic fragment; (c) separating the Fab or F(ab') portion of the Fc portion of said first monoclonal antibody by enzymatic cleavage; (d) combining said antigen and said Fab or F(ab') fragment of said first monoclonal antibody in a manner favoring the formation of an immunogenic complex; (e) isolating said immunogenic complex;

(f) injecting the immunogenic complex of step (e) into a host animal ; and

(g) obtaining said monoclonal antibodies directed against said immunogenic complex capable of recognizing the juncture between said antigen and said first antibody when complexed together.

2. The method of Claim 1 comprising the further step of subjecting said antigen to limited proteolytic digestion to produce at least one antigenic fragment capable of binding to an antibody directed against said antigen.

3. The method of Claim 2 wherein said antigen or said antigenic proteolytic digest fragment and said Fab or F(ab';i fragments of said monoclonal antibody are combined in a manner favoring the formation of an immunogenic complex between the antigenic determinant of said antigen or digest thereof and said Fab or F(ab') fragments.

4. The method of Claim 2 wherein said antigen fragment containing the desired antigenic determinant is identified comprising the further steps of:

(a) performing polyacrylamide gel electrophoresis upon the products of said limited proteolytic digestion;

(b) performing a Western blot on the product of step (a); and then

(c) performing an immunoassay technique on the product of step (b) with a monoclonal antibody.

5. The method of Claim 4 wherein the immunoassay technique of step (c) is an enzyme-linked immunosorbant assay.

6. The method of Claim 5 wherein said first monoclonal antibody is contacted with an effective amount of papain so as to cleave said first monoclonal antibody into at least three fragments such that the Fab fragments remain intact and retain their immunochemical activity.

7. The method of Claim 5 wherein said first monoclonal antibody is combined with an effective amount of pepsin so as to cleave said first monoclonal antibody into at least two fragments such that the F(ab')₂ fragment remains intact and retains its immunochemical activity.

8. The method of Claims 6 or 7 wherein said fragments are purified by an affinity chromatography method.

9. The method of Claims 6 or 7 wherein said fragment are purified by molecular exclusion chromatography.

10. The method of Claim 2 wherein said immunogenic complex is injected into a recipient animal so as to induce the production of antibodies directed against said immunogenic complex. ^•

11. The method of Claim 10 wherein immune lymphocytes producing said antibodies are removed and hybridoma cells producing monoclonal antibodies are prepared thereto.

12. The method of Claim 1 wherein a syngeneic system of animal recipients is used.

13. The method of Claim 12 wherein said monoclonal antibodies are prepared for a murine recipient.

14. A method for producing a monoclonal antibody directed against the juncture of the idiotypic determinant of a first monoclonal antibody and thyroxine when said first monoclonal antibody and said thyroxine are complexed together, comprising:

(a) contacting a first monoclonal antibody directed against thyroxine with an effective amount of pepsin in a manner so as to effect the separation of Fab from Fc fragments; (b) recovering the Fab fragment of step (a) by using Staphylococcus Protein A affinity chromatography; (c) combining said Fab fragment with an effective amount of thyroxine in a manner favoring the formation of an immunogenic complex; (d) purifying the immunogenic complex of step (c) by molecular exclusion chromatography; (c) contacting said MOMP with an effective amount of a protease under conditions favoring the limited proteolysis of said

MOMP while retaining at least one intact active antigenic determinant on said MOMP; (d) purifying the antigenic determinant of step (c);

(e) contacting the said purified Fab or F(ab') of step (b) with said purified fragment of said MOMP containing said antigenic determinant in a manner favoring the formation of an immunogenic complex; (f) purifying the immunogenic complex of step (e) by molecular exclusion chromatography; (g) chemically cross-linking with an effective amount of glutaraldeh de said immunogenic complex so as to enhance the immunogenicity of said immunogenic complex by formation of polymers of said immunogenic complex;

(h) immunizing a Balb/c mouse recipient syngeneic with said first monoclonal antibody of step (a) with the product of step (g);₄ (i) harvesting the spleen cells produced in response to said immunogenic complex;

(j) fusing said spleen cells of step (i) with an immortal myeloma cell line; and (k) identifying and purifying the monoclonal antibodies that are directed specifically against the juncture of said immune complex of said MOMP and said first monoclonal antibody rather than against said first monoclonal antibody or against said MOMP alone. (e) chemically crosslinking with glutaraldehyde the immunogenic complex of step (f) so as to enhance its immunogenicity by formation of polymers of said immunogenic complex.

(f) immunizing an animal recipient syngeneic with said first 5 monoclonal antibody with said immunogenic complex of step

(e);

(g) harvesting the immune spleen cells produced in response to said complex;

(h) fusing said spleen cells of step (g) with an immortal ₁0 myeloma cell line; and

(i) isolating and purifying the monoclonal antibodies formed that are directed specifically against the juncture of said complex rather than said first monoclonal antibody or said thyroxine alone. _l ~ 15. A method for producing a monoclonal antibody directed against the juncture of an antigenic determinant on the Chlamydia trachomatis major outer membrane protein ("MOMP") and the idiotypic determinant of a different monoclonal antibody directed against said MOMP when complexed together, comprising: ₂0 (^a) contacting a first monoclonal antibody directed against said MOMP and derived from a Balb/c mouse with an amount of papain so as to cleave the F(ab') fragments from the Fc fragment; (b) recovering the F(ab') fragments of step (a) by using

₂5 affinity chromatography;