CA1329545C - Immunological complex, its preparation and its use - Google Patents
Immunological complex, its preparation and its useInfo
- Publication number
- CA1329545C CA1329545C CA000535725A CA535725A CA1329545C CA 1329545 C CA1329545 C CA 1329545C CA 000535725 A CA000535725 A CA 000535725A CA 535725 A CA535725 A CA 535725A CA 1329545 C CA1329545 C CA 1329545C
- Authority
- CA
- Canada
- Prior art keywords
- antibodies
- animal species
- antigen
- immunological complex
- bifunctional
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
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- 108091007433 antigens Proteins 0.000 claims abstract description 62
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- 239000000126 substance Substances 0.000 claims abstract description 31
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- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 claims abstract description 26
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 claims abstract description 26
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6871—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting an enzyme
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- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6873—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting an immunoglobulin; the antibody being an anti-idiotypic antibody
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- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6875—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody being a hybrid immunoglobulin
- A61K47/6879—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody being a hybrid immunoglobulin the immunoglobulin having two or more different antigen-binding sites, e.g. bispecific or multispecific immunoglobulin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2812—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD4
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2833—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
- C07K16/4283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/46—Hybrid immunoglobulins
- C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
- G01N33/56977—HLA or MHC typing
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
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Abstract
Abstract The invention relates to an immunological complex of two primary antibodies, which have been conjugated to form a cyclic tetramer with two linker antibodies directed against the Fc-fragments of the primary antibodies. Preferably, the complex is bifunctional, that is to say, that it contains two different primary antibodies, one of which is preferably directed to a detectable substance, such as an enzyme, e.g. peroxidase, and the other is directed to any desired antigen. A tetrameric complex of this kind may be used as a labelled antibody in immunoassays.
Further, the invention relates to processes for preparing the complexes and for using the complexes in immunological reactions.
Finally, the invention relates to drugs containing a complex according to the invention, as the complexes may be used to direct certain active principles to target cells.
Further, the invention relates to processes for preparing the complexes and for using the complexes in immunological reactions.
Finally, the invention relates to drugs containing a complex according to the invention, as the complexes may be used to direct certain active principles to target cells.
Description
1 32q5~5 When carrying out immunological determinations, measurement of the extent to which an immunological reaction (antigen-antibody reaction) has proceeded, is necessary.
Generally, the antigen or the antibody has to be provided with a detectable group (label). For this purpose various methods are in use already, of which labelling with radioactive, fluorescent, chemiluminescent or enzymatically active atoms or groups may be mentioned. Generally, these detectable groups or atoms are introduced into the antigen or into the antibody by chemical means. Various chemical coupling methods, especially for enzymes, are discussed in Immunohistochemistry 20 (1983), pages 87-100. In chemical coupling of enzymes to antibodies bifunctional coupling agents reacting with the enzyme and with the antibody are generally used. The chemical coupling methods have various disadvantages. Thus, the antibody may be denatured by reaction with the coupling agent, and enzyme coupled with itself, as well as antibody coupled with itself are always obtained together with the desired labelled antibody.
Another approach to the labelling of antibodies is described in Nature 305 (1983), pages 537-540. This publication relates to the production of hybrid antibodies which are capable of binding with two different antigens.
The production of these hybrid antibodies is effected by means of hybrid hybridomas. Particularly, an anti-somatostatine-anti-peroxidase antibody is described which, consequently, is capable of reacting with a labeiY~S
9~
':
- ~ - t329~45 the enzyme peroxidase, as well as with somatostatine.
Although these bifunctional antibodies constitute an improvement with respect to the chemically labelled antibodies, they still have the disadvantage that a hybridoma has to be constructed for each separate bifunctional antigen, which is highly time-c:onsuming.
It has now been found that immunological reagents possessing two different functions may be prepared in a much simpler way. Namely, it was found that antibodies of a first animal species form a cyclic tetrameric complex with monoclonal antibodies of a second animal species the Fab-fragments of which are directed against the Fc fragments of the first animal species. The cyclic tetrameric complex is built from two molecules of primary antibodies and two molecules of linker antibodies specific for the Fc fragments of the primary antibodies. These cyclic tetrameric complexes possess high stability. When the primary antibodies are different from each other the immunological complex is bifunctional and may be used in the same way as the above-mentioned bifunctional antibodies which may be produced with hybridomas.
In accordance with the invention an immunological complex is provided comprising two primary antibodies, which are conjugated to form a cyclic tetramer with two linker antibodies directed against the Fc fragments of the primary antibodies. A broader aspect of the invention relates to i~munological complexes comprising two primary antibodies which are conjugated to form a cyclic tetramer with two linker antibodies directed against selected antigenic E
. . .
. ~., . . ~
- 3 ~ ~ 329 5 ~ 5 determinants on either the Fab or Fc fragments of the primary antibodies.
In one embodiment of the invention an immunological complex is provided comprising two primary antibodies of a first animal species which are conjugated to form a cyclic tetramer with two linker antibodies which are monoclonal antibodies of a second animal species which are directed against the Fc fragments of the primary antibodies.
In a second embodiment of the invention an immunological complex is provided comprising two primary antibodies of different animal species which are conjugated to form a cyclic tetramer with two linker antibodies which are hybrid bispecific antibodies directed against both the Fc fragments of the primary antibodies.
In a third embodiment of the invention an immunological complex is provided comprising two primary antibodies of different immunoglobulin isotypes conjugated to form a cyclic tetramer with two linker antibodies which are hybrid bispecific antibodies directed against both the Fc fragments of the primary antibodies.
Further details of the invention are described below with the help of the examples illustrated in the accompanying drawings in which:
Figure 1 shows the results of agar gel electrophoresis showing the formation of the cyclic tetrameric complexes of the invention;
~ Figure 2 shows a schematic diagram of one embodiment of the immunological complexes of the invention;
, - -. ~
and Figure 3 shows a schematic diagram of second and third embodiments of the immunological complexes of the invention.
In a preferred embodiment of the invention an immunological complex is provided which is bifunctional and in which two primary antibodies having different specificities are conjugated to form a cyclic tetramer with two linker antibodies directed against the Fc fragments of the primary antibodies.
The formation of the cyclic tetrameric complexes according to the invention has been shown by means of electrophoxesis on agar plates and by means of electron microscopy.
Mouse antibodies of the immunoglobulin gamma-l (IgGl) subclass against the enzyme peroxidase were mixed with a monoclonal rat antibody specific to mouse IgGl. The last-mentioned antibody was selected for its capability to bind two different mouse antibodies. On reaction of the anti-enzyme antibody with an equimolar amount of the rat antibody practically all of the enzyme binding activity appeared to migrate, in electrophoresis, as a single band with a mobility different from that of the separate antibodies. A similar test was carried out with mouse antibodies against the enzyme alkaline phosphatase. Also in this test a similar complex was formed.
The formation of the complexes proceeds extremely quickly, in any case within the time necessary to mix the antibodies, to apply a sample on the agar plate and to begin "
_ 5 ~ 1 3~95~5 with the electrophoresis. The minimum time necessary for this is 20 seconds.
When the two anti~enzyme antibodies are first ~ixed with each other and then the monoclonal rat antibody is added three different complexes are formed, namely a complex containing exclusively the anti-peroxidase, a complex containing exclusively the anti-alkaline phosphatase and a complex containing both of the anti-enzyme antibodies. When the anti-enzyme antibodies are first mixed separately with the monoclonal rat antibody and then both of the so formed homologous complexes are mixed, a bifunctional complex is not formed. Apparently the complexes formed are so stable that no mutual exchange of antibodies takes place. When a lower than equivalent amount of the rat antibody is added to a mixture of the anti-enzyme antibodies the above- mentioned three complexes are also formed, but unbonded anti-enzyme antibody is left.
The structure of the complexes according to the invention was investigated with scanning transmission electronmicroscopy (STEM). In images of negatively stained complexes, structures could be observed which correspond to cyclic tetramers of immunoglobulins~ Dominating in the image are tetrameric complexes of which the Fab-fragments of the mouse and rat antibodies together with the Fc-fragment of the rat antibodies are clearly visible. Certain details of the complexes, such as the domains of the two chains of the mouse-Fab-fragments, as well as the spatial orientation of the Fab- and Fc fragments could be easily distinguished.
It appears clearly from the results of the agar-gel E
,. , , . ~ .. . ...
. .
.
. ;- ~ ..... .
.. . , ` .~ ` .
.. ..
. . .
..
- 6 - 1 32~ 5 4 5 electrophoresis ~hich are illustrated in figure 1 and in example I, and from the elect~on micxoscopy that the c~clic tetrameric complexes are formed. The stable complexes according to the invention apparently have the structure as schematically indicated in figure 2.
When it is desired to prepare the bifunctional complexes, for example from antibodies against peroxidase, and from antibodies against a surface antigen on human blood platelets, both of the antibodies may be mixed in any desired ratio and the mixture may be reacted with the monoclonal antibody against the Fc fragments of the two first mentioned antibodies. The highest yield of bifunctional complex is obtained, however, when the antibodies against peroxidase and the antibodies against a surface antigen on human blood platelets are mixed in equimolar ratio. As discussed above the reaction product always additionally contains homologous complexes, in the case of the example complexes containing only antibodies against peroxidase, and complexes containing only antibodies against the surface antigen on human blood platelets. These homologous complexes should not always interfere in the use of the reaction product as an immunological reagent. For example, when the above-mentioned reaction product is reacted with human blood platelets the complexes containing exclusively antibodies against peroxidase will not adhere to the blood platelets. The other complexes will, but only the bifunctional complexes will give a reaction with platelets and a reaction with peroxidase.
If desired, the bifunctional complex may be r~
.. ..
' ~~ - 7 - 1329545 ,.
isolated in pure condition, for example by means of anion exchange chromatography. The ratio between the bifunctional complex and both of the homologous complexes in the reaction product can be influenced by changing the molar ratio of the ~ntibodies of the first animal species us2d. Thus, when using the anti-peroxidase antibody and the antibody against a surface antigen on blood platelets in a higher ratio than 1:1, for example 5:1, a reaction product is obtained in which the ratio of the homologous complex containing exclusively anti-blood platelets antibodies, to the bifunctional complex is changed in favour of the bifunctional complex.
An alternative approach to produce exclusively bifunctional complexes is to use hybrid antibodies with dual specificity for two different determinants on Fc fragments of two different primary antibodies for the preparation of complexes. Preparation of bispecific antibodies produced by hybrid hybridomas was described in Nature 305 (1983), pages 537-540, whereas preparation of bispecific antibodies by chemical recombination was described in Science 229 (1985), pages 81-83. For both these procedures two suitable, non-cross-reactive monoclonal antibodies with specificity for determinants of the Fc fragments of the two different primary antibodies have to be developed.
It i~ expected that the antigenic determinant~ ~an be present not only on the Fc fragments but also on the Fab fragments of the primary antibodies. The formation of bifunctional complexes depends on many different actors in addition to the bivalency of the antibodies and antigens, for E
- . ."" .. . .: `. ..... ' . .
, .~ ~ ~...... .. . .
,. .; ,: ,. ~ . . ,.; - ~
- 8 - ~ 3~9 5 45 example the stereochemi~try of the antibody-antigan interaction and the flexibility of the antibodies and antigens. Thus, only those monoclonal anti-immunoglobulin antibodies that will preferentially yield tetra~eric complexes when mixed with the appropriate antigen are useful as linker antibodies in the present invention. With two sui~able individual linker antibodies bispecific hybrid antibodies can be developed for use in the production of exclusively biu~ctional complexes.
The above approach could be used to prepare exclusively bifunctional tetrameric complexes of primary antibodies of different immunoglobulin subclasses (using purified hybrid antibodies with dual specificity for the two different immunoglobulin subclasses of the two primary antibodies) or species (using purified hybrid antibodies with dual specificity for immunoglobulins of the two species). A schematic diagram of such tetrameric complexes is given in figure 3.
Among the bifunctional immunological complexes according to the invention those complexes are preferred in which the Fab-fragments of one of the primary antibodies is directed against a substance having a desired property, such as detectability, toxicity, therapeutic activity. In this connection such substances should provoke the production of antibodies in an animal, including humans. Examples of detectable substances, which may provoke the production of antibodies are enzymes, such as horse-radish peroxidase, alkaline phosphatase, glucose oxidase and galactosidase.
The other primary antibody which is formed into a bifunctional complex together with the primary antibody E
:
.
r~
~ 9~ ` 1329545 against a substance having a desired property, may be directed to any desired antigen. As examples of antigens against which the second antibody may be directed, there may be mentioned: surface membrane antigens, such as antigens on the surface of blood cells, for example erythrocytes or blood platelets, or viral antigens. Further, the second antibody can be directed to a specific antigen which is produced by a malignant cell. sifunctional complexes containing such primary antibodies can be used not only for the detection of malignant cells, but also for directing substances which are toxic with respect to these cells towards the malignant cells. In the first case one of the primary antibodies is directed against a detectable substance, for example, an enzyme, and in the second case against a substance inhibiting the growth of the malignant cells.
The animal species from which the antibodles of the present tetrameric complexes are derived, are not important.
It is often convenient to combine mouse antibodies with rat antibodies. The llnker antibodies have to be direc-ted against the Fc fragments of the primary antibodies, and each of both of the Fab-fragments of the linker antibodies have to be capable of binding with the Fc fragments of one of the primary antibodies.
The presence of an Fc fragment in the linker antibodies is not essential. In certain cases it may even be advantageous to split off the Fc-fragment from the linker antibodies before formation of the cyclic complexes. This can be effected in a known way by means of an enzyme, for .
.
~\
lo - ` 1 329545 -example pepsin. This result is also achieved when hybrid bispecific antibodies are prepared as described in Science 229 (1985) page 81-83.
The invention further relates to a bifunctional immunological complex as described above, which is coupled with the antigen against which one of the primary antibodies is directed. This antigen may be, for example, a detectable substance, such as an enzyme. In that case the complex has the properties of a labelled antibody. When the antigen is a substance having another desirable property, for example toxicity with respect to malignant cells, the complex can be used, for example, as a drug.
The invention also relates to a process for preparing immunological complexes, which comprises reacting the primary antibodies with an equivalent amount of linker antibodies which are directed against the Fc fragments of the primary antibodies, and wherein a tetrameric complex is formed, and the tetrameric complex may be isolated.
Immunological complexes which are bifunctional complexes may also be prepared by reacting primary antibodies with ~wo different specificities with an equivalent amount of linker antibodies which are directed against the Fc fragments of the primary antibodies.
The preparation of a bifunctional complex coupled to an antigen, which is also the subject of this invention, is effected by reaction of the bifunctional complex with the antigen in ques-tion.
The invention further relates to a process for carrying out immunological reactions, in which the -ll-y 132q5~5 bifunctional immunological complexes according to the invention are used. An example of such a reaction is the detection of human antibodies bonded to viral antigens which have been transferred to nitrocellulose paper after electro-phoresis, by means of bispecific anti-human IgG/anti-alkaline phosphatase/alkaline phosphatase complexes.
The invention provides a simple, specific and efficient way to couple antibodies with each other and, for example, to label antibodies. Due to the high specificity of the linker antibodies the use of non-purified antibodies is possible. The process for preparing tetrameric complexes is very simple and takes extremely little time. This is a large improvement in comparison with chemical labelling of antibodies. Also in comparison with the bifunctional monoclonal antibodies produced by hybrid cell lines, the invention offers the advantage that it is not necessary to select cell lines, and the complexes may be built from bivalent monoclonal antibodies having known properties.
The bifunctional complexes according to the invention may also be used for specific coupling of antigens. Suitable combinations of monoclonal antibodies may be used for identification and separation of antigens.
:
': : . , :
.
~ 1 329~5 Further, specific interactions which are induced by bifunctional or homologous tetrameric complexes may generally improve the effectivity of fusions, transfections and interactions between cells. Thus, for example, erythrocytes may be labelled with bifunctional complexes built from anti-erythrocyte antibodies and anti-HLA-DR
antibodies. In this way bifunctional complexes may be used to direct or to improve interactions between cells. In this connection the use, in vivo, to direct certain effector cells to micro-organisms, virus-infec~ed cells and tumor cells, may be mentioned.
The homologous cyclic tetrameric complexes according to the in~ention may be used, for example, for detecting human antibodies on human red blood cells, for example Rhesus antibodies. In this case homologous complexes of anti-human IgG-antibodies are used as anti-immunoglobulin (Coomb's reagent) for the agglutination of sensitized erythrocytes.
The invention is illustrated in the following examples which, however, do not limit the invention in any way whatsoever.
Example I:
Detection of the formation of cyclic tetrameric antibody complexes by means of gel electrophoresis a. Mouse anti-peroxidase antibody.
Monoclonal mouse antibodies against horse-radish peroxidase (CLB-HRP-l) were used for the gel electrophoresis. A stock solution (0.2 mg/ml, in phosphate E
~.
buffered saline (PBS; pH 7.2)) o~ ~e~ 9a~t4b5Ody, which was purified over a protein-A-Sepharose column was diluted to the desired concentration with PBS.
b. Mouse anti-alkaline phosphatase antibody.
Monoclonal antibodies against alkaline phosphatase (CLB-AP-4) were used. Starting material was the 20 x concentrated supernatant of the tissue culture containing 0.2 mg antibody/ml. This was diluted to the desired concentration with PBS.
c. Monoclonal rat anti-mouse antibodies.
In the gel electrophoresis monoclonal rat antibodies against mouse IgG1 were used. These were obtained by fusion (Nature 266, 550-553 (1977)) of mouse-Sp2/0-Agl4 cells (Nature 274, 917-919 (1978)) with spleen cells of a Brown Norway Rat which was immunized three times with various preparations of purified monoclonal mouse IgGl. The supernatants of the hybridoma cells were tested for the property to couple monoclonal mouse peroxidase-antiperoxidase (PAP) complexes to mouse IgGl layered upon microtiter plates, by means of the ELISA technique. For further research a hybridoma cell line (CLB-M 1-1) was selected from a number of positively reacting cell lines, and was used for larger scale culturing. The IgGl-kappa-antibody produced by this cell line was purified by affinity chromatography on a mouse IgGl-Sepharose column.
d. Gel electrophoresis was carried out on agar plates according to Wieme "GelElectrophoresi8" Elsevier Publ. Co., Amsterdam (1976).
The following products were introduced upon the E~
.
.. ; ~.
. . .
' .
. ~ . .
~ 1 329545 lanes of three agar plates:
Lane 1: Monoclonal anti-peroxidase (anti-POD),
Generally, the antigen or the antibody has to be provided with a detectable group (label). For this purpose various methods are in use already, of which labelling with radioactive, fluorescent, chemiluminescent or enzymatically active atoms or groups may be mentioned. Generally, these detectable groups or atoms are introduced into the antigen or into the antibody by chemical means. Various chemical coupling methods, especially for enzymes, are discussed in Immunohistochemistry 20 (1983), pages 87-100. In chemical coupling of enzymes to antibodies bifunctional coupling agents reacting with the enzyme and with the antibody are generally used. The chemical coupling methods have various disadvantages. Thus, the antibody may be denatured by reaction with the coupling agent, and enzyme coupled with itself, as well as antibody coupled with itself are always obtained together with the desired labelled antibody.
Another approach to the labelling of antibodies is described in Nature 305 (1983), pages 537-540. This publication relates to the production of hybrid antibodies which are capable of binding with two different antigens.
The production of these hybrid antibodies is effected by means of hybrid hybridomas. Particularly, an anti-somatostatine-anti-peroxidase antibody is described which, consequently, is capable of reacting with a labeiY~S
9~
':
- ~ - t329~45 the enzyme peroxidase, as well as with somatostatine.
Although these bifunctional antibodies constitute an improvement with respect to the chemically labelled antibodies, they still have the disadvantage that a hybridoma has to be constructed for each separate bifunctional antigen, which is highly time-c:onsuming.
It has now been found that immunological reagents possessing two different functions may be prepared in a much simpler way. Namely, it was found that antibodies of a first animal species form a cyclic tetrameric complex with monoclonal antibodies of a second animal species the Fab-fragments of which are directed against the Fc fragments of the first animal species. The cyclic tetrameric complex is built from two molecules of primary antibodies and two molecules of linker antibodies specific for the Fc fragments of the primary antibodies. These cyclic tetrameric complexes possess high stability. When the primary antibodies are different from each other the immunological complex is bifunctional and may be used in the same way as the above-mentioned bifunctional antibodies which may be produced with hybridomas.
In accordance with the invention an immunological complex is provided comprising two primary antibodies, which are conjugated to form a cyclic tetramer with two linker antibodies directed against the Fc fragments of the primary antibodies. A broader aspect of the invention relates to i~munological complexes comprising two primary antibodies which are conjugated to form a cyclic tetramer with two linker antibodies directed against selected antigenic E
. . .
. ~., . . ~
- 3 ~ ~ 329 5 ~ 5 determinants on either the Fab or Fc fragments of the primary antibodies.
In one embodiment of the invention an immunological complex is provided comprising two primary antibodies of a first animal species which are conjugated to form a cyclic tetramer with two linker antibodies which are monoclonal antibodies of a second animal species which are directed against the Fc fragments of the primary antibodies.
In a second embodiment of the invention an immunological complex is provided comprising two primary antibodies of different animal species which are conjugated to form a cyclic tetramer with two linker antibodies which are hybrid bispecific antibodies directed against both the Fc fragments of the primary antibodies.
In a third embodiment of the invention an immunological complex is provided comprising two primary antibodies of different immunoglobulin isotypes conjugated to form a cyclic tetramer with two linker antibodies which are hybrid bispecific antibodies directed against both the Fc fragments of the primary antibodies.
Further details of the invention are described below with the help of the examples illustrated in the accompanying drawings in which:
Figure 1 shows the results of agar gel electrophoresis showing the formation of the cyclic tetrameric complexes of the invention;
~ Figure 2 shows a schematic diagram of one embodiment of the immunological complexes of the invention;
, - -. ~
and Figure 3 shows a schematic diagram of second and third embodiments of the immunological complexes of the invention.
In a preferred embodiment of the invention an immunological complex is provided which is bifunctional and in which two primary antibodies having different specificities are conjugated to form a cyclic tetramer with two linker antibodies directed against the Fc fragments of the primary antibodies.
The formation of the cyclic tetrameric complexes according to the invention has been shown by means of electrophoxesis on agar plates and by means of electron microscopy.
Mouse antibodies of the immunoglobulin gamma-l (IgGl) subclass against the enzyme peroxidase were mixed with a monoclonal rat antibody specific to mouse IgGl. The last-mentioned antibody was selected for its capability to bind two different mouse antibodies. On reaction of the anti-enzyme antibody with an equimolar amount of the rat antibody practically all of the enzyme binding activity appeared to migrate, in electrophoresis, as a single band with a mobility different from that of the separate antibodies. A similar test was carried out with mouse antibodies against the enzyme alkaline phosphatase. Also in this test a similar complex was formed.
The formation of the complexes proceeds extremely quickly, in any case within the time necessary to mix the antibodies, to apply a sample on the agar plate and to begin "
_ 5 ~ 1 3~95~5 with the electrophoresis. The minimum time necessary for this is 20 seconds.
When the two anti~enzyme antibodies are first ~ixed with each other and then the monoclonal rat antibody is added three different complexes are formed, namely a complex containing exclusively the anti-peroxidase, a complex containing exclusively the anti-alkaline phosphatase and a complex containing both of the anti-enzyme antibodies. When the anti-enzyme antibodies are first mixed separately with the monoclonal rat antibody and then both of the so formed homologous complexes are mixed, a bifunctional complex is not formed. Apparently the complexes formed are so stable that no mutual exchange of antibodies takes place. When a lower than equivalent amount of the rat antibody is added to a mixture of the anti-enzyme antibodies the above- mentioned three complexes are also formed, but unbonded anti-enzyme antibody is left.
The structure of the complexes according to the invention was investigated with scanning transmission electronmicroscopy (STEM). In images of negatively stained complexes, structures could be observed which correspond to cyclic tetramers of immunoglobulins~ Dominating in the image are tetrameric complexes of which the Fab-fragments of the mouse and rat antibodies together with the Fc-fragment of the rat antibodies are clearly visible. Certain details of the complexes, such as the domains of the two chains of the mouse-Fab-fragments, as well as the spatial orientation of the Fab- and Fc fragments could be easily distinguished.
It appears clearly from the results of the agar-gel E
,. , , . ~ .. . ...
. .
.
. ;- ~ ..... .
.. . , ` .~ ` .
.. ..
. . .
..
- 6 - 1 32~ 5 4 5 electrophoresis ~hich are illustrated in figure 1 and in example I, and from the elect~on micxoscopy that the c~clic tetrameric complexes are formed. The stable complexes according to the invention apparently have the structure as schematically indicated in figure 2.
When it is desired to prepare the bifunctional complexes, for example from antibodies against peroxidase, and from antibodies against a surface antigen on human blood platelets, both of the antibodies may be mixed in any desired ratio and the mixture may be reacted with the monoclonal antibody against the Fc fragments of the two first mentioned antibodies. The highest yield of bifunctional complex is obtained, however, when the antibodies against peroxidase and the antibodies against a surface antigen on human blood platelets are mixed in equimolar ratio. As discussed above the reaction product always additionally contains homologous complexes, in the case of the example complexes containing only antibodies against peroxidase, and complexes containing only antibodies against the surface antigen on human blood platelets. These homologous complexes should not always interfere in the use of the reaction product as an immunological reagent. For example, when the above-mentioned reaction product is reacted with human blood platelets the complexes containing exclusively antibodies against peroxidase will not adhere to the blood platelets. The other complexes will, but only the bifunctional complexes will give a reaction with platelets and a reaction with peroxidase.
If desired, the bifunctional complex may be r~
.. ..
' ~~ - 7 - 1329545 ,.
isolated in pure condition, for example by means of anion exchange chromatography. The ratio between the bifunctional complex and both of the homologous complexes in the reaction product can be influenced by changing the molar ratio of the ~ntibodies of the first animal species us2d. Thus, when using the anti-peroxidase antibody and the antibody against a surface antigen on blood platelets in a higher ratio than 1:1, for example 5:1, a reaction product is obtained in which the ratio of the homologous complex containing exclusively anti-blood platelets antibodies, to the bifunctional complex is changed in favour of the bifunctional complex.
An alternative approach to produce exclusively bifunctional complexes is to use hybrid antibodies with dual specificity for two different determinants on Fc fragments of two different primary antibodies for the preparation of complexes. Preparation of bispecific antibodies produced by hybrid hybridomas was described in Nature 305 (1983), pages 537-540, whereas preparation of bispecific antibodies by chemical recombination was described in Science 229 (1985), pages 81-83. For both these procedures two suitable, non-cross-reactive monoclonal antibodies with specificity for determinants of the Fc fragments of the two different primary antibodies have to be developed.
It i~ expected that the antigenic determinant~ ~an be present not only on the Fc fragments but also on the Fab fragments of the primary antibodies. The formation of bifunctional complexes depends on many different actors in addition to the bivalency of the antibodies and antigens, for E
- . ."" .. . .: `. ..... ' . .
, .~ ~ ~...... .. . .
,. .; ,: ,. ~ . . ,.; - ~
- 8 - ~ 3~9 5 45 example the stereochemi~try of the antibody-antigan interaction and the flexibility of the antibodies and antigens. Thus, only those monoclonal anti-immunoglobulin antibodies that will preferentially yield tetra~eric complexes when mixed with the appropriate antigen are useful as linker antibodies in the present invention. With two sui~able individual linker antibodies bispecific hybrid antibodies can be developed for use in the production of exclusively biu~ctional complexes.
The above approach could be used to prepare exclusively bifunctional tetrameric complexes of primary antibodies of different immunoglobulin subclasses (using purified hybrid antibodies with dual specificity for the two different immunoglobulin subclasses of the two primary antibodies) or species (using purified hybrid antibodies with dual specificity for immunoglobulins of the two species). A schematic diagram of such tetrameric complexes is given in figure 3.
Among the bifunctional immunological complexes according to the invention those complexes are preferred in which the Fab-fragments of one of the primary antibodies is directed against a substance having a desired property, such as detectability, toxicity, therapeutic activity. In this connection such substances should provoke the production of antibodies in an animal, including humans. Examples of detectable substances, which may provoke the production of antibodies are enzymes, such as horse-radish peroxidase, alkaline phosphatase, glucose oxidase and galactosidase.
The other primary antibody which is formed into a bifunctional complex together with the primary antibody E
:
.
r~
~ 9~ ` 1329545 against a substance having a desired property, may be directed to any desired antigen. As examples of antigens against which the second antibody may be directed, there may be mentioned: surface membrane antigens, such as antigens on the surface of blood cells, for example erythrocytes or blood platelets, or viral antigens. Further, the second antibody can be directed to a specific antigen which is produced by a malignant cell. sifunctional complexes containing such primary antibodies can be used not only for the detection of malignant cells, but also for directing substances which are toxic with respect to these cells towards the malignant cells. In the first case one of the primary antibodies is directed against a detectable substance, for example, an enzyme, and in the second case against a substance inhibiting the growth of the malignant cells.
The animal species from which the antibodles of the present tetrameric complexes are derived, are not important.
It is often convenient to combine mouse antibodies with rat antibodies. The llnker antibodies have to be direc-ted against the Fc fragments of the primary antibodies, and each of both of the Fab-fragments of the linker antibodies have to be capable of binding with the Fc fragments of one of the primary antibodies.
The presence of an Fc fragment in the linker antibodies is not essential. In certain cases it may even be advantageous to split off the Fc-fragment from the linker antibodies before formation of the cyclic complexes. This can be effected in a known way by means of an enzyme, for .
.
~\
lo - ` 1 329545 -example pepsin. This result is also achieved when hybrid bispecific antibodies are prepared as described in Science 229 (1985) page 81-83.
The invention further relates to a bifunctional immunological complex as described above, which is coupled with the antigen against which one of the primary antibodies is directed. This antigen may be, for example, a detectable substance, such as an enzyme. In that case the complex has the properties of a labelled antibody. When the antigen is a substance having another desirable property, for example toxicity with respect to malignant cells, the complex can be used, for example, as a drug.
The invention also relates to a process for preparing immunological complexes, which comprises reacting the primary antibodies with an equivalent amount of linker antibodies which are directed against the Fc fragments of the primary antibodies, and wherein a tetrameric complex is formed, and the tetrameric complex may be isolated.
Immunological complexes which are bifunctional complexes may also be prepared by reacting primary antibodies with ~wo different specificities with an equivalent amount of linker antibodies which are directed against the Fc fragments of the primary antibodies.
The preparation of a bifunctional complex coupled to an antigen, which is also the subject of this invention, is effected by reaction of the bifunctional complex with the antigen in ques-tion.
The invention further relates to a process for carrying out immunological reactions, in which the -ll-y 132q5~5 bifunctional immunological complexes according to the invention are used. An example of such a reaction is the detection of human antibodies bonded to viral antigens which have been transferred to nitrocellulose paper after electro-phoresis, by means of bispecific anti-human IgG/anti-alkaline phosphatase/alkaline phosphatase complexes.
The invention provides a simple, specific and efficient way to couple antibodies with each other and, for example, to label antibodies. Due to the high specificity of the linker antibodies the use of non-purified antibodies is possible. The process for preparing tetrameric complexes is very simple and takes extremely little time. This is a large improvement in comparison with chemical labelling of antibodies. Also in comparison with the bifunctional monoclonal antibodies produced by hybrid cell lines, the invention offers the advantage that it is not necessary to select cell lines, and the complexes may be built from bivalent monoclonal antibodies having known properties.
The bifunctional complexes according to the invention may also be used for specific coupling of antigens. Suitable combinations of monoclonal antibodies may be used for identification and separation of antigens.
:
': : . , :
.
~ 1 329~5 Further, specific interactions which are induced by bifunctional or homologous tetrameric complexes may generally improve the effectivity of fusions, transfections and interactions between cells. Thus, for example, erythrocytes may be labelled with bifunctional complexes built from anti-erythrocyte antibodies and anti-HLA-DR
antibodies. In this way bifunctional complexes may be used to direct or to improve interactions between cells. In this connection the use, in vivo, to direct certain effector cells to micro-organisms, virus-infec~ed cells and tumor cells, may be mentioned.
The homologous cyclic tetrameric complexes according to the in~ention may be used, for example, for detecting human antibodies on human red blood cells, for example Rhesus antibodies. In this case homologous complexes of anti-human IgG-antibodies are used as anti-immunoglobulin (Coomb's reagent) for the agglutination of sensitized erythrocytes.
The invention is illustrated in the following examples which, however, do not limit the invention in any way whatsoever.
Example I:
Detection of the formation of cyclic tetrameric antibody complexes by means of gel electrophoresis a. Mouse anti-peroxidase antibody.
Monoclonal mouse antibodies against horse-radish peroxidase (CLB-HRP-l) were used for the gel electrophoresis. A stock solution (0.2 mg/ml, in phosphate E
~.
buffered saline (PBS; pH 7.2)) o~ ~e~ 9a~t4b5Ody, which was purified over a protein-A-Sepharose column was diluted to the desired concentration with PBS.
b. Mouse anti-alkaline phosphatase antibody.
Monoclonal antibodies against alkaline phosphatase (CLB-AP-4) were used. Starting material was the 20 x concentrated supernatant of the tissue culture containing 0.2 mg antibody/ml. This was diluted to the desired concentration with PBS.
c. Monoclonal rat anti-mouse antibodies.
In the gel electrophoresis monoclonal rat antibodies against mouse IgG1 were used. These were obtained by fusion (Nature 266, 550-553 (1977)) of mouse-Sp2/0-Agl4 cells (Nature 274, 917-919 (1978)) with spleen cells of a Brown Norway Rat which was immunized three times with various preparations of purified monoclonal mouse IgGl. The supernatants of the hybridoma cells were tested for the property to couple monoclonal mouse peroxidase-antiperoxidase (PAP) complexes to mouse IgGl layered upon microtiter plates, by means of the ELISA technique. For further research a hybridoma cell line (CLB-M 1-1) was selected from a number of positively reacting cell lines, and was used for larger scale culturing. The IgGl-kappa-antibody produced by this cell line was purified by affinity chromatography on a mouse IgGl-Sepharose column.
d. Gel electrophoresis was carried out on agar plates according to Wieme "GelElectrophoresi8" Elsevier Publ. Co., Amsterdam (1976).
The following products were introduced upon the E~
.
.. ; ~.
. . .
' .
. ~ . .
~ 1 329545 lanes of three agar plates:
Lane 1: Monoclonal anti-peroxidase (anti-POD),
2.5 ~1; 50 ~g/ml.
Lane 2: Monoclonal anti-alkaline phosphatase (anti-AP), 2.5 ~1; 50 ~g/ml.
Lane 3: Anti-POD complexed with equal amount of monoclonal rat anti-mouse IgGl (anti-mIgGl). Final concentration 50 ~g/ml, cal-culated on anti-POD. 2.5 ~ll of the solution of the complex was introduced.
Lane 4: Anti-AP complexed with equal amount of anti-IgGl. Final concentration 50 ~g/ml, calculated on anti-AP. 2.5 ~1 of the solution of the complex was introduced.
Lane 5: Anti-POD, first mixed with an equal amount of anti-AP and then the mixture complexed with an amount of anti mIgGl equivalent with the total of the mouse antibodies.
Final concentration 50 ~g/ml calculated on the total of the mouse antibodies. 2.5 of the solution was introduced.
Lane 6: Complex of anti-POD with equal amount of anti-mIqGI mixed with same amount of complex of anti-AP with anti-mIgGl. Final concentration 50 ~g/ml calculated on the total of the mouse antibodies. 2.5 ~1 of the solution was introduced.
Lane 7: Anti-POD first mixed with an equal amount of anti-AP and the mixture then complexed E
..
. ~
. . .;
`
1 32~545 with 40~ of the amount of anti-mIgGl equl~alent with the total of anti-POD and anti-AP. Final concentration 50 Mg/ml calculated on the total of the mouse antibodies. 2~5 ~1 of the solution was introduced.
The electrophoresis was carried out during 45 minutes at pH 8.5 and 300 volts. Then the agar plates were covered with nitroceliulose paper and filter paper and a pressure of about 0.1 kg/cm2 was applied during 2 hours. The blots obtained were incubated in PBS, pH 7.2 with 3%
(weight/volume) bovine serum albumin (BSA) during 16 hours.
The three blots so obtained were treated in different ways with enzymes, and developed. The results are shown in figure 1.
Blot a (figure l) was incubated for 2 h with PBS-B~A containing peroxidase labelled human kappa-chain specific rabbit immunoglobulins (DAKO, Copenhagen, Denmark, codenumber p-129j in a dilution of 1:100. These labelled immunoglobulins gave a stronger colour reaction with the monoclonal anti-peroxidase antibody bonded to the nitrocellulose than purified (monovalent) peroxidase. The blots were washed three times during five minutes with PBS
containing 0.1 percent by weight of Tween 20, and were incubated once during ~0 minutes with 0.5 mg/ml diaminobenzidine (Sigma, Grade II) in 0.05 M tris-buffer, pH
7.5 containing 0.01 percent by weight of H202. Then the result was photographed.
Blot b (figure l) was incubated during 2 h with PBS-BSA containing alkaline phosphatase (Sigma, Grade VII, E ` catalogue number p 5521) in a concentration of 10 ~g/ml. The f^ - 16 ~ 1 32 9 5 4 5 blots were washed three times during 5 minutes with PBS
containing 0.1 percent by weight of Tween 20 and were incubated once during 20 minutes with 0.2 mg/ml naphthol-AS-MX-phosphate (Sigma) in 0.1 M tris buffer, pH 8.2 containing 1 mg/ml Fast Red TR (Sigma). Then the result was photographed.
Blot c (figure 1) was obtained by first carrying out the same steps as with blot a and, after the treatment with the peroxidase substrate, washing three times with PBS
containing 0.1 percent Tween 20, then incubating with the phosphatase substrate.
The results show that the mouse antibodies form a stable complex with the monoclonal rat-antimouse antibodies.
Namely, in lane 6 of figure la and figure lb one sees only one reaction product. In lane 6 of figure lc two reaction products are shown, namely the homologous complex of the anti-POD with the rat antibodies and the homologous complex of the anti-AP with the rat antibodies. This shows that no bifunctional complex is formed when two homologous complexes are mixed with each other.
Example II:
Preparation of homologous cyclic tetrameric complex of anti-immunoglobulin To 100 ~1 of a solution of monoclonal antibodies having specificity for human IgG of the mouse IgGl-class in a concentration of 1 mg/ml in phosphate buffered saline having a pH of 7.2 with 0.1~ (weight/volume) NaN3 is added 100 ~1 of rat-anti-mouse antibody (see example Ic) having a ' '` ; ~
` I 32~545 concentration of 1 mg/ml. The latter solution also contains 0.1~ (weight/volume) NaN3.
The mixture can be used in a dilution of 1:1000 as Coombs reagent in an indirect agglutination technique to detect binding of Rhesus antibodies to human blood cells~
Example III:
Preparation of a labelled bifunctional complex To 100 ~1 of a solution of monoclonal mouse antibodies anti-leu-3a (Becton and Dickinson, commercially available) having a concentration o 0.1 mg/ml in PBS (pH
7.2) with 0.2~ (weight/volume) gelatin and 0.1%
(weight/volume) NaN3 there are added 200 ~1 of a solution of monoclonal mouse-anti-alkaline phosphatase antibodies (see example Ib) having a concentration of 0.2 mg/ml. Then 100 ~1 of a solution of rat-anti-mouse antibodies (see Example Ic) having a concentration of 1 mg~ml and finally 200 ~1 of a solution of alkaline phosphatase(sigma~ Grade VII) having a concentration of 2.5 mg/ml in PBS (pH 7.2) are added to the mixture of mouse IgGl antibodies.
This mixture can be used in a dilution of 50 to 100 times for detection of leu-3a positive cells in cryostat sections of, for example, human skin.
Example IV:
Monoclonal mouse IgGl-anti-erythrocyte antibodies and mouse IgGl-anti-HLA-DR antibodies were mixed in a molar ratio of 1:4. Then the monoclonal rat-anti-mouse antibodies (see example Ic) were added in an amount equivalent to the E
.. .. ~ , .
' ~ - 18 - 9545 total of the mouse antibodies. The mixture was used for labelling erythrocytes. For that purpose the above-mentioned mixture of antibodies was added in such an amount to an erythrocyte suspension havlng a concentration of 108 cells/ml that the final concentration calculated on the mouse-antibodies used was 50 ~g/ml. After incubation for 60 minutes at 0C, the labelled erythrocytes were washed with PBS-BSA, the mixture was centrifuged (200 g; 10 min) and was again suspended in the original volume of PBS-BSA. Then human mononuclear peripheral blood cells were added in a ratio of erythrocytes to human mononuclear peripheral blood cells of 10:1, and centrifuged for 10 min. at 200g. After incubation for 60 minutes at 0C, the sediment was carefully suspended in PBS-BSA. Cytocentrifuge preparations of the suspension were stained with May-Grunwald/Giemsa. Clear rosettes appeared to have been formed, which consist of erythrocyte coated HLA-DR-positive cells. The percentage of rosette forming, nucleus containing cells (25~) agrees well with the percentage of HLA-DR-positive cells (29~o) which is determined by means of a step-wise amplified immuno-peroxidase staining (J. Histochemistry and Cytochemistry 32, Nr. 2, pages 172-178 (1984)). When the same test is carried out, but the anti-HLA-DR antibodies are replaced with anti-peroxidase antibodies, no rosettes are formed.
:`
Lane 2: Monoclonal anti-alkaline phosphatase (anti-AP), 2.5 ~1; 50 ~g/ml.
Lane 3: Anti-POD complexed with equal amount of monoclonal rat anti-mouse IgGl (anti-mIgGl). Final concentration 50 ~g/ml, cal-culated on anti-POD. 2.5 ~ll of the solution of the complex was introduced.
Lane 4: Anti-AP complexed with equal amount of anti-IgGl. Final concentration 50 ~g/ml, calculated on anti-AP. 2.5 ~1 of the solution of the complex was introduced.
Lane 5: Anti-POD, first mixed with an equal amount of anti-AP and then the mixture complexed with an amount of anti mIgGl equivalent with the total of the mouse antibodies.
Final concentration 50 ~g/ml calculated on the total of the mouse antibodies. 2.5 of the solution was introduced.
Lane 6: Complex of anti-POD with equal amount of anti-mIqGI mixed with same amount of complex of anti-AP with anti-mIgGl. Final concentration 50 ~g/ml calculated on the total of the mouse antibodies. 2.5 ~1 of the solution was introduced.
Lane 7: Anti-POD first mixed with an equal amount of anti-AP and the mixture then complexed E
..
. ~
. . .;
`
1 32~545 with 40~ of the amount of anti-mIgGl equl~alent with the total of anti-POD and anti-AP. Final concentration 50 Mg/ml calculated on the total of the mouse antibodies. 2~5 ~1 of the solution was introduced.
The electrophoresis was carried out during 45 minutes at pH 8.5 and 300 volts. Then the agar plates were covered with nitroceliulose paper and filter paper and a pressure of about 0.1 kg/cm2 was applied during 2 hours. The blots obtained were incubated in PBS, pH 7.2 with 3%
(weight/volume) bovine serum albumin (BSA) during 16 hours.
The three blots so obtained were treated in different ways with enzymes, and developed. The results are shown in figure 1.
Blot a (figure l) was incubated for 2 h with PBS-B~A containing peroxidase labelled human kappa-chain specific rabbit immunoglobulins (DAKO, Copenhagen, Denmark, codenumber p-129j in a dilution of 1:100. These labelled immunoglobulins gave a stronger colour reaction with the monoclonal anti-peroxidase antibody bonded to the nitrocellulose than purified (monovalent) peroxidase. The blots were washed three times during five minutes with PBS
containing 0.1 percent by weight of Tween 20, and were incubated once during ~0 minutes with 0.5 mg/ml diaminobenzidine (Sigma, Grade II) in 0.05 M tris-buffer, pH
7.5 containing 0.01 percent by weight of H202. Then the result was photographed.
Blot b (figure l) was incubated during 2 h with PBS-BSA containing alkaline phosphatase (Sigma, Grade VII, E ` catalogue number p 5521) in a concentration of 10 ~g/ml. The f^ - 16 ~ 1 32 9 5 4 5 blots were washed three times during 5 minutes with PBS
containing 0.1 percent by weight of Tween 20 and were incubated once during 20 minutes with 0.2 mg/ml naphthol-AS-MX-phosphate (Sigma) in 0.1 M tris buffer, pH 8.2 containing 1 mg/ml Fast Red TR (Sigma). Then the result was photographed.
Blot c (figure 1) was obtained by first carrying out the same steps as with blot a and, after the treatment with the peroxidase substrate, washing three times with PBS
containing 0.1 percent Tween 20, then incubating with the phosphatase substrate.
The results show that the mouse antibodies form a stable complex with the monoclonal rat-antimouse antibodies.
Namely, in lane 6 of figure la and figure lb one sees only one reaction product. In lane 6 of figure lc two reaction products are shown, namely the homologous complex of the anti-POD with the rat antibodies and the homologous complex of the anti-AP with the rat antibodies. This shows that no bifunctional complex is formed when two homologous complexes are mixed with each other.
Example II:
Preparation of homologous cyclic tetrameric complex of anti-immunoglobulin To 100 ~1 of a solution of monoclonal antibodies having specificity for human IgG of the mouse IgGl-class in a concentration of 1 mg/ml in phosphate buffered saline having a pH of 7.2 with 0.1~ (weight/volume) NaN3 is added 100 ~1 of rat-anti-mouse antibody (see example Ic) having a ' '` ; ~
` I 32~545 concentration of 1 mg/ml. The latter solution also contains 0.1~ (weight/volume) NaN3.
The mixture can be used in a dilution of 1:1000 as Coombs reagent in an indirect agglutination technique to detect binding of Rhesus antibodies to human blood cells~
Example III:
Preparation of a labelled bifunctional complex To 100 ~1 of a solution of monoclonal mouse antibodies anti-leu-3a (Becton and Dickinson, commercially available) having a concentration o 0.1 mg/ml in PBS (pH
7.2) with 0.2~ (weight/volume) gelatin and 0.1%
(weight/volume) NaN3 there are added 200 ~1 of a solution of monoclonal mouse-anti-alkaline phosphatase antibodies (see example Ib) having a concentration of 0.2 mg/ml. Then 100 ~1 of a solution of rat-anti-mouse antibodies (see Example Ic) having a concentration of 1 mg~ml and finally 200 ~1 of a solution of alkaline phosphatase(sigma~ Grade VII) having a concentration of 2.5 mg/ml in PBS (pH 7.2) are added to the mixture of mouse IgGl antibodies.
This mixture can be used in a dilution of 50 to 100 times for detection of leu-3a positive cells in cryostat sections of, for example, human skin.
Example IV:
Monoclonal mouse IgGl-anti-erythrocyte antibodies and mouse IgGl-anti-HLA-DR antibodies were mixed in a molar ratio of 1:4. Then the monoclonal rat-anti-mouse antibodies (see example Ic) were added in an amount equivalent to the E
.. .. ~ , .
' ~ - 18 - 9545 total of the mouse antibodies. The mixture was used for labelling erythrocytes. For that purpose the above-mentioned mixture of antibodies was added in such an amount to an erythrocyte suspension havlng a concentration of 108 cells/ml that the final concentration calculated on the mouse-antibodies used was 50 ~g/ml. After incubation for 60 minutes at 0C, the labelled erythrocytes were washed with PBS-BSA, the mixture was centrifuged (200 g; 10 min) and was again suspended in the original volume of PBS-BSA. Then human mononuclear peripheral blood cells were added in a ratio of erythrocytes to human mononuclear peripheral blood cells of 10:1, and centrifuged for 10 min. at 200g. After incubation for 60 minutes at 0C, the sediment was carefully suspended in PBS-BSA. Cytocentrifuge preparations of the suspension were stained with May-Grunwald/Giemsa. Clear rosettes appeared to have been formed, which consist of erythrocyte coated HLA-DR-positive cells. The percentage of rosette forming, nucleus containing cells (25~) agrees well with the percentage of HLA-DR-positive cells (29~o) which is determined by means of a step-wise amplified immuno-peroxidase staining (J. Histochemistry and Cytochemistry 32, Nr. 2, pages 172-178 (1984)). When the same test is carried out, but the anti-HLA-DR antibodies are replaced with anti-peroxidase antibodies, no rosettes are formed.
:`
Claims (68)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A bifunctional immunological complex comprising two different antibodies of a first animal species which have been conjugated to form a cyclic tetramer with two antibodies of a second animal species which are directed against the Fc fragments of the antibodies of the first animal species.
2. The bifunctional immunological complex as claimed in claim 1 coupled with the antigen against which one of the antibodies of the first animal species is directed.
3. The bifunctional immunological complex as claimed in claim 1 characterised in that the Fab-fragments of one of the antibodies of the first animal species are directed against a detectable substance or a substance having toxic or therapeutic activity.
4. The bifunctional immunological complex as claimed in claim 3 wherein the substance is a detectable substance.
5. The bifunctional immunological complex as claimed in claim 4 wherein the detectable substance is an enzyme.
6. The bifunctional immunological complex as claimed in claim 5 wherein the enzyme is peroxidase, alkaline phosphatase, or glucose oxidase or galactosidase.
7. The bifunctional immunological complex as claimed in claim 6, wherein the enzyme is peroxidase.
8. The bifunctional immunological complex as claimed in claim 1, wherein the Fab-fragments of one of the antibodies of the first animal species are directed against a detectable substance, and the Fab-fragments of the other antibody of the first animal species are directed against a desired antigen.
9. The bifunctional immunological complex as claimed in claim 8 wherein the desired antigen is an antigen to be assayed in an immunoassay.
10. The bifunctional immunological complex as claimed in claim 8, wherein the desired antigen is a surface membrane antigen, a viral antigen, or an antigen produced by a malignant cell.
11. The bifunctional immunological complex as claimed in claim 8 wherein the desired antigen is a plasma protein.
12. The bifunctional immunological complex as claimed in claim 8 wherein the desired antigen is a protein present on the surface of a particle.
13. The bifunctional immunological complex as claimed in claim 12 wherein the protein is present on the surface of a blood cell.
14. The bifunctional immunological complex as claimed in claim 13 wherein the blood cell is an erythrocyte.
15. The bifunctional immunological complex as claimed in claim 13 wherein the blood cell is a platelet.
16. The bifunctional immunological complex as claimed in claim 8 wherein the antigen is a blood typing antigen.
17. The bifunctional immunological complex as claimed in claim 16 wherein the antigen is a rhesus antigen, HLA-DR or Leu-3a.
18. The bifunctional immunological complex as claimed in any one of claims 1 to 17 wherein the first animal species is the mouse and the second animal species is the rat.
19. The bifunctional immunological complex as claimed in any one of claims 1 to 17 wherein the first animal species is human and the second animal species is the mouse.
20. A process for carrying out an immunological reaction comprising treating a sample suspected of containing an antigen with an immunological complex of the invention as claimed in any one of claims 1 to 7 containing an antibody of a first animal species specific for the antigen, and determining the amount of antigen bound to the immunological complex.
21. The bifunctional immunological complex as claimed in any one of claims8 to 17 wherein the detectable substance is an enzyme.
22. The bifunctional immunological complex as claimed in any one of claims 8 to 17 wherein the detectable substance is peroxidase, alkaline phosphatase or glucose oxidase or galactosidase.
23. The bifunctional immunological complex as claimed in any one of claims 8 to 17 wherein the detectable substance is peroxidase.
24. The bifunctional immunological complex as claimed in claim 1 wherein the Fab-fragments of one of the antibodies of the first animal species are directed against a substance having toxic or therapeutic activity and the Fab-fragments of the other antibody of the first animal species are directed against a surface-membrane antigen.
25. The bifunctional immunological complex as claimed in claim 24 wherein the Fab-fragments directed against a substance having toxic or therapeutic activity are coupled with the substance having toxic or therapeutic activity.
26. The bifunctional immunological complex as claimed in claim 1 wherein the Fab-fragments of one of the antibodies of the first animal species are directed against an effector cell, and the Fab-fragments of the other antibody of the first animal species are directed against a surface-membrane antigen.
27. An immunological complex as claimed in any one of claims 24, 25 or 26 wherein the surface membrane antigen is an antigen on the surface of a tumor cell, a virus-infected ce11 or a microorganism.
28. A process for preparing a bifunctional immunological complex comprising reacting two different antibodies of a first animal species with an about equimolar amount of antibodies of a second animal species which are directed against the Fc-fragments of the antibodies of the second animal species, wherein a tetrameric complex is formed, and isolating the tetrameric complex formed.
29. The process as claimed in claim 28 which further comprises reacting the bifunctional complex formed with an antigen against which one of the antibodies of the first animal species is directed.
30. The process as claimed in claim 28 or 29, wherein the tetrameric complex is isolated by means of anion exchange chromatography.
31. The process as claimed in claim 28, wherein the Fab fragments of one of the antibodies of the first animal species are directed against a detectable substance or a substance having toxic or therapeutic activity.
32. The process as claimed in claim 31 wherein the substance is a detectable substance.
33. The process as claimed in claim 32 wherein the detectable substance is an enzyme.
34. The process as claimed in claim 33 wherein the enzyme is peroxidase, alkaline phosphatase, glucose oxidase or galactosidase.
35. The process as claimed in claim 28, wherein the Fab-fragments of one of the antibodies of the first animal species are directed against a detectable substance, and the Fab-fragments of the other antibody of the first animal species are directed against a desired antigen.
36. The process as claimed in claim 35 wherein the desired antigen is an antigen to be assayed in an immunoassay.
37. The process as claimed in claim 35, wherein the desired antigen is a surface membrane antigen, a viral antigen, or an antigen produced by a malignant cell.
38. The process as claimed in claim 35, wherein the desired antigen is a plasma protein.
39. The process as claimed in claim 35, wherein the desired antigen is a protein present on the surface of a particle.
40. The process as claimed in claim 39, wherein the protein is present on the surface of a blood cell.
41. The process as claimed in claim 40, wherein the blood cell is an erythrocyte.
42. The process as claimed in claim 40, wherein the blood cell is a platelet.
43. The process as claimed in claim 35, wherein the desired antigen is a blood typing antigen.
44. The process as claimed in claim 43, wherein the antigen is a rhesus antigen, HLA-DR or Leu-3a.
45. The process as claimed in any one of claims 28, 29 and 31 to 44 wherein the first animal species is the mouse and the second animal species is the rat.
46. The process as claimed in any one of claims 28, 29 and 31 to 44 wherein the first animal species is human and the second animal species is the mouse.
47. Use of the bifunctional immunological complex as claimed in any one of claims 1, 2, 3 to 7, 24 and 25 as a pharmaceutical.
48. Use of the immunological complex as claimed in any one of claims 1, 2, 3 to 7, 24 and 25 for treatment of cancer or infectious diseases.
49. A pharmaceutical composition comprising the immunological complex as claimed in any one of claims 1, 2, 3 to 7, 24 and 25 and one or more of a physiologically acceptable carrier, diluent or excipient.
50. A bifunctional immunological complex comprising two different antibodies of a first animal species, the Fab fragments of one of which are directed against a surface membrane antigen of an erythrocyte and the Fab-fragments of the other antibody are directed against HLA-DR, which have been conjugated to form a cyclic tetramer with two antibodies of a second animal species which are directed against the Fc-fragments of the antibodies of the first animal species.
51. Use of the immunological complex as claimed in claim 50 for determining the blood group in humans.
52. Use of the immunological complex as claimed in claim 50 in an agglutination reaction.
53. An immunological complex comprising two homologous antibodies of a first animal species which have been conjugated to form a cyclic tetramer with two antibodies of a second animal species which are directed against the Fc-fragments of the antibodies of the first animal species.
54. The immunological complex as claimed in claim 53, wherein the Fab fragments of the antibodies of the first animal species are directed against a surface membrane antigen.
55. The immunological complex as claimed in claim 54 wherein the surface membrane antigen is a protein present on the surface of a blood cell.
56. An immunological complex as claimed in claim 55 wherein the blood cell is an erythrocyte.
57. An immunological complex as claimed in claim 55 wherein the blood cell is a platelet.
58. An immunological complex as claimed in claim 55 wherein the surface membrane antigen is a blood typing antigen.
59. An immunological complex as claimed in claim 58 wherein the antigen is a rhesus antigen, HLA-DR or Leu-3a.
60. An immunological complex as claimed in any one of claims 50 and 53 to 59 wherein the first animal species is the mouse and the second animal species is the rat.
61. An immunological complex as claimed in any one of claims 53 to 59 wherein the first animal species is human and the second animal species is the mouse.
62. Use of the immunological complex as claimed in any one of claims 53 to 59 for determining the blood group in humans.
63. Use of the immunological complex as claimed in any one of claims 53 to 59 in an agglutination reaction.
64. Use of the immunological complex as claimed in any one claims 53 to 59 in a dilution of 1:1000 as Coombs reagent in an indirect agglutination reaction.
65. A process for preparing immunological complexes comprising reacting two homologous antibodies of a first animal species with an about equimolar amount of antibodies of a second animal species which are directed against the Fc-fragments of the antibodies of the first animal species, wherein a tetrameric complex is formed, and isolating the tetrameric complex formed.
66. The process as claimed in claim 65 which further comprises reacting the bifunctional complex formed with an antigen against which one of the antibodies of the first animal species is directed.
67. The process as claimed in claim 65 or 66, wherein the immunological complex is isolated by means of anion exchange chromatography.
68. The process as claimed in claim 65, wherein the Fab fragments of the antibodies of the first animal species are directed against a surface membrane antigen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000535725A CA1329545C (en) | 1987-04-28 | 1987-04-28 | Immunological complex, its preparation and its use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000535725A CA1329545C (en) | 1987-04-28 | 1987-04-28 | Immunological complex, its preparation and its use |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1329545C true CA1329545C (en) | 1994-05-17 |
Family
ID=4135519
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000535725A Expired - Lifetime CA1329545C (en) | 1987-04-28 | 1987-04-28 | Immunological complex, its preparation and its use |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1329545C (en) |
-
1987
- 1987-04-28 CA CA000535725A patent/CA1329545C/en not_active Expired - Lifetime
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