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
  • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
• • 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

Definitions

• Antibody preparation directed against neoantigens in human C3 (complement factor 3) and the use and manufacture thereof
• C3 refers to human C3 unless otherwise stated.
• Immunoglobulin is designated "Ig”.
• the complement system consists of about 20 components which have to react in a well-defined reaction sequence in order that destruction of e.g. invading microorganisms can take place in the final stage.
• the complement system is considered to be a fundamental element of the mammal's defense against infections caused by bacteria and by viruses.
• the components discovered first were called “factors" and were designated chronologically Cl, C2 C9 all along as they were being discovered.
• Complement activation can often be correlated to inflammatory processes, exposure of the factors to synthetic surfaces, surgery and diseases involving immune complex formation, e.g. infections, autoimmune diseases, cancer etc.
• C3 is composed of two different polypeptide chains. They are called the alpha chain and the beta chain, respectively, and are linked together by disulfide bonds.
• the alpha chain is believed to have a molecular weight of 115,000 daltons and the beta chain is believed to have a molecular weight of 70,000 daltons.
• the structure of C3 is at present believed to be as follows:
• the C3d region contains a thioester grouping.
• Fragmentation and/or binding to target surfaces will involve conformational changes so that new antigenic determinants are exposed with concomitant disappearance of original determinants.
• Antigenic determinants which are not to be found in native C3 but which do come forth in fragments thereof are called “neoantigens" (“neodeterminants”) .
• This subject matter is dealt with in numerous review articles (5) .
• deoxy- cholate sodium lauryl sulfate
• guanidine hydrochloride acidic or alkaline media (pH ⁇ 3 or pH >10, respectively).
• Polyclonal rabbit antibody preparations have been described which are directed against each respective type of determinant, i.e. C3 (D) alpha, C3 (D) beta, C3 (D) , C3 (N) , C3(S) and C3(S,N).
• alpha and “beta” stand for each respective peptide chain of C3, that is reduced forms have been
• Objects of the invention are to provide antibody preparations having an improved specificity against neoantigens (as compared to preparations previously known) , in particular against neoantigens exclusive for bound forms which are generated due to covalent binding of C3b to a target surface or due to further fragmentation of C3b.
• improved methods for detecting (a) soluble C3 fragments, (b) complement activation, (c) tissue deposition of C3 fragments, (d) C3 fragments bound to cells, (e) immune complexes containing C3 fragments etc.
• a third object is to provide alternative methods for diagnosticating the aforesaid conditions where complement is activated.
• a further object that may be mentioned, finally, is an improved method of obtaining antibody preparations directed against individual neoantigens.
• the antibody preparations of the invention are monospecific against the individual neoantigens that are present in reduced forms of C3, in the first place in the C3b region of C3, such as the C3c or C3d region.
• the neoantigens in question are present either (preferably) in the alpha chain thereof or in the beta chain.
• An antibody preparation of the invention will thus not react with native intact C3 homo ⁇ logous to the immunogen used for immunization, but will react with denatured and completely reduced forms thereof, that is, with at least one of the C3 polypeptide chains dissociated from the other chain.
• the preparation may also react specifically with at least one fragment (possibly bound to a target surface) selected from the group consisting of C3b, C3bi, C3c, C3d,g, C3d, C3g and other physiologically occurring C3b fragments.
• the preparation preferentially reacts with the fragments covalently bound to any one of the aforesaid target surfaces, but may in certain other cases also react with other forms. Particularly good are preparations with which it is possible to immunochemically assay for bound fragment in the presence of the corresponding free fragment.
• the preferred antibody preparations of the invention are not significantly inhibited in their reaction with C3(D) by soluble (free) native C3b, C3bi, C3d,g or C3d fragments. For instance under the conditions given in the experimental part a more than 25 times higher, such as 50 times higher dose of the soluble fragments compared to the corresponding SDS- denatured or covalently bound fragments (molar basis) is required to effect the identical inhibition in the inhibition- ELISA.
• the antibodies of the preparations may be present therein in the form of antibody-active fragments, for instance Fab, Fab', F(ab').-,. They may also be in the form of derivatized antibodies. The essential requirement is that the antibody fragments and the derivatives possess biospecific immune- type affinity in accordance with the present invention.
• the antibody-active components of the preparations may be provided with analytically detectable groups such as enzym- atically active, fluorogenic, chemiluminogenic, radioactive, biotinyl groups etc., or groups capable of acting as cofactors, coenzymes, substrates or cosubstrates etc.
• the components may be in a form where they are bound to a phase that is insoluble in the test medium - so-called solid phase; thus for example they may be physically or covalently bound to various polymers such as hydrophilic OH- or NH_-containing polymers or plastics surfaces like those of microtiter wells.
• the preparations may be in the form of a solution or suspension to which have been added various known per se chemicals as required for any special type of use. The preparations may be freeze-dried; they may be packaged in tightly sealed packs for being employed as components of a test kit etc.
• test-pack contemplated for the immunochemical assay of at least one type of C3 fragment.
• the test pack comprises an antibody preparation according to the invention.
• cells potentially capable of producing antibodies of the specificity as prescribed in conformity with the invention are caused to secrete such antibodies which are then isolated, purified and optionally fragmentized and/or derivatized in a manner known per se.
• the purification involves removal of those antibodies that fail to fulfill the prescribed specifications.
• a warm-blooded vertebrate e.g. mammal such as mouse
• secretion may take place in vivo as a result of immunization with an immunogen having the particular neoantigenic structures (neodeterminants) as contemplated here.
• the resultant immune response is polyclonal, so an antiserum obtained from the animal will contain antibodies directed against all the determinants of the immunogen, irrespective of whether or not these are neoantigenic.
• suitable selection methods the specificity of the immune response can be limited.
• a suitable im unosorbent technique it is potentially possible to obtain purified forms of the antibodies directed against the desired neoantigen.
• immunosorbent purification of polyclonal antibodies to obtain the preparation of the' invention is a laborious procedure which will always give low yields.
• the best method for selecting the antibodies in the immune response in order to obtain a good antibody preparation according to the invention is a so-called monoclonal technique (8) by which after immunization antibody-producing plasma cells are fused with cells of a suitable myeloma cell line so that they become capable of quick and uninterrupted growth.
• monoclonal technique by which after immunization antibody-producing plasma cells are fused with cells of a suitable myeloma cell line so that they become capable of quick and uninterrupted growth.
• Cultivation of the selected cell clones for pro ⁇ ducing the antibody preparations of the invention may be carried out in cell cultures in vitro or as ascites tumors in vivo. Purification and isolation may be performed in the same manner as purification and isolation of any antibodies in general - by salt precipitation or by means of various chromatographic methods like, for instance, ion exchange, affinity, gel etc. chromatography.
• Immunogens that may be employed are those that will exhibit the aforesaid particular determinants when exposed in vivo to the immune system.
• a surprisingly large proportion of antibodies of the specificity as prescribed according to the invention are obtained in the immune response if the immunogen is either the alpha chain or the beta chain in the denatured form, a mixture of them, or suitable immunogenic fragments of the chains. Examples of such fragments are denatured- reduced forms corresponding to the C3b, C3bi, C3d,g and C3c regions of these chains.
• the antibody preparations of the present invention are useful primarily in immunochemical assay methodology for C3 fragments, but they may also be used potentially both in vitro and in vivo for modulating complement activation.
• the assays comtemplated in the present context involve contacting a sample containing C3 fragment with the antibody preparations of this invention to thus form an immune complex the formation and amount of which are a quantitative measure and qualitative measure, respectively, of this C3 fragment in the sample.
• additional reactants may be used which will react with components present in the immune complex formed (anti C3 fragment - C3 fragment) . Practical considerations will decide whether these latter reactants are to be added before, after or simultaneously with the antibody preparation of the invention.
• at least one of the reactants used is provided with a suitable marker group, a so-called “analytically detectable" group. Proportions of the reactants are then chosen such that the amount of labeled reactant incorporated in the complex or the amount thereof remaining unincorporated is a measure of the aforesaid C3 fragment in the sample.
• the methods may be classed as being either homogeneous or heterogeneous methods. Homogeneous methods assay for a labeled reactant without involving any physical separation of the labeled reactant incorporated in the complex from the non-incorporated form. When heterogeneous methods are employed the two forms of the labeled reactant are separated physically from each other before the labeled reactant is assayed for in either or both of the two forms. For ease of separation it is helpful if one of the reactants is insoluble in the test medium. According to a second classification system, the methods may be classed as being either competitive or non-competitive methods.
• the arrangement is such that two immune reactants having a common epitope (determinant) are made to compete for an insufficient number of homologous binding sites on an immunological counterpart.
• the neoantigen of the sample is allowed to compete for the antibodies with an added reactant which carries the same neoantigen.
• the added neoantigen may be in a labeled, solid-phase-bound or soluble form.
• the methods may be classed as being either precipitation or non-precipitation methods.
• useful precipitating reagents may be mentioned precipitating antiserum and so-called solid-phase- bound antibodies, both of these directed against components of the complex, preferably against the antibody components. It may be mentioned in this context that neodeterminants in C3 as a rule are nonrepetitive; for this reason monoclonal antibodies directed against them are non-precipitating:
• the methods are classed according to the marker group employed; the methods are thus radio-, enzyme-, fluorescence-, chemiluminescence-, enzyme-substrate-, immunochemical methods etc.
• immunochemical methods may be mentioned also immuno- electrophoresis, particle agglutination, immunodiffusion and miscroscopy with labeled antibodies.
• the exact choice of method is decided by the form in which the fragment sought is present in the sample. If the fragment is dissolved in the sample and is not bound to a target surface a method may be chosen in accordance with the principles applying to soluble analytes in general. If on the other hand the particular fragment sought is bound to a certain special target surface then this fact has to be taken into account when the method to be employed is chosen.
• the simplest way of assaying for tissue-deposited fragments is with the aid of an antibody preparation in a labeled form, optionally in combination with microscopy. Fragments bound to blood cells may be assayed for in an analogous manner, although for quantification purposes practical advantages are gained if recourse is had to inhibition techniques.
• One of these reactants has to be of a type reacting specifically with a neoantigen in the fragment while the other reactant should react with a suitable epitope on the target surface. This implies method ⁇ ologically that a ternary complex is formed (reactant 1 C3-M reactant 2) . In the technical language this is called a "sandwich”, and the general principles applying to "sandwich” tests can be applied when the specific method is chosen.
• the invention is applicable in particular to a method of assaying for circulating immune complexes (CIC) having C3 fragments bound to them covalently.
• the complex thus contains (1) C3 fragment (C3b, C3bi, C3d,g, and C3d are those that are known up to now to exist as covalently bound forms)', (2) antibody which may be of the IgA, IgD, IgE, IgG or IgM type, and (3) antigen.
• Known techniques are generally applicable, with the exception that an antibody preparation is used which has the specificity as prescribed according to the invention. Procedures will differ according to what exactly is to be measured in each individual case.
• the general precipitation methods for immune complexes may be employed in combination with an antibody preparation of the invention which is specific against the particular neodeterminant present in the fragment sought. If an antibody preparation is employed which reacts with all conceivable C3 fragments potentially present in immune complexes, then the result obtained will be the total content of C3 fragments bound in the immune complex. If specific components of C3 fragment-containing CIC are to be assayed for, two reagents are required differing inter se by being specific for different components of CIC. The reagent required in that case in addition to an anti-C3-fragment- antibody preparation is a reagent that is specific for the antibody portion or antigen portion in the CIC.
• the reagent may be specific for one of the known Ig classes or for the antigen.
• the reagents still further 'reagents may be used.
• Assays for immune complexes containing C3 fragments have been described heretofore, (1, 21, 23, 31).
• the invention is applicable to assays in various types of samples which contain C3 and/or fragments thereof. It -has been known that C3 and its fragments are present in, for example, tissues and body fluids like blood, plasma, serum, urine, synovial fluid, cerebrospinal fluid etc. C3-forms having no C3 (D) -determinant can in certain cases be assayed, if they are denatured before reaction with the anti-C3- preparation.
• Conditions employed for carrying out the immunochemical reaction(s) are such as are usual in this type of assay methods. For instance, temperatures may be chosen within the range of 0 - 40 °C, especially 15 - 40 °C.
• a suitable pH is usually pH 4.5 - 10, preferably about 5 - 8.6.
• measures and steps have to be taken so as to avoid activation of complement or undesired denaturation of C3.
• Complement activation requires the presence of proteases and divalent calcium and/or magnesium ions. It is therefore suitable to add protease inhibitors or agents that will form complexes with the said ions, like EDTA, if C3-forms carrying C3 (D) -antigens are to be assayed in the presence of other forms. Detergents and buffer systems if added should then be of a kind that will not have a denaturing effect on C3.
• C3 (SN) Native C3
• C3 (SN) Native C3
• C3 (D) Denatured- reduced C3 and isolated C3 alpha and C3 beta chains
• C3 (D) Denatured- reduced C3 and isolated C3 alpha and C3 beta chains
• C3b was prepared in the presence of 1 % (w/v) trypsin (TPCK treated, Worthington, USA) for 2 min at room temperature (17) .
• C3bi was obtained by incubating C3b with 5 ,ug of factor H and
• Radiolabelling of native C3 with 125I was performed as described previously by a lactoperoxidase technique to a specific activity of
• Hybridomas were produced according to standard procedures (6,9) with the following minor modifications.
• Four different Sp2/0 lines were used.
• FCS fetal calf serum
• FCS fetal calf serum
• 25 ,ug/ml of Gentamycin cat no G-7507, Sigma Chem Co, USA
• Two different subclones selected for growth in serum-free medium and one subclone growing in low serum medium Hy-0.1 were also used.
• Standard DMEM containing 10 % FCS or HY-0.1 media were used for selection and cloning. The clones were
• a subclone of Sp2/O.Ag 14 myeloma line selected for growth in low serum medium Hy-0.1 were used in the fusions.
• Two different hybridoma lines were selected and cloned either in DMEM-5 % FCS or low serum medium Hy-0.1 %.
• the clones were selected for specificity in the direct-binding ELISA. 140 clones were obtained out of which 13 were randomly selected and further tested.
• EAC14 ox ⁇ 23b and EACl4 oxy 23bi cells were prepared and the uptake of C3 on the cells was estimated with 125I-labelled native C3 as described before (16) .
• Enzyme-linked immuno sorbent assay (ELISA)
• the enzyme reaction was started by the addition of 100 ,ul of colour reagent (20 mg of 1,2-phenylenediamin dihydrochloride (Fluka AG, Switzerland) and 10 ,ul 30 % ⁇ 0- in 75 ml of 0.1 m citrate/phosphate buffer pH 5.0) to the wells after step 3.
• the reaction was stopped by 100 ,ul of 1 M H 2 SO. after approximately 10 _min.
• the stain was quantified spectrophotometrically at _ 492_nm.
• step 1 100 ul of a standard dilution from 1/10 to 1/10240 or plasma samples diluted 1/26 were incubated in the wells for one hour at RT. Next followed an incubation with an affinity purified Fc-specific rabbit anti-human IgG which was beta-galactosidase-conjugated and diluted 1/200 (1 hour at RT) . The amount of enzyme bound to the wells was quantified with the aid of PRIST/RAST developing substance (100 ,ul, Pharmacia AB) which was allowed to react in the wells for two hours, whereupon the reaction was stopped after two hours with 50 ,ul of 0.66 M a 2 C0 3 .
• PRIST/RAST developing substance 100 ,ul, Pharmacia AB
• Aggregated IgG with bound C3 was prepared in that serum is activated with an addition of 600 ,ug of aggregated IgG/ml and was incubated at 37 °C (14) .
• SDS-PAGE was performed according to the method of Laemmli et al (13) and immunoblotting as described earlier (16) .
• immunoblotting technique anti-mouse immunoglobulin conjugated with HRP was employed to detect the monoclonal antibodies.
• the monoclonal antibodies of groups I - III were tested individually in the inhibition ELISA for binding to 25 pmol of C3 expressing C3 (D) , C3 (SD) or C3(SN) antigens in fluid- phase.
• group I was only inhibited by C3 (SN) and not by C3 (SD) or C3(D) antigens, which indicated that C3 (N) antigens of native C3 caused the inhibition.
• Group III was also clear-cut in that C3 (D) and C3 (SD) affected binding, due to the presence of C3(D) antigens.
• the limited inhibition caused by native C3 was probably due to a small fraction of molecules denatured during preparation and storage and the dose of antigen needed to obtain an identical degree of inhibition by C3(SN) as by C3 (D) was more than 32 times higher.
• Group II was more complex since the antibodies were inhibited by all forms of C3. Three of the antibodies were preferentially inhibited by C3(D) antigens while others were more affected by C3 (SN) and C3(SD) antigens, suggesting that the specificity of group II antibodies was for both C3(S) and C3 (D) antigens.
• the antibodies of groups I - III were tested individually by inhibition ELISA against 2.5 pmol of particle-bound C3 of EAC14 ox ⁇ 23b, EACl4 oxy 23bi and ZyC3bi (figure 4) .
• Particle- bound C3 effected inhibition ( ⁇ 90 % binding) of all anti ⁇ bodies of group I. The mean level of binding was lowest when EAC14 y 23bi was used.
• Eight to nine antibodies from group II were affected by particle-bound C3 and the mean values were identical.
• Eight antibodies of group III bound to bound C3. Of those only one reacted with both particle-bound C3b and C3bi and the rest specifically with C3bi.
• Group III anti ⁇ bodies that had been shown to be inhibited by soluble physiological fragments of C3 were preferentially inhibited by the bound form since a 64 - 256 times higher dose of the soluble fragments was needed to effect an identical level of inhibition in the inhibition ELISA.
• the antibodies of group I - III were tested by direct- binding ELISA for binding to preadsorbed native C3, SDS- denatured C3, protease-generated C3 fragments and C3 alpha and beta chain (Table I) . All of the antibodies of group I bound to native but only one to SDS-denatured C3, 12 bound to C3c and 2 to the C3 alpha or beta chain. In contrast to group I all antibodies of groups II and III bound to both native and denatured C3 and the majority was positive for binding to the C3 alpha chain, but the distribution of binding to the protease-generated fragments was similar to group I antibodies.
• C3 (D) antigens expressed by denatured and denatured-reduced C3 as shown in figure 2 were defined as anti-C3 (D) anti ⁇ bodies.
• Three monoclonal antibodies with anti-C3 (D) specificity from group II and 13 from group III were selected and their properties are summarized in Table III. All of them bound to alpha chain fragments. Those that bound to the 36 kd fragment of C3c were, with one exception, completely unreactive'with bound C3. The one that was positive bound to both C3b and C3bi.
• a minor group of patients whose plasma samples had been delivered for analysis of Clq-binding immune complexes were compared with plasma from a minor group of healthy blood donors.
• the group of patients is a heterogeneous mixture of suspected immunological disorders, so what may be expected are immune complexes of various compositions and in varying amounts. Since a positive correlation with the Clq analysis (15) was neither expected nor detected, all the plasma samples were analysed which had been delivered during a given period of time for Clq analysis. This was done irrespective of whether the Clq analysis was positive or negative.
• Three monoclonals were selected, adsorbed to microtiter wells, and employed for separating off immunocomplexed C3 from plasma.
• One of these monoclonals was directed against a neoantigen that is exposed in bound C3b and C3bi and is located in the C3c alpha fragment of 40 kd molecular weight.
• a second monoclonal was directed against a neoantigen that is exposed in bound C3bi but is not exposed in bound C3b, and is believed to be directed against the g portion of C3d,g. Both of these belong to the group III monoclonals.
• the third one of the monoclonals belonged to group II, was directed against the 20 kd fragment of C3c and reacted with bound C3bi.
• the variation in reactivity with respect to a plurality of epitopes is supported by the theory that the "immune complex" is a broad, summary concept encompassing a sizable amount of heterogeneity (15) .
• the pluridimensional presentation makes it possible to distinguish for instance (a) a high quota of immunocomplexed C3d in plasmas containing low immune complex levels and therefore having a relative accumulation of metabolites deriving from a later stage of the elimination process, from (b) a plasma in which the C3d quota is more normal.
• Figure 2 25 pmol of C3 (SN) , C3(SD) or C3 (D) was allowed to compete for binding to the individual monoclonal antibodies of groups I - III in the inhibition ELISA.
• Figure 3 2.5 pmol of C3b, C3bi, C3 of normal human serum (NH. ) and C3 fragments of aged human serum (AHS) was allowed to compete for binding to the individual monoclonal anti ⁇ bodies of groups I - III in the inhibition ELISA. The mean values + SEM for the different groups in response to each C3 preparation were presented.
• Figure 4 2.5 pmol of particle-bound C3 of EAC14 oxy 23b, EAC14 y 23bi and ZyC3bi was allowed to bind to the individual monoclonal antibodies of groups I - III in the inhibition ELISA.
• Figure 5 Histogram showing percent distribution of patients material (a) and normal material (b) when analyses are carried out using the three monoclonal examples mentioned in the text.
• the antibody in analysis I is directed against a neoantigen exposed on bound C3b and C3bi and located in the C3c portion.
• analysis II the antibody is directed against a neoantigen exposed on bound C3bi, located in C3d,g, and in analysis III the antibody is directed against a neoantigen exposed on bound C3bi located in the C3c portion.
• Figure 6 Analysis responses from the same analyses as in Fig. 5 plotted against each other. Normal material is represented by filled-in rectangles, and patients material is represented by empty circles.
• Figure 6.1 Analysis responses I and II plotted against each other.

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