WO1988006837A2

WO1988006837A2 - STRUCTURE OF IgG PLUS ANTIGEN AND COMPLEMENT, ITS MECHANISMS AND ITS IMPORTANT CONSEQUENCES

Info

Publication number: WO1988006837A2
Application number: PCT/EP1988/000331
Authority: WO
Inventors: Yuly Zagyansky
Original assignee: Yuly Zagyansky
Priority date: 1988-04-19
Filing date: 1988-04-19
Publication date: 1988-09-22
Also published as: FR2609398A1; GB8814505D0; AU1710188A; WO1988006837A3

Abstract

The new structure of the interaction of IgG with an antigen and a complement is proposed. According to this structure, the signal passes into the Fc fragment by a switch of the hinge region with its bend ''in two'' and the interaction of the changed hydrophobic patches of the Fab fragments with the Fc fragment. The changed conformation of the Fc fragment, especially in the pFc' region, became similar in shape to a scorpion's tail which could presumably interact by its hydrophobic patches in the outside face of the CH3 domain with the complement component C1 to activate the complement. The analysis of data from old literature gives strong evidence for this ''scorpion'' structure. It is proposed that the small protein (m.w. SIMILAR 2000) associated with IgG (Dudich, E.I. and Dudich, I. Dokl. Acad. Sci. USSR 260,1259, 1981) serves as the IgG protector against a strong interaction with the complement. The dissociation of this small protein, after a large distorsion during the precipitation reaction with an antigen, allows for a strong fixation of the globular heads of the C1q subcomponent. The proposed structure explains: the role of an affinity in the aggregation and in the complement activation; and consequently, the mechanisms of a great number of associated diseases. This ''scorpion'' structure explains the interaction between B and T-lymphocytes which is exemplified by both types of experimental results, that is, by direct interaction as well as by interaction with soluble substances. The examples with extremely important practical consequences are proposed.

Description

STRUCTURE OF IgG PLUS ANTIGEN AND COMPLEMENT,

ITS MECHANISMS AND ITS IMPORTANT CONSEQUENCES. SPECIFICATION Technical field of the invention. This invention relates to the fundamental point of immunology: the reaction of the antibody with the antigen and subsequent interaction with the complement and with the membrane structures. The knowledge allows for a new step in the understanding of immunological phenomena and principally new practical approaches in the struggle against infectious deseases. Summary of the invention.

The invention involves new knowledge which allows one to create a new structure for antibodies (IgG class), the structure of their function (the polyglobular structure of antibodies and its change during the interaction with an antigen and the complement), and the conclusions which were made from this new structure of IgG (with the structure of the activation of the

B-cells and with other important consequences). The invention is composed of the new "scorpion" structure created on the basis of the sum of new knowledge /the direct interaction of the Fab fragment with Fc; the bend of the hinge region "in two"; an appearance of the gene which begins only with the Mammalia, which makes a code in the hinge region and which, with the mechanisms of genetic evolution, makes the appearance of the means to permit the best regulations of the reaction of the antigen with the antibodies; a presence of the changes of the antibody structure which must be responsible for an increase in the change of the entropy value; the proposition of the role of the small protein of 2 kDa as a protector of the complement activation before the interaction with the antigen; the supposition of the role of the "tail" of Fc (like that of a scorpion after mentioned changes of conformation) in a specific and independent interaction during the complement activation/ and the practical consequences of this new structure (the explanation of the structural causes of different diseases which concern protein aggregation and complement activation against large particles; an injection of the small regulatory protein into patients with diseases concerning complement fixation, and with an antibody deficiency syndrome; an addition of the small protein of the same species (instead of the small protein in the foetal calf serum which serves to support the cells) to monoclonal antibodies; the creation and injection of antibodies prepared with special hinge regions; the proposition of the structure of the activation of the membrane immunoglobulins on the B-cell membrane and that of the interaction between B and T-cells;an injection of the membrane immunoglobulins (natural and special) with or without fragments of the antigen, and with or without lymphokines. Background of the invention.

The importance of the interaction structure of the antigen with the antibodies and the importance of the structure of the subsequent complement activation are difficult to underestimate because they play a significant role in the immune system and consequently their role is important in the pharmacological industry.

The three structures (allosteric, distortive and associative) were discussed for a long time in international literature. According to the allosteric structure, there are changes of conformation in the Fc fragment which were induced by the interaction of Fab fragment with the antigen. According to the distorsive structure, the changes of conformation in the Fc fragment take place due to the distortion of a semirigid antibody molecule induced by the dispersed distribution of antigenic determinants. And according to the accociative structure, the multivalent antigen provokes the Fc fragments of different molecules to approach one another. But now there are no results which "lead one to doubt the associative model of antibody action as the most likely one" (Metzger.H. Mol. Immunol.21,67, 1984; Cont.Top.Mol. Immunol.7, 119-152,1978; Adv. Immunol.18, 164, 1974) and "attempts to correlate changing in IgG on binding antigen with C1q binding have however to date been unconvincing" (Burton,D. Mol. immunol.22, 161-206,1985) or in other words "the evidence available at present appears to favor a model of C1q-IgG interaction in which C1q becames firmely attached to the multiple Fc regions, presented by aggregated IgG, without the requirement for conformational change to have taken place in the Fc regions (i.e. mediated by the interaction of the Fab regions of IgG antibody with a large molecular weight antigen" (Reid,K.B.M. Biochem.Soc.Tranc.11, 1-11, 1983). From all these prestigious citations one can conclude that the associative structure has taken the dominant position. And even if the allosteric and distortive structures were better accepted today "no clear answer could be qiven" to the questions (1) "What is the detailed structural expression of such conformational transition" and (2) "What is its functional significance" (Pecht.I. in Antigens vol.VI (ed.Sela.M.) 1-68 Academic Press, New York, 1982). Detailed description of the invention.

In this invention I not only give the proof ot the "nonconcrete" allosteric-distortive structure, but I advance the innovation much father. I propose a real structure for the conformation of the IgG molecule created from new findings: a new knowledge of the antibody structure (the IgG class) and the structure of their function (the polyglobular structure of antibodies and its change during the interaction with antigens like bacteria, viruses and dangerous toxins); a new knowledge of the structural role of the affinity of antibodies for the best aggregation and the complement fixation; a new knowledge of the appearance of the genetic structure which begins only with the Mammalia (the new small gene which code the hinge region of the IgG antibodies; a new knowledge of the structural role of the small protein of ~2 kDa like a protector for the complement activation before the interaction with an antigen; a new knowledge of the structural role of the increase in the change of the entropy value with the increase in the change of the free energy value which must be responsible for the structural changes of antibodies and, as a partial consequence of this, a new knowledge of the structural role of the "tail" of the Fc fragment which (like a scorpion's tail), after mentioned structural changes) can have the specific interaction in the complement activation.

I think that an intuitive reason for the associative structure is the idea that the signal from the Fab fragment cannot go directly through the polypeptide chain of the hinge region on account of a segmental flexibility of a link between the Fab and the Fc fragments (Burton,D. et al Monogr.

Allergy 19, 10, 1986), moreover the complement activation is usually best with a more flexible link). And even if one supposes that the signal can pass directly into Fc, the structure of this hinge region is too variable in length and in amino acid composition to be extremely important in the "transmissive" function of the signal (Metzger,H. Cont.Top.Mol. Immunol.7, 119-152, 1978). The discovery of another possibility for the direct interaction between the Fab and the Fc fragments could abolish these obvious objections, but in the opposite sense.

Here is my original structure. After the interaction with the first antigenie determinant, the signal passes by the Fab fragment, for instance, as described by Haber et al (Nature 264,415, 1976). It is not necessary that the signal be large. The fact that there is a change of conformation without a detailed structural expression was established by the spin label being located in the Fab fragment (Timofeev,V.P. et al FEBS Lett.102, 103, 1978) and also by the other methods (Metzger,H. Cont.Top.Mol. Immunol.7, 119, 1978;

Pecht,I. 1982). One may also suppose that there is a conformational change in the regions of the hydrophobic patches H1 (the value of the surface is 520 Å²) and L1 (the value of the surface is 320 Å²) of the Fab fragment (the symbols and the values of the surface here and below, according Burton,D. Mol. Immunol.22, 161, 1985). They are composed of different chains. This gives a "stereoamplification" of the signal which could be naturally small. According to Bennet and Haber the change in the elbow region can only concern some amino acids (C.R.C.Rev.Biochem.15,221, 1984). After the conformational change in the Fab fragment there is a switch in the hinge region (in the state which corresponds to the state of the occupation of the combining site, according to Haber et al). The results concerning an interaction of the F(ab')₂ with the monovalent and bivalent antigen indicate this possibility (Pecht,I. et al Biochem.Biophys.Res.Comm.74, 1302, 1977). I suppose that during this switch there is a bend in the hinge region. This suggestion was designed as a result of understanding: mechanisms of genetic evolution allowed the appearance of possibilities to regulate better the aggregation of an antigen by the antibodies. [Our results which were established by the different physical methods give the proof for the big change in the segmental flexibility of 300-400% between the immunoglobulins of cold-blooded vertebrates and the immunoglobulins of the Mammalia (Zagyansky,Y. and Ivannikova,E.I. Mol.Biol.Rept.1,301, 1974; Zagyansky,Y. Arch. Biochem.Biophys.166,371, 1975). The comparison of these old results and the new genetics results concerning the existance of the special gene for the hinge region of the Mammalia (Tonegawa and coworkers Nature 277,627, 1979; Nature 302,575, 1983) permitted one to conclude that this gene has only appeared in the Mammalia. This is the only type of mechanism of the progressive evolution which one can conclude from the theory of neutral molecular evolution (Kimura,M. Nature 217,624, 1968)]. Utilising the segmental flexibility (due to the new hinge region permitting large relative movements of two active sites in the best search of the antigen) the IgG molecule "returns" to its ancient state of evolution by an"abolishment"of the hinge region between the Fab and Fc fragments by its bend.

Our results obtained through the polarisation of fluorescence (Turnerman,L., Nezlin,R. and Zagyansky,Y. FEBS Lett.19,290, 1972) which concern the decrease of segmental flexibility during the interaction of antibodies with hapten prove the correctness of the proposed structure. This result was, in principle, confirmed by Timofeev and coworkers (FEBS Lett.102, 103, 1978), if one accounts for the dependence of the value of the rotational relaxation time on the concentration of antibodies in the IgG class without a hapten inside the active site and the absence of such dependence in the case of the antibodies with a hapten inside the active site (Dudich,E.I. et al FEBS Lett.89.89, 1977). Here one can propose the explanation of such a dependence on the concentration as a result of the twisting-untwisting of the hinge region of the IgG1 subclass of man which can have these two structures as calculated by a computer (Pamphrey,R. Immunol.today 17.174, 1986). The other subclasses of IgG do not have such dependence on the concentration (Dudich,E.I. and coworkers Haematologie 14,83, 1981). This fact corresponds to the absence of two different structures calculated for each of the other subclasses of human IgG. The observation (that the changes of conformation in IgG during the interaction with antigen have a duration of approximately 5 min) confirms this explanation (FEBS Lett.102,103,1978). This penults the antibodies to have a better efficiency during the interaction with an antigen. The presence of the same dependence on the concentration of the rabbit IgG preparations (Dudich, E.I. et al FEBS Lett.89,89,1977) which has a unique subclass, indicates the importance of this mechanism.

The presence of prolines, which change the direction of the polypeptide chain, indicates the same suggestion concerning the bend in the hinge region (in the structure of this hinge region of the different IgG subclasses and the different animal origins at the important points, for the bend of the hinge region "in two", with the corresponding cysteines at the appropriate places). For instance, in the case of the subclass IgG1 (with the presence of the biological activity in the complement activation) one can suggest that there is a bend of the hinge region near the prolines 227,228; and 230 which are situated near the cysteines 226 and 229. But if there is a reduction in the interchain disulfide bonds (with the Fab and Fc fragments resting in situ; during which there is no complement activation) there is no transmission of the signal into Fc fragment (Pecht et al Biochem.Biophys.Res. Comm.74,1302,1977) and I suppose that there is no bend mentioned above of the hinge region around the cysteines 226 and 229. In the following events Fab (by its changed patches H1 and L1) touches and reacts with the exposed hydrophobic patches of the C_H2 region of Fc (designed by Brunhouse and Cibra

Mol.Immunol.16,907,1979; Burton,D. et al Nature 288,338,1980; and Lucas et al J.Immunol.127,2555,1981). For the interactions between the fitting parts it should be the favorable steric conditions. The interaction with the first antigenic determinant changes IgG from very flexible state (Zagyansky, Y. et al Immunochemistry 7,787,1969), with a large freedom of rotation of the two combining sites, to a "semiflexible" state (with a "semi-T" geometric form and the large freedom of rotation of one free combining site). This permits the second free binding site of antibodies to have two advantages: (1) "the best search" of the antigenic determinant of another particle nearby (due to a "semi-T" form and consequently to make the best precipitation, (2) to stay in a more mobile state and consequently be better able to find "its" antigenic determinant.

One can see the confirmation of this structure in the thermodynamic resuits. According to Dudich and coworkers (FEBS Lett.89,89,1977) the antibodies making the best precipation are the most flexible antibodies. According to Archer and Krakauer (Biochemistry 16,615,1977) the horse IgG antibodies, which do not precipitate, have the mean angle between arms of the Fab fragments less than 60°. This geometry, as one can naturally expect, does not give them the possibility to find the antigenic determinant of the another particle easily. It follows, from the results of the thermodynamic obtained with the simple haptens, that there is a correlation between the increase of the changes of the values of the free energy, of the enthalpy and of the entropy and moreover, a change in the entropy value increase relatively more rapidly than the change of the enthalpy value (Archer,B.J. and Krakauer,H. Biochemistry 16,615, 1977). If one attributes the change of the entropy value during the reaction with simple haptens to a change of conformation of antibodies, there is, according to my structure, a larger change from the flexible state of antibodies to the "semiflexible" state (with a "semi-T" geometric form). This permits a better precipitation for the antibodies with more affinity. This is a fact which coincides with the numerous results in literature (Steward,M.W. Immunol. today 2,134,1981). The precipitation of the IgG functional antibodies of the class Amphibia (Jurd,R.D. et al Comp.Biochem.Physiol.50B,65, 1975) gives the conviction that these antibodies (like the precipitated antibodies of mammals after their interaction with the antigen) have a "T" form. In other words, through an "abolishment" in the hinge region between Fab and Fc by its "bend", the flexible IgG molecule of the Mammalia "returns" to its ancient evolutionary state. The IgG immunoglobulins of the Mammalia (which have no hinge region) have the same "T" form (Burton,D. et al Monogr.Allergy 19, 10, 1986). Of course, the rigid antibodies of the Amphibia class are less effective for the search of antigenic determinants, but one can expect their similarity during the "potential precipitation" (the interaction with an antigenic determinant of the another antigen particle). It seems that the conformational change of antibodies after interacion of the antigenic determinant with the second combining site est larger than the conformational change after the interaction with the first site (Pilz,I. et al Biochemistry 12,4998, 1973; Goers,J. et al J. Immunol.118,2182, 1977). Another chain of events must happen after the switch of the hinge region and the mentioned interaction between hydrophobic patches of the Fab and Fc fragments. In order to establish the following events it is necessary to take account of the new knowledge about a role of the small protein recently discovered by Dudich and coworkers (Dokl.Acad.Sci.USSR 260, 1259, 1981; Mol. Immunol.12, 1253, 1983) (which attaches stoichiometrically to the Fc fragment of IgG, which profoundly changes the state of segmental flexibility (Dudich,E.I. and Dudich,I. Dokl.Acad.Sci.USSR 260, 1259,1981; Tabl.1) and which detaches after the precipitation with the antigen). I think that it should be the regulatory molecule which plays an important role in the regulation of the complement fixation, and I propose its principal physiological role here: to preserve the complement activation before the interaction of IgG with the antigen. Apparently, this protein attaches to the ilexihle partof Fc because, according to the results of chromatography (Dudich and coworkers Mol. Immunol.20, 1267, 1983;Fig.7), the complex of the flexible Fc fragment (Nezlin.R., Zagyansky,Y. and Tumerman,L. J.Mol.Biol.50, 569, 1970) with the small protein passes more rapidly than the isolated Fc fragment [this fact corresponds to a decrease in the segmental flexibility according to Zagyansky (Arch.Biochem.Biophys.166,371, 1975)]. The fixation of this small protein of ~2 kDa to the Fc fragment is rather ionic because it does dissociate after the change of the pH value (Dudich,E.I. and Dudich, I. Dokl.Acad.Sci. USSR 260, 1259, 1981) and does not dissociate with the cationic proteins after a treatment with a solution of guanidinium C1 (5M) (Carlson,S.D. and Bowman,B.H. Biochim.Biophys.Acta 667,23, 198l;Fig.2).

It appears that the carbohydrates play a role in the conformational change because of the change in the surrounding of the spin label fixed by the covalent bond with these carbohydrates were registered during the interaction between the antibodies and the haptens (Timofeev,V.P. et al FEBS Lett.10.2, 103, 1978) and during the dissociation of the small protein (Dudich,E.I. and Dudich, I. Dokl.Acad.Sci.USSR 260, 1259, 1981). If the interactions are more strained (as, for instance, during the formation of the complexes of the precipitation), the small protein dissociates from the Fc fragment (Dudich et al Mol. Immunol.12, 1273, 1983).The detachment of this small protein profoundly changes the state of the segmental flexibility increasing the rotational relaxation time from 70 to 124 nsec (Dokl.Acad.Sci. USSR 260, 1259, 1981;Tabl.1). It appears that the immunoglobulins with the sedimentation constant S₂₀,W =8-9 (which, according to the authors, have no aggregates but which provoke a better activation of the complement (Isli- ker,H. and coworkers Vox Sang.7, 157,1962) are just the IgG molecules which lose their small regulatory protein during purification. The other analogous experiments were done by Yasuda and coworkers (Vox Sang.51,270, 1986). They studied the bactericidal actions of the treated IgG preparations ("pH 4") (with, as antigen, the bacteria E.coli) and they found that this preparation had a better complement activation than the preparation of untreated IgG. This donfirms my structure concerning the protective biological role of this small protein-regulator: to preserve the strong interaction between IgG and the complement (I underlined the word: strong).

After the dissociation of this small protein the complement factor C1q can have a strong interaction with Fc It appears that the interaction of the factor C1q with IgG is the sum of the two types of interactions: ionic and hydrophobic (Burton,D. Mol. Immunol.22, 161, 1985). I suggest that the com- plement, with its positive charge, take the place of the small regulatory and protective protein. The factor C1q can touch piobably the hydrophobic regions of IgG (as, for instance, a very large hydrophobic patch of 920 A at the inner part of the C_H3 domain) by the hydrophobic interactions after a change of conformation of the mentioned carbohydrates. I would like to underline the two types of conformational changes in my proposition. The first type of change can take place (without the dissociation of the small regulatory protein) after the interaction with the antigenic determinants (usually without an aggregation). In this case one can suggest the switch of the hinge region, the direct interaction between Fab and Fc (apparently, by means of hydrophobic patches) and the subsequent change of conformation in the Fc fragment (mainly in the pFc' region.).Old electron microscopic data after an interaction of the IgG antibody with a hapten already prove my proposed structure: one can see that the plane of Fc fragment is different from the plane of Fab fragments; and also, more clearly, when one looks at two antibodies in the side view (in profile), which interact with two bivalent antigens, the sign ∫ could be seen (Green,N.M. Adv. Immunol.2, 1-30, 1969). The finding of Zavialov and coworkers (Haematologie 14,83, 1981) indicates the same structure: one could see the conformational changes after an interaction of antibodies with a hapten near the pH value 4 (when the small regulatory protein is dissociated, according Dudich and coworkers). I have already mentioned that the change of rotational relaxation time value of IgG, the change of the spectrum of the s~in label (Dokl.Acad.Sci.USSR 260, 1259, 1981) and the change of the constant of sedimentation S_20,W (Vox Sang.7, 167, 1962) demonstrate the second type of change. The first type of change of conformation can have a correlation with the spectrum of the circular polarisation of luminescence and the second type of change of conformation can have a correlation with the complement activation (Metzger,H. Cont.Top.Mol. Immunol.7, 119-152, 1978). Of course, this structure does not deny an argument that the IgG aggregation favoratethe complement fixation by means of a simple increase of a local concentration of the sites of the complement fixation (an increase in the avidity), although the complex strucure during the IgG aggregation is not really known. It does not deny the results of other experiments with conditions very different from those described here. One could see a new and clear confirmation of my structure by an analysis of other old electron microscopic results. According to the estimation of Dimmock and coworkers (J.Cell Sci.10, 525, 1972) the molecule of ferritin (conjugated with IgG) must have the distance of 16.0-22.5 nm from the binding site of IgG. However the experimental results, with the human antibodies against the the A antigen of the blood groups ABO of the RK₁₃ cells, show that the distance of the ferritin molecules from the cell membrane is only from 8 to 15 nm. The analogous results are obtained with the same A antigen on the erythrocytes (the mean distance between the ferritin molecules and the cell membrane is 9 nm (Voak,D. and Williams,M.A. Br.J.Haem. 20,9,1971). But the results of the antibodies against the virus Hepatitis B are more impressive (Huang,S.N. Am.J.Med.Sci.270,131,1975) since the majority of these antibodies belongs to the IgG3 subclass (30-80%) (Skvaril,F. and coworkers Infect.Immunol.39,565,1983). The electron microscopic data (Pamphrey,R. Immunol.today 7,174,1986) show that the immunoglobulins of this subclass are longer (the difference is 9 nm) due to the unusual hinge region. According to the mentioned publication (Huang,S.N. Am.J.Med.Sci.270, 131,1975), even in the case of the long IgG3 molecules, the ferritin molecules and the particules of the virus Hepatitis B are superposed. One could conclude from these results that the IgG molecules bend after interacting with the antibodies and the antigen. These proofs certainly confirm my new structure. The complexes of the ferritin molecules with the lectin molecules (of 140kDa) Dolishos biflorus (which apparently have no hinge region) have a larger distance from the cell membrane: 18.7 nm (14.5-32.6 nm) (Williams,M.A. and Voak,D. Br.J.Haem.23,427,1972). This fact gives another piece of evidence for the previous interpretation of the results concerning the complexes between the antibodies and the ferritin molecules.

This bending of antibodies was deduced after their interaction with theantigen, even without the complement. But one could suggest that, during subsequent interaction with the complement, the "tail" of Fc, for instance, with its hydrophobic patch at the external face of C_H3 domain (320 Å²), touches the complement component C1 (probably C1r). The IgG molecule became flexed like a scorpion. The complement subcomponents C1r and C1s are situated at the "good site" which is not far from the globular C1q heads (Reid,K.L. Biochem.Soc.Tranc.11,1,83). The interaction of the modified (C_H3)Ɣ1 fragment with C1 component (Metzger,H. Cont.Top. Mol.Immunol.7,119, 1978) also indicates this possibility. The suggestion of an appearance of a new site after an interaction of the complement (probably C1r) with IgG (after an interaction of the Fab fragments with the special hapten) (Goers,J.W. et al J.Immunol.118,2182,1977) supports my new "scorpion" structure. It is probable that a direct interaction between C_H3 domains of Fc with the subcomponent C1r (with or without C1q and with/or C1q of the region nearby) switches the complement activation.

If one accepts the structure of the interaction of the receptor for antibody (FcƔR) on the B-lymphocytes with the membrane antibodies, as it was proposed by Holowka and Baird (Biochemistry 22,3475,1983) and Burton (Immu nol.today 7,165,1985), it is possible to present the structure of the interaction between the membrane antibodies of B-cells and the cell membrane. In this structure (after an interaction of Fab with hapten) the tail of the "scorpion" makes the hydrophobic interactions with the membrane components. The fact that the membrane immunoglobulin G has the "new tail" of 26 amino acids (Tyler,B.M. et al Proc.Natl.Acad.Sci.USA 79,2008,1982) and the fact that the conformation of membrane IgG is different from the conformation of secreted IgG (Patridge,E. et al J.Immunol.128,1,1982) will guarantee an absence of the complement activation on the B-lymphocyte surface. A presence of a large asymmetry between the two sides of this "tail" was estimated (Proc.Natl.Acad.Sci. USA 79,2008,1982).

This proposed structure allows one to deduce the structure of an interaction between the B and T-cells which produces the switch to sIgG. In this field there is an agreement concerning the functions of sIgG, as an antigen receptor "but its role in specific B-cell activation remains unclear" (Burton,D. et al Monogr.Allergy 19,133, 1986). There are receptors for antigens (slgM and sIgD)and receptors for antibodies on B-cell membrane (Dickler,H.Mol.Immunol.19,1301,1982). It seems that the switch to sIgG occurs in the germinative centres (Krael,G. et al Nature 298,377,1982). The T-cells present an antigen to the receptors for antigens of B-cells. This direct interaction approaches the B and T-lymphocytes. One could propose that the T-cells secrete the substances (Viletta,E. et al J.Exp.Med.162,1726,1985) which make an appearance of the corresponding receptors for antibodies (FcƔR and FCαR). I think that the same receptor could have a specific affinity to one or to another subclasses, but I cannot exclude that the different receptors have a specific affinity for different subclasses. The appearance of a type of these substances could depend on the source of T-lymphocytes, the surrounding of an interaction (Kawanishi,H. et al J.Exp.Med.157,433,1983) and, as I propose, it could depend on the type of antigen. The type of substances secreted by the T-cells can depend on the antigen because (1) there are the numerous correlations between the appearance of antibodies of a particular subclass and the antigen types (for the references see Mayumi,M. et al J.Immtmol.130,671,1983) and (2) the soluble factors of the helper T-lymphocytes (Th) for the particular subclass were described (Isakson,P.C. et al J.Exp.Med.155,734,1982. This new structure of the interaction between B and T-cells confirms not only the established fact that the T-cells could selectively increase the frequency of the subclasses of sIgG on the B-cells by the direct interaction, or by the soluble factors (J.Exp.Med.155,734,1982) but it also confirms all the results concerning mentioned increase of a frequence of the subclasses after direct interaction with the T-cells,as well as by their soluble factors. Numerous results concerning different Subclass distributions of the membrane immunoglobulins on the B-cells and results concerning different interactions of the secreted immunoglobulins of different subclasses with the B-cells indicate this possibility (Perelmutter,L. Monogr.Allergy 19,36, 1986; Mayumi.M. et al J. Immunol.130,671,1983; Zapanska.B. et al Vox Sang.49,67, 1985). The membrane immunoglobulins arrive to occupy the part of antigenic determinants which were not occupied. It seems that a small quantity of these immunoglobulins is always free. [Or because of their probable synthesis in the places which are different from the places of the switch to sIgG, like bone marrow (J.Exp.Med.157,433, 1983), or because of their insignificant synthesis by B-cells of a surrounding (one can suppose this synthesis from the experiments of Mayumi et al J. Immunol. 130,671,1983), or from the presence of a synthesis of the two types of immunoglobulins (membrane and secreted) in a culture of the tumor B-cells (lymphoma) (Oi,V.T. et al J.Exp.Med.151, 1260,1980), or it could be proposed that the soluble factors of the T-cells stimulate a more intensive synthesis of the free membrane immunoglobulins of a corresponding subclass in the surrounding area.] When the corresponding receptors for antibody (FcƔR and FCαR) of B-cells are "ready" the antibodies (the membrane immunoglobulins) react by means of their site of the receptor interaction (apparently situated in the C_H2 domain) (Burton,D. Immunol.today 7,165,1985) and by means of their "tail of the scorpion" with the membrane structures due to an occupation of antigenic determinants. A strong interaction must take place in the last case. This thesis is a consequence of the proposed structure-"scorpion".

According to Dickler (Mol. Immunol.19, 1301,1982) there is an interaction between the receptors for antigen and the receptors for antibody (if the two types of receptors are occupied by their ligands). This interaction facilitates the switch of the signal (Zagyansky,Y. and Jard.S. Nature 251,591, 1979; Exp.Cell Res.138,387, 1981) to sIgG in the case of the T-dependant antigens. A proliferation and a maturation of B-cells (for production of the soluble antibodies), after this switch, pass later (Kawanishi.H. et al J.Exp.Med.157.433.1983). The established fact that the curve of appearance of the IgG antibodies follows the curve of appearance of the IgM antibodies (Roit,I. and coworkers, Immunologie fondamental et appliquée, Medsi, Paris, 1985) confirms this structure since the switch to sIgG needs the presence of the receptors for antigen of the IgM class in situ. After interaction with the antigen attached to the B and T-cells the membrane IgG molecule helps to switch the signal, due to its "scorpion" structure. The practical consequences of this new "scorpion" structure are. 1)It follows from the proposed structure that this small regulatory protein protects the strong interactions between the IgG molecules and the complement component C1 before the specific interactions between the antibodies and the antigen. And it is possible to propose three very important innovations (maybe four): (1) apparently, the cause of a part of the numerous diseases, which concern the complement fixation, is the perturbation of a synthesis of this small protein, so one can propose an injection of the corresponding quantities of the small regulatory protein into patients; (2) to avoid pain after injection of the IgG preparation into patients (in the case of an antibody deficiency syndrome) one could propose an injection of this small protein together with IgG, even without special treatment of IgG; (3) "the immortal cells" in a plastic dish which produce the monoclonal antibodies against the desired antigen, became one of the greatest discoveries of the life science during last thirty years. But nobody can efficiently predict the behavior of these antibodies with the complement because of the creation of the chimeras of mouse IgG with the small regulatory protein of the cow embryo, and to avoid the creation of these chimeras it is neccesary to supply the small regulatory protein of the mouse to the monoclonal antibodies instead of the small protein of the cow embryo from the serum which supports cells (the consequences of this innovation must be grand); (4) it is probable that an injection of this small protein, during the immunal system's reply to infections, could facilitate recovery by stimulating, for instance, the synthesis of IgG. 2)The proposed structure explains why the high affinity antibodies have a better precipitation. If the antibodies have a higher affinity there is a stronger change of angle between the Fab fragments (of the first occupied antigenic determinant) and the axe of symmetry of Fc. It means that the second active site of Fab will more easily find an antigenic determinant of an another particle (than an antigenic determinant of the same particle) for the best aggregation of, for instance, dangerous toxins. Also, as one of the effects of the stronger distortion of the antibodies during the big particle aggregation (for instance, the viruses) there is a better complement activation in the case of the high affinity antibodies. This fact reflects the structural cause of diseases concerning the complement fixation. As a consequence, the proposed structure will allow the production of the numerous pharmacological preparations.

3)From the proposed structure because of the importance of the hinge region in aggregation and in complement activation one could predict that the creation of the antibodies with the special hinge region will play an important role in the struggle against infections

4)The proposed structure allows greater efficiency in the B-cell activation after an injection of the membrane immunoglobulins into the corresponding regions of the human body /with or without more effective hinge regions and more effective COOH-terminal regions, with or without fragments of an antigen and with or without the soluble products derived from lymphocytes (lymphokines)/.

5)As a concrete example, the described structure allows to propose the new ways in a struggle against the AIDS. One can see that in an organism there is a presence of the anti-virus antibodies accompanying the virus contamination (Gressentis,A. La Recherche 16,1534,1986). But the successful withdrawal of the viruses from an organism, after their interaction with the antibodies, does not usually happen. It could mean that the antibodies are not effective or to crosslink the corresponding antigenic space determinants of the virus particle (s) or to activate the complement. In this case the creation of new antibodies according to the structure described above could solve the problem: or the antiserum containing the antibodies with the special hinge regions described in (3) could be created, or the adequate vaccination (La Recherche 16,1534,1986), as described in (4), with the corresponding lymphokines which provoke the production of the most effective subclass (see above) of antibodies (IgG3, par example) against the virus, could be done.

6) In an example of the cancer, the described structure allows to propose the new ways for its prevention or for its destruction. There is an analogy between the "foreign" antigenic determinants of bacteria, viruses, toxins and the new antigenic determinants of the organism cells which have undergone the malignant transformation. And, like in the previous case concerning the AIDS, the antibodies cannot successfully withdraw the cancer cells from an organism (Jasmin,C. La Recherche 18,754,1987). So one can choose the same strategy as in the case of the AIDS according to (5): or (a) to create the antibodies with the special hinge regions according to (3), or (b) to make the adequate vaccination with the cancer antigen (La Recherche 18,1534, 1986) with the corresponding lymphokines (4) which provokes the production of the most effective subclasses of antibodies against antigens (see above). This vaccination could help the antibodies to appear because they are not always evoked by the own cancer cells. I think that in the both cases (a and b) this new technique will provoke the activation of the complement to destroy the cancer cells. When the immunisation against the cancer will be used for preventive purposes a destruction of the cancer cells will happen at the earliest stages of their appearance,

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Claims

CLAIMSThat what is claimed is:

1.The invention "Structure of IgG plus antigen and complement, its mechanisms and its important consequences" is explained, wherein the improvement comprises new knowledge (which allowed the creation of the new "scorpion" structure); the new structure-"scorpion" of the IgG antibodies with its structure of the action (the polyglobular structure of the antibodies, its change during the interaction with the antigen and the complement activation); and the conclusions which were made from this new structure of IgG (with important consequences).

2.The invention as defined in claim 1, wherein the sum of new knowledge (which permitted the creation of the new structure) composed of following elements: the understanding of the necessity of the direct interaction of Fab with Fc in the case of the signal switch during complement activation; the understanding of the presence in the hinge region structure of the prolines which change a direction of the polypeptide chain to bend the hinge region "in two"; the understanding of the necessity of the appearance of genetic evolution mechanisms (with the new gene coding the hinge region of IgG of the Mammalia) with the means to permit the best aggregation of the antigen by the antibodies; the awareness of a presence of structural changes in the antibodies responsible for an increase of the change of the entropy value with the increase of the change of the free energy value; the understanding of the regulatory role of the attached small protein (of 2 kDa) in the complement activation; the understanding of the role of the mentioned structural changes in the specific interaction of the tail of Fc (like scorpion's tail) with an independent complement site during the complement activation.

3.The invention as defined in claim 1, wherein the proposed structure of the interaction of the IgG antibodies with the antigen and the subsequent activation of the complement, composed of the following steps: the signal transmission (switched by an interaction of Fab with the antigen) into the presumed region of the hydrophobic patches composed of the light and heavy chains and into the hinge region; the bend of the hinge region "in two", which allows for an approach of the Fab and Fc fragments and their interaction by means of changed hydrophobic patches of Fab and the Fc hydrophobic patches; the subsequent change of conformation of the Fc fragment (C_H2 and C_H3 domains): the change of conformation in the C_H2 domain, nearby the region of carbohydrate attachment which causes a dissociation of the smal! regulatory protein, a subsequent strong fixation of the complement by ionic and hydrophobic forces (the presumed region of strong fixation is the hydrophobic patch situated in the inner face of the C_H3 domain regions) and the change of conformation in the C_H3 domains which interact with component Cl

(presumably with Clr, and may be with or without Cls and/or Clq), presumably by its patch in the outer face of the C_H3 domain.

4.The invention as defined in claim 1, wherein the conclusion concerning the structural mechanisms of a great number of the diseases like a passive cutaneous anaphylaxis, a virus neutralisation, a destruction of D⁺ erythrocytes, a complement fixation, an immune elimination of antigens, a protective capacity against bacterial infections (since proposed structure explains how the antibodies make the best aggregation of antigens and the best activation of the complement) which will lead to other preparations.

5.The invention as defined in claim 1, wherein the proposition of a biological role of the small protein: to preserve a strong interaction between the numerous IgG molecules in man and the complement Cl before a specific interaction between the antibodies and the antigen.

6.The invention as defined in claim 5, wherein the proposition of an injection of the adequate quantities of the normal regulatory protein into patients with the diseases concerning the complement fixation (in the case of the perturbations of the synthesis of this protein), and might be the injection of the adequates quantities of the same normal regulatory protein during a process of infectious deseases for the best recovery, or in case of an antibody deficiency syndrome.

7.The invention as defined in claim 5, wherein the proposition of the addition of the small regulatory protein of the same species (as the antibodies) (instead of the small protein of a foetal calf serum which serve to support cells) to "immortal cells" which produce the monoclonal antibodies for the normal function of the monoclonal antibodies or an addition (preferably with the molar excess and at a more acid pH) of the small protein of the same species to the monoclonal antibodies (preferably purified) obtained by classical methods (proferably with subsequent elemination at a normal pH of the small protein from the foetal calf serum dissociated with an acid pH).

8.The invention as defined in claim 1, wherein the proposition of an injection of the antibodies which are made with the special hinge regions, for the best struggle against infections.

9.The invention as described in claim 5, wherein the proposition of an injection into patients of the IgG preparation (treated or untreated) with the small regulatory protein (even of an animal origin).

10.The invention as described in claim 1, wherein the conclusion concerning the structure of an action of the membrane immunoglobulins on the B-cell membrane. According to this structure (after an interaction of the Fab fragment with an antigen) the hydrophobic tail of the COOH-terminal region make a hydrophobic interaction with membrane components (like the scorpion's tail). The structure of the interaction of the B and T-lymphocytes (which results in the switch to sIgG) was deduced from the structure indicated above. It is composed of following steps: an approach between the B-cells (by the receptors for antigens: sIgM and sIgD) and the T-cells ("by dividing" the fragment of an antigen containing several antigenic determinants) and the interaction of the membrane immunoglobulins (free sIgG) with the same antigen fragment; a subsequent appearance of the receptors (FeƔR) (which can react with sIgG of the particular subclass) under influence of the soluble factors of T-cells (which depend, in particular, on the antigen type); the interaction of sIgG with their receptors (FcƔR) and the membrane components (due to the "scorpion" structure described in claim 1); an interaction between the FcƔR receptors and the receptors for antigens (occupied by T-dependent antigens) which introduce a redistribution of the receptors and a switch to sIgG.

11.The invention as described in claims 1 and 10, wherein the proposition of an injection of one or several following substances: the fragments of an antigen with (or without) a carrier which stimulates a production of the lymphokines for the most effective subclass of immunoglobulins, the membrane immunoglobulins with (or without) more efficient hinge regions and COOH-terminal regions, the soluble products derived from the lymphocytes (lymphokines) for the best efficiency of the B-lymphocyte activation and the production of the most efficient antibodies.

12.The invention as described in claims 8 and 11, wherein, in the case of the recovery or the prevention of the AIDS, the proposition of an injection of the antibodies which are irade with special hinge regions, and the proposition of an injection of an ATDS-virus antigen (facultatively in the case of the recovery) with (or without) a carrier which stimulates a production of the lymphokines for the most effective subclass of immunoglobulins with (or without) the membrane immunoglobulins (with or without more efficient hinge regions and COOH-terminal regions) with (or without) the soluble products derived from the lymphocytes (lymphokines), and also with the possible simultaneous injection of the small regilatory protein (the claim 6) which could help in the struggle against AIDS.

13.The invention as described in claims 8,11,12, wherein, in the case of the prevention (or the recovery) of the cancer, the proposition of an injection of the antibodies are made with special hinge regions, and the proposition of an injection of the cancer antigen in the adequate form (facultatively in the case of the recovery) with (or without) a carrier which stimulates a production of the lymphokines for the most effective subclass of immunoglobulins with (or without) the membrane immunoglobulins (with or without more efficient hinge regions and COOH-terminal regions) with (or without) the soluble products derived from lympocytes (lymphokines) and also with possible simultaneous injection of the small regulatory protein (the claim 6) which could help in the struggle against cancer.