JPS61500846A - Anti-idiotypic antibodies elicited by synthetic polypeptides - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Description of anti-idiotypic antibodies elicited by synthetic polypeptides technical field The present invention relates to amino acid residue sequences of specific variable or hypervariable regions of immunoglobulins. Regarding chemically synthesized polypeptides with corresponding amino acid residue sequences, this It may be administered alone or as a polymer or as a conjugate bound to a carrier. inducing the production of anti-idiotype antibodies of a given specificity.

Antibodies are immunoglobulin molecules that have a specific amino acid sequence and therefore (its immunogen) or a closely related antigen (or immunogen). Combine only. Immunoglobulin molecules contain two types of polypeptide chains, each molecule The heavy M(Hl) polypeptide chains are linked by disulfide bonds and non-covalently. They are bonded to each other by conjugation and are primarily hydrophobic. H and L polypeptide immunoglobulin An understanding of the structure and ipotency of antibodies is based on fragments created by enzymatic cleavage of antibody molecules. research has been promoted. For example, treatment of antibody molecules with the enzyme papain , two antigen-binding fragments (referred to as Fabs) and a complement-fixing fragment. (referred to as Fc), the latter has no antigen binding ability but has complete immunity. Determine key biological features of the whole lobulin molecule.

On the other hand, treatment of antibody molecules with the enzyme pepsin produces a single antigen-binding fragment) ( F (referred to as ab'>t) and a somewhat smaller complement fixing fragment (also referred to as (referred to as Fc).

) 1 'II (7) Yo 8□ Gate, Plague ≦ Bu 1. a. 7. You are here. G-su-Yuhae.

Biological properties that allow differentiation and consist, for example, activate complement, cross the placenta and the ability to bind to polymorphonuclear leukocytes or macrophages.

(, five immunoglobulin classes (IgG, Tg^, IgM, 1.0 and (IgE and IgE) have structural differences in their heavy chains, including amino acid sequence and polypeptide chain length. Based on! ! be recognized. The antigenic determinants on the heavy chain allow for compatibility.

The amino-terminal half of the L chain of the immunoglobulin molecule and the amino-terminal quarter of HlX are , varies in its amino acid sequence and is called the variable region (part 7) of the polypeptide chain. . One H and one part of the polypeptide chain serve as a site for antigen binding. make up the rank. Considerable changes exist in the amino acid sequences of the variable regions of immunoglobulin molecules. It is possible to make many different antibodies - isomers. Extremely variable in the main array within the variable part The region is called the hypervariable region. Capra et al., Proc, Natl. , ^cad, sci, ll5AST, 845 (1974).

The specificity of an antibody's molecular binding site is called its idiotype. Idiotite The term protein refers to the unique variable (7-part) structure produced by each type of antibody-forming cell. An antibody with binding site specificity for another antibody's binding site is labeled as an anti-identifier. called otype antibodies.

The same amino acid sequence changes that make immunoglobulins antigen-binding specificity also Determine whether biotypic determinants are present. That is, a particular idiotype is Almost always associated with immunoglobulins of particular specificity. in this way, Idiotypes are for immunoglobulins with specific specificities and their surface Immunoglobulins act as markers for B lymphocytes of the same specificity. I can hear it.

The words cross-reactive and cross-reactive refer to Cross-reactive anti-idiotypic antibodies, which refer to the ability of an antibody to bind to foreign antigens, are They can be divided into two large groups.

One group is associated with specific amino acid sequences in the H and Lli variable regions. . This group includes anti-idiotypic antibodies that recognize idiotypic antigenic determinants. Anti-idiotypic antibodies often inhibit the production of inherited immunoglobulin structural genes. reflect the purpose. Therefore, these antibodies may cross-react in genetically dissimilar subjects. do not.

The second group is anti-idiotypic antibodies that are cross-reactive to the internal image of the antigen. includes. This type of anti-idiotype antibody is produced by complete immunoglobulins. It is generated by immunization with light and heavy chains, and usually involves specific quaternary g) recognize idiotypic antigenic determinants as a result of interaction; But this group The antigenic site recognized by the anti-idiotype antibody of The antibody binding site has an internal image of the antigen that cannot be associated with the amino acid sequence ; i.e. antigen size, shape, tr@ and/or van der Waalska 1 (Because they are fiL, this group of anti-idiotypic antibodies contains many different antibodies with the same specificity. binds to the antibody. The idiotype recognized by such antibodies may be different genes that can be produced by individuals with a genetic background and do not develop special associations. more controlled.

Anti-idiotypic immunotherapy treats autoimmune diseases by neutralizing emotional autoantibodies. It can be extremely useful in the treatment of patients. Furthermore, anti-idiotypic immunotherapy , or can be used in the treatment of B111ltfi malicious transformations.

8 recent treatments in this field! Treatments include chemotherapy, non-specific stimulation of the immune system, Active immunization through IJ awards and directed against specific markers including idiotypes. This includes passive immunization with antibodies that have been detected. The main drawback of the first two methods is that A lack of isomerism, which usually results in general tissue toxicity in the case of chemotherapy.

On the other hand, anti-idiotype therapy can be highly specific. But such The treatment suffers from drawbacks associated with passive immunization. Because the anti-idiotype This is because antibodies must be produced in a non-human species. Follow me, that's it Will treated individuals develop an immune response to passively administered antibodies? This response may negate any potential therapeutic effect.

This is because the antibody must be administered multiple times to achieve the desired result. , especially correct.

Additionally, all anti-idiotypic antibodies immunize the host with the target immunoglobulin. This has traditionally been caused by this. The resulting polyclonal antiserum is then used to must be significantly purified to produce antibodies with anti-idiotype specificity. The selected purified “monoclonal” antibody is then tested to determine its specificity. must be carefully tested to ensure

Idiotypic structural relatives are present in several well-defined antibody systems. has been pursued. Kunk@l et al., Science , 140.1218 (1963) i Capra et al., Proc, N atlJcad, Sci, USA, AE, 4032 (1974) iWager W@igcrt et al., J. Erp. Med, 139.137 (1974 ); Klapp@r et al., Ann, rmmunol, (Inst , Pa5teur).

Nature, 1 Eyu, 35 (1980): Kabra et al., Iasuno I, Dingoday, 3-1332 (19112) i and Capra et al., Iasun ol, Todays 4.177 (1983), these studies The hypervariable region (also called complementarity determining region or CDR) of Robulin is an idiotype. Suggests that it is a structural relative of the determinant.

and (in the mouse anti-dexran system, one private (i.e. individual a public (i.e., cross-reactive) idiot were assigned to the third and second hypervariable parts of Hli, respectively (Shiling ling) et al., supra).

However, in many systems, specific idiotypic determinants are associated with specific amino acid sequences. It has proven extremely difficult to link (Kapra et al., Iasunol). , 1 oday s 4, supra), but rather a determinant that depends on the complete immunity image. (Kapra et al., same).

Lerner et al. used short amino acids to moderate length as immunogens. Using vaccines against pathogens that use synthetic polypeptides with amino acid residue sequences By doing so, we were able to successfully protect the animals. Sutcliff e) et al., Science (Sci@nce), 2nd edition, 495 (1983). , such synthetic polypeptides are specific for a given determinant of the complete protein. induces strong antibodies.

As described herein, the synthetic polypeptide method is useful for conventional anti-idiotypic treatments. The difficulties traditionally associated with the law can be avoided, as detailed below. According to the invention, immunoglobulins forming idiotypes - pria + ar y) A polypeptide with a relatively short amino acid residue sequence that roughly corresponds to a portion of the sequence The resulting antiserum can be synthesized and inoculated into host animals, including humans. is an immunoglobulin (a part of which a polypeptide corresponds in amino acid residue sequence) and idiotype-specific, i.e., synthetic polypeptides. Such antisera produced by Tide have a certain specificity and are highly purified and characterized. The need for opposite sex testing is eliminated.

can be administered to that individual in order to develop antibodies against it.

Autologous anti-idiotypic antibodies are well-documented and Immune! pole in the lI clause Synthetic polypeptide-containing antigens (immunogens) are widely believed to be extremely important. The -9 advantage of use is to generate antibodies reactive with determinants that are otherwise non-immunogenic. It is possible.

Synthetic polypeptides with 11 and 1 characteristics are specific to an individual's specific idiotype. This can induce anti-idiotypic antibodies in the individual against which it is directed. could not be achieved by full immunoglobulin immunization.

Therefore, individuals can become actively immunized against the pathological idiotype. and the number of therapeutic interventions required is comparable to conventional immunization with intact immunoglobulins. can be significantly reduced. In addition, the possibility of an immune response to anti-idiotype antibodies nature can be essentially reduced.

In addition, because polypeptides mimic antigens under attack by emotional autoantibodies, Can be synthesized. These polypeptides are responsible for the interactions between antigens and undesired autoantibodies. can be prohibited, thereby significantly slowing down the disease process. hinder.・The idiotype that consists of Conceivable. Synthetic polypeptides corresponding to such idiotypes are can be used to generate antibodies of a given specificity for Una syndrome; and then can be used in the diagnosis of the syndrome.

Brief summary of the invention The present invention mimics idiotypic antigenic determinants on immunoglobulin molecules and Production of antibodies of a given specificity that are reactive with the idiotype (anti-idiotype antibodies) We are considering using synthetic polypeptides to induce this. Such resistance The body may be useful in the treatment of autoimmune diseases and diseases involving B lymphocytes.

Such antibodies may also be used in the diagnosis of diseases in which specific idiotypes occur. It can be done.

Synthetic polypeptides according to the invention represent idiotypic antigenic determinants of immunoglobulins. That is, a contiguous amino acid sequence that substantially corresponds immunologically to the amino acid sequence of the variable or hypervariable region. This polypeptide has an amino acid residue sequence of about 6 to about 40 amino acids. The polypeptide comprises acid residues, preferably about 8 to about 20 amino acid residues. Alone or as a polymer or with carriers such as keyhole limbentohemothia A physiologically tolerable vehicle as a conjugate bound to Nin (KLH) etc. Kaoru is effective during the lecture! Once administered to a host animal, it produces an anti-ideal drug in that host. Induces production of iotype antibodies.

As used herein, the terms peptide and polypeptide are used interchangeably. The term synthetic polypeptide refers to naturally occurring pallidum and its fragments. ) refers to chemically derived amino acid residues. Such a case The adult polypeptide is capable of inducing the production of anti-idiotypic antibodies in the host.

The expression "approximately immunologically corresponding" in various grammatical forms applies to polypeptide sequences. As used herein and in the claims, the polypeptide sequences described herein are production of antibodies that bind to peptides as well as idiotypic antigenic determinants. It means to provoke.

The expression "corresponds" in various grammatical forms refers to the present invention that corresponds to polypeptide sequences. The polypeptide sequences used and described in the specification and claims (plus or eggplant) up to three amino acids at one or both of the amino- and carboxy-termini conservative substitutions only at specific amino acid residues and along the polypeptide sequence. It means containing.

The term conservative substitution mentioned above refers to the substitution of one amino acid residue with another physiologically similar P It is meant to indicate that it is replaced by an i group. Examples of conservative substitutions and , by one hydrophobic residue such as IIs%Vat%Leu or Met. Substitution of one polar residue for another hydrophobic residue or one polar residue for another Substitutions such as = between A'rg and Lys, between Glu and Asp or between Gla and Asn One example is ``between.''

In some examples, the replacement of an ionic residue with an oppositely charged ionic residue, For example, substitution of Asp by lys indicates that these ionic groups provide solubility assistance to the vehicle. It has been said to be conservative in that it is thought to have a positive impact. However, in general, the The substitutions made are for synthetic polypeptide antigens that are relatively short compared to the entire protein. Therefore, replacing an ionic residue with another ionic residue of opposite charge is Substitutions between ionic residues and bulky residues such as Phe, T yr or Trp and relatively less bulky residues such as Gly, IIs and νal. Substitutions between, as well as substitutions between, are considered herein to be radical substitutions.

The terms nonionic and ionic residues respectively refer to normally charged residues at physiological pH ([ Used herein in its ordinary sense to refer to an uncharged or charged amino acid residue. used in Examples of nonionic residues are Thr and Gln, and examples of ionic residues are Thr and Gln. Examples include Arg and Asp.

In accordance with the methods of the invention, an effective amount of synthetic polypeptide in a physiologically acceptable vehicle is obtained. A suitable host is treated with Tide, and this polypeptide is a variable or variable immunoglobulin. corresponds immunologically to the amino acid sequence of the hypervariable region (idiotypic antigenic determinant) Therefore, each immunoglobulin has a variable or hyperactive amino acid sequence. Anti-idiotype antibodies are produced that can bind to the altered region.

In this method, the obtained anti-antibody or anti-idiotype antibody has a predetermined specificity. and has approximately the same configuration as the antigen that binds to the variable or hypervariable region of immunoglobulin. This type of antibody has been improved to define the idiotypic structure. and provide means for diagnosis and treatment.

The method of the invention mimics the idiotypic antigenic determinants of naturally occurring proteins and A synthetic polymer that results in a large fraction of induced serum reactivity toward natural and denatured proteins. Create antibodies against the lipeptid.

In a given anti-polypeptide serum, the same antibody molecule interacts with both native and denatured proteins. Is it possible that the antibody molecules in the antiserum are responsible for the interaction between the two protein states? It remains unclear whether they preferentially react with one or the other. Same or Whether different antibodies react with denatured and native proteins depends on the specific epitope or It will depend on the characteristics of the idiotope obtained.

Another aspect of the invention provides a method for making antibodies against synthetic polypeptides that can be used naturally in a host. used to raise antibodies against idiotypic antigenic determinants that are not immunogenic. It's scales. That is, the part of the macromolecule that has the ability to be bound by an antibody (i.e., is antigenic) but does not induce the production of antibodies (i.e., is immunogenic). idiotypes that are antigenic but not immunogenic This is an example of a fixed group. That is, the polypeptide of the invention terminates tolerance, Thereby, it can be used to target immune responses to restricted regions of self-proteins. It can be done.

Anti-idiotypic antibodies made according to the invention are conventionally produced by intact immunoglobulins. Several notable advantages over anti-idiotypic antibodies generated by immunization with have.

Conventional anti-idiotypic sera are based on the quaternary structure of immunoglobulins. Distinguish between types. That is, anti-idiotype antibodies Recognizes the three-dimensional protein structure created by the adjacent arrangement of non-adjacent regions of the and-order sequences. Ru.

Anti-idiotypic antibodies produced according to the invention are based on continuous, sequence-defining determinants. Differentiate between idiot types. The recognition site is located near the non-contiguous region of the -order sequence. This results in a high degree of specificity for a given region of the -order sequence. resulting in the ability to generate anti-idiotypic antibodies with. This is a conventional method was not possible.

Another advantage of the present invention over conventional techniques is that anti-idiotypic antibodies do not require purification. These scales are produced for specific idiotypes. anti-idiotype A conventional method for raising antibodies is to use the appropriate immunoglobulins or fragments thereof. This involves immunizing the host with various antibodies on immunoglobulins. A polyclonal response to diotypic determinants results.

The serum is then isolated and isolated for the specific anti-idiotope of interest. To make serum with specificity for Otype, it must be passed through an absorption column. No.

In contrast, the present invention provides highly specific Anti-idiotype antibodies are raised against a given predefined idiotype. can be

In addition, synthetic polypeptide methods have been shown to address questions about idiotypic structural relatedness. provide new analytical tools that can best serve to answer questions.

directed against idiotypic determinants consisting of located in the antigen-binding region of an antibody Anti-polypeptide antisera can be a means of relating protein structure to antigen binding.

For example, by eliciting a set of antibodies against different regions near the binding site, which It can be determined whether antigen-antibody binding is disrupted.

The word antigen has historically been used to mean something that is bound by an antibody, as well as to mean something that is bound by an antibody. It has been used to mean something that induces production in the body. More recent usage is , restricts the meaning of antigen to that which is bound by antibodies, while the term immunogen Used for those that induce antibody production. In some instances, synthetic polypeptides as when used to induce the production of antibodies that bind to a polypeptide. The antigen and immunogen are the same thing. However, the same polypeptide also has immunoglobulin It can be used to elicit antibodies that also bind to whole proteins such as phosphorus. , in this case the polypeptide is both an immunogen and an antigen, while an immunoglobulin is an antigen 0 If something mentioned herein is immunogenic and antigenic, then This would be called an antigen.

Brief description of the drawing In the drawings forming part of this disclosure: Figure 1 shows the amino acid sequence of myeloma protein M104 at positions 1t90-110. The D segment amino acids at positions 100-101 are shown in bold type. Bone The lines of the arrangement of the white matter J558, excluding the D segment, are as shown in the diagram. Polypeptides 3MN and 3JN, which show identity with the sequence of 2O3, are each having a shorter amino acid sequence than the corresponding sequences of peptides hV3M and hV3J; However, to separate the sequence from the carrier protein and to increase solubility, the Italian letter Contains additional amino acids as indicated.

Figure 2 shows sodium dodecyl sulfate (SDS)-polypeptide containing anti-polypeptide serum. Ray showing a nitrocellulose (Western) plot of an acrylamide gel. Counts 1 to 12 were obtained with individual antisera generated as described with reference to Figure 1. Lane 13 shows the results with pooled normal rabbit serum. Lane 14, on the other hand, shows the total protein in each lens.

The upper lane contains the M104 myeloma protein and the lower lane contains the J55B myeloma protein. Including white matter, electrophoresis was carried out from the top to the bottom giving 11 μg of protein per run. It was carried out until the end. Sera 1-8 and 13 were diluted with borate-buffered saline CB before use. diluted 1:1QO''?? with a 1% by weight solution of powdered milk in Johnson et al. Nfant Dry Milk Four Analysis of Proteins 7n Do Niekrei 7ri Acid Transfer Art Two Nitrocellulose (Improved Technique Utilizing Nonfat Dry 1ilk for Analysis of ProL@ins and Nucleic ＾aids transferred to N1troce llulose), Gene Analysis Technique (GaneAnalysis) (1983)], whereas serums 9 to 12 were prepared using the same medium. The 0 letters M and J diluted with 1 = 10 indicate which protein sequences are immunizing polypeptides. (See Table 1 and Figure 1).

Figure 3 shows a Western profile of a non-dissociated gel probed with anti-polypeptide serum. The details are the same as in Figure 2, except that 1 JNg of M104 opercular white matter and and 14 μg of J558 protein were given per lane. of the lower set of gel lanes The J558 protein band at the top indicates material that did not enter the resolved gel. This is an IgA (immunoglobulin A) dimer and possibly higher oligomers. - may include.

Figure 4 shows the port to polypeptide (A) and complete 1xM-RF (Sit:B). Figure 3 shows inhibition of lipeptidically induced anti-idiotypic antibody binding.

Antibody binding was determined by polypeptide (·):IgM-RF (Sl e:II) and a control polypeptide (mu).

FIG. 5 shows five monoclonal hydrinymatous factors and pooled human IK. Western plot of G (separate ports from two immune rabbits (Figures 1 and b)) Each antibody was primarily As, about FO, ooo Dal corresponding to Hll of 1tM-RF(See) Identify the t bands. Control studies with polyvalent anti-HIN antibodies showed that lower molecular weight The small band represented a small proteolytic degradation product of Hli. The markers used were: fluoresceinated bovine serum albumin; (F-BS^=68k), fluoresceinated gamma HfA (F-HC; 5 3k), fluoresceinized chain (F-LC, 28k) and LS (25k) .

Figure 6 shows the reported amino acid residue sequence of Neemati factor (corresponding to PSL2). ), the N region and the number of residues are from Kajt et al., Sequence Option. Proteins Op Immienological Interest (Sequence o f ProtIlins of Ia*unological Interes t).

U, S, Department of Health and) lauan Specified by S@rvices (1983). pub link or crossover The reactive idiotype is described by Menkel et al., J. Exp. Med. , Joichi Ko, 331 (1973). IgM-RF (S The amino acid residue sequences of Ie) and IgM-RF (Mol) are a-(Andrev) et al., Proc, Natl.

Reported by Acad, Sci, USA, AE, 3799 (1981), -On the other hand, for IgM-RF (Foe) and IgM-RF (Lay) The amino acid residue sequence of the first is according to Flapper (I[1apPer) et al., ^na, Imm. uno1. (last.

Pa5t@ar), 11 Kotan, 261 (1976).

FIG. 7 is a Western plot analysis of antibody activity of anti-PSL2 antiserum.

Approximately 20 pg of each indicated sample was loaded onto each gel. Sodium dodecyl sulfate Electrophoresis on thorium-polyacrylamide gels and electrophoresis on nitrocellulose paper After aerophoretic transfer, the samples were treated with anti-1 gM (A%D), anti-PSH3 (B), respectively. and anti-PSL2 (C%E) antiserum. VA (1181-Protein After development with A (Staphylococcus aureus), the paper was finally exposed to film overnight. . However, (D) and (E) were exposed for three days.

Figure 8 shows that PSL2 is connected to ItM-RF (Sie) C prisoner); - Suppressing the binding of PsL2m antibodies to isolated Lll of Glo (Oi Figure B) Also shown in Figure B is inhibition by control PSH3(mu).

Figure 9 shows IgM-R to bound polypeptide-induced anti-idiotype antibodies. PSL2 and control P, SH3 showing inhibition of FSie binding were affinity purified. Anti-PSL2 antibodies were added to pre-coated wells at the indicated concentrations.

After 1 hour incubation at room temperature (23°C), AP-1 M-RFS ie (10 μg per mj!) was added to each well and the plate was incubated for an additional 1.5 min at room temperature. Incubated for 5 hours. Thereafter, the plates were washed and stimulated with 405n-. Yield was measured 1 hour after adding substrate to the wells.

detailed description 1.1 person Idiotypic determinants are generally described by Jerne, Ann.

l5auno1. (Inst, Pa5teur), 1255.373 (19 74), and Binion et al., J. Exp. 860 (1982).

It is believed to be related to immunity tJ41B, as described by. child The control of the system is also described by Milburn et al., J. Exp.

Idiots as described by Med., 155.852 (1982). It appears to involve type-specific T cells. The evidence is that the main origin of the idiotype consists of Currently, Cosenza et al., rsnunological Rev. Restricted immunological repertoire as described by 3 Sat., 3 (1977) - indicates that it is more likely to be the result of the 1lillf process.

Since idiotype and anti-idiotype antibodies are associated with the immune y4 node, cosen immune response by inducing autologous anti-idiotypes as described by Zara et al. (supra). can be operated. This manipulation with anti-idiotype antibodies consists of It is thought to have considerable medical significance in cell malignant transformation and autoimmune diseases. Ru.

In these autoimmune diseases where harmful antibodies may be of restricted origin use synthetic immunogens to condition or even eliminate B cell clones that make antibodies. It would be possible to use it.

Such autologous anti-idiotypic antibodies for manipulating immune responses are of the present invention. can be produced in animals according to the method. If you want to adjust specific clone types Anti-idiotype anti-private idiotype anti-private idiotype body must be generated, but the private idiotype is Idiots found on only one or a few clones of an antibody of specificity It is bu. If all antibodies of a given specificity are desired to be prepared, Anti-idiotypic antibodies against link idiotobes must be generated. stomach.

Anti-idiotypic antibodies are the first line of defense for fighting autoimmune diseases and transplantation or prosthetic control. For example, anti-idiotype antibodies with bimodal function block the binding site. binding between a harmful antibody and its antigen can be eliminated.

An antigenic determinant is a structural configuration of a macromolecule that has the ability to be bound by an antibody. Department. Further explanation of antigenic determinants is best served by example. simple egg White matter consists of linear chains of amino acid residues. This chain is folded into a three-dimensional structure becomes. Some parts of H are inside and other parts are outside. Also, in the −order array Amino acid residues that are far apart are brought into close proximity by folding . This gives the protein structural configuration (steric configuration). Part of this configuration components have the ability to be bound by antibodies of appropriate specificity; i.e. are antigenic determinants. These parts of the configuration bind the antibody It is thought that it is in a suitable neighboring molecular environment such that it has the correct "shape" to One antigenic determinant of particular interest in the present invention is the immunoglobulin located in the idiotypic region and are therefore called idiotypic antigenic determinants.・ The presence of antigenic determinants on molecules is not limited to simple proteins; this ability It contains a wide range of proteins including glycoprotein, dextran, multi-polypeptide protein, etc. It is common in many natural macromolecules.

The term immunogenic elicits an immune response; i.e., the production of antibodies against a determinant. Used to describe the ability of an antigenic determinant to initiate life. I mentioned above in general However, not all antigenic determinants are immunogenic, and this may be due to several reasons. by. First, animals may have tolerance for antigenic determinants; Second, the molecular environment surrounding the determinant may not be correct to induce antibody production. I don't know. Third, there are certain factors that suppress antibody production, such as exfoliated Ill! l Transplant antigens may be present in the serum.

The antigen-binding site of immunoglobulins is not unlike any other macromolecule in many ways. That is, the present invention provides a Illustrating that some structural configurations can be antigenic and immunogenic . Such antigenic determinants found in the region of the binding site are called idiotopes. It will be revealed. Often, binding site regions have several idiotopes.

As mentioned above, the term idiotype refers to the type expressed at the binding site. Used to describe a set of idiotobes.

Idiotopes are found in the pocket of the binding site and on the surrounding surface. was shown. This discovery was made by Givol, Int, Review X-ray diffraction method performed by Biochew, 23.71 (1979) Confirmed by.

He revealed that he was classified as a gory. In 1968, Wi (01H1iams), antibodies with similar binding specificities are unique in their own right. specific idiotope as well as a common idiotope with another antibody of the same specificity. It was shown that Schilling et al., Nature ), 283.35 (1980) study on α-1,3-dextran. This study, which included 10 hybridoma clones and 3 bone marrow L1 fluorescent matter, revealed that more than half of the dextran antibodies share a single idiotope. I did it. This study also revealed that one or a few clones have a unique idiotype. revealed. In other words, idiotypes are divided into two categories based on this phenomenon. Suitable for classification as Lee.

The first category of idiothes are “cross-reactive” or “publink” ideas. It's a good idea. Cross-reactive idiotopes are antibodies that have the same specificity (some antibodies It is an ideology shared by all. Occurs in different strains of the same species or even different species Antibodies against a given antigen have been shown to have the same idiotonib . The second category of idiotobes is the 4 private 1 or 1 individual id. It's Otobe. This idiotope is a combination of several antibodies against a given antigen. Found in only one of the clones.

Changes in the amino acid composition of idiotopes do not necessarily change binding site specificity; Rajewsky et al., Ann, Rev.

lm5uno1.1.569 (1983), one amino acid in the D gene segment A amino acid change is the loss of one idiotype in the parent molecule and the degeneration of the second one. and six others, but the antibody still retains its binding site specificity. hold. This finding suggests that if the idiotope is located in or near the binding site pocket, If it is located, it is still valid.

Finally, animals have over a hundred antibodies with different binding site specificities.

- A set of idiotopes is associated with each binding site specificity. In other words, there is no animal There are several idiotobes.

The present invention uses short chemically synthesized polypeptides to synthesize naturally occurring animal proteins. It uses a technique that generates antibodies against white matter. The basic diagram of this technology is as follows It is.

First, the primary sequence of the protein or part thereof is determined. This is done in several ways It is done in one. First, the sequence information has already been determined and is available in the literature. Wear. Second, the protein itself can be isolated and directly delivered using methods well known in the art. Third, genes encoding proteins that can be sequenced are: Can be identified using genetic techniques well known in the art.

The identified genes are then cloned using recombinant DNA technology and! Can be separated. The primary sequence of a protein can be cloned in one of two ways. It can be determined from the genes. First, we will use all of the conventionally known techniques and information to The offspring themselves can be directly sequenced and this information translated into primary sequence. Second, the deceased The protein encoded by the gene can be sequenced.

-The next step after sequencing is to analyze the sequence for the region contained in the antigenic determinant. Is Rukoto. This includes general knowledge of biochemistry and the behavior of specific proteins. This is done using special information.

Next, a short polypeptide portion of the region associated with the idiotypic determinant is For example, Merrified et al., J. Aae'r.

As described in Chem, 5ocities 86.2149 (1963) It is chemically synthesized using various methods. Scientifically synthesized polypeptides are Typically, it is bound to a carrier to form a conjugate, and the conjugate is administered to a host animal as a vaccine. Injected in an effective amount. Conjugation where the specific antigenic determinant is a synthetic polypeptide Antiserum is generated against the body.

The present invention uses synthetic polypeptides to target specific idiotypes of antibodies. Provides a method for making antiserum. That is, using the present invention, anti-idiotype Synthetic polypeptide technology can be used to make antibodies. The antibodies formed are small. It has at least the following characteristics.

First, antibodies whose corresponding sequences differ by only two amino acid residues Ability to distinguish between immunizing polypeptides and determinants (idiotypes) on natural ligaments Second, antibodies can be raised against a given specific idiotype. . This was not previously possible.

Additionally, antisera are directed against idiotypes that are not naturally immunogenic in animals. It can be caused by That is, this idiotype has the ability to be bound by antibodies. (i.e., is antigenic) but does not initiate antibody production against itself. Fourth, the antiserum is sensitive to both native and denatured proteins. Fifth, anti-idiotypic serum has the ability to react with can occur.

One of the uses and benefits of anti-idiotypic antibodies is as a possible means of medical immunomodulation. be. Immunization using anti-idiotype antibodies! Ji'WJ and individual idioms The rationale behind the choice of options will be considered in more detail below.

As detailed below, vaccine production and anti-idiotypic antibodies of the invention! Although there are many steps using many materials in lit manufacturing, the present invention has some special features. The present invention is not limited to specific steps or reactants or conditions; rather, the invention conceptually and as defined by the details of the appended claims.

■, Peptide synthesis and selection A1 synthesis Polypeptides are described by Merrifie J. Redo et al., J. A+mer, Ches. Soc. , 85.2149 (1963) and Houghten et al., Int. .

J. Paptide Protein Res, Earthworks, 311 (1930). It was synthesized by solid phase method as described. The relatively short polypeptide used here The number corresponds approximately to the hypervariable region of the selected immunoglobulin molecule. [Also, Mar Marglin et al., AH, R@y, Biachew, 11.84 1 (1970)] A polypeptide is a polypeptide whose carboxylic acid is was bound to the operculum white matter carrier KLI (through a cysteine residue added to the end of the . The step of attaching a polypeptide to a protein carrier is described by Liu et al. Strii (Bioche+wiatry), 18.690-697 (19 79), the support and N-maleimidobenzoyl-N-hydride cis to the double bond of the reaction product between roxysuccinimide ester (MBS) This is done by adding a tin sulfur atom.

B, a polypeptide related to M2O3 and J558 proteins. These antibodies are produced by B cells that have been transformed into cancer-producing cells. it is are essentially the same as antibodies produced by normal, healthy cells. M2O3 and J 558 is bone marrow 11 produced by MOPC104E and J558 mouse myeloma This is the Pallor Award. Like any antibodies, they have binding site specificity. these The antibody binds to the α-1,3-glycosidic bond of B1355 dextran. Re Lean et al., Biochemistry, 9-11023-1030 (1982) and Londblaft (1982) d) et al., Immunochemistry, 9.5 35-544 (1972).

Both M2O3 and J58 Myeloma Pale Award Hli and Llm were sequenced (Se q++enced) 0 bimyeloma protein Lui is a lambda allotype. and are identical in sequence. Appella, Proc, Natl , Acad, Sci, USA, 6B-1540-544 (1971) and the same The M104 medullary gland is a mini-isotype, with the sequence of It must be stated that it is a fact that Cary et al., Pr. oc, Natl, ^cad, sci, UsAsKodan, 2932-2936 (1 9) 9) and Schilling et al., Natia, 283.35-40 (1980).

The D segment sequence is the individual protein expressed by M2O3 and J55B myeloma proteins. It was shown to determine the idiotype. Talepinger (CIeviBer) et al., ICN-UCL A5yI+p, Mo1ec, Ce11.

Bio! , 30.159-168 (1981), position 54 and in H’ll. Additional idiotypes determined by 55 and 55 amino acids are M2O3 and J5 5B myeloma and also expressed by Dextra in Ba1b/C mice. It is also expressed by many of the clonotypes induced by the clone BL355. Ta Levinger et al. (1981), supra; Flebinger et al., J. ExpJed, supra. Above, 1059-1070 (198α), and Promberg (B] os+ber z) et al. Science, 17? , 179-189 (1972).

Lll of M2O3 and J558 are identical in sequence. The H chain variable region is They are the same except for the component. Therefore, the individual idiots found in the third hypervariable part The type appears to be determined by the heavy chain. Nevertheless, this means that Ll[is This is not to suggest that LIN has no role in the expression of the otype; , Carlin ( Carson) et al., Proc, Natl, Acad, Sci, USAs Engineering 0. 235-239 (1973).

In order to induce the production of antibodies specific for a particular idiotype, several A synthetic polypeptide was used. In particular, M2O3 and J558 myeloma proteins The idiotype containing the D segment of the third hypervariable part of was studied. D segment consists of two amino acid residues located at positions 100 and 101 from the amino terminus. .

The D segment is a V segment at the amino terminal end and a F segment at the carboxy terminal end. contact with the fragment. (See Figure 1).

Two sets of two synthetic polypeptides were prepared in this study. The first set is The first seven contiguous amino acid residues in the M2O3 and J558 fragments and two that correspond in sequence to three adjacent amino acids in the V segment. These synthetic polypeptides, including two polypeptides, are highly effective against anti-polypeptide immune responses. 1 synthetic polypeptide designed to be long enough to ensure that it induces were named hV3M and hV3J and were derived from M2O3 and J558 osteova species, respectively. corresponded to the above-mentioned parts of the protein.

hV3M and hV3J polypeptides share the J1 gene segment common to both polypeptides. therefore, the anti-polypeptide represents a substantial portion of the sequence encoded by the Many of the responses could be directed to this common sequence. This is probably unknown We were able to show antibody activities specific to various D segments that were not identified.

Street table Sho 6l-50084G ('8) A second set of synthetic polypeptides used in the present invention is described by Lerner et al., Natia, 1. As described in Koshu, 592 (1982), almost everything necessary to form an antigenic determinant is These synthetic polypeptides contain minimal cognate sequences. , 3MN and 3JN, respectively, to M2O3 and J558 myeloma proteins. Correspondingly, it is shown in FIG.

To prevent stereochemical interference by the carrier of the immune response of the second set of polypeptide antigens. In addition, three proline residues are used as spacers between the recognition sequence and the amount of KLH carrier protein. Added. Proline, glycine and other groups are 5. Stevens s) et al., As, J. Reprod.

As a spacer, as described in Immunol, supra, 307 (1981). It has been used successfully. A glutamic acid residue is added to the sequence to increase solubility. It was done.

C, most of the polypeptides associated with TtM-RF, the antibody response is caused by the invading organism, directed against altered self cells or self antigens, e.g. rheumatoid factors (RF) is 1. 18M or f, G with specificity for the Fc fragment of G It is an antibody. Rheumatoid factor usually results from abnormal conditions and is caused by synovial inflammation and IjM-RF causes rheumatoid joint inflammation and vascoliLts. It is abundant in the serum of many individuals with flame. IjM-RF in rheumatoid serum , is polyclonal and reacts with many different antigenic determinants in the ticOFcjl region. do. 1 from an unrelated individual. M-RF indicates a cross-reactive idiotype.

Andrews et al., Proc, Natl, Acad, Sci. Monochrome according to the sequence reported by , USA Eng. 113799 (1981). Ronal I! M-RF (Sie) antibody Hli third variable region (amino acid residue A polypeptide (named CPSH3) corresponding to 99-111) was synthesized. Ta. This polypeptide is defined from left to right and from the amino terminus to the carboxy terminus. had the following sequence: G1u? rpLysG]yGlnValAsnVal AsnProPheAspTyr Green et al. Cell , 28.477 (1982) to facilitate binding to protein carriers. In order to CydoG1yGlyCys was added. The obtained polypeptides are sorted from left to right and It had the following sequence from the mino-terminus to the carboxy-terminus: G1uTrpLy sGlyGInVaIAsnVaIAsnProPheAspTyrG1yGl Monoclonal according to the sequence reported by ycys Antriase et al. (supra). IgM-RF (Sie) second hypervariable region of the second chain (amino acid residues 49-61 ) was synthesized (named PSL2). P SL2 contains the following amino acids from left to right and from amino terminus to carboxy terminus: with amino acid residue: τyrGlyA1aSerArgAlaThrG1ylls ProAspArz additional Cys facilitates conjugation to protein carriers as described above. was added to the C-terminus of the sequence to make it.

°■, Immunization procedure The vaccine used here consists of administering the indicated amount of polypeptide alone to glutaralcohol. as a polymer of individual polypeptides linked together by reaction with a dehyde, or as a supported The polymeric polypeptide that binds to the body and contains also Addition of cysteine residues and subsequent mild pH analogy due to atmospheric oxygen, for example. For example, it can be prepared by oxidation at about pH 7 to allIO. The indicated amount of pot When a carrier is used, the weight of the polypeptide excluding the weight of the carrier To tell.

The vaccine also includes a physiologically acceptable vehicle, such as water, saline, Furthermore, an complete adjuvant (IFA), which typically includes an adjuvant, is the It is a well-known material in the art and is commercially available from several sources.

A vaccine stock solution was prepared using IFA and CFA as follows.

- an amount of synthetic polypeptide, polypeptide, or polypeptide sufficient to provide the desired amount of polypeptide per inoculum; The mer-like polypeptide or conjugate is placed in borate buffered saline (BS). Next, the vaccine was made. The mixture is then homogenized for vaccine storage I made a solution.

The vaccine storage solution also contains Keyhole Limbett Hemocyanin (KLH), I KLH, Alum, KL) (-Alum) in FA (Incomplete Freund's Adjuvant) Alum absorption, KLH - Alum absorption - whooping cough, edestin, teroglobulin, tetanus It can be prepared using toxoid and tetanus toxoid in IFA.

After injection or other methods of introduction into a host with an antigen or vaccine, the host's system becomes responds to antigens by producing antibodies.

Man-made antigens, i.e., specific antigens formed from synthetic polypeptides and carriers. Biotypic antigenic determinants are the same as or immunogenic as those of the natural antigen of interest. Because it is an epidemiological surrogate, the host becomes immunized against the natural antigen6. This is the desired result when used as

- The effective amount of polypeptide per inoculum (considered in the attached table) by dispersing the polypeptide in water, saline or an adjuvant, or The polypeptide is attached to a carrier and the carrier-bound polypeptide (conjugate) is subjected to similar physiological prepared by dispersing it in an acceptable vehicle such as an adjuvant. .

The effective amount of polypeptide present in the vaccine depends on the carrier used, if any. except - can be about 20 ug to about 500 μg per inoculum.

Synthetic polypeptides are used to assist in attaching synthetic polypeptides to carriers to form conjugates. - or two additional amino acids at the amino or carboxy terminus of the repeptide. It is often advantageous to add As mentioned above, synthetic polypeptide covers The cysteine added to the carboxy terminus is associated with a disulfide bond and a Michael addition reaction. It has been found to be particularly useful for forming conjugates via reaction products.

However, there are cases where other conventionally known methods may be used to prepare the zygote. Exemplary coupling procedures include the use of dialdehydes such as glutaraldehyde, or Water-soluble carbodiimides such as l-ethyl-3-(3-dimethylaminopropyl ) including the use of carbodiimide methods, such as in the use of carbodiimides.

Useful carriers are well known in the art and are themselves generally proteins. Like that Examples of suitable carriers are keyhole limpet hemodiacin (KLH), edestin, and Loglobulin, albumin such as bovine serum albumin or human serum albumin (BSA or BSA, respectively), red blood cells such as sheep red blood cells (SRBC), stout toxoid, as well as poly(D-lysine: D-glutamic acid), etc. It is a polyamino acid.

Also, as is well known in the art, synthetic polypeptides can be attached to their carriers, with intermediate connections. It is often advantageous to link through groups.

As mentioned above, glutaraldehyde is one such connecting group, while , if cysteine is used, the intermediate connecting group is preferably m-maleimidobenzene. MBS is the standard Typically, it is first attached to the support by an ester-amidation exchange reaction. after that , the aforementioned Michael reaction is carried out, or the addition is followed by the blocked mercap The group crosses the maleimidoni double bond of, for example, thiol acetate M (CLCO3H). Additions can be made. Cleavage of the blocking group is then carried out and the disulfide linkage is The combination of the unblocked connecting group mercaptan and the added system of the synthetic polypeptide It is bonded between the mercaptans of the Stine residues.

The choice of carrier depends more on the intended end use of the antigen than on the determinants of the antigen. For example, if the vaccine is is to be used in animals, it may produce an adverse reaction in that particular animal. A carrier that is not similar will be selected if the vaccine is to be used in humans. An important consideration is the absence of immunochemical or other side reactions of the carrier and/or the antigen obtained. Safety, safety and efficacy - Appropriate for any vaccine intended for use in one human. Regarding the same consideration.

Determining whether a particular antigen is present as an aid can be done by For example, synthetic antigens, which are often highly desirable in the diagnosis of certain diseases, are of interest. is monospecific for a single specific antigenic determinant and therefore Antibodies are also monospecific for the antigen of interest. Complete monospecificity is always Although not always achieved, cross-referencing of the antigen with other antigenic parts (cros s-raferencing) is avoided. Because just one immune response This is because it is possible with antibodies.

The antibodies are isolated and produced by any conventional procedure used in diagnostic tests. . For example, it is common practice to label antibodies for identification and quantitative measurements.

Radioactive labeling by the method of Greenwood (Hunta) - (Hunter, Br, Med, Bull.

30.18 (1974)] and fluorescent dye labeling for immunological evaluation (immunological evaluation). (No. 77) is commonly used.

Vaccines contain physiologically tolerated polypeptides bound (conjugate) to a carrier. 11m by suspending in a vehicle! be done. Other acceptable! ! ! The turbid medium is , well known in the art, and include water, saline, and the like. @turbid carrier-bound synthesis Polypeptides are referred to below as vaccines.

■、Results A. Immunization vaccines with M2O3- and JSSO-related polypeptides stimulate the immune response. injected into a host animal in an amount effective to induce. Three immunization methods are shown in Table 1. used to generate anti-polypeptide serum against polypeptides as . Two rabbits were used throughout the immunization process for each synthetic polypeptide. It was done. Antisera against hV3M and hV3J polypeptides were 3FIN and 3JN polypeptides. Antiserum was raised in lefdonian zealand rabbits.

Table 1 1. Using synthetic polypeptides related to bone marrow IIW Shirosho M104 and J558 A variety of whites used to generate anti-polypeptide serum; Numbers 9 to 12 were female Nienoland reds. Each animal received each injection. and received 200 μg of peptide conjugated to KLH. Rabbits 1-8 are the best Rabbit 9-1.2 was bled 7 days after the last injection and rabbit 9-1.2 was bled 9 days after the last injection. was taken.

2. The divant used and the injection site are indicated for each injection. Abbreviations below Z CFA-Complete Freund's 7 dievant; IFA ■ Incomplete 7 0 Indian 7 Dievant: 4 to 8 subcutaneous injections of sc- into both hind paws of Hfpm. Injections; lfp or rfp - injections into the left or right hind paw, respectively; 1p-1t! Intramembrane injection.

3. The interval between consecutive injections is indicated in days.

The immunization procedure involves three or four doses of the vaccine; for any vaccination, the animal 2 of the synthetic polypeptide conjugated to Ihor limpet hemocyanin (KLH). 00 (200) μg, and the zygotes were placed in Freund's adjuvant or alum. emulsified into.

The vaccine was administered by multiple subcutaneous injections in the back and hind legs or intraperitoneally. inoculations were performed 7 to 62 days after the previous inoculation.

Rabbits immunized with hV3J and hV3M polypeptides were tested at 7 p.m. after the last inoculation. Rabbits bled after 1 day and immunized with 3MN and 3JN polypeptides were Blood was drawn 9 days after i post-inoculation.

1. Antiserum reactive with denatured M104 and J5581F white matter was first Regarding the ability to distinguish between sexually transmitted M104 myeloma protein and degenerated J558 bone marrow III white matter. It was evaluated as follows. The measurement was carried out by first measuring M2O3 and J558 myeloma protein. (Laemnli), Nature, 22nd grade, 680 (197 0) Vertical plane modification using a 5-10% polyacrylamide gradient as described Dodecyl Ha sodium polyacrylamide gel electrophoresis (SDS-PAGE) This was done by running the top. The protein is then converted to Towbin. et, Proc, Natl, Aead, Sci, uS^,? 6,4350 (19? 9) Nitrocellulose (Scherider and Schue) was electrophoretically purified as described. Schleider and 5haell, Keene , NH), the plots were colored with amino black and gel-rayed. cut into corresponding strips. Non-specific binding is caused by John John As described by Son et al. (cited above), a recently developed agent for this purpose Locked. Finally, the strips were exposed to anti-polypeptide serum for 2 hours at room temperature. Then, a positive reaction was performed as described by Johnson et al. (supra). color Staphylococcus) and visualized by autoradiography.

The results of this measurement are shown in FIG. Two rows of multiple strips are shown. vinegar The top row of trips shows the binding of various antisera to denatured MLO4 protein. The bottom row shows the binding of various antisera to the j558 protein.

Fourteen rows of strips are shown. Each column is marked with a 0 with a number above it. Columns 1-12 each correspond to antisera raised in 12 immunized rabbits. (Table 1) Column 13 is blank, column 14 is a standard indicating the migration position of myeloma protein. be.

There is a letter M or J under the column number 0 The letter M means Rabbit is M104 Bone 9N11 'J indicates immunization with a polypeptide containing the D segment of white matter; BIT was immunized with a polypeptide containing the D segment of the J558 bone marrow protein. to show that

Antisera raised in 8 of 12 rabbits (numbers 1.3.5.6.7.8 ．． 10 and 11) distinguish between M104 protein and J558 protein and It showed a high level of specificity for proteins corresponding to peptides. Additionally, reactive Many (if not all) were limited to the Hui band. Consistent with the predetermined specificity of the lipeptidase. Vagueness evident in lanes l and 6 The additional bands are probably destroyed products or groups.

Due to one or both of the differences in lycosylation.

raised against hV3M and hV3J polypeptides in rabbits 12.3 and 7. The antisera showed cross-reactivity between the two bone type I proteins. This cross-reactivity is , a large range of Jl gene segment sequences common to the two proteins (exten sive 5tret)).

Antisera raised in rabbits 9 and 11 reacted with denatured M104 myeloma protein. Attention is also drawn to the observation that there was no such thing.

think about. There, when the antisera raised in rabbits 9 and 11 combine, the antiserum Different antibody molecules in serum may react preferentially with one or the other of two protein states. There is a tendency to show that This finding will be described in more detail in the next section.

2. Reactivity of antiserum with natural MIQ4 and J558 proteins The antiserum is a natural M10 protein. The ability to distinguish between 4 and j558 myeloma proteins was evaluated. This measurement is First run M2O3 and J558 myeloma proteins on vertical plate gel electrophoresis. This was done by The same equipment as in the measurements on denatured gels was used.

The following gel system was used: 1. M104 protein was incubated with 10mM to 100mM trisphosphonate at 3mA for 2.5 hours. Urate pH 9.0! ran on a 7% agarose gel in a l buffer gradient.

2. J558 protein was incubated with 70mM Tris-borate pH 9.0 at 10mA for 2 hours. was run in a 5-12% polyacrylamide gradient.

Proteins were transferred to B using 70mM Trisborate p)19.0 as transfer buffer. io-Rad (Richmond, California) Trans-Blot Cell Nitrocellulose (Scherider and Schue) for non-dissociated gels by It was moved to Schleider and 5huell.

Next, the plots were colored with amide black and streaked corresponding to the gel lanes. I cut it into two pieces. Non-specific binding is caused by the reagents described in Jigenson et al. (supra). more blocked. Finally, strips were incubated with anti-polypeptide serum for 2 hours at room temperature. and then the positive reaction was 112% as described in Jinson et al. (supra). Visualized with white iA and autoradiography.

The results of this measurement are shown in FIG. Two rows of numerous strips are shown. Ru. The upper row of strips shows the binding of various antisera to native MLO4 protein. The bottom row shows the binding of various antisera to native J558. The letter designation and immunizing polypeptide designation (M or J) are the same as in FIG.

The bands in Figure 3 are more diffuse than those in the denaturing gel system (Figure 2). Ru. This is due to the absence of stacking forces in the non-denatured gel in Figure 3. . Denaturation gel is the protein used! Fi! ! The object contains only a small amount of foreign protein material. It has been shown that it does not include (if any). Therefore, the diffuse appearance of the band Nevertheless, the band in Figure 3 contains exactly the M2O3 or J558 protein.

Antihaemia produced by 6 of 12 rapids (numbers 1, 5, 6, 8, 9 and 10) The supernatant differentiates between M2O3 and J558 native protein and corresponds to the immunizing polypeptide. It showed specificity for the protein.

Antiserum raised in Rabi7) 1.3.5.6.7.8.10, 11 is a denatured protein. It must be recalled that the same competence has been demonstrated in terms of quality, i.e. At least half of the proteins, whether natural or denatured, Created an antibody that can distinguish between M2O3 and J558 protein. is.

These results are consistent with defined idiotypes elicited by synthetic immunogens. Demonstrates the first production of specific antibodies.

antiserum raised in rabbit 9 and antiserum raised in rabbit 11 (lower (to a lesser extent) reacts with native bone marrow 1 [i1F white matter. But these rabbits? Their antiserum did not react with the denatured protein (Figure 2), given the anti-polypeptide In serum, the same antibody molecule is responsible for interactions with both native and denatured proteins. whether different molecules in the antiserum are present in one or the other of the two protein states. It remains unclear whether they respond preferentially.

The exact situation may depend to a large extent on the particular epitope considered; The data described above tend to indicate that different antibody molecules may be involved.

against rhV3M and hV3J polypeptides in rabbits 1, 2.3.6 and 7. The antiserum generated showed cross-reactivity between the two myeloid membrane proteins. this Cross-reactions occur over a large range of J gene segment sequences ( This is thought to be due to the extended stretch.

B. Immunization with IgM-RF related polypeptide 1. Immunization Synthetic polypeptides PSL2 and PSH3 can be prepared by a separate reaction, as previously described. In , keyhole limpet hemocyanin (K L H) m-maleimidobenzoyl N-hydroxysuccinamide ester Joined by.

Green et al., Cell, 1st Edition, 477 (1982) and Liu et al. 0, Biochemistry, 1 Lord, 690 (1979), each of the two rabbits is a complete Freund adjunct. 2.5 μ of conjugate emulsified in a bunt was injected subcutaneously.

The injection was repeated two months later. Three weeks after the second immunization, the rabbits were again 2.5 ■ Glutaraldehyde cross-linked polypeptide in complete Freund's adjuvant It was promoted by The latter agent is a 50% polypeptide in isotonic phosphate-buffered saline. ■／Stomach! Add glutaraldehyde (final concentration 0.25% v/v) to the i solution, made by incubating for 1 hour at room temperature. Rabbit has blood drawn Serum was stored at 20°C until analysis.

2. Protein purification Plasma from tricolor individuals with monoclonal ItM cryopropurine or purified Proteins were precipitated repeatedly at 4°C, followed by 0.2M sodium acetate at pH <3.5. Chromatography on 5ephadex G-200 or old trogel AcA22 Purified by graphics. [,M and 1,G peaks identified by immunodiffusion The appropriate fractions were then pooled and stored at a temperature of -20°C. human IgG DEAE cellulose chromatography in 0.01 M sodium phosphate at pH 8.0. Cohn fractionation (Sigma, St. Louis) It was prepared from the following sources:

H and LIJ of IxM-RF protein 1iste after complete reduction and alkylation. , 1M 6M on a 5ephadex G-100 column. Yellowtail Bridges et al., Biochemistry, 10.2525 (197 1) Polypeptides were stored frozen at 1/ml. 1. MH chain or kappa chain using radioimmunoassay specific for HlI or It was estimated that Lii contains less than 2% Hli.

3. Enzyme-linked immunosorbent evaluation (ELISA) synthetic protein (100μg/a+1) , various purified monoclonal Ig M-RF (10itg/to0, and Ig Isolated H chains and Lg of M-RF membrane proteins [(to μg/ml) at pH 8 , 2 O, 1M borate and 0.2M NaCj! Borate buffered saline (B BS). The mixture is then poured into a PVC microtiter plate. (Coster # 3590) (in the hollow, about 100/7j per hollow! Added in concentration. After overnight incubation at 4°C, plates were treated with 0.5% Tween-20 containing BBS (Sigma #L379; -B B S/ Washed twice with Tween-20), BB containing 1% bovine serum albumin (BSA) Quench for 1 hour at room temperature with S/L/0 then 0.5% BS 100 μl of serum diluted in A-containing fBBs was dispensed into the wells in duplicate. pre The cells were incubated for 3 hours at room temperature. Ween -20 three times (after washing, human Iz G -5reph Alkaline phosphatase-labeled goat anti-alkaline adsorbed with arose 4B Rabbit Ig (Kirkegaard and Perry) d Perry, Gaitherspurg, MD) at a 1:80Q dilution of 100 μ1 divisions were distributed into the wells. After a further 1 hour incubation at room temperature, The plate was washed five times with BBS/Tween-20. Then 0.05 p-nitrophenyl phosphate (1111/ Add 100 μN of mj to the well and measure the absorbance at 405 nm for 1 hour at room temperature. After 16 hours at 4°C, Titertek Mu It was measured with a ltiscan) meter.

4. Suppression evaluation Ig M-RF (Sie) or polypeptide coated plate Inhibition of binding anti-polypeptide antibodies was achieved by previously described ELISA methods, but It was evaluated with the following changes: BBSlo, 1:1000 dilution in 5% BSA From Rabbit No. 2.

The antiserum of e, immunization polypeptide or control polypeptide), then 100 μl It was distributed in two ways. From left to right and from amino terminus to carboxy terminus G　uTyrG　1　yPheAs　pThrSerAs　pTy in the direction of Amino acid residue sequence of rTyrTyrTyrT yrG l yG 1 yCys against the third hypervariable region of HlI of monoclonal human IgM-RF (Mol) with The corresponding polypeptide was used as a control (Antriace (Andrew s) et al. (supra)).

5. It M-RF (Sie) binding activity adsorption and elution anti-polypeptide antiserum The globulin fraction of Digested with 3% (w/w) pepsin for 16 hours in H4,1. After neutralization, 1g undigested To remove G, protein-ASepharose 4 B column (Pharma Pharmacia Fine Ches+1cal s), biscantaway, two knee jersey) with recirculated digestate on the zero order , FCab')t fragment into cyanogen bromide-activated 5eph arose 4B (Sigma, St. Louis, Missouri)! Made of poly 5epharose4B affinity column bound with peptide (approximately 6.6 w/ wr 1 gel x 5111). Substances not bound by BBS After the removal of F(ab　’) 1, pH 6, protein blotting 1176 (1982), tested by the Western plot method. It was done. Briefly, samples added with 0.01% 2-mercaptoethanol Pull buffer 25μ! Among the individual molecules, the clonal IBM-RF protein [Calson ( Carsoa) et al., Mol.

Iau+uno1. '20.1081 (1983)) or pooled human I Approximately 20 μg of gG was added to 10% polyacrylate containing 0.1% sodium dodecyl sulfate. applied onto each slot of the Luamide plate gel (Laes@1li), Natia, 11th Engineering, 680 (1970) After 3 hours of electrophoresis at 3° and 30 mA. , Pallium cells in gel were electrophoretically transferred to nitrocellulose paper. The joint site was incubated with PBS containing both BSA (5%) and ovalbumin (5%). Quench at warm temperature for 1 hour. The paper was then coated with anti-polypeptide anti* and ovalbumin (1g1oo dilution in PBS containing 2% of both) were layered for 1 hour. Ta. After washing, IiS the paper! Labeled pallidum A (1 Ci/*, 2　X　I It was further developed for 1 hour at Q'cpm/ml). After washing thoroughly, dry the paper. Finally, XAR-5 film (Eastman Kodak, Rotinistar, New - Yoke) overnight at -70°C.

7. Induction of anti-peptide antibodies Two subcutaneous injections of polypeptide-KLH conjugate in CFA and incomplete Freund. received a single injection of grugulaldehyde-crosslinked polypeptide in an adjuvant of Later, the rabbits were bled and the serum was analyzed for anti-polypeptide activity using ELISA method. As shown in Table 2, sera from both immunized rabbits were analyzed at 1: Contains anti-polypeptide antibodies that can be detected at high dilutions such as 100,000. Control serum from regular rabbits was added to polypeptide-coated plates. It didn't come together as intended.・Table 2 Monoclonal 5t 18 Induction of anti-idiotype antibodies by polypeptides roughly corresponding to Absorbance at 405nm (XIO”) Normal rabbit 25 0 0 4 Immuno Rabbit /) 1 1 0 2 3 4 9 7 9 5 5 Immuno Rabbit 2 937 530 85 0 Polypeptide-induced anti-inflammatory response of serum from two immunized rabbits The activity of deiotypic antibodies (as described above) is Solidification of PVC coated PVC on microtitre plates. The phase was evaluated by ELISA. The two series of microtiter depressions are peptide C Rabbit coated with 10100u per BBSI at various dilutions Antiserum was added. Two control wells contained buffer only. 3 hours ink After washing and washing, the amount of bound antibody is quantified by alkaline phophatase treatment. It was measured using 1gG of synthetic rabbit. The absorbance of the enzyme substrate at 405 nm was measured after 1 hour. was measured.

8. Reaction between anti-polypeptide antibody and complete antibody molecule 11 M-RF (Sie) sex Anti-polypeptide antiserum coated with complete tort M-RF (Sit) Table 3 (A and B) evaluated for direct binding to the plate Anti-polypeptide antiserum reacted with intact antibody molecules but control serum did not This shows that. 1: Even at 100.000 dilution, this anti-polypeptide Serum bound significantly to intact 14M protein.

Polypeptide-induced anti-idiotype antibodies and complete Ig M-RF (Sie) antibodies Reactivity with molecules* 1 person Absorbance at 405f1m (XLQ”) (room temperature normal rabbit 35 13 3 1 4 Immuno Rabbit 1 292 106 47 46 Immuno Rabbit 2 307 164 75 0 yu Absorbance at 405 nm CXl0') Normal rabbit 9619314 Immuno Rabbit 7) 1 1041 376 78 14 Immuno Rabbit 2 824 38 G　75　9　Solid-phase ELISA was performed in the same manner as in Table 2, except that the depression was Coated with 10 pg of complete Ig M-RF per liter of BBSI. Absorbance readings at 405 nm should be taken at room temperature (23°C) for 1 hour. After And incubate overnight at 4℃! taken after

■Includes breath M-R F (Sta) (some Ig M-RF rose prote has been shown to interact with rabbit IgG.

The effect is similar to the specific binding effect of anti-polypeptide antibodies, and the IgM-RF Anti-polypeptide antibodies have a negative effect on non-specific interaction with Rabi-/) IgG in serum. From M, - R F (Si@)! Isolated H chain produced by Pl (this is a Rabbit (lack of detectable binding to IjG).

Table 4 Isolation of polypeptide-induced anti-idiotype antibodies and Ig M-RF (Sie) Reactivity with the H chain produced* Workers Absorbance at 405 nm (XIO') (room temperature normal rabbit 16 9 3 10 Immuno Rabbit 1 437 105 10 11 Immuno Rabbit 2 324 92 60゛manufactory Normal rabbit 40 13 4 11 Immuno Rabbit 1 1443 404 67 12 Immuno Rabbit 2 1084 3 19 50 5 * In this measurement, the depression is Is M-RF (Sit) A monolayer of pallidum was coated with HIM (BBSI 10 per a1.

μg of protein), otherwise the conditions were the same as described for the tests shown in Table 2. It was the same.

In addition, the F(ab') fragment of the anti-polypeptide antibody is a complete IgM- RF (Sie) bound to the pallidum, but those of normal Labift ljG did not bind. There were no cases (Table 5).

Table 5 It M-RF (Sie) binding activity from affinity column bound to polypeptide Dynamic @ monitoring and elution Coated antigen: BSA polypeptide It M-RF (Sie ) Effluent 33 374 47 Eluate 28 1900 459 Normal Rabint IzG 59 136 89 polypeptide anti-idiotype antibody The crude F(ab') 5μ/Ill gel). Ink bath for 15 minutes at room temperature (23°C) Afterwards, the effluent was collected. After washing, the bound substances were dissolved in O,lM glycine-11 cj (pf13) and neutralized. &! A sample of this is mentioned above! Hon fishing In the EL I SA method, BSA, polypeptide or complete Tg M-RF (Si A concentration of 25 μg/jet for microtiter cavities coated with e). Evaluated in degrees.

9. Anti-peptide antibodies detect peptide-determined epitopes on It M-RF (Sie) 1! to discuss Antibodies are attached to specific polypeptide-determining epitopes on intact Ix M-RF (Sie) molecules. Two types of studies were performed to demonstrate the binding to the tope. Primarily , as shown in Table 5, much of the 11M-RF (Sie) binding activity It was adsorbed by and eluted from a Tide-conjugated immunosorbent column. Second, 11 Antibody binding activity to the plate coated with M-RF (Sie) Under the same conditions, we wanted to be completely inhibited by free polypeptide in the solution (Fig. 4). Corresponds to the third hypervariable part of Hli of monoclonal It M-RF C@ol) The control polypeptide had a significant inhibitory effect even at l,000 times higher concentrations of Re3. Naka song.

10. Anti-peptide antibody is a private idiot on Ig M-RF (Sie) Recognize the type.

Anti-polypeptide antibodies are effective against isolated Ig M-RF (Sie) Hli The observation that human [gM-RF and rabbit IgG bound to Sensitive protein protein protein for detection of epitope-bearing El M-RF to avoid use This made it possible to develop the ing method.

It M-RF paraprotein and pooled human IgG (Cohn hn) panels of fluxin ■) are grown in SOS polyacrylamide gels under reducing conditions. It was fractionated by electrophoresis and then transferred to nitrocellulose paper. anti-polypeptide ink 1 and 17s by antibody [last by labeled protein-A] After development, an autoradiograph was made.

Figure 5 shows that the anti-polypeptide antibody is IgM-R-F paraprotein (the two lo and Tgh) are cross-reactive (publink) idiotys on the 5L41 molecule. (Carson et al., Mo. 1. Iwmunol (+ supra)) is shown to react with Ha.

As can be seen, anti-polypeptide antibodies can detect HM of pooled human zG. It didn't react as much as it did.

These results indicate that the anti-polypeptide antibody was detected on the HM of Ig M-RF (Sie). Demonstrates recognition of private idiotypes.

t1. Polypeptide-induced antibodies are the public idiotype of human RF! ! To be aware.

Two subcutaneous injections of PSL2-KLH conjugate and glutaraldehyde cross-linked polypep After receiving a single injection of Tide, serum was obtained one week after the last injection and herein Anti-polypeptide activity was analyzed by the EL I SA method described.

As shown in Table 6(A), both sera contained high titers of anti-PSL2 antibodies and were normal samples. Coated serum pooled from bits coated polypeptides It did not bind significantly to the tested plates. In addition, immune serum was used as a control. It did not react with the polypeptide PSH3.

The anti-polypeptide antiserum was then tested for reactivity with IgM-RF (5ie). Table 6 (B) shows that the two sera specifically interacted with intact antibody molecules. showed that the two antisera and I reacted, but the control serum did not. g Reactivity with M-RF (Sie) was relatively weak. This means that PSL Only a small fraction of the 2m antibodies reacted with intact immunoglobulin, or Extremely low affinity interaction between SL2-inducing antibody and Ig M-RF (Sie) It suggests that it is a sexual thing.

Table 6 Induction of anti-RF antibodies by polypeptides (A) Detection of polypeptide-induced anti-idiotype antibodies 1・寞to-310-' 10-' Contrast Normal rabbit 23 6 3 5 Immuno Rabbit zEL 1036 302 53 1S Immuno Rabbit 2 948 3 70 54 1101・1G-’10” Contrast Normal rabbit 35 13 3 4 Immune Rabbit 1 136 28 5 12 Immune Rabbit 2 92 21 13 141, polypeptide-induced anti-idiotypic antibody activity is associated with various dilutions were added to the wells in duplicate and incubated for 3 hours at room temperature (23°C). Bound antibodies were then quantified using enzyme-conjugated anti-corycet IgG and did. Absorption at Aaes was measured after 1 hour at room temperature.

B The specificity of the re-evaluation is that the immune serum is the control PSH3 (A) and the control Tg. Indicated by not reacting with M-RFLay (B) (data omitted) .

3. Same as footnote 1 above, but the depressions are coated with IgM-RFSie It was done.

PSL2 is shared by L1'JI of two human monoclonal ItM-RFs. Since it contains an amino acid residue sequence corresponding to a hypervariable region (Antriuse et al., supra), PSL2-induced antibody reactivity was measured against a panel of human monoclonal RFs. Table 7 (row 2.3) shows that PSL2-induced antibodies react with IgM-RFGlo. However, it reacts to a smaller extent with M-RF PO@, but IgM-RFL Again, the binding was extremely weak, indicating no reaction with ay.

Table 7 Human monoclonal PSL2-inducing antibody 1. Anti-1gM with M-RF”56 B 73.5, Ej59 315 immune serum 3 First blood draw 38'109 23 0 Next blood collection 116 ND' 23 NDl, same as Table 6(B) except for the following points :l) The RF shown in the table is BBS1mj! Coated with 2 μg RF per Ta.

Immune sera were evaluated at t:too dilutions and absorbance at 405 nm at 6°C. g3) Anti-1g M (Ig M- from rabbits immunized with RFSie) and the absorbance at 4Q5nm was measured at room temperature. Measurements were taken after ink bathing for 1 hour.

2. Anti-1 gM was used to determine the relative amount of each 1 gM-RF.

3. Rabbit (#1) received one of the first injections of glutaraldehyde @PSL2. The first rabbit bled after a week was further bled to increase the anti-RFSie titer. two more injections (one of cross-linked PSL2 and then of the PSL2-KLH conjugate) once).

and blood was drawn after the last injection.

4. The numbers shown are the immune serum after subtracting A4゜$ of control normal rabbit serum. It is A4°.

5, ND - Not measured It was first shown that some IxM-RF paraproteins reacted with rabbit IgG. (Kunkel et al., J. Exp. Med, 139.129 (19 74)), thus the interaction between PSL2-inducing antibodies and these reactive 1tM-RFs. This is probably due to the specific binding effect of PSL2-inducing antibodies, and human IgM-RF This proves that this is not due to non-specific interaction with IgG (Rabif in the antiserum). It was necessary to clarify. Therefore, psL2ts-expressing antibodies and these Igll-R The reactivity of F with isolated chains was determined by the Western Pronto method (Toubin et al., supra). Measured by. As shown in Figure 7(C), the PSL2tR antibody has 11 It reacted equally well with M-RFSie, Glo and Teh LSIs. I Because of the extremely small amount of IzM-'RFTeh that each person had, IgM- Only 115 equivalent IL of RFTeh! only was used.

Therefore, in order to confirm antibody reactivity with RF-Teh, another Wester A sample plot was performed [7th (D) and 7(E) Figure 3. One set of samples was different. (Figure 7(D)) was reacted with anti-LgM antiserum to show the relative amount of RF ;The autoradiograph was then developed for 3 days.

These results indicated that the pst,2g-expressed antibody reacted with RF-Teh.

In addition, this antibody interacts with LH of Ig M-RF Po@ and pooled IgG. I reacted very weakly. But most importantly, it It did not react with SI and also did not react with HthI of any IjM-RF. contrast Generally, PSH3-inducing antibodies reacted only with HIM of IBM-RFSie. this Collectively, their results demonstrate that PSL2-induced antibodies are associated with several human 11M-RF It was shown that the pub link idiotype on I was [k].

12. Structure of RF pubic idiotype defined by pst, z-induced antibodies creative interconnectedness PSL2-induced anti-slope response to specific PSL2-determined idiotypes on 1gM-RF To prove the binding, complete RF (Sie), and RF-Glo The inhibition of antibody binding to both single MLg by the 5PL2 peptide was measured.

Figure 8(A) shows that antibody binding to RF-3ie is due to free MPSL2 peptide in solution. showed that the binding was completely inhibited by the control PSH3 at the same concentration. was not affected at all (data not shown), and the 5000 ng/sJ control PSH3 completely suppressed the binding of PSH3-induced antibodies to RF-5ie.

In other words, these data indicate that PSL2-induced antibodies are associated with specific PSLs on RF-3ie. It was shown that the 2-determined idiotype was iffed.

Isolated Lll was then prepared from IgM-RFGlo. as expected , the psL2ts-expressed antibody reacted with the L of RF-Glo [Fig. 8(B)], and Furthermore, binding was completely suppressed by free 11PsL2 in solution, but was inhibited by PSH3. [Figure 8(B)], which indicates that RF-3is and R F-Glo light chains share a homologous second complementary determining region (CDR-2) , and RF public clones in which Lli-CDR-2 is defined by PSLZ-induced antibodies. Suggests that it is a structural relative of the idiotype.

13. Anti-RF activity is present in anti-peptide antibodies. PSL2yk-expressing antibodies are further characterized. In order to mark, 1. G fraction from antiserum containing high titer anti-RF activity! Made by Il (e.g., yet another injection of glutaraldehyde-crosslinked polypeptide) and to remove immunolabeling one month after a single injection of polypeptide-protein contact complex. was adsorbed with BSA. Specific antibodies were then purified on a PSL2 binding column. Table 8 shows that antibody RF activity was adsorbed by the PSL2 binding column and eluted from it. showed that anti-RF antibodies in immune serum were directly induced by the peptide, but It is assumed that this is not induced indirectly through some pathway in the immune network. It shows.

Table 8 Adsorption and dissolution of anti-RF activity from PSL2 binding affinity column Peptide IgM-RF Sample 8 Pumin (BSA) (PSL 2) Sie original 24 1319 303 BSL & arrival 0 1214 250 85＾, Peptide adsorption 0 38 25° Dissolved U substance 12 1859 730 Eluate (IJJg/ml) 0 1616 3751, antiserum I section G section i! 1 (100■) was first adsorbed with a 3mjtBSA binding column (5■/sj gel). and then applied onto a 3 w I PSL2 binding column (1,6 μ/11 gel). It was. The effluent was collected after a 15 minute ink wash at room temperature (23°C). Wash the column thoroughly and adjust the bound substances to pH 2.5 with 0.1M glycine-HCj. eluted.

2. Unless otherwise specified, all samples with a concentration of 10rg/sj are as per Table 6(B). It was evaluated in the same manner as described above.

Furthermore, Table 9 shows that affinity purified anti-PSL2 antibodies strongly interacted with Sie & Glo. It reacts, but it shows that it reacts strongly with Pom.

Table 9 Reaction with human monoclonal IgM-RF resulting in affinity purified anti-PSL2 antibody. Responsiveness 1 Coating RF Sie Glo Pot Anti-PSL2 antibody 1136 1900 256 Antibody depletion 1z S 7 393 56 Connection difference 1179 1507. 2001, anti-PSL2 antibody (r9 release in !!8 1 g of antibody depletion (peptide adsorption amount 1ii in Table 8) at 2 gg/so l. reaction with various rjM-RF coated on microtiter plates. The response was evaluated. Absorbance was measured at 405 nm.

The absorbance at 22-405n was determined in room A (23°C) after a night of ink evacuation. It was measured.

3. The binding difference is (Aae% of antibody - A4* of immunoglobulin depleted of antibody) s).

14. Anti-polypeptide antibodies react with intact antibody molecules in their natural form During this study, the recesses were coated with modified 1. M-RF is an anti-peptide antibody was suspected of being responsible for the interaction with. In order to exclude this possibility, in the liquid phase Table 0 shows that IiM-RFSiθ was still recognized by anti-peptide antibodies. 10 is enzyme-conjugated 11 M-RFSie (AP-1g M-RF 5ie) , anti-PSL2 antibody was bound to pre-coated wells, but anti-PSL2 antibody This shows that depleted rabbit IgG does not bind.

Table 10 1g of natural M-RF Sie' (in liquid phase) is added to the pot coated in the cavity. Reacting with lipeptidically induced anti-idiotype antibodies ζ2.5 603. 22 1. 8 μg/l polypeptide-induced anti-idiotype antibody (fii release in Table 8) (1gG) and antibody depletion (1gG with peptide adsorption in Table 8) used for coating.

2. St M-RF Sie is alkaline phosphatide by glutaraldehyde. labeled with tase.

3. AP-11M-RF Sie of specified concentration (distributed in two ways in the recess) , plates were incubated for 3 hours at room temperature (23°C). After washing, remove the substrate. was added to the well and the absorbance at 405 nm was measured after 1 hour at room temperature (23°C). It was done.

Furthermore, as shown in FIG. -RFS4e binding was specifically inhibited by PSL2, but control PSH3 Not restrained. This indicates that the complete 11M-R'F to anti-PSL2 antibody Sie binding is due to its PSL2 epitope and not through its RF activity. This shows that it is not the case that the Taken together, these data suggest that anti-PS The L2 antibody is a PSL2-determined idiotype on a complete 1 gM-RFSie in its native form. This indicates that the sample reacted specifically with the sample.

v6 diagnosis The polypeptides described above may also be used in diagnostics to detect the presence of antigenic proteins and antibodies. Can be used as part of a composition.

The diagnostic reaction agent system embodying the present invention is characterized by its pallor compared to the amount of protein normally present. Useful for measuring the presence of increased amounts of white matter.

This system contains (al first reactant and -) second reactant in separate containers, both of which are Biologically active form, with an indicating group.

The first reactant is a synthetic polypeptide as described above, a combination of such polypeptides, The second reactant contains a synthetic polypeptide, including one of the conjugates made therefrom. or a polyamide containing the idiotype region of an antibody raised against the conjugate thereof. can be mentioned.

The idiotype-containing polyamide of the second reactant is a nearly complete antibody or its preparation. It can be a Fab or F(ab')x antibody fraction as described above. Directive group system and can be initially bound to either of the two reactants. or separate from them.

Mixing a predetermined amount of the first and second reactants in the presence of a predetermined amount of the body component to be evaluated. is the degree or amount of immune response so produced that results in an immune response. differs from the known immune response amount when an increased amount of The amount of immune response is , when an increased amount of pallor is present in a body composition compared to the normal amount reduced to

Another diagnostic system includes the aforementioned anti-idiotypic antibodies in biochemically active form and an indicator means. In this system, anti-idiotype antibodies or their Fab or F(ab')2 fractions reacts with the antigen in the sample to be evaluated, forming an immune reactant and determining its presence. The presence is signaled by the indicating means.

The system described above also works against antibodies of the same class and of the same species as the anti-idiotype antibody. as part of the indicating means. For example, anti-idea If otype antibodies are raised in rabbits, commercially available goat anti-rabbit antibodies can be used. Such second antibodies are conveniently labeled, e.g. conjugated to an enzyme. For example l! Contains Sl as a signal indicator.

A typical diagnostic agent system is enzyme-linked immunosorbent assay (ELISA) (in which The indicator group can be an enzyme such as horseradish that binds to the above-mentioned antibody or another antibody. oxidase), and radioimmunoassay (in which the indicator group is radioactive elements present in synthetic polypeptides or antibodies raised against them; For example 1! ) is mentioned.

■、Consideration゛ The antigen-binding site of an antibody is formed from the three-dimensional folding of the variable regions of the heavy and light chains. It will be done. Padla et al., Natia (Hature), New! 1io1. ．． 245.169 (1973), diversity in antibody specificity is due to variable region Derived from changes in the primary (vA form) amino acid sequence. -The following amino acid sequence is Define a three-dimensional folding pattern. Therefore, different active residues in the binding site Different "shaped" binding sites with different shapes are generated from different primary amino acid sequences. Inside the variable part There are three regions of extreme variability in the primary sequence of . These areas are hypervariable areas It is called. Capra et al., Proc, Nat.

Acad, Sci, USA, 71, (1974).

As described herein, the anti-idiotypic antibodies of the invention are directed against specific immunoglobulins. The corresponding polypeptide in the third hypervariable region of the pair corresponds to the sequence of the heavy chain of phosphorus. I was able to recognize and differentiate between individual idiotypes. The data shows that the idiotobes are mainly H. This further strengthens the generally accepted conclusion that ut is defined by ut. Also Regardless of whether LtI plays a role in the expression of specific idiotopes, For example, the individual idiotobes of j558 cannot be excluded from the Carson was not expressed by J558H1ji and L. et al., Proe, Natl, Acad, Sci, LISA, 1st, 235 (1 J558HIm reconfigured by inappropriate LIj as described in 973). Therefore, natural immunoglobulin or Fab or F(ab') In the * fragment, the L-H1l interaction is caused by the expression of a specific idiotope. It may be necessary to stabilize the heavy chain variable region in the required configuration for this purpose. stomach.

The present invention can be used to manipulate the immune response to treat or treat autoimmunity. can. Similarly, the present invention provides that the present invention can be used to reduce transplant rejection. Such uses are further described below.

The entry of antigenic substances is generally described in Ilansburg et al., J. Is. munol, AE, 1406 (197?): Ilriles et al., J. Exp, Med, 152-1151 (1980) and C. eney) et al., J. Immunol, supra 28.1885 (1982). , resulting in a polyclonal response to the antigen.

Polyclonal responses appear to occur for two reasons: First, antibodies or BwA Receptors on cells have precise specificity, but antibodies and B cell receptors Although it does not exactly conform to form 1, it reacts with relevant antigenic determinants in form 1. This ability is known as cross-reactivity. That is, certain The origin appears to fall within the range of cross-reactivity of some B cell clones. Therefore , several antibodies with different variable regions are produced against one antigenic determinant. Second, antigens are likely to have several antigenic determinants, and each of these determinants is appears to induce its own antibody response, thereby creating antibodies containing different variable regions. Ru.

In summary, the invasion of antigenic foreign substances leads to polyclonal antibody responses. These antibodies do not all share the same variable region, which is why they are directed against the antigen. The diversity of idiotypes expressed by these antibodies was investigated.

As previously mentioned, the third hypervariability of j558 and M104 bone marrow 11 proteins The generation of anti-idiotype antibodies against individual idiotypes found in It was desirable. Two different sets of synthetic polypeptides were studied. The first group is The first seven neighboring amino acids IA groups in the J segment and the three in the V segment This synthetic polypeptide, containing neighboring amino acids, is capable of eliciting an anti-polypeptide immune response. It was designed to be long enough to guarantee release.

The synthetic polypeptides were named hV3M and hV3J, M2O3 and J Corresponds to 558 myeloma protein.

The amino acid residue sequences selected for hV3M and hV3J polypeptides are Essential parts of the sequence encoded by the Jl gene segment common to lipeptides result in minutes. Therefore, much of the anti-polypeptide response is directed against this common sequence. It was possible.

This probably explains the ambiguity of antibody activities specific to different D segments. I accomplished it.

The second set of synthetic polypeptides used in this study were Lerner ) et al., Natia, Inc., 592 (1982). These combinations contain the minimum cognat@ sequence necessary to form the constant. The adult polypeptides were named 3MN and 3JN and were derived from M104 and J55B bones, respectively. Corresponds to myeloma proteinacea.

Antisera were raised against various synthetic polypeptides. Two of these antisera The ability to discriminate between myeloma proteins was determined.

Eight of the 12 antisera were able to distinguish between the two modified forms of myeloma operculum. (Figure 2), and these antibodies also contain natural proteins corresponding to the immunizing polypeptide. It showed a high level of specificity with respect to white matter. Six of the 12 antisera were directed against natural myeloma proteins. (i.e., non-denatured) and high levels of the protein corresponding to the immunizing agent. The specificity of the method was demonstrated.

In summary, at least half of the immunized animals have either native or denatured proteins. Distinguish between individual idiotypes of M2O3 and J55B proteins, regardless of whether they are I made an antibody that can do it. Therefore, these results are valid for a given specific idiotype. Anti-idiotypic antibodies specific for primary determinants are generated using synthetic polypeptides. i.e., the antibody is a specific antibody that corresponds to the immunizing polypeptide. The method differentiated the two protein questions. Furthermore, these anti-idiotype antibodies The ability to distinguish between idiotypes containing only 112 amino acid residues. have

Three of the antisera (numbers 5.8 and 10) were tested with respect to their target idiotype. It appears to have absolute specificity within empirical limits.

Other antisera that distinguish between the two myeloma proteins are related to their target idiotype. showed a high level of specificity. Shorter cogaate sequences and spacing The frequency of antisera with absolute specificity can be increased using This was carried out using the polypeptides 3■ and 3JN. Enough to make a reasonable comparison No animals were immunized.

Also, antibodies raised against the shorter recognition sequence polypeptides, 3MN and 3JN, The body generally uses synthetic polypeptides with longer recognition series, hV3M and hV3J. It was noted that the antibody showed a lower titer than the antibody raised against Tide (data omitted). various anti-blood antibodies against their corresponding immunizing polypeptides, which must be The binding site affinity of the supernatant was determined by Green et al., Ce11, 1st Eng. (1982) as described by . All polypeptides elicited antibodies that bound to the immunizing polypeptide. deer and by 3MN and 3JN.'

Elicited antibodies generally have lower titers, which is why the use of shorter conjugates It is not clear whether this is due to

Also, as described herein, human monoclonal 1. M-IFSi@ HE1 Synthetic polypeptides corresponding to the third hypervariable region of 1 also stimulate the production of anti-hypervariable region antibodies. used to induce Anti-idiotype antibodies are complete immunoglobulin M molecule (Sit) and its isolated chain, but other 1 gM paraprotein In addition, antibodies to 1 gM Binding was specifically inhibited by free polypeptide.

These results also demonstrate that for a given specificity, i.e., for a particular idiotypic antigenic determinant, specific anti-idiotypic antibodies that bind to can be elicited by synthetic polypeptides and that such anti-idiotypic antibodies have antibodies formed by known hypervariable regions. Demonstrates recognition of diotypic antigenic determinants.

One implementation of the present invention:! ! In a single antibody, its predefined specificity is It does not go beyond recognition of the private idiotype of the molecule.

However, in preferred embodiments, in order to induce the production of anti-idiotype antibodies, The use of synthetic polypeptides for pubic links or cross-reactive idiotypes was expanded to generate anti-idiotypic antibodies. Human monoclonal 1g Two main cross-reactive idiots of M-rheumatoid factor (lx M-RF) (i.e., Wa and Po). [Kunkel et al., J, εXp.

Med., U. 1.331 (1973)), the Wa group is monoclonal 1.331 (1973)). M - Contains 60% of RF, expression of Wa cross-reactive idiotype is reactive 1 g M -It seems to depend on the light chain of RF. [Kunkel et al., J. Exp. Med, 1 39.128 (1974)) and Andrews et al., supra. ].

Two cross-reactive (+) idiotype Ig M-RF (e.g. Sie and Glo), but two Wa cross-reactive (=) idiotypes (and For example, mouse monoclonal antibodies (intestinal abL7) that do not react with Lay and Paw) -109) was prepared. (Calson et al., Mo1.Ismun ol, 20-1 (1983)), and 5ab17-109 is Wa cross-reactive. Sexual (+) idiotype It reacted with M-RF LSI, but reacted with Hffl I didn't. Wa cross-reactive (+) idiotype Ig M-RF Lllil Comparison of the reported amino acid sequences of i. the synthetic polypeptide corresponding to that region. prepared and cross-reactive hypervariable regions to induce the production of anti-idiotype antibodies. used.

The above is intended to be illustrative of the invention and not limiting. (Numerous changes and modifications may be made without departing from the spirit and novel concept of the invention.) No limitations with respect to the specific antibodies, compositions and uses described herein are intended; It should also be understood that no assumptions should be made.

Engraving (no changes to the content) 1234 567 B $11011121314RF (Sit: pg/m1) OO,42105000,421050ya)b,) l, IgM-RF (Sit) '7 ioa! 1.　Glo　-L　jl To0;3’! BeW”-’ry%P punishment” [ng/ml10.5 5 50 5005000 Procedural amendment (formality) %formula% 2. Title of the invention: “Anti-idiotype antibodies induced by synthetic polypeptides” 3. Person who makes corrections゛ Relationship to the case: Applicant 4. Agent Address: 3-3-1 Marunouchi, Chiyoda-ku, Tokyo Telephone: 211-8741 5. Date of amendment order: January 21, 1985. 1! report □aiAyutayutan PC? /1Jsai! 10102l1 Gangcheon “1ha0”1. , (71υ5a4102116 Continuation of 5a @Int, CI,' Identification symbol: Office serial number 0 Author: Houten Richa Amed 0 shots bright person Cheno Posien Bee Amemon 0 shots clear person Vaughn John H Ame, Ru 0 shots brighter Lehner Richard A. Amend M Ming person Huong German Ametri o Originated by McMillan Seamus L Ameh - United States of America California 92075 Soranabinichi Four Avenue 558 Lee Ton Avenue 6512 California, California 92037 La Jolla Cal Dela Brata 7936 United States California G2037 La Jolla Rose Rough 50 United States California 92126 Sandy Hentz Dora Eve 78g8 United States, California, 92126, Sandy Bay, New Salem Te Russ 10857

Claims (15)

[Claims] 1. Approximately corresponds to the amino acid residue sequence of idiotypic antigenic determinants of immunoglobulins In a synthetic polypeptide having an amino acid residue sequence, the synthetic polypeptide has an amino acid residue sequence of , alone, as a polymer or as a conjugate of the polypeptide bound to a carrier. When injected into a host in an effective amount in a physiologically acceptable vehicle, the immunoglucose described above A compound that has the ability to induce the production of antibodies against the above antigenic determinants of Robulin. polypeptide. 2. Synthetic polypeptides contain at least about 6 amino acid residues but less than about 40 amino acid residues. A synthetic polypeptide according to claim 1 comprising amino acid residues. 3. According to claim 1, the synthetic polypeptide comprises from about 8 to about 20 amino acid residues. compliant synthetic polypeptides. 4. Written from left to right and from the amino terminus to the carboxy terminus, [There is an array]; [There is an array]; [There is an array]; [There is an array]; and [There is an array] (Here each amino acid residue in parentheses is an alternative to the immediately preceding amino acid residue. a claim containing a sequence of amino acid residues represented by a formula selected from the group consisting of Synthetic polypeptides according to scope 1. 5. Claim 1, wherein the immunoglobulin is human rheumatoid factor of class IgM. Synthetic polypeptides according to Section. 6. An amino acid that immunologically corresponds approximately to the amino acid sequence of the antigenic determinant of immunoglobulin. an effective amount of a synthetic polypeptide having a amino acid residue sequence and a physiologically acceptable diluent pathogens that have immunoglobulins with specific idiotypic determinants, including When the vaccine is introduced into the host, the Induces the production of antibodies in the host that immunoreacts with antigenic determinants and protects the host from infection. Vaccine where it can be given. 7. Synthetic polypeptides include keyhole limpet hemocyanin, incomplete Freund Keyhole limpet hemocyanin, alum, keyhole in the adjuvant of Limpet hemocyanin - alum adsorption, keyhole limpet hemocyanin - light Adsorption pertussis, edestin, thyroglobulin, tetanus toxoid, and incomplete A carrier selected from the group consisting of tetanus toxoid in whole Freund's adjuvant. Vaccine according to claim 6, which is combined with. 8. The amino acid sequences of idiotypic antigenic determinants of immunoglobulins have immunological implications. produced in an animal host against a synthetic polypeptide with a corresponding amino acid sequence. In the antibody, the antibody immunoreacts with the antigenic determinant to produce the immunoglobulin. Antibodies that have the ability to protect the host. 9. comprising the antibody of claim 8 in a biochemically active form, the antibody being evaluated. immunoreact with the mixed sample, the presence of which is signaled by the indicating means. The presence of immunoglobulin antigenic determinants that form the immune response A diagnostic system for evaluating the current situation. 10. further comprising an indicating means comprising a second antibody conjugated with an enzyme, the second antibody raised against antibodies of the same class and from the same species as the first-mentioned antibodies, and Signals an immune response by binding to the first-mentioned antibodies present in the reactant. and the signal is directed by the reaction of the bound enzyme with the added substrate. A diagnostic system according to claim 9. 11. said indicating means comprises a radioactive element bound to said antibody, said immunoreaction is A diagnostic thread according to claim 9, which causes precipitation of an immunoreactant containing the radioactive element. 12. (a) about 6 to about 6 immunologically corresponding approximately to the amino acid residue sequence of the antigenic determinant; A first reaction product containing a synthetic polypeptide containing a sequence of 40 amino acid residues in a physiologically active form. agent, provided that the polypeptide is conjugated to a carrier and used as a vaccine. When introduced into a host animal in an effective amount, it immunoreacts with the antigenic determinants and produces the immunoglobulins. being able to induce in the host the production of antibodies that protect the host from Brin; as well as (b) Biochemically activate the anti-idiotypic antibody that immunoreacts with the synthetic polypeptide described above. a second reactant containing in the sexual form; indicating the presence of an immune reaction between said first and second reactant; contained in separate containers with means to indicate; The first and second reactants are mixed in a predetermined amount in the presence of a predetermined amount of the body component to be evaluated. gives the amount of immune response signaled by the above-mentioned indicating means when The amount of immunoglobulin containing this antigenic determinant is already present in the body components. The idiotype of immunoglobulin in body components differs from the known immune response amount. Diagnostic system for assessing the presence of primary determinants. 13. (a) sufficient to induce the production of antibodies against antigenic determinants of immunoglobulins; administering a synthetic polypeptide to a host in an amount that is The amino acid residue sequence corresponds approximately to the idiotypic antigenic determinant of the immunoglobulin described above. have an amino acid residue sequence; and (b) recovering the anti-idiotype antibodies so produced; A method of forming an anti-idiotypic antibody, comprising the steps of: 14. The idiotypic antigenic determinant is a variable or hypervariable region of an immunoglobulin. A method according to claim 13. 15. (a) providing a synthetic polypeptide according to claim 1; (b) Regarding the presence of idiotypic antigenic determinants to which the polypeptide binds. mixing a predetermined amount of the sample to be evaluated with a predetermined amount of the polypeptide. ; (c) the polypeptide binds to idiotypic antigenic determinants present in the mixed sample; maintaining the mixture for a sufficient time to combine; and (d) a linkage between a synthetic polypeptide and an idiotypic antigenic determinant of an immunoglobulin; identification of the idiotypic antibodies in the immunoglobulins in the sample, including determining the amount of Methods for assessing the presence of primary determinants.