Idiotypic-anti-idiotypic regulation of antibody synthesis in rabbits.

Anti-ribonuclease antibodies (Ab1) of one rabbit were used to prepare in two other rabbits anti-idiotypic antibodies (Ab2) against them. Anti-idiotypic antibodies (Ab3) were prepared against Ab2 of one rabbit in two other rabbits that had the same phenotype as the former three for the allotypic a and b systems. After a rest period of 4 months, the latter two rabbits were immunized against ribonuclease. The antibodies produced (Ab1') crossreacted with the two anti-idiotypic sera (Ab2) against Ab1, showing an idiotypic similarity between anti-ribonuclease Ab1' and anti-ribonuclease Ab1. No such crossreaction was observed between the anti-idiotypic sera (Ab2) against Ab1 and any of the 12 unrelated antisera against ribonuclease that were tested. These results suggest (i) that rabbits, at least with the same allotypes, possess a closely related idiotypic repertoire; and (ii) that the immune system is a network of variable (V)-domains.     

Idiotypic cascades in cancer patients treated with monoclonal antibody CO17-1A.

We have previously shown that gastrointestinal cancer patients treated with monoclonal antibody CO17-1A (Ab1) developed anti-idiotypic antibodies (Ab2) to the Ab1. We now demonstrate that patients produce anti-anti-idiotypic antibodies (Ab3) to their autologous Ab2. Ab3 were demonstrated in culture supernatants of peripheral blood mononuclear cells from five Ab1-treated patients after stimulation of the cells with heterologous Ab2 that functionally mimicked the tumor antigen (Ag) defined by Ab1 and immunologically cross reacted with the patients' Ab2. Ab3 shared idiotopes with Ab1 and were Ab1-like in their binding specificities to tumor cells, Ag, and Ab2. Such antibodies were also elicited by stimulating cells with Ag. However, they were not produced by stimulating posttreatment mononuclear cells with control proteins or by stimulating pretreatment cells with either Ag or Ab2. Our results demonstrate idiotypic cascades in cancer patients treated with monoclonal antibody. Ag-specific Ab3 responses may underlie delayed clinical responses often observed in cancer patients treated with monoclonal antibodies of various specificities.     

Idiotypic analysis of monoclonal antibodies to poly(Glu60Ala30Tyr10).

Fifteen hybridoma anti-poly(Glu60Ala30Tyr10) (anti-GAT) antibodies were analyzed for the presence of a common set of idiotypic specificities associated with murine anti-GAT antibodies, termed CGAT idiotype, which are present on the anti-GAT antibodies of all mouse strains. Thirteen of these monoclonal anti-GAT antibodies expressed a major fraction of CGAT idiotypic specificities. However, the remaining fraction of CGAT idiotypic specificities were not detected in individual or pooled hybridoma anti-GAT antibodies. Anti-idiotypic antisera made against each of the 15 hybridoma anti-GAT antibodies preferentially bound homologous ligand and showed minimal binding activity to specifically purified serum anti-GAT antibodies. Furthermore, the diversity of the hybridoma anti-GAT antibodies was demonstrated by the presence of individual idiotypic specificities on each of the hybridoma anti-GAT antibodies. However, relatedness among some of the hybridoma antibodies was also apparent since idiotypic analysis revealed that some hybridoma anti-GAT antibodies shared cross-reactive idiotypic specificities not associated with CGAT idiotype. The genetic mechanisms which could account for the generation of such antibody diversity are discussed.     

Molecular characterization of monoclonal CRIA-positive anti-arsonate antibodies derived from idiotype-negative mice bearing a light chain polymorphism.

We have elicited anti-arsonate antibodies bearing the major cross-reactive idiotype (CRIA) in a double congenic idiotype-negative strain (C.C58.AL-20) bearing a light chain polymorphism that has previously been shown serologically not to complement idiotype-positive heavy chains. Using the idiotype cascade (Ab1-->Ab2-->Ab3-->-->Ab1'), CRIA-positive antibodies were raised and monoclonal antibodies were isolated and characterized serologically and by nucleotide sequence analysis. Two types of idiotype-positive anti-arsonate antibodies were generated in the C.C58.AL-20 strain. One group of hybridomas used the canonical VH1.8 heavy chain gene segment with V kappa 10 variant light chains. A second group used a VHGAM3.8 heavy chain with V kappa 10 variant light chains. This latter heavy-light pairing has been observed in CRIA-like responses previously in BALB/c mice after idiotypic manipulation (or rarely after antigen alone). These studies demonstrate the plasticity of the immune response when manipulated with idiotype reagents as well as its structural variability. Additionally, they provide important insights into the potentials of idiotype vaccines.     

Antiidiotypic antibodies raised against anti-prolactin (PRL) antibodies recognize the PRL receptor

Antiidiotypic antibodies capable of recognizing the PRL receptor have been raised against antibodies to ovine PRL (oPRL) and rat PRL (rPRL). Anti-oPRL or anti-rPRL antibodies were induced in rats or rabbits, respectively, and the immunoglobulin G (IgG) fractions were isolated by affinity chromatography on a Protein A-Sepharose 4B column. Specific anti-oPRL antibodies were purified on an oPRL-Sepharose 4B affinity column. The specific antibodies (0.5 mg/rabbit) in Freund's complete adjuvant were injected into rabbits at 2- to 3-week intervals for 3 months. Antiidiotypic antibodies against rat anti-oPRL specifically inhibited [125I]iodo-oPRL binding to the immunizing anti-oPRL antibody. Membrane binding of the antiidiotypic antibodies was determined by [125I]iodo-Protein A precipitation. It was found to be significantly higher toward membrane preparations rich in PRL receptors, such as liver membranes from pregnant mice or from estradiol-treated male rats or prostate membranes from adult rats. This membrane binding by the antiidiotypic antibody was competitively inhibited by the immunizing anti-oPRL antibody, suggesting that the idiotypic antibody may share common determinants with the PRL receptor. Recognition of the PRL receptor by the antiidiotypic antibodies was also assayed by indirect binding studies. After preincubation of the antiidiotypic antibodies with the membranes, the resulting complex was precipitated and the amount of free antiidiotypic antibody remaining in the supernatant estimated according to its ability to inhibit [125I]iodo-oPRL binding to anti-oPRL IgG. The lowest degree of inhibition of [125I]iodo-oPRL binding was achieved after incubation of the anti-idiotypic IgG with liver membranes from pregnant mice, while the inhibitory capacity was about 5-fold higher subsequent to parallel incubations with the same membranes, which had previously been heated for 30 min at 65 C, or with membranes of male rat liver, demonstrating a direct correlation between the binding of the antiidiotypic antibody to the membranes and the PRL receptor content of the membranes. Furthermore, significant and dose-dependent inhibition of [125I]iodo-oPRL binding to its receptors in various PRL receptor-rich membrane preparations from rats and rabbits was demonstrated with antiserum of rabbits immunized with rabbit anti-rPRL IgG. These results suggest that effective and specific anti-PRL receptor antibodies can be produced using the antiidiotypic antibody procedure, thus avoiding the necessity of isolating and purifying the PRL receptor itself.     

Gene repertoire of the anti-poly(Glu60Ala30Tyr10) (GAT) immune response: comparison of VH, V kappa, and D regions used by anti-GAT antibodies and monoclonal antibodies produced after anti-idiotypic immunization.

Eight monoclonal antibodies were selected from BALB/c mice immunized with two different monoclonal anti-idiotypic antibodies recognizing two discrete idiotopes characteristic of the anti-poly(Glu60Ala30Tyr10) (GAT) antibody response. These monoclonal antibodies were previously classified as Ab1 (anti-GAT-like) and Ab3 (anti-anti-idiotype) on the basis of expression of the public idiotypic specificity (p.GAT) studied with a xenogeneic serum, anti-GAT activity, and expression of various public idiotopes. All the heavy chain variable region (VH) sequences from Ab1 are nearly identical to the VH sequences of Ab1 anti-GAT monoclonal antibodies. The same type of results has been found with the Ab1 kappa light chain variable region (V kappa) sequences. Confirming our classification, Ab3 VH and V kappa sequences were found to be completely different from Ab1 VH and V kappa sequences. The Ab1 diversity (D) regions are different from one another and different from the D regions found on monoclonal anti-GAT antibodies but function similarly. These D regions are not simply derived from already described D genes. Finally, our results suggest that in the anti-GAT response VH and V kappa sequence are mainly responsible for idiotype expression.     

Shared idiotopes among antibodies encoded by heavy-chain variable region (VH) gene members of the J558 VH family as basis for cross-reactive regulation of clones with different antigen specificity.

A wide idiotype cross-reactivity was observed among six groups of monoclonal antibodies specific for arsonate and nitrophenyl haptens, hemagglutinin of PR8 and X31 influenza viruses, dextran, A48-idiotype, and a set of six monoclonal antibodies with unknown antigenic specificity. All of these antibodies are encoded by heavy-chain variable region (VH) genes belonging to the J558 VH family. This idiotypic cross-reactivity was determined by studying the binding of these antibodies to a panel of six monoclonal anti-idiotype antibodies, each one raised against a member of the six groups of monoclonal antibodies. The administration at birth of two such monoclonal anti-idiotype antibodies induced a long-lasting suppression not only of the corresponding idiotype but also of VH-related idiotypes with different antigenic specificities. These results suggest that the idiotypes encoded by VH genes that belong to the same VH gene family are interactive one with another. The possible physiological consequences of this immunochemical cross-reactivity are discussed.     

抗人膀胱癌抗独特型抗体的制备和鉴定

目的：制备抗人膀胱癌抗独特型抗体（Ab2)并进行体外实验和鉴定，探讨通过免疫效应治疗或预防膀胱癌术后复发的可能性。方法：提取膀胱癌抗原（Ag)，应用膀胱癌抗原通过杂交瘤技术制备抗人膀胱癌单克隆抗体（Ab1)，Ab1经胃蛋白酶水解制备Ab1的F（ab‘)2片优，以F（ab‘）2片段免疫新西兰白兔，其血清经凝胶纯化后获抗人膀胱癌抗独特型抗体（Ab2），采用ELISA等方法进行鉴别。结果：Ab2与Ab1呈阳性反应，与BALB／C小鼠γ球蛋白呈阴极反应；Ab2与Ag竞争结合Ab1；Ab2与Ag的结合。结论：Ab1是针对膀胱移行细胞癌的单克隆抗体，Ab2是具有膀胱癌抗原的抗独特型抗体。     

Production and characterization of human monoclonal antibodies against Schistosoma mansoni

We have produced a panel of human monoclonal antibodies (MoAb) from patients infected with Schistosoma mansoni in order to analyse more carefully the human immune response to this helminth infection. This study describes the production, characterization and analysis of these MoAbs. Briefly, peripheral blood mononuclear cells from chronically infected patients were (1) isolated and stimulated with parasite antigens in vitro, (2) positively selected for B-cells on anti-Ig columns, and (3) then transformed with Epstein-Barr virus (EBV). Once EBV cell lines were established, they were selected for anti-S. mansoni antibodies using an ELISA, cloned, retested and then fused with the mouse-human heteromyeloma SHM-D33. In this study, we describe five MoAbs which have different antigenic specificities for life-cycle stages based on ELISA to soluble crude antigen preparations, membrane immunofluorescence on whole intact organisms, and immunofluorescent staining of cryostat frozen sections. The importance of these reagents with regard to the human immune response to S. mansoni is currently being evaluated.     

Structural bases for public idiotypic specificities of monoclonal antibodies directed against poly(Glu60Ala30Tyr10) and poly(Glu60Ala40) random copolymers.

NH2-terminal amino acid sequences of heavy and light chains of seven poly(Glu60Ala30Tyr10) (GAT) specific hybridoma products derived from DBA/2 and (DBA/2 X BALB/c)F1 hybrid mice and those of BALB/B polyclonal antibodies have been determined over the first 40 residues. Comparison of these sequences with those of nine other GAT or poly(Glu60Ala40) (GA) specific hybridoma products previously reported allowed the following conclusions. (i) Sequences of hybridoma H and L chains are present in the pool of polyclonal antibodies. (ii) The public CGAT (or pGAT) idiotypic specificities are strictly confined to antibodies exhibiting limited heterogeneity with regard to both the variable heavy (VH) and the variable kappa (V kappa) sequences that may be accounted for by one and two germ-line genes, respectively. (iii) The public idiotypic specificities GA-1, expressed by some anti-GAT and most anti-GA antibodies, make use of the same (or similar) VH germ-line genes as the CGAT or pGAT antibodies but possess a distinctive V kappa sequence. (iv) Antibodies expressing neither of the alternative public specificities mentioned above appear to be more heterogeneous and express VH and V kappa sequences that were found to differ from the basic structures defining the CGAT (pGAT) or GA-1 correlates. It is concluded that CGAT (or pGAT) and GA-1 public idiotypic specificities are germ-line markers of both VH and V kappa regions, an observation in agreement with previously reported serological data.     

Idiotypic analysis of antibodies to poly(Glu60Ala30Tyr10): interstrain and interspecies idiotypic crossreactions.

A common idiotype was defined by a guinea pig anti-idiotypic antiserum made against D1.LP antibodies specific to the synthetic terpolymer poly(Glu60Ala30Tyr10), referred to as GAT. This idiotype was found in the anti-GAT antibodies of all individuals of all inbred strains of mice tested. Due to its common (C) occurrence in the anti-GAT antibodies of all mouse strains, it was termed the CGAT idiotype. The presence of CGAT idiotype in mice is independent of the expression of various Ig-1, H-2, or Vg genetic markers. In contrast, antibodies produced in response to the crossreactive poly(Glu60-Ala40), termed GA, bound to the GAT ligand but did not bear the CGAT idiotype. This suggests that GAT, although bearing GA determinants, preferentially stimulates clones of CGAT idiotype. Some of the CGAT idiotypic specificities were also identified in sera of WF inbred rats after immunization with GAT. However, no significant levels of CGAT idiotype were detected in LEW rats, F344 rats, guinea pigs, or rabbits, although measurable quantities of anti-GAT antibody were present.     

Immunisation of guinea-pigs with circulating immune complexes from patients with rheumatoid arthritis.

Sixteen guinea-pigs were immunised with immune complexes isolated from serum of nine patients with rheumatoid arthritis. The resulting antisera were analysed by radioimmunoassays. All guinea-pig sera were extensively absorbed with normal human serum. After this absorption eight guinea-pig sera contained antibodies specific for immune complexes isolated from the sera of three patients. One of these antisera reacted not only with immune complexes (and serum) from the corresponding patient but also with immune complexes (and sera) from other patients with rheumatoid arthritis. The antigen(s) to which the guinea-pig antibodies were directed sedimented as IgM, and they bound to IgG Sepharose. Therefore the guinea-pig sera were absorbed with IgM-rheumatoid factors isolated from the serum of the corresponding patient. After this absorption, the guinea-pig sera had lost their reactivity with immune complexes. We conclude that these antisera did not detect an exogenous antigen in immune complexes from patients with rheumatoid arthritis. The positive reactions found were due to antibodies specific for (idiotypic?) antigenic determinants on IgM-rheumatoid factors.     

Anti-idiotype monoclonal antibody elicits broadly neutralizing anti-gp120 antibodies in monkeys.

Murine monoclonal antibodies (mAbs) were raised against human, polyclonal, anti-gp120 antibodies (Ab1) and were selected for binding to broadly neutralizing anti-gp120 antibodies in sera positive for human immunodeficiency virus (HIV). One anti-idiotype mAb (Ab2), 3C9, was found to be specific for human anti-gp120 antibodies directed against an epitope around the conserved CD4 attachment site of gp120. The 3C9 reactive human anti-gp120 antibodies (3C9+ Ab) neutralized MN, IIIB, RF, and four primary isolates of HIV type 1 (HIV-1). Cynomolgus monkeys were immunized with 3C9 in adjuvant to test whether this anti-idiotype mAb could induce neutralizing anti-gp120 antibodies. The results show that purified anti-anti-idiotype antibodies (Ab3) from 3C9 immune sera bind to an epitope around the CD4 attachment site of gp120SF and gp120IIIB. Furthermore, purified gp120-specific Ab3 neutralize MN, IIIB, and RF isolates. These results demonstrate that primates immunized with an anti-idiotype mAb produce broadly neutralizing anti-HIV-1 antibodies. Since this anti-idiotype mAb was selected by identifying a clonotypic marker, its biological activity can be explained as the results of clonotypic B-cell stimulation.     

Anti-idiotypic antibodies in autoimmune diseases

The variable region (V region) of an antibody, the part of the molecule that binds to antigen, is itself antigenic and can elicit anti-V region antibodies when injected into animals of a different or even of the same species. The antigens of the V region constitute its idiotype, and they are defined by anti-idiotypic antibodies in polyclonal antisera or by monoclonal anti-idiotypic antibodies. An idiotype actually consists of multiple antigenic determinants, each of which is an idiotope. The antigenic determinants or idiotopes can reside in the heavy chain component of the V region, in its light chain component, or they may consist of a surface made up of parts of both chains. The idiotype of an antibody is, therefore, a way to describe by serological means the variable region of an antibody molecule. Originally, idiotypes were defined by heterologous antisera made, for example, by immunizing a rabbit with a monoclonal human myeloma protein. After extensive absorption with normal human immunoglobulins, such antisera were found to bind only to the V region of the immunizing myeloma immunoglobulin. Thus, idiotypes were originally thought to be unique markers of individual antibodies (hence their name). However, it is now known that different antibodies can share the same, or similar, idiotype. Even antibodies with different antigen-binding specificities can share the same idiotype if they use the same heavy or light chain in forming their V regions. Such antibodies constitute parallel sets. Thus, idiotypes can be: private (confined to a particular immunoglobulin molecule), public (shared by different antibodies), or cross-reactive (different idiotypes containing similar antigenic structures).(ABSTRACT TRUNCATED AT 250 WORDS)     

Murine monoclonal anti-DNA antibodies with an absolute specificity for DNA have a large amount of idiotypic diversity.

The clonal heterogeneity of nine monoclonal antibodies with absolute specificities for deoxyribonucleic acid (DNA) was analyzed. These monoclonal anti-DNA antibodies were generated in three different fusion experiments using autoimmune (NZB X NZW)F1 mouse spleen cells. Isoelectric focusing analyses demonstrated different isoelectric points within the IgG2a and IgG2b subclasses. Three anti-idiotypic antisera were prepared (one in a rabbit and two in mice) against two monoclonal anti-DNA antibodies. These antisera detected idiotypic determinants uniquely associated with homologous hybridoma anti-DNA antibodies. Two of these idiotypes could be detected at low levels in the sera of (NZB X NZW)F1 mice. Anti-PME77 idiotypic antiserum had no effect in vitro on the total binding capacity of (NZB X NZW)F1 sera. Taken together these results demonstrate that, in (NZB X NZW)F1 mice, the anti-DNA antibody repertoire contains molecules that show similar antigen binding characteristics but are not structurally uniform.     

Evidence for a functional idiotypic network among natural antibodies in normal mice.

We monitored in normal adult BALB/c mice the serum concentrations of four natural IgM antibodies, two of which show idiotypic complementarity in in vitro assays. In each individual, serum concentration of all four idiotypes were found to fluctuate in complex dynamical patterns with low correlation. The spectral power of some such patterns was found to be compatible with the existence of a chaotic regime. Groups of normal adult mice were injected intravenously with low (10 ng) or moderate (10 micrograms) doses of either of the two complementary idiotypes in saline. This treatment resulted in a pronounced inhibition of the fluctuation in the serum concentration of both complementary idiotypes for periods up to 3 months. Such compensations were not detected for the two unrelated natural idiotypes and were specifically induced, for they did not occur following the injection of unrelated antibodies. These results indicate the functional operation of an idiotypic network among natural antibodies.     

Epstein-Barr virus/complement fragment C3d receptor (CR2) reacts with p53, a cellular antioncogene-encoded membrane phosphoprotein: detection by polyclonal anti-idiotypic anti-CR2 antibodies.

Epstein-Barr virus and the C3d fragment of the third component of complement are specific extracellular ligands for complement receptor type 2 (CR2). However, intracellular proteins that react specifically with CR2 and are involved in post-membrane signals remain unknown. We recently prepared polyclonal anti-idiotypic anti-CR2 antibodies (Ab2) by using the highly purified CR2 molecule as original immunogen. We showed that Ab2 contained anti-idiotypic specificities that mimicked extracellular domains of CR2 and detected two distinct binding sites on CR2 for its specific extracellular ligands, Epstein-Barr virus and C3d. We postulated that Ab2 might also contain specificities that could mimic intracellular domains of CR2. Here we report that Ab2, which did not react with Raji B-lymphoma cell surface components, detected specifically, among all components solubilized from Raji cell membranes, a single intracellular membrane protein of apparent molecular mass of 53 kDa. This protein was identified as the p53 cellular antioncogene-encoded membrane phosphoprotein by analyzing its antigenic properties with Pab1801, a monoclonal anti-p53 antibody, and by comparing its biochemical properties with those of p53. Additionally, solubilized and purified CR2 bound to solubilized p53 immobilized on Pab1801-Sepharose. p53, like CR2, was localized only in purified plasma membranes and nuclei of Raji cells. These data suggest strongly that p53, a cellular antioncogene-encoded phosphoprotein, reacted specifically with CR2 in Raji membranes. This interaction may represent one of the important steps through which CR2 could be involved in human B-lymphocyte proliferation and transformation.     

Specificities of antibodies to human growth hormone (hGH) in patients treated with hGH: longitudinal study and comparison with the specificities of animal antisera

The specificities of human and animal antibodies (Abs) against human GH (hGH) were analyzed using competition experiments with five monoclonal antibodies to hGH (MAbs). The results indicate that 1) the Abs produced by patients receiving long term hGH therapy as well as Abs of goat, rabbit, and mouse origin recognized the various hGH epitopes defined by the MAbs; 2) the proportion of each Ab population, with a given specificity, differed markedly in different patients and also with time in the same patient; 3) the titer of certain Ab populations was very low in some patients, and 4) polyclonal mouse Abs and some human Abs enhanced the binding of [125I] hGH to insolubilized MAbs.     

Monoclonal antibodies specific for globin chains

Six monoclonal antibodies specific for human globin chains are described. They are produced by stable clones obtained by raising hybridomas using cells of mice immunized with either adult or fetal hemoglobin. Characterization of the antibodies included testing against tetrameric human and other animal hemoglobins, isolated hemoglobin chains, and when indicated, cyanogen bromide fragments. Monoclonals 16- 2 and 37-8 are beta-chain specific. Antibody 31-2 recognizes an antigenic determinant common to the alpha and beta subunits. Monoclonal 30-3 recognizes determinants best expressed in the alpha 2 beta 2 tetramer. Antibody 45-1 recognizes a determinant common to beta and gamma subunits, while antibody 51-7 is gamma-chain specific. None of the monoclonal antibodies recognizes mouse hemoglobin, and they display significant differences in binding to hemoglobins of various species. The species-specific reactions and the knowledge of the primary structures of globins allowed deductions about the antigenic sites recognized by two of the monoclonals (16-2 and 45-1). These antihemoglobin monoclonal antibodies will provide useful probes for studying hemoglobin expression in vivo and in vitro.     

Cytotoxicity of anti-fab antibodies against b lymphocytes in rheumatoid arthritis

Anti-Fab antibody titers were positive in 70% of rheumatoid arthritis patients (54 of 77) and 3% (1 of 35) of healthy donors. Their specificity was examined by inhibition of the radioimmunoassay for anti-Fab antibodies, which demonstrated that they are against the Fd region of the intact immunoglobulin molecule. In addition, anti-Fab antibodies have broad specificities for IgG antigens, shown by inhibition with myeloma IgG. They are cytotoxic against B (20% of B cells from most healthy donors) but not T lymphocytes, with cytotoxicity greater at 5°C than 37°C. These studies show that the anti-Fab antibodies interact with allotypic or idiotypic determinants on subpopulations of B lymphocyte cell surface antigens.