Shared idiotope on monoclonal anti-Ia.7 antibodies reactive with determinants in a structural domain of the I-E molecules

In previous studies, heterologous anti-idiotypic (anti-Id) antisera against the C3H.SW 14-4-4S or the A.TH 41.A anti-Ia.7 monoclonal antibodies (mAb) were shown to identify an interstrain cross-reactive idiotypic specificity (IdX.Ia.7) expressed on monoclonal or conventional anti-Ia.7 alloantibodies. The objective of the present investigation was to characterize further this IdX at the idiotopic level. To this end, 11 hybridomas producing IgG1, IgG2a, or IgM anti-Id mAb were derived from a rat immunized with a mixture of 10 A.TH or A.BY anti-Ia.7 mAb. The specificity of the latter anti-Id mAb was determined by direct Id binding radioimmunoassay (RIA) with the use of a panel of 52 anti-Ia mAb derived from hybridomas produced in various inbred mouse strains. These rat anti-Id mAb recognized idiotopes expressed on i) all anti-Ia.7 mAb against determinants in the topographic domain I of the I-Ek molecule but not on 18 other anti-I-Ek mAb directed at epitopes in domains II or III; ii) three of 19 anti-I-Ak mAb; and iii) one A.TL-derived anti-I-As mAb. Competitive Id binding assays revealed that among the 14 IdX+ anti-Ia.7 mAb, one (81.B) was bound to a lesser extent by various rat anti-Id mAb, suggesting that heterogeneity probably exists in this antibody family. By contrast, two isologous (B10.S(7R)) anti-Id mAb to the IdX.Ia.7+ mAb 41.A displayed a specificity restricted to 41.A individual idiotopes (IdI). Rat anti-IdX.Ia.7 and mouse anti-41.A IdI mAb inhibited the binding of 125I-labeled mAb 41.A to CBA spleen cells. These two sets of mAb bound in a noncompetitive fashion to mAb 41.A-coated plates, indicating that their corresponding public or private idiotopes were spatially distinct. These data may have implications for in vivo manipulations of anti-Ia immune responses.     

A public idiotypic determinant is present on spontaneous cationic IgG antibodies to DNA from mice of unrelated lupus-prone strains

A monoclonal anti-idiotypic antibody (anti-Id) to a public idiotype (Id) present on spontaneous IgG antibodies to DNA from NZB/NZW F1 mice recognized similar determinants on polyclonal and monoclonal IgG anti-DNA antibodies from mice of the unrelated MRL/lpr and BXSB strains. Incubation of the anti-Id with four of five monoclonal Id in solid phase inhibited their ability to bind DNA; however, different Id+ antibodies recognized different epitopes within the DNA molecule. Therefore, the public Id was located close to the antigen-binding regions but did not comprise all of those regions. Analysis of multiple polyclonal and monoclonal antibodies to DNA showed the Id on all subclasses of IgG. However, antibodies bearing the Id carried a neutral or cationic charge (10 of 10 monoclonals with pI greater than 7 were Id+); the presence of the Id on anionic IgG (pI less than or equal to 7) was infrequent (one of 21 serums, one of eight monoclonal antibodies). Therefore, IgG autoantibodies to DNA are constructed from closely related public idiotypes in several mouse strains that spontaneously develop lupus, and that Id is restricted to antibodies with a pI of 7 or greater.     

Autoimmunity induced by HgCl2 in Brown-Norway rats. II. Monoclonal antibodies sharing specificities and idiotypes with mouse natural monoclonal antibodies

Spleen cells derived from BN rats receiving HgCl2 were fused with the nonsecreting rat myeloma cell line IR983F. We screened 59 supernatants from immunoglobulin-secreting hybrids for antibody activity against actin, tubulin, autologous and heterologous myosin, myoglobin, dsDNA, peroxidase, and the haptens TNP, NIP, NNP, and NBrP. Six monoclonal antibodies (mAb) were found to react with antigen(s) of the panel. At least three groups of antibody specificities were identified: clones reacting with TNP (1 IgM, 1 IgE); clones reacting with horseradish peroxidase (1 IgM); and clones possessing widespread reactivity for several antigens as found for mouse natural autoantibodies (2 IgM, 1 IgE). We also analyzed the idiotypic (Id) determinants of the 59 mAb by using anti-Id antibodies described elsewhere prepared in rabbits against the BALB/c D23 natural monoclonal autoantibody and recognizing a BALB/c recurrent Id (Id D23) of natural polyspecific autoantibodies. We found that all rat mAb that possessed widespread reactivities bore this Id. We performed similar studies in sera from normal and mercury-stimulated rats. The results indicate a role for HgCl2 in the stimulation of natural antibodies producing cells and the existence of interspecies cross-reactive Id among mouse and rat natural antibodies.     

Paratope- and framework-related cross-reactive idiotopes on anti-acetylcholine receptor antibodies

Cross-reactive idiotopes are a possible target for therapeutical interventions in autoimmune diseases. To investigate their role in the pathogenesis of experimental autoimmune myasthenia gravis (EAMG) we analyzed the Id of rat anti-AChR mAb 6, 35, 61, 65 and a control myeloma protein IR27. Anti-Id 6, 35, 61, 65 bound in a direct binding assay with various affinity to all rat anti-AChR mAb that were tested. Anti-Id IR27 recognized none of the anti-AChR mAb. The specificity of these crossreactions was confirmed by inhibition studies with anti-AChR mAb and two control rat myeloma proteins (IR27 and IR241). In addition, the Id expression on mAb D6, a mouse anti-human AChR mAb was recognized by anti-Id 6, 35, and 65. Anti-Id, except anti-Id IR27, bound to affinity purified IgG from the sera of rats with EAMG, but not to preimmune Lewis IgG. These results suggest extensive sharing of idiotopes among anti-AChR mAb, which are also present in EAMG serum. Anti-AChR mAb against the main immunogenic region (6, 35, 65) from different rat strains, shared at least one paratope-related cross-reactive idiotopes. In the view of the fact that anti-main immunogenic region antibodies might form a predominant fraction of the polyclonal response against AChR, it is conceivable that an anti-Id recognizing these antibodies could have therapeutical applications as for example a selective immune absorbent or in immunotoxin therapy.     

An idiotype shared by monoclonal antibodies to different peptides of human myelin basic protein.

A mAb of the IgG1/kappa isotype was raised against human myelin basic protein (MBP) peptide acetyl 1-9. This mAb, termed F23, reacted with human MBP and human MBP peptides acetyl 1-9, 1-14, and 1-44, but not with MBP peptides 10-19, 80-89, or 45-89. According to the guidelines of the molecular recognition theory, a complementary peptide to human MBP peptide 1-9 was synthesized and used to raise murine mAb with anti-Id activity. Two mAb anti-Id, F25F7 and F25C8, both of the IgM/kappa isotype, were selected for further study. These anti-Id reacted with F23, the mAb for which they were selected, and also reacted with another mAb, which was of the IgG1/kappa isotype and was raised to human MBP peptide 80-89. There was no reaction with another control mAb of the IgG1/kappa isotype or murine myeloma IgG1. By immunoblotting techniques, it was demonstrated that the Id on each of the mAbs to MBP peptides was located on the kappa L chain but also could be recognized in nonreduced IgG. The cross-reactive anti-Id suppressed antibody secretion of Id-producing hybridoma cells in an Id-specific manner, and kinetic studies suggest an intracellular mechanism for the suppression. These cross-reactive Id among antibodies to different MBP peptides imply that the same V region genes of kappa L chains are involved in the selection of antibodies to an autoantigen, like MBP, and may play a role in the modulation of immune responses against MBP in certain inflammatory demyelinating diseases.     

Idiotypic and antiidiotypic T and B lymphocytes in myasthenia gravis.

The prevalence of Id and anti-Id T and B cells as measured by their reactivities with two human mAb, one antiacetylcholine receptor mAb and one anti-Id mAb, was studied in 38 patients with myasthenia gravis and in 27 healthy individuals. Id and anti-Id T cells were estimated by enumerating the numbers of cells secreting IFN-gamma in response to 10 pg/ml of the human mAb. T cell stimulation, measured as numbers of IFN-gamma-secreting cells that exceeded the mean + 2 SD of controls, was induced by the Id mAb in 78.9% of the patients and in 7.4% of the controls, whereas the anti-Id mAb-stimulated T cells in 55.3% of the patients and in 3.7% of the controls. The mean value of the Id and anti-Id-reactive T cells in the patients was 18.3/10(5) and 10.1/10(5) PBMC, respectively. B cells secreting IgM antibodies binding to the human mAb were increased in patients with myasthenia gravis compared to healthy controls. Seventy-five percent of the patients and 12% of the controls had B cells secreting IgM antibodies binding to the Id mAb, although 89% of the patients and 16% of the controls had B cells secreting IgM antibodies binding to the anti-Id mAb. The mean value of B cells secreting IgM antibodies binding to Id or anti-Id mAb in the patients were 7.4 cells/10(6) and 5.5 cells/10(6) PBMC, respectively. We conclude that Id and anti-Id T and B cells are present in myasthenia gravis. These methods allow a quantitative estimation of T and B cells with defined specificities and thus a way of mapping the repertoire of lymphocytes.     

Suppression of murine lupus nephritis by administration of an anti-idiotypic antibody to anti-DNA

The suppression of pathogenic antibodies to DNA in NZB/NZW f1 female mice was achieved by repeated inoculation of the mice with a monoclonal anti-idiotypic antibody (anti-Id). The anti-Id, an IgG1, kappa, was directed against a major cross-reactive idiotype (Id) on NZB/NZW IgG antibodies to DNA. One hundred micrograms of the anti-Id were inoculated i.p. every 2 wk, beginning at 6 wk of age (nondiseased mice--no circulating anti-DNA or proteinuria) or 20 wk of age (diseased mice--all with circulating anti-DNA, one-third with proteinuria). As controls, littermates received an IgG, kappa non-DNA-binding myeloma or no treatment. In the young mice, nephritis and anti-DNA antibodies appeared at the same time in all groups, and their circulating antibodies to DNA did not bear the target Id. In the older (20-wk-old) mice, survival was significantly prolonged because of delay in the onset of nephritis; the total quantities of antibodies to DNA were diminished, and the target Id, initially present on circulating IgG, was deleted. These benefits were transient; the suppression of antibodies was followed by the appearance of large quantities of anti-DNA that did not bear the major Id. Therefore, although administration of anti-Id was effective in reducing an undesirable antibody response after the target Id was present on circulating antibodies, the benefits were limited, probably by Id "switch" or by increased synthesis of pathogenic antibodies bearing a minor Id.     

Xenogeneic antibodies with apparent public idiotypic specificity for anti-Ia.7 antibodies are directed in part against V kappa 21D and E subgroup marker

Public idiotypes (IdX) expressed on monoclonal antibodies (mAb) against a monomorphic alpha-chain determinant of the I-E molecule (Ia.7 epitope cluster I) have been studied by using xenogeneic anti-Id reagents derived from pig, rabbit, and rat. IdX+ anti-Ia.7 mAb were recently demonstrated to be structurally related by a high frequency expression of the V kappa 21E light chain subgroup. This raised the question of whether V region determinants of the IdX were related to V kappa 21E sequences or whether they were unique to hypervariable regions of Ia.7 binding antibodies. To clarify this question, the possible association between the expression of the public Id (IdX(s)Ia.7) and the presence of V kappa sequences (V kappa 21E and/or J kappa segment) was examined. The reactivity of the anti-Id reagents with a random panel of 28 myeloma products (each containing a light chain from one of the different V kappa 21 subgroups) was studied by assaying the ability of these mAb to inhibit the binding between the anti-Id and anti-Ia.7 mAb. This analysis demonstrates that what has previously been defined as IdX Ia.7 includes determinants shared by V kappa 21E and V kappa 21D light chain V regions. The structures recognized are expressed irrespective of the J kappa segment. In addition, this study demonstrates interspecies variation in immune responses to such V kappa 21E antigenic determinants. Additional IdX components are found on anti-Ia.7 mAb but not on other V kappa 21E or D proteins. Thus V region subgroup considerations have crucial implications for Id characterization. In addition, this work describes the first division of the V kappa 21 subgroup into component parts by a mAb.     

Anti-peptide antibodies detect a lupus-related interspecies idiotype that maps to H chain CDR2.

Antibodies to the small nucleoprotein Sm occur spontaneously in human and murine systemic lupus erythematosus. Human and mouse monoclonal anti-Sm autoantibodies designated 4B4 and Y2 share an idiotype (Id) determinant located on the Ig H chain. To understand the molecular basis of this cross-reactivity, the VH regions of both antibodies were sequenced and analyzed for homology. The antibodies showed only 49.6% homology. The second complementary determining region (CDR2) was the most likely candidate for the Id site. To investigate this possibility, rabbit antiserum was made against a peptide corresponding to the CDR2 of 4B4. This antiserum was specific for the immunizing peptide and reacted weakly to a peptide corresponding to the CDR2 of Y2. Anti-CDR2 antibody bound to 4B4 and Y2 but not to other human and mouse mAb. Binding was directed at the H chain when analyzed by Western blots. Anti-CDR2 antibody blocked anti-Id antibody binding to 4B4 and Y2 by 58% and 24%, respectively. These studies suggest that this interspecies Id maps to the H chain CDR2 and that a conserved Id can occur within molecules that are otherwise radically different.     

A conserved anti-DNA antibody idiotype associated with nephritis in murine and human systemic lupus erythematosus

In order to identify unique structural features of pathogenic autoantibodies to DNA in SLE, a murine anti-anti-DNA (anti-Id) mAb (mAb 1C7) was produced in response to immunization of lupus mice with a syngeneic anti-DNA mAb (mAb 3E10). Immunization of lupus mice with mAb 3E10 inhibited production of native anti-DNA antibodies, suppressed development of lupus kidney disease (nephritis), and induced production of anti-anti-DNA (anti-Id) antibodies. mAb 1C7 bound F(ab')2 fragments of mAb 3E10, and it bound other murine anti-DNA mAb, but not murine mAb or polyclonal serum antibodies unreactive with DNA. Moreover, binding of mAb 1C7 anti-Id to mAb 3E10 was inhibited by DNA, suggesting anti-Id binding within or near the binding site for DNA. Furthermore, mAb 1C7 bound serum IgG immunoglobulins from 9/12 patients with lupus nephritis and serum anti-DNA antibodies compared to only 3/12 SLE patients with comparable serum levels of anti-DNA antibodies, but without nephritis (p = 0.04), and only 1/53 SLE patients without serum anti-DNA antibodies, 0/49 patients with rheumatoid arthritis, and 1/47 healthy subjects (p less than 0.001). These results provide evidence that mAb 1C7 identifies a conserved Id associated with anti-DNA antibodies in murine and human SLE and may be useful as a structural probe to characterize pathogenic anti-DNA antibodies in SLE.     

Anti-idiotypic antibodies recognizing stable epitopes limit the emergence of idiotype variants in a murine B cell lymphoma.

The emergence of Id variants is a major escape mechanism from anti-Id therapy of human B cell malignancies and of the murine B cell lymphoma 38C13. To determine what impact the epitope specificity of anti-Id antibodies has on the prevention of emergence of such Id variants in the 38C13 lymphoma, anti-Id mAb of varying epitope specificity for the Id of 38C13 tumor cells were produced and studied. Some antibodies, produced by immunizing mice with both the wild-type 38C13 IgM and variant IgM, cross-reacted with wild-type 38C13 IgM and with all four members of a panel of variant IgM. These anti-Id did not react with separated 38C13 IgM H or L chains by Western blot, but did react with the cytoplasmic H chain of the surface Ig- variant cell line T2D that expresses the same H chain as wild-type 38C13 in its cytoplasm but does not express any associated L chain. In contrast, anti-Id of narrower specificity did not react with this H chain. This indicated that the broadly cross-reactive antibodies recognized a stable epitope on 38C13 H chain. When a broadly cross-reactive antibody MS11G6 was compared to S1C5, an antibody of narrower specificity, MS11G6, was superior at preventing tumor growth in mice inoculated with 38C13 cells. Moreover, no surface Ig+ variants emerged in escaping tumors in the MS11G6-treated group, whereas such variants were common in the S1C5 treated group. Both anti-Id were of equal efficacy in eliminating wild-type 38C13 cells by using 38C13 cells in tumor inoculums that had just been cloned in vitro, but MS11G6 was also capable of preventing the growth of several surface Ig+ variant cell lines in vivo. We conclude that anti-Id recognizing more stable Id determinants can limit the emergence of Id variants and therefore be more effective therapeutic agents. This finding is of additional importance as additional in vivo and immunophenotypic studies demonstrated that the generation of Id variants was an ongoing process both in cloned parental 38C13 cells and its variants.     

VH-related idiotopes detected by site-directed mutagenesis. A study induced by the failure to find CD4 anti-idiotypic antibodies mimicking the cellular receptor of HIV.

The function of the CD4 cell surface protein as coreceptor on T helper lymphocytes and as receptor for HIV makes this glycoprotein a prime target for an immune intervention with mAb. A detailed understanding of the structural determinants on the therapeutic CD4 mAb that are involved in Ag binding or are recognized by anti-idiotypic mAb (anti-Id) may be important for designing antibodies with optimal therapeutic efficacy. Seven anti-Id raised against the CD4 mAb M-T310 were selected from a large panel with the intention to obtain CD4 mimicking structures with specificity for HIV gp120. The selected anti-Id did not react with other CD4-specific mAb cross-blocking M-T310. Among these, mAb M-T404, although having the same L chain as M-T310 and a VH region sequence differing only at 14 amino acid positions, was not recognized by the anti-Id. M-T310 H chain complexed with the J558L L chain reacted with all anti-Id, thus demonstrating that the recognized idiotopes are located within the VH region. To identify the idiotopes of M-T310 seen by the anti-Id, variants of M-T404 containing one or more of the M-T310-derived substitutions were generated by oligonucleotide-directed mutagenesis. The reactivity pattern of the mutant proteins with the anti-Id demonstrated that the idiotopes reside within the complementarity determining region (CDR) 2 and CDR3 loops of the VH region. A major idiotope was defined by a single amino acid in CDR2 that was recognized by three anti-Id, whereas the four other anti-Id reacted with determinants of CDR3. Although the performed amino acid substitutions did influence the Id recognition, Ag binding was not significantly affected, suggesting that none of the anti-Id can be considered as a mimicry of the CD4 Ag.     

Idiotope mapping on the variable region of an antibody clonotype produced by normal (nonmalignant) human B cells

Human anti-N-acetyl-D-glucosamine (GlcNAc) antibodies were prepared by affinity chromatography from serum of a healthy donor (MSS). They were heterogeneous but contained a unique antibody clonotype (1A) representing 7% of all anti-GlcNAc antibodies. Out of a series of monoclonal anti-idiotopic antibodies (anti-Id mAb), we identified five antibodies that bound to clonotype 1A as shown by isoelectric focusing and Western blotting. Two of them were specific for clonotype 1A (10F59 and 13F15), thus indicating its clonal origin. However, three anti-Id mAb (16F433, 16F539, and 16F812) bound to various additional portions of anti-GlcNAc antibodies of donor MSS. With the exception of one mAb, all anti-Id mAb have very similar relative affinities to clonotype 1A, so results from competition experiments between the different antibodies and between each antibody and antigen should reveal spatial relationships between the corresponding Id and between each Id and the antigen-combining site. The results show a consistent topography of Id on the V-region of clonotype 1A. Id 59, 812, and 433 were found to be arranged in one cluster (cluster I), whereas Id 15 and 539 belonged to a second cluster (cluster II). Cluster I resides completely in the antigen-combining site, whereas only Id 15 of cluster II weakly overlaps with the binding site. Our study demonstrates an analysis of spatial relationships of Id expressed on a human antibody clonotype. To our knowledge, this is the first demonstration of Id mapping on antibodies produced by a normal (nonmalignant) B cell clone that should be accessible to regulatory signals. Such analysis may contribute to a more detailed characterization of anti-Id mAb, and may provide additional information for a better understanding of their immunoregulatory effects.     

Human immune response to group A streptococcal carbohydrate (A-CHO). II. Antigen-independent stimulation of IgM anti-A-CHO production in purified B cells by a monoclonal anti-idiotopic antibody

The paper describes the induction by a monoclonal anti-idiotopic antibody (anti-Id mAb) of specific antibody production to group A streptococcal carbohydrate (A-CHO) in purified human B cells of several unrelated individuals. The anti-Id mAb, designated 16F498 (anti-Id498), recognizes a recurrent idiotope (Id 498) associated with the combining site of human antibodies to N-acetyl-D-glucosamine (GlcNAc), the immunodominant group of A-CHO. Id498 is expressed on IgM anti-GlcNAc antibodies but does not occur on IgG antibodies with the same specificity. It occurs also on a minor population of IgM antibodies without specificity for A-CHO. Id498 was found in 19 of 27 sera from unselected healthy donors and thus seems to be frequently expressed within the adult B cell repertoire. The in vitro induction of anti-A-CHO antibodies was analyzed in human B cells extensively depleted for T cells. Specific antibody secretion required cross-linked anti-Id which was achieved by coupling the mAb to agarose beads. No antibody secretion could be induced by soluble anti-Id (1 and 10 micrograms/ml). An optimal response required soluble T cell-derived factors which were added as a mixture of recombinant interleukin 2 with a T cell hybridoma supernatant that augments B cell growth and differentiation. Under these conditions an antigen-independent specific increase of IgM anti-A-CHO production (2.6- to 10-fold, or up to 2000 ng/ml respectively) could be induced in blood B cell populations of four of six normal individuals expressing the Id498 at serum level.     

Angiotensin II (AII)-related idiotypic network. III. Comparative analysis of idiotopes and paratopes borne by monoclonal antibodies raised against AII (AB1) and its internal image (AB3)

We have previously produced mAb against angiotensin II (AII), a phylogenetically conserved vasopressive octapeptide, and shown that they identify four distinct epitopes on the AII molecule. In addition we used internal image bearing polyclonal antiidiotypic antibodies raised against rabbit anti AII to produce mAb3. In this study we analyze the segregation of the idiotypic and paratopic repertoires of the mAb1 and mAb3. Analysis of mAb1 carried out with polyclonal Ab2 raised against the four distinct paratopes permitted classification of the mAb1 into four categories: (p+, id+) comprises antibodies with shared paratopic and idiotypic specificities: (p+, id-) is made up of antibodies that fail to express the Id defined by Ab2 raised against other antibodies pertaining to the same paratopic group; (p-, id+) includes antibodies that express cross-reactive Id on distinct paratopes; (p-, id-) refers to antibodies unrelated by their paratopes and Id mAb2 confirmed these results and showed expression of identical or closely related Id on clearly distinct paratopes. At the Ab3 level, using polyclonal Ab4, there was a higher degree of Id cross-reactivity between the two paratopes available. These data suggest that the parallel set concept may apply to the immune response to a natural peptidic Ag and its internal image. Comparison of idiotypic repertoires of mAb1 and mAb3 (using Ab2 and Ab4 antibodies) confirmed the lack of public Id and showed the predominance on mAb3 of "new" idiotypes absent from mAb1 molecules, as expected for internal image-induced antibodies. Cross-reactive idiotypes defined on mAb1 and conserved on mAb3 were expressed on the two paratopes defined at the Ab3 level. They were located on the H chain of the homologous paratope and required the association of H and L chains on the heterologous paratope. Our analysis suggests that, in the AII system, the idiotypic and paratopic repertoires segregate at least in part independently. The paratopic repertoire is limited to a small number of phylogenetically conserved specificities and may be encoded by germline genes. In contrast, the idiotypic repertoire is broader with respect to specificities, species, and localization on H and L chains. This extended diversity may be generated by somatic mutations or use of various combinations of H and L chains and/or V, D, J segments.     

Evidence that anti-idiotype induced immunity to experimental African trypanosomiasis is genetically restricted and requires recognition of combining site-related idiotopes

The ability of anti-idiotypic (anti-Id) antibodies to immunize mice against African trypanosomiasis independent of antigen has been confirmed. Of three allogeneic anti-Id antibodies raised against three protective monoclonal antibodies, each with specificity for the variant surface antigen of a clone of Trypanosoma rhodesiense, only one (anti-7H11 Id) was effective in immunizing BALB/c mice against homologous challenge. The immunity was associated with the more rapid and enhanced expression of the corresponding Id after infection. The immunity was restricted to mice bearing genes linked to Igh-Ca, which appeared to control expression of this Id both in response to infection and anti-Id treatment. Another Id, 11D5, appeared to be under similar genetic control. Anti-11D5 Id, however, was ineffective in immunizing mice against infection despite inducing high levels of Id bearing molecules before challenge. The immunizing potential of the respective anti-Id antibodies appeared to be related to the relative concentrations of antibodies reactive with idiotopes near to or within the antigen-combining site, which, in turn, determined the relative proportion of Id-bearing clones activated that had antigen binding activity.     

Monoclonal anti-idiotype induces protection against the cytotoxicity of the trichothecene mycotoxin T-2

An IgG1 mAb, designated HD11, specific for the trichothecene mycotoxin T-2 and capable of neutralizing its cytotoxicity was used to generate a syngeneic monoclonal anti-Id antibody. The generated anti-Id mAb, designated DE8, specifically bound to HD11 anti-T-2 mAb, and not to IgG1 mAb of irrelevant specificity or to normal mouse Ig. DE8 inhibited the binding of HD11 anti-T-2 to T-2-BSA-coated plates, whereas a control anti-Id mAb did not, suggesting recognition of an Id determinant associated with the T-2 binding site of HD11. Moreover, the binding of HD11 to DE8 and that of DE8 to HD11 were specifically inhibited by free T-2 mycotoxin. DE8 mAb was efficient in abrogating the protective effect of HD11 in the cytotoxicity of T-2 on the human epidermoid carcinoma cell line Hep-2. In vivo immunization of BALB/c mice with DE8 conjugated to KLH induced an anti-T-2 antibody titer comparable to that obtained with T-2-OVA immunization, whereas immunization with unconjugated DE8 resulted in a lower titered anti-T-2 response. Immunization with DE8-keyhole limpet or with unconjugated DE8 induced anti-T-2 antibody responses characterized by expression of "HD11-like" Id and by protection against T-2 cytotoxicity. However, the T-2-OVA-induced anti-T-2 response lacked the HD11+ Id and was only partially protective against T-2 cytotoxicity. This represents the first demonstration of the use of an anti-Id based vaccine in the in vivo induction of a protective antibody response against the cytotoxicity of a nonproteinaceous, small m.w. biologic toxin, whose very toxic nature precludes its use as the immunogen.     

Tumor idiotype vaccines. VII. Analysis and correlation of structural, idiotypic, and biologic properties of protective and nonprotective Ab2

We have previously generated and used anti-Id mAb (Ab2) to induce protective immunity against the L1210 DBA/2 tumor and for immunotherapy of established tumors. Among various anti-Id that were typed serologically as internal image Ab2 of the mouse mammary tumor virus tumor-associated Ag gp52, only one induced protective immunity and was effective in immunotherapy. In this study we compared the structural, idiotypic, and network properties of the protective and nonprotective antiidiotypic antibodies. The DNA sequence of the variable regions of six anti-Id was determined. The VH sequence of four Ab2, including the protective Ab2, are highly homologous, whereas the VL sequences differ and were assigned to different Vk families. In addition, the DH sequence region of the same four Ab2 are identical, whereas one is highly homologous and another one without homology. Search for amino acid sequence homologies between the Ab2 and gp52 showed the strongest similarities in the CDR2 of the L chain from the protective Ab2. In addition, the CDR2 region also had homology with a T cell epitope on gp52. The biologic basis of effective idiotypic mimicry was studied at the level of Ab3 induced by the Ab2. Id inhibition analysis using Ab3 induced by either protective or nonprotective Ab2, revealed differences. Thus, there is evidence for differences among the Ab1-Ab2-Ab3 cascade induced by protective and nonprotective anti-Id.     

Thymus-dependent antiidiotype and anti-antiidiotype responses to a dinitrophenyl-specific monoclonal antibody

BALB/c mice were inoculated i.p. with graded doses of a DNP-specific, IgM mAb (designated 57.1). Injection with unmodified 57.1 in the absence of adjuvants resulted in the generation of an anti-Id response (Ab2) and an anti-anti-Id response (Ab3). The generation of serum anti-Id antibodies was found to be thymus dependent. Nude mice immunized with 57.1 were unable to produce a serum Ab2 response above nonimmunized controls whereas euthymic mice receiving identical doses of 57.1 produced strong Ab2 responses. To examine the specificity of serum anti-Id, sera from mice receiving 57.1 were screened against a panel of mAb representing at least five distinct VH gene families. Serum titers were significantly higher against 57.1 than against any of the other antibodies in the panel. Three of the antibodies in this panel bind FD5-1, a monoclonal anti-Id (Ab2) that also binds 57.1. However, sera from mice receiving 57.1 bound 57.1 only. Thus, the serum Ab2 response appears to be highly specific for idiotopes on 57.1. The predominant isotype of these anti-Id antibodies was IgG1. The number of isotypes detected increased in a dose dependent manner with all IgG subclasses having anti-Id specificity in sera from animals receiving the higher doses of 57.1. Further analysis of the serum demonstrated that approximately 8% of the Ab2 response was paratope-specific (inhibitable by the monovalent hapten DNP-lysine). The same sera were analyzed for the presence of Ab3 by binding to the monoclonal anti-Id antibody FD5-1. Lower serum titers of Ab3 were generated in comparison to serum titers of Ab2. Analysis of the binding specificity of the Ab3 response revealed that DNP-BSA was able to partially inhibit the binding of serum IgM and IgG Ab3 to FD5-1. A subset of the Ab3 response. Ab1' that is specific for DNP was observed in a direct binding assay where detectable amounts of DNP binding IgM, IgG1, and IgG3 isotypes were present. We have thus described a complete circuit (Ab1----Ab2----Ab3) of antibodies within the Id network by immunizing animals with an unmodified mAb in the absence of Ag or adjuvants.     

Collagen-Induced Arthritis Suppressed with Monoclonal Anti-Idiotypic Antibody

In foregoing work, we identified at least 5 distinct epitopes on human type II collagen (CII), using 8 murine monoclonal antibodies (mAb) against human CII, and suggested that a species-nonspecific epitope on CII recognized by anti-CII mAb termed 1-5 is an arthritogenic epitope. We also found that antibody response against a selected epitope of human CII could be induced by immunization with rabbit anti-idiotypic (Id) antibody against anti-CII mAb. The author developed and characterized monoclonal anti-Id antibodies against 1-5 mAb recognizing a putative arthritogenic epitope. The author also investigated whether the anti-Id mAb could regulate antibody response directed against a selected epitope recognized by 1-5 mAb, and the induction of collagen-induced arthritis in DBA/1J mice. DBA/1J mice intravenously preinjected with anti-Id mAb to 1-5, did not produce anti-CII antibody expressing 1-5 Id upon immunization with human CII. Furthermore, as the development of collagen-induced arthritis (CIA) in DBA/1J mice pretreated with anti-Id mAb to 1-5 was significantly suppressed, anti-Id mAb will be a useful tool for studying the regulation of antibody response to a selected epitope. This study lends support to our hypothesis that the 1-5 epitope is an arthritogenic epitope.