Gangliosides, Ab1 and Ab2 antibodies II. Light versus heavy chain: An idiotype-anti-idiotype case study

The antibody heavy chain is generally more important than the light chain for the interaction with the antigen, although many reports demonstrate the influence of the light chain in the antibody binding properties. The heavy chains of anti-N-glycolyl-ganglioside P3 mAb and anti-idiotypic 1E10 mAb display complementary charged residues in their H-CDRs, particularly in H-CDR3. A basic residue in P3 mAb H-CDR1 was shown to be crucial for the interaction with the antigen and 1E10 mAb. The immunogenetic features of three other P3 mAb anti-idiotypic mAbs are now analyzed. One of them bears the same heavy chain as 1E10 mAb and a different light chain, but differs in its binding to P3 mAb mutants where H-CDR basic residues were replaced and in the binding to 1E10-specific phagotopes. Chimeric hybrid antibodies with P3 and 1E10 mAb heavy chains and unrelated light chains were obtained to further determine the importance of heavy chains in P3 and 1E10 mAb binding properties. One of the P3 heavy chain hybrid antibodies retained the specificity of P3 mAb with slight affinity differences. The heavy chains appear to play the main role in these mAb interactions, with the light chains modulating the affinity to their ligands.     

A monoclonal antibody against NeuGc-containing gangliosides contains a regulatory idiotope involved in the interaction with B and T cells

P3 (IgM-kappa) is a monoclonal antibody (mAb) reacting with N-glycolyl neuraminic acid (NeuGc)-containing gangliosides and sulfated glycolipids. To explore the nature of the idiotope defined by 1E10, we used a phage-displayed random peptide library. After three rounds of selection, seven different phagotopes were isolated. Noteworthy, all the sequences were found to bear the basic amino acid-rich motifs KPPR (3) or RRPR/K (4). This recursive selection of basic sequences by 1E10 mAb confirmed previous suggestions of the involvement of charged residues in the interaction between gamma-type Ab2 and P3 mAb. The binding of 1E10 to phage peptides representing each group was completely inhibited by P3 mAb. In addition, other Ab2 to P3 were able to recognize these peptides. Thus, phage peptides seem to be mimotopes of the idiotope recognized by anti-idiotypic antibodies in P3. Phage motifs were represented in the lineal sequence of P3's heavy chain H-CDR3 and a 14-mer peptide representing this region was able to specifically inhibit 1E10 binding to P3. Previous studies showed that P3's idiotype was autoimmunogenic and shared by antibodies with different specificities. Now, we demonstrated that P3 mAb is able to activate a network cascade involving autologous anti-idiotypic and anti-anti-idiotypic T cells. Thus, P3's idiotype fulfill the three criteria previously established to define a "regulatory idiotype". Particularly, data presented here revealed the immunodominance of the H-CDR3 of this mAb as a T cell epitope. Thus, H-CDR3 is simultaneously involved in the interaction of P3 mAb with anti-idiotypic B and T cells, behaving as a potential regulatory idiotope.     

Gangliosides, Ab1 and Ab2 antibodies IV. Dominance of VH domain in the induction of anti-idiotypic antibodies by gene gun immunization

The heavy chain of anti-N-glycolyl-ganglioside P3 mAb plays the main role in its binding properties. At least one hybrid idiotype consisting on the P3 VH and an unrelated VL domain retains antigen recognition. Moreover, the unusual immunogenic properties of P3 idiotype could be modified by single mutations of H-CDR residues. Here, we show that DNA gene gun immunization with the P3 VH combined with an unrelated VL domain or with itself (VH dimer, VHD) is enough for inducing anti-idiotypic antibodies, independently of antigen recognition by the resulting molecule. The scFv fragment of P3 mAb was also able to induce an anti-idiotypic response. For both the P3 and the P3 anti-idiotypic 1E10 mAbs, heavy chains dominate the induction of antibodies against the respective idiotypes.     

Immunogenetic analysis of variable regions encoding AB1 and gamma-type AB2 antibodies from the NeuGc-containing ganglioside family

The variable regions from P3, a murine monoclonal antibody (MAb) against NeuGc-containing gangliosides, and two anti-idiotype MAbs directed to P3 MAb were cloned and sequenced. Comparisons with previously reported sequences showed that P3 is a germline antibody encoded by genes from the V(H)Q52 and V(kappa)19 families. Analysis of nucleotides at the heavy chain CDR3 (H-CDR3) showed the presence of an extensive 3' N region that contains almost 50% of the nucleotides of this CDR. In addition, amino acid sequence analysis of the H-CDRs of this MAb revealed the presence of three arginines, two of which are present in the H-CDR3, that could be involved in the interaction of P3 MAb with its electronegative epitope on gangliosides. Anti-idiotype 1E10, which seems to define a "regulatory" idiotope on P3 MAb (it induces Id+ Ab3), represents a germline Ab2 that belongs to the V(H)J558 and V(kappa)10 gene families. By contrary, the anti-idiotype 3B11 is an extensively mutated antibody that belongs to the V(H)3660 and V(kappa)4/5 gene families, defining a "private" idiotope on P3 MAb. Even when different V genes contribute to the variable regions of 1E10 and 3B11 MAbs, they share an acidic motif E/D-D-Y/D-Y-D in H-CDR3, suggesting that both Ab2s recognize paratope positive residues on the Ab1. Therefore, complementary electrostatic interactions involving H-CDR3 from both Ab1 and Ab2, might provide a clue to understand the molecular basis for the generation of gamma-type anti-idiotype antibodies to V regions recognizing glycolylated ganglioside antigens.     

Switching on cytotoxicity by a single mutation at the heavy chain variable region of an anti-ganglioside antibody

Gangliosides are sialic acid-containing glycosphingolipids present in the plasma membrane of most mammalian cells. In humans, the expression of the N-glycolylated (Neu5Gc) variant of the sialic acid has been associated with malignant transformation, constituting therefore an attractive target for cancer immunotherapy. P3 monoclonal antibody (mAb) recognizes Neu5Gc-containing gangliosides, as well as sulfatides. Heavy chain CDR3 (H-CDR3) arginine residues have been shown to be crucial for ganglioside recognition, but less important for anti-idiotypic antibody binding. Here, we describe the effect on antibody reactivity of different mutations involving a single H-CDR3 acid residue. Substitution of glutamate 99 (Kabat numbering) by arginine, aspartate or serine residues resulted in no differences in anti-idiotype binding. However, the first mutation caused increased reactivity with the antigen, including a cytotoxic effect of the antibody on ganglioside-expressing cells previously unseen for the wild type antibody. Another antibody that recognizes N-glycolyl-GM3 ganglioside (GM3(Neu5Gc)), but not other glycolipids, named 14F7, exhibits also an arginine-enriched H-CDR3 and a complement-independent cell death activity. Unlike 14F7 mAb, the cytotoxicity of the P3 E₉₉ → R mutant antibody did not exclusively depend on ganglioside expression on tumor cells.     

Anti-idiotypic immunization provides protection against lethal endotoxaemia in BALB/c mice.

Against lipid A (the conserved moiety of lipopolysaccharides from Gram-negative bacteria) neutralizing IgM monoclonal antibodies (mAb) 8-2 and 26-20 anti-idiotypic (Ab2) mAb were produced: Ab2 mAb KM-04 (IgG1) against mAb 8-2, and Ab2 mAb PW-1 (IgG2a) and PW-2 (IgG1) against mAb 26-20. The binding of Ab2 mAb KM-04 to 8-2 (Ab1) was strongly inhibited by a lipopolysaccharide (LPS) extract from either Salmonella minnesota R595 (Re LPS) or Escherichia coli J5 (Rc LPS), whereas the binding of Ab2 mAb PW-1 and PW-2 to 26-20 (Ab1) was only marginally inhibited by both Re LPS and Rc LPS. The results indicated that Ab2 mAb KM-04 recognizes a lipid A-binding site related idiotope on mAb 8-2 and therefore KM-04 might bear the internal image of a neutralization determining epitope of lipid A. Consequently Ab2 KM-04 might induce antibodies to lipid A. Indeed anti-idiotypic immunization of syngeneic BALB/c mice with Ab2 mAb KM-04 resulted in development of lipid A-binding anti-anti-idiotypic (Ab3) antibodies in serum. Similar immunizations with Ab2 mAb PW-1 and PW-2 were unsuccessful. However, induction of lipid A-binding Ab3 by mAb KM-04 proved to be genetically restricted to BALB/c mice. DBA/2 mice, Swiss mice and rabbits did not develop lipid A-binding antibodies upon immunization with mAb KM-04. In protection experiments, it was shown that BALB/c mice vaccinated with mAb KM-04 showed significantly enhanced survival from challenge with either rough (Re) LPS from Salmonella minnesota or smooth LPS from E. coli 0111:B4 when compared to BALB/c mice immunized with a non-relevant Ab2 mAb. The results suggest that mAb KM-04 constitutes a non-internal image vaccine to the lethal effect of lipid A in BALB/c mice. Furthermore an Ab3 mAb was prepared against Ab2 mAb KM-04 that showed reactivity with Re LPS. This Ab3 mAb, designated LE-21 (IgG2a) protected mice against an otherwise lethal challenge of Re LPS.     

Generation of anti-Neu-glycolyl-ganglioside antibodies by immunization with an anti-idiotype monoclonal antibody: A self versus non-self-matter

We have previously generated a murine anti-idiotype (Ab2) monoclonal antibody (mAb) to a murine Ab1 mAb, named P3, which selectively binds Neu-glycolyl (NeuGc)-sialic acid on several monosialo- and disialogangliosides, and also reacts with sulfatides and antigens expressed in human melanoma and breast tumors. This Ab2 mAb, designated as 1E10, induced anti-anti-idiotype antibodies (Ab3) in mice and cancer patients. These Ab3 generated by 1E10 mAb were characterized by bearing P3 mAb idiotopes (Ab3, Id +). But when the specificity of these Ab3 antibodies was tested, no specific humoral response against NeuGc-containing gangliosides was detected in sera from immunized mice. However, hyperimmune sera from melanoma and breast cancer patients vaccinated with this Ab2 mAb were able to react specifically with these gangliosides. The different expression of NeuGc-containing gangliosides in the normal tissues of mice and humans could explain these results. In order to demonstrate these findings in other animal species with a different NeuGc-sialic acid expression, we performed similar studies in monkeys and chickens. In monkeys, as in most mammals, NeuGc-containing gangliosides are self-antigens. In contrast, chickens, like humans, lack the expression of these antigens in normal tissues. Here we report that the antibody response against NeuGc-containing gangliosides induced by immunization with 1E10 mAb was completely different in both species. No specific antibody response against these gangliosides was detected in hyperimmune monkey sera. In contrast, a strong and specific Ab3 response against GM3(NeuGc) and GM2(NeuGc) gangliosides (Ab3, Ag+) was generated in chickens due to the administration of 1E10 mAb.     

Cross-reactive idiotopes among anti-foot and mouth disease virus neutralizing antibodies.

Foot and mouth disease virus (FMDV) viral protein 1 is the only one of the four viral proteins (VP) that induces neutralizing antibodies as an isolated protein. A 32 amino acid (AA) residue (32dimer) of FMDV subtype A12 Lp ab VP1 (AA 137-168) was immunogenic against the A12 subtype. Three antibody populations each recognizing different epitopes on 32dimer were isolated by affinity chromatography (AFC) from the serum of a steer which had been immunized with the 32dimer. The 32dimer contains an AA sequence that is recognized by a protective paratope carried on a murine monoclonal antibody (mAb) (7SF-3.H3.1). Polyclonal anti-7SF-3 idiotype antibodies specifically inhibited the binding activity of one of these anti-32dimer antibody populations suggesting the existence of cross-reactive paratopic-related idiotopes between mAb 7SF-3 and antibodies elicited by the 32dimer. These anti-idiotypic antibodies were used in AFC to purify antibodies from the anti-32dimer serum. The purified antibody population has characteristics that resemble those of the mAb 7SF-3, i.e. its reactivity with FMDV A subtypes in ELISA, radioimmunoassay (RIA), mouse neutralization and its lack of reactivity with a mAb 7SF-3 neutralizing escape virus variant. Furthermore, these antibodies were specifically inhibited by either anti-mAb 7SF-3 idiotypic antibodies or peptides containing the mAb 7SF-3 epitope. Using the same experimental approach, mAb 7SF-3 idiotope-bearing antibodies were shown to be present in serum from bovine and swine convalescent from FMDV A12 Lp ab infection. Thus, the highly immunogenic area between residues 137 and 168 of FMDV VP1 elicited a cross-reactive neutralizing idiotope response conserved amongst several animal species.     

Gangliosides, Ab1 and Ab2 antibodies III. The idiotype of anti-ganglioside mAb P3 is immunogenic in a T cell-dependent manner

P3 mAb is an IgM monoclonal antibody specific for N-glycolyl-containing gangliosides. The immunogenicity of the P3 idiotype has been previously described by immunizing syngeneic BALB/c mice with the purified murine IgM or the mouse-human chimeric IgG antibody. In the present work we study the antibody response against the idiotype of P3 mAb through immunization with DNA. We used small immune proteins (SIP) consisting on the idiotype in the scFv format, covalently linked to gamma1CH3, the self-dimerizing domain of murine IgG1. SIPs were previously shown to be appropriate to induce specific anti-idiotypic responses. By gene gun immunization, a polyspecific response was occasionally generated, particularly with the P3 idiotype. A single shot of DNA was sufficient to induce a strong and long-lasting anti-P3 idiotype response. In addition, by delivery of the same DNA construct with a recombinant adeno-associated virus the unique immunogenicity of the P3 idiotype was demonstrated. The requirement of T cells in the anti-P3 idiotype response was indicated by the lack of P3-specific anti-idiotypic antibodies following immunization of both, allogeneic C57BL/6 and athymic BALB/c mice.     

Dissection of an antibody paratope into peptides discloses the idiotope recognized by the cognate anti-idiotypic antibody

Using methods of parallel synthesis, the complete amino acid sequence of an Ab 1 antibody (Tg 10, an anti-human thyroglobulin monoclonal antibody) was made in the form of a set of 100 synthetic overlapping peptides. This set of immobilized peptides was allowed to react with the cognate Ab2 (AI 10, a highly purified rabbit anti-idiotypic polyclonal antibody to Tg 10). A dominant peptide idiotope, INTFSGVPTYA, was thus mapped, which corresponds mainly to the CDR2 region from the V(H) domain of the Tg 10 mAb. A synthetic peptide replica of this idiotope was found to bind to AI 10 with an affinity (K(D) in the 10(-8) M range, as measured using BIACORE technology) which represents a significant part of the affinity of the complete Tg 10 antibody (K(D) in the 10(-9) M range). The synthetic peptide also elicited anti-idiotypic antibodies in rabbits that recognized specifically the Ab1 antibody in an Ab1- and antigen-inhibitable manner. The peptide idiotope was further characterized chemically by the identification of residues important for binding to the Ab2 and by modelization of its structure. Our approach makes it readily possible to map and characterize functional, continuous-type idiotopes that could be further used to manipulate the immune response by peptide technologies.     

Induction of anti-progesterone immunity and pregnancy blocking by anti-progesterone anti-idiotypes. Variable efficacy of polyclonal Ab2 antibodies directed against a panel of closely related Ab1 antibodies.

Polyclonal rabbit anti-idiotypes (Ab2) have been raised against three mouse monoclonal antiprogesterone Ab1 antibodies (DB3, 11/32, 11/64) closely related in VH and VL sequences. The anti-idiotypes were characterized for specificity and used to immunize groups of female mice. The latter responded with production of anti-progesterone (Ab3) antibodies, confirming the ability of anti-idiotypes to mimic the immunogenicity of a steroid. The response to one of the anti-idiotypic reagents (anti-DB3-id) was 5-10 times stronger than those to the others, despite close sequence homology between the idiotypes. Moreover, immunization with anti-DB3-id led to a reduction in fertility rate from 90% (control) to 30%, whereas immunization with the other anti-idiotypes was without effect. Sequence and structural comparisons suggest that residues associated with VH CDR3 and VL CDR3 may have a key role in determining the efficiency of anti-idiotypic immunization against progesterone. The variability in outcome of using anti-idiotypic reagents against a defined panel of related antibodies is relevant to the use of anti-idiotypes as surrogate antigens.     

Anti-anti-idiotypic (Ab3) antibodies that bind progesterone-11alpha-bovine serum albumin differ in their combining sites from antibodies raised directly against the antigen

Polyclonal rabbit anti-idiotypic (Ab2) antibodies raised against the antiprogesterone mAb DB3 (Ab1) were used to induce an Ab3 antiprogesterone response in BALB/c mice. While the affinity of Ab3 sera for progesterone was 10-50-times lower than that of DB3, their steroid-binding specificity showed considerable similarity to DB3. Two immunoglobulin M (IgM) Ab3 monoclonal antibodies (mAbs), 1A4 and 3B11, were obtained, both of which bound progesterone conjugated to bovine serum albumin (progesterone-BSA). 1A4 also bound free progesterone, although with low affinity and very broad cross-reactivity. Like DB3, 1A4 is encoded by a heavy-chain variable region (VH) gene segment from the small VGAM3.8 family, a restriction that is characteristic of antibodies raised against progesterone-11alpha-BSA. In contrast, 3B11 binds progesterone-11alpha-BSA but not free progesterone and is encoded by an unrelated VH gene from the J558 family. The light chain variable region (VL) of 1A4 lacks the intradomain disulphide bridge owing to replacement of CysL23 by Tyr. Both the 1A4 and 3B11 heavy chains have extremely short complementarity determining region (CDR) H3 loops, comprising three and four amino acids, respectively. Modelling of the combining site of 1A4 from the X-ray crystallographic structure of DB3 indicates that the short H3 loop is a major factor in the loss of affinity and specificity for steroid.     

Exploring the feasibility of an anti-idiotypic cocaine vaccine: analysis of the specificity of anticocaine antibodies (Ab1) capable of inducing Ab2beta anti-idiotypic antibodies

Conventional vaccination with the cocaine molecule conjugated to a protein carrier is a new approach in the treatment of addiction. Experimentally, this strategy has been shown to alter the pharmacokinetics as well as the psychostimulant effect of a cocaine challenge. The purpose of this study was to investigate whether a more stable and less controversial molecule, an anti-idiotypic antibody, which mimics the configuration of the cocaine molecule (Ab2beta), could be successfully used instead of cocaine. Two cocaine conjugates that presented different areas of the cocaine molecule to the immune system were used to produce monoclonal antibodies specific for cocaine (Ab1). Several anti-idiotypic antibodies were then produced. Four were identified as Ab2beta, or internal images of the antigen; when injected into BALB/c mice, they elicited an anticocaine response. The anticocaine response elicited by one of the four Ab2beta (K1-4c) was sufficient to significantly reduce the level of cocaine that targeted the brain following cocaine challenge, compared with the level of cocaine found in the brain of control animals immunized with irrelevant antibody. In conclusion, the possibility of an anti-idiotypic vaccine seems to be worth pursuing.     

Idiotypic Restriction of Murine Monoclonal Antibodies to a Defined Antigenic Region of Human Thyroglobulin

In previous studies, we demonstrated that anti-human thyroglobulin (hTg) autoantibodies in patients with thyroid disorders exhibit a restricted epitopic specificity towards antigenic region II defined by its reactivity with four murine monoclonal antibodies (mAb 3, 6, 10, 15). To analyze the relationships between epitopic specificity and idiotypic expression of these mAb, two polyclonal anti-idiotypic sera were generated in rabbits by immunization with F(ab')₂ fragments of mAb 3 and mAb 10. These anti-idiotypic preparations (AI 3 and AI 10) were tested against a panel of hTg-mAb produced in different strains of mice (HR BIOZZI and BALB/c). The idiotypic analysis showed that AI 3 and AI 10 specifically recognized framework-associated idiotopes as well as paratope-associated idiotopes shared by region II mAb. These results demonstrate that specificity for region II was strongly associated with a restricted idiotype suggesting a high sequence homology between V regions. In addition, naïve BALB/c mice immunized with AI 3 or AI 10 produced anti-hTg (Ab3) antibodies that recognize region II epitopes. These latter findings reveal that anti-Id contain a population of Ab2β carrying the internal image of region II epitopes.     

Characterization of protection against coronavirus infection by noninternal image antiidiotypic antibody

Previously, we have reported protective vaccination of mice against a coronavirus infection using rabbit polyclonal noninternal image Ab2gamma anti-idiotypic (anti-Id) antibody specific for a virus-neutralizing and protective monoclonal antibody (mAb) 7-10A against the viral surface S glycoprotein. To characterize further the mechanisms involved in the induction of protective immunity by this noninternal image anti-Id, plasma and splenocytes from Ab2gamma-immunized BALB/c mice were passively transferred to naive BALB/c mice, followed by viral challenge. A reproducible significant delay in mortality observed in mice to which plasma was passively transferred, together with the presence of specific in vitro neutralizing antiviral Ab3 identified the humoral immune response as the major element responsible for protection. The activation of specific and cross-reactive T lymphocytes by both virus and anti-Id in immunized mice and the absence of adoptive transfer of protection by splenocytes suggested the participation of T helper activity in the induction of protective virus-neutralizing Ab3. To obtain more defined monoclonal reagents for a better understanding of anti-Id-induced protection, mAb2 were generated against the same mAb1 7-10A and characterized. We report the successful generation of mAb2 of the gamma type. However, unlike the polyclonal Ab2gamma, they were not capable of inducing a protective immune response.     

Two different methods result in the selection of peptides that induce a protective antibody response to Neisseria meningitidis serogroup C

Neisseria meningitidis is a leading cause of morbidity and mortality worldwide. The presently available capsular polysaccharide vaccine is poorly immunogenic in children under the age of 2 due to its T-independent (TI) nature. Efforts to overcome the TI response elicited by the polysaccharide vaccine have led to the development of polysaccharide-protein conjugate vaccines. Although a T-dependent (TD) response can be achieved in young children, the response to the polysaccharide still retains characteristics of a TI antibody response. An alternative method of potentially inducing a TD response to a carbohydrate antigen is through peptides that mimic the capsular polysaccharide. Our laboratory, through the production of an anti-idiotypic (anti-id) monoclonal antibody, designated 6F9, has previously identified a peptide mimic of the meningococcal serogroup C polysaccharide (MCPS). Using the same selecting monoclonal antibody (mAb), 1E4, we have screened a phage display library and identified 13 unique peptides that bound specifically to mAb1E4. Two peptides, Pep1C and Pep2C, that demonstrated the highest binding to mAb1E4, were selected, complexed to proteosomes, and used to immunize Balb/c mice. Of the 13 peptide motifs, only one peptide motif, that of Pep2C, was found to resemble the immunogenic peptide sequence of the anti-id selected with the same mAb, although many contained several similar amino acid residues. Immunization with Pep2C, but not Pep1C, induced a significant and functional anti-MCPS antibody response that conferred protection from a lethal challenge with meningococci. Our results indicate that immunization with a peptide of N. meningitidis serogroup C, screened with the same mAb that selected an anti-id of MCPS, induces a functional and protective anti-MCPS immune response similar to that of the anti-id. This study demonstrates that two different selection methods, production of an anti-id and biopanning using a phage display library, can be used to select functional and protective peptides of MCPS with similar moieties.     

Public and individual idiotopes in the anti-poly(Glu60, Ala30, Tyr10) response: analysis by monoclonal antibodies

From BALB/c mice immunized with anti-GAT monoclonal antibody (mAb) G5, we have obtained anti-idiotypic mAb against individual (or private) idiotopes, expressed by G5 as well as anti-GAT mAb, that are heteroclitic because they recognize poly-(Glu50, Tyr50) (GT) better than poly(Glu60, Ala30, Tyr10) (GAT). From BALB/c mice immunized with BALB/c polyclonal anti-GAT antibodies, anti-idiotypic mAb directed against public idiotopes expressed following GAT immunization in all individuals of all mouse strains tested have been obtained. Nine anti-idiotypic mAb were studied in detail. One of these mAb recognizes only polyclonal anti-GAT antibodies; the other eight recognize polyclonal anti-GAT antibodies and anti-GAT mAb. The distribution of the structures recognized by the different anti-idiotypic mAb on a battery of 20 anti-GAT mAb allows definition of two families of public idiotopes.     

用抗-(抗HBc)抗体在同系小鼠中诱导抗-抗Id抗体

用单克隆抗-(抗HBc)抗体(抗-Id)免疫BALB/c鼠,诱生出具有与抗-HBc相同反应性的抗-抗Id抗体(Ab_3)。这种抗体在ELISA试验中可与HBc-Ag特异性反应,并能抑制单克隆抗-HBc与HBcAg的结合。表明我们建立的抗-H-Bc的抗Id能模拟HBcAg刺激小鼠产生免疫反应。     

Insights into the immunogenetic basis of two ganglioside-associated idiotypic networks

The heavy-chain variable regions (VH) from 14F7 MAb, an IgG1 antibody specific for GM3(NeuGc) ganglioside, and its anti-idiotype, the 4G9 MAb, were cloned and sequenced. Comparison with previously reported sequences showed that VH 14F7 belongs to the J558(VHI) gene family and that it is highly mutated. VH 4G9 belongs to the Q52(VHII) gene family. The HCDR3 14F7 sequence contains three basic residues that could be involved in the binding to 4G9 MAb, which bears acidic residues in its HCDR3. Studies performed in the syngeneic model showed that 14F7 MAb requires both coupling to KLH and the use of Freund's adjuvant to induce an effective anti-idiotypic IgG (Ab2) response. In contrast, P3 MAb, a germline gene-encoded Ab1 that also recognizes the GM3(NeuGc) ganglioside through a basic motif in its H-CDRs, has been reported to be immunogenic in syngeneic mice, even when injected in saline. In addition, when Leghorn chickens were immunized with 14F7 or P3 MAbs emulsified in Freund's adjuvant, only P3-immunized animals were able to develop antibodies that recognized NeuGc-containing gangliosides, antigens which are not present in the normal tissues of this animal species. This phenomenon could be due to the lack of idiotypic connectivity of 14F7MAb.     

Induction of immunity to a human osteosarcoma-associated antigen in mice using anti-idiotypic antibodies

Polyclonal rabbit anti-idiotypic antibodies (Ab2) were produced and analyzed for their ability to stimulate humoral immunity against a human tumor-associated antigen (TAA) in BALB/c mice. Murine monoclonal antibody (Mab) OSA-1 recognizes an 85,000-Da TAA present on both human osteosarcoma tissue and osteosarcoma cell lines. Rabbits were immunized with OSA-1 (Ab1) to produce Ab2. The polyclonal Ab2 were shown to react against an idiotope located at or near the antigen combining site of Ab1. Ab2 were demonstrated to be potent inhibitors of TAA binding to Ab1. BALB/c mice were immunized with this Ab2 preparation and then tested for the presence of osteosarcoma TAA reactive antibodies. Sera from Ab2-immunized mice were shown by Western blot to contain antibodies whose specificity resembled Ab1. Thus, immunization with polyclonal rabbit Ab2 was shown to stimulate production of Ab3 in mice which reacted against a human osteosarcoma TAA.