Anti-human tumor antibodies induced in mice and rabbits by "internal image" anti-idiotypic monoclonal immunoglobulins

MBr1 is a murine monoclonal antibody, defining a saccharidic epitope [CaMBr1] of a human tissue-specific, tumor-associated globoside, present on the mammary carcinoma cell line MCF-7. The same epitope is shared by glycoproteins present on normal and neoplastic mammary epithelial cells, and by mucins from some ovarian cyst fluids. We have used MBr1 as the monoclonal antitumor antibody in an idiotypic sequence of immunizations in order to obtain and characterize "internal images" of the original epitope to be used as substitutes of the nominal antigen in serologic immunoassays. Two monoclonal anti-idiotypic antibodies (beta-1 and beta-2), which reacted with paratope-related idiotopes on MBr1, were obtained. The analysis of the antigenic and immunogenic properties of these molecules by both "antigen" and "antibody" competition assays provided evidence that both beta-1 and beta-2 bear "internal images" of the MBr1-defined epitope. Moreover, when injected in mice and rabbits both beta-1 and beta-2 induced anti anti-idiotypic antibodies, which mimicked MBr1 in binding MCF-7 as well as normal and neoplastic mammary gland epithelial cells. These data are discussed in terms of their possible application to the production of tumor-associated antigen substitutes and their use in serologic immunoassays.     

Expression profile of saccharide epitope CaMBr1 in normal and neoplastic tissue from dogs, cats, and rats: implication for the development of human-derived cancer vaccines

CaMBr1 is a blood group-related tumour-associated antigen, whose pattern of expression provides a therapeutic window for passive or active immunotherapy and points to the promise of a vaccine against carcinomas overexpressing this antigen. In this context, an animal model that closely mimics the human situation would be extremely useful. We, therefore, utilised the murine monoclonal antibody MBr1, which defines CaMBr1, as a useful probe to detect the molecule targeted for vaccine development on canine and feline spontaneous breast and uterus tumours and on their normal counterparts, and on rat normal tissues and carcinoma cell lines. Immunoperoxidase staining of cryostat sections revealed homogeneous CaMBr1 expression only in normal feline uterus and a uterus papilloma, whereas MBr1 reactivity was very weak and heterogeneous in normal (1/3 and 1/3) and tumour (1/10 and 1/6) breast tissues from dogs and cats, respectively. In contrast, the data obtained in rat tissues were reproducible in the strains tested and showed that CaMBr1 was expressed in all epithelial tissues of the digestive tract, although with variable intensities. Monoclonal antibody staining appeared to correspond to membrane-bound structures as well as mucinous secretions. Similarly, secretion products of lactating mammary glands expressed CaMBr1. The spectrum of expression on rat digestive tract was broader than that in humans but the specificity of MBr1 reactivity was confirmed by competition assay with a synthetic tetrasaccharide that mimics the CaMBr1 antigen. On FACS analysis, only one of two clonal derivatives of the rat breast carcinoma line RAMA 25 expressed CaMBr1, and a negative cell subset was evident in repeated experiments. By contrast, both colon carcinoma lines, DHD/K12 and CC531, showed staining with MBr1, albeit at different levels of intensity, and no evidence of a negative subset. The cell line CC531 maintained or even increased CaMBr1 expression levels following transplantation in syngeneic immunocompetent animals. Our data suggest the usefulness of the rat as a test model for vaccines against human cancers overexpressing the CaMBr1 antigen.     

In vitro mimicry of CaMBr1 tumor-associated antigen by synthetic oligosaccharides

The murine monoclonal antibody (Mab) MBr1, raised against thebreast cancer cell line MCF7, recognizes a saccharidic epitopeoverexpressed on a high percentage of human breast, ovary, andlung carcinomas. This antigen was originally identified on theimmunogen as a globo-series glycosphingolipid with an H-likedeterminant at its terminus (globo-H). We report here the biologicalcharacterization of the entire globo-H hexasaccharide and fivesynthetic oligosaccharides representing fragments of the entirestructure andlor different anomeric configurations. Using competitivebinding assays on live cells, we identified the residues andthe linkages essential for mimicry of the cellular antigensrecognized by Mab MBr1 on the breast carcinoma cell line MCF7and small cell lung cancer cell line POVD. The terminal tetrasaccharidicfragment of globo-H is the oligosaccharide that most resemblesthe MBr1-defined epitope both on glycolipids and on glycoproteins.This information will help in the rational design of a highlyspecific reagent for active specific immunotherapy of carcinomasoverexpressing the MBr1-defined antigen. CaMBr1 immunotherapy monoclonal antibody oligosaccharides tumor-associated antigen     

Angiotensin II (AII)-related idiotypic network. I. Common occurrence of AII internal image on rabbit anti-idiotypic antibodies

Internal images of foreign Ag have been demonstrated in a variety of systems as anticipated by the idiotypic network theory formulated by Jerne. However, they seem to be of rare occurrence. In order to estimate the actual frequency of antibodies bearing internal images (Ab2-beta) of angiotensin II (AII), a phylogenetically conserved peptide made up of eight amino acids, nine rabbits were immunized with affinity or protein A purified anti-AII antibodies (Ab1) from allotype-matched rabbits. Four of nine antiidiotypic antibodies (Ab2) exhibited internal image-like reactivity. They recognized all the polyclonal Ab1 tested, whatever the species (rabbit, mouse, guinea pig). In addition, they were strongly reactive with three mAb specific for a carboxy terminus epitope on AII (mAb 110, 199, and 211) and with a fourth monoclonal Ab1 (133) identifying a more central epitope. Advantage was taken of this reactivity with mAb1 to purify Ab2-beta by affinity chromatography of Ab2 on Sepharose 4B covalently linked to the three monoclonal Ab1 specific for the carboxy terminus epitope. The eluate displayed typical internal image properties: 1) it reacted with all the polyclonal Ab1 tested, 2) this reaction was completely abolished by AII, and 3) rabbits and mice immunized with the eluate all produced Ab1. The AII related idiotypic network is thus characterized by high frequency and immunogenicity of AII internal images. In addition, reactivity of the latter with monoclonal Ab1 indicates variable expression on Ab2-beta of the epitopes defined by the mAb on the nominal Ag.     

Generation of anti-idiotypic and anti-anti-idiotypic monoclonal antibodies in the same fusion. Support of Jerne's Network Theory

Upon immunization of mice with a mAb (290A-167) directed against an epitope of Lol p I (the major allergenic determinant of Lolium perenne), both anti-idiotypic (aId) mAb (Ab2) and anti-aId mAb (Ab3) were produced. The Ab2 displayed the following internal image properties of Lol p I: it can be affinity-purified on an immobilized Id column; its binding to the anti-Lol p I mAb (290A-167) is inhibited by Lol p I; it inhibits in a dose-response fashion the binding of the specific Id to Ag. It is recognized by anti-Lol p I antisera from different species such as mouse, human, and goat. The Ab3 which binds to Lol p I was also produced from the same fusion. This binding was inhibited significantly by aId mAb (Ab2), anti-Lol p I mAb (290A-167) and Lol p I. These data indicate that the two mAb with specificity for Lol p I (290A-167 and Ab3) share similar reactivity to the Ag and that aId mAb is the internal image of the epitope recognized by the Id. We showed also that the capacity of rabbit aId Ab directed against the 290A-167 Id to inhibit the binding of Ab1 and Ab3 to Ag was almost abolished by passage over a Ab3-coated Sepharose column. This would suggest that not only are the two mAb with reactivity to Lol p I (Ab1 and Ab3) directed against identical epitopes, but that they in fact shared identical idiotopes as well. The production of identical mAb upon immunization with either the Ag or the aId mAb supports that the conceptual framework proposed by Jerne finds its biologic application in the course of an immune response.     

Angiotensin II (AII)-related idiotypic network. II. Heterogeneity and fine specificity of AII internal images analyzed with monoclonal antibodies

Although the structural basis of internal images borne by beta type monoclonal anti-idiotypic antibody (Ab2) begins to be elucidated, there is little information on the repertoire of epitopes which make up the internal images expressed by polyclonal Ab2. We addressed this question by using a two-way approach in the angiotensin II (AII)-related idiotypic network, a system characterized by common occurrence of internal images on rabbit Ab2. First, two sets of internal images were purified in parallel by affinity chromatography on Sepharose 4B covalently linked to either mAb 110 (S4B-110), a mAb specific for a phenylalanine requiring carboxy terminus epitope (Phe8) on AII, or mAb 133 (S4B-133), reactive with a more central epitope also expressed on Phe8 substituted peptide analogs. The respective eluates, EL1 110 and E11 133, exhibited only partially overlapping reactivity, as demonstrated by 1) a different pattern of inhibition by various AII peptide analogues of EL1 110 and E11 133 binding to the same anti-AII antibody (Ab1) (either the homologous polyclonal Ab1 102 or mAb 133), 2) and a distinct profile of EL1 110 and EL1 133 binding to 12 biotinylated monoclonal Ab1 identifying a variety of epitopes on AII. To analyze further the respective distribution of mAb 110 and mAb 133 defined epitopes on Ab2-beta molecules, Ab2 were submitted to sequential affinity chromatography on S4B-110 followed by S4B-133, and the fractionated internal images were characterized by the pattern of binding to the various monoclonal Ab1. It was thus possible to purify two Ab2-beta subpopulations that exclusively imaged the determinant identified by mAb 110 (ii 110) or that identified by mAb 133 (ii 133). A third subpopulation which was successively retained on S4B-110 and S4B-133 expressed both internal images (ii 110 + 133), and was additionally reactive with all the other monoclonal Ab1 tested. In any case, monoclonal Ab1 binding to the different sets of internal images was totally inhibited by an excess of AII. These results indicate that the repertoire of internal epitopes is similar to that of the nominal Ag, but is scattered over distinct subpopulations of Ab2-beta molecules that can be fractionated by affinity chromatography. Some of the latter seem to bear several epitopes and resemble the whole nominal Ag, whereas others appear to image only one determinant. Second, we raised 7 anti-anti-idiotypic mAb (monoclonal Ab3) against affinity-purified Ab2-beta and analyzed their fine specificity for AII.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of antibodies to idiotypic determinants of monoclonal anti-sperm antibodies

Rabbit polyclonal antibodies to the idiotype of murine monoclonal anti-sperm antibodies were developed and characterized. M29.6 and M42.15 are monoclonal antibodies (mAbs) that inhibit fertilization in vivo and in vitro. Sera from rabbits inoculated with purified mAbs (Ab1) were absorbed with normal mouse and isotype-specific immunoglobulin (Ig); the anti-idiotype Ig fraction (Ab2) was isolated by protein A-chromatography or by chromatography on the corresponding idiotype column. Binding specificity of Ab2 was confirmed by measuring the reactivity of Ab2 with homologous and heterologous mAbs. Ab2 competitively inhibited 125I-labeled Ab1 binding to mouse sperm, suggesting that the Ab2 preparation possessed subpopulations directed against idiotopes similar or adjacent to the antigen-binding site of the mAb. Anti-idiotype antibodies reactive with the antigen-combining site of the anti-sperm mAb may contain subpopulations that mimic the mouse sperm epitope recognized by Ab1. Immunization with Ab2 induced anti-(anti-idiotype) antibodies (Ab3), which competitively inhibited binding of 125I-labeled Ab1 to immobilized Ab2. These results are consistent with the hypothesis that immunization of mice with antibodies to the idiotype of sperm-specific mAbs can induce antibodies that share structural similarities with the anti-sperm mAb used for their induction. Immunization with anti-idiotype antibodies that mimic sperm antigen structure represents a possible strategy for induction of immunity to sperm.     

Murine monoclonal anti-idiotype antibody as a potential network antigen for human carcinoembryonic antigen.

Carcinomas of the gastrointestinal tract are not curable by standard therapies. Thus, new therapeutic approaches for this disease are needed. This study proposes the use of anti-Id mAb as Ag substitutes to induce anti-tumor immunity in gastrointestinal cancer patients. Recently, we have generated and characterized one monoclonal anti-Id antibody, designated 3H1 (Ab2), which mimics biologically and antigenically a distinct and specific epitope of the 180,000 m.w. carcinoembryonic antigen (CEA) primarily expressed in high density by human pancreatic and colonic tumor cells. This epitope is unique to CEA and not present on other CEA-related lower m.w. members of the Ag family also found on normal tissues. The antigenic determinant as defined by the mAb 8019 (Ab1) against which the Ab2, 3H1 was raised, is absent on normal adult tissues by immunoperoxidase staining and haematopoietic cells including granulocytes by flow cytometry analysis. Anti-Id (Ab2) 3H1 induced CEA-specific antibodies in mice and rabbits. The immune sera from both mice and rabbits competed with Ab1 for binding to the colon carcinoma cell line LS174T and inhibited the binding of radioiodinated Ab1 to Ab2. This indicates that anti-anti-Id (Ab3) in mice and rabbits share idiotopes with Ab1 (8019). Furthermore, monoclonal Ab3 that bind to CEA have been generated from mice immunized with 3H1. The Ab3 (both polyclonal as well as monoclonal) immunoprecipitated the same 180,000 m.w. CEA as Ab1 (8019) by Western blotting analysis and showed almost identical immuno-staining patterns as Ab1 on colonic adenocarcinoma tissue sections from several patients. Collectively these data suggest that Ab2 3H1 could potentially be used clinically as a network Ag for immunotherapy of patients with CEA positive tumors.     

Induction of an anti-human type II collagen response by monoclonal anti-idiotypic antibody

Several distinct epitopes on human type II collagen were defined by using mAb. The presence of species-specific and species-nonspecific (common) epitopes was thus clarified. Anti-idiotypic mAb (Ab2) was developed against one of the antibodies (Ab1) reactive with species-specific epitopes. Thus Ab2 was demonstrated to recognize an idiotope expressed on the Ag-binding site (paratope) of Ab1, since the binding of Ab1 to human type II collagen was blocked by Ab2, and the binding of Ab2 to Ab1 was inhibited by soluble human type II collagen, but not by murine and bovine type II collagens. DBA/1 mice immunized with Ab2 coupled to keyhole limpet hemocyanin produced an antibody (Ab3) specifically reactive with human type II collagen. It was also demonstrated that Ab3 expressed an idiotype similar to that of Ab1. These findings indicate that anti-idiotypic antibody directed against mAb to human type II collagen mimics a species-specific epitope on human type II collagen. The anti-idiotypic antibody bearing internal image of type II collagen will open the way to isolation of the arthritogenic epitope on type II collagen.     

Synthesis and functional characterization of a recombinant monoclonal antibody directed against the alpha-chain of the human interleukin-2 receptor.

We have determined the sequence of the light and heavy chains of mAb 3G-10 (IgG1), a monoclonal antibody competing with interleukin 2 (IL2) for binding to the human IL2 receptor Tac protein. The antibody-encoding genes were chimerized by introducing splice donor and part of the intron sequences into the cDNA and subsequently linking it to the constant parts of the human IgG1 gene. The chimeric mAb was produced in mouse myeloma cells and purified. Murine and chimeric mAbs showed similar properties with respect to inhibition of T-cell proliferation. In contrast to its murine counterpart, the chimeric mAb exhibited Ab-dependent cellular cytotoxicity and, when combined with an Ab recognizing a different epitope on the IL2 receptor Tac protein, was able to activate human complement. The chimerized mAb might therefore have improved therapeutic efficacy.     

Bovine monoclonal anti-idiotypes induce antibodies specific for a synthetic peptide bearing a neutralizing epitope of bovine herpesvirus 1 glycoprotein gI (gB).

Bovine monoclonal anti-Id mimicking a neutralizing epitope of bovine herpesvirus-1 (BHV-1) glycoprotein gI were developed. An epitope present on the 74K subunit of gI identified by a murine mAb 1E11 was selected for this study. Bovine lymphocytes from the prefemoral lymph node of a heifer immunized with mAb 1E11 were fused with SP-2/0, a nonsecreting murine cell-line. Two bovine x murine hybridomas secreting bovine monoclonal anti-Id specific for the Id of 1E11 were stabilized. These anti-Id inhibited the binding of 1E11 to purified glycoprotein gI in a dose-dependent fashion. Naive mice immunized with the anti-Id produced anti-anti-Id (Ab3) that reacted with BHV-1 glycoprotein gI in a RIA, and neutralized BHV-1 infection in vitro. The Ab3 also showed reactivity to the 74K subunit of authentic gI glycoprotein in a Western blot analysis, and to the synthetic peptide bearing the 1E11 epitope in a RIA. These results substantiate the presence of the population of anti-Ab2 that functionally resemble antibodies specific for the immunizing Ag BHV-1 in Ab3, and demonstrate the ability of these anti-Id to elicit BHV-1-specific antibody response.     

Characterization of a glycosphingolipid antigen defined by the monoclonal antibody MBr1 expressed in normal and neoplastic epithelial cells of human mammary gland

The antigen defined by a monoclonal antibody, MBr1, was found to be expressed in normal human mammary gland epithelia and human mammary carcinoma cells (Ménard, S., Tagliabue, E., Canevari, S., Fossati, G., and Colnaghi, M. I. (1983) Cancer Res. 43, 1295-1300). The antigen has been isolated from breast cancer cell line MCF-7, which was used as immunogen, and its structure was determined by methylation analysis, NMR spectroscopy, direct probe mass spectrometry, and enzymatic degradation as identified below. Fuc alpha 1----2Gal beta 1----3GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1Cer The antibody cross-reacted weakly with fucosylasialo-GM1 (IV2FucGg4), which shares the same terminal sequence, Fuc alpha 1----2Gal beta 1----3GalNAc, with this antigen. However, various other structures, including lacto-series H structure (Fuc alpha 1----2 Gal beta 1----4/or 3GlcNAc beta 1----3Gal), did not show any reactivity with this antibody. Therefore, this antigen represents a blood group H antigen with a globo-series structure which is abundant in human teratocarcinoma (Kannagi, R., Levery, S. B., Ishigami, F., Hakomori, S., Shevinsky, L. H., Knowles, B. B., and Solter, D. (1983) J. Biol. Chem. 258, 8934-8942), although its presence must be limited in normal adult human tissue.     

Reactivity of a human monoclonal antibody against carcinomas and other lesions of the colon

Summary To identify tumor-associated antigens that may be immunogenic to man, human monoclonal antibodies (human mAb) were generated by fusing nonsecreting mouse myeloma cells with lymphocytes from regional mesenteric nodes of patients with adenocarcinomas of the colon. One IgG1 human mAb, designated as 14-31-10, was identified by its reactivity against human tumor xenografts. We have studied the reactivity of mAb 14-31-10 with formalin-fixed, paraffin-embedded specimens of human colon. A total of 86 cases were studied, including normal adult and fetal colons, adenocarcinomas of the colon, and a variety of colonic inflammatory diseases and preneoplastic lesions. Intense reactivity was found in 15 of 18 adenocarcinomas of the colon, but not in 10 specimens of normal adult or 4 specimens of fetal colonic mucosa. Interestingly, in four cases of carcinoma, reactivity was also observed in histologically normal mucosa situated 10 cm or more from the primary lesion. On the other hand, no staining was detected in any of the 16 inflammatory lesions. Of the 38 preneoplastic lesions, only 6 showed staining by the mAb: 1 of 5 benign tubular adenomatous polyps, 3 of 9 villous adenomas and tubovillous polyps, 1 of 5 specimens of ulcerative colitis and 1 of 19 specimens of familial polyposis. However, the intensity of staining was only moderate in those cases. Our data, therefore, suggest that the epitope identified by the human mAb 14-31-10 shows preferential expression in preneoplastic and neoplastic lesions of the colon, and in ostensibly normal mucosa at some distance from a primary colonic carcinoma. In all instances, the staining was cytoplasmic, suggesting a cytoplasmic or internal membrane location of the target antigen. This antigen appeared to be distinct from carcinoembryonic antigen, since staining by 14-31-10 was consistently different from that of a mouse monoclonal antibody to carcinoembryonic antigen in serial sections of the same specimens. The restricted reactivity of 14-31-10 suggests its potential application in immunohistochemistry. Moreover, the epitope identified by mAb 14-31-10 may be expressed during the progression of normal mucosa to neoplasia.This work was supported by USPHS Grants CA 43220 and CA 36233 awarded by the National Cancer Institute, National Institutes of Health, Department of Health and Human Services, by a grant from the Concern Foundation, and gifts from Mr. Alan Gleitsman and the Morey and Claudia Mirkin Foundation     

Analysis of anti-tumor antibodies in mice and rabbits induced by monoclonal anti-idiotope antibodies

Eight different mouse monoclonal anti-idiotope antibodies (mAb2) generated against a mouse monoclonal anti-human melanoma proteoglycan Ag (MPG) antibody (mAb1), MEM136, were tested for their ability to induce anti-MPG responses in mice and rabbits. All Ab2 were idiotypically cross-reactive and combining site-specific as demonstrated by competitive cross-inhibition studies and their ability to inhibit the binding of MEM136 to the melanoma cells, Colo38. However, only two Ab2, IM32 and IM06, were able to induce specific anti-TAA-specific (Ab1') responses in rabbits. When IM32 and IM06 were tested in allogeneic stains of mice for the induction of anti-MPG responses, only IM32 produced an Ab1' response. In mice, the Ab3 response induced by IM32 is idiotypically cross-reactive with its Ab1. Furthermore, the IM32-induced murine Ab3 and MEM136 recognized a similar MPG epitope on the melanoma cells because the Ab3 inhibited the binding of MEM136 to melanoma cells. The Ab3 induced by IM32 and IM06 in rabbits also recognized a similar epitope as the Ab1. In rabbits, the Ab3 response induced by IM32 and IM06 were idiotypically cross-reactive with each other. However, additional studies indicated that the majority of Ab3 induced by IM32 were IM32 Id-specific and lacked IM06 idiotopes. Further experimentation indicated that IM32-induced rabbit Ab3 were biologically active as demonstrated by the ability of the Ab3 to inhibit melanoma cell invasion in a Matrigel assay.     

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.     

Characterization of syngeneic rat monoclonal antibodies to the HSN tumor using syngeneic monoclonal anti-idiotopic antibodies

Twelve syngeneic anti-idiotopic mAb (anti-idiotypic/idiotopic antibodies Ab2)) were prepared from CBH/Cbi rats immunized with one of three monoclonal anti-HSN antibodies (Ab1) (11/160, ALN/11/53, or ALN/16/53) specific for the HSN tumor. The sera of the rats used for hybridoma production and all of the monoclonal Ab2 specifically inhibited the binding to HSN of the immunizing Ab1 only. It is concluded that, in this completely syngeneic system, only the private idiotopes associated with the antibody-combining site were immunogenic. Analyses using Western blotting showed that the Ab2 bound to intact Ab1 and to isolated H chains where the intra-strand disulfide bonds remained intact. The Ab2 did not bind to L chains or to fully reduced H chains of the Ab1. It is concluded that the idiotopes expressed on the H chain were conformational. When a panel of 13 monoclonal Ab1 (including the three used for immunization) were tested for reactivity with the Ab2, three reacted specifically with their respective Ab2 and 8 gave no binding suggesting that each Ab1 had a distinct idiotypic specificity despite the fact that all the Ab1 competed with each other for binding to Ag. However, the two remaining Ab1 (ALN/9/94 and ALN/12/17) generated from different tumor-bearing rats, were found to possess the same idiotypic specificity as 11/160. A detailed analysis using seven Ab2 raised against 11/160 showed that while the idiotype of ALN/9/94 and 11/160 were very similar, that of ALN/12/17 showed some clear differences. These three Ab1 have been shown previously to bind a sequential epitope on the HSN Ag in Western blots and it is postulated that the common idiotype of these Ab1 reflects their recognition of a sequential epitope. This may also account for the relatively frequent occurrence in tumor bearer sera of antibodies with this Id.     

Carbohydrates contribute to the interactions between cockroach allergen Bla g 2 and a monoclonal antibody

The crystal structure of a murine mAb, 4C3, that binds to the C-terminal lobe of the cockroach allergen Bla g 2 has been solved at 1.8 ? resolution. Binding of 4C3 involves different types of molecular interactions with its epitope compared with those with the mAb 7C11, which binds to the N-terminal lobe of Bla g 2. We found that the 4C3 surface epitope on Bla g 2 includes a carbohydrate moiety attached to Asn(268) and that a large number of Ag-Ab contacts are mediated by water molecules and ions, most likely zinc. Ab binding experiments conducted with an enzymatically deglycosylated Bla g 2 and a N268Q mutant showed that the carbohydrate contributes, without being essential, to the Bla g 2-4C3 mAb interaction. Inhibition of IgE Ab binding by the mAb 4C3 shows a correlation of the structurally defined epitope with reactivity with human IgE. Site-directed mutagenesis of the 4C3 mAb epitope confirmed that the amino acids Lys(251), Glu(233), and Ile(199) are important for the recognition of Bla g 2 by the 4C3 mAb. The results show the relevance of x-ray crystallographic studies of allergen-Ab complexes to identify conformational epitopes that define the antigenic surface of Bla g 2.     

Idiotypic analysis of anti-I-Ak monoclonal antibodies. III. T- and B-cell responses to anti-Ia idiotopes are not modulated by syngeneic anti-idiotypic monoclonal antibodies

To investigate whether anti-idiotypic (anti-Id) antibodies activate T cells either directly or indirectly, we examined the ability of syngeneic anti-Id monoclonal antibodies (mAbs) to regulate idiotype (Id) expression, antigen-binding antibody production, and T-cell reactivity to antigen. Our idiotypic system consists of an anti-I-A mAb that carries an infrequently expressed Id. Using three syngeneic anti-Id mAbs (Ab2), we previously defined the idiotype of the 11-5.2.1.9 (11-5) anti-I-Ak mAb. Two of these mAbs, IIID1 and IA2, recognize the same or closely related epitopes on 11-5 and cross react with two additional anti-I-Ak mAbs, 8B and 39J; the third anti-Id mAb, VC6, recognizes a distinct epitope shared by 11-5 and 8B. In the present study, BALB/c (H-2d) mice were primed with varying doses of these anti-Ids and were then boosted with C3H (H-2k) spleen cells. Among 130 such primed mice, the syngeneic anti-Ids when tested at priming doses between 10 ng and 10 micrograms were unable to induce Id production. The priming anti-Id mAbs persisted in the serum of the mice and were detectable as late as 40 days after priming. Ab1 expression was not modulated in BALB/c mice immunized with KLH-coupled Ab2, however, this immunization elicited the production of Ab3 which shared idiotypes with 11-5, 8B, and 39J. BALB/c anti-C3H alloreactive T-cell clones were also not induced by anti-Id priming, nor could they be shown to bind directly to the three Ab2 used. Nevertheless, the proliferative response of one anti-I-Ak specific T-cell clone that recognizes the same epitope as 11-5, 8B, and 39J, was inhibited by the IIID1 and IA2 Ab2. Thus, a T cell can express an idiotype shared by a B cell, but the linked recognition of an Id-associated carrier determinant(s) by an alloreactive T cell is required to elicit an anti-Id antibody response. These results favor the possibility that the activation of T cells is not dependent upon their ability to bind to anti-Id, but rather on their capacity to respond to epitopes of Id-anti-Id antigen-antibody complexes formed on B cells.     

Expression, Purification, and In Vivo Administration of a Promising Anti-Idiotypic HIV-1 Vaccine

To date only a few neutralizing antibodies against HIV-1 exist. Since these neutralizing antibodies are only rarely found in sera of HIV-1 infected individuals an active vaccine is required. We recently developed murine anti-idiotypic antibody Ab2/3H6 against monoclonal antibody (mAb) 2F5, which is one of the most prominent neutralizing antibodies. Anti-idiotypic antibody Ab2/3H6 has been partially humanized and expressed as whole immunoglobulin G in Chinese hamster ovary cells in order to minimize the human anti-mouse antibody response. Here we describe the expression, purification, and immunohistochemical characterization of the chimeric Ab2/3H6 Fab fragment, which was finally used beside the whole IgG1 as an antigen for immunization of guinea pigs. The crude sera were screened for specific antibodies against the epitope of mAb 2F5 ELDKWA as well as for reactivity against HIV-1 gp41.     

Cloning and sequence analysis of cDNAs encoding the heavy and light chain variable regions of an Ab2beta anti-idiotypic monoclonal antibody possessing an internal image of cocaine.

We report here the cloning and sequence analysis of cDNAs encoding the variable regions of an Ab2beta anti-idiotypic monoclonal antibody (K1-4c, gamma1kappa) that mimics the configuration of cocaine. The Ab2beta specifically binds to the human dopamine transporter as shown by confocal immunofluorescence microscopy. The sequence of the heavy chain complementarity-determining region 3 of K1-4c is strikingly similar to that of a monoclonal antibody (F11.2.32) specific for HIV-1 protease. Three or four amino acids in the epitope recognized by the anti-HIV-1 protease antibody are also present in the third extracellular loop of the dopamine transporter. This epitope is within the conserved region of the known transporters for dopamine, norepinephrine and serotonin in Homo sapiens, Rattus norvegicus, Caenorhabditis elegans and Drosophila melanogaster.