anti-Id mAb Research Articles

Anti-bevacizumab idiotype antibody vaccination is effective in inducing vascular endothelial growth factor-binding response, impairing tumor outgrowth.

Tumors require blood supply and, to overcome this restriction, induce angiogenesis. Vascular endothelial growth factor (VEGF) plays an important role in this process, which explains the great number of antiangiogenic therapies targeting VEGF. The research and development of targeted therapy has led to the approval of bevacizumab, a humanized anti‐VEGF monoclonal antibody (mAb), in clinical settings. However, side effects have been reported, usually as a consequence of bolus‐dose administration of the antibody. This limitation could be circumvented through the use of anti‐idiotype (Id) antibodies. In the present study, we evaluated the efficacy of an active VEGF‐binding immune response generated by an anti‐bevacizumab idiotype mAb, 10.D7. The 10.D7 anti‐Id mAb vaccination led to detectable levels of VEGF‐binding anti‐anti‐Id antibodies. In order to examine whether this humoral immune response could have implications for tumor development, 10.D7‐immunized mice were challenged with B16‐F10 tumor cells. Mice immunized with 10.D7 anti‐Id mAb revealed reduced tumor growth when compared to control groups. Histological analyses of tumor sections from 10.D7‐immunized mice showed increased necrotic areas, decreased CD31‐positive vascular density and reduced CD68‐positive cell infiltration. Our results encourage further therapeutic studies, particularly if one considers that the anti‐Id therapeutic vaccination maintains stable levels of VEGF‐binding antibodies, which might be useful in the control of tumor relapse.

Yeast killer toxin-like candidacidal Ab6 antibodies elicited through the manipulation of the idiotypic cascade.

A mouse anti-anti-anti-idiotypic (Id) IgM monoclonal antibody (mAb K20, Ab4), functionally mimicking a Wyckerhamomyces anomalus (Pichia anomala) killer toxin (KT) characterized by fungicidal activity against yeasts presenting specific cell wall receptors (KTR) mainly constituted by β-1,3-glucan, was produced from animals presenting anti-KT Abs (Ab3) following immunization with a rat IgM anti-Id KT-like mAb (mAb K10, Ab2). MAb K10 was produced by immunization with a KT-neutralizing mAb (mAb KT4, Ab1) bearing the internal image of KTR. MAb K20, likewise mAb K10, proved to be fungicidal in vitro against KT-sensitive Candida albicans cells, an activity neutralized by mAb KT4, and was capable of binding to β-1,3-glucan. MAb K20 and mAb K10 competed with each other and with KT for binding to C. albicans KTR. MAb K20 was used to identify peptide mimics of KTR by the selection of phage clones from random peptide phage display libraries. Using this strategy, four peptides (TK 1-4) were selected and used as immunogen in mice in the form of either keyhole limpet hemocyanin (KLH) conjugates or peptide-encoding minigenes. Peptide and DNA immunization could induce serum Abs characterized by candidacidal activity, which was inhibited by laminarin, a soluble β-1,3-glucan, but not by pustulan, a β-1,6-glucan. These findings show that the idiotypic cascade can not only overcome the barrier of animal species but also the nature of immunogens and the type of technology adopted.

Recombinant DNA technology for melanoma immunotherapy: anti-Id DNA vaccines targeting high molecular weight melanoma-associated antigen.

Anti-idiotypic MK2-23 monoclonal antibody (anti-Id MK2-23 mAb), which mimics the high molecular weight melanoma-associated antigen (HMW-MAA), has been used to implement active immunotherapy against melanoma. However, due to safety and standardization issues, this approach never entered extensive clinical trials. In the present study, we investigated the usage of DNA vaccines as an alternative to MK2-23 mAb immunization. MK2-23 DNA plasmids coding for single chain (scFv) MK2-23 antibody were constructed via the insertion of variable heavy (V H) and light (V L) chains of MK2-23 into the pVAC-1mcs plasmids. Two alternative MK2-23 plasmids format V H/V L, and V L/V H were assembled. We demonstrate that both polypeptides expressed by scFv plasmids in vitro retained the ability to mimic HMW-MAA antigen, and to elicit specific anti-HMW-MAA humoral and cellular immunoresponses in immunized mice. Notably, MK2-23 scFv DNA vaccines impaired the onset and growth of transplantable B16 melanoma cells not engineered to express HMW-MAA. This pilot study suggests that optimized MK2-23 scFv DNA vaccines could potentially provide a safer and cost-effective alternative to anti-Id antibody immunization, for melanoma immunotherapy.

Naive Idiotope-Specific B and T Cells Collaborate Efficiently in the Absence of Dendritic Cells

Anti-idiotope (anti-Id) Abs have a role in therapy against B cell lymphomas, as inhibitors of pathogenic autoantibodies, and as surrogate Ags for immunization. Despite these observations, the mechanism by which Id(+) Ig generates anti-Id Abs is essentially unknown. To address this issue, we generated a double knock-in mouse that expresses V regions of a somatically mutated anti-Id mAb with intermediate affinity (affinity constant [Ka] = 0.77 × 10(7) M(-1)) for the myeloma protein M315. The anti-Id mice have normal peripheral B cell populations, and allelic exclusion is efficient. Anti-Id B cells from BCR knock-in mice, together with Id-specific CD4(+) T cells from previously established TCR-transgenic mice, enabled us to study Id-specific T cell-B cell collaboration by dilution of transferred cells into syngeneic BALB/c recipients. We show that previously unstimulated (naive) Id-specific B and T cells collaborate efficiently in vivo, even at low frequencies and in the presence of low amounts of Id(+) Ig, resulting in germinal center formation, plasma cell development, and secretion of isotype-switched anti-Id Abs. We further demonstrate that Id-specific T cell-B cell collaboration occurs readily in the absence of adjuvant and is not dependent on Id-presentation by dendritic cells. The results underscore the potency of anti-Id B cells in MHC class II-restricted presentation of Id(+) Ig and suggest that Id-specific T cell-B cell collaboration is of physiological relevance.

Carbohydrate-associated epitope-based anti-cancer drugs and vaccines

RP215 is one of the three thousand monoclonal antibodies (Mabs) which were generated against the OC-3-VGH ovarian cancer cell line. RP215 was shown to react with a carbohydrate-associated epitope located specifically on glycoproteins, known as CA215, from cancer cells. Further molecular analysis by matrix adsorption laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) revealed that CA215 consists mainly of immunoglobulin super-family (IgSF) proteins, including immunoglobulins, T-cell receptors, and cell adhesion molecules, as well as several other unrelated proteins. Peptide mappings and glycoanalysis were performed with CA215 and revealed high-mannose and complex bisecting structures with terminal sialic acid in N-glycans. As many as ten O-glycans, which are structurally similar to those of mucins, were also identified. In addition, two additional O-linked glycans were exclusively detected in cancerous immunoglobulins but not in normal B cell-derived immunoglobulins. Immunizations of mice with purified CA215 resulted in the predominant generation of RP215-related Mabs, indicating the immunodominance of this carbohydrate-associated epitope. Anti-idiotype (anti-id) Mabs of RP215, which were generated in the rat, were shown to contain the internal images of the carbohydrate-associated epitope. Following immunizations of these anti-id Mabs in mice, the resulting anti-anti-id (Ab3) responses in mice were found to be immunologically similar to that of RP215. Judging from these observations, anti-id Mabs, which carry the internal image of the RP215-specific epitope, may be suitable candidates for anticancer vaccine development in humans.

Anti-Idiotypic Antibody Specific to GAD65 Autoantibody Prevents Type 1 Diabetes in the NOD Mouse

Overt autoantibodies to the smaller isoform of glutamate decarboxylase (GAD65Ab) are a characteristic in patients with Type 1 diabetes (T1D). Anti-idiotypic antibodies (anti-Id) directed to GAD65Ab effectively prevent the binding of GAD65 to GAD65Ab in healthy individuals. Levels of GAD65Ab-specific anti-Id are significantly lower in patients with T1D, leading to overt GAD65Ab in these patients. To determine the possible protective role of GAD65Ab-specific anti-Id in T1D pathogenesis, we developed the monoclonal anti-Id MAb 8E6G4 specifically targeting human monoclonal GAD65Ab b96.11. MAb 8E6G4 was demonstrated as a specific anti-Id directed to the antigen binding site of b96.11. MAb 8E6G4 recognized human antibodies in sera from healthy individuals, T2D patients, and T1D patients as established by ELISA. We confirmed these MAb 8E6G4-bound human antibodies to contain GAD65Ab by testing the eluted antibodies for binding to GAD65 in radioligand binding assays. These findings confirm that GAD65Ab are present in sera of individuals, who test GAD65Ab-negative in conventional detection assays. To test our hypothesis that GAD65Ab-specific anti-Id have an immune modulatory role in T1D, we injected young Non Obese Diabetic (NOD) mice with MAb 8E6G4. The animals were carefully monitored for development of T1D for 40 weeks. Infiltration of pancreatic islets by mononuclear cells (insulitis) was determined to establish the extent of an autoimmune attack on the pancreatic islets. Administration of MAb 8E6G4 significantly reduced the cumulative incidence rate of T1D and delayed the time of onset. Insulitis was significantly less severe in animals that received MAb 8E6G4 as compared to control animals. These results support our hypothesis that anti-Id specific to GAD65Ab have a protective role in T1D.

Anti-idiotypic antibodies as cancer vaccines: achievements and future improvements

Since the discovery of tumor-associated antigens (TAAs), researchers have tried to develop immune-based anti-cancer therapies. Thanks to their specificity, monoclonal antibodies (mAbs) offer the major advantage to induce fewer side effects than those caused by non-specific conventional treatments (e.g., chemotherapy, radiotherapy). Passive immunotherapy by means of mAbs or cytokines has proved efficacy in oncology and validated the use of immune-based agents as part of anti-cancer treatment options. The next step was to try to induce an active immune protection aiming to boost own’s host immune defense against TAAs. Cancer vaccines are thus developed to specifically induce active immune protection targeting only tumor cells while preserving normal tissues from a non-specific toxicity. But, as most of TAAs are self antigens, an immune tolerance against them exists representing a barrier to effective vaccination against these oncoproteins. One promising approach to break this immune tolerance consists in the use of anti-idiotypic (anti-Id) mAbs, so called Ab2, as antigen surrogates. This vaccination strategy allows also immunization against non-proteic antigens (such as carbohydrates). In some clinical studies, anti-Id cancer vaccines indeed induced efficient humoral and/or cellular immune responses associated with clinical benefit. This review article will focus on recent achievements of anti-Id mAbs use as cancer vaccines in solid tumors.

Inhibition of in vitro tumor cell growth by RP215 monoclonal antibody and antibodies raised against its anti-idiotype antibodies.

RP215 monoclonal antibody (Mab) was shown to recognize carbohydrate-associated epitope(s) in the heavy chains of cancer cell-expressed immunoglobulins, designated in general as CA215 pan cancer marker. Growth inhibitions of tumor cells in vitro by RP215 Mab and antibodies against its anti-idiotype (anti-id) antibodies were investigated. Polyclonal rabbit anti-id antibodies and the corresponding rat anti-id Mabs were generated and characterized. Following immunizations in mice, antisera raised against anti-id antibodies were analyzed by typical immunoassays. It was observed that mouse anti-anti-id sera (Ab3) revealed binding affinity and specificity to CA215 that are comparable to those of RP215. Both RP215 and Ab3 were shown to induce apoptosis of cultured cancer cells in vitro by TUNEL and MTT assays. These experimental observations were consistent with that of in vivo tumor growth inhibition by RP215 in previous nude mouse experiments. Therefore, heterologous or homologous anti-id antibodies of RP215 that contain the internal image of its specific epitope in CA215 may serve as effective anti-cancer vaccines for therapeutic treatments of various cancers in humans. The relative stability of RP215-specific carbohydrate-associated epitope was compared to that of human IgG at extreme pH's (<or=2 or >or=12) or following NaIO(4) treatments. The major molecular forms of CA215 were further documented with various enzyme immunoassays and found to have similar secondary structures to those of normal human immunoglobulin G.

Monoclonal Anti-idiotype Antibodies against Carbohydrateassociate Epitope for Anti-Cancer Vaccine Development

Background: RP215 monoclonal antibody (Mab) was shown to react specifically with the unique carbohydrate associated epitope located in the variable regions of cancer cell-expressed immunoglobulins known as CA215. This Mab was shown to inhibit the growth of a variety of cancer cells in vitro or in vivo. Anti-idiotype (anti-id) Mabs against RP215 were generated and characterized for future development of epitope-specific anti-cancer vaccines in humans. Results: Following successful immunization of rats with F(ab’)2 fragments of RP215, rat anti-id Mabs were established for biochemical and immunological characterization. Subsequent immunizations of mice with purified rat anti-id Mab revealed significant anti-anti-id antibody (Ab3) responses. In immunohistochemical studies, both Ab3 and RP215 reacted positively with various cancer cells lines and with comparable staining intensities. Both RP215 and Ab3 inhibited significantly the growth of cancer cells in vitro by means of TUNEL assay. To document that the RP215-specific epitope is carbohydrate-associated, the absence of RP215-epitope expressions upon culturing of cancer cells in serumfree medium was demonstrated. Experiments confirmed that the expression of RP215-specific epitope was dependent on the presence of carbohydrate precursors in culture media. Conclusion: Judging from these observations, anti-id Mabs which carry the internal image of RP215-specific carbohydrate-associated epitope can be suitable candidates for anti-cancer vaccine development in humans.

Characterization of the Antibody Response against NeuGcGM3 Ganglioside Elicited in Non-Small Cell Lung Cancer Patients Immunized with an Anti-Idiotype Antibody

1E10 mAb is an anti-Id murine mAb (Ab2 mAb) specific for an Ab1 mAb that reacts with NeuGc-containing gangliosides, sulfatides, and Ags expressed in some human tumors. In preclinical studies, this Ab2 Ab was able to mimic NeuGc-containing gangliosides only in animals lacking expression of these Ags in normal tissues. In this study, we report on the immune responses elicited in 20 non-small cell lung cancer patients treated with 1 mg of aluminum hydroxide-precipitated 1E10 mAb. In the hyperimmune sera from 16 of 20 patients, a strong specific Ab response of both IgM and IgG isotypes against NeuGcGM3 ganglioside was observed. Patient immune sera were able to induce complement-independent cell death of NeuGcGM3-expressing X63 murine myeloma target cells. Significant immunoreactivity to NeuGcGM3 was still detected after the complete abrogation of the reactivity against 1E10 mAb by the adsorption of patient sera with this Ab. We hypothesize that Id(-)Ag(+) Abs could reflect the activation of an autologous idiotypic cascade into the patients. Both Id(+)Ag(+) and Id(-)Ag(+) fractions were separated by affinity chromatography and characterized. Although IgG isotype Abs were found in both fractions, IgM isotype Abs were found only in the Id(-)Ag(+) fraction. Both Id(+)Ag(+) and Id(-)Ag(+) Abs were able to specifically recognize and induce cell death in NeuGcGM3-expressing X63 myeloma target cells. Patients that developed IgG and/or IgM Abs against NeuGcGM3 showed longer median survival times.

A Limited Number of Anti-Idiotypic Antibodies Is Required To Maintain FVIII Function in Presence of Polyclonal Anti-FVIII Antibodies of Haemophilia A Patients with Inhibitors.

Replacement therapy using either plasma-derived or recombinant FVIII elicits FVIII-specific Abs in about 25% of the patients. The majority of such antibodies are directed towards specific FVIII regions on which major epitopes have been identified. Most of these epitopes are located in the carboxy-terminal end of the C2 domain of the FVIII light chain and in the amino-terminal end of the A2 domain of the heavy chain, whereas, antibodies directed towards the C1 domain have been described in cases of mild/moderate haemophilia A. From a functional point of view, epitopes have been mapped to the phospholipid and von Willebrand binding sites in the C1 and C2 domains and to the FIX binding site in A2.We have derived human mabs (mabRhd5, mabBo2C11 and mabBoIIB2) that react with high affinity to the FVIII C1, C2 and A2 domains, respectively, and are representative of most of the specific inhibitors observed in haemophilia A patients.Recently, we have generated mouse anti-Id mab (mab14C12) against mabBo2C11 and provided the first demonstration that an anti-Id mab directed towards a C2-specific inhibitor restored FVIII activity both in vitro and in vivo in the presence of the inhibitor. Futher more, we demonstrated that FVIII inhibition obtained by a mixture of two anti-FVIII mabs (mabBo2C11 (anti-C2) and mabBoIIB2 (anti-A2)) was neutralized up to 100% when a mixture of the corresponding anti-Ids (mab14C12 and mab30D1) was added to the assay establishing the proof of concept that combination of anti-Id mabs will be able to neutralize the anti-FVIII immune response of haemophilia A patients. Based on this observation, We have also generated an anti-idiotypic antibody (mab18B6) towards an anti-C1 domain inhibitor (mabRHD5). In ELISA, mab18B6 specifically prevents, in a dose-dependent manner, mabRHD5 binding to C1 and fully neutralized mabRHD5 inhibitory properties in a coagulation assay. With a mixture of anti-Ids (mab14C12, mab30D1 and mab18B6) we were able to completely neutralized the cumulative anti-FVIII activity obtained in functional assay by a mixture of the corresponding monoclonal anti-FVIII antibodies. We next evaluated whether a combination of anti-Ids (anti-anti-A2,-C1,-C2) had the ability to neutralize the inhibitory properties of polyclonal antibodies obtained from haemophilia A patients. To this end, eight patients with severe haemophilia A and inhibitor were tested. An average of 70% neutralization of the inhibition was obtained in 6 out of 8 cases. These data allow to conclude that potent polyclonal high-affinity FVIII inhibitors can be neutralized with corresponding anti-idiotypic antibodies and that only a limited number of anti-idiotypic antibodies is required for inhibitor neutralization in a majority of patients.

120 POSTER Transduction of tumor cell lines with adenoviral vectors expressing TLR ligands and scFv anti-IL-10 activates dendritic cell maturation and IL-12 secretion in the presence of IL-10

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, IIIDI 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 μg 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 IIIDI 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.

Structural Basis of Antigen Mimicry in a Clinically Relevant Melanoma Antigen System

Although mimics of human tumor antigens are effective immunogens to overcome host unresponsiveness to the nominal antigen, the structural basis of this mimicry remains poorly defined. Therefore, in this study we have characterized the structural basis of the human high molecular weight-melanoma-associated antigen (HMW-MAA) mimicry by the mouse anti-idiotypic (anti-id) monoclonal antibody (mAb) MK2-23. Using x-ray crystallography, we have characterized the three-dimensional structure of the anti-id mAb MK2-23 Fab' and shown that its heavy chain complementarity-determining region (CDR3) (H3) and its light chain CDR1 (L1) are closely associated. These moieties are the source of HMW-MAA mimicry, since they display partial amino acid sequence homology along with a similar structural fold with the HMW-MAA core protein. Furthermore, a 15-residue peptide comprising the H3 loop of anti-id mAb MK2-23 demonstrates HMW-MAA-like in vitro and in vivo reactivity. This peptide in conjunction with the structural data will facilitate the characterization of the effect of the degree of antigen mimicry on the induction of a self-antigen-specific immune response by a mimic.

934 POSTER Phase II study of irinotecan and oral etoposide in patients with platinum/taxane-resistant ovarian carcinoma

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, IIIDI 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 μg 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 IIIDI 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.

Variable-Region-Identical Antibodies Differing in Isotype Demonstrate Differences in Fine Specificity and Idiotype

A central tenet of the current understanding of the relationship between Ab structure and function is that the variable region domain is solely responsible for Ag specificity. However, this view was recently challenged by the observation that families of mouse-human chimeric Abs with identical V regions demonstrate differences in fine specificity and by reports of changes in Ab Id structure with isotype switching. Here we revisited this question by evaluating the reactivity of two families of murine IgG switch variants that differed in V region usage for Cryptococcus neoformans glucuronoxylomannan, glucuronoxylomannan peptide mimetics, and anti-Id mAbs. The results reveal isotype-related differences in fine specificities and Id for two mAb isotype switched families, thus establishing the validity of this observation with sets of homologous Abs. The results suggest that the C region affects V region protein conformation, leading to differences in fine specificity and Id. The finding that isotype can affect fine specificity has major implications for current concepts of the generation of secondary responses, idiotypic network regulation, and isotype function. Given that isotype class switching and Ig gene somatic hypermutation share molecular mechanisms, these observations unify these processes in the sense that both can alter specificity and affinity.

Fine specificity of high molecular weight-melanoma-associated antigen-specific cytotoxic T lymphocytes elicited by anti-idiotypic monoclonal antibodies in patients with melanoma.

HLA-A2-restricted CTLs, which lysed high molecular weight (HMW)-melanoma-associated antigen (MAA)(+) melanoma cells, were induced in patients with melanoma immunized with MELIMMUNE, a combination of the murine anti-idiotypic (anti-id) monoclonal antibodies (mAb) MEL-2 and MF11-30 (MW Pride et al., Clin Cancer Res 1998;4:2363.). In the present study we investigated whether CTL epitopes are present in anti-id mAb MF-11-30 and activate T cells to recognize HMW-MAA on melanoma cells. One candidate epitope in the mAb MF11-30 VH chain, VH (3-11), was selected based on the presence of HLA-A2 anchor residues and partial homology with the HMW-MAA epitope, HMW-MAA (76-84). Lymphocytes from HLA-A2(+)-immunized patients proliferated to VH (3-11) peptide and to a variant HMW-MAA peptide to a significantly greater extent than autologous lymphocytes stimulated with an irrelevant peptide and lymphocytes from nonimmunized patients. No proliferative response was detected to the wild-type HMW-MAA peptide (76-84). Significant increase in IFN-gamma production but not in interleukin 10 production in response to VH (3-11) and to variant HMW-MAA peptide (76-84) was observed in lymphocytes from the immunized patients. Stimulation of lymphocytes from HLA-A2(+) patients with the two peptides induced CTL, which lysed HMW-MAA(+)/HLA-A2(+) A375SM melanoma cells. This is the first report documenting the presence of immunogenic peptides in a murine anti-id mAb for a defined epitope expressed by a human melanoma-associated antigen. These results may be relevant for development of novel vaccines based on homology between anti-id mAb and tumor-associated antigen amino acid sequences.

Electron microscopic and immunochemical analysis of the broadly neutralizing HIV-1-specific, anti-carbohydrate antibody, 2G12

2G12 is one of only a few cloned antibodies with broadly neutralizing specificity to HIV-1 envelope proteins. Crystallographic and electron microscopic (EM) data showed that the Fab arms are locked together via a novel VH domain exchange. Both the conventional and the unprecedented additional VH–VH antigen binding sites show specificity for high mannose oligosaccharides on the silent face of gp120. We have now extended the EM and biochemical analysis of 2G12. Unligated 2G12 IgG1 molecules clearly show paired (parallel attached) Fab arms in the “doughnut” configuration attached to the Fc both in individual and computationally averaged images. A minority of the IgG molecules in the 2G12 prep showed the open “Y” configuration of conventional IgG. The averaged EM image compares well to the atomic structure model of 2G12. Papain digests of 2G12 yielded paired Fab arms (Fab dimer), as observed by EM, which dissociated into Fab-sized fragments in non-reducing SDS-PAGE. Purified 2G12 reduced and alkylated H and L chains can reassociate to form IgG molecules with the Fab dimer configuration and can combine with L and H chains from conventional human IgG to form hybrid molecules. 2G12 is heavily aggregated following brief acid exposure possibly as a result of its unique structure. A model of the aggregation process is proposed. An anti-Id MAb was shown by EM to react with neither the conventional nor additional antigen binding sites, but bound to the lateral faces of the Fab arms of intact, reduced and alkylated, and reconstructed 2G12 molecules. Efforts to identify IgG molecules with a similar intertwined Fab dimer structure in a large IgG pool were unsuccessful.

B and T cell responses elicited by monoclonal anti-idiotypic antibody (Ab2beta) mimicking gp43 from Paracoccidioides brasiliensis.

Paracoccidioidomycosis is a systemic mycosis endemic in Latin America, with a high prevalence in Brazil, Argentina, Colombia and Venezuela. The aetiological agent of disease is the thermal dimorphic fungus, Paracoccidioides brasiliensis. A glycoprotein of 43 kD (gp43) is the major antigen of P. brasiliensis. Antibodies directed to this antigen are detected in the sera of all patients with paracoccidioidomycosis (PCM). Recently, it has been shown that mice immunized with anti-gp43 monoclonal antibodies (MAbs) (Ab1), induce the idiotypic cascade in the gp43 system, which produced both, anti-Id antibodies (Ab2) and anti-anti-Id antibodies (Ab3). To further characterize the idiotypic cascade modulation in mice immunized with anti-gp43 MAb 17c, hybridomas were produced. Ab2 MAbs named 7.B12 inhibited (>95%) the binding of gp43 to MAb 17c (Ab1), suggesting that this anti-Id MAb bind to the idiotope, thus fulfilling the internal image criteria. To elucidate whether Ab2 MAb could act as antigen in serological assays, instead of gp43, sera from PCM patients were tested. Using an ELISA test, it was observed that antibodies from patients and not normal serum bound to Ab2. However, the ELISA test using Ab2 bound to the solid phase made possible to serologically monitor the patients after antifungal therapy, showing an equivalent curve when compared with ELISA test employing purified gp43. Our results also showed that, when mice were immunized with Ab2beta and their cells were exposed to gp43 in vitro, a T cell proliferation response was observed.

Transgenic expression of a human polyreactive Ig expressed in chronic lymphocytic leukemia generates memory-type B cells that respond to nonspecific immune activation.

We generated transgenic mice, designated SMI, expressing unmutated H and L chain Ig genes encoding a low-affinity, polyreactive human (h)IgM/kappa rheumatoid factor. These animals were compared with control AB29 transgenic mice expressing a hIgM/kappa rheumatoid factor specific for human IgG, with no detectable reactivity with mouse proteins. SMI B cells expressed significantly lower levels of surface hIgM/kappa than did the B cells of AB29 mice, but still could be induced to proliferate by surface Ig cross-linking in vitro and could be deleted with anti-Id mAb in vivo. Transgene-expressing B cells of AB29 mice had a B-2 phenotype and were located in the primary follicle. In contrast, a relatively high proportion of hIgM-expressing B cells of SMI mice had the phenotype of B-1 B cells in the peritoneum or marginal zone B cells in the spleen, where they were located in the periarteriolar sheath, marginal zone, and interfollicular areas that typically are populated by memory-type B cells. Although the relative proportions of transgene-expressing B cells in both types of transgenic mice declined with aging, SMI mice experienced progressive increases in the serum levels of IgM transgene protein over time. Finally, SMI transgene-expressing B cells, but not AB29 transgene-expressing B cells, were induced to secrete Ab when cultured with alloreactive T cells. These results indicate that expression of polyreactive autoantibodies can allow for development of B cells that are neither deleted nor rendered anergic, but instead have a phenotype of memory-type or Ag-experienced B cells that respond to nonspecific immune activation.

A Putative Host Cell Receptor for Tick-Borne Encephalitis Virus Identified by Anti-Idiotypic Antibodies and Virus Affinoblotting

Anti-idotypic monoclonal antibodies (anti-ID MAbs) were made against two mouse MAbs that neutralize the infectivity of the tick-borne encephalitis (TBE) virus. Three of the anti-ID MAbs (1) inhibited the binding of respective idiotypic MAb to the TBE virus antigen, (2) inhibited the infectivity of TBE virus when preincubated with virus-susceptible cells, and (3) bound to the surface of virus-susceptible but not virus-nonsusceptible cells. They recognized a 35-kD protein in immunoblotting analysis. Identification of this protein as a component of a putative TBE virus receptor was supported by the viroblot technique. In this assay, two polypeptide signals of 35 and 18 kD were obtained after incubation of blotted cell membrane proteins with the TBE virion antigen.