Murine Anti-idiotypic Antibody Research Articles

Impact of Peri-Transplant Rituximab and Host/Donor Fc Receptor Polymorphisms for Patients with Relapsed or Refractory CD20+ B-Cell Malignancies

We previously reported results of allogeneic hematopoietic cell transplantation (HCT) in 93 older or medically unfit patients with non-Hodgkin's lymphoma (NHL) or chronic lymphocytic lymphoma (CLL) after conditioning with fludarabine (FLU) and 2-3 Gy total body irradiation (TBI). Three-year survival ranged from 45% to 52% depending on the underlying histology and disease stage. The major complication in those studies was disease relapse (24% at 1 year). The current prospective study in 63 NHL patients tested the hypothesis that disease relapse could be reduced, and overall survival (OS) improved, by peri-transplant rituximab (RTX). Treatment (Figure 1) consisted of RTX (375 mg/m²/day) on days -3, +10, +24 and +38 in addition to 2-3 Gy TBI ± FLU (30 mg/m² × 3 days). As pharmacokinetics (PK) and optimal dosing of RTX in allogeneic HCT are undefined, we collected PK data to optimize RTX dosing. RTX levels were assayed with murine anti-idiotype monoclonal antibody (18C9) binding to RTX. Median therapeutic RTX levels of >25 μg/mL were achieved through day 84 after HCT, with peak median level of 200.5 μg/mL at day 64. RTX levels were not correlated with relapse or graft-vs-host disease (GVHD) rates. NHL patients had significantly higher median RTX levels than CLL patients (day 84, p=0.003, Figure 2). We collected data on donor and recipient FCγRIIIa receptor polymorphisms expressed predominantly on natural killer cells, thought to affect responsiveness to RTX. This was done to identify patients or donor/recipient pairs most likely to benefit from peri-transplant RTX. FCγRIIIa polymorphisms were determined by PCR and single-strand confirmation polymorphism (SSCP) from DNA isolated from patient cells. Recipients with F/F and V/F FCγRIIIa polymorphisms experienced higher relapse rates compared to recipients with V/V polymorphism (p=0.15). Overall, almost a third of pre-HCT F/F patients relapsed. Probably due to small numbers, no difference in outcome was found with V/V donor pairing for these patients. Results are shown in Figure 3. Relapse rates at 5 years were similar among RTX-treated patients and historical controls (32% vs, 28%; P=0.94). RTX-treated patients experienced higher 5-year OS and progression-free survival; (47% vs, 38%; P=0.13) (41 vs, 32%; P=0.12), respectively, but none of the differences was statistically significant. Non-relapse mortality was lower among RTX-treated patients versus controls (27% vs, 40; P=0.05), respectively; incidences of grade 2-4 acute GVHD and grade 3-4 acute GVHD were similar (60% vs, 57%, 13% vs. 17%); and 5-year chronic GVHD was higher among RTX-treated patients (62% vs, 47%). These findings suggest that for patients with relapsed or refractory NHL, peri-transplant RTX neither reduced relapse nor improved OS or other post-HCT parameters. The role of donor-recipient pairing by FCγRIIIa polymorphisms in outcome remains to be determined.

Optimization and qualification of a functional anti-drug antibody assay for HIV-1 bnAbs

The recent identification of human monoclonal antibodies with broad and potent neutralizing activity against HIV-1 (bnAbs) has resulted in substantial efforts to develop these molecules for clinical use in the prevention and treatment of HIV-1 infection. As with any protein therapeutic drug product, it is imperative to have qualified assays that can accurately detect and quantify anti-drug antibodies (ADA) that may develop in patients receiving passive administration of HIV-1 bnAbs. Here, we have optimized and qualified a functional assay to assess the potential of ADA to inhibit the neutralizing function of HIV-1 bnAbs. Using a modified version of the validated TZM-bl HIV-1 neutralization assay, murine anti-idiotype antibodies were utilized to optimize and evaluate parameters of linearity, range, limit of detection, specificity, and precision for measuring inhibitory ADA activity against multiple HIV-1 bnAbs that are in clinical development. We further demonstrate the utility of this assay for detecting naturally occurring ADA responses in non-human primates receiving passive administration of human bnAbs. This functional assay format complements binding-antibody ADA strategies being developed for HIV-1 bnAbs, and when utilized together, will support a multi-tiered approach for ADA testing that is compliant with Good Clinical Laboratory Practice (GCLP) procedures and FDA guidance.

Cancer vaccine characterization: From bench to clinic

BackgroundThe development of safe, effective, and affordable vaccines has become a global effort due to its vast impact on overall world health conditions. A brief overview of vaccine characterization techniques, especially in the area of high-resolution mass spectrometry, is presented. It is highly conceivable that the proper use of advanced technologies such as high-resolution mass spectrometry, along with the appropriate chemical and physical property evaluations, will yield tremendous in-depth scientific understanding for the characterization of vaccines in various stages of vaccine development. This work presents the physicochemical and biological characterization of cancer vaccine Racotumomab/alumina, a murine anti-idiotypic antibody that mimics N-glycolyl-GM3 gangliosides. This antibody has been tested as an anti-idiotypic cancer vaccine, adjuvated in Al(OH)3, in several clinical trials for melanoma, breast, and lung cancer. MethodsRacotumomab was obtained from ascites fluid, transferred to fermentation in stirred tank at 10L and followed to a scale up to 41L. The mass spectrometry was used for the determination of intact molecule, light and heavy chains masses; amino acids sequence analysis, N- and C-terminal, glycosylation and posttranslational modifications. Also we used the DLS for the size distribution and zeta potential analysis. The biological analyses were performed in mice and chickens. ResultsWe observed differences in glycosylation pattern, charge heterogeneity and structural stability between in vivo-produced and bioreactor-obtained Racotumomab products. Interestingly, these modifications had no significant impact on the immune responses elicited in two different animal models. ConclusionsWe are demonstrated that this approach could potentially be more efficient and effective for supporting vaccine research and development.

Comparability Assessments of Process Changes Made during Development of Anti-Idiotype Vaccine

Racotumomab monoclonal antibody is a murine anti-idiotypic antibody. This monoclonal antibody mimics N-glycolyl-GM3 gangliosides has been tested in several clinical trials Phase I/II for breast, melanoma and non-small cell lung cancer patients as an anti-idiotypic cancer vaccine. The early production process was performed in vivo from mice ascites fluid. This process was transferred to bioreactor-based method at pilot scale followed to the scale-up of the fermentation. In this work we present a comprehensive molecular characterization of racotumomab MAb produced by the two different production scales in order to determine the impact of the manufacturing process in vaccine performance. We observed differences in glycosylation pattern and charge heterogeneity between racotumomab produced in both scales. Interestingly, these modifications had no significant impact on biological activity elicited in chickens. So, changes in primary structure like glycosylation, charge heterogeneity and oxidation did not affect biological activity of the vaccine.

Physicochemical and biological characterization of 1E10 Anti-Idiotype vaccine

Background1E10 monoclonal antibody is a murine anti-idiotypic antibody that mimics N-glycolyl-GM3 gangliosides. This antibody has been tested as an anti-idiotypic cancer vaccine, adjuvated in Al(OH)3, in several clinical trials for melanoma, breast, and lung cancer. During early clinical development this mAb was obtained in vivo from mice ascites fluid. Currently, the production process of 1E10 is being transferred from the in vivo to a bioreactor-based method.ResultsHere, we present a comprehensive molecular and immunological characterization of 1E10 produced by the two different production processes in order to determine the impact of the manufacturing process in vaccine performance. We observed differences in glycosylation pattern, charge heterogeneity and structural stability between in vivo-produced 1E10 and bioreactor-obtained 1E10. Interestingly, these modifications had no significant impact on the immune responses elicited in two different animal models.ConclusionsChanges in 1E10 primary structure like glycosylation; asparagine deamidation and oxidation affected 1E10 structural stability but did not affect the immune response elicited in mice and chickens when compared to 1E10 produced in mice.

Molecular Simulations to Rationalize Humanized Ab2/3H6 Activity

The murine anti-idiotypic antibody 3H6 (Ab2/3H6) is directed against the human 2F5 antibody, which is capable of neutralizing HIV-1. Recently, four humanized Ab2/3H6 models have been developed in order to reduce the risk of human anti-mouse antibody (HAMA) responses in case of administration to humans. In this study, molecular dynamics simulations were performed on these models as well as on the murine Ab2/3H6 in solution and bound to 2F5, in order to rationalize the differences in binding affinities of the models towards 2F5. Analysis of these simulations suggested that the orientation and dynamics of the residues TYR54 and TYR103 of the heavy chain of Ab2/3H6 play an important role in these differences. Subsequently, the contribution of these residues to the binding affinity was quantified by applying free energy calculations.

Immunogenicity of the hu14.18-IL2 immunocytokine molecule in adults with melanoma and children with neuroblastoma.

Immunocytokine (IC) hu14.18-IL2 is a fusion protein of humanized antidisialoganglioside (GD2) antibody (hu14.18) and interleukin (IL)-2. Sixty-one melanoma and neuroblastoma patients received IC in phase I/Ib studies. Patient sera were examined in ELISA to determine if an anti-IC antibody response occurred during treatment. Serum was assayed for anti-idiotypic antibody (anti-id Ab) based on ability to bridge biotinylated hu14.18 to plate-bound hu14.18 and ability to inhibit binding of hu14.18 to GD2 antigen and/or murine anti-idiotypic antibody. ELISA was also used to detect antibodies to the Fc-IL2 end of hu14.18-IL2. Thirty-two patients (52%) developed an anti-idiotypic antibody response (absorbance, >0.7) in the bridge ELISA. Twelve patients (20%) had an intermediate response, whereas 17 patients (28%) were negative (adsorbance, <0.3). The development of antibody to hu14.18-IL2 detected in the bridge ELISA was not related to the dose of hu14.18-IL2. Twenty of 33 adult patients (61%) demonstrated an anti-idiotypic antibody response based on binding inhibition ELISA. The anti-idiotypic response was inversely correlated (P < 0.002) with IC measured during the second course of treatment, indicating that development of anti-idiotypic antibodies interfered with detection of circulating hu14.18-IL2. All patients developed some inhibitory activity in the binding inhibition assay designed to detect antibodies to the Fc-IL2 region of the IC. There was a positive correlation between the peak serum level of IC in course 1 and the anti-Fc-IL2 response. Patients treated with hu14.18-IL2 developed anti-idiotypic antibodies and anti Fc-IL2 antibodies. No association was seen between development of anti-IC antibodies and clinical toxicity.

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.

Partial Humanization and Characterization of an Anti-idiotypic Antibody against Monoclonal Antibody 2F5, a Potential HIV Vaccine?

We recently developed a murine anti-idiotypic antibody (Ab2/3H6) versus the human monoclonal antibody 2F5, one of a few antibodies yet known to neutralize a broad range of HIV-1 primary isolates. Ab2/3H6 was not only able to bind to the paratope of mAb 2F5 but also significantly inhibited the binding of 2F5 to its synthetic epitope ELDKWA on gp41. In the present work we describe the partial humanization, expression, and characterization of Ab2/3H6 variants followed by several corresponding interaction studies with 2F5. The results of these studies support the high specificity of the recombinantly expressed Ab2s to the idiotype. Apparent affinities were designated by end point measurement and were similar compared to the murine Ab2/3H6. Moreover, the inhibition potency of chimeric Ab2/3H6 analyzed by in vitro studies could be shown to be the same as that detected for the hybridoma-derived murine Ab2/3H6.

Phase I Study of Abagovomab in Patients with Epithelial Ovarian, Fallopian Tube, or Primary Peritoneal Cancer

To the Editor: In their article, Sabbatini et al. ([1][1]) describe the phase I data of abagovomab, a murine anti-idiotype antibody to the CA125-specific monoclonal antibody OC125. In their Introduction, they compare abagovomab's mechanism of action to oregovomab's, a murine Ab1 antibody to CA125.

Preface: antibody therapies for cancer

It took over a century to validate what Behring (1890) and Ehrlich (1900) have reasoned and predicted the application of antibody-mediated therapies against infectious diseases and cancer. Then, Porter (1967) and Edelman et al. (1968) reported the structure of the immunoglobulin and Kohler and Milstein (1975) described the production of hybridoma cell lines capable of producing monoclonal antibodies (mAbs). This technique began to enter clinical trials. Nadler et al. (1980) treated a patient with relapsed lymphoma with an mAb and was found to be safe and well-tolerated. Subsequent studies failed treatment with this antibody and yielded only short-lived responses (Ritz and Schlossman, 1982). Miller et al. (1982) reported a patient with B-cell lymphoma achieving a complete response after treatment with murine anti-idiotypic antibodies. Further research with generic murine antibodies failed to produce desirable remissions. During the next 15 years, the search for the right target and overcoming previously encountered obstacles began to be accomplished. mAbs have established themselves as one of the most important and fastest growing classes of therapeutic drugs (over 20 approved by the Food and Drug Administration (FDA)) in the treatment of non-malignant and malignant diseases (see reviews by Stern and Herrmann, 2005; Booy et al., 2006). This issue of Oncogene brings together reviews of many of the FDA approved mAbs for cancer, and also reviews on novel mAb-mediated strategies for cancer.

The anti-idiotypic antibody abagovomab in patients with recurrent ovarian cancer. A phase I trial of the AGO-OVAR

Abagovomab is a murine anti-idiotypic antibody against the antigen CA-125 which has been shown to elicit humoral and cellular immune responses against ovarian cancer (oc). This phase I trial included 36 patients with recurrent oc comparing two subcutaneous (s.c.) vaccination schedules: nine (group L) versus six injections (group S), 18 patients in each group. Four injections of 2.0 mg abagovomab were administered every 2 weeks and then two or five additional doses monthly. Primary endpoint was drop-out rate due to toxicity, and the secondary endpoint was analysis of immunological response. Treatment was completed in eight (44%) and 16 (89%) patients in groups L and S, respectively. Premature termination occurred due to patient withdrawal or disease progression. No treatment-limiting toxicities occurred in either group. The most common toxicity related to the vaccine was grade 1/2 local injection site reaction. Induction of Ab3 was observed in all evaluable patients. There were no differences between the groups with regard to induction of human anti-mouse antibody (P = 0.1006). IFNgamma-expressing CA125-specific CD8+ T-cells were significantly more frequent in group L, while there was no significant difference between CD4+ T-cells in the two groups. Abagovomab s.c. vaccination is safe and well tolerated. The long vaccination schedule tended to be more effective with regard to AB3-induction and cellular cytotoxicity.

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.

Therapeutic Vaccines for Colorectal Cancer

A number of cancer vaccine strategies for the treatment of colorectal cancer have entered clinical trials. Whole tumor cell vaccines have been developed from both patients’ autologous tumor cells as well as established allogeneic tumor cell lines. A vaccine consisting of autologous tumor cells along with bacillus Calmette-Guerin (BCG) has shown a potential clinical benefit in patients with stage II colon cancer. Other approaches using autologous tumor cells have involved transfection of primary tumor cells with cytokine genes. Allogeneic tumor cell vaccines have also been modified to express cytokine genes. Vectors have been studied extensively as a means of vaccine strategy. One tumor-associated antigen (TAA) that has been extensively studied in viral vector vaccines is carcinoembryonic antigen (CEA). A recombinant vaccinia virus containing the CEA transgene (rV-CEA) has been shown to elicit CEA-specific immune responses in advanced carcinoma patients. However, patients receiving multiple vaccinations had limited increases in CEA-specific responses by the third vaccination. This problem may be overcome by the use of non-replicating poxviruses, which have been shown in clinical trials to be safe and to elicit CEA-specific responses. However, recent clinical studies have shown that the optimal use of poxviruses is to prime with vaccinia, followed by boosts with avipox vectors. A recent randomized clinical trial showed that patients primed with rV-CEA and boosted with avipox-CEA had greater immune responses compared with patients receiving three 1-monthly avipox-CEA vaccinations followed by an rV-CEA vaccination. Furthermore, a statistically significant survival advantage was noted in the prime/boost arm. Ongoing studies are now incorporating the genes for costimulatory molecules along with TAA in these vectors. Another vaccine strategy involving TAA that is currently in clinical trials for colorectal cancer is the peptide vaccine. Dendritic cells (DCs) are considered to be the most potent antigen-presenting cell, thus providing an attractive modality for cancer vaccines. In addition to using DCs for peptide-based vaccines, a number of other strategies, including transfection with messenger RNA, have produced specific T-cell responses in clinical trials. In addition, several clinical trials using murine anti-idiotype antibodies as vaccines for patients with advanced colorectal cancer have shown both immunologic responses as well as clinical responses.

Preparation of humanized ovarian carcinoma anti-idiotypic minibody.

Murine anti-idiotypic monoclonal antibody (MAb) 6B11 mimicking the tumor-associated antigen OC166-9 is used as a vaccine for the induction of an anti-tumoral immunity in experiments of in vitro and in vivo animal model with ovarian carcinoma. In this article, we have humanized 6B11 anti-idiotypic minibody using overlap polymerase chain reaction (PCR) and DNA recombinant technique, prokaryotic expression vector was produced by genetic fusion of 6B11V(L)-V(H) to human IgG1 hinge and CH3 region. Transformed E. coli BL21(DE3) were propagated and induced by isopropyl-beta D-thiogalactopyranoside (IPTG). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that a protein band with molecular weight of 50kD appeared as the expected size after transformation. Molecular weight of 100 kDa may be examined by electrophoresis in nondenaturing systems. The fusion protein was analyzed with enzyme-linked immunosorbant assay (ELISA), inhibition ELISA tests and Western blot, respectively. The humanized anti-idiotype minibody showed capacity of bivalent binding to ovarian cancer MAb COC166-9 and goat anti-human immunoglobulin IgG1. It is useful reagents for clinical use.

Monoclonal antibody therapy for lymphoma: an update.

The purpose of this article is to review the historical development and recent advances in the application of monoclonal antibodies for the treatment of lymphoma. The history of clinical applications of monoclonal antibodies has been intertwined with that of lymphomas. The first report of a complete remission in 1981 described a patient with follicular lymphoma who was treated with a murine anti-idiotype antibody. Later that decade there appeared additional encouraging reports of radiolabeled monoclonal antibodies, immunotoxins, and other antibodies with antitumor effects against lymphoma and chronic lymphocytic leukemia. Monoclonal antibodies as a treatment of malignancy became reality in late 1997 when the US Food and Drug Administration approved the anti-CD20 chimeric monoclonal antibody rituximab for the treatment of B-cell lymphoma. Since that time an anti-CD25 monoclonal antibody (dacliximab) and an anti-CD25 immunotoxin fusion product (denileukin diftitox) have become clinically available. Several radio- labeled antibodies, including the murine anti-CD20 products (131)I-tositumomab and (90)Y-ibritumomab tiuxetan, are in advanced stages of clinical testing as are other unlabeled monoclonal antibodies with antilymphoma activity. Other antilymphoma immunotoxins that react with CD25, CD19, and CD22 also have shown promise. The therapeutic arsenal against lymphoma has been significantly changed by the addition of these antibody products that are active as single agents, and are synergistic, additive, or both with other antilymphoma treatments.

Molecular characterization of the VH1-specific variable region determinants recognized by anti-idiotypic monoclonal antibodies G6 and G8.

Mutational analysis was used to determine the structural basis for the binding of the murine anti-idiotypic (anti-Id) monoclonal antibodies (MoAb) G6 and G8 to VH1-encoded antibodies. The MoAb G6 binds a cross-reactive idiotope present on the heavy (H) chains of immunoglobulins (Ig) encoded by 51p1-related gene segments, but not those encoded by hv1263-related gene segments. Gene segments 51p1 (DP-10) and hv1263 differ by only four amino acids; three in complementarity-determining region 2 (CDR2), and one in framework region 3 (FR3). The MoAb G8 also binds 51p1-related sequences, although it is undetermined whether G8 can bind hv1263-related sequences. In order to localise the Ids recognized by MoAbs G6 and G8 on 51p1-encoded antibodies, recombinant antibodies containing H-chain mutants were expressed in insect cells. Idiotypic analysis on the expressed recombinant proteins definitively localised the reactivity in each molecule. These studies should be important in the structural appreciation for critical serological reagents.

Production of a single-chain fragment of the murine anti-idiotypic antibody ACA125 as phage-displayed and soluble antibody by recombinant phage antibody technique.

The F(ab')2 fragment of the murine monoclonal anti-idiotypic antibody ACA125 mimicking the tumor-associated antigen CA125 is used as a vaccine for the induction of an anti-tumoral immunity in patients with ovarian carcinoma. We tried to generate a single-chain fragment (ScFv) composed of ACA125 heavy- and light-chain variable domains connected by a polypeptide linker as an alternative to the corresponding F(ab')2 fragment. Heavy- and light-chain genes of antibody-producing mouse hybridoma cell line were amplified separately and assembled into a ScFv gene with linker DNA by the polymerase chain reaction (PCR). The ScFv gene was ligated into the phagemid vector pCANTAB5E, which allows the production of both phage-displayed and soluble ScFv. Transformed Escherichia coli TG1 cells were infected with M13K07 helper phage to yield recombinant phage, which display ScFv fragments as a g3p fusion protein on the surface of the filamentous phage M13. Recombinant phages could be selected by binding to the idiotypic antibody OC125 after one round of panning and directly used to reinfect E. coli TG1 cells. The E. coli nonsuppressor strain HB2151 was infected with an antigen-positive phage clone, previously screened by enzyme-linked immunosorbent assay (ELISA), to express soluble ScFv fragments. Functional soluble ScFv binding to the idiotypic antibody OC125 F(ab')2 could be detected in the bacterial periplasm by Western blot and ELISA. The variable heavy- and light-chain genes of the ACA125 ScFv fragment were further sequenced and compared with known antibody sequences.

A monoclonal antiidiotypic antibody ACA 125 mimicking the tumor-associated antigen CA 125 for immunotherapy of ovarian cancer.

A new concept of oncological immunotherapy comprises the attempt to trigger the immune system of the host into a response against tumor cells. Antiidiotypic antibodies bearing the internal image of an antigen expressed on the surface of the tumor seem to be most suited for this purpose. We have generated a murine antiidiotypic antibody (ACA 125) functionally imitating the tumor-associated antigen CA 125, which can be detected in about 80% of ovarian carcinomas. The hybridoma cell was adapted to serum-free medium and antibody was produced in a hollow fiber cell culture system (Technomouse). ACA 125 (Ab2) shows high affinity for the paratope of Ab1 (affinity constant: 2.3 x 10(9) liters/mol) and binding of Ab2 to Ab1 is completely inhibited by the nominal antigen. Application of F(ab')2 fragments of ACA 125 to rats lead to an anti-CA 125 immunity by production of IgG and IgM antiantiidiotypic antibodies (Ab3) that bind to both ACA 125 and CA 125. Furthermore the induction of a non-MHC-restricted cell-mediated cytotoxicity for human ovarian adenocarcinoma cell line NIH-OVCAR3 (expressing CA 125 on its surface) could be proved; additionally complement-dependent cytotoxicity (CDC) as well as an antibody-dependent cellular cytotoxicity (ADCC) was observed. Thus, monoclonal antiidiotypic antibody ACA 125 fulfills recent criteria for an antibody, which might be successful in immunotherapy using the anti-idiotypic network approach.

Monoclonal Anti-idiotypic Antibodies against Human Anti-thyroglobulin Autoantibodies Recognize Idiotopes Shared by Disease-Associated and Natural Anti-thyroglobulin Autoantibodies

Monoclonal murine anti-idiotypic antibodies generated against purified human anti-thyroglobulin (TG) antibodies from a patient with Hashimoto's thyroidiris recognized two major clusters of idiotopes (1C2 and 1B1) on human anti-thyroglobulin antibodies which were framework and paratope-related. A survey of affinity-purified autoantibodies from Hashimoto's patients indicated that the 1C2 and 1B1 idiotopes are expressed on the disease-associated anti-TG antibodies from several patients. 1C2 and 1B1 idiotopes are also detected on affinity-purified anti-TG antibodies isolated from serum of healthy donors and with therapeutic preparations of immunoglobulins obtained from plasma pools of healthy individuals. The observation that both framework and paratope-related idiotopes are shared by natural and patients' autoantibodies suggests that the anti-TG autoantibodies found in sera of patients with Hashimoto's disease represent an abnormal expansion of normal autoreactive B cell clones rather than the emergency of an antigen-driven, disease-specific B cell response.