Idiotypic Determinants Research Articles

What Can We Learn from Research with Monoclonal 1F7?

1F7 is a monoclonal antibody that recognizes an idiotypic determinant expressed on primate antibodies binding to HIV-1 and hepatitis C proteins. This monoclonal antibody was used as a tool to dissect the immune response in humans infected with HIV-1 and hepatitis B. Furthermore, 1F7 was also used to manipulate the immune response against HIV-1 in macaques. The generation of a monoclonal antibody describing a network suggests similar antibodies could be developed as tools to dissect entangled networks in autoimmune diseases and allergic reactions. This review discusses the body of work done with 1F7 in the light of contemporary immunology.

New insights into B cells as antigen presenting cells

In addition to their role as antibody producing cells, B cells make a critical contribution to adaptive immune responses by functioning as professional antigen-presenting cells (APC). Distinctive features of B cells as APC include the expression of the B cell receptor (BCR) for antigen and regulated expression of HLA-DO. Here, we discuss recent progress in investigation of B cells as APC. We start with an update on the canonical MHC class II antigen presentation pathway in B cells and alternative pathways, including generation of extracellular vesicles. Turning to APC function, we highlight the roles of B cells as thymic APC, as APC for T follicular helper (TFH), as APC for CD4 memory T cells and as presenters of idiotypic BCR determinants. We also note recent examples that link B cell Ag-presentation to disease. Emerging evidence indicates that, in addition to unique features of B cells compared to other professional APC, there is appreciable heterogeneity among B cells, arising from, for example, B cell activation state or the microenvironment.

The Impact of the Hybridoma Technology on the R&D of Idiotypic Antibodies.

The PubMed data set was scanned with the title and abstract term "Idiotype" followed by secondary searches with "Vaccine" and "Clinical trial." The retrieved references were analyzed from the period before and after hybridoma technology (1975). In 1963, Oudin and Kunkel discovered that antibodies against antibodies can be raised to identify determinants unique to an antibody termed idiotype or individual antigenic determinant. Two laboratories reported that anti-idiotypic antibodies can suppress specific antibody responses in mice. In 1974, Jerne proposed a network of idiotypes and anti-idiotypes and the functionality of the idiotype network was confirmed. This prompted the proposal of a symmetrical regulatory immune response. By 1989, the concept and the functional parameters of the immune idiotype network were established in the prehybridoma period. It was not until 1981 that monoclonal anti-idiotypic antibodies were used as tools to study the expression of idiotypic determinants on antibodies and to categorize functional properties in the immune network as network antigens in 1989. Hybridoma-generated monoclonal anti-idiotypic antibodies provided the tools to precisely identify different idiotypic regions on antibodies and test these as targets to induce network cascades. The initial distinction of Ab2s as alpha and beta were expanded to include gamma and delta. The initial concept of Ab2beta being an antigen internal image, used as vaccine, was challenged showing that targeting all idiotopes on B cell receptors can induce specific antibodies. After the discovery of the hybridoma technology a wave of idiotype topic publications occurred, that declined by 2015. In 1985, in this wave of reports on anti-idiotypes, their importance to vaccines dominated. These vaccines targeted in animal models parasite, bacterial, and viral diseases, and cancer. The reported data indicated a therapeutic response in inbred mice. The issue of idiotype matching between mouse haplotypes of vaccine origin and treated mice were raised. In 1995, the human clinical trials in different cancers using anti-Id vaccines were reported. Only one such vaccine received conditional approval in Argentina and Cuba, whereas the other trials failed in phase II and III. The reasons for this failure were subsequently discussed. Although the use of the Milstein and Kohler hybridoma technology and subsequently alternative methods to produce monoclonal animal and human antibodies created a new class of drugs, commonly referred as "Biological," it failed on the promise therapeutic of anti-Id vaccines.

A Novel Therapeutic DNA Vaccine Elicits Reduction of Tumor Clones and Favorable Perturbations in the Immune Microenvironment in Patients (pts) with Untreated Smoldering Waldenström Macroglobulinemia (sWM)

A Novel Therapeutic DNA Vaccine Elicits Reduction of Tumor Clones and Favorable Perturbations in the Immune Microenvironment in Patients (pts) with Untreated Smoldering Waldenström Macroglobulinemia (sWM)

Phase I trial of a novel DNA vaccine in patients (pts) with smoldering Waldenstrom macroglobulinemia (sWM).

8050 Background: Idiotypic determinants of the surface immunoglobulin (Ig) associated with a given pt’s B-cell lymphoma are unique to that tumor, and thus are a tumor-specific marker. This study aims to use an idiotype DNA vaccine to lengthen the smoldering phase of WM without inducing cross-resistance to available therapies. Administered vaccine used recombinant plasmid DNA encoding a fusion protein, consisting of autologous lymphoma scFv (pt-specific idiotype) and human CCL20 (macrophage inflammatory protein-3 alpha - MIP-3α) chemokine. Targeted delivery of this fusion protein to APCs, and subsequent processing and presentation, is hypothesized to break tolerance and generate an immune response against the idiotype, promoting eradication of antigen-expressing B-cell lymphoma cells. Methods: Pts with sWM received 3 i.d. vaccinations of pt-specific DNA vaccine at 4-week (wk) intervals (wks 0, 4 and 8). Two dose levels (500µg; 2500µg) were evaluated in a 3+3 design. Primary objective: to evaluate the vaccine’s safety and identify it’s MTD. Secondary objectives: 1) to assess immunogenicity of the vaccine 2) to determine time to symptomatic WM. Results: Between 1/2016 - 1/2019, 9 pts (7 men) were treated (500 µg: n = 3; 2500µg: n = 6). Median age at enrollment was 67 yrs (range 56-78); median time from diagnosis to 1st vaccination was 26.5 mos (8.8-120.9). MYD88 L265P + (6 pts). CXCR4 WHIM + (1 pt). With median follow up of 26.5 months (range: 8-36.4), all pts remain alive. Seven have stable disease; 2 progressed to symptomatic WM (8 mos. (1pt) and 26 mos. (1pt) from 1st vaccination). All pts completed planned therapy. No DLTs or Grade 4 AEs occurred. Ten mos. after the 3rd vaccination, 1 pt had a grade 3 pleural effusion and leukopenia with an increase in rheumatoid factor (23.1 IU/mL [normal range 0.0-15.9]) and ANA titer of 1:80; all resolved within 2 mos. Grade 1-2 AEs ( > 3pts): leukopenia (6), nausea (5), anemia (4), increased creatinine (4), fatigue (4). Conclusions: Idiotype (scFv-CCL20) DNA vaccine therapy appears to be safe in pts with sWM. Results of immunogenicity assays are underway, and will inform whether tumor specific immune responses are induced. Additional follow up is required to determine time to symptomatic WM. Clinical trial information: NCT01209871.

Phase I study of an active immunotherapy for asymptomatic phase Lymphoplasmacytic lymphoma with DNA vaccines encoding antigen-chemokine fusion: study protocol

BackgroundThere is now a renewed interest in cancer vaccines. Patients responding to immune checkpoint blockade usually bear tumors that are heavily infiltrated by T cells and express a high load of neoantigens, indicating that the immune system is involved in the therapeutic effect of these agents; this finding strongly supports the use of cancer vaccine strategies. Lymphoplasmacytic lymphoma (LPL) is a low grade, incurable disease featuring an abnormal proliferation of Immunoglobulin (Ig)-producing malignant cells. Asymptomatic patients are currently managed by a “watchful waiting” approach, as available therapies provide no survival advantage if started before symptoms develop. Idiotypic determinants of a lymphoma surface Ig, formed by the interaction of the variable regions of heavy and light chains, can be used as a tumor-specific marker and effective vaccination using idiotypes was demonstrated in a positive controlled phase III trial.MethodsThese variable region genes can be cloned and used as a DNA vaccine, a delivery system holding tremendous potential for streamlining vaccine production. To increase vaccination potency, we are targeting antigen-presenting cells (APCs) by fusing the antigen with a sequence encoding a chemokine (MIP-3α), which binds an endocytic surface receptor on APCs. Asymptomatic phase LPL is an excellent model to test our vaccine since patients have not received chemotherapeutics that interfere with innate immune function and have low tumor burden. We are evaluating the safety of this next-generation DNA vaccine in a first-in-human clinical trial currently enrolling asymptomatic LPL patients. To elucidate the mode of action of this vaccine, we will assess its ability to generate tumor-specific immune responses and examine changes in the immune profile of both the peripheral blood and bone marrow.DiscussionThis vaccine could shift the current paradigm of clinical management for patients with asymptomatic LPL and inform development of other personalized approaches.Trial registrationClinicalTrials.gov identifier NCT01209871; registered on September 24, 2010.

Description and characterization of a unique human immunoglobulin G1 kappa idiotype found in placental tissue

Does maternal IgG found in placental tissue provide the fetus with more than just humoral immunity? To address this question, the IgGs from twelve placentas were studied and four of these samples were examined using mass spectrometry which revealed an IgG1k idiotype. A special dodecapeptide portion of the 3rd framework region of the VH chain sequence was identified as an idiotypic determinant in these placental- IgG1k (p-IgG1k) and referred to as peptideX2 and found to have biological activity. Antiserum to peptideX2 was made and then used with Western Immunoblotting to show that this unique H chain (containing peptideX2) appears to be present in all p-IgG tested and in all subjects tested. It appears that the placenta contains not only conventional polyclonal maternal IgGs but also an idiotypic population of maternal IgG1k which binds to TLR2>TLR4 via the epitope “peptideX2″ and promotes IL-6, TNFα, and IL-10 production and may play a role in maternal-fetal tolerance.

Immunization of sheep with DNA coding for the variable region of anti-idiotypic antibody generates humoral and cell mediated immune responses specific for peste des petits ruminants virus.

Peste des Petits Ruminants (PPR) is a highly contagious transboundary animal disease of wild and domestic small ruminants and notifiable byworld organization for animal health (OIE). It constitutes a serious threat to livestock production in many developing countries, in western Africa and southern Asia. The disease is caused by peste des petits ruminants virus (PPRV), a negative sense, single stranded RNAvirus belonging to the genus Morbillivirus of the family Paramyxoviridae (Gibbs et al. 1979). PPRV causes huge disease burden on agriculture across developing world, affecting small ruminant production and in turn increases poverty in some of the poorest parts of the world (Banyard et al. 2010). In PPR endemic regions of the world, conventional live attenuated vaccines are being used to vaccinate small ruminants (Diallo et al. 1989). Currently, the original Nigerian strain of PPRV (Nig 75/1) is used in the African countries (Diallo 2003). In India, three Indian strains of PPRV have been attenuated and shown to provide protective immunity (Saravanan et al. 2010). Although the live attenuated vaccines are efficacious, they suffer from the same draw back as that of live attenuated Rinderpest vaccine in terms of heat stability. This puts severe constraints on the coverage of small ruminant’s population during vaccination campaigns. Although there is one report of a method for making a heat stable live attenuated vaccine (Worrwall et al. 2000), such a thermo stable vaccine has not been put to use or field tested. Another major disadvantage of using live attenuated vaccine is that the antibody responses they induce in animals cannot be distinguished from those following a natural infection. This makes sero-epidemio surveillance of the disease impossible in endemic areas where a vaccination programme has been or is being implemented. The immune response to a given antigen can be regulated by a number of idiotypic determinants (Ids), and its counterpart, an anti-idiotypic antibody (anti-Id) or Ab2 (Jerne 1974). An idiotype is located on the variable regions of antibody molecules. An internal image anti-Id, Ab2 β recognizes an idiotypic determinant within the antigen combining site and bears a structural resemblance to the original antigen (Bona and Kohler 1984). The internal image Ab2 β antibodies have been shown to induce specific immune responses to hepatitis B surface antigen in chimpanzees (Kennedy et al. 1986) and as a vaccine candidate for the control of bluetongue infection by functionally mimicking VP7 antigen of bluetongue virus (Zhou and Lin 1997). We had earlier demonstrated that an internal image Ab2 against Rinderpest virus/Peste des petits Ruminants virus H/HN protein elicits virus neutralizing antibodies as well as T cell responses in the mouse model (Vani et al. 2007a). Further, using the mouse model, DNA encoding VH region of Ab2 elicited both neutralizing antibody and T cell responses, in the complete absence of viral antigen and these responses were long lasting (Vani et al. 2007b). * M. S. Shaila shaila@mcbl.iisc.ernet.in

Functional Analysis of the Anti-adalimumab Response Using Patient-derived Monoclonal Antibodies

The production of antibodies to adalimumab in autoimmune patients treated with adalimumab is shown to diminish treatment efficacy. We previously showed that these antibodies are almost exclusively neutralizing, indicating a restricted response. Here, we investigated the characteristics of a panel of patient-derived monoclonal antibodies for binding to adalimumab. Single B-cells were isolated from two patients, cultured, and screened for adalimumab specificity. Analysis of variable region sequences of 16 clones suggests that the immune response against adalimumab is broad, involving multiple B-cell clones each using different combinations of V(D)J segments. A strong bias for replacement mutations in the complementarity determining regions was found, indicating an antigen-driven response. We recombinantly expressed 11 different monoclonal antibodies and investigated their affinity and specificity. All clones except one are of high affinity (Kd between 0.6 and 233 pm) and compete with TNF as well as each other for binding to adalimumab. However, binding to a panel of single-point mutants of adalimumab indicates markedly different fine specificities that also result in a differential tendency of each clone to form dimeric and multimeric immune complexes. We conclude that although all anti-adalimumab antibodies compete for binding to TNF, the response is clonally diverse and involves multiple epitopes on adalimumab. These results are important for understanding the relationship between self and non-self or idiotypic determinants on therapeutic antibodies and their potential immunogenicity.

Abstract 2891: Bench-to-bedside development of a novel idiotype vaccine against lymphoma

Abstract Available therapies for lymphoplasmacytic lymphoma (LPL) provide no survival advantage if started before symptoms of end organ damage develop. Current recommendations are to follow a program of observation while patients are in the asymptomatic phase of disease. In this clinical study we will use idiotypic determinants of B-cell lymphoma surface immunoglobulins as tumor-specific antigens (idiotype) to develop a patient-specific vaccine against LPL. By activating the host immune system through vaccination to eradicate tumor cells, we postulate that disease control of asymptomatic phase lymphoplasmacytic lymphoma can be maintained. This novel 2nd generation idiotype vaccine was initially developed by genetic fusion of idiotype antigen in single-chain format with a pro-inflammatory chemokine. The central hypothesis is that antitumor immunity can be triggered by targeting antigen delivery to antigen-presenting cells in vivo by chemokine receptor-mediated binding, uptake and processing of idiotype antigens for more efficient presentation to T cells. A large body of preclinical data demonstrated that the immunogenecity of the idiotype antigen was considerably enhanced by such a genetic modification by a mechanism of facilitating antigen presentation. As a consequence, vaccine-induced prophylactic and therapeutic antitumor effects were significantly potentiated. To translate our vaccine therapy from bench to bedside, we established a CLP laboratory for vaccine preparation. Furthermore, we developed reliable technology to differentiate lymphoma idiotype antigens from idiotypic cell-surface immunoglobulins on normal B cells. Using this technology, we successfully cloned the cDNA encoding variable regions of heavy and light chains of LPL idiotype, and generated patient-specific plasmid constructs containing LPL idiotype single chain in fusion with macrophage inflammatory protein 3 alpha (MIP3a). All three engineering rounds of down-stream GMP amplification of clinical-grade plasmid DNA have been accomplished, which eventually paves the way for us to take a lab-grown agent to a first-in-human clinical trial. Citation Format: Hong Qin, Soungchul Cha, Sheetal S. Rao, Kunhwa Kim, Dongho Gwak, Sung-doo Kim, Sapna R. Parshottam, Sheeba K. Thomas, Larry W. Kwak. Bench-to-bedside development of a novel idiotype vaccine against lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2891. doi:10.1158/1538-7445.AM2014-2891

Lymphoma Vaccine Therapy: Next Steps After a Positive, Controlled Phase III Clinical Trial

Many of the efforts toward developing vaccines against human malignancies have been frustrated by the lack of identification of a tumor-specific antigen that would allow tumor cells to be distinguished from normal cells. Idiotypic determinants of the surface immunoglobulin (Ig) associated with a given patient's B-cell lymphoma are unique to that tumor, and can thus serve as a tumor-specific marker. When conjugated to the immune carrier keyhole limpet hemocyanin (KLH), vaccination with an idiotype protein vaccine has been able to improve length of freedom from disease relapse in patients with follicular lymphoma (FL) in a minimal residual disease (MRD) state after induction therapy, as demonstrated in a recent randomized, controlled phase III trial. In addition to predictive biomarker discovery, using residual autologous tumor and blood samples from patients vaccinated on the phase III trial, we have now developed a next generation idiotype DNA vaccine with the goal of reducing vaccine production time while maintaining efficacy. A first-in-human study is planned to evaluate its use in patients with asymptomatic phase lymphoplasmacytic lymphoma.

Design and Pre-Clinical Development of Epitope-based DNA Vaccines Against B-Cell Lymphoma

Optimally designed cancer vaccines should combine the best tumor antigens with the most effective immunotherapy agents and delivery strategies to achieve positive clinical results. The unique immunoglobulin (Ig) idiotype on the surface of each B-cell lymphoma represents an ideal tumor-specific antigen for use as a cancer vaccine. It has been theorized that effective cancer vaccines can be developed using the minimum essential subset of T cell and B cell epitopes that comprise the 'immunome', the universe of neoplasm-derived peptides that interface with B and T cells of the host immune system. Idiotypic antigenic determinants of a B-cell lymphoma lie within the hypervariable regions and mainly within the complementarity-determining regions (CDR)s 3. Thus, the CDR3s are considered a "hot spot" of particular interest for construction of subunit vaccines. DNA vaccines, whose safety and tolerability are substantiated in completed and ongoing clinical trials, have emerged as a novel lymphoma vaccine formulation for antigen-specific immunotherapy. The molecular precision tools offered by gene-based vaccines allow to explore the use of CDR3 sequence as an anti-lymphoma vaccine.

Vaccination with IgM but Not IgG Idiotype Prolongs Remission Duration In Follicular Lymphoma Patients

AbstractAbstract 429 Background:Tumor-derived idiotype (Id) protein conjugated to keyhole limpet hemocyanin (KLH) administered with granulocyte-monocyte colony-stimulating factor (GM-CSF) can induce follicular lymphoma (FL)-specific immune responses that target tumor-specific antigenic determinants within the tumor cell's unique immunoglobulin (Ig) variable region (Fv). While Fv idiotypic determinants serve as specific tumor antigens, preclinical evidence suggests that the isotype of the Ig constant region (Fc) may independently influence the immunogenicity of hybridoma-derived immunoglobulins. Whereas Ids that have switched to IgG were tolerogenic, Ids of their IgM progenitors were highly immunogenic (Reitan et al. Proc Natl Acad Sci U S A, 2002). Thus, we examined the clinical impact of tumor Ig isotype on disease-free survival (DFS) within a prospective randomized double-blind placebo-controlled multicenter phase III study of patient-specific tumor heterohybridoma-derived Id vaccine in advanced stage previously untreated FL patients with a lymph node adequate for vaccine production (≥ 2cm). Methods:Patients achieving complete response (CR) or complete response unconfirmed (CRu) after chemotherapy (PACE: prednisone, doxorubicin, cyclophosphamide, etoposide) were stratified by International Prognostic Index risk group and number of cycles of PACE and randomized 2:1 to receive tumor isotype-matched Id vaccine (Id-KLH/GM-CSF) or control (KLH/GM-CSF). The primary endpoint was DFS. Results:234 patients were enrolled; 177 (76%) achieved CR/CRu and were randomized. Of 177 randomized patients, 117 maintained CR/CRu ≥ 6 months per protocol requirement and then received at least one dose of vaccine, 55 relapsed before vaccination, 4 were vaccine manufacturing failures, and 1 violated protocol. Patients who received ≥ 1 vaccine dose constituted the modified intent-to-treat population for determination of efficacy. 76 patients received Id vaccine and 41 patients received the control. At a median follow-up of 56.6 months (range 12.6 – 89.3), median DFS after randomization for the Id vaccine arm was 44.2 months versus 30.6 months for the control arm (P = 0.048; HR = 0.62; 95% CI: 0.39–0.99). Among 76 patients receiving Id vaccine, 36 received IgM-Id vaccines and 40 received IgG-Id vaccines corresponding to their tumor Ig isotype. Of 41 patients receiving control, 25 had tumors with IgM isotype and 15 had tumors with IgG isotype; 1 patient had a tumor with mixed IgM/IgG isotypes. Among 36 patients with IgM tumor isotype receiving an IgM-Id vaccine, median time to relapse after randomization was 50.6 months, versus 27.1 months in the IgM tumor isotype control-treated patients (log-rank p=0.002; HR = 0.36 (p=0.003); [95% CI: 0.19–0.71]) [Figure 1]. Among 40 patients with IgG tumor isotype receiving an IgG-Id vaccine, median time to relapse after randomization was 35.1 months, versus 32.4 months in control-treated patients with IgG tumor isotype (log-rank p = 0.807; HR = 1.1 (p=0.807); [95% CI: 0.50–2.44]) [Figure 2]. It must be noted that although this trial was not powered to address such subsetting, the dramatically different results suggest that the treatment effect is different in the two groups, with a surprisingly small p-value of 0.085. [Display omitted] Conclusions:Our results suggest that the isotype of an Id vaccine may influence DFS in FL patients vaccinated in first complete remission. Unexpectedly, we observed that the IgM-Id vaccine significantly prolonged remission duration in comparison to the IgG-Id vaccine. Compared to other phase III Id vaccine trials that used recombinant Id vaccines with IgG constant regions for all patients, the positive outcome of our study may reflect the use of hybridomas to produce Id protein with variable and constant regions identical to patient tumor Ig. Additional studies are expected to further evaluate the effect of Id vaccine isotype on clinical outcome in FL and other B-cell malignancies. If confirmed, our findings will have profound implications on Id vaccine production strategies and clinical development. Disclosures: Schuster:Biovest International, Inc.: Research Funding. Santos:Biovest International, Inc.: Employment. Neelapu:Biovest International, Inc.: Research Funding. Popa:Biovest International, Inc.: Employment. McCord:Biovest International, Inc.: Employment. Kwak:Biovest International, Inc.: Consultancy.

In vivo treatment with a monoclonal chimeric anti-CD4 antibody results in prolonged depletion of circulating CD4+ cells in chimpanzees.

Chimeric M-T412 (cM-T412), an anti-CD4 antibody, was tolerated in chimpanzees at a dosage of 5 mg/kg per day for up to 7 consecutive days, or 5 mg/kg per dose, twice weekly for 4 weeks. All cM-T412-treated chimpanzees showed a prolonged CD4-cell depression. Weak chimpanzee antibody responses to chimeric M-T412 were observed. One of the chimpanzees on the biweekly dosage regimen exhibited a hypersensitivity reaction immediately after receiving its seventh dose. Following supportive treatment, the animal recovered and remained asymptomatic during the non-treatment observation period. The hypersensitivity reaction was not an unexpected response considering the animal received repeated intermittent i.v. administration of a foreign protein. This animal also showed a chimpanzee antibody response to chimeric M-T412 after the seventh dose. Chimeric M-T412 also induced an anti-cM-T412 response in some of the other animals. The level of this response was lower than the anti-mouse responses observed in animals treated with murine anti-CD4. Moreover, the anti-cM-T412 response was mainly directed to idiotypic determinants. The decrease in CD4+ cells observed for all chimeric M-T412-treated chimpanzees is an expected effect of the anti-CD4 antibody. The duration of this CD4+ cell decrease is, however, much longer than observed for other CD4-specific MoAbs described. No selective loss of either memory or naive CD4+ cells was observed after either the single, 7-day or twice-weekly treatments. The CD4+ cell depression was reversible, although individual variation in time to recovery was observed. Therefore, cM-T412 could be a good candidate for clinical use in autoimmune conditions.

Cross-reactive idiotypes in sera from patients with leprosy, lupus and Lyme disease and from healthy individuals

Monoclonal IgM autoantibodies have previously been generated from a patient with lepromatous leprosy. Polyclonal anti-idiotypes raised against two of these monoclonal antibodies (8E7 and TH9) were used in an immunoassay to detect the presence of idiotype in human serum. The anti-idiotypes recognize different but overlapping sets of idiotypic determinants, some of which are present on antibodies which bind to Mycobacterium leprae. Sera were tested from 16 individuals with leprosy, 45 with systemic lupus erythematosus, 20 with Lyme disease, and 80 healthy subjects. Positive sera were detected in all groups (seven, two, three, and four, respectively). In most cases the serum bound to both anti-idiotypes, the idiotype being present in the IgM and/or IgG fraction. Levels of the two idiotypes varied independently of total serum IgG concentration and, in serial samples from one patient, independently of each other. The results indicate that 8E7 and TH9 may be representative of serum antibodies which are commonly expressed in leprosy, but may also be expressed in other diseases and in health; and they suggest that such serum antibodies are encoded by a widely shared set of variable region genes.

Anti-idiotypic Immunotherapy in Follicular Lymphoma Patients

A specific immune response against the individual idiotypic determinants of clonal immunoglobulins can be elicited in patients with B-cell malignancies. We analyze the clinical outcome and the presence of tumor cells in the blood of 8 patients with follicular lymphoma, vaccinated with autologous idiotype protein from their lymphoma cells. After a median follow-up of 90 months (range: 54 to 128), all patients, except 1, remain in complete clinical remission and 5 are in complete molecular remission. Our results suggest that idiotype vaccination induces long-lasting clinical and molecular remissions and constitutes a potential curative treatment in follicular lymphoma patients.

Etiology of sperm immunity in women

Sperm immunity in females can reduce the likelihood of natural conception, and sperm antibodies from female sera have been shown to inhibit IVF in humans and in several animal models. The etiology of sperm immunity in human females is unknown, but several possible mechanisms have been proposed, including cross-reactivity with microbial antigens and interferon gamma-mediated potentiation of the antisperm immune response in women whose male partners have sperm autoantibodies in their semen. This article reviews these ideas and postulates a novel hypothesis based on the potential for the generation of anti-idiotype antibodies in women whose partners have sperm antibodies in their semen.

In Vivo Depletion of Cynomolgus Monkey B Cells Targeted by a Novel Monoclonal Antibody (Xi-20H5) Specific for a Shared Portion of the Human Immunoglobulin Variable Light Chain Lambda 1.

Genitope is developing a novel, personalized treatment for surface immunoglobulin (Ig) positive B cell malignancies. This treatment is based on a panel of monoclonal antibodies (Mabs) directed against shared epitopes expressed on different subsets of the variable regions of human Ig. The concept of targeting surface tumor Ig was explored in a series of clinical trials performed by Dr. Ronald Levy and his colleagues at Stanford. In these studies, Mabs directed against patient specific (idiotypic) determinants of the surface tumor Ig produced significant clinical benefit in relapsed follicular non-Hodgkin's lymphoma patients. A number of these patients have had long term remissions. In Genitope's planned clinical use, each patient will receive a single Mab from the panel selected based on its reactivity with the patient's tumor. The selected Mab will react with the patient's tumor and a minority of normal B cells, leaving the majority of the normal B cell repertoire intact. The ability of the panel members to provide therapeutic effect requires binding to tumor surface Ig in the presence of serum containing soluble Ig molecules. Mab Xi-20H5, a member of this panel of antibodies, is specific for a shared determinant on the human Ig variable light chain lambda 1. It binds to 25 to 35% of normal human B cells from peripheral blood and to 20 to 30% of normal cynomolgus monkey B cells from peripheral blood. In this study, we sought to demonstrate that, despite the presence of serum Ig, Mab Xi-20H5 would bind to surface Ig expressed on monkey B cells in vivo, resulting in specific depletion of target B cells. Six naïve cynomolgus monkeys received 8 intravenous infusions of the Mab Xi-20H5 at a dose of 40 mg/kg on days 1, 2, 3, 4, 7, 10, 14 & 17. Two naïve control animals received 8 infusions of vehicle only following the same schedule. The frequencies of lymphocyte sub-populations and of target B cells were monitored by flow cytometry on plasma-depleted whole blood samples. Samples were collected 23 hours after each infusion. In addition, two baseline samples were collected prior to treatment. The frequencies of lymphocyte sub-populations and of target B cells were compared to the average of the two baseline measurements. Frequencies of target B cells bound by Mab Xi-20H5 decreased in all treated animals while no significant change was detectable in the control animals. The bulk of the reduction in target B cell frequencies was observed 23 hours after the first infusion (range: 22% – 62%, average 41%). Frequencies of target B cells continued to decrease moderately with additional daily infusions (days 2 – 4), resulting in maximum reduction in target B cell frequency at 23 h post infusion 4 (range: 39% – 78%, average 54%). The frequencies of total B and T lymphocytes did not significantly change during the treatment. In vivo administration of Mab Xi-20H5 results in depletion of target B cells in a manner consistent with the expectation of an immunotherapeutic Mab aimed at treating surface Ig expressing B cell malignancies.

Designed Ankyrin Repeat Proteins as Anti‐Idiotypic‐Binding Molecules

According to the network theory antibodies may act as antigens thus generating anti-idiotypic antibodies that can function as regulators of immune responses. Designed ankyrin repeat proteins (DARPins) are a new class of binding proteins and may serve as an alternative to antibodies. Selections from large DARPin libraries against the variable regions of a murine monoclonal anti-human IgE antibody, termed BSW17, yielded two highly specific anti-idiotypic DARPins both with high affinity. Their binding characteristics were comparable with these of a previously selected anti-idiotypic antibody. In vitro cell assays showed that the anti-idiotypic DARPins were able to inhibit the binding of BSW17 to cell-bound IgE and prevented BSW17 functional activity. These experiments demonstrate the possibility to isolate anti-idiotypic DARPins recognizing idiotypic determinants analogous to antibodies. In the future these DARPins may be further analyzed for their potential as putative vaccine candidates.

MERCURY-INDUCED ACCELERATION OF SPONTANEOUS AUTOIMMUNITY IN PRE-NEPHRITIC (NZBxSWR)F1 (SNF1) FEMALE MICE (B90)

Abstract Mercury exposure in susceptible mice has been known to induce autoimmunity, including the proliferation of autoantibodies, Th2-dominated antibody responses and immune complex disease. We investigated whether the immunostimulating effects of mercury can accelerate the induction of autoimmunity in SNF1 mice, a model that closely resembles human SLE nephritis. Previous studies from our laboratory have shown that dysregulation of the idiotypic network is key in the development of renal immune complex deposits in SNF1 females. In this study, six week-old pre-nephritic SNF1 females were exposed to 10 mg/L of either mercuric chloride or methylmercury in drinking water and examined for modulations in pathogenic immunophenotypes, autoantibody titers and clinical evidence of disease. We found increased populations of splenic T-cells and B-cells reactive with a pathogenic idiotypic determinant (IdLNF1) in mice exposed to mercury which, interestingly, also showed reductions in the percentage and absolute numbers of regulatory phenotypes (CD4+CD25+GITR+). Splenic lymphocytes from exposed mice showed increased proliferative responses to IdLNF1. LPS-induced proliferation was also increased in exposed mice, regardless of mercurial species used. We found modulations in serum antibody levels, including increased titers of anti-double stranded DNA autoantibodies. Clinically, mice exposed to mercury showed increased proteinuria and are currently being evaluated for glomerular damage and nephritis. The results of this study support the hypothesis that low-level exposure to metal xenobiotics such as mercury may trigger or accelerate autoimmune disease especially in a background of genetic susceptibility.