What Can We Learn from Research with Monoclonal Antibody 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.

Transfer of natural auto-antibodies via egg yolk in chickens divergently selected for natural antibodies binding keyhole limpet hemocyanin.

Barcodes of natural auto-antibody (NAAb) profiles based on staining intensity of isotypes binding numbers of self-(tissue) antigen fragments were suggested as parameters for immune diversity, and related to genetic background and health status in man, rodents and poultry. Here, hens, eggs and hatchlings from chicken lines divergently selected and bred for high (H line) or low (L line) total natural antibodies (NAb) levels in plasma binding keyhole limpet hemocyanin (KLH) at 16 weeks of age were tested for their NAAb repertoire binding chicken liver homogenate (CLH) fragments using quantitative Western immunoblotting. The aims of this study were 1. to detect line differences between the H and L line adult hens, eggs and hatchlings for the IgM and IgG isotypes binding CLH fragments, 2. study the presence of NAAb of both isotypes in yolk and albumen, as well as in hatchlings to detect a maternal NAAb transfer route via the egg to the hatchling, and 3. study whether new self-antigen binding isotypes and idiotypes are present in the hatchling. NAAb binding CLH fragments were found in plasma of adult hens (both IgM and IgG), in yolk (IgG only), and hatchlings (mostly IgG, but low levels of IgM). Auto-profiles of IgM showed homogeneity, while IgG profiles were heterogenic between individual hens and individual hatchlings. Significant higher levels as indicated by staining intensity and number of stained CLH fragments were found in plasma of hens genetically selected for high levels of NAb binding KLH. Lines could be clustered based on their auto-profiles indicating that profiles of self-binding IgM and IgG antibodies are genetically based. Visual comparison, clustering and correlation of hens and their hatchlings showed similarities for the IgG, but not the IgM isotype, indicating maternal transfer of IgG NAAb via the yolk. The IgM profile in the hatchlings on the other hand might represent neonatal self-binding antibody formation. As a consequence, hatchlings initially depend for self-binding antibodies on maternal IgG provision during early life.

Monitoring multiple myeloma by idiotype-specific peptide binders of tumor-derived exosomes.

Tumor-derived exosomes (TDEs) play a pivotal role in tumor establishment and progression, and are emerging biomarkers for tumor diagnosis in personalized medicine. To date, there is a lack of efficient technology platforms for exosome isolation and characterization. Multiple myeloma (MM) is an incurable B-cell malignancy due to the rapid development of drug-resistance. MM-released exosomes express the immunoglobulin B-cell receptor (Ig-BCR) of the tumor B-cells, which can be targeted by Idiotype-binding peptides (Id-peptides). In this study, we analyzed the production of MM-released exosomes in the murine 5T33MM multiple myeloma model as biomarkers of tumor growth. To this end, we selected Id-peptides by screening a phage display library using as bait the Ig-BCR expressed by 5T33MM cells. By FACS, the FITC-conjugated Id-peptides detected the MM-released exosomes in the serum of 5T33MM-engrafted mice, levels of which are correlated with tumor progression at an earlier time point compared to serum paraprotein. These results indicate that Id-peptide-based recognition of MM-released exosomes may represent a very sensitive diagnostic approach for clinical evaluation of disease progression.

Monomeric IgA can be produced in planta as efficient as IgG, yet receives different N-glycans.

The unique features of IgA, such as the ability to recruit neutrophils and suppress the inflammatory responses mediated by IgG and IgE, make it a promising antibody isotype for several therapeutic applications. However, in contrast to IgG, reports on plant production of IgA are scarce. We produced IgA1κ and IgG1κ versions of three therapeutic antibodies directed against pro-inflammatory cytokines in Nicotiana benthamiana: Infliximab and Adalimumab, directed against TNF-α, and Ustekinumab, directed against the interleukin-12p40 subunit. We evaluated antibody yield, quality and N-glycosylation. All six antibodies had comparable levels of expression between 3.5 and 9% of total soluble protein content and were shown to have neutralizing activity in a cell-based assay. However, IgA1κ-based Adalimumab and Ustekinumab were poorly secreted compared to their IgG counterparts. Infliximab was poorly secreted regardless of isotype backbone. This corresponded with the observation that both IgA1κ- and IgG1κ-based Infliximab were enriched in oligomannose-type N-glycan structures. For IgG1κ-based Ustekinumab and Adalimumab, the major N-glycan type was the typical plant complex N-glycan, biantennary with terminal N-acetylglucosamine, ß1,2-xylose and core α1,3-fucose. In contrast, the major N-glycan on the IgA-based antibodies was xylosylated, but lacked core α1,3-fucose and one terminal N-acetylglucosamine. This type of N-glycan occurs usually in marginal percentages in plants and was never shown to be the main fraction of a plant-produced recombinant protein. Our data demonstrate that the antibody isotype may have a profound influence on the type of N-glycan an antibody receives.

Idiotype vaccine production using hybridoma technology.

Non-Hodgkin's lymphoma (NHL) is the most common hematological malignancy both in Europe and in the United States. Follicular lymphoma (FL), a tumor comprised of mature B cells, represents one fourth of all NHL and, despite good response rates to standard treatments, tends to frequently relapse to such an extent that it is still considered incurable. Among several alternative therapeutic options actively being pursued, immunotherapy by idiotypic vaccination is in the forefront of clinical experimental medicine. The idiotype vaccine consists of the tumor-specific immunoglobulin conjugated with keyhole limpet hemocyanin (KLH) and administered together with an adjuvant. Over the last 20 years, researchers have proven that this vaccine can induce specific immune responses. Too, those patients with such responses experience a disease-free survival longer than normally achievable, although these latter results require further confirmation in large clinical trials. Traditionally, idiotype vaccines have been produced through hybridoma technology. In this chapter this technology is described.

Types of tolerance seen in autoreactive phosphocholine-specific B cells are dependent on the idiotype of the receptors expressed.

Phosphocholine (PC) is the immunodominant epitope found on the surface of a number of microorganisms, including Streptococcus pneumoniae (SPn), and is thought to play a vital role in the pathogenesis of SPn. B cells expressing M167Hκ24L immunoglobulin receptors specific for PC have been shown to be autoreactive in that they undergo clonal deletion in both X-linked immune-deficient and Rag(-/-) mice. We have now shown that B cells expressing M603Hκ8L PC-specific receptors also delete in Rag(-/-) mice, whereas those expressing T15Hκ22L transgenes do not delete. However, T15Hκ22L B cells are lost in normal heterozygous transgenic mice because they cannot compete with normal B cells. These data indicate that M167Hκ24L and M603Hκ8L PC-specific B cells are recognizing an autoantigen expressed on membranes which causes them to downregulate their receptors and clonally delete, while T15Hκ22L B cells are tolerized by a soluble form of PC-antigen which results in their being trapped in the spleen. Thus, the types of tolerance seen in autoreactive PC-specific B cells are dependent on the idiotype of the receptors expressed.

A small-molecule screen yields idiotype-specific blockers of neuromyelitis optica immunoglobulin G binding to aquaporin-4.

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system caused by binding of anti-aquaporin-4 (AQP4) autoantibodies (NMO-IgG) to AQP4 on astrocytes. A screen was developed to identify inhibitors of NMO-IgG-dependent, complement-dependent cytotoxicity. Screening of 50,000 synthetic small molecules was done using CHO cells expressing human AQP4 and a human NMO recombinant monoclonal antibody (rAb-53). The screen yielded pyrano[2,3-c]pyrazoles that blocked rAb-53 binding to AQP4 and prevented cytotoxicity in cell culture and spinal cord slice models of NMO. Structure-activity analysis of 82 analogs yielded a blocker with IC(50) ∼ 6 µm. Analysis of the blocker mechanism indicated idiotype specificity, as (i) pyrano[2,3-c]pyrazoles did not prevent AQP4 binding or cytotoxicity of other NMO-IgGs, and (ii) surface plasmon resonance showed specific rAb-53 binding. Antibody structure modeling and docking suggested a putative binding site near the complementarity-determining regions. Small molecules with idiotype-specific antibody targeting may be useful as research tools and therapeutics.

Differential idiotype utilization for the in vivo type 14 capsular polysaccharide-specific Ig responses to intact Streptococcus pneumoniae versus a pneumococcal conjugate vaccine.

Murine IgG responses specific for the capsular polysaccharide (pneumococcal capsular polysaccharide serotype 14; PPS14) of Streptococcus pneumoniae type 14 (Pn14), induced in response to intact Pn14 or a PPS14-protein conjugate, are both dependent on CD4(+) T cell help but appear to use marginal zone versus follicular B cells, respectively. In this study, we identify an idiotype (44.1-Id) that dominates the PPS14-specific IgG, but not IgM, responses to intact Pn14, isolated PPS14, and Group B Streptococcus (strain COH1-11) expressing capsular polysaccharide structurally identical to PPS14. The 44.1-Id, however, is not expressed in the repertoire of natural PPS14-specific Abs. In distinct contrast, PPS14-specific IgG responses to a soluble PPS14-protein conjugate exhibit minimal usage of the 44.1-Id, although significant 44.1-Id expression is elicited in response to conjugate attached to particles. The 44.1-Id elicited in response to intact Pn14 was expressed in similar proportions among all four IgG subclasses during both the primary and secondary responses. The 44.1-Id usage was linked to the Igh(a), but not Igh(b), allotype and was associated with induction of relatively high total PPS14-specific IgG responses. In contrast to PPS14-protein conjugate, avidity maturation of the 44.1-Id-dominant PPS14-specific IgG responses was limited, even during the highly boosted T cell-dependent PPS14-specific secondary responses to COH1-11. These results indicate that different antigenic forms of the same capsular polysaccharide can recruit distinct B cell clones expressing characteristic idiotypes under genetic control and suggest that the 44.1-Id is derived from marginal zone B cells.

Surface functionalization of virus-like particles by direct conjugation using azide-alkyne click chemistry.

We present a cell-free protein synthesis (CFPS) platform and a one-step, direct conjugation scheme for producing virus-like particle (VLP) assemblies that display multiple ligands including proteins, nucleic acids, and other molecules. Using a global methionine replacement approach, we produced bacteriophage MS2 and bacteriophage Qß VLPs with surface-exposed methionine analogues (azidohomoalanine and homopropargylglycine) containing azide and alkyne side chains. CFPS enabled the production of VLPs with yields of ~ 300 µg/mL and with 85% incorporation of methionine analogues without requiring a methionine auxotrophic production host. We then directly conjugated azide- and alkyne-containing proteins (including an antibody fragment and the granulocyte-macrophage colony stimulating factor, or GM-CSF), nucleic acids and poly(ethylene glycol) chains to the VLP surface using Cu(I) catalyzed click chemistry. The GM-CSF protein, after conjugation to VLPs, was shown to partially retain its ability to stimulate the proliferation of cells. Conjugation of GM-CSF to VLPs resulted in a 3-5-fold reduction in its bioactivity. The direct attachment scheme facilitated conjugation of three different ligands to the VLPs in a single step, and enabled control of the relative ratios and surface abundance of the attached species. This platform can be used for the production of novel VLP bioconjugates for use as drug delivery vehicles, diagnostics, and vaccines.

Control of the specificity of T cell-mediated anti-idiotype immunity by natural regulatory T cells.

The idiotypes of B cell lymphomas represent tumor-specific antigens. T cell responses induced by idiotype vaccination in vivo are directed predominantly against CDR peptides, whereas in vitro T cells also recognize framework-derived epitopes. To investigate the mechanisms regulating the specificity of idiotype-specific T cells, BALB/c or B10.D2 mice were immunized with mature dendritic cells loaded with H-2K(d)-restricted peptides from influenza hemagglutinin, or from shared (J region) or unique (CDR3) structures of the A20 lymphoma idiotype. Antigen-specific T cells were induced in vivo by the CDR3 and influenza epitopes, but not by the J peptide. Gene expression profiling of splenic regulatory T cells revealed vaccination-induced Treg activation and proliferation. Treg activity involved J epitope-dependent IL-10 secretion and functional suppression of peptide-specific effector T cells. Vaccination-induced in vivo proliferation of transgenic hemagglutinin-specific T cells was suppressed by co-immunization with the J peptide and was restored in CD25-depleted animals. In conclusion, Treg induced by a shared idiotype epitope can systemically suppress T cell responses against idiotype-derived and immunodominant foreign epitopes in vivo. The results imply that tumor vaccines should avoid epitopes expressed by normal cells in the draining lymph node to achieve optimal anti-tumor efficacy.

Comparative analysis of predicted HLA binding of immunoglobulin idiotype sequences indicates T cell-mediated immunosurveillance in follicular lymphoma.

Active immunization with the idiotype of follicular lymphoma induces tumor-specific immunity. T cells induced in vivo by idiotype vaccination recognize human leukocyte antigen (HLA)--restricted hypervariable but not conserved idiotype peptides. We hypothesized that idiotype-directed T-cell immunity occurs naturally and performed a reverse immunology analysis of idiotype HLA binding in 39 follicular lymphoma patients. For every idiotype, the sum of HLA-A or -B binding scores of the 20 highest-scoring peptides was calculated for all 39 HLA types through the BIMAS algorithm. The idiotype sum score of every patient's lymphoma was compared on the respective patient's HLA type to the mean of the sum scores of the remaining 38 idiotypes. Autologous idiotypes had lower immunogenicity than allogeneic idiotypes. Differential immunogenicity resided predominantly in all 3 complementarity-determining regions rather than in framework peptides. Idiotype immunogenicity was not changed by somatic hypermutation. These findings indicate T cell-mediated immunosurveillance of follicular lymphoma directed specifically against individual idiotype epitopes.

Rapid, high-yield production in plants of individualized idiotype vaccines for non-Hodgkin's lymphoma.

BACKGROUND: Animal and clinical studies with plant-produced single-chain variable fragment lymphoma vaccines have demonstrated specific immunogenicity and safety. However, the expression levels of such fragments were highly variable and required complex engineering of the linkers. Moreover, the downstream processing could not be built around standard methods like protein A affinity capture. DESIGN: We report a novel vaccine manufacturing process, magnifection, devoid of the above-mentioned shortcomings and allowing consistent and efficient expression in plants of whole immunoglobulins (Igs). RESULTS: Full idiotype (Id)-containing IgG molecules of 20 lymphoma patients and 2 mouse lymphoma models were expressed at levels between 0.5 and 4.8 g/kg of leaf biomass. Protein A affinity capture purification yielded antigens of pharmaceutical purity. Several patient Igs produced in plants showed specific cross-reactivity with sera derived from the same patients immunized with hybridoma-produced Id vaccine. Mice vaccinated with plant- or hybridoma-produced Igs showed comparable protection levels in tumor challenge studies. CONCLUSIONS: This manufacturing process is reliable and robust, the manufacturing time from biopsy to vaccine is <12 weeks and the expression and purification of antigens require only 2 weeks. The process is also broadly applicable for manufacturing monoclonal antibodies in plants, providing 50- to 1000-fold higher yields than alternative plant expression methods.

Role of idiotype-anti-idiotype interactions in the induction of collagen-induced arthritis in rats.

The mechanism of autoantibodies (rheumatoid factor (RF) and anti-collagen autoantibodies) induction in collagen-induced arthritis (CIA) is unknown. The study assessed the hypothesis that activation of autoantibody-producing clones is mediated by idiotype-anti-idiotype (IAI) interactions with lymphocytes on heterologous collagen. It was demonstrated that RF-containing serum of rats immunized with bovine collagen (BCII) in ELISA competes with BCII for binding to anti-BCII antibodies. Immunization of rats with Fc fragments of IgG caused not only an increase in RF levels, but also induction of antibodies to BCII and anti-collagen autoantibodies. Taken together, these facts suggest that activation of RF-producing lymphocytes in CIA model occurs through IAI interactions with anti-BCII lymphocytes. Three populations of antibodies were detected in the blood of arthritic rats: a population of antibodies reacting only with BCII, a population of antibodies reacting only with rat collagen (RCII) and a population of antibodies that can bind to both bovine and rat collagen. It was shown that RF in relation to anti-collagen autoantibodies act as anti-idiotype antibodies, and a comparative analysis of antibody production in arthritic and resistant rats demonstrated that the level of anti-collagen autoantibody production depends on the level of RF production. This suggests that RF and RF-producing lymphocytes are involved in regulation of anti-collagen autoreactive lymphocyte activity through an IAI interaction mechanism. A direct activation of autoreactive anti-RCII lymphocytes by BCII cannot be excluded, but it can be supposed that induction of anti-collagen autoreactive lymphocytes needs a signal generated in IAI interactions by RF-producing lymphocytes. This hypothesis is based on the data obtained, and not only explains the mechanism of autoreactive lymphocytes activation in the rat CIA model, but also indicates that the key regulatory element in the development of arthritis in animals is RF-producing lymphocytes. The results allow a new insight on the role of RF in the pathogenesis of rheumatoid arthritis and on seeking more effective therapeutic means.

A phase 2 trial of immunotherapy with mitumprotimut-T (Id-KLH) and GM-CSF following rituximab in follicular B-cell lymphoma.

We evaluated the efficacy and safety of patient-specific immunotherapy with mitumprotimut-T idiotype keyhole limpet hemocyanin and granulocyte-monocyte colony-stimulating factor (GM-CSF) following rituximab in patients with follicular B-cell lymphoma. Patients with previously untreated or relapsed/refractory CD20+ follicular lymphoma received 4 weekly infusions of rituximab and those with a complete response (CR), partial response (PR), or stable disease received mitumprotimut-T and GM-CSF injections subcutaneously. Courses were given monthly for 6 doses, every 2 months for 6 doses, and then every 3 months until disease progression. Computed tomography scans were obtained every 3 to 6 months and reviewed centrally. The primary endpoint was event-free survival (EFS). Among 103 patients treated with rituximab, 92 (54 relapsed/refractory and 38 previously untreated) received mitumprotimut-T/GM-CSF; median age was 53 years, 91% had stage III to IV disease, and 59% had failed earlier therapy. The premitumprotimut-T objective response rate was 47% (2 CRs, 41 PRs). During the mitumprotimut-T treatment phase, 16 patients converted to CR resulting in an overall objective response rate of 60% (18 CRs, 37 PRs). Median EFS was 15.2, 20.8, and 13.5 months for all, treatment-naive, and relapsed/refractory disease patients, respectively. Anti-Id cellular immune responses were detected in 13 of 18 (72%) patients and humoral immune responses in 17 of 83 (20%) patients. Adverse events were usually mild-to-moderate. The most common adverse event was injection site reactions. Mitumprotimut-T/GM-CSF-induced anti-Id cellular immune responses in most patients. The occurrence of late CRs and favorable EFS suggested a clinical benefit of active immunotherapy and led to a placebo-controlled phase 3 trial.

Review: to what extent are T cells tolerant to immunoglobulin variable regions?

During the last 25 years it has become increasingly clear that short peptides derived from Ig V-regions are displayed on MHC class II molecules. Recognition of such idiotypic(Id)-peptide/MHC class II complexes by Id-specific CD4(+) T cells plays a role in (1) Id-driven T-B collaboration, (2) immunosurveillance of B cell cancers and (3) Id-vaccination. A crucial question is then: to what extent are T cells tolerized to Ig V-region sequences? Or rephrased: how large is the T-cell repertoire for Ig V-region sequences presented by MHC class II molecules? We argue that T cells are to a large extent tolerant to germline-encoded V-region sequences but that there is a T-cell repertoire for rare Id-sequences that arise as a consequence of somatic hyper mutation or N-region diversity. Moreover, when otherwise rare Id-sequences increase in concentration, T-cell tolerance is induced (Fig. 1). For these reasons, T cells that recognize rare Id-peptides, arising as a consequence of somatic genetic events unique to each B cell, may play a special importance in Id-driven T-B collaboration, immunosurveillance of B-cell malignancies, and Id-vaccination.

The 1F7 idiotype is selectively expressed on CD5+ B cells and elevated in chronic hepatitis C virus infection.

Antibodies against different chronic viruses, including hepatitis C virus (HCV), express a public cross-reactive idiotype (Id) designated as 1F7. The prominence of this Id may reflect selective engagement of B1 B cells by chronic pathogens. We investigated this by comparing 1F7 Id expression on CD5(+) and CD5(-) B cells, total IgG, total IgM and anti-HCV core antibodies in different HCV exposure settings. By flow cytometry, we observed a selective increase in 1F7 Id(+)CD5(+) B cells in chronic HCV infection. 1F7 Id levels in different immunoglobulin compartments were measured by enzyme-linked immunosorbent assay. 1F7 Id expression was prominent in anti-HCV core antibodies of approximately 90% of 141 HCV-exposed individuals tested. In the Canadian and Armenian study groups, participants who spontaneously cleared HCV infection had lower median 1F7 Id levels on total plasma IgG and anti-HCV core antibodies. Armenian spontaneous clearers, who were younger and more recently infected than their Canadian counterparts, also had had lower median 1F7 Id levels on total plasma IgM. Engagement by HCV of B-cell receptors within, or overlapping with the CD5(+) B1 B-cell repertoire is reflected in the production of 1F7 Id(+) anti-HCV antibodies and expansion of 1F7 Id(+)CD5(+) B cells. Higher 1F7 Id expression levels are associated with chronic infection.

Enhanced immune stimulation by a therapeutic lymphoma tumor antigen vaccine produced in insect cells involves mannose receptor targeting to antigen presenting cells.

Therapeutic vaccination of lymphoma patients with tumor-specific immunoglobulin (idiotype, Id) coupled to the carrier protein keyhole limpet hemocyanin (Id-KLH) is undergoing clinical investigation, and methods to improve the immunogenicity of these and other protein tumor antigen vaccines are being sought. Id proteins can be produced via tumor-myeloma hybridomas or recombinant methods in mammalian, bacteria, or insect cells. We now demonstrate that terminal mannose residues, characteristic of recombinant proteins produced in insect cells, yield Id proteins with significantly enhanced immunostimulatory properties compared to Id proteins derived from mammalian cells. Recombinant baculovirus-infected insect cell-derived Id showed higher binding to and activation of human dendritic cells mediated by mannose receptors. In vivo, insect cell-derived Id elicited higher levels of tumor-specific CD8+ cytotoxic T lymphocyte (CTL) and improved eradication of pre-established murine lymphoma. Insect cell and mammalian Id generated similar levels of tumor-specific antibodies, showing no impairment in antibody responses to native tumor antigen despite the glycoslylation differences in the immunogen. Combining insect cell production and maleimide-based KLH conjugation offered the highest levels of anti-tumor immunity. Our data comparing sources of recombinant Id protein tumor antigens used in therapeutic cancer vaccines demonstrate that insect cell-derived antigens can offer several immunologic advantages over proteins derived from mammalian sources.

Binding of monosodium urate crystals with idiotype protein efficiently promote dendritic cells to induce cytotoxic T cells.

Monosodium urate (MSU) crystals have been reported to evoke specific cell immunity and to work as an adjuvant in a mouse model. The crystals also have another unique characteristic to bind with positively charged proteins, which could help to deliver some antigens into human dendritic cells (DC). We focused on the application of MSU crystals as not only an adjuvant but also as a carrier of positively charged antigenic protein to induce human cytotoxic T cells (CTL) efficiently in vitro. We selected human leukocyte antigen (HLA)-A2 expressing the multiple myeloma IM-9 cell line and its product idiotype (Id) protein as one of the best pairs of target cells and positively charged tumor-specific antigen, respectively. Following the sensitization of DC derived from HLA-A2-positive volunteers pulsed with tumor-specific monoclonal immunoglobulin G-Fab fragments (IM-9 Fab) attached to MSU crystals, the DC-stimulated CD8(+) T cells killed significantly more target cells (40.1 +/- 1.7%) than those stimulated by DC pulsed with MSU crystals alone (6.2 +/- 8.6%, P < 0.01) or IM-9 Fab alone (4.7 +/- 8.1%, P < 0.01). These cytotoxic effects of the DC-stimulated CD8(+) cells were reduced when MSU crystals were precoated with fetal bovine serum. In addition, we confirmed that MSU crystals facilitated human DC to express the maturation marker, CD83 and deliver (Fab')(2), attaching to the crystals by flow cytometer analysis. MSU crystals have distinct advantages of a protein carrier binding with positively charged proteins and delivering antigenic protein into DC, as well as an adjuvant promoting DC maturation and inducing CTL.

Sulfhydryl-based tumor antigen-carrier protein conjugates stimulate superior antitumor immunity against B cell lymphomas.

Therapeutic vaccination of B cell lymphoma patients with tumor-specific Ig (idiotype, or Id) chemically coupled to the immunogenic foreign carrier protein keyhole limpet hemocyanin (KLH) using glutaraldehyde has shown promising results in early clinical trials, and phase III trials are underway. However, glutaraldehyde Id-KLH vaccines fail to elicit anti-Id immune and clinical responses in many patients, possibly because glutaraldehyde reacts with lysine, cysteine, tyrosine, and histidine residues, damaging critical immunogenic epitopes. A sulfhydryl-based tumor Ag-carrier protein conjugation system using maleimide chemistry was used to enhance the efficacy of Id-KLH vaccines. Maleimide Id-KLH conjugates eradicated A20 lymphoma from most tumor-bearing mice, whereas glutaraldehyde Id-KLH had little efficacy. Maleimide Id-KLH elicited tumor-specific IgG Abs and T cells, with CD8(+) T cells being the major effectors of antilymphoma immunity. Maleimide Id-KLH vaccines also demonstrated superior efficacy in 38C13 and BCL-1 lymphoma models, where Abs were shown to be critical for protection. Importantly, standard glutaraldehyde Id-KLH conjugation procedures could result in "overconjugation" of the tumor Ag, leading to decreased efficacy, whereas the heterobifunctional maleimide-based conjugation yielded potent vaccine product regardless of conjugation duration. Under lysosomal processing conditions, the Id-carrier protein linkage was cleavable only after maleimide conjugation. Maleimide KLH conjugation was easily performed with human Igs analogous to those used in Id-KLH clinical trials. These data support the evaluation of sulfhydryl-based Id-KLH vaccines in lymphoma clinical trials and possibly the use of tumor Ag-carrier protein vaccines for other cancers.

Influence of Fas on the regulation of the response of an anti-nuclear antigen B cell clonotype to foreign antigen.

A peripheral B cell tolerance checkpoint appears to be operative during the germinal center (GC) reaction. We previously showed that a transgenic BCR clonotype that is 'dual reactive' for the hapten arsonate (Ars) and nuclear auto-antigens is stimulated to enter the GC response via Ars immunization. However, the participation of this clonotype in this response wanes with time and it gives rise to few memory B cells capable of mounting a secondary anti-Ars IgG response. Enforced expression of Bcl-2 partially rescues the GC and memory B cell responses of this clonotype, suggesting that apoptotic pathways are involved in the action of the GC tolerance checkpoint. Since GC B cells substantially up-regulate levels of expression of the Fas apoptotic death receptor, we determined whether an intrinsic Fas deficient could rescue the participation of this clonotype in the GC response. It could not, strongly indicating that Fas expression by autoreactive GC B cells is not necessary for their elimination. In addition, experiments in which Fas-sufficient dual reactive clonotype B cells were transferred to Fas-deficient hosts revealed an absence of participation of these B cells in the GC and IgG anti-Ars responses. We present data consistent with the idea that T cells in Fas-deficient hosts are primed to express elevated levels of FasL and eliminate antigen-activated B cells that up-regulate Fas.