Low-affinity Antibodies Research Articles

Effect of IVIG Formulation on IgG Binding to Self- and Exo- Antigens In Vitro and In Vivo.

In relation to the recent trials of Intravenous Immunoglobulin (IVIG) in Alzheimer’s Disease (AD) it was demonstrated that different IgG preparations contain varying amounts of natural anti-amyloid β (Aβ) antibodies as measured by ELISA. We therefore investigated the relevance of ELISA data for measuring low-affinity antibodies, such as anti-Aβ. We analysed the binding of different commercial Immunoglobulin G (IgG) preparations to Aβ, actin and tetanus toxoid in different binding assays to further investigate the possible cause for observed differences in binding to Aβ and actin between different IgG preparations. We show that the differences of commercial IgG preparations in binding to Aβ and actin in ELISA assays are artefactual and only evident in in vitro binding assays. In functional assays and in vivo animal studies the different IVIG preparations exhibited very similar potency. ELISA data alone are not appropriate to analyse and rank the binding capacity of low-affinity antibodies to Aβ or other endogenous self-antigens contained in IgG preparations. Additional analytical methods should be adopted to complement ELISA data.

Increased expression of TACI on NOD B cells results in germinal centre reaction anomalies, enhanced plasma cell differentiation and immunoglobulin production.

B cells have an important pathogenic role in the development of type 1 diabetes in the non-obese diabetic (NOD) mouse. We have previously reported that NOD mice display an increased percentage of TACIhigh -expressing B cells compared with C57BL/6 mice and this trait is linked to chromosomes 1 and 8. In this paper the genetic association of the transmembrane activator, calcium modulator and cyclophilin ligand interactor (TACI) trait was confirmed using double congenic NOD.B6C1/Idd22 mice. TACI ligation by a proliferation-inducing ligand (APRIL) has been shown to influence plasma cell differentiation, immunoglobulin production and isotype switch. Hence, the functional consequence of the up-regulation of TACI on NOD B cells was analysed both in vitro and in vivo. NOD B cells stimulated with APRIL showed an enhanced plasma cell differentiation and class switch to IgG and IgA compared with B cells from C57BL/6 mice. Moreover, flow cytometry analyses revealed that germinal centre B cells in NOD failed to down-regulate TACI. Availability of the TACI ligand B-cell activating factor (BAFF) has been shown to be a limiting factor in the germinal centre reaction. In line with this, upon immunization with 4-hydroxy-3-nitrophenylacetyl hapten-conjugated hen egg lysozyme, NOD mice produced higher titres of low-affinity antibodies compared with C57BL/6 mice. This observation was supported by the detection of increased levels of BAFF in NOD germinal centres after immunization compared with C57BL/6 by immunofluorescence. Our results support the hypothesis that increased TACI expression on NOD B cells contributes to the pathogenesis of type 1 diabetes in the NOD mouse.

Failure of gut-associated pDCs to express membrane bound APRIL and BAFF prevents their ability to promote low-affinity IgA expression in ME/CFS

Abstract Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a heterogeneous illness characterized by a number of comorbid conditions; gastrointestinal (GI) dysregulation make up one subgroup of this disease. IgA is the most abundant antibody isotype found in mucosal secretions including the gut. In a process of class switch recombination (CSR), that relies on the interaction of plasmacytoid dendritic cells (pDCs) with B cells, in a T cell independent (TI) manner, low-affinity IgA are produced that limit the adhesion of commensal bacteria to intestinal epithelia without neutralizing them. These low-affinity antibodies also limit bacterial overgrowth and potential bacterial translocation thus maintaining gut homeostasis. This process is known as “immune exclusion”. Two ligands on the surface of pDCs that are obligatory for the process; the membrane bound form of APRIL and BAFF. The upregulation of APRIL and BAFF on the surface of pDCs is dependent on low-level expression of type I interferon (IFN) which is produced by intestinal stromal cells in response to Toll-like receptor (TLR) engagement. Previous studies suggest that peripheral pDCs are significantly lower in subjects with ME/CFS when compared to controls and studies conducted by us further suggest these cells likely redistribute from the periphery to the gut. We have observed that, in contrast to controls, gut-associated pDCs in subjects with ME/CFS lack APRIL and BAFF expression. These data support a model of gut pathology in ME/CFS whereby dysregulated pDCs fail to promote the production of low-affinity IgA through the process of TI activation of B cells, thereby leading to bacterial overgrowth, dysbiosis, bacterial translocation and systemic immune activation.

Plasmablasts induced by experimental Plasmodium infection suppress protective humoral immunity through PD-L1

Abstract Plasmodium infections cause malaria, which remains a global public health crisis. Germinal center derived anti-Plasmodium antibody responses are essential for control of blood-stage parasite replication and clearance. However, antibody responses are dominated by extrafollicular, short-lived plasmablasts that secrete low-affinity antibodies. The factors that regulate plasmablast expansion and the contribution of plasmablasts to protective anti-Plasmodium humoral immunity are not well understood. Utilizing experimental murine Plasmodium yoelii infection, we identified that early, splenic plasmablast responses (CD138hi B cells) are dominated by innate B1b (CD43+CD5−) B cells and are comprised of both parasite- and host-specific cells. We also found that CD138hi B cell responses are largely governed by IFN-γ inflammation, not parasite burden. Strikingly, adoptive transfer of CD138hi B cells reduced germinal center B cell class-switching and parasite-specific IgG secretion in Plasmodium infection-matched recipients. Moreover, T follicular helper cells recovered from CD138hi B cell recipient mice were inefficient at promoting germinal center B cell differentiation. In vitro, Plasmodium infection-induced CD138hiB cells suppressed CD4 T cell proliferation. Mechanistically, we linked the in vivo and in vitro suppressive activity of CD138hi B cells to their expression of the inhibitory ligand PD-L1. Together, our data identify that Plasmodium infection-induced plasmablasts suppress the induction of protective humoral immunity during experimental malaria. This knowledge can be used to develop immune-based therapies that limit or prevent innate plasmablast responses.

B-cell memory against factor VIII

The development of persistent neutralizing antibodies against factor VIII (FVIII) is the most severe complication in the treatment of congenital hemophilia A patients with FVIII replacement therapies. Recently, we presented data which indicate that neutralizing antibodies are high-affinity antibodies which are mostly of the IgG1 and IgG4 subclasses. However, there are also FVIII-specific antibodies of low to moderate affinity which are found in some patients without neutralizing antibodies and in some healthy individuals. The underlying immune mechanisms which regulate the development of these different populations of FVIII-specific antibodies are poorly understood.In this review, we discuss potential mechanisms for the development of low-affinity and high-affinity antibodies. In particular, we discuss the role of memory B cells which are essential in maintaining long-lasting antibody responses and in driving the rapid anamnestic antibody response after re-exposure to the same antigen. We also present available data on the potential role of FVIII-specific memory B cells in the maintenance of neutralizing antibodies against FVIII.

Human B-cell isotype switching origins of IgE

B cells expressing IgE contribute to immunity against parasites and venoms and are the source of antigen specificity in allergic patients, yet the developmental pathways producing these Bcells in human subjects remain a subject of debate. Much of our knowledge of IgE lineage development derives from model studies in mice rather than from human subjects. We evaluate models for isotype switching to IgE in human subjects using immunoglobulin heavy chain (IGH) mutational lineage data. We analyzed IGH repertoires in 9 allergic and 24 healthy adults using high-throughput DNA sequencing of 15,843,270 IGH rearrangements to identify clonal lineages of Bcells containing members expressing IgE. Somatic mutations in IGH inherited from common ancestors within the clonal lineage are used to infer the relationships between Bcells. Data from 613,641 multi-isotype B-cell clonal lineages, of which 592 include an IgE member, are consistent with indirect switching to IgE from IgG- or IgA-expressing lineage members in human subjects. We also find that these inferred isotype switching frequencies are similar in healthy and allergic subjects. We found evidence that secondary isotype switching of mutated IgG1-expressing Bcells is the primary source of IgE in human subjects, with lesser contributions from precursors expressing other switched isotypes and rarely IgM or IgD, suggesting that IgE is derived from previously antigen-experienced Bcells rather than naive Bcells that typically express low-affinity unmutated antibodies. These data provide a basis from which to evaluate allergen-specific human antibody repertoires in healthy and diseased subjects.

Clinical application of clustered-AChR for the detection of SNMG.

Myasthenia gravis (MG) is an autoantibody-mediated disease of the neuromuscular junction (NMJ). However, accumulating evidence has indicated that MG patients whose serum anti-acetylcholine receptor (AChR) antibodies are not detectable (serumnegative MG; SNMG) in routine assays share similar clinical features with anti-AChR antibody-positive MG patients. We hypothesized that SNMG patients would have low-affinity antibodies to AChRs that would not be detectable using traditional methods but that might be detected by binding to AChR on the cell membrane, particularly if they were clustered at the high density observed at the NMJ. We expressed AChR subunits with the clustering protein rapsyn (an AChR-associated protein at the synapse) in human embryonic kidney (HEK) cells, and we tested the binding of the antibodies using immunofluorescence. With this approach, AChR antibodies to rapsyn-clustered AChR could be detected in the sera from 45.83% (11/24) of SNMG patients, as confirmed with fluorescence-activated cell sorting (FACS). This was the first application in China of cell-based AChR antibody detection. More importantly, this sensitive (and specific) approach could significantly increase the diagnosis rate of SNMG.

Atg7 is critical for the functional maturation of germinal center B cells (LYM7P.625)

Abstract The role of autophagy, especially individual components of the autophagy machinery, in antibody response has not been well established. Atg7 is an integral part in autophagesome formation and elongation. Using lineage- and stage-specific conditional knockout (CKO) mice, we have found that, in GC B cell-specific Atg7 CKO mice, although GC formation and structure were not affected by Atg7 deficiency, both primary and memory antibody responses were severely impaired in Atg7 CKO mice. Class-switch recombination (CSR) and Ig somatic hypermutation (SHM) were defective in Atg7-/- GC B-cells, leading to a severely inhibited production of class-switched high-affinity antibodies. Analysis of GC B-cell clones showed that Atg7-deficiency in GC B-cells causes a dramatic change in Ig gene composition in the responding B-cell repertoire, which is dominated by non-canonical Ig genes encoding lower affinity antibodies. Thus, our study demonstrated for the first time that a single gene mutation affects GC function without inhibiting GC formation. The underlying molecular mechanism for impaired GC function in Atg7 mutant mice is that Atg7 is critical in regulating activation-induced deaminase (AID). Atg7-deficiency in GC B cells led to delayed onset and reduced disease severity in collagen-induced arthritis (CIA), demonstrating that Atg7 is critical in the development and pathogenesis of autoimmune disease.

High-Throughput Kinetic Screening of Hybridomas to Identify High-Affinity Antibodies Using Bio-Layer Interferometry

Kinetic analysis of antibodies is crucial in both clone selection and characterization. Historically, antibodies in supernatants from hybridomas are selected based on a solid-phase enzyme-linked immunosorbent assay (ELISA) in which the antigen is immobilized on the assay plate. ELISA selects clones based on a combination of antibody concentration in the supernatant and affinity. The antibody concentration in the supernatant can vary significantly and is typically unknown. Using the ELISA method, clones that express high levels of a low-affinity antibody can give an equivalent signal as clones that express low levels of a high-affinity antibody. As a consequence, using the ELISA method, superior clones can be overshadowed by inferior clones. In this study, we have applied Bio-Layer Interferometry to screen hybridoma clones based on disassociation rates using the OctetRED 384 platform. Using the OctetRED platform, we were able to screen 2000 clones within 24 hours and select clones containing high-affinity antibodies for further expansion and subsequent characterization. Using this method, we were able to identify several clones producing high-affinity antibodies that were missed by ELISA.

Dynamic quantification of antigen molecules with flow cytometry

Traditional methods for estimating the number of expressed molecules, based on the detection of target antigens bound with fluorescently labeled antibodies, assume that the antigen–antibody reaction reaches equilibrium. A calibration procedure is used to convert the intensity of the fluorescence signal to the number of target molecules. Along with the different limitations of every calibration system, this substantially limits the applicability of the traditional approaches especially in the case of low affinity antibodies.We address this problem here with studies in which we demonstrate a new approach to the antigen molecule quantification problem. Instead of using a static calibration system, we analyzed mean fluorescence values over time by flow cytometry during antibody–antigen binding. Experimental data obtained with an LSRII cytometer were fitted by a diffusion–reaction mathematical model using the Levenberg–Marquardt nonlinear least squares curve-fitting algorithm in order to obtain the number of target antigen molecules per cell. Results were compared with the Quanti-BRITE calibration system.We conclude that, instead of using experiment-specific calibration, the value of the binding rate constant for each particular antibody–antigen reaction can be used to quantify antigen molecules with flow cytometry. The radius of CD8 antibody molecule binding site was found, that allows recalculating the binding rate constant for other conditions (different sizes of reagent molecules, fluorescent label, medium viscosity and temperature). This approach is independent of specially prepared calibration beads, antibody reagents and the specific dye and can be applied to both low and high affinity antibodies, under both saturating and non-saturating binding conditions. The method was demonstrated on a human blood sample dataset investigating CD8α antigen on T cells in stable binding conditions.

Pharmacological profile of a bifunctional ligand of the formyl peptide receptor1 fused to the myc epitope

In human peripheral blood neutrophils or in myeloid PLB-985 cells differentiated towards a neutrophil-like phenotype, the peptide N-formyl-L-norleucyl-L-leucyl-L-phenylalanyl-L-norleucyl-L-tyrosyl-L-leucyl-fluorescein isothiocyanate (f-Nle-Leu-Phe-Nle-Tyr-Lys-FITC) binds to and activates formyl peptide receptor1 (FPR1) and is submitted to receptor-mediated endocytosis (microscopy, cytofluorometry). This peptide may be considered a C-terminally extended version of f-Met-Leu-Phe which carries a fluorescent cargo into cells. By analogy to other peptide hormones for which we have evaluated epitope-tagged agonists as carriers of antibody cargoes, we have designed and evaluated f-Nle-Leu-Phe-Nle-Tyr-Lys-myc, C-terminally extended with the 10-residue myc tag. This peptide is as potent as f-Met-Leu-Phe to compete for f-Nle-Leu-Phe-Nle-Tyr-Lys-FITC uptake by PLB-985 cells, but did not mediate (10-1000nM) the internalization of the fluorescent anti-myc monoclonal antibody 4A6 added to the extracellular fluid at ~7nM (microscopy). The nonfluorescent version of the antibody (28nM) acts as a pre-receptor antagonist of f-Nle-Leu-Phe-Nle-Tyr-Lys-myc, but not of f-Met-Leu-Phe (superoxide release assay in differentiated PLB-985 cells). A further prolonged analog, f-Nle-Leu-Phe-Nle-Tyr-Lys-(Asn-Gly)5-myc, designed to decrease the possible steric hindrance between FPR1 and the bound anti-myc antibody, has little affinity for the receptor, precluding a direct assessment of this issue. Thus, the relatively low-affinity anti-myc antibody used at a high concentration functionally behaves as a selective pre-receptor antagonist of the agonist f-Nle-Leu-Phe-Nle-Tyr-Lys-myc.

Generation of Monoclonal Antibodies against Defined Oligosaccharide Antigens.

Unique carbohydrate antigens are expressed on the surface of various pathogens, including bacteria, parasites, and viruses, and aberrant glycosylation is a frequent feature of cancer cells. Antibodies recognizing such carbohydrate antigens may be used for the specific detection of potentially harmful cells, immunohistochemistry, and diagnostic and therapeutic applications. The generation of specific and strongly binding antibodies against defined carbohydrate epitopes is challenging, since isolated carbohydrates often suffer from low purity, usually have limited immunogenicity, and induce antibodies of low affinity. We describe a protocol to generate highly affine monoclonal antibodies (mAbs) against pure and defined synthetic carbohydrate antigens. First, an oligosaccharide is covalently coupled to an immunogenic carrier protein to obtain a glycoconjugate. This glycoconjugate is used to raise oligosaccharide-specific antibodies in mice, and splenocytes are fused with myeloma cells to form hybridomas. Hybridoma clones producing oligosaccharide-specific mAbs are selected by glycan microarray screening. Selected clones are expanded and mAbs are purified from the cell culture supernatant. This protocol is suitable to procure carbohydrate-specific mAbs of high specificity, selectivity, and affinity that may be useful for a variety of biochemical and medical applications.

A high-affinity native human antibody neutralizes human cytomegalovirus infection of diverse cell types.

Human cytomegalovirus (HCMV) is the most common infection causing poor outcomes among transplant recipients. Maternal infection and transplacental transmission are major causes of permanent birth defects. Although no active vaccines to prevent HCMV infection have been approved, passive immunization with HCMV-specific immunoglobulin has shown promise in the treatment of both transplant and congenital indications. Antibodies targeting the viral glycoprotein B (gB) surface protein are known to neutralize HCMV infectivity, with high-affinity binding being a desirable trait, both to compete with low-affinity antibodies that promote the transmission of virus across the placenta and to displace nonneutralizing antibodies binding nearby epitopes. Using a miniaturized screening technology to characterize secreted IgG from single human B lymphocytes, 30 antibodies directed against gB were previously cloned. The most potent clone, TRL345, is described here. Its measured affinity was 1 pM for the highly conserved site I of the AD-2 epitope of gB. Strain-independent neutralization was confirmed for 15 primary HCMV clinical isolates. TRL345 prevented HCMV infection of placental fibroblasts, smooth muscle cells, endothelial cells, and epithelial cells, and it inhibited postinfection HCMV spread in epithelial cells. The potential utility for preventing congenital transmission is supported by the blockage of HCMV infection of placental cell types central to virus transmission to the fetus, including differentiating cytotrophoblasts, trophoblast progenitor cells, and placental fibroblasts. Further, TRL345 was effective at controlling an ex vivo infection of human placental anchoring villi. TRL345 has been utilized on a commercial scale and is a candidate for clinical evaluation.

Risky business of inhibitors: HLA haplotypes, gene polymorphisms, and immune responses

Abstract The development of neutralizing antibodies against factor VIII (FVIII inhibitors) and factor IX (FIX inhibitors) is the major complication in hemophilia care today. The antibodies neutralize the biological activity of FVIII and FIX and render replacement therapies ineffective. Antibodies are generated as a result of a cascade of tightly regulated interactions between different cells of the innate and the adaptive immune system located in distinct compartments. Any event that modulates the repertoire of specific B or T cells, the activation state of the innate and adaptive immune system, or the migration pattern of immune cells will therefore potentially influence the risk for patients to develop inhibitors. This chapter reviews our current understanding of different pathways of antibody development that result in different qualities of antibodies. Potential differences in differentiation pathways leading to high-affinity neutralizing or low-affinity non-neutralizing antibodies and the potential influence of gene polymorphisms such as HLA haplotype, FVIII haplotype, and polymorphisms of immunoregulatory genes are discussed.

Immunoglobulin A as an anti-inflammatory agent.

Immunoglobulin A as an anti-inflammatory agent.

Risky business of inhibitors: HLA haplotypes, gene polymorphisms, and immune responses.

The development of neutralizing antibodies against factor VIII (FVIII inhibitors) and factor IX (FIX inhibitors) is the major complication in hemophilia care today. The antibodies neutralize the biological activity of FVIII and FIX and render replacement therapies ineffective. Antibodies are generated as a result of a cascade of tightly regulated interactions between different cells of the innate and the adaptive immune system located in distinct compartments. Any event that modulates the repertoire of specific B or T cells, the activation state of the innate and adaptive immune system, or the migration pattern of immune cells will therefore potentially influence the risk for patients to develop inhibitors. This chapter reviews our current understanding of different pathways of antibody development that result in different qualities of antibodies. Potential differences in differentiation pathways leading to high-affinity neutralizing or low-affinity non-neutralizing antibodies and the potential influence of gene polymorphisms such as HLA haplotype, FVIII haplotype, and polymorphisms of immunoregulatory genes are discussed.

Epstein-Barr virus-specific intrathecal oligoclonal IgG production in relapsing-remitting multiple sclerosis is limited to a subset of patients and is composed of low-affinity antibodies.

BackgroundThe purpose of this study was to investigate intrathecal production and affinity distributions of Epstein-Barr virus (EBV)-specific antibodies in multiple sclerosis (MS) and controls.MethodsCerebrospinal fluid (CSF) and serum concentrations, quantitative intrathecal synthesis, oligoclonal bands (OCB) patterns and affinity distributions of anti-Epstein Barr virus (EBV) antibodies were evaluated in 100 relapsing-remitting MS (RRMS) patients and 200 age- and sex-matched controls with other inflammatory neurological disorders (OIND) and other noninflammatory neurological disorders (NIND).ResultsLevels of anti-EBNA-1 and anti-viral capsid antigen (VCA) IgG were different in both the CSF (P <0.0001 and P <0.01, respectively) and serum (P <0.001 and P <0.05, respectively) among the RRMS, OIND and NIND. An intrathecal synthesis of anti-EBNA-1 IgG and anti-VCA IgG, as indicated by the antibody index, was underrepresented in the RRMS, OIND and NIND (range 1 to 7%). EBV-specific OCB were detected in 24% of the RRMS patients and absent in the controls. High-affinity antibodies were more elevated in the RRMS and in the OIND than in the NIND for CSF anti-EBNA-1 IgG (P <0.0001) and anti-VCA IgG (P <0.0001). After treatment with increasing concentrations of sodium thiocyanate, the EBV-specific IgG OCB had low affinity in all 24 RRMS patients analyzed.ConclusionsOur findings do not support the potential role of an EBV persistent brain chronic infection in MS and suggest that an EBV-specific intrathecal oligoclonal IgG production can occur in a subset of MS patients as part of humoral polyreactivity driven by chronic brain inflammation.

Bi-epitope SPR surfaces: a solution to develop robust immunoassays.

Surface plasmon resonance (SPR)-based immunoassays have numerous applications and require high affinity reagents for sensitive and reliable measurements. We describe a quick approach to turn low affinity antibodies into appropriate capture reagents. We used antibodies recognizing human ephrin type A receptor 2 (EphA2) and a ProteOn XPR36 as a model system. We generated so-called ‘bi-epitope’ sensor surfaces by immobilizing various pairs of anti-EphA2 antibodies using standard amine coupling. The apparent binding affinities to EphA2 and EphA2 detection sensitivities of the bi-epitope and ‘single-epitope’ surfaces were then compared. For all antibody pairs tested, bi-epitope surfaces exhibited an ∼10–100-fold improvement in apparent binding affinities when compared with single-epitope ones. When pairing 2 antibodies of low intrinsic binding affinities (∼10−8 M) and fast dissociation rates (∼10−2 s−1), the apparent binding affinity and dissociation rate of the bi-epitope surface was improved up to ∼10–10 M and 10−4 s−1, respectively. This led to an ∼100–200-fold enhancement in EphA2 limit of detection in crude cell supernatants. Our results show that the use of antibody mixtures in SPR applications constitutes a powerful approach to develop sensitive immunoassays, as previously shown for non-SPR formats. As SPR-based assays have significantly expanded their reach in the last decade, such an approach promises to further accelerate their development.

Applications for an engineered Protein-G variant with a pH controllable affinity to antibody fragments

Immunoglobulin binding proteins (IBPs) are broadly used as reagents for the purification and detection of antibodies. Among the IBPs, the most widely used are Protein-A and Protein-G. The C2 domain of Protein-G from Streptococcus is a multi-specific protein domain; it possesses a high affinity (KD ~10nM) for the Fc region of the IgG, but a much lower affinity (KD~low μM) for the constant domain of the antibody fragment (Fab), which limits some of its applications. Here, we describe the engineering of the Protein-G interface using phage display to create Protein-G-A1, a variant with 8 point mutations and an approximately 100-fold improved affinity over the parent domain for the 4D5 Fab scaffold. Protein-G-A1 is capable of robust binding to Fab fragments for numerous applications. Furthermore, we isolated a variant with pH-dependent affinity, demonstrating a 1,000-fold change in affinity from pH7 to 4. Additional rational mutagenesis endowed Protein-G with significantly enhanced stability in basic conditions relative to the parent domain while maintaining high affinity to the Fab. This property is particularly useful to regenerate Protein-G affinity columns. Lastly, the affinity-matured Protein-G-A1 variant was tethered together to produce dimers capable of providing multivalent affinity enhancement to a low affinity antibody fragment–antigen interaction. Engineered Protein-G variants should find widespread application in the use of Fab-based affinity reagents.

A subtraction tolerization method of immunization allowed for Wilms' tumor protein-1 (WT1) identification in melanoma and discovery of an antitumor peptide sequence

On searching for melanoma transcription factors in a project focusing on internal antitumor peptide sequences from transcription factors, we found that a highly immunogenic component emerged upon using a subtraction tolerization method of immunization. While several conventional immunization procedures using whole melanoma cells induced a plethora of low affinity antibodies of various specificities, the subtraction tolerization method efficiently elicited mono-specific antibodies that recognized Wilms' tumor protein 1 (WT1), which is known as an important marker in melanoma prognosis and treatment. For the tolerization step, pre-immunization of Balb/c mice with a membrane-rich preparation of glioblastoma U87 cells was used. The subsequent immunizations with SK-MEL-28 melanoma cells elicited antibodies strongly reacting with 50 and 55kDa proteins, identified as WT1. Remarkably, this was the only component strongly reactive with these antibodies in a melanoma cell lysate. WT1 was then chosen as a target for selecting internally bioactive peptides. A hydrophilic Trojan peptide containing most of the zinc finger-2 domain of WT1 was synthesized and shown to inhibit SK-MEL-28 melanoma growth in vitro. The peptide WT1-pTj was also protective in vivo in a metastatic melanoma model and peptide-stimulated syngeneic dendritic cells reproduced the anti-melanoma effect of the unprotected peptide. Identification of antitumor peptides derived from major transcription factors represents a new tool to be explored in cancer research aiming at new therapeutic drugs.