Multispecific monoclonal antibodies]

Immunization of BALB/c mice with Rickettsia prowazekii antigens, Bordetella pertussis toxin and Legionella pneumophila cytolysin induces the synthesis of IgM autoantibodies of different specificity. Among monoclonal antibodies, multispecific antibodies with a wide reactivity spectrum have been found to make up high percentage (30-80%). Monoclonal antibodies interact with different bacterial antigens and tissue substances. A hypothesis has been put forward that normally the injection of the antigen is followed by the appearance of antigen-nonspecific "immunological noise", including the synthesis of both tissue-specific and multispecific autoantibodies. Such antigen nonspecific "immunological noise" must have a certain threshold level which can be determined with the use of hybridoma techniques. This problem is particularly topical for bacterial antigens, as many of them are used in the development of vaccinal preparations, which may lead to an increase in the synthesis of autoantibodies and induce different autoimmune disturbances in the body.     

A Systems Immunology Approach to the Host-Tumor Interaction: Large-Scale Patterns of Natural Autoantibodies Distinguish Healthy and Tumor-Bearing Mice

Traditionally, immunology has considered a meaningful antibody response to be marked by large amounts of high-affinity antibodies reactive with the specific inciting antigen; the detection of small amounts of low-affinity antibodies binding to seemingly unrelated antigens has been considered to be beneath the threshold of immunological meaning. A systems-biology approach to immunology, however, suggests that large-scale patterns in the antibody repertoire might also reflect the functional state of the immune system. To investigate such global patterns of antibodies, we have used an antigen-microarray device combined with informatic analysis. Here we asked whether antibody-repertoire patterns might reflect the state of an implanted tumor. We studied the serum antibodies of inbred C57BL/6 mice before and after implantation of syngeneic 3LL tumor cells of either metastatic or non-metastatic clones. We analyzed patterns of IgG and IgM autoantibodies binding to over 300 self-antigens arrayed on slides using support vector machines and genetic algorithm techniques. We now report that antibody patterns, but not single antibodies, were informative: 1) mice, even before tumor implantation, manifest both individual and common patterns of low-titer natural autoantibodies; 2) the patterns of these autoantibodies respond to the growth of the tumor cells, and can distinguish between metastatic and non-metastatic tumor clones; and 3) curative tumor resection induces dynamic changes in these low-titer autoantibody patterns. The informative patterns included autoantibodies binding to self-molecules not known to be tumor-associated antigens (including insulin, DNA, myosin, fibrinogen) as well as to known tumor-associated antigens (including p53, cytokeratin, carbonic anhydrases, tyrosinase). Thus, low-titer autoantibodies that are not the direct products of tumor-specific immunization can still generate an immune biomarker of the body-tumor interaction. System-wide profiling of autoantibody repertoires can be informative.     

Latent autoantibodies to cardiolipin in the blood serum of healthy subjects

Pooled health blood donors' sera were fractionated by gel filtration and/or ion exchange chromatography on strong anion-exchanger. Measurement of anticardiolipin antibodies levels by ELISA method shows that gel filtration at pH 4.05 and 9.2 (complex-degradation conditions) leads to increase in summary levels while after elution at neutral pH such an effect did not appear. Light increasing of anticardiolipin levels was also noted after fractionation on QAE-Sephadex. Data obtained state that anticardiolipin autoantibodies amount in sera is greater than it can be detected by direct measurement in untreated serum samples. Existence of "hidden" anticardiolipin autoantibodies supposes the hypothesis about alternative way of humoral immunity regulation by blocking anti-self antigens activities with serum biopolymers.     

Autoantibodies against spectrin in rats

Purified, homologous spectrin, inner component of red blood cell (RBC) membrane, injected intraperitoneally in rats determines the appearance in the serum of precipitating autoantibodies against spectrin. These have been purified and characterized according to their classes and anti spectrin activity. Immunochemical and immunocytochemical tests, including immunoelectronmicroscopy (colloidal gold method) have been employed. They belong principally to IgG1 and IgG2a subclasses and react in electro-immunodiffusion and ELISA tests with purified spectrin showing a more elevated level of antibodies than that of control rats (normal and adjuvant treated). They also mark in immunoelectronmicroscopy assays purified RBC membranes. The ease in inducing precipitating anti spectrin autoantibodies, as resulted in many experiments, and the appearance, also in control rats, of anti spectrin antibodies, even if at a very low level, suggest they rise as consequence of an enhancement of a "natural" immunological system normally operating at low and controlled degree, presumably intervening in the elimination of effete or damaged RBCs.     

Systemic sclerosis immunoglobulin G autoantibodies bind the human cytomegalovirus late protein UL94 and induce apoptosis in human endothelial cells

Systemic sclerosis is an autoimmune disease characterized by immunological and vascular abnormalities. Autoantibodies against intracellular antigens are associated with particular clinical features of the disease, whereas autoantibodies against cell surface antigens may be pathogenic by inducing endothelial cell damage, considered the primary event in the pathogenesis of the disease. Latent human cytomegalovirus infection may contribute to progression of systemic sclerosis through its ability to infect endothelial cells; however, direct links between human cytomegalovirus infection and systemic sclerosis are still lacking. Molecular mimicry is one of the mechanisms that account for the link between infection and autoimmunity. Here we have identified an immunodominant peptide using systemic sclerosis serum screening of a random peptide library; such peptide shares homology with autoantigens and with the human cytomegalovirus late protein UL94 (ref. 9). Immunoglobulin G antibodies against the peptide affinity-purified from the sera of patients with systemic sclerosis specifically recognized the viral product and autoantigens; moreover, such antibodies induced endothelial cell apoptosis through specific interaction with the cell surface integrin-NAG-2 protein complex. Our results provide evidence that antibodies against human cytomegalovirus cause apoptosis of endothelial cells, considered the initial pathogenic event of systemic sclerosis, and indicate a previously unknown mechanism for the etiological link between human cytomegalovirus infection and autoimmunity.     

Self-reactive repertoire of tight skin (TSK/+) mouse: immunochemical and molecular characterization of anti-cellular autoantibodies.

The tight skin (TSK/+) mouse has been proposed as an experimental model for progressive systemic sclerosis because of the biochemical alterations in collagen synthesis and pathological similarities to the human disease. Here, we report the analysis of tight skin mice sera for the presence of anti-cytoplasmic and anti-nuclear autoantibodies and determination of the frequency of hybridomas producing anti-cellular autoantibodies. The binding specificity of TSK mAbs to nuclear and cytoplasmic antigens such as keratin, actin, vimentin, and mitochondria was determined. Of 71 monoclonal antibodies that we have studied, only 3 appear to bind to foreign as well as self-antigens, indicating that the majority of these antibodies do not belong to the class of natural autoantibodies. Our results also showed that the frequency of hybridomas producing anti-nuclear and anti-cytoplasmic antibodies was higher in TSK mice than in C57BL/6 pa/pa, the control mouse strain, used in these studies. The results of the analysis of V gene usage showed that the majority of anti-cytoplasmic and anti-nuclear antibodies are encoded by genes from a restricted number of VH and VK genes families. In the sera of TSK mice we have detected the presence autoantibodies specific for cytoplasmic antigens in addition to anti-nuclear and anti-topoisomerase I antibodies which are characteristic of scleroderma. Since the presence of anti-cytoplasmic antibodies has not been described in scleroderma, the significance of their production in tight skin mice is not clear. However, the presence of such autoantibodies in the animal model provides a basis for investigation of this type of antibodies in human disease.     

Anti-DNA antibodies and the problem of autoimmunity

Several different classes of autoreactive antibodies are known to exist: those that are stimulated by bacterial infection (e.g., streptococci/rheumatic fever), those that react with tissue-specific antigens (e.g., thyrotropin receptor/Graves' disease), and those that bind to ubiquitous autoantigens (e.g., DNA/systematic lupus). The origin of the last kind of autoantibody is unknown, but it now seems that their production is an inherent property of the normal immune system. Indeed, it would appear that autoantibodies of the lupus variety actually have a physiological role in normal immunity. The development of the autoimmune disease may occur when there is an "escape" from the normal function of lupus autoantibodies.     

Antibodies Specific for Carbamylated Proteins Precede the Onset of Clinical Symptoms in Mice with Collagen Induced Arthritis

Objective

The immune response to post-translationally modified antigens is a key characteristic of rheumatoid arthritis. Carbamylation is such a posttranslational modification. Recently, we demonstrated that autoantibodies recognizing carbamylated proteins are present in sera of rheumatoid arthritis. The molecular mechanisms underlying the break of tolerance and hence the induction of anti-CarP antibody responses are unknown as well as their appearance in mouse models for systemic arthritis. Therefore we analyzed their appearance in the mouse collagen-induced arthritis model.

Methods

collagen induced arthritis was induced by immunization with type II collagen in complete Freund''s adjuvant. Arthritis severity was monitored by clinical scoring and anti-CarP antibody levels were determined by ELISA.

Results

Anti-CarP antibodies were detectable in mice with collagen induced arthritis. We did not detect ACPA in mice with collagen induced arthritis. The specificity of the antibodies for carbamylated proteins was confirmed by inhibition assays and immunoblotting. Injection with complete Freund''s adjuvant without type II collagen could also induce anti-CarP antibodies, however, in mice with arthritis, the anti-CarP antibody response was stronger and developed more rapidly. The onset of collagen induced arthritis was preceded by an increase of anti-CarP IgG2a levels in the serum.

Conclusion

In mice with collagen induced arthritis we did not observe an immune response against citrullinated antigens, but we did observe an immune response against carbamylated antigens. This anti-CarP response already appeared before disease onset, indicating that collagen induced arthritis can be used as an in vivo model to study anti-CarP antibodies. Our data also indicate that the tolerance to carbamylated proteins, in contrast to the response to citrullinated proteins, is easily broken and that arthritis boosts the immune response against these proteins. The anti-CarP response in mice with CIA can be used as a model for immune responses to post-translationally modified proteins.     

Cancer-associated antigens and antigen arrays in serological diagnostics of malignant tumors

The appearance of antibodies to cancer-associated antigens in biological fluids (particularly, in blood sera) of cancer patients is now a well-established fact, and their detection by immunochemical methods is a promising approach to diagnostics of malignant neoplasms. In this review, we consider some immunobiological aspects of the most extensively studied cancer-associated B-cell antigens, various applications of autoantibodies as cancer biomarkers, and prospects for the use of antigen arrays for improving diagnostic sensitivity.     

B-cell epitopes of autoantigenic DNA-binding proteins

Conclusions Autoantibodies to chromatin-associated proteins are frequently present in sera from patients with SLE, and related disorders. Autoantibodies to conformational epitopes may constitute the majority of the immune response to chromatin-associated antigens, suggesting that intact chromatin may be the immunogen in SLE as well as in certain forms of drug-induced lupus (eg. in procainamide-induced lupus). The preferential reactivity of autoantibodies to histones, PCNA, and Ku with antigenic determinants that are exposed on the surface of the native antigens is consistent with this interpretation.Strikingly, autoantibodies to these antigens frequently bind within or near active or functional sites, such as the DNA binding site of Ku [29], the site of PCNA critical for its role in enhancing DNA synthesis by polymerase delta [52], the posttranslational modification sites of the histones [68], and the catalytic site of poly(ADP-ribose) polymerase [69]. The explanation for the frequent observation that autoantibodies inhibit function is not yet known. It is possible that this phenomenon is related to the generation of autoantibodies by molecular mimicry, and that the functional sites of foreign antigens may crossreact with self antigens having similar functional sites [9]. Alternatively, the targeting of functional sites by autoantibodies may reflect merely a similar requirement for active sites and antibody-recognition sites to be exposed on surface. Features that make a site suitable for interacting with other proteins (eg. enzymes) or nucleic acids (eg DNA binding sites) may also make it more easily recognized by antibodies.The amino acids critical for autoantibody binding have not, in any of these cases, been shown to be critical to function. Further mapping and/ or mutagenesis studies will be necessary to determine the significance of the targeting of active or functional sites by autoantibodies.This work was supported by Public Health Service grant AR40391 from the National Institutes of Health     

Antinuclear Antibodies and Nuclear Antigens

The lupus erythematosus (LE) cell factor is one of a variety of heterogeneous antibodies directed against many nuclear antigens. These antinuclear antibodies are found in a wide variety of clinical disorders. As detected by immunofluorescence techniques, they are so frequently found in association with systemic lupus erythematosus that their absence essentially excludes that diagnosis.It is suggested that in certain situations other than systemic lupus, antinuclear antibodies may be the result of some inflammatory and destructive processes. Antinuclear antibodies may be detected in certain lower animals frequently in association with disease. In lower animals, immunization with nuclear antigens appropriately complexed^* to protein carriers consistently results in the induction of antinuclear antibodies. In both man and animals, antinuclear antibodies are often detected without associated disease. Furthermore, antinuclear antibodies are not injurious to intact tissue culture cells. Nonetheless, antigen-antibody complexes consisting of nuclear antigens and antinuclear antibodies may contribute to the propagation of certain diseases, particularly lupus nephritis.The use of antinuclear antibodies as immunochemical tools holds great promise for the better understanding of such nuclear antigens as are found in viruses and in the nuclei of certain malignant cells.     

Application of protein microarrays for multiplexed detection of antibodies to tumor antigens in breast cancer

There is strong preclinical evidence that cancer, including breast cancer, undergoes immune surveillance. This continual monitoring, by both the innate and the adaptive immune systems, recognizes changes in protein expression, mutation, folding, glycosylation, and degradation. Local immune responses to tumor antigens are amplified in draining lymph nodes, and then enter the systemic circulation. The antibody response to tumor antigens, such as p53 protein, are robust, stable, and easily detected in serum; may exist in greater concentrations than their cognate antigens; and are potential highly specific biomarkers for cancer. However, antibodies have limited sensitivities as single analytes, and differences in protein purification and assay characteristics have limited their clinical application. For example, p53 autoantibodies in the sera are highly specific for cancer patients, but are only detected in the sera of 10-20% of patients with breast cancer. Detection of p53 autoantibodies is dependent on tumor burden, p53 mutation, rapidly decreases with effective therapy, but is relatively independent of breast cancer subtype. Although antibodies to hundreds of other tumor antigens have been identified in the sera of breast cancer patients, very little is known about the specificity and clinical impact of the antibody immune repertoire to breast cancer. Recent advances in proteomic technologies have the potential for rapid identification of immune response signatures for breast cancer diagnosis and monitoring. We have adapted programmable protein microarrays for the specific detection of autoantibodies in breast cancer. Here, we present the first demonstration of the application of programmable protein microarray ELISAs for the rapid identification of breast cancer autoantibodies.     

Cellular and molecular pathogenesis of systemic lupus erythematosus: lessons from animal models

Systemic lupus erythematosus (SLE) is a complex disease characterized by the appearance of autoantibodies against nuclear antigens and the involvement of multiple organ systems, including the kidneys. The precise immunological events that trigger the onset of clinical manifestations of SLE are not yet well understood. However, research using various mouse strains of spontaneous and inducible lupus in the last two decades has provided insights into the role of the immune system in the pathogenesis of this disease. According to our present understanding, the immunological defects resulting in the development of SLE can be categorized into two phases: (a) systemic autoimmunity resulting in increased serum antinuclear and antiglomerular autoantibodies and (b) immunological events that occur within the target organ and result in end organ damage. Aberrations in the innate as well as adaptive arms of the immune system both play an important role in the genesis and progression of lupus. Here, we will review the present understanding - as garnered from studying mouse models - about the roles of various immune cells in lupus pathogenesis.     

Anti-cytokine autoantibodies: Epiphenomenon or critical modulators of cytokine action

Low amounts of high-affinity autoantibodies to various cytokines have been detected in sera from healthy donors. Their levels, although highly variable, are increased in the circulation of patients subjected to cytokine therapy or suffering from a variety of immunoinflammatory diseases. It has been suggested that these autoantibodies play a regulatory role in the intensity and duration of an immune response. The antibodies may prevent the binding of a cytokine to its specific cell surface receptor thereby neutralizing its biological activityin vivo. They may also act as carrier proteins preventing the rapid elimination of a cytokine from the circulation and thus increase its bioactivity. Additionally or alternatively, autoantibodies may modulate cytokine-induced intracellular signal transduction pathways or trigger complement-mediated cytotoxicity towards cells carrying membrane-bound cytokines. The autoantibodies may exert their regulatory role in compliance with other factors that control cytokine activity, including soluble cytokine receptors, cell surface decoy receptors, and receptor antagonists. Although not favored by many investigators, a less sophisticated role for naturally occurring anti-cytokine autoantibodies should be considered as well. Recent evidence has shown that autoantibodies are generated at a high frequency as part of a response to foreign antigens. These antibodies are produced by B cells arising from the process of somatic mutation. Thus anti-cytokine autoantibodies may be the result of a “leaky” B cell response triggered by immunoinflammatory processes. High-titered autoantibodies induced by cytokine therapy are of clinical concern since their occurrence is often associated with the loss of response to treatment. Moreover, they may also neutralize endogenously produced cytokines with possible pathological consequences. In this paper we have reviewed the available information on the biological and clinical significance of both naturally occurring and therapeutically induced anti-cytokine autoantibodies in animals and man with the emphasis on antibodies directed to interferons.     

The impact of symbolism on immunogenetics: an application to HLA

The genes coding for the class I human lymphocyte antigens (HLA) are located on chromosome 6. These antigens are involved with the immunological interaction between cells. In some immunogenetic systems, such as HLA in humans, genes are defined by antibody/antigen reaction and are denoted by single symbolic identifiers. This symbolization assumes a one-to-one correspondence between antibodies, antigens and genes. Recent molecular studies, however, suggest that HLA antibody/antigen reaction is complex and most HLA class I specific antibodies may not uniquely identify a single allelic product. Where cross-reactivity is present in an immunogenetic system it is important to label each reagent with symbols corresponding to all genes coding for antigens with which the reagent will react. The problems of cross-reactive groups and unexplained linkage relations may be elucidated by the redefinition and clarification of certain HLA antigens. A computer program can suggest such labelling schemes using input given by phenotype reaction patterns with a panel of reagents. When this program was applied to data on the class I HLA antigens a genetic model was suggested that differs somewhat from the currently accepted model. The new model is able to predict what would appear as linkage relations in the accepted model. Our methodology can provide alternate models to guide in typing cloned genes in terms of the HLA locus and alleles.     

Identification of Ki (Ku, p70/p80) autoantigens and analysis of anti-Ki autoantibody reactivity

Anti-Ki (Ku, p70/p80) autoantibodies, named after the prototype patient Kikuta by Tojo et al., occur in approximately 10% of patients with SLE, often in association with anti-Sm autoantibodies. The immunofluorescent staining pattern characteristic of anti-Ki antibodies is diffuse speckled nuclear, although some substrates show nucleolar staining as well. Anti-Ki sera specifically immunoprecipitated two protein antigens, Ki86 (Mr 86,000) and Ki66 (Mr 66,000), from radiolabeled cell extracts. The Ki system was found to be immunologically identical to the Ku system described by Mimori et al. and the p70/p80 system described by Reeves. The Ki primary in vitro translation products were identified and proved similar in size to the cellular antigens. The Ki antigens were purified from human spleen by immunoaffinity chromatography followed by SDS-PAGE. The purified Ki antigens proved to be closely related by amino acid composition, and did not appear to be phosphorylated, glycosylated, or associated with RNA. The Ki antigens were found to bind to DNA, in agreement with the observations on the Ku and p70/p80 antigens. They were found to be widely conserved in mammals and were coordinately expressed in all tissues tested. Anti-Ki autoantibodies were purified by antigen-affinity chromatography and were tested by immunoblotting. The antibodies were classified as class I, II, or III, depending on their reactivity with the Ki antigens in immunoblots. Class I antibodies cross-reacted with both Ki antigens, class II antibodies reacted solely with Ki66, and class III antibodies reacted solely with Ki86. These results suggest that at least three different epitopes are present on the Ki autoantigens and that patients differ in their autoantibody response to each epitope.     

大疱性类天疱疮IgG型抗基底膜带抗体的靶抗原初步分析

目的　研究大疱性类天疱疮 Ｉｇ Ｇ 型抗基底膜带抗体的靶抗原。方法　应用 Ｄ Ｉ Ｆ与 Ｉ Ｉ Ｆ检测的 Ｉｇ Ｇ型抗体阳性 Ｂ Ｐ 血清进行 Ｗ ｅｓｔｅｒｎ 免疫印迹分析。结果　初步研究发现 Ｉｇ Ｇ 阳性的 Ｂ Ｐ 血清能结合抗原１８０ Ｋ Ｄ,１６０ Ｋ Ｄ 以及 １０５ Ｋ Ｄ 与 ７７ Ｋ Ｄ 的多肽。结论　 Ｉｇ Ｇ 型 Ｂ Ｐ自身抗体可能靶向基底膜带不同层出现的不同抗原并提示该病的发生可能涉及不同的免疫机制。     

Human monoclonal autoantibodies to characterize platelet antigens in immune-mediated thrombocytopenia

Idiopathic thrombocytopenic purpura is characterized by antiplatelet antibodies which mediate the rapid destruction of these cells by the reticuloendothelial cell system. Low serum titers of autoantibodies and the polyclonal nature of human serum make it difficult to identify platelet target antigens with plasma antibodies. To circumvent these problems, we have utilized the techniques of EBV transformation and somatic cell hybridization in order to isolate human monoclonal antibodies from patients with ITP. In this paper we describe the use of human monoclonal autoantibodies to characterize an activation specific antigen on GPIIIa and an autoantigen on the GPIb complex. Ultimately, we hope to determine whether these autoantibodies emerge from a pool of naturally occurring antibodies to activation or senescence antigens, or are triggered by environmental agents such as bacteria or virus, which are comprised of antigens similar to those found on the platelet membrane.     

Expression of epidermis-specific antigens during embryogenesis of the ascidian, Halocynthia roretzi

We have produced two monoclonal antibodies (Epi-1 and Epi-2) which specifically recognize epidermal cells and their derivative, the larval tunic, of developing embryos of the ascidian Halocynthia roretzi. The antigens, examined by indirect immunofluorescence staining, first appear at the early tailbud stage and are present until at least the swimming larval stage. There were distinct and separate puromycin and actinomycin D sensitivity periods for each antigen. Aphidicolin, a specific inhibitor of DNA synthesis, prevented the appearance of each antigen when embryos were exposed to the drug continuously from cleavage stages. These results suggest that the antigens are synthesized during embryogenesis by developing epidermal cells and that several rounds of DNA replication are required for the antigen expression. Early cleavage stage embryos, including fertilized but unsegmented eggs, in which cytokinesis had been blocked with cytochalasin B expressed the antigens, and blastomeres exhibiting the antigens were always of the epidermis lineage. In partial embryos produced by four separated blastomere pairs of the 8-cell embryos, the expression of antigens was seen only in those developed from the animal blastomere pairs, which are progenitors of epidermal cells. These observations indicate that differentiation of epidermal cells in ascidian embryos takes place in a typical "mosaic" fashion.     

Tumor-infiltrating T cells correlate with NY-ESO-1-specific autoantibodies in ovarian cancer

Background

Tumor-infiltrating CD8+ T cells are correlated with prolonged progression-free and overall survival in epithelial ovarian cancer (EOC). A significant fraction of EOC patients mount autoantibody responses to various tumor antigens, however the relationship between autoantibodies and tumor-infiltrating T cells has not been investigated in EOC or any other human cancer. We hypothesized that autoantibody and T cell responses may be correlated in EOC and directed toward the same antigens.

Methodology and Principal Findings

We obtained matched serum and tumor tissue from 35 patients with high-grade serous ovarian cancer. Serum samples were assessed by ELISA for autoantibodies to the common tumor antigen NY-ESO-1. Tumor tissue was examined by immunohistochemistry for expression of NY-ESO-1, various T cell markers (CD3, CD4, CD8, CD25, FoxP3, TIA-1 and Granzyme B) and other immunological markers (CD20, MHC class I and MHC class II). Lymphocytic infiltrates varied widely among tumors and included cells positive for CD3, CD8, TIA-1, CD25, FoxP3 and CD4. Twenty-six percent (9/35) of patients demonstrated serum IgG autoantibodies to NY-ESO-1, which were positively correlated with expression of NY-ESO-1 antigen by tumor cells (r = 0.57, p = 0.0004). Autoantibodies to NY-ESO-1 were associated with increased tumor-infiltrating CD8+, CD4+ and FoxP3+ cells. In an individual HLA-A2+ patient with autoantibodies to NY-ESO-1, CD8+ T cells isolated from solid tumor and ascites were reactive to NY-ESO-1 by IFN-γ ELISPOT and MHC class I pentamer staining.

Conclusion and Significance

We demonstrate that tumor-specific autoantibodies and tumor-infiltrating T cells are correlated in human cancer and can be directed against the same target antigen. This implies that autoantibodies may collaborate with tumor-infiltrating T cells to influence clinical outcomes in EOC. Furthermore, serological screening methods may prove useful for identifying clinically relevant T cell antigens for immunotherapy.