SSX2-4 expression in early-stage non-small cell lung cancer.

The expression of cancer/testis antigens SSX2, SSX3, and SSX4 in non-small cell lung cancers (NSCLC) was examined, since they are considered promising targets for cancer immunotherapy due to their immunogenicity and testis-restricted normal tissue expression. We characterized three SSX antibodies and performed immunohistochemical staining of 25 different normal tissues and 143 NSCLCs. The antibodies differed in binding to two distinctive splice variants of SSX2 that exhibited different subcellular staining patterns, suggesting that the two splice variants display different functions. SSX2-4 expression was only detected in 5 of 143 early-stage NSCLCs, which is rare compared to other cancer/testis antigens (e.g. MAGE-A and GAGE). However, further studies are needed to determine whether SSX can be used as a prognostic or predictive biomarker in NSCLC.

Limited SP17 expression within tumors diminishes its therapeutic potential.

In this study, we have investigated the expression of the tumor antigen sperm protein 17 (SP17) in a large panel of human cancers and compared it with the expression of two well-characterized families of tumor antigens, melanoma-associated antigen-A (MAGE-A) and G antigen (GAGE). We found that SP17 was expressed in many cancer types with an overall frequency of 12%. SP17 was most frequently expressed in a different set of cancer types than MAGE-A and GAGE antigens and rarely overlapped with these proteins. Importantly, SP17 expression was limited to a small number of scattered cancer cells in most positive tumors in contrast to MAGE-A and GAGE proteins, which were homogenously expressed in large foci. Our results suggest that SP17 may not be an optimal target for cancer vaccines.

SSX2 promotes the formation of a novel type of intranuclear lamin bodies.

SSX proteins are normally restricted to spermatogenic cells, but ectopic expression can be observed in many types of human cancer. We recently demonstrated that SSX family members may contribute to tumorigenesis by modifying chromatin structure and, in specific settings, compromise chromatin stability. Here, we used normal and tumorigenic breast epithelial cell line models to further study the effect of ectopic expression of SSX2 on nuclear organization. We show that SSX2 induces the formation of a novel type of nucleoplasmic lamin bodies. Ectopic expression of SSX2 in various breast epithelial cell lines led to the formation of a previously undescribed type of intranuclear bodies containing both A and B type lamins but no other components of the nuclear lamina. SSX2-expressing cells contained a highly variable number of lamin bodies distributed throughout the nuclear space. SSX2-mediated establishment of intranuclear lamin bodies could not be linked to previous molecular interactions of SSX proteins, including polycomb proteins and the Mediator complex, but was, however, dependent on S-phase progression. These results reveal a novel interaction between SSX2 and lamins in the nucleoplasmic space. They further suggest that SSX2 promotes the formation of chromatin neighborhoods supporting the organization of lamins into nuclear bodies. We speculate that this may have implications for the organization and functional regulation of chromatin in cancer cells. Our study contributes to the further understanding of the biology of SSX proteins in tumorigenesis.

Correction: Gene expression profiling identifies FYN as an important molecule in tamoxifen resistance and a predictor of early recurrence in patients treated with endocrine therapy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Correction: Gene expression profiling identifies FYN as an important molecule in tamoxifen resistance and a predictor of early recurrence in patients treated with endocrine therapy.

Since the online publication of the above article, the authors have noted errors in subfigures 1c and 3b. Therefore, new images of the original immmunocytochemistry stainings have been obtained for Fig. 1c, and the Western blots for siRNA-mediated FYN knockdown in Fig. 3b were repeated. The amended versions of Figs. 1c and 3b are now provided.

Mapping the protein surface of human immunodeficiency virus type 1 gp120 using human monoclonal antibodies from phage display libraries.

Panels of hybridoma-derived monoclonal antibodies against diverse epitopes are widely used in defining protein surface topography, particularly in the absence of crystal or NMR structural information. Here we show that recombinant monoclonal antibodies from phage display libraries provide a rapid alternative for surface epitope mapping. Diverse epitopes are accessed by presenting antigen to the library in different forms, such as sequential masking of epitopes with existing antibodies or ligands prior to selection and selection on peptides. The approach is illustrated for a recombinant form of the human immunodeficiency virus type 1 (HIV-1) surface glycoprotein gp120 which has been extensively mapped by rodent and human monoclonal antibodies derived by cellular methods. Human recombinant Fab fragments to most of the principal epitopes on gp120 are selected including Fabs to the C1 region, a C1/C5 epitope, a C1/C2 epitope, the V2 loop, the V3 loop and the CD4 binding domain. In addition an epitope linked to residues in the V2 loop and CD4 binding domain is identified. Most of these specificities are associated with epitopes presented poorly on native multimeric envelope, consistent with the notion that these antibodies are associated with immunization by forms of gp120 differing in conformation from that found on whole virus or infected cells.

Gene expression profiling identifies FYN as an important molecule in tamoxifen resistance and a predictor of early recurrence in patients treated with endocrine therapy.

To elucidate the molecular mechanisms of tamoxifen resistance in breast cancer, we performed gene array analyses and identified 366 genes with altered expression in four unique tamoxifen-resistant (TamR) cell lines vs the parental tamoxifen-sensitive MCF-7/S0.5 cell line. Most of these genes were functionally linked to cell proliferation, death and control of gene expression, and include FYN, PRKCA, ITPR1, DPYD, DACH1, LYN, GBP1 and PRLR. Treatment with FYN-specific small interfering RNA or a SRC family kinase inhibitor reduced cell growth of TamR cell lines while exerting no significant effect on MCF-7/S0.5 cells. Moreover, overexpression of FYN in parental tamoxifen-sensitive MCF-7/S0.5 cells resulted in reduced sensitivity to tamoxifen treatment, whereas knockdown of FYN in the FYN-overexpressing MCF-7/S0.5 cells restored sensitivity to tamoxifen, demonstrating growth- and survival-promoting function of FYN in MCF-7 cells. FYN knockdown in TamR cells led to reduced phosphorylation of 14-3-3 and Cdc25A, suggesting that FYN, by activation of important cell cycle-associated proteins, may overcome the anti-proliferative effects of tamoxifen. Evaluation of the subcellular localization of FYN in primary breast tumors from two cohorts of endocrine-treated ER+ breast cancer patients, one with advanced disease (N=47) and the other with early disease (N=76), showed that in the former, plasma membrane-associated FYN expression strongly correlated with longer progression-free survival (P<0.0002). Similarly, in early breast cancer patients, membrane-associated expression of FYN in the primary breast tumor was significantly associated with increased metastasis-free (P<0.04) and overall (P<0.004) survival independent of tumor size, grade or lymph node status. Our results indicate that FYN has an important role in tamoxifen resistance, and its subcellular localization in breast tumor cells may be an important novel biomarker of response to endocrine therapy in breast cancer.

Protection against HIV-1 infection in hu-PBL-SCID mice by passive immunization with a neutralizing human monoclonal antibody against the gp120 CD4-binding site.

OBJECTIVE: Mice with severe combined immunodeficiency (SCID) transplanted with human peripheral blood lymphocytes (hu-PBL) have been shown to be useful as an animal model for HIV-1 infection. This model was used to assess the ability of a human anti-gp120 antibody to protect against HIV-1 infection. DESIGN AND METHODS: hu-PBL-SCID mice were injected with an HIV-1 broadly neutralizing human monoclonal antibody against the gp120 CD4-binding site prior to challenge with HIV-1SF2. The antibody b12, employed for these studies, was isolated from an antibody phage-display library prepared from bone-marrow of a long-term asymptomatic HIV-1-seropositive donor. Both Fab fragments and whole immunoglobulin (Ig) G1 b12 antibody were assessed for protection. RESULTS: Fab b12, tested at a dose approximately 1.9 mg/kg, was able to protect 25% of hu-PBL-SCID mice from HIV-1 infection. IgG1 b12, which displayed favorable pharmacokinetic properties, showed a dose-dependent protection that was complete with a regimen of two injections of 100 micrograms per mouse. The in vivo protective dose of antibody at the time of virus challenge was estimated to be 4.5-7 mg/kg from antibody clearance data. CONCLUSIONS: This study demonstrates for the first time that complete protection against HIV-1 infection can be achieved in the hu-PBL-SCID model by passive immunization with physiologically relevant doses of a human gp120 CD4-binding site antibody derived from natural infection.

Human antibodies in cancer and autoimmune disease.

In recent years, a number of novel human autoantigens and tumor-associated antigens have been identified using patient sera. Several of these antigens have been used as diagnostic markers, but defining their role in disease pathogenesis has been hampered by the lack of cloned human antibodies and antigens. Focusing on the solid cancers of the breast and colon and on autoimmune hematologic diseases, we are studying the role of human antibodies in disease pathogenesis. We have generated several human monoclonal autoimmune and cancer-associated antibodies, using antibody phage display technology, and have identified, cloned, and expressed their corresponding (novel) antigens. Using the monoclonal human antibodies as probes, we are elucidating the processes that lead to the generation of these antibodies and their possible pathogenic or protective effect. These studies may lead to the development of reagents for diagnosis and therapeutic intervention of these important diseases.

Antibodies in human infectious disease.

Investigation of human antibody responses to viral pathogens at the molecular level is revealing novel aspects of the interplay of viruses with the humoral immune system. In viral infection, at least two types of human antibody responses exist: a response to mature envelope on virions that is neutralizing and a response to immature forms of envelope (viral debris) that is not. Many pathogens have, to varying degrees, evolved envelopes to minimize antibody responses against epitopes exposed on the virion. In this article, we review recent studies on human immunodeficiency virus type 1, Ebola virus, and respiratory syncytial virus. Prion diseases are diseases of protein conformation. We have generated a large panel of antibodies recognizing the cellular prion protein (PrP(c)), some of which also react with the abnormally folded infectious prion protein (PrP(Sc)). These antibodies are being used to gain insight into both the molecular events leading to the formation of infectious PrP and the physiologic role played by PrP in normal and prion-infected cells.

Pilot scale purification of human monoclonal IgM (COU-1) for clinical trials.

No standard procedure is available for the purification of human monoclonal antibodies for human i.v. administration. Here we describe the procedure developed for pilot scale purification of the human IgM monoclonal antibody COU-1 directed against a cancer-associated antigen. The hybridoma cells were grown in protein-free medium and purification from the clarified culture supernatant was carried out in 4 simple chromatographic steps: (1) hydroxylapatite chromatography; (2) hydrophobic interaction chromatography on phenyl-Sepharose: (3) cation-exchange chromatography on sulphonyl-Sepharose; and (4) anion-exchange chromatography on tetraethylamino-Sepharose. The product was substantially pure with regard to protein after step 3, but contained DNA which was removed in step 4. The average recovery of the IgM was 54% with a range of 40-65%. Importantly, the ability of the antibody to bind to its antigen in ELISA was fully maintained during the purification. Subsequently, the purified antibody was isotope labelled and successfully used for in vivo detection of colon, rectal and pancreas carcinomas in patients. The purification procedure described appears to compare favourably with previously published methods, but a critical comparison is not possible due to the lack of necessary information in the available literature.

Human antibody responses to HIV type 1 glycoprotein 41 cloned in phage display libraries suggest three major epitopes are recognized and give evidence for conserved antibody motifs in antigen binding.

A large panel of human Fab fragments against the gp41 subunit of the HIV-1 envelope glycoprotein was isolated by panning six phage-displayed antibody libraries against recombinant gp41. The libraries were prepared from HIV-1-seropositive donors. Twenty-three Fabs recognizing conformation-dependent determinants on gp41 were isolated. Further selection of libraries against (1) gp41 ligated with Fabs from the initial selection and against (2) a recombinant gp41-containing gp140 protein yielded five additional Fabs. Competition of members of the Fab panel with one another and with previously described antibodies revealed a series of overlapping specificities that were conveniently grouped into three major epitope clusters. The majority of Fabs recognized epitopes involving residues 649-668 (previously known as the cluster II region), numbered using the Los Alamos LAI sequence. A second set of Fabs reacted with an epitope involving residues 584-609 (known as the cluster I region). Another set of Fabs appeared to recognize a third conformational epitope that has been termed the cluster III region. This third Fab epitope group demonstrated some overlap with both clusters I and II in binding assays. None of the Fabs neutralized HIV-1 laboratory strains at biologically significant concentrations. This tends to support the opinion that a vaccine based on the gp41 molecule has the drawback that neutralizing epitopes of gp41 are rare and/or unfavorably presented to the immune system. Analysis of heavy chain sequences revealed common CDR3 motif sequences in several antibodies, which appears to be an interesting consequence of a persistent immune response to conserved antigen structures.

An overview of the GAGE cancer/testis antigen family with the inclusion of newly identified members.

GAGE cancer/testis antigens are frequently expressed in many different types of cancer, whereas their expression in normal tissues is limited to the germ cells of the immune-privileged organs, testis and ovary. Thus, GAGE proteins may be attractive candidates for immunotherapy of cancer. This review describes the structure and phylogeny of the GAGE family members and presents a revised nomenclature, which will enable a more clear distinction of genes and gene products. The GAGE gene locus at chromosome X p11.23 consists of at least 16 genes, each of which is located in one of an equal number of highly conserved tandem repeats, and more genes remain to be identified. These genes are likely the creation of unequal replication under positive selection after the divergence of primates from other mammals. The encoded products are predicted to be highly similar small acidic proteins involved in germ cell biology. When expressed in tumor cells, GAGE proteins can elicit both cellular and humoral immune responses, indicating that they are appropriate targets for cancer immunotherapy. The potential use of GAGE proteins in cancer immunotherapy, including possible limitations, is also discussed.

Restriction of GAGE protein expression to subpopulations of cancer cells is independent of genotype and may limit the use of GAGE proteins as targets for cancer immunotherapy.

The GAGE cancer testis antigen gene family encodes products that can be recognized by autologous T cells, and GAGE proteins have been suggested as potential targets for cancer immunotherapy. Analysis of GAGE expression in tumours has primarily been performed at the level of gene transcription, whereas little is known about GAGE expression at the protein level. To evaluate the potential of GAGE proteins as targets for cancer-specific immunotherapy, we studied the expression of these proteins in normal and malignant cells/tissues using a novel panel of monoclonal antibodies. Immunohistochemical analysis of more than 250 cancer specimens demonstrated that GAGE proteins were frequently expressed in numerous cancer types and correlated with the expression of the cancer testis antigens MAGE-A1 and NY-ESO-1. Significant intercellular and subcellular differences in GAGE protein levels were observed, and most GAGE-positive tumours also contained cancer cells lacking GAGE expression. Studies of genetically homogenous cell lines with similar intercellular heterogeneous GAGE expression showed that GAGE expression was not associated with a specific genotype, but defined a phenotypically distinct population of cells. Surprisingly, in normal tissues we found that GAGE proteins were not restricted to testis, but were also present in a subset of oocytes of resting primordial follicles and in maturing oocytes. This is the first time that a cancer testis antigen has been reported in postfoetal oocytes. The lack of GAGE expression in a subset of cancer cells within GAGE-positive tumours has decisive implications for the development of GAGE-targeted cancer therapy.

Raised levels of anti-glucose-6-phosphate isomerase IgG in serum and synovial fluid from patients with inflammatory arthritis.

BACKGROUND: In K/BxN mice, anti-glucose-6-phosphate isomerase (GPI) antibodies (Abs) are arthritogenic, and their transfer into naive mice induces arthritis. Anti-GPI Abs develop in many human patients with RA and are associated with more severe forms of the disease. OBJECTIVE: To elucidate the serum and synovial fluid (SF) anti-GPI IgG profiles among different patient groups with a variety of arthritides. METHODS: Blood and SF obtained concomitantly from 91 patients with clinically well defined arthritis were tested for concentrations of total anti-GPI IgG, anti-GPI IgG subclasses, B lymphocyte stimulator (BLyS), and APRIL by ELISA. RESULTS: Anti-GPI IgG was detected in sera and SF of patients with many arthritic diseases, but was preferentially associated with inflammatory arthritis, in general, and RA, in particular. The anti-GPI IgG subclass usage was skewed and varied among the different arthritic disease groups. Inverse correlations between serum levels of BLyS and anti-GPI IgG and positive correlations between serum levels of APRIL and anti-GPI IgG were seen among immune based arthritic patients and patients with RA but not among non-immune based patients. No correlations were found in SF from any group of arthritic patients. CONCLUSION: Raised circulating anti-GPI Abs are not unique to patients with RA but are present in many patients with inflammatory arthritis. The difference in anti-GPI IgG subclass usage among disease groups may influence effector function and disease outcome. The inverse correlation between serum BLyS and anti-GPI IgG levels suggests that anti-GPI B cells may be regulated differently from other autoantibody producing B cells. Anti-GPI Abs may serve a pathogenic function in humans by promoting the maintenance of existing disease.

The tumor-infiltrating B cell response in medullary breast cancer is oligoclonal and directed against the autoantigen actin exposed on the surface of apoptotic cancer cells.

Medullary carcinoma of the breast (MCB) is a morphologically and biologically distinct subtype of human breast cancer that, despite cytologically anaplastic features, has a more favorable prognosis than other types of breast cancer at similar stages of differentiation. It has been proposed that the improved clinical outcome is due, at least in part, to the presence of a prominent lymphoplasmacytic cell infiltrate in the tumor stroma. We studied the B lymphoplasmacytic cell infiltrates in MCB to determine the role of the antibody response produced by the local infiltrating cells. Oligoclonal predominance among tumor-infiltrating B cells in a panel of MCB patients was observed, suggesting that certain B cell clones were expanded, possibly in response to specific tumor-associated stimuli. IgG antibody phage-display libraries were generated from MCB-infiltrating lymphoplasmacytic cells of two patients, and MCB-reactive monoclonal antibodies were retrieved by selection on fresh-frozen MCB tissue sections. Analysis by mass spectrometry revealed that the antigen targeted by the dominant clones in the oligoclonal B lymphoplasmacytic response in both patients was not a cancer-specific antigen but the cytoskeletal protein beta-actin. MCB exhibits an increased rate of apoptosis, and apoptotic MCB cells were shown to expose actin on the cell surface, permitting its recognition by the humoral immune system. Further, actin fragments, similar to those observed after cleavage with the apoptotic protease granzyme B, were observed in MCB tissue. Our results indicate that the major antibody response produced by tumor-infiltrating B lymphoplasmacytic cells are autoimmune in nature and a consequence of the perturbed state of increased MCB apoptosis caused by granzyme B-induced T cell cytotoxicity and/or intrinsic cellular factors of MCB cells.

Determinants of polyreactivity in a large panel of recombinant human antibodies from HIV-1 infection.

A considerable part of the Ab repertoire is given over to polyreactive Abs capable of interacting with multiple antigenic species. Neither the function of these Abs nor the molecular basis for their activity is known. To address the latter problem, we have compared the amino acid sequences of a large panel (n = 70) of polyreactive human monoclonal Fab fragments and conducted a series of engineering experiments on a prototype polyreactive Fab. The Fab fragments were retrieved from combinatorial IgG libraries prepared from the bone marrow of long term asymptomatic HIV-1 seropositive donors. The general features displayed by the panel of IgG polyreactive Abs include 1) skewed VH family usage with a predominance of VH1 and VH4 clones and a paucity of the normally prevalent VH3 family; 2) use of a variety of different VH germ-line genes within the context of the family usage and no restriction in D or JH gene usage; 3) skewed VL gene usage: 75% of Fabs used one of two germ lines; and 4) extensive somatic modification of both heavy and light chains. The importance of the heavy chain, in particular the heavy chain CDR3 (HCDR3), in dictating the polyreactive phenotype was demonstrated for the prototype Fab by chain shuffling and CDR transplantation experiments. in addition, and most strikingly, a constrained peptide based on the HCDR3 sequence was shown to be polyreactive and to inhibit binding of the parent Ab to a panel of Ags. A role for conformational flexibility in polyreactivity was suggested by a marked temperature dependence of Ab recognition of Ag. One Ab was shown to be polyreactive at 37 degrees C, but was apparently monoreactive at 4 degrees C. We hypothesize that Ab polyreactivity is associated with conformationally flexible HCDR3 regions in the context of certain favorable framework configurations.

Autoantibodies to GPI in rheumatoid arthritis: linkage between an animal model and human disease.

In K/BxN T cell receptor-transgenic mice, spontaneous inflammatory arthritis exhibiting many of the features of human rheumatoid arthritis (RA) is initiated by T cells, but is almost entirely sustained by antibodies to the self-antigen glucose-6-phosphate isomerase (GPI). The relevance of these observations to human disease has been questioned. Here we show that 64% of humans with RA, but not controls, had increased concentrations of anti-GPI immunoglobulin G (IgG) in serum and synovial fluid. In addition, the concentrations of soluble GPI in the sera and synovial fluids of RA patients were also elevated, which led to immune complex formation. Using phage-display methods, we cloned a panel of specific high-affinity human monoclonal anti-GPI IgGs from a patient with RA. These antibodies were highly somatically mutated, which was indicative of an affinity-matured response that was antigen driven. Immunohistochemistry of RA synovium showed high concentrations of GPI on the surface of the synovial lining and on the endothelial cell surface of arterioles; this indicated a mechanism by which antibodies to GPI may precipitate joint disease. The results indicate that the immunological events that lead to the development of autoimmune disease in the K/BxN mouse model may also occur in human RA. This data may be used to develop new strategies for therapeutic intervention.

The first dose of a Haemophilus influenzae type b conjugate vaccine reactivates memory B cells: evidence for extensive clonal selection, intraclonal affinity maturation, and multiple isotype switches to IgA2.

The Ab response of a healthy adult to the first dose of a Haemophilus influenzae type b capsular polysaccharide (HibCP) conjugate vaccine was studied at the level of Ig gene usage by circulating Ab-secreting cells. Forty-one IgA and 17 IgG mRNA sequences were obtained. The major part of the response was confined to IgA Ab-secreting cells, and 72% of the IgA sequences were derived from the progeny of a single rearranged B cell. These sequences could be arranged in a genealogical tree showing multiple somatic mutations and at least two intraclonal isotype switches to IgA2. Fourteen somatic mutations were shared by this clonal progeny, indicating that extreme clonal selection had occurred early in the clonal development. Taking into account the frequency of somatic mutations and the clone size, it was evident that the responding cell population must have originated from a mutated, highly selected, and expanded population of cells existing before vaccination, i.e., memory B cells. The dominating heavy and light chains of the response were combined in a Fab that bound HibCP. It was shown that the shared heavy and light chain mutations increased the affinity for HibCP considerably, indicating that the clonal selection had been driven by affinity. Pre-existing memory cells in unvaccinated adults may explain several features of Ab responses to polysaccharide vaccines and may play a role in acquiring the ability to respond to pure polysaccharides during infancy.