Klaus-Dieter Preuss

Analysis of the B-cell repertoire against antigens expressed by human neoplasms.

Summary: The screening of tumor‐derived expression libraries for antigens detected by high‐titer immunoglobulin G antibodies from the sera of patients with cancer by SEREX (serological identification of antigens by recombinant expression cloning) allows a systematic search for antigens of human cancers. SEREX has led to the identification of a multitude of new tumor antigens in many different tumor entities. According to their specificities, the antigens can be grouped into different classes, of which the cancer testis antigens appear to be the most attractive candidates for vaccine development. Serologically defined human tumor antigens facilitate the identification of antigenic peptides recognized by tumor‐specific T lymphocytes, thus providing a molecular basis for polyvalent peptide‐based and gene‐therapeutic vaccine strategies in a wide variety of human neoplasms. Moreover, many of the SEREX‐identified antigens seem to play a functional role in the pathogenesis of malignant disease. With more than 2000 antigens listed in the SEREX database, it appears that tumor antigens that have resisted discovery to date are expressed in only a small minority of tumors, thus limiting their clinical usefulness. Novel strategies are necessary to identify antigens that can serve as a vaccine target in a broad spectrum of non‐Hodgkins lymphoma patients.

Prospective study on the expression of cancer testis genes and antibody responses in 100 consecutive patients with primary breast cancer.

To determine the expression of cancer testis (CT) genes and antibody responses in a nonselected population of patients with primary breast cancer, we investigated the composite expression of 11 CT genes by RT‐PCR in fresh biopsies of 100 consecutive cases of primary breast carcinoma and by immunohistology in selected RT‐PCR‐positive cases. Antibody responses against 7 CT antigens were analyzed using recombinant antigen expression on yeast surface. In 98 evaluable cases, SCP‐1 and SSX‐4 were expressed most frequently (both 65%), followed by HOM‐TES‐85/CT‐8 (47%), GAGE (26%), SSX‐1 (20%), NY‐ESO‐1 (13%), MAGE‐3 (11%), SSX‐2 (8%), CT‐10 (7%), MAGE‐4 (4%) and CT‐7 (1%). One CT gene was expressed by 90% of the cases; 79% expressed ≥2, 48% ≥3, 29% ≥4, 12% ≥5, 6% ≥6, 3% ≥7, 2% ≥8 and one case coexpressed 9 antigens. Of 100 serum samples screened for CT antigen‐specific antibodies, antibodies against NY‐ESO‐1 were detected in 4 patients, against SCP‐1 in 6 patients and against SSX‐2 in 1 patient, while no antibodies were detected against MAGE‐3, CT‐7 and CT‐10. Expression of CT genes or antibody responses was not correlated with clinical parameters (menopausal status, tumor size, nodal involvement, grading, histology and estrogen receptor status) or the demonstration of CT gene expression at the protein level, by immunohistology. Our results show that breast carcinomas are among the tumors with the most frequent expression of CT antigens, rendering many patients potential candidates for vaccine trials.

Analysis of the antibody repertoire of astrocytoma patients against antigens expressed by gliomas

The molecular characterization of antigens preferentially or exclusively expressed by astrocytomas and recognized by the autologous immune system are a prerequisite for the development of specific vaccines. To identify such antigens, we screened 5 cDNA expression libraries derived from astrocytomas and other gliomas for reactivity with high‐titered IgG antibodies in the sera of astrocytoma patients using SEREX, the serologic identification of antigens by recombinant cDNA expression cloning. Autologous and allogeneic SEREX analysis of >5 × 106 clones with the sera of 18 astrocytoma patients revealed 10 antigens: the differentiation antigen glial fibrillary acidic protein (GFAP), Bax‐inhibitor 1 (which was overexpressed in all glioma samples tested), 3 other molecules involved in the regulation of gene expression and proliferation (the nm23‐H2‐encoded nucleoside diphosphate kinase B, the Ran binding protein‐2 and a DNA binding protein encoded by the son gene), SP40,40 (a complement inhibitory molecule), the chaperonin TCP‐1, calnexin and 2 new gene products. No immune responses were detected against the “shared tumor” or “cancer testis antigens” that are regularly expressed in gliomas. Antibody responses in astrocytoma patients against antigens expressed by gliomas were rare and, with the exception of Bax‐inhibitor 1 and the product of the son gene, were also found in apparently healthy controls. We conclude that although astrocytomas express a broad spectrum of antigens, they elicit antibody responses only rarely, most likely because of their intrinsic immunosuppressive effects.

EXPRESSION OF CANCER TESTIS ANTIGENS IN PANCREATIC CARCINOMA CELL LINES, PANCREATIC ADENOCARCINOMA AND CHRONIC PANCREATITIS

In order to define antigens that might be suitable as vaccines for pancreatic carcinoma, we investigated the composite expression of 10 cancer testis (CT) antigens (SCP‐1, NY‐ESO‐1, SSX‐1, SSX‐2, SSX‐4, GAGE, MAGE‐3, MAGE‐4, CT‐7 and CT‐8) by Reverse Transcriptase‐PCR (RT‐PCR) in fresh biopsies of human pancreatic adenocarcinoma, chronic pancreatitis and pancreatic carcinoma cell lines. While all CT genes were frequently expressed in cell lines derived from pancreatic cancer, no expression of MAGE‐3, SSX‐1, SSX‐2, NY‐ESO‐1 and CT‐7 was detected in fresh tumor biopsies, and MAGE‐4 (1/52), SSX‐4 (1/39) and CT‐8 (2/41) were only rarely expressed. In contrast, HOM‐TES‐14/SCP‐1 was expressed in 48% (29/61) and GAGE in 21% (13/61) of cases, respectively. One CT gene was expressed by 59% (75% in male, 46% in female patients; p = 0.05) and 2 or more CT genes by 15% of the samples. SCP‐1 protein expression correlated well with mRNA expression. While SCP‐1 and GAGE were absent in normal pancreas, they were found in 2/8 (SCP‐1) and 1/8 (GAGE) samples of chronic pancreatitis, respectively, supporting the concept of chronic pancreatitis as a premalignant condition. SCP‐1 and GAGE represent promising candidates for vaccine development in pancreatic carcinoma. Whether SCP‐1 and GAGE expression identify cases of chronic pancreatitis with a high risk of malignant transformation remains to be shown.

The impact of Fc-γ receptor polymorphisms in elderly patients with diffuse large B-cell lymphoma treated with CHOP with or without rituximab.

Fcγ receptor (FcγR) polymorphisms have been shown to affect rituximab-mediated antibody-dependent cellular cytotoxicity. Of 512 patients with diffuse large B-cell lymphoma treated in the RICOVER-60 trial, carriers of FcγRIII 158 valine homozygous receptors (V/V) presented with a slightly decreased incidence of B-symptoms (158 V/V: 26%, V/F: 35%, phenylalanine receptors [F/F]: 42%; P = .037). Survival curves of all FcγR single nucleotide polymorphisms were superimposable after cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP); but after CHOP with rituximab (R-CHOP), event-free survival (EFS) and progression-free survival (PFS), but not overall survival, of FcγRIIIa 158 F/F had a trend to be lower than those of 158 V/F and 158 V/V: 3-year EFS: FcγRIIIa 158 F/F: 64.5%, 158 V/F: 70.2%, 158 V/V: 76.9% (log-rank test: P = .224 F/F vs V/V; P = .285 F/F vs V/F + V/V); 3-year PFS: FcγRIIIa 158 F/F: 68.3%, V/F: 76.1%, V/V: 80.5% (log-rank test: P = .233 for F/F vs V/V; P = .185 for F/F vs V/F + V/V). By multivariate analysis adjusting for International Prognostic Index factors, relative risk of F/F compared with V/F plus V/V was 1.80 (P = .052) for PFS and 1.55 (P = .120) for EFS. The interaction of R-CHOP, but not CHOP with FcγRIIIa polymorphisms, indicates a window of opportunity for CD20 antibodies designed to mediate enhanced antibody-dependent cellular cytotoxicity.

Association of a dominantly inherited hyperphosphorylated paraprotein target with sporadic and familial multiple myeloma and monoclonal gammopathy of undetermined significance: a case–control study

BACKGROUND Chronic antigenic stimulation might have a role in the pathogenesis of monoclonal gammopathy of unknown significance (MGUS) and multiple myeloma. The aim of this study was to search for factors underlying the autoimmunogenicity of paratarg-7, a frequent antigenic target of paraproteins in MGUS and multiple myeloma. METHODS Between January, 2005, and February, 2009, serum and peripheral blood cells were obtained from consecutive patients with MGUS or multiple myeloma and healthy blood donors, and paratarg-7 was analysed by DNA sequencing, SDS-PAGE, isoelectric focusing, and western blotting. FINDINGS Mutations or polymorphisms of paratarg-7 were not noted, but hyperphosphorylation was detected in 35 (13.9%) of 252 patients with MGUS or multiple myeloma, all of whom had an anti-paratarg-7-specific paraprotein. Analysis of eight families showed that hyperphosphorylated paratarg-7 is inherited in a dominant fashion, and that carriers of hyperphosphorylated paratarg-7 have an increased risk of developing MGUS and multiple myeloma (odds ratio [OR] 7.9, 95% CI 2.8-22.6; p=0.0001). INTERPRETATION Familial MGUS and multiple myeloma were associated with a dominant inheritance of hyperphosphorylated paratarg-7, enabling family members at increased risk for MGUS or multiple myeloma to be identified. That only patients with MGUS or multiple myeloma who are carriers of hyperphosphorylated paratarg-7 had a paratarg-7-specific paraprotein suggests that the hyperphosphorylation of paratarg-7 induces auto-immunity and is involved in the pathogenesis of MGUS and multiple myeloma; for example, by chronic antigenic stimulation. FUNDING Förderverein Krebsforschung Saar-Pfalz-Mosel e.V. (eingetragener Verein: officially registered charity) and HOMFOR (the research programme of the Saarland University Faculty of Medicine).

Progranulin antibodies in autoimmune diseases

Systemic vasculitides constitute a heterogeneous group of diseases. Autoimmunity mediated by B lymphocytes and their humoral effector mechanisms play a major role in ANCA-associated vasculitis (AAV) as well as in non-ANCA associated primary systemic vasculitides and in the different types of autoimmune connective tissue disorders and rheumatoid arthritis. In order to detect autoantibodies in systemic vasculitides, we screened protein macroarrays of human cDNA expression libraries with sera from patients with ANCA-associated and ANCA-negative primary systemic vasculitides. This approach led to the identification of antibodies against progranulin, a 88 kDA secreted glycoprotein with strong anti-inflammatory activity in the course of disease of giant-cell arteritis/polymyalgia rheumatica (14/65), Takayasus arteritis (4/13), classical panarteritis nodosa (4/10), Behcets disease (2/6) and in the course of disease in granulomatosis with polyangiitis (31/75), Churg-Strauss syndrome (7/23) and in microscopic polyangiitis (7/19). In extended screenings the progranulin antibodies were also detected in other autoimmune diseases such as systemic lupus erythematosus (39/91) and rheumatoid arthritis (16/44). Progranulin antibodies were detected only in 1 of 97 healthy controls. Anti-progranulin positive patients with systemic vasculitides, systemic lupus erythematosus or rheumatoid arthritis had significant lower progranulin plasma levels, indicating a neutralizing effect. In light of the anti-inflammatory effects of progranulin, progranulin antibodies might exert pro-inflammatory effects thus contributing to the pathogenesis of the respective autoimmune diseases and might serve as a marker for disease activity. This hypothesis is supported by the fact that a positive progranulin antibody status was associated with active disease in granulomatosis with polyangiitis.

Analysis of the antibody repertoire of lymphoma patients.

Abstract. Cancer testis or cancer germline antigens (CGA) are promising vaccine candidates because they are expressed only in malignant but not in normal tissues, except for germ cells in the testis. Since non-Hodgkins lymphomas (NHL) express the known CGA at low frequencies, we aimed at increasing the number of CGA with frequent expression in NHL by screening a cDNA expression library derived from normal testis for reactivity with high-titered IgG antibodies in the sera of lymphoma patients using SEREX, the serological identification of antigens by recombinant cDNA expression cloning. The analysis of 1.6×106 clones with the sera of 25 lymphoma patients revealed 42 clones which coded for 23 antigens, 12 of which had already been included in the SEREX databank. Four cDNA clones coded for unknown and 19 for known genes. Three antigens reacted only with the serum by which they had been detected, 9 antigens reacted with the sera of several NHL patients, but not with that of healthy controls, and 11 antigens reacted with both normal and NHL sera. Most of the antigens were ubiquitously expressed. Only HOM-NHL-6, HOM-NHL-8, HOM-NHL-21 and HOM-NHL-23 showed a restricted expression pattern. HOM-NHL-6 and HOM-NHL-8 were homologous to the previously described CGA NY-ESO-1 and HOM-TES-14/SCP-1, respectively. HOM-NHL-21 was expressed in rare cases of lymphomas, but not in normal tissues except for testis and brain, while HOM-NHL-23 appeared to be a testis-specific antigen. In summary, using the antibody repertoire of these 25 NHL patients, no new CGA were detected. The number of CGA detectable by the classical SEREX approach appears to be limited, and novel strategies are necessary to identify antigens that can serve as a vaccine target in a broad spectrum of NHL patients.

A frequent target of paraproteins in the sera of patients with multiple myeloma and MGUS

Antigenic targets of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) paraproteins might play a role in the pathogenesis of these neoplasms. We screened a human fetal brain‐derived macroarray with the IgA or IgG containing sera of 192 consecutive MGUS and MM patients. Twenty‐nine of 192 (15.1%) paraproteins reacted with paratarg‐7, a protein of unknown function which is expressed in all human tissues. Paratarg‐7 reactivity was similarly frequent among IgA and IgG paraproteins, but all paratarg‐7 reactive IgG paraproteins belonged to the IgG3 subtype with 24/57 IgG3 (42.1%) paraproteins displaying this specificity. Sequence analysis of paratarg‐7 derived from patients having a paraprotein with specificity for paratarg‐7 revealed no differences to paratarg‐7 derived from patients with paraproteins of other specificities or healthy controls, excluding mutations or polymorphisms as a reason for its autoimmunogenicity. Similarly, Western‐blot analysis showed identical bands for paratarg‐7 derived from patients and controls. The above‐random frequency of paratarg‐7 as a paraprotein target suggests that paratarg‐7 might be involved in the development of the respective clonal proliferations. The identification of paratarg‐7 as an antigenic target enables the more detailed analysis of tumor–host interactions in these patients and their role in the pathogenesis of MM and MGUS.

Analysis of the B cell repertoire against autoantigens in patients with giant cell arteritis and polymyalgia rheumatica

The analysis of the antibody repertoire of patients with giant cell arteritis (GCA) and polymyalgia rheumatica (PMR) might identify target antigens of the autoimmune response with potential relevance to our understanding of the pathogenesis of the disease and to the development of serodiagnostic tests. To detect such antigens, we screened a cDNA library derived from normal human testis for antigens reacting with IgG antibodies in the 1 : 250 diluted sera of three patients with untreated GCA using SEREX, the serological identification of antigens by recombinant cDNA expression cloning. Of 100 000 clones screened with each serum, six, 28 and six clones, respectively, were positive, representing a total of 33 different antigens. Most of the antigens reacted only with the serum used for identification and/or at a similar frequency with normal control sera. However, lamin C and the nuclear antigen of 14 kD reacted specifically with 32% of GCA/PMR, but with none of the control sera, while human cytokeratin 15, mitochondrial cytochrome oxidase subunit II, and a new gene product were detected preferentially, but not exclusively by sera from GCA/PMR patients. We conclude that patients with GCA/PMR develop antibodies against a broad spectrum of human autoantigens. Antibodies against human lamin C, the nuclear autoantigen of 14 kD as well as human cytokeratin 15, mitochondrial cytochrome oxidase subunit II and the product of a new gene should be investigated further to determine their value as tools for the diagnosis and/or the definition of clinical subgroups of patients with GCA/PMR.