Target validation for genomics using peptide-specific phage antibodies: a study of five gene products overexpressed in colorectal cancer

Genomic approaches are providing a wealth of information on differential gene expression in cancer. To identify the most interesting genes amongst the many identified, high-throughput methods for analysis of genes at the translational level are required. We have used a rapid method for the in vitro selection of antibodies to peptide antigens for the generation of probes to 5 gene products that we have found to be overexpressed in colorectal cancer. The rationale of our study was to select a non-immune phage displayed human antibody library on peptides designed from the coding regions of the gene sequences and to verify whether such antibodies would be suitable probes for the parental protein in immunohistochemical and Western blot analysis. After the generation of a profile of genes overexpressed in primary colorectal cancer (CRC) we selected 5 genes, Ese-3b, Fls353, PBEF, SPARC and Smad5 for a more detailed analysis using phage display-derived antibodies. For these 5 antigens we designed 14-20 amino acid peptides predicted to be exposed on the surface of the parental protein. Selection of a large phage displayed antibody library resulted in specific antibodies for 6 of 8 different peptides with between 2 and 15 different antibodies isolated per peptide. Of 20 antibodies tested, 2 antibodies recognized the putative parental protein from primary CRC tissue. An antibody specific for a PBEF-derived peptide (Fab/PBEF-D4) was shown to recognize a protein product of the expected molecular weight in Western blotting and showed overexpression in n = 6/8 matched tumor/normal protein lysates. Furthermore, in immunohistochemistry this antibody showed restricted staining of the tumor stromal compartment with no detectable staining of epithelial cells. The discovery that PBEF is overexpressed in cancer is unexpected given that the normal function of PBEF is as a cytokine required for the maturation of B cell precursors. We also report on the isolation of an antibody (Fab/SMAD-50) specific for a Smad5-derived peptide that showed cytoplasmic staining of epithelial cells in both CRC tumor and matched normal mucosa. Fab/SMAD-50 also bound to a group of proteins in Western blotting with molecular weights consistent with belonging to the Smad family. These antibodies may be suitable probes for further investigation of the roles of PBEF and Smad5 in cancer. The amenability of phage display to automation suggests that this approach may be developed for implementation on a genomics scale. Indeed, the large-scale generation of antibody probes that can be used to study protein expression in situ would be of great value in target validation for functional genomics.     

Selective antibody response to Streptococcus gallolyticus pilus proteins in colorectal cancer patients

Streptococcus gallolyticus subsp. gallolyticus (previously called Streptococcus bovis biotype I) infections have long been associated with colorectal cancer (CRC). This work aimed to investigate the CRC-associated humoral immune response to four pilus proteins of this bacterium by newly developed ELISAs. Pilus proteins are interesting diagnostic targets as they are the building blocks of pilin-like structures that mediate bacterial virulence and are readily exposed to the host immune system upon infection. The presence of serum antibodies against these pilus proteins was evaluated in Dutch and American populations. These analyses showed that an immune response to these antigens was specific for clinical S. gallolyticus subsp. gallolyticus infections, but that increased serum antibody titers to multiple pilus proteins in single individuals were rarely observed. However, a multiplex approach based on antibody titers against any of these four antigens resulted in assay sensitivities between 16% and 43% for the detection of early-stage CRC. Together these findings underscore the potential of a multi-antigen approach to complement diagnosis of S. gallolyticus subsp. gallolyticus-associated CRC.     

Novel technologies for cancer biomarker discovery: humoral proteomics

The repertoires of serum autoantibodies differ between healthy people and cancer patients. While in healthy individuals these autoantibodies are directed against a limited number of self-proteins, in cancer patients the antibody repertoires are much further expanded with a wider range of reactivities against other proteins. Although cancer patients clearly mount humoral immune responses, they are not very effective in preventing the progression of the disease. However, the implication from the presence of these new and abnormal antibody specificities relates to their potential as novel tools for early detection before clinical manifestations. Proteomics technologies, with their unique ability to identify both tumor antigens and their cognate serum autoantibodies, hold great promise in facilitating the development of early detection kits and possibly also as conduits for the isolation of tumor antigens for immunotherapy.     

An HSP90-mimic peptide revealed by fingerprinting the pool of antibodies from ovarian cancer patients

To gain insight into the mechanisms of molecular recognition and humoral immune response in ovarian cancer, we used fingerprinting, a phage display-based combinatorial selection to isolate peptide ligands to tumor-related antibodies present in ascites from patients with advanced disease. First, we have isolated a consensus motif (sequence CVPELGHEC) in 86% of the peptides screened; this enriched motif was selected from a total of 10(8)-10(9) unique random sequences present in the library. Next, we identified the heat-shock protein 90 kDa (HSP90) as the native antigen mimicked by the motif. Finally, we evaluated the expression of HSP90 and the presence of antibodies against the HSP90-mimic peptide in a large panel of ovarian cancer patients and controls. In tissue microarrays, we show that the expression of HSP90 is ubiquitous. However, the corresponding humoral immune response against HSP90 is restricted to a subset of patients with stage IV disease. Together, these results show that screening humoral response can identify tumor antigens that may serve as molecular targets in ovarian cancer. Recognition of such relevant proteins in the immunobiology of malignant tumors may lead to the development of therapies     

Recombinant NY-ESO-1 protein with ISCOMATRIX adjuvant induces broad antibody responses in humans, a RAYS-based analysis

Antibody responses to tumor antigens play an important role in initiating a cellular antitumor response with respect to antigen cross-presentation and T cell cross-priming. Successful vaccination strategies rely on an optimally timed activation of the humoral and cellular immune system by using appropriate adjuvant stimulation. The LUD99-008 trial used the cancer testis antigen NY-ESO-1 formulated with ISCOMATRIX adjuvant injected into patients intramuscularly. It was shown that this vaccination strategy is a safe and highly potent activator of the humoral and cellular immune system. Using the RAYS technology, we analyzed in detail the humoral immune response in these patients before and after vaccination: during the course of repeated vaccinations with the adjuvant, antibody titers against NY-ESO-1 and cross-reactivity to LAGE 1A and B increased as an indicator of an enhanced immune response, whereas no antibody response could be detected after vaccination without the adjuvant. Analysis of single fragments of the NY-ESO-1 protein revealed that the humoral response was almost exclusively directed against the N-terminal fragments and the number of fragments and their length being recognized by the NY-ESO-1-specific antibodies increased during the course of vaccination. The humoral immune response mainly consisted of antibodies of the IgG1 and IgG3 subclass. We rarely found IgG2 and IgG4 subclass antibodies. Our results support the implication that target-specific antibodies raised after vaccination contribute to the stimulation of an effective T cell response against the target antigen.     

Mitofilin and titin as target antigens in melanoma-associated retinopathy

Melanoma-associated retinopathy (MAR) is a rare paraneoplastic syndrome in patients with melanoma. Since the onset of MAR symptoms is often associated with tumor progression or recrudescence of metastases, MAR-related symptoms are prognostic relevant. The pathomechanism underlying MAR is supposed to result from antibody production against yet unknown melanoma-associated antigens that are also expressed in retinal tissue, leading to the destruction of retinal cells and resulting in defective signal transduction. Only a 35 kDa protein in Müller glial cells, a 22 kDa neuronal antigen and retinal transducin have been identified as MAR-associated antigens to date. To identify additional antigens potentially involved in the pathogenesis of MAR, we screened a retina cDNA phage library for reactivity with antibodies in the sera from 9 patients with MAR or subclinical MAR using the serological analysis of recombinantly expressed clones (SEREX) approach. Six sera from melanoma patients without evidence of MAR and 10 sera from healthy donors served as controls. Mitofilin and titin were identified as antigens against which antibodies were found exclusively in sera of MAR patients, but not in the sera of MM patients without MAR or healthy donors. This is the first study to demonstrate that titin is highly expressed from retinal tissue and melanoma. The fact that none of the MAR-associated antigens detected to date by their capacity to elicit a humoral immune response is located on the cell surface questions a major pathogenetic role of the respective antibodies and suggests that cellular, rather than humoral mechanisms are operative in the primary immune attack against the retina in MAR.     

Serum antibodies against frameshift peptides in microsatellite unstable colorectal cancer patients with Lynch syndrome

High level microsatellite instability (MSI-H) occurs in about 15% of colorectal cancer (CRCs), either as sporadic cancers or in the context of hereditary non-polyposis cancer or Lynch syndrome. In MSI-H CRC, mismatch repair deficiency leads to insertion/deletion mutations at coding microsatellites and thus to the translation of frameshift peptides (FSPs). FSPs are potent inductors of T cell responses in vitro and in vivo. The present study aims at the identification of FSP-specific humoral immune responses in MSI-H CRC and Lynch syndrome. Sera from patients with history of MSI-H CRC (n = 69), healthy Lynch syndrome mutation carriers (n = 31) and healthy controls (n = 52) were analyzed for antibodies against FSPs using peptide ELISA. Reactivities were measured against FSPs derived from genes frequently mutated in MSI-H CRCs, AIM2, TGFBR2, CASP5, TAF1B, ZNF294, and MARCKS. Antibody reactivity against FSPs was significantly higher in MSI-H CRC patients than in healthy controls (P = 0.036, Mann–Whitney) and highest in patients with shortest interval between tumor resection and serum sampling. Humoral immune responses in patients were most frequently directed against FSPs derived from mutated TAF1B (11.6%, 8/69) and TGFBR2 (10.1%, 7/69). Low level FSP-specific antibodies were also detected in healthy mutation carriers. Our results show that antibody responses against FSPs are detectable in MSI-H CRC patients and healthy Lynch syndrome mutation carriers. Based on the high number of defined FSP antigens, measuring FSP-specific humoral immune responses is a highly promising tool for future diagnostic application in MSI-H cancer patients.     

Stratégies d’immunothérapie dans les cancers colorectaux

Recent studies have underlined the close link between immune response and prognosis of patients with colorectal cancer (CRC). Immune response understanding combined with biotechnology progress of the last years has allowed development of immunotherapy strategies in CRC. Immunotherapy strategies are divided in “active” or “passive” strategies (patients immune system stimulation or not) and considering the activation of antigen specific immune response or not. These immunotherapy strategies are well tolerated and induced cellular and humoral response correlated with clinical response. Many monoclonal antibodies targeting signalisation pathways or angiogenic growth factors have demonstrated their efficacy in CRC. Multiple vaccine strategies, using different tumour associated antigens, have demonstrated a biological efficacy but with poor clinical results. Results are more promising in adjuvant setting but need to be confirmed by randomized trials. Adoptive immunotherapy with transfer of tumour associated antigen specific T cell is probably the most promising strategy. Actually, except monoclonal antibodies, immunotherapy is not used in clinical practice in CRC due to the lack of results and absence of standardisation.     

A study of the humoral immune response of breast cancer patients to a panel of human tumor antigens identified by phage display

OBJECTIVE: In this article we provide evidence of a significant spontaneous humoral response in cancer patients. METHODS: A panel of tumor-associated antigens, previously identified through serological screening of phage-displayed cDNA libraries from solid human tumors, breast carcinoma cell lines and human testis by employing breast cancer patient sera, was used in this study to survey sera from 182 patients with known disease histories and clinical stages. RESULTS: This analysis reveals a statistically significant association between tumor disease and presence in peripheral blood of IgG antibodies against four autoantigens. One of these antigens (D7-1) is particularly interesting in that the antibody response against it grows with cancer progression from stages I through IV, with an incidence of 13.2, 13.5, 18.2 and 27%, respectively. The significance of this stage-dependent increase in the incidence is confirmed by the Mantel-Haenszel Chi-squared test (P=0.001). CONCLUSIONS: Our data confirm association between breast cancer diagnosis of patients and presence in their peripheral blood of antibodies against several autoantigens identified by phage display.     

Identification of immunogenic antigens using a phage-displayed cDNA library from an invasive ductal breast carcinoma tumour.

The identification of immunogenic antigens for serological testing and vaccine development is a major challenge facing cancer immunology research. To study the humoral immune response in patients with breast cancer, a T7 phage display cDNA library from an invasive ductal breast carcinoma was panned on patient serum IgG antibodies. By monitoring the selection with an immunoscreening technique, positive phage-displayed cDNA products reacting with breast cancer patient IgG antibodies were selected. Sequence analysis identified immunogenic antigens such as the cytochrome oxidase I, sp100 and Ran GTPase activating protein. Additionally, immunogenic uncharacterized gene products were also identified. Both the known and unknown immunoselected gene products should offer an additional source for cancer gene discovery for diagnostic testing and vaccine development.     

Antigens recognized by autologous antibody in patients with renal-cell carcinoma.

The screening of cDNA expression libraries derived from human tumors with autologous antibody (SEREX) is a powerful method for defining the structure of tumor antigens recognized by the humoral immune system. Sixty-five distinct antigens (NY-REN-1 to NY-REN-65) reactive with autologous IgG were identified by SEREX analysis of 4 renal cancer patients and were characterized in terms of cDNA sequence, mRNA expression pattern, and reactivity with allogeneic sera. REN-9, -10, -19, and -26 have a known association with human cancer. REN-9 (LUCA-15) and REN-10 (gene 21) map to the small cell lung cancer tumor suppressor gene locus on chromosome 3p21.3. REN-19 is equivalent to LKB1/STK11, a gene that is defective in Peutz-Jeghers syndrome and cancer. REN-26 is encoded by the bcr gene involved in the [t(9:22)] bcr/abl translocation. Genes encoding 3 of the antigens in the series showed differential mRNA expression; REN-3 displays a pattern of tissue-specific isoforms, and REN-21 and REN-43 are expressed at a high level in testis in comparison to 15 other normal tissues. The other 62 antigens were broadly expressed in normal tissues. With regard to immunogenicity, 20 of the 65 antigens reacted only with autologous sera. Thirty-three antigens reacted with sera from normal donors, indicating that their immunogenicity is not restricted to cancer. The remaining 12 antigens reacted with sera from 5-25% of the cancer patients but not with sera from normal donors. Seventy percent of the renal cancer patients had antibodies directed against one or more of these 12 antigens. Our results demonstrate the potential of the SEREX approach for the analysis of the humoral immune response against human cancer.     

Characterization of human colon cancer antigens recognized by autologous antibodies

The screening of cDNA expression libraries derived from human tumors with autologous antibody (SEREX) has proven to be a powerful method for defining the structure of tumor antigens recognized by the humoral immune system. In the present study, 48 distinct antigens (NY-CO-1–NY-CO-48) reactive with autologous IgG were identified by SEREX analysis in 4 patients with colon cancer. Sequencing analysis showed that 17 of the cDNA clones were previously uncharacterized molecules and 31 represented known gene products. The individual cDNA clones were analyzed in the following manner: a search for mutations or other structural changes; an analysis of mRNA expression in a panel of normal tissues; and a frequency analysis of the antibody response to the expressed product in the sera of colon cancer patients and normal individuals. The initial analysis showed NY-CO-13 to be a mutated version of the p53 tumor suppressor gene. Three of the 48 antigens showed a differential pattern of mRNA expression, with NY-CO-27 (galectin-4) expressed primarily in gastrointestinal tract, and NY-CO-37 and -38 showing a pattern of tissue-specific isoforms. With regard to immunogenicity, 20 of the 48 antigens were detected by allogeneic sera; 14 of these were reactive with sera from both normal donors and cancer patients, and 6 other clones (NY-CO-8, -9, -13, -16, -20 and -38) reacted exclusively with sera from colon cancer patients (ranging from 14% to 27%). Our results on colon cancer illustrate both the complexity and the potential of the SEREX approach for analysis of the humoral immune response against human cancer. Int. J. Cancer76:652–658, 1998.© 1998 Wiley-Liss, Inc.     

Modulation of the immune response by systemic targeting of antigens to lymph nodes.

Factors that determine the immunogenicity of an antigen in vivo are still largely unknown. Direct administration of antigens into lymphatic organs appears to enhance immune response. We hypothesized that systemically targeting antigens to lymphatic tissue in vivo might modulate immunity. To test this hypothesis, we measured the humoral immune response elicited by bacteriophage vaccination. We show that the responses against a lymph node-targeted phage are significantly higher than those against control untargeted phage; the effect is specific because it is inhibited by coadministration of the cognate synthetic peptides displayed. Our data suggest that systemic targeting of antigens to lymph nodes through the circulation modulates humoral immune response. This strategy may have broad applications in the development of vaccines, production of antibodies, and immunotherapy.     

Identification of colon tumour-associated antigens by phage antibody selections on primary colorectal carcinoma

Immunotargeting of solid tumours using antibodies has become a valuable tool for the detection of cancer metastases and the treatment of minimal residual disease. However, only few tumour antigens useful for targeting have been described to date. To identify cell-surface targets on colorectal carcinoma (CRC), we selected a large, human phage antibody repertoire on freshly isolated colon tumour cells. Two antibodies were identified that reacted with epithelial cell-restricted cell-surface antigens, whereas one clone preferentially reacted with stromal cells. These antigens are tumour-associated antigens, as shown by their uniform expression in tumours of different patients and of different differentiation stages and by their limited expression on normal tissues. The pattern of reactivity in immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA) suggests that these antigens are different from previously identified tumour-associated antigens (e.g. Ep-CAM or c-ERB-2). This phage antibody-based method may lead to the cloning of novel tumour antigens that are useful for the immunotargeting of solid tumours.     

Immunogenicity of recombinant GA733-2E antigen (CO17-1A, EGP, KS1-4, KSA, Ep-CAM) in gastro-intestinal carcinoma patients

Targeting the GA733 antigen (also known as CO17-1A, EGP, KS1-4, KSA, Ep-CAM) by monoclonal antibody CO17-1A or anti-idiotypic antibodies mimicking the CO17-1A or GA733 epitope has induced prolonged survival and specific immune responses to the antigen, respectively, in colorectal cancer (CRC) patients. In pre-clinical studies in mice and rabbits, recombinant baculovirus-derived GA733-2E antigen was superior to anti-idiotypic antibodies at modulating specific immune responses. Our aim was to evaluate the immunogenicity and potential toxicity of alum-precipitated GA733-2E in a phase I trial in patients with resected CRC or pancreatic cancer. Six patients with advanced pancreatic carcinoma and 6 with CRC Dukes' stage A, B or C received between 4 and 7 doses of alum-precipitated GA733-2E at 50, 200 or 800 microg/dose at monthly intervals. Antibody binding to GA733-2E or antigen-positive CRC cells was determined, as were antigen-specific proliferative, cytolytic T-lymphocyte and delayed-type hypersensitivity responses. Six of the 12 patients developed antigen-specific humoral immune responses after immunotherapy, and 8 developed cellular immune responses. The overall immune response rate, including patients with humoral and/or cellular immune responses, was 83%. Median overall survival of the CRC and pancreatic cancer patients was 39.8 and 11.2 months, respectively. Following 18 years of single-epitope targeting of the GA733 antigen, immunization of patients against multiple epitopes of the antigen frequently induces an immune response in the absence of significant toxicity, despite relatively widespread expression of this antigen on normal epithelial cells.     

Réponse immunitaire et cancers colorectaux

Immune response has a crucial role in the control of tumoral progression in colorectal cancer (CRC). A close link between the rate of tumor infiltrating lymphocytes and prognosis has been found. Indeed, recent studies have shattered our dogmas as the rate of tumor infiltrating lymphocytes seems to be a better prognostic factor than TNM classification. This review is focused on immune response in CRC. Specific cell-mediated immunity is important for the control of tumoral growth. Specific T cell activation is induced by tumoral antigens process by dendritic cells. Among the numerous tumoral antigens that could induce an immune response in CRC, the carcinoembryonic antigen is the most studied but its immunogenicity remains low. In CRC with microsatellite instability, several immunogenic neo-antigens have been identified and could explain the high level of tumor infiltrating lymphocytes and the better prognosis of this type of tumour. However, CRC can escape immune response by immune response modulation or tumoral cells modifications conducting to immune resistance. These data should be considered for the treatment of CRC or the development of immunotherapy strategies.     

Comparison of the techniques used for monitoring humoral immunity in cancer patients

The humoral immune response to tumor-associated antigens (TAA) in cancer patients undergoing immunotherapy can vary considerably. This variability is inherent from patient to patient and is also dependent on the type of assay used to measure patient antibody response to tumors. In this investigation four serological techniques, complement-dependent antibody-mediated cytotoxicity, immune adherence, complement fixation, and indirect membrane immunofluorescence, were used to assess the humoral immune response in melanoma patients receiving specific immunization with cultured allogeneic melanoma cell vaccine. Two different patterns of antibody response were elicited. One was directed against TAA and oncofetal antigens (OFA) and the other was against HLA antigens. The sensitivity and reactivity varied among the different techniques suggesting that one test or technique may not be sufficient to evaluate critically a patient's response to immunotherapy.     

Isolation and characterization of the human monoclonal antibodies C10 in single-chain fragment variable (scFv) format to glucose oxidase from Aspergillus niger

Despite biotechnological and clinical applications very few monoclonal antibodies (MAbs) directed to the enzyme glucose oxidase, have been produced so far because of the heavy side effects of the immunization schedule for conventional MAb preparation. In contrast, the phage display method allows for the selection of monoclonal human antibody fragments against any antigens, including toxic proteins. Furthermore, cDNA sequences encoding selected antibody fragments are readily identified, facilitating various molecular targeting approaches. In order to obtain such human fragments recognizing glucose oxidase, we used the large synthetic ETH-2 library based on the principle of protein design. Phage displaying glucose oxidase reactive scFvs were obtained after three rounds of selection on glucose oxidase-coated immunotubes and subsequent amplification in TG1 E. coli cells. Eventually, one high reactive scFv clone was selected and further examined. The anti-glucose oxidase scFv C10 was found suitable for Western blot; Biacore analysis showed that the binding affinity of the glucose oxidase-reactive scFv is almost equal that of MAbs prepared with conventional hybridoma technology. Finally, the cDNA sequence of this human scFv may be exploited to generate bispecific antibodies to target in the tumor environment-specific toxic enzymatic reaction.     

Identification of tumor-associated antigens in human hepatocellular carcinoma by autoantibodies

Hepatocellular carcinoma (HCC) is a major cause of death in Asian countries. The false-negative rate with serum alpha-fetaprotein level alone can reach 40% for early stage HCC patients. Due to the lack of sensitive and specific tumor markers for early diagnosis, it is impossible for HCC patients to receive effective therapy. However, tumor antigens can be recognized by immune cells and be rejected in immune responses. In order to identify antigens which may be used as new markers and immunotherapy targets for HCC, a cDNA expression library derived from an HCC sample was constructed, which was screened with mixed autologous and allogenic serum of HCC patients. Seventeen different HCC antigens were obtained, which are classified as tumor-associated antigens. A panel of allogenic sera from patients with chronic hepatitis, liver cirrhosis, HCC and other tumor entities and sera from health volunteers, was used for frequency analysis of antibody responses. Four of 17 antigens, including eukaryotic translation initiation factor 3, subunit I, lactate dehydrogenase 1, A chain, replication factor C2, 40 kDa and mitochondrial carrier triple repeat 1, reacted predominantly with sera from patients with HCC (31.8, 45.5, 27.3 and 50.0% respectively). Patients (81.8%) with HCC had the antibody against at least one of these four antigens, which indicates that disease-specific humoral response against these antigens was induced in HCC patients and the corresponding antibodies may be used as tumor markers for HCC.