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Discover how PEPperPRINT Peptide Microarray products have been used in different fields of research.

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Discovery of a novel highly specific, fully human PSCA antibody and its application as an antibody-drug conjugate in prostate cancer

Chu, Xiaojie; Shin, Seungmin; Baek, Du-San; Zhang, Liyong; Conard, Alex; Shi, Megan; Kim, Ye-Jin; Adams, Cynthia; Hines, Maggie; Liu, Xianglei; Chen, Chuan; Sun, Zehua; Jelev, Dontcho V.; Mellors, John W.; Dimitrov, Dimiter S.; Li, Wei
mAbs.
Aug 2024
10.1080/19420862.2024.2387240
Prostate stem cell antigen (PSCA) is expressed in all stages of prostate cancer, including in advanced androgen-independent tumors and bone metastasis. PSCA may associate with prostate carcinogenesis and lineage plasticity in prostate cancer. PSCA is also a promising theranostic marker for a variety of other solid tumors, including pancreatic adenocarcinoma and renal cell carcinoma. Here, we identified a novel fully human PSCA antibody using phage display methodology. The structure-based affinity maturation yielded a high-affinity binder, F12, which is highly specific and does not bind to 6,000 human membrane proteins based on a membrane proteome array assay. F12 targets PSCA amino acids 63–69 as tested by the peptide scanning microarray, and it cross-reacts with the murine PSCA. IgG1 F12 efficiently internalizes into PSCA-expressing tumor cells. The antimitotic reagent monomethyl auristatin E (MMAE)-conjugated IgG1 F12 (ADC, F12-MMAE) exhibits dose-dependent efficacy and specificity in a human prostate cancer PC-3-PSCA xenograft NSG mouse model. This is a first reported ADC based on a fully human PSCA antibody and MMAE that is characterized in a xenograft murine model, which warrants further optimizations and investigations in additional preclinical tumor models, including prostate and other solid tumors.

Diagnostic Profiling of the Human Public IgM Repertoire With Scalable Mimotope Libraries

Pashov, Anastas; Shivarov, Velizar; Hadzhieva, Maya; Kostov, Victor; Ferdinandov, Dilyan; Heintz, Karen-Marie; Pashova, Shina; Todorova, Milena; Vassilev, Tchavdar; Kieber-Emmons, Thomas; Meza-Zepeda, Leonardo A.; Hovig, Eivind
Front. Immunol..
Dec 2019
10.3389/fimmu.2019.02796
Specific antibody reactivities are routinely used as biomarkers, but the antibody repertoire reactivity (igome) profiles are still neglected. Here, we propose rationally designed peptide arrays as efficient probes for these system level biomarkers. Most IgM antibodies are characterized by few somatic mutations, polyspecificity, and physiological autoreactivity with housekeeping function. Previously, probing this repertoire with a set of immunodominant self-proteins provided a coarse analysis of the respective repertoire profiles. In contrast, here, we describe the generation of a peptide mimotope library that reflects the common IgM repertoire of 10,000 healthy donors. In addition, an appropriately sized subset of this quasi-complete mimotope library was further designed as a potential diagnostic tool. A 7-mer random peptide phage display library was panned on pooled human IgM. Next-generation sequencing of the selected phage yielded 224,087 sequences, which clustered in 790 sequence clusters. A set of 594 mimotopes, representative of the most significant sequence clusters, was shown to probe symmetrically the space of IgM reactivities in patients’ sera. This set of mimotopes can be easily scaled including a greater proportion of the mimotope library. The trade-off between the array size and the resolution can be explored while preserving the symmetric sampling of the mimotope sequence and reactivity spaces. BLAST search of the non-redundant protein database with the mimotopes sequences yielded significantly more immunoglobulin J region hits than random peptides, indicating a considerable idiotypic connectivity of the targeted igome. The proof of principle predictors for random diagnoses was represented by profiles of mimotopes. The number of potential reactivity profiles that can be extracted from this library is estimated at more than 1070. Thus, a quasi-complete IgM mimotope library and a scalable representative subset thereof are found to address very efficiently the dynamic diversity of the human public IgM repertoire, providing informationally dense and structurally interpretable IgM reactivity profiles.

A Low‐Cost Laser‐Based Nano‐3D Polymer Printer for Rapid Surface Patterning and Chemical Synthesis of Peptide and Glycan Microarrays

Eickelmann, Stephan; Tsouka, Alexandra; Heidepriem, Jasmin; Paris, Grigori; Zhang, Junfang; Molinari, Valerio; Mende, Marco; Loeffler, Felix F.
Adv. Mater. Technol..
Nov 2019
10.1002/admt.201900503
A low-cost laser-based printing setup is presented, which allows for the spot-wise patterning of surfaces with defined polymer nanolayers. These nanolayer spots serve as a “solid solvent,” embedding different chemicals, chemical building blocks, materials, or precursors and can be stacked on top of each other. By melting the spot pattern, the polymer-embedded molecules are released for chemical reaction. This enables researchers to quickly pattern a surface with different molecules and materials, mixing them directly on the surface for high-throughput chemical synthesis to generate and screen diverse microarray libraries. In contrast to expensive ink-jet or contact printing, this approach does not require premixing of inks, which enables in situ combinatorial mixing. Easy access and versatility of this patterning approach are shown by generating microarrays of various biomolecules, such as glycans for the first time, to screen interactions of antibodies and lectins. In addition, a layer-by-layer solid-phase synthesis of peptides directly on the microarray is presented. Amino acid–containing nanolayers are repeatedly laser-transferred and reacted with the functionalized acceptor surface in defined patterns. This simple system enables a reproducible array production, down to spot-to-spot distances of 100 µm, and offers a flexible and cheap alternative to expensive spotting robot technology.

Characterization of a sandwich ELISA for quantification of total human soluble neuropilin‐1

Gadermaier, Elisabeth; Tesarz, Manfred; Wallwitz, Jacqueline; Berg, Gabriela; Himmler, Gottfried
J Clin Lab Anal.
Sep 2019
10.1002/jcla.22944
Background Neuropilin-1 (NRP1) is a highly interactive molecule that exists as transmembrane and soluble isoforms. Measurement of circulating levels of soluble NRP1 (sNRP1) in human serum and plasma has proven to be difficult due to present matrix interferences and due to the lack of a reliable technique. Methods We developed a highly specific and sensitive sandwich ELISA assay for total sNRP1 quantification in peripheral blood, and we validated the test according to ICH guidelines. The linear epitopes of the employed polyclonal and monoclonal anti-human NRP1 antibodies were mapped with microarray technology. We included a sample pre-treatment step with guanidine hydrochloride (GuHCl) to release sNRP1 from existing interferants. Results The ELISA assay which is calibrated with sNRP1 isoform 2 and covers a calibration range from 0.375 to 12 nmol/L detects sNRP1 in human serum and plasma (heparin, EDTA, and citrate). Multiple linear epitopes recognized by the polyclonal coating antibody are distributed over the whole sNRP1 sequence. The monoclonal detection antibody binds to a linear epitope which is in the N-terminal region of the a1 domain of human sNRP1. Assay parameters like precision (intra-assay: 6%), dilution linearity (95%-115%), specificity (98%), and spike recovery (81%-109%) meet the international standards of acceptance. Conclusion Our novel sandwich ELISA provides a reliable tool for the quantitative determination of total human sNRP1. The assay detects free and previous ligand-bound total NRP1.

LRPAP1 is a frequent proliferation-inducing antigen of BCRs of mantle cell lymphomas and can be used for specific therapeutic targeting

Thurner, Lorenz; Hartmann, Sylvia; Fadle, Natalie; Kemele, Maria; Bock, Theresa; Bewarder, Moritz; Regitz, Evi; Neumann, Frank; Nimmesgern, Anna; von Müller, Lutz; Pott, Christiane; Kim, Yoo-Jin; Bohle, Rainer Maria; Wasik, Mariusz; Schuster, Stephen J.; Hansmann, Martin-Leo; Preuss, Klaus-Dieter; Pfreundschuh, Michael
Leukemia.
Jun 2019
10.1038/s41375-018-0182-1
The predominant usage of VH4-34 and V3-21 and reports of stereotyped CDR3s suggest a shared antigenic target of B-cell receptors (BCR) from mantle cell lymphomas (MCL). To identify the target antigens of MCL–BCRs, BCRs from 21 patients and seven MCL cell lines were recombinantly expressed and used for antigen screening. The BCRs from 8/21 patients and 2/7 MCL cell lines reacted specifically with the autoantigen low-density lipoprotein receptor-related protein-associated protein 1 (LRPAP1). High-titered and light chain-restricted anti-LRPAP1 serum antibodies were found in MCL patients, but not in controls. LRPAP1 induced proliferation by BCR pathway activation, while an LRPAP1–ETA′ toxin-conjugate specifically killed MCL cells with LRPAP1-specific BCRs. Our results suggest a role of LRPAP1 in lymphomagenesis and maintenance of a considerable proportion of MCL cases by chronic autoantigenic stimulation, likely evolving from a chronic autoreactive B-cell response. Importantly, LRPAP1 can be used for a novel therapeutic approach that targets MCL with LRPAP1-reactive BCRs with high specificity.

Miniaturized and Automated Synthesis of Biomolecules—Overview and Perspectives

Mattes, Daniela S.; Jung, Nicole; Weber, Laura K.; Bräse, Stefan; Breitling, Frank
Adv. Mater..
Jun 2019
10.1002/adma.201806656
Chemical synthesis is performed by reacting different chemical building blocks with defined stoichiometry, while meeting additional conditions, such as temperature and reaction time. Such a procedure is especially suited for automation and miniaturization. Life sciences lead the way to synthesizing millions of different oligonucleotides in extremely miniaturized reaction sites, e.g., pinpointing active genes in whole genomes, while chemistry advances different types of automation. Recent progress in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging could match miniaturized chemical synthesis with a powerful analytical tool to validate the outcome of many different synthesis pathways beyond applications in the life sciences. Thereby, due to the radical miniaturization of chemical synthesis, thousands of molecules can be synthesized. This in turn should allow ambitious research, e.g., finding novel synthesis routes or directly screening for photocatalysts. Herein, different technologies are discussed that might be involved in this endeavor. A special emphasis is given to the obstacles that need to be tackled when depositing tiny amounts of materials to many different extremely miniaturized reaction sites.

A high-sensitivity enzyme immunoassay for the quantification of soluble human semaphorin 4D in plasma

Laber, Anna; Gadermaier, Elisabeth; Wallwitz, Jacqueline; Berg, Gabriela; Himmler, Gottfried
Analytical Biochemistry.
Jun 2019
10.1016/j.ab.2019.03.004
Human semaphorin 4D (SEMA4D), a type I integral membrane glycoprotein, regulates key cellular functions (e.g. cell-cell communication, platelet activation). Its 120 kDa extracellular region can be shed from the membrane to release soluble SEMA4D (sSEMA4D). Studies on circulating sSEMA4D levels are mostly performed with poorly characterized assays and use serum and plasma as matrix. We developed and validated a sandwich ELISA utilizing two monoclonal antibodies with resolved epitopes and determined affinities. Human serum and plasma samples were analyzed, and the influence of protease activity on sSEMA4D concentration was tested by collecting samples in the presence of the protease inhibitor TAPI-1. Both antibodies recognize conformational epitopes in the sema domain. Validation for plasma (EDTA, citrate, heparin) showed valid specificity, precision, accuracy, dilution linearity, and robustness. The assay shows a calibration range from 62.5 to 2000 pmol/L with a quantification limit of 31 pmol/L. sSEMA4D was significantly higher in serum than in plasma, whereas serum and plasma levels from samples collected in the presence of TAPI-1 showed no statistical difference. This ELISA provides a reliable tool for the quantification of sSEMA4D in human plasma. Serum is not recommended as matrix due to the accumulation of shed SEMA4D during blood coagulation altering serum sSEMA4D levels.

Non-invasive glioblastoma immunoprofiling by printed peptide arrays

Mock, Andreas; Herold-Mende, Christel
OncoImmunology.
Feb 2016
10.1080/2162402X.2015.1069941
Immune monitoring assays for patient stratification and treatment efficacy in clinical trials are in demand. We have recently described a cost-effective non-invasive assay to determine the immune status of glioblastoma patients. Profiling antitumor serum antibodies by customized printed peptide arrays identified response against a tenascin-C (TNC) peptide as a robust prognostic biomarker.

Autoantibodies specific to estrogen receptor alpha act as estrogen agonists and their levels correlate with breast cancer cell proliferation

Maselli, Angela; Capoccia, Sara; Pugliese, Patrizia; Raggi, Carla; Cirulli, Francesca; Fabi, Alessandra; Malorni, Walter; Pierdominici, Marina; Ortona, Elena
OncoImmunology.
Feb 2016
10.1080/2162402X.2015.1074375
Estrogen receptors have recently been demonstrated at the cell surface. Unlike nuclear receptors, they are able to trigger rapid responses inside the cells. In this study, we evaluated the presence and the possible role of autoantibodies specific to estrogen receptor (anti-ER Abs) in the peripheral blood of breast cancer patients. Anti-ERα Abs were detectable in 22/48 (46%) patients’ sera and their levels positively correlated with the percentage of Ki-67-positive breast cancer cells. Anti-ERα Abs purified from breast cancer patients’ sera were able: (i) to recognize ERα epitopes expressed at the cell surface of ER-positive breast cancer cells, (ii) to trigger rapid extracellular signal-regulated kinase (ERK) phosphorylation, and (iii) to induce cell proliferation. Our results suggest that anti-ERα Abs can act as estrogen agonists playing a pathogenetic role as breast cancer-promoting factors. These autoantibodies could also be considered as possible peripheral blood biomarkers indicative of the breast cancer growth potential.

Combinatorial Synthesis of Peptide Arrays onto a Microchip

Beyer, M.; Nesterov, A.; Block, I.; Konig, K.; Felgenhauer, T.; Fernandez, S.; Leibe, K.; Torralba, G.; Hausmann, M.; Trunk, U.; Lindenstruth, V.; Bischoff, F. R.; Stadler, V.; Breitling, F.
Science.
Dec 2007
10.1126/science.1149751
Arrays promise to advance biology through parallel screening for binding partners. We show the combinatorial in situ synthesis of 40,000 peptide spots per square centimeter on a microchip. Our variant Merrifield synthesis immobilizes activated amino acids as monomers within particles, which are successively attracted by electric fields generated on each pixel electrode of the chip. With all different amino acids addressed, particles are melted at once to initiate coupling. Repetitive coupling cycles should allow for the translation of whole proteomes into arrays of overlapping peptides that could be used for proteome research and antibody profiling.

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