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

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Human Antibody Domains and Fragments Targeting Neutrophil Elastase as Candidate Therapeutics for Cancer and Inflammation-Related Diseases

Chu, Xiaojie; Sun, Zehua; Baek, Du-San; Li, Wei; Mellors, John W.; Shapiro, Steven D.; Dimitrov, Dimiter S.
Int J Mol Sci.
Oct 2021
10.3390/ijms222011136
Neutrophil elastase (NE) is a serine protease released during neutrophil maturation. High levels of NE are related to lung tissue damage and poor prognosis in cancer; thus, NE is a potential target for therapeutic immunotherapy for multiple lung diseases and cancers. Here, we isolate and characterize two high-affinity, specific, and noncompetitive anti-NE antibodies Fab 1C10 and VH 1D1.43 from two large phage-displayed human Fab and VH libraries. After fusion with human IgG1 Fc, both of them (VH-Fc 1D1.43 and IgG1 1C10) inhibit NE enzymatic activity with VH-Fc 1D1.43 showing comparable inhibitory effects to that of the small molecule NE inhibitor SPCK and IgG1 1C10 exhibiting even higher (2.6-fold) activity than SPCK. Their epitopes, as mapped by peptide arrays combined with structural modeling, indicate different mechanisms for blocking NE activity. Both VH-Fc and IgG1 antibodies block NE uptake by cancer cells and fibroblast differentiation. VH-Fc 1D1.43 and IgG1 1C10 are promising for the antibody-based immunotherapy of cancer and inflammatory diseases.

HSP70iQ435A to subdue autoimmunity and support anti-tumor responses

Jaishankar, Dinesh; Cosgrove, Cormac; Ramesh, Prathyaya; Mahon, James; Shivde, Rohan; Dellacecca, Emilia R.; Yang, Shiayin F.; Mosenson, Jeffrey; Guevara-Patiño, José A.; Le Poole, I. Caroline
Cell Stress and Chaperones.
Sep 2021
10.1007/s12192-021-01229-x
Developing immunosuppressive therapies for autoimmune diseases comes with a caveat that immunosuppression may promote the risk of developing other conditions or diseases. We have previously shown that biolistic delivery of an expression construct encoding inducible HSP70 (HSP70i) with one amino acid modification in the dendritic cell (DC) activating moiety 435–445 (HSP70iQ435A) to mouse skin resulted in significant immunosuppressive activity of autoimmune vitiligo, associated with fewer tissue infiltrating T cells. To prepare HSP70iQ435A as a potential therapeutic for autoimmune vitiligo, in this study we evaluated whether and how biolistic delivery of HSP70iQ435A in mice affects anti-tumor responses. We found that HSP70iQ435A in fact supports anti-tumor responses in melanoma-challenged C57BL/6 mice. Biolistic delivery of the HSP70iQ435A-encoding construct to mice elicited significant anti-HSP70 titers, and anti-HSP70 IgG and IgM antibodies recognize surface-expressed and cytoplasmic HSP70i in human and mouse melanoma cells. A peptide scan revealed that the anti-HSP70 antibodies recognize a specific C-terminal motif within the HSP70i protein. The antibodies elicited surface CD107A expression among mouse NK cells, representative of antibody-mediated cellular cytotoxicity (ADCC), supporting the concept, that HSP70iQ435A-encoding DNA elicits a humoral response to the stress protein expressed selectively on the surface of melanoma cells. Thus, besides limiting autoimmunity and inflammation, HSP70iQ435A elicits humoral responses that limit tumor growth and may be used in conjunction with immune checkpoint inhibitors to not only control tumor but to also limit adverse events following tumor immunotherapy.

Serum Peptide Immunoglobulin G Autoantibody Response in Patients with Different Central Nervous System Inflammatory Demyelinating Disorders

Lee, Hye Lim; Park, Jin-Woo; Seok, Jin Myoung; Jeon, Mi Young; Kim, Hojin; Lim, Young-Min; Shin, Ha Young; Kang, Sa-Yoon; Kwon, Oh-Hyun; Lee, Sang-Soo; Seok, Hung Youl; Min, Ju-Hong; Lee, Sung-Hyun; Kim, Byung-Jo; Kim, Byoung Joon
Diagnostics.
Jul 2021
10.3390/diagnostics11081339
Previous efforts to discover new surrogate markers for the central nervous system (CNS) inflammatory demyelinating disorders have shown inconsistent results; moreover, supporting evidence is scarce. The present study investigated the IgG autoantibody responses to various viral and autoantibodies-related peptides proposed to be related to CNS inflammatory demyelinating disorders using the peptide microarray method. We customized a peptide microarray containing more than 2440 immobilized peptides representing human and viral autoantigens. Using this, we tested the sera of patients with neuromyelitis optica spectrum disorders (NMOSD seropositive, n = 6; NMOSD seronegative, n = 5), multiple sclerosis (MS, n = 5), and myelin-oligodendrocyte glycoprotein antibody-associated disease (MOGAD, n = 6), as well as healthy controls (HC, n = 5) and compared various peptide immunoglobulin G (IgG) responses between the groups. Among the statistically significant peptides based on the pairwise comparisons of IgG responses in each disease group to HC, cytomegalovirus (CMV)-related peptides were most clearly distinguishable among the study groups. In particular, the most significant differences in IgG response were observed for HC vs. MS and HC vs. seronegative NMOSD (p = 0.064). Relatively higher IgG responses to CMV-related peptides were observed in patients with MS and NMOSD based on analysis of the customized peptide microarray.

Non-invasive immunoPET imaging of PD-L1 using anti-PD-L1-B11 in breast cancer and melanoma tumor model

Bansal, Aditya; Pandey, Mukesh K.; Barham, Whitney; Liu, Xin; Harrington, Susan M.; Lucien, Fabrice; Dong, Haidong; Park, Sean S.; DeGrado, Timothy R.
Nuclear Medicine and Biology.
May 2021
10.1016/j.nucmedbio.2021.05.004
Introduction Immunotherapy targeting PD-1/PD-L1 immune checkpoint inhibition (ICI) is efficacious in various solid and hematologic malignancies. However, the response rate to PD-1/PD-L1 therapy is only 15–35%. To obtain optimal clinical response to ICI therapies, a reliable assessment of tumor PD-L1 expression is needed to select appropriate patients, and a non-invasive imaging-based assessment of PD-L1 expression is critically needed. Although radiolabeled PET probes based on PD-L1 targeted therapeutic antibodies (e.g. atezolizumab) have shown encouraging results, there is concern that residual therapeutic antibody may compete for binding with the radiotracer thereby compromising imaging studies that follow treatment. Methods and results In this study, we used novel anti-PD-L1-B11 clone antibody known to bind to a different epitope of PD-L1 than the therapeutic antibodies to avoid potential saturation effects. The anti-PD-L1-B11 clone was radiolabeled with zirconium-89 and evaluated to detect PD-L1 expression in various in vitro and in vivo cancer model systems in comparison with [89Zr]Zr-DFO-NCS-atezolizumab. In vitro binding parameters of anti-PD-L1-B11 were like those of atezolizumab. [89Zr]Zr-DFO-NCS-anti-PD-L1-B11 clone showed favorable properties to [89Zr]Zr-DFO-NCS-atezolizumab in an in vivo breast cancer tumor model system with higher uptake in PD-L1 expressing tumors. Conclusion Our data demonstrates that [89Zr]Zr-DFO-NCS-anti-PD-L1-B11 exhibits excellent imaging properties for the assessment PD-L1 expression. The independent binding site of anti-PD-L1-B11 relative to therapeutic anti-PD-L1 antibodies may be advantageous for anti-PD-L1 therapy monitoring.

Analysis of chronic inflammatory lesions of the colon for BMMF Rep antigen expression and CD68 macrophage interactions

Bund, Timo; Nikitina, Ekaterina; Chakraborty, Deblina; Ernst, Claudia; Gunst, Karin; Boneva, Boyana; Tessmer, Claudia; Volk, Nadine; Brobeil, Alexander; Weber, Achim; Heikenwalder, Mathias; Zur Hausen, Harald; de Villiers, Ethel-Michele
Proc Natl Acad Sci U S A.
Mar 2021
10.1073/pnas.2025830118
Consumption of Eurasian bovine meat and milk has been associated with cancer development, in particular with colorectal cancer (CRC). In addition, zoonotic infectious agents from bovine products were proposed to cause colon cancer (zur Hausen et al., 2009). Bovine meat and milk factors (BMMF) are small episomal DNA molecules frequently isolated from bovine sera and milk products, and recently, also from colon cancer (de Villiers et al., 2019). BMMF are bioactive in human cells and were proposed to induce chronic inflammation in precancerous tissue leading to increased radical formation: for example, reactive oxygen and reactive nitrogen species and elevated levels of DNA mutations in replicating cells, such as cancer progenitor cells (zur Hausen et al., 2018). Mouse monoclonal antibodies against the replication (Rep) protein of H1MSB.1 (BMMF1) were used to analyze BMMF presence in different cohorts of CRC peritumor and tumor tissues and cancer-free individuals by immunohistochemistry and Western blot. BMMF DNA was isolated by laser microdissection from immunohistochemistry-positive tissue regions. We found BMMF Rep protein present specifically in close vicinity of CD68+ macrophages in the interstitial lamina propria adjacent to CRC tissues, suggesting the presence of local chronic inflammation. BMMF1 (modified H1MSB.1) DNA was isolated from the same tissue regions. Rep and CD68+ detection increased significantly in peritumor cancer tissues when compared to tissues of cancer-free individuals. This strengthens previous postulations that BMMF function as indirect carcinogens by inducing chronic inflammation and DNA damage in replicating cells, which represent progress to progenitor cells for adenoma (polyps) formation and cancer.

Comparison of Monoclonal Gammopathies Linked to Poliovirus or Coxsackievirus vs. Other Infectious Pathogens

Harb, Jean; Mennesson, Nicolas; Lepetit, Cassandra; Fourny, Maeva; Louvois, Margaux; Bosseboeuf, Adrien; Allain-Maillet, Sophie; Decaux, Olivier; Moreau, Caroline; Tallet, Anne; Piver, Eric; Moreau, Philippe; Salle, Valéry; Bigot-Corbel, Edith; Hermouet, Sylvie
Feb 2021
10.3390/cells10020438
Chronic stimulation by infectious pathogens or self-antigen glucosylsphingosine (GlcSph) can lead to monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM). Novel assays such as the multiplex infectious antigen microarray (MIAA) and GlcSph assays, permit identification of targets for >60% purified monoclonal immunoglobulins (Igs). Searching for additional targets, we selected 28 purified monoclonal Igs whose antigen was not represented on the MIAA and GlcSph assays; their specificity of recognition was then analyzed using microarrays consisting of 3760 B-cell epitopes from 196 pathogens. The peptide sequences PALTAVETG and PALTAAETG of the VP1 coat proteins of human poliovirus 1/3 and coxsackievirus B1/B3, respectively, were specifically recognized by 6/28 monoclonal Igs. Re-analysis of patient cohorts showed that purified monoclonal Igs from 10/155 MGUS/SM (6.5%) and 3/147 MM (2.0%) bound to the PALTAVETG or PALTAAETG epitopes. Altogether, PALTAV/AETG-initiated MGUS are not rare and few seem to evolve toward myeloma.

Immunity to Influenza is dependent on MHC II polymorphism: study with 2 HLA transgenic strains

Luckey, David; Weaver, Eric A.; Osborne, Douglas G.; Billadeau, Daniel D.; Taneja, Veena
Sci Rep.
Dec 2019
10.1038/s41598-019-55503-1
Major histocompatibility complex II (MHC II) molecules are involved in antigen presentation and the development of a functional adaptive immune response. Evolutionary selection for MHC molecules that effectively clear infectious agents provides an advantage to humans. However, certain class II molecules are associated with autoimmune diseases. In this study we infected autoimmune-susceptible DRB1*0401.AEo and non-susceptible *0402.AEo mice with H1N1 influenza and determined clearance and protective immunity to H3N2 virus. *0401 mice generated a robust TLR-triggered immune response and cleared H1N1 influenza virus infection. After vaccination and challenge with H1N1, *0401 mice, when challenged with H3N2, generated cross-protective immunity to heterosubtypic H3N2 influenza strain whereas *0402 mice cleared the H1N1 infection but did not generate cross-protective immunity against the H3N2 influenza strain. The intracellular trafficking route of MHCII revealed that *0401 molecules traffic through the late endosome/lysosomes while *0402 molecules traffic into early endosomes. This suggested that trafficking of MHCII could affect the functional output of the innate immune response and clearance of viral infections. Also, DRB1*0401 mice live longer than HLA-DRB1*0402 mice. The study provides a potential hypothesis for evolutionary selection of *0401 molecule, even though it is associated with autoreactivity, which may be dependent on the availability of peptide repertoire of self-antigens.

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.

A Monoclonal Antibody to M-Phase Phosphoprotein 1/Kinesin-Like Protein KIF20B

Fritzler, Marvin J.; Brown, Rachael D.; Zhang, Meifeng
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy.
Aug 2019
10.1089/mab.2019.0016
Kinesin-like protein KIF20B, originally named M-phase phosphoprotein 1 (MPP1), is a plus-end-directed kinesin-related protein that exhibits in vitro microtubule-binding and -bundling properties as well as microtubule-stimulated ATPase activity. It has been characterized as a slow molecular motor that moves toward the plus-end of microtubules. Human autoantibodies directed against KIF20B have been described in up to 25% of patients with idiopathic ataxia and less commonly in other neuropathies and autoinflammatory conditions. One of the limitations of research into the structure and function of KIF20B has been a reliable monoclonal antibody that can be used in a variety of applications. To establish a reference standard for anti-KIF20B immunoassays and facilitate studies on the role of KIF20B in developmental cell biology, we developed an IgG1 monoclonal antibody, 10C7, which reacts with the cognate KIF20B protein in Western immunoblots and in addressable laser bead immunoassays. In HEp2 cells, leptomeningeal pericytes, and transfected HEK293T cells, indirect immunofluorescence studies showed that reactivity was mainly localized to a proportion of interphase nuclei, but during metaphase, it was redistributed throughout the cytoplasm and perichromatin mass. Later in telophase/anaphase, KIF20B was localized to the stem body and midzone of the midbody. 10C7 also showed remarkable staining of a subset of cells in the cerebellum, ovary, and testis tissues. KIF20B was shown to have extensive coiled-coil domains. The monoclonal antibody, 10C7, will be of value to diagnostic laboratory scientists interested in having a reliable reference standard for anti-KIF20B immunoassays as well as cell, molecular, and developmental biology researchers.

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.

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.

Autoantibodies to a novel Rpp38 (Th/To) derived B-cell epitope are specific for systemic sclerosis and associate with a distinct clinical phenotype

Koenig, Martial; Bentow, Chelsea; Satoh, Minoru; Fritzler, Marvin J; Senécal, Jean-Luc; Mahler, Michael
Apr 2019
10.1093/rheumatology/kez123
Abstract Objective Detection of antinuclear antibodies and specific autoantibodies is important in the diagnosis and classification of SSc. Several proteins of the Th/To complex, including Rpp25, Rpp38 and hPop1 are the target of autoantibodies in SSc patients. However, very little is known about the epitope distribution of this autoantigen. Consequently, we screened Rpp25, Rpp38 and hPop1 for B cell epitopes and evaluated their clinical relevance. Methods Serum pools with (n = 2) and without (n = 1) anti-Th/To autoantibodies were generated and used for epitope discovery. Identified biomarker candidate sequences were then utilized to synthesize synthetic, biotinylated, soluble peptides. The peptides were tested to determine reactivity with sera from SSc cohorts (n = 202) and controls (n = 159) using a chemiluminescence immunoassay. Additionally, samples were also tested for antibodies to full-length recombinant Rpp25 antibodies by chemiluminescence immunoassay. Results Several immunodominant regions were found on the three proteins. The strongest reactivity was observed with an Rpp38 peptide (aa 229–243). Autoantibodies to the Rpp38 peptide were detected in 8/149 (5.4%) limited cutaneous SSc patients, but not in any of 159 controls (P = 0.003 by two-sided Fisher’s exact probability test). Although reactivity to the novel antigenic peptide was correlated with the binding to Rpp25 (rho = 0.44; P < 0.0001), subsets of patient sera either reacted strongly with Rpp25 or with the novel Rpp38-derived peptide. Conclusion A novel Rpp38 epitope holds promise to increase the sensitivity in the detection of anti-Th/To autoantibodies, thus enhancing the serological diagnosis of SSc.

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