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

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Targeting FLT3 by new-generation antibody-drug-conjugate in combination with kinase inhibitors for treatment of AML

Roas, Maike; Vick, Binje; Kasper, Marc-André; Able, Marina; Polzer, Harald; Gerlach, Marcus; Kremmer, Elisabeth; Hecker, Judith S.; Schmitt, Saskia; Stengl, Andreas; Waller, Verena; Hohmann, Natascha; Festini, Moreno; Ludwig, Alexander Edmund; Rohrbacher, Lisa; Herold, Tobias; Subklewe, Marion; Götze, Katharina S.; Hackenberger, Christian P.R.; Schumacher, Dominik; Helma-Smets, Jonas; Jeremias, Irmela; Leonhardt, Heinrich; Spiekermann, Karsten
Aug 2022
10.1182/blood.2021015246
Fms like tyrosine kinase 3 (FLT3) is often overexpressed or constitutively activated by internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations in acute myeloid leukemia (AML). Despite the use of receptor tyrosine kinase inhibitors (TKI) in FLT3-ITD positive AML, the prognosis of patients is still poor and further improvement of therapy is required. Targeting FLT3 independent of mutations by antibody‑drug‑conjugates (ADCs) is a promising strategy for AML therapy. Here, we report the development and preclinical characterization of a novel FLT3‑targeting ADC, 20D9-ADC, which was generated by applying the innovative P5 conjugation technology. In vitro, 20D9‑ADC mediated potent cytotoxicity to Ba/F3 cells expressing transgenic FLT3 or FLT3-ITD, to AML cell lines and to FLT3-ITD positive patient derived xenograft AML cells. In vivo, 20D9‑ADC treatment led to a significant tumor reduction and even durable complete remission in AML xenograft models. Further, 20D9‑ADC demonstrated no severe hematotoxicity in in vitro colony formation assays using concentrations that were cytotoxic in AML cell line treatment. The combination of 20D9-ADC with the TKI midostaurin showed strong synergy in vitro and in vivo, leading to reduction of aggressive AML cells below the detection limit. Our data indicate that targeting FLT3 with an advanced new-generation ADC is a promising and potent antileukemic strategy, especially when combined with FLT3-TKI in FLT3‑ITD positive AML.

IFx-Hu2.0 phase I first in human study for unresectable melanoma for an intralesional “in-situ vaccine” approach.

Markowitz, Joseph; Shamblott, Michael; Brohl, Andrew Scott; Sarnaik, Amod; Eroglu, Zeynep; Khushalani, Nikhil I.; Chen, Pei-Ling; De-Aquino, Deanryan B.; Sondak, Vernon K.; Tarhini, Ahmad A.; Kim, Youngchul; Pilon-Thomas, Shari
Jun 2022
10.1200/JCO.2022.40.16_suppl.e21542
e21542 Background: Many melanoma patients do not respond to anti-PD1 therapy due to lack of antigen specific responses. IFx-Hu2.0 (plasmid DNA encoding the streptococcal membrane protein, Emm55, contained within a cationic polymer) primes innate and antigen dependent responses in murine/equine melanoma models to produce an environment needed for checkpoint inhibitor efficacy. We describe the first in human study utilizing IFx-Hu2.0 in unresectable melanoma – NCT03655756. Methods: Melanoma patients (unresectable stage III/IV) had cutaneous lesions injected with IFx-Hu2.0 to test safety and feasibility. Patients were refractory to standard of care (anti-PD1, BRAF/MEK) or did not wish these treatments. 1-3 lesions (> 3 mm – 0.1 mg/0.2 mL) were injected, pre/post treatment biopsies obtained, and the primary endpoint of 5/6 patients without dose limiting toxicity (DLT) was assessed at 28 days. Retreatment was permitted. ≥2 lesions were needed: one for injection and uninjected lesion for biopsy. Tissue samples were analyzed for mRNA profiles, antigen responses (PEPperPRINT assay), and multiplex immunofluorescence (markers: CD3, CD8, FOXP3, PD1, PDL1, SOX10, DAPI). Results: The primary endpoint was met in 6 evaluable patients out of 7 enrolled. Observed toxicities included: G1-2 Injection site reactions – 5/7; G1 Bleeding – 1/7; G1-2 Pain – 2/7, G1 Lymphopenia – 1/7, G1 Pruritis – 1/7; with no ≥ G3 toxicities related to study drug observed. One G5 toxicity (Clostridium septicum infection 20 days post injection) was deemed unlikely related to study drug. 5/6 patients received 1 cycle prior to post-protocol immune-based therapy. One treatment naïve patient retreated once with IFx-Hu2.0 required no additional therapy > 9 months. Available paired tissue and plasma sampling revealed increased T cell infiltration into treated lesions, increase in IgM and IgG epitope recognition to melanoma associated antigens in the plasma (detected by PEPperPRINT assay), an increase in mRNA associated with innate immune responses in the injected lesion (CXCL13, LAG3, CXCL11, CXCL10, ICOS) and an adaptive immune response (IL-12, HLA-DRB5, WNT4, CD3D, Arg I) in uninjected lesions associated with downregulation of known melanoma antigens. Of 4 anti-PD1 refractory patients, three patients had clinical benefit to post-protocol retreatment with anti-PD1 based therapy (Stable Disease (SD) lasting > 2 years followed by surgical resection, Partial Response (PR) lasting > 9 months, PR subsequently surgical resected and rendered no evidence of disease). Conclusions: In this pilot study, intralesional IFx-Hu2.0 demonstrated a favorable safety profile. These data support encouraging immunological correlative responses and further study of IFx-Hu2.0 as a priming agent to enhance or restore sensitivity to immune checkpoint inhibitor therapy in melanoma. Clinical trial information: NCT03655756.

Spot peptide arrays and SPR measurements: throughput and quantification in antibody selectivity studies: Peptide Arrays for Antibody Selectivity Studies

Vernet, Thierry; Choulier, Laurence; Nominé, Yves; Bellard, Laure; Baltzinger, Mireille; Travé, Gilles; Altschuh, Danièle
J. Mol. Recognit..
Oct 2015
10.1002/jmr.2477
Antibody selectivity represents a major issue in the development of efficient immuno-therapeutics and detection assays. Its description requires a comparison of the affinities of the antibody for a significant number of antigen variants. In the case of peptide antigens, this task can now be addressed to a significant level of details owing to improvements in spot peptide array technologies. They allow the high-throughput mutational analysis of peptides with, depending on assay design, an evaluation of binding stabilities. Here, we examine the cross-reactive capacity of an antibody fragment using the PEPperCHIP® technology platform (PEPperPRINT GmbH, Heidelberg, Germany; >8800 peptides per microarray) combined with the surface plasmon resonance characterization (Biacore® technology; GE-Healthcare Biacore, Uppsala, Sweden) of a subset of interactions. ScFv1F4 recognizes the N-terminal end of oncoprotein E6 of human papilloma virus 16. The spot permutation analysis (i.e. each position substituted by all amino acids except cysteine) of the wild type decapeptide (sequence 6TAMFQDPQER15) and of 15 variants thereof defined the optimal epitope and provided a ranking for variant recognition. The SPR affinity measurements mostly validated the ranking of complex stabilities deduced from array data and defined the sensitivity of spot fluorescence intensities, bringing further insight into the conditions for cross-reactivity. Our data demonstrate the importance of throughput and quantification in the assessment of antibody selectivity.

Monoclonal antibodies to HLA-E bind epitopes carried by unfolded β 2 m-free heavy chains: Molecular immunology

Tremante, Elisa; Lo Monaco, Elisa; Ingegnere, Tiziano; Sampaoli, Camilla; Fraioli, Rocco; Giacomini, Patrizio
Eur. J. Immunol..
Aug 2015
10.1002/eji.201545446
Since HLA-E heavy chains accumulate free of their light β2-microglobulin (β2m) subunit, raising mAbs to folded HLA-E heterodimers has been difficult, and mAb characterization has been controversial. Herein, mAb W6/32 and 5 HLA-E-restricted mAbs (MEM-E/02, MEM-E/07, MEM-E/08, DT9, and 3D12) were tested on denatured, acid-treated, and natively folded (both β2m-associated and β2m-free) HLA-E molecules. Four distinct conformations were detected, including unusual, partially folded (and yet β2m-free) heavy chains reactive with mAb DT9. In contrast with previous studies, epitope mapping and substitution scan on thousands of overlapping peptides printed on microchips revealed that mAbs MEM-E/02, MEM-E/07, and MEM-E/08 bind three distinct α1 and α2 domain epitopes. All three epitopes are linear since they span just 4–6 residues and are “hidden” in folded HLA-E heterodimers. They contain at least one HLA-E-specific residue that cannot be replaced by single substitutions with polymorphic HLA-A, HLA-B, HLA-C, HLA-F, and HLA-G residues. Finally, also the MEM-E/02 and 3D12 epitopes are spatially distinct. In summary, HLA-E-specific residues are dominantly immunogenic, but only when heavy chains are locally unfolded. Consequently, the available mAbs fail to selectively bind conformed HLA-E heterodimers, and HLA-E expression may have been inaccurately assessed in some previous oncology, reproductive immunology, virology, and transplantation studies.

Printed peptide arrays identify prognostic TNC serumantibodies in glioblastoma patients

Mock, Andreas; Warta, Rolf; Geisenberger, Christoph; Bischoff, Ralf; Schulte, Alexander; Lamszus, Katrin; Stadler, Volker; Felgenhauer, Thomas; Schichor, Christian; Schwartz, Christoph; Matschke, Jakob; Jungk, Christine; Ahmadi, Rezvan; Sahm, Felix; Capper, David; Glass, Rainer; Tonn, Jörg-Christian; Westphal, Manfred; von Deimling, Andreas; Unterberg, Andreas; Bermejo, Justo Lorenzo; Herold-Mende, Christel
Oncotarget.
May 2015
10.18632/oncotarget.3791
Liquid biopsies come of age offering unexploited potential to monitor and react to tumor evolution. We developed a cost-effective assay to non-invasively determine the immune status of glioblastoma (GBM) patients. Employing newly developed printed peptide microarrays we assessed the B-cell response against tumor-associated antigens (TAAs) in 214 patients. Firstly, sera of long-term (36+ months, LTS, n=10) and short-term (6-10 months, STS, n=14) surviving patients were screened for prognostic antibodies against 1745 13-mer peptides covering known TAAs (TNC, EGFR, GLEA2, PHF3, FABP5, MAGEA3). Next, survival associations were investigated in two retrospective independent multicenter validation sets (n=61, n=129, all IDH1-wildtype). Reliability of measurements was tested using a second array technology (spotted arrays). LTS/STS screening analyses identified 106 differential antibody responses. Evaluating the Top30 peptides in validation set 1 revealed three prognostic peptides. Prediction of TNC peptide VCEDGFTGPDCAE was confirmed in a second set (p=0.043, HR=0.66 [0.44-0.99]) and was unrelated to TNC protein expression. Median signals of printed arrays correlated with pre-synthesized spotted microarrays (p<0.0002, R=0.33). Multiple survival analysis revealed independence of age, gender, KPI and MGMT status. We present a novel peptide microarray immune assay that identified increased anti-TNC VCEDGFTGPDCAE serum antibody titer as a promising non-invasive biomarker for prolonged survival.

Single-Molecule Detection on a Protein-Array Assay Platform for the Exposure of a Tuberculosis Antigen

Schmidt, Ronny; Jacak, Jaroslaw; Schirwitz, Christopher; Stadler, Volker; Michel, Gerd; Marmé, Nicole; Schütz, Gerhard J.; Hoheisel, Jörg D.; Knemeyer, Jens-Peter
J. Proteome Res..
Jan 2011
10.1021/pr101070j

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