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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.

Optimised ‘on demand’ protein arraying from DNA by cell free expression with the ‘DNA to Protein Array’ (DAPA) technology

Schmidt, Ronny; Cook, Elizabeth A.; Kastelic, Damjana; Taussig, Michael J.; Stoevesandt, Oda
Journal of Proteomics.
Aug 2013
10.1016/j.jprot.2013.02.002
We have previously described a protein arraying process based on cell free expression from DNA template arrays (DNA Array to Protein Array, DAPA). Here, we have investigated the influence of different array support coatings (Ni-NTA, Epoxy, 3D-Epoxy and Polyethylene glycol methacrylate (PEGMA)). Their optimal combination yields an increased amount of detected protein and an optimised spot morphology on the resulting protein array compared to the previously published protocol. The specificity of protein capture was improved using a tag-specific capture antibody on a protein repellent surface coating. The conditions for protein expression were optimised to yield the maximum amount of protein or the best detection results using specific monoclonal antibodies or a scaffold binder against the expressed targets. The optimised DAPA system was able to increase by threefold the expression of a representative model protein while conserving recognition by a specific antibody. The amount of expressed protein in DAPA was comparable to those of classically spotted protein arrays. Reaction conditions can be tailored to suit the application of interest. Biological significance: DAPA represents a cost effective, easy and convenient way of producing protein arrays on demand. The reported work is expected to facilitate the application of DAPA for personalized medicine and screening purposes.

Purification of High-Complexity Peptide Microarrays by Spatially Resolved Array Transfer to Gold-Coated Membranes

Schirwitz, Christopher; Loeffler, Felix F.; Felgenhauer, Thomas; Stadler, Volker; Nesterov-Mueller, Alexander; Dahint, Reiner; Breitling, Frank; Bischoff, F. Ralf
Adv. Mater..
Mar 2013
10.1002/adma.201203853
A method for the one-step purification of high-complexity peptide microarrays is presented. The entire peptide library is transferred from the synthesis support to a gold coated polyvinylidenfluoride (PVDF) membrane, whereby only full-length peptides covalently couple to the receptor membrane via an N-terminally added cysteine. Highly resolved peptide transfer and purification of up to 10 000 features per cm2 is demonstrated.

Combinatorial Peptide Synthesis on a Microchip

Schirwitz, Christopher; Block, Ines; König, Kai; Nesterov, Alexander; Fernandez, Simon; Felgenhauer, Thomas; Leibe, Klaus; Torralba, Gloria; Hausmann, Michael; Lindenstruth, Volker; Stadler, Volker; Breitling, Frank; Bischoff, F. Ralf
Current Protocols in Protein Science.
Aug 2009
10.1002/0471140864.ps1802s57
Microchips are used in the combinatorial synthesis of peptide arrays by means of amino acid microparticle deposition. The surface of custom-built microchips can be equipped with an amino-modified poly(ethylene glycol)methacrylate (PEGMA) graft polymer coating, which permits high loading of functional groups and resists nonspecific protein adsorption. Specific microparticles that are addressed to the polymer-coated microchip surface in a well defined pattern release preactivated amino acids upon melting, and thus allow combinatorial synthesis of high-complexity peptide arrays directly on the chip surface. Currently, arrays with densities of up to 40,000 peptide spots/cm2 can be generated in this way, with a minimum of coupling cycles required for full combinatorial synthesis. Without using any additional blocking agent, specific peptide recognition has been verified by background-free immunostaining on the chip-based array. This unit describes microchip surface modification, combinatorial peptide array synthesis on the chip, and a typical immunoassay employing the resulting high-density peptide arrays.

Particle-Based Synthesis of Peptide Arrays

Breitling, Frank; Felgenhauer, Thomas; Nesterov, Alexander; Lindenstruth, Volker; Stadler, Volker; Bischoff, F. Ralf
ChemBioChem.
Mar 2009
10.1002/cbic.200800735
Lithographic methods allow for the combinatorial synthesis of >50,000 oligonucleotides per cm(2), and this has revolutionized the field of genomics. High-density peptide arrays promise to advance the field of proteomics in a similar way, but currently lag behind. This is mainly due to the monomer-by-monomer repeated consecutive coupling of 20 different amino acids associated with lithography, which adds up to an excessive number of coupling cycles. Combinatorial synthesis based on electrically charged solid amino acid particles resolves this problem. A color laser printer or a chip addresses the different charged particles consecutively to a solid support, where, when completed, the whole layer of solid amino acid particles is melted at once. This frees hitherto immobilized amino acids to couple all 20 different amino acids to the support in one single coupling reaction. The method should allow for the translation of entire genomes into sets of overlapping peptides to be used in proteome research.

High-density peptide arrays

Breitling, Frank; Nesterov, Alexander; Stadler, Volker; Felgenhauer, Thomas; Bischoff, F. Ralf
Mol. BioSyst..
Jan 2009
10.1039/b819850k
Arrays promise to advance biology by allowing parallel screening for many different binding partners. Meanwhile, lithographic methods enable combinatorial synthesis of >50 000 oligonucleotides per cm2, an advance that has revolutionized the whole field of genomics. A similar development is expected for the field of proteomics, provided that affordable, very high-density peptide arrays are available. However, peptide arrays lag behind oligonucleotide arrays. This review discusses recent developments in the field with an emphasis on methods that lead to very high-density peptide arrays.

Combinatorial Synthesis of Peptide Arrays with a Laser Printer

Stadler, Volker; Felgenhauer, Thomas; Beyer, Mario; Fernandez, Simon; Leibe, Klaus; Güttler, Stefan; Gröning, Martin; König, Kai; Torralba, Gloria; Hausmann, Michael; Lindenstruth, Volker; Nesterov, Alexander; Block, Ines; Pipkorn, Rüdiger; Poustka, Annemarie; Bischoff, F. Ralf; Breitling, Frank
Angew. Chem. Int. Ed..
Sep 2008
10.1002/anie.200801616
Special delivery: The “freezing” of activated amino acid derivatives within solid particles enables a laser printer to deliver these “postal packages” to defined locations on a solid support with high resolution. Subsequent parallel coupling is initiated simply by melting a whole layer of 20 different amino acid particles (see schematic representation; Fmoc=9-fluorenylmethoxycarbonyl).

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