Tumour-targeting properties of antibodies specific to MMP-1A, MMP-2 and MMP-3

Purpose: Matrix metalloproteinases (MMPs), a group of more than 20 zinc-containing endopeptidases, are upregulated in many diseases, but several attempts to use radiolabelled MMP inhibitors for imaging tumours have proved unsuccessful in mouse models, possibly due to the limited specificity of these agents or their unfavourable pharmacokinetic profiles. In principle, radiolabelled monoclonal antibodies could be considered for the selective targeting and imaging of individual MMPs. Methods: We cloned, produced and characterized high-affinity monoclonal antibodies specific to murine MMP-1A, MMP-2 and MMP-3 in SIP (small immunoprotein) miniantibody format using biochemical and immunochemical methods. We also performed comparative biodistribution analysis of their tumour-targeting properties at three time points (3h, 24h, 48h) in mice bearing subcutaneous F9 tumours using radioiodinated protein preparations. The clinical stage L19 antibody, specific to the alternatively spliced EDB domain of fibronectin, was used as reference tumour-targeting agent for in vivo studies. Results: All anti-MMP antibodies and SIP(L19) strongly stained sections of F9 tumours when assessed by immunofluorescence methods. In biodistribution experiments, SIP(SP3), specific to MMP-3, selectively accumulated at the tumour site 24 and 48h after intravenous injection, but was rapidly cleared from other organs. By contrast, SIP(SP1) and SIP(SP2), specific to MMP-1A and MMP-2, showed no preferential accumulation at the tumour site. Conclusion: Antibodies specific to MMP-3 may serve as vehicles for the efficient and selective delivery of imaging agents or therapeutic molecules to sites of diseas

Expression, engineering and characterization of the tumor-targeting heterodimeric immunocytokine F8-IL12

Proinflammatory cytokines have been used for several years in patients with advanced cancer but their administration is typically associated with severe toxicity hampering their application to therapeutically active regimens. This problem can be overcome by using immunocytokines (cytokines fused to antibody or antibody fragments) which selectively deliver the active cytokine to the tumor environment. Preclinical and recent clinical results confirmed that this approach is a very promising avenue to go. We designed an immunocytokine consisting of the scFv(F8) specific to extra-domain A of fibronectin and the very potent human cytokine interleukin-12 (IL12). The heterodimeric nature of IL12 allows the engineering of various immunocytokine formats, based on different combinations of the two subunits (p35 and p40) together with the scFv. In comparison to monomeric or homodimeric cytokines, the construction of a heterodimeric immunocytokine poses many challenges, e.g. gene dosing, stable high-yield expression as well as good manufacture practice (GMP) purification and characterization. In this paper, we describe the successful construction, characterization and production of the heterodimeric immunocytokine F8-IL12. The positive outcome of this feasibility study leads now to GMP production of F8-IL12, which will soon enter clinical trial

MALDI-TOF and nESI Orbitrap MS/MS identify orthogonal parts of the phosphoproteome

Phosphorylation is a reversible posttranslational protein modification which plays a pivotal role in intracellular signaling. Despite extensive efforts, phosphorylation site mapping of proteomes is still incomplete motivating the exploration of alternative methods that complement existing workflows. In this study, we compared tandem mass spectrometry (MS/MS) on matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) and nano-electrospray ionization (nESI) Orbitrap instruments with respect to their ability to identify phosphopeptides from complex proteome digests. Phosphopeptides were enriched from tryptic digests of cell lines using Fe-IMAC column chromatography and subjected to LC-MS/MS analysis. We found that the two analytical workflows exhibited considerable orthogonality. For instance, MALDI-TOF MS/MS favored the identification of phosphopeptides encompassing clear motif signatures for acidic residue directed kinases. The extent of orthogonality of the two LC-MS/MS systems was comparable to that of using alternative proteases such as Asp-N, Arg-C, chymotrypsin, Glu-C and Lys-C on just one LC-MS/MS instrument. Notably, MALDI-TOF MS/MS identified an unexpectedly high number and percentage of phosphotyrosine sites (∼20% of all sites), possibly as a direct consequence of more efficient ionization. The data clearly show that LC-MALDI MS/MS can be a useful complement to LC-nESI MS/MS for phosphoproteome mapping and particularly so for acidic and phosphotyrosine containing peptides

Tumour-targeting properties of antibodies specific to MMP-1A, MMP-2 and MMP-3

ISSN:1619-7070ISSN:1619-708

Expression, engineering and characterization of the tumor-targeting heterodimeric immunocytokine F8-IL12

ISSN:1741-0126ISSN:1741-0134ISSN:0269-2139ISSN:1460-213

Using enhanced development tools offered by analytical Quality by Design to support switching of an analytical quality control method

Quality by Design (QbD) principles play an increasingly important role in pharmaceutical industry. Here, we used an analytical QbD (AQbD) approach to develop a capillary electrophoresis method under reducing conditions (rCE-SDS), with the aim of replacing SDS-PAGE as release and stability test method for a commercialized monoclonal antibody product. Method development started with defining analytical method performance requirements as part of an analytical target profile (ATP), followed by a systematic risk assessment of method input parameters and their relation to defined method outputs. Based on this, design of experiments (DoE) studies were performed to identify a method operable design region (MODR). The MODR could be leveraged to improve method robustness. In a bridging study, it was demonstrated that the rCE-SDS method is more sensitive than the legacy SDS-PAGE method, and a correlation factor could be established to compensate for an off-set due to the higher sensitivity, without losing the correlation to the historical data acquired with the former method. Overall, systematic application of AQbD principles for designing and developing a new analytical method helped to elucidate the complex dependency of method outputs on its input parameters. The link of the method to critical quality attributes and the definition of method performance requirements were found to be most relevant for de-risking the analytical method switch, regarding impact on the control strategy

Expression, engineering and characterization of the tumor-targeting heterodimeric immunocytokine F8-IL12

ISSN:1741-0126ISSN:1741-0134ISSN:0269-2139ISSN:1460-213