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Physiological and functional consequences of BTK‐inactivation by αCD20‐mAB‐protamine/free protamine‐ibrutinib‐Cy3.5 treatment in DLBCL cell lines. A: HBL1 cells were treated by the respective conjugates shown for 72 hrs, lysed and subjected to SDS‐PAGE and Western blotting for phospho‐BTK (pBTK), total BTK (tBTK), phospho‐ERK (p‐ERK), total‐ERK (t‐ERK) and actin as a loading control. Here, untargeted ibrutinib‐Cy3.5 inhibited the phosphorylation of BTK a bit less than αCD20‐mAB‐P/P‐ibrutinib‐Cy3.5, the difference of expected downstream phosphorylation targets such as ERK was more pronounced: Here, only αCD20‐mAB‐P/P‐ibrutinib‐Cy3.5 treatment was able to reduce ERK phosphorylation. B: In colony formation assays, untargeted ibrutinib‐Cy3.5 modestly reduced colony growth of HBL1 cells, while the specific targeting of ibrutinib‐Cy3.5 by αCD20‐mAB‐P/P boosted the colony growth reduction to below 50 %. In order to demonstrate the significance of the free protamine in the conjugate construct, we depleted it from the conjugate mixture, the application of this combination revealed no more colony forming reduction than the single application of ibrutinib‐Cy3.5, so the antibody conjugate has lost its targeting ability (B, rightmost bar). α, anti.
Source publication
Ibrutinib is an inhibitor of Bruton's tyrosine kinase that has been approved for the treatment of patients with chronic lymphocytic leukemia, mantle cell lymphoma and Waldenstrom's macroglobulinemia and is connected with toxicities. To minimize its toxicities, we linked ibrutinib to a cell‐targeted, internalizing antibody. To this end, we synthesiz...
Citations
... Blocking macrophage molecular mechanisms, boosting macrophage signaling pathways, or combinations of these may prove fruitful and provide opportunities to relieve the paucity of proven effective treatments [137] and meet the clinical needs of CTD-ILD. The literature shows that aiming at specific cell types is a viable choice for several disease entities [138][139][140], especially CTDs [141,142]. Several systemic reviews also suggest the therapeutic potential of targeting specific cells in CTD-ILD [143,144]. ...
Connective tissue disease-associated interstitial lung disease (CTD-ILD) is a severe manifestation of CTD that leads to significant morbidity and mortality. Clinically, ILD can occur in diverse CTDs. Pathologically, CTD-ILD is characterized by various histologic patterns, such as nonspecific interstitial pneumonia, organizing pneumonia, and usual interstitial pneumonia. Abnormal immune system responses have traditionally been instrumental in its pathophysiology, and various changes in immune cells have been described, especially in macrophages. This article first briefly overviews the epidemiology, clinical characteristics, impacts, and histopathologic changes associated with CTD-ILD. Next, it summarizes the roles of various signaling pathways in macrophages or products of macrophages in ILD, helped by insights gained from animal models. In the following sections, this review returns to studies of macrophages in CTD-ILD in humans for an overall picture of the current understanding. Finally, we direct attention to potential therapies targeting macrophages in CTD-ILD in investigation or in clinical trials, as well as the future directions regarding macrophages in the context of CTD-ILD. Although the field of macrophages in CTD-ILD is still in its infancy, several lines of evidence suggest the potential of this area.
... Here, we introduce a modular adjustable antibody-protamine nanocarrier platform, which can electrostatically bind anionic components and transport them via target-cell specific receptor-internalization into leukemia cells. The anionic components can be oncogene-specific small interfering (si)RNAs [3][4][5] or anionic small molecule inhibitors [6]. The use of RNA interference (RNAi) has been proposed for many cancers, but so far no RNAi-based therapeutic is approved for oncological diseases. ...
... For the targeted treatment of Diffuse-Large B-Cell Lymphoma (DLBCL), we established a targeted chemically modified ibrutinib, ibrutinib-Cy3.5, which was incorporated into a nanocarrier specifically internalizing in CD20-positive cells [6]. Ibrutinib is a covalent binder of BTK, which also has been proposed to be a relevant drug target in AML [23]. ...
... Anionic charged ibrutinib-Cy3.5 was synthesized as published recently [6] and complexed in 20 times molar excess to αCD33-mAB-P/P, if not stated otherwise, for ibrutinib at room temperature in the dark for 30-60 min. ...
Background
Acute myeloid leukemia (AML) is a fatal clonal hematopoietic malignancy, which results from the accumulation of several genetic aberrations in myeloid progenitor cells, with a worldwide 5-year survival prognosis of about 30%. Therefore, the development of more effective therapeutics with novel mode of action is urgently demanded. One common mutated gene in the AML is the DNA-methyltransferase DNMT3A whose function in the development and maintenance of AML is still unclear. To specifically target “undruggable” oncogenes, we initially invented an RNAi-based targeted therapy option that uses the internalization capacity of a colorectal cancer specific anti-EGFR-antibody bound to cationic protamine and the anionic siRNA. Here, we present a new experimental platform technology of molecular oncogene targeting in AML.
Methods
Our AML-targeting system consists of an internalizing anti-CD33-antibody–protamine conjugate, which together with anionic molecules such as siRNA or ibrutinib-Cy3.5 and cationic free protamine spontaneously assembles into vesicular nanocarriers in aqueous solution. These nanocarriers were analyzed concerning their physical properties and relevant characteristics in vitro in cell lines and in vivo in xenograft tumor models and patient-derived xenograft leukemia models with the aim to prepare them for translation into clinical application.
Results
The nanocarriers formed depend on a balanced electrostatic combination of the positively charged cationic protamine-conjugated anti-CD33 antibody, unbound cationic protamine and the anionic cargo. This nanocarrier transports its cargo safely into the AML target cells and has therapeutic activity against AML in vitro and in vivo. siRNAs directed specifically against two common mutated genes in the AML, the DNA-methyltransferase DNMT3A and FLT3-ITD lead to a reduction of clonal growth in vitro in AML cell lines and inhibit tumor growth in vivo in xenotransplanted cell lines. Moreover, oncogene knockdown of DNMT3A leads to increased survival of mice carrying leukemia patient-derived xenografts. Furthermore, an anionic derivative of the approved Bruton’s kinase (BTK) inhibitor ibrutinib, ibrutinib-Cy3.5, is also transported by this nanocarrier into AML cells and decreases colony formation.
Conclusions
We report important results toward innovative personalized, targeted treatment options via electrostatic nanocarrier therapy in AML.
The CRISPR‐Cas9 system has revolutionized the field of genetic engineering, but targeted cellular delivery remains a central problem. The delivery of the preformed ribonuclease‐protein (RNP) complex has the advantages of fewer side effects and avoidance of potential permanent effects. We reasoned that an internalizing IgG antibody as a targeting device could address the delivery of Cas9‐RNP. We opted for protein trans ‐splicing mediated by a split intein to facilitate posttranslational conjugation of the two large protein entities. We recently described the cysteine‐less CL split intein that efficiently performs under oxidizing conditions and does not interfere with disulfide bonds or thiol bioconjugation chemistries. Using the CL split intein, we report for the first time the ligation of monoclonal IgG antibody precursors, expressed in mammalian cells, and a Cas9 precursor, obtained from bacterial expression. A purified IgG‐Cas9 conjugate was loaded with sgRNA to form the active RNP complex and introduced a double‐strand break in its target DNA in vitro. Furthermore, a synthetic peptide variant of the short N‐terminal split intein precursor proved useful for chemical modification of Cas9. The split intein ligation procedure reported here for IgG‐Cas9 provides the first step towards a novel CRISPR‐Cas9 targeting approach involving the preformed RNP complex.
