Antibody-Drug Conjugates for Tumor Targeting-Novel Conjugation Chemistries and the Promise of non-IgG Binding Proteins

Antibody-drug conjugates (ADCs) have emerged as a promising class of anticancer agents, combining the specificity of antibodies for tumor targeting and the destructive potential of highly potent drugs as payload. An essential component of these immunoconjugates is a bifunctional linker capable of reacting with the antibody and the payload to assemble a functional entity. Linker design is fundamental, as it must provide high stability in the circulation to prevent premature drug release, but be capable of releasing the active drug inside the target cell upon receptor-mediated endocytosis. Although ADCs have demonstrated an increased therapeutic window, compared to conventional chemotherapy in recent clinical trials, therapeutic success rates are still far from optimal. To explore other regimes of half-life variation and drug conjugation stoichiometries, it is necessary to investigate additional binding proteins which offer access to a wide range of formats, all with molecularly defined drug conjugation. Here, we delineate recent progress with site-specific and biorthogonal conjugation chemistries, and discuss alternative, biophysically more stable protein scaffolds like Designed Ankyrin Repeat Proteins (DARPins), which may provide such additional engineering opportunities for drug conjugates with improved pharmacological performance.

A combined biomaterial and cellular approach for annulus fibrosus rupture repair

Recurrent intervertebral disc (IVD) herniation and degenerative disc disease have been identified as the most important factors contributing to persistent pain and disability after surgical discectomy. An annulus fibrosus (AF) closure device that provides immediate closure of the AF rupture, restores disc height, reduces further disc degeneration and enhances self-repair capacities is an unmet clinical need. In this study, a poly(trimethylene carbonate) (PTMC) scaffold seeded with human bone marrow derived mesenchymal stromal cells (MSCs) and covered with a poly(ester-urethane) (PU) membrane was assessed for AF rupture repair in a bovine organ culture annulotomy model under dynamic load for 14 days. PTMC scaffolds combined with the sutured PU membrane restored disc height of annulotomized discs and prevented herniation of nucleus pulposus (NP) tissue. Implanted MSCs showed an up-regulated gene expression of type V collagen, a potential AF marker, indicating in situ differentiation capability. Furthermore, MSCs delivered within PTMC scaffolds induced an up-regulation of anabolic gene expression and down-regulation of catabolic gene expression in adjacent native disc tissue. In conclusion, the combined biomaterial and cellular approach has the potential to hinder herniation of NP tissue, stabilize disc height, and positively modulate cell phenotype of native disc tissue.

Non-Viral Gene Therapy to Cells of the bovine Intervertebral Disc

Background. Low back pain is an increasing global health problem, which is associated with intervertebral disc (IVD) damage and degeneration. Major changes occur in the nucleus pulposus (NP), with the degradation of the extracellular matrix (ECM).1 Further studies showed that growth factors from transforming growth factor β (TGFβ) and bone morphogenic proteins (BMP) family may induce chondrogenic differentiation of mesenchymal stem cells (MSC).2 Focusing on non-viral gene therapies and their possible translation into the clinics, we investigated if GDF6 (syn. BMP13 or CDMP2) can induce regeneration of degraded NP. We hypothesized that IVD transfected with plasmid over-expressing GDF6 also up-regulates other NP- and chondrogenic cell markers and enhances ECM deposition. Methods. Bovine nucleus pulposus (bNPC) and annulus fibrosus cells (bAFC) were harvested from bovine coccygeal IVD. Primary cells were then electroporized with plasmid GDF6 (Origene, vector RG211366) by optimizing parameters using the Neon Transfection system (Life Technologies, Basel). After transfection, cells were cultured in 2D monolayer or 3D alginate beads for 7, 14 or 21 days. Transfection efficiency of pGDF6 was analyzed by immunohistochemistry and fluorescent microscopy. Cell phenotype was quantified by real-time RT-PCR. To test a non-viral gene therapy applied directly to 3D whole organ culture, coccygeal bovine IVDs were harvested as previously described. Bovine IVDs were transfected by injection of plasmid GDF6 into the center. Electroporation was performed with ECM830 Square Wave Electroporation System (Harvard Apparatus, MA) using 2-needle array electrode or tweezertrodes. 72 h after tranfection discs were fixed and cryosectioned and analyzed by immunofluorescence against GDF6. Results. RT-PCR and immunohistochemistry confirmed up-regulation of GFP and GDF6 in the primary bNPC/bAFC culture. The GFP-tagged GDF6 protein, however, was not visible, possibly due to failure of dimer formation as a result of fusion structure. Organ IVD culture transfection revealed GDF6 positive staining in the center of the disc using 2-needle array electrode. Results from tweezertrodes did not show any GDF6 positive cells. Conclusion. Non-viral transfection is an appealing approach for gene therapy as it fulfills the translational safety aspects of transiency and lacks the toxic effects of viral transduction. We identified novel parameters to successfully transfect primary bovine IVD cells. For transfection of whole IVD explants electroporation parameters need to be further optimized. Acknowledgements. This project was funded by the Lindenhof Foundation (Funds “Research & Teaching”) Project no. 13-02-F. The imaging part of this study was performed with the facility of the Microscopy Imaging Center (MIC), University of Bern. References. Roughly PJ (2004): Spine (Phila), 29:2691-2699 Clarke LE, McConell JC, Sherratt MJ, Derby B, Richardson SM, Hoyland JA (2014), Arthritis Research & Therapy, 16:R67

A synthetic biology-based device prevents liver injury in mice

BACKGROUND & AIMS The liver performs a panoply of complex activities coordinating metabolic, immunologic and detoxification processes. Despite the liver's robustness and unique self-regeneration capacity, viral infection, autoimmune disorders, fatty liver disease, alcohol abuse and drug-induced hepatotoxicity contribute to the increasing prevalence of liver failure. Liver injuries impair the clearance of bile acids from the hepatic portal vein which leads to their spill over into the peripheral circulation where they activate the G-protein-coupled bile acid receptor TGR5 to initiate a variety of hepatoprotective processes. METHODS By functionally linking activation of ectopically expressed TGR5 to an artificial promoter controlling transcription of the hepatocyte growth factor (HGF), we created a closed-loop synthetic signalling network that coordinated liver injury-associated serum bile acid levels to expression of HGF in a self-sufficient, reversible and dose-dependent manner. RESULTS After implantation of genetically engineered human cells inside auto-vascularizing, immunoprotective and clinically validated alginate-poly-(L-lysine)-alginate beads into mice, the liver-protection device detected pathologic serum bile acid levels and produced therapeutic HGF levels that protected the animals from acute drug-induced liver failure. CONCLUSIONS Genetically engineered cells containing theranostic gene circuits that dynamically interface with host metabolism may provide novel opportunities for preventive, acute and chronic healthcare. LAY SUMMARY Liver diseases leading to organ failure may go unnoticed as they do not trigger any symptoms or significant discomfort. We have designed a synthetic gene circuit that senses excessive bile acid levels associated with liver injuries and automatically produces a therapeutic protein in response. When integrated into mammalian cells and implanted into mice, the circuit detects the onset of liver injuries and coordinates the production of a protein pharmaceutical which prevents liver damage.