Better by design: What to expect from novel CAR-engineered cell therapies?

Chimeric antigen receptor (CAR) technology, and CAR-T cells in particular, have emerged as a new and powerful tool in cancer immunotherapy since demonstrating efficacy against several hematological malignancies. However, despite encouraging clinical results of CAR-T cell therapy products, a significant proportion of patients do not achieve satisfactory responses, or relapse. In addition, CAR-T cell applications to solid tumors is still limited due to the tumor microenvironment and lack of specifically targetable tumor antigens. All current products on the market, as well as most investigational CAR-T cell therapies, are autologous, using the patient's own peripheral blood mononuclear cells as starting material to manufacture a patient-specific batch. Alternative cell sources are, therefore, under investigation (e.g. allogeneic cells from an at least partially human leukocyte antigen (HLA)-matched healthy donor, universal "third-party" cells from a non-HLA-matched donor, cord blood-derived cells, immortalized cell lines or cells differentiated from induced pluripotent stem cells). However, genetic modifications of CAR-engineered cells, bioprocesses used to expand cells, and improved supply chains are still complex and costly. To overcome drawbacks associated with CAR-T technologies, novel CAR designs have been used to genetically engineer cells derived from alpha beta (αß) T cells, other immune cells such as natural killer (NK) cells, gamma delta (γδ) T cells, macrophages or dendritic cells. This review endeavours to trigger ideas on the next generation of CAR-engineered cell therapies beyond CAR-T cells and, thus, will enable effective, safe and affordable therapies for clinical management of cancer. To achieve this, we present a multidisciplinary overview, addressing a wide range of critical aspects: CAR design, development and manufacturing technologies, pharmacological concepts and clinical applications of CAR-engineered cell therapies. Each of these fields employs a large number of ground-breaking scientific advances, where coordinated and complex process and product development occur at their interfaces.

Intracellular drug delivery: Potential usefulness of engineered Shiga toxin subunit B for targeted cancer therapy.

A treasure trove of intracellular cancer drug targets remains hidden behind cell membranes. However, engineered pathogen-derived toxins such as Shiga toxins can deliver small or macromolecular drugs to specific intracellular organelles. After binding to ganglioglobotriaosylceramide (Gb3, CD77), the non-toxic subunit B (StxB) of the Shiga-holotoxin is endocytosed and delivers its payload by a unique retrograde trafficking pathway via the endoplasmic reticulum to the cytosol. This review provides an overview of biomedical applications of StxB-based drug delivery systems in targeted cancer diagnosis and therapy. Biotechnological production of the Stx-material is discussed from the perspective of developing efficacious and safe therapeutics.

In vitro and in vivo investigation of bisphosphonate-loaded hydroxyapatite particles for peri-implant bone augmentation.

Locally applied bisphosphonates, such as zoledronate, have been shown in several studies to inhibit peri-implant bone resorption and recently to enhance peri-implant bone formation. Studies have also demonstrated positive effects of hydroxyapatite (HA) particles on peri-implant bone regeneration and an enhancement of the anti-resorptive effect of bisphosphonates in the presence of calcium. In the present study, both hydroxyapatite nanoparticles (nHA) and zoledronate were combined to achieve a strong reinforcing effect on peri-implant bone. The nHA-zoledronate combination was first investigated in vitro with a pre-osteoclastic cell assay (RAW 264.7) and then in vivo in a rat model of postmenopausal osteoporosis. The in vitro study confirmed that the inhibitory effect of zoledronate on murine osteoclast precursor cells was enhanced by loading the drug on nHA. For the in vivo investigation, either zoledronate-loaded or pure nHA were integrated in hyaluronic acid hydrogel. The gels were injected in screw holes that had been predrilled in rat femoral condyles before the insertion of miniature screws. Micro-CT-based dynamic histomorphometry and histology revealed an unexpected rapid mineralization of the hydrogel in vivo through formation of granules, which served as scaffold for new bone formation. The delivery of zoledronate-loaded nHA further inhibited a degradation of the mineralized hydrogel as well as a resorption of the peri-implant bone as effectively as unbound zoledronate. Hyaluronic acid with zoledronate-loaded nHA, thanks to its dual effect on inducing a rapid mineralization and preventing resorption, is a promising versatile material for bone repair and augmentation. Copyright © 2015 John Wiley & Sons, Ltd.

(Metallo)porphyrins as potent phototoxic anti-cancer agents after irradiation with red light.

Novel photoactive (metallo)porphyrins were synthesised and characterised. When irradiated with light at a wavelength greater than 600 nm, these porphyrins act as photosensitisers and show high cytotoxicity towards two different human cancer cell lines with IC50 values down to 0.4 µM. A paramagnetic copper(II) porphyrin is the first photosensitiser to display excellent phototoxicity, explained by the electron paramagnetic resonance (EPR) spin trapping of hydroxy radicals and experimentally confirmed by the discovery of elevated levels of reactive oxygen species (ROS) inside A2780 cells after irradiation with red light. This finding indicates that paramagnetic compounds should be considered for photodynamic therapy (PDT). Furthermore, an additive effect of cisplatin and a zinc porphyrin, both at subtherapeutic concentrations of 0.22 µM, was observed.

Physiological oxygen concentration alters glioma cell malignancy and responsiveness to photodynamic therapy in vitro.

OBJECTIVES: The partial pressure of oxygen (pO2) in brain tumors ranges from 5 to 15%. Nevertheless, the majority of in vitro experiments with glioblastoma multiforme (GBM) cell lines are carried out under an atmospheric pO2 of 19 to 21%. Recently, 5-aminolevulinic acid (5-ALA), a precursor of protoporphyrin IX (PpIX), has been introduced to neurosurgery to allow for photodynamic diagnosis and photodynamic therapy (PDT) in high-grade gliomas. Here, we investigate whether low pO2 affects GBM cell physiology, PpIX accumulation, or PDT efficacy. METHODS: GBM cell lines (U-87 MG and U-251 MG) were cultured under atmospheric (pO2  =  19%) and physiological (pO2  =  9%) oxygen concentrations. PpIX accumulation and localization were investigated, and cell survival and cell death were observed following in vitro PDT. RESULTS: A physiological pO2 of 9% stimulated GBM cell migration, increased hypoxia-inducible factor (HIF)-1 alpha levels, and elevated resistance to camptothecin in U-87 MG cells compared to cultivation at a pO2 of 19%. This oxygen reduction did not alter 5-ALA-induced intracellular PpIX accumulation. However, physiological pO2 changed the responsiveness of U-87 MG but not of U-251 MG cells to in vitro PDT. Around 20% more irradiation light was required to kill U-87 MG cells at physiological pO2, resulting in reduced lactate dehydrogenase (LDH) release (one- to two-fold) and inhibition of caspase 3 activation. DISCUSSION: Reduction of oxygen concentration from atmospheric to a more physiological level can influence the malignant behavior and survival of GBM cell lines after in vitro PDT. Therefore, precise oxygen concentration control should be considered when designing and performing experiments with GBM cells.

Impact of IGF-I release kinetics on bone healing: a preliminary study in sheep.

Spatiotemporal release of growth factors from a delivery device can profoundly affect the efficacy of bone growth induction. Here, we report on a delivery platform based on the encapsulation of insulin-like growth factor I (IGF-I) in different poly(D,L-lactide) (PLA) and poly(D,L-lactide-co-glycolide) (PLGA) microsphere (MS) formulations to control IGF-I release kinetics. In vitro IGF-I release profiles generally exhibited an initial burst (14-36% of total IGF-I content), which was followed by a more or less pronounced dormant phase with little release (2 to 34 days), and finally, a third phase of re-increased IGF-I release. The osteoinductive potential of these different IGF-I PL(G)A MS formulations was tested in studies using 8-mm metaphyseal drill hole bone defects in sheep. Histomorphometric analysis at 3 and 6 weeks after surgery showed that new bone formation was improved in the defects locally treated with IGF-I PL(G)A MS (n=5) as compared to defects filled with IGF-I-free PL(G)A MS (n=4). The extent of new bone formation was affected by the particular release kinetics, although a definitive relationship was not evident. Local administration of IGF-I resulted in down-regulation of inflammatory marker genes in all IGF-I treated defects. The over-expression of growth factor genes in response to IGF-I delivery was restricted to formulations that produced osteogenic responses. These experiments demonstrate the osteoinductive potential of sustained IGF-I delivery and show the importance of delivery kinetics for successful IGF-I-based therapies.

Phenytoin reduces 5-aminolevulinic acid-induced protoporphyrin IX accumulation in malignant glioma cells.

Epileptic seizures are among the presenting clinical signs of malignant glioma patients, frequently necessitating treatment with antiepileptic drugs (AEDs). The efficacy of 5-aminolevulinic acid (5-ALA)-based intraoperative fluorescence-guided surgery and photodynamic therapy (PDT) in glioblastoma multiforme (GBM) patients depends on the specific accumulation and total amount of intracellularly synthesized protoporphyrin IX (PpIX) in tumour cells. In this study, we investigated the effect of the AEDs phenytoin (PHY) and levetiracetam (LEVE) on 5-ALA-induced PpIX accumulation in two glioma cell lines (U373 MG and U-87 MG) and primary GBM cells isolated from a human biopsy. After treatment with PHY and LEVE for three days cells were incubated with 1 mM: 5-ALA for 4 h and PpIX accumulation was determined by fluorescence measurement. We observed a decrease in PpIX synthesis of up to 55 ± 12 % in primary GBM cells after incubation with phenytoin. This reduction was dose-dependent for all tested cell lines and primary GBM cells. LEVE on the other hand did not alter PpIX concentration in GBM cells. PDT was performed in vitro by irradiating the GBM cells with light doses from 0.5 to 10 J cm(-2) at 627 nm after AED and 5-ALA treatment. Although less PpIX accumulated in PHY-treated cells, efficacy of PDT was not affected. We assume that damage to the mitochondrial membrane by PHY inhibits PpIX synthesis in vitro, because we showed mitochondrial dysfunction as a result of reduced mitochondrial membrane potential in PHY-treated cells. No change in glutathione status was observed. To evaluate further the effect of PHY on PpIX fluorescence, and to establish its significance in clinical practice, animal and clinical studies are required, because the results presented here imply PHY may reduce intracellular accumulation of PpIX in patients with high-grade gliomas.

Susceptibility to 5-aminolevulinic acid based photodynamic therapy in WHO I meningioma cells corresponds to ferrochelatase activity.

5-Aminolevulinic acid (5-ALA) is a natural precursor of protoporphyrin IX (PpIX), which can be used as a photosensitizer in photodynamic therapy (PDT). Accumulation of PpIX in benign meningioma cells has been observed previously, its exploitation for PDT, however, was discouraged by inconsistent results. To evaluate PDT for benign meningiomas, we investigated PpIX synthesis in two human meningioma cell lines (HBL-52 and BEN-MEN-1), their respective extracellular loss of PpIX and corresponding ferrochelatase (FECH) activity as well as their susceptibility to PDT. We demonstrated PpIX production after 5-ALA administration and minor loss to the extracellular space in both cell lines. However, significantly more (up to five times) PpIX was accumulated in BEN-MEN-1 as compared with HBL-52 cells. FECH activity was 2.7-fold higher in HBL-52 compared with BEN-MEN-1 cells and accordingly higher FECH levels could be shown in HBL-52 cells by Western blot analysis. BEN-MEN-1 cells were much more sensitive to PDT and cells could be almost completely killed by irradiation doses of 2 J cm(-2) , whereas HBL-52 showed only an insufficient response at this irradiation dose. We conclude that differences in intracellular PpIX concentrations between HBL-52 and BEN-MEN-1 benign meningioma cells were mainly due to differences in FECH activity and that these differences correspond to their susceptibility to 5-ALA-induced PDT.

Preparing Poly (Lactic-co-Glycolic Acid) (PLGA) Microspheres Containing Lysozyme-Zinc Precipitate Using a Modified Double Emulsion Method.

Lysozyme, as a model protein, was precipitated through the formation of protein-Zn complex to micronize for subsequent encapsulation within poly (lactic-co-glycolic acid) (PLGA) microspheres. Various parameters, including pH, type and concentration of added salts and protein concentration, were modified to optimize the yield of protein complexation and precipitation. The resulting protein particles (lysozyme-Zn complex as a freshly prepared suspension or a freeze-dried solid) were then loaded into PLGA (Resomer(®) 503H) microspheres, using a double emulsion technique and microspheres encapsulation efficiency and their sizes were determined. It was observed that salt type could significantly influence the magnitude of protein complexation. At the same conditions, zinc chloride was found to be more successful in producing pelletizable lysozyme. Generally, higher concentrations of protein solution led also to the higher yields of complexation and at the optimum conditions, the percentage of pelletizable lysozyme reached to 80%. Taking advantage of this procedure, a modified technique for preparation of protein-loaded PLGA microspheres was established, although it is also expected that this technique increases the protein drugs stabilization during the encapsulation process.

Controlled release of tetracycline from biodegradable beta-tricalcium phosphate composites.

For use in the prevention of bone infections, a novel controlled release system composed of beta-tricalcium phosphate (TCP) granules with biodegradable coatings incorporating the antibiotic drug tetracycline (TC) was developed. Six formulations using poly(D,L-lactide) and poly(D,L-lactide-co-glycolide) as coating materials to incorporate TC were prepared and tested in vitro and in vivo. Release of TC from TCP composites was dependent on the biodegradability of the used polymers and on physical-chemical interactions of TC with the polymer materials. Three characteristic release profiles were obtained: slow release lasting up to 67 days, intermediate release with 60% of the total dose released up to day 20, and fast release with a high initial burst and 90% of TC released within 4 days. Even though TC decomposition products had formed during in vitro release, no cytotoxic effects on osteoblast-like cells were observed. The biological activity of TC after incorporation into PL(G)A films was confirmed using a TC-repressible promoter system in genetically engineered Chinese Hamster Ovary (CHO) cells. TC-loaded TCP composites implanted in ovine cancellous bone defects showed good biocompatibility and new bone formation in the histological evaluation. No differences in the cellular reactions were seen between antibiotic-loaded composites and the control group. These experimental results indicate the potential of coated TCP composites to be used as local carrier system for controlled TC delivery with different release kinetics and good in vitro and in vivo biocompatibility.

Insulin-like growth factor I-releasing alginate-tricalciumphosphate composites for bone regeneration.

PURPOSE: Development and characterization of an in situ-forming, osteoconductive, and growth factor-releasing bone implant. METHODS: Injectable in situ-forming scaffolds were prepared from a 2% (m/v) alginate solution, tricalciumphosphate (TCP) granules, and poly(lactide-co-glycolide) microspheres (MS), loaded with the osteoinductive growth factor insulin-like growth factor I (IGF-I). Scaffolds were prepared by mixing the components followed by hydrogel formation through calcium carbonate-induced physical cross-linking of the alginate at slightly acidic pH. Physical-chemical properties and cell biocompatibility using osteoblast-like cells (MG-63 and Saos-2) of these scaffolds were investigated. RESULTS: The addition of TCP to the alginate resulted in reduced swelling and gelation time and an increase in stiffness. Osteoblast-like cells (MG-63 and Saos-2) did not show toxic reactions and adhered circumferentially to the TCP granules surface. The addition of the IGF-I MS resulted in an up to sevenfold increased proliferation rate of MG-63 cells as compared to scaffold preparations without IGF-I MS. The alkaline phosphate (ALP) activity-a parameter for osteblastic activity-increased with increasing amounts of TCP in Saos-2 loaded composite scaffolds. CONCLUSIONS: A prototype in situ-hardening composite system for conformal filling of bone defects supporting osteoblastic activity for further clinical testing in relevant fracture models was developed and characterized.

Localized delivery of growth factors for bone repair.

Delivery of growth factors for tissue (e.g. bone, cartilage) or cell repair (e.g. nerves) is about to gain important potential as a future therapeutic tool. Depending on the targeted cell type and its state of differentiation, growth factors can activate or regulate a variety of cellular functions. Therefore, strictly localized delivery regimens at well-defined kinetics appear to be logical prerequisites to assure safe and efficacious therapeutic use of such factors and avoid unwanted side effects and toxicity, a major hurdle in the clinical development of growth factor therapies so far. This review summarizes various approaches for localized growth factor delivery as focused on bone repair. Similar considerations may apply to other growth factors and therapeutic indications. Considering the vast number of preclinical studies reported in the area of growth factor-assisted bone repair, it surprises though that only two medical products for bone repair have so far been commercialized, both consisting of a collagen matrix impregnated with a bone morphogenetic protein. The marked diversity of the reported growth factors, delivery concepts and not yet standardized animal models adds to the complexity to learn from past preclinical studies presented in the literature. Nonetheless, it is now firmly established from the available information that the type, dose and delivery kinetics of growth factors all play a decisive role for the therapeutic success of any such approach. Very likely, all of these parameters have to be adapted and optimized for each animal model or clinical case. In the future, systems for localized growth factor delivery thus need to be designed in such a way that their modular components are readily adaptable to the individual pathology. To make such customized systems feasible, close cooperative networks of biomedical and biomaterials engineers, pharmaceutical scientists, chemists, biologists and clinicians need to be established.

Localized insulin-like growth factor I delivery to enhance new bone formation.

Insulin-like growth factor I (IGF I) exerts an important role during skeletal growth and bone formation. Therefore, its localized delivery appears attractive for the treatment of bone defects. To prolong IGF I delivery, we entrapped the protein into biodegradable poly(lactide-co-glycolide) microspheres (PLGA MS) and evaluated the potential of this delivery system for new bone formation in two defect models of ovine long bones, i.e., a 8-mm methaphyseal drill hole and a 10-mm segmental tibia defect. Administration of 100 microg of IGF I in PLGA MS resulted in new bone formation within 3 weeks in the drill hole and bridging of the segmental defect within 8 weeks. The observed increase of 12% newly formed bone in the drill hole defect after 3 weeks was substantial, compared to the measured morphometric bone-to-total area ratio of 31% bone in normal cancellous bone. Bone regeneration was further explored by measuring gene expression of typical markers for local mediators and growth factors by real-time polymerase chain reaction. Inflammation was reduced in presence of IGF I and this in vivo observation was corroborated in vitro by quantifying gene expression of inflammatory proteins and by assessing the activation of the NF-kappaB pathway, playing an important role in the regulation of inflammation. Administration of the IGF I delivery system downregulated inflammatory marker gene expression at the site of bone injury, induced new bone formation and reduced bone resorption, and resulted in bridging of 10-mm segmental tibial defects within 8 weeks.