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.

Scale-up manufacturing of gelatin-based microcarriers for cell therapy.

Microcarriers, including crosslinked porous gelatin beads (Cultispher G) are widely used as cell carriers for cell therapy applications. Microcarriers can support a range of adherent cell types in stirred tank bioreactor culture, which is scalable up to several thousands of liters. Cultispher G in particular is advantageous for cell therapy applications because it can be dissolved enzymatically, and thus cells can be harvested without the need to perform a large-scale cell-bead filtration step. This enzymatic dissolution, however, is challenged by the slow degradation of the carriers in the presence of enzymes as new extracellular matrix is being deposited by the proliferating cells. This extended dissolution timelimits the yield of cell recovery while compromising cellular viability. We report herein the development of crosslinked porous gelatin beads that afford rapid, stimuli-triggered dissolution for facile cell removal using human mesenchymal stem cells (hMSC) as a model system. We successfully fabricated redox-sensitive beads (RS beads) and studied their cell growth, dissolution time and cell yield, compared to regular gelatin-based beads (Reg beads). We have shown that RS beads allow for much faster dissolution compared to Reg beads, supporting better hMSC detachment and recovery following 8 days of culture in spinner flasks, or in 3L bioreactors. These newly synthesized RS beads show promise as cellular microcarriers and can be used for scale-up manufacturing of different cell types while providing on-demand degradation for facile cell retrieval.

End-to-End Platform for Human Pluripotent Stem Cell Manufacturing.

Industrialization of stem-cell based therapies requires innovative solutions to close the gap between research and commercialization. Scalable cell production platforms are needed to reliably deliver the cell quantities needed during the various stages of development and commercial supply. Human pluripotent stem cells (hPSCs) are a key source material for generating therapeutic cell types. We have developed a closed, automated and scalable stirred tank bioreactor platform, capable of sustaining high fold expansion of hPSCs. Such a platform could facilitate the in-process monitoring and integration of online monitoring systems, leading to significantly reduced labor requirements and contamination risk. hPSCs are expanded in a controlled bioreactor using perfused xeno-free media. Cell harvest and concentration are performed in closed steps. The hPSCs can be cryopreserved to generate a bank of cells, or further processed as needed. Cryopreserved cells can be thawed into a two-dimensional (2D) tissue culture platform or a three-dimensional (3D) bioreactor to initiate a new expansion phase, or be differentiated to the clinically relevant cell type. The expanded hPSCs express hPSC-specific markers, have a normal karyotype and the ability to differentiate to the cells of the three germ layers. This end-to-end platform allows a large scale expansion of high quality hPSCs that can support the required cell demand for various clinical indications.

Platforms for Manufacturing Allogeneic, Autologous and iPSC Cell Therapy Products: An Industry Perspective.

As cell therapy processes mature from benchtop research protocols to industrial processes capable of manufacturing market-relevant numbers of doses, new cell manufacturing platforms are required. Here we give an overview of the platforms and technologies currently available to manufacture allogeneic cell products, such as mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), and technologies for mass production of autologous cell therapies via scale-out. These technologies include bioreactors, microcarriers, cell separation and cryopreservation equipment, molecular biology tools for iPSC generation, and single-use controlled-environment systems for autologous cell production. These platforms address the challenges of manufacturing cell products in greater numbers while maintaining process robustness and product quality.

Matrix-Embedded Endothelial Cells Attain a Progenitor-Like Phenotype.

Culture of endothelial cells (ECs) embedded in 3D scaffolds of denatured collagen has shown tremendous therapeutic potential in clinical trials of tissue repair. It is postulated that these matrix-embedded ECs (MEECs) attain a differential phenotype similar to early progenitor forms, which cannot be attained in 2D culture. MEECs are compared to 2D-ECs and endothelial progenitor cells (EPCs) by secretome, phenotype, and genetic fingerprint, and are found to be altered from 2D-ECs on all levels, adopting an EPC-like phenotype. This manifests in elevation of CD34 expression-a progenitor cell marker-and protein secretion and gene expression pro-files that are similar to EPCs. Even more striking is that EPCs in 2D lose their phenotype, evident by the loss of CD34 expression, but are able to regain expression over time when embedded in the same 3D matrices, suggesting that future in vitro EPC work should use ME-EPCs to recapitulate in vivo phenotype. These findings elucidate the relationship between EPCs and the substratum-dependent regulation imparted by ECs which is critical to understand in order to optimize MEEC therapy and propel it into the clinic.

A consensus introduction to serum replacements and serum-free media for cellular therapies.

The cell therapy industry is a fast-growing industry targeted toward a myriad of clinical indications. As the cell therapy industry matures and clinical trials hit their pivotal Phase 3 studies, there will be a significant need for scale-up, process validation, and critical raw material quality assurance. Part of the well discussed challenges of upscaling manufacturing processes there is a less discussed issue relating to the availability of raw materials in the needed quality and quantities. The FDA recently noted that over 80% of the 66 investigational new drug (IND) applications for mesenchymal stem cell (MSC) products analyzed described the use of FBS during manufacturing. Accumulated data from the past years show an acceleration in serum consumption by at least 10%-15% annually, which suggests that the global demand for serum may soon exceed the supply. Ongoing concerns of safety issues due to risks of various pathogen contaminations, as well as issues related to the aforementioned serum variability that can affect final product reproducibility, are strong motivators to search for serum substitutes or serum-free media. it is important to note that there are no accepted definitions for most of these terms which leads to misleading's and misunderstandings, where the same term might be defined differently by different vendors, manufacturer, and users. It is the drug developer's responsibility to clarify what the supplied labels mean and to identify the correct questions and audits to ensure quality. The paper reviews the available serum replacements, main components, basic strategies for replacement of serum and suggests definitions.

Managing particulates in cell therapy: Guidance for best practice.

The intent of this article is to provide guidance and recommendations to cell therapy product sponsors (including developers and manufacturers) and their suppliers in the cell therapy industry regarding particulate source, testing, monitoring and methods for control. This information is intended to help all parties characterize the processes that generate particulates, understand product impact and provide recommendations to control particulates generated during manufacturing of cell therapy products.

Human placenta-derived stromal cells decrease inflammation, placental injury and blood pressure in hypertensive pregnant mice.

Pre-eclampsia, the development of hypertension and proteinuria or end-organ damage during pregnancy, is a leading cause of both maternal and fetal morbidity and mortality, and there are no effective clinical treatments for pre-eclampsia aside from delivery. The development of pre-eclampsia is characterized by maladaptation of the maternal immune system, excessive inflammation and endothelial dysfunction. We have reported that detection of extracellular RNA by the Toll-like receptors (TLRs) 3 and 7 is a key initiating signal that contributes to the development of pre-eclampsia. PLacental eXpanded (PLX-PAD) cells are human placenta-derived, mesenchymal-like, adherent stromal cells that have anti-inflammatory, proangiogenic, cytoprotective and regenerative properties, secondary to paracrine secretion of various molecules in response to environmental stimulation. We hypothesized that PLX-PAD cells would reduce the associated inflammation and tissue damage and lower blood pressure in mice with pre-eclampsia induced by TLR3 or TLR7 activation. Injection of PLX-PAD cells on gestational day 14 significantly decreased systolic blood pressure by day 17 in TLR3-induced and TLR7-induced hypertensive mice (TLR3 144-111 mmHg; TLR7 145-106 mmHg; both P<0.05), and also normalized their elevated urinary protein:creatinine ratios (TLR3 5.68-3.72; TLR7 5.57-3.84; both P<0.05). On gestational day 17, aortic endothelium-dependent relaxation responses improved significantly in TLR3-induced and TLR7-induced hypertensive mice that received PLX-PAD cells on gestational day 14 (TLR3 35-65%; TLR7 37-63%; both P<0.05). In addition, markers of systemic inflammation and placental injury, increased markedly in both groups of TLR-induced hypertensive mice, were reduced by PLX-PAD cells. Importantly, PLX-PAD cell therapy had no effects on these measures in pregnant control mice or on the fetuses. These data demonstrate that PLX-PAD cell therapy can safely reverse pre-eclampsia-like features during pregnancy and have a potential therapeutic role in pre-eclampsia treatment.

Tuning of collagen scaffold properties modulates embedded endothelial cell regulatory phenotype in repair of vascular injuries in vivo.

Perivascularly implanted matrix embedded endothelial cells (MEECs) are potent regulators of inflammation and intimal hyperplasia following vascular injuries. Endothelial cells (ECs) in collagen scaffolds adopt a reparative phenotype with significant therapeutic potential. Although the biology of MEECs is increasingly understood, tuning of scaffold properties to control cell-substrate interactions is less well-studied. It is hypothesized that modulating scaffold degradation would change EC phenotype. Scaffolds with differential degradation are prepared by cross-linking and predegradation. Vascular injury increases degradation and the presence of MEECs retards injury-mediated degradation. MEECs respond to differential scaffold properties with altered viability in vivo, suppressed smooth muscle cell (SMC) proliferation in vitro, and altered interleukin-6 and matrix metalloproteinase-9 expression. When implanted perivascularly to a murine carotid wire injury, tuned scaffolds change MEEC effects on vascular repair and inflammation. Live animal imaging enables real-time tracking of cell viability, inflammation, and scaffold degradation, affording an unprecedented understanding of interactions between cells, substrate, and tissue. MEEC-treated injuries improve endothelialization and reduce SMC hyperplasia over 14 d. These data demonstrate the potent role material design plays in tuning MEEC efficacy in vivo, with implications for the design of clinical therapies.

Critical elements in the development of cell therapy potency assays for ischemic conditions.

A successful potency assay for a cell therapy product (CTP) used in the treatment of ischemic conditions should quantitatively measure relevant biological properties that predict therapeutic activity. This is especially challenging because of numerous degrees of complexity stemming from factors that include a multifactorial complex mechanism of action, cell source, inherent cell characteristics, culture method, administration mode and the in vivo conditions to which the cells are exposed. The expected biological function of a CTP encompasses complex interactions that range from a biochemical, metabolic or immunological activity to structural replacement of damaged tissue or organ. Therefore, the requirements for full characterization of the active substance with respect to biological function could be taxing. Moreover, the specific mechanism of action is often difficult to pinpoint to a specific molecular entity; rather, it is more dependent on the functionality of the cellular components acting in a in a multifactorial fashion. In the case of ischemic conditions, the cell therapy mechanism of action can vary from angiogenesis, vasculogenesis and arteriogenesis that may activate different pathways and clinical outcomes. The CTP cellular attributes with relation to the suggested mechanism of action can be used for the development of quantitative and reproducible analytical potency assays. CTPs selected and released on the basis of such potency assays should have the highest probability of providing meaningful clinical benefit for patients. This White Paper will discuss and give examples for key elements in the development of a potency assay for treatment of ischemic disorders treated by the use of CTPs.

Natural tissue microenvironmental conditions modulate adhesive material performance.

We designed and optimized tissue-responsive adhesive materials by matching material and tissue properties. A two-component material based on dextran aldehyde and dendrimer amine provides a cohesive gel through aldehyde-amine cross-linking and an adhesive interface created by a dextran aldehyde-selective reaction with tissue amines. By altering aldehyde-amine chemistry, we examined how variations in tissue surfaces (serosal amine density in the duodenum, jejunum, and ileum) affect interactions with adhesive materials of varied compositions (aldehyde content). Interestingly, the same adhesive formulation reacts differentially with the three regions of the small intestine as a result of variation in the tissue amine density along the intestinal tract, affecting the tissue-material interfacial morphology, adhesion strength, and adhesive mechanical properties. Whereas tissues provide chemical anchors for interaction with materials, we were able to tune the adhesion strength for each section of the small intestine tissue by altering the adhesive formulation using a two-component material with flexible variables aimed at controlling the aldehyde/amine ratio. This tissue-specific approach should be applied to the broad spectrum of biomaterials, taking into account specific microenvironmental conditions in material design.

Regulation of temporal and spatial organization of newborn GnRH neurons by IGF signaling in zebrafish.

When and how newborn neurons are organized to form a functional network in the developing brain remains poorly understood. An attractive model is the gonadotropin-releasing hormone (GnRH) neuron system, master regulator of the reproductive axis. Here we show that blockage of IGF signaling, a central growth-promoting signaling pathway, by the induced expression of a dominant-negative form of IGF1 receptor (IGF1R) or specific IGF1R inhibitors delayed the emergence of GnRH2 neurons in the midbrain and GnRH3 neurons in the olfactory bulb region. Blockage of IGF signaling also resulted in an abnormal appearance of GnRH3 neurons outside of the olfactory bulb region, although it did not change the locations of other olfactory neurons, GnRH2 neurons, or brain patterning. This IGF action is developmental stage-dependent because the blockade of IGF signaling in advanced embryos had no such effect. An application of phosphatidylinositol 3-kinase (PI3K) inhibitors phenocopied the IGF signaling deficient embryos, whereas the MAPK inhibitors had no effect, suggesting that this IGF action is mediated through the PI3K pathway. Real-time in vivo imaging studies revealed that the ectopic GnRH3 neurons emerged at the same time as the normal GnRH3 neurons in IGF-deficient embryos. Further experiments suggest that IGF signaling affects the spatial distribution of newborn GnRH3 neurons by influencing neural crest cell migration and/or differentiation. These results suggest that the IGF-IGF1R-PI3K pathway regulates the precise temporal and spatial organization of GnRH neurons in zebrafish and provides new insights into the regulation of GnRH neuron development.

Targeted gonadotropin-releasing hormone-3 neuron ablation in zebrafish: effects on neurogenesis, neuronal migration, and reproduction.

Hypophysiotropic GnRH neurons are located in the preoptic area and ventral hypothalamus of sexually mature vertebrates. In several species, the embryonic origin of hypophysiotropic GnRH neurons remains unclear. Using the Tg(GnRH3:EGFP) zebrafish line, in which GnRH3 neurons express EGFP, GnRH3 neurons in the olfactory region were specifically and individually ablated during early development using laser pulses. After ablation, the olfactory region maintained the capacity to regenerate GnRH3 neurons. However, this capacity was time-limited. When ablation of GnRH3 cells was conducted at 2 d after fertilization, high regeneration rates were observed, but regeneration capacity significantly decreased when ablation was performed at 4 or 6 d after fertilization. Unilateral GnRH3 neuron ablation results in unilateral soma presence. These unilateral somata are capable of projecting fiber extensions bilaterally. Successful bilateral GnRH3 soma ablation during development resulted in complete lack of olfactory, terminal nerve, preoptic area, and hypothalamic GnRH3 neurons and fibers in 12-wk-old animals. Mature animals lacking GnRH3 neurons exhibited arrested oocyte development and reduced average oocyte diameter. Animals in which GnRH3 neurons were partially ablated exhibited normal oocyte development; however, their fecundity was significantly reduced. These findings demonstrate that the hypophysiotropic GnRH3 populations in zebrafish consist of neurons that originate in the olfactory region during early development. The presence of GnRH3 neurons of olfactory region origin in reproductively mature zebrafish is a prerequisite for normal oocyte development and reproduction.

Cxcl12a-Cxcr4b signaling is important for proper development of the forebrain GnRH system in zebrafish.

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons control pituitary gonadotropin secretion and gametogenesis. In the course of development, these neurons migrate from the olfactory placode to the hypothalamus. The precise molecular mechanism of this neuronal migration is unclear. Here, we investigated whether the chemokine receptor, Cxcr4b, and its cognate ligand, Cxcl12a, are required for proper migration of GnRH3 neurons in zebrafish. Deviated GnRH3 axonal projections and neuronal migration were detected in larvae that carry a homozygote cxcr4b mutation. Similarly, knockdown of Cxcr4b or Cxcl12a led to the appearance of abnormal GnRH3 axonal projections and cell migration, including absence of the characteristic lateral crossing of GnRH3 axons at the anterior commissure and optic chiasm. Double-labeling analysis has shown that cxcr4b and cxcl12a are expressed along the GnRH3 migration pathway (i.e. olfactory placode, terminal nerve and the optic chiasm). The results of this study suggest that the Cxcl12a-Cxcr4b ligand-receptor pair are involved in the migration of GnRH3 neurons in zebrafish, and are therefore crucial for the development of this system.

The zebrafish as a model system for forebrain GnRH neuronal development.

Development and function of the forebrain gonadotropin-releasing hormone (GnRH) neuronal system has long been the focus of study in various vertebrate species. This system is crucial for reproduction and an important model for studying tangential neuronal migration. In addition, the finding that multiple forms of GnRH exist in the CNS as well as in non-CNS tissues, coupled with the fact that GnRH fibers project to many CNS regions, implies that GnRH has a variety of functions in addition to its classic reproductive role. The study of the GnRH system and its functions is, however, limited by available model systems and methodologies. The transgenic (Tg) GnRH3:EGFP zebrafish line, in which GnRH3 neurons express EGFP, allows in vivo study of the GnRH3 system in the context of the entire animal. Coupling the use of this line with the attributes and molecular tools available in zebrafish has expanded our ability to study the forebrain GnRH system. Herein, we discuss the use of the Tg(GnRH3:EGFP) zebrafish line as a model for studying forebrain GnRH neurons, both in developing larvae and in sexually mature animals. We also discuss the potential use of this line to study regulation of GnRH3 system development.

Nasal embryonic LHRH factor plays a role in the developmental migration and projection of gonadotropin-releasing hormone 3 neurons in zebrafish.

The initiation of puberty and the functioning of the reproductive system depend on proper development of the hypophysiotropic gonadotropin-releasing hormone (GnRH) system. One critical step in this process is the embryonic migration of GnRH neurons from the olfactory area to the hypothalamus. Using a transgenic zebrafish model, Tg(gnrh3:EGFP), in which GnRH3 neurons and axons are fluorescently labeled, we investigated whether zebrafish NELF is essential for the development of GnRH3 neurons. The zebrafish nelf cDNA was cloned and characterized. During embryonic development, nelf is expressed in GnRH3 neurons and in target sites of GnRH3 projections and perikarya, before the initiation of their migration. Nelf knockdown resulted in a disruption of the GnRH3 system which included absence or misguiding of GnRH3 axonal outgrowth and incorrect or arrested migration of GnRH3 perikarya. These results suggest that Nelf is an important factor in the developmental migration and projection of GnRH3 neurons in zebrafish.

Ontogeny of the GnRH systems in zebrafish brain: in situ hybridization and promoter-reporter expression analyses in intact animals.

The ontogeny of two gonadotropin-releasing-hormone (GnRH) systems, salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II), was investigated in zebrafish (Danio rerio). In situ hybridization (ISH) first detected sGnRH mRNA-expressing cells at 1 day post-fertilization (pf) anterior to the developing olfactory organs. Subsequently, cells were seen along the ventral olfactory organs and the olfactory bulbs, reaching the terminal nerve (TN) ganglion at 5-6 days pf. Some cells were detected passing posteriorly through the ventral telencephalon (10-25 days pf), and by 25-30 days pf, sGnRH cells were found in the hypothalamic/preoptic area. Continuous documentation in live zebrafish was achieved by a promoter-reporter expression system. The expression of enhanced green fluorescent protein (EGFP) driven by the sGnRH promoter allowed the earlier detection of cells and projections and the migration of sGnRH neurons. This expression system revealed that long leading processes, presumably axons, preceded the migration of the sGnRH neuron somata. cGnRH-II mRNA expressing cells were initially detected (1 day pf) by ISH analysis at lateral aspects of the midbrain and later on (starting at 5 days pf) at the midline of the midbrain tegmentum. Detection of red fluorescent protein (DsRed) driven by the cGnRH-II promoter confirmed the midbrain expression domain and identified specific hindbrain and forebrain cGnRH-II-cells that were not identified by ISH. The forebrain DsRed-expressing cells seemed to emerge from the same site as the sGnRH-EGFP-expressing cells, as revealed by co-injection of both constructs. These studies indicate that zebrafish TN and hypothalamic sGnRH cell populations share a common embryonic origin and migratory path, and that midbrain cGnRH-II cells originate within the midbrain.