TEG001 Insert Integrity from Vector Producer Cells until Medicinal Product.

Despite extensive usage of gene therapy medicinal products (GTMPs) in clinical studies and recent approval of chimeric antigen receptor (CAR) T cell therapy, little information has been made available on the precise molecular characterization and possible variations in terms of insert integrity and vector copy numbers of different GTMPs during the complete production chain. Within this context, we characterize αßT cells engineered to express a defined γδT cell engineered to express a defined γδT receptor (TEG) currently used in a first-in-human clinical study (NTR6541). Utilizing targeted locus amplification in combination with next generation sequencing for the vector producer clone and TEG001 products, we report on five single-nucleotide variants and nine intact vector copies integrated in the producer clone. The vector copy number in TEG001 cells was on average a factor 0.72 (SD 0.11) below that of the producer cell clone. All nucleotide variants were transferred to TEG001 without having an effect on cellular proliferation during extensive in vitro culture. Based on an environmental risk assessment of the five nucleotide variants present in the non-coding viral region of the TEG001 insert, there was no altered environmental impact of TEG001 cells. We conclude that TEG001 cells do not have an increased risk for malignant transformation in vivo.

Discovery of GLPG2737, a Potent Type 2 Corrector of CFTR for the Treatment of Cystic Fibrosis in Combination with a Potentiator and a Type 1 Co-corrector.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) protein. This epithelial anion channel regulates the active transport of chloride and bicarbonate ions across membranes. Mutations result in reduced surface expression of CFTR channels with impaired functionality. Correctors are small molecules that support the trafficking of CFTR to increase its membrane expression. Such correctors can have different mechanisms of action. Combinations may result in a further improved therapeutic benefit. We describe the identification and optimization of a new pyrazolol3,4-bl pyridine-6-carboxylic acid series with high potency and efficacy in rescuing CFTR from the cell surface. Investigations showed that carboxylic acid group replacement with acylsulfonamides and acylsulfonylureas improved ADMET and PK properties, leading to the discovery of the structurally novel co-corrector GLPG2737. The addition of GLPG2737 to the combination of the potentiator GLPG1837 and C1 corrector 4 led to an 8-fold increase in the F508del CFTR activity.

Acylated Gly-(2-cyano)pyrrolidines as inhibitors of fibroblast activation protein (FAP) and the issue of FAP/prolyl oligopeptidase (PREP)-selectivity.

A series of N-acylated glycyl-(2-cyano)pyrrolidines were synthesized with the aim of generating structure-activity relationship (SAR) data for this class of compounds as inhibitors of fibroblast activation protein (FAP). Specifically, the influence of (1) the choice of the N-acyl group and (2) structural modification of the 2-cyanopyrrolidine residue were investigated. The inhibitors displayed inhibitory potency in the micromolar to nanomolar range and showed good to excellent selectivity with respect to the proline selective dipeptidyl peptidases (DPPs) DPP IV, DPP9 and DPP II. Additionally, selectivity for FAP with respect to prolyl oligopeptidase (PREP) is reported. Not unexpectedly, the latter data suggest significant overlap in the pharmacophoric features that define FAP or PREP-inhibitory activity and underscore the importance of systematically evaluating the FAP/PREP-selectivity index for inhibitors of either of these two enzymes. Finally, this study forwards several compounds that can serve as leads or prototypic structures for future FAP-selective-inhibitor discovery.

Discovery and Optimization of Orally Bioavailable Phthalazone and Cinnolone Carboxylic Acid Derivatives as S1P2 Antagonists against Fibrotic Diseases.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease. Current treatments only slow down disease progression, making new therapeutic strategies compelling. Increasing evidence suggests that S1P2 antagonists could be effective agents against fibrotic diseases. Our compound collection was mined for molecules possessing substructure features associated with S1P2 activity. The weakly potent indole hit 6 evolved into a potent phthalazone series, bearing a carboxylic acid, with the aid of a homology model. Suboptimal pharmacokinetics of a benzimidazole subseries were improved by modifications targeting potential interactions with transporters, based on concepts deriving from the extended clearance classification system (ECCS). Scaffold hopping, as a part of a chemical enablement strategy, permitted the rapid exploration of the position adjacent to the carboxylic acid. Compound 38, with good pharmacokinetics and in vitro potency, was efficacious at 10 mg/kg BID in three different in vivo mouse models of fibrotic diseases in a therapeutic setting.

Novel Small Molecule-Derived, Highly Selective Substrates for Fibroblast Activation Protein (FAP).

Fibroblast activation protein (FAP) is a proline-selective serine protease. It is hardly expressed in healthy adult tissue but upregulated in tissue remodeling sites associated with several diseases including epithelial cancer types, atherosclerosis, arthritis and fibrosis. Ongoing research aims at clinical implementation of FAP as a biomarker for these diseases. Several immunochemical methods that quantify FAP expression have been reported. An alternative/complementary approach focuses on quantification of FAP's enzymatic activity. Developing an activity-based assay for FAP has nonetheless proven challenging because of selectivity issues with respect to prolyl oligopeptidase (PREP). Here, we present substrate-type FAP probes that are structurally derived from a FAP-inhibitor (UAMC1110) that we published earlier. Both cleavage efficiency and FAP-selectivity of the best compounds in the series equal or surpass the most advanced peptide-based FAP substrates reported to date. Finally, proof-of-concept is provided that 4-aminonaphthol containing probes can spatially localize FAP activity in biological samples.

GMP-Grade Manufacturing of T Cells Engineered to Express a Defined γδTCR.

γ9δ2T cells play a critical role in daily cancer immune surveillance by sensing cancer-mediated metabolic changes. However, a major limitation of the therapeutic application of γ9δ2T cells is their diversity and regulation through innate co-receptors. In order to overcome natural obstacles of γ9δ2T cells, we have developed the concept of T cells engineered to express a defined γδT cell receptor (TEGs). This next generation of chimeric antigen receptor engineered T (CAR-T) cells not only allows for targeting of hematological but also of solid tumors and, therefore, overcomes major limitations of many CAR-T and γδT cell strategies. Here, we report on the development of a robust manufacturing procedure of T cells engineered to express the high affinity Vγ9Vδ2T cell receptor (TCR) clone 5 (TEG001). We determined the best concentration of anti-CD3/CD28 activation and expansion beads, optimal virus titer, and cell density for retroviral transduction, and validated a Good Manufacturing Practice (GMP)-grade purification procedure by utilizing the CliniMACS system to deplete non- and poorly-engineered T cells. To the best of our knowledge, we have developed the very first GMP manufacturing procedure in which αßTCR depletion is used as a purification method, thereby delivering untouched clinical grade engineered immune cells. This enrichment method is applicable to any engineered T cell product with a reduced expression of endogenous αßTCRs. We report on release criteria and the stability of TEG001 drug substance and TEG001 drug product. The GMP-grade production procedure is now approved by Dutch authorities and allows TEG001 to be generated in cell numbers sufficient to treat patients within the approved clinical trial NTR6541. NTR6541 will investigate the safety and tolerability of TEG001 in patients with relapsed/refractory acute myeloid leukemia, high-risk myelodysplastic syndrome, and relapsed/refractory multiple myeloma.

Vasopressin immunoreactivity, but not vasoactive intestinal polypeptide, correlates with expression of circadian rhythmicity in the suprachiasmatic nucleus of voles.

In common voles (Microtus arvalis), natural variation in locomotor behavior can be exploited to study the mechanism of pacemaker control over circadian timing of behavior. Here we studied daily patterns in numbers of neuropeptide immunoreactive suprachiasmatic nucleus neurons in rhythmic, weakly rhythmic, and non-rhythmic voles. Circadian rhythmic voles showed circadian variation in numbers of vasoactive intestinal polypeptide and vasopressin immunoreactive suprachiasmatic nucleus neurons with a peak at zeitgeber time 0. In contrast, voles with weak or no circadian rhythmicity exhibited similar fluctuations for vasoactive intestinal polypeptide, but a continuous, non-rhythmic high profile for vasopressin. Vole suprachiasmatic nucleus neurons do not produce somatostatin or substance P. We conclude that the vasopressin system in the common vole suprachiasmatic nucleus acts as a principal correlate with expression of circadian behavior, in contrast to vasoactive intestinal polypeptide, somatostatin, and substance P. We also conclude that high levels of vasopressin immunoreactivity in the non-rhythmic vole suprachiasmatic nucleus is in line with previously demonstrated hampered release, probably resulting in vasopressin accumulation in the suprachiasmatic nucleus. Vasopressin could be a candidate in mediating output of the vole circadian clock, leading to circadian expression of locomotor behavior.

Scaling-up of a HepaRG progenitor cell based bioartificial liver: optimization for clinical application and transport.

A new generation of bioartificial livers, based on differentiated proliferative hepatocyte sources, has been developed. Several practicable and regulatory demands have to be addressed before these can be clinically evaluated. We identified three main hurdles: (1) expansion and preservation of the biocomponent, (2) development of scaled-up culture conditions and (3) transport of the device to the bedside. In this study we address these three issues for the HepaRG-progenitor cell line-loaded AMC-Bioartificial Liver. (1) HepaRG cells were expanded in large quantities and then cryopreserved or loaded directly into bioreactors. After 3 weeks of culture, key hepatic functions (ammonia/lactate elimination, apolipoprotein A1 synthesis and cytochrome P450 3A4 activity) did not differ significantly between the two groups. (2) Bioartificial livers were scaled up from 9 ml to 540 ml priming volume, with preservation of normalized hepatic functionality. Quantification of amino acid consumption revealed rapid depletion of several amino acids. (3) Whole-device cryopreservation and cooled preservation induced significant loss of hepatic functionality, whereas simulated transport from culture-facility to the bedside in a clinical-grade transport unit with controlled temperature maintenance, medium perfusion and gas supply did not affect functionality. In addition, we assessed tumorigenicity of HepaRG cells in immune-incompetent mice and found no tumor formation of HepaRG cells (n = 12), while HeLa cells induced formation of carcinomas in eight out of 12 mice in 140 days.

Not only vasopressin, but also the intracellular messenger protein kinase Calpha in the suprachiasmatic nucleus correlates with expression of circadian rhythmicity in voles.

The suprachiasmatic nucleus (SCN) is the locus of the main pacemaker for circadian behavioral rhythms. In common voles, variation in circadian behavioral rhythmicity correlates with vasopressin (AVP) immunoreactive cells in the SCN. Here we studied the immunostaining of four AVP linked Ca(2+)-dependent protein kinase C (PKC) isoforms (PKCalpha, PKCbeta1, PKCbeta2, and PKCgamma) at the beginning of the light period, and conclude that PKCalpha is highly expressed in the vole SCN compared to the other isozymes. Voles, characterized as strongly circadian rhythmic showed circadian variation in numbers of PKCalpha immunoreactive SCN neurons, while voles with weak or no circadian rhythmicity did not reveal such a circadian profile. PKCalpha immunoreactivity in acute SCN slices that were treated with a physiological dose of AVP was significantly lowered when compared with control slices. The intracellular messenger PKCalpha may reflect variation in locomotor behavior via the AVP system in the vole SCN. This system could play a key role in the vole SCN by mediating output of its circadian clock.

Vasopressin immunoreactivity and release in the suprachiasmatic nucleus of wild-type and tau mutant Syrian hamsters.

Despite the prominent role of the Syrian hamster (Mesocricetus auratus) in studies of circadian rhythms, there are no data available on the temporal dynamics of the neuropeptide vasopressin (AVP), a major output system of the suprachiasmatic nucleus (SCN). We studied the hamster SCN-AVP system in vivo across the light period and in vitro using long-term organotypic SCN cultures. Additionally, we compared wild-type and tau mutant hamsters with an endogenous circadian period of approximately 24 h and approximately 20 h, respectively. The in vivo study revealed no differences in the number of SCN-AVP neurons between the two genotypes of hamsters studied at three time points across the light period of the circadian cycle. A significantly higher level of AVP-immunoreactivity, however, was found in the SCN of wild-type compared to tau mutant hamsters at the beginning and in the middle of the light period, but not at the end of the light period. SCN-AVP cell number and immunostaining decreased significantly across the light period in wild-type hamsters, but not in tau mutants. The in vitro study revealed a significantly higher rate of AVP release per 24 h from the tau mutant SCN compared to the wild-type SCN. Robust circadian oscillations in AVP release were not found in either type of hamster. These results may suggest that the SCN-AVP system of hamsters, irrespective of genotype, is relatively weak compared to other species. Moreover, the tau mutation seems to influence the SCN-AVP system by enhancing the rate of AVP release and by reducing AVP content and its daily fluctuation.

Long-term regeneration of the rat sciatic nerve through a biodegradable poly(DL-lactide-epsilon-caprolactone) nerve guide: tissue reactions with focus on collagen III/IV reformation.

Long-term studies on nerve-guide regeneration are scarce. Therefore, in rats, long-term (16 months) sciatic nerve regeneration through poly(DL-lactide-epsilon-caprolactone) [poly(DLLA-epsilon-CL)] nerve guides was studied and compared with the nonoperated control side. Poly(DLLA-epsilon-CL) degradation and possible long-term foreign body reaction against poly(DLLA-epsilon-CL) nerve guides, as well as the distribution of both collagen type III and IV were studied. In vivo poly(DLLA-epsilon-CL) studies have been performed before but not for such long time points; also, a detailed analysis of collagen III/IV has not been presented before. The results demonstrate that biodegradable poly(DLLA-epsilon-CL) nerve guides yield good nerve regeneration and collagen III/IV deposition relative to the anatomy of the control side. Regenerated nerve showed almost similar collagen type III/IV distribution patterns as compared with the nonoperated control side, although the delineation of matrix was clearer in the control side. The relative amount of collagen III and IV immunostaining in nerve cross-sections did not, however, differ between the control nerve tissue and the operated side after 16 months. After 16 months of implantation, however, some very small fragments of biomaterial could still be found on the edge of the epineurium of the regenerated nerve, indicating remnants of a secondary foreign body reaction. The biomaterial fragments and foreign body reaction did not influence the nerve regeneration process after 16 months. Biodegradable poly(DLLA-epsilon-CL) nerve guides are useful for long-term bridging of short peripheral nerve gaps.

In vitro degradation and biocompatibility of poly(DL-lactide-epsilon-caprolactone) nerve guides.

Bridging nerve gaps by means of autologous nerve grafts involves donor nerve graft harvesting. Recent studies have focused on the use of alternative methods, and one of these is the use of biodegradable nerve guides. After serving their function, nerve guides should degrade to avoid a chronic foreign body reaction. The in vitro degradation, in vitro cytotoxicity, hemocompatibility, and short-term in vivo foreign body reaction of poly((65)/(35) ((85)/(15) (L)/(D)) lactide-epsilon-caprolactone) nerve guides was studied. The in vitro degradation characteristics of poly(DLLA-epsilon-CL) nerve guides were monitored at 2-week time intervals during a period of 22 weeks. Weight loss, degree of swelling of the tube wall, mechanical strength, thermal properties, and the intrinsic viscosity of the nerve guides were determined. Cytotoxicity was studied by measuring the cell proliferation inhibition index (CPII) on mouse fibroblasts in vitro. Cell growth was evaluated by cell counting, while morphology was assessed by light microscopy. Hemocompatibility was evaluated using a thrombin generation assay and a complement convertase assay. The foreign body reaction against poly(DLLA-epsilon-CL) nerve guides was investigated by examining toluidine blue stained sections. The in vitro degradation data showed that poly(DLLA-epsilon-CL) nerve guides do not swell, maintain their mechanical strength and flexibility for a period of about 8-10 weeks, and start to lose mass after about 10 weeks. Poly(DLLA-epsilon-CL) nerve guides were classified as noncytotoxic, as cytotoxicity tests demonstrated that cell morphology was not affected (CPII 0%). The thrombin generation assay and complement convertase assay indicated that the material is highly hemocompatible. The foreign body reaction against the biomaterial was mild with a light priming of the immunesystem. The results presented in this study demonstrate that poly((65)/(35) ((85)/(15) (L)/(D)) lactide-epsilon-caprolactone) nerve guides are biocompatible, and show good in vitro degradation characteristics, making these biodegradable nerve guides promising candidates for bridging peripheral nerve defects up to several centimeters.

Extended structure-activity relationship and pharmacokinetic investigation of (4-quinolinoyl)glycyl-2-cyanopyrrolidine inhibitors of fibroblast activation protein (FAP).

Fibroblast activation protein (FAP) is a serine protease related to dipeptidyl peptidase IV (DPPIV). It has been convincingly linked to multiple disease states involving remodeling of the extracellular matrix. FAP inhibition is investigated as a therapeutic option for several of these diseases, with most attention so far devoted to oncology applications. We previously discovered the N-4-quinolinoyl-Gly-(2S)-cyanoPro scaffold as a possible entry to highly potent and selective FAP inhibitors. In the present study, we explore in detail the structure-activity relationship around this core scaffold. We report extensively optimized compounds that display low nanomolar inhibitory potency and high selectivity against the related dipeptidyl peptidases (DPPs) DPPIV, DPP9, DPPII, and prolyl oligopeptidase (PREP). The log D values, plasma stabilities, and microsomal stabilities of selected compounds were found to be highly satisfactory. Pharmacokinetic evaluation in mice of selected inhibitors demonstrated high oral bioavailability, plasma half-life, and the potential to selectively and completely inhibit FAP in vivo.

Selective Inhibitors of Fibroblast Activation Protein (FAP) with a (4-Quinolinoyl)-glycyl-2-cyanopyrrolidine Scaffold.

Fibroblast activation protein (FAP) is a serine protease that is generally accepted to play an important role in tumor growth and other diseases involving tissue remodeling. Currently there are no FAP inhibitors with reported selectivity toward both the closely related dipeptidyl peptidases (DPPs) and prolyl oligopeptidase (PREP). We present the discovery of a new class of FAP inhibitors with a N-(4-quinolinoyl)-Gly-(2-cyanopyrrolidine) scaffold. We have explored the effects of substituting the quinoline ring and varying the position of its sp(2) hybridized nitrogen atom. The most promising inhibitors combined low nanomolar FAP inhibition and high selectivity indices (>10(3)) with respect to both the DPPs and PREP. Preliminary experiments on a representative inhibitor demonstrate that plasma stability, kinetic solubility, and log D of this class of compounds can be expected to be satisfactory.

Two years after in vivo implantation of poly(DL-lactide-epsilon-caprolactone) nerve guides: has the material finally resorbed?

Long-term nerve guide studies on nerve regeneration in vivo are scarce. Previously, we showed that small fragments of biomaterial could still be found on the edge of the epineurium of the regenerated nerve after implantation of poly(DL-lactide-epsilon-caprolactone) [poly(DLLA-epsilon-CL), Neurolac(R)] nerve guides. As these nerve guides are assumed to fully resorb, we studied the 2-year degradation and possible long-term foreign body reaction against the nerve guides after implantation in the sciatic nerve of the rat. In addition, the distribution of both collagen type III and IV, and nerve regeneration through the nerve guides were studied and compared with the non-operated control side. The results demonstrate that nerve regeneration took place through the poly(DLLA-epsilon-CL) nerve guides. After 2 years of implantation, biomaterial could not be found macroscopically. Biomaterial fragments in company of multi-nucleated giant cells and macrophages were found along the regenerated nerve tissue. Collagen III and IV were both found around the epineurium and perineurium in the regenerated nerve, the organization of these layers resembled that of the contra-lateral non-operated nerve. Although sufficient nerve regeneration was obtained after long-term implantation in the rat sciatic nerve, biomaterial fragments and foreign body reactions against these fragments, even after 24 months of implantation, could still be found. The poly(DLLA-epsilon-CL), Neurolac nerve guides do resorb, however, not complete up to 2 years of implantation. It is not known whether the remaining biomaterial fragments and foreign body reactions may cause granulomas or other complications after longer implantation periods.

Circadian rhythms of C-FOS expression in the suprachiasmatic nuclei of the common vole (Microtus arvalis).

The suprachiasmatic nuclei of the hypothalamus (SCN) are the master circadian clock in mammals. Transcriptional activity in this master clock has a marker in the immediate-early gene c-Fos. Within the SCN, distinct differences in c-Fos in the ventrolateral and the dorsomedial SCN have been reported for rodent species such as rats, mice, and hamsters. We studied C-FOS expression in the common vole (Microtus arvalis) SCN under LD 12:12 h and under constant dim light conditions. In the vole dorsomedial SCN, rhythmic C-FOS expression was seen in LD with a clear peak in the middle of the light period. Under constant dim light, we report constitutive, non-rhythmic expression of C-FOS in the dorsomedial SCN. This pattern is consistent with the circadian organization of behavioral activity, which is weak in voles and may be lost under constant dim-light conditions. In the ventrolateral SCN, we observed a rise in C-FOS expression under LD conditions prior to lights-on, followed by peak expression at lights-on. Another peak was seen at lights-off. In an additional experiment, we subjected animals to LD 16:8 to test the hypothesis that the dawn and dusk peaks in ventrolateral C-FOS expression change phase along with the photoperiod. The peak in C-FOS expression did not shift with the time of lights on, but remained at the same external time 6. The results are consistent with the interpretation that in the vole, c-Fos expression reports transcriptional activity associated more likely with an internal, gating process than with an external effect of light.