An ultra-high-affinity small organic ligand of fibroblast activation protein for tumor-targeting applications

We describe the development of OncoFAP, an ultra-high-affinity ligand of fibroblast activation protein (FAP) for targeting applications with pan-tumoral potential. OncoFAP binds to human FAP with affinity in the subnanomolar concentration range and cross-reacts with the murine isoform of the protein. We generated various fluorescent and radiolabeled derivatives of OncoFAP in order to study biodistribution properties and tumor-targeting performance in preclinical models. Fluorescent derivatives selectively localized in FAP-positive tumors implanted in nude mice with a rapid and homogeneous penetration within the neoplastic tissue. Quantitative in vivo biodistribution studies with a lutetium-177–labeled derivative of OncoFAP revealed a preferential localization in tumors at doses of up to 1,000 nmol/kg. More than 30% of the injected dose had already accumulated in 1 g of tumor 10 min after intravenous injection and persisted for at least 3 h with excellent tumor-to-organ ratios. OncoFAP also served as a modular component for the generation of nonradioactive therapeutic products. A fluorescein conjugate mediated a potent and FAP-dependent tumor cell killing activity in combination with chimeric antigen receptor (CAR) T cells specific to fluorescein. Similarly, a conjugate of OncoFAP with the monomethyl auristatin E-based Vedotin payload was well tolerated and cured tumor-bearing mice in combination with a clinical-stage antibody-interleukin-2 fusion. Collectively, these data support the development of OncoFAP-based products for tumor-targeting applications in patients with cancer.

Small organic ligands which selectively bind with high affinity to tumor-associated antigens are increasingly applied as targeting delivery vehicles of small payloads such as radionuclides (1, 2), drugs (3–5), and fluorophores (6, 7) to tumor sites. In principle, the use of small ligands for targeting applications offers several advantages compared to intact immunoglobulins, including superior penetration of solid neoplastic lesions (8), lower immunogenicity (9), and a reduced cost of goods (10). Low molecular weight compounds may reach their target in vivo in a matter of seconds, thanks to rapid extravasation after intravenous administration (8). A strikingly selective accumulation of small ligands in neoplastic masses has been demonstrated for a small number of targets including somatostatin receptor type 2 (SSTR-2) (11), prostate-specific membrane antigen (PSMA) (12), and carbonic anhydrase IX (CAIX) (13), for which high-affinity small organic ligands are available. Those ligands are typically specific for defined tumor entities, such as neuroendocrine tumors (11), prostate cancer (3), and clear cell renal cell carcinoma (2).¹⁷⁷Lu-DOTATATE (Lutathera), a small-molecule product targeting SSTR-2, has been approved based on phase III data in which a clinically meaningful 82% reduction in the risk of disease progression or death was demonstrated in patients with gastroenteropancreatic neuroendocrine tumors (GEP-NETs) (14). Similar data are expected from the currently ongoing phase III VISION trial for ¹⁷⁷Lu-PSMA-617 (clinical trial no. {"type":"clinical-trial","attrs":{"text":"NCT03511664","term_id":"NCT03511664"}}NCT03511664), a radiolabeled small molecule that binds with high affinity to PSMA and that enables targeted beta particle therapy in metastatic castration-resistant prostate cancer patients (15). PHC-102, a ^99mTc-labeled small-molecule derivative targeting CAIX, exhibited favorable uptake in primary and metastatic lesions in patients with renal cell carcinoma (RCC) (2). In light of the promising performance of small organic ligands, it would be desirable to discover and develop small molecules with a broader tumor-targeting potential, therefore covering multiple cancer types.Fibroblast activation protein (FAP) is a type II integral membrane serine protease which is abundantly expressed in the stroma of more than 90% of the epithelial cancers, including malignant breast, colorectal, skin, prostate, and pancreatic cancers (16, 17), while exhibiting a restricted expression in normal adult tissues (18, 19). Haberkorn and coworkers (1, 20, 21) have recently described a series of FAP ligands capable of selective accumulation in FAP-positive tumors in mice and in patients. One of these products (named FAPI-04) showed impressive tumor to background ratios at early time points (i.e., few hours after administration) in a broad range of different cancer types in patients. More than 28 tumor types including breast, lung, pancreatic, head and neck, esophagus, and colorectal cancer presented a remarkably high uptake of a FAP-targeted small molecule labeled with gallium-68 (1, 20, 21). For this reason, FAP has recently been dubbed as “the next billion-dollar target for theranostic products” (22).Here, we describe how the chemical modification of a quinoline moiety in position 8 led to the discovery of OncoFAP, a small organic FAP ligand with a dissociation constant in the subnanomolar concentration range. OncoFAP exhibited a strikingly selective and efficient tumor-targeting performance when equipped with various types of payloads, including radionuclides, fluorophores, and cytotoxic drugs. The targeting delivery of radionuclides to solid tumors is rapidly gaining in popularity, as it may open theranostic opportunities, associated with the use of gallium-68 for positron emission tomography (PET) imaging and of lutetium-177 for therapeutic applications (23). The delivery of fluorescein to tumors enables the conditional activation of chimeric antigen receptor (CAR) T cells, which display a potent biocidal activity only in the presence of fluorescein-labeled adaptor molecules specific to a tumor antigen (24, 25). Finally, small-molecule–drug conjugates (SMDCs) promise to represent a valid alternative to antibody–drug conjugates for cancer therapy, with better tumor penetration and a lower cost of goods (8, 26, 27).     

Reprogramming of primordial germ cells begins before migration into the genital ridge, making these cells inadequate donors for reproductive cloning

Germ cells undergo epigenetic modifications as they develop, which suggests that they may be ideal donors for nuclear transfer (cloning). In this study, nuclei from confirmed embryonic germ cells were used as donors to determine whether they are competent for cloning and at which stage they are most competent. Embryos cloned from migrating 10.5-days-postcoitum (dpc) primordial germ cells (PGCs) showed normal morphological development to midgestation but died shortly thereafter. In contrast, embryos cloned from later-stage germ cells were developmentally delayed at midgestation. Thus, donor germ cell age inversely correlated with the developmental stage attained by cloned embryos. The methylation status of the H19- and Snrpn-imprinting control regions in germ cell clones paralleled that of the donors, and revealed that demethylation, or erasure of imprints, was already initiated in PGCs at 10.5 dpc and was complete by 13.5 dpc. Similarly, clones derived from male 15.5-dpc germ cells showed increased methylation correlating with the initiation of de novo methylation that resets imprints at this stage, and clones from neonatal germ cells showed nearly complete methylation in the H19 imprinting control region. These results indicate that the epigenetic state of the donor nucleus is retained in cloned embryos, and that germ cells are therefore inadequate nuclear donors for cloning because they are either erasing or resetting epigenetic patterns.     

Liver frailty and all-cause mortality in the older participants of the Salus in Apulia Study

The liver contribution to the biological network underlying physical frailty in aging is underestimated. How best to measure this contribution magnitude and impact on health risk trajectories in frail individuals is not yet entirely clear. We analyzed the association of a novel liver frailty phenotype with the risk of death in older participants of the Salus in Apulia Study cohort. Clinical and physical examination, routine biomarkers, medical history, and anthropometry were analyzed in 1929 older adults (65?+). Physical frailty was classified by Cardiovascular Health Study criteria, and liver fibrosis risk by fibrosis-4 (FIB-4). The liver frailty phenotype was defined as physical frailty plus high-risk liver fibrosis (score?>?2.67). Physical frailty, high-risk liver fibrosis, and liver frailty subjects were compared to subjects without these conditions (non-frail). Proportional Cox regression tested the adjusted association between liver frailty and all-cause mortality for each category. The liver frailty prevalence was relatively low (3.8%), but higher in men (58.1%). Compared to non-frail older subjects, liver frailty subjects were significantly older (effect size (ES)???1.11, 95% confidence interval (CI)???1.35 to???0.87), with a lower education (ES 0.48, 95%CI 0.24 to 0.71) and higher multimorbidity (ES 15.81, 95%CI 4.20 to 27.41). Cox multivariate analyses showed a two-fold increased risk of overall mortality (hazard ratio 2.09, 95%CI 1.16–3.74) even after the adjustment for age, sex, education, and alcohol consumption. The liver frailty phenotype runs twice the risk of overall mortality compared with the non-frail population. This clinical tool, validated in a Southern Italian population, is based on simple sets of measures that can conveniently be assessed also in the primary care setting.

     

Novel de novo heterozygous loss-of-function variants in MED13L and further delineation of the MED13L haploinsufficiency syndrome

MED13L haploinsufficiency has recently been described as responsible for syndromic intellectual disability. We planned a search for causative gene variants in seven subjects with intellectual disability and overlapping dysmorphic facial features such as bulbous nasal tip, short mouth and straight eyebrows. We found two de novo frameshift variants in MED13L, consisting in single-nucleotide deletion (c.3765delC) and duplication (c.607dupT). A de novo nonsense variant (c.4420A>T) in MED13L was detected in a further subject in the course of routine whole-exome sequencing. By analyzing the clinical data of our patients along with those recently described in the literature, we confirm that there is a common, recognizable phenotype associated with MED13L haploinsufficiency, which includes intellectual disability and a distinctive facial appearance. Congenital heart diseases are found in some subjects with various degree of severity. Our observation of cleft palate, ataxia, epilepsy and childhood leukemia observed in single cases broadens the known clinical spectrum. Haploinsufficiency for MED13L should be considered in the differential diagnosis of the 1p36 microdeletion syndrome, due to overlapping dysmorphic facial features in some patients. The introduction of massive parallel-sequencing techniques into clinical practice is expected to allow for detection of other causative point variants in MED13L. Analysis of genomic data in connection with deep clinical evaluation of patients could elucidate genetic heterogeneity of the MED13L haploinsufficiency phenotype.     

Growth-related oncogene alpha induction of apoptosis in osteoarthritis chondrocytes

OBJECTIVE: To evaluate the apoptotic effect of the chemokine growth-related oncogene alpha (GROalpha), which we recently reported to be up-regulated in osteoarthritis (OA) chondrocytes. Chondrocyte apoptosis is considered to be a major determinant of cartilage damage in OA, a disease resulting from the aberrant production of inflammatory mediators (cytokines and chemokines) and effectors (matrix metalloproteinases and reactive oxygen and nitrogen species) by chondrocytes. METHODS: We investigated the apoptotic effect of GROalpha on isolated human cells and on in vitro-cultured cartilage explants by conventional methods (morphology, detection of DNA fragmentation in situ and in solution, exposure of phosphatidylserine) and by analysis of "early" biochemical events (plasma membrane depolarization, activation of caspase 3, and phosphorylation of c-Jun N-terminal kinase/stress-activated protein kinase). RESULTS: We clearly demonstrated that GROalpha was able to initiate a series of morphologic, biochemical, and molecular changes that led to chondrocyte apoptosis. Moreover, we found that additional signals delivered from the extracellular matrix (ECM) were essential in the control of chondrocyte susceptibility to GROalpha-induced apoptosis, since cell death was detected only when cells were stimulated after reestablishment of their proper interactions with the ECM, or in cartilage explant samples with reduced ECM, as indicated by decreased Safranin O staining. CONCLUSION: GROalpha can induce apoptosis in articular chondrocytes, and the induction is dependent upon additional signals from the ECM. These findings are relevant to understanding the pathogenesis of OA, in view of the availability of the GROalpha chemokine in the joint space in the course of this rheumatic disease.     

Interchangeability of biosimilar and biological reference product: updated regulatory positions and pre- and post-marketing evidence

Introduction: Since 2006, biosimilars have been available in several countries worldwide, thus allowing for potential savings in pharmaceutical expenditure. However, there have been numerous debates about the interchangeability of biosimilars and reference products based on concerns of immunogenicity by switching between biological products, which may cause lack of effect and toxicity.

Areas covered: The authors provide the reader with an overview of the different positions of regulatory authorities on the interchangeability and automatic substitution of biosimilars and reference products. Presently, the FDA allows automatic substitution without prescriber intervention if the biosimilar is interchangeable with reference products, while the European Medicines Agency delegate to each single EU member state.

Expert opinion: Different approaches in defining interchangeability and automatic substitution call for harmonization to increase confidence of healthcare professionals and patients about the clinical impact of switching. Networks of electronic healthcare records and administrative databases, potentially linkable to clinical charts and registries may rapidly assess frequency and benefit-risk profile of different switching patterns in routine care at different levels, thus integrating and strengthening pre-marketing evidence.     

Non-coronary cardiac events,younger age,and IVIG unresponsiveness increase the risk for coronary aneurysms in Italian children with Kawasaki disease

Clinical Rheumatology - Kawasaki disease (KD) is the most frequent cause of acquired heart disease in children in high-income countries because of coronary artery involvement. Risk factors for...