European Conditional Marketing Authorization in a Rapidly Evolving Treatment Landscape: A Comprehensive Study of Anticancer Medicinal Products in 2006-2020

Since 2006, the European conditional marketing authorization (CMA) aims to facilitate timely patient access to medicinal products for which there is an unmet medical need by accepting less comprehensive data than normally required. The granting of CMA requires a positive benefit-risk balance, unmet medical needs to be fulfilled, likely submission of comprehensive data postauthorization, and the benefit of immediate availability to outweigh the risks of data noncomprehensiveness. Since its first use, more than half of all CMAs represent (hemato-)oncology indications. Therefore, we aimed to investigate the conditions in which CMA has been applied for anticancer medicinal products and whether they have changed over time. We retrospectively assessed the European public assessment reports of the 30 anticancer medicinal products granted CMA in 2006-2020 (51% of all 59 CMAs). Comparison of 2006-2013 to 2014-2020 highlighted increased proportions of proactively requested CMAs (+40%), medicinal products that addressed unmet medical needs by providing a major therapeutic advantage over authorized treatments (+38%), and orphan designated indications (+32%). In contrast, it showed decreased proportions of medicinal products for which a scientific advisory group was consulted (-55%) and phase III randomized controlled trial data were available (-38%). This suggests that applicants and the European Medicines Agency have learned how to use the CMA as a regulatory tool, among others, through better planning and proactive interaction. However, the increasing number of granted CMAs complicates the establishment of unmet medical need and the benefit-risk balance, especially in crowded indications and when only phase II uncontrolled trials are available

A Comparison of Post-marketing Measures Imposed by Regulatory Agencies to Confirm the Tissue-Agnostic Approach

There are currently four anti-cancer medicinal products approved for a tissue-agnostic indication. This is an indication based on a common biological characteristic rather than the tissue of origin. To date, the regulatory experience with tissue-agnostic approvals is limited. Therefore, we compared decision-making aspects of the first tissue-agnostic approvals between the Food and Drug Administration (FDA), European Medicines Agency (EMA) and Pharmaceuticals and Medical Devices Agency (PMDA). Post-marketing measures (PMMs) related to the tissue-agnostic indication were of specific interest. The main data source was the publicly available review documents. The following data were collected: submission date, approval date, clinical trials and datasets, and PMMs. At the time of data collection, the FDA and PMDA approved pembrolizumab, larotrectinib, and entrectinib for a tissue-agnostic indication, while the EMA approved larotrectinib and entrectinib for a tissue-agnostic indication. There were differences in analysis sets (integrated vs. non-integrated), submission dates and requests for data updates between agencies. All agencies had outstanding issues that needed to be addressed in the post-market setting. For pembrolizumab, larotrectinib and entrectinib, the number of imposed PMMs varied between one and eight, with the FDA requesting the most PMMs compared to the other two agencies. All agencies requested at least one PMM per approval to address the remaining uncertainties related to the tissue-agnostic indication. The FDA and EMA requested data from ongoing and proposed trials, while the PMDA requested data from use-result surveys. Confirmation of benefit in the post-marketing setting is an important aspect of tissue-agnostic approvals, regardless of agency. Nonetheless, each approach to confirm benefit has its inherent limitations. Post-marketing data will be essential for the regulatory and clinical decisions-making of medicinal products with a tissue-agnostic indication

The EMA Assessment of Asciminib for the Treatment of Adult Patients With Philadelphia Chromosome-Positive Chronic Myeloid Leukemia in Chronic Phase Who Were Previously Treated With at Least Two Tyrosine Kinase Inhibitors

Asciminib is an allosteric high-affinity tyrosine kinase inhibitor (TKI) of the BCR-ABL1 protein kinase. This kinase is translated from the Philadelphia chromosome in chronic myeloid leukemia (CML). Marketing authorization for asciminib was granted on August 25, 2022 by the European Commission. The approved indication was for patients with Philadelphia chromosome-positive CML in the chronic phase which have previously been treated with at least 2 TKIs. Clinical efficacy and safety of asciminib were evaluated in the open-label, randomized, phase III ASCEMBL study. The primary endpoint of this trial was major molecular response (MMR) rate at 24 weeks. A significant difference in MRR rate was shown between the asciminib treated population and the bosutinib control group (25.5% vs. 13.2%, respectively, P=.029). In the asciminib cohort, adverse reactions of at least grade 3 with an incidence≥5% were thrombocytopenia, neutropenia, increased pancreatic enzymes, hypertension, and anemia. The aim of this article is to summarize the scientific review of the application which led to the positive opinion by the European Medicines Agency's Committee for Medicinal Products for Human Use.</p

A Rac1 inhibitory peptide suppresses antibody production and paw swelling in the murine collagen-induced arthritis model of rheumatoid arthritis

Introduction: The Rho family GTPase Rac1 regulates cytoskeletal rearrangements crucial for the recruitment, extravasation and activation of leukocytes at sites of inflammation. Rac1 signaling also promotes the activation and survival of lymphocytes and osteoclasts. Therefore, we assessed the ability of a cell-permeable Rac1 carboxy-terminal inhibitory peptide to modulate disease in mice with collagen-induced arthritis (CIA). Methods: CIA was induced in DBA/1 mice, and in either early or chronic disease, mice were treated three times per week by intraperitoneal injection with control peptide or Rac1 inhibitory peptide. Effects on disease progression were assessed by measurement of paw swelling. Inflammation and joint destruction were examined by histology and radiology. Serum levels of anti-collagen type II antibodies were measured by enzyme-linked immunosorbent assay. T-cell phenotypes and activation were assessed by fluorescence-activated cell sorting analysis. Results were analyzed using Mann-Whitney U and unpaired Student t tests. Results: Treatment of mice with Rac1 inhibitory peptide resulted in a decrease in paw swelling in early disease and to a lesser extent in more chronic arthritis. Of interest, while joint destruction was unaffected by Rac1 inhibitory peptide, anti-collagen type II antibody production was significantly diminished in treated mice, in both early and chronic arthritis. Ex vivo, Rac1 inhibitory peptide suppressed T-cell receptor/CD28-dependent production of tumor necrosis factor a, interferon. and interleukin-17 by T cells from collagen-primed mice, and reduced induction of ICOS and CD154, T-cell costimulatory proteins important for B-cell help. Conclusions: The data suggest that targeting of Rac1 with the Rac1 carboxy-terminal inhibitory peptide may suppress T-cell activation and autoantibody production in autoimmune disease. Whether this could translate into clinically meaningful improvement remains to be show

The chemorepellent Slit3 promotes monocyte migration

Directional migration is an essential step for monocytes to infiltrate sites of inflammation, a process primarily regulated by chemoattractants. Slits are large matrix proteins that are secreted by endothelial cells; they were reported to inhibit the chemoattractant-induced migration of different cell types, including leukocytes. The aim of this study was to determine the effect of Slit3 on primary monocyte migration and to address the underlying mechanisms. We show that Roundabout (Robo)1, one of the Robo receptors that recognize Slit3, is the only Robo homolog expressed by CD14+ monocytes. Interestingly, we found that stimulation with Slit3 increased the spontaneous and chemoattractant-induced migration of primary monocytes in vitro and increased the myeloid cell recruitment during peritoneal inflammation in vivo. In addition, Slit3 did not seem to act as a chemoattractant itself; it promoted directed migration triggered by chemoattractants, such as CXCL12, by inducing a chemokinetic effect. We further show that Slit3 prevented monocyte spreading and induced rounding of spread monocytes without affecting monocyte adhesion. Stimulation with Slit3 was not associated with changes in the levels of phosphorylated p38, p42/p44, or Src, known regulators of monocyte migration, but it directly acts on molecular pathways involved in basal leukocyte migration by activating RhoA. These findings show an unexpected response of monocytes to Slit3 and add insights into the possible role of Slit proteins during inflammatory cell recruitment

Nucleophosmin1 Is a Negative Regulator of the Small GTPase Rac1

The Rac1 GTPase is a critical regulator of cytoskeletal dynamics and controls many biological processes, such as cell migration, cell-cell contacts, cellular growth and cell division. These complex processes are controlled by Rac1 signaling through effector proteins. We have previously identified several effector proteins of Rac1 that also act as Rac1 regulatory proteins, including caveolin-1 and PACSIN2. Here, we report that Rac1 interacts through its C-terminus with nucleophosmin1 (NPM1), a multifunctional nucleo-cytoplasmic shuttling protein with oncogenic properties. We show that Rac1 controls NPM1 subcellular localization. In cells expressing active Rac1, NPM1 translocates from the nucleus to the cytoplasm. In addition, Rac1 regulates the localization of the phosphorylated pool of NPM1 as this pool translocated from the nucleus to the cytosol in cells expressing activated Rac1. Conversely, we found that expression of NPM1 limits Rac1 GTP loading and cell spreading. In conclusion, this study identifies NPM1 as a novel, negative regulator of Rac1

Rac1 interacts through its C-terminus with NPM1.

<p>(A) Schematic representation of the Rho-like GTPase C-terminal peptides fused to a protein transduction domain as used in this study. (B) Pull-down (PD) experiments were performed using lysates from HeLa cells (upper panel) or Jurkat T-cells (lower panel) with a control peptide, wild-type and mutant Rac1 C-terminal peptides, Rac2, RhoA and RhoG C-terminal peptides. Association of endogenous NPM1 was detected by immunoblotting (IB) with an NPM1 specific monoclonal antibody (representative example out of three independent experiments is shown). (C) Pull-down (PD) experiment was performed using lysates from HeLa cells with a control peptide, wild-type and mutant Rac1 C-terminal peptides. Association of phosphorylated NPM1 (pNPM1) was detected by immunoblotting (IB) with a phospho-specific NPM1 antibody. (representative example out of two independent experiments is shown). (D) Pull-down (PD) experiment using full-length Rac1 and Rac1 lacking the C-terminus (ΔC) both fused to GST or GST alone was performed with lysates from HeLa cells exogenously expressing GFP-NPM1. Association of NPM1 was detected by immunoblotting (IB) with a GFP specific monoclonal antibody. The ponceau staining shows the presence of the different GST constructs. ED: effector domain of Rac1, HV: hypervariable domain of Rac1, PTD: protein transduction domain, Rac1 PPP→AAA, Rac1 RKR→AAA: Rac1 C-terminal peptide mutants where the three prolines, or RKR sequence were replaced by alanine residues, respectively, TCL: total cell lysates, PD: pull-down, IB: immunoblotting.</p

Rac1 activity alters phospho-NPM1 distribution.

<p>HeLa cells were grown on glass cover slips and transfected with either mCherry Rac1Q61L (A) or mCherry Rac1V12G (B). After 24 hours, cells were fixed and stained with a phospho-specific rabbit antibody against NPM1 followed by a goat anti-Rabbit IgG Alexa488, the nuclear dye DAPI and the F-actin binding toxin Phalloidin fluorescently labeled with Alexa 633 and analyzed by confocal laser scanning microscopy. Higher magnification images of the boxed areas are included. (C) Hela cells were transfected with an empty vector, myc-tagged Rac1 WT or two different myc-tagged constitutively active Rac1 mutants; Rac1V12G and Rac1Q61L and after 24 hours lysates were made and endogenous NPM1 as well as phospho-NPM1 (pNPM1) were detected by immunoblotting (IB) with a NPM1 specific or a NPM1 phospho-specific antibody. EV: empty vector. Scale bars, 20 µm.</p

NPM1 is a negative regulator of Rac1.

<p>(A) Rac1 GTP loading was measured by biotinylated Pak-CRIB peptide-based pull-down (PD) with lysates of GFP control-transfected HeLa cells or HeLa cells transfected with GFP-NPM1. Rac1, Rac1 GTP and GFP-NPM1 were detected by immunoblotting with Rac1 and GFP specific monoclonal antibodies respectively. The bar graph shows the relative levels of Rac1 GTP levels compared to that in control as determined by quantification of Western blots (representative example out of three independent experiments is shown). TCL: total cell lysates, PD: pull-down. (B) Cell spreading on fibronectin coated ECIS-electrodes was determined for mock-transfected HeLa cells or HeLa cells expressing GFP-tagged NPM1. The results are depicted as normalized mean resistance of three independent experiments. (n = 3) *p<0.05, **p<0.01. (C) Rac1 GTP loading was measured by biotinylated Pak-CRIB peptide-based pull-down (PD) with lysates of GFP control-transfected HeLa cells or HeLa cells transfected with GFP-NPM1 in the absence or presence of the GEF protein Tiam-C-1199 tagged with HA. Rac1 and Rac1 GTP were detected by immunoblotting with Rac1 specific monoclonal antibody. GFP-NPM1 and Tiam-C-1199-HA were detected by immunoblotting with GFP and HA specific monoclonal antibodies, respectively.</p