Do the Outcomes of Clinical Efficacy Trials Matter in Regulatory Decision-Making for Biosimilars?

BACKGROUND: There is an increasing body of evidence supporting a more flexible approach in clinical data requirements for the approval of more complex biosimilar substances such as monoclonal antibodies (mAbs). OBJECTIVE: The aim of this paper is to further analyse the role of quality/chemistry, manufacturing and controls (CMC) and clinical data for the conclusion on biosimilarity and the decision on marketing authorisation (MA). METHODS: In the present study, we analysed the MA applications (MAAs) of all 33 mAbs and three fusion proteins evaluated by the European Medicines Agency (EMA) between July 2012 and November 2022 with special emphasis on all submitted rituximab (four products) and trastuzumab (seven products) biosimilar candidates, including withdrawn applications. For the two withdrawn applications, the comparative efficacy trials suggested biosimilarity, but the quality/CMC package was not accepted by EMA. We therefore investigated whether a negative MAA outcome could have been predicted based on the evidence generated in the quality/CMC packages, regardless of clinical trial data. For this purpose, we reviewed the respective European Public Assessment Reports (EPARs) or withdrawal assessment reports, and the first regulatory assessments for all these 36 MAAs (i.e. day 120 of the centralized procedure), which are not publicly available. During EMA review, where significant issues are identified which would preclude a marketing authorisation, these issues are raised as questions to the applicant and are classified as major objections (MO). RESULTS: In 67% of cases, the outcome of the quality and clinical assessment was the same, i.e. both the quality and clinical assessments either supported approval or did not support approval. In 11% of cases, MO were identified in the quality part of the submission but not in the clinical data. In 22% of cases, MO were raised on the clinical data package but not on the quality data. However, we found no instance where seemingly negative clinical data, including failed efficacy trials, led to a negative overall decision. In each instance, the failure to confirm similar clinical performance in all investigated aspects was eventually viewed as not being related to the biosimilar per se but as being due to imbalances in the trial arms, immaturity of secondary endpoint results, change in the reference product, or even chance findings. Furthermore, when performing an in-depth analysis of the quality and clinical packages of trastuzumab and rituximab biosimilars, we found that in no case were clinical trial data necessary to resolve residual uncertainties regarding the quality part. CONCLUSION: The results further support the argument that sufficient evidence for biosimilarity can be obtained from a combination of analytical and functional testing and pharmacokinetic studies which may also generate immunogenicity data. This calls into question the usefulness of comparative efficacy studies for the purposes of regulatory decision-making when approving biosimilar mAbs and fusion proteins.

A Data Driven Approach to Support Tailored Clinical Programs for Biosimilar Monoclonal Antibodies.

Biosimilar monoclonal antibodies (mAbs) have been approved in the European Union since 2013 and have been demonstrated to reduce healthcare costs and to expand patient access. Biosimilarity is mainly established on the basis of demonstrated similarity of relevant quality attributes (QAs), determined by comprehensive physiochemical and functional analyses, and demonstration of bioequivalence. In addition, comparative efficacy/safety studies have been requested for all approved biosimilar mAbs so far, although the European Medicines Agency (EMA) Guidelines state that such confirmatory clinical trials may not be necessary in specific circumstances. In order to evaluate the degree of analytical similarity, how residual uncertainty regarding biosimilarity was resolved, and the value of clinical data, we analyzed the quality and clinical data packages for authorized adalimumab (7 products) and bevacizumab (5 products) biosimilars. The percentage of biosimilar batches meeting the similarity range for QAs, as defined by the biosimilar manufacturer based on a comprehensive characterization of the EU reference product (RP), was determined and clinical data were reviewed. Our analyses show that QAs of approved adalimumab and bevacizumab biosimilars have varying concordance with the EU-RP similarity range. In this study, we found that clinical efficacy data played a limited role in addressing quality concerns. Therefore, we encourage a regulatory review of the standards for clinical data requirements for mAb and fusion protein biosimilars. This study outlines a quality data driven approach for facilitating tailored clinical programs for biosimilars.

Regulatory Evaluation of Biosimilars: Refinement of Principles Based on the Scientific Evidence and Clinical Experience.

The World Health Organization (WHO) guidelines on evaluation of similar biotherapeutic products (SBPs; also called biosimilars) were adopted by the WHO Expert Committee on Biological Standardization (ECBS) in 2009. In 2019, the ECBS considered that a more tailored and potentially reduced clinical data package may be acceptable in cases where this was clearly supported by the available scientific evidence. The goal of this publication is to review the current clinical experience and scientific evidence and to provide an expert perspective for updating the WHO guidelines to provide more flexibility and clarity. As the first step, the relevant guidelines by other regulatory bodies were reviewed in order to identify issues that might help with updating the WHO guidelines. Next, a literature search was conducted for information on the long-term efficacy, safety, and immunogenicity of biosimilars to identify possible long-term problems. Finally, a search for articles concerning the role of clinical studies in the benefit-risk evaluation of biosimilars was conducted. The analysis of other guidelines suggested that the WHO guidelines may need more emphasis on the importance of the state-of-the-art physicochemical and structural comparability exercise and in vitro functional testing. The use of "foreign" reference product will also need clarifications. The value of in vivo toxicological tests in the development of biosimilars is questionable, and the non-clinical part needs revisions accordingly. The concepts of "totality of evidence," "stepwise development," and "residual uncertainty" were applied in the evaluation of the clinical sections of the guideline. The review of long-term safety and efficacy demonstrated the robustness of the current biosimilar development concept. The analysis of the roles of different development phases suggested that the large efficacy, safety, and immunogenicity studies are, in most cases, redundant. The residual uncertainty of safety, immunogenicity, and efficacy of biosimilars that has shaped the current regulatory guidelines is now substantially reduced. This will allow the re-evaluation of the non-clinical and clinical requirements of the current WHO main guideline. The shift of the relative impact of the development phases towards physico-chemical and in vitro functional testing will provide a relief to the manufacturers and new challenges to the regulators.

Biosimilar regulation in the EU.

In the EU, the EMA has been working with biosimilars since 1998. This experience is crystallized in the extensive set of guidelines, which range from basic principles to details of clinical trials. While the guidance may appear complicated, it has enabled the development of biosimilars, of which 21 have managed to get marketing authorization. Currently marketed biosimilars in the EU have a good track record in safety and traceability. No biosimilars have been withdrawn from the market because of safety concerns. The most controversial issues with biosimilars are immunogenicity and extrapolation of therapeutic indications. The available data for these topics do not raise concerns among EU regulators. Interchangeability and substitution are regulated by individual EU member states.

Biosimilars: what clinicians should know.

Biosimilar medicinal products (biosimilars) have become a reality in the European Union and will soon be available in the United States. Despite an established legal pathway for biosimilars in the European Union since 2005 and increasing and detailed regulatory guidance on data requirements for their development and licensing, many clinicians, particularly oncologists, are reluctant to consider biosimilars as a treatment option for their patients. Major concerns voiced about biosimilars relate to their pharmaceutical quality, safety (especially immunogenicity), efficacy (particularly in extrapolated indications), and interchangeability with the originator product. In this article, the members and experts of the Working Party on Similar Biologic Medicinal Products of the European Medicines Agency (EMA) address these issues. A clear understanding of the scientific principles of the biosimilar concept and access to unbiased information on licensed biosimilars are important for physicians to make informed and appropriate treatment choices for their patients. This will become even more important with the advent of biosimilar monoclonal antibodies. The issues also highlight the need for improved communication between physicians, learned societies, and regulators.

LKB1 in endothelial cells is required for angiogenesis and TGFbeta-mediated vascular smooth muscle cell recruitment.

Inactivation of the tumor suppressor kinase Lkb1 in mice leads to vascular defects and midgestational lethality at embryonic day 9-11 (E9-E11). Here, we have used conditional targeting to investigate the defects underlying the Lkb1(-/-) phenotype. Endothelium-restricted deletion of Lkb1 led to embryonic death at E12.5 with a loss of vascular smooth muscle cells (vSMCs) and vascular disruption. Transforming growth factor beta (TGFbeta) pathway activity was reduced in Lkb1-deficient endothelial cells (ECs), and TGFbeta signaling from Lkb1(-/-) ECs to adjacent mesenchyme was defective, noted as reduced SMAD2 phosphorylation. The addition of TGFbeta to mutant yolk sac explants rescued the loss of vSMCs, as evidenced by smooth muscle alpha actin (SMA) expression. These results reveal an essential function for endothelial Lkb1 in TGFbeta-mediated vSMC recruitment during angiogenesis.

LKB1 signaling in mesenchymal cells required for suppression of gastrointestinal polyposis.

Germline mutations in STK11 (also known as LKB1) are found in individuals with Peutz-Jeghers syndrome (PJS) manifesting with gastrointestinal polyps that contain a prominent stromal component. Epithelia in polyps of Stk11(+/-) mice can retain a functional copy of Stk11 (refs. 2,3), and loss of heterozygosity is not an obligate feature of human polyps, raising the possibility of non-epithelial origins in tumorigenesis. Here we show that either monoallelic or biallelic loss of murine Stk11 limited to Tagln-expressing mesenchymal cells results in premature postnatal death as a result of gastrointestinal polyps indistinguishable from those in PJS. Stk11-deficient mesenchymal cells produced less TGFbeta, and defective TGFbeta signaling to epithelial cells coincided with epithelial proliferation. We also noted TGFbeta signaling defects in polyps of individuals with PJS, suggesting that the identified stromal-derived mechanism of tumor suppression is also relevant in PJS.

The LKB1 tumor suppressor kinase in human disease.

Inactivating germline mutations in the LKB1 gene underlie Peutz-Jeghers syndrome characterized by hamartomatous polyps and an elevated risk for cancer. Recent studies suggest the involvement of LKB1 also in more common human disorders including diabetes and in a significant fraction of lung adenocarcinomas. These observations have increased the interest towards signaling pathways of this tumor suppressor kinase. The recent breakthroughs in understanding the molecular functions of the LKB1 indicate its contribution as a regulator of cell polarity, energy metabolism and cell proliferation. Here we review how the substrates and cellular functions of LKB1 may be linked to Peutz-Jeghers syndrome and other diseases, and discuss how some of the molecular changes associated with altered LKB1 signaling might be used in therapeutic approaches.

Multiple angiopoietin recombinant proteins activate the Tie1 receptor tyrosine kinase and promote its interaction with Tie2.

The Tie1 receptor tyrosine kinase was isolated over a decade ago, but so far no ligand has been found to activate this receptor. Here, we have examined the potential of angiopoietins, ligands for the related Tie2 receptor, to mediate Tie1 activation. We show that a soluble Ang1 chimeric protein, COMP-Ang1, stimulates Tie1 phosphorylation in endothelial cells with similar kinetics and angiopoietin dose dependence when compared with Tie2. The phosphorylation of overexpressed Tie1 was weakly induced by COMP-Ang1 also in transfected cells that do not express Tie2. When cotransfected, Tie2 formed heteromeric complexes with Tie1, enhanced Tie1 activation, and induced phosphorylation of a kinase-inactive Tie1 in a ligand-dependent manner. Tie1 phosphorylation was also induced by native Ang1 and Ang4, although less efficiently than with COMP-Ang1. In conclusion, we show that Tie1 phosphorylation is induced by multiple angiopoietin proteins and that the activation is amplified via Tie2. These results should be important in dissecting the signal transduction pathways and biological functions of Tie1.

Bmx tyrosine kinase transgene induces skin hyperplasia, inflammatory angiogenesis, and accelerated wound healing.

The Bmx gene, a member of the Tec family of nonreceptor protein tyrosine kinases, is expressed in arterial endothelium and in certain hematopoietic and epithelial cells. Previous in vitro studies have implicated Bmx signaling in cell migration and survival and suggested that it contributes to the progression of prostate carcinomas. However, the function of Bmx in normal tissues in vivo is unknown. We show here that Bmx expression is induced in skin keratinocytes during wound healing. To analyze the role of Bmx in epidermal keratinocytes in vivo, we generated transgenic mice overexpressing Bmx in the skin. We show that Bmx overexpression accelerates keratinocyte proliferation and wound reepithelialization. Bmx expression also induces chronic inflammation and angiogenesis in the skin, and gene expression profiling suggests that this occurs via cytokine-mediated recruitment of inflammatory cells. Our studies provide the first data on Bmx function in vivo and form the basis of evaluation of its role in epithelial neoplasia.

Etk/Bmx transactivates vascular endothelial growth factor 2 and recruits phosphatidylinositol 3-kinase to mediate the tumor necrosis factor-induced angiogenic pathway.

Tumor necrosis factor (TNF), via its receptor 2 (TNFR2), induces Etk (or Bmx) activation and Etk-dependent endothelial cell (EC) migration and tube formation. Because TNF receptor 2 lacks an intrinsic kinase activity, we examined the kinase(s) mediating TNF-induced Etk activation. TNF induces a coordinated phosphorylation of vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and Etk, which is blocked by VEGFR2-specific inhibitors. In response to TNF, Etk and VEGFR2 form a complex resulting in a reciprocal activation between the two kinases. Subsequently, the downstream phosphatidylinositol 3-kinase (PI3K)-Akt signaling (but not signaling through phospholipase C-gamma) was initiated and directly led to TNF-induced EC migration, which was significantly inhibited by VEGFR2-, PI3K-, or Akt-specific inhibitors. Phosphorylation of VEGFR2 at Tyr-801 and Tyr-1175, the critical sites for VEGF-induced PI3K-Akt signaling, was not involved in TNF-mediated Akt activation. However, TNF induces phosphorylation of Etk at Tyr-566, directly mediating the recruitment of the p85 subunit of PI3K. Furthermore, TNF- but not VEGF-induced activation of VEGFR2, Akt, and EC migration are blunted in EC genetically deficient with Etk. Taken together, our data demonstrated that TNF induces transactivation between Etk and VEGFR2, and Etk directly activates PI3K-Akt angiogenic signaling independent of VEGF-induced VEGFR2-PI3K-Akt signaling pathway.

p130Cas Couples the tyrosine kinase Bmx/Etk with regulation of the actin cytoskeleton and cell migration.

Bmx/Etk, a member of the Tec/Btk family of nonreceptor kinases, has recently been shown to mediate cell motility in signaling pathways that become activated upon integrin-mediated cell adhesion (Chen, R., Kim, O., Li, M., Xiong, X., Guan, J. L., Kung, H. J., Chen, H., Shimizu, Y., and Qiu, Y. (2001) Nat Cell Biol. 3, 439-444). The molecular mechanisms of Bmx-induced cell motility have so far remained unknown. Previous studies by us and others have demonstrated that a complex formation between the docking protein p130Cas (Cas) and the adapter protein Crk is instrumental in connecting several stimuli to the regulation of actin cytoskeleton and cell motility. We demonstrate here that expression of Bmx leads to an interaction between Bmx and Cas at membrane ruffles, which are sites of active actin remodeling in motile cells. Expression of Bmx also enhances tyrosine phosphorylation of Cas and Cas.Crk complex formation, and coexpression of Bmx with Cas results in an enhanced membrane ruffling and haptotactic cell migration. Importantly, a mutant form of Bmx that fails to interact with Cas also fails to induce cell migration. Furthermore, expression of a dominant-negative form of Cas that is incapable of interacting with Crk inhibits Bmx-induced membrane ruffling and cell migration. These studies suggest that Bmx-Cas interaction, phosphorylation of Cas by Bmx, and subsequent Cas.Crk complex formation functionally couple Bmx to the regulation of actin cytoskeleton and cell motility.

Synthesis of Optically Active endo,endo Bicyclo[2.2.2]octane-2,5-diol, Bicyclo[2.2.2]octane-2,5-dione, and Related Compounds.

Optically active C(2)-symmetric (1S,2S,4S,5S)-bicyclo[2.2.2]octane-2,5-diol ((+)-12; 98% ee) and several selectively protected optically active intermediates useful for synthetic transformations were synthesized via a 1,2-carbonyl transposition route starting from the easily available optically active (1R,4S,6S)-6-hydroxybicyclo[2.2.2]octan-2-one ((-)-2). The synthetic route also allowed the preparation of optically active (1S,4S)-bicyclo[2.2.2]octane-2,5-dione ((+)-14; 98% ee).