Options for imaging cellular therapeutics in vivo: a multi-stakeholder perspective.

Cell-based therapies have been making great advances toward clinical reality. Despite the increase in trial activity, few therapies have successfully navigated late-phase clinical trials and received market authorization. One possible explanation for this is that additional tools and technologies to enable their development have only recently become available. To support the safety evaluation of cell therapies, the Health and Environmental Sciences Institute Cell Therapy-Tracking, Circulation and Safety Committee, a multisector collaborative committee, polled the attendees of the 2017 International Society for Cell & Gene Therapy conference in London, UK, to understand the gaps and needs that cell therapy developers have encountered regarding safety evaluations in vivo. The goal of the survey was to collect information to inform stakeholders of areas of interest that can help ensure the safe use of cellular therapeutics in the clinic. This review is a response to the cellular imaging interests of those respondents. The authors offer a brief overview of available technologies and then highlight the areas of interest from the survey by describing how imaging technologies can meet those needs. The areas of interest include imaging of cells over time, sensitivity of imaging modalities, ability to quantify cells, imaging cellular survival and differentiation and safety concerns around adding imaging agents to cellular therapy protocols. The Health and Environmental Sciences Institute Cell Therapy-Tracking, Circulation and Safety Committee believes that the ability to understand therapeutic cell fate is vital for determining and understanding cell therapy efficacy and safety and offers this review to aid in those needs. An aim of this article is to share the available imaging technologies with the cell therapy community to demonstrate how these technologies can accomplish unmet needs throughout the translational process and strengthen the understanding of cellular therapeutics.

Mining scientific advice reports on cell-based products: Insight into the nonclinical development program.

AIMS: The field of cell-based therapies for human diseases is currently evolving from promising treatment options to established therapeutic concepts. The design of the nonclinical development program for cell-based products, intended to provide a rationale for treatment and to gain insight into the safety profile, is challenging because of limitations caused by species-specificity. The elements of the nonclinical package for cell-based products were evaluated using advice reports from the European Medicines Agency database from 2013 to 2018 to identify the approach followed for nonclinical development of these products. METHODS: The number and purpose of proposed and performed in vivo studies was recorded, as well as the type and design of in vitro and in vivo studies addressing biodistribution and tumorigenicity. Subsequently, the nonclinical development program was analysed for consistency across products. RESULTS: In vivo studies for cell-based therapies were primarily aimed at proof-of-concept (75/86), followed by addressing safety (64/86), biodistribution (49/86) and tumourigenicity (46/86). No animal studies were performed or proposed by sponsors or regulators for 6/86 products which contained cell types that have been studied in humans for a relatively long time. For one-third of the products in vivo biodistribution and/or tumourigenicity studies were not considered necessary. in vivo tumourigenicity studies were regarded as having limited value. CONCLUSIONS: Compared to more conventional medicinal products, the nonclinical development program for cell-based products was more tailored and focused on proof-of-concept. For tumourigenicity an in vitro approach may suffice. Total omission of in vivo studies appears to be possible for products with sufficient clinical experience.

Radiation modulates the peptide repertoire, enhances MHC class I expression, and induces successful antitumor immunotherapy.

Radiotherapy is one of the most successful cancer therapies. Here the effect of irradiation on antigen presentation by MHC class I molecules was studied. Cell surface expression of MHC class I molecules was increased for many days in a radiation dose-dependent manner as a consequence of three responses. Initially, enhanced degradation of existing proteins occurred which resulted in an increased intracellular peptide pool. Subsequently, enhanced translation due to activation of the mammalian target of rapamycin pathway resulted in increased peptide production, antigen presentation, as well as cytotoxic T lymphocyte recognition of irradiated cells. In addition, novel proteins were made in response to gamma-irradiation, resulting in new peptides presented by MHC class I molecules, which were recognized by cytotoxic T cells. We show that immunotherapy is successful in eradicating a murine colon adenocarcinoma only when preceded by radiotherapy of the tumor tissue. Our findings indicate that directed radiotherapy can improve the efficacy of tumor immunotherapy.

Risk factors in the development of stem cell therapy.

Stem cell therapy holds the promise to treat degenerative diseases, cancer and repair of damaged tissues for which there are currently no or limited therapeutic options. The potential of stem cell therapies has long been recognised and the creation of induced pluripotent stem cells (iPSC) has boosted the stem cell field leading to increasing development and scientific knowledge. Despite the clinical potential of stem cell based medicinal products there are also potential and unanticipated risks. These risks deserve a thorough discussion within the perspective of current scientific knowledge and experience. Evaluation of potential risks should be a prerequisite step before clinical use of stem cell based medicinal products.The risk profile of stem cell based medicinal products depends on many risk factors, which include the type of stem cells, their differentiation status and proliferation capacity, the route of administration, the intended location, in vitro culture and/or other manipulation steps, irreversibility of treatment, need/possibility for concurrent tissue regeneration in case of irreversible tissue loss, and long-term survival of engrafted cells. Together these factors determine the risk profile associated with a stem cell based medicinal product. The identified risks (i.e. risks identified in clinical experience) or potential/theoretical risks (i.e. risks observed in animal studies) include tumour formation, unwanted immune responses and the transmission of adventitious agents.Currently, there is no clinical experience with pluripotent stem cells (i.e. embryonal stem cells and iPSC). Based on their characteristics of unlimited self-renewal and high proliferation rate the risks associated with a product containing these cells (e.g. risk on tumour formation) are considered high, if not perceived to be unacceptable. In contrast, the vast majority of small-sized clinical trials conducted with mesenchymal stem/stromal cells (MSC) in regenerative medicine applications has not reported major health concerns, suggesting that MSC therapies could be relatively safe. However, in some clinical trials serious adverse events have been reported, which emphasizes the need for additional knowledge, particularly with regard to biological mechanisms and long term safety.

Dawn of Monitoring Regulatory T Cells in (Pre-)clinical Studies: Their Relevance Is Slowly Recognised.

Regulatory T cells (Tregs) have a prominent role in the control of immune homeostasis. Pharmacological impact on their activity or balance with effector T cells could contribute to (impaired) clinical responses or adverse events. Monitoring treatment-related effects on T cell subsets may therefore be part of (pre-)clinical studies for medicinal products. However, the extent of immune monitoring performed in studies for marketing authorisation and the degree of correspondence with data available in the public domain is not known. We evaluated the presence of T cell immunomonitoring in 46 registration dossiers of monoclonal antibodies indicated for immune-related disorders and published scientific papers. We found that the depth of Treg analysis in registration dossiers was rather small. Nevertheless, data on treatment-related Treg effects are available in public academia-driven studies (post-registration) and suggest that Tregs may act as a biomarker for clinical responses. However, public data are fragmented and obtained with heterogeneity of experimental approaches from a diversity of species and tissues. To reveal the potential added value of T cell (and particular Treg) evaluation in (pre-)clinical studies, more cell-specific data should be acquired, at least for medicinal products with an immunomodulatory mechanism. Therefore, extensive analysis of T cell subset contribution to clinical responses and the relevance of treatment-induced changes in their levels is needed. Preferably, industry and academia should work together to obtain these data in a standardised manner and to enrich our knowledge about T cell activity in disease pathogenesis and therapies. This will ultimately elucidate the necessity of T cell subset monitoring in the therapeutic benefit-risk assessment.

Dynamics of measles virus protein expression are reflected in the MHC class I epitope display.

Following measles virus (MV) infection, viral peptides are presented to CTL by MHC class I molecules on infected antigen presenting cells at widely different epitope densities. Whereas three MV epitopes (MV-M(211-219), MV-F(438-446) and MV-H(30-38)) derived from different structural proteins occur at regular densities, one peptide derived from the non-structural C protein (MV-C(84-92)) fully dominates the MV peptide display in HLA class I molecules on end-stage-infected human B cells. Here we demonstrate that this hierarchy in MV epitope density is not a constant, but varies with progression of infection. While MV-M(211-219), MV-F(438-446) and MV-H(30-38) epitopes were already presented by HLA class I molecules early in infection, expression of MV-C(84-92) was restricted to the later phases of infection. These dynamics in epitope densities correlated with features of MV protein expression. Synthesis of C protein mainly focused towards the final stages of infection, while the other MV proteins were more readily synthesised from earlier time points on, in line with the emergence of their respective epitopes. Furthermore, the most abundant MV epitope was derived from the most unstable viral protein and vice versa, suggesting that the stability of viral proteins may be an indicator for the final abundance of their epitopes. Thus, even though many other factors may influence the generation of peptide-MHC class I complexes, we here report that the regulation of viral protein expression seems closely linked to the viral MHC class I epitope display. Finally, the observed dynamics in viral epitope hierarchy may have important implications for the induction of antiviral T cell immunity.

Autoreactivity against induced or upregulated abundant self-peptides in HLA-A*0201 following measles virus infection.

Infectious agents have been implied as causative environmental factors in the development of autoimmunity. However, the exact nature of their involvement remains unknown. We describe a possible mechanism for the activation of autoreactive T cells induced by measles virus (MV) infection. The display of HLA-A*0201 associated peptides obtained from MV infected cells was compared with that from uninfected cells by mass spectrometry. We identified two abundant self peptides, IFI-6-16(74-82) and Hsp90beta(570-578), that were induced or upregulated, respectively, following infection. Their parental proteins, the type I interferon inducible protein IFI-6-16, and the beta chain of heat shock protein 90, have not been involved in MV pathogenesis. MV infection caused minor and major changes in the intracellular expression patterns of these proteins, possibly leading to altered peptide processing. CD8+ T cells capable of recognizing the self-peptides in the context of HLA-A*0201 were detectable at low basal levels in the neonatal and adult human T cell repertoire, but were functionally silent. In contrast, peptide-specific producing IFN-gamma producing effector cells were present in MV patients during acute infection. Thus, MV infection induces an enhanced display of self-peptides in MHC class I, which may lead to the temporary activation of autoreactive T cells.

Cutting edge: HLA-B27 acquires many N-terminal dibasic peptides: coupling cytosolic peptide stability to antigen presentation.

Ag presentation by MHC class I is a highly inefficient process because cytosolic peptidases destroy most peptides after proteasomal generation. Various mechanisms shape the MHC class I peptidome. We define a new one: intracellular peptide stability. Peptides with two N-terminal basic amino acids are more stable than other peptides. Such peptides should be overrepresented in the peptidome of MHC class I-associated peptides. HLA-B27 binding peptides use anchor residue R at P2 and, although most amino acids are allowed, particular amino acids are overrepresented at P1, including R and K. We show that such N-terminal dibasic peptides are indeed more efficiently presented by HLA-B27. This suggests that HLA-B27 can present peptides from Ags present in fewer copies than required for successful peptide generation for other MHC class I molecules.

MHC class I alleles and their exploration of the antigen-processing machinery.

At the cell surface, major histocompatibility complex (MHC) class I molecules present fragments of intracellular antigens to the immune system. This is the end result of a cascade of events initiated by multiple steps of proteolysis. Only a small part of the fragments escapes degradation by interacting with the peptide transporter associated with antigen presentation and is translocated into the endoplasmic reticulum lumen for binding to MHC class I molecules. Subsequently, these newly formed complexes can be transported to the plasma membrane for presentation. Every step in this process confers specificity and determines the ultimate result: presentation of only few fragments from a given antigen. Here, we introduce the players in the antigen processing and presentation cascade and describe their specificity and allelic variation. We highlight MHC class I alleles, which are not only different in sequence but also use different aspects of the antigen presentation pathway to their advantage: peptide acquaintance.

Vigorous but short-term gamma interferon T-cell responses against a dominant HLA-A*02-restricted measles virus epitope in patients with measles.

The absolute and relative abundance of major histocompatibility complex class I-presented viral epitopes is important in the induction and maintenance of antiviral cytotoxic-T-lymphocyte (CTL) responses. We demonstrate that the supra-abundant HLA-A*0201-restricted peptide KLWESPQEI of the measles virus nonstructural C protein induces strong gamma interferon CD8(+)-T-cell responses in children with acute measles. However, longitudinal analysis indicates that these responses are only short-lived. Thus, some viral epitopes that can be immunodominant during primary infection may fail to establish memory CTL responses.

A measles virus glycoprotein-derived human CTL epitope is abundantly presented via the proteasomal-dependent MHC class I processing pathway.

Peptides derived from measles virus (MV) are presented by MHC class I molecules at widely divergent levels, but it is currently unknown how functional these levels are. Here, for the first time, we studied the natural occurrence and the underlying processing events of a known MV CTL epitope derived from the fusion glycoprotein (MV-F) and restricted via HLA-B*2705. Using MHC-peptide elution of MV-infected cells followed by sensitive mass spectrometry we determined the naturally occurring sequence to be RRYPDAVYL, corresponding to MV-F(438-446). Its level of expression was enumerated at approximately 1500 copies per cell, which is considered to be abundant, but lies within the range described for other viral CTL epitopes in human MHC class I molecules. We found that processing of the MV-F(438-446) epitope occurs primarily via the classic MHC class I loading pathway, since presentation to CTL depends on both the transporter associated with antigen presentation (TAP) and the proteasome. Even though it is cotranslationally inserted into the ER, a major part of MV-F is located in the cytosol, where it accumulates rapidly in the presence of proteasome inhibitors. We therefore conclude that a substantial cytosolic turnover of MV-F, together with some excellent processing features of MV-F(438-446) precursors, such as precise C-terminal excision by proteasomes, efficient TAP transport and strong HLA binding, dictate the abundant functional expression of the MV-F(438-446) CTL epitope in HLA-B*2705 at the surface of MV-infected cells.