Accelerated Development of COVID-19 Vaccines: Technology Platforms, Benefits, and Associated Risks.

Multiple preventive COVID-19 vaccines have been developed during the ongoing SARS coronavirus (CoV) 2 pandemic, utilizing a variety of technology platforms, which have different properties, advantages, and disadvantages. The acceleration in vaccine development required to combat the current pandemic is not at the expense of the necessary regulatory requirements, including robust and comprehensive data collection along with clinical product safety and efficacy evaluation. Due to the previous development of vaccine candidates against the related highly pathogenic coronaviruses SARS-CoV and MERS-CoV, the antigen that elicits immune protection is known: the surface spike protein of SARS-CoV-2 or specific domains encoded in that protein, e.g., the receptor binding domain. From a scientific point of view and in accordance with legal frameworks and regulatory practices, for the approval of a clinic trial, the Paul-Ehrlich-Institut requires preclinical testing of vaccine candidates, including general pharmacology and toxicology as well as immunogenicity. For COVID-19 vaccine candidates, based on existing platform technologies with a sufficiently broad data base, pharmacological-toxicological testing in the case of repeated administration, quantifying systemic distribution, and proof of vaccination protection in animal models can be carried out in parallel to phase 1 or 1/2 clinical trials. To reduce the theoretical risk of an increased respiratory illness through infection-enhancing antibodies or as a result of Th2 polarization and altered cytokine profiles of the immune response following vaccination, which are of specific concern for COVID-19 vaccines, appropriate investigative testing is imperative. In general, phase 1 (vaccine safety) and 2 (dose finding, vaccination schedule) clinical trials can be combined, and combined phase 2/3 trials are recommended to determine safety and efficacy. By applying these fundamental requirements not only for the approval and analysis of clinical trials but also for the regulatory evaluation during the assessment of marketing authorization applications, several efficacious and safe COVID-19 vaccines have been licensed in the EU by unprecedentedly fast and flexible procedures. Procedural and regulatory-scientific aspects of the COVID-19 licensing processes are described in this review.

[Combination therapies in immuno-oncology: differentiated regulatory approaches]. / Kombinationstherapien in der Immunonkologie: Differenzierte regulatorische Herangehensweisen.

Combination therapies of different drugs are an integral part of medicine. However, there is a scientific and regulatory need to understand the contribution of each drug to the overall effect, i.e., nonclinical and clinical development programs have to consider these aspects and need to be designed accordingly. Many drugs are currently under development that attempt to control malignant diseases in the long term by using and activating components of the patient's own immune system. The term immuno-oncology is often used in this context. Medicines that are developed and used for immuno-oncology can be assigned to completely different classes of medicines.This article provides an analysis of combination therapies in immuno-oncology with medicinal products produced by biotechnological manufacturing. This encompasses checkpoint inhibitors, genetically modified cell therapies, tumor vaccines, and oncolytic viruses. The challenges in clinical development are demonstrated on the basis of this heterogenous group of approved immuno-oncological drugs that have been investigated in combination therapies. Due to the different characteristics and number of combination partners, an individually tailored program must be designed for each development program and there is no standard solution.

Extrapolation in the development of paediatric medicines: examples from approvals for biological treatments for paediatric chronic immune-mediated inflammatory diseases.

The European Union (EU) Paediatric Regulation requires that all new medicinal products applying for a marketing authorisation (MA) in the EU provide a paediatric investigation plan (PIP) covering a clinical and non-clinical trial programme relating to the use in the paediatric population, unless a waiver applies. Conducting trials in children is challenging on many levels, including ethical and practical issues, which may affect the availability of the clinical evidence. In scientifically justified cases, extrapolation of data from other populations can be an option to gather evidence supporting the benefit-risk assessment of the medicinal product for paediatric use. The European Medicines Agency (EMA) is working on providing a framework for extrapolation that is scientifically valid, reliable and adequate to support MA of medicines for children. It is expected that the extrapolation framework together with therapeutic area guidelines and individual case studies will support future PIPs. Extrapolation has already been employed in several paediatric development programmes including biological treatment for immune-mediated diseases. This article reviews extrapolation strategies from MA applications for products for the treatment of juvenile idiopathic arthritis, paediatric psoriasis and paediatric inflammatory bowel disease. It also provides a summary of extrapolation advice expressed in relevant EMA guidelines and initiatives supporting the use of alternative approaches in paediatric medicine development.

No association of IL-12p40 pro1.1 polymorphism with juvenile idiopathic arthritis.

BACKGROUND: IL-12p40 plays an important role in the activation of the T-cell lines like Th17 and Th1-cells. Theses cells are crucial in the pathogenesis of juvenile idiopathic arthritis. A polymorphism in its promoter region and the genotype IL12p40 pro1.1 leads to a higher production of IL-12p40. We studied whether there is a difference in the distribution of the genotype in patients with JIA and the healthy population. METHODS: In 883 patients and 321 healthy controls the IL-12p40 promoter genotype was identified by ARMS-PCR. RESULTS: There is no association of IL-12p40 pro polymorphism neither in patients with JIA compared to controls nor in subtypes of JIA compared to oligoarthritis. We found a non-significant tendency of a higher prevalence of the genotype pro1.1 in systemic arthritis (32.4%) and in rheumatoid factor negative polyarthritis (30.5%) and a lower pro1.1 genotype in persistent oligoarthritis (20.7%) and in enthesitis-related arthritis (17%). Likelihood of the occurrence of genotype IL12-p40 pro1.1 in patients with systemic arthritis (OR 1.722, CI 95% 1.344-2.615, p 0.0129) and RF-negative polyarthritis (OR 1.576, CI 95% 1.046-2.376, p 0.0367) compared to persistent oligoarthritis was significantly higher. This was also true for comparison of their homozygous genotypes IL-12p40 pro 1.1 and 2.2 in systemic arthritis (OR 1.779, CI 95 % 1.045-3.029, p 0.0338). However, in Bonferroni correction for multiple hypothesis this was not significant. CONCLUSION: A tendency of a higher prevalence of the genotype IL-12p40 pro1.1 in systemic arthritis and in rheumatoid factor negative polyarthritis was observed but not significant. Further investigations should be done to clarify the role IL-12p40 in the different subtypes of JIA.

ICOS-LICOS interaction is critically involved in TGN1412-mediated T-cell activation.

TGN1412, a superagonistic CD28-specific antibody, was shown to require Fc-cross-linking or immobilization as a prerequisite to mediate T-cell proliferation and cytokine release in vitro. We used primary human umbilical vein endothelial cells (HUVECs) to study their ability to induce activation of TGN1412-treated T cells. We confirmed that peripheral primary human T cells do not show activation upon stimulation with soluble TGN1412 alone. Nevertheless, cocultivation of TGN1412-treated T cells with HUVECs induced T-cell activation that was further enhanced using cytokine prestimulated HUVECs. Unexpectedly, Fc-FcγR interaction was dispensable for endothelial cell-mediated proliferation of TGN1412-treated T cells. Transwell-culture assays showed that TGN1412-treated T cells need direct cell-to-cell contact to HUVECs to induce proliferation. We found that costimulatory ICOS-LICOS interaction between T cells and endothelial cells is critically involved in TGN1412-mediated effects. Blocking LICOS reduced TGN1412-mediated T-cell proliferation significantly, whereas recombinant LICOS fully conferred TGN1412-mediated T-cell proliferation. Of note, cytokine stimulation enhanced LICOS expression on HUVECs and ICOS-LICOS interaction up-regulated ICOS expression on TGN1412-treated T cells. Hence, we provide a model of positive feedback conferred by ICOS-LICOS interaction between TGN1412-treated T cells and endothelial cells.

Toward experimental assessment of receptor occupancy: TGN1412 revisited.

In March 2006, 6 healthy volunteers experienced serious adverse reactions during a first-in-human clinical trial of the superagonistic anti-CD28 mAb TGN1412. A first investigation excluded contaminations of the drug product or protocol irregularities as the root cause. Later, an expert scientific group convened in the United Kingdom to develop recommendations pertinent to minimizing risks of first-in-human clinical trials. The expert scientific group concluded from in silico calculations that at the initial dose of 0.1 mg/kg, which was adjusted on the basis of the no observed adverse effect level, approximately 86.2% to 90.9% CD28 receptor occupancy was obtained. Here we developed a flow cytometric method that revealed receptor occupancy of approximately 45% to 80% under the above conditions. Thus we present a method to experimentally determine receptor occupancy that can be taken as one parameter to define the minimal anticipated biological effect level as the basis for calculating safer starting doses for first-in-human clinical trials for products in which a potential risk has been identified. Additional measures are being discussed that will help to significantly improve safety of first-in-human clinical trials.

The clinical course of steroid-sensitive childhood nephrotic syndrome is associated with a functional IL12B promoter polymorphism.

BACKGROUND: Steroid-sensitive nephrotic syndrome (NS) of childhood is the most common glomerular disease in children. The type and duration of response to corticosteroid therapy are used for clinical classification, and especially patients with steroid dependence often have a complicated course, requiring intensified immunosuppressive treatment. Its cause is still unknown although a cytokine-mediated course of disease has been implicated. Interleukin 12 (IL-12) is critical in determining the type of immune response. The ability of dendritic cells to secrete bioactive IL-12 is associated with a bi-allelic polymorphism within the promoter region of IL12B, the gene encoding the IL-12 p40 subunit. We hypothesized that this genotype may be involved in steroid-sensitive INS. METHODS: Using allele-specific PCR, 79 children with relapsing NS were genotyped for the IL12Bpro polymorphism, and genotype was correlated with clinical phenotype (presence/absence of steroid dependence). RESULTS: Children with the steroid-dependent course are at a significantly higher frequency homozygous for one IL12B allele compared to children without steroid dependence (46.7% and 17.6%, respectively). This genotype has previously been shown to be associated with impaired IL-12 secretion. CONCLUSION: Polymorphisms in the IL12B promoter region associate with two different clinical courses of NS. The IL12Bpro polymorphism may therefore define molecular subgroups with different prognosis. Further studies are needed to evaluate the prognostic value.

Identification of HLA class I dependent immunogenic peptides from clonotypic TCRbeta expressed in cutaneous T-cell lymphoma.

The clonotypic T-cell receptor (TCR) is a potential target antigen for specific immunotherapy of cutaneous T-cell lymphoma (CTCL). We identified T-cell epitopes from the rearranged TCR beta chain of the malignant T-cell population by the "reverse immunology" approach. Peptide-specific T-cell lines were generated against predicted epitopes and tested for the recognition of tumor cells and cells transfected with the full-length DNA coding for TCRV beta chain. Two peptides derived from the clonotypic TCRVbeta of a HLA-A2 positive patient could induce peptide-specific T cells from peripheral blood mononuclear cells of healthy donors and the patient as assessed by IFN-gamma ELISpot assay. Furthermore, the reactive CTLs efficiently recognized autologous Sézary tumor cells, as well as HLA-A2 positive 293 cells transfected with recombinant plasmid expressing the corresponding TCRVbeta29 protein. Similar results were obtained in a HLA-A3+ patient for TCRVbeta7-Jbeta2.7. In conclusion, our experiments show that the TCR beta chain harbors epitopes suitable as targets for specific vaccination which might be a promising approach for the specific immunotherapy of cutaneous T-cell lymphoma patients.

Melanoma-reactive T cells in the bone marrow of melanoma patients: association with disease stage and disease duration.

Using IFN-gamma enzyme-linked immunospot, we investigated reactivity of T cells from bone marrow and peripheral blood to melanoma lysate-pulsed autologous dendritic cells in 40 melanoma patients. Melanoma-reactive T cells were present in the bone marrow of seven patients and in peripheral blood of four patients. In the bone marrow, melanoma-reactive T cells were present in 6 of 21 stage IV patients and in 1 of 10 stage III patients, whereas none were detected in stage I to II patients (0 of 9). The occurrence of tumor-reactive bone marrow T cells in melanoma patients was associated with advanced disease stage, disease duration and tumor load, and independent of treatment. These findings provide new insights into the generation of T-cell responses in melanoma patients.

IL-12 production by human monocyte-derived dendritic cells: looking at the single cell.

Dendritic cells (DCs) are under investigation as immunotherapeutic agents in the treatment of cancer and infectious diseases. One of the important factors in skewing the immune response toward clinically beneficial TH1-type immunity is interleukin-12p70. IL-12p70 is synthesized and secreted in response to inflammatory cytokines, bacterial/viral components, and CD40 ligation. This study investigated the production of IL-12 by DCs at the single-cell level using a sensitive intracellular cytokine flow cytometry-based assay system. The authors observed that immature DCs could be stimulated with several compounds to produce IL-12, but that IL-12 production was a feature of a minority of activated DCs. IL-12+ DCs were characterized as being partially matured (ie, absent or low CD83 expression, with variable expression of CD1a and CD64). Interestingly, activated DCs lacked expression of the CD16 and CD64 Fc gammaR, which may have important implications for exogenous antigen-loading strategies.

Melanoma patients respond to a new HLA-A*01-presented antigenic ligand derived from a multi-epitope region of melanoma antigen TRP-2.

Tyrosinase-related protein-2 (TRP-2) is a known target antigen of spontaneous cytotoxic T cell responses in melanoma patients. Its frequent expression in metastatic tumors suggests that it might be an ideal candidate antigen for T cell-based immunotherapy. To provide knowledge about TRP-2-derived T cell epitopes useful for immunotherapy we applied a "reverse immunology strategy" based on repeated in vitro peptide stimulation of peripheral blood lymphocytes (PBL) from normal donors with predicted HLA-A*01 ligands. This led to the identification of TRP-2(181-190) as the first HLA-A*01-presented TRP-2-derived epitope. T-cell lines specific for peptide TRP-2(181-190) could be established from PBL of 50% of the normal HLA-A*01(+) donors tested. Such T cells responded specifically to autologous dendritic cells transduced virally with TRP-2, as well as to HLA-A*01(+), TRP-2(+) melanoma cells, although tumor cells had to be pretreated with IFN-gamma to become susceptible to T cell recognition. Interestingly, short-term in vitro peptide stimulation of PBL from HLA-A*01(+) melanoma patients showed the presence of TRP-2(181-190)-reactive CD8(+) T cells in some donors, suggesting their in vivo sensitization. Because TRP-2(181-190) overlaps with the known HLA-A*0201-presented epitope TRP-2(180-188), an 11mer peptide encompassing both epitopes might be of specific value for vaccination of a broad population of melanoma patients.

Effect of common B-RAF and N-RAS mutations on global gene expression in melanoma cell lines.

We studied global gene expression in three melanoma cell lines with the most common and potent V600E mutation in the B-RAF gene-four cell lines with a common Q61R mutation in the N-RAS gene and three cell lines with no mutations using human HG-U133A 2.0 micro-arrays with 22 277 transcripts. Data analysis using stringent criteria revealed several upregulated and downregulated genes in cell lines with B-RAF and N-RAS mutations compared with cell lines without mutations. We found 29 genes specifically upregulated and 32 genes downregulated in cell lines with B-RAF mutations, whereas 70 genes were upregulated and 39 downregulated in cell lines with N-RAS mutations; 11 genes showed overlapping upregulation and 45 downregulation. The micro-array data for nine selected genes were validated by the real-time PCR technique. Expression of a large number of genes, that encode members or regulators of the RAS/RAF/MEK/ERK pathways or are involved in metastasis or invasion, was affected in cell lines with mutations in B-RAF and N-RAS. Upregulated genes in cell lines with mutations included dual-specificity phosphatase 6 (DUSP6), sprouty 2 (SPRY2), v-akt murine thymoma viral oncogene homolog 3 (AKT3) and matrix metalloproteinase 14 (MMP14); downregulated genes included interleukin 18 (IL18), Krüppel-like factor 5 (KLF5) and inhibitor of DNA binding 2 (ID2). Our results, though carried on cell lines, provide a novel insight into the effect of mutations in the B-RAF and N-RAS genes on global gene expression in melanoma and highlight the complexity of mechanisms involved in tumour initiation and maintenance.

Differential regulation of maturation and apoptosis of human monocyte-derived dendritic cells mediated by MHC class II.

Antigen-driven interaction of dendritic cells (DC) with CD4(+) T(h) cells results in the exchange of bidirectional activating signals. Cross-linking of TCR by MHC class II-bound antigen activates T(h) cells, resulting in their up-regulation of CD40 ligand. Here we show that MHC class II molecules, in addition to their passive role in DC-T(h) cell interaction, can also actively induce DC maturation. Cross-linking of MHC class II molecules on human monocyte-derived DC results in the up-regulation of the surface expression of CD83, CD80, CD86, CD54, CD1a and CD40 molecules, the typical DC maturation-associated markers. It also promotes a rapid homotypic aggregation of DC paralleled by the up-regulation of such adhesion molecules as VLA-4, tissue transglutaminase, CD54 and CD11c. The impact of MHC class II cross-linking upon DC was context dependent. The outcome of MHC class II signaling depends on the maturation status of DC. While the cross-linking of MHC class II on immature DC promoted their maturation, the dominant effect upon the DC that were previously matured was the induction of DC apoptosis. Our current observations indicate that, in addition to the previously reported negative impact of MHC class II-mediated signaling on DC function, it also promotes DC maturation, participating in the enhancement of DC stimulatory function. Importantly, MHC class II-induced DC maturation and apoptosis are mediated by different signaling pathways, sensitive to different sets of inhibitors. This opens the possibility of differential regulation of each of these events in immunotherapy.

Reduction in the circulating pDC1/pDC2 ratio and impaired function of ex vivo-generated DC1 in chronic hepatitis B infection.

Dendritic cells (DCs) induce and regulate T-cell-mediated immune responses. Circulating precursor (p)DC1 and pDC2 from patients with chronic hepatitis B virus (HBV) infection were quantified by flow cytometry. To assess their function, DC1 were cultured from patients and compared to those of healthy volunteers. HBV patients exhibited a significant decrease in the proportion of freshly isolated pDC1 to pDC2. DC1 propagated from patients showed lower expression of costimulatory molecules and impaired allostimulatory capacity in comparison to controls. After exposure to proinflammatory cytokines, expression of costimulatory molecules, secretion of interleukin-12 (IL-12) and allostimulatory properties increased, but capacity for T-cell stimulation and IL-12 production remained inferior to that of control DCs. HBV-DNA was amplified by polymerase chain reaction in DC1 cultured from all patients. Viral particles were visible in DC1 by electron microscopy. These results suggest that intracellular presence of HBV impairs DC1 functional maturation and subsequent deficits in T-lymphocyte activation may contribute to viral persistence.

Alterations in the frequency of dendritic cell subsets in the peripheral circulation of patients with squamous cell carcinomas of the head and neck.

Patients with advanced squamous cell carcinomas of the head and neck (SCCHN) are frequently immunocompromised. Dendritic cells (DCs) are potent antigen-presenting cells that play a role in antitumor immune responses. Using multicolor flow cytometry, the percentages of lineage-negative (LIN(-)) and DR(+) DC precursors, as well as their LIN(-)DR(+)CD11c(+) (myeloid) and LIN(-)DR(+)CD123(+) (lymphoid) subsets, were determined in the peripheral blood of 36 patients with SCCHN before surgery. Peripheral blood mononuclear cells of 28 age- and sex-matched healthy individuals were used as controls. The proportions of LIN(-)DR(+) cells were found to be comparable in the circulation of patients and controls. However, the relative level of DR expression in LIN(-)DR(+) DC was lower in patients than in controls, suggesting a difference in the maturity of DC. The relative proportion of LIN(-)DR(+)CD123(+) cells in the LIN(-)DR(+) subset of DC did not differ significantly in patients compared with normal individuals. However, the percentage of myeloid-derived LIN(-)DR(+)CD11c(+) DCs was significantly lower (P < 0.002) in SCCHN patients than in controls. Of the 13 patients who were restudied 6 weeks after surgery, 9 showed an increase of the myeloid-derived LIN(-)DR(+)CD11c(+) DC subset postoperatively. This observation suggests that deficiency in the myeloid-derived DC precursors in patients with SCCHN is related to the presence of tumor and is reversible. An overall decrease in the myeloid-derived subset of DC could contribute to the failure of SCCHN patients to develop effective antitumor immune responses.

Proinflammatory Cytokines and CD40 Ligand Enhance Cross-Presentation and Cross-Priming Capability of Human Dendritic Cells Internalizing Apoptotic Cancer Cells.

SUMMARY: Human monocyte-derived dendritic cells (DC) can ingest apoptotic tumor cells (ATC) and present tumor-associated antigens (TAA) to T cells, leading to the generation of tumor-specific cytotoxic effector cells (Cancer Res 2000;60:3542-9). To further augment antitumor effector cell responses, attempts were made to modify antigen presentation and cross-priming of T cells by DC fed with ATC. Proinflammatory cytokines (PC), CD40 ligand (CD40L) and/or interferon-gamma (IFN-gamma) were found to markedly enhance the immunogenicity of TAA presented by DC. While PC upregulated expression of major histocompatibility complex class I/II and costimulatory molecules on the surface of DC, CD40L +/- IFN-gamma increased interleukin (IL)-12 and to a lesser extent, IL-15 production by DC. Additionally, lactacystin, a specific proteasome inhibitor, significantly abrogated the effects of IFN-gamma and, in part, also those of CD40L or PC. The ability of DC + ATC to cross-prime TAA-inexperienced ("naive") T cells was significantly enhanced by PC and CD40L or CD40L + IFN-gamma, but not by IFN-gamma alone. These results indicate that future vaccines for patients with cancer incorporating DC fed with ATC could be made more effective by the addition of proinflammatory cytokines or CD40L +/- IFN-gamma to improve the DC function.