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Stage III-IV non-small cell lung cancer (NSCLC) is a devastating disease characterized by a poor prognosis. NSCLC tumors carry genetic mutations, which can lead to the expression of altered protein sequences. Peptides originating from mutated proteins and bound to MHC molecules on the tumor cell surface are referred to as neoantigens, as they are tumor-specific and not expressed in normal cells. Due to their tumor specificity, neoantigens have a strong potential to induce an anti-tumor immune response and have been investigated for development of personalized therapeutic cancer vaccines. The current study describes the development of a clinical grade neoantigen vaccine formulation (FRAME-001) intended as immunotherapy in advanced NSCLC in combination with the immune checkpoint inhibitor pembrolizumab. The detection of aberrant tumor-specific transcripts as well as an algorithm to select immunogenic neoantigen peptides are described. Subsequently, selected neoantigen peptides were synthesized with a high throughput synthesis platform and aseptically formulated under good manufacturing practice (GMP) conditions into four aqueous peptides mixtures that each contained six neoantigen peptides. A validated stability-indicating analytical method was developed in which we considered the personalized nature of the formulation. An extensive stability study performed either at -25 °C or -80 °C showed that the formulation was stable for up to 32 weeks. The formulation was mixed with the vaccine adjuvant Montanide ISA 51 VG, which yielded the final vaccine emulsion. The stability of the vaccine emulsion was demonstrated using microscopic examination, differential light scattering, and the water-drop test. The presented data show that FRAME-001 is a feasible personalized vaccine formulation for the treatment of stage III-IV NSCLC. The presented data may give guidance in the development of novel personalized therapeutic vaccines since this formulation strategy could be used for any cancer indication., Competing Interests: K.F., M.V.M., and W.P.K are employees and stock option holders of CurveVac Netherlands B.V.; B.G., L.T.O., L.A.G., J.A.J., M.W.d.J., D.P.A., J.v.Z.: declare no conflict of interest. The present study was a multidisciplinary project between Frame Therapeutics B.V. (now CureVac Netherlands B.V.; genetic analyses of tumor material and neoantigen peptide selection), Almac Sciences Scotland Ltd. (manufacturing of the freeze-dried peptide mixtures), 3D-PharmXchange (CMC regulatory affairs and compliance), and the University Medical Center Groningen (development of the stability-indicating analytical method; manufacturing of the aqueous peptide mixtures; aqueous peptide mixture as well as vaccine emulsion stability study). The different individual authors combined their expertise to design a scientifically sound study protocol. The individual authors collected, analyzed, and interpreted the data independently from each other after which these data were discussed with the entire team. The analyses and interpretation of the data gathered by one group was not influenced by the other groups. The four independent groups together decided to write the manuscript and publish the results.

Swallowing problems and the required dose adaptations needed to obtain optimal pharmacotherapy may be a hurdle in the use of tablets in daily clinical practice. Tablet splitting, crushing, or grinding is often applied to personalise medication, especially for the elderly and children. In this study, the performance of different types of (commercially available) devices was studied. Included were splitters, screwcap crushers, manual grinders, and electric grinders. Unscored tablets without active ingredient were prepared, with a diameter of 9 and 13 mm and a hardness of 100-220 N. Tablets were split into two parts and the difference in weight was measured. The time needed to pulverise the tablets (crush time) was recorded. The residue remaining in the device (loss) was measured. The powder was sieved to obtain a particle fraction >600 µm and <600 µm. The median particle size and particle size distribution of the later fraction were determined using laser diffraction analysis. Splitting tablets into two equal parts appeared to be difficult with the devices tested. Most screwcap grinders yielded a coarse powder containing larger chunks. Manual and especially electric grinders produced a finer powder, making it suitable for administration via an enteral feeding tube as well as for use in individualised preparations such as capsules. In conclusion, for domestic and incidental use, a screwcap crusher may provide sufficient size reduction, while for the more demanding regular use in hospitals and nursing residences, a manual or electric grinder is preferred.

Monoclonal antibodies are an important addition to the medicinal treatment paradigm for IBD patients. While effective, these agents show a high degree of primary and secondary non-response, and methods to predict response are highly desired. Information on drug distribution at the target level is often lacking. Fluorescent endoscopic imaging using labelled antibody drugs may provide insight regarding drug distribution, target engagement and drug response, but these assessments require stable and functional fluorescently-conjugated probes. Infliximab, vedolizumab, adalimumab and ustekinumab were conjugated to IRDye 800CW, IRDye 680LT and ZW800-1. The resulting 12 tracer candidates were analysed and characterised on SE-HPLC, SDS-PAGE, iso-electric focussing (IEF) and ELISA in order to evaluate their feasibility as candidate clinical tracers for cGMP development. Major differences in the conjugation results could be seen for each conjugated drug. For Infliximab, 2 conjugates (800CW and 680LT) showed formation of aggregates, while conjugates of all drugs with ZW800-1 showed reduced fluorescent brightness, reduced purification yield and formation of fragments. All 6 of these candidates were considered unfeasible. From the remaining 6, ustekinumab-680LT showed reduced binding to IL23, and was therefore considered unfeasible. Out of 12 potential tracer candidates, 5 were considered feasible for further development: vedolizumab-800CW, vedolizumab-680LT, adalimumab-800CW, adalimumab-680LT and ustekinumab-800CW. Infliximab-680LT and ustekinumab-680LT failed to meet the standards for this panel, but may be rendered feasible if tracer production methods were further optimized.

D,L-3-hydroxybutyrate (D,L-3-HB, a ketone body) treatment has been described in several inborn errors of metabolism, including multiple acyl-CoA dehydrogenase deficiency (MADD; glutaric aciduria type II). We aimed to improve the understanding of enantiomer-specific pharmacokinetics of D,L-3-HB. Using UPLC-MS/MS, we analyzed D-3-HB and L-3-HB concentrations in blood samples from three MADD patients, and blood and tissue samples from healthy rats, upon D,L-3-HB salt administration (patients: 736-1123 mg/kg/day; rats: 1579-6317 mg/kg/day of salt-free D,L-3-HB). D,L-3-HB administration caused substantially higher L-3-HB concentrations than D-3-HB. In MADD patients, both enantiomers peaked at 30 to 60 minutes, and approached baseline after 3 hours. In rats, D,L-3-HB administration significantly increased C max and AUC of D-3-HB in a dose-dependent manner (controls vs ascending dose groups for C max : 0.10 vs 0.30-0.35-0.50 mmol/L, and AUC: 14 vs 58-71-106 minutes*mmol/L), whereas for L-3-HB the increases were significant compared to controls, but not dose proportional (C max : 0.01 vs 1.88-1.92-1.98 mmol/L, and AUC: 1 vs 380-454-479 minutes*mmol/L). L-3-HB concentrations increased extensively in brain, heart, liver, and muscle, whereas the most profound rise in D-3-HB was observed in heart and liver. Our study provides important knowledge on the absorption and distribution upon oral D,L-3-HB. The enantiomer-specific pharmacokinetics implies differential metabolic fates of D-3-HB and L-3-HB., (© 2021 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

A first-in-human phase I trial of Vvax001, an alphavirus-based therapeutic cancer vaccine against human papillomavirus (HPV)-induced cancers was performed assessing immunological activity, safety, and tolerability. Vvax001 consists of replication-incompetent Semliki Forest virus replicon particles encoding HPV16-derived antigens E6 and E7. Twelve participants with a history of cervical intraepithelial neoplasia were included. Four cohorts of three participants were treated per dose level, ranging from 5 × 10 5 to 2.5 × 10 8 infectious particles per immunization. The participants received three immunizations with a 3-week interval. For immune monitoring, blood was drawn before immunization and 1 week after the second and third immunization. Immunization with Vvax001 was safe and well tolerated, with only mild injection site reactions, and resulted in both CD4 + and CD8 + T cell responses against E6 and E7 antigens. Even the lowest dose of 5 × 10 5 infectious particles elicited E6/E7-specific interferon (IFN)-γ responses in all three participants in this cohort. Overall, immunization resulted in positive vaccine-induced immune responses in 12 of 12 participants in one or more assays performed. In conclusion, Vvax001 was safe and induced immune responses in all participants. These data strongly support further clinical evaluation of Vvax001 as a therapeutic vaccine in patients with HPV-related malignancies., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: Multiple acyl-CoA dehydrogenase deficiency (MADD) is a life-threatening, ultrarare inborn error of metabolism. Case reports described successful D,L-3-hydroxybutyrate (D,L-3-HB) treatment in severely affected MADD patients, but systematic data on efficacy and safety is lacking., Methods: A systematic literature review and an international, retrospective cohort study on clinical presentation, D,L-3-HB treatment method, and outcome in MADD(-like) patients., Results: Our study summarizes 23 MADD(-like) patients, including 14 new cases. Median age at clinical onset was two months (interquartile range [IQR]: 8 months). Median age at starting D,L-3-HB was seven months (IQR: 4.5 years). D,L-3-HB doses ranged between 100 and 2600 mg/kg/day. Clinical improvement was reported in 16 patients (70%) for cardiomyopathy, leukodystrophy, liver symptoms, muscle symptoms, and/or respiratory failure. D,L-3-HB appeared not effective for neuropathy. Survival appeared longer upon D,L-3-HB compared with historical controls. Median time until first clinical improvement was one month, and ranged up to six months. Reported side effects included abdominal pain, constipation, dehydration, diarrhea, and vomiting/nausea. Median D,L-3-HB treatment duration was two years (IQR: 6 years). D,L-3-HB treatment was discontinued in 12 patients (52%)., Conclusion: The strength of the current study is the international pooling of data demonstrating that D,L-3-HB treatment can be effective and safe in MADD(-like) patients.

Therapeutic vaccination is being explored as a treatment strategy for the treatment of patients with primary or metastatic tumours. We developed a vaccine targeted to Human papillomavirus (HPV)-induced tumours based on recombinant Semliki Forest virus (rSFV) encoding a fusion protein of the E6 and E7 proteins of HPV type 16. To enable a phase I clinical trial with this vaccine, Vvax001, a Good Manufacturing Practice (GMP)-compliant manufacturing process was set up and clinical material was produced. Upstream production of the clinical material resulted in viral titers from 2.4 × 10 7 to 1.3 × 10 9 infectious particles/ mL in the harvest. The total volume of 6.0 liter crude virus was purified in 13 consecutive downstream purification runs. The mean titer after purification was 4.0 × 10 8 infectious particles/ mL and the mean recovery was 19%. Finally, clinical material was filled at a target concentration of 1.25 × 10 8 infectious particles/mL. Release testing included tests for viral titer and virus identity, biological activity, sterility, bacterial endotoxins, adventitious viruses and absence of replication competent virus. The product complied with all specifications and was released for use as an investigational medicinal product. This is the first GMP production process developed for a SFV-based therapeutic vaccine. The vaccine, Vvax001 is targeted to HPV and has shown promising results in preclinical studies. The GMP-produced Vvax001 material met the quality criteria and was of sufficient quantity to enable assessment of its immunogenicity, safety and efficacy in a clinical setting., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

Optical molecular imaging using fluorescently labeled monoclonal antibodies is of significant added value in guiding surgical or endoscopic procedures. However, development of tracers for clinical trials is complex, and implementation in the clinic is therefore slow. We present a roadmap for development and translation of monoclonal antibody tracers into a drug product compliant with current good manufacturing processes (cGMPs). Methods: The production process for cetuximab-800CW and trastuzumab-800CW was optimized with regard to dye-to-protein ratio and formulation buffer. Promising formulations were produced under cGMP conditions and advanced to a full-scale stability study. Tracers were analyzed for stability by size-exclusion high-pressure liquid chromatography, pH measurement, osmolality, visual inspection, and sterility, as required by the European Pharmacopeia and cGMP guidelines. Results: Seven formulations were investigated for cetuximab-800CW and 10 for trastuzumab-800CW. On the basis of the formulation study results, we chose 2 formulations per antibody for investigation during the full-scale stability study. These formulations all performed well, showing good compliance with the acceptance criteria set for each product. Conclusion: We designed a roadmap to standardize the development, formulation, and cGMP translation of molecular fluorescent tracers. Using our standardized approach, we developed 2 stable antibody-based tracers for clinical use. The proposed roadmap can be used to efficiently develop a cGMP-compliant formulation and improve the translation of newly developed optical tracers to first-in-human use., (© 2019 by the Society of Nuclear Medicine and Molecular Imaging.)

In HIV-seronegative haemophiliac patients abnormal immune parameters have been demonstrated. In this review data on these abnormalities, their aetiology and clinical consequences are summarized and discussed. The data reviewed show abnormalities at different levels of the adaptive immune system. Most of the reported abnormalities regard lymphocyte subsets and their function, both in vivo and in vitro testing. Strong evidence has not been found for a causal relation between abnormalities and the consumption of factor VIII concentrates nor the purity of the concentrates. It seems likely that certain contaminants in the factor VIII concentrates have an inhibiting effect on lymphocytes and monocytes. Two clinical consequences of the abnormalities have been suggested: a higher susceptibility for infections and a greater risk to develop malignancies. Data on these consequences, however, are contradicting and not in agreement with the good results of long-term treatment of HIV-seronegative haemophiliac patients with factor VIII concentrates. The studies reviewed give no convincing evidence that more pure concentrates are advantageous in HIV-negative haemophiliacs.

Chronic substitution therapy of HIV-negative haemophiliacs with factor VIII products can result in abnormalities of ex-vivo measured immune parameters. To assess a possible relation between these abnormalities and product purity, we analyzed two groups of HIV-negative HCV-positive haemophiliacs, one treated with cryoprecipitate exclusively, the other with more purified factor VIII concentrates. Compared to age matched non-transfused male controls, increased numbers of white cells, granulocytes, IgG and IgM levels and decreased CD4+/CD8+ ratios were found in both patient groups. In the concentrate receivers, the numbers of mononuclear cells, CD4+, CD8+ and CD3+/HLA-DR+ cells indicating activated T-cells, were higher than in the cryoprecipitate group. In conclusion, both cryoprecipitate and intermediate/high purity concentrate recipients showed immune parameter abnormalities. These abnormalities tended to be somewhat more pronounced in patients treated with concentrates. By now there is no indication of the clinical relevance of the abnormalities in previously treated HIV seronegative haemophiliacs.

Depending on logistics, whole blood has to be stored for several hours after collection. If storage time exceeds 8 h, storage has to be at 1-6 degrees C to comply with FDA regulations. In the Netherlands, however, whole blood is also stored for 12-15 h at 20-24 degrees C using butane-1,4-diol cooling devices. We compared these two storage methods for factor VIII recovery in plasma and cryoprecipitate. At laboratory scale a significantly higher factor VIII recovery was found in plasma prepared from whole blood stored at ambient temperature; in cryoprecipitate this was confirmed but differences were not significant. However, as a result of in-efficient cooling in routine procedure, no significantly different factor VIII recoveries were found in plasma prepared from whole blood stored at either of the two temperatures. From our study we conclude that overnight storage of whole blood at ambient temperature using butane-1,4-diol cooling devices has to be favoured.

International and national documents on and standards for quality control have been introduced for Blood Banks. Recently, the Standard Registration Document and a National Health Authority Licence for factor VIII preparations based on the Document were introduced in the Netherlands. In the course of developing a preparation of lyophilized heat-treated cryoprecipitates in our Blood Bank, we used this Standard Registration Document and the good pharmaceutical manufacturing practices to design a quality control protocol. The aim of this protocol was to provide documented evidence on the quality of both our product and the production process. The protocol included the validation or revalidation of individual installations and procedures used during production, validation of the whole process and quality control of routine production. With our quality protocol we have been able to demonstrate that our preparation is consistently of the intended quality.

A method was developed for the batch-wise production of small-pool lyophilized heat-treated cryoprecipitates in a regional Blood Bank. Ten vials of final product were derived from twenty 600 g apheresis plasma donations. We found 384 IU of factor VIII per vial and a specific activity of 0.20 IU per mg total protein. Production recovery was 340 IU of factor VIII per kg plasma. The method was easy to perform and gave a product of good quality.