Development of a Personalized Tumor Neoantigen Based Vaccine Formulation (FRAME-001) for Use in a Phase II Trial for the Treatment of Advanced Non-Small Cell Lung Cancer

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 degrees C or -80 degrees 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

Immunity to human cytomegalovirus after organstransplantation

This thesis is written on the immune response to cytomegalovirus in organ transplant recipients. In this summary, the issue of this thesis will be explained, the study design and the results will be described, and the conclusions will be given. Cytomegalovirus (CMV) is a member of the herpesvirus familiy. ... Zie: Summary

Optimization of the Production Process of Clinical-Grade Human Salivary Gland Organoid-Derived Cell Therapy for the Treatment of Radiation-Induced Xerostomia in Head and Neck Cancer

Head and neck cancer is a common cancer worldwide. Radiotherapy has an essential role in the treatment of head and neck cancers. After irradiation, early effects of reduced saliva flow and hampered water secretion are seen, along with cell loss and a decline in amylase production. Currently, there is no curative treatment for radiation-induced hyposalivation/xerostomia. This study aimed to develop and optimize a validated manufacturing process for salivary gland organoid cells containing stem/progenitor cells using salivary gland patient biopsies as a starting material. The manufacturing process should comply with GMP requirements to ensure clinical applicability. A laboratory-scale process was further developed into a good manufacturing practice (GMP) process. Clinical-grade batches complying with set acceptance and stability criteria were manufactured. The results showed that the manufactured salivary gland-derived cells were able to self-renew, differentiate, and show functionality. This study describes the optimization of an innovative and promising novel cell-based therapy.</p

Long-term biocompatibility, chemistry, and function of microencapsulated pancreatic islets

Transplantation of encapsulated living cells is a promising approach for the treatment of a wide variety of diseases. Large-scale application of the technique, however, is hampered by insufficient biocompatibility of the capsules. In the present study, we have implemented new as well as previously reported technologies to test biocompatibility issues of immunoisolating microcapsules on the long term (i.e. 2 years) instead of usually reported short time periods. When transplanted empty, the capsules proved to be highly biocompatible not only for short periods (i.e. 1 month) but also on the long term as evidenced by the absence of any significant biological response up to 2 years after implantation in rats. The immunoprotective properties of the capsules were confirmed by prolonged survival of encapsulated islet allografts up to 200 days. The surface of the applied capsule was analyzed and provides new insight in the chemical structure of true biocompatible and immunoprotective capsules applicable for transplantation of encapsulated islets in type I diabetes.

Factors influencing functional survival of microencapsulated islet grafts

Graft function of encapsulated islets is restricted in spite of the fact that inflammatory responses against capsules are limited to a portion less than 10%. It has been shown that dysfunction is accompanied by a gradual decrease in the glucose-induced insulin response (GIIR), a hyperproliferation of islet cells, and gradual necrosis. Also, limited survival is associated with the presence of macrophages in the overgrowth. In the present study, we investigate whether macrophages are the inducers of dysfunction of encapsulated grafts. Four weeks after successful transplantation of microencapsulated rat allografts we determined the GIIR, the rate of islet cell replication, and islet cell death. Also, we quantified the number of macrophages on the overgrown capsules. This assessment was applied to set up an in vitro coculture system of macrophages and encapsulated islets. We retrieved 93 +/- 6.2% of the capsules of which 9.2 +/- 0.3% was overgrown. The GIIR of the retrieved nonovergrown islets was reduced when compared with freshly encapsulated islets. The replication rate of the retrieved islet cells was eightfold higher than in the normal pancreas. Apoptosis was rarely observed but 37 +/- 4% of the total islet surface was composed of necrosis. We found a mean of 1542 +/- 217 macrophages per capsule. Coculture of 1500 NR8383 macrophages per encapsulated islets induced a substantial reduction in GIIR but a decrease instead of increase in replication. Necrosis was restricted to 13 +/- 1.3% of the islet cells and was not increased by the presence of macrophages. Our observations indicate that we should focus on reduction of macrophage activation and on improving the nutrition of encapsulated islets to prevent islet cell death

Corpus Christi HTC

The human pancarcinoma-associated epithelial cell adhesion molecule (EpCAM) (EGP-2, CO17-1A) is a well-known target for carcinoma-directed immunotherapy. Mouse-derived mAbs directed to EpCAM have been used to treat colon carcinoma patients showing well-tolerable toxic side effects but limited antitumor effects. Humanized or fully human anti-EpCAM mAbs may induce stronger antitumor activity, but proved to produce severe pancreatitis upon use in patients. To evaluate treatment-associated effects before a clinical trial, we have generated a transgenic mouse tumor model that expresses human EpCAM similar to carcinoma patients. In this study, we use this model to study the in vivo behavior of two humanized and one mouse-derived anti-EpCAM mAb, i.e., MOC31-hFc, UBS54, and MOC31. The pharmacokinetics and tissue distribution of the fully human mAb UBS54 and the mouse-derived MOC31 were largely the same after injection in tumor-bearing transgenic mice, whereas the molecularly engineered, humanized MOC31-hFc behaved differently. Injection of UBS54 and MOC31 resulted in significant, dose-dependent uptake of mAb in EpCA.M-expressing normal and tumor tissues, accompanied by a drop in serum level, whereas injection of MOC31-hFc resulted in uptake in tumor tissue, limited uptake by normal tissues, and slow blood clearance. It is concluded that the EpCAM-transgenic mouse model provides valuable insights into the potential behavior of humanized antiEpCAM mAbs in patients. mAbs sharing the same epitope and isotype but constructed differently were shown to behave differently in the model, indicating that the design of mAbs is important for eventual success in in vivo application