Nykode Therapeutics ASA is a biotech company that has developed a unique DNA vaccine technology that may be used for both therapeutic and prophylactic vaccine purposes against oncology and infectious diseases. Through pre-clinical experiments, Nykode will gain scientific data to aid the preparation of applications to implement in clinical trials with vaccines against various diseases. The immune system plays a vital role in inhibiting several diseases, such as cancer and infectious diseases. Nykode developed a plasmid DNA vaccine platform that induces protective antibodies and T-cell responses against oncology and infectious diseases. Nykode’s platform can identify cancer antigens based on DNA and RNA sequencing data. Multiple antigens/epitopes can be incorporated into a single vaccine and can be used to drive an immune response against the tumor. Cells transfected with Nykode plasmids secretes dimeric fusion proteins encoded by the plasmid nucleic acids. Each protein chain contains a targeting unit, a dimerization unit, and an antigenic unit. These proteins may target antigens towards antigen presenting cells (APCs), which results in a more efficient stimulation of T and B cells. The different targeting units may skew the immune response in different directions thus, influence the properties and strength of the immune response. Previous studies have demonstrated the effect and protection with Vaccibodies containing different targeting units and different antigens in mice (Fredriksen & Bogen 2007, Schjetne et al. 2007, Ruffini et al. 2010, Grodeland G et al. 2013, Norheim et al. 2020, Gudjonsson et al. 2019, Beraas et al. 2022). Nykode vaccines may work as prophylactic (FOTS3727) or therapeutic (FOTS4025, 4726 + 7891). The vaccines give strong, and long-lasting immune responses (FOTS 25797, 23816, 10622, 10910, 15606, 15608), and utilizing different antigens, the Nykode platform demonstrated protection in several animal models against various cancer types and infectious diseases. (FOTS7888, 7890 + 7900, Fredriksen et al. 2006, Fredriksen & Bogen 2007, Ruffini 2010, Grødeland et al. 2013, Lambert et al. 2016). We aim to investigate whether our optimized vaccines with various tumor antigens can provide therapeutic effect against the listed tumor models. The experimental data obtained will provide opportunities to submit and conduct DNA vaccinee-based immunotherapy in clinical trials, which can provide a new line of treatment for cancer patients.

Tumor cell injection: once. The animals are anesthetized with isoflurane prior to s.c. injection on the thigh. The injection site is shaved and disinfected with 70% ethanol. The skin is lifted with a forceps and tumor cell suspension (100 µl) is injected in subcutaneously (s.c.) in the flank, using a 25G syringe. For i.v. injection the mice will be restrained in a mechanical restrainer and the tail will be heated up for easier visibility of the lateral tail vein. The injection site is disinfected with 70% ethanol. For i.p. injection of cells, the animal is securely restrained by their scruff. Tumor cells are injected (500 µl, PBS) with a 25G needle syringe into the lower left abdominal quadrant. After i.p., i.v., or s.c. injection of cells (day 0), animals are earmarked with an ear-puncher. Vaccination: once weekly, 4 weeks total. The animals are anesthetized as described. The tibialis region on both hind legs is shaved and disinfected with 70% ethanol, and DNA solution (50 µg DNA in 25 µl PBS) is injected into each (both) tibialis muscles with an insulin syringe (total dose 100 µg). Immediately after injection of the DNA vaccine solution, electroporation is applied to the site with the AgilePulse (BTX Harvard Apparatus, USA), which generates short electrical pulses to increase pDNA uptake. After vaccination, the mice are placed back into their cage and monitored. IVIS: twice weekly, until the end of the experiment. For IVIS imaging, the mice is first injected with 200 μl D-luciferin substrate i.p.(150 mg/kg). After 8 minutes, the animals are anesthetized in a gas chamber with sevoflurane, and then placed on a multimask in the IVIS machine (up to 5 mice). The animals are anesthetized for the whole imaging procedure which lasts approximately 5 minutes. When euthanizing an animal, blood can be collected by terminal cardiac puncture for flow cytometry analysis. The animal is placed under deep anesthesia (sevoflurane, or ZRF mix), and blood is collected from the heart with a 25G needle syringe. The animal is immediately euthanized afterwards by cervical dislocation. Tumor tissue/organs may be harvested surgically after the animal is euthanized (e.g. for immunohistochemistry, flow cytometry, weighing of tumor nodules).

This study will evaluate the therapeutic efficacy of DNA vaccines against tumors. Unfortunately, animal models are the only option to test novel potential DNA vaccines in vivo, as there are no other alternative models that exist which can represent the complexity of the cancer immunity cycle and the anti-tumor immune responses. To be able to estimate the effect of a vaccine on a patient's immune system, it is necessary to give the vaccine to a multicellular organism with biology, physiology and anatomy as similar to a human as possible. The immune system is a complex system of different cell types that circulates between different organs. Therefore limited knowledge is obtained from studying the induction of actual immune responses outside (in vitro) an experimental animal. Validation of constructs will be performed in vitro prior to use in animals. However, in vitro models cannot provide insight into crucial aspects of vaccinations, such as antigen loading and -competition, functional development of immune cells, activation/exhaustion and anti-tumor effect of T cells induced by vaccination and their interplay in the tumor microenvironment.