Virtually all recombinant proteins are now prepared using fusion domains also known as "tags". The use of tags helps to solve some serious problems: to simplify procedures of protein isolation, to increase expression and solubility of the desired protein, to simplify protein refolding and increase its efficiency, and to prevent proteolysis. In this review, advantages and disadvantages of such fusion tags are analyzed and data on both well-known and new tags are generalized. The authors own data are also presented.

Genetic instability of tumors leads to the appearance of numerous tumor-specific somatic mutations that could potentially result in the production of mutated peptides that are presented on the cell surface by the MHC molecules. Peptides of this kind are commonly called neoantigens. Their presence on the cell surface specifically distinguishes tumors from healthy tissues. This feature makes neoantigens a promising target for immunotherapy. The rapid evolution of high-throughput genomics and proteomics makes it possible to implement these techniques in clinical practice. In particular, they provide useful tools for the investigation of neoantigens. The most valuable genomic approach to this problem is whole-exome sequencing coupled with RNA-seq. High-throughput mass-spectrometry is another option for direct identification of MHC-bound peptides, which is capable of revealing the entire MHC-bound peptidome. Finally, structure-based predictions could significantly improve the understanding of physicochemical and structural features that affect the immunogenicity of peptides. The development of pipelines combining such tools could improve the accuracy of the peptide selection process and decrease the required time. Here we present a review of the main existing approaches to investigating the neoantigens and suggest a possible ideal pipeline that takes into account all modern trends in the context of neoantigen discovery.

The melanoma origin of cell lines obtained from the axillary lymph node (mel Kas, mel Pet, and mel Lap from patients with a verified diagnosis) was confirmed by the detection of the Melan A melanocyte marker expression. A hyperdiploid (2n+) for the mel Kas line; near-diploid (2n), and in some cells near-tertaploid (4n), and even hypo-octaploid (8n) set (172–179 chromosomes) in the mel Pet cell line; and a hypotetraploid (4n−) for the mel Lap line were detected by karyotypic analysis. All three cell lines are tumorigenic; however, mel Pet demonstrates tumor growth in Balb/c nude mice only in the presence of matrigel. All three lines showed a high expression of TUBB3 and PD-L1 markers, while ERa was low (minimum for mel Pet). Significant differences in the expression level were shown for the Cyt molecular marker. In the transplantation of cells to Balb/c nude mice, a stable expression level is observed only for TUBB3. For the rest of the markers, a decrease in their expression level of varying degrees was noted when the cells were growing in solid tumors in vivo. Mutations were detected in oncogenes (BRAF, EZH2, KIT, KRAS, NRAS, ROS1) and tumor suppressor genes (CDKN2A, FAT4, KMT2C, LRP1B, PTEN, PTPRB, TP53). The detailed characterization of the cell lines makes them valuable for various scientific and regulatory experiments, particularly those involving preclinical data on antiproliferative drugs for malignant melanoma or investigations into melanoma cell properties and progression.

Bioresource collection of the N. N. Blokhin National Medical Research Center of Oncology is a unique structured biobank that combines different types of primary and subsidiary samples and ensures its comprehensive characterization, including both generally accepted indicators and specific types of research. Samples of paraffin blocks, frozen material, blood and its derivatives are deposited in the clinical department of the center. The research institute of the center has collected commercial and unique human cell lines and transplantable strains obtained from clinical material, as well as cells from laboratory animals, mainly of mouse origin. The provided cell lines undergo multi- stage quality control, including confirmation of authenticity, assessment of viability and determination of optimal cultivation conditions, analysis of interspecific and intraspecific contamination, study of tumorigenicity and reproducibility in athymic Balb/ c-nude mice, etc. In addition, the Bioresource Collection has hybridoma clones that produce antibodies to a wide range of targets. The Center provides the opportunity to conduct preclinical research using samples from the Bioresource Collection to obtain scientific and regulatory data on the antiproliferative activity of new agents or methods for cancer treatment.

Introduction. One of the promising approaches to cancer immunotherapy is the generation of personal antitumor vaccines that provides immune system recognition of mutant tumor neoantigenes.The aim of this study was to develop bioinformatic approaches for melanoma NGS-sequencing data analysis. On the basis of data obtained, to predict the peptides capable of inducing an immune response against B16F10 mouse melanoma.Materials and methods. Exom and transcriptom of B16/F10 melanoma tumor tissue and normal tissue of C57BL/6 mice were sequenced. Library sequencing procedure was performed on Illumina HiSeq 2500platform, quality analysis of all obtained libraries was performed using FastQC and MultiQC. The obtainedfiles were usedfor further bioinformatics analysis. The GATKMuTect2 and Strelka were used for searching the mutations in tumor samples.Results. Identification of somatic mutations specific for tumor was based on the analysis of few mice tumors. Here we show that mutations in different tumor samples significantly overlap, but are not identical. Prediction of short peptides affinity to the mouse main histocompatibility complex (MHC) H2 was performed using netMHCpan version 3.0. Mutations such as missense and frameshift were used to predict short peptides that could trigger an immune response. Transcriptome data confirm that mutated alleles are expressed in tumors. Vaxrank pipeline predicted immunogenic peptides with a length of25—27. We also present the synthesis and solubility of these peptides. Conclusion. A bioinformatic approach has been developed to predict peptides capable of increasing immune response of mouse melanoma B16/F10.

Introduction. Personalized vaccine therapy that stimulates immune system to recognize mutant tumor neoantigens is one of the promising approaches of cancer treatment.Objective. Evaluation of immunogenicity and antitumor efficacy of synthetic neoantigen peptides and poly(I:C) adjuvant against murine melanoma B16F10.Materials and methods. 43 synthetic mutant neoantigen peptides selected earlier according to the bioinformatics analysis of the results of melanoma B16F10 sequencing were investigated. The study was performed in mice C57Bl / 6J with subcutaneously transplanted melanoma B16F10 after double vaccination by peptide groups with or without adjuvant poly(I:C). Immunogenicity of peptides was evaluated by ELISPOT that detected the number of interferon-gamma producing cells. Antitumor activity was evaluated on the base of tumor growth inhibition and the increase of the survival rate.Results. The number of interferon-gamma producing cells increased after in vitro stimulation by the peptides of the model vaccine in the group of mice which had been vaccinated by these peptides with the adjuvant. The number of interferon-gamma producing cells in vitro did not change after addition of vaccine peptides or any other peptides in the groups of mice that had been vaccinated by the peptides without the adjuvant as compared to the control group. Double vaccination by the peptides with the adjuvant induced a potent antitumor effect in some cases, three of the eight groups of the peptides with the adjuvant induced significant inhibition of tumor growth and the increase of survival rate in mice.Conclusion. Thereby, it was shown that vaccination of mice by peptides in combination with adjuvant poly(I:C) stimulated the cell immunity response, however, the increase of the number of interferon-gamma producing cells is not an indicator of the antitumor efficacy of neoantigen peptides.