We report, in brief, the results of a phase I, non-randomized study of idiotypic DNA vaccination in patients with B-cell non-Hodgkin’s lymphoma (ISRCTN31090206). The DNA sequence of lymphoma-derived immunoglobulin variable regions was used as a tumor-specific antigen fused to the potato virus X coat protein. A conjugate of plasmid DNA with polyethylenimine was used for the intramuscular injections, followed by a boost with an oral live-attenuated Salmonella vaccine carrying the same plasmid. The patients with a complete or partial response to previous chemotherapy received one or two courses of vaccination, including four injections at monthly intervals. The vaccine was well tolerated, with low-grade adverse events. The T-cell immune responses were assessed by ELISpot, at last vaccine, one week and one month post-vaccination, and were detected in 11/14 (78.6%) of the patients. In cases of progression requiring chemotherapy, or the presence of a positive MRD after the first course of vaccination, the patients underwent a second course of vaccination. At the end point, 6/19 vaccinated patients had disease stabilization, while 13/19 were in complete remission. The overall survival was 100% at follow-up, of a median of 2.3 years.

The potential of immune-evasive mutation accumulation in the SARS-CoV-2 virus has led to its rapid spread, causing over 600 million confirmed cases and more than 6.5 million confirmed deaths. The huge demand for the rapid development and deployment of low-cost and effective vaccines against emerging variants has renewed interest in DNA vaccine technology. Here, we report the rapid generation and immunological evaluation of novel DNA vaccine candidates against the Wuhan-Hu-1 and Omicron variants based on the RBD protein fused with the Potato virus X coat protein (PVXCP). The delivery of DNA vaccines using electroporation in a two-dose regimen induced high-antibody titers and profound cellular responses in mice. The antibody titers induced against the Omicron variant of the vaccine were sufficient for effective protection against both Omicron and Wuhan-Hu-1 virus infections. The PVXCP protein in the vaccine construct shifted the immune response to the favorable Th1-like type and provided the oligomerization of RBD-PVXCP protein. Naked DNA delivery by needle-free injection allowed us to achieve antibody titers comparable with mRNA-LNP delivery in rabbits. These data identify the RBD-PVXCP DNA vaccine platform as a promising solution for robust and effective SARS-CoV-2 protection, supporting further translational study.

Background: Neuroblastoma is a childhood tumor that is usually fatal after relapse in high-risk patients. Most clinical trials of CAR-T therapy for neuroblastoma are based on targeting the disialoganglioside GD2. B7-H3, a protein from the immunoglobulin superfamily, is a specific marker for neuroblastoma and a number of other solid tumors. We conducted a preclinical study of three variants of anti-B7-H3 CAR-T cells in order to justify the selection of the best candidate for subsequent clinical trials. Methods: The expression level of B7-H3 was measured in a number of cell lines and neuroblastoma tissue samples via flow cytometry. The functional activity of CAR-T cells was compared using an NFAT-inducible reporter assay, a cytotoxicity test, cytokine production, and a repeated stimulation assay. Results: The obtained CAR-T cells carrying all resulting CAR variants specifically recognized and killed B7-H3-positive tumor cells in vitro. Nevertheless, TE9-28z and 8H9-28BBz demonstrated superior activation and cytokine production compared to the second-generation 8H9-BBz construct. TE9-28z and 8H9-28BBz exhibited functional differences in expansion, exhaustion markers, and cytokine secretion in co-cultures with target cells in vitro. In particular, TE9-28z induced higher IFNγ production, while 8H9-28BBz showed increased TNFα release. Despite comparable cytotoxicity, TE9-28z and 8H9-28BBz CAR-T cells exhibited varying persistence depending on the tumor type, and showed signs of functional exhaustion upon prolonged exposure to the target antigen. Conclusion: TE9-28z and 8H9-28BBz were selected for further preclinical development as promising candidates for the effective targeting of B7-H3-expressing malignancies.

Neuroblastoma (NB) is the most common extracranial solid tumor in children comprising, 8 to 10% of all pediatric tumors, with incidence of about 1-1.3 cases per 100,000 children under 15 years of age. Despite the use of intensive treatment with surgery, high-dose chemotherapy and radiotherapy, the 5-year event-free survival rate is 25-50% and 10-40% after relapse. Over the past decade, a new type of cell therapy with chimeric antigen receptor (CAR) modification of lymphocytes has been rapidly developed. Among the main promising antigens for development of CAR-T therapy against NB is the disialoganglioside GD2, the expression of which is shown in 100% of NB cases. Most clinical variants of anti-GD2 CAR-T cells are based on scFv 14.G2, originating from chimeric antibody 14.18 (denutuximab). In 2020, the FDA approved a new anti-GD2 humanized antibody, 3F8 (naxitamab) with a better safety profile. Despite partial success, the results of anti-GD2 CAR-T therapy remain modest. One option to increase receptor specificity is based on targeting O-acetyl-GD2, a disialoganglioside derivative in which the external sialic acid residue is modified with an O-acetyl ester. Acetylation of GD2 occurs only in tumor cells, and it is not detected in peripheral nerves. An 8B6 antibody is known to target O-acetyl-GD2. Thus, at least 3 therapeutic antibodies, 14G2a, hu3F8 and 8B6, compete with each other for targeting GD2 with CAR-T cells. In all these cases, the anti-GD2 CAR contains insertion domains in extracellular portion of the molecule. Results of separate clinical trials have been published for CAR-T based on 14G2a and hu3F8. So far, however, there are no data on the usage of 8B6 antibody in CARs. The aim of the present study is to obtain 2^nd generation chimeric antigenic receptors based on three antibodies with different lengths of extracellular domain, and to evaluate their functional activity against a number of cell lines to justify further clinical trials.