ONC201, the anticancer drug, targets and activates mitochondrial ATP-dependent caseinolytic peptidase P (ClpP), a serine protease located in the mitochondrial matrix. Given the promise of ONC201 in cancer treatment, we evaluated its effects on the breast ductal carcinoma cell line (BT474). We showed that the transient single-dose treatment of BT474 cells by 10 µM ONC201 for a period of less than 48 h induced a reversible growth arrest and a transient activation of an integrated stress response indicated by an increased expression of CHOP, ATF4, and GDF-15, and a reduced number of mtDNA nucleoids. A prolonged exposure to the drug (>48 h), however, initiated an irreversible loss of mtDNA, persistent activation of integrated stress response proteins, as well as cell cycle arrest, inhibition of proliferation, and suppression of the intrinsic apoptosis pathway. Since Natural Killer (NK) cells are quickly gaining momentum in cellular anti-cancer therapies, we evaluated the effect of ONC201 on the activity of the peripheral blood derived NK cells. We showed that following the ONC 201 exposure BT474 cells demonstrated enhanced sensitivity toward human NK cells that mediated killing. Together our data revealed that the effects of a single dose of ONC201 are dependent on the duration of exposure, specifically, while short-term exposure led to reversible changes; long-term exposure resulted in irreversible transformation of cells associated with the senescent phenotype. Our data further demonstrated that when used in combination with NK cells, ONC201 created a synergistic anti-cancer effect, thus suggesting its possible benefit in NK-cell based cellular immunotherapies for cancer treatment.

In recent years, the arrival of the immunotherapy industry has introduced the possibility of providing transformative, durable, and potentially curative outcomes for various forms of malignancies. However, further research has shown that there are a number of issues that significantly reduce the effectiveness of immunotherapy, especially in solid tumors. First of all, these problems are related to the protective mechanisms of the tumor and its microenvironment. Currently, major efforts are focused on overcoming protective mechanisms by using different adoptive cell therapy variants and modifications of genetically engineered constructs. In addition, a complex workforce is required to develop and implement these treatments. To overcome these significant challenges, innovative strategies and approaches are necessary to engineer more powerful variations of immunotherapy with improved antitumor activity and decreased toxicity. In this review, we discuss recent innovations in immunotherapy aimed at improving clinical efficacy in solid tumors, as well as strategies to overcome the limitations of various immunotherapies.

Therapeutic antibodies are implicated into the very promising and fast growing area of pharmaceutics. Human hybridoma technology, allowing generation of natural human antibodies in a native form, seems to be the most direct way that require no additional modifications for production of therapeutic antibodies. However, technical difficulties in human hybridoma creation discovered in the 80s of the last century have switched the mainstream therapeutic antibody development into new directions like display and transgenic mice techniques. These approaches have provided remarkable achievements in antibody engineering within last 15 years, but also revealed other limitations. Thus, it is time to turn back to forgotten human hybridoma technology. In this review, we describe new advances in all components of human hybridoma technology and discuss challenges in generating novel therapeutic mABs based on hybridoma technologies.

The translocator protein (TSPO; 18 kDa) is a high-affinity cholesterol-binding protein located in the outer membrane of mitochondria. A domain in the C-terminus of TSPO was characterized as the cholesterol recognition/interaction amino acid consensus (CRAC). The ability of the CRAC domain to bind to cholesterol led us to hypothesize that this peptide may participate in the regulation of mitochondrial membrane permeability. Herein, we report the effect of the synthetic CRAC peptide, VLNYYVW, on mitochondrial permeability transition pore (mPTP) opening. It was found that the CRAC peptide alone prevents the mPTP from opening, as well as the release of apoptotic factors (cytochrome c, AIF, and EndoG) in rat brain mitochondria (RBM). Co-incubation of CRAC, together with the TSPO drug ligand, PK 11195, resulted in the acceleration of mPTP opening and in the increase of apoptotic factor release. VLNYYVW did not induce swelling in rat liver mitochondria (RLM). 3,17,19-androsten-5-triol (19-Atriol; an inhibitor of the cholesterol-binding activity of the CRAC peptide) alone and in combination with the peptide was able to stimulate RLM swelling, which was Ca²⁺- and CsA-sensitive. Additionally, a combination of 19-Atriol with 100 nM PK 11195 or with 100 µM PK 11195 displayed the opposite effect: namely, the addition of 19-Atriol with 100 µM PK 11195 in a suspension of RLM suppressed the Ca²⁺-induced swelling of RLM by 40%, while the presence of 100 nM PK 11195 with 19-Atriol enhanced the swelling of RLM by 60%. Taken together, these data suggest the participation of the TSPO’s CRAC domain in the regulation of permeability transition.

The aim. To study possible allergenic and immunotoxic properties of the recombinant non-immunogenic staphylokinase molecule with the amino acids replacement Lys₇₄, Glu₇₅ and Arg₇₇ with alanine in preclinical and clinical studies.Materials and methods. The allergenic and immunotoxic properties of the recombinant non-immunogenic staphylokinase drug were studied using standard methods in accordance with the Guidelines for the Preclinical Study of New Substances in guinea pigs (n=15) and mice (n=45) at doses 5, 10 and 20 times higher than therapeutic (for humans). A clinical study was conducted in 100 patients with acute ST-segment elevation myocardial infarction after a single intravenous injection of the drug. The study included the determination of titers of specific antibodies to recombinant non-immunogenic staphylokinase and the study of plasma neutralizing activity.Results. During the complete set of preclinical studies, it was found that the drug does not affect the cellular and humoral immune response in guinea pigs and mice at doses many times higher than therapeutic doses for humans. It was found that the drug did not cause an immediate-type of hypersensitivity reaction (Weigle index 0) and a delayed type IV (0 points according to S.V. Suvorov) in guinea pigs, did not affect the cellular capacity of popliteal lymph nodes (reaction index 0.91), did not affect the number of nucleated and antibody-forming cells in the spleen of mice. As a result of a clinical study of recombinant non-immunogenic staphylokinase, no allergic reactions were registered. Assessment of the neutralizing activity of the plasma of patients who were administered recombinant non-immunogenic staphylokinase showed that 70% samples did not have neutralizing activity: 30% of the patients’ samples were characterized by a minimum neutralizing activity of 0.33±0.02 μg/mL, which is 30–310 times lower than after the use of native staphylokinase. These values are 7.8 times lower than the determined concentration of recombinant non-immunogenic staphylokinase in the blood (2.59 μg/mL). Thus, the drug does not lead to the anti-staphylokinase neutralizing antibodies formation capable to neutralize its effect upon repeated administration.Conclusion. According to the results of the trials, the absence of allergenic and immunotoxic properties of the recombinant non-immunogenic staphylokinase and its safety in relation to the immune system have been proven.

The aim. To investigate the pharmacokinetic parameters of non-immunogenic staphylokinase (Fortelyzin®, SuperGene LLC, Russia) in patients with acute myocardial infarction with ST-segment elevation (STEMI) on the electrocardiogram and in patients with ischemic stroke.Materials and methods. The clinical study was conducted in 50 patients with STEMI after a single intravenous administration of non-immunogenic staphylokinase at a dose of 15 mg and in 50 patients with ischemic stroke after a single intravenous administration of the drug at a dose of 10 mg. The main pharmacokinetic parameters were determined: half-life, initial concentration, volume of distribution, clearance, and area under the pharmacokinetic curve.Results. As a result of the study of the pharmacokinetics of non-immunogenic staphylokinase, it was found that after a single intravenous administration of the drug at a dose of 15 mg, the initial concentration was 7.1 ± 2.7 μg/mL, the half-life was 5.77 ± 0.72 min, the clearance was 0.33 ± 0.04 l/min, and the area under the pharmacokinetic curve (AUC_0-t) was 42.9 ± 3.2 μg/mL*min. After administration of the drug at a dose of 10 mg, the initial concentration was 2.8 ± 0.3 μg/ml, the half-life was 5.11 ± 0.56 min, the clearance was 0.35 ± 0.06 l/min, and the area under the pharmacokinetic curve (AUC_0-t) was 28.5 ± 3.6 μg/mL*min. The terminal half-life was 32 min in both dosage regimens.Conclusion. It was found that non-immunogenic staphylokinase is characterized by a short half-life and high clearance, which ensures the safety of the drug in clinical practice. The peculiarities of the pharmacodynamics of non-immunogenic staphylokinase, associated with its interaction with plasmin in the thrombus and subsequent recirculation of released drug molecules, allow it to be used in low doses, regardless of the patient’s body weight, despite the short half-life.