Chimeric antigen receptor T-lymphocytes (CAR-T) therapy is a highly effective method in treating refractory B-cell lymphomas and acute lymphoblastic leukemias (ALL). However, CAR-T therapy can be associated with the development of severe life-threatening complications. Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) are the most serious toxicities. The aim of this study is to evaluate the nature, frequency and treatment methods of CAR-T therapy complications in adult patients.

Materials and methods

The prospective analysis included 9 CAR-T therapy procedures, which were performed in 5 patients (3 women, 2 men) with B-cell lymphomas and B-ALL at the age from 19 to 38 years (median – 29 years). 1 patient with relapse B-ALL underwent 4 CAR-T-therapy procedures, 1 patient with refractory diffuse large B-cell lymphoma underwent 2 procedures. In 3 patients with extramedullary relapses of B-ALL (1 patient with neuroleukemia relapse), CAR-T therapy procedures were performed once. The dose and type of CAR-T therapy were determined individually. All patients before CAR-T therapy received tocilizumab and were observed in the intensive care unit (ICU). The timing of complications, the types of complications based on the accepted criteria1 and the effectiveness of the treatment of complications of CAR-T therapy were recorded.

Results

CRS developed in 4 of 9 CAR-T therapy procedures. The incidence of severe CRS (grade ≥3) was 25%. All patients with CRS had a fever >39°C. Hypotension was presented in 3 of 4 cases of CRS. Shock with multiple organ dysfunction developed in 1 case, in which vasopressor agents, extracorporeal cytokine adsorption and hemodiafiltration were used. Hypoxemia was registered in 2 of 4 cases, none of patients required mechanical ventilation. The median time to the onset of CRS was +6.5 days (range 4-7) after the transfusion of CAR-T cells, the median duration of CRS was 4.5 days (range 2-5 days). Tocilizumab (8 mg/kg) was used in 2 cases of CRS as single injections. Hypofibrinogenemia was observed in 8 of 9 cases of CAR-T therapy (median 1.2 g/l, range 0.8-1.9 g/l). In 6 of 9 cases, hyponatremia was noted (median 124 mmol/l, range 122-132 mmol/l). 1 patient with neuroleukemia developed ICANS grade 3 on +7 day. Cerebrospinal fluid (CSF) cytosis was 77 cells/ml (CAR-T cells were detected in the CSF by immunophenotyping). ICANS was resolved within 24 hours after the initiation of dexamethasone in dose 20 mg/m²/day. Tumor lysis syndrome developed in 2 of 9 cases, septic shock developed in 1 case and caused the patient’s death. The overall survival rate after CAR-T therapy was 89%.

Conclusions

The incidence of serious CAR-T therapy complications (CRS, ICANS, septic) requiring intensive differential diagnosis and treatment can reach 55%.

Reference

Lee DW, Santomasso BD, Locke FL, et al. ASTCT Consensus Grading for Cytokine Release Syndrome and Neurologic Toxicity Associated with Immune Effector Cells. Biol Blood and Marrow Transplant. 2019; 25 (4): 625-38. doi: 10.1016/j.bbmt.2018.12.758

Keywords

CAR-T therapy, cytokine release syndrome, neurotoxicity syndrome.

A number of workers demonstrated an ATP-dependent activity of moving hematopoietic stem cells from immunopurified FLAG-labeled RecQL1 eluate using the example of the human gene RAD54L. Also in the UCSC genome browser presented many publications related to mutant gene activity, which indicates the biomedical potential of the study of this gene.

Materials and methods

Data were collected and maintained by the UCSC Genome Browser. For the gene under consideration, most matches with Human mRNA were found in the NCBI RefSeq, UCSC genes, and GENCODE databases. The ratio of most introns and exons (only the initial ones have different lengths), their length corresponds to Human mRNA and the Human expressed sequence tag (EST). When considering the RAD54L gene with different annotations in RefSeq, it is impossible to give an accurate assessment of the comparison with the experimental and predicted RNAs with PolyA end (there are no such RNAs). According to the RAD54L gene, sequencing in the cell culture K562 PolyA + predominates, found many pseudogenes. Most of the transcript located in the cytosol and nucleosome. The expression level for CAGE is most observed in the nucleosome and cytosol.

Results

The study revealed that this gene in the annotations was reasonably correctly calculated and interpreted. A large number of peptides are observed for the RAD54L gene, a high level of ribosomal profiling signals that coincide with the exon structure of the gene, therefore it is a protein-coding gene. According to COSMIC, multiple mutations were identified that correspond to all exons of the gene in hematopoietic, endometrial and lung tumor diseases. Adenocarcinoma, colonic, somatic, Lymphoma, non-Hodgkin, Breast cancer, pathologies of stem cells were found in the OMIM database.

Conclusions

Based on the results obtained, it can be argued that this RAD54L gene encodes a protein, which is an important link in many physiological, functional and pathological processes in the human body. The mutational activity of this gene is highly likely to lead to the development of pathologies of different genesis: the analysis showed an effect on the oncogenic process, the development of somatic diseases. It has a large role in the development of hereditary diseases. In conclusion, there is a sufficient number of publications on the information of this gene, which indicates possible promising studies of biomedicine in terms of prevention and treatment of somatic and oncogenic diseases of humans.

Keywords

Bioinformatics analysis, hematopoietic stem cells, RAD54L gene.

Chimeric antigen receptor T cells (CAR-T) and bispecific antibody technologies (BsAbs) became a breakthrough in the therapy of relapsed/refractory B-cell malignancies. CAR-T cells possess not negligible toxicity that can be fatal in some patients, while their efficiency may be hampered by mechanisms of resistance, such as antigen escape and CAR-T exhaustion. Most of the BsAbs bind CD3 epsilon (CD3ε) protein on T-cells alongside with tumor-associated antigen, lacking important co-stimulatory signals present in CAR structure, which may limit their potential. Introducing CD3 epsilon as an extracellular portion of CAR allows to engage CAR in immune synapse formation using BsAbs. Our approach addresses limitations of both methods, combining potential for control of toxicity by controlling BsAbs infusion, ability to modify antigen-specificity by changing the BsAbs as well as enabling dual or multi-targeting. Moreover, the possibility of making CAR-T cells “rest” by stopping BsAbs infusion may presumably prevent their exhaustion [1].

Materials and methods

cDNA of extracellular domain (ECD) of CD3E gene was derived from healthy donor peripheral blood mononuclear cells (PBMC) mRNA and cloned to pIRES. Then CD3E ECD was inserted in pCDH lentiviral plasmid containing FMC63-CD8stalk-41bbz CAR with NGFR as reporter gene substituting FMC63 (clone 7). Lentiviral preparations were made using HEK293T cells and pMD2.G and psPAX2 packaging plasmids. Lentiviral preparation was titrated using HEK293 cell line. PBMCs of a healthy donor were separated using Ficoll density gradient centrifugation. Cells were plated at the density of 1-2*10⁶ cells/ml and activated with 0.1 mcg/mL OKT3 antibody with 50 IE/mL of IL-2 for 3 days. On day 3 cells were transduced with lentiviral vector for 2 days. Cells were assessed for NGFR expression on day 5-8 post-activation using flow cytometry. On the next day after determining the percentage of NGFR-positive cells, cytotoxicity test was conducted. For cytotoxicity test Raji cell line stained with CFSE dye were plated with CAR-T cells at different effector:target (E:T) ratio in duplicates; BsAbs – blinatumomab or glofitamab – was added at different concentrations. Percentage of living target cells were determined by flow cytometry after 4 or 24 hours. Cytotoxicity was determined by the formula: 100 – cells alive/control cells alive (no effectors). Results. Lentiviral titer was 8×10⁴/µL using HEK293T cells. Efficiency of T-cell transduction ranged from 30 to 90%. Cytotoxicity tests conducted for 4 and 24 hours with blinatumomab at concentrations 0, 10, 100 and 200 ng/mL at E:T ratio of 0.3:1, 1:1, 3:1, 5:1 did not reveal the difference of cytotoxicity from control T cells. Cytotoxicity with glofitamab was tested at concentrations 0, 1, 10 pM/L with E:T ratio of 0.3:1, 1:1, 5:1 for 24 hours. The most evident difference was seen in 5:1 E:T ratio with increase of cytotoxicity in CAR-T compared to untransduced T-cells at glofitamab concentration 1 pM/L (93% vs 59%) and 10 pM/L (96% vs 74%).

Conclusion

Novel chimeric antigen receptors with extracellular portion of CD3E redirected by bispecific antibody glofitamab, but not blinatumomab may have activity against B-cell lymphoma cells in vitro. These may be explained by the fact that OKT3 antibody fragment, on which blinatumomab is based, can’t recognize CD3E as monomer, only as a dimer [2-4]. Therefore, design of CARs with CD3epsilon/CD3gamma and CD3epsilon/CD3delta extracellular domains is warranted.

References

1. Weber EW, Parker KR, Sotillo E, et al. Transient rest restores functionality in exhausted CAR-T cells through epigenetic remodeling. Science. 2021.

2. Salmerón A, Sánchez-Madrid F, Ursa MA, Fresno M, Alarcón B. A conformational epitope expressed upon association of CD3-epsilon with either CD3-delta or CD3-gamma is the main target for recognition by anti-CD3 monoclonal antibodies. J Immunol. 1991.

3. Law CL, Hayden‐Ledbetter M, Buckwalter S, McNeill L, Nguyen H, Habecker P, Thorne BA, Dua R, Ledbetter JA. Expression and characterization of recombinant soluble human CD3 molecules: presentation of antigenic epitopes defined on the native TCR-CD3 complex. International Immunology, 2002.

4. Trinklein ND, Pham D, Schellenberger U, et al. Efficient tumor killing and minimal cytokine release with novel T-cell agonist bispecific antibodies. MAbs. 2019.

Keywords

CAR-T, chimeric antigen receptor, cell therapy, B-cell malignancies, bispecific antibodies.

Cryopreservation is a crucial step in the cell lines culturing, as well as in the production of cellular therapy products (CTP). Currently the elimination of xenogenic components of cryopreservation media is considered as a necessary condition for CTP development. The aim of this study is to compare a set of cryopreservation conditions for cell line and primary cell cultures and their effect on the viability and proliferative activity of cells.

Materials and methods

The study was carried out on the K 562 cell line and primary murine mesenchymal stromal cells (mMSC). The study included 5 groups differing in the composition of the cryopreservation medium: a) standard conditions – complete medium (CM, RPMI/®-MEM+10%FBS)+10%DMSO using a cryo container (Mr. Frosty, Nalgene, USA), b) CM+10%DMSO without use of a cryocontainer, c) commercially available xeno-free medium CryoStor® CS10 (Stem Cell Technologies, USA), d) SSP+ solution (MacoPharma, France) with 10%DMSO, e) negative control CM without DMSO. Each sample from the group was tested in three repeats, subjected to cryopreservation for 7 days at -80°C, in three independent experiments. After thawing, the cell viability was assessed by flow cytometry with the 7AAD vital staining. The dynamics of the proliferative activity of 1×10^4 cells of each of the studied groups was analyzed by the spectrophotometric method using the alamar blue vital dye after 24, 72 and 168 hours. Statistical assesment of the groups was performed with the Man-Whitney U test.

Results

Assessment of K562 cells viability after thawing, demonstrated the median proportion of 7-AAD(+) cells in group a) of 1.5% (1.2-2.1), group b) 1.6% (1.1-1, 8) group c) 1.4% (1.2-1.9), group d) 1.4% (1.1-1.8), group e) 4.5% (2.7-5,6). Comparison of viability showed no differences between conditions a, b, c, d. The listed conditions had a statistically significant advantage over the negative control (e) (p=0.02). When assessing the viability of mMSC cells, the median proportion of 7-AAD + cells in group a) was 43.8% (16-62), b) 46.0% (22-65), c) 30.2% (17-46), d) 19.7% (16-26), e) 90.1% (86-97). The comparison revealed a statistically significant advantage of conditions a, b, c, d compared to control e (p <0.001). The number of 7AAD+ cells after conservation using SSP+ based medium was significantly lower compared to other conditions of conservation (p <0.05). Assessment of K562 cell line proliferative activity, showed an increase in the median value of the fluorescence intensity of resorufin after 168 hours compared to the value after 24 hours in group a) by 39% b) by 61% c) by 67% d) by 44% e) by 23 %. All conditions of conservation had an advantage over the negative control group (p <0.0001). The fluorescence score was significantly higher in the Cryostor group compared to standard conditions (complete medium + DMSO).

Conclusion

The performed analysis demonstrates no advantages of a cryo container use for cryopreservation of the studied cell types. Cryopreservation using Cryostor® CS10 medium and SSP+ based medium demonstrated the greatest potential in terms of maintaining the viability and proliferative activity of the studied cultures.

Keywords

Cryopreservation, cell cultures, cell products, DMSO.

For the effective delivering of genetic material to target cells, it’s necessary to provide sustainable conditions of transfection and protect it from intracellular microenvironment, pH, enzymes. It’s possible with a method of creating micro-sized carriers by new method of “soft lithography”, allowing to stamp polymer carriers with precise size and shape. This way passes undesirable stage of fabrication of polyelectrolyte carriers, such as synthesis of template, application of polymers on this template, deletion of the template. The process is becoming more controlled and effective, packing the cargo and the medium does not depend on affinity of the cargo to the template, charge of the polymers. In the research process hydrophobic and hydrophilic structures from 10nm to 5µm were packed, which tend to be unstable in external environment. Also sterile carriers were fabricated. The purpose of the study is to assess stability of the micro-carriers, toxicity for living system and ability to save the cargo over definite time.

Materials and methods

Silicon PDMS mold for micro-carriers fabrication was used. Stability was assessed with a confocal microscope and specrofluorometry. The following polymers were tested: polylactid PLA, Polycaprolaction PCL, polymethylmethacrylate PMMA. We used several test mediums: water, phosphate-buffered saline, human serum. Toxicity was determined with CT-26 cell line.

Results

Experimental results showed that polymer micro-carriers are not toxic for cells. Micro-carriers were able to 10 nm structures, while maintaining their medium.

Conclusions

Fabrication of micro-sized carriers by “soft lythograpy” is possibly effective for packing and saving of genetic material with its microenvironment.

Keywords

Microcarriers, micro-sized carriers, microcapsules, delivery, lythography, genetic material, polymer.

Doxorubicin (DOX) is a water-soluble anthracycline antibiotic with high anti-cancer efficacy. Reducing the concentration of DOX in the blood below the cardiotoxic level during therapy can be achieved by forming a depot containing DOX delivery systems with prolonged drug release. For these purposes, calcium carbonate porous vaterites coated with polyanion dextran sulfate (DexS) were used. The submicron size of the carriers does not allow them to be freely included in the bloodstream. The toxic drug spreads through the body only after it enters the blood as a result of release from the delivery systems (DS). Intraperitoneal administration of DOX-containing DS to rats with transplanted Seidel’s hepatoma allowed us to evaluate the effective concentration of DOX, which inhibits tumor growth and reduces the volume of ascitic fluid.

Methods and results

The dynamics of DOX intake into the blood of healthy rats after intraperitoneal administration of DOX in DS based on submicron DexS-coated porous CaCO₃ cores was determined by HPLC. The reaction of experimental rats with Seidel’s hepatoma inoculated within 21 days after DS administration was controlled on the basis of physical examination results, as well as autopsy of rats.

Results

It was shown that intraperitoneal administration of 2 mg of DOX in a DS in rats increases the life expectancy by 1.75 times and reduces the volume of ascites in experimental animals by half. A more effective suppression of the tumor can be expected by increasing the dose of DOX to 4 mg per animal. The dynamics of the appearance of DOX in blood plasma after intraperitoneal administration of the drug in DS to healthy rats was compared with free DOX dynamics. 2 days after the administration of free DOX to rats, it disappears from the blood plasma. As can be seen from Figure 1, the use of DS allows us to prolong the presence of the drug in the blood. Porous carbonate cores coated with dextran sulfate showed DOX release at theconcentrations below cardiotoxic within two weeks.

Figure 1. DOX concentrations in rats’ plasma after intraperitoneal administration. 1 – free DOX; 2 – CaCO₃+DexS+DOX

Conclusion

No negative reactions were detected in rats using DS, which was confirmed by the behavior and physical condition of the experimental animals, as well as the results of autopsy. Therefore, the studied DS based on CaCO₃ carbonate cores coated by DexS can be used for prolonged regional delivery of the DOX antitumor drug.

Keywords

Doxorubicin, drug delivery system, CaCO₃, dextran sulfate, Seidel’s hepatoma.

Targeted insertion of protein-coding sequences regions into the genome of hematopoietic stem cells (HSCs) mediated by engineered nucleases represents a promising platform for gene therapy of monogenic diseases. Homologous recombination (HR) represents the key mechanism for the introduction of genetic material into the double-stranded breaks (DSBs) generated by the targeted nuclease. Increasing the HR efficiency may enable the insertion of large DNA fragments into the HSC genome more efficiently upon delivery of donor repair templates by both viral and non-viral carriers. The mechanisms of DNA damage-induced innate immune response represent an important factor influencing the outcomes of DSB formation induced by engineered nucleases. The aim of this work is to evaluate the effects of adding small-molecule inhibitors of TLR9/AIM2/cGAS, STING and caspase antagonists A151, H151, and Z-VAD-FMK to cultures of primary human HSCs on the HR rate at the CCR5 locus after CCR5-Uco-TALEN mRNA transfection.

Materials and methods

Uridine-depleted CCR5-Uco-TALEN mRNA was synthesized by Trilink. Magnetic selection of CD34 + HSCs from the bone marrow of healthy donors was performed using the CD34 MicroBead kit (Miltenyi Biotec). After the activation phase of cultivation, HSCs were transfected with 25 µg/ml CCR5-Uco-TALEN mRNA using Gene Pulser Xcell (BioRad) device. After transfection, HSCs were cultured for 24 hours at 32°C in a StemMACS HSC Expansion Media XF (Miltenyi Biotech) medium. Small molecule inhibitors A151, H151 and FMK were added at concentrations 4 mkg/ml, 0.5 mkg/ml, and 25 mkg/ml respectively at 3 hours before the electroporation. The proportion of non-homologous end joining (NHEJ) events at the CCR5 locus was estimated by digital droplet PCR (ddPCR) on a QX200 System (Bio-Rad) according to the previously described protocol (Mock et al., 2015). In order to count the burden of HR-repaired CCR5 alleles the copy number of the reference gene EPOR was additionally estimated by the method of multiplex ddPCR (Schwarze et al., 2021). The difference between the copy number of the reference gene and the sum of HR-repaired and wild-type alleles was considered the proportion of alleles repaired by HR.

Results

The total average efficiency of the CCR5 gene knockout, calculated as the sum of the NHEJ- and HR-repaired alleles, ranged from 9 to 53.5%. The addition of a FMK small molecule to the HSCs culture significantly affected not only the CCR5 gene knockout overall efficiency (53.5%), but the frequency of both NHEJ (27.2%) and HR (26.3%) as well. The addition of H151 did not affect the overall efficiency of the CCR5 gene knockout (33.5%), as well as the ratio of NHEJ (18.3%) and HR (15.2%) events in the samples. In the presence of A151 small molecule, the CCR5 knockout efficiency was significantly reduced (9%), and 95.6% of knockout alleles were the result of NHEJ events at DSB formed by CCR5-Uco-TALEN.

Conclusion

The small molecules contribution to the stimulation of HR-mediated DSB repair after the CCR5-Uco-TALEN mRNA transfection of primary hematopoietic stem cells was studied. Z-VAD-FMK seems to be the most promising.

Acknowledgments

K.V. Lepik thanks the Russian Foundation for Basic Research for the support, grant No. 19-29-04025mk.

Keywords

TALEN, homology directed repair, STING inhibitors, HSC.

Various methods of gene therapy demonstrate great prospects in the treatment of various hereditary, infectious and oncological diseases. However, now, the effectiveness of their use is limited by the lack of effective and safe methods of delivering genetic material to cells. To solve this problem, a method consisting in the delivery of genetic material using polymer particles can be used. One of the important advantages of this method is the structure of the capsule, which allows it to protect the contents from the aggressive effects of biological environments of the body. However, for further use of these carriers in clinical practice, it is necessary to study in detail their bio-distribution after introduction into the body. The purpose of this work is to study the bio-distribution of polymer particles on mouse model by various methods, as well as histopathological analysis of tissues after the introduction of carriers.

Materials and methods

In this work, polymer particles obtained by applying polyarginine and dextran sulfate (PARG/DEXS) using Layer-by-Layer technology to calcium carbonate nuclei were used. The nuclei were obtained by co-precipitation of aqueous solutions of salts: sodium carbonate and calcium chloride with the addition of fluorescent dyes FITC and Cy5, as well as magnetite particles. The synthesized carriers were evaluated by light and confocal microscopy, as well as dynamic light scattering. Then the particles were injected into the tail vein of the mice, after which their organs (heart, lungs, liver, spleen and kidneys) were analyzed on the IVIS Spectrum CT device for in vivo fluorescence imaging on days 2, 5, 7, 10 and 15. The data of histological sections were also obtained using imaging methods on a confocal laser scanning microscope (CLSM) and a light microscope.

Results

The results obtained by the methods of CLSM (particles labeled with FITC), light microscopy (labeled with magnetite) and IVIS bioluminograph (labeled with Cy5) correlate with each other, but the assessment of biodistribution using particles labeled with magnetite is not optimal due to the characteristics of the liver and spleen tissue. After administration, the particles are found in large quantities in the lungs, presumably due to the small size of the capillaries and in the liver. Then there is a general decrease in the number of particles in the organs. On day 10, the number of particles in the lungs becomes minimal, while a sufficient amount is recorded in the liver. Also, on days 10 and 15, particles were registered in the spleen. Histopathological analysis illustrates the absence of pathological changes when using polymer micron particles. There is a significant accumulation of particles in tissues with a highly developed reticuloendothelial system (liver, spleen and lungs).

Conclusion

The presented data allow us to better understand the distribution of particles in the animal’s body over a long time and provide information about which organs can potentially be delivered therapeutic agents using this delivery system. In the future, it is planned to conduct experiments on the delivery of clinically relevant genetic material in vivo.

Acknowledgments

The work was carried out with the support of the project of the Russian Science Foundation “19-75-10010”.

Keywords

Polymer carriers, delivery of genetic material, bio-distribution, histological examination, in vivo visualization.

Currently, gene therapy is a promising approach to the treatment of a wide range of diseases. Low efficiency of genetic material delivery is the main limiting factor for development of gene therapy, since the use of “naked” nucleic acids does not provide the desired result. Polymeric carriers protect the genetic material during its transport to the target cells, but it is also necessary to protect it after its entrance to the cell. Background activity of nucleases is considered a significant obstacle to efficient gene delivery using non-viral vectors. To resolve this problem, it is necessary to select an inhibitor that protects the genetic material from degradation and has a positive effect on the efficiency of transfection. The aim of this work was to evaluate the effectiveness of the peptide DNase II inhibitor (SLRLLQWFLWAC) in increasing the efficiency of transfection of mammalian cells.

Materials and methods

In this work, polyelectrolyte particles have been used, being obtained by applying layers of differently charged polymers (Polyarginine/Dextran sulfate) on calcium carbonate cores prepared in advance by co-precipitation of aqueous sodium carbonate solutions and calcium chloride salts. The model genetic material was plasmid DNA encoding green fluorescent protein. Variants of pDNA encapsulation into the core, between polymer layers and simultaneous packing into the core and into the layer were considered. The peptide inhibitor was added to the DNA at the 1:1 ratio. The in vitro experiments were carried out with HEK 293 cell culture. Different cell inoculation techniques were tested with the particles: they were added either to adherent cells, or were incubated with suspended cells, followed by seeding on culture plastic surface. Effects of fetal bovine serum (FBS) upon transfection efficiency was studied. The particles were added to the cells at a ratio of 100:1. Efficiency of transfection was assessed qualitatively by means of laser scanning confocal microscopy as well as quantitatively, using flow cytometry.

Results

Analysis of the experimental results showed that the use of DNase II peptide inhibitor significantly increases the efficiency of transfection. The best results were obtained by incorporating genetic material both into the core and as a layer of a polymer particle. We have found that addition of particles to adherent cells, as well as usage of FBS-free media exerted positive effects upon the level of transfection.

Conclusions

In summary, the effect of a peptide inhibitor of DNase II upon transfection efficiency was investigated, and optimal conditions were determined for inoculation of mammalian cells with the particles containing genetic material.

Acknowledgments

This work was supported by the project of the Russian Science Foundation No. 19-75-10010.

Keywords

Gene therapy, DNase inhibitor, transfection efficiency.

Impressive clinical activity of CD19-specific CAR T-cell therapy has bolstered active research in the field of adoptive cellular immunotherapy of cancer. To date, FDA has approved 5 CAR T-cell products. However, it becomes clear that further improvements are needed to ensure CAR T-cell homing and long-term persistence, broader range of targetable epitopes, reduced costs, and switching to an allogeneic format. In the present work, we compared in vitro and in vivo activities of three different CD20-specific CARs vs a reference FMC63-based CD19-specific CAR (Kymriah), the first CAR approved for clinical practice.

Materials and methods

We produced lentiviral constructs encoding two CARs based on the mAbs 1F5 and Leu16, as well as the CAR based on the sequence of a fully human mAb ofatumumab (2F2). These constructs differ only in the antigen-recognition domain. Based on these constructs and T cells obtained from peripheral blood of healthy donors, the CAR T-cells were produced and their performance was compared using several assays.

Results

First, we used FACS to measure surface expression of CARs on T-cells. Then, we observed that all CAR T-cells displayed similar level of cytotoxicity in a 4-hour co-incubation test against Nalm6 target cells expressing CD20 (Fig. 1). The proliferation rate of CAR T-cells with antigen-recognition regions from 1F5 and FMC63 was approximately 1.5 times higher than that of other CAR T-cells. In the replating assay, CAR T-cells based on 1F5 and Leu16 likewise showed more promising results (Fig. 2). All the CAR T-cells obtained controlled xenografted tumors in NOD. Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ mice significantly better than irrelevant CAR T-cells (specific to PSMA) (Fig. 3).

Figure 1. Level of momentary cytotoxicity of CAR-T cells against Nalm6-CD20+ after 4 hours of incubation

Figure 2. Level of cytotoxicity of CAR-T cells against Nalm6-CD20+ in replating test

Figure 3. CAR-T cells effectively control the progression of B-ALL in a model experiment in vivo

Conclusion

CAR T-cells obtained display pronounced activity both in vitro and in vivo warranting their analysis in the clinic as well as the design and pre-clinical analysis of bi-specific CARs.

This study was supported by the RFBR grant №19-415-543015 р_мол_а.

Keywords

Т-cells, chimeric antigen receptor, CAR T-cell therapy, ofatumumab.

Despite common views that the early-stage prostate cancer is usually well treatable with surgery and hormonal therapy, the disorder remains risky, due to a tendency to develop into the chemo- and castration-resistant metastatic forms. Thus, development and testing of effective therapy for prostate cancer are urgent tasks of modern oncology. Hence, we proposed a combination therapy for prostate cancer based on the two powerful anticancer platforms: CAR-T and oncolytic virotherapy. The first component is T cells expressing chimeric antigen receptor which specifically directed towards the main prostate cancer antigen PSMA (aPSMA-CAR-T). Together with aPSMA-CAR-T, it is proposed to use a recombinant vaccinia virus strain L-IVP (VV-GMCSF-CXCL11) which have been modified to express GM-CSF and CXCL11 cytokines. This should result in efficient lysis of PC3/PSMA prostate cancer cells, as well as boost the tumor homing activity and cytotoxicity of the host immune cells and adoptively transferred aPSMA-CAR-T. This project aims to develop an in vivo model of therapy for prostate cancer and to explore the antitumor potential of aPSMA-CAR-T and VV-GMCSF-CXCL11 platforms separately or in combination.

Materials and methods

We used NOD/Scid mice for human prostate cancer xenograft model. The mice were subcutaneously injected with 2*10^6 PSMA-expressing PC3 cells (PSMA-PC3) and divided into 5 groups. At the 16^th day after tumor administration mice from the 1^st group received intratumoral injections of VV-GMCSF-CXCL11 at a dose of 10^7 PFU/mouse. Each mouse from the 2^nd group was intravenously treated with 2*10^7 human aPSMA-CAR-T cells at the 18^th and 23^rd day after PSMA-PC3 engraftment. Third group received both virus and CAR-T: mice were intratumorally treated with 10^7 PFU/mouse of VV-GMCSF-CXCL11 at the day 16 followed by the two intravenous injections of 2*10^7 aPSMA-CAR-T at the 18^th and 23^rd days. Negative control groups were intratumorally injected with saline or intravenously treated with untransduced human T-cells.

Results

We have shown that the single dose of oncolytic VV-GMCSF-CXCL11 virus inhibit tumor growth up to 90% at the 15th day after treatment, whereas mice that received aPSMA CAR-T only or virus with aPSMA CAR-T didn’t control tumors. The probable reason was insufficient immunodeficiency of NOD/Scid mice, since even at the 7th day after second injection no human CAR-T cells were already presented in mice blood. Such rapid elimination of CAR-T may be due to the “host versus graft” reaction. Thus, we repeated the experiments using more immunocompromised NSG mouse model. We showed that CAR-T cells were able to persist and proliferate in NSG mice and, in contrast to NOD/Scid model, the highest antitumor activity was observed in aPSMA-CAR-T only group. However, mice that received combined therapy or virus only were not able to control tumors, which may be due to the spread of viral infection over the bodies of severely immunodeficient NSG mice. High concentration of viral particles in the blood of animals was confirmed by qPCR. The NSG model could be optimized by lowering the dose of VV-GMCSF-CXCL11.

Conclusion

Overall, it seems that both NOD/Scid and NSG-based mice models are not optimal translational models of combination therapy for prostate cancer. Apparently, it is necessary to use an immunocompetent mouse model with syngeneically transplanted PSMA+ tumors, as well as mouse CAR-T cells as a therapy.

This work was supported by the RFBR grant mk18-29-09044.

Keywords

Oncolytic virus, CAR-T, prostate cancer, PSMA.

The development of genome editing methods and the discovery of the RfxCas13d endonuclease (CasRx) allow to target not only protein-coding genes but also non-coding RNAs. Regulators of the processes of transcription and translation, long noncoding RNAs (lncRNAs) become promising targets for gene therapy. Glioblastoma is one of the oncological diseases, which develop involving lncRNAs. An incomplete understanding of the pathogenesis of glioblastoma makes it difficult to develop an effective therapy. The study of pathogenesis is usually carried out on model cell lines. The models of glioblastoma are the cell lines U87MG, U251MG, U343MG.

Materials and methods

In this work, bioinformatic analysis of the transcriptomic data of the cell lines U87MG, U251MG, and U343MG was carried out in comparison with the lines WI-38, HEK293FT, HEB, BEAS2B, IMR90.

Results

Based on the data of current analysis, three lncRNA have an increased level of expression in glioma lines compared to the group of control cell lines and were selected as potential targets. A high level of LINC00461 is observed in glioma cell lines [1] and in patients with multiple myeloma [2]. LINC01152 is localized mainly in the nucleus and is involved both in the regulation of gene transcription, in particular, IL-23 [3], and in post-transcriptional regulation, for example, by interacting with the 3’-UTR of MAML2 [4]. In glioblastoma, increased expression of LINC01152 promotes epithelial-mesenchymal transition (EMT) [5]. It is shown that LINC01272 also promotes EMT in gastric and colorectal cancer [6].

Using the CasRx system, we will get and characterize cell lines with knockdown of selected lncRNAs. This will allow a deeper study of the role of the selected targets in oncogenesis, as well as improve the strategy of finding and suppressing target genes for gene therapy of oncology.

This work was carried out with the support of the Russian Science Foundation (project No. 21-14-00195), as well as with partial support of the project of basic budgetary financing of the Ministry of Education and Science of the Russian Federation (0245-2019-0001).

References

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Keywords

CasRx, lncRNA, glioblastoma.

Lung cancer is the most common malignant tumor, one of the most frequent causes of cancer-related deaths worldwide and the most common cause of death from oncological pathology. In fact, existing methods of treatment, such as сhemotherapy, radiotherapy and immunotherapy, still are able to evoke side effects in normal tissues when administered alone. That is why it is necessary to improve the effectiveness of lung cancer therapy by developing new approaches. Methods based on the combined use of several approaches of treatment have great potential. Radionuclide and chemotherapy can be considered the most promising methods of treating lung cancer. The aim of this project is to study effectiveness of combined radiotherapy and chemotherapy in lung cancer.

Materials and methods

This study developed a potentially promising treatment with a combination of ¹⁷⁷Lu-MPs as polymeric micro-carrier radionuclide, and Cisplatin (a cytostatic for chemotherapy). Balb/c mice with lung metastases received this treatment followed by harvesting and examination of the mice lungs. In the course of experiments, effectiveness of treatment was evaluated by measuring mass of lung tissues and counting the number of metastatic pulmonary nodules in the H&E-stained images calculated by means of Image-Pro Plus.

Results

Histological analysis revealed that the number of metastatic foci in the lungs was significantly decreased. Thus, higher efficiency of the combined therapy was shown, as compared to local radiation therapy with ¹⁷⁷Lu-MPs, or Cisplatin chemotherapy.

Conclusion

According to the results of the study, obtained data showed the effectiveness of combined radiotherapy and chemotherapy during in vivo experiments, thus requiring more detailed further studies in the area.

Acknowledgment

The study was performed with the support of the V. A. Almazov NMHC of the Ministry of Health of the Russian Federation.

Keywords

Lung cancer, nanobiotechnologies, nanoparticles, combined therapy, micro-carriers.

The alpha emitting radionuclide ²²⁵Ac is currently one of the most promising isotopes in alpha therapy due to its high linear energy transfer during four consecutive alpha decays. However, the main obstacle preventing the full implementation of ²²⁵Ac in clinical practice is the lack of stable retention of daughter radioactive isotopes (²²¹Fr and ²¹³Bi), which leads to their free circulation in the body and the destruction of healthy cells in the body.

Materials and methods

In this work, the surface of silica nanoparticles (SiO₂) obtained by the sol-gel method was modified with metal shells consisting of nanostructures of titanium butoxide (Ti(C₄H₉O)₄) and hydrogen tetrachloroaurate (HAuCl₄) to retain ²²⁵Ac and its decomposition products in developed nanocarriers.

Results

In vitro and in vivo studies in healthy mice show that a metal shell applied to the surface of SiO₂ nanoparticles contributes to enhanced inhibition of the release of radionuclides (²²⁵Ac and its daughter isotopes) compared to unmodified SiO₂ nanoparticles for a long period of time. Unmodified titanium/gold-coated silicon nanoparticles exhibited a ²²⁵Ac leakage of 20±3% in the first 5 days and 60%-70% after 15-30 days of incubation. In contrast, only 0.3% and 2.6% ²²⁵Ac release was found in the case of titanium and gold coated nanoparticles during 30 days of incubation. Histological analysis has shown that the developed nanocarriers do not have a significant toxic effect on the organ systems of laboratory mice within 3-10 days after injections. At the same time, practically no accumulation of released radionuclides was found in non-target organs (for example, in the kidneys). In contrast, unmodified carriers demonstrated the release of free radionuclides, which were distributed throughout the animal’s body with subsequent morphological changes in the tissues of the lungs, liver and kidneys.

Conclusion

These results highlight the potential of the developed nanocarriers for use as radionuclide delivery systems and propose a technology for the manufacture of new nanotherapeutic agents. This work was supported by the project of the Russian Science Foundation “19-75-10010”.

Keywords

Radiotherapy, nanoparticles, radionuclides, alpha radiation, silica, titanium, gold.

Nanoparticles have many advantages when used for various types of imaging. This type of drug carriers offers a wide range of material choices and are easily subject to various types of surface modifications. That is why there is currently a great deal of interest in this area and many organic and inorganic nanosystems have been developed that provide imaging signal, directionality and correction of the pharmacokinetics of particles. Despite a wide range of studies on combinations of various types of nanoparticles with fluorescent dyes and their widespread use in many types of research, the need of using new technologies and the appearance of particles that are more complex in their structure impose limitations on the already developed working protocols. Purpose of this project is to develop methods for modifying nano- and microcarriers with fluorescent dyes for in vitro and in vivo visualization.

Materials and methods

Particles of micron and submicron sizes were investigated in this work. To obtain studied carriers, CaCO₃ cores were used for polymer microparticles with a Polyarginine/Dextran sulfate shell and SiO₂ cores with a TiO₂ metal shell as nanoparticles were used as a matrix of the multilayer structure. The modification was performed by the method of layer-by-layer deposition of polyelectrolytes; the adsorption of the layers was proved by measuring the change in ζ-potentials. To optimize fluorescence imaging techniques, the optical properties of Cyanine5, Cyanine7, and Rhodamine 800 dyes were investigated. To modify particles with fluorescent agents, these dyes were incorporated into the structure of carriers at the stage of matrix formation of nano- and microparticles. The efficiency of the developed protocols for labeling carriers with fluorescent agents was tested using confocal scanning laser microscopy (CLSM) and bioluminescence studies.

Results

According to the results of the analysis by the СSLM methods and bioluminescence studies, it was proved that the developed protocols for fluorescent labeling of nano- and microcarriers demonstrated their effectiveness in preventing the release of the fluorescent label from the structure of the carriers. Experimental data have shown that the Cyanine5 fluorescent label is the most effective due to its high radiation intensity and long-term retention of fluorescent properties.

Conclusions

According to the results of the study, methods for modifying nano- and imicroparticles with fluorescent agents for in vitro and in vivo visualization were obtained and experimentally tested.

Acknowledgments

The work was done with the support of the Federal State Budgetary Institution “V. A. Almazov National Medical Research Center” of the Ministry of Health of the Russian Federation.

Keywords

Nanobiotechnologies, polymeric microparticles, silica nanoparticles, modification, fluorescent visualization.