Antigen-binding fragments of antibodies specific to the tumor-associated ganglioside GD2 are well poised to play a substantial role in modern GD2-targeted cancer therapies, however, rapid elimination ...from the body and reduced affinity compared to full-length antibodies limit their therapeutic potential. In this study, scFv fragments of GD2-specific antibodies 14.18 were produced in a mammalian expression system that specifically bind to ganglioside GD2, followed by site-directed pegylation to generate mono-, di-, and tetra-scFv fragments. Fractionated pegylated dimers and tetramers of scFv fragments showed significant increase of the binding to GD2 which was not accompanied by cross-reactivity with other gangliosides. Pegylated multimeric di-scFvs and tetra-scFvs exhibited cytotoxic effects in GD2-positive tumor cells, while their circulation time in blood significantly increased compared with monomeric antibody fragments. We also demonstrated a more efficient tumor uptake of the multimers in a syngeneic GD2-positive mouse cancer model. The findings of this study provide the rationale for improving therapeutic characteristics of GD2-specific antibody fragments by multimerization and propose a strategy to generate such molecules. On the basis of multimeric antibody fragments, bispecific antibodies and conjugates with cytotoxic drugs or radioactive isotopes may be developed that will possess improved pharmacokinetic and pharmacodynamic properties.
Liver diseases, characterized by high morbidity and mortality, represent a substantial medical problem globally. The current therapeutic approaches are mainly aimed at reducing symptoms and slowing ...down the progression of the diseases. Organ transplantation remains the only effective treatment method in cases of severe liver pathology. In this regard, the development of new effective approaches aimed at stimulating liver regeneration, both by activation of the organ’s own resources or by different therapeutic agents that trigger regeneration, does not cease to be relevant. To date, many systematic reviews and meta-analyses have been published confirming the effectiveness of mesenchymal stromal cell (MSC) transplantation in the treatment of liver diseases of various severities and etiologies. However, despite the successful use of MSCs in clinical practice and the promising therapeutic results in animal models of liver diseases, the mechanisms of their protective and regenerative action remain poorly understood. Specifically, data about the molecular agents produced by these cells and mediating their therapeutic action are fragmentary and often contradictory. Since MSCs or MSC-like cells are found in all tissues and organs, it is likely that many key intercellular interactions within the tissue niches are dependent on MSCs. In this context, it is essential to understand the mechanisms underlying communication between MSCs and differentiated parenchymal cells of each particular tissue. This is important both from the perspective of basic science and for the development of therapeutic approaches involving the modulation of the activity of resident MSCs. With regard to the liver, the research is concentrated on the intercommunication between MSCs and hepatocytes under normal conditions and during the development of the pathological process. The goals of this review were to identify the key factors mediating the crosstalk between MSCs and hepatocytes and determine the possible mechanisms of interaction of the two cell types under normal and stressful conditions. The analysis of the hepatocyte–MSC interaction showed that MSCs carry out chaperone-like functions, including the synthesis of the supportive extracellular matrix proteins; prevention of apoptosis, pyroptosis, and ferroptosis; support of regeneration; elimination of lipotoxicity and ER stress; promotion of antioxidant effects; and donation of mitochondria. The underlying mechanisms suggest very close interdependence, including even direct cytoplasm and organelle exchange.
Neuroblastoma is a pediatric solid cancer of heterogeneous clinical behavior. The unique features of this type of cancer frequently hamper the process of determining clinical presentation and ...predicting therapy effectiveness. The tumor can spontaneously regress without treatment or actively develop and give rise to metastases despite aggressive multimodal therapy. In recent years, immunotherapy has become one of the most promising approaches to the treatment of neuroblastoma. Still, only one drug for targeted immunotherapy of neuroblastoma, chimeric monoclonal GD2-specific antibodies, is used in the clinic today, and its application has significant limitations. In this regard, the development of effective and safe GD2-targeted immunotherapies and analysis of other potential molecular targets for the treatment of neuroblastoma represents an important and topical task. The review summarizes biological characteristics of the origin and development of neuroblastoma and outlines molecular markers of neuroblastoma and modern immunotherapy approaches directed towards these markers.
Ganglioside GD2 is expressed on plasma membranes of various types of malignant cells. One of the most promising approaches for cancer immunotherapy is the treatment with monoclonal antibodies ...recognizing tumor-associated markers such as ganglioside GD2. It is considered that major mechanisms of anticancer activity of anti-GD2 antibodies are complement-dependent cytotoxicity and/or antibody-mediated cellular cytotoxicity. At the same time, several studies suggested that anti-GD2 antibodies are capable of direct induction of cell death of number of tumor cell lines, but it has not been investigated in details. In this study we investigated the functional role of ganglioside GD2 in the induction of cell death of multiple tumor cell lines by using GD2-specific monoclonal antibodies.
Expression of GD2 on different tumor cell lines was analyzed by flow cytometry using anti-GD2 antibodies. By using HPTLC followed by densitometric analysis we measured the amount of ganglioside GD2 in total ganglioside fractions isolated from tumor cell lines. An MTT assay was performed to assess viability of GD2-positive and -negative tumor cell lines treated with anti-GD2 mAbs. Cross-reactivity of anti-GD2 mAbs with other gangliosides or other surface molecules was investigated by ELISA and flow cytometry. Inhibition of GD2 expression was achieved by using of inhibitor for ganglioside synthesis PDMP and/or siRNA for GM2/GD2 and GD3 synthases.
Anti-GD2 mAbs effectively induced non-classical cell death that combined features of both apoptosis and necrosis in GD2-positive tumor cells and did not affect GD2-negative tumors. Anti-GD2 mAbs directly induced cell death, which included alteration of mitochondrial membrane potential, induction of apoptotic volume decrease and cell membrane permeability. This cytotoxic effect was mediated exclusively by specific binding of anti-GD2 antibodies with ganglioside GD2 but not with other molecules. Moreover, the level of GD2 expression correlated with susceptibility of tumor cell lines to cytotoxic effect of anti-GD2 antibodies.
Results of this study demonstrate that anti-GD2 antibodies not only passively bind to the surface of tumor cells but also directly induce rapid cell death after the incubation with GD2-positive tumor cells. These results suggest a new role of GD2 as a receptor that actively transduces death signal in malignant cells.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
CD133 is an extensively studied marker of the most malignant tumor cell population, designated as cancer stem cells (CSCs). However, the function of this glycoprotein and its involvement in cell ...regulatory cascades are still poorly understood. Here we show a positive correlation between the level of CD133 plasma membrane expression and the proliferative activity of cells of the Caco-2, HT-29, and HUH7 cancer cell lines. Despite a substantial difference in the proliferative activities of cell populations with different levels of CD133 expression, transcriptomic and proteomic profiling revealed only minor distinctions between them. Nonetheless, a further in silico assessment of the differentially expressed transcripts and proteins revealed 16 proteins that could be involved in the regulation of CD133 expression; these were assigned ranks reflecting the apparent extent of their involvement. Among them, the TRIM28 transcription factor had the highest rank. The prominent role of TRIM28 in CD133 expression modulation was confirmed experimentally in the Caco2 cell line clones: the knockout, though not the knockdown, of the TRIM28 gene downregulated CD133. These results for the first time highlight an important role of the TRIM28 transcription factor in the regulation of CD133-associated cancer cell heterogeneity.
Ganglioside GD2 is a well-established target expressed on multiple solid tumors, many of which are characterized by low treatment efficiency. Antibody-drug conjugates (ADCs) have demonstrated marked ...success in a number of solid tumors, and GD2-directed drug conjugates may also hold strong therapeutic potential. In a recent study, we showed that ADCs based on the approved antibody dinutuximab and the drugs monomethyl auristatin E (MMAE) or F (MMAF) manifested potent and selective cytotoxicity in a panel of tumor cell lines and strongly inhibited solid tumor growth in GD2-positive mouse cancer models. Here, we employed two different GD2-binding moieties-minibodies and scFv fragments that carry variable antibody domains identical to those of dinutuximab, and site-directly conjugated them to MMAE or MMAF by thiol-maleimide chemistry with drug-to-antibody ratios (DAR) of 2 and 1, respectively. Specific binding of the antibody fragment-drug conjugates (FDCs) to GD2 was confirmed in direct ELISA, flow cytometry, and confocal microscopy. Selective cytotoxic and cytostatic effects of the conjugates were observed in GD2-positive but not GD2-negative neuroblastoma and melanoma cell lines. Minibody-based FDCs demonstrated more pronounced cytotoxic effects and stronger antigen binding compared to scFv-based FDCs. The developed molecules may offer considerable practical benefit, since antibody fragment-drug conjugates are capable of enhancing therapeutic efficacy of ADCs by improving their pharmacokinetic characteristics and reducing side effects.
Over the past two decades, mesenchymal stem cells (MSCs) have shown promising therapeutic effects both in preclinical studies (in animal models of a wide range of diseases) and in clinical trials. ...However, the efficacy of MSC-based therapy is not always predictable. Moreover, despite the large number of studies, the mechanisms underlying the regenerative potential of MSCs are not fully elucidated. Recently, it has been reliably established that transplanted MSCs can undergo rapid apoptosis and clearance from the recipient’s body, still exhibiting therapeutic effects, especially those associated with their immunosuppressive/immunomodulating properties. The mechanisms underlying these effects can be mediated by the efferocytosis of apoptotic MSCs by host phagocytic cells. In this concise review, we briefly describe three types of MSC-generated extracellular vesicles, through which their therapeutic functions can potentially be carried out; we focused on reviewing recent data on apoptotic MSCs and MSC-derived apoptotic bodies (MSC-ApoBDs), their functions, and the mechanisms of their therapeutic effects.
Oligomerization of antibody fragments via modification with polyethylene glycol (pegylation) may alter their function and properties, leading to a multivalent interaction of the resulting constructs ...with the target antigen. In a recent study, we generated pegylated monomers and multimers of scFv fragments of GD2-specific antibodies using maleimide–thiol chemistry. Multimerization enhanced the antigen-binding properties and demonstrated a more efficient tumor uptake in a syngeneic GD2-positive mouse cancer model compared to monomeric antibody fragments, thereby providing a rationale for improving the therapeutic characteristics of GD2-specific antibody fragments. In this work, we obtained pegylated conjugates of scFv fragments of GD2-specific antibodies with maytansinoids DM1 or DM4 using tetravalent PEG-maleimide (PEG4). The protein products from the two-stage thiol–maleimide reaction resolved by gel electrophoresis indicated that pegylated scFv fragments constituted the predominant part of the protein bands, and most of the scFv formed pegylated monomers and dimers. The conjugates retained the ability to bind ganglioside GD2 comparable to that of the parental scFv fragment and to specifically interact with GD2-positive cells. Both induced significant inhibitory effects in the GD2-positive B78-D14 cell line, in contrast to the GD2-negative B16 cell line. The decrease in the B78-D14 cell viability when treated with scFv-PEG4-DM4 was more prominent than that for scFv-PEG4-DM1, and was characterized by a twofold lower half-maximal inhibitory concentration (IC50). Unlike the parental scFv fragment, the product of scFv and PEG4 conjugation (scFv–PEG4), consisting predominantly of pegylated scFv multimers and monomers, induced direct cell death in the GD2-positive B78-D14 cells. However, the potency of scFv–PEG4 was low in the selected concentration range, thus demonstrating that the cytotoxic effect of DM1 and DM4 within the antibody fragment–drug conjugates was primary. The suggested approach may contribute to development of novel configurations of antibody fragment–drug conjugates for cancer treatment.
Mesenchymal stem cells (MSCs) have a pronounced therapeutic potential in various pathological conditions. Though therapeutic effects of MSC transplantation have been studied for a long time, the ...underlying mechanisms are still not clear. It has been shown that transplanted MSCs are rapidly eliminated, presumably by apoptosis. As the mechanisms of MSC apoptosis are not fully understood, in the present work we analyzed MSC sensitivity to Fas-induced apoptosis using MSCs isolated from the biopsies of liver fibrosis patients (L-MSCs). The level of cell death was analyzed by flow cytometry in the propidium iodide test. The luminescent ATP assay was used to measure cellular ATP levels; and the mitochondrial membrane potential was assessed using the potential-dependent dye JC-1. We found that human L-MSCs were resistant to Fas-induced cell death over a wide range of FasL and anti-Fas mAb concentrations. At the same time, intrinsic death signal inducers CoCl2 and staurosporine caused apoptosis of L-MSCs in a dose-dependent manner. Despite the absence of Fas-induced cell death treatment of L-MSCs with low concentrations of FasL or anti-Fas mAb resulted in a cellular ATP level decrease, while high concentrations of the inducers caused a decline of the mitochondrial membrane potential. Pre-incubation of L-MSCs with the pro-inflammatory cytokine TNF-α did not promote L-MSC cell death. Our data indicate that human L-MSCs have increased resistance to receptor-mediated cell death even under inflammatory conditions.
Chronic liver diseases constitute a significant economic, social, and biomedical burden. Among commonly adopted approaches, only organ transplantation can radically help patients with end-stage liver ...pathologies. Cell therapy with hepatocytes as a treatment for chronic liver disease has demonstrated promising results. However, quality human hepatocytes are in short supply. Stem/progenitor cells capable of differentiating into functionally active hepatocytes provide an attractive alternative approach to cell therapy for liver diseases, as well as to liver-tissue engineering, drug screening, and basic research. The application of methods generally used to isolate mesenchymal stem cells (MSCs) and maintain them in culture to human liver tissue provides cells, designated here as liver MSCs. They have much in common with MSCs from other tissues, but differ in two aspects-expression of a range of hepatocyte-specific genes and, possibly, inherent commitment to hepatogenic differentiation. The aim of this review is to analyze data regarding liver MSCs, probably another type of liver stem/progenitor cells different from hepatic stellate cells or so-called hepatic progenitor cells. The review presents an analysis of the phenotypic characteristics of liver MSCs, their differentiation and therapeutic potential, methods for isolating these cells from human liver, and discusses issues of their origin and heterogeneity. Human liver MSCs are a fascinating object of fundamental research with a potential for important practical applications.
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