Progress of antibody-drug conjugates (ADCs) targeting c-Met in cancer therapy; insights from clinical and preclinical studies.

The cell-surface receptor tyrosine kinase c-mesenchymal-epithelial transition factor (c-Met) is overexpressed in a wide range of solid tumors, making it an appropriate target antigen for the development of anticancer therapeutics. Various antitumor c-Met-targeting therapies (including monoclonal antibodies [mAbs] and tyrosine kinases) have been developed for the treatment of c-Met-overexpressing tumors, most of which have so far failed to enter the clinic because of their efficacy and complications. Antibody-drug conjugates (ADCs), a new emerging class of cancer therapeutic agents that harness the target specificity of mAbs to deliver highly potent small molecules to the tumor with the minimal damage to normal cells, could be an attractive therapeutic approach to circumvent these limitations in patients with c-Met-overexpressing tumors. Of great note, there are currently nine c-Met-targeting ADCs being examined in different phases of clinical studies as well as eight preclinical studies for treating various solid tumors. The purpose of this study is to present a broad overview of clinical- and preclinical-stage c-Met-targeting ADCs.

Clinical perspective: Antibody-drug conjugates for the treatment of HER2-positive breast cancer.

Antibody-drug conjugates (ADCs) are a promising class of cancer biopharmaceuticals that exploit the specificity of a monoclonal antibody (mAb) to selectively deliver highly cytotoxic small molecules to targeted cancer cells, leading to an enhanced therapeutic index through increased antitumor activity and decreased off-target toxicity. ADCs hold great promise for the treatment of patients with human epidermal growth factor receptor 2 (HER2)-positive breast cancer after the approval and tremendous success of trastuzumab emtansine and trastuzumab deruxtecan, representing a turning point in both HER2-positive breast cancer treatment and ADC technology. Additionally and importantly, a total of 29 ADC candidates are now being investigated in different stages of clinical development for the treatment of HER2-positive breast cancer. The purpose of this review is to provide an insight into the ADC field in cancer treatment and present a comprehensive overview of ADCs approved or under clinical investigation for the treatment of HER2-positive breast cancer.

The role of B cells in the immunopathogenesis of multiple sclerosis.

There is ongoing debate on how B cells contribute to the pathogenesis of multiple sclerosis (MS). The success of B-cell targeting therapies in MS highlighted the role of B cells, particularly the antibody-independent functions of these cells such as antigen presentation to T cells and modulation of the function of T cells and myeloid cells by secreting pathogenic and/or protective cytokines in the central nervous system. Here, we discuss the role of different antibody-dependent and antibody-independent functions of B cells in MS disease activity and progression proposing new therapeutic strategies for the optimization of B-cell targeting treatments.

IL-21 and IL-21-producing T cells are involved in multiple sclerosis severity and progression.

Multiple sclerosis is a common neuroinflammatory disease of the central nervous system causing nervous system defects and severe physical disability. IL-21 is a proinflammatory cytokine produced mainly by Th-17 and Tfh cells which its exact role in MS was not yet clearly understood. In the present study we aimed to investigate the possible correlation of IL-21 gene expression, methylation, and its serum levels with MS severity and progression. The results showed that IL-21 mRNA level and serum level were significantly increased in patient group compared with control group (p = 0.02 and p < 0.0001 respectively). Moreover, we found a strong positive correlation between IL-21 mRNA levels and EDSS scores (r = 0.637, P < 0.0001), IL-21 mRNA levels and Progression Index (r = 0.540, P < 0.0001), IL-21 serum levels and EDSS scores (r = 0.617, P < 0.0001), and IL-21 serum levels and Progression Index (r = 0.527, P < 0.0001) in MS patients. Additionally, we found that the methylation level of IL-21 promoter region was decreased in patient group compared with the control group (p < 0.0001). We also found that methylation level of IL-21 gene promoter is negatively correlated with the IL-21 mRNA level (r = -0.263, p = 0.02), serum level (r = -0.249, p = 0.03), EDSS scores (r = -0.276, p = 0.01) and Progression Index (r = -0.430, p = 0.0001). Data showed that the increased percentages of IL-21-producing Tfh-like, Th-17 and Th1 cells in patients are positively correlated with MS severity and progression. The results of our study suggest a pro-inflammatory and booster role for IL-21 in the MS pathogenesis and progression.

Anti-inflammatory effects of human embryonic stem cell-derived mesenchymal stem cells secretome preconditioned with diazoxide, trimetazidine and MG-132 on LPS-induced systemic inflammation mouse model.

Systemic inflammatory response syndrome is a complex pathophysiologic and immunologic response to an insult. Sepsis is a life-threatening condition happening when the body's response to infection causes injury to its own tissues and organs. Stem cell therapy is a new approach to modulate immune responses. Mesenchymal stem cells (MSCs) establish a regenerative niche by secreting secretome and modulating immune responses. MSC secretome can be leveraged for therapeutic applications if production of secretary molecules were optimized. Pharmacological preconditioning using small molecules can increase survival of MSCs after transplantation. The aim of this study was to investigate the effect of secretome of human embryonic-derived mesenchymal stem cells (hESC-MSCs) preconditioned with MG-132,Trimetazidine (TMZ) and Diazoxide (DZ) on immunomodulatory efficiency of these cells in Lipo polysaccharide (LPS) challenged mice models. Mice were injected intraperitoneally with LPS and groups of animals were intraperitoneally given 1 ml 30× secretome 6 h after LPS injection. Serum levels of biochemical parameters were then measured by an auto analyser and serum inflammatory cytokine levels were analysed using commercially available RayBio Mouse Inflammation Antibody Array. Ultimately, histopathology and survival studies were conducted. The results showed that TMZ and DZ-conditioned medium significantly increasing the survival and improvement of histopathological score. We found that MG-132-conditioned medium failed to show significant outcomes. This study demonstrated that human MSC secretome has the potential to control inflammation.

Secretome of Aggregated Embryonic Stem Cell-Derived Mesenchymal Stem Cell Modulates the Release of Inflammatory Factors in Lipopolysaccharide-Induced Peripheral Blood Mononuclear Cells

Background: Bone marrow mesenchymal stem cells (BM-MSCs) have emerged as a potential therapy for various inflammatory diseases. Because of some limitations, several recent studies have suggested the use of embryonic stem cell-derived MSCs (ESC-MSCs) as an alternative for BM-MSCs. Some of the therapeutic effects of the ESC-MSCs are related to the secretion of a broad array of cytokines and growth factors, known as secretome. Harnessing this secretome for therapeutic applications requires the optimization of production of secretary molecules. It has been shown that aggregation of MSCs into 3D spheroids, as a preconditioning strategy, can enhance immunomodulatory potential of such cells. In this study, we investigated the effect of secretome derived from human ESC-MSCs (hESC-MSCs) spheroids on secretion of IL-1ß, IL-10, and tumor necrosis factor α (TNF-α) from lipopolysaccharide (LPS)-induced peripheral blood mononuclear cells (PBMCs). Methods: In the present study, after immunophenotyping and considering mesodermal differentiation of hESC-MSCs, the cells were non-adherently grown to prepare 3D aggregates, and then conditioned medium or secretome was extracted from the cultures. Afterwards, the anti-inflammatory effects of the secretome were assessed in an in vitro model of inflammation. Results: Results from this study showed that aggregate-prepared secretome from hESC-MSCs was able to significantly decrease the secretion of TNF-α (301.7 ± 5.906, p < 0.0001) and IL-1ß (485.2 ± 48.38, p < 0.001) from LPS-induced PBMCs as the indicators of inflammation, in comparison with adherent culture-prepared secretome (TNF-α: 166.6 ± 8.04, IL-1ß: 125.2 ± 2.73). Conclusion: Our study indicated that cell aggregation can be an appropriate strategy to increase immunomodulatory characteristics of hESC-MSCs.

The effect of Trimetazidine and Diazoxide on immunomodulatory activity of human embryonic stem cell-derived mesenchymal stem cell secretome.

Comprehensive proteome profiling of the factors secreted by mesenchymal stem cells (MSCs), referred to as secretome, revealed that it consists of cytokines, chemokines, growth factors, extracellular matrix proteins, and components of regeneration, vascularization, and hematopoiesis pathways. Harnessing this MSC secretome for therapeutic applications requires the optimization of production of secretary molecules. A variety of preconditioning methods have been introduced, which subject cells to stimulatory molecules to create the preferred response and stimulate persistent effects. Pharmacological preconditioning uses small molecules and drugs to increase survival of MSCs after transplantation or prolong release of effective secretary factors such as cytokines that improve immune system responses. In this study, we investigated the effect of secretome of human embryonic-derived mesenchymal stem cells (hESC-MSCs) preconditioned with Trimetazidine (TMZ) and Diazoxide (DZ) on immunomodulatory efficiency of these cells in LPS-induced peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from human peripheral blood and treated with concentrated hESC-MSC-derived conditioned medium and then, the secreted levels of IL-10, TNFα and IL-1ß were assessed by ELISA after induction with LPS. The results showed that TMZ and DZ-conditioned medium significantly enhanced immunomodulatory potential of hESC-MSCs by increasing the secretion of IL-10, TNFα and IL-1ß from LPS- induced PBMCs. We also found that hESC-MSCs did not secrete mentioned cytokines prior to or after the preconditioning with TMZ and DZ. In conclusion, our results implied that TMZ and DZ can be used to promote the immunomodulatory effects of hESC-MSC secretome. It is obvious that for applying of these findings in clinical demands, the potency of different pre-conditioned MSCs secretome on immune response needs to be more clarified.

Evaluation of Factors Influencing Antibody Reduction for Development of Antibody Drug Conjugates

Background: Reduction/alkylation is one of the leading strategies for the development of antibody drug conjugates (ADCs). Precise control of the reduction process would not only yield a defined number of free thiols per antibody but also result in development of more homogenous conjugates. Methods: In the present study, we investigated the effect of various dithiothreitol (DTT) concentrations, temperature conditions, and DTT exposure times on antibody reduction. After antibody reduction, the Ellman's test and SDS-PAGE analysis were used to evaluate free thiols produced and confirm the reduction process, respectively. Results: DTT concentration seems to be a potential factor in the reduction process. Concentrations of 0.1, 1, 5, 10, 20, 50, and 100 mM DTT at 37°C for 30 minutes resulted in approximately 0.4, 1.2, 5.4, 7, 8, 8, and 8 thiols per antibody, respectively. Conclusion: Optimized site-specific conjugation can provide better process control and reproducibility for the development of disulfide-based ADCs.