Human mesenchymal stem cell secretomes: Factors affecting profiling and challenges in clinical application.

The promising field of regenerative medicine is thrilling as it can repair and restore organs for various debilitating diseases. Mesenchymal stem cells are one of the main components in regenerative medicine that work through the release of secretomes. By adopting the use of the secretome in cell-free-based therapy, we may be able to address the challenges faced in cell-based therapy. As one of the components of cell-free-based therapy, secretome has the advantage of a better safety and efficacy profile than mesenchymal stem cells. However, secretome has its challenges that need to be addressed, such as its bioprocessing methods that may impact the secretome content and its mechanisms of action in clinical settings. Effective and standardization of bioprocessing protocols are important to ensure the supply and sustainability of secretomes for clinical applications. This may eventually impact its commercialization and marketability. In this review, the bioprocessing methods and their impacts on the secretome profile and treatment are discussed. This improves understanding of its fundamental aspects leading to potential clinical applications.

Selection and evaluation of single domain antibody against p19 subunit of IL-23 by phage display for potential use as an autoinflammatory therapeutic.

IL-23 is a double-subunit cytokine that plays an important role in shaping the immune response. IL-23 was found to be associated with several autoinflammatory diseases by generating sustained inflammatory loops that lead to tissue damage. Antibody neutralization of IL-23 was proven to be effective in ameliorating associated diseases. However, antibodies as large proteins have limited tissue penetration and tend to elicit anti-drug antibodies. Additionally, anti-IL-23 antibodies target only one subunit of IL-23 leaving the other one unneutralized. Here, we attempted to isolate a recycling single domain antibody by phage display. One of IL-23 subunits, p19, was expressed in E. coli fused to Gamillus protein to stabilize the α-helix-only p19. To remove Gamillus binders, two biopanning methods were investigated, first, preselection with Gamillus and second, challenge with IL-23 then on the subsequent round challenge with p19-Gam. The isolation of calcium-dependent and pH-dependent recycling binders was performed with EDTA and citrate buffers respectively. Both methods of panning failed to isolate high-affinity and specific p19 recycling binders, while from the second panning method, a high affinity and specific p19 standard binder, namely H11, was successfully isolated. H11 significantly inhibited the gene expression of IL-17 and IL-22 in IL-23-challenged PBMCs indicating H11 specificity and neutralizing ability for IL-23. The new binder due to its small size can overcome antibodies limitations, also, it can be further engineered in the future for antigen clearance such as fusing it to cell penetrating peptides, granting H11 the ability to clear excess IL-23 and enhancing its potential therapeutic effect.

The Future of TCR-like Antibodies in Diagnosis and Potential Application Targets.

The human leukocyte antigen (HLA, also known as the major histocompatibility complex or MHC) system, is responsible for immune monitoring of the intracellular proteome of all nucleated cells. The presentation of antigen peptides separates malignant or infected cells from their healthy counterparts and forms aberrant cells tagged as the foundation for identification. Therefore, peptide-MHC molecules can give potential diagnostic targets for cancer or infection. TCR-like antibodies recognize specific peptides that bind to MHC molecules, allowing them to target Such inaccessible cytoplasmic or nuclear tumors or virus-associated antigens. It binds to MHC, presenting peptides found on the surface of target cells. These antibodies have shown promising clinical applications in diagnosing and imaging cancer and infected cells. This review presents the current situation of TCR-like antibodies and its prospects for application in the field of intracellular antigen diagnostics. It also lists the potential application targets of TCR, like antibodies in various disease diagnoses, providing valuable information for developing diagnostic reagents and selecting targets in the future.

Designing molecules: directing stem cell differentiation.

Stem cells have been widely applied in regenerative and therapeutic medicine for their unique regenerative properties. Although much research has shown their potential, it remains tricky in directing stem cell differentiation. The advancement of genetic and therapeutic technologies, however, has facilitated this issue through development of design molecules. These molecules are designed to overcome the drawbacks previously faced, such as unexpected differentiation outcomes and insufficient migration of endogenous or exogenous MSCs. Here, we introduced aptamer, bacteriophage, and biological vectors as design molecules and described their characteristics. The methods of designing/developing discussed include various Systematic Evolution of Ligands by Exponential Enrichment (SELEX) procedures, in silico approaches, and non-SELEX methods for aptamers, and genetic engineering methods such as homologous recombination, Bacteriophage Recombineering of Electroporated DNA (BRED), Bacteriophage Recombineering with Infectious Particles (BRIP), and genome rebooting for bacteriophage. For biological vectors, methods such as alternate splicing, multiple promoters, internal ribosomal entry site, CRISPR-Cas9 system and Cre recombinase mediated recombination were used to design viral vectors, while non-viral vectors like exosomes are generated through parental cell-based direct engineering. Besides that, we also discussed the pros and cons, and applications of each design molecule in directing stem cell differentiation to illustrate their great potential in stem cells research. Finally, we highlighted some safety and efficacy concerns to be considered for future studies.

Investigating the Diagnostic and Therapeutic Potential of a T Cell Receptor (TCR)-like single Domain Antibody (sDAb)-Human IgG1 Antibody against Heat Shock Protein (HSP) 16KDa/HLA-A2 for Latent Tuberculosis.

Heat shock protein 16-kDa (HSP 16-kDa) is essential for the survival of Mycobacterium tuberculosis (M. tuberculosis) during the latent period; hence, a peptide-MHC presentation of HSP 16-kDa could be a potential diagnostic and therapeutic target for latent tuberculosis (LTB). This study aimed to generate a TCR-like single-domain antibody (sDAb)-human IgG1 antibody and subsequently investigate its diagnostic and therapeutic potential in LTB, utilizing a model cell presenting the target peptide. A previously generated TCR-like sDAB that can bind to HSP 16-kDa was first fused to a human IgG1 Fc-receptor via a linker. The fusion product, sDAb-IgG1, was expressed with HEK293-F and was subsequently purified. Its diagnostic potential was investigated via cell-based ELISA utilizing MCF-7 cells peptide-pulsed with HSP 16-kDa peptides. Investigation into the antibody-dependent cell-mediated cytotoxicity (ADCC) of MCF-7 cells was also conducted to investigate its therapeutic potential. Finally, TCR-like sDAb-IgG1 was successfully produced transiently with HEK-293F and was purified using protein A chromatography. The generated antibody was tested using cell-based ELISA, which demonstrated the effective binding of the TCR-like sDAb-IgG1 to the 16-kDa peptide-MHC on the cell surface. The ADCC assay also showed that the antibody effectively mediated the ADCC of MCF-7 cells with the help of 16-kDa peptide-MHC. This allows us to hypothesize the possible utility of the said antibody for both diagnostics and therapeutics of latent tuberculosis after more investigations with clinical samples.