Most of the hosts used to produce the 151 recombinant pharmaceuticals so far approved for human use by the Food and Drug Administration (FDA) and/or by the European Medicines Agency (EMEA) are ...microbial cells, either bacteria or yeast. This fact indicates that despite the diverse bottlenecks and obstacles that microbial systems pose to the efficient production of functional mammalian proteins, namely lack or unconventional post-translational modifications, proteolytic instability, poor solubility and activation of cell stress responses, among others, they represent convenient and powerful tools for recombinant protein production. The entering into the market of a progressively increasing number of protein drugs produced in non-microbial systems has not impaired the development of products obtained in microbial cells, proving the robustness of the microbial set of cellular systems (so far Escherichia coli and Saccharomyces cerevisae) developed for protein drug production. We summarize here the nature, properties and applications of all those pharmaceuticals and the relevant features of the current and potential producing hosts, in a comparative way.
Understanding the structure, functionalities and biology of functional amyloids is an issue of emerging interest. Inclusion bodies, namely protein clusters formed in recombinant bacteria during ...protein production processes, have emerged as unanticipated, highly tunable models for the scrutiny of the physiology and architecture of functional amyloids. Based on an amyloidal skeleton combined with varying amounts of native or native-like protein forms, bacterial inclusion bodies exhibit an unusual arrangement that confers mechanical stability, biological activity and conditional protein release, being thus exploitable as versatile biomaterials. The applicability of inclusion bodies in biotechnology as enriched sources of protein and reusable catalysts, and in biomedicine as biocompatible topographies, nanopills or mimetics of endocrine secretory granules has been largely validated. Beyond these uses, the dissection of how recombinant bacteria manage the aggregation of functional protein species into structures of highly variable complexity offers insights about unsuspected connections between protein quality (conformational status compatible with functionality) and cell physiology.
One-third of diffuse large B-cell lymphoma patients are refractory to initial treatment or relapse after rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone chemotherapy. In ...these patients, CXCR4 overexpression (CXCR4
) associates with lower overall and disease-free survival. Nanomedicine pursues active targeting to selectively deliver antitumor agents to cancer cells; a novel approach that promises to revolutionize therapy by dramatically increasing drug concentration in target tumor cells. In this study, we intravenously administered a liganded protein nanocarrier (T22-GFP-H6) targeting CXCR4
lymphoma cells in mouse models to assess its selectivity as a nanocarrier by measuring its tissue biodistribution in cancer and normal cells. No previous protein-based nanocarrier has been described as specifically targeting lymphoma cells. T22-GFP-H6 achieved a highly selective tumor uptake in a CXCR4
lymphoma subcutaneous model, as detected by fluorescent emission. We demonstrated that tumor uptake was CXCR4-dependent because pretreatment with AMD3100, a CXCR4 antagonist, significantly reduced tumor uptake. Moreover, in contrast to CXCR4
subcutaneous models, CXCR4
tumors did not accumulate the nanocarrier. Most importantly, after intravenous injection in a disseminated model, the nanocarrier accumulated and internalized in all clinically relevant organs affected by lymphoma cells with negligible distribution to unaffected tissues. Finally, we obtained antitumor effect without toxicity in a CXCR4
lymphoma model by administration of T22-DITOX-H6, a nanoparticle incorporating a toxin with the same structure as the nanocarrier. Hence, the use of the T22-GFP-H6 nanocarrier could be a good strategy to load and deliver drugs or toxins to treat specifically CXCR4-mediated refractory or relapsed diffuse large B-cell lymphoma without systemic toxicity.
BACKGROUND:Insulin-like growth factor 1 (IGF-1) was found to stimulate Schwann cell mitosis. Exogenous IGF-1 may improve nerve regeneration after cryopreservation.
OBJECTIVE:To evaulate the effect of ...intraneural administration of IGF-1 in cryopreserved nerve isografts.
METHODS:Eighteen millimeter grafts were used for bridging an 18-mm defect in the rat sciatic nerve. A total of 57 rats were randomly divided into three groups(1) autograft (Group 1); (2) cryopreserved isograft (Group 2); (3) cryopreserved isograft with intraneural IGF-1 administration (Group 3). 12 weeks after surgery, functional recovery (Sciatic functional index SFI, Swing speed SS, nerve conduction velocity NCV, amplitude of compound motor action potentials CMAP, and gastrocnemius muscle index GMI) and nerve regeneration (myelin sheath area, total fiber counts, fiber density, and fiber width) were all evaluated.
RESULTS:The intraneural injection of IGF-1 significantly improved SFI and SS at weeks 10 and 12. There were no statistical differences between Groups 1 and 3 in any of the SFI or SS evaluations. CMAP and NCV in Group 1 were significantly higher than in Groups 2 and 3, and Group 3 had significantly higher CMAP and NCV compared to Group 2. No significant differences were found in fiber width. The number of nerve fibers, percentage of myelinated fibers, fiber density, and GMI was significantly higher in Group 1 compared to Group 2, but no significant differences were found between Groups 1 and 3.
CONCLUSION:The results show that intraneural injection of IGF-1 in an 18 mm cryopreserved isograft improve axonal regeneration and functional recovery.
Flavobacterium psychrophilum affects many cultured fish species and is considered one of the most important bacterial pathogens causing substantial economic losses in salmonid aquaculture worldwide. ...Here, F. psychrophilum was identified by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) and nested PCR as the aetiological agent causing mortality in diseased juvenile Siberian sturgeons (Acipenser baerii) reared on a freshwater fish farm. Diseased sturgeons were lethargic and displayed dark skin pigmentation, increased mucus production and the presence of skin ulcerations and haemorrhages specially on the ventral side and the base of fins. The histological examination of fish revealed proliferative branchitis, ulcerative and necrotizing dermatitis and myositis, lymphoid tissue atrophy, liver and kidney degeneration and thrombosis. To the best of our knowledge, this is the first report describing the infection of Siberian sturgeons by F. psychrophilum. The detection of F. psychrophilum in diseased Siberian sturgeons and the description of the pathological findings observed during the outbreak may contribute to a better understanding of the bacterium pathogenicity and the range of fish species susceptible to infection.
The last big outbreaks of Ebola fever in Africa, the thousands of avian influenza outbreaks across Europe, Asia, North America and Africa, the emergence of monkeypox virus in Europe and specially the ...COVID-19 pandemics have globally stressed the need for efficient, cost-effective vaccines against infectious diseases. Ideally, they should be based on transversal technologies of wide applicability. In this context, and pushed by the above-mentioned epidemiological needs, new and highly sophisticated DNA-or RNA-based vaccination strategies have been recently developed and applied at large-scale. Being very promising and effective, they still need to be assessed regarding the level of conferred long-term protection. Despite these fast-developing approaches, subunit vaccines, based on recombinant proteins obtained by conventional genetic engineering, still show a wide spectrum of interesting potentialities and an important margin for further development. In the 80's, the first vaccination attempts with recombinant vaccines consisted in single structural proteins from viral pathogens, administered as soluble plain versions. In contrast, more complex formulations of recombinant antigens with particular geometries are progressively generated and explored in an attempt to mimic the multifaceted set of stimuli offered to the immune system by replicating pathogens. The diversity of recombinant antimicrobial vaccines and vaccine prototypes is revised here considering the cell factory types, through relevant examples of prototypes under development as well as already approved products.
Therapy resistance, which leads to the development of loco-regional relapses and distant metastases after treatment, constitutes one of the major problems that head and neck squamous cell carcinoma ...(HNSCC) patients currently face. Thus, novel therapeutic strategies are urgently needed. Targeted drug delivery to the chemokine receptor 4 (CXCR4) represents a promising approach for HNSCC management. In this context, we have developed the self-assembling protein nanotoxins T22-PE24-H6 and T22-DITOX-H6, which incorporate the de-immunized catalytic domain of Pseudomonas aeruginosa (PE24) exotoxin A and the diphtheria exotoxin (DITOX) domain, respectively. Both nanotoxins contain the T22 peptide ligand to specifically target CXCR4-overexpressing HNSCC cells. In this study, we evaluate the potential use of T22-PE24-H6 and T22-DITOX-H6 nanotoxins for the treatment of HNSCC.
T22-PE24-H6 and T22-DITOX-H6 CXCR4-dependent cytotoxic effect was evaluated in vitro in two different HNSCC cell lines. Both nanotoxins cell death mechanisms were assessed in HNSCC cell lines by phase-contrast microscopy, AnnexinV/ propidium iodide (PI) staining, lactate dehydrogenase (LDH) release assays, and western blotting. Nanotoxins antitumor effect in vivo was studied in a CXCR4
HNSCC subcutaneous mouse model. Immunohistochemistry, histopathology, and toxicity analyses were used to evaluate both nanotoxins antitumor effect and possible treatment toxicity. GSMDE and CXCR4 expression in HNSCC patient tumor samples was also assessed by immunohistochemical staining.
First, we found that both nanotoxins exhibit a potent CXCR4-dependent cytotoxic effect in vitro. Importantly, nanotoxin treatment triggered caspase-3/Gasdermin E (GSDME)-mediated pyroptosis. The activation of this alternative cell death pathway that differs from traditional apoptosis, becomes a promising strategy to bypass therapy resistance. In addition, T22-PE24-H6 and T22-DITOX-H6 displayed a potent antitumor effect in the absence of systemic toxicity in a CXCR4
subcutaneous HNSCC mouse model. Lastly, GSDME was found to be overexpressed in tumor tissue from HNSCC patients, highlighting the relevance of this strategy.
Altogether, our results show that T22-PE24-H6 and T22-DITOX-H6 represent a promising therapy for HNSCC patients. Remarkably, this is the first study showing that both nanotoxins are capable of activating caspase-3/GSDME-dependent pyroptosis, opening a novel avenue for HNSCC treatment.
Selective elimination of metastatic stem cells (MetSCs) promises to block metastatic dissemination. Colorectal cancer (CRC) cells overexpressing CXCR4 display trafficking functions and ...metastasis‐initiating capacity. We assessed the antimetastatic activity of a nanoconjugate (T22‐GFP‐H6‐FdU) that selectively delivers Floxuridine to CXCR4+ cells. In contrast to free oligo‐FdU, intravenous T22‐GFP‐H6‐FdU selectively accumulates and internalizes in CXCR4+ cancer cells, triggering DNA damage and apoptosis, which leads to their selective elimination and to reduced tumor re‐initiation capacity. Repeated T22‐GFP‐H6‐FdU administration in cell line and patient‐derived CRC models blocks intravasation and completely prevents metastases development in 38–83% of mice, while showing CXCR4 expression‐dependent and site‐dependent reduction in foci number and size in liver, peritoneal, or lung metastases in the rest of mice, compared to free oligo‐FdU. T22‐GFP‐H6‐FdU induces also higher regression of established metastases than free oligo‐FdU, with negligible distribution or toxicity in normal tissues. This targeted drug delivery approach yields potent antimetastatic effect, through selective depletion of metastatic CXCR4+ cancer cells, and validates metastatic stem cells (MetSCs) as targets for clinical therapy.
Synopsis
Control of metastatic spread is an unmet need. This study reports on the therapeutic effect of a nanoconjugate (NC) that selectively delivers a drug to metastatic stem cells overexpressing surface CXCR4 receptor (CXCR4+ MetSCs) (Targeted drug delivery) after its intravenous injection in colorectal cancer (CRC) mouse models.
Selective NC internalization, leading to a high genotoxic damage and elimination of CXCR4+ CRC cells, in a CXCR4‐dependent way, both in vitro and in vivo.
Reduction of tumor re‐initiation capacity and CXCR4+ tumor emboli intravasation in colonic peri‐tumoral vessels after NC treatment.
Potent prevention of metastases, in cell line and patient‐derived orthotopic CRC mouse models, yielding a high percent of metastasis‐free mice after repeated‐dose therapy, as compared to the free drug.
Metastatic sites with high CXCR4 expression show higher response to the NC and higher reduction in CXCR4+ cancer cell fraction at the end of treatment.
Absence of accumulation or toxicity in normal tissues and high therapeutic index achieved by the NC, which exploits the high CXCR4 overexpression in target MetSCs in comparison to non‐tumor cells.
Control of metastatic spread is an unmet need. This study reports on the therapeutic effect of a nanoconjugate (NC) that selectively delivers a drug to metastatic stem cells overexpressing surface CXCR4 receptor (CXCR4+ MetSCs) (Targeted drug delivery) after its intravenous injection in colorectal cancer (CRC) mouse models.
Colorectal cancer (CRC) remains the third cause of cancer-related mortality in Western countries, metastases are the main cause of death. CRC treatment remains limited by systemic toxicity and ...chemotherapy resistance. Therefore, nanoparticle-mediated delivery of cytotoxic agents selectively to cancer cells represents an efficient strategy to increase the therapeutic index and overcome drug resistance. We have developed the T22-PE24-H6 therapeutic protein-only nanoparticle that incorporates the exotoxin A from Pseudomonas aeruginosa to selectively target CRC cells because of its multivalent ligand display that triggers a high selectivity interaction with the CXCR4 receptor overexpressed on the surface of CRC stem cells. We here observed a CXCR4-dependent cytotoxic effect for T22-PE24-H6, which was not mediated by apoptosis, but instead capable of inducing a time-dependent and sequential activation of pyroptotic markers in CRC cells in vitro. Next, we demonstrated that repeated doses of T22-PE24-H6 inhibit tumor growth in a subcutaneous CXCR4
+
CRC model, also through pyroptotic activation. Most importantly, this nanoparticle also blocked the development of lymphatic and hematogenous metastases, in a highly aggressive CXCR4
+
SW1417 orthotopic CRC model, in the absence of systemic toxicity. This targeted drug delivery approach supports for the first time the clinical relevance of inducing GSDMD-dependent pyroptosis, a cell death mechanism alternative to apoptosis, in CRC models, leading to the selective elimination of CXCR4
+
cancer stem cells, which are associated with resistance, metastases and anti-apoptotic upregulation.
Two human proteins involved in the inflammatory cell death, namely Gasdermin D (GSDMD) and the Mixed Lineage Kinase Domain-Like (MLKL) protein have been engineered to accommodate an efficient ligand ...of the tumoral cell marker CXCR4, and a set of additional peptide agents that allow their spontaneous self-assembling. Upon production in bacterial cells and further purification, both proteins organized as stable nanoparticles of 46 and 54 nm respectively, that show, in this form, a moderate but dose-dependent cytotoxicity in cell culture. In vivo, and when administered in mouse models of colorectal cancer through repeated doses, the nanoscale forms of tumor-targeted GSDMD and, at a lesser extent, of MLKL promoted CD8+ and CD20+ lymphocyte infiltration in the tumor and an important reduction of tumor size, in absence of systemic toxicity. The potential of these novel pharmacological agents as anticancer drugs is discussed in the context of synergistic approaches to more effective cancer treatments.
•Engineered forms of GSDMD and MLKL can be produced as self-assembling nanoparticles.•Being tumor targeted, they promote potent lymphocyte infiltration in tumor tissue.•Targeted GSDMD and MLKL nanoparticles arrest tumor growth.
PDF
