Advances in immunotoxin engineering: precision therapeutic strategies in modern oncology.

Immunotoxins (ITs) are specialized therapeutic agents designed for targeted treatment, particularly in cancer therapy. They consist of a monoclonal antibody or antibody fragment linked to a potent cytotoxic agent, such as bacterial- or plant-derived toxins like diphtheria toxin, ricin, or pseudomonas exotoxin. The monoclonal antibody component specifically binds to antigens expressed on the surface of target cells, facilitating the internalization of the IT. Once inside the cell, the cytotoxic agent is released, disrupting essential cellular processes and leading to cell death. This targeted approach minimizes damage to healthy tissues while effectively eliminating diseased cells. The production of ITs involves two primary methods: recombinant fusion and chemical conjugation. In recombinant fusion, genetic engineering is used to create a fusion protein that combines the antibody and toxin, ensuring precise control over their ratio and functionality. In chemical conjugation, pre-existing antibodies are chemically linked to toxins, allowing for greater flexibility in combining different antibodies and cytotoxic agents. Each method has its advantages and challenges, influencing the specificity, production complexity, and therapeutic potential of the resulting ITs. As research advances, ITs continue to show promise not only in oncology but also in treating other diseases, including inflammatory conditions and atherosclerosis. The precise targeting and potent effects of ITs make them a valuable tool in the development of new therapeutic strategies.

Development and Characterization of an Anti-PD-L1 Immunotoxin for Targeted Cancer Therapy.

BACKGROUND: Immunotoxins (ITs) represent a novel class of therapeutics with bifunctional structures that facilitate their penetration through cell membranes to induce target cell destruction. Programmed cell death ligand-1 (PD-L1), a human cell surface protein, is overexpressed in various cancers. This study aimed to construct a novel IT by genetically fusing an anti-PD-L1 Nanobody (Nb) to a truncated diphtheria toxin (DT). METHODS: The IT construct comprised a 127-amino acid anti-PD-L1 Nb fused to a 380-amino acid fragment of DT, with an N-terminal 6x-His tag. Molecular cloning techniques were employed, followed by transformation and verification through colony-PCR, enzyme digestion, and sequencing. The anti-PD-L1 Nb was expressed in WK6 E. coli cells induced by Isopropyl ß-D-1- Thiogalactopyranoside (IPTG) and purified from periplasmic extracts using immobilized Metal Ion Affinity hromatography (IMAC). The IT was similarly expressed, purified, and validated via SDS-PAGE and Western blot analysis. RESULTS: ELISA confirmed the binding activity of both Nb and IT to immobilized PD-L1 antigen, whereas truncated DT exhibited no binding. MTT assays demonstrated significant cytotoxicity of IT on A-431 cell lines compared to Nb and truncated DT controls. Statistical analyses underscored the significance of these findings. CONCLUSION: This study provides a thorough characterization of the constructed IT, highlighting its potential as a therapeutic agent targeting PD-L1-expressing cancer cells. The results support the potential of this IT in cancer immunotherapy, emphasizing the need for further investigation into its efficacy and safety profiles.

Development and characterization of nanobody against envenomation by Naja naja oxiana.

Snakebites are considered a significant health issue. Current antivenoms contain polyclonal antibodies, which vary in their specificity against different venom components. Development and characterization of next generation antivenoms including nanobodies against Naja naja oxiana was the main aim of this study. Crude venom was injected into the Sephadex G50 filtration gel chromatography column and then toxic fractions were obtained. Then the corresponding fraction was injected into the HPLC column and the toxic peaks were identified. N. naja oxiana venom was injected into a camel and specific nanobodies screening was performed against the toxic peak using phage display technique. The obtained results showed that among the 12 clones obtained, N24 nanobody was capable of neutralizing P1, the most toxic peak obtained from HPLC chromatography. The molecular weight of P1 was measured with a mass spectrometer and was found to be about seven kDa. The results of the neutralization test of crude N. naja oxiana venom with N24 nanobody showed that 250 µg of recombinant nanobody could neutralize the toxic effects of 20 µg equivalent to LD50 × 10 of crude venom in mice. The findings indicate the potential of the developed nanobody to serve as a novel antivenom therapy.

Development of a Functional Nanobody Targeting Programmed Cell Death Protein-1 as Immune Checkpoint Inhibitor.

BACKGROUND: Programmed cell death protein 1 (PD-1) is a membrane receptor that is expressed on the surface of various immune cells, such as T cells, B cells, monocytes, natural killer T cells, and dendritic cells. In cancer, the interaction between PD-1 and its ligand PD-L1 suppresses the activation and function of T lymphocytes, leading to the impairment and apoptosis of tumor-specific T cells. This mechanism allows cancer cells to evade the immune response and promotes tumor progression. METHODS: Recombinant PD-1 protein was produced and used to immunize a camel. A nanobody library was generated from the camel's peripheral blood lymphocytes and screened for PD-1 binding. A specific nanobody (3PD9) was selected and characterized by affinity measurement, western blotting, and flow cytometry analysis. The ability of the selected nanobody to block the inhibitory signal of PD-1 in peripheral blood mononuclear cells (PBMCs) was evaluated by measuring the level of interleukin-2 (IL-2). RESULTS: The selected nanobody showed high specificity and affinity for human PD-1. Western blot and flow cytometry analysis confirmed that 3PD9 could recognize and bind to human PD-1 on the cell surface. It was demonstrated that the level of IL-2 was significantly increased in PBMCs treated with 3PD9 compared to the control group, indicating that the nanobody could enhance the T cell response by disrupting the PD-1/PD-L1 interaction. CONCLUSION: The results suggested that the anti-PD-1 nanobody could be a promising candidate for cancer immunotherapy.

Development of polyclonal heavy chain antibodies targeting programmed death ligand-1.

Programmed death ligand-1 (PD-L1, CD274 and B7-H1) has been described as a ligand for immune inhibitory receptor programmed death protein 1 (PD-1). With binding to PD-1 on activated T cells, PD-L1 can prevent T cell responses via motivating apoptosis. Consequently, it causes cancers immune evasion and helps the tumor growth; hence, PD-L1 is regarded as a therapeutic target for malignant cancers. The anti-PD-L1 monoclonal antibody targeting PD-1/PD-L1 immune checkpoint has attained remarkable outcomes in clinical application and has turned to one of the most prevalent anti-cancer drugs. The present study aimed to develop polyclonal heavy chain antibodies targeting PD-L1via Camelus dromedarius immunization. The extra-cellular domain of human PD-L1 (hPD-L1) protein was cloned, expressed, and purified. Afterwards, this recombinant protein was utilized as an antigen for camel immunization to acquire polyclonal camelid sera versus this protein. Our outcomes showed that hPD-L1 protein was effectively expressed in the prokaryotic system. The antibody-based techniques, such as enzyme-linked immunosorbent assay, western blotting, and flow cytometry displayed that the hPD-L1 protein was detected by generated polyclonal antibody. Due to the advantages of multi-epitope-binding ability, our study exhibited that camelid antibody is effective to be applied significantly for detection of PD-L1 protein in essential antibody-based studies.

In vivo solid tumor targeting with recombinant VEGF-diphtheria immunotoxin.

Objectives: A variety of signaling molecules have been identified that play a role in angiogenesis, of prime importance, vascular endothelial growth factor (VEGF) and its resceptor (VEGFR), which is highly expressed in most human solid tumors. Targeting VEGF or/and VEGFR with immunotoxin may be a promising approach to directly affect cancer cells. Immunotoxins are for targeted treatment comprising two functional moieties, an antibody that binds to target cells along with toxin that kills molecules. Materials and Methods: In this study, an immunotoxin comprising domain of diphtheria toxin subunit A (DT386) genetically fused to mouse VEGF (mVEGF-DT) was developed. The second construct, which contains the DT386 domain, was made to investigate the action of the DT386 domain on tumor cells. Both gene constructs were cloned, expressed, and were further purified. The biological activity of mVEGF-DT and DT386 proteins was assessed on the TC1 cell line bearing mouse model. Proteins were injected intra-tumoral in mice, in separate groups. Results: Tumors in the mVEGF-DT group started to dwindle after six injections, but tumor size in both control groups (DT386 and PBS), continued to grow. Conclusion: Successful targeting of solid tumor cells by mVEGF-DT immunotoxin demonstrates the therapeutic potential utility of these conjugates for tumor targeting.

Tumor Suppression by PD-1/PD-L1 Interaction Blockage in Mice Model.

Background: Overexpression of programmed cell death ligand 1 (PD-L1) in tumor cells and subsequent interaction with the programmed cell death protein 1 (PD-1) in tumor-infiltrating T cells cause an immune evasion of the tumor from cytotoxic T-cells. Therefore, inhibiting such interaction by a recombinant PD-1 can hinder tumor growth and extend the survival rate. Methods: The mouse extracellular domain of PD-1 (mPD-1) was expressed in E. coli BL21 (DE3) strain and purified using nickel affinity chromatography. The binding ability of the purified protein to human PD-L1 was studied using ELISA. Finally, the tumor-bearing mice were used to evaluate the potential antitumor effect. Results: The recombinant mPD-1 showed a significant binding capacity to human PD-L1 at the molecular level. The tumor size significantly decreased in the tumor-bearing mice after the intra-tumoral injections of mPD-1. Moreover, the survival rate increased significantly after eight weeks of monitoring. The histopathology revealed the necrosis in the tumor tissue of the control group compared to the mPD-1 received mice. Conclusions: Our outcomes propose that interaction blockade between PD-1 and PD-L1 is a promising approach for targeted tumor therapy.

Nanobodies as novel therapeutic agents in envenomation.

BACKGROUND: An effective therapy against envenoming should be a priority in view of the high number scorpion stings and snakebites. Serum therapy is still widely applied to treat the envenomation victims; however this approach suffers from several shortcomings. The employment of monoclonal antibodies might be an outcome as these molecules are at the core of a variety of applications from protein structure determination to cancer treatment. The progress of activities in the twilight zone between genetic and antibody engineering have led to the development of a unique class of antibody fragments. These molecules possess several benefits and lack many possible disadvantages over classical antibodies. Within recombinant antibody formats, nanobodies or single domain antigen binding fragments derived from heavy chain only antibodies in camelids occupy a privileged position. SCOPE OF REVIEW: In this paper we will briefly review the common methods of envenomation treatment and focus on details of various in vivo research activities that investigate the performance of recombinant, monoclonal nanobodies in venom neutralization. MAJOR CONCLUSIONS: Nanobodies bind to their cognate target with high specificity and affinity, they can be produced in large quantities from microbial expression systems and are very robust even when challenged with harsh environmental conditions. Upon administering, they rapidly distribute throughout the body and seem to be well tolerated in humans posing low immunogenicity. GENERAL SIGNIFICANCE: Scorpion and snake envenomation is a major issue in developing countries and nanobodies as a venom-neutralizing agent can be considered as a valuable and promising candidate in envenomation therapy.