Specific Targeting of Zinc Transporter LIV-1 with Immunocytokine Containing Anti-LIV-1 VHH and Human IL-2 and Evaluation of its In vitro Antitumor Activity.

BACKGROUND: Interleukin 2 (IL-2) is a vital cytokine in the induction of T and NK cell responses, the proliferation of CD8+ T cells, and the effective treatment of human cancers such as melanoma and renal cell carcinoma. However, widespread use of this cytokine is limited due to its short half-life, severe toxicity, lack of specific tumor targeting, and activation of Treg cells mediated by high-affinity interleukin-2 receptors. OBJECTIVE: In this study, a tumor-targeting LIV-1 VHH-mutIL2 immunocytokine with reduced CD25 (α chain of the high-affinity IL-2 receptor) binding activity was developed to improve IL-2 half-life by decreasing its renal infiltration in comparison with wild and mutant IL-2 molecules. METHODS: The recombinant immunocytokine was designed and expressed. The biological activity of the purified fusion protein was investigated in in vitro and in vivo experiments. RESULTS: The fusion protein represented specific binding to MCF7 (the breast cancer cell line) and more efficient cytotoxicity than wild-type IL-2 and mutant IL-2. The PK parameters of the recombinant immunocytokine were also improved in comparison to the IL-2 molecules. CONCLUSION: The observed results showed that LIV1-mIL2 immunocytokine could be considered as an effective agent in the LIV-1-targeted treatment of cancers due to its longer half-life and stronger cytotoxicity.

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.

Receptor tyrosine kinase inhibitors in cancer.

Targeted therapy is a new cancer treatment approach, involving drugs that particularly target specific proteins in cancer cells, such as receptor tyrosine kinases (RTKs) which are involved in promoting growth and proliferation, Therefore inhibiting these proteins could impede cancer progression. An understanding of RTKs and the relevant signaling cascades, has enabled the development of many targeted drug therapies employing RTK inhibitors (RTKIs) some of which have entered clinical application. Here we discuss RTK structures, activation mechanisms and functions. Moreover, we cover the potential effects of combination drug therapy (including chemotherapy or immunotherapy agents with one RTKI or multiple RTKIs) especially for drug resistant cancers.

Urtica dioica agglutinin (UDA) as a potential candidate for inhibition of SARS-CoV-2 Omicron variants: In silico prediction and experimental validation.

BACKGROUND: The high number of mutations and consequent structure modifications in a Receptor-Binding Domain (RBD) of the spike protein of the Omicron variant of SARS-CoV-2 increased concerns about evading neutralization by antibodies induced by previous infection or vaccination. Thus, developing novel drugs with potent inhibitory activity can be considered an alternative for treating this highly transmissible variant. Considering that Urtica dioica agglutinin (UDA) displays antiviral activity against SARS-CoV-2, the potency of this lectin to inhibit the Receptor Binding Domain of the Omicron variant (RBDOmic) was examined in this study. PURPOSE: This study examines how UDA inhibits the Omicron variant of SARS-CoV-2 by blocking its RBD, using a combination of in silico and experimental methods. METHODS: To investigate the interaction between UDA and RBDOmic, the CLUSPRO 2.0 web server was used to dock the RBDOmic-UDA complex, and molecular dynamics simulations were performed by the Gromacs 2020.2 software to confirm the stability of the selected docked complex. Finally, the binding affinity (ΔG) of the simulation was calculated using MM-PBSA. In addition, ELISA and Western blot tests were used to examine UDA's binding to RBDOmic. RESULTS: Based on the docking results, UDA forms five hydrogen bonds with the RBDOmic active site, which contains mutated residues Tyr501, Arg498, Arg493, and His505. According to MD simulations, the UDA-RBDOmic complex is stable over 100 ns, and its average binding energy during the simulation is -87.201 kJ/mol. Also, the ELISA test showed that UDA significantly binds to RBDOmic, and by increasing the concentration of UDA protein, the attachment to RBDOmic became stronger. In Western blotting, RBDOmic was able to attach to and detect UDA. CONCLUSION: This study indicates that UDA interaction with RBDOmic prevents virus attachment to Angiotensin-converting enzyme 2 (ACE2) and, therefore, its entry into the host cell. Altogether, UDA exhibited a significant suppression effect on the Omicron variant and can be considered a new candidate to improve protection against severe infection of this variant.

In Vitro and In Vivo Studies of a Heminecrolysin Toxin-VEGF Fusion Protein as a Novel Therapeutic for Solid Tumor Targeting.

Angiogenesis, the formation of new vessels, is a critical step in the malignancy progression of solid tumors. Many investigations have demonstrated the usefulness of immunotoxins to halt angiogenesis in solid tumors. Pharmaceutically, Vascular Endothelial Growth Factor (VEGF) can deliver coupled toxins to the tumor vessels through VEGF Receptors. In the current study, we designed, expressed, and assessed the in vitro and in vivo toxicities of a novel immunotoxin consisting of mouse VEGF and heminecrolysin toxin (mVEGF-HNc). The fusion protein was expressed in E. coli and purified via Ni+2 affinity chromatography. The biological activity of immunotoxin was evaluated on NIH/3T3 cells and TC1-tumorized mouse model. The mVEGF-NHc showed significant cytotoxicity on the cells as VEGFR-expressing cells. Moreover, the size of the tumor in the mVEGF-HNc-treated group started to reduce after six injections, while it continued to grow in the PBS-received mice. Efficacious targeting of solid tumor cells via mVEGF-HNc suggests its prospective therapeutic potential for cancer therapy.

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.

Development of camelid monoclonal nanobody against SLC39A6 zinc transporter protein.

Objectives: SLC39A6 (solute carrier family 39) or LIV-1, is a zinc-transporter protein associated with estrogen-positive breast cancer and its metastatic spread. Significantly there is a direct relation between high zinc intake and unregulated cell proliferation and cancers. Blocking SLC39A6 protein may result in reduced metastasis and proliferation in many malignant tumors. This study aimed to develop an anti-SLC39A6 nanobody that is able to detect and block the SLC39A6 protein on the surface of cancerous cells. Materials and Methods: The recombinant SLC39A6 was expressed and used for camel immunization. The VHH library was constructed and screened for SLC39A6-specific nanobody. Then, the strength of nanobody in SLC39A6 detection was evaluated by Western blotting and flow cytometry. Results: We showed the ability of SLC39A6 specific Nanobody (C3) to detect SLC39A6 by Western blotting and flow cytometry. Furthermore, the C3 nanobody potently inhibits cell proliferation in MTT assay. Conclusion: These data show the potential of SLC39A6-specific nanobody for the blockade of zinc transporter protein and provide a basis for the development of novel cancer therapeutics.

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.