Enhancing cancer immunotherapy with Anti-NKG2D/IL-15(N72D)/Sushi fusion protein: Targeting cytotoxic immune cells and boosting IL-15 efficacy.

BACKGROUND: There are a number of distinct challenges and complexities associated with administering IL-15 for cancer immunotherapy that must be taken into consideration. OBJECTIVE: The purpose of this study was to design a fusion protein for targeting cytotoxic immune cells and enhance IL-15 efficiency. METHODS: A fusokine that contains IL-15(N72D), a Sushi domain, and anti-NKG2D scFv was designed. The fusion protein was in-silico modeled using the Swiss model server, followed by docking and molecular dynamics simulations. The in-vitro purified fusokine was evaluated using dot blot and Western blot. Then, flow cytometry was employed to evaluate biological properties such as proliferation, cytotoxicity, and degranulation. RESULTS: Fusokine and IL-15(N72D)/Sushi, which had molecular weights of about 52 kDa and 26 kDa, respectively, were expressed in CHO-K1 cells. The fusokine binds 69.6 % of the CHO-NKG2D+ cells that express 83.1 % NKG2D. Both the fusokine and the IL-15(N72D)/Sushi significantly stimulate the proliferation of lymphocytes. After 14 days of growth, the vitality of untreated cells decreased to about 17.5 %, but 82.2 % and 56.6 % of cells were still alive when fusokine and IL-15(N72D)/Sushi were present. Furthermore, administration of fusokine was associated with the highest rates of target tumor cell cytotoxicity. Additionally, although it was not statistically significant, fusokine increased the expression of CD107a and granzyme B by 1.25 times and 2.4 times, respectively. CONCLUSION: The fusokine possesses the capability to stimulate the survival and multiplication of lymphocytes, as well as their ability to eliminate tumors. These characteristics have led to its consideration as a potential treatment for immunotherapy.

Anti-CD19/CD8 bispecific T cell engager for the potential treatment of B cell malignancies.

The administration of blinatumomab was accompanied by several adverse effects, including activation of regulatory T-cells and cytokine storm. The objective of this study was to produce and evaluate a novel αCD8/CD19 BiTE (αCD8/CD19) with the potency to directly target CD8+T-cells. In-silico studies were utilized for determining proper folding, receptor binding, and structural stability of αCD8/CD19 protein. Western blotting and indirect surface staining were used to evaluate the size accuracy and binding potency of the purified protein. Functionality was assessed for granzyme B production, cytotoxicity, and proliferation. TheαCD8/CD19recombinant protein was produced in the CHO-K1 cell line with a final concentration of 1.94 mg/l. The αCD8/CD19 bound to CD8+and CD19+cell lines and induced significant granzyme B production, cytotoxic activity and proliferation potential in the presence of IL-2 and tumor target cells. The maximum CD8+T-cell biological activity was observed on the 10th day with 10:1 effector-to-target ratio.

Production of a biosimilar version of aflibercept to improve VEGF blocker cytotoxicity on endothelial cells.

This project aimed to produce a biosimilar version of aflibercept (AFL) and evaluate the effect of the co-treatment of AFL with other vascular endothelial growth factor (VEGF) blocker drugs. For this purpose, the optimized gene was inserted into the pCHO1.0 plasmid and transfected into the CHO-S cell line. The final concentration of biosimilar-AFL for the selected clone was 782 mg/L. Results revealed that the inhibition potential of the biosimilar-AFL on HUVEC cells was significant at 10 and 100 nM concentrations and in a dose-dependent manner. Furthermore, co-treatment of biosimilar-AFL with Everolimus (EVR), Lenvatinib (LEN), and Sorafenib (SOR) could reduce HUVEC cell viability/proliferation, more than when used alone. When LEN and SOR were co-treated with biosimilar-AFL, their cytotoxicity increased 10-fold. The most and least efficient combination was seen when biosimilar-AFL combined with LEN and EVR, respectively. Finally, biosimilar-AFL may improve the efficiency of LEN, EVR, and SOR in reducing the VEGF effect on endothelial cells.

Triggering of lymphocytes by CD28, 4-1BB, and PD-1 checkpoints to enhance the immune response capacities.

Tumor infiltrating lymphocytes (TILs) usually become exhausted and dysfunctional owing to chronic contact with tumor cells and overexpression of multiple inhibitor receptors. Activation of TILs by targeting the inhibitory and stimulatory checkpoints has emerged as one of the most promising immunotherapy prospectively. We investigated whether triggering of CD28, 4-1BB, and PD-1 checkpoints simultaneously or alone could enhance the immune response capacity of lymphocytes. In this regard, anti-PD-1, CD80-Fc, and 4-1BBL-Fc proteins were designed and produced in CHO-K1 cells as an expression host. Following confirmation of the Fc fusion proteins' ability to bind to native targets expressed on engineered CHO-K1 cells (CHO-K1/hPD-1, CHO-K1/hCD28, CHO-K1/hCTLA4, and CHO-K1/h4-1BB), the effects of each protein, on its own and in various combinations, were assessed in vitro on T cell proliferation, cytotoxicity, and cytokines secretion using the Mixed lymphocyte reaction (MLR) assay, 7-AAD/CFSE cell-mediated cytotoxicity assay, and a LEGENDplex™ Human Th Cytokine Panel, respectively. MLR results demonstrated that T cell proliferation in the presence of the combinations of anti-PD-1/CD80-Fc, CD80-Fc/4-1BBL-Fc, and anti-PD-1/CD80-Fc/4-1BBL-Fc proteins was significantly higher than in the untreated condition (1.83-, 1.91-, and 2.02-fold respectively). Furthermore, anti-PD-1 (17%), 4-1BBL-Fc (19.2%), anti-PD-1/CD80-Fc (18.6%), anti-PD-1/4-1BBL-Fc (21%), CD80-Fc/4-1BBL-Fc (18.5%), and anti-PD-1/CD80-Fc/4-1BBL-Fc (17.3%) significantly enhanced cytotoxicity activity compared to untreated condition (7.8%). However, concerning the cytokine production, CD80-Fc and 4-1BBL-Fc alone or in combination significantly increased the secretion of IFN-γ, TNF-α, and IL-2 compared with the untreated conditions. In conclusion, this research establishes that the various combinations of produced anti-PD-1, CD80-Fc, and 4-1BBL-Fc proteins can noticeably induce the immune response in vitro. Each of these combinations may be effective in killing or destroying cancer cells depending on the type and stage of cancer.

Introducing a New Method for Purification of Human IL-4 by Substitution of a Single Amino Acid in IL-4 Protein Sequence.

BACKGROUND: It is advantageous to develop an effective purification procedure to produce recombinant protein drugs (rPDs) without any tags. To remove N- or C-terminus tags from the rPDs, several cleavage site-based endopeptidases were used. Separating the endopeptidase enzyme from the rPDs is a time-consuming and costly process. OBJECTIVE: To design and develop a new method for the purification of human interleukin (IL)-4 with potential application for other cytokines. METHODS: Met-like amino acids were substituted at position 120 to reduce the possibility of alteration in the structure of IL-4 and its biological activity. Based on the in silico analysis, isoleucine was chosen as an alternative amino acid, and the M120I mutant IL-4 (mIL-4) model was selected for the downstream analysis. Recombinant mIL-4 was produced in the E.coli BL21 host and purified with CNBr. Then in vitro evaluations of the native and mutant IL-4 were performed. RESULTS: The results showed that both the native and mutant IL-4 had the same effect on TF-1 cell proliferation. On the other hand, there was no significant difference between the effects of native IL-4 (nIL-4) and mIL-4 on the expression of IL-4 and IL-10 in activated peripheral blood mononuclear cells. Native and mutant IL-4 have similar biological activities. CONCLUSION: Here, an efficient and straightforward system is introduced to purify IL-4 cytokine using CNBr, which could be applied to other rPDs.

Molecular Cloning, Expression and Purification of G-CSF Isoform D, an Alternative Splice Variant of Human G-CSF.

Granulocyte colony-stimulating factor (G-CSF) is the major regulator of hemopoiesis and granulopoiesis. However, overexpression of G-CSF has been implicated in several important processes in tumor biology such as tumor growth, angiogenesis, and metastasis. Four different mRNA isoforms resulting from alternative splicing have been reported for G-CSF (transcript variants 1, 2, 3 and 4). The mRNAs and protein products of splice variants 1 and 2 have been isolated for the first time, from tumor cell lines. In the present study for the first time we isolated the G-CSF transcript variant 4 encoding G-CSF isoform D from a highly malignant tumor cell line (Mehr80) with overexpression of G-CSF. Both the full-length G-CSF isoform B and G-CSF isoform D were cloned from Mehr80 cell line, overexpressed in Escherichia coli as N-terminal glutathione-S-transferase fusion proteins in the form of inclusion bodies and affinity purified by the batch method using glutathione-Sepharose 4B resin. Both fusion proteins were successfully cloned and expressed. Folded recombinant proteins were solubilized from inclusion bodies using sarkosyl, Triton X-114 and CHAPS and purified. The purity of G-CSF isoforms was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and they were clearly detected in western blot analysis using anti-G-CSF polyclonal antibody. The G-CSF plays various roles in physiological and pathological conditions, however to date, the differential function of G-CSF isoforms remains unknown. Considering the fact that G-CSF isoform D was isolated from a highly malignant tumor cell line with overexpression of G-CSF, the role of this splice variant in tumorigenesis requires further investigation.

Killer Cell Immunoglobulin-Like Receptors (KIRs) Genotype and Haplotype Analysis in Iranians with Non-Melanoma Skin Cancers

Background: The innate immune system against malignancies is mainly orchestrated by natural killer cells, which carry out killing mechanisms by using their receptors, such as killer immunoglobulin-like receptors (KIRs). This study was designed to determine the diversity of KIR genes in non-melanoma skin cancers. Methods: A total of 160 subjects with skin cancer, including 60 cases of squamous cell carcinoma and 100 cases of basal cell carcinoma (BCC), and 270 healthy subjects formed the study groups. The sequence-specific polymerase chain reaction was carried out to detect the presence or absence of 16 KIR genes. Results: KIR3DL1 (p = 0.0381, OR = 4.78, 95% CI = 1.108 to 20.62) increased in BCC patients compared to healthy controls. Conclusion: We concluded that the higher frequency of KIR3DL1 in BCC patients compared with healthy controls may increase the probability of developing BCC in Iranians.

Expression Analysis of MALAT1, GAS5, SRA, and NEAT1 lncRNAs in Breast Cancer Tissues from Young Women and Women over 45 Years of Age.

Breast cancer, as the most common cancer in women worldwide, represents about 30% of all cancers affecting women. Long non-coding RNAs (lncRNAs) have been implicated in the regulation of several biological processes, and their dysregulation in cancer has well been documented. To investigate possible age-dependent variations in expression profiles of lncRNAs, we evaluated the expression levels of four lncRNAs, i.e., MALAT1, GAS5, SRA, and NEAT1, in breast cancer (BC) samples obtained from younger (<45 years) and older (>45 years) women. Tumor samples (n = 23) and 15 normal tissues were collected from BC patients. All tumor and normal samples were morphologically confirmed by a pathologist. RNA was extracted from the tissues and cDNAs were then synthesized. The lncRNA expression levels were evaluated by qRT-PCR. The changes in the expression levels were determined using the ΔΔCt method. Compared to normal tissues, BC tissues from both age groups (women under 45 years of age and women above 45 years of age) showed upregulation of MALAT1 (p = 0.003 and p = 0.0002), SRA (p = 0.005 and p = 0.0002), and NEAT1 (p = 0.010 and p = 0.0002) and downregulation of GAS5 (p = 0.0002 and p = 0.0005). Additionally, our analysis showed significant and direct correlation between the age and the expression levels of three of the four lncRNAs studied in this work. All four lncRNAs were overexpressed in both MDA-MB-231 and MCF7 cell lines (p = 0.1000). Our data show that MALAT1, GAS5, SRA, and NEAT1 lncRNAs are dysregulated in BC samples. However, except for MALAT1, the expression levels of all of these lncRNAs were significantly lower in cancers developed in younger cases, where poorer prognosis is suggested. Of note, GAS5 reduced expression has been documented to correlate with tumor progression.

Cloning and Expression of Recombinant Human Interleukin-7 in Chinese Hamster Ovary (CHO) Cells.

BACKGROUND: The critical role of interleukin-7 (IL-7) in homeostatic proliferation and T cell survival has made it a promising cytokine for the treatment of various clinical conditions, especially those associated with lymphopenia. METHODS: In the present study we expressed recombinant human interleukin-7 (rhIL-7) in Chinese hamster ovary (CHO)-K1 cells. CHO-K1 cells were stably transfected with both circular and linear forms of the pBud-hIL-7 recombinant by electroporation. Expression of rhIL-7 in CHO-K1 cells was confirmed by enzyme-linked immunosorbent assay (ELISA) and dot and western blots. RESULTS: On western blots of transformed cells, a single 25 kDa band was observed, consistent with the expected molecular weight of glycosylated hIL-7. No significant expression difference was observed between cells transfected with circular or linear plasmids. CONCLUSION: We established a stable CHO-K1 cell line expressing rhIL-7, which we consider to be a promising system for the production of rhIL-7 as a biopharmaceutical.

Improving Pertuzumab production by gene optimization and proper signal peptide selection.

Using proper signal peptide and codon optimization are important factors that must be considered when designing the vector to increase protein expression in Chinese Hamster Ovary (CHO) cells. The aim of the present study is to investigate how to enhance Pertuzumab production through heavy and light chain coding gene optimization and proper signal peptide selection. First, CHO-K1 cells were transiently transfected with whole-antibody-gene-optimized, variable-regions-optimized and non-optimized constructs and then we employed five different signal peptides to improve the secretion efficiency of Pertuzumab. Compared to the native antibody gene, a 3.8 fold increase in Pertuzumab production rate was achieved with the whole heavy and light chain sequence optimization. Although an overall two fold increase in monoclonal antibody production was achieved by human albumin signal peptide compared to the control signal peptide, this overproduction was not statistically significant. Selected signal peptides had no effect on the binding of Pertuzumab to the ErbB2 antigen. The combined data indicate that human albumin signal peptide along with whole antibody sequence optimization can be used to improve Pertuzumab production rates. This sequence was used to produce Pertuzumab producing CHO-K1 stably transfected cells. This result is useful for producing Pertuzumab as a biosimilar drug.

Establishment a CHO Cell Line Expressing Human CD52 Molecule.

BACKGROUND: CD52 is a small glycoprotein with a GPI anchor at its C-terminus. CD52 is expressed by Normal and malignant T and B lymphocytes and monocytes. There are detectable amounts of soluble CD52 in plasma of patients with CLL and could be used as a tumor marker. Although the biological function of CD52 is unknown but it seems that CD52 may be involved in migration and activation of T-cells .The aim of this study was to clone and express human CD52 gene in CHO cell line and studying its function in more details. METHODS: Based on GenBank databases two specific primers were designed for amplification of cd52 gene. Total RNA was extracted from Raji cell line and cDNA synthesized. Amplified fragment was cloned in pBudCE4.1 vector. The new construct was transfected to CHO-K1 cell line using electroporation method. Expression of recombinant CD52 protein was evaluated by Real time PCR and flow cytometry methods. RESULTS: Amplification of CD52 gene using specific primers on Raji cDNA showed a 209 bp band. New construct was confirmed by PCR and restriction pattern and sequence analysis. The new construct was designated as pBudKT1. RT-PCR analysis detected cd52 mRNAs in transfected cells and Flow cytometry Results showed that 78.4 % of cells represented CD52 in their surfaces. CONCLUSION: In conclusion, we established a human CD52 positive cell line, CHO-CD52, and the protein was expressed on the membrane. Cloning of the CD52 gene could be the first step for the production of therapeutic monoclonal antibodies and detection systems for soluble CD52 in biological fluids.

Construction of CTLA-4-Ig Fusion Gene in pBudCE4.1 Expression Vector.

BACKGROUND: CTLA-4 inhibitory signals prevent cell cycle progression and IL-2 production, leading to a halt on an ongoing immune response. CTLA4-Ig fusion proteins contain the extracellular domain of CTLA-4 and Fc fragment of human IgG antibody. In this study we aimed to fuse the ctla-4 gene encoding the extracellular domain of CTLA-4 molecule with igg1 gene encoding Fc region of human IgG. METHODS: After primer design, PCR reaction was performed using pfu polymerase enzyme and specific primers. PCR amplified fragment was ligated into the vector containing the human igg1 gene. The resulting fusion fragment of ctla-4 and human igg1 genes was ligated to pBudCE4.1 expression vector. RESULTS: Extracellular domain of ctla-4 gene was ligated to igg1 gene and then ctla4-ig fragment was cloned into pBudCE4.1 vector. Construction of the expression vector was confirmed by restriction pattern analysis and sequencing. CONCLUSION: By confirming the construct, in the next step, the recombinant DNA will be used to produce CTLA4-Ig recombinant protein for the clinical uses.