Natural Killer Cells and Anti-Cancer Therapies: Reciprocal Effects on Immune Function and Therapeutic Response.

Natural Killer (NK) cells are innate immune cells with the unique ability to recognize and kill virus-infected and cancer cells without prior immune sensitization. Due to their expression of the Fc receptor CD16, effector NK cells can kill tumor cells through antibody-dependent cytotoxicity, making them relevant players in antibody-based cancer therapies. The role of NK cells in other approved and experimental anti-cancer therapies is more elusive. Here, we review the possible role of NK cells in the efficacy of various anti-tumor therapies, including radiotherapy, chemotherapy, and immunotherapy, as well as the impact of these therapies on NK cell function.

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.

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.