| Malignant gliomas rank as the most common and aggressive forms of primary brain tumor, and more than half of all gliomas are GBM. Patients with primary GBM have a median survival of less than15months, whereas those with recurrent GBM have a median survival of only approximately8weeks, even with therapeutic intervention. The current mainstay therapeutic modalities, including surgical resection (microneurosurgery), radiotherapy and chemotherapy, provide short-term benefit and largely fail to improve the poor prognosis of the patients.Epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family. This transmembrane receptor tyrosine kinase (TK) consists of an extracellular ligand-binding domain, a transmembrane lipophilic region, and an intracellular TK domain. EGFR signaling mediates a broad range of normal biological processes, including motility, adhesion, proliferation and differentiation; as such, uncontrolled EGFR signaling can have significant detrimental effects, and has been associated with many tumoral processes, including cells invasion, inhibition of apoptosis, resistance to treatment, and induction of pathogenesis. EGFR overexpression has been clinically observed in approximately70%of solid tumors, and has been reported as abnormally activated in more than60%of glioblastomas (GBMs). Tumor diagnosis and monitoring are often carried out by imaging techniques based on response to treatment with EGFR inhibitors, such as anti-EGFR antibodies and insulin-like growth factor. Unfortunately, exposure to these EGFR inhibitors has been demonstrated to complicate the cancer process, reinforcing innate and acquired resistance to tumorigenesis.Immunotherapy has proven an effective strategy to selectively treat a broad range of diseases; however, the selection of molecular targets for any immunotherapeutic approach requires a thorough understanding of the underlying molecular mechanisms of pathogenesis. Recent progress in the understanding of GBM initiation and progression has provided critical insights into biological pathways and molecules that may be exploited by a molecularly-targeted immunotherapy approach. For example, EGFR signaling plays an important role in the pathogenesis of GBM and is clinically associated with increased tumor growth rate and shorter survival. EGFR and the EGFR-mediated oncogenic signaling system have arisen as particularly attractive targets for antitumor therapy. Several IgG-based anti-EGFR agents that have been developed are currently in use, including Cetuximab, Nimotuzumab, Panitumumab, Matuzumab and Zalutumumab. The application of anti-EGFR antibody therapy has unique advantages, but the challenge of penetrance remains. IgG-based antibodies have a molecular weight of about150kDa, necessitating an auxiliary passage strategy for delivery to brain tumor lesions. To overcome this issue, the anti-EGFR scFv used in this study is the smallest fragment that retains the full binding structure of a native antibody; it is expected that this size will favor the brain delivery procedure to treat GBM in vivo.The plant enzyme ribosome inactivating protein (RIP) has recently been described for its ability to inhibit protein synthesis by depurinating the large ribosomal RNA (rRNA) and thereby blocking ribosome binding to elongation factor2(EF-2). RIPs have since been tested extensively for their immunotoxin properties as anti-tumor and anti-HIV agents. Traditional RIPs have molecular weights ranging from26to31kDa; a subclass of small RIPs, having molecular weight of less than10kDa, have been discovered but are not well-characterized. The smallest RIP known to date is the5kDa ribosome-inactivating polypeptide Luffin P1(LP1), isolated from the seeds of Luffa cylindrica. Similar to most RIPs, LP1displays translation-inhibiting potency in cell-free systems. In addition, LP1in vitro treatment significantly inhibits tumor cell proliferation, while in vivo treatment effectively prolongs survival of allograft models.Anti-EGFR antibody efficiency is another challenge to EGFR-based immunotherapy. Therefore, we fused the LP1RIP immunotoxin to the C-terminus of the anti-EGFR scFv. The RIP family members are classified among three types, based on primary structure, biological properties and their corresponding genes. Type I RIPs are composed of a single enzymatically-active chain and have molecular weights ranging from26to31kDa. Type Ⅱ RIPs are highly toxic heterodimeric proteins with molecular weights～30kDa. Type Ⅲ RIPs are uniquely synthesized as zymogens. As with ricin, all RIPs are immunogenic and allergenic. Administration of RIPs induces the humoral immune response (IgE response) with cross-reaction occurring only with RIPs belonging to the same family. RIPs are also known to enhance the response against other antigens administered at the same time. Interestingly, RIPs also prohibit immunosuppressive effects of both the humoral and cellular arms of the immune response by preventing the formation of antibodies and inhibiting the graft rejection process. It seems that RIPs can exert their immune effects by eliminating the antigen-presenting cells, which are critical components of the early immune response. However, the suppression effect only impacts the antigen given after the RIPs while the existing immunity is intact. Traditional RIPs with strong immunogenic capacity and large molecular size are not suitable for the preparation of exogenous immunotoxins because of their inability to effectively penetrate tissues and excessive stimulation of the host immune response, leading to subsequent rapid clearance from the host. LP1is the smallest of the type III RIPs, having a molecular weight of5226.8Da and possessing highly inhibitory activity towards cell-free protein synthesis. Many studies have been published on the immunogenicity of Luffin PI and its potent immunotoxin properties, and LP1has been used successfully in a broad range of laboratory research and clinical human therapies of cancer, autoimmune diseases, and infectious diseases. Furthermore, a fusion protein conjugate of Luffin PI with hIL-2had been previously developed and demonstrated as effective in prolonging survival of animals with MHC-mismatched skin and kidney allografts, while eliciting very few side effects. AntiEGFR/LP1assembled by fusing anti-EGFR scFv and RIP LP1via a linker encoding a short peptide may be suitable for cancer therapy applications based on their small size.Here, we described the expression, purification and characterization of a recombinant protein composed of the anti-EGFR scFv and RIP LP1. It is known that the reforming of disulfide bonds in the scFv play a major role in renaturation. In the present study, we used a Ni2+-chelating column to refold, purify, and reform the disulfide bonds of antiEGFR/LPl in a single step. The results indicate that this process of renaturation and purification of antiEGFR/LP1is suitable and effective. This fused antiEGFR/LP1is biologically active and specifically binds EGFR-positive tumor cells in vitro and mediates their killing. Importantly, this cytotoxic property was restricted to the EGFR-expressing cells and had minimal effects on EGFR-negative cells, indicating that antiEGFR/LP1may be an effective immunotoxin in vivo and elicit minimal side-effects on non-tumor cells proximal to the EGFR-overexpressing tumor cells. We also probed the antitumoral effects of antiEGFR/LP1in vivo. The results showed that the recombinant protein antiEGFR/LP1suppressed the growthe of the U251solid tumors successfully. Furthermore, the inoculation of the recombinant protein antiEGFR/LP1prolonged the mean survival time, tumor doubling time and tumor delay time.In conclusion, this newly-developed toxin-linked conjugate may represent a novel antibody-based immunotherapeutic agent to treat GBM or an adjuvant to enhance the current radiotherapy and chemotherapy treatments. |
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