Nimotuzumab,a promising therapeutic monoclonal for treatment of tumors of epithelial origin

Nimotuzumab is a humanized therapeutic monoclonal antibody against epidermal growth factor receptor (EGFR). Clinical trials are ongoing globally to evaluate nimotuzumab in different indications. Nimotuzumab has been granted approval for use in squamous cell carcinoma of head and neck (SCCHN), glioma and nasopharyngeal cancer in different countries. This review focuses on the unique functional characteristics of nimotuzumab. Also, it discusses the safety and efficacy data obtained from the Phase IIb clinical trial conducted in India in SCCHN. Post Marketing Surveillance data from Cuba for the use of nimotuzumab in pediatric and adult glioma is also discussed. Overall, nimotuzumab has immense therapeutic potential in cancers of epithelial origin.     

Comparative in vitro and experimental in vivo studies of the anti–epidermal growth factor receptor antibody nimotuzumab and its aglycosylated form produced in transgenic tobacco plants

A broad variety of foreign genes can be expressed in transgenic plants, which offer the opportunity for large‐scale production of pharmaceutical proteins, such as therapeutic antibodies. Nimotuzumab is a humanized anti–epidermal growth factor receptor (EGFR) recombinant IgG1 antibody approved in different countries for the treatment of head and neck squamous cell carcinoma, paediatric and adult glioma, and nasopharyngeal and oesophageal cancers. Because the antitumour mechanism of nimotuzumab is mainly attributed to its ability to interrupt the signal transduction cascade triggered by EGF/EGFR interaction, we have hypothesized that an aglycosylated form of this antibody, produced by mutating the N₂₉₇ position in the IgG₁ Fc region gene, would have similar biochemical and biological properties as the mammalian‐cell‐produced glycosylated counterpart. In this paper, we report the production and characterization of an aglycosylated form of nimotuzumab in transgenic tobacco plants. The comparison of the plantibody and nimotuzumab in terms of recognition of human EGFR, effect on tyrosine phosphorylation and proliferation in cells in response to EGF, competition with radiolabelled EGF for EGFR, affinity measurements of Fab fragments, pharmacokinetic and biodistribution behaviours in rats and antitumour effects in nude mice bearing human A431 tumours showed that both antibody forms have very similar in vitro and in vivo properties. Our results support the idea that the production of aglycosylated forms of some therapeutic antibodies in transgenic plants is a feasible approach when facing scaling strategies for anticancer immunoglobulins.     

Treatment of children with high grade glioma with nimotuzumab: A 5-y institutional experience

Brain tumors are a major cause of cancer-related mortality in children. Overexpression of epidermal growth factor receptor (EGFR) is detected in pediatric brain tumors and receptor density appears to increase with tumor grading. Nimotuzumab is an IgG1 antibody that targets EGFR. Twenty-three children with high-grade glioma (HGG) were enrolled in an expanded access program in which nimotuzumab was administered alone or with radio-chemotherapy. The mean number of doses was 39. Nimotuzumab was well-tolerated and treatment with the antibody yielded a survival benefit: median survival time was 32.66 mo and the 2-y survival rate was 54.2%. This study demonstrated the feasibility of prolonged administration of nimotuzumab and showed preliminary evidence of clinical benefit in HGG patients with poor prognosis.     

Nimotuzumab Enhances the Radiosensitivity of Cancer Cells In Vitro by Inhibiting Radiation-Induced DNA Damage Repair

Background

Nimotuzumab is a humanized IgG1 monoclonal antibody specifically targeting EGFR. In this study, we aimed to investigate the molecular mechanisms of nimotuzumab in its effects of enhancing cancer cell radiosensitivity.

Principal Finding

Lung cancer A549 cells and breast cancer MCF-7 cells were pretreated with or without nimotuzumab for 24 h before radiation to perform the clonogenic survival assay and to analyze the cell apoptosis by flow ctyometry. γ-H2AX foci were detected by confocal microscopy to assess the effect of nimotuzumab on radiation induced DNA repair. EGFR activation was examined and the levels of DNA damage repair related proteins in A549 cells at different time point and at varying doses exposure after nimotuzumab and radiation treatment were examined by Western blot. Pretreatment with nimotuzumab reduced clonogenic survival after radiation, inhibited radiation-induced EGFR activation and increased the radiation-induced apoptosis in both A549 cells and MCF-7 cells. The foci of γ-H2AX 24 h after radiation significantly increased in nimotuzumab pretreated cells with different doses. The phosphorylation of AKT and DNA-PKcs were remarkably inhibited in the combination group at each dose point as well as time point.

Conclusions

Our results revealed that the possible mechanism of nimotuzumab enhancing the cancer radiosensitivity is that nimotuzumab inhibited the radiation-induced activation of DNA-PKcs through blocking the PI3K/AKT pathway, which ultimately affected the DNA DSBs repair.     

Characterization and in vitro evaluation of nimotuzumab conjugated with cisplatin-loaded liposomes

In this paper, we report the conjugation of the humanized monoclonal antibody nimotuzumab with cisplatin-loaded liposomes and the in vitro evaluation of its affinity for tumor cells. The conjugation procedure was performed through derivatization of nimotuzumab with N-succinimidyl S-acetylthioacetate (SATA) followed by a covalent attachment with maleimide groups at the end of PEG-DSPE chains located at the membrane of pre-formed liposomes. Confocal microscopy was performed to evaluate the immunoliposome affinity for EGFR antigens from human epidermoid carcinoma (A-431) and normal lung (MRC-5) cell lines. Results showed that the procedures implemented in this work do not affect the capability of the nimotuzumab-immunoliposomes to recognize the tumor cells, which overexpress the EGFR antigens.     

Structural characterization of N-linked oligosaccharides on monoclonal antibody Nimotuzumab through process development

Nimotuzumab (TheraCIM, CIMAher, h-R3, humanized anti-EGF-R antibody), monoclonal antibody (mAb) manufactured at the Center of Molecular Immunology (Havana, Cuba) is currently being tested in several clinical trials. Nimotuzumab has a single N-glycosylation site in the Fc-CH2 fragment but no N-glycosylation site in the Fab region. The current study reports the full characterization of the mAb N-glycosylation and the consistency observed in several production batches from a perfusion mode culturing system that lasted between 68 and 150 days. It confirms that the N-glycan structures of Nimotuzumab expressed in the NS0 murine myeloma cell line are of the murine type. They consist mainly of fucosylated G0, G1 and G2 oligosaccharides, which are normally found in the CH2 region of IgG. Other minor species found were high mannose and sialylated structures. A small portion of the glycans were sialylated (～12%) and the only type of sialic acid detected was N-glycolyl-sialic acid, α2,6-linked to Gal. No Galα1-3Gal moieties were detected.     

Anti-(epidermal growth factor) receptor monoclonal antibodies for the induction of antibody-dependent cell-mediated cytotoxicity against squamous cell carcinoma lines of the head and neck

 Squamous cell carcinomas of the head and neck (SCCHN) frequently display high levels of the epidermal growth factor receptor (EGFR). Since EGFR is expressed on the cell surface it may form a suitable target for anticancer therapy with anti-receptor monoclonal antibodies (mAb). Besides the interference with receptor/ligand interactions, binding of mAb to EGFR leads to immunoglobulin-coated tumour cells that may induce or enhance non-specific immune effector mechanisms like antibody-dependent cell-mediated cytotoxicity (ADCC). In established cell lines of SCCHN (UM-SCC 11B, 14C, 22B, and 8029 NA) we investigated the antitumour activity of allogeneic peripheral blood mononuclear cells (PBMC) in combination with rat (ICR 62), mouse (EMD 55900), and humanized (EMD 72000) anti-EGFR mAb. In addition, autologous PBMC were available for tumour line UD-SCC 4. The EGFR protein content of the tumour cell lines ranged between 170 fmol/mg protein and 8100 fmol/mg protein, and MCF-7 cells served as receptor-negative controls. PBMC activity against SCCHN targets was determined in 96-well microtitre-plate monolayer cultures by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after coincubation for 4 h, 24 h and 72 h at effector target ratios of 1:1, 5:1, 10:1 and 20:1. PBMC subpopulations were obtained by macrophage depletion (plastic adherence) or natural killer (NK) cell enrichment (magnetic bead negative selection). Prolonged time of exposure and increased effector:target ratios revealed marked antitumour activity of PBMC alone. This non-specific immune destruction was enhanced considerably by humanized and rat, but not mouse anti-EGFR mAb. Increased EGFR protein in tumour cells partly correlated with an intensification of ADCC but was accompanied by decreased primary PBMC cytotoxicity. The utilization of PBMC subpopulations suggested a mainly NK-cell-mediated ADCC, which appeared to benefit directly or indirectly, e.g. via the secretion of cytokines, from other PBMC components. In conclusion, humanized (EMD 72000) and rat (ICR 62) anti-EGFR mAb were able to generate strong antitumour ADCC in target monolayers of SCCHN. Received: 5 December 1997 / Accepted: 15 January 1998     

Delineating the functional map of the interaction between nimotuzumab and the epidermal growth factor receptor

Molecular details of epidermal growth factor receptor (EGFR) targeting by nimotuzumab, a therapeutic anti-cancer antibody, have been largely unknown. The current study delineated a functional map of their interface, based on phage display and extensive mutagenesis of both the target antigen and the Fv antibody fragment. Five residues in EGFR domain III (R353, S356, F357, T358, and H359T) and the third hypervariable region of nimotuzumab heavy chain were shown to be major functional contributors to the interaction. Fine specificity differences between nimotuzumab and other anti-EGFR antibodies were revealed. Mapping information guided the generation of a plausible in silico binding model. Knowledge about the epitope/paratope interface opens new avenues for the study of tumor sensitivity/resistance to nimotuzumab and for further engineering of its binding site. The developed mapping platform, also validated with the well-known cetuximab epitope, allows a comprehensive exploration of antigenic regions and could be expanded to map other anti-EGFR antibodies.     

Delineating the functional map of the interaction between nimotuzumab and the epidermal growth factor receptor

Molecular details of epidermal growth factor receptor (EGFR) targeting by nimotuzumab, a therapeutic anti-cancer antibody, have been largely unknown. The current study delineated a functional map of their interface, based on phage display and extensive mutagenesis of both the target antigen and the Fv antibody fragment. Five residues in EGFR domain III (R353, S356, F357, T358, and H359T) and the third hypervariable region of nimotuzumab heavy chain were shown to be major functional contributors to the interaction. Fine specificity differences between nimotuzumab and other anti-EGFR antibodies were revealed. Mapping information guided the generation of a plausible in silico binding model. Knowledge about the epitope/paratope interface opens new avenues for the study of tumor sensitivity/resistance to nimotuzumab and for further engineering of its binding site. The developed mapping platform, also validated with the well-known cetuximab epitope, allows a comprehensive exploration of antigenic regions and could be expanded to map other anti-EGFR antibodies.     

靶点药物Cetuximab(C225)研究新进展

在许多肿瘤组织中均有表皮生长因子受体(epidermal growthfactor receptor,EGFR)的过表达,它的失调与肿瘤对化疗和放疗的耐受以及不良预后相关,为肿瘤的治疗提供了一个理想的分子靶点.Cetuximab(C225)是特异性EGFR单克隆抗体,与化疗或放疗联合应用时具有协同作用,具有毒副作用少、靶向性好等优点.Cetuximab(C225)已被批准用于对伊利替康抵抗的结直肠癌和头颈部鳞癌的治疗,对非小细胞肺癌、乳腺癌、胰腺癌等具有EGFR高表达肿瘤治疗的临床试验正在进行之中,为肿瘤治疗开辟了一个全新的领域.     

人源化抗体研究历程及发展趋势

单克隆抗体从问世到目前广泛应用于临床,经历了一段曲折的发展历程。其中人源化抗体是一个重要的里程碑,并伴随着一系列重大的技术革新,如PCR技术、抗体库技术、转基因动物等。人源化抗体的形式也从最初的嵌合抗体、改型抗体等逐步发展为今天的人抗体。抗体人源化已经成为治疗性抗体的发展趋势,同时各种抗体衍生物也不断涌现,它们从不同角度克服抗体本身的应用局限,也为治疗人类疾病提供了更多利器。对单克隆抗体进行改造使之应用于临床治疗,不仅需要对抗体效应机制进行更细致深入的研究,同时还有赖于对人类免疫系统调控机制的全面精确认识。     

Current perspectives on therapeutic antibodies

Since the first monoclonal antibody, muromonab-CD3, was approved for therapeutic use in 1986, numerous molecules have been targeted using therapeutic antibody technology, resulting in 26 therapeutic antibodies being approved by the US FDA as of November, 2009. Initial concerns regarding antibody drugs focused on immunogenicity, short serum half-life, and weak efficacy. As the types of antibodies progressed from murine to chimeric, humanized, and fully human antibodies, great progress has been made in immunogenicity and in vivo instability issues. For example, humanized antibodies, such as bevacizumab, exhibit less than 0.2% immunogenicity and a 20 day serum half-life, which is comparable to native immunoglobulin. Some recently developed antibodies are exceedingly efficacious and have become first-line therapy for their target diseases. Here, we address and analyze all clinically approved therapeutic antibodies to date by discussing immunogenicity, half-life, and efficacy.     

Design of humanized antibodies: from anti-Tac to Zenapax

Since the introduction of hybridoma technology, monoclonal antibodies have become one of the most important tools in the biosciences, finding diverse applications including their use in the therapy of human disease. Initial attempts to use monoclonal antibodies as therapeutics were hampered, however, by the potent immunogenicity of mouse (and other rodent) antibodies in humans. Humanization technology has made it possible to remove the immunogenicity associated with the use of rodent antibodies, or at least to reduce it to an acceptable level for clinical use in humans, thus facilitating the application of monoclonal antibodies to the treatment of human disease. To date, nine humanized monoclonal antibodies have been approved for use as human therapeutics in the United States. In this paper, we describe procedures for antibody humanization with an emphasis on strategies for designing humanized antibodies with the aid of computer-guided modeling of antibody variable domains, using as an example the humanized anti-CD25 monoclonal antibody, Zenapax.     

Humanization of Murine Monoclonal anti-hTNF Antibody: The F10 Story

Tumor necrosis factor (TNF) is a proinflammatory cytokine implicated in pathogenesis of multiple autoimmune and inflammatory diseases. Anti-TNF therapy has revolutionized the therapeutic paradigms of autoimmune diseases and became one of the most successful examples of the clinical use of monoclonal antibodies. Currently, anti-TNF therapy is used by millions of patients worldwide. At the moment, fully human anti-TNF antibody Adalimumab is the best-selling anti-cytokine drug in the world. Here, we present a story about a highly potent anti-TNF monoclonal antibody initially characterized more than 20 years ago and further developed into chimeric and humanized versions. We present comparative analysis of this antibody with Infliximab and Adalimumab.     

Development of humanized antibodies as cancer therapeutics

Recent success in the development of monoclonal antibody-based anti-cancer drugs has largely benefitted from the advancements made in recombinant technologies and cell culture production. These reagents, derived from the antibodies of mouse origin, while maintaining the exquisite specificity and affinity to the tumor antigens, have low immunogenicity and toxicity in human. High-level expressing cell clones are generated and used to produce large quantities of the recombinant antibodies in bioreactors in order to meet the clinical demand for therapeutic applications. In this report, the systems and general methodologies developed by us to construct and produce humanized antibodies from the parent mouse antibodies are described. Once the humanized antibodies are available, they can be applied in three principal forms for cancer therapy: (1) naked antibodies, (2) drug- or toxin conjugates, and (3) radioconjugates. Using the humanized anti-CD22 (epratuzumab) and anti-carcinoembryonic antigen (ant-CEA; labetuzumab) antibody prototypes, clinical applications of naked and radiolabeled humanized monoclonal antibodies are described.     

Production and Characterization of Single Chain Nimotuzumab: An In Vitro Study

Recombinant expression and EGFR-binding activity assay of single chain nimotuzumab (nimotuzumab scFv) is reported in this study. The scFv was produced in VH-linker-VL format in Origami^? 2(DE3)pLysS bacterial cells and purified using Ni-TNA resin column. 3-D structure prediction using I-TASSER (Iterative Threading Assembly Refinement) server and analyzing the predicted models using YASARA (Yet Another Scientific Artificial Reality Application) viewer revealed that VH and VL domains assemble into a correctly-folded single chain antibody that is able to bind EGFR. The scFv was evaluated in ELISA and western blot tests and proven to be able to bind EGFR-overexpressing cancer cells (A-431 cells in an efficient manner but unable to recognize cancer cells expressing low levels of EGFR (MCF-7 breast cancer cells).     

Have we overestimated the benefit of human(ized) antibodies?

The infusion of animal-derived antibodies has been known for some time to trigger the generation of antibodies directed at the foreign protein as well as adverse events including cytokine release syndrome. These immunological phenomena drove the development of humanized and fully human monoclonal antibodies. The ability to generate human(ized) antibodies has been both a blessing and a curse. While incremental gains in the clinical efficacy and safety for some agents have been realized, a positive effect has not been observed for all human(ized) antibodies. Many human(ized) antibodies trigger the development of anti-drug antibody responses and infusion reactions. The current belief that antibodies need to be human(ized) to have enhanced therapeutic utility may slow the development of novel animal-derived monoclonal antibody therapeutics for use in clinical indications. In the case of murine antibodies, greater than 20% induce tolerable/negligible immunogenicity, suggesting that in these cases humanization may not offer significant gains in therapeutic utility. Furthermore, humanization of some murine antibodies may reduce their clinical effectiveness. The available data suggest that the utility of human(ized) antibodies needs to be evaluated on a case-by-case basis, taking a cost-benefit approach, taking both biochemical characteristics and the targeted therapeutic indication into account.Key words: immunogenicity, human anti-mouse antibody, cytokine release syndrome     

Generation of recombinant antibodies

Recombinant antibody technology is opening new perspectives for the development of novel therapeutic and diagnostic agents. In this review we focus on advances in the generation of both genetically engineered humanized and fully human monoclonal antibodies. Methods for their production in different expression systems are also discussed.     

Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus

Neutralization of West Nile virus (WNV) in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Using random mutagenesis and yeast surface display, we defined individual contact residues of 14 newly generated monoclonal antibodies against domain III of the WNV E protein. Monoclonal antibodies that strongly neutralized WNV localized to a surface patch on the lateral face of domain III. Convalescent antibodies from individuals who had recovered from WNV infection also detected this epitope. One monoclonal antibody, E16, neutralized 10 different strains in vitro, and showed therapeutic efficacy in mice, even when administered as a single dose 5 d after infection. A humanized version of E16 was generated that retained antigen specificity, avidity and neutralizing activity. In postexposure therapeutic trials in mice, a single dose of humanized E16 protected mice against WNV-induced mortality, and may therefore be a viable treatment option against WNV infection in humans.