Emerging antibody combinations in oncology

The use of monoclonal antibodies (mAbs) has become a general approach for specifically targeting and treating human disease. In oncology, the therapeutic utility of mAbs is usually evaluated in the context of treatment with standard of care, as well as other small molecule targeted therapies. Many anti-cancer antibody modalities have achieved validation, including the targeting of growth factor and angiogenesis pathways, the induction of tumor cell killing or apoptosis and the blocking of immune inhibitory mechanisms to stimulate anti-tumor responses. But, as with other targeted therapies, few antibodies are curative because of biological complexities that underlie tumor formation and redundancies in molecular pathways that enable tumors to adapt and show resistance to treatment. This review discusses the combinations of antibody therapeutics that are emerging to improve efficacy and durability within a specific biological mechanism (e.g., immunomodulation or the inhibition of angiogenesis) and across multiple biological pathways (e.g., inhibition of tumor growth and induction of tumor cell apoptosis).Key words: antibody combination, receptor tyrosine kinase, angiogenesis, immunomodulation, apoptosis, CD20     

Negative regulators of cell death pathways in cancer: perspective on biomarkers and targeted therapies

Cancer is a primary cause of human fatality and conventional cancer therapies, e.g., chemotherapy, are often associated with adverse side-effects, tumor drug-resistance, and recurrence. Molecularly targeted therapy, composed of small-molecule inhibitors and immunotherapy (e.g., monoclonal antibody and cancer vaccines), is a less harmful alternative being more effective against cancer cells whilst preserving healthy tissues. Drug-resistance, however, caused by negative regulation of cell death signaling pathways, is still a challenge. Circumvention of negative regulators of cell death pathways or development of predictive and response biomarkers is, therefore, quintessential. This review critically discusses the current state of knowledge on targeting negative regulators of cell death signaling pathways including apoptosis, ferroptosis, necroptosis, autophagy, and anoikis and evaluates the recent advances in clinical and preclinical research on biomarkers of negative regulators. It aims to provide a comprehensive platform for designing efficacious polytherapies including novel agents for restoring cell death signaling pathways or targeting alternative resistance pathways to improve the chances for antitumor responses. Overall, it is concluded that nonapoptotic cell death pathways are a potential research arena for drug discovery, development of novel biomarkers and targeted therapies.     

Antibodies in oncology

Monoclonal antibodies (mAbs) have become one of the largest classes of new therapeutic agents approved for use in oncology, and have revolutionised the treatment of many human malignancies. Clinically useful mAbs can function through several different mechanisms, including inhibition of tumour-related signalling, induction of apoptosis, inhibition of angiogenesis, enhancing host immune response against cancer and targeted delivery of payloads (such as toxins, cytotoxic agents or radioisotopes) to the tumour site. The increasing knowledge of key molecules and cellular pathways involved in tumour induction and progression has led to a rise in the proportion of therapeutic mAbs entering clinical trials. These mAbs consist of various conventional or recombinant, murine, humanised, chimeric or fully human and fusion constructs. In this review, we provide an overview of mAbs approved for use in clinical oncology and those currently in clinical development. We also discuss the mechanisms of action of anti-cancer mAbs, as well as the antigen targets recognised by these antibodies.     

MicroRNAs in cancer drug resistance: Basic evidence and clinical applications

Development of drug resistance has considerably limited the efficacy of cancer treatments, including chemotherapy and targeted therapies. Hence, understanding the molecular mechanisms underpinning the innate or the acquired resistance to these therapies is critical to improve drug efficiency and clinical outcomes. Several studies have implicated microRNAs (miRNA) in this process. MiRNAs repress gene expression by specific binding to complementary sequences in the 3' region of target messenger RNAs (mRNAs), followed by target mRNA degradation or blocked translation. By targeting molecules specific to a particular pathway within tumor cells, the new generation of cancer treatment strategies has shown significant advantages over conventional chemotherapy. However, the long-term efficacy of targeted therapies often remains poor, because tumor cells develop resistance to such therapeutics. Targeted therapies often involve monoclonal antibodies (mAbs), such as those blocking the ErB/HER tyrosine kinases, epidermal growth factor receptor (cetuximab) and HER2 (trastuzumab), and those inhibiting vascular endothelial growth factor receptor signaling (e.g., bevacizumab). Even though these are among the most used agents in tumor medicine, clinical response to these drugs is reduced due to the emergence of drug resistance as a result of toxic effects in the tumor microenvironment. Research on different types of human cancers has revealed that aberrant expression of miRNAs promotes resistance to the aforementioned drugs. In this study, we review the mechanisms of tumor cell resistance to mAb therapies and the role of miRNAs therein. Emerging treatment strategies combine therapies using innovative miRNA mimics or antagonizers with conventional approaches to maximize outcomes of patients with cancer.     

Single domain antibodies: a new concept for epidermal growth factor receptor and EGFRvIII targeting

Epidermal growth factor receptor (EGFR) is one of the major molecular targets for cancer diagnosis and therapy. EGFR and EGFRvIII, mutated form of EGFR, have been identified as participating in pathogenesis of some forms of human cancers. Monoclonal antibodies (mAbs) targeting EGFR/EGFRvIII have been shown to suppress the signal transduction pathways controlling tumor cell growth, proliferation, and apoptosis. Until now, different types of mAbs or antibody fragments against EGFR family have been established. Some of these antibodies have been used clinically for treating various forms of human malignancies. More recently, a single domain antibody (sdAb) targeting this family of receptors has been introduced. The heavy chain antibodies (HCAbs) that made up variable regions of heavy chain, CH2, and CH3 domains are shown in camelids. SdAbs derived from camel HCAbs are the smallest known natural building parts for binding to antigen. They also possess a longer antigen recognizing region, which increases their capability for being more specific in target antigen enhancement. Camelid antibodies are highly valuable for their special characteristics, including heat resistance, small size, high solubility in an aqueous environment, and non-immunogenicity in a human environment. Due to these abilities, research on biotechnological production and treatment applications of recombinant smaller fragments of these only HCAbs is widely in progress. In this article, we will discuss the challenges and successes of different types of mAbs targeting EGFR/EGFRvIII in human cancer.     

A dual-targeting PDGFRβ/VEGF-A molecule assembled from stable antibody fragments demonstrates anti-angiogenic activity in vitro and in vivo

Targeting angiogenesis is a promising approach to the treatment of solid tumors and age-related macular degeneration (AMD). Inhibition of vascularization has been validated by the successful marketing of monoclonal antibodies (mAbs) that target specific growth factors or their receptors, but there is considerable room for improvement in existing therapies. Combination of mAbs targeting both the VEGF and PDGF pathways has the potential to increase the efficacy of anti-angiogenic therapy without the accompanying toxicities of tyrosine kinase inhibitors and the inability to combine efficiently with traditional chemotherapeutics. However, development costs and regulatory issues have limited the use of combinatorial approaches for the generation of more efficacious treatments. The concept of mediating disease pathology by targeting two antigens with one therapeutic was proposed over two decades ago. While mAbs are particularly suitable candidates for a dual-targeting approach, engineering bispecificity into one molecule can be difficult due to issues with expression and stability, which play a significant role in manufacturability. Here, we address these issues upstream in the process of developing a bispecific antibody (bsAb). Single-chain antibody fragments (scFvs) targeting PDGFRβ and VEGF-A were selected for superior stability. The scFvs were fused to both termini of human Fc to generate a bispecific, tetravalent molecule. The resulting molecule displays potent activity, binds both targets simultaneously, and is stable in serum. The assembly of a bsAb using stable monomeric units allowed development of an anti-PDGFRB/VEGF-A antibody capable of attenuating angiogenesis through two distinct pathways and represents an efficient method for rapid engineering of dual-targeting molecules.     

Anticancer activity of TTAC-0001, a fully human anti-vascular endothelial growth factor receptor 2 (VEGFR-2/KDR) monoclonal antibody,is associated with inhibition of tumor angiogenesis

Vascular endothelial growth factor (VEGF) and its receptors are considered the primary cause of tumor-induced angiogenesis. Specifically, VEGFR-2/kinase insert domain receptor (KDR) is part of the major signaling pathway that plays a significant role in tumor angiogenesis, which is associated with the development of various types of tumor and metastasis. In particular, KDR is involved in tumor angiogenesis as well as cancer cell growth and survival. In this study, we evaluated the therapeutic potential of TTAC-0001, a fully human antibody against VEGFR-2/KDR. To assess the efficacy of the antibody and pharmacokinetic (PK) relationship in vivo, we tested the potency of TTAC-0001 in glioblastoma and colorectal cancer xenograft models. Antitumor activity of TTAC-0001 in preclinical models correlated with tumor growth arrest, induction of tumor cell apoptosis, and inhibition of angiogenesis. We also evaluated the combination effect of TTAC-0001 with a chemotherapeutic agent in xenograft models. We were able to determine the relationship between PK and the efficacy of TTAC-0001 through in vivo single-dose PK study. Taken together, our data suggest that targeting VEGFR-2 with TTAC-0001 could be a promising approach for cancer treatment.     

Anticancer activity of TTAC-0001, a fully human anti-vascular endothelial growth factor receptor 2 (VEGFR-2/KDR) monoclonal antibody,is associated with inhibition of tumor angiogenesis

Vascular endothelial growth factor (VEGF) and its receptors are considered the primary cause of tumor-induced angiogenesis. Specifically, VEGFR-2/kinase insert domain receptor (KDR) is part of the major signaling pathway that plays a significant role in tumor angiogenesis, which is associated with the development of various types of tumor and metastasis. In particular, KDR is involved in tumor angiogenesis as well as cancer cell growth and survival. In this study, we evaluated the therapeutic potential of TTAC-0001, a fully human antibody against VEGFR-2/KDR. To assess the efficacy of the antibody and pharmacokinetic (PK) relationship in vivo, we tested the potency of TTAC-0001 in glioblastoma and colorectal cancer xenograft models. Antitumor activity of TTAC-0001 in preclinical models correlated with tumor growth arrest, induction of tumor cell apoptosis, and inhibition of angiogenesis. We also evaluated the combination effect of TTAC-0001 with a chemotherapeutic agent in xenograft models. We were able to determine the relationship between PK and the efficacy of TTAC-0001 through in vivo single-dose PK study. Taken together, our data suggest that targeting VEGFR-2 with TTAC-0001 could be a promising approach for cancer treatment.     

Large‐scale production,purification, and function of a tumor multi‐epitope vaccine: Peptibody with bFGF/VEGFA

In tumor tissue, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor A (VEGFA) promote tumorigenesis by activating angiogenesis, but targeting single factor may produce drug resistance and compensatory angiogenesis. The Peptibody with bFGF/VEGFA was designed to simultaneously blockade these two factors. We were aiming to produce this Fc fusion protein in a large scale. The biological characterizations of Peptibody strains were identified as Escherichia coli and the fermentation mode was optimized in the shake flasks and 10‐L bioreactor. The fermentation was scaled up to 100 L, with wet cell weight (WCW) 126 g/L, production 1.41 g/L, and productivity 0.35 g/(L·h) of IPTG induction. The target protein was isolated by cation‐exchange, hydrophobic and Protein A chromatography, with total recovery of 60.28% and HPLC purity of 86.71%. The host cells protein, DNA, and endotoxin residues were within the threshold. In mouse model, immunization of Peptibody vaccine could significantly suppressed the tumor growth and angiogenesis, with inhibition rate of 57.73 and 39.34%. The Peptibody vaccine could elicit high‐titer anti‐bFGF and anti‐VEGFA antibodies, which inhibited the proliferation and migration of Lewis lung cancer cell cells by decreasing the Akt/MAPK signal pathways. Therefore, the Peptibody with bFGF/VEGFA might be used as a therapeutic tumor vaccine. The large‐scale process we developed could support its industrial production and pre‐clinical study in the future.     

The mechanism underlying the effects of the cell surface ATP synthase on the regulation of intracellular acidification during acidosis

The F1F0 ATP synthase has recently become the focus of anti‐cancer research. It was once thought that ATP synthases were located strictly on the inner mitochondrial membrane; however, in 1994, it was found that some ATP synthases localized to the cell surface. The cell surface ATP synthases are involved in angiogenesis, lipoprotein metabolism, innate immunity, hypertension, the regulation of food intake, and other processes. Inhibitors of this synthase have been reported to be cytotoxic and to induce intracellular acidification. However, the mechanisms by which these effects are mediated and the molecular pathways that are involved remain unclear. In this study, we aimed to determine whether the inhibition of cell proliferation and the induction of cell apoptosis that are induced by inhibitors of the cell surface ATP synthase are associated with intracellular acidification and to investigate the mechanism that underlines the effects of this inhibition, particularly in an acidic tumor environment. We demonstrated that intracellular acidification contributes to the cell proliferation inhibition that is mediated by cell surface ATP synthase inhibitors, but not to the induction of apoptosis. Intracellular acidification is only one of the mechanisms of ecto‐ATP synthase‐targeted antitumor drugs. We propose that intracellular acidification in combination with the inhibition of cell surface ATP generation induce cell apoptosis after cell surface ATP synthase blocked by its inhibitors. A better understanding of the mechanisms activated by ecto‐ATP synthase‐targeted cancer therapies may facilitate the development of potent anti‐tumor therapies, which target this enzyme and do not exhibit clinical limitations. J. Cell. Biochem. 114: 1695–1703, 2013. © 2013 Wiley Periodicals, Inc.     

A dual-targeting PDGFRβ/VEGF-A molecule assembled from stable antibody fragments demonstrates anti-angiogenic activity in vitro and in vivo

Targeting angiogenesis is a promising approach to the treatment of solid tumors and age-related macular degeneration (AMD). Inhibition of vascularization has been validated by the successful marketing of monoclonal antibodies (mAbs) that target specific growth factors or their receptors, but there is considerable room for improvement in existing therapies. Combination of mAbs targeting both the VeGF and pDGF pathways has the potential to increase the efficacy of anti-angiogenic therapy without the accompanying toxicities of tyrosine kinase inhibitors and the inability to combine efficiently with traditional chemotherapeutics. However, development costs and regulatory issues have limited the use of combinatorial approaches for the generation of more efficacious treatments.The concept of mediating disease pathology by targeting two antigens with one therapeutic was proposed over two decades ago. While mAbs are particularly suitable candidates for a dual-targeting approach, engineering bispecificity into one molecule can be difficult due to issues with expression and stability, which play a significant role in manufacturability. Here, we address these issues upstream in the process of developing a bispecific antibody (bsAb). Single-chain antibody fragments (scFvs) targeting pDGFRβ and VeGF-A were selected for superior stability. the scFvs were fused to both termini of human Fc to generate a bispecific, tetravalent molecule. resulting molecule displays potent activity, binds both targets simultaneously, and is stable in serum. assembly of a bsAb using stable monomeric units allowed development of an anti-pDGFRB/VeGF-A antibody capable of attenuating angiogenesis through two distinct pathways and represents an efficient method for rapid engineering of dual-targeting molecules.Key words: bispecific, antibody, PDGFRβ, VEGF-A, stability, angiogenesis     

A bi-functional antibody-receptor domain fusion protein simultaneously targeting IGF-IR and VEGF for degradation

Bi-specific antibodies (BsAbs), which can simultaneously block 2 tumor targets, have emerged as promising therapeutic alternatives to combinations of individual monoclonal antibodies. Here, we describe the engineering and development of a novel, human bi-functional antibody-receptor domain fusion molecule with ligand capture (bi-AbCap) through the fusion of the domain 2 of human vascular endothelial growth factor receptor 1 (VEGFR1) to an antibody directed against insulin-like growth factor – type I receptor (IGF-IR). The bi-AbCap possesses excellent stability and developability, and is the result of minimal engineering. Beyond potent neutralizing activities against IGF-IR and VEGF, the bi-AbCap is capable of cross-linking VEGF to IGF-IR, leading to co-internalization and degradation of both targets by tumor cells. In multiple mouse xenograft tumor models, the bi-AbCap improves anti-tumor activity over individual monotherapies. More importantly, it exhibits superior inhibition of tumor growth, compared with the combination of anti-IGF-IR and anti-VEGF therapies, via powerful blockade of both direct tumor cell growth and tumor angiogenesis. The unique “capture-for-degradation” mechanism of the bi-AbCap is informative for the design of next-generation bi-functional anti-cancer therapies directed against independent signaling pathways. The bi-AbCap design represents an alternative approach to the creation of dual-targeting antibody fusion molecules by taking advantage of natural receptor-ligand interactions.     

Generation of functional scFv intrabodies for triggering anti-tumor immunity

Intracellular expression of recombinant antibodies (intrabodies) allows to interfere with the functions of oncogenic or viral molecules expressed in different cell compartments and has therefore a vast clinical potential in therapy. Although the use of phage-display libraries has made it possible to select Fab or single chain Fv (scFv) antibody fragments usable for intracellular targeting, a major source of recombinant antibodies for therapeutic use still remains hybridoma B cells producing well-characterized monoclonal antibodies (mAbs). However, the cloning and the intracellular expression of antibody fragments derived from mAbs can be markedly hampered by a number of technical difficulties that include failure of cloning functional variable regions as well as lack of binding of the antibody fragments to the targeted molecule in an intracellular environment. We discuss herein various molecular methods that have been developed to generate functional recombinant antibody fragments usable as anti-tumor triggering agents when expressed in tumor cells. Such antibodies can neutralize or modify the activity of oncogenic molecules when addressed in specific subcellular compartments and/or they can be used to trigger anti-tumor immunity when expressed on tumor cell surface.     

Inhibition of subcutaneous growth of Ehrlich ascites carcinoma (EAC) tumor by post-immunization with EAC-cell gangliosides and its anti-idiotype antibody in relation to tumor angiogenesis, apoptosis, cell cycle and infiltration of CD4+, CD8+ lymphocytes, NK cells, suppressor cells and APC-cells in tumor

Both EAC-tumor associated gangliosides and its anti-idiotype antibody inhibited growth of this tumor significantly. Immuno-histological studies with von Willebrand Factor (vWF) antibody indicated that tumor angiogenesis as determined by expression of vWF decreased in tumors of mice, post-immunized with EAC-cell gangliosides as well as its anti-idiotype antibody. Infiltration of various immune cells of the host in the tumor correlated to some extent with tumor-growth inhibition. Apoptosis study using AnnexinV-FITC and propidium iodide indicated that tumor growth inhibition in mice post-immunized with EAC-gangliosides and its anti-idiotype antibody were due to enhanced apoptosis and cell death. Cell cycle analysis by FACS indicated that EAC-cell associated gangliosides and its anti-idiotype antibody were acting both at the M2 i.e. S and M3 i.e. G2/M phases of the cell cycle to arrest tumor growth.     

Human anti-EGFL7 recombinant full-length antibodies selected from a mammalian cell-based antibody display library

Epidermal growth factor-like domain 7 (EGFL7) has been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. The advent of antibody display technology (phage, bacteria, and yeast) led to an enormous revival in the use of antibodies as diagnostic and therapeutic tools for fighting cancer. However, problems with protein folding, posttranslational modification, and codon usage still limit the number of improved antibodies that can be obtained. We describe here the isolation of an EGFL7-specific antibody from a mammalian cell-based full-length antibody display library generated from peripheral blood mononuclear cells of patients with hepatocellular carcinoma. Using a novel vector, contained glycosylphosphatidylinositol anchor and restriction enzyme sites NheI and ClaI, antibody libraries are displayed as whole IgG molecules on the cell surface and screened for specific antigen binding by a combination of magnetic beads and measured by cell ELISA. Anti-EGFL7 antibody was successfully isolated from the library. The mammalian cell-based full-length antibody display library is a great potential application for rapid identification and cloning of human mAbs of targeting hepatocellular carcinoma.     

Linking cell structure to gene regulation: Signaling events and expression controls on the model genes PAI-1 and CTGF

The microtubule and microfilament cytoskeletal systems as well as cell-to-cell contacts and cell–matrix interactions are critical regulators of cell structure and function. Alterations in cell shape profoundly influence signaling events and gene expression programs that impact a spectrum of biological responses including cell growth, migration and apoptosis. These same pathways also contribute to the progression of several important pathologic conditions (e.g., arteriosclerosis, vascular fibrosis, and endothelial dysfunction). Indeed, hemodynamic forces in the vascular compartment are established modifiers of endothelial and smooth muscle cell cytoarchitecture and orchestrate complex genetic and biological responses in concert with contributions from the extracellular matrix (ECM), growth factors (e.g., EGF, and TGF-β) and cell adhesion receptors (e.g., integrins, and cadherins). The profibrotic matricellular proteins plasminogen activator inhibitor-1 (PAI-1) and connective tissue growth factor (CTGF) are prominent members of a subset of genes the expression of which is highly responsive to cell shape-altering stimuli (i.e., disruption of the actin-based and microtubule networks, shear strain and cyclic stretch). Since both PAI-1 and CTGF are major mediators of cardiovascular fibrotic disease, understanding cell structure-linked signaling cascades provides potential avenues for focused therapy. It is increasingly evident that growth factor receptors (EGFR) are activated by changes in cytoarchitecture and that the “repressive state” of certain signaling proteins (e.g., SMAD, and Rho-GEFs) is maintained by sequestration on cell structural networks. Functional repression can be relieved by cytoskeletal perturbations (e.g., in response to treatment with network-specific drugs) resulting in activation of signaling cascades (e.g., Rho, and MAPK) with associated changes in gene reprogramming. Recent studies document a complex network of both similar and unique signaling control elements leading to the induction of PAI-1 and CTGF in response to modifications in cell shape. The purpose of this review is to highlight our current understanding of “cell deformation”-responsive signaling cascades focusing on the potential value of targeting such pathways, and their model response genes (e.g., PAI-1, and CTGF), as a therapeutic option for the treatment of fibrotic diseases.     

Selective blockade of tumor angiogenesis

Blocking tumor angiogenesis is an important goal of cancer therapy, but clinically approved anti-angiogenic agents suffer from limited efficacy and adverse side effects, fueling the need to identify alternative angiogenesis regulators. Tumor endothelial marker 8 (TEM8) is a highly conserved cell surface receptor overexpressed on human tumor vasculature. Genetic disruption of Tem8 in mice revealed that TEM8 is important for promoting tumor angiogenesis and tumor growth but dispensable for normal development and wound healing. The induction of TEM8 in cultured endothelial cells by nutrient or growth factor deprivation suggests that TEM8 may be part of a survival response pathway that is activated by tumor microenvironmental stress. In preclinical studies, antibodies targeted against the extracellular domain of TEM8 inhibited tumor angiogenesis and blocked the growth of multiple human tumor xenografts. Anti-TEM8 antibodies augmented the activity of other anti-angiogenic agents, vascular targeting agents and conventional chemotherapeutic agents and displayed no detectable toxicity. Thus, anti-TEM8 antibodies provide a promising new tool for selective blockade of neovascularization associated with cancer and possibly other angiogenesis-dependent diseases.     

Selective blockade of tumor angiogenesis

Blocking tumor angiogenesis is an important goal of cancer therapy, but clinically approved anti-angiogenic agents suffer from limited efficacy and adverse side effects, fueling the need to identify alternative angiogenesis regulators. Tumor endothelial marker 8 (TEM8) is a highly conserved cell surface receptor overexpressed on human tumor vasculature. Genetic disruption of Tem8 in mice revealed that TEM8 is important for promoting tumor angiogenesis and tumor growth but dispensable for normal development and wound healing. The induction of TEM8 in cultured endothelial cells by nutrient or growth factor deprivation suggests that TEM8 may be part of a survival response pathway that is activated by tumor microenvironmental stress. In preclinical studies, antibodies targeted against the extracellular domain of TEM8 inhibited tumor angiogenesis and blocked the growth of multiple human tumor xenografts. Anti-TEM8 antibodies augmented the activity of other anti-angiogenic agents, vascular targeting agents and conventional chemotherapeutic agents and displayed no detectable toxicity. Thus, anti-TEM8 antibodies provide a promising new tool for selective blockade of neovascularization associated with cancer and possibly other angiogenesis-dependent diseases.     

非小细胞肺癌靶向治疗的临床前研究进展

针对表皮生长因子受体（EGFR）和血管生成（angiogenesis）信号通路的靶向治疗已经在晚期非小细胞肺癌的治疗上取得成功,但由于抗药性的存在,大多数晚期患者的生存时间仍然提高有限。继发性的EGFR T790M突变和原癌基因肝细胞生长因子受体（MET）的扩增被鉴定为两种主要的抗药机制。最近转化生长因子-β（TGF-β）/白介素-6信号通路被报道能介导选择性和适应性地对erlotinib的抗药。另一方面,Kras突变所致肺癌的靶向治疗方面也取得了一些进展。双重抑制磷脂酰肌醇3-激酶（PI3K）和促分裂素原活化蛋白激酶激酶（MEK）信号通路可导致Kras突变肿瘤的显著消退,联合抑制SRC、PI3K和MEK可使丝氨酸/苏氨酸蛋白激酶11（Lkb1）缺失,Kras突变的肺癌小鼠的肿瘤明显消退,抑制核因子-κB（NF-κB）信号通路导致p53缺失,Kras突变的肿瘤发展显著减慢。这些发现都为发展非小细胞肺癌患者的靶向治疗提供了有力的支持。     

Efficient growth inhibition of EGFR over-expressing tumor cells by an anti-EGFR nanobody

Epidermal growth factor receptor (EGFR) is deemed to be one of the main molecular targets for diagnosis and treatment of cancer. It has been identified that EGFR involves in pathogenesis of some forms of human cancers. Monoclonal antibodies targeting EGFR could control the tumor cell growth, proliferation, and apoptosis by suppressing the signal transduction pathways. Nanobodies can be regarded as the smallest intact antigen binding fragments, derived from heavy chain-only antibodies existing in camelids. Here, we describe the identification of an EGFR-specific nanobody, referred to as OA-cb6, obtained from immunized camel with a cell line expressing high levels of EGFR. Utilizing flow cytometry (FACS) and blotting methods, we demonstrated that OA-cb6 nanobody binds specifically to EGFR expressing on the surface of A431 cells. In addition, OA-cb6 nanobody potently causes the inhibition of EGFR over expression, cell growth and proliferation. The antibody fragments can probably be regarded as worthwhile binding block for further rational design of anti-cancer therapy.