HER3 intracellular domains play a crucial role in HER3/HER2 dimerization and activation of downstream signaling pathways

Dimerization among the EGFR family of tyrosine kinase receptors leads to allosteric activation of the kinase domains of the partners. Unlike other members in the family, the kinase domain of HER3 lacks key amino acid residues for catalytic activity. As a result, HER3 is suggested to serve as an allosteric activator of other EGFR family members which include EGFR, HER2 and HER4. To study the role of intracellular domains in HER3 dimerization and activation of downstream signaling pathways, we constructed HER3/HER2 chimeric receptors by replacing the HER3 kinase domain (HER3-2-3) or both the kinase domain and the C-terminal tail (HER3-2-2) with the HER2 counterparts and expressed the chimeric receptors in Chinese hamster ovary (CHO) cells. While over expression of the intact human HER3 transformed CHO cells with oncogenic properties such as AKT/ERK activation and increased proliferation and migration, CHO cells expressing the HER3-2-3 chimeric receptor showed significantly reduced HER3/HER2 dimerization and decreased phosphorylation of both AKT and ERK1/2 in the presence of neuregulin-1 (NRG-1). In contrast, CHO cells expressing the HER3-2-2 chimeric receptor resulted in a total loss of downstream AKT activation in response to NRG-1, but maintained partial activation of ERK1/2. The results demonstrate that the intracellular domains play a crucial role in HER3’s function as an allosteric activator and its role in downstream signaling.     

Ligand stimulation of ErbB4 and a constitutively-active ErbB4 mutant result in different biological responses in human pancreatic tumor cell lines

Pancreatic cancer is the fourth leading cause of cancer death in the United States. Indeed, it has been estimated that 37,000 Americans will die from this disease in 2010. Late diagnosis, chemoresistance, and radioresistance of these tumors are major reasons for poor patient outcome, spurring the search for pancreatic cancer early diagnostic and therapeutic targets. ErbB4 (HER4) is a member of the ErbB family of receptor tyrosine kinases (RTKs), a family that also includes the Epidermal Growth Factor Receptor (EGFR/ErbB1/HER1), Neu/ErbB2/HER2, and ErbB3/HER3. These RTKs play central roles in many human malignancies by regulating cell proliferation, survival, differentiation, invasiveness, motility, and apoptosis. In this report we demonstrate that human pancreatic tumor cell lines exhibit minimal ErbB4 expression; in contrast, these cell lines exhibit varied and in some cases abundant expression and basal tyrosine phosphorylation of EGFR, ErbB2, and ErbB3. Expression of a constitutively-dimerized and -active ErbB4 mutant inhibits clonogenic proliferation of CaPan-1, HPAC, MIA PaCa-2, and PANC-1 pancreatic tumor cell lines. In contrast, expression of wild-type ErbB4 in pancreatic tumor cell lines potentiates stimulation of anchorage-independent colony formation by the ErbB4 ligand Neuregulin 1β. These results illustrate the multiple roles that ErbB4 may be playing in pancreatic tumorigenesis and tumor progression.     

Inhibition of HER3 activation and tumor growth with a human antibody binding to a conserved epitope formed by domain III and IV

Human epidermal growth factor receptor 3 (HER3, also known as ErbB3) has emerged as relevant target for antibody-mediated tumor therapy. Here, we describe a novel human antibody, IgG 3–43, recognizing a unique epitope formed by domain III and parts of domain IV of the extracellular region of HER3, conserved between HER3 and mouse ErbB3. An affinity of 11 nM was determined for the monovalent interaction. In the IgG format, the antibody bound recombinant bivalent HER3 with subnanomolar affinity (K_D = 220 pM) and HER3-expressing tumor cells with EC₅₀ values in the low picomolar range (27 - 83 pM). The antibody competed with binding of heregulin to HER3-expressing cells, efficiently inhibited phosphorylation of HER3 as well as downstream signaling, and induced receptor internalization and degradation. Furthermore, IgG 3–43 inhibited heregulin-dependent proliferation of several HER3-positive cancer cell lines and heregulin-independent colony formation of HER2-overexpressing tumor cell lines. Importantly, inhibition of tumor growth and prolonged survival was demonstrated in a FaDu xenograft tumor model in SCID mice. These findings demonstrate that by binding to the membrane-proximal domains III and IV involved in ligand binding and receptor dimerization, IgG 3–43 efficiently inhibits activation of HER3, thereby blocking tumor cell growth both in vitro and in vivo.     

Insights into HER2 signaling from step-by-step optimization of anti-HER2 antibodies

HER2, a ligand-free tyrosine kinase receptor of the HER family, is frequently overexpressed in breast cancer. The anti-HER2 antibody trastuzumab has shown significant clinical benefits in metastatic breast cancer; however, resistance to trastuzumab is common. The development of monoclonal antibodies that have complementary mechanisms of action results in a more comprehensive blockade of ErbB2 signaling, especially HER2/HER3 signaling. Use of such antibodies may have clinical benefits if these antibodies can become widely accepted. Here, we describe a novel anti-HER2 antibody, hHERmAb-F0178C1, which was isolated from a screen of a phage display library. A step-by-step optimization method was employed to maximize the inhibitory effect of this anti-HER2 antibody. Crystallographic analysis was used to determine the three-dimensional structure to 3.5 Å resolution, confirming that the epitope of this antibody is in domain III of HER2. Moreover, this novel anti-HER2 antibody exhibits superior efficacy in blocking HER2/HER3 heterodimerization and signaling, and its use in combination with pertuzumab has a synergistic effect. Characterization of this antibody revealed the important role of a ligand binding site within domain III of HER2. The results of this study clearly indicate the unique potential of hHERmAb-F0178C1, and its complementary inhibition effect on HER2/HER3 signaling warrants its consideration as a promising clinical treatment.     

Insights into HER2 signaling from step-by-step optimization of anti-HER2 antibodies

HER2, a ligand-free tyrosine kinase receptor of the HER family, is frequently overexpressed in breast cancer. The anti-HER2 antibody trastuzumab has shown significant clinical benefits in metastatic breast cancer; however, resistance to trastuzumab is common. The development of monoclonal antibodies that have complementary mechanisms of action results in a more comprehensive blockade of ErbB2 signaling, especially HER2/HER3 signaling. Use of such antibodies may have clinical benefits if these antibodies can become widely accepted. Here, we describe a novel anti-HER2 antibody, hHERmAb-F0178C1, which was isolated from a screen of a phage display library. A step-by-step optimization method was employed to maximize the inhibitory effect of this anti-HER2 antibody. Crystallographic analysis was used to determine the three-dimensional structure to 3.5 Å resolution, confirming that the epitope of this antibody is in domain III of HER2. Moreover, this novel anti-HER2 antibody exhibits superior efficacy in blocking HER2/HER3 heterodimerization and signaling, and its use in combination with pertuzumab has a synergistic effect. Characterization of this antibody revealed the important role of a ligand binding site within domain III of HER2. The results of this study clearly indicate the unique potential of hHERmAb-F0178C1, and its complementary inhibition effect on HER2/HER3 signaling warrants its consideration as a promising clinical treatment.     

帕妥珠单抗生物类似药SMMU-27 静脉注射对食蟹猴的重复给药毒性探索研究

目的:观察帕妥珠单抗生物类似药SMMU-27四周静脉注射对食蟹猴的安全性。方法:20只健康食蟹猴按体重随机分为阳性对照组、SMMU-27低、中、高剂量组和辅料对照组,每组4只,雌雄各半。低、中、高剂量组剂量分别为15、150和450 mg/kg,阳性对照组给予150 mg/kg帕妥珠单抗(Pertuzumab),辅料对照组给予空白溶剂(0 mg/kg)。各组动物按相应体重慢速静脉注射给药,给药体积为15 m L/kg,给药速度约5 m L/min。每周给药1次,共给药4周,恢复期4周,期间进行各项毒理学指标检测。结果:一般症状结果显示给药期间与给药后,低、中、高剂量组和阳性对照组陆续有动物出现腹泻症状。高剂量组1只动物在d40时濒死剖解,其最早出现稀便,停药后腹泻状态也未见好转,生化指标显示在d28时碱性磷酸酶(Alkaline Phosphatase,ALP)升高,在d14和d40时尿素(Blood Urea,BU)升高,总蛋白(Total Protein,TP)、白蛋白(Albumin,ALB)降低。低、高剂量组和阳性对照组均有部分动物白细胞(White Blood Cell,WBC)给药后数值降低,各给药组在d14时及高剂量组和阳性对照组在d28时BU升高或有升高的趋势,恢复期时有恢复趋势。高剂量濒死动物骨髓检查发现核红细胞较多,各阶段粒细胞减少,出现较多裸核;病理检查发现肾脏可见散在多发的中度肾小管扩张,近曲小管上皮轻度变性。其余指标包括一般症状、体重、尿液、心电图、免疫学指标等未见明显与供试品相关的异常变化。结论:SMMU-27主要毒性靶部位是胃肠道(腹泻)、肾脏(血清BU升高)和血液系统(WBC下降),应与这些部位表达供试品结合的相关受体有关,属供试品的药理作用放大和延伸。因此本实验条件下食蟹猴的安全剂量(NOAEL)为150 mg/kg,致死剂量为450 mg/kg。SMMU-27与等剂量阳性对照药物毒性反应基本类似。     

The ErbB/HER receptor protein-tyrosine kinases and cancer

The ErbB/HER protein-tyrosine kinases, which include the epidermal growth factor receptor, consist of a growth-factor-binding ectodomain, a single transmembrane segment, an intracellular protein-tyrosine kinase catalytic domain, and a tyrosine-containing cytoplasmic tail. The genes for the four members of this family, ErbB1-ErbB4, are found on different human chromosomes. Null mutations of any of the ErbB family members result in embryonic lethality. ErbB1 and ErbB2 are overexpressed in a wide variety of tumors including breast, colorectal, ovarian, and non-small cell lung cancers. The structures of the ectodomains of the ErbB receptors in their active and inactive conformation have shed light on the mechanism of receptor activation. The extracellular component of the ErbB proteins consists of domains I-IV. The activating growth factor, which binds to domains I and III, selects and stabilizes a conformation that allows a dimerization arm to extend from domain II to interact with an ErbB dimer partner. As a result of dimerization, protein kinase activation, trans-autophosphorylation, and initiation of signaling occur. The conversion of the inactive to active receptor involves a major rotation of the ectodomain. The ErbB receptors are targets for anticancer drugs. Two strategies for blocking the action of these proteins include antibodies directed against the ectodomain and drugs that inhibit protein-tyrosine kinase activity. A reversible ATP competitive inhibitor of ErbB1 (ZD1839, or Iressa) and an ErbB1 ectodomain directed antibody (IMC-C225, or Erbitux) have been approved for the treatment of non-small cell lung cancer and colorectal cancer, respectively. An ErbB2/HER2 ectodomain directed antibody (trastuzumab, or Herceptin) has also been approved for the treatment of breast cancer. Current research promises to produce additional agents based upon these approaches.     

Structural and functional characterization of MBS301, an afucosylated bispecific anti-HER2 antibody

MBS301, a glyco-engineered bispecific anti-human epidermal growth factor receptor 2 (HER2) antibody with a typical IgG1 monoclonal antibody structure, was developed through dual-cell expression and in vitro assembling process. MBS301 consists of two half antibodies engineered from trastuzumab and pertuzumab, respectively. Integrity and purity profiles of MB301 indicated that the heterodimerization of the two half antibodies was successful. The high and similar melting temperatures (Tm1,72.0°C and Tm2, 84.8°C) of MBS301 compared with those of its parental monoclonal antibodies trastuzumab and pertuzumab (in-house made T-mab and P-mab, respectively) revealed its structural compactness. With computer-modeling experiments and Biacore binding and competition kinetics studies, the binding stoichiometry between MBS301 and HER2-ECD was determined to be 1:1 and the two arms of MBS301 were shown to bind to domains II and IV of HER2-ECD antigen simultaneously. MBS301 displayed synergistic bioactivities as the combination of T-mab and P-mab in vitro in multiple cancer cell lines and in vivo in xenograft mouse model studies, and showed more effective activity than T-mab or P-mab used individually. Moreover, fucose-knockout dramatically increased MBS301’s binding affinity to low affinity FcγRIIIa allotype 158F (K_D = 2.35 × 10^?7M) to near the high affinity level of allotype V158 (K_D = 1.17 × 10^?7M). This resulted in far more effective ADCC activity of MBS301 than the combination of T-mab and P-mab in killing HER2-positive cancer cells. Hence, a novel fully afucosylated anti-HER2 bispecific antibody with improved antitumor activities was generated and shown to have the potential to be used for treating HER2-positive but trastuzumab-resistant solid tumors.     

Gefitinib induces epidermal growth factor receptor dimers which alters the interaction characteristics with ¹²⁵I-EGF

The tyrosine kinase inhibitor gefitinib inhibits growth in some tumor types by targeting the epidermal growth factor receptor (EGFR). Previous studies show that the affinity of the EGF-EGFR interaction varies between hosting cell line, and that gefitinib increases the affinity for some cell lines. In this paper, we investigate possible mechanisms behind these observations. Real-time interaction analysis in LigandTracer® Grey revealed that the HER2 dimerization preventing antibody pertuzumab clearly modified the binding of ¹²⁵I-EGF to EGFR on HER2 overexpressing SKOV3 cells in the presence of gefitinib. Pertuzumab did not affect the binding on A431 cells, which express low levels of HER2. Cross-linking measurements showed that gefitinib increased the amount of EGFR dimers 3.0–3.8 times in A431 cells in the absence of EGF. In EGF stimulated SKOV3 cells the amount of EGFR dimers increased 1.8–2.2 times by gefitinib, but this effect was cancelled by pertuzumab. Gefitinib treatment did not alter the number of EGFR or HER2 expressed in tumor cell lines A431, U343, SKOV3 and SKBR3. Real-time binding traces were further analyzed in a novel tool, Interaction Map, which deciphered the different components of the measured interaction and supports EGF binding to multiple binding sites. EGFR and HER2 expression affect the levels of EGFR monomers, homodimers and heterodimers and EGF binds to the various monomeric/dimeric forms of EGFR with unique binding properties. Taken together, we conclude that dimerization explains the varying affinity of EGF – EGFR in different cells, and we propose that gefitinib induces EGFR dimmers, which alters the interaction characteristics with ¹²⁵I-EGF.