共查询到20条相似文献,搜索用时 13 毫秒
1.
Daniela Bumbaca Anne Wong Elizabeth Drake Arthur E Reyes II Benjamin C Lin Jean-Philippe Stephan Luc Desnoyers Ben-Quan Shen Mark S Dennis 《MABS-AUSTIN》2011,3(4):376-386
Off-target binding can significantly affect the pharmacokinetics (PK), tissue distribution, efficacy and toxicity of a therapeutic antibody. Herein we describe the development of a humanized anti-fibroblast growth factor receptor 4 (FGFR4) antibody as a potential therapeutic for hepatocellular carcinoma (HCC). A chimeric anti-FGFR4 monoclonal antibody (chLD1) was previously shown to block ligand binding and to inhibit FGFR4-mediated signaling as well as tumor growth in vivo. A humanized version of chLD1, hLD1.vB, had similar binding affinity and in vitro blocking activity, but it exhibited rapid clearance, poor target tissue biodistribution and limited efficacy when compared to chLD1 in a HUH7 human HCC xenograft mouse model. These problems were traced to instability of the molecule in rodent serum. Size exclusion high performance liquid chromatography, immunoprecipitation and mass spectral sequencing identified a specific interaction between hLD1.vB and mouse complement component 3 (C3). A PK study in C3 knock-out mice further confirmed this specific interaction. Subsequently, an affinity-matured variant derived from hLD1.vB (hLD1.v22), specifically selected for its lack of binding to mouse C3 was demonstrated to have a PK profile and in vivo efficacy similar to that of chLD1 in mice. Although reports of non-specific off-target binding have been observed for other antibodies, this represents the first report identifying a specific off-target interaction that affected disposition and biological activity. Screens developed to identify general non-specific interactions are likely to miss the rare and highly specific cross-reactivity identified in this study, thus highlighting the importance of animal models as a proxy for avoiding unexpected clinical outcomes.Key words: antibody humanization, non-specific binding, fibroblast growth factor receptor 4, affinity maturation, off-target binding, complement C3Antibodies are an attractive source of biotherapeutic agents due to their high affinity, exquisite target selectivity and extended half-life in vivo. Their development for therapeutic applications has been facilitated by hybridoma technology, antibody humanization and numerous in vitro antibody selection technologies that enable antibodies with desired biological properties to be engineered at will.Co-incident with this increase in the in vitro development of antibodies for therapeutic applications has been the recognition of how antibodies evolve in vivo. Several studies have pointed to the relationship between antibody affinity and antigen specificity.1–6 The conformational flexibility of initial recombined antibodies is considered to be an important feature of the immune system''s ability to generate antibodies against a broad spectrum of antigens. During antibody maturation, this structural plasticity is thought to be restricted through somatic hypermutation in vivo (and perhaps affinity maturation in vitro) leading to a reduced entropy cost for specific antigen binding and a corresponding increase in antigen specificity. The increase in antigen specificity helps to eliminate undesired off-target antibody interactions, and serves as part of immune system checkpoints designed to prevent autoimmune disease.In contrast, antibodies that are generated in vitro lack any regulatory immune surveillance. For these, various screens utilizing protein chips and microarrays have been developed in order to evaluate or anticipate off-target interactions.7–9 In one study comparing antibodies against TNFα, for example, multiple off-target interactions were found for adalimumab, an antibody derived in vitro from a cloned human antibody phage library.7 However, such screens are artificial and whether any observed non-specific off-target binding events result in adverse side effects in vivo or actually take place in vivo has yet to be demonstrated.Recently, affinity matured variants of palivizumab were found to have a less than anticipated increase in potency as a prophylactic treatment in a rat model of respiratory syncytial virus (RSV). The variants unexpectedly exhibited broad tissue binding that led to their rapid clearance and low target tissue bioavailability.10 Reversion of some of the amino acid changes incorporated during affinity maturation diminished much of the non-specific tissue binding and improved efficacy and pharmacokinetics. Direct interactions leading to the broad non-specific tissue binding were thus identified and removed, enabling the development of a more effective variant of palivizumab. This is the first report correlating broad tissue cross-reactivity in vitro with rapid clearance and poor tissue bioavailability.The likelihood of identifying an off-target binding event is a function of the size of the protein repertoire and the affinity that is considered relevant.11 Given the huge complexity of an in vivo system and the typically high therapeutic dosing in a clinical setting, the odds of an off-target event that affects efficacy, clearance, tissue bioavailability or toxicity are greatly increased. Such antigen promiscuity in an antibody may arise from the recognition of structurally related epitopes (molecular mimicry), the utilization of overlapping or independent antibody paratopes or through conformational flexibility that enables the complementary-determining regions (CDRs) or side chains of an antibody to adapt to more than one antigen.12,13In contrast to the broad non-specific off-target recognition and antibody polyspecificity described above, here we report an unexpected specific off-target binding event we identified for a humanized antibody directed toward fibroblast growth factor receptor (FGFR) 4. The FGFR signaling system plays critical roles in a variety of normal developmental and physiological processes, and aberrant signaling may lead to tumor development and progression.14 FGFR4 has been shown to play a modulatory role in the development and progression of hepatocellular carcinoma (HCC) in mice, and potentially in humans. A chimeric anti-FGFR4 monoclonal antibody (chLD1) was previously shown to block ligand binding, inhibit FGFR4-mediated signaling and inhibit HCC tumor growth in vivo.14 Following the humanization of chLD1, we observed an unexpected loss of efficacy in a mouse tumor model, although the humanized variant had identical affinity for FGFR4. This variant was found to cross-react with an abundant mouse serum protein. This specific off-target interaction interfered with FGFR4 binding, altered antibody clearance, impacted target tissue distribution, resulting in reduced therapeutic activity. Subsequently we eliminated this off-target binding through affinity maturation of the humanized antibody leading to the full restoration of the in vivo properties inherent in chLD1.This work illustrates some of the challenges that extend well beyond simple antibody-antigen binding and serves as a cautionary tale to therapeutic antibody development. 相似文献
2.
Sharmila Rajan Danielle Mandikian Amos Baruch Thomas R. Gelzleichter Dale Stevens Junichiro Sonoda 《MABS-AUSTIN》2017,9(8):1379-1388
Target receptor levels can influence pharmacokinetics (PK) or pharmacodynamics (PD) of monoclonal antibodies (mAbs), and can affect drug development of this class of molecules. We generated an effector-less humanized bispecific antibody that selectively activates fibroblast growth factor receptor (FGFR)1 and βKlotho receptor, a FGF21 receptor complex highly expressed in both white and brown adipocytes. The molecule shows cross-species binding with comparable equilibrium binding affinity (Kd) for human, cynomolgus monkey, and mouse FGFR1/βKlotho. To understand the PK/PD relationship in non-obese and obese animals, we evaluated the adipose tissue distribution of the antibody, serum exposures, and an associated PD marker (high-molecular-weight adiponectin), in both non-obese and obese mice and monkeys. Antibody uptake into fat tissue was found to be higher on a per gram basis in non-obese animals compared to obese animals. Since obesity has been reported to be associated with reduced expression of FGFR1 and βKlotho receptor in white adipose tissues in mice, our results suggest that the distribution in adipose tissues was influenced by target expression levels. Even so, the overall dose-normalized serum exposures were comparable between non-obese and obese mice and monkeys, suggesting that adipose tissue uptake plays a limited role in overall systemic PK determination. It remains to be determined if and how obesity and receptor expression in humans influence the PK and PD profile of this novel therapeutic candidate. 相似文献
3.
Bing Li Devin Tesar C Andrew Boswell Hendry S Cahaya Anne Wong Jianhuan Zhang Y Gloria Meng Charles Eigenbrot Homer Pantua Jinyu Diao Sharookh B Kapadia Rong Deng Robert F Kelley 《MABS-AUSTIN》2014,6(5):1255-1264
Pharmacokinetic (PK) testing of a humanized (κI, VH3 framework) and affinity matured anti-hepatitis C virus E2-glycoprotein (HCV-E2) antibody (hu5B3.κ1VH3.v3) in rats revealed unexpected fast clearance (34.9 mL/day/kg). This antibody binds to the rat recycling receptor FcRn as expected for a human IgG1 antibody and does not display non-specific binding to baculovirus particles in an assay that is correlated with fast clearance in cynomolgus monkey. The antigen is not expressed in rat so target-dependent clearance does not contribute to PK. Removal of the affinity maturation changes (hu5B3.κ1VH3.v1) did not restore normal clearance. The antibody was re-humanized on a κ4, VH1 framework and the non-affinity matured version (hu5B3.κ4VH1.v1) was shown to have normal clearance (8.5 mL/day/kg). Since the change in framework results in a lower pI, primarily due to more negative charge on the κ4 template, the effect of additional charge variation on antibody PK was tested by incorporating substitutions obtained through phage display affinity maturation of hu5B3.κ1VH3.v1. A variant having a pI of 8.61 gave very fast clearance (140 mL/day/kg) whereas a molecule with pI of 6.10 gave slow clearance (5.8 mL/kg/day). Both antibodies exhibited comparable binding to rat FcRn, but biodistribution experiments showed that the high pI variant was catabolized in liver and spleen. These results suggest antibody charge can have an effect on PK through alterations in antibody catabolism independent of FcRn-mediated recycling. Furthermore, introduction of affinity maturation changes into the lower pI framework yielded a candidate with PK and virus neutralization properties suitable for clinical development. 相似文献
4.
《MABS-AUSTIN》2013,5(5):1255-1264
Pharmacokinetic (PK) testing of a humanized (κI, VH3 framework) and affinity matured anti-hepatitis C virus E2-glycoprotein (HCV-E2) antibody (hu5B3.κ1VH3.v3) in rats revealed unexpected fast clearance (34.9 mL/day/kg). This antibody binds to the rat recycling receptor FcRn as expected for a human IgG1 antibody and does not display non-specific binding to baculovirus particles in an assay that is correlated with fast clearance in cynomolgus monkey. The antigen is not expressed in rat so target-dependent clearance does not contribute to PK. Removal of the affinity maturation changes (hu5B3.κ1VH3.v1) did not restore normal clearance. The antibody was re-humanized on a κ4, VH1 framework and the non-affinity matured version (hu5B3.κ4VH1.v1) was shown to have normal clearance (8.5 mL/day/kg). Since the change in framework results in a lower pI, primarily due to more negative charge on the κ4 template, the effect of additional charge variation on antibody PK was tested by incorporating substitutions obtained through phage display affinity maturation of hu5B3.κ1VH3.v1. A variant having a pI of 8.61 gave very fast clearance (140 mL/day/kg) whereas a molecule with pI of 6.10 gave slow clearance (5.8 mL/kg/day). Both antibodies exhibited comparable binding to rat FcRn, but biodistribution experiments showed that the high pI variant was catabolized in liver and spleen. These results suggest antibody charge can have an effect on PK through alterations in antibody catabolism independent of FcRn-mediated recycling. Furthermore, introduction of affinity maturation changes into the lower pI framework yielded a candidate with PK and virus neutralization properties suitable for clinical development. 相似文献
5.
William J.J. Finlay James E. Coleman Jonathan S. Edwards Kevin S. Johnson 《MABS-AUSTIN》2019,11(1):26-44
Monoclonal anti-programmed cell death 1 (PD1) antibodies are successful cancer therapeutics, but it is not well understood why individual antibodies should have idiosyncratic side-effects. As the humanized antibody SHR-1210 causes capillary hemangioma in patients, a unique toxicity amongst anti-PD1 antibodies, we performed human receptor proteome screening to identify nonspecific interactions that might drive angiogenesis. This screen identified that SHR-1210 mediated aberrant, but highly selective, low affinity binding to human receptors such as vascular endothelial growth factor receptor 2 (VEGFR2), frizzled class receptor 5 and UL16 binding protein 2 (ULBP2). SHR-1210 was found to be a potent agonist of human VEGFR2, which may thereby drive hemangioma development via vascular endothelial cell activation. The v-domains of SHR-1210’s progenitor murine monoclonal antibody ‘Mab005? also exhibited off-target binding and agonism of VEGFR2, proving that the polyspecificity was mediated by the original mouse complementarity-determining regions (CDRs), and had survived the humanization process. Molecular remodelling of SHR-1210 by combinatorial CDR mutagenesis led to deimmunization, normalization of binding affinity to human and cynomolgus PD1, and increased potency in PD1/PD-L1 blockade. Importantly, CDR optimization also ablated all off-target binding, rendering the resulting antibodies fully PD1-specific. As the majority of changes to the paratope were found in the light chain CDRs, the germlining of this domain drove the ablation of off-target binding. The combination of receptor proteome screening and optimization of the antibody binding interface therefore succeeded in generating novel, higher-potency, specificity-enhanced therapeutic IgGs from a single, clinically sub-optimal progenitor. This study showed that highly-specific off-target binding events might be an under-appreciated phenomenon in therapeutic antibody development, but that these unwanted properties can be fully ameliorated by paratope refinement. 相似文献
6.
Agas D Marchetti L Menghi G Materazzi S Materazzi G Capacchietti M Hurley MM Sabbieti MG 《Journal of cellular physiology》2008,214(1):145-152
In this study, we investigated the role of prostaglandin F2alpha (PGF2alpha) in mouse osteoblast survival and the function of fibroblast growth factor 2 (FGF-2) and fibroblast growth factor receptor 1 (FGFR1) in this process. In particular, for the first time, we demonstrated that PGF2alpha increased osteoblast survival in a dose-dependent manner and we showed that the effect is correlated with an increase in Bcl-2/Bax ratio. Furthermore, we demonstrated that PGF2alpha caused a decrement of the active caspases 9 and 3. By blocking FGF-2 with the specific neutralizing antibody and by depletion of FGFR1 gene with a specific siRNA, we showed that FGFR1 and FGF-2 are critical for the increment of Bcl-2/Bax ratio and the decrement of the active caspases 9 and 3, induced by PGF2alpha. Moreover, transmission electron microscopy studies showed that PGF2alpha increased binding of FGF-2 and FGFR1 and co-localization of reactive sites at plasma membrane level. In conclusion, we report a novel mechanism in which PGF2alpha induces FGF-2 binding to its specific cell surface receptor 1 leading to a cascade pathway that culminates with increased mouse osteoblast survival. 相似文献
7.
Isabel Figueroa Doug Leipold Steve Leong Bing Zheng Montserrat Triguero-Carrasco Aimee Fourie-O'Donohue 《MABS-AUSTIN》2018,10(5):738-750
For antibody-drug conjugates (ADCs) that carry a cytotoxic drug, doses that can be administered in preclinical studies are typically limited by tolerability, leading to a narrow dose range that can be tested. For molecules with non-linear pharmacokinetics (PK), this limited dose range may be insufficient to fully characterize the PK of the ADC and limits translation to humans. Mathematical PK models are frequently used for molecule selection during preclinical drug development and for translational predictions to guide clinical study design. Here, we present a practical approach that uses limited PK and receptor occupancy (RO) data of the corresponding unconjugated antibody to predict ADC PK when conjugation does not alter the non-specific clearance or the antibody-target interaction. We used a 2-compartment model incorporating non-specific and specific (target mediated) clearances, where the latter is a function of RO, to describe the PK of anti-CD33 ADC with dose-limiting neutropenia in cynomolgus monkeys. We tested our model by comparing PK predictions based on the unconjugated antibody to observed ADC PK data that was not utilized for model development. Prospective prediction of human PK was performed by incorporating in vitro binding affinity differences between species for varying levels of CD33 target expression. Additionally, this approach was used to predict human PK of other previously tested anti-CD33 molecules with published clinical data. The findings showed that, for a cytotoxic ADC with non-linear PK and limited preclinical PK data, incorporating RO in the PK model and using data from the corresponding unconjugated antibody at higher doses allowed the identification of parameters to characterize monkey PK and enabled human PK predictions. 相似文献
8.
A humanized monoclonal antibody constructed from intronless expression vectors targets human hepatocellular carcinoma cells 总被引:4,自引:0,他引:4
Chan KT Cheng SC Xie H Xie Y 《Biochemical and biophysical research communications》2001,284(1):157-167
An anti-human hepatocellular carcinoma (HCC) monoclonal antibody, hHP-1, was genetically humanized from a murine monoclonal antibody. In this study, a concept of positional template approach was applied to design the amino acid sequence of hHP-1's variable region, and synthetic DNA fragments for protein expression were produced through overlapping PCR from single strand oligonucleotides. Synthetic DNA fragments and human antibody constant region cDNA were used to construct two CMV promotor-based expression vectors for the antibody light and heavy chains, in which the variable region was connected directly to the constant region without an intron sequence. Completely assembled humanized antibody was successfully expressed in mammalian cells as IgG1 kappa molecules and purified using protein A affinity column. The immunogenicity of the hHP1 was estimated by the amino acid sequence and determined through a HAMA (human anti-murine antibody) serum reaction assay. Results indicated that the immunogenicity of hHP-1 was significantly reduced. In vitro binding activity assay showed that the hHP-1 had retained its binding function to a human HCC SMMC-7721 cell-line, without cross binding to other human normal tissues. Immunofluorescence staining showed that hHP-1 had a strong binding activity to SMMC cells. A competitive binding assay showed that the relative binding activity of hHP-1 was approximately 25% binding activity of the original murine antibody. Our results indicate that a humanized antibody could be produced using intronless vectors and expressed as a complete IgG1 kappa antibody. Hence we believe that hHP-1 could be a potential candidate for HCC treatment. 相似文献
9.
French DM Lin BC Wang M Adams C Shek T Hötzel K Bolon B Ferrando R Blackmore C Schroeder K Rodriguez LA Hristopoulos M Venook R Ashkenazi A Desnoyers LR 《PloS one》2012,7(5):e36713
The fibroblast growth factor (FGF)-FGF receptor (FGFR) signaling system plays critical roles in a variety of normal developmental and physiological processes. It is also well documented that dysregulation of FGF-FGFR signaling may have important roles in tumor development and progression. The FGFR4-FGF19 signaling axis has been implicated in the development of hepatocellular carcinomas (HCCs) in mice, and potentially in humans. In this study, we demonstrate that FGFR4 is required for hepatocarcinogenesis; the progeny of FGF19 transgenic mice, which have previously been shown to develop HCCs, bred with FGFR4 knockout mice fail to develop liver tumors. To further test the importance of FGFR4 in HCC, we developed a blocking anti-FGFR4 monoclonal antibody (LD1). LD1 inhibited: 1) FGF1 and FGF19 binding to FGFR4, 2) FGFR4-mediated signaling, colony formation, and proliferation in vitro, and 3) tumor growth in a preclinical model of liver cancer in vivo. Finally, we show that FGFR4 expression is elevated in several types of cancer, including liver cancer, as compared to normal tissues. These findings suggest a modulatory role for FGFR4 in the development and progression of hepatocellular carcinoma and that FGFR4 may be an important and novel therapeutic target in treating this disease. 相似文献
10.
Amrita V Kamath Victor Yip Priyanka Gupta C Andrew Boswell Daniela Bumbaca Peter Haughney Joni Castro Siao Ping Tsai Glenn Pacheco Sarajane Ross Minhong Yan Lisa A Damico-Beyer Leslie Khawli Ben-Quan Shen 《MABS-AUSTIN》2014,6(6):1631-1637
Delta-like-4 ligand (DLL4) plays an important role in vascular development and is widely expressed on the vasculature of normal and tumor tissues. Anti-DLL4 is a humanized IgG1 monoclonal antibody against DLL4. The purpose of these studies was to characterize the pharmacokinetics (PK), tissue distribution, and anti-tumor efficacy of anti-DLL4 in mice over a range of doses. PK and tissue distribution of anti-DLL4 were determined in athymic nude mice after administration of single intravenous (IV) doses. In the tissue distribution study, radiolabeled anti-DLL4 (mixture of 125Iodide and 111Indium) was administered in the presence of increasing amounts of unlabeled anti-DLL4. Dose ranging anti-DLL4 anti-tumor efficacy was evaluated in athymic nude mice bearing MV522 human lung tumor xenografts. Anti-DLL4 had nonlinear PK in mice with rapid serum clearance at low doses and slower clearance at higher doses suggesting the involvement of target mediated clearance. Consistent with the PK data, anti-DLL4 was shown to specifically distribute to several normal tissues known to express DLL4 including the lung and liver. Maximal efficacy in the xenograft model was seen at doses ≥ 10 mg/kg when tissue sinks were presumably saturated, consistent with the PK and tissue distribution profiles. These findings highlight the importance of mechanistic understanding of antibody disposition to enable dosing strategies for maximizing efficacy. 相似文献
11.
《MABS-AUSTIN》2013,5(6):1631-1637
Delta-like-4 ligand (DLL4) plays an important role in vascular development and is widely expressed on the vasculature of normal and tumor tissues. Anti-DLL4 is a humanized IgG1 monoclonal antibody against DLL4. The purpose of these studies was to characterize the pharmacokinetics (PK), tissue distribution, and anti-tumor efficacy of anti-DLL4 in mice over a range of doses. PK and tissue distribution of anti-DLL4 were determined in athymic nude mice after administration of single intravenous (IV) doses. In the tissue distribution study, radiolabeled anti-DLL4 (mixture of 125Iodide and 111Indium) was administered in the presence of increasing amounts of unlabeled anti-DLL4. Dose ranging anti-DLL4 anti-tumor efficacy was evaluated in athymic nude mice bearing MV522 human lung tumor xenografts. Anti-DLL4 had nonlinear PK in mice with rapid serum clearance at low doses and slower clearance at higher doses suggesting the involvement of target mediated clearance. Consistent with the PK data, anti-DLL4 was shown to specifically distribute to several normal tissues known to express DLL4 including the lung and liver. Maximal efficacy in the xenograft model was seen at doses ≥ 10 mg/kg when tissue sinks were presumably saturated, consistent with the PK and tissue distribution profiles. These findings highlight the importance of mechanistic understanding of antibody disposition to enable dosing strategies for maximizing efficacy. 相似文献
12.
Guided selection of human antibody light chains against TAG-72 using a phage display chain shuffling approach 总被引:1,自引:0,他引:1
To enhance therapeutic potential of murine monoclonal antibody, humanization by CDR grafting is usually used to reduce immunogenic mouse residues. Most humanized antibodies still have mouse residues critical for antigen binding, but the mouse residues may evoke immune responses in humans. Previously, we constructed a new humanized version (AKA) of mouse CC49 antibody specific for tumor-associated glycoprotein, TAG-72. In this study, to select a completely human antibody light chain against TAG-72, guided selection strategy using phage display was used. The heavy chain variable region (VH) of AKA was used to guide the selection of a human TAG-72-specific light chain variable region (VL) from a human VL repertoire constructed from human PBL. Most of the selected VLs were identified to be originated from the members of the human germline VK1 family, whereas the VL of AKA is more homologous to the VK4 family. Competition binding assay of the selected Fabs with mouse CC49 suggested that the epitopes of the Fabs overlap with that of CC49. In addition, they showed better antigen-binding affinity compared to parental AKA. The selected human VLs may be used to guide the selection of human VHs to get completely human anti-TAG72 antibody. 相似文献
13.
4C8 is a new mouse anti-human CD34 monoclonal antibody (mAb), which recognizes class II CD34 epitopes and can be used for clinical hematopoietic stem/progenitor cell selection. In an attempt to improve its safety profiles, we have developed a humanized antibody of 4C8 by complementarity-determining region (CDR) grafting method in this study. Using a molecular model of 4C8 built by computer-assisted homology modelling, framework region (FR) residues of potential importance to the antigen binding were identified. A humanized version of 4C8, denoted as h4C8, was generated by transferring these key murine FR residues onto a human antibody framework that was selected based on homology to the mouse antibody framework, together with the mouse CDR residues. The resultant humanized antibody was shown to possess antigen-binding affinity and specificity similar to that of the original murine antibody, suggesting that it might be an alternative to mouse anti-CD34 antibodies routinely used clinically. 相似文献
14.
为探讨肝癌特异性鼠源及其人源化单链抗体基因的表达策略并比较二者的结合能力,在3种载体中分别以融合、分泌及胞内表达的方式进行了研究;对复性后的单链抗体以抗原捕获ELISA法进行检测。结果表明,在3种载体中表达的鼠源及人源化单链抗体都是包含体,诱导物浓度及培养温度不影响表达形式;抗原捕获细胞ELISA表明人源化的单链抗体和鼠源单链抗体有相近的抗原结合能力。结论是:在大肠杆菌中表达的基因工程单链抗体的可溶性可能主要由自身氨基酸一级序列决定;先前的设计所采取的人源化方案没有影响到鼠源抗体的CDRs的天然构象,表达的人源化单链抗体提供了免疫原性评价及临床应用的基础。 相似文献
15.
Sasaki S Ishida T Toyota M Ota A Suzuki H Takaoka A Yasui H Yamamoto H Takagi H Maeda M Seito T Tsujisaki M Shinomura Y Imai K 《PloS one》2011,6(5):e19618
Background
Hepatocellular carcinoma (HCC) is the most commonly occurring primary liver cancer and ranks as the fifth most frequently occurring cancer, overall, and the third leading cause of cancer deaths, worldwide. At present, effective therapeutic options available for HCC are limited; consequently, the prognosis for these patients is poor. Our aim in the present study was to identify a novel target for antibody therapy against HCC.Methodology/Principal Findings
We used Western blot and flow cytometric and immunocytochemical analyses to investigate the regulation of FGFR1 expression by interferon-α/β in several human hepatic cancer cell lines. In addition, we tested the efficacy of combined treatment with anti-FGFR1 monoclonal antibody and interferon-α/β in a murine xenograft model of human HCC. We found that interferon-α/β induces expression of FGFR1 in human HCC cell lines, and that an anti-FGFR1 monoclonal antibody (mAb) targeting of the induced FGFR1 can effectively inhibit growth and survival of HCC cells in vitro and in vivo. Moreover, the combination of interferon-α, anti-FGFR1 mAb and peripheral blood mononuclear cells (PBMCs) exerted a significant antitumor effect in vitro.Conclusions
Our results suggest that the combined use of an anti-FGFR1 antibody and interferon-α/β is a promising approach to the treatment of HCC. 相似文献16.
Liming Liu 《蛋白质与细胞》2018,9(1):15
There are many factors that can influence the pharmacokinetics (PK) of a mAb or Fc-fusion molecule with the primary determinant being FcRn-mediated recycling. Through Fab or Fc engineering, IgG-FcRn interaction can be used to generate a variety of therapeutic antibodies with significantly enhanced half-life or ability to remove unwanted antigen from circulation. Glycosylation of a mAb or Fc-fusion protein can have a significant impact on the PK of these molecules. mAb charge can be important and variants with pI values of 1–2 unit difference are likely to impact PK with lower pI values being favorable for a longer half-life. Most mAbs display target mediated drug disposition (TMDD), which can have significant consequences on the study designs of preclinical and clinical studies. The PK of mAb can also be influenced by anti-drug antibody (ADA) response and off-target binding, which require careful consideration during the discovery stage. mAbs are primarily absorbed through the lymphatics via convection and can be conveniently administered by the subcutaneous (sc) route in large doses/volumes with co-formulation of hyaluronidase. The human PK of a mAb can be reasonably estimated using cynomolgus monkey data and allometric scaling methods. 相似文献
17.
Yan Li Yuhua Wan Peipei Liu Jincun Zhao Guangwen Lu Jianxun Qi Qihui Wang Xuancheng Lu Ying Wu Wenjun Liu Buchang Zhang Kwok-Yung Yuen Stanley Perlman George F Gao Jinghua Yan 《Cell research》2015,25(11):1237-1249
The newly-emerging Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe and fatal acute respiratory disease in humans. Despite global efforts, the potential for an associated pandemic in the future cannot be excluded. The development of effective counter-measures is urgent. MERS-CoV-specific anti-viral drugs or vaccines are not yet available. Using the spike receptor-binding domain of MERS-CoV (MERS-RBD) to immunize mice, we identified two neutralizing monoclonal antibodies (mAbs) 4C2 and 2E6. Both mAbs potently bind to MERS-RBD and block virus entry in vitro with high efficacy. We further investigated their mechanisms of neutralization by crystallizing the complex between the Fab fragments and the RBD, and solved the structure of the 4C2 Fab/MERS-RBD complex. The structure showed that 4C2 recognizes an epitope that partially overlaps the receptor-binding footprint in MERS-RBD, thereby interfering with the virus/receptor interactions by both steric hindrance and interface-residue competition. 2E6 also blocks receptor binding, and competes with 4C2 for binding to MERS-RBD. Based on the structure, we further humanized 4C2 by preserving only the paratope residues and substituting the remaining amino acids with the counterparts from human immunoglobulins. The humanized 4C2 (4C2h) antibody sustained similar neutralizing activity and biochemical characteristics to the parental mouse antibody. Finally, we showed that 4C2h can significantly abate the virus titers in lungs of Ad5-hCD26-transduced mice infected with MERS-CoV, therefore representing a promising agent for prophylaxis and therapy in clinical settings. 相似文献
18.
肝癌特异性鼠源及人源化单链抗体基因的构建及大肠杆菌中的表达 总被引:12,自引:1,他引:12
为探讨一株肝细胞癌特异性鼠源及其人源化单链抗体基因在大肠杆菌中的可溶性表达策略并比较二者对抗原的结构能力,在三种载体中分别以融合、分泌及胞内表达的方式进行了研究,表达产物均以包涵体形式存在;对复性后的单链抗体以细胞ELISA及竞争抑制流式细胞仪法进行检测,表明人源化单链抗体和鼠源单链抗体有相近的抗原结合能力。结论是:大肠杆菌中表达的基因工程单链抗体的可溶性可能主要由自身氨基酸一级序列决定;先前的设 相似文献
19.
Zhang Y McKeehan K Lin Y Zhang J Wang F 《Molecular endocrinology (Baltimore, Md.)》2008,22(1):167-175
Binding of the fibroblast growth factor (FGF) to the FGF receptor (FGFR) tyrosine kinase leads to receptor tyrosine autophosphorylation as well as phosphorylation of multiple downstream signaling molecules that are recruited to the receptor either by direct binding or through adaptor proteins. The FGFR substrate 2 (FRS2) family consists of two members, FRS2alpha and FRS2beta, and has been shown to recruit multiple signaling molecules, including Grb2 and Shp2, to FGFR1. To better understand how FRS2 interacted with FGFR1, in vivo binding assays with coexpressed FGFR1 and FRS2 recombinant proteins in mammalian cells were carried out. The results showed that the interaction of full-length FRS2alpha, but not FRS2beta, with FGFR1 was enhanced by activation of the receptor kinase. The truncated FRS2alpha mutant that was comprised only of the phosphotyrosine-binding domain (PTB) bound FGFR1 constitutively, suggesting that the C-terminal sequence downstream the PTB domain inhibited the PTB-FGFR1 binding. Inactivation of the FGFR1 kinase and substitutions of tyrosine phosphorylation sites of FGFR1, but not FRS2alpha, reduced binding of FGFR1 with FRS2alpha. The results suggest that although the tyrosine autophosphorylation sites of FGFR1 did not constitute the binding sites for FRS2alpha, phosphorylation of these residues was essential for optimal interaction with FRS2alpha. In addition, it was demonstrated that the Grb2-binding sites of FRS2alpha are essential for mediating signals of FGFR1 to activate the FiRE enhancer of the mouse syndecan 1 gene. The results, for the first time, demonstrate the specific signals mediated by the Grb2-binding sites and further our understanding of FGF signal transmission at the adaptor level. 相似文献
20.
Jianxin Dai Bohua Li Jinping Shi Ling Peng Dapeng Zhang Weizhu Qian Sheng Hou Lei Zhao Jie Gao Zhiguo Cao Jian Zhao Hao Wang Yajun Guo 《Cancer immunology, immunotherapy : CII》2010,59(3):355-366
Osteopontin (OPN) has been implicated as an important mediator of breast cancer progression and metastasis and has been investigated for use as a potential therapeutic target in the treatment of breast cancer. However, the in vivo antitumor effect of anti-OPN antibodies on breast cancer has not been reported. In this study, a mouse anti-human OPN antibody (1A12) was humanized by complementarity-determining region grafting method based on computer-assisted molecular modeling. A humanized version of 1A12, denoted as hu1A12, was shown to possess affinity comparable to that of its parental antibody. The ability of hu1A12 to inhibit cell migration, adhesion, invasion and colony formation was assessed in a highly metastatic human breast cancer cell line MDA-MB-435S. The results indicated that hu1A12 was effective in inhibiting the cell adhesion, migration, invasion and colony formation of MDA-MB-435S cells in vitro. hu1A12 also showed significant efficacy in suppressing primary tumor growth and spontaneous metastasis in a mouse lung metastasis model of human breast cancer. The specific epitope recognized by hu1A12 was identified to be 212NAPSD216, adjacent to the calcium binding domain of OPN. Our data strongly support that OPN is a potential target for the antibody-based therapies of breast cancer. The humanized anti-OPN antibody hu1A12 may be a promising therapeutic agent for the treatment of human breast cancer. 相似文献