Biodistribution and tumor imaging of an anti-CEA single-chain antibody-albumin fusion protein

Albumin fusion proteins have demonstrated the ability to prolong the in vivo half-life of small therapeutic proteins/peptides in the circulation and thereby potentially increase their therapeutic efficacy. To evaluate if this format can be employed for antibody-based imaging, an anticarcinoembryonic antigen (CEA) single-chain antibody(scFv)-albumin fusion protein was designed, expressed and radiolabeled for biodistribution and imaging studies in athymic mice bearing human colorectal carcinoma LS-174T xenografts. The [125 I]-T84.66 fusion protein demonstrated rapid tumor uptake of 12.3% injected dose per gram (ID/g) at 4 h that reached a plateau of 22.7% ID/g by 18 h. This was a dramatic increase in tumor uptake compared to 4.9% ID/g for the scFv alone. The radiometal [111 In]-labeled version resulted in higher tumor uptake, 37.2% ID/g at 18 h, which persisted at the tumor site with tumor: blood ratios reaching 18:1 and with normal tissues showing limited uptake. Based on these favorable imaging properties, a pilot [64 Cu]-positron emission tomography imaging study was performed with promising results. The anti-CEA T84.66 scFv-albumin fusion protein demonstrates highly specific tumor uptake that is comparable to cognate recombinant antibody fragments. The radiometal-labeled version, which shows lower normal tissue accumulation than these recombinant antibodies, provides a promising and novel platform for antibody-based imaging agents.     

Interaction of a monoclonal antibody against hEGF with a receptor site for EGF

Epidermal growth factor (EGF) has been detected by radioimmunoassay (RIA) in different body fluids such as serum, amniotic fluid, and urine. Human tumor tissues with EGF receptors (EGF-Rc) may be saturated with EGF, which may be of prognostic value. An RIA was envisaged to measure human epidermal growth factor (hEGF) levels using EGF-Rc as capture agent and a monoclonal antibody anti-hEGF (MAb anti-hEGF) labeled with ¹²⁵Iodine as a marker for this binding. The purpose of this work was to study the feasibility of MAb anti-hEGF to detect the receptor binding sites and to investigate the interaction between MAb anti-hEGF and the EGF-Rc. Various binding experiments were performed to study possible interference and interactions in the complex MAb anti-hEGF and the receptor. Affinity constants were determined by means of Scatchard plot analysis to interpret the complex stability challenged with other compounds for a better understanding of the interaction process. Binding constants were of the same order for all the ligands tested separately involving the EGF-Rc, but were significantly higher ( t=15.7, p < 0.05) for hEGF in its binding to MAb anti-hEGF. It was possible with equilibrium studies and competition experiments to evaluate the interaction of EGF and MAb anti-hEGF with the EGF receptor. This observation makes the MAb anti-hEGF a potential tracer for the quantitation of receptors in vitro, and possibly for the detection of membrane receptors on tumor cells in vivo.     

Scintigraphic imaging of P-glycoprotein expression with a radiolabelled antibody

Purpose P-glycoprotein (P-gp) is a membrane efflux pump protein that is involved in multidrug resistance (MDR). Tumour cells with high P-gp expression show poor response to cancer treatment with several chemotherapeutics. In vivo targeting and visualisation of P-gp expression would allow MDR to be evaluated non-invasively prior to treatment. The aim of this study was to investigate the feasibility of visualising P-gp expression in tumours using a monoclonal anti-P-gp antibody, 15D3.Methods Nude BALB/c mice with subcutaneously growing human uterine sarcoma cell tumours with either high (MES-SA/D×5 1977) or low (MES-SA 1976) P-gp expression were used. When tumours were 0.2–0.4 g, mice received ¹³¹I-15D3 or ¹¹¹In-DTPA-15D3 monoclonal anti-P-gp antibody intravenously. Images were acquired up to 3 days p.i. and radioactivity concentration in various tissues was determined after euthanisation of the animals.Results The images demonstrated that radioactivity accumulated to a higher concentration in high P-gp expressing tumours than in the low P-gp expressing MES-SA 1976 tumour. Furthermore, visualisation of the P-gp expressing tumours was superior with ¹¹¹In-DTPA-15D3 than with ¹³¹I-15D3. After injection of ¹¹¹In-DTPA-15D3, the high P-gp expressing MES-SA/D×5 1977 tumours were clearly visualised at 3 days p.i. The biodistribution data indicated that radioactivity concentration in the high P-gp expressing tumours was higher than in the tumours with low P-gp expression (20.78±1.42 %ID/g for MES-SA/Dx5 1977 tumours and 8.39±3.78 %ID/g for MES-SA 1976 tumours for ¹¹¹In-DTPA-15D3).Conclusion The ¹¹¹In-labelled monoclonal anti-P-gp antibody clearly visualised P-gp expression in a human uterine sarcoma tumour in nude mice.     

Pretargeted radioimmunotherapy of mesothelin-expressing cancer using a tetravalent single-chain Fv-streptavidin fusion protein.

Mesothelin is a glycoprotein that is overexpressed in several human tumors, including mesotheliomas and ovarian cancers, and has been identified as a potential target for therapy. We evaluated the biodistribution and tumor-targeting ability of an antimesothelin tetravalent single-chain Fv-streptavidin fusion protein (SS1scFvSA) in mice. METHODS: SS1scFvSA was labeled with 125I or 111In for evaluation of internalization in vitro and for optimization of its biodistribution. The A431-K5 mesothelin transfected cell line was used as the target. We used a 3-step pretargeting approach consisting of injections of (i) SS1scFvSA, followed 20 h later by (ii) a synthetic clearing agent, and (iii) 4 h later, radiolabeled (111In, 88Y/90Y, or 177Lu) 1,4,7,10-tetraazacyclododecane-N,N',N',N'-tetraacetic acid (DOTA)-biotin. To optimize the tumor uptake, the effect of the specific activity of 111In-DOTA-biotin was evaluated. RESULTS: Approximately 60% of SS1sc FvSA internalized within 6 h. The optimal dose of SS1scFvSA for pretargeting was 600 microg. Decreasing the specific activity of DOTA-biotin by administering 0.1-5 microg of DOTA-biotin resulted in tumor uptake decreasing from 31.8 to 5.5 %ID/g (percentage injected dose per gram) at 2 h. Pretargeted therapy of A431-K5 tumor with 90Y doses of 11.1-32.4 MBq resulted in a dose-dependent tumor response. With 32.4 MBq, 86% of mice survived tumor free for 110 d. All nontreated mice died, with a median survival of 16 d. CONCLUSION: SS1scFvSA localized in the mesothelin-expressing tumor, resulting in a high accumulation of radiolabeled DOTA-biotin. The specific activity of DOTA-biotin had a significant effect on its tumor uptake. Therapeutic tumor doses were obtained without dose-limiting toxicity.     

抗癌胚抗原单链抗体与核心链霉亲和素融合蛋白在荷瘤裸鼠体内的预定位显像

目的：研究^153Sm-生物素和抗癌胚抗原单链抗体与核心链霉亲和素融合蛋白（CEA ScFv-core-streptavidin）在荷人直肠癌裸鼠体内的两步法预定位显像和体内分布。方法：对23只荷人结直肠癌裸鼠腹腔注射CEA ScFv-core-streptavidin进行预定位，24h后腹腔注射^153Sm-生物素，其中20只于1、4、8和24h进行体内分布研究，余3只于8和24h进行定位显像。结果：在荷人结直肠癌裸鼠腹腔注射CEA ScFv-core-streptavidin预定位后24h，注射^153Sm-生物素后1h，肿瘤/血比值为0.49，4和8h达1.21和1.56，24h达最高，为3.09。裸鼠定位显像示，8h肿瘤部位放射性明显浓聚，24h本底明显降低，肿瘤呈放射性“热“区。结论：^153Sm-生物素和CEA ScFv-core-streptavidin预定位显像能提高靶/非靶比值，缩短显像时间，改善图像质量。     

EGF receptor targeted tumor imaging with biotin-PEG-EGF linked to 99mTc-HYNIC labeled avidin and streptavidin

IntroductionAs direct radiolabeled peptides suffer limitations for in vivo imaging, we investigated the usefulness of radioloabeled avidin and streptavidin as cores to link peptide ligands for targeted tumor imaging.MethodsHuman epidermal growth factor (EGF) was site specifically conjugated with a single PEG-biotin molecule and linked to ^99mTc-HYNIC labeled avidin-FITC (Av) or streptavidin-Cy5.5 (Sav). Receptor targeting was verified in vitro, and in vivo pharmacokinetic and biodistribution profiles were studied in normal mice. Scintigraphic imaging was performed in MDA-MB-468 breast tumor xenografted nude mice.ResultsWhereas both ^99mTc-Av-EGF and ^99mTc-Sav-EGF retained receptor-specific binding in vitro, the two probes substantially diverged in pharmacokinetic and biodistribution behavior in vivo. ^99mTc-Av-EGF was rapidly eliminated from the circulation with a T1/2 of 4.3 min, and showed intense hepatic accumulation but poor tumor uptake (0.6%ID/gm at 4 h). ^99mTc-Sav-EGF displayed favorable in vivo profiles of longer circulation (T1/2β, 51.5 min) and lower nonspecific uptake that resulted in higher tumor uptake (3.8 %ID/gm) and clear tumor visualization at 15 h.Conclusion^99mTc-HYNIC labeled streptavidin linked with growth factor peptides may be useful as a protein-ligand complex for targeted imaging of tumor receptors.     

抗NI-35重组单链抗体表达载体的构建与融合表达

重新改计并人工合成抗NI-35重组单链抗体(anti-NI-35-sefv)的cDNA克隆,构建其表达载体,在原核系统中实现初步表达。认为抗NI-35重组单链抗体(IN-I-seFv)基因表达载体的构建和表达,为深入研究anli-NT-35-seFv应用于神经系统损伤和修复奠定了基础。     

靶向表皮生长因子受体单克隆抗体预定位免疫PET显像的实验研究

目的:采用预定位技术在表皮生长因子受体(EGFR)阳性/阴性荷瘤鼠中探索西妥昔单克隆抗体(简称单抗;Cetuximab)靶向EGFR免疫PET显像的可行性。方法:以反式环辛烯(TCO)- N-羟基琥珀酰亚胺(NHS)修饰Cetuximab获得Cetuximab-TCO。以2,2′-((6-氨基-1-(4,7...     

PET imaging of CD105/endoglin expression with a 61/64Cu-labeled Fab antibody fragment

Purpose

The goal of this study was to generate and characterize the Fab fragment of TRC105, a monoclonal antibody that binds with high affinity to human and murine CD105 (i.e., endoglin), and investigate its potential for PET imaging of tumor angiogenesis in a small-animal model after ^61/64Cu labeling.

Methods

TRC105-Fab was generated by enzymatic papain digestion. The integrity and CD105 binding affinity of TRC105-Fab was evaluated before NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) conjugation and ^61/64Cu labeling. Serial PET imaging and biodistribution studies were carried out in the syngeneic 4T1 murine breast cancer model to quantify tumor targeting efficiency and normal organ distribution of ^61/64Cu-NOTA-TRC105-Fab. Blocking studies with unlabeled TRC105 were performed to confirm CD105 specificity of the tracer in vivo. Immunofluorescence staining was also conducted to correlate tracer uptake in the tumor and normal tissues with CD105 expression.

Results

TRC105-Fab was produced with high purity through papain digestion of TRC105, as confirmed by SDS-PAGE, HPLC analysis, and mass spectrometry. ^61/64Cu labeling of NOTA-TRC105-Fab was achieved with about 50 % yield (specific activity about 44 GBq/μmol). PET imaging revealed rapid uptake of ⁶⁴Cu-NOTA-TRC105-Fab in the 4T1 tumor (3.6?±?0.4, 4.2?±?0.5, 4.9?±?0.3, 4.4?±?0.7, and 4.6?±?0.8 %ID/g at 0.5, 2, 5, 16, and 24 h after injection, respectively; n?=?4). Since tumor uptake peaked soon after tracer injection, ⁶¹Cu-labeled TRC105-Fab was also able to provide tumor contrast at 3 and 8 h after injection. CD105 specificity of the tracer was confirmed with blocking studies and histological examination.

Conclusion

We report PET imaging of CD105 expression using ^61/64Cu-NOTA-TRC105-Fab, which exhibited prominent and target-specific uptake in the 4T1 tumor. The use of a Fab fragment led to much faster tumor uptake (which peaked at a few hours after tracer injection) compared to radiolabeled intact antibody, which may be translated into same-day immunoPET imaging for clinical investigation.     

胎儿皮肤EGF、EGFR基因表达特征及与汗腺形成的关系研究

目的 探讨在不同发育胎龄的胎儿皮肤中,表皮细胞生长因子（epidermal growth factor,EGF）和其受体EGFR基因表达特征及其与附件形成的关系。方法 根据病理学技术,检测不同发育阶段胎儿皮肤的结构特征,并提取不同胎龄（EGA12-32周）胎儿皮肤的总RNA,用RT-PCR方法检测EGF、EGFR基因在不同胎龄标本中的表达变化。结果 在发育早期的胎儿皮肤中,EGF和EGFR基因表达较弱;随着胎龄的增长,特别在皮肤汗腺诱导形成阶段,此两种基因表达量增加最明显;在胎儿皮肤发育后期,此2种基因表达量增加缓慢。结论 EGF与其受体EGFR基因在不同胎龄胎儿皮肤中的表达方式及结合后引起的信号通路对胎儿皮肤汗腺的发生和结构的形成以及皮肤生理功能的维持具有重要意义。     

Small-animal PET imaging of human epidermal growth factor receptor type 2 expression with site-specific 18F-labeled protein scaffold molecules.

Human epidermal growth factor receptor type 2 (HER2) is a well-established tumor biomarker that is overexpressed in a wide variety of cancers and that serves as a molecular target for therapeutic intervention. HER2 also serves as a prognostic indicator of patient survival and as a predictive marker of the response to antineoplastic therapy. The development of (18)F-labeled biomolecules for PET imaging of HER2 (HER2 PET) is very important because it may provide a powerful tool for the early detection of HER2-positive tumor recurrence and for the monitoring of HER2-based tumor treatment. METHODS: In this study, anti-HER2 monomeric and dimeric protein scaffold molecules [Z(HER2:477) and (Z(HER2:477))(2), respectively] were radiofluorinated at a reasonable radiochemical yield (13%-18%) by use of site-specific oxime chemistry. The resulting radiofluorinated protein scaffold molecules were then evaluated as potential molecular probes for small-animal HER2 PET by use of a SKOV3 tumor-bearing mouse model. RESULTS: The 4-(18)F-fluorobenzaldehyde conjugated aminooxy-protein scaffolds [(18)F-N-(4-fluorobenzylidene)oxime (FBO)-Z(HER2:477) and (18)F-FBO-(Z(HER2:477))(2)] both displayed specific HER2-binding ability in vitro. Biodistribution and small-animal PET imaging studies further revealed that (18)F-FBO-Z(HER2:477) showed rapid and high SKOV3 tumor accumulation and quick clearance from normal tissues, whereas (18)F-FBO-(Z(HER2:477))(2) showed poor in vivo performance (low tumor uptake and tumor-to-normal tissue ratios). The specificity of (18)F-FBO-Z(HER2:477) for SKOV3 tumors was confirmed by its lower uptake on pretreatment of tumor-bearing mice with the HER2-targeting agents Z(HER2) and trastuzumab. Moreover, small-animal PET imaging studies revealed that (18)F-FBO-Z(HER2:477) produced higher-quality tumor imaging than (18)F-FBO-(Z(HER2:477))(2). (18)F-FBO-Z(HER2:477) could clearly identify HER2-positive tumors with good contrast. CONCLUSION: Overall, these data demonstrate that (18)F-FBO-Z(HER2:477) is a promising PET probe for imaging HER2 expression in living mice. It has a high potential for translation to clinical applications. The radiofluorination method developed can also be used as a general strategy for the site-specific labeling of other proteins with (18)F. The protein scaffold molecules used here are attractive for the further development of PET probes for other molecular targets.     

霍乱毒素B亚基融合蛋白表达载体的构建

采用ＰＣＲ技术，将霍乱弧菌ＣＴ－Ｂ基因的终止密码子定点突变并引入一个ＥｃｏＲ Ｉ位点，然后，与人工合成的接头连接，构建成了新的融合蛋白表达载体。在ＣＴ－Ｂ’的接头上有四个单一的限制性酶切位点，在其中任何限制性位点上插入处源基因序列后，其转录产物中都有终止密码子。     

Development of an immunoassay for fluvoxamine detection using a recombinant single-chain variable fragment antibody

In forensic toxicology, immunoassays for drug screening are widely used because of the simple test procedures and instantaneous outcome of results. However, commercial immunoassay products are available for only a limited number of drugs. Preparation of antidrug antibodies is a crucial, but time-consuming in creating an immunoassay system. In this study, we focused on the application of a single-chain variable fragment (scFv) antibody for drug screening and developed a fluvoxamine (FLV) detection system for indirect competitive enzyme-linked immunosorbent assay (icELISA) using the scFv against FLV. To clarify the influence of domain order on the scFv binding activities, we prepared two kinds of scFv with different domain orders (HL, V_H-linker-V_L, and LH, V_L-linker-V_H) and examined their kinetic parameters against FLV. The scFvs showed sufficient FLV binding activities (K _D = 3.8 and 7.6 nM), and the HL scFv was slightly more favorable for FLV binding than the LH scFv. The developed icELISA using the HL scFv could detect FLV in the range of 10–200 ng/mL, and the scFv has no cross-reactivity below 100 μM except for chlorpromazine and imipramine. We also quantified the plasma FLV concentrations in forensic autopsy cases, and the results showed that this method could be applied effectively for FLV quantification without the need for extraction steps. Although recombinant antibodies against small molecule drugs for immunoassays have not yet been commonly used, we can predict that they could be a powerful tool to screen drugs in the near future, because of their advantages.     

Hexa-histidine tag as a novel alternative for one-step direct labelling of a single-chain Fv antibody fragment with 99m Tc

From genetic material of hybridoma cells, we have generated a recombinant single-chain antibody fragment (scFv antibody) specific to carcinoembryonic antigen (CEA), which can substitute an intact murine monoclonal immunoglobulin G1 (IgG1) antibody, also developed by our group, and used in clinical practice for many years. In this paper, we examine a novel one-step method for direct 99mTc labelling of a recombinant anti-CEA scFv fragment through a C-terminal peptide tag containing a six-histidine sequence. This C-terminal peptide tag does not affect antigen binding, and was employed as a strategy for the one-step method of direct 99mTc labelling of a recombinant antibody fragment, based on the criteria of Zamora and Rhodes (Zamora PO, Rhodes BA. Imidazoles as well as thiolates in proteins bind technetium-99m. Bioconj Chem 1992; 3: 493-498). This is a novel technique for the rapid labelling of molecules, suitable for in vivo trials. The method yields >95% labelling efficiency without major effects on biological or in vitro stability.