Deregulation of epidermal growth factor receptor (EGFR) signaling has been correlated with the development of a variety of human carcinomas. EGF-induced receptor dimerization and consequent trans- ...auto-phosphorylation are among the earliest events in signal transduction. Binding of EGF is thought to induce a conformational change that consequently unfolds an ectodomain loop required for dimerization indirectly. It may also induce important allosteric changes in the cytoplasmic domain. Despite extensive knowledge on the physiological activation of EGFR, the effect of targeted therapies on receptor conformation is not known and this particular aspect of receptor function, which can potentially be influenced by drug treatment, may in part explain the heterogeneous clinical response among cancer patients. Here, we used Förster resonance energy transfer/fluorescence lifetime imaging microscopy (FRET/FLIM) combined with two-color single-molecule tracking to study the effect of ATP-competitive small molecule tyrosine kinase inhibitors (TKIs) and phosphatase-based manipulation of EGFR phosphorylation on live cells. The distribution of dimer on-times was fitted to a monoexponential to extract dimer off-rates (koff). Our data show that pretreatment with gefitinib (active conformation binder) stabilizes the EGFR ligand-bound homodimer. Overexpression of EGFR-specific DEP-1 phosphatase was also found to have a stabilizing effect on the homodimer. No significant difference in the koff of the dimer could be detected when an anti-EGFR antibody (425 Snap single-chain variable fragment) that allows for dimerization of ligand-bound receptors, but not phosphorylation, was used. These results suggest that both the conformation of the extracellular domain and phosphorylation status of the receptor are involved in modulating the stability of the dimer. The relative fractions of these two EGFR subpopulations (interacting versus free) were obtained by a fractional-intensity analysis of ensemble FRET/FLIM images. Our combined imaging approach showed that both the fraction and affinity (surrogate of conformation at a single-molecule level) increased after gefitinib pretreatment or DEP-1 phosphatase overexpression. Using an EGFR mutation (I706Q, V948R) that perturbs the ability of EGFR to dimerize intracellularly, we showed that a modest drug-induced increase in the fraction/stability of the EGFR homodimer may have a significant biological impact on the tumor cell’s proliferation potential.
Antibody-based diagnostic and therapeutic agents play a substantial role in medicine, especially in cancer management. A variety of chemical, genetic and enzymatic site-specific conjugation methods ...have been developed for equipping antibodies with effector molecules to generate homogeneous antibody conjugates with tailored properties. However, most of these methods are relatively complicated and expensive and require several reaction steps. Self-labeling proteins such as the SNAP-tag are an innovative solution for addressing these challenges. The SNAP-tag is a modified version of the human DNA repair enzyme alkylguanine-DNA alkyltransferase (AGT), which reacts specifically with O(6)-benzylguanine (BG)-modified molecules via irreversible transfer of an alkyl group to a cysteine residue. It provides a simple, controlled and robust site-specific method for labeling antibodies with different synthetic small effector molecules. Fusing a SNAP-tag to recombinant antibodies allows efficient conjugation of BG-containing substrates by autocatalytic, irreversible transfer of the alkyl group to a cysteine residue in the enzyme's active site under physiological conditions and with a 1:1 stoichiometry. This protocol describes how to generate site-specific SNAP-tag single-chain antibody fragment (scFv) conjugates with different types of BG-modified effector molecules. A specific example is included for the design and production of an scFv-photosensitizer conjugate and its characterization as an immuno-theranostic agent. This protocol includes DNA sequences encoding scFV-SNAP-tag fusion proteins and outlines strategies for expression, purification and testing of the resulting scFv-SNAP-tag-based immuno-conjugates. All experiments can be performed by a graduate-level researcher with basic molecular biology skills within an 8-week time frame.
Hexahistidine tags (His-tags), incorporated into recombinant proteins to facilitate purification using metal-affinity chromatography, are useful binding sites for radiolabeling with 99mTc(CO)3+ and ...188Re(CO)3+ for molecular imaging and radionuclide therapy. Labeling efficiencies vary unpredictably, and the method is therefore not universally useful. To overcome this, we have made quantitative comparisons of radiolabeling of a bespoke Celluspots array library of 382 His-tag-containing peptide sequences with 99mTc(CO)3+ and 188Re(CO)3+ to identify key features that enhance labeling. A selected sequence with 10-fold enhanced labeling efficiency compared to the most effective literature-reported sequences was incorporated into an exemplar protein and compared biologically with non-optimized analogues, in vitro and in vivo. Optimal labeling with either 99mTc(CO)3+ or 188Re(CO)3+ required six consecutive His residues in the protein sequence, surrounded by several positively charged residues (Arg or Lys), and the presence of phosphate in the buffer. Cys or Met residues in the sequence were beneficial, to a lesser extent. Negatively charged residues were deleterious to labeling. His-tags with adjacent positively charged residues could be labeled as much as 40 times more efficiently than those with adjacent negatively charged residues. 31P NMR of Re(CO)3(H2O)3+ and electrophoresis of solutions of 99mTc(CO)3(H2O)3+ suggest that phosphate bridges form between cationic residues and the cationic metal synthon during labeling. The trial optimized protein, a scFv targeted to the PSMA antigen expressed in prostate cancer, was readily labeled in >95% radiochemical yield, without the need for subsequent purification. Labeling occurred more quickly and to higher specific activity than comparable non-optimized proteins, while retaining specific binding to PSMA and prostate cancer in vivo. Thus, optimized His-tags greatly simplify radiolabeling of recombinant proteins making them potentially more widely and economically available for imaging and treating patients.
Development of effective polymer-based nanocarriers for the successful application in cancer therapy still remains a great challenge in current research. In the present study we present a dendritic ...polyglycerol-based multifunctional drug immunoconjugate that specifically targets and kills cancer cell lines expressing epidermal growth factor receptor (EGFR). The nanocarrier was provided with a dendritic core as a multifunctional anchoring point, doxorubicin (Doxo) coupled through a pH-sensitive linker, a fluorescence marker, poly(ethylene glycol), as solubilizing and shielding moiety, and a scFv antibody conjugated through the SNAP-Tag technology. The study provides the proof of principle that SNAP-tag technology can be used to generate drug-carrying nanoparticles efficiently modified with single-chain antibodies to specifically target and destroy cancer cells.
Cancer cells can be killed by photosensitizing agents that induce toxic effects when exposed to nonhazardous light, but this also causes significant damage to surrounding healthy cells. The ...specificity of photodynamic therapy can be increased by conjugating photosensitizing agents to antibodies and antibody fragments that bind specifically to tumor cell antigens. However, standard conjugation reactions produce heterogeneous products whose targeting specificity and spectroscopic properties can be compromised. In this study, we used an antibody fragment (scFv-425) that binds to the epidermal growth factor receptor (EGFR) as a model to investigate the use of SNAP-tag fusions as an improved conjugation strategy. The scFv-425-SNAP-tag fusion protein allowed the specific conjugation of a chlorin e6 photosensitizer modified with O(6)-benzylguanine, generating a homogeneous product that was delivered specifically to EGFR+ cancer cells and resulted in significant, tumor cell-specific cytotoxicity. The impact of our results on the development of photodynamic therapy is discussed.
Recombinant antibodies are promising tools for a wide range of bioanalytical and medical applications. However, the chemical modification of such molecules can be challenging, which limits their ...broader utilization. Here we describe a universal method for the site-specific labeling of antibody fragments and protein ligands by genetically fusing them to an engineered version of the human DNA-repair enzyme O(6)-alkyllguanine DNA alkyltransferase (AGT), known as SNAP-Tag − . Substrates containing O(6)-benzylguanine are covalently bound to the fusion proteins via a stable thioether bond in a rapid and highly specific self-labeling reaction. The coupling is site-directed, allowing the design and synthesis of antibody conjugates with predefined stoichiometry. We cloned a series of ligand SNAP-Tag fusion proteins and expressed them in HEK 293T cells. The antibody/ligand-fusions were characterized by labeling with different fluorophores, labeling with biotin, or by coupling them to fluorescent nanobeads, followed by analysis by flow cytometry and confocal microscopy. All ligands retained their original antigen-binding properties when fused to the SNAP-Tag. The combination of recombinant antibodies or protein ligands with the SNAP-Tag facilitates simple and efficient covalent modification with a broad range of substrates, thus providing a useful and advantageous alternative to existing coupling strategies.
Purpose
The epidermal growth factor receptor (EGFR) is overexpressed in several types of cancer and its inhibition can effectively inhibit tumour progression. The purpose of this study was to design ...an EGFR-specific imaging probe that combines efficient tumour targeting with rapid systemic clearance to facilitate non-invasive assessment of EGFR expression.
Methods
Genetic fusion of a single-chain antibody fragment with the SNAP-tag produced a 48-kDa antibody derivative that can be covalently and site-specifically labelled with substrates containing
0
6
-benzylguanine. The EGFR-specific single-chain variable fragment (scFv) fusion protein 425(scFv)SNAP was labelled with the near infrared (NIR) dye BG-747, and its accumulation, specificity and kinetics were monitored using NIR fluorescence imaging in a subcutaneous pancreatic carcinoma xenograft model.
Results
The 425(scFv)SNAP fusion protein accumulates rapidly and specifically at the tumour site. Its small size allows efficient renal clearance and a high tumour to background ratio (TBR) of 33.2 ± 6.3 (
n
= 4) 10 h after injection. Binding of the labelled antibody was efficiently competed with a 20-fold excess of unlabelled probe, resulting in an average TBR of 6 ± 1.35 (
n
= 4), which is similar to that obtained with a non-tumour-specific probe (5.44 ± 1.92,
n
= 4). When compared with a full-length antibody against EGFR (cetuximab), 425(scFv)SNAP-747 showed significantly higher TBRs and complete clearance 72 h post-injection.
Conclusion
The 425(scFv)SNAP fusion protein combines rapid and specific targeting of EGFR-positive tumours with a versatile and robust labelling technique that facilitates the attachment of fluorophores for use in optical imaging. The same approach could be used to couple a chelating agent for use in nuclear imaging.
Purpose
Preclinical in vivo analyses of treatment responses are an important prerequisite to evaluate new therapeutics. Molecular in vivo imaging in the far red (FR)/near infra red (NIR) is a ...promising method, as it enables measurements at different time points in individual animals, thereby reducing the number of animals required, while increasing statistical significance. Here, we show the establishment of a method to monitor response to treatment using fluorescent cells, expressing the epidermal growth factor receptor (EGFR), a target already used in therapy.
Methods
We transfected A-431 tumour cells with the far red–emitting protein Katushka (Kat2), resulting in strong fluorescence allowing for the monitoring of tumour growth when implanted in BALB/c
nu/nu
mice with a CRi Maestro in vivo imager. We targeted A-431 cells with a previously reported immunotoxin (IT), consisting of the anti-EGFR antibody single-chain variable fragment (scFv) 425, fused to
Pseudomonas
aeruginosa
Exotoxin A’ (ETA’). In addition, EGFR expression was verified using the 425(scFv) conjugated to a NIR dye BG-747 through a SNAP-tag linker.
Results
The results show the feasibility to evaluate response to treatment in vivo by FR imaging, while at the same location detecting EGFR expression. Treatment with 425(scFv)-ETA’ resulted in decelerated tumour growth, while not affecting the overall health of the animals. This is in contrast to treatment with Doxorubicin, which, although decreasing the tumour size, resulted in poor health.
Conclusions
We developed a novel method to non-invasively determine treatment responses by in vivo imaging of multiple parameters which showed the efficacy of 425(scFv)-ETA’.
Complement activation is a recognised mediator of myocardial ischaemia-reperfusion-injury (IRI) and cardiomyocytes are a known source of complement proteins including the central component C3, whose ...activation products can mediate tissue inflammation, cell death and profibrotic signalling. We investigated the potential to detect and quantify the stable covalently bound product C3d by external body imaging, as a marker of complement activation in heart muscle in a murine model of myocardial IRI. We used single-photon-emission-computed-tomography (SPECT) in conjunction with
Technecium-labelled recombinant complement receptor 2 (
Tc-rCR2), which specifically detects C3d at the site of complement activation. Compared to control imaging with an inactive CR2 mutant (
Tc-K41E CR2) or an irrelevant protein (
Tc-PSMA) or using
Tc-rCR2 in C3-deficient mice, the use of
Tc-rCR2 in complement-intact mice gave specific uptake in the reperfused myocardium. The heart to skeletal muscle ratio of
Tc-rCR2 was significantly higher than in the three control groups. Histological analysis confirmed specific uptake of
Tc-rCR2. Following therapeutic inhibition of complement C3 activation, we found reduced myocardial uptake of
Tc-rCR2. We conclude, therefore that
Tc-rCR2 imaging can be used for non-invasive detection of activated complement and in future could be exploited to quantify the severity of myocardial damage due to complement activation.
Background
Sensitive and specific detection of nodal status, sites of metastases and low-volume recurrent disease could greatly improve management of patients with advanced prostate cancer. ...Prostate-specific membrane antigen (PSMA) is a well-established marker for prostate carcinoma with increased levels of expression in high-grade, hormone-refractory and metastatic disease. The monoclonal antibody (mAb) J591 is directed against an extracellular epitope of PSMA and has been shown to efficiently target disseminated disease including metastases in lymph nodes and bone. Its use as a diagnostic imaging agent however is limited due to its slow pharmacokinetics. In this study a diabody derived from mAb J591 was developed as a single photon emission computed tomography (SPECT) tracer with improved pharmacokinetics for the detection of PSMA expression in prostate cancer.
Methods
A diabody in V
H
-V
L
orientation and with a C-terminal cysteine was expressed in HEK293T cells and purified by a combination of metal ion affinity and size exclusion chromatography. Specificity and affinity were determined in cell binding studies. For SPECT imaging, the diabody was site-specifically labelled with
99m
Tc(CO)
3
+
via the C-terminal His tag and evaluated in a subcutaneous DU145/DU145-PSMA prostate carcinoma xenograft model.
Results
J591C diabody binds to PSMA-expressing cells with low nanomolar affinity (3.3 ± 0.2 nM). SPECT studies allowed imaging of tumour xenografts with high contrast from 4 h post injection (p.i.).
Ex vivo
biodistribution studies showed peak tumour uptake of the tracer of 12.1% ± 1.7% injected dose (ID)/g at 8 h p.i. with a tumour to blood ratio of 8.0. Uptake in PSMA-negative tumours was significantly lower with 6.3% ± 0.5% at 8 h p.i. (
p
< 0.001).
Conclusion
The presented diabody has favourable properties required to warrant its further development for antibody-based imaging of PSMA expression in prostate cancer, including PSMA-specific uptake, favourable pharmacokinetics compared to the parental antibody and efficient site-specific radiolabelling with
99m
Tc.
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