Veerle Kersemans

Cell Penetrating Peptides for In Vivo Molecular Imaging Applications

Cell penetrating peptides (CPPs) are a relatively new class of peptides that have the promising capability to cross cell membranes. While details remain to be resolved, various non-receptor-mediated endocytic pathways likely contribute most to the cell penetrating properties of these peptides. CPPs have been used to deliver many different cargos - ranging from radionuclides and other peptides to antibodies and nanoparticles - into cells. Besides many different drug delivery applications, CPPs have also seen a limited use in molecular imaging. Molecular imaging of intracellular and intranuclear targets, by techniques such as SPECT, PET, optical imaging, and MRI, relies heavily on the delivery of contrast agents to the cytoplasm and/or nuclei of the target tissue. Therefore, the number of applications in molecular imaging of intracellular targets has remained relatively low, because of the effective barrier presented by the cell membrane. One of the key strategies to overcome this challenge is the introduction of membrane-transducing peptides in the design of new contrast agents. This review presents an overview of the literature on CPPs, focusing on their use for molecular imaging. Applications using proteins and peptides, DNA/RNA, and CPP-loaded cells as the imaging agents will be looked at. Moreover, the difficulties and pitfalls regarding the use of CPPs in molecular imaging will be discussed.

Drug-Resistant AML Cells and Primary AML Specimens Are Killed by 111In-Anti-CD33 Monoclonal Antibodies Modified with Nuclear Localizing Peptide Sequences

Multidrug resistance (MDR) is a major challenge to the successful treatment of acute myeloid leukemia (AML). Our purpose was to determine whether 111In-HuM195 anti-CD33 antibodies modified with peptides harboring nuclear localizing sequences (NLS) could kill drug-resistant AML cell lines and primary AML patient specimens expressing MDR transporters through the emission of Auger electrons. Methods: HuM195, M195, and irrelevant mouse IgG (mIgG) were conjugated to 10 ± 3 NLS peptides and then labeled with 111In by diethylenetriaminepentaacetic acid substitution to a specific activity of 1–8 MBq/μg. The binding affinity of HuM195 and M195 was determined for HL-60 and mitoxantrone-resistant HL-60-MX-1 cells. Nuclear localization of 111In-NLS-HuM195, 111In-NLS-M195, 111In-HuM195, and 111In-M195 was measured by subcellular fractionation. The antiproliferative effects of 111In-NLS-HuM195, 111In-NLS-M195, 111In-HuM195, and 111In-M195 (2.5–250 kBq/well) on HL-60 and HL-60-MX-1 were studied using the WST-1 assay. Clonogenic survival of HL-60 and HL-60-MX-1 leukemic cells and 10 primary AML specimens with MDR phenotype (assessed by flow cytometry) was determined after exposure for 3 h at 37°C to 2.5–250 mBq/cell of 111In-NLS-HuM195, 111In-HuM195, or 111In-NLS-mIgG. Clonogenic survival versus the amount of radioactivity incubated with the cells (mBq/cell) was plotted, and the mean lethal amount of radioactivity and the lower asymptote of the curve (plateau) were determined. Results: The 111In-labeled anti-CD33 monoclonal antibodies HuM195 and M195 modified with NLS were efficiently routed to the nucleus of HL-60 cells and their mitoxantrone-resistant clone after CD33-mediated internalization. The following are the principal findings of our study: 111In-NLS-HuM195 was more effective at killing HL-60 and HL-60-MX-1 cells than was 111In-HuM195, a strong correlation between the specific activity of the 111In-labeled radioimmunoconjugates and their cytotoxicity toward AML cells existed, and leukemic cells from patients were killed by 111In-NLS-M195 or 111In-M195, but the cytotoxic response among specimens was heterogeneous. Conclusion: NLS conjugation enhanced the nuclear uptake and cytotoxicity of 111In-HuM195 and 111In-M195 toward drug-resistant AML cell lines as well as patient specimens expressing a diversity of MDR phenotypes, including Pgp-170, BCRP1, or MRP1 transporters. Targeted Auger electron radioimmunotherapy using 111In-labeled anti-CD33 monoclonal antibodies modified with NLS may be able to overcome MDR and provide a means of treating chemotherapy-resistant myeloid leukemias in patients.

Peptide and nonpeptide antagonist interaction with constitutively active human AT1 receptors

Wild type human AT(1) receptors (WT-AT(1)) and mutant receptors, in which Asn(111) was replaced by glycine (N111G), alanine (N111A) and serine (N111S), or in which Asp(281) was replaced by alanine (D281A) or in which N111G and D281A replacements were combined, were transiently expressed in CHO-K1 cells. While the biphenyltetrazole compound candesartan dissociated slowly and behaved as an insurmountable antagonist for WT-AT(1), it dissociated swiftly and only produced a rightward shift of the angiotensin Ang II- and -IV dose-response curves for inositol phosphate (IP) accumulation in cells expressing N111G. [3H]candesartan competition binding yielded the same potency order of the related biphenyltetrazoles for WT-AT(1) and mutated receptors, i.e. candesartan>EXP3174>irbesartan>losartan. Affinities were equal for WT-AT(1) and D281A and 40- to 400-fold lower for all Asn(111) mutants. Mutations did not affect the affinity of the peptide antagonist [Sar(1)Ile(8)]Ang II (SARILE). Basal IP accumulation in cells with WT-AT(1) was not affected by any biphenyltetrazole antagonists and was increased by SARILE to 19% of the maximal Ang II stimulation. Basal IP accumulation was higher for cells expressing the Asn(111)-mutated receptors. For N111G, this accumulation was partially inhibited by all the biphenyltetrazoles upon long-term (18hr) exposure. In these cells SARILE produced the same maximal stimulation as Ang II. Asn(111)-mutated AT(1) receptors are thought to mimic the pre-activated state of the wild type receptor and comparing the efficacy and affinity of ligands for such mutated receptors facilitate the distinction of partial (SARILE) and inverse (biphenyltetrazoles) agonists from true antagonists.

I-123/125-labelled 2-iodo-L-phenylalanine and 2-iodo-D-phenylalanine : comparative uptake in various tumour types and biodistribution in mice

PurposeIn vitro in the R1M cell model and in vivo in the R1M tumour-bearing athymic model, both [123I]-2-iodo-L-phenylalanine and [123I]-2-iodo-D-phenylalanine have shown promising results as tumour diagnostic agents for SPECT. In order to compare these two amino acid analogues and to examine whether the observed characteristics could be generalised, both isomers were evaluated in various tumour models.MethodsTransport type characterisation in vitro in A549, A2058, C6, C32, Capan2, EF43fgf4, HT29 and R1M cells with [123I]-2-iodo-L-phenylalanine was performed using the method described by Shotwell et al. Subsequently, [123I]-2-iodo-L-phenylalanine and [123I]-2-iodo-D-phenylalanine tumour uptake and biodistribution were evaluated using dynamic planar imaging and/or dissection in A549, A2058, C6, C32, Capan2, EF43fgf4, HT29 and R1M inoculated athymic mice. Two-compartment blood modelling of the imaging results was performed.ResultsIn vitro testing demonstrated that [123I]-2-iodo-L-phenylalanine was transported in all tumour cell lines by LAT1. In all tumour models, the two amino acid analogues showed the same general biodistribution characteristics: high and specific tumour uptake and renal tracer clearance. Two-compartment modelling revealed that the D-isomer showed a faster blood clearance together with a faster distribution to the peripheral compartment in comparison with [123I]-2-iodo-L-phenylalanine.Conclusion[123I]-2-iodo-L-phenylalanine and its D-isomer are promising tumour diagnostic agents for dynamic planar imaging. They showed a high and similar uptake in all tested tumours. [123I]-2-iodo-D-phenylalanine showed better tracer characteristics concerning radiation dose to other organs.

111In-Labeled Immunoconjugates (ICs) Bispecific for the Epidermal Growth Factor Receptor (EGFR) and Cyclin-Dependent Kinase Inhibitor, p27Kip1

OBJECTIVE Probing intranuclear proteins in breast cancer (BC) cells by using radiolabeled antibodies is restricted by delivery barriers presented by cell and nuclear membranes. Our aim was to construct immunoconjugates (ICs) bispecific for epidermal growth factor receptors (EGFRs) and the intranuclear cyclin-dependent kinase inhibitor (CDKI) p27(Kip1) modified with nuclear-localizing sequences (NLSs) to facilitate their nuclear uptake following EGFR-mediated internalization. METHODS Bispecific ICs were constructed by first modifying EGF with peptides [GGPKKKRKVGYGCG] harboring NLS from SV-40 large T-antigen (underlined), then conjugating NLS-EGF to anti-p27(Kip1) antibodies through an extended PEO(12)-maleimide linker (Compound 1). Analogous ICs were constructed by using mouse IgG (Compound 2), a disrupted NLS (Compound 3) or omission of the EGF moiety (Compound 4). Binding to EGFR on MDA-MB-468, H2N, or HR2 BC cells and to p27(Kip1) in HELA cell lysate was measured. Internalization and nuclear importation were evaluated. Retention of the ICs in H2N or trastuzumab (Herceptin)-resistant HR2 cells exposed to trastuzumab to modulate p27(Kip1) expression with/without coexposure to the IGF-1 receptor kinase inhibitor, AG1024, was determined. RESULTS Trastuzumab (10 microg/mL) unexpectedly decreased p27(Kip1) expression by 1.7-2.4-fold in H2N or HR2 cells. Conjugation of EGF to anti-p27(Kip1) antibodies (Compound 1) decreased the binding affinity of the ICs 7-fold toward EGFR and p27(Kip1). All ICs bound EGFR on MDA-MB-468 cells except Compound 4. Compound 1 was internalized into H2N cells over 48 hours and Compound 2 exhibited 1.6-fold greater nuclear importation in H2N or MDA-MB-468 cells than Compound 3. There was a significantly lower retention of Compound 1 in H2N cells exposed to trastuzumab, compared to unexposed cells, corresponding to decreased p27(Kip1), but in HR2 cells, diminished retention was observed only when these cells were coexposed to trastuzumab and AG]024. CONCLUSION We conclude that (111)In-labeled bispecific ICs were constructed that specifically bound EGFR and p27(Kip1). These ICs were internalized into BC cells expressing EGFR and HER2 and imported into the nucleus. Their decreased retention by cells with trastuzumab-modulated p27(Kip1) suggests that they may be useful for probing this CDKI by imaging.

Cd11b+ myeloid cells support hepatic metastasis through down‐regulation of angiopoietin‐like 7 in cancer cells

Myeloid cells are known to mediate metastatic progression. Here, we attempted to elucidate the mechanisms underlying these effects by identifying gene expression alterations in cancer cells forming hepatic metastases after myeloid cell depletion. Hepatic metastases are heavily infiltrated by CD11b+ myeloid cells. We established hepatic metastases in transgenic CD11b‐diphtheria toxin receptor mice by intrasplenic injection of MC38 colon and Lewis lung carcinoma cells before depleting myeloid cells with diphtheria toxin. Myeloid cell depletion inhibited metastatic growth with a marked diminishment of tumor vasculature. Expression of ANGPTL7 (angiopoietin‐like 7), a protein not previously linked to metastasis, was highly up‐regulated in cancer cells after myeloid cell depletion. This effect was duplicated in tissue culture, where coculture of cancer cells with tumor‐conditioned myeloid cells from liver metastases or myeloid cell conditioned media down‐regulated ANGPTL7 expression. Analogous to myeloid cell depletion, overexpression of ANGPTL7 in cancer cells significantly reduced hepatic metastasis formation and angiogenesis. We found that ANGPTL7 itself has strong antiangiogenic effects in vitro. Furthermore, analysis of The Cancer Genome Atlas colorectal and breast cancer data sets revealed striking ANGPTL7 underexpression in cancerous compared to normal tissues. Also, ANGPTL7 was down‐regulated in metastatic liver colonies of colorectal cancer patients compared to their adjacent liver tissue. Conclusion: Myeloid cells promote liver metastasis by down‐regulating ANGPTL7 expression in cancer cells; our findings implicate ANGPTL7 as a mediator of metastatic progression and a potential target for interference with liver metastases. (Hepatology 2015;62:521–533

ErbB-2 Blockade and Prenyltransferase Inhibition Alter Epidermal Growth Factor and Epidermal Growth Factor Receptor Trafficking and Enhance 111 In-DTPA-hEGF Auger Electron Radiation Therapy

The intracellular distribution of Auger electron–emitting radiopharmaceuticals is a determinant of cytotoxicity. However, the mechanisms by which these agents are routed through the cell are ill understood. The aim of this study was to investigate how trafficking of 111In-labeled human epidermal growth factor (111In-DTPA-hEGF) relates to that of the EGF receptor (EGFR) and whether coadministration of agents that modulate EGFR signaling alters the efficacy of 111In-DTPA-hEGF. Methods: The spatiotemporal interaction between AlexaFluor488-EGF (AF488-EGF) and Cy3-conjugated anti-EGFR antibody (Cy3-anti-EGFR) was studied in the breast cancer cell line MDA-MB-468 using fluorescence resonance energy transfer and 2-photon fluorescence lifetime imaging. 111In internalization and nuclear fractionation assays were performed to investigate the effect of the ErbB-2–blocking antibody trastuzumab and a prenyltransferase inhibitor, L-778,123, on the subcellular localization of 111In-DTPA-hEGF in MDA-MB-468 (1.3 × 106 EGFR per cell; ErbB-2 negative) and 231-H2N (0.2 × 106 EGFR per cell; 0.4 × 105 ErbB-2 per cell) cell lines. The cytotoxicity of 111In-DTPA-hEGF (0–64 nM) plus trastuzumab (0–50 μg/mL) or L-778,123 (0–22.5 μM) was measured using clonogenic assays in a panel of breast cancer cell lines that express different levels of EGFR and ErB-2. Clonogenic survival data were used to calculate combination indices. Tumor growth inhibition was measured in vivo in 231-H2N xenograft–bearing mice treated with 111In-DTPA-hEGF plus trastuzumab or L-788,123. Results: Using fluorescence resonance energy transfer, we showed that EGF interacts with EGFR in the cytoplasm and nucleus after internalization of the ligand–receptor complex in MDA-MB-468 cells. Nuclear localization of 111In-DTPA-hEGF is enhanced by trastuzumab and L-788,123. Trastuzumab and L-788,123 sensitized 231-H2N cells to 111In-DTPA-hEGF. Nuclear localization and cytotoxicity of 111In-DTPA-hEGF were significantly increased in 231-H2N xenografts by cotreatment with L-788,123 (P < 0.0001). Conclusion: The therapeutic efficacy of 111In-DTPA-hEGF is increased through the coadministration of selected molecularly targeted drugs that modulate EGFR signaling and trafficking.

Synthesis, biodistribution and effects of farnesyltransferase inhibitor therapy on tumour uptake in mice of 99mTc labelled epidermal growth factor.

ObjectiveThe goal of this study was to develop a 99mTc labelled human epidermal growth factor (hEGF) for the in-vivo prediction of cancer cell response to farnesyltransferase inhibitor (FTI) therapy. This is based on the observation that internalization of EGF receptors is inhibited by FTIs. MethodsWe describe the radiolabelling of 99mTc-hEGF using the hydrazinonicotinamide (HYNIC) linker. Binding characteristics of 99mTc-HYNIC-hEGF to the EGF receptor are explored using an in-vitro binding assay. Biodistribution data of the compound in mice and tumour uptake in LoVo tumour bearing athymic mice before and after farnesyltransferase inhibitor therapy are presented. ResultsNo colloid formation was observed. Binding parameters and LoVo tumour uptake of 99mTc-HYNIC-hEGF did not differ significantly from directly labelled 123I-hEGF values. However, the biodistribution data of the 99mTc-HYNIC-hEGF showed higher uptake in liver and intestines and decreased stomach uptake compared to its 123I analogue. Eight hours after farnesyltransferase inhibitor therapy with R115777, LoVo tumour uptake of 99mTc-HYNIC-hEGF decreased significantly, as shown using planar gamma scintigraphy (the ratio tumour vs. thigh dropped from 2.54±0.83 to 0.99±0.18). These data confirm the results obtained using 123I-hEGF. ConclusionThese data suggest that 99mTc-HYNIC-hEGF is a promising and selective new radiotracer for in-vivo monitoring of the EGF receptor with SPECT. Moreover, 99mTc-HYNIC-hEGF is a possible tool for early therapy response prediction of farnesyltransferase inhibitors.

18F-Trifluoromethylation of Unmodified Peptides with 5-18F-(Trifluoromethyl)dibenzothiophenium Trifluoromethanesulfonate

The 18F-labeling of 5-(trifluoromethyl)-dibenzothiophenium trifluoromethanesulfonate, commonly referred to as the Umemoto reagent, has been accomplished applying a halogen exchange 18F-fluorination with 18F-fluoride, followed by oxidative cyclization with Oxone and trifluoromethanesulfonic anhydride. This new 18F-reagent allows for the direct chemoselective 18F-labeling of unmodified peptides at the thiol cysteine residue.

Low dose angiostatic treatment counteracts radiotherapy-induced tumor perfusion and enhances the anti-tumor effect.

The extent of tumor oxygenation is an important factor contributing to the efficacy of radiation therapy (RTx). Interestingly, several preclinical studies have shown benefit of combining RTx with drugs that inhibit tumor blood vessel growth, i.e. angiostatic therapy. Recent findings show that proper scheduling of both treatment modalities allows dose reduction of angiostatic drugs without affecting therapeutic efficacy. We found that whilst low dose sunitinib (20 mg/kg/day) did not affect the growth of xenograft HT29 colon carcinoma tumors in nude mice, the combination with either single dose RTx (1x 5Gy) or fractionated RTx (5x 2Gy/week, up to 3 weeks) substantially hampered tumor growth compared to either RTx treatment alone. To better understand the interaction between RTx and low dose angiostatic therapy, we explored the effects of RTx on tumor angiogenesis and tissue perfusion. DCE-MRI analyses revealed that fractionated RTx resulted in enhanced perfusion after two weeks of treatment. This mainly occurred in the center of the tumor and was accompanied by increased tissue viability and decreased hypoxia. These effects were accompanied by increased expression of the pro-angiogenic growth factors VEGF and PlGF. DCE-MRI and contrast enhanced ultrasonography showed that the increase in perfusion and tissue viability was counteracted by low-dose sunitinib. Overall, these data give insight in the dynamics of tumor perfusion during conventional 2 Gy fractionated RTx and provide a rationale to combine low dose angiostatic drugs with RTx both in the palliative as well as in the curative setting.