Nanobody-targeted photodynamic therapy induces significant tumor regression of trastuzumab-resistant HER2-positive breast cancer, after a single treatment session

Rationale: A substantial number of breast cancer patients with an overexpression of the human epidermal growth factor receptor 2 (HER2) have residual disease after neoadjuvant therapy or become resistant to trastuzumab. Photodynamic therapy (PDT) using nanobodies targeted to HER2 is a promising treatment option for these patients. Here we investigate the in vitro and in vivo antitumor efficacy of HER2-targeted nanobody-photosensitizer (PS) conjugate PDT. Methods: Nanobodies targeting HER2 were obtained from phage display selections. Monovalent nanobodies were engineered into a biparatopic construct. The specificity of selected nanobodies was tested in immunofluorescence assays and their affinity was evaluated in binding studies, both performed in a panel of breast cancer cells varying in HER2 expression levels. The selected HER2-targeted nanobodies 1D5 and 1D5-18A12 were conjugated to the photosensitizer IRDye700DX and tested in in vitro PDT assays. Mice bearing orthotopic HCC1954 trastuzumab-resistant tumors with high HER2 expression or MCF-7 tumors with low HER2 expression were intravenously injected with nanobody-PS conjugates. Quantitative fluorescence spectroscopy was performed for the determination of the local pharmacokinetics of the fluorescence conjugates. After nanobody-PS administration, tumors were illuminated to a fluence of 100 J∙cm-2, with a fluence rate of 50 mW∙cm-2, and thereafter tumor growth was measured with a follow-up until 30 days. Results: The selected nanobodies remained functional after conjugation to the PS, binding specifically and with high affinity to HER2-positive cells. Both nanobody-PS conjugates potently and selectively induced cell death of HER2 overexpressing cells, either sensitive or resistant to trastuzumab, with low nanomolar LD50 values. In vivo, quantitative fluorescence spectroscopy showed specific accumulation of nanobody-PS conjugates in HCC1954 tumors and indicated 2 h post injection as the most suitable time point to apply light. Nanobody-targeted PDT with 1D5-PS and 1D5-18A12-PS induced significant tumor regression of trastuzumab-resistant high HER2 expressing tumors, whereas in low HER2 expressing tumors only a slight growth delay was observed. Conclusion: Nanobody-PS conjugates accumulated selectively in vivo and their fluorescence could be detected through optical imaging. Upon illumination, they selectively induced significant tumor regression of HER2 overexpressing tumors with a single treatment session. Nanobody-targeted PDT is therefore suggested as a new additional treatment for HER2-positive breast cancer, particularly of interest for trastuzumab-resistant HER2-positive breast cancer. Further studies are now needed to assess the value of this approach in c

Тенденції розвитку національної інноваційної системи в Україні

Проаналізовано національну інноваційну систему України. Розглянуто галузі промисловості України за ознаками інноваційної активності та досліджено темпи зростання показників, враховуючи індекс інфляції. Встановлено, що спад темпів зростання динаміки реалізованої продукції призводить до зменшення витрат на інноваційну діяльність.Дан анализ национальной инновационной системы Украины. Рассмотрены отрасли промышленности Украины по признакам инновационной активности и исследованы темпы роста показателей, учитывая индекс инфляции. Установлено, что спад темпов роста динамики реализованной продукции приводит к уменьшению затрат на инновационную деятельность.This article analyses national innovation system of Ukraine. Examined the industry of Ukraine based on innovative activity and investigated the growth indicators, taking into account inflation-index. It is established that the slowdown in the dynamics realized production leads to a decrease in the cost of innovation

Nanobody-based cancer therapy of solid tumors

The development of tumor-targeted therapies using monoclonal antibodies has been successful during the last 30 years. Nevertheless, the efficacy of antibody-based therapy is still limited and further improvements are eagerly awaited. One of the promising novel developments that may overcome the drawbacks of monoclonal antibody-based therapies is the employment of nanobodies. Current nanobody-based therapeutics can be divided into three different platforms with nanobodies functioning as: receptor antagonists; targeting moieties of effector domains; or targeting molecules on the surface of nanoparticles. In this article, we describe factors that affect their performance at three different stages: their systemic circulation upon intravenous injection; their extravasation and tumor penetration; and, finally, their interaction with target molecules

MAGE-A antigens as targets for cancer immunotherapy

Targeted anti-cancer therapies aim at reducing side effects while retaining their anti-cancer efficacy. Immunotherapies e.g. monoclonal antibodies, adoptive T cell therapy and cancer vaccines are used to combat cancer, but the number of available cancer specific targets is limited and new approaches are needed to generate more effective and patient tailored treatments. Unique cancer intracellular epitopes can be presented on the cell surface by MHC class I molecules, which can function as epitopes for targeted therapies. The intracellular MAGE proteins belong to a sub-class of Cancer Testis (CT) antigens which are expressed in germline cells and a wide variety of tumors of different histological origin. Evidence has emerged that their expression is linked to pro-tumorigenic activities like increased cell motility, resisting cell death, and tumor promoting inflammation. Intracellular MAGE proteins are processed by the proteasome and their peptides are presented by MHC class I molecules on the cell surface of cancer cells thereby making them ideal cancer specific antigens. Here we review the previous and ongoing (pre-) clinical studies on the use of surface expressed MAGE antigens for their employment in targeted anti-cancer therapies. We present and analyze study outcomes and discuss possible future directions and improvements for MAGE directed anti-cancer immunotherapies

Nanobody-Targeted Photodynamic Therapy: Nanobody Production and Purification

Nanobodies have recently been introduced to the field of photodynamic therapy (PDT) as a very promising strategy to target photosensitizers selectively to cancer cells. Nanobodies are known for their characteristic small size (15 kDa), high specificity, and high binding affinities. These features allow rapid accumulation of nanobody-photosensitizer conjugates at the tumor site and rapid clearance of unbound fractions, and thus illumination for activation is possible 1 or 2 h postinjection. Preclinical studies have shown extensive tumor damage after nanobody-targeted PDT . This chapter addresses the first steps toward preparing nanobody-photosensitizer conjugates, which are the nanobody production and purification. The protocol for nanobody production addresses either medium- or large-scale bacterial expression, while the nanobody purification is described for two main strategies: affinity chromatography and ion-exchange chromatography. For the first strategy, protocols are described for different affinity tags and purification from either medium-scale or large-scale productions. For the second strategy, the protocol given is for purification from a large-scale production

Nanobody-Targeted Photodynamic Therapy: Nanobody Production and Purification

Nanobodies have recently been introduced to the field of photodynamic therapy (PDT) as a very promising strategy to target photosensitizers selectively to cancer cells. Nanobodies are known for their characteristic small size (15 kDa), high specificity, and high binding affinities. These features allow rapid accumulation of nanobody-photosensitizer conjugates at the tumor site and rapid clearance of unbound fractions, and thus illumination for activation is possible 1 or 2 h postinjection. Preclinical studies have shown extensive tumor damage after nanobody-targeted PDT . This chapter addresses the first steps toward preparing nanobody-photosensitizer conjugates, which are the nanobody production and purification. The protocol for nanobody production addresses either medium- or large-scale bacterial expression, while the nanobody purification is described for two main strategies: affinity chromatography and ion-exchange chromatography. For the first strategy, protocols are described for different affinity tags and purification from either medium-scale or large-scale productions. For the second strategy, the protocol given is for purification from a large-scale production

Optical imaging of pre-invasive breast cancer with a combination of VHHs targeting CAIX and HER2 increases contrast and facilitates tumour characterization

BACKGROUND: Optical molecular imaging is an emerging novel technology with applications in the diagnosis of cancer and assistance in image-guided surgery. A high tumour-to-background (T/B) ratio is crucial for successful imaging, which strongly depends on tumour-specific probes that rapidly accumulate in the tumour, while non-bound probes are rapidly cleared. Here, using pre-invasive breast cancer as a model, we investigate whether the use of combinations of probes with different target specificities results in higher T/B ratios and whether dual-spectral imaging leads to improvements in tumour characterization. METHODS: We performed optical molecular imaging of an orthotopic breast cancer model mimicking ductal carcinoma in situ (DCIS). A combination of carbonic anhydrase IX (CAIX)- and human epidermal growth factor receptor 2 (HER2)-specific variable domains of the heavy chain from heavy-chain antibodies (VHHs) was conjugated either to the same fluorophore (IRDye800CW) to evaluate T/B ratios or to different fluorophores (IRDye800CW, IRDye680RD or IRDye700DX) to analyse the expression of CAIX and HER2 simultaneously through dual-fluorescence detection. These experiments were performed non-invasively in vivo, in a mimicked intra-operative setting, and ex vivo on tumour sections. RESULTS: Application of the CAIX- and HER2-specific VHH combination resulted in an increase of the T/B ratio, as compared to T/B ratios obtained from each of these single VHHs together with an irrelevant VHH. This dual tumour marker-specific VHH combination also enabled the detection of small metastases in the lung. Furthermore, dual-spectral imaging enabled the assessment of the expression status of both CAIX and HER2 in a mimicked intra-operative setting, as well as on tumour sections, which was confirmed by immunohistochemistry. CONCLUSIONS: These results establish the feasibility of the use of VHH 'cocktails' to increase T/B ratios and improve early detection of heterogeneous tumours and the use of multispectral molecular imaging to facilitate the assessment of the target expression status of tumours and metastases, both invasive or non-invasively

Optical imaging of pre-invasive breast cancer with a combination of VHHs targeting CAIX and HER2 increases contrast and facilitates tumour characterization

Background: Optical molecular imaging is an emerging novel technology with applications in the diagnosis of cancer and assistance in image-guided surgery. A high tumour-to-background (T/B) ratio is crucial for successful imaging, which strongly depends on tumour-specific probes that rapidly accumulate in the tumour, while non-bound probes are rapidly cleared. Here, using pre-invasive breast cancer as a model, we investigate whether the use of combinations of probes with different target specificities results in higher T/B ratios and whether dual-spectral imaging leads to improvements in tumour characterization. Methods: We performed optical molecular imaging of an orthotopic breast cancer model mimicking ductal carcinoma in situ (DCIS). A combination of carbonic anhydrase IX (CAIX)- and human epidermal growth factor receptor 2 (HER2)-specific variable domains of the heavy chain from heavy-chain antibodies (VHHs) was conjugated either to the same fluorophore (IRDye800CW) to evaluate T/B ratios or to different fluorophores (IRDye800CW, IRDye680RD or IRDye700DX) to analyse the expression of CAIX and HER2 simultaneously through dual-fluorescence detection. These experiments were performed non-invasively in vivo, in a mimicked intra-operative setting, and ex vivo on tumour sections. Results: Application of the CAIX- and HER2-specific VHH combination resulted in an increase of the T/B ratio, as compared to T/B ratios obtained from each of these single VHHs together with an irrelevant VHH. This dual tumour marker-specific VHH combination also enabled the detection of small metastases in the lung. Furthermore, dual-spectral imaging enabled the assessment of the expression status of both CAIX and HER2 in a mimicked intra-operative setting, as well as on tumour sections, which was confirmed by immunohistochemistry. Conclusions: These results establish the feasibility of the use of VHH ‘cocktails’ to increase T/B ratios and improve early detection of heterogeneous tumours and the use of multispectral molecular imaging to facilitate the assessment of the target expression status of tumours and metastases, both invasive or non-invasively

Rapid optical imaging of human breast tumour xenografts using anti-HER2 VHHs site-directly conjugated to IRDye 800CW for image-guided surgery

<p>Purpose Molecular optical imaging using monoclonal antibodies is slow with low tumour to background ratio. We used anti-HER2 VHHs conjugated to IRDye 800CW to investigate their potential as probes for rapid optical molecular imaging of HER2-positive tumours by the determination of tumour accumulation and tumour to background levels.</p><p>Three anti-HER2 VHHs (11A4, 18C3, 22G12) were selected with phage display and produced in Escherichia coli. Binding affinities of these probes to SKBR3 cells were determined before and after site-specific conjugation to IRDye 800CW. To determine the potential of VHH-IR as imaging probes, serial optical imaging studies were carried out using human SKBR3 and human MDA-MB-231 xenograft breast cancer models. Performance of the anti-HER2 VHH-IR was compared to that of trastuzumab-IR and a non-HER2-specific VHH-IR. Image-guided surgery was performed during which SKBR3 tumour was removed under the guidance of the VHH-IR signal.</p><p>Site-specific conjugation of IRDye 800CW to three anti-HER2 VHHs preserved high affinity binding with the following dissociation constants (K-D): 11A4 1.9 +/- 0.03, 18C3 14.3 +/- 1.8 and 22G12 3.2 +/- 0.5 nM. Based upon different criteria such as binding, production yield and tumour accumulation, 11A4 was selected for further studies. Comparison of 11A4-IR with trastuzumab-IR showed similar to 20 times faster tumour accumulation of the anti-HER2 VHH, with a much higher contrast between tumour and background tissue (11A4-IR 2.5 +/- 0.3, trastuzumab-IR 1.4 +/- 0.4, 4 h post-injection). 11A4-IR was demonstrated to be a useful tool in image-guided surgery.</p><p>VHH-IR led to a much faster tumour accumulation with high tumour to background ratios as compared to trastuzumab-IR allowing same-day imaging for clinical investigation as well as image-guided surgery.</p>

Nanobody-targeted photodynamic therapy induces significant tumor regression of trastuzumab-resistant HER2-positive breast cancer, after a single treatment session

RATIONALE: A substantial number of breast cancer patients with an overexpression of the human epidermal growth factor receptor 2 (HER2) have residual disease after neoadjuvant therapy or become resistant to trastuzumab. Photodynamic therapy (PDT) using nanobodies targeted to HER2 is a promising treatment option for these patients. Here we investigate the in vitro and in vivo antitumor efficacy of HER2-targeted nanobody-photosensitizer (PS) conjugate PDT. METHODS: Nanobodies targeting HER2 were obtained from phage display selections. Monovalent nanobodies were engineered into a biparatopic construct. The specificity of selected nanobodies was tested in immunofluorescence assays and their affinity was evaluated in binding studies, both performed in a panel of breast cancer cells varying in HER2 expression levels. The selected HER2-targeted nanobodies 1D5 and 1D5-18A12 were conjugated to the photosensitizer IRDye700DX and tested in in vitro PDT assays. Mice bearing orthotopic HCC1954 trastuzumab-resistant tumors with high HER2 expression or MCF-7 tumors with low HER2 expression were intravenously injected with nanobody-PS conjugates. Quantitative fluorescence spectroscopy was performed for the determination of the local pharmacokinetics of the fluorescence conjugates. After nanobody-PS administration, tumors were illuminated to a fluence of 100 J∙cm-2, with a fluence rate of 50 mW∙cm-2, and thereafter tumor growth was measured with a follow-up until 30 days. RESULTS: The selected nanobodies remained functional after conjugation to the PS, binding specifically and with high affinity to HER2-positive cells. Both nanobody-PS conjugates potently and selectively induced cell death of HER2 overexpressing cells, either sensitive or resistant to trastuzumab, with low nanomolar LD50 values. In vivo, quantitative fluorescence spectroscopy showed specific accumulation of nanobody-PS conjugates in HCC1954 tumors and indicated 2 h post injection as the most suitable time point to apply light. Nanobody-targeted PDT with 1D5-PS and 1D5-18A12-PS induced significant tumor regression of trastuzumab-resistant high HER2 expressing tumors, whereas in low HER2 expressing tumors only a slight growth delay was observed. CONCLUSION: Nanobody-PS conjugates accumulated selectively in vivo and their fluorescence could be detected through optical imaging. Upon illumination, they selectively induced significant tumor regression of HER2 overexpressing tumors with a single treatment session. Nanobody-targeted PDT is therefore suggested as a new additional treatment for HER2-positive breast cancer, particularly of interest for trastuzumab-resistant HER2-positive breast cancer. Further studies are now needed to assess the value of this approach in clinical practice