Comparative Preclinical Evaluation of HYNIC-Modified Designed Ankyrin Repeat Proteins G3 for the 99mTc-Based Imaging of HER2-Expressing Malignant Tumors.

HER2 status determination is a necessary step for the proper choice of therapy and selection of patients for the targeted treatment of cancer. Targeted radiotracers such as radiolabeled DARPins provide a noninvasive and effective way for the molecular imaging of HER2 expression. This study aimed to evaluate tumor-targeting properties of three 99mTc-labeled DARPin G3 variants containing Gly-Gly-Gly-Cys (G3C), (Gly-Gly-Gly-Ser)3-Cys ((G3S)3C), or Glu-Glu-Glu-Cys (E3C) amino acid linkers at the C-terminus and conjugated to the HYNIC chelating agent, as well as to compare them with the clinically evaluated DARPin G3 labeled with 99mTc(CO)3 using the (HE)3-tag at the N-terminus. The labeling of DARPin G3-HYNIC variants provided radiochemical yields in the range of 50-80%. Labeled variants bound specifically to human HER2-expressing cancer cell lines with affinities in the range of 0.5-3 nM. There was no substantial influence of the linker and HYNIC chelator on the binding of 99mTc-labeled DARPin G3 variants to HER2 in vitro; however, [99mTc]Tc-G3-(G3S)3C-HYNIC had the highest affinity. Comparative biodistribution of [99mTc]Tc-G3-G3C-HYNIC, [99mTc]Tc-G3-(G3S)3C-HYNIC, [99mTc]Tc-G3-E3C-HYNIC, and [99mTc]Tc-(HE)3-G3 in healthy CD1 mice showed that there was a strong influence of the linkers on uptake in normal tissues. [99mTc]Tc-G3-E3C-HYNIC had an increased retention of activity in the liver and the majority of other organs compared to the other conjugates. The tumor uptake of [99mTc]Tc-G3-(G3S)3C-HYNIC and [99mTc]Tc-(HE)3-G3 in Nu/j mice bearing SKOV-3 xenografts was similar. The specificity of tumor targeting in vivo was demonstrated for both tracers. [99mTc]Tc-G3-(G3S)3C-HYNIC provided comparable, although slightly lower tumor-to-lung, tumor-to spleen and tumor-to-liver ratios than [99mTc]Tc-(HE)3-G3. Radiolabeling of DARPin G3-HYNIC conjugates with 99mTc provided the advantage of a single-step radiolabeling procedure; however, the studied HYNIC conjugates did not improve imaging contrast compared to the 99mTc-tricarbonyl-labeled DARPin G3. At this stage, [99mTc]Tc-(HE)3-G3 remains the most promising candidate for the clinical imaging of HER2-overexpressing cancers.

Direct Intra-Patient Comparison of Scaffold Protein-Based Tracers, [99mTc]Tc-ADAPT6 and [99mTc]Tc-(HE)3-G3, for Imaging of HER2-Positive Breast Cancer.

Previous Phase I clinical evaluations of the radiolabelled scaffold proteins [99mTc]Tc-ADAPT6 and DARPin [99mTc]Tc-(HE)3-G3 in breast cancer patients have demonstrated their safety and indicated their capability to discriminate between HER2-positive and HER2-negative tumours. The objective of this study was to compare the imaging of HER2-positive tumours in the same patients using [99mTc]Tc-ADAPT6 and [99mTc]Tc-(HE)3-G3. Eleven treatment-naïve female patients (26-65 years) with HER2-positive primary and metastatic breast cancer were included in the study. Each patient was intravenously injected with [99mTc]Tc-ADAPT6, followed by an [99mTc]Tc-(HE)3-G3 injection 3-4 days later and chest SPECT/CT was performed. All primary tumours were clearly visualized using both tracers. The uptake of [99mTc]Tc-ADAPT6 in primary tumours (SUVmax = 4.7 ± 2.1) was significantly higher (p < 0.005) than the uptake of [99mTc]Tc-(HE)3-G3 (SUVmax = 3.5 ± 1.7). There was no significant difference in primary tumour-to-contralateral site values for [99mTc]Tc-ADAPT6 (15.2 ± 7.4) and [99mTc]Tc-(HE)3-G3 (19.6 ± 12.4). All known lymph node metastases were visualized using both tracers. The uptake of [99mTc]Tc-ADAPT6 in all extrahepatic soft tissue lesions was significantly (p < 0.0004) higher than the uptake of [99mTc]Tc-(HE)3-G3. In conclusion, [99mTc]Tc-ADAPT6 and [99mTc]Tc-(HE)3-G3 are suitable for the visualization of HER2-positive breast cancer. At the selected time points, [99mTc]Tc-ADAPT6 has a significantly higher uptake in soft tissue lesions, which might be an advantage for the visualization of small metastases.

Feasibility of Co-Targeting HER3 and EpCAM Using Seribantumab and DARPin-Toxin Fusion in a Pancreatic Cancer Xenograft Model.

Pancreatic cancer (PC) is one of the most aggressive malignancies. A combination of targeted therapies could increase the therapeutic efficacy in tumors with heterogeneous target expression. Overexpression of the human epidermal growth factor receptor type 3 (HER3) and the epithelial cell adhesion molecule (EpCAM) in up to 40% and 30% of PCs, respectively, is associated with poor prognosis and highlights the relevance of these targets. Designed ankyrin repeat protein (DARPin) Ec1 fused with the low immunogenic bacterial toxin LoPE provides specific and potent cytotoxicity against EpCAM-expressing cancer cells. Here, we investigated whether the co-targeting of HER3 using the monoclonal antibody seribantumab (MM-121) and of EpCAM using Ec1-LoPE would improve the therapeutic efficacy in comparison to the individual agents. Radiolabeled 99mTc(CO)3-Ec1-LoPE showed specific binding with rapid internalization in EpCAM-expressing PC cells. MM-121 did not interfere with the binding of Ec1-LoPE to EpCAM. Evaluation of cytotoxicity indicated synergism between Ec1-LoPE and MM-121 in vitro. An experimental therapy study using Ec1-LoPE and MM-121 in mice bearing EpCAM- and HER3-expressing BxPC3 xenografts demonstrated the feasibility of the therapy. Further development of the co-targeting approach using HER3 and EpCAM could therefore be justified.

Direct In Vivo Comparison of 99mTc-Labeled Scaffold Proteins, DARPin G3 and ADAPT6, for Visualization of HER2 Expression and Monitoring of Early Response for Trastuzumab Therapy.

Non-invasive radionuclide molecular visualization of human epidermal growth factor receptor type 2 (HER2) can provide stratification of patients for HER2-targeting therapy. This method can also enable monitoring of the response to such therapies, thereby making treatment personalized and more efficient. Clinical evaluation in a phase I study demonstrated that injections of two scaffold protein-based imaging probes, [99mTc]Tc-(HE)3-G3 and [99mTc]Tc-ADAPT6, are safe, well-tolerated and cause a low level of radioactivity in healthy tissue. The goal of this preclinical study was to select the best probe for stratification of patients and response monitoring. Biodistribution of both tracers was compared in mice bearing SKOV-3 xenografts with high HER2 expression or MDA-MB-468 xenografts with very low expression. Changes in accumulation of the probes in SKOV-3 tumors 24 h after injection of trastuzumab were evaluated. Both [99mTc]Tc-ADAPT6 and [99mTc]Tc-(HE)3-G3 permitted high contrast imaging of HER2-expressing tumors and a clear discrimination between tumors with high and low HER2 expression. However, [99mTc]Tc-ADAPT6 has better preconditions for higher sensitivity and specificity of stratification. On the other hand, [99mTc]Tc-(HE)3-G3 is capable of detecting the decrease of HER2 expression on response to trastuzumab therapy only 24 h after injection of the loading dose. This indicates that the [99mTc]Tc-(HE)3-G3 tracer would be better for monitoring early response to such treatment. The results of this study should be considered in planning of further clinical development of HER2 imaging probes.

Switchable targeting of solid tumors by BsCAR T cells.

The development of chimeric antigen receptor (CAR) T cell therapy has become a critical milestone in modern oncotherapy. Despite the remarkable in vitro effectiveness, the problem of safety and efficacy of CAR T cell therapy against solid tumors is challenged by the lack of tumor-specific antigens required to avoid on-target off-tumor effects. Spatially separating the cytotoxic function of CAR T cells from tumor antigen recognition provided by protein mediators allows for the precise control of CAR T cell cytotoxicity. Here, the high affinity and capability of the bacterial toxin-antitoxin barnase-barstar system were adopted to guide CAR T cells to solid tumors. The complementary modules based on (1) ankyrin repeat (DARPin)-barnase proteins and (2) barstar-based CAR (BsCAR) were designed to provide switchable targeting to tumor cells. The alteration of the DARPin-barnase switches enabled the targeting of different tumor antigens with a single BsCAR. A gradual increase in cytokine release and tunable BsCAR T cell cytotoxicity was achieved by varying DARPin-barnase loads. Switchable BsCAR T cell therapy was able to eradicate the HER2+ ductal carcinoma in vivo. Guiding BsCAR T cells by DARPin-barnase switches provides a universal approach for a controlled multitargeted adoptive immunotherapy.

Comparative Preclinical Evaluation of Peptide-Based Chelators for the Labeling of DARPin G3 with 99mTc for Radionuclide Imaging of HER2 Expression in Cancer.

Non-invasive radionuclide imaging of human epidermal growth factor receptor type 2 (HER2) expression in breast, gastroesophageal, and ovarian cancers may stratify patients for treatment using HER2-targeted therapeutics. Designed ankyrin repeat proteins (DARPins) are a promising type of targeting probe for radionuclide imaging. In clinical studies, the DARPin [99mTc]Tc-(HE)3-G3 labeled using a peptide-based chelator His-Glu-His-Glu-His-Glu ((HE)3), provided clear imaging of HER2 expressing breast cancer 2-4 h after injection. The goal of this study was to evaluate if the use of cysteine-containing peptide-based chelators Glu-Glu-Glu-Cys (E3C), Gly-Gly-Gly-Cys (G3C), and Gly-Gly-Gly-Ser-Cys connected via a (Gly-Gly-Gly-Ser)3-linker (designated as G3-(G3S)3C) would further improve the contrast of imaging using 99mTc-labeled derivatives of G3. The labeling of the new variants of G3 provided a radiochemical yield of over 95%. Labeled G3 variants bound specifically to human HER2-expressing cancer cell lines with affinities in the range of 1.9-5 nM. Biodistribution of [99mTc]Tc-G3-G3C, [99mTc]Tc-G3-(G3S)3C, and [99mTc]Tc-G3-E3C in mice was compared with the biodistribution of [99mTc]Tc-(HE)3-G3. It was found that the novel variants provide specific accumulation in HER2-expressing human xenografts and enable discrimination between tumors with high and low HER2 expression. However, [99mTc]Tc-(HE)3-G3 provided better contrast between tumors and the most frequent metastatic sites of HER2-expressing cancers and is therefore more suitable for clinical applications.

Effect of Surface Modification of Multifunctional Nanocomposite Drug Delivery Carriers with DARPin on Their Biodistribution In Vitro and In Vivo.

We present a targeted drug delivery system for therapy and diagnostics that is based on a combination of contrasting, cytotoxic, and cancer-cell-targeting properties of multifunctional carriers. The system uses multilayered polymer microcapsules loaded with magnetite and doxorubicin. Loading of magnetite nanoparticles into the polymer shell by freezing-induced loading (FIL) allowed the loading efficiency to be increased 5-fold, compared with the widely used layer-by-layer (LBL) assembly. FIL also improved the photoacoustic signal and particle mobility in a magnetic field gradient, a result unachievable by the LBL alone. For targeted delivery of the carriers to cancer cells, the carrier surface was modified with a designed ankyrin repeat protein (DARPin) directed toward the epithelial cell adhesion molecule (EpCAM). Flow cytometry measurements showed that the DARPin-coated capsules specifically interacted with the surface of EpCAM-overexpressing human cancer cells such as MCF7. In vivo and ex vivo biodistribution studies in FvB mice showed that the carrier surface modification with DARPin changed the biodistribution of the capsules toward epithelial cells. In particular, the capsules accumulated substantially in the lungs─a result that can be effectively used in targeted lung cancer therapy. The results of this work may aid in the further development of the "magic bullet" concept and may bring the quality of personalized medicine to another level.

Epithelial cell adhesion molecule­targeting designed ankyrin repeat protein­toxin fusion Ec1­LoPE exhibits potent cytotoxic action in prostate cancer cells.

Targeted anticancer therapeutics offer the advantage of reducing cytotoxic side effects to normal cells by directing the cytotoxic payload selectively to cancer cells. Designed ankyrin repeat proteins (DARPins) are promising non­immunoglobulin­based scaffold proteins for payload delivery to cancer­associated molecular targets. Epithelial cell adhesion molecule (EpCAM) is overexpressed in 40­60% of prostate cancers (PCs) and is associated with metastasis, increased risk of PC recurrence and resistance to treatment. Here, we investigated the use of DARPin Ec1 for targeted delivery of Pseudomonas exotoxin A variant (LoPE) with low immunogenicity and low non­specific toxicity to EpCAM­expressing prostate cancer cells. Ec1­LoPE fusion protein was radiolabeled with tricarbonyl technetium­99m and its binding specificity, binding kinetics, cellular processing, internalization and cytotoxicity were evaluated in PC­3 and DU145 cell lines. Ec1­LoPE showed EpCAM­specific binding to EpCAM­expressing prostate cancer cells. Rapid internalization mediated potent cytotoxic effect with picomolar IC50 values in both studied cell lines. Taken together, these data support further evaluation of Ec1­LoPE in a therapeutic setting in a prostate cancer model in vivo.

Phase I Trial of 99mTc-(HE)3-G3, a DARPin-Based Probe for Imaging of HER2 Expression in Breast Cancer.

Radionuclide molecular imaging of human epidermal growth factor receptor type 2 (HER2) expression may enable a noninvasive discrimination between HER2-positive and HER2-negative breast cancers for stratification of patients for HER2-targeted treatments. DARPin (designed ankyrin repeat proteins) G3 is a small (molecular weight, 14 kDa) scaffold protein with picomolar affinity to HER2. The aim of this first-in-humans study was to evaluate the safety, biodistribution, and dosimetry of 99mTc-(HE)3-G3. Methods: Three cohorts of patients with primary breast cancer (each including at least 4 patients with HER2-negative and 5 patients with HER2-positive tumors) were injected with 1,000, 2,000, or 3,000 µg of 99mTc-(HE)3-G3 (287 ± 170 MBq). Whole-body planar imaging followed by SPECT was performed at 2, 4, 6, and 24 h after injection. Vital signs and possible side effects were monitored during imaging and up to 7 d after injection. Results: All injections were well tolerated. No side effects were observed. The results of blood and urine analyses did not differ before and after studies. 99mTc-(HE)3-G3 cleared rapidly from the blood. The highest uptake was detected in the kidneys and liver followed by the lungs, breasts, and small intestinal content. The hepatic uptake after injection of 2,000 or 3,000 µg was significantly (P < 0.05) lower than the uptake after injection of 1,000 µg. Effective doses did not differ significantly between cohorts (average, 0.011 ± 0.004 mSv/MBq). Tumor-to-contralateral site ratios for HER-positive tumors were significantly (P < 0.05) higher than for HER2-negative at 2 and 4 h after injection. Conclusion: Imaging of HER2 expression using 99mTc-(HE)3-G3 is safe and well tolerated and provides a low absorbed dose burden on patients. This imaging enables discernment of HER2-positive and HER2-negative breast cancer. Phase I study data justify further clinical development of 99mTc-(HE)3-G3.

Barnase encapsulation into submicron porous CaCO3 particles: studies of loading and enzyme activity.

The present study focuses on the immobilization of the bacterial ribonuclease barnase (Bn) into submicron porous calcium carbonate (CaCO3) particles. For encapsulation, we apply adsorption, freezing-induced loading and co-precipitation methods and study the effects of adsorption time, enzyme concentration and anionic polyelectrolytes on the encapsulation efficiency of Bn. We show that the use of negatively charged dextran sulfate (DS) and ribonucleic acid from yeast (RNA) increases the loading capacity (LC) of the enzyme on CaCO3 particles by about 3-fold as compared to the particles with Bn itself. The ribonuclease (RNase) activity of encapsulated enzyme depends on the LC of the particles and transformation of metastable vaterite to stable calcite, as studied by the assessment of enzyme activities in particles.

Genetic Variations and Haplotypic Diversity in the Myostatin Gene of Different Cattle Breeds in Russia.

The myostatin gene (MSTN) in cattle has a number of polymorphisms associated with increased muscle mass. The aim of the current study was to determine the haplotype frequencies of F94L and nt821(del11) MSTN polymorphisms among cattle bred for meat in Russia, using DNA analysis. Using the earlier created test systems based on the AS-PCR and PCR-RFLP methods, six populations of Aberdeen Angus (n = 684), two populations of Limousin (n = 54), one population of Simmental (n = 55), and one population of Belgian Blue (n = 137) belonging to Russian farms were genotyped on nt821(del11) and F94LMSTN polymorphisms. The animal carriers of the mutant allele of nt821(del11)MSTN associated with the double-muscling genetic defect were found in one Aberdeen Angus population at a frequency of 2.18%, but were not found in the Limousin and Simmental populations. However, 100% of the Belgian Blue population were heterozygous carriers of nt821(del11)MSTN. The frequencies of the A allele F94LMSTN desirable for productivity traits in the Limousin populations were the highest and accounted for 0.97 and 1 in populations one and two, while in the Aberdeen Angus, Simmental, and Belgian Blue populations, these figures were considerably lower at 0.04-0.08, depending on the population. The obtained data show the high genetic potential of Russian beef cattle, and facilitate an improvement in meat productivity by preserving the health of animals.

Imaging-Guided Therapy Simultaneously Targeting HER2 and EpCAM with Trastuzumab and EpCAM-Directed Toxin Provides Additive Effect in Ovarian Cancer Model.

Efficient treatment of disseminated ovarian cancer (OC) is challenging due to its heterogeneity and chemoresistance. Overexpression of human epidermal growth factor receptor 2 (HER2) and epithelial cell adhesion molecule (EpCAM) in approx. 30% and 70% of ovarian cancers, respectively, allows for co-targeted treatment. The clinical efficacy of the monoclonal antibody trastuzumab in patients with HER2-positive breast, gastric and gastroesophageal cancers makes it readily available as the HER2-targeting component. As the EpCAM-targeting component, we investigated the designed ankyrin repeat protein (DARPin) Ec1 fused to a truncated variant of Pseudomonas exotoxin A with reduced immunogenicity and low general toxicity (LoPE). Ec1-LoPE was radiolabeled, evaluated in ovarian cancer cells in vitro and its biodistribution and tumor-targeting properties were studied in vivo. The therapeutic efficacy of Ec1-LoPE alone and in combination with trastuzumab was studied in mice bearing EpCAM- and HER2-expressing SKOV3 xenografts. SPECT/CT imaging enabled visualization of EpCAM and HER2 expression in the tumors. Co-treatment using Ec1-LoPE and trastuzumab was more effective at reducing tumor growth and prolonged the median survival of mice compared with mice in the control and monotherapy groups. Repeated administration of Ec1-LoPE was well tolerated without signs of hepatic or kidney toxicity. Co-treatment with trastuzumab and Ec1-LoPE might be a potential therapeutic strategy for HER2- and EpCAM-positive OC.

Influence of the Position and Composition of Radiometals and Radioiodine Labels on Imaging of Epcam Expression in Prostate Cancer Model Using the DARPin Ec1.

The epithelial cell adhesion molecule (EpCAM) is intensively overexpressed in 40-60% of prostate cancer (PCa) cases and can be used as a target for the delivery of drugs and toxins. The designed ankyrin repeat protein (DARPin) Ec1 has a high affinity to EpCAM (68 pM) and a small size (18 kDa). Radiolabeled Ec1 might be used as a companion diagnostic for the selection of PCa patients for therapy. The study aimed to investigate the influence of radiolabel position (N- or C-terminal) and composition on the targeting and imaging properties of Ec1. Two variants, having an N- or C-terminal cysteine, were produced, site-specifically conjugated to a DOTA chelator and labeled with cobalt-57, gallium-68 or indium-111. Site-specific radioiodination was performed using ((4-hydroxyphenyl)-ethyl)maleimide (HPEM). Biodistribution of eight radiolabeled Ec1-probes was measured in nude mice bearing PCa DU145 xenografts. In all cases, positioning of a label at the C-terminus provided the best tumor-to-organ ratios. The non-residualizing [125I]I-HPEM label provided the highest tumor-to-muscle and tumor-to-bone ratios and is more suitable for EpCAM imaging in early-stage PCa. Among the radiometals, indium-111 provided the highest tumor-to-blood, tumor-to-lung and tumor-to-liver ratios and could be used at late-stage PCa. In conclusion, label position and composition are important for the DARPin Ec1.

The Molecular Bases Study of the Inherited Diseases for the Health Maintenance of the Beef Cattle.

The article highlighted the problem of meat cattle genetic defects. The aim was the development of DNA tests for some genetic defects diagnostics, the determination of the animal carriers and their frequencies tracking in time. The 1490 DNA samples from the Aberdeen Angus (n = 701), Hereford (n = 385), Simmental (n = 286) and Belgian Blue (n = 118) cattle have been genotyped on the genetic defects by newly created and earlier developed DNA tests based on AS-PCR and PCR-RFLP methods. The Aberdeen Angus cattle genotyping has revealed 2.38 ± 0.31% AMC-cows and 1.67 ± 0.19 % AMC-bulls, 0.65 ± 0.07% DDC-cows and 0.90 ± 0.10% DDC-bulls. The single animals among the Hereford cattle were carriers of MSUD and CWH (on 0.27 ± 0.05%), ICM and HY (on 0.16 ± 0.03%). The Simmental cattle were free from OS. All Belgian Blue livestock were M1- and 0.84%-CMD1-carriers. The different ages Aberdeen Angus cattle genotyping has shown the tendency of the AMC- and DDC frequencies to increase in the later generations. The statistically significant increase of DDC of 1.17% in the cows' population born in 2019 compared to those born in 2015 allows concluding the further development of the DNA analysis-based measures preventing the manifestation of the genetic anomalies in meat cattle herds is necessary.

Dual Targeting of Cancer Cells with DARPin-Based Toxins for Overcoming Tumor Escape.

We report here a combined anti-cancer therapy directed toward HER2 and EpCAM, common tumor-associated antigens of breast cancer cells. The combined therapeutic effect is achieved owing to two highly toxic proteins-a low immunogenic variant of Pseudomonas aeruginosa exotoxin A and ribonuclease Barnase from Bacillus amyloliquefaciens. The delivery of toxins to cancer cells was carried out by targeting designed ankyrin repeat proteins (DARPins). We have shown that both target agents efficiently accumulate in the tumor. Simultaneous treatment of breast carcinoma-bearing mice with anti-EpCAM fusion toxin based on LoPE and HER2-specific liposomes loaded with Barnase leads to concurrent elimination of primary tumor and metastases. Monotherapy with anti-HER2- or anti-EpCAM-toxins did not produce a comparable effect on metastases. The proposed approach can be considered as a promising strategy for significant improvement of cancer therapy.

Radionuclide Molecular Imaging of EpCAM Expression in Triple-Negative Breast Cancer Using the Scaffold Protein DARPin Ec1.

Efficient treatment of disseminated triple-negative breast cancer (TNBC) remains an unmet clinical need. The epithelial cell adhesion molecule (EpCAM) is often overexpressed on the surface of TNBC cells, which makes EpCAM a potential therapeutic target. Radionuclide molecular imaging of EpCAM expression might permit selection of patients for EpCAM-targeting therapies. In this study, we evaluated a scaffold protein, designed ankyrin repeat protein (DARPin) Ec1, for imaging of EpCAM in TNBC. DARPin Ec1 was labeled with a non-residualizing [125I]I-para-iodobenzoate (PIB) label and a residualizing [99mTc]Tc(CO)3 label. Both imaging probes retained high binding specificity and affinity to EpCAM-expressing MDA-MB-468 TNBC cells after labeling. Internalization studies showed that Ec1 was retained on the surface of MDA-MB-468 cells to a high degree up to 24 h. Biodistribution in Balb/c nu/nu mice bearing MDA-MB-468 xenografts demonstrated specific uptake of both [125I]I-PIB-Ec1 and [99mTc]Tc(CO)3-Ec1 in TNBC tumors. [125I]I-PIB-Ec1 had appreciably lower uptake in normal organs compared with [99mTc]Tc(CO)3-Ec1, which resulted in significantly (p < 0.05) higher tumor-to-organ ratios. The biodistribution data were confirmed by micro-Single-Photon Emission Computed Tomography/Computed Tomography (microSPECT/CT) imaging. In conclusion, an indirectly radioiodinated Ec1 is the preferable probe for imaging of EpCAM in TNBC.

UCNP-based Photoluminescent Nanomedicines for Targeted Imaging and Theranostics of Cancer.

Theranostic approach is currently among the fastest growing trends in cancer treatment. It implies the creation of multifunctional agents for simultaneous precise diagnosis and targeted impact on tumor cells. A new type of theranostic complexes was created based on NaYF4: Yb,Tm upconversion nanoparticles coated with polyethylene glycol and functionalized with the HER2-specific recombinant targeted toxin DARPin-LoPE. The obtained agents bind to HER2-overexpressing human breast adenocarcinoma cells and demonstrate selective cytotoxicity against this type of cancer cells. Using fluorescent human breast adenocarcinoma xenograft models, the possibility of intravital visualization of the UCNP-based complexes biodistribution and accumulation in tumor was demonstrated.

Feasibility of Imaging EpCAM Expression in Ovarian Cancer Using Radiolabeled DARPin Ec1.

Epithelial cell adhesion molecule (EpCAM) is overexpressed in 55%-75% of ovarian carcinomas (OC). EpCAM might be used as a target for a treatment of disseminated OC. Designed ankyrin repeats protein (DARPin) Ec1 is a small (18 kDa) protein, which binds to EpCAM with subnanomolar affinity. We tested a hypothesis that Ec1 labeled with a non-residualizing label might serve as a companion imaging diagnostic for stratification of patients for EpCAM-targeting therapy. Ec1 was labeled with 125I using N-succinimidyl-para-iodobenzoate. Binding affinity, specificity, and cellular processing of [125I]I-PIB-Ec1 were evaluated using SKOV-3 and OVCAR-3 ovarian carcinoma cell lines. Biodistribution and tumor-targeting properties of [125I]I-PIB-Ec1 were studied in Balb/c nu/nu mice bearing SKOV-3 and OVCAR-3 xenografts. EpCAM-negative Ramos lymphoma xenografts served as specificity control. Binding of [125I]I-PIB-Ec1 to ovarian carcinoma cell lines was highly specific and had affinity in picomolar range. Slow internalization of [125I]I-PIB-Ec1 by OC cells confirmed utility of non-residualizing label for in vivo imaging. [125I]I-PIB-Ec1 provided 6 h after injection tumor-to-blood ratios of 30 ± 11 and 48 ± 12 for OVCAR-3 and SKOV-3 xenografts, respectively, and high contrast to other organs. Tumor targeting was highly specific. Saturation of tumor uptake at a high dose of Ec1 in SKOV-3 model provided a rationale for dose selection in further studies using therapeutic conjugates of Ec1 for targeted therapy. In conclusion, [125I]I-PIB-Ec1 is a promising agent for visualizing EpCAM expression in OC.

DNA analysis of the Russian populations of Aberdeen Angus, Hereford and Belgian Blue cattle.

The use of specialized meat breeds in cattle breeding programs is considered very promising for improving herds' productivity. However, in animal genotype, along with genes that positively affect the productivity signs, there are genes whose mutations, known as genetic defects, negatively affect the health of animals. The aim of the study was the screening of the Russian populations of Aberdeen Angus, Hereford and Belgian Blue cattle on gene mutations associated with the genetic defects of arthrogryposis multiplex (AM), osteopetrosis (OS), developmental duplication (DD), double muscling (M1), hypotrichosis (HY) and maple syrup urine disease (MSUD) as well as the F94L polymorphism of myostatin gene (MSTN) linked with the gene responsible for less fat content in the carcass by means of DNA analysis. In the article, test systems based on the polymerase chain reaction method are presented. The analysis of the Aberdeen Angus ( n = 4480 ) population has revealed 0.19  ±  0.09 % animal M1 carriers, 0.53  ±  0.03 % OS carriers, 1.92  ±  0.09 % AM carriers and 9.00  ±  0.20 % DD carriers. The genotyping of Hereford cattle of Russian populations ( n = 525 ) has not revealed any individual carriers of MSUD or HY genetic defects. All of the Belgian Blue population ( n = 92 ) animals were heterozygous M1 carriers. The study of the F94L MSTN polymorphism has demonstrated extremely high frequencies of the desirable A allele (0.93 and 0.90) in two Aberdeen Angus populations with an average mean of 0.63  ±  0.08, which was 32 % higher compared to the Belgian Blue population. The results suggest the high genetic potential of the Aberdeen Angus and Belgian Blue cattle, but the existence in the genotypes of the mutant alleles associated with hereditary diseases indicates the risk of uncontrolled use of these breeds.

Effect of a radiolabel biochemical nature on tumor-targeting properties of EpCAM-binding engineered scaffold protein DARPin Ec1.

Radionuclide-based imaging of molecular therapeutic targets might facilitate stratifying patients for specific biotherapeutics. New type of imaging probes, based on designed ankyrin repeat proteins (DARPins), have demonstrated excellent contrast of imaging of human epidermal growth factor type 2 (HER2) expression in preclinical models. We hypothesized that labeling approaches, which result in lipophilic radiometabolites (non-residualizing labels), would provide the best imaging contrast for DARPins that internalize slowly after binding to cancer cells. The hypothesis was tested using DARPin Ec1 that binds to epithelial cell adhesion molecule (EpCAM). EpCAM is a promising therapeutic target. Ec1 was labeled with 125I using two methods to obtain the non-residualizing labels, while residualizing labels were obtained by labeling it with 99mTc. All labeled Ec1 variants preserved target specificity and picomolar binding affinity to EpCAM-expressing pancreatic adenocarcinoma BxPC-3 cells. In murine models, all the variants provided similar tumor uptake. However, 125I-PIB-H6-Ec1 had noticeably lower retention in normal tissues, which provided appreciably higher tumor-to-organ ratios. Furthermore, 125I-PIB-H6-Ec1 demonstrated the highest imaging contrast in preclinical models than any other EpCAM-imaging agent tested so far. In conclusion, DARPin Ec1 in combination with a non-residualizing label is a promising probe for imaging EpCAM expression a few hours after injection.