Noninvasive Evaluation of Tumoral PD-L1 Using a Novel 99mTc-Labeled Nanobody Tracer with Rapid Renal Clearance.

The expression level of PD-L1 in tumor tissue is considered one of the effective biomarkers to guide PD-1/PD-L1 therapy. Quantifying whole-body PD-L1 expression by SPECT imaging may help in selecting patients that potentially respond to PD-1/PD-L1 therapy. Nanobody is the smallest antibody fragment with antigen-binding ability that is well suited for radionuclide imaging. Nevertheless, high retention of radioactivity in the kidney may limit its clinical translation. The present study aimed to screen, design, and prepare a nanobody-based SPECT probe with rapid renal clearance to evaluate the PD-L1 expression level in vivo noninvasively. A phage library was constructed by immunizing alpaca with recombinant human PD-L1 protein, and 17 anti-PD-L1 nanobodies were screened by the phage display technique. After sequence alignment and flow cytometry analysis, APN09 was selected as the candidate nanobody, and a GGGC chelator was attached to its C-terminus for 99mTc labeling to prepare a SPECT imaging probe. The affinity and specificity of 99mTc-APN09 were evaluated by protein and cell-binding experiments, and SPECT imaging and biodistribution were performed in a mouse model with bilateral transplantation of A549 and A549PD-L1 tumors. The ability of 99mTc-APN09 to quantify the PD-L1 expression level in vivo was validated in tumor models with different PD-L1 expression levels. 99mTc-APN09 had a radiochemical purity higher than 99% and a binding equilibrium dissociation constant of 21.44 ± 1.65 nM with hPD-L1, showing high affinity. SPECT imaging results showed that 99mTc-APN09 could efficiently detect PD-L1-positive tumors within 0.5 h, and the quantitative results of SPECT were well correlated with the expression level of PD-L1 in cell lines. SPECT imaging and biodistribution results also showed that 99mTc-APN09 was rapidly cleared from the kidney in 2 h postinjection. 99mTc-APN09 was a simple and stable tool for visualizing PD-L1 expression in the whole body. In addition, due to its significant reduction in renal retention, it has better prospects for clinical translation.

HER2-targeted dual radiotracer approach with clinical potential for noninvasive imaging of trastuzumab-resistance caused by epitope masking.

Rationale: The decreased HER2-accessibility by epitope masking is a primary trastuzumab-resistance mechanism. In this study, we developed a HER2-targeted dual radiotracer approach to predict the HER2-trastuzumab engagement noninvasively. Methods: Two novel HER2-specific VHHs, MIRC208 and MIRC213, were acquired by immunizing alpaca with human HER2 protein, and were site-specifically labeled with 99mTc. Biodistribution and SPECT/CT imaging studies were performed in mice bearing HER2-positive and HER2-negative tumors. The HER2 binding sites of 99mTc-MIRC208 and 99mTc-MIRC213 were investigated by cell binding and SPECT/CT imaging studies. We evaluated the therapeutic predictive ability of our dual-radiotracer imaging approach for trastuzumab treatment in mice bearing MUC4-positive tumors (trastuzumab-resistant JIMT-1 and 87MUC4) and MUC4-negative tumors (trastuzumab-sensitive 7HER2 and NCI-N87). The preliminary clinical studies of 99mTc-MIRC208 were performed in two patients with HER2-positive breast tumors. Results:99mTc-MIRC208 and 99mTc-MIRC213 clearly visualized HER2-positive tumors, but not HER2-negative tumors. 99mTc-MIRC208 competes with trastuzumab for HER2-binding while 99mTc-MIRC213 recognizes HER2 on an epitope that is not masked by MUC4. The SPECT/CT studies with 99mTc-MIRC208 and 99mTc-MIRC213 clearly showed that the MUC4-negative and trastuzumab-sensitive 7HER2 and NCI-N87 tumors had very similar tumor uptake with the SUV208/SUV213 (2 h) ratios of 1.11 ± 0.17 in 7HER2 and 1.25 ± 0.22 in NCI-N87. However, the MUC4-positive JIMT-1 tumors showed the decreased SUV208/SUV213 (2 h) ratio (0.63 ± 0.07), which correlated well with the low response rate to trastuzumab therapy. The SUV208/SUV213 (2 h) ratio was reduced to 0.72 ± 0.02 in MUC4-expressing NCI-N87 cells, and resulting in the decreased trastuzumab sensitivity, further supporting the correlation between the SUV208/SUV213 (2 h) ratio and trastuzumab-sensitivity. The primary and metastatic HER2-positive lesions of patients were clearly visualized by 99mTc-MIRC208 SPECT at 2 h post injection. Conclusion: Overall, we demonstrated that the dual radiotracer imaging strategy is a valid noninvasive approach for the cancer patient selection before trastuzumab therapy. 99mTc-MIRC213 SPECT is utilized to quantify the tumor HER2 expression and screen HER2-positive cancer patients, while 99mTc-MIRC208 SPECT is used to determine the HER2-accessibility of trastuzumab. The SUV208/SUV213 (2 h) ratio is an important biomarker to determine the responsiveness of trastuzumab therapy.

IgG-Binding Nanobody Capable of Prolonging Nanobody-Based Radiotracer Plasma Half-Life and Enhancing the Efficacy of Tumor-Targeted Radionuclide Therapy.

Nanobodies have been developed rapidly as targeted probes for molecular imaging owing to their high affinity, outstanding tissue penetration, and rapid blood clearance. However, the short retention time at the tumor site limits their application in targeted radionuclide therapy. In this study, we designed a dual-targeting nanobody referred to as MIRC213-709, which can specifically bind to the HER2 receptor in tumor cell lines with high affinity (by nanobody MIRC213) and endogenous IgG in plasma to prolong the half-life by the MIRC213 C-terminal fusion nanobody, MIRC709. The nanobodies were site-specifically radiolabeled with 99mTc and 177Lu, and radiochemical purity was >95% after purification. The long blood circulation time and tumor retention property of 99mTc/177Lu-MIRC213-709 were confirmed by a blood clearance assay, single-photon emission computed tomography (SPECT), and a biodistribution study. The blood clearance assay showed that the distribution phase half-life (T1/2α) and elimination phase half-life (T1/2ß) of 99mTc-MIRC213-709 were 6.74- and 19.04-fold longer than those of 99mTc-MIRC213, respectively. The SPECT/CT and biodistribution results showed that the highest uptake of 177Lu-MIRC213 in the NCI-N87 model was 5.24 ± 0.95% ID/g at 6 h p.i., while the highest uptake of 177Lu-MIRC213-709 in the NCI-N87 model was 30.82 ± 7.29% ID/g at 48 h p.i. Compared with 177Lu-MIRC213, 177Lu-MIRC213-709 had a 16.9-fold increased tumor cumulative uptake (2606 ± 195.1 vs 153.9 ± 22.37% ID/g·h). The targeted radionuclide therapy assay was performed in the NCI-N87 tumor model, and treatment monitoring ended on day 32. The post-treatment/pretreatment tumor volumes were 12.99 ± 1.66, 3.58 ± 0.96, 1.26 ± 0.17, and 1.54 ± 0.50 in the 0, 9, and 18 MBq single-dose groups and the two 9 MBq divided dose group (14 days apart), respectively. All treatment groups showed significant therapeutic effects (P < 0.0001). Thus, fusion with the IgG-binding nanobody MIRC709 provides MIRC213 derivatives with improved metabolic properties for targeted radionuclide therapy.

99mTc-CD3813: A Nanobody-Based Single Photon Emission Computed Tomography Radiotracer with Clinical Potential for Myeloma Imaging and Evaluation of CD38 Expression.

Daratumumab (DARA) is an anti-CD38 monoclonal antibody for the treatment of multiple myeloma (MM). The tumor CD38 expression level is one of the important factors in determining the efficacy of DARA treatment. Therefore, there is an urgent clinical need for a noninvasive tool to evaluate the CD38 levels in cancer patients before, during, and after DARA treatment. In this study, we prepared a new molecular imaging probe 99mTc-CD3813, the 99mTc-labeled nanobody CD3813, for noninvasive imaging of CD38 expression by single photon emission computed tomography (SPECT). We evaluated 99mTc-CD3813 for its CD38 affinity and specificity and its capacity to image the CD38 expression in the MM and lymphoma xenografts models. 99mTc-CD3813 SPECT/CT is able to visualize subcutaneous/orthotopic myeloma lesions in animal models and has advantages over 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography. Excess DARA has less impact on its tumor uptake (3.14 ± 0.83 vs 2.29 ± 0.91 %ID/g, n.s.), strongly suggesting that there is no competition between 99mTc-CD3813 and DARA in binding to CD38. 99mTc-CD3813 SPECT/CT revealed significant reduction in CD38 expression in the Ramos-bearing mice under DARA treatment, as evidenced by their reduced tumor uptake (3.04 ± 0.70 vs 1.07 ± 0.28 %ID/cc, P < 0.001). 99mTc-CD3813 SPECT/CT was also able to detect the increased tumor uptake (0.79 ± 0.29 vs 2.12 ± 0.12 %ID/cc, P < 0.001) due to the upregulation of CD38 levels caused by all-trans retinoic acid infection. 99mTc-CD3813 is a promising SPECT radiotracer for imaging the CD38-positive tumors and has clinical potential as a molecular imaging tool for evaluation of the CD38 expression level in patients before, during, and after DARA treatment.

Preclinical evaluation of [99mTc]Tc-labeled anti-EpCAM nanobody for EpCAM receptor expression imaging by immuno-SPECT/CT.

PURPOSE: Overexpression of epithelial cell adhesion molecule (EpCAM) plays essential roles in tumorigenesis and tumor progression in almost all epithelium-derived cancer. Monitoring EpCAM expression in tumors can be used for the diagnosis, staging, and prognosis of cancer patients, as well as guiding the individualized treatment of EpCAM-targeted drugs. In this study, we described the synthesis and evaluation of a site-specifically [99mTc]Tc-labeled EpCAM-targeted nanobody for the SPECT/CT imaging of EpCAM expression. METHODS: We first prepared the [99mTc]Tc-HYNIC-G4K; then, it was site-specifically connected to EpCAM-targeted nanobody NB4. The in vitro characteristics of [99mTc]Tc-NB4 were investigated in HT-29 (EpCAM positive) and HL-60 (EpCAM negative) cells, while the in vivo studies were performed using small-animal SPECT/CT in the subcutaneous tumor models and the lymph node metastasis model to verify the specific targeting capacity as well as the potential applications of [99mTc]Tc-NB4. RESULTS: [99mTc]Tc-NB4 displayed a high EpCAM specificity both in vitro and in vivo. SPECT/CT imaging revealed that [99mTc]Tc-NB4 was cleared rapidly from the blood and normal organs except for the kidneys, and HT-29 tumors were clearly visualized in contrast with HL-60 tumors. The uptake value of [99mTc]Tc-NB4 in HT-29 tumors was increased continuously from 3.77 ± 0.39%ID/g at 0.5 h to 5.53 ± 0.82%ID/g at 12 h after injection. Moreover, the [99mTc]Tc-NB4 SPECT/CT could clearly image tumor-draining lymph nodes. CONCLUSION: [99mTc]Tc-NB4 is a broad-spectrum, specific, and sensitive SPECT radiotracer for the noninvasive imaging of EpCAM expression in the epithelium-derived cancer and revealed a great potential for the clinical translation.

Arabidopsis N6-methyladenosine reader CPSF30-L recognizes FUE signals to control polyadenylation site choice in liquid-like nuclear bodies.

The biological functions of the epitranscriptomic modification N6-methyladenosine (m6A) in plants are not fully understood. CPSF30-L is a predominant isoform of the polyadenylation factor CPSF30 and consists of CPSF30-S and an m6A-binding YTH domain. Little is known about the biological roles of CPSF30-L and the molecular mechanism underlying its m6A-binding function in alternative polyadenylation. Here, we characterized CPSF30-L as an Arabidopsis m6A reader whose m6A-binding function is required for the floral transition and abscisic acid (ABA) response. We found that the m6A-binding activity of CPSF30-L enhances the formation of liquid-like nuclear bodies, where CPSF30-L mainly recognizes m6A-modified far-upstream elements to control polyadenylation site choice. Deficiency of CPSF30-L lengthens the 3' untranslated region of three phenotypes-related transcripts, thereby accelerating their mRNA degradation and leading to late flowering and ABA hypersensitivity. Collectively, this study uncovers a new molecular mechanism for m6A-driven phase separation and polyadenylation in plants.

Monoclonal-Antibody-Templated Gold Nanoclusters for HER2 Receptors Targeted Fluorescence Imaging.

HER2 receptor-specific monoclonal-antibody-templated gold nanoclusters, Herceptin-templated Au NCs (Her-Au NCs), have been successfully obtained via "green" synthesis. This strategy allows the fluorescent gold nanoclusters (Au NCs) formed in the three-dimensional structure of Herceptin without destroying the high specificity and affinity to HER2 receptors. The Her-Au NCs have been found to be superior compared to Cy3-Herceptin in the fluorescence emission (λem = 645 nm) and the photostability under high-intensity UV irradiation or long-time storage. Moreover, Her-Au NCs can achieve receptor-specific imaging without targeted modification owing to the HER2-binding ability of the Herceptin scaffold. For imaging applications, Her-Au NCs can be utilized as effective optical probes for not only fluorescence imaging of HER2-positive cancer cells but also imaging of HER2-positive tumors in vivo.

Lectin-Mediated pH-Sensitive Doxorubicin Prodrug for Pre-Targeted Chemotherapy of Colorectal Cancer with Enhanced Efficacy and Reduced Side Effects.

Doxorubicin (DOX) has been clinically used as a broad-spectrum chemotherapeutic agent for decades, but its clinical application is hindered by the lack of tumour specificity, severe cardiotoxicity and haematotoxicity. Pre-targeted strategies are highly tumour-specific, therapeutic approaches. Herein, a novel pre-targeted system was constructed, aiming to enhance anticancer efficacy of DOX and maximally reduce its side effects. Methods: The DOX prodrug (bDOX) was first synthesized by conjugating DOX with mini-PEGylated (mPEGylated) biotin through a pH-sensitive bond. During the pre-targeted treatment, avidin was first administrated. After an optimized interval, bDOX was second administrated. The nontoxic prodrug bDOX was eventually transformed into the toxic anticancer form (DOX) by a pH-triggered cleavage specifically in tumour cells. The drug efficacy and side effect of the two-step, pre-targeted treatment were fully compared with free DOX in vitro and in vivo. Results: The prodrug bDOX was quite stable under neutral conditions and nearly nontoxic, but was immediately transformed into the toxic anticancer form (DOX) under acidic conditions. Compared to free DOX, the pre-targeted bDOX exhibited a higher cellular uptake by human colorectal tumour cells (LS180 and HT-29 cells). In vivo evaluation performed on LS180 xenograft animal model demonstrated that the pre-targeted bDOX achieved a much more significant tumour inhibition than free DOX. The largely decreased, unwanted bystander toxicity was demonstrated by changes in body weight, cardiomyocyte apoptosis, blood routine examination and splenic pathological changes. Conclusion: The high therapeutic efficacy, together with the minimal side effects, of this easily synthesized, pre-targeted system exhibited immense potentiality for the clinical application of DOX delivery.

Fusion Peptide Improves Stability and Bioactivity of Single Chain Antibody against Rabies Virus.

The combination of rabies immunoglobulin (RIG) with a vaccine is currently effective against rabies infections, but improvements are needed. Genetic engineering antibody technology is an attractive approach for developing novel antibodies to replace RIG. In our previous study, a single-chain variable fragment, scFv57R, against rabies virus glycoprotein was constructed. However, its inherent weak stability and short half-life compared with the parent RIG may limit its diagnostic and therapeutic application. Therefore, an acidic tail of synuclein (ATS) derived from the C-terminal acidic tail of human alpha-synuclein protein was fused to the C-terminus of scFv57R in order to help it resist adverse stress and improve the stability and halflife. The tail showed no apparent effect on the preparation procedure and affinity of the protein, nor did it change the neutralizing potency in vitro. In the ELISA test of molecular stability, the ATS fusion form of the protein, scFv57R-ATS, showed an increase in thermal stability and longer half-life in serum than scFv57R. The protection against fatal rabies virus challenge improved after fusing the tail to the scFv, which may be attributed to the improved stability. Thus, the ATS fusion approach presented here is easily implemented and can be used as a new strategy to improve the stability and half-life of engineered antibody proteins for practical applications.