On Demand Bioorthogonal Switching of an Antibody-Conjugated SPECT Probe to a Cytotoxic Payload: from Imaging to Therapy.

Antibody-drug conjugates (ADCs) for the treatment of cancer aim to achieve selective delivery of a cytotoxic payload to tumor cells while sparing normal tissue. In vivo, multiple tumor-dependent and -independent processes act on ADCs and their released payloads to impact tumor-versus-normal delivery, often resulting in a poor therapeutic window. An ADC with a labeled payload would make synchronous correlations between distribution and tissue-specific pharmacological effects possible, empowering preclinical and clinical efforts to improve tumor-selective delivery; however, few methods to label small molecules without destroying their pharmacological activity exist. Herein, we present a bioorthogonal switch approach that allows a radiolabel attached to an ADC payload to be removed tracelessly at will. We exemplify this approach with a potent DNA-damaging agent, the pyrrolobenzodiazepine (PBD) dimer, delivered as an antibody conjugate targeted to lung tumor cells. The radiometal chelating group, DOTA, was attached via a novel trans-cyclooctene (TCO)-caged self-immolative para-aminobenzyl (PAB) linker to the PBD, stably attenuating payload activity and allowing tracking of biodistribution in tumor-bearing mice via SPECT-CT imaging (live) or gamma counting (post-mortem). Following TCO-PAB-DOTA reaction with tetrazines optimized for extra- and intracellular reactivity, the label was removed to reveal the unmodified PBD dimer capable of inducing potent tumor cell killing in vitro and in mouse xenografts. The switchable antibody radio-drug conjugate (ArDC) we describe integrates, but decouples, the two functions of a theranostic given that it can serve as a diagnostic for payload delivery in the labeled state, but can be switched on demand to a therapeutic agent (an ADC).

Underwater biomimetic orientation method using imaging polarization sensor based on direct sunlight compensation.

This paper addresses the challenge of significant interference caused by direct sunlight, which adversely affects the orientation accuracy of underwater imaging polarization sensors (IPS). A novel underwater polarization orientation method is proposed based on direct sunlight compensation. Firstly, based on the polarization transmission model at the water-air interface, the interference mechanism of the underwater direct sunlight polarization detection model was analyzed. The underwater IPS detection model based on direct sunlight compensation is constructed, which uses the weight coefficient of underwater direct sunlight to compensate for the interference on the polarization channel and improve the accuracy of underwater polarization detection models. Furthermore, the analytical solution method for the polarization state information of underwater IPS is proposed, employing the augmented Stokes vectors to construct a linear equation for solving the weight coefficients of direct sunlight and improving the computational efficiency. Finally, an underwater polarization orientation experimental platform is established, and both simulation and actual underwater experiments are conducted. Compared with the traditional methods, the proposed method reduces heading error by an average of 92.53% at different solar altitudes.

Attitude and heading measurement based on adaptive complementary Kalman filter for PS/MIMU integrated system.

The bionic polarization sensor (PS)/MEMS inertial measurement unit (MIMU) integrated system can provide reliable attitude and heading information for unmanned vehicles in the case of GNSS rejection. However, the existing measurement methods have poor adaptability to inclining, sheltering, and other harsh environments, and do not make full use of the complementary characteristics of the gyroscopes, accelerometers, and PS, which seriously affects the system performance. Therefore, this paper proposes an attitude and heading measurement method based on an adaptive complementary Kalman filter (ACKF), which corrects the gyroscopes according to the gravity measured by the accelerometers to improve the attitude accuracy and fuses the IMU heading and tilt-compensated polarization heading by Kalman optimal estimation. On this basis, the maximum correlation entropy of the measured gravity and the theoretical gravity is used to construct an adaptive factor to realize the adaptive complementary of the gyroscopes and the accelerometers. Finally, the effectiveness of the method is verified by the outdoor rotation test without occlusion and the vehicle test with occlusion. Compared with the traditional Kalman filter, the pitch, roll, and heading RMSE of the vehicle test are reduced by 89.3%, 93.2% and, 9.6% respectively, which verifies the great advantages.

An Improved Bio-Orientation Method Based on Direct Sunlight Compensation for Imaging Polarization Sensor.

Direct sunlight in complex environmental conditions severely interferes with the light intensity response for imaging Polarization Sensor (PS), leading to a reduction in polarization orientation accuracy. Addressing this issue, this article analyzes the impact mechanism of direct sunlight on polarization sensor detection in a complex environment. The direct sunlight interference factor is introduced into the intensity response model of imaging polarization detection, enhancing the accuracy of the polarization detection model. Furthermore, a polarization state information analytical solution model based on direct sunlight compensation is constructed to improve the accuracy and real-time performance of the polarization state information solution. On this basis, an improved bio-orientation method based on direct sunlight compensation for imaging polarization sensor is proposed. The outdoor dynamic reorientation experiment platform is established to validate the effectiveness of the proposed method. Compared with the traditional methods, the experimental results demonstrate a 23% to 47% improvement in the polarization orientation accuracy under various solar zenith angles.

The HER2-directed antibody-drug conjugate DHES0815A in advanced and/or metastatic breast cancer: preclinical characterization and phase 1 trial results.

Approved antibody-drug conjugates (ADCs) for HER2-positive breast cancer include trastuzumab emtansine and trastuzumab deruxtecan. To develop a differentiated HER2 ADC, we chose an antibody that does not compete with trastuzumab or pertuzumab for binding, conjugated to a reduced potency PBD (pyrrolobenzodiazepine) dimer payload. PBDs are potent cytotoxic agents that alkylate and cross-link DNA. In our study, the PBD dimer is modified to alkylate, but not cross-link DNA. This HER2 ADC, DHES0815A, demonstrates in vivo efficacy in models of HER2-positive and HER2-low cancers and is well-tolerated in cynomolgus monkey safety studies. Mechanisms of action include induction of DNA damage and apoptosis, activity in non-dividing cells, and bystander activity. A dose-escalation study (ClinicalTrials.gov: NCT03451162) in patients with HER2-positive metastatic breast cancer, with the primary objective of evaluating the safety and tolerability of DHES0815A and secondary objectives of characterizing the pharmacokinetics, objective response rate, duration of response, and formation of anti-DHES0815A antibodies, is reported herein. Despite early signs of anti-tumor activity, patients at higher doses develop persistent, non-resolvable dermal, ocular, and pulmonary toxicities, which led to early termination of the phase 1 trial.

Analysis of sleep for the American population: Result from NHANES database.

OBJECTIVES: To assess the contemporary prevalence and decade-long trends of sleep duration, sleep disorders and trouble sleeping among adults in the United States, as well as their risk factors, from 2005 to 2018. MATERIALS AND METHODS: We used National Health and Nutrition Examination Survey data to calculate the sleep duration and weighted prevalence of sleep disorders and trouble sleeping in adults aged 20 years or older. Sleep duration, sleep disorders and trouble sleeping were assessed by questionnaire. RESULTS: A total of 27,399 people were included in the survey on sleep duration, with a weighted percentage of normal sleep (7-8 h/night) of 56.33 % (95 % CI, 53.06-59.60 %) and a weighted percentage of short sleep (5-6 h/night) of 31.73 %. In stratified descriptions, participants aged 40-49 years were more likely to sleep less than five hours, while women aged 80 years and older were more likely to sleep longer and blacks were more likely to sleep shorter. A total of 27,406 participants were included in the survey for sleep disorders. The weighted proportion of the population with sleep disorders was 8.44 % (95 % CI, 7.79-9.8 %). Independent risk factors for sleep disorders were being 40-69 years old, being white, having a high education level, smoking, having hypertension, diabetes, heart disease, and BMI ≥ 25. From 2005 to 2014, the prevalence of sleep disorders increased year by year, from 7.44 % in 2005-2006 to 10.40 % in 2013-2014 (P for Trend<0.001). A total of 38,165 participants were included in the survey on trouble sleeping. The weighted proportion of the population with troubled sleeping was 27.30 % (25.70-28.90 %). Independent risk factors for troubled sleeping were being 30-79 years old, being white, having a high education level, smoking, drinking, having hypertension, diabetes, heart disease and BMI ≥ 25. From 2005 to 2018, the prevalence of trouble sleeping increased annually, from 24.44 % in 2005-2006 to 30.58 % in 2017-2018 (P for trend<0.001). CONCLUSION: Adults in the United States are likely to have abnormal sleep durations, and the prevalence of sleep disorders and troubled sleeping is on the rise.

A high-sensitivity fiber Bragg grating sensor for displacement measurement in structural health monitoring.

Displacement measurement is of great significance to monitor the crack variation and ensure the health of building structures. Aiming at the problems of low sensitivity and high temperature error of fiber Bragg grating (FBG) displacement sensors in displacement monitoring, this paper presents an adjustable cantilever beam displacement sensor with the FBGs as the sensing element. The sensor adds double FBGs on the relative surfaces of the equal-strength cantilever beam, which increases the bending deformation on the FBG of the beam surface to improve the sensitivity and realize the temperature compensation of the sensor. By adding an adjustable external rod structure between a flexible spring and a fixed foot stand, the sensor can regulate the range of initial crack width for different occasions. A theoretical analysis of the displacement sensor is performed, and the simulation analysis and optimization design for the structural parameters of the cantilever beam elastic sensitive element are implemented by adopting SolidWorks and ANSYS software. Finally, a displacement testing platform is constructed to test its performance. Experimental results show that this design has a high sensitivity coefficient of 39.47 pm/mm and a temperature coefficient of 1.04 pm/°C in the range of initial crack width from 0 to 110 mm or from 0 to 130 mm depending on different monitoring situations. Furthermore, good linearity, hysteresis delay, repeatability, and temperature compensation performance have also been demonstrated.

High-Accuracy Relative Pose Measurement of Noncooperative Objects Based on Double-Constrained Intersurface Mutual Projections.

Relative pose measurement for noncooperative objects is an important part of 3D shape recognition and motion tracking. The methods based on scanning point clouds have better environmental adaptability and stability than image-based methods. However, the discrete points obtained from a continuous surface are sparse, which leads to point-to-point dislocations in the overlapping area and seriously reduces the accuracy. Therefore, this paper proposed a relative-pose-measurement algorithm based on double-constrained intersurface mutual projections. First, the initial corresponding set was constructed using mutual projections between the areas with similar feature descriptors, and then the final corresponding set was determined through the rigid-transformation-consistency constraint to improve the accuracy of the matchings and achieve a high-accuracy relative pose measurement. In the Stanford dataset, the rotation error and translation error were reduced by 19.3% and 13.4%, respectively. Furthermore, based on the proposed evaluation method, which separated the error of the pose-measurement algorithm from that of the instrument, the experiments were carried out with a self-made swept-frequency interferometer. The rotation error was reduced by 39.8%, and the surface deviation was reduced by 4.9%, which further proved the advancement of the method.

Chiral FA Conjugated CdTe/CdS Quantum Dots for Selective Cancer Ablation.

Inducing apoptosis in cancer cells is considered a potential therapeutic mechanism underlying cancers. Here, chiral folic acid (FA) conjugated Cys-CdTe/CdS quantum dots (QDs) conjugated with a cancer-targeting ligand were fabricated to induce apoptosis in vivo. Ligand-induced chirality mechanism for FA-Cys-CdTe/CdS QDs was discussed, which is verified by density functional theory (DFT) simulation. Interestingly, we found that the circular dichroism (CD) signals of chiral QDs can effectively distinguish breast cancer cells from normal cells, where a sharp decrease in CD signal and absorption intensity can be seen. Notably, chiral FA-Cys-CdTe/CdS QDs showed significant apoptosis-inducing ability after the release of mitochondrial apoptotic factors. Furthermore, in vivo experiments showed that chiral FA-Cys-CdTe/CdS QDs provide an efficient cancer ablation through the apoptosis process with negligible toxicity, demonstrating their great potential utility in targeted anticancer agent for future clinic application.

A homogeneous high-DAR antibody-drug conjugate platform combining THIOMAB antibodies and XTEN polypeptides.

The antibody-drug conjugate (ADC) is a well-validated modality for the cell-specific delivery of small molecules with impact expanding rapidly beyond their originally-intended purpose of treating cancer. However, antibody-mediated delivery (AMD) remains inefficient, limiting its applicability to targeting highly potent payloads to cells with high antigen expression. Maximizing the number of payloads delivered per antibody is one key way in which delivery efficiency can be improved, although this has been challenging to carry out; with few exceptions, increasing the drug-to-antibody ratio (DAR) above ∼4 typically destroys the biophysical properties and in vivo efficacy for ADCs. Herein, we describe the development of a novel bioconjugation platform combining cysteine-engineered (THIOMAB) antibodies and recombinant XTEN polypeptides for the unprecedented generation of homogeneous, stable "TXCs" with DAR of up to 18. Across three different bioactive payloads, we demonstrated improved AMD to tumors and Staphylococcus aureus bacteria for high-DAR TXCs relative to conventional low-DAR ADCs.

Identification and elimination of cancer cells by folate-conjugated CdTe/CdS Quantum Dots Chiral Nano-Sensors.

The specific identification and elimination of cancer cells has been a great challenge in the past few decades. In this study, the circular dichroism (CD) of cells was measured by a self-designed special system through the folate-conjugated chiral nano-sensor. A novel method was established to recognize cancer cells from normal cells according to the chirality of cells based on their CD signals. After a period of interaction between the nano-sensor and cells, the sharp weakening of CD signals was induced in cancer cells but normal cells remained unchanged. The biocompatibility of the nano-sensor was evaluated and the result showed that it exhibited significant cytotoxic activity against cancer cells while no obvious damage on normal cells. Notably, the research indicated that the nano-sensor may selectively cause apoptosis in cancer cells, and thus, have the potential to act as an antitumor agent.

Systematic Variation of Pyrrolobenzodiazepine (PBD)-Dimer Payload Physicochemical Properties Impacts Efficacy and Tolerability of the Corresponding Antibody-Drug Conjugates.

Cytotoxic pyrrolobenzodiazepine (PBD)-dimer molecules are frequently utilized as payloads for antibody-drug conjugates (ADCs), and many examples are currently in clinical development. In order to further explore this ADC payload class, the physicochemical properties of various PBD-dimer molecules were modified by the systematic introduction of acidic and basic moieties into their chemical structures. The impact of these changes on DNA binding, cell membrane permeability, and in vitro antiproliferation potency was, respectively, determined using a DNA alkylation assay, PAMPA assessments, and cell-based cytotoxicity measurements conducted with a variety of cancer lines. The modified PBD-dimer compounds were subsequently incorporated into CD22-targeting ADCs, and these entities were profiled in a variety of in vitro and in vivo experiments. The introduction of a strongly basic moiety into the PBD-dimer scaffold afforded a conjugate with dramatically worsened mouse tolerability properties relative to ADCs derived from related payloads, which lacked the basic group.

Fluorescence and Optical Activity of Chiral CdTe Quantum Dots in Their Interaction with Amino Acids.

Ligand-induced chirality in semiconducting nanocrystals has been the subject of extensive study in the past few years and shows potential applications in optics and biology. Yet, the origin of the chiroptical effect in semiconductor nanoparticles is still not fully understood. Here, we examine the effect of the interaction with amino acids on both the fluorescence and the optical activity of chiral semiconductor quantum dots (QDs). A significant fluorescence enhancement is observed for l/d-Cys-CdTe QDs upon interaction with all the tested amino acids, indicating suppression of nonradiative pathways as well as the passivation of surface trap sites brought via the interaction of the amino group with the CdTe QDs' surface. Heterochiral amino acids are shown to weaken the circular dichroism (CD) signal, which may be attributed to a different binding configuration of cysteine molecules on the QDs' surface. Furthermore, a red shift of both CD and fluorescence signals in l/d-Cys-CdTe QDs is only observed upon adding cysteine, while other tested amino acids do not exhibit such an effect. We speculate that the thiol group induces orbital hybridization of the highest occupied molecular orbital (HOMOs) of cysteine and the valence band of CdTe QDs, leading to the decrease of the energy band gap and a concomitant red shift of CD and fluorescence spectra. This is further verified by density functional theory calculations. Both the experimental and theoretical findings indicate that the addition of ligands that do not "directly" interact with the valence band (VB) of the QD (noncysteine moieties) changes the QD photophysical properties, as it probably modifies the way cysteine is bound to the surface. Hence, we conclude that it is not only the chemistry of the amino acid ligand that affects both CD and PL but also the exact geometry of binding that modifies these properties. Understanding the relationship between the QD's surface and chiral amino acid thus provides an additional perspective on the fundamental origin of induced chiroptical effects in semiconductor nanoparticles, potentially enabling us to optimize the design of chiral semiconductor QDs for chiroptic applications.

Development, Optimization, and Structural Characterization of an Efficient Peptide-Based Photoaffinity Cross-Linking Reaction for Generation of Homogeneous Conjugates from Wild-Type Antibodies.

Site-specific conjugation of small molecules to antibodies represents an attractive goal for the development of more homogeneous targeted therapies and diagnostics. Most site-specific conjugation strategies require modification or removal of antibody glycans or interchain disulfide bonds or engineering of an antibody mutant that bears a reactive handle. While such methods are effective, they complicate the process of preparing antibody conjugates and can negatively impact biological activity. Herein we report the development and detailed characterization of a robust photoaffinity cross-linking method for site-specific conjugation to fully glycosylated wild-type antibodies. The method employs a benzoylphenylalanine (Bpa) mutant of a previously described 13-residue peptide derived from phage display to bind tightly to the Fc domain; upon UV irradiation, the Bpa residue forms a diradical that reacts with the bound antibody. After the initial discovery of an effective Bpa mutant peptide and optimization of the reaction conditions to enable efficient conjugation without concomitant UV-induced photodamage of the antibody, we assessed the scope of the photoconjugation reaction across different human and nonhuman antibodies and antibody mutants. Next, the specific site of conjugation on a human antibody was characterized in detail by mass spectrometry experiments and at atomic resolution by X-ray crystallography. Finally, we adapted the photoconjugation method to attach a cytotoxic payload site-specifically to a wild-type antibody and showed that the resulting conjugate is both stable in plasma and as potent as a conventional antibody-drug conjugate in cells, portending well for future biological applications.

Exploration of Pyrrolobenzodiazepine (PBD)-Dimers Containing Disulfide-Based Prodrugs as Payloads for Antibody-Drug Conjugates.

A number of cytotoxic pyrrolobenzodiazepine (PBD) monomers containing various disulfide-based prodrugs were evaluated for their ability to undergo activation (disulfide cleavage) in vitro in the presence of either glutathione (GSH) or cysteine (Cys). A good correlation was observed between in vitro GSH stability and in vitro cytotoxicity toward tumor cell lines. The prodrug-containing compounds were typically more potent against cells with relatively high intracellular GSH levels (e.g., KPL-4 cells). Several antibody-drug conjugates (ADCs) were subsequently constructed from PBD dimers that incorporated selected disulfide-based prodrugs. Such HER2 conjugates exhibited potent antiproliferation activity against KPL-4 cells in vitro in an antigen-dependent manner. However, the disulfide prodrugs contained in the majority of such entities were surprisingly unstable toward whole blood from various species. One HER2-targeting conjugate that contained a thiophenol-derived disulfide prodrug was an exception to this stability trend. It exhibited potent activity in a KPL-4 in vivo efficacy model that was approximately three-fold weaker than that displayed by the corresponding parent ADC. The same prodrug-containing conjugate demonstrated a three-fold improvement in mouse tolerability properties in vivo relative to the parent ADC, which did not contain the prodrug.

Mechanisms of Acquired Resistance to Trastuzumab Emtansine in Breast Cancer Cells.

The receptor tyrosine kinase HER2 is overexpressed in approximately 20% of breast cancer, and its amplification is associated with reduced survival. Trastuzumab emtansine (Kadcyla, T-DM1), an antibody-drug conjugate that is comprised of trastuzumab covalently linked to the antimitotic agent DM1 through a stable linker, was designed to selectively deliver DM1 to HER2-overexpressing tumor cells. T-DM1 is approved for the treatment of patients with HER2-positive metastatic breast cancer following progression on trastuzumab and a taxane. Despite the improvement in clinical outcome, many patients who initially respond to T-DM1 treatment eventually develop progressive disease. The mechanisms that contribute to T-DM1 resistance are not fully understood. To this end, we developed T-DM1-resistant in vitro models to examine the mechanisms of acquired T-DM1 resistance. We demonstrate that decreased HER2 and upregulation of MDR1 contribute to T-DM1 resistance in KPL-4 T-DM1-resistant cells. In contrast, both loss of SLC46A3 and PTEN deficiency play a role in conferring resistance in BT-474M1 T-DM1-resistant cells. Our data suggest that these two cell lines acquire resistance through distinct mechanisms. Furthermore, we show that the KPL-4 T-DM1 resistance can be overcome by treatment with an inhibitor of MDR1, whereas a PI3K inhibitor can rescue PTEN loss-induced resistance in T-DM1-resistant BT-474M1 cells. Our results provide a rationale for developing therapeutic strategies to enhance T-DM1 clinical efficacy by combining T-DM1 and other inhibitors that target signaling transduction or resistance pathways. Mol Cancer Ther; 17(7); 1441-53. ©2018 AACR.

Discovery of Peptidomimetic Antibody-Drug Conjugate Linkers with Enhanced Protease Specificity.

Antibody-drug conjugates (ADCs) have become an important therapeutic modality for oncology, with three approved by the FDA and over 60 others in clinical trials. Despite the progress, improvements in ADC therapeutic index are desired. Peptide-based ADC linkers that are cleaved by lysosomal proteases have shown sufficient stability in serum and effective payload-release in targeted cells. If the linker can be preferentially hydrolyzed by tumor-specific proteases, safety margin may improve. However, the use of peptide-based linkers limits our ability to modulate protease specificity. Here we report the structure-guided discovery of novel, nonpeptidic ADC linkers. We show that a cyclobutane-1,1-dicarboxamide-containing linker is hydrolyzed predominantly by cathepsin B while the valine-citrulline dipeptide linker is not. ADCs bearing the nonpeptidic linker are as efficacious and stable in vivo as those with the dipeptide linker. Our results strongly support the application of the peptidomimetic linker and present new opportunities for improving the selectivity of ADCs.

Pyrrolobenzodiazepine Dimer Antibody-Drug Conjugates: Synthesis and Evaluation of Noncleavable Drug-Linkers.

Three rationally designed pyrrolobenzodiazepine (PBD) drug-linkers have been synthesized via intermediate 19 for use in antibody-drug conjugates (ADCs). They lack a cleavable trigger in the linker and consist of a maleimide for cysteine antibody conjugation, a hydrophilic spacer, and either an alkyne (6), triazole (7), or piperazine (8) link to the PBD. In vitro IC50 values were 11-48 ng/mL in HER2 3+ SK-BR-3 and KPL-4 (7 inactive) for the anti-HER2 ADCs (HER2 0 MCF7, all inactive) and 0.10-1.73 µg/mL (7 inactive) in CD22 3+ BJAB and WSU-DLCL2 for anti-CD22 ADCs (CD22 0 Jurkat, all inactive at low doses). In vivo antitumor efficacy for the anti-HER2 ADCs in Founder 5 was observed with tumor stasis at 0.5-1 mg/kg, 1 mg/kg, and 3-6 mg/kg for 6, 8, and 7, respectively. Tumor stasis at 2 mg/kg was observed for anti-CD22 6 in WSU-DLCL2. In summary, noncleavable PBD-ADCs exhibit potent activity, particularly in HER2 models.

Attachment Site Cysteine Thiol pKa Is a Key Driver for Site-Dependent Stability of THIOMAB Antibody-Drug Conjugates.

The incorporation of cysteines into antibodies by mutagenesis allows for the direct conjugation of small molecules to specific sites on the antibody via disulfide bonds. The stability of the disulfide bond linkage between the small molecule and the antibody is highly dependent on the location of the engineered cysteine in either the heavy chain (HC) or the light chain (LC) of the antibody. Here, we explore the basis for this site-dependent stability. We evaluated the in vivo efficacy and pharmacokinetics of five different cysteine mutants of trastuzumab conjugated to a pyrrolobenzodiazepine (PBD) via disulfide bonds. A significant correlation was observed between disulfide stability and efficacy for the conjugates. We hypothesized that the observed site-dependent stability of the disulfide-linked conjugates could be due to differences in the attachment site cysteine thiol pKa. We measured the cysteine thiol pKa using isothermal titration calorimetry (ITC) and found that the variants with the highest thiol pKa (LC K149C and HC A140C) were found to yield the conjugates with the greatest in vivo stability. Guided by homology modeling, we identified several mutations adjacent to LC K149C that reduced the cysteine thiol pKa and, thus, decreased the in vivo stability of the disulfide-linked PBD conjugated to LC K149C. We also present results suggesting that the high thiol pKa of LC K149C is responsible for the sustained circulation stability of LC K149C TDCs utilizing a maleimide-based linker. Taken together, our results provide evidence that the site-dependent stability of cys-engineered antibody-drug conjugates may be explained by interactions between the engineered cysteine and the local protein environment that serves to modulate the side-chain thiol pKa. The influence of cysteine thiol pKa on stability and efficacy offers a new parameter for the optimization of ADCs that utilize cysteine engineering.

JS-K, a nitric oxide prodrug, induces DNA damage and apoptosis in HBV-positive hepatocellular carcinoma HepG2.2.15 cell.

Hepatocellular carcinoma (HCC) is the most important cause of cancer-related death, and 85% of HCC is caused by chronic HBV infection, the prognosis of patients and the reduction of HBV DNA levels remain unsatisfactory. JS-K, a nitric oxide-releasing diazeniumdiolates, is effective against various tumors, but little is known on its effects on HBV positive HCC. We found that JS-K reduced the expression of HBsAg and HBeAg in HBV-positive HepG2.2.15 cells. This study aimed to further examine anti-tumor effects of JS-K on HepG2.2.15 cells. The MTT assay and colony forming assay were used to study the cell growth inhibition of JS-K; scratch assay and transwell assay were performed to detect cell migration. The cell cycle was detected by flow cytometry. The immunofluorescence, flow cytometry analysis, and western blot were used to study DNA damage and cell apoptosis. JS-K inhibited HepG2.2.15 cell growth in a dose-dependent manner, suppressed cell colony formation and migration, arrested cells gather in the G2 phase. JS-K (1-20µM) increased the expression of DNA damage-associated protein phosphorylation H2AX (γH2AX), phosphorylation of checkpoint kinase 1 (p-Chk1), phosphorylation of checkpoint kinase 2 (p-Chk2), ataxia-telangiectasia mutated (ATM), phosphorylation of ataxia-telangiectasia mutated rad3-related (p-ATR) and apoptotic-associated proteins cleaved caspase-3, cleaved caspase-7, cleaved poly ADP-ribose polymerase (cleaved PARP). The study demonstrated JS-K is effective against HBV-positive HepG2.2.15 cells, the mechanisms are not only related to inhibition of HBsAg and HBeAg secretion, but also related with induction of DNA damage and apoptosis. JS-K is a promising anti-cancer candidate against HBV-positive HCC.