High Intrinsic Affinity Research Articles

Convergent High O2 Affinity but Distinct ATP-Mediated Allosteric Regulation of Hemoglobins in Oviparous and Viviparous Eremias Lizards from the Qinghai-Tibet Plateau.

The functional adaptation and underlying molecular mechanisms of hemoglobins (Hbs) have primarily concentrated on mammals and birds, with few reports on reptiles. This study aimed to investigate the convergent and species-specific high-altitude adaptation mechanisms of Hbs in two Eremias lizards from the Qinghai-Tibet Plateau. The Hbs of high-altitude E. argus and E. multiocellata were characterized by significantly high overall and intrinsic Hb-O2 affinity compared to their low-altitude populations. Despite the similarly low Cl- sensitivities, the Hbs of high-altitude E. argus exhibited higher ATP sensitivity and ATP-dependent Bohr effects than that of E. multiocellata, which could facilitate O2 unloading in respiring tissues. Eremias lizards Hbs exhibited similarly low temperature sensitivities and relatively high Bohr effects at lower temperatures, which could help to stably deliver and release O2 to cold extremities at low temperatures. The oxygenation properties of Hbs in high-altitude populations might be attributed to varying ratios of β2/β1 globin and substitutions on the β2-type globin. Notably, the Asn12Ala in lowland E. argus could cause localized destabilization of the E-helix in the tetrameric Hb by elimination of hydrogen bonds, thereby resulting in its lowest O2 affinity. This study provides a valuable reference for the high-altitude adaptation mechanisms of hemoglobins in reptiles.

Nitrogen-doped carbon nanotube encapsulated Co9S8 composite cathode for high-selective capacitive extraction of uranium (VI) from radioactive wastewater

Selective extraction of uranium [U(VI)] from uranium containing nuclear wastewater is of great significance in alleviating the uranium resources shortages and eliminating the radioactive radiation. Benefited by the merits of cost-effectiveness and environment-friendliness, capacitive deionization (CDI) shows a great promise towards selective removal of U(VI). However, exploring novel electrode materials with high intrinsic affinity rather than the excessive assistance of external means faces challenges. Herein, a nitrogen-doped carbon tube encapsulated Co9S8 composite (CS-NCNT) was developed as a Faradic cathode for selective electrosorption of U(VI) from radioactive wastewater. This work mainly emphasized on these following results: (i) The electrochemical redox activity and high surface-controlled ratios endowed the optimized CS-NCNT-2 electrode a maximum electrosorption capacity (189 mg g−1) towards U(VI). (ii) Except that CS-NCNT-2 exhibited excellent selective electrosorption performance in U(VI)-containing spiked real seawater, an ultrahigh selectivity coefficient of 707 (UO22+ over Na+) was also achieved at a Na+: UO22+ molar ratio of 200. (iii) The valence state analysis and molecular dynamic simulation revealed the essence of the reversible electrosorption-desorption process and the preferable extraction towards UO22+ compared with Na+ ions, respectively. Overall, this work can be potentially expanded to other metal chalcogenides-based electrode materials for selective removal of UO22+ even other radionuclide in U(VI)-containing radioactive wastewater.

Manganese-doped albumin-gelatin composite nanogel loaded with berberine applied to the treatment of gouty arthritis in rats via a SPARC-dependent mechanism

In this study, manganese-doped albumin-gelatin composite nanogels (MAGN) were prepared and used to load berberine (Ber) for the treatment of gouty arthritis (GA). The nanodrug delivery system (Ber-MAGN) can target inflammatory joints due to the intrinsic high affinity of albumin for SPARC, which is overexpressed at the inflammatory site of GA. Characterization of the pharmaceutical properties in vitro showed that Ber-MAGN had good dispersion, and the particle size was 121 ± 10.7 nm. The sustained release effect significantly improved the bioavailability of berberine. In vitro and in vivo experimental results showed that Ber-MAGN has better therapeutic effects in relieving oxidative stress and suppressing inflammation. Therefore, Ber-MAGN, as a potential pharmaceutical preparation for GA, provides a new reference for the clinical treatment plan of GA.

A novel mechanism for high-altitude adaptation in hemoglobin of black-spotted frog (Pelophylax nigromaculatus)

Understanding how animals living in highland adapt to extreme conditions is critical to evolutionary biology. In contrast to birds and mammals, little information was available on the adaptation mechanisms for O2 transport in high-altitude ectothermic vertebrates. Here we report for the first time on hematological parameters, amino acid sequences of α and β chains of hemoglobin (Hb), O2 affinity of purified hemoglobins (Hbs) and their sensitivities to anion allosteric effector (H+, Cl−, ATP) and temperature in the high-altitude (2,292 m) black-spotted frogs (Pelophylax nigromaculatus) from the Qinghai-Tibet plateau (QTP) compared with the low-altitude (135 m) population. Our results showed that high-altitude black-spotted frogs exhibit significantly increased relative lung mass, hematocrit, and hemoglobin concentration, but significantly decreased body mass and erythrocyte volume, which could improve the blood O2 carrying capability. Compared with the low-altitude population, the purified Hbs of high-altitude black-spotted frogs possessed significantly higher intrinsic Hb-O2 affinity, similar low anion allosteric effector sensitivities, Bohr effects and temperature sensitivities. The elevated Hb-O2 affinity of highland frogs could maximize the O2 extraction from the lungs. Molecular dynamics simulations showed that the Gln123Glu substitution on α2 chain in highland frogs could form a hydrogen bond with 127Lys on α2 chain, resulting in the elimination of a hydrogen bond between 127Lys on α2 chain and 141Arg on α1 chain. This could weaken the interaction between two semirigid dimers (α1β1 and α2β2) and then lead to the high intrinsic Hb-O2 affinity in high-altitude black-spotted frogs.

Synthesis and 68Ga radiolabelling of calcium alginate beads for positron emission particle tracking (PEPT) applications

Positron emission particle tracking (PEPT) relies on the back-to-back γ-rays generated through a positron annihilation to track a single radiolabelled particle in three dimensions and in dense, multiphase, and opaque systems. The spectrum of applications of PEPT such as batch mixing systems in chemical, food and pharmaceutical sectors or granulation, die-filling and fluidisation in industrial processes, is directly related to the development of efficient radiolabelled materials and often restricted due to the lack of suitable materials with adequate radiochemical properties. In this work, we report a straightforward synthesis and radiolabelling of calcium alginate beads of different size, specific surface, concentration, and density, with the positron emitter gallium-68 (68Ga) to cover a broad range of possibilities in PEPT. We demonstrate a high intrinsic affinity between alginate and 68Ga providing directly radiolabelled particles with excellent radiochemical properties and excellent performance for tracking complex trajectories. These results, in combination with the availability and relatively simple chemistry of 68Ga, provide a high versatile and straightforward platform for the future development and advance of PEPT in different applications.

Arsenal of nanobodies shows broad-spectrum neutralization against SARS-CoV-2 variants of concern in vitro and in vivo in hamster models

Nanobodies offer several potential advantages over mAbs for the control of SARS-CoV-2. Their ability to access cryptic epitopes conserved across SARS-CoV-2 variants of concern (VoCs) and feasibility to engineer modular, multimeric designs, make these antibody fragments ideal candidates for developing broad-spectrum therapeutics against current and continually emerging SARS-CoV-2 VoCs. Here we describe a diverse collection of 37 anti-SARS-CoV-2 spike glycoprotein nanobodies extensively characterized as both monovalent and IgG Fc-fused bivalent modalities. The nanobodies were collectively shown to have high intrinsic affinity; high thermal, thermodynamic and aerosolization stability; broad subunit/domain specificity and cross-reactivity across existing VoCs; wide-ranging epitopic and mechanistic diversity and high and broad in vitro neutralization potencies. A select set of Fc-fused nanobodies showed high neutralization efficacies in hamster models of SARS-CoV-2 infection, reducing viral burden by up to six orders of magnitude to below detectable levels. In vivo protection was demonstrated with anti-RBD and previously unreported anti-NTD and anti-S2 nanobodies. This collection of nanobodies provides a potential therapeutic toolbox from which various cocktails or multi-paratopic formats could be built to combat multiple SARS-CoV-2 variants.

Metal‐free phthalimide‐labeled peptide nucleic acids for electrochemical biosensing applications

AbstractPeptide nucleic acids (PNAs) are neutral mimics of natural DNA and RNA biopolymers that have caught the attention of researchers working on the identification of specific sequences of nucleobases in DNA/RNA strands. For this purpose, specific analytical protocols need to be developed to optimize the nucleic acid recognition ability of PNAs, exploiting both the high intrinsic affinity of PNA/DNA(RNA) couples as well as suitable markers, either linked to the PNA backbone or properly interacting with it in the working medium. In this context, the paper reports on phthalimide and 4‐nitrophthalimide as two cheap, metal‐free electroactive markers covalently bound to the pseudo‐peptide backbone of a PNA decamer. After a preliminary characterization of the markers, as such and in PNA conjugates, attention has been moved toward the optimization of the detectability of the labeled PNA decamers in aqueous solutions. Exploiting the potentiometric stripping analysis on hanging mercury drop electrode it has been possible to reach satisfactory detection limits of ca. 10 nM avoiding the use of expensive transition metal complexes as labels and/or of co‐reagents.

Strategies to Maximize Anthracycline Drug Loading in Albumin Microbubbles.

Human serum albumin (HSA) microbubbles (MBs) are attracting increasing attention as image-guided and stimuli-responsive drug delivery systems. To better understand and maximize drug encapsulation in HSA MBs, we investigated the impact of the loading strategy and the drugs' physicochemical properties on their entrapment in the MB shell. Regarding loading strategy, we explored preloading, i.e., incubating drugs with HSA prior to MB formation, as well as postloading, i.e., incubating drugs with preformed MB. Both strategies were utilized to encapsulate six anthracyclines with different physicochemical properties. We demonstrate that drug loading in the HSA MB shell profits from preloading as well as from employing drugs with high intrinsic HSA binding affinity. These findings exemplify the potential of exploiting the natural bioconjugation interactions between drugs and HSA to formulate optimally loaded MBs, and they promote the development of HSA MBs for ultrasound-triggered drug delivery.

Conformational equilibria in allosteric control of Hsp70 chaperones

Heat-shock proteins of 70kDa (Hsp70s) are vital for all life and are notably important in protein folding. Hsp70s use ATP binding and hydrolysis at a nucleotide-binding domain (NBD) to control the binding and release of client polypeptides at a substrate-binding domain (SBD); however, the mechanistic basis for this allostery has been elusive. Here, we first characterize biochemical properties of selected domain-interface mutants in bacterial Hsp70 DnaK. We then develop a theoretical model for allosteric equilibria among Hsp70 conformational states to explain the observations: a restraining state, Hsp70R-ATP, restricts ATP hydrolysis and binds peptides poorly, whereas a stimulating state, Hsp70S-ATP, hydrolyzes ATP rapidly and has high intrinsic substrate affinity but rapid binding kinetics. We support this model for allosteric regulation with DnaK structures obtained in the postulated stimulating state S with biochemical tests of the S-state interface and with improved peptide-binding-site definition in an R-state structure.

HbA2 induction: the merit of pancellularity in sickle cell disease.

HbA2 induction: the merit of pancellularity in sickle cell disease.

Discovery and Functional Characterization of hPT3, a Humanized Anti-Phospho Tau Selective Monoclonal Antibody.

As a consequence of the discovery of an extracellular component responsible for the progression of tau pathology, tau immunotherapy is being extensively explored in both preclinical and clinical studies as a disease modifying strategy for the treatment of Alzheimer's disease. Describe the characteristics of the anti-phospho (T212/T217) tau selective antibody PT3 and its humanized variant hPT3. By performing different immunization campaigns, a large collection of antibodies has been generated and prioritized. In depth, in vitro characterization using surface plasmon resonance, phospho-epitope mapping, and X-ray crystallography experiments were performed. Further characterization involved immunohistochemical staining on mouse- and human postmortem tissue and neutralization of tau seeding by immunodepletion assays. Various in vitro experiments demonstrated a high intrinsic affinity for PT3 and hPT3 for AD brain-derived paired helical filaments but also to non-aggregated phospho (T212/T217) tau. Further functional analyses in cellular and in vivo models of tau seeding demonstrated almost complete depletion of tau seeds in an AD brain homogenate. Ongoing trials will provide the clinical evaluation of the tau spreading hypothesis in Alzheimer's disease.

Build-up functionalization of anti-EGFR \xd7 anti-CD3 bispecific diabodies by integrating high-affinity mutants and functional molecular formats

Designing non-natural antibody formats is a practical method for developing highly functional next-generation antibody drugs, particularly for improving the therapeutic efficacy of cancer treatments. One approach is constructing bispecific antibodies (bsAbs). We previously reported a functional humanized bispecific diabody (bsDb) that targeted epidermal growth factor receptor and CD3 (hEx3-Db). We enhanced its cytotoxicity by constructing an Fc fusion protein and rearranging order of the V domain. In this study, we created an additional functional bsAb, by integrating the molecular formats of bsAb and high-affinity mutants previously isolated by phage display in the form of Fv. Introducing the high-affinity mutations into bsDbs successfully increased their affinities and enhanced their cytotoxicity in vitro and in vivo. However, there were some limitations to affinity maturation of bsDb by integrating high-affinity Fv mutants, particularly in Fc-fused bsDb with intrinsic high affinity, because of their bivalency. The tetramers fractionated from the bsDb mutant exhibited the highest in vitro growth inhibition among the small bsAbs and was comparable to the in vivo anti-tumor effects of Fc-fused bsDbs. This molecule shows cost-efficient bacterial production and high therapeutic potential.

The art of suicidal molecular seduction for targeting drug resistance

The development of drug resistance is one of the most significant challenges of the current century in the pharmaceutical industry. Superinfections, cancer chemoresistance, and resistance observed in many non-infectious diseases are nullifying the efforts and monetary supplies, put in the advent of new drug molecules. Millions of people die because of this drug resistance developed gradually through extensive use of the drugs. Inherently, some drugs are less prone to become ineffective by drug resistance than others. Covalent inhibitors bind to their targets via a biologically permanent bound with their cognate receptor and therefore display more potent inhibiting characteristics. Suicide inhibitors or mechanism-based inhibitors are one of the covalent inhibitors, which require a pre-activation step by their targeting enzyme. This step accrues their selectivity and specificity with respect to other covalent inhibitors. After that pre-activation step, they produce an analogue of the transition state of the catalytic enzyme, which is practically incapable of dissociating from the enzyme. Suicide inhibitors, due to their high intrinsic affinity toward the related enzyme, are resistant to many mechanisms involved in the development of drug resistance and can be regarded as one of the enemies of this scientific hurdle. These inhibitors compete even with monoclonal antibodies in terms of their cost-effectiveness and efficacy.

Theory of Allosteric Regulation in Hsp70 Molecular Chaperones.

Heat-shock proteins of 70 kDa (Hsp70s) are ubiquitous molecular chaperones that function in protein folding as well as other vital cellular processes. They bind and hydrolyze ATP in a nucleotide-binding domain (NBD) to control the binding and release of client polypeptides in a substrate-binding domain (SBD). However, the molecular mechanism for this allosteric action has remained unclear. Here, we develop and experimentally quantify a theoretical model for Hsp70 allostery based on equilibria among Hsp70 conformational states. We postulate that, when bound to ATP, Hsp70 is in equilibrium between a restraining state (R) that restricts ATP hydrolysis and binds peptides poorly, if at all, and a stimulating state (S) that hydrolyzes ATP relatively rapidly and has high intrinsic substrate affinity but rapid binding kinetics; after the hydrolysis to ADP, NBD and SBD disengage into an uncoupled state (U) that binds peptide substrates tightly, but now with slow kinetics of exchange.

Secreted Protein Acidic and Rich in Cysteine Mediated Biomimetic Delivery of Methotrexate by Albumin-Based Nanomedicines for Rheumatoid Arthritis Therapy.

Rheumatoid arthritis (RA)is one of the most common chronic autoimmune diseases. Despite considerable advances in clinical treatment of RA, suboptimal response to therapy and treatment discontinuation are still unresolved challenges due to systemic toxicity. It is of crucial importance to actively target and deliver therapeutic agents to inflamed joints in order to promote in situ activity and decrease systemic toxicity. In this study, we found that SPARC (secreted protein acidic and rich in cysteine) was overexpressed in the synovial fluid and synovium of RA patients as well as mice with collagen-induced arthritis (CIA), which has been scarcely reported. Building upon the SPARC signature of RA joint microenvironment and the intrinsic high affinity of SPARC for albumin, we fabricated methotrexate-loaded human serum albumin nanomedicines (MTX@HSA NMs) and explored them as biomimetic drug delivery systems for RA therapy. Upon intravenous injection of chlorin e6-labeled MTX@HSA NMs into CIA mice, the fluorescence/magnetic resonance dual-modal imaging revealed higher accumulations and longer retention of MTX@HSA NMs in inflamed joints with respect to free MTX molecules. In vivo therapeutic evaluations suggested that the MTX@HSA NMs were able to attenuate the progression of RA with better efficacy and fewer side effects even at half dose of administrated MTX in comparison with free MTX. By unraveling the mechanism driving the efficient accumulation of MTX@HSA NMs in RA joints and showing their ability to improve the safety and therapeutic efficacy of MTX, our work sheds light on the development of innovative anti-RA nanomedicines with a strong potential for clinical translation.

Melanin-Concentrating Hormone Receptor 1 Antagonists Lacking an Aliphatic Amine: Synthesis and Structure-Activity Relationships of Novel 1-(Imidazo[1,2-a]pyridin-6-yl)pyridin-2(1H)-one Derivatives.

Aiming to discover melanin-concentrating hormone receptor 1 (MCHR1) antagonists with improved safety profiles, we hypothesized that the aliphatic amine employed in most antagonists reported to date could be removed if the bicyclic motif of the compound scaffold interacted with Asp123 and/or Tyr272 of MCHR1. We excluded aliphatic amines from our compound designs, with a cutoff value of pK(a) < 8, and explored aliphatic amine-free MCHR1 antagonists in a CNS-oriented chemical space limited by four descriptors (TPSA, ClogP, MW, and HBD count). Screening of novel bicyclic motifs with high intrinsic binding affinity for MCHR1 identified the imidazo[1,2-a]pyridine ring (represented in compounds 6a and 6b), and subsequent cyclization of the central aliphatic amide linkage led to the discovery of a potent, orally bioavailable MCHR1 antagonist 4-[(4-chlorobenzyl)oxy]-1-(2-cyclopropyl-3-methylimidazo[1,2-a]pyridin-6-yl)pyridin-2(1H)-one 10a. It exhibited low potential for hERG inhibition and phospholipidosis induction as well as sufficient brain concentration to exert antiobesity effects in diet-induced obese rats.

SGN-CD33A in Combination with Hypomethylating Agents Is Highly Efficacious in Preclinical Models of AML

Acute myeloid leukemia (AML) remains a therapeutic challenge. Long-term survival rates, in particular for older AML patients, remain poor, highlighting the need for improved and well-tolerated treatment options. AML patients who are unfit for high dose chemotherapy in the US are often prescribed hypomethylating agents (azacitidine or decitabine) although the efficacy of these agents in this population was modest (Estey, Leukemia 2013).SGN-CD33A is a CD33-directed antibody drug conjugate (ADC) that is comprised of a cysteine-engineered anti-CD33 antibody, a cleavable dipeptide linker that is highly stable in circulation, and a PBD dimer that binds DNA with high intrinsic affinity. The ADC is active as a single agent in preclinical models of AML that are characteristically resistant to chemotherapy (multi-drug-resistant, MDR-positive) (Sutherland et al. Blood 2013). In the present study, we tested the activity of SGN-CD33A in combination with hypomethylating agents (HMA), azacitidine or decitabine. We hypothesized that the combination of SGN-CD33A with an HMA will have greater impact on the DNA repair pathway in leukemic cells, furthering the processes of apoptosis and cell death. MDR-positive AML cells were treated for 96 hours with SGN-CD33A and each of the HMAs alone and in combination, evaluating both simultaneous as well as sequential administration. Enhanced tumor cell killing was observed when AML cells were treated concomitantly with the combination of SGN-CD33A and an HMA or pre-treated with HMAs prior to the addition of SGN-CD33A.Superior anti-leukemic activity of subtherapeutic doses of SGN-CD33A in combination with HMAs was observed in mouse xenograft models generated with MDR-positive AML cell lines. In the difficult to treat HEL9217 model, decitabine or a single dose of 200 mcg/kg SGN-CD33A delayed tumor growth, while significant reductions in tumor growth were observed for the combination treatment (p=0.0001). Improved antitumor activity in this model was also observed for SGN-CD33A in combination with azacitidine. SGN-CD33A in combination with decitabine significantly reduced tumor burden compared to either agent alone in the TF1-α AML model (p=0.0002). Similarly, sequenced dosing of azacitidine followed by a subtherapeutic dose of 30 mcg/kg SGN-CD33A in mice bearing KG-1 xenografts delivered greater antitumor activity compared to the individual agents (p=0.0001).To investigate the mechanism underlying the enhancement in antileukemic activity, we looked at the impact of HMAs alone and in combination with SGN-CD33A on myeloid marker expression, PBD drug release, and impact on the DNA damage and apoptotic pathways. In AML cell lines that did not show improved cytotoxic activity with the combination of SGN-CD33A and HMA, HMA treatment appeared to have no positive effect on the cell surface levels of CD33. However in responsive cell lines such as TF1-α, HMA treatment resulted in time- and dose-dependent increases in the levels of CD33. Significant increases in expression were observed between 2 and 4 days with decitabine and after 4 days with azacitidine. In addition, more PBD dimer drug from SGN-CD33A was incorporated into DNA in HMA treated cells. Concomitant, the treatment of AML cells with the combination of SGN-CD33A and HMA resulted in greater DNA damage and apoptosis as shown by the increased levels of the early DNA damage marker, γH2AX, and the formation of cleaved PARP, an apoptosis marker, compared to either agent alone. Studies are in progress to investigate the effects on other components of the DNA repair and cell cycle pathways.These findings demonstrate that SGN-CD33A can be successfully combined with hypomethylating agents to deliver marked antitumor activity in preclinical drug-resistant models of AML. DisclosuresSutherland:Seattle Genetics, Inc.: Employment. Yu:Seattle Genetics, Inc.: Employment. O'Day:Seattle Genetics,Inc: Employment. Alley:Seattle Genetics,Inc.: Employment. Anderson:Seattle Genetics, Inc.: Employment. Emmerton:Seattle Genetics, Inc.: Employment. Zeng:Seattle Genetics, Inc.: Employment. O'Meara:Seattle Genetics, Inc: Employment, Equity Ownership. Feldman:Seattle Genetics,Inc: Employment. Kennedy:Seattle Genetics,Inc: Employment, Equity Ownership, Honoraria, Speakers Bureau. Ryan:Seattle Genetics, Inc.: Employment. Benjamin:Seattle Genetics, Inc: Employment.

Abstract DDT02-04: SGN-CD33A: Preclinical and phase 1 interim clinical trial results of a CD33-directed PBD dimer antibody-drug conjugate for the treatment of acute myeloid leukemia (AML)

Abstract CD33 is expressed on the surface of myeloblasts in 85 to 90% of patients with AML and represents a promising target regardless of age, risk factors, or underlying mutational heterogeneity. SGN-CD33A is an antibody-drug conjugate (ADC) composed of an anti-CD33 antibody with engineered cysteines enabling uniform site-specific conjugation, conjugated to a pyrrolobenzodiazepine (PBD) dimer which binds DNA with high intrinsic affinity. The antibody and PBD dimer are conjugated via a cleavable dipeptide linker that is highly stable in circulation. Upon binding to the cell surface, SGN-CD33A is internalized and transported to the lysosomes where PBD dimer is released via proteolytic cleavage of the linker and diffuses inside the cell. PBD dimer crosslinks DNA, overwhelms DNA repair mechanisms, and triggers a cascade of events leading to cell death. SGN-CD33A is active as a single agent against a broad panel of primary AML samples and in multidrug-resistant preclinical models of AML (Sutherland 2013). Additionally, significant synergism in tumor cell killing was demonstrated in cytotoxicity assays when SGN-CD33A was combined with cytarabine, azacitidine, or decitabine (Sutherland 2014). Consistent with these observations, greater antitumor activity and reduced tumor growth were observed with these agents in combination with a single noncurative dose of SGN-CD33A in mouse xenograft models of multidrug resistant AML. The synergism with hypomethylating agents was associated with upregulation of CD33 expression on the cell surface of the AML cells as well as greater activation of the DNA damage sensor and apoptoticpathways. A first-in-human, phase 1, dose-escalation study of SGN-CD33A is ongoing to investigate the safety, tolerability, pharmacokinetics, and antitumor activity of SGN-CD33A in patients with CD33-positive AML (NCT# 01902329). Patients must have either relapsed disease following initial complete remission (CR) of &amp;gt;3 months, or have declined conventional induction/consolidation. SGN-CD33A is administered outpatient IV every 3 weeks for up to 4 cycles (Part A), followed by optional maintenance treatment for patients achieving a CR/CRi (Part B). Investigator assessment of response is per IWG criteria (Cheson 2003). Another arm of the study will investigate the safety of SGN-CD33A in combination with either azacitidine or decitabine. Data have been reported on the first 56 patients who received SGN-CD33A as a single agent at doses ranging from 5−60 mcg/kg (presented by E. Stein at ASH 2014). Median age of patients was 75 years (range 27−86) and approximately 90% of patients had an ECOG performance score of 1. About half of the patients had received prior intensive therapy for AML, while the other half had declined intensive therapy. The most common adverse events considered related to SGN-CD33A included febrile neutropenia, fatigue, thrombocytopenia, anemia, and pyrexia. The 30-day mortality rate at the time of data presentation was 2% (1 patient whose death was not considered associated with SGN-CD33A). Preliminary pharmacokinetic data demonstrate rapid clearance of the ADC, suggesting target-mediated drug disposition. Among 52 patients evaluated for antitumor activity, 9 achieved CR or CR with incomplete hematologic recovery (CRi), 11 patients had clearance of blasts in their bone marrow (morphologic leukemia-free state, MLFS), and 29 patients had residual blasts in the bone marrow. The CR/CRi rate at 40 mcg/kg was 29% (5/17). The ongoing phase 1 study is evaluating SGN-CD33A as monotherapy and in combination with hypomethylating agents. Efforts are ongoing to refine a recommended dosing regimen for future study. Also enrolling is a phase 1b study of SGN-CD33A in combination with 7+3 chemotherapy in the frontline treatment of AML (NCT# NCT02326584). To date, preclinical data combined with emerging clinical safety and antitumor activity data form a foundation for the development of SGN-CD33A as a novel, targeted CD33-directed therapy for the treatment of AML and potentially other myeloid malignancies. Citation Format: Dana A. Kennedy, Stephen C. Alley, Baiteng Zhao, Eric J. Feldman, Megan O'Meara, May Sutherland. SGN-CD33A: Preclinical and phase 1 interim clinical trial results of a CD33-directed PBD dimer antibody-drug conjugate for the treatment of acute myeloid leukemia (AML). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr DDT02-04. doi:10.1158/1538-7445.AM2015-DDT02-04

N-Terminal Hypervariable Region of Muscle Type Isoforms of Troponin T Differentially Modulates the Affinity of Tropomyosin-Binding Site 1.

The troponin complex plays a central role in the contraction and relaxation of striated muscle by enacting Ca²⁺-regulated allosteric changes in the sarcomeric thin filaments. The troponin T subunit (TnT) contains two binding sites for tropomyosin (Tm) and is responsible for anchoring the troponin complex to the thin filament. While the amino acid sequences of the regions containing the Tm-binding sites are highly conserved among the three muscle type isoforms of TnT, previous studies have observed significant discrepancies in the affinity of Tm-binding site 1 in the chymotryptic fragment T1 of different TnT isoforms. Here we cross-examined the Tm-binding affinity of TnT isoforms and molecular engineered fragments using affinity chromatography and microplate protein binding assays to investigate the effects of the evolutionarily diverged N-terminal region that is significantly variable among muscle type isoforms. The results demonstrated that the C-terminal T2 fragment of TnT containing the Tm-binding site 2 had similarly high affinity across isoforms. In the absence of the N-terminal variable region, Tm-binding site 1 in the conserved middle region of TnT also exhibited high intrinsic affinity. The presence of isoform specific N-terminal variable region differentially reduced the binding affinity of TnT for Tm, primarily at binding site 1 in the middle region. These findings indicate that the N-terminal variable region of TnT plays a pivotal role in the functional difference of muscle type-specific isoforms and the developmental and pathogenic splice variants by modulating the interaction with Tm during Ca²⁺ regulation of cardiac and skeletal muscle contraction and relaxation.

Preparation of abiotic polymer nanoparticles for sequestration and neutralization of a target peptide toxin.

Synthetic polymer nanoparticles (NPs) with intrinsic affinity for target biomacromolecules hold great promise in the development of novel tools for biological and biomedical research. We recently reported the design and synthesis of abiotic, synthetic polymer NPs with high intrinsic affinity for a peptide toxin melittin. The NP was selected by screening a small library of NPs (∼100 nm) composed of various ratios of monomers that contain functional groups complementary to the peptide melittin. The selected polymer NP, a co-polymer of acrylic acid (AAc), N-tert-butylacrylamide (TBAm), N-isopropylacrylamide (NIPAm) and N,N'-methylenebisacrylamide (BIS), effectively captures and neutralizes the toxicity of the peptide through a combination of electrostatic and hydrophobic interactions. This protocol describes a step-by-step procedure for the preparation and evaluation of synthetic polymer NPs for sequestration and neutralization of the target peptide toxin. The polymer NPs can be synthesized in a one-step polymerization reaction using commercially available reagents. The polymerization reaction for the synthesis of polymer NPs takes several hours, and the total protocol including subsequent purification and characterization by dynamic light scattering, NMR and toxicity neutralization assays takes 1-2 weeks in total.