Systematic synthetic and biophysical development of mixed sequence DNA binding agents.

It is now well established that, although only about 5% of the human genome codes for protein, most of the DNA has some function, such as synthesis of specific, functional RNAs and/or control of gene expression. These functional sequences open immense possibilities in both biotechnology and therapeutics for the use of cell-permeable, small molecules that can bind mixed-base pair sequences of DNA for regulation of genomic functions. Unfortunately very few types of modules have been designed to recognize mixed DNA sequences and for progress in targeting specific genes, it is essential to have additional classes of compounds. Compounds that can be rationally designed from established modules and which can bind strongly to mixed base pair DNA sequences are especially attractive. Based on extensive experience in design of minor-groove agents for AT recognition, a small library of compounds with two AT specific binding modules, connected through linkers which can recognize the G·C base pairs, were prepared. The compound-DNA interactions were evaluated with a powerful array of biophysical methods and the results show that some pyridyl-linked compounds bind with the target sequence with sub-nanomolar KD, with very slow dissociation kinetics and 200 times selectivity over the related sequence without a G·C base pair. Interestingly, a set of compounds with AT module connected by different linkers shows cooperative dimer recognition of related sequences. This type of design approach can be expanded to additional modules for recognition of a wide variety of sequences.

Mixed up minor groove binders: Convincing A·T specific compounds to recognize a G·C base pair.

DNA minor-groove-binding compounds have limited biological applications, in part due to problems with sequence specificity that cause off-target effects. A model to enhance specificity has been developed with the goal of preparing compounds that bind to two AT sites separated by G·C base pairs. Compounds of interest were probed using thermal melting, circular dichroism, mass spectrometry, biosensor-SPR, and molecular modeling methods. A new minor groove binder that can strongly and specifically recognize a single G·C base pair with flanking AT sequences has been prepared. This multi-site DNA recognition mode offers novel design principles to recognize entirely new DNA motifs.

Selective G-quadruplex DNA recognition by a new class of designed cyanines.

A variety of cyanines provide versatile and sensitive agents acting as DNA stains and sensors and have been structurally modified to bind in the DNA minor groove in a sequence dependent manner. Similarly, we are developing a new set of cyanines that have been designed to achieve highly selective binding to DNA G-quadruplexes with much weaker binding to DNA duplexes. A systematic set of structurally analogous trimethine cyanines has been synthesized and evaluated for quadruplex targeting. The results reveal that elevated quadruplex binding and specificity are highly sensitive to the polymethine chain length, heterocyclic structure and intrinsic charge of the compound. Biophysical experiments show that the compounds display significant selectivity for quadruplex binding with a higher preference for parallel stranded quadruplexes, such as cMYC. NMR studies revealed the primary binding through an end-stacking mode and SPR studies showed the strongest compounds have primary KD values below 100 nM that are nearly 100-fold weaker for duplexes. The high selectivity of these newly designed trimethine cyanines for quadruplexes as well as their ability to discriminate between different quadruplexes are extremely promising features to develop them as novel probes for targeting quadruplexes in vivo.

Binding to the DNA Minor Groove by Heterocyclic Dications: from AT Specific to GC Recognition Compounds.

Compounds that bind in the DNA minor groove have provided critical information on DNA molecular recognition, have found extensive uses in biotechnology, and are providing clinically useful drugs against diseases as diverse as cancer and sleeping sickness. This review focuses on the development of clinically useful heterocyclic diamidine minor groove binders. These compounds show that the classical model for minor groove binding in AT DNA sequences must be expanded in several ways: compounds with nonstandard shapes can bind strongly to the groove, water can be directly incorporated into the minor groove complex in an interfacial interaction, compounds can be designed to recognize GC and mixed AT/GC base pair sequences, and stacked dimers can form to recognize specific sequences. © 2023 Wiley Periodicals LLC.

"Stapling" scFv for multispecific biotherapeutics of superior properties.

Single-chain fragment variable (scFv) domains play an important role in antibody-based therapeutic modalities, such as bispecifics, multispecifics and chimeric antigen receptor T cells or natural killer cells. However, scFv domains exhibit lower stability and increased risk of aggregation due to transient dissociation ("breathing") and inter-molecular reassociation of the two domains (VL and VH). We designed a novel strategy, referred to as stapling, that introduces two disulfide bonds between the scFv linker and the two variable domains to minimize scFv breathing. We named the resulting molecules stapled scFv (spFv). Stapling increased thermal stability (Tm) by an average of 10°C. In multiple scFv/spFv multispecifics, the spFv molecules display significantly improved stability, minimal aggregation and superior product quality. These spFv multispecifics retain binding affinity and functionality. Our stapling design was compatible with all antibody variable regions we evaluated and may be widely applicable to stabilize scFv molecules for designing biotherapeutics with superior biophysical properties.

Correlation of local effects of DNA sequence and position of ß-alanine inserts with polyamide-DNA complex binding affinities and kinetics.

To improve our understanding of the effects of ß-alanine (ß) substitution and the number of heterocycles on DNA binding affinity and selectivity, we investigated the interactions of an eight-ring hairpin polyamide (PA) and two ß derivatives as well as a six-heterocycle analogue with their cognate DNA sequence, 5'-TGGCTT-3'. Binding selectivity and the effects of ß have been investigated with the cognate and five mutant DNAs. A set of powerful and complementary methods have been employed for both energetic and structural evaluations: UV melting, biosensor surface plasmon resonance, isothermal titration calorimetry, circular dichroism, and a DNA ligation ladder global structure assay. The reduced number of heterocycles in the six-ring PA weakens the binding affinity; however, the smaller PA aggregates significantly less than the larger PAs and allows us to obtain the binding thermodynamics. The PA-DNA binding enthalpy is large and negative with a large negative ΔC(p) and is the primary driving component of the Gibbs free energy. The complete SPR binding results clearly show that ß substitutions can substantially weaken the binding affinity of hairpin PAs in a position-dependent manner. More importantly, the changes in the binding of PA to the mutant DNAs further confirm the position-dependent effects on the PA-DNA interaction affinity. Comparison of mutant DNA sequences also shows a different effect in recognition of T·A versus A·T base pairs. The effects of DNA mutations on binding of a single PA as well as the effects of the position of ß substitution on binding tell a clear and very important story about sequence-dependent binding of PAs to DNA.

Dynamics of bleomycin interaction with a strongly bound hairpin DNA substrate, and implications for cleavage of the bound DNA.

Recent studies involving DNAs bound strongly by bleomycins have documented that such DNAs are degraded by the antitumor antibiotic with characteristics different from those observed when studying the cleavage of randomly chosen DNAs in the presence of excess Fe·BLM. In the present study, surface plasmon resonance has been used to characterize the dynamics of BLM B(2) binding to a strongly bound hairpin DNA, to define the effects of Fe(3+), salt, and temperature on BLM-DNA interaction. One strong primary DNA binding site, and at least one much weaker site, were documented. In contrast, more than one strong cleavage site was found, an observation also made for two other hairpin DNAs. Evidence is presented for BLM equilibration between the stronger and weaker binding sites in a way that renders BLM unavailable to other, less strongly bound DNAs. Thus, enhanced binding to a given site does not necessarily result in increased DNA degradation at that site; i.e., for strongly bound DNAs, the facility of DNA cleavage must involve other parameters in addition to the intrinsic rate of C-4' H atom abstraction from DNA sugars.

A novel series of G-quadruplex ligands with selectivity for HIF-expressing osteosarcoma and renal cancer cell lines.

A series of naphthalene derivatives with disubstituted triazole side-arms have been assembled by click chemistry. Lead compounds show a high level of selectivity for renal, osteo- and Ewing's sarcomas that express the HIF-1α transcription factor. They also interact selectively with the quadruplex DNAs located in the promoter of the HIF genes and it is suggested that the mechanism of action involves inhibition of transcription by drug-mediated quadruplex stabilization in these regions.

Halogenated pentamethine cyanine dyes exhibiting high fidelity for G-quadruplex DNA.

Design and optimization of quadruplex-specific small molecules is developing into an attractive strategy for anti-cancer therapeutics with some promising candidates in clinical trials. A number of therapeutically favorable features of cyanine molecules can be effectively exploited to develop them as promising quadruplex-targeting agents. Herein, the design, synthesis and evaluation of a series of dimethylindolenine cyanine dyes with varying halogen substitutions are reported. Their interactions with telomeric and c-myc quadruplexes as well as a reference duplex sequence have been evaluated using thermal melting, biosensor-surface plasmon resonance, circular dichroism, isothermal titration calorimetry and mass spectrometry. Thermal melting analysis indicates that these ligands exhibit significant quadruplex stabilization and a very low duplex binding, with the dimethyl incorporation of paramount importance for decreased duplex affinity. Circular dichroism studies showed that the interaction of cyanines with quadruplex structures are primarily through stacking at one or both ends of the terminal tetrads with the two (trimethylammonium)propyl groups interacting in the accessible quadruplex grooves. Surface plasmon resonance and mass spectral studies shows the formation of an initial strong 1:1 complex followed by a significantly weaker secondary binding. Isothermal calorimetry studies show that the interaction of cyanines is predominantly entropy driven. In line with the design principles, this work provides new insights for further developing potent, highly selective cyanines as promising quadruplex-specific agents.

Discovery and pharmacological characterization of cetrelimab (JNJ-63723283), an anti-programmed cell death protein-1 (PD-1) antibody, in human cancer models.

PURPOSE: Preclinical characterization of cetrelimab (JNJ-63723283), a fully humanized immunoglobulin G4 kappa monoclonal antibody targeting programmed cell death protein-1 (PD-1), in human cancer models. METHODS: Cetrelimab was generated by phage panning against human and cynomolgus monkey (cyno) PD-1 extracellular domains (ECDs) and affinity maturation. Binding to primate and rodent PD-1 ECDs, transfected and endogenous cell-surface PD-1, and inhibition of ligand binding were measured. In vitro activity was evaluated using cytomegalovirus recall, mixed lymphocyte reaction, staphylococcal enterotoxin B stimulation, and Jurkat-PD-1 nuclear factor of activated T cell reporter assays. In vivo activity was assessed using human PD-1 knock-in mice implanted with MC38 tumors and a lung patient-derived xenograft (PDX) model (LG1306) using CD34 cord-blood-humanized NSG mice. Pharmacodynamics, toxicokinetics, and safety were assessed in cynos following single and/or repeat intravenous dosing. RESULTS: Cetrelimab showed high affinity binding to human (1.72 nM) and cyno (0.90 nM) PD-1 and blocked binding of programmed death-ligand 1 (PD-L1; inhibitory concentration [IC] 111.7 ng/mL) and PD-L2 (IC 138.6 ng/mL). Cetrelimab dose-dependently increased T cell-mediated cytokine production and stimulated cytokine expression. Cetrelimab 10 mg/kg reduced mean MC38 tumor volume in PD-1 knock-in mice at Day 21 (P < 0.0001) versus control. In a PDX lung model, 10 mg/kg cetrelimab (every 5 days for six cycles) increased frequency of peripheral T cells and reduced (P < 0.05) mean tumor volume versus control. Activity was consistent with that of established PD-1 inhibitors. Cetrelimab dosing was well tolerated in cynos and mean drug exposure increase was dose-dependent. CONCLUSION: Cetrelimab potently inhibits PD-1 in vitro and in vivo, supporting its clinical evaluation.

Dicationic phenyl-2,2'-bichalcophenes and analogues as antiprotozoal agents.

A series of phenyl-2,2'-bichalcophene diamidines 1a-h were synthesized from the corresponding dinitriles either via a direct reaction with LiN(TMS)2, followed by deprotection with ethanolic HCl or through the bis-O-acetoxyamidoxime followed by hydrogenation in acetic acid and EtOH over Pd-C. These diamidines show a wide range of DNA affinities as judged from their ΔT(m) values which are remarkably sensitive to replacement of a furan unit with a thiophene one. These differences are explained in terms of the effect of subtle changes in geometry of the diamidines on binding efficacy. Five of the eight compounds were highly active (below 6 nM IC50) in vitro against Trypanosoma brucei rhodesiense (T. b. r.) and four gave IC50values less than 7 nM against Plasmodium falciparum (P. f.). Only one of the compounds was as effective as reference compounds in the T. b. r. mouse model for the acute phase of African trypanosomiasis.

Teclistamab is an active T cell-redirecting bispecific antibody against B-cell maturation antigen for multiple myeloma.

B-cell maturation antigen (BCMA), a member of the tumor necrosis factor family of receptors, is predominantly expressed on the surface of terminally differentiated B cells. BCMA is highly expressed on plasmablasts and plasma cells from multiple myeloma (MM) patient samples. We developed a BCMAxCD3 bispecific antibody (teclistamab [JNJ-64007957]) to recruit and activate T cells to kill BCMA-expressing MM cells. Teclistamab induced cytotoxicity of BCMA+ MM cell lines in vitro (H929 cells, 50% effective concentration [EC50] = 0.15 nM; MM.1R cells, EC50 = 0.06 nM; RPMI 8226 cells, EC50 = 0.45 nM) with concomitant T-cell activation (H929 cells, EC50 = 0.21 nM; MM.1R cells, EC50 = 0.1 nM; RPMI 8226 cells, EC50 = 0.28 nM) and cytokine release. This activity was further increased in the presence of a γ-secretase inhibitor (LY-411575). Teclistamab also depleted BCMA+ cells in bone marrow samples from MM patients in an ex vivo assay with an average EC50 value of 1.7 nM. Under more physiological conditions using healthy human whole blood, teclistamab mediated dose-dependent lysis of H929 cells and activation of T cells. Antitumor activity of teclistamab was also observed in 2 BCMA+ MM murine xenograft models inoculated with human T cells (tumor inhibition with H929 model and tumor regression with the RPMI 8226 model) compared with vehicle and antibody controls. The specific and potent activity of teclistamab against BCMA-expressing cells from MM cell lines, patient samples, and MM xenograft models warrant further evaluation of this bispecific antibody for the treatment of MM. Phase 1 clinical trials (monotherapy, #NCT03145181; combination therapy, #NCT04108195) are ongoing for patients with relapsed/refractory MM.

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

BACKGROUND: 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. OBJECTIVE: Describe the characteristics of the anti-phospho (T212/T217) tau selective antibody PT3 and its humanized variant hPT3. METHODS: 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. RESULTS AND CONCLUSION: 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.

Biosensor-Surface Plasmon Resonance: Label-Free Method for Investigation of Small Molecule-Quadruplex Nucleic Acid Interactions.

Biosensor-surface plasmon resonance (SPR) technology is now well established as a quantitative approach for the study of nucleic acid interactions in real time, without the need for labeling any components of the interaction. The method provides real-time equilibrium and kinetic characterization for quadruplex DNA interactions and requires small amounts of materials and no external probe. A detailed protocol for quadruplex-DNA interaction analyses with a variety of binding molecules using biosensor-SPR methods is presented. Explanations of the SPR method with basic fundamentals for use and analysis of results are described with recommendations on the preparation of the SPR instrument, sensor chips, and samples. Details of experimental design, quantitative and qualitative data analyses, and presentation are described. Some specific examples of small molecule-DNA quadruplex interactions are presented with results evaluated by both kinetic and steady-state SPR methods.

Tri- and tetra-substituted naphthalene diimides as potent G-quadruplex ligands.

A series of tri- and tetra-substituted naphthalene diimides have been designed and synthesized. Several compounds show exceptional affinity for telomeric G-quadruplex DNA in classical and competition FRET assays and SPR studies. They inhibit telomerase in the TRAP assay, and show potent senescence-based short-term anti-proliferative effects on MCF7 and A549 cancer cell lines, and localize in the nucleus and particularly the nucleolus of MCF7 cells.

Human IgG subclass cross-species reactivity to mouse and cynomolgus monkey Fcγ receptors.

In therapeutic antibody discovery and early development, mice and cynomolgus monkey are used as animal models to assess toxicity, efficacy and other properties of candidate molecules. As more candidate antibodies are based on human immunoglobulin (IgG) subclasses, many strategies are pursued to simulate the human system in the test animal. However, translation rate from a successful preclinical trial to an approved drug is extremely low. This may partly be due to differences in interaction of human IgG based candidate molecules to endogenous Fcγ receptors of model animals in comparison to those of human Fcγ receptors. In this study, we compare binding characteristics of human IgG subclasses commonly used in drug development (IgG1, IgG2, IgG4) and their respective Fc silent versions (IgG1σ, IgG2σ, IgG4 PAA) to human, mouse, and cynomolgus monkey Fcγ receptors. To control interactions between Fab and Fc domains, the test IgGs all have the same variable region sequences. We found distinct variations of interaction of human IgG subclasses to model animal Fcγ receptors in comparison to their human counterparts. Particularly, cynomolgus monkey Fcγ receptors showed consistently tighter binding to human IgGs than human Fcγ receptors. Moreover, the presumably Fc silent human IgG4 PAA framework bound to cynomolgus monkey FcγRI with nanomolar affinity while only very weak binding was observed for the human FcγRI. Our results highlighted the need for a thorough in vitro affinity characterization of candidate IgGs against model animal Fcγ receptors and careful design of preclinical studies.

Unique pharmacology of a novel allosteric agonist/sensitizer insulin receptor monoclonal antibody.

OBJECTIVE: Insulin resistance is a key feature of Type 2 Diabetes (T2D), and improving insulin sensitivity is important for disease management. Allosteric modulation of the insulin receptor (IR) with monoclonal antibodies (mAbs) can enhance insulin sensitivity and restore glycemic control in animal models of T2D. METHODS: A novel human mAb, IRAB-A, was identified by phage screening using competition binding and surface plasmon resonance assays with the IR extracellular domain. Cell based assays demonstrated agonist and sensitizer effects of IRAB-A on IR and Akt phosphorylation, as well as glucose uptake. Lean and diet-induced obese mice were used to characterize single-dose in vivo pharmacological effects of IRAB-A; multiple-dose IRAB-A effects were tested in obese mice. RESULTS: In vitro studies indicate that IRAB-A exhibits sensitizer and agonist properties distinct from insulin on the IR and is translated to downstream signaling and function; IRAB-A bound specifically and allosterically to the IR and stabilized insulin binding. A single dose of IRAB-A given to lean mice rapidly reduced fed blood glucose for approximately 2 weeks, with concomitant reduced insulin levels suggesting improved insulin sensitivity. Phosphorylated IR (pIR) from skeletal muscle and liver were increased by IRAB-A; however, phosphorylated Akt (pAkt) levels were only elevated in skeletal muscle and not liver vs. control; immunochemistry analysis (IHC) confirmed the long-lived persistence of IRAB-A in skeletal muscle and liver. Studies in diet-induced obese (DIO) mice with IRAB-A reduced fed blood glucose and insulinemia yet impaired glucose tolerance and led to protracted insulinemia during a meal challenge. CONCLUSION: Collectively, the data suggest IRAB-A acts allosterically on the insulin receptor acting non-competitively with insulin to both activate the receptor and enhance insulin signaling. While IRAB-A produced a decrease in blood glucose in lean mice, the data in DIO mice indicated an exacerbation of insulin resistance; these data were unexpected and suggested the interplay of complex unknown pharmacology. Taken together, this work suggests that IRAB-A may be an important tool to explore insulin receptor signaling and pharmacology.

Anti-Tau Monoclonal Antibodies Derived from Soluble and Filamentous Tau Show Diverse Functional Properties in vitro and in vivo.

The tau spreading hypothesis provides rationale for passive immunization with an anti-tau monoclonal antibody to block seeding by extracellular tau aggregates as a disease-modifying strategy for the treatment of Alzheimer's disease (AD) and potentially other tauopathies. As the biochemical and biophysical properties of the tau species responsible for the spatio-temporal sequences of seeding events are poorly defined, it is not yet clear which epitope is preferred for obtaining optimal therapeutic efficacy. Our internal tau antibody collection has been generated by immunizations with different tau species: aggregated- and non-aggregated tau and human postmortem AD brain-derived tau fibrils. In this communication, we describe and characterize a set of these anti-tau antibodies for their biochemical and biophysical properties, including binding, tissue staining by immunohistochemistry, and epitope. The antibodies bound to different domains of the tau protein and some were demonstrated to be isoform-selective (PT18 and hTau56) or phospho-selective (PT84). Evaluation of the antibodies in cellular- and in vivo seeding assays revealed clear differences in maximal efficacy. Limited proteolysis experiments support the hypothesis that some epitopes are more exposed than others in the tau seeds. Moreover, antibody efficacy seems to depend on the structural properties of fibrils purified from tau Tg mice- and postmortem human AD brain.

Novel Monoclonal Antibody Is an Allosteric Insulin Receptor Antagonist That Induces Insulin Resistance.

A hallmark of type 2 diabetes is impaired insulin receptor (IR) signaling that results in dysregulation of glucose homeostasis. Understanding the molecular origins and progression of diabetes and developing therapeutics depend on experimental models of hyperglycemia, hyperinsulinemia, and insulin resistance. We present a novel monoclonal antibody, IRAB-B, that is a specific, potent IR antagonist that creates rapid and long-lasting insulin resistance. IRAB-B binds to the IR with nanomolar affinity and in the presence of insulin efficiently blocks receptor phosphorylation within minutes and is sustained for at least 3 days in vitro. We further confirm that IRAB-B antagonizes downstream signaling and metabolic function. In mice, a single dose of IRAB-B induces rapid onset of hyperglycemia within 6 h, and severe hyperglycemia persists for 2 weeks. IRAB-B hyperglycemia is normalized in mice treated with exendin-4, suggesting that this model can be effectively treated with a GLP-1 receptor agonist. Finally, a comparison of IRAB-B with the IR antagonist S961 shows distinct antagonism in vitro and in vivo. IRAB-B appears to be a powerful tool to generate both acute and chronic insulin resistance in mammalian models to elucidate diabetic pathogenesis and evaluate therapeutics.

Discovery of new G-quadruplex binding chemotypes.

We report here on the discovery and preliminary evaluation of a novel non-macrocyclic low molecular weight quadruplex-stabilizing chemotype. The lead compounds, based on a furan core, show high G-quadruplex stabilisation and selectivity as well as potent in vitro anti-proliferative activity.