Structural Characterization of a Therapeutic Anti-Methamphetamine Antibody Fragment: Oligomerization and Binding of Active Metabolites

Vaccines and monoclonal antibodies (mAb) for treatment of (+)-methamphetamine (METH) abuse are in late stage preclinical and early clinical trial phases, respectively. These immunotherapies work as pharmacokinetic antagonists, sequestering METH and its metabolites away from sites of action in the brain and reduce the rewarding and toxic effects of the drug. A key aspect of these immunotherapy strategies is the understanding of the subtle molecular interactions important for generating antibodies with high affinity and specificity for METH. We previously determined crystal structures of a high affinity anti-METH therapeutic single chain antibody fragment (scFv6H4, K_D = 10 nM) in complex with METH and the (+) stereoisomer of 3,4-methylenedioxymethamphetamine (MDMA, or “ecstasy”). Here we report the crystal structure of scFv6H4 in homo-trimeric unbound (apo) form (2.60Å), as well as monomeric forms in complex with two active metabolites; (+)-amphetamine (AMP, 2.38Å) and (+)-4-hydroxy methamphetamine (p-OH-METH, 2.33Å). The apo structure forms a trimer in the crystal lattice and it results in the formation of an intermolecular composite beta-sheet with a three-fold symmetry. We were also able to structurally characterize the coordination of the His-tags with Ni²⁺. Two of the histidine residues of each C-terminal His-tag interact with Ni²⁺ in an octahedral geometry. In the apo state the CDR loops of scFv6H4 form an open conformation of the binding pocket. Upon ligand binding, the CDR loops adopt a closed formation, encasing the drug almost completely. The structural information reported here elucidates key molecular interactions important in anti-methamphetamine abuse immunotherapy.     

Design considerations for PAMAM dendrimer therapeutics

We have previously shown that methotrexate (MTX) conjugated to a cancer-specific poly amido amine (PAMAM) dendrimer has a higher therapeutic index than MTX alone. Unfortunately, these therapeutics have been difficult to advance because of the complicated syntheses and an incomplete understanding of the dendrimer properties. We wished to address these obstacles by using copper-free click chemistry to functionalize the dendrimer scaffolds and to exploring the effects of two dendrimer properties (the targeting ligand and drug linkage) on cytotoxicity. We conjugated either ester or amide-linker modified MTX to dendrimer scaffolds with or without folic acid (FA). Because of multivalency, the FA and MTX functionalized dendrimers had similar capacities to target the folate receptor on cancer cells. Additionally, we found that the ester- and amide-linker modified MTX compounds had similar cytotoxicity but the dendrimer–ester MTX conjugates were much more cytotoxic than the dendrimer–amide MTX conjugates. These results clarify the impact of these properties on therapeutic efficacy and will allow us to design more effective polymer therapeutics.     

GPCR ligand dendrimer (GLiDe) conjugates: adenosine receptor interactions of a series of multivalent xanthine antagonists

Previously, G protein-coupled receptor (GPCR) agonists were tethered from polyamidoamine (PAMAM) dendrimers to provide high receptor affinity and selectivity. Here, we prepared GPCR ligand--dendrimer (GLiDe) conjugates from a potent adenosine receptor (AR) antagonist; such agents are of interest for treating Parkinson's disease, asthma, and other conditions. Xanthine amine congener (XAC) was appended with an alkyne group on an extended C8 substituent for coupling by Cu(I)-catalyzed click chemistry to azide-derivatized G4 (fourth-generation) PAMAM dendrimers to form triazoles. These conjugates also contained triazole-linked PEG groups (8 or 22 moieties per 64 terminal positions) for increasing water-solubility and optionally prosthetic groups for spectroscopic characterization and affinity labeling. Human AR binding affinity increased progressively with the degree of xanthine substitution to reach K(i) values in the nanomolar range. The order of affinity of each conjugate was hA(2A)AR > hA(3)AR > hA(1)AR, while the corresponding monomer was ranked hA(2A)AR > hA(1)AR ≥ hA(3)AR. The antagonist activity of the most potent conjugate 14 (34 xanthines per dendrimer) was examined at the G(i)-coupled A(1)AR. Conjugate 14 at 100 nM right-shifted the AR agonist concentration--response curve in a cyclic AMP functional assay in a parallel manner, but at 10 nM (lower than its K(i) value), it significantly suppressed the maximal agonist effect in calcium mobilization. This is the first systematic probing of a potent AR antagonist tethered on a dendrimer and its activity as a function of variable loading.     

In vitro targeting of synthesized antibody-conjugated dendrimer nanoparticles

This study reports the synthesis and in vitro biological properties of dendrimer-antibody conjugates. The polyamidoamine dendrimer platform was conjugated to fluorescein isothiocyanate as a means to analyze cell binding and internalization. Two different antibodies, 60bca and J591, which bind to CD14 and prostate-specific membrane antigen (PSMA), respectively, were used as model targeting molecules. The binding of the antibody-conjugated dendrimers to antigen-expressing cells was evaluated by flow cytometry, confocal microscopy, and a new two-photon-based optical fiber fluorescence detection system. The conjugates specifically bound to the antigen-expressing cells in a time- and dose-dependent fashion, with affinity similar to that of the free antibody. Confocal microscopic analysis suggested at least some cellular internalization of the dendrimer conjugate. Dendrimer-antibody conjugates are a suitable platform for targeted molecule delivery into antigen-expressing cells.     

Systematic investigation of polyamidoamine dendrimers surface-modified with poly(ethylene glycol) for drug delivery applications: synthesis, characterization, and evaluation of cytotoxicity

Surface modification of amine-terminated polyamidoamine (PAMAM) dendrimers by poly(ethylene glycol) (PEG) groups generally enhances water-solubility and biocompatibility for drug delivery applications. In order to provide guidelines for designing appropriate dendritic scaffolds, a series of G3 PAMAM-PEG dendrimer conjugates was synthesized by varying the number of PEG attachments and chain length (shorter PEG 550 and PEG 750 and longer PEG 2000). Each conjugate was purified by size exclusion chromatography (SEC) and the molecular weight (MW) was determined by (1)H NMR integration and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). NOESY experiments performed in D 2O on selected structures suggested no penetration of PEG chains to the central PAMAM domain, regardless of chain length and degree of substitution. CHO cell cultures exposed to PAMAM-PEG derivatives (< or =1 microM) showed a relatively high cell viability. Generally, increasing the degree of PEG substitution reduced cytotoxicity. Moreover, compared to G3 PAMAM dendrimers that were N-acetylated to varying degrees, a lower degree of surface substitution with PEG was needed for a similar cell viability. Interestingly, when longer PEG 2000 was fully incorporated on the surface, cell viability was reduced at higher concentrations (32 muM), suggesting increased toxicity potentially by forming intermolecular aggregates. A similar observation was made for anionic carboxylate G5.5 PAMAM dendrimer at the same dendrimer concentration. Our findings suggest that a lower degree of peripheral substitution with shorter PEG chains may suffice for these PAMAM-PEG conjugates to serve as efficient universal scaffolds for drug delivery, particularly valuable in relation to targeting or other ligand-receptor interactions.     

Characterization of site-specific ScFv PEGylation for tumor-targeting pharmaceuticals

New radiopharmaceuticals are possible using site-specific conjugation of small tumor binding proteins and poly(ethylene glycol) (PEG) scaffolds to provide modular multivalent, homo- or heterofunctional cancer-targeting molecules having preferred molecular size, valence, and functionality. Residence time in plasma can be optimized by modification of the size, number, and charge of the protein units. However, random PEG conjugation (PEGylation) of these small molecules via amine groups has led to variations of structural conformation and binding affinity. To optimize PEGylation, scFvs have been recombinantly produced in a vector that adds an unpaired cysteine (c) near the scFv carboxy terminus (scFv-c), thus providing a specific site for thiol conjugation. To evaluate the general applicability of this unpaired cysteine for PEGylation of scFv-c, conjugation efficiency was determined for four different scFvs and several PEG molecules having thiol reactive groups. The effect of the PEG molecular format on scFv-c PEG malignant cell binding was also addressed. ScFvs produced as scFv-c and purified by anti E-TAG affinity chromatography were conjugated using PEG molecules with maleimide (Mal) or o-pyridyl disulfide (OPSS). Conjugations were performed at pH 7.0, with 2 molar excess TCEP/scFv and PEG-(Mal) or PEG-OPSS, using 5:1 (PEG/scFv). PEG-Mal conjugation efficiency was also evaluated with 1:5 (PEG/scFv). PEGylation efficiency was determined for each reaction by quantitation of the products on SDS-PAGE. ScFv-c conjugation with unifunctional maleimide PEGs resulted in PEG conjugates incorporating 30-80% of the scFv-c, but usually above 50%. Efficiency of scFv-c conjugation to both functional groups of the bifunctional PEG-(Mal)2 varied between the PEG and scFv-c molecules studied. A maximum of 45% of scFv-c protein was conjugated as PEG- (scFv-c)2 using the smallest PEG-(Mal)2 (2 kDa). No significant increase in scFv-c conjugation was observed by the use of greater than a 5 molar excess of PEG/scFv-c. Under the same conjugation conditions, PEG as OPSS yielded less than 10% PEG-scFv-c. PEG-(scFv)2 conjugates had increased binding in ELISA using malignant cell membranes, when compared with unmodified scFv-c. PEGylated-scFv binding was comparable with unmodified scFv-c. In summary, scFv-c can be PEGylated in a site-specific manner using uni- or bivalent PEG-Mal, either linear or branched. ScFv-c was most efficiently conjugated to smaller PEG-Mal molecules, with the smallest, 2 kDa PEG-Mal, usually PEGylating 60-90% of the scFv-c. ScFv-c conjugation to form PEG-(scFv-c)2 reached greatest efficiency at 45%, and its purified form demonstrated greater binding than the corresponding scFv-c.     

Crystal structures of a therapeutic single chain antibody in complex with two drugs of abuse—Methamphetamine and 3,4‐methylenedioxymethamphetamine

Methamphetamine (METH) is a major drug threat in the United States and worldwide. Monoclonal antibody (mAb) therapy for treating METH abuse is showing exciting promise and the understanding of how mAb structure relates to function will be essential for future development of these important therapies. We have determined crystal structures of a high affinity anti-(+)-METH therapeutic single chain antibody fragment (scFv6H4, K_D= 10 nM) derived from one of our candidate mAb in complex with METH and the (+) stereoisomer of another abused drug, 3,4-methylenedioxymethamphetamine (MDMA), known by the street name “ecstasy.” The crystal structures revealed that scFv6H4 binds to METH and MDMA in a deep pocket that almost completely encases the drugs mostly through aromatic interactions. In addition, the cationic nitrogen of METH and MDMA forms a salt bridge with the carboxylate group of a glutamic acid residue and a hydrogen bond with a histidine side chain. Interestingly, there are two water molecules in the binding pocket and one of them is positioned for a C—H⋯O interaction with the aromatic ring of METH. These first crystal structures of a high affinity therapeutic antibody fragment against METH and MDMA (resolution = 1.9 Å, and 2.4 Å, respectively) provide a structural basis for designing the next generation of higher affinity antibodies and also for carrying out rational humanization.     

Biophysical characterization of a riboflavin-conjugated dendrimer platform for targeted drug delivery

The present study describes the biophysical characterization of generation-five poly(amidoamine) (PAMAM) dendrimers conjugated with riboflavin (RF) as a cancer-targeting platform. Two new series of dendrimers were designed, each presenting the riboflavin ligand attached at a different site (isoalloxazine at N-3 and d-ribose at N-10) and at varying ligand valency. Isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) were used to determine the binding activity for riboflavin binding protein (RfBP) in a cell-free solution. The ITC data shows dendrimer conjugates have K(D) values of ≥ 465 nM on a riboflavin basis, an affinity ~93-fold lower than that of free riboflavin. The N-3 series showed greater binding affinity in comparison with the N-10 series. Notably, the affinity is inversely correlated with ligand valency. These findings are also corroborated by DSC, where greater protein-conjugate stability is achieved with the N-3 series and at lower ligand valency.     

Synthesis, characterization, and in vitro activity of dendrimer-streptokinase conjugates

Dendrimer conjugation with low molecular weight drugs has been of increasing interest recently for improving pharmacokinetics, targeting drugs to specific sites, and facilitating cellular uptake. Opportunities for increasing the performance of relatively large therapeutic proteins such as streptokinase (SK) using dendrimers are being explored in this study. Using the active ester method, a series of streptokinase-poly(amido amine) (PAMAM) G3.5 conjugates were synthesized with varying amounts of dendrimer-to-protein molar ratios. Characterization of these conjugates by GPC, IEC, and native-PAGE suggested that the conjugation reaction was successful, resulting in relatively pure SK-dendrimer conjugates. The conjugate made with an equimolar ratio of dendrimer to streptokinase (1:1) exhibited the highest enzymatic activity retention ( approximately 80% retained) that has been reported so far for conjugated streptokinase with macromolecules such as PEG or dextran. SK conjugates with higher streptokinase-to-dendrimer molar ratios (1:10 and 1:20) exhibited lower initial enzymatic activities. However, these conjugates showed sustained thrombolytic activity in plasma, perhaps due to the release of SK from the conjugate. All of the SK conjugates displayed significantly improved stability in phosphate buffer solution, compared to free SK. The high coupling reaction efficiencies and the resulting high enzymatic activity retention achieved in this study could enable a desirable way for modifying many bioactive macromolecules with dendrimers.     

Dendrimer versus linear conjugate: Influence of polymeric architecture on the delivery and anticancer effect of paclitaxel

The relative difference in polymeric architectures of dendrimer and linear bis(poly(ethylene glycol)) (PEG) polymer in conjugation with paclitaxel has been described. Paclitaxel, a poorly soluble anticancer drug, was covalently conjugated with PAMAM G4 hydroxyl-terminated dendrimer and bis(PEG) polymer for the potential enhancement of drug solubility and cytotoxicity. Both conjugates were characterized by 1NMR, HPLC, and MALDI/TOF. In addition, molecular conformations of dendrimer, bis(PEG), paclitaxel, and its polymeric conjugates were studied by molecular modeling. Hydrolysis of the ester bond in the conjugate was analyzed by HPLC using esterase hydrolyzing enzyme. In vitro cytotoxicity of dendrimer, bis(PEG), paclitaxel, and polymeric conjugates containing paclitaxel was evaluated using A2780 human ovarian carcinoma cells. Cytotoxicity increased by 10-fold with PAMAM dendrimer-succinic acid-paclitaxel conjugate when compared with free nonconjugated drug. Data obtained indicate that the nanosized dendritic polymer conjugates can be used with good success as anticancer drug carriers.     

Bifunctional PAMAM dendrimer conjugates of folic acid and methotrexate with defined ratio

Our group previously developed a multifunctional, targeted cancer therapeutic based on Generation 5 (G5) polyamidoamine (PAMAM) dendrimers. In those studies we conjugated the targeting molecule folic acid (FA) and the chemotherapeutic drug methotrexate (MTX) sequentially. This complex macromolecule was shown to selectively bind and kill KB tumor cells that overexpress folate receptor (FR) in vitro and in vivo. However, the multistep conjugation strategy employed in the synthesis of the molecule resulted in heterogeneous populations having differing numbers and ratios of the functionally antagonistic FA and MTX. This led to inconsistent and sometimes biologically inactive batches of molecules, especially during large-scale synthesis. We here resolved this issue by using a novel triazine scaffold approach that reduces the number of dendrimer conjugation steps required and allows for the synthesis of G5 conjugates with defined ratios of FA and MTX. Although an unoccupied γ-glutamyl carboxylate of FA has been previously suggested to be nonessential for FR binding, the functional requirement of an open α-carboxylate still remains unclear. In an attempt to also address this question, we have synthesized isomeric FA dendrimer conjugates (α-carboxyl or γ-carboxyl linked). Competitive binding studies revealed that both linkages have virtually identical affinity toward FR on KB cells. Our studies show that a novel bifunctional triazine-based conjugate G5-Triazine-γMTX-αFA with identical numbers of FA and MTX binds to FR through a polyvalent interaction and induces cytotoxicity in KB cells through FR-mediated cellular internalization, inducing higher toxicity as compared to conjugates synthesized by the multistep strategy. This work serves as a proof of concept for the development of bifunctional dendrimer conjugates that require a defined ratio of two functional molecules.     

Antibody-dendrimer conjugates: the number, not the size of the dendrimers, determines the immunoreactivity

Radioimmunotherapy using antibodies with favorable tumor targeting properties and high binding affinity is increasingly applied in cancer therapy. The potential of this valuable cancer treatment modality could be further improved by increasing the specific activity of the labeled proteins. This can be done either by coupling a large number of chelators which leads to a decreased immunoreactivity or by conjugating a small number of multimeric chelators. In order to systematically investigate the influence of conjugations on immunoreactivity with respect to size and number of the conjugates, the anti-EGFR antibody hMAb425 was reacted with PAMAM dendrimers of different size containing up to 128 chelating agents per conjugation site. An improved dendrimer synthesis protocol was established to obtain compounds of high homogeneity suitable for the formation of defined protein conjugates. The quantitative derivatization of the PAMAM dendrimers with DOTA moieties and the characterization of the products by isotopic dilution titration using (111)In/(nat)In are shown. The DOTA-containing dendrimers were conjugated with high efficiency to hMAb425 by applying Sulfo-SMCC as cross-linking agent and a 10- to 25-fold excess of the thiol-containing dendrimers. The determination of the immunoreactivities of the antibody-dendrimer conjugates by FACS analysis revealed a median retained immunoreactivity of 62.3% for 1.7 derivatization sites per antibody molecule, 55.4% for 2.8, 27.9% for 5.3, and 17.1% for 10.0 derivatization sites per antibody but no significant differences in immunoreactivity for different dendrimer sizes. These results show that the dendrimer size does not influence the immunoreactivity of the derivatized antibody significantly over a wide molecular weight range, whereas the number of derivatization sites has a crucial effect.     

In silico experiments of single-chain antibody fragment against drugs of abuse

Three sets of in silico experiments have been conducted to elucidate the binding mechanics of two drugs, (+)-methamphetamine (METH) and amphetamine (AMP) to the single-chain variable fragment (scFv) recently engineered from anti-METH monoclonal antibody mAb6H4 (IgG, κlight chain, K_d = 11 nM). The first set of in silico experiments are long time equilibration runs of scFv:drug complexes and of drug-free scFv both in the solution. They demonstrate how the solution structures of scFv deviate from its crystallographic form with or without drug molecules bound to it. They lead to the prediction that the Arrhenius activation barrier is nearly zero for transitions from the dissociated state to the bound state. The second set of in silico experiments are nonequilibrium dynamics of pulling the drug molecules out of the binding pocket of scFv and the equilibration runs for drugs to fall back into the binding pocket. They demonstrate that extra water molecules (in addition to the two crystallographic waters) exist inside the binding pocket, underneath the drug molecules. These extra waters must have been evaporated from the binding pockets during the crystallization process of the in vitro experiments of structural determination. The third set of in silico experiments are nonequilibrium steered molecular dynamics simulations to determine the absolute binding free energies of METH and AMP to scFv. The center of mass of a drug molecule (METH or AMP) is steered (pulled) towards (forward) and away from (reverse) the binding site, sampling forward and reverse pulling paths. Mechanic work is measured along the pulling paths. The work measurements are averaged through the Brownian dynamics fluctuation dissipation theorem to produce the free-energy profiles of the scFv:drug complexes as a function of the drug-scFv separation. These experiments lead to the theoretical prediction of absolute binding energies of METH and AMP that are in agreement with the in vitro experimental results.     

Cytotoxicity of polypropylenimine dendrimer conjugates on cultured endothelial cells

The cytotoxicity and time-dependent membrane disruption by polypropylenimine dendrimer conjugates on cultured human umbilical vein endothelial cells (HUVEC) is reported. Fluorescently labeled derivatives of generation 5 polypropylenimine dendrimers were prepared via conversion of amines to acetamides or through the covalent attachment of high molecular weight poly(ethylene glycol) (PEG) chains. Direct interactions between the fluorescent dendrimer conjugates and HUVEC were monitored using confocal fluorescence microscopy to track dendrimer movement across the plasma membrane and the fluorescent staining of cell nuclei. Propidium iodide and lactate dehydrogenase cytotoxicity assays confirmed that chemical modification of the surface amines of the parental dendrimer to neutral acetamide or PEG functionalities eliminated their acute cytotoxicity. Cationic primary-amine-containing dendrimers demonstrated drastic time-dependent changes in the plasma membrane permeability and prominent cytotoxicity. However, complete removal of the primary amines or masking of the cationic surface via PEGylation decreased dendrimer cytotoxicity. Thus, preventing electrostatic interactions of dendrimers with cellular membranes apparently is a necessary step toward minimizing the toxicity of delivery vehicles to the endothelium.     

Application of the functionalized congener approach to dendrimer-based signaling agents acting through A2A adenosine receptors

As a continued effort to develop multivalent ligands to enhance the pharmacological effects of monomeric drugs, DITC-APEC, a chemically reactive nucleoside A(2A) adenosine receptor (AR) agonist, was employed to derivatize the surface of third-generation (G3) polyamidoamine (PAMAM) dendrimers. The resulting conjugates carried multiple copies of the agonist attached through a thiourea linkage and differed in the number of attachments and in the presence of a fluorophore or additional surface modification. Computer modeling studies suggested that these DITC-APEC-loaded dendrimers extended the overall diameter of the previously reported PAMAM-CGS21680 dendrimer derivatives (Kim et al., Bioconjugate Chem 2008 19:406-411) by ca. 20 A, potentially increasing the conformational flexibility of the appended ligands to achieve optimal geometry for efficient binding at A(2A) ARs. Increased affinity and selectivity in binding in comparison to the CGS21680 conjugate were envisioned, due to the presence of an extended linker, i.e., a dithioureylenephenyl functionality. In vitro radioligand competition experiments showed effective binding of these PAMAM-DITC-APEC dendrimer conjugates at the human A(2A) and A(3) ARs with submicromolar K (i) values and selectivity in comparison to the human A(1) AR. Furthermore, these nucleoside-loaded dendrimers exhibited an A(2A) AR-mediated inhibitory effect on ADP-induced aggregation of human platelets. The present study demonstrates the potential of applying the functionalized congener concept to engineer dendrimer-based multivalent ligands for G protein-coupled receptors.     

PAMAM dendrimers for the delivery of the antibacterial Triclosan

Many oral care products incorporate an antibacterial compound to prevent the formation of dental plaque which predisposes teeth to dental caries or periodontal disease. Triclosan (TCN) is a commonly used antiplaque agent in toothpastes. Strategies to increase the delivery efficiency of antibacterials using formulation aids such as polyamidoamine (PAMAM) dendrimers are of interest. Solubilisation studies over the pH range 5-12 demonstrated an increase in the level of TCN solubilised with increasing dendrimer concentration (1 mM-5 mM). However, the dendrimer was unable to enhance TCN solubility at lower pH values and the solubilising effect observed was attributed to the ionization of TCN (pKa 8.14) resulting from dendrimer induced pH changes. End group modification of G3 PAMAM dendrimer with phenylalanine in order to promote solubility through pi-pi stacking between TCN and the amino acid has been carried out. Phenylalanine:G3 PAMAM conjugates of different ratios (32:1, 21:1, 16:1) were synthesized. The fully conjugated dendrimer (32:1) had poor aqueous solubility, whereas the 21:1 and 16:1 dendrimer conjugates were water soluble. The 21:1 conjugate was tested for its ability to solubilise TCN, however, again there was no increase over control buffer solutions of the same pH. An alternative approach under investigation is to directly conjugate TCN to PAMAM dendrimers via a hydrolysable linkage.     

PAMAM dendrimers for the delivery of the antibacterial Triclosan

Many oral care products incorporate an antibacterial compound to prevent the formation of dental plaque which predisposes teeth to dental caries or periodontal disease []. Triclosan (TCN) is a commonly used antiplaque agent in toothpastes []. Strategies to increase the delivery efficiency of antibacterials using formulation aids such as polyamidoamine (PAMAM) dendrimers are of interest.

Solubilisation studies over the pH range 5-12 demonstrated an increase in the level of TCN solubilised with increasing dendrimer concentration (1 mM–5 mM). However, the dendrimer was unable to enhance TCN solubility at lower pH values and the solubilising effect observed was attributed to the ionization of TCN (pKa 8.14) resulting from dendrimer induced pH changes.

End group modification of G3 PAMAM dendrimer with phenylalanine in order to promote solubility through π–π stacking between TCN and the amino acid has been carried out. Phenylalanine:G3 PAMAM conjugates of different ratios (32:1, 21:1, 16:1) were synthesized. The fully conjugated dendrimer (32:1) had poor aqueous solubility, whereas the 21:1 and 16:1 dendrimer conjugates were water soluble. The 21:1 conjugate was tested for its ability to solubilise TCN, however, again there was no increase over control buffer solutions of the same pH. An alternative approach under investigation is to directly conjugate TCN to PAMAM dendrimers via a hydrolysable linkage.     

High-generation polycationic dendrimers are unusually effective at disrupting anionic vesicles: membrane bending model

The membrane disruption properties of high generation (G4 to G7) poly(amidoamine) (PAMAM) dendrimers are evaluated and compared to linear poly(lysine). The G6 and G7 dendrimers are unusually effective at inducing leaky fusion of anionic, large unilamellar vesicles, as determined by standard fluorescence assays for lipid mixing, leakage, and contents mixing. Both G7 dendrimer and poly(lysine) are able to disrupt sterically stabilized vesicles that are coated with poly(ethylene glycol). A G7 dendrimer/DNA complex with a 1:1 concentration ratio of dendrimer surface amines to DNA phosphate groups is unable to induce leakage of 3:7 POPA-PE vesicles; however, extensive leakage is observed when the surface amine to phosphate stoichiometry is >/=3:1. Thus, the DNA/dendrimer complexes that typically induce high levels of cell transfection are also able to induce high levels of vesicle leakage. The G7 dendrimer does not induce membrane phase separation in 3:7 POPA-PE vesicles, but an inverse hexagonal phase is observed by (31)P NMR. The enhanced membrane disruption is interpreted in terms of a membrane bending model. A rigid, polycationic dendrimer sphere uses electrostatic forces to bend a malleable, anionic membrane and induce bilayer packing stresses. This bending model is biomimetic in the sense that protein-induced membrane bending is currently thought to be an important factor in the fusion mechanism of influenza virus.     

Impact of linker and conjugation chemistry on antigen binding,Fc receptor binding and thermal stability of model antibody-drug conjugates

Antibody-drug conjugates (ADCs) with biotin as a model cargo tethered to IgG1 mAbs via different linkers and conjugation methods were prepared and tested for thermostability and ability to bind target antigen and Fc receptor. Most conjugates demonstrated decreased thermostability relative to unconjugated antibody, based on DSC, with carbohydrate and amine coupled ADCs showing the least effect compared with thiol coupled conjugates. A strong correlation between biotin-load and loss of stability is observed with thiol conjugation to one IgG scaffold, but the stability of a second IgG scaffold is relatively insensitive to biotin load. The same correlation for amine coupling was less significant. Binding of antibody to antigen and Fc receptor was investigated using surface plasmon resonance. None of the conjugates exhibited altered antigen affinity. Fc receptor FcγIIb (CD32b) interactions were investigated using captured antibody conjugate. Protein G and Protein A, known inhibitors of Fc receptor (FcR) binding to IgG, were also used to extend the analysis of the impact of conjugation on Fc receptor binding. H10_NPEG4 was the only conjugate to show significant negative impact to FcR binding, which is likely due to higher biotin-load compared with the other ADCs. The ADC aHIS_NLC and aHIS_TPEG8 demonstrated some loss in affinity for FcR, but to much lower extent. The general insensitivity of target binding and effector function of the IgG1 platform to conjugation highlight their utility. The observed changes in thermostability require consideration for the choice of conjugation chemistry, depending on the system being pursued and particular application of the conjugate.     

Impact of linker and conjugation chemistry on antigen binding,Fc receptor binding and thermal stability of model antibody-drug conjugates

Antibody-drug conjugates (ADCs) with biotin as a model cargo tethered to IgG1 mAbs via different linkers and conjugation methods were prepared and tested for thermostability and ability to bind target antigen and Fc receptor. Most conjugates demonstrated decreased thermostability relative to unconjugated antibody, based on DSC, with carbohydrate and amine coupled ADCs showing the least effect compared with thiol coupled conjugates. A strong correlation between biotin-load and loss of stability is observed with thiol conjugation to one IgG scaffold, but the stability of a second IgG scaffold is relatively insensitive to biotin load. The same correlation for amine coupling was less significant. Binding of antibody to antigen and Fc receptor was investigated using surface plasmon resonance. None of the conjugates exhibited altered antigen affinity. Fc receptor FcγIIb (CD32b) interactions were investigated using captured antibody conjugate. Protein G and Protein A, known inhibitors of Fc receptor (FcR) binding to IgG, were also used to extend the analysis of the impact of conjugation on Fc receptor binding. H10_NPEG4 was the only conjugate to show significant negative impact to FcR binding, which is likely due to higher biotin-load compared with the other ADCs. The ADC aHIS_NLC and aHIS_TPEG8 demonstrated some loss in affinity for FcR, but to much lower extent. The general insensitivity of target binding and effector function of the IgG1 platform to conjugation highlight their utility. The observed changes in thermostability require consideration for the choice of conjugation chemistry, depending on the system being pursued and particular application of the conjugate.