Monoclonal antibodies are used in numerous therapeutic and diagnostic applications; however, their efficacy is contingent on specificity and avidity. Here, we show that presentation of antibodies on the surface of nonspherical particles enhances antibody specificity as well as avidity toward their targets. Using spherical, rod-, and disk-shaped polystyrene nano-and microparticles and trastuzumab as the targeting antibody, we studied specific and nonspecific uptake in three breast cancer cell lines: BT-474, SK-BR-3, and MDA-MB-231. Rods exhibited higher specific uptake and lower nonspecific uptake in all cells compared with spheres. This surprising interplay between particle shape and antibodies originates from the unique role of shape in determining binding and unbinding of particles to cell surface. In addition to exhibiting higher binding and internalization, trastuzumab-coated rods also exhibited greater inhibition of BT-474 breast cancer cell growth in vitro to a level that could not be attained by soluble forms of the antibody. The effect of trastuzumab-coated rods on cells was enhanced further by replacing polystyrene particles with pure chemotherapeutic drug nanoparticles of comparable dimensions made from camptothecin. Trastuzumab-coated camptothecin nanoparticles inhibited cell growth at a dose 1,000-fold lower than that required for comparable inhibition of growth using soluble trastuzumab and 10-fold lower than that using BSA-coated camptothecin. These results open unique opportunities for particulate forms of antibodies in therapeutics and diagnostics.morphology | nanomedicine | nanotechnology | drug delivery A ntibodies are used routinely in various analytical, diagnostic, and therapeutic applications, including cell and protein sorting (1), in vitro assays (2), in vivo imaging (3), and targeted delivery of therapeutics for the treatment of various diseases, including cancer (4), arthritis (5), and allergies (6). Significant attention has been given to understanding the molecular basis of antibody-antigen interactions (7) as well as to molecular engineering of antibodies to enhance their functions (8). An increasingly larger number of emerging applications of antibodies, however, are based on particulate systems in which antibodies are chemically or physically immobilized on the surface of microor nanoparticles (9-11). Such particulate antibodies are being pursued for targeted drug delivery or imaging. Although the benefits of antibodies in delivering therapeutic carriers to tissues have long been recognized, the effect of carriers themselves on antibody function has been relatively less studied. The function of antibodies that are immobilized on particles depends on the physicochemical properties of underlying particles, including the choice of material, size, surface modification, and shape. Commonly used nanoparticles for immobilizing antibodies include gold (9), iron oxide (12), quantum dots (13, 14), silica (15), polymers including poly(lactide-coglycolic acid) (PLGA) (11, 16) and polystyrene (...
Antibody-drug conjugates (ADCs), formed through the chemical linkage of a potent small molecule cytotoxin (drug) to a monoclonal antibody, have more complex and heterogeneous structures than the corresponding antibodies. This review describes the analytical methods that have been used in their physicochemical characterization. The selection of the most appropriate methods for a specific ADC is heavily dependent on the properties of the linker, the drug, and the choice of attachment sites (lysines, inter-chain cysteines, Fc glycans). Improvements in analytical techniques such as protein mass spectrometry and capillary electrophoresis have significantly increased the quality of information that can be obtained for use in product and process characterization, and for routine lot release and stability testing.
In the manufacture of the antibody-drug conjugate Trastuzumab-DM1 (T-DM1), the lysine residues on the antibody trastuzumab (Tmab) are modified to form the intermediate Tmab-MCC (T-MCC) and then conjugated with the drug DM1. Our goal is to understand the effects of modification and conjugation steps on the physicochemical stability of the antibody. The structural stability of Tmab relative to its modified and conjugated forms was assessed, employing thermally induced stress conditions to formulations containing Tmab, T-MCC, and T-DM1. DSC, SEC, CE-SDS, and LC-MS were used to study the stability of Tmab, T-MCC, and T-DM1 to thermal stress. The DSC thermograms show a decrease in melting temperature for the CH2 transition, in the order Tmab > T-MCC > T-DM1. As per SEC analysis, a significant increase in level of aggregation was detected in T-MCC (∼32%) and T-DM1 (∼5%) after 14 days at 40 °C. Tmab did not show significant aggregate formation. CE-SDS and LC-MS data demonstrate that the aggregation in the case of T-MCC is largely covalent and involves mechanisms other than formation of intermolecular disulfide cross-links. The aggregation observed for T-MCC was significantly inhibited upon addition of amino acids with nucleophilic side chains containing thiol (Cys) and hydroxyl moieties (Ser, Tyr). The covalent aggregation observed for T-MCC and the ability of nucleophilic amino acids, particularly Cys, to inhibit it indicate that the maleimide moiety in the MCC linker may react to form intermolecular covalent cross-links between T-MCC molecules, possibly through a Michael addition mechanism. In addition, DSC results demonstrate that the conjugation of the drug moiety DM1 to Tmab results in destabilization of the CH2 domain of the antibody.
The aspartic acid residues (Asp) present in the complementarity-determining regions (CDRs) of the light chains of two recombinant monoclonal antibodies (MAbs), MAb I and MAb II, are highly susceptible to isomerization due to the presence of glycine residues (Gly) on their C-terminal ends. Asp isomerization in these MAbs leads to formation of the isoaspartate (IsoAsp) and the cyclic imide (Asu) variants of these MAbs. Both MAb I and MAb II, employed in this study, elicit their pharmacological responses through binding human IgE. The formation of the MAb variants as a result of Asp isomerization significantly reduces the binding affinities of these antibodies to IgE, thereby reducing their potencies. Here we report on significant differences in the susceptibility of the MAb I and the MAb II to Asp isomerization. The molecular basis for these differences in rates of Asp isomerization was elucidated. The effect of primary sequence on Asp isomerization was evaluated using pentapeptide models of the MAbs, which included the labile Asp residues and their neighboring amino acid residues. The separation of the parent MAbs and pentapeptides from their isomerization products was achieved using hydrophobic interaction chromatography (HIC) and rp-HPLC, respectively. Structural characterization of the MAbs was performed using differential scanning calorimetry (DSC), circular dichroism (CD), and X-ray crystallography. Our investigations demonstrate that the differences in the Asp isomerization rates between MAb I and MAb II can be attributed to structural factors including the conformational flexibility and the extent of solvent exposure of the labile Asp residue.
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