Reduction In Net Positive Charge Research Articles

Balancing charge in the complementarity-determining regions of humanized mAbs without affecting pI reduces non-specific binding and improves the pharmacokinetics

Lowering the isoelectric point (pI) through engineering the variable region or framework of an IgG can improve its exposure and half-life via a reduction in clearance mediated through non-specific interactions. As such, net charge is a potentially important property to consider in developing therapeutic IgG molecules having favorable pharmaceutical characteristics. Frequently, it may not be possible to shift the pI of monoclonal antibodies (mAbs) dramatically without the introduction of other liabilities such as increased off-target interactions or reduced on-target binding properties. In this report, we explored the influence of more subtle modifications of molecular charge on the in vivo properties of an IgG1 and IgG4 monoclonal antibody. Molecular surface modeling was used to direct residue substitutions in the complementarity-determining regions (CDRs) to disrupt positive charge patch regions, resulting in a reduction in net positive charge without affecting the overall pI of the mAbs. The effect of balancing the net positive charge on non-specific binding was more significant for the IgG4 versus the IgG1 molecule that we examined. This differential effect was connected to the degree of influence on cellular degradation in vitro and in vivo clearance, distribution and metabolism in mice. In the more extreme case of the IgG4, balancing the charge yielded an ∼7-fold improvement in peripheral exposure, as well as significantly reduced tissue catabolism and subsequent excretion of proteolyzed products in urine. Balancing charge on the IgG1 molecule had a more subtle influence on non-specific binding and yielded only a modest alteration in clearance, distribution and elimination. These results suggest that balancing CDR charge without affecting the pI can lead to improved mAb pharmacokinetics, the magnitude of which is likely dependent on the relative influence of charge imbalance and other factors affecting the molecule's disposition.

Phospholipase Cζ binding to PtdIns(4,5)P2 requires the XY-linker region

Phospholipase C-zeta (PLCζ) is a strong candidate for the mammalian sperm-derived factor that triggers the Ca2+ oscillations required for egg activation at fertilization. PLCζ lacks a PH domain, which targets PLCδ1 to the phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) substrate in the plasma membrane. Previous studies failed to detect PLCζ in the plasma membrane, hence the means of PLCζ binding to PtdIns(4,5)P2 is unclear. We find that the PLCζ XY linker, but not the C2 domain, exhibits robust binding to PtdIns(4,5)P2 or to liposomes containing near-physiological levels of PtdIns(4,5)P2. The role of positively charged residues within the XY linker was addressed by sequentially substituting alanines for three lysine residues, K374, K375 and K377. Microinjection of these mutants into mouse eggs enabled their Ca2+ oscillation-inducing activities to be compared with wild-type PLCζ. The XY-linker mutant proteins were purified and the in vitro PtdIns(4,5)P2 hydrolysis and binding properties were monitored. Successive reduction of net positive charge within the PLCζ XY linker significantly affects both in vivo Ca2+-oscillation-inducing activity and in vitro PtdIns(4,5)P2 interaction of mouse PLCζ. Our data suggest that positively charged residues within the XY linker play an important role in the PLCζ interaction with PtdIns(4,5)P2, a crucial step in generating the Ca2+ activation signal that is essential for fertilization in mammals.

Effect of Phosphorylation of Myelin Basic Protein By MAPK on its Interactions with Actin and Actin Binding to a Lipid Membrane in Vitro

Myelin basic protein (MBP) binds to negatively charged lipids on the cytosolic surface of oligodendrocyte membranes and is most likely responsible for adhesion of these surfaces in the multilayered myelin sheath. It can also polymerize actin, bundle F-actin filaments, and bind actin filaments to lipid bilayers through electrostatic interactions. MBP consists of a number of posttranslationally modified isomers of varying charge, some resulting from phosphorylation at several sites by different kinases, including mitogen-activated protein kinase (MAPK). Phosphorylation of MBP in oligodendrocytes occurs in response to various extracellular stimuli. Phosphorylation/dephosphorylation of MBP also occurs in the myelin sheath in response to electrical activity in the brain. Here we investigate the effect of phosphorylation of MBP on its interaction with actin in vitro by phosphorylating the most highly charged unmodified isomer, C1, at two sites with MAPK. Phosphorylation decreased the ability of MBP to polymerize actin and to bundle actin filaments but had no effect on the dissociation constant of the MBP-actin complex or on the ability of Ca2+-calmodulin to dissociate the complex. The most significant effect of phosphorylation on the MBP-actin complex was a dramatic reduction in its ability to bind to negatively charged lipid bilayers. The effect was much greater than that reported earlier for another charge isomer of MBP, C8, in which six arginines were deiminated to citrulline, resulting in a reduction of net positive charge of 6. These results indicate that although average electrostatic forces are the primary determinant of the interaction of MBP with actin, phosphorylation may have an additional effect due to a site-specific electrostatic effect or to a conformational change. Thus, phosphorylation of MBP, which occurs in response to various extracellular signals in both myelin and oligodendrocytes, attenuates the ability of MBP to polymerize and bundle actin and to bind it to a negatively charged membrane.

Identification of the Tissue Inhibitor of Metalloproteinases-2 (TIMP-2) Binding Site on the Hemopexin Carboxyl Domain of Human Gelatinase A by Site-directed Mutagenesis: THE HIERARCHICAL ROLE IN BINDING TIMP-2 OF THE UNIQUE CATIONIC CLUSTERS OF HEMOPEXIN MODULES III AND IV

Cell surface activation of progelatinase A occurs in a quaternary complex with the tissue inhibitor of metalloproteinases-2 (TIMP-2) and two membrane-type matrix metalloproteinases. We have mutated the unique cationic clusters found in hemopexin modules III and IV of the carboxyl domain (C domain) of human gelatinase A to determine their role in binding TIMP-2. Twelve single, double, and triple site-directed mutations were produced that exhibited different TIMP-2 binding properties. Notably, single alanine substitutions at Lys547 and Lys617 reduced TIMP-2 binding by an order of magnitude from that of the recombinant wild-type C domain. Mutations that completely disrupted the C domain.TIMP-2 interaction were K558A/R561A, K610T/K617A, and K566A/K568A/K617A. A triple mutation, K566A/K568A/K575A, having TIMP-2 binding indistinguishable from the wild-type C domain (Kd 3.0 x 10(-8) M), showed that simple reduction of net positive charge does not reduce TIMP-2 affinity. Because the double mutation K566A/K568A also did not alter TIMP-2 binding, these data do not confirm previously reported chimera studies that indicated the importance of the triple lysine cluster at positions 566/567/568 in TIMP-2 binding. Nonetheless, a subtle role in TIMP-2 interaction for the 566/567/568-lysine triad is indicated from the enhanced reduction in TIMP-2 binding that occurs when mutations here were combined with K617A. Thus, these analyses indicate that the TIMP-2 binding surface lies at the junction of hemopexin modules III and IV on the peripheral rim of the gelatinase A C domain. This location implies that considerable molecular movement of the TIMP-2. C domain complex would be needed for the bound TIMP-2 to inhibit in cis the gelatinase A active site.

Refocusing neutralizing antibody response by targeted dampening of an immunodominant epitope.

Immunodominant epitopes are known to suppress a primary immune response to other antigenic determinants by a number of mechanisms. Many pathogens have used this strategy to subvert the immune response and may be a mechanism responsible for limited vaccine efficacies. HIV-1 vaccine efficacy appears to be complicated similarly by a limited, immunodominant, isolate-restricted immune response generally directed toward determinants in the third variable domain (V3) of the major envelope glycoprotein, gp120. To overcome this problem, we have investigated an approach based on masking the V3 domain through addition of N-linked carbohydrate and reduction in net positive charge. N-linked modified gp120s were expressed by recombinant vaccinia virus and used to immunize guinea pigs by infection and protein boosting. This modification resulted in variable site-specific glycosylation and antigenic dampening, without loss of gp120/CD4 binding or virus neutralization. Most importantly, V3 epitope dampening shifted the dominant type-specific neutralizing Ab response away from V3 to an epitope in the first variable domain (V1) of gp120. Interestingly, in the presence of V3 dampening V1 changes from an immunodominant non-neutralizing epitope to a primary neutralizing epitope with broader neutralizing properties. In addition, Ab responses were also observed to conserved domains in C1 and C5. These results suggest that selective epitope dampening can lead to qualitative shifts in the immune response resulting in second order neutralizing responses that may prove useful in the fine manipulation of the immune response and in the development of more broadly protective vaccines and therapeutic strategies.