Bruce Charles Gomes

Increasing Serum Half-life and Extending Cholesterol Lowering in Vivo by Engineering Antibody with pH-sensitive Binding to PCSK9

Background: An antagonistic anti-PCSK9 antibody exhibits target-mediated clearance, resulting in a dose-dependent PK. Results: Engineering of an antibody with pH-sensitive binding to PCSK9 decreases target-mediated clearance, resulting in increased PK and efficacy in vivo. Conclusion: pH-sensitive anti-PCSK9 antibodies are excellent candidates for therapeutic development. Significance: pH-sensitive antibodies may enable less frequent or lower dosing of antibodies hampered by target-mediated clearance and high antigen load. Target-mediated clearance and high antigen load can hamper the efficacy and dosage of many antibodies. We show for the first time that the mouse, cynomolgus, and human cross-reactive, antagonistic anti-proprotein convertase substilisin kexin type 9 (PCSK9) antibodies J10 and the affinity-matured and humanized J16 exhibit target-mediated clearance, resulting in dose-dependent pharmacokinetic profiles. These antibodies prevent the degradation of low density lipoprotein receptor, thus lowering serum levels of LDL-cholesterol and potently reducing serum cholesterol in mice, and selectively reduce LDL-cholesterol in cynomolgus monkeys. In order to increase the pharmacokinetic and efficacy of this promising therapeutic for hypercholesterolemia, we engineered pH-sensitive binding to mouse, cynomolgus, and human PCSK9 into J16, resulting in J17. This antibody shows prolonged half-life and increased duration of cholesterol lowering in two species in vivo by binding to endogenous PCSK9 in mice and cynomolgus monkeys, respectively. The proposed mechanism of this pH-sensitive antibody is that it binds with high affinity to PCSK9 in the plasma at pH 7.4, whereas the antibody-antigen complex dissociates at the endosomal pH of 5.5–6.0 in order to escape from target-mediated degradation. Additionally, this enables the antibody to bind to another PCSK9 and therefore increase the antigen-binding cycles. Furthermore, we show that this effect is dependent on the neonatal Fc receptor, which rescues the dissociated antibody in the endosome from degradation. Engineered pH-sensitive antibodies may enable less frequent or lower dosing of antibodies hampered by target-mediated clearance and high antigen load.

The RIP‐like kinase, RIP3, induces apoptosis and NF‐κB nuclear translocation and localizes to mitochondria

A RIP‐like protein, RIP3, has recently been reported that contains an N‐terminal kinase domain and a novel C‐terminal domain that promotes apoptosis. These experiments further characterize RIP3‐mediated apoptosis and NF‐κB activation. Northern blots indicate that rip3 mRNA displays a restricted pattern of expression including regions of the adult central nervous system. The rip3 gene was localized by fluorescent in situ hybridization to human chromosome 14q11.2, a region frequently altered in several types of neoplasia. RIP3‐mediated apoptosis was inhibited by Bcl‐2, Bcl‐xL, dominant‐negative FADD, as well as the general caspase inhibitor Z‐VAD. Further dissection of caspase involvement in RIP3‐induced apoptosis indicated inhibition by the more specific inhibitors Z‐DEVD (caspase‐3, ‐6, ‐7, ‐8, and ‐10) and Z‐VDVAD (caspase‐2). However, caspase‐1, ‐6, ‐8 and ‐9 inhibitors had little or no effect on RIP3‐mediated apoptosis. Mutational analysis of RIP3 revealed that the C‐terminus of RIP3 contributed to its apoptotic activity. This region is similar, but distinct, to the death domain found in many pro‐apoptotic receptors and adapter proteins, including FAS, FADD, TNFR1, and RIP. Furthermore, point mutations of RIP3 at amino acids conserved among death domains, abrogated its apoptotic activity. RIP3 was localized by immunofluorescence to the mitochondrion and may play a key role in the mitochondrial disruptions often associated with apoptosis.

Tumor necrosis factor-α induces the expression of DR6, a member of the TNF receptor family, through activation of NF-κB

The tumor necrosis factor (TNF) receptor family are ligand-regulated transmembrane proteins that mediate apoptosis as well as activation of the transcription factor NF-κB. Exogenous expression of DR6, a recently identified member of the TNF receptor family, induced apoptosis in untransformed or tumor-derived cells and the apoptotic function of DR6 was inhibited by co-expression of Bcl-2, Bcl-xL or the inhibitor-of-apoptosis (IAP) family member, survivin. Expression of a dominant negative mutant of FADD failed to protect from DR6-mediated apoptosis indicating that unlike TNFR1 and Fas, DR6 induced apoptosis via a FADD-independent mechanism. Despite the ability of exogenous DR6 expression to induce apoptosis, DR6 mRNA and protein were found to be elevated in prostate tumor cell lines and in advanced stages of prostate cancer. Analysis of several anti-apoptotic proteins revealed that Bcl-xL levels and serine 32 phosphorylation of IκB, the natural inhibitor of NF-κB, were similarly elevated in cells expressing high levels of DR6, suggesting that NF-κB-regulated survival proteins may protect from DR6-induced apoptosis and that DR6 is a target of NF-κB regulation. Treatment of LnCAP cells with TNF-α resulted in increases in both DR6 mRNA and protein levels, and this induction was suppressed by inhibitors of NF-κB. Similarly, treatment of cells expressing high levels of DR6 with indomethacin and ibuprofen, compounds also known to perturb NF-κB function, resulted in a dose-dependent decrease in DR6 protein and mRNA levels. These results demonstrate that TNF-α signaling induces the expression of a member of its own receptor family through activation of NF-κB.