A potent, long-lasting form of interferon alpha-2a mono-pegylated with a 40 kilodalton branched poly(ethylene glycol) was designed, synthesized, and characterized. Mono-pegylated interferon alpha-2a was comprised of four major positional isomers involving Lys31, Lys121, Lys131, and Lys134 of interferon. The in vitro anti-viral activity of pegylated interferon alpha-2a was found to be only 7% of the original activity. In contrast, the in vivo antitumor activity was severalfold enhanced compared to interferon alpha-2a. Pegylated interferon alpha-2a showed no immunogenicity in mice. After subcutaneous injection of pegylated interferon alpha-2a, a 70-fold increase in serum half-life and a 50-fold increase in mean plasma residence time concomitant with sustained serum concentrations were observed relative to interferon alpha-2a. These preclinical results suggest a significantly enhanced human pharmacological profile for pegylated interferon alpha-2a. Results of Phase II/III hepatitis C clinical trials in humans confirmed the superior efficacy of pegylated interferon alpha-2a compared to unmodified interferon alpha-2a.
Complement is a key component of the innate immune system. Inappropriate complement activation underlies the pathophysiology of a variety of diseases. Complement component 5 (C5) is a validated therapeutic target for complement-mediated diseases, but the development of new therapeutics has been limited by a paucity of preclinical models to evaluate the pharmacokinetic (PK) and pharmacodynamic (PD) properties of candidate therapies. The present report describes a novel humanized C5 mouse and its utility in evaluating a panel of fully human anti-C5 antibodies. Surprisingly, humanized C5 mice revealed marked differences in clearance rates amongst a panel of anti-C5 antibodies. One antibody, pozelimab (REGN3918), bound C5 and C5 variants with high affinity and potently blocked complement-mediated hemolysis in vitro. In studies conducted in both humanized C5 mice and cynomolgus monkeys, pozelimab demonstrated prolonged PK and durable suppression of hemolytic activity ex vivo. In humanized C5 mice, a switch in dosing from in-house eculizumab to pozelimab was associated with normalization of serum C5 concentrations, sustained suppression of hemolytic activity ex vivo, and no overt toxicity. Our findings demonstrate the value of humanized C5 mice in identifying new therapeutic candidates and treatment options for complement-mediated diseases. OPEN ACCESS Citation: Latuszek A, Liu Y, Olsen O, Foster R, Cao M, Lovric I, et al. (2020) Inhibition of complement pathway activation with Pozelimab, a fully human antibody to complement component C5. PLoS ONE 15(5): e0231892. https://doi.org/10.
Recombinant human serum albumin (HSA) conjugates of a 15-amino-acid truncated peptide YY (PYY) analogue were prepared using three heterobifunctional linkers [succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC), 6-maleimidohexanoic acid N-hydroxysuccinimide ester (MHS), and N-[γ-maleimidobutyryloxy]sulfosuccinimide ester (GMBS)] in 2 synthetic steps involving (1) reaction of succinimidyl ester on linker with ε-amine of Lys2 on the peptide and (2) reaction of maleimide on peptide linker with free thiol of Cysteine 34 (Cys34) on albumin. In-process controls using ESI LC-MS were used to follow reactions and identify reaction products. Proteolytic digests of the conjugate revealed that peptide conjugation occurs at Cys34 on HSA. Conjugates were assayed in cell-based assays to determine potency at the human Y2-receptor, and selectivity at the human Y1-, Y4-, and Y5-receptors using a calcium flux assay. All three conjugates assayed were selective agonists of the Y2-receptor, and displayed nanomolar potencies. MCC and MH conjugates were selected for acute PK/PD studies in DIO mice. Significant reduction in food intake was observed with the MH conjugate, which lasted for 24 h at the 10 mg (or 4 μmol)/kg dose. While the MCC conjugate exhibited greater potency in vitro, it was slightly less effective than the MH conjugate in vivo with respect to reduction in food intake. Both conjugates were significantly less active than the peptide coupled to a 30 kDa PEG. The observed T1/2 (8-9 h) for both conjugates was significantly lower than that observed for the PEGylated peptide (∼25 h). These results suggest that, as compared with the unmodified and PEGylated peptide, the extended circulation half-life of albumin conjugates is mediated through uptake and recirculation by FcRn, and allometric scaling methods are necessary to account for interspecies variation in pharmacokinetic properties.
Harnessing complement-mediated cytotoxicity by therapeutic antibodies has been limited because of dependency on size and density of antigen, structural constraints resulting from orientation of antibody binding, and blockade of complement activation by inhibitors expressed on target cells. We developed a modular bispecific antibody platform that directs the complement-initiating protein C1q to target cells, increases local complement deposition and induces cytotoxicity against target antigens with a wide-range of expression. The broad utility of this approach to eliminate both prokaryotic and eukaryotic cells was demonstrated by pairing a unique C1q-recruiting arm with multiple targeting arms specific for
Staphylococcus aureus
,
Pseudomonas aeruginosa
, B-cells and T-cells, indicating applicability for diverse indications ranging from infectious diseases to cancer. Generation of C1q humanized mice allowed for demonstration of the efficacy of this approach to clear disease-inducing cells
in vivo
. In summary, we present a novel, broadly applicable, and versatile therapeutic modality for targeted cell depletion.
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