Identification of fibrinogen as a plasma protein binding partner for lecanemab biosimilar IgG

Abstract

Objective: Recombinant monoclonal therapeutic antibodies like lecanemab, which target amyloid beta in Alzheimer's disease, offer a promising approach for modifying the disease progression. Due to its relatively short half-life, lecanemab administered as a bi-monthly infusion (typically 10 mg/kg) has a relatively brief half-life. Interaction with abundant plasma proteins binder in the bloodstream can affect pharmacokinetics of drugs, including their half-life. In this study, we investigated potential plasma protein binding (PPB) interaction to lecanemab using lecanemab biosimilar.

Methods: Lecanemab biosimilar used in this study was based on publicly available sequences. ELISA and western blotting were used to assess lecanemab biosimilar immunoreactivity in the fractions of human plasma obtained through size exclusion chromatography. The binding of lecanemab biosimilar to candidate plasma binders was confirmed by western blotting, ELISA, and surface plasmon resonance analysis.

Results: Using a combination of equilibrium dialysis, ELISA, and western blotting in human plasma, we first describe the presence of likely PPB partners to lecanemab biosimilar and then identify fibrinogen as one of them. Utilizing surface plasmon resonance, we confirmed that lecanemab biosimilar does bind to fibrinogen, although with lower affinity than to monomeric amyloid beta.

Interpretation: In the context of lecanemab therapy, these results imply that fibrinogen levels could impact the levels of free antibodies in the bloodstream and that fibrinogen might serve as a reservoir for lecanemab. More broadly, these results indicate that PPB may be an important consideration when clinically utilizing therapeutic antibodies in neurodegenerative disease.

Figure 1

(A) Equilibrium dialysis experiments comprise a semipermeable membrane with an MWCO of ~300 kDa that separates two liquid‐proof compartments of equal size. In a simple experimental set up with only Lec‐bs IgGs, the experiment started with a pre‐equilibrium state when Compartment 1 (C1) was filled with PBS containing IgG, and Compartment 2 (C2) was filled with PBS only. Driven by Brownian motion, IgGs will freely diffuse into C2 because their MW (150 kDa) is lower than the MWCO of the semipermeable membrane. After several hours of incubation at RT, a thermodynamic steady state is reached, and the same concentration of IgG is observed in both compartments. (B) In another experimental setting, Lec‐bs IgGs are in solution with another large molecule (green‐colored) that cannot diffuse freely into C2 due to a molecular size exceeding the MWCO of the semipermeable membrane. Schematics on the right show the possible steady states reached after several hours of incubation at RT. When IgGs do not bind to macromolecules, IgG concentrations in C1 and C2 are equal, and equilibrium is observed. When IgGs bind to the macromolecule, IgG concentration in C1 is higher than in C2, and a disequilibrium is observed. (C) Graph showing disequilibrium ratios of biotinylated Lec‐bs IgG between C1 and C2. “PBS” and “Plasma” columns show ratios after 12 h incubation when IgGs are spiked in PBS (n = 4) or in human donor's plasmas (n = 15), respectively. Error bars indicate the 95% CI, and dotted lines indicate the median value. ns, nonsignificantly different from the ratio at equilibrium (0%). (****P < 0.0001).

Figure 2

Identification of Lec‐bs IgG‐immunoreactive human donor's plasma fractions after size exclusion chromatography (SEC: Superose‐6 10‐300GL column). (A) Schematic of the SEC system equipped with a UV detector to monitor UV‐absorbance, and a fraction collector. (B) SEC profile of a typical donor's plasma as recorded by the UV detector. The collected fractions are indicated as upward ticks on the x‐axis. The black dot indicates the position of the highest Lec‐bs IgG‐immunoreactive peak seen in (C). (C) Indirect ELISA using Lec‐bs IgG on the human plasma SEC fractions; the black dot indicates the position of the most immunoreactive peak. (D) Western blot using Lec‐bs IgG (upper boxes) and anti‐human fibrinogen (lower box) after electrophoresis of every other SEC fraction (from 9 to 29) by SDS‐PAGE in nonreducing or reducing conditions. (E) Schematic diagram of fibrinogen structure.

Figure 3

Binding of Lec‐bs IgG to purified fibrinogen. (A) WB using Lec‐bs IgG on samples of purified plasminogen, purified fibrinogen, and donor's plasma. Lec‐bs IgG‐immunoreactive bands are observed only in lanes containing purified fibrinogen and donor plasma. (B) Sandwich ELISA for purified human fibrinogen and plasminogen using lecanemab as a capture antibody and anti‐human fibrinogen antibody as the detection antibody coupled with an anti‐mouse IgG peroxidase‐labeled antibody. A dynamic response is observed with purified fibrinogen, which is statistically significant and different from the control (purified plasminogen) at any concentration (P < 0.0001). (C) Graph showing disequilibrium ratios of biotinylated Lec‐bs IgG between C1 and C2 after 12 h of incubation (as in Fig. 1) when IgGs were spiked into PBS (n = 3) or in a purified fibrinogen solution (n = 6). Error bars, 95% CI; dotted lines, median value. Nonsignificantly different from equilibrium (ns); significantly different from equilibrium (*P = 0.0312). (D) Determination of K _D of lecanemab for Aβ2‐42 peptide using SPR. (E) Determination of K _D of lecanemab for human fibrinogen using SPR.