Albumin fusion at the N-terminus or C-terminus of human lactoferrin leads to improved pharmacokinetics and anti-proliferative effects on cancer cell lines.

Human lactoferrin (hLF), a soluble factor of the innate immune system, exhibits various biological functions and therefore has potential as a therapeutic protein. However, the clinical applications of hLF are limited by its low stability in blood. We therefore attempted to resolve this by producing recombinant hLF fused to human serum albumin (HSA). Two HSA-fused hLFs with different fusion orientations (hLF-HSA and HSA-hLF) were produced in Chinese hamster ovary (CHO) DG44 cells. hLF-HSA revealed higher thermal stability, resistance to peptic degradation, and stability during the process of cellular uptake and release in an intestinal enterocyte model (Caco-2 cells) than HSA-hLF. The lower stability of HSA-hLF is presumably due to the steric hindrance imposed by HSA fusion to the N-terminus of hLF. Both HSA fusion proteins, especially HSA-hLF, displayed improved pharmacokinetic properties despite the lower protein stability of HSA-hLF. hLF-HSA and HSA-hLF exhibited approximately 3.3- and 20.7-fold longer half-lives (64.0 and 403.6 min), respectively, than holo-rhLF (19.5 min). Both HSA fusion proteins were found to exert enhanced growth inhibition effects on cancer cells in vitro, but not normal cells. Their enhanced growth inhibitory activities were considered to be due to the synergetic effects of hLF and HSA because hLF alone or HSA alone failed to exert such an effect. Altogether, Fusion of HSA to hLF yielded superior pharmacokinetics and anti-proliferative activities against cancer cells. HSA-fused hLF is a novel candidate for further application of hLF as biopharmaceuticals for intravenous administration.

Hinge-Deficient IgG1 Fc Fusion: Application to Human Lactoferrin.

Fusion of therapeutic proteins with the antibody Fc domain is a strategy widely applied to increase protein half-life in plasma. In our previous study, we generated a recombinant human lactoferrin (hLF)-immunoglobulin G1 Fc fusion protein (hLF-hinge-CH2-CH3) with improved stability, biological activity, and pharmacokinetics ( Shiga , Y. et al. Eur J Pharm Sci. , 2015 , 67 , 136 - -143 ). However, the Fc domain in fusion proteins can potentially induce antibody-dependent and complement-dependent cytotoxicity and serious side effects. To overcome these drawbacks, we engineered an hLF-Fc fusion protein (hLF-CH2-CH3) without the Fc hinge region which is essential for engaging Fc receptors on immune cells and inducing complement-mediated cell lysis. The hLF-CH2-CH3 protein was stably expressed in Chinese hamster ovary (CHO) DG44 cells and compared for in vitro activities, thermal stability, pharmacokinetics, and attenuation of Fc-mediated immune effector functions with the conventional hinge-containing Fc fusion protein. Both hLF-hinge-CH2-CH3 and hLF-CH2-CH3 exhibited iron-binding activity, superior uptake by Caco-2 cells, similar thermal stability, and longer plasma half-life compared to recombinant hLF. However, in contrast to conventional hLF-hinge-CH2-CH3, hinge-deficient hLF-CH2-CH3 did not elicit Fc-mediated effector response potentially damaging for the target cells. Our findings demonstrate that conjugation of hinge-deficient Fc to therapeutic proteins is a promising strategy for improving their pharmacokinetic properties without enhancing effector functions. Cell-expressed hinge-deficient hLF-CH2-CH3 is a potential drug candidate with improved plasma half-life for parenteral administration.

Estimating the probability of exceeding the maximum residue limit for Japanese tea using a crop residue model.

Maximum residue limits (MRLs) for pesticides in export countries from Japan often become a trade barrier for Japanese tea. The purpose of this study is to develop a probabilistic risk estimation method for pesticide residues in green tea. First, we developed a model to estimate the pesticide residue level in green tea. Second, we introduced a regression model for pesticide half-lives on plants, one of the most critical parameters in the model. Finally, we estimated the time-course change of the distribution of the residue level by setting the probability distribution to the half-lives on tea leaves. Applying the model to three pesticides, acetamiprid, dinotefuran, and thiamethoxam, we suggested that the pre-harvest interval of thiamethoxam should be increased by three weeks for export to Taiwan. For EU nations, the MRL excess probabilities of acetamiprid and dinotefuran were measured as 99.6% and 99.5%, respectively, even 28 days after spraying.

Recombinant human lactoferrin-Fc fusion with an improved plasma half-life.

Lactoferrin (LF), an 80-kDa iron-binding glycoprotein found in mammalian exocrine secretions, has potential therapeutic efficacy due to its extensive health-promoting effects. However, LF is rapidly cleared from the circulation (∼12.6min half-life for recombinant human LF [rhLF] in rats), which limits its therapeutic potential. Therefore, to improve plasma stability, we developed a recombinant human LF (hLF)-immunoglobulin G1 (IgG1) fragment crystallizable domain (Fc) fusion (hLF-hinge-CH2-CH3) expressed in a Chinese Hamster Ovary cell (CHO) expression system and evaluated the in vitro bioactivities and pharmacokinetic properties of the purified fusion. CHO DG44 cells were transfected with an expression vector coding for recombinant hLF-hinge-CH2-CH3. Iron binding, Caco-2 uptake, and thermal stability were investigated in vitro, and pharmacokinetic parameters were investigated in vivo. hLF-hinge-CH2-CH3 was significantly expressed in CHO cells (∼100mg/l culture), was readily purified, and exhibited 98.3% of the non-fused rhLF iron-binding activity. Caco-2 uptake and thermal stability were improved for hLF-Fc fusion relative to rhLF. Moreover, hLF-hinge-CH2-CH3 demonstrated a plasma half-life that was 9.1-fold longer than that of rhLF as well as longer than that of the PEGylated bovine LFs that we previously developed. Thus, CHO-derived hLF-hinge-CH2-CH3, with enhanced pharmacokinetic properties, is a promising candidate drug for potential parenteral administration.