Hinge-Deficient IgG1 Fc Fusion: Application to Human Lactoferrin

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Hinge-deficient IgG1 Fc fusion: Application to human lactoferrin Yuki Shiga, Daisuke Murata, Akinori Sugimoto, Yuta Oshima, Minoru Tada, Akiko IshiiWatabe, Kenichiro Imai, Kentaro Tomii, Takashi Takeuchi, Shinji Kagaya, and Atsushi Sato Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00221 • Publication Date (Web): 01 Aug 2017 Downloaded from http://pubs.acs.org on August 2, 2017

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Molecular Pharmaceutics

Molecular pharmaceutics Title Page Hinge-deficient IgG1 Fc fusion: Application to human lactoferrin

Yuki Shiga,† Daisuke Murata,† Akinori Sugimoto,† Yuta Oshima,† Minoru Tada,‡ Akiko Ishii-Watabe,‡ Kenichiro Imai,§,∥ Kentaro Tomii,§,∥ Takashi Takeuchi,⊥ Shinji Kagaya,# and Atsushi Sato†,*

†

School of Bioscience and Biotechnology, Tokyo University of Technology, Hachioji, Tokyo,

Japan ‡

Division of Biological Chemistry and Biologicals, National Institute of Health Sciences,

Setagaya-ku, Tokyo, japan §

Artificial Intelligence Research Center, National Institute of Advanced Industrial Science

and Technology (AIST), Koto-ku, Tokyo, Japan ∥

Biotechnology Research Institute for Drug Discovery, National Institute of Advanced

Industrial Science and Technology (AIST), Koto-ku, Tokyo, Japan ⊥

Department of Veterinary Medicine, Tottori University, Koyama-Minami, Tottori, Japan

#

NRL Pharma, Inc., Kawasaki, Kanagawa, Japan

*

Corresponding author. Atsushi Sato, School of Bioscience and Biotechnology, Tokyo

University of Technology, 1404-1 Katakura, Hachioji, Tokyo 192-0982, Japan Tel.: +81-42-637-2197; fax: +81-42-637-2129 E-mail address: [email protected]

ACS Paragon Plus Environment


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ABSTRACT: 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.

KEY WORDS: IgG1 CH2-CH3, fusion protein, lactoferrin, pharmacokinetics, effector function


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ABBREVIATIONS ADCC, antibody-dependent cellular cytotoxicity; AUC, area under the curve; bLF, bovine lactoferrin; CD, circular dichroism; CDC, complement-dependent cytotoxicity; CHO, Chinese hamster ovary; Fc, fragment crystallizable; FcγR, Fcγ receptor; FcRn, neonatal Fc receptor; hLF, human lactoferrin; hLF-CH2-CH3, human lactoferrin conjugated to human IgG1 CH2-CH3;

hLF-hinge-CH2-CH3,

human

lactoferrin

conjugated

hinge-CH2-CH3; RU, resonance units; SPR, Surface Plasmon Resonance.


to

human

IgG1


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INTRODUCTION Many therapeutic proteins, including human lactoferrin (hLF) are not stable in vivo.1 To overcome this limitation, several U.S. Food and Drug Administration (FDA)-approved drugs have been developed by fusing biologically active proteins to the immunoglobin (Ig) fragment crystallizable (Fc) domain.2 These so-called Fc fusion proteins interact with the neonatal Fc receptor (FcRn) in a pH-dependent manner, which prolongs their plasma half-lives.3 After Fc fusion proteins enter cells by pinocytosis, they can bind to FcRn within acidic (pH 6.0) endosomes and are transported back to circulation at neutral pH (7.4), whereas proteins that do not bind FcRn are subjected to lysosomal degradation.3,4 However, Fc-fused proteins have serious drawbacks, which limit their wide application as therapeutic agents. First, Fc mediates effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) which can result in adverse events,2 and second, the flexible hinge of Fc is susceptible to proteolytic degradation in vitro.5-6 Although attempts have been made to minimize effector functions and eliminate ADCC and CDC by introducing various mutations into the Fc region7,8, there are safety concerns about their potential immunogenicity.9 Therefore, Fc-based platforms with improved safety and efficacy profiles are needed. Lactoferrin (LF) is an iron-binding glycoprotein secreted in many body fluids, including tears, saliva, milk, semen, and mucus, and within specific granules of polymorphonuclear leukocytes.1 As a component of the innate immune system, LF exerts a number of biological effects, including immunomodulatory10, antioxidant11, antimicrobial12, antiviral13, anti-inflammatory1, antitumor14, and analgesic15 activities, and enhancement of lipid metabolism.16 This wide range of potential biological properties explains considerable interest in the pharmaceutical development of LF. However, the therapeutic potential of LF may be limited by its short half-life which is about 12.6 min for recombinant human (h)LF in


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rats.17 Here, we developed a strategy to increase the stability and improve the biological activity of hLF through its fusion to a non-immunostimulatory Fc domain. Since the entire hinge region is essential for eliciting immune effector response, we replaced the full Fc domain with the hinge-deficient (CH2-CH3) Fc and evaluated the in vitro biological functions, thermal stability, in vivo pharmacokinetics, and binding to Fcγ receptors (FcγRs) and complement component 1q (C1q) of the resulting hLF-CH2-CH3 in comparison with those of conventional hLF-hinge-CH2-CH3.17



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MATERIALS AND METHODS

Cell culture Chinese hamster ovary (CHO) DG44 (DHFR-) cells (Invitrogen, Carlsbad, CA, USA) were grown in CD DG44 medium (Invitrogen). Human colon carcinoma Caco-2 cells (DS Pharma Biomedical, Osaka, Japan) were maintained in high-glucose Dulbecco’s Modified Eagle’s medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS), 1% (w/v) non-essential amino acids, and 1% (w/v) sodium pyruvate (all from Wako Pure Chemicals, Osaka, Japan). CHO-K1 cells (Health Science Research Resources Bank, Osaka, Japan) were cultured in Ham’s F12 medium (Wako) supplemented with 10% (v/v) FBS. All cells were incubated in a 5%-CO2 atmosphere at 37°C.

Expression of IgG1 CH2-CH3 The cDNA coding for the CH2-CH3 domain of human IgG1 was amplified by PCR from human IgG1 genomic sequence18 using primers containing BamHI and XbaI sites (underlined): mIgGS, 5′-GGATCCCGCACCTGAACTCCTGGGGGGA-3′ mIgGA, 5′-TCTAGAGTCGCGGCCGTCGCAC-3′. The fragment was subcloned into BamHI and XbaI sites of pBluescript II SK- (Agilent Technologies, Inc., Santa Clara, CA, USA) yielding pBSII-CH2-CH3 which was used to isolate the CH2-CH3 domain after BamHI and NotI digestion. The CH2-CH3 fragment was then inserted into mammalian expression vector pSecTag2A (Invitrogen) to obtain pSecTag2A-CH2-CH3 which was transferred into CHO-K1 cells using Lipofectamine 2000 (Invitrogen). Zeocin (Invitorgen) was used to select stably transfected clones. Stable transfectants were then cultured in Hybridoma SFM medium (Invitrogen). IgG1 CH2-CH3


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was purified from culture medium using Protein G-Sepharose 4 Fast Flow (GE Healthcare UK Ltd., Amersham, UK), and its concentration was determined using Protein assay CBB solution (Nacalai Tesque, Kyoto, Japan).

Cloning and expression of hLF-CH2-CH3 The pBSIILfAL/Bam vector carrying hLF sequence17 was digested with XhoI and BamHI, and pBSIICH2-CH3 was digested with BamHI and XbaI; the fragments were inserted into XhoI and XbaI sites of pOptiVEC MCS17 to obtain expression vector pOptiVEC/hLF-mFc which was used to transfect DG44 cells. Clones with high hLF-CH2-CH3 expression were selected as described using the OptiCHO Express kit (Invitrogen)17.

hLF-CH2-CH3 purification The hLF-CH2-CH3 protein was purified from culture medium by cation exchange chromatography using a MacroCap SP column (GE Healthcare) as described for hLF-hinge-CH2-CH3.17 hLF-CH2-CH3 was eluted with 1 M NaCl in 10 mM sodium phosphate (pH 7.6) and concentrated using a Pellicon XL 50 ultrafiltration cassette (Millipore, Billerica, MA, USA). For all experiments except SDS-PAGE and circular dichroism (CD), hLF-CH2-CH3 was used according to hLF content.

SDS-PAGE analysis Purified recombinant (r)hLF expressed in Aspergillus niger (NRL Pharma Inc., Kawasaki, Japan), IgG1 CH2-CH3, hLF-CH2-CH3, and hLF-hinge-CH2-CH3 (3 µg) were dissolved in SDS sample buffer with or without a reducing agent and heated or not at 95°C for 5 min. Proteins were loaded onto SDS-polyacrylamide (7.5%) gels, separated by electrophoresis, and stained with Coomassie Brilliant Blue.



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Gel filtration chromatography Purified rhLF, hLF-CH2-CH3, and hLF-hinge-CH2-CH3 were subjected to gel filtration chromatography using a Superdex 200 10/300 GL column connected to the AKTA prime plus system (GE Healthcare); 10 mM sodium phosphate (pH 7.6) containing 0.5 M NaCl was applied at a flow rate of 0.5 mL/min, and elution was monitored at 280 nm. Protein molecular weight was determined according to molecular mass standards (Gel Filtration Calibration Kit; GE Healthcare).

Circular dichroism spectrometry Secondary structures of the holo and apo forms of rhLF, hLF-CH2-CH3, and hLF-hinge-CH2-CH3 were assessed by circular dichroism (CD) spectroscopy. Iron contents in holo-rhLF,

holo-hLF-CH2-CH3,

holo-hLF-hinge-CH2-CH3,

apo-rhLF,

and

apo-hLF-CH2-CH3 determined by the Fe C-test kit (Wako) were 1806, 2596, 2112, 38, and 32 ng/mg of hLF equivalent, respectively, whereas that in apo-hLF-hinge-CH2-CH3 ranged from very low to undetectable levels. Proteins were diluted to 0.1 mg/ml in PBS and subjected to CD analysis in a JASCO J-1500 spectropolarimeter (JASCO, Tokyo, Japan). To evaluate thermostability, samples were heated from 30°C to 90°C at 1°C intervals, and CD peaks at 225 nm were recorded. The data were analyzed using the Spectra Manager Ver.2 software (Model JWTDA-519, JASCO) to determine the melting temperature (Tm).

Iron-binding assay Apo-hLF-CH2-CH3 was prepared by ferric ion removal from hLF-CH2-CH3 (5 mg/ml) by dialysis in 0.1 M citric acid (pH 2.1), and iron-rebinding (holo) hLF-CH2-CH3 was obtained from apo-hLF-CH2-CH3 by dialysis against 50 mM bicarbonate and 0.001% (w/v) ferric


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ammonium citrate in 50 mM phosphate buffer (pH 7.5) as described.17 The iron content was determined using the Fe C-test kit (apo-form of hLF-CH2-CH3, n = 6; holo-form of hLF-CH2-CH3, n = 5). The iron concentrations of rhLF and hLF-hinge-CH2-CH3 have been reported previously17.

Protein uptake by Caco-2 cells The uptake of rhLF and hLF-Fcs by Caco-2 cells was evaluated as described previously.17 rhLF, hLF-CH2-CH3, and hLF-hinge-CH2-CH3 were labeled with Alexa Fluor 488 carboxylic acid, 2,3,5,6-tetrafluorophenyl ester (Alexa Fluor 488 5-TFP), 5-isomer (Invitrogen) with labeling efficiencies of 5.4, 4.0, and 3.1 moles of Alexa Fluor 488/mole of protein, respectively, and dissolved in PBS. Caco-2 cells were seeded at a density of 5 × 104 cells per well in 12-well culture plates in high-glucose DMEM supplemented with 10% (v/v) FBS, 1% (w/v) non-essential amino acids, and 1% (w/v) sodium pyruvate, grown for 7 days, and then treated with 15 µg/ml of labeled rhLF, hLF-CH2-CH3, or hLF-hinge-CH2-CH3 for 1 h at 4°C or 37°C with or without unlabeled bovine (b)LF (1.5 mg/ml) or 0.2% (w/v) sodium azide (ATPase inhibitor). After rinsing with cold PBS, cells were detached with 0.25% (w/v) trypsin-1 mM EDTA, collected into 1.5-ml tubes, and fixed with 4% (w/v) phosphate-buffered paraformaldehyde; cell nuclei were counterstained with Hoechst 33258 (Wako). Then, cells were visualized under a laser scanning confocal microscope LSM 510 (Carl Zeiss Co. Ltd, Oberkochen, Germany).

Western blotting analysis of protein uptake Caco-2 cells cultured on 12-well plates for 7 days were treated with rhLF (20 µg/ml), hLF-CH2-CH3 (20 µg hLF equivalent/ml), or hLF-hinge-CH2-CH3 (20 µg hLF equivalent/ml) for 1 h at 37°C, and cell lysates were subjected to western blotting as



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described.17 Intracellular LF content was analyzed using an anti-human LF antibody (2B8, AbD Serotec, Oxford, UK) and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Promega, Madison, WI, USA). Positive signals were detected by enhanced chemiluminescence (ImmunoStar Zeta, Wako).

Surface Plasmon Resonance (SPR) analysis of protein binding to FcRn and FcγRs Receptor binding was analyzed by SPR using a Biacore T200 SPR biosensor (GE Healthcare). The affinity to FcRn was evaluated as described previously.19 Recombinant hFcRn was immobilized on a CM5 sensor chip and 335 nM of rhLF, hLF-Fcs, or infliximab (an anti-human tumor necrosis factor-alpha (TNF-α) monoclonal antibody; Janssen Biotech, Inc., Horsham, PA, USA) was injected into flow cells. Association and dissociation was monitored for 120 s and 150 s, respectively. The affinity to Fcγ receptors was examined by capturing hLF-Fcs or infliximab on a protein A-immobilized sensor chip. Each protein was immobilized at the following densities: for FcγRI binding - infliximab, 72 resonance units (RU); hLF-hinge-CH2-CH3, 70 RU; and hLF-CH2-CH3, 76 RU; for FcγRIIa - infliximab, 348 RU; hLF-hinge-CH2-CH3, 410 RU; and hLF-CH2-CH3, 422 RU; and for FcγRIIIa - infliximab, 348 RU; hLF-hinge-CH2-CH3, 397 RU; and hLF-CH2-CH3, 436 RU. The ectodomains of recombinant human FcγRI (0.02 µM), IIa (0.2 µM), or IIIa (0.2 µM) were injected into flow cells, and association/dissociation were recorded.

FcγRIIIa reporter assay As FcγR activation by hLF-Fcs can lead to ADCC, it was assessed using a reporter assay.20 In a cell-based luciferase reporter assay, FcγRIIIa signal activation through the nuclear factor of activated T cells (NFAT) induction can be detected by luciferase reporter activity.


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FcγRIIIa-expressing Jurkat/FcγRIIIa/NFAT-Luc cells were seeded in Opti-MEM I Reduced Serum Medium (Invitrogen) at a density of 2 × 104 cells per well in a 96-well plate and incubated with different concentrations of hLF-Fcs for 5 h at 37 °C. Luciferase reporter activity in cell lysates was evaluated using ONE-Glo Luciferase Assay System (Promega).

ELISA of protein binding to human C1q LF proteins were added to 96-well microtiter plates (Immuno Module F8 Maxisorp, Nunc, Roskilde, Denmark) at different concentrations (1.25, 1.5, 2.0, 2.5, and 5 µg/ml) at 4°C overnight. Plates were blocked with 1% (w/v) BSA, 0.05% (v/v) Tween 20 in PBS for 1 h, and 100 µl of 2 µg/ml human C1q (Merck Millipore, Billerica, MA, USA) in blocking buffer was added to each well for 2 h at room temperature. After washing with 0.05% (v/v) Tween 20 in PBS, 100 µl/well of HRP-conjugated anti-human C1q antibody (AbD Serotec, Oxford, UK) in blocking buffer at a dilution of 1:500 was added and incubated for 2 h at room temperature. Plates were washed again, and the reaction was developed by adding 100 µl of TMB One Solution (Promega) and stopped by 100 µl of 1 N H2SO4; then, the absorbance at 450 nm was determined using a microplate reader.

Pharmacokinetic analyses by sandwich ELISA for measuring hLF All animal procedures were approved by the Animal Research Committee of Tottori University. Male Wistar rats (8-week-old, 250–280 g; Institute for Animal Reproduction, Ibaraki, Japan) were maintained at 22 ± 2°C in a 12-h/12-h light/ dark cycle and received standard chow (CE-2, Nihon CLEA, Tokyo, Japan). Pharmacokinetic studies were conducted, as described previously17. Animals (n = 5) were anesthetized with urethane (4 g/kg, s.c.) and sodium pentobarbital (0.1 ml, s.c.) and injected with 1 mg hLF equivalent/kg of hLF-CH2-CH3 through the femoral vein. Blood was drawn through the cannulated external



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jugular vein at 1, 5, 10, 15, 30, 60, 120, 180, and 240 min after hLF-CH2-CH3 administration, and plasma hLF-CH2-CH3 content was measured using the Assay Max Human Lactoferrin ELISA kit (ASSAYPRO, St. Charles, MO, USA). A standard curve was obtained using purified hLF-CH2-CH3. Pharmacokinetic parameters for the distribution of hLF-CH2-CH3 were calculated based on plasma content using GraphPad Prism 4 (San Diego, CA, USA), and Welch’s test was performed to analyze the half-life and area under the curve (AUC); p < 0.01 was considered statistically significant.

Pharmacokinetic analyses by a new ELISA for detecting intact hLF-Fcs Commercial sandwich ELISA used for pharmacokinetic studies of LF can also detect partially degraded hLF-Fcs. Therefore, to capture intact hLF-Fcs and evaluate their degradation in vivo, we established a new assay (LF-Fc ELISA) in which HRP-conjugated anti-human IgG (A80-219P, Bethyl Laboratories, Inc., Montgomery, AL, USA) was used instead of a biotinylated anti-hLF antibody and streptavidin-HRP conjugate. A commercial ELISA plate was used to capture hLF-Fcs in rat plasma according to the standard protocol provided by the manufacturer; then, 50 µl of HRP-conjugated anti-human IgG (1:1,250) was added for 2 h to detect bound hLF-Fcs. The plate was then washed, and 50 µl of TMB One Solution was added to each well. The reaction was stopped and analyzed as described above, and pharmacokinetic parameters were calculated using GraphPad Prism 4.

Structural models of FcγRIIIa-hLF-CH2-CH3 and FcγRIIIa-hLF-hinge-CH2-CH3 complexes Modeling of hLF-CH2-CH3 was performed under the following assumptions: hLF directly interacts with CH2-CH3 as its linker is very short (only two residues) and does not affect the association between CH2-CH3 and FcRn, as evidenced by the experimental data on


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hLF-CH2-CH3 binding to FcRn. Therefore, we first sampled plausible hLF-CH2-CH3 complexes using ZDOCK21 with hLF (PDB ID: 1FCK_A) and the rat FcRn-Fc complex (PDB ID: 1FRT) to maintain interactions between CH2-CH3 and FcRn. Through protein-protein docking, we designated sub-structural regions that allowed hLF and CH2-CH3 interaction around the C-terminal region of hLF and the N-terminal region of CH2-CH3, and masked all other regions. Then, we selected an hLF-CH2-CH3 complex with the highest ZDOCK score among the complexes without intermolecular clashes between hLFs in the hLF-CH2-CH3 dimer, as a template for comparative modeling. The modeling of hLF-hinge-CH2-CH3 was performed by superposing CH2-CH3 and hLF over the CH2-CH3 region and light chain, respectively, of mouse Ig (PDB ID: 1IGY) in the Molecular Operating Environment

(MOE)

(Chemical

Computing

Group,

MOE,

version

2014.09,

www.chemcomp.com) using the Amber10:EHT force field. The FcγRIIIa-hLF-CH2-CH3 and FcγRIIIa-hLF-hinge-CH2-CH3 models were obtained by superposing CH2-CH3 over the CH2-CH3 region of the FcγRIIIa-CH2-CH3 complex (PDB ID: 3SGK).



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RESULTS Molecular design and production of the hinge-deficient hLF-Fc protein To diminish possible problems caused by effector functions of the conventional hLF-hinge-CH2-CH3 (Figure 1A)17, hLF was fused to hinge-deficient human Fc with the aim to generate hLF-CH2-CH3 (Figure 1B), which was expressed in CHO DG44 cells at the level similar to that of hLF-hinge-CH2-CH3 (about 100 mg/l). Molecular composition analysis indicated that hLF-CH2-CH3 dimerized in solution as its elution profile was comparable to that of hLF-hinge-CH2-CH3, which is known to form homodimers via two disulfide bridges in the hinge region (Figure 2A). This result was confirmed by hLF-CH2-CH3 and hLF-hinge-CH2-CH3 migration pattern in SDS-PAGE with or without boiling samples. Heat treatment denatures the protein by breaking non-covalent bonding such as hydrophobic interactions. SDS-PAGE analysis showed that heat-treated hLF-CH2-CH3 migrated as a 105-kDa band, which corresponds to the calculated molecular mass of hLF (~80 kDa) plus CH2-CH3 (~ 25 kDa) (Figure 2B, lane 5 and 13). At the same time, non-heat treated hLF-CH2-CH3 and hLF-hinge-CH2-CH3 migrated as a ~180-kDa band (Figure 2B, lane 6, 14 and 16), suggesting dimerization. Notably, heat denaturation did not change the apparent molecular mass of recombinant hLF (rhLF) (Figure 2B, lane 1, 2, 9 and 10), but did change that of recombinant CH2-CH3 (from 49 to 32 kDa and from 52 to 35 kDa under non-reducing and reducing conditions, respectively) (Figure 2B). These results suggest that hLF-CH2-CH3 dimerizations, was mediated by hydrophobic interactions between CH2-CH3 domains, especially between CH3 because a recombinant human CH3 is dimeric under reducing conditions as reported previously.22

hLF-CH2-CH3 retains full iron-binding activity


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A bilobate hLF molecule binds two Fe3+ ions and two synergistic CO32- ions.23 As iron chelation is essential for antioxidant24 and antimicrobial1 properties of hLF, we measured the iron content in hLF-CH2-CH3 and compared it to that in rhLF and hLF-hinge-CH2-CH317 (Table 1). hLF-CH2-CH3 bound iron at the concentration of 1,429 ng per mg hLF equivalent (apo-form, n = 6; holo-form, n = 5), which is comparable to the iron-binding capacity of conventional hLF-hinge-CH2-CH3, indicating that the removal of the hinge region did not affect Fe3+ chelation.

Thermal stability of holo- and apo-hLF-CH2-CH3 There was no difference in the far-UV CD spectra (200–250 nm) between the holo and apo forms of hLF proteins (Figure 3A). The holo-rhLF exhibited a much higher melting transition temperature (Tm) (Tm > 90°C) than that of the apo form (Tm=64.7°C), as reported previously25. As is the case with rhLF, both holo-hLF-Fcs also showed higher Tm values than those of the corresponding apo forms. Based on the Tm values of their apo forms, the thermal stability of hLF-CH2-CH3 (Tm=67.8°C) turns out to be comparable with those of rhLF (Tm=64.7°C) and hLF-hinge-CH2-CH3 (Tm=65.4°C).

hLF-CH2-CH3 uptake by Caco-2 cells Human intestinal epithelial Caco-2 cells mimic the absorption of intact LF in the intestinal tract26; therefore, we evaluated the uptake of the LF proteins by Caco-2 cells. Similar to rhLF (Figure 4A–D) and hLF-hinge-CH2-CH3 (Figure 4I–L), hLF-CH2-CH3 was internalized by Caco-2 cells in a temperature- and energy-dependent manner (Figure 4E–G). Protein uptake at 37°C was fully inhibited by 100-fold excess of unlabeled bLF (Figure 4H), indicating hLF-mediated endocytosis. Immunoblotting analyses of cell lysates revealed that inside Caco-2 cells, hLF-CH2-CH3 demonstrated the same stability as hLF-hinge-CH2-CH3,



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although rhLF was prone to aggregation during endocytosis (Figure 4M) as reported previously.17

hLF-CH2-CH3 binding to FcRn is pH-dependent The stability of Fc fusion proteins relies on pH-dependent binding of Fc to FcRn.3 The affinity of hLF-CH2-CH3 and hLF-hinge-CH2-CH3 to human FcRn was assessed by SPR. At pH 6.0, there was no difference between the proteins in binding to immobilized FcRn (Figure 5A), suggesting that they had similar affinities to FcRn. However, at pH 7.4 both hLF-Fcs exhibited poor binding to FcRn (Figure 5B). Infliximab, a chimeric monoclonal antibody used as a positive control showed pH-dependent binding to FcRn, whereas rhLF had poor affinity to FcRn at both pH (Figure 5A and B). These results indicate that the hinge region is not involved in pH-dependent interaction between Fc and FcRn.

Improved pharmacokinetic properties of hLF-CH2-CH3 Similar to hLF-hinge-CH2-CH3, hLF-CH2-CH3 showed pH-dependent FcRn binding, which accounts

for

improved

pharmacokinetic

properties.17

To

evaluate

hLF-CH2-CH3

pharmacokinetics, we intravenously injected rats with 1 mg/kg hLF-CH2-CH3, measured circulating hLF-CH2-CH3 by ELISA, and then compared its pharmacokinetic profile to those of rhLF and hLF-hinge-CH2-CH3 that have been reported previously (Figure 6).17 The maximum

plasma

concentration

(Cmax)

values

for

rhLF,

hLF-CH2-CH3,

and

hLF-hinge-CH2-CH3 were 12.3 ± 1.1 µg/ml, 22.0 ± 0.7 µg/ml, and 9.8 ± 0.5 µg/ml detected at 1 min, 1 min, and 10 min, respectively, post-injection, indicating much higher Cmax and shorter time to achieve it (Tmax), for hLF-CH2-CH3 compared to hLF-hinge-CH2-CH3. Half-lives of hLF-CH2-CH3 and hLF-hinge-CH2-CH3 (67.7 ± 1.1 min and 114.3 ± 1.6 min, respectively) were significantly (5.4-fold and 9.1-fold, respectively) increased compared to


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that of rhLF (12.6 ± 0.3 min; p = 0.004 vs hLF-CH2-CH3). The area under the curve (AUC) values for hLF-CH2-CH3 and hLF-hinge-CH2-CH3 were 1,706.0 ± 88.2 µg·min·ml-1 and 1,024.0 ± 83.6 µg·min·ml-1, respectively, exceeding that of rhLF (229.9 ±7.4 µg·min·ml-1) by 7.4-fold (p = 0.0002) and 4.5-fold (p = 0.0002), respectively. Overall, hinge-deficient hLF-CH2-CH3 showed better pharmacokinetic properties than rhLF and, although it exhibited a shorter half-life than hLF-hinge-CH2-CH3, its AUC value was increased (p=0.0002 vs hLF-hinge-CH2-CH3) because of a higher Cmax.

hLF-CH2-CH3 does not bind Fcγ receptors and C1q For pharmaceutical applications of hLF, the enhancement of immune effector functions, including ADCC and CDC should be avoided. As Fc binding to FcγRs and C1q is the first steps in eliciting ADCC and CDC, respectively7, we examined the affinity of hLF-Fcs to FcγRs I, IIa, and IIIa by SPR (Figure 7A) and to C1q by ELISA (Figure 7B). As expected, conventional hLF-hinge-CH2-CH3, similar to infliximab (positive control), interacted with all the examined FcγRs, whereas hinge-deficient hLF-CH2-CH3 showed no significant binding to any of them in the SPR assay. Furthermore, conventional hLF-hinge-CH2-CH3 demonstrated concentration-dependent binding to C1q, but hinge-deficient hLF-CH2-CH3 did not. As FcγRIIIa expressed on natural killer (NK) cells is a key player in ADCC elicited by IgG1 Fc20, we assessed FcγRIIIa activation by hLF-Fcs in the reporter assay. Consistent with the affinity data, hLF-hinge-CH2-CH3, but not hLF-CH2-CH3, activated FcγRIIIa-dependent signaling in a dose-dependent manner (Figure 7C), indicating that hinge-deficient hLF-CH2-CH3 did not stimulate immune effector reactions. Thus, hinge deletion in Fc fusion proteins eliminates immune effector functions and could be a good strategy to develop therapeutic proteins with improved safety profiles.



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Hinge degradation in hLF-hinge-CH2-CH3 in vivo is minor Several issues regarding in vitro degradation of the hinge region in IgGs and Fc fusion proteins have been reported.5-6 Cleavage of the hinge region in Fc fusion drugs in vivo most likely contributes to their poor pharmacokinetic profiles and consequently leads to lower efficacy. Therefore, we evaluated proteolytic degradation of hLF-Fcs in vivo using a newly established LF-Fc ELISA in which plasma hLF-Fcs are sandwiched between anti-hLF and anti-human IgG antibodies to detect non-degraded hLF-Fc (Figure 8A). Overall, for both hLF-Fcs the new assay revealed pharmacokinetic profiles similar to those detected using the commercial ELISA (Figure 6). However, lower plasma concentrations were detected by LF-Fc ELISA (Figure 8B). Thus, the AUC values for hLF-CH2-CH3 and hLF-hinge-CH2-CH3 were 1,422.8 ± 142.6 µg·min·ml-1 and 789.3 ± 77.4 µg·min·ml-1, which constituted 83.4% and 77.1%, respectively, of those obtained using the commercial assay (Figure 8B, inset). The results indicate that decrease in the AUC values was comparable for both proteins, suggesting that hinge degradation in hLF-hinge-CH2-CH3 may not be significant in vivo.


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DISCUSSION Recently, fusion of bioactive peptides to the Fc domain has become a focus of increasing attention as a strategy to improve therapeutic activity of protein drugs.2, 27 Several Fc fusion proteins have already been developed and approved by the FDA for the treatment of some diseases.2 In most cases, Fc fusion increases plasma half-life of the attached protein, most likely because of pH-dependent interaction between Fc and FcRn.3 However, the limitation of this approach is that Fc may enhance effector functions such as ADCC and CDC, causing particular problems for the application of agonistic proteins such as hLF, as they interact with cell surface receptors28 and may potentially result in significant dysfunction of the target cells.29 To decrease host effector response, several Fc fusion proteins with modified Fc backbones have been developed and evaluated. In one of them, three amino acid substitutions (L234F, L235E, and P331S) were introduced into human IgG1, which reduced FcγRs and C1q binding.7 In the other study, Fc backbones of human IgG2 and IgG4 isotypes were considered, because they are known to have relatively low binding affinities for FcγRs and C1q.30 An engineered Fc variant of IgG2 carrying four amino acid substitutions (H268Q, V309L, A330S, and P331S) introduced based on IgG4 isotype elicited low effector responses.8 It was also found that in IgG2σ, simultaneous amino acid substitutions V234A, G237A, P238S, H268A, V309L, A330S, and P331S eliminated immune effector functions.29 An alternative approach is to utilize hybrid IgG2/IgG4-based31 or IgD/IgG4-based32 Fc backbones. However, in such mutagenesis- and hybrid-based strategies immunogenicity may be an important safety concern.9 The hinge domain of human IgG1 plays a key role in Ig effector functions. Thus, the upper and middle hinge regions were shown to induce significant immune effector activities33 , and simultaneous mutation of residues 234 and 235 in the lower hinge



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completely abolished both ADCC and CDC.34 Therefore, we hypothesized that elimination of the entire hinge region in Fc fusion proteins would completely abrogate immune effector functions. Indeed, hinge-deficient hLF-CH2-CH3 did not show any detectable FcγR and C1q binding which causes ADCC and CDC (Figure 7). Regarding the molecular mechanism underlying the lack of interaction between FcγRIIIa and CH2-CH3 in hLF-CH2-CH3 (Figures 7A and C), the 3-D models indicate that hLF in hLF-CH2-CH3 may cause steric hindrance between the glycosylated extracellular domain of FcγRIIIa involved in Fc recognition35 and CH2-CH3 in hLF-CH2-CH3 (intermolecular clashes circled orange in Figure 9A), while hLF in hLF-hinge-CH2-CH3 may not (Figure 9B). In addition to eliminating the direct interaction between FcγRIIIa and CH2-CH3 in hLF-CH2-CH3, this steric hindrance may also contribute to inhibiting FcγRIIIa binding to hLF-CH2-CH3. There are three potential concerns associated with hinge deletion in Fc fusion proteins. First, it may result in a decrease of biological effects because the hinge serves as a flexible spacer minimizing or preventing undesired steric hindrance between Fc and proteins of interest.36,32 However, we showed that hLF-CH2-CH3 retained its biological activities despite the absence of the hinge region. Consistent with this observation, monomeric IgG1 CH2-CH3 conjugated to human heavy chain variable domain (VH) targeting HIV-1 also retained full binding to gp120Bal-CD437, suggesting that hinge flexibility may not be critical for the biological activity of CH2-CH3 conjugates. Second, hinge deficiency may result in lower structural stability because the hinge connects the two heavy chains of conventional Fc fusion proteins through two disulfide bonds.38 However, hinge-deficient hLF-CH2-CH3, which dimerized via hydrophobic interactions between CH3 domains, showed thermal stability comparable to that of conventional hLF-hinge-CH2-CH3 (Figure 3). In addition, both hLF-CH2-CH3 and hLF-hinge-CH2-CH3 remained intact after internalization by Caco-2 cells despite the tendency of hLF itself to aggregate (Figure 4). Thus, low structural stability is not


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expected to be a major issue in the application of hinge-deficient Fc fusion proteins. Finally, hinge deletion may reduce the interaction of Fc with FcRn. However, we found that hinge-deficient hLF-CH2-CH3 showed pH-dependent binding to FcRn similar to that of conventional hLF-hinge-CH2-CH3 in vitro (Figure 5). An X-ray crystallographic study revealed that the binding site for FcRn is located at the interface between CH2 and CH3 regions.39 In agreement with these data, an IgG1 Fc (CH2-CH3) dimer expressed in E. coli retained pH-dependent FcRn binding affinity.40 In our study, hLF-CH2-CH3 showed extended plasma half-life and increased AUC in rats compared to rhLF, which is consistent with pH-dependent interaction of hLF-CH2-CH3 with FcRn. Although both hinge-deficient hLF-CH2-CH3 and conventional hLF-hinge-CH2-CH3, exhibited superior pharmacokinetics compared to rhLF, they differed in pharmacokinetic parameters such as Tmax, Cmax, and half-life (Figure 6). Thus, hLF-CH2-CH3 showed much higher Cmax (22.0 ± 0.7 µg/ml) with a shorter Tmax compared to hLF-hinge-CH2-CH3 (9.8 ± 0.5 µg/ml), which may be attributed to possible differences in hepatic uptake, as circulating hLF is reported to be eliminated mainly by the liver.41, 42 In addition, given that hLF-Fc internalization by various immune cells occurs through FcγR43 and that hLF-CH2-CH3 does not bind FcγR, it may account for the shorter hLF-CH2-CH3 Tmax and higher Cmax. The decreased half-life of hLF-CH2-CH3 compared to hLF-hinge-CH2-CH3 is consistent with a similar previous finding that the presence of the intact hinge in murine IgG1 provided longer IgG1 half-life.44 Both hLF-Fcs exhibited much shorter half-lives compared to human IgG1 in rats45, which was probably due to very rapid clearance of hLF.17 In addition to eliciting effector functions, the flexible hinge region of Fc is prone to proteolytic degradation in vitro5-6, which may cause decreased production of Fc constructs in cell culture. The degradation of the hinge region in Fc fusion proteins in vivo most likely contributes to their poor pharmacokinetic properties and, consequently, low efficacy. However,



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contrary to our expectation, hinge degradation in conventional hLF-hinge-CH2-CH3 did not seem to impact its pharmacokinetic profile in vivo, as indicated by LF-Fc ELISA (Figure 8). This result is consistent with earlier findings that IgG1 in which one of hinge peptides is cleaved exhibited a pharmacokinetic profile similar to that of intact IgG46 and that hinge degradation in IgG1 induced by radical reactions does not affect its pharmacokinetics.6 As beneficial health effects of LF have been established, its potential therapeutic applications have recently attracted much attention.47, 48 However, a phase III trial of talactoferrin alfa, a recombinant form of hLF, did not revealed therapeutic effects against advanced stage IIIB/IV non-small-cell lung cancer after application of a high dose of 1.5 g twice daily49, probably because of low bioavailability of non-modified rhLF through the oral route.50 Thus, parenteral administration of hLF conjugated to hinge-deficient Fc may provide a possible solution to the problem by increasing the potency of lactoferrin as an effective and safe therapeutic agent.

CONCLUSIONS We generated hLF conjugated to the non-immunostimulatory Fc region of IgG1. Hinge-deficient hLF-CH2-CH3 demonstrates robust biological activity and improved intracellular stability in vitro and increased plasma half-life compared to rhLF, without stimulation of effector functions observed for conventional hLF-hinge-CH2-CH3. Therefore, fusion to hinge-deficient Fc presents a feasible and safe strategy to develop therapeutic proteins, and hLF-CH2-CH3 produced in CHO cells may be used as an effective hLF derivative for intravenous administration.

AUTHOR INFORMATION Corresponding Author


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(A.S) Tel.: +81-42-637-2197; Fax: +81-42-637-2129 E-mail address: [email protected] Author Contribution All authors have given approval to the final version of the manuscript. Conflict of Interest This study was partially funded by a grant from NRL Pharma, Inc. S.K. is an employee of NRL Pharma, Inc.

ACKNOWLEDGMENTS We are grateful to J. Baba (School of Bioscience and Biotechnology, Tokyo University of Technology) for technical assistance. This research is partially supported by the Platform Project for Supporting in Drug Discovery and Life Science Research from Japan Agency for Medical Research and Development (AMED). Editorial support, in the form of medical writing was provided by Editage.



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Conesa, C.; Sanchez, L.; Perez, M. D.; Calvo, M. A calorimetric study of thermal



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4 (1), 17-23. 39. Burmeister, W. P.; Huber, A. H.; Bjorkman, P. J. Crystal structure of the complex of rat neonatal Fc receptor with Fc. Nature 1994, 372 (6504), 379-383. 40. Ying, T.; Ju, T. W.; Wang, Y.; Prabakaran, P.; Dimitrov, D. S. Interactions of IgG1 CH2 and CH3 Domains with FcRn. Front. Immunol. 2014, 5, 146. 41. Bennett, R. M.; Kokocinski, T. Lactoferrin turnover in man. Clin. Sci. (Lond) 1979, 57 (5), 453-460. 42. Peen, E.; Johansson, A.; Engquist, M.; Skogh, T. Hepatic and extrahepatic clearance of circulating human lactoferrin: an experimental study in rat. Eur. J. Haematol. 1998, 61 (3), 151-159. 43. Fleit, H. B.; Lane, B. P. FC gamma receptor mediated phagocytosis by human neutrophil cytoplasts. Inflammation 1999, 23 (3), 253-262. 44. Kim, J. K.; Tsen, M. F.; Ghetie, V.; Ward, E. S. Evidence that the hinge region plays a role in maintaining serum levels of the murine IgG1 molecule. Mol. Immunol. 1995, 32 (7), 467-75. 45. Bazin-Redureau, M. I.; Renard, C. B.; Scherrmann, J. M. Pharmacokinetics of heterologous and homologous immunoglobulin G, F(ab')2 and Fab after intravenous administration in the rat. J. Pharm. Pharmacol. 1997, 49 (3), 277-81. 46. Brezski, R. J.; Vafa, O.; Petrone, D.; Tam, S. H.; Powers, G.; Ryan, M. H.; Luongo, J. L.; Oberholtzer, A.; Knight, D. M.; Jordan, R. E. Tumor-associated and microbial proteases compromise host IgG effector functions by a single cleavage proximal to the hinge. Proc. Natl. Acad. Sci. U S A 2009, 106 (42), 17864-17869. 47. Mulder, A. M.; Connellan, P. A.; Oliver, C. J.; Morris, C. A.; Stevenson, L. M. Bovine lactoferrin supplementation supports immune and antioxidant status in healthy human males. Nutr. Res. 2008, 28 (9), 583-589. 48. Ishikado, A.; Imanaka, H.; Kotani, M.; Fujita, A.; Mitsuishi, Y.; Kanemitsu, T.; Tamura, Y.; Makino, T. Liposomal lactoferrin induced significant increase of the interferon-alpha (IFN-alpha) producibility in healthy volunteers. Biofactors 2004, 21 (1-4), 69-72.49. 49. Ramalingam, S.; Crawford, J.; Chang, A.; Manegold, C.; Perez-Soler, R.; Douillard, J. Y.; Thatcher, N.; Barlesi, F.; Owonikoko, T.; Wang, Y.; Pultar, P.; Zhu, J.; Malik, R.; Giaccone, G. Talactoferrin alfa versus placebo in patients with refractory advanced non-small-cell lung cancer (FORTIS-M trial). Ann. Oncol. 2013, 24 (11), 2875-80. 50. Falchook, G. F.; Varadhachary, G. R.; Smith, D. P.; Davis, L. D.; Dhingra, H. M.; Hayes, B. P.; Varadhachary, A. Phase I trial of oral talactoferrin alfa in refractory solid tumors. Invest. New Drugs 2006, 24 (3), 233-240.



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Table 1. Iron-binding activities of hLF-Fcs Fe concentration, ng/mg hLF equivalent (mean ± S.E.) Bound Fe*) Apo-form Holo-form (holo-apo) rhLF hLF-CH2-CH3 hLF-hinge-CH2-CH3

188.2 ± 21.3 211.2 ± 34.1 255.9 ± 30.0

1488.2 ± 97.5 1640.0 ± 62.1 1534.0 ± 80.0

1300.0 1428.8 1278.1

Relative binding ratio (% of rhLF) 100% 100% 98.3%

*) The concentration of bound Fe was calculated by subtracting that of apo-protein from that of holo-protein. Data for hLF-CH2-CH3 represent means ± S.E. (apo-form, n = 6; holo-form, n = 5). The iron concentrations of rhLF and hLF-hinge-CH2-CH3 have been reported previously17.


Page 29 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure legends Figure 1. Structure of human lactoferrin (hLF)-hinge-CH2-CH3 and hLF-CH2-CH3. Signal peptide-containing full-length human LF sequence was fused with the human IgG1 hinge-CH2-CH3

domain

by

Asp-Pro-Glu

(DPE)

linkage

(underlined)

yielding

hLF-hinge-CH2-CH317 (A) or with the CH2-CH3 domain by Asp-Pro (DP) linkage (underlined) yielding hLF- CH2-CH3 (B).

Figure 2. Homodimerization of the hLF-CH2-CH3 protein. (A) Gel filtration chromatography of rhLF, hLF-CH2-CH3, and hLF-hinge-CH2-CH3; inset shows a standard curve for molecular weight (MW) markers. (B) Separation of heat-treated and non-heat-treated proteins by SDS-PAGE in non-reducing and reducing conditions. Lanes 1, 3, 5, 7, 9, 11, 13, and 15 show heated samples, and lanes 2, 4, 6, 8, 10, 12, 14, and 16 show unheated samples after Coomassie Brilliant Blue staining.

Figure 3. Thermostability of hLF-CH2-CH3 and hLF-hinge-CH2-CH3. (A) Circular dichroism spectra for holo and apo forms of rhLF, hLF-CH2-CH3, and hLF-hinge-CH2-CH3. (B) Thermal stability of rhLF, hLF-CH2-CH3, and hLF-hinge-CH2-CH3 determined in the temperature range of 30°C–90°C by circular dichroism (225 nm). Temperature-induced unfolding of holo forms (left) and apo forms (right).

Figure 4. Internalization of intact hLF-CH2-CH3 by Caco-2 cells. (A–L) Caco-2 cells were treated with Alexa Fluor 488-labeled proteins at 37°C (A, E, and I) or 4°C (B, F, and J) in the presence of 0.2% sodium azide (ATPase inhibitor; C, G, and K) or unlabeled bovine (b)LF (D, H, and L). (M) Western blotting analysis of rhLF and hLF-Fcs uptake by Caco-2 cells.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5. Hinge-deficient hLF-CH2-CH3 retains pH-dependent affinity to FcRn. Binding of rhLF, hLF-CH2-CH3, and hLF-hinge-CH2-CH3 to FcRn at pH 6.0 (A) and pH 7.4 (B) measured by SPR. Infliximab was used as positive control.

Figure 6. Improved pharmacokinetics of hLF-CH2-CH3 compared to rhLF. Rats were injected with hLF-CH2-CH3 (n = 5), rhLF (n= 5), and hLF-hinge-CH2-CH3 (n = 6) and analyzed for plasma content of LF proteins; the data are presented as the mean ± S.E. The pharmacokinetic parameters of hLF-CH2-CH3 were compared with those of rhLF and hLF-hinge-CH2-CH317 (inset). The half-live of hLF-CH2-CH3 half-life was longer compared to that of rhLF (p = 0.004) and the AUC was higher compared to that of rhLF (p = 0.0002) and hLF-hinge-CH2-CH3 (p = 0.0002) by Welch’s test.

Figure 7. Complete abrogation of immune effector functions by the deletion of the hinge region in hLF-CH2-CH3. (A) Representative SPR sensorgrams showing interaction of hLF-Fcs with FcγRI (top left), FcγRIIa (top right), and FcγRIIIa (bottom left); infliximab was used as positive control. (B) Binding of hLF-Fcs to human C1q measured by ELISA; rhLF was used as negative control. Data are expressed as mean ± S.D. (n = 3). (C) Activation of FcγRIIIa by hLF-Fcs evaluated in a luciferase reporter assay. Data represented as the mean ± S.E. (n = 3).

Figure 8. Hinge degradation may not affect in vivo pharmacokinetics of Fc fusion proteins. (A) Intact hLF-Fcs were detected by a newly developed LF-Fc ELISA where proteins were sandwiched between anti-hLF and anti-human IgG antibodies. (B) Pharmacokinetic profiles of hLF-Fcs determined by LF-Fc ELISA. The data are presented as the mean ± S.E. (n = 5 for hLF-CH2-CH3 group, and n = 6 for the hLF-hinge-CH2-CH3 group). The inset shows


Page 30 of 41

Page 31 of 41

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comparison of AUC values obtained by commercial ELISA (open bars) and LF-Fc ELISA (gray bars).

Figure 9. Docking of hLF-Fcs to the FcγRIIIa structure. (A) FcγRIIIa–hLF-CH2-CH3 complex. (B) FcγRIIIa–hLF-hinge-CH2-CH3 complex. hLF-Fcs are indicated by light pink and light blue, respectively, and the FcγRIIIa extracellular domain is colored light green. Oligosaccharides of FcγRIIIa (dark green) and CH2-CH3 (magenta and blue) are represented as stick models, and orange circles indicate intermolecular clashes between hLF and FcγRIIIa (including oligosaccharides).


Fig.1 Shiga,Y. Page 32 et of al. 41


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

A ・・・・・EFLRKDPEEPKSC・・・・・

NH2

hinge

hLF

CH2

CH3

CH2

CH3

CH2

CH3

COOH

hLF S

S

hLF

B

・・・・・EFLRKDPAPELL・・・・・

NH2

hLF

CH2

CH3

CH2

CH3

CH2

CH3

hLF

hLF


COOH

Page 33 of 41

A

B Non-reducing

Reducing

1000,000

MW (Da)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Fig.2 Shiga,Y. et al.


100,000

10,000

9

11

13

15

17

19

Elution volume (mL)

hLF-CH2-CH3

21

(KDa) M

12

34

192 rhLF

118 91 64

hLF-hinge-CH2-CH3

50 37

10.0

15.0

29

Elution volume (mL)


56

78

9 10 1112 1314 1516



Wavelength (nm)

Θ (mdeg)

hLF-hinge-CH2-CH3

4 2 0 -2 -4 -6 -8 -10 200 210 220 230 240 250

Wavelength (nm)

Holo-form

-4

-7

Θ (mdeg)

-9

-11

-6 -7 -8

Tm=64.7

Tm＞90

-9 30 40 50 60 70 80 90

-12 30 40 50 60 70 80 90 -6

Apo-form

-5

-8

-10

Temperature (℃)

-6

-7

Temperature (℃)

-7

-8 -9

-10

-8 -9

-10

-11

-11

Tm＞90

-12 30 40 50 60 70 80 90

Wavelength (nm)

Wavelength (nm) 6 4 2 0 -2 -4 -6 -8 -10 -12 200 210 220 230 240 250

-6

Θ (mdeg)

Θ (mdeg)

hLF-CH2-CH3

Wavelength (nm) 6 Wavelength (nm) 6 4 4 2 2 0 0 -2 -2 -4 -4 -6 -6 -8 -8 -10 -10 200 210 220 230 240 250 200 210 220 230 240 250

Θ (mdeg)

rhLF

Θ (mdeg)

Θ (mdeg)

8 6 5 4 2 0 0 -2 -4 -5 -6 -8 -10 -10 -12 -15 -14 200 210 220 230 240 250 200 210 220 230 240 250

B

Temperature (℃)

Temperature (℃) -2 -3 -4 -5 -6 -7 -8 Tm＞90 -9 30 40 50 60 70 80 90

Temperature (℃)


Tm=67.8

-12 30 40 50 60 70 80 90

-5 -6 -7 -8 -9 -10 -11 Tm=65.4 -12 30 40 50 60 70 80 90

Θ (mdeg)

Apo-form

Θ (mdeg)

Holo-form

Θ (mdeg)

10

Θ (mdeg)

A

Θ (mdeg)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Temperature (℃)

Page 35 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42



M Control

rhLF Nuclei

Uptake

hLF-CH2-CH3 Nuclei

hLF-hinge-CH2-CH3

Uptake

Nuclei

A

E

I

B

F

J

C

G

K

D

H

L

Uptake

37℃

4℃

+0.2% sodium azide

+Unlabeled bLF Scale bar=50 µm


Lysate Sample

Fig.5 Shiga,Y. Page 36et of al. 41


B

A 800

800

700

700 rhLF Infliximab hLF-CH2-CH3 hLF-hinge-CH2-CH3

600 500 400 300

Response (RU)

Response (RU)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

600 500 400 300

200

200

100

100

0

rhLF Infliximab hLF-CH2-CH3 hLF-hinge-CH2-CH3

0 0

100

200

300

0

Time (sec)


100 200 Time (sec)

300

Page 37 of 41

140

Half-life

2000

AUC

1800

120

1600

30

μg・min/ml

Time (min)

100 80 60

1400 1200 1000 800 600

40

25

400 20

Concentration (μg/ml)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42



200 0

0

20 15 10 5 0 0

50

100

150

200

Time (min) rhLF (n=5) hLF-CH2-CH3 (n=5)

hLF-hinge-CH2-CH3 (n=6) ACS Paragon Plus Environment

250

A

20 Response (RU)

Response (RU)

FcγR IIa

25

25 20 15 10

15 10 5 0

5 0 0

200

100

100

0

300

Response (RU)

200

300

Time (sec)

Time (sec)

FcγR IIIa

35 30 25 20 15 10 5 0

Infliximab hLF-CH2-CH3 hLF-hinge-CH2-CH3

0

100

300

200

B

C

0.8 0.6 rhLF hLF-CH2-CH3

0.4

hLF-hinge-CH2-CH3

0.2 0

0

1

2

3

4

5

Luciferase activity (fold increase)

Time (sec)

A 450 nm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Fig.7 Shiga,Y. Page 38et of al. 41


FcγR I

30

35 30 25 20

hLF-CH2-CH3

15

hLF-hinge-CH2-CH3

10 5

0 Environment ACS Paragon Plus

Immobilized protein conc. (hLF equivalent) µg/ml

-3

-2

-1

0

Log [hLF-Fc] (µg/ml)

1

Page 39 of 41

A

AUC

B

2000 1800 1600 1400 1200

Undetectable

HRP-conjugated Anti human IgG Ab

μg・min/ml

Detectable

30 25

hLF-Fcs

Immobilized Anti hLF Ab

Concentration (μg/ml)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42



1000 800 600 400 200 0

20 15 Commercial ELISA LF-Fc ELISA

10 5 0 0

50

100

150

200

Time (min) Intact hLF-CH2-CH3 (n=5) Intact hLF-hinge-CH2-CH3 (n=6)


250


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

A

B



Conventional IgG Fc fusion protein (hLF-hinge-CH2-CH3) hLF hinge COOH CH3 CH2 NH2 hLF SS hLF

CH2

CH3

CH2

CH3

140

2000

Half-life

1800

120 100 80 60

1600

μg・min/ml

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


Time (min)

Page 41 of 41

40

Hinge-deficient IgG Fc fusion protein (hLF-CH2-CH3) NH2 CH3 hLF CH2

20

COOH 0

hLF

hLF

CH2

CH3

CH2

CH3


1400 1200 1000 800 600 400 200 0

AUC

Hinge-Deficient IgG1 Fc Fusion: Application to Human Lactoferrin

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