MONOMERIC Fc-FUSION PROTEINS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 106

7.3.1 Interferon Beta-Fc Monomer after pulmonary administration of the same dose of 3 m g/mL

of IFN- b Fc monomer. However, at approximately equal
molar IFN- b doses of 0.135 nmol/kg (10 m g/kg) IFN- b Fc
monomer and 0.125 nmol/kg (3 m g/kg) IFN- b , the C max was
approximately 4.6-fold higher

Interferon- b (IFN- b ) is a 166 amino acid glycoprotein used
in the treatment of multiple sclerosis (MS) [38]. The mech-
anism of action of IFN- b in MS is not clear, but IFN- b is
known to protect cells from viral challenge, and an in vitro
assay was developed in which the protective effect of IFN- b
is measured in cells challenged with a cytopathic virus [39].
IFN- b 1a is approved for the treatment of MS and is typically
administered subcutaneously or intramuscularly several
times per week due to its relatively short plasma half-life
[40]. Thus, an IFN- b Fc fusion monomer that could be
administered less frequently and by pulmonary inhalation
could be an important advance in the treatment of MS.
The IFN- b Fc monomer and dimer were constructed and
expressedsimilarlytothatdescribedinSection7.5.Ina
cytopathic effect assay, the IFN- b Fc monomer was demon-
strated to be approximately fourfold more active than IFN- b Fc
dimer [41] suggesting that having two IFN- b molecules in the
fusion protein may hinder the ability of both IFN- b molecules
to bind to IFN- b receptor, whereas an Fc-fusion protein with a
single IFN- b molecule may have better access to the receptor.
IFN- b Fc monomer was administered to cynomolgus
monkeys by pulmonary delivery at deposited doses of 1,
3, or 10 m g/kg and the PKs were compared to a subcutaneous
dose of 3 m g/kg IFN- b [42]. The PKs of IFN- b Fc were dose
dependent with C max values of 1.15

for IFN- b Fc monomer

(17.92 0.92 ng/mL, respectively). In addi-

tion, the AUC of IFN- b Fc monomer was approximately 6.8-
fold higher and the half-life was approximately threefold
longer than for IFN- b [42].

6.13 and 3.66

7.3.2 Interferon a-Fc Monomer

Interferon- a (IFN- a ) is an 18 kDa glycoprotein used in the
treatment of hepatitis C and B and several forms of cancer
[43]. The half-life of IFN- a is short (

5 h) [44], resulting in
subcutaneous dosing three times per week as typical therapy.
Similar to IFN- b , a monomeric Fc-fusion of IFN- a that
reduces dosing frequency and provides a more tolerable
alternate route of administration could be an important
advance in hepatitis therapies.
IFN- a Fc monomer and dimer constructs with 15 amino
acid linkers of three repeats of the sequence GGGGS inserted
between the IFN- a and Fc moieties were generated in a
similar manner to that described in Section 7.5 and tested
in a cytopathic effect assay [41]. IFN- a Fc monomer was
demonstrated to be approximately threefold more active than
IFN- a Fc dimer [41] suggesting that similar to IFN- b ,having
two IFN- a molecules in the fusion protein causes steric
hindrance between the two IFN- a moieties in the dimeric
IFN- a Fc (Table 7.1). Consistent with this, reducing the linker
length between the IFN- a and Fcmoieties reduced the activity

0.73, 3.9 0.65, and

17.92 6.13 ng/mL for the 1, 3, and 10 m g/kg doses, respec-

tively [42]. The maximum serum concentration of IFN- b
administered as a single 3 m g/kg subcutaneous dose was
3.66 0.92 ng/mL which was similar to that value attained

TABLE 7.1 Summary of Bioactivity and Pharmacokinetics for Dimeric and Monomeric Fc-Fusion Proteins

Pharmacokinetics

In Vitro Bioactivity

Fusion
Protein

Dose
( m g/kg)

C max
( m g/mL)

AUC
( m g h/mL)

t 1/2
(h) Species and Route Construct Result

Assay

EpoFc Monkey/pulmonary Dimer 20 0.017 0.557 16 TF-1 proliferation

assay a (EC 50 )

0.07 nM

Monomer 20 0.086 5.279 25 0.09 nM

CPE assay b
(specific activity)

0.45 10 6 IU/nmol IFN- b -Fc Monkey/pulmonary Dimer 20 0.0038 0.123 11

1.22 10 6 IU/nmol Monomer 20 0.022 0.987 27

IFN- a -Fc c 107 CPE assay b

(specific activity)

2.3 10 5 IU/nmol Monkey/pulmonary Dimer 15 0.107 18.760

5.2 10 5 IU/nmol

Monomer 15 0.102 17.325 87

FIXFc Neonatal rat/oral Dimer 1,370 0.590 9,570 14 Not determined

Monomer 1,000 5.830 121,250 19

Adult rat/IV Dimer 5,000 7.500 109 22

Monomer 5,000 33.000 509 35

Adult FIX-deficient
mouse/IV

Dimer 5,000 10.100 167 53

Monomer 5,000 33.400 761 46

AUC, area under the serum or plasma concentration versus time curve; C max , maximum serum or plasma concentration; CPE, cytopathic effect; EC 50 ,
concentration of drug that stimulates growth to 50% of maximum; EpoFc, erythropoietin-Fc; FIXFc, factor IX-Fc; IFN, interferon; IU, international units; t 1/2 ,
terminal half-life in plasma or serum; TF-1, human erythroleukemia cells.
a Growth of human erythroleukemia cells were measured for 48 h. Values represent the concentration of drug that stimulated growth by 50% of maximum (EC 50 ).
b Antiviral activity was measured in a standard CPE assay using human lung A549 cells and encephalomyocarditis virus.
c Constructs with 15 amino acid linkers.