AAPS NBC, May 19, 2010
PK of Protein Therapeutics:
Implications of Immunogenicity
Bernd Meibohm, PhD, FCP
Professor & Associate Dean for Graduate Programs and Research
College of Pharmacy
The University of Tennessee Health Science Center
Memphis, TN, U.S.A.
2
Dose Conc
Efficacy
Toxicity
Pharmacokinetics
Pharmacodynamics
Central Paradigm of Clinical
Pharmacology
3
Pharmacokinetics of
Protein Therapeutics
4
Absorption of Proteins
Mostly IV, IM, SC
(e.g. etanercept, insulin, pegfilgrastim)
Presystemic metabolism after IM, SC possible
Apparent absorption rate constant k
app
:
Parenteral Administration
C
1
,V
1
k
a
D
k
deg
F
k
k k k
a
deg a app
= + =
deg
k k
k
F
a
a
+
=
5
Distribution of Proteins
Distribution by Convective Extravasation rather than Diffusion
Vascular space
L
y
m
p
h
a
t
i
c
s
y
s
t
e
m
Convection
Osmotic pressure, paracellular pores,
sieve effect
Interstitial tissue space
Low conc. since
CL
lymph
>> CL
extravasation
Meibohm, Chin J Clin Pharmacol 2007, 12, 1089-98
6
Elimination of Proteins
Same catabolic pathways as endogenous or dietetic
proteins Amino acid re-utilized in endogenous pool
Proteolysis
Either unspecific or limited to specific organ/tissues:
endothelial cells as major contributor of endocytosis
(>1000 m
2
in adult human)
Liver
Major site of protein metabolism for larger proteins
Intracellular uptake as prerequisite
o Receptor-mediated endocytosis via membrane receptors (e.g. LDLR, LRP,
sugar-recognizing receptors (mannose/fucose) etc.)
Kidneys
Major site of protein metabolism for smaller proteins that
undergo glomerular filtration.
Glomerular filtration as rate-limiting step
o Size-selective cut-off 60 kDa
7
Renal Protein Metabolism
Glomerular filtration and
reabsorption by endocytosis
and subsequent lysosomal
degradation
(large complex peptides and
proteins: IL-2, IL-11,
growth hormone, insulin)
Peritubular extraction
(Receptor- and non-receptor-
mediated; growth hormone,
insulin)
Glomerular filtration and
intraluminal metabolism
(small linear peptides: LH-RH,
glucagon)
P
e
r
i
t
u
b
u
l
a
r
b
l
o
o
d
v
e
s
s
e
l
P
r
o
x
i
m
a
l
t
u
b
u
l
e
Lumen
Glomerular
Filtration
P
AA
P
AA AA
P
P AA
Small, linear
peptides
P Protein
AA Amino acid
P
Filtrate
PEPT2
(PEPT1)
Endocytosis
Tang & Meibohm,
In: Meibohm (ed)
Pharmacokinetics
and Pharmaco-
dynamics of
Biotech Drugs,
Wiley-VCW 2006
8
Target-Mediated Drug Disposition
Small molecule drugs
o Receptor interaction
negligible for mass action
in PK model
Proteins
o Receptor interaction
contributes substantially
to disposition of drug (e.g.
receptor-mediated
distribution or elimination)
Nonlinear PK
Effect
Interaction between
drug & pharmacologic
target
CL
3
RC
k
on
k
off
Drug-target complex
degradation
C
2
,V
2
C
1
,V
1
CL
1
D
Q
Proteolysis
Renal metabolism
CL
2
Proteolysis
in tissue
+
k
syn
k
deg
R
Receptor
turnover
9
mAb Binding
Specific binding Non-specific binding Specific binding Non-specific binding
Fab
Antigen
Fc receptor
Fc Fc
Two binding types:
Non-specific via the Fc region
Specific via the Fab region
o Cell surface receptor
o Receptor/target in solution (shed)
o Combination
Almost irreversible binding of antigen
Affinity constant 10
10
10
11
M
Kuester & Kloft, In: Meibohm
(ed), Pharmacokinetics and
Pharmacodynamics of Biotech
Drugs, Wiley-VCW 2006
10
Neonatal Fc Receptor (FcRn)
Protection of IgG from
catabolism by
endocytosis and
recycling; effect
dependent on affinity
to FcRn
IgG1, IgG2, IgG4: t =
18-21 days
IgG3: t = 7 days
Murine IgG in humans:
t = 1-2 days
Not saturable at
therapeutic
concentrations for
mAbs
pH-dependent binding:
increase in binding
affinity at lower pH
Roopenian & Akilesh, Nature Rev Immunol 2007, 7, 715-25
11
Impact of
Anti-Drug Antibodies on PK
of Protein Therapeutics
12
Immunogenicity
Immune response to a biological drug can occur in
nonclinical animal species or in clinical trial subjects
and patients
The more the structure and amino acid sequence of
the protein drug differ from the native protein, the
greater the immunogenic potential of the drug
Immunogenicity less likely in biological products with a
high degree of sequence homology to the native human
protein
Antibody responses with biological therapeutics that are
identical or nearly identical to the native human protein.
General Considerations
Immunogenicity
Schellekens, Nat Rev Drug Discov 2002, 1, 457-62
Immuno-
genicity
A
L
C
N
A
T
F
K
K
T
K
A
L
S
N
A
I
F
K
K
F
K
Sequence variation
Glycosylation
human
non-human
Contaminants &
impurities Formulation
Application
route Dose
Length of
treatment
Assay technology Patient features
Unknown
factors
February
Immunogenicity
Dogma: Protein aggregates are immunogenic
Schellekens & Jiskoot, In: Crommelin, Sindelar, Meibohm (eds.),
Pharmaceutical Biotechnology, 3
rd
ed, Informa Healthcare 2007
15
Immune Complex Formation
Formation of immune complexes in equilibrium with
unbound therapeutic protein and anti-drug antibodies
In pre-clinical and clinical studies, anti-Abs can affect
drug exposure, thereby complicating the
interpretation of the toxicity, efficacy, PK and PD
Antibody-antigen complex formation and deposition in
various tissues
Can inactivate protein therapeutic
Can lead to immune complex-mediated toxicity
Glomerulonephritis observed in Cynomolgus monkeys
after IM rHuIFN-
o Deposition of anti-rHuIFN- antibody complexes in renal
glomeruli
Consequences & Toxicity
16
Immune Complex Formation
The electron micrograph shows
a. unreacted molecules
b. chains of three
c. rings of four
d. a ring of six
e. a ring of 10
TS1 and its monoclonal anti-idiotype, TS1
Johansson et al., Cancer 2002, 94, 130613
Electron micrograph
of TS1/TS1
immune complexes
(0.1 mg/mL) 1:1
mixed, incubated for
20 min, and diluted
10-fold just prior to
mounting and
staining
17
Circulating immune complexes trigger regular
endogenous elimination processes
Uptake and lysosomal degradation by
reticuloendothelial system (phagocytic cells
[monocytes and macrophages])
Primarily in liver and spleen
Internalized ICs are degraded with slow kinetics
Mediated via Fc receptors, primarily FcRIIb2
(in rat liver sinusoidal endothelial cells)
o used as both a recycling receptor and a receptor for
efficient IC clearance
Clearance
Immune Complex Formation
Ali Mousavi et al., Hepatology 2007, 46, 871-84
Immunogenicity
Meibohm & Braeckman, In: Crommelin, Sindelar, Meibohm (eds.),
Pharmaceutical Biotechnology, 3
rd
ed, Informa Healthcare 2007
Possible Scenarios
19
Neutralizing Abs bind to or near the target-binding
domain of the biological drug
Interfere with its ability to bind its target receptor
Clearing or neutralizing Abs in humans and animals
(tox): lower exposure of target organs to the biological
drug product
Cross-reactive Abs
Bind and neutralize the biological therapeutic
Can also bind and neutralize the biological function of the
endogenous protein
Neutralizing vs. Cross-reactive Abs
Neutralizing Anti-Drug Antibodies
20
Neutralizing Anti-Drug Antibodies
Neutralizing Ab
bind IFN-
hinder activation of IFN- receptors, signal
transduction and regulation of interferon
stimulated genes
Impair biological activity
Loss of bioactivity can be overcome by IV
application of high-dose IFN-
Saturation of circulating antibodies and binding of
excess IFN- to its receptor
Stochiometric interaction
High-dose IV INF- in MS patients
with neutralizing antibodies
Millonig et al., Mult Scler 2009; 15; 977-83
21
Clearing Ab
Bind to drug and alter PK
Increase clearance of the drug, resulting in a reduced
systemic exposure and decreased distribution to target
organs
Immune complex formation triggers RES
For therapeutic proteins immune complex formation
can constitute an additional elimination pathway,
thereby reducing the elimination half-life.
Immune complex formation may also limit drug tissue
penetration
Example:
The terminal half-life of the anti-infliximab IgG antibody
immune complex was approximately 38 h compared with 86 h
for the non-immune antibody in Cynomolgus monkeys
Effect on Disposition
Clearing Anti-Drug Antibodies
22
Clearing Abs
Preclinical Evaluation of Lenercept
Representative individual
plasma concentrations of
lenercept (closed symbols)
and antibodies against
lenercept (open symbols)
following a single i.v. dose
to dogs (0.1 mg/kg).
Antibody levels are
reported as the
concentration of lenercept
that can be neutralized
Richter et al. Drug Metab Disp 1999, 27, 21-54
Lenercept is a recombinant fusion protein consisting of the
extracellular domain of two human TNF receptors and the hinge as
well as the constant domain C2 and C3 sequences of the human IgG1
heavy chain
23
Sustaining Ab
Bind to drug and alter PK
Immune complex formation does not trigger regular
endogenous elimination process, but serves as storage depot
for the therapeutic protein
Reduces clearance of the drug, resulting in prolonged systemic
exposure and increased distribution to target organs
t of the therapeutic protein often approaches that of IgG
Often observed for small protein therapeutics:
cytokines and hormones
Immune complex formation can be stabilizing and extend
elimination half-life, e.g. via prevention of glomerular filtration
and subsequent tubular metabolism
Possibly also mediated through FcRn-mediated recycling
(similar to fusion proteins using Fc fragments)
Effect on Disposition
Sustaining Anti-Drug Antibodies
24
Effect of anti-antibody on
the clearance of IL-3
Balb/c mice were injected
intravenously with either
1,000 U recombinant IL-3
() or recombinant IL-3
preincubated with 10 pg
rabbit anti-antibody () for
30 minutes at room
temperature
Mean values SE
Outcomes
Nine-fold reduction in the
total body clearance
Enhanced in vivo activity
IL-3 Example
Tomlinson Jones & Ziltener, Blood 1993, 82, 1133-41
Sustaining Anti-Drug Antibodies
25
Clearing vs. Sustaining Antibodies
At low molar ratios of cytokine/anti-antibody
(2:1), enhanced and prolonged in vivo IL-4
activity (also IL-3, IL-7)
Neutralizing antibodies as carrier proteins
Stimulatory effect of IL-4 only after dissociating
from the anti-cytokine antibodies
Can be blocked by increasing the ratio (1:100)
of anti-IL-4 mAb to IL-4, by injection of anti-
IL-4R mAb, and by in vivo aggregation of the
complexes
PK effect as function molar ratios
Finkelman et al., J Immunol 1993, 151, 1335-44
26
Clearance of human IL-6 in mice treated with either one or with
several murine antibodies of very high affinity for human IL-6.
Single Ab: Sustaining effect
Monomeric complexes of about 180 kD
10 x higher MRT for IL-6
Stabilization of the circulating cytokine
Three Abs binding three distinct epitopes: Clearing effect
Ternary immune complexes with enhanced affinity Fc receptors
Enhanced IL-6 clearance
Influential factors:
Physicochemical properties of IC: size, antibody class, antibody
antigen ratio, characteristics of antigens, location of binding epitopes
Interaction with the RES: Binding to FcR, receptors for complement
components
Example: IL-6
Montero Julian et al., Blood 1995,85, 917-24
Clearing vs. Sustaining Antibodies
27
PK and Anti-Protein Drug Abs
IC formation may result in decrease or
increase in systemic exposure of protein
therapeutics
Effect of sustaining vs. clearing antibodies is
largely determined by the formed ICs and the
size of protein therapeutic
Fc receptor-mediated endocytosis is main
pathway for immune complex clearance, FcRn
recycling of ICs may contribute to effect
sustaining Abs
Summary
28
Meibohm, B (Ed.), 2006. Pharmacokinetics and Pharmacodynamics of
Biotech Drugs. Weinheim, Wiley-VCH.
Crommelin, DJA, Sindelar, RD, Meibohm, B (Eds.), 2007.
Pharmaceutical Biotechnology: Fundamentals and Applications. Third
Edition. New York, Informa Healthcare.
Literature on PK/PD of Biologics
29
Memphis, Tennessee
