AAPS NBC, May 19, 2010

PK of Protein Therapeutics: Implications of Immunogenicity

Bernd Meibohm, PhD, FCPProfessor & Associate Dean for Graduate Programs and Research

College of PharmacyThe University of Tennessee Health Science Center

Memphis, TN, U.S.A.

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Dose Conc

Efficacy

Toxicity

Pharmacokinetics Pharmacodynamics

Central Paradigm of Clinical Pharmacology

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Pharmacokinetics of Protein Therapeutics

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Absorption of Proteins

Mostly IV, IM, SC (e.g. etanercept, insulin, pegfilgrastim)

Presystemic metabolism after IM, SC possible Apparent absorption rate constant kapp:

Parenteral Administration

C1,V1kaD

kdeg Fkkkk a

degaapp =+=

degkkkF

a

a

+=

5

Distribution of ProteinsDistribution by Convective Extravasation rather than Diffusion

Vascular space

Lym

phat

ic s

yste

m

ConvectionOsmotic pressure, paracellular pores,

sieve effect

Interstitial tissue space

Low conc. since CLlymph >> CLextravasation

Meibohm, Chin J Clin Pharmacol 2007, 12, 1089-98

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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 m2 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

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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)

Perit

ubul

ar b

lood

ves

sel

Prox

imal

tubu

le

Lumen

GlomerularFiltration

P AA

P

AAAA

PP AA

Small, linear peptides

P ProteinAA Amino acid

P

Filtrate

PEPT2(PEPT1)

Endocytosis

Tang & Meibohm, In: Meibohm (ed) Pharmacokinetics and Pharmaco-dynamics of Biotech Drugs, Wiley-VCW 2006

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Target-Mediated Drug Disposition

Small molecule drugso Receptor interaction

negligible for mass action in PK model

Proteinso Receptor interaction

contributes substantially to disposition of drug (e.g. receptor-mediated distribution or elimination)

Nonlinear PKEffect

Interaction between drug & pharmacologic target

CL3 RC

konkoff

Drug-target complex degradation

C2,V2C1,V1CL1

D

Q

ProteolysisRenal metabolism

CL2

Proteolysisin tissue

+

ksyn

kdeg

R

Receptor turnover

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mAb BindingSpecific binding Non-specific bindingSpecific binding Non-specific binding

Fab

Antigen Fc receptor

FcFc

Two binding types: Non-specific via the Fc region Specific via the Fab region

o Cell surface receptoro Receptor/target in solution (shed)o Combination

Almost irreversible binding of antigen Affinity constant 1010 – 1011 M

Kuester & Kloft, In: Meibohm (ed), Pharmacokinetics and Pharmacodynamics of Biotech Drugs, Wiley-VCW 2006

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Neonatal Fc Receptor (FcRn)

Protection of IgG from catabolism by endocytosis and recycling; effect dependent on affinity to FcRn IgG1, IgG2, IgG4: t½ =

18-21 days IgG3: t½ = 7 days Murine IgG in humans:

t½ = 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

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Impact of Anti-Drug Antibodies on PK

of Protein Therapeutics

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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

ALCNA

T F KKTK

ALSNA

I F KKFK

Sequence variation Glycosylation

human non-human

Contaminants & impurities Formulation

Application route Dose

Length of treatment Assay technology Patient features Unknown

factorsFebruary

ImmunogenicityDogma: Protein aggregates are immunogenic

Schellekens & Jiskoot, In: Crommelin, Sindelar, Meibohm (eds.), Pharmaceutical Biotechnology, 3rd ed, Informa Healthcare 2007

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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-γo Deposition of anti-rHuIFN-γ antibody complexes in renal

glomeruli

Consequences & Toxicity

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Immune Complex Formation

The electron micrograph shows a. unreacted molecules b. chains of threec. rings of fourd. a ring of sixe. a ring of 10

TS1 and its monoclonal anti-idiotype, αTS1

Johansson et al., Cancer 2002, 94, 1306–13

Electron micrograph of TS1/αTS1 immune complexes (0.1 mg/mL) 1:1 mixed, incubated for 20 min, and diluted 10-fold just prior to mounting and staining

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Circulating immune complexes trigger regular endogenous elimination processes

Uptake and lysosomal degradation by reticuloendothelial system (phagocytic cells [monocytes and macrophages]) Primarily in liver and spleenInternalized ICs are degraded with slow kineticsMediated via Fcγ receptors, primarily FcγRIIb2

(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, 3rd ed, Informa Healthcare 2007

Possible Scenarios

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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

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Neutralizing Anti-Drug Antibodies

Neutralizing Abbind IFN-βhinder activation of IFN-β 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-βSaturation of circulating antibodies and binding of

excess IFN-β to its receptor⇒ Stochiometric interaction

High-dose IV INF-β in MS patientswith neutralizing antibodies

Millonig et al., Mult Scler 2009; 15; 977-83

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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

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Clearing AbsPreclinical 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

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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

t½ 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

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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

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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 proteinsStimulatory 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

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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 FcγR, receptors for complement

components

Example: IL-6

Montero Julian et al., Blood 1995,85, 917-24

Clearing vs. Sustaining Antibodies

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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

Fcγ receptor-mediated endocytosis is main pathway for immune complex clearance, FcRn recycling of ICs may contribute to effect sustaining Abs

Summary

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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

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Memphis, Tennessee