ADAPTING PHARMACOKINETIC PROPERTIES OF A HUMANIZED ANTI-INTERLEUKIN-8 ANTIBODY FOR THERAPEUTIC APPLICATIONS USING SITE-SPECIFIC PEGYLATION

doi:10.1006/cyto.2001.0936

Cytokine

Volume 16, Issue 3, November 2001, Pages 106-119

https://doi.org/10.1006/cyto.2001.0936 Get rights and content

Abstract

A neutralizing anti-interleukin-(IL-)8 monoclonal antibody was humanized by grafting the complementary determining regions onto the human IgG framework. Subsequent alanine scanning mutagenesis and phage display enabled the production of an affinity matured antibody with a >100-fold improvement in IL-8 binding. Antibody fragments can be efficiently produced inEscherichia coli but have the limitation of rapid clearance rates in vivo. The Fab′ fragment of the antibody was therefore modified with polyethylene glycol (PEG) in order to obtain a more desirable pharmacokinetic profile. PEG (5–40 kDa) was site-specifically conjugated to the Fab′ via the single free cysteine residue in the hinge region. In vitro binding and bioassays showed little or no loss of activity. The pharmacokinetic profiles of the 20 kDa, 30 kDa, 40 kDa, and 40 kDa branched PEG-Fab′ molecules were evaluated in rabbits. Relative to the native Fab′, the clearance rates of the PEGylated molecules were decreased by 44–175-fold. In a rabbit ear model of ischemia/reperfusion injury, all PEGylated Fab′ molecules were as efficacious in reducing oedema as the original monoclonal antibody. These studies demonstrate that it is possible to customize the pharmacokinetic properties of a Fab′ while retaining its antigen binding activity.

References (44)

SC Donnelly et al.
Interleukin-8 and development of adult respiratory distress syndrome in at-risk patient groups
Lancet
(1993)
T Tsuruma et al.
Induction of heat shock protein-73 reduces ischemia-reperfusion injury in rat small intestine
Transplantation Proc
(1998)
EM Boyle et al.
Inhibition of interleukin-8 blocks myocardial ischemia-reperfusion injury
J Thorac Cardiovasc Surg
(1998)
MJ Glennie et al.
Clinical trials of antibody therapy
Immunol Today
(2000)
IK Koumenis et al.
Modulating pharmacokinetics of an anti-interleukin-8 F(ab′)(2) by amine-specific PEGylation with preserved bioactivity
Int J Pharm
(2000)
R Clark et al.
Long-acting growth hormones produced by conjugation with polyethylene glycol
J Biol Chem
(1996)
MJ Knauf et al.
Relationship of effective molecular size to systemic clearance in rats of recombinant interleukin-2 chemically modified with water-soluble polymers
J Biol Chem
(1988)
DK Pettit et al.
Structure-function studies of interleukin 15 using site-specific mutagenesis, polyethylene glycol conjugation, and homology modeling
J Biol Chem
(1997)
A Abuchowski et al.
Effect of covalent attachment of polyethylene glycol on immunogenicity and circulating life of bovine liver catalase
J Biol Chem
(1977)
P Chomczynski et al.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction
Anal Biochem
(1987)

C Eigenbrot et al.

X-ray structures of the antigen-binding domains from three variants of humanized anti-p185HER2 antibody 4D5 and comparison with molecular modeling

J Mol Biol

(1993)

HB Lowman et al.

Affinity maturation of human growth hormone by monovalent phage display

J Mol Biol

(1993)

SR Leong et al.

Complete mutagenesis of the extracellular domain of interleukin-8 (IL-8) type A receptor identifies charged residues mediating IL-8 binding and signal transduction

J Biol Chem

(1994)

WP Lee et al.

A P-selectin-immunoglobulin G chimera is protective in a rabbit ear model of ischemia-reperfusion

Surgery

(1995)

Y Ashihara et al.

Modification of E. coli L-asparaginase with polyethylene glycol: disappearance of binding ability to anti-asparaginase serum

Biochem Biophys Res Commun

(1978)

T Maack et al.

Renal filtration, transport, and metabolism of low-molecular-weight proteins: a review

Kidney Int

(1979)

A Abuchowski et al.

Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol

J Biol Chem

(1977)

CA Hebert et al.

Interleukin-8: a review

Cancer Invest

(1993)

SJ Weiss

Tissue destruction by neutrophils

N Engl J Med

(1989)

CR Welbourn et al.

Pathophysiology of ischaemia reperfusion injury: central role of the neutrophil

Br J Surg

(1991)

ACJ Windsor et al.

Role of the neutrophil in adult respiratory distress syndrome

Br J Surg

(1993)

T Matsumo et al.

Pivotal role of interleukin-8 in the acute respiratory distress syndrome and cerebral reperfusion injury

J Leukoc Biol

(1997)

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^f2: Correspondence to: Steve Leong, Maxygen, Inc., 515 Galveston Dr, Redwood City, CA 94063, USA. E-mail:[email protected]

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Regular ArticlesADAPTING PHARMACOKINETIC PROPERTIES OF A HUMANIZED ANTI-INTERLEUKIN-8 ANTIBODY FOR THERAPEUTIC APPLICATIONS USING SITE-SPECIFIC PEGYLATION

Abstract

Lancet

Transplantation Proc

J Thorac Cardiovasc Surg

Immunol Today

Int J Pharm

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

Anal Biochem

J Mol Biol

J Mol Biol

J Biol Chem

Surgery

Biochem Biophys Res Commun

Kidney Int

J Biol Chem

Interleukin-8: a review

Cancer Invest

Tissue destruction by neutrophils

N Engl J Med

Pathophysiology of ischaemia reperfusion injury: central role of the neutrophil

Br J Surg

Role of the neutrophil in adult respiratory distress syndrome

Br J Surg

Pivotal role of interleukin-8 in the acute respiratory distress syndrome and cerebral reperfusion injury

J Leukoc Biol

Regular Articles
ADAPTING PHARMACOKINETIC PROPERTIES OF A HUMANIZED ANTI-INTERLEUKIN-8 ANTIBODY FOR THERAPEUTIC APPLICATIONS USING SITE-SPECIFIC PEGYLATION