ADAPTING PHARMACOKINETIC PROPERTIES OF A HUMANIZED ANTI-INTERLEUKIN-8 ANTIBODY FOR THERAPEUTIC...

doi:10.1006/cyto.2001.0936, available online at http://www.idealibrary.com on

ADAPTING PHARMACOKINETIC PROPERTIESOF A HUMANIZED ANTI-INTERLEUKIN-8

ANTIBODY FOR THERAPEUTIC APPLICATIONSUSING SITE-SPECIFIC PEGYLATION

Steven R. Leong,1* Laura DeForge,2 Leonard Presta,1 Tania Gonzalez,1

Audrey Fan,1 Marcel Reichert,2 Anan Chuntharapai,1 K. Jin Kim,1

Daniel B. Tumas,5 Wyne Pun Lee,1 Peter Gribling,1 Brad Snedecor,3 Han Chen,3

Vanessa Hsei,4 Monika Schoenhoff,4 Victoria Hale,4 James Deveney,6

Iphigenia Koumenis,7 Zahra Shahrokh,7 Patrick McKay,7 Walter Galan,7

Brian Wagner,7 Daljit Narindray,7 Caroline Hebert,1 Gerardo Zapata7*

A neutralizing anti-interleukin-(IL-)8 monoclonal antibody was humanized by grafting thecomplementary determining regions onto the human IgG framework. Subsequent alaninescanning mutagenesis and phage display enabled the production of an affinity matured antibodywith a >100-fold improvement in IL-8 binding. Antibody fragments can be efficiently producedin Escherichia coli but have the limitation of rapid clearance rates in vivo. The Fab� fragment ofthe antibody was therefore modified with polyethylene glycol (PEG) in order to obtain a moredesirable 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 showedlittle or no loss of activity. The pharmacokinetic profiles of the 20 kDa, 30 kDa, 40 kDa, and40 kDa branched PEG-Fab� molecules were evaluated in rabbits. Relative to the native Fab�, theclearance rates of the PEGylated molecules were decreased by 44–175-fold. In a rabbit earmodel of ischemia/reperfusion injury, all PEGylated Fab� molecules were as efficacious inreducing oedema as the original monoclonal antibody. These studies demonstrate that it ispossible to customize the pharmacokinetic properties of a Fab� while retaining its antigen bindingactivity.

� 2001 Academic Press

From the Departments of 1Immunology, 2Assay & AutomationTechnology, 3Fermentation, 4Pharmacokinetics, 5Pathology,6Analytical Chemistry, and 7Process Sciences, Genentech, Inc.,1 DNA Way, South San Francisco, CA 94080, USA

Correspondence to: *Gerardo Zapata, 709 Swedeland Road,UE3838, P.O. Box 1539, King of Prussia, PA 19406, USA.E-mail: [email protected]; or: Steve Leong,Maxygen, Inc., 515 Galveston Dr, Redwood City, CA 94063,USA. E-mail: [email protected]

Received 5 April 2001; received in revised form 30 June 2001;accepted for publication 3 July 2001

� 2001 Academic Press1043–4666/01/210106+14 $35.00/0

KEY WORDS: DNA shuffling/heterodimer formation/interleukin12/product homogeneity/proliferation/differentiation

Interleukin-8 (IL-8) is a member of the CXCfamily of chemokines that functions as a potent neu-trophil chemoattractant and activator.1 Activatedneutrophils release oxygen radicals and degradativeenzymes which contribute to the destruction of normaltissue architecture and thereby lead to the patho-physiological changes associated with inflammatory

106

disease.2–4 By virtue of its ability to recruit and activateneutrophils, IL-8 has been implicated in a wide rangeof disease states, including ischemia/reperfusion injuryand adult respiratory distress syndrome.5,6 Neutraliz-ing antibodies that bind to IL-8 and prevent it frominteracting with its receptors (CXCR1 and CXCR2) onthe surface of neutrophils can effectively block IL-8’sbiological activity. Indeed, neutralization of IL-8 inseveral animal models of inflammatory disease hasbeen reported to result in significant reduction inneutrophil influx6–12 as well as amelioration ofinflammation and tissue damage.6,8–13

Several monoclonal antibodies (mAbs) are cur-rently used clinically as therapeutic agents and manymore are in late-stage clinical trials.14 In order toprevent the development of human anti-mouse anti-bodies (HAMA), therapeutic mAbs are typicallyhumanized.14 This can be accomplished by grafting thecomplementary-determining regions (CDRs) of theoriginal mAb onto the human antibody framework or,

CYTOKINE, Vol. 16, No. 3 (7 November), 2001: pp 106–119

Efficacy of PEGylated variants of a humanized anti-IL-8 Fab� / 107

alternatively, by using phage display or transgenicanimals.15 In designing mAb therapeutics, anotherimportant consideration is the form of the antibody tobe used; intact IgG and the various fragments such asF(ab�)2, Fab�, Fab, and scFv are possible candi-dates.15,16 However, antibody fragments have substan-tially shorter half-lives than full length IgG, andtherefore this pharmacokinetic characteristic must beconsidered in the context of the desired duration ofdrug activity and the constraints of viable dosingregimens. Another issue in choosing the form of anantibody is the means of antibody production. Fulllength IgGs are typically expressed using mammaliancells, while antibody fragments can be produced bybacterial cultures. Advantages of bacterial expressionsystems include shorter expression times, higher yields,and lower cost than mammalian systems. Numerousreports have demonstrated the ability of polyethyleneglycol (PEG) conjugation to increase the residence timeof various proteins17–28—including antibodies made bybacterial fermentation. Therefore, PEGylation of anti-body fragments produced in Escherichia coli could beused as a means of generating antibody therapeuticswith the desired pharmacokinetic characteristics.

In this report, we describe the development of anaffinity matured humanized anti-IL-8 antibody and theuse of PEGylation to slow the clearance rate whileretaining the IL-8 binding activity of the molecule.Specifically, PEGs with molecular weights rangingfrom 5 kDa to 40 kDa were conjugated to the singlefree cysteine in the hinge region of a Fab� form of theanti-IL-8 antibody. These studies show that PEGyla-tion had little effect on the in vitro IL-8-neutralizingactivity of the antibody and that, by altering the size ofthe PEG conjugated to the Fab�, it was possible tocustomize the pharmacokinetic profile of the antibodywith no loss of in vivo efficacy.

EXPERIMENTAL PROCEDURES

Molecular cloning of the variable light and heavyregions of the murine 6G4.2.5 monoclonalantibody

Total RNA was isolated from the hybridomacell line, 6G4.2.5,7 using the procedure described byChomczynski and Sacchi.29 First strand cDNA wassynthesized by priming the mRNA with syntheticDNA oligonucleotides designed to hybridize withregions of the murine RNA encoding the constantregion of the kappa light chain or the IgG2a heavychain.30 Amplification of the first strand cDNA todouble-stranded DNA was accomplished using for-ward primers that were derived from the N-terminalsequence of the first eight amino acids of either thelight or heavy chains of 6G4.2.5. The polymerase chain

reaction (PCR) products were cloned into a plasmidcontaining a sequence encoding a murine-human anti-body chimera downstream of the alkaline phosphatasepromoter and heat-stable enterotoxin II signalsequence of E. coli. The murine DNA sequences at thevariable-constant junction of both light and heavychains were converted to their corresponding humanconstant domain sequence by site-directed muta-genesis31 to maintain the proper conformation ofthe �-strands. The authenticity of the subunits wasconfirmed following transfection into 293 cells andassaying the cell supernatants for their ability to blockIL-8 binding to human neutrophils or to 293 cellstransfected with CXCR1 (data not shown).

Construction of humanized versions of anti-IL-8antibody 6G4.2.5

The murine cDNA sequence information obtainedfrom the hybridoma cell line, 6G4.2.5, was used toconstruct recombinant humanized variants of themurine anti-IL-8 antibody. The first humanized vari-ant, Fab-1, was made by grafting synthetic DNAoligonucleotide primers encoding the murine CDRs ofthe heavy and light chains onto a phagemid vector,pEMX1.31 Amino acids comprising the framework ofthe antibody that were potentially important for highaffinity binding to IL-8 by the CDRs were identified bycomparing molecular models of the murine andhumanized 6G4.2.5 Fab-1 variable domains using pre-viously described methods.32,33 Additional humanizedframework variants Fab-2 through Fab-9 were con-structed from the information obtained from thesemodels and are presented in Table 1. In these variants,site-directed mutagenesis was used to exchange specifichuman framework residues with their corresponding6G4.2.5 murine counterparts. Subsequently, the entirecoding sequence of each variant was confirmed byDNA sequencing. Expression and purification of eachFab variant was performed as previously described31

with the exception that hen egg white lysozyme wasomitted from the purification protocol. The variantantibodies were analyzed by SDS-polyacrylamidegel electrophoresis (PAGE), electrospray mass spec-troscopy, and amino acid analysis. The variants weresubsequently tested for their ability to block IL-8binding to human neutrophils.

Construction of an anti-IL-8-gene III fusionprotein

An expression plasmid, pPh6G4.V11, encoding afusion protein (heavy chain of the humanized 6G4.2.5version 11 antibody and the M13 phage gene-III coatprotein) and the light chain of the humanized 6G4.2.5version 11 antibody was assembled to produce amonovalent display of the anti-IL-8 antibody onphage particles. The construct was made by digesting a

108 / Leong et al. CYTOKINE, Vol. 16, No. 3 (7 November, 2001: 106–119)

modified version of phGHam-3,34 pFPHX, withEcoRV and ApaI to remove the existing irrelevantantibody coding sequence and replacing it with a1305 bp EcoRV-ApaI fragment from the plasmid,p6G4.V11, encoding the humanized 6G4.2.5 version 11anti-IL-8 antibody. The final product, pPh6G4.V11,was used as the template for the alanine scanningmutagenesis of the CDRs and for the construction ofrandomized CDR libraries of the humanized 6G4.V11antibody.

TABLE 1. Humanization and affinity maturation of the murine anti IL-8 Fab. The first humanizedvariant Fab-1 was a variant containing an unaltered CDR swap in which all the murine CDR amino acidswere transferred to the human framework. The IC50 values of the Fab-1 and the subsequent Fab variantswere determined by the ability of the antibodies to competitively inhibit binding of 125I-IL-8 to humanneutrophils

Variant Template Changesa

IC50b (nM)

nMean SD

Fab-1 CDR swap 63.0 12.3 4Fab-2 Fab-1 PheH68Ala 106.0 17.0 2Fab-3 Fab-1 ArgH72Val 79.8 42.2 4Fab-4 Fab-1 IleH70Leu 44.7 9.0 3Fab-5 Fab-1 LeuH79Ala 52.7 31.0 9Fab-6 Fab-1 IleH70Leu/LeuH79Ala 34.6 6.7 7Fab-7 Fab-6 LeuH81Val 38.4 9.1 2Fab-8 Fab-6 ArgH38Lys 14.0 5.7 2Fab-9 (6G4V11) Fab-6 GluH6Gln 19.0 5.1 7Chimeric Fabc 11.4 7.0 3rhu4D5 Fabd >200 �M 5

aAmino acid changes made relative to the template used. Murine residues are in bold italics and residue numbering is according toKabat et al.30; bThe nM concentration of variant necessary to cause 50% inhibition of binding of 125I-IL-8 to human neutrophils inthe competitive binding assay; cChimeric Fab contains the murine heavy and light chain variable domains fused to the human lightchain kI constant domain and the human heavy chain subgroup III constant domain I, respectively; drhu4D5 Fab is the same isotypeas the humanized 6G4.2.5 Fab. It is a humanized antibody directed against anti-HER2 and should not bind to IL-8.Key: IL-8, interleukin-8; CDR, complementary determining regions.

Alanine scanning mutagenesis of the humanizedantibody

Amino acid residues that were solvent exposed inall the CDRs of the humanized anti-IL-8 6G4.2.5version 11 antibody (h6G4V11) were changed toalanine for determining which amino acids in theCDRs improve or weaken the binding to IL-8. Theamino acids selected for the alanine scan were deter-mined by viewing a three dimensional computer-generated model of the anti-IL-8 antibody. The alaninescan was performed as previously described.35

The concentrations of the phage carrying the wildtype and mutated antibodies were normalized by titer-ing the phage on IL-8 coated plates and establishingtheir half maximal effective concentration (EC50) val-ues. Sulfhydryl coated 96-well plates (Corning-Costar,Wilmington, MA, USA) were coated with an IL-8variant in which Lys64 was converted to Cys. Theformation of a disulfide bond between Cys64 and thesulfhydryl groups on the plate allowed for orientationof the IL-8 receptor binding domains toward thesolution interface (unpublished observations). Plates

were incubated with a 0.1 mg/ml solution of Lys64CysIL-8 in phosphate buffered saline (PBS), 1 mM EDTA,pH 6.5, for 1 h at 25�C followed by three washes withPBS. Remaining free reactive sulfhydryl groups on theplate were blocked by incubating with PBS containing1.75 mg/ml L-cysteine-HCl and 0.1 M NaHCO3. Phagedisplaying the wild type or mutant h6G4V11 anti-bodies were resuspended in 500 �l 10 mM Tris-HCl,1 mM EDTA, 100 mM NaCl, pH 7.5, and held at 4�Cfor no longer than 3 h. The plate was blocked for atleast 2 h with 50 mg/ml skim milk powder in 25 mMcarbonate buffer (pH 9.6), and an aliquot of eachphage was serially diluted across the plate. After a 1 hincubation and a wash step, the plates were incubatedwith a rabbit anti-phage antibody. Following anadditional wash step, a horseradish peroxidase-conjugated goat anti-rabbit Fc antibody was added.The plates were incubated and washed a final time.Bound phage was detected using o-phenylene diamine(Sigma, St. Louis, MO, USA), and the plates wereread at 492 nm using a microplate reader (SLT LabInstruments, Research Triangle Park, NC, USA). TheEC50 value for each phage was calculated by perform-ing a four-parameter fit of the data. The phage weremaintained at 4�C and immediately diluted in PBS toachieve final concentrations corresponding to theirrespective EC50 or target optical density at wave length492 (OD492) values.

The relative half maximal inhibitory concen-tration (IC50) values of the wild type and mutanth6G4V11 antibodies were established using a modifiedcompetition phage ELISA.36 in which the ability ofindividual antibodies to bind to the IL-8 coated onto


the plates was assessed in the presence of competingsoluble IL-8 ranging in concentration from 0.2 to1 �M. The phage samples were added to a 96-well platecontaining serial dilutions of soluble IL-8, and aliquotsof the mixture (100 �l) were then transferred to anIL-8-coated 96-well plate. The remainder of the assaywas performed as described above. Each sample wasanalyzed in triplicate.

Screening of CDR-L1 Asn35 randomizedantibody-phage library

Initially, the wild type Asn35 codon (AAC) wassubstituted with a stop codon (TAA) to prevent recov-ery of the parent clone. Subsequently, an antibody-phage library expressing all twenty known amino acidsincluding the amber stop codon (TAG) at positionAsn35 in CDR-L1 was made by site-directed mutagen-esis using a degenerate oligonucleotide primer with thesequence NNS (N=A/G/T/C; S=G/C).34 Selection ofhu6G4V11 Fab variants with high affinity binding wasaccomplished by panning the library using 96-wellplates coated with IL-8 as described above. Prior topanning, the number of phage was normalized to1.1�1013 phage/ml. The plates were washed andblocked, and phage (100 �l) were added. After a 1 hincubation at 25�C, the non-specifically bound phagewere removed by washing with PBS containing 0.5%Tween-20 (PBST). For sort 1, a low stringency wash(100 �l PBST/well for 10 min at 25�C) was employed tocapture the small proportion of antibody-phage tightlybound to the immobilized IL-8. The antibody-phagevariants specifically bound to IL-8 were eluted with100 �l/well of 200 mM glycine, pH 2.0, for 5 min at25�C and immediately neutralized with a 1/3 volume of1 M Tris-HCl, pH 8.0. The harvested phage weretitrated and re-propagated as before. The stringency ofthe washes was successively increased with eachadditional round of panning depending upon the per-cent recovery of phage at the end of a sort. Thewashing conditions were as follows: sort 2 (4�15 minintervals; total time=60 min) and sort 3 (either 3a:8�15 min intervals or 3b: 12�10 min intervals; totaltime=120 min). The total number of phage recoveredwas progressively reduced after each sort suggestingthat non-specific or weaker binders were selectedagainst. The recovery of the antibody-phage stop vari-ant negative control was constant throughout thepanning (�10�4–10�5 %).

Kinetic exclusion assayEquilibrium and kinetic measurements of vari-

ants AsnL35Ala and AsnL35Glu were determinedusing the KinExA� automated immunoassay system(Sapidyne Instruments Inc., Idaho City, ID, USA) asdescribed.37

Characterization of the humanized anti-il-8antibodies

Soluble AsnL35Ala Fab antibody was made bytransforming an amber non-suppressor strain of E.coli, 34B8, with pPh6G4.V11 and growing the culturein low phosphate medium for 24 h. The periplasmicfraction was collected and passed over a Hi-Trapprotein G column (Pharmacia, Piscataway, NJ, USA)followed by a desalting and concentration step. Theprotein was analyzed by SDS-PAGE, mass spec-trometry, and amino acid analysis. Antibodies of thecorrect size and amino acid composition were tested inbinding and functional assays.

IL-8 binding and functional assaysAll murine-human chimeras and humanized anti-

body variants [IgG1, Fab, and F(ab�)2] in either theirnative or PEGylated forms were compared to eithermurine 6G4.2.5 full length IgG2a or humanized6G4V11 Fab or F(ab�)2 in assessing their ability toblock 125I-IL-8 binding to neutrophils, IL-8-mediatedneutrophil chemotaxis, and IL-8-stimulated neutrophildegranulation. The methodology for these assays hasbeen described previously.17

Fab�-SH purificationFab�-SH antibody fragments of the affinity

matured antibodies AsnL35Ala and AsnL35Gluwere expressed in E. coli and fermented to high celldensity as described.34 E. coli cells were harvested bycentrifugation from fermentation cultures and resus-pended in three volumes of 20 mM 2-(N-morholino)ethanesulfonic acid (MES), 5 mM EDTA, pH 6.0 con-taining 10.7 mg of 4�,4�-dithiodipyridine per gram offermentation paste. The suspension was homogenizedand 5% PEI (W/V), pH 6, was added to achieve a finalconcentration of 0.25%. The mixture was then centri-fuged to remove solids, and the conductivity of theclarified supernatant was adjusted to 3 mS (unit ofmeasure for conductivity) by addition of cold water.The conditioned supernatant was loaded onto an ABX(J.T. Baker, Phillipsburg, NJ, USA) column equili-brated in 20 mM MES, pH 6.0, and the Fab�-SH waseluted with a 15 column volume linear NaCl gradient(0–350 mM). The column was monitored by absorb-ance at 280 nm, and the fractions containing proteinwere combined for the deprotection step. The ABXpool was adjusted to pH 4.0 by the addition of diluteHCl, and the -SH groups were deprotected by addingDTT to a final concentration of 0.2 mM. The solutionwas incubated for about 30 min and then applied to aSephadex G25 gel filtration column equilibrated with15 mM sodium phosphate, 25 mM MES, pH 4.0. ThepH of the pooled sample was subsequently raisedto pH 5.5 and immediately derivatized with PEG-maleimide as described below.


Preparation of Fab�-S-PEGThe concentration of free thiol was determined

using the method described by Creighton38 and calcu-lated from the increase in absorbance at 412 nm, usinge412=14 150 cm�1 M�1 for the thionitrobenzoateanion and e280=1.5 for the Fab�-SH antibody fragment(molecular weight [MW]=48 690 Da). Two to fivemolar equivalents of PEG-maleimide (ShearwaterPolymers, Huntsville, AL, USA) were added to thedeprotected Fab�-SH gel permeation pool and the pHwas immediately adjusted to pH 6.5 with 10% NaOH.The Fab�-S-PEG product was purified using a2.5�20 cm cation exchange column (Poros 50-HS).The column was equilibrated with 20 mM MES buffer,pH 5.5. The coupling reaction containing thePEGylated antibody fragment was diluted with deion-ized water to a conductivity of approximately 2.0 mS.The conditioned coupling reaction mixture was thenloaded onto the equilibrated Poros 50 HS column. Un-reacted PEG-maleimide was washed from the columnwith two column volumes of 20 mM MES, pH 5.5. TheFab�-SH-PEG product was eluted from the columnusing a gradient from 20 mM MES, pH 5.5, to 0.5 MNaCl, 20 mM MES, pH 5.5, over 15 column volumes.

Size exclusion chromatographyThe hydrodynamic effective size of each molecule

was determined using a Pharmacia Superose-6 HR10/30 column (10�300 mm). The mobile phase was50 mM sodium phosphate, 200 mM NaCl, pH 6.0. Theflow rate was 0.5 ml/min, the column was kept atambient temperature, and the absorbance was moni-tored at 280 nm. Molecular weight standards (Biorad,Hercules, CA, USA) containing cyanocobalamin,myoglobin, ovalbumin, IgG, and thyroglobulin mono-mer and dimer were used to generate a standard curvefrom which the effective size of the PEGylated anti-bodies was estimated. Protein concentrations weredetermined by amino acid composition analysis andabsorbance at 280 nm using a extinction coefficient of1.26 for all PEGylated Fab� constructs.

Tryptic mapping of anti-IL-8 Fab� andPEGylated-anti-IL-8 Fab�

A 100 �g aliquot of each antibody was transferredto a microcentrifuge tube and dried. A 0.5 ml volumeof unfolding buffer (6M guanidine HCl in 30 mMTris–HCl, pH 7.5) and 10 �l of 1 M dithiothreitol(DTT) were added and incubated for 4 h at roomtemperature. At the end of this time, 10 �l of aniodoacetic acid solution (0.54 g/ml in 1 M NaOH) wasadded, and incubation continued for an additional30 min. This solution was then dialyzed overnightagainst 10 mM N-(2-acetamido)-2-aminoethane-sulfonic acid (ACES), 20 mM CaCl , pH 7.0. The
2
samples were then transferred to microcentrifugetubes and digested at 37�C with N-tosylphenylalaninechloromethyl ketone (TPCK)-trypsin (Sigma-Aldrich,St. Louis, MO, USA) at an initial 2% (W/W) ratio.After 4 h, a second addition of 2% (W/W) enzyme wasmade. The digestion proceeded for an additional 4 hand was then terminated by freezing. Half of thedigested protein, 50 �g was loaded onto a reverse-phase high performance liquid chromatography(HPLC) column, (Vydac C18, 250�2.1 mm, 300 Apore size, 5 �m particle size; The Separations Group,Hesperia, CA, USA) on a Hewlett-Packard (Palo Alto,CA, USA) 1090 HPLC system controlled by a Chem-station. The initial mobile phase was aqueous 0.1%Trifluroacetic acid (TFA). Following a 5 min isocratichold, there was a linear gradient from 0–45% TFA inacetonitrile (Burdich and Jackson, Muskegon, MI,USA) over 115 min (0.39%/min). The flow rate was0.25 ml/min, the column temperature was maintainedat 45�C, and the absorbance was monitored at both214 and 295 nm. All peaks were collected, and theamino acid sequence of the peaks was determine usingN-terminal sequencing methods.

Pharmacokinetic and safety studyFive groups of male New Zealand White (NZW)

rabbits (2–3 animals per group) received a 2 mg/kg i.v.bolus dose of unmodified or PEGylated AsnL35AlaFab� antibody in a marginal ear vein. Blood sampleswere collected via a cannula placed in the artery of thecontralateral ear. Rabbits in group 1 were injected withunmodified Fab�, and blood samples were drawn at 0,5, 30 min, 1, 2, 3, 4, 6, 8, 10, 14, 20, 24, and 360 h.Animals in groups 2 and 3 received 20 kDa PEG-Fab�and 40 kDa PEG-Fab�, respectively, and blood wasdrawn at 0, 5, 30 min, 1, 2, 4, 6, 8, 10, 12, 24, 28, 32, 48,72, 96, 168, 216, 264, 336, and 360 h. Groups 4 and 5were injected with 40 kDa branched PEG-Fab� and30 kDa PEG-Fab�, respectively; blood samples weredrawn as for groups 2 and 3, with additional samplestaken at 17, 21, and 25 days post-administration.Group 6 was injected with humanized 6G4.2.5F(ab�)2,17 and blood samples were drawn as for thePEGylated groups through the 48 h time point;additional samples were taken at 52 and 56 h. Serumwas isolated, stored at �70�C, and later analyzed todetermine the circulating concentrations unmodifiedor PEGylated Fab� using an IL-8 binding ELISA.17

Non-compartmental pharmacokinetic analysis wasconducted on concentration vs time data up to 168 h.

In vivo efficacy testing of anti-IL-8 antibodyreagents in rabbit ear model of tissue ischemiaand reperfusion

To evaluate the in vivo efficacy of the PEGylatedand control anti-IL-8 antibodies, a rabbit model of


tissue ischemia-reperfusion39,40 was used with minoralterations. General anesthesia was achieved by i.m.injections of ketamine (50 mg/kg) plus xylazine (5 mg/kg) and acepromazine (2 mg/kg). The right external earwas prepared for surgery, and under sterile procedurethe ear was partially transected at its base, leaving onlythe central artery and vein intact. Nerves to the iso-lated ear were transected at the surgical site to producecomplete loss of sensory innervation and permanentanesthesia of target tissue. A straight microaneurismclip (1.5�10 mm) was placed across the artery toproduce complete ischemia. The ear was reattachedwith the microaneurism clip protruding through thewound. The rabbits were then housed at 26�C, and 6 hlater, the clip was removed to start reperfusion. Foreach animal, reperfusion was noted visually by thepresence of an obvious vascular blush. Untreated rab-bits (n=11) received an i.v. injection of vehicle (10 mMsodium acetate, 8% trehalose, and 0.01 polysorbate-20,pH 5.5) immediately prior to reperfusion. Treatedanimals received either the full length IgG murineanti-rabbit IL-8 monoclonal antibody 6G4.2.5 (5 mg/kg, i.v., n=4), the 20 kDa linear PEG AsnL35GluFab� (5 mg/kg, i.v., n=3), the 30 kDa linear PEGAsnL35Glu Fab� (5 mg/kg, i.v., n=3), or the 40 kDabranched PEG AsnL35Glu Fab� (5 mg/kg, i.v., n=3)immediately prior to reperfusion.

The ear volume, a quantitative measure of tissueswelling and oedema, was calculated for each animal asdescribed previously.40 Briefly, the ear was completelysubmerged in a beaker of sterile water containing1.2% povidone iodine (VWR/Scientific Products,San Francisco, CA, USA), and the volume of fluiddisplacement determined the ear tissue volume. Earvolume was monitored daily in this manner and thepercent change was calculated (% change in earvolume=100�[(ear vol. at day N�ear vol. at day0)/ear vol. at day 0]. The relative efficacy determined byear volume changes was verified by histological evalu-ation. Routine haematoxylin and eosin stained 3 �mthick cross sections of each test ear from each animalwere taken at a point 3 cm distal to the surgical wound.Partial transections were evaluated. The sections weregraded on a 0–4 scale (0 normal, 1 minimal, 2 mild, 3moderate, 4 severe) for severity of tissue oedema,inflammation, and necrosis.

RESULTS

Humanization and affinity maturation of 6G4.2.5The first humanized variant, Fab-1, was an un-

altered CDR swap in which all the murine CDR aminoacids defined by both x-ray crystallography andsequence hyper-variability were transferred to thehuman framework. When the purified Fab was tested

for its ability to inhibit binding of 125I-IL-8 to IL-8receptors on freshly isolated human neutrophils, a5.5-fold increase in IC50 was evident as compared tothe chimeric Fab control indicating a decreased abilityof the antibody to bind to IL-8 (Table 1). Subsequentversions of Fab-1 were engineered to alter the threedimensional structure of the CDR loops into a morefavorable conformation for binding IL-8. The IC50

values of these additional Fab variants are presented inTable 1. Slight increases in IC50 (<2-fold) wereobserved for Fab-2 and Fab-3, while slight decreases inIC50 were noted for Fab-4 and Fab-5. Variant Fab-6showed the greatest improvement in IL-8 binding, withan IC50 reduced by approximately two-fold (34.6 nM)compared to Fab-1 (63 nM). Further framework sub-stitutions were made using the Fab-6 variant as atemplate to bring the IC50 values closer to that of thechimeric Fab. Neutrophil binding experiments revealedno change in the IC50 for Fab-7 (38.4 nM), but asignificant improvement was obtained for both Fab-8and Fab-9, with IC50 values of 14 nM and 19 nM,respectively. The amino acid changes introduced intoFab-8 and Fab-9 were ArgH38Lys and GluH6Gln,respectively (heavy chain residue numbers are prefixedwith H and light chain residues are prefixed with an L).Examination of the human antibody sequences withrespect to amino acid variability revealed that thefrequency of Arg at residue H38 was >99%, whereasresidue H6 was �20% Gln and �80% Glu. Therefore,to reduce the likelihood of inducing an immuneresponse to the antibody because of a change in ahighly conserved amino acid, Fab-9 (6G4V11) waschosen over Fab-8 for further affinity maturationstudies.

Selected amino acid residues in the CDR regionsof 6G4V11 were substituted with alanine to locatepositions that may be suitable targets for randomiza-tion to engineer potential improvements in IL-8 bind-ing. For the most part, the mutations had little or noimpact on the ability of the antibodies to inhibit125I-IL-8 binding to neutrophils. Other mutationsresulted in complete loss of binding activity, presum-ably due to conformational perturbations that affectedeither crucial structural or buried amino acids in theCDR (Table 2). The alanine scan identified CDR-H3(heavy chain, third CDR) as the dominant IL-8 bind-ing epitope. Alanine substitutions in this region gener-ally resulted in large increases in the IC50 values.TyrH101, TyrH103, and AspH106 were of particularinterest because the computer generated model of theanti-IL-8 antibody predicted that these residues weresolvent exposed and might participate in hydrogenbonding or charge interactions with IL-8 or otheramino acids of the antibody that influence eitherbinding to IL-8 or the conformation of the CDR-H3loop structure. Another key finding from these studies


was the significant decrease in IC50 observed whenalanine was substituted into some positions in CDR-1of the variable light chain. The lowest IC50 was seenfor AsnL35Ala (3.3 nM), and this amino acid becamethe primary candidate for affinity maturation of theanti-IL-8 mAb.

In order to construct an improved version of thehumanized 6G4V11 mAb, studies were conducted inwhich position Asn35 of CDR-L1 was randomly muta-genized to any of the twenty known amino acids, andan antibody-phage library was constructed. Eighteenrandom variants from the third round of sorting wereanalyzed by DNA sequencing to look for an aminoacid consensus at position 35 of CDR-L1. The datashowed that Gly occupied position 35 in 33% of thevariants sequenced. However, after correcting for thenumber of NNS codon combinations/amino acid,the frequency of Gly was reduced to 16.6%. Glu wasrepresented with the highest frequency (22%) followedby Asp and Gly (16.6%). The frequencies of recoveryof the wild type Asn and substituted Ala were only5.6%. Soluble Fab of the affinity matured variantssubstituted with Ala, Glu, Asp or Gly were evaluatedfor their ability to inhibit 125I-IL-8 binding to humanneutrophils. The resulting IC50 values were 0.2 nM,0.1 nM, 0.9 nM, and 3.0 nM, respectively, rangingfrom 6–190-fold less than that of the humanized

6G4V11 mAb (19.0 nM). Moreover, the Glu variantwas two-fold more potent in blocking IL-8 binding tohuman neutrophils than the Ala variant (IC50 values of0.1 nM vs 0.2 nM, respectively). In further characteri-zation studies, affinity measurements confirmed thatthe Glu substitution was more favorable for bindingIL-8. The equilibrium constants (Kd) for the affinitymatured Glu variant were 54pM (Fab�) and 49pM(IgG1) as compared to 114pM (Fab�) and 88pM(IgG1) for the Ala variant (Table 3). In addition,chemotaxis assays demonstrated that AsnL35Glu wassomewhat more effective than AsnL35Ala in blockingneutrophil chemotaxis stimulated by 2 nM human orrabbit IL-8. Average IC50 values of 2.8 nM (humanIL-8) and 1.2 nM (rabbit IL-8) were observed forAsnL35Glu as compared to 3.0 nM (human IL-8) and5.0 nM (rabbit IL-8) for AsnLAla. An irrelevantisotype control Fab had no effect on neutrophilmigration.

TABLE 2. Relative affinities for alanine scan versions of theanti-IL-8 6G4V11 CDR mutants. Selective amino acid residuesin the CDR regions of 6G4V11 were substituted with alanine tolocate suitable amino acid targets for randomization andimprovement in IL-8 binding. The IC50 values were determinedby experiments involving competitive inhibition of 125I-IL-8binding to human neutrophils

CDR Amino Acid residueAverage

IC50 (nM)a SD

CDR-L1 Ser26 6.3 2.9Gln27 10.2 2.4Ser28 14.2 5.2Val30 29.1 12.3His31 580.3 243Ile33 64.2 14.6

Asn35 3.3 0.7Tyr36 138 ndc

Tyr37 NBDb ndCDR-H3 Asp100 NBD nd

Tyr101 NBD ndArg102 36.6 15.3Tyr103 199.5 ndAsn104 278.3 169.4Asp106 159.2 44Trp107 NBD ndPhe108 NBD ndPhe109 209.4 72.3Asp110 25.3 21.7

aCompare to the 19.0 nM IC50 of the parent Fab-9 (6G4V11) (Table 1);bNBD, no binding detected; cnd, not determined.Key: IL-8, interleukin-8; CDR, complementary determining regions.

TABLE 3. Equilibrium and kinetic measurements of bindingof 6G4V11 anti-IL-8 variants Asn35Ala and Asn35Glu toIL-8. Equilibrium and kinetic measurements were determinedusing the KinExA automated immunoassay system.AsnL35Glu was more efficient than AsnL35Ala in binding IL-8

Sample ka kdKd

(pM)

6G4V11Asn35Ala-Fab nd nd 1146G4V11Asn35Glu-Fab 4.7�106 2.6�10�4 546G4V11Asn35Ala-IgG1 8.7�105 7.7�10�4 886G4V11Asn35Glu-IgG1 3.0�106 1.4�10�4 49Murine 6G4.2.5 IgG2a 8.3�105 2.9�10�4 350

Key: nd, not determined; IL-8, interleukin-8.

Physical characterization of PEGylated antibodyfragments

A PEGylated Fab� was produced by attaching amolecule of PEG-maleimide to the single free -SH onthe hinge of the humanized anti-IL-8 6G4V11 mol-ecule. A small molar excess of PEG-maleimide resultedin a rapid and efficient coupling reaction, and thePEG-Fab� fragments were easily purified from con-taminating E. coli proteins and unreacted PEG-maleimide in a single step using cation exchangechromatography. Attachment of the larger MW PEGmoieties appeared to affect the overall charge of themolecule as indicated by the fact that the antibodyfragments with higher MW PEGs eluted from thecation exchange column at lower concentrations of saltduring the gradient elution as compared to the identi-cal antibody fragment coupled to a lower MW PEG(data not shown).

Anti-IL-8 Fab� fragments were modified withPEG-maleimide with MWs of 5–100 kDa (linear) or40 kDa (branched). Figure 1 shows the theoretical MW


of the molecules along with their corresponding appar-ent MW determined by HPLC size exclusion chroma-tography. The addition of successively higher MWPEG moieties dramatically increased the apparentMW of the Fab� fragments relative to their theoreticalMW. For example, attachment of a lower MW PEG(10 kDa) to the antibody resulted in a theoretical MWof 59 kDa and an apparent MW of 180 kDa, a differ-ence of three-fold. Attachment of a high MW PEG(100 kDa), however, resulted in theoretical and appar-ent MW values of 158 kDa and 2000 kDa, respectively,a difference of 12-fold. Further characterization ofthese molecules by SDS-PAGE under reducing con-ditions demonstrated that the PEG was specificallycoupled to the heavy chain of the Fab� molecules.Figure 2A shows that the heavy and light chains of theunmodified Fab� had similar MWs of approximately30 kDa while the PEGylated Fab� molecules consistedof two bands of different size. The MW of the putativeheavy chain (upper band) was observed to migrate atsizes of 40 kDa, 45 kDa, 70 kDa, 110 kDa, 125 kDa,150 kDa, and 300 kDa for the molecules modified with5 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 40 kDabranched, and 100 kDa PEG, respectively. The MWof the light chain (lower band) remained constantat approximately 30 kDa. Under non-reducing con-ditions, the PEGylated fragments exhibited MWs ofapproximately 70 kDa, 115 kDa, 120 kDa, 140 kDa,200 kDa, and 300 kDa for the molecules modified with5 kDa, 20 kDa, 30 kDa, 40 kDa, 40 kDa branched,and 100 kDa PEG, respectively (Fig. 2B). The resultsof SDS-PAGE gels confirmed that all PEG-Fab� mol-ecules were purified to homogeneity and that themolecules were different only with respect to the size ofthe PEG molecule attached to the heavy chain. Furtheranalysis of the PEG-Fab� molecules by triptic mapping

demonstrated that the PEG was specifically attached tothe hinge peptide containing the free cysteine (Fig. 3).

0100 K

2.5

Molecule

Theoretical MWEffective MW by SEC-HPLC

MW

(×1

06 )

FAB'

2.0

40 KBR

40 K30 K20 K10 K5 K

1.5

1.0

0.5

Figure 1. Apparent and theoretical MWs of anti-IL-8 Fab� fragmentsmodified with 5–100 kDa PEG-maleimide.

The apparent MW dramatically increased relative to the theoreticalMW with the attachment of larger sized PEG moieties to the Fab�molecule.

Figure 2. SDS-PAGE of purified PEG-Fab� antibody fragments ofdifferent MWs.

Panel A, reducing conditions. The heavy and light chains of theunmodified Fab� had similar MWs of approximately 30 kDa whilethe PEGylated Fab� molecules resulted in two bands of different size.The MW of the putative heavy chain increased with the size ofthe PEG molecule attached. Panel B, non-reducing conditions. ThePEGylated fragments exhibited increasing MWs proportional tothe size of the PEG molecule attached.

In vitro characterization of PEGylatedAsnL35Ala Fab� fragments

The anti-IL-8 Ala variant modified with PEG-maleimide molecules of different size were tested fortheir ability to inhibit binding of 125I-IL-8 to neu-trophils. The IC50 values resulting from the bindingstudies are presented in Table 4. Due to the largenumber of neutrophils required, analysis of thePEGylated variants was divided into three separateexperiments, with the unPEGylated Ala variant as acontrol for each experiment. Assay-to-assay variationin the IC values of the control Fab� was due to the
50


1 h 30 minTime

Abs

orba

nce

0 1 h30 min

PE

Fal

2

1

Figure 3. Tryptic mapping of Anti-IL-8-Fab� and PEGylated anti-IL-8-Fab�.

The two tryptic maps are comparable with the exception of peptide 1 in the Anti-IL-8-Fab map. Peptide 1 contains the single hinge sulfhydroin the Anti-IL-8-Fab�. Specific PEGylation of the peptide containing the hinge sulfhydro, changes the retention time of the peptide to elute atthe end of the peptide map. Peptide 2 in the tryptic map of the PEGylated-Fab� is the PEGylated peptide containing the hinge sulfhydro.

TABLE 4. Binding of PEGylated Fab� anti-IL-8 variants to125I-IL-8. The PEGylated and control unPEGylated Fab�fragments were tested for their ability to block binding of125I-IL-8 to freshly isolated human neutrophils. The analyseswere divided into separate experiments using neutrophils fromdifferent donors. The control Fab� was run in each experiment,and all measurements were performed in triplicate

Fab variantsIC50

(pM)Standarddeviation

RatioIC50 PEGFab�/IC50

control)

6G4V11Asn35Ala-Fab control 366 �102 —5 kDa PEG-Fab� 350 �40.8 110 kDa PEG-Fab� 537 �82 1.520 kDa PEG-Fab� 732 �81 26G4V11Asn35Ala-Fab control 802 �314 —30 kDa PEG-Fab� 624 �89 0.840 kDa PEG-Fab� 1077 �207 1.36G4V11Asn35Ala-Fab control 294 �113 —40 kDa Branched PEG-Fab� 734 �225 2.5

Key: PEG, polyethylene glycol; IL-8, interleukin-8.

50

TABLE 5. Ability of PEGylated Fab� fragments to inhibitIL-8-stimulated neutrophil chemotaxis. The PEGylated andcontrol unPEGylated Fab� fragments were tested in a neu-trophil chemotaxis assay to assess their ability to blockIL-8-stimulated chemotaxis. The analyses were divided intoseparate experiments using freshly isolated neutrophils fromdifferent donors. The control Fab� was run in each experiment,and all measurements were performed in triplicate

Fab variantsIC50

(nM)

Ratio(IC50 PEG Fab�/IC50

control)

6G4V11Asn35Ala-Fab control 1.2 —5 kDa PEG-Fab� 3.7 3.010 kDa PEG-Fab� 2.5 2.020 kDa PEG-Fab� 2.0 1.66G4V11Asn35Ala-Fab control 0.8 —30 kDa PEG-Fab� 2.5 3.040 kDa PEG-Fab� 3.7 4.6

Key: PG, polyethylene glycol; IL-8, interleukin-8.

use of neutrophils from different donors. Within aparticular assay, however, the variation in the IC50

values for the control Fab� was negligible (data notshown). All samples were analyzed in triplicate. Ascompared to the control, the IC50 values for thePEG-Fab� variants differed by �2.5-fold, with thegreatest increase in IC50 being seen for the 40 kDabranched PEG-Fab� (IC values of 734 nM and

294 nM for the 40 kDa branched PEG-Fab� and thecontrol Fab�, respectively).

Additional studies were performed to evaluate theability of the PEG-Fab� molecules to block the biologi-cal effects of IL-8 on human neutrophils using botha neutrophil chemotaxis assay and a degranulationassay. The results of the chemotaxis assay are shown inTable 5. As described for the experiments shown inTable 4, the analysis of the PEGylated molecules wasdivided into separate experiments. The molecules


modified with 5 kDa, 10 kDa, 20 kDa, and 30 kDawere able to block IL-8-mediated chemotaxis within2–3 fold of the unmodified Fab� control. This level ofdifference was not significant given the inherent vari-ation of up to two-fold for this type of assay. The40 kDa linear PEG-Fab� however, showed a somewhatlesser ability to block the neutrophil chemotacticactivity of IL-8 with an IC50 4.6-fold higher than thatof the control Fab�.

Degranulation of human neutrophils as measuredby the release of �-glucuronidase was used as anothermeans of assessing IL-8 biological activity. The IC50

values of the antibodies were determined using 10 nMIL-8 as the stimulus. Although a trend was observedtoward increasing IC50 values with increasing MW ofPEG, the overall effect of PEGylation on antibodyefficacy was minor; a maximal increase in IC50 ofonly 1.4-fold was observed for the 40 kDa branchedPEG-Fab� (Table 6).

Pharmacokinetic study using PEGylated anti-IL-8Fab� fragments in normal rabbits followingintravenous administration

The mean serum concentration vs time profiles ofthe PEGylated anti-IL-8 constructs in normal rabbitsare depicted in Figure 4, and the pharmacokineticparameters are summarized in Table 7. For thePEGylated Fab� molecules, clearance was reduced44–175-fold, terminal half-life was increased by 15–35-fold, and the mean residence time was increased by52–230-fold as compared to the unmodified Fab�.Attachment of PEG-maleimide with increasing MWresulted in a pharmacokinetic profile for the Fab�approaching that of the full length anti-IL-8 antibody(terminal half-life of 74 h, mean residence time of 99 h,and clearance rate of 0.47 ml/h/kg).

In vivo efficacy testing of anti-IL-8 antibodyreagents in rabbit ear model of tissue ischemiaand reperfusion

Full length murine anti-rabbit IL-8 monoclonalantibody 6G4.2.5, and affinity matured Glu variantFab� coupled to 20 kDa linear, 30 kDa linear, or40 kDa branched PEG were administered i.v. by asingle dose (5 mg/kg) at the time of reperfusion in arabbit ear model of tissue ischemia-reperfusion injury.This model induces significant reperfusion injurycharacterized by neutrophilic inflammation, oedema,necrosis, and ulceration. In a previous report, we havedemonstrated that inhibition of neutrophil traffickingwith an anti-CD18 mAb affords significant protectionin this model.39 Treatment with all PEGylated affinitymatured Fab� molecules effectively reduced ear swell-ing and oedema compared to animals in the placebogroup and was statistically indistinguishable from thatof the full length IgG murine anti-rabbit IL-8 mono-clonal antibody 6G4.2.5 at all time points observed(Fig. 5). The relative efficacy demonstrated by changesin ear volume was verified by histological evaluation(data not shown).

TABLE 6. Ability of PEGylated Fab� fragments to inhibit IL-8-stimulated neutrophil degranulation. ThePEGylated and control unPEGylated Fab� fragments were tested in a neutrophil degranulation assay toassess their ability to block IL-8-stimulated release of �-glucuronidase. The analyses were divided intoseparate experiments using freshly isolated neutrophils from different donors. The control Fab� was run ineach experiment, and all measurements were performed in triplicate. All PEGylated Fab� antibodies wereable to inhibit IL-8-stimulated release of �-glucuronidase with IC50 values similar to the unPEGylated Fab�control

Fab variantsIC50

(nM) SDRatio

(IC50 PEG Fab�/IC50 control)

6G4V11Asn35Ala-Fab control 10.2 0.3 —5 kDa PEG-Fab� 7.2 0.2 0.710 kDa PEG-Fab� 10.8 0.3 1.120 kDa PEG-Fab� 13.0 0.3 1.36G4V11Asn35Ala-Fab control 8.6 0.3 —30 kDa PEG-Fab� 10.4 0.4 1.240 kDa PEG-Fab� 10.7 0.4 1.26G4V11Asn35Ala-Fab control 8.3 0.2 —40 Branched kDa PEG-Fab� 11.4 0.3 1.4

Key: PEG, polyethylene glycol; IL-8, interleukin-8.

DISCUSSION

Numerous researchers have used murine mAbsdirected against IL-8 to study the role of IL-8 in thepathophysiology of neutrophil-mediated disease.6–13

Here we report the development of a humanized highaffinity antibody to IL-8 and modification of its Fab�fragment with high MW PEG moieties to improve thepharmacokinetic profile. In a model of ischemia-reperfusion injury in rabbits, the efficacy of thesePEGylated molecules was found to be equivalent tothat of the full length murine monoclonal antibody.


0.1192

1000

Time (h)

An

ti-I

L8

con

cen

trat

ion

(µg

/mL

)

0 1449648

100

10

1

IgG (C)

20 K Fab' (H)Fab'2 (H)

Fab' (H)

40 K Fab' (H)

Branched Fab' (H)

Figure 4. Mean concentration vs time profiles of anti-IL-8PEG-Fab� constructs in the serum of normal rabbits.

Six groups of male rabbits (n=2 or 3) received a single i.v. bolus dose of 2 mg/kg of one of the PEGylated Fab� molecules or one of the antibodycontrols. Serum samples were collected and the anti-IL-8 protein concentrations were determined by ELISA.

TABLE 7. Pharmacokinetic parameters obtained for anti-IL-8 Fab� molecules modified with differentMW PEGs. Rabbits were injected with PEG-Fab� or the unmodified Fab� control; serum samples werecollected over time and circulating antibody levels were quantitated using a specific ELISA. Non-compartmental pharmacokinetic analysis was conducted on concentration vs time data up to 168 h. Thepharmacokinetic parameters of the higher MW PEG-Fab� molecules approached those of the full-lengthanti-IL-8 antibody (terminal half-life of 74 h, MRT of 99 h, and CL of 0.47 ml/h/kg)

Group no. 1 2 3 4 5

Molecule Fab� Fab� Fab� Fab� Fab�PEG structure — linear linear linear branchedPEG MW — 20 kDa 30 kDa 40 kDa 40 kDaNo. of PEGs 0 1 1 1 1Dose (mg/kg) 2 2 2 2 2Vc (ml/kg)a 58�3 36�3 35�1 34 44�1Vss (ml/kg)b 68�8 80�8 110�15 79 88�21Cmax (�g/ml)c 35�1 58�3 57�1 60 45�1Tmax (min)d 5 5 5 5 5T1/2 term (h)e 3.0�0.9 44�2 43�7 50 105�11AUC00-� (h·mg/ml)f 18�3 80�74 910�140 1600 3400�1300CL (ml/h/kg)g 110�17 2.5�0.2 2.2�0.4 1.3 0.63�0.20MRT (h)h 0.61�0.15 32�2 45�9 63 140�18No. of animals 3 3 3 2 3

aInitial volume of distribution; b Volume of distribution at steady state; cObserved maximum concentration; dObserved time to Cmax;eHalf-life associated with the terminal phase of the concentration vs time profile; fArea under the concentration vs. time curve(extrapolated to infinity); gSerum clearance h Mean residence time; PEG, polyethylene glycol; IL-8, interleukin-8.

Murine mAb 6G4.2.5 was raised against rabbitIL-8 but also binds with high affinity to human IL-8.In order to humanize this mAb, the CDRs of thelight and heavy chains were transferred to a humanframework.31–33 Subsequently, changing four frame-

work positions (IleH70Leu, LeuH79Ala, ArgH38Lys,and GluH6Gln) from the human to the murinesequence was found to improve binding (Table 1),most likely by altering the packing of the buriedresidues that interact with the CDRs and thereby


0

500

Ear

vol

um

e (%

ch

ange

)300

Anti-IL8 formulations:Single Dose (5 mg/kg)administered i.v. attime of reperfusion

Days post ischemia-reperfusion

400

100

200

31 2

Figure 5. Efficacy of PEGylated anti-IL-8 in the rabbit ear model of ischemia-reperfusion injury.

Treatment with 20 kDa linear PEG-Fab� (— —); 30 kDa linear PEG-Fab� (– – – –); and 40 kDa branched PEG-Fab� (· · · ·); effectivelyreduced ear oedema compared to placebo-treated animals (— —). The efficacy of all three PEGylated Fab� molecules was statisticallyindistinguishable from that of the full length IgG murine anti-rabbit IL-8 monoclonal antibody 6G4.2.5 (· · · ·) at all time points evaluated.

influence the conformation of the CDR loops. Sinceposition H38 is partially exposed and highly conservedas Arg, this amino acid was left unchanged while theother three amino acids (IleH70, LeuH79, and GluH6)were changed to the murine sequence in the finalhumanized antibody. Alanine scanning mutagenesis ofthe CDR-H3 region resulted in many variants withsubstantial losses of binding activity, indicating thatthis region is important in IL-8 binding. In contrast, analanine scan of CDR-L1 allowed for the identificationof positions which resulted in an improvement in IL-8binding, with the lowest IC50 being observed forAsnL35. When AsnL35 was substituted with otheramino acids using a phage display library, four aminoacids (glutamic acid, aspartic acid, alanine, and gly-cine) displayed increased affinity to IL-8; specificallythe faster on-rates of the ala- and glu-variants com-pared to murine 6G4.2.5 antibody (Table 3 and unpub-lished data). The latter two amino acids lack sidechains and may thereby influence binding to IL-8 byallowing for greater conformational flexibility of theCDR-L1 possibly promoting better interactions withneighboring CDRs and/or with IL-8 as compared tothe parent AsnL35. Improvement in binding forAsnL35Glu suggests formation of a charge interactionbetween the Fab and IL-8 that is not present in theparent AsnL35 Fab.

The anti-IL-8 Fab� was site-specifically PEGylatedon the single free sulfhydryl group in the hinge regionof the molecule in order to minimize the possibility ofaffecting the CDRs through conformational alterationsor steric hindrance. Although modification of the Fab�with the bulkier PEG molecules (particularly thebranched 40 kDa PEG) tended to cause some reduc-tion in biological activity by some analyses, the overallactivity of the PEGylated-Fab� molecules was notsubstantially compromised as shown in Tables 2, 3,and 6. Moreover, these molecules were effective inabrogating neutrophil-mediated inflammation in vivo(Fig. 5). In studies by Chapman et al.,18 site-specificattachment of a 25 kDa PEG molecule to the hingeregion of a Fab� allowed for complete retention ofbinding ability in vitro; however, in vivo activity of themolecule was not addressed. As compared to targetedcoupling of PEG, PEGylation of proteins using ran-dom chemistries can result in heterogeneously sizedproducts17,19,22 and substantial alterations in biologicalactivity. Studies have shown increasing losses ofactivity as greater numbers of PEG molecules arecoupled to proteins18–21 and that extensive modifica-tion with PEG can result in substantial losses ofprotein activity.28,41 PEGylation can also have un-anticipated effects on protein activity depending onthe particular residues that are solvent exposed and


available for modification. PEGylation of IL-2, forexample, increased its activity in vivo as a result ofimproving the protein’s solubility at neutral pH.24,25

Modification of IL-15 with PEG, however, changedthe molecule from an agonist to a specific antagonistby disrupting its ability to bind to the IL-15 receptor �subunit.26 In addition, an altered spectrum of biologi-cal activity was observed for PEGylated GM-CSF inthat its ability to prime for an oxidative burst wasenhanced while colony stimulating activity was eitherdecreased or unaffected.42 In a study evaluating thepharmacokinetics of various PEGylated forms of IL-2,Knauf et al.24 observed that the rate of clearancedecreased rapidly up to the MW cut-off for glomerularfiltration (�70 kDa43); beyond this cutoff point, theclearance rates decreased more slowly. Knauf et al.observed no further decrease in clearance rates whenthe apparent MW of the protein was increased above200 kDa. These observations were substantially differ-ent from the data reported by Koumenis et al.17 instudies using PEGylated versions of the F(ab�)2 frag-ment of the same anti-IL-8 antibody examined in thepresent study. Data compiled from the Fab� moleculesused in this study and the F(ab�)2 molecules evaluatedby Koumenis et al. demonstrate a continual and sub-stantial decrease in the rate of clearance for PEGylatedmolecules with apparent MWs increasing fromapproximately 200 kDa–1900 kDa.17

In the present experiments, the PEGylated Fab�molecules evaluated in vivo had MWs above theglomerular filtration cutoff, and multiple mechanismsare likely responsible for the decreasing rate of clear-ance with increasing MW. Alterations in the shape andcharge of the protein as a result of PEGylation are stillpossible factors affecting the clearance rates. As notedin the Results section, addition of successively higherMW PEG molecules to the Fab� resulted in a progres-sive alteration in charge as suggested by the elutionof the PEGylated Fab� molecules from the cationexchange column at lower concentrations of salt. Simi-lar effects have been observed for other PEGylatedmolecules.22 In addition, while the 40 kDa linear PEG-Fab� and the 40 kDa branched PEG-Fab� have thesame theoretical and effective MWs as assessed bySEC, the clearance rate of the branched PEG-Fab� wasreduced by two-fold relative to the linear PEG- Fab�(1.3 vs 0.63 ml/h/kg, respectively), suggesting that theshape of the molecule is important in determining theclearance rate. Other factors affecting the half-life ofPEGylated proteins include the ability of PEG toshield sites of proteolysis20,27,41 thereby slowing thecatabolism of the protein and resulting in an increasein half-life.19,20 Lastly, PEGylation has been shownto alter the distribution of proteins in vivo. WhilePEGylation increases the penetration of proteins intotumors, it has the overall effect of decreasing uptake

into other tissues, providing a means for increasing themolecule’s life span in the circulation.20,23

The use of a PEGylated Fab� fragment has severalpotential advantages over a full-length antibody. First,production of antibody fragments using E. coli istypically faster and more cost effective than productionof IgG using mammalian cells. In addition, PEGyla-tion not only prolongs the half-life of the proteins,17–28

but is also has been repeatedly shown to reduce immu-nogenicity27,44 as well as susceptibility to proteo-lysis.20,27,41 Lastly, use of PEGylated Fab� moleculesmay be advantageous when Fc-mediated effector func-tions are undesirable. The results presented here showthat by site-specific attachment, and variations in MWand configuration of the PEG, it is possible to engineerPEGylated-Fab� molecules with the desired half-lives.The ability to control the half-life of an antibody andthus its duration of action offers an obvious safetybenefit in the therapeutic neutralization of cytokinessuch as IL-8 which play an important role in hostdefense and control of infection.

Acknowledgements

We would like to thank Jackie Curnutte forassistance with IL-8 binding and chemotaxis assays,Peter Ng and Pakash Ghurani for producing syntheticoligonucleotides, and David Wood for the graphic arts.

REFERENCES

1. Hebert CA, Baker JB (1993) Interleukin-8: a review. CancerInvest 11:743–750.

2. Weiss SJ (1989) Tissue destruction by neutrophils. N Engl JMed 320:365–376.

3. Welbourn CR, Goldman G, Paterson IS, Valeri CR, SheproD, Hetchman HB (1991) Pathophysiology of ischaemia reperfusioninjury: central role of the neutrophil. Br J Surg 78:651–655.

4. Windsor ACJ, Mullen PG, Fowler AA, Sugerman HJ(1993) Role of the neutrophil in adult respiratory distress syndrome.Br J Surg 80:10.

5. Donnelly SC, Strieter RM, Kunkel SL, Walz A, RobertsonCR, Carter DC, Grant IS, Pollock AJ, Haslett C (1993) Interleukin-8and development of adult respiratory distress syndrome in at-riskpatient groups. Lancet 341:643–647.

6. Matsumo T, Yokoi K, Mukaida N, Harada A, YamashitaJ, Watanabe Y, Matsushima K (1997) Pivotal role of interleukin-8 inthe acute respiratory distress syndrome and cerebral reperfusioninjury. J Leukoc Biol 62:581–587.

7. Broaddus VC, Boylan AM, Hoeffel JM, Kim KJ, Sadick M,Chuntharapai A, Hebert CA (1994) Neutralization of IL-8 inhibitsneutrophil influx in a rabbit model of endotoxin-induced pleurisy.J Immunol 152:2960–2967.

8. Sekido N, Mukaida N, Harada A, Nakanishi I, WatanabeY, Matsushima K (1993) Prevention of lung reperfusion injury inrabbits by a monoclonal antibody against interleukin-8. Nature365:654–657.

9. Nakamura M, Fujishima S, Sawafugi M, Ishizaka A,Oguma T, Soeijima K, Matsubara H, Tasaka S, Kikuchi K,Kobayashi K, Ikeda E, Sadick M, Hebert CA, Aikawa N, Kanazawa


M, Yamaguchi K (2000) Importance of interleukin-8 in the develop-ment of reexpansion lung injury in rabbits. Am J Respir Crit CareMed 161:1030–1036.

10. Yokoi K, Mukaida N, Harada A, Watanabe Y,Matsushima K (1997) Prevention of endotoxemia-induced acuterespiratory distress syndrome-like lung injury in rabbits by a mono-clonal antibody to IL-8. Lab Invest 76:375–384.

11. Tsuruma T, Yagihashi A, Tarumi K, Sasaki K, WatanabeN, Hirata K (1998) Induction of heat shock protein-73 reducesischemia-reperfusion injury in rat small intestine. TransplantationProc 30:3449–3451.

12. Mukaida N, Matsumoto T, Yokoi K, Harada A,Matsushima K (1998) Inhibition of neutrophil-mediated acuteinflammation injury by an antibody against interleukin-8 (IL-8).Inflam Res 47(Suppl 3):S151–S157.

13. Boyle EM Jr, Kovacich JC, Hebert CA, Canty TG Jr,Chi E, Morgan EN, Pohlman TH, Verrier ED (1998) Inhibitionof interleukin-8 blocks myocardial ischemia-reperfusion injury.J Thorac Cardiovasc Surg 116:114–121.

14. Glennie MJ, Johnson PW (2000) Clinical trials of antibodytherapy. Immunol Today 21:403–410.

15. Vaughan TJ, Osbourn JK, Tempest PR (1998) Humanantibodies by design. Nature Biotechnol 16:535–539.

16. Colcher D, Pavlinkova G, Beresford G, Booth BJ,Choudhury A, Batra SK (1998) Pharmacokinetics and biodistribu-tion of genetically-engineered antibodies. J Nucl Med 42:225–241.

17. Koumenis IK, Shahrokh Z, Leong S, Hsei V, DeForge L,Zapata G (2000) Modulating pharmacokinetics of an anti-interleukin-8 F(ab�)(2) by amine-specific PEGylation with preservedbioactivity. Int J Pharm 198:83–95.

18. Chapman AP, Antoniw P, Spitali M, West S, Stephens S,King DJ (1999) Therapeutic antibody fragments with prolonged invivo half-lives. Nature Biotechnol 17:780–783.

19. Kitamura K, Takahashi T, Yamaguchi T, Noguchi A,Noguchi A, Takashina K, Tsurumi H, Inagake M, Toyokuni T,Hakomori S (1991) Chemical engineering of the monoclonal anti-body A7 by polyethylene glycol for targeting cancer chemotherapy.Cancer Res 51:4310–4315.

20. Tsutsumi Y, Kihira T, Tsunoda S, Kamada H, NakagawaS, Kaneda Y, Kanamori T, Mayumi T (1996) Molecular design ofhybrid tumor necrosis factor-alpha III: polyethylene glycol-modifiedtumor necrosis factor-alpha has markedly enhanced antitumorpotency due to longer plasma half-life and higher tumor accumu-lation. J Pharmacol Exp Ther 278:1006–1011.

21. Clark R, Olson K, Fuh G, Marian M, Mortensen D,Teshima G, Chang S, Chu H, Mukku V, Canova-Davis E, Somers T,Cronin M, Winkler M, Wells JA (1996) Long-acting growth hor-mones produced by conjugation with polyethylene glycol. J BiolChem 271:21969–21977.

22. Chamow SM, Kogan TP, Venuti M, Gadek T, Harris RJ,Peers DH, Mordenti J, Shak S, Ashkenazi A (1994) Modificationof CD4 immunoadhesin with monomethoxypoly(ethylene glycol)aldehyde via reductive alkylation. Bioconjug Chem 5:133–140.

23. Delgado C, Pedley RB, Herraez A, Boden R, Boden JA,Keep PA, Chester KA, Fisher D, Begent RH, Francis GE (1996)Enhanced tumour specificity of an anti-carcinoembrionic antigenFab� fragment by poly(ethylene glycol) (PEG) modification. Br JCancer 73:175–182.

24. Knauf MJ, Bell DP, Hirtzer P, Luo Z-P, Young JD, KatreNV (1988) Relationship of effective molecular size to systemicclearance in rats of recombinant interleukin-2 chemically modifiedwith water-soluble polymers. J Biol Chem 263:15064–15070.

25. Katre NV, Knauf MJ, Laird WJ (1987) Chemical modifi-cation of recombinant interleukin 2 by polyethylene glycol increasesits potency in the murine Meth A sarcoma model. Proc Natl AcadSci USA 84:1487–1491.

26. Pettit DK, Bonnert TP, Eisenman J, Srinivasan S, PaxtonR, Beers C, Lynch D, Miller B, Yost J, Grabstein KH, Gomotz WR(1997) Structure-function studies of interleukin 15 using site-specific

mutagenesis, polyethylene glycol conjugation, and homology mod-eling. J Biol Chem 272:2312–2318.

27. Abuchowski A, McCoy JR, Palczuk NC, van Es T, DavisFF (1977) Effect of covalent attachment of polyethylene glycol onimmunogenicity and circulating life of bovine liver catalase. J BiolChem 252:3582–3586.

28. Pyatak PS, Abuchowski A, Davis FF (1980) Preparation ofa polyethylene glycol: superoxide dismutase adduct, and an exami-nation of its blood circulation life and anti-inflammatory activity.Res Commun Chem Pathol Pharmacol 29:113–127.

29. Chomczynski P, Sacchi N (1987) Single-step method ofRNA isolation by acid guanidinium thiocyanate-phenol-chloroformextraction. Anal Biochem 162:156–159.

30. Kabat EA, Wu TT, Perry HM, Gottesman KS, Foeller C(1991) Sequences of Proteins Immunological Interest, 5th Ed.. PublicHealth Service, National Institutes of Health, Bethesda, MD.

31. Werther WA, Ganzalez TN, O’Connor SJ, McCabe S,Chan B, Hotaling T, Champe M, Fox JA, Jardieu PM, Berman PW,Presta LG (1996) Humanization of an anti-lymphocyte function-associated antigen (LFA)-1 monoclonal antibody and reengineeringof the humanized antibody for binding to rhesus LFA-1. J Immunol157:4986–4995.

32. Carter P, Presta L, Gorman CM, Ridgway JBB, Henner D,Wong WLT, Rowland AM, Kotts C, Carver ME, Shepard HM(1992) Humanization of an anti-p185HER2 antibody for humancancer therapy. Proc Natl Acad Sci USA 89:4285–4289.

33. Eigenbrot C, Randal M, Presta L, Carter P, Kossiakoff AA(1993) X-ray structures of the antigen-binding domains from threevariants of humanized anti-p185HER2 antibody 4D5 and compari-son with molecular modeling. J Mol Biol 229:969–995.

34. Lowman HB, Wells JA (1993) Affinity maturation ofhuman growth hormone by monovalent phage display. J Mol Biol234:564–578.

35. Leong SR, Kabakoff RC, Hibert CA (1994) Completemutagenesis of the extracellular domain of interleukin-8 (IL-8) typeA receptor identifies charged residues mediating IL-8 binding andsignal transduction. J Biol Chem 269:19343–19348.

36. Cunningham BC, Lowe DG, Li B, Bennett BD, Wells JA(1994) Production of an atrial natriuretic peptide variant that isspecific for type A receptor. EMBO J 13:2508–2515.

37. Chuntharapai A, Gibbs V, Lu J, Ow A, Marsters S,Ashkenazi A, De Vos A, Jin Kim KJ (1999) Determination ofresidues involved in ligand binding and signal transmission in thehuman IFN-alpha receptor 2. J Immunol 163:766–773.

38. Creighton TE (1990) In: Creighton TE (ed.) Protein struc-ture: a practical approach. IRL Press, Oxford, pp 155–167.

39. Lee WP, Gribbling P, DeGuzman D, Ehsani N, Watson SR(1995) A P-selectin-immunoglobulin G chimera is protective in arabbit ear model of ischemia-reperfusion. Surgery 117:458–465.

40. Vedder NB, Winn RK, Rice CL, Chi EY, Arfors KE,Harlan JM (1990) Inhibition of leukocyte adherence by anti-CD18monoclonal antibody attenuates reperfusion injury in the rabbit ear.Proc Natl Acad Sci USA 87:2643–2646.

41. Ashihara Y, Kono T, Yamazaki S, Inada Y (1978) Modi-fication of E. coli L-asparaginase with polyethylene glycol: disap-pearance of binding ability to anti-asparaginase serum. BiochemBiophys Res Commun 83:385–391.

42. Knusli C, Delgado C, Malik F, Damine M, Tejedor MC,Irvine AE, Fisher D, Francis GE (1992) Polyethylene glycol (PEG)modification of granulocyte-macrophage colony stimulating factor(GM-CSF) enhances neutrophil priming activity but not colonystimulating activity. Br J Haematol 82:654–663.

43. Maack T, Johnson V, Kau ST, Figueiredo J, Sigulem D(1979) Renal filtration, transport, and metabolism of low-molecular-weight proteins: a review. Kidney Int 16:251–270.

44. Abuchowski A, van Es T, Palczuk NC, Davis FF (1977)Alteration of immunological properties of bovine serum albumin bycovalent attachment of polyethylene glycol. J Biol Chem 252:3578–3581.

ADAPTING PHARMACOKINETIC PROPERTIES OF A HUMANIZED ANTI-INTERLEUKIN-8 ANTIBODY FOR THERAPEUTIC...

Documents

Transcript of ADAPTING PHARMACOKINETIC PROPERTIES OF A HUMANIZED ANTI-INTERLEUKIN-8 ANTIBODY FOR THERAPEUTIC...