www.elsevier.com/locate/ymgme

Molecular Genetics and Metabolism 91 (2007) 325–334

Structure-based epitope and PEGylation sites mapping ofphenylalanine ammonia-lyase for enzyme substitution

treatment of phenylketonuria

Alejandra Gamez a, Lin Wang a, Christineh N. Sarkissian b, Dan Wendt c, Paul Fitzpatrick c,Jeffrey F. Lemontt c, Charles R. Scriver b, Raymond C. Stevens a,*

a Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USAb Departments of Biology, Human Genetics, and Pediatrics, McGill University, and DeBelle Laboratory,

McGill University-Montreal Children’s Hospital Research Institute, 2300 Tupper Street, A-717, Montreal, Que., Canada H3H 1P3c BioMarin Pharmaceutical Inc., 105 Digital Drive, Novato, CA 94949, USA

Received 20 March 2007; received in revised form 18 April 2007; accepted 18 April 2007Available online 8 June 2007

Abstract

Protein and peptide therapeutics are of growing importance as medical treatments but can frequently induce an immune response.This work describes the combination of complementary approaches to map the potential immunogenic regions of the yeast Rhodospo-

ridium toruloides phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and to engineer the protein as a human therapeutic agent for the treat-ment of phenylketonuria (PKU), an inherited metabolic disorder. The identification of B and T cell epitopes on the PAL protein wasperformed by computational predictions based on the antigenicity and hydrophilicity of proteins, as well as by experimental epitopemapping using a PepSpots� peptide array (Jerini AG). Human T cell epitope mapping was performed by applying the computationalEpiMatrix� algorithm (EpiVax, Inc.) for MHC Class I and Class II associated T cell epitopes on PAL, which predicts which sequencesare associated with binding to several different HLA alleles, a requirement for antigen presentation and subsequent primary immuneresponse. By chemical modification through PEGylation of surface lysine residues, it is possible to cover the immunogenic regions ofa protein. To evaluate this strategy, we used mass spectrometry to determine which of the immunogenic epitopes are covered by the cova-lent PEGylation modification strategy. This approach has allowed us to determine whether additional lysines are needed in specific res-idue locations, or whether certain lysine residues can be removed in order to accomplish complete molecular coverage of the therapeuticenzyme.� 2007 Elsevier Inc. All rights reserved.

Keywords: Phenylketonuria; Phenylalanine ammonia-lyase; Epitope mapping; Immunodominant; PEGylation; Enzyme therapy; Protein engineering;Mass spectroscopy

Immunogenicity of protein drugs is a significant chal-lenge in the biological therapeutics development process.The risk is particularly high for chronically administeredproducts [1–3]. Immunogenicity could have a dramaticeffect on the potency and efficacy of the therapeutic; inaddition, it can cause adverse side reactions, such as throm-bocytopenia, aplastic anemia and anaphylactic responses

1096-7192/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.ymgme.2007.04.015

* Corresponding author. Fax: +1 858 784 9483.E-mail address: stevens@scripps.edu (R.C. Stevens).

among others. Apart from the extrinsic factors such as for-mulation, route, dose, and frequency of the protein drug,the intrinsic parameters, protein primary structure andthree-dimensional structure determine the potential immu-nogenicity of certain protein therapeutics [1,4]. In order toreduce immunogenicity, different strategies have beendeveloped based on protein structural information. Proteinhumanization [5], B-cell mediated epitopes removal [6,7],PEGylation [8–11], and class II MHC agretopes engineer-ing [2,12,13] are strategies that have been proven to be

Fig. 1. 1.6 A of Rhodosporidium toruloides phenylalanine ammonia lyase,PAL, tetramer determined by high-resolution X-ray crystallography (PDBIDCODE: 1Y2M).

326 A. Gamez et al. / Molecular Genetics and Metabolism 91 (2007) 325–334

successful to varying extents. The basic rule above all, is tominimize the immune response without perturbing thestructure, function and stability of the protein therapeutic.In order to de-immunize therapeutic proteins rationally,the first essential step is to identify their epitopes.

We have developed a combinatorial strategy to reducethe immunogenicity of PEGylated phenylalanine ammo-nia lyase (PAL), the chosen protein therapeutic for phen-ylketonuria (PKU). PKU is a metabolic disorder (OMIM261600) that results from impaired activity of hepaticphenylalanine hydroxylase (PAH), the enzyme responsiblefor metabolism of the majority of phenylalanine intake[14]. Patients with PAH mutations that lead to PKU dis-play impaired neurophysiological functioning and reducedcognitive development. There is the potential for irrevers-ible mental retardation if dietary restriction is not used tokeep L-phenylalanine (Phe) levels in a near-normal range.Approximately 1 in 10,000 Caucasians are born withPKU, making PAH deficiency among the most commonof the autosomal recessive inborn errors of metabolism[14]. Current treatment for PKU requires patients toadhere to a restricted semi-synthetic diet low in the aminoacid Phe for life [15]. This dietary therapy is difficult tomaintain [16] and does not always eliminate the damagingneurological effects that can be caused by elevated Phelevels [17]. In addition, less than ideal dietary controlduring pregnancy can lead to birth defects [14,16]. There-fore, an alternative treatment is needed to accompany thecurrent dietary regimen and prevent the neurologicaldamage inflicted on those individuals with PKU, particu-larly for those patients with the most severe forms of thedisorder.

PAL is an appealing alternative treatment of PKU[8,18–22]. Both pharmacological and physiological proofsof principle have been established using recombinantPAL for enzyme substitution therapy in orthologousmutant mouse strains with hyperphenylalanemia [17,20].PAL is a robust autocatalytic protein without need for acofactor, an enzyme that stoichiometrically converts Pheto trans-cinnamic acid and ammonia by non-oxidativedeamination. The trans-cinnamic acid that would be gener-ated with complete dietary Phe conversion by PAL is pre-dicted to be harmless to both PKU and normal individuals,and is excreted as hippurate in urine [23] along with smallamounts of cinnamic and benzoic acids [24]. The ammoniaformed would be metabolically insignificant. Previous stud-ies [20] demonstrated that intraperitoneal injections ofunmodified PAL lowered blood Phe levels in the PKUmouse model, but repeated injections led to immunogenicreductions in efficacy. Recently, we reported the successin suppressing the immunogenicity of PAL from yeast Rho-

dosporidium toruloides using the administration of a modi-fied PAL protein conjugated with a 20 kDa linearpolyethylene glycol (PEG) [8]. Among different formula-tions, the 1:16 (PAL:PEG) formulation displayed barelydetectable in vivo immunogenicity 42 days after repeatedPEG–PAL challenge, while retaining near full enzymatic

activity towards Phe, suggestive of its possible use as atherapeutic treatment for PKU. PEGylated formulationsat lower ratios did not lead to significant lowering of theimmune response [8]. Further development of a therapeuticdrug using structure-based approaches is now possible withthe availability of the recently determined high-resolutionPAL crystal structures (Fig. 1) [22,25,26]. With thesehigh-resolution three-dimensional protein crystal struc-tures, protein engineering methods can be applied toimprove the in vivo effectiveness of PAL [8,22]. Identifica-tion of the regions within the native structure that are themost flexible allows engineering of a more compact andstable form of the protein; residues located near the activesite can be mutated to enhance activity and/or substratespecificity; and surface locations close to sensitive immuno-genic and/or proteolytic sites can be mutated to disrupt thesensitivity. Alternatively, surface PEGylation or otherchemical derivatization can be applied to mask the sites ofsensitivity. The PAL protein is a homotetramer (Fig. 1),

A. Gamez et al. / Molecular Genetics and Metabolism 91 (2007) 325–334 327

with each monomer consisting of mainly alpha-helicesand subdivided into three domains—a central catalyticdomain, an N-terminal domain similar to the histidineammonia-lyase (HAL) structure [27], and a C-terminaldomain that protrudes from the ends of the intact tetramermolecule. The active site contains the important electro-philic group, 4-methylidene-imidazole-5-one (MIO),formed auto-catalytically by cyclization and dehydrationof a tri-peptide Ala-Ser-Gly [22,25–27], that helps theenzyme achieve the unusual non-oxidative deaminationreaction.

Information about the epitopes of proteins recognizedby antibodies is important for their use as biological tools.This article describes the identification of the B and T cellepitopes on the PAL protein. Further, we determinedwhich lysine residues were readily PEGylated, which ofthe PEGylation sites were in a position where their cova-lent modification could potentially effect a reduction inthe immune response, and which other sites might bemutated to lysine and further PEGylated in order to pro-tect the protein.

These computational and experimental studies to iden-tify sensitive epitope sites provide a possible basis forunderstanding the results of our previously reportedPEGylation experiments, as well as for engineering themolecule to an improved therapeutic form [8]. PEG mod-ification sites have now been identified using mass spec-troscopy, and the results have been mapped onto tothe three-dimensional protein structure, thus, providinga unique opportunity to assess the susceptibility andavailability of each lysine residue to modification.

Protein and peptide therapeutics are becoming increas-ingly attractive options in the treatment of disease, buttheir utility can be limited by therapy-induced immuneresponses. By combining complementary approaches,using consensus scoring to map the potential immunogenicregions of a molecule of interest, in this case PAL, it maybe possible to significantly improve therapeutic efficacyfor treatment of PKU using rational engineering strategies[8,20].

Materials and methods

PAL expression, purification and PEGylation

The Escherichia coli expression plasmid containing full-length Rhodos-

poridium toruloides PAL coding sequence has been previously reported [20]as well as the protein purification and activity assay protocols [22]. PAL–PEG conjugates were produced by coupling linear 20 kDa methoxy-PEG-SPA (mPEG-SPA, Nektar Therapeutics) to PAL using an establishedreaction protocol [8].

Prediction of B cell epitopes using antigenicity scales

Using the Peptide companion program (http://www.5z.com/csps/index.html) from CSPS Pharmaceuticals Inc. on the 716 amino acid R. tor-

uloides PAL sequence (GenBank Accession Number X51513), epitopesthat were likely to elicit a B cell response were determined based on anti-genicity parameters [28].

Peptide scan for antibody binding

Experimental epitope mapping was carried out using PepSpots� tech-nology [29] by JPT Peptide Technologies GmbH (www.jpt.com), a subsidi-ary of Jerini AG. To cover all possible epitopes within the 716-amino acidR. toruloides PAL sequence, an overlapping peptide library was synthe-sized on a single membrane (353 13-mer PAL peptides with 11 amino-acidoverlap) [30,31]. Synthesis was initiated by covalent binding of C-terminalamino acids for each peptide to a membrane solid support in a definedarray, followed by solid-phase synthesis of the remaining 12 amino acids.All cysteine residues were replaced with serine and all N-termini were acet-ylated. The mapping experiments are divided into two parts; first, a nega-tive-control incubation with a horseradish peroxidase (HRP)-labeled anti-mouse IgG secondary antibody (Sigma) to evaluate nonspecific binding,and then the test incubation performed with purified total mouse IgGraised against normal recombinant PAL protein, followed by the HRP-labeled secondary antibody. The detection of HRP was performed bythe Chemiluminescent Blotting Substrate (Roche). The data were analyzedwith an imaging system showing the chemiluminescence signal intensity(Boehringer Light units, BLU) as single measurements for each peptide.The negative-control incubation step did not exhibit any nonspecific bind-ing to the conjugated secondary antibody.

Prediction of T cell epitopes with EpiMatrix�

T cell epitopes were predicted using EpiVax tools and methodology(www.epivax.com). The 716 amino acid R. toruloides PAL sequence wasparsed into 708 overlapping 9-mer frames where each frame overlapsthe last by 8 amino acids. Each of the derived 9-mer frames were thenscreened for predicted affinity against a panel of 8 representative MHCClass II alleles using the EpiMatrix� program and the data were normal-ized to results of random sequences [32]. The resulting EpiMatrix�Z-scores fall on a common scale that can be directly compared acrossHLA alleles. Z-scores above 1.64 (approximately the top 5% of all 9-mersderived from any given protein) are expected to have a significant chanceof binding to MHC molecules and scores above 2.32 (approximately thetop 1% of all 9-mers derived from any given protein) are highly likely tobind to MHC molecules. For the purposes of the analysis all EpiMatrix�Z-Scores equal to or above 1.64 were defined as ‘‘Hits’’ (potentially immu-nogenic regions requiring further investigation). Each frame was analyzedagainst a panel of eight common MHC Class II HLA alleles that are rep-resentative of the human population. In total 5664 assessments were per-formed; from among these, 303 potential epitopes or ‘‘hits’’ wereidentified, representing approximately 5% of the sample.

A search was also made of regions of the protein where high-scoringepitopes clustered as these regions have been shown to be relevant to stud-ies attempting to reduce immunogenicity. Previous studies have found thatpotential T cell epitopes may not always be randomly distributed through-out protein sequences, but instead could tend to clump together [33].These regions of unusually high potential are referred to as ‘‘T cell epitopeclusters’’. Clusters range from 9 to roughly 25 amino acids in length and,considering their affinity to multiple alleles and across multiple frames, cancontain anywhere from 4 to 40 binding motifs.

Identification of PEGylated lysines by mass spectroscopy

Samples of 1:4, 1:8, and 1:16 20 kDa linear PEGylated PAL and non-PEGylated control PAL (50 lg in 100 ll reaction volume) were digestedwith 2.5 lg of trypsin (Promega) in 50 mM Tris, pH 8.2 buffer containing1 mM CaCl2 and 1 M urea (final). The samples were incubated overnight(�20 h) at 37 �C. Tryptic digests were then reduced by adding 2 ll of a1 M DTT stock solution in H2O. The samples were incubated an addi-tional 30 min at 37 �C. Afterwards the reaction was quenched with theaddition of 6 ll 15% TFA and stored at �20 �C overnight. The trypticdigest (98 ll) was then injected onto a C18-RP column (Phenomenex;250 mm · 2 mm; 00G-4396-B0) using an Agilent 1100 Series HPLC sys-tem, equipped with an electrospray ionization source single quadrupole

328 A. Gamez et al. / Molecular Genetics and Metabolism 91 (2007) 325–334

mass detector (ESI-MSD). Peptides were eluted by increasing the acetoni-trile in a water/acetonitrile/0.1% formic acid mobile phase. Percent PEGy-lation was determined by differential peptide mapping calculations basedon the reduction in mass quantity of the unPEGylated parent peptide.Quantification by mass spectrometry was done manually by extractingthe calculated m/z species, deconvoluting the mass, and reporting the totalion abundance at the peak. Since trypsin no longer recognizes lysines mod-ified by PEG the penultimate peptide also decreases in abundance afterPEGylation. In some cases, where the lysine containing peptide was toosmall for mass spectroscopy analysis, the penultimate peptide was usedto calculate the percent PEGylation. In other cases, both sets of data wereused, and the data were averaged. The data set was protein normalized tono fewer than three non-lysine containing tryptic peptides. Masses of theactual PEGylated peptides could not be obtained by ESI-MSD. Mass specdata were considered significant when the total ion chromatogram signalwas higher than 20 k. The coefficient of variability in this assay is 20%.

Table 1aPredicted and experimental epitopes of PAL

Method B cell epitopemapping

B cell epitopemapping

T cell epitopemapping

Bionexus Jerini EpiVax

Approach Computationalpredictions based onhydrophilicity andantigenicity

Experimentalanti-PALantibodybinding to PAL

Computationalpredictions forbinding toMHC II

Results and discussion

To study potential improvements in use of the non-human PAL protein for treatment of PKU, three epitopemapping approaches were used in this work. The firstmethod employed the computational Peptide companionprogram from CSPS Pharmaceuticals, which predicts B cellantigenicity and hydrophilicity of proteins depending uponthe chemical composition and conformation of the protein[28]. The second method involved experimental B cell epi-tope mapping with a custom PepSpots� peptide arrayfrom JPT Peptide Technologies, a subsidiary of JeriniAG [30,31]. The third method used computational humanT cell epitope mapping with the EpiMatrix� system fromEpiVax, Inc. to map the protein sequence for MHC ClassII-associated T cell epitopes [33]. Based on existing dat-abases of MHC Class II sequence binding motifs, EpiMa-trix� predicts which sequences have an increasedprobability of binding and subsequent presentation onantigen-presenting cells of the immune system for the eightmost abundant MHC Class II alleles carried in the generalhuman population.

parameters peptides

Technology Peptide companion PepSpot� arrays EpiMatrix�

Residues 70–7895–112

147–157b

162–184180–202

226–243a

307–317b

337–356a 354–376c

396–413517–542541–559

569–589 571–596c

597–601b

619–636 616–638c

654–677

a Sequence regions with higher scores in the B cell predicted epitopemapping.

b Sequence regions by B cell experimental epitope mapping using anti-PAL antibodies. The 307–317 and 597–601 regions are located inside thePAL structure.

c Immunodominant T cell epitopes with EpiVax� scores above 20.

B cell epitopes mapping

Based on the hypothesis that antigenic determinants aresurface-located and often contain charged and polar resi-dues, a computational antigenicity scale was applied toPAL to predict its B cell epitopes using the Peptide com-panion program (CSPS Pharmaceuticals). This programpredicts B cell epitopes by identifying hydrophilic patchesin the PAL sequence, as these are expected to be surfaceaccessible, and then calculates the predicted antigenicityusing the Willing’s antigenicity index [28]. This index pro-vides a value for each amino acid based on a database ofoccurrences of these amino acids in epitopes. The analysisis based on antigenicity and hydrophilicity predictions thatdetermine which parts of the protein are susceptible forantibody binding depending upon the chemical composi-tion and conformation of the protein. The Peptide com-panion program assigns a ‘‘hydrophilic index’’ to eachamino acid in a protein and then plots out a profile. It also

predicts the antigenicity of the protein. The predictions arenot sequential and use several parameters to assess antige-nicity. The results of this analysis mapped the followingPAL sequences as potentially immunogenic 70–78, 226–243, 337–356, 396–413, 569–589, 619–636 (Table 1a,Fig. 2a, and Fig. 3a), among which 226–243 and 337–356received the higher scores.

Peptide scan for antibody binding

Experimental identification of specific synthetic peptidesable to bind to polyclonal antibody raised in PAH-deficientmice against native PAL protein was performed using Pep-tide Scan technology. Fig. 2b shows which synthetic pep-tides exhibit the greatest binding to the mouse anti-PALIgG antibody. The most intense signals and the corre-sponding peptide sequences are listed in Table 1b. The anti-body binds most strongly to three major peptide clusters,which correspond to PAL amino acid positions 147–157,307–317, and 597–601. This in vitro test result indicates thatthe antibody exhibits sequence specificity in three differentregions of PAL (Table 1a). Based on the PAL structure(PDB code 1Y2M) [22], epitope 145–159 presents a mobileloop with average higher B factor (18.15) compared to thatof the whole molecule (12.65), indicating a flexibility featureof this region, which implies high risk of immunogenicity.

Fig. 2. (a) Bionexus cluster score from the Peptide companion programfor the PAL sequence. Calculated average antigenicity for PAL using asliding window of 15 amino acids. Shown are averages for which thecalculated average hydrophilicity of the sequence segment in the window isgreater than 0. Antigenicity values for each amino acids are taken fromWelling et al. [28]. while the hydrophilicity values are from Hopp et al.[36]. (b) Results of a PepSpots� analysis for PAL sequence. Peptides aregenerated from the PAL protein amino acid sequences as 13-mers with 11amino acid overlap. Numbers in labeled spikes represent the correspond-ing amino acid sequence in the protein (see Table 1a). (c) EpiMatrix�Cluster Score for the PAL sequence. In red ( ) are depicted the nine PALclusters with scores above 10 which have a significant potential forimmunogenicity. In order to improve the capture ratio the eight additionalframes with lower cluster scores (above 7) are also shown in blue forinformation purposes.

Table 1bPepSpots� peptides with higher scores in the B cell predicted epitopemapping

Peptide # Peptide amino acid sequence Corresponding residuenumber of amino acidsequence in PAL protein

73 SSFDSFRLGRGLE 147–15774 FDSFRLGRGLENS

153 VGHAGSFHPFLHD 307–317154 HAGSFHPFLHDVT

295 NSYDLVPRWHDAF 597–601296 YDLVPRWHDAFSF297 LVPRWHDAFSFAA298 PRWHDAFSFAAGT299 WHDAFSFAAGTVV

Overlapping PAL amino acid residues are marked bold.

A. Gamez et al. / Molecular Genetics and Metabolism 91 (2007) 325–334 329

Both epitope 305–319 and 588–609 have helix secondarystructure showing a rigid structural feature with similaraverage B factor as that of the whole molecule. The sidechain of residues Ser146, Phe147, Asp148, Ser149,

Arg151 and Glu157 in epitope 145–159, His312, Pro313,Asp317 and Val318 in epitope 305–319, Val594 andHis598 in epitope 588–609 are full or partially exposedon the surface of the molecule, based on PAL structureand most likely are involved in antibody recognition andbinding, as well as providing potential modification candi-dates for linear epitope removal.

Fig. 3a shows the position of the affected amino acid res-idues in the crystal structure of PAL and, for improvedvisualization, only subunit A of the complete molecule isshown. These regions are located on the interior of thethree-dimensional PAL protein structure, which suggeststhat they may not be recognized by the antibodies in thefolded state, but may be recognized in unfolded, proteo-lyzed, or aggregated PAL.

T cell epitopes mapping

An in silico immunogenicity evaluation of the PALamino-acid sequence for clusters of potential T cell epi-topes was conducted by the EpiMatrix� program devel-oped by EpiVax. The results (Fig. 4) illustrate how PALcompares with a set of different proteins previously ana-lyzed by the EpiMatrix� program. This figure plots, onthe left side of the scale, a series of proteins with low scoresknown to engender little to no immunogenicity, while theright side of the scale includes a number of known immu-nogens. EpiMatrix� considers proteins scoring above 10contain an unusually high number of potential T cell epi-topes and proteins scoring above 20 have a significantchance of engendering an immune response. Each of theknown immunogens listed scores above the threshold valueof 20. By testing a large number of randomly generatedprotein sequences, an expected value of zero is establishedwith a standard deviation of approximately seven. ForPAL, when all of the ‘‘hits’’ are summed and the lengthof the protein is considered, an overall immunogenicityscore is calculated as �2.93. This would indicate that thePAL protein contains an average level of immunogenicity.

Fig. 3. (a) Results of the epitope mapping by the three methods used in this study, Bionexus (left; red); Jerini (center; green); and EpiVax (right; blue).(b) Highlights the surface lysine residues of the PAL molecule.

Fig. 4. EpiVax� Cluster Summary in relation to other human proteins.

330 A. Gamez et al. / Molecular Genetics and Metabolism 91 (2007) 325–334

The epitope algorithm employed was configured to findimmunodominant T cell epitopes, which are sequences withstrong MHC affinity that can be presented in the context ofmore than one HLA (i.e. MHC Class II) molecule. TheEpiMatrix� ‘‘clustering’’ function determines from thedatabase the MHC binding potential of each 9-aminoacid-long snapshot (8 amino-acid overlap) for 8 different

human HLA genotypes, those that best represent thehuman population and can be used, therefore, to identifyregions of high-density clusters of potential immunodomi-nant MHC-binding sequences [25].

EpiVax epitope mapping identified nine clusters scoringabove 10 (Table 1 and Fig. 2c). Taken collectively, thesenine clusters contain 118 EpiMatrix� ‘‘hits’’ out of 303

A. Gamez et al. / Molecular Genetics and Metabolism 91 (2007) 325–334 331

‘‘hits’’ comprising the complete protein. A protein whichscores ‘‘average’’ or even ‘‘below average’’ on the overallEpiMatrix� scale may still create problems if it containsone or more clustered regions. In total, 39% of all EpiMa-trix� hits are contained in nine regions. These regions span124 9-mer frames, or 17% of the total frames analyzed. Thepreference is to locate at least 50% of the EpiMatrix�‘‘hits’’ within our target clusters for deimmunization. Inorder to improve our capture ratio, eight additional frameswith cluster scores above seven were included in the analy-sis. As a group, the eight additional clusters contain 51 hits,17% of the total. Combining the two groups gives 17 tar-gets covering 169 hits, 56% of the total depicted inFig. 2c which shows the locations of the 17 target regionswithin the complete linear PAL sequence. The Cluster sum-mary (Fig. 4) shows how these clusters stack up againstseveral immunodominant peptides. In order to ensure goodin vitro performance of the N- and C-terminal clusters,three amino acids to the N-terminal and three amino acidsto the C-terminal have been added to each of the clustersidentified. The cluster scores were calculated with and with-out the leading/trailing amino acids. The 9-mer frames con-tained in the PAL protein were screened against theEpiVax database of known MHC ligands and T cell epi-topes, and no significant homologies were found.

The only 9-mer frame sequences that show homology tohuman sequences are peptide 109–117, 611–619 and 654–622 (Fig. 3a), which may cause autoimmune reaction bybreaking immune tolerance. These results suggest that thePAL sequence presents a limited homology with the humangenome containing an average level of immunogenicity,which represents an advantage for the further developmentof the PAL protein as a potential human therapeutic agentto treat PKU.

Correlation of the three epitope mapping results and

conclusions

The results obtained by computational and experimentalmapping of the B cell epitopes of PAL were conflicting inseveral regions for the sequences identified by the antige-nicity index of the Peptide companion program and theantibody-binding regions mapped using PepSpots� tech-nology. Antibodies, as effectors of the humoral immuneresponse, are often directed at three-dimensional epitopeson the surface of the protein. Thus, whole proteins in theirnative conformation may be required to stimulate thehumoral immune response. As a result, B cell epitope map-ping cannot be predicted accurately solely by experimentalmapping of linear epitopes or by bioinformatics toolsbecause of the three-dimensional interaction between theantibody molecule and its epitope. Moreover, it is impor-tant to note that production of the final humoral antibodyis dependent upon early events in T cell development.These involve antigen presentation of small peptides (9–15 amino acids) on cell-surface MHC Class II molecules,followed by binding and activation of certain T cells carry-

ing sequence-specific T cell receptors. Then, activationleads to T cell replication and further B cell developmentof a plasma cell lineage secreting a specific antibody. Aftera plasma cell lineage has been established, additional expo-sure to the protein stimulates (boosts) further antibodyproduction by means of a ‘‘memory B cell’’ responseinvolving recognition of antigen-antibody complexes byFc receptors. Therefore, although the primary immuneresponse is dependent upon the interaction of small peptidefragments with MHC, a continuing memory B cellresponse is dependent upon the interaction of the final anti-body with the entire folded protein, for which the so-called‘‘B cell epitopes’’, both linear and conformational, areimportant.

The computational predictions of a B cell immuneresponse were intended to map the conformational epi-topes created in the protein once it is folded. These predic-tions were based on antigenicity of surface polar andcharged exposed residues, and on chemical compositionand conformation of the protein, which allows a mappingfor the protein orientation in its natural environment. Inorder to map these linear epitopes, PepSpots� technologywas applied to PAL using mouse anti-PAL antibodies andsynthetic PAL peptides. It is interesting to note that twoout of three of these peptides, experimentally identified asbinding strongly to antibody, are located inside the PALstructure and are likely not part of any conformational epi-topes. This might partly explain the different results of thetwo B cell mapping approaches.

By contrast, T cell computational epitope predictionshave proven to be a highly effective means of identifyingT cell epitopes from primary protein sequences. Short pep-tides proteolytically processed from the foreign protein inantigen-presenting cells are required to stimulate a primaryT cell response. T cell epitope predictions are of value andare currently used in many preclinical studies of vaccinesand protein therapeutics.

The information obtained by these different approachesis likely to be very useful for the development of a PAL-based therapeutic drug in conjunction with rational proteinengineering methods and design, followed by further test-ing in vitro and in vivo. The results show that both B celland T cell epitopes can be identified in the PAL protein.In silico T cell epitope mapping predicts that the PAL mol-ecule should have an average level of immunogenicity inhumans. Yet, PAL does contain some regions of higherimmunogenic potential, and these could be modified toreduce the likelihood for an immune reaction of the proteinto the same level as other, safe, less immunogenic proteins.This process would allow us to achieve a better proteintherapeutic molecule suitable for treatment of PKU. Over-all, the purpose of epitope identification efforts is to gener-ate a rational deimmunization PAL engineering strategy.This strategy will be implemented in order to protect, byprotein modification (as PEGylation), the most immuno-genic regions of PAL, especially those regions that arenot already covered by the surface lysines and are not

Table 2Site specific % PEGylation of PAL

Position (1:4) %PEGylation

(1:8) %PEGylation

(1:16) %PEGylation

(n-term)a 91 99 100K20a 70 90 97K50a 0 0 0K78b

K85 15 17 34K93b

K96 26 35 47K132 46 60 74K232b

K240 28 38 80K242a 0 0 0K263 4 1 2K334 50 27 29K350a 35 44 56K352a 32 38 48K405a 4 15 41K424 38 34 51K434 14 4 0K468 0 0 0K545 0 0 0K546 33 46 52K576 15 9 28K579 54 63 77K583 0 0 0K626 0 0 0K676 0 0 0K686 42 49 73K697 48 68 78K703 28 2 8K713 0 0 0

The identification of the PEGylated lysine residues was performed by massspectroscopy after tryptic digestion and location of the lysine residues inthe PAL crystal structure. This table shows sites that get >20%PEGylated.

a Residues that showed ambiguous patterns due to the proximity ofadjacent or close lysine residues. This effect is statistically insignificant andis shown for trend purposes only. This effect is common in tryptic peptidesthat are flanked by multiple lysines.

b Residues that produced inconsistent measurements.

Fig. 5. Plot of the percent of PEGylation of the PAL lysine residues forthree different PAL:PEG ratios (1:4, 1:8, 1:16). The three major B cellepitope regions identified experimentally, 147–157, 307–317 and 597–601.

332 A. Gamez et al. / Molecular Genetics and Metabolism 91 (2007) 325–334

already PEGylated as determined in mass spectroscopystudies. Further, it is important to consider the recentlydescribed PEG immunogenicity, which could be a potentialproblem in humans as reported in studies of patients trea-

ted with PEG-uricase [34], even when PEG-specific IgGantibodies in humans are very rarely seen [35]. This pointwill be further investigated in the next cycle of our in vivo

research. In the design of a new protein therapeutic drugit is necessary to consider several key aspects that shouldbe accomplished once the route of administration is estab-lished. Among these factors are the effects on activity,immunogenicity, and pharmacokinetic and pharmacody-namic properties. The strategy described here will ulti-mately be tested in vivo to demonstrate that a PEGylatedPAL mutant developed using the information of thesecombinatorial approaches is enzymatically active and lessimmunogenic after repeated administration than thePEGylated wild-type PAL and the non-PEGylated PAL.

Identification of PEG substitution sites

In order to direct PEGylation toward the most immuno-genic parts of the PAL protein, identification of the PEGy-lated lysine residues at various ratios was performed todetermine whether the identified epitopes of PAL were cov-ered by this covalent modification. The PAL monomersequence contains 29 Lys residues. Based on the PAL tetra-mer structure, each monomer presents 10 surface lysines(K78, K96, K240, K405, K545, K546, K579, K676,K686, K713; Fig. 3b) and 3 disordered lysines (K20,K350, K352) that are directly available for PEGylation,and another 12 partially exposed lysines (K50, K85, K93,K132, K232, K242, K334, K468, K576, K583, K697,K703) which are also potential candidates for PEGylationmodification (Fig. 3b) [22]. Based on the structure, K424 isburied and the nitrogen atom in its amino group can formthree hydrogen bonds with waters and other residues, so itshould not be available for PEGylation. However, the sidechain of K424 has a higher B factor and the steric hinder-ance that prevents it from solvent access is the N-terminalloop, which is flexible and may move around. Therefore, atcertain buffer conditions, K424 might be available forPEGylation, as the mass spec PEGylation data suggests(Table 2). In the process of identifying PEGylated lysineresidues by mass spectrometry, when two residues are men-tioned (e.g. K350/K352) this is because the sensitivity ofthe method could not distinguish between the two (i.e.either or both residues may be PEGylated) due to a closeproximity in the sequence. For therapeutic purposes thisclose proximity is not relevant since we pursued a modifica-tion method to assure that the protein will be regionallycovered by a certain spatial ratio provided by the attachedPEG molecules. Table 2 summarizes the results of the site-specific percent of PEGylation for the different PAL–PEGspecies produced with input reaction molar ratios (lysinesto PEG) of 1:4, 1:8 and 1:16. Interestingly, the N-terminusof the protein appears to be PEGylated quite easily, most

A. Gamez et al. / Molecular Genetics and Metabolism 91 (2007) 325–334 333

likely due to its open access to lysine residues. By compar-ing the increase of PEGylation attained when the PAL:-PEG ratio increased for a given residue, we observed anexpected tendency that corroborates the mechanism ofthe PEGylation reaction to be directed toward the mostaccessible lysine residues first (Fig. 5). This observationdemonstrates the possibility of directing the reaction withthe most accessible residues and/or mutation of other resi-dues located on the surface of the protein to lysine. Fromthe mass spectrometric identification results of PEGylatedlysine residues, we can conclude that specific residues(e.g. K20, K132, K240, K579, K686, K697) are more sus-ceptible to modification due to their accessibility (Fig. 5).

Correlation between epitope mapping results and PEGylatedlysines in PAL molecule

From the combination of the complementary epitopemapping techniques and the PEGylation identificationexperiments it is possible to predict which regions in PALcould potentially benefit from further engineering, andwhich do not require further modification since they willnot be easily recognized by the immunological machinery.The uniform coverage of the protein by PEG will help tomitigate an immune response, even in areas of the proteinthat do not appear to be epitopes by these experimentaland computational approaches (Fig. 3a). The identifiedimmunogenic regions of PAL could be covered through aprocess of site-specific mutagenesis to introduce additionallysine residues followed by PEGylation (if they are on thesurface of the protein).

From the Peptide companion analysis, the regions withhigher scores for B cell epitopes are for amino acids 226–243 and 337–356. Within these sequences, the first onehas as a closest linear lysine the residue 240, which is highlyPEGylated; the second region showed a PEG coverage pat-tern by mass spectrometry. The B cell epitopes identified byanti-PAL antibodies are 147–157, 307–317 and 597–601.The two last sequences are located inside the PAL struc-ture; however, some of the residues are partially exposedfor the region 307–317. The sequence 597–601 did not con-tain any lysine in the native PAL protein and, therefore,was not PEGylated. The immunodominant T cell epitopeswith higher EpiMatrix� scores are 354–376, 571–596 and616–638. From these three regions only the one comprisedbetween the residues 616–638 showed no PEGylation pat-tern in the species tested.

The availability of the PAL crystal structure, identifica-tion of B and T cell epitope regions, and the location of thePEG molecules attached to the lysine amino groups havebeen useful tools in understanding the in vivo results inthe PKU mice model we previously reported [8]. Thoseresults established the success of the PAL–PEG species at1:16 ratio (PAL:PEG), which strongly reduced in vivo

immunogenicity after repeated challenge, yet retained fullenzymatic activity against Phe, thus demonstrating poten-tial as a PKU therapeutic. In conclusion, the prospect of

successful PKU treatment with PAL or PAL–PEG hasbeen enhanced greatly by combining different approachesto analyses of structure, function, and immunogenicity,leading to development of a safer and more effective thera-peutic enzyme.

Acknowledgments

The authors thank Angela Walker for her assistancewith manuscript preparation, Holly Archer and MaryStraub for excellent technical support and Jeremiah S. Jo-seph and Kumar Saikatendu for figures creation. We alsowant to thank Bionexus and EpiVax for the help on thecomputational epitope mapping predictions. We are grate-ful to the Michaux Family for funding the Tia Piziali Fel-lowship (A.G.) and to the Piziali Family Foundation foradditional financial support. Funding from the Michauxand Piziali families and MACPAD has been critical tothe advancement of our PKU program.

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