of March 4, 2016.This information is current as

AntibodyLess Immunogenic Humanized MonoclonalEssential for Ligand Contact to Engineer a Containing Specificity-Determining ResiduesComplementarity-Determining Regions Grafting of ''Abbreviated''

and Syed V. S. KashmiriSantos, Mariateresa Giuliano, Peter Schuck, Jeffrey SchlomEduardo A. Padlan, Noreen R. Gonzales, Ameurfina D. Roberto De Pascalis, Makoto Iwahashi, Midori Tamura,

http://www.jimmunol.org/content/169/6/3076doi: 10.4049/jimmunol.169.6.3076

2002; 169:3076-3084; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/169/6/3076.full#ref-list-1

, 17 of which you can access for free at: cites 55 articlesThis article

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2002 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

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Grafting of “Abbreviated” Complementarity-DeterminingRegions Containing Specificity-Determining Residues Essentialfor Ligand Contact to Engineer a Less ImmunogenicHumanized Monoclonal Antibody

Roberto De Pascalis,* Makoto Iwahashi,1* Midori Tamura, 2* Eduardo A. Padlan,3†

Noreen R. Gonzales,* Ameurfina D. Santos,4† Mariateresa Giuliano,5* Peter Schuck,‡

Jeffrey Schlom,6* and Syed V. S. Kashmiri*

Murine mAb COL-1 reacts with carcinoembryonic Ag (CEA), expressed on a wide range of human carcinomas. In preclinicalstudies in animals and clinical trials in patients, murine COL-1 showed excellent tumor localization. To circumvent the problemof immunogenicity of the murine Ab in patients, a humanized COL-1 (HuCOL-1) was generated by grafting the complementarity-determining regions (CDRs) of COL-1 onto the frameworks of the variable light and variable heavy regions of human mAbs. Tominimize anti-V region responses, a variant of HuCOL-1 was generated by grafting onto the human frameworks only the “ab-breviated” CDRs, the stretches of CDR residues that contain the specificity-determining residues that are essential for the surfacecomplementarity of the Ab and its ligand. In competition RIAs, the recombinant variant completely inhibited the binding ofradiolabeled murine and humanized COL-1 to CEA. The HuCOL-1 and its variant showed no difference in their binding abilityto the CEA expressed on the surface of a CEA-transduced tumor cell line. Compared with HuCOL-1, the HuCOL-1 variantshowed lower reactivity to patients’ sera carrying anti-V region Abs to COL-1. The final variant of the HuCOL-1, which retainsits Ag-binding reactivity and shows significantly lower serum reactivity than that of the parental Ab, can serve as a prototype forthe development of a potentially useful clinical reagent. The Journal of Immunology, 2002, 169: 3076–3084.

T he mAbs against tumor Ag hold promise for diagnosisand therapy of human cancers (reviewed in Refs. 1–3). Amajor impediment to the clinical use of murine mAbs is

the human anti-murine Ab (HAMA)7 response these mAbs elicit inpatients (4–7). To obviate the potential HAMA response, human-ized Abs have been developed by grafting the complementarity-

determining regions (CDRs) of the murine Ab onto the frame-works of the variable light (VL) and variable heavy (VH) regionsof human mAbs (reviewed in Ref. 8). Humanization of a xeno-geneic Ab, however, does not necessarily eliminate the immuno-genicity of the molecule, because the humanized molecule canevoke anti-V region response (7, 9–13). It has been proposed thatbinding of an Ab to an Ag involves only a small number of resi-dues within the CDRs. The latter residues have been designated asspecificity-determining residues (SDRs) (14). In an attempt tocircumvent anti-V region response in patients, a murine Ab hasbeen humanized by grafting only its SDRs onto human frame-works (13).

The carcinoembryonic Ag (CEA), a cell surface glycoprotein of180-kDa molecular mass, is one of the most widely used human tu-mor markers. It is expressed at high levels in the embryonic and fetaldigestive epithelial tissue and, to a lesser extent, in normal adult colonand stomach epithelium (15). CEA is overexpressed in�95% of gas-trointestinal and pancreatic cancers, as well as in most non-small-celllung carcinomas. Also, it is expressed in breast carcinoma and squa-mous cell carcinoma of the head and neck (reviewed in Refs. 11 and16). A number of murine anti-CEA mAbs have been generated (17)and used for measuring CEA levels in blood (18) and for immuno-histopathology of tissues from cancer patients (11, 19). In addition,Abs directed to CEA were among the first to be used in clinical trialsto successfully localize tumors (20–23). Also, several clinical trialshave been conducted to evaluate the anti-tumor activity of anti-CEAmAbs (24–27). CEA has some degree of cross-reactivity with severalproteins, including nonspecific cross-reacting Ag-1 (NCA-1), normalfecal Ag-1, and the NCA-related proteins present in human granulo-cytes (17).

*Laboratory of Tumor Immunology and Biology, Center for Cancer Research, Na-tional Cancer Institute,†Laboratory of Molecular Biology, National Institute of Di-abetes and Digestive and Kidney Diseases, and‡Division of Bioengineering andPhysical Sciences, Office of Research Services, Office of the Director, National In-stitutes of Health, Bethesda, MD 20892

Received for publication April 12, 2002. Accepted for publication July 3, 2002.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 Current address: Wakayama Medical School, 27 Shichibancho, Wakayama 640, Japan.2 Current address: Tohoku University School of Medicine, 1-1 Seiro-Machi, Aoba-Ku, Sendai 980-77, Japan.3 Current address: Marine Science Institute, College of Science, University of thePhilippines, Diliman, Quezon City, Philippines.4 Current address: Institute of Molecular Biology and Biotechnology, College of Sci-ence, University of the Philippines, Diliman, 1101 Quezon City, Philippines.5 Current address: Dipartimento Medicina Sperimentale, Sezione di Biotecnologie eBiologia Molecolare, Facolta’ di Medicina e Chirurgia, Seconda Universita’ degliStudi di Napoli, Via Costantinopoli 16, 80128 Naples, Italy.6 Address correspondence and reprint requests to Dr. Jeffrey Schlom, Laboratory ofTumor Immunology and Biology, Center for Cancer Research, National Cancer In-stitute, National Institutes of Health, Bethesda, MD 20892. E-mail address:js141c@nih.gov7 Abbreviations used in this paper: HAMA, human anti-murine Ab; CDR, comple-mentarity-determining region; SDR, specificity-determining residue; CEA, carcino-embryonic Ag; NCA, nonspecific cross-reacting Ag; m, murine; c, chimeric; Hu,humanized; pBSc, pBluescript II S/K�; SPR, surface plasmon resonance.

The Journal of Immunology

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The mAb COL-1, an IgG2a, has a high affinity for CEA and hasno detectable reactivity to granulocytes or to the CEA-related AgsNCA-1 and normal fecal Ag-1 (28). Murine COL-1 (mCOL-1)reacts with biopsies of colon and gastric carcinomas (80 and 88%,respectively) and with biopsies of human mammary and non-small-cell lung carcinomas (17, 29). Except for some reactivity toskin and gastric and colon mucosa, mCOL-1 does not react withnormal tissues (17). In preclinical studies, 125I-labeled mCOL-1achieved good tumor radiolocalization using LS-174T colon car-cinoma xenograft in athymic mice (30). In patients, 131I-labeledmCOL-1 showed good tumor targeting of human gastrointestinalcarcinomas in a phase I clinical trial (24). Not unexpectedly,mCOL-1 was found to induce HAMA response in patients (24,31). Yu et al. (24) reported that 61 and 83% of patients treated withthe Ab developed elevated HAMA levels by days 20 and 40, re-spectively. It was observed by Meredith et al. (31) that 93% of thepatients developed Ig response, with most patients developingHAMA by day 14 and some as early as days 7–11 after mCOL-1administration. The peak of Ab response generally occurred at4–6 wk after exposure. An attempt has now been undertaken toreduce the immunogenicity of the mCOL-1 mAb by progressivelyreducing its murine content by genetic manipulation. This reportdescribes the development and characterization of a mouse-humanchimeric COL-1 (cCOL-1) mAb, a humanized COL-1 (HuCOL-1)mAb, and variants of HuCOL-1. A final variant of HuCOL-1 wasdeveloped by a previously suggested humanization protocol (14)based on transplantation of “abbreviated” xenogeneic CDRs ontothe human Ab frameworks. This variant, which retains significantAg-binding reactivity, is minimally reactive to sera from patientswho were earlier administered mCOL-1 during clinical trials.

Materials and MethodsSynthetic oligonucleotides

The long overlapping oligomers and oligonucleotide primers used for DNAamplifications were supplied by Lofstrand Labs (Gaithersburg, MD) and Mid-land Certified Reagent (Midland, TX). The sequences of the four primers that

were used to generate DNA fragments encoding the VH and VL domains of themCOL-1 mAb were as follows: 1) 5� VH: 5�-AGTAAGCTTCCACCATGGAGTGGTCCTGGGTCTTCCTCTTCTTCCTGTCCGTGACTACTGGAGTGCACTCCGAGGTTCAGCTGCAGCA-3�; 2) 3� VH: 5�-CGATGGGCCCGTAGTTTTGGCAGAGGAGACGGCGACCG-3�; 3) 5� VL: 5�-TAGCAAGCTTCCACCATGGATAGCCAGGCCCAGGTGCTCATGCTCCTGCTGCTGTGGGTGAGCGGCACATGCGGCGACATTGTGCTGACACA-3�; 4)and 3� VL: 5�-TGCAGCCGCGGCCCGTTTGATTTCCAGCTTGG-3�.

Each of the 5� primers carries a HindIII site followed by a sequenceencoding a signal peptide. The 3� VH primer carries an ApaI, whereas the3� VL primer has a SacII site. The four 119- to 133-bp-long oligonucleo-tides that were used to generate each of the VH and VL genes of HuCOL-1are shown by long arrows in Fig. 1. The sequence of the 20- to 21-bp-longend primers used for DNA amplification were as follows: 5) 5� VH: 5�-CGTAAGCTTCCACCATGGAG-3�; 6) 3� VH: 5�-TGGGCCCTTGGTGGAGGCTGA-3�; 7) 5� VL: 5�-GCAAGCTTCCACCATGGATA-3�; and 8)3� VL: 5�-TGCAGCCGCGGTACGTTTGAT-3�.

The 5� primers (nos. 5 and 7) carry a HindIII site. Whereas a site for ApaIhas been incorporated in the 3� VH primer (no. 6), the sequence for the SacIIsite has been included in the 3� VL primer (no. 8). The sequences of twoadditional mutagenic primers (supplied by Milligen/Bioresearch, Burlington,VT) that were used for the generation and amplification of the genes encodingthe V domains of HuCOL-1 variants were as follows: 9) 3� VH: 5�-TGCCCTGGAACTTCTGGGCATATTCAGTA-3�; and 10) 3� VL: 5�-GCACTGACACTTTGGCTGGACTTGCAGTTGATGGTGGCCCCTC-3�.

The sequences recognized by the restriction endonucleases are in italics,and the mutagenic changes are underlined.

DNA amplification

All PCRs were conducted in a final volume of 100 �l of PCR buffercontaining 200 �M dNTPs, 3 U of Taq polymerase (Boehringer Mann-heim, Indianapolis, IN), 0.2 �M each of the end primers, and 100 ng ofDNA template. Initial denaturation at 94°C for 2 min was followed by 30cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, andextension at 72°C for 1 min. It was followed by a final primer extensionstep at 72°C for 10 min. The VL and VH genes of the HuCOL-1 weresynthesized by the overlap extension PCR that has previously been de-scribed (32). Primer-induced mutagenesis was conducted by a dual-stepPCR as described by Landt et al. (33).

Expression vectors

Two different baculovirus transfer vectors, pAcUW51 (BD PharMingen,San Diego, CA) and pBAC4x-1 (Novagen, Madison, WI), have been used

FIGURE 1. Nucleotide sequence of the genes encoding the V regions of HuCOL-1 and their leader peptides. Nucleotide sequences of the humanizedVL (A) and VH (B) genes were generated and amplified by PCR amplification, using four overlapping synthetic oligonucleotides (indicated by arrows) thattogether encompass, on alternating strands, the entire sequence of each of the genes and its leader. Sequences on the flanks of the genes encoding thevariable region domains and their leader peptides are shown by lower case letters. The VL region (A) is comprised of nucleotides from positions 73–402,whereas the VH region starts from position 70 and ends at 441. The restriction enzyme sites incorporated in the oligomers to facilitate cloning are shownin italics.

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for generating the recombinant viruses and the subsequent coexpression ofthe Ig H and L chains. In pAcUW51 vector, one of the target genes can becloned at the BamHI site located downstream of the polh promoter,whereas the other gene can be driven by the p10 promoter by inserting thegene at either the BglII or the EcoRI site located 3� to the promoter. ThepBAC4x-1 baculovirus transfer plasmid contains two of each of the polhand p10 promoters, with a unique cloning site placed downstream of eachpromoter.

Assembly of the V and C region genes and generation ofexpression constructs

To generate constructs encoding the chimeric H and L chains of mAbCOL-1, the V region sequences of the H and L chain genes were PCRamplified using the phagemid constructs of the cDNAs encoding the Fdand the L chain of mCOL-1 as templates. Primers no. 3 and no. 4 were usedas forward and reverse primers, respectively, to amplify a 420-bp sequenceencoding the VL domain along with the signal peptide located upstream.The 3� primer was designed to extend the 3� end of the amplified sequenceto a unique SacII site located 10 bp downstream from the start of the human� C region. A DNA fragment encoding the human � C region was excisedfrom a pre-existing construct pLNCXHuCC49HuK (32) by SacII/ClaItreatment. The construct carried an EcoRI site immediately upstream ofClaI site. The V and the C regions of the L chain were joined to theHindIII/ClaI linearized pBluescript II S/K� (pBSc) plasmid (Stratagene,La Jolla, CA) by three-way ligation. Taking advantage of an EcoRI siteupstream of the HindIII site in pBSc, the entire L chain sequence was thenreleased from the construct by EcoRI digestion and inserted into the bac-ulovirus expression vector pAcUW51 at the EcoRI site located down-stream from the p10 promoter.

For the assembly of the chimeric H chain, a 460-bp sequence encodingthe VH domain and its leader peptide was PCR amplified using primers nos.1 and 2 as 5� and 3� primers, respectively. The design of the 3� primerfacilitated amplification of the VH sequence to extend to the ApaI sitelocated 17 bp downstream from the start of the CH1 domain. To assemblethe V and C regions, an ApaI/ClaI DNA fragment carrying the human �1C region was excised from a pre-existing construct pLgpCXHuCC49HuG1(32). The ApaI/ClaI fragment along with the 460-bp PCR product wasinserted into the HindIII/ClaI linearized pBSc. The DNA encoding theentire H chain was released by HindIII/ClaI treatment of the pBSc con-struct. The termini of the target DNA were filled in using Klenow frag-ments of the DNA polymerase, and the DNA fragment was subcloned inthe L chain construct of pAcUW51 at the blunt-ended BamHI site locateddownstream of the polh promoter.

DNA manipulations similar to those described for cCOL-1 were con-ducted to assemble the humanized and the variant V regions and theirrespective C regions into pBSc for the subsequent subcloning in baculo-virus expression vector pBAC4x-1. After joining the V regions of the Hand L chains to their respective C regions in pBSc, the assembled L chainof the HuCOL-1 or its variant was released from the pBSc construct andcloned at the EcoRI site, downstream from the p10 promoter. The entire Hchain of the HuCOL-1 or its variant was excised from its pBSc constructby HindIII/XhoI treatment, and it was cloned unidirectionally in the L chainconstruct of pBAC4x-1 at the HindIII/XhoI site, downstream of polh pro-moter. Three expression constructs were generated: one containing thevariant L chain and the parental humanized H chain, the second containingthe variant H chain and the parental humanized L chain, and the thirdcarrying variants of both the L and H chains (Table I).

Insect cell culture and production of recombinant Abs

Serum-free-adapted Sf9 insect cells (Life Technologies, Rockville, MD)were cultured at 27°C in Sf900-II medium (Life Technologies) with 50�g/ml gentamicin. To develop transfectomas secreting cCOL-1, insectcells were cotransfected with the pAcUW51-derived expression construct

and the linearized BaculoGold Baculovirus DNA (BD PharMingen).Transfectomas producing HuCOL-1 and its variants were generated bytransfecting insect cells with one of the pBAC4x-1-derived expression con-structs and the linearized BacVector2000 Baculovirus DNA (Novagen). Acationic liposome-mediated system (DOTAP; Boehringer Mannheim) wasused for all transfections. Harvesting of the recombinant virus, screeningfor Ig expression, and Ag binding by ELISA have previously been de-scribed (13).

ELISA

ELISAs were conducted by coating 96-well polyvinyl microtiter plateswith CEA (100 ng/well; Research Diagnostic, Flanders, NJ) or with Fc�-fragment-specific goat anti-human IgG (100 ng/well) (Jackson Immuno-Research Laboratories, West Grove, PA). Anti-human IgG or the CEA-coated plates were used to test for the production of Ig by the insect cellsor to assess its Ag reactivity, respectively. The details of the assay proce-dure have been reported earlier (34).

Purification of recombinant Abs

Three days after infection, the supernatants were collected and made freeof cellular debris and any contaminating proteins, before using protein Gagarose column (Life Technologies) to purify the desired protein as de-scribed earlier (13). The protein was concentrated using Centricon 30(Amicon, Beverly, MA) and dialyzed in PBS buffer using a Slide-A-Lyzercassette (Pierce, Rockford, IL). The protein concentration was determinedby the method of Lowry et al. (35), and the purity of the eluted proteins wasevaluated by SDS-PAGE under reducing and nonreducing conditions, us-ing precast 4–20% Tris-glycine gel (Novex, San Diego, CA). The proteinbands were visualized by Coomassie blue staining (Novex).

Competition RIA

The relative Ag binding of the mCOL-1 and the recombinant Abs derivedfrom it were determined using competition RIA. Twenty-five microliters ofserial dilutions of the Abs to be tested as well as the mCOL-1, prepared in1% BSA in PBS, were added to microtiter plates containing 200 ng of CEAsaturated with 5% BSA in PBS. 125I-labeled mCOL-1 or 125I-labeled Hu-COL-1 (100,000 cpm in 25 �l) was then added to each well. After anovernight incubation at 4°C, the plates were washed and counted in agamma-scintillation counter. The relative affinity constants were calculatedby a modification of the Scatchard method (36).

Flow cytometric analysis

A previously described method (37) has been used for FACS analysis. Toevaluate the ability of HuCOL-1 and its variants to bind to cell-surfaceCEA, 1 � 106 retrovirally transduced MC38 cells expressing CEA (38)were resuspended in cold Ca2�- and Mg2�-free Dulbecco’s PBS and in-cubated with the mCOL-1-derived Abs for 30 min on ice. A human IgGwas used as an isotype control. After one washing cycle, the cell suspen-sion was stained with FITC-conjugated mouse anti-human Ab (BD Phar-Mingen) for 30 min on ice. A second washing cycle was performed, andthen the samples were analyzed with a FACScan (BD Biosciences, Moun-tain View, CA) using CellQuest for Macintosh. Data from analysis of10,000 cells were obtained.

Immunoadsorption of patient serum and detection of anti-Vregion Abs

Stored patients’ sera, from a phase I clinical trial (24) that involved theadministration of 131I-labeled mCOL-1 to gastrointestinal carcinoma pa-tients, were used to assess serum reactivity of the mCOL-1-derived Abs.Several sera were tested for the presence of anti-V region Abs to mAbCOL-1. The sera, however, contain circulating Ag and anti-murine Fc Abs,which could interfere with the binding of mAb COL-1 and its derivative

Table I. Affinity constants of HuCOL-1 and its variants

mAbDesignation

L Chain VariableDomain

H Chain VariableDomain Manipulated CDR

PositionsSubstituteda Ka (�108 M�1)

HuCOL-1 Humanized Humanized – – 2.8224,25,27L Variant Humanized LCDR1 24, 25, 27 1.2061H Humanized Variant HCDR2 61 2.6424,25,27L/61H Variant Variant LCDR1/HCDR2 24, 25, 27/61 1.03

a Numbering convention of Kabat et al. (47).

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Abs to the anti-V region Abs. To circumvent this problem, the circulatingCEA and anti-murine Fc Abs were removed by sequential preadsorption ofthe sera with purified mCOL-6 and mCOL-4 mAbs, two Abs that react withepitopes of CEA different from the one recognized by mCOL-1 (28). ThemAb mCOL-4 has the same isotype as that of mCOL-1. For preadsorption,serum samples were added to mCOL-6 coupled to Reacti-gel according tothe method of Hearn et al. (39). The mixtures were incubated overnight at4°C with end-to-end rotation and were centrifuged at 1000 � g for 5 min.Preadsorption was repeated until the supernatants displayed no detectableanti-murine Fc activity. The procedure was then repeated using mCOL-4coupled to Reacti-gel. To detect anti-V region Abs by surface plasmonresonance (SPR), the preadsorbed serum was used as a mobile reactant.Proteins were immobilized on carboxymethylated dextran CM5 chips(BIAcore, Piscataway, NJ) by amine coupling using standard procedure(40, 41). HuCOL-1 was immobilized on the surface of flow cell 1, whereasthe surface of flow cell 2 was coated with an unrelated protein, rabbit �globulin (Bio-Rad, Hercules, CA).

Sera reactivity

The reactivity of COL-1 variants to anti-V region Abs was determinedusing a recently developed SPR-based competition assay (58). Competitionexperiments were performed at 25°C using a CM5 sensor chip containingeither mCOL-1 or HuCOL-1 in flow cell 1 and rabbit � globulin (Bio-Rad),as a reference, in flow cell 2. Typically, mCOL-1, HuCOL-1, or its variantswere used at different concentrations, to compete with the Ab immobilizedon the sensor chip for binding to serum anti-V region Abs. Patient’s serumwith or without the competitor (mCOL-1, HuCOL-1, or its variants) wasapplied across the sensor surface using a recently developed sample ap-plication technique (42) at the unidirectional flow of 1 �l/min. After thebinding was measured for 1000 s, the samples were washed from the sur-faces with running buffer using a flow rate of 100 �l/min, and the surfaceswere regenerated with 10 mM glycine (pH 2.0) for the HuCOL-1 sensorchip or with HCl (pH 2.3) for the mCOL-1 sensor chip. The percent bind-ing at each Ab concentration was calculated as follows: % binding �[slope of the signal obtained with competitor (serum � mCOL-1, Hu-COL-1, or HuCOL-1 variants)/slope of the signal obtained without com-petitor (serum only)] � 100. IC50 for each Ab, the concentration requiredfor 50% inhibition of the binding of the serum to either mCOL-1 or Hu-COL-1, was calculated.

ResultsIsolation of COL-1 H and L chain genes

The genes encoding the L chain and the Fd region of the H chainof mAb COL-1 were generated by repertoire cloning methodology(43), using synthetic oligonucleotides described in Materials andMethods. The PCR products of the appropriate size were cloned in

� phage vector, and phagemids carrying the target genes weresubsequently excised. The cloned genes were sequenced (data notshown) before the phagemids were used as templates for the sub-sequent PCR amplification.

Generation of genes encoding humanized COL-1 VL and VH

domains

The mCOL-1 was humanized by grafting the CDRs of the L andH chains onto the VL and VH frameworks of the appropriate hu-man Abs, but retaining those framework residues that weredeemed essential for preserving the structural integrity of the com-bining site (44–46). The Ig CDRs have been defined as compris-ing residues 31–35b, 50–65, and 95–102 in the H chain and res-idues 24–34, 50–56, and 89–97 in the L chain (47). Theframework residues that were deemed critical were identified onthe basis of the atomic coordinates of the Abs of known structuresavailable in the database (for example, see Ref. 48). The human Absequences that are most similar to mCOL-1 are VJI�CL (49) (Gen-Bank accession number Z00022) for VL and MO30 (50) (GenBankaccession number A32483) for VH. The alignment of the VL se-quences of mCOL-1 and VJI’CL, and the VH sequences ofmCOL-1 and MO30 are shown in Fig. 2. Also indicated in Fig. 2are the locations of the framework residues that are critical for Agbinding. The humanization protocols for the VL and VH genes,shown in Fig. 2, are based on putting the CDR sequences of mAbCOL-1 together with the frameworks of the human VL and VH

templates, while replacing some of the human framework residueswith those murine framework residues that may be critical for Agbinding.

A nucleotide sequence was then deduced from the amino acidsequence of each of the designed humanized VL and VH domains.The nucleotide sequences were refined to provide high frequencyusage of codons and by eliminating, with the help of programsFOLD and MAPSORT (51), any self-annealing regions and anysites for restriction endonucleases that might complicate cloning ofthe designed genes in the desired vectors. Using the four overlap-ping oligonucleotides (shown by long arrows in Fig. 1) that en-compassed, on alternating strands, the entire sequence of either theVL or VH region and its leader and the respective end primers

FIGURE 2. Humanization protocols for mAb COL-1. A, Amino acid sequences of the VL regions of mCOL-1, human Ab VJI’CL, HuCOL-1 derivedfrom mCOL-1 and VJI’CL, and the HuCOL-1 variant 24,25,27L. B, Amino acid sequences of the VH regions of mCOL-1, human Ab MO30, HuCOL-1derived from mCOL-1 and MO30, and the variant 61H. Dashes indicate residues that are identical in mCOL-1, MO30, HuCOL-1, and variant. Asterisksmark framework residues that are deemed essential for maintaining the combining site structure of mCOL-1. Murine framework residues retained in theHuCOL-1 are shown in bold.

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described in Materials and Methods, DNA fragments encoding thehumanized VL or VH regions were generated and amplified byoverlap extension PCR technique (32). The humanized VL or VH

regions thus generated were extended to the ApaI and SacII siteslocated 10 and 17 bp downstream from the 5� end of the C regionsof the H and L chains, respectively. The PCR products were clonedin pBSc to generate pBScHuCOL-1VL and pBScHuCOL-1VH

constructs. The inserts were sequenced to check the fidelity of thePCR products.

Generation of genes encoding variants of HuCOL-1 VL and VH

domains

Examination of the known structures of Ab-Ag complexes revealsthat only one-third of the CDR residues are involved in the inter-action with the Ag (48). This led to the proposal to redefine theboundaries of the CDRs to 31–35b, 50–58, and 95–101 in the Hchain and 27d–34, 50–55, and 89–96 in the L chain (14). Accord-ingly, we have developed variants of HuCOL-1 H and L chains inwhich these abbreviated CDRs have been grafted onto the VJI’CLand MO30 frameworks (Table I).

Genes encoding the humanized VL and VH domains of the vari-ants 24,25,27L and 61H were generated by primer-induced mutagen-esis, using pBScHuCOL-1VL and pBScHuCOL-1VH constructs,respectively, as templates. Variant 61H was generated by replacingresidue 61 of mCOL-1 H chain CDR2 (numbering convention ofKabat et al. (47)) with the corresponding residues of mAb MO30H chain CDR2. For the generation of 24,25,27L variant, residues 24,25, and 27 of mCOL-1 L chain CDR1 were replaced with thecorresponding residues in L chain CDR1 of the human mAbVJI’CL (Table I). The V region sequences were synthesized by adual-step PCR procedure according to Landt et al. (33). For eachL and H chain V region, the mutagenic primer, containing thedesired nucleotide changes in the targeted CDR, was used as a 3�primer, whereas a 20-mer end primer served as a 5� primer. Theresulting PCR product was gel purified and used as a 5� primer forthe subsequent step of the PCR in which a 21-mer oligonucleotidewas used as a 3� primer. The PCR products were cloned in pBSCvector and sequenced. The amino acid sequences of the VL of thevariant 24,25,27L and VH of the variant 61H are shown in Fig. 2, Aand B, respectively.

Immunoreactivity of the mAbs expressed in insect cells

The genes encoding the VL and VH domains of mCOL-1, Hu-COL-1, and the variants 61H and 24,25,27L were assembled with therespective human C region genes (�1 for the H and � for the Lchain). The expression constructs were introduced into Sf9 insectcells, and the supernatants harvested from the transfectants wereassayed for Ig production and Ag-binding reactivity by ELISA, asdescribed in Materials and Methods. All the transfectants and theviral plaques, generated by infecting Sf9 cells with the infectioussupernatants, were found to be positive for Ig production as as-sayed by ELISA. Results of an ELISA for Ag binding also showedthat all culture supernatants and the viral plaques were reactivewith CEA, albeit with varying degrees. To examine whether thedifferent constructs were expressing comparable levels of Ig mol-ecules, viral plaques were expanded and a large batch of Sf9 cellswas freshly infected, at a multiplicity of infection of 5, with in-fectious supernatant derived from the highest producing clone ofeach construct, and the infected cells were cultured under identicalconditions. The secreted Abs were purified from equal volumes ofthe culture supernatants. The concentration of the secreted Abswas comparable (2–3 �g/ml) in culture supernatants of all fivetransfectants.

SDS-PAGE of the secreted mAbs

The apparently lower Ag-binding reactivities of the variant mAbs24,25,27L and 24,25,27L/61H than those of cCOL-1, HuCOL-1, andthe variant 61H could be attributed either to any possible detrimen-tal effect of genetic manipulations of the combining site of thesecreted Abs or to some structural abnormality of the expressed Igmolecules. The latter may be detected on SDS-PAGE by a changein size or mobility of the molecules. To this end, the purified Absfrom the culture supernatants and the murine mAb COL-1 wereanalyzed by SDS-PAGE. The gel profile under nonreducing con-ditions (data not shown) showed that the mobility of all five re-combinant Abs was identical with that of mCOL-1 mAb, whichhas a molecular mass of �160 kDa. Under reducing conditions, allthe recombinant COL-1 Abs, like that of mCOL-1, yielded twoprotein bands of �25–28 and 50–55 kDa (data not shown). Thesemolecular masses are in conformity with those of the Ig L and Hchains. The results of the SDS-PAGE analysis, together with theELISA for CEA reactivity of the serially diluted Abs, suggest thatthe reduced Ag reactivity of the variants 24,25,27L and 24,25,27L/61Hmay be due to some detrimental effect of the amino acid substi-tutions in the combining site of HuCOL-1.

Relative CEA-binding affinities of mAbs derived from COL-1

A competition RIA was performed to determine the relative CEA-binding affinities of the COL-1-derived mAbs and the parentalmCOL-1 mAb. Serial dilutions of unlabeled Abs (murine, chi-meric, humanized COL-1 and its variants) were used to competewith the binding of 125I-labeled HuCOL-1 (Fig. 3) or 125I-labeledmCOL-1 (data not shown) to CEA. All of the COL-1-derived re-combinant Abs, like the parental mCOL-1, were able to com-pletely inhibit the binding of 125I-labeled mCOL-1 and 125I-la-beled HuCOL-1 to CEA. The competition profiles of cCOL-1,HuCOL-1, and the variant 61H were comparable to that of themCOL-1. In contrast, the competition profiles of mAbs 24,25,27Land 24,25,27L/61H, although of slopes similar to that of the parentalmCOL-1 mAb, were shifted to the right. The values of the relativeKa of cCOL-1, HuCOL-1, and 61H mAbs, calculated from the lin-ear parts of the competition curves in Fig. 3, were 3.45 � 108 M�1,

FIGURE 3. Competition RIA of mCOL-1-derived Abs. Increasing con-centrations of mAbs mCOL-1 (�), cCOL-1 (■ ), HuCOL-1 (Œ), 24,25,27L(E), 61H (�), 24,25,27L/61H (●), and HuIgG (‚) were used to compete forthe binding of 125I-labeled HuCOL-1 to 200 ng of CEA coated in each well.Dashed line indicates competitor HuCOL-1. The assay was done in trip-licate and the error bars denote the SD from the mean value of the data intriplicate.

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2.82 � 108 M�1, and 2.64 � 108 M�1, respectively. These relativeaffinities are �1.5- to 2-fold less than that of mCOL-1 (Table I).The relative Ka values of 24,25,27L and 24,25,27L/61H were 1.2 � 108

M�1 and 1.03 � 108 M�1, respectively, �4.3- to 5-fold lower thanthat of parental mCOL-1 (Table I).

Flow cytometric analysis

Flow cytometric analysis was used to measure the binding of Hu-COL-1 and its variants (61H, 24, 25, 27L, and 24, 25, 27L/61H) to theCEA expressed on the cell surface of a tumor cell line, MC38, thatwas retrovirally transduced with CEA (38). No significant differ-ences were found in the mean fluorescence intensity or in the per-centage of cells that was reactive with HuCOL-1 and its variants(Fig. 4). The percentages of gated cells, calculated after exclusionof irrelevant binding, were indeed between 54 and 56, whereas themean fluorescence intensities were between 15 and 16 when 1 �gof each Ab was used.

Reactivity of HuCOL-1 and HuCOL-1 variants to patients’ sera

The immunogenicity of an Ab variant can be assessed onlythrough its clinical trial. A reasonable measure of the potentialimmunogenicity of the variant Ab, however, is its in vitro reac-tivity to sera from patients who were administered the parental Abin a clinical trial. To assess the potential immunogenicity of themCOL-1, HuCOL-1, and its variants in patients, the Abs werecharacterized for their reactivity to sera from gastrointestinal car-cinoma patients who were administered 131I-labeled mCOL-1 in aphase I clinical trial (24). As described in Materials and Methods,any circulating CEA and anti-murine Fc Abs were removed fromthe sera by immunoadsorption with mCOL-6 and mCOL-4, twomurine anti-CEA Abs of IgG1 and IgG2a isotypes, respectively. Ithas been suggested on the basis of epitope mapping of CEA (28)that mCOL-4 and mCOL-6 may react with CEA epitopes that aredifferent from each other and different from the one recognized bymAb COL-1. Preadsorbed sera were tested for the presence ofanti-V region Abs to mAb COL-1. Specific binding profiles ofHuCOL-1 to the sera from patients EM, JS, and MB (data notshown) show that all three sera have Abs against the variable re-

gions of mCOL-1. Serum reactivity of HuCOL-1 and HuCOL-1variants was determined by their ability to compete with mCOL-1or HuCOL-1 immobilized on a sensor chip for binding to the anti-variable region Abs to mCOL-1 present in the serum. IC50, theconcentration of the competitor Ab required for 50% inhibition ofthe binding of mCOL-1 or HuCOL-1 to the patient’s serum, wascalculated by plotting the percent inhibition as a function of com-petitor concentration. A higher IC50 value indicates a decreasedreactivity to the serum, suggesting potentially reduced immunoge-nicity of the Abs in patients. Fig. 5 shows the competition profilesgenerated by HuCOL-1 and its variants when they were used tocompete with the HuCOL-1 immobilized on the sensor chip forbinding to the anti-V region Abs to COL-1 present in the sera ofpatients EM (A), JS (B), and MB (C). The competition profileswere used to calculate IC50 values that are presented in Table II.For serum MB, the IC50 values of all three variants are 2- to 3-foldhigher than that of HuCOL-1. Studies with the serum from patientEM show that the variants 24,25,27L and 24,25,27L/61H have 50%higher IC50 values, whereas the variant 61H has a significantlylower IC50 value than that of HuCOL-1. For the serum from pa-tient JS, the IC50 of the variant 24,25,27L/61H is twice as much asthat of HuCOL-1, whereas the IC50 values of the variant 61H and24,25,27L are comparable to that of parental HuCOL-1. WhenmCOL-1 was immobilized on the sensor chip and mCOL-1, Hu-COL-1, and the variant 24,25,27L/61H were used to compete with itfor binding to the anti-V region Abs in the serum of patient MB,the competition profiles shown in Fig. 6 were generated. The datashow that the concentration of HuCOL-1 required for 50% inhi-bition of the binding of the patient’s serum to mCOL-1 is �3-foldhigher than that of mCOL-1, whereas the concentration of the vari-ant 24,25,27L/61H required to attain 50% inhibition of the binding ofmCOL-1 to the patient’s serum is �5.5- and 17-fold higher thanthose of HuCOL-1 and mCOL-1, respectively. Moreover, it shouldbe pointed out that the slope of the competition profile of thevariant 24,25,27L/61H is quite different from that of mCOL-1. In-deed, there was �2 log differential in the concentrations of the24,25,27L/61H and mCOL-1 mAbs required for the 60% inhibition

FIGURE 4. Flow cytometric analysis of the binding of HuCOL-1 and its variants to cells expressing cell surface CEA. Binding profiles of 1 �g ofHuCOL-1 (A), 24,25,27L (B), 61H (C), and 24,25,27L/61H (D) mAbs, to MC38 cells engineered to express CEA on their cell surface. Binding of an irrelevantmAb, human IgG (dashed line), is shown in each panel and represents �2% of the cell population.

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of the binding of the sera anti-V region Abs to mCOL-1 immobi-lized on the sensor chip (Fig. 6). Sera from two other patients, JSand EM, were used to compare serum reactivity of mCOL-1 andHuCOL-1. Although the IC50 value of HuCOL-1 was �6-foldhigher than that of mCOL-1 for JS serum, it was not possible toevaluate the difference in the reactivity of the two Abs to EMserum; even 1000 nM HuCOL-1 was unable to attain 50% inhi-bition of the binding of the serum to mCOL-1 (data not shown).

DiscussionThe HAMA response is a major impediment to the clinical use ofthe murine mAbs, particularly for the dose fractionation clinicalprotocols that are likely to be used for greater therapeutic efficacyand reduced toxicity. The advantage of dose fractionation proto-

cols for therapeutic efficiency of the radiolabeled mCOL-1 hasbeen demonstrated in an animal model (30). In addition to causinga possible allergic reaction, the HAMA response results in rapidclearance of the Ab from the circulation, preventing the Ab fromreaching the targeted tumor sites. In a phase I clinical trial of131I-labeled mCOL-1, the onset of HAMA response prevented ad-ministration of a second dose of the radiolabeled Ab in two pa-tients who were otherwise eligible for further treatment. HAMAresponse was also reported in a phase II clinical trial, when IFNwas combined with 131I-labeled COL-1 and 131I-labeled CC49, amurine Ab against another pancarcinoma Ag, tumor-associatedglycoprotein-72, to achieve more efficient targeting of human colo-rectal cancer (31).

Development of a mouse-human chimeric mAb is an approachthat has been widely used to minimize HAMA response (52). Ac-cordingly, a cCOL-1 mAb was developed by replacing the C re-gions of the L and H chains of the mCOL-1 with the human � and�1 constant regions. The Ag-binding affinity of the cCOL-1(3.45 � 108 M�1) was found to be comparable to that of murineAb (5.17 � 108 M�1). A chimeric Ab, though likely to be lessimmunogenic than the murine Ab, may evoke anti-V region re-sponse in patients, because the V region of a murine Ab is poten-tially immunogenic. Anti-V region responses have been reportedafter the administration of chimeric Abs in patients (53, 54; re-viewed in Ref. 55). To attempt to reduce this problem, mAbCOL-1 has been humanized following a procedure that involvesgrafting of the CDRs of a xenogeneic Ab onto the human Ig frame-works (reviewed in Ref. 8).

The most important consideration in humanizing an Ab is thepreservation of its Ag-binding property, which depends on the

FIGURE 5. Serum reactivity, by SPR, of HuCOL-1 and its variants.Increasing concentrations of HuCOL-1 (Œ), 24,25,27L (E), 61H (�), and24,25,27L/61H (F) mAbs were used to compete with the anti-V region Absto COL-1 present in sera from patients EM (A), JS (B), and MB (C) forbinding to HuCOL-1 immobilized on a sensor chip. Percent binding of thesera to HuCOL-1 was calculated from the sensograms and plotted as afunction of the concentration of the competitor.

FIGURE 6. Serum reactivity, by SPR, of mCOL-1 and the engineeredAbs derived from it. Increasing concentrations of mCOL-1 (f), HuCOL-1(Œ), and 24,25,27L/61H (F) were used to compete with the anti-V region Absto COL-1 present in serum from patient MB for binding to mCOL-1 im-mobilized on a sensor chip. Percent binding of the sera to mCOL-1 wascalculated from the sensograms and plotted as a function of the concen-tration of the competitor.

Table II. Reactivity of the variant mAbs with patients’ seraa

Competitor AbEM

(nM)JS

(nM)MB(nM)

HuCOL-1 6.6 2.0 5.524,25,27L 10.7 2.5 15.261H 4.9 2.8 10.524,25,27L/61H 9.1 4.1 14.4

a Competitor Ab concentrations required for IC50 of binding of serum from pa-tients EM, JS, and MB to HuCOL-1 were calculated.

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structural integrity of the combining site. This requires selectingthe most appropriate human templates for humanization and graft-ing the CDRs of the target Ab onto the human scaffold, whileretaining those murine framework residues that may be involved inligand contact directly or through their interaction with the CDRs.In the absence of a three-dimensional structure of mCOL-1, theidentification of the crucial frameworks residue was facilitated bya search of the database that yielded the V regions of two humanAbs, VJI’CL and MO30, respectively, whose sequences were mostsimilar to the mCOL-1 VL and VH sequences. There are 76 iden-tities among the 112 overall residues of the VL of the VJI’CL andmCOL-1, and when comparing the 80 framework residues only,there are 65 identities. Among the 127 overall VH residues of theMO30 and COL-1 mAbs, there are 75 identities, while there are 57identities in their 87 frameworks residues. Because the VL frame-works of VJI’CL and mCOL-1 are so extensively homologous,they differ only by five residues among those deemed crucial forAg binding. The VH frameworks of COL-1, however, differ fromMO30 at 17 positions that are most probably essential for the li-gand binding. This approach of selecting the human frameworks tobe used as templates had been successful for the humanization ofseveral Abs, including mAb AUK12–2 (56), mAb 1B4 (57), andmAb CC49 (32). Based on these considerations, humanization ofmCOL-1 was conducted using VJI’CL and MO30 as human tem-plates, and the resulting HuCOL-1 showed only 2.5-fold loweraffinity than that of the murine COL-1.

Because the murine CDRs of a humanized Ab could still evokean anti-V region response in patients, CDR grafting is not an ul-timate solution of the potential immunogenicity of a xenogeneicAb. Padlan et al. (14) suggested that not all of the CDR residuesare involved in the ligand contact. Tamura et al. (13) humanizedthe anti-tumor-associated glycoprotein-72 mAb CC49 by graftingonly its SDRs onto the human Ab frameworks. The HuCC49 vari-ant, designated HuCC49V10, that was developed by this approachretained the Ag-binding properties of the parental mAb, while re-acting with the patients’ sera only minimally. The immunogenicityof an Ab, therefore, could be reduced by transplanting only thoseparts of the CDRs that contain the SDRs. The “abbreviated” CDRshave been defined (14). Based on this rationale, a variant of eachof the L and H chains of the HuCOL-1 was developed. In L chainvariant, residues 24, 25, and 27 of the L chain CDR1 were replacedwith the corresponding residues of the human Ab VJI’CL, whereasin H chain variant, residue 61 of the H-chain CDR2 was replacedwith the residue of the human Ab MO30, located at the sameposition. The Ag-binding affinity of the H chain variant was com-parable to that of the parental HuCOL-1, whereas that of the Lchain variant was �2.4-fold lower. The slight loss in the affinity ofthe L chain variant was reflected in the affinity of the HuCOL-1variant (2.7-fold lower than that of the parental HuCOL-1) thatwas generated by combining the H and the L chain variants. Flowcytometric analysis of the CEA-transduced MC38 cells that weretreated with the variant Abs, however, shows that the slight lossesin the relative affinities of the variants had no effect on their bind-ing ability to the CEA expressed on the cell surface.

Humanization by transplanting the “abbreviated” CDRs was un-dertaken to eliminate any possible idiotopes, present in the CDR-grafted HuCOL-1, that could be the potential targets of patients’immune response. However, it should be pointed out that engi-neering of this variant may have generated new idiotopes on thecombining site of HuCOL-1 that may be targeted by the patient’simmune response. The immunogenicity of the COL-1-derived Abscan be evaluated only through their clinical trials in patients.Whether the HuCOL-1 variants, generated by grafting of the“abbreviated” CDRs, have reduced the potential to evoke anti-V

region response in patients compared with the CDR-graftedHuCOL-1 was assessed by comparing their reactivity to sera fromgastrointestinal cancer patients who were administered 131I-labeled mCOL-1 in a phase I clinical trial (24). The sera wereshown to carry anti-V region Abs to COL-1. In lieu of clinicaltrials, serum reactivity of the variant Ab is as good a measure of itspotential immunogenicity as one can get, without administeringthe variant in a patient. The results confirm an earlier observationusing another Ab (13), that the pattern of the anti-V regionresponses differs from patient to patient. Some patients may elicitmore vigorous response to certain idiotopes than other patients;nevertheless, the variant 24,25,27L/61H, compared with HuCOL-1,shows 1.5-, 2-, and 3-fold lower reactivity to EM, JS, and MB sera,respectively. The reduction in serum reactivity of the variant ismuch more significant than these numbers suggest, because thereactivity of HuCOL-1 to serum MB is 3-fold lower than that ofmCOL-1. The lower serum reactivity of the variant 24,25,27L/61Hmay be due to the elimination of the immunogenic idiotopes fromthe L chain variant 24,25,27L, which shows 2- to 3-fold lowerreactivity to EM and MB sera than does HuCOL-1. These resultssuggest that the variant 24,25,27L/61H, compared with HuCOL-1, issignificantly less reactive to sera from patients who were admin-istered 131I-labeled mCOL-1 in a clinical trial and, hopefully,substantially less immunogenic in patients.

AcknowledgmentsWe thank Debra Weingarten for her editorial assistance in the preparationof this manuscript.

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