Proc. Natl. Acad. Sci. USAVol. 91, pp. 12957-12961, December 1994Immunology

Specifi'city analysis of blood group Lewis-y (Ley) antibodiesgenerated against synthetic and natural Ley determinants

(anti-LeY antibody/humanized antibody/synthetic neoglycoprotein)

KUNIO KITAMURA*, ELISABETH STOCKERT*, PILAR GARIN-CHESA*t, SYDNEY WELT*t, KENNETH 0. LLOYD§,KATHRYN L. ARMOUR¶, THOMAS P. WALLACE1, WILLIAM J. HARRIS$, FRANCIS J. CARR¶,AND LLOYD J. OLD* 11*Ludwig Institute for Cancer Research, New York Unit, Departments of tPathology and *Medicine, and §Immunology Program, Memorial Sloan-KetteringCancer Center, New York, NY 10021; and ¶Scotgen Biopharmaceuticals, Aberdeen AB22 8GU, Scotland, United Kingdom

Contributed by Lloyd J. Old, July 11, 1994

ABSTRACT LeY-reactive monoclonal antibodies (mAbs)were generated in mice by immunization with synthetic Leyneoglycoproteins or with LeY-expressing cells. Serological anal-ysis indicated that mAbs raised against synthetic Ley (0) reactedstrongly with synthetic Ley but poorly with natural LeY, (it)cross-reacted with Lex or H-type 2 structures, and (iii) wereIgGl, IgG2a, or IgG2b. mAbs raised against LeY-expressingcells (a) reacted with both synthetic Lev and natural LeY, (it)were of two types: cross-reactive with Lex or H-type 2 struc-tures or specific for Ley, and (iiM) were IgM or IgG3. One of themAbs raised against natural LeY, mAb 3S193 (IgG3), showedhigh specificity for Lev in ELISA tests with synthetic Lev andLey containing glycoproteins and glycolipids; it also reactedstrongly in rosetting assays and cytotoxic tests with LeY-expressing cells. mAb 3S193 did not lyse 0, A, AB, and Bhuman erythrocytes in the presence of human complement. Inflow cytometry, there was weak reactivity with granulocytes, areactivity also observed with two previously described highlyspecific Lev mouse mAbs-BR55-2 (IgG3) and B3 (IgGl). Ahumanized version ofmAb 3S193 has been constructed, and thespecificity pattern and reactivity for LeY remain very similar tomouse mAb 3S193.

The serological analysis of human cancer cells with mousemonoclonal antibodies (mAbs) has identified a number ofcarbohydrate determinants, linked either to lipids (glycolip-ids) or to proteins (glycoproteins) (1-5). Blood group-related(BGR) antigens-mainly, the lacto- (type 1) and neolacto-(type 2) structures-have been the focus of much attentionbecause of their strong expression on tumors of epithelialorigin (1-5). Although BGR antigens are also expressed innormal tissues, there is evidence for altered expression incertain tumor types (1-6). We and others showed that Ley{Fuc(al -* 2)Gal(,81-4)[Fuc(a1 -- 3)]GlcNAc} antigen accu-mulates to a higher level in colonic cancer than in the adjacentnormal colon epithelium (7, 8). Because Ley is expressed in>70% of epithelial cancers, such as breast, ovary, colon, andlung cancer, there is considerable interest in its use as a targetformAb imaging and therapy. A large number of Ley-reactivemAbs have been generated and subjected to various degreesof specificity analysis, particularly in relation to their reac-tivity with Lex {Gal(,/1 -- 4)[Fuc(al -* 3)]GlcNAc} andH-type 2 [Fuc(al -- 2)Gal(,B1 -- 4)GlcNAc] structures, twodeterminants structurally related to Ley (9-16). The impor-tance of a detailed serological analysis before an anti-Leyreagent is used in humans is illustrated by our findings witha number of Ley antibodies originally thought to be specificfor Ley but later shown to cross-react with Ley-related

structures-especially Lex or H-type 2-and to agglutinatehuman erythrocytes (14). For this reason, we initiated aneffort to generate Ley reagents with improved reactivity andspecificity. The availability of a wide range of syntheticcarbohydrate structures (17) greatly facilitates the specificitytesting of mAbs to Ley and other BGR antigens. In addition,synthetic Ley used as an immunogen offers opportunities forgenerating anti-Ley reagents. In the present study, we haveanalyzed in detail the specificity of newly derived mAbsraised against synthetic or natural Ley. One of the mostspecific and reactive Ley mAbs derived in this series, mAb3S193, has been humanized, and the reactivities of thehumanized mAb 3S193 and mouse mAb 3S193 are compared.

MATERIAL AND METHODSTissue Culture and mAb. Tumor cell lines were obtained

from the tumor cell bank in the Ludwig Unit at. MemorialSloan-Kettering Cancer Center and maintained as described(18). mAbs 118 and F-12 have been described (14, 15). mAbsBR55-2 (IgG3) (12) and B3 (IgGl) (16) were provided by Z.Steplewski (Wistar Institute, Philadelphia) and I. Pastan(National Institutes of Health, Bethesda, MD), respectively.

Synthetic Neoglycoproteins. Ley-human serum albumin(HSA), LeY-keyhole limpet hemacyanin (KLH), H-type 2-bo-vine serum albumin (BSA), Lex-BSA, lacto-N-neotetraose(LNneoT)-BSA, A type 1-BSA, ALeb-BSA, B type 1-BSA,B type 2-BSA, and BLeb-BSA were obtained from Chem-biomed (Edmonton, Canada). Ley-BSA, Leb-BSA, H-type1-BSA, and Lea-BSA were purchased from BioCarb (Lund,Sweden).Blood Group Glycoproteins and Glycolipids. Blood group-

active glycoproteins-i.e., A(MSS), B(Beach), LeY/Leb(Tighe), and Lex/Lea (N-1) from ovarian cyst fluids and hoggastric A+H mucin-and purified standard glycolipids-i.e.,H-type 1, H-type 2, Lea, Leb, Lex, and Ley were prepared asdescribed (14, 19).

Immunizations. mAbs were produced by using the standardhybridoma technique after four to six immunizations ofBALB/c mice with 2.5-50 ug of Ley-HSA or 3 to 10 x 106of MCF-7 breast or HCT-15 colon adenocarcinoma cells.Culture supernatants from hybridomas were screened bymixed hemadsorption (MHA) rosetting assays and cytotoxictests with tumor cells and ELISA with synthetic Ley neo-

Abbreviations: mAb, monoclonal antibody; ADCC, antibody-dependent cellular cytotoxicity; MHA, mixed hemadsorption; BGR,blood group-related; BSA, bovine serum albumin; KLH, keyholelimpet hemacyanin; HSA, human serum albumin."To whom reprint requests should be addressed at: Ludwig Institutefor Cancer Research, New York Unit, Memorial Sloan-KetteringCancer Center, 1275 York Avenue, New York, NY 10021.

12957

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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glycoprotein. The immunoglobulin subclass of mAbs wasdetermined by the Ouchterlony immunodiffusion method.

Serological Assays. MHA assays. Protein A-MHA andrabbit anti-mouse immunoglobulin-MHA assays for the de-tection of cell surface antigens were done as described (18,20).

Cytotoxic tests for complement-dependent cytotoxicity.Ten microliters of a cell suspension (1.5 x 104/ml) wasdistributed into wells of microtiter plates (Nunclon; Nunc)and incubated for 20 hr at 37°C in 5% C02/95% air. Themedium was removed, 10 Al of serially diluted antibody wasadded to each well, and incubation was for 45 min. Then 10Al of complement (human serum diluted 1:3) was added.Tests were done in duplicate with medium, antibody, andcomplement controls. After 4 hr, plates were fixed withmethanol for 10 min, rinsed in distilled water, stained with 2%Giemsa stain in phosphate-buffered saline for 25 min, andrinsed. Plates were analyzed under the light microscope, andthe percentage cytotoxicity of a given antibody dilution wascalculated as follows: [1 - (number of cells in well treatedwith antibody and complement/number of cells in welltreated with medium only)] x 100.ELISA. ELISA was done with natural glycoproteins and

glycolipids and synthetic neoglycoproteins adsorbed to thewells of microtiter plates as described (21).

Tests for antibody-dependent cellular cytotoxicity(ADCC). A short-term 51Cr-release test was done with somemodification (22). Target cells (106) were labeled with 100 ,Ci(3.7 MBq; 1 Ci = 37 GBq) of 51Cr for 1 hr at 37°C; labeled cellswere seeded (104 cells per well in 50 ,l) into 96-well flat-bottom plates and incubated for 2 hr. Then, antibody (50 Alper well) and human lymphocytes (50 ul per well) wereadded. After incubation for 6 hr at 37°C, supernatants wereharvested, and radioactivity was measured with a y counter.Spontaneous release was defined as the cpm released inmedium alone instead of in mAb and lymphocytes, andmaximum release was defined as the cpm released by Noni-det P-40. Percentage cytotoxicity was calculated as follows:[(sample release - spontaneous release)/(maximum release- spontaneous release)] x 100. Spontaneous release was<35% of maximum release in all experiments.Hemolysis test. The 51Cr-release assay also was used to

test hemolysis by mAbs. Briefly, 0.1% 51Cr-labeled erythro-cytes were incubated with purified mAb and autologouscomplement for 30 min, and the radioactivity in the super-natants was measured. Percentage lysis was calculated ac-cording to the same formula used in ADCC assays. Sponta-neous release was <1% of maximum release in all experi-ments.Flow Cytometry Analysis (Peripheral Blood Leukocyte As-

say). The reactivity with peripheral blood leukocytes wasanalyzed with purified mAbs using a FACStarflow cytometer(Becton Dickinson) as described (14).Immunohistochemical Procedures. Tissues were obtained

through the Tumor Procurement Service of the Departmentof Pathology, Memorial Hospital, embedded in OCT com-pound (Miles), snap-frozen in isopentane precooled in liquidN2, and stored at -70°C. Sections (5 ,um thick) were cut,mounted on poly(L-lysine)-coated slides, air-dried, and fixedin acetone (4°C, 10 min). mAbs were used at 0.5-20 ,ug/ml,and the avidin-biotin immunoperoxidase procedure wasdone as described (23).

Generation of Humanized and Chimeric Antibodies. mAb3S193 was humanized by the principles of Riechmann et al.(24). The humanized heavy-chain variable regions containedthe complementarity-determining regions of mAb 3S193 andthe framework regions of the human KOL heavy chain,incorporating one (HuVH) or two (HuVHT) mouse frame-work residues at positions 28 and 24/28, respectively [num-bering according to Kabat et al. (25)]. The humanized K chain

was based on the human REI K-chain frameworks andincluded no mouse framework residues (HuVK) or a substi-tution at Kabat position 71 (HuVKF). DNAs encoding thesedomains were expressed with human IgGl and K constant-region genes using a vector system based on that of Orlandiet al. (26). Similar constructs, containing mouse mAb 3S193variable-region DNAs, allowed synthesis of chimeric anti-body chains, termed MuVH and MuVK. Antibodies contain-ing different combinations of heavy and light chains wereproduced by cotransfection of myeloma cells as described(27); antibodies composed of a mixture of one chimeric andone humanized chain are hereafter referred to as "hybrid"antibodies. The antibodies described here are numbered asfollows: 3S193#5, MuVH/MuVK; 3S193#6, MuVH/HuVK; 3S193#7, HuVH/HuVK; 3S193#11, HuVHT/HuVKF.

RESULTSGeneration of Mouse Anti-LeY Antibodies. Two approaches

to generate mouse anti-Ley antibodies were compared: oneusing chemically synthesized Ley neoglycoproteins as theimmunogen and the other using cultured human tumor linesexpressing Ley on the cell surface. Culture supematants fromhybridomas were initially screened for (i) Ley-KLH andKLH reactivity using ELISA and (ii) cell-surface reactivityusing MHA rosetting assays and cytotoxic tests on three celllines, MCF-7 (LeY+), HCT-15 (LeY+), and SK-MEL-28(Ley-). On the basis of these tests, five mAbs generatedagainst synthetic Ley and five mAbs generated against Ley-expressing cells were selected for detailed serological anal-ysis. These results are summarized in Tables 1 and 2.

Anti-synthetic LeY mAbs. The isotypes of the anti-syntheticLey mAbs were IgGl, IgG2a, or IgG2b. These mAbs showedstrong and equal reactivity with Ley conjugated to HSA,BSA, or KLH and no reactivity with a number of othersynthetic oligosaccharides conjugated to BSA with the samelinker, excluding a significant contribution of carrier proteinor linker moiety. However, none of the five mAbs werespecific for LeY; four ofthem strongly cross-reacted with Lex,and one cross-reacted with H-type 2 determinants. Theanti-synthetic Ley mAbs did not react with Ley containingnatural glycoproteins or glycolipids in ELISA and werepoorly reactive with cells expressing Ley in rosetting andcytotoxic assays.

Anti-natural Lev mAbs. The isotypes ofthe anti-natural LeymAbs were IgM or IgG3. Their reactivity with synthetic Leywas weaker than mAbs to synthetic Ley, but they reactedstrongly with LeY-containing natural glycolipids and glyco-proteins and showed 2- to 4-logarithmic-higher titers inassays using LeY-expressing cells. As with anti-synthetic LeymAbs, the main cross-reactions of anti-natural Ley mAbswere with Lex and with H-type 2 structures. However, twomAbs in this series, 3S193 and 8S202, showed a high degreeof specificity and reactivity for Ley determinants. In hemol-ysis tests, mAb 8S202 was strongly hemolytic for 0 eryth-rocytes and to a lesser degree for A, AB, and B erythrocytes.In contrast, mAb 3S193 did not lyse erythrocytes. This stronghemolytic activity of mAb 8S202 cannot be accounted for byits weak cross-reactivity with H-type 2 (at a mAb concen-tration of 100 mg/ml) because mAb 118 (see below), which isless hemolytic, has a stronger cross-reactivity with H-type 2(6.25 ,g/ml). In flow cytometry, mAb 8S202 showed noreactivity with granulocytes, whereas mAb 3S193 showedlow granulocyte reactivity (mean positive count, 19.7). Thislevel of reactivity is >10-fold lower than the reactivity ofanti-Lex mAb P12 on granulocytes (mean positive count, 267)in fluorescence intensity.Immunohistochemical Staining. The reactivity of mAbs

with normal and malignant tissues was examined. Of three

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Table 1. Specificity of mAbs with synthetic neoglycoproteins, glycoproteins, and glycolipids

Synthetic antigen* GlycolipidtA-1, Glycoproteint H-1,

Lea, ALe", B-1, Lea,Antibody Ley H-2 Lex LNneoT H-1 Leb BLeb B-2 A B Tighe N-1 Hog Ley H-2 Lex Leb

Anti-synthetic Ley2A1 (IgG2b) 0.025 100 0.025 - + - - - - -.-2A4 (IgG2b) 0.025 ± 0.10 - - - - - NT NT NT NT NT NT NT NT NT2A7 (IgG2a) 0.025 ± 0.10 - - - - - - - -.-2A19 (IgGi) 0.025 100 0.10 100 ± + - - NT NT NT NT NT NT NT NT NT2A37 (IgG2b) 0.39 1.56 + + + + + + - - -.-

Anti-natural Ley3A5 (IgG3) 0.10 - 6.25 - - _ _ _ _ - ++ - - ++ _ ++ -3S193 (IgG3) 0.10 - 100 - - - - - - ++ - ++ - -3S209 (IgM) 0.10 100 25 100 100 100 100 00 - + ++ + + ++ - ++ -8S202 (IgG3) 0.39 100 - - - - - - - + + + - -8A15 (IgG3) 0.10 25 - - - - - + ++ _ ++ _

Previouslydescribed anti-LeyBR55-2 (IgG3) 0.10 - 100 - - - - - ++ - ++ -B3 (IgGi) 0.10 - 25 - - - - - - ++ - ++ -118 (IgG3) 0.39 6.25 25 25 25 25 25 25 - ± ++ + ++ + + -F12 (IgM) 0.39 ± + + + + + - - + ++ NT NT NT NT

Engineered 3S193Chimeric #5 0.10 50 100 - - - - - - - ++ - + NT NT NT NTHybrid #6 0.025 6.25 25 100 100 + + - - + ++ - + NT NT NT NTHumanized #7 1.56 - + - - - - - - - ++ - - NT NT NT NTHumanized #11 0.39 + - - - - - - - ++ - - NT NT NT NT

*Values are minimum antibody concentrations (,g/ml) that show OD >0.6 in ELISA. Synthetic oligosaccharides conjugated to BSA were usedas antigens. In addition, Ley-HSA and Ley-KLH were also tested and gave the same results as Ley-BSA. Each mAb was titrated from 100,ug/ml. -, Negative; ±, weakly positive at highest concentration tested (100 ,ug/ml).

tAntibody concentration of 5 or 10 pg/ml was used for extracted antigens. -, Negative; +, trace; ±, weakly positive; +, positive; + +, stronglypositive. NT, not tested.

mAbs generated with synthetic Ley, mAbs 2A1 and 2A37showed weak reactivity with some normal and malignantepithelial tissues, and mAb 2A7 was completely unreactive.By contrast, the mAbs generated with natural Ley reactedstrongly with a high proportion of carcinomas and severalnormal tissues. mAb 3S193 was selected for detailed analysisand stained tumor cells in 142 of the 192 human epithelialtumors tested, including tumors of the colon, stomach,breast, lung, prostate, bladder, and pancreas. Among normaltissues, prominent immunostaining with mAb 3S193 wasfound in the gastrointestinal mucosa and, with a heteroge-neous pattern, in several other histologic types of epithelia,including breast, bronchus, pancreas, and genitourinary sys-tem. Two tissues with strong Lex expression-namely, nor-mal brain and spleen (28)-were also tested with mAbs 3S193and 3A5. Both mAbs were unreactive with brain tissue. Theyshowed no staining with spleen tissue when tested at 0.5-1,g/ml, the concentration used for all other tissues, andstaining limited to a subset of perifollicular cells, predomi-nantly granulocytes, when tested at 10-fold higher immuno-globulin concentration.Comparison of mAb 3S193 with Previously Described Anti-

LeY mAbs. A detailed specificity analysis was carried out onfour previously described mAbs with Ley reactivity: BR55-2(IgG3) (12), B3 (IgGl) (16), 118 (IgG3) (14), and F-12 (IgM)(15). mAbs BR55-2 and 3S193 showed a virtually identicalpattern with synthetic oligosaccharides, natural glycopro-teins and glycolipids, and granulocytes. However, in roset-ting assays and cytotoxicity tests, mAb 3S193 has a 10- to100-fold-higher reactivity than mAb BR55-2. mAb B3 alsoshowed a high degree of specificity for Ley. mAb B3 has beenreported to react with difucosylated Lex and trifucosylatedLey but not with Lex at 10 pg/ml (16). At a higher concen-tration of mAb B3 (25 ,ug/ml), we find a cross-reaction ofmAb B3 with Lex. mAb 118 reacted with H-type 2 at 6.25

,ug/ml, reacted with other BGR determinants at 25 pg/ml,and lysed 0 erythrocytes in the presence of complement.mAb F-12 showed highly restricted Ley reactivity but wassignificantly weaker in cytotoxic tests with Ley-expressingcells.

Construction and Analysis of Humanized mAb 3S193. Be-cause of its specificity and strong reactivity for Ley, mAb3S193 was selected for humanization. The reactivity patternsfor one chimeric, one hybrid, and two humanized versions ofmAb 3S193 are shown in Tables 1 and 2. In tests withsynthetic neoglycoproteins, chimeric mAb 3S193#5 showedthe same titers as mouse mAb 3S193 with synthetic Ley, butin contrast to mouse mAb 3S193, it reacted with H-type 2structure (50 ug/ml). Hybrid mAb 3S193#6 was 5-fold morereactive than mouse mAb 3S193 with Ley, but its cross-reactivity with H-type 2 (6.25 ,ug/ml) and Lex (25 ,ug/ml)increased greatly. Comparison of humanized mAbs 3S193#7and 3S193#11 shows that their Ley specificity is identical, but#11 has higher reactivity in ELISA, protein A-MHA assay,and cytotoxic test and lower reactivity for granulocytes than#7. Fig. 1 illustrates ADCC tests using five colon and threebreast cancer cell lines in the presence of mAb 35193 11 andhuman effector cells. LeY-expressing cell lines show variousdegrees of lysis ranging from 64% (MCF-7) to 11% (SK-CO-10) with antibody concentrations as low as 0.1 ug/ml. Lyticactivity correlated with Ley expression as tested in MHAassays. Significant lysis could still be demonstrated at a mAbconcentration of 0.01 ,tg/ml or at effector-to-target ratio of<10:1. LeY-negative colon cancer cell line SW1222 was notlysed by humanized mAb 3S193#11 but could be lysed by anunrelated humanized mAb. A direct comparison of human-ized mAb 3S193#11 and mouse mAb 3S193 showed that theADCC activity of the humanized antibody was 100-foldgreater.

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Table 2. Reactivity of mAbs in rosetting assays, cytotoxic test, hemolysis test, and flow cytometry analysis

Flow cytometry analysistPA-MHA* Rb a mIg-MHA* Cytotoxic test* Hemolysis test,t % Lymph. Mono. Granulo.

Antibody MCF-7 HCT-15 MCF-7 HCT-15MCF-7 HCT-15 0 A B AB MPC, %+ MPC, %+ MPC, %+Anti-synthetic Ley2A1 (IgG2b) 1.56 1.56 0.39 1.56 12.5 50 1 - _ - 0.2 9 0.4 14 6.9 932A4 (IgG2b) 12.5 50 6.25 12.5 25 100 NT NT NT NT NT NT NT2A7 (IgG2a) 25 100 25 25 25 100 - - - - 0.1 2 0.3 14 1.0 22A19 (IgGl) - - 12.5 25 - - - - - - NT NT NT2A37 (IgG2b) 1.56 6.25 1.56 3.13 25 100 - - - - 0.1 1 0.3 13 1.4 5

Anti-natural Ley3A5 (IgG3) 0.0016 0.0016 0.0016 0.0125 0.78 1.56 1 - - - 0.2 6 0.3 15 11.5 913S193 (IgG3) 0.0016 0.0016 0.0016 0.0063 0.10 0.20 - - - - 0.2 11 0.3 18 19.7 993S209 (IgM) - - 0.0063 0.025 0.10 0.10 - - - - 0.3 27 0.4 30 25.3 998S202 (IgG3) 0.0063 0.0125 0.0063 0.10 0.39 0.39 100 37 22 28 0.3 30 0.4 32 0.9 68A15 (IgG3) 0.0063 0.025 0.0063 0.10 1.56 1.56 79 28 NT 11 0.3 19 0.4 34 0.8 6

Previouslydescribed anti-LeYBR55-2 (IgG3) 0.10 0.10 0.10 0.39 0.78 1.56 - - - - 0.1 1 0.2 11 11.8 98B3 (IgGl) 1.56 6.25 0.0063 0.025 - - - - - - 0.2 6 0.3 12 24.7 98118 (IgG3) 0.0063 0.0063 0.0063 0.025 1.56 6.25 58 9 2 5 NT NT NTF-12 (IgM) - - 0.39 0.39 12.5 25 - - - - 0.1 1 0.2 4 1.2 54

Engineered 3S193Chimeric #5 0.0016 0.0125 0.025 25 0.39 0.39 - - - - 0.4 43 0.8 47 46.2 99Hybrid #6 0.0016 0.025 0.10 50 0.20 0.39 - - - - 0.5 47 1.4 61 98.7 99Humanized #7 0.0125 0.39 12.5 - 1.56 3.13 - - - - 0.2 10 0.5 24 70.0 99Humanized #11 0.0063 0.39 25 - 0.78 1.56 - - - - 0.2 18 0.9 41 36.0 98

*Values are the minimum antibody concentrations (,ug/ml) that show at least 50% rosette formation in rosetting assays or 50% lysis in cytotoxictests. Each antibody was titrated at 2- or 4-fold serial dilutions from 100 lAg/ml. -, No reactivity.tFor hemolysis test, the concentration ofmAb was 70 Mg/ml. -, Negative result. NT, not tested. PA-MHA, protein A-MHA; Rb a-mIg MHA,rabbit anti-mouse immunoglobulin-MHA.tMPC, mean positive count indicating fluorescence intensity. %+, percentage of positive cells. The concentration ofmAb was 100 tig/ml. NT,not tested. Lymph., lymphocytes; Mono., monocytes; Granulo., granulocytes.

DISCUSSION

There are a number of reasons for selecting Ley as anantigenic target for antibody-based therapeutic strategies inhumans: high frequency of LeY-expressing human tumors,homogenous Ley expression in primary and metastatic le-sions, and high density of Ley determinants represented onthe cell surface. In addition, Ley antibodies of suitableisotypes mediate strong complement-dependent cytotoxicityand antibody-dependent cellular cytotoxicity. In fact, wehave found that Ley and Ley-related structures appear to bethe predominant cellular antigens eliciting cytotoxic antibod-ies in mice immunized with MCF-7 human breast cancercells; cotyping initial hybridoma supernatants by cytotoxictests and ELISA with synthetic Ley indicated that 70% ofthewells with cytotoxic antibody showed Ley reactivity. Adrawback of Ley as an antigenic target in human cancers,shared with all tumor antigens identified to date, is theexpression of Ley in normal tissues. Epithelial cells in colon,stomach, breast, lung, and pancreas express Ley, but certaincancers have been reported to express higher levels of Leythan normal tissues (6-8). In addition to the degree of Leyexpression in normal and malignant tissues, the relativeaccessibility of normal vs. tumor tissue to circulating Leyantibodies needs to be ascertained, information that willcome from antibody biodistribution studies in patients.Although it is well known that antibodies generated against

peptides often do not react with the native protein, we had notexpected this to be so with carbohydrate determinants suchas Ley. However, several key features distinguished anti-bodies raised against synthetic Ley determinants and thoseraised against natural Ley. (i) The anti-synthetic Ley mAbsreacted well against synthetic Ley determinants but poorlyagainst natural Ley; (ii) the isotypes of the antibodies raisedagainst synthetic Ley were IgGl, IgG2a, or IgG2b, in contrast

to the IgM or IgG3 isotypes of antibodies to natural Ley; and(iii) even the most specific of the anti-synthetic Ley mAbscross-reacted with Lex or H-type 2 structures, whereasanti-natural Ley mAbs could be isolated that showed appar-ent exclusive specificity for Ley. These distinctions may beaccounted for by differences in the density/concentration ofLey determinants on the synthetic Ley-neoglycoproteins ascompared with natural Ley products and by the influence ofcarrier protein and linker on the immunogenicity, conforma-tion, and accessibility of Ley epitopes. We have made at-tempts to modify the immune response to synthetic Ley usingdifferent adjuvants and different immunization procedures,but these have not succeeded in changing specificity or mAbisotypes. Despite this inability of synthetic Ley to generateantibodies that react efficiently against natural Ley, Ley andother synthetic oligosaccharide determinants are extremelyuseful in specificity testing of antibodies raised against nat-ural carbohydrate determinants.

In addition to mAb 3S193 (the IgG3 anti-Ley mAb dis-cussed here), a number of other Ley mAbs have beendescribed (9-16). Two of the best characterized anti-LeymAbs are BR55-2 and B3. Both mAbs have high specificityfor LeY; however, they have been reported to react withclosely related structures, such as BLey for mAb BR55-2 (12)and dimeric Lex and extended Lex in the case ofmAb B3 (16).It will be interesting to determine the reactivity patterns ofmAb 3S193 with these and other Ley-related structures, suchas trifucosylated Ley and extended forms ofLey (29). Knowl-edge of these cross-reactions is of more than academicinterest, as illustrated by the hemolytic activity of mAbll8for 0 erythrocytes, an antibody originally thought to behighly specific for Ley but subsequently found to havereactivity for the H-type 2 structure (14). To assess cross-reactivity with BGR determinants, a range of antibody con-

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A

10 0.1 0.001

B

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mAb concentration ( ,g I ml)Dc

50 12.5 3.1

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Effector to target cell ratio

FIG. 1. ADCC mediated by humanized mAb 3S193#11. Fivecolon cancer cell lines (o, SW837; o, DLD-1; A, SW620; *, SK-CO-10; m, SW1222) (A and C) and three breast cancer cell lines (o,MCF-7; o, BT-20; m, ZR-75-1) (B and D) were incubated with.humanlymphocytes and humanized mAb 3S193#11. A and B show per-centage of specific release of 51Cr at 10-fold serial dilutions ofhumanized mAb 3S193#11. The effector/target cell ratio was 50:1.Cytotoxicity by effector cells alone was 0-21%, depending on targetcells, and these values are subtracted from the data given in eachexperiment. In C and D, humanized mAb 3S193#11 was tested at 1.0pg/ml (C) or 0.1 ;ug/ml (D) at different effector/target cell ratios.Effector cells alone gave 0-23% cytotoxicity at each ratio, and datashown are obtained by subtracting these background values. Anti-body alone gave -1% cytotoxicity.

centrations needs to be tested; cross-reactivity may not beobserved at 10 ,ug/ml but it may be seen at 25 ,ug/ml, a levelthat will probably be exceeded in clinical trials of anti-Leyantibodies. Further knowledge of the structures seen by Leyreagents on cells should help explain the basis of the lowreactivity of highly specific LeY reagents for granulocytes inflow cytometry and the strong hemolysis oferythrocytes withmAb 8S202, a mAb that shows excellent Ley specificity by allother tests.Chimeric and humanized forms of mAb 3S193 were gen-

erated in an attempt to recreate the specificity ofmouse mAb3S193 in a form more acceptable for clinical use. Cross-reaction with Ley-related antigens was seen for the chimericantibody (3S193#5); this may be a consequence of the

presence of nonauthentic residues at the termini of thevariable regions derived from the expression vectors. Sub-stitution of HuVK for the MuVK chain, giving mAb3S193#6, exacerbated the cross-reactivity, whereas conver-sion to a fully humanized antibody (mAb 3S193#7) restoredthe specificity, suggesting that the nature of the heavy- andK-chain variable-region interface influences antigen binding.Inclusion of an additional mouse framework residue in eachchain produced a molecule, mAb 3S193#11, with improvedreactivity and serological properties closely approximatingthose of its mouse counterpart. The availability of thishumanized Ley reagent with high specificity and strongbiological functions will facilitate clinical exploration of Leyas a therapeutic target in human cancer.

We thank Drs. Y. Noguchi, D. Rafter, G. Ritter and W. J. Rettigfor helpful discussions, and Mrs. S. King and Mrs. M. Fernie fortechnical assistance. This work was supported, in part, by grantsfrom the U.S. Public Health Service (CA 08748, CA 33049, and CA52477) and the Avon Program in Ovarian Cancer.

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