`Vol. 233, No. 2, September, pp. 501-506, 1984
`
`Monoclonal Antibodies PMN 6, PMN 29, and PM-81 Bind Differently
`
`to Glycolipids Containing a Sugar Sequence Occurring
`Ill
`in Lacto-N-Fucopentaose
`
`JOHN L. MAGNANI,* EDWARD D. BALL,t MICHAEL W. FANGER,t
`SEN-ITIROH HAKOMORI,t AND VICTOR GINSBURG*
`
`•National Institute of Arthriti.s, Diabetes, and Digestive and Kidney Disease, National Institutes of lfealih,
`Bethesda, Maryland 20205; tDepartments of Medicine and Microbio/,ogy, Dartmauth Medical. ScJwol,
`of Biochemical OtzcokJgy, Fred Hutchinson Cancer Research
`Hanover, New Ha111pshire 09756; a.nd fl)ivision
`Center and Department of Pathobiclo(ly, University of Washington, Seattle, Washingtqn 98104
`
`Received March 29, 1984, and in revised form May 7, 1984
`
`Three monoclonal antibodies, PMN 6, PMN 29, and PM-81, bind myeloid cells. An
`tibodies PMN 6 and PMN 29 bind specifically to granulocytes but differ in their ability
`to bind some other cell lines [E. D. Ball, R. F. Graziano, L. Shen, and M. W. Fanger
`(1982) Proc. Natl Acad. Sci USA 79, 5374-5378]. Antibody PM-81, in addition to gran
`ulocytes, also binds to eosinophils, monocytes, and most acute myelocytic leukemia cells
`[E. D. Ball, R. F. Graziano, and M. W. Fanger (1983) J. ImmunoL 130, 2937-2941). Despite
`these differences, the binding of all three antibodies to cells was inhibited by the
`oligosaccharide, lacto-N-fucopentaose III [Gal/jl-4(Fucal-3)GlcNAc/jl-3Gal/jl-4Glc].
`Solid-phase radioimmunoassays using purified glycolipids containing sugar sequences
`found in lacto-N-fucopentaose III demonstrated different binding characteristics for
`each antibody. PM-81 bound lower concentrations of glycolipids than PMN 29, while
`PMN 6 required the highest concentration of glycolipids for binding. Autoradiography
`of thin-layer chromatograms of glycolipid antigens supported these results. The binding
`of these monoclonal antibodies to cells probably depends on the density of antigens on
`the cell surface, each antibody requiring a different density. Thus, cells containing
`antigen below a certain threshold concentration may not bind low-affinity antibodies.
`
`Many monoclonal antibodies with ap
`parent specificities for various cells are di
`rected against the sugar sequence
`
`Gal/jl-4GlcNAcf11-3Gal · · ·
`3
`I
`Fucal
`
`which occurs in the human milk oligosac
`charide, lacto-N-fucopentaose III (1). This
`sequence is very immunogenic in mice, and
`is a marker for human adenocarcinoma of
`the colon, stomach (2, 3), and lung (4), as
`well as granulocytes and granulocyte pre
`cursors (5-8). It is also the murine em
`bryonic antigen known as SSEA-1 (9, 10).
`
`Although the antigen is restricted to
`myeloid cells among hemopoetic cells as
`evidenced by immunoftuorescence studies
`(5-8), small amounts of antigen were de
`tected in glycolipids from erythrocytes by
`immunostaining of thin-layer chromato
`grams (2). Recently, three monoclonal an
`tibodies, PMN 6, PMN 29, and PM-81, have
`been described which bind differently to
`cells of the myeloid series, including gran
`ulocytes, monocytes, and blasts from pa
`tients with acute myelogenous leukemia
`(11, 12). Despite these differences, the
`binding of all three antibodies is inhibited
`by lacto-N-fucopentaose III. The data in
`the present paper suggest that differences
`
`501
`
`0003-9861/84 $3.00
`Copyright e 1984 by Academie Pres.•. Inc.
`All righl.8 of reproduetion in any form res-erved.
`
`1 of 6
`
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`
`MAGNANI ET AL.
`
`in the binding affinity of these antibodies
`for their antigen may explain their differ
`ential reactivities.
`
`manner were analyzed for fluorescence on the Ortho
`
`Cytolluorograph System 50H.
`&I.id-Phase radicnm»it•noassay. The binding of an
`
`li body to purified glycolipids was measured by solid
`
`
`
`phase radioimmunoassay as previously described (18,
`EXPERIMENTAL PROCEDURES
`
`
`19) with minor modifications. Glycolipids in 30 µI
`methanol were added to the wells of a round-bottom
`
`Ma.terials. Monoclonal antibodies P.MN 6 and PMN
`
`
`
`polyvinylchloride microtiter plate (Dynatech, Alex
`
`29 are produced by hybridoma.s prepared from spleen
`
`andria, Va.), and the solutions were dried by evap
`
`cells obtained from mice immunized with neutrophils
`
`oration. The wells were then filled with 0.05 M Tris
`from normal donors (11); PM-81 is produced by a
`HCI, pH 7.8, containing0.15MNaCl,1% bovine serum
`hybridoma prepared from spleen cells obtained from
`albumin, and 0.1 % NaN8 (Buffer A). After 30 min, the
`
`a mouse immunized with the promyelocytic leukemia
`wells were emptied and to each was added 30 141 buffer
`
`cell line, HL-60 (12). Monoclonal anlibody AML-1-
`
`A containing5 µg/ml monoclonal antibody. The wells
`
`201 binds {J-2 microglobulin (12), and was used as a
`
`were covered with paralilm, incubated for 3 h at 22°C
`
`
`control antibody for these studies. All four antibodies
`washed once with buffer A, and then to each w�
`are of the lgM isotype.
`
`added 100,000 cpm of '"'I-labeled goal anti-mouse lgM
`
`Lacto-N-fucopen taosyl(III)ceramide (ITrFucnLc
`(40-50 µCi/µg) in 30 µl buffer A. After 3 h, the wells
`Ose,Cer) was prepared from human colonic adeno
`were washed six times with cold phosphate-buffered
`
`
`carcinoma as previously described (3). The glycolipid
`
`saline (0.15 M NaCl, 0.01 M sodium phosphate, pH 7.4),
`
`
`was further purified by rechromatography on HPLC
`cut from the plate, and assayed for 1"'I in an Auto
`and was freed from lacto-N-fucopentaosyl(II)ce
`Gamma spectrometer.
`
`ramide (Le• glycolipid; m• FucLc0se4Cer). The Yt
`Atdoradiographv of gl'l/IX)lipid antigen& Glycolipid
`
`glycolipid (V'FucnLc()sesCer) was prepared from hu
`
`
`
`antigens were detected on thin-layer chromatograms
`
`man erythrocytes as previously described (13). Di
`
`
`
`
`
`by autoradiography as previously described (19) with
`
`fucosyl lacto-N-1Wrhexaosylceramide (bands 4a-e;
`
`minor modifications. Glycolipids were chromato
`
`III'V'Fu�Os�Cer) was prepared from a human
`
`graphed on aluminum-backed high-performance thin
`
`colonic cancer metastasis in the liver (14).
`
`
`layer chromatography plates (silica gel 60, E. Merck,
`Globoside was purchased from Supelco Inc., Bel
`
`Darmstadt, West Germany) in chloroform/methanol/
`
`
`lefonte, Pennsylvania. Sialylated lacto-N-fucopen
`
`0.25% KCI (50/50/12, by volume). The dried chro
`
`taosyl(III)ceramide was kindly provided by Dr. H.
`
`matogram was soaked for 1 min in a 0.1% solution
`
`
`RauvaJa (Unive.rsity of Helsinki, Helsinki, Finland).
`
`of polyisobutylmetbacrylate beads (Polysciences, Inc.,
`
`
`Oligosaccharides, lacto-N-fucopentaose Ill, and lato
`
`
`Warrington, Pa.) dissolved in hexane. After drying
`
`
`N-fucopentaose I were isolated Crom human milk as
`in air, the chromatogram was sprayed with phos
`
`previously described (15).
`
`phate-buffered saline (0.15 M NaCl, 0.01 M sodium
`
`Affinity-purified goat anti-mouse JgM (Kirkegaard
`
`
`phosphate, pH 7.4) and immediately soaked in buffer
`
`
`and Perry Laboratories, Inc., Gaithersburg, Md.) was
`A until all of the silica gel was wet (about 15 min).
`
`
`iodinated with NaUGJ (ICN Biochemicals,lrvine, Calif.)
`
`The plate wa.s then removed and laid horizontally on
`
`to a specific activity of about 40 µCi/ µg using Iodogen
`
`a slightly smaller glass plate in a large Petri dish.
`(16) (Pierce Chemical Co., Rockford, nl.).
`
`Monoclonal antibody (5 µg/ml) diluted in buffer A
`
`Total lipid extracts were prepared by homogeni
`waa layered on the plate (60 µl/cm1 chromatogram
`zation of cells in chloroform/methanol/H20
`(301601
`
`
`surface). After incubation at 22° for 2 h, the chro
`4, final ratio) (17).
`matogram was washed by dipping in four successive
`Inhibiti-On of binding of antibodies to cells by oligo
`
`changes of cold phosphate-buffered saline at 1-min
`saccho.ride& Monoclonal antibodies PMN 6, PMN 29,
`
`
`intervals, and overlayed with buffer A containing 2
`
`PM-81, and AML-1-201 (5 µg/ml) were preincubated
`
`
`X 10' cpm/ml •2'SJ-labeled goat anti-mouse IgM. After
`
`with 5.4 mM lacto-N-fucopentaose I or lacto-N-fu
`
`1 h at 22°C, the chromatogram was washed as before
`
`
`copentaose III for 30 min at room temperature. This
`
`
`in cold phosphate-buffered saline, dried, and exposed
`
`mixture was added to 10' neutrophils previously
`
`to Xar-5 X-ray film (Eastman-Kodak, Rochester,
`
`
`washed with phosphate-buffered saline, pH 7.4, con
`N. Y.) for 10 h at 22°C.
`
`taining 0.1 % bovine serum albumin anti 0.05% sodium
`azide, and incubated for 30 min at4°C. After washing
`
`with the same buffer, fluorescein isothiocyanate-con
`RESULTS
`jugated goat F(ab'}2 antibody directed to mouse im
`
`
`munoglobulin (Boehringer-Mannheim, Indianapolis,
`EJf eds of Oligosaccharides on
`
`Ind.) was added and incubated for 30 min at 4•c.
`Cell Binding
`
`
`Controls in which each monoclonal antibody was in
`
`cubated with neutrophils in the absence of oligosac
`Monoclonal antibodies PMN 6, PMN 29,
`
`charides were run in parallel. Cells treated in this
`and PM-81 bound to most neutrophils, and
`
`2 of 6
`
`BI Exhibit 1007
`
`
`
`DIFFERENTIAL PMN 6, PMN 29, AND PM-81 BINDING TO GLYCOLIPIDS
`
`503
`
`this binding was completely inhibited by
`5.4 mM lacto-N-fucopentaose III but not by
`5.4 mM lacto-N-fucopentaose I (Fig. 1).
`Neither oligosaccharide
`inhibited
`the
`binding of monoclonal antibody AML-1-
`201, an lgM which binds {3-2 microglobu
`lin (12).
`
`Autoradiography of Glycolipid Antigens
`
`Glycolipid antigens were detected by au
`toradiography of thin-layer chromato
`grams as described under Experimental
`Procedures. Purified glycolipids Y2 and 4c,
`which contain a carbohydrate sequence
`found in lacto-N-fucopentaose III (see Ta
`ble 1), bound PMN 6. PMN 29, and PM-81
`(Figs. 2A, B, C, lanes 1). The smaller
`glycolipid, lacto-N-fucopentaosyl(IIl)cer-
`
`amide, bound only PMN 29 and PM-81 un
`der these conditions.
`The reactivity of these antibodies, par
`ticularly PMN6, resembled that of SSEA-
`1 (9, 10); ZWG 13, ZWG 14, and ZWG 111
`(2); and Fll-1and1'�H-5 (20); which do not
`bind as well to lacto-N-fucopentaosyl
`(IIl)ceram ide as to glycolipids with longer
`carbohydrate chains, including di- and tri
`fucosylated derivatives.
`All three antibodies detected glycolipid
`antigens from total lipid extracts of gran
`ulocytcs and HL-60 cells (Figs. 2A, B, and
`C; lanes 6 and 7). Both of these cell types
`have high concentrations of glycolipids
`containing a carbohydrate sequence found
`in lacto-N-fucopentaose III (5). Antigen
`comigrating with lacto-N-fucopentaosyl
`(Ill)ceramide, however, was not detected
`
`A
`
`... . ,
`
`D
`
`G
`
`PWH t
`
`Allill 12'01
`
`i......i ...... __ _JI �-.... --.1
`
`8
`
`, .. .. ,
`• LNr I
`
`E
`
`H
`
`,MN I
`• ltrf, I
`
`AML 1 201
`• LHF I
`
`«
`...
`
`; :>
`z
`� ..
`
`u
`
`c
`
`F
`
`AlllL 1.2Qi
`• LHF trl
`
`FLUOllESCENCE INTENSITY
`
`FIG. 1. The effect of lacto-N-fucopentaose I {LNF I) and lacto-N-fucopentaose Ill (LNF III) on
`the binding of monoclonal antiborues PM-81, PMN 29, PMN 6, and AML-1-201 was determined by
`cytoftuorography as described under Experimental Procedures.. The fluorescence of neutrophils
`stained with PM-81, PMN 29, PMN 6, and AML-1-201 is shown in panels A, 0, G, and J, respectively.
`The effect of lacto-N-fucopentaose I and lacto-N-fucopentaose III on this fluorescence is shown in
`panels B, E, H, and K, and panels C, F, I, and L, respectively.
`
`3 of 6
`
`BI Exhibit 1007
`
`
`
`504
`
`MAGNANI ET AL.
`
`TABLE I
`STRUCl'URE OF CARBOHYDRATES
`
`Name
`
`Structure
`
`Glycolipids
`Lacto-N-fucopentaosyl(Ill)ceramide Galtll-4GlcNActll-3Galtll-4G lctll-1 Cer
`3
`I
`Fucal
`Galtll-4GlcNActll-3Galtll-4GlcNActll-3GalPl-4GlcPl-1Cer
`3
`3
`I
`I
`Fucal
`Fucal
`
`Galtll-4G lcN Actll-3Galtll-4G lcNActll-3Galtll-4G lctll-1 Cer
`3
`I
`Fucal
`
`Globoside
`Oligosaccharides
`
`Lacto-N-fucopentaose III
`
`I
`Lacto-N-fucopentaose
`
`
`
`GalNActll-3Galal-4Gal,11-4Glctll-1Cer
`
`Galtll-4G lcNActll-3Galtll-4G le
`3
`I
`Fucal
`Fucal-2Galtll-3GlcNAc,11-3Gal,11-4Glc
`
`by PMN 6. The same chromatographic pat
`lipid extracts of acute myelocytic leukemia
`
`
`cells or monocytes by antibodies PMN 6
`
`tern was obtained by all three antibodies;
`and PMN 29 (Figs. 2A and B, lanes 2, 3, 4,
`however, the intensity of staining in
`creased from PMN 6 to PMN 29 to PM-81.
`
`5). Under the same conditions PM-81 de
`tected low levels of antigen in both extracts
`No antigens were detected in the total
`
`A
`
`B
`
`c
`
`LNF • cer -
`
`-
`
`-
`
`4'c- -
`
`Origin-
`
`1 2 345
`
`6 7 1 2 3 " 5 6 7
`
`FIG. 2. Autoradiography of glycolipid antigens. Autoradiography of glycolipid antigens was per
`
`
`
`
`
`
`
`formed as described under Experimental Procedures. (A) was stained with antibody PMN 6, (BJ
`
`with PMN 29, and (C) with PM-81, each at 5 µg/ml. Purified glycolipids (30 ng) 4c, Y2, and lacto·
`
`
`N-fucopentaosyl(III)ceramide (LNF III cer) were chromatographed in lane 1. The amount of extract
`is lane 2, 5
`
`
`chromatographed expressed as the volume of packed cells from which it was obtained
`
`µI AML blasts; Jane 3, 2 µI AML blasts; lane 4, 5 µI monocytes; lane 5, 2 µI monocytes; lane 6, 2 µI
`
`
`
`
`granulocytes; and lane 7, 2 µI HL-60 cells. The positions of the purified glycolipids are shown on
`the left.
`
`4 of 6
`
`BI Exhibit 1007
`
`
`
`DIFFERENTIAL PMN 6, PMN 29, AND PM-81 BINDING TO CL YCOLIPIDS
`
`505
`
`(Fig. 2C, lanes 2, 3, 4, 5). These data support
`the previous findings that PMN 6 and PMN
`29 bind to neutrophils (11) while PM-81
`binds to neutrophils, monocytes, and acute
`myelocytic leukemia cells (12).
`
`Solid-Phase Radiaimmunoassay
`
`Monoclonal antibodies PMN 6, PMN 29,
`and PM-81 were assayed for binding to pu
`rified glycolipids by solid-phase radioim
`munoassay as described under Experi
`mental Procedures. Differences in binding
`were found for each .antibody as shown in
`Fig. 3. PM-81 bound to the lowest concen
`tration of glycolipids containing sugar se
`quences found in lacto-N-fucopentaose III.
`Higher concentrations of glycolipids were
`required for binding antibody PMN 29.
`PMN 6 showed the least binding to high
`concentrations of glycolipids Y2 and 4c,
`and did not bind to lacto-N-fucopeotao
`syl(IIl)ceramide at the concentrations
`tested. These results agree with the inten
`sity of staining of glycolipid antigens
`shown in Fig. 2.
`Differences in binding were also found
`for each purified glycolipid. All three an
`tibodies bound to glycolipids Y2 and 4c at
`lower concentrations than to lacto-N-fu
`copentaosyl(Ill)ceramide (Fig. 3). These
`results also agree with the chromato
`graphic patterns of glycolipid antigens
`shown in Figure 2. None of these antibodies
`bound to a monosialoganglioside contain
`ing sialic acid linked a2-3 to the termi
`nal galactose of lacto-N-fucopentaosyl
`(III)ceramide (data not shown).
`
`.......
`.. .
`
`*'!ll
`' .. .. . ,,. ..
`' - - • � &i
`Cil_Yta._Al)�U
`
`' • •
`
`Frc. 3. Binding of antibodies to purified glycolipids.
`Solid-phase radioimmunoassays were performed as
`described under Experimental Procedures. Antibody
`PMN 6 was used for assays in (A), PMN 29 for (B).
`and PM-81 for (C). Purified glycolipids tested were
`4c, t:.; Y1, O; lacto-N-fucopentaosyl(UI)ceramide, O;
`and globoside, o. Structures of these glycolipids are
`depicted in Table I.
`
`DISCUSSION
`
`The carbohydrate sequence
`
`Gal/H-4GlcNAcfJ1-3Gal · · ·
`3
`I
`Fucal
`
`is a potent antigen to the mouse. Out of
`325 monoclonal antibodies from different
`laboratories that have been analyzed in our
`laboratory, 55 are directed against this se
`quence (21).
`Some antibodies directed against the
`same antigen, as judged by hapten binding
`or hapten inhibition studies, have different
`cell specificities. For example, a rabbit anti
`paragloboside antibody (22) and a Wal
`denstrom cold agglutinin (cold agglutinin
`McC) (23) both bind to paragloboside
`(GalfJ1-4GlcNAcfJ1-3GalfJl-4GlcfJ1-1Cer),
`yet react differently with cells: the rabbit
`antibody reacts equally well with human
`cord and adult erythrocytes (22) while the
`cold agglutinin reacts strongly with cord
`cells but weakly or not at all with adult
`cells (23). This differential r-eactivity might
`be explained in some cases by the fact that
`some antibodies bind to different parts or
`to different sides of the same sugar chain
`(18, 20, 24, 25). If the adult erythrocyte
`antigen were actually substituted para
`globoside and the two antibodies bound to
`different parts of the paragloboside sugar
`chain, the antibodies would react differ
`ently with the substituted paragloboside
`depending on where the substitution oc
`curred. This hypothesis, however, is not
`likely to explain the differential reactivity
`of antibodies PMN 6, PMN 29, and PM-81
`with various cell types, as the three an
`tibodies appear to bind to the same gly
`colipid antigens (Fig. 2). It is more likely
`that their differential reactivity is ex
`plained by their different affinities for an
`tigen (Fig. 3). PM-81 has the highest af
`finity and binds to more cell types than do
`PMN 6 or PMN 29. It is the only antibody
`that binds monocytes (11, 12) which contain
`little glycolipid antigen (Fig. 2C, lane 4).
`PMN 29 detects intermediate concentra
`tions of glycolipid antigen and binds to
`some cell lines that PMN 6 does not (11).
`Thus, cells that contain antigen below a
`
`i :t A l . If' L L
`A: f
`
`
`-
`
`..
`
`I �
`r
`
`5 of 6
`
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`
`
`
`506
`
`MAGNANI ET AL.
`
`certain threshold concentration may bind
`high-affinity but not low-affinity antibod
`ies. Glycoproteins containing the same
`carbohydrate sequence may also be in
`volved in antibody binding (26, 27).
`
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`
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`244, 5496.
`2. BROCKHAUS, M., MAGNANI, J. L., HERLYN, M.,
`8!.AsZC'LYK, M., Sn:PLEWSKJ, Z., KOPROWSKI, H.,
`AND GINSBURG, V. (1982) Arch. Bi.ochem... BiJ>.
`ph'IJS. 217, 647-651.
`3. HAKOMORI, S., NUDELMAN, E., KANNAGl, R., AND
`LEVERY, S. B. (1982) Bi.ochem. Biophys. Res.
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`CUTI'ITTA, F .• ROSEN, s., MINNA, J. D .• AND
`GINSBURG, V. (1983) Arch. Bi.ochem. Bioph11s.
`220, 318-320.
`5. HUANG, L. C., CIVIN, C. I., MAGNANI, J. L., SHAPER,
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`6. GJRARDET, C., LADISCH, S., HEUMANN, D., MACH,
`J. P., AND CARREL, s. (1983) Im J. Cancer 32,
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`HAKOMORI, S. (1982) J. Bid Chem. 257, 14865-
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`15. KOBATA, A. (1972) in Methods in Enzymology
`(Ginsburg, V., ed.), Vol. 28, p. 262, Academic
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`16. SALACINSKI, P. R. P., MCLEAN, C., SYKES, J.E. C.,
`CLEMENT-JONES, V. V., AND LoWRY, P. J. (1981)
`Anal Bi.ochem. 117, 136-146.
`17. SVENNERHOLM, L., AND FREDMAN, P. (1980)
`Bi.ochim .. Bi&phys. Acta 617, 97-109.
`18. YOUNG, w. w., JR., MACDONALD, E. M. s .. Now
`INSKY, R. C., AND HAKOMOR:I, S. (1979) J. EX'f).
`Med. 150, 1008-1019.
`19. MAGNANC, J. L., NILSSON, B., BROCKHAUS, M .. ZoPF,
`D., STEPLEWSKI, Z., KOPROWSKI, H., AND GINS·
`BURG, V. (1982) J. Bid Chem.. 257, 14365-14369.
`20. FuKUSHI, Y., HAKOMORI, S., NUDELMAN, E., AND
`CocHRAN, N. (1984) J. Bid. Chem. 259, 4681-
`4685.
`21. GINSBURG, V., FREDMAN, P., AND MAGNANI,
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`
`6 of 6
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`BI Exhibit 1007
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