OF BIOCHEMISTRY
`ARCHIVES
`Vol. 233, No. 2, September,
`
`BIOPHYSICS
`AND
`pp. 501-506,
`1984
`
`Monoclonal Antibodies PMN 6, PMN 29, and PM-81 Bind Differently
`to Glycolipids Containing
`a Sugar Sequence Occurring
`in Lacto-N-Fucopentaose
`III
`
`JOHN
`
`L. MAGNANI,*
`SEN-ITIROH
`
`EDWARD
`HAKOMORI,$
`
`W. FANGER,t
`D. BALL,-/. MICHAEL
`AND VICTOR GINSBURG*
`
`Institutes of Health,
`Institute of Arthritis, Diabetes, and Digestive and Kidney Disease, National
`*National
`Bethesda, Maryland
`tDepartments
`of Medicine and Microbiology, Dartmouth Medical School,
`20205;
`New Hampshire
`03756; and $Div-ision of Biochemical 0ncology, Fred Hutchinson Cancer Research
`Hanover,
`Center and Department of Path&o&p,
`University of Washington, Seattle, Washington 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. Soi. USA 79,5374-53’781. 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 130,293’7-29411. Despite
`these differences,
`the binding of all three antibodies
`to cells was inhibited by the
`oligosaccharide,
`lacto-N-fucopentaose
`III
`[Gal~l-4(FuccY1-3)GlcNAc~l-3Gal~l-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
`
`* .
`
`Galal-4GlcNAc/31-3Gal.
`3
`I
`Fuca 1
`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).
`
`to
`is restricted
`the antigen
`Although
`myeloid cells among hemopoetic cells as
`evidenced by immunofluorescence 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
`
`$3.00
`0003-9861/84
`Press,
`Copyright
`0 1984 by Academic
`All
`rights
`of reproduction
`in any
`form
`
`Inc.
`reserved.
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1007 Page 1 of 6
`
`

`
`502
`
`MAGNANI
`
`ET AL.
`
`in the binding affinity of these antibodies
`for their antigen may explain their differ-
`ential reactivities.
`
`EXPERIMENTAL
`
`PROCEDURES
`
`on
`
`the Ortho
`
`fluorescence
`for
`analyzed
`were
`manner
`50H.
`System
`Cytofluorograph
`Solid-Phase
`of an-
`The binding
`radioimmunoassay.
`was measured
`by solid-
`tibody
`to purified
`glycolipids
`as previously
`described
`(18,
`phase
`radioimmunoassay
`19) with minor
`modifications.
`Glycolipids
`in 30 ~1
`methanol
`were
`added
`to the wells
`of a round-bottom
`polyvinylchloride
`microtiter
`plate
`(Dynatech,
`Alex-
`and
`andria,
`Va.),
`the
`solutions
`were
`dried
`by evap-
`oration.
`The wells were
`then
`filled with
`0.05 M Tris-
`HCI,
`pH 7.8, containing
`0.15 M NaCl,
`1% bovine
`serum
`albumin,
`and 0.1% NaN3
`(Buffer
`A). After
`30 min,
`the
`wells were
`emptied
`and
`to each was added
`30 ~1 buffer
`A containing
`5 rig/ml monoclonal
`antibody.
`The wells
`were
`covered
`with
`parafilm,
`incubated
`for 3 h at 22’C,
`washed
`once with
`buffer
`A, and
`then
`to each was
`added
`100,000
`cpm of lw’I-labeled
`goal anti-mouse
`IgM
`(40-50
`&i/&g)
`in 30 (~1 buffer
`A. After
`3 h,
`the wells
`were washed
`six
`times with
`cold phosphate-buffered
`saline
`(0.15 M NaCl,
`0.01 M sodium
`phosphate,
`pH 7.4),
`cut
`from
`the plate,
`and assayed
`for
`‘%I
`in an Auto-
`Gamma
`spectrometer.
`antigens. Glycolipid
`of glycolipid
`Autoradiography
`chromatograms
`antigens
`were
`detected
`on
`thin-layer
`described
`(19) with
`by autoradiography
`as previously
`were
`chromato-
`minor
`modifications.
`Glycolipids
`graphed
`on aluminum-backed
`high-performance
`thin-
`layer chromatography
`plates
`(silica
`gel 60, E. Merck,
`Darmstadt,
`West Germany)
`in chloroform/methanol/
`chro-
`0.25%
`KC1
`(50/50/12,
`by
`volume).
`The
`dried
`matogram
`was
`soaked
`for
`1 min
`in a 0.1%
`solution
`of polyisobutylmethacrylate
`beads
`(Polysdencea,
`Inc.,
`Warrington,
`Pa.)
`dissolved
`in hexane.
`After
`drying
`in air,
`the
`chromatogram
`was
`sprayed
`with
`phos-
`phate-buffered
`saline
`(0.15 M NaCl,
`0.01 M sodium
`phosphate,
`pH 7.4) and
`immediately
`soaked
`in buffer
`A until
`all of
`the silica
`gel was wet
`(about
`15 min).
`The plate was
`then
`removed
`and
`laid horizontally
`on
`a slightly
`smaller
`glass
`plate
`in a large
`Petri
`dish.
`Monoclonal
`antibody
`(5 pg/ml)
`diluted
`in buffer
`A
`was
`layered
`on
`the plate
`(60
`).d/cm’
`chromatogram
`surface).
`After
`incubation
`at 22’
`for
`2 h,
`the
`chro-
`matogram
`was washed
`by dipping
`in four
`successive
`changes
`of cold phosphate-buffered
`saline
`at 1-min
`buffer
`intervals,
`and overlayed
`with
`A containing
`X lo6 cpm/ml
`?-labeled
`goat anti-mouse
`IgM. After
`1 h at 22”C,
`the chromatogram
`was washed
`as before
`in cold phosphate-buffered
`saline,
`dried,
`and exposed
`to Xard
`X-ray
`film
`(Eastman-Kodak,
`Rochester,
`N. Y.)
`for 10 h at 22°C.
`
`2
`
`6 and PMN
`PMN
`antibodies
`Monoclonal
`Materials.
`from
`spleen
`29 are produced
`by hybridomas
`prepared
`neutrophils
`cells obtained
`from mice
`immunized
`with
`from
`normal
`donors
`(11); PM-81
`is produced
`by a
`hybridoma
`prepared
`from
`spleen
`cells obtained
`from
`a mouse
`immunized
`with
`the promyelocytic
`leukemia
`cell
`line, HL-60
`(12). Monoclonal
`antibody
`AML-l-
`201 binds
`p-2 microglobulin
`(12),
`and was used as a
`control
`antibody
`for
`these
`studies.
`All
`four
`antibodies
`are of
`the
`IgM
`isotype.
`(IIPFucnLc-
`Lacto-N-fucopentaosyl(III)ceramide
`colonic
`adeno-
`human
`Ose&er)
`was
`prepared
`from
`carcinoma
`as previously
`described
`(3). The glycolipid
`by
`was
`further
`purified
`rechromatography
`on HPLC
`and
`was
`freed
`from
`lacto-N-fucopentaosyl(II)ce-
`yz
`The
`ramide
`(Le”
`glycolipid,
`III’
`FucLcOse&er).
`hu-
`from
`glycolipid
`(V*FucnLcOse&er)
`was prepared
`(13). Di-
`man
`erythrocytes
`as previously
`described
`fucosyl
`lacto-N-norhexaosylceramide
`(bands
`4a-e;
`II18V3FucznLcOse&er)
`was
`prepared
`from
`a human
`colonic
`cancer metastasis
`in
`the
`liver
`(14).
`Inc., Bel-
`Globoside
`was
`purchased
`from
`Supelco
`lefonte,
`Pennsylvania.
`Sialylated
`Iacto-N-fucopen-
`taosyl(III)ceramide
`kindly
`provided
`by Dr. H.
`was
`Rauvala
`(University
`of Helsinki,
`Helsinki,
`Finland).
`Oligosaccharides,
`la&o-N-fucopentaose
`III, and
`lato-
`N-fucopentaose
`I were
`isolated
`from
`human
`milk
`as
`previously
`described
`(15).
`IgM
`Affinity-purified
`goat
`anti-mouse
`and Perry
`Laboratories,
`Inc., Gaithersburg,
`iodinated
`with Nam’I
`(ICN Biochemicals,Irvine,
`to a specific
`activity
`of about
`40 pCi/Fg
`using
`(16)
`(Pierce
`Chemical
`Co., Rockford,
`Ill.).
`were prepared
`Total
`lipid
`extracts
`by homogeni-
`zation
`of cells
`in chloroform/methanol/Hz0
`(30/60/
`4, final
`ratio)
`(17).
`to ceL% bg oligo
`Inhibition
`of binding of antibodies
`PMN
`6, PMN
`29,
`saccharides.
`Monoclonal
`antibodies
`were
`preincubated
`PM-81,
`and AML-l-201
`(5 fig/ml)
`I or
`lacto-hr-fu-
`with
`5.4 mM
`la&o-N-fucopentaose
`temperature.
`This
`copentaose
`III
`for 30 min at room
`mixture
`was
`added
`to
`lo6
`neutrophils
`previously
`washed
`with
`phosphate-buffered
`saline,
`pH 7.4, con-
`taining
`0.1% bovine
`serum
`albumin
`and 0.05%
`sodium
`azide, and
`incubated
`for 30 min at 4°C. After
`washing
`with
`the same buffer,
`fluorescein
`isothiocyanate-con-
`jugated
`goat F(ab’)Z
`antibody
`directed
`to mouse
`munoglobulin
`(Boehringer-Mannheim,
`Indianapolis,
`Ind.)
`was
`added
`and
`incubated
`30 min
`at 4°C.
`Controls
`in which
`each monoclonal
`antibody
`was
`in-
`cubated
`with
`neutrophils
`in
`the absence
`of oligosac-
`charides
`were
`run
`in parallel.
`Cells
`treated
`in
`this
`
`(Kirkegaard
`Md.) was
`Calif.)
`Iodogen
`
`for
`
`im-
`
`RESULTS
`
`Eflects of Oligosaccharides
`Cell Binding
`
`on
`
`Monoclonal antibodies PMN 6, PMN 29,
`and PM-81 bound to most neutrophils, and
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1007 Page 2 of 6
`
`

`
`DIFFERENTIAL
`
`PMN
`
`6, PMN
`
`29, AND
`
`PM-81
`
`BINDING
`
`TO GLYCOLIPIDS
`
`503
`
`by
`inhibited
`this binding was completely
`III but not by
`5.4 mM lacto-iV-fucopentaose
`I (Fig. 1).
`5.4 mM
`lacto-N-fucopentaose
`inhibited
`the
`Neither
`oligosaccharide
`antibody
`AML-l-
`binding
`of monoclonal
`201, an IgM which binds p-2 microglobu-
`lin (12).
`
`Autwadiography
`
`of Glyco1ip.d Antigens
`
`antigens were detected by au-
`Glycolipid
`of
`thin-layer
`chromato-
`toradiography
`grams as described under Experimental
`Procedures. Purified glycolipids
`Yz and 4c,
`which contain
`a carbohydrate
`sequence
`III
`found
`in lacto-N-fucopentaose
`(see Ta-
`ble I), bound PMN 6, PMN 29, and PM-81
`(Figs. 2A, B, C, lanes 1). The smaller
`glycolipid,
`lacto-N-fucopentaosyl(III)cer-
`
`amide, bound only PMN 29 and PM-81 un-
`der these conditions.
`par-
`The reactivity
`of these antibodies,
`ticularly
`PMNG,
`resembled
`that of SSEA-
`1 (9,10); ZWG 13, ZWG 14, and ZWG 111
`(2); and FH-1 and FH-5 (20); which do not
`bind
`as well
`to
`lacto-N-fucopentaosyl-
`(1II)ceramide
`as to glycolipids with
`longer
`carbohydrate
`chains,
`including
`di- and tri-
`fucosylated
`derivatives.
`detected glycolipid
`All
`three antibodies
`antigens
`from
`total
`lipid extracts of gran-
`ulocytes 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-
`(III)ceramide,
`however, was not detected
`
`A
`
`PM 81
`
`D
`
`PIN 19
`
`FLUORESCENCE
`
`INTENSITY
`
`FIG. 1. The effect
`III)
`(LNF
`III
`lacto-N-fucopentaose
`I) and
`I (LNF
`lacto-N-fucopentaose
`of
`was determined
`6, and AML-l-201
`29, PMN
`PMN
`PM-81,
`the binding
`of monoclonal
`antibodies
`Procedures.
`The
`fluorescence
`of neutrophils
`Experimental
`cytofluorography
`as described
`under
`is shown
`in panels A, D, G, and J, respectively.
`stained
`with PM-81,
`PMN
`29, PMN
`6, and AML-1-201
`The effect
`of
`lacto-N-fucopentaose
`I and
`lacto-N-fucopentaose
`III
`on
`this
`fluorescence
`is shown
`panels
`B, E, H, and K, and panels
`C, F,
`I, and L, respectively.
`
`on
`by
`
`in
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1007 Page 3 of 6
`
`

`
`MAGNANI
`
`ET AL.
`
`STRUCNRE
`
`I
`TABLE
`OF CARBOHYDRATES
`
`Structure
`
`Gal~l-4GleNAc~1-3Gal~l-4Glc~1-1Cer
`3
`I
`Fuccu 1
`Gal~l-4GlcNAc~1-3Gal~l-4GlcNAc~l-3Gal~l-4Glc~1-1Cer
`3
`3
`I
`I
`Fuccu 1
`Fuccu 1
`Gal~1-4GlcNAc~1-3Gal~l-4GlcNAc~l-3Gal~l-4Glc~1-1Cer
`3
`I
`Fuccu 1
`
`GalNAc~l-3Galal-4Gal~1-4Glc~l-1Cer
`
`Galj31-4GlcNAc/31-3Gal~l-4Glc
`3
`I
`FUW 1
`FuccY1-2Gal~l-3GlcNAcj3l-3Gal~l-4Glc
`
`Name
`
`Glycolipids
`Lacto-N-fucopentaosyl(III)ceramide
`
`Globoside
`Oligosaccharides
`Lacto-N-fucopentaose
`
`La&o-N-fucopentaose
`
`III
`
`I
`
`pat-
`by PMN 6. The same chromatographic
`tern was obtained
`by all three antibodies;
`however,
`the
`intensity
`of staining
`in-
`creased
`from PMN 6 to PMN 29 to PM-81.
`No antigens were detected
`in
`the
`total
`
`leukemia
`lipid extracts of acute myelocytic
`PMN
`cells or monocytes
`by antibodies
`and PMN 29 (Figs. 2A and B, lanes 2,3,4,
`5). Under
`the same conditions
`PM-81 de-
`tected
`low levels of antigen
`in both extracts
`
`6
`
`LNF
`
`III cer
`
`-
`
`Y2-
`
`4c-
`
`Origin
`
`-
`
`1234567
`
`1234567
`
`1234567
`
`was per-
`antigens
`of glycolipid
`Autoradiography
`antigens.
`of glycolipid
`FIG. 2. Autoradiography
`stained
`with
`antibody
`PMN
`6, (B)
`Procedures.
`(A) was
`Experimental
`under
`formed
`as described
`PM-81,
`each at 5 pg/ml.
`Purified
`glycolipids
`(30 ng) 4c, YZr and
`lacto-
`(C) with
`with
`PMN
`29, and
`(LNF
`III
`cer) were
`chromatographed
`in lane 1. The amount
`of extract
`N-fucopentaosyl(III)ceramide
`as the volume
`of packed
`cells
`from
`which
`it was obtained
`is lane 2, 5
`chromatographed
`expressed
`~1 AML
`blasts;
`lane 3, 2 ~1 AML
`blasts;
`lane 4, 5 pl monoeytes;
`lane 5.2 ~1 monocytes;
`lane 6,2 ~1
`granulocytes;
`and
`lane 7, 2 ~1 HL-66
`cells. The positions
`of
`the purified
`glycolipids
`shown
`on
`the
`left.
`
`are
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1007 Page 4 of 6
`
`

`
`DIFFERENTIAL
`
`PMN
`
`6, PMN
`
`29, AND
`
`PM-81
`
`BINDING
`
`TO GLYCOLIPIDS
`
`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 Radioimmunoassay
`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
`Yz and 4c,
`and did not bind
`to
`lacto-N-fucopentao-
`syl(III)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
`Yz and 4c at
`lower concentrations
`than
`to lacto-N-fu-
`copentaosyl(III)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).
`
`I A
`
`IC
`
`glycolipids.
`to purified
`of antibodies
`FIG. 3. Binding
`performed
`were
`Solid-phase
`radioimmunoassays
`Procedures.
`Antibody
`described
`under
`Experimental
`in (A), PMN
`29
`for
`(B),
`PMN
`6 was used
`for assays
`glycolipids
`tested
`were
`and PM-81
`for
`(C). Purified
`4c, a, Ya, 0,
`lacto-N-fucopentaosyl(III)ceramide,
`0;
`and globoside,
`O. Structures
`of
`these
`are
`depicted
`in Table
`I.
`
`glycolipids
`
`as
`
`DISCUSSION
`
`The carbohydrate
`
`sequence
`
`Gal@l-4GlcNAc/31-3Gal.
`3
`
`. *
`
`Fuccu 1
`
`to the mouse. Out of
`is a potent antigen
`325 monoclonal
`antibodies
`from different
`laboratories
`that have been analyzed
`in our
`laboratory,
`55 are directed against
`this se-
`quence (21).
`the
`against
`directed
`Some antibodies
`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
`(Gal~l-4GlcNAc~l-3Gal~1-4Glc~1-1Cer),
`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
`reactivity 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
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1007 Page 5 of 6
`
`

`
`506
`
`MAGNANI
`
`ET AL.
`
`concentration may bind
`threshold
`certain
`high-affinity
`but not
`low-affinity
`akibod-
`ies. Glycoproteins
`containing
`the same
`carbohydrate
`sequence may also be
`in-
`volved
`in antibody
`binding
`(26, 27).
`
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`PETITIONER'S EXHIBITS
`
`Exhibit 1007 Page 6 of 6