Hybridomas producing monoclonal antibodies to mono-, di-, and trifucosylated type 2 chain

Hybridoma cell lines that produce monoclonal antibodies that differentially recognize glycolipids with mono-, di-, and trifucosylated type 2 chain structures are disclosed. The monoclonal antibodies can be used to detect specific types of tumor cells that are characterized by enrichment in mono-, di-, or trifucosylated type 2 chain structure. As such, the antibodies produced by the hybridoma cell lines are useful for diagnosis and treatment of human cancer. Also disclosed is an improved method of raising hybridoma cell lines by selecting the hybridomas by positive reactivity with one or more fucosylated type 2 chain structures selected from the group consisting of III.sup.3 FucnLc.sub.4, V.sup.3 FucnLc.sub.6, III.sup.3 FucnLc.sub.6, III.sup.3 V.sup.3 Fuc.sub.2 nLc.sub.6, and III.sup.3 V.sup.3 VII.sup.3 Fuc.sub.n nLc.sub.8.

TECHNCIAL FIELD 
This invention relates to hybridoma cell lines that produce monoclonal 
antibodies useful for the detection and treatment of human cancers. 
BACKGROUND OF THE INVENTION 
Some tumor cells have been characterized by the presence of unique 
glycolipid markers which are absent or expressed minimally at normal celll 
surfaces. Accumulation of a series of fucolipids having the X determinant 
(Gal.beta.1.fwdarw.4[Fuc.alpha.1.fwdarw.3]GlcNAc) at the terminus and 
Fuc.alpha.1.fwdarw.3 at the internal GlcNAc residue of unbranched type 2 
chain (Gal .beta.1.fwdarw.4[GlcNAc.beta.1.fwdarw.3 Gal].sub.n 
.beta.1.fwdarw.4GlcNAc.fwdarw.R) is one of the most characteristic 
membrane phenotypes detected in various human adenocarcinomas. III.sup.3 
FucnLc.sub.4 (Formula a, Table I) was found to accumulate in some 
adenocarcinomas. J. Biol. Chem. 246: 1192-1200 (1971); Biochem. Biophys. 
Res. Commun. 100: 1578-1586 (1981). Monofucosylated type 2 chain, 
previously designated as y.sub.2 (V.sup.3 FucnLc.sub.6) (Formula b, Table 
I), z.sub.1 (VII.sup.3 FucnLc.sub.8), and z.sub.2 (V.sup.3 VII.sup.3 
Fuc.sub.2 nLc.sub.8) were isolated and characterized as normal cell 
components. J. Biol. Chem. 252: 14865-14874 ( 1982). Difucosylated 
lacto-N-norhexaosylceramide (Formula d, Table I) was implicated in 
adenocarcinoma of liver but absent in normal liver cells. Biochem. 
Biophys. Res. Comm. 109 (1): 36-44 (1982). All these cell surface 
components have the X determinant structure at the terminus and, 
therefore, have been detected by immunostaining with monoclonal antibodies 
directed to the X determinant, such as anti-SSEA-1, WGHS 29, ZWG 13, 14, 
111, 538 F12, 538 F8, VEP8, VEP9, My-1, etc. None of these previously 
established monoclonal antibodies, however, can distinguish among various 
fucosylated type 2 chain structures such as those associated with human 
adenocarcinoma cells. 
SUMMARY OF THE INVENTION 
The present invention provides hybridoma cell lines that produce monoclonal 
antibodies that differentially recognize glycolipids with mono-, di-, and 
trifucosylated type 2 chain structures. The monoclonal antibodies of this 
invention can be used to detect specific types of tumor cells that are 
characterized by enrichment in mono-, di-, or trifucosylated type 2 chain 
structure. As such, the antibodies produced by the hybridoma cell lines of 
this invention are useful for diagnosis and treatment of human cancer. The 
invention also provides an improved method of raising hybridoma cell lines 
by selecting the hybridomas by positive reactivity with one or more 
fucosylated type 2 chain structures selected from the group consisting of 
III.sup.3 FucnLc.sub.4, V.sup.3 FucnLc.sub.6, III.sup.3 FucnLc.sub.6, 
III.sup.3 V.sup.3 Fuc.sub.2 nLc.sub.6, and III.sup.3 V.sup.3 VII.sup.3 
Fuc.sub.3 nLc.sub.8. 
The antibody FH4 produced by hybridoma cell line ATCC No. HB8775 shows a 
remarkable preferential reactivity towards di- or trifucosylated type 2 
chain, i.e., it does not react with monofucosylated structures, including 
lactofucopentaosyl (III) ceramide (III.sup.3 FucnLc.sub.4), monofucosyl 
neolactonorhexaosylceramide (y.sub.2, V.sup.3 FucnLc.sub.6), and 
monofucosyl neolactonoroctaosylceramide (z.sub.1, VII.sup.3 FucnLc.sub.8), 
but reacts well with di- and trifucosylated type 2 chain structures such 
as difucosyl neolactonorhexaosylceramide (III.sup.3 V.sup.3 Fuc.sub.2 
nLc.sub.6) and trifucosyl neolactonoroctaosylceramide (III.sup.3 V.sup.3 
VII.sup.3 Fuc.sub.3 nLc.sub.8). 
Two and other monoclonal antibodies, FH5 (produced by hybridoma cell line 
ATCC No. HB8770) and ACFH18, preferentially react with trifucosylated type 
2 chain structure (III.sup.3 V.sup.3 VII.sup.3 Fuc.sub.3 nLc.sub.8), 
although cross-reactivity with difucosylated type 2 chain (III.sup.3 
V.sup.3 Fuc.sub.2 nLc.sub.6) was observed. Both FH5 and ACFH18 showed a 
minimal cross-reaction with monofucosylated type 2 chain. 
In contrast, the antibody FH1 dos not react with III.sup.3 FucnLc.sub.4 but 
reacts with V.sup.3 FucnLc.sub.6, III.sup.3 V.sup.3 Fuc.sub.2 nLc.sub.6, 
and III.sup.3 V.sup.3 VII.sup.3 Fuc.sub.3 nLc.sub.8. 
Two monoclonal antibodies, FH2 and FH3, do not discriminate among various 
glycolipids having fucosylated type 2 chain, and their reactivities are 
essentially similar to previously established antibodies directed to the 
terminal X determinant, such as anti-SSEA-1, WGHS 29, VEP8 and 9, My-1, 
etc.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The glycolipid designations used herein, according to the recommendations 
of the Nomenclature Committee of the International Union of Pure and 
Applied Chemistry, are as follows: III.sup.3 FuncLchd 4, 
lactofucopentaosyl (III) ceramide; III.sup.3 V.sup.3 Fuc.sub.2 nLc.sub.6, 
difucosyl neolactonorhexaosylceramide; III.sup.3 V.sup.3 VII.sup.3 
Fuc.sub.3 nLc.sub.8, trifucosyl neolactonoroctaosylceramide; V.sup.3 
FucnLc.sub.6, monofucosyl neolactonorhexaosylceramide, VII.sup.3 
FucnLc.sub.8, monofucosyl neolactonoroctaosylceramide; and V.sup.3 
VII.sup.3 Fuc.sub.2 nLc.sub.8, difucosyl neolactonoroctaosylceramide. 
The representative antibodies FH4 and FH5 are produced by hybridoma cell 
lines ATCC Nos. HB 8775 and HB 8770, respectively, which were deposited on 
Apr. 2 and Mar. 26, 1985, at the American Type Culture Collection, 12301 
Parklawn Drive, Rockville, MD 20852. 
A series of glycolipids having the X determinant 
(Gal.beta.1.fwdarw.4[Fuc.alpha.1.fwdarw.3]GlcNAc) at the terminus and a 
fucosyl .alpha.1.fwdarw.3 residue at the internal GlcNAc residue have been 
isolated and characterized from tumor tissues. Specifically, the 
glycolipids III.sup.3 V.sup.3 Fuc.sub.2 nLc.sub.6 (Formula d, Table I) and 
III.sup.3 V.sup.3 VII.sup.3 Fuc.sub.3 nLc.sub.8 (Formula e, Table I) have 
been isolated and chemically characterized as the major components 
accumulating in human primary liver adenocarcinoma and colonic 
adenocarcinoma, as described in Hakomori, S., et al., J. Biol. Chem. 259: 
4672-4680 (1984), hereby incorporated in its entirety by reference. Since 
accumulation of difucosyl neolactonorhexaosylceramide and trifucosyl 
neolactonoroctaosylceramide is a characteristic membrane phenotype of 
various human cancers, it is highly desirable to establish hybridoma 
antibodies that react specifically to these tumor-associated structures 
and that do not react to monofucosylated type 2 chain with the X 
determinant at the terminus. 
FIRST SERIES OF EXPERIMENTS 
Selective isolation of hybridoma antibodies that differently recognize 
mono-, di-, and trifucosylated type 2 chain 
The general procedure described by Kohler and Milstein for establishing 
hybridomas producing monoclonal antibodies was followed, and specifically 
a modification of that procedure was employed as described in J. Exp. Med. 
150: 1008-1009 (1979). The glycolipids designated III.sup.3 FucnLc.sub.4, 
III.sup.3 V.sup.3 Fuc.sub.2 nLc.sub.6, and III.sup.3 V.sup.3 VII.sup.3 
Fuc.sub.3 nLc.sub.8 were prepared from human adenocarcinoma as described 
in J. Biol. Chem. 259: 4672-4680 (1984). The y.sub.2 (V.sup.3 
FucnLc.sub.6) and z.sub.1 (VII.sup.3 FucnLc.sub.8) glycolipids were 
prepared from human adenocarcinoma and human erythrocytes as described in 
J. Biol. Chem. 257: 14865-14874 (1982). 
Immunization of Balb/c mice was performed in one series of experiments with 
a membrane fraction and in another series of experiments with glycolipids 
adsorbed to Salmonella minnesota. The membrane fraction that was used for 
immunization was prepared by the following procedure. 1.5-2 g of tumor 
tissue or cells (colonic cancer metastatic to liver tumor cell line TG115 
or gastric cancer cell line MKN74) were homogenized in a Dounce 
homogenizer with 10 ml of distilled water containing 10 kallikrein 
inhibitor units of "aprotinin" (protease inhibitor; Sigma). After about 50 
strokes in an ice-water bath, the homogenate was centrifuged at 2000 rpm 
for 10 min, and the supernatant was separated and centrifuged at 35,000 
rpm for 1 h. The pellet was suspended in 10 ml of distilled water 
containing aprotinin, and the protein concentration was adjusted to 2.5 
mg/ml. An aliquot of 0.5 ml containing 1.25 mg of protein was injected 4 
times intraperitoneally on every 4th day. Fusion of the host spleen cells 
with commercially available mouse myeoloma SP/2 cells was performed on the 
3rd day after the last injection. In one experiment, only one intravenous 
injection with the membrane preparation was made, followed by fusion with 
SP/2 after 3 days. Cloning of hybridomas was performed on plates coated 
with purified III.sup.3 V.sup.3 Fuc.sub.2 nLc.sub.6, III.sup.3 
FucnLc.sub.4, and V.sup.3 FucnLc.sub.6. 
In the other series of experiments, a pure glycolipid was used as immunogen 
with S. minnesota as the carrier. J. Exp. Med. 150: 1008-1019 (1979); Eur. 
J. Biochem. 24: 116-122 (1971). An ethanol solution (50 .mu.l) containing 
20 .mu.g of a purified glycolipid, III.sup.3 V.sup.3 Fuc.sub.2 nLc.sub.6, 
was mixed with 800 .mu.l of phosphate-buffered saline (pH 7.4). The 
solution was further mixed with 250 .mu.g of acid-treated S. minnesota 
suspended in 250 .mu.l of phosphate-buffered saline. The whole mixture was 
thoroughly mixed at 40.degree. C. The suspension containing 5 .mu.g of 
glycolipid was intravenously injected on the 1st day; subsequently, an 
aliquot containing 2 .mu.g of glycolipid was injected 3 times on every 4th 
day. The fusion of the host spleen cells with mouse myeloma SP/2 cells was 
performed on the 3rd day after the last injection. The hybridoma was 
cloned on 96-well plates (Dynatech Immunolon, Dynatech Laboratories, Inc., 
Alexandria, VA) coated with purified glycolipid (10 ng/well) and 
cholesterol (30 ng/well) and lecithin (50 ng/well). Cloning was performed 
repeatedly. 
Various type 2 chain glycolipids with X determinant structure were fully 
characterized by methylation analysis and .sup.1 H-NMR spectroscopy as 
described in J. Biol. Chem. 259: 4672-4680 (1984). Hybridomas were 
selected by positive reactivity with desired glycolipid antigen and 
negative reactivity with undesired glycolipid antigen as stated in Table 
I. For example, FH4 was selected by positive reactivity (+) and di- and 
trifucosyl type 2 chain with X structure (III.sup.3 V.sup.3 Fuc.sub.2 
nLc.sub.6 and III.sup.3 V.sup.3 VII.sup.3 Fuc.sub.3 nLc.sub.8) and 
negative reactivity (-) with various other monofucosyl type 2 chain 
structures. The reactivity of the antibody was based on solid phase 
radioimmunoassay, i.e., antibody binding to glycolipids adsorbed on 
plastic surfaces with cholesterol and lecithin, as described in Cancer 
Res. 43: 4997-5005 (1983). 
Of many hybridomas produced, six clones were established which produce 
ascites with high antibody titer. The monoclonal antibodies that are 
capable of distinguishing among various fucosylated type 2 chain 
glycolipids and their specificities and immunoglobulin class are listed in 
Table I. 
3 TABLE I 
Properties of monoclonal antibodies directed to various fucosylated 
type 2 chain structures Reactivities with III.sup.3 V.sup.3 - 
III.sup.3 V.sup.3 VII.sup.3 - VI.sup.2 FucnLc.sub.6 Hybridoma Ig class 
Method of preparation III.sup.3 FucnLc.sub.4.sup.a V.sup.3 
FucnLc.sub.6.sup.b III.sup.3 FucnLc.sub.6.sup.c Fuc.sub.2 
nLc.sub.6.sup.d Fuc.sub.3 
nLc.sub.8.sup.e (H.sub.2 glycolipid).sup.f FH1 IgM TG115 membrane; 
intraperitoneal injection; - + - + + - 4 .times. selected by glycolpid 
FH2 IgM As above + + - + + - FH3 IgG3 III.sup.3 V.sup.3 Fuc.sub.2 
nLc.sub.6 and S. minnesota; intrave- + + - + + - nous injection; 4 
.times. selected by glycolipid FH4 IgG3 As above - - - ++ ++ - FH5 IgM 
TG115 membrane; intravenous injection; 1 - .+-. - .+-. ++ - .times. 
selected by glycolipid ACFH18 IgM MKN74 cell membrane; intraperitoneal 
in- - .+-. - + ++ - jection; 4 .times. 
##STR1## 
? 
##STR2## 
? 
##STR3## 
? 
##STR4## 
? 
##STR5## 
? 
##STR6## 
? 
##STR7## 
The clones that produce the monoclonal antibodies FH4 and FH5 listed in 
Table I have been deposited in the American Type Culture Collection, 1230 
Parklawn Drive, Rockville, Md., USA 20852. The deposited hybridoma cell 
lines and the monoclonal antibodies they respectively produce are as 
follows: ATCC No. HB8775, FH4; ATCC No. HB8770, FH5. 
Methods for culturing and harvesting the hybridomas 
No special methods are employed for culturing and harvesting antibodies 
from the abovereferenced hybridomas. The hybridoma cell can be grown very 
well in regular hybridoma medium, e.g., RPMI medium supplemented with 15% 
fetal calf serum. However, immunization after cells are thawed from stock 
vial requires special care to start cell proliferation, particularly for 
hybridoma ATCC No. HB8775 that produces antibody FH4. Procedures for 
thawing and growing the hybridoma producing FH4 and the other hybridomas 
are described below. For production of ascites, 5.times.10.sup.5 cells ca 
be injected into the peritoneal cavity of Balb/c mice that have been 
pretreated with 0.5% Pristane in accordance with standard methodology. 
Cells can also be grown in chemicallydefined medium established for 
hybridomas such as described in Anal. Biochem. 102: 255-270 (1980). 
IgG3 antibody either in culture medium or ascites can be directly purified 
by protein ASepharose column, and IgM antibody can be purified by gel 
filtration on Sepharose 4B column; both in accordance with established 
methodology. 
Procedures for thawing and growing the hybridoma cell line producing 
antibody FH4 
Each vial typically contains about 5.times.10.sup.5 cells. Thaw a vial 
rapidly at 37.degree. C., then centrifuge at 800 rpm for one minute to 
separate cells from freezing media. Cells in pellet are suspended in RPMI 
media supplemented with 15% fetal calf serum, and transferred in 8 ml 
volume of the same media with fetal calf serum placed in a small flask (2 
ml) to which 1/4 part of thymocytes derived from a single whole thymus of 
Balb/c mice are added. Fetal calf serum should be proven to be suitable 
for growing hybridoma cells. Thymocytes are prepared from a thymus excise 
from 2-3 week old Balb/c mice (male or female). Hybridoma cells and 
thymocytes in a 25 ml flask are incubated in a CO.sub.2 incubator for one 
day and 8 ml of new media added, and further incubated for three days. 
Observe cells are proliferating during this time. The culture is divided 
into two flasks of the same size, and to each flask 8 ml of new media is 
added. Cells are thereby passed to a new medium at every three days. 
Within ten days after cells are thawed, cells growing in one or a few 
flasks are injected into pristaneprimed peritoneal cavity of Balb/c mice. 
Procedures for thawing and growing the hybridoma cell lines producing 
antibodies FH1, 2, 3, 5, or ACFH18 
The procedure is the same as above but can be simplified without addition 
of thymocytes. The culture schedule is the same as above recommendation. 
Injection of cells into peritoneal cavity to obtain ascites is not 
necessary within ten days after thawing. Injection can be made at any 
time. 
Reactivities of the antibodies with various types of glycolipids having 
fucosylated type 2 chain structure 
The reactivities of the antibodies with various types of glycolipids havin 
fucosylated type 2 chain structure are shown in FIGS. 1 and 2. The 
reactivity of varying concentrations of the antibodies with constant 
quantities of glycolipids is shown in FIG. 1, and the reactivity of the 
antibodies with varying quantities of glycolipid antigens is shown in FIG 
2. Solid phase radioimmunoassay was performed on a detachable vinyl strip 
(Costar, Cambridge, MA) as described in Cancer Res. 43: 4997-5005 (1983). 
Specifically, FIG. 1 shows the reactivity of the monoclonal antibodies FH1 
2, 3, 4, and 5 with various glycolipid antigens at different 
concentrations of antibodies. The assay was made on vinyl strips coated 
with glycolipids, cholesterol, and lecithin. The quantity of glycolipid 
per well was 10 ng with 30 ng of cholesterol and 50 ng of lecithin. FIG. 
shows the reactivity of the monoclonal antibodies FH1, 2, 3, 4, 5, and 
ACFH18 at different concentrations of glycolipid antigens. The initial 
concentration of glycolipid coated on a vinyl strip was 30 ng with 90 ng 
of cholesterol and 150 ng of lecithin. The antibody concentration applied 
to each well was constant at a 1:100 dilution of the culture supernatant. 
Referring to FIG. 1A and FIG. 2A, the antibody FH1 reacted with tri, di, 
and monofucosylated type 2 chain equally well, although it did not react 
with III.sup.3 FucnLc.sub.4. 
Referring to FIG. 1, B and C, and FIG. 2, B and C, the antibodies FH2 and 
FH3, in contrast to FH1, reacted with all fucosylated type 2 chain 
glycolipids, including lactofucopentaosyl(III)ceramide, although a subtle 
difference in the order of reactivity was observed between FH2 and FH3. 
Referring to FIG. 1D and FIG. 2D, a remarkable selective reactivity with 
trifucosylated and difusosylated type 2 chain was observed for the 
antibody FH4, which did not react with monofucosylated type 2 chain 
V.sup.3 FucnLc.sub.6, VII.sup.3 FucnLc.sub.8, or III.sup.3 FucnLc.sub.4. 
The antibody FH4, however, did not discriminate between di and 
trifucosylated type 2 chain glycolipids. 
Referring to FIG. 1E and FIG. 2E, the antibody FH5 showed a preferential 
reactivity with trifucosylated neolactonoroctaosylceramide, but a 
crossreaction with di and monofucosyl neolactonorhexaosylceramide was 
observed. The antibody FH5 did not crossreact with 
lactofucopentaosyl(III)ceramide. 
Referring to FIG. 2F, a similar, but more obvious, preferential reactivity 
with trifucosyl neolactonoroctaosylceramide was observed for the antibody 
clone ACFH18, which did not react with monofucosyl 
neolactonorhexaosylceramide or neolactonoroctaosylceramide (y.sub.2 ; 
z.sub.1) or lactofucopentaosyl(III)ceramide. The antibody ACFH18, however 
crossreacted minimally with difucosyl neolactonorhexaosylceramide. 
Referring to FIG. 1, A-E, and specifically to the lines with solid 
triangles therein, none of the abovestated FH series and ACFH18 monoclona 
antibodies reacted with a glycolipid having an internal fucosyl residue 
(III.sup.3 FucnLc.sub.6 ; Formula c, Table I) which was obtained by 
desialylation of a unique ganglioside. Biochem. Biophys. Res. Commun. 113 
791-798 (1983). 
None of the abovestated FH series and ACFH18 antibodies reacted with 
glycolipids carrying the H structure (Biochem. 14: 2725-2733, 1975), 
specifically the H.sub.2 glycolipid structure (Formula f, Table I), even 
in high concentration (2 .mu.g/well) (data not shown). 
Confirmation of the preferential reactivity of FH4 with difusocyl 
neolactonorhexaosylceramide 
The preferential reactivity of FH4 with difucosyl 
neolactonorhexaosylceramide was confirmed by inhibition of antibody 
binding to solid phase glycolipidlecithin-cholesterol on vinyl strips. 
Solid phase radioimmunoassay was performed on a detachable vinyl strip as 
referenced above. The reactivity of FH3 and FH4 to the solid phase 
III.sup.3 V.sup.3 Fuc.sub.2 nLc.sub.6 coated on a vinyl strip with 
cholesterol and lecithin was inhibited by liposomes containing III.sup.3 
V.sup.3 Fuc.sub.2 nLc.sub.6 or V.sup.3 FucnLc.sub.6. The initial 
concentration of inhibitory glycolipid in liposomes was 1 .mu.g/well. The 
glycolipid concentration on vinyl strips was 10 ng with 50 ng of lecithin 
and 30 ng of cholesterol/well. The concentration of antibody was a 1:50 
dilution of the culture supernatant. 
Referring to FIG. 3, the reactivity of the FH4 antibody with the solid 
phase difucosyl neolactonorhexaosylceramide was specifically inhibited by 
incubation of the antibody with the same glycolipid antigen in liposome. 
The selective reactivity of FH4 with certain types of tumor cells in 
contrast to an antibody directed to the X determinant 
The reactivity of FH4 was further tested with various cell lines in 
comparison to antiSSEA-1, which recognizes the X determinant irrespective 
of the internal structure. Biochem. Biophys. Res. Commun. 100: 1578-1586 
(1981); Proc. Natl. Acad. Sci. U.S.A. 75: 5565-5569 (1978); Nature (Lond. 
292: 156-158 (1981). As shown in FIG. 4, the reactivity of FH4 was much 
more restricted than that of antiSSEA-1. 
The cell lines listed on the ordinate of FIG. 4 were of the following 
types: gastric cancer cell lines MKN series (Acta Med. Biol. 27: 49-63, 
1979); lung cancer cell lines QG56, QG90, PC1, 3, 7, 9, and 10 
(Tampakushitsu-Kakusan-Koso (Protein-Nucleic Acid-Enzyme) 23: 697-711, 
1978); epidermoid tumor cells RT4; ovarial adenocarcinoma SKOV3 (J. Natl. 
Cancer Inst. 59: 221-226, 1977); monocytic leukemia cell line THP1 (Int. 
J. Cancer 26: 171-176, 1980); myelocytic leukemia KG1; erythroleukemia 
K562; B cell lines Prent, Crow, and Kasner; human fibroblasts WI38 and 
L5; human lung cancer cell line LX1; human cervical carcinoma HeLa; human 
teratocarcinoma 2102; promyelocytic leukemia HL60; and human bladder 
carcinoma cell line EJ23. 
PC7, PC9, KG1, HL60, K562, THP1, and B cell lines were cultured in 
suspension in RPMI 1640 medium supplemented with 10% fetal calf serum. Al 
other cell lines were cultured in Dulbecco's modified Eagle's medium 
supplemented with 10% calf serum. Trypsinized cells were washed and 
resuspended in phosphatebuffered saline, and 5.times.10.sup.4 cells/well 
were seeded in Linbro plates which were precoated and 0.5 mg/ml of 
polylysine. Plates were centrifuged, and cells were fixed with 0.1% 
glutaraldehyde and used for antibodybinding assay. 
The reactivity was expressed relative to that of gastric cancer cell line 
MKN74 which is shown as a shadowed bar in FIG. 4 and is regarded as 100%. 
Cells fixed on plates were treated with 5% bovine serum albumin in 
phosphatebuffered saline, pH 7.4, for 1 h, followed by incubation for 18 
with FH4 or antiSSEA-1. Both antibodies were ascites form and 100 times 
diluted. The cells on plates were then successively treated with the 
second antibody followed by .sup.125 Ilabeled protein A as described in 
Cancer Res. 43: 4997-5005 (1983). The second antibody for FH4 was 1000 
times diluted rabbit antibody directed to mouse IgG.sub.3, and that for 
antiSSEA-1 was 1000 times diluted rabbit antibody directed to mouse IgM. 
Referring to FIG. 4, a selective reactivity of FH4 with certain types of 
tumor cells, including gastric cancer cell line MKN74, lung cancer cell 
line PC7, and monocytic leukemia cell line THP1, was observed, in contras 
to antiSSEA-1 which reacts with a large variety of cells including 
nonmalignant B cell lines. 
Discussion 
The carbohydrate structure with the X determinant, in either glycolprotein 
or glycolipids, is highly immunogenic in mice. Since the established 
KohlerMilstein procedure depends solely on immunization of mice with 
cells, a great number of monoclonal antibodies, originally claimed to be 
"tumorspecific," have been found to be directed to the X determinant, 
i.e., Gal.beta.1.fwdarw.4[Fuc.alpha.1.fwdarw.3]GlcNAc.beta.1.fwdarw.R. Th 
first monoclonal antibody indentified as being directed to this X 
determinant was antiSSEA-1. J. Biol. Chem. 257: 14865-14874 (1982); Proc. 
Natl. Acad. Sci. U.S.A. 75: 5565-5569 (1978); Nature (Lond.) 292: 156-158 
(1981). With the antiSSEA-1 reagent a number of glycolipids with the X 
determinant have been detected in normal and tumor tissue. However, some 
adenocarcinomas accumulate lactofucopentaosyl(III)ceramide, difucosyl 
neolactonorhexaosylceramide, and trifucosyl neolactonoroctaosylceramide. 
These structures were not detected in normal erythrocytes, granulocytes, 
normal colonic mucosa, or normal liver, although various other bands with 
X determinant structure are present in those normal cells and tissues. 
Granulocytes are particularly rich in glycolipids with the X determinant. 
Eur. J. Immunol. 13: 306-312 (1983); Blood 61: 1020-1023 (1983); Dev. 
Biol. 93: 54-58 (1982). 
In order to distinguish among various structures with fucosylated type 2 
chain, hybridoma cell lines have been selected by a chemically well 
defined structure rather than by cells. For example, the hybridoma 
producing antibody FH4 was obtained by immunization with pure difucosyl 
neolactonorhexaosylceramide and selection by various purified glycolipids 
as listed in Table I. As a result, monoclonal antibodies directed to 
defined structures, and specifically those that can distinguish between 
mono and di and trifucosylated type 2 chain structures, were selected. Th 
antibody FH4 is only reactive with di and trifucosylated type 2 chain, bu 
is not reactive with neolactonorhexaosylceramide that is monofucosylated 
at either the V.sup.3 or III.sup.3 positions. Namely, the antibody FH4 
recognizes two adjacent fucosyl structures at the III.sup.3 and V.sup.3 
positions. The antibody FH4 shows a preferential reactivity with some 
human tumor cell lines and shows no reaction with various B cell lines 
which were highly reactive with antiSSEA-1. The restricted reactivity 
demonstrated by antibody FH4 indicates a restricted distribution of such 
structures with adjacent fucosyl residues in the type 2 chain. It is 
assumed that the structures could be synthesized by a mechanism of type 2 
chain elongation coupled with .alpha.1.fwdarw.3 fucosylation at every 
GlcNAc residue, as discussed in J. Biol. Chem. 259: 4672-4680 (1984). In 
contrast to the specificity of the FH4 antibody, the FH5 and ACFH18 
monoclonals showed a preferential reactivity to trifucosylated type 2 
chain (trifucosyl neolactonorhexaosylceramide), although a crossreaction 
with difucosylated type 2 chain was also observed. Therefore, the 
monoclonal antibodies FH4, FH5, and ACFH18 are specific reagents that 
recognize di or trifucosyl residues linked to type 2 chain, excluding the 
terminal Xhapten structure. 
A possible epitope recognized by the FH series, ACFH18, and previously 
established monoclonals directed to the X determinant is shown in FIG. 5. 
Various monoclonal antibodies including FH2 and FH3 are directed to X 
determinant, in contrast to FH1 which recognizes internal repeating type 
chain as well. FH4 recognizes two fucosyl residues (shown as a solid 
zone), and FH5 and ACFH18 recognize three fucosyl residues (shown as 
shadowed zone) linked to type 2. 
It is predicted that these antibodies will be useful in detecting specific 
types of cells such as tumor cells and undifferentiated cells that are 
characterized by enrichment in di or trifucosylated type 2 chain structur 
of human cancer. In order to evaluate this possibility the following 
experiments were performed. 
SECOND SERIES OF EXPERIMENTS 
Because it was noticed that the di or trifucosylated type 2 chains (or 
multimeric X antigens) defined by antibody FH4 were much more restricted 
than the X hapten structure defined by FH3, and because both FH3 and FH4 
antibodies are IgG3 with comparable reactivities, a systematic study of 
the distribution of the antigens defined by these two antibodies in 
various stages of human development and in human cancer in comparison wit 
adult normal tissues was undertaken using immunohistological techniques. 
Tissue sections were stained by monoclonal antibody FH3, which defines X 
determinant, and by monoclonal antibody FH4, which defines di or trimeric 
X determinant. The following general trends in the expression of the 
antigens defined by FH3 and FH4 have been observed: (a) A wellorganized, 
orderly appearance and disappearance of the antigens was observed during 
the histogenesis of various epithelia of gastrointestinal and other 
organs. The development stage exhibiting the maximum antigen expression i 
different for each organ. (b) The X determinant defined by FH3 is 
expressed about 2 wk earlier than the di or trimeric X determinant define 
by FH4, and the antigen defined by FH4 regressed more rapidly and more 
completely than the X determinant defined by FH3 on further development o 
epithelial tissue. Thus, expression of the FH4 antigen is highly limited 
to specific types of cells in newborn and adult epithelial tissues. (c) 
The antigen defined by FH4 was strongly expressed in the majority of 
tubular and papillary adenocarcinoma of stomach, adenocarcinoma of colon, 
and infiltrating ductal carcinoma of breast and its metastatic lesions. N 
antigen defined by FH4 was found in poorly differentiated stomach 
adenocarcinoma, squamous lung carcinoma, and many other types of tumors 
from ovary, testis, prostate, skin, and muscle. The presence of the 
antigen defined by FH4 is therefore limited to carcinoma of the stomach, 
colon, and breast and can be regarded as a retrograde expression of the 
antigen to a certain stage of fetal development in which expression of 
this antigen was maximal. 
Materials and Methods 
Tissues. 27 human embryos and fetuses (aged 1-8 wk and 9-38 wk after 
fertilization, respectively) were collected at the Divison of Human 
Embryology and Teratology, Department of Pediatrics, Universitiy of 
Washington. The ages of the embryos and fetuses were between 38 and 127 d 
and were organized as described in Monitoring Birth Defects and 
Environment: The problem of surveillance. E. B. Hook, et al., eds., 
Academic Press, N.Y., pp. 29-44 (1971). Two newborn tissue samples were 
donated from the Department of Pathology, Children's Orthopedic Hospital, 
Seattle. Various normal adult tissues, including colon, stomach, ovary, 
lung, skin, breast, liver, spleen, and skeletal muscle, were obtained fro 
specimens associated with the surgical removal of tumors. In total, 65 
cases of various cancer tissues were obtained from the Department of 
Surgery, Swedish Hospital, Seattle, and from Japan Immunoresearch 
Laboratories, Takasaki, Japan. Formalinfixed, paraffinembedded sections o 
normal bladder, urethra, and testis were obtained from the Department of 
Urology, Tohoku University School of Medicine, Sendai, Japan. 
Antibodies. IgG3 monoclonal antibodies FH3 and FH4 were purified from 
culture supernatant or from ascites fluid by affinity chromatography on a 
protein ASepharose column. Antibody adsorbed on protein ASepharose was 
eluted with 0.1M citrate buffer, pH 4.2, dialyzed against 
phosphatebuffered saline (PBS: 10 mM sodium phosphate buffer, pH 7.2, 
containing 0.9% NaCl), and stabilized by addition of 0.1% bovine serum 
albumin. The final concentration of antibody was adjusted to 100 .mu.g/ml 
which was approximately equivalent to a sixfold dilution of ascites. The 
secondary antibody (rabbit Ig directed to mouse Ig) conjugated with 
horseradish peroxidase was purchased from Accurate Chemicals Co., 
Westbury, NY. 
Preparation of Tissue Sections. Tissues were embedded in OCT compound 
(TissueTek II Division, Miles Laboratories, Inc., Naperville, IL), frozen 
in dry iceacetone, and stored in a Revco freezer at -80.degree. C. until 
use. Frozen sections (4-6 .mu.m thick) were prepared on a cryostat. Each 
section was dried on objective glass for 30 min at room temperature, fixe 
in acetone at 4.degree. C. for 10 min, and washed with PBS at 4.degree. C 
Tissue sections (6-8 .mu.m thick) were also prepared fom formalinfixed, 
paraffinembedded specimens according to established procedure. Sections 
were deparaffinized in xylene for 5 min at 4.degree. C., dehydrated in 
ethanol, and washed with PBS. Before antibody labeling, frozen sections 
and paraffinembedded sections were blocked by incubation with 15% normal 
rabbit serum in PBS for 1 h at room temperature. 
Immunostaining Procedure. After blocking with normal rabbit serum, section 
were incubated at room temperature with the primary antibody solution (FH 
or FH4 as prepared above) for 18 h in a moist chamber. Sections were 
washed three times with PBS at 4.degree. C. (5 min per washing). Sections 
were then incubated with the peroxidaseconjugated secondary antibody 
(diluted 1:30) for 1 h at room temperaturein a moist chamber and washed 
three time in PBS at 4.degree. C. as above. Bound antibodies were detecte 
by incubating tissue sections in 0.05M Tris/HCl buffer, pH 7.6, containin 
0.03% 3,3'-diaminobenzidine (Sigma Chemical Co., St. Louis, MO) and 0.008 
hydrogen peroxide. After 10 min, the sections were washed with distilled 
water, counterstained with hematoxylin, dehydrated in ethanol, washed wit 
xylene, and mounted. 
Two controls were performed for each staining experiment: sections treated 
without the primary antibody and sections treated with normal mouse serum 
Specific tissue labeling was not observed after either of the above 
control treatments. 
Comparison of Immunostaining of Frozen Sections and ParaffinEmbedded 
Sections. In view of a possible deletion of glycolipid antigens during 
preparations of sections from paraffinembedded specimens, staining of the 
antigens in these sections was carefully compared with cryostat sections 
from frozen samples. There were no significant differences in 
immunoreactivity between frozen sections and paraffin sections for both 
fetal and newborn specimens. 
General developmental changes of antigens defined by FH3 and FH4 antibodie 
Strong staining of embryonic and fetal tissue by both FH3 and FH4 
antibodies was limited to gastrointestinal and urogenital epithelia, and 
the patterns of antibody reactivity showed dramatic changes depending on 
the stage of the embryo. The X determinant defined by the FH3 antibody 
showed a wider distribution than the multimeric X antigen defined by the 
FH4 antibody. Both antigens were absent or present in relatively low 
concentration during early embryonic development (up to 40-50 d), showed 
maximum expression at a specific stage of development (mostly 50-70 d), 
and regressed upon further differentiation and development. The antigen 
defined by FH3 appeared about 2 wk earlier than the antigen defined by FH 
and either regressed later than that defined by FH4 or was continuously 
expressed after birth. Thus, the FH4 antigen was often limited to a 
specific type of cell in certain epithelial tissues, as discussed further 
below. 
Weak staining was also observed in the pulmobronchial epithelia, adrenal 
medulla, and entire layers of the epidermis by both FH3 and FH4 in 
embryonic stages. The reactivities of FH4 regressed completely in newborn 
tissues. A weak staining with FH3 remained, however, in the adrenal 
medulla as well as in sebaceous and sweat glands of the epidermis. 
The antigens recognized by FH3 and FH4 were not found in connective 
tissues, nervous tissues (brain and spinal cord), tissues of the 
circulatory system (heart, arteries, and veins), skeletal tissue (bones 
and muscles), or other parenchymatous organs, such as liver and spleen, a 
any stage of development. Distribution of the antigen defined by FH4 in 
tissues of newborns and adults was limited to specific types of cells in 
gastric and intestinal epithelia, and the antigen was completely absent i 
colonic epithelia. 
The reactivities of the FH3 and FH4 antibodies with fetal, newborn, and 
adult tissues are summarized in Tables II, III, and IV. 
3 TABLE II 
Reactivities of Digestive Organs of Human Embryo with FH3 and FH4 
Stages of embryo (in days of gestation) 38 40 42 52 53 53 54 57 58 59 6 
7 69 72 84 110 127 Newborn Antibody Organ P* F P P F P P F P P P P P 
F P F F F P P P P P 
Esophagus + - + + + + Stomach ++ ++ ++ ++ ++ ++ 
++ ++ ++ ++. + -,+ -,+,++ -,+,++ Small intestine + - - ++ ++ 
- + + + -,++ + -,+ -,+ -,+ -,+ -,.+-. -,+ -,++** -,++** FH3 Colon 
.+-. - .+-. + .+-. .+-. - - .+-. - - - Liver - 
- - Gall bladder .+-. Pancreas .+-. - + .+-. .+-. 
- .+-. -,.+-.# Esophagus - - .+-. - .+-. - 
Stomach ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + -,+ -,+,++ -,+,++ 
Small intestine - - - - .+-. - .+-. ++ + -,++ + -,+ -,++ -,++ -,+ 
-,.+-. -,+ -,++** -,++** FH4 Colon - - - + - + - .+-. - - - - 
Liver - - - Gall bladder .+-. Pancreas - 
- - .+-. .+-. - - - 
*Paraffin section. 
Frozen section. 
+, Deeply folded portion; -, superficial portion. 
-, chief cells; +, surface mucous cells; ++, parietal cells and other 
epithelial cells. 
Some unidentified cells are strongly positive. 
**++, Paneth's cell, basal granular cells; -, other epithelial cells. 
#.+-., Cells of Islets of Langerhaus; -, other cells. 
TABLE III 
__________________________________________________________________________ 
Reactivities of Circulatory, Respiratory Organs, Adrenal, Skin, and 
Nervous System of Human 
Embryo with FH3 and FH4 Antibodies 
Stage of embryo (in gestation days) 
Anti- 38 40 
52 
53 54 58 59 64 67 69 
72 84 110 
127 
Newborn 
body 
Organ P F F P F P P P P F F P F P P P P F 
__________________________________________________________________________ 
Heart, 
- - - - - - - - - - 
artery 
and vein 
Lung + + - - - .+-. 
.+-. - - 
airway 
Trachea + + + 
FH3 
Cerebrum, - - - - - - - 
cerebellum 
and pons 
Spinal cord 
- - - - - - - - 
Adrenal 
- - - - -,.+-.* 
-,.+-.* 
-,.+-.* 
-,.+-.* 
-,.+-.* -,.+-.* 
-,.+-.* -,.+-. 
Spleen - - 
Skin + + + + + 
Bone and 
- - - - - - - - 
Muscle 
Heart, 
- - - - - - - - - - 
artery 
and vein 
Lung - .+-. - - - - - - 
airway 
Trachea + .+-. 
- - 
FH4 
Cerebrum, - - - - - - - 
cerebellum 
and pons 
Spinal cord 
- - - - - - - 
Adrenal 
- - - - -,.+-.* 
-,.+-.* 
-,.+-.* 
-,.+-.* 
-,.+-.* -,.+-.* 
-,.+-.* -,.+-.* 
Spleen - - 
Skin + + + + - - 
Bone and 
- - - - - - - - 
muscle 
__________________________________________________________________________ 
*Adrenal cortex, -; medulla, .+-.. 
Adrenal cortex, -; medulla, +. 
Entire layers of skin, +. 
Duct of ecrine sweat glanc, +; epidermis, +; corium, -. 
Entire layer, -. 
TABLE IV 
______________________________________ 
Reactivities of Human Adult Tissues with FH4 Antibody 
Reactivity (number of cases) 
Paraffin Frozen 
Tissue section section 
______________________________________ 
Stomach 
Surface mucous cells 
-,.+-.* .+-. 
Chief cells - - 
Parietal cells ++ (4) + (3) 
Pyrolic gland ++ + 
Other cells -,+.sup. .+-. 
Small intestine 
Paneth's cells ++ ++ 
Basal granular cell 
++ ++ 
Cuticular or brush border cells 
- (2) - (1) 
Globlet cell - - 
Colon 
Crypt cells - -,+.sctn. 
Cuticular cell - (4) - (7) 
Globlet cells - - 
Mammary gland 
Parenchyma and duct cells 
- (3) - (4) 
Lung - (3) - (4) 
Skin - (2) - (1) 
Testis - (3) 
Prostate - (4) 
Muscle - (2) 
______________________________________ 
A single grading indicates that all cases examined showed the same degree 
of reactivity. 
*-, Three of four cases; .+-., one of four cases. 
.sup. -, three of four cases; +, one of four cases. 
.sctn.-, Six of seven cases; +, one of seven cases. 
Distribution of antigens defined by FH3 and FH4 antibodies in 
gastrointestinal tissues of the human embryo 
The strongest expression of the antigens defined by FH3 and FH4 antibodies 
was found in stomach epithelia. The reactivity was intense in all cell 
populations of stomach epithelia at 35 d of development (FIGS. 6 and 7, 
arrows a). This intense reactivity continued up to 90 d. After 100 d, the 
reactivity of many stomach cell populations regressed, became limited to a 
deep layer at later stages (FIG. 8, arrows a), and was finally limited to 
the parietal cells (FIG. 9, arrows a) and pyloric glands (data not shown) 
of newborn and adult gastric epithelia. The chief cells and other 
epithelia cells in stomach epithelia became completely negative in newborn 
and adult epithelia (see Table IV and FIG. 9). 
Small intestinal epithelial cells were not stained by FH4 at 40 d of 
development (FIG. 6, arrows b) but were strongly stained by FH3 (FIG. 7, 
arrows b). At later stages, the majority of small intestine epithelial 
cells became negative; however, a strong reactivity with both FH3 and FH4 
was observed in some unidentified cells of fetal tissue (FIG. 10). The 
antigens defined by both FH3 and FH4 in the majority of cell populations 
regressed subsequently, becoming restricted only to Paneth's cells and 
basal granular cells in the cryptic region of the newborn and adult small 
intestine (FIG. 11, arrows a; Table IV). 
The reactivity of FH3 and FH4 with colonic epithelial cells showed a 
similar pattern. These cells were weakly positive in the early embryo, 
became positive and maximally expressed between 50 and 60 d (FIG. 13, 
arrows a; the same section was not stained by FH4), and subsequently 
regressed. At later stages, FH3 antigen expression was limited to crypt 
cells in the newborn and adult (FIG. 14, arrows a; the same section was 
not stained by FH4). Crypt cells in newborn and adult tissues were not 
stained by FH4 (data not shown). The antigen defined by FH3 was present in 
esophageal epithelia in the early fetus (FIG. 12) as well as in the 
newborn and adult (data not shown), but the antigen defined by FH4 was 
absent in esophageal epithelia throughout all stages of embryonic 
development examined. A similar distribution was found in the fetal 
pancreas; the ductal epithelium was weakly stained by FH3, but not by FH4, 
and became completely negative in the newborn. It is interesting to note 
that the antigen defined by FH 3 was present in cells of Langerhan's 
islets, but that defined by FH4 was absent (data not shown). Neither 
antigen was detected in the various stages of liver development (data not 
shown). 
Respiratory organs and other tissues 
The epithelial cells of trachea and secretory glands of bronchus were 
stained by FH3, but not by FH4 in newborns. No fetal tissue of bronchus 
was available. The epithelial cells of the lung airway found in fetal lung 
bud were positive with FH3, but were negative with FH4 (data not shown). 
The entire layer of epidermal tissue of fetal age was positive with both 
FH3 and FH4, but the reactivity with FH4 became negative in adult tissue. 
A weakly positive reaction was observed with FH3 in sebaceous glands and 
in ecrine sweat glands; no reactivity was observed with FH4 (data not 
shown). Heart, aorta, arteries, veins, cerebrum, cerebellum, pons, spinal 
cord, muscles, and bones were all negative with both FH3 and FH4 
throughout all developmental stages (data not shown). 
Distribution of the antigen defined by FH4 in human cancer in comparison 
with adult normal tissues 
TABLE V 
______________________________________ 
Reactivities of Human Cancers with FH4 Antibody 
Reactivity 
(number of cases) 
paraffin Frozen 
Tumor tissue section section 
______________________________________ 
Stomach cancer 
Adenocarcinoma ++ (5/11) ++ (2/5) 
+ (3/11) + (2/5) 
- (4/11) - (1/5) 
Colon cancer 
Adenocarcinoma ++ (1/3) ++ (5/8) 
+ (2/3) + (2/8) 
- (0/3) - 
Ovary Clear cell carcinoma - (1/1) 
Serous - (1/1) 
cystoadenocarcinoma 
Mucinary - (1/1) 
cystoadenocarcinoma 
Testis Seminoma - (3/3) 
Breast Infiltrating ductal 
++ (3/4) ++ (3/4) 
carcinoma - (1/4) - (1/4) 
Lymph node ++ (2/2) ++ (2/2) 
metastasis 
Lung Squamous cell - (2/2) - (4/4) 
carcinoma 
Gall bladder 
Adenocarcinoma + (1/1) 
Prostate Adenocarcinoma - (3/3) 
Benign adenoma - (3/3) - (1/1) 
Skin Malignant melanoma 
- (1/1) - (1/1) 
Muscle Leronmyosarcoma - (1/1) 
______________________________________ 
Gastric cancer. 8 out of 11 cases of paraffin-embedded sections and four 
out of five frozen sections of gastric cancers were strongly or clearly 
positive with FH4 (see Table V). All the positive cases were tubular or 
papillary adenocarcinoma, while all the negative cases were 
undifferentiated adenocarcinoma (see Table V). A typical positive example 
is shown in FIG. 17. In normal adult gastric epithelia, only parietal 
cells and pyloric gland cells were consistently positive in both paraffin 
and frozen sections (see Table IV). 
Colonic cancer. Three out of three cases of paraffin-embedded sections and 
seven out of eight cases of frozen sections were strongly or clearly 
positive with FH4 (see Table V). A typical section from colonic cancer is 
shown in FIG. 15. Only one case was negative, which was not correlated 
with the histological characteristics of the case. Normal parts of colonic 
epithelia were all negative, including crypt cells in both paraffin and 
frozen sections, except for one case that showed a positive reaction in 
the crypt cells. 
Breast cancer. Three out of four cases of both paraffin-embedded and frozen 
sections of infiltrating ductal carcinoma were positive with FH4. A 
typical case is shown in FIG. 16. Metastatic lesions in lymph nodes were 
also strongly positive (see Table V). 
Kidney cancer. A great deal of variation in the staining of kidney tumors 
was observed, which may appropriately reflect the normal variation in the 
reactivity of FH3 and FH4 with urogenital epithelia during their 
development from pronephros to mesonephros to metanephros. 
Other cancers. Various types of ovarial carcinoma, seminoma, lung squamous 
cell carcinoma, prostrate adenocarcinoma, malignant melanoma, and 
leiomyosarcoma were all negative with FH4. 
Discussion 
The reactivity of FH3 and FH4 with the developing human embryo and fetus 
can be summarized as follows: (a) The antigens detected by both FH3 and 
FH4 are most strongly expressed in the epithelial cells of the 
gastrointestinal and urogenital organs at specific stages of development. 
Expression of these antigens, particularly FH4, regressed upon further 
development with functional differentiation and disappeared from most of 
the epithelial cell populations of those tissues with the exception of a 
few specific types of cells in normal adult tissue. (b) The antigen 
defined by FH3 appeared at an earlier stage of fetal development than the 
antigen defined by FH4; however, the antigen defined by FH4 regressed 
rapidly and completely at later stages of development and its expression 
became highly limited in adult epithelial tissues. The antigen defined by 
FH3 remained in a wider variety of cells than the antigen defined by FH4 
in developed tissues. (c) In adult epithelial tissue, the antigen defined 
by FH4 was found to be limited to parietal cells and pyloric glands of 
stomach epithelia, Paneth's cells and basal granular cells of the 
intestine, and proximal convoluted tubules of the kidney. The antigen was 
not detected by FH4 in the entire colonic epithelia, including crypt 
cells. The crypt cells in colonic epithelia were positive with FH3. (d) A 
clear differential reactivity was found between the FH3 and FH4 antibodies 
in sebaceous and sweat glands of the epidermis, Langerhans' islet of the 
pancreas, adrenal medulla, esophageal epithelia, bronchial epithelia, 
airway of lung buds, and vaginal epithelia. Cells in these tissues were 
clearly or strongly stained by FH3, but were not reactive with FH4 
throughout fetal development as well as in newborn and adult tissues. 
The antigens defined by FH3 and FH4 may not be expressed or may be 
expressed weakly in preimplantation human embryos, in contrast to mouse 
embryos. This possibility is suggested by the absence of SSEA-1 in 
undifferentiated human teratocarcinoma and its appearance on 
differentiation (EMBO (Eur. Mol. Biochem. Org.) J. 2: 2355, 1983), in 
contrast to a strong expression of SSEA-1 in undifferentiated mouse 
teratocarcinoma and its decline on differentiation (Proc. Natl. Acad. Sci. 
U.S.A. 75: 5565, 1978; Dev. Biol. 83: 391, 1981). There were no cases in 
which tissues were negative at fetal stages, followed by increasing 
expression in newborn or adult tissues. 
Maximum expression of the antigens defined by FH3 and FH4 was found in the 
epithelia of tissues at a specific developmental stage, mostly 40-80 d. 
This may indicate that these structures are essential signals for cell 
adhesion and recognition, which could be an essential step for further 
differentiation of fetal epithelial cells into a variety of functionally 
differentiated adult epithelia cells. Despite our lack of knowledge of a 
functional role of these structures, such a dramatic change, with maximum 
expression at a defined stage followed by orderly disappearance, suggests 
a vital function for these structures in "chemical conversation" (Dev. 
Biol. 18: 250, 1968) between embryonic cells during epitheliogenesis. 
Since a majority of human cancers are derived from gastrointestinal, 
urogenital, and pulmobronchial epithelia, in which X or multimeric X 
antigens are strongly expressed at embryonic to fetal age, an intense 
reexpression of these antigens in a large variety of human cancers 
suggests that these structures are essentially oncofetal antigens. Only 
those cancer cells derived from epithelial tissues that express a high 
level of these antigens during a certain stage of development showed a 
strong reactivity with FH3 and FH4 antibodies. Interestingly, 
differentiated papillary adenocarcinoma of stomach expresses the antigen 
defined by FH4, whereas undifferentiated adenocarcinoma does not express 
this antigen. Thus, reexpression of the structure defined by FH4 in tumors 
could be associated with retrogenesis of tumor cells to a certain stage of 
organogenesis rather than to a stage of the very early embryo. If 
retrogenesis of cells occurs to a very early, undifferentiated stage of 
embryonic tissue, tumor cells may not express the FH4 antigen. 
Referring to FIG. 18, the stage-dependent expressions of X antigen (defined 
by FH3) and di- or trimeric X antigen (defined by FH4) in gastrointestinal 
epithelia during human development, and the retrogenetic expression of 
these antigens in gastrointestinal tumors, are compared and contrasted. X 
and di- or trimeric X antigens may not be expressed in preimplantation 
embryo, although the X antigen is highly expressed in mouse 
preimplantation embryo. Proc. Natl. Acad. Sci. U.S.A. 75: 5565 (1978); 
Biochem. Biophys. Res. Commun. 100: 1578 (1981); Nature (Lond.) 292-156 
(1981). This possibility is suggested by the absence of the X antigen in 
undifferentiated human teratocarcinoma and the induction of antigen 
synthesis on differentiation, which is the opposite of mouse 
teratocarcinoma. The antigens, however, are expressed in various tissues 
of human postimplantation embryos and fetuses. Curves a and c illustrate 
the change in FH4 antigen expression in gastric and colonic epithelia, 
respectively. Curve b represents the change in FH3 antigen expression in 
colonic epithelia. FH4 expression in colonic epithelia reaches its maximum 
between 7 and 9 wk, then regresses almost completely, while the FH3 
antigen does not regress and remains in the crypt cells. The FH4 antigen 
is strongly expressed in differentiated gastic cancer, suggesting that 
antigen retrogenesis occurs to the point at which FH4 expression is at its 
maximum (arrow 2). However, FH4 antigen expression is negative in 
undifferentiated gastric cancer because antigen retrogenesis occurs to a 
point at which FH4 is not yet expressed at the very early stages of 
embryogenesis (arrow 1). Both FH3 and FH4 antigen expression in colonic 
cancer could be strong if retrogenesis of the antigen expression occurs to 
the point at which the FH3 antigen is active. 
Immunostaining of both X antigen and multimeric X antigen in 
paraffin-embedded sections gave similar or identical results to those from 
immunostaining of frozen cryostat sections. This may indicate that (a) 
glycolipid antigens are not diminished during preparation of paraffin 
sections, of (b) the antigens may be carried by glycoproteins that are not 
diminished by preparation of paraffin sections. Since many lacto-series 
carbohydrates are carried by both glycolipids and glycoproteins (J. Biol. 
Chem. 254: 5458, 1979), it is reasonable to assume that the antigens 
detected by immunostaining of fetal tissue sections are, in fact, 
glycoproteins with properties similar to "embryoglycan" or 
lactosaminoglycans (Cell 18: 183, 1979). 
Oncofetal expression of both FH3 and FH4 should be based on a common 
mechanism for activation of .alpha.1.fwdarw.3 fucosyltransferase in fetal 
epithelial tissue and in certain types of human cancer. The 
fucosyltransferase that makes FH4 antigen can be distinguished from that 
for synthesis of FH3 antigen, and the genetic regulation of these enzymes 
is a crucial mechanism controlling embryogenesis and oncogenesis as well. 
THIRD SERIES OF EXPERIMENTS 
The antigens defined by monoclonal antibodies FH1, 2, 3, 4, 5, and ACFH18 
were not detectable by regular radioimmunoassay methods in serum or plasma 
of patients with various cancers. 
Tumor detection method using these antibodies 
Because the antigens were not detectable by regular radioimmunoassay, the 
above-stated antibodies may not be immediately useful for diagnosis of 
human cancer by simple application of existing in vitro radioimmunoassay 
methodology. However, there is a strong possibility that the antigens are 
present in body fluids of cancer patients but at low concentration. 
On the other hand, tumor antigens with great releasability are less useful 
for imaging than those antigens localized in tumor tissue. New England J. 
Medicine 298: 1384-1388 (1978). The antigens defined by FH3 and FH4 are 
highly restricted to differentiated human cancer, and their restricted 
presence in normal tissue is well defined. Thus it is contemplated that 
radiolabeled antibodies FH3 and FH4 will be particularly useful for 
imaging tumor location in vivo. For example, a radionuclide such as I-123 
can be coupled to the antibody using standard methodologies, such as those 
employing the Bolton-Hunter reagent. The radiolabeled antibody can be 
admixed in a suitable carrier solution and intravenously injected into the 
body of a mammal. The body can be thereafter scanned with scintillation 
detector means, such as a gamma camera, to localize tumor tissue bearing 
antigens reactive with the radiolabeled antibody. 
Immunotherapy of human cancer with these antibodies 
The FH series and ACFH18 antibodies, particularly the IgG3 antibodies FH3 
and FH4, are also suitable for cancer immunological therapy. Any of these 
antibodies can be coupled to a radionuclide or antitumor drug and 
intravenously injected into the body of a mammal to differentially deliver 
the radionuclide or drug to tissues bearing antigens reactive with the 
antibody. In view of the recent application of IgG3 antibody directed to a 
glycolipid antigen that suppresses tumor growth in vivo, (Proc. Natl. 
Acad. Sci. USA 82: 1242-1246, 1985), the IgG3 antibodies FH4 and FH3 are 
considered to be very good candidates for immunotherapy via direct 
intravenous injection of unconjugated antibody. Although the antigens 
reactive with FH4 and FH3 are present in kidney tubules, there is a 
possibility that the antigen may be cryptic in normal tissues, in view of 
recent reports as cited immediately above and also in J. Immunol. 132: 
2111-2116 (1984). 
While the present invention has been described in conjunction with 
preferred embodiments, one of ordinary skill after reading the foregoing 
specification will be able to effect various changes, substitutions of 
equivalents, and alterations to the compositions and methods set forth 
herein. It is therefore intended that the protection granted by Letters 
Patent hereon be limited only by the definitions contained in the appended 
claims and equivalents thereof.