Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WO93/14775 2 1 0 6 l~ ~ 7 PCT/US92/008S2
MONOCLONAL ANTIBODY PD41 AND ANTIGEN
ASSOCIATED WITH PRosTATE ADENOCARCINOMAS
1. Field of the Invention
5The invention relates to a novel monoclonal
antibody that shows preferential binding to prostate
carcinoma tissue with little or no cross-reactivity to
benign prostatic hyperplasia or to normal prostate
epithelium, as well as a hybridoma cell line and
10 method for producing the antibody. Additionally, the
p~sent invention relates to a novel ant gen with
_~h the antibody reacts specifically.
2. Backqround of the Invention
15Several laboratories have developed
monoclonal antibodies (MAbs) or immunoassays for
monitoring the expression of the two well-
characterized prostate antigens, i.e., prostatic acid
phosphatase (PAP) (Chu et al., }n T. M. Chu, ed.,
~ 20 Biochemical Markers for Cancer, pp. 117-128, New York,
¦~ Marcel Dekker, Inc., 1982; Taga et al., 1983, Prostate
4:141-150) and prostate-specific antigen (PSA) (Wang
et al., 1979, Invest. Urol. 17:159-163). Neither of
these prostate markers, however, has been found useful
for early detection or for distinguishing benign from
malignant prostate tumors. Moreover, these prostate
markers do not provide meaningful information
regarding the progressive nature or aggressiveness of
30 the tumor.
Additional MAbs that have shown potential
for detecting circulating prostate antigens in patient
' serum include TURP-27 (Starling et al., 1986, Cancer
Res. 46:367-374), 7Ell-C5 (Horoszewicz et al., 1987,
Anticancer Res. 7:92?-936) and PR92 (Kim et al., 1988, ~-
Canc~: Res. 48:4543-4548). These antibodies recogni2e
. ~ ~ ''' ~" " .
... . .
. ~ . .
. . . :. .
A . .. .. .. . , ,. ~ . , ,. " , ~ ., ,, ",
WO93/1477~ PC~/US92/0~852
tumor antigens that are either prostate-organ
specific, such that they react with normal and benign
prostate antigens as well as carcinoma, i.e., PSA,
PAP, 7Ell~C5 and TURP-27, or they cross-react with
non-prostate cells or carcinoma, i.e., PR92 cross-
reacts with breast carcinomas.
United States Patent No. 5,055,404 issued
October 8, l99l to Ueda et al., describes monoclonal
antibodies which recognize "differentiated antigen"
10 specifically found on epithelial cells of human
pros .at2 including normal prostate, benign prostatic
hyperplasia and prostatic cancer.
Two monoclonal antibodies described by
Bazinet et al., (1988, Cancer Res. 48:6938-6942)
recognize an antigen that is selectively expressed on
malignant prostatic epithelium. However, these MAbs
appear to identify only a small reactive subset of
prostate carcinomas provided the tissue specimens have
not been exposed to fixatives.
At present, there still remains a definite ~ -
need for the identification of other prostate tumor
associated antigens. In particular, there is a need
for MAbs which preferentially bind to prostatic
carcinoma and show little or no cross-reactivity to
25 benign prostatic hyperplasia and normal prostatic
epithelia. Such antibodies will be useful for both
diagnosis and therapy of prostate carcinoma.
Additionally, there is a need for MAbs specific for
prostate carcinoma, that will be useful for early
30 detection and monitoring of prostatic carcinoma
disease and progression and/or which can provide
additional clinical and pathological information with
respect to aggressiveness or metastatic potential of
prostate carcinoma.
., - ,
.. ~: . . . . .
- , - . . . ; :, . - .~ . . -
W093/1477~ 2 1 ~ ~ ~ 8 7 PCT/US92/00852
- 3 -
3. Summary of the Invention
The present invention encompasses hybridoma
cell lines that produce novel monoclonal antibodies
and the monoclonal antibodies (and fragments thereof)
that show pr2ferential specific binding to prostate
carcinoma with little to no binding to benign
prostatic hyperplasia or to normal prostate
epithelium.
In a specific embodiment, the invention is
directed to a hybridoma cell line PD41, having ATCC
Accession ,~o. HB and the PD41 monoclonal
antiboày produced by this cell line. The present
invenlion further encompasses other monoclonal
antibodies that bind to or recognize the PD41 antigen,
designated the prostate mucin antigen (PMA), as well
as monoclonal antibodies that competitively inhibit
the binding of the PD41 monoclonal antibody produced
by the hybridoma cell line ATCC Accession No. HB
to PMA, as measured by an enzyme immunoassay, a
20 radioimmunoassay or other competitive inhibition
immunoassay.
The present invention additionally
encompasses a novel antigen, PMA, in isolated or
substantially pure form, to which the monoclonal
25 antibodies of the invention bind, as well as methods
and kits for using the antigen and/or the antibodies
for detection or treatment or prophylaxis of prostate
carcinoma.
The present invention also encompasses kits
30 for using the monoclonal antibodies and/or antigen for
in vitro or ln vlvo applications for diagnosis,
monitoring, prophylaxis or therapy of prostate
carcinoma.
In other embodiments, the invention
3 encompasses compounds comprising the antigen binding
..... . . , ~ ,.
.. . ~ ................... .. , .. . .- - . -
, ~ !,, : . . .
'7
WO93/14775 PCT/US92/00852
region of the monoclonal antibodies of the invention
or portions thereof, including Fv, F(ab' )2~ Fab
fragments, chimeric antibodies, humanized antibodies,
single chain antibodies, complementarity determining
regions (CDRs), etc.
In yet other embodiments, the invention
encompasses the use of the hybridoma cell lines as a
source of DNA or mRNA encoding for the rearranged,
activated immunoglobulin genes, which ~ay be isolated,
10 cloned by recombinant DNA techniaues and transferred
to other cells for the product,o.n cf spec fis
immunoglobulin specific for pros~ate carcino~.a. By
isolating rearranged DNA or preparing cDNA from the
messenger RNA of the hybridoma cell line of the
15 invention, a sequence free of introns may be
obtained.
In still other embodiments, the invention
encompasses the nucleotide sequence encoding the PMA
antigen of this invention.
3.l. Abbreviations
The following abbreviations have the
meanings indicated:
MAb = monoclonal antibody;
PAP = prostatic acid phosphatase;
PSA = prostate-specific antigen;
PMA = prostate mucin antigen; -
PBS = phosphate-buffered saline
(136 mM NaCl; 2.7 mM XCl; 8
mM Na7 HPO~; l.5 mM XH7PO4
0.9 mM CaCl7; 0.5 mM MgCl7);
RIA = radioimmunoassay
TBS = Tris-buffered saline
(20 mM Tris; 0.9% NaCl; 0.3%
Tween 20; S% bovine serum
albumin);
BPH = benign prostatic hyperplasia;
W O 93/14775 2 ~ ~ 6 ~ ~ 7 P ~ /US92/00852
-- 5
CDR = comple.entarity determining
region
CaP = prostate adenocarcinoma;
NCA = non-cross-reacting antigen;
TAA = tumor-associated antigen;
TCC = transitional cell carcinoma;
and
BSM = bovine submaxillary mucin.
:
4. Brief Descri~tion of the Fiqures
FIG. l(A-F) illustrates staining patterns o.
prostate carcinoma tissue sections with MAb PD4l.
FIG. lA, well- to moderately-differentiated prostate
adenocarcinoma showing cytoplasmic staining of
epithelial cells of neoplastic ducts as well as
5 luminal secretions (upper left) and no staining of
, benign and normal ducts (arrows) X lO0. FIG. lB,
well- to moderately-differentiated prostate
; adenocarcinoma showing intense staining of all tumor
f cells in a large cribriform neoplastic duct and little
to no staining in smaller neoplastic ducts. X 200 FIG.
lC, a large duct from a well differentiated prostate
carcinoma with strong staining of epithelial cells and
luminal contents. X 400. FIG. lD, poorly
differentiated prostate carcinoma with intense
staining of tumor cells in most neoplastic ducts. X
lO0. FIG. lE, undifferentiated prostate carcinoma
with very few tumor cells (arrows) staining. X 200.
FIG. lF, section from bone metastasis showing a large
30 nest of intensely stained prostate tumor cells and no
staining in cartilage and non-tumor tissue. X 400
FIG. 2 is a scattergram representing the
percentage of cells staining with PD4l MAb in fixed or
frozen well differentiated (WD), moderately
35 differentiated (MD), poorly differentiated (PD), and
undifferentiated (UD) prostate carcinomas. ~ymbols
~, . . . . .. .... ... .. . .
WO93/1~77~ 2 1 0 6 ~ ~ I PCT/US92/00852
indicate, percent of positive cells in a given tumor
sample.
FIG. 3 is a representative Western
immunoblot of normal, benign and carcinoma tissues.
5 FIG . 3A is a blot of tissues or fluid samples reacted
with MAb PD4 1: prostate carcinoma membrane extracts
(Lanes 1, 2 and 3); CaP seminal plasma (Lane 4);
membrane extracts from breast carcinoma (Lane 5);
colon carcinoma (Lane 6); no~.~al prostate (Lanes 7 and
1C 8); BPH (Lanes 9 and 10); and normal seminal plasma
(Lane ~), respectively. FIG. 3B is a blot as in FIG.
3A, but react~d with an isotype-matched negative
contro antibody. Blots were transferred from a 3-15%
gradient SDS-PAGE gel (50 ~g of p-otein loaded per
Lane) onto i~mobilon-P transfer membrane and blotted.
Blots were exposed to x-ray film for 72 hours. SG
indicates top of separating gel. See text for
details. ~--
FIG. 4 is a graphic representation of a -
20 competitive binding assay of I-125 labeled PD41 MAb
against MAbs B72.3, anti-CEA, HMFG-2 and PD41. Each
curve represents the mean cpm of triplicate
determinations performed in duplicate experiments.
FIG. 5 ls graphic representation of a double
25 determinant immunoradiometric assay. See text for
details and Table 9 for identification of the tumor-
associated MAbs (competing MAbs).
FIG. 6 is a graphic representation of a
reciprocal blocking experiment. See text for details
30 and Table 9 for identification of the mucin tumor-
associated MAbs. Symbols are as follows: where
"Block" represents blocking MAb and "Trace" represents
radiolabelled MAb: O - O Block: B72.3, Trace: B72.3;
35 - ~ Block: PD41, Trace: B72.3, CEA, M344, OC125;
- - . ~ . . - . .: .: ~ .. ., - :
WO93/14775 2 ~ O ~ 7 PCT/US92/00852
~ - ~ Block: CEA, Trace: CEA; O - O Block: M344,
Trace: M344; and V - V Block: OC125, Trace OC125.
FIG. 7 is a graphic representation of a
double determinant immunoradiometric assay to assess
epitope co-expression. Symbols indicate: O Antigen
1; C' Antigen 2. See text for details and Table 9 for
identification of the tumor-associated MAbs.
5. Dctailcd Descri~tion of the Invention
The present invention is directed to
hybridoma csll lines, that produce monoclonal
antibodies, and monocl~n21 antibodies specific for
prostate carcinoma that are advantageously useful for
5 detection, diagnosis and/or monitoring and for
prophylaxis or treatment of pathological disease
associated with prostate carcinoma. More
particularly, the present invention encompasses a
novel hybridoma cell line, which produces a monoclonal
20 antibody that shows preferential binding to prostate
carcinoma, with little or no specific binding to
benign prostatic hyperplasia or to normal prostate
epithelium. The invention further encompasses the
monoclonal antibody specific for prostate carcinoma,
which shows little or no binding to benign hyperplasia
or normal prostate. The invention also encompasses
other prostate carcinoma specific monoclonal
antibodies that bind specifically with the novel
30 antigen of the invention as well as antibodies that
competitively inhibit binding of the antibody to the
novel antigen of the invention.
In addition, the present invention is
directed to a novel antigen, PMA, with which.the
35 monoclonal antibodies of the invention react, as well
as methods and ~its for using the antigen and/or
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- . . . - . -
- WO93/14775 ~ 1~3 ~ 7 PCT/US92/00852
antibodies for detection, prophylaxis or treatment of
prostate carcinoma.
For ease of explanation, the description of
the invention is divided into the following sections:
ta) preparation of hybridoma cell lines and monoclonal
antibodies; (b) characterization of the monoclonal
antibodies and the novel antigen; znd (c) applic2tions
for which the hybridoma cell lines, monoclonal
antibodies and the antigen are suited.
5.l Hvbridoma Cell Line and Antiboay ~o~uced
In the embodiment of the presen~ invention
described in the Examples which follow, a crude
membrane preparation of a moderately to poorly
differentiated prostate adenocarcinoma was used as the
"antigen" or immunogen. Based on results obtained and
described in the Examples, the epitope recognized by
the antibody of this invention is present in primary
prostate carcinomas; including poorly-, moderately-
20 and well-differentiated tumors; in metastatic prostate
carcinomas; in seminal plasma; split ejaculates; and
J in prostatic fluids of prostate carcinoma patients as
well as in a cultured colorectal carcinoma cell line
; LSl80, dialyzed spent culture medium and the
25 glycopeptide fraction digest of the LSl80 cell line
and in bovine submaxillary gland. Thus, such cells or
fluids and/or membrane fractions or extracts thereof
also represent potential "antigen" or sources of
immunogen with which to immunize animals or cells to
30 obtain somatic cells for fusion to generate antibodies
of the invention.
Somatic cells with the potential for
producing antibody and, in particular B lymphocytes,
are suitable for fusion with a B-cell myeloma line.
Those antibody-producing cells that are in the
' ' .
'
, . . ,. ,, . ; ... . . . ..
WO93/14775 2 1 ~ 7 PCT/~IS92/00852
dividing plasmablast stage fuse preferentially.
Somatic cells may be derived from the lymph nodes,
spleens and peripheral blood of primed animals and the
lymphatic cells of choice depend to a large extent on
5 their empirical usefulness in the particular fusion
system. Once-primed or hyperimmunized animals can be
used as a source of antibody-producing lymphocytes.
Mouse lymphocytes give a higher percentage of stable
fusions with the mouse myelo~a lines described below.
10 Of these, the BALB/c mouse is preferred. Ho-~ever, 'ho
use of rat, rabbit, sheep and frog ceils is also
possible. As reviewed by Goding (ln Monoclonal
Antibodies: Principles and Practice, 2d e~., pp. 60-
61, Orlando, Fla, Academic Press, 1986) use of rat
lymphocytes may provide several advantages.
Alternatively, human somatic cells capable
of producing antibody, specifically B lymphocytes, are
suitable for fusion with myeloma cell lines. While B
lymphocytes from biopsied spleens, tonsils or lymph
20 nodes of individual may be used, the more easily
accessible peripheral blood B lymphocytes are
- preferred. The lymphocytes may be derived from
patients with diagnosed prostate carcinomas.
Myeloma cell lines suited for use in
25 hybridoma-producing fusion procedures preferably are
non-antibody-producing, have high fusion efficiency,
and enzyme deficiencies that render then incapable of
growing in certain selective media which support the
growth of the desired hybridomas.
Séveral myeloma cell lines may be used for
the production of fused cell hybrids of the invention,
including P3-X63/Ag8, X63-Ag8.653, NS1ll.Ag 4.1,
Sp210-Agl4, FO, NSO/U, MPC-11, MPCll-X~5-GTG 1.7,
S194/SXX0 8ul, all derived from mice; R210.RCY3, Y3-Ag
1.2.3, IR983F and 4B210 derived from rats and U-266,
... . .
.: . .. . . :
~iO6~7
WO93/l4775 PCT/US92/00852
-- 10 --
GM1500-GRG2, LlCR-LON-HMy2, UC729-6, all derived from
- humans (Goding ln Monoclonal Antibodies: Principles
and Practice, 2d ed., pp. 65-66, Orlando, Fla,
Academic Press, 1986; Campbell, ln Monoclonal Antibody
5 Technology, Laboratory Techniques in Biochemistry and
Molecular Biology Vol. 13, Burden and Von Knippenberg,
eds. pp. ,~-~3, Amsterdam, Elseview, 1984).
Methods for generating hybrids of antibody-
producing spleen or Iymph node cells and myeloma cells
10 usually co~prise mixing somatic cells with myeloma
cells in a 2:1 proportion as in the example below,
(though the proportion may vary from about 20:1 to
about 1:1), respectively, in the presence of an agent
or agents (chemical or electrical) that promote the
fusion of cell membranes. It is often preferred that
the same species of animal serve as the source of the
somatic and myeloma cells used in the fusion
procedure. Fusion methods have been described by -~
Kohler and Milstein ~Nature 256:495-497 (1975) and
- 20 Eur. J. Immunol. 6:511-519 (1976)], and by Gefter et
al. [Somatic Cell Genet. 3:231-236 1977)]. The
; fusion-promotion agents used by those investigators
; were Sendai virus and polyethylene glycol (PEG),
respectively. Fusion methods reviewed by Goding (in
25 Monoclonal Antibodies: Principles and Practice, 2d
ed., pp. 71-74, Orlando, Fla, Academic Press, 1986)
including the above as well as electrically induced ~, -
fusion are also suitable to generate monoclonal
antibodies of the invention.
Fusion procedures usually produce viable
hybrids at very low frequency, about 1 x 10~ to 1 x
108 somatic cells. Because of the low frequency of
obtaining viable hybrids, it is essential to have a
means to select fused cell hybrids from the remaining
unfused cells, particularly the unfused myeloma cells.
. . .
.. ' .. ." ' ~ . , .:. ,': ' " " : ., `
: . ' . '
2 ~ ~ 6 ~ 8 ~
W0~3/1477~ PCT/US92/00852
~ 11 -
A means of detecting the desired antibody-producing
hybridomas among the other resulting fused cell
hybrids is also necessary.
- Generally, the fused cells are cultured in
selective media, for instance HAT medium containing
hypoxanthine, aminopterin and thymidine. HAT medium
permits the proliferation of hybrid cells and prevents
growth of unfused myeloma cells which normally would
continue to divid2 indefinitely. Aminopterin blocks
10 ~e novo purine and pyrimidine synthesis by inhibiting
the production o,~ tetrahydrofolate. The addition of
thymidine bypasses the block in pyrimidine synthesis,
while hv~oxanthine is included in the media so that
inhibited cells synthesize purine using the nucleotide
salvage pathway. The myeloma cells employed are
mutants lacking hypoxanthine phosphoribosyl
transferase (HPRT) and thus cannot utilize the salvage
pathway. In the surviving hybrid, the B lymphocyte
supplies genetic information for production of this
20 enzyme. Since B lymphocytes themselves have a limited
life span in culture (approximately two weeks), the
only cells which can proliferate in HAT media are
hybrids formed from myeloma and spleen cells.
To facilitate screening of antibody secreted
25 by the hybrids and to prevent individual hybrids from
overgrowing others, the mixture of fused myeloma and B
lymphocytes is diluted in HAT medium and cultured in
multiple wells of microtiter plates. In two to three
weeks, when hybrid clones become visible
30 microscopically, the supernatant fluid of the
individual wells containing hybrid clones is assayed
for specific antibody. The assay must be sensitive,
simple and rapid. Assay techniques include
radioimmunoassays, enzyme immunoassays, cytotoxicity
.. . .
-: . . . . . . ................ .
: , ' ': :' ' ' ' '' ~ ' .` ~' ; " '
W093/14775 ~ 7 PCT/US92/00852
assays, plaque assays, dot immunobinding assays, and
the like.
Once the desired fused cell hybrids have
been selected and cloned into individual an~ibody-
producing cell lines, each cell line may be propaga~ed
in either of two standard ways. A sample of the -
hybridoma can be injected into a histocompatible
animal of the type that was used to provide the
somatic and myeloma cells for the original fusion.
0 The injected animal develops tumors sec-o__..g the
specific monoclonal antibody produced by tno fused
cell hybrid. The body fluids of the animal, such as
serum or ascites fluid, can be tapped to provide
monoclonal antibodies in high concentration.
Alternatively, the individual cell lines may be
propagated ln vitro in laboratory culture vessels; the .
culture medium, also containing hiqh concentrations of
a single specific monoclonal antibody, can be
harvested by decantation, filtration or
20 centrifugation. ,
5.2 Characterization of MAb PD41
and P~A
Using techniques described generally in
25 Section 5.1 and illustrated in the Examples, a new
hybridoma cell line, PD41, that produces a novel MAb,
PD41, that reacts selectively with prostate
adenocarcinoma was generated. The present invention
encompasses not only the PD41 MAb, but also, other
30 monoclonal antibodies that bind specifically to PMA as ~ -
well as any MAbs that competitively inhibit the
binding of the PD41 MAb to PMA as assessed in an
enzyme immunoassay, a radioimmunoassay or any other
competitive binding immunoassay.
As demonstrated in the Examples which
follow, the immunohistochemical reactivity of ~Ab PD41
- . .. : - : , :, . . .
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WOg3/l4775 ~ 1 C 6 '~ (~ 7 PCT/US92/00852
is highly restricted to prostate carcinoma tissues, in
partlcular ductal epithelia and secretions of prostate
adenocarcinoma tissues. Sixty-five percent of the
prostate tumor specimens examined stained with MAb
PD41, whereas no staining of fetal or benign prostate
specimens was observed. MAb PD41 reacts only
minimally with normal prostate tissues, as less than
1% of the epithelial cells of normal tissue specimens
appear to stain, and then, only weakly. Moreover, M~b
10 PD41 does not react with nonprostate car_ nomas or a
variety of normal non-prostate human tissues, althouch
it is reactive with metas~atic prostate carcinoma,
e.a., in lymph nodes.
Additionally, preliminary experimental
evidence demonstrates that MAb PD41 reacts with about
53% of prostatic intraepithelial neoplasia (PIN)
lesions adjacent to PD41 positive staining tumor areas
in primary prostate carcinoma. PIN is presently
thought to be a precursor ("premalignant") to prostate
20 carcinoma. Hence, PD41-positive reactivity in areas
` of PIN could be predictive of disease potential.
Also as demonstrated in the Examples, MAb
PD41 does not react with available cultured human
prostate tumor cell lines, including DU145, PC3, PC3-
25 P, LNCaP and PPC-1, with human blood cells, or with
purified antigens, including prostate-specific antigen
(PSA) and prostatic acid phosphatase (PAP), using both
radioimmunoassay and immunofluorescence procedures.
MAb PD41 binds specifically to target
30 antigen present in seminal plasma obtained from
prostate carcinoma patients, but not to seminal plasma
from normal donors.
MAb PD41 can also be distinguished from
other MAbs that bind specifically to prostate `
carcinoma, such as the recently described MAbs P25.48
W093/l4775 2 :1 0 bA ~ ~ 7 PCT/US92/00852
- 14 -
and P25.sl (Bazinet et al., 1988, Cancer Res. 48:6438-
6442). I~ contrast to MAb PD41 antigen, the P25 MAbs
of Bazinet react with an antigen which is completely
destroyed after chemical fixation of tissues and
cannot be restored by enzymatic dlgestion. Further
dissimilarity is evident on the basis of molecular
weight o. the respective antigens: P25.48 and P25.91
bind to a M, 58,000 protein (Bazinet et al., 1g89, J.
Urol. 1~1:203A), quite distinct from the antigen
10 recognized by M~b PD41. Moreover,
i~unohistochemically the P25 MAbs do not react with
well-differentiated CaPs as does PD41, but rather do
react with a subpopulation of cells within higher
grade tumors, i.e., poorly to undifferentiated tumors.
MAb PD41 also can be shown to be distinct
from the anti-prostate MAbs PR92 (Kim et al., 1988,
Cancer Res. 48:4543-4548) and 7Ell-C5 (Horoszewicz et
al., Anticancer Res. 7:927-936) on the basis of their
respective antigens and cell-line reactivity. The
20 PR92 antigen is reported to be a glycoprotein with an
approximate molecular weight of 470,000 (unreduced)
and 44,000 (reduced), whereas the antigen defined by
7E11-CS consists of a single M, lOO,OO0 band ~reduced) -
(Wright et al., 1990, Radiopharm. 3:89, Abst. 193).
25 PD41 also can be distinguished from the PR92 and 7Ell-
C5 MAbs, as well as several other prostate-directed
MAbs, i.e., TURP-73 and TURP-27 (Starling et al.,
1986, Cancer Res. 46:367-374), MAbs 35 and 24 (Frankel
et al., 1982, Proc. Nat'l Acad. Sci. USA 79:903-907),
30 KR-P8 (Raynor et al., 1981, J. Nat'l Cancer Inst.
73:617-625), the 3 F77 MAbs (Carroll et al., 1984,
Clin. Immunol. Immunopathol. 33:268-281), and ~-Pro-3
(Ware et al., 1982, Cancer Res. 42:1215-1222) on the
basis of the reactivity of these MAbs to cultured cell
.. . ~ .' . . !
WO~3/14775 2 i a 6 -~ 8 I PCT/US92/00852
- 15 -
lines andlor various normal, benign and neoplastic
tissues.
As demonstrated in the Examples, the
immunoblots of gel-separated components of prostate
carcinoma lissue extracts indicate that the molecular
weight of the proteins carrying the PD41 antigenic
deterlinant differ among individual tumors, ranging
from about M, so,ooo to greater than about 400,000. In
seminal plasma from prostate carcinoma patients, the
10 predominan. compo~ent car-ying the PD41 antigenic
determinant i5 the diffuse Mr ~ 400,000 band.
The variability in molecular size of the
PD41 antigen in prostate carcinoma extracts is similar
to observations described from other carcinomas in
which the reactive antigen is a high molecular weight
glycoprotein or mucin-like tumor-associated antigen.
(See Johnson et al., 1986, Cancer Res. 46:850-857; Lan
et al., 1990, Cancer Res. 50:2993-3001; Burchell et
al., 1983, J. Immunol. 131:508-513; Davis et al.,
20 1986, Cancer Res. 46:6143-6148; Itkowitz et al., 1988,
Cancer Res. 48:3834-3842; Kuroki et al., 1989, Int. J.
Cancer 44:208-218; as well as a review article by
Feller et al., 1990, Immunol. Series 53:631-672).
Data obtained in this study also suggest
25 that the PMA antigen recognized by MAb PD41 is
different from other mucin-like antigens identified on
many non-prostate tumors. MAbs to these non-prostate
mucins not only react with the tumor tissues or cell
lines used for their generation, but also exhibit a
30 spectrum of reactivity against other carcinomas and
normal tissues. Preferential binding of PD41 to CaP
and the virtual absence of reactivity with normal and
non-prostate malignant tissues, particularly breast,
colon, stomach, ovary, and pancreas, provide further
evidence that MAb PD41 is distinct from other mucin-
- . ~ . .
. . - : . .
: ' -.- : ' -: : . . . :, , ' :- ' ~. ' :
~.i~,`li~X7
W0~3/14775 PCT/US92/00852
- 16 -
directed MAbs and that PMA is distinct from other
mucin antigens. Additional support is provided by the
inability of MAbs to other mucin-like TAAs to bloc~
PD41 binding to its target antigen. (See Section 6.4,
below).
.
5.3 Applications
5.3.1 Immunohistological and
Immunocytological A~lications
0 Monoclonal antibodies of the p-es~r.l
invention can be used to detect potential proscate
carcinoma cells in histo'ogical and cytological
specimens, and, in particular, to distinguish
malignant tumors from normal tissues and non-malignant
tumors. For example, using the indirect
immunoperoxidase assay described in Section 6.2.4, it
has been observed that monoclonal antibodies of this
invention stain (1) strongly to very strongly in well
and moderately differentiated primary prostate
20 carcinomas; (2) moderately to very strongly in poorly
differentiated primary prostate carcinoma; and (3)
moderately to strongly in undifferentiated primary
prostate carcinomas. In addition, strong staining was
observed in metastatic prostate carcinoma in lymph
25 node, bone, breast and lung metastases. The PD41 MAb
did not bind to fetal prostate tissues, frozen normal
prostate tissues and fixed BPH tissues. One of 68
-frozen BPH specimens and 3 of 22 fixed normal prostate
specimens were PD41 positive; however, only weak
30 staining was observed, in less than 1% of the ductal
epithelial cells. With the exceptions noted above, no
specific staining was observed in non-malignant
prostate epithelial tissues nor in normal human organs
and tissues examined.
, ' ' ~ ', ~''. ` '`''' '`'' , ' ,' ' :
- : :
- . -, .
. . . :,
, . ,. . .
WO93/14775 2 :L ~ S 7 Pcr/uss2/ooss2
As an alternative to immunoperoxidase
staining, im~unofluorescence techniques can use the
monoclonal antibodies of the present invention to
examine human specimens. In a typical protocol,
slides containing cryostat sections of frozen, unfixed
tissue biopsy samples or cytological smears are air
dried, formalin or acetone fixed, and incubated with
the monoclonal antibody preparation in a humidifled
chamber at room temperature.
The slides are then washed and further
incubated with a preparation of antibody directed
against the monoclonal antibody, usually some type of
anti-mouse immunoglobulin if the monoclonal antibodi2s
used are derived from the fusion of a mouse spleen
lymphocyte and a mouse myeloma cell line. This anti-
mouse immunoglobulin is tagged with a compound, for
instance rhodamine or fluorescein isothiocyanate, that
fluoresces at a particular wavelength. The staining
pattern and intensities within the sample are then
determined by fluorescent light microscopy and
optionally photographically recorded.
As yet another alternative, computer
enhanced fluorescence image analysis or flow cytometry
can be used to examine tissue specimens or exfoliated
cells, i.e., single cell preparations from aspiration
biopsies of prostate tumors using the monoclonal
antibodies of the invention.
As shown in the Examples, preliminary
evidence demonstrates that MAb PD4l reacts with about
53~ of prostatic intraepithelial neoplasia (PIN)
lesions adjacent to PD41 positive staining areas. PIN
is presently thought to be a precursor of prostate
carcinoma. Thus, in another embodiment of the
invention, monoclonal antibodies of the invention can
be used to examine PIN areas in histological and
.. .... . ... .. . .
,: - - , - - . . . ,. . ~.
'' ' ' ~, ,' ~, '' . . ' ' ' . ' . ', . ' ' .
, ' . ' . .. ~, . ' ', . ' . ~ . ' . ' ' ' ' '
.' ' ' ' ' ' ' . . .' ' ~' ,, " . ' .
,' .' .'' '' ' ". '" " ', ',~ ' ~' ''. ': ' ~ . '',' '
. . ~ ,' '
W093/14775 2 1 ~ 6 ll 8 7 PCT/US92/00852
- 18 -
cytological specimens. Positive PD41 reactivity ln
such PIN areas may be useful to predict disease
progression. By way of example, and not limitation,
the monoclonal antibodies of the invention could be
used in quan.itation of the fluorescing tumor cells on
tissue slides or exfoliated cells, i.e., single cell
preparations from aspiration biopsies of prostate
tumors by computer enhanced fluorescence image
analyzer or ~ith a flow cytometer. Use of MAb PD41 in
such assavs would be valuable to differentiate benign
fro~ malignan. prostat2 tumors since the PMA antigen
~o which the ronoclonal antibody binds is expressed
only DV malignant tumors. The percent PMA reactive
cell population, alone or in conjunction with
determination of the DNA ploidy of these cells, may,
additionally, provide very useful prognostic
information by providing an early indicator of disease
progression. (See, Wright et al., 1990, Cancer
' 66:1242-1252, McGowan et al., 1990, Amer. J. Surg.
159:172_177).
In yet another alternative embodiment, the
monoclonal antibodies of the present invention can be
used in combination with other known prostate MAbs to
provide additional information regarding the malignant
phenotype of a prostate carcinoma. For example, the
monoclonal antibody of the invention can be used in
immunohistological or immunocytological tests as part
of a panel of MAbs including such as the P25 MAbs of
Bazinet et al. (su~ra) to distinguish early evidence
of neoplastic change (PD41 MAb staining pattern) from
potentially aggressive tumors (P25 MAb staining
pattern).
,
- - , . :
- : - :. :
.
WO93/14775 2 1 0 ~ 7 PCT~US92/00852
-- 19 --
5.'.2 Immunoseroloqical Applications
The use of the monoclonal antibodies and/or
the PD41 antigen described herein can be extended to
the screenlng of human biological fluids for the
presence of ~he specific antigenic determinant
recognized. In vitro immunoserological evaluation of
biological -luids withdrawn from patients thereby
permits non-invasive diagnosis of cancers. By way of
illustration, human fluids, such as prostatic fluid,
seminal fluid, seru.m or u-ine can be taken from a
patien, and assayed for the specific epitope, either
as released antigen or membrane-bound on cells in the
sample rluid, using the anti-prostate carcinoma
monoclonal antibodies in standard radioimmunoassays or
enzyme-linked immunoassays known in the art,
competitive binding enzyme-linked immunoassays, dot
blot or Western blot, or other assays known in the
art.
Kits containing the PD41 MAb or fragments of
MAbs (as well as conjugates thereof) or PMA antigen of
the invention can be prepared for in vitro diagnosis,
prognosis and/or monitoring prostate carcinoma by the
immunohistological, immunocytological and
immunoserological methods described above. The
components of the kits can be packaged either in
aqueous medium or in lyophilized form. When the
monoclonal antibodies (or fragments thereof) are used
in the kits in the form of conjugates in which a label
moiety is attached, such as a radioactive metal ion,
the components of such conjugates can be supplied
either in fully conjugated form, in the form of
intermediates or as separate moieties to be conjugated ~ -
by the user of the kit.
A kit may comprise a carrier being
compartmentalized to receive in close confinement
:;
W093/14775 ~ 7 Pcr/~s92/ooxs2
- 20 -
therein one or more container means or series of
container means such as test tubes, vials, flasks,
bottles, syringes, or the like. A first of said
container means or series of container means may
contain the monoclonal antibody (or fragmen- ~herPo-)
or the PMA antigen of the invention. A second
container means or series of container means may
contain a label or linker-label intermediate capable
of binding to the monoclonal (or fragment thereof) or
PMA of the invention.
.3.3 In Vivo Diagnostic, Prophylac~ic
and Thera~eutic Uses
The monoclonal antibodies or f-a~monts
thereof of this invention are particularly useful for
targeting carcinoma cells in vivo. Thus, they can be
used for tumor localization for detection and
monitoring (enhancing patient management) as well as
for therapy of primary prostate carcinoma and
metastases. For these in vivo applications, it is
preferable to use purified monoclonal antibodies or
purified fragments of the monoclonal antibodies having
at least a portion of an antigen binding region,
including such as Fv, F(ab')~, Fab fragments, single
chain antibodies, chimeric or humanized antibodies,
CDRs, etc. Purification of the antibodies or
fragments can be accomplished by a variety of methods
known to those of skill including, precipitation by
ammonium sulfate or sodium sulfate followed by
dialysis against saline, ion exchange chromatography,
affinity or immunoaffinity chromatography as well as
gel filtration, zone electrophoresis, etc. (see Goding
ln, Monoclonal Antibodies: Principles and Practice, 2d
ed., pp 104-126, Orlando, Fla, Academic Press).
For use in in vivo detection and/or
monitoring of prostate carcinoma, the purified
., . . : . .: :
. . . ,. : . . . . ,.:
- . :: : ': : . '
- : . ~ : . ..
.. ~ , . . .
W093/14775 PCT/US92/00852
- 21 -
monoclonal antibodies can be covalently attached,
either directly or via a linker, to a compound which
serves as a reporter group to permit imagin~ of
specific tissues or organs following administration
and localiza~ion of the conjugates or complexes.
A variety of different types of substances
can serve as the reporter group, including such as
radiopaque dyes, radioactive metal and non-metal
isotopes, fluorogenic compounds, fluorescent
compounds, positron emitting isotopes, non-
paramagnetic metals, etc.
For use in ln vivo therapy of prostate
carcinoma, the purified monoclonal antibodies can be
covalently attached, either directly or via a linker,
to a compound which serves as a therapeutic agent to
kill and/or prevent proliferation of the malignant
cells or tissues following administration and
localization of the conjugates. A variety of
different types of substances can serve as the
therapeutic agent including radioactive metal and non-
metal isotopes, chemotherapeutic drugs, toxins, etc.
Methods for preparation of antibody
conjugates of the antibodies (or fragments thereof) of
the invention useful for detection, monitoring and/or
therapy are described in U.S. Patent Nos. 4,671,958;
4,741,900 and 4,867,973.
Kits for use with such ln vivo tumor
localization and therapy methods containing the
monoclonal antibodies (or fragments thereof) -
conjugated to any of the above types of substances can
be prepared. The components of the kits can be
packaged either in aqueous medium or in lyophilized
form. When the monoclonal antibodies (or fragments
thereof) are used in the kits in the form of ~ -
conjugates in which a label or a therapeutic moiety is
~ -
-' ' '' ' ' ': '' - .' ' '.' ,~ : ,, .
. . '- i - ~.:
. . ~............................ ~ . ... ... .
WO93/14775 2 1 ~ X ~ PCT~US92/00852
- 22 -
attached, such as a radioactive metal ion or a
therapeutic drug moiety the components of such
conjugates can be supplied either in fully conjugated
form, in the form of intermediates or as separate
~oieties tO be conjugated by the user of the kit.
Other components of the kits can include
such as those mentioned in Section 5.3.3 above.
5.3.4 Other Uses
The ~MA antigen of the present invention is
a uni~u_ 2.._ig2n selec.ively expressed bi prostate
carcinomas. Tt is envlsaged that PM~ and the PD41 MAb
will addi.ionally be valuable to study the natural
history, development, effect of hormone/drug
manipulation, etc. of prostate carcinoma.
In another embodiment of the invention, the
PMA may be used to prepare a vaccine formulation for
prostate carcinoma. Either purified native PMA (see,
Kaufman et al., 1991, Int. J. Can. 48:900-907) or the
nucleotide sequence encoding PMA inserted into a virus
vector such as vaccinia virus (see, Moss., 1991, Sci.
2S2:1662-1667) can serve as the immunogen for the
vaccine formulation of this embodiment.
In other embodiments, the hybridoma cell
lines, including, in particular, the PD41 hybridoma
cell line, of the present invention may be used to
produce compositions comprising an antigen binding
site or antibody variants which combine the murine
variable or hypervariable regions with the human
constant region or constant and variable framework
regions, i.e., chimeric or humanized antibodies as
well as humanized antibodies that retain only the
antigen-binding CDRs from the parent PD41 MAb in
association with human framework regions (see,
Waldmann, 1991, Sci. 252:1657, 1662, particularly
.. -. .. - ... ~ .
- ' . '~: : '
': '' ,: ' :
W093/~477~ 6 1 ,~,~ PCT/US92/00852
- 23 -
1658-59 and references cited therein). Such chimeric
or humanized antibodies retaining binding specificity
of the anti~odies of the lnvention would be expected
to have reduced immunogenicity when administered ln
vivo for diagnostic, prophylactic or therapeutic
applications according to the invention.
In vet other embodiments, the invention
encompasses the use of the hybridoma cell lines as a
source of DNA or mRNA encoding for the rearranged,
activated immunoglobulin genes, which may be isolated,
cloned _y- ~n~w~ recom~inant DNA techniques and
transferr2d to othPr cells for the production of
antigen binding fragments specific for prostate
carcinoma. By isolating rearranged DNA or preparing
cDNA from the messenger RNA of the hybridoma cell line
of the invention, a sequence free of introns may be
; obtained.
To illustrate, and not by way of limitation,
an immunoexpression library can be prepared and
screened for antibody binding fragments for PMA as
follows (See, Huse et al., 1989, Sci. 246:1275-1281;
Mullinax et al., 1990, Proc. Nat'l Acad. Sci. USA
87:8045-8099). Total RNA can be purified (e.a., using
commercially available kits) and converted to cDNA
using an oligo (dT) primer for the light (L) chain and
a specific primer for the heavy (H) chain using
reverse transcriptase. Polymerase chain reaction
(PCR) amplification of the immunoglobulin H and L
chain sequences can be done separately with sets of
primer pairs. Upstream primers can be designed to
hybridize to partially conserved sequences in the
leader andtor framework regions of VH or VL and
downstream primers can be designed to hybridize to
constant domain sequences. Such primers would
preserve full length L chain and provide H chains
' - ~ . .
:: , ,
,
~: - : ,-,
~ ' : - . ' ` , ::
W093/14775 21 f3 ~ 7 Pcr/lJss2/noss2
- 24 -
corresponding to the Fd of IgG and conserving the H-L
disulfide bonds. The PCR amplified L and H DNA
fragments are then digested and separately ligated
into H and L chain vectors. Such vectors contain a
pelB leader sequence, a ribosome binding site and StOp
codons. Suitable ~ phage vectors for expression in E.
coli can be prepared from commercially available
vectors (ImmunoZAP L, ImmunoZAP H; Stratacyte, La
Jolla, CA). The ligated recombinant phage DNA is
incorporated into bacteriophage with i vitro
packaging extract and used to infect E. coli. The
immunoexpression library thus created is screened for
antigen binding fragments using PMA. Positive clones
can be screened and identified as described by
Mullinax et al. (supra).
- In still other embodiments, the invention
encompasses the nucleotide sequence encoding the PMA
antigen of this invention. The PMA antigen of the
invention may be isolated and purified using only
methods known in the art based on binding to the PD41
MAb of the present invention. For example, and not by
way of limitation, PMA may be isolated from extracts
of prostate carcinoma either by affinity
chromatography, in which the PD41 MAb is bound to a
solid support, or by preparative SDS-polyacrylamide
gel electrophoresis, in which gel slices containing
PMA are identified by allowing iabeled PD41 MAb to
bind to the antigen. The PMA may require further
purification and is subjected to amino acid sequencing
using known techniques. Oligonucleotide probes
corresponding to the amino acid sequence thus obtained
may be generated by standard techniques and then used
to identify DNA or genomic clones encoding PMA using
standard techniques lncluding PCR (see generally,
Sambrook et al., ln Molecular Cloning: A Laboratory
., , ~ ,~
.~ ` ,
W093/l4775 2 ~ ~ 6 1 8 7 PCT/US92/00852
- 25 -
Manual, 2d. ed., Cold Spring Harbor Laboratory Press,
1989). Once the gene encoding PMA is cloned, it can
be produced in large quantity using standard
expresslon systems. Alternatively, the PMA gene can
be cloned by a "shotgun" approach in which genomic DNA
or, preferably, cDNA obtained from prostate carcinoma
cells may be used to create an expression library in
which clones expressing PMA are identified by binding
to labelled PD41 MAb using standard techniques.
The following examples are intended as ~on-
limiting illustrative examples of certain embodimen_s
of the present invention.
6. Exam~les
Some of the results described below are -
described in Bec~ett et al., l991, Cancer Res.
51:1326-1333.
6.1 Preparation of Antibodies Specific
for Prostate Carcinoma
Production of MAbs. Three female BALB/c
mice (8 weeks old; Harlan Sprague-Dawley,
Indianapolis, IN) were hyperimmunized ip at monthly -
intervals with 100 ~g of a crude membrane preparation
from a moderately to poorly differentiated prostate
adenocarcinoma surgical specimen mixed 1:1 with
Freunds Complete Adjuvant (FCA), initial injection, or
Freunds Incomplete Adjuvant (FICA), subsequent 2
monthly injections. Following a final iv injection of
the membrane preparation alone, immunized spleen cells
were harvested and fused at a 2:1 ratio with ;
NS1/l.Ag4.1 mouse myeloma cells in the presence of 50%
PEG. Hybridoma production was performed as described
previously. (Starling et al., 1986, Cancer Res.
46:367-374).
"'' ' ' ''''
'
. ., . , - - - ' , . ' ' , - ', : ~ ; ' ' ' .'; .~ ' ~ , . ' . ' : . . .
2 1 ~ 7
W O 93/1477S PCT/US92/00852
- 26 - .
Screening of Hybridoma Supernatants.
- Supernatants from actively growing hybrldomas were
screened for reactivity against the immunizing
material and for negative reactivity against PSA and
PAP ~ntigens and 2 panel of normal cells (AB+ and O
RBC, WBC, and fibroblasts) by a solid-phase RIA.
(Starling et al., 1982, Cancer Res. 42:3084-3089) One
hybrid, designated PD41.84 (PD41), was selected for
further analysis, subcloned using a Coulter Epics 5
flow cytomete~, and isotyped (IgGl,k) usina an enzyme-
linked in~."unGs~r~en. assay and ~it (Hyclone
Laboralories, Logan, "T).
~ nus, following the procedure of the
invention, hybridoma cell line PD41 was obtained. The
hybridoma cell line was cultured to produce monoclonal
antibody PD41 in sufficient quantity for
characterization and further analysis as described
below.
- 20 6.2 Materials and Methods
6.2.1 Cell Lines
- Human tumor cell lines were grown in
nutrient medium supplemented with serum additives as
recommended by the supplier of the line. The source
of many cell lines used in this study has been
described in earlier publications. (Starling, et al.,
1986, Cancer Res. 46:367-374; Starling et al., 1982,
Cancer Res. 42:3084-3089). The cell lines evaluated,
include the following: T24, CUBl, SW733, 253J,
SCaber, J82, RT4, EJ, TCCSUP, SW780, HT1376, CUB3, and
HU609 (bladder); DU145, PC3, PC3-P, LNCaP, and PPC-1
(Brothman et al., 1989, Int. J. Cancer 44:898-903)
(prostate); Calu-1, A-427, Plano-1, SKLU-1, A549,
SKMES-1, OH-1, and SKLC-2 (lung); VAMT-1, JMN, and
r - , . - . - . - - :.-- : :
~1~6~1~7
WO93/14775 PCT/US92/00852
- 27 -
NCI28 (mesothelioma); MCF-7, SKBr3, and ZR-75-1
(breast); LS174, CX-1, SW480, SW1463, and LS180
(colorectal); A37s, WM56, and H1477 (melanoma); PANC-1
and MIA (pancreatic); CMVMJHEL-1 (cytomegalovirus
trans~ormed fibroblast); and CCRF-HSB2 (human T-cell
lymphocytic leukemia).
6.2.2 Tissues
~resh surgical or autopsy specimens as well
10 as .or.ualin-~ixed, paraffin-embedded blocks of various
human tlssuos were obtained from the Department of
Pathology of Sentara Norfolk General ~ospital,
Norfolk, VA; the Department of Pathology of the
Veterans Administration Hospital, Hampton, VA; the
Cooperative Human tissue Network, University of
Alabama at Birmingham; and the National Disease
Research Interchange, Philadelphia, PA. Tissues for
paraffin blocks were fixed in neutral buffered 10%
formalin, whereas those for frozen sections were
20 embedded in Tissue-Tek OCT Compound (10.24% w/w
polyvinyl alcohol, 4.26% w/w carbowax, and 85.50% w/w
non-reactive ingredients) and snap-frozen in
isopentane over li~uid nitrogen. Paraffin blocks of
various passages of the prostatic carcinoma xenografts
25 PC-82, PC-EW, and PC-EG were kindly provided by Dr.
Gert Jan Van Steenbrugge, University of Rotterdam, and
Dr. Zoltan Csapo, Department of Urology,
Zentralklinikum, Augsburg, Federal Republic of
Germany. The origin and establishment of these
30 xenografts have been described previously. (Hoehn, et
al., 1980, Prostate 1:95-104; ln G.B.A Bastert et al.,
eds., Thymus Aplastic Nude Mice and Rats in Clinical
Oncology, pp. 413-415, Stuttgart, New York, Gustav
Fischer Verlag, 1981; and 1984, Prostate 5:445-452).
:; - . :. .. - :,, . : : : . ... , ~: : :: ~ .
? /~
W093/14775 PCT/US92/00852
- 28 -
6.2.3 Tumor Tissue Pre~aration
Crude membrane preparations were prepared
from prostate carcinoma specimens or other tissues by
first finely mincing the tissue in 10 ml of 1.0 mM
5 NaHCO3 buffer containing 200 ~1 of a 50X protease
cocktail (antipain, 3.4 mg; pepstatin, 10.0 mg; EDTA,
0.372 g (Sigma Chemical Co., St. Louis, M0) dissolved
in 20.0 ml of DDH.0). The minced tumor tissue was then
homogenized in a Polytron (Brinkman Instruments,
10 Westbury, NY) and further disrupted using Wheaton
glass homogenizers. The homogenate was centrifuged at
2000 X g for 5 min, and the supernatant resulting from
this spin was further centrifuged (2 h, 138,000 X g,
4C). The resulting pellet was resuspended in a
minimum volume of PBS and stored at -70C. Protein
concentrations were determined using the Bicinchoninic
Acid (BCA) Protein Assay (Pierce Chemical Co.,
Rockford, IL).
6.2.4 ImmunoDeroxidase Stainina
The staining reactivity on frozen or fixed
tissues was evaluated by the avidin-biotin peroxidase
assay using the ABC Elite Vectastain kit (Vector
Laboratories, Burlingame, CA) as described previously.
(Wright et al., 1983, Cancer ~es. 43:5509-5516; Wahab
et al., 1985, Int. J. Cancer 36:677-683). Following
development with the chromogen 3,3'-diaminobenzidine
tetrahydrochloride (Sigma), the tissues were
counterstained with Mayer's hematoxylin and mounted in
30 aquamount (Learner Laboratories, Pittsburgh, PA).
Tissues were scored independently by 2 investigators
for both intensity of reactivity, using a scale of 0
(absence of staining) to 4+ (most intense staining) as
well as for numbers of cells positive in each
specimen.
.... .. .. , . -, . . , - ~ .
WO93/14775 2 ~ 7 PCT/US9~/00852
6.2.5 Immunofluorescent Staininq
Cells for immunofluorescence analysis were
re~loved from culture flasks either by scraping or with
PBS-EDTA; PBS-washed cells were pelleted for use in
either live-cell or fixed-cell t25% ethanol, on ice,
15 min) indirect immunofluorescence. MAb PD4l culture
supernatant (l00 ul) was added to test wells and
incubated at 4C for 60 min. After the cells were
washed in PBS, they were incubated for 30 min at 4C
10 with 50 ~l of fluorescein-conjugated goat anti-mouse
IgGs (Organon leknika-Cappel, Malvern, PA) at 50
~g/ml. The cells were again washed and observed for
fluorescent staining using an Olympus microscope
equipped with a fluorescence vertical illuminator
system. The percent of intact cells showing
fluorescence and their staining intensity (scale 0 to
+4), out of a total of 300 cells analyzed, was
determined.
6.2.6 Sodium Dodecyl Sulfate-Polyacrylamide
Gel Electrophoresis/Western Blotting
Procedures
Electrophoresis was carried out in
polyacrylamide gels under reducing conditions [Laemmli
et al., 1970, Nature (London) 227:680-685], and
protein migration in gels was determined using Rainbow
protein molecular weight markers (Amersham, Arlington
Heights, IL). Proteins separated by electrophoresis
were transferred to an immobilon-P transfer membrane
30 (Millipore, Bedford, MA) at 25 V for 12 h (Tobin et
al., 1979, Proc. Nat'l Acad. Sci. USA 76:4350-4354)
using transfer buffer withou. methanol. Following
transfer of the proteins, the membrane was placed in
TBS blocking buffer (l h at 37C). The membrane was
35 incubated with PD41 MAb culture supernatant (20 ~g/ml,
2 h, 25C) or an isotype matched control antibody.
, ; ., ,,, - , ,, ~ , . ...... .. ... . .... . . .. .
- , ,, , : . i. .
W093/14775 2 ~ PCT/US92/00852
- 30 -
Membranes were washed in dilute TBS to remove unbound
antibody, then incubated with ~25I-labeled rabbit antl-
mouse IgG (secondary antibody, specific activity 1.25
~ci/~g) at 1 x 107 cpm/50 ml of TBS for an additional 2
h at 2~oc. rollowing incubation with the secondary
antibody, the membrane was further washed, air dried,
and exposed to Kodak XAR X-ray film (-70C, 48-72 h).
The membranes were gently agitated during all
incubations.
~.2./ Comoetitive Bindinq Assav
?urifie~ PD41 M~b was labeled with l-5I-
labeled soàium iodide (specific activity, 1.25 ~Ci/~g)
by the conven~ional chloramine-T method. Unbound
iodine was removed from the ~25I-labeled MAb product by
a membrane ultrafiltration technique established in
our laboratory (Lipford et al., l99O, Anal. Biochem.
187:133-135). Twenty-five ~l of unlabeled antibody
(blocking antibody) at varying concentrations from O
20 to lOO ~g/ml were incubated with the antigen-coated
wells (3 ~g/well) for 2 h at 25C. After a series of
washes, 25 ~l of ~25I-labeled PD41 MAb were added at
approximately 50% bindinq activity and incubated an
additional 2 h. Following additional wash steps, the
25 wells were dried and cut, and the radioactivity
remaining in the wells was determined. The blocking
antibodies used were MAb B72.3 (a gift from Dr.
Jeffrey Schlom, Laboratory of Tumor Immunology and
Biology, National Cancer Institute Bethesda, MD see
30 U.S. Pat. No. 4,522,918), anti-carcinoembryonic
antigen (CEA) MAb (Zymed, San Francisco, CA) that had
been adsorbed to eliminate reactivity to non-cross-
reacting antigen, and anti-HMFG-2 MAb (Unipath
Limited, Bedford, United Kingdom).
- : .:, ......... . . - ...................... .. . .
. ~ . . : , , ' , . . . ,.. - .
WO93/147752 ~ ~ 6 ~ 7 PCT/US92/00852
- 31 -
6.3 Results
6.~.l Reactlvity of MAb PD4l With Human
Cell Lines and Blood Cells
Monoclonal antibody PD4l was screened
against a panel of 44 cultured human tumor cell lines
(see Section 6.l.l above) and normal human blood cells
using both fixed-cell indirect radioimmunoassay (RIA)
and live anà fixed-cell indirect immunofluorescence.
Results are shown in Table l.
:.
.~
~ -
:
.
3S
. ~ ' ` ` ' ' .' ' ;; ' ' ' ' : ' .',` `. .' ' ` ' ' ''.
'` `'.'`. .` `' ' ' ` . ~ ' `. ' .: '. '.': ' ` . : . ' ' ' '
2 ~31~l~87
W093/14775 PCT/~'S92/OOX52
- 32 -
Table l
Reactivity of MAb PD4l to Malignant Cell
Lines and Human Blood Cells by Indirect
RIA and Indirect Immunofluorescence
No. Positive / No. Tested
Immuno- Immuno-
Target cell RIA fluorescenceb fluorescence'
TCC (~ladder) 0/12 0/3 0/3
Prostate 0/~ 0/5 0/_
Breast O/l 0/3 0/3
Lung O/lO o/4 o/4
Adeno- 0/6 0/3 o/4
carcinoma
Small cell O/l O/l O/l
Meso- 0/3 NTd NT
thelioma
Colorectal 0/3 l/5' 0/3
` Pancreatic 0/2 NT NT
.: Melanoma NT 0/3 0/3
Blood cells
RBC (AB+/-) 0/3 0/3 0/3
WBC 0/4 0/4 o/4
Values given are for a fixeà-cell radioimmunoassay
as described in Section 6.2.
b,~ Values given are for a live-cell (b) and a fixed
-cell (c) indirect immunofluorescence assay as
described in Section 6.2.
d NT = not tested.
35 ' Reactivity to LSl80, a colorectal carcinoma.
. ' . -, :~ . ~ . .. ..
.: . . : . . . - ;
2 ~ 8 7
W093/t4775 PCT/US92/00852
- 33 -
As demonstrated in Table 1, only one
cultured tumor cell line, LS180, a colorectal
carcinoma, reacted with the PD41 MAb. PD41 reactivity
was also detected in the concentrated, dialyzed spent
culture medium and the glycopeptide fraction digest
from the L5180 cell line (data not shown). No other
cell type, including the 5 prostate cell lines, i.e.,
DUl45, PC3, PC3-P, LNCaP, and PPC-1, expressed the
PD41 antigen.
6.3.2 Tissue SpecificitY of MAb PD41
The tissue specificity of monoclonal
antibody PD~1 was determined using an avidin biotin
complex immunoperoxidase assay (see Section 6.2.4
above). The results are presented in Table 2 and
FIGS. l (A-F) and 2.
; 20
,' - '
' '', , '' ~ ' ' . '
.. . . . . .
~1~lj4Y7
W O 93/14775 P ~ /Us92/00852
- 34 - -
Table 2
Reactivity of Monoclonal Antibody PD41
~o Formalin-Fi.;ed or Frozen Prostate
T:s~ues by Indirect Immunoperoxidase Asgay
No. Positive/No. Tested
(~ Positive~
Staining Positive
Tissue Type Frozen Fixed intensity~ Cells~
Primary 52/S5 (80) 43/81 (53) 3-4+ 2-95
Carcinoma
WD~ 14/15 (93) 12/20 (60) 3-4+ 3-go
MD 13/15 (87) 9/12 (75) 3-4+ 3-95
PD 24/2q (83) 20/25 (80) 2-4+ 2-95
VD 1/~ ~17) 2/24 (8) 2-3+ 2-10
Metas.at;~
Bladder 0/1 (0) 0/2 (0) 0 0
Bone 0/1 (0) 1/2 (50) 3-4+ 80-90
Breast l/l (lO0) l/2 (50) 2+ <1
Liver NTd 0/3 (0) 0 0
Lung NT 1/3 (33) 3+ 30-40
Lymph node NT 0/1 (0) 0 0
8PH 1/68 (1) 0/48 (0) 1-2+ <1
Normal 0/18 (0) 3/22 (14) 1-3+ <l
Fetal' 0/3 (0) 0/5 (0)
i Nude Mouse
Xenografts
PC-82 NAd l/2 (50) 2+ 2
PC-EW NA 1/2 (50) 3-4+ 23
PC-EG NA 0/2 (0) 0 0
Staining intensity: 0 = no staining, l+ = weak staining,
2+ = moderate staining, 3+ = strong staining, and 4+ = very
strong staining.
30 D Intensity and per~ent po~itive oells apply to both
fixed or frozen tissue~.
WD, MD, PD, UD: well, moderately, poorly, and
undifferentiated prostate caro:noma.
~ NT, NA: not tested, not available.
35 ' Fetal tissue obtai~ed from second ar.d third
trimester specimens.
2 :10 fi ~
W O 93/14775 PCT/US92/00852
- 35 -
As demonstrated ln Table 2, PD41 MAb reacts
to both frozen and flxed primary prostate carcinoma.
Considering only those cases in which 10% or more of
the tumor cells are identified by PD41, the
sensitivity for pros.ate cancer specimens is 47%
(fixed tissues) and 65% (frozen tissues). In
instances in which both fixed and frozen specimens
from the same case could be evaluated, 26% of the
flxed specimens failed to stain, suggesting the
10 possibility ~ha scme den2tll~2tion of the target
antigen may occur during eien~r the formalin fixation
or deparaffiniz2tion procedures. In either case,
s~rong staining of tne tumor cells was observed in the
majority of the PD41-positive prostate carcinoma
specimens (FIG. 1). The PD41 staining appears
confined to an antigen expressed by prostatic
epithelial cells, although staining of luminal
secretions and the borders of the ductal epithelial
cells was also observed (FIG. 1). The staining
20 pattern was, however, very heterogeneous with the
number of PD41-positive tumor cells ranging from 2 to
95% (Table 2) irrespective of specimen preparation
(FIG. 2), and this pattern of expression remained
fairly constant for the differentiated carcinomas
(i.e. well, moderately, and poorly differentiated)
(Table 2; FIG. 2). Undifferentiated primary prostate
carcinomas (those with no glandular elements present),
on the other hand, appeared to lac~ PD41 expression
(FIG. lE; FIG.2).
PD41 antigen detection was observed in bone,
breast, and lung metastases tested (Table 2), with
intense staining in the majority of the tumor cells of
one metastatic bone specimen (FIG. lF).
PD41 MAb did not bind substantially to fetal
3 prostate tissues, frozen normal prostate tissues, and
.. . .... , . . ~,; ~.
-: . ,~ ~ - . -, . .
- : ~ - :
' 7
W093/14775 PCT/IJS92/00852
- 36 -
fixed BPH tissues (~able 2). One of 6~ frozen BPH
specimens and 3 of 22 fixed normal prostate specimens
were PD41-positive, however only weak staining was
observed in less than 1% of the ductal epithelial
cells.
The PD41 target antigen also was expressed
in the tumor cells of 2 nude mouse prostate xenografts
(Table 2), and the staining pattern observed,
especially in the PC-EW heterotransplant, was similar
to that described above for human prostato surgic~1
specimens.
Both the number and intensity of the stained
cells, in PMA-positive specimens, could be enhanced by
exposure to neuraminidase (0.1 unit, 30 min), whereas
15 trypsin or Pronase treatment had no effect on PD41
expression (data not shown). Pretreatment with
neuraminidase could not convert a PMA-negative
specimen to a PMA-positive specimen.
Reactivity of normal human tissues, fixed or
20 frozen and of non-prostate human carcinoma tissues
with MAb PD41 was assessed by immunoperoxidase assay.
Results are presented in Tables 3 and 4.
- ,: . , ., . , , ,.. ~ . . - ,. , .: . . : .. ~
WO 93tl4775 2 i G ~ ~ ~ 7 PCT/US92/00852
Table 3
Reactivity of MAb PD41 to Fixed or Frozen Normal
Human Tissues by Indirect Immunoperoxidase Assay
No. positive/no. tested
Tissue type Frozen Fixed
- Bladder 0/3 0/3 :
Blood vessels 0/3 0/4
Brain 0/6 0/5
Breasta 0/5 0/3
Bronchus 0/3 0/3
Colona 0/4 0/3
Esophagus 0/3 0/3
Heart 0/3 NT~
Kidney 0/4 o/3
Liver 0/4 0/3
Lung 0/3 0/4
r 20 Lymph node O/4 0/4
Ovary 0/3 0/3 ~
Pancreas O/3 O/5 ~ -
Peripheral 0/3 0/
nerve
Salivary NT 0/2
gland
Skin 0/2 0/3
Spleen 0/6 0/3
Stomach NT 0/1
Testicle 0/3 0/4
Uterus 0/3 0/3
a Epithelial cells negative, an occasional gland or
duct showed positive staining of secretory
material.
' NT, not tested.
2 L ~3 ~
WO93/14775 PCT/US92/00852
- 38 -
Table 4
Immunoperoxidase Reactivity of MAb PD41
to Fixed or Frozen Non-Prostate Human Carcinomaa
No. ~ositive/no. tested
Tumor type Frozen Fixed
TCC (urinar~ 0/5 0/3
bladder)
Breast o/~ 0/6
Colon 0/10 0/7
Laryngeal 0/1 N~
Liver NT 0/1
15 Lung 0/4 0/1
adenocarcinoma)
; Lung o/7 o/7
(mesothelioma)
Lymphomas 0/4 0/2
20 Ovarian . 0/3 O/4
Pancreatic 0/1 O/2
Gastric 0/2 O/5
Carcinoid NT 0/1
25 " MAb B72.3 or HMFG~ was used as a positive control
MAb where appropriate.
NT, not tested.
As demonstrated in Table 3, 21 normal tissue
30 types (represented by 131 specimens) did not react
with PD41 MAb. As seen in Table 4, 80 non-prostate
carcinoma tissues (12 different types) did not react
with the PD41 MAb.
Preliminary experiments demonstrated that : . -
35 the PMA antigen was expressed in 53% of PIN lesions
adjacent to PMA-positive staining tumor areas in 73
" '
~ '
,, .. , .. ~ .. . .,, , . ,. ~ ., . , .,, , . ... , ", . .,; ., ,. ,,, .,, , , ",. . ..
2 ~ 1 8 7
WO93/14775 PCT/US92/00852
- 39 -
primary prostate carcinoma tissues tested. Such
results indicate that PMA-positlve reactivity in areas
of PIN could be predictive of disease progression.
- Addi.ionally, PMA was found in 79 of 88
(90%) lymph nodes ~i'h pathologically diagnosed
prostate metastasis. These results also suggest that
PMA may be useful in predic.ing progression of
disease.
Reactivitv of normal seminal plasma and
semin21 pl~s~2 cb'ained from patients with a
definitive ~iaanosis of pros.ate carcinoma was
assessed by indirect RIA. Results are presented in
Table 5.
'~ .
~ 20
, '
.. , ,.. .. .. . ., ~ . . , ~ . . , . , ", .... . .
. ~ ., . . , :. ., :
; . : -: -
-,
- - ' , ~ ' . . ~ . , - .. ' :
WO 93/14775 ~ ' PCT/US92/00852
-- 40 --
Table 5
Comparison of Reactivity of Monoclonal
Antibodies PD41, anti-PSA, and Anti-PAP in
Human Seminal Plasma by Indirect Radioimmunoassay
- Reactivity~
PD41.84 anti-PSAanti-PAP
Specimenb(IgG1) (IgG1) (IgG2a)
10 Normal seminal - +
plasma pool
Seminal plasma
(carcinoma) d
1. WD + +
2. MD ~ + +
3. MD
4. MD - +
5. WD ~ +
6. PD + + +
7. MD + + +
8. WD - + +
.. .
Values greater than 3 times the values for the isotype
-matched control antibodies were considered to ~e positive
(+). Isotype-matched negative control MAb, either IgG1 or
IgG2a.
b For all seminal plasma samples tested, a sample dilution of
1:8 was determined to be the optimal concentration.
Seminal plasma ~amples were obtain~ad from patients with a
definitive diagnosis of prostate carcinoma but who had not
yet undergone treatment. WD = well-differentiated; MD =
moderately differentiated; PD = poorly differentiated.
Table 5 shows that the PMA antigen could be
detected in three of eight CaP seminal plasma samples
by RIA, although it was not detected in normal seminal
plasma. Comparison of MAbs to PSA and PAP (generated
35 in our labcratory) indicated tha~ these antigens were
. - ;'. ' ' . ' ' ' . ' , ' ,. ' ': ': ': :' . ' , ', ' : . :' . . .. : . '
21G6~ 7
W093/14775 PCT/US92/00852
- 41 -
detectable, as expected, in both types of seminal
plasma specimens.
Strong PMA reactivity was also observed in
immunoblot studies of split ejaculates and prostatic
fluids obtained from prostate carcinoma patients. A
sample of ejaculate or prostatic fluid was spotted on
PVDF membrane, dried, reacted with MAb PD~1 and I-125
labeled sheep anti-mouse IgG and exposed to x-ray
film. Strong PMA reactivity was seen in both split
ejaculate and prostatic fluids.
In similar preliminary immunoblot studies,
of urine of prostate carcinoma patients some
indication of the presence of PMA in urine has been
demonstrated.
6.4. Characterization of PMA Antiaen
Preliminary characterization of the PMA
antigen and molecular weight determination of the PD41
; reactive bands were obtained by Western blot analysis
20 of normal, benign and prostate carcinoma tissues as
well as other normal and non-prostate carcinoma
tissues. Results are presented in FIG. 3.
FIG. 3A is a representative immunoblot
developed with the PD41 MAb. The most prominent band
25 observed, a large diffuse band (Mr ~ 400,000) is seen
in both the CaP tissue extracts (Fig. 3A, Lanes 1 and
3) and CaP seminal plasma (FIG. 3A, Lane 4), although
this band was not observed in all CaP tissue extracts
FIG. 3A, Lane 2). Two other prominent components
30 reactive with MAb PD41, a Mr 166,000 and a M, 91,000
band, were usually observed in all the CaP extracts
examined (FIG.3 A, Lanes 1-3), however, occasionally,
additional minor bands were observed in some CaP
tissues. It also can be noted that the pattern (i.e.,
number and mobility of bands) reacting with the PD41
.. . :
.. ~ . .. .. . . .
~,
,.. ' - . . . .
: . .
: ' :
WO93/14775 ~ 8 7 PCT/US92/00852
- 42 -
MAb, especially the high molecular weight components
(Mr ~ 400,000), differed among individual tumors.
However, the absence of MAb PD41 reactive bands in
tissue extracts from normal prostate, BPH, breast and
5 colon carcinomas, and normal seminal plasma (FIG. 3 A,
Lanes 7 and 8, 9 and 10, 5 and 6) clearly confirmed
the restrict2d distribution of PMA to prostate
carcinoma.
E~idence that the reactivity observed on the
10 PD~ ~b irlmunoblot was specific was provided by an
identical l~unoblot stained with an isotype-matched
control antibody (FIG. 3B). A non-specific Mr 55,000
band seen on both blots (FIGs. 3A and 3B) was
identified as endogenous human IgG heavy chain (data
15 not shown). The control blot showed no other positive
; bands. All ~embrane preparations used in this assay,
: when tested for the presence of residual endogenous
protease activity (Endoproteinase Biochemical Test
Combination kit; Boehringer Mannheim GmbH, Federal
20 Republic of Germany), were found to be neqative (data
not shown). This assures that the multiple banding
pattern was not the result of enzymatic cleavage
during membrane extract preparation.
A series of biochemical assays were
25 conducted to further characterize the PMA antigen
having a molecular weight of > 400 kd under reducing,
conditions. Sensitivity of the PMA antigen to
physiochemical and enzymatic treatment was
investigated. In view of the relationship between an
30 epitope found in bovine submaxillàry mucin (BSM) and
another mucin-reactive MAb, i.e., MAb B72.3, Kjeldsen
et al., 1988, Cancer Res. 48:2214-2220. BSM was
included in the experiments. Results are presented in
Table 6.
i ` - , - - . '.:: ',. ~ , . .. ' ' , ', '~ ' . `- -, : ' . ' . '~ . . ' :
2 ~
WO 93/14775 PCI/US92/00852
-- 43 --
Table 6
Percent Control Bindlng of PD41 MAb to
~-s Taraet Antigen Following Enzymatic
or Chemleal Treatment of the PMA Antiaen
Treatments of ~.~A Antigen % Contr~l Binding
CaP BSM~
_
Physioal/Che~ical:
Heat ('C0C) 95 98
Alkali-30rohv-ide 10 13
0 DH 4.~ 139 266
pH 1'.0 77 85
Sodium Meta-?e oidate 2 7
Glycosid~se ~-sataents of
PMA Ant~gen:
Neuraminidase 145 160
Chondroltinase ABC 100 104
Alpha Fucosidase 103 106
N-Glycanase 104 146
t Endo-F 104 106
20 Mixed Glycosidases 4 N~
0-Glycanase 9 24
Beta-Galactosidase 30 45
Proteolytic Digestion of
PMA Antigen:
Pronase 20 0.2
25 Protease Type XIV 7 N
Protease Type XXI 15 8
Protease Type XXVII 14 7
Trypsin 5 4
Alpha Chymotrypsin 14 10
a Percent control binding of the P~41 antibody to its
target antigen (prostate carcinoma _issue memDrane
extract or bovine submaxillary mucir.) was
determined by comparing the cpms of the untreated
antigen with those obtained after various antigen
treatments in a standard solid phase
radioimmunoassav. Binding of the antibody to the
untreated antigen is considered to ae 100%.
~ 80vine submaxillary mucin.
W093/l4775 ~ (Y 7 PCT/~S92/008S2
As shown in Table 6, the PMA antigen is
sensitive to treatment with various proteolytic
enzymes. Additionally, the PMA antigen is sensitive
to agents which affect carbohydrate moieties,
including sodium borohydride, sodium meta-periodate,
O-glycanase, and beta-galactosidase. Such results
indicate that carbohydrate forms an important portion
of the PD41 proteinaceous antigen.
Additional experiments were conducted to
investigate the ca-bohydrate moiety of the PM~ antigen.
using standard RIA procedures. (See, e.a., Starling e~
al., 1982, Cancer Res. 42: 3084-3089; Riott et al. ~n
Immunology, Ch. 25, Section 25.9, St. Louis, MO, Mosby
Co., 1985; Goding in Monoclonal Antibodies: Principles
and Practice, 2d ed., pp 75-78, Orlando, Fla, Academic
Press, 1986). Results of blocking experiments with
MAb PD41 with a variety of carbohydrates and lectins
are presented in Tables 7 and 8.
...... ~ . - -
WO93/1477S 2 ~ O ~ PCT/US92/00852
- 45 -
Table 7
Carbohydrate Blocking
of PD41 MAb Binding to PMA
Carbohydrate Percent Control Binding
Galactose 87
Mannose 79
Maltose 83
N-Acetyl Galactosamine 70
N-Acetyl Glucosamine 9~
N-Acetyl-Lactosamine 81
Sialic Acid 98
Stachyose 8Q
2-Acetamido-2-Deoxy-3- 93
15 O-~-Galactopyranosyl
Galactopyranose
; Raffinose 70
~, a PD41 binding in the absence (100%) and presence of
various mono-, di- and polysaccharides was
determined by standard radioimmunoassay.
.
~,
, 30
.
,. .. . .
,:. : :. :
- , . . : .
.. .. ~ . .
:. . .. .: ~ ~. " :.
~lOb ~7
W093/l4775 PCT/~S92/00852
- 46 -
Table 8
Lectin Blocking of
PD41 MAb Bindinq to PMA
% Control
LectinBindingb Specificity
.
PBS 100 CONTROL
BP.~ 95 D-GalNAc
CON A 112 alpha man
0 DBA 95 alpha GalNAc
GS--~ 110 alpha Gal; GalNAc
GS-II 97 beta D-GlcNAc
LPA 100 sialic acid
MPA 115 alpha Gal
PNA 106 Gal 1-> 3GalNAc
UEA 98 Fuc 1-> 2Gal
WGA 108 GLcNAc, sialic acid ~ ~ -
S-WGA 107 beta 1-> 4-D-GlcNAc
SBA 46 GalNAc, Gal
Abbreviations: PBS, phosphate buffered saline; BPA,
Bauhimina purpurea agglutinin; Con A, concanavalin
A; DBA, Dolichos biflourus agglutinin, GS-I,
Griffonia simplicifolia I agglutinin; GS-II,
Griffonia simplicifolia II agglutinin; LPA, Limulus
polyphemus agglutinin; MPA, Maclura pomifora
agglutinin; PNA, Arachis hypogea agglutinin; UEA,
Ulex europaeus agglutinin; WGA, Triticum vulgaris
agglutinin; S-WGA, succinyl-Triticum vulgaris
agglutinin; SBA, Glycine max.
30 ~ Percent control binding was determined by comparing
PD41 binding in the absence (100%) and presence of
various lectins using a standard radioimmunoassay
procedure.
.:- ' . ' , : ', '.''
2 ~ 7
W093/14775 PCT/US92/00852
- 47 -
.~s de~onstrated in Table 7, sialic acid does
not inhibit binding of the PD41 MAb to PMA, despite
the fact that treatment of the antigen with
neuraminidase appears to enhance PD4l MAb binding
(Table 6). Thus, sialic acid does not appear to be a
component of the antigenic determinant of the PMA
antigen, although removal of sialic acid residues may
expose more of the PD4l antigenic determinant to the
PD4l ,~Ab.
Æs also demonslrated in Table 7, n-acetyl
galactosamine znd rafCinose bo~h blocked binding of
PD4l MAb ~o the PMA antigen by about 30~.
Results of the lectin binding experiments
(Table ~) sup?ort the glycoprotein nature of the PMA
antigen. Only soy bean agglutinin (SBA), which has
specificity for GalNAc, Gal, inhibited binding of the
- PMA antigen to the PD41 MAb (54%). None of the other
eleven lectins tested showed any significant
~- inhibition.
Based on all the above evidence the PMA
antigen appears to encompass an O-linked glycoprotein
of molecular weight >400kd with N-acetyl galactosamine
as the predominant amino sugar of the antigenic
epitope, which is most probably mucin in nature.
A series of competitive binding and
reciprocal binding experiments were conducted to
determine whether the PMA antigen was related to any
previously identified mucin tumor associated marker or
to any of the human blood group antigens.
Results of a competitive binding assay (as
described above in Section 6.2) presented in FIG. 4,
demonstrate that, except for unlabeled PD41, none of
the unlabeled antibodies, i.e., B72.3, anti-CEA or
HMFG-2, which are known to bind to high molecular
,;: . ;:. :: .:. ... ~ . . ,.. . ., : . - . - . ,
: - . : -: . - : ... . ;. .: : ~ . - . . .;, : , . ..
.. . . - , ~- - - - . .. : .,
;- .. . . ~. :.. ;. .. .
WO93/14775 PCT/US92/()0852
- 48 -
weight TAA~s, could compete with I-125 labeled MAb
PD41 for binding with PMA.
Double determinant immunoradiometric
inhibition experiments were conducted as follows. A
series of unlabelled MAbs, (listed in Table 9 together
with antigens with which such MAbs react) were then
added, at a concentration of lO0 ~g/ml to the target
PMA bound to unlabeled "capture" PD41 MAb. Non-
saturating amounts of the l25I-labeled PD41 MAb were
then added.
The percent inhibition or blocking of ,he
labelled PD41 MAb to its target antigen was calculated
as:
: 100 - sDec1fic bindina in Dresence of blockina antibodv x 100
specific binding in absence of blocking antibody
Results are presented in FIG. 5.
.' '
i ~ .
::
:~
~ '' ..
' ~
2la~s7
WO93/14775 PCT/US92/00852
- 49 -
Table 9
Tumor Associated Antigens and Blood Group
Antigens Recognized by Monoclonal Antibodies
Antigen/Antibody Type of Cancer or Antiaen
- DF3 Breast
OC125 Ovarian
HMFG2 Breast
B72.3 Breast, ovarian, colon
CEA GI, lung, breast, etc.
TURP 73 Prostate, breast, colon
SM3 Breast (Peptide of HMGF2)
AR3 Pancreas, stomach, colon,
uterus, ovarian
M344 Bladder
3F8 Melanoma, neuroblastoma
BG-l* Precursor (type 1 chain)
BG-4* H-l
BG-5* Le' (type 1 chain)
BG-6* Leb (type 1 chain)
BG-7* X (type 2 chain)
GB-8* Y (type 2 chain)
25 * Refers to monoclonal antibodies related to blood
gro~p antigens.
As illustrated in FIG 5, only unlabelled
PD41 MAb was able to block (73%) of the binding of the
30 labelled PD41 MAb.
A series of reciprocal blocking experiments
using B72.3, CEA, M344, OC125 as well as PD41 as
blocking MAbs, were performed as described below: A
range of concentrations (0-100 ~g/ml) of unlabeled
35 MAbs (blocking) were added to target antigens
appropriate for each antibody tested and incubated for
:-, . - , . , . . ~ ;, . . .
-' . ': , ' - . . . : :-., . ', . : ' :
~ rl
W093/1~775 PCT/~S92/00852
- 50 -
1 h. After washing, non-saturating amounts of I-125
labeled MAb (tracer), as indicated, were added to each
well, inc~bated and the radioactivity remaining in the
cells after washing was determined. Results are
presented in FIG. 6.
As shown in FIG. 6, unlabeled PD41 MAb did
not block the binding of the selected I-125 labeled
MAbs to their respective mucin tumor associated
antigen targets. Blocking occurred only with the
1o homologous ~b-
E?itope c~-expression was determined using a
radioimmunometric assay as described above. In this
experiment, the ability of the radiolabelled PD41 ~Ab
to bind target antigen bound by a different capture
mucin tumor associated MAb was determined. A series
of MAbs which bind to different TAAs were immobilized
on the plates to serve as "capture" MAb. Two
different prostate carcinoma Surgical specimens
previously shown to contain PMA antigen, served as the
20 target antigen to be "captured" by the capture MAbs.
These preparations were designated "Antigen 1" and
~ "Antigen 2". Radiolabelled PD41 MAb was then added to
determine whether it would bind to whatever antigen
bound to the capture MAbs. Results are presented in
25 FIG. 7.
As seen in FIG. 7, radiolabelled PD41 MAb
showed minimal to no binding to whatever antigen bound
to the different tumor mucin "capture" Mabs. Thus,
the PMA epitope is not coexpressed on the same
30 glycoproteins recognized by the other tumor Mabs.
In summary, a series of selected Mabs
specific for tumor-associated antigen did not block
Mab PD41 binding to the PMA target antigen (FIG. 5);
and PD41 MAb did not block the tumor-associated MAbs
in reciprocal blocking experiments (FIG. 6). MAb PD41
. , ~ . : - . ~ . . ,., . , . .. . . , -
: ... , . -, . ............. . .
WO93/1477~ 2 1 ~ ~ A 8 7 PCT/US92/00852
did not bind to target antigens bound to other MAbs
which recognize TAA's antigens. Radiolabelled PD41
bound only to the antigen ~ound to MAb PD41 as
"capture" antibody.
~r,other double determinant immunoradiometric
competitive inhibitor experiment was conducted using
radiolabelled PD41 MAb 2gainst a series of MAbs
specific for human blood group antigens listed in
Table 9. All of the human blood group MAbs failed to
10 block binding of PD41 MAb to the target PMA antigen
(resules no-~ shown).
Alter2tion in glycosylation, particularly
incomplele glycosylation, has been observed as a
frequent event in tumor cells leading to expression of
Tn, sialosyl-Tn and T antigen expression or unmasking
of these blood group related antigens in tumor cells.
tSee, Springer, 1984, Sci 224:1198-1206; Itkowitz et
; al., 1989, Cancer Res. 49: 197-204; Kjeldsen et al.,
1988, Cancer Res. 48:2214-2220). A comparison of the
20 reactivity of PD41 MAb and B72.3 MAb (breast - -
carcinoma) using a standard solid phase RIA was made
using bovine and ovine submaxillary mucins and "T"
antigen. Results are presented in Table 10.
., . ~ ., . . . . , . . - .... .
., ,,. . , - ., . . . . - . . . - - . . : .
-: : ~ . ~ . . . -
WO93/14775 ~ 8 7 PCT/US92/0~852
- 52 -
Table 10
PD41 Antigen and its Relationship
to TN, SIALOSYL-TN, and T Antigens'b
Monoclonal Antibody
Submaxillary Mucins:PD41 B72.3
Bovine (untreated)5060 cpm 6335 cpm
- (0.1 U neur.) 7793 cpm 633 cpm
0Ovine (untreated)103 cpm 6341 cpm
(0.1 U neur.) 125 cpm 712 _~m
"T" Antigen 201 cpm 210 c~r,
5 a "T" antisen: Galbl-3GalNAc - R; Tn antigen: GalNAc -
R; Sialosyl Tn antigen: Sia 2,6GalNAc - R.
b The bindin~ of MAb PD41 and B72.3 to bovine and
ovine submaxillary mucin and synthetic "T" antigen
linked to human serum albumin were determined using
a standard solid phase radioimmunoassay as -
described in Section 6.2. ~
.. - .. ..
As shown in Table 10, PD41 MAb does not
react with any of these T,TN and sialosyl-TN antigens.
As can be seen from the data, neither
25 antibody reacts with the "T" antigen. B72.3 reacts
with both bovine (with approximately 50~ of the
carbohydrate chains consisting of sialosyl-Tn) and
ovine (90% sialosyl-Tn) submaxillary mucin in the
native state, but not after desialylation with
30 neuraminidase, thus indicating B72.3 recognizes the
sialosyl-Tn form. PD41 reactivity with bovine
submaxillary mucin occurs in the native state and is
enhanced following neuraminidase treatment. PD41 does
not react with ovine submaxillary mucin in the native
35 state (sialosyl-Tn) or in the neuraminidase treated
state (Tn). Immunoperoxidase staining of tissues :
....
, '
. A, . . ~ . '
~ ' ', '. , ' ; ~ ' ' ' ' . " .
2 i (~ 7
WO93/14775 PCT/US92/00852
- 53 -
showed that PMA was expressed by bovine (confirming
the above finding) but not by ovine, porcine, monkey
or human submaxillary tissues (data not shown).
Reactlvity of MAb PD41 with BSM was
confirmed by immunoblotting which indicated a similar
antigenic molecular weight distribution as that seen
for the PMA antigen obtained from prostate carcinoma
tissues. Results of biochemical and immunochemical
experiments indicate that the antigen detected in BSM
10 had the same or substantially similar biochQmical
characteristics as the PMA antigen present in extracts
of prostate carcinoma tissues or fluids from prostate
cancer patients.
Based on the biochemical and immunochemical
evidence derived from the above experiments, the PD41
monoclonal antibody reacts with a distinct and novel
mucin antigen selectively expressed by human prostate
carcinoma cells and by the bovine submaxillary gland,
and is not related to any previously described mucin-
20 associated tumor antigen. The PMA antigen, therefore,
is a new and unique mucin that has not been previously
described.
:~
Deposit of Cell Lines
A cell line, PD41, as described herein has
been deposited with the American Type Culture
? Collection, Rockville, Maryland and been assigned
accession number ATCC No. _ . The invention
described and claimed herein is not to be limited in
30 scope by the cell lines deposited since the deposited
embodiment is intended as an illustration of one
aspect of 'he invention and any equivalent cell lines
j which produce functionally equivalent monoclonal
antibodies are within the scope of this invention.
5 Indeed, various modifications of the invention in
.~' "
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WO93/14775 P~T~US92/00852
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addition to those shown and described herein will
become apparent to those skilled in the art from the
foregoing description. Such modifications are also
intended to fall within the scope of the appended
claims.
It is apparent that many modifications and
variations of this invention as set forth above may be
made without departing from the spirit and scope
thereof. The specific embodiments described are given
10 by way c' _~ample cnly and the invention is limited
only by the terms or the appended claims.
A numD~r of references are cited in the
present specification, the entire disclosure of each
of which is incorporated by reference herein, in its
entirety.
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