Note: Descriptions are shown in the official language in which they were submitted.
WO 92/16~;58 PC~/US92/02109
2~0~3
CELL SURFACE RECEPTORS HOMOLOGOUS
TO COAGUI~TION FACTORS V AND VIII
,Description
Technical Field
The present invention relates to a new class of
extracellular receptor molecules homologous to human
coagulation factors V and VIII, and activated forms ~,
thereof. Amino acid residue sequences of the
receptors are described, as are antibodies
immunoreactive with the receptor molecules and
associated diagnostic kits.
Backaround
The assembly of proteolytic activities on
cellular surfaces initiates a variety of essential
biologic responses. Specific high affinity receptors ,
coordinate such interactions, protect the protease
from inactivation by ubiquitous extracellular
inhibitors, and provide optimal spatial alignment for
the catalytic efficiency of the enzyme. The regulated
association of coagulation and fibrinolytic proteins
with a variety of cells may well exemplify these
mechanisms of specialized protease-cell interactions
tMiles, L. A., et al., Fibrinolysis, 2:61 (1988);
Morrissey, et al., ~ell, 50:129 (1987); Nesheim, N~
E., et al., J. Biol Chem., 254:10952 (1979)].
It has become increasingly clear, howe~er, that
the same enzymes that participate in blood coagulation
and f ib,rinolysis also mediate additional and disparate
blologic functions. With surprising analogies with
the mechanisms of hormone-mediated growth factor
activity, thrombin exerts a potent mitogenic effect on
various cell ~ypes in a reaction exquisitely
coordinated by specific cellular receptors ~Chen, L.
3S B., et al., Proc. Natl. Acad. SCi. USA, 72:131 (1975);
WO92/16558 PCT/US92/02109
2~0S993
Glenn, K. C., et al., Nature, 278:711 (1979); Baker,
J. B., et al., Nature, 278:743 (1979)]. Similarly,
the delicate balance between mitogenesis, malignant
transformation, protooncogene expression, and cell
differentiation has been shown to be profoundly
influenced by protease activity [Unkeless, J. C., et
al., ~. Exp. Med., 137:85 tl973); Sullivan, L. M., et
al., Cell, 45:905 (1986); and Fenner, F. et al. in The
Orthopoxviruses Academic Press (San Diego) (1989)~.
Various immune-inflammatory reactions are no
exceptions. The binding of urokinase as well as of
thrombin to their complementary cellular receptors
produces a potent chemotactic reaction with local
accumulation of neutrophils and monocytes in ~lvo
tBovie, M.D.P., et al., J. Immunol., 139:169 (1987);
Bar-Shavit, R., et al., Science, 220:728 (1983)].
Moreover, synthetic protease inhibitors have been
shown to decrease or abolish NK- and CTL-mediated
target cell ly?~is, as well as monocyte synthesis and
release of TNF-~ tRedelman, D., et al., J. Immunol.,
124:870 (1980); Chang, T. W., et al., J. Immunol.,
124:1028 (1980); Suffys, P., et al., Eur. J. Biochem.,
178:257 (1988); Scuderi, P., J. Immunol., 143:168
(1989)~. `
This concept of a more direct participation of
protea~es in specific cellular immune effector
functions has been recently further enforced by the
identification of a family of related serine proteases
in cytotoxic NK and ~TL clones ~Masson, D., et al.,
Cell, 49:679 (1987~]. These serine proteases, termed
granzymes tJenne, D., et al., Proc. Natl. Acad. Sci.
USA, 85:4814 (1988)], are compartmentalized in
subcellular granules together with the pore-forming
protein perforin and are locally released during the
polarized exocytosis associated with the formation of
WO92/165~ PCT/US92/02109
210~993
endothelial:T cell conjugates tMasson, D., et al., J.
Biol. Chem., 260:9069 (1985); Pasternack, M. S., et
al., Nature, 322:740.12 (1986); Podack, E. R., et al.,
J. Exp. Med., 160:695 (1984)].
As revealed by molecular cloning, several
granzymes share a remarkable degree of homology with
other ~erine proteases involved in coagulation and
fibrinolysis, and particularly with the plasma
coagulation protea~es factors IXa and Xa tJenne, D.,
et al., Proc. Natl. Acad. Sci. USA, 85:4814 (1988);
Ger~henfeld, H. K., et al., Science, 232:854 (1986);
Jenne, D., et al., J. I D unol., 140:318 (1988); Lobe,
C. G., et al., Science, 232:858 (1986); Gershenfeld,
H. K., et al., Proc. Natl. Acad. Sci. USA, 85:1184
(1988)]. While compelling evidence has accumulated
for a direct role of perfor~n in target cell injury
tMasson, D., et al., J. Biol. Chem., 260:9069 (1985),
Duke, R. C., et al., J. Exp. Med., 170:1451 (1989)],
the participation and mechanistic role of the
granzymes or other serine proteases in the lytic ~-
process remains unclear tDennert, G., et al., Proc.
Natl. Acad. Sci. USA, 84:5004 (1987)]. -~
Elucidation of the structures and reactive
properties of granzymes and other serine proteases
2~ likely would lead to a better understanding of the
biologic roles played by these entities. Such
knowledge could afford improved diagnostic probes for
abnormal cellular conditions and new therapeutic tools
that reverse associated diseases.
3~
Brief Summary of the Xnvention
The present invention relates to a new class of
cellular receptor molecules. The receptor molecules
are homologous with certain coagulation cofactors,
such as human coagulation factors V and VIII.
WO92/165~8 PCT/US92/02109
olj9~3
Functionally, the receptor molecules bind serine
protease ligands, such as the circulating proteins
factor Xa, factor IX/IXa and plasmin(ogen). A
preferred receptor molecule, referred to herein as
EPR-1, is homologous to but different from human
factor V and binds factor Xa. Polypeptides containing
an amino acid residue sequence homologous to EPR-l are
also contemplated.
A preferred embodiment of the invention, is a
purified protein that has a molecular weight of about
78 kDa and an amino acid residue seguence comprising
the following amino acid residue sequences: ~.
(1) Thr-Leu-Lys-Gly-Gln-Thr-Gln-Gly-Ala-Val-Met-Ile;
(2) Pro-Xaa-Ile-Xaa-Gln-Met-Asp-Leu-Leu;
(3) Ala-Cys-Lys-Leu-Arg-Glu-Glu-Leu-His-Lys;
(4) Val-Asp-Lys-Leu-Ala-Pro-Arg-Asp-Pro-Leu-Ala;
(5) Gly-Val-Pro-Pro-Val-Val-Thr; .
(6) Gly-Asn-Ser-Asp-Ala-Xaa-Tyr-Val-Lys-Xaa-Val; and
~7) Val-Gln-Lys-Leu-Ala-Glu-Asp-Glu-Asn-Asn-Ala-Lys-
Lys-His-Val-Glu-Pro-His-Ala-Thr,
wherein Xaa is an unspecified amino acid, which aan be
in the protein as any naturally-occurring amino acid
residue, including a modified or unusual, e.g., ~-
glycosylated, residue. The Xaa re~idue can be
2S iden~ical to or other than another Xaa residue in the
mole~uIe. ln a further preferred aspect of the
invention, the protein immunoreacts with the antibody
produced by the hybridoma designated 12Hl (ATCC
Accession No. HB 10637). In a still further preferred
aspect of the invention, the protein is the EPR-1
protein isolated from the cell line designated MOLT13
~3 (A~CC Accession No. CRL 10638).
Another preferred embodiment of the invention is
a polypeptide comprising up to about 600 amino acid
residues which includes an amino acid residue sequence
WO 92/16558 r ~ !3 3PCr/US92/02109
-.
selected from the group of sequences (1)-(7) listed
above.
Also contemplated within the invention are a DNA
segment that codes for a protein or polypeptide as
S defined previously and a vector, i.e., self-
replicating DNA molecule, including the DNA segment.
In a preferred aspect of the invention, an
antibody composition i8 contemplated which
immunoreacts with an instant protein or polypeptide.
A particularly preferred antibody composition is the
monoclonal antibody produced by the 12Hl hybridoma.
Another preferred antibody composition immunoreacts
with a protein isolated from the MOLT13 #3 cell line,
which has a molecular weight of about 78 kDa, and
includes a before-listed amino acid residue sequence.
A method of assaying for the presence of an
instant receptor molecule on a cell surface is also ~ ~-
contemplated. The method comprises the steps of:
(a) admixing a cell or cell lysate suspected of
expressing the receptor molecule with an antibody
composition described before, such as hybridoma 12Hl;
(b) maintaining the admixture for a time
sufficient to form an immunoreaction product; and
(c) determining the presence of immunoreaction
2S product and thereby detecting presence of the receptor
molecuIe.
Also contemplated is a method of monitoring the
response to treatment of a patient having a disease
associated with a before-described protein localized
on cells in a body sample withdrawn from the patient
that is used as a marker for the disease state. The
method comprises assaying for the marker ~sing an
antibody composition described before, repeating the
assay after a course of treatment, and determining the
patient's response to treatment as a function of the
WO92~165~8 PCT/US92/02109
~10 i~3 6
amount of that cell surface protein present after
treatment. An exemplary disease state monitored is
chronic lymphocytic leukemia (CLL).
~rief Description of the Drawinas
Figure l depicts FMF ( f low microf luorometry)
analysis of monocytes or PMN (polymorphonuclear
leucocytes) with anti-EPR-l mAb 9D4. Suspensions of
monocytes were prepared from PBMC (peripheral blood
mononuclear cells) by adherence to plastic precoated
with autologous serum for l hour at 37C. PNN were
isolated by dextran sedimentation. The l x lO6 cells
were stained with lO ~g/ml of mAb 9D4 for 30 minutes
in ice, washed, and further incubated with l/20
dilutions of fluorescein-conjugated goat anti-mouse
IgG. Cells were washed and immediately analyzed by
FMF. Control mAb was the irrelevant V82A6 and is
shown with a dotted line. The abscissa indicates
fluorescence intensity on a 4 log scale. The ordinate
indicates the cell number.
Figure 2 depicts modulation of EPR-l expression
during T cell activation~ Aliquots of freshly
isolated PBMC (lO x lO6) were polyclonally activated
by culture in the presence of 1 ~g/ml PHA or Con A for
7 days at 37C. For antigen-specif~c expansion,
~uspensions of PBMC at lO x lO6 were separately
cultivated in unidirectional mixed lymphocyte culture
(NIC) with irradiated (lO,000 rad) Daudi or Raji cells
(lO x lO~) for 7 days in 5% CO2 at 37C. At the end
of the incubation, responder T cells were harvested,
recovered by centrifugation over Ficoll-Hypague,
washed, stained with aliquots of mAb 12Hl plus
fluorescein-conjugated goat anti-mouse IgG + IgM, and
analyzed by FMF. The dotted line indicates the
background staining with the irrelevant mAb V82A6.
WO92/16558 PCT/US92/0210g
2 11)~3
The percentage of 12H1+ cells analyzed before each
incubation mixture was 6.7 ~ 1.4%.
Figure 3 depicts the effects of long term
alloreactive stimulation on EPR-l expression. ~`
Unidirectional MLC was set up against irradiated
(10,000 rad) Daudi cells and maintained with weekly
transfers and 10% T-cell growth factor (TCGF).
Aliquots of responder T cells were harvested after
various time intervals (Days = 0, 7, 15 and 32),
recovered by centrifugation on Ficoll-Hypaque, and
analyzed by FNF using anti-EPR-l mAb 12H1 or the
polyclonal antiserum B78.9.
Figure 4 depicts expression of EPR-l on
transformed T cell lines, FMF analysis of a panel of
continuous T cell lines in culture with anti-EPR-l mAb
7G12 was performed as described above for Figure 1.
The irrelevant mAb V82A6 was used as a control, and is ~;
shown as a dotted line.
~etailed Descri~tion Qf the Invention
A. Definitions
Amino ~cid Residue Sequence: a series of two or
more amino acid residues joined via peptide linkages ,-
between adjacent residues to form a peptide or
polypeptide. An amino acid residue sequence is
conveniently represented by the one or three letter
abbreviations for its constituent amino acids. The
abbr~viations used herein for amino acids are those
provided at 37 C.F.R. 1~822(b)(2) and are reproduced
in the following Table of Correspondence:
WO92/165~ PCT/VS92~02109
210~3
TABLE OF CORRESPONDENCE
ABBREVIATION AMINO ACID
1-Lette~ 3-Letter :
Y Tyr tyrosine
G Gly glycine
F Phe phenylalanine :
M ~et methionine
A Ala alanine
S Ser serine
I Ile isoleucine
L Leu leucine
T Thr threonine
V ~al valine
P Pxo proline
K Lys lysine
His histidine
Q Gln glutamine
E Glu glutamic acid
Z Glx Glu and/or Gln
W Trp tryptophan
R Arg arginine
D Asp aspartic acid
N Asn asparagine ,~
B AsX Asn and/or Asp
C Cys cysteine
J Xaa Unspecifisd
Th~ indi~idual residues comprising an amino acid
residue seguence herein may be in the D or L isomeric
for~ as long as the desired functional property is
retained by molecule(s) incorporating the amino acid
residue sequence. Also, the amino acid residue
sequence may include post-translationally modified
amino acids, e.g., hydroxylated, glycosylated amino
acid residues, or residues lin~ed via disulfide bonds.
WO92/165~8 PCT/VS92/02109
2 1 ~
In addition, an amino acid residue sequence can
include one or more modified or unusual amino acids,
such as those listed in 37 C.F.R. 1.822(b)(4), which
are incorporated herein by reference. An amino acid
residue sequence can be represented by the `
abbreviations corresponding to its constituent amino
acids in which a hyphen between two adjacent
abbreviations indicates a peptide linkage between the
corre~ponding residues.
a~tibody: a polypeptide which chemically binds
to a haptenic group, i.e., ligand. Antibodies, as
used herein, are immunoglobulin molecules and
immunologically active fragments of immunoglobulin
molecules. Such portions known in the art as Fab,
Fab'; F(ab' )2 and Fv are included. Typically,
antibodies bind ligands that range in size from about
6 to about 34 ~ with association constants in the
range of about lO~ to lO10 M-', and as high as lO13 N-1.
Ant~bodies can bind a wide range of ligands, including
fiDall molecules such as steroids and prostaglandins,
biopolymers such as nucleic acids, proteins and
polysaccharides, and synthetic polymers such as
polypropylene. An "antibody combining site" i~ that -
structural portion of an antibody molecule comprised
of a heavy and light chain variable and hypervariable
regions that specifically binds (immunoreacts with)
~ntigen. The term "immunoreact" in its various forms
ifi used herein to re~er to binding between an
antigenic determinant-containing molecule (antigen)
and a molecule containing an antibody combining site
guch as a whole antibody molecule or a portion
thereof. An "antigenic determinant" is the structural
por~ion of the antigen that is immunologically bound
by an antibody combining site. The term is also used
interchangeably with "epitope". Antibodies can bind a
WO92/16558 PCT/US92/0210s
21~5993
10
single epitope of an antigen (monoclonal) or multiple
epitopes (polyclonal). `~
Ligand: a molecule having a structural region ;
that binds specifically to a particular receptor s
molecule, usually via electrostatic forces and/or
hydrogen bonds.
Pe~tide/Polv~eptide: a polymer comprising at
least two amino acid residues in which adjacent
residues are connected by a peptide bond between the
alpha-amino group of one residue and the alpha-
carbonyl group of an adjacent residue. The primary
structure of a polypeptide has a primary amine group
at one terminus and a carboxylic acid group at the
other terminus of the polymer. Thus, a polypeptide
may be represented by the formula:
H--tNH--CH-C (O) 1 l--OH
R
where R is a side chain characteristic of a given
amino acid residue and i indicates the number of amino
acid residues comprising the polymer which number is
two or more. A polypeptide can comprise one or more
amino acid residue sequences. Also, a polypeptide in ,~,
aqueous solution is usually in one or more
zwitterionic forms depending on the pH of the
solution.
Protein: a single polypeptide or set of cross-
~0 linked polypeptides comprising more than about 50
amino acid residues. Proteins can have chemical
crosslinking, i.e., via disulf ide bridges , within the
same polypeptide chain or between adjacent
polypeptides. Proteins can be glycosylated in which
case they are called glycoproteins.
Rece~tor: a biologically active proteinaceous
molecule having a structural region that specifically
WO92/16558 PCT/US92/02109
210~3
11 -
binds to (or with) another molecule (ligand).
B. The Polvpeptides
A polypeptide of the present invention is derived
from a new class of cell surface receptors, called
effector cell protease receptors (EPRs) because
members of the class bind protease ligands and are
also found on many types of inflammatory effector
cells. The first member of this class tEPR-l) is
shown to bind protease ligands of which human factor
Xa is prototypic.
A polypeptide of the present invention
corresponds in amino acid residue sequence to one or
more amino acid residue sequence of EPR-l. Moreover,
a polypeptide of the invention is shown to have
pronounced homologies with the amino acid residue
sequence of human coagulation factor V, suggesting a
common e~olutionary origin of factor V and EPR-l. As
factor V includes the residue sequence of factor Va,
an instant polypeptide also can have homologies with
factor Va. A polypeptide of the invention also can
exhibit homology in sequence to a polypeptide portion
of factor VIII, as well as to a polypeptide of the
murine protein denominated NFG E-8 ~Stubbs et al.,
Proc Natl Acad_S~i USA 87:8417 (1990~. A polypeptide
of this invention is nonetheleæs distinct from factor
V; factor VIII or murine MFG E-8.
In a preferred embodiment of the inventi~n, the
polypeptide is a protein having a molecular weight of
about 78 kDa which includes the following amino acid
residue seguences~
(l) Thr-Leu-Lys-Gly-Gln-Thr-Gln-Gly-Ala-Val-Met-Ile;
(2) Pro-Xaa-Ile-Xaa-Gln-Met-Asp-Leu-Leu;
(3) Ala-Cys-Lys-Leu-Arg-Glu-Glu-Leu-His-Lys;
(4) Val-Asp-Lys-Leu-Ala-Pro-Arg-Asp-Pro-Leu-Ala;
WO92/165~ PCT/US92/02109
210~9~3 12
(5) Gly-Val-Pro-Pro-Val-Val-Thr;
(6) Gly-Asn-Ser-Asp-Ala-Xaa-Tyr-Val-Lys-Xaa-Val; and
~7) Val-Gln-Lys-Leu-Ala-Glu-Asp-Glu-Asn-Asn-Ala-Lys-
Lys-His-Val-Glu-Pro-His-Ala-Thr,
s wherein Xaa is present in the molecule as an
unspecified amino acid residue. The Xaa residue can
be any naturally-occurring amino acid residue
including a modified or unusual residue (cf. 37 C.F.R.
S1-822(b)(4)). Also, the Xaa residue need not be the
same as another Xaa residue in a particular amino acid
residue sequence or in another sequence of the
molecule having an Xaa residue. Characteristically,
the protein has a molecular weight of 78+4 kDa, as
determined by polyacrylamide gel electrophoresis.
(The number associated with each amino acid residue
sequence refers to its Sequence I.D. No. as presented
in the sequence listing provided herewith pursuant to
the requirements of 37 C~F.R. 1.821~c)).
In a further preferred embodiment, the protein is
isolated from the cell line designated NOLTl3 #3 and
has a molecular weight of about 78 kDa. The MOLTl3 #3
cell line was deposited at the American Type Culture
Collection (ATCC) 12301 Parklawn Drive, Rockville, ~,
Maryland, USA 20B52. This cell line was deposited on
January 11, 1991 and received Accession Number CRL
10638.
The present deposit was made in compliance with
the Budapest Treaty requirements that the duration of
the deposits should be for 30 years from the date of
deposit or for 5 years after the last request for the
deposit at the depository or for the enforceable life
of a U.S. patent that matures from this application,
whichever is longer. The cell line will be
replenished should it become non-viable at the
depository.
WO92/165~ PCT/US92/02109
2 1 ~
13
In another embodiment of the invention, the
protein is immunoreactive with certain antisera to
human factor V as well as with polyclonal antibodies
purified from the antisera, e.g., by immunoadsorption
on immobilized, purified human factor V. In a further
aspect of the invention, the protein immunoreacts with
antibodies to human factor VIII. Hence, although the
instant EPR protein is not a human factor V or VIII
protein ~er se, it possesses epitopes that are cross-
reactive with ligands for certain epitopes of factorsV and VIII.
In a furt~er preferred embodiment of the
invention, the isolated protein immunoreacts with a
small set of antibodies such as those produced by the
hybridoma designated 12Hl, which was deposited on
January 11, 1991, at the ATCC pursuant to the Budapest
Treaty as described above. The hybridoma was given
the designation ATCC HB 10637.
In another embodiment, a polypeptide of this
invention has an amino acid residue sequence
represented by the following formula:
H-Xn-Y-X~-OH
where Y is an amino acid residue sequence selected
from the group of a~ino acid residue sequences (1)-(7)
listed above which are present in EPR-1; Xn is absent
when n-0 and is an N-terminal (leader segment and
mature protein) amino acid residue sequence containing
up to about 5~0 residues when n=1; and X~ is absent
when m=0 and is a C-terminal (tail segment) amino acid
residue sequence containing up to about 550 residues
when m=1. The polypeptide comprises up to about 600
amino acid residues.
Preferably, when either X~ or Xm is an amino acid
residue sequence, in or Xm contains up to about 200
3~ residues, more preferably, up to about 50 residues,
WO92/16558 PCT/US92/02109
210~993 14
and most preferably, up to about 20 amino acid
residues. Typically, Xn and X~ each contain one or
more of the before-listed amino acid residue
sequences.
In a further preferred aspect of the invention,
X~ and X~ are selected so that the polypeptide
immunoreacts with sera containing antibodies raised to
human factor V. More preferably, Xn and X~ are
selected so that the polypeptide is a protein having a
molecular weight of about 78 kDa. Most preferably,
the isolated protein also binds to human factor Xa.
In a still further preferred embodiment, a
polypeptide of the invention has the formula:
H-Y-OH
~5 where Y i8 as defined previously.
A polypeptide of the present invention can be
used to generate a variety of useful antibodies by
~e~ns described herein. The utilities of the
polypeptides will be apparent from the discussion
provided hereinbelow.
Typically an instant polypeptide is not
glycosylated, i.e., it is synthesized either directly
by standard peptide synthesis techniques or by
procaryotic host expression of a recombinant DNA
molecule of the present invention. A eucaryotically
produced polypeptide of the present inyention is
typically glycosylated.
An instant polypeptide can incorporate a variety
of changes, such as insertions, deletions, and
substitutions of amino acid residues which are either
conservative or nonconservative as long as the
resulting polypeptide molecule exhibits the desired
properties. The "desired properties" as referred to
herein include that the polypeptide is immunogenic in
3s a suitable host and able to generate antibodies to the
.
WO92~16558 PCT/US92/02109
15 2 ~
EPR-l molecule or a polypeptide homologous to EPR-l,
at least in the denatured state as is found in an SDS-
PAGE gel, but frequently also in the natural state as
expressed on cells. Additionally, the polypeptide is
s antigenic when expressed on cells or in its denatured -
state so that antibodies immunoreactive with the EPR-l
molecule also immunoreact with the instant ~-
polypeptide.
When an instant polypeptide incorporates
conservative substitution~ of the sequences
corresponding to EPR-l depicted above, the substituted
amino acid residues are replaced by another,
biologically similar amino acid residue such that the
resulting polypeptide has an amino acid residue
~equence that is different from (other than) a
sequence of factor V, factor VIII or sequence MFG E-8.
Some examples of conservative substitutions include
substitution of a hydrophobic residue such as
isoleucine, valine, leucine or methionine for another
hydrophobic residue. Also, a polar residue such as
arginine, glycine, glutamic acid, aspartic acid,
glutamine, asparagine, and the like, can be
conservatively substituted for another member of this ~-
group. ætill another aspect of a polypeptide
2~ incorporating conserv~ive substitutions occurs when a
substituted amino acid residue replaces an
uncubstituted parent amino acid residue. Examples of
substituted amino acids may be found at 37 C.F.~.
1.822(b)(4), which species are incorporated herein by
reference. When the polypeptide has an amino acid
residue sequence that oorresponds to the sequence of
EPR-l but has nne or more conservative substitutions,
preferably no more than about 40%, and more preferably
no more than about 20%, of the amino acid residues of
the native protein are substituted.
WO92/16558 PCT/US92/02109
2105993 16
A polypeptide of the present invention can be ~;
synthesized by any of the peptide synthetic techniques
known to those skilled in the art. A summary of some
of the techniques available can ~e found in J.M.
Stuard and J. D. Young, "Solid Phase Peptide
Synthesis", W. H. Freeman, Co., San Francisco (1969),
J. Meinhofer," Hormonal Proteins and Peptides" Vol. 2,
pp. 46, Academic Press (New York~ 1983, and U.S.
Patent No. 4,631,211, which description is
incorporated herein by reference. When a polypeptide
desired for use in the present invention is relatively
short ~less than about 50 amino acid residues in
length) direct peptide synthetic techniques are
generally favored, usually by employing a solid phase
technique such as that of Merrifield [Merrifield JACS,
85:2149 (1963)].
- An instant polypeptide can also be synthesized by
recombinant DNA techniques. Such recombinant
techniques are favored especially when the desired
polypeptide is relatively long (greater than about 50
amino acids residues in length). When recombinant DNA
techniques are employed to prepare an instant
polypeptide, a DNA se~ment coding for the desired ~.
polypeptide is incorporated into a preselected vector
that is subsequently expressed in a suita~le host.
The expresced polypeptide, containing at least one of
amino acid residue sequences (1)-(7) corr~sponding to
EPR-1 identified above, is preferably purified by a
routine method such as gel electrophoresis,
i~munosorbent chromatography, and the like.
C. DNA Seqments
When recombinant DNA techniques are employed to
prepare a polypeptide of the present invention, a DNA
segment encoding the polypeptide is used. A DNA
WO92/16558 PCT/US92/02109
~ 10 ~ 9 v 3
17
segment contemplated within the invention is
operatively linked to a vector that is subsequently
expressed in a suitable host. The segment is
"operatively linked" to the vector as used herein when
it is ligated (covalently bonded) thereto, as is well
known. Also contemplated is an RNA segment eguivalent
to an instant DNA segment.
The present DNA segment is a molecule that can be
readily synthesized by chemical techniques, e.g., by
the well-known phosphotriester method ~Matteuci et
al., JACS, 103:3185 (1981)]. By chemically
synthesizing the DNA segments, any desired
substitution, insertion or deletion of an amino acid
residue or sequence from a template polypeptide, e.g.,
the native protein, can be readily provided by simply
making the corresponding changes in the nucleotide
sequence of the DNA segment.
Whenever an RNA segment coding for the instant
polypeptide is used, the RNA molecule including the
polypeptide coding segment is transcribed into
complementary DNA (cDNA) via a reverse transcriptase.
The cDNA molecule can then be transcribed and
translated as described herein to generate a desired
polypeptide.
In a preferred ~spect of the invention, a DNA
nucleotide sequence (segment) coding for at least one
of the amino acid residue sequences (1)-(7) of EPR-l
identified above is operatively linked to a larger DNA
molecule. The resultant DNA molecule is then
trans~ormed in a suitable host and expressed therein.
The DNA segment coding for an amino acid residue
sequence ~ (7) listed above can be provided with
~tart and stop codons or one or both of the start and
stop codons can be provided by the larger DNA
~olecule, e.g~, vector, operatively linked to the DNA
W O 92/16558 P(~r/US92/02109
~ S~ ~ 3 18
segment so that only the corresponding polypeptide is
generated. Alternatively, a nucleotide sequence
coding for additional amino acid residues can be
provided at the 3' and/or 5' ends of the DNA segment
S so that a larger polypeptide is expressed having an
amino acid residue sequence at either or both of its
N-t~rminal and C-terminal ends in addition to an amino
acid residue sequence (1)-(7) listed above of the EPR-
1 molecule.
A DNA molecule of the invention can encode a
polypeptide having an amino acid residue sequence
represented by the formula:
H-X~-Y-X~-OH
where Xn, Y, and X,are as defined previously.
Preferably, the DNA segment encodes a polypeptide up
to about 600 amino acid residues in length. When the
DNA segment encodes a polypeptide having either Xn or
X, as an amino acid residue sequence, X~ or X~ contains
up to about 200 residues, more preferably up to about
50 residues, and most preferably, up to about 20 amino
acid residues. Typiaally, one or both of the flanking
regions to the DNA segment encoding the Y sequence of
the polypeptide encodes one or more of the amino acid
residue sequences (1)-(7) listed above.
An instant DNA molecule can also be produced by
enzymatic techniques. Thus, restriction enzymes which
cleave DNA molecules at predefined recognition
sequences can be used to isolate DNA fragments from
larger DNA molecules containing the desired DNA
segments such as the DNA (or RNA) that codes for the
EPR-l protein. Typioally, DNA fragments produced in
this manner will have cohesive, "overhanging~' termini,
in which single-stranded nucleotide sequences extend
beyond the double-stranded portion of the molecule.
3s The presence of such cohesive termini is generally
W092/165~ 2 1~ 3 PCT/US92/02109
19
preferred over blunt-ended DNA molecules. The
isolated fragments containing the desired coding
sequence can then be ligated (cloned) into a suitable
vector for amplification and expression.
Additionally, an instant DNA segment can be
generated by polymerase chain reaction (PCR)
techniques, which amplify targeted DNA sagments of a
template nucleic acid. In PCR, a specific polynucleic
acid target is transcribed by a reaction in which a
primer molecule complementary to a particular section
of a nucleic acid template is used to form an
extension product of the primer including a nucleic
acid region complementary to the target. After
separation of template and extended primer, each
primer extension product acts as a template and
specifically anneals with a complementary primer
molecule. The resulting primed template acts as a
substrate for further extension reactions. These
steps are repeated, preferably using an automated
cycling procedure, thereby exponentially amplifying
the initial polynucleic acid target to which the
primer hybridizes. Procedures for conducting PCR have
been extensively described, see, e.g., U.S. Patent
Nos. 4,683,195 and 4,683,202, which descriptions are
incorporated herein by reference.
In using PCR technology herein, a DNA primer
molecule coding for one or more of the before-
enumerated amino acid residue sequences (1)-(7) is
preferably utilized. However, additional nucleotide
3~ sequences can be revealed by cloning the cDNA or
genomic DNA encoding EPR-1 and smaller amino acid
residue sequences thereof. A DNA probe molecule
encoding an EPR-l amino acid residue sequence having
minimal homology to either of factors V or VIII is
usually employed in order to maximize specificity of
WO92~165~ PCT/US92/02109
Z l05l~93 20
hybridization with DNA or RNA encoding the targeted
EPR-l amino acid residue sequence and to minimize
hybridization with DNA or RNA coding for either factor
V, factor VIII, or homologous, nontargeted molecules.
S However, such "rule of thumb" regarding the homology
of a probe molecule is not critical in identifying a
suitable probe, and indeed, may not be appropriate at
all, as when examined cells do not express factors V
or VIII. The use of mixed, redundant primers that
encode a targeted amino acid residue sequence ;`
utilizing different codons for the same amino acid
residue is of course contemplated.
D. Vectors
Also contemplated within the present invention is
a vector that can be operatively linked to an instant
DNA segment to provide a self-replicating reco~binant
DNA molecule that encodes an instant polypeptide,
preferably expressing the EPR-l protein itself. The
recombinant molecule can be used to transform suitable
host cells so that the host cells express the desired
polypeptide. Hence, the DNA molecule can be regarded
as self-replicating.
The choice of vector to which a DNA segment of
the present invention is operatively linked depends,
as i8 well known in the art, on the functional
properties desired, e.g., efficiency of expression,
the transformation host cell, and the like. However,
a vector of the present invention is at least capable
of directing the replication, and preferably also
expression, of a DNA se~ment coding for an instant
polypeptide.
Preferably, a chosen vector includes a
procaryotic replicon, i.e., a DNA sequence, having the
ability to direct autonomous replication and
WO92/16558 ~ ~CT/US92/02109
2105~
21
maintenance of the recombinant DNA molecule
extrachromosomally in a procaryotic host cell
transformed therewith. Such replicons are well known
in the art. In addition, a vector that includes a
procaryotic replicon preferably also includes a drug
resistance gene so that hosts transformed with a
vector can be readily screened. Typical bacterial
drug resistance genes are those that confer resistance
to ampicillin or tetracycline.
Vectors that include a procaryotic replicon
preferably include a procaryotic promoter capable of
directing the transcription of the instant polypeptide
genes. A promoter is an expression control element
formed by a DNA sequence that promotes binding of RNA
polymerase and transcription of single-stranded DNA
into messenger RNA (mRNA) molecules. Promoter
sequences compatible with bacterial hosts, such as a
tac promoter, are typically provided in plasmid
vectors having convenient restriction sites for
insertion of a DNA segment of the present invention.
Typical of such vector plasmids are pUC8, pUC9, pBR322
and pBR329 available from Biorad Laboratories,
~Richmond, CA) and pPL and pKK223 available from --
Pharmacia (Piscataway, NJ).
Expression vectors compatible with eucaryotic
cells, preferably those compatible with vertebrate
cells, can also be used to form a recombinant DNA
molecule described before. Eucaryotic cell expression
vectors are well known in the art and are available
from several commercial ~ources. Typically, such
vectors are provided with convenient restriction sites
for insertion of the desired D~A segment. 1ypical of
such vectors are pSVL and pKSV-lO (Pharmacia), pBPV-
~ lpML2d (International Biotechnologies, Inc.), and
pTDTl (ATCC, #31255). A preferred drug resistance
WO92~165~ PCT/VS92/02109
2ib5393 22
marker for use in vectors compatible with eucaryotic
cells is the neomycin phosphotransferase (neo) gene.
tSouthern et al., J. Mol. Appl. Genet., 1:327-341 ;
(1982)~. ^
Retroviral expression vectors capable of ~;
generating the recombinant DNA of the present
invention are also contemplated. The construction and
use of retroviral vectors for generating desired DNA
molecules have been described by Sorge, et al.,
Cell. Biol., 4:1730-37 (1984).
A number of methods are available to operatively
link DNA to vectors via complementary cohesive
termini. For instance, complementary homopolymer
tracts can be added to the DNA segment to be inserted
and to the vector DNA. The vector and DNA segment are
then allowed to hybridize by hydrogen bonding between
the complementary homopolymer tails to form
recombinant duplex DNA molecules.
Alternatively, synthetic linkers containing one
or more restriction sites can be used to join the DNA
segment to vectors. When the DNA segment is generated
by endonuclease restriction digestion, as described
earlier, it is treated with bacteriophage T4 DNA
polymerase of E. coli DNA polymerase I which removes
2~ protruding 3' single-stranded termini and fills in
rece~sed 3' ends. Blunt-ended DNA segments are
thereby generated.
Blunt-ended DNA segments are incubated with a
large molar excess of linker mo~ecules in the presence
of an enzyme that is able to catalyze the ligation of
blunt-ended DNA molecules, such as bacteriophage T4
DNA ligase. Thus, the products of the reaction are
DNA segments bonded at their ends to linker sequences
having restriction sites therein. The restriction
sites of these DNA segments are then cleaved with the
WO92/16558 PCT/US92/02109
23
appropriate restriction enzyme and the segments
ligated to an expression vector having termini
compatible with those of the cleaved DNA segment.
Synthetic linkers containing a variety of restriction
endonuclease sites are commercially available from a
number of sources including International
Biotechnologies, Inc. (New Haven, CT).
E. Transformation of Hosts
The present invention also relates to host cells
transfor~ed with a recombinant DNA molecule of the
present invention. The host cell can be either
procaryotic or eucaryotic. Preferred procaryotic host
cells are strains of E. coli, e.g., the E. coli strain
lS DHS available from Bethesda Research Laboratories,
Inc., Bethesda, MD. Preferred eucaryotic host cells
include yeast a~d mammalian cells, preferably
vertebrate cells such as those from mouse, rat, monkey
or hu~an fibroblastic cell line. Preferred eucaryotic
host cells include Chinese hamster ovary (CHO) cells
available from the ATCC as CCL61 and NIH Swiss mouse
embry~ cells NIH/3T3 available from the ATCC as CRL
1658. Transformation of appropriate cell hosts with a~
recom~inant DN~ molecule of the present invention is
2~ accoqplished by well known methods that typically
depend on the type of vector used. With regard to
~ransformation of procaryotic host cells, see, for
example, Maniatis et al., Molecular Clonina. A
Laboratorv Manual, Cold Spring Harbor Laboratory, Cold
Spring Harbor, NY (1982). With regard to
transformation of vertebrate cells with retroviral
vectors containing RNA encoding the instant
polypeptides and a reverse transcriptase, see, e.g.,
Sorge et al., Mol. Cell. Biol., 4:1730-37 (1984).
Successfully transformed cells, i.e., those
WO92/165~ PCT/US92/02109
21053~3 24
containing a recombinant DNA molecule of the present
invention, can be identified by well known techniques.
For example, transformed cells can be cloned to
produce monoclonal colonies. Cells from those
colonies can be harvested, lysed and their DNA content
examined for the presence of the desired DNA segment
using a method such as that described by Southern, J.
Mol. Biol., 98:503 (1975).
In addition to directly assaying for the presence
of the desired DNA segment, successful transformation ;
can be confirmed by well known immunological methods
when the DNA directs expression of the polypeptides of
the present invention. Samples of cells suspected of
being transformed are harvested and assayed for
antigenicity by antibodies that specifically bind to
the instant polypeptides.
In addition to the transformed host cells
themselves, also contemplated ~y the present invention
are cultures of those cells. Nutrient media useful
for culturing transformed host cells are well known in
the art and can be obtained from several commercial ~-
sources. In embodiments wherein the host cell is
mammalian a "serum-free" medium is preferably used.
Methods for recovering an expressed protein from -
a culture are well known in the art. For instance,
gel filtration, gel chromatography, ultrafiltration,
electrophoresis, ion exchange, affinity chromatography
and related technigues can be used to isolate the
expressed proteins found in the culture. In addition,
immunochemical methods, such as immunoaffinity,
immunoadsorption, and the like, can be performed using
well kn~wn methods, as exemplified by the methods
described herein.
3S F. Antibody Compositions
W092/16~58 2 ~ o ~ ~ ~ 3 PCT/US9~0~109
Also contemplated within the present invention is
an antibody composition that immunoreacts with an
instant polypeptide. An antibody composition
immunoreacts with the polypeptide either associated
with cellular surfaces or free from cellular
structures. Thus, an antibody composition binds to
one or more epitopes presented by the polypeptide on
the exterior surface of cells or to the epitopes of
cell-free polypeptides.
A preferred antibody composition ~f the invention
immunoreacts with an EPR-l protein molecule presented
on the cell surface or free of cellular components as
when the EPR-l molecule is isol~ted upon lysis of
cells carrying the molecule. Particularly preferred
antibody compositions in this regard are the
monoclonal antibodies (mAbs) designated 7Gl2, 9D4, and
12Hl. Such mAbs are obtained as described herein and
in Altieri et al., J. Biol. Chem., 264(S):2969 (1989)
and Altieri et al., J. Immun. 145:246 (l990). Of
course, polyclonal antibodies are also contemplated.
Brieflyt a preferred antibody composition is
generated by immunizing mice with human factor V,
factor VIII or a polypeptide of this invention. The
~ntibodies generated are screened for binding affinity
2S for a polypeptide of the instant invention, such as
EPR-l. Isolated EPR-l or EPR-l on washed lymphocytes
free o~ ~actor V or factor VIII can be used for
screening the antibodies.
Typically the instant mAbs immunoreact both with
factor V and with the target polypeptide of this
invention having homology with factor V. However,
when a polypeptide homologous but not identical to
factor V is used to obtain the instant mAbs, the mAbs
preferably immunoreact with the target polypeptide but
not with the blood coagulation factor. The mAbs can
WO92/165~ PCT/US92/02109
~105~93 26
also immunoreact with factor VIII proteins similar to
the reaction with factor V.
Since the antibodies of the present invention can
bind to receptors for coagulation factor Xa, they can
s be used to competitively inhibit factor Xa from
binding to sites on cellular surfaces. Thus, the
protease activity of factor Xa in the region of the
cell surfaces can be curtailed. Methods for
inhibiting such binding are well known to tho~e
skilled in the art.
A preferred antibody composition as contemplated
herein is typically produced by immunizing a mammal
with an inoculum containing human factor V or a
polypeptide of the present invention, thereby inducing
lS in the mammal antibody molecules having the
appropriate immunospecificity for the immunogenic
polypeptide. The antibody molecules are then
collected from the mammal, screened and purified to
the extent desired by well known techniques such as,
for example,-by immunoaffinity for the immunogen
immobilized on a solid support. The antibody
composition so produced can be used inter_alia, in the
diagnostic methods and systems of the present
invention to detect expression of the instant
polypeptides on the surface of cells, e.g., leukocytes
of pat~ents with chronic lymphocytic leuk~mia (CLL).
A monoclonal antibody composition (mAb) is also
contemplated by the present invention, as noted
before. The phrase "monoclonal antibody composition"
in its various grammatical forms refers to a
population of antibody molecules that contain only one
species of antibody combining site capable of
immunoreacting with a particular antigen. The instant
mAb composition thus typically displays a single
3s binding affinity for any antigen with which it
W092/16558 2 1 0 S ~ ~ ~3PCI/llS92/02109
27
immunoreacts. However, a given monoclonal antibody
composition may contain antibody molecules having two
different antibody combining ~ites, each
immunospecific for a different antigenic determinant,
i.e., a bispecific monoclonal antibody.
An instant mAb is typically composed of
antibodies produced by clones of a single cell called
a hybridoma that secretes (produces) but one kind of
antibody molecule. The hybridoma cell is formed by
fusing an antibody-producing cell and a myeloma or
other self-perpetuating cell line. Such antibodies
were first described by Xohler and Milstein, Nature
256:495-497 (1975). A particularly preferred
hybridoma is designated 12H1 (ATCC Accession No. HB
10637).
A monoclonal antibody can also be produced by
methods well known to those skilled in the art of
producing chimeric antibodies. Those methods include
i801ating, manipulating, and expressing the nucleic
acid that codes for all or part of an immunoglobulin
variable region including both the portion of the
variable region comprising the variabl2 region of
immunoglobulin light c:hain and the portion of the ,~
variable region comprising the variable region of
im~unoglobulin heavy chain. Methods for isolating,
manipulating, and expressing the variable region
- coding r;ucleic acid in procaryotic and eucaryotic
hosts are disclosed in Robinson et al., PCT
Publication No~ WO 89/0099; Winter et al., European
Pa~ent Publication No. 0239400; Reading, U.S. Patent
No. 4,714,681; Cabilly et al., European Patent
Publication No. 0125023; Sorge et al., Mol. Cell ~`
Biol., 4:1730-1737 (1984); Beher et al., Science,
240:1041-1043 (1988); Skerra et al., Science,
3~ 240:1030-1041 (1988); and Orlandi et al., Proc. Natl.
WO92/16558 PCT/US92/02109
3 28
Acad. Sci.. U.S.A., 86: 3833-3837 (1989). Typically
the nucleic acid codes for all or part of an
immunoglobulin variable region that binds a
preselected antigen (ligand). Sources of such nucleic
acid are well known to one skilled in the art and, for
example, can be obtained from a hybridoma producing a
monoclonal antibody that binds the preselected
antigen, or the preselected antigen can be used to ;~
~creen an expression library coding for a plurality of
immunoglobulin variable regions, thus isolating the
nucleic acid.
The present invention contemplates a method of
forming a monoclonal antibody molecule that
immunoreacts with a polypeptide of the present
invention, and optionally a factor V or VIII protein
obtained from a mammal. The method comprises the
steps of:
(a) Immunizing an animal with a polypeptide of
thi~ invention or a protein homologous thereto, such
as a factor V or VIII protein. Conveniently, the
im~unogen is a protein taken directly from a subject
ani~al species. However, the antigen can also be ~`
linked to a carrier protein such as keyhole limpet ~ -
hemocyanin, particularly when the antigen is small,
~ch as a polypeptide consisting essentially of an
aaino acid residue sequence (1~-(7~ listed above. The
immunization is typically performed by administering
th8 sample to an immunologically competent mammal in
an immunologically effective amount, i.e., an amount
sufficient to produce an immune response. Preferably,
the Dammal is a rodent such as a rabbit, rat or mouse.
The mammal is then maintained for a time period
sufficient for the ma~mal to produce cells secreting
antibody molecules that immunoreact with the
3~ i~munogen.
WO92/16558 2 1 ~ 5 ~ 9 3 PCT/US92/02109
2g
(b) A suspension of antibody-producing cells
removed from the immunized mammal is then prepared.
This is typically accomplished by removing the spleen
of the mammal and mechanically separating the
individual spleen cells in a physiologically tolerable
medium using methods well known in the art.
(c) The suspended antibody-producing cells are
treated with a transforming agent capable of producing
a transformed ("immortalized") cell line.
Transforming agents and their use to produce
immortalized cell lines are well known in the art and
include DNA viruses such as Epstein-Barr virus (EBV),
simian virus 40 (SV40), polyoma virus and the like,
RNA viruses such as Moloney murine leukemia virus (Mo-
MuLV), Rous sarcoma virus and the like, myeloma cells
such as P3X63-Ag8.653, Sp2/O-Agl4 and the like.
In preferred embodiments, treatment with the
transforming agent results in the production of an
"immortalized" hybridoma by fusing the suspended ~;
spleen cells with mouse myeloma cells from a suitable
cell line, e.g., SP-2, by the use of a suitable fusion
promoter. The preferred ratio is about 5 spleen
cells per myeloma cell in a suspension containing
about 108 splenocytes. A preferred fusion promoter is
polyethylene glycol having an average molecule weight `~
from about 1000 to about 4000 (commercially available
as PEG lO00, etc.); however, other fusion promoters -
known in the art may be employed.
The cell line used should preferably be of the
so-called "drug resistant" type, so that unfused
myeloma cells will not survive in a selective medium,
while hybrids will survive. The most common claæs is
8-azaguanine resistant cell lines, which lack the
enzyme hypoxanthine-guanine phosphoribosyl transferase
and hence will not be supported by HAT (hypoxanthine,
WO92/165~ PCT/US92/02109
'~ 10~9~3 30
aminopterin, and thymidine) medium. It is also
generally preferred that the myeloma celi line used be
of the so-called ^'non-secreting" type which does not ;
itself produce any antibody. In certain cases, -
however, secreting myeloma lines may be preferred.
(d) The transformed cells are then cloned,
preferably to monoclonality. The cloning is
preferably performed in a tissue culture medium that
does not sustain (support) non-transformed cells.
When the transformed cells are hybridomas, this is
typically performed by diluting and culturing in
separate containers the mixture of unfused spleen
cells, unfused myeloma cells, and fused cells `~
(hybridomas) in a selective medium which will not
sustain the unfused myeloma cells. The cells are
cultured in this medium for a time sufficient to allow
death of the unfused cells (about one week). The
dilution can be a limiting dilution, in which the ~;
volume of diluent is statistically calculated to
isolate a certain number of cells (e.g., 0.3-0.5) in
each separate container (e.g., each-well of a
microtiter plate). The medium is one (e.g., HAT ~-
medium) that does not sustain the drug-resistant
(e.g., 8-azaguanine resistant) unfused myeloma cell
line.
(e) The tissue culture medium of the cloned
transformants is analyzed (immunologically assayed) to
detect the presence of antibody molecules that
preferentially react with the instant polypeptides or
cells bearing the EPR-l receptox molecule. This is
accomplished using well known immunological
techniques.
(f) A desired transformant is then selected and
grown in an appropriate tissue culture medium for a
suitable length of time, followed by recovery
W O 92/16558 PC~r/US92/02109
21~
(harvesting) of the desired antibody from the culture
supernatant by well known techniques. A suitable
medium and length of culturing time are also well
known or are readily determined.
It is noted that monoclonal antibodies to EPR-l
induced by immunization with factor V are relatively
rare. Indeed, only about one percent of the
monoclonal antibodies induced by the above method
immunoreact with EPR-1.
To produce a much greater concentration of
slightly less pure monoclonal antibody, the desired
hybridoma can be transferred by injec~ion into mice,
preferably syngenic or semisyngenic mice. The ~ -
hybridoma causes formation of antibody-producing
tumors after a suitable incubation time, which results
in a high concentration of the desired antibody (about
5-20 mg/ml) in the bloodstream and peritoneal exudate
(ascites) of the host mouse.
Media and animals useful for the preparation of
these ~ompositions are both well known in the art and
commercially available and include synthetic culture
media, inbred mice and the like. An exemplary
synthetic medium i8 Dulbecco's minimal essential ,~
medium [DMEM; Dulbecco et al., Virol. 8:396 (1959)~
supplemented with 4.5 gm/l glucose, 20 mM glutamine,
and 20% fetal calf serum. A preferred inbred mouse
strain is Balb/c.
Methods for producing the instant hybridomas
which generate (æecrete~ the antibody molecules of the
present invention, are well known in the art and are
descri~ed further herein. Particularly applicable
descriptions of relevant hybridoma technology are
presented by Niman et al., Proc. Natl. Acad. Sci. USA,
80:4949-4953 (1983), and by Galfre et al., Meth.
EnzYmol., 73:3-46 (1981), which descriptions are
WO92/1~ ~ PCT/US92/02109
210599.3.. 32
incorporated herein by reference.
A further preferred method for forming the
instant antibody compositions involves the generation
of libraries of Fab molecules using the method of Huse
et al., Science, 246:1275 (l9~9). In this method,
mRNA molecules for heavy and light antibody chains are
isolated from the immunized animal. The mRNAs are
amplified using polymerase chain reaction (PCR)
techniques. The nucleic acids are then randomly
cloned into lambda phages to generate a library of
recombined phage particles. The phages are used to
infect an expression host such as E. coli. The E.
coli colonies and corresponding phage recombinants can
then be screened for those producing the desired Fab
fragments. Preferred lambda phage vectors are gtll
and zap 2. -~
The antibody molecule-containing composition
employed in the present invention can take the form of -
a solution or suspension. The preparation of a
composition that contains antibody molecules as active
ingredients is well understood in the art. Typically,
such compositions are prepared as liquid solutions or
suspensions, however, solid forms suitable for
solution in, or suspension in, liquid can also be
prepared. The preparation can also be emulsifie~.
The active therapeutic ingredient is often mixed with
excipients which do not interfere with the assay and
are compatible with the active ingredient. Suitable
excipients are, for example, water, ~aline, dextrose,
glycerol, ethanol, or the like and combinations
thereof. In addition, if desired, the composition
can contain minor amounts of auxiliary substances such
as wetting or emulsifying agents, pH buffering agents
and the like which enhance the effectiveness of the
active ingredient.
WO92/165~ PCT/US92/02109
210599~-~
33
An antibody molecule composition can be
formulated into a neutralized acceptable salt form.
Acceptable salts include the acid addition salts
(formed with the free amino groups of the antibody
s molecule) that are formed with inorganic acids such
as, for example, hydrochloric or phosphoric acids, or
such organic acids as acetic, tartaric, mandelic, and
the like. Salts formed with the free carboxyl groups
can also be derived from inorganic bases such as, for
example, sodium, potassium, ammonium, calcium, or `
ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, 2-ethylamino ethanol,
histidine, procaine, and the like.
-',
G. Diaanostic Assav Methods
The present invention contemplates a method ~or
detecting an EPR-1 molecule, or polypeptide portion
thereof. The assay can be for cell surface receptors
homologous with EPR-l, including EPR-l itself. The
as~ay can be specific for EPR-l îtself by a proper
selection of antibody ~pecificity. Also, an assay of
the invention can be for polypeptide receptors
homologous to portions of EPR-1 as well as "free", ~.
i.e., unassociated with any particular cell structure,
polypeptides ho~ologous to EPR-1, or polypeptide
portions thereof. Typically, the asæay methods
involve detecting EPR-l exposed on cell surfaces,
e.g., CLL cells.
The relative binding affinity of a reagent
molecule for its target species is conveniently
determined as described herein using the method of
flow microfluorimetry (FMF). Thus, cells expressing
the target antigen, e.g., EPR-1, are indicated
whenever the fluorescence intensity associated with
the cells due to binding of the instant fluorescent-
WOY2/165~ PCT/US92/02109
,~lO5993 34
labelled antibodies to cell surface antigens exceeds apredefined threshold level. The labelled antibodies
are typically fluorescein isothiocyanate-conjugated
(FITC), although other well known fluorescent labels
can be used.
The method for detecting an antigenic polypeptide
of the present invention preferably comprises
formation of an immunoreaction product between the
polypeptide and an anti-polypeptide antibody molecule,
as disclosed herein. The antigen to be detected can
be present in a vascular fluid sample or in a body
tissue sample. The immunoreaction product is detected
by methods well-known to those skilled in the art.
Numerous clinical diagnostic chemistry procedures can ;~
be utilized to form the detectible immunocomplexes.
Alternatively, a polypeptide ligand (non-antibody
composition) for an instant EPR-l receptor or
polypeptide can be used in the assay méthod. An
exemplary ligand in this aspect of the invention is a
labelled factor Xa enzyme. Thus, while exemplary
assay methods are described herein, the invention is
not so limited.
A preferred assay method of the present invention
involves determining the presence of EPR-l cell
surface receptors. Various heterogeneous and
homogeneous assay protocols can be employed, either
competitive or non-competitive for detecting the
presence and preferably amount of cell surface
receptors in a body sample, preferably cell-containing
sample. The receptor molecules are homologous with
human coagulation factors V and VIII or substantial
polypeptide portion thereof, so that some care is
required in distinguishing an EPR-l molecule or its
polypeptide portion from factors V and VIII. A
3S particularly preferred receptor for assay is EPR-l, as
WO92/16558 PCT/US92/02109
21~5~3
expressed on CLL cells.
The method comprises admixing a body sample,
preferably human, containing cells to be analyzed with
a before-described antibody composition that
S immunoreacts with the receptor molecules. Pre~erably,
the cell cample is washed free of coagulation factors
V and VIII prior to the admixing step. The
i"~unoreaction admixture thus formed is maintained
under biological assay conditions for a time period
sufficient for any cells expressing the antigen to
Lmmunoreact with antibodies in the antibody
composition to form an antibody-receptor
immunocomplex. The immunoreaction product
(i ~unocomplex) is then separated from any unreacted
antibodies present in the admixture. The presence,
and if ~esired, the amount of immunoreaction product
forned is then determined`. The amount of product
formed can then be correlated with the amount of
receptors expressed by the cells.
Determination of the presence or amount of
immunoreaction product formed depends upon the method
selected for identifying the product. For instance, a
labelled antibody can be used to form a labelled
i~munocomplex with a receptor molecule of the present
invention. The labelled immunocomplex can be
quantitated by ~ethods appropriate for detecting the
respective label, e.g., fluorescent, radioactive,
biotin labels and the like as discussed hereinbelow.
Alternatively, an unlabelled antibody can be used to
form an unlabelled immunocomplex, which is
subsequently detected by immunoreacting a labelled
antibody recognizing the unlabelled antibody with the
unlabelled immunocomplex. The immunocomplex thereby
becomes labelled and can be detected as described
above.
W O 92/16558 PC~r/US92/02109
9 ~ 3 36
Biological conditions used in the instant assays
are those that maintain the biological activity of the
antibody, EPR-1 cell surface molecule and polypeptide
molecules of this invention. Those conditions include
a temperature range of about 4~C to about 45C,
preferably about 37OC, at a pH value range of about 5
to about 9, preferably about 7, and an ionic ætrength
varying from that of distilled water to that of about
one molar sodium chloride, preferably about that of
physiological saline. Methods for optimizing such
conditions are well known in the art.
In a preferred embodiment, a body sample to be
analyzed is withdrawn from a patient and apportioned
into aliquots. At least one aliquot is used for the ~-
determination of antigen expression using an antibody
composition of the present invention. If desired, a
second aliquot can be used for determining reactivity
of a control antibody with the sample. The analysis
can be performed concurrently but is usually performed
sequentially.
In a further aspect of the invention, data
obtained in the instant assays are recorded via a
tangible medium, e.g., computer storage or hard copy
versions. Th~ data can be automatically input and
stored by standard analog/digital (A/D)
in~trumentation that is commercially available. Also,
the data can be recalled and reported or displayed as
desired for best presenting the instant correla ions
of data. Accordingly, instrumentation and software
suitable for use with the present methods are
contemplated as within the scope of the present
invention.
The antibody compositions and methods of the
invention afford a method of monitoring treatment of
patients afflicted with chronic lymphocytic leukemia
WO92/165~ PCT/US92/02109
21~ ~ 9 93
37 ``
(CLL), and other diseases in which expression of
receptors homologous to factors V and VIII is
correlated with the disease state. For instance, it -
is found that the frequency of cells expressing an
EPR-l marker is inversely related to the response to
treatment of patients suffering from CLL. -
Additionally, patients afflicted with hairy cell
leukemia (HCL) of the EPR-l+ type expreæs markers
detected by an instant antibody composition, thereby
permitting monitoring of treatment.
Accordingly, a method of monitoring a patient's
response to treatment is contemplated in which a
marker for the disease is detected. The method
comprises admixing a body sample containing cells to
be assayed for EPR-l marker with an instant antibody
composition according to an assay method described
above. The admixture is maintained for a time period
sufficient to form an i D unoreaction product under
predefined reaction conditions. The amount of
immunoreaction product formed is correlated to an
initial disease state. These steps are repeated at a
later time during the treatment regimen thereby
permitting determination of the patient's response to -
treatment, with a decrease in the number of EPR-l
molecules expressed on cell surfaces indicating an
improvement in the disease state.
H. Piaanostic SYstems
Also contemplated within the instant invention is
a diagnostic system for performing the described
ascays. A diagnostic system in kit form of the
present invention includes, in an amount sufficient
for at least one assay, a composition containing
antibody molecules or fragments thereof of the present
invention, as a separately packaged reagent, and
WO92/16558 PCT/US92/02109
~ ~Or~9 ~ ~ 38
preferably with a label able to indicate the presence
of an immunoreaction product. Instructions for use of
the packaged reagent are also typically included.
"Instructions for use" typically include a tangible
S expression describing the reagent concentration or at
least one assay method parameter such as the relative
amounts of reagent and sample to be admixed,
maintenance time periods for reagent/sample
admixtures, temperature, buffer conditions and the
like. In one embodiment, a diagnostic system is
contemplated for assaying for the presence of EPR-l
receptors expressed on cells in a cell-containing
sample.
A preferred kit is provided as an enclosure
lS (package) that comprises a container for anti-EPR-l
antibodies that immunoreact with recept~r molecules on
cells in the cell sample. Typically, the kit also
contains a labelled antibody probe that immunoreacts
with the immunocomplex of the anti-EPR-l antibody and
the EPR-l receptor.
The label can be any of those commonly available,
e.g., fluorescein, phycoerythrin, rhodamine, ~25I, and
the like. Other exemplary labels include lllIn, 99Tc,
6~Ga, and ~32I and nonradioactive labels such as biotin
and enzyme-linked antibodies. Any label or indicating
means that can be linked to or incorporated in an
antibody molecule is contemplated as part of an
antibody or monoclonal antibody composition of the
present invention. A contemplated label can also be
used separately, and those atoms or molecules can be
used alone or in conjunction with additional reagents.
Such labels are themselves well-known in clinical
diagnostic chemistry and constitute a part of this
invention only insofar as they are utilized with
otherwise novel methods and/or systems.
WO92/165~ PCT/VS92/02109 ~
3 g ~ ,
39
The linking of labels to polypeptides and
proteins is well known. For instance, antibody
molecules produced by a hybridoma can be labelled ~y
metabolic incorporation of radioisotope-containing
amino acids provided as a component in the culture
medium. See, for example, Galfre et al., Meth.
Enzvmol., 73:3-46 (1981). The techniques of protein
conjugation or coupling throuqh activated functional
groups are particularly applicable. See, for example,
Aurameas, et al., Scand. J. Immunol., Vol. 8, Suppl.
7:7-23 (1978), Rodwell et al., Biotech., 3:889-894
(1984), and U.S. Pat. No. 4,493,795.
An instant diagnostic system can also include a
specific binding agent. A "specific binding agent" is
a chemical species capable of selectively binding a
reagent species of the present invention but is not
itself an antibody molecule of the present invention.
Exemplary specific binding agents are antibody
molecules, complement proteins or fragments thereof,
protein A and the like that react with an antibody
molecule of this invention when the antibody is
present as part of the immunocomplex described above.
In preferred embodiments the specific binding
agent is labelled. However, when the diagnostic
~ystem includes a cpecific binding agent that is not
labelled, the agent is typically used as an amplifying
~eanæ or reagent. In these embodiments, a labelled
specific binding agent is capable of specifically
binding the amplifying means when the amplifying means
is bound to a complex containing one of the instant
reagents.
For example, a diagnostic kit of the present
invention can be used in an "ELISA" format to detect
the presence or quantity of an EPR-l polypeptide in a
3S body sample or body fluid sample such as serum, plasma
W092/16558 PCT/US92/02109
210~3~393 40
or urine or a detergent lysate of cells, e.g., a lOmM
CHAPS lysate. "ELISA" refers to an enzyme-linked
immunosorbent assay that employs an antibody or
antigen bound to a solid phase and an enzyme-antigen
or enzyme-antibody conjugate to detect and quantify
the amount of antibody or antigen present in a sample.
A description of the ELISA technique is found in
Chapter 22 of the 4th Edition of Basic and CLinical
I~munoloav by D.P. Sites et al., published by Lange
Medical Publications of Los Altos, CA in 1982 and in
U.S. Patents No. 3,654,090; No. 3,850,752; and No.
4,016,043, which patents are incorporated herein by
reference.
In preferred embodiments, the antibody or antigen
reagent component can be affixed to a solid matrix to
form a ~olid support that is separately packaged in ;~
the subject diagnostic systems. The reagent is
typically affixed to the solid matrix by adsorption
from an aqueous medium, although other modes of
affixation well known to those skilled in the art can
be used. For example, an instant anti-EPR-l antibody
can be affixed to a surface and used to assay a
solution containing EPR-l molecules or cells
expressing EPR-1 receptors. Alternatively, EPR-l,
polypeptide fragments thereof, and whole or partially
lysed cells expressing EPR-l can be affixed to the
surface and used to screen a solution for antibody
co~positions that immunoreact with the affixed
species.
Useful solid matrix materials in this regard
include the derivatized cross-linked dextran available
under the trademark SEPHADEX from Pharmacia Fine
Chemicals (Piscataway, NJ), agarose in its derivatized
and/or cross-linked form, polystyrene beads about 1
micron to about 5 millimeters in diameter available
W092~1~ ~ PCT/US92/02109
a 9 3 3
41
from Abbott Laboratories of North Chicago, IL,
polyvinyl chloride, polystyrene, cross-linked
polyacrylamide, nitrocellulose- or nylon-based webs 'A
such as sheets, strips or paddles, tubes, plates, the
wells of a microtiter plate such as those made from
polystyrene or polyvinylchloride, and the like.
The reagent species, labelled specific binding
agent or amplifying reagent of any diagnostic system ~`~
described herein can be provided in solution, as a
liquid dispersion or as a substantially dry powder,
e.g., in lyophilized form. Where the indicating means
i5 an enzyme, the enzyme' 8 substrate can also be -
provided in a separate package of a system. Usually,
the reagents are packaged under an inert atmosphere.
A solid support such as the before-described
microtiter plate and one or more buffers can also be
included as separately packaged elements in this `
diagnostic assay system.
The diagnostic system is contained in a
conventional package. Such packages include glass and
pla~tic (e.g., polyethylene, polypropylene and ~;
polycarbonate) bottles, vials, plastic and plastic-
foil laminated envelopes and the liJce.
Examples
The following examples illustrate but do not
limit the invention.
1. Cells and Cell Culture. PMN
(polymorphonuclear leucocytes) were isolated by
dextran sedimentation from acid-citrate dextrose
anticoagulated blood. PBMC (peripheral blood
mononuclear cells) were separated after platelet-rich
plasma was removed by low speed differential
centrifugation over Ficoll-Hypague (Sigma Chemical
3~ Co., St. Louis, MO) (density - 1.077 g/ml~ at 400 x g
WO g2/16558 PCr/USg2/02109
~lO'i'~93
42
for 18 minutes at 22C. PBMC were extensively washed
in S mM EDTA-PBS, pH 7.2, and incubated twice in
autologous serum containing S mM EDTA for 30 minutes
at 370C to prevent platelet-monocyte rosetting.
Monocytes were isolated from PBMC by adherence to
plastic petri dishes precoated with autologous serum
for 1 hour at 37C. Cells were suspended at 1 to 1.5
x 107/ml in detectable endotoxin-free RPNI 1640 medium
(Irvine Scientific, Santa Ana, CA) and 10% heat
inactivated FCS (fetal calf serum; Gemini Bioproducts
Inc., Calabasas, CA), 2 mM L-glutamine (Irvine), 25 mM ~:
HEPES (Calbiochem Boehring Diagnostic, La Jolla, CA),
100 ~g/ml gentamicin (Geramycin, S~hering Corp.,
Kenilworth, NJ).
The monocytic cell line THP-l (ATCC) was
maintained in continuous culture in the above media
further supplemented with 10 ~M 2-ME (Eastman Kodak,
Rochester, NY). The transformed human leukemic-
lymphoma T cell lines HuT 78, MOLT 4, CCR~-CEM, CCRF-
HSB-2, and the human B lymphoma cell lines Raji and
Daudi (ATCC) were maintained in culture as
recommended. The human leukemia-lymphoma T cell lineæ
Jurkat, MLT, PEER, and MOLT 13 were the generous gift~
of Dr. D. P. Dialynas, Research Institute of Scripps
Clinic, La Jolla, CA.
For mixed lymphocyte response, 2Q x 106 freshly
isolated PBMC were cultivated in T-25 vented flasks
(Costar Corp., Cambridge, MA) in the presence of 20 x
106 irradiated (10,000 rad) Raji or Daudi cells.
Cultures were maintained in RPMI 1~40, 10% FCS, 25 mM
HEPES, 10 ~M 2-ME (mixed lymphocyte culture (MLC)
medium) in a 5% CO2 humidified incubator for 7 days at
37~C. Responder cells were harvested, isolated by
centrifugation on Ficoll-Hypaque at 400 x g for 18
minutes at 22C, washed in complete NLC medium, then
WO92/165~ PCT/US92~02109
2~, 3~
cultured in 24-well plates (Costar) at 2 x 105/well in
the presence of 2 x 106 irradiated Daudi or Raji
cell~.
For long term culture of alloreactive cells
stimulated by irradiated Daudi or Raji, responder T
cells were transferred every 6 days according to the
protocol described above and recultured in MLC medium
- containing 10% T cell growth ~actor (Cellular Product
Inc., Buffalo, NY). In some studies, suspensions of
freshly isolated PBMC at 1 x 106/ml were treatad with ~:~
1 ~g/ml Con A (Calbiochem) or 1 ~g/ml PHA
(Phytohemagglutinin; Calbiochem) for 7 days in 5% CO2
at 37C. Aliquots of cells from these cultures were
harvested after various time intervals, washed, and
analyzed by FMF (flow microfluorometry).
2. mAb. The experimental procedures for
purification and characterization of human V have been
reported previously in Altieri, D. et al., J~ Biol.
Chem., 264:2969 (1989). Briefly, B~LB/c mice were
immunized intraperitoneally with 50 ~g of V in CFA
(~omplete Freund's adjuvant; Calbiochem) and
hybridomas generated as de~cribed previously tAltieri,
D. et al. J. Biol. Chem.~ 264:2969 (1989)J. Screening
strategy for antibody selection was to analyze by FMF
the reactivity of hybridoma culture fluids with THP-1
~ells ~Altieri, D. et al., J. Biol. Chem., 264:2969
(1989)]. Six hybridomas reacting with >98% of THP-l
cells were selected for antibody produ~tion in solid
phase RIA and immunoblotting using immobilized V~ and
finally established by two to four times sequential
subclonings by limiting dilution. A rabbit polyclonal
antiserum raised by multiple immunizations with
puri~ied V was also screened by FMF and characterized
according to the strategy described above. In
W092/16558 PCT/US92~0210s ~
~ ~o~9t3~ 44
addition, a second panel of mAb elicited by
immunization with V, reactive with V by Western blot
but nonreactive with THP-l cells by FMF was selected
and established.
Purified Ig fractions of mAb 7Gl2 (IgG2a), 9D4
(IgGl), ~nd 12Hl ~IgM) (ATCC Accession No. HB 10637)
were prepared by chromatography on Affi-Gel MAPS II or
hydroxylapatite columns (Bio-Rad, Richmond, CA).
Purified Ig fractions of anti-V rabbit polyclonal
anti~erum B78.9 were prepared by ammonium sulfate
fractionation and chromatography on DE~E Sephadex.
I D unopurified B78.9 antibodies were isolated from
purified factor V immobilized on Affigel 15 (Biorad,
Richmond, CA) according to the manufacturer's
directions.
Anti-CDl6 mAb we~e ~eu llb (Becton Dickinson,
Mountain View, CA) B73.l and 3G8, the kind gift of Dr.
G. TrLnchieri, the Wistar Institute, Philadelphia PA.
Anti-CD56 mAb NKH-l (Leu l9) was purchased from
Coulter Immunology, Hialeah, ~L. Anti-CDllb and anti-
CD18 ~Abs were OKMl and 60.3, respectively. mAbs to
CDS7 (ENK-l), CD3 (OKT3), CD4 tOXT4), CD8 (OKT8), CD2
~OKTll), HLA class I (W6/32) were acquired from ATCC.~.
Anti-~tB T cell ~eceptor (TCR) mAb WT31 was purchased
2S fr~m Becton Dickînson, anti- /~TCR ~Ab ~l was kindly
provided by Dr. M.B. Brenner, Harvard Medical School,
Boston, ~a.
3. Bindina Reactions. The interaction of
various mAb with different cell types was evaluated by
FMFo Briefly, 1 x lO6 cells were incubated in V-
bottomed microtiter plates (Costar) with saturating
concentrations of each mAb for 30 minutes at 4C~ ;
After ~ashes in MLC media, l/20 dilution aliquots of
fluorescein-conjugated goat (F(ab1)2 anti-mouse IgG +
~.
WO92/16558 PCT/US92/02109
'~ 1a ~ 33
IgM (Tago Inc., Burlingame, CA) were added for an
additional 30 minutes at 4C. Cells were washed and
immediately analyzed on a Becton Dickinson IV/40 FACS.
Simultaneous two-color FNF analyses were performed as ~-~
described previously [Altieri, D. et al., J. Biol.
Chem., 264:2969 (1989)] using mAb 7Gl2 or 9D4
previously conjugated with biotin (N- ;
hydroxysuccinimido-biotin, Sigma) and revealed by l/20
dilutions of phycoerythrin-conjugated streptavidin
reagent (Tago).
To confirm the accuracy of the two-color FMF ~;
analysis performed on the various cell populations,
two additional sets of studies were also carried out.
First, to avoid possible cross-reaction of the second
lS FITC-conjugated anti-mouse reagent with the
biotinylated mAb, these studies were repeated by using
biotin-conjugated aliquots of the rabbit polyclonal
antibody B78.9 in association with the various anti-T
cell or anti-NK (natural killer) cell-related markers
~Ab.
In a further series of studies, directly FITC-
conjugated mA~ 7Gl2 or 9D4 (Chromaprobe, Inc., Redwood
City, CA) were also used in combination with biotin-
conjugated mAb) OKT3, OKT4, OKT8. For cell sorting
studies, HuT 78 cells (1.5 x 107/ml) were incubated
with the anti-V polyclonal ~ntiserum B78.9 followed by
fluorescein-conjugated goat anti-rabbit IgG (Tago).
B78.9~ HuT 78 cells (HuT 78*, 34% of the
unfractionated population) were isolated on a Becton
Dickinson Facstar under negative pressure with a sweep
rate of 2000 cells/s, washed in complete MLC medium,
and cloned by limiting dilution in 96-well-round-
bottomed plates (Costar) at 0.3, l, 3 cells/well in
HuT 78 conditioned medium supplemented with 20% FCS.
After 3 weeks, proliferating cells of single cell ~;
WO92/165~ PCT/US92/02109
2 lrj993 46
clonal origin on the basis of Poisson distribution
were subcloned, established, and further
phenotypically characterized by FMF.
The procedures for the isolation,
S characterization, and l25I-labelling of factor Xa were
as described previously by Altieri, D. et al., J.
Biol. Chem., 264:2969 (1989). The interaction of
5I-Xa with HuT 78* cells was analyzed by incubating
increasing concentrations of l25I-Xa (0.45 to 36 nM)
with cell suspension at l.5 to 2 x lO7/ml in the
presence of 2.S mM CaCl2 for 20 minutes at room
temperature. At the end of the incubation, the
reaction was terminated by centrifugation of aliquots
of the cell suspension at 12,000 x g for 2 minutes
through a mixture of ~ilicone oil to separate free
from cell-associated radioactivity. Nonspecific
binding was quantified in the presence of S0-fold
molar excess of unlabelled factor Xa added at the
start of the incubation reaction, and was subtracted
from the total to yield net specific binding. In some
studies, aliquots of HuT 78* cells were preincubated
with S0 ~g/ml of mAb 9D4 for 30 minutes at room
temperature before the addition of serial
concentrations of l2sI-Xa.
4. Cell Surface Labelling and Immuno~recipi-
tation. Suspensions of PMN at 1 x 108/ml, were
surface iodinated with 5 mCil25I-Na by the Iodogen
method rFraker, P. J., et al., Biochem. Biophvs. ~es.
Commun., 80:849 (1978)]. After extensive. washes in
HEPES saline buffer p~ 7.35 cells were lysed in buffer
containing 0.5% Triton X-lO0 or lOmM CHAPS, o.OS M
Tris HCl, 0.15 M NaCl, 1 mN benzamidine, 0.1 mM (PPACK
= D-phe-pro-Arg chloromethylketone; Calbiochem), 25
~g/ml leupeptin, lmM PMSF (phenylmethyl sulfonyl
WO92/16558 PCT/~S92/02109
2105~3
47
fluoride; Calbiochem), pH 8.3 (lysis buffer), for 30
minutes at 4C. The iodinated lysate was cleared of
nuclei and other cellular debris by centrifugation at
14,000 x g for 30 minutes at 4OC, and extensively
preabsorbed with aliquots of goat anti-mouse IgG ~ IgN
conjugated with sepharose CL4B (Calbiochem). Aliquots -
of the l~I-labelled PMN lysate were separately
incubated with mAb 12H1 or 60.3 for 14 hours at 4C
under agitation. The immune complexes were
precipitated by the addition of gcat anti-mouse IgG +
IgM conjugated with sepharose CL4B for an additional 6
hours at 4C, extensively washed in the above lysis
buffer, and finally resuspended in 2% SDS sample
buffer, pH 6.8, containing 50 mM 2-dithiothreitol as a
lS reducing agent. The samples were immediately boiled
for 5 minutes, clarified by centrifugation at 14,000 x
g for 5 minutes and finally electrophoresed on 7.5%
SDS polyacrylamide slab gels in 0.1% SDS. Gels were
stained in Coomassie blue R 250, destained in 5%
acetic acid, dried and exposed for autoradiography at ~
-70C by using Kodak X-Omat AR X-Ray film and -
intensifying screens (Cronex E.I., DuPont de Nemours,
Wilmington, DE). ~.
5. Isolation o~_~PR-l Molecule. The isolation
to homogeneity of the EPR-l molecule required the
identification and/or establishment of cell types that
constitutively express high levels of this surface
antigen. These studies were conducted primarily on
peripheral blood polymorphonuclear leukocytes (PMN)
and on a specifically selected T cell clonal
derivative from the parental T cell line MOLT13 #3
~Altieri, et al., J Immun. 145:246 (1990)].
The subline MOLT13 #3 was established by two
sequential cycles of fluorescence sorting of the
WO92/165~ PCT/US92/02109
` ~10~99~ 48
parental line MOLT 13 using the anti-EPR-l mAb 12H1.
Only MOLT 13 cells expressing the highest levels of
reactivity with mAb 12Hl by fluorescence analysis were
isolated, cloned by limiting dilutions at 1 or 3
cells/well, grown to confluency, and finally re-
screened again by flow cytometry for reactivity with
mAb 12Hl as well as with a panel of mAbs directed
against various T cell-related markers. The subline
MOLT13 #3, established as described above, expressed
7-10 fold higher levels of EPR-1 as compared with the
parental line.
MOLT13 #3 cells (ATCC Accession Number CRL 10638)
were grown continuously in suspension at 37C in T150
tissue culture flasks, 5% CO2. Cells were harvested,
washed twice in ice cold phosphate buffered saline
supplemented with S mM EDTA~ pH 7.2, and lysed in
lysis buffer for 1 hour at 4C in continuous
agitation.
The composition of the lysis buffer was found to
ba critical in obtaining satisfactory recovery of the
isolated EPR-1 molecule. The detergent used was 0.3%
CHAPS (3-t(3-cholamidopropyl)-dimethylammonio]-1-
propanesulfonate; Calbiochem) in the preFence of lmM
CaCl2 and a cocktail of protease inhibitors, including
PMSE (1 mM), p-APMSF (amidinophenylmethyl sulfonyl
fluori~e; Calbiochem~ (1 mM)~ PPACK (0.25 mg/ml),
soybean trypsin inhibitor (0.1 ~g/ml), benzamidine (1
mM), leupeptin (0.25 mg/ml~, aprotinin (10,000 U/ml).
The ratio between volume of lysis buffer and cell
number ~ubjected to lysis depends on the cell type
selected for the analysis. For MOLT13 #3 cells,
approximately 120 ml of lysis buffer were required to
effectively solubilize 109 cells. Under the same
conditions, 4xlO9 PMN was effectively solubilized
using 200 ml of EPR-l lysis buffer. The solubilized
WO92/165~ PCT/US92/02109
210a993 ~
cell extract was then cleared of nuclei and other
insoluble material by centrifugation at 6,000 rpm for
30 minutes at 4C, and stored at -70C until ready to
use.
The immunoaffinity isolation of the EPR-l
molecule from the detergent-solubilized cell extracts
prepared as described above, was based on the use of
the anti-EPR-l mAb 12H1 (IgM isotype). Approximately
4xlO9 cells solubilized as described were incubated
with mAb 12Hl for 16 hours at 4C in agitation. The
ratio of mAb 12H1 used was 1 ml of ascites/100 ml of
EPR-1 lysate.
At the end of the incubation, the EPR-1 molecule
bound to mAb 12H1 was isolated by the addition of 2 mi
aliquots (5 mg) of goat anti-mouse IgM covalently -~
conjugated to Sepharose CL4B ~solid phase immuno-
absorbent) for an additional 6 hours at 4C in ~-
agitation. At the end of that time period, the
immunoprecipitate was washed five times in EPR-1 lysis
buffer by centrifugation at 3,000 rpm for 20 minutes
at 4C.
After the washes, the pellets from various tubes
were pooled, resuspended in 0.4 ml of nonreduced SDS
sample buffer, pH 6.8, boiled for 5 minutes to
separate the antigen from the antibody complex, and
finally separated by electrophoresis on a 7.5%
preparative SDS polyacrylamide gel, applying 25
mAmps/gel constant current. After electrophoretic
migration, the gels were fixed in methanol, stained in
0.08% Coomassie blue, and destained overnight in 10%
methanol and 5% acetic acid.
This isolation procedure allowed visualization of
a prominent 78 kDa band stained by Coomassie blue and
a second specific component migrating with a 66-68 kDa
molecular size and frequently appearing as a dsublet
WO92/165~ PCT/US92/02109
210~993
of 66/68 kDa. The 66/68 kDa species are believed due
to limited proteolysis or underglycosylation. The 78
kDa stained band was excised from the preparative gel
using a razor blade, macerated in small fragments with
s a spatula, and mixed with a digestion buffer
containing 20% glycerol, 0.1% bromophenol blue, 125 mM
Tris-HCl~ 1 mM EDTA, 0.1% SDS, pH 6 .8. The macerated
bands were loaded in 6 wells on a 15% SDS-
polyacrylamide gel and each sample was overlaid with
40 ~1 of Endoproteinase G, sequencing grade (v8) at
100 ~g/ml final concentration.
The samples were prerun at 50 mAmps until they
entered the separating gel, digested during a 30 ;-
minute incubation time, and the generated fragments
were finally separated during the remaining
electrophoretic run. The gels were quickly soaked in
water after the migration, then in CAPS (t3-
(cyclohexylamino)-propanesulfonic acid~; Calbiochem) ~;
transfer buffer containing 10 mM CAPS and 20%
methanol, and finally assembled onto I D obilon
membrane in a gel transfer apparatus. Protein
transfer was carried out at 450 mAmps for hours at
room temperature. The Immobilon membranes were then
removed, soaked in water, stained for 30-60 minutes in
Coomassie blue 50% methanol, rinsed in water and
destained in 10% acetic acid and 40% methanol. This
procedure permitted the direct identification of a
number of heavily stained bands by internal cleavage
of the 78 kDa band EPR-l molecule, and subsequential
electrophoretic separation of the corresponding
fragments.
Bands of interest were excised from the stained
transfer on the basis of their relative molecular
weight and subjected to NH2 microsequence analysis
using an Applied Biosystems Vapor Phase Sequencer with
WO92/16558 PCT/US92/02109
2105~93
51
on-line HPLC available in the Microchemistry core of
the Research Institute of Scripps Clinic. The derived
peptide sequences from a number of studies conducted
as described above on both PMN and MOLTl3 #3 T cells
S were analyzed by the GENALIGN computer program
(Intelligenetics, Inc.) on a SUN computer and by the
WORDSEARCH programs (Univ. of Wisconsin Genetics
Computer Group) on a VAX 750 computer. Aæ indicated
in Table l, all the deduced peptide sequences derived
from the EPR-l molecule showed a significant degree of
homology exclusively with the coagulation proteins,
factor V and VIII, and with a recently described
murine cell-surface molecule denominated MFG E-8 -
tStubbs et al., Proc. Natl. Acad. Sci. U.S.A. 87:8417
(l990)], that shares remarkable homologies with these
twn coagulation proteins and the EPR-l molecule.
W092/16558 PCT/~S92/02109
21059()3 52
TABLE 1
EPR-l SEQUENCE ALIGNMENTS*
RES. RES.
# SEOUENCE ~ SOURCE
EPR-l SEQUENCE #l
1 TLKG.QTQGAVMI 12 EPR-l #l
:-1 1111 .
215 RV5GYMTQGASRA 227 MFG E-8
2099 IIHGIKTQGARQR 2111 factor VIII (Cl)
1972 IITGlQTQGAKHY 1984 factor V (Cl)
2033 KITiIITQGKDSI 2044 factor V (C2) (Preferred)
2259 KVTGVTTQGVKSL 2271 factor VIII ~C2) (Preferred)
376 QVTGIITQGARDF 388 MFG E-8 (Preferred)
1 TLKG.QTQGAVMI 12 EPR-l #l
EPR-l SEQUENCE #2
1 PXIXQNDLL 9 EPR-l #2
204 PWl QVNLl 211 ~FG E-8
2089 .WI KVDLL 2095 factor VIII (Cl)
35 1963 PWI QVDMQ 1967 factor V (C13
204 PWI QVNLL 211 MFG E-8
EPR-l SEQUENCE #3
1 ACKLREELHKX 11 EPR-l #3
293 GCTLRFELLGC 303 MFG E~8
d,5 :111.11.
2178 RSTLRMELMGC 2188 factor VIII (Cl)
I 111-11 .
2Q51 RPTLRLELQGC 2061 factor V (Cl)
I I I I
1 ACKLREELHKX 11 EPR-l #3
WO92/16558 PCT/US92/02109
53
EPR-1 SEQUENCE #4
1 VDKLAPRDP.LA 11 EPR-1 #4
173 FMG1QRWQPELA 184 MFG E-8
2059 SGQYGQWAPKLA 2070 faCtOr VIII (C1)
1926 SEFLGYWEPRLA 1937 faCtOr V (Cl)
10: 1--::1 11
1 VDKLAPRDP.LA 11 EPR-1 #4
EPR-1 SEQUENCE #5
-~
274 FNPTLEAQ 281 MFG E-8
1 GVPPVVT 7 EPR-1 #5
202032 FDPPIVARY 2040 faCtOr V ~C1)
2159 FNPPIIAR1 . 2167 faCtOr VIII (C1)
2316 LDPPLLTR1 2324 faCtOr VIII (C2) (Preferred)
1 GVPPVVT 7 EPR-1 ~5
2~92 FNPPIISRF 2200 faCtOr V (C2) (Preferred)
30434 FEKPFMARY 442 MFG E-8 (Preferred)
EPR-l SEQUENCE #6
35262 GNLDNNSLXVN 272 MFG E-8 (Prefer~d)
1 GNSDAXYVKXV 9 ~PR-1 #6
2020 GNSDASTIREN ~028 faCtOr V (C1) (Preferr~d)
2147 GNVDSSGIKH1 2158 faCtOr VIII (C1) (Preferred)
2181 G1TNTKGHVXN 2191 faCtOr V (C2)
452304 G1QDSFTPVVN 2314 faCtOr VIII (C~)
422 1NLDNNSHKKN 430 MFG E-8
1 11SDAXYVKXV 9 EPR-1 #6
WO92/1~58 PCT/US92/02109
. .
21()5993 54 `
EPR~l SEQUENCE #7 ;
353 INAWTAQSNSAKEWLQVD 370 MFG E-8
.. . I 11 ...
51 VQKLAEDENNAKKHVEPH 18 EPR-l #7
1- :~---11-1- :1 .
2109VNAWQAKANNNKQWLEID 2126 factor V (C2
353INAWTAQSNSAKEWLQVD 370 MFG E-8
2109VNA~QAXANNNXQWLEID 2125 factor V (C2) ``
111-:--11 1:11::1 `
2235SNAWRPQVNNPKEWLQVD 2251 factor VIII
I I 1: -
~51 YQKLAEDENNAKXHVEPH 18 EPR-l #7
*Numbering nomenclature for human factor V and
factor VII~ is from NBRF Protein Data Bank and
includes the leader peptides in the sequence (29
residues for factor V and 19 residues for factor
VIII). Numbering of MFG E-8 is from Stubbs et al.
Proc. Nat. Acad. Sci., 87:8417-8421 (1990), but
includes the 22 residue leader peptide. The factor V -;
and factor VIII sequence numbering in Stubbs et al. is
the NBRF scheme minus 29 for factor V and minus 19 for
factor VIII. ¦ = identity; : = csnservative
substitution; . = semi-conservative substitution. X is
a variable amino acid re~idue. A space, where present
in a sequenae, is a gap introduced to improve sequence
alignments. Note that factor V and factor VIII have
two repeated highly homologous C domains and
sequences, thus there are duplicate homology matches.
The domain matches with Cl or C2 and the preferred
homology are indicated whe~ both must be considered. ,~
6. EPR-1 ~xpression Correlate~s with_Response to
CLL ~hera~y. The reactivity of anti-EPR-1 mAbs with
peripheral blood cells isolated from patients with
hematopoietic malignanci~s was explored with flow
cytometry. It was found that in 28 out of 37 CLL
patients (75%), the number of EPR-lt cells was
increased 5-6 fold thereby including most of the
circulating population, as compared with normal
controls (EPR-lt cells in normal donors: 16.5+3.2%,
n-12 versus EPR-l~ cells in CLL: 89.1+2.5%, n-28).
WO92/1~ ~ PCT/US92/02109
2 10~3
~:
The number of EPR-l molecules expressed on CLL cells
also showed a mean increase of 2.5 fold as compared
with normal controls (mean fluorescence of EPR-l+ -
normal cells: 85.6+16.1 versus EPR-1~ CLL: :~
215.6+50.3). Roughly 98% of PMC cell were positive to -:
this marker. Two-color flow cytometry studies
confirmed that in CLL patients both CD5 and EPR-l were
simultaneously co-expressed in the ~ame cell
population. Finally, sequential analysis of a group
of CLL patients carried out over a 4-month period
(starting at day 0) showed that positive biologic
response to the therapy was frequently associated with
drastic reduction (67-90% reduction) in the number of
EPR-1' cells detected. Representative patient data
are presented in Table 2, below, and illustrate this
trend.
EPR-l therefore representæ a novel cellular
marker in CLL and its surface expression inversely ~:
correlates`with the patient's biologic response to the
therapy. The data further emphasize the possible
participation of protease-mediated mechanisms in the
development and/or establishment of selected
hematopoietic malignancies. ,- :
TABLE 2
EPR-l Expression on Leukemia Cells*
Patient # Day % 12H1 % B78.9
1 0 91.5 74.4
95.3 11.4
9.2 49.6
2 0 46.8 52.8
3s 41 98.7 98.6
5.2 32.8
0 98.3 92.6
36 66.7 83.7
WO92/16558 PCT/US92/02109
. .
2 10~ 99g 56
4 0 25.4 32 :
28 46.3 51.~
56 2.9 1.4 ~,
84 63 41.2
0 99.7 98.9
21 43.8
6 0 94 31.2
9.6 8~.9
7 0 97 96.8
34 75.8 87
8 0 96.7 88.4
34 92.6 77.2
69 8~.2 1~.2
* 12Hl and B78.9 are monoclonal and polyclonal
antibodies, respectively, as described above. The
data presented are percentages of cells examined which
expressed suprathreshold amount of EPR-l.
7. PCR Generation of EPR-1 DNA. Using the
amino acid residue sequences obtained for EPR-l
presented in Table 1, primer molecul s were identified
for PCR amplification of nucleic acids coding for the
EPR-1 polypeptides, including ~PR-l itsel~. For
instance, oligonucleotides that anneal with the (~
strand of DNA and that code ~or EPR-l #1 and ~3 amino
acid residue sequences are:
#1 5'-A~AGGICAGACICA~GGIGCIGTIAT~AT-3';
#3 5'-TGCAAAITIIGIGAAGAAITICACA~A 3'.
Probes complementary to those identi~ied above
can be used to hybridize with a (+) ~trand of RNA or
DNA. Generally, one of the primers listed above
should be used with a primer complementary to the
other primer listed above so that extension products
on both the (+) and ~-) strands are generated
multaneously. Also, the primers can be extended to
include convenient restriction and cloning sites as
WO92~16558 PCT/US92/02109
,~10~i~9X
57
desired. Since a #1 primer can anneal 5' and/or 3' to
the #3 primer, usually both sets of primers and primer
complements will be used in a PCR protocol. The
reaction conditions and cycling protocol for PCR are
well known and are described above. In these
nucleotide probes, A is adenine, G is quanine, C is
cytosine, T is thymine, and I is inosine.
8. Discussion of Examples 1-7.
Cellular Distribution of EPR-1. Monoclonal antibodies
against V, the circulating plasma protein that binds
the Xa serine protease of coagulation cascade Xa were
prepared [Nesheim, N., et al., J. 8iol. Che~
254:10952 (1979)]. In a previous study, it was shown
that a minor fraction of these mAb (mAb panel I 7G12,
9D4, and 12Hl) also reacts with a surface molecule
expressed on various myeloid monocytic cell lines
~Altieri, D. et al., J. Biol. Chem., 264:2969 (1989)].
Using mAb inhibition studies and receptor-ligand
chemical cross-linking, it has been demonstrated that
this cell-associated immunoreactive molecule functions
as a high affinity (Xd - 30 nM, n about 150,000)
receptor for Xa [Altieri, D. et al., J. BiQlo Chem.,
264:2969 (1989)]. In thi study, mAb panel I was
exploited to characterize the cellular di tribution
and identity of ~he putative membrane serine protease
receptor (EPR-l).
As illustrated in Figur@ 1, the reactivity of
panel I anti-V mAb is not an eccentric characteristic
of transfo~med in vitro cell lines. mAb 9D4,
recognizing a different epitope from the one
previously identified by mAb 7G12 ~Altieri, D. C., J.
Biol. Chem., 264:2969 (1989)], reacted with peripheral
blood monocytes and dextran-isolated PMN, although
with considerable heterogeneity in the latter
WO92/16558 PCT/US92/02109
~l~S~93 58
population (Fig. 1).
When suspensions of PBMC were analyzed by FMF,
mAb 7G12, 9D4, and 12Hl consistently reacted with a
population of cells (5 to 20%) with forward light
scatter characteristic of lymphocytes. Simultaneous
two-color FMF analyses were performed to further
dissect the phenotype of this lymphoid population.
For these studies, suspensions of PBMC were
preparatively depleted of adherent cells by either
adherence to plastic or by nylon wool fractionation to
yield populations enri~hed in PBL (peripheral blood
lymphocyte). Approximately 50 percent of the lymphoid
subset identified by mAbs 7G12, 9D4, or 12Hl was OKT3-
and expressed the NK-associated markers CD16 and CD56,
as revealed by the simultaneous binding of mAb Leu
llb, 3G8, B73.1, and NKH-l, respectively.
Furthermore, when enriched populations of NK cells
(<3%CD3~,~85%CD16~) prepared from PBMC by nylon wool
fractionation, SRBC (sheep red blood cells) rosetting,
and negative selection with mAb OKT3, were analyzed by
FMF, mAb 7G12 and 9D4 reacted with 68 and 72% of these
cells.
The remaining EPR-l+ PBL were phenotypically
established as CD3~ lymphocytes. Table 3,
hereinafter, shows a representative study of two-color
FMF characterization o~ this EPR-lt subset. Although
double positive cells coexpressing either CD4 or CD8
were identified, the latter fraction consistently
exhibited a higher frequency and a far greater
intensity of reaction with EPR-l marker mA~.
Virtually all EPR-l' T cells also coexpressed
CDllb and CD57~Leu 7), as revealed by mAb OKMl and
HNX-l respectively, and approximately 70 to 80 percent
were CD2~(0KTll)(Table 3). Although the EPR-l~ subset
was predominantly WT31~, approximately 10% of EPR-1~
W092/165~8 PCT/US92/0210g
21iD~9~3
59
cells (2% of unfractionated PBL, n = 3) were found to
be reactive with anti- /~TCR mAb ~1. Quantitatively
comparable results were also obtained when two-color
FMF analyses of PBL were carried out using biotin-
conjugated aliquots of the rabbit polyclonal antibodyB78.9, or the directly FITC-conju~ated mAb 7G12, or
9D4, in combination with the various anti-T cell or
anti-NK cell related markers mAb.
TABLE 3 :
Two-color FMF characterization of EPR-1+
subset of T cells'
Relative Percent
Percent Coexpressing
Coexpressing Cells in EPR-l~
~Ab Specificity PBL Subset ~;
OKT3 CD3 9.6 73
ORT4 CD4 3.6 27
0KT8 CD8 7.4 56
OXTll CD2 10.5 79
OKNl CDllb 10.8 82
HNX-l CD57 11.5 88
60.3 CD18 10.7 82
WT31 ~/B TCR 8.1 61
~ TCR 1.4 10
W6/32 Class I ~HC12.0 91
.
~rwo-color FMF analysis of adherent cell-depleted
PBL was ~arried out as fcllows: Suspensions of PBL
~ere depleted of adherent cells and ~sparately stained
with aliquots of anti-CD16 mAb Leu llb, ~73.1, or 3G8,
or with anti-CD56 mAb NKH-1 (Leu 19) for 30 minutes at
4C. Cells were washed and incubated with
fluorescein-conjugated soat (F(ab' )2 anti-mouse IgG +
IgM for additional 30 minutes at 4C. After extensive
washes, cells were equilibrated with 10 ~g/ml of
~iotinylated mAb 7G12, washed, and incubated with 1/20
4~ dil~tion of phycoerythrin-streptavidin conjugated
reagent. Double-positive cells from a representative
study are indicated for the unfractionated PBL
population and relative to the EPR-1~ subset (13.1%).
EPR-1 is di$tin~t from CDllb~CD18. The
WO92/16S58 PCT/US92/02109
210~993
expression of EPR-l on monocytes, PMN, NK cells, and a
fraction of T cells that is also predominantly CD8+,
appears to mimic the cellular distribution of the
leukocyte integrin CDllb/CD18 (Mac-l) [Sanchez Madrid,
F., et al., J. Exp~ed., 158:1785 (198~)].
Therefore, additional studies were designed to
establish the reciprocal structure and ~unctional
properties of CDllb/CD18 and EPR-l. For these
studies, suspensions of PMN that express abundant
levels of the CD11/CD18 molecules [Sanchez Madrid, F.,
et al., J. Ex. Med., 158:1785 (1983)~ were surface
labelled with l2sI, detergent-solubilized, and
~ubjected to immunoprecipitation using either the
anti-CD18 mAb 60.3 or the anti~EPR-l mAb 12H1.
From ~ labelled PMN lysate, mAb 60.3
im~unoprecipitated the polypeptides corresponding to
the ~ subunits of the leukocyte integrins CDlla,
CDlllb, and CDllc in association with the common B-
sub~nit CD18, in agreement with previous observations
tSanchez Madrid, F., et al., J. Exp~_Med., 158:1785
(1983)]. In contrast, under the same conditions, mAb -~s
12Hl immunoprecipitated a major surface component
having a molecular mass of about 78+~4 kDa.
Functionally, CDllb/CD18 and EPR-l have different
ligand recognition specificities. Although CDllb/CD18
has been recognized as an oligo-specific receptor for
C3bi, fibrinogen, and factor X ~Sanchez Nadrid, F., et
al., J. Exp. Med., 158:1785 (1983); Altieri, D. et
al., J. Cell Biol., 107:1893 (1988); Wright, S. D., et
al., ~roc. Natl. Acad~ Sci. USA, 85:7734 (1988);
Altieri, D. et al., J. Biol~ Chem , 263:7007 (1988)],
EPR-l binds the activated serine protease Xa tAltieri,
D. et al. J. Biol. Chem., 264:2969 (1989)].
Antî-CDllb/CD18 mAb do not inhibit EPR-l receptor
function and the reverse also applies for EPR-l mAb on
WOs2/16558 PCT/US92/021~
210~9~3
61
CDllb/Cdl8 ligand recognition. Similarly, soluble
CDllb/CD18 ligands such as fibrinogen tAltieri, D~ et
al., J. Cell Biol., 107:1893 (1988); Wright, S. D., et
al., Proc. Natl. Acad. Sci. USA, 85:7734 (1988)], and
factor X [Altieri, D. C., et al., J. Biol. Chem.,
263:7007 (1988)], do not compete or inhibit EPR-l
receptor recognition of Xa.
Dvnamic Requlated Ex~ression of EPR-l on PBL.
Additional studies were designed to explore the
possibility of a dynamic modulation of EPR-l
expression under conditions of antigen-specific or
mitogen-driven T cell activation. Freshly isolated
PBMC were set up in unidirectional mixed lymphocyte
culture (MLC) against irradiated allogeneic B cells,
i.e., Raji (MHC class I and II driven) or Daudi (MHC
class II driven). After 7 days culture, responder T
cells were harvested, washed, and phenotypically
characterized by FMF using mAb 7G12, 9D4, and 12H1.
In another series of studies, PBMC were
separately cultivated for 7 days in the presence of 1
~g/ml of the polyclonal activators (PHA) or Con A then
subjected to FMF analysis. As shown in Figure 2, both
allogeneic expansion of normal PBMC or lectin ~,
activation resulted in a consistent three- to four-
fold increase in EPR-l~ T cells, as recogniæed by mAb
12H1.
To exclude the possibility that the observed
expansion of 12H1~ cells resulted from a selective
redistribution of T cell subsets occurring upon
activation, Con A-stimulated PBMC were seguentially
analyzed by FMF after various time intervals of
culture. Con A-mediated quantitative expansion of the
EPR-l~ subset occurred in cells with forward light
scatter characteristic of proliferating, activated
blasts. The number of these cells increased
WO92/16~ PCTJUS92/02109
~10~9~3 62
approximately four-fold between day 6 and 7 of culture
and when these cells were phenotypically characterized
by two-color FMF they were CD3', CD4-, CD8+, CD2~.
Additional studies were carried out to
investigate the effects of long term alloreactive
stimulation on EPR-l expression. Unidirectional MLC
against irradiated Daudi cells was maintained in
continuous culture with weekly transfers in the
presence of 10% T-cell growth factor (TCGF). At
various time intervals, aliquots of responder T cells
were harvested, recovered by centrifugation over
Ficoll-Hypaque, and finally analyzed for EPR-l marker
expression by FMF using mAb 12H1 or the polyclonal
antiserum B78.9. These data are summarized in Figure
~5 3. The number of EPR-1~ cells detected by mAb 12Hl
increased approximately ninefold during antigen-
mediated activation after one month of culture.
Similar results were also obtained using the
polyclonal antiserum B78.9, which shows a larger
reactivity consistent with the greater number of EPR-l
epitopes detected by this reagent. ~
To distinguish between selective expansion of ~;
EPR-l~ cells or de novo expression of this marker ~ -
resulting from polyclGnal or antigen stimulation, an
additional set of s~udies was carried out. ~-
Suspensions of freshly isolated PBL were preparatively
~ractionated in EPR-l~ and EPR-1- subsets by FMF
sorting with mAb 12Hl. These resulting populations
were then separately cultivated for 10 days with 1
~g/ml Con A, 5 ~g/ml PHA, or stimulated in mixed
lymphocyte response with irradiated Daudi in the
presence of 10% TCGF before FMF analysis of EPR-l
expression. The results of these experiments are
shown in Table 4, hereinafter. Both polyclonal- or
antigen-stimulation of the negatively selected EPR-1-
WO92/165S8 PCT/US92/02J09
63 2 1 0~ g ~3
subset was associated with de novo expression of EPR-1
as detected by binding of mAb 12Hl.
TABLE 4
E~ novo EPR-1 expression on negatively selected
EPR~ ubset activated after short term culture'
EPR-l- Subset EPR~ Subset _
Stimulation Positive Fluorescence Positive Fluorescence
Cells (%) ~U) Cells (%) (U)
--- 0.2 5.5 77.5 102.1
PHA 6.8 78.3 ND ND
Con A 47.2 218.5 ND ND
Daudi NLC 45 82 91.8 83.1
_
Freshly isolated PBL were fractionated in EPR-l- and
EPR-1~ subsets by FMF using mAb 12H1. The resulting
populations were cultivated in the presence of 1 ~g/ml Con
A, 5 ~g/ml PHA or stimulated in allogeneic MHC with Daudi
cells for 10 days before FMF analy is with anti-EPR-l mAb
12Hl. U = arbitrary units. .
EPR-1 Ex~ressed on T Cells is FunctionallY Actiye :
Prote~se ReceptQr. To further substantiate the
expression of EPR-l on discrete lymphoid populations, -~
a number of transformed ~a vitro T cell lines were
screened by FMF using the panel of m~b described ~:
above~ As shown in Figure 4, of the various T cell ~.
lines assayed only a subpopulation of HuT 78 cells w~s
reactive with mAb 7G12. These cells were isolated to
~90% purity by fluorescence sorting using the
polyclonal antiserum B78.g to yield the subpopulation
HuT 78*, which was then cloned by limiting dilution.
Three clones were established, subcloned,
phenotypically characterized by ~MF as OKT3+, OKT4~,
OKT8-, 12Hlt, B78.9~, and one of them was selected for
further investigations.
When suspensions of HuT 78* were equilibrated
with increasing concentrations of l25I-Xa in the
presence of 2.5 mM CaCl2, these cells bound the
WO92/165~8 PCT/US92/02109
~105~393 64
offered ligand in a specific and concentration-
dependent reaction, approaching steady saturation at
30 to 36 nM of added l25I-Xa (Table 5). Quantitatively
similar to the results previously obtained with THP-l
cells [Altieri, D. et al., J. Biol. Chem., 264:2969
(1989)], this reaction was regulated by an apparent K~
on the order of 10 to 20 nM, and was saturated when
194,000 ~ 26,000 molecules of 125I-Xa were specifically
associated with the surface of each HuT 78* cell.
Finally, preincubation of HuT 78* cells with
saturating amounts of mAb 9D4 inhibited specific ~i~
binding of 125I-Xa to these cells.
TABLE 5 ~-
1Z5I-Factor Xa 125I-Xa bound(molecules/cell Xl0-3)
added (nM)* mAb 9D4 No mAb ~;
1.5 1 6 -~
8 26
8 20
18 33 140
27 58 170
36 76 200
* lZ5I-factor Xa binding to HuT 78* cells. HuT 78*
cells reacting with the rabbit polyclonal antiserum
B78.9 were isolated to 94.2% purity by fluore~cence
sorting and cloned by limiting dilution. Three clones ~--
were established, phenotypically characterized and one
{HuT 78*-3} selected for further investi~ations.
Suspensions of HuT 7~*-3 cells at 1 X 10 /ml were
separately incubated with control antibody or with
50 ~g/ml of the anti-EPR-l mAb 9D4 for 30 minutes at
room temperature, before the addition of increasing
concentrations of l25I-factor Xa (0.45 to 36 nM) and
2.5 mM CaCl2 for additional 20 min at room
temperature. The reaction was terminated by
centrifugation through mixture of silicone oils and
L~I-Xa specific binding to HuT 78* cells was
calculated in the presence or absence of anti-EPR-l
mAb 9D4.
WO92/16558 2 1 ~ 5 9 ~ 3 PCT/US92/02l09
6s
Conclusion
The reactivity of a panel of mAb with a cell
surface protease receptor expressed on some leukocytes
has been characterized. In previous studies, it was
S shown that a mAb originally raised against the plasma
coagulation protein V (7Gl2) bound in specific and
saturable reaction to the monocytic-myeloid cell lines
THP-l, U937, and HL-60 tAltieri, D. et al., J. Biol.
Chem., 264:2969 (1989)~. Further, by analogy with the
known acceptor/cofactor function of the plasma protein
Va tNesheim, M.-E., et al., J. Biol. Chem., 254:10952
(1979)], the molecule recognized by mAb 7Gl2 on these
cells appeared to be implicated in a specific receptor
function for the serine protease Xa.
lS A panel of anti-V mAb has now been raised. The
hybridomas secreting those mAb were selected by FMF
analysis of THP-l cells, and the mAb were-used as `~
probes to search for expression of the V cell surface
cross-reacting molecule on peripheral blood cells.
The first conclusion that can be drawn from these
studies is that the molecule recognized by these mAb,
operatively defined as EPR-l, is not inappropriately
expressed only by transfoxmed cell lines in culture.
Rather, it has a broad cellular distribution and a
2~ remarkable association with cells of myeloid and
lymphoid lineage. Although with considerable
heterogeneity among the various populations examined,
these mAb defining EPR-l were found to be reactive
with peripheral blood monocytes, PMN, and CD3-
CDl6~CD56+ NK cells.
Intere~tingly, a small fraction of circulating T
cells was also identified as EPR-l+. Phenotypic
characterization of this subset by FMF suggested that
the expression of EPR l does not appear to be
segregated into a unique subpopulation defined by
W092/16558 PCT/US92/02109
~ lo5 ~93 66
currently known markers of T cells. Although the
majority of EPR-1~ T cells isolated from various
donors was also CD8~ or ~/BTCR+, cells coexpressing
CD4 or /~TCR were identified as well. In agreement
with this finding, FMF analysis of various transformed
T cell lines in vitro, revealed expression of EPR-1
markers on MOLT 13 cells that were further
phenotypically established as CD4+ and TCR /~+,
respectively, in agreement with previous observations ~
tLefranc, M. P., et al., Nature, 316:464 (1985); ;
Brenner, M. B., et al., Nature, 325:689 (1987)].
Within the CD8+ fraction of normal PBL, EPR-l
expression was consistently associated with
coexpression of CDllb(Leu 15) and CD57(Leu 7~, as
identified by mAbs OKM1 and HNK-l. In earlier
studies, this pattern of markers has been associated
with suppressor function ~Clement, L. T., et al., J.
Immunol., 133:2461 (1984); Fox, E. J., et al., J. Exp.
~ , 166:404 (1987); Takeuchi, T., et al., Cell.
I~munol., 111:398 (1988)] and LAK activity tDianzani,
et al., Eur. J. Immunol!, 19:1037 (1989)]. However,
at variance with the previously repor~ed poor
proliferative response of this T cell subset [Fox, E. -
J., et al., J. Exp. Med., 166:404 (1987~], EPR-l
expression is observed as strongly increased by both
mitogen and antigen stimulation.
This finding appeared to be particularly
emphasized in studies using long term cultures of
alloreactive-stimulated T cells, where the anti-EPR-1
rabbit polyclonal antibody B78.9 reacted with
virtually all responder cells after one month culture.
Similarly, de novo EPR-l expression was also observed
after short term polyclonal or antigen stimulation of
preparatively sorted EPR-1- populations. Although
these data would appear to be compatible with the
WO92/16558 PCT/USg2/02109
?~ 1~5993
67
hypothesis that EPR-1 is a true T cell activation
responsive molecule, further investigations at the
sinqle clonal cell level are necessary to conclusively
address this possibility. Finally, in agreement with
the expression of both CD4 or CD8, no preferential
expansion of EPR-l~ cells was observed by either clasc
I or class II NHC allogeneic stimulation.
Although the cellular distribution of EPR-l
closely resembles that of the leukocyte integrin
CDllb/CD18 [Sanchez Madrid, F., et al., J. Exp. Med.,
158:1785 (1983)], structure/function analyses revealed
by immunoprecipitation studies and l25I-labelled ligand
binding assays clearly demonstrate that these are two
different molecules implicated in distinct and
different receptor recognition functions ~Altieri, D.
et al., J. Biol. Chem., 264:2969 (1989); Sanchez
Madrid, F., et al., J. Exp. Med., 158:1785 (1983);
Altieri, D. C., et al., J. Cell Biol., 107:1893
(19B8); Wright, S. D., et al., Proc. Natl. Acad. Sci.
SA, 85:7734 (1988); Altieri, D. C., et al., J. Biol.
Che~., 263:7007 (198B)~.
~his study has not been designed to address the
reciprocal relationship between cellular EPR-1 and the~.
plasma protein V, that originally served as an
immunogen to raise the anti-EPR-l mAb used. However,
it is important to note that the anti-EPR-l m~b panel
described (panel Il, constitutes only a minor fraction
of the anti-V hybridomas elicited by immunization with
factor V. In fact, a second panel of anti-V mAb
3~ raised and established under identical protocols and
selected for production of mAb immunoreactive with
factor V did not exhibit cross-reactivity with THP-l
cells. Furthermore, the size (78+4 kDa) and
structural organization of EPR-l resolved in
immunoprecipitation studies exhibits remarkable size
WO9~16558 PCT/USg2/02109
68
to the light chain of the plasma protein
tor Va ~Nesheim, M. E., et al., J. Biol. Chem.,
254:10952 (1979)]. On the basis of these
considerations, it is thought that EPR-l represents a
cell surface molecule homologous to the plasma
coagulation protein V maintaining some conserved
immunoreactive epitopes functionally associated with
ligand recognition.
Whether the expression of EPR-l on various
leukocyte populations implies its involvement in
specific immune effector functions is presently not
known. However, the observation that NX cells and
CD8~ T cells express a high affinity serine protease
receptor is provocative in view of the identification
of a family of closely related serine proteases
(granzymes~ contained in the granules of human and
mouse NK and CTL clones tMasson, D., et al., Cell, ~
49:679 (1987)]. These enzymes share significant ~-
homology with a number of serine proteases, -~
particularly with the coagulation proteases factor
IXa, Xa, and plasmin rJenne, D., et al., Proc. Natl.
Acad. Sci. USA, 85:4814 (1988); Gershenfeld, H. K., et
al., Science, 232:854 (1986); Jenne, D., et al., J.
Immunol., 140:318 (1988); Lobe, C. G., et al.,
Science, 232:858 (1986); Gershenfeld, H. K., et al.,
Proc. Natl. Acad. Sci. USA, 85:1184 (1988)~. It is -
also noteworthy that dynamic modulation of gene
expression and secretion of the granzymes is increased
by the same stimuli that are associated with increased
EPR-1 expression n vitro, i.eO, long term response to
antigen and IL-2 tManyak, C. L., et al., J. Immunol.,
142:3707 (1989); Masson, D., et al., EMBO J., 4:2533
tl985)]. Although the role of the cellular granzymes
in NK or CTL killing remains to be elucidated
tDennert, G., et al., Proc. Natl. Acad. Sci. USA,
WO 92/16558 ~ ~ 9PF~ /US92/02109
69
84:5004 (1987)], a putative role for serine proteases
in the lytic process has been suggested by experiments
using serine proteases inhibitors [Redelman, D., et
al., J Immunol., 124:870 (1980); Chang, T. W., et
al., J. Immunol., 124:1028 (lg80); Suffys, P~, et al.,
Eur. J. Biochem., 178:257 (1988); Scuderi, P., J. `
Immunol., 143:168 (1989)].
By analogy with the general concept of receptor-
mediated amplification of proteolytic activities
tMiles, L. A., et al., Fibrinolysis, 2:61 (1988);
Morrissey, et al., Cell, 50:129 (1987); Nesheim, M.
E., et al., J. Biol. Chem., 254:10952 (1979)~, it is
thought that locally released granzymes might interact
with a membrane component on the effector cell to
deliver optimal catalytic efficiency, protected from
neutralization by circulating protease inhibitors. In
this context, EPR-1 would embody the requirements for
a surface receptor expressed by immune effector cells,
displaying ligand recognition for a prototypical and
highly conserved serine protease such as factor Xa,
and dynamically up-regulated by antigenic stimulation.
In conclusion, by using an uncommon strategy for
mAb selection, a new leukocyte marker, a serine
protease receptor, and an apparent cell-surface
homologue of the plasma coagulation protein V have
been identified. Because of its remarkable
distribution on immune effector cells, the name "EPR-
1" is proposed to tentatively identify this molecule.
Although the role of EPR-l in the mechanism of cell-
mediated formation of fibrin is highlighted by its
recognition for Xa fNesheim, M. E., et al., J. Biol.
Chem., 254:10952 (1979)~, it is thought that the wide
spectrum of biologic activities mediated by serine
proteases implicates the involvement of EPR-1 in
additional ligand(s) recognition and cell-mediated
WOg2/16558 PCT/US92/02109
~ ~Qs9 93 ` 70 :;~
functions. :
While the present invention is described in some
detail by way of illustration and example for purposes
of clarity, certain obvious modifications can be
S practiced within the scope of the appended claims.
