Note: Descriptions are shown in the official language in which they were submitted.
.:
i WO91~1~85 PCT/US91/00~0
CYTOTOXIC CELL-SPECIFIC PROTEASE-REL~TED
MOLECULES AND METHODS
Backqround of the Invention
This is a continuation-in-part of copending
application serial number 002,960 filed on January 13, 1987.
This invention relates to protease inhibitors.
Thymus derived (T) lymphocytes play a major role in
the immune system. Maturation of the T cell lineage
involves three distinct stages: (a) generation of a T cell
precursor from a pluripotent stem cell, (b) differentiation
in the thymus, and (c) migration of mature cells to the
peripheral tissues. Maturation of T cells within the thymus
is antigen independent. ~owever, once they have left the
thymus, upon interaction with an antigen they are driven
through the final steps of differentiation to become mature
cells. These final steps are complex and involve
interactions with other cells and soluble effector
molecules.
Several subsets of T cells have been recognized
among activated peripheral T cells. There are three main
classes: helper, suppressor, and cytotoxic. Helper T
lymphocytes potentiate immune responses (both humoral and
cell-mediated) either by cell-cell contact or by synthesis
and secretion of factors. These factors, although
synthesized in response to an antigen-specific signal, can
be either antigen-specific or antigen-nonspecific.
Suppressor T lymphocytes, inhibit the functions of other
lymphocytes, again either directly or via soluble factors.
cytotoxic T lymphocytes are the effector cells in cell
mediated immune reactions. They specifically recognize
foreign antigens on the surface of cells, bind to them, and
cause the target cell to lyse. Cytotoxic T lymphocytes are
.: .
,
2Q~8~
WO91/1~85 PCT/US91/00
known to cause or exacerbate autoimmune diseases such as
rheumatoid arthritis, and are also involved in allograft
rejection and graft-versus-host disease.
The various steps in the process of cytotoxic T
lymphocyte induced lysis have been analyzed in some detail,
e.g., Berke, (1983) Immunol. Rev. 72:5; Nabholz & MacDonald,
(1983) Ann. Rev. Immunol. 1:273. Recent studies by Padack &
Konigsberg, (1984) J. Exp. Med. 160:695 and Henkart et al.,
(1984) J. Exp. Med. l~Q:75 have suggested that the dense
cytoplasmic granules seen in CTL and natural killér cells
-~ are directly involved in target cell lysis by a mechanism
involving transmembrane channels.
A general description of cytotoxic T lymphocytes,
natural killer cells, and killer (K) cells is contained in
;; 15 Stites et al., Basic & Clinical Immunology 227-31 (Lange
Medical Publications, Los Altos, Ca., 1984).
Su~marv of the Invention
In general, the invention features a vector
containing a DNA sequence encoding the CCP1 protein.
In another aspect the invention features a vector
containing a DNA sequence encoding the CCP2 protein.
In another aspect the invention features a vector
containing a DNA sequence encoding the hCCP1 protein.
In another aspect the invention features a vector
containing a DNA seguence encoding the hCCPX protein.
In another aspect the invention features
substantially pure CCPl protein expressed from a vector
containing a D~A sequence encoding the CCPl protein.
Substantially pure means a preparation with a purity of 95
or greater by weight, and free of the proteins, lipids, and
carbohydrates with which the protein is-naturally
associated.
' .:
;: ' - - . . . . , ~
: . . : -- ~ - .
.. . . . .
:: , , ~ . . :~, ,
~ WO91/10685 2 b 7 ~ ~ 8 ~ PCT/US91/00~0
In another aspect the invention features
substantially pure CCP2 protein expressed from a vector
containing a DNA sequence encoding the CCP2 protein.
In another aspect the inve~tion features
S substantially pure hCCP1 protein expressed from a vector
containing a DNA sequence encoding the hCCP1 protein.
In another aspect the invention features
substantially pure hCCPX protein expressed from a vector
containing a DNA sequence encoding the hCCPX protein.
In another aspect, the invention features a peptide
of the formula: Asp-Val-Asp-Ala; Ala-Pro-Asp-Ala; Ala-Asn-
Pro-Ala; Phe-Pro-Arg-Phe; Ala-Pro-Arg-Phe; Phe-Pro-Asp-Phe;
Phe-Pro-Asn-Phe; Phe-Asn-Pro-Phe; or Phe-Asp-Pro-Phe.
The term competitive inhibition, as used herein,
refers to inhibition in which the inhibitor combines with
the free protease such that it competes with the normal
substrate of the protease. Competitive inhibition is
described, e.g., in Lehninger, Biochemistry 197-200 ~Worth,
2d ed. 1975).
The term protease, as used herein, refers to an
enzyme that hydrolyzes, and thus cleaves, peptide bonds.
Cytotoxic lymphocytes, e.g. cytotoxic T lymphocytes
(sometimes called T killer cells) and natural killer cells
are described in Jandl, Blood: Textbook of Hematology
(Little, Brown and Co., Boston, 1987) hereby incorporated by
reference.
The term serine protease, as used herein, refers to
a protease which has a serine residue at the active site of
the enzyDe.
The term peptide, as used herein, includes proteins
as well as peptides too short to be characterized as
proteins. Generally those peptides having a molecular
weight of greater than 5,000 are characteriZed as proteins.
.
WO91/1~5 2 0 7 4~0 ~-~ P~T/USgl/~O ~
4 --
The term cytotoxic cell protease, as used herein,
refers to any protease, preferably a serine protease, that
has 30% or greater homology, more preferably 50% or greater
homology, with the protein encoding sequence of the murine
C11 gene, and which cleaves at different sites than does
; plasmin. Preferably the cytotoxic cell protease is
expressed by cytotoxic lymphocytes, more preferably
exclusively by cytotoxic lymphocytes.
Cytotoxic lymphocytes produce, as part of their
cytotoxic activity, proteases, some of which, we have
discovered, cleave proteins at sites different from the
sites cleaved by proteases such as plasmin produced by other
cells of the body. These proteases are members of the
cytotoxic cell protease family. The inhibitory molecules of
the invention, since they mimic the unique cleavage sites
recognized by cytotoxic cell proteases, can exclusively
inhibit cytotoxic cell proteases e.g., those produced by
cytotoxic lymphocytes. Thus a person suffering from an
i~mune disorder, or experiencing allograft rejection, c:an be
administered a molecule of the invention to inhibit the
cytotoxic lymphocytes involved in the disease or rejection
process, and the administered molecule will not interfere
with, for example, lysis of blood clots, or other normal
protease-dependent functions.
Other features and advantages of the invention will
be apparent from the following description of the preferred
embodiments and from the claims.
Description of the Preferred Embodiment
The structure, synthesis, and use of the preferred
embodiments are discussed next, after the drawings are
briefly described.
' . ' . ' ,, , ~ . : . ' ~ . . . , : .
:,'. " ',, , '.-~. ~ ,' ~ ' . ' ',, ' ' ' "' ,' ` ',: ' ' . ':
... . . ~ . .
,", ' ' ' ' " ' ': '' ' " ;., , ' ' ' ' ' ' '`
~ 1~ 2 ~ 7 ~
WOgl/10685 PCT/US91/~ ~0
Drawinas
Fig. 1 is a graph showing the correlation of a
protease mRNA expression () with cell activation in a mixed
lymphocyte culture.
Fig. 2 is a partial nucleotide sequence comparison
of two protease-encoding cDNA's.
Fig. 3 is the nucleotide sequence of one of said
cDNA's and the predicted protein structure it encodes.
Fig. 4 is a partial amino acid sequence comparison
of five serine proteases.
Fig. 5 is the sequence of CCP2.
Fig. 6 is the sequence of hCll, the human analog of
the murine C11 gene.
Fig. 7 is a restriction map of the hCCPX gene.
Fig. 8 is the nucleotide sequence of the hCCPX gene.
Fig. 9 is the predicted cDNA sequence encoded by the
hCCPX gene.
Fig. 10 is the amino acid sequence of proteins
encoded by hCCPX and the CCP genes.
Fig. 11 is the amino acid sequence of some protease
inhibitors of the invention.
Table 1 shows the expression of Cll mRNA in
infiltrating cells of tissue grafts.
Table 2 shows the degree of homology between CCPl
and various proteins.
Table 3 shows the effect of peptides of the
invention on the cytotoxicity of cells from a cyclosporine-
A mixed lymphocyte reaction.
Table 4 shows the effect of peptides of the
invention on the cytotoxicity of cytotoxic T-cells activated
with ConA and interleukin 2.
The Appendix is a copy of Murphy et al. (1988)
Proteins: Structure, Function, and Genetics 4:190-204 which
i
- . ~ - i
.
,, '' : : ' ~
.
, . .
-
207~33~
WO91/10685 PCTIUS~1/00~0
provides a detailed example ~f computer aided analysis of
enzyme and substrate structure.
Structure
The inhibitory molecules of the invention
competitively inhibit the activity of cytotoxic cell
proteases produced e.g. by cytotoxic lymphocytes, while not
inhibiting the activity of proteases produced by other cell
types or any other proteases produced by the cells producing
the cyto`toxic cell proteases. Preferably the inhibitory
molecules are peptides.
Cytotoxic lymphocytes synthesize a characteristic
set of cytotoxicity-related proteases which are expressed at
much reduced levels, if at all, in other subsets of
lymphocytes. The cytotoxicity- related proteases can be
divided into two groups, effector proteases and non-effector
proteases. Effector proteases are released by a cytotoxic
lymphocyte when it comes in contact with a target cell, and
break down proteins in the membrane of the target cell or
enter the target cell and hydrolyze intracellular proteins,
leading to the cell's destruction. Non-effector proteases
are involved in the enzymatic processes that lead to the
production and/or release of the effector proteases (or
other effector molecules) from the lymphocyte. Inhibiting
the action of either an effector protease or a non-effector
protease inhibits the ability of cytotoxic lymphocytes to
destroy a target cell.
The preferred peptides contain the two amino acids
that constitute the cleavage site recognized by the
protease, and have between 3 and 20 (more preferably between -
3 and S) amino acids residues. Shorter peptides are
preferred because they are, in general, readily taken up
cells. The peptides should not contain a cleavage site
recognized ~y other proteases, for example, those sites
.. . . . . . .
'"'-'`` 20~b8l';
~^ WO91t10685 PCT/US91/00~0
described by Zreighton, Proteins: Structure and Molecular
Properties 336-37, 427-38 (W.H. Friedman, N.Y., 1983).
Described in Example l below is the isolation,
cloning, and characterization of two genes expressed
5 exclusively in the cytotoxic T lymphocytes of mice.
(Exclusively means that either the genes are not expressed,
or are only expressed in very low (less than 5 molecules of
mRNA per cell) levels, in other types of cells in the
organism). Example 2 describes the sequencing of the two
lO genes, the determination of the amino acid sequencé of the
protease which one of the genes encodes, and the
characterization of the protease. Example 3 describes the
identification and isolation of a human gene (hCII)
encoding a cytotoxic cell protease (hCCPl) produced
15 exclusively by human cytotoxic T lymphocytes. Example 4
describes the isolation, cloning, and characterization of a
gene encoding another human cytotoxic cell protease, human
cytotoxic cell protease X (hCCPX). Example 5 describes the
sequencing of the hCCPX gene, the determination of the amino
20 acid sequence of the hCCPX protease, and the
characterization of the protease. Example 6 describes the
determination of three dimensional structure of a cytotoxic
; cell protease and the structure of a peptide that can act as
a competitive inhibitor of that protease. Example 7
25 describes several inhibitors of the invention. Example 8
~ describes the production of substantially pure proteases and
f their use in the design of inhibitors.
Example l
Cells - The cytotoxic T-cell lines MTL2.8.2 and
30 MTLll.l were generated from CBA/J mice as described by
; Bleackley et al., (1982) J. Immunol. 128:758. EL4.El is an
interleukin 2 (IL-2)-producing variant of the EL4 cell line
described by Farr et al., (1980) J. Immunol. 125:2555. CHl
.
': , ~ ' ' '
. /
2i~7~81
WO91/1~85 PCT/US91/00~0
-- 8 --
is a CBA/J ~ CBA/J X BALB/c antigen- specific helper T-cell
line. It was produced from a 2-day mixed lymphocyte ~ulture
by continuous restimulation with irradiated Fl spleen cells
in RPMI 1640 medium supplemented with 10% fetal bovine serum
and 100 ~M 2-mercaptoethanol (RHFM). To generate human
cytotoxic T lymphocytes (CTL), peripheral blood lymphocytes
were incubated in RHFM and stimulated with irradiated
allogeneic cells at days 0 and 7 and harvested at day 10.
The fetal-derived cells used are described by Teh et al.,
10 (1985) J. Immunol. 135:1582. For the time course of cell
activation, spleen cells from CBA/J mice were incubated in
RHFM (106 cells per ml) and purified IL-2 (described by
Riendeau et al., (1~8~) J. Biol. Chem. 25~:12114), either
with an equal number of mitomycin C-treated EL4.El cells or
Con A (2 ~qtml). Samples were removed at day 1 through day
6, assayed for cytotoxic activity by the procedure described
in Shaw et al., (1978) J. Immunol. l~Q:1974, and analyzed by
cytodot hybridization.
cDNA Library Construction - Double-stranded cDNA was
synthesized from 4 ~g of MTL.2.8.2 mRNA as described by
Gubler and Hoffman, (1983) Gene 25:263. Following repair
with the Klenow fragment of DNA polymerase and T4 DNA
polymerase to maximize flush ends, phosphorylated ~_RI
linkers (P-L Biochemicals) were ligated to the cDNA in 70 mM
Tris-HCl, pH 7.6/10 mM MgC12/5 m~ dithiothreitol/l mM ATP/1
unit of ~4 DNA ligase at 14C overnight (Goodman &
MacDonald, (1979) Methods Enzymol. 68:75). After digestion
with EcoRI, the product was run on a 5-ml Sepharose 4B
column, and the excluded fractions were pooled and ethanol-
precipitated. The cDNA was ligated to FcoRI/bacterialalkaline phosphatase-treated pUC13 (P-L 8iochemicals) in 66
mM Tris-HCl, pH 7.6/6.6 mM MgC12tlO mM dithiothreitol/1 mM
ATP. Reactions were heated to 37C for 5 min, quick-chilled
, , : : .
20~.~0:g~
`~ WO91/1~85 PCT/US91/~ ~0
_ g _
before the addition of 1 unit of T4 DNA ligase, and
incubated at 14C for 2 hr. Escherichia ~li JM83 cells were
made competent by using the CaC12/RbCl procedure described
by Maniatis et al. in Molecular Cloning: A Laboratory
S Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y., 1982) and were transformed with the ligated cDNA.
White colonies (those containing inserts) were ordered in
96-well microtiter plates and stored in LB medium containing
20% glycerol at -70C.
Differential Screening - Colonies were replicated in
triplicate onto nitrocellulose filters, grown for 6 hr, and
then amplified on chloramphenicol (100 ~g/ml) for 12 hr.
Bacteria were lysed, and the filters were prewashed to
remove bacterial debris, as described by Maniatis, supra.
Prehybridization at 42C for 12-20 hr was done in 50%
(vol/vol) formamide containing 2x Denhardt's solution (lx
Denhardt's solution = 0.02% polyvinylpyrrolidone/0.02%
Ficoll/0.02% bovine serum albumin), 4x SET buffer (lx SET
buffer = 0.6 M NaCl/0.12 M Tris-HCl, pH 8/1 mM EDTA), 0.1%
NaDodS04, 100 ~g of yeast tRNA per ml, and 125 ~g of poly(A)
per ml (Sigma). Hybridization in the same buffer included
1-5 x 105 cpm of cDNA probe per m} synthesized from mRNA
with 20 ~g of T-primers per ml (Collaborative Research,
Waltham, MA); 50 mM Tris-HCl (pH 8.3); 10 mM MgC12; 5 mM
dithiothreitol, 500 ~M each of dGTP, dATP, and dTTP; 70 mM
KCl; 30 ~Ci (1 Ci = 37 GBq) of ~-32P~dCTP (New England
Nuclear, 300 Ci/mmol); and 15 units of avian myeloblastosis
virus reverse transcriptase at 42C for 60 min. Template
RNA was hydrolyzed by the addition of NaOH to 1.5 M.
Samples were boiled for 3 min and fractionated by Sephadex
G-50 column chromatography. Filters were washed in 5x SET
buffer for 15 min at 22C and then in 2x SET buffer/50%
formamide for 20 min at 42C and were exposed to film (Kodak
. ., ",: .
.
..
. - .
3~ ~
WO 91tlO~5 PCT/US91/00~0
-- 10 -- ::
X-Omat AR) with an intensifying screen for 1 to 3 days at -
70C. Hybridized probe was removed by boiling the filters
for 10 min in distilled water.
Blot Analysis - Cytodots were prepared as described
by White and Bancroft (1982) J. Biol. Chem. 257:8569. For
blot-hybridization analysis, total cytoplasmic RNA (10 ~g)
or poly(A)+ mRNA (2 ~g) was denatured in 6.3%
formaldehyde/50% formamide at 55C and size fractionated on
a O.8~ agarose gel containing O.66% formaldehyde. RNA was
transferred to nitrocellulose as described by Thomas (1980)
Proc. Natl. Acad. Sci. USA 77:5201. Plasmid DNA was
digested with EcoRI, run on a 0.7% agarose gel, and
transferred to nitrocellulose, as described by Southern
(1975) J. Mol. Biol. 26:365. Filters were baked at 80C for
2 hr, then prehybridized at 42C for 6-12 hr in 50%
formamide containing 20 mM phosphate buffer (pH 6.8), 2 mM
pyrophosphate, 100 ~M ATP, 5x Denhardt's solution, 0.75 M
NaCl, 0.075 M sodium citrate (pH 7), 100 ~g of salmon sperm
DNA per ml, 0.1% NaDodS04, 50 ~g of poly(A) per ml, and 2.5
mM EDTA. Hybridization was carried out in the same buffer
with a nick-translated plasmid of specific activity 1 x 108
cpm/~g (Bethesda Research Laboratories kit) at 1 x 106
cpm/ml.
Results - Triplicate copies of the library were
hybridized first with cDNA synthesized from MTL2.8.2 mRNA,
then, after autoradiography and washing, with helper T-cell
cDNA, and finally with thymocyte cDNA. Colonies that gave a
higher hybridization signal with killer cell mRNA in at
least two of the three copies of the library were picked.
Upon rescreening, again in triplicate, 36 of these 121
colonies appeared to be clearly CTL-specific. Plasmid DNA
isolated from these colonies was cut with EcoRI, and a
series of cross-hybridizations was performed. Two clones
.
; ~
~ WO91/10685 2 0 ~ ~ ~ 8 ~ PCT/USg1/~340
- 11 - . -
were chosen for more extensive analysis: clone BlO because
it appeared to be the most abundant in the library, cross-
hybridizing strongly with eight other inserts, and clone Cll
because it weakly cross-hybridized with BlO but not with all
BlO-related clones (one other Cll-related sequence was
found).
Cytodots prepared from a variety of cells and
tissues were hybridized with nick-translated BlO and Cll.
The number of cells per dot was 104. The data with probe
Cll are similar and are not discussed. The highest signal
was detected in MTL2.8.2--i.e., the killer cell line that
was used to generate the cDNA library. A weaker but
positive signal was observed with MTL-III, a variant of
MTL2.8.2 that had a low level of cytotoxicity and had
become IL-2 and antigen independent. A similar level of
expression was observed in a novel T-cell clone derived from
murine fetal thymus of Teh, supra. In all over 20 cytotoxic
T cell lines and cultures have been tested and all have been
positive for BlO and Cll expression.
Natural killer (NK) and T killer (T~) cells were
purified, cultured, and tested for the expression of Cll
mRNA by the methods described in Manyak et al. (1989)
J. Immunol. 142:3707-3713. Culturing NK cells in IL-2
induced: i) lytic activity, ii) chymase and tryptase
enzymatic activities and iii) the total ~RNA levels of the
Cll gene in a dose-dependent manner. Cll ~RNA reached peak
activity on days 5 to 7 of culture. Similar resu~ts were
seen with TK cells.
There was no evidence for expression of BlO or Cll
in either mouse thymocytes or a helper T-cell line (CHl)
that secretes IL-~ in response to antigen. Mouse brain,
mouse liver, and a human CTL line were similarly negative
under the high-stringency conditions of this experiment. In
.
WO91/10~85 2 $ 7 ~ ~ 8 ~ pcr/us91~oo34o ~
addition, no evidence for expression of BlO or Cll was found
in a helper T-cell hybridoma that secretes an antigen-
specific factor (Kwong et al. (1984)) J. Immunol. 133:653.
To ensure that the negative samples did contain hybridizable
RNA, all of the cytodots were reprobed with either a
lymphocyte-specific probe or oligo(dT) or the T-cell antigen
receptor ~-chain gene (Hendrick et al. (1984) Nature
308:153). Although the level of signal varied, all samples
were positive.
To enrich for the B cells of a spleen cell
suspension, lymphocytes were separated from adherent cells
on Petri dishes and then treated with anti-Thy-l.2
antiserum. The enriched B cells were then incubated with
lipopolysaccharide (LPS) or Con A or RHFM medium. After 24
hr, the cells were harvested, cytodots were prepared and the
filter was probed with BlO or Cll. No expression of either
sequence could be detected in any sample. However, when the
blot was hybridized with an immunoglobulin ~ heavy chain
probe (Calame et al. (1980) Nature 284:452) a strong
positive signal was seen in the LPS-stimulated cells.
Poly(A) RNA was isolated from a variety of cell
sources, run on a denaturing agarose gel, and transferred to
nitrocellulose. The same filter was probed first with nick-
translated BlO, then with Cll, and finally with probe lO, a
cloned gene that detects mRNA in a variety of cell types
(Paetkau et al., in Contemporary Topics in Molecular Biology
10:35 (S. Gillis ed., Plenum, N.Y., 1984)). Probe BlO
detected a single band (approximately 900 bases) in two
different murine cytotoxic T cell clones, MTL2.8.2 and
MTLll.l. No bands were detected in RNA from thymocytes, an
antigen-specific helper cell line, or murine thymoma EL4. -
When the blot was reprobed with Cll, again only the two
cytotoxic T cell clones showed bands. However, in contrast
. ; . , :, .
"
. ' ' '.,. .~
' ' '.':.
2 ~ 8 ~
W091/1~85 PCT/US91/00~0
to BlO, this probe hybridized to two bands, one of
approximately 900 bases and the other of 1200 bases. Probe
lO detected a band in all cell samples. In addition, blot-
hybridization analysis was performed on poly(A)~ RNA from a
number of murine cells including eleven CTL lines, two
helper lymphocyte lines, brain cells, liver cells, three
helper T-cell lines, unstimulated and LPS-stimulated B
lymphocytes, and one ~-cell myeloma. Of these, only the
actively cytotoxic T cells expressed mRNAs that hybridized
with BlO and Cll. To ensure that all tracks contained
hybridizable RNA, the blot was rehybridized with probe lO.
A band of the expected size was seen in all tracks.
The results from the cytodots and blot-hybridization
analysis indicates that both BlO and Cll are murine
cytotoxic T lymphocyte specific.
CBA/J (H-2k) spleen cells were stimulated with
either mitomycin C-treated EL4 cells (Fig. lA) or Con A
(Fig. lB). On each of the 6 days after stimulation, the
level of cytotoxicity was measured in a chromium-release
assay against EL4 (H-2b) (~ , S194(H-2d) (~), and RI(~=~k)
(O) cell lines. Cytodots were also prepared on each of
these days, and the blots were hybridized with nick-
translated BlO and Cll. Data are presented only for BlO, as
Cll gave indistinguishable results. Relative BlO mRNA
levels () were determined by scanning densitometry on an
ELISA plate reader. In the allo-specific response
(Fig. lA), the peak of cytotoxicity was observed on day 4,
while the peak of BlO or Cll mRNA expression appeared to be
on days 3 and 4. The peak of killing activity in the Con A-
stimulated cells (Fig. lB) was also at day 4; however, thepeak of mRNA expression was very sharply on day 3. In both
experiments, the mRNA expression was reduced to background
levels by day 6, while there were still significant levels
... . . . . .. .
.. . . .
- -
.:: . .
, ~ ,: :
. ' ' ' :' ~ '
2~7~08~`:
WO91/10685 PCT/US91/0
- 14 -
of cytotoxicity on this day. When the cytodots were
hybridized with 32P-end- labelled oligo(dT), the peak of
total mRNA was seen on day 2. (data not shown)
The experimental results illustrated in Fig. 1
indicates that the maximum expression of B10 and Cll mRNAs
precedes the peak of cytotoxicity in'an n vlvo allogenic or
mitogen-induced cytotoxic response by 24hrs; thus, they both
fulfill the primary prerequisite for genes encoding proteins
that are important in the lytic process.
In situ hybridization experi~ents indicate that a
high proportion of T lymphocytes that infiltrate
incompatible heart allografts in vivo express the Cll gene.
Complete details of the in situ hybridization procedure, and
all related techniques, are found in Mueller et al., (1988)
15 J. Exp. Med. 167:1124-1136,
Transplantations in these experiments were performed
as described in Mueller et al. (1988) J. Exp.
Med. 167:1124-1136 and Billingham et al. (1977)
Transplantation 23:171. In short, the myocardium of newborn
20 (12-36 h) BALB/cJ (H-2d) donor mice were diced into 0.1-
0.2-cm fragments and subsequently transplanted under the
kidney capsule of adult (6-8 wk) sex-matched C57 Bl/Ka
recipients (H-2b; experimental animals). As a control,
adult BALB/cJ (H-2d) mice received grafts from the same
donor animals under the kidney capsule. On days 2, 4, 6, 8,
10, and 12 after transplantation, three experimental and two
control animals were killed and 5-~m frozen sections through
the graft were prepared. Labelled probe for in situ
hybridization was prepared as described in Mueller et
al. (1988) J. Exp. Med. 167:1124-1136 and as follows.
A 1.1-kb fragment of the C11 gene was subcloned into
the polylinker of the transcription vector pSPT 672 using
standard techniques. This vector ha a SP6 and a T7 promotor
: . . . .
- : ,
: . . - . . .
:. . . ..
-'
~..
~ . . . ' ' - , . ~ ' ,
:' ' ..
~ WO9l/1~5 2 ~ 7 ~ 0 8 ~ PCT/US91/oo~o
- 15 -
at the 5' and 3' end of the multicloning site,
respectively. After linearization of the vector with an
appropriate restriction enzyme, sense and antisense probes
were prepared using SP6-polymerase and T7-polymerase (both
from New England Biolab, Beverly, MA) reactions and (S-35)
UTP No. SJ 1303, Amersham Corp., Arlington Heights, IL) at a
final concentration of 12 ~M. The labelled nucleotide was
dried down before adding the other reagents of the reaction
mixture. A typical reaction (35 ~l) contained 7 ~l 5X SP6
buffer (final concentration; 40mM Tris-HCl, pH 7.9, 5mM
MgCl2; 2 mM spermidine); 3.5 ~l 100 mM dithiothreitol (DTT);
3.5 ~l ribonucleotides (CTP, ATP, and GTP; 10 mM each, in lo
mM Hepes, pH 7.4); 3.5 ~l bovine serum albumin (BSA), 5
mg/ml; 1 ~l Rnasin, 40 U/~l (New England Biolab); 1 ~l
linearized DNA template, 1 ~g/~l; 13.5 ~l H20. SP6 and T7
reactions were incubated for 90 min at 40C and 37C,
respectively. DNA template was digested with DNase I (2U/~g
DNA; Worthington) for 15 min at 37C. The RNA probe was
subsequently extracted with phenol/chloroform, separated on
a Bio-Gel P-60 spin column, and ethanol precipitated after
adding 7.5 ~g of yeast tRNA per lo6 cpm-labelled probe. The
probe was subsequently resuspended at 2 X 109 cpm/~l in
Tris-EDTA (TE), boiled for 2 min. and stored frozen at -
70C. For the hybridization, this probe was mixed with
formamide (final concentration 50%), dextran sulfate (10%),
DTT (lOOmM), NaCl (300mM), Tris-HCl, pH 7.5 (20mM), EDTA
(5mM) Denhardt's solution (lX) at a concentration of 2 X lo6
cpm/~l hybridization solution.
In situ hybridizations were performed according to
Angerer et al. (1987) In In Situ hybridization:
Applications to the CNS, K. Valentino, J. Eberwine, and
J. 8archus, eds. Oxford University Press, New York pp. 42-
; 70 as modified in Mueller et al. (1988) J. Exp. Med.
.
. ,.=~., . - - -- . .
.
.,
: . , ,. . . . , - .,- :.: . . -
.. ~. . . , , . ~ . . .. .. ..
WOsl/1~5 PCT/US91/00~0 ~
2 0~
- 16 -
167:1124-1136. 5-~m-thick cryostat sections were placed on
poly-L-lysine (Sigma Chemical Co.)-coated glass slides and
fixed in 4% paraformaldehyde dissolved in lX phosphate
buffered saline (PBS) for 20 min., rinsed in PBS, and
dehydrated through graded ethanol. Slides were stored at
this stage at 4C before being used for in situ
hybridization. In situ hybridizations on different cell
populations were done on sorted cells that were spun onto
poly-L-lysine-coated glass slides with a Shandon
cytocentrifuge. These cytospin preparations were fixed and
hybridized as described for cryostat sections. The fixed
sections or cytospin preparations were treated with
proteinase K (Boehringer Mannheim, Federal Republic of
Germany), 1 ~g/ml in lOo mM Tris-HCl, pH 8.0, and S0 mM EDTA
15 at 37C for 30 min. The slides were postfixed again with 4%
paraformaldehyde for 20 min. Free amino groups on tissue
sections were acetylated by treatment with 0.25% acetic
anhydride in 0.1 M triethanolamine for 10 min. Eor the
hybridization step, 10 ~l of the hybridization solution
20 (described above) containing 106 cpm S-35 UTP-labelled RNA
probe were placed on each section, covered with a
siliconized coverslip (18 x 18 mm), and sealed with rubber
cement. The sections were hybridized at 46C for 16-18 h.
Thereafter, the slides were washed in a solution containing
25 50~ formamide, 2x SSC ~SSC = 0.15M sodium chloride, 0.3M
sodium citrate at pH 7), 20 mM Tris at pH 7.5, and 5 mM EDTA
in four changes for a total of 2 h at 56C. After the first
wash a digestion step with RNase A (20 ~g/ml) and RNase (1
U/ml) (both obtained from Sigma Chemical Co.) for 30 min at
37C was included. The slides were dipped into NTB-2
nuclear track emulsion ~Eastman Kodak, Rochester, NY), 1:2
diluted with 600 mM ammonium acetate, and exposed at 4C for
8 days. The slides were developed with Kodak developer D-
.~,., . - : .
'', . :~ , . ,
'.
,.. ,. ~ ' , . .
': ' .. ' ' ' '
' ~: . , ' - . , ' ' ' . . .
, - ' '.. '
' : ' ' ' , ' ,
2~08~
WO91/t~5 PCT/US91/~ ~0
19 for 2.5 min and fixed with Kodak fixer for 5 min.
Counterstaining was done with 4% Giemsa stain (Fisher
Scientific Co., Orangeburg, NY) for 10-15 min. From each
animal, two sections were each hybridized with a labelled
S Cll antisense probe (complementary sequence to the
cytoplasmic Cll mRNA) and one section was each hybridized
with a labelled C11 sense probe.
The results of in situ hybridizations with Cll-
specific probes demonstrated that the cellular infiltrate in
rejecting allografts contains a high proportion of cells
expressing Cll transcripts. See Table 1 which shows the
frequency of infiltrating cells with detectable levels of
Cll mRNA. Cryostat sections of the graft were hybridized
with radiolabelled RNA antisense probe of the Cll qene. The
results in Table 1 are expressed as the number of positive
cells per unit area (1 mm2) of infiltration area. Three
animals with an allograft and two animals with a syngeneic
graft were examined and two sections of each animal and each
probe were used for evaluation.
The first cells with detectable levels of C11 mRNA
were found on day 2 after transplantation both in animals
with an allogeneic and those with a syngeneic graft. These
positive cells, however, were extremely rare at this
timepoint and were normally not found on every section of
2S the same animal. On day 4 after transplantation, the
experimental animals showed a 5-10-fold higher frequency of
... . ., : , ., . ~, ., :-.. . . ~: . .... ., , - ... . .
2~ LO~ ~
WO91/10685 PCT/US91/00~0
- 18 -
TABLE 1
Frequency of Infiltrating Cells with
Detectable Levels of Cll mRNA
5 Days after Allogeneic graft Syngeneic graft
transplan- -
tation C11 C11
2 3 + 4 4 + 1 -
4 44 + 69 3 + 4
10 6 205 + 84 7 + 4
8 313 + 56 21 + 12
323 + 112 15 + 1
12 350 + 189 3 + 1
C11 cells than the control group with a syngeneic graft.
The frequency of inflammatory cells expressing the gene
increased dramatically between day 4 and 12 after
allotransplantation and was at least eight times higher than
in the control animals during this period.
In one of the control animals, the syngeneic graft
became necrotiG and no viable syngeneic graft cells could be
detected 8 d after transplantation. This animal, which was
; not included in Table 1, had 5-10 times more C11 mRNA+ cells
than other control animals at the sa~e timepoint. However,
2~ compared to the experimental animals 8 days after
transplantation, the frequency of positive cells was still
-50% lower. In the ~irst 4 days after the mice received the
allograft, about equal numbers of Cll+ cells were found
among the infiltrating cells.
The amount of Cll specific ~RNA per cell, as
measured as the number of silver grains over a single cell,
increased steadily during the entire observation period in
- . - . . . . . . ~ .
.. . . .:
: ~
: , . ~ ' ' ~ " ' '
~ WO91/1~85 2 ~ 7 ~ ~ $ 1 PCT/US91/00~0
-- 19 --
experimental animals, indicating that the gene was expressed
over long periods, perhaps after local induction by
alloantigen and/or mediators. In control animals, the
expression level increased only slightly after more than 4
days after transplantation.
The phenotype of the Cll transcript positive cells
was determined as detailed in Mueller et al. (1988)
J. Exp. Med. 167:1124-1136. Briefly, small pieces of the
allograft were digested with collagenase, and the resulting
suspension of the isolated infiltrating T cells wére sorted
on a fluorescence activated cell sorter for subsequent in
situ hybridization. The infiltrating cells of the allograft
and the splenocytes of six animals that received a heart
muscle graft 6 days before were pooled and sorted according
to their phenotype. The frequency of C11-positive cells in
the CD8+ subset was generally 10-20 times higher in the
infiltrate of the allograft than in the spleen of the same
animals. ~he recovery of CD4+ cells from the infiltrate was
always very low and the frequency of positive cells in this
subpopulation was at least 10-fold lower than in the CD8
subset of infiltrating cells; of 84 C11 mRNA + cells
analyzed, 82 were CD8+ (98%) and 2 CD4+ (2%). On cytospin
preparations from sorts cells, C11-transcript positive cells
were mainly found among the blast-like CD8+ cells. In
double stainings of cell suspensions and tissue sections, no
evidence for a significant contribution of CD4 , CD8 or
CD4+, CD8+ T cells among the allograft infiltrating cells
and the Cll transcript positive cells were found~
Example 2
Clones B10 and Cll were sequenced according to the
dideoxy method of Stanger et al. (1980) J. ~ol.
Biol. 143:161. Sequence analysis of B10 and C11 (Fig. 2)
reveals that they are related to each other and that the
- - , . - .: . . ~ : . ~ ,. . .
:.. . - . : . : ; . . . : . .
'- . - ' : . ~ ......................... , ................... : ' ' '
.
æ~
W091/1~5 PCT/US91/00~0
- 20 -
hypothetical proteins they encode contain a short region
characteristic of serine proteases, Asp-Ser-Gly-Gly (a
sequence homologous to that surrounding Ser195 of
chymotrypsin).
With B10 and Cll as probes, another CTL
complementary DNA (cDNA) library was screened, in which
inserts greater than 1000 base pairs were cloned in AgtlO.
Forty thousand recombinants were screened and 39 plaques
corresponding to C11 were isolated.
A cDNA insert of 1400 base pairs, which hybridized
with C11, was selected for sequence analysis. The predicted
protein sequence encoded, of molecular weight 25,319, is
shown in Fig. 3. The putative start codon is preceded by a
potential ribosome binding site CCW CCG (Hagenbuchle et
al. (1978) Cell 13:551) and a polyadenylation signal
sequence AAUAAA (Proudfoot & Brownlee, (1966) Nature
263:211) occurs just upstream from the poly(A) tract. Of
the first 12 amino acids predicted, ten are hydrophobicl and
the amino acid in position 2 (Lys) is basic, suggesting that
this sequence may act as a signal to direct secretion or
intracellular organelle location. A search of the National
Biomedical Research Foundation (NBRF) protein sequence data
bank revealed that the protein encoded by Cll resembles a
number of serine proteases (Table 2).
Table 2
_ __ _ _ _
p~ ;" EC Sp~ Rc~u~ a~ed p~
~ Ca~l hn~ p~on ho~noloQ
7S no~e ~h ~or 3.4.21 Munne 29 22 ~ 26-229 ~o
amr~ ,~3.',21.1 Ib~ine 1 200 16 216 35
a-_ B3.~21.1 Ib~inc 1200 16-216 36
Coi ~,d~ Clr 3.~,.21.~1 H~nun 52-22~. 56 238 3s
_i-e 3A21.11 Po~inc 3-220 3-233 33
3.~.2~ -225 192- 2~ 33
I~MCr~ 3.~21 R-t 121~. 1213 51
~in 3.~21~ R-c 26-225 S1 262 36
1~ 3.~21.~ n 3 22~ 563 ~7 3
CD~ 3.~2131 Hum-n 72 22~ 339 560 3s
~pin3.~ 21.'. S.~niw 19 220 22-al~, 33
S 3.'.. 21.' R~29 226 31 223 39 ,
:
':
.
.
~ WO91~1~85 2 ~ ~ I 0 81 PCT/US91/00340
- 21 -
When the sequences were optimally aligned according to the
Dayhoff algorithm (Dayhoff, in Atlas of Protein Sequencing
and Structure 5:l (Supp. 3) (National Biomedical
Res. Found., Washington, D.C., 1979)), the homologies
generally varied between 30 and 40 percent. The greatest
homology was found with rat mast cell protease type II
(RMCPII), which had amino acids identical to lO9 of 215
amino acids encoded by Cll, giving a match per length of 51
percent. The amino acid residues known to form the
catalytic triad of the active site in serine proteases
~His57, Aspl02, and Serl95) were all found in the protein
encoded by Cll (Fig. 3, ~). The sequences around these
residues, which are highly conserved among serine proteases,
are also conserved in the Cll gene product. Indeed, largely
because of conservation around this region, the protein
encoded by Cll appears to be somewhat homologous (about 30
percent of 209 residues) even to the prokaryotic proteases
trypsin and type B from Stre~tomvces ariseus.
The cytotoxic T lymphocyte-specific proteins (CCP"3)
encoded by Cll and BlO will be referred to as CCPl and CCP2,
respectively. In Fig. 4 the optimal protein alignment with
CCPl is presented for RMCPII, bovine chymotrypsin, bovine
trypsin, and CCP2 (not numbered, as the full sequence is not
presented). The full sequence of CCP2 can be obtained by
application of the procedures applied to Cll and CCPl. The
full sequence of CCP2 is presented in Fig. 5.
RMCPII is an intracellular serine protease found in
the granules of atypical mast cells. The high level of
homology of CCPl with RMCPII is particularly intriguing as
RMCPII has a num~er of structural features that make it
exceptional in the serine protease superfamily. Protein
CCPl contains cysteines in precisely the same positions as
RMCPII which, by analogy with RMCPII, form three disulfide
~- . .: . ~ . : ,
.. - :, . : .,; , " , . , - : .
: . . . . . . .
WO91/1~85 PCT/US91/00~0
- 22 -
bonds. These occur in the same positions in chymotrypsin,
trypsin, and elastase. Both CCP1 and RMCPII lack a
disulfide bond that is present in all other known serine
proteases, including several from prokaryotes, and that
links Cysl91 with Cys220 in chymotrypsin. In both CCPl and
RMCPII the first of these two half-cysteines is replaced by
a phenylalanine, while the second half-cysteine has been
deleted along with other residues. Linkage of Cysl91 to
Cys220 is thought to b~ important in stabilizing the
conformation of the substrate binding site (Woodbury et al.,
(1978) Biochem. 17:811). Its absence in CCP1 and RMCPII may
lead to significant changes in that site and, hence, in
substrate specificity.
Two other primary structure changes previously seen
only in RMCPII and thought to alter substrate binding are
also present in the predicted CCP1 protein. In RMCPII and
CCP1 the amino aci~ six residues before the active-site
serine is alanine. In chymotrypsin-like proteases it is
serine and in trypsin-like proteases, aspartic acid. The
residue in this position lies at the bottom of the S1
binding site, so the change to a less polar residue would
indicate a preference for a hydrophobic amino acid at the
Pl position in the substrate. Furthermore, the sequence
Ser-Trp-Gly216 in chymotrypsin, which forms hydrogen bonds
with the Pl and P3 residues of the substrate, is replaced by
Ser-Tyr-Gly in CCP1 and RMCPII, again suggesting altered
substrate specificity. Both of these changes are also seen
with CCP2.
One of the few RMCPII-specific differences that is
not present in CCP1 is the substitution of isoleucine at
position 99 in chymotrypsin for asparagine. In most
mammalian serine proteases this residue is hydrophobic, and
indeed in CCP1 it appears to be phenylalanine. However,
.-
. ~ ' : ' ~ . . .
:.: , . ' . ': : ' ,. ' .
. :.
~WO91/1~85 2 0 7 ~ PCT/USg1/oo~o
most of the RMCPII-specific changes are present in CCPl
protein, suggesting that the substrate binding site of CCPl
resembles that of RMCPII and is significantly different from
those of other mammalian serine proteases.
Example 3
The initial step in determining the structure of a
protease expressed exclusively by human cytotoxic T
lymphocytes and recognizing a unique protein cleavage site
is to clone human cytotoxic T lymphocyte specific cDNAs.
lOPolyA RNA from a human cytotoxic T lymphocyte cell
line, e.g., one of the lines on deposit at the Coriel
Institute for Medical Research, Copewood and Davis Street,
Camden, NJ, is used as a template for the synthesis, by
standard procedures, of double stranded complementary DNA.
EcoRI recognition sequences are then ligated onto the ends
of the dscDNA by standard methods, and the resultant
molecules are size selected on low melt agarose and then
inserted into the EÇ_RI site of ~gtll, all by conventional
procedures. These recombinant molecules are then packaged -
into A phage heads (Gigapack plus, Stragene) and used to
infect E. ~1i Yl088. DNA from plaques harboring
recombinant molecules are hybridized with radioactive probes
generated from BlO and Cll by standard procedures to
identify corresponding human genes. The screening is
conducted in duplicate to minimize the possibility of false
positives. hCll, a human counterpart of Cll, was found
using the above procedures.
The phage DNA from any positive plaques are isolated
and immediately recloned, using conventional procedures, in
the plasmid vector pUC13. Large amounts of these
recombinant plasmid DNAs are then isolated for further
analysis. The human cytotoxic T lymphocyte specific clones
can be characterized by restriction enzyme digestions and,
~ .
- - . .. .... .. ... ,. .. : .... . . . .
. - : ' : , . . . ' ~ , . . . : . . ' . . . . .
' . . ' ' ' '.' : .'' `'. ~ : ' .
WO91/10685 PCT/US91/~
ultimately, sequence analysis. In addition, their
relationships to one another can be investigated by standard
cross-hybridization and heteroduplex mapping.
Tissue specific expression and transcript sizes of
isolated genes can be established using the same methods as
described for B10 and C11. Using Northern ~lot analysis, as
described above, a number of different cell lines (all
obtained from ATCC) were tested for expression of hC11.
CEM-CM3 (acute lymphoblastic leukemia), CCRF-CEM (acute
lymphoblastic leukemia), CCRF-SB (acute lymphoblastic
leukemia), RPMI 7666 (B lymphoblast), DLD-1 (colon
adenocarcinoma), and CRL-7123 (spleen line) all failed to
express hC11. Human thymocytes and peripheral blood
lymphocytes were also negative. Cytolytic T cells,
activated by mitogen, interleukin 2, anti-T cell-receptor
antibody, or fucose, were all positive for hC11 expression.
A human cytotoxic T cell line was also positive. Thus,
expression of hC11 appears to be specific to cytotoxic T
cells.
The correlation between the level of cytotoxicity
and the expression of the human genes also can be examined
using the above-described methods. The expression of hC11
was found to correlate with the cytolytic activity of the
cells in which it was expressed. Expression of hCll was
detected in lymphokine activated killer (LAX) cells. ~he
procedure for generating LAX cells is essentially that of
Rosenberg et al. (1985) N.E.d. Med ~ 1485.
When expression of a human cytotoxic T lymphocyte-
specific gene correlates with toxicity, the gene is
sequenced by standard methods (as was done with the B10 and
Cll genes). From the gene seguence, the structure of the
protease can be determined, and a computer analysis of the
structure of protease performed, as with the C11 gene.
~..... . . :
-' ~
. . ~
, 2~7~81
~i WO91/1~85 PCTtUS91/00~0
- 25 -
Further computer analysis can show the location of the
active site of the enzyme, and the appropriate sequence of a
peptide that can act as a competitive inhibitor can be
determined.
hC11 was sequenced, as described above, and found to
be very similar to murine gene C11 (Fig. 6). The active
site of hCCPl, the protein encoded by hCll, resembles the
active site of the murine protein, CCP1, very closely. Most
importantly, like CCPl, hCCPl appears to have an Arg at Sl,
imparting the unusual specificity of Asp at Pl. The only
other difference is the substitution of an aromatic amino -
acid two residues downstream from the Arg. Due to the
similarity of the proteins encoded by hCll and Cll
inhibitors synthesized to inhibit one should inhibit the -
other.
A partially purified preparation of hCCPl does not
cleave at sites recognized by trypsin and chy~otrypsin.
Analysis of hCll gene expression, by in situ
hybridization to biopsy sample, indicates that hCll is
expressed in cardiac tissue of a patient that rejected a
transplanted heart. In situ hybridization and related
procedures were performed as described above.
Example 4
A human placental genomic library, in ~ charon 4A,
was screened by hybridization in 20% formamide and 6 x SSC
(l x SSC is 0.15 M sodium chloride, 0.3 M sodium citrate,
pH7) at 41C with a mixture of radioactivity labelled cDNAs
corresponding to the murine cytotoxic cell proteases CCPl-
4, Bleackley et al., (1988) FEBS Letters 234: 153-159 and
Lobe et al. (1976) Science 232: 858-861.
Phage DNA from one of the positive plaques gave a
6.3 kb EcoRI fragment (and ultimately a l.5 kb Bam fragment)
(Fig. 7) that hybridized with the murine probes but failed,
. .
WO91/1~85 PCT/US91/00~0
- 26 -
under conditions of high stringency, to hybridize with
hCCPl. Preliminary sequence analysis revealed that the 1.5
kb fragment encoded a protein which was highly homologous to
the murine cytotoxic proteases. Thus this gene is a new
member of the human CCP family but is different from hCCP1.
hCCPX is expressed in cytotoxic cells. Poly A+ RNA
was purified from resting and activated peripheral blood
lymphocytes and subjected to Northern blot analysis using
the l.S kb genomic fragment as a probe. A transcript is
clearly present in the activated cells that is absent in RNA
from the unstimulated control. Sometimes a small amount of
transcrlpt ls seen in the unstimulated cells, perhaps due to
cell~ar contamlnation, however, the transcript is always
induced upon sti~ulation.
Beaause of the hl~h le-~el of homologs~ between the
var~ous ~P ~amlly mem~ers cross-hy~rldlzatlon can occur.
In the ca~e of the murine genes, C~P1 can be disting~shed
from the others because of a difference in transcript size.
; However, the transcripts detected by hCCP1 and HCCPX are
very similar in mobility. Therefore, high stringency
was~ing conditions were used to minimize cross-
hybridization. With washing at 41C the 1.5 kb probe
detects transcripts in both human and mouse cytotoxic
cells. However at 550 the signal due to the cross-
hybridization with the mouse transcripts is markedly lessthan that seen for the human RNA, even though this ~ouse
cell line expresses extremely high levels of the protease
transcripts. The human-human and human-mouse identities are
both approximately 70%, thus we believe that the signal seen
under high stringency washing conditions in the RNA from
activated human cells is due to specific hybridization With
hCCPX transcripts.
.'.~."': .
.
': ' . ; ,. ~'.. .. .
~ 2~7`~31
~ WO91/10685 PCT/US91/00~0
In addition, no detectable signal was detected using
this probe on RNA samples from a number of human cell lines
obtained from the ATCC including CEM-CM3, CCRF-CAM, CCRF-SB
(acute lymphoblastic leukemias), RPMI 7666 (EBV-transformed
B lymphoblast), DLD-l (colon, adenocarcinoma), CRL-7020
(thymus), CRL-7123 (spleen) and freshly isolated human
splenocytes and thymocytes.
Example 5
The nucleotide sequence of the region indicated by
the heavy line in Fig. 7 is presented in Fig. 8. A
comparison of this sequence with those of the murine ccP
genes revealed high levels of homology (-70% identity) in
regions which correspond to exons and dissimilarity in
reqions which correspond to introns. By placing the introns
(the underlined regions in Fig. 8) in exactly the same
places that they occur in the murine sequences (all four
murine genes have introns in precisely the same positions,
Lobe et al. (1988) Biochemistry 27: 6941-6946), the sequence
of a cDNA could be determined (Fig. 9). A cDNA
corresponding to exons 3, 4 and 5 has been isolated and
confirms the positioning of the introns. The predicted
protein which would be encoded by this gene is 246 amino
acids in length (molecular weight = 27,318). The amino acid
sequence is shown below the nucleotide sequence in Fig. 9.
This protein was not found in the GenBank data base. It is
however, homologous to a wide variety of serine proteases.
The highest level of identity was with the cytotoxic cells
proteases (human 70%), murine (61%), cathepsin G (human
57%), and mast cell proteases (40-50%). In addition, a
significant level of identity (-30%) was found with many
other trypsin and chymotrypsin like enzymes. This protein
is a serine protease and is related to the cytotoxic cell
- - ,. , . . ,- .
.
2~7~08~ ~
WO91/1~ PCT/US91/00~0
- 28 -
proteins, it will be referred to as human cytotoxic cell
protease-X (hCCPX).
An alignment of the hCCPX sequence with those
predicted from the murine genes (Fig. 10) illustrates the
high degree of primary se~lence similarity and also reveals
that hCCPX shares many features in common with the CCP
genes, Bleackley et al. (1988) FEBS Letters ~ 153-159.
hCCPX is very basic (14% basic, 6% acidic amino acids) and
contains a hydrophobic leader sequence of 18 residues
followed by a putative zymogen dipeptide which precedes the
mature protease amino terminal Ile residue. It is believed
that the basic nature of the proteins may play a role in
sequestering them within granules bound to proteoglycans,
Stevens et al. (1988) Current Topics in Microbiology and
Immunology 140: 93-108. The two sequences +21 to l24 (Ile
Ile Gly Gly) and +29 to +36 (Pro His Ser Arg Pro ~yr Met
Ala) which are fGund in all the CCPs, granzymes, RMCPI and
II, and cathepsin G are also conserved in hCCPX as are the
six cysteine residues which form disulfide bonds, Jenne et
al., (1988) Current Topics in Microbiology and Immunology
140:33-48. The catalytic triad residues (marked with an "*"
in Fig. 10) which form the active site of the serine
proteases are all present in the correct positions, Neurath
(1984) Science ~ 350. The sequences surrounding these,
which are highly conserved in serine proteases, are also
conserved.
CCP1 and 2 both contain unusual residues in regions
that are believed to be important in defining substrate
specificity, Lobe et al. (1986) Science 232: 858-861 and
Murphy et al. (1988) Proteins 4:190-204. In addition, they
lack a disulfide bond which in other serine proteases is
important in restricting the size of the substrate binding
pocket. Si~ilar results were subsequently found for the
'
.
.~ ''
~ WO91/1~85 2 0 7 ~ O ~ 1 PCT/US91t~ ~0
- 29 -
other CCPs and granzymes, Bleackley et al. (1988) FE~S
Letters 234:153-159 and Masson et al. (1987) Cell 49:679-
685. The protease described here also has unusual residues
in these same sites and lacks the disulfide bond. However,
the pattern of amino acids seen in thi6 protein, namely Thr,
Ser-Tyr-Gly, and Gly at positions -6, +15 to +17, and +25
relative to the active site Ser, does not correspond to any
of the murine proteases characterized to date. It would
appear then that hCCPX would also have an unusual substrate
specificity.
Purified insert from the cDNA containing plas~id was
labelled by rando~ priming and used as a probe for in situ -
hybridization on human metaphase spreads. The gene is
present at a single locus on chromosome 14 at qll.2. The
human gene encoding hCCP1 maps to the same region. In mice
the genes encoding CCPl, CCP2, CCP3, and CCP4 are all
located on chromosome 14 close to the ~-chain of the T cell
antigen receptor locus Brunet et al. (1986) Nature 322:268-
271.
Exa~Fple 6
The three-dimensional structure of CCPl, the
protease encoded by C11, was predicted by computer
analysis. The use of comparative molecular modeling to
predict the structure of a protease and its characteristic
substrate is particularly reliable when the protein of
unknown structure is relatively homologous with a protein of
known three dimensional structure. The existence of a large
database of known three dimensional structures of related
proteins and their substrate is also very helpful. In the
case of the cytotoxic cell proteases both of these criteria
are met.
The model building procedure (as applied to CCP1 and -
another unrelated serine protease) is described in detail in
.
, , , ' ' ~
WO91/1~85 ` PCT/US91/00~0
- 30 -
Murphy et al. (1988) Proteins: Structure, Function, and
Genetics 4:190-204 which is included herein as an appendix.
(The computer program MUTATE referred to in the appendix is
available from Dr. R. Read, Department of Medical
Microbiology, University of Alberta, Canada). Briefly, the
process begins with aligning the sequence of the protein of
unknown structure with the sequence of a template protein, a
protein of known three-dimensional structure. In the case
of highly homologous proteins the alignment is
lo straightforward: the sequences are ali~ned and a computer
generated model of the template protein is modified to yield
a model of the structure of the unknown protein. The side
chain of each amino acid of the template is then replaced
with the side chain of the corresponding amino acid of the
protein of unknown structure. The replacement side chain
conformations are adjusted to follow the conformation of the
replaced, i.e., template, side chain conformations when
possible. When this is not possible preferred side chain
angles are selected from a dictionary of preferred side
chain conformations.
Subsequent refinements include adjusting the model
to remove unacceptably close non-bonded intramolecular
contacts and adjusting the placement of deletion and
insertion loops. In the final step, the deduced structure
is adjusted to relieve any remaining unacceptably close non-
bonded contents.
The prediction of substrate structure is drawn from
several types of information. This procedure begins with an
examination of the deduced three dimensional structure of
the protease and an analysis of the identity of amino acid
residues in key positions on the catalytic site of the
protease. This information is compared to the reactive site
on the substrate of a closely related protease. The
. :
,
~ .
~ WO91/10685 2 ~ 7 ~ ~ 8 i PCT/US91/00~0
- 31 -
sequence of that substrate can then be altered to achieve a
sequence complementary to the catalytic site of the modeled
protein.
Analysis of CCP1 indicates that the active 6ite has
histidine, aspartic acid, and serine residues at the back,
meaning that it is a serine protease. Computer analysis
further indicated that this active site cleaves proteins at
a cleavage site (between the C-linkage of an Asp residue and
an N-linkage of an adjacent amino acid, Phe) different from
the cleavage sites recognized by any other known eukaryotic
serine proteases. This deduced cleavage site permits the
synthesis of synthetic peptides which, by mimicking all or a
portion of the natural cleavage site, can bind to the active
site of the protease and competitively inhibit it.
Exam~le 7
The amino acid residues of a substrate are
4P3P2P1Pl P2 P3'P4' with cleavage by the
protease occurring between P1 and Pl'. The corresponding
interacting amino acids of the binding pocket of an enzyme
are designated S4S3S2slsl's2's3~s4~ with S1 for e-g-
~example interacting with Pl.
The computer generated three-dimensional structure
of CCPl indicates that the residues of the binding pocket
which might interact with a substrate are: Pro 28-Cys 42;
His 57-Asn 65; Leu 32; Ile 41; Ile 73; Tyr 151; Gly 153; Phe
99; Ser 214-Asp 219; Phe l91-Ser 195; Arg 226; and Asn 174-
Arg 175. (See pages 198-200 of Murphy et al., Appendix).
The most important prediction is that Sl equals Arg 226.
This predicts an acid substrate specificity (probably Asp)
at Pl, the site of cleavage. ~his specificity is unique
among eukaryotic serine proteases. S2 appears from the
computer analysis to be Phe 99, indicating a small amino
acid e.g., Val, at P2. The presence of basic residues in S3
- .: .:; . :
:,' . . :.
WO91/1068S 2~7~ PCT/USgl/oo~o ~
- 32 - -
and S4 predict acidic residues at P3 and P4. Guided by
these considerations the inhibiting peptides, corresponding
to residues P3-P1' of the substrate, were synthesized.
These peptides are shown in Fig. 11. The effect of the
inhibitors on the cytotoxic properties of cytotoxic T
lymphocytes is shown in Tables 3 and 4.
TABLE 3
% Lysis
2368 2369 2372 2373
Control cytotoxicity 15% 15% 15% 15%
+ 100 ~g/ml peptide8% 13% 12% 9%
Control cytotoxicity 30~ 30% 30% 30%
+ 50 ~g/ml peptide 19% 17% 17% 23%
Cytotoxicity was measured with cells from a cyclosporine-A
induced mixed lymphocyte reaction mixed lymphocyte reaction
(CsA-MIR). Spleen cells were obtained aseptically by
pressing the spleen through a wire mesh into a medium of
RPMI 1640 (GIBCO Laboratories, Grand Island, NY), 10~ (v/v)
fetal bovine serum (GIBCO Laboratories~, 10-4M 2-
mercaptoethanol, and 10 mM HEPES buf~er (Sig~a, St. Louis,
MO) (RHFM). Responder cells (1-2x10 /ml) were cocultured
with e ~ ~ numbers of allogenic stimulator cells (1500 rad
from a Cs source) in RHFM plus 300mg/ml CsA and 200
units/ml interleukin 2 in a final vo~ume of 4 ml (Costar 6
well cluster) or 25 ml (Costar 75 cm tissue culture
flask). The cultures were incubated at 37C in 5% CO and
90% relative humidity. Cells from the primary MLR cu~tures
were harvested, washed in RHFM and then recultured with
cytokines at a cell density of 2-5 x 105 cells/ml for 24 or
48 hours. In some experiments, viable cells were isolated
by gradient density centrifugation. 4For cytotoxicity
assays,5~ells were incubated with 10 target cells labelled
with Na CrOI (New England Nuclear, Boston, MA) in a round-
bottom microt~ter plate (final volume of 200 ~L). After 4hours at 37, 100 ~L of supernatant was removed from each
well for counting. Specific lysis was calculated as:
.
~ . :
'
.
'~`.` WO9l/10685 2 ~ 3 1 PCT/US91/00~0
- 33 -
lysis = çx~erimental - s~ontaneous release x 100
total release - spontaneous release
Spontaneous release was obtained by incubating 51Cr-labelled
targets alone, and total release from target cells incubated
with 1% Zap-Isoton lytic agent (Coulter Electronics of
Canada, Ltd., Mississauga, Ontario).
TABLE 4
Untreated Pretreated
PeptideControl effectors effectors
10 EF2394 54% 90% 58%
EF2395 61% 86% 78
EF2396 54% 73% 50%
EF2397 54% 87% 50%
EF2398 54% 107S 89%
15 EF2368 54% 100% 75%
EF2369 54% 100% 28%
EF2372 54% 97% 35%
EF2373 54% 87~ 40%
Cytotoxicity in cytotoxic T-cells activated with ConA and - .
interleukin 2 (I12). Cytotoxic T Cells were actiYated using
10 ~g/ml ConA and 10 U/ml IL2. The cytolytic activity was
measured in a standard chromium release assay. Targets were
pretreated for 3 hr with 100 ~g/ml peptide and were then
mixed with either pretreated lOO ~g/ml peptide and were then
mixed with either pretreated 100 ~g/ml peptide) or untreated
effectors. All results are at an effector to target ratio
of 5:1. Results are calculated as described in the legend
of Table 1.
Example 8
The cDNA clones of the invention can be used to
generate copious quantities of purified cytotoxic cell
proteases by inserting the coding sequence of a cytotoxic
cell protease gene into an expression vector and expressing
the desired proteins in an expression system. These
procedures are well known to those skilled in the art.
'. ' ..
.- ' :' : '
.. - : ~
a~
W091/10685 Pcr/us91/oo34
-- 34 --
The possession of purified protease allows for a
greatly simplified alternative approach to the design of
inhibitor molecules. Rather than the extremely cumbersome
and complex immunologically based assays used to produce the
5 results in Tables 3 and 4, the enzymatic action of the
purified protease on a given substrate can be followed
directly, by cleavage of the substrate, when the purified
protease is available. (Sequence specific protease cleavage
can be followed with standard thioester-based assays such as
that described in Harper et al. (1984~ Biochem. 23:2995-
3002). This allows a large number of potential inhibitors
to be tested with relative ease. The purified protease
based assay can be used alone, or in conjunction with the
rational design factors obtained by computer analysis, to
15 screen large numbers of potential inhibitors. Positive
compounds could then be tested for their immunosuppressive
properties.
Inhibitor Peptide Synthesis
The inhibitory peptides of the invention can be
20 prepared by standard solid phase synthesis, for example, a
method in which a tert-butyloxycarbonylamino acid is
attached to either chloromethyl resin containing 0.75 mM Cl
g 1, or the p-methylbenzhydrylamine resin containing 0.35 mM
NH2 g ~ followed by the sequential addition of desired
25 amino acid residues to produce the desired peptide.
Synthetic reactions are performed in 70 ml polypropylene
syringes fitted with a polyethylene frit using appar~tus and
techniques described in Burton et al., (1975) Biochemistry
14:3892, and Merrifield, (1963) J. Amer. Chem. Soc. 85:2149.
30 Completeness of coupling is determined by the standard
ninhydrin test. The C-terminal amino acid is attac:hed using
procedures described in Stewart et al., Solid Phase Peptide
Synthesis (W.~. Freeman ed. 1970), or Pietta et al., 1970
.
' ' ; .
. ~ . .
,
"~ J WO 91/1~8~ 2 ~ PCT/US91/00~0
- 35 -
Chem. Comm. 650. Hplc purifications of the synthetic
peptides are carried out using a Beckman ODS colu~n (10 x
250 mm).
Amino acid analyses of the synthetic peptides are,
if desired, performed using a Durrum D-500 analyzer.
Cysteinyl residues in the peptides are quantitated as
cysteic acid using a modification of the method of Moore
(196~) in which loo mM peptide is oxidized with 2.0 ml
performic acid (1 ml 30% H202 + 9 ml 88% HCOOH) for 2 -
hrs. at 0. Performic acid is removed in a ~eacti-Therm at
40 using N2, and 0.5 ml distilled water is then added to
the residue and re-evaporated. The product is then
hydrolyzed using 6 N HCl. Free sulfhydryl groups are
determined using the method of Ellman et al. ~1959).
Use
The inhibitory molecules are effective inhibitors of -
cytotoxic cells, e.g., cytotoxic lymphocytes. The
inhibition of the target cell destroying activity of such
cells can be used to treat patients suffering of autoimmune
diseases such as Hashimoto's thyroiditis, primary myxedema,
thyrotoxicosis, pernicious anaemia, autoimmune atrophic
gastritis, Addison's disease, myasthenia gravis, juvenile
diabetes, Goodpasture's syndrome, pemphigus vulgaris,
pemphigoid, sympathetic ophthalmia, phacogenic uveitis,
autoimmune haemolytic anaemia, idiopathic thrombocytopenic
purpura, idiopathic leucopenia, primary biliary cirrhosis,
active chronic hepatitis HBs-ve, cryptogenic cirrhosis (some
cases), ulcerative colitis, Sjogren's syndrome, systemic
lupus erythematosus (SLE), discoid LE, dermatomyositis,
30 scleroderma, rheumatoid arthritis, and possibly multiple ~-
sclerosis, and similar diseases in other mammals, for
example, various types of livestock such as cows. Such
inhibition can also be used to treat allograft (a tissue or
.. , . , . . .. . ~ .
, , ~ , : ; ' : ':
,: ! '
" ' ~ ' ' ' ' ' ', , , ~
~' '~., . ' ` '
' ~
2 ~ 7 ~ ~ 3 ~
wo9l/lo68s PCT/US91/00~0
organ graft from a donor who is a genetically dissimilar
member of the same species as the receptor) rejecti~n, and
graft v. host disease.
The peptides can be administered to a mammal in a
dosage of 25 to 500 mg/kg/day, preferably 50 to 100
mg/kg/day. When administered to mammals (e.g., orally,
intravenously, parenterally, nasally, or by suppository),
the peptides inhibit the ability of cytotoxic T lymphocytes
to destroy cells, thus inhibiting the cell-mediated immune
response to provide an effective treatment for thé above
listed disorders.
Nucleic acid probes (prepared by standard methods)
capable of hybridizing to a gene encoding a protease
expressed only by cytotoxic lymphocytes can be used in a
variety of useful hybridization assays. For example, such
probes can be used to monitor cytotoxic T lymphocytes in
transplanted tissue, e.g., by the n situ hybridization
methods of Cox et al. (1984) Dev. Biol. 101:485. The
presence of the lymphocytes in the transplanted tissue is an
indication that the tissue is being rejected by the host
organism and that appropriate immunotherapy should be
undertaken.
The probes can also be used to assess the potential
cytotoxicity of lymphokine activated killer cells. The
generation and use of such cells to treat tumor patients is
described by Rosenberg et al. (1985) N.E.J. Med. 313:1485.
Rosenberg describe how human peripheral-blood lymphocytes
are treated with interleukin-2 (a lymphokine) to generate
killer cells that will attack tumor cells when reintroduced
into the host. The probes can be used in a hybridization
assay with the nucleic acid of the treated lymphocytes by
standard methods; the assay monitors the degree to which
the activated killer cells have been generated by
. . ,, ~ : ' ~ .,
i- WO91/1068~ 2 ~ A PCT/VS91/00
- 37 -
determining the level of expression of the protease-encoding
gene in the cells.
Other embodiments are within the following claims.
What is claimed is: -
.. ...
:. -,-- - ,' ~ ' . - . ' ~ . ',
. . : ~: i , . . .
' , '~' ' ': ~
