IE84906B1 - Cytotoxic Lymphocyte Maturation Factor - Google Patents
Cytotoxic Lymphocyte Maturation FactorInfo
- Publication number
- IE84906B1 IE84906B1 IE1999/0007A IE990007A IE84906B1 IE 84906 B1 IE84906 B1 IE 84906B1 IE 1999/0007 A IE1999/0007 A IE 1999/0007A IE 990007 A IE990007 A IE 990007A IE 84906 B1 IE84906 B1 IE 84906B1
- Authority
- IE
- Ireland
- Prior art keywords
- clmf
- subunit
- leu
- cells
- glu
- Prior art date
Links
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Abstract
ABSTRACT The present invention relates to a novel subunit of the cytokine protein called Cytotoxic Lymphocyte Maturation Factor (CLMF) which is produced and synthesized by a human B lymphoblastoid cell line. CLMF synergistically induces in the presence of low concentrations of IL-2 the cytolytic activity of Lymphokine Activated Killer (LAK) cells. CLMF is also capable of stimulating T-cell growth. The present invention also relates to cloned genes coding for the novel proteins and derivatives thereof, to recombinant vectors comprising a polynucleotide encoding said proteins, to microorganisms transformed with the said recombinant vectors, to antibodies directed to the said proteins as well as to processes for the preparation of the said proteins, vectors and antibodies.
Description
The present invention relates to the field of cytokines,
in particular to those cytokines which synergize with
interleukin-2 (IL-2) to activate cytotoxic lymphocytes such
as the cytokine Cytotoxic Lymphocyte Maturation Factor
(CHfl‘). The present invention also relates to monoclonal
antibodies directed to cunx
'Cytokine' is one term for a group of protein cell
regulators. variously called lymphokines, monokines,
interleukins and interferons, which are produced by a wide
variety of cells in the body. These cytokines play an
important role in many physiological responses, are involved
in the pathophysiology of a range of diseases, and have
therapeutic potential. They are a heterogeneous group of
proteins having the following characteristics in common.
They are low molecular weight (580 kDa) secreted proteins
which are often glycosylated; they are involved in immunity
and inflammation where they regulate the amplitude and
duration of a response; and are usually produced transiently
and locally. acting in a paracrine or autocrine, rather than
endocrine manner. Cytokines are extremely potent, generally
acting at picomolar concentrations; and interact with high
affinity cell surface receptors specific for each cytokine
or cytokine group. Their cell surface binding ultimately
leads to a change in the pattern of cellular RNA and protein
synthesis, and to altered cell behavior. Individual
cytokines have multiple overlapping cell regulatory actions.
‘it is concomitantly exposed.
The response of a cell to a given cytokine is dependent
upon the local concentration of the cytokine, upon the cell
type it is acting on and upon other cell regulators to which
The overlapping regulatory
actions of these structurally unrelated proteins which bind
to different cell surface receptors is at least partially
accounted for by the induction of common proteins which can
have common response elements in their DNA.
interact in a network by:
Cytokines
first. inducing each other:
second, transmodulating cytokine cell surface receptors and
third. by synergistic. additive or antagonistic interactions
on cell function. [Immunology Today lg: 299 (1989)).
The potential utility of cytokines in the treatment of
neoplasia and as immunoenhancing agents has recently been
demonstrated in studies using human recombinant
interleukin-2 (rIL—2). Natural interleukin-2 (IL-2) is a
lymphokine which is produced and secreted by T—lymphocytes.
This glycoprotein molecule is intimately involved in the
induction of virtually all immune responses in which T—cells
play a role. B cell responses in vitro are also enhanced by
the presence of IL-2. IL-2 has also been implicated as a
differentiation inducing factor in the control of B and T
lymphocyte responses.
Administration of human rIL—2 has been shown in some
of established tumors in both
The anti—tumor effects of
animal models suggest that rIL-2 might also have value in
In addition, results from
in ameliorating chemotherapy-induced immunosuppression
the treatment of certain infectious diseases [J.
[Immunol. Lett. ;g:307-314 (1985)).
However, the clinical use of rIL—2 has been complicated
‘by the serious side effects which it may cause [N. Engl. J.
Med. 3l3:148S-1492 (1985) and N. Engl. J. Med. 3l6:889—897
Kobayashi et al. (J. Exp. Med. (1989) 170, 827-845)
relates to the identification and purification of natural
killer cell stimulatory factor (NKSF), a cytokine with
multiple biologic effects on human lymphocytes.
Thus, the present invention provides a 35 kDa subunit of a
cytokine protein called Cytotoxic Lymphocyte Maturation Factor (CLMF)
which is produced and synthesized by cells capable of
secreting CLMF. Examples for such cells are mammalian cells
particularly human lymphoblastoid cells. In the presence of
low concentrations of IL-2 CLMF synergistically induces the
cytolytic activity of Lymphokine Activated Killer (LAK)
cells. CLMF is also capable of stimulating T—cell growth.
CLMF can be isolated in a substantially pure form by the
following steps:
a) stimulating B lymphoblastoid cells such as NC—37
cells to produce and secrete cytokines into a
supernatant liquid:
b) collecting the supernatant liquid produced by
the stimulated cells;
c) separating the supernatant liquid into protein
fractions;
d) testing each protein fraction for the presence
of CLMF:
e) retaining the protein fractions which are able
to stimulate T—cell growth, said fractions containing an
active protein which is responsible for the T—cell
stimulating activity of the protein fractions:
f) isolating said active protein into a
substantially pure form, said protein being Cytolytic
Lymphocyte Maturation Factor (CLM).
The CLMF protein obtained in this way is free from other
cytokine proteins. The natural CLMF protein is a 75
kilodalton (kDa) heterodimer comprised of two polypeptide
subunits. a 40 kDa subunit and a 35 kDa subunit which are
bonded together via one or more disulfide bonds. The present
invention also provides the nucleotide sequence.of the 35 kDa
subunit of the CLMF gene and the amino acid sequence of the 35
kDa subunit of the CLMF protein encoded by the said gene. The
present invention relates to a protein which exhibits CLMF
activity and contains a biologically active portion of the
amino acid sequence of CLMF or which contains an amino acid
sequence of CLMF as well as other amino acids or proteins
containing analogous sequences to CLMF or its biologically
active fragments which proteins exhibit CLMF activity.
The above process steps c) to f) may be used to purify
CLMF from any liquid or fluid which contains CLMF together
with other proteins. The present invention relates also to
protein fractions having CLMF activity and being capable of
stimulating T-cell growth. to a substantially purified
active CLMF protein. obtained by the above described
process. to the isolated cloned gene encoding
the 35 kDa subunit, to vectors containing this gene to host
cells transformed with the vector containing the said gene
and to CLMF proteins prepared in such a transformed host
cell. Furthermore the present invention relates to isolated
polyclonal or monoclonal antibodies capable of binding to
CLMF.
Monoclonal antibodies prepared against a partially
purified preparation of CLMF have been identified and
characterized by 1: immunoprecipitation of 125I—labelled
CLMF, 2: immunodepletion of CLMF bioactivity. 3: western
blotting of CLMF. 4: 12Sl—CLMF binding to
its cellular receptor and 5: neutralization of CLMF
Twenty hybridomas secreting anti—CLMF
antibodies were found to
inhibition of
bioactivity.
antibodies were identified. The
I—label1ed
CLMF bioactivity as assessed in
immunoprecipitate CLMF and to immunodeplete
the T—cel1 proliferation and
LAK cell induction assays. western blot analysis showed
that each antibody binds to the 70 kDa heterodimer and to
one of the subunits. Each of the above-mentioned 20
anti—CLMF monoclonal antibodies were specific for CLMF and
in particular for the 40 kDa subunit of CLMF. A CLMF
receptor binding assay has been developed to evaluate the
ability of individual antibodies to inhibit CLMF binding to
its cellular receptor.
The assay measures the binding of
125
I—labelled CLMF to PHA activated PBL blast cells in the
presence and absence of each antibody. Of the 20 antibodies
tested, L2 antibodies were found to inhibit greater than 60%
. .
of the I-labelled CLMF binding to the blast cells. Two
inhibitory antibodies, viz. 7B2 and 4A1, neutralize CLMF
bioactivity while one non—inhibitory antibody, SE3, does not
neutralize CLMF bioactivity.
I—labelled CLMF binding to its
cellular receptor will neutralize CLMF bioactivity as
assessed by the T—cell proliferation and LAK cell induction
assays. The ability of the antibodies specific for the 40
kDa subunit of CLMF to neutralize CLMF bioactivity indicates
that determinants on the 40 kDa subunit are necessary for
binding to the CLM cellular receptor.
These data confirm that
antibodies which block
The monoclonal anti—CLMF antibodies
provide powerful analytical, diagnostic and
therapeutic reagents for the immunoaffinity purification of
natural and recombinant human CLMF, the development of human
CLMF immunoassays. the identification of the active site of
the 40 KDa subunit of CLMF and may be used in therapeutic
treatments of patients which require selective
immunosuppression of cytotoxic T cells. such as in
transplantation. Monoclonal antibodies which recognize
different epitopes on human CLMF can be used as reagents in
a sensitive two—site immunoassay to measure levels of CLMF
in biological fluids, cell culture supernatants and human
cell extracts.
The monoclonal antibodies against CLMF exhibit a
number of utilities including but not limited to:
. Utilizing the monoclonal antibodies as affinity
reagents for the purification of natural and recombinant
human CLMF:
. Utilizing the monoclonal antibodies as reagents to
configure enzyme-immunoassays and radioimmunoassays to
measure natural and recombinant CLMF in biological fluids,
cell culture supernatahts, cell extracts and on plasma
membranes of human cells and as reagents for a drug
screening assay;
. Utilizing the monoclonal antibodies as reagents to
construct sensitive two-site immunoassays to measure CLMF in
biological fluids, cell culture supernatants and human cell
extracts:
. Utilizing the monoclonal antibodies as reagents to
identify determinants of the 40 kDa subunit which
participate in binding to the 35 kDa subunit and which
participate in binding to the CLMF cellular receptor:
. Utilizing the intact IgG molecules, the Fab
fragments or the humanized IgG molecules of the inhibitory
monoclonal antibodies as therapeutic drugs for the selective
blockade of proliferation and activation of cytotoxic T
cells, such as in transplantation.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a plot of a supernatant solution obtained
from cultured NC37 lymphoblastoid cells applied to a Nu-Gel
P-SP column showing the protein fraction containing TGF
activity being eluted with a salt gradient.
Figure 2 is a plot of the material containing TGF
activity obtained from the separation shown in Figure 1 as
it was being eluted with a salt gradient through a
Blue-B—Agarose Column.
Figure 3 shows the plot of the material containing TGF
activity obtained from the separation shown in Figure 2 as
it was being eluted with a Nacl gradient through a Mono Q
column.
Figure 4 shows a SDS—polyacrylamide gel electrophoresis
(SDS—PAGE) analysis of the fractions 30 to 45, 48 and 50
obtained from the step illustrated in Figure 3. The numbers
on the left side, 44 and 68,
molecular weight of standard proteins of 44 and 68 kDa in
i.e. refer to the apparent
lane 5.
Figure 5 shows the elution profile through a Vydac
Diphenyl column of fraction 38 from the Mono Q
Chromatography separation (reversed—phase HPLC) shown in
Figure 3.
Figure 6 shows SDS—PAGE analysis of protein purity of
the protein fractions 85-90 recovered from the separation
process depicted in Figure 5.
Figure 7 shows a SDS—PAGE analysis of fractions 87 and
88 from the reversed-phase HPLC separation under
non—reducing (lane A; without B—mercaptoethanol) and
reducing (lane B; in the presence of B-mercaptoethanol)
conditions showing the 75,000 molecular weight CLMF
The
remaining lanes in the gel shown in this Figure contain
separated into two subunits of 40 kDa and 35 kDa.
standard proteins comprising the 44 and 68 kDa marker
protein.
Figure 8 shows the elution pattern of the proteins from
the supernatant solution from NC—37 cells applied to a Nu-
Gel P-SP column and eluted with a salt gradient.
Figure 9 is a Blue—B—Agarose column salt gradient
elution profile of the active fractions obtained from the
Nu-Gel P-SP column elution shown in Figure 8.
Figure 10 is a Mono-Q column salt gradient elution
profile of the active fractions obtained from the elution
shown in Figure 9.
Figure 11 is the elution pattern through a Vydac
Diphenyl column of active fractions 39 and 40 obtained from
the Mono Q Chromatography shown in Figure 10.
Figure 12 shows a SDS-PAGE analysis under reducing
conditions of the active fractions obtained from the
separation process shown in Figure 11.
Figure 13 is a schematic diagram depicting the
separation of the 40 kDa subunit from the 35 kDa subunit of
the CLMF cytokine.
Figure 14 is a schematic diagram depicting the
determination of the amino acid composition, the N—terminal
sequencing. the proteolytic digestion and the complete
sequencing of the 40 kDa subunit of the CLMF cytokine.
Figure 15 shows a separation of the tryptic peptides of
the digested 40 kDa subunit of the CLMF cytokine.
Figure 16 shows a separation of the proteolytic peptides
of the Staphylococcus aureus V8 protease digested 40 kDa
subunit CLMF.
Figure 17 is a chart which summarizes the information on
the protein structure obtained from the analysis of the
proteolytic peptides of the 40 kDa subunit of CLMF. The
following abbreviations and symbols are used:
_ lo _
N~t — N—termina1 sequencing on intact protein
Tr — tryptic peptides from map HP2383 numbered by
"fraction number
V8 —
V8 protease peptides from map HP2412 numbered by
fraction number
— indicates probable glycosylation site: boxes
indicate potential sites
Figure 18 shows the SDS—PAGE analysis of Fraction 39
from the Mono Q FPLC elution profile shown in Figure 3. Lane
A: Standardproteins without B—mercaptoethano1: lane B:
Fraction 39 without B—mercaptoethanol; lane C: Fraction 39
with B-mercaptoethanolz
lane D: Standard proteins with
B—mercaptoethano1.
Figure 19 relates to the purification of the 35 kDa
subunit by reversed—phase HPLC and depicts the elution
pattern through a Vydac C-18 column of fraction 39 of the
Mono Q chromatography which was reduced in 5% B-mercapto—
ethanol.
Figure 20 shows a SDS—PAGE gel analysis under
non—reducing conditions of the fractions which were
fluorescamine positive from the Vydac C-l8 column elution
profile shown in Figure 19. S: = protein—standard; F:‘=
flow-through; numbers refer to the fraction number.
Figure 21 depicts the elution pattern of a tryptic
digest of fractions 36 and 37 of the Mono Q Chromatography
through a YMC ODS column.
Figure 22 shows the stained PVDF membrane with the
smeared bands comprising the CNBr cleaved CLMF before
22B) and after (Fig. 22A) excising the regions of
about 29, 25, 14. 12, and 9 kDa.
contain the CNBr fragments having the following sequences:
(Fig.
respectively. The regiones
I (P?)—P-K-N-L—Q-L-K-P-L—K-N-?-V-(Q?)—
(New sequence from 40 kDa protein)
?—Q—K—A—(R?)—Q—T—L-E—F—Y—P—?—T—
(New sequence starting at residue no. 30 of 35 kDa
protein)
III V—V-L-T-?-D-T-P—E-E—D—G-I—T—
(Starts at residue no. 24 of 40 KDa protein)
IV V—D—A—V—(H?)—K—L—K—Y—E—?—Y—T-?—?—F—F—I—
(Starts at residue no. 190 of 40 kDa protein)
note: it is assumed or known that the above sequences
are preceeded by a Met residue.
Figure 23 shows a reverse—phase HPLC separation of the
peptide fragments obtained by cleaving CLMF with CNBr.
Figure 24 shows an SDS—PAGE of pure CLMF and "free"
unassociated 40 kDa subunit of CLMT purified by affinity
chromatography using the monoclonal antibody 782 covalently
attached to an agarose resin. Lane A: molecular weight
marker proteins; lane B: starting material: lane C: flow-
through; lane D: acid eluate; lane E: potassium thiocyanate
eluate.
Figure 25 a. b, c and d show the DNA sequence and the
deduced amino
CLMF.
acid sequence of the 40 kDa subunit of human
Figure 26 a, b and c show the cDNA sequence and the
deduced amino acid sequence of the 35 kDa subunit of CLMF
Figure 27 depicts the inhibition of CLMF bioactivity by
serum from rats immunized with CLMF and from non—immunized
rats (control).
Figure 28 shows a SDS-PAGE analysis of
immunoprecipitates of l25I—CLMF by monoclonal antibodies
A1 (lane 1), 4D1 (lane 2). 8E3 (lane 3) and 9C8 (lane 4),
by a control antibody (lane 5), by immune rat serum (lanes 6
and 8) and by normal rat serum (lanes 7 and 9). On the left
side the molecular weight in kDa is indicated.
Figure 29 shows the immunodepletion of CLMF bioactivity
(TGF activity) by monoclonal anti—CLMF antibodies (a—CLMF).
Figure 30 shows the immunodepletion of CLMF bioactivity
(LAK induction activity) by monoclonal anti—CLMF antibodies
(a—CLMF).
Figure 31 shows a Western blot analysis of the
reactivity of the monoclonal antibodies (mAbs) 7B2.
8B3, 6A3,
A1,
9F5 and 2A3 and of rat polyclonal anti—CLMF
antibodies (RS1) with the CLMP 75 kDa heterodimer. NR8: —
normal rat serum.
Figure 32 shows a Western blot analysis of the
reactivity of monoclonal and rat polyclonal anti—CLMF
antibodies with the CLMF 40 kDa subunit.
the following mAbs were used: 4A1. 4Dl, 7B2, 7A1, 2A3, 1C1,
8B4, 8A2, 8B3. 1B8. 4A6, 6A2. 8C4, 9P5, 6A3. 9C8. QAI and
E7, respectively. In lane 19 a control antibody, in lane
In lanes 1 to 18
a fusion rat serum and in lane 21 a normal rat serum was
used.
l25I—CLMF to
PHA-activated peripheral blood lymphocyte (PBL)
Figure 33 shows the binding of
lymphoblasts.
I—CLMF binding to
PHA—activated PBL blast cells by rat anti-CLMF serum. The
1 I—CLMF
binding to the cells in the presence of the indicated
Figure 34 shows the inhibition of
data are expressed as amount (% bound) of
concentrations of serum when compared to the total specific
binding in the absence of serum.
Figure 35 shows the inhibition of the binding of
-CLMF to PHA-activated PBL blast cells by monoclonal
antibody supernatants. The data are expressed as %
.
I—CLMF to the cells in the
presence of a 1:1 dilution of supernatant when compared to
inhibition of the binding of
the total specific binding in the absence of antibody
supernatant.
Figure 36 shows the inhibition of the binding of
. » .
I-CLMF to PHA—act1vated PBL blast cells by various
concentrations of purified monoclonal antibodies. The data
-CLMF
bound to the cells in the presence of the indicated
are expressed as the amount (% cpm bound) of
concentrations of antibody when compared to the total
specific binding in the absence of antibody.
Figure 37 shows a western blot analysis of the
reactivity of a rabbit polyclonal anti-CLMF antibody with
the 75 KDa CLMF (nonreduced) and with the 35 kDa CLMF
subunit (reduced). The antibody was prepared against a
synthetic peptide fragment of the 35 kDa CLMF subunit. Lanes
1 to 5 are without B-mercaptoethanol: lanes 6 to 10 with
B-mercaptoethanol.
Lane
ul CLMF
ul CLMF
ul CLMF
Blank
Blank
ul prestained molecular weight standards
7 1 ul CLMF
ul CLMF
ul CLMF
ul prestained molecular weight standards
The CLMF biological
activity of all of the proteins of the present invention
including the fragments and analogues may be determined by
using a standard T—cell growth factor assay.
In accordance with the present invention, natural CLMF
is obtained in pure form. The amino acid sequences of the
kDa subunit and the 40 kDa subunit of the CLMF protein is
depicted in Figures 25 and 26.
Thus, the present invention relates to a protein having
Cytotoxic Lymphocyte Maturation Factor (CLMF) activity in a
substantially pure form, such as the CLMF protein per se, or
to a 35 kDa subunit of the said protein which exhibits CLMF
activity if combined with the 40 kDa subunit and comprises at
least a part of the amino acid sequence of the natural form of
CLMF.
The present invention also relates to cloned genes coding
for 35 kDa subunit of CLMF and to isolated polynucleotides
encoding a subunit as defined above, which polynucleotide
contains a sequence corresponding to the CDNA encoding 35 kDa
subunit of CLMF, to recombinant vectors comprising a
polynucleotide encoding a 35 kDa subunit of the CLMF protein,
to microorganisms transformed with the said recombinant
vectors, to antibodies directed to the said subunits as well
as to processes for the preparation of the said subunits,
govectors and antibodies.
The practice of the present invention will employ,
unless otherwise indicated, conventional techniques of
molecular biology. microbiology, recombinant DNA and
immunology, which are within the skills of an artisan in the
field. Such techniques are explained fully in the
literature. See e.g., Maniatis, Fitsch & Sambrook,
MOLECULAR CLONING: A LABORATORY MANUAL (1982); DNA CLONING,
VOLUMES I AND II (D.N Glover ed., 1935); OLIGONUCLEOTIDE
SYNTHESIS (M.J. 1984); NUCLEIC ACID HYBRIDIZATION
(B.D. Hames & S.J. Higgins eds., 1984); TRANSCRIPTION AND
TRANSLATION (B.D. Harnes & S.J. Higgins eds., 1984); ANIMAL
CELL CULTURE (R.I. Freshney ed.. 1986): IMMOBILIZED CELLS
AND ENZYMES (IRL Press, 1986); B. Perbal. A PRACTICAL GUIDE
TO MOLECULAR CLONING (1984); the series, METHODS IN
ENZYMOLOGY (Academic Press. Inc.): GENE TRANSFER VECTORS FOR
MAMMALIAN CELLS (J.H. Miller and M.P. Calos eds.. 1987, Cold
Spring Harbor Laboratory), Methods in Enzymology Vol. 154
Gait ed.,
- 17 _
and Vol. 155 (Wu and Grossman, and Wu. eds., respectively);
IMMUNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY (Mayer
and walker. 1987. London), Scopes,
PROTEIN PURIFICATION: PRINCIPLES AND PRACTICE. second
Edition (1987. Springer—Verlag, N.Y.), and HANDBOOK OF
EXPERIMENTAL IMMUNOLOGY, VOLUMES I—IV (D.M. Weir and C.C.
Blackwell eds., 1986).
eds., Academic Press,
The DNA sequences and DNA molecules of the present
invention may be expressed using a wide variety of
host/vector combinations. For example, useful vectors may
consist of segments of chromosomal, non—chromosoma1 and
synthetic DNA sequences[ Examples of such vectors are viral
vectors, such as the various known derivatives of SVQO,
bacterial vectors. such as plasmids from E. coli including
pCRl, pBR322, pMB9 and RP4. phage DNAs, such as the numerous
derivatives of phagex, M13 and other filamentous
single-stranded DNA phages, as well as vectors useful in
yeasts, such as the Zn plasmid, vectors useful in
eukaryotic cells more preferably vectors useful in animal
cells, such as those containing SV40. adenovirus and/or
retrovirus derived DNA sequences. Useful vectors may be also
derived from combinations of plasmids and phage DNA's, such
as plasmids which have been modified to comprise phage DNA
or other derivatives thereof.
Expression vectors which may be used for the preparation of
recombinant 35 kDa CLMF subunits are characterized by comprising
at least one expression control sequence which is operatively linked
to the 35 kDa CLMF subunit DNA sequence inserted in the vector in
order to control and to regulate the expression of the cloned 35 kDa
CLMF subunit DNA sequence. Examples of useful expression
control sequences are the lac system, the
major operator and promoter
the trp system.
tac system, the trc system.
regions of phage X, the control region of fd coat protein.
the glycolytic promoters of yeast. e.g., the promoter for
—phosphoglycerate kinase, the promoters of yeast acid
_ 13 _
phosphatase. e.g., Pho 5,
a—mating factors. and promoters derived from polyoma
the promoters of the yeast
virus, adenovirus, retrovirus, and simian virus. e.g., the
early and late promoters or SV40. and other sequences known
to control the expression of genes of prokaryotic or
eukaryotic cells and of their viruses as well as
combinations of the said promoter/operator sequences.
Among such useful expression vectors are known vectors
that enable the expression of the cloned CLMF—related DNA
sequences in eukaryotic hosts, such as in animal and human
P. J. Southern and P. Berg, J. Mol. Appl.
-41 (1982): S. Subramani et al., Mol. Cell.
854-64 (1981): R. J. Kaufmann and P. A. Sharp, Mol.
Biol. lggz 601-64 (1982); S. I. Scahill et al.,
"Expression and Characterization of The Product of A Human
cells [e.g.,
Genet. ;:
Biol. lz
Cell.
Proc. Sci.
Furthermore, within each specific expression vector.
various sites may be selected for insertion of the
CLMF—related DNA sequences of the present invention. These
sites are usually designated by the restriction endonuclease
which cut them. They are well recognized by those of skill
in the art. It is. of course to be understood that an
expression vector useful in this invention need not have a
restriction endonuclease site for insertion of the chosen
DNA fragment. Instead. the vector could be joined to the
fragment by alternative means. The site chosen in the
expression vector for the insertion of a selected DNA
fragment and the operative linking of the DNA fragment to an
expression control sequence is determined by a variety of
factors. such as the number of sites susceptible to a
particular restriction enzyme. the location of start and
stop codons relative to the vector sequence and the desired
-19..
selection method for the host transformed with the
recombinant vector. The choice of a vector and an insertion
site for a DNA sequence is determined by a balance of these
factors, not all selections being equally effective for a
given case.
The host cell used for the expression of the
CLMF-related DNA sequence
known hosts.
may be selected from a variety of
Examples for such hosts are prokaryotic or
eukaryotic cells. A large number of such hosts are available
from various depositories such as the American Type Culture
Collection (ATCC) or the Deutsche Sammlung fur
Mikroorganismen (DSM). Examples for prokaryotic cellular
hosts are bacterial strains such as E.coli, B.subtilis and
others. Preferred hosts are mammalian cells such as the SV4O
transformed African Green monkey kidney cell line COS.
Not all host/expression vector combinations function
with equal efficiency in expressing a given DNA sequence.
However, a particular selection of a host/expression vector
combination may be made by those of skill in the art after
due consideration of the principles set forth herein without
departing from the scope of this invention. For example,
the selection should be based on a balancing of a number of
factors, These include, for example, compatibility of the
host and vector. susceptibility of the protein to
proteolytic degradation by host cell enzymes, possible
contamination of the protein to be expressed by host cell
proteins difficult to remove during purification, toxicity
of the proteins encoded by the DNA sequence to the host,
ease of recovery of the desired protein, expression
characteristics of the DNA sequence and the expression
control sequence operatively linked to them, biosafety.
costs and the folding, form or any other necessary
post—expression modifications of the desired protein.
_ 20 _
The host organisms which contain the expression vector
comprising the 35 kDa CLMF subunit DNA are usually grown up under
conditions which are optimal for the growth of the host
organism. Towards the end of the exponential growth. when
the ihcrease in the number of cells per unit time decreases,
the expression of the CLMF subunit is induced, i.e. the DNA
coding for the subunit is transcribed and the transcribed
mRNA is translated. The induction can be effected by adding
an inducer or a derepressor to the growth medium or by
altering a physical parameter, e.g. by a temperature change.
The CLMF subunit produced in the host organism can be
secreted by the cell by special transport mechanisms or can
be isolated by breaking open the cell. The cell can be
broken open by mechanical means [Charm et al., Meth. Enzmol.
ggz 476-556 (1971)). by enzymatic treatment (e.g. lysozyme
treatment) or by chemical means (e.g. detergent treatment.
urea or guanidine-HCl treatment, etc.) or by a combination
thereof.
In eukaryotes, polypeptides which are secreted from the
cell are synthesized in the form of a precursor molecule.
The mature polypeptide results by cleaving off the so-called
signal peptide. As prokaryotic host organisms are not
capable of cleaving eukaryotic signal peptides from
precursor molecules. eukaryotic polypeptides must be
expressed directly in their mature form in prokaryotic host
organisms. The translation start signal AUG, which
corresponds to the codon ATG on the level of the DNA. causes
that all polypeptides are synthesized in a prokaryotic host
organism with a methionine residue at the N-terminus. In
certain cases, depending on the expression system used and
possibly depending on the polypeptide to be expressed this
N—terminal methionine residue is cleaved off.
The 35 kDa CLMF subunit produced by fermentation of the
prokaryotic and eukaryotic hosts transformed with the DNA
sequences of this
_ 21 _
invention can then be purified to essential homogeneity by
known methods such as. for example, by centrifugation at
different velocities, by precipitation with ammonium
sulphate, by dialysis (at normal pressure or at reduced
pressure), by preparative isoelectric focusing, by
preparative gel electrophoresis or by various
chromatographic methods such as gel filtration, high
performance liquid chromatography (HPLC), ion exchange
chromatography, reverse phase chromatography and affinity
chromatography (e.g. on Sepharose” Blue CL-6B or on
carrier—bound monoclonal antibodies directed against CLMF).
The purified CLMF subunit of the present invention can
be employed for the preparation of LAK cell and T cell
activator and antitumor compositions and in methods for
stimulating LAK cell, T—cells or Natural Killer Cells.
The 35 kDa CLMF subunit of the present invention can also be
analyzed to determine the active sites for CLMF activity. The
information from this analysis may be used to predict and
produce fragments or peptides, including synthetic peptides.
Among the known techniques for
determining such active sites are x—ray crystallography.
UV
spectroscopy and site specific mutagenesis. Accordingly. the
fragments obtained in this way may be employed in methods
for stimulating T-cells or LAK cells.
having the activity of CLMF.
nuclear magnetic resonance, circular dichroism,
The CLMF subunits prepared in accordance with
this invention or pharmaceutical compositions
comprising the 35 kDa CLMF subunit may be
administered to warm blooded mammals for the clinical uses
indicated above. The administration may be by any
conventional modes of administration of agents which exhibit
antitumor activity auch as by intralesional or parenteral
application either intravenously. subcutaneously or
intramuscularly. Obviously, the required dosage will vary
_ 22 _
with the particular condition being treated, the severity of
the condition, the duration of the treatment and the method
for administration. A suitable dosage form for pharmaceuti-
cal use may be obtained from sterile filtered, lyophilized
protein reconstituted prior to use in a conventional manner.
It is also within the skill of the artisan in the field to
prepare pharmaceutical Compositions comprising 35 kDa CLMF subunit
of the present invention by mixing the said CLMF subunit
with compatible pharmaceutically acceptable carrier
materials such as buffers. stabilizers, bacteriostats and
other excipients and additives conventionally employed in
pharmaceutical parenteral dosage forms. The present
invention also relates to such pharmaceutical compositions.
The preferred form of administration depends on the
intended mode of administration and therapeutic
application. The pharmaceutical compositions comprising a
CLMF protein or peptide derivative of the present invention
also will preferably include conventional pharmaceutically
acceptable carriers and may include other medicinal agents
(e.g.
e.g., human serum albumin or plasma preparations.
interleukin—2), carriers. adjuvants. excipients, etc.,
Preferably, the compositions of the invention are in the
form of a unit dose and will usually be administered one or
more times a day. The unit dose is preferably packed in 1 ml
vials containing an effective amount of the 35 kDa CLMF
subunit and if desired of interleukin—2 in lyophilized
form. The vials containing the CLMF subunit
and if desired the interleukin-2 are preferably packed in a
container together with written instructions describing the
correct use of the pharmaceutical composition. The present
invention relates also to such a unit dose packed in a
container, preferably together with a separate unit dose of
inter1eukin—2, most preferably together with the appropriate
instructions. Furthermore the present invention relates to a
process for the preparation of the said unit dose.
_ 23 _
In order that our invention herein described may be more
fully understood, the following examples are set forth. It
should be understood that these examples are for
illustrative purposes only and should not be construed as
limiting this invention in any way to the specific
embodiments recited therein. It has to be noted that the
specific product names and suppliers mentioned below are not
meant to be mandatory. The person skilled in the art is in a
position to select alternative products from other suppliers.
EXAMPLE
PURIFICATION AND CHARACTERIZATION OF CYTOTOXIC LYMPHOCYTE
MATURATION FACTOR (CLMF)
Production of Supernatant Liquid Containing_CLMF.
Human NC-37 B lymphoblastoid cells (ATCC CCL 214,
American Type Culture Collection. Rockville, MD) were used
for production of CLMF. These cells were maintained by
serial passage in RPMI 1640 medium supplemented with 5%
heat-inactivated (56°C. 30 min.) fetal bovine serum,
and 100 ug/ml
streptomycin (all cell culture media were from GIBCO
Grand Island. NY).
mM
L—glutamine, 100 units/ml penicillin,
Laboratories,
Higher producer sublines of NC-37 cells were derived by
limiting dilution cloning in liquid microcultures. Each
well of three Costar 3596 microplates (Costar Co.,
Cambridge, MA) received 100 ul of a cell suspension
containing five NC—37 cells/ml. The medium used for the
cloning was a 1:1 mixture of fresh passage medium and
filtered, conditioned medium from stock cultures of the
parent NC-37 cells. One week and two weeks after culture
initiation each of the microcultures was fed with 50 ul of
the 1:1 mix of fresh and conditioned medium. Between 3 and
weeks after culture initiation the contents of wells
_ 24 _
containing clones of NC—37 cells were harvested and passed
into larger cultures.
when the number of cells in a given subline exceeded 1.4
X 106, one million cells were stimulated to produce CLMT
in 1 ml cultures containing 3 ng/ml phorbol l2—myristate
l3~acetate (PMA) (Sigma Chemical Co., St.
ng/ml calcium ionophore A23lB7 (Sigma).
Louis, MO) and 100
Supernatants were
harvested from the cultures after 2 days. dialyzed against
about so volumes of Dulbecco's phosphate buffered saline
(Gibco) using e.g. SPECTROPOR® #1 tubing (Fisher
Scientific) overnight with one change of buffer and then for
4 hours against 50 volumes of RPMI 1640 medium with 50
ug/ml of gentamicin (both from Gibco) and tested for CLMF
by means of the T cell growth factor assay (see below).
Three sublines, NC—37.89, NC—37.98, and NC—37.lO2, werei
identified which routinely produced CLMF at titers 3 4 times
the titers produced by the parental NC-37 cell line. Since
cells from these three sublines produced CLMF at similar
titers (3 800 units/ml), culture supernatants derived from
the three sublines were pooled for use as starting material
for the purification of CLMF.
Bulk production of CLMF was carried out in roller bottle
cultures on a roller apparatus set at about 38 rpms (wheaton
Cell Production Roller Apparatus Model 11, Wheaton
Millville. NJ).
containing l-1.5 X 106 NC~37;B9, NC—37.98 or NC—37.l02
Instruments, Cell suspensions were prepared
cells/ml in RPMI 1640 medium supplemented with 1%
Nutridoma—SP (Boehringer Mannheim Biochemicals.
IN), 100 units/ml
penicillin, 100 ug/ml streptomycin. 10 ng/ml PMA and 20-25
ng/ml calcium ionophore A23l87.
Indianapolis, 2 mM L—glutamine,
Two hundred fifty to three
hundred fifty ml aliquots of the cell suspensions were added
to Falcon 3027 tissue culture roller bottles (Becton
Dickinson, Lincoln Park. NJ) which had been gassed with a
mixture of 5% CO2, 95% air. The roller bottles were then
_ 25 -
capped tightly and incubated at 37°C with continuous rolling
for three days. At the end of this time,
supernatants were harvested.
the culture
EDTA and phenylmethylsulfonyl
fluoride (both from Boehringer Mannheim) were added to the
culture supernatants at final concentrations of 1 mM and 0.1
mM. to retard proteolytic degradation.
supernatants were stored at 4°C.
respectively, The
Lympokine Activated Killer (LAK) Cell Induction (LCI)
Assay.
Culture supernatants and chromatographic fractions were
tested for their ability to synergize with rIL-2 to induce
the generation of cytolytic LAK cells as follows. Human
peripheral blood mononuclear cells (PBMC) were isolated by
the following method. Blood from normal volunteer donors
was drawn into syringes containing sufficient sterile
preservative—free heparin (Sigma) to give a final
The blood was
balanced salt solution (HBSS)
without calcium or magnesium (GIBCO). The diluted blood was
then layered over 15 ml aliquots of Ficoll/sodium
concentration of approximately 5 units/ml.
diluted lzl with Hanks’
diatrizoate solution (Lymphocyte Separation Medium, organon
Teknika Corp.. Durham, NC) in 50 ml Falcon 2098 centrifuge
tubes. The tubes were centrifuged for 30 minutes at room
temperature at 500 x g. Following centrifugation, the cells
floating on the Ficoll/sodium diatrizoate layer were
collected and diluted by mixing with 3 2 volumes of HBSS
without calcium or magnesium. The resulting cell suspension
was then layered over 15 ml aliquots of 20% sucrose (Fisher)
in RPMI 1640 medium with 1% human AB serum (Irvine
Scientific, Santa Ana, CA) in Falcon 2098 centrifuge tubes.
The tubes were centrifuged for 10 minutes at room
temperature at 500 x g. and the supernatant fluids were
discarded. The cell pellets were resuspended in 5 ml of
HBSS without calcium or magnesium, repelleted by
centrifugation, and finally resuspended in the appropriate
- 25 _
culture medium. Accessory cells were removed from the PBMC
by treatment with 5 mM L—glutamic acid dimethyl ester
(Sigma) using the same conditions as described by Thiele et
al. J. Immunol. l31:2282—229O (1983) for accessory cell
depletion by L-leucine methyl ester except that the glutamic
acid ester was substituted for the leucine ester.
The accessory ce1l—depleted PBMC were further
fractionated by centrifugation on a discontinuous Percoll
density gradient (Pharmacia, Piscataway, NJ) as described by
Wong et al.. Cell Immunol. ;;l:39—54 (1988).
cells recovered 41. 45, and 58% Percoll layers
used as a source of LAK cell precursors in
cells recovered from the Percoll gradient
suspended in tissue culture medium (TCM)
composed of a 1:1 mixture of RPMI 1640 and Dulbecco's
modified Eagle's medium.
Mononuclear
from the 38.
were pooled and
The
were washed and
the assay.
supplemented with 0.1 mM
nonessential amino acids} 60 ug/ml arginine HCl, 10 mM
HEPES buffer. 2 mM L—g1utamine. .
ng/ml streptomycin (all available from GIBCO), 5 x 10-5
M 2—mercaptoethanol (Fisher Scientific. Fair Lawn, NJ).
units/ml penicillin, 100
mg/ml dextrose (Fisher),
CA). These cells were incubated in
24-well tissue culture plates (costar, Cambridge, MA) in
ml cultures (7.5 x 105
and 5% human AB serum (Irvine
Scientific, Santa Ana,
, -4
cells/culture) to which 10 M
hydrocortisone sodium succinate (Sigma) was added to
minimize endogenous cytokine production. some cultures also
received human rIL-2 (supplied by Hoffmann-La Roche, Inc.,
Nutley, NJ) at a final concentration of S units/ml and/or
supernatants to be assayed for CLMF activity. All cultures
were incubated for 3~4 days at 37°C in a humidified
atmosphere of 5% CO2. 95% air.
At the end of this incubation, the contents of each
culture were harvested. and the cells were pelleted by
centrifugation and resuspended in 0.5 ml of fresh TCM. One
tenth ml aliquots of these cell suspensions were mixed with
_ 27 -
lCr—labelled K562 or Raji cells (both
cell lines may be obtained from the ATCC) and tested for
.1 ml aliquots of
their lytic activity in 5 hour Cr release assays. The
. . 51
method for labelling target cells with Cr and performing
the cytolytic assays have been described by Gately et a1.,
release was calculated as [(g - g)/(100 — 5)] X 100, where g
. 51
the percentage of Cr released from target cells
Slct
released spontaneously from target cells incubated alone.
The total releasable 51
incubated with lymphocytes and g is the percentage of
see Gately et
al..
LAK Cell Induction Microassay. The microassay for
measuring synergy between rIL—2 and CLMF-containing
solutions in the induction of human LAK cells was similar to
the LAK cell induction assay described above but with the
following modifications. Human peripheral blood mononuclear
cells which had been depleted of accessory cells and
fractionated by Percoll gradient centrifugation as described
above were added to the wells of Costar 3596 microplates (5
x 104 cells/well).
(5 units/ml final concentration) and/or purified CLMF or
Some of the wells also received rIL-2
immunodepleted CLMF—containing solutions. All cultures
M hydrocortisone sodium succinate (Sigma)
and were brought to a total volume of 0.1 ml by addition of
TCM with
3 days at 37°C,
, -4
contained 10
% human AB serum. The cultures were incubated for
lCr—labelled K562
cells (5 x 104 cells/ml in TCM with 5% human AB serum)
were added to each well.
after which 0.1 ml of
The cultures were then incubated
overnight at 37°C. Following this, the cultures were
centrifuged for 5 minutes at 500 x g. and the supernatant
solutions were harvested by use of a skatron supernatant
collection system (Skatron.
. .
Cr released into each supernatant solution was measured
Sterling, VA). The amount of
with a gamma counter (Packard, Downer's Grove, IL), and the
. . 1
% specific Cr release was calculated as described
above. All samples were assayed in quadruplicate.
Cytolytic T Lymphocyte (CTL) Generation Assay.
Methods used for generating and measuring the lytic
activity of human CTL have been described in detail by
Gately et al. in J. Immunol. lggz l274—l282 (1986) and by
Wong et al. in Cell. Immunol. lll: 39-54 (1988). Human
peripheral blood mononuclear cells were isolated from the
blood of normal volunteer donors, depleted of accessory
cells by treatment with L-glutamic acid dimethyl ester. and
fractioned by Percoll gradient centrifugation as described
above. High density lymphocytes recovered from the
interface between the 45% and 58% Percoll layers were used
as responder lymphocytes in mixed lymphocyte—tumor cultures
(MLTC). CTL were generated in MLTC in 24-well tissue
culture plates (Costar #3424) by incubation of Percoll
gradient—derived high density lymphocytes (7.5 x 105
culture) together with l x 105 uv—irradiated melanoma
cells e.g. HTl44 (obtainable from ATCC) or with 5 x 104
gamma—irradiated melanoma cells e.g. HTl44 in TCM with 5%
human AB serum (1.2 ml/culture). For uv—irradiation, HTl44
cells were suspended at a density of l-1.5 x 106 cells/ml
in Hanks’ balanced salt solution without phenol red (GIBCO)
containing 1% human AB serum. One ml aliquots of the cell
suspension were added to 35 x 10 mm plastic tissue culture
dishes (Falcon #3001), and the cells were then irradiated
(960 uw/cmz for 5 min) by use of a 254 nm uv light
(model UVG—54 MINERAL1GHT® lamp, U1tra—violet Products,
CA). For gamma irradiation, HTI44 cells
were suspended at a density of l—5 x 106 cells/ml in TCM
with 5% human AB serum and irradiated (10,000 rad) by use of
a cesium source irradiator (model 143, J.L. Shepherd and
CA).
HTl44 were centrifuged and resuspended in TCM with 5% human
Inc.. San Gabriel,
Associates, San Fernando, Uv— or gamma-irradiated
_ 29 -
AB serum at the desired cell density for addition to the
MLTC. In addition to lymphocytes and melanoma cells, some
MLTC received human rlL-2 and/or purified human CLMF at the
concentrations indicated. Hydrocortisone sodium succinate
(sigma) was added to the MLTC at a final concentration of
-4 M (cultures containing uv-irradiated melanoma cells)
or 1o"S M (cultures containing gamma—irradiated melanoma
cells) to supress endogenous cytokine production [s. Gillis
et al.. J. 13;: 1624-1631 (1979)) and to reduce the
generation of nonspecific LAK cells in the cultures [L.M.
Muul and M.K. Gately, J. 1202-1207 (1984)].
at 37°C in a humidified
for 6 days. At the end of
replicate cultures were pooled.
Immunol.
Immunol. 132:
The cultures were incubated
atmosphere of 5% CO2 in air
this time, lymphocytes from
centrifuged, resuspended in 1.2 ml TCM containing 5% human
AB serum, and tested for their ability to lyse HTl44
melanoma cells. and. as a specificity control, K562
erythroleukemia cells (obtainable from ATCC) in overnight
Cr release assays.
Slcr
[JNCI Q3:
measurement of lympocyte—
Melanoma cells and K562 cells were labeled with
sodium chromate as described by Gately et al.
1245-1254 (l982)].
mediated lysis of
Likewise,
Slcr-labeled melanoma cells was
performed in a manner identical to that described by Gately
et al. (ibid.) for quantitating lysis of glioma target
Slcr-labeled K562 cells,
0.1 ml aliquots of lymphocyte suspensions were mixed with 25
ul aliquots of 51Cr—labe1ed K562 (2 X 105 cells/ml in
TCM with 5% human AB serum) in the wells of costar 3696
cells. For assaying the lysis of
“half—area" microtest plates. After overnight incubation at
°C. the plates were centrifuged for 5 min at 1400 x g, and
50 ul of culture medium was aspirated from each well. The
amount of lcr in each sample was measured with a gamma
counter (Packard), and the %
calculated as described above.
. . 51
specific Cr release was
All assays were performed in
quadruplicate, and values in the table (see below) represent
_ 30 _
the means 1 1 S.E.M. of replicate samples.
T cell growth factor (TGF) assay.
The ability of culture supernatants and chromatographic
fractions to stimulate the proliferation of PHA~activated
human T lymphoblasts was measured as follows. Human PBMC
were isolated by centrifugation over discontinuous Ficoll
and sucrose gradients as described above for the LCI assay.
The PBMC (5 x 105 cells/ml) were cultured at 37°C in TCM
containing 0.1% phytohemagglutinin—P (PHA-P) (Difco
Laboratories, Detroit, MI). After 3 days.
split 1:1 with fresh TCM. and human rIL-2 was added to each
culture to give a final concentration of 50 units/ml. The
cultures were then incubated for an additional 1 to 2 days.
at which time the cells were harvested. washed, and
resuspended in TCM at 4 x 105 cells/ml. To this cell
suspension was added heat—inactivated goat anti-human rlL-2
the cultures were
antiserum (final dilution: 1/200) to block any potential
lL~2—induced cell proliferation in the assay. This
antiserum may be prepared using methods well—known in the
art or may be obtained from Genzyme Co.. Boston, MA. The
antiserum used was shown to cause 50% neutralization of 2
units/ml rIL—2 at a serum dilution of 1/20,000.
Fifty ul aliquots of the cell suspension containing
anti~IL—2 antiserum were mixed with 50 ul aliquots of
serial dilutions of culture supernatants or chromatographic
The
cultures were incubated for 1 day at 37°C in a humidified
fractions in the wells of costar 3596 microplates.
. . 3 . .
atmosphere of 5% CO2 in air. and 50 ul of H—thym1d1ne
(New England Nuclear, Boston, MA), 10 uCi/ml in TCM, were
then added to each well. The cultures were further
incubated overnight, the culture contents
Subsequently.
were harvested onto glass fiber filters by means of a cell
harvester (Cambridge Technology Inc., Cambridge. MA), and
. . . . .
H—thymidine incorporation into cellular DNA was measured
_ 31 _
by liquid scintillation counting. All samples were assayed
in triplicate.
In purifying CLMF it was necessary to define units of
activity in order to construct chromatographic elution
profiles and to calculate the percent recovery of activity
To do
this, a partially purified preparation of human cytokines
produced by coculturing PHA-activated human PBMC with NC—37
cells was used as a standard. The preparation was assigned
an arbitrary titer of 2000 units/ml. Several dilutions of
and the specific activity of the purified material.
this preparation were included in each TGF or LAK induction
assay. The results obtained for the standard preparation
were used to construct a dose-response curve from which
could be interpolated units/ml of activity in each unknown
sample at the dilution tested. Multiplication of this value
by the dilution factor yielded the activity of the original
sample expressed in units/ml.
For antibody neutralization studies, the TGF assay was
modified as follows. Twenty—five ul aliquots of
CLMF—containing medium were mixed with 50 ul aliquots of
serial dilutions of antiserum or antibody solutions in the
wells of COSTAR 3S96® microplates.
incubated for 30 minutes at 37°C, and 25 ul aliquots of a
suspension of PHA—activated lymphoblasts (8 x 105/ml in
TCM plus 1:100 anti—rIL—2) were then added to each well.
The cultures were further incubated, pulsed with
The mixtures were
H—thymidine, harvested, and analyzed for 3H—thymidine
incorporation as described above.
Natural killer LNK) cell activation assay.
Purified CLMF was tested for its ability to activate NK
cells when added alone or in combination with rIL-2 as
follows. Human PBMC were isolated by centrifugation over
discontinuous Ficoll and sucrose gradients as described
_ 32 _
above and were suspended in RPMI 1640 medium supplemented
with 10% heat—inactivated fetal bovine serum, 100 units/ml
penicillin, 100 ug/ml streptomycin, and 2.mM L—glutamine.
The PBMC were incubated overnight at 37°C in 1 ml cultures
6
(5 x 10
purified CLMF at various concentrations.
cells/culture) together with rIL-2 and/or
After l8—20 hours,
the contents of the cultures were harvested and centrifuged,
and the cells were resuspended in the same medium used for
the overnight cultures. The cytolytic activity of the
cultured PBMC was then assessed in 51
described above.
Cr release assays as
Concentration of cell supernatant solutions
Stored. frozen crude human CLMF supernatant solutions
totaling 60 liters prepared from several batches of induced
NC-37 cells were pooled and concentrated 30-fold using the
Pellicon Cassette System (30,000 NMWL PTTKOOOOS; Millipore
Corp.. Bedford. MA). After concentrating to the desired
volume of approximately 1.9 liters, a buffer exchange was
performed with 10 mM MES, pH adjusted to 6.0 with 10 N
NaOH. The concentrate was centrifuged at 10,000 X g for
minutes at 4°C and the precipitate discarded.
Ion—Exchange Chromatography on NuGel P—SP Column
The concentrated supernatant solution was applied at a
flow rate of 120 ml/hr to a Nu—Gel P-SP (separation
Industries, Metuchen, NJ) column (5 x 5 cm), equilibrated in
l0mM MES, pH 5.0.
absorbance monitoring at 280 nm was obtained.
The column was washed until baseline
Absorbed
proteins were then eluted with a 500 ml salt gradient from
O to 0.5 M NaCl/l0 mM MES, pH 6.0 at a flow rate of 2 ml/min
(Fig. l). Aliquots of fractions were assayed for T cell
growth factor (TGF) activity. Fractions containing TGF
activity were pooled and dialyzed (Spectra/Por 7, Fisher
Scientific) against 50 volumes 20 mM Tris/Hcl, pH 7.5 in
r 33 _
order to reduce the salt concentration of the preparation by
50-fold.
Q1e—Affinity Chromatography on Blue B-Agarose Column
The dialyzed sample was centrifuged at 10,000 x g for 10
The
supernatant solution was applied at a flow rate of 20 ml/hr
minutes at 4°C and the precipitate discarded.
to a Blue B~Agarose (Amicon. Danvers, MA) column (2.5 x 10
cm) equilibrated in 20 mM Tris/HCl, pH 7.5. The column was
washed with this same buffer until baseline absorbance
monitoring at 280 nm was obtained. Absorbed proteins were
then eluted with a 500 ml salt gradient from 0 to 0.5 M
Nacl/20 mM Tris/Hcl, pH 7.5 at a flow rate of 15 ml/hr
(Fig. 2). Aliquots of fractions were assayed for TGF
activity. Fractions containing TGF activity were pooled and
dialyzed (Spectra/Por 7, Fisher Scientific) against 100
volumes 20 mM Tris/HCl, pH 7.5 in order to reduce the salt
concentration of the preparation by 100-fold.
Ion—Exchanqe Chromatography on Mono Q Chromatography
The dialyzed sample was filtered through a 0.45 um
cellulose acetate filter (Nalgene Co., Rochester, NY) and
the filtrate applied at a flow rate of 60 ml/hr to a Mono Q
HR 5/5 (Pharmacia LKB Biotechnology, Inc.. Piscataway, NJ)
column (5 X 50mm) equilibrated in 20mM Tris/HCl, pH 7.5.
The column was washed with this same buffer until baseline
Absorbed
proteins were then eluted with a 1 hr linear salt gradient
from O to 0.25 M NaC1/20 mM Tris/HCI, pH 7.5 at a flow rate
of so ml/hr (Fig. 3).
absorbance monitoring at 280 nm was obtained.
Aliquots of fractions were assayed for
TGF activity and protein purity was assessed without
reduction by SDS—PAGE [Laemm1i. Nature (London) g;1:680—685
(1970)] using 12% slab gels. Gels were silver stained
[Morrissey, Anal. Biochem. l;1:307-310 (l98l)] to visualize
protein (Fig. 4). Fractions 36 and 37 were of greater than
_ 34 _
% purity and revealed a major band at 75.000 molecular
weight. Fractions 38 through 41 containing TGF activity,
revealed the 75 kDa protein by SDS-PAGE with major
contaminants at 55,000 and 40,000 molecular weight.
Therefore. to eliminate these contaminating proteins,
fraction 38 of the previous Mono Q chromatography was
diluted 1:1 vol/vol with 8 M urea and pumped onto a Vydac
diphenyl column using a reversed—phase HPLC enrichment
technique. The column was then washed with 5 ml of 0.1%
trifluoroacetic acid. Elution of the proteins was
accomplished with a gradient of 0—70% acetonitrile over 7
hrs in 0.1% trifluoroacetic acid (Fig. 5). Aliquots of
fractions were assayed for TGF activity. Protein purity of
the fractions containing TGF activity was assessed by
sDS—PAGE under non—reducing conditions using a 10% slab
gel.
6).
and revealed protein of 75,000 molecular weight.
The gel was silver stained to visualize protein (Fig.
Fractions 86 through 90 were of greater than 95% purity
Fractions
87 and 88 were pooled and aliquots were analyzed by SDS—PAGE
under reducing (in the presence of B—mercaptoethanol) and
non—reducing conditions (in the absence of B-mercapto—
ethanol). Under the reducing conditions. the 75,000
molecular weight CLMF was separated into two subunits of
40,000 and 35.000 daltons (Fig. 7). Thus it was concluded
that CLMF is a 75 kDa heterodimer composed of disulfide—
-bonded 40 kDa and 35 kDa subunits.
The overall purification of CLMF that was achieved is
shown in Table l. The protein content of the Mono Q— and
Vydac diphenyl—purified material was calculated on the basis
boa X ~.m
mm+nm zowuucuh
oHo.o moo.o meg x -.m mod x vn.m H.H Hzcozaflo
~vAxmm cowuomuu
nod x m.m mov.o Hmo.o boa x mv.m mod x oa.o m o ocoz
hm :o..3u.our._
nofi x m.m m>o.o mno.o woa x v.o coa x oe.e A 0 ocoz
boa x m.H dd vm.o mod x v.~ ooa x HH.m me mmoumm<»m-m:Hn
oofi x o.m mo o>.o wed x m.H ooa x oo.~ ca mmsm fimozz
munduzmocov
vofi x m.m ommm mm.H mod n c.m noa x >m.~ ovm.fi cmumuflfiumuyfis
L
mpcmuwcummsw
oz oz oz aofi x m.~ moa x am.~ ooo.oo Hawo cofioom
Amsxav Aoav Adsxosv As. AHE\:v AHEV
»u«>«n_u< cflmuoum cwwuoum mu..H:: .3fi>W_u<
ofiwommm Hauom. cmfioom Hanan. cofioom ®E:.mO>
H mam
mmum
of amino acid analysis.
. 7 .
units/mq and 5.2 x 10 units/mg for Mono Q- and Vydac
diphenyl—purified material respectively,
A specific activity of 8.5 x 107
was obtained. The
fact that the diphenyl-purified protein has a slightly lower
specific activity than the Mono Q—purified material may be
due to inactivation or denaturation of some or the molecules
of CLMF in the HPLC elution solvents (i.e..
0.1% trifluoroacetic acid).
acetonitrile in
Chemical Characterization
j\:
The ability to prepare homogeneous CLMF allowed for the
first time the determination of the amino acid composition
and a partial sequence analysis of the naturally occurring
CLMF protein. Between 10 and 20 picomoles of
Mono—Q—purified CLMF was subjected to hydrolysis.
amino acid composition was determined (Table 2).
cysteine and tryptophan were not determined (ND).
Quantitation of histidine was not possible due to a large
artifact peak.
and its
Proline,
associated with Tris. coeluting with His (*1.
Between 5 and 30 picomoles of diphenyl—purified CLMF was
subjected to hydrolysis with and without pre-treatment with
performic acid. Complete amino acid composition was thus
obtained (Table 3) with the exception of tryptophan.
Amino—terminal sequence determination was attempted by
automated Edman degradation on 100 pmol of the Mono
Q-purified CLMF. Data from the first 22 cycles indicated
two sequences present. as would be expected from the
hetetodimeric structure of CLMF.
summarized as follows:
These results may be
Claims (14)
- (CLMF) protein characterized in that (a) the subunit comprises the amino acid sequence if combined with comprising the amino the second subunit of acid sequence
- The subunit of claim 1, wherein the combined CLMF protein displays a specific activity of at least 5.2 x 107 Units/mg when determined in a T cell growth factor assay, and when combined with the protein as defined in claim l(b).
- 3. The sequence Arg Leu Gln Glu Glu Asn Se: Ty: Ala Asn Asn Phe Arg Ala Asn His Lys IIe Ala Se: AC9 Glu Lys MET Se: Ty: IIe Se: subunit of claim 1 or 2 comprising the amino acid Leu His Ala Asp CY5 Arg Lys Asp Leu Len Glu Lys Arg Pro Se: Arg His Leu Glu Th: Len Len Ala Th: Th: Ala Val Cln Gln Glu Pro Th: Se: Lys Val Val Lys Val Ala Asn Th: Asp Leu Se: Phe MET Asp Ile Pro Ile Th: Th: Leu Leu Ile Glu Phe MET Ty: Pro Asp Gln Lys Ile Pro Leu Glu Th: Leu Ile Gln Lys Glu Lys Leu Asp. Asp Arg Phe Lys Th: Th: Ala Val A:q Leu Se: Cys Arg Pro Ala TY: Asp Lys Asn Leu Glu Gln Se: Ire Val Gly Val Pro Lys Asn Gly Cys Phe Ile Gln Leu Léu Th: MET Se: Cys Th: Glu Se: Leu Lys Phe Ala Glu Leu Se: Phe Asn Th: Se: Pro MET Se: Th: Cys Cys Leu Glu Val Leu Ala IIe Asn Gln Phe Asp Phe Asn Se: Cys Leu Set Set Th: Leu Asp Asn Pro Ala Leu Leu Glu His Ty:
- 4. A polynucleotide encoding a subunit as claimed in any of claims 1 to 3.
- 5. A polynucleotide encoding a subunit as claimed in any one of claims 1 to 4 which polynucleotide comprises the nucleotide S €qU€1’1C€ ATG TGT CCA GCC CCC AGC CTC CTC CTT GTG GCT ACC CTG GAC CAC CTC AGT TTG CCC AGA AAC CTC CCC CTG GAC CCA GGA ATG TTC CCA TCC CTT CTG GTC cac CAC TCC cm AAC‘ cm cm AGG GCC GTC AGC AAC ATG CTC CAG AAG GCC AGA CAA ACT CTA GAA TTT TAC CCT TCC ACT TCT GAA GAG ATT CAT CAT GAA GAT ATC ACA AAA CAT AAA ACC AGC ACA GTG GAG GCC TGT TTA CCA TIC GAA TTA ACC AAG AAT GAG AGT TCC CTA AAT TCC AGA GAG ACC TCT TTC ATA ACT AAT GGG ACT TGC CTG GCC TCC AGA AAG ACC TCT TIT ATC ATG GCC CTG TGC CTT ACT ACT ATT TAT GAA GAC TTG AAG ATG TAC CAG CTG GAG TTC AAG ACC ATG AAT GCA AAG CTT CTG ATG GAT CCT RAG AGG CAG ATC TTT CTA CAT CAA AAC ATG CTG GCA GTT ATT CAT GAG CTG ATG CAG CCC CTG AAT TTC AAC ACT GAG ACT GTG CCA CAA AAA TCC TCC CTT GAA GAA CCG CAT TTT TAT AAA ACT AAA ATC RAG CTC TGC ATA CTT CTT CAT GCT TTC AGA ATT CGG GCA CTG ACT ATT GAC AGA GTG ACG AGC TAT CTG AAT GCT TCC.
- 6. A recombinant vector comprising a polynucleotide encoding a subunit as claimed in any one of claims 1 to 3 or comprising all or parts of the polynucleotide of claim 5.
- 7. A microorganism transformed with a recombinant vector comprising a polynucleotide encoding a subunit as claimed in any one of claims 1 to 3 or all or parts of the polynucleotide of claim 5.
- 8. A polyclonal or monoclonal antibody directed to a subunit as claimed in any one of claims 1 to 3.
- 9. A process for producing a subunit according to any one of claims 1 to 3 which process comprises culturing a microorganism transformed with a recombinant vector comprising a polynucleotide encoding the said subunit in a culture medium under conditions permitting the expression of the encoded subunit.
- 10. A process for producing a subunit according to any one of claims 1 to 3 which process comprises (a) preparing sub—unit peptides of the said subunit by conventional peptide synthesis methods; and (b) coupling the sub—unit peptides under conditions favouring the formation of peptide bonds.
- 11. A process for producing the CLMF protein which comprises a process as claimed in any one of claims 9 to 10.
- 12. A pharmaceutical composition comprising a subunit as claimed in any one of claims 1 to 3 and a pharmaceutically acceptable diluent, adjuvant or carrier.
- 13. Use of a subunit as claimed in any one of claims 1 to 3 for the manufacture of a medicament for antitumor therapy.
- 14. Use of a subunit as claimed in any one of claims 1 to 3 for the preparation of a CLMF protein.
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
USUNITEDSTATESOFAMERICA22/12/19894 | |||
US45570889A | 1989-12-22 | 1989-12-22 | |
US52093590A | 1990-05-09 | 1990-05-09 | |
US57228490A | 1990-08-27 | 1990-08-27 |
Publications (3)
Publication Number | Publication Date |
---|---|
IE19990007A1 IE19990007A1 (en) | 2000-11-01 |
IE990007A1 IE990007A1 (en) | 2000-11-01 |
IE84906B1 true IE84906B1 (en) | 2008-06-11 |
Family
ID=
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