Note: Descriptions are shown in the official language in which they were submitted.
1
_1_
Title
EXPRESSION OF HIV PROTEINS IN DROSOPHIL~A CELLS
Fi~ld of Tnventian
The present invention relates generally ~o
expression of HIV proteins in Drosophila cells and
purification of the expressed gene products. More
specifically, this invention relates to the production of
novel mutant gp160 and gp120 gene products by this
expression system.
Background of the Invention
Human immunodeficiency virus type d (HIV--1) is
the etiological agent of acquired immune deficiency
syndrome, also known as AIDS. This retrovirus has a
complex genetic organization, including the long germinal
repeats (LTRs); the gag, ~, and env genes, and other
genes. This retrovirus carries a number of viral antigens
which are potential candidates either alone or in concert
as vaccinal agents capable of inducing a protective immune
response.
Among the more p~omisin.g of the HIV-l antigens
is the viral envelope glycoprotein (gp160) or specific
fragments thereof. The env gene encodes the 160
kilodalton (kd) precursor glycoprotein of the viral
CA 02003794 1998-12-07
-2-
1 envelope. gp160 is cleaved posttranslationally into a
120 kd glycoprotein (gp120) and a 41 kd glycoprotein
(gp41), which are present at the virus surface.
gpI20, a 511 amino acid glycoprotein, is located
on the amino terminal two-thirds of the gp160 glycoprotein
and is exposed on the outside of the virus. gp120 is
crucial to the interaction of the virus with its cellular
receptor, the CD4 protein present on the surface of helper
T4 lymphocytes, macrophages, and other cells of the
immune system. gpI20 thus determines the tissue
selectivity of viral infection and contributes to the
cytopathogenicity of HIV through its involvement in
syncytium formation.
gp4l, a 345 amino acid protein derived from the
~-5 carboxyl terminus of gp160, is an integral membrane
protein of HIV-1. gp41 contains a series of hydrophobic
amino acids which anchor the protein in the lipid bilayer
of the cellular plasma membrane. The carboxyl end of gp41
is believed to protrude into the viral particle. gp41 or
a portion thereof is believed to be responsible for fusion
between the HIV glycoproteins expressed at the surface of
the cell with cells displaying surf ace T4 receptors.
The portion of gp41 which is believed to be responsible
for this fusion is located at the amino terminal. Such
fusion is believed to play a role in viral replication.
See, e.g., M. Kowalski et al, Science, 237: 1351-55
(1987); D.M. Knight et al, Science, 236: 837-36 (1987).
These viral glycoproteins assume a tertiary
structure as viral spikes protruding outwards from the
surface of the viral particle. About 70 to 80 spikes are
believed to be associated with each newly synthesized
viral particle. As the viral particle ages, the spikes
disappear, apparently because the association between the
gp120 and gp41 is weak. Thus, for newly synthesized viral
particles, this viral glycoprotein spike is believed to be
the most immediate target accessible to the immune system
following infection.
ia~~~ i':~~='~
1 Virus neutralizing antibodies have been reported
directed against gp120 and gp41 epitopes. It has been
specifically noted that a 'target site for type specific
neutralizing antibodies is located in the 3' half of the
gp120 glycoprotein molecule.
The env gene of HIV-1 has thus been the target
a~ numeraus recent investigations. Expressian of
glycosylated gp160 has previously been obtained in
mammalian cells and pertain baculavirus insect cells by
groups which have also reported the induction of both
humoral and cellular immune responses to these antigens.
gp120 has been expressed recombinantly with the use of
heterologous promoters in several systems. See, e.g.,
S. Chakrabarti et al, Nature (London), 320: 535 (1986);
S.I. Hu et al, Nature (London), 320: 537 (1986); and
M.P. Kieny et al, Biotechnology, _4: 790 (1986).
L.A. Lasky et al, Science, 233: 209-212 (1986)
constructed a number of plasmids containing mutant env
genes for tranfectian into mammalian cells, specifically
Chinese hamster ovary (CHO) cells. These researchers
secreted a gene product encoded in a plasmid containing
the first 50 amino acids of the glycoprotein D (gD)
protein joined in phase to an amino acid sequence
(#61-#531) of the env protein, an HBsAg polyA signal, a
DHFR gene and the SV40 origin of replication . A
recombinant envelope antigen was produced containing 25
amino acids of gD at its amino terminus and lacking 30
residues from the mature processed from of gp120, and also
having a deletion of the gp41 sequence (about 20 amino
acids of the carboxyl terminus to the actual 160 kd
precursor processing site). The resulting gene was 520
amino acids in length. When transfected into CHO cells,
the cell-conditioned supernatants contained a 130 kd
protein, Balled gp130.
A later report, L.A. Lasky et al, Cell, _50:
975-986 (1987), discussed the interaation between the
_4_
1 gp120 glycoprotein and its cellular receptor, CD4. By
deleting 12 amino acids contained within amino acids
#410-421 of gp120, a complete loss of binding resulted.
Similarly, a single amino acid substitution at position
417 resulted in decreased binding,
Kawalski et al, cited above, introduced deletion
and insertion mutations into a plasmid that encodes the
envelope glycoprotein derived from the HTLV-IIIB strain
of HIV-1. The plasmid also contained the art gene.
Mammalian CD4+ and CD4- cell lines expressing the tat gene
product were used as transfection recipients to study
ability of the transfected cells tn fuse.
Knight et al, cited above, describe expression
of the art/trs transactivator protein of HIV in mammalian
cells. The mammalian cell line used for expression of
these HTV proteins was the COS-~-7 monkey cell line. These
plasmids utilized the HIV LTR as a promoter and RNA
processing signals from SV40 to express the inserted DNA
as a functional messenger RNA. To express gp120, a
plasmid pENV160 was developed which contains the entire
coding region of the env gene fused to the HIV LTR.
S.W. Pyle, Aids Research and Human Retroviruses,
3(4): 387 (1987) disclose the purification of the gp120
glycoprotein from culture fluids of HIV infected H9 cells
by immunoaffinity chromatdgraphy.
U.S. Patent 4,725,669 also discloses
glycoproteins of approximately 160 kd and 120 kd obtained
from the human H9/HTLV-III cell line, each having an
approximately 90 kd unglycosylated moiety.
Fox, Hiotechnoloqy, _6: 116 (1988) reports the
VAXSYN HIV-1 vaccine developed by MicroGeneSys. This
report does not disclose any details of this vaccine.
D.L. Lynn, et al, in "Mechanisms of Control of
Gene Expression", Eds. Allan R. Liss Inc., pp. 359-368
(1988) disclose the cloning of the entire gp160 gene
behind the polyhedron promoter of the baculovirus
, CA 02003794 1998-12-07
-5-
1 Autographa californica. These insect cells infected with
the recombinant virus express a protein that is released
from the cell upon lysis. This protein co-migrates with
gpl6o, is not cleaved into gp120 and gp4l, and is
glycosylated and associated with the cell membrane. When
deglycosylated with N-glycanase, the protein had a
molecular weight of approximately 96 kd. The recombinant
protein was immunoreactive with protein from HIV-infected
H9 cells, with antisera to a recombinant fraction of
gp120, with gp120 itself, with a peptide fragment of gp4l,
and with human AIDS sera.
R.L. Willey, et al, Proc. Nat'1 Acad. Sci., USA,
83: 5038 (1986) discusses hypervariable regions of amino
acids in gp120 protein. A later report, R.L. Willey et
al, J. Virol., 62(1): 139 (1988), studied a region within
the env gene necessary for infectivity. Specifically
disclosed herein are amino acid substitutions at the Asp
codons of four N-linked glycosylation sites within the
gp120 gene.
W.R. Gallagher, Cell, 50: 327 (1987) discusses a
fusion peptide sequence of the transmembrane protein gp41
of HIV.
S.D. Putney et al, Science, 234: 1392 (1986)
discloses the production of neutralizing antibodies to an
E, coli-produced fragment'of the HIV env gene.
B.J. Bond et al, Mol. Cell. Biol., 6(6): 2080
(1986) disclose the structure of the Drosophila
melanoqaster actin 5C gene. The report discusses the two
transcription start sites of the actin 5C gene and fusions
between the promoter sequences and bacterial
chloramphenicol acetyltransferase gene inserted into D
melanoqaster host cells.
P.J. Barr et al, Vaccine, 5: 90 (1987) discloses
the expression of HIV proteins in the yeast Saccharomyces
cerevisiae.
-
1 H. Johansen et al, 28th Annual Drosophila
Conference, p. 41 (1987) is an abstract by the inventors
of the present application which briefly states that _E.
call gal K genes regulated by a D_rosophila metallothionein
promoter were expressed in DrOSOphila cell lines.
A. Vanderstraten et al, Proceedings of the 7th
International Conference on Tnvertebrate and Fish Tissue
Culture, Abstract, University of Tokyo Press, Japan,
(1987) arid A. Vanderstraten et a1, in "invertebrate and
Fish Tissue Culture", Eds. Y. Kuroda et al, Japan
Scientific Societies Press, Tokyo, pp. 131-134, (1988) are
also publications by the present inventors which discuss a
hygromycin B selection system for use in expressing
foreign genes in D. m_elanoaaster cells in culture. The
abstract notes that the system was used to co-introduce
and overexpress the E. coli gal K gene and other genes of
mammalian origin,
There thus remains need in the art for
high-level production of HIV proteins for use as vaccinal
and diagnostic agents.
Summary of Invention
In one aspect, the present invention is an HIV
env gene expression unit which includes a DNA coding
sequence for the desired protein and regulatory seguences
necessary for transcription of the protein coding sequence
and subsequent translation within a Drosophila cell.
In related aspects, this invention is a DNA
vector which comprises the gene expression unit of the
present invention.
In yet another related aspect, this invention is
a Drosophila cell transfected with the DNA vector of this
invention.
_7_
1 In further related aspects, this invention is an
HIV env protein, or a derivative thereof produced by the
transfected insect cells of this invention. The
derivative encompasses any HIV env protein such as
deletions, additions, substitutions or rearrangement of
amino acids or chemical modifications thereof which retain
the ability to be recognized by antibodies raised to the
wild-type HIV env protein.
In another aspect, this invention is a vaccine
far stimulating protection against HIV infection, which
comprises an immunaprotective and non-toxic quantity of
the HIV env protein produced by this invention.
Also provided by this invention is a diagnostic
agent useful in detecting presence of HIV infection in a
sample of biological fluid which contains a Drosophila
cell-produced HIV protein of the invention. Additionally,
the env protein of the present invention may be employed
to identify or isolate HIV binding proteins or
proteinaceous substances, such as CD4 or derivatives
thereof.
This invention also relates to a method for
production of an HIV env protein, or an immunogenic
derivative thereof. The method ewtails culturing
Drosophila cells transfected with an HIV env gene
expression unit in a medium suitable for growth of the
cells. The transfected cells, cultured in said suitable
medium, are capable of expressing said protein of
interest. The protein may thereafter be collected from
the cell or cell culture medium.
The present invention also provides a method for
purifying HIV proteins or fragments thereof which bind to
a monoclonal antibody reactive with an epitope present on
mature gp120 and also within the gp160 unprocessed
intracellular protein. Among antibodies of this class is
the mouse monoclonal antibody designated 178.1.
~(3~; j'°1~~~
1 Other aspects and details of the present
invention are disclosed in the following description:
Detailed Description of the Invention
The method and expression system of the present
invention facilitate high-level production of HIV
proteins, particularly gp120, gp160 and derivatives
thereof, in a Dros~ila cell structure. The Drosophila
cells are transfected by using standard cloning techniques
which permit introduction of foreign DNA into a host cell
without adversely affecting the foreign DNA or the host
cell. The recombinant Drosophila cells so constructed
produce HIV proteins.
In contrast to the Baculovirus system of the
prior art an which the HIV protein is provided only upon
lysis of the infected insect cells, the method of this
invention provides a continuous cell expression system for
HIV prateins. Upon secretion, the protein is available by
purification from the culture medium using conventional
techniques. Alternatively, the protein may be produced
intracellularly or membrane-bound. The protein may be
extracted from the cells using conventional techniques.
Alternatively, membrane-bound protein may be employed in a
variety of cell-associated assays.
A preferred Dr~hila cell line for use in the
practice of the invention is the D_. melanoctaster S2
line. S2 cells [Schneider, ,3. Embryol. Exp. Morph. 27:
353 (1972)] are stable cell cultures of polyploid
embryonic Drosophila cells. Introduction of the cDNA
coding sequence for gp160, or its subunits gp120 or gp41
or derivatives thereof into Drosophila S2 cells by DNA
trans~ection techniques produces unexpectedly large
amounts of the glycoprotein. Use of the S2 Drosophila
cell has many advantages, inoluding, but not limited to,
CA 02003794 1998-12-07
_g_
1 its ability to grow to a high density at room
temperature. Stable integration of the selection system
has produced up to 1000 copies of the transfected gene
expression unit into the cell chromosomes.
Other Drosophila cell culture systems may also
useful in the present invention. Some possibly useful
cells are, for example, the KC-O Drosophi~ melanoaast- r
cell line which is a serum-free cell Line [Schulz et al,
Proc. Nat'1 Acad. Sci. USA, _83: 9428 (1986)]. Preliminary
studies using the KC-O line have suggested that
transfection is more difficult than with S2 cells.
Another cell Iine which may be useful is a cell line from
Drosophila hydei. Protein expression can be obtained
using the hydei cell line; however, transfection into this
cell line can result in the transfected DNA being
expressed with very low efficiency [Sinclair et al, Mol.
Cell. Biol., 5: 3208 (1985)]. Other available Drosophila
cell lines which may be used in this invention include
S1 and S3.
The Drosophila cells selected for use in the
present invention can be cultured in a variety of suitable
culture media, including, e.g., M3 medium. The M3
medium consists of a formulation of balanced salts and
essential amino acids at a pH of 6.6. Preparation of the
media is-substantially as'described by Lindquiat, DIS, 58:
163 (1982). Other conventional media for growth of
Drosophila cells may also be used.
A recombinant DNA molecule or vector containing
an HIV protein gene expression unit can be used to
transfect the selected Drosophila cells, according to the
invention. The gene expression unit contains a DNA coding
sequence for a selected HIV protein or for a derivative
thereof. Such derivatives may be obtained by manipulation
of the gene sequence using traditional genetic engineering
techniques, e.g., mutagenesis, restriction endonuclease
treatment, ligation of other gene sequences including
y
~ -lo-
1 synthetic sequences and the like. See, e.g., T. Maniatis
et al, Molecular Cloning, A Laboratory Manual., Cold
Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).
The HIV DNA coding sequence has been recently
published. See, Rather et al, Nature 313:277-284 (1985)
or Wain-Hobson et al, Cell 40:9-17 (1985). The nucleotide
sequence is also available from GenBank (clone BH10, .
Rather et al, su ra).
DNA molecules comprising the coding sequence of
this invention can be derived from HTLV-III infected cells
using known techniques (see, Hahn et al, Nature
312:166-169 (1984)), or, in the alternative, can be
synthesized by standard oligonucleotide techniques.
Moreover, there are numerous recombinant host cells
containing the cloned DNA coding sequences, which are
widely available.
Derivatives can then be prepared by standard
techniques, including DNA synthesis. Such derivatives may
include, e.g., gp120 or gp160 molecules in which. one or
ZO more amino acids have been substituted, added or deleted
without significantly adversely affecting the binding
capacity or biological characteristics of the protein.
Derivatives of these proteins may also be prepared by.
standard chemical modification techniques, e.g.,
acylation, methylation.
Also included in the gene expression unit are
regulatory regions necessary or desirable for
transcription of the HIV protein coding sequence and its
subsequent translation and expression in the host cell.
The regulatory region typically contains a promoter region
which functions in the binding of RNA polymerase and in
the initiation of RNA transcription: The promoter region
is typically found upstream from the HIV protein coding
sequence.
Preferred promoters are of Drosophila origin,
e.g., the Drosophila metallothionein promoter
-11-
1 CLastowski-Perry et al, J. Biol. Chem., _260: 1527
(1985)]. This inducible promoter directs high-level
transcription of the gene in the presence of metals, e.g.,
CuSO~. Use of the Drasophila metallothionein promoter
results in the expression system of the invention
retaining full regulation even at very high copy number.
This is an direct contrast to the use of the mammalian
metallothionein promoter in mammalian cells in which the
regulatory effect of the metal is diminished as copy
n~ber increases. In the Drosophila expression system,
this retained inducibility effect increases expression of
the gene product in the Drosophila cell at high copy
number.
The Drosophila actin 5C gene promoter CB. J. Bond
et al, Mol. Cell. Biol., 6: 2080 (1986)] is also a
desirable promoter sequence. The actin 5C promoter is a
constitutive promoter and does not require addition of
metal. Therefore, it is better-suited for use in a large
scale production system, like a perfusion system, than is
the Drasophila metallothionein promoter. An additional
advantage is that the absence of a high concentration of
copper in the media maintains the cells in a healthier
state for longer periods of time.
Examples of other known Drosophila promoters
include, e.g., the inducible heatshock (Hsp70) and COPIA
LTR promoters. The SV40 early promoter gives lower levels
of expression than the Drosophila metallothionein
promoter. Promoters which are commonly employed in the
cell expression vectors including, e.g., avian Rous
sarcoma virus LTR and simian virus (SV40 early promoter)
demonstrate poor function and expression in the Drosophila
system.
A desirable gene expression unit or expression
vector for the HIV protein may be constructed by fusing
the HIV protein coding sequence to a desirable signal
sequence. The signal sequence functions to direct
, CA 02003794 1998-12-07
-12-
1 secretion of the protein from the host cell. Such a
signal sequence may be derived from the sequence of tissue
plasminogen activator (tPA). Other available signal
sequences include, e.g., those derived from Herpes Simplex
virus gene HSV-I gD [Lasky et al, Science, supra.].
The HIV DNA coding sequence may also be followed
by a polyadenylation (poly A) region, such as an SV40
early poly A region. The poly A region which functions in
the polyadenylation of RNA transcripts appears to play a
role in stabilizing transcription. A similar poly A
region can be derived from a variety of genes in which it
is naturally-present. This region can also be modified to
alter its sequence provided that polyadenylation and
transcript stabilization functions are not significantly
adversely affected.
The recombinant DNA molecule may also carry a
genetic selection marker, as well as the HIV protein gene
functions. The selection marker can be any gene or genes
which cause a readily detectable phenotypic change in a
transfected host cell. Such phenotypic change can be, for
example, drug resistance, such as the gene for hygromycin
. B resistance.
Alternatively, a selection system using the drug
methotrexate, and prokaryotic dihydrofolate reductase
(DHFR) gene, can be used with Drosophila cells. The
endogenous eukaryotic DHFR of the cells is inhibited by
methotrexate. Therefore, by transfecting the cells with a
plasmid containing the prokaryotic DHFR which is
insensitive to methotrexate and selecting with
methotrexate, only cells transfected with and expressing
the prokaryotic DHFR will survive. Unlike methotrexate
selection of transformed mammalian and bacterial cells, in
the Drosophila system, methotrexate can be used to
initially select high-copy number transfectants. Only cells
which have incorporated the protective prokaryotic DHFR
gene will survive. Concomitantly, these cells have the
gene expression unit of interest.
i~~~ ~~~~
-13-
1 An illustrative plasmid produced, according to
'the present invention, is pgp160032, which contains a
gp160 derivative replacing the N-terminal 32 amino acid
sequence of gp160 with the first amino acid of tPA,
serine. This plasmid is further described in Example 1.
Another such plasmid vector is pgp120Fd32
which contains gp160 sequence having the first 32 amino
acids replaced with serine and containing a carboxyl
deletion of 216 amino acids. This plasmid is also
l0 described in Example 1.
Still another plasmid which illustrates the
derivative proteins of the present invention is
pgp120~32, which contains the entire coding sequence for
gp120 minus the first 32 amino acids at the N-terminal
which are replaced with serine. Additionally, plasmid
pgpl2onz74 contains a gp120 protein sequence which has
replaced the first 274 amino terminal amino acids with the
first amino acid of tPA, serine, and containing the
remaining amino acids of gp120 up to the processing site
of gp160. These vector constructions are described more
completely in Example 1.
Once a recombinant DNA molecule or expression
vector containing the HIV protein gene expression unit has
been constructed, it can be transfected into the selected
Drosophila cell using standard transfection techniques.
Such techniques are known to those of skill in the art and
include, for example, calcium phosphate co-precipitation,
cell fusion, 2lectroporation, microinjection and viral
~transfection.
A two-vector system can be used in the present
invention to co-transfect into the Drosophila cell a gene
expression unit for the desired HTV protein or derivative
and the coding region for the selection system to be
used. A preferred illustrative embodiment of this
invention is the production of an HIV protein employing a
vector containing an HIV protein expression unit, e.g.;
' CA 02003794 1998-12-07 -
-14-
1
pgp120~32, and a vector containing a hygromycin B gene
expression unit, e.g., pCOHYGRO. pgp120~32 contains an
expression unit comprising the Drosophila metallothionein
promoter, a derivative of the gpI20 gene, and the SV40
poly A site. This gp120 expression unit in combination
with the pCOHYGRO vector system will produce a gp120
derivative in S2 Drosophila cells by maximizing the
advantage of hygromycin B resistance for selection. With
this system, the antibiotic hygromycin B can be used to
select for those cells containing the transfected
vectors. A more complete description of this embodiment
is described in Example 2.
As another example, an expression system
employing the DHFR gene/methotrexate selection system,
consisting of the vectors pgp120032 and pHGCO, can be
used to select methotrexate-resistant cells expression
gp120 or a derivative thereof. The vector pgp120a32
comprises a gp120 gene expression unit in which the
promoter is the Drosophila metallothionein promoter. The
2C
pHGCO vector comprises a DHFR gene expression unit and is
co-transfected with the pgp120~32 vector, thereby
providing the DHFR gene necessary for selection. These
selectable markers along with cotransfection of Drosophila_
cells is -further described by Johansen et al, U.S. Patent 1
5,681 713
. issued October 28, 1997.
According to the invention, the two vectors are
co-transfected into the S2 Drosophila cell using the
method as described by Wigler et al, Cell, 16: 777
-
(1979). The vectors are co-transfected in varying ratios
depending upon the particular copy number desired. The
transfected cells are then selected, such as in M3
medium containing serum and the appropriate selection
agent, e.g., hygromycin B or methotrexate.
Once an appropriate vector has been constructed
and transfected into the selected Drosophila cell line,
~~~ ~''~;~-~
-i5-
1 the expression of gp120 is induced by the addition of an
appropriate inducing agent for the inducible promoter.
For example, cadmium or copper are inducing agents for the
metallothionein promoter. Heat is the inducing agent for
the Hsp70 promoter. For constitutive promoters, such as
'the actin 5C promoter, no inducing agent i~, required for
expression.
Transcription and expression of the HIV protein
coding sequence in the above-described systems can be
monitored. For example, Southern blot analysis can be
used to determine copy number of the gp120 gene. Northern
blot analysis provides information regarding the size of
the transcribed gene sequence [see, e.g., Maniatis et a1,
cited above]. The level of transcription can also be
quantitated. Expression of the selected HIV protein in
the recombinant cells can be further verified through
Western blot analysis and activity tests on the resulting
glycoprotein [see Example 5].
Drosophila S2 cells are especially suited to
20,high-yield production of protein in the method of the
present invention. The cells can be maintained in
suspension cultures at room temperature (24+i°C). Culture
medium is M3 supplemented with between 5 and 10% (v/v)
heat-inactivated fetal bovine serum (FBS). In the
preferred embodiment of the invention, the culture medium
contains 5% FBS. After induction, the cells are cultured
in serum-free media. When the pCOHYGRO vector system is
used, the media is also supplemented with 3OO ug/ml
hygromycin B. In this media, the S~ cells can be grown
30-in suspension cultures, for example, in 250 ml to 2000 ml
spinner flasks, with stirring at 50-60 rpm. Cell
densities are typically maintained between 105 and 10~
cells per ml. In one embodiment of this invention, the
cells axe grown prior to induction in 1500 m1 spinner
flasks in media containing 5% serum.
' CA 02003794 1998-12-07
-16-
1 Following cell culture, the HIV protein can be
isolated from the spent media, e.g., by use of a
monoclonal antibody affinity column. Other known protein
purification steps, e.g., metal chelates, various affinity
chromatography steps or absorption chromatography, can be
used to purify the HIV protein from the culture media.
The use of the cell line S2 which secretes the gene
product directly into the media is an important feature of
the present invention. Direct secretion into the media
allows utilization of an efficient one-step purification
system. Using a monoclonal antibody column directed
against the HIV protein, the spent culture media can be
added directly to the column and the protein eluted using
1.5 _M KSCN in phosphate-buffered saline (PHS).
A preferred purification technique enabling
large-scale efficient production of the HIV proteins of
the invention employs an immunoaffinity column containing
a monoclonal antibody directed against an epitope present
in gp160 and present in mature secreted gp120 proteins.
Such a,monoclonal is advantageous because of its capacity
to recognize the protein sequence in more than one
configuration. An antibody having these characteristics
and useful in immunoaffinity columns for various HIV
proteins, derivatives or fragments thereof is designated
178.1. This monoclonal aritibody is described in greater
detail in Example 3. Such a column of the invention may
be made by coupling an antibody with the characteristics
of 178.1 to a conventional absorbant carrier, such as
SEPHADEX, Wider appropriate conventional conditions of pH,
temperature and the like. Such a purification column and
procedure may be utilized to separate the HIV proteins and
fragments of the present invention.
Other monoclonal antibodies may be used in this
purification procedure. A variety of monoclonal
antibodies which are capable of binding to HIV proteins,
particularly gp160 or gp120, have been described in the
-17-
1 art and are available. Other new monoclonal antibodies
useful in this invention may be developed by
now-conventional techniques.
The glycoproteins produced by Drosotahila cells,
according to this invention, are completely free of
contaminating materials, e.g., mammalian, yeast, bacterial
and more importantly, other HIV viral materials.
Drosophila-produced HTV proteins have also been
demonstrated to possess different pattern of glycosylation
than that reported by other systems, e.g., mammalian
systems.
The HIV proteins and derivatives produced,
according to the present invention, may be useful in a
variety of products. For example, these recombinant
proteins may be used in pharmaceutical compositions for
the treatment of HIV-infected subjects. Such a
pharmaceutical composition, according to the present
invention, comprises a therapeutically effective amount of
the HIV protein or derivative of the invention in
admixture with a pharmaceutically acceptable carrier. The
composition can be systemically administered either
. parenterally, intravenously or subcutaneously. When
systemically administered, the therapeutic composition for
use in this invention is in the form of a pyrogen-free,
z5 parenterally acceptable aqueous solution. The preparation
of such a parenterally acceptable protein solution, having
due regard to pH, isotonicity, stability and the like, is
within the skill of the art.
The dosage regimen will be determined by-the
attending physician, considering various factors which
modify the action of drugs, e.g., the condition, body
weight, sex and diet of the patient, the severity of any
infection, time of administration and other clinical
factors. The pharmaceutical carrier and other components
of a pharmaceutical formulation would be selected by one
of skill in the art.
-18-
Additionally, the recombinant proteins of the
present invention may be used as components of vaccines to
innoculate mammalian subjects against HIV infection.
These proteins may be used alone or with other recombinant
proteins or therapeutic vaccinal agent s, Components of
such a vaccine would be determined,by one of skill in the
art.
Finally, the proteins of the present invention
may be useful as diagnostic agents for the detection of
the presence of HIV infection or antibodies to an HTV
infective agent in biological fluids, such as blood,
sertun, saliva and the like. These proteins of the
invention may also be employed in methods to identify
and/or isolate HIV-binding proteins or other HIV-binding
substances in biological fluids and tissues, e.g., sCD4 or
derivatives thereof. The proteins may thus be components
in kits to perform such methods. To identify an
HIV-binding substance, a protein, according to the
invention, is employed to contact the substance or an
impure mixture containing 'the substance under conditions
to promote binding between the protein and the I-IIV-binding
. substance. A conventional assay to detect the occurrence
of binding, e.g " detection of radioactive labels or the
like, is also part of the method. The presence of binding
between the protein and the binding substance is,
therefore, indicative of HIV binding.
Similarly, in a method to isolate an HIV-binding
substance from the mixture, the binding event could be
followed by a conventional procedure to purify the bound
entity formed by the protein of the present invention and
the HIV-binding substance from the mixture. Other
components of such diagnostic systems and kits may be
conventional components of diagnostic kits and may be
selected by those of skill in the art.
The following examples illustrate the
construction of exemplary vectors and transformants of the
~(~~ ~'~~~
-19-
1 invention, the preferred purification system and assays
for determination of the production level of the
glycoproteins gp120 and gpl6o. These examples are not to
be considered as limiting the scope of this invention.
5 Restriction enzymes and other reagents were used
subst ant:ially in accordance with 'the vexxdars' instructions.
Examples
example 1. Vector Constructions
a) Mp ~'tPA
As the basic vector for gene expression in
Drosophila, the tPA expression vector pMTtPA (also called
pDMtPA) was used. This vector is a derivative o~ vector
pMLI, a small pER322 vector containing the beta-lactamase
gene which has deleted the poison sequences [Melton et al,
Cell, 27: 297 (1982)]. These sequences are inhibitory to
amplification of the vector. This vector was digested
with Sall and Aat2 which removes a small piece of pBR322,
and the digested ends were filled in. The missing piece
of pBR322 is then replaced with a cassette containing the
Drosophila metallothioneir~ promoter on an end-filled
EcoRl-Stul fragment, followed by a filled-in _HindIII-Sacl
fragment from pDSPI [D.S. Pfarr et al, DNA, _4(6): 461
(1985)] containing a tPA sequence containing the signal
sequence, prepeptide and the entire coding region of tPA.
The tPA gene an this fragment is followed by an SV40 early
polyadenylatian site.
b) p_p1~0~32
A HindIII-Xbal fragment containing the entire
env gene was isolated from an HIV-isolate clone BH10 [L.
-20-
1 Ratner et al, cited above; GenBank]. The entire gp160
sequence was then inserted into a N_co_1-Xbal digested
vector pDSPl. The resulting vector, _SU2, was digested
with Ndel, followed by treatment with mung bean nuclease
and subsequently digested with Sacl, thus isolating the
gp160 gene. The digestion with Ndel cwt the gp160
sequence at amino acid #32. The Sacl digestion cuts 3' of
the gp160 gene, including in the sequence part of the
original pDSPl vector containing a polylinker. This
fragment was inserted into the above-described expression
vector pMTtPA which had been digested with Hc~lII,
end-filled, and subsequently cut with Sacl, which deletes
the mature tPA sequence, The Bg_lII site is positioned at
the first amino acid of tPA. Consequently, the resulting
vector pgp160~32 codes for a modified gp160 protein
which has replaced the N-terminal 32 amino acids of gp160
with serine.
c) ~apl2oF~32
Another vector containing a modified gene
sequence coding for HIV-1 surface glycoprotein gp160 was
constructed by digesting pgp160d32 with H_indIII and
Sacl, thereby removing the carboxyl terminal of gp160.
Approximately two-thirds of the sequence coding for gp41
is removed by this digestion. Thus, this gp160 sequence
is missing the first 32 amino acids and the last 216 amino
acids of the natural gp160 sequence. The deleted sequence
was replaced by a short synthetic linker sequence coding
for a stop colon on an HindIII-_Sacl fragment. The
6-amino-acid linker sequence is as follows:
5'AGCTTTGACTGACTGAGCT 3'.
d) pap120A32
Yet another vector containing a mutant gp160
_21-
gene was constructed by digesting pgp160~32 with S~1
and Xbal, thereby deleting all of the sequence of protein
gp41 and about 30 amino acids at the carboxyl terminus of
the gp120 glycoprotein sequence. This fragment was
replaced by a synthetic S~1-Xbal linker sequence coding
for the correct carboxyl terminus from the S~1 site to
the processing site of gp120-gp4l. This sequence was
followed by a stop colon. This sequence thereby contained
all of 'the coding sequence for gp120 minus the first 32
amino acids and vane of the gp41 coding sequence.
e) pai~1200274
Still another exemplary vector containing a
mutant gp120 gene was constructed as follows: a 720-base
pair carboxyl terminal fragment of gp120 was isolated by
partial digestion of pgp120~32 with Bc~lII followed by
XbaI digestion. This fragment was now inserted in place
of the tPA gene into the ~qlII-Xbal cut pMTtPA expression
vector. The resulting vector p120~274 codes for a gp120
protein which has replaced the first 274 amino terminal
amino acids with the first amino acid of tPA, serine.
f) pCOHxGRO
A commercially available plasmid, pUCl8 [BRL]
containing a BamHI arid SmaT site was used. The S' LTR
from an integrated COPIA element (357 base pairs) was
cloned into the BamHI site of vector pUCl8, resulting in
the vector designated pUCOPIA COPTA is a representative
member of the disperse middle repetition sequences found
scattered through the Drosophila genome (Rubin et al, in
Cold Sprina Harbor Symp Quant ~ Biol , _45: 619 (1980)].
The vector pUCOPIA was cut at the SmaI site and the E.
coli gene coding for hygromycin B phosphotransferase~
(hygromycin B cassette) was cloned into pUCOPTA using
' CA 02003794 1998-12-07
-22-
1 standard cloning techniques. The hygromycin B cassette
was isolated on a HindIII-BamHI fragment of 1481 base
pairs from the vector DSP-hygro [Gertz et al, Gene, _25:
179 (1983)]. The hygromycin B cassette contains the
sequence coding for the hygromycin B phosphotransferase
gene and the SV40 early poly A region. The HindIII and
BamHI sites were filled in using T4 DNA polymerase, and
the hygromycin B cassette was ligated into the Smal site
of the vector pUCOPIA producing vector pCOHYGRO.
Example 2. Transfection into Drosophila S2 Cells
pCOHYGRO was co-transfected into S2 Drosophila
cells together with one of the vectors carrying a gp160
mutant gene under the control of the Drosophila
metallothionein promoter as described above. For purposes
of this example, the vector employed is pgp120~32. The
transfected cells were selected in M3 medium containing
5% serum and 300 ug/ml of hygromycin B. After 2 to 3
days under identical conditions, the untransfected cells
stop dividing and begin to die. The time of selection in
order to obtain stable, growing hygromycin B-resistant
cells in the transfected cultures is approximately two to
three weeks.
To obtain cultures having integrated into their
chromosomes different copy numbers of the gp120 mutant
gene, the ratios of the two vectors were varied. The
ratio in this example was 20:1. Similar ratios have been
employed for other gpI60 mutant vectors of this
invention. This ratio is the same when any of the gp160
mutant vectors are used.
Expression of the pgp120~32 gene product was
verified after induction of the metallothionein promoter
with 500 uM CuS04. Western blot analysis of the spent
supernatant from the induced cell cultures revealed a
single band at approximately 100 kd.
e~~%~li ~',~~
_23_
1 The level of the mutant gp160 gene product in
the cell supernatants was measured using the gp120 ELISA
assay, described in Example 4, and using purified viral
gp120 as standards. 5 x 106 cells/m1 were seeded in
S M3 medium without serum and induced for 3 to 4 days.
The level of gp120 measured in the supernatant is
approximately lw2 mg/l.
Cells were maintained as suspension. cultures in
250 ml to 2000 ml spinner flasks. Culture medium was M3
supplemented with 300 ~g/ml hygromycin B. Cultures were
incubated at 24+1°C and stirred at 50-~60 rpm. Cell
densities were typically maintained between 106 and
10~ cells per ml in M3 medium supplemented with
hygromycin B. CuSO~ was added to a final concentration
1S of 500 NM, and the cultures were allowed to grow for 3
to 4 days in serum-free media prior to harvesting the
modified gp120 glycoprotein.
The proteins, according to this method produced,
were approximately 100 MW, and the level of expression was
higher than any other reported gg120/gp160 expression in
any eukaryotic cell system. Tn standard biological
activity assays, the purified modified gp120 expressed, as
described above, is capable of inhibiting virus infection
in tissue culture, binds T~ and reacts to antibodies to
gp120.
It is expected that one of skill in the art
could express the other gp160 and gp120 proteins and
fragments thereof, described by the present invention,
using substantially the same systems and procedures as
exemplified above for the protein fragment encoded in
pgpI20~32.
Example 3. Monoclonal Antibody 178.1
An affinity purification column employing a
novel monoclonal antibody was used in the purification
' CA 02003794 1998-12-07
-24-
1 scheme applied to the above-described mutant gp160/gp120
proteins. This monoclonal antibody may be characterized
as being capable of reacting with non-denatured HIV
glycoprotein products present in cell lysate and with
mature gp120 as secreted into the supernatant of a yeast
culture. One such-monoclonal antibody specific for the
epitope which is contained both in the unprocessed gp160
recombinant molecule and in the full-size processed gp120
protein is a mouse monoclonal antibody 178.1.
An expression system employing the _C. _albicans
glucoamylase promoter and signal peptide was employed to
produce partially purified yeast-recombinant gp160 for
production of 178.1. The production of this yeast-derived
production of 178.1. The production of this yeast-derived
15-gp160 is described in European patent application No. 362,179,
published April 4, 1990.
Eight-week-old Balb/c mice were injected three
times subcutaneously and intraperitonally with the
partially purified (1.5 - 3% purity) yeast-recombinant
gp160 in Freund's adjuvant at 4-week intervals. After a
resting period of 3 months-, one mouse was sacrificed, and
its spleen cells were fused with myeloma cells [see, e.g.,
R.P. Siraganian et al, Meth. Enz., 92: 17 (1983); yMBp
Course on:Hybridoma Production, Basel Inst. for Immunol.
(1980)]. The myeloma cells used are a subclone of the
Sp2/O-Agl4 line previously selected for optimal growth
in agar medium and high fusion efficiency [J. D. Franssen
et al, Proc. XXIX Collog Protids Biol Fluids, _29:
645-649 (1981)]. After about ten days, supernatants were
withdrawn for screening in a capture ELISA, using a
commercial monospecific anti-gp120 reagent [Biochorm,
Seromed Ref. D7324) as capture antibody.
Briefly, NUNC IMMUNOPLATE*'I (nr 4-39454) were
coated overnight at 4°C with 50 ul of a solution of
5 ug/ml of sheep anti-gp120 IgGs in PBS. The plates
*Trade-mark
' CA 02003794 1998-12-07
-25-
1 were washed with washing buffer (PBS, TWEEN* 20 0.1%) and
saturated with 100 ul of saturation buffer [PBS, Newborn
Calf Serum 4%, bovine serum albumen (BSA) 1%, TWEEN 20
0.1%] for 1 hour at 37°C. Fifty ul/well of crude-
Molt3/HTLV-IIIB,or Molt3 cell lysate (107 cells/ml
in PBS, TRITON x-100* 1$) or of the supernatant fraction
(S2-30) of the recombinant-yeast gp160 (or similarly-
treated negative control) were used as antigen and
incubated in the plates for 3 to 5 hours at room
temperature. The plates were washed extensively, and
50 ul of hybridoma supernatants were added to each well
and incubated overnight at 4°C. After a washing step,
50 ul/well of a 1/500 dilution of biotinylated
anti-mouse immunoglobulins (Igs) (Amersham Ref. RPN 1021)
in saturation buffer were incubated in the plates for 1
hour at 37°C. The plates were washed again, and 50
ul/well of a 1/1000 dilution of streptavidin
biotinylated horseradish peroxidase complex (Amersham Ref.
RPN 1051) in saturation buffer were added to each well.
After an additional washing step, 50 ul of a
solution of 0.4 mg/ml of orthophenylene diamine
dihydrochloride (OPD, Sigma P1526) and 1 ul/ml of
H202 (30% in citric/Na citrate 0.1 M pH 5)
supplemented with O.ls TWEEN 20 were added to each well.
The plates were then incubated for 20 minutes at room
temperature in the dark, and the reaction was stopped by
addition of 50 ul/well of 2M H2S04. The optical
density at lambda = 492 nm was monitored, and 50 positive
clones were selected for further subcloning in soft
agarose, according to P. Herion et al, Proc. XXIX Colloq.
Protids Biol. Fluids, _29: 627 (1981). The cloned
hybridomas were then grown in vivo by injecting 2 to
5 x 106 hybridoma cells in the peritoneal cavity of
Halb/c mice pretreated by intraperitoneal injection of
pristane (2, 6, 10, 14-tetramethyl pentadecane).
*Trade-mark
' CA 02003794 1998-12-07
-26-
1 The monoclonal antibodies selected from the
above procedure were characterized by Western blot
analysis (WB), radioimmuno precipitation assay (RIPA),
purification, biotin-labeling and competition assays.
Resulting monoclonal antibodies were further characterized
by analysis of. their reactivity toward various recombinant
and native antigens.
A high yield of hybridomas was obtained by this
procedure. More than 200 wells were positive in the
screening assays. However, among them, only 50 wells were
selected and after cloning, the cells were expanded in
ascitic acid. All the ascitic fluids were tested in WB
and RIPA. Among the 39 monoclonals tested, 37 showed a
gp160 band in RIPA. None reacted with the gp120 form in
the same assay. Those monoclonal antibodies that
displayed only gpl6o recognition in RLPA, while being
clearly reactive to gp120 in WB, were analyzed by
subclass. Three monoclonals that were IgG2A were purified
on a protein A-SEPHAROSE* column and biotin-labelled.
Competition assays using vaccinia gp160 as antigen were
performed, and the obtained result defined at least five
different groups of epitope recognition with the gp160
protein. Monoclonal 178.1 was selected for an epitope
present on mature gp120 and unprocessed intracellular
gp160.
A Western blot (WB) analysis was performed
according to conventional techniques to demonstrate that
178.1 is capable of binding HIV virus isolated from human
cells infected with HTLV-IIIB [Molt3/HTLV-IIIB].
Radio immuno precipitation assays (RIPA) were
performed, as described in P.J. Kanki et al, Science, 228:
1199 (1985) to demonstrate that 178.1 could
immunoprecipitate the human cells infected with HTLV-III
virus strains.
The reactivity of the monoclonal antibodies
recognizing non-overlapping epitopes towards a large panel
*Trade-mark
' CA 02003794 1998-12-07
-27-
1 of antigens was assessed using a sandwich ELISA involving
sheep anti-gp120 as capture reagant. Monoclonal antibody
178.1 was negative in ELISA on divergent HIV isolates
Molt3/HTLV-IIIB, H9/HTLV-III~1 and Hut78/ARV2,
while clearly positive when tested on HTLV-IIIB in RIPA,
WB, or ELISA using recombinant antigens. This monoclonal
recognizes an epitope that is apparently conserved between
gp160 and gp120 and thus, when used in the purification
technique described in Example 4 below, provides an added
advantage for the production of gp160/gp120 glycoproteins
in various constructs.
Example 4. Purification of gp120032 from Drosophila
conditioned Cell Culture Medium
The recombinant gp120 protein from Example 2 was
purified as follows: 30 liters of Drosophila-conditioned
media (CM) containing gp120~32 was made with 1 mM
phenylmethylsulfonyl fluoride (PMSF), 10 mM
ethylenediamine tetraacetic acid (EDTA) and 70 Kallikrein
inhibitor units. CM was filtered through a .45 um
Durapore membrane using a pellicon (Millipore) device.
Filtered CM was applied to S-SEPHAROSE* fast flow
(Pharmacia) (5 liters; 25.2 cm x 11'cm) at a linear flow
rate (LFR) of 37 ml/cm2hr~equilibrated in Buffer A,
containing 20 mM 2-[N-morpholino]ethanesulfonic acid
(~S), pH 6Ø After application of all CM, the column
was eluted .in one step with Buffer B, containing 20 mM
MES, pH 6.0, 0.4M NaCl.
The S-SEPHAROSE-eluted gp120~32 was applied to
an anti-gp120 mouse monoclonal-SEPHAROSE 4B column (60 ml;
3.2 cm x 6.5 cm) at a LFR of 10 ml/cmZhr. This column was
equilibrated in Buffer B. After application of one-half S-
SEPHAROSE pool, the column was washed with 1 column volume of
Buffer B, 2 column volumes of 20 mM MES, pH 6.0, 1. OM NaCl
(Buffer C), and 2 column volumes of
*Trade-mark
' CA 02003794 1998-12-07
-28-
1 Buffer A. gp120032 was eluted with O.1M acetic acid,
pH 2.8, and fractions were immediately neutralized by
addition of 0.1 volumes of 1M Tris
(hydroxymethyl)aminomethane (Tris), pH 10.4.
Mouse anti-gp120 monoclonal antibody hybridoma
178.1 was produced according to Example 3 above. This
hybridoma was seeded at 2 x 105 cells/ml and cultured
for four days in Dulbecco's Modified Eagle Medium
[Hazelton Research Productsl supplemented with
4.5 grams/liter glucose, 2 uM glutamine and loo serum.
CM containing 178.1 antibody was filtered (0.2 um
membrane) and applied to a protein A-SEPHAROSE (Pharmacia)
(I7 ml; 1.5 cm x 10 cm) equilibrated in O.1M Tris, pH 8.2.
Antibody was eluted with O.1M sodium citrate, pH 3.5 and
immediately neutralized with Tris.
Purified anti-gp120 monoclorial antibody was
coupled to CNBr-activated SEPHAROSE 4B* (Pharmacia),
according to manufacturer's instructions at a density of
2 mg antibody/ml resin and with a coupling efficiency of
98~, resulting in an anti-gp120-SEPHAROSE-affinity resin.
This affinity resin will specifically bind gp120 protein
through the interaction of the antibody with a unique
structural epitope on gp120.
The purity of the final gp120 protein product,
according to this purification technique, is 80-90% with
an estimated yield of 8.5 mg/30 liters conditioned media.
Recovery is estimated at between 25-500.
This purification technique and affinity resin
is also believed to be effective with other HIV proteins
or fragments thereof having this epitope.
Example 5. Assay
The assay described below is a non-isotopic
assay utilizing an enzyme and a substrate for the
detection of gp120 or fragments thereof, which was
*Trade-mark
' CA 02003794 1998-12-07
-29-
1 employed in detecting the gp120 proteins produced by the
methods and compositions of the present invention.
In the assay, the criteria for detecting gp120
is dependent on antibody specificity. An anti-gp120
monoclonal antibody [DuPont, Cat. No. 9284 diluted in
O.1M sodium carbonate buffer (pH 9.5) to two ugs/ml, is
used to capture the gp120 protein. 100 ul of this
antibody dilution is added to each well in duplicate in an
assay plate, except for those wells designated as
controls. The plates were incubated at 4°C overnight.
The antibody was washed-out the following day and the
plate blocked by adding 300 ul of blocking buffer
consisting of to BSA in PBS to each well for 1 hour at
room temperature.
The viral gp120 standards were diluted to
1 ~g/ml, 0.5 ug/ml, 0.25 ug/mI, 0.1 ug/ml, and
0.2 ug/ml in a washing buffer consisting of PBS and
0.05 TWEEN 20. 100 ~1 of the diluted standards are
added to each well in\duplicate. The plates were
incubated on a plate shaker for 2 hours at room
temperature, and thereafter, each plate was washed four
times with washing buffer.
To each well, 100 ul of rabbit anti-~p120
antibody (described by DeBouck et al, European
Application No. 293,184 published November 30, 1988)
diluted 1/1000 in washing buffer was added and each plated
incubated on a shaker for 1 hour. This second antibody
'sandwiches the gp120 between the two antibodies. The
plates were, thereafter, washed four times with washing
buffer. To detect this complex, a third antibody, 100
ul of peroxidase (POD) labeled goat anti-rabbit antibody
(mostly IgG and IgM antibody) diluted in washing buffer
with no azide, is added to each well. The plates were
then incubated for 2 hours on a sha'_~er at room temperature.
After the plates were washed four times,
100 ul of a colorless substrate (1 mg/ml of OPD in
v~~~ ~ s ~:'~~
-30-
1 citrate buffer with 4 ul of 35% hydrogen peroxide per
ZO ml of buffer) was added. The hydrogen peroxide was
added just prior to adding substrate to the wells. These
plates were incubated ~or 8 minutes on a shaker and the
reaction stopped by adding 100 ~l of 0.1 M sodium
flyoride to each well, rn the presence of
peroxidase-conjugated antibodies, the substrate turns deep
yellaw. Optical density, or intensity of the color, which
is proportional to the amaunt of gp120 captured, was read
on a plate reader at 450 manometers, and a standard curve
was constructed with concentrations of unknowns
aalcula~ted. The amount of gp120 in the supernatant
culture was determined by comparison to this standard
curve.
The above description and examples fully
disclose the invention, including preferred embodiments
thereof. Modifications of the methods described, e.g,
employing other truncated gp160/gp120 sequences that are
obvious to one of ordinary skill in the art of molecular
genetics and related sciences, are intended to fall within
the scope of the following claims:
30
