U.S. patent application number 10/073118 was filed with the patent office on 2003-03-20 for albumin derivatives with therapeutic functions.
This patent application is currently assigned to Rhone-Poulenc Sante. Invention is credited to Becquart, Jerome, Fleer, Reinhard, Hirel, Philippe, Klatzmann, David, Landais, Didier, Mayaux, Jean-Francois, Yeh, Patrice.
Application Number | 20030054554 10/073118 |
Document ID | / |
Family ID | 9384443 |
Filed Date | 2003-03-20 |
United States Patent
Application |
20030054554 |
Kind Code |
A1 |
Becquart, Jerome ; et
al. |
March 20, 2003 |
Albumin derivatives with therapeutic functions
Abstract
Utilization of albumin as a stable plasma transporter with a
therapeutic function that is derived from a membrane receptor. The
present invention is exemplified by the description of new
therapeutic agents that can be used in the treatment of Acquired
Immunodeficiency Syndrome: hybrid macromolecules composed of
albumin derivatives coupled to derivatives of the CD4 receptor
having a normal or a higher affinity for the HIV-1 virus.
Inventors: |
Becquart, Jerome; (Paris,
FR) ; Fleer, Reinhard; (Gif Sur Yvette, FR) ;
Hirel, Philippe; (Paris, FR) ; Klatzmann, David;
(Paris, FR) ; Landais, Didier; (Paris, FR)
; Mayaux, Jean-Francois; (Fontenay Aux Roses, FR)
; Yeh, Patrice; (Paris, FR) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT AND DUNNER
1775 K Street, N.W.
Washington
DC
20006
US
|
Assignee: |
Rhone-Poulenc Sante
Antony
FR
|
Family ID: |
9384443 |
Appl. No.: |
10/073118 |
Filed: |
February 12, 2002 |
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09551635 |
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Current U.S.
Class: |
435/456 ;
530/350 |
Current CPC
Class: |
C07K 2319/32 20130101;
C07K 2319/02 20130101; A61P 31/18 20180101; C07K 14/00 20130101;
C07K 14/70514 20130101; A61P 31/12 20180101; C07K 14/82 20130101;
A61P 35/00 20180101; C12N 15/67 20130101; C07K 2319/31 20130101;
C12N 15/62 20130101; C07K 2319/73 20130101; C12N 15/815 20130101;
C07K 14/4716 20130101; A61K 38/00 20130101; C07K 2319/00 20130101;
C07K 14/765 20130101 |
Class at
Publication: |
435/456 ;
530/350 |
International
Class: |
C07K 014/705; C12N
015/86 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 3, 1989 |
FR |
89 10480 |
Claims
1/ Hybrid macromolecule characterized by the fact that it carries
either the active domain of a receptor for a given virus, or the
active domain of a molecule which can bind to the virus, or the
active domain of a receptor of a ligand intervening in a
pathological process, coupled to albumin or a variant of
albumin.
2/ Macromolecule according to claim 1, in which the receptor is a
membrane receptor.
3/ macromolecule according to claims 1 and 2, characterized by the
fact that such a macromolecule is substantially proteinic:
4/ Macromolecule according to claims 1, 2 or 3, in which the
coupling is covalent.
5/ Macromolecule according to claim 4, in which the covalent
coupling is accomplished by a peptide linkage.
6/ Macromolecule according to claims 1, 2, 3, 4 or 5, in which the
active domain of the receptor is the active domain of a receptor
normally used by a virus for its propagation in the host
organism.
7/ Macromolecule according to claims 1, 2, 3, 4 or 5, in which the
active domain of the receptor is the active domain of a receptor
intervening in the internalization of infectious virions complexed
to immunoglobulins.
8/ Macromolecule according to claim 7, in which the active domain
of the receptor is the active domain of a receptor of the type
Fc.gamma. RIII.
9/ Macromolecule according to claim 8, in which the active domain
of the receptor is the active domain of the receptor CD16.
10/ Macromolecule according to claim 1, 2, 3, 4 or 5 in which the
active domain of the receptor is the active domain of a receptor of
a factor intervening in an oncogenic process.
11/ Macromolecule according to claim 10, in which the active domain
of the receptor is the active domain of a tyrosine kinase-type
receptor.
12/ Macromolecule according to claim 11, in which the active domain
of the receptor is the active domain of the proto-oncogene
c-erbB2.
13/ Macromolecule according to one of the claims 1 through 12,
characterized by the fact that the albumin used is of human
origin.
14/ Macromolecule according to one of the claims 1 through 6, in
which the receptor is all or part of the CD4 molecule used by the
HIV-1 virus for its propagation in the host organism, including all
artificial variations of the region of interaction with the virus
which have a higher than normal molecular affinity for the
virus.
15/ Macromolecule according to claim 14 in which the receptor is
made up of the V1 and V2 domains of the CD4 molecule.
16/ Macromolecule according to one of the claims 1 through 15,
characterized by the fact that it carries more than one active
domain of the receptor or of the molecule capable of binding the
ligand.
17/ Macromolecule according to one of the claims 1 through 16, in
which albumin or the variant of albumin is localized at the
N-terminal end.
18/ Macromolecule according to claim 17 in which a dimerization or
polymerization function is incorporated to permit an increase in
the local concentration of the active domain of the receptor of the
virus or of the receptor of the ligand associated with an oncogenic
process.
19/ Macromolecule according to claims 1 to 18 characterized in that
it is devoid of proteolytic cleavage sites between the active
domain of the receptor or of the molecule capable of binding said
ligands, and albumin or said variant of albumin.
20/ Macromolecule according to one of the claims 1 through 19,
characterized by the fact that it is obtained by cultivating cells
that have been transformed, transfected, or infected by a vector
expressing such macromolecule.
21/ Macromolecule according to claim 20, in which the transformed
cell is a yeast.
22/ Macromolecule according to claim 21, in which the yeast is a
strain of the genus Kluyveromyces.
23/ Macromolecule according to claim 21, in which the vector is an
expression vector derived from plasmid pKD1 in which the genes A, B
and C, the origin of replication, and the inverse repeats have been
conserved.
24/ A macromolecule according to one of the claims 1 through 23,
for use as a pharmaceutical.
25/ For use as a pharmaceutical according to claim 24, a
macromolecule composed of human albumin or an albumin variant, and
the V1 domain of the CD4 molecule.
26/ For use as a pharmaceutical according to claim 25, a
macromolecule composed of human albumin or an albumin variant, and
the V1V2 domains of CD4.
27/ Cells that have been transformed, transfected, or infected by a
vector expressing a macromolecule according to one of the claims 1
through 19.
28/ Cells according the claim 27, characterized by the fact that
these cells are yeasts.
29/ Cells according to claim 28, characterized by the fact that the
yeast is of the genus Kluyveromvces.
Description
[0001] The present invention involves the utilization of albumin
derivatives in the fabrication of therapeutic agents that can be
used in the treatment of certain viral diseases and cancers. More
precisely, this invention involves hybrid macromolecules
characterized by the fact that they carry either the active domain
of a receptor for a virus, or the active domain of a molecule which
can bind to a virus, or the active domain of a molecule able to
recognize the Fc fragment of immunoglobulins bound to a virus, or
the active domain of a molecule able to bind a ligand that
intervenes in a pathologic process, coupled to albumin or a variant
of albumin. In the text that follows, the terms albumin derivatives
or albumin variants are meant to designate all proteins with a high
plasma half-life obtained by modification (mutation, deletion,
and/or addition) via the techniques of genetic engineering of a
gene encoding a given isomorph of albumin, as well as all
macromolecules with high plasma half-life obtained by the in vitro
modification of the protein encoded by such genes. Such albumin
derivatives can be used as pharmaceuticals in antiviral treatment
due to the high affinity of a virus or of an immunoglobulin bound
to a virus for a site of fixation present on the albumin
derivative. They can be used as pharmaceuticals in the treatment of
certain cancers due to the affinity of a ligand, for example a
growth factor, for a site of fixation present on the albumin
derivative, especially when such a ligand is associated with a
particular membrane receptor whose amplification is correlated with
a transforming phenotype (proto-oncogenes). It should be understood
in the text that follows that all functionally therapeutic albumin
derivatives are designated indifferently by the generic term of
hybrid macromolecules with antiviral function, or hybrid
macromolecules with anticancer function, or simply hybrid
macromolecules. In particular, the present invention consists in
the obtention of new therapeutic agents characterized by the
coupling, through chemical or genetic engineering techniques, of at
least two distinct functions:
[0002] (i) a stable plasma transporter function provided by any
albumin variant, and in particular by human serum albumin (HSA).
The genes coding for HSA are highly polymorphic and more than 30
different genetic alleles have been reported (Weitkamp L. R et al.,
Ann. Hum. Genet. 37(1973) 219-226). The albumin molecule, whose
three-dimensional structure has been characterized by X-ray
diffraction (Carter D. C. et al. Science 244(1989) 1195-1198), was
chosen to provide the stable transporter function because it is the
most abundant plasma protein (40 g per liter in humans), it has a
high plasma half-life (14-20 days in humans, Waldmann T. A., in
"Albumin Structure, Function and Uses", Rosenoer V.M. et al. (eds),
Pergamon Press, Oxford, (1977) 255-275), and above all it has the
advantage of being devoid of enzymatic function, thus permitting
its therapeutic utilization at high doses.
[0003] (ii) an antiviral or anticancer therapeutic function. The
antiviral function is to serve as a decoy for the specific binding
of a virus, or as a decoy for the binding of a virus-immunoglobulin
complex. For example, the antiviral function can be provided by all
of part of a specific receptor normally used by a virus for its
propagation in the host organism, or by any molecule capable of
binding such a virus with an affinity high enough to permit its
utilization in vivo as an antiviral agent. The antiviral function
can also be provided by all or part of a receptor capable of
recognizing immunoglobulins complexed with a virus, or by any
molecule capable of binding such complexes with an affinity high
enough to permit its utilization in vivo as an antiviral agent. The
anti-cancer function is to serve as a decoy for the binding of a
ligand and in particular a growth factor implicated in an oncogenic
process, and is provided by all or part of a cellular
proto-oncogene, or by any molecule capable of binding such a ligand
With an affinity high enough to allow its utilization in vivo as an
anticancer agent.
[0004] (iii) in cases where a high local concentration of the
therapeutic function is desirable, for example because it
synergizes an inhibition of the infectivity of a virus in vivo a
third function allowing the dimerization or the polymerization of
the therapeutically active hybrid macromolecule can be added,
possibly in a redundant fashion. For example, such a function could
be provided by a "leucine zipper" motif (Landschulz W. H. et al.,
Science 240(1988) 1759-1764), or by protein domains known to be
necessary for homodimerization of certain proteins such as the
domain of the product of the tat gene coded by the HIV-1 viral
genome (Frankel A. D. et al., Science 240(1988) 70-73; Frankel A.
D. et al., Proc. Natl. Acad. Sci. USA 85(1988) 6297-6300).
[0005] In the present invention, the plasma transporter function,
the therapeutic function, and a potential polymerization function,
are integrated into the same macromolecule using the techniques of
genetic engineering.
[0006] One of the goals of the present invention is to obtain
hybrid macromolecules derived from HSA which can be useful in the
fight against certain viral diseases, such as Acquired
Immunodeficiency Syndrome (AIDS). Another goal is to obtain hybrid
HSA macromolecular derivatives useful in the treatment of certain
cancers, notably those cancers associated with genomic
amplification and/or overexpression of human proto-oncogenes, such
as the proto-oncogene c-erbB-2(Semba K. et al., Proc. Natl. Acad.
Sci. USA. 82(1985) 6497-6501; Slamon D. J. et al., Science
235(1987) 177-182; Kraus M. H. et al., EMBO J. 6(1987) 605610).
[0007] The HIV-1 virus is one of the retroviruses responsible for
Acquired Immunodeficiency Syndrome in man. This virus has been well
studied over the past five years; a fundamental discovery concerns
the elucidation of the role of the CD4(T4) molecule as the receptor
of the HIV-1 virus (Dalgleish A. G. et al., Nature 312(1984)
763-767; Klatzmann D. et al., Nature 312(1984) 767-768). The
virus-receptor interaction occurs through the highly specific
binding of the viral envelope protein (gp120) to the CD4 molecule
(McDougal et al., Science 231(1986) 382-385). The discovery of this
interaction between the HIV-1 virus and certain T lymphocytes was
the basis of a patent claiming the utilization of the T4 molecule
or its antibodies as therapeutic agents against the HIV-1 virus
(French patent application FR 2 570 278).
[0008] The cloning and the first version of the sequence of the
gene encoding human CD4 has been described by Maddon et al. (Cell
42(1985) 93-104), and a corrected version by Littmann et al. (Cell
55(1988) 541): the CD4 molecule is a member of the super-family of
immunoglobulins and specifically, it carries a V1 N-terminal domain
which is substantially homologous to the immunoglobulin heavy chain
variable domain (Maddon P. J. et al., Cell 42(1985) 93-104).
Experiments involving in vitro DNA recombination, using the gene
coding for the CD4 molecule, have provided definite proof that the
product of the CD4 gene is the principal receptor of the HIV-1
virus (Maddon P. J. et al., Cell 47(1986) 333-348). The sequence of
this gene as well as its utilization as an anti-HIV-1 therapeutic
agent are discussed in International patent application WO 88 013
040 A1.
[0009] The manipulation of the CD4 gene by the techniques of DNA
recombination has provided a series of first generation soluble
variants capable of antiviral action in vitro (Smith D. H. et al.,
Science 238(1987) 1704-1707; Traunecker A. et al., Nature 331(1988)
84-86; Fischer R. A. et al., Nature 331(1988) 76-78; Hussey R. E.
et al., Nature 331(1988) 78-81; Deen K. C. et al., Nature 331(1988)
82-84), and in vivo (Watanabe M. et al., Nature 337(1989) 267-270).
In all cases, it was observed during various in vivo assays in
animals (rabbit, monkey) as well as during phase I clinical trials,
that the first generation soluble CD4 variant consisting of the CD4
molecule lacking the two domains in the C-terminal region has a
very short half-life: approximately 15 minutes in rabbits (Capon et
al., Nature 337(1989) 525-531), while 50% of first generation
soluble CD4 administered intramuscularly to Rhesus monkeys remained
bioavailable for 6 hours (Watanabe et al., Nature 337(1989)
267-270). In addition, Phase 1 clinical trials conducted on 60
patients presenting AIDS or ARC ("Aids Related Complex") indicated
that the half-life of the Genentech product varied between 60
minutes (intraveinous administration) and 9 hours (intramuscular
administration) (AIDS/HIV Experimental Treatment Directory, AmFAR,
May 1989). Clearly, a therapeutic agent with such a weak stability
in vivo constitutes a major handicap. In effect, repeated
injections of the product, which are costly and inconvenient for
the patient, or an administration of the product by perfusion,
become necessary to attain an efficient concentration in plasma. It
is therefore especially important to find derivatives of the CD4
molecule characterized by a much higher in vivo half-life.
[0010] The part of the CD4 molecule which interacts with the HIV-1
virus has been localized to the N-terminal region, and in
particular to the V1 domain (Berger E. A. et al., Proc. Natl. Acad.
Sci. USA 85(1987) 2357-2361). It has been observed that a
significant proportion (about 10%) of HIV-1infected subjects
develop an immune response against the CD4 receptor, with
antibodies directed against the C-terminal region of the
extra-cellular portion of the receptor (Thiriart C. et al., AIDS
2(1988) 345-352; Chams V. et al., AIDS 2(1988) 353-361). Therefore,
according to a preferred embodiment of the present invention, only
the N-terminal domains V1 or V1 V2 of the CD4 molecule, which carry
all the viral binding activity, will be used in fusion with the
stable transporter function derived from albumin.
[0011] On the basis of the homology observed with the variable
domain of immunoglobulins, several laboratories have constructed
genetic fusions between the CD4 molecule and different types of
immunoglobulins, generating hybrid immunoglobulins with antiviral
action in vitro (Capon D. J. et al., Nature 337(1989) 525-531;
Traunecker A. et al., Nature 339(1989) 68-70; also see
International patent application WO 89 02922). However, the
implication of the FcyRIII receptor (type 3 receptor for the Fc
region of IgG's), which in humans is the antigen CD16(Unkeless J.
C. and Jacquillat C., J. Immunol. Meth. 100(1987) 235-241), in the
internalization of the HIV-1 virus (Homsy J. et al., Science
244(1989) 1357-1360) suggests an important role of these receptors
in viral propagation in vivo. The receptor, which has been recently
cloned (Simmons D. and Seed B., Nature 333(1988) 568-570), is
mainly located in the membranes of macrophages, polynuclear cells
and granulocytes, but in contrast to CD4, the CD16 receptor also
exists in a soluble state in serum (Khayat D. et al., J. Immunol.
132(1984) 2496-2501; Khayat D. et al., J. Immunol. Meth. 100(1987)
235-241). It should be noted that the membraneous CD16 receptor is
used as a second route of entry by the HIV-1 virus to infect
macrophages, due to the presence of facilitating antibodies (Homsy
J. et al., Science 244(1989) 1357-1360). This process of infection
which involves an "Fc receptor" at the surface of target cells (for
example the CD16 receptor), and the Fc region of antibodies
directed against the virion, is named ADE ("Antibody Dependent
Enhancement"); it has also been described for the flavivirus
(Peiris J. S. M. et al., Nature 289(1981) 189-191) and the
Visna-Maedi ovine lentivirus (Jolly P. E. et al., J. Virol.
63(1989) 1811-1813). Other "Fc receptors" have been described for
IgG's (FcyRI and FcyRII for example) as well as for other classes
of immunoglobulins, and the ADE phenomenon also involves other
types of "Fc receptors" such as that recognized by the monoclonal
antibody 3 G8(Homsy J. et al., Science 244(1989) 1357-1360; Takeda
A. et al., Science 242(1988) 580-583). One can thus call into
question the efficiency of hybrid antiviral macromolecules which
depend uniquely on fusions between immunoglobulins and all or part
of a receptor normally used by a virus such as HIV-1 for its
propagation in the host; in effect, the presence of a functional Fc
fragment on such molecules could actually facilitate viral
infection of certain cell types. It is also important to obtain CD4
derivatives that can be used at high therapeutic
concentrations.
[0012] A different type of chimeric construction involving the
bacterial protein MalE and the CD4 molecule has been studied
(Clement J. M. et al., C. R Acad. Sci. Paris 308 series III (1989)
401-406). Such a fusion allows one to take advantage of the
properties of the MalE protein, in particular regarding the
production and/or purification of the hybrid protein. In addition,
the construction of a genetic fusion between the CD4 molecule and a
bacterial toxin has also been described (Chaudhary V. K. et al.,
Nature 335(1988) 369-372). In these cases, utilization of a genetic
fusion involving a bacterial protein for therapy in humans can be
questionable.
[0013] The discovery of the role of the ADE phenomenon in the
propagation of certain viruses, in particular lentiviruses
including HIV-1, justifies the search for alternatives to both the
development of an anti-AIDS vaccine, and to the development of
therapeutic agents based solely on fusions between immunoglobulins
and molecules capable of binding the virus. This is why the
anti-AIDS therapeutic agents described in the present invention are
based on the fusion of all or part of a receptor used directly or
indirectly by the HIV-1 virus for its propagation in vivo, with a
stable plasma protein, devoid of enzymatic activity, and lacking
the Fc fragment.
[0014] In particular, the present invention concerns the coupling,
mainly by genetic engineering, of human albumin variants with a
binding site for the HIV-1 virus. Such hybrid macromolecules
derived from human serum albumin are characterized by the presence
of one or several variants of the CD4 receptor arising from the
modification, particularly by in vitro DNA recombination techniques
(mutation, deletion, and/or addition), of the N-terminal domain of
the CD4 receptor, which is implicated in the specific interaction
of the HIV-1 virus with target cells. Such hybrid macromolecules
circulating in the plasma represent stable decoys with an antiviral
function, and will be designated by the generic term HSA-CD4.
Another goal of this invention concerns the coupling of human
albumin variants with variants of the CD16 molecule, which is
implicated in the internalization of viruses including HIV-1(to be
designated by the generic term HSA-CD16), and in general the
coupling of albumin variants with olecules capable of mimicking the
cellular receptors responsible for the ADE phenomenon of certain
viruses, and in particular the lentiviruses.
[0015] The principles of the present invention can also be applied
to other receptors used directly or indirectly by a human or animal
virus for its propagation in the host organism. For example:
[0016] 1/ intercellular adhesion molecule 1(ICAM-1), shown to be
the receptor for human rhinovirus HRV14 (Greve J. M. et al., Cell
56(1989) 839-847; Staunton D. E. et al., Cell 56(1989)
849-853);
[0017] 2/ poliovirus receptor, recently doned by Mendelsohn et al.
(Cell 56(1989) 855-865);
[0018] 3/ the receptor of complement factor C3 D which is the
receptor of Epstein-Barr virus (EBV) in human cells (Fingeroth J.
D. et al., Proc. Natl. Acad. Sci. USA 81(1984) 4510-4514), this
virus being responsible for infectious mononucleosis and for
certain lymphomas in man;
[0019] 4/ human T cell leukemia virus HTLV-I and HTLV-II receptors,
recently mapped to chromosome 17(Sommerfelt M. A. et al., Science
242(1988) 1557-1559), these viruses being responsible for adult T
cell leukemia as well as for tropical spastic paraparesie (HTLV-I)
and tricholeucocytic leukemia (HTLV-II);
[0020] 5/ the receptor of the ecotropic murine leukemia virus
MuLV-E, mapped to chromosome 5 of the mouse by Oie et al. (Nature
274(1978) 60-62) and recently cloned by Albritton et al. (Cell
57(1989) 659-666).
[0021] Another goal of the present invention concerns the
development of stable hybrid macromolecules with an anticancer
function, obtained by the coupling of albumin variants with
molecules able to bind growth factors which, in certain pathologies
associated with the amplification of the corresponding membraneous
proto-oncogenes, can interact with their target cells and induce a
transformed phenotype. An example of such receptors is the class of
receptors with tyrosine kinase activity (Yarden Y. and Ulrich A.,
Biochemistry 27(1988) 3113-3119), the best known being the
epidermal growth factor (EGF) and the colony stimulating factor I
(CSF-I) receptors, respectively coded by the proto-oncogenes
c-erbB-1(Downward J. et al., Nature 307(1984) 521-527) and c-fms
(Sherr C. J. et al., Cell 41(1985) 665-676). Another example of
such receptors includes the human insulin receptor (HIR), the
platelet-derived growth factor (PDGF) receptor, the insulin-like
growth factor I (IGF-I) receptor, and most notably the
proto-oncogene c-erbB-2, whose genomic amplification and/or
overexpression was shown to be strictly correlated with certain
human cancers, in particular breast cancer (Slamon D. J. et al.,
Science 235(1987) 177-182; Kraus M. H. et al., EMBO J. 6(1987)
605-610). Furthermore, the principles put forth in the present
invention can be equally applied to other receptors, for example
the interleukin 6(IL-6) receptor, which has been shown in vitro to
be an autocrine factor in renal carcinoma cells (Miki S. et al.,
FEBS Lett., 250(1989) 607-610).
[0022] As indicated above, the hybrid macromolecules of interest
are substantially preferably proteinic and can therefore be
generated by the techniques of genetic engineering. The preferred
way to obtain these macromolecules is by the culture of cells
transformed, transfected, or infected by vectors expressing the
macromolecule. In particular, expression vectors capable of
transforming yeasts, especially of the genus Kluyveromyces, for the
secretion of proteins will be used. Such a system allows for the
production of high quantities of the hybrid macromolecule in a
mature form, which is secreted into the culture medium, thus
facilitating purification.
[0023] The preferred method for expression and secretion of the
hybrid macromolecules consists therefore of the transformation of
yeast of the genus Kluyveromyces by expression vectors derived from
the extrachromosomal replicon pKD1, initially isolated from K.
marxianus var. drosophilarum. These yeasts, and in particular K.
marxianus (including the varieties lactis, drosophilarum and
marxianus which are henceforth designated respectively as K. lactis
K. drosophilarum and K. fragilis), are generally capable of
replicating these vectors in a stable fashion and possess the
further advantage of being included in the list of G.R.A.S.
("Generally Recognized As Safe") organisms. The yeasts of
particular interest include industrial strains of Kluyveromyces
capable of stable replication of said plasmid derived from plasmid
pKD1 into which has been inserted a selectable marker as well as an
expression cassette permitting the secretion of the given hybrid
macromolecule at high levels.
[0024] Three types of cloning vectors have been described for
Kluyveromyces:
[0025] i) Integrating vectors containing sequences homologous to
regions of the Kluvveromyces genome and which, after being
introduced into the cells, are integrated in the Kluvveromyces
chromosomes by in vivo recombination (International patent
application WO 83/04050). Integration, a rare event requiring an
efficient selection marker, is obtained when these vectors do not
contain sequences permitting autonomous replication in the cell.
The advantage of this system is the stability of the transformed
strains, meaning that they can be grown in a normal nutritive
medium without the need for selection pressure to maintain the
integrated sequences. The disadvantage, however, is that the
integrated genes are present in only a very small number of copies
per cell, which frequently results in a low level of production of
a heterologous protein.
[0026] ii) Replicating vectors containing Autonomously Replicating
Sequences (ARS) derived from the chromosomal DNA of Kluyveromyces
(Das S. and Hollenberg C.P., Current Genetics 6(1982) 123-128;
International patent application WO 83/04050). However these
vectors are of only moderate interest, since their segregation in
mitotic cell division is not homogeneous, which results in their
loss from the cells at high frequency even under selection
pressure.
[0027] iii) Replicating vectors derived from naturally occurring
yeast plasmids, either from the linear "killer" plasmid kl isolated
from K. lactis (de Louvencourt L. et al., J. Bacteriol. 154(1983)
737-742; European patent application EP 0 095 986 A1, publ. Dec. 7,
1983), or from the circular plasmid pKD1 isolated from K.
drosophilarum (Chen X.J. et al., Nucl. Acids Res. 14(1986)
4471-4480; Falcone C. et al., Plasmid 15(1986) 248-252; European
patent application EP 0 241 435 A2, publ. Oct. 14, 1987). The
vectors containing replicons derived from the linear "killer"
plasmid require a special nutrient medium, and are lost in 40-99%
of the cells after only 15 generations, even under selection
pressure (European patent application EP 0 095 986 A, 1983), which
limits their use for mass production of heterologous proteins. The
vectors derived from plasmid pKD1 described in European patent
application EP 0 241 435 A2 are also very unstable since even the
most performant vector (P3) is lost in approximately 70% of the
cells after only six generations under nonselective growth
conditions.
[0028] An object of the present invention concerns the utilization
of certain plasmid constructions derived from the entire pKD1
plasmid; such constructions possess significantly higher stability
characteristics than those mentioned in European patent application
EP 0 241 435 A2. It will be shown in the present invention that
these new vectors are stably maintained in over 80% of the cells
after 50 generations under nonselective growth conditions.
[0029] The high stability of the vectors used in the present
invention was obtained by exploiting fully the characteristics of
plasmid pKD1. Besides an origin of replication, this
extrachromosomal replicon system possesses two inverted repeats,
each 346 nucleotides in length, and three open reading frames
coding for genes A, B et C, whose expression is crucial for plasmid
stability and high copy number. By analogy with the 2 .mu. plasmid
of S. cerevisiae. which is structurally related to plasmid pKD1
(Chen X.J. et al., Nud. Acids Res. 14(1986) 4471-4480), the
proteins encoded by genes B et C are probably involved in plasmid
partitioning during mitotic cell division, and may play a role in
the negative regulation of gene A which encodes a site-specific
recombinase (FLP). It has been shown that the FLP-mediated
recombination between the inverted repeats of the 2.mu. plasmid of
S. cerevisiae is the basis of a mechanism of autoregulation of the
number of plasmid copies per cell: when copy number becomes too low
to permit the production of sufficient quantities of the products
of genes B and C which act as repressors of gene A, the FLP
recombinase is induced and the plasmid replicates according to a
rolling circle type model, which amplifies copy number to about 50
copies per cell (Futcher A.B., Yeast 4(1988) 27-40).
[0030] The vectors published in European patent application EP 0,
241, 435 A2 do not possess the above-mentioned structural
characteristics of plasmid pKD1 of K. drosophilarum: vector A15
does not carry the complete sequence of pKD1, and vectors P1 and P3
carry an interrupted A gene, thereby destroying the system of
autoregulated replication of resident plasmid pKD1. In contrast,
the pKD1-derived constructs used in the present invention maintain
the structural integrity of the inverted repeats and the open
reading frames A, B and C, resulting in a notably higher stability
of the plasmid as well as an increased level of secretion of the
therapeutically active hybrid macromolecules.
[0031] The expression cassette will include a transcription
initiation region (promoter) which controls the expression of the
gene coding for the hybrid macromolecule. The choice of promoters
varies according to the particular host used. These promoters
derive from genes of Saccharomyces or Kluyveromyces type yeasts,
such as the genes encoding phosphoglycerate kinase (PGK),
glyceraldehyde-3phosphate dehydrogenase (GPD) , the lactase of
Kluyveromyces (LAC4), the enolases (ENO), the alcohol
dehydrogenases (ADH), the acid phosphatase of S. cerevisiae(PHO5),
etc... These control regions may be modified, for example by in
vitro sitedirected mutagenesis, by introduction of additional
control elements or synthetic sequences, or by deletions or
substitutions of the original control elements. For example,
transcription-regulating elements, the so-called "enhancers" of
higher eukaryotes and the "upstream activating sequences" (UAS) of
yeasts, originating from other yeast promoters such as the GAL1 and
GAL10 promoters of S. cerevisiae or the LAC4 promoter of K. lactis,
or even the enhancers of genes recognized by viral transactivators
such as the E2 transactivator of papillomavirus, can be used to
construct hybrid promoters which enable the growth phase of a yeast
culture to be separated from the phase of expression of the gene
encoding the hybrid macromolecule. The expression cassette used in
the present invention also includes a transcription and translation
termination region which is functional in the intended host and
which is positioned at the 3' end of the sequence coding for the
hybrid macromolecule.
[0032] The sequence coding for the hybrid macromolecule will be
preceded by a signal sequence which serves to direct the proteins
into the secretory pathway. This signal sequence can derive from
the natural N-terminal region of albumin (the prepro region), or it
can be obtained from yeast genes coding for secreted proteins, such
as the sexual pheremones or the killer toxins, or it can derive
from any sequence known to increase the secretion of the so-called
proteins of pharmaceutical interest, including synthetic sequences
and all combinations between a "pre" and a "pro" region.
[0033] The junction between the signal sequence and the sequence
coding for the hybrid macromolecule to be secreted in mature form
corresponds to a site of cleavage of a yeast endoprotease, for
example a pair of basic amino acids of the type
Lys.sup.-2-Arg.sup.-1 or Arg .sup.-2-Arg.sup.-1 corresponding to
the recognition site of the protease coded by the KEX2 gene of S.
cerevisiae or the KEXI gene of K. lactis (Chen X. J. et al., J.
Basic Microbiol. 28(1988) 211-220; Wesolowski-Louvel M. et al.,
Yeast 4(1988) 71-81). In fact, the product of the KEX2 gene of S.
cerevisiae cleaves the normal "pro" sequence of albumin in vitro
but does not cleave the sequence corresponding to the pro-albumin
"Christchurch" in which the pair of basic amino acids is mutated to
Arg.sup.-2-Glu.sup.-1(Bathurst I. C. et al., Science 235(1987)
348-350).
[0034] In addition to the expression cassette, the vector will
include one or several markers enabling the transformed host to be
selected. Such markers include the URA3 gene of yeast, or markers
conferring resistance to antibiotics such as geneticin (G418), or
any other toxic compound such as certain metal ions. These
resistance genes will be placed under the control of the
appropriate transcription and translation signals allowing for
their expression in a given host.
[0035] The assembly consisting of the expression cassette and the
selectable marker can be used either to directly transform yeast,
or can be inserted into an extrachromosomal replicative vector. In
the first case, sequences homologous to regions present on the host
chromosomes will be preferably fused to the assembly. These
sequences will be positioned on each side of the expression
cassette and the selectable marker in order to augment the
frequency of integration of the assembly into the host chromosome
by in vivo recombination. In the case where the expression cassette
is inserted into a replicative vector, the preferred replication
system for Kluyveromyces is derived from the plasmid pKD1 initially
isolated from K. drosophilarum. while the preferred replication
system for Saccharomyces is derived from the 2 .mu. plasmid. The
expression vector can contain all or part of the above replication
systems or can combine elements derived from plasmid pKD1 as well
as the 2 .mu. plasmid.
[0036] When expression in yeasts of the genus Kluyveromyces is
desired, the preferred constructions are those which contain the
entire sequence of plasmid pKD1. Specifically, preferred
constructions are those where the site of insertion of foreign
sequences into pKD1 is localized in a 197 bp region lying between
the SacI (SstI) site and the MstII site, or alternatively at the
SphI site of this plasmid, which permits high stability of the
replication systems in the host cells.
[0037] The expression plasmids can also take the form of shuttle
vectors between a bacterial host such as Escherichia coli and
yeasts; in this case an origin of replication and a selectable
marker that function in the bacterial host would be required. It is
also possible to position restriction sites which are unique on the
expression vector such that they flank the bacterial sequences.
This allows the bacterial sequences to be eliminated by restriction
deavage, and the vector to be religated prior to transformation of
yeast, and this can result in a higher plasmid copy number and
enhanced plasmid stability. Certain restriction sites such as
5'-GGCCNNNNNGGCC-3'(SfiI) or 5'-GCGGCCGC-3'(NotI) are particularly
convenient since they are very rare in yeasts and are generally
absent from an expression plasmid.
[0038] The expression vectors constructed as described above are
introduced into yeasts according to dassical techniques described
in the literature. After selection of transformed cells, those
cells expressing the hybrid macromolecule of interest are
inoculated into an appropriate selective medium and then tested for
their capacity to secrete the given protein into the extracellular
medium. The harvesting of the protein can be conducted during cell
growth for continuous cultures, or at the end of the growth phase
for batch cultures. The hybrid proteins which are the subject of
the present invention are then purified from the culture
supernatant by methods which take into account their molecular
characteristics and pharmacological activities.
[0039] The present invention also concerns the therapeutic
application of the hybrid macromolecules described therein, notably
in the treatment and the prevention of AIDS, as well as the cells
which are transformed, transfected, or infected by vectors
expressing such macromolecules.
[0040] The examples which follow as well as the attached figures
show some of the characteristics and advantages of the present
invention.
DESCRIPTION OF FIGURES
[0041] The diagrams of the plasmids shown in the figures are not
drawn to scale, and only the restriction sites important for the
constructions are indicated.
[0042] FIG. 1: Oligodeoxynucleotides used to generate the MstII and
HindIII-SmaI restriction sites, situated respectively upstream and
downstream of the V1 V2 domains of the CD4 molecule.
[0043] FIG. 2: Nucleotide sequence of the MstII-SmaI restriction
fragment including the V1 and V2 domains of the CD4 receptor of the
HIV-1 virus. The recognition sites for MstII, HindIII and SmaI are
underlined.
[0044] FIG. 3: Construction of plasmid pXL869 coding for
prepro-HSA.
[0045] FIG. 4: Construction of plasmids pYG208 and pYG210.
[0046] FIG. 5: Construction of plasmid pYG11.
[0047] FIG. 6: Construction of plasmid pYG18.
[0048] FIG. 7: Restriction map of plasmid pYG303.
[0049] FIG. 8: Nucleotide sequence of restriction fragment HindIII
coding for the protein fusion prepro-HSA-V1 V2. Black arrows
indicate the end of the "pre" and "pro" regions of HSA. The MstII
site is underlined.
[0050] FIG. 9: Restriction map of plasmid pYG306.
[0051] FIG. 10: Construction of plasmid pUC-URA3.
[0052] FIG. 11: Construction of plasmid pCXJ1.
[0053] FIG. 12: Construction of plasmid pkl-PS1535-6.
[0054] FIG. 13: Construction of plasmids pUC-kan1 and
pUC-kan202.
[0055] FIG. 14: Construction of plasmid pKan707.
[0056] FIG. 15: Stability curve of plasmid pKan707in strain MW98-8
C under nonselective growth conditions.
[0057] FIG. 16: Construction of plasmid pYG308 B.
[0058] FIG. 17: Construction of plasmid pYG221 B.
[0059] FIG. 18: Characterization of the material secreted after 4
days in culture by strain MW98-8 C transformed by plasmids pYG221 B
(prepro-HSA) and pYG308 B (prepro-HSA-V1 V2). A, Coomassie staining
after electrophoretic migration in an 8.5% polyacrylamide gel.
Molecular weight standards (lane 1); supernatant equivalent to 300
.mu.l of the culture transformed by plasmid pYG308 B (lane 2);
supernatant equivalent to 100.mu.l of the culture transformed by
plasmid pYG221 B (lane 3); 500 ng of HSA (lane 4). B, immunologic
characterization of the secreted material subject to
electrophoretic migration in an 8.5% polyacrylamide gel, followed
by transfer to a nitrocellulose membrane and utilization of primary
antibodies directed against human albumin: 250 ng of HSA standard
(lane 1); supernatant equivalent to 100.mu.l of the culture
transformed by plasmid pYG308 B (lane 2); supernatant equivalent to
10.mu.l of the culture transformed by plasmid pYG221 B (lane 3). C,
exactly as in B except that polyclonal antibodies directed against
the CD4 molecule were used in place of antibodies directed against
HSA.
[0060] FIG. 19: Titration of the protein HSA-V1 V2(1 .mu.g/ml) by
mouse monoclonal antibody Leu3 A (Becton Dickinson, Mountain View,
Calif., U.S.A.) (panel A), by mouse monoclonal antibody OKT4 A
(Ortho Diagnostic Systems, Raritan, N.J., USA) (panel B), or by
polyclonal goat anti-HSA coupled to peroxidase (Nordic, Tilburg,
Netherlands) (panel C). After using antibodies Leu3 A and OKT4 A, a
secondary rabbit anti-mouse antibody coupled to peroxidase (Nordic)
is used. Titration curves for the three primary antibodies used in
parts A, B and C were determined by measuring optical density at
405 nm after addition of a chromogenic substrate of peroxidase
(ABTS, Fluka, Switzerland). Ordinate: OD at 405 nm, abscissa:
dilution factor of the primary antibody used.
[0061] FIG. 20: Assay of protein HSA-V1 V2 by the ELISA sandwich
method: rabbit polydonal anti-HSA (Sigma)/HSA-V1 V2/ mouse
monoclonal antibody Leu3 A (Becton Dickinson) (panel A), or rabbit
polyclonal anti-HSA (Sigma)/HSA-V1 V2/mouse monocdonal antibody
OKT4 A (Ortho Diagnostic Systems) (panel B). After incubation of
each antibody with the HSA-15 V1 V2 protein, a secondary rabbit
anti-mouse antibody coupled to peroxidase (Nordic) is added.
Titration curves were determined by measuring optical density at
405 nm after addition of the peroxidase substrate ABTS. Ordinate:
OD at 405 nm; abscissa: concentration of HSA-V1 V2 in .mu.g/ml.
[0062] FIG. 21: Soluble phase inhibition of binding to CD4 by 125
femtomoles of recombinant gp160 protein (Transgene, Strasbourg,
France). Optical density at 492 nm is represented on the ordinate
(the value 2 is the saturation optical density of the system) and
the quantities of HSA (control), HSA-CD4, and soluble CD4 are shown
on the abscissa (picomoles of protein).
[0063] FIG. 22: Inhibition of the binding of inactivated HIV-1
virus to cell line CEM13. A, preliminary analysis of cell
populations sorted as a function of their fluorescence. Ordinate:
cell number; abscissa: fluorescence intensity (logarithmic scale).
B, histogram of cell populations sorted as a function of their
fluorescence. Column 1, negative control; Column 2, HIV-1 virus;
Column 3, HIV-1 virus preincubated with 116 picomoles of CD4
recombinant protein; Column 4, HIV-1 virus preincubated with 116
picomoles of HSA-V1 V2; Column 5, HIV-1 virus preincubated with 116
picomoles of HSA.
[0064] FIG. 23: Inhibition of infection in cell culture. Reverse
transcriptase activity was measured for 19 days after infection of
CEM13 cells. Assays were performed on microtitration plates
according to the following protocol: into each well, 10 .mu.l of
Buffer A (0.5 M KCl, 50 mM DTT, 0.5% Triton X-100), then 40.mu.l of
Buffer B (10 .mu.l 5 mM EDTA in 0.5 M Tris-HCl pH 7.8, 1 .mu.l 0.5
M MgCl.sub.2, 3 .mu.l .sup.3H-dTTP, 10 .mu.l poly rA-oligodT at 5
OD/ml, 16 .mu.l H.sub.2O) were added to 50 .mu.l culture
supernatant removed at different times after infection. The plates
were incubated for 1 hour at 37.degree. C., then 20 .mu.l of Buffer
C (120 mM Na.sub.4 P.sub.2 O.sub.7 in 60% TCA) was added and
incubation was continued for 15 minutes at 4.degree. C. The
precipitates formed were passed through Skatron filters using a
Skatron cell harvester, and washed with Buffer D (12 mM Na.sub.4
P.sub.2 O.sub.7 in 5% TCA). Filters were dried 15 minutes at
80.degree. C. and the radioactivity was measured in a scintillation
counter. Three independent samples were tested for each point.
[0065] FIG. 24: Changes in the in vivo concentrations of CD4, HSA
and HSA-CD4 over time.
[0066] FIG. 25: Construction of plasmids pYG232, pYG233 and
pYG364.
[0067] FIG. 26: Construction of plasmid pYG234.
[0068] FIG. 27: Construction of plasmids pYG332 and pYG347.
[0069] FIG. 28: Construction of plasmids pYG362, pYG363 and
pYG511.
[0070] FIG. 29: Restriction maps of plasmids pYG371, pYG374 and
pYG375.
[0071] FIG. 30: Restriction map of expression plasmid pYG373 B.
[0072] FIG. 31: Construction of plasmid pYG537.
[0073] FIG. 32: Construction of expression plasmid pYG560.
[0074] FIG. 33: Intracellular expression of hybrid proteins HSA-V1
(plasmid pYG366 B; lane b), V1-HSA (plasmid pYG373B; lane c),
V1-HSA-V1V2 (plasmid pYG380B; lane d), V1-HSA-V1(plasmid pYG381B,
lane e) and HSA-V1V2 (plasmid pYG308 B, lane f) in K. lactis strain
MW98-8C. Detection was performed by the Western Blot method using
polyclonal rabbit serum directed against HSA as primary antibody.
10 .mu.g of protein from the insoluble fraction was loaded in each
case.
[0075] FIG. 34: Introduction of the "Leucine Zipper" of c-jun(Bg1
II-AhaII fragment) in a hybrid protein HSA-CD4.
[0076] FIG. 35: Secretion in strain MW98-8C of truncated HSA
variants coupled to the V1V2 domains of the CD4 receptor. Panel 1:
Coomassie blue staining. Each lane was loaded with the equivalent
of 400 .mu.l of culture supernatant from the early stationary
phase. Molecular weight markers (lane a), strain transformed by
control vector pKan707 (lane b), HSA standard (lane c), strain
transformed by expression plasmids pYG308B (HSA.sub.585-V1V2, lane
d), pYG334B (HSA.sub.312-V1V2, lane e), and pYG335B
(HSA.sub.300-V1V2, lane f).
[0077] Panel 2: Western Blot detection using rabbit polyclonal
anti-HSA. Each lane was loaded with the equivalent of 100 .mu.l of
culture supernatant from the early stationary phase. Biotinylated
molecular weight markers (Bio-Rad, lane a), strain transformed by
control vector pKan707 (lane b), HSA standard (lane f), strain
transformed by expression plasmids pYG308B (HSAs.sub.585-V1V2, lane
c), pYG334B (HSA.sub.312-V1V2, lane d), and pYG335B
(HSA.sub.300-V1V2, lane e). Panel 3: Western Blot detection using a
rabbit polyclonal anti-CD4 serum; same legend as in Panel 2.
[0078] FIG. 36: Panel a: representation of several HindIII
(-25)-MstII restriction fragments corresponding to deletions in
HSA. Amino acid position (numbered according to mature HSA) is
indicated in parentheses. Panel b: detail of the position of the
MstII site in one of the deletants (clone YP63, linker insertion at
amino acid 495).
[0079] FIG. 37: Examples of the hinge regions between the HSA and
CD4 moieties. The amino acid pairs that are potential targets of
endoproteases involved in the secretory pathway are boxed.
[0080] Panel 1: hinge region of protein HSA.sub.585-CD4. Panel 2:
hinge region of HSA.sub.Ba131-CD4 proteins obtained by Ba131
deletion of the C-terminal portion of HSA (in this representation
the Lys-Lys pairs situated at the beginning of the CD4 moiety have
been modified by site-directed mutagenesis as exemplified in
E.13.2.). Panel 3: hinge region obtained by insertion of a
polypeptide (shown here a fragment of troponin C), obtained after
site directed mutagenesis using oligodeoxynucleotide Sq1445. Panel
4: general structure of the hinge region between the HSA and CD4
moieties.
[0081] FIG. 38: Panel 1: structure of the in-frame fusion between
the prepro region of HSA and the CD4 receptor, present notably in
expression plasmids pYG373B, pYG380B, pYG381B and pYG560. Panel 1a:
the amino acid pairs that are potential targets of endoproteases
involved in the secretory pathway are boxed. Panel 1b: These amino
acid pairs can be modified by mutating the second lysine of each
pair such that the pair is no longer a target for such
endoproteases. Panel 2: Examples of hinge regions between the CD4
and HSA moieties present notably in hybrid proteins V1-HSA (panel 2
a) or V1V2-HSA (panels 2b and 2c). Panel 3: general structure of
the hinge region between the CD4 and HSA moieties.
EXAMPLES
[0082] GENERAL CLONING TECHNIQUES.
[0083] The classical methods of molecular biology such as
preparative extractions of plasmid DNA, the centrifugation of
plasmid DNA in cesium chloride gradients, agarose and
polyacrylamide gel electrophoresis, the purification of DNA
fragments by electroelution, the extraction of proteins by phenol
or phenol/chloroform, the precipitation of DNA in the presence of
salt by ethanol or isopropanol, transformation of Escherichia coli
etc... have been abundantly described in the literature (Maniatis
T. et al., "Molecular Cloning, a Laboratory Manual", Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y., 1982; Ausubel F.M. et
al. (eds), "Current Protocols in Molecular Biology", John Wiley
& Sons, New York, 1987), and will not be reiterated here.
[0084] Restriction enzymes are furnished by New England Biolabs
(Biolabs), Bethesda Research Laboratories (BRL) or Amersham and are
used according to the recommendations of the manufacturer.
[0085] Plasmids pBR322, pUC8, pUC19 and the phages M13mp8 and
M13mp18 are of commercial origin (Bethesda Research
Laboratories).
[0086] For ligations, the DNA fragments are separated by size on
agarose (generally 0.8%) or polyacrylamide (generally 10%) gels,
purified by electroelution, extracted with phenol or
phenol/chloroform, precipitated with ethanol and then incubated in
the presence of T4 DNA ligase (Biolabs) according to the
recommendations of the manufacturer.
[0087] Filling in of 5' ends is carried out using the Klenow
fragment of E. coli DNA polymerase I (Biolabs) according to
manufacturer recommendations. Destruction of 3' protruding termini
is performed in the presence of T4 DNA polymerase (Biolabs) as
recommended by the manufacturer. Digestion of 5' protruding ends is
accomplished by limited treatment with S1 nuclease.
[0088] In vitro site-directed mutagenesis is performed according to
the method developed by Taylor et al. (Nucleic Acids Res. 13 (1985)
8749-8764) using the kit distributed by Amersham.
[0089] Enzymatic amplification of DNA fragments by the PCR
technique (Polymerase-catalyzed Chain Reaction, Saiki R.K. et al.,
Science 230 (1985) 1350-1354; Mullis K. B. and Faloona F. A., Meth.
Enzyme. 155(1987) 335-350) is carried out on a "DNA thermal
cycler"(Perkin Elmer Cetus) according to manufacturer
specifications.
[0090] Nucleotide sequencing is performed according to the method
developed by Sanger et al. (Proc. Natl. Acad. Sci. USA, 74 (1977)
5463-5467), using the Amersham kit.
[0091] Transformation of K. lactis with foreign DNA as well as the
purification of plasmid DNA from K. lactis are described in the
text.
[0092] Unless indicated otherwise, the bacterial strains used are
E. coli MC1060 (lacIPOZYA, X74, galU, galK, strA.sup.r), or E. coli
TG1(lac proA,B, supE, thi hsdD5/F'traD36, proA .sup.+B.sup.+,
ladI.sup.q, LacZ, M15).
[0093] All yeast strains used are members of the family of budding
yeasts and in particular of the genus Kluyveromyces. Examples of
these yeasts are given in the text. The K. lactis strain MW98-8 C
(.alpha., uraA arg, lys, K.sup.+, pKD1.degree.) was often used; a
sample of this strain has been deposited on Sep. 16, 1988 at the
Centraalbureau voor Schimmelkulturen (CBS) at Baarn (Netherlands)
under the registration number CBS 579.88.
EXAMPLE 1
CONSTRUCTION OF A MSTII/HINDIII-SMAI RESTRICTION FRAGMENT CARRYING
THE V1V2 DOMAINS OF THE RECEPTOR OF THE HIV-1 VIRUS.
[0094] An MstII-SmaI restriction fragment corresponding to the V1V2
domains (where V1 and V2 designate the first two N-terminal domains
of the CD4 molecule) was generated by the technique of enzymatic
amplification (PCR) according to the following strategy: the
lymphoblastic cell line CEM13, which expresses high quantities of
CD4 receptor, was used as the source of messenger RNAs coding for
the receptor. Total RNA was first purified from 3.times.10.sup.8
cells of this line by extraction with guanidium thiocyanate as
originally described by Cathala et al. (DNA 4(1983) 329-335);
[0095] 50 .mu.g of RNA prepared in this manner then served as
matrix for the synthesis of complementary DNA (cDNA) using the
Amersham kit and the oligodeoxynucleotide Xol27 as primer (FIG. 1).
The resulting cDNA was subjected to 30 cycles of enzymatic
amplification by the PCR technique at a hybridization temperature
of 62.degree. C., using 1.mu.g each of oligodeoxynucleotides Xol26
and Xol27 as primer, as shown in FIG. 1. The amplified fragment was
directly cloned into the SmaI site of M13mp8 which had been
previously dephosphorylated, to generate vector M13/CD4. This
vector is an intermediate construction containing the restriction
fragment MstII-SmaI which itself is the source of the MstII-HindIII
fragment carrying the V1V2 domains of the CD4 molecule; the
nucleotide sequence of this fragment is shown in FIG. 2.
EXAMPLE 2
CONSTRUCTION OF THE EXPRESSION CASSETTE FOR PREPRO-HSA.
[0096] E.2.1. Construction of plasmid pXLS69 coding for
prepro-HSA.
[0097] The Ndel site of plasmid pXL322 (Latta M. et al.,
Bio/Technology 5(1987) 1309-1314) including the ATG translation
initiation codon of prepro-HSA was changed to a HindIII site by
oligodeoxynucleotide-directed mutagenesis using the following
strategy: the HindIII-BgIII fragment of pXL322 containing the 5'
extremity of the prepro-HSA gene was cloned into vector M13mp18 and
mutagenized with oligodeoxynucleotide
5'-ATCTAAGGAAATACAAGCTT-ATGAAGTGGGT-3' (the HindIII site is
underlined and the ATG codon of prepro-HSA is shown in bold type);
the phage obtained after this mutagenesis step is plasmid pXL855
whose restriction map is shown in FIG. 3. After verification of the
nucleotide sequence, the complete coding sequence for prepro-HSA
was reconstituted by ligation of the HindIII-PvuII fragment derived
from the replicative form of the mutagenized phage and coding for
the N-terminal region of prepro-HSA, with the PvuII-HindIII
fragment of plasmid pXL322 containing the C-terminal of HSA,
thereby generating a HindIII fragment coding the entire prepro-HSA
gene. This HindIII fragment, which also contains a 61 bp
nontranslated region at its 3' extremity, was cloned into the
corresponding site of plasmid pUC8 to generate plasmid pXL869(FIG.
3).
[0098] E.2.2. Construction of expression cassettes for prepro-HSA
expressed under the control of the PGK promoter of S.
cerevisiae.
[0099] Plasmid pYG12 contains a 1.9 kb SalI-BamHI restriction
fragment carrying the promoter region (1.5 kb) and terminator
region (0.4 kb) of the PGK gene of S. cerevisiae(FIG. 4). This
fragment is derived from a genomic HindIII fragment (Mellor J. et
al., Gene 24(1983) 1-14) from which a 1.2 kb fragment corresponding
to the structural gene has been deleted, comprising a region
between the ATG translation initiation codon and the BgIII site
situated 30 codons upstream of the TAA translation termination
codon. The HindIII sites flanking the 1.9 kb fragment were then
destroyed using synthetic oligodeoxynucleotides and replaced by a
SalI and a BamHIII site respectively upstream of the promoter
region and downstream of the transcription terminator of the PGK
gene. A unique HindIII site was then introduced by site-directed
mutagenesis at the junction of the promoter and terminator regions;
the sequence flanking this unique HindIII site (shown in bold
letters) is as follows:
[0100] 5'-TAAAAACAAAAGATCCCCAAGCTGGGGATCTCCCATGTCTCTACT-3'
[0101] Plasmid pYG208 is an intermediate construction generated by
insertion of the synthetic adaptor BamHI/SalI/BamHI
(5'-GATCCGTCGACG-3') into the unique BamHI site of plasmid
pYG12;
[0102] plasmid pYG208 thereby allows the removal of the promoter
and terminator of the PGK gene of S. cerevisiae in the form of a
SalI restriction fragment (FIG. 4).
[0103] The HindIII fragment coding for prepro-HSA was purified from
plasmid pXL869 by electroelution and cloned in the "proper"
orientation (defined as the orientation which places the N-terminal
of the albumin prepro region just downstream of the PGK promoter)
into the HindIII site of plasmid pYG208 to generate plasmid pYG210.
As indicated in FIG. 4, plasmid pYG210 is the source of a SalI
restriction fragment carrying the expression cassette (PGK promoter
/ prepro-HSA / PGK terminator).
[0104] E.2.3 Optimization of the expression cassette.
[0105] The nucleotide sequence located immediately upstream of the
ATG translation initiation codon of highly expressed genes
possesses structural characteristics compatible with such high
levels of expression (Kozak M., Microbiol. Rev. 47(1983) 1-45;
Hamilton R. et al., Nucl. Acid Res. 15(1987) 3581-3593). The
introduction of a HindIII site by site-directed mutagenesis at
position-25 (relative to the ATG initiation codon) of the PGK
promoter of S. cerevisiae is described in European patent
application EP N.sup.o 89 10480.
[0106] In addition, the utilization of oligodeoxynucleotides Sq451
and Sq452 which form a HindIII-BstEII adaptor is described in the
same document and permits the generation of a HindIII restriction
fragment composed of the 21 nucleotides preceding the ATG initiator
codon of the PGK gene, followed by the gene coding for prepro-HSA.
The nucleotide sequence preceding the ATG codon of such an
expression cassette is as follows (the nucleotide sequence present
in the PGK promoter of S. cerevisiae is underlined):
[0107] 5'-AAGCTTTACAACAAATATAAAAACAATG-3'.
EXAMPLE 3
IN-FRAME FUSION OF PREPRO-HSA WITH THE V1V2 DOMAINS OF THE CD4
RECEPTOR.
[0108] The cloning strategy used for the in-frame construction of
the hybrid molecule prepro-HSA-V1V2 is illustrated in FIGS. 5
through 9. Plasmid pYG11 is an intermediate construction in which
the HindIII fragment coding for prepro-HSA has been purified from
plasmid pXL869 and cloned into the HindIII site of plasmid
pYG12(FIG. 5). The construction of plasmid pYG18 is represented in
FIG. 6; this plasmid corresponds to the SalI-BamHI fragment coding
for the expression cassette (PG K promoter/prepro-HSA/PGK
terminator) purified from plasmid pYG11 and cloned into the
corresponding sites of plasmid pIC20R (Marsh F. et al., Gene 32
(1984) 481-485).
[0109] The MstII-SmaI restriction fragment carrying the V1V2
domains of the CD4 receptor, obtained as described in Example 1,
was cloned into plasmid pYG18 cut by the same enzymes to generate
recombinant plasmid pYG303 whose restriction map is shown in FIG.
7. Plasmid pYG303 therefore carries a HindIII fragment
corresponding to the in-frame fusion of the entire prepro-HSA gene
followed by the V1V2 domains of the CD4 receptor; FIG. 8 shows the
nucleotide sequence of this fragment. This fragment was then cloned
into the HindIII site of plasmid pYG208:
[0110] insertion of this fragment, which codes for the gene
prepro-HSA-V1V2, in the proper orientation into plasmid pYG208,
generates plasmid pYG306 whose restriction map is shown in FIG. 9.
Plasmid pYG306 carries a SalI restriction fragment containing the
expression cassette (PGK promoter / prepro-HSA-V1V2/ PGK
terminator).
EXAMPLE 4
CONSTRUCTION OF STABLE CLONING VECTORS DERIVED FROM REPLICON
pKD1.
[0111] E4.1. Isolation and purification of plasmid pKD1.
[0112] Plasmid pKD1 was purified from K. drosophilarum strain UCD
51-130 (U.C.D. collection, University of California, Davis, Calif.
95616) according to the following protocol: a 1 liter culture in
YPD medium (1% yeast extract, 2% Bacto-peptone, 2% glucose) was
centrifuged, washed, and resuspended in a solution of 1.2 M
sorbitol, and cells were transformed into spheroplasts in the
presence of zymolyase (300 .mu.g/ml), 25 mM EDTA, 50 mM phosphate
and .beta.-mercaptoethanol (1 .mu.g/ml). After washing in a
solution of 1.2 M sorbitol, spheroplasts corresponding to 250 ml of
the original culture were resuspended in 2.5 ml of 1.2 M sorbitol
to which was added the same volume of buffer (25 mnM Tris-HCl, pH
8,0; 50 mM glucose; 10 mM EDTA). The following steps correspond to
the alkaline lysis protocol already described (Birnboim H. C. and
Doly J. C., Nucleic Acids Res. 7 (1979) 1513-1523). DNA was
purified by isopycnic centrifugation in a cesium chloride
gradient.
[0113] E4.2. Construction of plasmid pCXJ1.
[0114] The intermediate construction pUC-URA3(FIG. 10) consists of
a 1.1 kb fragment containing the URA3 gene of S. cerevisiae
inserted in the unique NarI site of plasmid pUC19 as follows: the
HindIII fragment coding for the URA3 gene was purified by HindIII
digestion of plasmid pG63(Gerbaud C. et al., Curr. Genet. 3 (1981)
173-180); the fragment was treated with the Klenow fragment of E.
coli DNA polymerase I to generate blunt ends, purified by
electroelution, and inserted into plasmid pUC19 which had been
cleaved by NarI and treated with the KIenow fragment of E. coli DNA
polymerase I.
[0115] Plasmid PCXJ1(FIG. 11) contains the complete sequence of
plasmid pKD1 inserted into the unique AatII site of pUC-URA3 as
follows: plasmid pKDl was linearized by cleavage with EcoRI, then
blunt-ended with the Kienow fragment of E. coli DNA polymerase I.
This fragment was then ligated with plasmid pUC-URA3 which had been
cut by AatII and blunt-ended with T4 DNA polymerase: cloning of a
blunt-ended EcoRI fragment into a blunt-ended AatII site
reconstitutes two EcoRI sites. It should be noted that
linearization of plasmid pKD1 at the EcoRI site does not inactivate
any of the genes necessary for plasmid stability and copy number,
since the EcoRI site is located outside of genes A, B, and C, and
outside of the inverted repeats of pKD1. In fact, plasmid pCXJ1
transforms K. lactis uraA cir.degree. at high frequency, is
amplified to 70-100 copies per cell, and is maintained in a stable
fashion in the absence of selection pressure. Due to the origin of
replication carried by plasmid pUC-URA3, plasmid pCXJ1 can also
replicate in E. coli, and thus constitutes a particularly useful
shuttle vector between E. coli and several yeasts of the genus
Kluyveromyces in particular K. lactis, K. fragilis and K.
drosophilarum. However, the utilization of pCXJ1 as a vector for
the transformation of Kluyveromvces remains limited to those
auxotrophic strains carrying a chromosomal uraA mutation.
[0116] E.4.3. Construction of an in-frame fusion between ORF1 of
the killer plasmid of K. lactis and the product of the bacterial
gene aph[3']-I of transposon Tn903.
[0117] Plasmid pKan707 was constructed as a vector to be used in
wild type yeasts. This plasmid was generated by insertion of the
aph[3']-I gene of bacterial transposon Tn 903 coding for
3'-aminoglycoside phosphotransferase (APH), expressed under control
of a yeast promoter, into the SalI of plasmid pCXJ1.
[0118] In the first step, the bacterial transcription signals of
the aph[3']-I gene were replaced by the P.sub.k1 promoter isolated
from the killer plasmid k1 of K. lactis as follows: the 1.5 kb
ScaI-PstI fragment of plasmid k1 was cloned into the corresponding
sites of vector pBR322, to generate plasmid pk1-PS1535-6 (FIG. 12);
this 1.5 kb fragment contains the 5' region of the first open
reading frame (ORF1) carried by plasmid k1 as well as approximately
220 bp upstream (Sor F. and Fukuhara H., Curr. Genet. 9 (1985)
147-155). The purified ScaI-PstI fragment probably contains the
entire promoter region of ORF1, since the ScaI site is situated
only 22 nucleotides from the extremity of plasmid k1(Sor F. and
al., Nucl. Acids. Res. 11 (1983) 5037-5044). Digestion of
pk1-PS1535-6 by DdeI generates a 266 bp fragment containing 17 bp
from pBR322 at the extremity close to the ScaI site, and the first
11 codons of ORF1 at the other extremity.
[0119] Plasmid pUC-kan1 is an intermediate construction obtained by
insertion of the 1.25 kb EcoRI fragment carrying the aph[3']-I gene
of Tn903 (Kanamycin Resistance Gene Block TM, Pharmacia), into the
EcoRI site of plasmid pUC19(FIG. 13). The 266 bp DdeI fragment from
plasmid pk1-5 PS1535-6 was treated with the Klenow fragment of E.
coli DNA polymerase I, purified by electroelution on a
polyacrylamide gel, then inserted into the XhoI site of plasmid
pUC-kan1 treated by S1 nuclease to generate blunt ends;
[0120] this generated plasmid pUC-kan202(FIG. 13). This cloning
strategy creates an in-frame fusion of the ORF1 gene of plasmid k1
with the N-terminal extremity of the aph[3']-I gene of Tn903: in
the fusion, the first 11 amino acids of the aph[3']-I gene product
have been replaced by the first 11 amino acids of ORF1, and the
expression of this hybrid gene is under the control of a K. lactis
promoter. The nucleotide sequence surrounding the initation codon
of the fusion protein ORF1-APH is as follows (codons originating
from ORF1 are underlined, and the first codons from APH are
italicized):
1 5'-TTACATTATTAATTTAAAA ATG GAT TTC AAA GAT AAG GCT TTA AAT GAT
CTA AGG CCG CGA TTA AAT TCC AAC . . . 3'
[0121] E4.4. Construction and stability of plasmid pKan707 in K.
lactis.
[0122] Plasmid pCXJ1 was cleaved by HindIll, treated with the
Klenow fragment of E. coli DNA polymerase I, then ligated with the
1.2 kb ScaI-HincII fragment coding for the ORF1-APH fusion
expressed under control of the K. lactis P.sub.k1 promoter deriving
from plasmid pUC-Kan202. The resulting plasmid (pKan707, FIG. 14)
confers very high levels of resistance to G418(Geneticin, GIBCO,
Grand Island, N.Y.) in strains of K. lactis(>2,5 g/l), is able
to transform K. lactis strains cir.degree. due to the functional
integrity of replicon pKD1, can be amplified to 70-100 copies per
cell, and can be stably maintained in the absence of selection
pressure (FIG. 15). This high stability, coupled with the presence
of a dominant marker permitting the transformation of industrial
strains of Kluyveromyces, make plasmid pKan707 a high performance
vector for the expression of proteins in yeasts of the genus
Kluyveromyces.
EXAMPLE 5
CONSTRUCRION OF EXPRESSION PLASMIDS pYG221B (PREPRO-HSA) AND
pYG308B (PREPRO-HSA-V1V2).
[0123] The SalI restriction fragment coding for the hybrid protein
prepro-5 HSA-V1V2 expressed under control of the PGK promoter of S.
cerevisiae was purified by electrolution from plasmid pYG306 cut by
the corresponding enzyme, and then cloned into the SalI site of
plasmid pKan707, to generate plasmids pYG308A and pYG308B which are
distinguished only by the orientation of the SalI fragment in
relation to the vector pKan707. A restriction map of plasmid
pYG308B is shown in FIG. 16.
[0124] Plasmid pYG221B is a control construction coding for
prepro-HSA alone; this plasmid was constructed as for plasmid
pYG308B (prepro-HSA-V1 V2): the SalI fragment coding for prepro-HSA
expressed under control of the PGK promoter was purified from
plasmid pYG210 and cloned into the SalI site of plasmid pKan707 to
generate plasmid pYG221B (FIG. 17).
[0125] Plasmids pYG221B (prepro-HSA) and pYG308B (prepro-HSA-V1V2)
possess the same orientation of the SalI expression cassettes in
relation to the vector and are strictly isogenic except for the
difference of the MstII-HindIII fragment located immediately
upstream of the PGK terminator. The nucleotide sequence of the
MstII-HindIII fragment in plasmid pYG221B (prepro-HSA) is as
follows (the translation stop codon for the prepro-HSA gene is in
bold type):
2 5'-CCTTACGCTTATAACATCACATTTAAAAGCATCTCAGCCTA
CCATGAGAATAAGAGAAAGAAAATGAAGATCAAAAGCTT-3'
[0126] The nucleotide sequence of the MstII-HindIII fragment of
plasmid pYG308B is included in the sequence of the MstII-SmaI
fragment shown in FIG. 2.
EXAMPLE 6
TRANSFORMATION OF YEASTS.
[0127] Transformation of yeasts of the genus Kluyveromyces, and in
particular K. lactis strain MW98-8C, was performed by treating
whole cells with lithium acetate (Ito H. et al., J. Bacteriol.
153(1983) 163-168), adapted as follows. Cells were grown in shaker
flasks in 50 ml of YPD medium at 28.degree. C., until reaching an
optical density of 0.6-0.8, at which time they were harvested by
low speed centrifugation, washed in sterile TE (10 mM Tris HCl pH
7,4; 1 mM EDTA), resuspended in 3-4 ml of lithium acetate (0.1 M in
TE) to give a cell density of 2.times.108 cells/ml, then incubated
1 hour at 30.degree. C. with moderate agitation. Aliquots of 0.1 ml
of the resulting suspension of competent cells were incubated 1
hour at 30.degree. C. in the presence of DNA and polyethylene
glycol (PEG.sub.4000, Sigma) at a final concentration of 35%. After
a 5 minute thermal shock at 42.degree. C., cells were washed twice,
resuspended in 0.2 ml sterile water, and incubated 16 hours at
28.degree. C. in 2 ml YPD to allow for phenotypic expression of the
ORF1-APH fusion protein expressed under control of promoter
P.sub.k1; 200 .mu.l of the resulting cell suspension were spread on
YPD selective plates (G418, 200 .mu.g/ml). Plates were incubated at
28.degree. C. and transformants appeared after 2 to 3 days
growth.
EXAMPLE 7
SECRETION OF ALBUMIN AND ITS VARIANTS BY YEASTS OF THE GENUS
KLUYVEROMYCES.
[0128] After selection on rich medium supplemented with G418,
recombinant clones were tested for their capacity to secrete the
mature form of albumin or the hybrid protein HSA-V1V2. Certain
clones corresponding to strain MW98-8C transformed by plasmids
pYG221B (prepro-HSA) or pYG308B (prepro-HSA-V1V2) were incubated in
selective liquid rich medium at 28.degree. C. Culture supernatants
were prepared by centrifugation when cells reached stationary
phase, then concentrated by precipitation with 60% ethanol for 30
minutes at 20.degree. C. Supernatants were tested after
electrophoresis through 8.5% polyacrylamide gels, either by direct
Coomassie blue staining of the gel (FIG. 18, panel A), or by
immunoblotting using as primary antibody a rabbit polyclonal
anti-HSA serum (FIG. 18, panel B) or a rabbit polyclonal anti-CD4
serum (FIG. 18, panel C). For immunoblot experiments, the
nitrocellulose filter was first incubated in the presence of
specific rabbit antibodies, then washed several times, incubated
with a biotinylated goat anti-rabbit Ig's serum, then incubated in
the presence of an avidin-peroxidase complex using the "ABC" kit
distributed by Vectastain (Biosys S.A., Compigne, France). The
immunologic reaction was then revealed by addition of diamino-3,3'
benzidine tetrachlorydrate (Prolabo) in the presence of oxygenated
water, according to the kit recommendations. The results shown in
FIG. 18 demonstrate that the hybrid protein HSA-V1V2 is recognized
by both the anti-HSA antibodies and the anti-CD4 antibodies,
whereas HSA is only recognized by the anti-HSA antibodies.
EXAMPLE 8
PURIFICATION AND MOLECULAR CHARACTERIZATION OF SECRETED
PRODUCTS.
[0129] After ethanol precipitation of the culture supernatants
corresponding to the K. lactis strain MW98-8C transformed by
plasmids pYG221B (prepro HSA) and pYG308B (prepro-HSA-V1V2), the
pellet was resolubilized in a 50 mM Tris-HCl buffer, pH 8.0. The
HSA-CD4 and HSA proteins were purified by affinity chromatography
on Trisacryl-Blue (IBF). An additional purification by ion exchange
chromatography can be performed if necessary. After elution,
protein-containing fractions were combined, dialyzed against water
and lyophylized before being characterized. Sequencing (Applied
Biosystem) of the hybrid protein secreted by K. lactis strain
MW98-8C revealed the expected N-terminal sequence of albumin
(Asp-Ala-His . . . ), demonstrating the proper maturation of the
protein.
[0130] The isoelectric point was determined by
isoelectrofocalization to be 5.5 for the HSA-V1V2 protein and 4.8
for HSA.
[0131] The HSA-V1V2 protein is recognized by the monoclonal mouse
antibodies OKT4A and Leu3A directed against human CD4, as well as
by a polyclonal anti-HSA serum (FIG. 19), and can be assayed by the
ELISA method (Enzyme-Linked Immuno-Sorbent Assay, FIG. 20). The
substrate for the peroxidase used in these two experiments is
2-2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium
salt (ABTS) (Fluka, Switzerland).
EXAMPLE 9
CHARACTERIZATION OF THE ANTI-VIRAL PROPERTIES OF THE HSA-CD4
VARIANTS.
[0132] The proteins corresponding to albumin (negative control) and
to the HSA-V1V2 fusion purified from culture supernatants of K.
lactis strain MW98-8C transformed respectively by plasmids pYG221B
(prepro-HSA) and pYG308B (prepro-HSA-V1V2) as in examples 7 and 8,
were tested in vitro for antiviral activity and compared to the
entire soluble CD4 molecule purified from CHO (Chinese Hamster
Ovary) cells. Protein concentrations are expressed in molarity and
were determined both by methods to measure proteins in solution as
well as by comparison of successive dilutions of each protein after
electrophoretic migration in polyacrylamide gels followed by silver
nitrate staining.
[0133] FIG. 21 shows that the HSA-V1V2 fusion is able to inhibit in
vitro the binding of the viral glycoprotein gp160 (uncleaved
precursor of gp120) to the CD4 receptor in soluble phase. In this
experiment, the ELISA plates were covered with purified recombinant
CD4 and incubated with recombinant gp160 (125 femtomoles) and
having been preincubated with varying quantities of CD4, albumin,
or the hybrid protein HSA-V1V2. The residual binding of gp160 to
CD4 was then revealed by the successive addition of mouse
monoclonal anti-gp160(110.4), followed by the binding of a goat
serum linked to peroxidase and directed against mouse antibodies.
After addition of a chromogenic substrate (orthophenyldialenine) in
the presence of oxygenated water, optical density was measured at
492 nm. The results reported in FIG. 21 demonstrate that the hybrid
protein HSA-V1V2 is able to inhibit the binding of gp160 to CD4 in
soluble phase, in a manner indistinguishable from the positive
control corresponding to the entire CD4 molecule. In contrast, the
albumin molecule is almost completely inactive in this regard. This
experiment indicates that the inhibition by the hybrid protein is
due to the presence of the V1V2 domains in a conformation and
accessibility similar to the complete CD4 receptor.
[0134] FIG. 22 shows that the HSA-V1V2 hybrid is able to inhibit
the in vitro binding of the HIV-1 virus to cells expressing the CD4
receptor on their membranes. In this experiment, a cell line that
expresses high quantities of CD4 receptor (lymphoblastic cell line
CEM13) was incubated with 2.mu.g of heat-inactivated viral
particles that had been preincubated with 116 picomoles of either
HSA-V1V2(10.7 .mu.g), HSA (7.5 .mu.g), or recombinant entire CD4
purified from CHO cells (5 .mu.g). The binding of the inactivated
viral particles to cell membranes was revealed by successive
incubations of a mouse monoclonal anti-gp120 antibody and a goat
anti-mouse IgG serum marked with phycoerythrin. The negative
control corresponds to cell line CEM13 incubated successively with
these two antibodies. Fluorescence was measured with a cell sorter
(FIG. 22, panel A) and the results are presented in the form of a
histogram (FIG. 22, panel B). This experiment shows that the
HSA-V1V2 protein is able to inhibit the binding of the HIV-1 virus
to CEM13 cells almost completely. Furthermore, this inhibition is
slightly higher than that of the complete CD4 molecule; this can be
explained by the fact that albumin, known for its adhesive
properties, is able to inhibit the binding of the virus to the
target cells in a nonspecific manner and with a low efficiency.
[0135] The HSA-CD4 protein is also able to inhibit viral infection
of permissive cells in cell culture. This inhibition was measured
either by assaying the production of viral antigens (viral p24)
using the kit ELAVIA-AG1(Diagnostics Pasteur), or the kit p24-ELISA
(Dupont), or by measuring the reverse transcriptase activity by the
technique of Schwartz et al. (Aids Research and Human Retroviruses
4 (1988) 441-448). The experimental protocol was as follows: the
product of interest at a final concentration X was first
preincubated with supernatants of CEM13 cells infected by the
isolate LAV-Brul of virus HIV-1(dilution 1/250, 1/2500 and 1/25000)
in a total volume of 1 ml of culture medium (RPMI1640 containing
10% fetal calf serum, 1% L-glutamine and 1%
penicillin-streptomycin-neomycin). The mixture was then transfered
onto a pellet of 5.times.10.sup.5 permissive cells (e.g. MT2,
CEM13, or H9) and incubated in tubes for 2 hours at 37.degree. C.
for infection to occur. The infection could also be carried out on
microtitration plates with 10.sup.4 cells per well in 100 .mu.l of
complete medium. A volume of 100 .mu.l of the virus that had been
preincubated with the product to be tested was then added, followed
by 50 .mu.l of the product at 5X concentration. Cells were then
washed twice with 5 ml RPMI 1640 and resuspended in culture medium
at a density of 2:5.times.10.sup.5 cells/ml. 100 .mu.l of this
suspension was then aliquoted into each well of microtitration
plates which already contain 100 .mu.l of the product at 2X
concentration, and the plates were incubated at 37.degree. C. in a
humid atmosphere containing 5% CO.sub.2. At different days
(D3-D4-D6-D8-D10-D12-D14-D16-D19-D21 and D25), 100 .mu.l of
supernatant was removed and the p24 viral production as well as the
reverse transcriptase activity were assayed. Cells were then
resuspended and distributed onto microtitration plates for assays
of cell viability (MTT) as described above. To the 50 .mu.l
remaining on the original plates, 200 .mu.l of culture medium
containing the product to be tested at concentration X were added,
and infection was allowed to progress until the next sampling. For
the cell viability test, 10 .mu.l of MTT at 5 mg/ml filtered on 0.2
.mu.m filters was added to each well and plates were incubated 4
hours at 37.degree. C. in a humid atmosphere containing 5%
CO.sub.2. Then to each well was added 150 .mu.l of an
isopropanol/0.04 N HCl mixture, and the Formazan crystals were
resuspended. Optical density from 520 to 570 nm was measured on a
Titertek plate reader; this measure reflects cell viability
(Schwartz et al., Aids Research and Human Retroviruses 4 (1988)
441-448).
[0136] FIG. 23 shows an example of inhibition of infectivity in
cell culture (cell line CEM13) as measured by reverse transcriptase
activity. This demonstrates that the HSA-V1V2 hybrid is able to
reduce the infectivity of the HIV-1 virus to the same extent as the
soluble CD4 molecule.
EXAMPLE 10
STABILITY OF THE HYBRID PROTEINS IN VIVO.
[0137] It has been shown that first generation soluble CD4
possesses a half-life of 20 minutes in rabbits (Capon D. J. et al.;
Nature 337 (1989) 525-531). We have therefore compared the
half-life in rabbits of the HSA-CD4 hybrid to soluble CD4 and to
recombinant HSA produced in yeast and purified in the same manner
as HSA-CD4. In these experiments, at least 2 male NZW(Hy/Cr)
rabbits weighing 2.5-2.8 kg were used for each product. Rabbits
were kept in a room maintained at a temperature of
18.5-20.5.degree. C. and a humidity of 45-65%, with 13 hours
light/day. Each product was administered in a single injection
lasting 10 seconds in the marginal vein of the ear. The same molar
quantity of each product was injected: 250 .mu.g of CD4 per rabbit,
400 .mu.g of HSA per rabbit, or 500 .mu.g of HSA 4 per rabbit, in 1
ml physiologic serum. Three to four ml blood samples were taken,
mixed with lithium heparinate and centrifuged 15 min at 3500 rpm;
samples were then divided into three aliquots, rapidly frozen at
-20.degree. C., then assayed by an ELISA method. Blood samples from
rabbits injected with CD4 were taken before injection (To), then 5
min, 10 min, 20 min, 30 min, 1 h, 2 h, 4 h and 8 h after injection.
Blood samples from rabbits injected with HSA-CD4 or HSA were taken
at To, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, 32 h, 48 h, 56 h, 72 h, 80
h, 96 h, 104 h and 168 h after injection.
[0138] Assays of the CD4 molecule were carried out on Dynatech
M129B microtitration plates previously covered with the HSA-CD4
hybrid protein. Increasing concentrations of CD4 or the samples to
be assayed were then added in the presence of the mouse monoclonal
antibody OKT4A (Ortho-Diagnostic, dilution 1/1000); after
incubation and washing of the plates, the residual binding of
antibody OKT4A was revealed by addition of antibodies coupled to
peroxidase (Nordic, dilution 1/1000) and directed against mouse
IgG. Measurements were made at OD 405 nm in the presence of the
peroxidase substrate ABTS (Fluka).
[0139] Assays of recombinant HSA were carried out on Dynatech M129B
microtitration plates previously covered with anti-HSA serum (Sigma
Ref. A0659, dilution 1/1000); increasing concentrations of HSA or
samples to be measured were then added, followed by addition of
anti-HSA serum coupled to peroxidase (Nordic, dilution 1/1000).
Measurements were made at OD 405 nm as above.
[0140] Two different assays were done for the HSA-CD4 hybrid:
either the assay for the HSA moiety alone, using the same methods
as for recombinant HSA, or an assay for the HSA moiety coupled with
an assay for the CD4 moiety. In the latter case, microtitration
plates were covered first with anti-HSA serum (Sigma Ref. A0659,
dilution 1/1000), then incubated with the samples to be assayed.
The mouse monoclonal antibody Leu3A directed against CD4 was then
added, followed by antibodies coupled to peroxidase (Nordic,
dilution 1/1000) and directed against mouse antibodies.
Measurements were made at 405 nm as described above.
[0141] The curves for each of these assays are given in FIG. 24.
Interpretation of these results allows the evaluation of the
pharmacokinetic characteristics of each product in the rabbit. The
half-lives measured for each product are as follows:
3 CD4: 0.25 .+-. 0.1 h HSA: 47 .+-. 6 h HSA-CD4: 34 .+-. 4 h
[0142] These results underscore the following points:
[0143] 1/ The coupling of CD4 to albumin allows a significant
increase in the stability of CD4 in the organism since the
half-life of elimination is increased 140-fold.
[0144] 2/ The half-life of elimination of the HSA-CD4 hybrid is
comparable to that of HSA.
[0145] 3/ The clearance of CD4 is approximately 3 ml/min/kg while
that of HSA and HSA-CD4 is approximately 0.02 ml/min/kg.
[0146] 4/ The CD4 moiety of the HSA-CD4 hybrid apparently retains
an active conformation (i.e. able to bind gp120) since the assay
for CD4 involves the Leu3A monoclonal antibody which recognizes an
epitope close to the binding site of gp120 (Sattentau Q. J. et al.,
Science 234 (1986) 1120-1123; Peterson A. and Seed B., Cell 54
(1988) 65-72). Furthermore, the two independent assay methods for
the HSA-CD4 hybrid gave essentially the same result, which suggests
that the CD4 moiety is not preferentially degraded in vivo.
[0147] It is noteworthy that the volume of distribution of HSA and
HSA-CD4 is close to that of the blood compartment, and therefore
suggests a distribution of the product limited to the extracellular
compartment.
EXAMPLE 11
GENERIC CONSTRUCTIONS OF THE TYPE HSA-CD4.
[0148] E.11.1. Introduction of AHaII and BglII sites at the end of
the prepro region of HSA.
[0149] Introduction of the AhaII restriction site was carried out
by site-directed mutagenesis using plasmid pYG232 and
oligodeoxynucleotide Sq1187, to generate plasmid pYG364. Plasmid
pYG232 was obtained by cloning the HindII fragment coding for
prepro-HSA into the vector M13 mp9. The sequence of
oligodeoxynudeotide Sq1187 is (the AhaII site is in bold type):
[0150] 5'- GTGTTTCGTCGAGACGCCCACAAGAGTGAGG-3'.
[0151] It should be noted that creation of the AhalI site does not
modify the protein sequence of the N-terminal of mature HSA. The
construction of plasmid pYG364 is shown in FIG. 25.
[0152] Plasmid pYG233 was obtained in analogous fashion, after
site-directed mutagenesis of plasmid pYG232 using
oligodeoxynucleotide Sq648(the codons specificying the amino acid
pair Arg-Arg situated at the end of the prepro region of HSA are in
bold type, and the BglII site is underlined):
[0153] 5'-GGTGTGTTTCGTAGATCTGCACACAAGAGTGAGG-3'
[0154] The creation of this restriction site does not change the
protein sequence of the prepro region of HSA. In contrast, the
first amino acid of the mature protein is changed from an aspartate
to a serine; plasmid pYG233 therefore codes for a mature HSA
modified at its N-terminal (HSA*, FIG. 25).
[0155] E.11.2. Introduction of the prepro region of HSA upstream of
the CD4 receptor.
[0156] The introduction of the prepro region of HSA upstream of the
V1V2 domains of the CD4 receptor was accomplished by site-directed
mutagenesis, to generate plasmid pYG347 as shown in FIGS. 26 and
27. Plasmid pYG231 (FIG. 26) is an intermediate construction
corresponding to a pUC-type replicon into which has been doned a
SalI fragment carrying the expression cassette for HSA (yeast
promoter/prepro-HSA/PGK terminator of S. cerevisiae). Plasmid
pYG234 is isogenic to plasmid pYG231 except that
oligodeoxynucleotide Sq648 was used to carry out the in vitro
mutagenesis (E11.1.).
[0157] Plasmid pYG347 was obtained by site-directed mutagenesis of
plasmid pYG332 with oligodeoxynucleotide Sq1092 (FIG. 27) whose
sequence is as follows (HSA sequence is in italics and CD4 sequence
is in bold type):
[0158] 5'-CCAGGGGTGTGTTTCGTCGAAAGAAAGTGGTGCTGGGC-3'
[0159] Plasmid pYG347 therefore carries a HindIII fragment composed
of the 21 nucleotides preceding the ATG codon of the PGK gene of S.
cerevisiae, the ATG translation initiation codon, and the prepro
region of HSA (LP.sub.HSA) immediately followed by the V1 V2
domains of the CD4 receptor.
[0160] E.11.3. Introduction of an AhaII site at the end of the V1
domain of the CD4 receptor.
[0161] The introduction of an AhaII site at the end of the V1
domain of the CD4 receptor was accomplished by site-directed
mutagenesis using oligo-deoxynucleotide Sq1185 and a derivative of
plasmid pYG347(pYG368, FIG. 28), to generate plasmid pYG362. The
sequence of oligodeoxynucleotide Sq1185 is (the AhaII site is shown
in bold type):
[0162] 5'-CCAACTCTGACACCGACGCCCACCTGCTTCAGG3'.
[0163] Plasmid pYG362 therefore carries a Hindm-AhaII fragment
composed of the 21 nucleotides preceding the ATG codon of the PGK
gene of S. cerevisiae followed by the coding sequence of the HSA
prepro region fused to the V1 domain of the CD4 receptor, according
to example E.11.2. In a fusion such as the example given here, the
V1 domain of the CD4 receptor carries 106 amino acids and includes
the functional binding site of the HIV-1 viral glycoprotein
gp120.
[0164] E.11.4. Introduction of an AhaII site at the end of the V2
domain of the CD4 receptor.
[0165] The introduction of an AhaII site at the end of the V2
domain of the CD4 receptor was accomplished by site-directed
mutagenesis using oligo-deoxynucleotide Sq1186 and plasmid pYG368,
to generate plasmid pYG363 (FIG. 28). The sequence of
oligodeoxynucleotide Sq1186 is (the AhaII site is shown in bold
type): 5'-GCTAGCTTTCGACGCCGGGGGAATTCG-3'. Plasmid pYG363 therefore
carries a HindIII-AhaII fragment composed of the 21 nucleotides
preceding the ATG codon of the PGK gene of S. cerevisiae followed
by the coding sequence for the HSA prepro region fused to the V1V2
domains of the CD4 receptor. In this particular fusion, the V1V2
domains contain 179 amino acids.
[0166] Other variants of plasmid pYG363 were generated by
site-directed mutagenesis in order to introduce an AhaII at
different places in the V2 domain of the CD4 receptor. In
particular, plasmid pYG511, shown in FIG. 28, does not contain the
amino acid pair Lys-Lys at positions 166-167 of the V2 domain; this
is due to the oligodeoxynucleotide used (Sq1252; the AhaII site is
shown in bold type):
[0167] 5'-GCAGAACCAGAAGGACGCCAAGGTGGAGTTC-3'.
[0168] E.11.5. Generic constructions of the type V1-HSA.
[0169] The plasmids described in the preceding examples allow for
the generation of HindIII restriction fragments coding for hybrid
proteins in which the receptor of the HIV-1 virus (fused to the
signal sequence of HSA) precedes HSA. For example, plasmids pYG362
and PYG364 are respectively the source of a HindIII-AhaII fragment
(fusion of the HSA prepro region to the V1 domain of the CD4
receptor), and an AhaII-NcoI fragment (N-terminal region of mature
HSA obtained as in example E.11.1.). The ligation of these
fragments with the NcoI-KpnI fragment (C-terminal region of HSA and
terminator of the PGK gene of S. cerevisiae) in an analogue of
plasmid pYG18 cut by HindIII and KpnI generates plasmid pYG371
whose structure is shown in FIG. 29. In this plasmid, the gene
coding for the hybrid protein V1-HSA fused to the HSA prepro region
is cloned into an expression cassette functional in yeasts. This
cassette can then be cloned into a replicative vector that can be
selected in yeasts, for example the vector pKan707, which generates
expression plasmid pYG373B (FIG. 30).
[0170] .E11.6. Generic constructions of the type V1V2-HSA.
[0171] Hybrid proteins of the type V1V2-HSA were generated by the
following strategy: in a first step, plasmids pYG511(FIG. 28) and
pYG374 (FIG. 29) were respectively the source of the restriction
fragments BglII-AhaII (fusion of the HSA prepro region and the V1V2
domains of the CD4 receptor) and AhaII-KpnI (in-frame fusion
between mature HSA and the V1V2 domains of the CD4 receptor as
exemplified in E.12.2.). Ligation of these fragments in a
chloramphenicol resistant derivative of pBluescript II SK(+) vector
(plasmid pSCBK(+), Stratagene) generates plasmid pYG537 (FIG. 31).
This plasmid contains a HindIII fragment coding for the hybrid
bivalent molecule CD4-HSA-CD4 fused in-frame with the signal
peptide of HSA as exemplified in E.11.2. Plasmid pYG547 which
contains a HindIII fragment coding for the hybrid protein V1V2-HSA
fused in-frame with the prepro region of HSA, was then derived by
substitution of the PstI-KpnI fragment of pYG537 by the PstI-KpnI
fragment from plasmid pYG371. The HindIII fragment carried by
plasmid pYG547 can then be expressed under control of a functional
yeast promoter cloned in a vector that replicates, for example, in
yeasts of the genus Kluyveromyces. One example is the expression
plasmid pYG560 whose structure and restriction map are shown in
FIG. 32. Vector pYG105 used in this particular example corresponds
to plasmid pKan707 whose HindIII site has been destroyed by
site-directed mutagenesis (oligodeoxynudeotide Sq1053,
5'-GAAATGCATAAGCTCTTGCCATTCTCACCG-3') and whose SalI-SacI fragment
coding for the URA3 gene has been replaced by a SalI-SacI fragment
carrying a cassette made up of a promoter, a terminator, and a
unique HindIII site.
EXAMPLE 12.
BIVALENT HYBRID PROTEIN COMPLEXES.
[0172] E.12.1. Introduction of a stop codon downstream of the V1
domain of the CD4 receptor.
[0173] Conventional techniques permit the introduction of a
translation stop codon downstream of the domain of the CD4 receptor
which is responsible for the binding of the HIV-1 viral
glycoprotein gp120. For example, a TAA codon, immediately followed
by a HindIII site, was introduced by site-directed mutagenesis
downstream of the V1 domain of the CD4 receptor. In particular, the
TAA codon was placed immediately after the amino acid in position
106 of the CD4 receptor (Thr.sup.106) using oligodeoxynucleotide
Sq1034 and a plasmid analogous to plasmid M13 CD4 as matrix. The
sequence of oligodeoxynucleotide Sq1034 is (the stop codon and the
HindIII site are in bold type):
[0174]
5'-ACTGCCAACTCTGACACCTAAAAGCTTGGATCCCACCTGCTTCAGGGGCAG-3'
[0175] E12.2. Constructions of the type CD4-HSA-CD4.
[0176] The plasmids described in examples E.11.5. et E.11.6. which
exemplify generic constructions of the type CD4-HSA allow for the
easy generation of bivalent constructions of the type CD4-HSA-CD4.
Plasmids pYG374(V1-HSA-V1V2) or pYG375 (V1-HSA-V1) illustrate two
of these generic constructions: for example, the small
MstII-HindIII fragment of plasmid pYG371 which codes for the last
amino acids of HSA can be replaced by the MstII-HindIII fragment
coding for the last 3 amino adds of HSA fused to the V1V2 domains
of the CD4 receptor (plasmid pYG374, FIG. 29), or to the V1 domain
alone (plasmid pYG375, FIG. 29). The genes coding for such bivalent
hybrid proteins can then be expressed under control of a functional
yeast promoter that replicates, for example, in yeasts of the genus
Kluyveromyces. Examples of such expression plasmids are the
plasmids pYG380B (V1-HSA-V1V2) and pYG381B (V1-HSA-V1) which are
strictly isogenic to plasmid pYG373B (V1-HSA) except for the
structural genes encoded in the HindIII fragments. The bivalent
hybrid proteins described here are expressed at levels comparable
to their monovalent equivalents, indicating a very weak level of
recombination of the repeated sequences resulting from genetic
recombination in vivo (FIG. 33).
[0177] The construction of HindIII fragments coding for bivalent
hybrid proteins of the type V1V2-HSA-V1V2 has already been
described in FIG. 31 (plasmid pYG537). The genes coding for such
bivalent hybrid proteins of the type CD4-HSA-CD4 can then be
expressed under control of a functional yeast promoter in a vector
that replicates, for example, in yeasts of the genus Kluyveromyces.
Such expression plasmids are generated by the strategy described in
FIG. 32 (cloning of a HindIII fragment into plasmids analogous to
plasmid pYG560).
[0178] E.12.3. Introduction of a dimerization domain.
[0179] For a given hybrid protein derived from albumin and carrying
one or several binding sites for the HIV-1 virus, it may be
desirable to include a polypeptide conferring a dimerization
function, which allows for the agglomeration of trapped virus
particles. An example of such a dimerization function is the
"Leucine Zipper"(LZ) domain present in certain transcription
regulatory proteins (JUN, FOS...). In particular, it is possible to
generate a BglII-AhaII fragment coding, for example, for the LZ of
JUN, by the PCR technique by using the following
oligodeoxynucleotides and the plasmid pTS301(which codes for an
in-frame fusion between the bacterial protein LexA and the LZ of
JUN, T. Schmidt and M. Schnar, unpublished results) as matrix
(BglII and AhaII sites are underlined):
4 5'-GGTAGGTCGTGTGGACGCCAGATCTTTGGAAAGAATTGCCCGTCTG GAAG-3'
5'-CTGCAGGTTAGGCGTCGCCAACCAGTTGCTTCAGCTGTGC-3'
[0180] This BglII-AhaII fragment (FIG. 34) can be ligated to the
HindIII-BglII fragment of plasmid pYG233(HSA prepro region, FIG.
25) and the AhaII-HindIII fragment as shown in one of the examples
E.11. to generate a HindIII fragment coding for hybrid proteins of
the type LZ-HSA-CD4, fused to the signal sequence of HSA. To
prevent a possible dimerization of these molecules during their
transit through the yeast secretory pathway, it may be desirable to
utilize a LZ domain which cannot form homodimers. In this case the
"Leucine Zipper" of FOS is preferred; dimerization would then
result when these proteins are placed in the presence of other
hybrid proteins carrying the LZ of JUN.
[0181] The introduction of carefully selected restriction sites
that permit the construction of genes coding for hybrid proteins of
the type LZ-CD4-HSA or LZ-CD4-HSA-CD4 is also possible, using
conventional in vitro mutagenesis techniques or by PCR.
EXAMPLE 13
GENETIC ENGINEERING OF THE HINGE REGION BETWEEN THE CD4 AND HSA
MOIETIES.
[0182] E.13.1. Strategy using Bal31 exonuclease.
[0183] Proteins secreted by strain MW98-8C transformed by
expression plasmids for HSA-CD4 hybrid proteins in which the CD4
moiety is carried on the MstII-HindIII fragment in the natural
MstII site of HSA (plasmid pYG308B for example), were analyzed.
FIG. 35 demonstrates the presence of at least two cleavage products
comigrating with the albumin standard (panel 2), which have a
mature HSA N-terminal sequence, and which are not detectabe using
polyclonal antibodies directed against human CD4 (panels 2 and 3).
It is shown that these cleavage products are generated during
transit through the yeast secretory pathway, probably by the KEX1
enzyme of K. lactis(or another protease with a specificity
analogous to the endoprotease YAP3 of S. cerevisiae whose gene has
been cloned and sequenced (Egel-Mitani M. et al. Yeast 6 (1990)
127-137). Therefore, the peptide environment of the hinge region
between the HSA and CD4 moieties was modified, notably by fusion of
the CD4 molecule (or one of its variants capable of binding the
gp120 protein of HIV-1) to HSA N-terminal regions of varying
length, according to the following strategy: plasmid pYG400 is an
intermediate plasmid carrying the prepro-HSA gene, optimized with
respect to the nucleotide sequence upstream of the ATG codon, on a
HindIII fragment. This plasmid was linearized at its unique MstII
site and partially digested by Bal31 exonudease. After inactivation
of this enzyme, the reaction mixture was treated with the Klenow
fragment of E. coli DNA polymerase I and then subjected to ligation
in the presence of an equimolar mixture of oligodeoxynucleotides
Sq1462(5'-GATCCCCTAAGG-3') and Sq1463(5'-CCTTAGGG-3') which
together form a synthetic adaptor containing a MstII site preceding
a BamHI site. After ligation, the reaction mixture was digested
with HindIII and BamHI and fragments between 0.7 and 2.0 kb in size
were separated by electroelution and cloned into an M13 mp19 vector
cut by the same enzymes. 10.sup.6 lytic plaques were thus obtained
of which approximately one-third gave a blue color in the presence
of IPTG and XGAL. Phage clones which remained blue were then
sequenced, and in most cases contained an in-frame fusion between
the HSA N-terminal moiety and .beta.-galactosidase. These composite
genes therefore contain HindIII-MstII fragments carrying sections
of the N-terminal of HSA; FIG. 36 shows several examples among the
C-terminal two-thirds of HSA. These fragments were then ligated
with a MstII-HindIII fragment corresponding to the CD4 moiety (for
example the V1V2 domains of FIG. 2, or the V1 domain alone), which
generates HindIII fragments coding for hybrid proteins of the type
HSA-CD4 in which the HSA moiety is of varying length. These
restriction fragments were then cloned in the proper orientation
into an expression cassette carrying a yeast promoter and
terminator, and the assembly was introduced into yeasts. After
growth of the culture, the hybrid proteins HSA-CD4 can be obtained
in the culture medium; certain of these hybrids have an increased
resistance to proteolytic cleavage in the hinge region (FIG.
35).
[0184] E132. Mutation of dibasic amino acid pairs.
[0185] Another way to prevent cleavage by endoproteases with
specificity for dibasic amino acid pairs is to suppress these sites
in the area of the hinge region between the HSA and the CD4
moieties (FIG. 37), or in the area of the hinge region between CD4
and HSA (FIG. 38). As an example, the hinge region present in the
hybrid protein HSA-V1V2 coded by plasmid pYG308B is represented in
FIG. 37 (panel 1), and points out the presence of a Lys-Lys pair in
the C-terminal of HSA and two such pairs in the N-terminal of the
V1 domain of CD4. Using site-directed mutagenesis, these potential
endoprotease cleavage sites can be suppressed by changing the
second lysine in each pair to a glutamine (Risler J.L et al., J.
Mol. Biol. 204 (1988) 1019-1029), for example by using plasmid
M13-ompA-CD4 as matrix and the oligodeoxynucleotides Sq1090 and
Sq1091(the codons specifying glutamine are in bold type):
5 5'-GTCCTGCGCAAACAAGGGGATACAG-3' 5'-GGCTTAAAGCAAGTGGTGCTG-3'
[0186] Plasmid M13-ompA-CD4 is a derivative of plasmid M13-CD4 in
which the signal sequence of the ompA gene of E. coli is fused in
frame to the CD4 receptor using the MstII site generated by PCR
upstream of the V1 domain (example 1).
[0187] E13.3. Introduction of a synthetic hinge region.
[0188] In order to promote an optimal interaction between the CD4
moiety fused to HSA, and the gp120 protein of the HIV-1 virus, it
may be desirable to correctly space the two protein moieties which
form the building blocks of the hybrid protein HSA-CD4. For
example, a synthetic hinge region can be created between the HSA
and CD4 moieties by site directed mutagenesis to introduce a
fragment of troponin C between amino acids 572 and 582 of mature
HSA (FIG. 37, panel 3). In this particular example, the junction
peptide was introduced via site-directed mutagenesis by using a
recombinant M13 phage (carrying the PstI-SacI fragment coding for
the in-frame fusion between the C-terminal portion of HSA and the
C-terminal part of the CD4 receptor) as matrix and
oligodeoxynucleotide Sq1445:
6 5'-TGCTTTGCCGAGGAGGGTAAGGAAGACGCTAAGGG-
TAACTCTGAACAAGAAGCCTTAGGCTTAAAGAAA-3'.
[0189] Similar techniques also permit the introduction of such a
synthetic hinge region between the HSA and CD4 moieties (junction
peptide, FIG. 38, panel 3).
EXAMPLE 14
EXPRESSION OF HYBRID PROTEINS UNDER THE CONTROL OF DIFFERENT
PROMOTERS.
[0190] For a given protein secreted by cells at high levels, there
exists a threshold above which the level of expression is
incompatible with cell survival. Hence there exist certain
combinations of secreted protein, promoter utilized to control its
expression, and genetic background that are optimal for the most
efficient and least costly production. It is therefore important to
be able to express the hybrid proteins which are the object of the
present invention under the control of various promoters. The
composite genes coding for these proteins are generally carried on
a HindIII restriction fragment that can be cloned in the proper
orientation into the HindIII site of a functional expression
cassette of vectors that replicate in yeasts. The expression
cassette can contain promoters that allow for constitutive or
regulated expression of the hybrid protein, depending on the level
of expression desired. Examples of plasmids with these
characteristics include plasmid pYG105 (LAC4 promoter of K. lactis,
FIG. 32), plasmid pYG106 (PGK promoter of S. cerevisiae), or
plasmid pYG536 (PHO5 promoter of S. cerevisiae) etc... In addition,
hybrid promoters can be used in which the UAS regions of tightly
regulated promoters have been added, such as the hybrid promoters
carried by plasmids pYG44 (PGK/LAC hybrid, European patent
application EP N.sup.o89 10480), pYG373 B (PGK/GAL hybrid), pYG258
(PHO5/LAC hybrid) etc....
* * * * *