Note: Descriptions are shown in the official language in which they were submitted.
W O 92/10506 2 ~ 9 7 7 0 5 PC~r/US91/09382
HUMAN RETROVIRUS RECEPTOR AND DNA CODING THEREFOR
B~CRGROUND OF THE INVENTION
Field of the Invention
The invention in the field of virology and molecular
genetics relates to the H13 protein molecule, which is a human
protein highly homologous in sequence to a murine retrovirus
receptor molecule, DNA coding therefore, methods of preparing
the protein molecule, and methods of use of the protein to
10 prevent or treat retrovirus infection. The invention also
concerns substitutio~s in the human retrovirus receptor of
amino acid residues from the murine homologue and human cells
expressing this DNA which are rendered susceptible to
infection and thus gene transfer by murine retroviral
15 vectors.
Description of the Backaround Art
Viruses infect cells by first attaching to the cell.
This requires specific interactions between molecules on the
surface of the virus and receptor molecules on the susceptible
20 cell. A number of virus-specific cellular receptors have been
identified, and most of these receptor molecules have other
known cellular functions. Human immunodeficiency virus (HIV-
1) binds to the CD4 molecules (Dalgleish et al., Nature.
312:763-767 (1984)); Klatzmann, D., Nature 312:767 (1984);
25 Maddon et al., Cell, 42: 93-104 (1986)). The Epstein-Barr
virus (EBV) binds to the complement receptor protein, CR2
(Fingeroth et al., Proc. Natl. Acad. Sci. USA, 81: 4510-4514
(1984)). Human rhinoviruses bind to the cell adhesion
molecule, ICAM-l (Greve, J.M. et al., Cell 56:839 (1989);
30 Staunton, D.E. et al., Cell 56:849 (1989)). Rabies virus
binds to the acetylcholine receptor (Lentz, T.L., Science 215:
wo 92/105n6 ~ 7 V 5 PCT/U591/09382
182 (1082)). Reoviruses bind to beta-adrenergic receptors
(Co, M.S. et al., Proc. Natl. Acad. Sci. USA 82:1494 (1985).
Herpes simplex virus appears to use the fibroblast growth
factor receptor as a binding site (Kaner, R.J. et al.,
5 Science 248:1410-1413 (1990)).
The expression of these virus binding proteins or
receptors is a strong determinant of susceptibility to virus
infection. Binding is required for fusion of the virus
envelope to the target cell, an event that may occur at the
10 cell surface or within an acidified endosome after receptor-
mediated endocytosis (White et al., Ouant. Rev. Bio~hvs. 16:
151-195 (1983)). After fusion, the virion core enters the
cytoplasm and the viral replication process is initiated.
In the case of HIV, recent studies suggested that
15 cell surface molecules other than CD4 ~ay also be important
for virus entry into human cells. First, cells lacking the
CD4 molecule, including human fibroblasts and cells derived
from human brain, can be infected in vitro by HIV, suggesting
an alternate virus receptor. Furthermore, murine cells which
Z0 have been transfected with the CD4 gene and express this
molecule on their surface are o~ten resistant to HIV,
indicating that the mere presence of CD4 is not sufficient for
HIV infection. A major target cell for HIV is the CD4+ T
lymphocyte. A majority of circulating T lymphocytes are non-
25 dividing quiescent cells; for infection by HIV in vitro, thesecells must be "activated," for example, by a mitogenic lectin.
This observation further supports the notion that the presence
of the CD4 molecule on a cell is not sufficient for
susceptibility to HIV infection.
30 Murine Retrovirus Rece~tors
As with HIV and EBV, susceptibility of cells to
infection with ecotropic murine leukemia virus ~E-MuLV) may
also be determined by binding of the virus envelope to a
membrane receptor. The E-MuLV envelope protein, gp70,
35 encoded by the env gene, binds avidly to the membranes of
murine cells but poorly to those of other mammals that are not
permissive for E-MuLV (DeLarco et al., Cell 8:365-371 (1976)).
WO92/10~06 2 0 9 ~ I 0 5 PCT/US9l/09382
3 --
Furthermore, the gp70 molecule of ecotropic MuLV is
structurally distinct from the envelope proteins of other
MuLV subgroup viruses with different patterns of infectivity
(Levy, J.A., Science, 182:1151-1153 (1973); Elder et al.,
5 Nature 267:23-28 (1977)~. Chimeric viruses constructed
between E-MuLV and other MuLV subgroups acquire the host range
of the env gene donor (Cone and Mulligan, Proc. Natl. Acad.
Sci. USA, 81:6349-6353 (1984)).
Based on viral interference assays, four types of
10 specific MuLV receptors have been postulated: (a) receptors
for E-MuLV; (b) receptors for wild-type amphotropic MuLV; (c)
receptors for recombinant viruses derived from E-MuLV, such as
the "mink cell focus-inducing" or MCF virus; and (d) receptors
for a recombinant virus derived from an amphotropic MuLV
15 (Rein, A. et al., Virolooy 136:144-152 (1984)).
Hybrid cells which were created by fusion of primary
mouse lymphocytes with nonpermissive Chinese hamster lung
cel~s retained susceptibility to E-MuLV infection and bound
gp70 to the membrane (Gazdar, A.F., Cel.l 11:949-956 (1977)).
20 Analysis of the chromosome content of a large number of these
hybrids has permitted the assignment of putative E-MuLV
receptor gene(s) to the Rec-l locus on mouse chromosome 5
(Oie et al., Nature 274: 60-62 (1978); Ruddle et al., J. Exp.
~ 148:451-465 (1978)). Purification of the protein encoded
25 by Rec-1 has not been achieved because specific gp70 binding
activity was lost upon detergent solubilization of the cell
membrane (Johnson et al., J. Virol. 58:900-908 ~1986)).
Assignment of a single genetic locus for
susceptibility to virus infection is consistent with the
30 hypothesis that a single gene encodes the receptor protein.
~urthermore, successful MuLV infection of the hybrid cell
lines demonstrates that expression of the receptor gene in
nonpermissive cells can confer MuLV susceptibility. These two
observations suggest that it might be possible to transfer
35 murine DNA into nonpermissive cells by transfection and then
recover the putative receptor gene from recipient cells that
had acquired susceptibility to E-MuLV infection. A similar
strategy has been employed in cloning genes encoding other
WO92/10506 2 ~ 9 r~ rl 0 5 PCT/US91/09382
- 4 -
cell membrane proteins such as the nerve growth factor
receptor (Chao et al., science, 232:418-421 (1966)), CD8
(Littman et al., Cell 40 237-246 (1985)), and CD4 (Maddon et
al., Cell 42:93-104 (1985)).
Recently, a cDNA clone (termed Wl) encoding the
murine ecotropic _etroviral Eeceptor (ERR) was identified
(Albritton, L.W. et al., Cell 57:659-666 (1989)). This study
demonstrated that susceptibility to E-~uLV infection was
ac~uired by the expression of a single mouse gene in human EJ
10 cells. Furthermore, this gene appears to define Rec-1, the
genetic locus on mouse chromosome 5 associated with ecotropic
virus infectivity tOie et al., Nature 274:60-62 (1978);
Ruddle et al., J. Exp. Med. 148:451-465 (1978)).
The hydropathy plot of the predicted amino acid
15 sequence of the ERR (SEQ ID NO:4) protein revealed an
extremely hydrophobic protein containing 14 potential
transmembrane domains (Eisenberg et al., J. Mol. Biol.
179:125-142 (1984)). Such structure strongly implies that
the protein resides in the membrane. This protein may permit
20 infection by functioning as a true receptor that binds
specifically to E-MuLV gp70 in analogous fashion to HIV gpl20
binding to the CD4 protein (Maddon et al., Cell 47: 333-348
(1986); McDougal et al., Science 237:382-385 (1986)).
Demonstration of a physical association between the protein
25 and the virus envelope gp70 would strongly support its
proposed role as a virus receptor.
Independent from its role in viral attachment to the
cell surface, the ERR protein could also be important for
virus envelope fusion to the membrane of the target cell.
30 Evidence for the existence of membrane proteins that mediate
virus fusion comes from studies of Sendai virus (Richardson et
al., Virology 131:518-532 (1983)) and HIV (Maddon et al.,
Cell 42:93-104 (1985)), two viruses that fuse to the plasma
membrane (Harris et al., Nature 205:640-646 (1965); Stein et
35 al., Cell 49:659-668 (1987): Maddon et al., Cell 47:333-348
(1986)) through a mechanism that may be similar to that
employed by E-MuLV (Pinter et al., J. Virol. 57:1048-105~
(1986)). The potential role for a protein in virus fusion
WO92/10506 2 ~ 9 7 7 9 5 PCT/US91/09382
- 5 -
could be distinct from, or in addition to, the act of binding
viral gp70.
Computer searches through the GenBank and NBRF
databases did not reveal any sequences similar to the
5 predicted protein that might help classify or identify the
function of the ERR protein in normal cell metabolism.
Proteins with multiple membrane-spanning domains that function
as gated channels or pumps to transport ions or sugars across
the lipid bilayer have been identified, but a direct
lO comparison of the predicted amino acid sequences of several of
these proteins ~o the ERR protein using the BestFit algorithm
(Devereux et al., Nucl. Acids Res. 12: 387-395 (1984)) also
did not reveal any significant sequence similarity
((Albritton, L.W. et al., 1989, suDra).
15 ~ Cell Earlv Activation Gene Resembles Retrovirus Receptor
Gene
Many genes that encode products which function in T
cell development, homing, or immune responsiveness remain to
be identified. In an effort to isolate novel T cell cDNA
20 clones which identify new functions, MacLeod, C.L. et al.
(Mol. Cell. Biol. 10:3663-3674 (l990)) used two closely
related T lymphoma cell clones obtained from a single
individual which differed in a limited number of
characteristics and had defined and stable phenotypes. This
25 model system is known as the SL12 T lymphoma (Hays et al.,
Int. J. Cancer 38:597-601 (1986)); MacLeod et al., Cancer Res.
44:1784-1790 (1984)); ~ 74:875-882 (1985); Proc. Natl.
Acad. Sci. USA 83:6989-6993 (1986); Cell Growth & Differ.
1:271-279 (1990)). The two cell clones derived from a single
30 SL12 T lymphoma cell line were chosen based on their known
differences in gene expression and their different capacities
to cause tumors in syngeneic host animals (MacLeod, C.L. et
al., 1990, supra). SL12.3 cells express very few of the genes
required for T cell function, and are highly tumorigenic in
35 syngeneic animals (MacLeod et al., 1985, 1986, supra). In
contrast, the cells of sister clone SLl2.4 express mRNAs for
all the components of the T cell receptor (TCR)-CD3 comple~
WO92/10506 2 0 9 7 7 ~ ~ PCT/US91/09382
- 6 -
except TCR-~ and resemble thymocytes at an intermediate stage
of development (MacLeod et al., 1986, supra; Wilkinson et al ,
EMBO J. 7:101-109 (1988)). SL12.4 cells are much less
tumorigenic than SL12.3 cells (MacLeod et al., 1985, su~ra).
A combination of subtraction hybridization-enriched
probes (Hedrick et al., Nature 308:149-153 (1984); MacLeod et
al., J. BioL. Chem. 1:271-279 tlg9o); Timberlake, Dev. Biol.
78:497-S03 (19~0)) and classical differential screening
Sambrook et al., Mo~ecular Clonina: A Laboratorv Manual, 2nd
10 Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY,
1989) was used by MacLeod et al. to obtain cDNA clones
representing genes which were preferentially expressed in the
SL12.4 T cell clone and undetectable in sister clone SL12.3.
One cDNA clone, 20.5, identified transcripts found in only a
15 limited number of tissues. The gene expressed by this clone
was designated TEA (T cell early activation, SEQ ID NO:5).
1~ transcripts were induced in Balb/c mouse spleen cells
activated in vitro with the T cell mitogen, concanavalin A
(Con A). The TEA gene appears to encode a protein which
20 traverses the membrane multiple times (SEQ ID NO:6), in
contrast to the large number of known integral membrane
proteins induced during T cell activation which are single-
membrane-spanning proteins (see, for review, Crabtree, G.R.,
Science 243:355-361 (1989)).
Seventy genes or gene products are known to increase
in expression when T cells are activated in response to either
antigens (in combination with self-histocompatibility
molecules) or polyclonal activators such as lectins, calcium
ionophores, or antibodies to the TCR (Crabtree, 1989, su~ra).
30 Some of these activation genes are involved in cell cycle
progression, others encode cytokines and cytokine receptors,
nuclear regulatory proteins, and still others are involved in
the transport of ions and nutrients into the cells to prepare
them for growth. At least 26 T cell activation gene products
35 have been localized to the cell membrane (Crabtree, supra).
The TEA gene, as exemplified by clone 20.5 (MacLeod
et al., 1990, supra), is the first example of a cloned gene or
cDNA that has the potential to encode a multiple
WO92/10506 2 ~ 9 7 7 0~ PCT/US91/09382
-- 7
transmembrane-spanning protein which is induced durin~ T cell
activation (Crabtree, Science 243:355-361 (1989)). TEA is an
early gene because TEA mRNA is virtually undetectable in
normal quiescent T cells, increases to detectable levels
5 within 6 hours, and peaks at about 24 hours after Con A
stimulation of spleen cells. The function of the tea gene is
not yet known; it could function to transduce signals or
transport small molecules which are signal transducers, or it
could function as a receptor for an unidentified ligand. The
10 rather long carboxy terminus of the putative tea protein might
function as a signal transducer. Since numerous T and B tumor
cell lines do not express TEA, its expression is clearly not
absolutely required for cell growth, although normal (non-
tumor) T cells might require tea expression for normal
15 proliferation in an immune response.
The sequence of 20.5 cDNA (SEQ ID NO:5) was found to
be strikingly homologous to the murine ERR cDNA clone (SEQ ID
NO:3) discussed above (the Rec-l gene). This finding suggests
that the ~ gene product might function as a murine
20 retroviral receptor. In contrast to the Rec-l gene (encoding
ERR), which is ubiquitously expressed in mouse tissues
(Albritton et al., 1989, supra), expression of the TEA gene
has a much more limited tissue distribution. If the TEA gene
product is a retroviral receptor, this limited tissue
25 distribution could be responsible for the tissue specificity
of retroviruses which are restricted to cells of the lymphoid
lineage tQuint et al., J. Virol. 39:1-10 (1981)). Recent
studies indicate that retroviruses use cell-membrane permease
proteins to gain entry to target cells. The transmembrane
30 topology of ERR is reminiscent of that of several membrane
transporter proteins, the permeases for arginine, histidine
and choline of yeast (Vile, R.G. et al., Nature 352:666-667
(1991). Indeed, two groups have found that the ERR protein
functions as a cationic amino acids transporter (Kim, J. W. et
35 al., Nature 352:752-728 (1991); Wang, H. et al., Nature
352:729-731 (1991)). In fact, this was the fist mammalian
amino acid transporter to be cloned and the first example of a
virus exploiting a transmembrane channel protein as a
WO92/10506 ~ o~ PCT/US91/09382
-- 8
receptor. Related to these findings is the fact that a human
cDNA which confers susceptibility to infe~tion by gibbon ape
leukemia virus has a sequence similar to a phosphate
transporter protein in fungi (Vile et al., supra). It is not
5 known whether Tea also encodes a permease.
~ IV is an example of a virus exhibiting receptor-
mediated tissue restriction, apparently based on its use of
the CD4 protein as its primary receptor. However, cell-
specific receptors are unlikely to be the sole determinant of
10 tissue specificity. The tissue tropism of retroviruses is
likely to result from a complex series of factors, such as the
tissue specificity of long terminal repeats, variations in
viral env proteins, cellular factors, and the expression of
appropriate cell surface receptors (Kabat, Curr. ToP.
15 Microbiol. Immunol. 148:1-31 (1989)). Viral binding and
infection studies are required to determine whether the TEA-
encoded protein functions as a viral receptor (Rein et al.,
Viroloay 136:144-152 (1984)).
Despite the high degree of similarity between TEA
20 and ERR, the two genes differ in chromosomal location, and
their predicted protein products differ in tissue expression
patterns. However, the identification of this new gene family
and of regions of DNA sequence which are highly conserved
between the two members of this family permits searches for
25 new family members.
~ enetically engineered chimeric receptors are known
in the art (see, for example, Riedel, H. et al., Nature
324:628-670 (1986)). However, there are no known examples of
genetically-engineered chimeric receptors for retroviruses
30 which permit, for example, infection of a human cell with a
murine retrovirus. Such a chimeric receptor would be useful
in the gene therapy setting. With the growing interest in
gene therapy, there is a constant search for more efficient
and safer means for introducing exogenous genes into human
35 cells. One relatively efficient means for achieving transfer
of genes is by retrovirus-mediated gene transfer (Gilboa, E.,
Bio-Essavs 5:252-258 (1987): Williams, D.A. et al., Nature
310:476-480 (1984); Weiss, R.A. et al., RNA Tumor Viruses,
WO92/10506 PCT/US91/09382
2~977~
g
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New
York, 1985). One class of retroviruses, recombinant
amphotropic retroviruses, have been studied with greater
intensity as vector for the transfer of genes into human cells
5 (Cone, R.D. et al., Proc. Natl. Acad. Sci. USA 81:6349-6353
(1984); Danos, O. et al., Proc. Natl. Acad. Sci. USA 85:6460-
6464 (1988)). One of the safety problems inherent in this
approach, which may preclude progress in the clinic, is the
fact that even retroviruses that have been rendered
10 replication-defective are sometimes capable of generating
wild-type variants through recombinational events. Such an
alteration could lead to the possibility of widespread
retroviral infection in cells and tissues which were not
intended to be genetically modified. This could result in
15 generalized disease. It is to these needs and problems that
the present invention is also directed.
SUMMARY OF THE INVENTION
The present inventors have discovered and cloned a
novel human DNA sequence, H13, which is highly homologous to
20 murine ERR and TEA, and is therefore considered to encode a
cell membrane protein which acts as a human retrovirus
receptor. Such a human retrovirus receptor can serve as a
target for therapeutic intervention in retrovirus-induced
disease such as AInS. See Yoshimoto, T. et al., Viroloay
25 185:10-17 (1991), and Meruelo et al., U.S. Patent Application
Serial No. 07/627,950, filed December 14, 1990 ~which
references are hereby incorporated by reference in their
entirety).
The partial sequence of the H13 gene was described
30 in U.S. Patent Application Serial No. 07/627,950, filed
December 14, 1990, and is presented herein as SEQ ID NO:1 (and
the putative amino acid sequence is SEQ ID NO:2). This
sequence was s obtained by sequencing a cDNA clone designated
7-2.
The full length sequence of H13 (SEQ ID NO:7) was
obtained by sequencing overlapping clones 1-1 and 3-2
(schematically illustrated in Figure 15). The H13 DNA bears
WO92t10506 PCT/US91/09382
-- 10 --
extensive nucleotide and amino acid sequence similarity with
ERR (SEQ ID NO:3) and with TEA (SEQ ID NO:5). The cDNA
sequence predicts a highly hydrophobic protein which contains
several putative membrane spanning domains. The predicted
5 amino acid sequence of the full-length Hl3 molecule (SEQ ID
NO:8) is homologous to the amino acid sequence of ERR (SEQ ID
NO:4) and TEA (SEQ ID NO:6). The human gene maps to
chromosome 13 and appears to be conserved among mammalian and
avian species. The predicted Hl3 protein has 629 amino acids
lO and an expected molecular weight of about 68 kDa. This
protein has 7 more amino acids than the homologous murine ERR
protein.
The present invention is directed to a recombinant
DNA molecule (SEQ ID NO: 7) comprising a genetic sequence
15 which encodes the Hl3 molecule, or a functional derivative
thereof.
The present invention is further directed to an
expression vector containing the recombinant DNA molecule and
a host transformed or transfected with the vector.
The present invention is also directed to a human
retroviral receptor molecule termed Hl3, the seguence of SEQ
ID NO:8 or close homology thereto, or a functional derivative
thereof, substantially free from impurities of human origin
with which it is natively associated.
Another embodiment of the invention relates to a
method for inhibiting the infection of a cell by a retrovirus,
such as HIV-l, comprising contacting the virus with an
effective amount of the Hl3 protein molecule or functional
derivative thereof and allowing the molecule to prevent the
30 virus from attaching to the cell thereby inhibitinq infection.
The present invention includes a method for
preventing, suppressing, or treating a retrovirus infection,
such as HIV-l, in a subject comprising providing to that
subject an effective amount of the Hl3 molecule or functional
35 derivative.
The invention is further directed to an antibody
specific for the Hl3 molecule or an epitope thereof, including
polyclonal, monoclonal, and chimeric antibody. An additional
W092/10506 2 0 9 7 7 0 ~ PCT/US91/09382
-- 11 --
embodiment involves a method for preventing, suppressing, or
treating a retrovirus infection, such as HIV-l in a subject
comprising providing to that subject an effective amount of
the antibody.
The present invention includes a method for
producing a composition useful for preventing, suppressing, or
treating a retrovirus infection in a subject comprising the
steps of:
(a) providing a recombinant DNA molecule encoding
the Hl3 protein or a functional derivative
thereof in expressible form;
(b) expressing the protein or functional derivative
in a host cell in culture; and
(c) obtaining the protein or functional derivative
from the culture.
The method preferably also i~cludes the additional
purification of the protein or functional derivative. This
method can be carried out in bacterial or eukaryotic,
preferably mammalian, host cells.
The invention also provides a pharmaceutical
composition useful for preventing, suppressing or treating a
retrovirus infection, comprising the Hl3 protein molecule or a
functional derivative thereof or an antibody specific for the
Hl3 protein, and a pharmaceutically acceptable carrier.
It is yet a further object of the present invention
to provide a transgenic experimental animal which has been
transformed by the gene carrying the DNA sequences encoding
the Hl3 protein, alone or in combination with the ~D4 gene.
This transgenic animal serves as a ~odel for human retrovirus
30 infection and allows testing of anti-viral therapies.
The methods of the present invention which identify
normal or mutant Hl3 genes or measure the presence or amount
of Hl3 protein associated with a cell or tissue can serve as
methods for identifying susceptibility to human retrovirus
35 infection, as in AIDS or certain forms of leukemia.
The present invention further provides a DNA
molecule encoding a chimeric retroviral receptor protein,
comprising:
WO92~10506 2 0 ~ PCT/US91/09382
- 12 -
(a) a first nucleotide sequence which encodes retroviral
receptor protein I of a first animal species; and
(b) substituted therein, a sufficient number of nucleotides
from a second nucleotide sequence encoding retroviral
receptor protein II of a second animal species,
wherein the substituting nucleotides confer on the chimeric
retroviral receptor protein the ability to bind a retrovirus
which binds to receptor protein II but not to receptor protein
I, allowing the chimeric protein to function as a retroviral
10 receptor for the retrovirus.
In the above DNA molecule, the first nucleotide
sequence preferably comprises the coding portion of human H13
DNA (SEQ ID NO:7). More preferably, the DNA molecule encodes
a chimeric H13/ERR chimeric protein wherein the second
15 nucleotide sequence comprises the coding portion of murine ERR
DNA (SEQ ID ~0:3), and the substituting ERR nucleotides are
those encoding an amino acid residue selected from the group
consisting of Ile214, Lys222, Asn223, SerZ25, Asn227, Asn232,
Val233, Tyr235, Glu237, Ile313, Asp314, Gly319, Gln324, Glu328
20 and any combination of the above.
The above DNA molecule may be an expression vector.
The present invention also includes a host transformed or
transfected with this vector. Preferably, the host is a
mammalian cell.
Also provided are chimeric retroviral receptor
protein molecules encoded by the above DNA molecules.
The present invention includes a method for
rendering a cell of species I susceptible to infection by, and
retrovirus-mediated gene transfer by, a retroviral vector
WO92/10506 2 0 9 7 7 0 ~ PCT/US91/09382
- 13 -
normally incapable of infeoting a cell of species I,
comprising the steps of:
(a) transforming a cell of species I with an expressible DNA
molecule encoding a chimeric receptor as above,
5 (b) expressing the chimeric retroviral receptor protein on
the surface of the cell in culture,
thereby rendering the cell susceptible to infection by the
retroviral vector. In this method, the cell is preferably a
human cell, the retrovirus is preferably a murine retrovirus,
lO most preferably an ecotropic murine leukemia virus, and the
chimeric receptor is preferably a chimeric Hl3/ERR protein.
In another embodiment, the present invention
provides a method for transferring a gene to a cell of species
I for use in gene therapy, comprising: .
15 (a) culturing a cell intended to receive the transferred
gene;
(b) transforming the cell with a DNA molecule encoding a
chimeric retroviral receptor as described above, thereby
providing the cell with a chimeric retroviral receptor
protein:
(c) infecting the cell with a retroviral vector normally
incapable of infecting a cell of species I, the
retroviral virus being capable of infecting the cell
expressing the chimeric receptor, the retroviral vector
further carrying the gene to be transferred; and
(d) allowing the gene carried by the retroviral vector to be
expressed in the ell,
thereby transferring the gene. In this method, the cell is
preferably a human cell, the retrovirus is preferably a murine
WO92/10506 2 V 9 7 7 0 5 PCT/US91/09382
retrovirus, most preferably an ecotropic murine leukemia
virus, and the chimeric receptor is preferably a chimeric
H13/ERR protein.
BRIEF DESCRIPTION OF THE_DRAWINGS
Figure 1 shows the H13 DNA sequence (SEQ ID NO:7),
including coding and noncoding sequences, and the predicted
protein sequence (SEQ ID NO:8) of the H13 protein.
Figure 2 is a schematic diagram of the alignment of
one strand of the ~13 and ERR cDNA sequence (SEQ ID No:7 and
10 3, respectively). The sequences were analyzed using the
Genetics computer group sequence analysis software package
(Devereux, J. çt al., Nuçl. Acids Res. 12:387-395 (1984)).
Figure 3 shows the alignment of H13, ERR and TEA
deduced amino acid sequences. Vertical lines indicate
15 sequence identity. Dots indicate lack of identity, with
double dots representing conservative amino acid changes. The
sequences were analyzed as in Figure 2. Shown in brackets are
the sequences of H13 corresponding to Extracellular Domain 3
- (residues 210-249) and Extracellular Domain 4 (residues 310-
20 337).
Figure 4 is an autoradiogram showing the
hybridization pattern of EcoRI-digested DNA of human (CCL120,
CCL 119, SupTl, H9, MOLT4), hamster (CHO-Kl) and mouse (Balb/c
thymocytes, BIOT6R) origin, probed with the KpnI-KpnI fragment
25 (390 bp) of murine ERR cDNA.
Figure 5 is an autoradiogram showing Southern blot
analysis DNA from various species with H13 cDNA (SEQ ID NO:l).
DNA hybridized was EcoRI-digested DNA of human (CCL120, CCL
WO92/10506 2 ~ 3 7 7 0 ~ PCT/US91/09382
119, SupTl, H9, MOLT4), hamster (C~O-Kl) and mouse thymocytes
(Balb/c or BIOT6R) origin.
Figure 6 is an autoradiogram showing H13 gene
expression. RNA from the indicated human cell lines was
5 hybridized with the H13 cDNA (SEQ ID NO:1).
Figure 7 is an autoradiogram showing the
hybridization pattern of RNA of human (CEM, Hg, MOLT4, SupTl,
CCL120, CCLll9), hamster (CHO Kl) and mouse (RL12) origin,
probed with the KpnI-KpnI fragment (390 bp) of murine ERR
10 cDNA.
Figure 8 shows the acquisition of susceptibility to
infection with murine ecotropic retrovirus by transfection of
a resistant cell with ERR cDNA. After transfection of ERR
cDNA into hamster CHO Rl cells, the transfectants expressing
15 the murine retroviral receptor gene were infected with murine
radiation leukemia virus (RadLV). Two weeks later, Northern
blot analysis was performed using a viral probe, and reverse
transcriptase (RT) activity of the cell supernatants was
measured.
Figure 9 shows hydropathy plots of H13, ERR and TEA
predicted proteins. The vertical axis gives the
hydropathicity values from the PEPTIDESTRUCTURE program
(Jameson et al., CABIOS 4: 181-186 (1988)).
Figure 10 is a graph indicating the antigenicity of
25 H13 predicted protein, analyzed using the PEPTIDESTRUCTURE
program. One of the highly antiqenic peptides (amino acid
residues 309-367) was prepared using an AccI-EcoRI ~ragment as
shown in Figure 14.
W092/10506 2 0 9 7 7 0 ~ PCT/US91/09382
- 16 -
Figure ll depicts a polyacrylamide gel
electropherogram showing the synthesis of a fusion protein
including the Hl3 protein with glutathione-S-transferase
(GST). The fusion protein was prepared by ligating the 180 bp
5 AccI-EcoRI fragment of H13 cDNA to the plasmid pGEX-2T, which
expresses antigens as fusion proteins, was induced by addition
of isopropyl-beta-thiogalacto-pyranoside (IPTG), and was
purified using glutathione-Sepharose chromatography.
Figure 12 shows the genetic mapping of the H13 gene
lO to human chromosome 13. The autoradiogram (Figure 12A) shows
the hybridization pattern of EcoRI-digested DNA from human-
hamster somatic cell hybrids probed with Hl3 cDNA (SEQ ID
NO:l). Lane l and 11 contain DNA from human and hamster,
respectively. Lanes 2-10 contain DNA whi~h is derived from the
15 chromosomes as designated in the table in Figure 12B.
Figure 13 is a schematic diagram of the genetic
structure of the Hl3 and ERR genes, and four chimeric
constructs therebetween. The infectivity of E-MuLV on human
cells transfected with the various constructs is also
20 indicated.
Figure 14 shows a comparison of sequences
(nucleotide and amino acid) of the region of Hl3 and ERR
termed Extracellular Domain 3 (see also SEQ ID NO:7, SEQ ID
NO:8 and Figure l). This region of the receptor protein is
25 most diverse between the human and mouse sequences. The
sequences were aligned using Genetics computer group sequence
analysis software package (Devereux, J. et al., Nucl. Acids
Res. 12:387-395 (1984)).
Figure 15 shows a schematic illustration of severa
WO92/10506 PCT/US91/09382
2097705
- 17 -
cDNA clones from which the H13 sequence was derived, and their
general structural relationship to the murine ERR homologue.
Clone 7-2 (H-13.7-2) represents a part of the complete H13 DNA
sequence; this was the first H13 clone sequenced, yielding
5 SEQ ID NO:1 and SEQ ID NO:2. Clones 1-1 (H13.1-1) and 3-2
(H13.3-2) each contain parts of the H13 sequence. The
combined sequencing of these three clones resulted in the full
H13 DNA and amino acid sequences (SEQ ID NO:7 and SEQ ID NO:8,
respectively).
DE$5~IPT~ON OF TRE~ PREFERRED EHBODIMENTS
The present invention is directed to a DNA molecule
discovered by the inventors which is homologous to murine
endogenous retrovirus receptor (ERR) and T cell early
activation antigen genes (TEA) and encodes a protein termed
15 H13. The present inventors have conceived of a method of use
of the H13 protein, or a functional derivative thereof,
preferably a soluble form of the protein, to bind human
retroviruses in a manner that prevents their entry into
- susceptible cells.
The methods of the present invention which identify
normal or mutant H13 genes or measure the presence or amount
of H13 protein associated with a cell or tissue can serve as
methods for identifying susceptibility to human retrovirus
infection, as in AIDS or certain forms of leukemia.
In one embodiment, the invention is directed to a
naturally occurring H13 protein substantially free from
impurities of human origin with which it is natively
associated. In another embodiment, the invention is directed
W092/10506 ~0 9 7 7 ~ ~ PCT/US91/09382
- 18 -
to a recombinant H13 encoded protein. "Substantially free of
other proteins" indicates that the protein has been purified
away from at least 90 per cent (on a weight basis), and from
even at least 99 per cent, if desired, of other proteins and
5 glycoproteins with which it is natively associated, and is
therefore substantially free of them. That can be achieved by
subjecting the cells, tissue or fluids containing the H13
protein to protein purification techniques such as
immunoadsorbent columns bearing monoclonal antibodies reactive
10 against the protein. Alternatively, the purification can be
achieved by a combination of standard methods, such as
ammonium sulfate precipitation, molecular sieve
chromatography, and ion exchange chromatography.
It will be understood that the H13 protein of the
15 present invention can be purified biochemically or
physicochemically from a variety of cell or tissue sources.
For preparation OL naturally occurring H13 protein, tissues
such as human lymphatic organs and cells such as human
lymphoid cells are preferred. Alternatively, methods are well
20 known for the synthesis of polypeptides of desired sequence on
solid phase supports and their subsequent separation from the
support.
Because the H13 gene can be isolated or synthesized,
the H13 polypeptide, or a functional derivative thereof, can
25 be synthesized substantially free of other proteins or
glycoproteins of mammalian origin in a prokaryotic organism or
in a non-mammalian eukaryotic organism, if desired. As
intended by the present invention, an H13 protein molecule
produced by recombinant means in mammalian cells, such as
WO92/10506 2 0 9 7 7 0 5 PCT/US91/09382
-- 19 --
transfected COS, NIH-3T3, or CHO cells, for example, is either
a naturally occurring protein sequence or a functional
derivative thereof. Where a naturally occurring protein or
glycoprotein is produced by recombinant means, it is provided
5 substantially free of the other proteins and glycoproteins
with which it is natively associated.
A preferred use of this invention is the production
by chemical synthesis or recombinant DNA technology of
fragments of the H13 molecule, preferably as small as
10 possible, while still retaining sufficiently high affinity in
binding to HIV to inhibit i~fection. Preferred fragments of
H13 include extracellular domain 3 and extracellular domain 4.
Due to its function as a virus receptor, an extracellular
fragment of the H13 protein is expected to bind to a human
15 retrovirus. By production of smaller fragments of this
peptide, one skilled in the art, using known binding and
inhibition assays, will readily be able to identify the
minimal peptide capable of binding a retrovirus with
sufficiently high affinity to inhibit infectivity without
20 undue experimentation. Shorter peptides are expected to have
two advantages over the larger proteins: (1) greater stability
and diffusibility, and (2) less immunogenicity.
The identification of the H13 as a potential
receptor or site of entry of a retrovirus into target cells
25 establishes a critical mechanism to explain how the retrovirus
enters the cell. The availability of specific H13 receptor
"mimics" or "decoys" that can prevent retrovirus uptake
provides promise in controlling the spread of retrovirus
infection and related pathologies.
WO92/10506 ~0 ~,~ r~ ~ ~ PCT/US91/09382
- 20 -
~ espite the degree of similarity in amino acid
sequence (87.6% identity) and structure (14 transmembrane
spanning domains), between the human Hl3 protein of the
present invention and the murine ERR, H13 fails to bind
5 detectably to E-MuLV.
The present inventors have taken advantage of this
species difference in susceptibility in infection to construct
and analyze DNA molecules encoding chimeric retrovirus
receptor proteins by substituting bases encoding amino acids
10 of murine ERR for bases encoding H13 amino acid residues.
This has resulted in the identification of critical amino
acids which must be present for susceptibility o~ a cell to
infection by E-MuLV.
The chimeric mouse-human E-MuLV receptor of the
15 present invention can be synthesized substantially free of
other proteins or glycoproteins of mammalian origin in a
prokaryotic or non-~ammalian eukaryotic cell. Preferably,
however, a chimeric ~13/ERR protein molecule is produced by
recombinant means and expressed in ~ammalian cells, most
20 preferably in human cells.
Due to the presence of critical amino acid residues
from the murine ERR sequence, the chimeric receptor of the
present invention endows human cells or other non-murine cells
expressing the receptor with the ability to be infected by
` 25 murine E-MuLV.
By appropriate substitutions of one or more of the
amino acid residues of ERR in the corresponding site in H13,
one skilled in the art, using known binding and inhibition
assays, Will be able, without undue experimentation, to
20~770~
WO92/10506 PCT/US91/09382
- 21 -
identify the single or multiple amino acid substitutions which
result in a chimeric retroviral receptor capable of binding E-
MuLV with sufficiently high affinity to permit infection of a
non-murine cell, preferably a human cell, with E-MuLV.
Hl3 extracellular domain 3 and Hl3 extracellular
domain 4, appear to be the most sensitive sites for modifying
virus binding. Thus, to confer E-MuLV susceptibility on a
cell, it is preferred to substitute amino acid residues of
these domains of Hl3. Domain 3 comprises residues between
lO positions 210 and 250 (SEQ ID N0:7). Preferred substitution
is with one or more amino acid residues from the corresponding
domain of ERR, between amino acid residues 210 and 242 (SEQ ID
N0:4~. Domain 4 of Hl3 comprises residues 31-337 (SEQ ID
NO:7). Preferred substitution is with one or more amino acid
15 residues from the corresponding domain of ERR, between amino
acid residues 303 and 330 (SEQ ID NO:4).
Substitution of between l and 4 residues is
preferred. Substitution of as few as one amino acid may alter
the virus specificity of the chimeric receptor protein. The
20 residues and positions which differ in Extracellular Domain 3
and Domain 4 of Hl3 and ERR are listed below in Table l.
WO92/lOS06 ~ 0 9 7 7 o ~ PCT/US91tO9382
- 22 -
Table 1
Possible Substitutions in H13 Extracellular Domain 3 and
Domain 4 for E-MuLV Binding
Domain 3 Domain 4
Original SubstitutingOriginal Substituting
H13 ERR H13 ERR
Residue Residue Residue Residue
V 214 I 214 N 320 I 313
E 222 K 222 N 321 D 314
E 223 N 223 D 32~ G 319
G 225 S 225 V 331 Q 324
L 233 N 227 G 335 E 328
E 239 N 232
G 240 V 233
P 242 Y 235
V 244 E 237
Another means for modifying the virus binding
specificity of H13 is by deletion of one or more of the
"extra" amino acid residues in H13 (in extracellular domain 4)
~hat do not correspond to residues of ERR. Preferred
5 deletions are of between one and six residues from H13
positions 326 to 331 (SEQ ID NO:l), ~ost preferably, deletion
of all six of these residues.
A major advantage of transfecting non-murine cells
with a chimeric receptor or substituted receptor of the
10 present invention, râther than with ~he ERR protein, is the
major decrease in immunogenicity. Thus, for example, human
cells to be infected with a murine retrovirus are made to
express a chimeric receptor comprising virtually all human
sequence, but having only a few necessary amino acid residues
15 of the murine retroviral receptor sequence needed to confer
infectibility by the murine retrovirus. If cells bearing a
receptor to which E-MuLV can bind are to be introduced on
multiple occasions into the same human subject, thè fact that
the chimeric receptor is largely of human origin decreases
20 the chances of an undesirable immune response directed to the
sequences derived from receptor sequence of non-human origin.
If large portions of the ER~ protein were used for this
purpose, the human subject would respond immunologically to
WO92/10506 2 0 ~ 7 7 0 ~ PCT/US91/09382
- 23 -
the foreign epitopes on the injected cells, diminishing the
utility of these cells in gene therapy.
This lacX of immunogenicity is important for
survival and therapeutic efficacy of the infused retrovirally
5 infected cells. In gene therapy using bone marrow stem cells
or hepatocytes, for example, it is common to manipulate the
cells in vitro with cytokines and then to infect them with the
vector bearing the gene of interest. Such cells are very
short lived an have yielded very short lived therapeutic
10 changes (see, for example, Wilson, J.M. et al., Proc. Natl.
Acad. Sci. USA 87:8437-8441 (1990)). The addition of
immunogenic epitopes on such cells would further shorten their
half-life in vivo.
The H13 protein as well as the ERR/H13 chimeric
15 protein can be expressed on the cell surface as an integral
membrane protein in a number of cell types, particularly cells
of the T lymphocyte and monocyte/macrophage lineages,
consistent with in vitro tropism of known human retroviruses
such as HIV-l and HTLV-1. Thus, the chimeric receptor will
20 permit cells of these lineages in the human, which are
normally resistant to murine retrovirus infection, to be
infected with E-MuLV.
The virus infections for which the present invention
is useful include HIV-l, HIV-2, human T lymphotropic viruses
25 that induce leukemia (HTLV-l, HTLV-2, etc.) and other human
retroviruses (~eiss, R.A. et al.j RNA Tumor Viruses, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, New York,
1985). The present invention is intended to encompass all the
retroviruses which attach to the H13 as their cellular
30 receptor or enter the cell via an H-13-dependent mechanism.
Genetic constructs encoding H13 functional deriva-
tives thereof such as those described above, can be used in
gene therapy. An abnormal H13 molecule which results in
enhanced susceptibility to disease, may be replaced by
35 infusion of cells of the desired lineage (such as hemopoietic
cells, for example) transfected with a modified H13 protein,
under conditions where the infused cells will preferentially
replace the endogenous cell population.
W O 92/10~06 PC~r/US91/09382
~V9'`170~
- 24 -
Genetic constructs encoding a recombinant chimeric
ERR/H13 molecule are particularly useful in gene therapy.
Recombinant amphotropic retroviruses have been recognized as
useful vectors for transferring genes efficiently into human
5 cells, for example to correct enzyme deficiencies (Cone, R.D.
et al., Proc. Natl. Acad. Sci. USA 81:6349-6353 (1984); Danos,
O. et al., Proc. Natl. Acad. Sci. USA 85:6460-6464 tl988)).
Depending on the receptor specificity of the viral envelope
protein gp70, such viruses have varying host ranges; some
10 recognize human cells. Cone et al. (supra) and Danos et al.
(supra) produced packaging cell lines which produced
amphotrophic retroviruses which could infect human cells and
integrate randomly into human genomic DNA. Such vectors have
been used to transfer a histochemically detectable marker qene
15 into neurons ttPrice, J. et al., Proc. Natl. Acad. Sci. USA
84:156-160 tl987)).
For safety reasons, it is important that a
retroviral vector used for gene therapy be capable of
infecting only desired cells and not cause generalized
20 infection of cells throughout the body of the individual being
treated. In the past, this has generally been accomplished by
using helper-defective virus preparations, or mutants lacking
the Dsi packaging sequence, etc. The present invention
provides an improved measure of safety compared to the prior
; 25 art approaches in that it permits use of an competent E-MuLV
vector, having a limited murine host range. Only those cells
to be infected with the vector are given the capacity by
virtue of their expression of the chimeric receptor of the
present invention.
While gene transfer using retroviruses is generally
more efficient than transfection with naked DNA, some cells
are not easily infectible by retroviruses, making it difficult
to use retroviruses as vectors for introducing new genes into
such cells. According to the present invention, a human cell
35 which is not infectable by a human retrovirus or is infectable
only at very low efficiency due to lack of sufficient
retroviral receptor protein on its surface is transfected with
the H13 gene or a functional derivative, and the H13 protein
2~9770~
WO92/10506 PCT/US91/09382
- 25 -
expressed, resulting in retrovirus receptor appearing on the
cell surface. Such a transfected cell can then be infected
with a human retroviral vector carrying a gene of interest, in
order to transfer the gene of interest permanently into the
5 cell. This type of manipulation has been accomplished to
render hamster cells, which are not susceptible to infection
with MuLV, susceptible to this virus (see Example IV, below).
Following transfection with and expression of the ERR gene
- (the H13 homolog) in hamster cells, these cells could be
10 infected with MuLV, and could serve as targets for MuLV-
mediated gene transfer. For a general discussion of
retrovirus-mediated gene transfer, see, for example (Gilboa,
E., Bio-Essays 5:252-258 tl987); Williams, D.A. et al.,
Nature 310:476-480 (1984)).
The present invention is intended to encompass any
ecotropic murine retroviruses, or any other mammalian
retroviruses with si~ilar receptor specificity, which attach
to ERR and to the chimeric H13/ERR molecule of the present
invention as their cellular receptor or enter the cell via an
20 ERR-dependent mechanism.
More broadly, the invention is directed to the
general concept of generating a chimeric retroviral receptor
which will allow selected cells of one animal species to be
infected with a retrovirus which normally does not infect
25 cells of that species. Thus, for example, murine retroviruses
can infect chimpanzee cells if they express a chimpanzee
retrovirus-murine retrovirus chimeric receptor which allows
binding of the murine retrovirus. By specific changes in the
amino acid sequence of, for example, the H13 molecule, it
30 would be possible to create a receptor molecule that would
confer susceptibility to any of a number of viruses. Thus a
different chimeric H13-based construct can be tailor made for
any given human or non-human retrovirus. One of ordinary
skill in the art will readily be able to apply this teaching
35 to any of a number of retroviruses and chimeric receptors
without undue experimentation.
one of ordinary skill in the art will know how to
obtain DNA encoding a retroviral receptor homologous to ERR or
2097~5
W092t10506 PCT/US91/09382
- 26 -
Hl3 from any species or cell type without undue
experimentation. First, one will screen (using methods
routine in the art) a cDNA library of the species or cell type
of interest, for example, a chimpanzee T cell cDNA library,
5 using a probe based on the sequence of ERR or Hl3. Next, one
will clone and sequence the hybridizing DNA to obtain the
sequence of the "new" retroviral receptor. By visual
inspection or with the aid of a computer program (as described
herein) it is possible to identify the regions in which the
lO sequence of the new retroviral receptor protein differs from
ERR or Hl3. In particular, one will concentrate on the
extracellular domain regions 3 or 4. Based on the sequence
differences observed, it is possible, using the teachings
provided herein, to create a sequence having one or more amino
15 acid substitutions such that a chimeric receptor between the
new receptor and a known receptor is created. The chimeric
receptor can then be expressed in a cell of choice and its
function can easily be tested using conventional virus
binding assays or virus infectivity assays.
Furthermore, according to the present invention, it
is possible to modify the receptor attachment site of a virus
so that it will not bind to its natural receptor. For
example, changes in the sequence of the HIV-l CD4-binding
domain will render this virus non-infective for CD4-bearing
25 cells. Corresponding changes may be introduced into Hl3, so
that this mutant HIV will bind to it. In this way, a safe HIV
preparation can be generated which binds only to select cells
bearing the appropriate variant receptor, but not to the
normal targets of HIV-l.
In another embodiment, the methods and constructs of
the present invention can be used to produce a bone marrow
stem cells which are infectible via CD4 without disrupting the
normal cellular developmental and maturational process that
depend on intact CD4 expression. In this embodiment, a
35 chimeric Hl3/CD4 molecule is expressed in a bone marrow stem
cell, which is then infected by a E-MuLV retroviral vector
having a CD4-binding domain of HIV engineered into the murine
gp70 molecule.
2~77 0~
WO92/10506 PCT/US91/09382
Expression of the intact or a chimeric human H13
sequence in a chimpanzee cell or cell lineage may allow an HIV
infection in chimpanzees to develop into and AIDS-like
syndrome, providing an i~proved animal model for AIDS than the
5 simian immunodeficiency virus infects of chimpanzees.
It is also within the scope of the present invention
to express more than one intact or chimeric retroviral
receptor molecule on the surface of the same cell. Thus, by
virtue of a first retroviral receptor, e.g. ERR, a human cell
10 can be infected with one virus strain in vitro in a transient
fashion, and can be manipulated by the judicious use of
cytokine growth or differentiation factors. Such cells can be
introduced into a recipient. At the desired time, a second
virus which binds to a second genetically engineered receptor
15 can be introduced into the individual to infect stably alter
only those introduced cells bearing the second retroviral
receptor.
The preferred animal subject of the present
invention is a mammal. By the term "mammal" is meant an
20 individual belonging to the class Mammalia. The invention is
particularly useful in the treatment of human subjects,
although it is intended for veterinary uses as well.
Also included are soluble forms of H13 or of a
chimeric receptor, as well as functional derivatives thereof
25 having similar bioactivity for all the uses described herein.
Also intended are all active forms of H13 deri~ed from the H13
transcript, and all muteins with H13 activity. Methods for
production of soluble forms of receptors which are normally
transmembrane proteins are well known in the art (see, for
30 example, Smith, D.H. et al., Science 238:1704-1707 (1987);
Fisher, R.A. et al., Nature 331:76-78 (1988); Hussey, R.E. et
al., Nature 331:78-81 (1988); Deen, K.C. et al., Nature
331:82-84 (1988); Traunecker, A. et al., Nature 331:84-86
(1988); Gershoni, J.M. et al., Proc. Natl. Acad. Sci. USA
35 85:4087-4089 (1988), which references are hereby incorporated
by reference). Such methods are generally based on truncation
of the DNA encoding the receptor protein to exclude the
transmembrane portion, leaving intact the extracellular domain
W092/lOS06 ~ ~ 7 ~ O ~ PCT/US91/09382
- 28 -
(or domains) capable of interacting with specific ligands,
such as an intact retrovirus or a retroviral protein or
glycoprotein.
For the purposes of the present invention, it is
5 important that the soluble Hl3, or a functional derivative of
Hl3, comprise the elements of the binding site of the Hl3 that
permits binding to a retrovirus. An Hl3 molecule has many
amino acid residues, only a Pew of which are critically
involved in virus recognition and binding.
As discussed herein, the Hl3 proteins or peptides of
the present invention may be further modified for purposes of
drug design, such as, for example, to reduce immunogenicity,
to promo~e solubility or enhance delivery, or to prevent
clearance or degradation.
In a further embodiment, the invention provides
"functional derivatives" of the Hl3 protein. By "functional
derivative" is meant a "fragment," "variant," "analog," or
"chemical derivative" of the Hl3 protein. A functional
derivative retains at least a portion of the function of the
20 Hl3 protein which permits its utility in accordance with the
present invention.
A "fragment" of the Hl3 protein is any subset of the
molecule, that is, a shorter peptide.
A "variant" of the Hl3 refers to a molecule sub-
25 stantially similar to either the entire peptide or a fragmentthereof. Variant peptides may be conveniently prepared by
direct chemical synthesis of the variant peptide, using
methods well- known in the art.
Alternatively, amino acid sequence variants of the
30 peptide can be prepared by mutations in the DNA which encodes
the synthesized peptide. Such variants include, for example,
deletions from, or insertions or substitutions of, residues
within the amino acid sequence. Any combination of deletion,
insertion, and substitution may also be made to arrive at the
35 final construct, provided that the final construct possesses
the desired activity. Obviously, the mutations that will be
made in the DNA encoding the variant peptide must not alter
the reading frame and preferably will not create complementary
WO92/10506 2 0 9 7 7 0 ~ PCT/US91/09382
- 29 -
regions that could produce secondary mRNA structure (see
European Patent Publication No. EP 75,444).
At the genetic level, these variants ordinarily are
prepared by site-directed mutagenesis (as exemplified by
5 Adelman et al., DNA 2:183 (1983)) of nucleotides in the DNA
encoding the peptide molecule, thereby producing DNA encoding
the variant, and thereafter expressing the DNA in recombinant
cell culture. The variants typically exhibit the same
qualitative biological activity as the nonvariant peptide.
10 In particular, the Hl3 molecule having critical amino acid
residues derived from ERR can be produced using site-directed
mutagenesis.
In general, site-directed mutagenesis in accordance
herewith is performed by first obtaining a single-stranded
15 vector that includes within its sequence a DNA sequence that
encodes the relevant peptide. An oligonucleotide primer
bearing the desired mutated sequence is prepared, generally
synthetically, for example, by the method of Crea et al.,
Proc. Natl. Acad. SCi. (USA) 75:5765 (1978). ~his primer is
20 then annealed with the single-stranded protein-sequence-
containing vector, and subjected to DNA-polymerizing enzymes
such as E. coli polymerase I Xlenow fragment, to complete the
synthesis of the mutation-bearing strand. Thus, a mutated
sequence in the second strand bears the desired mutation.
25 This heteroduplex vector is then used to transform appropriate
cells and clones are selected that include recombinant vectors
bearing the mutated sequence arrangement. The mutated protein
region may be removed and placed in an appropriate vector for
protein production, generally an expression vector of the type
30 that may be employed for transformation of an appropriate
host.
An example of a terminal insertion includes a fusion
of a signal sequence, whether heterologous or homologous to
the host cell, to the N-terminus of the peptide molecule to
35 facilitate the secretion of mature peptide molecule from
recombinant hosts.
Another group of variants are those in which at
least one amino acid residue in the protein molecule, and
W092/]0506 2 ~ 9 r~ ~f o 5 PCT/US91/09382
- 30 -
preferably, only one, has been removed and a different residue
inserted in its place. For a detailed description of protein
chemistry and structure, see Schulz, G.E. et al., Principles
of Protein Structure, Springer-Verlag, New York, 1978, and
5 Creighton, T.E., Proteins: Structure and Molecular
Pro~erties, W.H. Freeman & Co., San Francisco, 1983, which are
hereby incorporated by reference. The types of substitutions
which may be made in the protein or peptide molecule of the
present invention may be based on analysis of the frequencies
lO of amino acid changes between a homologous protein of
different species, such as those presented in Table 1-2 of
Schulz et al. (su~ra) and Figure 3-9 of Creighton (su~ra).
Base on such an analysis, conservative substitutions are
defined herein as exchanges within one of the following five
15 groups:
1. Small aliphatic, nonpolar or slightly polar
residues: ala, ser, thr (pro, gly);
2. Polar, negatively charged residues and their
amides: asp, asn, glu, gln;
3. Polar, positively charged residues:
his, arg, lys;
4. Large aliphatic, nopolar residues:
met, leu, ile, val (cys); and
5. Large aromatic residues: phe, tyr, trp.
The three amino acid residues in parentheses above
have special roles in protein architecture. Gly is the only
residue lacking any side chain and thus imparts flexibility to
the chain. Pro, because of its unusual geometry, tightly
constrains the chain. Cys can participate in disulfide bond
30 formation which is important in protein folding. Note the
Schulz et al. would merge Groups 1 and 2, above. Note also
that Tyr, because of its hydrogen bonding potential, has some
kinship with Ser, Thr, etc. Substantial changes in
functional or immunological properties are made by selecting
35 substitutions that are less conservative, such as between,
rather than within, the above five groups, which will differ
more significantly in their effect on maintaining (a) the
W092/lOS06 2 ~ 9 7 7 0 ~ PCT/US91/09382
- 31 -
structure of the peptide backbone in the area of the
substitution, for example, as a sheet or helical
conformation, (b) the charge or hydrophobicity of the molecule
at the target site, or (c) the bulk of the side chain.
5 Examples of such substitutions are (a) substitution of gly
and/or pro by another amino acid or deletion or insertion of
gly or pro; (b) substitution of a hydrophilic residue, e.g.,
ser or thr, for (or by) a hydrophobic residue, e.g., leu, ile,
phe, val or ala; (c) substituion of a cys residue for (or by)
10 any other residue; (d) substitution of a residue having an
electropositive side chain, e.g., lys, arg or his, for (or by)
a residue having an electronegative charge, e.g., glu or asp;
or (e) substitution of a residue having a bulky side chain,
e.g., phe, for (or by) a residue not having such a side chain,
15 e.g., gly.
Most deletions and insertions, and substitutions
according to the present invention are those which do not
produce radical changes in the characteristics of the protein
or peptide molecule. However, when it is difficult to predict
20 the exact effect of the substituti~n, deletion, or insertion
in advance of doing so, one skilled in the art will appreciate
that the effect will be evaluated by routine screening assays,
either immunoassays or bioassays. For example, a variant
typically is made by site-specific mutagenesis of the peptide
25 molecule-encoding nucleic acid, expression of the variant
nucleic acid in recombinant cell culture, and, optionally,
purification from the cell culture, for example, by
immunoaffinity chromatography using a specific antibody on a
column (to absorb the variant by binding to at least one
30 epitope).
The activity of the cell lysate containing H13 or a
chimeric H13 protein, or of a purified preparation of H13, a
variant thereof, of of chimeric H13, can be screened in a
suitable screening assay for the desired characteristic. For
35 example, a change in the immunological character of the
protein molecule, such as binding to a given antibody, is
measured by a competitive type immunoassay (see below).
WO92/10506 ~ ~ 7 7 ~ PCT/US91/09382
Biological activity is screened in an appropriate bioassay,
such as virus infectivity, as described herein.
Modifications of such peptide properties as redox or
thermal stability, hydrophobicity, susceptibility to
5 proteolytic degradation or the tendency to aggregate with
carriers or into multimers are assayed by methods well known
to the ordinarily skilled artisan.
An "analog" of the Hl3 protein refers to a non-
natural molecule substantially similar to either the entire
10 molecule or a fragment thereof.
A "chemical derivative" of the H13 protein contains
additional chemical moieties not normally a part of the
protein. Covalent modifications of the peptide are included
within the scope of this invention. Such modifications may be
15 introduced into the molecule by reacting targeted amino acid
residues of the peptide with an organic derivatizing agent
that is capable of reacting with selected side chains or
terminal residues.
Cysteinyl residues most commonly are reacted with
20 alpha-haloacetates (and corresponding amines), such as 2-
chloroacetic acid or chloroacetamide, to give carboxymethyl or
carboxyamidomethyl derivatives. Cysteinyl residues also are
derivatized by reaction with bromotrifluoroacetone, alpha-
bromo- beta-(5-imidozoyl)propionic acid, chloroacetyl
25 phosphate, N- alkylmaleimides, 3-nitro-2-pyridyl disulfide,
methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-
chloromercuri-4- nitrophenol, or chloro-7-nitrobenzo-2-oxa-
l,3-diazole.
Histidyl residues are derivatized by reaction with
30 diethylprocarbonate at pH 5.5-7.0 because this agent is
relatively specific for the histidyl side chain. Para-
bromophenacyl bromide also is useful; the reaction is
preferably performed in 0.1 M sodium cacodylate at pH 6Ø
Lysinyl and amino terminal residues are reacted with
35 succinic or other carboxylic acid anhydrides. Derivatization
with these agents has the effect of reversing the charge of
the lysinyl residues. Other suitable reagents for
derivatizing alpha-amino-containing residues include
WO92/10506 2 0 9 7 7 ~ ~ PCTtUS91/09382
- 33 -
imidoesters such as methyl picolinimidate; pyridoxal
phosphate; pyridoxal; chloroborohydride;
trinitrobenzenesulfonic acid; O-methylisourea; 2,4
pentanedione; and transaminase-catalyzed reaction with
5 glyoxylate.
Arginyl residues are modified by reaction with one
or several conventional reagents, among them phenylglyoxal,
2,3- butanedione, 1,2-cyclohexanedione, and ninhydrin.
Derivatization of arginine residues re~uires that the reaction
10 be performed in alXaline conditions because of the high PXa f
the guanidine functional group. Furthermore, these reagents
may react with the groups of lysine as well as the arginine
epsilon-amino group.
The specific modification of tyrosyl residues per se
15 has been studied extensively, with particular interest in
introducing spectral labels into tyrosyl residues by reaction
with aromatic diazonium compounds or tetranitromethane. Most
commonly, N-acetylimidizol and tetranitromethane are used to
form O-acetyl tyrosyl species and 3-nitro derivatives,
20 respectively.
Carboxyl side groups (aspartyl or glutamyl) are
selectively modified by reaction with carbodiimides (R'-N-C-N-
R') such as 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)
carbodiimide or 1- ethyl-3-(4-azonia-4,4-dimethylpentyl)
25 carbodiimide. Furthermore, aspartyl and glutamyl residues are
converted to asparaginyl and glutaminyl residues by reaction
with ammonium ions.
Glutaminyl and asparaginyl residues are frequently
deamidated to the corresponding glutamyl and aspartyl
30 residues. Alternatively, these residues are deamidated under
mildly acidic conditions. Either form of these residues falls
within the scope of this invention.
Derivatization with bifunctional agents is useful
for cross-linking the peptide to a water-insoluble support
35 matrix or to other macromolecular carriers. Commonly used
cross-linking agents include, e.g., l,1-bis(diazoacetyl)-2-
phenylethane, glutaraldehyde, N-hydroxysuCCinimide esters, for
example, esters with 4-azidosalicylic acid, homobifunctional
WO92/10506 2 0 ~ r~ r~ ~ 5 PCT/VS91/09382
- 34 -
imidoesters, including disuccinimidyl esters such as 3,3'-
dithiobis(succinimidylpropionate), and bifunctional maleimides
such as bis-N-maleimido-1,8-octane. Derivatizing agents such
as methyl-3-[(p-azidophenyl)dithio]propioimidate yield
5 photoactivatable intermediates that are capable of forming
crosslinks in the presence of light. Alternatively, reactive
water-insoluble matrices such as cyanogen bromide-activated
carbohydrates and the reactive substrates described in U.S.
Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642;
10 4,229,537; and 4,330,440 are employed for protein
immobilization.
Other modifications include hydroxylation of proline
and lysine, phosphorylation of hydroxyl groups of seryl or
threonyl residues, methylation of the alpha-amino groups of
15 lysine, arginine, and histidine side chains (T.E. Creighton,
Proteins: Structure and Molecule Properties, W.H. Freeman &
Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-
terminal amine, and, in some instances, amidation of the C-
terminal carboxyl groups.
Such derivatized moieties may improve the
solubility, absorption, biological half life, and the like.
The moieties may alternatively eli.minate or attenuate any
undesirable side effect of the protein and the like. Moieties
capable of mediating such effects are disclosed, for example,
25 in Reminqton's Pharmaceutical Sciences, 16th ed., Mack
Publishing Co., Easton, PA (1980).
Standard reference works setting forth the general
principles of recombinant DNA technology include Watson, J.D.
et al., Molecular ~3ioloay o~ ~he Gene, Volumes I and II, The
30 Benjamin/Cummings Publishing Company, Inc., publisher, Menlo
Park, CA (1987); Darnell, J.E. et al., Molecular Cell Bioloay,
Scientific American Books, Inc., publisher, New York, N.Y.
~1986); Lewin, B.M., Genes II, John Wiley & Sons, publishers,
New York, N.Y. (1985); Old, R.W., et al., Principles of Gene
35 Manipulation: An Introduction to Genetic Enqineerinq, 2d
edition, University of California Press, publisher, Berkeley,
CA (1981); and Sambrook, J. et al., Molecular Clonina. A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
2a9770~
WO92/10506 PCT/US91/09382
- 35 -
Harbor, NY (1989). These references are hereby incorporated
by reference. The recombinant DNA molecules of the present
invention can be produced through any of a variety of means,
such as, for example, DNA or RNA synthesis, or more
5 preferably, by application of reco~binant DNA techniques.
By "cloning" is meant the use of in vitro
recombination techniques to insert a particular gene or other
DNA sequence into a vector molecule. In order to successfully
clone a desired gene, it is necessary to employ methods for
10 generating DNA fragments, for joining the fragments to vector
molecules, for introducing the composite DNA molecule into a
host cell in which it can replicate, and for selecting the
clone having the target gene from amongst the recipient host
cells.
By "cDNA" is meant complementary or copy DNA
produced from an RNA template by the action of RNA-dependent
DNA polymerase (reverse transcriptase). Thus a "cDNA clone"
means a duplex DNA sequence complementary to an RNA molecule
of interest, carried in a cloning vector.
By "cDNA library" is meant a collection of
recombinant DNA ~olecules containing cDNA inserts which
together comprise the entire expressible genome of an
organism. Such a cDNA library may be prepared by methods
known to those of skill, and described, for example, in
25 Sambrook et al., Molecular Clonin~: A Laboratory Manual,
supra. Generally, RNA is first isolated from the cells of an
organism from whose genome it is desired to clone a particular
gene. Preferred for the purposes of the present invention are
mammalian cell lines.
Oligonucleotides representing a portion of the H13
sequence are useful for screening for the presence of
homologous genes and for the cloning of such qenes.
Techniques for synthesizing such oligonucleotides are
disclosed by, for example, Wu, R., et al., Pro~. Nucl. Acid.
35 Res. Molec. Biol. 21:101-141 (1978)).
Because the genetic code is degenerate, more than
o~e codon may be used to encode a particular amino acid
(Watson, J.D. et al., supra). Using the genetic code, one or
WO92/10506 2 0 3 ~ 7 0 ~ PCT/US91/09382
- 36 -
more different oligonucleotides can be identified, each of
which would be capable of encoding the amino acid. The
probability that a particular oligonucleotide will, in fact,
constitute the actual XXX-encoding sequence can be estimated
S by considering abnormal base pairing relationships and the
frequency with which a particular codon is actually used (to
encode a particular amino acid) in eukaryotic cells. Such
"codon usage rules" are disclosed by Lathe, R., et al., J.
Molec. Biol. 183:1-12 (1985). Using the "codon usage rules"
lO of Lathe, a single oligonucleotide, or a set of
oligonucleotides, that contains a theoretical "most probable"
nucleotide sequence capable of encoding the Hl3 sequences is
identified.
Although occasionally an amino acid sequence may be
15 encoded by only a single oligsnucleotide, frequently the amino
acid sequence may be encoded by any of a set of similar
oligonucleotides. Importantly, whereas all of the members of
this set contain oligonucleotides which are capable of
encoding the peptide fragment and, thus, potentially contain
20 the same oligonucleotide sequence as the gene which encodes
the peptide fragment, only one member of the set contains the
nucleotide sequence that is identical to the nucleotide
sequence of the gene. Because this member is present within
the set, and is capable of hybridizing to DNA even in the
25 presence of the other members of the set, it is possible to
employ the unfractionated set of oligonucleotides in the same
manner in which one would employ a single oligonucleotide to
clone the gene that encodes the protein.
The oligonucleotide, or set of oligonucleotides,
30 containing the theoretical "most probable" sequence capable of
encoding the Hl3 fragment is used to identify the sequence of
a complementary oligonucleotide or set of oligonucleotides
which is capable of hybridizing to the "most probable"
sequance, or set of sequences. An oligonucleotide containing
35 such a complementary sequence can be employed as a probe to
identify and isolate the Hl3 genP (Sambrook et al., supra).
A suitable oligonucleotide, or set of
oligonucleotides, which is capable of encoding a fragment of
2097705
WO92~10506 PCT/US91/09382
the H13 gene (or which is complementary to such an
oligonucleotide, or set of oligonucleotides) is identified
(using the above-described procedure), synthesized, and
hybridized by means well known in the art, against a DNA or,
5 more preferably, a cDNA preparation derived from cells which
are capable of expressing the H13 gene. Single stranded
oligonucleotide molecules comple~entary to the "most probable"
H13 peptide coding sequences can be synthesized using
procedures which are well known to those of ordinary skill in
10 the art (Belagaje, R., et al., J. Biol. Chem. 254:5765-5780
(1979); Maniatis, T., et al., n: Molecular Mechanisms in the
Control of Gene Expression, Nierlich, D.P., et al., Eds.,
Acad. Press, NY (1976); Wu, R., et al., Proa. Nucl. Acid Res.
Molec. Biol. 21:101-141 tl978); Khorana, R.G., Science
15 203:614-625 (1979)). Additionally, DNA synthesis may be
achieved through the use of automated synthesizers.
Techniques of nucleic acid hybridization are disclosed by
Sambrook et al. (suDra), and by ~aymes, B.D., et al. (In:
Nucleic Acid Hybridization. A Practical Approach, IRL Press,
20 Washington, DC (1985)), which references are herein
incorporat~d by reference. Techniques such as, or similar to,
those described above have successfully enabled the cloning of
genes for human aldehyde dehydrogenases (Hsu, L.C., et al.,
Proc. Natl. Acad. Sci. USA 82:3771-3775 (198S)), fibronectin
25 (Suzuki, S., et al., Eur. Mol. Biol. oraan. J. 4:2519-2524
(1985)), the human estrogen receptor gene (Walter, P., et al.,
Proc. Natl. Acad. Sci. USA 82:7889-7893 (1985)), tissue-type
plasminogen activator (Pennica, D., et al., Nature 301:214-221
(1983)) and human term placental alkaline phosphatase
30 complementary DNA (Kam, W., et al., Proc. Natl. Acad. Sci.
USA 82:8715-8719 (1985)).
In an alternative way of cloning the H13 gene, a
library of expression vectors is prepared by cloning DNA or,
more preferably, cDNA (from a cell capable of expressing H13)
35 into an expression vector. The library is then screened for
members capable of expressing a protein which binds to anti-
H13 antibody, and which has a nucleotide sequence that is
capable of encoding polypeptides that have the same amino acid
WO92~10506 PCT/US91/09382
2 0977~5
- 38 -
sequence as H13 proteins or peptides, or fragments thereof.
In this embodiment, DNA, or more preferably cDNA, is extracted
and purified from a cell which is capable of expressing H13
protein. The purified cDNA is fragmentized (by shearing,
5 endonuclease digestion, etc.) to produce a pool of DNA or cDNA
fragments. DNA or cDNA fragments from this pool are then
cloned into an expression vector in order to produce a genomic
library of expression vectors whose members each contain a
unique cloned DNA or cDNA fragment.
By "vector" is meant a DNA molecule, derived from a
plasmid or bacteriophage, into which fragments of DNA may be
inserted or cloned. A vector will contain one or more unique
restriction sites, and may be capable of autonomous
replication in a defined host or vehicle organism such that
15 the cloned sequence is reproducible.
An "expression vector" is a vector which (due to the
presence of appropriate transcriptional and/or translational
control sequences) is capable of expressing a DNA (or cDNA)
molecule which has been cloned into the vector and of thereby
20 producing a polypeptide or protein. Expression of the cloned
sequences occurs when the expression vector is introduced into
an appropriate host cell. If a prokaryotic expression vector
is employed, then the appropriate host cell would be any
prokaryotic cell capable of expressing the cloned sequences.
25 Similarly, if a eukaryotic expression vector is employed, then
the appropriate host cell would be any eukaryotic cell capable
of expressing the cloned sequences. Importantly, since
eukaryotic D~A may contain intervening sequences, and since
such sequences cannot be correctly processed in prokaryotic
30 cells, it is preferable to employ cDNA from a cell which is
capable of expressing H13 in order to produce a prokaryotic
genomic expression vector library. Procedures for preparing
cDNA and for producing a genomic library are disclosed by
Sambrook et al. (supra).
By "substantially pure" is meant any protein or
peptide of the present invention, or any gene encoding any
such protein or peptide, which is essentially free of other
proteins or genes, respectively, or of other contaminants with
WO92/1050~ 2 ~ ~ 7 7 0 ~ PCT/US91/09382
- 39 -
which it might normally be found in nature, and as such exists
in a form not found in nature.
By "functional derivative" of a polynucleotide
molecule is meant a polynucleotide molecule encoding a
5 "fragment" "variant" or "analogue" of the H13 protein. Such a
functional derivative may be "substantially similar" in
nucleotide sequence to the H13-encoding sequence and thus
encode a protein possessing similar activity to the ~13
protein. Alternatively, a "functional derivative" of a
10 polynucleotide can be a chemical derivative which retains its
functions, such as the capability to express the protein, or
the ability to hybridize with a complementary polynucleotide
molecule. Such a chemical derivative is useful as a molecular
probe to detect H13 sequences through nucleic acid
15 hybridization assays.
A molecule is said to be "substantially similar" to
another molecule if the sequence of amino acids in both
molecules is substantially the same. Substantially similar
amino acid molecules will possess a similar ~iological
20 activity. Thus, provided that two molecules possess a similar
activity, they are considered variants as that term is used
herein even if one of the molecules contains additional amino
acid residues not found in the other, or if the sequence of
amino acid residues is not identical.
A DNA sequence encoding the H13 protein or a
chimeric ERR/H13 protein of the present invention, or a
functional derivative thereof, may be recombined with vector
DNA in accordance with conventional techniques, including
blunt-ended or staggered-ended termini for ligation,
30 restriction enzyme digestion to provide appropriate termini,
filling in of cohesive ends as appropriate, alkaline
phosphatase treatment to avoid undesirable joining, and
ligation with appropriate ligases. Techniques for such
manipulations are disclosed by Sambrook, J. et al., supra, and
35 are well known in the art.
A nucleic acid molecule, such as DNA, is said to be
"capable of expressing" a polypeptide if it contains
nucleotide sequences which contain transcriptional and
WO92/10506 ~ ~ 7 7 ~ ~ PCT/US91/09382
- 40 -
translational regulatory information and such sequences are
"operably linked~ to nucleotide sequences which encode the
polypeptide. An operable linkage is a linkage in which the
regulatory DNA sequences and the DNA sequence sought to be
5 expressed are connected in such a way as to permit gene
expression The precise nature of the regulatory regions
needed for gene expression may vary from organism to organism,
but shall in general include a promoter region which, in
prokaryotes, contains both the promoter (which directs the
10 initiation of RNA transcription) as well as the DNA sequences
which, when transcribed into RNA, will signal the initiation
of protein synthesis. Such regions will normally include
those 5'- non-coding sequences involved with initiation of
transcription and translation, such as the TATA box, capping
15 sequence, CAAT sequence, and the like.
If desired, the non-coding region 3' to the gene
sequence coding for the protein may ~e obtained by ~he above-
described methods. This region may be retained for its
transcriptional termination regulatory sequences, such as
20 termination and polyadenylation. Thus, by retaining the 3'-
region naturally contiguous to the DNA sequence coding for the
protein, the transcriptional termination signals may be
prcvided. Where the transcriptional termination signals are
not satisfactorily functional in the expression host cell,
25 then a 3' region functional in the host cell may be
substituted.
Two sequences of a nucleic acid molecule are said to
be "operably linked" when they are linked to each other in a
manner which either permits both sequences to be transcribed
30 onto the same RNA transcript, or permits an RNA transcript,
begun in one sequence to be extended into the second sequence.
Thus, two sequences, such as a promoter sequence and any other
"second" sequence of DNA or RNA are operably linked if
transcription commencing in the promoter sequence will produce
35 an RNA transcript of the operably linked second sequence. In
order to be "operably linked" it is not necessary that two
sequences be immediately adjacent to one another.
WO92/10506 2 0 9 7 ~ O ~ PCT/US91/09382
- 41 -
As used herein, a "promoter" is a region of a DNA or
RNA molecule which is capable of binding RNA polymerase and
promoting the transcription of an "operably linked" nucleic
acid sequence. A "promoter sequence" is the sequence of the
5 promoter which is found on that strand of the DNA or RNA which
is transcribed by the RNA polymerase. This functional
promoter will direct the transcription of a nucleic acid
molecule which is operably linked to that strand of the
double-stranded molecule which contains the "promoter
10 sequence."
Certain RNA polymerases exhibit a high specificity
for such promoters. The RNA polymerases of the bacteriophages
T7, T3, and SP-6 are especially well characterized, and
exhibit high promoter specificity. The promoter sequences
15 which are specific for each of these RNA polymerases also
direct the polymerase to utilize (i.e. transcribe) only one
strand of the two strands of a duplex DNA template. The
selection of which strand is transcribed is determined by the
orientation of the promoter sequence. This selection
20 determines the direction of transcription since RNA is only
polymerized enzymatically by the addition of a nucleotide 5'
phosphate to a 3' hydroxyl terminus. The sequences of such
polymerase recognition sequences are disclosed by Watson, J.D.
et ~1., supra). The promoter sequences of the present
25 invention may be either prokaryotic, eukaryotic or viral.
Suitable promoters are repressible, or, more preferably,
constitutive. Strong promoters are preferred.
The present invention encompasses the expression of
the H13 protein (or a functional derivative thereof) or a
30 chimeric H13 protein in either prokaryotic or eukaryotic
cells, although preferred expression is in eukaryotic cells,
expression is preferred, most preferably in human cells. To
express the chimeric protein of the present invention in a
prokaryotic cell (such as, for example, E. coli, B. subtilis,
35 Pseudomonas, Streptomyces, etc.), it is necessary to operably
link the H13 encoding sequence to a functional prokaryotic
promoter, examples of which are well-known in the art. Proper
expression in a prokaryotic cell also requires the presence of
W092/10506 ~ ~ 9 ~`~ rl ~ ~ PCT/USg1/09382
- 42 -
a ribosome binding site upstream of the gene-encoding sequence
(see, for example, Gold, L. et al. (Ann. Rev. Microbiol.
35:365-404 (1981)).
To express the H13 protein (or a functional
5 derivative thereof) or a chimeric H13 protein in a prokaryotic
cell (such as, for example, ~. coli, B. subtilis, Pseudomonas,
Streptomyces, etc.), it is necessary to operably link the H13
encoding sequence to a functional prokaryotic promoter.
Examples of constitutive promoters include the int promoter of
10 bacteriophage lambda, the kl~ promoter of the ~-lactamase gene
of pBR322, and the CAT promoter of the chloramphenicol acetyl
transferase gene of pBR325, etc.- Examples of inducible
prokaryotic promoters include the major right and left
promoters of bacteriophage 1 (PL and PR), the trp, recA,
15 lacZ, lacI, and aal promoters of E. coli, the ~-amylase
(Ulmanen, I., et al., ~. Bac~e~ol. 162:176-182 ~1985)) and
the s-28-specific promoters o~ B. subtilis (Gilman, M.Z., et
al., Gene 32:11-20 (1984)), the promoters of the
bacteriophages of Bac~llus (Gryczan, T.J., In: The Molecular
20 Biology of the Bacilli, Academic Press, Inc., NY (1982)), and
Stre~tomvces promoters (Ward, J.M., et al., Mol. Gen. Genet.
203:468-478 (1986)). Prokaryotic promoters are reviewed by
Glick, B.R., (J. Ind. Microbiol. 1:277-282 (1987));
Cenatiempo, Y. (Biochimie 68:505-516 ~1986)); and Gottesman,
25 S. (Ann. Rev. Genet. 18:415-442 (1984)).
Proper expression in a prokaryotic cell also
reguires the presence of a ribosome binding site upstream of
the gene- encoding sequence. Such ribosome binding sites are
disclosed, for example, by Gold, L., et al. (Ann. Rev.
30 Microbiol. 35:365-404 (1981)).
Eukaryotic hosts include yeast, insects, fungi, and
mammalian cells either in vivo, or in tissue culture.
Mammalian cells provide post-translational modifications to
protein molecules including correct folding or glycosylation
35 at correct sites. Mammalian cells which may be useful as
hosts include cells of fibroblast origin such as VER0 or CH0,
or cells of lymphoid origin, such as the hybridoma SP2/0-Agl4
or the murine myeloma P3-X63Ag8, and their derivatives.
W~92/]0~06 2~77a5 PCT/US91/09382
Preferred mammalian cells are cells which are intended to
replace the function of the genetically deficient cells in
vivo. Bone marrow s~em cells are preferred for gene therapy
of disorders of the hemopoietic or immune system.
For a mammalian cell host, many possible vector
systems are available for the expression of Hl3. A wide
variety of transcriptional a~d translational regulatory
sequences may be employed, depending upon the nature of the
host. The transcriptional and translational regulatory
lO signals may be derived from viral sources, such as adenovirus,
bovine papilloma virus, Simian virus, or the like, where the
regulatory signals are associated with a particular gene which
has a high level of expression. Alternatively, promoters from
mammalian expression products, such as actin, collagen,
15 myosin, etc., may be employed. Transcriptional initiation
regulatory signals may be selected which allow for repression
or activation, so that expression of the genes can be
modulated. Of interest are regulatory signals which are
temperature-sensitive so that by varying the temperature,
20 expression can be repressed or initiated, or are subject to
chemical regulation, e.q., metabolite.
For yeast host cells, any of a series of yeast gene
expression systems can be utilized which incorporate promoter
and termination elements from the actively expressed genes
25 coding for glycolytic enzymes produced in large guantities
when yeast are grown in glucose-rich medium. Known glycolytic
genes can also provide very efficient transcriptional control
signals. For example, the promoter and terminator signals of
the phosphoglycerate kinase gene can be utilized.
Production of Hl3 or chimeric Hl3 molecules in
insects can be achieved, for example, by infecting the insect
host with a baculovirus engineered to express Hl3 by methods
known to those of skill. Thus, in one embodiment, sequences
encoding Hl3 may be operably linked to the regulatory regions
35 of the viral polyhedrin protein (Jasny, Science 238: 1653
(1987)). Infected with the recombinant baculovirus, cultured
insect cells, or the live insects themselves, can produce the
Hl3 protein in amounts as great as 20 to 50~ of total protein
WO92/10506 PCT/US91/09382
~9770~
production. When live insects are to be used, caterpillars
are presently preferred hosts for large scale H13 production
according to the invention.
As discussed above, expression of the H13 protein in
5 eukaryotic hosts requires the use of eukaryotic regulatory
regions. Such regions will, in general, include a promoter
region sufficient to direct the initiation of RNA synthesis.
Preferred eukaryotic promoters include the promoter of the
mouse metallothionein I gene (Hamer, D., et al., J. Mol. A~
10 Gen. 1:273_288 (1982)); the TX promoter of Herpes virus
(McKnight, S., Cell 31:355-365 (1982)); the SV40 early
promoter (Benoist, C., et al., Nature rLondon) 290:304-310
(1981)); the yeast aal4 gene promoter (Johnston, S.A., et al.,
Proc._Natl. Acad. Sci. (USA) 79:6971-6975 (1982); Silver,
15 P.A., et al., Proc. Natl. ~cad. Sci. rUSA) 81:5951-5955
(1984)).
As is widely known, translation of eukaryotic mRNA
is initiated at the codon which encodes the first methionine.
For this reason, it is preferable to ensure that the linkage
20 between a eukaryotic promoter and a DNA sequence which encodes
the H13 or chimeric protein does not contain any intervening
codons which are capable of encoding a methionine (i.e., AUG).
The presence of such codons results either in a formation of a
fusion protein (if the AUG codon is in the same reading frame
25 as H13 encoding DNA sequence) or a frame-shift mutation (if
the AUG codon is not in the same reading frame as the H13
encoding sequence).
The H13 or chimeric receptor coding sequence and an
operably linked promoter may be introduced into a recipient
30 prokaryotic or eukaryotic cell either as a non-replicating DNA
(or RNA) molecule, which may either be a linear molecule or,
more preferably, a closed covalent circular molecule. Since
such molecules are incapable of autonomous replication, the
expression of the H13 protein may occur through the transient
35 expression of the introduced sequence. Alternatively,
permanent expression may occur through the integration of the
introduced sequence into the host chromosome.
W0~2/10506 PCT/US91/09382
2a~ ~05
- 45 -
In one embodiment, a vector is employed which is
capable of integrating the desired gene sequences into the
host cell chromosome. Cells which have stably integrated the
introduced DNA into their chromosomes can be selected by also
5 introducing one or more markers which allow for selection of
host cells which contain the expression vector. The marker
may provide for prototropy to an auxotrophic host, biocide
resistance, e.g., antibiotics, or heavy metals, such as copper
or the like. The selectable marker gene can either be
10 directly linked to the DNA gene sequences to be expressed, or
introduced lnto the same cell by co-transfection. Additional
elements may also be needed for optimal synthesis of single
chain binding protein mRNA. These elements may include splice
signals, as well as transcription promoters, enhancers, and
15 termination signals. cDNA expression vectors incorporating
such elements include those described by Okayama, H., Mol.
Cell. Biol. 3:280 (1983).
In another embodiment, the introduced sequence will
be incorporated into a plasmid or viral vector capable of
20 autonomous replication in the recipient host. Any of a wide
variety of vectors may be employed for this purpose.
Preferred eukaryotic plasmids include BPV, vaccinia, SV40, 2-
micron circle, etc., or their derivatives. Such plasmids are
well known in the art (Botstein, D., et al., iami Wntr. Sym~.
25 19:265-274 (1982); Broach, J.R., In: The Molecular Biology of
the Yeast Saccharomyces: Life_Cycle and Inheritance, Cold
Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470
(1981); Broach, J.R., Cell 28:203-204 (1982); Bollon, D.P., et
aL~, J. Clin. Hematol. Oncol. 10:39-48 (1980); Maniatis, T.,
30 In: Cell Biolooy: A Comprehensive Treatise Vol. 3, Gene
Expression, Academic Press, NY, pp. 563-608 (1980)).
Preferred eukaryotic plasmids include BPV, vaccinia,
SV40, 2-micron circle, etc., or their derivatives. Such
plasmids are well known in the art (Botstein, D., et al.,
35 Miami Wntr. Sym~. 19:265-274 (1982); Broach, J.R., In: The
Molecular Biology of the Yeast Saccharomvces: Life Cvcle and
Inheritance, Cold Spring Harbor Laboratory, Cold Spring
Harbor, NY, p. 445-470 (1981); Broach, J.R., Cell 28:203-20
WO92/10506 ~ V9 7 7 ~ ~ PCT/US91/09382
(1982); Bollon, D.P., et al., J. Clin. Hematol. Oncol. 10:39-
48 (1980); Maniatis, T., In: Cell Biology- A Comprehensive
Treatise. Vol. 3. Gene Ex~ression, Academic Press, NY, pp.
563-608 (1980)).
Preferred vectors for transient expression of the
H13 or chimeric receptor protein of the present invention in
CHO cells is the pSG5 or pCDM8 expression vector.
once the vector or DNA se~uence containing the
construct(s) has been prepared for expression, the vector or
10 DNA construct(s) may be introduced into an appropriate host
cell by any of a variety of suitable means, including such
biochemical means as transformation, transfection,
conjugation, protoplast fusion, calcium phosphate-
precipitation, and application with polycations such as
15 diethylaminoethyl (DEAE) dextran, and such mechanical means as
electroporation, direct microinjection, and microprojectile
bombardment (Johnston ~_~1., Science ?40(4858~: 1538 (1988)),
etc.
After the introduction of the vector, recipient
20 cells are grown in a selective medium, which selects for the
growth of vector-containing cells. Expression of the cloned
gene sequence(s) results in the production of H13 or the
chimeric H13 protein and its expression on the cell surface.
If so desired, the expressed H13 or chimeric protein
25 may be isolated and purified in accordance with conventional
conditions, such as extraction, precipitation, chromatography,
affinity chromatography, electrophoresis, or the like. For
example, the cells may be collected by centrifugation, or with
suitable buffers, lysed, and the protein isolated by column
30 chromatography, for example, on DEAE-cellulose,
phosphocellulose, polyribocytidylic acid-agarose,
hydroxyapatite or by electrophoresis or immunoprecipitation.
Alternatively, the chimeric proteins may be isolated by the
use of specific antibodies, such as an anti-H13 antibody that
35 still reacts with the protein containing ERR-derived amino
acid substitutions. Such antibodies may be obtained by well-
known methods.
WO92/10506 PCT/US91/09382
2~.~7705
- 47 -
Furthermore, manipulation of the genetic constructs
of the present invention allow the grafting of a particular
virus-binding domain onto the transmembrane and
intracytoplasmic portions of the H13 protein, or grafting the
5 retrovirus binding domain of ~13 onto the transmembrane and
intracytoplasmic portions of another molecule, resulting in
yet another type of chimeric molecule.
The present invention is also directed to a
transgenic non-human eukaryotic animal (preferably a rodent,
lO such as a mouse) the germ cells and somatic cells of which
contain genomic DNA according to the present invention which
codes for the H13 protein or a functional derivative thereof
capable as serving as a human retrovirus receptor. The H13
~NA is introduced into the animal to be made transgenic, or an
15 ancestor of the animal, at an embryonic stage, preferably the
one-cell, or fertilized oocyte, stage, and generally not later
than about the 8-cell stage. The term "transgene," as used
herein, means a gene which is incorporated into the genome of
the animal and is expressed in the animal, resulting in the
20 presence of protein in the transgenic animal.
There are several means by which such a gene can be
introduced into the genome of the animal embryo so as to be
chromosomally incorporated and expressed. One method is to
transfect the embryo with the gene as it occurs naturally, and
25 select transgenic animals in which the gene has integrated
into the chromosome at a locus which results in expression.
Other methods for ensuring expression involve modifying the
gene or its control sequences prior to introduction into the
embryo. One such method is to transfect the embryo with a
30 vector (see above) containing an already modified gene. Other
methods are to use a gene the transcription of which is under
the control of a inducible or constitutively acting promoter,
whether synthetic or of eukaryotic or viral origin, or to use
a gene activated by one or more base pair substitutions,
35 deletions, or additions (see above).
Introduction of the desired gene sequence at the
fertilized oocyte stage ensures that the transgene is present
in all of the germ cells and somatic cells of the transgenic
WO92/10506 ~ 7 ~ 5 PCT/US91/09382
- 48 -
animal and has the potential to be expressed in all such
cells. The presence of the transgene in the germ cells of the
transgenic "founder" animal in turn means that all its progeny
will carry the transgene in all of their germ cells and
5 somatic cells. Introduction of the transgene at a later
embryonic stage in a founder animal may result in limited
presence of the tr~nsgene in some somatic cell lineages of the
founder; however, all the progeny of this founder animal
that inherit the transgene conventionally, from the founder's
lO germ cells, will carry the transgene in all of their germ
cells and somatic cells.
Chimeric non-human mammals in which fewer than all
of the somatic and germ cells contain the Hl3 DNA of the
present invention, such as animals produced when fewer than
15 all of the cells of the morula are transfected in the process
of producing the transgenic ~ammal, are also intended to be
within the scope of the pre6ent invention.
The techniques described in Leder, U.S. Patent
4,736,866 (hereby incorporated by reference) for producing
20 transgenic non-human mammals may be used for the production of
the transgenic non-human mammal of the present invention. The
various techniques described in Palmiter, R. et al., Ann. Rev.
Genet. 20:465-99 (1986), the entire contents of which are
hereby incorporated by reference, may also be used.
The animals carrying the Hl3 gene can be used to
test compounds or other treatment modalities which may
prevent, suppress or cure a human retrovirus infection or a
disease resulting from such infection for those retroviruses
which infect the cells using the Hl3 molecule as a receptor.
30 These tests can be extremely sensitive because of the ability
to adjust the virus dose given to the transgenic animals of
this invention. Such animals will also serve as a model for
testing of diagnostic methods for the same human retrovirus
diseases. Such diseases include, but are not limited to AIDS,
35 HTLV-induced leukemia, and the like. Transgenic animals
according to the present invention can also be used as a
source of cells for cell culture.
W092/10506 2 0 9 7 ~ ~ ~ PCT/US91/09382
- 49 -
The transgenic animal model of the present invention
has numerous economic advantages over the "SCID mouse" model
(McCune, J.M et al., Science 241:1632-1639 (1988)) wherein it
is necessary to repopulate each individual mouse with the
5 appropriate cells of the human immune system at great cost.
This invention is also directed to an antibody
specific for an epitope of H13 protein. In additional
embodiments, the antibody of the present invention is used to
prevent or treat retrovirus infection, to detect the presence
10 of, or measure the quantity or concentration of, H13 protein
in a cell, or in a cell or tissue extract, or a biological
fluid.
The term "antibody" is meant to include polyclonal
antibodies, monoclonal antibodies (mAbs), chimeric antibodies,
15 and anti-idiotypic (anti-Id) antibodies.
An antibody is said to be "capable of binding" a
molecule if it is capable o~ specifically reacting with the
molecule to thereby bind ~he molecule to the antibody. The
term "epitope" is ~eant to refer to that portion of any
20 molecule capable of being bound by an antibody which can also
be recognized by that antibody. Epitopes or~"antigenic
determinants~' usually consist of chemically active surface
groupings of molecules such as amino acids or sugar side
chains and have specific three dimensional structural
25 characteristics as well as specifiG charge characteristics.
An "antigen" is a molecule or a portion of a
molecule capable of being bound by an antibody which is
additionally capable of inducing an animal to produce antibody
capable of binding to an epitope of that antigen. An antigen
30 may have one, or more than one epitope. The specific reaction
referred to above is meant to indicate that the antigen will
react, in a highly selective manner, with its corresponding
antibody and not with the multitude of other antibodies which
may be evoked by other antigens.
Polyclonal antibodies are heterogeneous populations
of antibody molecules derived from the sera of animals
immunized with an antigen.
W092/lOS06 ~ 0 9 7 r) ~ 5 PCT/US91/09382
- 5~ -
Monoclonal antibodies are a substantially
homogeneous population of antibodies to specific antigens.
MAbs may be obtained by methods known to those skilled in the
art. See, for example Kohler and Milstein, Nature 256:495-497
5 (1975~ and U.S. Patent No. 4,376,110. Such antibodies may be
of any immunoglobulin class including IgG, IgM, IgE, IgA, and
any subclass thereof. The hybridoma producing the mAbs of
this invention may be cultivated in vitro or in vivo.
Production of high titers of mAbs in vivo production makes
10 this the presently preferred method of production. Briefly,
cells from the individual hybridomas are injected
intraperitoneally into pristane-primed Balb/c mice to produce
ascites fluid containing high concentrations of the desired
mAbs. M~bs of isotype IgM or IgG may be purified from such
15 ascites fluids, or from culture supernatants, using column
chromatography methods well known to those of skill in the
art.
Chimeric antibodies are molecules different portions
of which are derived from different animal species, such as
20 those having a variable region derived from a murine mAb and a
human immunoglobulin constant region. Chimeric antibodies and
methods for their production are known in the art (see, for
example, Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-
6855 (1984); Neuberger et al., Na~e ~14:268-270 (1985); Sun
25 et al., Proc. Natl. Acad. Sci. USA 84:214-218 (1987); Better
et al., Science 240:1041- 1043 (1988); Better, M.D.
International Patent Publication WO 9107494, which references
are hereby incorporated by reference).
An anti-idiotypic (anti-Id) antibody is an antibody
30 which recognizes unique determinants generally associated with
the antigen-binding site of an antibody. An Id antibody can
be prepared by immunizing an animal of the same species and
genetic type (e.g. mouse strain) as the source of the mAb with
the mAb to which an anti-Id is being prepared. The immunized
35 animal will recognize and respond to the idiotypic
determinants of the immunizing antibody by producing an
antibody to these idiotypic determinants (the anti-Id
antibody).
W092/10506 2 0 9 7 ~ ~ 5 PCT/US91/09382
- 51 -
The anti-Id antibody may also be used as an
"immunogen" to induce an immune response in yet another
animal, producing a so-called anti-anti-Id antibody. The
anti-anti-Id may bear structural similarity to the original
5 mAb which induced the anti-Id. Thus, by using antibodies to
the idiotypic determinants of a mAb, it is possible to
identify other clones expressing antibodies of identical
specificity.
Accordingly, mAbs generated against the H13 protein
10 of the present invention may be used to induce anti-Id
antibodies in suitable animals, such as Balb/c mice. Spleen
cells from such immunized mice are used to produce anti-Id
hybridomas secreting anti-Id mAbs. Further, the anti-Id mAbs
can be coupled to a carrier such as keyhole limpet hemocyanin
15 (KLH) and used to immunize additional Balb/c mice. Sera from
these mice will contain anti-anti-Id antibodies that have the
binding properties of the original mAb specific for an H13
protein epitope.
The anti-Id mAbs thus have their own idiotypic
20 epitopes, or "idiotopes" structurally similar to the epitope
being evaluated, such as an epitope of the H13 protein.
The term "antibody" is also meant to include both
intact molecules as well as fragments thereof, such as, for
example, Fab and F(ab')2, which are capable of binding
25 antigen. Fab and F(ab')2 fragments lack the Fc fragment of
intact antibody, clear more rapidly from the circulation, and
may have less non-specific tissue binding than an intact
antibody (Wahl et al., J. Nucl. ~ed. 24:316-325 (1983)).
It will be appreciated that Fab and F(ab')2 and
30 other fragments of the antibodies useful in the present
invention may be used for the detection and quantitation of
H13 protein according to the methods disclosed herein for
intact antibody molecules. Such fragments are typically
produced by proteolytic cleavage, using enzymes such as papain
35 (to produce Fab fragments) or pepsin (to produce F(ab')2
fragments).
The antibodies, or fragments of antibodies, of the
present invention may be used to quantitatively or qualita-
WO92/10506 2 ~ 9 7 7 0 5 PCT/US91/09382
- 52 -
tively detect the presence of cells which express the Hl3
protein (or a chimeric receptor having an Hl3-derived epitope)
on their surface or intracellularly. This can be
accomplished by immunofluorescence techniques employing a
5 fluorescently labeled antibody (see below) coupled with light
microscopic, flow cytometric, or fluorimetric detection.
The antibodies of the present invention may be
employed histologically, as in immunofluorescence or
immunoelectron microscopy, for in situ detection of Hl3
lO protein. In situ detection may be accomplished by removing a
histological (cell or tissue) specimen from a subject and
providing the a labeled antibody of the present invention to
such a specimen. The antibody (or fragment) is preferably
provided by applying or by overlaying on the biological
15 sample. Through the use of such a procedure, it is possible
to determine not only the presence of the Hl3 protein but also
its distribution on the examined tissue. Using the present
invention, those of ordinary skill will readily perceive that
any of a wide variety of histological methods (such as
20 staining procedures) can be modified in order to achieve such
in situ detection.
Additionally, the antibody of the present invention
can be used to detect the presence of soluble Hl3 molecules in
a biological sample. Used in this manner, the antibody can
25 serve as a means to monitor the,presence and ~uantity of Hl3
proteins or derivatives used therapeutically in a subject to
prevent or treat human retrovirus infection.
Such immunoassays for Hl3 protein typically comprise
incubating a biological sample, such as a biological fluid, a
30 tissue extract, freshly harvested cells such as lymphocytes or
leucocytes, or cells which have been incubated in tissue
culture, in the presence of a detectably labeled antibody
capable of identifying Hl3 protein, and detecting the antibody
by any of a number of techniques well-known in the art.
The biological sample may be treated with a solid
phase support or carrier (which terms are used interchangeably
herein) such as nitrocellulose, or other solid support which
is capable of immobilizing cells, cell particles or soluble
WO92/10506 PCT/US91/09382
7 ~ ~
- 53 -
proteins. The support may then be washed with suitable
buffers followed by treatment with the detectably labeled H13-
specific antibody. The solid phase support may then be washed
with the buffer a second time to remove unbound antibody. The
5 amount of bound label on said solid support may then be
detected by conventional means.
By "solid phase support" or "carrier" is intended
any support capable of binding antigen or antibodies. Well-
known supports, or carriers, include glass, polystyrene,
10 polypropylene, polyethylene, dextran, nylon, amylases, natural
and modified celluloses, polyacrylamides, gabbros, and
magnetite. The nature of the carrier can be either soluble to
some extent or insoluble for the purposes of the present
invention. The support material may have virtually any
15 possible structural configuration so long as the coupled
molecule is capable of binding to an antigen or antibody.
Thus, the support configuration may be spherical, as in a
bead, or cylindrical, as in the inside surface of a test tube,
or the external surface of a rod. Alternatively, the surface
20 may be flat such as a sheet, test strip, etc. Those skilled
in the art will know many other suitable carriers for binding
antibody or antigen, or will be able to ascertain the same by
use of routine experimentation.
The binding activity of a given lot of anti-H13
25 antibody may be determined according to well known methods.
Those skilled in the art will be able to determine operative
and optimal assay conditions for each determination by
employing routine experimentation.
Other such steps as washing, stirring, shaking, fil-
30 tering and the like may be added to the assays as is customaryor necessary for the particular situation.
One of the ways in which the H13-specific antibody
can be detectably labeled is by linking the same to an enzyme
and use in an enzyme immunoassay (EIA). This enzyme, in turn,
35 when later exposed to an appropriate substrate, will react
with the substrate in such a manner as to produce a chemical
moiety which can be detected, for example, by
spectrophotometric, fluorimetric or by visual means. ~nzymes
W092/10506 2 0 9 ~ 7 0 S PCT/US91/09382
- 54 -
which can be used to detectably label the antibody include,
but are not limited to, malate dehydrogenase, staphylococcal
nuclease, delta-5-steroid isomerase, yeast alcohol
dehydrogenase, alpha-glycerophosphate dehydrogenase, triose
5 phosphate isomerase, horseradish peroxidase, alkaline
phosphatase, asparaginase, glucose oxidase, beta-
galactosidase, ribonuclease, urease, catalase, glucose-6-
phosphate dehydroqenase, glucoamylase and
acetylcholinesterase. The detection can be accomplished by
lO colorimetric methods which employ a chromogenic substrate for
the enzyme. Detection may also be accomplished by visual
comparison of the extent of enzy~atic reaction of a substra~e
in comparison with similarly prepared standards.
Detection may be accomplished using any of a variety
15 of other immunoassays. For example, by radioactively labeling
the antibodies or antibody fragments, it is possible to detect
Hl3 protein through the use of a radioimmunoassay (RIA). A
good description of RIA may be found in Labo~atory Techniques
and Biochemistry in Molecular BioloqY, by Work, T.S., et al.,
20 North Holland Publishing Company, New York (1978) with
particular reference to the chapter entitled "An Introduction
to Radioimmune Assay and Related Techniques" by T. Chard,
incorporated by reference herein. The radioactive isotope can
be detected by such means as the use of a gamma counter or a
25 liquid scintillation counter or by autoradiography.
It is also possible to label the antibody with a
fluorescent compound. When the fluorescently labeled antibody
is exposed to light of the proper wave length, its presence
can then be detected due to fluorescence. Among the most
30 commonly used fluorescent labelling compounds are fluorescein
isothiocyanate, rhodamine, phycoerythrin, phycocyanin,
allophycocyanin, o- phthaldehyde and fluorescamine.
The antibody can also be detectably labeled using
fluorescence emitting metals such as l52Eu, or others of the
35 lanthanide series. These metals can be attached to the
antibody using such metal chelating groups as
diethylenetriaminepentaacetic acid (DTPA) or
ethylenediaminetetraacetic acid (EDTA).
W O 92/10506 2 ~ 3 7 7 o 5 P ~ /US91/09382
- 55 -
The antibody also can be detectahly labeled by
coupling it to a chemiluminescent compound. The presence of
the chemiluminescent-tagged antibody is then determlned by
detecting the presence of luminescence that arises during the
5 course of a chemical reaction. Examples of particularly
useful chemiluminescent labeling compounds are luminol,
isoluminol, theromatic acridinium ester, imidazole, acridinium
salt and oxalate ester.
Likewise, a bioluminescent compound may be used to
10 label the antibody of the present invention. Bioluminescence
is a type of chemiluminescence found in biological systems in
which a catalytic protein increases the efficiency of the
chemiluminescent reaction. The presence of a bioluminescent
protein is determined by detecting the presence of
15 luminescence. Important bioluminescent compounds for purposes
of labeling are luciferin, luciferase and aequorin.
The antibody molecules of the present invention may
be adapted for utilization in an immunometric assay, also
known as a "two-site" or "sandwich" assay. In a typical
20 immunometric assay, a quantity of unlabeled antibody (or
fragment of antibody) is bound to a solid support and a
quantity of detectably labeled soluble antibody is added to
permit detection and/or quantitation of the ternary complex
formed between solid-phase antibody, antigen, and labeled
25 antibody.
Typical, and preferred, immunometric assays include
"forward" assays in which the antibody bound to the solid
phase is first contacted with the sample being tested to
"extract" the antigen from the sample by formation of a binary
30 solid phase antibody-antigen complex. After a suitable
incubation period, the solid support is washed to remove the
residue of the fluid sample, including unreacted antigen, if
any, and then contacted with the solution containing an
unknown quantity of labeled antibody (which functions as a
35 "reporter molecule"). After a second incubation period to
permit the labeled antibody to complex with the antigen bound
to the solid support through the unlabeled antibody, the solid
WO92/10506 PCT/US91/09382
2 ~ 7 0 ~
- 56 -
support is washed a second time to remove the unreacted
labeled antibody.
In another type of "sandwich" assay, which may also
be useful with the antigens of the present invention, the so-
5 called "simultaneous" and "reverse" assays are used. Asimultaneous assay involves a single incubation step as the
antibody bound to the solid support and labeled antibody are
both added to the sample being tested at the same time. After
the incubation is completed, the solid support is washed to
10 remove the residue of fluid sample and uncomplexed labeled
antibody. The presence of labeled antibody associated with
the solid support is then determined as it would be in a
conventional "forward" sandwich assay.
In the "reverse" assay, stepwise addition first of a
15 solution of labeled antibody to the fluid sample followed by
the addition of unlabeled antibody bound to a solid support
after a suitable incubation period is utilized. After a
se~ond incubation, the solid phase is washed in conventional
fashion to free it of the residue of the sample being tested
20 and the solution of unreacted labeled antibody. The
determination of labeled antibody associated with a solid
support is then determined as in the "simultaneous" and
"forward" assays.
According to the present invention, it is possible
25 to diagnose circulating antibodies in a subject which are
specific for the H13 protein. This is accomplished by means
of an immunoassayl as described above, using the protein of
the invention or a functional derivative thereof.
Based on similar principles, since a retrovirus
30 binds to its cellular receptor with detectable affinity, it is
possible to detect the presence of a human retrovirus capable
of binding to H13 in a biological sample, using the H13
protein or a functional derivative thereof as a ligand. In
such an assay, the protein or functional derivative may be
35 bound to an insoluble support or carrier, as in an
immunoassay. The biological sample, e.g. serum, suspected of
having a retrovirus is then contacted with the H13-containing
support and the virus allowed to bind to its receptor
W092/10506 2 0 ~ 7 7 ~ ~ PCT/US91/09382
- 57 -
material. The presence of the bound virus is then revealed in
any of a number of ways well known in the art, for example, by
addition of a detectably-labelled antibody specific for the
virus. The same assay can be used to detect the presence in a
5 biological sample of a viral component such as a viral
protein or ~lycoprotein which has affinity for the H13
protein. Alternatively, the virus or viral protein may
be labelled and binding measured in a competitive assay using
an antibody specific for the virus-binding portion of the H13
lO molecule.
As used herein, the term "prevention" of infection
involves administration of the Hl3 protein, peptide
derivative, or antibody ~see above) prior to the clinical
onset of the disease. Thus, for example, successful
15 ad~inistration of a composition prior to initial contact with
a retrovirus results in "prevention" of the disease.
Administration may be after initial contact with the virus,
but prior to actual development of the disease.
"Treatment" involves administration of the
20 protective composition after the clinical onset of the
disease. For example, successful administration of a Hl3
- protein or peptide or anti-~13 antibody according to the
; invention after development of a retrovirus infection in order
to delay or suppress further virus spread comprises
25 "treatment" of the disease.
The H13 protein, peptides or antibody of the present
invention may be administered by any means that achieve their
intended purpose, for example, to treat local infection or to
treat systemic infection in a subject who has, or is
30 susceptible to, such infection. For example, an
immunosuppressed individual is particularly susceptible to
retroviral infection and disease.
For example, administration may be by various
parenteral routes such as subcutaneous, intravenous,
~5 intradermal, intramuscular, intraperitoneal, intranasal,
intracranial, transdermal, or buccal routes. Alternatively,
or concurrently, administration may be by the oral route.
W092/10506 PCT/US91/09382
~ o 9 r~ 7 o ~i
- 58 -
Parenteral administration can be by bolus injection or by
gradual perfusion over time.
An additional mode of using the compositions of the
present invention is by topical application. This route of
5 administration is particularly important in treating some
types of retrovirus infections. The proteins, peptides and
pharmaceutical compositions of the present invention may be
incorporated into topically applied vehicles such as salves or
ointments, which have both a soothing effect on the skin as
lO well as a means for administering the active ingredient
directly to the affected area.
The carrier for the active ingredient may be either
in sprayable or nonsprayable form. Non-sprayable forms can be
semi-solid or solid forms comprising a carrier conducive to
15 topical application and having a dynamic viscosity preferably
greater than that of water. Suitable formulations include,
but are not limited to, solution, suspensions, emulsions,
creams, ointments, powders, liniments, salves, and the like.
If desired, these may be sterilized or mixed with auxiliary
20 agents, e.g., preservatives, stabilizers, wetting agents,
buffers, or salts for influencing osmotic pressure and the
like. Preferred vehicles f~r non-sprayable topical
preparations include ointment bases, e.g., polyethylene
glycol-lOOO (PEG-lOOO); conventional creams such as HEB cream;
25 gels; as well as petroleum jelly and the like.
Also suitable for systemic or topical application,
in particular to the mucus membranes and lungs, are sprayable
aerosol preparations wherein the active ingredient, preferably
in combination with a solid or liquid inert carrier material.
30 The aerosol preparations can contain solvents, buffers,
surfactants, perfumes, and.or antioxidants in addition to the
proteins or peptides of the present invention. For aerosol
administration, the active principles in accordance with the
present invention may be packaged in a squeeze bottle, or in a
35 pressurized container with an appropriate system of valves and
actuators. Preferably, metered valves are used with the valve
chamber being recharged between actuation or dose, all as is
well known in the art.
WO92/10506 2 ~ 9 7 7 0 ~ PCT/US91/09382
- 59 -
For topical applications, it is preferred to
administer an effective amount of a compound according to the
present invention to an infected area, e.g., skin surfaces,
mucous membranes, etc. This amount will generally range from
5 about O.OOl mg to about l g per application, depending upon
the area to be treated, whether the use is prophylactic or
therapeutic, the severity of the symptoms, and the nature of
the topical vehicle employed. A preferred topical preparation
is an ointment wherein about O.Ol to about 50 mg of active
lO ingredient is used per cc of ointment base, the latter being
preferably PEG-lOO0.
A typical regimen for preventing, suppressing, or
treating retrovirus infection comprises administration of an
effective amount of the Hl3 protein or functional derivative
15 thereof, administered over a period of one or several days, up
to and including between one week and about six months.
It is understood that the dosage administered n
vivo or in vitro will be dependent upon the age, sex, health,
and weight of the recipient, kind of concurrent treatment, if
20 any, frequency of treatment, and the nature of the effect
desired. The ranges of effective doses provided below are not
intended to be limiting and represent preferred dose ranges.
However, the most preferred dosage will be tailored to the
individual subject, as is understood and determinable by one
25 of skill in the art.
~ he total dose required for each treatment may be
administered by multiple doses or in a single dose. The
protein, functional derivative thereof or antibody may be
administered alone or in conjunction with other therapeutics
30 directed to the viral infection, or directed to other symptoms
of the viral disease.
Effective amounts of the Hl3 protein, functional
derivative thereof, or antibody thereto, are from about O.Ol
~g to about lO0 mg/kg body weight, and preferably from about
35 lO ~g to about 50 mg/kg body weight.
In one embodiment, the peptides of the present
invention are provided to expectant mothers suspected of
having a retrovirus infection, by either systemic or
WO92/10506 PCT/US91/09382
~ ~Y ~7~60
intrauterine administration. This treatment is designed to
protect the fetus ~rom spread of HIV, for example.
Alternatively, the compositions of the invention can be used
intravaginally, especially during the birth process, to
5 protect the newborn from infectious retrovirus which may be
present in the birth canal.
Preparations for parenteral administration include
sterile aqueous or non-aqueous solutions, suspensions, and
emulsions, which may contain auxiliary agents or excipients
lO which are known in the art. Pharmaceutical compositions such
as tablets and capsules can also be prepared according to
routine methods.
Pharmaceutical compositions comprising the
proteins, peptides or antibodies of the inventioninclude all
15 compositions wherein the protein, peptide or antibody is
contained in an amount effective to achieve its intended
purpose. In addition, the pharmaceutical compositions may
contain suitable pharmaceutically acceptable carriers
comprising excipients and auxiliaries which facilitate
20 processing of the active compounds into preparations which can
be used pharmaceutically.
Pharmaceutical compositions include suitable
solutions for administration by injection or orally, and
contain from about O.Ol to 99 percent, preferably from about
25 20 to 75 percent of active component (i.e., the Hl3 protein or
antibody) together with the excipient. Pharmaceutical
compositions for oral administration include tablets and
capsules. Compositions which can be administered rectally
include suppositories.
The present invention provides methods for
evaluating the presence and the level of normal or mutant Hl3
protein or mRNA in a subject. Absence, or more typically, low
expression of the Hl3 gene or presence of a mutant Hl3 in an
individual may serve as an important predictor of resistance
35 to retrovirus infection and thus to the development of AIDS or
certain types of leukemia or other retrovirus-mediated
diseases. Alternatively, over-expression of Hl3, may serve
WO 92tlO506 2 0 9 7 7 0 5 Pcr/~ls91/09382
-- 61 --
as an important predictor of enhanced susceptibility to
retrovirus infection.
In addition, ERR or H13 mRNA expression is increased
in virally-induced tumor cell lines, indicating that the level
5 of mRNA or receptor protein expression may serve as a useful
indicator of a viral infection not otherwise detectable.
Therefore, by providing a means to measure the quantity of H13
mRNA (see below) or protein (using an immunoassay as described
above), the present invention provides a means for detecting a
10 human retrovirus-infected or retrovirus-transformed cell in a
subject.
Oligonucleotide probes encoding various portions of
the H13 DNA sequence are used to test cells from a subject for
the presence H13 DNA or mRNA. A preferred probe would be one
15 directed to the nucleic acid sequence encoding at least 12 and
preferably at least 15 nucleotides of the H13 sequence.
Qualitative or quantitative assays can be performed using such
probes. For example, Northern analysis (see below) is used to
measure expression of an H13 mRNA in a cell or tissue
20 preparation.
Such methods can be used even with very small
amounts of DNA obtained from an individual, following use of
selective amplification techniques. Recom`binant DNA
methodologies capable of amplifying purified nucleic acid
25 fragmen~s have long been recognized. Typically, such
methodologies involve the introduction of the nucleic acid
fragment into a DNA or RNA vector, the clonal amplification of
the vector, and the recovery of the amplified nucleic acid
fragment. Examples of such methodologies are provided by
30 Cohen et al. tU.S. Patent 4,237,224), Sambrook et al. (supra),
etc.
Recently, an in vitro enzymatic method has been de-
scribed which is capable of increasing the concentration of
such desired nucleic acid molecules. This method has been
35 referred to as the "polymerase chain reaction" or "PCR"
(Mullis, K. et al., Cold Sprin~ Harbor S~mp. Ouant. Biol.
51:263-273 (1986); Erlich H. et al., EP 50,424; EP 84,796, EP
258,017, EP 237,362, Mullis, K., EP 201,184; Mullis K. et al.,
WO92/10506 PCT/US91/09382
~09770.~
- 62 -
US 4,683,202; Erlich, H., US 4,582,788; and Saiki, R. et al.,
US 4,683,194).
The polymerase chain reaction provides a method for
selectively increasing the concentration of a particular
5 nucleic acid sequence even when that sequence has not been
previously purified and is present only in a single copy in a
particular sample. The method can be used to amplify either
single- or double-stranded DNA. The essence of the method
involves the use of two oligonucleotide probes to serve as
10 primers for the template-dependent, polymerase mediated
replication of a desired nucleic acid molecule.
The precise nature of the two oligonucleotide probes
of the PCR method is critical to the success of the method.
As is well known, a molecule of DNA or RNA possesses
15 directionality, which is conferred through the 5'-3' linkage
of the phosphate groups of the molecule. Sequences of DNA or
RNA are linked together through the formation of a
phosphodiester bond between the terminal 5' phosphate group of
one seguence and the terminal 3' hydroxyl group of a second
20 sequence. Poly~erase dependent amplification of a nucleic acid
molecule proceeds by the addition of a 5' nucleotide
triphosphate to the 3' hydroxyl end of a nucleic acid
molecule. Thus, the action of a p~lymerase extends the 3' end
of a nucleic acid molecule. These inherent properties are
25 exploited in the selection of the oligonucleotide probes of
the PCR. The oligonucleotide sequences of the probes of the
PCR method are selected such that they contain sequences
identical to, or complementary to, sequences which flank the
particular nucleic acid sequence whose amplification is
30 desired.
More specifically, the oligonucleotide sequences of
the "first" probe is selected such that it is capable of
hybridizing to an oligonucleotide sequence located 3' to the
desired sequence, whereas the oligonucleotide sequence of the
35 "second" probe is selected such that it contains an
oligonucleotide sequence identical to one present 5' to the
desired region. Both probes possess 3' hydroxy groups, and
therefore can serve as primers for nucleic acid synthesis.
WO92/10506 2 a ~ 7 7 0 ~ PCT/US91/09382
- 63 -
In the PCR, the reaction conditions are cycled
between those conducive to hybridization and nucleic acid
polymerization, and those which result in the denaturation of
duplex molecules. In the first step of the reaction, the
5 nucleic acids of the sample are transiently heated, and then
cooled, in order to denature any double-stranded molecules
which may be present. The "first" and "second" probes are
then added to the sample at a concentration which greatly
exceeds that of the desired nucleic acid molecule. When the
lO sample is incubated under conditions conducive to
hybridization and polymerization, the "first" probe will
hybridize to the nucleic acid molecule of the sample at a
position 3' to the sequence to be a~plified. If the nucleic
acid molecule of the sample was initially double-stranded,
15 the "second" probe will hybridize to the compl~mentary strand
of the nucleic acid molecule at a position 3' to the seguence
which is the complement of the sequence whose amplification is
desired. Upon addition of a polymerase, the 3' ends of the
"first" and (if the nucleic acid molecule was double-stranded)
2~ "second" probes will be extended. The extension of the
"first" probe will result in the synthesis of an
oligonucleotide having the exact sequence of the desired
nucleic acid. Extension of the "second" probe will result in
the synthesis of an oligonucleotide having the exact sequence
25 of the complement of the desired nucleic acid.
The PCR reaction is capable of exponential
amplification of specific nucleic acid sequences because the
extension product of the "first" probe, of necessity, contains
a sequence which is complementary to a sequence of the
30 "second" probe, and thus can serve as a template for the
production of an extension product of the "second" probe.
Similarly, the extension product of the "second" probe, of
necessity, contains a sequence which is complementary to a
sequence of the l'first" probe, and thus can serve as a
35 template for the production of an extension product of the
"first" probe. Thus, by permitting cycles of polymerization,
and denaturation, a geometric increase in the concentration of
the desired nucleic acid molecule can be achieved. Reviews of
W092/10506 ~ 0~,~ 7 PCT/US91/09382
- 64 -
the PCR are provided by Mullis, K.B. (Cold Sprlnq Harbor SYm~.
Ouant. 8iol. 51:263-273 (1986)); Saiki, R.K., et al.
(Bio/Technoloay 3:1008-1012 (1985)); and Mullis, K.B., et al.
(Meth. Enzvmol. 155:335-350 (1987)).
Having now generally described the invention, the
same will be more readily understood through reference to the
following example which is provided by way of illustration,
and is not intended to be limiting of the present invention,
unless specified.
EXAMPLE I
General Mate~als and Methods
Cell Lines
The following cell lines were used in the studies
described below: CCL120 (ATCC~ CCL120), a human B
15 lymphoblastoid cell line; CCLll9 (CEM, ATCC# CCLll9), a human
T lymphoblastoid cell line; SupTl, a human non-Hodgkin's T
lymphoma cell line; H9, a single cell clone derived from
HUT78,a human cutaneous T cell lymphoma cell line; MOLT4
(ATCC# CRL1582), a human acute lymphoblastic leukemia cell
20 line; HOS (ATCC# CRL1543), a huma~ osteosarcoma cell line;
HeLa (ATCC# CCL2), a human epithelioid carcinoma cell line;
CHO-Kl (ATCC #61~, a Chinese hamster ovary cell line; BlOT6R,
a radiation-induced thymoma of BlO.T(6R) mice; and RL12, a
radiation-induced thymoma of C57BL/6Ka mice.
25 Screeninq
Human CEM and HUT 78 T-cell cDNA library (lambda
gtll~ was obtained from Clontech Laboratories Inc. (Palo Alto,
California). The human lymphocyte cosmid library (pWE15) was
obtained from Stratagene (LaJolla, CA). The libraries were
30 screened by the method of Maniatis et al. (Maniatis, T. et al.
Cell 15:887_701 (1978)). The BamHl-EcoRI fragment, containing
the entire open reading frame of ERR cDNA (pJET) was provided
by Drs. Albritton and Cunningham (Harvard Medical School,
Boston, MA). This DNA was labelled with 32p by nick
35 translation to a specific activity of about 2 x 106 cpm/~g and
used as a hybridization probe.
WO92/10~06 2 0 9 7 7 0 ~ PCT/US91/09382
- 65 -
Southern Blot Analvsis
High relative mass DNA was prepared from cells as
described by Blin, N. et al. (Nucl. Acids Res. 3:2303-2308
(1976)) and modified by Pampeno and Meruelo (Pampeno, C. L. et
5 al. J. Virol. 58:296-306 (1986)). Restriction endonuclease
digestion, agarose gel electrophoresis, transfer to
nitrocellulose (Schleicher & Schuell, lnc., Reene, New
Hampshire), hybridization and washing was as described
(Pampeno, C. L. et al. supra; Brown, G. D. et al.
10 Immunogenetics 27:239-251 (1988)).
Northern Blot Analysis
Total cellular RNA was isolated from cells by the
acid guanidinium thiocyanate-phenol-ChlorofQrm method
(Chomczynski, P. et al. Anal. Biochem. 162:156-159 (1987)).
15 The DNA was electrophoresed in 1% formaldehyde agarose gels
and transferred to Nytran filters (Schleicher & Schuell, Inc.,
Keene, New Hampshire). The hybridization and washing was
performed according to Amari, N. M. B. et al. (Mol. Cell.
Biol. 7:4159-4168 (1987)).
20 DNA Sequence Analysis
cDNA clones from positive phages were recloned into
the EcoRI site of plasmid vector pBluescript (Stratagene).
Unidirectional deletions of the plasmids were constructed by
using exonuclease III and Sl nuclease, and sequenced by the
25 dideoxy chain termination methods (Sanger, F. S. et al. Proc.
Natl. Acad. Sci. USA 74:5463- 5467 (1977)) with Sequenase
reagents (U.S. Biochemical Corp., Cleveland, Ohio).
Restriction maps of positive cosmid inserts were determined
using T3 or T7 promoter-specific oligonucleotides to probe
30 partially digested cosmid DNA as described elsewhere (Evans,
G.A. et al., Meth. Enzymol. 152:604-610 (1987)). EcoRI-EcoRI
or EcoRI-HindIII fragments in the cosmids were subcloned into
pBluescript or pSport 1 (GIBCO BRL, Gaithersburg, MD). The
exons and exon-intron junctions were sequenced using synthetic
35 oligonucleotides as primers. Sequences were compiled and
analyzed using the Genetics computer group sequence analysis
software package (Devereux, J. et al., Nucl. Acids Res.
12:387-395 (1984)).
W092/lOS06 'JO 9 7 7 o 5 PCT/US91/09382
- 66 -
EXAMPLE II
DNA and Predicted Protein Seouence of H13
The complete nucleotide sequence of H13 (SEQ ID
NO:7) including non-coding sequences at the 5' and 3' end of
5 the coding sequence are shown in Figure 1. This sequence
includes the partial sequence originally obtained from clone
7-2 (SEQ ID NO:l); nucleotides 1-6 and 1099-1102 of SEQ ID
NO:1 were originally incorrectly determined. Figure 1 also
shows the complete amino acid sequence predicted from the
10 nucleotide sequence (SEQ ID NO:8). This sequence includes the
originally described partial amino acid sequence (SEQ ID NO:2)
with the exception of the N-terminal Pro-Gly and the C-
terminal Pro, which were originally incorrectly predicted from
the nucleotide sequence.
The nucleotide sequence comparison between H13, ERR
and TEA is shown in Figure 2 and the amino acid sequence
comparison is shown in Figure 3.
The homology between the compared sequences is very
high, for example 87.6% homology between H13 and ERR DNA, and
20 52.3~ homology between H13 and TEA amino acids.
EXa~PlE I~
Presence and Expression of_~he ~13 Gene in Human Cells
By Southern analysis of DNA taken from cells of
various species, it was shown that DNA capable of hybridizing
5 with a murine ERR cDNA probe (Figure 4) and with the H13 cDNA
(Figure 5) was present in cells of 5 human cell lines,
including CCL120, CCL119, SupTl, H-9 and MOLT-4, and also in
hamster cells (CHO-Kl) and murine cells (normal Balb/c mouse
thymocytes). H13 gene expression was examined using Northern
10 analysis, using the H13 cDNA probe. The probe detected a
transcript of approximately 9kb in RNA from HeLa, SupTl, HOS
and CCL119 cells (Figure 6). This RNA could also be detected
using a murine ERR cDNA probe (Figure 7).
WO92/10506 2 0 9 7 7 0 5 PCT/US91/09382
- 67 -
~XA~PLE IV
Transfection of Mu~ine Retroviral Rece~tor
cDNA into Hamster Cells
Murine retroviral receptor (ERR) cDNA was
5 cotransfected into hamster CHO cells, which can not be
infected by murine ecotropic retroviruses, with the selectable
marker plasmid DNAP, pSV2Neo, using calcium phosphate (Wigler,
M. et al., Cell 14: 725-731 (1978)). The transfectant
expressing the receptor gene was, then, infected by murine
10 radiation leukemia virus (RadLV). Two weeks later after the
infection the reverse transcriptase (RT) activity of the
supernatant was measured (Stephenson, J.R. et al., Virologv
48: 749-756 (1972)), and Northern Blot analysis was performed
using a viral probe after preparing its RNA. As shown in
15 Figure 8, the RT activity detected in untransfected CHO cells
which do not express the receptor gene was indistinguishable
from the activity of tissue culture medium (background). This
indicates that the cells were not infected by MuLV.
Following transfection with the ERR cDNA, the RT
20 activity of the transfected cell supernatant was much higher
than background (Figure 8).
The ~uLV viral probe detected transcripts in RNA
prepared from the transfectant, but not in RNA prepared from
untransfected CHO cells. The results indicate that the cells
25 transfected with the ERR cDNA can acquire the susceptibility
to ecotropic murine leukemia virus.
EXAMPLE V
Preparation and Use of Antibodies to H-13
It is very difficult to make an H-13-containing
30 fusion protein having the whole predicted protein (SEQ ID
NO:2) since the predicted protein is highly hydrophobic, as
shown in Figure 9. In order to predict antigenic epitopes
present in the protein, therefore, the computer analysis was
carried out using the program of PEPTIDESTRUCTURE (Jameson et
35 al., CABIOS 4: 181-186 (1988)). Figure 10 shows the
antigenicity profile of the H-13 protein sequence.
W092/10506 2 ~ 9 7 7 ~ S PCT/US91/09382
- 68 -
The DNA sequence encoding a highly antigenic portion
(SEQ ID NO:2, amino acid residues 309-367) was prepared by
cutting with the restriction enzymes AccI and EcoRI yielding a
180 bp AccI-EcoRI fragment. This fragment of H13 cDNA was
5 ligated to the cloning sites of pGEX-2T plasmid vector
(Pharmacia LXB Biotechnology), which can express antigens as
fusion proteins with glutathione-S-transferase (GST), in the
orientation that permit[s] the expression of the open reading
frames (Smith, D.B. et al., Gene 67: 31-40 (1988)).
The fusion protein was induced by addition of
isopropyl-beta-thiogalactopyranoside (IPTG) to cultures, and
was purified using glu~athione Sepharose 4B chromatography
(Pharmacia LKB Biotechnology) (see Figure 11). The purified
fusion protein injected intramuscularly and subcutaneously
15 into rabbits with Freund's complete adjuvant to obtain
antisera.
The antisera are shown to bind specifically to the
H-13 protein and epitopic fragments thereof.
Membrane proteins from human cells are prepared
20 according to standard technigues and are separated by
polyacrylamide gel electrophoresis, an blotted onto
nitrocellulose for Western Blot analysis. The H-13 specific
antibodies are shown to bind to proteins on these blots.
EX~LE Vl
Genetic ~ap~in~ of H13
Chromosomal location of the H13 gene was determined
using Chromosome Blots (Bios Corp., New Haven, Connecticut)
containing DNA from a panel of human-hamster somatic cell
hybrids (Kouri, R. E. et al., Cytoqenet. Cell Genet. 51:1025
30 (1989)). By comparison of which human chromosomes remained in
the human-hamster hybrid cell and the expression of H13 cDNA,
the H13 gene was mapped to human chromosome 13 (see Figure
12). Human genes (or diseases caused by mutations therein )
linked to chromosome 13 include: retinoblastoma,
35 osteosarcoma, Wilson's disease, Letterer-Siwe disease, Dubin-
Johnson syndrome, clotting factor Vii and X, collagen IV ~1
and ~2 chains, X-ray sensitivity, lymphocyte cytosolic
WO92/10506 2 0 9 ~ 7 0 5 PCT/US91/09382
- 69 -
protein-l, carotid body tumor-l, propionyl CoA carboxylase (~
subunit), etc.
EXAMPL~ VII
Chimeric H13/ERR DNA and Protein Molecules
Several chimeric molecules between the mouse ERR
sequence and the human H13 sequence were produced, and have
been designated ChimeraI - ChimeraIV). Specifically, four
regions in H13 cDNA were substituted based on the use of
common restriction sites as shown in Figure 13.
These DNA sequences were transiently transfected
into Chinese hamster ovary tCHO) cell lines using pSG5 or
pCDM8 expression vectors.
Two days later, these transfectants were tested for
their ability to support E-MuLV infection. Cells were
15 infected with a recombinant Moloney E-MuLV designated 2BAG
(Price, J. et al., Proc. Natl. Acad. Sci. USA 84:156-160
(1987)). This recombinant virus also contained ~-
galactosidase and neomycin phosphotransferase (neoR) genes
~; which provide a selectable marker and a detectable product.
20 The cells were then grown under selective conditions in the
presence of the antibiotic G418 at a concentration of 0.6
mg/ml to select n~_R-expressing transfectants. After two
weeks, numbers of G418-resistant colonies were counted.
These results indicate that portion of the ERR gene
25 essential for E-MuLV infection is located within NcoI-BstXI
restriction sites, and included extracellular Domain 3.
Extracellular Domain 3 (as shown in the upper line of Figure
13) is the region of the receptor protein which is most
diverse between the human and mouse sequences, as shown in
30 Figure 14. The sequences in Figure 14 (derived from the
sequences shown in Figure 1-3) were aligned using Genetics
computer group sequence analysis software package (Devereux,
J. et al., Nucl. Acids Res. 12:387-395 (1984)).
Next, oligonucleotide-directed mutagenesis was
35 employed to produce chimeric molecules containing individual
amino acid substitutions within extracellular domain 3. These
were transfected as a~ove and the transfectant cells are
W O 92/~0506 PC~r/US91/09382 20~7705
- 70 -
tested for susceptibility to infection by E-MuLV as shown
above.
The results of the above studies show that the human
H13 molecule acquires ability to bind to E-MuLV by
5 substituting the native amino acid sequence with between 1 and
4 amino acids from corresponding positions in the murine ERR
protein.
Having now fully described this invention, it will
be appreciated by those skilled in the art that the same can
10 be performed within a wide range of equivalent parameters,
concentrations, and conditions without departing from the
spirit and scope of the invention and without undue
experimentation.
While this invention has been described in
15 connection with specific embodiments thereof, it will be
understood that it is capable of further modifications. This
application is intended to cover any variations, uses, or
adaptations of the inventions following, in general, the
principles of the invention and including such departures from
20 the present di-~closure as co~e within known or customary
practice within the art to which the invention pertains and as
may be applied to the essential features hereinbefore set
forth as follows in the scope of the appended claims
The foregoing description of the specific
25 embodiments will so fully reveal the general nature of the
invention that others can, by applying current knowledge,
readily modify and/or adapt for various applications such
specific embodiments without departing from the generic
concept, and, therefore, such adaptations and modifications
30 should and are intended to be comprehended within the meaning
and range of equivalents of the disclosed em~odiments. It is
to be understood that the phraseology or terminology employed
herein is for the purpose of description and not of
limitation.
WO 92/10506 2 0 ~ 7 1 0 ~i PCr/US91/09382
S~NOE IISlING
(1) OENER~ lNFORM~llCN:
Yo6~rol q~YU~
(ii) ll~rlE OF ~ON: }~nan Retn~vi~ Receptor ar~ ~ Coding
I5~erefor
(iii) N~ OF S~OE:S: 8
(iv) ~REæS~NDENOE ADE~S:
(A) A~i~x B~ and Ne~nark
(B) Sl~;~r: 419 Seventh Street, N.W.
(C) CI'rY: Wa~i~
(D) Sl~E: DC
(E) ~RY: ~SA
(F) ZIP: 20004
(v) a~WER RE~ F~:
(A) MEDIt~l TY~: ~o~y disk
(B) C~II~: IEM PC npatible
(D) SOFIW~ PatentIn Release #1.24
(vi) a~EæNr A~rC~rION n~:
(B) ~ U~E:
(C) CLAssIFlt~oN
(viii) A~/AOE2~r IN~ON:
(A) NPME: Li~mat, 5hnuel
(B) REX;ISTRAI~ON Nt~: 33,949
(C) ~;F~OE/DC~l NU~: M~J~l
(ix) ~?~CP~ON ~IION:
(A) 1 = E: 202 628-5197
(2) INF~RM~O~ FCR SE~ ID NO:l:
(i) SEX~ENOE CHPRACIERI~
(A) ~æI~I: 1102 base pairs
(B) TYPE: nucleic acid
(C) S~: single
(D) I~POIDGY: linear
(ii) r~I~:CULE TYPE: c~NA
( ix) ~lURE:
(A) NAME/ÆY: C~;
(B) I~ON: 1..1102
W O 92/10506 PCT/US9l/09382
~ ~ 9 ~ ~U~
- 72 -
(Xl) SEQUENOE DESCK~ ON: SEQ ID ND:1:
CCG GGC GCC ACC ITC GAC GAG CTG ATA GGC AGA CCC ATC GGG GAG TTC
Pr~ Gly Ala qhr Phe Asp Glu Leu Ile Gly Arg Pr~ Ile Gly Glu Phe 48
1 5 10 15
TCA CGG ACA CAC ATG ACT CTG AAC GCC CCC GGC GTG ~1~ GCT GAA AAC 96
Ser Arg Ihr His Met qhr Leu Asn Ala Pro Gly Val Leu Ala Glu As~
CCC GAC AlA TrC GCA GIG ATC ATA AIT CTC ArC Tl~ ACA GGA CIT IIA 144
Prc Asp Ile Phe Ala Val Ile Ile Ile Leu Ile T~- Ihr Gly Leu Leu
ACT CIT GGT GTG AAA GAG T~ GCC ATG GTC AAC M A ATA TTC ACT TGT 192
q~r Leu Gly Val Lys Glu Ser Ala Met Val Asn Lys Ile Phe Ihr Cys
AIT AAC GTC CrG GTC CTG GGC ITC ATA AIG GTG TCA GGA m GTG A~A 240
Ile Asn Val L~u Val Leu Gly Phe Ile Met Val Ser Gly Phe Val Lys
GGA TCG GIT AAA AAC ~GG CAG CTC ACG G~G GAG G~T m GGG AAC AC~ 288
Gly Ser Val Lys Asn Trp Gln Leu Thr Glu Glu Asp Phe Gly Asn Thr
TC~ GGC CGT CTC TGT TTG AAC AAT G~C ACA AAA GAA GGG AAG CCC GGT 336
Ser Gly Arg Leu Cys Leu Asn Asn Asp IThr Lys Glu Gly Lys PrD Gly
100 105 110
GIT GGT GGA TT~ ATG OOC TTC GGG TrC TCT GGT GTC CTG TCG GGG GCA 384
Val Gly Gly Phe Met Pro Phe Gly Phe Ser Gly Val Leu Ser Gly Ala
115 120 125
GCG ACT TGC TTC TAT GCC TTC GTG GGC TTT GAC IGC ATC GCC ACC ACA 432
Ala Thr Cys Phe Tyr Ala Fhe Val Gly Phe Asp Cy~ Ile Ala Thr Thr
130 135 140
GGT GAA GAG GTG AAG AAC CCA CAG A~G GCC A~C CCC OEG GGG ATC GT~ 480
Gly Glu Glu Val Lys Asn Pr~ Gln Lys Ala Ile Pr~ Val Gly Ile Val
145 150 155 160
GCG TCC crc TTG ATC TGC TTC ATC GCC TAC TTT GGG GTG TCG GCT GCC 528
Ala Ser Leu Leu Ile CY5 Phe Ile Ala Tyr Phe Gly Val Ser Ala Ala
165 170 175
CTC ACG CTC A~G ATG ~ TAC TTC TGC CTG GAC AAT AAC AGC CCC CTG 576
Leu Thr Leu Met Met ~ ~r ~e Cys Leu Asp Asn Asn Ser Pr~ Leu
180 185 190
CCC GAC GCC TTT A~G CAC GTG GGC T~G GAA GGT GCC AAG TAC GCA GTG 624
Pr~ Asp Ala Phe Lys His Val Gly Trp Glu Gly Ala Lys Tyr Ala Val
195 200 205
W O 92/10506 2 0 3 7 7 0 5 PCT/US91/09382
- 73 -
GCC GIG GGC TCC CTC rGC GCT ~T TCC GCC AGT CTr Cl~ GGT TCC ATG 672
Ala Val Gly Ser Leu Cys Ala Leu Ser Ala Ser Leu Leu Gly Ser Met
210 215 220
m ccc AIG CCT CGG GTT ATC lAr GCC ATG GCT GAG GAr GGA CTG CI~ 720
Phe Pr~ Met Pr~ Arg Val Ile Tyr Ala Met Ala Glu Asp Gly T~ll Leu
225 230 235 240
TTT AAA TTC q ~ GCC A~C GTC MT G~T AGG ACC MA ACA CCA A~ AIC 768
Phe Lys Phe T~- Ala Asn Val Asn Asp Arg Thr Lys Thr Pro Ile Ile
245 250 255
GCC ACA TTA GCC ICG GCr GCC GlT GCT GCT GTG Al~; GCC ITC 1~ m 816
Ala Thr Leu Ala Ser Gly Ala V 1 Ala Ala Val Met Ala Phe LPI1 Phe
260 265 270
GAC ~ AAG G~C TTG ~rG G~C CTC ATG T~C Al'r GGC ACT CTC CTG GCT 864
Asp Leu Lys Asp Leu Val Asp Leu Met Ser Ile Gly Thr Leu Leu Ala
275 280 285
TAC TCG TTG GTG GCT GCC TGT GTG TTG GTC TTA CGG TAC CAG CCA GAG 912
Tyr Ser Leu Val Ala Ala Cys Val Leu Val T~l Arg Tyr Gln PrD Glu
290 295 300
CæG CCT AAC CIG GTA ~hC CAG ATG GCC AGT ACT TCC G~C G~G TTA G~T 960
Gln PrD Asn Leu Val Tyr Gln Met Ala Ser Thr Ser Asp Glu Leu Asp
305 310 315 320
CC~ GCA GAC CA~ A~T GAA TTG GC~ AGC ACC AA~ GAT TCC CAG CTG GGG 1008
Pr~ Ala Asp Gln Asn Glu T~l Ala Ser Thr Asn Asp Ser Gln T~l Gly
325 330 335
m TI~ CC~ GAG GC~ G~G A~G TTC TCT TTG AAA ACC ATA C~C TC~ CCC 1056
Phe T~U Pro Glu Ala Glu Met Fhe Ser L2U Lys m r Ile Leu Ser Pro
3~0 345 350
A~A AAC AlG G~G CCT TCC A~A AIC TCT GGG CTA AIT GTG AAC COG G 1102
Lys Asn Met Glu Pr~ Ser Lys Ile Ser Gly Leu Ile Val Asn PrD
355 360 365
(2) INFORM~IION FOR SEQ ID N0:2:
(i) SEÇUENCE CH~RA~l~KlSTICS:
(A) LENGIH: 367 aminD acids
(B) TYPE: amlnD acid
tD) TOPOLDGY: linear
(ii) MOLECULE TYFE: protein
(xi) SEÇpENOE DESCRIPIION: SEQ ID NO:2:
Pro Gly Ala Thr Phe Asp Glu Leu Ile Gly Arg Pro Ile Gly Glu Phe
1 5 10 15
Ser Arg Thr His Met Thr Leu Asn Ala Pro Gly Val Leu Ala Glu Asn
W O 92/10506 ~ U ~ PC~r/US91/09382
- 74 -
Pr~ A~p Ile Phe Ala Val Ile Ile Ile Leu Ile Leu qhr Gly T~l Leu
Ihr Leu Gly Val Lys Glu S~r Ala Mk~t Val PY~n Lys Ile Phe Thr Cys
Ile A~n Val Leu Val Leu Gly Fhe Ile ~t Val Ser Gly Phe Val Lys
ly S~r Val Lys Asn Trp Gln Leu Thr Glu Glu ~Y~p Phe Gly Asn Ihr
~Yr Gly A~ng Leu Cys Leu A~;n Asn P~p I~Lr Lys Glu Gly Lys Pro Gly
100 105 110
~l Gly Gly Phe Met PrD Phe Gly Phe S~r Gly Val Leu Ser Gly Ala
115 120 125
Ala q~lr Cys Fhe Tyr Ala Phe Val Gly Phe AY~P Cys Ile Ala Thr Thr
130 135 140
Gly Glu Glu Val Lys AY3n Pr~ Gln Lys Ala Ile Pr~ Val Gly Ile Val
145 150 155 160
la Sb~r Leu Leu Ile Cys Fhe Ile Ala q~nr Fhe Gly Val Ser Ala Ala
165 170 175
eu q~Lr Ifau Met Mbt Pso q~r Phe Cys Leu A~p A~;n Asn Ser Pro Leu
180 185 190
ro A~p Aaa Phe Lys His Val Gly q~p Glu Gly Ala Lys Tyr Ala Val
195 200 205
Ala Val Gly Ser leu ~s Ala Leu Ser Ala Ser Leu Leu Gly Ser Met
210 215 220
Phe Pro ~ t Pro Arg Val Ile q~r Ala ~ t Ala Glu ~ p Gly Leu T~
225 230 235 240
he Lys Fhe T~ Ala Asn V 1 ~n A ~ Arg Thr Lys Thr Pro Ile Ile
245 250 255
la Thr Leu Ala Ser Gly Ala Val Ala Ala Val Met Ala Phe Leu Fhe
260 265 270
~p Leu Lys A~p Leu Val A~p Leu Met Ser Ile Gly Thr Leu Leu Ala
275 280 285
Tyr Ser Leu Val Aaa Ala Cys Val Leu Val Leu A ~ ~r Gln Pro Glu
290 295 300
Gln Pro A~n Leu Val Tyr Gln Met Ala Ser qllr Ser ~p Glu Leu Asp
305 310 315 320
Pro Ala Asp Gln Asn Glu Leu ~aa Ser mr Asn Asp Ser Gln Leu Gly
325 330 335
W O 92/10506 2 ~ ~ 7 7 0 ~ PCT/US91/09382
- 75 -
Phe Leu Pro Glu Ala Glu Met Fhe Ser Leu Lys Thr Ile Leu Ser Pr~
340 345 350
~ys Asn Met Glu Pro Ser Lys Ile Ser Gly Leu Ile Val Asn PrD
355 360 365
4) DNFOR~aIlCN FOR SEQ ID ND:3:
(A) IENGTH: 2425 base pairs
(B) TYPE: nucleic acid
(C) SlRANDELNESS: s mgle (D) TDPOLDGY: lLnear
(ii) MDLECUIE IYPE: cDNA
(ix) P~:
(A) N~ME/XEY: CDS
(B) IDChIIoM: 199..2064
(xi) SE~UENCE DeSCRIPq5QN: SEQ ID N0:3:
G~ITCCECCC GCCIOCGCC~ qCCCClC~GC IaoCaGGTGT G~Ga~GCTTr CT~CCCECGG 60
I~TCC~C~C~ GCIC~ACArC IIGCCGCCIC CTCCGAGCCr G~AGCI~CCG IGCPCICIGC 120
qGTG.~GT:r IG30000CAG GrGOE G~IOC I~C~aLIGA G~AGrCCCAC G~GTCTT~C~ 180
GCALo~l~CC TCAGC~CA AIG GGC TGC AA~ AAC CIG CTC CTG GGC C~G 231
Met Gly Cys Lys Asn Leu T~l Gly Leu Gly Gln
1 5 10
CAG ATG CTG CGC CGG A~G GIG GTG GAC TGC AGC CGG GAG G~G AGC CGG 279
Gln Met T~7 Arg Arg Lys Val Val Asp Cys Ser Arg Glu Glu Ser Arg
2S
CTG TCC CGC TGC crc AAC ACC TAT GAC CTG GTA GCT CTT GGG G~G GGC 327
T~- Ser Arg Cys Leu Asn Thr Tyr Asp Leu Val Ala Leu Gly Val Gly
AGC ACC ~1~ GGC GCT GGT GTC TAT GTC CTA GCC GGT GCC GTG GCC CGl~ 375
Ser Thr Leu Gly Ala Gly Val Tyr Val Leu Ala Gly Ala Val Ala Arg
GAA AAT GCT GGC CCT GC~ ATC GTC ATC TC~ TIC TrG ATT GCT GCT CT~ 423
Glu Asn Ala Gly Pr~ Ala Ile Val Ile Ser Phe Leu Ile Ala Ala Leu
GCC TCC GTG CrG GCC G C CTG TGC TAC GGC G~G TTT GGT GCC CGT GTC 471
Ala Ser Val Leu Ala Gly Leu Cys Tyr Gly Glu Phe Gly Ala Arg Val
go
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- 76 -
CCC AAG ACG GGC TCA GCC TAC CTC TAC A~C TAC GTG ACG GIG GGG GAG 519
Pro Lys Thr Gly Ser Ala Tyr Leu Tyr Ser Tyr Val Thr Val Gly Glu
100 105
CTr TGG GCC TTC AIC ACT GGC TGG AAC CTG ATT CTC TC~ TAC ATC ATC 567
T~- Trp Ala Phe Ile Thr Gly Trp Asn Leu Ile Leu Ser Tyr Ile Ile
110 115 ~.20
GGT ACT TC'A AGC GIG GCA AGA GCC TGG AGT GCG ACT m GAC GAG CIG 615
Gly qhr Ser Ser Val Ala Ar3 Ala Trp Ser Ala Thr Phe Asp Glu Leu
125 130 135
A~A GGC AAG CCC ATC GGA GAG TTC TCA CGT C~G C~C ATG GCC CIG A~T 663
Ile Gly Lys Pro Ile Gly Glu Fhe Ser Arg Gln His Met Ala Leu Asn
140 145 150 155
GCT C T G G GTG CTG GC~C C~AA AOC CCG GAC ATA m GCT ~1~ ATT ATA 711
Ala Pro Gly Val Leu Ala Gln Thr Pr~ Asp Ile Phe Ala Val Ile Ile
160 165 170
ATT A~C ATC TTA ACA GGA CTG TTA ACT CTT G~C GTG AAG GAG TCA GC~ 759
Ile Ile Ile ~ Thr Gly Leu Leu Thr Leu Gly Val Lys Glu Ser Ala
1~5 180 185
AT~ GTC AAC AAA AIT TTC ACC TCT ATC AAT ~1~ CTG G~C ITG TGC ITC 807
Mbt Val Asn Lys Ile Phe Thr Cys Ile Asn Val Leu Val T~l Cys Phe
190 195 200
ATC G~G ~1~ TCC GGG TrC GTG AAA GGC TCC A~T M A AAC I~G CAG CTC 855
Tle Val Val Ser Gly Phe Val Lys Gly Ser Ile Lys Asn Tr,o Gln Leu
205 210 215
ADG GAG AAA AAT ITC TCC T&T AAC AAC AAC G~C AC~ AAC GTG AAA I~C 903
Ihr Glu Lys Asn Phe Ser Cys Asn Asn Asn Asp Thr Asn V~l Lys Tyr
220 225 230 235
G~T G~G G~ GGG TTT A~G CCC m GGA TTC TCT GGT GTC CTG TCA GGG 951
Gly Glu Gly Gly Phe Met Pro Phe Gly Phe Ser Gly Val T~ Ser Gly
240 245 250
GCA GCG ACC TGC m T~T GCC TTC GTG GGC m GAC TGC ATC GCC ACC 999
Ala Ala Thr Cys Phe Tyr Ala Phe Val Gly Phe Asp Cys Ile Ala Thr
255 260 265
ACA G~G GAA GAA GTC AAG AAC CCC CAG AAG GCC ATT c~r GTG GGC ATC 1047
Thr Gly Glu Glu Val Lys Asn PrD Gln Lys Ala Ile Pr~ Val Gly Ile
270 275 280
GTG GCG TCC CTC CTC ATT TGC TTC ATA GCG TAC m GGC GTG TCC GCC 1095
Val Ala Ser Leu Leu Ile Cys Phe Ile Ala Tyr Phe Gly Val Ser Al~
285 290 295
GCT CTC ACG crc ATG ATG cc~r TAC TTC TGC CTG GAC A'TC GAC AGC C~G 1143Ala Leu Thr Leu Met Met Pro Tyr Phe Cys Leu Asp Ile Asp Ser PrD
300 305 310 315
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- 77 -
CTG CCT GGT GCC TTC A~G CAC CAG GGC TGG GAA GAA GCT AAG TAC GCA 1191
T~l Pr~ Gly Ala Phe Lys His Gln Gly Trp Glu Glu Ala Lys Tyr Ala
320 325 330
GTG GCC ATT GGC TCT CTC TGC GCA CTT TCC ACC AGT CTC CrA GGC TCC 1239
Val Ala Ile Gly Ser Leu Cys Ala Leu Ser Thr Ser Leu Leu Gly Ser
335 340 345
ATG m ccc ATG CCC CGA GIT ATC TA~ GCC ATG GCT GAA GAT GGA CTA 1287
Met Phe Pro Met Pr~ Arg Val Ile Tyr Ala Met Ala Glu Asp Gly Leu
350 355 360
~1~ m AAA m TTG GCC AAA ATC AAC A~T AGG ACC AAA AC~ CCC Gl~ 1335
Leu Fhe Lys Phe Leu Ala Lys Ile Asn Asn Arg lhr Lys Thr Pro Val
365 370 375
ATC GCC ACT GIG ACC TCA GGC GCC AIT GCT GCT GTG ATG GCC TrC CTC 1383
Ile Ala Thr Val Thr Ser Gly Ala Ile Ala Ala Val Met Ala Phe Leu
380 385 390 395
m G~A CIG AAG GAC CTG GTG GAC CTC ATG TCC A~T GGC ACT crc CTG 1431
Fhe Glu Leu Lys Asp T~l Val Asp Leu Met Ser Ile Gly Thr Leu Leu
400 405 410
GCT TAC TCT TTG GTG GCT GCC TGr GIT TTG GTC TTA CGG TAC CC~ 1479
Ala Tyr Ser T~l Val Ala Ala Cys Val Leu Val T~- Arg Iyr Gln Pr~
415 420 425
GAA CAA CCT AAT C~G GTA TAC A'rG GCC ACA ACC ACC GAG G~G CI~ 1527
Glu Gln Pro Asn Leu Val Tyr Gln Met Ala Arg qhr Thr Glu Glu Leu
430 435 440
GAT oG~ GTA GAT AAT GAG C~G GTC A~T GCC AGT GAA TCA C~G ACA 1575
Asp Arg Val Asp Gln Asn Glu T~l Val Ser Ala Ser Glu Ser Gln Thr
445 450 455
GG~ TTT TTA CCG GTA GCC GAG AAG m TCT ~1~ A~A TCC ATC crc TCA 1623
Gly Phe Leu Pro Val Ala Glu Lys Phe Ser Leu Lys Ser Ile Leu S r
460 465 470 475
CCC AAG AAC GTG GAG OOC TCC AAA TTC TC~ ~GG CTA ATT GTG AAC ATT 1671
Pro Lys Asn Val Glu Pro Ser Lys Phe Ser Gly Leu Ile Val Asn Ile
480 485 490
TC~ G C GGC crc crA GCC GCT CTT ATC ATC ACC GIG TGC ATT GTG GOC 1719
Ser Ala Gly Leu Leu Ala Ala Leu Ile Ile Thr Val Cys Ile Val Ala
495 500 505
Gl~ CTT GGA AGA GAG GCC CTG GCC GAA GGG ACA CTG TGG GCA GTC TTT 1767
Val L~u Gly Arg Glu Ala Leu Ala Glu Gly Ihr Leu Trp Ala Val Phe
510 515 520
GTA ATG ACA GGG TC~ GTC CTC CI~- TGC ATG CTG G~G ACA GGC AIC AIC 1815
Val Met Thr Gly Ser Val Leu Leu Cys Met Leu Val Thr Gly Ile Ile
525 530 535
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- 78 -
TGG AGA C~G CCT GAG AGC AAG ACC AAG CTC TCA m AAG GTA CCC m 1863
Trp Arg Gln Pro Glu Ser Lys Thr Lys Leu Ser Phe Lys Val Pro Phe
540 545 550 555
GrC CCC GTA CIT CCT ~-~ TIG AGC ATC TTC GIG AAC AIC T~r CTC ATG 1911
Val Pro Val Leu Pr~ Val Leu Ser Ile Phe Val Asn Ile Tyr Leu Met
560 565 570
ATG CAG CIG GAC C~G GGC ASG IGG GIC CGG m GC~ GTG TGG ATG CTG 1959
Met Gln T~- Asp Gln Gly Ihr Trp Val Arg Phe Ala Val Trp Met Leu
575 580 585
AIA GGT TrC ACC ATC TAT TrC GGT TAT G~G ATC IGG CAC AGT G~G G~A 2007
Ile Gly Phe Thr Ile Tyr Phe Gly Tyr Gly Ile Trp His Ser Glu Glu
590 595 600
GCG TCC CTG GCT GCT GGC C~G GC~ AAG Acr CCT GAC bGC AAC TTG GAC 2055
Ala Ser Leu Ala Ala Gly Gln Ala Lys Thr E~o Asp Ser Asn Leu Asp
605 610 615
CAG TGC AAA ~GPoGIGCA~ c~Acc~ac CAGGGIGACA GCGGTTGACG 2104
G}n Cys Lys
620
GGIGC~ OE ra GPACc~nGCG A~ICAChA T~TCTCCACT car~OCTC~G GATcAGcqC~ 2164
CaC~rDAr GTCACCAAAG clGGrrIacr GC~ALCTCGT GAG~5CCrGG ICarTICTGG 2224
ALPGICC~Tr GCTTIPC~CA l~lCDClCr: AACA~AGAAA GCAGCOCIrC TC~TTGCCGG 2284
IGCGGC~OCA GCaEAAGGGA GGCC~CCTTC ICCTCTCACT 2344
loGGAAGIAG GC~IC~CTOC CIOOCTGaG~ CC~CCCTGGC AICGC~IG TGCAC~CTCC 2404
ALP3CCCTAG IGPGC~TCT~ C 2425
(5) INF~Na~ION FOR SEQ ID N0:4:
ti) SE~y~NOE CH~RACTERISTIC5:
(A) LENGIH: 622 a~ acids
(B) TYPE: amino acid
(C) STFANDer~ESS: s mgle
(D) TOPOLDGY: linear
(ii) MDLECULE TYPE: protein
(xi) SEÇUENOE DESCRIPqION: SEQ ID NO:4:
Met Gly Cys Lys Asn Leu Leu Gly Leu Gly Gln Gln Met Leu Arg Arg
1 5 10 15
Lys Val Val Asp Cys SOE Ar~ 51U G1U SOE Ar~ Leu Ser Ar~ Cys Leu
~0~7705
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- 79 -
Asn Thr Tyr Asp Leu Val Ala Leu Gly Val Gly Ser Thr Leu Gly Ala
Gly Val Tyr Val Leu Ala Gly Ala Val Ala ~ Glu Asn Ala Gly Pro
Ala Ile Val Ile Ser Phe Leu Ile Ala Ala Leu Ala Ser Val Leu Ala
7S 80
Gly Leu Cys Tyr Gly Glu Fhe Gly Ala Arg Val Pro Lys Thr Gly Ser
Ala Tyr Leu Tyr Ser Tyr Val Thr Val Gly Glu Leu Trp Ala Phe Ile
10~ 105 110
qhr Gly Irp Asn Leu Ile Leu Ser Tyr Ile Ile Gly Thr Ser Ser Val
115 120 125
Ala Arg Ala Trp Ser Ala Ihr Phe Asp Glu Leu Ile Gly Lys Pro Ile
130 135 140
Gly Glu Phe Ser Arg Gln His Met Ala Leu Asn ~la Pro Gly Val Leu
145 150 155 160
Ala Gln Thr Pro Asp Ile Phe Aaa Val Ile Ile Ile Ile Ile Leu Thr
165- 170 175
Gly Leu Leu Thr Leu Gly V~l Lys Glu Ser Ala Met Val Asn Lys Ile
180 lB5 190
Fhe Thr Cys Ile Asn Val leu Val Leu Cys Phe Ile Val Val Ser Gly
195 200 205
Phe Val Lys Gly Ser Ile Lys Asn Trp Gln Leu Thr Glu Lys Asn Phe
210 215 220
Ser Cys Asn Asn Asn Asp Thr Asn Val Lys I~r Gly Glu Gly Gly Phe
225 230 235 240
Met Pro Phe Gly Phe Ser Gly Val Leu Ser Gly Ala Ala Ihr Cys Phe
245 250 255
Tyr Ala Phe Val Gly Phe Asp Cys Ile Ala Thr Thr Gly Glu Glu Val
260 265 270
Lys Asn Pro Gln Lys Ala Ile Pro Val Gly Ile Val Ala Ser Leu Leu
275 280 285
Ile Cys Phe Ile Ala Tyr Phe Gly Val Ser Ala Ala Leu Thr Leu Met
290 295 300
Met Pro Tyr Phe Cys Leu Asp Ile Asp Ser Pro L u Pro Gly U a Phe
305 310 315 320
Lys His Gln Gly Trp Glu Glu Ala Lys Tyr Ala Val Ala Ile Gly Ser
325 330 335
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- 80 -
Leu Cys Ala Leu Ser Thr Ser Leu Leu Gly Ser Met Phe PrD Met PrD
340 345 350
Arg Val Ile Tyr Ala Met Ala Glu Asp Gly T~l Leu ~he Lys Phe Leu
355 360 365
Ala Lys Ile Asn Asn Arg Ihr Lys Thr Pro Val Ile Ala Thr Val Thr
370 375 380
Ser Gly Ala Ile Ala Ala Val Met Ala Phe Leu Phe Glu Leu Lys Asp
385 390 395 400
Leu Val Asp Leu Met Ser Ile Gly Thr Leu Leu Ala Tyr Ser Leu Val
405 410 415
Ala Ala Cys Val Leu Val Leu Arg Tyr Gln Pro Glu Gln PrD Asn Leu
420 425 430
Val Tyr Gln Met Ala Arg Thr Thr Glu Glu Leu Asp Arg Val Asp Gln
435 ~ 440 445
Asn Glu Leu Val Ser Ala C~r Glu Ser Gln Thr Gly Phe Leu PrD Val
450 455 460
Ala Glu Lys Phe Ser Leu Lys Ser Ile Leu Ser Pr3 Lys Asn Val Glu
465 470 475 480
PrD Ser Lys Phe Ser Gly Leu Ile Val Asn Ile Ser Ala Gly Leu Leu
485 490 495
Ala Ala Leu Ile Ile Thr Val Cys Ile Val Ala Val Lsu Gly Arg Glu
500 505 510
Ala T~l Aaa Glu Gly Ihr Leu ~ Ala Val Phe Val Met Thr Gly Ser
515 520 525
Val Leu T~ll Cys Met Leu Val Ihr Gly Ile Ile Trp Arg Gln Pr~ Glu
530 535 540
. Ser Lys Thr Lys Leu Ser Phe Lys Val Pro Phe Val PrD Val Leu PrD
545 550 555 560
Val Leu Ser Ile Phe Val Asn Ile Tyr T~l Met Met Gln Leu ~ Gln
565 570 575
Gly Thr Trp Val Arg Phe Ala Val Trp Met Leu Ile Gly Phe Thr Ile
580 585 590
Tyr Phe Gly Tyr Gly Ile Trp His Ser Glu Glu Ala Ser Leu Ala Ala
595 600 605
Gly Gln Ala Lys Thr Pro Asp Ser Asn Leu Asp Gln Cys Lys
610 615 620
WO 92/10506 2 0 9 7 7 0 ~ PCr/US91/09382
-- 81 --
(2) ~FORM~ON FOR S}~Q ID NO:5:
(i) SE~IOE a~ACrERIS~CS:
(A) ~I~: 2397 base pairs
(B) I'Y~: nucleic acid
(C) ~s: s~le
(D) IOPO~GY: l~near
(ix) E~lU~:
(A) tlP~: CDS
(B) IDCl~llON: 410 . .1768
(xi) SE~ENOE I~ESCRI~r[ON: SE~ :5:
GGG~ A~;Cr mn:GCCr a3~I~XC a~CITn.C I~lGCm~AT 60
llGa~: AGIA~; ACI~ ~= CAClTACGIC AX~I~G 120
AGCI~I ~ Cl ~ ~ TC l ~ ~ I~rC ~ TA GGrACGI~C~ 180
GTGTCGCAAG AGcaIGGAGr GGCAC~IITG ACGhbCTTCr TAAI~A~CAG A7lGGccaoT 240
mDc;AAAc G~ACIICYAA A5GAAlTACA CqGGTClGGC AGAoTATccA GACTTCTTTG 300
CCG~GrGCCT IGIATTACr~ ClGGCAGGrC mrYrcm IGEACTPAAA Gprcl~cTT 360
rGTGpAT~A AmrrhcaG CIATI~UTAT CCn3aTCCTT CTCTIDCTC AT& GTG 415
Met Val
GCT GGG m GIb A~A GG~ AAT GTG GCT A~C T æ AAG ATC AGT GAA GAG 463
Ala Gly Phe Val Lys Gly Asn Val Ala Asn Trp Lys Ile Ser Glu Glu
TIT CTC AAA AAT ATA TCA GCA AGT GCT ALA GAA CCA C'CT ~ GAG AAC 511Phe Leu Lys Asn Ile Ser Ala Ser Ala Arg Glu Pro Pro Sex Glu Asn
GGA ACA AGC ATC TAC GGG GCT GGC GGC TIT ATG CCC TAT GGC m ACA 559
Gly Thr Ser Ile Tyr Gly Ala Gly Gly Phe Met Pr~ Tyr Gly Phe Thr
GGG ACG TrG GCT GGT GCT GCA ACG TGC m TAT GC~ m GTG GGC m 607
Gly Thr Leu Ala Gly Ala Ala Thr Cys Phe Tyr Ala Phe Val Gly Phe
GAC T&C ATT GCA ACA ACC GGT GAA GAG G~T oGG AAT C~A CAA AAG GOG 655
Asp Cys Ile Ala Thr Thr Gly Glu Glu Val Arg Asn Pr~ Gln Lys Ala
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- 82 -
ATC CCC ATC GGA ATA GTG AOG TCC T~A CIT GTC TGC m AT~ GCT TAC 703
Ile Pro Ile Gly Ile Val Thr Ser Leu Leu Val Cys Phe Met Ala Tyr
m GGG GIT TCT GCA GCT TTA ACG CIT ATG ATG CCT TAC TAC crc CTG 751
Phe Gly Val Ser Ala Ala Leu Thr Leu Met Met Pro Tyr Tyr Leu Leu
100 105 110
G~T GAG AAA AGT CC~ CTC CC~ GTC GCG m GAG TAT GTC AGA TGG G~C 799
Asp Glu Lys Ser Pr~ Leu Pro Val Ala Phe Glu Tyr Val Arg Trp Gly
115 120 ~75 130
CCC GCC AAA TAC GIT GTC GCA GCA GGC TCC CTC TGC GCC Tr~ TC~ ACA 847
Pr~ Ala Lys Tyr Val Val Ala Ala Gly Ser Leu Cys Ala Leu Ser Thr
135 140 145
AGT Cl'l' CIT GGA TC~ AIT TTC OCA ATG CCT C~T G~ AIC TAT GCr ATG 895Ser Leu Leu Gly Ser Ile Phe Pro Mbt Pr~ Arg Val Ile Tyr Ala Met
150 155 160
GC~ GAG GAT GGG TTG CIT TTC AAA TGT CTA GCT CAA ATC AAT TCC AAA 943
Ala Glu Asp Gly T~u T~l ~e Lys Cys Leu Ala Gln Ile Asn Ser Lys
165 170 175
ACX~ AAG AC~ ~rA ATT GCT ACT ~G ~ TC; GGT GC2~ GTG GC~ GCT 991
q~r Lys l~r ~ro Val Ile Ala Ihr Le~ Ser Ser Gly Ala Val Ala Ala
180 185 190
GTG AIIG GOC m CTT I~CT G~C CTG AAG GOC CTC GTG G~C AIG A~rG TCT 039
V~l ~et Ala Phe L~u Phe Asp Leu ~rs Ala T~l Ual Asp Met Met Ser
195 200 205 210
AIT GGC ACC CTC A~ GCC TAC ICT CTG GrG GC~ GOC IGT GTG ~11 A~ 087
Ile Gly fflr L~u Met Ala l~r Ser Leu Val Ala Ala Cys Val Leu Ile
215 220 225
AGG TAC CAA CCT GGC qTG TGT q~C GPY; CP~G COC AAA I~C AOC CCT 1135
Leu Ar~ ~r Gln }~ ly ~u ~ys l~yr Glu Gln ~ ~s Tyr Ihr ~ro
230 235 240
GAG AAA GAA ACT CTG GAA T~A IGT ACC AAT GCG ACT TIG AAG AGC GAE 1183
Glu Lys Glu Thr Leu Glu Ser Cys Thr Asn Ala Thr Leu Lys Ser Glu
245 250 255
TCC CAG GTC ACC AIG CTG CAA GGA CAG GGT TTC AGC CTA CGA ACC CTC 1231
Ser Gln Val Thr Met Leu Gln Gly Gln Gly Fhe Ser keu Arg mr Leu
260 265 270
TTC AGC OCC TCT GCC CTG CCC ACA CGA CAG TCG ECT TCC CTT GTG AGC 1279
~e Ser E~ro Ser Ala Lu ~ Ihr Ar~ Gln Ser Ala Ser Leu Val Ser
275 280 285 290
TTT ~1~ GTG GGA TTC CrG GCT TTC CIC ATC CTG GGC TTG AGT AIT CTA 1327
E~e Leu Val Gly Phe Leu Ala Phe Leu Ile Leu Gly Leu Ser Ile Leu
295 300 305
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ACC ACG lAr GGC GTC CAG GCC Alr GCC AGA CT~ GAA GCC TGG AGC CTG 1375
Thr Thr Tyr Gly Val Gln Ala Ile Ala Arg Leu Glu Ala Trp Ser Leu
310 315 320
GCT CTT CTC GCC CrG TTC W l GTC ~ C TGC GCT GCC GTC ~TT CTG ACC 1423
Ala Leu T~l Ala Le~ Phe Leu Val Le~ Cys Ala Ala Val Ile Leu Thr
325 330 335
AIT TGG A~G CAG CCA CAG AAr CAG C~ AAA GIA GCC TT~ ATG GTC CCG 1471
Ile Irp Arg Gln PrD Gln Asn Gln Gln Lys Val Ala Phe Met Val Pro
340 345 350
TTC TTA CCG m CTG OCG GCC TTC AGC AIC CTG GTC AAC ATT IAC TTG 1519
Fhe Leu Pr~ ~he Leu PrD Ala Fhe Ser Ile Leu Val Asn Ile Tyr Leu
3S5 360 365 370
AIG GTC C~G TT~ A~T GaG GAC ACT TGG ATC AG~ TTC AGC ATC TGG ATG 1567
Mbt V~l Gln Leu Ser Ala Asp Thr Trp Ile Arg Phe Ser Ile Trp Met
375 380 385
GCG CIT GGC TTr CTG AIC TAT TrC GCC I~T GGC AIT AGA C~C AGC TIG 1615
Ala Leu Gly ~he T~- Ile Tyr Phe Ala Tyr Gly Ile Arg His Ser Leu
390 395 400
G~G GGT AAC CCC AEG G~C G~A GAA G~C G~T G~G G~T GCC TTT TCA GAA 1663
Glu Gly Asn PrD Arg Asp Glu Glu Asp Asp Glu Asp Ala Phe Ser Glu
405 410 415
A~C ATC AAr GTA GCA ACA GAA GAA AAG TCC GTC A~G CAA GCA AAT GAC 1711
Asn Ile Asn Val Ala Thr Glu Glu Lys Ser Val Met Gln Ala Asn AsF
420 425 430
CAT CAC CA~ hGA A~C crc AGC TT~ CCT TTC ATA CTT CAT G~A AA5 ACA 1759
His His Gln Arg Asn Leu Ser Leu PrD Phe Ile T~1 HiS Glu Lys Thr
435 440 445 450
A~T G~A IGT TG nGcIaGc CCICEGTCIT AC~ACGC~TA CCII~ACAAT 1808
Ser Glu Cys
GAGI~CACIG T&GCCGGATG CCACCAICGT GC3GGGCTGT CGnG3GTCqa CTGT~G~CAT 1868
GGClTGCCIa ACITGTACIT CrTCCqCC2a ACAGCrTCnC TrcaGaDGaT GGATrCTGlG 1928
ICIG2GGAGA cTGccIGAGa GCACTCCTCA GCTATATGTA TCo~C~AA~C AGTATCICCG 1988
TGIGOGTACA TGTAT~lw ~ CGArCIG~T GTT~PPICIT GTCCGITATT A ~ AC 2048
AT~ATICCAG CATGGIAATT GGT~GCATAT ACTGCACACA CTAGTAAACA GTATATIGCT 2108
GA~TAGAGAT GT;TTCTGTA TATGTCCTAG GTGGCIGGGG AA~TAGIGGT GGTTTCTITA 2168
TIa3GT~IAT GACCATCAGT TIGGACATAC T&AAAIGCCA TCCO~IaIC~ GGAIGIITAA 2228
C~GTGGTcaT GGGDGGGGAA GGGaIaAEGA AIGGGCATrG TCT~IAAATT GI~ATGCATA 2288
TATCCITCTC CTACTIGCTA AGACAGCTTT cTTAAAaGGc C~GGGAGAGT GrTnCrTTcC 2348
WO 92/10506 ~ o ~ r~ t~ ~J 5 PCI/US91/09382
~ 84 --
I~l~.l~C MG~ a~i~5r (a3c~Ga~ ~ 2397
(7) INFORM~lION F~R SEQ ID N0:6:
(i) SE~ENCE CH~RACrERISrICS:
(A) ~: 453 am~no acids
(B) TY~: amino acid
(C) Sl~: single
(D) lOPOLOGY: linear
(ii) ~IE0I.E T~: pr~tein
(xi) SE~ENCE DESC~:ON: SE~Q ID N0:6:
Met Val Ala Gly }~e V~l Lys Gly Asn Val Ala Asn l~p Lys Ile Ser
Glu Glu E~e Leu Lys Asn Ile Ser Ala Ser Ala Ar~ Glu E~o E~ Ser
Glu Asn Gly Ihr Ser Ile ~r Gly Ala Gly Gly ~e Met ~ l~rr Gly
~e q~r Gly Ihr Leu Ala Gly Ala Ala mr ~ys ~e Tyr Ala l~e Val
Gly E~e Asp C~ys Ile Ala n,r ~hr Gly Glu Glu Val Ar~ Asn ~ro Gln
Lys Ala Ile ~ Ile Gly Ile Val mr Ser Leu Leu Val Cys Phe Met
Ala l~ e Gly Val Ser Ala Ala Leu mr Leu Met Met ~7 Tyr Tyr
100 105 110
Leu T~ Asp Glu Lys Ser ~ro Leu Pro Val Ala Ehe Glu Tyr Val Arg
115 120 125
Trp Gly ~ro Ala Lys Tyr Val Val Ala Ala Gly &r Leu Cys Ala Leu
130 135 140
Ser Ihr Ser T~77 Leu Gly Ser Ile ~e ~ Met ~ro Ar~ Val Ile Tyr
145 150 15S 160
Ala Met Ala Glu Asp Gly Leu Leu ~e Lys Cys Leu Ala Gln Ile Asn
165 170 175
&r Lys mr Lys Ihr ~ro Val Ile Ala mr Leu Ser Ser Gly Ala Val
180 185 190
Ala Ala Val Met Ala ~e Leu ~e Asp Leu Lys Ala Leu Val Asp Met
195 200 205
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Met Ser Ile Gly Thr Leu Met Ala Tyr Ser Leu Val Ala Ala Cys Val
210 215 220
Leu Ile T ~ Arg Tyr Gln Pro Gly Leu Cys Tyr Glu Gln Pro Lys Tyr
225 230 235 240
Thr Pro Glu Lys Glu Thr Leu Glu Ser Cys Thr Asn Ala Thr Leu Lys
245 250 255
Ser 51u Ser Gln Val Thr Met Leu Gln Gly Gln Gly Fhe Ser Leu Arg
260 265 270
Thr Leu Ehe Ser Pro Ser Ala Leu Pro Thr Arg Gln Ser Ala Ser Leu
275 280 285
V~l Ser Phe Leu Val Gly Phe Leu Ala Phe Leu Ile T~l Gly Leu 5er
290 295 300
Ile Leu Ihr Thr Tyr Gly Val Gln Ala Ile Ala Arg Leu Glu Ala Trp
305 310 315 320
Ser Leu Ala Leu Leu Ala Leu ~he leu Val Leu Cys Ala Ala V~l Ile
325 330 335
L~u Thr Ile Trp Arg Gln Pro Gln Asn Gln Gln Lys Val Ala Phe Met
340 345 350
VA1 Pro Phe Leu Er~ E~e Leu F~o Ala he Ser Ile T~l Val A~n Ile
355 360 365
Tyr Leu Met V~l Gln Leu Ser Ala Asp Thr Trp Ile Arg Phe Ser Ile
370 375 380
Trp Met Ala Leu Gly E!he Leu Ile Tyr Phe Ala Tyr Gly Ile Arg His
385 390 395 400
Ser Lsu Glu Gly Asn PrD Arg Asp Glu Glu Asp Asp Glu Asp Ala Ehe
405 410 415
Ser Glu Asn Ile Asn Val Ala mr Glu Glu Lyæ Ser Val Met Gln Ala
420 425 430
Asn Asp His His Gln Ary Asn Leu Ser Leu Pro Phe Ile Leu His Glu
435 440 445
Lys Thr 5er Glu Cys
450
(2) INF~RXPIION F~R SEQ ID N0:7:
(i) 5EQUEN OE CH~R~l~KlSTYCS:
(A) LENGTH: 2157 base pairs
(B) TYPE: nucleic acid
(C) STRaN~EINESS: single
(D) T~POLDGY: linear
W O 92/10506 PCT/US91/09382
~) ()9 770~
~ 86 -
(:~x) ~:
(A) N~ME/KEY: CDS
(B) LDC~IION: 1482034
(Xi) SE~UENOE ~ESCRIPqIQN: SEQ ID NO:7:
COalCCTGOC G~AECCrCGC CGCCGCrGaC IIGG~rlC~G AAA ~ TGIATCCCTC 60
CTG~G~C~rC Tq1GCIGChA Ga~CEaGGCT G~CCIC53Gr GaG~GGrGG I~aGGCqIC~ 120
CGTCAIarIC CPGCTCIG;; C~GCAAC AIG GGG IGC A~A GTC CTG CTC AAC AIT 174
Met G1Y CYS VA1 T~- L u Asn Ile Ile
GGG CAG CAG ATG CTG CGG CGG AAG GTG GTG G~C IGT AGC CGG GAG GhG 222
Gly G1n G1n Met LeU Arg Arg Lys Val Val Asp Cys Ser Arg G1U G1U
ACG CGG CTG TCT OGC TGC CTG AAC ACT TTT GAT CIG GTG GCC crc GGG 270
Thr Arg T~ Ser Arg Cys Leu Asn Thr Phe Asp LRU Val Ala L~u Gly
GIG GGC AGC AC~ CTG GGr GCT GGr GTC TAC GIC C~G Gcr GG~ GCT GTG 318
Val Gly Ser Thr T~l Gly Ala Gly Val Tyr V~ T~l Ala Gly Ala Val
G C OGT G~G AAT GCA GGC OCT GCC ATT GTC AIC TCC TTC CIG A~C GCT 366
Ala Arg Glu Asn Ala Gly PrD Ala Ile Val Ile Ser Phe Leu Ile Ala
GCG CrG GCC TCA GIG CTG G T GGC CrG TCC IAT GGC GAG TIT GGT GCT 414
Ala Leu Ala Ser Val Leu Ala Gly Leu C~s Tyr Gly Glu Fhe Gly Ala
~ GrC CCC AAG AOG GGC TCA Gcr TAC CTC TAC AGC TAT GTC ACC' GTT 462
Arg Val Pro Lys Thr Gly Ser Ala Tyr Leu ~yr Ser Tyr Val Thr Val
100 105
GGA GAG CTC TGG GCC TTC ATC ACC GGC TGG AAC TTA ATC CTC TCC TAC 507
Gly Glu Leu Trp Ala Phe Ile Thr Gly Trp Asn Leu Ile Leu Ser Tyr
110 115 120
ATC ATC GGr ACT TCA AGC GTA GCG AGG GCC T~G AGC GCC ACC TrC GAC 558
Ile Ile Gly Thr Ser Ser Val Ala Ary Ala Trp Ser Ala Thr Phe Asp
125 130 135
GAG CTG ATA GGC AGA CCC AIC GGG GAG TTC TCA CGG ACA CAC ATG ACT 605
Glu Leu Ile Gly Arg Fr~ Ile Gly Glu Fhe Ser Arg Thr His Met Thr
140 145 150
20~770~
W O 92/10506 PCT/US91/09382
- 87 -
CTG AAC GCC CCC GGC GTG CTG GCT GAA AAC CCC GAC ATA ITC GCA Gl~ 654
Leu Asn Ala Pro Gly Val Leu Ala Glu Asn Pro Asp Ile Phe Ala Val
155 160 165
ATC ATA ATT CTC ATC ITG ACA GGA CTT TrA ACT CIT GGT GIG AAA GAG 702
Ile Ile Ile Leu Ile Leu Thr Gly Leu Leu Thr T~l Gly Val Lys Glu
170 175 180 185
TCG GCC AT~ GTC AAC AAA ATA ITC ACT TGT ATT AAC GTC CIG GTC CTG 750
Ser Ala Met Val Asn Lys Ile Phe Thr Cys Ile Asn Val Leu Val Leu
190 195 200
GGC TTC ATA ATG GIG TCA GGA TIT GTG AAA GGA TCG GIT AAA AAC TGG 798
Gly Phe Ile Met Val Ser Gly Phe Val Lys Gly Ser Val Lys Asn Trp
205 210 215
CAG CTC ACG GAG GAG GAr TTT GGG AAC A~ TC,A GGC CGT CTC TGT TrG 846
Gln Leu Thr Glu Glu Asp Phe Gly Asn Thr Ser Gly Arg Leu Cys Leu
220 225 230
AAC AAT GAC ACA AAA GAA GGG AAG CCC GGT GIT GGT GGA ITC ATG CCC 894
Asn Asn Asp ~hr Lys Glu Gly Lys Pro Gly Val Gly Gly Phe Met PrD
235 240 245
TTC GGG TTC TCT GGT GTC CTG qCG G~G GC~ G~G Acr T~C TTC ~T GC~ 942
Phe Gly Fhe Ser Gly Val Leu Ser Gly Ala Ala Thr Cys Yhe Tyr Ala
250 255 260 265
TTC GTG GGC ITT GAC TGC ATC GCC AOC ACA GGT GAA G~G GqG AAG AAC 990
Fhe Val Gly Ehe Asp Cys Ile Ala Thr Thr Gly Glu Glu Val Lys Asn
270 275 280
CCA CAG AAG.GCC ATC CCC G~l~ GGG AIC G$G GCG TCC CTC ITG A~C TGC 1038Pro Gln Lys Ala Ile Pro Val Gly Ile Val Ala Ser Leu Leu Ile Cys
285 290 295
ITC ATC G C TAC TIT GGG GTG TCG GrT GCC CTC ACG CTC ATG AIG CCC 1086
Phe Ile Ala Tyr Phe Gly V~l Ser Ala Ala T~- Thr Leu Met ~et Pro
300 305 310
TAC TTC TGC CTG GAC A~T AAC AGC CCC CTG CCC GAC GrC TTT A~G CAC 1134
Tyr Phe Cys Leu Asp Asn Asn Ser Pro Leu Pro Asp Ala Phe Lys His
315 320 325
GTG GGC TGG GAA GGT GCC AAG TAC GCA GTG GCC ~1~ GGC TCC CTC TGC 1182
Val Gly Trp Glu Gly Ala Lys Tyr Ala Val Ala Val Gly Ser Leu Cys
330 335 340 345
GCT CTT TCC GCC A~T CTT CIA GGT TCC ATG m ccc ATG CCT CGG GIT 1230
Ala Leu Ser Ala Ser Leu Leu Gly Ser Met Phe Pro Met Pro Arg Val
350 355 360
A'TC TAT GCC ATG GCT GAG GAT GGA. C~G CTA m AAA TTC TTA GCC A~C 1278
Ile Tyr Ala Mbt Ala Glu Asp Gly Leu Leu Phe Lys Phe Leu Ala Asn
365 370 375
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GTC AAT G~T AGG ACC AAA ACA CCA ATA ATC GCC AC~ TTA GCC TCG GGT 1326
Val Asn Asp Arg Thr Lys Thr Pr~ Ile Ile Ala Thr Leu Ala Ser Gly
380 385 390
GCC GTT G~l GCT GTG ATG GCC TTC CTC TTT GAC CTG AhG GAC TTG GIG 1374
Ala Val Ala Ala Val ~et Ala Phe Leu Phe Asp Leu Lys Asp Leu Val
395 400 405
G~C CTC AIG T~ ATT G~C AST CTc CIG GCT TAC TCG TIG GTG GCT GCC 1422
Asp Leu Met Ser Ile Gly Thr Leu L~u Ala Tyr Ser Leu Val Ala Ala
410 415 420 425
TGT G~G TIG GTC TIA u~ C~G CCA G~G CAG CCT AAC CTG GIA T~C 1470
Cys Val Leu Val Leu Arg Tyr Gln PrD Glu Gln Pr~ Asn Leu Val Tyr
430 435 440
C~G AT~ GCC AGT ACT TCC G~C G~G TT~ GAT CC~ GC~ GAC CAA A~T GAA 1518
Gln Met Ala Ser Thr Ser Asp Glu Leu Asp Pro Ala Asp Gln Asn Glu
445 450 455
TTG GCA AGC ACC AAT GAT TCC C~G C~G GGG m Tl~ cc~ GAG GCA GAG 1566
T~ Ala Ser Thr Asn Asp Ser Gln Leu Gly Phe Leu Pr~ Glu Ala Glu
460 465 470
AIG TTC TCT llG AAA A~C ATA CTC TCA CCC AAA AAC A~G G~G CCT TCC 1614
Met Phe Ser Leu Lys Thr Ile Leu Ser PrD Lys Asn Met Glu Pr~ Ser
475 480 485
AA~ ATC TCT GGG CI~ ATT GTG AAC AIT TC~ ACC AGC CTT AT~ GCT GIT 1662
Lys Ile Ser Gly T~l Ile Val Asn Ile Ser Thr Ser Leu Ile Ala Val
490 495 500 505
CTC ATC AIC ACC TTC T~C AIT GTG ACC GIG CTT GGA AGG G~G G T CTC 1710
T~l Ile Ile Thr Phe Cys Ile Val Thr Val Leu Gly Arg Glu Ala Leu
510 515 520
A~C AAA GGG GCG CTG TGG GC~ G~C TTT Cl~ crc GC~ GGG TCT GOC CIC 1758
Thr Lys Gly Ala Leu Trp Ala Ual Fhe Leu Leu Ala Gly Ser Ala Leu
525 530 535
CTC T~r GCC GTG G~ A~G GGC GTC AIC ~GG A~;G CAG CCC GAG A~;C AAG 1806
I~ Cys Ala Val Val qhr Gly V~l Ile qrp An~ Gln ~ro Glu Ser Lys
540 545 550
ACC AAG CTC TCA TTT AAG G~T CCC TTC CrG CCA GrG C~C COC ATC c~rG 1854
Thr Lys T~l Ser Phe Lys Val Pr~ Phe Leu Pro Val Leu Pro Ile Leu
555 560 565
AGC ATC TTC C~l~ AAC GTC TAT crc ArG A~ CAG C~ G~C CAG G5C ACC 1902
Ser Ile Phe Val Asn Val Tyr Leu Met Met Gln Leu Asp Gln Gly Thr
570 575 580 585
Tæ GTC ~;G m G T GTG TGG ATG CIG ATA GGC TrC ATC ATC TAC m 1950
Trp Val Ary Phe Ala Val Trp Met Leu Ile Gly Phe Ile Ile Tyr Phe
590 59S 600
W O 92/10506 2 0 9 7 7 0 5 PCT/USg1/09382
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GGC TAT GGC CIG TGG CAC AGC GAG GAG GCG TCC CTG GAr GCC GAC CAA 1998
Gly Tyr Gly Leu Trp His Ser Glu Glu Ala Ser Leu Asp Ala Asp Gln
605 610 615
GCA AGG ACT CCT GAC GGC AAC TTG GAC CAG T&C AAG T&ACGCACAG 2044
Ala Arg Thr Pro Asp Gly Asn Leu Asp Gln Cys Lys
620 625
COCDGCCrCC CEGPGGIGGC PGCa~CCCCG AGYGaLGCCC CC~GAGGACC GGG~GGCACC 2104
CCACCCTCCC CACC~GTGCA ACAG~AAOCA CCIaCGICC~ CACCCTCACT GCA 2157
(2) INFORMAIICN FOR SEQ ID ND:8:
(i) SE~UENOE CH~RACTERISTICS:
(A) LENGTff: 629 amuno acids
- (B) TYPE: a ~ acid
(D) IOFOLDGY: linear
(ii) MOL ~ TYPE: prokein
(xi) Sæ~UENOE DESCRIPqION: SEQ ID NO:8:
Met Gly Cys Val Leu Leu Asn Ile Ile Gly Gln Gln Met Leu Arg Arg
5 10 15
Lys Val Val Asp Cys Ser Arg Glu Glu Thr Arg Leu Ser Arg Cys Leu
20 25 30
Asn Thr Phe Asp Leu Val Ala Leu Gly Val Gly Ser Thr heu Gly Ala
35 40 45
Gly Val Tyr Val T~l Ala Gly Ala Val Ala Arg Glu Asn Ala Gly Pro
50 55 60
Ala Ile Val Ile Ser Phe Leu Ile Ala Ala Leu Ala Ser Val Ieu Ala
65 70 75 80
Gly Leu Cys Tyr Gly Glu Phe Gly Aaa Arg Val Pro Lys Thr Gly Ser
85 90 95
Ala Tyr Leu Tyr Ser Tyr Val Thr Val Gly Glu Leu Trp Ala Phe Ile
100 105 110
Thr Gly Trp Asn Leu Ile Leu Ser Tyr Ile Ile Gly Thr Ser Ser Val
llS 120 125
Ala Arg Ala Trp Ser Ala Thr Phe Asp Glu Leu Ile Gly Arg Pro Ile
130 135 140
Gly Glu Phe Ser Arg Thr His Met m r Leu Asn Ala Pro Gly Val Leu
145 150 155 160
W O 92/10506 2 0 9 7 7 0 5 PCT/US91/09382
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Ala Glu Asn PrD Asp Ile Phe Ala -Val Ile Ile Ile Leu Ile Leu Thr
165 170 175
Gly Leu Leu ~hr Leu Gly Val Lys Glu Ser Ala Met Val Asn Lys Ile
180 185 190
Phe Thr Cys Ile Asn Val Leu Val Leu Gly Phe Ile Met Val Ser Gly
195 200 205
Fhe Val Lys Gly Ser Val Lys Asn Trp Gln Leu Thr Glu Glu Asp Phe
210 215 220
Gly Asn Thr Ser Gly Arg T~l Cys Leu Asn Asn Asp Ihr Lys Glu Gly
225 230 235 240
Lys Pr~ Gly Val Gly Gly Phe Met Pr~ Phe Gly Phe Ser Gly Val Leu
245 250 255
Ser Gly Ala Ala ffl r Cys Phe Tyr Ala Phe Val Gly Fhe Asp Cys Ile
260 265 270
Ala Thr Thr Gly Glu Glu V~l Lys Asn Pr~ Gln Iys Ala Ile PrD Val
275 - 280 285
Gly Ile Val Ala Ser L u L~u Ile Cys Phe Ile Ala Tyr Phe Gly Val
230 295 300
Ser Ala Ala T~l Thr T~l Met Met Pro Tyr Phe Cys Leu Asp Asn Asn
305 310 315 320
Ser Pr~ Leu Pr~ Asp Ala Phe Lys His Val Gly TSp Glu Gly Ala Ly~
325 330 335
Tyr Ala Val Ala Val Gly Ser Ieu Cys Ala Leu Ser Ala Ser Leu T~
340 345 350
Gly Ser Met Phe Pro Met Pro Arg Val Ile Tyr Ala Met Ala Glu Asp
355 360 365
Gly Leu Leu Phe Lys Phe Leu Ala Asn Val Asn Asp Arg Thr Lys Thr
370 375 380
Pro Ile Ile Ala Thr L u Ala Ser Gly Ala Val Ala Ala Val Met Ala
385 390 395 400
Phe Leu Phe Asp Leu Lys Asp T~l Val Asp Leu Met Ser Ile Gly Thr
405 410 415
Leu Leu Ala Tyr Ser Leu Val Ala Ala Cys Val Leu Val Leu Arg Tyr
420 425 430
Gln PrD Glu Gln Pr~ Asn Leu Val Tyr Gln Met Ala Ser Thr Ser Asp
435 440 445
Glu Leu Asp Pro Ala Asp Gln Asn Glu Leu Ala Ser Ihr Asn Asp Ser
450 455 460
W O 92/10506 2 0 9 7 7 ~ 5 PCT/US91/09382
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Gln Leu Gly Fhe Leu Pro Glu Ala Glu Met Phe Ser Leu Lys Thr Ile
465 470 475 480
T~l Ser Pro Lys Asn Met Glu Pro Ser Lys Ile Ser Gly Leu Ile Val
485 490 495
Asn Ile Ser Thr Ser Leu Ile Ala Val Leu Ile Ile Ihr Fhe Cys Ile
500 505 510
Val Thr V~l Leu Gly Arg Glu Ala Leu Ihr Lys Gly Ala LPU Trp Ala
515 520 525
V 1 Phe Leu Leu Ala Gly Ser Ala T~7 Leu Cys Ala Val Val Thr Gly
530 535 540
Val Ile Trp Arq Gln Pro Glu Ser Lys Thr Lys Leu Ser Phe Lys Val
545 550 555 560
Pro Phe Leu Pro Val Leu Pro Ile T~- Ser Ile Phe Val Asn Val Tyr
565 570 575
T~l Met M~t Gln Leu Asp Gln Gly Thr Trp Val Arg Phe Ala Val Trp
580 585 590
xet T~- Ile Gly Phe Ile Ile ~ Phe Gly Tyr Gly Leu ~ His Ser
595 600 605
Glu Glu Ala Ser Leu Asp Ala Asp Gln Ala Arq Thr Pro Asp Gly Asn
610 615 620
Leu Asp Gln Cys Lys
625
