U.S. patent application number 13/831464 was filed with the patent office on 2013-07-25 for a novel chimeric protein for prevention and treatment of hiv infection.
This patent application is currently assigned to Department of Health and Human Service. The applicant listed for this patent is The Government of the United States of America as represented by the Secretary of the Department of Health and Hu. Invention is credited to Edward A. Berger, Christie M. Del Castillo.
Application Number | 20130189264 13/831464 |
Document ID | / |
Family ID | 37037216 |
Filed Date | 2013-07-25 |
United States Patent
Application |
20130189264 |
Kind Code |
A1 |
Berger; Edward A. ; et
al. |
July 25, 2013 |
A NOVEL CHIMERIC PROTEIN FOR PREVENTION AND TREATMENT OF HIV
INFECTION
Abstract
This invention relates to bispecific fusion proteins effective
in viral neutralization. More specifically, such proteins have two
different binding domains, an inducing-binding domain and an
induced-binding domain, functionally linked by a peptide linker.
Such proteins, nucleic acid molecules encoding them, and their
production and use in preventing or treating viral infections are
provided. One prototypical bispecific fusion protein is
sCD4-SCFv(17b), in which a soluble CD4 fragment (containing domains
D1 and D2) is fused to a single chain Fv portion of antibody 17b
via a linker.
Inventors: |
Berger; Edward A.;
(Rockville, MD) ; Del Castillo; Christie M.; (San
Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
the Secretary of the Department of Health and Hu; The Government of
the United States of America as represented by |
Rockville |
MD |
US |
|
|
Assignee: |
Department of Health and Human
Service
Rockville
MD
The Government of the United States of America as represented by
the Secretary of the
|
Family ID: |
37037216 |
Appl. No.: |
13/831464 |
Filed: |
March 14, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11535957 |
Sep 27, 2006 |
8420099 |
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13831464 |
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09936702 |
Sep 13, 2001 |
7115262 |
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PCT/US00/06946 |
Mar 16, 2000 |
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11535957 |
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60124681 |
Mar 16, 1999 |
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Current U.S.
Class: |
424/136.1 ;
435/325; 435/375; 435/69.7; 530/387.3; 536/23.4 |
Current CPC
Class: |
C07K 16/1045 20130101;
Y10S 435/975 20130101; A61K 39/395 20130101 |
Class at
Publication: |
424/136.1 ;
530/387.3; 536/23.4; 435/325; 435/69.7; 435/375 |
International
Class: |
C07K 16/10 20060101
C07K016/10 |
Claims
1. A neutralizing bispecific fusion protein capable of binding to
two sites on a target protein, comprising a first binding domain
capable of binding to an inducing site on the target protein,
thereby exposing an induced epitope; a second binding domain
capable of forming a neutralizing complex with an induced epitope
of the target protein; and a linker connecting the first domain to
the second domain.
2. The protein according to claim 1, wherein the first binding
domain comprises a binding domain of a variable region of an
antibody heavy or light chain.
3. The protein according to claim 2, wherein the antibody binding
domain mimics a biological activity of a CD4 molecule in binding to
the inducing site and exposing the inducing epitope.
4. The protein according to claim 1, wherein the first binding
domain is derived from a CD4 molecule.
5. The protein according to claim 1, wherein the second binding
domain comprises a binding domain of a variable region of an
antibody heavy or light chain.
6. The protein according to claim 1, wherein the target protein is
a viral envelope protein of a virus.
7. The protein according to claim 6, wherein the viral envelope
protein is gp120.
8. The protein according to claim 7, wherein the second binding
domain mimics a biological activity of an HIV coreceptor molecule
in binding to gp120.
9. The protein according to claim 8, wherein the second binding
domain is a peptide fragment of a chemokine receptor selected from
the group consisting of CXCR4, CCR2B, CCR3, and CCR5, CCR8, CCR9,
CX.sub.3CR1, US28, or the chemokine receptor related proteins
including STRL33, GPR15, APJ, ChemR23, and BLTR.
10. The protein according to claim 5, wherein the binding domain of
the antibody is capable of binding to at least one coreceptor
binding determinant of gp120.
11. The protein according to claim 1, wherein the linker maintains
the second binding domain in binding proximity to the induced
epitope when the first binding domain is bound to the inducing
site.
12. The protein according to claim 11, wherein the linker is: (a)
substantially flexible; (b) 15-100 angstroms (.ANG.) long; (c)
10-100 amino acid residues in length; or (d) any two or more of
(a), (b), and (c).
13. An isolated nucleic acid molecule encoding a protein according
to claim 1.
14. A transgenic eukaryotic cell comprising the isolated nucleic
acid molecule according to claim 13.
15. A method for producing in a eukaryotic cell a functional
recombinant bispecific fusion protein capable of binding two sites
on a target protein, comprising the steps of: a) transfecting the
eukaryotic cell with a recombinant nucleic acid molecule according
to claim 13; b) culturing the transfected eukaryotic cell under
conditions that cause production of the protein; and c) recovering
the protein produced by the cultured eukaryotic cell.
16. The method of claim 15, wherein the eukaryotic cell is a
mammalian cell.
17. The method of claim 15, wherein recovering the protein
comprises: a) identifying the protein by the presence of a
molecular tag; and b) separating the protein having the molecular
tag so identified from molecules without the tag, so as to recover
the protein produced by the cultured eukaryotic cell.
18. A method for inactivating a gp120 protein and/or for blocking
and preventing the binding of a viral or recombinant gp120 protein
to soluble CD4 or lymphocyte CD4, comprising the step of:
contacting the gp120 protein with a protein according to claim 7,
or a variant protein, analog or mimetic thereof.
19. A method for neutralizing a human immunodeficiency virus,
comprising the step of: contacting the human immunodeficiency virus
with a protein according to claim 7, or a variant protein, analog
or mimetic thereof.
20. A method for inhibiting HIV virus replication or infectivity in
a subject, comprising administering to the subject an amount of the
protein according to claim 7, or a variant protein, analog or
mimetic thereof, sufficient to inhibit HIV virus replication or
infectivity.
21. The method according to claim 20, wherein the protein is
administered in the form of a pharmaceutical composition.
22. A composition comprising the protein of claim 1, or a variant
protein, analog or mimetic thereof.
23. A pharmaceutical composition comprising the protein according
to claim 1, or a variant protein, analog or mimetic thereof, and a
pharmaceutically acceptable carrier.
24. A kit for treatment and/or prevention of HIV infection,
comprising a clinically effective dose of the neutralizing
bispecific fusion protein of claim 1.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional of co-pending U.S. patent application
Ser. No. 11/535,957, filed Sep. 27, 2006; which is a continuation
of U.S. patent application Ser. No. 09/936,702, filed Sep. 13,
2001, now U.S. Pat. No. 7,115,262, issued Oct. 3, 2006; which is
the U.S. National Stage of International Application No.
PCT/US00/06946, filed Mar. 16, 2000; and claims the benefit of U.S.
Provisional Application No. 60/124,681, filed Mar. 16, 1999, all of
which are incorporated herein in their entirety.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS AN ASCII TEXT
FILE
[0002] A Sequence Listing is submitted herewith as an ASCII
compliant text file named "Sequence_Listing.txt", created on Mar.
12, 2013, and having a size of .about.9.90 kilobytes, as permitted
under 37 CFR 1.821(c). The material in the aforementioned file is
hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] This invention relates to proteins useful in the prevention
and treatment of human immunodeficiency virus (HIV) infection. More
specifically, it relates to fusion proteins that bind to two sites
on a single target protein, especially when one binding domain of
the fusion protein binds to an induced site (on the target protein)
that is exposed by the binding of the other binding domain of the
fusion protein.
BACKGROUND OF THE INVENTION
[0004] Acquired immune deficiency syndrome (AIDS) is a fatal
disease of growing prevalence in the modern world. The agent
responsible for this disease, human immunodeficiency virus (HIV),
was first identified in 1983. HIV is a T-lymphotropic retrovirus
that invades and replicates in cells of the immune system,
primarily helper T-lymphocytes. The consequent dysfunction in
T-lymphocyte-mediated immunity results in an immuno-compromised
condition. Patients usually die of associated opportunistic viral,
bacterial or fungal infections. A characteristics laboratory
finding in AIDS is the decrease in helper T lymphocytes (CD4), and
particularly a steady decrease in the ratio of CD4 to suppressor T
lymphocytes CD8 as the disease progresses. Virus binding is
primarily mediated by interaction of gp120, the external subunit of
the HIV envelope glycoprotein (Env) with CD4 protein and various
coreceptor molecules (one of several alternative chemokine
receptors). These interactions then activate the gp41 transmembrane
subunit of the envelope glycoprotein, to cause fusion between the
virus and cell membranes. See Retroviruses, Coffin et al. (eds.)
(1997) CSHP, New York, Ch. 11.
[0005] The humoral immune system is triggered by HIV infection,
though it generally does not provide sufficient protection to ward
off the infection. Env is the major target of anti-HIV neutralizing
antibodies (Wyatt et al. Nature 393:705-711, 1998). However, Env
has evolved so that its relatively invariant neutralizing
determinants are protected from the humoral immune system.
Antibodies to these regions therefore are generated at a low
frequency and their neutralizing activities in vivo are generally
weak. Certain variable regions (e.g., the V3 loop) are targets for
potent neutralizing antibodies, but these are typically restricted
to a limited number of HIV-strains (in other words, they are not
broadly cross-reactive). For a list of several gp120 antigenic
epitopes and consensus definitions of the conserved and variable
regions of gp120, see published PCT application PCT/US98/02766
(publication number WO 98/36087) and Coffin et al. (eds.) (1997)
CSHP, New York, Ch. 12.
[0006] A neutralizing monoclonal antibody (MAb) with potent and
broadly cross-reactive activity would have great potential value in
protocols aimed at preventing HIV infection before or immediately
after exposure, for example in neonatal transmission, post-exposure
prophylaxis, and as a topical inhibitor. Such a MAb may also be
useful in treating chronic infection (D'Souza et al. J. Infect.
Dis. 175:1056-1062, 1997). However only a handful of MAbs with the
desired broadly cross-reactive neutralizing activities have been
described. Because of limited potency and cross-reactivity of these
molecules, even the three most promising candidates have
questionable clinical value (D'Souza et al., 1997).
[0007] Extensive efforts are underway to provide immunological or
pharmacological approaches to controlling HIV infection (Coffin et
al., 1997, Ch. 12). The specific interaction between gp120 and CD4
has been exploited in efforts to provide a possible treatment for
HIV infection. See, e.g., U.S. Pat. No. 5,817,767; Capon et al.,
Nature 337:525-531, 1989. A soluble fragment of CD4 (sCD4),
comprising the first and second domains of this protein (D1D2) has
been generated, and this molecule interacts specifically with
gp120, essentially serving as a molecular decoy. sCD4 has been
shown to block the spread of HIV between cultured cells (Moore et
al., Science 250:1139-1142, 1990). However, clinical trials with
sCD4 were inconclusive as to the effects on human viral load
(Schooley et al., Ann. Internal Med. 112:247-253, 1990; Kahn et
al., Ann. Internal Med. 112:254-261, 1990). Subsequent studies
indicated that, unlike laboratory-adapted HIV strains, isolates
obtained directly from infected patients (primary isolates) are
resistant to neutralization by sCD4 (Darr et al., Proc. Natl. Acad.
Sci. 87:6574-6578, 1990).
[0008] In another approach, researchers have generated an
antibody-like molecule by fusing the binding portion of CD4 to the
constant region (Fc) of a human IgG heavy chain (see, e.g., Capon
et al., Nature 337:525-531, 1989; and Byrn et al., Nature
344:667-670, 1990). This molecule, termed CD4 immunoadhesin,
exploits the native functions of immunoglobulin Fc, such as its
ability to fix complement, its ability to mediate
antibody-dependent cytotoxicity, and its transfer across the
placental barrier. There are significant drawbacks to using Fc
receptors in association with CD4, because such a construct may be
responsible for targeting HIV to Fc-receptor bearing cells (e.g.
macrophages), and might lead to increased transmission of HIV-1
across the placental barrier.
[0009] A complementary recombinant molecule has also been made,
wherein the binding portion of CD4 is fused to the Fv region of an
antibody directed to human CD3; this "Janusin" molecule may be able
to re-target cytotoxic T-lymphocytes onto HIV-infected cells
(Traunecker et al., Embo J. 10:3655-3659, 1991; Traunecker et al.,
Int. J. Cancer: Supp. 7:51-52, 1992). Janusin has been reported to
inhibit HIV-mediated cell fusion when administered in vitro with
neutralizing antibody to either gp41 or the V3 loop of gp120
(Allaway et al., AIDS Res. Hum. Retroviruses 9:581-587, 1993; U.S.
Pat. No. 5,817,767). This system is inherently complicated and
inefficient because multiple molecules must be co-administered to
the subject.
[0010] This invention is directed to proteins that address key
failures of the prior art.
SUMMARY OF THE INVENTION
[0011] The present invention takes advantage of the finding that
the neutralizing activities of MAbs against certain highly
conserved determinants of the coreceptor-binding region of gp120
are revealed only when CD4 first binds to gp120 (as in an
sCD4-activated fusion assay). Although some MAbs to CD4-induced
epitopes (e.g., the human MAbs 17b and 48d, Thali et al., J. Virol.
67:3978-3988, 1993) are broadly cross-reactive with Envs from
diverse HIV genetic subtypes (Clades), these neutralizing epitopes
are only briefly exposed in vivo, and therefore have provided poor
targets for clinically protective antibody binding.
[0012] The inventors have overcome these difficulties by creating a
fusion protein containing a fragment of CD4 attached via a linker
to a human single chain Fv directed against an induced (for
example, a CD4-induced) neutralizing epitope on gp120, for instance
a coreceptor-binding determinant of gp120. CD4-binding exposes
highly conserved gp120 determinants involved in binding to
coreceptor; therefore the provided fusion protein will have the
properties of a highly potent, broadly cross-reactive neutralizing
antibody with high in vivo activity and no Fc-mediated undesirable
targeting properties. When the fusion protein is substantially
derived from human proteins, it has minimal immunogenicity and
toxicity in humans. Such an agent has great value in the prevention
of infection during or immediately after HIV exposure
(mother/infant transmission, post-exposure prophylaxis, topical
inhibitor), and also in the treatment of chronic infection.
[0013] Accordingly, a first embodiment of the current invention is
a neutralizing bispecific fusion protein capable of binding to two
sites on a target protein. This protein has two different binding
domains, an inducing-binding domain and an induced-binding domain,
functionally linked by a peptide linker. Nucleic acid molecules
encoding such fusion proteins are further aspects of this
invention. Also encompassed in the invention are protein analogs,
derivatives, or mimetics of such neutralizing bispecific fusion
proteins. The arrangement of the inducing- and induced-binding
domains need not be organized in binding sequence; the
amino-proximal or carboxy-proximal binding domain of the fusion
protein may be either the induced-binding or the inducing-binding
domain.
[0014] In certain embodiments, the linker of this invention is of
such length and secondary structure that the linker allows the
second binding domain to be in binding proximity to the induced
epitope of the target protein when the first binding domain is
bound to the inducing site of the target protein. The linker may
for instance be substantially flexible. Linkers of about 25-100
angstroms (.ANG.), or about 15-100 amino acid residues in length,
are examples of linkers of a sufficient length to maintain the
second binding domain in binding proximity to the induced epitope.
Specific examples of linkers will include one or more occurrences
of the amino acid sequence represented by SEQ ID NO: 1. For
instance, the invention encompasses bispecific fusion proteins
wherein the two binding domains are functionally linked by the
amino acid sequence represented by SEQ ID NO: 2.
[0015] Targets for bispecific fusion proteins according to this
invention include viral envelope proteins. For instance, viral
envelope proteins from the human immunodeficiency virus (HIV) are
targets for the disclosed invention. In a specific embodiment of
the invention, the viral envelope protein target is gp120.
[0016] In further aspects of the invention, the first binding
domain is capable of binding to an inducing site on the target
protein, thereby exposing an induced epitope. For instance, the
first binding domain can be a ligand such as CD4 or fragments
thereof. Alternatively, such a first binding domain may be a
binding portion of a variable region of an antibody heavy or light
chain. The first binding domain may include, for example, an
antibody-binding domain, a single-chain Fv (SCFv), or binding
fragments thereof.
[0017] The second binding domain, which is capable of forming a
neutralizing complex with an induced epitope of the target protein,
may be for example an antibody or fragments thereof, such as the
variable region, Fv, Fab or antigen-binding domain of an antibody.
Another example of the second binding domain of the fusion protein
is an engineered single-chain Fv (SCFv).
[0018] In some particular examples where HIV gp120 is the target,
and the inducing site is the gp120 CD4 binding site, the induced
epitope may be a coreceptor-binding determinant of gp120.
Accordingly, aspects of this invention include proteins in which
the first binding domain binds to gp120 in such a way as to cause a
CD4-induced conformational change in the complexed gp120 that
exposes the second binding domain. The first binding domain may be
derived from a CD4 molecule, and include CD4 and soluble fragments
thereof (sCD4, e.g. D1, D1D2 and other such fragments), and
proteins that mimic the biological activity of a CD4 molecule in
binding to the inducing site of gp120. In another embodiment of the
invention, the first domain of the gp120-targeted bispecific fusion
protein is derived from a CD4 anti-idiotypic antibody, or
antibodies that mimic CD4 in exposing epitopes.
[0019] The second domain of the gp120-targeted bispecific fusion
protein, which binds to an epitope induced by binding of the first
fusion domain, may be chosen from domains and fragments of proteins
that bind to such CD4 induced epitopes. Antibodies directed to the
induced epitopes, as well as the HIV coreceptor (e.g. a chemokine
receptor), HIV coreceptor mimics, and fragments of HIV coreceptor
proteins, are examples of sources for the second binding domain of
a gp120-target bispecific fusion protein of this invention.
Examples of chemokine receptors with HIV coreceptor activity
include CXCR4, CCR5, CCR2B, and CCR3. Neutralizing antibodies,
including 17b and 48d, are examples of antibodies. Fusion proteins
wherein the second domain is an engineered single chain Fv (SCFv)
derived from such a neutralizing antibody are also encompassed.
[0020] A particular embodiment of this invention is a functional
recombinant bispecific fusion protein capable of binding to two
sites on gp120, wherein the inducing-binding domain is sCD4; the
induced-binding domain is SCFv(17b); and these two domains are
linked by a linker of a length sufficient to maintain the SCFv(17b)
in binding proximity an SCFv(17b) epitope when sCD4 is bound to
gp120. A prototypical bispecific fusion protein has the amino acid
sequence shown in SEQ ID NO: 3. Nucleic acid molecules encoding
such a fusion protein are also encompassed; the prototypical
nucleic acid molecule has the sequence shown in SEQ ID NO: 4.
Vectors and cells comprising this nucleic acid molecule are also
encompassed in the current invention, as are transgenic plants and
animals that express the nucleic acid molecule.
[0021] This invention also provides methods for producing
functional recombinant bispecific fusion proteins capable of
binding two sites on a target protein. Such a protein can be
produced in a prokaryotic or eukaryotic cell (e.g., yeast, insect
and mammalian cells), for instance by transforming or transfecting
such a cell with a recombinant nucleic acid molecule comprising a
sequence which encodes a disclosed bispecific fusion protein. Such
transformed cells can then be cultured under conditions that cause
production of the fusion protein, which is then recovered through
protein purification means. The protein can include a molecular
tag, such as a six-histidine tag, to facilitate its recovery. In
particular embodiments, the protein has a hexa-histidine (hexa-his)
tag, and a thrombin cleavage site.
[0022] The invention further provides methods for inactivating a
target protein, for instance a gp120 protein, by contacting the
target protein with a fusion protein according to this invention.
Where the target protein is gp120, this method involves contacting
gp120 with a gp120-targeted bispecific fusion protein, for instance
sCD4-SCFv(17b). Proteins according to the current invention can
also be used to neutralize a human immunodeficiency virus, by
contacting the human immunodeficiency virus with a gp120-targeted
fusion protein according to this invention. Binding of a viral or
recombinant gp120 protein to soluble CD4 or lymphocyte CD4 can also
be blocked and/or prevented by contacting the gp120 protein with
gp120-targeted fusion protein. In any of these methods, a variant
protein, analog or mimetic of the fusion protein as provided herein
may also be used.
[0023] Proteins of the current invention can be used to inhibit
virus replication or infectivity in a subject by administering to
the subject an amount of the fusion protein (for example the
sCD4-SCFv(17b) fusion protein), or a variant protein, analog or
mimetic thereof, sufficient to inhibit HIV virus replication or
infectivity. The fusion protein can be administered in a
pharmaceutical composition, and given therapeutically to a person
who is known to be infected with HIV, or prophylactically to help
prevent infection in someone who has been exposed to the virus, or
is at high risk for exposure. Proteins of this invention can also
be administered in combination with another compound for the
treatment or prevention of HIV infection, such as an HIV reverse
transcriptase (RT), integrase, or protease inhibitor, another HIV-1
neutralizing antibody, or an Env-targeted toxin. The other drug may
be an HIV antiviral agent, an HIV anti-infective agent, and/or an
immunomodulator, or combinations thereof.
BRIEF DESCRIPTION OF THE FIGURES
[0024] FIG. 1 is a bar graph illustrating relative HIV-1
Env-mediated fusion, in the presence (+) or absence (-) of soluble
CD4, between effector cells expressing Env (Ba-L) and target cells
expressing CCR5 (co-receptor), but no CD4 (primary receptor).
[0025] FIG. 2 is a graph showing that antibody 17b does not inhibit
HIV-1 Env-mediated fusion in the conventional assay (open box:
CXCR4 and CD4 on target cell), but strongly inhibits cell fusion in
the sCD4-activated assay (filled circle: only CXCR4 on target cell,
sCD4 provided). Additional experiments indicate that this
phenomenon occurs with diverse Envs using either CXCR4 or CCR5, and
that 17b has broad cross-reactive activity with Envs from
genetically diverse HIV-1 isolates.
[0026] FIG. 3 is a schematic diagram of the CD4-SCFv(17b) genetic
construct. The genetic construct encodes sCD4 (D1D2, plus the
native CD4 N-terminal signal sequence), followed by the L1 linker
(Gly.sub.4Ser).sub.7, which attaches the 17b SCFv (VH attached to
VL via the L2 linker (Gly.sub.4Ser).sub.3), followed by the
thrombin cleavage site and hexa-his tag. There is a BamH I site in
the middle of L1 to facilitate production of constructs of
different lengths.
[0027] FIG. 4 is a drawing of mechanisms of binding of a
sCD4-SCFv(17b) to gp120, and the resulting neutralization of HIV
Env function (fusion and infectivity).
[0028] FIGS. 4A, 4B, and 4C depict the proposed interaction of HIV
(mediated by gp120) with the cell surface receptor CD4 and
co-receptor CCR5, and the beginning of fusion (mediated by gp41).
Interaction between gp120 and CD4 (FIG. 4A) causes a change in the
conformation of gp120 (FIG. 4B), which enables interaction between
gp120 and CCR5 (FIG. 4B). This triggers a conformational change in
gp41 (FIG. 4C), and leads to fusion. Antibody (for instance, MAb
17b) binds poorly to the transiently exposed epitope on gp120 (FIG.
4B), and thus results in only weak neutralization of fusion or
infection.
[0029] FIGS. 4D and 4E depict a proposed mechanism of
sCD4-SCFV(17b) neutralization of fusion. In the presence of the
bispecific chimeric fusion protein, the sCD4 domain can bind to
gp120 and induce a conformational change in this protein sufficient
to permit binding of the SCFV(17b) (FIG. 4D). This effectively
blocks fusion between the HIV and infection and the target
cell.
SEQUENCE LISTING
[0030] The nucleic and amino acid sequences listed in the
accompanying sequence listing are shown using standard letter
abbreviations for nucleotide bases, and three letter code for amino
acids. Only one strand of each nucleic acid sequence is shown, but
the complementary strand is understood as included by any reference
to the displayed strand.
[0031] SEQ ID NO: 1 shows the basic repeat cassette for a linker
polypeptide.
[0032] SEQ ID NO: 2 shows a seven-repeat polypeptide linker
[0033] SEQ ID NO: 3 shows the amino acid sequence of the
CD4-SCFv(17b) chimeric protein.
[0034] SEQ ID NO: 4 shows the nucleic acid sequence of
CD4-SCFv(17b).
[0035] SEQ ID NO: 5 shows the pair of synthetic oligonucleotides
used to form the second half of the Stu I site near the 3' end of
CD4 and to produce an Spe I overhang at the 3' end of an
intermediate construct (site to be destroyed upon ligation into
pCB-3); the oligonucleotide sequences reconstruct the remainder of
the second domain of CD4 (through ser.sub.183), and encode an amino
acid sequence including ala.sub.182ser.sub.183 of CD4 D2 plus an
intermediate 37 residue linker
(gly.sub.4ser).sub.6gly.sub.4thr.sub.2ser, followed directly by the
universal translational termination sequence (UTS).
[0036] SEQ ID NO: 6 shows the peptide sequence encoded for by the
nucleotide sequences in SEQ ID NO: 5.
[0037] SEQ ID NO: 7 shows the forward (5') primer used to amplify
and attach the 17b SCFv sequence to the CD4-linker sequence in
pCD2. Italics show the region of the primer that overlaps with
17b.
[0038] SEQ ID NO: 8 shows the amino acid sequence encoded by the
oligonucleotide primer in SEQ ID NO:7. This sequence includes the
GlySer residues at the third (Gly.sub.4Ser) repeat within L1
(encoded by the BamH I site, followed by the remaining four
(Gly.sub.4Ser) repeats, followed by the first ten residues of the
17b SCFv (shown in italics).
[0039] SEQ ID NO: 9 shows the 3' primer used to amplify and attach
the 17b SCFv sequence plus the thrombin cleavage site and the
hexa-his tag to the CD4-linker sequence in pCD2.
[0040] SEQ ID NO: 10 shows the peptide encoded for by the
nucleotide sequence in SEQ ID NO: 9.
DETAILED DESCRIPTION OF THE INVENTION
I. Abbreviations and Definitions
A. Abbreviations
[0041] HIV: human immunodeficiency virus gp120: the external
subunit of the envelope glycoprotein complex of HIV Env: the
envelope glycoprotein complex of HIV MAb: monoclonal antibody Fv:
antibody "fragment variable", the variable region of an antibody
SCFv: single-chain antibody variable region
B. Definitions
[0042] Unless otherwise noted, technical terms are used according
to conventional understanding. Definitions of common terms in
molecular biology may be found in Benjamin Lewin, Genes V, Oxford
University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.),
The Encyclopedia of Molecular Biology, Blackwell Science Ltd., 1994
(ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology
and Biotechnology: a Comprehensive Desk Reference, VCH Publishers,
Inc., 1995 (ISBN 1-56081-569-8).
[0043] In order to facilitate review of the various embodiments of
the invention, the following definitions of terms are provided:
[0044] Animal: Living multi-cellular vertebrate organisms, a
category that includes, for example, mammals and birds. The term
mammal includes both human and non-human mammals. Similarly, the
term "subject" includes both human and veterinary subjects.
[0045] Bispecific fusion protein: Proteins that have at least two
domains fused together, each domain comprising a binding region
capable of forming a specific complex with a target protein. In
general, the two domains are genetically fused together, in that
nucleic acid molecules that encode each protein domain are
functionally linked together, for instance by a linker
oligonucleotide, thereby producing a single fusion-encoding nucleic
acid molecule. The translated product of such a fusion-encoding
nucleic acid molecule is the bispecific fusion protein.
[0046] The two binding regions of such a bispecific protein may
associate with two different binding determinants or epitopes on a
single target molecule. One binding domain may bind first to such a
target and thereby induce a conformational change in the target
such that the binding of the second binding domain to the target is
enabled, facilitated, or otherwise increased in affinity. In such
an instance, the domain that binds first to the target can be
referred to as the inducing-binding domain, while the domain that
binds second is the induced-binding domain. These fusion protein
domains need not be organized in binding sequence; the
amino-proximal binding domain of the fusion protein may be either
the induced-binding or the inducing-binding domain; likewise for
the carboxy-proximal binding domain.
[0047] Bispecific fusion proteins can be further labeled according
to the target protein they bind to and neutralize. For instance, a
bispecific fusion protein according to the current invention that
binds to two specific sites on HIV gp120 protein may be referred to
as a gp120-targeted bispecific fusion protein.
[0048] CD4: Cluster of differentiation factor 4, a T-cell surface
protein that mediates interaction with the MHC class II molecule.
CD4 also serves as the primary receptor site for HIV on T-cells
during HIV infection.
[0049] Molecules that are derived from CD4 include fragments of
CD4, generated either by chemical (e.g. enzymatic) digestion or
genetic engineering means. Such a fragment may be one or more
entire CD4 protein domains (for example, extracellular domains D1,
D2, D3, and D4), as defined in the immunological literature, or a
portion of one or more of these well-defined domains. For instance,
a binding molecule or binding domain derived from CD4 would
comprise a sufficient portion of the CD4 protein to mediate
specific and functional interaction between the binding fragment
and a native or viral binding site of CD4. One such binding
fragment includes both the D1 and D2 extracellular domains of CD4
(CD4 D1D2), though smaller fragments may also provide specific and
functional CD4-like binding. The gp120-binding site has been mapped
to D1 of CD4.
[0050] The term "CD4-derived molecules" also encompasses analogs
(non-protein organic molecules), derivatives (chemically
functionalized protein molecules obtained starting with the
disclosed protein sequences) or mimetics (three-dimensionally
similar chemicals) of the native CD4 structure, as well as proteins
sequence variants or genetic alleles, that maintain the ability to
functionally bind to a target molecule.
[0051] CD4-induced conformational change: A change induced in the
three-dimensional conformation of the interacting gp120 protein
when CD4 specifically interacts with gp120 to form a complex. One
characteristic of such a change is the exposure of at least one
induced epitope on the interacting gp120 molecule. An epitope
induced by such a change is called a CD4-induced epitope. Such a
CD4-induced epitope may for instance include gp120 epitopes at or
near the co-receptor-binding region of the protein.
[0052] In addition to CD4 binding, the binding of other molecules
may induce the exposure of induced epitopes on gp120. Such other
inducing molecules are considered CD4-like in terms of their
epitope-inducing ability, to the extent that they expose epitopes
congruent with or equivalent to those induced epitopes exposed upon
the binding of native CD4. These other inducing molecules include,
but in no way are limited to, fragments of CD4, for instance sCD4,
or a fragment containing the D1 or D1 and D2 domains of native CD4.
A mannose-specific lectin (SC) may also serve to expose a
CD4-induced epitope (see U.S. Pat. No. 5,843,454), as can certain
anti-gp120 MAbs.
[0053] Complex (complexed): Two proteins, or fragments or
derivatives thereof, are said to form a complex when they
measurably associate with each other in a specific manner. Such
association can be measured in any of various ways, both direct and
indirect. Direct methods may include co-migration in non-denaturing
fractionation conditions, for instance. Indirect measurements of
association will depend on secondary effects caused by the
association of the two proteins or protein domains. For instance,
the formation of a complex between a protein and an antibody may be
demonstrated by the antibody-specific inhibition of some function
of the target protein. In the case of gp120, the formation of a
complex between gp120 and a neutralizing antibody to this protein
can be measured by determining the degree to which the antibody
inhibits gp120-dependent cell fusion or HIV infectivity. Cell
fusion inhibition and infectivity assays are discussed further
below.
[0054] Exposing an induced epitope: The process by which two
proteins interact specifically to form a complex (an inducing
complex), thereby causing a conformational change in at least one
of the two proteins (the target protein) such that at least one
previously poorly accessible epitope (an induced epitope) is made
accessible to intramolecular interaction. The formation of such an
inducing complex will generally cause the exposure of more than one
induced epitope, each of which may be thereby rendered accessible
for intramolecular interaction.
[0055] HIV coreceptor: A cell-surface protein other than CD4
involved in the interaction of HIV virus and its subsequent entry
into a target cell. These proteins may also be referred to as
fusion coreceptors for HIV. Examples of such coreceptor proteins
include, for instance, members of the chemokine receptor family
(e.g. CXCR4, CCR5, CCR3, and CCR2B).
[0056] HIV coreceptor proteins interact with coreceptor binding
determinants of gp120. In general, it is believed that some of
these determinants are exposed on gp120 only after the specific
interaction of gp120 with CD4, and the consequent CD4-induced
conformational change in the interacting gp120. Thus certain HIV
coreceptor binding determinants are, or overlap with, CD4-induced
epitopes.
[0057] Neutralization of gp120 can be achieved by the specific
binding of neutralizing proteins or protein fragments or domains to
one or more coreceptor binding determinants of gp120, thereby
blocking interaction between complexed gp120 and the native
coreceptor.
[0058] HIV neutralizing ability: The measurable ability of a
molecule to inhibit infectivity of HIV virus, either in vivo or in
vitro. The art is replete with methods for measuring the
neutralizing ability of various molecules. Techniques include in
vitro peripheral blood mononuclear cell (PBMC) based assays
(D'Souza et al., 1997); measurement of virion attachment (Mondor et
al., J. Virol. 72:3623-3634, 1998); neutral red dye uptake and
antigen capture assays (U.S. Pat. No. 5,695,927); vaccinia-based
reporter gene cell fusion assay (Nussbaum et al., J. Virol.
68:5411-5422, 1994) (standard and sCD4 activated assays);
productive infection assays (measuring gag antigen p24 or RT
synthesis) (Karn, HIV: a practical approach. Oxford Univ. Press,
Cambridge, 1995); and infectivity titer reduction assays (Karn,
1995).
[0059] In addition, physical interaction between gp120 and CD4 or
other CD4-like molecules can be examined by various methods. See,
for instance U.S. Pat. No. 5,843,454 (measuring conformational
changes of gp120 on binding of various proteins by virus release
and susceptibility of gp120 to thrombin-mediated cleavage of the V3
loop). Alternately, the ability of the CD4-like molecule to compete
for binding to gp120 with either native CD4 or antibody that
recognizes the CD4 binding site on gp120 (CD4BS) can be measured.
This will allow the calculation of relative binding affinities
through standard techniques.
[0060] The invention also includes analogs, derivatives or mimetics
of the disclosed fusion proteins, and which have HIV neutralizing
ability. Such molecules can be screened for HIV neutralizing
ability by assaying a protein similar to the disclosed fusion
protein, in that it has one or more conservative amino acid
substitutions, or analogs, derivatives or mimetics thereof, and
determining whether the similar protein, analog, derivative or
mimetic provides HIV neutralization. The HIV neutralization ability
and gp120 binding affinity of these derivative compounds can be
measured by any known means, including those discussed in this
application
[0061] Injectable composition: A pharmaceutically acceptable fluid
composition comprising at least one active ingredient, e.g. a
bispecific fusion protein. The active ingredient is usually
dissolved or suspended in a physiologically acceptable carrier, and
the composition can additionally comprise minor amounts of one or
more non-toxic auxiliary substances, such as emulsifying agents,
preservatives, and pH buffering agents and the like. Such
injectable compositions that are useful for use with the fusion
proteins of this invention are conventional; formulations are well
known in the art.
[0062] Isolated: An "isolated" biological component (such as a
nucleic acid molecule, protein or organelle) is one that has been
substantially separated or purified away from other biological
components in the cell of the organism in which the component
naturally occurs, i.e., other chromosomal and extra-chromosomal DNA
and RNA, proteins and organelles. Nucleic acids and proteins that
have been "isolated" include nucleic acids and proteins purified by
standard purification methods. The term also embraces nucleic acids
and proteins prepared by recombinant expression in a host cell as
well as chemically synthesized nucleic acids.
[0063] Neutralizing antibodies: An antibody that is able to
specifically bind to a target protein in such a way as to inhibit
the subsequent biological functioning of that target protein is
said to be neutralizing of that biological function. In general,
any protein that can perform this type of specific blocking
activity is considered a neutralizing protein; antibodies are
therefore a specific class of neutralizing protein. The complex
formed by binding of a neutralizing protein to a target protein is
called a neutralizing complex.
[0064] Antibodies that bind to viruses and bacteria and thereby
prevent the binding of these pathogens to target host cells are
said to neutralize the pathogen. Therefore, antibodies that bind to
HIV proteins and measurably reduce the ability of the virus to bind
to or enter target cells (e.g., T-cells or macrophages) are
HIV-neutralizing antibodies. In general, HIV neutralizing
antibodies can be broken down into several different classes
dependent on what region of the viral envelope protein the antibody
binds to. Broad classes of such antibodies include anti-gp41 and
anti-gp120 antibodies. There are several antigenic regions on the
gp120 protein that provide epitopes for the natural or laboratory
generation of HIV neutralizing antibodies (see WO 98/36087).
Broadly cross-reactive neutralizing antibodies usually interact
with relatively invariant regions of Env.
[0065] A primary source of neutralizing antibodies is the
peripheral blood of patients infected with the HIV virus. Such
primary isolates can be cloned and/or immortalized using standard
techniques. In addition to the isolation of naturally-occurring
neutralizing antibodies, procedures specifically directed toward
their production are known in the art. See U.S. Pat. Nos.
5,843,454; 5,695,927; 5,643,756; and 5,013,548 for instance.
[0066] Linker: A peptide, usually between two and 150 amino acid
residues in length that serves to join two protein domains in a
multi-domain fusion protein. Examples of specific linkers can be
found, for instance, in Hennecke et al. (Protein Eng. 11:405-410,
1998); and U.S. Pat. Nos. 5,767,260 and 5,856,456.
[0067] Depending on the domains being joined, and their eventual
function in the fusion protein, linkers may be from about two to
about 150 amino acids in length, though these limits are given as
general guidance only. The tendency of fusion proteins to form
specific and non-specific multimeric aggregations is influenced by
linker length (Alfthan et al., 1998 Protein Eng. 8:725-731, 1998).
Thus, shorter linkers will tend to promote multimerization, while
longer linkers tend to favor maintenance of monomeric fusion
proteins. Aggregation can also be minimized through the use of
specific linker sequences, as demonstrated in U.S. Pat. No.
5,856,456.
[0068] Linkers may be repetitive or non-repetitive. One classical
repetitive linker used in the production of single chain Fvs
(SCFvs) is the (Gly.sub.4Ser).sub.3 (or (GGGGS).sub.3 or
(G.sub.4S).sub.3) linker. More recently, non-repetitive linkers
have been produced, and methods for the random generation of such
linkers are known (Hennecke et al., Protein Eng. 11:405-410, 1998).
In addition, linkers may be chosen to have more or less secondary
character (e.g. helical character, U.S. Pat. No. 5,637,481)
depending on the conformation desired in the final fusion protein.
The more secondary character a linker possesses, the more
constrained the structure of the final fusion protein will be.
Therefore, substantially flexible linkers that are substantially
lacking in secondary structure allow flexion of the fusion protein
at the linker.
[0069] A linker is capable of retaining a binding domain of a
protein in binding proximity of a target binding site when the
linker is of sufficient length and flexibility to allow specific
interaction between the binding domain and the target binding site.
In the case of the bispecific fusion proteins of this invention, a
linker that maintains binding proximity permits the sequential
binding with the target of first the inducing-binding domain of the
fusion protein, then the induced-binding domain. A linker that
maintains the domains of a bispecific fusion protein in binding
proximity to a target can be considered an operable or functional
linker as relates to such a bispecific fusion protein.
[0070] Oligonucleotide: A linear polynucleotide sequence of between
six and 300 nucleotide bases in length.
[0071] Operably linked: A first nucleic acid sequence is operably
linked with a second nucleic acid sequence when the first nucleic
acid sequence is placed in a functional relationship with the
second nucleic acid sequence. For instance, a promoter is operably
linked to a coding sequence if the promoter affects the
transcription or expression of the coding sequence. Generally,
operably linked DNA sequences are contiguous and, where necessary
to join two protein-coding regions, in the same reading frame.
[0072] ORF (open reading frame): A series of nucleotide triplets
(codons) coding for amino acids without any internal termination
codons. These sequences are usually translatable into a
peptide.
[0073] Parenteral: Administered outside of the intestine, e.g., not
via the alimentary tract. Generally, parenteral formulations are
those that will be administered through any possible mode except
ingestion. This term especially refers to injections, whether
administered intravenously, intrathecally, intramuscularly,
intraperitoneally, or subcutaneously, and various surface
applications including intranasal, intradermal, and topical
application, for instance.
[0074] Pharmaceutically acceptable carriers: The pharmaceutically
acceptable carriers useful in this invention are conventional.
Remington's Pharmaceutical Sciences, by E. W. Martin, Mack
Publishing Co., Easton, Pa., 15th Edition (1975), describes
compositions and formulations suitable for pharmaceutical delivery
of the fusion proteins herein disclosed.
[0075] In general, the nature of the carrier will depend on the
particular mode of administration being employed. For instance,
parenteral formulations usually comprise injectable fluids that
include pharmaceutically and physiologically acceptable fluids such
as water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol or the like as a vehicle. For solid compositions
(e.g., powder, pill, tablet, or capsule forms), conventional
non-toxic solid carriers can include, for example, pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. In
addition to biologically-neutral carriers, pharmaceutical
compositions to be administered can contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate.
[0076] Purified: The term purified does not require absolute
purity; rather, it is intended as a relative term. Thus, for
example, a purified fusion protein preparation is one in which the
fusion protein is more enriched than the protein is in its
generative environment, for instance within a cell or in a
biochemical reaction chamber. In some embodiments, a preparation of
fusion protein is purified such that the fusion protein represents
at least 50% of the total protein content of the preparation.
[0077] Recombinant: A recombinant nucleic acid molecule is one that
has a sequence that is not naturally occurring or has a sequence
that is made by an artificial combination of two otherwise
separated segments of sequence. This artificial combination can be
accomplished by chemical synthesis or, more commonly, by the
artificial manipulation of isolated segments of nucleic acids,
e.g., by genetic engineering techniques.
[0078] Similarly, a recombinant protein is one encoded for by a
recombinant nucleic acid molecule.
[0079] Sequence identity: The similarity between two nucleic acid
sequences, or two amino acid sequences is expressed in terms of the
similarity between the sequences, otherwise referred to as sequence
identity. Sequence identity is frequently measured in terms of
percentage identity (or similarity or homology); the higher the
percentage, the more similar the two sequences are. Homologs of the
bispecific fusion protein will possess a relatively high degree of
sequence identity when aligned using standard methods.
[0080] Methods of alignment of sequences for comparison are well
known in the art. Various programs and alignment algorithms are
described in: Smith and Waterman (Adv. Appl. Math. 2: 482, 1981);
Needleman and Wunsch (J. Mol. Biol. 48: 443-453, 1970); Pearson and
Lipman (Proc. Natl. Acad. Sci., USA 85:2444-2448, 1988); Higgins
and Sharp (Gene, 73:237-244, 1988); Higgins and Sharp (CABIOS
5:151-153, 1989); Corpet et al. (Nuc. Acids Res. 16: 10881-10890,
1988); Huang et al. (Comp. Appls. Biosci. 8:155-165, 1992); and
Pearson et al. (Methods in Molecular Biology 24: 307-331, 1994).
Altschul et al. (Nature Genet., 6:119-129, 1994) presents a
detailed consideration of sequence alignment methods and homology
calculations.
[0081] The alignment tools ALIGN (Myers and Miller, CABIOS 4:11-17,
1989) or LFASTA (Pearson and Lipman, Proc. Natl. Acad. Sci., USA
85:2444-2448, 1988) may be used to perform sequence comparisons
(Internet Program .COPYRGT. 1996, W. R. Pearson and the University
of Virginia, "fasta20u63" version 2.0u63, release date December
1996). ALIGN compares entire sequences against one another, while
LFASTA compares regions of local similarity. These alignment tools
and their respective tutorials are available on the Internet.
[0082] Orthologs of the disclosed bispecific fusion proteins are
typically characterized by possession of greater than 75% sequence
identity counted over the full-length alignment with the amino acid
sequence of bispecific fusion protein using ALIGN set to default
parameters.
[0083] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul
et al., J Mol. Biol. 1990 215:403-410, 1990) is available from
several sources, including the National Center for Biotechnology
Information (NCBI, Bethesda, Md.) and on the Internet, for use in
connection with the sequence analysis programs blastp, blastn,
blastx, tblastn and tblastx. It can be accessed at the NCBI BLAST
website. A description of how to determine sequence identity using
this program is also available at the NCBI website BLAST
tutorial.
[0084] For comparisons of amino acid sequences of greater than
about 30 amino acids, the "Blast 2 sequences" function is employed
using the default BLOSUM62 matrix set to default parameters, (gap
existence cost of 11, and a per residue gap cost of 1). When
aligning short peptides (fewer than around 30 amino acids), the
alignment should be performed using the Blast 2 sequences function,
employing the PAM30 matrix set to default parameters (open gap 9,
extension gap 1 penalties). Proteins with even greater similarity
to the reference sequences will show increasing percentage
identities when assessed by this method, such as at least 90%, at
least 92%, at least 94%, at least 95%, at least 97%, at least 98%,
or at least 99% sequence identity. In addition, sequence identity
can be compared over the full length of one or both binding domains
of the disclosed fusion proteins. In such an instance, percentage
identities will be essentially similar to those discussed for
full-length sequence identity.
[0085] When significantly less than the entire sequence is being
compared for sequence identity, homologs will typically possess at
least 80% sequence identity over short windows of 10-20 amino
acids, and may possess sequence identities of at least 85%, at
least 90%, at least 95%, or at least 99% depending on their
similarity to the reference sequence. Sequence identity over such
short windows can be determined using LFASTA; methods are described
on the Internet. One of skill in the art will appreciate that these
sequence identity ranges are provided for guidance only; it is
entirely possible that strongly significant homologs could be
obtained that fall outside of the ranges provided. The present
invention provides not only the peptide homologs that are described
above, but also nucleic acid molecules that encode such
homologs.
[0086] An alternative indication that two nucleic acid molecules
are closely related is that the two molecules hybridize to each
other under stringent conditions. Stringent conditions are
sequence-dependent and are different under different environmental
parameters. Generally, stringent conditions are selected to be
about 5.degree. C. to 20.degree. C. lower than the thermal melting
point (T.sub.m) for the specific sequence at a defined ionic
strength and pH. The T.sub.m is the temperature (under defined
ionic strength and pH) at which 50% of the target sequence
hybridizes to a perfectly matched probe. Conditions for nucleic
acid hybridization and calculation of stringencies can be found in
Sambrook et al. (In Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor, N.Y., 1989) and Tijssen (Laboratory Techniques in
Biochemistry and Molecular Biology Part I, Ch. 2, Elsevier, New
York, 1993). Nucleic acid molecules that hybridize under stringent
conditions to the disclosed bispecific fusion protein sequences
will typically hybridize to a probe based on either the entire
fusion protein encoding sequence, an entire binding domain, or
other selected portions of the encoding sequence under wash
conditions of 0.2.times.SSC, 0.1% SDS at 65.degree. C.
[0087] Nucleic acid sequences that do not show a high degree of
identity may nevertheless encode similar amino acid sequences, due
to the degeneracy of the genetic code. It is understood that
changes in nucleic acid sequence can be made using this degeneracy
to produce multiple nucleic acid sequences, each encoding
substantially the same protein.
[0088] Specific binding agent: An agent that binds substantially
only to a defined target. Thus a gp120-specific binding agent binds
substantially only the gp120 protein. As used herein, the term
"gp120-specific binding agent" includes anti-gp120 antibodies and
other agents that bind substantially only to a gp120 protein.
[0089] Anti-gp120 antibodies may be produced using standard
procedures described in a number of texts, including Harlow and
Lane (Using Antibodies, A Laboratory Manual, CSHL, New York, 1999,
ISBN 0-87969-544-7). In addition, certain techniques may enhance
the production of neutralizing antibodies (U.S. Pat. Nos.
5,843,454; 5,695,927; 5,643,756; and 5,013,548). The determination
that a particular agent binds substantially only to gp120 protein
may readily be made by using or adapting routine procedures. One
suitable in vitro assay makes use of the Western blotting procedure
(described in many standard texts, including Harlow and Lane,
1999). Western blotting may be used to determine that a given
protein binding agent, such as an anti-gp120 monoclonal antibody,
binds substantially only to the MSG protein. Antibodies to gp120
are well known in the art.
[0090] Shorter fragments of antibodies can also serve as specific
binding agents. For instance, FAbs, Fvs, and single-chain Fvs
(SCFvs) that bind to gp120 would be gp120-specific binding
agents.
[0091] Therapeutically effective amount of a bispecific fusion
protein: A quantity of bispecific fusion protein sufficient to
achieve a desired effect in a subject being treated. For instance,
this can be the amount necessary to inhibit viral proliferation or
to measurably neutralize disease organism binding mechanisms. In
general, this amount will be sufficient to measurably inhibit virus
(e.g. HIV) replication or infectivity.
[0092] An effective amount of bispecific fusion protein may be
administered in a single dose, or in several doses, for example
daily, during a course of treatment. However, the effective amount
of fusion protein will be dependent on the fusion protein applied,
the subject being treated, the severity and type of the affliction,
and the manner of administration of the fusion protein. For
example, a therapeutically effective amount of fusion protein can
vary from about 0.01 mg/kg body weight to about 1 g/kg body
weight.
[0093] The fusion proteins disclosed in the present invention have
equal application in medical and veterinary settings. Therefore,
the general term "subject being treated" is understood to include
all animals (e.g. humans, apes, dogs, cats, horses, and cows) that
are or may be infected with a virus or other disease-causing
microorganism that is susceptible to bispecific fusion
protein-mediated neutralization.
[0094] Transformed: A transformed cell is a cell into which has
been introduced a nucleic acid molecule by molecular biology
techniques. As used herein, the term transformation encompasses all
techniques by which a nucleic acid molecule might be introduced
into such a cell, including transfection with viral vectors,
transformation with plasmid vectors, and introduction of naked DNA
by electroporation, lipofection, and particle gun acceleration.
[0095] Vector: A nucleic acid molecule as introduced into a host
cell, thereby producing a transformed host cell. A vector may
include nucleic acid sequences that permit it to replicate in a
host cell, such as an origin of replication. A vector may also
include one or more selectable marker genes and other genetic
elements known in the art.
II. Construction, Expression, and Purification of Bispecific Fusion
Proteins
[0096] A. Selection of Component Domains.
[0097] This invention provides generally a bispecific fusion
protein that binds to two different sites on a target protein. As
such, any target protein that has two different binding sites is an
example of a target for a bispecific fusion protein. Particular
targets include proteins on which one of the two binding sites (the
induced-binding site) is exposed/induced by the binding of the
fusion protein to a first binding site (the inducing-binding site)
on the target. The choice of protein binding domains for
incorporation into the disclosed bispecific fusion protein will be
dictated by the target protein chosen. The choice of linker will
also be influenced by the target protein and binding sites chosen.
In general, the linker used in any bispecific fusion will be of a
length and secondary character to hold the induced-binding domain
within binding proximity of the target protein induced binding
site, once the inducing-binding domain of the fusion protein has
formed a specific complex with the target.
[0098] In certain embodiments, the target protein is an HIV
envelope glycoprotein, for instance HIV-1 gp120. In certain of
these and other embodiments, the inducing-binding site is the CD4
binding site on gp120. As such, the inducing-binding domain of the
disclosed bispecific fusion protein can be a binding fragment of
CD4, for instance sCD4. Alternately, any other molecule that
specifically interacts with gp120 in such a way as to expose one or
more induced epitopes would also serve as the source of an
inducing-binding protein domain. The specific fragments used to
construct the fusion protein should be chosen so that the
conformation of the final fusion provides functional and inducing
binding to gp120; this can be assayed either directly (e.g.,
affinity measurements) or indirectly (e.g., neutralization
assays).
[0099] Non-CD4-derived CD4 mimics may also be employed as sources
for inducing-binding domains of the disclosed fusion proteins. For
instance, a mannose-specific lectin (SC) may serve to induce CD4
induced conformational changes (see U.S. Pat. No. 5,843,454).
Alternatively, antibodies that bind the CD4-binding site or another
epitope of gp120 and thereby induce a CD4-like conformational
change on the complexed protein can also be used.
[0100] Non-peptide CD4 analogs can also be used in this invention,
for instance organic or non-organic structural analog of the
gp120-interacting domain(s) of the CD4 molecule.
[0101] Induced-binding domains of a gp120-targeted fusion protein
will include antibodies (or fragments thereof) that recognize
induced epitopes of the complexed gp120. In some embodiments, such
antibodies are broadly cross-reactive against diverse HIV-1
isolates. Induced epitopes include all of those referred to as
CD4-induced (CD4i) epitopes, and in particular those which overlap
coreceptor-binding determinants of gp120. Previously identified
neutralizing monoclonal antibodies can be used, and include but are
not limited to human monoclonal antibodies 17b, 48d, and CG10.
[0102] Likewise, induced binding domains of the disclosed chimeric
molecules can be non-peptide molecules, for instance organic or
non-organic structural analogs of SCFv(17b).
[0103] In addition to antibodies that bind induced epitopes of
gp120, other sources for induced-binding domains include fragments
of coreceptors that specifically interact with a coreceptor binding
domain(s) of gp120.
[0104] The construction of a gp120-specific bispecific fusion
protein can be aided by review of the X-ray crystallographic
structure of the ternary complex containing the gp120 core, a
two-domain fragment of CD4 (D1D2), and an FAb from a broadly
cross-reactive human MAb (17b) directed against the
coreceptor-binding determinants of gp120 (Kwong et al., Nature
393:648-659, 1998). Computer-based examination of the structural
coordinates of this ternary complex, using FRODO (Jones et al.,
Meth. Enzymol. 115:157-171, 1985; Jones, J. Appl. Cryst.
11:268-272, 1978; Pflugrath et al. Methods and Applications in
Crystallography, pages 407-420, Clarendon Press, Oxford), has
revealed choices for constructing the chimeric protein. The
shortest distance between free termini of CD4 and the 17b FAb is 56
.ANG., i.e. from the free C-terminus of the D1D2 sCD4 fragment to
the N-terminus of the 17b FAb heavy chain. A linker connecting
these termini would be essentially free of steric hindrance from
CD4 and the N-terminus of the 17b light chain. Possible connections
could also be made between the N-terminus of CD4 and the C-termini
of the 17b heavy or light chains; such connections would require
linkers of about 65 .ANG. and about 86 .ANG., respectively. In the
latter two connections the linker is required to circumvent other
portions of the complex, including the bulky variable loops.
[0105] B. Assembly.
[0106] The construction of chimeric molecules, in particular fusion
proteins, from domains of known proteins is well known. In general,
a nucleic acid molecule that encodes the desired protein domains
are joined using standard genetic engineering techniques to create
a single, operably linked fusion oligonucleotide. Molecular
biological techniques may be found in Sambrook et al. (In Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989).
Specific examples of genetically engineered multi-domain proteins,
especially those based on molecules of the immunoglobulin
superfamily, joined by various linkers, can be found in the
following patent documents: [0107] U.S. Pat. No. 5,856,456 ("Linker
for linked fusion polypeptides"); [0108] U.S. Pat. No. 5,696,237
("Recombinant antibody-toxin fusion protein"); [0109] U.S. Pat. No.
5,767,260 ("Antigen-binding fusion proteins"); [0110] U.S. Pat. No.
5,587,455 ("Cytotoxic agent against specific virus infection"); and
[0111] WO 98/36087 ("Immunological tolerance to HIV epitopes").
[0112] Non-peptide analogs that serve as inducing-binding or
induced binding domains of the invention can be linked to the
opposite domain of the chimeric molecules using known chemical
linking techniques, including chemical cross-linking. Cross-linkers
are well known, and examples of molecules used for cross-linking
can be found, for instance, in U.S. Pat. No. 6,027,890 ("Methods
and compositions for enhancing sensitivity in the analysis of
biological-based assays").
[0113] C. Expression.
[0114] One skilled in the art will understand that there are myriad
ways to express a recombinant protein such that it can subsequently
be purified. In general, an expression vector carrying the nucleic
acid sequence that encodes the desired protein will be transformed
into a microorganism for expression. Such microorganisms can be
prokaryotic (bacteria) or eukaryotic (e.g., yeast). One example
species of bacteria that can be used is Escherichia coli (E. coli),
which has been used extensively as a laboratory experimental
expression system. An eukaryotic expression system can be used
where the protein of interest requires eukaryote-specific
post-translational modifications such as glycosylation. Also,
protein can be expressed using a viral (e.g., vaccinia) based
expression system.
[0115] Protein can also be expressed in animal cell tissue culture,
and such a system can be used where animal-specific protein
modifications are desirable or required in the recombinant
protein.
[0116] The expression vector can include a sequence encoding a
targeting peptide, positioned in such a way as to be fused to the
coding sequence of the bispecific fusion protein. This allows the
bispecific fusion protein to be targeted to specific sub-cellular
or extra-cellular locations. Various prokaryotic and eukaryotic
targeting peptides, and nucleic acid molecules encoding such, are
known. In a prokaryotic expression system, a signal sequence can be
used to secrete the newly synthesized protein. In an eukaryotic
expression system, the targeting peptide would specify targeting of
the hybrid protein to one or more specific sub-cellular
compartments, or to be secreted from the cell, depending on which
peptide is chosen. Through the use of an eukaryotic secretion
signal sequence, the bispecific fusion protein can be expressed in
a transgenic animal (for instance a cow, pig, or sheep) in such a
manner that the protein is secreted into the milk of the
animal.
[0117] Vectors suitable for stable transformation of culturable
cells are also well known. Typically, such vectors include a
multiple-cloning site suitable for inserting a cloned nucleic acid
molecule, such that it will be under the transcriptional control of
5' and 3' regulatory sequences. In addition, transformation vectors
include one or more selectable markers; for bacterial
transformation this is often an antibiotic resistance gene. Such
transformation vectors typically also contain a promoter regulatory
region (e.g., a regulatory region controlling inducible or
constitutive expression), a transcription initiation start site, a
ribosome binding site, an RNA processing signal, and a
transcription termination site, each functionally arranged in
relation to the multiple-cloning site. For production of large
amounts of recombinant proteins, an inducible promoter can be used.
This permits selective production of the recombinant protein, and
allows both higher levels of production than constitutive
promoters, and enables the production of recombinant proteins that
may be toxic to the expressing cell if expressed
constitutively.
[0118] In addition to these general guidelines, protein
expression/purification kits are produced commercially. See, for
instance, the QIAEXPRESS.TM. expression system from QIAGEN
(Chatsworth, Calif.) and various expression systems provided by
INVITROGEN (Carlsbad, Calif.). Depending on the details provided by
the manufactures, such kits can be used for production and
purification of the disclosed bispecific fusion proteins.
[0119] D. Purification.
[0120] One skilled in the art will understand that there are myriad
ways to purify recombinant polypeptides, and such typical methods
of protein purification may be used to purify the disclosed
bispecific fusion proteins. Such methods include, for instance,
protein chromatographic methods including ion exchange, gel
filtration, HPLC, monoclonal antibody affinity chromatography and
isolation of insoluble protein inclusion bodies after over
production. In addition, purification affinity-tags, for instance a
six-histidine sequence, may be recombinantly fused to the protein
and used to facilitate polypeptide purification. A specific
proteolytic site, for instance a thrombin-specific digestion site,
can be engineered into the protein between the tag and the fusion
itself to facilitate removal of the tag after purification.
[0121] Commercially produced protein expression/purification kits
provide tailored protocols for the purification of proteins made
using each system. See, for instance, the QIAEXPRESS.TM. expression
system from QIAGEN (Chatsworth, Calif.) and various expression
systems provided by INVITROGEN (Carlsbad, Calif.). Where a
commercial kit is employed to produce a bispecific fusion protein,
the manufacturer's purification protocol is a particularly
disclosed protocol for purification of that protein. For instance,
proteins expressed with an amino-terminal hexa-his tag can be
purified by binding to nickel-nitrilotriacetic acid (Ni-NTA) metal
affinity chromatography matrix (The QIAexpressionist, QIAGEN,
1997).
[0122] Alternately, the binding specificities of either the first
or second binding domains, or both, of the disclosed fusion protein
may be exploited to facilitate specific purification of the
proteins. One method of performing such specific purification would
be column chromatography using column resin to which the target
molecule, or an epitope or fragment or domain of the target
molecule, has been attached.
[0123] If the bispecific fusion protein is produced in a secreted
form, e.g. secreted into the milk of a transgenic animal,
purification will be from the secreted fluid. Alternately,
purification may be unnecessary if the fusion protein can be
applied directly to the subject in the secreted fluid (e.g.
milk).
III. Variation of a Bispecific Fusion Protein
[0124] A. Sequence Variants
[0125] The binding characteristics and therefore neutralizing
activity of the fusion proteins disclosed herein lies not in the
precise amino acid sequence, but rather in the three-dimensional
structure inherent in the amino acid sequences encoded by the DNA
sequences. It is possible to recreate the binding characteristics
of any of these proteins or protein domains of this invention by
recreating the three-dimensional structure, without necessarily
recreating the exact amino acid sequence. This can be achieved by
designing a nucleic acid sequence that encodes for the
three-dimensional structure, but which differs, for instance by
reason of the redundancy of the genetic code. Similarly, the DNA
sequence may also be varied, while still producing a functional
neutralizing protein.
[0126] Variant neutralizing bispecific binding proteins include
proteins that differ in amino acid sequence from the disclosed
sequence, but that share structurally significant sequence homology
with any of the provided proteins. Variation can occur in any
single domain of the fusion protein (e.g. the first or second
binding domain, or the linker). Variation can also occur in more
than one of such domains in any particular variant protein. Such
variants may be produced by manipulating the nucleotide sequence of
the, for instance a CD4-SCFv(17b)-encoding sequence, using standard
procedures, including site-directed mutagenesis or PCR. The
simplest modifications involve the substitution of one or more
amino acids for amino acids having similar biochemical properties.
These so-called conservative substitutions are likely to have
minimal impact on the activity of the resultant protein, especially
when made outside of the binding site of each domain. Table 1 shows
amino acids that may be substituted for an original amino acid in a
protein, and which are regarded as conservative substitutions.
TABLE-US-00001 TABLE 1 Original Residue Conservative Substitutions
Ala ser Arg lys Asn gln; his Asp glu Cys ser Gln asn Glu asp Gly
pro His asn; gln Ile leu; val Leu ile; val Lys arg; gln; glu Met
leu; ile Phe met; leu; tyr Ser thr Thr ser Trp tyr Tyr trp; phe Val
ile; leu
[0127] More substantial changes in protein structure may be
obtained by selecting amino acid substitutions that are less
conservative than those listed in Table 1. Such changes include
changing residues that differ more significantly in their effect on
maintaining polypeptide backbone structure (e.g., sheet or helical
conformation) near the substitution, charge or hydrophobicity of
the molecule at the target site, or bulk of a specific side chain.
The following substitutions are generally expected to produce the
greatest changes in protein properties: (a) a hydrophilic residue
(e.g., seryl or threonyl) is substituted for (or by) a hydrophobic
residue (e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl);
(b) a cysteine or proline is substituted for (or by) any other
residue; (c) a residue having an electropositive side chain (e.g.,
lysyl, arginyl, or histadyl) is substituted for (or by) an
electronegative residue (e.g., glutamyl or aspartyl); or (d) a
residue having a bulky side chain (e.g., phenylalanine) is
substituted for (or by) one lacking a side chain (e.g.,
glycine).
[0128] Variant binding domain or fusion protein-encoding sequences
may be produced by standard DNA mutagenesis techniques, for
example, M13 primer mutagenesis. Details of these techniques are
provided in Sambrook (In Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor, N.Y., 1989), Ch. 15. By the use of such
techniques, variants may be created which differ in minor ways from
the bispecific fusion protein-encoding sequences disclosed. DNA
molecules and nucleotide sequences which are derivatives of those
specifically disclosed herein and that differ from those disclosed
by the deletion, addition, or substitution of nucleotides while
still encoding a protein that binds twice to gp120, thereby
neutralizing HIV virus infectivity, are comprehended by this
invention. In their most simple form, such variants may differ from
the disclosed sequences by alteration of the coding region to fit
the codon usage bias of the particular organism into which the
molecule is to be introduced.
[0129] Alternatively, the coding region may be altered by taking
advantage of the degeneracy of the genetic code to alter the coding
sequence such that, while the nucleotide sequence is substantially
altered, it nevertheless encodes a protein having an amino acid
sequence substantially similar to the disclosed fusion sequences.
For example, the 18th amino acid residue of the CD4-SCFv(17b)
protein (after cleavage of the N-terminal signal sequence) is
alanine. The nucleotide codon triplet GCT encodes this alanine
residue. Because of the degeneracy of the genetic code, three other
nucleotide codon triplets--(GCG, GCC and GCA)--also code for
alanine. Thus, the nucleotide sequence of the disclosed
CD4-SCFv(17b) encoding sequence could be changed at this position
to any of these three alternative codons without affecting the
amino acid composition or characteristics of the encoded protein.
Based upon the degeneracy of the genetic code, variant DNA
molecules may be derived from the cDNA and gene sequences disclosed
herein using standard DNA mutagenesis techniques as described
above, or by synthesis of DNA sequences. Thus, this invention also
encompasses nucleic acid sequences which encode a neutralizing
bispecific fusion protein, but which vary from the disclosed
nucleic acid sequences by virtue of the degeneracy of the genetic
code.
[0130] B. Peptide Modifications
[0131] The present invention includes biologically active molecules
that mimic the action of the bispecific fusion proteins of the
present invention, and specifically neutralize HIV Env. The
proteins of the invention include synthetic embodiments of
naturally-occurring proteins described herein, as well as analogues
(non-peptide organic molecules), derivatives (chemically
functionalized protein molecules obtained starting with the
disclosed peptide sequences) and variants (homologs) of these
proteins that specifically bind with and neutralize HIV gp120. Each
protein of the invention is comprised of a sequence of amino acids,
which may be either L- and/or D-amino acids, naturally occurring
and otherwise.
[0132] Proteins may be modified by a variety of chemical techniques
to produce derivatives having essentially the same activity as the
unmodified proteins, and optionally having other desirable
properties. For example, carboxylic acid groups of the protein,
whether carboxyl-terminal or side chain, may be provided in the
form of a salt of a pharmaceutically-acceptable cation or
esterified to form a C.sub.1-C.sub.16 ester, or converted to an
amide of formula NR.sub.1R.sub.2 wherein R.sub.1 and R.sub.2 are
each independently H or C.sub.1-C.sub.16 alkyl, or combined to form
a heterocyclic ring, such as a 5- or 6-membered ring. Amino groups
of the protein, whether amino-terminal or side chain, may be in the
form of a pharmaceutically-acceptable acid addition salt, such as
the HCl, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric
and other organic salts, or may be modified to C.sub.1-C.sub.16
alkyl or dialkyl amino or further converted to an amide.
[0133] Hydroxyl groups of the protein side chains may be converted
to C.sub.1-C.sub.16 alkoxy or to a C.sub.1-C.sub.16 ester using
well-recognized techniques. Phenyl and phenolic rings of the
protein side chains may be substituted with one or more halogen
atoms, such as fluorine, chlorine, bromine or iodine, or with
C.sub.1-C.sub.16 alkyl, C.sub.1-C.sub.16 alkoxy, carboxylic acids
and esters thereof, or amides of such carboxylic acids. Methylene
groups of the protein side chains can be extended to homologous
C.sub.2-C.sub.4 alkylenes. Thiols can be protected with any one of
a number of well-recognized protecting groups, such as acetamide
groups. Those skilled in the art will also recognize methods for
introducing cyclic structures into the proteins of this invention
to select and provide conformational constraints to the structure
that result in enhanced stability.
[0134] It also may be advantageous to introduce one or more
disulfide bonds to connect the frameworks of the heavy and light
chains in the SCFv domain. This modification often enhances the
stability and affinity of SCFvs (Reiter et al., Protein Engineering
7:697-704, 1994). Here too, the X-ray crystal structure containing
the 17 FAb (Kwong et al., Nature 393:648-659, 1998) can be used to
assess optimal sites for engineering cysteine residues of the heavy
and light chains.
[0135] Peptidomimetic and organomimetic embodiments are also within
the scope of the present invention, whereby the three-dimensional
arrangement of the chemical constituents of such peptido- and
organomimetics mimic the three-dimensional arrangement of the
protein backbone and component amino acid side chains in the
bispecific neutralizing fusion protein, resulting in such peptido-
and organomimetics of the proteins of this invention having
measurable or enhanced neutralizing ability. For computer modeling
applications, a pharmacophore is an idealized, three-dimensional
definition of the structural requirements for biological activity.
Peptido- and organomimetics can be designed to fit each
pharmacophore with current computer modeling software (using
computer assisted drug design or CADD). See Walters,
"Computer-Assisted Modeling of Drugs", in Klegerman & Groves,
eds., 1993, Pharmaceutical Biotechnology, Interpharm Press: Buffalo
Grove, Ill., pp. 165-174 and Principles of Pharmacology Munson
(ed.) 1995, Ch. 102, for descriptions of techniques used in CADD.
Also included within the scope of the invention are mimetics
prepared using such techniques that produce neutralizing fusion
proteins.
[0136] C. Domain Length Variation.
[0137] It will be appreciated that the protein domains of the
current invention may be combined to produce fusion protein
molecules without necessarily splicing the components in the same
place. It is believed to be possible to use shorter or longer
fragments of each component domain, linked by a functional linker.
For instance, any component which is spliced within about 10 amino
acid residues of the residue specified, and which still provides a
functional binding fragment, comprises about the same domain.
However, domains of substantially longer or substantially shorter
length can be used. For instance, in certain embodiments, the
protein can include a leader sequence plus a four-domain CD4
(D1-D4, amino acid residues 1-372), or just the first domain of CD4
(D1 residues 1-113).
IV. Activity of Fusion Proteins
[0138] It is important to assess the chemical, physical and
biological activity of the disclosed bispecific fusion proteins.
Among other uses, such assays permit optimization of the domains
chosen, as well as optimization of the length and conformation of
the linkers used to connect them. Control molecules should be
included in each assay; usually such will include each domain
alone, as well as the two domains as separate molecules mixed in
the reaction, for instance in a 1:1 molar ratio. In the case of a
CD4-SCFv(17b) bispecific fusion protein, such controls would
include sCD4 and SCFv(17b), for instance.
[0139] A. Fusion Protein Affinity for Target Protein
[0140] Fusion protein affinity for the target protein can be
determined using various techniques. For instance,
co-immunoprecipitation analyses with metabolically labeled proteins
can be employed to determine binding of sCD4-SCFv proteins, e.g.
sCD4-SCFv(17b) to soluble HIV-1 gp120, using anti-gp120 MAbs that
do not interfere with CD4 binding (e.g. MAb D47 that binds to V3),
or polyclonal antibody to the C-terminus of gp120. ELISA can also
be used to examine the binding characteristics of each domain of
the chimera.
[0141] B. Neutralization Assays
[0142] Various assays can be used to measure the ability of the
disclosed fusion proteins to inhibit function of the target
protein. Individual components of the fusion protein will serve as
controls. In general, assays will be specific for the target/fusion
protein. For instance, many functional analyses can test the
ability of sCD4-SCFv fusions to neutralize the HIV Env. It is
particularly advantageous to use Envs from diverse HIV-1 strains to
test the breadth of inhibition (neutralizing ability) of each
fusion protein for different HIV-1 genetic subtypes and different
phenotypes (i.e. coreceptor usage). In addition, it is advantageous
to test such fusion proteins in the standard and sCD4-activated
assays for Env-mediated cell fusion. Known HIV-1 neutralizing MAbs
and MAbs against CD4-induced epitopes on gp120 are examples of
controls for such experiments. Possible synergistic inhibition with
other known broadly cross-reactive neutralizing MAbs should be
tested (e.g. b12, 2F5, F105, 2G12).
[0143] In the case of gp120-targeted fusion proteins, the
vaccinia-based reporter gene cell fusion assay may be used to
assess fusion inhibition (Nussbaum et al., J. Virol. 68:5411-5422,
1994). One population of tissue culture cells (e.g. BS-C-1, HeLa,
or NIH 3T3) uniformly expressing vaccinia virus-encoded binding and
fusion-mediating viral envelope glycoprotein(s) is mixed with
another population expressing the corresponding cellular
receptor(s). In the case of sCD4-SCFv fusions, where the target
protein is HIV-1 gp120, one cell population expresses HIV-1 Env,
while the other expresses necessary HIV-1 receptors (e.g. CD4 and a
chemokine receptor). The cytoplasm of either cell population also
contains vaccinia virus-encoded bacteriophage T7 DNA polymerase;
the cytoplasm of the other contains a transfected plasmid with the
E. coli lacZ gene linked to the T7 promoter. Upon mixing of the two
populations, cell fusion results in activation of the lacZ gene,
through the introduction of the T7 RNA polymerase into proximity
with the transfected T7 promoter-lacZ in the cytoplasm of the fused
cells. The resultant .beta.-galactosidase (.beta.-gal) activity is
proportional to the amount of fusion that occurs, and can be
measured by colorimetric assay of detergent cell lysates or in situ
staining. Cell-fusion neutralizing activity of bispecific fusion
proteins is therefore assessed by measuring their inhibition of
.beta.-gal production.
[0144] The gp120-targeted fusions (e.g. sCD4-SCFv) can also be
tested for ability to block HIV-1 infection using single round
assays (e.g. using indicator cell lines, Vodicka et al., Virology
233:193-198, 1997). Target cells expressing CD4 and a specific
coreceptor, and containing the lacZ reporter gene linked to the
HIV-1 long terminal repeat (LTR), are infected with specific HIV-1
strains (Vodicka, 1997). Integration of an HIV provirus in these
cells leads to production of the viral transactivator, Tat, which
then turns on expression of the .beta.-gal gene via interaction
with LTR. The activity of sCD4-SCFv is assessed by its inhibition
of production of .beta.-gal-positive cells (stained blue with
X-gal), which is proportional to its ability to block HIV-1
infection.
V. Incorporation of Bispecific Fusion Proteins into Pharmaceutical
Compositions
[0145] Pharmaceutical compositions that comprise at least one
bispecific fusion protein as described herein as an active
ingredient will normally be formulated with a solid or liquid
carrier, depending upon the particular mode of administration
chosen. The pharmaceutically acceptable carriers and excipients
useful in this invention are conventional. For instance, parenteral
formulations usually comprise injectable fluids that are
pharmaceutically and physiologically acceptable fluid vehicles such
as water, physiological saline, other balanced salt solutions,
aqueous dextrose, glycerol or the like. Excipients that can be
included are, for instance, other proteins, such as human serum
albumin or plasma preparations. If desired, the pharmaceutical
composition to be administered may also contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate.
[0146] Other medicinal and pharmaceutical agents, for instance
nucleoside derivatives (e.g. AZT) or protease inhibitors, also may
be included. It may also be advantageous to include other fusion
inhibitors, for instance one or more neutralizing antibodies.
[0147] The dosage form of the pharmaceutical composition will be
determined by the mode of administration chosen. For instance, in
addition to injectable fluids, topical and oral formulations can be
employed. Topical preparations can include eye drops, ointments,
sprays and the like. Oral formulations may be liquid (e.g., syrups,
solutions or suspensions), or solid (e.g., powders, pills, tablets,
or capsules). For solid compositions, conventional non-toxic solid
carriers can include pharmaceutical grades of mannitol, lactose,
starch, or magnesium stearate. Actual methods of preparing such
dosage forms are known, or will be apparent, to those skilled in
the art.
[0148] The pharmaceutical compositions that comprise bispecific
fusion protein may be formulated in unit dosage form, suitable for
individual administration of precise dosages. One possible unit
dosage contains approximately 100 .mu.g of protein. The amount of
active compound administered will be dependent on the subject being
treated, the severity of the affliction, and the manner of
administration, and is best left to the judgment of the prescribing
clinician. Within these bounds, the formulation to be administered
will contain a quantity of the active component(s) in an amount
effective to achieve the desired effect in the subject being
treated.
VI. Clinical Use of Bispecific Fusion Proteins
[0149] The potent viral-neutralizing activity exhibited by the
disclosed bispecific fusion proteins makes them useful for treating
viral infections in human and other animal subjects. Possibly
susceptible viruses include the immunodeficiency viruses, such as
HIV and similar or related viruses in simians and other animals. In
addition, other viral or microbial systems that involve the
interaction of a first inducing and second induced binding site of
a single protein will also be susceptible to neutralization using
bispecific fusion proteins of the current invention. The bispecific
fusion proteins disclosed herein can also be used in highly
sensitive detection or purification of target protein.
[0150] The bispecific fusion proteins of this invention may be
administered to humans, or other animals on whose cells they are
effective, in various manners such as topically, orally,
intravenously, intramuscularly, intraperitoneally, intranasally,
intradermally, intrathecally, and subcutaneously. The particular
mode of administration and the dosage regimen will be selected by
the attending clinician, taking into account the particulars of the
case (e.g., the subject, the disease, and the disease state
involved, and whether the treatment is prophylactic or
post-infection). Treatment may involve daily or multi-daily doses
of bispecific fusion protein(s) over a period of a few days to
months, or even years.
[0151] If treatment is through the direct administration of cells
expressing the bispecific fusion protein to the subject, such cells
(e.g. transgenic pluripotent or hematopoietic stem cells or B
cells) may be administered at a dose of between about 10.sup.6 and
10.sup.10 cells, on one or several occasions. The number of cells
will depend on the patient, as well as the fusion protein and cells
chosen to express the protein.
[0152] A general strategy for transferring genes into donor cells
is disclosed in U.S. Pat. No. 5,529,774, which is incorporated by
reference. Generally, a gene encoding a protein having
therapeutically desired effects is cloned into a viral expression
vector, and that vector is then introduced into the target
organism. The virus infects the cells, and produces the protein
sequence in vivo, where it has its desired therapeutic effect. See,
for example, Zabner et al., Cell 75:207-216, 1993. As an
alternative to adding the sequences encoding the bispecific fusion
protein or a homologous protein to the DNA of a virus, it is also
possible to introduce such a gene into the somatic DNA of infected
or uninfected cells, by methods that are well known in the art
(Sambrook et al., In Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor, N.Y., 1989). These methods can be used to introduce
the herein disclosed fusion proteins to human cells to provide
long-term resistance to HIV-1 infection or AIDS. For example, gene
therapy can be used to secrete the protein at mucosal surfaces,
such as the vaginal, rectal, or oral mucosa.
[0153] HIV-1 gp120-targeted bispecific fusion proteins, for
instance sCD4-SCFv(17b), are particularly useful in the prevention
of infection during or immediately after HIV exposure (e.g.,
mother/infant transmission, post-exposure prophylaxis, and as a
topical inhibitor). In such instances, one or more doses of the
bispecific fusion protein are administered before or soon after the
triggering event. To prevent or ameliorate mother/infant
transmission of viral infection, for instance, it may be beneficial
to administer the gp120-targeted bispecific fusion protein to the
mother intermittently throughout pregnancy, and/or immediately
before or following delivery, and/or directly to the newborn
immediately after birth. Post-exposure prophylactic treatments may
be particularly beneficial where there has been accidental exposure
(for instance, a medically related accidental exposure), including
but not limited to a contaminated needle-stick or medical exposure
to HIV-1 contaminated blood or other fluid.
[0154] The present invention also includes combinations of chimeric
bispecific fusion proteins with one or more other agents useful in
the treatment of disease, e.g. HIV disease. For example, the
compounds of this invention may be administered, whether before or
after exposure to the virus, in combination with effective doses of
other anti-virals, immunomodulators, anti-infectives, and/or
vaccines. The term "administration in combination" refers to both
concurrent and sequential administration of the active agents.
[0155] Examples of antiviral agents that can be used in combination
with the chimeric bispecific fusion proteins of the invention are:
AL-721 (from Ethigen of Los Angeles, Calif.), recombinant human
interferon beta (from Triton Biosciences of Alameda, Calif.),
Acemannan (from Carrington Labs of Irving, Tex.), gangiclovir (from
Syntex of Palo alto, CA), didehydrodeoxythymidine or d4T (from
Bristol-Myers-Squibb), EL10 (from Elan Corp. of Gainesville, Ga.),
dideoxycytidine or ddC (from Hoffman-LaRoche), Novapren (from
Novaferon labs, Inc. of Akron, Ohio), zidovudine or AZT (from
Burroughs Wellcome), ribaririn (from Viratek of Costa Mesa,
Calif.), alpha interferon and acyclovir (from Burroughs Wellcome),
Indinavir (from Merck & Co.), 3TC (from Glaxo Wellcome),
Ritonavir (from Abbott), Saquinavir (from Hoffmann-LaRoche), and
others.
[0156] Examples of immuno-modulators that can be used in
combination with the chimeric bispecific fusion proteins of the
invention are AS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn),
gamma interferon (Genentech), GM-CSF (Genetics Institute), IL-2
(Cetus or Hoffman-LaRoche), human immune globulin (Cutter
Biological), IMREG (from Imreg of New Orleans, La.),
SK&F106528, and TNF (Genentech).
[0157] Examples of some anti-infectives with which the chimeric
bispecific fusion proteins can be used include clindamycin with
primaquine (from Upjohn, for the treatment of Pneumocystis
pneumonia), fluconazlone (from Pfizer for the treatment of
cryptococcal meningitis or candidiasis), nystatin, pentamidine,
trimethaprim-sulfamethoxazole, and many others.
[0158] The combination therapies are of course not limited to the
lists provided in these examples, but includes any composition for
the treatment of HIV disease (including treatment of AIDS).
VII. Kits
[0159] The chimeric proteins disclosed herein can be supplied in
the form of a kit for use in prevention and/or treatment of
diseases (e.g., HIV infection and AIDS). In such a kit, a
clinically effective amount of one or more of the chimeric
bispecific fusion proteins is provided in one or more containers.
The chimeric bispecific fusion proteins may be provided suspended
in an aqueous solution or as a freeze-dried or lyophilized powder,
for instance. In certain embodiments, the chimeric proteins will be
provided in the form of a pharmaceutical composition.
[0160] Kits according to this invention can also include
instructions, usually written instructions, to assist the user in
treating a disease (e.g., HIV infection or AIDS) with a chimeric
bispecific fusion protein. Such instructions can optionally be
provided on a computer readable medium.
[0161] The container(s) in which the protein(s) are supplied can be
any conventional container that is capable of holding the supplied
form, for instance, microfuge tubes, ampoules, or bottles. In some
applications, chimeric proteins may be provided in pre-measured
single use amounts in individual, typically disposable, tubes or
equivalent containers.
[0162] The amount of a chimeric bispecific fusion protein supplied
in the kit can be any appropriate amount, depending for instance on
the market to which the product is directed. For instance, if the
kit is adapted for research or clinical use, the amount of each
chimeric protein provided would likely be an amount sufficient for
several treatments.
[0163] Certain kits according to this invention will also include
one or more other agents useful in the treatment of disease, e.g.
HIV disease. For example, such kits may include one or more
effective doses of other anti-virals, immunomodulators,
anti-infectives, and/or vaccines.
Example 1
Construction of a CD4-SCFv(17b) Encoding Sequence
[0164] A gp120-targeted fusion protein, sCD4-SCFv(17b), is
constructed by linking the C-terminus of CD4 (D1D2, 183 amino acid
residues) to the N-terminus of the heavy chain of the 17b SCFv,
which contains the heavy chain at its N-terminus, linked via its
C-terminus to the N-terminus of the light chain (see schematic
diagram of the construct, FIG. 3). The 17b SCFv DNA was obtained
from R. Wyatt and J. Sodroski, Dana Farber Cancer Institute,
Boston, Mass. The 17b-MAb producing-hybridoma was obtained from J.
Robinson, Tulane University.
[0165] Linkers were chosen to have sufficient length and
flexibility to connect the desired protein segments without
inducing unacceptable torsion. For the SCFv, the 15 amino acid
residue sequence (Gly.sub.4Ser).sub.3 (designated L2) was chosen,
which has been employed successfully for production of SCFvs. This
sequence confers excellent flexibility with minimal aggregation.
The linker between the C-terminus of CD4 and the N-terminus of the
SCFv (designated L1; SEQ ID NO: 2), is seven repeats of the same
Gly.sub.4Ser sequence. Conservative estimates indicate that this 35
amino acid residue linker is sufficiently long to allow CD4 and
SCFv to bind simultaneously to their respective binding sites on
gp120. A schematic of the genetic construct is shown in FIG. 3. A
unique BamH I restriction site has been introduced within L1 to
enable the production of constructs with shorter or longer linkers,
and especially to provide negative controls (linkers too short,
thereby not allowing both the CD4 and SCFv moieties of a single
molecule to bind simultaneously to their respective binding sites
on gp120).
[0166] The starting CD4 plasmid is pCB-3, which contains the
full-length CD4 cDNA (including its natural 5' signal sequence) in
the vaccinia expression plasmid pSC59 (Broder & Berger, J.
Virol. 67:913-926, 1993). This plasmid was digested with Stu I,
which cuts near the end of the 2nd domain of sCD4, and with Spe I,
which cuts within the vector downstream of the CD4 insert and
leaves a 5' overhang.
[0167] Synthetic oligonucleotides (SEQ ID NO: 11) were annealed
together to recapitulate the 5' end of the second half of the Stu I
site (CCT) and the next two bases (CC) of the CD4 cDNA, and to
produce an Spe I overhang at the 3' end (this site to be destroyed
upon ligation into pCB-3). The oligonucleotide sequence
reconstructs the remainder of the second domain of CD4 (through
ser.sub.183), and encodes the 37 amino acid intermediate linker
(gly.sub.4ser).sub.6gly.sub.4thr.sub.2ser, followed directly by the
universal translational termination sequence (UTS) (SEQ ID NO: 6).
A BamH I site has been deliberately included within the linker near
the end of the third (gly.sub.4ser) repeat, to enable subsequent
linkage to the 17b SCFv with the exact L1 sequence, and to enable
modification of linker length. The resulting intermediate plasmid
is designated pCD1. This construct was confirmed by DNA sequence
analysis using standard techniques. To facilitate subsequent
procedures, the sCD4-linker sequence was recloned into a pSC59
derivative lacking a BamH I site, forming intermediate plasmid
pCD2.
[0168] The starting 17b plasmid containing the 17b SCFv cDNA in a
plasmid vector (pmt del 0) was donated by Dr. Richard Wyatt (Dana
Farber Cancer Institute, Boston, Mass.). The SCFv cDNA is
constructed with the heavy chain at the 5' segment and light chain
at the 3' segment, attached via DNA encoding the L2 linker
(gly.sub.4ser).sub.3. The 17b SCFv construct has a TPA signal
sequence at the 5' end, and sequences corresponding to a thrombin
cleavage site and a hexa-his tag (to facilitate purification) at
the 3' end, followed by a stop codon. A comparable construct
without the thrombin cleavage site and hexa-his tag can also be
produced.
[0169] PCR technology was used to attach the 17b SCFv sequence to
the CD4-linker sequence in pCD2. Suitable primers are represented
in SEQ ID NOs: 7 and 9. The forward (5') primer (SEQ ID NO: 7)
contains a BamH I site near the 5' end (preceded by an overhang),
followed by nucleotides that reconstruct the third (gly.sub.4ser)
plus four additional (gly.sub.4ser) repeats; this is followed by
nucleotides exactly corresponding to the start of the 17b heavy
chain (excluding the 5' signal sequence, beginning at CAG GTG). The
3' primer (SEQ ID NO: 9) begins with convenient restriction sites
for cloning into pCD2 (Spe I and others), followed by nucleotides
exactly complementary to the 3' end of the 17b SCFv sequence in pmt
del 0 (stop codon, hexa-his tag, and thrombin cleavage site).
[0170] These primers are used to prime the plasmid vector
containing the 17b SCFv sequence in pmt del 0, and the resultant
PCR product digested with BamH I plus a restriction enzyme that
cleaves at the opposite 3' end (e.g., Spe I). This digested
fragment is then force-cloned into pCD2 that has been digested with
the same enzymes (BamH I and Spe I). The resulting plasmid
(designated herein as pCD3) contains the final sCD4-SCFv(17b)
construct (with the thrombin cleavage site and hexa-his tag)
downstream from the strong, synthetic early/late vaccinia promoter
in pSC59. There are convenient, unique restriction sites on each
side of the sCD4-SCFv sequence for possible future cloning
steps.
[0171] The 17b SCFv cDNA (including the 5' signal sequence) also
has been excised from the pmt del 0 vector by restriction enzyme
digestion or PCR, and cloned into the vaccinia expression plasmid
pSC59 to provide a control construct.
Example 2
Expression and Purification of CD4-SCFv(17b) Fusion Protein
A. Expression
[0172] For small amounts of protein expression, vaccinia expression
technology can be used to produce the sCD4-SCFv(17b) (as well as
the control 17b SCFv protein). The plasmid containing the construct
in the vaccinia expression plasmid pSC59 is used to produce a
vaccinia recombinant, using standard technology. For such
expression, suitable cells (HeLa, BSC-1, etc.) are infected with
the recombinant vaccinia virus; after incubation for 24-36 hours at
37.degree. C., the recombinant protein is present in the culture
supernatant. Initial biochemical and functional studies can be done
with unfractionated supernatant; where necessary, the sCD4-SCFv
protein may be purified (see below). Small scale, initial
experiments can be performed with small amounts of material (5-20
micrograms, obtained from 1-5.times.10.sup.7 cells). The
preparation can be scaled up; for such large-scale production, it
may be advantageous to employ higher yield technologies for
expression of the recombinant proteins (e.g., baculovirus, yeast,
or E. coli).
[0173] Expression of the pCD1 secreted protein product (the first
two domains of CD4 through ser.sub.183, plus the 37 amino acid
linker) was analyzed. BSC-1 cells were transfected with pCD1 and
infected with wild type vaccinia virus, then incubated overnight at
37.degree. C. Supernatants were analyzed by Western (immunoblot)
analysis, using antibodies against CD4. As expected, the protein
encoded by pCD1 migrated slightly more slowly than standard
purified two-domain sCD4 (Upjohn-Pharmacia, Kalamazoo, Mich.).
[0174] The pCD3 full-length sCD4-SCFv(17b) (sCD4-17b) fusion
protein has been expressed and tested similarly, and 17b SCFv
domain (as cloned into pSC59) can be examined likewise. The
sCD4-17b fusion protein (at least a portion of which is secreted)
has the expected molecular size (approximately 55 kD) when analyzed
by SDS PAGE and Western blotting. The protein reacted strongly with
antibodies against CD4 or the hexa-his tag, confirming the presence
of these N-terminal and C-terminal moieties, as well as the correct
reading frame.
B. Purification
[0175] Expressed fusion protein as constructed above with an
amino-terminal hexa-his tag was purified using this molecular tag.
The tag enables the specific binding and purification of the fusion
protein by binding to nickel-nitrilotriacetic acid (Ni-NTA) metal
affinity chromatography matrix (see, for instance, The
QIAexpressionist, QIAGEN, 1997). A hexa-his tag was used in the
present examples.
[0176] Alternative purification methods include a combination of
HPLC and conventional liquid column chromatography (gel filtration;
ion exchanger; isoelectric focusing).
C. Primary Characterization
[0177] In order to test gp120 binding to the 17b domain of the
sCD4-17b fusion protein, 96-well ELISA plates were first coated
with the 13B8.2 anti-CD4 MAb (Beckman Coulter, Chaska, Minn.,
Catalogue no. 1M0398), whose epitope on CD4 overlaps determinants
involved in binding to gp120. The plates were then incubated with
either the purified sCD4-17b or control buffer. When the chimeric
protein was captured this way, the 17b moiety remained available to
bind gp120 complexed to sCD4; however the sCD4 moiety could not
bind free gp120, since it was captured on the plate by the anti-CD4
MAb that blocks the binding site. The plates were incubated with
gp120 (MB isolate, Ratner et al., Nature 313:277-284, 1985)
complexed to sCD4. Binding of gp120 was detected by a polyclonal
anti-gp120 antiserum, followed by anti-rabbit IgG conjugated to
horseradish peroxidase. The plates were washed and incubated with
ABTS substrate, and the oxidized product was quantitated by
measuring absorbance at 405 nm. The results indicated specific
binding: absorbance values were 0.15 with the sCD4-17b chimeric
protein, compared to 0.05 with the control buffer.
[0178] For testing functionality of the sCD4 region of the chimeric
protein, the ELISA plates were first coated with an anti-His tag
MAb (QIAGEN Inc., Valencia, Calif., Catalog no. 34670), then
incubated with either the purified chimeric protein or control
buffer. With the chimeric protein captured in this way, the sCD4
moiety was available to bind free gp120; however the 17b moiety
could not bind gp120 that was not complexed to sCD4. The plates
were incubated with free gp120, and binding was detected as
detailed above. The results indicated specific binding: absorbance
values were 0.46 with sCD4-17b, compared to 0.05 with the control
buffer. Thus, the ELISA assays confirmed the expected functional
binding properties for each moiety of the chimeric protein: 17b
moiety bound the gp120/sCD4 complex, and the CD4 moiety bound free
gp120.
Example 3
HIV-Envelope Neutralization Measurements
A. Vaccinia-Based Reporter Gene Cell Fusion
[0179] Env-mediated cell fusion activated by CD4 was measured using
the vaccinia-based reporter gene assay (Nussbaum et al., J. Virol.
68:5411-5422, 1994). For the experiment shown in Table 2 and FIG.
1, effector cells (HeLa) were transfected with plasmid
pG1NT7-.beta.-gal (lacZ linked to T7 promoter), then infected with
vaccinia recombinants encoding either the mutant uncleaved Unc Env
or the wildtype (WT) SF162 Env (Broder & Berger, Proc. Natl.
Acad. Sci., USA 92:9004-9008, 1995). Target cells were created by
transfecting NIH 3T3 cells with plasmid pGA9-CKR5, containing the
CCR5 cDNA linked to a vaccinia promoter (Alkhatib et al., Science
272:1955-1958, 1996), then infecting these cells with wild type
vaccinia virus WR. Target cells also carry and express a
bacteriophage T7 RNA polymerase. Prior to fusion assays,
transfected cells were incubated overnight at 31.degree. C. to
allow expression of recombinant proteins, then washed.
[0180] For each fusion assay, mixtures of effector and target cells
(1.times.10.sup.5 of each cell type per well, duplicate wells) were
prepared in the absence or presence of sCD4 (100 nM final). After
2.5 hours at 37.degree. C., cells were lysed with NP-40 and
.beta.-gal activity was quantitated using standard procedures
(Table 2 and FIG. 1). Relative fusion (specific .beta.-gal
activity) was determined from the mean of duplicate samples, and
calculated as WT-Unc.
TABLE-US-00002 TABLE 2 Vaccinia-based reporter gene cell fusion
assay using soluble CD4 Total .beta.-gal (Raw data) Unc Env
(Control) WT Env (SF162) Relative fusion duplicates mean duplicates
mean (WT - Unc) -sCD4 0.50 0.40 0.45 0.50 0.40 0.45 0.0 +sCD4 0.40
0.50 0.45 6.60 5.20 5.90 5.45
[0181] This vaccinia-based fusion assay can be used to assess the
neutralizing ability of the herein disclosed bispecific fusion
proteins. The neutralizing ability of MAb 17b was demonstrated to
be dependent on the addition of soluble CD4 as follows (see Table 3
and FIG. 2). Effector cells were created by co-infecting HeLa cells
with a vaccinia recombinant encoding HIV-1 Env (LAV) (Broder &
Berger, Proc. Natl. Acad. Sci., USA 92:9004-9008, 1995), and
another encoding T7 RNA polymerase. Target cells were created by
co-transfecting NIH 3T3 cells with plasmids pYF1-fusin (Feng et
al., Science 272:872-877, 1996) encoding CXCR4, and
pG1NT7-.beta.-gal (lacZ linked to the T7 promoter). The target
cells were then infected with vaccinia viruses vCB-3 (encoding CD4,
standard assay) (Broder et al., Virology 193:483-491, 1993), or WR
(wild type virus, sCD4 assay). As background controls, target cells
were transfected with pG1NT7-.beta.-gal only (i.e., no coreceptor).
Transfected cells were incubated overnight at 31.degree. C. to
allow expression of recombinant proteins, then washed. Effector
cells were incubated 30 minutes at 37.degree. C. with the indicated
concentration of MAb 17b (Table 3).
[0182] For fusion assays, mixtures were prepared between effector
and indicated target cells (2.times.10.sup.5 of each cell type per
well, duplicate wells); in the standard assay, target cells
expressed CXCR4 and CD4, and no soluble CD4 added; in the sCD4
assay, target cells expressed CXCR4 alone, and soluble CD4 was
added (200 nM final). After 2.5 hours at 37.degree. C., cells were
lysed and .beta.-gal activity was quantitated. Background control
.beta.-gal values (standard assay, 0.6; sCD4 assay, 0.2), obtained
with target cells lacking coreceptor, were subtracted to give the
data presented in Table 3. Data represent percentage of control (no
MAb) for each assay.
TABLE-US-00003 TABLE 3 MAb-mediated inhibition of fusion assay
[17b] Standard Assay sCD4 Assay (.mu.g/ml) .beta.-gal % control
.beta.-gal % control none 42.3 100.0 11.89 100.0 0.1 39.5 93.4
13.55 113.9 0.5 43.9 103.8 4.66 39.2 1 39.8 94.1 1.68 14.1 5 50.5
119.4 0 0
[0183] The effectiveness of the herein described bispecific fusion
proteins for neutralizing fusion is tested in a similar manner, by
adding varying amounts of the bispecific fusion protein, e.g.
sCD4-SCFv(17b), to the above assay. Exogenous sCD4 and SCFv(17b) or
other gp120-binding proteins need not be added, though they can be
used as controls as above, or to determine relative inhibitory
efficiencies compared to the bispecific fusion protein. Using this
assay, the effects of media from control cells infected with
wild-type vaccinia virus WR, were compared with media from cells
infected with the recombinant vaccinia virus encoding sCD4-17b. The
relative specific .beta.-galactosidase values were 23.4 with the
control media and <1 with sCD4-SCFv media. Thus, the chimeric
sCD4-17b protein strongly inhibited HIV-1 Env-mediated cell
fusion.
Example 4
Large Scale Production and Analysis of sCD4-17b
[0184] To produce large amounts of the sCD4-17b protein, the DNA
construct has been transferred to the pET11b plasmid vector
(Novagen, Madison, Wis., Catalog no. 69437-3), which is suitable
for high level inducible expression in E. coli. This system
involves cloning of target genes under control of strong
bacteriophage T7 transcription signal. Once established in a
non-expression host bacterial cell, plasmids are then transferred
into expression hosts containing a chromosomal copy of the T7 RNA
polymerase gene under lacUV5 control, and expression of the
recombinant protein of interest (here, sCD4-17b) is induced by the
addition of IPTG. The expressed protein is produced at a very high
level, and may constitute more than 50% of the total cell protein
in the induced culture within a few hours after induction. Western
blot results indicate high level expression of the sCD4-17b from
the pET11b plasmid.
[0185] The protein produced can be denatured and renatured from
inclusion bodies to provide a large quantity of functional sCD4-17b
protein. This protein can be used for in vitro studies to test
inhibition in assays of both Env-mediated cell fusion and HIV
infection (p24 production).
[0186] In addition, the sCD4-17b protein can be used for in vivo
studies. One in vivo model involves SCID mice reconstituted with
human thymus plus liver (Pettoello-Montovani et al., J. Infect.
Dis. 177:337-346, 1998); this system will be used to test whether
sCD4-17b inhibits (and to what extent), or prevents, acute HIV-1
infection. This system has been successfully used to demonstrate
potent blocking activities of other anti-HIV agents (e.g., protease
inhibitors and reverse transcriptase inhibitors, and Env-targeted
toxins) (Pettoello-Montovani et al., J. Infect. Dis. 177:337-346,
1998).
[0187] A second example of an in vivo system for testing sCD4-17b
activity involves rhesus macaques challenged with SHIV viruses
(recombinant viruses containing SIV gag and pol, plus an HIV
envelope; Li et al., J. Virol. 69:7061-7071, 1995). This system
will be used to test whether the sCD4-17b protein inhibits (and to
what extent), or prevents, acute SHIV infection.
[0188] The effects of sCD4-17b against chronic infection will also
be examined, again using the SCID-hu/HIV-1 mouse system and the
macaque/SHIV system.
[0189] Both in vitro and in vivo study systems also will be used to
test the potency of sCD4-17b protein when used in combination with
other anti-HIV agents (e.g., RT and protease inhibitors or other
HIV-1 neutralizing MAbs).
[0190] The foregoing examples are provided by way of illustration
only. Numerous variations on the biological molecules and methods
described above may be employed to make and use bispecific fusion
molecules capable of binding to two sites on a single protein, and
especially two sites on the HIV envelope protein gp120, and for
their use in detection, treatment, and prevention of HIV infection.
We claim all such subject matter that falls within the scope and
spirit of the following claims.
Sequence CWU 1
1
1115PRTArtificial SequenceDescription of Artificial Sequence linker
peptide 1Gly Gly Gly Gly Ser 1 5235PRTArtificial
SequenceDescription of Artificial Sequence seven repeat polypeptide
linker 2Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly 20 25 30 Gly Gly Ser 353508PRTArtificial
SequenceDescription of Artificial Sequence CD4-scFv(17b) 3Met Asn
Arg Gly Val Pro Phe Arg His Leu Leu Leu Val Leu Gln Leu 1 5 10 15
Ala Leu Leu Pro Ala Ala Thr Gln Gly Lys Lys Val Val Leu Gly Lys 20
25 30 Lys Gly Asp Thr Val Glu Leu Thr Cys Thr Ala Ser Gln Lys Lys
Ser 35 40 45 Ile Gln Phe His Trp Lys Asn Ser Asn Gln Ile Lys Ile
Leu Gly Asn 50 55 60 Gln Gly Ser Phe Leu Thr Lys Gly Pro Ser Lys
Leu Asn Asp Arg Ala 65 70 75 80Asp Ser Arg Arg Ser Leu Trp Asp Gln
Gly Asn Phe Pro Leu Ile Ile 85 90 95 Lys Asn Leu Lys Ile Glu Asp
Ser Asp Thr Tyr Ile Cys Glu Val Glu 100 105 110 Asp Gln Lys Glu Glu
Val Gln Leu Leu Val Phe Gly Leu Thr Ala Asn 115 120 125 Ser Asp Thr
His Leu Leu Gln Gly Gln Ser Leu Thr Leu Thr Leu Glu 130 135 140 Ser
Pro Pro Gly Ser Ser Pro Ser Val Gln Cys Arg Ser Pro Arg Gly 145 150
155 160Lys Asn Ile Gln Gly Gly Lys Thr Leu Ser Val Ser Gln Leu Glu
Leu 165 170 175 Gln Asp Ser Gly Thr Trp Thr Cys Thr Val Leu Gln Asn
Gln Lys Lys 180 185 190 Val Glu Phe Lys Ile Asp Ile Val Val Leu Ala
Phe Gln Lys Ala Ser 195 200 205 Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly 210 215 220 Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly 225 230 235 240Gly Gly Ser Gln
Val Gln Leu Leu Glu Ser Gly Ala Glu Val Lys Lys 245 250 255 Pro Gly
Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Asp Thr Phe 260 265 270
Ile Arg Tyr Ser Phe Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 275
280 285 Glu Trp Met Gly Arg Ile Ile Thr Ile Leu Asp Val Ala His Tyr
Ala 290 295 300 Pro His Leu Gln Gly Arg Val Thr Ile Thr Ala Asp Lys
Ser Thr Ser 305 310 315 320Thr Val Tyr Leu Glu Leu Arg Asn Leu Arg
Ser Asp Asp Thr Ala Val 325 330 335 Tyr Phe Cys Ala Gly Val Tyr Glu
Gly Glu Ala Asp Glu Gly Glu Tyr 340 345 350 Asp Asn Asn Gly Phe Leu
Lys His Trp Gly Gln Gly Thr Leu Val Thr 355 360 365 Val Thr Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 370 375 380 Gly Ser
Glu Leu Glu Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser 385 390 395
400Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Ser
405 410 415 Ser Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Arg Leu 420 425 430 Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Val
Pro Ala Arg Phe 435 440 445 Ser Gly Ser Gly Ser Gly Ala Glu Phe Thr
Leu Thr Ile Ser Ser Leu 450 455 460 Gln Ser Glu Asp Phe Ala Val Tyr
Tyr Cys Gln Gln Tyr Asn Asn Trp 465 470 475 480Pro Pro Arg Tyr Thr
Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Leu 485 490 495 Val Pro Arg
Gly Ser Gly His His His His His His 500 505 41440DNAArtificial
SequenceDescription of Artificial Sequence CD4-scFv(17b)
4atgaaccggg gagtcccttt taggcacttg cttctggtgc tgcaactggc gctcctccca
60gcagccactc agggaaagaa agtggtgctg ggcaaaaaag gggatacagt ggaactgacc
120tgtacagctt cccagaagaa gagcatacaa ttccactgga aaaactccaa
ccagataaag 180attctgggaa atcagggctc cttcttaact aaaggtccat
ccaagctgaa tgatcgcgct 240gactcaagaa gaagcctttg ggaccaagga
aacttccccc tgatcatcaa gaatcttaag 300atagaagact cagatactta
catctgtgaa gtggaggacc agaaggagga ggtgcaattg 360ctagtgttcg
gattgactgc caactctgac acccacctgc ttcaggggca gagcctgacc
420ctgaccttgg agagcccccc tggtagtagc ccctcagtgc aatgtaggag
tccaaggggt 480aaaaacatac agggggggaa gaccctctcc gtgtctcagc
tggagctcca ggatagtggc 540acctggacat gcactgtctt gcagaaccag
aagaaggtgg agttcaaaat agacatcgtg 600gtgctagctt tccagaaggc
ctccggaggt ggcggtagtg ggggaggcgg ttcaggcgga 660ggtggatccg
gtggcggagg gtcgggcggg ggtggaagcg ggggtggcgg ctccggaggc
720ggaggttcac aggtgcagct gctcgagtct ggggctgagg tgaagaagcc
tgggtcctcg 780gtgaaggtct cctgcaaggc ctctggagac accttcatca
gatatagttt tacctgggtg 840cgacaggccc ctggacaagg ccttgagtgg
atgggaagga tcatcactat ccttgatgta 900gcacactacg caccgcacct
ccagggcaga gtcacgatta ccgcggacaa gtccacgagc 960acagtctacc
tggagctgcg gaatctaaga tctgacgata cggccgtata tttctgtgcg
1020ggagtgtacg agggagaggc ggacgagggg gaatatgata ataatgggtt
tctgaaacat 1080tggggccagg gaaccctggt cacggtcacc tcaggtggcg
gtggctccgg aggtggtggg 1140agcggtggcg gcggatctga actcgagttg
acgcagtctc cagccaccct gtctgtgtct 1200ccaggggaaa gagccaccct
ctcctgcagg gccagtgaga gtgttagtag cgacttagcc 1260tggtaccagc
agaaacctgg ccaggctccc aggctcctca tatatggtgc atccaccagg
1320gccaccggtg tcccagccag gttcagtggc agtgggtctg gggcagaatt
cactctcacc 1380atcagcagcc tgcagtctga agattttgca gtttattact
gtcagcagta caataactgg 14405127DNAArtificial SequenceDescription of
Artificial Sequence synthetic oligonucleotide 5cctccggagg
tggcggtagt gggggaggcg gttcaggcgg aggtggatcc ggaggcggag 60ggtcgggcgg
gggtggaagc gggggtggcg gctctggtgg cggaggtacc actagttaag 120tgagtag
127639PRTArtificial SequenceDescription of Artificial Sequence
peptide encoded by SEQ ID NO 5 6Ala Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly 1 5 10 15 Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 30 Gly Gly Gly Gly Thr
Thr Ser 35 7103DNAArtificial SequenceDescription of Artificial
Sequence primer 7ttttatggat ccggtggcgg agggtcgggc gggggtggaa
gcgggggtgg cggctccgga 60ggcggaggtt cacaggtgca gctgctcgag tctggggctg
agg 103832PRTArtificial SequenceDescription of Artificial Sequence
peptide encoded for by SEQ ID NO 7 8Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly 1 5 10 15 Ser Gly Gly Gly Gly Ser
Gln Val Gln Leu Leu Glu Ser Gly Ala Glu 20 25 30 965DNAArtificial
SequenceDescription of Artificial Sequence primer 9taatttatcg
atcacgtgac tagtcctagg cccgggtcaa tgatgatgat gatgatggcc 60gctgc
65108PRTArtificial SequenceDescription of Artificial Sequence
peptide encoded for by SEQ ID NO 9 10Ser Gly His His His His His
His 1 5 11131DNAArtificial SequenceDescription of Artificial
Sequence reverse oligonucleotide 11ctagctactc acttaactag tggtacctcc
gccacctgag ccgccacccc cgcttccacc 60ccccgcccga ccctccgcct ccggatccac
ctccgcctga accgcctccc cactaccgcc 120acctccggag g 131
* * * * *