U.S. patent application number 11/418940 was filed with the patent office on 2006-12-14 for trimeric ox40-immunoglobulin fusion protein and methods of use.
This patent application is currently assigned to Providence Health System. Invention is credited to Nicholas P. Morris, Carmen Peters, Andrew D. Weinberg.
Application Number | 20060280728 11/418940 |
Document ID | / |
Family ID | 37397109 |
Filed Date | 2006-12-14 |
United States Patent
Application |
20060280728 |
Kind Code |
A1 |
Weinberg; Andrew D. ; et
al. |
December 14, 2006 |
Trimeric OX40-immunoglobulin fusion protein and methods of use
Abstract
Compositions including a trimeric OX-40 fusion protein are
disclosed. Also disclosed are methods for enhancing the immune
response of a mammal to an antigen by engaging the OX-40 receptor
on the surface of T-cells involving administering to the mammal a
composition comprising a trimeric OX-40 fusion protein and a
pharmaceutically acceptable carrier.
Inventors: |
Weinberg; Andrew D.;
(Portland, OR) ; Morris; Nicholas P.; (Portland,
OR) ; Peters; Carmen; (Anchorage, AK) |
Correspondence
Address: |
KLARQUIST SPARKMAN, LLP
121 SW SALMON STREET
SUITE 1600
PORTLAND
OR
97204
US
|
Assignee: |
Providence Health System
|
Family ID: |
37397109 |
Appl. No.: |
11/418940 |
Filed: |
May 4, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60678420 |
May 6, 2005 |
|
|
|
Current U.S.
Class: |
424/93.21 ;
424/185.1; 435/320.1; 435/325; 435/456; 435/69.7; 530/350;
536/23.5 |
Current CPC
Class: |
A61K 39/39 20130101;
C07K 14/52 20130101; A61P 37/02 20180101; A61K 38/191 20130101;
A61P 37/04 20180101; C07K 2319/30 20130101; A61K 2039/55516
20130101 |
Class at
Publication: |
424/093.21 ;
530/350; 435/069.7; 435/320.1; 435/325; 536/023.5; 424/185.1;
435/456 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61K 39/00 20060101 A61K039/00; C07H 21/04 20060101
C07H021/04; C12P 21/04 20060101 C12P021/04; C07K 14/705 20060101
C07K014/705; C12N 15/86 20060101 C12N015/86 |
Goverment Interests
ACKNOWLEDGMENT OF GOVERNMENT SUPPORT
[0002] Aspects of the invention disclosed herein were made with
support from the Government of the United States of America,
pursuant to grants 5RO1CA102577 and 5R01CA109563 from the National
Institutes of Health. The United States Government has certain
rights in this invention.
Claims
1. A fusion polypeptide comprising in an N-terminal to C-terminal
direction: an immunoglobulin domain; a trimerization domain; and a
receptor binding domain, wherein the fusion polypeptide
self-assembles into a trimeric fusion protein.
2. The fusion polyeptide of claim 1, wherein the receptor binding
domain comprises a domain of a Tumor Necrosis Family ligand.
3. The fusion polypeptide of claim 2, wherein the receptor binding
domain is an OX-40L receptor binding domain.
4. The fusion polypeptide of claim 3, wherein the fusion protein is
capable of binding to the OX-40 receptor and stimulating at least
one OX-40 mediated activity.
5. The fusion polypeptide of claim 4, OX-40 mediated activity is
CD4.sup.+ T cell proliferation.
6. The fusion polypeptide of claim 3, wherein the OX-40 receptor
binding domain comprises an extracellular domain of OX-40 ligand
(OX-40L).
7. The fusion polypeptide of claim 6, wherein the OX-40 receptor
binding domain comprises a polynucleotide sequence at least 95%
identical to SEQ ID NO:2.
8. The fusion polypeptide of claim 1, wherein the trimerization
domain comprises an isoleucine zipper domain.
9. The fusion polypeptide of claim 1, wherein the isoleucine zipper
domain comprises a polynucleotide sequence at least 95% identical
to SEQ ID NO:4.
10. The fusion polypeptide of claim 1, wherein the immunoglobulin
domain comprises an Fc domain.
11. The fusion polypeptide of claim 1, comprising an amino acid
sequence at least 95% identical to SEQ ID NO:8.
12. The fusion polypeptide of claim 11, wherein the polypeptide is
encoded by a nucleic acid that hybridizes under highly stringent
conditions to a nucleic acid with the polynucleotide sequence of
SEQ ID NO:7.
13. A fusion protein comprising a plurality of the fusion
polypeptides of claim 1.
14. The fusion protein of claim 13, consisting of three or six
fusion polypeptides.
15. The fusion protein of claim 13, comprising an OX-40L fusion
protein.
16. A recombinant nucleic acid comprising a polynucleotide sequence
that encodes the fusion polypeptide of claim 1.
17. The recombinant nucleic acid of claim 16, the polynucleotide
sequence comprising in a 5' to 3' direction: a polynucleotide
sequence encoding an immunoglobulin domain; a polynucleotide
sequence encoding a trimerization domain; and a polynucleotide
encoding a ligand receptor binding domain.
18. The recombinant nucleic acid of claim 17, wherein the
polynucleotide encoding a ligand receptor binding domain encodes an
OX-40L domain.
19. A pharmaceutical composition comprising the fusion polypeptide
of claim 1, or a nucleic acid encoding the fusion polypeptide.
20. A fusion polypeptide comprising in an N-terminal to C-terminal
direction: a dimerization domain; a trimerization domain; and an
OX-40 receptor binding domain, wherein the fusion polypeptide
self-assembles into a trimeric fusion protein.
21. The fusion polypeptide of claim 20, wherein the dimerization
domain is an immunoglobulin domain.
22. The fusion polypeptide of claim 21, wherein the immunoglobulin
domnain is an Fc domain.
23. The fusion polypeptide of claim 20, wherein the trimerization
domain is an isoleucine zipper domain.
24. The fusion polypeptide of claim 20, wherein the trimeric fusion
protein consists of three or six fusion polypeptides.
25. A method of enhancing an immune response in a subject, the
method comprising: administering to a subject exposed to an
antigen, a therapeutically effective amount of the fusion protein
of claim 1, thereby enhancing the immune response to the antigen by
the subject.
26. The method of claim 25, wherein the subject is a human
subject.
27. The method of claim 25, wherein the subject is exposed to the
antigen prior to administering theusion protein.
28. The method of claim 25, wherein the antigen and the trimeric
OX-40L fusion protein are administered at the same time.
29. The method of claim 25, wherein the fusion protein is
administered by expressing a recombinant nucleic acid encoding an
OX-40L fusion polypeptide, which fusion polypeptide assembles into
the fusion protein in at least one cell of the subject.
30. The method of claim 29, wherein the recombinant nucleic acid
encoding the OX-40L fusion polypeptide is introduced ex vivo into
at least one cell, and the at least one cell comprising the
recombinant nucleic acid is introduced into the subject.
31. The method of claim 29, comprising administering the fusion
protein by introducing into the subject a viral or plasmid vector
comprising the nucleic acid encoding an OX-40L fusion polypeptide,
which fusion polypeptide assembles into the fusion protein.
32. The method of claim 31, wherein the viral vector is an
adenovirus vector, a retrovirus vector or a herpesvirus vector.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional
Application No. 60/678/420, filed May 6, 2005, the disclosure of
which is incorporated herein in its entirety.
FIELD
[0003] This disclosure relates to methods and compositions for
generating enhanced immune responses in animals, particularly in
human and non-human mammals. In particular, this disclosure relates
to a trimeric OX-40 ligand fusion protein and to methods for its
use. More generally, this disclosure relates to trimeric fusion
proteins including a receptor binding (ligand) domain, a
trimerization domain and a dimerization domain, such as an
immunoglobulin Fc domain.
BACKGROUND
[0004] Numerous receptor-ligand interactions are involved in the
induction, establishment and modulation of immune responses
directed against antigens. At least two signals are necessary to
activate a CD4 or CD8 T-cell response to antigen (Lenschow et al.
(1996) Ann. Rev. Immunol. 14:233-258). The first signal is
delivered through the T-cell receptor (TCR) by an antigen
(typically a peptide) bound to a major histocompatibility (MHC)
class I or II molecule present on the surface of an antigen
presenting cell (APC). The second signal involves the binding of a
ligand present on the surface of the APC to a second receptor
molecule on the surface of the T-cell. This second signal is termed
co-stimulation, and the APC ligand is often referred to as a
co-stimulatory molecule.
[0005] During activation of CD4.sup.+ T-cells important
co-stimulation is delivered by OX-40 receptor/OX-40 ligand
interaction. The OX-40 receptor ("OX-40") (Paterson et al. (1987)
Mol. Immunol. 24:1281-1290; Calderhead et al. (1993) J. Immunol.
151:5261-5271) has been shown to be present only on antigen
activated CD4.sup.+ T-cells in vivo (Weinberg et al. (1994) J.
Immunol. 152:4712-4721; Wienberg et al. (1996) Nature Medicine
2:183-189) unlike the CD28 receptor, which is present on the
surface of many sub-classes of T-cells (irrespective of whether
they are activated or not). For example, OX-40 is present on
activated CD4.sup.+ T-cells that recognize autoantigen at the site
of inflammation in autoimmune disease, but not in the periphery.
OX-40 has also been shown to be present on the surface of a
percentage of CD4.sup.+ T-cells isolated from tumor infiltrating
lymphocytes and draining lymph node cells removed from patients
with squamous cell tumors of the head and neck and melanomas (Vetto
et al. (1997) Am. J. Surg. 174:258-265). The OX-40 ligand, a member
of the tumor necrosis factor (TNF) superfamily, has been shown to
co-stimulate T-cells which have been activated with an anti-CD3
antibody (i.e., in a nonantigen-specific manner) (Godfrey et al.
(1994) J. Exp. Med. 180:757-762). Despite the recognition of the
costimulatory properties of the OX-40 ligand, its benefits have not
previously been fully exploited to enhance an antigen specific
immune response.
SUMMARY
[0006] This disclosure relates to fusion polypeptides that include
a ligand domain, a trimerization domain and an immunoglobulin Fc
domain, which are capable of forming stable multimeric fusion
proteins. Compositions and methods are provided that are useful for
enhancing and maintaining an immune response of a mammal to an
antigen. More specifically, this disclosure provides novel
multimeric OX-40 ligand ("OX-40L") fusion proteins, as well as
nucleic acids encoding polypeptides that form multimeric OX-40
ligand fusion proteins. This disclosure also provides methods of
using trimeric OX-40 ligand fusion proteins to enhance and/or
maintain an antigen specific immune response in a subject.
[0007] The invention is further detailed in the description,
drawings and non-limiting examples set forth below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 schematically illustrates an exemplary multimeric
protein, namely an OX-40L fusion protein.
[0009] FIG. 2 is an image of an agarose gel illustrating the
correctly sized Fc/ILZ/OX-40L insert and vector.
[0010] FIG. 3A is a line graph illustrating the quantity of fusion
polypeptide eluted in each elution fraction after binding to a
Protein G column. FIG. 3B is an image of a 10% acrylamide gel run
under reducing conditions and stained with Coomassie blue,
illustrating maximal elution in peaks 6-7.
[0011] FIGS. 4A-D are western blots illustrating binding of
anti-human IgG (A and B) and anti-human OX-40L antibodies (C and D)
to purified protein. A and C illustrate blots of gels run under
reducing conditions, whereas B and D illustrate blots of gels run
under non-reducing conditions. A serial dilution is shown in each
panel.
[0012] FIG. 5 is a digital image of a coomassie stained acrylamide
gel illustrating the elution profile in ActiSep elution medium.
[0013] FIG. 6 is a graph illustrating results of size exclusion
chromatography comparing human OX-40L fusion proteins with and
without a trimerization domain.
[0014] FIG. 7 is a graph illustrating proliferation of T cells in
response to exposure to multimeric human OX-40L fusion protein.
BRIEF DESCRIPTION OF SEQUENCE LISTING
[0015] The nucleic and amino acid sequences listed in the
accompanying sequence listing are shown using standard letter
abbreviations for nucleotide bases, and one letter code for amino
acids, as defined in 37 C.F.R.1.822. 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.
All sequences designated herein by GENBANK.RTM. Accession No. refer
to nucleic and amino acid sequences electronically accessible as of
May 6, 2005.
[0016] SEQ ID NO:1 is the polynucleotide sequence of a human OX-40
receptor binding domain.
[0017] SEQ ID NO:2 is the amino acid sequence of a human OX-40
receptor binding domain.
[0018] SEQ ID NO:3 is the polynucleotide sequence of an isoleucine
zipper (ILZ) trimerization domain.
[0019] SEQ ID NO:4 is the amino acid sequence of a yeast mutant
Gcn4 isoleucine zipper (ILZ) trimerization domain.
[0020] SEQ ID NO:5 is the polynucleotide sequence of a human
immunoglobulin Fc domain.
[0021] SEQ ID NO:6 is the amino acid sequence of a human
immunoglobulin Fc domain.
[0022] SEQ ID NO:7 is the polynucleotide sequence of a human OX-40
ligand fusion polypeptide.
[0023] SEQ ID NO:8 is the amino acid sequence of a human OX-40
ligand fusion polypeptide.
[0024] SEQ ID NO:9 is the sequence of the OX-40 ligand denoted as
GENBANK.RTM. Accession No. NM 003326.
[0025] SEQ ID NO:10 is the nucleotide sequence of the primer
hFc.gamma.1-5'.
[0026] SEQ ID NO:11 is the nucleotide sequence of the primer
hOX-40L-3'.
[0027] SEQ ID NO:12 is the polynucleotide sequence of the human
BM40 protein secretory signal.
[0028] SEQ ID NO:13 is the amino acid sequence of the human BM40
protein secretory signal.
DETAILED DESCRIPTION
Introduction
[0029] Engagement of the OX-40 receptor on CD4+ T-cells during, or
shortly after, priming by an antigen results in an increased
response of the CD4.sup.+ T-cells to the antigen. In the context of
the present disclosure, the term "engagement" refers to binding to
and stimulation of at least one activity mediated by the OX-40
receptor. For example, engagement of the OX-40 receptor on antigen
specific CD4.sup.+ T-cells results in increased T cell
proliferation as compared to the response to antigen alone. The
elevated response to the antigen can be maintained for a period of
time substantially longer than in the absence of OX-40 receptor
engagement. Thus, stimulation via the OX-40 receptor enhances the
antigen specific immune response and increases resistance to
disease by boosting T-cell recognition of antigens presented by
infectious agents, such as bacteria and viruses, as well as tumor
cells.
[0030] OX-40 receptor binding agents enhance antigen specific
immune responses in a subject, such as a human subject, when
administered to the subject during or shortly after priming of
T-cells by an antigen. OX-40 receptor binding agents include OX-40
ligand ("OX-40L"), such as soluble extracellular ligand domains and
OX-40L fusion proteins; anti-OX-40 antibodies (for example,
monoclonal antibodies such as humanized monoclonal antibodies); and
immunologically effective portions of anti-OX-40 antibodies. A
specific example is a novel OX-40L fusion polypeptide that
self-assembles into a multimeric (e.g., trimeric or hexameric)
OX-40L fusion protein. The multimeric OX-40L fusion protein
exhibits increased efficacy in enhancing antigen specific immune
response in a subject, particularly a human subject, relative to
previously described OX-40L fusion polypeptides. This increased
activity results from the novel ability of this OX-40L fusion
polypeptide to spontaneously assemble into highly stable trimers
and hexamers. Also described are nucleic acids including
polynucleotide sequences that encode such fusion polypeptides. This
disclosure also provides methods for enhancing an antigen specific
immune response in a subject using the multimeric OX-40L fusion
polypeptides. The compositions and methods disclosed herein with
respect to OX-40L fusion proteins can be more generally applied to
the production and use of multimeric (e.g., trimeric and hexameric)
receptor-binding fusion proteins.
Summary of Specific Embodiments
[0031] This disclosure relates to a multimeric OX-40L fusion
protein that is useful for enhancing an antigen specific immune
response in a subject, such as a human subject. A trimeric OX-40L
fusion protein is composed of three OX-40L fusion polypeptides,
each of which includes an OX-40 ligand domain, a trimerization
domain and a dimerization domain, such as an immunoglobulin Fc
domain. The trimerization domain promotes self-assembly of the
expressed polypeptide by associating with two other trimerization
domains to form a trimer. Upon assembly of the OX-40L fusion
protein into a trimer, two Fc domains dimerize, and one Fc domain
remains unpaired. The unpaired Fc domain associates with an
unpaired Fc domain of a second OX-40L fusion protein trimer giving
rise to a stable OX-40L fusion protein hexamer. For convenience,
because the basic unit of this fusion protein is an assembly of
three OX-40L fusion polypeptides, both the OX-40L fusion protein
trimer and hexamer (formed from two OX-40L fusion protein trimers)
are referred to herein as a "trimeric OX-40L fusion protein."
[0032] In an embodiment, the present disclosure provides a fusion
polypeptide that includes in an N-terminal to C-terminal direction:
an immunoglobulin Fc domain; a domain that induces trimerization of
the fusion polypeptide (a "trimerization domain"); and an OX-40
receptor binding domain (FIG. 1). The fusion polypeptide forms a
trimeric OX-40L fusion protein upon expression, which assembles
into an active hexameric complex including two trimeric OX-40L
fusion proteins. Within the trimeric OX-40L fusion protein, the Fc
domain dimerizes, leaving one unpaired Fc polypeptide. The unpaired
Fc domain in the fusion protein trimer is capable of interacting
with the unpaired Fc domain of another OX-40L trimer acting as a
dimerization domain between two OX-40L trimers and resulting in the
formation of a hexamer (FIG. 1, and Holler et al., Mol. Cell. Biol.
23:1428, 2003). Thus, embodiments of the present disclosure include
OX-40L fusion polypeptides that include in an N-terminal to
C-terminal direction: a dimerization domain; a trimerization
domain; and an OX-40 receptor binding domain. The fusion protein
produced by assembly of this fusion polypeptide is capable of
binding to, and stimulating at least one activity of, the OX-40
receptor. A particularly favorable attribute of this trimeric
OX-40L fusion protein is its increased ability (as compared to
previously described OX-40L fusion polypeptides) to stimulate
activity, for example cellular proliferation, mediated via the
OX-40 receptor.
[0033] Generally (but not necessarily), the OX-40 receptor binding
domain and the immunoglobulin Fc domain are selected from a species
that corresponds to that of the subject to which the fusion protein
is to be administered. For example, if the subject is a human,
optimal efficacy and minimal immunogenicity ("antigenicity") of the
fusion protein can be achieved by administering a fusion protein
with a human OX-40 receptor binding domain and a human Fc domain.
Similarly, for example, if the subject is a non-human animal (e.g.,
a mammal), such as a mouse, a fusion protein made up of
polypeptides that include a murine OX-40 receptor binding domain
and a murine Fc domain can be administered. Likewise, for any other
mammalian subject (e.g., veterinary subjects, including dogs, cats,
horses, cows, pigs, sheep, goats, and non-human primates), the
appropriate species specific OX-40 and immunoglobulin domains are
included in a trimeric OX-40L fusion protein.
[0034] In an embodiment, the fusion polypeptide includes a
trimerization domain that is an isoleucine zipper domain, for
example, the isoleucine zipper domain represented by the amino acid
sequence of SEQ ID NO:4. In an embodiment, the OX-40 receptor
binding domain is an extracellular domain of an OX-40 ligand. For
example, the OX-40 receptor binding domain can be the extracellular
domain of the human OX-40 ligand.
[0035] In addition to the receptor binding domain and the
trimerization domain, the fusion polypeptides disclosed herein also
include an immunoglobulin constant region domain. The constant
region domain is typically an Fc domain. For example, the
immunoglobulin constant region domain can include a human IgG
constant region domain (e.g., the CH2 and CH3 domains), such as a
human IgG1 Fc region. An exemplary amino acid sequence of an
immunoglobulin Fc domain is provided in SEQ ID NO:6.
[0036] In an embodiment, the fusion polypeptide is a polypeptide
with the amino acid sequence represented by SEQ ID NO:8. Fusion
polypeptides with at least 95% sequence identity to SEQ ID NO:8 are
also included among the fusion polypeptides disclosed herein. For
example, a fusion polypeptide encompassed by the present disclosure
includes a fusion polypeptide with a sequence that is at least 96%
identical to SEQ ID NO:8. In an embodiment, the fusion polypeptide
is at least 97% identical. In certain embodiments, the fusion
polypeptide is as much as 98%, or even as much or greater than 99%
identical to SEQ ID NO:8. For example, a fusion polypeptide that
forms a trimeric OX-40L fusion protein can include at least one
amino acid deletion, addition or substitution relative to SEQ ID
NO:8, (or at most 2, 5 or 10 amino acid deletions, additions or
substitutions relative to SEQ ID NO:8). That is, a fusion
polypeptide can include one amino acid deletion, addition or
substitution relative to SEQ ID NO:8, or it can include more than
one (such as two, three, four or five) amino acid deletions,
additions or substitutions relative to SEQ ID NO:8. Typically,
where a fusion polypeptide has an amino acid alteration (deletion,
addition or substitution) relative to SEQ ID NO:8, the function or
activity of the polypeptide is not substantially altered with
respect to the activity of the fusion polypeptide represented by
SEQ ID NO:8. For example, where an amino acid substitution is
present, the amino acid substitution is most commonly a
conservative amino acid substitution.
[0037] Another feature of the disclosure includes recombinant
nucleic acids that encode an OX-40L fusion polypeptide, such as the
polypeptide represented by SEQ ID NO:8. The nucleic acids described
herein encode OX-40L fusion polypeptides that possess the desirable
characteristic of assembling into a trimeric OX-40L fusion protein
that is capable of binding to and stimulating activity of the OX-40
receptor. In an embodiment, the fusion polypeptide is encoded by a
nucleic acid with the polynucleotide sequence represented by SEQ ID
NO:7. In other embodiments, the fusion polypeptide is encoded by a
related polynucleotide sequence that differs from SEQ ID NO:7 by
the deletion, addition or substitution of one or more nucleotides.
For example, a nucleic acid that hybridizes under highly stringent
conditions to a nucleic acid with the polynucleotide sequence of
SEQ ID NO:7. Typically, the nucleic acids are at least 95%
identical to SEQ ID NO:7. For example, a nucleic acid that encodes
an OX-40L fusion polypeptide can be at least 96%, or at least 97%,
or frequently at least 98%, or even 99% identical to SEQ ID
NO:7.
[0038] A recombinant nucleic acid that encodes an OX-40L fusion
polypeptide in accordance with the present disclosure generally
includes in a 5' to 3' direction: a polynucleotide sequence that
encodes an immunoglobulin Fc domain; a polynucleotide sequence that
encodes a trimerization domain; and a polynucleotide sequence that
encodes an OX-40 receptor binding domain. The nucleic acids encode
an OX-40L fusion polypeptide that includes in an N-terminal to
C-terminal direction: an immunoglobulin Fc domain; a trimerization
domain; and an OX-40 receptor binding domain.
[0039] As discussed above, it is generally desirable to select
polynucleotide sequences that encode polypeptides (polypeptide
domains) that correspond to the species of the subject to whom the
encoded fusion proteins are to be administered. Thus,
polynucleotide sequences encoding polypeptides having the amino
acid sequence of human protein domains, for example, the human
OX-40L receptor binding domain and a human immunoglobulin Fc domain
are selected for administration to a human subject. In a similar
manner, polynucleotide sequences that encode the polypeptide
sequence found in any other species can be selected for
administration to a subject of that species.
[0040] For example, in one embodiment, the nucleic acid encoding
the OX-40L fusion polypeptide includes a polynucleotide sequence
that encodes a human Ig Fc domain, such as a human IgG1 Fc domain.
Typically, the polynucleotide sequence encodes one or both of a CH2
domain and a CH3 domain. For example, the polynucleotide sequence
encoding the immunoglobulin domain can be the polynucleotide
sequence represented by SEQ ID NO:5.
[0041] The trimerization domain can be encoded by a polynucleotide
sequence that encodes an isoleucine zipper domain, as indicated
above. In an embodiment, the trimerization domain is an isoleucine
zipper domain encoded by the polynucleotide sequence represented by
SEQ ID NO:3.
[0042] Typically, the OX-40 receptor binding domain is encoded by a
polynucleotide sequence that encodes an extracellular domain of
OX-40L. For example, the recombinant nucleic acid can include the
polynucleotide sequence represented by SEQ ID NO: 1.
[0043] More generally, the disclosure can be applied to the
production and use of trimeric fusion proteins that incorporate a
receptor binding (e.g., ligand) domain, a trimerization domain and
an immunoglobulin Fc domain. Such fusion proteins self-assemble
into stable timers (and hexamers) with enhanced biological
activities relative to other soluble forms of the ligand. For
example, trimeric fusion proteins that include in an N-terminal to
C-terminal direction: an immunoglobulin Fc domain; a trimerization
domain; and a receptor binding domain. Typically, the receptor
binding domain includes one or more domain (such as an
extracellular domain) of a ligand that specifically binds to the
receptor. Exemplary receptor binding domains that can be included
in trimeric fusion proteins include TNF ligand domains, such as
domains from the following ligands: TNF-a, TNF-b, Lymphotoxin-b,
CD40L, FasL, CD27L, CD30L, 4-1BBL, TRAIL, RANK ligand, TWEAK,
APRIL, BAFF, LIGHT, GITR ligand, EDA-A1, EDA-A2. Nucleic acids
encoding these trimeric fusions can be produced and introduced into
vectors as discussed below.
[0044] Another aspect of the disclosure relates to a method of
enhancing an immune response in a subject. The method disclosed
herein involves administering a trimeric OX-40L fusion protein to a
subject who (or which) has been exposed to an antigen.
Administration of the trimeric OX-40L fusion protein serves to
enhance the antigen specific immune response (e.g., the antigen
specific T-cell response) to the antigen. The subject can be a
human subject, or a non-human subject. Typically, the non-human
subject is a mammal (a veterinary subject), such as a dog, a cat, a
horse, a cow, a pig, a sheep, a goat, or a non-human primate.
[0045] In an embodiment, the subject is exposed to the antigen
prior to administration of the trimeric OX-40L fusion protein.
Typically, if the subject is exposed to a soluble antigen prior to
administration of the trimeric OX-40L fusion protein, the fusion
protein is administered within about 10 days of exposure to the
antigen. For example, the OX-40L fusion protein can be administered
within about 7 days, for example within 24 to 48 hours, or within 3
days, or within about 7 days after exposure to the antigen. The
exposure to the antigen can be brought about intentionally, for
example, in the form of a vaccine. Alternatively, the exposure can
be unintended, such as an environmental exposure to a pathogen
(such as a bacterium, a virus, or a cellular or extracellular
parasite), or the occurrence of a tumor. In another embodiment, the
exposure to the antigen and administration of trimeric OX-40L
fusion protein occur at the same time. If the exposure to the
antigen and the administration of the trimeric OX-40L fusion
protein occur simultaneously, the exposure (e.g., the intentional
exposure) and administration of the trimeric fusion protein can be
effected in a single formulation or pharmaceutical composition.
Alternatively, the antigen and the trimeric OX-40L fusion protein
can be administered in separate formulations.
[0046] In one embodiment, the trimeric OX-40L fusion protein is
administered by expressing a recombinant nucleic acid encoding an
OX-40L fusion polypeptide capable of trimerization in at least one
cell of the subject. Upon expression in the cell(s) of the subject,
the fusion polypeptides assemble into the trimeric OX-40L fusion
protein. For example, a nucleic encoding the fusion polypeptide can
be introduced into a cell (such as a cell, a mixed population of
cells, or a purified population of cells removed from the subject)
ex-vivo. The cell(s) comprising the recombinant nucleic acid are
then introduced into the subject where the trimeric OX-40L fusion
protein is expressed. The cell can be an autologous cell removed
from the subject, or the cell can be a heterologous cell, such as a
cell line (e.g., a cell line catalogued by the American Type
Culture Collection, "ATCC").
[0047] In another embodiment, the OX-40L fusion protein is
administered by introducing a vector (such as a bacterial plasmid
or viral vector) including a nucleic acid encoding the fusion
polypeptide, which assembles into the trimeric OX-40L fusion
protein. For example, the vector can be an adenovirus vector, a
retrovirus vector or a herpesvirus vector. If a viral vector is
employed, it can be an attenuated or disabled virus, incapable of
autonomous replication in the cells of the subject, thus, unable to
cause a pathologic infection in the subject.
[0048] In some cases, the cell into which the recombinant nucleic
acid encoding the trimeric OX-40L fusion protein is introduced is
an antigen presenting cell (e.g., a B cell, a macrophage, a
dendritic cell, etc.). The antigen can be an antigen of a
pathogenic agent, such as a viral antigen, a bacterial antigen or
an antigen of a parasite, or the antigen can be a tumor antigen. If
the antigen is a tumor antigen, that is, an antigen expressed by or
on a tumor cell, then the cell into which the recombinant nucleic
acid encoding the trimeric OX-40L fusion protein is introduced can
be a tumor cell (such as an autologous tumor cell obtained, e.g.,
following surgical removal or biopsy of a primary or metastatic
tumor). Alternatively, a tumor cell line can be utilized, such as
an immortalized or established tumor cell line. Typically, the cell
line is selected to correspond to the type (i.e., origin, cell or
tissue type) of tumor to be treated in the subject.
[0049] Additional details regarding the various embodiments are
provided below.
Terms
[0050] Unless otherwise explained, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this disclosure belongs.
Definitions of common terms in molecular biology can be found in
Benjamin Lewin, Genes V, published by Oxford University Press, 1994
(ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of
Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN
0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and
Biotechnology: a Comprehensive Desk Reference, published by VCH
Publishers, Inc., 1995 (ISBN 1-56081-569-8).
[0051] The singular terms "a," "an," and "the" include plural
referents unless context clearly indicates otherwise. Similarly,
the word "or" is intended to include "and" unless the context
clearly indicates otherwise. It is further to be understood that
all base sizes or amino acid sizes, and all molecular weight or
molecular mass values, given for nucleic acids or polypeptides are
approximate, and are provided for description. Although methods and
materials similar or equivalent to those described herein can be
used in the practice or testing of this disclosure, suitable
methods and materials are described below. The term "comprises"
means "includes." The abbreviation, "e.g." is derived from the
Latin exempli gratia, and is used herein to indicate a non-limiting
example. Thus, the abbreviation "e.g." is synonymous with the term
"for example."
[0052] In order to facilitate review of the various embodiments of
this disclosure, the following explanations of specific terms are
provided:
[0053] The "OX-40 receptor" is a protein (also variously termed
ACT-4 and ACT-35) expressed on the surface of antigen-activated
mammalian CD4.sup.+ T-cells (Weinberg et al. (1994) J. Immunol.
152:4712-4721; Weinberg et al. (1996) Nature Medicine 2:183-189; WO
95/12673; Latza et al. (1994) Eur. J. Immunol. 24:677-683). DNA
sequences encoding mouse, rat and human OX-40 receptor homologs
have been cloned and sequenced (Mallet et al. (1990) EMBO J.
9:1063-1068; Calderhead et al. (1993) J. Immunol. 151:5261-5271;
Latza et al. (1994) Eur. J. Immunol. 24:677-683; WO 95/12673).
Additionally, nucleotide and amino acid sequences for the human and
mouse OX-40 receptors can be found in GENBANK.RTM. as Accession
Nos. NM 003327 and NM 011659, respectively.
[0054] The "OX-40 ligand" ("OX-40L") (also variously termed gp34
and ACT-4-L) has been found expressed on the surface of certain
mammalian cells, such as antigen presenting cells ("APCs"). OX-40L
specifically binds to the OX-40 receptor. The human protein is
described in PCT Publication No. WO 95/21915. The mouse OX-40L is
described in U.S. Pat. No. 5,457,035. Polynucleotide and amino acid
sequences of the human and mouse OX-40L are available in
GENBANK.RTM. as Accession Nos. NM 003326 and NM 009452,
respectively. The naturally occurring OX-40 ligand includes
intracellular, transmembrane and extracellular domains. A
functionally active soluble form of OX-40 ligand ("soluble OX-40
ligand") can be produced by deleting the intracellular and
transmembrane domains as described, e.g., in U.S. Pat. Nos.
5,457,035 and 6,312,700, and WO 95/21915, the disclosures of which
are incorporated herein for all purposes. A functionally active
form of OX-40 ligand is a form that retains the capacity to bind
specifically to the OX-40 receptor, that is, that possesses an
OX-40 "receptor binding domain." Methods of determining the ability
of an OX-40 ligand molecule or derivative to bind specifically to
the OX-40 receptor are discussed below. Methods of making and using
the OX-40 ligand and its derivatives (such as derivatives that
include an OX-40 receptor binding domain) are described in WO
95/21915 (supra), which also describes proteins comprising the
soluble form of OX-40 ligand linked to other peptides, such as
human immunoglobulin ("Ig") Fc regions, that can be produced to
facilitate purification of OX-40 ligand from cultured cells, or to
enhance the stability of the molecule after in vivo administration
to a mammal (see also, U.S. Pat. No. 5,457,035).
[0055] As used herein, the term "OX-40L" includes the entire OX-40
ligand, soluble OX-40 ligand, and functionally active portions of
the OX-40 ligand. Also included within the definition of OX-40L are
OX-40 ligand variants which vary in amino acid sequence from
naturally occurring OX-40 ligand molecules but which retain the
ability to specifically bind to an OX-40 receptor. Such variants
are described in U.S. Pat. No. 5,457,035 and WO 95/21915
(supra).
[0056] An "OX-40 receptor binding agent" is an agent that binds
substantially only to an OX-40 receptor, e.g., an OX-40 receptor
present on the surface of antigen activated mammalian T-cells, such
as activated CD4.sup.+ T-cells. As used herein, the term "OX-40
receptor binding agent" includes anti-OX-40 antibodies and OX-40L.
An OX-40 "receptor binding domain" is a domain that binds
specifically to an OX-40 receptor.
[0057] A "trimerization domain" is an amino acid sequence within a
polypeptide that promotes assembly of the polypeptide into trimers.
For example, a trimerization can promote assembly into trimers via
associations with other trimerization domains (of additional
polypeptides with the same or a different amino acid sequence). The
term is also used to refer to a polynucleotide that encodes such a
peptide or polypeptide.
[0058] The term "Fc" domain refers to a portion of an antibody
constant region. Traditionally, the term Fc domain refers to a
protease (e.g., papain) cleavage product encompassing the paired
CH2, CH3 and hinge regions of an antibody. In the context of this
disclosure, the term Fc domain or Fc refers to any polypeptide (or
nucleic acid encoding such a polypeptide), regardless of the means
of production, that includes all or a portion of the CH2, CH3 and
hinge regions of an immunoglobulin polypeptide.
[0059] The term "anti-OX-40 antibodies" encompasses monoclonal and
polyclonal antibodies which are specific for OX-40, that is, which
bind substantially only to OX-40 when assessed using the methods
described below, as well as immunologically effective portions
("fragments") thereof. Immunologically effective portions of
antibodies include Fab, Fab', F(ab').sub.2, Fabc and Fv portions
(for a review, see Better and Horwitz (1989) "Advances in Gene
Technology: The Molecular Biology of Immune Disease and the Immune
Response" (ICSU Short Reports, Streilein et al. (eds.) vol. 10). In
the present disclosure, immunologically effective portions of
antibodies commonly include a heavy chain domain. Humanized forms
of anti-OX-40 antibodies, e.g., monoclonal antibodies, and
immunologically effective portions of anti-OX-40 antibodies are
described in PCT Publication Nos. WO 95/12673 and WO 95/21915
(supra), along with methods which can be employed to produce such
antibodies. Anti-OX-40 antibodies can also be produced using
standard procedures described in a number of texts, including
Antibodies: A Laboratory Manual by Harlow and Lane, Cold Spring
Harbor Laboratory (1988).
[0060] More generally, an "antibody" or "immunoglobulin" (or an
immunologically active portions of an immunoglobulin molecule) is a
molecule that contains an antigen binding site that specifically
binds (immunoreacts with) an antigen. A naturally occurring
antibody (e.g., IgG, IgM, IgD) includes four polypeptide chains,
two heavy (H) chains and two light (L) chains interconnected by
disulfide bonds. However, it has been shown that the
antigen-binding function of an antibody can be performed by
fragments of a naturally occurring antibody. Thus, these
antigen-binding fragments are also intended to be designated by the
term "antibody." Specific, non-limiting examples of binding
fragments encompassed within the term antibody include (i) a Fab
fragment consisting of the V.sub.L, V.sub.H, C.sub.L and C.sub.H1
domains; (ii) an F.sub.d fragment consisting of the V.sub.H and
C.sub.H1 domains; (iii) an Fv fragment consisting of the V.sub.L
and V.sub.H domains of a single arm of an antibody, (iv) a dAb
fragment (Ward et al. (1989) Nature 341:544-546) which consists of
a V.sub.H domain; (v) an isolated complimentarity determining
region (CDR); and (vi) a F(ab').sub.2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the
hinge region.
[0061] Methods of producing polyclonal and monoclonal antibodies
are known to those of ordinary skill in the art, and many
antibodies are available. See, for example, Coligan (1991) Current
Protocols in Immunology Wiley/Greene, NY; Harlow and Lane (1989)
Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY;
Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange
Medical Publications, Los Altos, Calif., and references cited
therein; Goding (1986) Monoclonal Antibodies: Principles and
Practice (2d ed.) Academic Press, New York, N.Y.; and Kohler and
Milstein (1975) Nature 256: 495497. Other suitable techniques for
antibody preparation include selection of libraries of recombinant
antibodies in phage or similar vectors. See, Huse et al. (1989)
Science 246: 1275-1281; and Ward et al., (1989) Nature 341:
544-546. "Specific" monoclonal and polyclonal antibodies and
antisera (or antiserum) will usually bind with a K.sub.D of at
least about 0.1 .mu.M, preferably at least about 0.01 .mu.M or
better, and most typically and preferably, 0.001 .mu.M or
better.
[0062] Immunoglobulins and certain variants thereof are known and
many have been prepared in recombinant cell culture (e.g., see U.S.
Pat. No. 4,745,055; U.S. Pat. No. 4,444,487; PCT Publication No. WO
88/03565; European Patent Nos. EP 256,654; EP 120,694; EP 125,023;
Faoulkner et al. (1982) Nature 298:286; Morrison (1979) J. Immunol.
123:793; and Morrison et al. (1984) Ann Rev. Immunol. 2:239).
Detailed methods for preparation of chimeric (humanized) antibodies
can be found in U.S. Pat. No. 5,482,856. Additional details on
humanization and other antibody production and engineering
techniques can be found in Borrebaeck (ed) (1995) Antibody
Engineering, 2.sup.nd Edition Freeman and Company, NY; McCafferty
et al. (1996) Antibody Engineering, A Practical Approach IRL at
Oxford Press, Oxford, England; and Paul (1995) Antibody Engineering
Protocols Humana Press, Towata, N.J.
[0063] The abbreviation "DNA" refers to deoxyribonucleic acid. DNA
is a long chain polymer which comprises the genetic material of
most living organisms (some viruses have genes comprising
ribonucleic acid ("RNA"). The units in DNA polymers are four
different nucleotides, each of which comprises one of the four
bases, adenine, guanine, cytosine and thymine bound to a
deoxyribose sugar to which a phosphate group is attached. Triplets
of nucleotides (referred to as codons) code for each amino acid in
a polypeptide. The term codon is also used for the corresponding
(and complementary) sequences of three nucleotides in the mRNA into
which the DNA sequence is transcribed.
[0064] A "cDNA" or "complementary DNA" is a piece of DNA lacking
internal, non-coding segments (introns) and transcriptional
regulatory sequences. cDNA can also contain untranslated regions
(UTRs) that are responsible for translational control in the
corresponding RNA molecule. cDNA is synthesized in the laboratory
by reverse transcription from messenger RNA extracted from
cells.
[0065] A "transformed" cell, or a "host" cell, is a cell into which
a nucleic acid molecule has been introduced by molecular biology
techniques. As used herein, the term transformation encompasses all
techniques by which a nucleic acid molecule can 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. A
transformed cell or a host cell can be a bacterial cell or a
eukaryotic cell.
[0066] An "isolated" biological component (such as a nucleic acid
or protein) has been substantially separated or purified away from
other biological components in the cell of the organism in which
the component naturally occurs, such as, other chromosomal and
extrachromosomal DNA and RNA, and proteins. Isolated nucleic acids
and proteins 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.
[0067] The term "purified" does not require absolute purity;
rather, it is intended as a relative term. Thus, for example, a
purified OX-40 ligand preparation is one in which the OX-40 ligand
is more pure than the ligand in its natural environment within a
cell. Preferably, a preparation of an OX-40 ligand is purified such
that the OX-40 ligand protein represents at least 50% of the total
protein content of the preparation.
[0068] A "recombinant" nucleic acid is one that has a sequence that
is not naturally occurring or that has a sequence that is made by
an artificial combination of two otherwise separated segments of
sequence. This artificial combination is often accomplished by
chemical synthesis or, more commonly, by the artificial
manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques.
[0069] The term "polynucleotide" or "nucleic acid" refers to a
polymeric form of nucleotide at least 10 bases in length. The term
polynucleotide "sequence" refers to the series of constituent
nucleotides that make up a polynucleotide. The term polynucleotide
sequence is also used to refer to the series of letters, e.g., a,
c, g, t, that are used to represent a nucleic acid. A "recombinant"
nucleic acid (e.g., a recombinant DNA) includes a genetic element
(a polynucleotide sequence) that is not immediately contiguous with
both of the genomic elements with which it is immediately
contiguous (one on the 5' end and one on the 3' end) in the
naturally occurring genome of the organism from which it is
derived. The term therefore includes, for example, a recombinant
DNA which is incorporated into a vector; into an autonomously
replicating plasmid or virus; or into the genomic DNA of a
prokaryote or eukaryote, or which exists as a separate molecule
(e.g., a cDNA) independent of other sequences. The nucleotides can
be ribonucleotides, deoxyribonucleotides, or modified forms of
either nucleotide. The term includes single- and double-stranded
forms of DNA.
[0070] A "vector" is nucleic acid molecule as introduced into a
host cell, thereby producing a transformed host cell. A vector can
include nucleic acid sequences that permit it to replicate in a
host cell, such as an origin of replication. A vector can also
include one or more selectable marker gene and other genetic
elements known in the art.
[0071] A nucleic acid that regulates the expression of a
heterologous polynucleotide sequence to which it is operably linked
is referred to as an "expression control sequence" or a
"transcription regulatory sequence." A transcription regulatory
sequence is operably linked to a nucleic acid sequence when the
regulatory sequence controls and regulates the transcription and,
as appropriate, translation of the nucleic acid sequence. Thus,
transcription regulatory sequences can include appropriate
promoters, enhancers, transcription terminators, a start codon
(typically, ATG) in front of a protein-encoding gene, splicing
signal for introns, maintenance of the correct reading frame of
that gene to permit proper translation of mRNA, and stop codons.
The term "control sequences" is intended to include, at a minimum,
components whose presence can influence expression, and can also
include additional components whose presence is advantageous, for
example, leader sequences and fusion partner sequences.
[0072] A "promoter" is a minimal sequence sufficient to direct
transcription of a nucleic acid. Also included are those promoter
elements which are sufficient to render promoter-dependent gene
expression controllable for cell-type specific, tissue-specific, or
inducible by external signals or agents; such elements can be
located in the 5' or 3' regions of the gene. Both constitutive and
inducible promoters are included (see, e.g., Bitter et al. Methods
in Enzymology (1987) 153:516-544). For example, when cloning in
bacterial systems, inducible promoters such as pL of bacteriophage
lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like
can be used. In one embodiment, when cloning in mammalian cell
systems, promoters derived from the genome of mammalian cells (for
example, metallothionein promoter) or from mammalian viruses (for
example, the cytomegalovirus immediate early promoter, the
retrovirus long terminal repeat; the adenovirus late promoter; the
vaccinia virus 7.5K promoter) can be used. Promoters produced by
recombinant DNA or synthetic techniques can also be used to provide
for transcription of the nucleic acid sequences.
[0073] A first nucleic acid sequence is "operably linked" to 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, for example, two polypeptide domains or
components of a fusion protein.
[0074] A polynucleotide is said to "encode" a polypeptide if, in
its native state or when manipulated by methods well known to those
skilled in the art, it can be transcribed and/or translated to
produce the mRNA for and/or the polypeptide or a fragment thereof.
The anti-sense strand is the complement of such a nucleic acid, and
the encoding sequence can be deduced therefrom.
[0075] A "polypeptide" is any chain of amino acids, regardless of
length or post-translational modification (for example,
glycosylation or phosphorylation), such as a protein or a fragment
or subsequence of a protein. The term "peptide" is typically used
to refer to a chain of amino acids of between 3 and 30 amino acids
in length. For example an immunologically relevant peptide can be
between about 7 and about 25 amino acids in length, e.g., between
about 8 and about 10 amino acids.
[0076] In the context of the present disclosure, a polypeptide can
be a fusion polypeptide comprising a plurality of constituent
polypeptide (or peptide) elements. Typically, the constituents of
the fusion polypeptide are genetically distinct, that is, they
originate from distinct genetic elements, such as genetic elements
of different organisms or from different genetic elements (genomic
components) or from different locations on a single genetic
element, or in a different relationship than found in their natural
environment. Nonetheless, in the context of a fusion polypeptide
the distinct elements can be translated as a single polypeptide.
The term monomeric fusion polypeptide (or monomeric fusion protein)
is used synonymously with a single fusion polypeptide molecule to
clarify reference to a single constituent subunit where the
translated fusion polypeptides assume a multimeric tertiary
structure or protein, e.g., a trimeric OX-40L fusion protein.
[0077] The term "mammal" includes both human and non-human mammals.
Similarly, the term "subject" or "patient" includes both human and
veterinary subjects or patients.
Trimeric OX-40L Fusion Proteins
[0078] Various formulations of OX-40 receptor binding agents have
been described, including antibodies to the OX-40 receptor and a
variety of OX-40L molecules. Such OX-40 receptor binding agents are
useful for enhancing and maintaining an antigen specific immune
response in a subject. For example, fusion proteins in which one or
more domains of OX-40L are covalently linked to one or more
additional protein domains can be administered to a subject with or
following administration of (or exposure to) an antigen, to enhance
the strength and/or duration of the antigen specific immune
response. Exemplary OX-40L fusion proteins that can be used as
OX-40 receptor binding agents are described in U.S. Pat. No.
6,312,700, the disclosure of which is incorporated herein for all
purposes.
[0079] The present disclosure relates more specifically to an
OX-40L fusion polypeptide that has the advantageous property of
assembling into a trimeric form with an increased ability to
stimulate human T cells relative to previously described OX-40L
fusion polypeptides. An exemplary embodiment is illustrated
schematically in FIG. 1. The OX-40L fusion polypeptide described
herein possesses an OX-40L receptor binding domain 101, a
trimerization domain 102, and a dimerization domain 103, such as an
immunoglobulin (e.g., Fc) domain. Typically, the immunoglobulin
domain, the trimerization domain and the OX-40L receptor binding
domain are arranged in an N-terminal to C-terminal direction. An
exemplary OX-40L fusion polypeptide is represented by SEQ ID NO:8.
Optionally, the fusion polypeptide can include one or more
additional polypeptide sequence, such as a signal sequence (e.g., a
secretory signal sequence), a linker sequence, an amino acid tag or
label, or a peptide or polypeptide sequence that facilitates
purification.
[0080] In an exemplary embodiment, the OX-40L receptor binding
domain is an extracellular domain of a human OX-40L. The sequence
of one such a domain is represented by SEQ ID NO:2. However, any
OX-40L polypeptide sequence that retains the desired property of
binding to the OX-40 receptor is suitable in the fusion
polypeptides and methods described herein.
[0081] Adjacent to (and most typically, contiguous with) the OX-40L
receptor binding domain is a trimerization domain. The
trimerization domain serves to promote self-assembly of individual
OX-40L fusion polypeptide molecules into a trimeric protein. Thus,
an OX-40L fusion polypeptide with a trimerization domain
self-assembles into a trimeric OX-40L fusion protein. In one
embodiment, the trimerization domain is an isoleucine zipper
domain. An exemplary isoleucine zipper domain is the engineered
yeast GCN4 isoleucine variant described by Harbury et al. (1993)
Science 262:1401-1407, the disclosure of which is incorporated
herein for all purposes. The sequence of one suitable isoleucine
zipper domain is represented by SEQ ID NO:4, although variants of
this sequence that retain the ability to form a coiled-coil
trimerization domain are equally suitable. Alternative coiled coil
trimerization domains include: TRAF2 (GENBANK.RTM. Accession No.
Q12933 [gi:23503103]; amino acids 299-348); Thrombospondin 1
(Accession No. PO7996 [gi: 135717]; amino acids 291-314);
Matrilin-4 (Accession No. 095460 [gi:14548117]; amino acids
594-618; CMP (matrilin-1) (Accession No. NP.sub.--002370
[gi:4505111]; amino acids 463-496; HSF1 (Accession No. AAX42211
[gi:61362386]; amino acids 165-191; and Cubilin (Accession No.
NP.sub.--001072 [gi:4557503]; amino acids 104-138.
[0082] In addition to the OX-40L receptor binding domain and the
trimerization domain, the fusion polypeptide includes an
immunoglobulin domain, such as a constant region or "Fc" domain.
The amino acid sequence of an exemplary immunoglobulin domain is
provided in SEQ ID NO:6, although numerous other immunoglobulin
domain sequences can be used. In certain embodiments, the
immunoglobulin domain serves as a dimerization domain that promotes
assembly between two trimeric fusion polypeptides into a stable
hexamer (that is a multimer that contains six OX-40L fusion
polypeptides) via interactions between unpaired immunoglobulin
domains (as shown schematically in FIG. 1). Optionally, alternative
dimerization domains capable of forming stable interactions between
the polypeptides that remain unpaired following trimerization of
OX-40L fusion polypeptides can be used in place of the
immunoglobulin domain.
[0083] The additional protein domains of the OX-40L fusion protein
can serve a number of functions, including enhancing the activity
of OX-40L, facilitating purification, and/or increasing the
stability of the protein in the body of a subject. In the fusion
proteins described herein, OX-40L, e.g., an extracellular domain of
OX-40L, or other active fragment thereof, or a conservative or
other variant of such a domain or fragment, can be fused with an
immunoglobulin domain or other fusion protein domain that is
selected to correspond to the subject to whom the OX-40L fusion
polypeptide is to be administered. For example, if the intended
subject is a human subject, it is desirable to select the
Immunoglobulin domain from a human immunoglobulin protein or
polypeptide. The specific example described below involves a fusion
between OX-40L extracellular domain, a trimerization domain and a
polypeptide including a constant domain of human IgG. Typically,
the fusion polypeptide includes at least one immunoglobulin
constant region domain. For example, the OX-40L fusion polypeptide
can include the CH2 and CH3 domains of IgG. In some embodiments,
the fusion polypeptide includes a hinge amino acid sequence region
corresponding to all or part of a hinge region of the IgG.
Optionally, one or more cysteine residues can be mutated to
non-sulfur amino acid residues, such as alanine or glycine. For
example, by introducing altering the nucleotides "tgt" to "acc"
(e.g., at position 8 in SEQ ID NO:5 and SEQ ID NO:7), a cysteine to
threonine substitution can be introduced into the beginning of the
Fc domain.
[0084] An exemplary OX-40L fusion polypeptide that assembles into a
trimeric OX-40L fusion protein is further described in the
Examples. The amino acid sequence of this fusion polypeptide is
provided in SEQ ID NO:8. Nonetheless, one of ordinary skill in the
art will recognize that numerous other sequences also fulfill the
criteria set forth herein for multimeric OX-40L fusion
polypeptides. Thus, although multimeric OX-40L fusion polypeptides
are predominantly described with respect to the polypeptide of SEQ
ID NO:8, numerous additional embodiments are encompassed by this
disclosure.
[0085] In addition to the trimeric OX-40L fusion polypeptides and
proteins described herein, functional fragments and variants are
also a feature of this disclosure. A functional fragment or variant
is a fragment or variant that maintains one or more functions of
the reference polypeptide. The terms fragment and variant are not
necessarily mutually exclusive. Functional fragments and variants
can be of varying length. For example, some fragments have at least
10, 25, 50, 75, 100, or 200 amino acid residues. In general, the
term "fragment" is used to refer to a subsequence of a polypeptide
less than its entirety. The term "variant" is used to designate a
polypeptide with one or more alterations or modifications with
respect to a reference polypeptide, such as, the OX-40L fusion
polypeptide explicitly described in detail in the examples. A
variant can be identical in length to the reference polypeptide, or
it can have one or more deletions or additions of amino acids. The
variant can include deletions or additions of one or several amino
acids, as long as the desired functional attribute (e.g., binding
to the OX-40 receptor) is maintained. Additionally, a variant can
include one or more amino acid substitutions. Generally, an amino
acid substitution is a conservative substitution that replaces a
naturally occurring amino acid with similar functional
attributes.
[0086] One of ordinary skill in the art will recognize that a
nucleic acid encoding a OX-40L fusion polypeptide can be altered or
modified without materially altering one or more of the fusion
protein's functions. As a preliminary matter, the genetic code is
degenerate, and different codons can encode the same amino acid.
More importantly, with respect to the encoded protein, even where
an amino acid substitution is introduced, the mutation can be
"conservative" and have no material impact on the essential
functions of a protein. See Stryer (1988) Biochemistry 3rd Ed.
[0087] Modifications of a polypeptide that involve the substitution
of one or more amino acids for amino acids having similar
biochemical properties that do not result in change or loss of a
biological or biochemical function of the polypeptide are
designated "conservative" substitutions. These conservative
substitutions are likely to have minimal impact on the activity of
the resultant protein. Table 1 shows amino acids that can be
substituted for an original amino acid in a protein, and which are
regarded as conservative substitutions based on a BLOSUM similarity
matrix. TABLE-US-00001 Amino Conservative Acid Substitutions G
A,S,N P E D S, K, Q, H, N, E E P, D, S, R, K, Q, H. N N G, D, E, T,
S, R, K, Q, H H D, E, N, M, R, Q Q D, E, N, H, M, S, R, K K D, E,
N, Q, R R E, N, H, Q, K S G, D, E, N, Q, A, T T N, S, V, A A G, S,
T, V M H, Q, Y, F, L, I, V V T, A, M, F, L, I I M, V, Y, F, L L M,
V, I, Y, F F M, V, I, L, W, Y Y H, M, I, L, F, W W F, Y C None
[0088] One or more conservative changes, or up to ten conservative
changes (e.g., two substituted amino acids, three substituted amino
acids, four substituted amino acids, or five substituted amino
acids, etc.) can be made in the polypeptide without changing a
biochemical function of the OX-40L fusion polypeptide. Accordingly,
OX-40L fusion polypeptides with one, two, three, four or five
conservative amino acid substitutions are equivalents of the fusion
polypeptide represented in SEQ ID NO:8, or one or more domains or
subportions thereof, such as SEQ ID NO:2, SEQ ID NO:4 and/or SEQ ID
NO:6. Thus, equivalent OX-40L fusion polypeptides include
polypeptides with amino acid sequences that are at least 95%
identical, such as 96%, or more than 97%, or even 98%, or 99%
identical to SEQ ID NO:8, or one or more domain thereof, such as
SEQ ID NO:2, SEQ ID NO:4 and/or SEQ ID NO:6. One of ordinary skill
in the art will understand that the amino acid changes can be
distributed throughout the length of SEQ ID NO:8, or can be
distributed within one or more subportions, e.g., domains of the
fusion polypeptide.
[0089] For example, one or more conservative changes can be made in
an OX-40L fusion polypeptide (including a trimeric OX-40L fusion
polypeptide without changing its ability to bind to the OX-40
receptor. Similarly, one or more conservative changes can be made
in an OX-40L fusion polypeptide without altering its ability to
trimerize. More substantial changes in a biochemical function or
other protein features can be obtained by selecting amino acid
substitutions that are less conservative than those listed in Table
1. Such changes include, for example, 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).
[0090] Additionally, part of a polypeptide chain can be deleted
without impairing or eliminating all of its functions. Similarly,
insertions or additions can be made in the polypeptide chain, for
example, adding epitope tags, without impairing or eliminating its
functions (Ausubel et al. (1997) J. Immunol. 159:2502). Other
modifications that can be made without materially impairing one or
more functions of a polypeptide include, for example, in vivo or in
vitro chemical and biochemical modifications that incorporate
unusual amino acids. Such modifications include, for example,
acetylation, carboxylation, phosphorylation, glycosylation,
labeling, e.g., with radionuclides, and various enzymatic
modifications, as will be readily appreciated by those of ordinary
skill in the art. A variety of methods for labeling polypeptides
and labels useful for such purposes are well known in the art, and
include radioactive isotopes such as .sup.32P, fluorophores,
chemiluminescent agents, enzymes, and antiligands.
[0091] More generally, stable multimeric fusion proteins that
include a domain selected from a ligand that binds a biologically
relevant receptor can be produced in a manner analogous to that
described herein with respect to OX-40 ligand. Such fusion proteins
assemble into stable timers (and hexamers) with enhanced biological
activity relative to other soluble forms of the ligand. The fusion
proteins are characterized by the inclusion, in an N-terminal to
C-terminal orientation, of an immunoglobulin (e.g., Fc) domain; a
trimerization domain; and a receptor binding domain. While such
fusion proteins can be made from essentially any ligand, they are
especially useful for producing soluble counterparts for ligands
that are multimeric (e.g., trimeric) in their active form. For
example, trimeric fusion proteins can be favorably produced and
employed that correspond to ligands that bind to receptors for
members of the Tumor Necrosis Factor (TNF) family of proteins, such
as: TNF-a, TNF-b, Lymphotoxin-b, CD40L, FasL, CD27L, CD30L, 4-1BBL,
TRAIL, RANK ligand, TWEAK, APRIL, BAFF, LIGHT, GITR ligand, EDA-A1,
EDA-A2.
Polynucleotides Encoding OX-40L Fusion Proteins
[0092] The OX-40L fusion polypeptides disclosed herein (such as the
polypeptide represented by SEQ ID NO:8) are encoded by novel
polynucleotide sequences. Polynucleotide sequences that encode an
OX-40L fusion polypeptide capable of trimerization include at least
a first polynucleotide subsequence that encodes an immunoglobulin
domain, at least a second polynucleotide subsequence that encodes a
trimerization domain, and at least a third polynucleotide
subsequence that encodes an OX-40L receptor binding domain. An
exemplary polynucleotide sequence that encodes an OX-40L fusion
polypeptide is represented by SEQ ID NO:7. Typically, the
polynucleotides encoding the immunoglobulin domain, the
trimerization domain and the OX-40L receptor binding domains are
joined in a 5' to 3' orientation. In one embodiment, the
polynucleotides that encode the immunoglobulin (e.g., Fc) domain,
the trimerization domain and the OX-40L domain are contiguously
linked in a 5' to 3' orientation. Optionally, the polynucleotide
encodes a signal sequence, e.g., a secretory signal sequence or a
membrane localization sequence. In an embodiment, a polynucleotide
sequence that encodes an amino acid linker sequence (e.g., a
flexible linker sequence) is included in the polynucleotide that
encodes the OX-40L fusion polypeptide.
[0093] For example, the nucleic acid that encodes the OX-40L fusion
polypeptide favorably includes a polynucleotide sequence that
encodes an OX-40 receptor binding domain that is an extracellular
domain of a human OX-40L. An exemplary polynucleotide sequence is
represented by SEQ ID NO:1. The extracellular domain of the OX-40L
represented by GENBANK.RTM. Accession No. NM 003326 (SEQ ID NO:9),
is equivalently suitable in the context of an OX-40L fusion
polypeptide. SEQ ID NO:1 and SEQ ID NO:9 represent functionally
equivalent polynucleotide sequences of the human OX-40L. SEQ ID
NO:1 possesses two nucleotide substitutions, each of which is an A
to T substitution. The polypeptide represented by SEQ ID NO:2
includes a substitution of a phenylalanine for an isoleucine at
amino acid position 9 with respect to the GENBANK.RTM. sequence.
Similarly, any polynucleotide sequence that encodes a functionally
equivalent OX-40L domain can be employed in the fusion polypeptides
described herein.
[0094] Adjacent to the polynucleotide sequence encoding the OX-40L
receptor binding domain is a polynucleotide sequence encoding a
trimerization domain. As indicated above, one favorable
trimerization domain is an isoleucine zipper domain. In one
favorable embodiment, the nucleic acid encoding the OX-40L fusion
polypeptide includes a polynucleotide sequence that encodes an
isoleucine zipper domain. An exemplary polynucleotide sequence is
provided in SEQ ID NO:3. Alternative trimerization domains include
those of TRAF2, Thrombospondin 1, Matrilin-4, CMP, HSF1 and
Cubilin.
[0095] In addition to polynucleotide sequences that encode an
OX-40L receptor binding domain and a trimerization domain, the
nucleic acid that encodes the OX-40L fusion polypeptide also
includes a polynucleotide sequence that encodes an immunoglobulin
constant region domain ("Fc domain"). Typically the polynucleotide
encodes the CH2, CH3 and hinge domains of a human immunoglobulin Fc
region, although other constant region domains, e.g., the CH2 and
CH1 domains, could be substituted. In an exemplary embodiment, the
polynucleotide encodes an IgG1 Fc domain. Favorably, the
immunoglobulin domain is capable of promoting dimerization (e.g.,
with another polypeptide including an immunoglobulin domain). An
exemplary polynucleotide sequence that encodes a human IgG1 Fc
domain is provided in SEQ ID NO:5.
[0096] Polynucleotides encoding the OX-40L fusion polypeptides
include deoxyribonucleotides (DNA, cDNA) or ribodeoxynucletides
(RNA) sequences, or modified forms of either nucleotide, which
encode the fusion polypeptides described herein. The term includes
single and double stranded forms of DNA and/or RNA.
[0097] Polynucleotide sequences described herein include
polynucleotide sequences, such as the sequences represented by SEQ
ID NO:7, which encode OX-40L fusion polypeptides, as well as
polynucleotide sequences complementary thereto. For example, a
polynucleotide that encodes an OX-40L fusion polypeptide sequence
represented by SEQ ID NO:8 is a feature of this disclosure.
[0098] In addition to SEQ ID NOs:1, 3, 5, 7 and 9, polynucleotide
sequences that are substantially identical to these polynucleotide
sequences can be used in the compositions and methods of the
disclosure. Fore example, a substantially identical polynucleotide
sequence can have one or a small number of deletions, additions
and/or substitutions. Such polynucleotide changes can be contiguous
or can be distributed at different positions in the nucleic acid. A
substantially identical polynucleotide sequence can, for example,
have 1, or 2, or 3, or 4, or even more nucleotide deletions,
additions and/or substitutions. Typically, the one or more
deletions, additions and/or substitutions do not alter the reading
frame encoded by the polynucleotide sequence, such that a modified
("mutant") but substantially identical polypeptide is produced upon
expression of the nucleic acid.
[0099] The similarity between amino acid (and/or polynucleotide)
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); the higher the percentage, the more similar are the
primary structures of the two sequences. Thus, a polynucleotide
that encodes an OX-40L fusion polypeptide can be at least about
95%, or at least 96%, frequently at least 97%, 98%, or 99%
identical to SEQ ID NO:7 (or SEQ ID NO:9) or to at least one
subsequence thereof, such as SEQ ID NO:1, SEQ ID NO:3 and/or SEQ ID
NO:5). Methods of determining sequence identity are well known in
the art. Various programs and alignment algorithms are described
in: Smith and Waterman, Adv. Appl. Math. (1981) 2:482; Needleman
and Wunsch (1970) J. Mol. Biol. 48:443; Higgins and Sharp (1988)
Gene 73:237; Higgins and Sharp (1989) CABIOS 5:151; Corpet et al.
(1988) Nucleic Acids Research 16:10881; and Pearson and Lipman
(1988) Proc. Natl. Acad. Sci. USA 85:2444. Altschul et al. (1994)
Nature Genet. 6:119, presents a detailed consideration of sequence
alignment methods and homology calculations.
[0100] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul
et al., J. Mol. Biol. (1990) 215:403) 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. A description of how to determine
sequence identity using this program is available on the NCBI
website on the internet.
[0101] Thus, a sequence (that is a polynucleotide or polypeptide
sequence) that is substantially identical, or substantially similar
polynucleotide to a polynucleotide of SEQ ID NO:1, 3, 5, 7 or 9 (or
to a polypeptide sequence of SEQ ID NO:2, 4, 6, or 8) is
encompassed within the present disclosure. A sequence is
substantially identical to one of SEQ ID NOs:1-9 if the sequence is
identical, on a nucleotide by nucleotide basis, with at least a
subsequence of the reference sequence (e.g., SEQ ID NOs:1-9). Such
polynucleotides can include, e.g., insertions, deletions, and
substitutions relative to any of SEQ ID NOs:1, 3, 5, 7, and/or 9.
For example, such polynucleotides are typically at least about 70%
identical to a reference polynucleotide (or polypeptide) selected
from among SEQ ID NO:1 through SEQ ID NO:9. That is, at least 7 out
of 10 nucleotides (or amino acids) within a window of comparison
are identical to the reference sequence selected SEQ ID NO:1-9.
Frequently, such sequences are at least about 80%, usually at least
about 90%, and often at least about 95%, or more identical to a
reference sequence selected from SEQ ID NO:1 to SEQ ID NO:9. For
example, the amino acid or polynucleotide sequence can be 96%, 97%,
98% or even 99% identical to the reference sequence, e.g., at least
one of SEQ ID NO:1 to SEQ ID NO:9
[0102] Another indicia of sequence similarity between two nucleic
acids is the ability to hybridize. The more similar are the
sequences of the two nucleic acids, the more stringent the
conditions at which they will hybridize. Substantially similar or
substantially identical nucleic acids to SEQ ID NO:7 (and to
subsequences thereof, such as SEQ ID NO:1, SEQ ID NO:3 and SEQ ID
NO:5) include nucleic acids that hybridize under stringent
conditions to any of these reference polynucleotide sequences.
Thus, a nucleic acid that hybridizes under stringent conditions to
a reference polynucleotide sequence selected from among SEQ ID
NOs:1, 3, 5, and/or 7 is substantially identical or substantially
similar to the polynucleotides encoding OX-40L fusion polypeptides
described herein.
[0103] The stringency of hybridization conditions are
sequence-dependent and are different under different environmental
parameters. Thus, hybridization conditions resulting in particular
degrees of stringency will vary depending upon the nature of the
hybridization method of choice and the composition and length of
the hybridizing nucleic acid sequences. Generally, the temperature
of hybridization and the ionic strength (especially the Na.sup.+
and/or Mg.sup.++ concentration) of the hybridization buffer will
determine the stringency of hybridization, though wash times also
influence stringency. 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,
for example, in Sambrook et al. (2001) Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.; Tijssen (1993) Hybridization With Nucleic Acid
Probes, Part I: Theory and Nucleic Acid Preparation, Laboratory
Techniques in Biochemistry and Molecular Biology, Elsevier Science
Ltd., NY and Ausubel et al. (1999) Short Protocols in Molecular
Biology, 4.sup.th ed., John Wiley & Sons, Inc.
[0104] For purposes of the present disclosure, "stringent
conditions" encompass conditions under which hybridization will
only occur if there is less than 25% mismatch between the
hybridization molecule and the target sequence. "Stringent
conditions" can be broken down into particular levels of stringency
for more precise definition. Thus, as used herein, "moderate
stringency" conditions are those under which molecules with more
than 25% sequence mismatch will not hybridize; conditions of
"medium stringency" are those under which molecules with more than
15% mismatch will not hybridize, and conditions of "high
stringency" are those under which sequences with more than 10%
mismatch will not hybridize. Conditions of "very high stringency"
are those under which sequences with more than 6% mismatch will not
hybridize. In contrast nucleic acids that hybridize under "low
stringency conditions include those with much less sequence
identity, or with sequence identity over only short subsequences of
the nucleic acid.
[0105] For example, in nucleic acid hybridization reactions, the
conditions used to achieve a particular level of stringency will
vary depending on the nature of the nucleic acids being hybridized.
The length, degree of complementarity, nucleotide sequence
composition (e.g., GC v. AT content), and nucleic acid type (e.g.,
RNA versus DNA) of the hybridizing regions of the nucleic acids all
influence the selection of appropriate hybridization conditions.
Additionally, whether one of the nucleic acids is immobilized, for
example, on a filter can impact the conditions required to achieve
the desired stringency.
[0106] A specific example of progressively higher stringency
conditions is as follows: 2.times.SSC/0.1% SDS at about room
temperature (hybridization conditions); 0.2.times.SSC/0.1% SDS at
about room temperature (low stringency conditions);
0.2.times.SSC/0.1% SDS at about 42.degree. C. (moderate stringency
conditions); and 0.1.times.SSC at about 68.degree. C. (high
stringency conditions). One of skill in the art can readily
determine variations on these conditions (e.g., with reference to
Sambrook, Tjissen and/or Ausubel, cited above). Washing can be
carried out using only one of these conditions, e.g., high
stringency conditions, or each of the conditions can be used, e.g.,
for 10-15 minutes each, in the order listed above, repeating any or
all of the steps listed. However, as mentioned above, optimal
conditions will vary, depending on the particular hybridization
reaction involved, and can be determined empirically.
[0107] Additionally, the nucleic acid encoding the OX-40L fusion
polypeptides can also include polynucleotide sequences, such as
expression regulatory sequences and/or vector sequences that
facilitate the expression or replication of the nucleic acids.
Similarly, the nucleic acid encoding the OX-40L fusion polypeptide
can include additional coding sequences that confer functional
attributes on the encoded polypeptide. Such sequences include
secretory signal sequences and membrane localization signals.
[0108] Nucleic acids encoding OX-40L fusion polypeptides can be
manipulated with standard procedures such as restriction enzyme
digestion, fill-in with DNA polymerase, deletion by exonuclease,
extension by terminal deoxynucleotide transferase, ligation of
synthetic or cloned DNA sequences, site-directed
sequence-alteration via single-stranded bacteriophage intermediate
or with the use of specific oligonucleotides in combination with
PCR or other in vitro amplification. These procedures are well
known to those of ordinary skill in the art, and exemplary
protocols can be found, e.g., in Sambrook and Ausubel (supra).
[0109] A polynucleotide sequence (or portions derived from it) such
as a cDNA encoding an OX-40L fusion polypeptide can be introduced
into a vector, such as a eukaryotic expression vector, by
conventional techniques. An expression vector is designed to permit
the transcription of the polynucleotide sequence encoding the
OX-40L fusion polypeptide in cells by providing regulatory
sequences that initiate and enhance the transcription of the cDNA
and ensure its proper splicing and polyadenylation. Numerous
expression vectors are known to those of skill in the art, and are
available commercially, or can be assembled from individual
components according to conventional molecular biology procedures,
such as those described in, e.g., Sambrook and Ausubel, cited
above. The pCEP D4-7 vector described in the Examples is one such
suitable expression vector.
[0110] For example, the cytomegalovirus ("CMV") immediate early
promoter can favorably be utilized to regulate transcription of an
OX-40L fusion polypeptide upon introduction of an expression vector
containing a polynucleotide encoding the OX-40L fusion polypeptide
operably linked to the CMV promoter. Additionally, vectors
containing the promoter and enhancer regions of the SV40 or long
terminal repeat (LTR) of the Rous Sarcoma virus and polyadenylation
and splicing signal from SV40 are readily available (Mulligan et
al. (1981) Proc. Natl. Acad. Sci. USA 78:1078-2076; Gorman et al.
(1982) Proc. Natl. Acad. Sci USA 78:6777-6781). The level of
expression of the polynucleotide that encodes a polypeptide can be
manipulated with this type of vector, either by using promoters
that have different activities (for example, the baculovirus pAC373
can express cDNAs at high levels in S. frugiperda cells (Summers
and Smith (1985) In Genetically Altered Viruses and the
Environment, Fields et al. (Eds.) 22:319-328, CSHL Press, Cold
Spring Harbor, N.Y.) or by using vectors that contain promoters
amenable to modulation, for example, the glucocorticoid-responsive
promoter from the mouse mammary tumor virus (Lee et al. (1982)
Nature 294:228).
[0111] In addition, some vectors contain selectable markers such as
the gpt (Mulligan and Berg (1981) Proc. Natl. Acad. Sci. USA
78:2072-2076) or neo (Southern and Berg (1982) J. Mol. Appl. Genet.
1:327-341) bacterial genes. These selectable markers permit
selection of transfected cells that exhibit stable, long-term
expression of the vectors (and therefore the cDNA). The vectors can
be maintained in the cells as episomal, freely replicating entities
by using regulatory elements of viruses such as papilloma (Sarver
et al. (1981) Mol. Cell Biol. 1:486) or Epstein-Barr (Sugden et al.
(1985) Mol. Cell Biol. 5:410). Alternatively, one can also produce
cell lines that have integrated the vector into genomic DNA. Both
of these types of cell lines produce the gene product on a
continuous basis. One can also produce cell lines that have
amplified the number of copies of the vector (and therefore of the
cDNA as well) to create cell lines that can produce high levels of
the gene product (Alt et al. (1978) J. Biol. Chem. 253:1357).
[0112] Vector systems suitable for the expression of
polynucleotides encoding fusion proteins include, in addition to
the specific vectors described in the examples, the pUR series of
vectors (Ruther and Muller-Hill (1983) EMBO J. 2:1791), pEX1-3
(Stanley and Luzio (1984) EMBO J. 3:1429) and pMR100 (Gray et al.
(1982) Proc. Natl. Acad. Sci. USA 79:6598). Vectors suitable for
the production of intact native proteins include pKC30 (Shimatake
and Rosenberg (1981) Nature 292:128, 1981), pKK177-3 (Amann and
Brosius (1985) Gene 40:183) and pET-3 (Studiar and Moffatt (1986)
J. Mol. Biol. 189:113).
[0113] The present disclosure, thus, encompasses recombinant
vectors that comprise all or part of the polynucleotides encoding
trimeric OX-40L fusion proteins or cDNA sequences encoding OX-40L
fusion polypeptides, for expression in a suitable host, either
alone or as a labeled or otherwise detectable protein. The DNA is
operably linked in the vector to an expression control sequence in
the recombinant DNA molecule so that the fusion polypeptide or
protein can be expressed. The expression control sequence can be
selected from the group consisting of sequences that control the
expression of genes of prokaryotic or eukaryotic cells and their
viruses and combinations thereof. The expression control sequence
can be specifically selected from the group consisting of the lac
system, the trp system, the tac system, the trc system, major
operator and promoter regions of phage lambda, the control region
of fd coat protein, the early and late promoters of SV40, promoters
derived from polyoma, adenovirus, retrovirus, baculovirus and
simian virus, the promoter for 3-phosphoglycerate kinase, the
promoters of yeast acid phosphatase, the promoter of the yeast
alpha-mating factors and combinations thereof.
[0114] The nucleic acid encoding an OX-40L fusion polypeptide can
also be transferred from its existing context to other cloning
vehicles, such as other plasmids, bacteriophages, cosmids, animal
viruses and yeast artificial chromosomes (YACs) (Burke et al.
(1987) Science 236:806-812). These vectors can then be introduced
into a variety of hosts including somatic cells, and simple or
complex organisms, such as bacteria, fungi (Timberlake and Marshall
(1989) Science 244:1313-1317), invertebrates, plants (Gasser and
Fraley (1989) Science 244:1293), and animals (Pursel et al. (1989)
Science 244:1281-1288), which cell or organisms are rendered
transgenic by the introduction of the heterologous cDNA.
[0115] For expression in mammalian cells, a cDNA sequence can be
ligated to heterologous promoters, such as the simian virus (SV) 40
promoter in the pSV2 vector (Mulligan and Berg (1981) Proc. Natl.
Acad. Sci. USA 78:2072-2076), and introduced into cells, such as
monkey COS-1 cells (Gluzman (1981) Cell 23:175-182), to achieve
transient or long-term expression. The stable integration of the
chimeric gene construct can be maintained in mammalian cells by
biochemical selection, such as neomycin (Southern and Berg (1982)
J. Mol. Appl. Genet. 1:327-341) and mycophenolic acid (Mulligan and
Berg (1981) Proc. Natl. Acad. Sci. USA 78:2072-2076).
Production of Recombinant OX-40L Fusion Proteins
[0116] OX-40L fusion proteins can be made in any suitable
heterologous expression system, and, where appropriate, the DNA
encoding the fusion protein can also encode a known secretory
signal sequence suitable for the host cell system employed so that
the DNA is translated into a protein that at first includes the
secretory signal and the cleavage sequence but is then transported
out of the cell without such ancillary sequences.
[0117] The expression and purification of proteins, such as a
trimeric OX-40L fusion protein, can be performed using standard
laboratory techniques. Examples of such methods are discussed or
referenced herein. After expression, purified proteins have many
uses, including for instance functional analyses, antibody
production, and diagnostics, as well as the prophylactic and
therapeutic uses described below. Partial or full-length cDNA
sequences, which encode the fusion proteins, can be ligated into
bacterial expression vectors. Methods for expressing large amounts
of protein from a cloned sequence introduced into Escherichia coli
(E. coli) or baculovirus/Sf9cells can be utilized for the
purification, localization and functional analysis of proteins, as
well as for the production of antibodies and vaccine compositions.
For example, fusion proteins consisting of an OX-40L fusion
polypeptide can be used in various procedures, for instance to
prepare polyclonal and monoclonal antibodies against these
proteins. Thereafter, these antibodies can be used to purify
proteins by immunoaffinity chromatography, in diagnostic assays to
quantitate the levels of protein and to localize proteins in
tissues and individual cells by immunofluorescence. More
particularly, the fusion proteins and the polynucleotides encoding
them described herein can be used to produce pharmaceutical
compositions, including vaccine compositions suitable for
prophylactic and/or therapeutic administration.
[0118] Methods and additional plasmid vectors for producing the
polynucleotides encoding fusion proteins and for expressing these
polynucleotides in bacterial and eukaryotic cells are well known in
the art, and specific methods are described in Sambrook (supra).
Such fusion proteins can be made in large amounts, are easy to
purify, and can be used to enhance an immune response, including an
antibody response or a T-cell response. Native proteins can be
produced in bacteria by placing a strong, regulated promoter (such
as the CMV promoter) and an efficient ribosome-binding site
upstream of the cloned gene. If low levels of protein are produced,
additional steps can be taken to increase protein production; if
high levels of protein are produced, purification is relatively
easy. Suitable methods are presented in Sambrook (supra), and are
well known in the art. Often, proteins expressed at high levels are
found in insoluble inclusion bodies. Methods for extracting
proteins from these aggregates are described by Sambrook (supra).
Proteins, including fusion proteins, can be isolated from protein
gels, lyophilized, ground into a powder and used as an antigen.
[0119] The transfer of DNA into eukaryotic, in particular human or
other mammalian cells, is now a conventional technique known to
those of ordinary skill in the art. The vectors are introduced into
the recipient cells as pure DNA (transfection) by, for example,
precipitation with calcium phosphate (Graham and vander Eb (1973)
Virology 52:466) or strontium phosphate (Brash et al. (1987) Mol.
Cell Biol. 7:2013), electroporation (Neumann et al. (1982) EMBO J.
1:841), lipofection (Felgner et al. (1987) Proc. Natl. Acad. Sci
USA 84:7413), DEAE dextran (McCuthan et al. (1968) J. Natl. Cancer
Inst. 41:351), microinjection (Mueller et al. (1978) Cell 15:579),
protoplast fusion (Schafner, (1980) Proc. Natl. Acad. Sci. USA
77:2163-2167), biolistics, e.g., pellet guns (Klein et al. (1987)
Nature 327:70) or Gene guns. Alternatively, the cDNA, or fragments
thereof, can be introduced by infection with virus vectors. Systems
are developed that use, for example, retroviruses (Bernstein et al.
(1985) Gen. Entr'g 7:235), adenoviruses (Ahmad et al. (1986) J.
Virol. 57:267), or Herpes virus (Spaete et al. (1982) Cell 30:295).
Polynucleotides that encode proteins, such as fusion proteins, can
also be delivered to target cells in vitro via non-infectious
systems, such as liposomes.
[0120] Using the above techniques, the expression vectors
containing a polynucleotide encoding a monomeric fusion polypeptide
as described herein or cDNA, or fragments or variants or mutants
thereof, can be introduced into human cells, mammalian cells from
other species or non-mammalian cells as desired. The choice of cell
is determined by the purpose of the treatment. For example, monkey
COS cells (Gluzman (1981) Cell 23:175-182) that produce high levels
of the SV40 T antigen and permit the replication of vectors
containing the SV40 origin of replication can be used. Similarly,
Chinese hamster ovary (CHO), mouse NIH 3T3 fibroblasts or human
fibroblasts or lymphoblasts can be used.
Methods of Enhancing an Antigen Specific Immune Response
[0121] The enhancement of an antigen-specific immune response in a
subject (e.g., a mammalian subject, such as a human subject) by
engaging the OX-40 receptor on CD4.sup.+ T-cells during or after
antigen activation can be accomplished using a wide variety of
methods. The method of choice will primarily depend upon the type
of antigen against which it is desired to enhance the immune
response, and various methods available are discussed below.
Whatever method is selected, the trimeric OX-40L fusion protein
should be administered to the animal such that it is presented to
T-cells of the subject during or shortly after priming of the
T-cells by the antigen. In an exemplary method a trimeric OX-40L
fusion protein comprising the polypeptide represented by SEQ ID
NO:8 is administered.
[0122] Since the activation of T-cells generally takes place within
about 7 days after an antigen is presented to the immune system
(and often within about 24-48 hours of exposure to antigen), it is
generally preferable to administer the trimeric OX-40L fusion
protein to the subject by the selected method within about 10 days
after the subject's immune system is exposed to the antigen.
Typically, the trimeric OX-40L fusion protein is administered
either concurrently with, or within about 24 hours of exposure to
antigen. Nonetheless, later administration, e.g., within about 48
hours, within about 72 hours, up to within about 4-10 days of
exposure to antigen is possible. Where the trimeric OX-40L fusion
protein is administered simultaneously with the antigen, it is
generally advantageous to administer a form of the agent which has
enhanced stability (such as, increased half-life, resistance to
proteolysis, etc.) in the body so that the agent will remain in the
circulatory system for a sufficient period of time to engage with
OX-40 receptor during or after antigen priming. Favorably, the
trimeric OX-40L fusion protein described herein, including a
trimerization domain and an immunoglobulin domain, exhibits such
enhanced stability as compared to an isolated extracellular OX-40L
domain or a monomeric OX-40L fusion polypeptide. Within the purview
of the present disclosure, a polypeptide domain can be substituted
for the immunoglobulin domain so long as the selected polypeptide
domain maintains a similar increase in stability.
[0123] One of ordinary skill in the art can determine the half-life
of any selected OX-40L fusion polypeptide using standard methods.
For example, after administration of the fusion polypeptide by
intravenous injection, a small blood sample can be removed from the
subject, with subsequent samples being taken every 6-24 hours over
the period of about 10 days. Thereafter, the concentration of the
fusion polypeptide present in each sample is determined (e.g.,
using standard immunological quantification methods, such as those
discussed in Harlow & Lane (1988), e.g., ELISA). The half-life
of the fusion polypeptide is defined as that time point at which
the concentration of the agent falls to 50% of that in the first
sample measurement.
[0124] In some situations, for example where the antigen is
presented to the immune system over an extended duration (for
example, in cancer patients), the trimeric OX-40L fusion protein
can be administered more than 7 days after the immune system is
first exposed to the antigen. For example, following surgical
removal of a primary tumor from a patient, a trimeric OX-40L fusion
protein can be administered to enhance the immune response to tumor
antigens present on metastases, thereby promoting the clearance of
such metastases from the body. In such a situation, administration
of the trimeric OX-40L fusion protein will usually occur more than
7 days after the immune system of the patient was first exposed to
the tumor antigens, but will nevertheless be present subsequently
when the antigens are being presented to T-cells.
[0125] In contrast, when the antigen to which an immune response is
desired is a soluble antigen, it is generally desirable to
administer the trimeric OX-40L fusion protein simultaneously with,
or within approximately 24 to 48 hours of, exposure to the
antigen.
[0126] While the molecule which engages the OX-40 receptor will be
in the form of a protein, that is, as an assembled hexameric
complex including two trimeric OX-40L fusion proteins, the
preparation administered to the mammal can take a number of forms,
including a preparation of a purified trimeric OX-40L fusion
protein, preparation of a purified OX-40L fusion polypeptide,
preparation of a nucleic acid molecule which encodes the trimeric
OX-40L fusion protein, a cell or a virus which expresses the
trimeric OX-40L fusion protein, or a preparation derived from such
a cell or virus.
[0127] In its simplest form, the preparation administered to the
mammal is a hexameric OX-40L fusion protein (e.g., made up of
"dimerized" trimers), administered in conventional dosage form, and
preferably combined with a pharmaceutical excipient, carrier or
diluent. Suitable pharmaceutical carriers can be solids or liquids,
and can include buffers, anti-oxidants such as ascorbic acid, other
polypeptides or proteins such as serum albumin, carbohydrates,
chelating agents and other stabilizers and excipients. Suitable
solid carriers include lactose, magnesium stearate, terra alba,
sucrose, talc, stearic acid, gelatin, agar, pectin, acacia and
cocoa butter. The amount of a solid carrier will vary widely
depending on which carrier is selected, but preferably will be from
about 25 mg to about 1 g per dose of active agent. Suitable liquid
carriers include normal saline and neutral buffered saline,
optionally with suitable preservatives, stabilizers and excipients.
The carrier or diluent can also include time delay material well
known to the art such as, for example, glycerol distearate, either
alone or with a wax. The foregoing examples of suitable
pharmaceutical carriers are only exemplary and one of skill in the
art will recognize that a very wide range of such carriers can be
employed. Liposome-based delivery systems can also be employed to
deliver trimeric OX-40L fusion proteins. Liposome-based systems,
which can be employed to provide a measured release of the agent
over time into the bloodstream, are well known in the art and are
exemplified by the systems described in U.S. Pat. Nos. 4,356,167;
5,580,575; 5,595,756; and 5,188,837, and documents cited
therein.
[0128] The formulation of the trimeric fusion protein, such as a
trimeric OX-40L fusion protein, with a pharmaceutical carrier can
take many physical forms, but is preferably a sterile liquid
suspension or solution, suitable for direct injection. Preferably,
the subject will be administered the trimeric OX-40L fusion protein
in a formulation as described above (for example, in combination
with a pharmaceutical carrier), wherein the formulation includes a
clinically effective amount of the fusion protein.
[0129] As used herein, "a therapeutically effective amount" is an
amount that results in a therapeutically significant effect. This
nature of this effect will vary with the context in which the
trimeric OX-40L fusion protein is being used, for example, whether
the fusion protein is being administered to treat an existing
condition (for example, to treat an infectious disease, or cancer)
or as a prophylactic (to prevent or reduce the risk of disease or
cancer, e.g, recurrence of a tumor or metastasis of a tumor) agent.
If the trimeric OX-40L fusion protein is being administered to a
cancer patient, it will be appreciated that any improvement in the
patient's condition is therapeutically significant. Hence, in such
a situation, "a therapeutically effective amount" encompasses
amounts of the trimeric OX-40L fusion protein that result in at
least partial remission of the cancer as well as amounts which slow
or limit the further progression of the cancer. Similarly, in the
therapeutic context where the agent is being used to enhance the
immune response of a patient to an infectious agent, such as a
virus or a bacterium, where the patient is already infected with
the agent, a therapeutically effective amount can produce a
therapeutic effect, meaning an effect which results in some degree
of recovery from the infection or amelioration of the clinical
symptoms.
[0130] In the prophylactic context, such as vaccination, a
therapeutically effective amount of a trimeric OX-40L fusion
protein can provide an enhancement of the immune response to the
target antigen, that is, produce an immune response greater than
would be presented absent administration of the trimeric OX-40L
fusion protein. Quantification of the immune response arising from
a vaccination can be achieved in any standard way, e.g.,
measurement of serum antibody titer for level and/or duration
against any convenient test antigen, and/or lymphoproliferation in
response to test antigen in vitro.
[0131] It will be appreciated that a therapeutically effective dose
of a trimeric OX-40L fusion protein will vary depending on the
clinical context (e.g., whether the agent is being used
therapeutically or prophylactically), the characteristics of the
subject (age, weight, other medications being taken, etc.) and the
severity of the condition. Thus, the assessment of a
therapeutically effective dosage will ultimately be decided by a
physician, veterinarian, or other health care worker familiar with
the subject. Typically, administering a trimeric OX-40L fusion
protein to a subject according to the methods of the present
disclosure will involve administration of from about 10 ng to 1 g
of trimeric OX-40L fusion protein per dose, with single dose units
of from about 10 ng to 100 mg being commonly used, and specific
dosages of up to 1 mg or 10 mg also being within the commonly used
range.
[0132] The trimeric OX-40L fusion protein can be administered to a
subject through a number of routes, including subcutaneously or
intravenously or, where the subject has a tumor, directly into the
tumor site. The agent can be the sole active ingredient in the
composition, or it can be combined with other agents having a
beneficial effect, such as an interferon or other
immune-stimulatory molecules.
[0133] In the prophylactic (vaccine) context, the trimeric OX-40L
fusion protein is often administered to a subject in combination
with a conventional vaccine preparation or formulation, such as a
vaccine preparation comprising bacterial or viral antigens. The
trimeric OX-40L fusion protein can be combined with the
conventional vaccine, or can be administered as a separate
preparation along with the conventional vaccine. For example, where
the trimeric OX-40L fusion protein is administered separately, it
is typically administered within about a week of the vaccine being
administered. Conventional vaccine preparations suitable for use in
the present disclosure include those prepared with purified
bacterial antigens, heat killed bacteria, subunit vaccines and
viral vaccines based on live or attenuated virus. A vaccine
preparation can include a pharmaceutical carrier and/or
adjuvant.
[0134] Where the trimeric OX-40L fusion protein is administered to
the subject in a single preparation with the vaccine antigens, the
preparation can be formulated simply by mixing a therapeutically
effective amount of a trimeric OX-40L fusion protein with the
antigen preparation. Alternatively, the trimeric OX-40L fusion
protein can be produced along with the antigen. For example, where
the antigen to be administered as a vaccine is a bacterial antigen
or a mixture of bacterial antigens, the bacterium from which the
antigen preparation is prepared can be a transgenic bacterium which
expresses the trimeric OX-40L fusion protein. In such a situation,
the trimeric OX-40L fusion protein is directly obtained in
combination with the bacterial antigens. Similarly, vaccines
comprising tumor antigens and trimeric OX-40L fusion protein can be
prepared from tumor cells which express the trimeric OX-40L fusion
protein. Methods of expressing proteins such as OX-40L fusion
polypeptides in transgenic prokaryotic and eukaryotic cells are
well known to those of ordinary skill in the art, and are described
in standard laboratory texts such as Sambrook and Ausubel, cited
above.
[0135] In other embodiments, the immune response of a subject to a
particular antigen is enhanced by administering to the subject a
nucleic acid molecule that encodes an OX-40L fusion polypeptide
that is capable of forming a trimeric OX-40L fusion protein. Such a
nucleic acid molecule is preferably administered either as a
component of a cell, or as part of a viral genome. Alternatively,
the nucleic acid encoding the OX-40L fusion polypeptide can be
administered to the subject as a "naked" nucleic acid molecule.
[0136] For example, a nucleic acid molecule encoding an OX-40L
fusion polypeptide can be introduced into an attenuated bacterium
(that is, a form of the bacterium that does not cause significant
disease when administered to a subject) in a plasmid vector such
that the trimeric OX-40L fusion protein is secreted by the
bacterium. The bacterium can be administered to the mammal in the
same manner as a conventional attenuated bacterial vaccine.
[0137] Alternatively, the nucleic acid molecule encoding the
trimeric fusion protein, such as nucleic acids encoding trimeric
OX-40L fusion proteins, can be introduced into the genome of a
virus that is used as a live attenuated vaccine. Attenuated viruses
include those in which an essential gene has been deleted, as
described in U.S. Pat. Nos. 5,665,362 and 5,837,261. Viruses
suitable for this purpose include DNA viruses, such as adeno,
herpes, papova, papilloma and parvo viruses, as well as RNA viruses
such as poliovirus and influenza virus. Methods of preparing
viruses carrying heterologous nucleic acid sequences that can be
used as viral vaccines are described in U.S. Pat. Nos. 5,665,362
and 5,837,261 (supra); U.S. Pat. Nos. 5,338,683 and 5,494,807.
[0138] In another embodiment, a nucleic acid encoding an OX-40L
fusion polypeptide capable of forming a trimeric OX-40L fusion
protein can be introduced into a tumor cell. In many cancer
patients, tumor cells escape detection by the immune system by
mechanisms such as down-regulating MHC and/or co-stimulatory
molecule expression. Accordingly, one method of treatment is to
remove tumor cells from the patient and introduce into them nucleic
acids encoding, for example, MHC class II, the co-stimulatory
molecule B7 and the stimulatory/adhesion molecule CD2 (see, for
example, European Patent Application publication number EP 733,373,
and references cited therein). Similarly, a nucleic acid encoding a
OX-40L fusion polypeptide can be introduced into tumor cells to
increase the immunogenicity of the tumor cells.
[0139] All types of tumor are potentially amenable to treatment by
this approach including, for example, carcinoma of the breast,
lung, pancreas, ovary, kidney, colon and bladder, as well as
melanomas, sarcomas and lymphomas. Nucleic acid molecules encoding
an OX-40L fusion polypeptide capable of forming a trimeric OX-40L
fusion protein are incorporated into a vector suitable for
expression of the OX-40L fusion polypeptide in tumor cells.
Suitable vectors include plasmid, cosmid and viral vectors, such as
retroviruses, adenoviruses and herpesviruses. Disabled viruses,
such as those described in U.S. Pat. Nos. 5,665,362 and 5,837,261
can be employed for this purpose.
[0140] In addition to a nucleic acid molecule encoding a trimeric
OX-40L fusion protein polypeptide, other nucleic acid molecules can
also be introduced into the vector to further enhance the
immunogenic effect. By way of example, such other nucleic acid
molecules include nucleic acids encoding MHC class II proteins
(including .alpha. and .beta. subunits), and other co-stimulatory
molecules, such as B7.1 and B7.2. If desired, a nucleic acid
molecule encoding a selectable marker can also be introduced into
the vector, such that those tumor cells successfully transformed
with the vector can be readily selected.
[0141] The vector is then introduced into the tumor cell by one of
a range of techniques, such as electroporation, lipofection,
co-cultivation with virus-producing cells, or other standard means.
In an exemplary embodiment, the tumor cells are cells removed from
the subject (patient) to be treated. Alternatively the tumor cells
can be cells from a tumor cell line, such as the human tumor cell
lines available from the American Type Culture Collection
(ATCC).
[0142] Optionally, the cells can be screened to identify those
cells into which the vector was introduced. Screening can be
accomplished by any of a variety of procedures, including selecting
for expression of the selectable marker if one is used, or
screening for expression of the trimeric OX-40L fusion protein on
the surface of the cells. This latter procedure can be conveniently
performed by flow cytometry using a labeled antibody specific for
the extracellular portion of OX-40L or for the Ig domain.
[0143] The tumor cells are subsequently administered to the subject
in combination with a suitable carrier such as buffered water,
saline, or glycine. In one embodiment, where the tumor cells are
cells originally removed from the patient, they are attenuated
before being administered to the subject. An attenuated cell is one
which is metabolically active but which is no longer able to
proliferate. Methods for attenuating tumor cells are well known and
include those described in EP 733,373.
[0144] In an alternative embodiment, cell membranes from the tumor
cells, which include the trimeric OX-40L fusion protein can be
administered to the patient instead of intact tumor cells. A cell
membrane preparation can readily be prepared by disrupting or
lysing the cells using standard techniques, such as a French Press,
freeze-thawing, or sonication. Following disruption of the cells, a
membrane enriched fraction is obtained by centrifugation.
[0145] Alternatively, nucleic acid molecules encoding an OX-40L
fusion polypeptide that is capable of assembly into a trimeric
OX-40L fusion protein can be administered directly to a subject in
the form of "naked" DNA, such that expression of the OX-40L fusion
polypeptide occurs in the subject's body. Methods of administering
naked DNA to animals in a manner to cause expression of that DNA in
the body of the animal are well known, and are described, for
example, in U.S. Pat. Nos. 5,620,896; 5,643,578 and 5,593,972, and
references cited therein.
[0146] The present disclosure also encompasses other immunotherapy
methods for treating conditions such as cancer, including adoptive
immunotherapy. As is known in the art, adoptive immunotherapy
involves obtaining lymphoid cells exposed to a particular antigen,
culturing those cells ex vivo under conditions whereby the activity
of the cells is enhanced, and then administering the cells to an
individual. The lymphoid cells are preferably T-cells removed from
a cancer patient, for example T-cells from a draining lymph node.
As discussed above, engagement of the OX-40 receptor on these cells
with a trimeric OX-40L fusion protein will stimulate these cells
and enhance memory T cell generation. Accordingly, the methods
provide a form of adoptive immunotherapy in which the incubation of
lymphoid cells ex vivo is performed in a medium containing a
trimeric OX-40L fusion polypeptide prior to administration of the
cells to a patient. The technical details of methods for obtaining
lymphoid cells, ex vivo cultivation of such cells with immune
stimulants, and administration to patients are known in the field
and are described, for example in U.S. Pat. Nos. 4,690,915;
5,229,115; 5,631,006 and 4,902,288, and references cited
therein.
EXAMPLES
Example 1
Production of an OX-40L Fusion Polypeptide
[0147] An exemplary multimeric human Ig:OX-40L fusion protein
(shown schematically in FIG. 1) was prepared in the following
manner. The construct involved assembling four domains: a signal
sequence, the Fc domain of human IgG1, an isoleucine zipper derived
from yeast GCN4 transcription factor, and finally at the
C-terminus, the complete extracellular domain of human OX-40L.
[0148] The starting point was a pCMVFlag.1-TriZP-BAFF. TriZP is the
isoleucine zipper and is referred to as ILZ. The BAFF domain in
this plasmid is flanked by Eco RI (5') and Xho I (3') restriction
sites. The complete extracellular domain (C-terminal to the
transmembrane domain) of OX-40L was amplified from a plasmid
containing the full-length human OX-40L coding sequence by PCR. For
this reaction, the 5' primer contained a flanking Eco RI site and
an A>T change in the coding sequence to remove an interfering
Eco RI site 26 bases down stream. The 3' primer contained a
flanking Xho I site, a stop codon and an A>T change 13 bases
from the Xho I site to remove another interfering Eco RI site at
this position. The first A to T mutation resulted in the
substitution of isoleucine with phenylalanine (e.g., as shown in
SEQ ID NO:2, 9.sup.th amino acid). The second A to T mutations did
not alter the amino acid sequence of the encoded OX-40L domain. The
amplified OX-40L extracellular domain was cleaved with Eco RI and
Xho I, purified by agarose gel electrophoresis (gel purified) and
cloned into the Eco RI/Xho I site vacated by the BAFF domain in the
pCMV vector. The now contiguous ILZ:OX-40L domains were amplified
by PCR from this new pCMV plasmid using a 5' primer containing a
flanking Sac I site and the same 3' primer used to amplify the
OX-40L domain initially. TOPO TA cloning was used to ligate, via
topoisomerase, the amplified product into the pCR 2.1 plasmid
(Invitrogen, Carlsbad, Calif.). The same strategy was employed to
amplify and clone the human Fc-.gamma. domain from IgG1 into pCR
2.1. The Fc-.gamma. fragment of IgG1 was previously modified by
converting the Cys residue (tgt) in the hinge region to Thr (acc)
corresponding to base 799 in BC 041037. The 5' primer included a
flanking Nhe I site, an additional base, A, to maintain reading
frame for the next step in cloning and the coding sequence started
with the mutated Thr codon. The 3' primer contains a flanking Sac I
site and the coding sequence ends with the C-terminal Lys (aaa) of
the IgG1. The ILZ-OX-40L insert was excised from pCR 2.1 by
cleavage with Sac I and Xho I, gel purified, and cloned into the
Fc-.gamma. pCR2.1 also cut with Sac I and Xho I. This results in
the contiguous positioning of Fc-gamma, ILZ and OX-40L and the
insertion of the dipeptide, Leu-Gln, encoded by the added Sac I
site between Fc-.gamma. and ILZ. For expression in mammalian cells
the construct, FC-ILZ-OX-40L, was cloned into a modified version of
the pCEP4 expression vector (Invitrogen). The plasmid, designated
pCEP D4-7, was modified to include the signal sequence of the
basement membrane protein BM40 adjacent to the multiple cloning
site. pCEP4 controls transcription from the CMV promoter.
Expression of the EBNA gene from Epstein Barr virus promotes
autosomal replication of the plasmid resulting in high copy number.
The Fc-ILZ-OX-40L insert was cleaved from pCR2.1 using Nhe I and
Xho I, gel purified and ligated into pCEP D4-7 also cut with Nhe I
and Xho I. The final construct was analyzed by restriction
analysis, as shown in FIG. 2. The insert was sequenced to confirm
the authenticity of the encoded fusion protein.
Example 2
Production of an OX-40L Fusion Polypeptide
[0149] In order to produce recombinant multimeric OX-40L fusion
protein, the Fc-ILZ-OX-40L fusion construct was introduced into HK
293 cells by transfection with lipofectamine. The HK 293 cell line
is a well-established culture line used extensively for mammalian
protein expression. The pCEP D4-7 contains a hygromycin resistance
gene permitting selection of stably transfected colonies of 293
cells in the presence of hygromycin. Because pCEP D4-7 replicates
autosomally, all of the hygromycin resistant cells were pooled and
expanded in cell culture to monitor Fc-ILZ-OX-40L synthesis. For
protein production the cells were cultured in a laboratory scale
bioreactor (Cell-Max). The fusion protein was purified by Protein G
affinity chromatography. An exemplary protein G elution profile is
shown in FIGS. 3A and B. As shown in FIG. 3B, maximal elution was
observed in fractions 6 and 7. The identity of the eluted protein
was confirmed by immunoreactivity using anti human IgG (FIGS. 4A
and B) and anti-human OX-40 ligand antibodies (FIGS. 4C and D).
Under reducing conditions, the predominant product was observed to
migrate at approximately 43 kD, consistent with a monomeric fusion
polypeptide. Under non-reducing conditions, higher molecular weight
species were observed. A strong band was observed at 86 kD
consistent with formation of dimers linked by disulfide bonds
between two Fc domains. Assembly into trimers involves noncovalent
interactions between OX-40L and trimerization domains, and leaves
one unpaired Fc domain. Association between unpaired Fc domains in
two trimeric OX-40L fusion proteins results in the formation of
hexamer under native conditions. However, on non-reducing SDS PAGE
gels nothing larger than dimers is observed following elution in
acid pH.
[0150] Although analysis of F-ILZ-OX-40L after elution at acid pH
indicated appropriate covalent assembly of subunits, analysis by
size exclusion chromatography under non-denaturing conditions
indicated that acid pH induced non-covalent aggregation of the
protein into higher order structures. To prevent this aggregation,
the fusion protein was eluted from the protein-G column using
ActiSep Elution Medium (Sterogene, Carlsbad, Calif.), buffered at a
pH of between 4 and 7. This single step yielded a high degree of
purification (FIG. 5) and generated the material subsequently
analyzed for structure and for biological activity.
[0151] The contribution of the ILZ domain to the folding
recombinant Fc:OX-40L fusion protein was demonstrated by comparing
the elution profile from size exclusion chromatography of
Fc:ILZ:OX-40L and Fc:OX-40L as shown in FIG. 6. Fc:ILZ:OX-40L
elutes as a largely homogeneous and symmetrical peak at about 20
ml, corresponding to an equivalent sphere with a mass of about 570
kDa. This is about twice the expected mass but this is likely due
to the asymmetric structure imparted by the three domains of the
fusion protein. In contrast, in the absence of the ILZ domain, very
little of the purified protein elutes at 20 ml and instead elutes
as large aggregates in the void volume or as low molecular weight
components likely to be unassembled monomers. This indicates that
for the human molecule, the ILZ trimerization domain is involved in
productive folding of the recombinant extracellular
receptor-binding domain of OX-40L.
Example 3
Trimeric OX-40L Fusion Protein Induced T-Cell Proliferation
[0152] The functional contribution of the ILZ domain was tested by
comparing the costimulatory activity of Fc:ILZOX-40L with Fc:OX-40L
in a proliferation assay in vitro (FIG. 7). FIG. 7 illustrates the
biological activity of recombinant human Fc:ILZOX-40L with and
without the ILZ domain. The recombinant protein was tested for
biological activity in vitro by costimulation of CD4.sup.+ T-cell
proliferation in response to anti-CD3. Ninety-six-well culture
plates were coated with goat anti-human Ig and goat anti-mouse Ig
capture antibodies, both at 2 .mu.g/ml. The plates were incubated
with mouse anti-human CD3 at 2 ng/ml followed by serial two-fold
dilutions of recombinant OX-40L fusion protein (1600 to 3 ng/ml).
Purified human CD4 T-cells that had been activated with PHA and
cultured for four days with IL2 (10 U/ml) were washed and added to
each well at 5.times.10.sup.4 cells per well. The cells were
labeled with .sup.3H-thymidine for the last 16 hours of a 62 hour
culture, harvested and counted. The results, shown in FIG. 7, are
presented as mean CPM with standard deviation calculated from
triplicate wells. The results indicate that the trimeric OX-40L
fusion protein containing the ILZ domain produced a dose-dependent
costimulation/stimulation (mitogenesis) of the CD4.sup.+ T-cells
while the construct lacking the ILZ domain was essentially
inactive.
[0153] In view of the many possible embodiments to which the
principles of the disclosed invention may be applied, it should be
recognized that the illustrated embodiments are only preferred
examples of the invention and should not be taken as limiting the
scope of the invention. Rather, the scope of the invention is
defined by the following claims. We therefore claim as our
invention all that comes within the scope and spirit of these
claims.
Sequence CWU 1
1
13 1 408 DNA Homo sapiens misc_feature (1)..(408) OX-40 receptor
binding domain 1 caggtatcac atcggtatcc tcgatttcaa agtatcaaag
tacaatttac cgaatataag 60 aaggagaaag gtttcatcct cacttcccaa
aaggaggatg aaatcatgaa ggtgcagaac 120 aactcagtca tcatcaactg
tgatgggttt tatctcatct ccctgaaggg ctacttctcc 180 caggaagtca
acattagcct tcattaccag aaggatgagg agcccctctt ccaactgaag 240
aaggtcaggt ctgtcaactc cttgatggtg gcctctctga cttacaaaga caaagtctac
300 ttgaatgtga ccactgacaa tacctccctg gatgacttcc atgtgaatgg
cggagaactg 360 attcttatcc atcaaaatcc tggtgaattt tgtgtccttt aactcgag
408 2 133 PRT Homo sapiens misc_feature (1)..(133) OX-40 receptor
binding domain 2 Gln Val Ser His Arg Tyr Pro Arg Phe Gln Ser Ile
Lys Val Gln Phe 1 5 10 15 Thr Glu Tyr Lys Lys Glu Lys Gly Phe Ile
Leu Thr Ser Gln Lys Glu 20 25 30 Asp Glu Ile Met Lys Val Gln Asn
Asn Ser Val Ile Ile Asn Cys Asp 35 40 45 Gly Phe Tyr Leu Ile Ser
Leu Lys Gly Tyr Phe Ser Gln Glu Val Asn 50 55 60 Ile Ser Leu His
Tyr Gln Lys Asp Glu Glu Pro Leu Phe Gln Leu Lys 65 70 75 80 Lys Val
Arg Ser Val Asn Ser Leu Met Val Ala Ser Leu Thr Tyr Lys 85 90 95
Asp Lys Val Tyr Leu Asn Val Thr Thr Asp Asn Thr Ser Leu Asp Asp 100
105 110 Phe His Val Asn Gly Gly Glu Leu Ile Leu Ile His Gln Asn Pro
Gly 115 120 125 Glu Phe Cys Val Leu 130 3 135 DNA Artificial
sequence isoleucine zipper domain 3 cttggtggcg gaagtatcaa
acagatcgaa gataagattg aagagatctt gagcaaaatc 60 taccacattg
aaaacgagat cgcgcgcatt aagaaactga tcggcgaacg tggccatggc 120
ggtgggtcga attca 135 4 32 PRT Artificial sequence isoleucine zipper
4 Ile Lys Gln Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys Ile Tyr 1
5 10 15 His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly Glu
Arg 20 25 30 5 691 DNA Homo sapiens misc_feature (1)..(691)
immunoglobulin Fc domain 5 gctagcaacc gacaaaactc acacatgccc
accgtgccca gcacctgaac tcctgggggg 60 accgtcagtc ttcctcttcc
ccccaaaacc caaggacacc ctcatgatct cccggacccc 120 tgaggtcaca
tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg 180
gtacgtggac ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa
240 cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac caggactggc
tgaatggcaa 300 ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc
cccatcgaga aaaccatctc 360 caaagccaaa gggcagcccc gagaaccaca
ggtgtacacc ctgcccccat cccgggagga 420 gatgaccaag aaccaggtca
gcctgacctg cctggtcaaa ggcttctatc ccagcgacat 480 cgccgtggag
tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt 540
gctggactcc gacggctcct tcttcctcta tagcaagctc accgtggaca agagcaggtg
600 gcagcagggg aacgtcttct catgctccgt gatgcatgag gctctgcaca
accactacac 660 gcagaagagc ctctccctgt ctccgggtaa a 691 6 230 PRT
Homo sapiens misc_feature (1)..(230) immunoglobulin Fc domain 6 Leu
Ala Thr Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 1 5 10
15 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
20 25 30 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp 35 40 45 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly 50 55 60 Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn 65 70 75 80 Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp 85 90 95 Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 100 105 110 Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 115 120 125 Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 130 135 140
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 145
150 155 160 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr 165 170 175 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys 180 185 190 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys 195 200 205 Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu 210 215 220 Ser Leu Ser Pro Gly Lys
225 230 7 1240 DNA Artificial sequence polynucleotide encoding an
OX-40 ligand fusion polypeptide 7 gctagcaacc gacaaaactc acacatgccc
accgtgccca gcacctgaac tcctgggggg 60 accgtcagtc ttcctcttcc
ccccaaaacc caaggacacc ctcatgatct cccggacccc 120 tgaggtcaca
tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg 180
gtacgtggac ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa
240 cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac caggactggc
tgaatggcaa 300 ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc
cccatcgaga aaaccatctc 360 caaagccaaa gggcagcccc gagaaccaca
ggtgtacacc ctgcccccat cccgggagga 420 gatgaccaag aaccaggtca
gcctgacctg cctggtcaaa ggcttctatc ccagcgacat 480 cgccgtggag
tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt 540
gctggactcc gacggctcct tcttcctcta tagcaagctc accgtggaca agagcaggtg
600 gcagcagggg aacgtcttct catgctccgt gatgcatgag gctctgcaca
accactacac 660 gcagaagagc ctctccctgt ctccgggtaa agagctcctt
ggtggcggaa gtatcaaaca 720 gatcgaagat aagattgaag agatcttgag
caaaatctac cacattgaaa acgagatcgc 780 gcgcattaag aaactgatcg
gcgaacgtgg ccatggcggt gggtcgaatt cacaggtatc 840 acatcggtat
cctcgatttc aaagtatcaa agtacaattt accgaatata agaaggagaa 900
aggtttcatc ctcacttccc aaaaggagga tgaaatcatg aaggtgcaga acaactcagt
960 catcatcaac tgtgatgggt tttatctcat ctccctgaag ggctacttct
cccaggaagt 1020 caacattagc cttcattacc agaaggatga ggagcccctc
ttccaactga agaaggtcag 1080 gtctgtcaac tccttgatgg tggcctctct
gacttacaaa gacaaagtct acttgaatgt 1140 gaccactgac aatacctccc
tggatgactt ccatgtgaat ggcggagaac tgattcttat 1200 ccatcaaaat
cctggtgaat tttgtgtcct ttaactcgag 1240 8 410 PRT Artificial sequence
OX-40 ligand fusion polypeptide 8 Leu Ala Thr Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu 1 5 10 15 Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 20 25 30 Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 35 40 45 Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 50 55 60
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 65
70 75 80 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp 85 90 95 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro 100 105 110 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu 115 120 125 Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met Thr Lys Asn 130 135 140 Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 145 150 155 160 Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 165 170 175 Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 180 185
190 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
195 200 205 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu 210 215 220 Ser Leu Ser Pro Gly Lys Glu Leu Leu Gly Gly Gly
Ser Ile Lys Gln 225 230 235 240 Ile Glu Asp Lys Ile Glu Glu Ile Leu
Ser Lys Ile Tyr His Ile Glu 245 250 255 Asn Glu Ile Ala Arg Ile Lys
Lys Leu Ile Gly Glu Arg Gly His Gly 260 265 270 Gly Gly Ser Asn Ser
Gln Val Ser His Arg Tyr Pro Arg Phe Gln Ser 275 280 285 Ile Lys Val
Gln Phe Thr Glu Tyr Lys Lys Glu Lys Gly Phe Ile Leu 290 295 300 Thr
Ser Gln Lys Glu Asp Glu Ile Met Lys Val Gln Asn Asn Ser Val 305 310
315 320 Ile Ile Asn Cys Asp Gly Phe Tyr Leu Ile Ser Leu Lys Gly Tyr
Phe 325 330 335 Ser Gln Glu Val Asn Ile Ser Leu His Tyr Gln Lys Asp
Glu Glu Pro 340 345 350 Leu Phe Gln Leu Lys Lys Val Arg Ser Val Asn
Ser Leu Met Val Ala 355 360 365 Ser Leu Thr Tyr Lys Asp Lys Val Tyr
Leu Asn Val Thr Thr Asp Asn 370 375 380 Thr Ser Leu Asp Asp Phe His
Val Asn Gly Gly Glu Leu Ile Leu Ile 385 390 395 400 His Gln Asn Pro
Gly Glu Phe Cys Val Leu 405 410 9 408 DNA Homo sapiens 9 caggtatcac
atcggtatcc tcgaattcaa agtatcaaag tacaatttac cgaatataag 60
aaggagaaag gtttcatcct cacttcccaa aaggaggatg aaatcatgaa ggtgcagaac
120 aactcagtca tcatcaactg tgatgggttt tatctcatct ccctgaaggg
ctacttctcc 180 caggaagtca acattagcct tcattaccag aaggatgagg
agcccctctt ccaactgaag 240 aaggtcaggt ctgtcaactc cttgatggtg
gcctctctga cttacaaaga caaagtctac 300 ttgaatgtga ccactgacaa
tacctccctg gatgacttcc atgtgaatgg cggagaactg 360 attcttatcc
atcaaaatcc tggtgaatta tgtgtccttt aactcgag 408 10 28 DNA Artificial
sequence oligonucleotide primer 10 gctagcaacc gacaaaactc acacatgc
28 11 24 DNA Artificial sequence oligonucleotide primer 11
ctcgagttaa agcacacaaa attc 24 12 63 DNA Homo sapiens misc_feature
(1)..(63) secretory signal 12 atgagggcct ggatcttctt tctcctttgc
ctggccggga gggctctggc agccccgcta 60 gcn 63 13 21 PRT Homo sapiens
misc_feature (1)..(21) secretory signal 13 Met Arg Ala Trp Ile Phe
Phe Leu Leu Cys Leu Ala Gly Arg Ala Leu 1 5 10 15 Ala Ala Pro Leu
Ala 20
* * * * *