U.S. patent application number 13/243966 was filed with the patent office on 2012-03-15 for dendritic cell co-stimulatory molecules.
This patent application is currently assigned to The Johns Hopkins University. Invention is credited to Kevin S. Gorski, Drew M. Pardoll, Su-Yi Tseng, Haruo Tsuchiya.
Application Number | 20120065374 13/243966 |
Document ID | / |
Family ID | 26895892 |
Filed Date | 2012-03-15 |
United States Patent
Application |
20120065374 |
Kind Code |
A1 |
Pardoll; Drew M. ; et
al. |
March 15, 2012 |
DENDRITIC CELL CO-STIMULATORY MOLECULES
Abstract
A novel costimulatory protein molecule, B7-DC, which is a member
of the B7 family, is described as is DNA coding therefor and
expression vectors comprising this DNA. B7-DC protein, fragments,
fusion polypeptides/proteins and other functional derivatives, and
transformed cells expressing B7-DC are useful in vaccine
compositions and methods. Compositions and methods are disclosed
for inducing potent T cell mediated responses that can be harnessed
for anti-tumor and anti-viral immunity.
Inventors: |
Pardoll; Drew M.;
(Brookville, MD) ; Tsuchiya; Haruo; (Baltimore,
MD) ; Gorski; Kevin S.; (Baltimore, MD) ;
Tseng; Su-Yi; (Baltimore, MD) |
Assignee: |
The Johns Hopkins
University
|
Family ID: |
26895892 |
Appl. No.: |
13/243966 |
Filed: |
September 23, 2011 |
Related U.S. Patent Documents
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11931653 |
Oct 31, 2007 |
8053558 |
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13243966 |
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11361057 |
Feb 24, 2006 |
7560540 |
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11931653 |
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09794210 |
Feb 28, 2001 |
7030219 |
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11361057 |
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60240169 |
Oct 13, 2000 |
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60200580 |
Apr 28, 2000 |
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Current U.S.
Class: |
530/350 |
Current CPC
Class: |
A61P 37/06 20180101;
A61K 2039/5156 20130101; A61K 39/39558 20130101; A61K 2039/55516
20130101; A61P 37/02 20180101; A61P 35/00 20180101; A61K 2039/5158
20130101; C07K 14/70532 20130101; C07K 2319/00 20130101; A61P 31/00
20180101; A61K 38/00 20130101; A61K 2039/505 20130101; A61K
2039/5152 20130101; A61P 37/04 20180101; A61K 2039/57 20130101;
C07K 16/2827 20130101; C07K 2319/30 20130101 |
Class at
Publication: |
530/350 |
International
Class: |
C07K 14/47 20060101
C07K014/47 |
Goverment Interests
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH
[0001] This invention was funded in part by grants from National
Institutes of Health, which provides to the United States
government certain rights in this invention.
Claims
1.-31. (canceled)
32. A polypeptide that is selectively expressed on dendritic cells
as compared to activated macrophages and has the following
functional properties: (a) binds to a binding partner on T cells;
and (b) costimulates T cells to proliferate and/or to produce and
secrete cytokines.
33.-120. (canceled)
Description
BACKGROUND OF TIM INVENTION
[0002] 1. Field of the Invention
[0003] The invention in the field of biochemistry and medicine
relates to novel proteins that are selectively expressed on the
surface of dendritic cells and can be used as cell surface
molecules or in soluble form in vaccine compositions to stimulate
immune responses.
[0004] 2. Description of the Background Art
[0005] The generation of a T lymphocyte response is a complex
process involving cell-cell interactions and production of soluble
mediators (cytokines or lymphokines). This response is regulated by
several T-cell surface molecules acting as "receptors," including
the T-cell receptor (TCR) complex and other "accessory" surface
molecules many of which are cell surface "differentiation antigens"
that were first defined by monoclonal antibodies ("CD
molecules")
[0006] Optimal activation of all lymphocytes is believed to require
two signals: an antigen specific or clonal signal, as well as a
second, antigen non-specific signal (Janeway, C., Cold Spring
Harbor Symp. Quant. Biol. 54:1-14 (1989)). If a lymphocyte
encounters an antigen alone, without co-stimulation by so-called
co-stimulatory molecules (such as B7 described below), it will
respond with either clonal inactivation also called "anergy"
(Schwartz, R. Science 248:1349 (1990)) or apoptosis (programmed
cell death); if the co-stimulatory signal is provided it will
respond with clonal expansion specific for the stimulating antigen.
No significant amplification of an immune response against a given
antigen occurs without co-stimulation (June et al. (Immunology
Today 15:321-331, 1994); Chen et al. (Immunology Today 14:483-486);
Townsend, S E and Allison, J P (1993) Science 259:368-370).
[0007] The quality and potency of an immune response depends in
large part on the type of antigen presenting cells (APC) that
process and present the antigen to T cells. The density of the
peptide antigen/MHC ligand available for engagement of the TCR and
the provision of soluble and/or membrane-bound co-stimulatory
signals by APCs at the time of T cell engagement and activation is
critical. It is for these reasons that immunotherapeutic strategies
have begun to focus on providing (a) the target antigen to the
appropriate APC types and (b) appropriate co-stimulatory molecules
to enhance T cell activation.
[0008] APCs that provide the signals required for activation of T
cells include monocytes/macrophages, B lymphocytes, and, most
importantly dendritic cells (DCs). In the past, activated
macrophages were believed to be the critical APCs that initiated T
cell responses in vivo. This notion was based on their ability to
phagocytose antigens effectively and process them for surface
display and presentation. More recently, attention has shifted to
DC as the major initiator in vivo of antigen-specific T cell
responses. DCs have a distinct phenotype from activated macrophages
and are classified into different subtypes capable of initiating
distinct immune responses. A functional hallmark of DCs is their
approximately 100-fold greater potency then macrophages to activate
naive T cells in vitro. To date, the explanation of this potency
has been based on quantitative differences in molecules known to be
important for antigen presentation. The present invention is based
on the discovery of an important qualitative difference.
[0009] The first signal in antigen presentation is initiated by
interaction of the TCR with antigen presented in the context of
class II major histocompatibility complex (MHC) molecules on the
APC (Allen, Immunol. Today 8:270 (1987)). Co-stimulatory signals
come from other molecules, the best characterized of which is the
B7 family (namely B7.1, B7.2, and possibly B7.3) which are also
present on APCs.
[0010] Two proteins expressed on the surface of T cells are the
best-characterized ligands or counter-receptors for co-stimulatory
molecules such as B7. CD28 is a homodimeric glycoprotein of the
immunoglobulin (Ig) superfamily (Aruffo and Seed, Proc. Natl. Acad.
Sci. 84:8573-8577 (1987)) found on most mature human T cells that
functions in T cell activation. CD28, is constitutively expressed
on resting T cells and increases after activation. After signaling
through the T cell receptor, ligation of CD28 induces T cells to
proliferate and secrete IL-2 (Linsley, P S, et al. (1991) J. Exp.
Med. 173, 721-730; Gimmi, C D, et al. (1991) Proc. Natl. Acad. Sci.
USA. 88, 6575-6579; Thompson, C. B., et al. (1989) Proc. Natl.
Acad. Sci. USA. 86, 1333-1337; June, C. H., et al. (1990) Immunol.
Today. 11, 211-6; Harding, F. A., et al. (1992) Nature. 356,
607-609.). CD28 mediates cell-cell contact ("intercellular
adhesion"), a form of antigen-independent intercellular interaction
that is essential for immune responses (Springer et al., Ann. Rev.
Immunol. 5:223-252 (1987)).
[0011] CTLA4 is a T cell surface molecule highly homologous to CD28
but is not expressed on resting T cells and appears following T
cell activation (Brunet, J. F., et al., (1987) Nature 328,
267-270). CTLA-4 was originally identified by differential
screening of a murine cytolytic T cell cDNA library, Brunet et al.
supra. The role of CTLA-4 as a second receptor for B7 is discussed
in Linsley et al. (1991) J. Exp. Med. 174:561-569, which also noted
that B7 has a higher affinity for CTLA4 than for CD28. Freeman et
al. (1993) Science 262:907-909 discussed CTLA-4 in B7 deficient
mice. Ligands for CTLA-4 are described in Lenschow et al. (1993)
Proc. Nat'l. Acad. Sci. 90:11054-11058.
[0012] Th cells secrete growth and differentiation-inducing
cytokines such as IL-2, IL-4 and IL-6 possibly in a focused manner
in the area of Th-B cell contact which serves to ensure activation
of only B cells presenting antigen to Th cells and avoid activation
of bystander B cells.
[0013] CD28 and CTLA-4 interact with a co-stimulatory molecule
generally known as B7. B7 was originally described as a B cell
activation antigen because it was found on B cells and was termed
B7/BB-1 (Linsley et al, Proc. Natl. Acad. Sci. USA 87:5031-5035
(1990). Hereafter, this molecule will be referred to as B7, B7-1 or
B7.1). B7 and more recently described B7 homologues are also
members of the Ig superfamily. In contrast to CD28 and CTLA-4, B7
comprises two extracellular Ig domains, an N-terminal variable
(V)-like domain followed by a constant (C)-like domain.
[0014] B7 family members are generally expressed on APCs and, as
noted, are of critical importance to the activation of naive T
cells. These family members include B7-1 (=B7, also designated
CD80) and B7-2 (also designated CD86). References describing B7-1
include Schwartz, R. H. Cell 71:1065-1068, 1992; Chen, L. et al.
Cell 71:1093-1102, 1992; Freeman, G. J. et al. J. Immunol
143:2714-2722, 1989; and Freeman, G. J. et al. J. Exp. Med.
174:625-631, 1991)). References describing B7-2 include (Freeman,
G. J. et al, Science 262:909-911 813-960, 1993). To date, both
murine B7-1 and B7-2 and human B7-1 and B7-2 have been described
(Freeman et al., 1989, supra; 1991, supra; and 1993, supra).
Activated human B lymphocytes express CTLA4/CD28 binding
counter-receptors B7-2 and B7-3, both of which can deliver
costimulatory signals to T cells via either CD28 or CTLA4.
[0015] B7-2 is expressed by B cells at about 24 hours following
stimulation with either anti-Ig or anti-MHC class II mAbs. B7-2
induces detectable IL-2 secretion and T cell proliferation. At
about 48 to 72 hours post activation, B cells express both B7-1 and
a third CTLA4 counter-receptor identified by a mAb BB-1 (Yokochi,
T, et al. (1982) J. Immunol. 128, 823-827), termed B7-3. B7-3 is
also expressed on B7-negative activated B cells and can costimulate
T cell proliferation without detectable IL-2 production, indicating
that the B7-1 and B7-3 molecules are distinct. B7-3 is expressed on
a wide variety of cells including activated B cells, activated
monocytes, dendritic cells, Langerhans cells and keratinocytes. At
72 hours post B cell activation, the expression of B7-1 and B7-3
begins to decline. The presence of these CTLA4/CD28 binding
counter-receptors on the surface of activated B lymphocytes
indicates that T cell costimulation is regulated, in part, by the
temporal expression of these molecules following B cell
activation.
[0016] The importance of the B7:CD28/CTLA4 costimulatory pathway(s)
has been demonstrated in vitro and in vivo. A direct relationship
exists between increased T cell activity and increased B7
expression (Razi-Wolf et al., Proc. Natl. Acad. Sci. USA,
89:4210-4214 (1992)). T cells are rendered anergic when they,
encounter peptides antigens on cells lacking a costimulatory ligand
that binds CD28 Blockade of this costimulatory pathway results in
the development of antigen specific tolerance in murine and humans
systems (Harding et al., supra; Lenschow, D. J. et al. (1992)
Science. 257, 789-792; Turka, L A et al. (1992) Proc. Natl. Acad.
Sci. USA. 89, 11102-11105; Gimmi, C D et al. (1993) Proc. Natl.
Acad. Sci USA 90, 6586-6590; Boussiotis, V. et al. (1993) J. Exp.
Med. 178, 1753-1763). Conversely, expression of B7 by B7-negative
murine tumor cells induces T-cell mediated specific immunity
accompanied by tumor rejection and long lasting protection to tumor
challenge (Chen, L, et al. (1992) Cell 71:1093-1102; Townsend et
al., supra; Baskar, S, et al. (1993) Proc. Natl. Acad. Sci. 90,
5687-5690.). Therefore, manipulation of the B7:CD28/CTLA4 pathway
offers great potential to stimulate or suppress immune responses in
humans.
[0017] Interactions between CD28 and B7 have been characterized
using genetic fusions of the extracellular portions of B7 or CD28
with Ig C.gamma.1 chains (Linsley et al, J. Exp. Med. 173:721-730
(1991)). When B7Ig fusion proteins are immobilized, or when B7 is
expressed on the surface of a cell, such as a transfected CHO cell,
they costimulate T cell proliferation. T cell stimulation with
B7+CHO cells also specifically stimulates increased levels of
transcripts for IL-2.
[0018] U.S. Pat. No. 5,521,288 describes a method for regulating
immune responses by contacting CD28 positive T cells with fragments
encoded by parts of DNA encoding B7, primarily corresponding to the
extracellular domain (BCD) of B7. Immune responses were also
regulated by derivatives of B7 that were are fusion protein
constructs including at least a portion of B7 ECD and another
protein, such as the human IgC.gamma.1 domain that altered the
solubility, binding affinity and/or valency of B7. For example DNA
encoding amino acid residues from positions 1-215 of the B7 ECD was
joined to DNA encoding amino acid residues of the sequences
corresponding to the hinge, CH2 and CH3 regions of human IgC.gamma.
1 to form a DNA fusion product which encoded a B7Ig fusion protein.
Also disclosed was a method for treating an immune system disease
mediated by T cells by administering B7 or B7Ig fusion protein to
react with T cells by binding the CD28 receptor. T cell
proliferation in graft versus host disease was inhibited by
reacting CD28+T cells with B7 antigen or B7Ig fusion protein in
combination with an immunosuppressant.
[0019] U.S. Pat. No. 5,861,310 discloses tumor cells modified to
express One or more T cell costimulatory molecules, including B7-2
and B7-3. One embodiment includes further expression of B7.
Modification was by transfection with nucleic acid encoding the
B7-2, B7-3 or B7 proteins. Tumor cells could also be genetically
modified in vivo. Such modified tumor cells said to be useful for
treating a patient with a tumor, to prevent or inhibit metastatic
spread or inhibit recurrence of the tumor. This document disclosed
a method for specifically inducing a CD4+ T cell response against a
tumor.
[0020] U.S. Pat. No. 5,942,607 discloses isolated nucleic acids
encoding novel CTLA4/CD28 ligands which costimulate T cell
activation. In one embodiment, the isolated nucleic acid encoded
B7-2. Also disclosed was a nucleic acid comprising at least a
portion of the disclosed full length B7-2 sequence. According to
this document, the nucleic acid sequences could be integrated into
various expression vectors which could direct the synthesis of the
corresponding proteins or peptides in a variety of host cells
including mammalian and insect cells. Also disclosed were host
cells transformed to produce proteins or peptides encoded by these
nucleic acid sequences and isolated proteins and peptides which
comprise at least a portion of the B7-2 sequence.
[0021] Dong H et al., Nat Med 1999 5:1365-1399, described a third
member of the B7 family, designated B7-H1 that does not bind CD28,
CTLA4 or ICOS (inducible co-stimulator). Ligation of B7-H1
co-stimulated T-cell responses to polyclonal stimuli and
alloantigens, and preferentially stimulated the production of
interleukin-10. IL-2, produced in small amounts, was required for
the effect of B7-H1 co-stimulation. This study defined a previously
unknown co-stimulatory molecule that may be involved in the
negative regulation of cell-mediated immune responses. The same
laboratory (Wang S et al., Blood. 2000;96:2808-2813) described a
new human B7-like gene designated B7-H2, the expression of which
was detected on the surface of monocyte-derived immature DCs.
Soluble B7-H2 and an Ig fusion protein, B7-H2Ig, bound to
activated, but not resting, T cells. This binding was inhibited by
a soluble form of ICOS (ICOSIg) but not by CTLA4Ig. ICOSIg stained
CHO cells transfected with the B7-H2 gene. Using suboptimal
cross-linking of CD3 as a stimulus, costimulation of T-cell
proliferation by B7-H2Ig was found to be dose-dependent and
correlated with secretion of IL-2, whereas optimal CD3 ligation
preferentially stimulated IL-10 production. The authors concluded
that B7-H2 is a putative ligand for the ICOS T-cell molecule.
[0022] Swallow M M et al., Immunity, 1999, 11:423-432 reported
cloning of a novel gene, b7h, a is a close homolog of B7 molecules
that are expressed on APCs. B7h costimulated proliferation of
purified T cells by acting on a receptor distinct from CD28 or
CTLA-4. Surprisingly, although B7h was expressed in unstimulated B
cells, its expression was induced in nonlymphoid cells (3T3 cells;
embryonic fibroblasts) treated with TNF.alpha. and was upregulated
nonlymphoid tissue of mice treated with LPS, a potent activator of
TNF.alpha.. These studies defined a novel costimulatory ligand of T
cells and suggested that induction of B7h by TNF.alpha. may
directly augment recognition of self during inflammation.
[0023] Yoshinaga S K et al., Nature, 1999, 402:827-832, described a
new murine costimulatory receptor-ligand pair. The receptor,
related to CD28, was the murine homologue of the human protein
ICOS, and was expressed on activated T cells and resting memory T
cells. The ligand, which was homologous to B7 molecules was
designated B7-related protein-1 (B7RP-1). B7RP-1 is a type 1
transmembrane protein with 20% and 19% amino acid identity to
murine B7.1 (CD80) and B7.2 (CD86), respectively. This homology is
significant as B7.1 and B7.2 share only 27% amino acid identity
(Freeman, G J et al., J. Exp. Med. 178:2185-2192 (1993)). This
homology includes the cysteines that are important for Ig loop
formation at conserved locations (residues 62, 138,185 and 242 from
the initiating methionine). The overall length and relative
position of the transmembrane domain of B7RP-1 are similar to those
of the B7 molecules (Greenfield, E A et al., Crit. Rev. Immunol.
18:389-418 (1998)). B7RP-1 was shown to be expressed on B cells and
macrophages. ICOS and B7RP-I did not interact with proteins in the
CD28-B7 pathway, and B7RP-1 co-stimulated T cells independently of
CD28. Transgenic mice expressing a fusion protein between B7RP-1
and the Fc portion of Ig ("B7-RP1-Fc") had lymphoid hyperplasia in
spleen, lymph nodes and Peyer's patches. Co stimulatory activity of
B7RP-1 in vivo was found by demonstrating enhanced delayed-type
hypersensitivity in antigen-presensitized mice treated with
B7RP-1-Fc at the time of antigen challenge. The authors concluded
that ICOS and B7RP-1 define a distinct new receptor-ligand pair
that is structurally related to CD28-B7 and is involved in the
adaptive immune response.
[0024] Yoshinaga S K et al., Int Immunol, 2000 Oct. 12: 1439-1447,
reported co-stimulation of human T cells through the human B7RP-1
and ICOS interaction. This ligand-receptor pair interacted with a
K.sub.D of approximately 33 nM and an off-rate having a t.sub.(1/2)
of >10 min. TNF.alpha. differentially regulated expression of
human B7RP-1 on B cells, monocytes and DC. TNF.alpha. enhanced
B7RP-1 expression on B cells and monocytes, but inhibits expression
on DC. A human B7RP-1-Fc protein, or cells that expressed
membrane-bound B7RP-1, co-stimulated T cell proliferation in vitro.
Specific cytokines, such as IFN.gamma. and were induced by B7RP-1
co-stimulation. Although IL-2 levels were not significantly
increased, B7RP-1-induced co-stimulation was dependent on IL-2.
These studies defined the human ortholog to murine B7RP-1 and
characterized its interaction with human ICOS.
[0025] PD-1 is an immuno-inhibitory receptor expressed by activated
T, B and myeloid cells. Mice deficient in PD-1 showed multiple
forms of autoimmunity due to the loss of peripheral tolerance.
Freeman, G J et al., J. Exp. Med. 192:1027-1034(2000) reported that
the ligand of PD-1 (PD-L1) is a member of the B7 gene family.
Engagement of PD-1 by PD-L1 resulted in inhibition of TCR-mediated
lymphocyte activation (proliferation, cytokine secretion). In
addition, PD-1 signaling inhibited suboptimal levels of
CD28-mediated costimulation. PD-L1 is expressed by APCs (human
monocytes stimulated with IPN.gamma., activated human DCs). In
addition, PD-L1 was shown to be is expressed in heart and lung. The
authors speculated that relative magnitude of inhibitory PD-L1
signals and costimulatory B7-1/B7-2 signals on APCs may determine
the extent of T cell activation and the threshold between tolerance
and autoimmunity. The presence of PD-L1 on nonlymphoid tissues may
contribute to the magnitude of immune responses at sites of
inflammation.
[0026] Citation of the above documents is not intended as an
admission that any of the foregoing is pertinent prior art. All
statements as to the date or representation as to the contents of
these documents is based on the information available to the
applicant and does not constitute any admission as to the
correctness of the dates or contents of these documents.
SUMMARY OF THE INVENTION
[0027] In order to identify genes encoding novel dendritic cell
(DC) specific costimulatory molecules for T cell activation, the
inventors screened a subtracted cDNA library between DCs and
activated macrophages. This cDNA subtraction approach defines genes
expressed by DCs but not by activated macrophages. Use of this
approach has led to discover of several novel DC-specific genes
that are useful in enhancing the potency of vaccines that depend on
activation of T cells. The present application focuses on one such
gene.
[0028] Based on presence in the DC library and absence from the
activated macrophage library, a novel coding sequence, termed
"B7-DC" was identified. The B7-DC gene is a member of the B7 family
of genes encoding costimulatory molecules. B7-DC is the first B7
family member with DC-specific expression and different receptor
specificity. The product of this gene has an important role in
mediating the unique ability of DCs to stimulate T cells.
Functional analysis indicated that B7-DC is more active than B7-1
in stimulating IFN.gamma. production by T cells. B7-DC DNA and
polypeptides are therefore useful in compositions and methods to
enhance the efficacy of cellular and molecular vaccine
compositions, whether antigen-specific or not.
[0029] In one embodiment, the present invention provides an
isolated nucleic acid molecule that encodes a mammalian protein
termed B7-DC that is selectively expressed on dendritic cells as
compared to activated macrophages. The nucleic acid molecule
preferably comprises a nucleotide sequence selected from. SEQ ID
NO:1 (of human origin) or SEQ ID NO:5. (of murine origin). The
invention is also directed to an isolated nucleic acid that
hybridizes with the above nucleic acid molecule under stringent
hybridization conditions. Preferred stringent conditions include
incubation in 6.times. sodium chloride/sodium citrate (SSC) at
about 45.degree. C., followed by a wash in about 0.2.times.SSC at a
temperature of about 50.degree. C. Preferably the above nucleic
acid molecule comprises the nucleotide sequence SEQ ID NO:1. A
preferred nucleic acid molecule as above encodes a protein having
an amino acid sequence selected from SEQ ID NO:2 and SEQ ID NO:4 or
encodes a biologically active fragment, homologue or other
functional derivative of the protein. Preferably, the nucleic acid
molecule encodes the protein having the sequence SEQ ID NO:2 (B7-DC
of human origin) or encodes the biologically active fragment,
homologue or other functional derivative of SEQ ID NO:2.
[0030] In a preferred embodiment, the nucleic acid molecule encodes
the extracellular domain of the B7-DC protein, which includes
residues 26-221, which encodes a co-stimulatory homologue, fragment
or other functional derivative thereof.
[0031] In another embodiment, the above nucleic acid molecule of
encodes a B7-DC fusion protein which comprises: [0032] (a) a first
nucleic acid sequence encoding a first polypeptide that is all or a
part of a B7-DC protein (preferably SEQ 1D NO:2 or SEQ ID NO:4);
[0033] (b) optionally, fused in frame with the first nucleic acid
sequence a linker nucleic acid sequence encoding a linker peptide;
and [0034] (c) a second nucleic acid sequence that is linked in
frame to the first nucleic acid sequence or to the linker nucleic
acid sequence and that encodes a second polypeptide. The second
polypeptide preferably consists of one or more domains of an Ig
heavy chain constant region, preferably the two C domains of human
IgG, preferably IgG1.
[0035] Also provided is an expression vector comprising any of the
above nucleic acid molecules operatively linked to [0036] (a) a
promoter and [0037] (b) optionally, additional regulatory sequences
that regulate expression of the nucleic acid in a eukaryotic
cell.
[0038] The above expression vector may be a plasmid or a viral
vector. These vectors include self replicating RNA replicons
(DNA-launched or RNA), suicide RNA vectors DNA viruses (such as
adenovirus, vaccina virus, etc.) and RNA virions grown on packaging
cell lines.
[0039] The vector DNA or RNA may be complexed to gold particles for
gene gun-mediated introduction to a host or complexed with other
polymers, for example, in controlled release formulations, that
enhance delivery to the desired target cells and tissues.
[0040] Also included is a vector composition which comprises:
[0041] (a) a first recombinant expression vector having
incorporated in its sequence a nucleotide sequence encoding an
antigen of interest against which an immune response is to be
induced; and [0042] (b) a second recombinant expression vector
having incorporated in its nucleic acid sequence or more nucleotide
sequences encoding a co-stimulator polypeptide, at least one of
which polypeptides is B7-DC, or a biologically active fragment,
homologue or other functional derivative thereof, wherein the
expression vectors are able to co-infect or co-transfect a host
cell resulting in co-expression of the antigen and the costimulator
polypeptide, fragment, homologue or derivative.
[0043] In a modification of the above embodiment, the invention
provides a third nucleic sequence encoding a targeting protein that
(i) promotes spread of the expressed product (antigen) between
cells, preferably APCs, (ii) increases the display of the antigen
on APCs in which the nucleic acid is expressed, and/or (iii)
promotes the re-presentation (cross-priming) and display of the
antigen in APCs of a host into which the vector is introduced. The
targeting protein-encoding nucleic acid may be used to the nucleic
acid encoding the antigen or the co-stimulator or both acid the
first or the second vector includes nucleic acid. In one
embodiment, the vector composition combines thee antigen-encoding
nucleic acid, the co-stimulator-encoding nucleic acid (preferably
B7-DC) and a "targeting" protein-encoding nucleic acid into a
single fused construct.
[0044] This invention includes a cell transformed or transfected
with any of the above nucleic acid molecules or expression vectors.
The cell is preferably a eukaryotic cell, more preferably a
mammalian cell, most preferably a human cell. The cell may be a
dendritic cell or a progenitor thereof. In another embodiment, the
cell is a tumor cell, preferably a tumor cell that bears an antigen
that is the same as, or cross-reactive with, an antigen on a tumor
in the host against which an immune response is desired.
[0045] A preferred embodiment is an isolated mammalian tumor cell
transfected with an exogenous nucleic acid molecule encoding a
mammalian B7-DC protein (preferably SEQ ID NO:2 or SEQ ID NO:4) or
a biologically active fragment, homologue or other functional
derivative thereof, such that when the protein, fragment, homologue
or derivative is expressed by the tumor cell, and the tumor cell is
contacted with T cells [0046] (i) the B7-DC protein, fragment),
homologue or derivative binds to the T cells; and [0047] (ii) the
tumor cell costimulates the T cells to proliferate and/or to
produce and secrete cytokines.
[0048] The present invention is also directed to a polypeptide that
is selectively expressed on dendritic cells as compared to
activated macrophages and has the following functional properties:
[0049] (a) binds to a binding partner on T cells; and [0050] (b)
costimulates T cells to proliferate and/or to produce and secrete
cytokines.
[0051] Also included are biologically active fragments, homologues
or other functional derivatives of the polypeptide.
[0052] The polypeptide, fragment, homologues or functional
derivative is preferably encoded by a nucleic acid molecule having
the sequence SEQ ID NO:1 or SEQ ID NO:5, or a fragment, homologue
or equivalent of the nucleic acid molecule. A preferred polypeptide
has the amino acid sequence SEQ ID NO:2 or SEQ NO:4.
[0053] The polypeptide or a biologically active fragment,
homologues or other functional derivative of the polypeptide may be
produced by recombinant expression of one of the above nucleic
acids.
[0054] A preferred polypeptide comprises the extracellular domain
of the B7-DC protein, preferably [0055] (a) amino acid residues
26-221 of SEQ ID NO:2 (human) or [0056] (b) amino acid residues
26-221 of SEQ ID NO:4 (mouse). The above polypeptide may consist
essentially of the extracellular domain of B7-DC
[0057] Also provided is a B7-DC fusion polypeptide having a first
fusion partner comprising all or a part of a B7-DC protein fused
[0058] (i) directly to a second polypeptide or, [0059] (ii)
optionally, fused to a linker peptide sequence that is fused to the
second polypeptide.
[0060] The above A B7-DC fusion protein may also be fused to a
second polypeptide, preferably one or more domains of an Ig heavy
chain constant region, preferably having an amino acid sequence
corresponding to the hinge, C.sub.H2 and C.sub.H3 regions of a
human immunoglobulin C.gamma.1 chain.
[0061] In one embodiment of the above fusion protein, the first
fusion partner is the extracellular domain of a B7-DC protein, the
full length sequence of which is SEQ ID NO:2 or SEQ ID NO:4.
[0062] The fusion protein preferably binds to a binding partner on
T cells and co-stimulates T cells in the presence of an adequate
stimulus to the T cell receptor.
[0063] Also provided is a dimeric or trimeric fusion protein which
is a dimer or trimer of the above fusion proteins. Preferably, the
chains are tandemly linked via disulfide bonds or other interchain
covalent bonds.
[0064] In a preferred dimeric fusion protein, the dimer results
from the covalent bonding of Cys residue in the C.sub.H regions of
two of the Ig heavy chains that are the same Cys residues that are
disulfide linked in dimerized normal Ig H chains.
[0065] The fusion protein of the invention may comprise a multimer
of two or more repeats of the first fusion partner linked end to
end, directly or with a linker sequence between one or more
monomers.
[0066] The present invention also provides an antibody that is
specific for an epitope of a B7-DC protein, which epitope is not
present in a known member of a B7 family protein. The epitope may
be a linear or conformational epitope of a polypeptide of SEQ ID
NO:2 or SEQ ID NO:4. The antibody is preferably a monoclonal
antibody, more preferably a human or humanized (via engineering)
monoclonal antibody.
[0067] Also provided is a method of using the above antibody to
identify or quantitate cells expressing a B7-DC polypeptide on
their surface in a cell population, comprising [0068] (a)
contacting cells of the population with the above antibody so that
the antibody binds to cells expressing the epitope; [0069] (b)
assessing the presence of or quantitating the number of cells to
which the antibody is bound.
[0070] Another method is provided for isolating cells expressing a
B7-DC polypeptide on their surface from a cell population,
comprising [0071] (a) contacting the population with the above
antibody so that the antibody binds to cells expressing the
epitope; [0072] (b) positively selecting cells to which the
antibody has bound or negatively selecting cells to which the
antibody has not bound.
[0073] Also provided is a method of detecting the presence or
quantitating a B7-DC polypeptide, fragment or homologue in a
sample, comprising the steps of: [0074] (a) contacting the sample
with the antibody of claim 43 such that the antibody binds to any
polypeptides or fragments bearing the epitope; [0075] (b) detecting
the presence of or quantitating the polypeptides or fragments bound
to the antibody.
[0076] The present invention is also directed to a method of
inducing or increasing the expression of a B7-DC polypeptide in an
antigen presenting cell or a progenitor thereof to increase the
ability of the cell to co-stimulate T cells in vitro or in vivo in
the presence of an adequate stimulus to the T cell receptor,
comprising transforming or transfecting the antigen presenting cell
or progenitor cell with the expression vector as described above,
such that the expression of the B7-DC polypeptide is induced or
increased on the cells. The antigen presenting cells are preferably
dendritic cells and the progenitors are dendritic cell
progenitors.
[0077] The present invention provides method for stimulating immune
responses using cellular co-stimulatory compositions as well as
polypeptide co-stimulators. One method for increasing the T cell
response of a mammalian subject to antigenic stimulation comprises
administering to the subject an effective amount of cells as
described above, preferably tumor cells, in conjunction with an
antigenic stimulus, wherein the cells are effective to increase the
T cell response of the subject to the antigenic stimulation. The
foregoing is preferably accomplished by co-injection of the antigen
and the co-stimulatory composition.
[0078] A method for increasing the T cell response of a mammalian
subject to antigenic stimulation with a tumor-associated antigen,
comprises administering to the subject an effective amount of tumor
cells as described above, wherein the tumor cells express the
antigen, the administration of the tumor cells being effective to
increase the T cell response of the subject to the tumor antigen
stimulation.
[0079] A method for increasing the T cell response of a mammalian
subject to antigenic stimulation, comprising administering to the
subject an effective amount of a polypeptide, fragment, homologue
or functional derivative as above, or a fusion polypeptide or
protein as above, in conjunction with an antigenic stimulus,
wherein the administration of the polypeptide is effective to
increase the T cell response of the subject to the antigenic
stimulation.
[0080] This invention also provides a method for inhibiting a T
cell response of a mammalian subject to antigenic stimulation,
comprising administering to the subject an effective amount of an
antibody as described, wherein the administration of the antibody
is effective to block stimulation of T cells or to eliminate
antigen-reactive T cells, thereby inhibiting the T cell response.
These methods are particularly useful for treating a subject with a
tissue or organ transplant to inhibit transplant rejection and/or
to promote engraftment. In the case of an autoantigen, the method
blocks or diminishes autoimmune reactions and their pathologic
sequelae.
[0081] The present invention provides therapeutic methods using T
cells that have undergone ex vivo stimulation with the compositions
of this invention. One method for increasing the immune response of
a mammalian subject to antigenic stimulation comprises: [0082] (a)
obtaining T cells from the subject, from an immunologically
compatible donor for said subject, or from an immunologically
acceptable cultured cell line; [0083] (b) contacting the T cells ex
vivo with an effective amount of cells as described above, wherein
the contacting is effective to increase the response of the T cells
to antigenic stimulation; and [0084] (c) administering the T cells
of step (b) to the subject, thereby increasing the immune response
of the subject.
[0085] In another embodiment, the method for increasing the immune
response of a mammalian subject to antigenic stimulation comprises:
[0086] (a) obtaining T cells from obtaining T cells from the
subject, from an immunologically compatible donor for said subject,
or from an immunologically acceptable cultured cell line; [0087]
(b) contacting the T cells ex vivo with an effective amount of (i)
a polypeptide, fragment, homologue or functional derivative as
described above, or (ii) a fusion polypeptide as above, wherein the
contacting is effective to increase the response of the T cells to
antigenic stimulation; and [0088] (c) administering the T cells of
step (b) to the subject, thereby increasing (or generating) an
immune response of the subject.
[0089] Also provided herein is a vaccine composition comprising
[0090] (a) (i) cells as described above that express a B7-DC
construct, (ii) s B7-DC polypeptide, fragment, homologue or
functional derivative, (iii) a B7-DC fusion polypeptide or protein
[0091] (b) generally, an additional source of antigen to which an
immune response is desired--though this may not be required in the
case of the cell-based vaccine wherein the cells themselves
expresses the antigen (as in the case of tumor antigen-bearing
tumor cells); [0092] (c) optionally, a general immunostimulatory
agent or adjuvant; and [0093] (d) a pharmaceutically and
immunologically acceptable excipient or carrier for (a), b) and
(c).
[0094] A method for inducing or enhancing an immune response to an
antigen in a mammalian subject comprises administering to the
subject an effective amount of the above vaccine composition.
[0095] Also provided is a co-stimulatory composition for use with
an antigen or a vaccine, comprising: [0096] (a) a B7-DC polypeptide
(preferably SEQ ID NO:2 or SEQ ID NO:4), a fragment, a homologue or
a functional derivative thereof, or a B7-DC fusion polypeptide, and
[0097] (b) a pharmaceutically and immunologically acceptable
excipient or carrier.
[0098] A method for potentiating an immune response to an antigen
or a vaccine in a mammalian subject, comprises administering to the
subject, in combination with the antigen or vaccine, the above
costimulatory composition.
[0099] A method of stimulating a systemic immune response to a
tumor in a subject, comprises administering to the subject
genetically altered tumor cells which cells [0100] (a) are derived
from a tumor in the subject, and [0101] (b) are genetically altered
by introduction ex vivo of a B7-DC nucleic acid as described above,
the expression of which provides a costimulatory signal in the
subject, wherein the administering results in stimulation of the
systemic immune response directed to the tumor.
[0102] The tumor cells are preferably treated, preferably by
irradiation, to prevent their growth after they have been
administered.
[0103] The subject may be subjected to a tumor-reducing regimen of
chemotherapy, irradiation or surgical resection prior to the
administering of the above therapeutic compositions.
[0104] Also provided is a method of inducing an antitumor response
in a mammal having an antigen-positive tumor, comprising: [0105]
(a) providing cells of the tumor or of a tumor cell line that
[0106] (i) express antigens shared with the tumor of the mammal;
[0107] (ii) are transfected with a B7-DC-encoding nucleic acid
vector as above, that when expressed, s a B7-DC molecule causes the
cells to co-stimulate a T cell response to antigens of the tumor;
[0108] (iii) optionally, are irradiated prior to step (b); [0109]
(b) administering an effective number of the cells to the mammal,
which cells express the antigens and the B7-DC molecule; thereby
inducing an antitumor response
[0110] In the above method, the antitumor response is characterized
by: [0111] (A) at least a 50% decrease in the sum of the products
of maximal perpendicular diameters of all measurable lesions;
[0112] (B) no evidence of new lesions, and [0113] (C) no
progression of any preexisting lesions.
[0114] Also provided is a method of inducing regression or
attenuation of primary growth or regrowth of a tumor in a mammal
bearing the tumor, comprising: [0115] (a) providing cells of the
tumor or of a tumor cell line that [0116] (i) express antigens
shared with the tumor of the mammal; [0117] (ii) are transfected
with a B7-DC-encoding nucleic acid vector as above, that, when
expressed, as a B7-DC molecule causes the cells to co-stimulate a T
cell response to antigens of the tumor; [0118] (iii) optionally,
are irradiated prior to step (b); [0119] (b) administering an
effective number of the cells to the mammal, which cells express
the antigens and the B7-DC molecule; thereby inducing a systemic
immune response specific to the tumor antigens of the melanoma,
thereby inducing the regression or the attenuation
[0120] A method of inhibiting recurrent growth of an
antigen-positive tumor in a mammal, comprises: [0121] (a) providing
cells of the tumor or of a tumor cell line that [0122] (i) express
antigens shared with the tumor of the mammal; [0123] (ii) are
transfected with a B7-DC-encoding nucleic acid vector as above,
that, when expressed, causes the cells to co-stimulate a T cell
response to antigens of the tumor; [0124] (iii) optionally, are
irradiated prior to step (b); [0125] (b) administering an effective
number of the cells to the mammal, which cells express the antigens
and the B7-DC molecule; thereby inducing a systemic immune response
specific to the tumor antigens in the mammal, which immune response
inhibits the recurrent growth of the tumor.
[0126] Another embodiment is directed to a method of providing a
co-stimulatory signal in the vicinity of locally-administered
antigen in a mammalian subject to promote the local generation of
an inflammatory and immune response that results in a state of
systemic immunity to the antigen, the method comprising
administering to a local site in the subject [0127] (a) cells that
express a costimulation-effective amount of B7-DC polypeptide,
fragment, homologue or functional derivative as above, and [0128]
(b) the antigen such that costimulation in physical proximity with
the antigen promotes the local generation of the response and
results in the state of systemic immunity.
[0129] In the above method, the antigen is preferably a tumor
antigen that is administered in (b) in the form of tumor cells or
subcellular antigenic material. The tumor cells may also be the
cells that express the B7-DC polypeptide, fragment, homologue or
derivative in (a).
BRIEF DESCRIPTION OF THE DRAWINGS
[0130] FIG. 1 is a diagram showing shows the map of hB7-DC which is
localized on human chromosome 9p24. hB7-DC maps to BAC clone
RPCI-11.2.
[0131] FIG. 2 shows that B7-DC is differentially expressed between
DCs and macrophages. Distribution of B7-DC mRNA in bone marrow DCs,
splenic DCs, macrophages, macrophage lines and tissues was assessed
by virtual Northern blot analysis using 0.5 .mu.g/lane purified DNA
run on a 1% agarose gel. G3PDH was used as control. J774A1,
Raw264.7, Pu5-1.8 and WEHI cells are macrophage cell lines. BM:
bone marrow.
[0132] FIG. 3 shows a virtual Northern blot of B7-DC expression on
human DCs. Lane 1 shows human DCs cultured with GM-CSF+Flt-3L, lane
2 shows human placenta and lane 3 shows human DCs cultured with
GM-CSF+IL4. Oligonucleotides from the 5' and 3' UTR of human B7-DC
were used to make PCR DNA probe for virtual Northern analysis of
total RNA of human DCs. .beta.-actin was used as control to ensure
the quality of mRNA.
[0133] FIG. 4 represents is a flow cytometric analysis showing
surface expression of B7-DC on mature BM-DCs. Day 9 murine BM-DCs
were Fc-blocked and stained with control antibody or B7-DC
antisera. Specificity of binding was demonstrated by adding
B7-DC-Ig to compete for the binding of anti-B7-DC to the surface of
DCs.
[0134] FIG. 5 shows the binding of B7-DC to PD-1 but not CTLA-4 or
CD28. 293T cells were transiently transfected with pCAGGS-B7.1 o
pCAGGS-B7-DC. Transfectants were stained with PD-1-Ig, 28-Ig and
CTLA-4-Ig fusion molecules followed by PE-labeled secondary
antibody. Staining of pCAGGS (empty vector) transfectants was
negative (not shown)
[0135] FIG. 6 (left and right panel) shows the costimulation of T
cell proliferation by anti-CD3 and B7-DC-Ig. Left graph: purified T
cells (CD4+CD8) were cultured in wells pre-coated with increasing
concentrations of anti-CD3 (mAb 2C11) and a fixed concentration
(0.1 .mu.g/ml) of immobilized B7.1-Ig (.diamond-solid.), B7-DC-Ig (
) or isotype control (.tangle-solidup.). Results depict one
representative experiment of three. Cells were incubated for 72 h
and labeled with .sup.3H-thymidine. CPM, counts per minute. Right
Graph: purified CD8 T cells were cultured in wells pre-coated with
increasing concentrations of anti-CD3 and fixed concentration of
immobilized B7.1-Ig (.diamond-solid.), B7-DC-Ig ( ) or isotype
control (.tangle-solidup.) as in (a). Results are of one
representative experiment of two. Cells were incubated for 72 h and
labeled with .sup.3H-thymidine. CPM, counts per minute.
[0136] FIG. 7 shows the costimulation of antigen-specific T cell
proliferative responses RENCA cells were treated with IFN.gamma.
for 72 hrs to induce MHC class II expression and incubated with
12.5 .mu.g/ml of HA110-120 peptide. Purified HA+I-E.sup.d specific
transgenic T cells were added together with increasing
concentrations of either B7.1-Ig (.diamond-solid.), B7-DC-Ig ( ) or
Isotype control (.tangle-solidup.) in soluble form. Cells were
incubated for 48 h and labeled with .sup.3H-thymidine. CPM, counts
per minute. Results are one representative experiment of three.
[0137] FIG. 8 shows cytokine secretion of T cells costimulated by
B7-DC. Upper panels: purified T cells were cultured in wells
pre-coated with anti-CD3 (0.12 .mu.g/ml) and 0.1 .mu.g/ml of
immobilized B7.1-Ig (.diamond-solid.), B7-DC-Ig ( ) or isotype
control (.tangle-solidup.) as in FIG. 6 (left). Results depict one
representative experiment of three. Lower panels: .gamma.-IFN
treated RENCA cells loaded with 12.5 .mu.g/ml HA (110-120) peptide
were incubated with purified HA+I-E.sup.d specific transgenic T
cells together with 2 .mu.g/ml of soluble B7.1-Ig, B7-DC-Ig or
isotype control (symbols as above). Results depict one
representative experiment of two. Supernatants were collected after
24 h and 48 h culture and assayed for the indicated lymphokines
using ELISA.
[0138] FIG. 9 shows that B7-DC-Ig greatly enhances antigen specific
proliferation after in vivo co-stimulation. After adoptive transfer
of 2.5.times.10.sup.6 TCR transgenic cells specific for HA, three
groups of mice were immunized s.c., in their hind footpads with
either HA peptide (110-120), incomplete Freund's adjuvant (IFA)
alone or in combination with either B7-DC-Ig+IFA or an isotype
control antibody with IFA. Draining lymph nodes were harvested on
day 7. 1.5.times.10.sup.5 LN cells were incubated with the HA
peptide for 48 h, pulsed with 1 .mu.Ci[.sup.3H]thymidine and the
radioactivity incorporated after 12 h was determined.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0139] The present inventors have now identified new proteins and
nucleic acids that serve as the basis for improved
immunotherapeutic compositions and methods. Human and murine forms
of a novel costimulatory protein named B7-DC have been discovered
and are disclosed herein.
[0140] DNA encoding human B7-DC has the nucleotide sequence SEQ ID
NO:1, show below.
TABLE-US-00001 1
atgatcttcctcctgctaatgttgagcctggaattgcagcttcaccagatagcagcttta 61
ttcacagtgacagtccctaaggaactgtacataatagagcatggcagcaatgtgaccctg 121
gaatgcaactttgacactggaagtcatgtgaaccttggagcaataacagccagtttgcaa 181
aaggtggaaaatgatacatccccacaccgtgaaagagccactttgctggaggagcagctg 241
cccctagggaaggcctcgttccacatacctcaagtccaagtgagggacgaaggacagtac 301
caatgcataatcatctatggggtcgcctgggactacaagtacctgactctgaaagtcaaa 361
gcttcctacaggaaaataaacactcacatcctaaaggttccagaaacagatgaggtagag 421
ctcacctgccaggctacaggttatcctctggcagaagtatcctggccaaacgtcagcgtt 481
cctgccaacaccagccactccaggacccctgaaggcctctaccaggtcaccagtgttctg 541
cgcctaaagccaccccctggcagaaacttcagctgtgtgttctggaatactcacgtgagg 601
gaacttactttggccagcattgaccttcaaagtcagatggaacccaggacccatccaact 661
tggctgcttcacattttcatcccctcctgcatcattgctttcattttcatagccacagtg 721
atagccctaagaaaacaactctgtcaaaagctgtattcttcaaaagacacaacaaaaaga 781
cctgtcaccacaacaaagagggaagtgaacagtgctatc 819
[0141] The human B7-DC protein has the amino acid sequence SEQ ID
NO:2, shown below (with leader sequence, transmembrane domain and
cytoplasmic tail annotated):
TABLE-US-00002 putative leader sequence 1 MIFLLLMLSL ELQLHQIAAL
FTVTVPKELY IIEHGSNVTL ECNFDTGSHV 50 51 NLGAITASLQ KVENDTSPHR
ERATLLEEQL PLGKASFHIP QVQVRDEGQY 100 101 QCIIIYGVAW DYKYLTLKVK
ASYRKINTHI LKVPETDEVE LTCQATGYPL 150 151 AEVSWPNVSV PANTSHSRTP
EGLYQVTSVL RLKPPPGRNF SCVFWNTHVR 200 putative TM domain 201
ELTLASIDLQ SQMEPRTHPT W LLHIFIPSC IIAFIFIATV IALRKQLCQKL 250 251
LYSSKDTTKR PVTTTKREVN SAI 273 cytoplasmic tail
The extracellular domain of this protein is from residue P.sup.26
through residue W.sup.221.
[0142] A DNA clone that includes the coding sequence encoding
murine B7-DC has the nucleotide sequence SEQ ID NO:3, show below.
The coding sequence (underscored, set off in triplets) begins from
the methionine codon atg (bolded) beginning at nucleotide 210 and
terminates with the tag stop codon (bolded) (nucleotides
951-953)
TABLE-US-00003
gaattcggcacgaggtcaaatgtggcatatctttgttgtctccttctgtctcccaactag 60
agagaacacacttacggctcctgtcccgggcaggtttggttgtcggtgtgattggcttcc 120
agggaacctgatacaaggagcaactgtgtgctgccttttctgtgtctttgcttgaggagc 180
tgtgctgggtgctgatattgacacagacc 209 atg ctg ctc ctg ctg ccg ata ctg
aac ctg agc tta caa ctt cat cct 257 gta gca gct tta ttc acc gtg aca
gcc cct aaa gaa gtg tac acc gta 305 gac gtc ggc agc agt gtg agc ctg
gag tgc gat ttt gac cgc aga gaa 353 tgc act gaa ctg gaa ggg ata aga
gcc agt ttg cag aag gta gaa aat 401 gat acg tct ctg caa agt gaa aga
gcc acc ctg ctg gag gag cag ctg 449 ccc ctg gga aag gct ttg ttc cac
atc cct agt gtc caa gtg aga gat 497 tcc ggg cag tac cgt tgc ctg gtc
atc tgc ggg gcc gcc tgg gac tac 545 aag tac ctg acg gtg aaa gtc aaa
gct tct tac atg agg ata gac act 593 agg atc ctg gag gtt cca ggt aca
ggg gag gtg cag ctt acc tgc cag 641 gct aga ggt tat ccc cta gca gaa
gtg tcc tgg caa aat gtc agt gtt 689 cct gcc aac acc agc cac atc agg
acc ccc gaa ggc ctc tac cag gtc 737 acc agt gtt ctg cgc ctc aag cct
cag cct agc aga aac ttc agc tgc 785 atg ttc tgg aat gct cac atg aag
gag ctg act tca gcc atc att gac 833 cct ctg agt cgg atg gaa ccc aaa
gtc ccc aga acg tgg cca ctt cat 881 gtt ttc atc ccg gcc tgc acc atc
gct ttg atc ttc ctg gcc ata gtg 929 ata atc cag aga aag agg atc tag
953 gggaagctgtattacggaagaagtggtctcttcttcccagatctggacctgcggtcttgg
1013 gagttggaaggatctgatgggaaaccctcaagagacttctggactcaaagtgagaatctt
1073 gcaggacctgccatttgcacttttgaaccctttggacggtgacccagggctccgaagagg
1133 agcttgtaagactgacaatcttccctctgtctcaagactctctgaacagcaagaccccaa
1193 tggcactttagacttacccctgggatcctggaccccagtgagggcctaaggctcctaatg
1253 actttcagggtgagaacaaaaggaattgctctccgccccacccccacctcctgctttccg
1313 cagggagacatggaaattcccagttactaaaatagattgtcaatagagttatttatagcc
1373 ctcatttcctccggggacttggaagcttcagacagggtttttcataaacaaagtcataac
1433 tgatgtgttttacagcatcctagaatcctggcagcctctgaagttctaattaactggaag
1493 catttaagcaacacgtcaagtgcccctgctgtggtatttgcttctacttttctgttttta
1553 aagtgtgagtcacaaggtaattgttgtaacctgtgatatcactgtttcttgtgtctcttc
1613 tttcaactacatcttttaaaacaaaaaaaaaaaaaaaaaaaa 1655
[0143] SEQ ID NO:5 is the coding sequence part of SEQ ID NO:3.
[0144] The murine B7-DC protein, encoded by the coding region of
SEQ ID NO:3, (i.e., by SEQ ID NO:5) has the amino acid sequence SEQ
NO:4 shown below (with leader sequence, transmembrane domain and
cytoplasmic tail annotated):
TABLE-US-00004 putative leader sequence 1 MLLLLPILNL SLQLHPVAAL
FTVTAPKEVY TVDVGSSVSL ECDFDRRECT 50 51 ELEGIRASLQ KVENDTSLQS
ERATLLEEQL PLGKALFHIP EVQVRDSGQY 100 101 RCLVICGAAW DYKYLTVKVK
ASYMRIDTRI LEVPGTGEVQ LTCQARGYPL 150 151 AEVSWQNVSV PANTSHIRTP
EGLYQVTSVL RLKPQPSRNF SCMFWNAHMK 200 putative TM domain 201
ELTSAIIDPL DRMEPKVPRT W PLHVFIPAC TIALIFLAIV IIQRKRI 247 cyto.
tail
The extracellular domain of this protein is from residue P.sup.26
through residue W.sup.221
[0145] The complete DNA sequence of murine B7-DC (originally termed
"butyrophilin-like protein" or "Btdc") has the Genbank accession
number AF142780.2
Basic Molecular Approach
[0146] This approach is described in more detail in the Examples.
The inventors utilized the PCR Select approach which incorporate
two important features. First, the initial PCR reaction prior to
the hybridization steps requires only small quantity of RNA. This
technique allows the use of highly purified mature DCs that have
been rendered substantially free of contaminating macrophages,
progenitor cells or other potential contaminating cells. Such
highly purified DCs are known to be difficult to obtain in very
large numbers. Second, the PCR Select procedure enabled cloning of
low copy number, differentially expressed genes.
[0147] To identify genes differentially expressed by DCs relative
to their cellular counterpart, the activated macrophage, and to
identify genes associated with DC-specific functions, the present
inventors applied a cDNA subtraction approach. They used a modified
PCR-based "representative differential analysis" (RDA) combined
with suppression PCR (PCR Select.TM.) (Diatchenko, L. et al., Proc.
Natl. Acad. Sci USA 93:66025-6030 (1996)).
General Recombinant DNA Methods
[0148] Basic texts disclosing general methods of molecular biology,
all of which are incorporated by reference, include: Sambrook, J et
al, Molecular Cloning: A Laboratory Manual, 2.sup.nd Edition, Cold
Spring Harbor Press, Cold Spring Harbor, N.Y., 1989; Ausubel, F M
et al. Current Protocols in Molecular Biology, Vol. 2,
Wiley-Interscience, New York, (current edition); Kriegler, Gene
Transfer and Expression: A Laboratory Manual (1990); Glover, D M,
ed, DNA Cloning: A Practical Approach, vol. I & II, IRL Press,
1985; Albers, B. et al., Molecular Biology of the Cell, 2.sup.nd
Ed., Garland Publishing, Inc., New York, N.Y. (1989); Watson, J D
et al., Recombinant DNA, 2.sup.nd Ed., Scientific American Books,
New York, 1992; and Old, R W et al., Principles of Gene
Manipulation: An Introduction to Genetic Engineering, 2.sup.nd Ed.,
University of California Press, Berkeley, Calif. (1981).
[0149] Unless otherwise indicated, a particular nucleic acid
sequence is intended to encompasses conservative substitution
variants thereof (e.g., degenerate codon substitutions) and a
complementary sequence. The term "nucleic acid" is synonymous with
"polynucleotide" and is intended to include a gene, a cDNA
molecule, an mRNA molecule, as well as a fragment of any of these
such as an oligonucleotide, and further, equivalents thereof
(explained more fully below). Sizes of nucleic acids are stated
either as kilobases (kb) or base pairs (bp). These are estimates
derived from agarose or polyacrylamide gel electrophoresis (PAGE),
from nucleic acid sequences which are determined by the user or
published. Protein size is stated as molecular mass in kilodaltons
(kDa) or as length (number of amino acid residues). Protein size is
estimated from PAGE, from sequencing, from presumptive amino acid
sequences based on the coding nucleic acid sequence or from
published amino acid sequences.
[0150] Specifically, cDNA molecules encoding the amino acid
sequence corresponding to B7-DC or fragments or derivatives thereof
can be synthesized by the polymerase chain reaction (PCR) (see, for
example, U.S. Pat. No. 4,683,202) using primers derived the
sequence of the protein disclosed herein. These cDNA sequences can
then be assembled into a eukaryotic or prokaryotic expression
vector and the resulting vector can be used to direct the synthesis
of B7-DC, or its fragment or derivative by appropriate host cells,
for example COS or CHO cells.
[0151] This invention includes isolated nucleic acids having a
nucleotide sequence encoding the novel B7-DC, fragments thereof or
equivalents thereof. The term nucleic acid as used herein is
intended to include such fragments or equivalents. The nucleic acid
sequences of this invention can be DNA or RNA. A preferred nucleic
acid is cDNA encoding human B7-DC having the sequence SEQ ID NO:1
or equivalents thereof.
[0152] Preferably, the nucleic acid of the present invention is a
cDNA molecule encoding at least a portion of B7-DC. This cDNA can
be made from mRNA extracted from mature DCs or other cells
naturally expressing this protein. A nucleic acid sequence encoding
B7-DC is obtainable from DC genomic DNA. Thus, DNA encoding B7-DC
can be cloned from a cDNA or a genomic library in accordance with
known protocols.
[0153] A cDNA nucleotide sequence encoding B7-DC can be obtained by
isolating total mRNA from an appropriate cell line. Double stranded
cDNA is prepared from total mRNA. cDNA can be inserted into a
suitable plasmid, bacteriophage or viral vector using any one of a
number of known techniques.
[0154] In reference to a nucleotide sequence, the term "equivalent"
is intended to include sequences encoding structurally homologous
and/or a functionally equivalent proteins. For example, a natural
polymorphism of the B7-DC nucleotide sequence (especially at the
third base of a codon) may be manifest as "silent" mutations which
do not change the amino acid sequence. However, polymorphisms that
involve amino acid sequence changes in B7-DC may exist in a human
(or other mammalian) population. Those of skill in the art will
appreciate that these allelic variants that have changes in one or
more nucleotides (up to about 3-4% of the total coding sequence)
will likely be found in a human population due to natural allelic
variation. Any and all such allelic variations and resulting
nucleic acid and polypeptide polymorphisms in the DNA encoding
B7-DC are within the scope of the invention.
[0155] Furthermore, there may be one or more naturally occurring
isoforms or related, immunologically cross-reactive family members
of the B7-DC protein described herein. Such isoforms or family
members are defined as proteins that share function amino acid
sequence similarity to B7-DC, even if they are encoded by genes at
different loci.
[0156] Fragment of Nucleic Acid
[0157] A fragment of the nucleic acid sequence is defined as a
nucleotide sequence having fewer nucleotides than the nucleotide
sequence encoding the full length B7-DC protein. This invention
includes such nucleic acid fragments that encode polypeptides which
retain (1) the ability of B7-DC to bind to its natural ligand(s) on
T cells and (2) to enhance or inhibit (depending on how they are
presented) activated T cell mediated immune responses (measured as
cytokine production and/or T cell proliferation by T cells that
have received a primary activation signal).
[0158] For example, a nucleic acid fragment as intended herein
encodes a B7-DC polypeptide that retains the ability to bind to the
surface of T cells to a receptor that has not yet been identified
(but is does not appear to be CD28 or CTLA-4) and deliver a
costimulatory signal to T lymphocytes. By another criterion, the
present nucleic acid fragment is one that hybridizes with a nucleic
acid from another animal species and is therefore useful in
screening assays to detect novel proteins that are "cross-reactive"
with B7-DC.
[0159] Generally, the nucleic acid sequence encoding a fragment of
the B7-DC polypeptide comprises of nucleotides from the sequence
encoding the mature protein. However, in some instances it may be
desirable to include all or part of the leader sequence portion of
the nucleic acid. Nucleic acid sequences of this invention may also
include linker sequences, natural or modified restriction
endonuclease sites and other sequences that are useful for
manipulations related to cloning, expression or purification of
encoded protein or fragments. These and other modifications of
nucleic acid sequences are described herein or are well-known in
the art.
[0160] In one embodiment, DNA encoding the amino acid sequence
corresponding to the ECD of B7-DC, containing amino acids from
about position 26-221, is joined to DNA encoding the amino acid
sequences corresponding to the hinge, C.sub.H2 and C.sub.H3 regions
of human Ig C.gamma.1, using PCR, to form a construct that is
expressed as B7-DC-Ig fusion protein.
[0161] An analogous DNA molecule encoding a B7-Ig fusion protein
was disclosed in U.S. Pat. No. 5,521,288 and deposited with the
American Type Culture Collection in Rockville, Md., under accession
number 68627.
[0162] The techniques for assembling and expressing DNA encoding
B7-DC and soluble B7-DC fusion proteins such as synthesis of
oligonucleotides, PCR, transforming cells, constructing vectors,
expression systems, and the like are well-established in the art.
Those of ordinary skill are familiar with the standard resource
materials for specific conditions and procedures.
[0163] In other embodiments, the DNA encoding a domain or fragment
of B7-DC is fused with a nucleic acid encoding most or all of the
remaining portion of another B7 family protein, sch as B7.1, B7.2
or B7.3. The complete DNA sequence of human B7.1 (CD80) has the
Genbank accession number X60958; the accession number for the mouse
sequence is X60958; the accession number for the rat sequence is
U05593. The complete cDNA sequence of human B7.2 (CD86) has the
Genbank accession number L25259; the accession number for the mouse
sequence is L25606.
Expression Vectors and Host Cells
[0164] This invention includes an expression vector comprising a
nucleic acid sequence encoding a B7-DC polypeptide operably linked
to at least one regulatory sequence. "Operably linked" means that
the coding sequence is linked to a regulatory sequence in a manner
that allows expression of the coding sequence. Known regulatory
sequences are selected to direct expression of the desired protein
in an appropriate host cell. Accordingly, the term "regulatory
sequence" includes promoters, enhancers and other expression
control elements. Such regulatory sequences are described in, for
example, Goeddel, Gene Expression Technology. Methods in
Enzymology, vol. 185, Academic Press, San Diego, Calif.
(1990)).
[0165] Those skilled in the art appreciate that the particular
design of an expression vector of this invention depends on
considerations such as the host cell to be transfected and/or the
type of protein to be expressed.
[0166] The present expression vectors comprise the full range of
nucleic acid molecules encoding the various embodiments of B7-DC:
full length protein and its functional derivatives (defined herein)
including polypeptide fragments, variants, fusion proteins, etc.
Thus, in one embodiment, the expression vector comprises a nucleic
acid encoding at least a portion of the B7-DC protein such as the
ECD, alone or fused to another protein.
[0167] Such expression vectors are used to transfect host cells for
expression of the DNA and production of the encoded proteins which
include fusion proteins or peptides. It will be understood that a
genetically modified cell expressing the B7-DC polypeptide may
transiently express the exogenous DNA for a time sufficient for the
cell to be useful for its stated purpose. Thus, if the cell is to
serve as an immunogen having an augmented costimulatory capacity in
vivo or ex vivo, the length of time that expression is required, or
that the cell remain alive, is the time necessary for the cell to
exert its immunogenic and/or costimulatory function. For example,
in the case of a transduced tumor cell expressing the B7-DC of the
present invention, expression of B7-DC may be for as little as 6
hours, preferably 24 hours, more preferably for at least 2-4 days.
Of course, expression may also be stable (i.e., for the life of the
cell). Appropriate expression vectors and regulatory elements
(e.g., (e.g., inducible or constitutive promoters) discussed below
are selected in accordance with the desired or required stability
of expression.
[0168] The present in invention provides methods for producing the
B7-DC protein, fragments and derivatives. For example, a host cell
transfected with a nucleic acid vector that encodes at least a
portion of the B7-DC protein is cultured under appropriate
conditions to allow expression of B7-DC polypeptide.
[0169] Host cells may also be transfected with one or more
expression vectors that singly or in combination comprise DNA
encoding at least a portion of the B7-DC protein and DNA encoding
at least a portion of a second protein, so that the host cells
produce fusion polypeptides that include both the portions.
[0170] When the recombinant expression vector comprises DNA
encoding a portion of B7-DC and DNA encoding another protein, such
as human. IgC.gamma.1, the resulting fusion protein may have
altered solubility, binding amity and/or valency. A B7-DC Ig fusion
protein, for example, is preferably secreted by transfected host
cells in cultures and is therefor isolated from the culture medium.
Alternatively, if protein is retained in the cytoplasm, the cells
are harvested and lysed and the protein isolated from this
lysate.
[0171] A culture typically includes host cells, appropriate growth
media and other byproducts. Suitable culture media are well known
in the art. B7-DC protein can be isolated from medium or cell
lysates using conventional techniques for purifying proteins and
peptides, including ammonium sulfate precipitation, fractionation
column chromatography (e.g. ion exchange, gel filtration, affinity
chromatography, etc.) and/or electrophoresis (see generally,
"Enzyme Purification and Related Techniques", Methods in
Enzymology, 22: 233-577 (1971)). Once purified, partially or to
homogeneity, the recombinant B7-DC proteins of the invention can be
utilized in pharmaceutical compositions as described in more detail
herein.
[0172] Prokaryotic or eukaryotic host cells transformed or
transfected to express B7-DC or a homologue or functional
derivative thereof are within the scope of the invention. For
example, B7-DC may be expressed in bacterial cells such as E. coli,
insect cells (baculovirus), yeast, or mammalian cells such as
Chinese hamster ovary cells (CHO) or human cells. Other suitable
host cells may be found in Goeddel, (1990) supra or are otherwise
known to those skilled in the art.
[0173] Expression in eukaryotic cells leads to partial or complete
glycosylation and/or formation of relevant inter- or intra-chain
disulfide bonds of the recombinant protein.
[0174] Examples of vectors for expression in yeast S. cerevisiae
include pYepSec1 (Baldari et al., (1987) EMBO J. 6:229-234), pMFa
(Kurjan et al. (1982) Cell 30:933-943), pJRY88 (Schultz et al.,
(1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San
Diego, Calif.). Baculovirus vectors available for expression of
proteins in cultured insect cells (SF 9 cells) include the pAc
series (Smith et al., (1983) Mol. Cell Biol. 3: 2156-2165,) and the
pVL series (Lucklow, V. A., and Summers, M. D., (1989) Virology
170: 31-39). Generally, COS cells (Gluzman, Y., (1981) Cell 23:
175-182) are used in conjunction with such vectors as pCDM 8
(Aruffo A. and Seed, B., supra, for transient
amplification/expression in mammalian cells, while CHO
(dhfr-negative CHO) cells are used with vectors such as pMT2PC
(Kaufman et al. (1987), EMBO J. 6: 187-195) for stable
amplification/expression in mammalian cells. The NS0 myeloma cell
line (a glutamine synthetase expression system.) is available from
Celltech Ltd.
[0175] Often, in fusion expression vectors, a proteolytic cleavage
site is introduced at the junction of the reporter group and the
target protein to enable separation of the target protein from the
reporter group subsequent to purification of the fusion protein.
Proteolytic enzymes for such cleavage and their recognition
sequences include Factor Xa, thrombin and enterokinase.
[0176] Typical fusion expression vectors include pGEX (Amrad Corp.,
Melbourne, Australia), pMAL (New England Biolabs, Beverly, Mass.)
and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione
S-transferase, maltose E binding protein, or protein A,
respectively, to the target recombinant protein.
[0177] Inducible non-fusion expression vectors include pTro (Amann
et al., (1988) Gene 69: 301-315) and pET 11d (Studier et al., Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990) 60-89). While target gene expression
relies on host RNA polymerase transcription from the hybrid trp-lac
fusion promoter in pTrc, expression of target genes inserted into
pET 11d relies on transcription from the T7 gn10-lacO fusion
promoter mediated by coexpressed viral RNA polymerase (T7gn1). This
viral polymerase is supplied by host strains BL21(DE3) or
HMS174(DE3) from a resident .lamda. prophage harboring a T7gn1
under the transcriptional control of the lacUV 5 promoter.
[0178] One embodiment of this invention is a transfected cell which
expresses novel B7-DC de novo. In the case of a cell already
expressing B7-DC, such as a mature DC, the transfected cell
expresses increased amounts of B7-DC proteins or fragments thereof
on the cell surface.
[0179] For example, a tumor cell such as a sarcoma, melanoma,
leukemia, lymphoma, carcinoma or neuroblastoma is transfected with
an expression vector directing the expression of B7-DC on the tumor
cell surface. Such transfected tumor cells can be used as
immunogens to induce therapeutic antitumor immunity as described
herein.
Vector Construction
[0180] Construction of suitable vectors containing the desired
coding and control sequences employs standard ligation and
restriction techniques which are well understood in the art.
Isolated plasmids, DNA sequences, or synthesized oligonucleotides
are cleaved, tailored, and re-ligated in the form desired.
[0181] The DNA sequences which form the vectors are available from
a number of sources. Backbone vectors and control systems are
generally found on available "host" vectors which are used for the
bulk of the sequences in construction. For the pertinent coding
sequence, initial construction may be, and usually is, a matter of
retrieving the appropriate sequences from cDNA or genomic DNA
libraries. However, once the sequence is disclosed it is possible
to synthesize the entire gene sequence in vitro starting from the
individual nucleotide derivatives. The entire gene sequence for
genes of sizeable length, e.g., 500-1000 bp may be prepared by
synthesizing individual overlapping complementary oligonucleotides
and filling in single stranded nonoverlapping portions using DNA
polymerase in the presence of the deoxyribonucleotide
triphosphates. This approach has been used successfully in the
construction of several genes of known sequence. See, for example,
Edge, M. D., Nature (1981) 292:756; Nambair, K. P., et al., Science
(1984) 223:1299; and Jay, E., J Biol Chem (1984) 259:6311.
[0182] Synthetic oligonucleotides are prepared by either the
phosphotriester method as described by references cited above or
the phosphoramidite method as described by Beaucage, S. L., and
Caruthers, M. H., Tet Lett (1981) 22:1859; and Matteucci, M. D.,
and Caruthers, M. H., J Am Chem Soc (1981) 103:3185 and can be
prepared using commercially available automated oligonucleotide
synthesizers. Kinase treatment of single strands prior to annealing
or for labeling is achieved using an excess, e.g., about 10 units
of polynucleotide kinase to 1 nmole substrate in the presence of 50
mM Tris, pH 7.6, 10 mM MgCl.sub.2, 5 mM dithiothreitol, 1-2 mM ATP,
1.7 pmoles .gamma.-.sup.32P-ATP (2.9 mCi/mmole), 0.1 mM spermidine,
0.1 mM EDTA.
[0183] Once the components of the desired vectors are thus
available, they can be excised and ligated using standard
restriction and ligation procedures. Site-specific DNA cleavage is
performed by treating with the suitable restriction enzyme (or
enzymes) under conditions which are generally understood in the
art, and the particulars of which are specified by the manufacturer
of these commercially available restriction enzymes. See, e.g., New
England Biolabs, Product Catalog. In general, about 1 mg of plasmid
or DNA sequence is cleaved by one unit of enzyme in about 20 ml of
buffer solution; in the examples herein, typically, an excess of
restriction enzyme is used to insure complete digestion of the DNA
substrate. Incubation times of about one hour to two hours at about
37.degree. C. are workable, although variations can be tolerated.
After each incubation, protein is removed by extraction with
phenol/chloroform, and may be followed by ether extraction, and the
nucleic acid recovered from aqueous fractions by precipitation with
ethanol. If desired, size separation of the cleaved fragments may
be performed by polyacrylamide gel or agarose gel electrophoresis
using standard techniques. A general description of size
separations is found in Methods in Enzymology (1980)
65:499-560.
[0184] Restriction cleaved fragments may be blunt ended by treating
with the large fragment of E. coli DNA polymerase I (Klenow) in the
presence of the four deoxynucleotide triphosphates (dNTPs) using
incubation times of about 15 to 25 min at 20.degree. to 25.degree.
C. in 50 mM Tris pH 7.6, 50 mM NaCl, 6 mM MgCl.sub.2, 6 mM. DTT and
0.1-1.0 mM dNTPs. The Klenow fragment fills in at 5'
single-stranded overhangs but chews back protruding 3' single
strands, even though the four dNTPs are present. If desired,
selective repair can be performed by supplying only one of the, or
selected, dNTPs within the limitations dictated by the nature of
the overhang. After treatment with Klenow, the mixture is extracted
with phenol/chloroform and ethanol precipitated. Treatment under
appropriate conditions with S1 nuclease or BAL-31 results in
hydrolysis of any single-stranded portion.
[0185] Ligations are typically performed in 15-50 ml volumes under
the following standard conditions and temperatures: for example, 20
mM Tris-HCl pH 7.5, 10 mM MgCl.sub.2, 10 mM DTT, 33 .mu.g/mi BSA,
10-50 mM NaCl, and either 40 .mu.M ATP, 0.01-0.02 (Weiss) units T4
DNA ligase at 0.degree. C. (for "sticky end" ligation) or 1 mM ATP,
0.3-0.6 (Weiss) units T4 DNA ligase at 14.degree. C. (for "blunt
end" ligation). Intermolecular "sticky end" ligations are usually
performed at 33-100 .mu.g/ml total DNA concentrations (5-100 nM
total end concentration). Intermolecular blunt end ligations are
performed at 1 mM total ends concentration.
[0186] In vector construction employing "vector fragments", the
fragment is commonly treated with bacterial alkaline phosphatase
(BAP) or calf intestinal alkaline phosphatase (CIAP) in order to
remove the 5' phosphate and prevent self-ligation. Digestions are
conducted at pH 8 in approximately 10 mM Tris-HCl, 1 mM EDTA using
BAP or CIAP at about 1 unit/mg vector at 60.degree. for about one
hour. The preparation is extracted with phenol/chloroform and
ethanol precipitated. Alternatively, re-ligation can be prevented
in vectors which have been double digested by additional
restriction enzyme and separation of the unwanted fragments.
[0187] Any of a number of methods are used to introduce mutations
into the coding sequence to generate the variants of the invention.
These mutations include simple deletions or insertions, systematic
deletions, insertions or substitutions of clusters of bases or
substitutions of single bases.
[0188] For example, modifications of B7-DC DNA sequence (cDNA or
genomic DNA) are created by site-directed mutagenesis, a well-known
technique for which protocols and reagents are commercially
available (Zoller, M J et al., Nucleic Acids Res (1982)
10:6487-6500 and Adelman, J P et al., DNA (1983) 2:183-193)).
Correct ligations for plasmid construction are confirmed, for
example, by first transforming E. coli strain MC1061 (Casadaban,
M., et al., J Mol Biol (1980) 138:179-207) or other suitable host
with the ligation mixture. Using conventional methods,
transformants are selected based on the presence of the
ampicillin-, tetracyclines or other antibiotic resistance gene (or
other selectable marker) depending on the mode of plasmid
construction. Plasmids are then prepared from the transformants
with optional chloramphenicol amplification optionally following
chloramphenicol amplification ((Clewell, D B et al., Proc Natl Acad
Sci USA (1969) 62:1159; Clewell, D. B., J Bacterial (1972)
110:667). Several mini DNA preps are commonly used. See, e.g.,
Holmes, D S, et al., Anal Biochem (1981) 114:193-197; Bimboim, H C
et al., Nucleic Acids Res (1979) 7:1513-1523. The isolated DNA is
analyzed by restriction and/or sequenced by the dideoxy nucleotide
method of Sanger (Proc Natl Acad Sci USA (1977) 74:5463) as further
described by Messing, et al., Nucleic Acids Res (1981) 9:309, or by
the method of Maxam et al. Methods in Enzymology (1980) 65:499.
[0189] Vector DNA can be introduced into mammalian cells via
conventional techniques such as calcium phosphate or calcium
chloride co-precipitation, DEAE-dextran-mediated transfection,
lipofection, or electroporation. Suitable methods for transforming
host cells can be found in Sambrook et al. supra and other standard
texts.
[0190] Often, in fusion expression vectors, a proteolytic cleavage
site is introduced at the junction of the reporter group and the
target protein to enable separation of the target protein from the
reporter group subsequent to purification of the fusion protein.
Proteolytic enzymes for such cleavage and their recognition
sequences include Factor Xa, thrombin and enterokinase.
[0191] Typical fusion expression vectors include pGEX (Amrad Corp.,
Melbourne, Australia), pMAL (New England Biolabs, Beverly, Mass.)
and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione
S-transferase, maltose E binding protein, or protein A,
respectively, to the target recombinant protein.
[0192] Inducible non-fusion expression vectors include pTrc (Amann
et al., (1988) Gene 69: 301-315) and pET 11d (Studier et al., Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990) 60-89). While target gene expression
relies on host RNA polymerase transcription from the hybrid trp-lac
fusion promoter in pTrc, expression of target genes inserted into
pET 11d relies on transcription from the T7 gn10-lacO fusion
promoter mediated by coexpressed viral RNA polymerase (T7gn1). Th
is viral polymerase is supplied, by host strains BL21(DE3) or
HMS174(DE3) from a resident .lamda. prophage harboring a T7gn1
under the transcriptional control of the lacUV 5 promoter.
Promoters and Enhancers
[0193] A promoter region of a DNA or RNA molecule binds RNA
polymerase and promotes the transcription of an "operably linked"
nucleic acid sequence. As used herein, a "promoter sequence" is the
nucleotide sequence of the promoter which is found on that strand
of the DNA or RNA which is transcribed by the RNA polymerase. Two
sequences of a nucleic acid molecule, such as a promoter and a
coding sequence, are "operably linked" when they are linked to each
other in a manner which permits both sequences to be transcribed
onto the same RNA transcript or permits an RNA transcript begun in
one sequence to be extended into the second sequence. Thus, two
sequences, such as a promoter sequence and a coding sequence of DNA
or RNA are operably linked if transcription commencing in the
promoter sequence will produce an RNA transcript of the operably
linked coding sequence. In order to be "operably linked" it is not
necessary that two sequences be immediately adjacent to one another
in the linear sequence.
[0194] The preferred promoter sequences of the present invention
must be operable in mammalian cells and may be either eukaryotic or
viral promoters. Suitable promoters may be inducible, repressible
or constitutive. An example of a constitutive promoter is the viral
promoter MSV-LTR, which is efficient and active in a variety of
cell types, and, in contrast to most other promoters, has the same
enhancing activity in arrested and growing cells. Other preferred
viral promoters include that present in the CMV-LTR (from
cytomegalovirus) (Bashart, M. et al., Cell 41;521(1985)) or in the
RSV-LTR (from Rous sarcoma virus) (Gorman, C. M., Proc. Natl. Acad.
Sci. USA 79:6777 (1982). Also useful are the promoter of the mouse
metallothionein I gene (Ranier, D., et al., J. Mol. Appl. Gen.
1:273-288 (1982)); the TK promoter of Herpes virus (McKnight, S.,
Cell 31:355-365 (1982)); the SV40 early promoter (Benoist, C., et
al., Nature 290:304-310 (1981)); and the yeast gal4 gene promoter
(Johnston, S. A., et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975
(1982); Silver, P. A., et al., Proc. Natl. Acad. Sci. (USA)
81:5951-5955 (1984)). Other illustrative descriptions of
transcriptional factor association with promoter regions and the
separate activation and DNA binding of transcription factors
include: Keegan et al., Nature (1986) 231:699; Fields et al.,
Nature (1989) 340:245; Jones, Cell (1990) 61:9; Lewin, Cell (1990)
61:1161; Ptashne et al., Nature (1990) 346:329; Adams et al., Cell
(1993) 72:306. The relevant disclosure of all of these above-listed
references is hereby incorporated by reference.
[0195] The promoter region may further include an octamer region
which may also function as a tissue specific enhancer, by
interacting with certain proteins found in the specific tissue. The
enhancer domain of the DNA construct of the present invention is
one which is specific for the target cells to be transfected, or is
highly activated by cellular factors of such target cells. Examples
of vectors (plasmid or retrovirus) are disclosed in (Roy-Burman et
al., U.S. Pat. No. 5,112,767). For a general discussion of
enhancers and their actions in transcription, see, Lewin, B. M.,
Genes IV, Oxford University Press, Oxford, (1990), pp. 552-576.
Particularly useful are retroviral enhancers (e.g., viral LTR). The
enhancer is preferably placed upstream from the promoter with which
it interacts to stimulate gene expression. For use with retroviral
vectors, the endogenous viral. LTR may be rendered enhancer-less
and substituted with other desired enhancer sequences which confer
tissue specificity or other desirable properties such as
transcriptional efficiency on the B7-DC-encoding DNA molecule of
the present invention.
[0196] The nucleic acid sequences of the invention can also be
chemically synthesized using standard techniques. Various methods
of chemically synthesizing polydeoxy-nucleotides are known,
including solid-phase synthesis which, like peptide synthesis, has
been fully automated with commercially available DNA synthesizers
(See, e.g., Itakura et al. U.S. Pat. No 4,598,049; Caruthers et al.
U.S. Pat. No. 4,458,066; and Itakura U.S. Pat. Nos. 4,401,796 and
4,373,071, incorporated by reference herein).
Proteins and Polypeptides
[0197] The present invention includes an "isolated" B7-DC protein
having the sequence SEQ ID NO:2 or SEQ ID NO:4. While the present
disclosure exemplifies the full length human and murine B7-DC
protein (and DNA), it is to be understood that homologues of B7-DC
from other mammalian species and mutants thereof that possess the
characteristics disclosed herein are intended within the scope of
this invention.
[0198] Also included is a "functional derivative" of B7-DC which is
means an amino acid substitution variant, a "fragment," or a
"chemical derivative" of B7-DC, which terms are defined below. A
functional derivative retains measurable B7-DC activity, preferably
that of binding to a receptor on T cells and costimulating T cell
activity, which permits its utility in accordance with the present
invention. "Functional derivatives" encompass "variants" and
"fragments" regardless of whether the terms are used in the
conjunctive or the alternative herein.
[0199] A functional homologue must possess the above biochemical
and biological activity. In view of this functional
characterization, use of homologous proteins B7-DC from other
species, including proteins not yet discovered, fall within the
scope of the invention if these proteins have sequence similarity
and the recited biochemical and biological activity.
[0200] To determine the percent identity of two amino acid
sequences or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). In a
preferred method of alignment, Cys residues are aligned.
[0201] In a preferred embodiment, the length of a Sequence being
compared is at least 30%, preferably at least 40%, more preferably
at least 50%, even more preferably at least 60%, and even more
preferably at least 70%, 80%, or 90% of the length of the reference
sequence. For example, when aligning a second sequence to the human
B7-DC protein amino acid sequence (SEQ ID NO:2) having 276 amino
acid residues, at least 83, preferably at least 110, more
preferably at least 138, even more preferably at least 166, and
even more preferably at least 193, 221 or 248 amino acid residues
are aligned). The amino acid residues (or nucleotides) at
corresponding amino acid positions (or nucleotide) positions are
then compared. When a position in the first sequence is occupied by
the same amino acid residue (or nucleotide) as the corresponding
position in the second sequence, then the molecules are identical
at that position (as used herein amino acid or nucleic acid
"identity" is equivalent to amino acid or nucleic acid "homology").
The percent identity between the two sequences is a function of the
number of identical positions shared by the sequences, taking into
account the number of gaps, and the length of each gap, which need
to be introduced for optimal alignment of the two sequences.
[0202] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In a preferred embodiment, the percent
identity between two amino acid sequences is determined using the
Needleman and Wunsch. (J. Mol. Biol. 48:441 453 (1970) algorithm
which has been incorporated into the GAP program in the GCG
software package (available at http://www.gcg.com), using either a
Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14,
12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In
yet another preferred embodiment, the percent identity between two
nucleotide sequences is determined using the GAP program in the GCG
software package (available at http://www.gcg.com), using a
NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and
a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the
percent identity between two amino acid or nucleotide sequences is
determined using the algorithm of E. Meyers and W. Miller (CABIOS,
4:11-17 (1989)) which has been incorporated into the ALIGN program
(version 2.0), using a PAM120 weight residue table, a gap length
penalty of 12 and a gap penalty of 4.
[0203] The nucleic acid and protein sequences of the present
invention can further be used as a "query sequence" to perform a
search against public databases, for example, to identify other
family members or related sequences. Such searches can be performed
using the NBLAST and XBLAST programs (version 2.0) of Altschul et
(1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be
performed with the NBLAST program, score=100, wordlength=12 to
obtain nucleotide sequences homologous to human or murine B7-DC
nucleic acid molecules. BLAST protein searches can be performed
with the XBLAST program, score=50, wordlength=3 to obtain amino
acid sequences homologous to human or murine B7-DC protein
molecules of the invention. To obtain gapped alignments for
comparison purposes, Gapped BLAST can be utilized as described in
Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. When
utilizing BLAST and Gapped BLAST programs, the default parameters
of the respective programs (e.g., XBLAST and NBLAST) can be used.
See http://www.ncbi.nlm.nih.gov.
[0204] Thus, a homologue of the B7-DC protein described above is
characterized as having (a) functional activity of native B7-DC,
and (b) sequence similarity to a native B7-DC protein (such as SEQ
ID NO:2 or SEQ ID NO:4, when determined above, of at least about
30% (at the amino acid level), preferably at least about 50%, more
preferably at least about 70%, even more preferably at least about
90%.
[0205] It is within the skill in the art to obtain and express such
a protein using DNA probes based on the disclosed sequences of
B7-DC. Then, the protein's biochemical and biological activity can
be tested readily using art-recognized methods such as those
described herein, for example, a standard T cell proliferation or
cytokine secretion assay. A biological assay of T cell
co-stimulation will indicate whether the homologue has the
requisite activity to qualify as a "functional" homologue.
[0206] Preferred assays measure the functional characteristics of
B7-DC such as stimulating T cells synthesis of cytokines, which
depends on binding or cross-linking of the TCR ("primary activation
signal"), as well as delivery of a costimulatory signal. The
binding of B7-DC to its natural ligand(s) on T cells transmits a
signal that induces increased cytokine production, such as IL-2,
which in turn stimulates proliferation which can also be measured
routinely.
[0207] A "variant" of B6-DC refers to a molecule substantially
identical to either the full protein or to a fragment thereof in
which one or more amino acid residues have been replaced
(substitution variant) or which has one or several residues deleted
(deletion variant) or added (addition variant). A "fragment" of
B6-DC refers to any subset of the molecule, preferably one that
includes the ECD, that is, a shorter polypeptide of the full-length
protein.
[0208] A number of processes can be used to generate fragments,
mutants and variants of the isolated DNA sequence. Small subregions
or fragments of the nucleic acid encoding the B7-DC protein, for
example 1-30 bases in length, can be prepared by standard, chemical
synthesis. Antisense oligonucleotides and primers for use in the
generation of larger synthetic fragment.
[0209] A preferred functional derivative is a fusion protein, a
polypeptide that includes a functional fragment of B7-DC. For
example, a useful derivative of B7 is a B7-DC-Ig fusion protein
that comprises a polypeptide corresponding to the ECD of B7-DC and
an Ig C region. The presence of the fusion partner can alter the
solubility, affinity and/or valency (defined here as the number of
binding sites available per molecule) of the B7-DC protein. A
soluble B7-DC fusion protein, while still binding to a receptor on
T cells, may have a different biological, effect than of the native
protein expressed on an APC, i.e., inhibition of T cell stimulation
by competitive binding rather than costimulation.
[0210] As used herein an extracellular domain (ECD) of B7-DC is the
entire extracellular portion of the protein or any fragment thereof
that recognizes and binds to PD-1 or to another receptor on T cells
that is not CD28 or CTLA-4. Preferably, an ECD of B7-DC is that
portion encoded by amino acid residues from about position 26 to
about position 221 of SEQ ID NO:2 or SEQ. ID NO:4.
[0211] By "soluble B7-DC" is intended a cell-free form of B7-DC
that may be shed, secreted or otherwise extracted from the
producing cells. Soluble B7-DC includes, but is not limited to,
soluble fusion proteins such as B7-DC-Ig, free ECD of B7-DC, or the
B7-DC BCD fused (genetically or chemically) to a biologically
active molecule.
[0212] As indicated earlier, this invention also includes hybrid
fission proteins between a B7-DC domain and a domain or fragment of
another B7 family protein, preferably expressed on the cell surface
in costimulatory form.
[0213] A preferred group of B7-DC variants are those in which at
least one amino acid residue and preferably, only one, has been
substituted by different residue. For a detailed description of
protein chemistry and structure, see Schulz, G E et al, Principles
of Protein Structure, Springer-Verlag, New York, 1978, and
Creighton, T. E., Proteins: Structure and Molecular Properties,
W.H. Freeman & Co., San Francisco, 1983, which are hereby
incorporated by reference. The types of substitutions that may be
made in the protein molecule may be based on analysis of the
frequencies of amino acid changes between a homologous protein of
different species, such as those presented in Table 1-2 of Schulz
et al. (supra) and FIG. 3-9 of Creighton (supra). Based on such an
analysis, conservative substitutions are defined herein as
exchanges within one of the following five groups:
TABLE-US-00005 1 Small aliphatic, nonpolar or slightly Ala, Ser,
Thr (Pro, Gly); polar residues 2 Polar, negatively charged residues
Asp, Asn, Glu, Gln; and their amides 3 Polar, positively charged
residues His, Arg, Lys; 4 Large aliphatic, nonpolar residues Met,
Leu, Ile, Val (Cys) 5 Large aromatic residues Phe, Tyr, Trp.
[0214] The three amino acid residues in parentheses above have
special roles in protein architecture. Gly is the only residue
lacking a side chain and thus imparts flexibility to the chain.
Pro, because of its unusual geometry, tightly constrains the chain.
Cys can participate in disulfide bond formation, which is important
in protein folding.
[0215] More substantial changes in biochemical, functional (or
immunological) properties are made by selecting substitutions that
are less conservative, such as between, rather than within, the
above five groups. Such changes will differ more significantly in
their effect on maintaining (a) the structure of the peptide
backbone in the area of the substitution, for example, as a sheet
or helical conformation, (b) the charge or hydrophobicity of the
molecule at the target site, or (c) the bulk of the side chain.
Examples of such substitutions are (i) substitution of Gly and/or
Pro by another amino acid or deletion or insertion of Gly or Pro;
(ii) substitution of a hydrophilic residue, e.g., Ser or Thr, for
(or by) a hydrophobic residue, e.g., Leu, Ile, Phe, Val or Ala;
(iii) substitution of a Cys residue for (or by) any other residue;
(iv) substitution of a residue having an electropositive side
chain, e.g., Lys, Arg or His, for (or by) a residue having an
electronegative charge, e.g., Glu or Asp; or (v) substitution of a
residue having a bulky side chain, e.g., Phe, for (or by) a residue
not having such a side chain, e.g., Gly.
[0216] Most acceptable deletions, insertions and substitutions
according to the present invention are those that do not produce
radical changes in the characteristics of the B7-DC protein in
terms of its T cell costimulatory activity. However, when it is
difficult to predict the exact effect of the substitution, deletion
or insertion in advance of doing so, one skilled in the art will
appreciate that the effect can be evaluated by routine screening
assays such as those described here, without requiring undue
experimentation.
[0217] Whereas shorter chain variants can be made by chemical
synthesis, for the present invention, the preferred longer chain
variants are typically made by site-specific mutagenesis of the
nucleic acid encoding the B7-DC polypeptide, expression of the
variant nucleic acid in cell culture, and, optionally, purification
of the polypeptide from the cell culture, for example, by
immunoaffinity chromatography using specific antibody immobilized
to a column (to absorb the variant by binding to at least one
epitope).
Chemical Derivatives of B7-DC
[0218] "Chemical derivatives" of B7-DC contain additional chemical
moieties not normally a part of the protein. Covalent modifications
of the polypeptide are included within the scope of this invention.
Such derivatized moieties may improve the solubility, absorption,
biological half life, and the like. Moieties capable of mediating
such effects are disclosed, for example, in Remington's
Pharmaceutical Sciences, 16.sup.th ed., Mack Publishing Co.,
Easton, Pa. (1980).
[0219] Such modifications may be introduced into the molecule by
reacting targeted amino acid residues of the polypeptide with an
organic derivatizing agent that is capable of reacting with
selected side chains or terminal residues. Another modification is
cyclization of the protein.
[0220] Examples of chemical derivatives of the polypeptide
follow.
[0221] Lysinyl and amino terminal residues are derivatized with
succinic or other carboxylic acid anhydrides. Derivatization with a
cyclic carboxylic anhydride has the effect of reversing the charge
of the lysinyl residues. Other suitable reagents for derivatizing
amino-containing residues include imidoesters such as methyl
picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride;
trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione;
and transaminase-catalyzed reaction with glyoxylate.
[0222] Carboxyl side groups, aspartyl or glutamyl, may be
selectively modified by reaction with carbodiimides
(R--N.dbd.C.dbd.N--R') such as
1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide or
1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore,
aspartyl and glutamyl residues can be converted to asparaginyl and
glutaminyl residues by reaction with ammonia.
[0223] Other modifications include hydroxylation of proline and
lysine, phosphorylation of hydroxyl groups of seryl or threonyl
residues, methylation of the amino group of lysine (Creighton,
supra, pp. 79-86), acetylation of the N-terminal amine, and
amidation of the C-terminal carboxyl groups.
[0224] Also included are peptides wherein one or more D-amino acids
are substituted for one or more L-amino acids.
[0225] Multimeric Peptides
[0226] The present invention also includes longer polypeptides in
which a basic peptidic sequence obtained from the sequence of B7-DC
is repeated from about two to about 100 times, with or without
intervening spacers or linkers. It is understood that such
multimers may be built from any of the peptide variants defined
herein. Moreover, a peptide multimer may comprise different
combinations of peptide monomers and the disclosed substitution
variants thereof. Such oligomeric or multimeric peptides can be
made by chemical synthesis or by recombinant DNA, techniques as
discussed herein. When produced chemically, the oligomers
preferably have from 2-8 repeats of the basic peptide sequence.
When produced recombinantly, the multimers may have as many repeats
as the expression system permits, for example from two to about 100
repeats.
[0227] In tandem multimers, preferably dimers and turners, of the
B7-DC peptide or polypeptide, the chains bonded by interchain
disulfide bonds or other "artificial" covalent bonds between the
chains such that the chains are "side-by-side" rather than "end to
end." Preferred dimers and trimers are those between fusion
proteins of B7-DC such as B7-DC-Ig, as described herein.
Antibodies Specific for Epitopes of B7-DC
[0228] In the following description, reference will be made to
various methodologies known to those of skill in the art of
immunology, cell biology, and molecular biology. Publications and
other materials setting forth such known methodologies to which
reference is made are incorporated herein by reference in their
entireties as though set forth in full. Standard reference works
setting forth the general principles of immunology include A. K.
Abbas et at., Cellular and Molecular Immunology (Fourth Ed.), W.B.
Saunders Co., Philadelphia, 2000; C. A. Janeway et al.,
Immunobiology. The Immune System in Health and Disease, Fourth ed.,
Garland Publishing Co., New York, 1999; Roitt, I. et al.,
Immunology,
[0229] (current ed.) C.V. Mosby Co., St. Louis, Mo. (1999); Klein,
J., Immunology, Blackwell Scientific Publications, Inc., Cambridge,
Mass., (1990).
[0230] Monoclonal antibodies (mAbs) and methods for their
production and use are described in Kohler and Milstein, Nature
256:495-497 (1975); U.S. Pat. No. 4,376,110; Hartlow, E. et al.,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1988); Monoclonal Antibodies and
Hybridomas: A New Dimension in Biological Analyses, Plenum Press,
New York, N.Y. (1980); H. Zola et al., in Monoclonal Hybridoma
Antibodies: Techniques and Applications, CRC Press, 1982)).
[0231] Immunoassay methods are also described in Cagan, J. E. et
al., eds., Current Protocols in Immunology, Wiley-Interscience, New
York 1991(or current edition); Butt, W. R. (ed.) Practical
Immunoassay: The State of the Art, Dekker, New York, 1984;
Bizollon, Ch. A., ed., Monoclonal Antibodies and New Trends in
Immunoassays, Elsevier, New York, 1984; Butler, J. E., ELISA
(Chapter 29), In: van Oss, C. J. et al., (eds); IMMUNOCHEMISTRY,
Marcel Dekker, Inc., New York, 1994, pp. 759-803; Butler, J. E.
(ed.), Immunochemistry of Solid--Phase Immunoassay, CRC Press, Boca
Raton, 1991; Weintraub, B., Principles of Radioimmunoassays,
Seventh Training Course on Radioligand Assay Techniques, The
Endocrine Society, March, 1986; Work, T. S. et al., Laboratory
Techniques and Biochemistry in Molecular Biology, North Holland
Publishing Company, NY, (1978) (Chapter by Chard, T., "An
Introduction to Radioimmune Assay and Related Techniques").
[0232] Anti-idiotypic antibodies are described, for example, in
Idiotypy in Biology and Medicine, Academic Press, New York, 1984;
Immunological Reviews Volume 79, 1984; Immunological Reviews Volume
90, 1986; Curr. Top. Microbiol., Immunol. Volume 119, 1985; Bona,
C. et al., CRC Crit. Rev. Immunol., pp. 33-81 (1981); Jerne, N K,
Ann. Immunol. 125C:373-389 (1974); Jerne, N K, In:
Idiotypes--Antigens on the Inside, Westen-Schnurr, I., ed.,
Editiones Roche, Basel, 1982, Urbain, J et al., Ann. Immunol.
133D:179- (1982); Rajewsky, K. et al., Ann. Rev. Immunol. 1:569-607
(1983)
[0233] The present invention provides antibodies, both polyclonal
and monoclonal, reactive with novel epitopes of B7-DC that are
absent from known B7 family proteins. The antibodies may be
xenogeneic, allogeneic, syngeneic, or modified forms thereof, such
as humanized or chimeric antibodies. Antiidiotypic antibodies
specific for the idiotype of an anti-B7-DC antibody are also
included. The term "antibody" is also meant to include both intact
molecules as well as fragments thereof that include the
antigen-binding site and are capable of binding to a B7-DC epitope.
These include, Fab and F(ab').sub.2 fragments which lack the Fc
fragment of an intact antibody, clear more rapidly from the
circulation, and may have less non-specific tissue binding than an
intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)).
Also included are Fv fragments (Hochman, J. et al. (1973)
Biochemistry 12:1130-1135; Sharon, J. et al. (1976) Biochemistry
15:1591-1594).). These various fragments are be produced using
conventional techniques such as protease cleavage or chemical
cleavage (see, e.g., Rousseaux et al., Meth. Enzyma, 121:663-69
(1986))
[0234] Polyclonal antibodies are obtained as sera from immunized
animals such as rabbits, goats, rodents, etc. and may be used
directly without further treatment or may be subjected to
conventional enrichment or purification methods such as ammonium
sulfate precipitation, ion exchange chromatography, and affinity
chromatography (see Zola et al., supra).
[0235] The immunogen may comprise the complete B7-D6 protein, or
fragments or derivatives thereof. Preferred immunogens comprise all
or a part of the ECD of human B7-DC (amino acid residues 26-221),
where these residues contain the post-translation modifications,
such as glycosylation, found on the native B7-DC. Immunogens
comprising the extracellular domain are produced in a variety of
ways known in the art, e.g., expression of cloned genes using
conventional recombinant methods, isolation from cells of origin,
cell populations expressing high levels of B7-DC, etc.
[0236] The mAbs may be produced using conventional hybridoma
technology, such as the procedures introduced by Kohler and
Milstein (Nature, 256:495-97 (1975)), and modifications thereof
(see above references). An animal, preferably a mouse is primed by
immunization with an immunogen as above to elicit the desired
antibody response in the primed animal.
[0237] B lymphocytes from the lymph nodes, spleens or peripheral
blood of a primed, animal are fused with myeloma cells, generally
in the presence of a fusion promoting agent such as polyethylene
glycol (PEG). Any of a number of murine myeloma cell lines are
available for such use: the P3-NS1/1-Ag4-1, P3-x63-k0Ag8.653,
Sp2/0-Ag14, or HL1-653 myeloma lines (available from the ATCC,
Rockville, Md.). Subsequent steps include growth in selective
medium so that unfused parental myeloma cells and donor lymphocyte
cells eventually die while only the hybridoma cells survive. These
are cloned and grown and their supernatants screened for the
presence of antibody of the desired specificity, e.g. by
immunoassay techniques using the B7-DC-Ig fusion protein Positive
clones are subcloned, e.g., by limiting dilution, and the mAbs are
isolated.
[0238] Hybridomas produced according to these methods can be
propagated in vitro or in vivo (in ascites fluid) using techniques
known in the art (see generally Fink et al., Prog. Clin. Pathol.,
9:121-33 (1984)). Generally, the individual cell line is propagated
in culture and the culture medium containing high concentrations of
a single mAb can be harvested by decantation, filtration, or
centrifugation.
[0239] The antibody may be produced as a single chain antibody or
say instead of the normal multimeric structure. Single chain
antibodies include the hypervariable regions from an Ig of interest
and recreate the antigen binding site of the native Ig while being
a fraction of the size of the intact Ig (Skerra, A. et al. (1988)
Science, 240: 1038-1041; Pluckthun, A. et al. (1989) Methods
Enzymol. 178: 497-515; Winter, G. et al. (1991) Nature, 349:
293-299); Bird et al., (1988) Science 242:423; Huston et al. (1988)
Proc. Natl. Acad. Sci. USA 85:5879; Jost C R. et al., J Biol Chem.
1994 269:26267-26273; U.S. Pat. No, 4,704,692, 4,853,871,
4,94,6778, 5,260,203, 5,455,0 Kn contacted with the solution
containing an unknown quantity of labeled antibody (which functions
as a "reporter molecule"). After a second incubation period to
permit the labeled antibody to complex with the antigen bound to
the solid support through the unlabeled antibody, the solid support
is washed a second time to remove the unreacted labeled antibody.
This type of forward sandwich assay may be a simple "yes/no" assay
to determine whether antigen is present or may be made quantitative
by comparing the measure of labeled antibody with that obtained for
a standard sample containing known quantities of antigen.
[0240] In another type of "sandwich" assay the so-called
"simultaneous" and "reverse" assays are used. A simultaneous assay
involves a single incubation step as the antibody bound to the
solid support and labeled antibody are both added to the sample
being tested at the same time. After the incubation is completed,
the solid support is washed to remove the residue of fluid sample
and uncomplexed labeled antibody. The presence of labeled antibody
associated with the solid support is then determined as it would be
in a conventional "forward" sandwich assay.
[0241] In the "reverse" assay, stepwise addition first of a
solution of labeled antibody to the fluid sample followed by the
addition of unlabeled antibody bound to a solid support after a
suitable incubation period is utilized. After a second incubation,
the solid phase is washed in conventional fashion to free it of the
residue of the sample being tested and the solution of unreacted
labeled antibody. The determination of labeled antibody associated
with a solid support is then determined as in the "simultaneous"
and "forward" assays.
[0242] The foregoing antibodies are useful in method for inhibiting
T cell stimulation and treating diseases associated with undesired
T cell activation, such as transplant rejection and autoimmunity.
This method involves administering a subject in need of such
treatment an effective amount of an antibody, preferably a mAb,
more preferably a human or humanized mAb specific for a
costimulatory epitope of B7-DC. The administration of antibody must
be effective in blocking stimulation of T cells or in eliminating
antigen-reactive T cells, thereby inhibiting the targeted T cell
response. Relevant dose ranges are described below.
Uses of Nucleic Acids that Encode B7-DC Protein
[0243] The nucleic acids of this invention are used diagnostically
to monitor the progress of a disease, by measuring the expression
of B7-DC in cells from biological samples or for assaying the
effect of an agent on the expression of B7-DC. This is preferably
accomplished by measurement of cellular mRNA levels. For use in
such diagnostic methods, the nucleic acid sequence is detectably
labeled, e.g., with a radioactive or fluorescent label or biotin
and used in a conventional dot blot or Northern hybridization
procedure to probe mRNA molecules present in, for example, a
preparation of total kor poly(A+) RNA from a biological sample.
Therapeutic Compositions and their Administration
[0244] The B6-DC polypeptide or a cell expressing this polypeptide
such as a DC or a tumor cell is administered to a mammalian
subject, preferably a human. Cell-associated, immobilized or
otherwise aggregated forms of the polypeptide are used to enhance T
lymphocyte reactivity and the resultant immunity. The B6-DC-Ig
fusion protein assembles as a dimer and, as shown in the examples,
co-stimulates T cells. Soluble monomeric forms of the B6-DC
polypeptide can bind to the receptor on T cells without stimulating
activity and can therefore be considered competitive inhibitors or
antagonists of T cell co-stimulation by a stimulatory form of the
molecule. Binding of a such a B6-DC antagonist may suppress ongoing
T cell reactivity or may interfere with the effect of a
costimulatory signal presented by endogenous B6-DC or even by other
B7 family members acting via their receptors (e.g., CD28 or
CTLA-4).
[0245] A composition having the activity of B7-DC as described
herein is administered in a pharmaceutically acceptable carrier in
a biologically effective or a therapeutically effective amount. The
B7-DC polypeptide (or cell expressing the polypeptide) may be given
alone or in combination with another protein or peptide such as one
having the activity of another member of the B7 family or another
immunostimulatory molecule Treatment may include administration of
an adjuvant, used in its broadest sense to include any nonspecific
immune stimulating compound such as an interferon. Adjuvants
contemplated herein include resorcinols, non-ionic surfactants such
as polyoxyethylene oleyl ether and n-hexadecyl polyethylene
ether.
[0246] The following doses and amounts also pertain to the
antibodies of the invention when administered to a subject.
[0247] A therapeutically effective amount is a dosage that, when
given for an effective period of time, achieves the desired
immunological or clinical effect.
[0248] A therapeutically active amount of a polypeptide having
B7-DC activity (or an anti-B7-DC antibody) may vary according to
factors such as the disease state, age, sex, and weight of the
individual, and the ability of the peptide to elicit a desired
response in the individual. Dosage regimes may be adjusted to
provide the optimum therapeutic response. For example, several
divided doses may be administered daily or the dose may be
proportionally reduced as indicated by the exigencies of the
therapeutic situation. A therapeutically effective amounts of the
protein, in cell associated form may be stated in terms of the
protein or cell equivalents.
[0249] Thus an effective amount is between about 1 ng and about 1
gram per kilogram of body weight of the recipient, more preferably
between about 1 .mu.g and 100 mg/kg, more preferably, between about
100 .mu.g and about 100 mg/kg. Dosage forms suitable for internal,
administration preferably contain (for the latter dose range) from
about 0.1 mg to 500 mg of active ingredient per unit. The active
ingredient may vary from 0.5 to 95% by weight based on the total
weight of the composition. Alternatively, an effective dose of
cells expressing B7-DC, such preferably transduced cells such as
DC's or inactivated tumor cells, is between about 10.sup.4 and
10.sup.9 cells, more preferably between about 10.sup.6 and 10.sup.8
cells per subject, preferably in split doses. Those skilled in the
art of immunotherapy will be able to adjust these doses without
undue experimentation.
[0250] The active compound (e.g., B6-DC polypeptide or cell
transduced with B6-DC DNA) may be administered in a convenient
manner, e.g., injection by a convenient and effective route.
Preferred routes include subcutaneous, intradermal, intravenous and
intramuscular routes. Other possible routes include oral
administration, intrathecal, inhalation, transdemial application,
or rectal administration. For the treatment of tumors which have
not been completely resected, direct intratumoral injection is also
intended.
[0251] Depending on the route of administration, the active
compound may be coated in a material to protect the compound from
the action of enzymes, acids and other natural conditions which may
inactivate the compound. Thus, to a administer a polypeptide or
peptide having B7-DC activity by an enteral route, it may be
necessary to coat the composition with, or co-administer the
composition with, a material to prevent its inactivation. For
example, a peptide may be administered to an individual in an
appropriate carrier, diluent or adjuvant, co-administered with
enzyme inhibitors (e.g., pancreatic trypsin inhibitor,
diisopropylfluorophosphate (DEP) and trasylol) or in an appropriate
carrier such as liposomes (including water-in-oil-in-water
emulsions as well as conventional liposomes (Strejan et al., (1984)
J. Neuroimmunol 7:27).
[0252] As used herein "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active compound, use thereof in the therapeutic compositions is
contemplated. Supplementary active compounds can also be
incorporated into the compositions.
[0253] Preferred pharmaceutically acceptable diluents include
saline and aqueous buffer solutions. Pharmaceutical compositions
suitable for injection include sterile aqueous solutions (where
water soluble) or dispersions and sterile powders for the
extemporaneous preparation of sterile injectable solutions or
dispersion. Isotonic agents, for example, sugars, polyalcohols such
as mannitol, sorbitol, sodium chloride may be included in the
pharmaceutical composition. In all cases, the composition should be
sterile and should be fluid. It should be stable under the
conditions of manufacture and storage and must include
preservatives that prevent contamination with microorganisms such
as bacteria and fungi. Dispersions can also be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations may contain a preservative to prevent the growth of
microorganisms.
[0254] The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants.
[0255] Prevention of the action of microorganisms can be achieved
by various antibacterial and antifungal agents, for example,
parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the
like.
[0256] Prolonged absorption of the injectable compositions can be
brought about by including in the composition an agent which delays
absorption, for example, aluminum monostearate and gelatin.
[0257] Parenteral compositions are preferably formulated in dosage
exit form for ease of administration and uniformity of dosage.
Dosage unit form refers to physically discrete units suited as
unitary dosages for a mammalian subject; each unit contains a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on (a) the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the treatment
of sensitivity in individuals.
[0258] For lung instillation, aerosolized solutions are used. In a
sprayable aerosol preparations, the active protein may be in
combination with a solid or liquid inert carrier material. This may
also be packaged in a squeeze bottle or in admixture with a
pressurized volatile, normally gaseous propellant. The aerosol
preparations can contain solvents, buffers, surfactants, and
antioxidants in addition to the protein of the invention.
[0259] For topical application, the proteins of the present
invention may be incorporated into topically applied vehicles such
as salves or ointments, which have both a soothing effect on the
skin as well as a means for administering the active ingredient
directly to the affected area.
[0260] The carrier for the active ingredient may be either in
sprayable or nonsprayable form. Non-sprayable forms can be
semi-solid or solid forms comprising a carrier indigenous to
topical application and having a dynamic viscosity preferably
greater than that of water. Suitable formulations include, but are
not limited to, solution, suspensions, emulsions, creams,
ointments, powders, liniments, salves, and the like. If desired,
these may be sterilized or mixed with auxiliary agents, e.g.,
preservatives, stabilizers, wetting agents, buffers, or salts for
influencing osmotic pressure and the like. Examples of preferred
vehicles for non-sprayable topical preparations include ointment
bases, e.g., polyethylene glycol-1000 (PEG-1000); conventional
creams such as HEB cream; gels; as well as petroleum jelly and the
like.
[0261] Other pharmaceutically acceptable carriers for the B7-DC
polypeptide according to the present invention are liposomes,
pharmaceutical compositions in which the active protein is
contained either dispersed or variously present in corpuscles
consisting of aqueous concentric layers adherent to lipidic layers.
The active protein is preferably present in the aqueous layer and,
in the lipidic layer, inside or outside, or, in any event, in the
non-homogeneous system generally known as a liposomic suspension.
The hydrophobic layer, or lipidic layer, generally, but not
exclusively, comprises phospholipids such as lecithin and
sphingomyelin, steroids such as cholesterol, more or less ionic
surface active substances such as dicetylphosphate, stearylamine or
phosphatidic acid, and/or other materials of a hydrophobic
nature.
Modification of Tumor Cells to Express B7-DC and Multiple
Costimulaty Molecules
[0262] Another aspect of the invention is a cell, preferably a
tumor cell, modified to express multiple costimulatory molecules.
The temporal expression of costimulatory molecules on activated B
cells is different for B7, B7-2 and B7-3. For example, B7-2 is
expressed early following B cell activation, whereas B7-3 is
expressed later. The different costimulatory molecules may thus
serve distinct functions during the course of an immune response.
An effective T cell response may require that the T cell receive
costimulatory signals from multiple costimulatory molecules.
[0263] Accordingly, the invention encompasses a tumor cell which is
genetically modified or to express more than one costimulatory
molecule. For example, a tumor cell can be modified to express
B7-DC and one or more of B7, B7-2 and B7-3.
[0264] Before modification, a cell such as a tumor cell may not
express any costimulatory molecules, or may express certain
costimulatory molecules but not others. As described herein, tumor
cells can be modified by transfection with nucleic acid encoding
B7-DC alone or with another costimulatory molecule(s). For example,
a tumor cell transfected with nucleic acid encoding B7-DC can be
further transfected with nucleic acid encoding B7. The sequence of
cDNA molecules encoding human or mouse B7-DC proteins are SEQ ID
NO:1 and the coding portion of SEQ ID NO:3, respectively.
Alternatively, more than one type of modification can be used. For
example, a tumor cell transfected with a nucleic acid encoding
B7-DC can be stimulated Owith an agent which induces expression of
B7-1, B7-2 or B7-3.
Antigens Associated with Pathogens
[0265] A major utility for the present invention is the use of the
present compositions in therapeutic vaccine for cancer and for
major chronic viral infections that cause morbidity and mortality
worldwide. Such vaccines are designed to eliminate infected
cells--this requires T cell responses as antibodies are
ineffective. The vaccines of the present invention, include, in
addition to the antigenic epitope itself: [0266] (a) a vector such
as naked DNA, naked RNA, self replicating RNA replicons and viruses
including vaccinia, adenoviruses, adeno-associaged virus (AAV),
lentiviruses and RNA alphavimses; [0267] (b) an antigen targeting
or processing signal such as HSP70, calreticulin, the extracellular
domain of Flt-3 ligand, domain II of Pseudomonas exotoxin ETA,
herpes simplex VP22 targeting protein, and the like. (See commonly
assigned U.S. patent application Ser. Nos. 09//421,608; 09/501,097;
09/693,450; 60/222,9002; 60/222,985; 60/268,575 and Chang, W-F et
al., J. Virol. 75;2368-2376 (2001), which are hereby incorporated
by reference in their entirety); and [0268] (c) a costimulatory
signal, preferably the B7-DC protein of the present invention or a
fusion protein, fragment or functional derivative thereof (alone or
in combination with other known costimulatory proteins such as
B7.1, B7.2, soluble CD40, etc.).
[0269] Tumor cells or other types of host cells, including APCs,
are transformed, transfected or otherwise transduced with a nucleic
encoding an antigen to which an immune response is desired. Such
antigens are preferably epitopes of pathogenic microorganisms
against which the host is defended by effector T cells responses,
including cytotoxic T lymphocyte (CTL) and delayed type
hypersensitivity. These typically include viruses, intracellular
parasites such as malaria, and bacteria that grow intracellularly
such as mycobacteria and listeria. Thus, the types of antigens
included in the vaccine compositions of this invention are any of
those associated with such pathogens (in addition, of course, to
tumor-specific antigens). It is noteworthy that some viral antigens
are also tumor antigens in the case where the virus is a causative
factor in cancer.
[0270] In fact, the two most common cancers worldwide, hepatoma and
cervical cancer, are associated with viral infection. Hepatitis B
virus (HBV) (Beasley, R. P. et al., Lancet 2, 1129-1133 (1981) has
been implicated as etiologic agent of hepatomas. 80-90% of cervical
cancers express the E6 and E7 antigens from one of four "high risk"
human papillomavirus types: HPV-16, HPV-18, HPV-31 and HPV-45
(Gissmann, L. et Ciba Found Symp. 120, 190-207 (1986); Beaudenon,
S., et al. Nature 321, 246-249 (1986). The HPV E6 and E7 antigens
are the most promising targets for virus associated cancers in
immunocompetent individuals because of their ubiquitous expression
in cervical cancer. In addition to their importance as targets for
therapeutic cancer vaccines, virus associated tumor antigens are
also ideal candidates for prophylactic vaccines. Indeed,
introduction of prophylactic HBV vaccines in Asia have decreased
the incidence of hepatoma (Chang, M. H., et al, New Engl. J. Med.
336, 1855-1859 (1997), representing a great impact on cancer
prevention.
[0271] Among the most important viruses in chronic human viral
infections are human papillomavirus (HPV) hepatitis B virus (HBV),
hepatitis C Virus (HCV), human immunodeficiency virus (HIV),
Epstein Barr Virus (EBV) and herpes simplex virus (HSV).
[0272] In addition to its applicability to human cancer and
infectious diseases, the present invention is also intended for use
in treating animal diseases in the veterinary medicine context.
Thus, the approaches described herein may be readily applied by one
skilled in the art to treatment of veterinary herpesvirus
infections including equine herpesviruses, bovine herpesviruses,
Marek's disease virus in chickens and other fowl; animal retroviral
diseases; pseudorabies and rabies and the like.
[0273] The following references set forth principles and current
information in the field of basic, medical and veterinary virology
and are incorporated by reference: Fields Virology, Fields, B N et
al., eds., Lippincott Williams & Wilkins, NY, 1996;
[0274] Principles of Virology: Molecular Biology, Pathogenesis, and
Control, Flint, S. J. et al., eds., Amer Society for Microbiology,
Washington, 1999; Principles and Practice of Clinical Virology, 4th
Edition, Zuckerman. A. J. et al., eds, John Wiley & Sons, NY,
1999; The Hepatitis C Viruses, by Hagedorn, C H et al., eds.,
Springer Verlag, 1999; Hepatitis B Virus: Molecular Mechanisms in
Disease and Novel Strategies for Therapy, Koshy, R. et al., eds,
World Scientific Pub Co, 1998; Veterinary Virology, Murphy, F. A.
et al., eds., Academic Press, NY, 1999; Avian Viruses: Function and
Control, Ritchie, B. W., Iowa State University Press, Ames, 2000;
Virus Taxonomy: Classification and Nomenclature of Viruses: Seventh
Report of the International Committee on Taxonomy of Viruses, by M.
H. V. Van Regenmortel, MHV et al., eds., Academic Press; NY,
2000.
Targeting Molecules
[0275] A number of proteins that have various modes of action have
been implicated as "targeting" molecules to be used in conjunction
with antigens, preferably as fusion polypeptides, to target the
antigen to cells and subcellular compartments that promote
presentation of the antigen to T cells in a more potent and
effective manner.
[0276] Linkage of antigens to heat shock proteins (HSPs) represents
a potential approach for increasing the potency of nucleic
acid-based (and other) vaccines. HSPs appear to act as natural
biologic adjuvants in cancer and viral vaccination. Both the gp96
HSP resident in the endoplasmic reticulum (ER) and the cytosolic
Hsp70 act as immunologic adjuvants (Srivastava, P K et al., Semin.
Immunol. 3, 57-64 (1991); Udono, H et al., Proc. Natl. Acad. Sci.
USA 91, 3077-3081 (1994)). These HSPs, or chaperonins, bind a wide
array of peptides (Lanunert, E., et al. Eur. J. Immunol. 27,
923-927 (1997)). Hsp70 is a chaperonin that can target associated
proteins to the proteosorne--the primary cellular protease complex
that generates peptides for association with MHC class I molecules.
Therefore, antigens directly linked to Hsp70 are more efficiently
presented by MHC class I (leading, inter alia, to CTL responses).
Two features appear to be responsible for HSP adjuvanticity: (1) in
vitro, peptide loaded gp96 effectively introduce antigens into the
MHC class I processing pathway; (2) binding of gp96 to macrophages
induces secretion of proinflammatory cytokines, thus augmenting the
function of the cells to which the peptide antigen has been
targeted.
[0277] Immunization with HSP complexes isolated from tumor or from
virus-infected cells induces potent antitumor immunity (Srivastava,
P K et al, Int J Cancer. 33: 417-22, 1984; Srivastava, P K et al.,
Proc Natl Acad Sci USA. 83: 3407-11, 1986; Udono, H et al, J.
Immunol 152: 5398-5403, 1994; Blachere, N E et al, J Immunother.
14: 352-6, 1993; Udono, H et al, supra; Tamura, Y et al, Science.
278: 117-20, 1997; Janetzki, S et al, J Immunother. 21: 269-76,
1998)) or antiviral immunity (Heikema, A et al., Immunol Lett 57:
69-74, 1997; Suto, R et al., Science. 269: 1585-8, 1995). Mixing
peptides with HSPs in vitro generated immunogenic HSP-peptide
complexes (Ciupitu, A M et al., J Exp Med. 187: 685-91, 1998;
Blachere, N E et al., J Exp Med. 186: 1315-22, 1997). Some
HSP-based protein vaccines involved fusion of the antigen to the
HSP (Suzue, K et al., J Immunol. 156: 873-9, 1996; Suzue, K. et
al., Proc Natl Acad Sci USA 94: 13146-51, 1997). More recently, the
present inventors and their colleagues (e.g., Chen, C-H et al.,
Canc. Res. 60:1035-1042 (2000)) used HSPs in the form of chimeric
DNA or RNA replicon vaccines. They used HPV-16 E7 as antigen fused
to Mycobacterium tuberculosis HSP70 and showed increased expansion
and activation of E7-specific CD8+ T cells which resulted in potent
antitumor immunity against established tumors (Lin, K.-Y. et al.,
Cancer Res. 56: 21-26, 1996).
[0278] Another useful targeting molecule is the translocation
domain of a Pseudomonas exotoxin A (ETA), e.g., domain II (dII) of
ETA (spanning residues 253-364). A translocation domain is a
polypeptide that induces translocation of protein or polypeptide to
which it is linked into the cytosol of a cell. For example,
similarly applicable polypeptide are derived from a Diphtheria,
Clostridia (botulinum, tetani), Anthrax, Yersinia, Vibrio cholerae,
or Bordetella pertussis toxin. The toxic domain of the DNA encoding
the toxin is preferably mutated or deleted in the preparation of
such compositions.
[0279] Calreticulin (CRT) is an abundant 46 kDa protein located in
the endoplasmic reticulum (ER) lumen that displays lectin activity
and is known to be involved in the folding and assembly of nascent
glycoproteins (Nash (1994) Mol. Cell. Biochem. 135:71-78; Hebert
(1997) J. Cell Biol. 139:613-623; Vassilakos (1998) Biochemistry
37:3480-3490; Spiro (1996) J. Biol. Chem. 271:11588-11594. CRT
associates with peptides transported into the ER by transporters
associated with antigen processing, such as TAP-1 and TAP-2 (Spee
(1997) Eur. J. Immunol. 27:2441-2449). CRT forms complexes in vitro
with peptides. These complexes, when administered to mice, elicited
peptide-specific CD8+ T cell responses (Basu (1999) J. Exp. Med.
189:797-802; Nair (1999) J. Immunol. 162:6426-6432). CRT purified
from mouse tumors elicited immunity specific to the tumor used as
the source of CRT, but not to an antigenically distinct tumor
(Basu, supra). By pulsing DCs in vitro with a CRT bound to a
peptide, the peptide was re-presented in the context of DC Class I
molecules and stimulated peptide-specific CTLs (Nair, supra).
[0280] The Flt-3 ligand stimulates growth of DC precursors and can
promote generation of large numbers of DCs in vivo (Maraskovsky, E.
et al., J Exp Med. 184: 1953-62, 1996; Shurin, M R. et al., Cell
Immunol. 179: 174-84, 1997). Flt3, a murine tyrosine kinase
receptor (Rosnet, O. et al., Oncogene 6: 1641-50, 1991) is a member
of the III receptor kinase family (for review see Lyman, S D, Curr
Opin Hematol. 5: 192-6, 1998). In hematopoietic tissues, the
expression of Flt3 is restricted to the CD34-positive progenitors.
Flt3 was used to identify and subsequently clone, the corresponding
ligand, Flt3-ligand (Lyman, S D et al., Cell 75: 1157-67, 1993;
Hannum, C et al., Nature 368: 643-8, 1994). The predominant form of
Flt3-ligand is synthesized as a transmembrane protein from which
the functionally similar soluble ECD is generated by proteolytic
cleavage (Lyman et al., supra). These proteins bind to and
activating unique tyrosine kinase receptors. Among hematopoietic
cells, expression of the Flt3 receptor is primarily restricted to
the most primitive progenitor cells, including DC precursors. The
ECD of Flt3-ligand generated strong anti-tumor effects against
several murine model tumors including fibrosarcoma, breast cancer,
liver cancer, lung cancer, melanoma and lymphoma (Lynch, D H et
al., Nat Med. 3: 625-631, 1997;Chen, K et al., Cancer Res. 57:
3511-3516, 1997; Braun, S E et al., Hum Gene Ther. 10: 2141-2151,
1999; Peron, J M et al., J Immunol. 161: 6164-6170, 1998;
Chakravarty, P K et al., Cancer Res. 59: 6028-6032, 1999; Bache, C
et al., Cancer Res. 58: 380-383, 1998.) (19). The present
inventors' colleagues linked DNA encoding HPV Ek7 protein to DNA
encoding Flt3-ligand ECD. Immunization with this construct
dramatically increased expansion and activation of E7
antigen-specific CD8.sup.+ T cells, resulting in potent anti-tumor
immunity against established E7-expressing metastatic tumors.
[0281] The HSV-1 protein VP22 is a prototype protein that
contributes, among other things, to enhanced spread of antigen due
to its remarkable property of intercellular transport (Elliott, G.,
and P. O'Hare. 1997. Cell 88:223-33) can be used. For example, VP22
linked to p53 (Phelan, A. et al., 1998, Nat Biotechnol 16:440-443)
or thymidine kinase (Dilber, M S et al., 1999, Gene Ther 6:12-21),
facilitated the spread of linked protein to surrounding cells in
vitro and the treatment of model tumors. VP22 linked to HPV-16 E7
antigen in the context of a DNA vaccine led to a dramatic increase
in the number of E7-specific CD8.sup.+ T cell precursors in
vaccinated mice (around 50-fold) and converted a less effective DNA
vaccine into one with significant potency against E7-expressing
tumors. Anon-spreading VP22 mutant failed to enhance vaccine
potency. VP22 and proteins that may have a similar mode of action,
contribute in several ways to enhanced vaccine potency: (1)
facilitate spreading of antigen from transfected cells to
surrounding APCs, thereby increasing the number of APCs that
present antigen through MHC class I pathway; (2) present antigen
more efficiently in transfected cells (3) carryout "cross-priming"
whereby release of a VP22/antigen fusion protein leads to uptake
and processing by DCs (or other APCs) for presentation via the
MHC-I restricted pathway (Huang, A Y et al., 1994, Science
264:961-965)
[0282] Those skilled in the art will know how to identify
appropriate epitopes, e.g., CTL epitopes, of the relevant proteins
from the pathogens for use in accordance with this invention.
Delivery of B7-DC DNA to Cells and Animals
[0283] DNA delivery, for example to effect what is generally known
as "gene therapy" involves introduction of a "foreign" DNA into a
cell and ultimately, into a live animal. Several general strategies
for gene therapy have been studied and have been reviewed
extensively (Yang, N-S., Crit. Rev. Biotechnol. 12:335-356 (1992);
Anderson, W. F., Science 256:808-813 (1992); Miller, A. S., Nature
357:455-460 (1992); Crystal, R. G., Amer. J. Med. 92(suppl
6A):44S-52S (1992); Zwiebel, J. A. et al., Ann. N.Y. Acad. Sci.
618:394-404 (1991); McLachlin, J. R. et al., Prog. Nucl. Acid Res.
Molec. Biol. 38:91-135 (1990); Kohn, D. B. et al., Cancer Invest.
7:179-192 (1989), which references are herein incorporated by
reference in their entirety).
[0284] One approach comprises nucleic acid transfer into primary
cells in culture followed by autologous transplantation of the ex
vivo transformed cells into the host, either systemically or into a
particular organ or tissue.
[0285] For accomplishing the objectives of the present invention,
nucleic acid therapy would be accomplished by direct transfer of a
the functionally active DNA into mammalian somatic tissue or organ
in vivo. DNA transfer can be achieved using a number of approaches
described below. These systems can be tested for successful
expression in vitro by use of a selectable marker (e.g., G418
resistance) to select transfected clones expressing the DNA,
followed by detection of the presence of the B7-DC expression
product (after treatment with the inducer in the case of an
inducible system) using an antibody to the product in an
appropriate immunoassay. Efficiency of the procedure, including DNA
uptake, plasmid integration and stability of integrated plasmids,
can be improved by linearizing the plasmid DNA using known methods,
and co-transfection using high molecular weight mammalian DNA as a
"carrier".
[0286] Examples of successful "gene transfer" reported in the art
include: (a) direct injection of plasmid DNA into mouse muscle
tissues, which led to expression of marker genes for an indefinite
period of time (Wolff, J. A. et al., Science 247:1465 (1990);
Acsadi, G. et al., The New Biologist 3:71 (1991)); (b) retroviral
vectors are effective for in vivo and in situ infection of blood
vessel tissues; (c) portal vein injection and direct injection of
retrovirus preparations into liver effected gene transfer and
expression in vivo (Horzaglou, M. et al., J. Biol. Chem. 265:17285
(1990); Koleko, M. et al., Human Gene Therapy 2:27 (1991); Ferry,
N. et al., Proc. Natl. Acad. Sci. USA 88:8387 (1991)); (d)
intratracheal infusion of recombinant adenovirus into lung tissues
was effective for in vivo transfer and prolonged expression of
foreign genes in lung respiratory epithelium (Rosenfeld, M. A. et
al., Science 252:431 (1991); (e) Herpes simplex virus vectors
achieved in vivo gene transfer into brain tissue (Ahmad, F. et al.,
eds, Miami Short Reports--Advances in Gene Technology: The
Molecular Biology of Human Genetic Disease, Vol 1, Boerringer
Manneheim Biochemicals, USA, 1991).
[0287] Retroviral-mediated human therapy utilizes amphotrophic,
replication-deficient retrovirus systems (Temin, H. M., Human Gene
Therapy 1:111(1990); Temin et al., U.S. Pat. No. 4,980,289; Temin
et al., U.S. Pat. No. 4,650,764; Temin et al., U.S. Pat. No.
5,124,263; Wills, J. W. U.S. Pat. No. 5,175,099; Miller, A. D.,
U.S. Pat. No. 4,861,719). Such vectors have been used to introduce
functional DNA into human cells or tissues, for example, the
adenosine deaminase gene into lymphocytes, the NPT-II gene and the
gene for tumor necrosis factor into tumor infiltrating lymphocytes.
Retrovirus-mediated gene delivery generally requires target cell
proliferation for gene transfer (Miller, D. G. et al., Mol. Cell.
Biol. 10:4239 (1990). This condition is met by certain of the
preferred target cells into which the present DNA molecules are to
be introduced, i.e., actively growing tumor cells. Gene therapy of
cystic fibrosis using transfection by plasmids using any of a
number of methods and by retroviral vectors has been described by
Collins et al., U.S. Pat. No. 5,240,846.
[0288] The DNA molecules encoding the B7-DC sequences may be
packaged into retrovirus vectors using packaging cell lines that
produce replication-defective retroviruses, as is well-known in the
art (see, for example, Cone, R. D. et al., Proc. Natl. Acad. Sci.
USA 81:6349-6353 (1984); Mann, R. F. et al., Cell 33:153-159
(1983); Miller, A. D. et al., Molec. Cell. Biol. 5:431-437 (1985),;
Sorge, J., et al., Molec. Cell. Biol. 4:1730-1737 (1984); Hock, R.
A. et al., Nature 320:257 (1986); Miller, A. D. et al., Molec.
Cell. Biol. 6:2895-2902 (1986). Newer packaging cell lines which
are efficient an safe for gene transfer have also been described
(Bank a al., U.S. Pat. No. 5,278,056.
[0289] This approach can be utilized in a site specific manner to
deliver the retroviral vector to the tissue or organ of choice.
Thus, for example, a catheter delivery system can be used (Nabel, E
G et al., Science 244:1342 (1989)). Such methods, using either a
retroviral vector or a liposome vector, are particularly useful to
deliver the nucleic acid to be expressed to a blood vessel wall, or
into the blood circulation of a tumor.
[0290] Other virus vectors may also be used, including recombinant
adenoviruses (Horowitz, M. S., In: Virology, Fields, B N et al.,
eds, Raven Press, New York, 1990, p. 1679; Berkner, K. L.,
Biotechniques 6:616 9191988), Strauss, S. E., In: The Adenoviruses,
Ginsberg, H S, ed., Plenum Press, New York, 1984, chapter 11),
herpes simplex virus (HSV) for neuron-specific delivery and
persistence. Advantages of adenovirus vectors for human gene
therapy include the fact that recombination is rare, no human
malignancies are known to be associated with such viruses, the
adenovirus genome is double stranded DNA which can be manipulated
to accept foreign genes of up to 7.5 kb in size, and live
adenovirus is a safe human vaccine organisms. Adeno-associated
virus is also useful for human therapy (Samulski, R. J. et al.,
EMBO J. 10:3941 (1991) according to the present invention.
[0291] Another vector which can express the DNA molecule of the
present invention, and is useful in the present therapeutic
setting, particularly in humans, is vaccinia virus, which can be
rendered non-replicating (U.S. Pat. Nos. 5,225,336; 5,204,243;
5,155,020; 4,769,330; Sutter, G et al., Proc. Natl. Acad. Sci. USA
(1992) 89:10847-10851; Fuerst, T. R. et al., Proc. Natl. Acad. Sci.
USA (1989) 86:2549-2553; Falkner F. G. et al.; Nucl. Acids Res
(1987) 15:7192; Chakrabarti, S et al., Molec. Cell. Biol. (1985)
5:3403-3409). Descriptions of recombinant vaccinia viruses and
other viruses containing heterologous DNA and their uses in
immunization and DNA therapy are reviewed in: Moss, B., Curr. Opin.
Genet. Dev. (1993) 3:86-90; Moss, B. Biotechnology (1992) 20:
345-362; Moss, B., Curr Top Microbiol Immunol (1992) 158:25-38;
Moss, B., Science (1991) 252:1662-1667; Piccini, A et al., Adv.
Virus Res. (1988) 34:43-64; Moss, B. et al., Gene Amplif Anal
(1983) 3:201-213.
[0292] In addition to naked DNA or RNA, or viral vectors,
engineered bacteria may be used as vectors. A number of bacterial
strains including Salmonella, BCG and Listeria monocytogenes (LM)
(Hoiseth & Stocker, Nature 291, 238-239 (1981); Poirier, T P et
al. J. Exp. Med. 168, 25-32 (1988); (Sadoff, J. C., et al., Science
240, 336-338 (1988); Stover, C. K., et al., Nature 351, 456-460
(1991); Aldovini, A. et al., Nature 351, 479-482 (1991); Schafer,
R., et al., J. Immunol. 149, 53-59 (1992); Ikonomidis, G. et at, J.
Exp. Med. 180, 2209-2218 (1994)). These organisms display two
promising characteristics for use as vaccine vectors: (1) enteric
routes of infection, providing the possibility of oral vaccine
delivery; and (2) infection of monocytes/macrophages thereby
targeting antigens to professional APCs.
[0293] In addition to virus-mediated gene transfer in vivo,
physical means well-known in the art can be used for direct
transfer of DNA, including administration of plasmid DNA (Wolff et
al., 1990, supra) and particle-bombardment mediated gene transfer
(Yang, N.-S., et al., Proc. Natl. Acad. Sci. USA 87:9568 (1990);
Williams, R. S. et al., Proc. Natl. Acad. Sci. USA 88:2726 (1991);
Zelenin, A. V. et al., FEBS Lett. 280:94 (1991); Zelenin, A. V. et
al., FEBS Lett. 244:65 (1989); Johnston, S. A. et al., In Vitro
Cell. Dev. Biol. 27:11(1991)). Furthermore, electroporation, a
well-known means to transfer genes into cell in vitro, can be used
to transfer DNA molecules according to the present invention to
tissues in vivo (Titomirov, A. V. et al., Biochim. Biophys. Acta
1088:131 ((1991)).
[0294] "Carrier mediated gene transfer" has also been described
(Wu, C. H. et al., J. Biol. Chem. 264:16985 (1989); Wu, G. Y. et
al., J. Biol. Chem. 263:14621 (1988); Soriano, P. et al., Proc.
Natl. Acad. Sci. USA 80:7128 (1983); Wang, C-Y. et al., Proc. Natl.
Acad. Sci. USA 84:7851 (1982); Wilson, J. M. et al., J. Biol. Chem.
267:963 (1992)). Preferred carriers are targeted liposomes
(Nicolau, C. et al., Proc. Natl. Acad. Sci. USA 80:1068 (1983);
Soriano et al., supra) such as immunoliposomes, which can
incorporate acylated mAbs into the lipid bilayer (Wang et al.,
supra). Polycations such as asialoglycoprotein/polylysine (Wu et
al., 1989, supra) may be used, where the conjugate includes a
molecule which recognizes the target tissue (e.g.,
asialoorosomucoid for liver) and a DNA binding compound to bind to
the DNA to be transfected. Polylysine is an example of a DNA
binding molecule which binds DNA without damaging it. This
conjugate is then complexed with plasmid DNA according to the
present invention for transfer.
[0295] Plasmid DNA used for transfection or microinjection may be
prepared using methods well-known in the art, for example using the
Quiagen procedure (Quiagen), followed by DNA purification using
known methods, such as the methods exemplified herein.
[0296] Again, as noted above, for the utility of transduced. B7-DC
molecules according to this invention may not require stable
expression. Rather, transient expression of the polypeptide may be
sufficient for transduced cells to perform their immunogenic and/or
costimulatory function.
[0297] Having now generally described the invention, the same will
be more readily understood through reference to the following
examples which are provided by way of illustration, and are not
intended to be limiting of the present invention, unless
specified.
EXAMPLE I
Materials and Methods
Cell Preparation and Culture
[0298] 6-12 week old female BALB/c mice were purchased from NCI and
used for DC and macrophage preparation.
[0299] Bone marrow-derived DCs were cultured in RPMI1640 (Gibco
BRL) medium supplemented with 5% fetal calf serum (FCS) (Hyclone),
Penicillin/Streptomycin (JRH Biosciences), Gentamycin (Sigma),
Nonessential amino acids (JRH Biosciences), L-Glutamate (JRH
Biosciences), Sodium Pyruvate (Sigma), 2 mercaptoethanol (Sigma)
and 1000 units/ml recombinant murine GM-CSF (Immunex) as previously
described (26). Day 8 bone marrow-derived DCs were stained with
monoclonal antibodies by conventional methods. Monoclonal antibody
against MHC class II, 14-4-4s, was purified from hybridoma
supernatant. Dr. William Baldwin, Johns Hopkins University, kindly
supplied CTLA4-Ig fusion molecule. Antibodies for MEC class I
(28-14-8), F4/80 (C1.A3-1), B7.1 (1G10), B7.2 (GL1),
Fc.gamma.RII/III (2.4G2) and Mac-1 (M1/70) were purchased from
PharMingen. For tester cDNA preparation, day 8 cells were purified
by cell sorter using 14-4-4s and CTLA4-Ig at the Johns Hopkins
University Oncology Center. The purity of MHC class II.sup.hi and
B7.sup.hi population after sorting was 93-98%.
[0300] Bone marrow-derived macrophages were cultured with RPMI-1640
medium supplemented with 10% FCS, Penicillin/Streptomycin,
non-essential amino acids, sodium pyruvate, L glutamine,
2-mercaptoethanol and 250 units/ml recombinant murine M-CSF, and
they were treated with 500 units/ml. .gamma.-IFN (Pharmingen) and 5
.mu.g/ml LPS (Sigma) as previously described (27). After
stimulation, cell surface expression of MHC class II and B7 were
confirmed using flow cytometric analysis on day 10 of culture.
[0301] Macrophage cell lines WEHI-3, RAW264.7, J774.A.1, PU5-1.8
were kindly provided by Dr. Joshua Farber of the NIAID, National
Institutes of Health. They were cultured with ATCC recommended
medium.
Allogeneic Mixed Lymphocyte Reaction
[0302] Day 8 BM-derived DCs characterized as MHC class II.sup.hi
and B7.sup.hi were tested for their ability to stimulate allogeneic
T cells in MLC. MLC reactions were performed in 96 well flat bottom
microplates by adding increasing number of BALB/c stimulator cells
s to 3.times.10.sup.5 allogeneic C57BL/6 lymphocytes. After 3 days
of culture, T cell proliferation was assessed by addition of 1
.mu.Ci of [.sup.3H]-methyl-thymidine (Amersham) to each well for
the final 18 h of culture. Cells were then harvested, and
incorporation of radioactivity was determined using a counter
(Packard 96),
cDNA Subtractive Hybridization
[0303] Total RNA from sorted DCs and activated macrophages was
extracted with TRIZOL (Gibco BRL). Messenger RNA was purified by
Oligotex mRNA purification kit (Qiagen). We used the PCR based
SMART cDNA synthesis system (Clonetech) to amplify cDNA followed by
the PCR based subtraction system. PCR Select (Clonetech).
Subtraction was performed following the manufacturer's protocol.
After final subtractive PCR, DNA fragments were ligated into
plasmid vectors pCR2.1 (Invitrogen) or pCR Blunt (Invitrogen).
After transformation, each clone was grown for plasmid DNA
amplification and miniprep DNA and then digested with EcoRI to
confirm the presence of inserts. Plasmid dot blot was then
performed to confirm that the cDNA cloned is dendritic cell
specific. Alkaline denatured miniprep DNAs were spotted on Hybond
N+ membrane (Amersham) and hybridized with SMART cDNA probe-derived
from sorted DCs or activated macrophages. These cDNA probes were
.sup.32P labeled using random primer labeling method (Stratagene
Prime-It II). Hybridizations and washing were done as previously
described (28). Membranes were exposed to a film (Amersham) for 1-2
days and developed.
Plasmid Dot Blot Analysis
[0304] Alkaline denatured miniprep DNA samples were spotted on
Hybond N+ membrane (Amersham) and hybridized with SMART.RTM. cDNA
probe-derived from sorted DCs or activated macrophages. These cDNA,
probes were .sup.32P labeled using a random primer labeling method
(Stratagene Prime It II). Hybridizations and washing were done as
previously described [cites??]. For autoradiography, membranes were
exposed to a film (Amersham) for 1-2 days and developed.
cDNA Library Construction and Screening--Cloning of B7-DC
[0305] Bone marrow-derived DCs were harvested on day 8 without
sorting. About 20% to 40% of these cells expressed high MHC class
II and B7. Total RNA extraction followed by poly A RNA purification
was done as described above. For oligo dT primed DC library
construction, we used Lambda ZAP Express cDNA synthesis system
(Stratagene). The PCR DNA fragment of B7-DC was probed and used for
screening. Membrane transfer, denaturation, renaturation, were
performed using Stratagene's protocol. Radiolabeling of probes,
hybridization, washing, and autoradiography were done as described
above. Positive clones were isolated and 2nd screening was
performed. After 2nd screening, plasmids were excised by in vivo
excision and tested by dot blotting and sequencing. Sequencing was
done by the Core Facility at the Johns Hopkins University School of
Medicine. BLAST program was used to do homology search of the
nucleotide sequence against Genbank (NCBI) for similarity to
previously reported genes. The full length B7-DC cDNA clone was
pulled out from the DC cDNA library. 5' RACE was performed suing
SMART RACE cDNA amplification kit (Clontech). 5'-RACE products were
cloned into pCR2.1 vector and sequenced. Two more full length B7-DC
clones were obtained by RT-PCR and their sequence were compared to
avoid sequence error.
[0306] Human B7-DC was cloned as follows: human DCs were obtained
from normal peripheral blood mononuclear cells by culture in either
GM-CSF+IL-4 or GM-CSF+Flt-3L as described previously (29). RNA was
extracted as described above. A BLAST search identified an
overlapping EST clone, GenBank accession number AK001879, with
homology to mouse B7-DC. 5' RACE was performed as described above.
We sequenced a 5'-RACE PCR fragment and designed a primer
corresponding to 5'-UTR of human B7DC. The following primers in the
5'-UTR and 3'-UTR of B7-DC was used to amplify full-length human
B7-DC:
TABLE-US-00006 5'-GGAGCTACTGCATGTTGATTGTTTTG-3' [SEQ ID NO: 6] and
5'-TGCAAACTGAGGCACTGAAAAGTC-3' [SEQ ID NO: 7]
The full length cDNA sequences of the human and murine B7-DC cDNAs
have been deposited with EMBL/GenBank/DDBS under accession number
AF329193 and AF142780.
BAC (129SVJ) Library Screening/Genomic Cloning and Mapping
[0307] BAC library screening, followed manufacturer's protocol
(Genome Systems, Inc.) Primers used:
TABLE-US-00007 5'-TTGTTGTCTCCTTCTGTCTCCCAAC-3' [SEQ ID NO: 8] and
5'-ACAGTTGCTCCTTGTATCAGGTTC-3' [SEQ ID NO: 9]
[0308] BAC library screening obtained 3 positive clones. Chromosome
location mapping was done by fluorescence in situ hybridization
(Genome Systems Inc.). A total of 80 metaphase cells were analyzed
with 79 exhibiting specific labeling. The human B7-DC mapping was
done by using available bioinformatic tools, NCBI's BLAST program
and the International RH Mapping Consortium. The hB7-DC sequence
was searched in htsg and was found to map to two BAC clones
RP11-574F11 (AL162253) and Rp11-635N21 (AL354744) localizing on
chromosome 9.
Virtual Northern Blotting
[0309] 4-6 weeks old female Balb/c mice were purchased from NCI and
used for tissue RNA preparation. RNA extraction and SMART cDNA
synthesis for tissues, sorted DCs and activated macrophages were
performed as described above. SMART PCR cDNAs were purified by PCR
purification kit (Qiagen). 0.5 .mu.g/lane purified DNAs were run on
a 1% agarose gel and transferred on a Nytran nylon membrane
(Schleier and Schuell). To make radioactive probes, we amplified
subtracted library derived plasmid DNAs as templates. We amplified
DNA by PCR using primer sets just adjacent to the cloning site of
plasmid DNA and used purified PCR DNA of each of the clones for
hybridization probes. The nucleotide sequences of these primers are
as follows.
TABLE-US-00008 5'-GTAACGGCCGCCAGTGTGCTG-3' [SEQ ID NO: 10] and
5'-CGCCAGTGTGATGGATATCTGCA-3' [SEQ ID NO: 11]
Virtual Northern analysis of total RNA of human DCs and control
placenta was also performed. The probes used and RNA preparation
were described above. Radiolabeling of probes, hybridization,
washing and autoradiography were done as described above.
Hamster Anti-mB7-DC Ab Production
[0310] Stable transfectants of B7-DC in DC2.4, RAW246.7 and RENCA
cell lines were used to immunize Armenian Hamsters. The B7-DC was
cloned into the modified pCAGGS vector (30). The hamsters were
boosted three times with plasmids containing B7-DC (Rockland). The
anti-B7-DC antibody used in this study was from one the sera of one
of the three hamsters immunized.
CD28-Ig, CTLA4-Ig and PD-1-IG Binding Assay
[0311] 293T cells were transfected with B7.1-pCAGGS, B7-DC-pCAGG,
PD-1-pCAGGS or vector alone using Lipofectamine 2000 (Gibco BRL).
After 24 hrs, cells were resuspended in FACS buffer (1.times.HBSS,
2% calf serum, 10 mM HEPES and 0.1% NaN.sub.3) and spun at 1000 rpm
for 5 min at 4.degree. C. The buffer was then decanted, antibody
added to the tubes, incubated at 4.degree. C. for 20 min, washed
2.times. with FACS buffer, and repeated this for secondary
antibody. The samples were run on FACScan. B7.1 antibody was used
1:5 dilution, 10 .mu.l/sample (Cal-Tag). Recombinant CD28-Ig,
CTLA-4-Ig and PD-1-Ig chimeras were used at 2 .mu.g/ml, 10
.mu.l/sample (R&D System, Inc.). Goat F(ab').sub.2 anti-human
IgG-PE was used at 1:20 dilution (Southern Biotechnology
Associates, Inc.).
B7-DC-Ig Dimer Synthesis
[0312] The B7-DC-Ig construct was made by fusing the sequence
encoding the N-terminal amino acids of B7-DC without the
transmembrane domain in-frame to the sequence encoding the
C-terminal amino acids of the human IgG.sub.1 Fc-in the pIg-Tail
Plus vector (R &D systems). COS-7 cells were transiently
transfected with pIg/B7-DC using LipofectAMINE 2000 (GIBCO BRL) or
GINE JAMMER (Stratagene). The B7-DC-Ig fusion protein was purified
from the serum-free supernatants using the saturated ammonium
sulfate precipitation. SDS-PAGE and silver staining demonstrated a
purity >90%.
T Cell Proliferation and Cytokine Assays
[0313] For costimulation assays with anti-CD3, 96 well flat bottom
plates (Immulon 4 from Dynex) were precoated with anti-CD3
antibodies (2C11, Pharmingen) and B7.1-Ig (R&D System),
B7-DC-Ig or Isotype control (Sigma) at 100 ng/ml were diluted in
1.times.PBS (Gibco) re 7.4 for two hours at 37.degree. C. The
plates were then washed 3.times. with 1.times.PBS and blocked with
RPMI1640 medium supplemented with 10% FCS, Penicillin/Streptomycin,
non-essential amino acids, sodium pyruvate, L glutamine,
2-mercaptoethanol for one half hour before adding T cells. Spleens
and lymph nodes were obtained from 6-10 weeks old BALB/c mice. RBCS
were lysed using ACK buffer and T cells were purified using
dynabeads M-280 (Dynex), with the indirect method. The beads were
washed 2.times. with PBS pH 7.4+1% FCS before adding the cells and
an antibody cocktail composed of anti-IE.sup.d and B220/CD45RO or
CD8.alpha. (Pharmingen) was added to the cells and incubated at
4.degree. C. with bi-directional mixing for 30'. The cells were
isolated by placing the tube in a Dynal MPC for 5', centrifuged at
1500 rpm for 5' and washed 2.times. with PBS pH 7.4+1% FCS to
remove unbound Abs. The same procedure was repeated with 15'
incubation, and the cells plated at 2.times.10.sup.5 cells/well.
After 72 h of incubation, 10 .mu.l of .sup.3H-thymidine (1
.mu.Ci/well) was added to each well and incubated for 18 hrs. Cells
were harvested with a Packard Micromate Cell Harvester, and filters
were read on a Packard Matrix 96 direct .beta. counter.
[0314] For costimulation assays using the RENCA system to present
HA antigen, RENCA cells were cultured with RPMI-1640 medium
supplemented with 10% FCS, Penicillin/Streptomycin, non-essential
amino acids, sodium pyruvate. It was induced with IFN-.gamma. (75
U/ml) for 72 hours for MHC class II expression. They were then
irradiated for 13,200 Rad, and plated at 2.times.10.sup.4
cells/well (96 well flat bottom plates). HA110-120 peptide was then
added at 2.5 ug/well and various concentrations of the Ig-fusion
molecules were added. Transgenic I-E.sup.d+HA specific T cells
(kind gift of H. von Boehmer, Harvard University) were isolated as
described above and plated at 4.times.10.sup.5 cells/well. After 48
hr of incubation, 10 .mu.l of .sup.3H thymidine (1 .mu.Ci/well) was
added to each well and incubated for 18 hrs. Cells were harvested
with a Packard Micromate Cell Harvester and filters were read on a
Packard Matrix 96 Direct .beta. counter.
[0315] For analysis of cytokine production by ELISA, cultures were
set up as described above and supernatants were harvested at the
indicated times. IL-2, IL-10 and IFN-.gamma. concentrations were
determined using commercially available ELISA kits (Endogen), and
IL-4 and IL-6 (R&D System).
In Vivo Costimulation
[0316] Pooled axillary, inguinal, cervical, and mesenteric LNs from
the TCR transgenic mouse line 6.5 that expresses a TCR recognizing
an I-E.sup.d restricted HA epitope (.sup.110SFERFEIFPKE.sup.120
[SEQ ID NO:12]) on a B10.D2 genetic background were dissociated in
RPMI media (GIBCO BRL), passed over 100 .mu.m nylon cell strainer,
and washed in sterile Hank's buffer (GIBCO BRL). After FACS.RTM.
staining to determine the proportion of clonotypic CD4 cells, a
cell preparation containing 2.5.times.10.sup.6 clonotypic cells in
0.2 ml sterile Hank's buffer was injected intravenously (i.v.) into
the tail vein of recipient B10.D2 mice. Three days after this
adoptive transfer, the animals were vaccinated via subcutaneous
(s.c.) injection into the hind footpads. Each mouse received
bilateral injections of one of three preparations: [0317] (A) 10
.mu.g synthetic HA (per footpad) (HA peptide (110-120) combined in
a 1:1 v/v ratio with incomplete Freund's Adjuvant (IFA) (Sigma),
[0318] (B) the HA-IFA mixture with 25 .mu.g of B7-DC-Ig, or [0319]
(C) the HA-IFA mixture with 25 .mu.g of an isotype control
antibody. 7 days later, draining LNs nodes were harvested;
1.5.times.10.sup.5 LN cells were incubated in round-bottom 96-well
tissue culture plates with the indicated concentration of synthetic
HA peptide. Proliferation assays were performed by pulsing 48 h
cultures with 1 .mu.Ci [.sup.3H]thymidine and incubating an
additional 12 h before harvest and determination of the amount of
incorporated radioactivity.
EXAMPLE II
Identification and Characterization of B7-DC
[0320] B7-DC was isolated from a subtracted library between DCs and
activated macrophages. The two populations used for cDNA
subtraction were bone marrow-derived GM-CSP cultured DCs as the
"tester" population and .gamma.-interferon+LPS activated adherent
bone marrow-derived M-CSF macrophages as the "driver" population.
Day 8 MHC class II.sup.hiB7.sup.hi "mature" DCs were sorted to
>93% purity as the source of tester cDNA. DCs were characterized
by flow cytometry as having roughly 50 fold higher MHC class II
levels than macrophages. Both populations expressed B7.1 and B7.2
although B7.2 levels were significantly higher in the DCs. F4/80
and CD16 were expressed at higher levels on the macrophage
population. Functional comparison of the two populations
demonstrated that the DC population was roughly 100 fold more
potent than the macrophage population in stimulating an allogeneic
mixed lymphocyte reaction.
[0321] After RNA extraction from both populations, we used a PCR
based cDNA synthesis system followed by the PCR based subtraction
procedure, PCR Select. One of the differentially expressed clones
encoded a novel immunoglobulin supergene family member, which we
name B7-DC. The murine B7-DC cDNA is 1.7 kb in length encoding a
247 amino acid (aa) precursor protein with a 23 aa N-terminal
signal peptide and a predicted molecular weight of .about.25 kd
(Table 1). The putative leader sequence and transmembrane domain
were identified using the SOSUI program (31). Two charged aa are
found within the 23 aa transmembrane domain of mB7-DC, suggesting a
possible binding partner. At the aa level, murine B7-DC is 70%
identical to the human B7-DC indicating that they are orthologues
(See Tables below)
TABLE-US-00009 TABLE 1 Amino acid sequence comparison of murine
B7-DC and human B7-DC. The mB7-DC putative leader and transmembrane
domain are overlined. The alignment was done using Clustalw-Gonnet
Pam250 matrix. The [*] indicates identical amino acids and [:]
shows conservative substitutions. Cysteine residues that may be
important in the formation of disulfide bonds inside the
immunoglobulin V or C domains are italicized.. Putative leader
sequence mB7-DC
MLLLLPILNLSLQLHPVAALFTVTAPKEVYTVDVGSSVSLECDFDRRECTELEGIRASLQ hB7-DC
MIFLLLMLSLELQLHQIAALFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQ *::**
:*.*,**** :*******.***:* :: **.*:***:** . .:* .* **** mB7-DC
KVENDTSLQSERATLLEEQLPLGKALFHIPSVQVRDSGQYRCLVICGAAWDYKYLTVKVK hB7-DC
KVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVK
******* : *************** ****.*****.***:*::* *.********:*** mB7-DC
ASYMRIDTRILEVPGTGEVQLTCQARGYPLAEVSWQNVSVPANTSHIRTPEGLYQVTSVL hB7-DC
ASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVL ***
:*:*:**:** *.**:***** ********* ********** ************* Putative
TM domain mB7-DC RLKPQPSRNFSCMFWNAHMKELTSAIIDPLSRMEPKVPRTW
PLHVFIPACTIALIFLAIV hB7-DC
RLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPTWLLHIFIPSCIIAFIFIATV ****
*.*****:***:*::*** * ** *:***:. ** **:***:* **:**:* * mB7-DC
IIQRKRI-------------------------- hB7-DC
IALRKQLCQKLYSSKDTTKRPVTTTKREVNSAI * **::
TABLE-US-00010 TABLE 2 Amino acid sequence comparison of mB7-IDC
and mB7-H1. mB7-DC
MLLLLPILNLSLQLHPVAALFTVTAPKEVYTVDVGSSVSLECDFDRRECTELEGIRASLQ mB7-H1
-MRIFAGIIFTACCH-LLRAFTITAPKDLYVVEYGSNVTMECFRPVERELDLLALVVYWE : ::.
: :: * : **:****::*.*: **.*::** * .. :* .: . : mB7-DC
K----------VENDTSLQSE----RATLLEEQLPLGKALFHIPSVQVRDSGQYRCLVIC mB7-H1
KEDEQVIQFVAGEEDLKPQHSNFRGRASLPKDQLLKGNAALQITDVKLQDAGVYCCIISY * *:*
. * . **:* ::** :*: ::*..*:::*:* * *:: mB7-DC
GAAWDYKYLTVKVKASYMRIDTRILEVPGTGEVQLTCQARGYPLAEVSWQN-----VSVP mB7-H1
GGA-DYKRITLKVNAPYRKINQRISVDPATSEHELICQAEGYPEAEVIWTNSDHQPVSGK *.*
*** :*:**:8.* :*: ** *.*.* :* ***.*** *** * *. ** mB7-DC
ANTSHIRTPEGLYQVTSVLRLKPQPSRNFSCMFWWAH--MKELTSAIIDPLSRMEKKVPR mB7-H1
RSVTTSRTEGNLLNVTSSLRVNATANDVFYCTFWRSQPGQNHTAELIIPELPATHPPQNR ..: **
* :*** **::. .. * * **.:: :. :. ** *. .* * mB7-DC
T-WPLHVFIPACTIALIFLAIVIIQRKRI------------------------ mB7-H1
THWVLLGSILLFLIVVSTVLLFLRKQVRMLDVEKCGVEDTSSKNRNDTQFEET * * * * *.: :
:.: :: *:
The hB7-DC differs slightly from the murine B7-DC in that it has a
longer cytoplasmic tail.
TABLE-US-00011 TABLE 3 Amino acid sequence comparison of B7-DC to
the B7 family members. Reference B7 % % Protein Compared to:
identity.sup.1 similarity.sup.2 mB7-DC hB7-DC 70 80 hBT3.1 25 41
mB7-DC mB7-H1 34 48 mBT.sup.4 30 45 mB7.1 -- --.sup.3 mB7.2 -- --
mB7RP-1/B7h -- -- MBT mB7.1 24 48 mB7.2 24 40 MB7.1 mB7.2 27 45
MB7-H1.sup.5 mB7.1 23 40 mB7.2 25 49 mB7RP-1/B7h 24 41 mBT 24 45
Comparison were done using NCBI blast2 search (matrix BLOSUM62).
.sup.1Identical amino acids at corresponding positions
.sup.2Similar amino acids at corresponding position - grouped as
follows: (A, G); (S, T); (E, D); (R, K, H); (Q, N); (V, I, L, M);
(Y, F); (W); (C); (P) .sup.3no significant similarity was found
using matrix BLOSUM62 .sup.4BT = butyrophilin .sup.5=PDL-1
[0322] Through a homology search, it was found that B7-DC has
significant homology to B7-H1 (34% identity, 48% similarity) (Table
2), to a lesser extent butyrophilin (30% identity, 45% similarity),
and <20% identity to B7.1 and B7.2 (Table 3). Phylogenetic
studies indicate that butyrophilin is likely related to the B7
family through exon shuffling (32, 33). They each possess the
canonical IgV-IgC structure and a transmembrane domain. In contrast
to the other B7 family members, murine B7-DC has an extremely short
cytoplasmic tail (4 aa).
[0323] To determine the genomic structure of mB7-DC, the present
inventors isolated a genomic clone by screening a pooled Bacterial
Artificial Chromosome (BAC) library using probes from the 5' and 3'
UTRs. Chromosome location mapping was performed using the BAC
clones. Chromosome localization of B7-DC was done using fluorescent
in situ hybridization (FISH). Measurements of 10 specifically
labeled chromosomes 19 demonstrated that mB7-DC is located at a
position which is 47% of the distance from the
heterochromatic-euchromatic boundary to the telomere of chromosome
19, an area that corresponds to the interface between bands 19C2
and 19C3. Specific hybridization signals were detected by
incubating the hybridized slides in fluoresceinated antidigoxigenin
antibodies followed by counter staining with DAPI. This locus
corresponds to a region of human chromosome 9, where hB7-H1 has
been mapped.
[0324] hB7-DC was found to be located on two chromosome 9 BAC
clones. In addition, both hB7-DC and hB7-H1 were found to be
located on a single chromosome 9 BAC clone with an insertion size
of approximately 164 kb (FIG. 1). The genomic proximity of B7-DC
and B7-H1 is reminiscent of the B7.1/B7.2 pair, which map to within
one megabase of each other.
EXAMPLE III
B7-DC is Selectively Expressed in Dendritic Cells
[0325] In order to determine the expression pattern of B7-DC,
virtual Northern analysis was performed using RNA extracted from
multiple tissues, macrophage cell lines, macrophage cultures and
dendritic cells derived from both bone marrow and spleen. While
strong hybridization was detected using a B7-DC probe in immature
(day 4,6) and mature (day 8 and sorted MHC II.sup.hiB7.sup.hi) bone
marrow derived DC and splenic DC, no signal was detected in any of
4 macrophage cell lines, activated BM macrophages or peritoneal
macrophages (FIG. 2). Strong expression of hB7-DC was detected in
human DCs grown from peripheral blood mononuclear cells with GM-CSF
plus either IL-4 or Flt-3L (FIG. 3). In order to verify cell
surface expression of B7-DC protein, anti-mB7-DC antibodies were
used to stain DCs. Staining, blockable with soluble B7-DC-Ig, was
observed on DC (FIG. 4).
B7-DC Does Not Bind to CD28 or CTLA-4 But Does Bind to PD-1
[0326] Although B7-DC has structural and sequence homology to the
B7 family, it does not contain the putative CD28/CTLA-4 binding
sequence, SQDXXXELY [SEQ ID NO:13] or XXXYXXRT [SEQ ID NO:14] (34)
(where X=any amino acid). To directly assess binding, the ability
of dimeric CD28-Ig and CTLA-4-Ig to stain 293T cells transfected
with either B7-DC or B7.1 was investigated. Whereas strong binding
was observed with B7.1 transfectants, there was no binding to B7-DC
transfectants (FIG. 5). Based on homlogy and genomic proximity
between B7-DC and B7-H1/PD-L1, experiments were conducted to test
PD-1 as a candidate binding partner for B7-DC. Ideed, PD-100IG
bbound to B7-DC transfectants but not to B7 . . . I
trransffectants. The binding of BPD-1-Ig too B7-DC transfectants
was lower than thg bidig of CTL-4-It and CD28-Ig to B7.1
trransfectants, although it was specific. Further confirmation of
the binding of PD-1 to B7-DC was obtained from positive staining of
stablee B7-DC-GFP transfectants with PD-1-Ig. It was concluded
that, as with B7-H1 and B7h/B7RP-1, B7-DC does not use CD28 or
CTLA-4 as receptors. Rather, PD-1 appears to be a receptor for
B7-DC.
EXAMPLE IV
B7-DC Functions as a Costimulatory Molecule for T Cells
[0327] A soluble B7-DC-Ig fusion protein which could be added to T
cell stimulation assays was produced for use in testing whether
B7-DC possessed costimulatory activity. The proliferative response
of T cells was measured to stimulation by increasing amounts of
immobilized anti-CD3 in the presence of either B7-DC-Ig, B7.1-Ig or
an isotype control. FIG. 6 (left) shows that, in the presence of
suboptimal anti-CD3 concentration, B7-DC costimulated a greater T
cell proliferative response than did B7.1. Furthermore, B7-DC
costimulated proliferative responses were higher in CD4 than in CD8
cells (FIG. 6, right). B7-DC failed to stimulate T cells in the
absence of a TCR-focused stimulus, indicating that B7-DC was
providing a true costimulatory signal.
[0328] B7-DC also costimulated a proliferative responses when
"signal 1" was provided by an MHC-peptide complex, RENCA cells
(which express no endogenous B7.1, B7.2 or B7-DC by RT-PCR
analysis) were treated with .gamma.-IFN to induce MHC class II
expression. These cells were loaded with the I-E.sup.d restricted
HA 110-120 peptide (FERFEIFPKE)(35) [SEQ ID NO:15]. Purified
splenic T cells from an I-E.sup.d+HA 110-120 specific TCR
transgenic mouse line were added and the proliferative response was
measured in the presence of either B7-DC-Ig, B7.1-Ig or an isotype
control. FIG. 7 shows that B7-DC possessed greater costimulatory
activity than did B7.1.
Patterns of Lymphokine Production Costimulated by B7-DC
[0329] The best characterized T cell responses to costimulation by
the B7 family molecules is lymphokine production. These lymphokines
are important mediators of T cell effects. Studies were done to
analyze production of a number of different lymphokines by T cells
that had been stimulated with anti-CD3 or HA antigen (FIG. 8)
costimulated with either B7-DC-Ig, B7.1-Ig or an isotype control.
Patterns of lymphokine costimulation were fairly consistent whether
anti-CD3 or an MHC-peptide complex was utilized as "signal 1".
Significantly, B7-DC costimulated greater levels of .gamma.-IFN
than did B7.1. B7-DC also costimulated significant amounts of IL-6
production whereas B7.1 costimulated virtually none. While both
molecules costimulated IL-2 production, B7.1 did so more
efficiently than did B7-DC. Thus, the patterns of costimulation by
B7-DC and B7.1 are distinct, with B7-DC being more efficient in
costimulating important proinflammatory lymphokines.
EXAMPLE V
B7-DC Enhances In Vivo Immune Responses
[0330] In order to determine whether B7-DC possesses in vivo
biologic activity, the inventors asked whether B7-DC-Ig enhanced
immune responses to peptide vaccines. B7-DC-Ig or an isotype
control antibody was added to the immunogenic cocktail of HA
110-120 peptide and IFA. To permit enumeration of HA-specific CD4 T
cells in vivo, 2.5.times.10.sup.6 anti-HA 6.5 T cells were
transferred into the mice 3 days before immunization. Seven days
after immunization, draining LN cells were harvested and the cells
stimulated in vitro for 2 days with varying amounts of HA110-120
peptide. FIG. 9 shows that addition of B7-DC-Ig indeed dramatically
enhanced the proliferative response to HA. The total number of
HA-specific T cells in draining LN was increased by roughly 2 fold
in groups receiving B7-DC-Ig relative to isotype antibody controls.
It was therefore concluded that B7-DC had the ability to enhance
antigen-specific responses even on a per cell basis.
EXAMPLE VI
Discussion and Conclusions
[0331] The present inventors have discovered and characterized a
new B7 family member with expression highly restricted to DCs and
having unique costimulatory properties for T cells. The human
orthologue of B7-DC is also expressed in DCs.
[0332] This restricted expression pattern contrasts with the
previously described B7 family members, suggesting that B7-DC
participates in different immune responses than the known B7.1/2
pathways. While a weak B7-DC signal was detected by RT-PCR in
activated macrophages, preliminary realtime RT-PCR analysis
indicated that B7-DC mRNA expression in DCs was >15-fold higher
than in activated macrophages. Antibody staining likewise detected
very low levels of B7-DC on the surface of activated macrophages.
It is unclear whether this is sufficient for significant T cell
activation.
[0333] The unusual pattern of lymphokine production that B7-DC
costimulates implies a unique biologic role compared to other B7
family members. The traditional classification of cytokines is as
follows: Th1 cytokines include IL-2, .gamma.-IFN and lymphotoxin;
Th2 cytokines include IL-4, IL-5, IL-6 and IL-13 (36). B7-DC does
not induce either a classic Th1 or Th2 lymphokine profile, B7-DC
induces very little IL-4 and no IL-10. However, IL-6 is considered
a Th2 cytokine. The lower IL-2 and higher .gamma.-IFN costimulated
by B7-DC relative to B7.1 does not conform to a classic Th1
pattern. Nonetheless, the high .gamma.-IFN production suggests that
B7-DC evokes important T cell effector function.
[0334] B7-DC is noteworthy in its ability to costimulate IL-6. The
robust proliferative response of T cells induced by B7-DC is
explained in part by its strong costimulation of IL-6 production,
which is not observed with B7.1. IL-6 is a potent amplifier of T
cell proliferation in conjunction with other proliferative stimuli)
(37, 38). IL-6 is a multifunctional cytokine that regulates not
only T cell function but also proinflammatory responses, monocyte
differentiation, B cell differentiation, thrombopoiesis, bone
resorption, and the growth of certain hematopoietic tumors (39,
40). IL-6 can function in concert with soluble IL6 receptors
(sIL-6R) in the induction of chemokines and leukocyte recruitment
(41). It can mediate potent antiapoptotic effects via Stat-3
activation. IL-6 dependent Stat-3 activation in T cells has been
reported to be an important pathway for the survival of activated T
cells (42, 43) although other reports suggest that Stat-3 exerts
its effect on resting T cells.
[0335] While B7-DC fails to bind CD28 or CTLA-4, it does bind PD-1,
a receptor for B7-H1/PD-L1 (22, 47, 48). It has not yet been
determined whether it binds ICOS, a receptor for B7h/B7RP-1 (23-25,
44-46). The marked homology between B7-DC and B7-H1/PDL-1 (greater
than that between B7.1 and B7.2), the close physical linkage of
hB7-H1/PD-L1 and hB7-DC and their binding to a common receptor,
suggests that they are related by a relatively recent gene
duplication event. This is analogous to the relationship between
B7.1 and B7.2, which both map to within one megabase on mouse
chromosome 16 and on human chromosome 3 (49).
[0336] It will be important to discern the relative biologic roles
of B7-DC vs B7-H1/PD-L1 as mediated by PD-1 and other putative
receptor(s). PD-1 is expressed subsequent to, and appears to
inhibit, T cells activation. PD-1 induces apoptosis under
conditions of T cell stimulation with high concentrations of
anti-CD3. PD-1 knockout mice develop an autoimmune syndrome (22)
characterized by clinical manifestations of hypertrophic
cardiomyopathy. In contrast, Dong et al. (21) reported that
B7-H1/PD-L1 co-stimulated T cell proliferation and cytokine release
at lower concentrations of anti-CD3. By analogy to the relationship
of CD28/CTLA-4, PD-L1 may be a counterreceptor for an as yet
unidentified activating receptor. Despite sharing the property of
binding to PD-1, B7-DC and B7-H1 are distinct in their lymphokine
costimulation patterns; B7-H1 costimulated T cell IL-10 production
whereas B7-DC does not. The distinct cellular expression patterns
and costimulatory functions of B7-DC suggest a unique role in
immune function.
[0337] The references cited above and below are all incorporated by
reference herein, whether specifically incorporated or not.
[0338] Having now fully described this invention, it will be
appreciated by those skilled in the art that the same can be
performed within a wide range of equivalent parameters,
concentrations, and conditions without departing from the spirit
and scope of the invention and without undue experimentation.
[0339] While this invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications. This application is intended to
cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth as follows in the scope of the appended
claims.
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Sequence CWU 1
1
161819DNAHomo sapiensCDS(1)..(819) 1atg atc ttc ctc ctg cta atg ttg
agc ctg gaa ttg cag ctt cac cag 48Met Ile Phe Leu Leu Leu Met Leu
Ser Leu Glu Leu Gln Leu His Gln1 5 10 15ata gca gct tta ttc aca gtg
aca gtc cct aag gaa ctg tac ata ata 96Ile Ala Ala Leu Phe Thr Val
Thr Val Pro Lys Glu Leu Tyr Ile Ile 20 25 30gag cat ggc agc aat gtg
acc ctg gaa tgc aac ttt gac act gga agt 144Glu His Gly Ser Asn Val
Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser 35 40 45cat gtg aac ctt gga
gca ata aca gcc agt ttg caa aag gtg gaa aat 192His Val Asn Leu Gly
Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn 50 55 60gat aca tcc cca
cac cgt gaa aga gcc act ttg ctg gag gag cag ctg 240Asp Thr Ser Pro
His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu65 70 75 80ccc cta
ggg aag gcc tcg ttc cac ata cct caa gtc caa gtg agg gac 288Pro Leu
Gly Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp 85 90 95gaa
gga cag tac caa tgc ata atc atc tat ggg gtc gcc tgg gac tac 336Glu
Gly Gln Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr 100 105
110aag tac ctg act ctg aaa gtc aaa gct tcc tac agg aaa ata aac act
384Lys Tyr Leu Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr
115 120 125cac atc cta aag gtt cca gaa aca gat gag gta gag ctc acc
tgc cag 432His Ile Leu Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr
Cys Gln 130 135 140gct aca ggt tat cct ctg gca gaa gta tcc tgg cca
aac gtc agc gtt 480Ala Thr Gly Tyr Pro Leu Ala Glu Val Ser Trp Pro
Asn Val Ser Val145 150 155 160cct gcc aac acc agc cac tcc agg acc
cct gaa ggc ctc tac cag gtc 528Pro Ala Asn Thr Ser His Ser Arg Thr
Pro Glu Gly Leu Tyr Gln Val 165 170 175acc agt gtt ctg cgc cta aag
cca ccc cct ggc aga aac ttc agc tgt 576Thr Ser Val Leu Arg Leu Lys
Pro Pro Pro Gly Arg Asn Phe Ser Cys 180 185 190gtg ttc tgg aat act
cac gtg agg gaa ctt act ttg gcc agc att gac 624Val Phe Trp Asn Thr
His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp 195 200 205ctt caa agt
cag atg gaa ccc agg acc cat cca act tgg ctg ctt cac 672Leu Gln Ser
Gln Met Glu Pro Arg Thr His Pro Thr Trp Leu Leu His 210 215 220att
ttc atc ccc tcc tgc atc att gct ttc att ttc ata gcc aca gtg 720Ile
Phe Ile Pro Ser Cys Ile Ile Ala Phe Ile Phe Ile Ala Thr Val225 230
235 240ata gcc cta aga aaa caa ctc tgt caa aag ctg tat tct tca aaa
gac 768Ile Ala Leu Arg Lys Gln Leu Cys Gln Lys Leu Tyr Ser Ser Lys
Asp 245 250 255aca aca aaa aga cct gtc acc aca aca aag agg gaa gtg
aac agt gct 816Thr Thr Lys Arg Pro Val Thr Thr Thr Lys Arg Glu Val
Asn Ser Ala 260 265 270atc 819Ile2273PRTHomo sapiens 2Met Ile Phe
Leu Leu Leu Met Leu Ser Leu Glu Leu Gln Leu His Gln1 5 10 15Ile Ala
Ala Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile 20 25 30Glu
His Gly Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser 35 40
45His Val Asn Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn
50 55 60Asp Thr Ser Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln
Leu65 70 75 80Pro Leu Gly Lys Ala Ser Phe His Ile Pro Gln Val Gln
Val Arg Asp 85 90 95Glu Gly Gln Tyr Gln Cys Ile Ile Ile Tyr Gly Val
Ala Trp Asp Tyr 100 105 110Lys Tyr Leu Thr Leu Lys Val Lys Ala Ser
Tyr Arg Lys Ile Asn Thr 115 120 125His Ile Leu Lys Val Pro Glu Thr
Asp Glu Val Glu Leu Thr Cys Gln 130 135 140Ala Thr Gly Tyr Pro Leu
Ala Glu Val Ser Trp Pro Asn Val Ser Val145 150 155 160Pro Ala Asn
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val 165 170 175Thr
Ser Val Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys 180 185
190Val Phe Trp Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp
195 200 205Leu Gln Ser Gln Met Glu Pro Arg Thr His Pro Thr Trp Leu
Leu His 210 215 220Ile Phe Ile Pro Ser Cys Ile Ile Ala Phe Ile Phe
Ile Ala Thr Val225 230 235 240Ile Ala Leu Arg Lys Gln Leu Cys Gln
Lys Leu Tyr Ser Ser Lys Asp 245 250 255Thr Thr Lys Arg Pro Val Thr
Thr Thr Lys Arg Glu Val Asn Ser Ala 260 265 270Ile31655DNAMurinae
gen. sp.CDS(210)..(953) 3gaattcggca cgaggtcaaa tgtggcatat
ctttgttgtc tccttctgtc tcccaactag 60agagaacaca cttacggctc ctgtcccggg
caggtttggt tgtcggtgtg attggcttcc 120agggaacctg atacaaggag
caactgtgtg ctgccttttc tgtgtctttg cttgaggagc 180tgtgctgggt
gctgatattg acacagacc atg ctg ctc ctg ctg ccg ata ctg 233 Met Leu
Leu Leu Leu Pro Ile Leu 1 5aac ctg agc tta caa ctt cat cct gta gca
gct tta ttc acc gtg aca 281Asn Leu Ser Leu Gln Leu His Pro Val Ala
Ala Leu Phe Thr Val Thr 10 15 20gcc cct aaa gaa gtg tac acc gta gac
gtc ggc agc agt gtg agc ctg 329Ala Pro Lys Glu Val Tyr Thr Val Asp
Val Gly Ser Ser Val Ser Leu25 30 35 40gag tgc gat ttt gac cgc aga
gaa tgc act gaa ctg gaa ggg ata aga 377Glu Cys Asp Phe Asp Arg Arg
Glu Cys Thr Glu Leu Glu Gly Ile Arg 45 50 55gcc agt ttg cag aag gta
gaa aat gat acg tct ctg caa agt gaa aga 425Ala Ser Leu Gln Lys Val
Glu Asn Asp Thr Ser Leu Gln Ser Glu Arg 60 65 70gcc acc ctg ctg gag
gag cag ctg ccc ctg gga aag gct ttg ttc cac 473Ala Thr Leu Leu Glu
Glu Gln Leu Pro Leu Gly Lys Ala Leu Phe His 75 80 85atc cct agt gtc
caa gtg aga gat tcc ggg cag tac cgt tgc ctg gtc 521Ile Pro Ser Val
Gln Val Arg Asp Ser Gly Gln Tyr Arg Cys Leu Val 90 95 100atc tgc
ggg gcc gcc tgg gac tac aag tac ctg acg gtg aaa gtc aaa 569Ile Cys
Gly Ala Ala Trp Asp Tyr Lys Tyr Leu Thr Val Lys Val Lys105 110 115
120gct tct tac atg agg ata gac act agg atc ctg gag gtt cca ggt aca
617Ala Ser Tyr Met Arg Ile Asp Thr Arg Ile Leu Glu Val Pro Gly Thr
125 130 135ggg gag gtg cag ctt acc tgc cag gct aga ggt tat ccc cta
gca gaa 665Gly Glu Val Gln Leu Thr Cys Gln Ala Arg Gly Tyr Pro Leu
Ala Glu 140 145 150gtg tcc tgg caa aat gtc agt gtt cct gcc aac acc
agc cac atc agg 713Val Ser Trp Gln Asn Val Ser Val Pro Ala Asn Thr
Ser His Ile Arg 155 160 165acc ccc gaa ggc ctc tac cag gtc acc agt
gtt ctg cgc ctc aag cct 761Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser
Val Leu Arg Leu Lys Pro 170 175 180cag cct agc aga aac ttc agc tgc
atg ttc tgg aat gct cac atg aag 809Gln Pro Ser Arg Asn Phe Ser Cys
Met Phe Trp Asn Ala His Met Lys185 190 195 200gag ctg act tca gcc
atc att gac cct ctg agt cgg atg gaa ccc aaa 857Glu Leu Thr Ser Ala
Ile Ile Asp Pro Leu Ser Arg Met Glu Pro Lys 205 210 215gtc ccc aga
acg tgg cca ctt cat gtt ttc atc ccg gcc tgc acc atc 905Val Pro Arg
Thr Trp Pro Leu His Val Phe Ile Pro Ala Cys Thr Ile 220 225 230gct
ttg atc ttc ctg gcc ata gtg ata atc cag aga aag agg atc tag 953Ala
Leu Ile Phe Leu Ala Ile Val Ile Ile Gln Arg Lys Arg Ile 235 240
245gggaagctgt attacggaag aagtggtctc ttcttcccag atctggacct
gcggtcttgg 1013gagttggaag gatctgatgg gaaaccctca agagacttct
ggactcaaag tgagaatctt 1073gcaggacctg ccatttgcac ttttgaaccc
tttggacggt gacccagggc tccgaagagg 1133agcttgtaag actgacaatc
ttccctctgt ctcaagactc tctgaacagc aagaccccaa 1193tggcacttta
gacttacccc tgggatcctg gaccccagtg agggcctaag gctcctaatg
1253actttcaggg tgagaacaaa aggaattgct ctccgcccca cccccacctc
ctgctttccg 1313cagggagaca tggaaattcc cagttactaa aatagattgt
caatagagtt atttatagcc 1373ctcatttcct ccggggactt ggaagcttca
gacagggttt ttcataaaca aagtcataac 1433tgatgtgttt tacagcatcc
tagaatcctg gcagcctctg aagttctaat taactggaag 1493catttaagca
acacgtcaag tgcccctgct gtggtatttg tttctacttt tctgttttta
1553aagtgtgagt cacaaggtaa ttgttgtaac ctgtgatatc actgtttctt
gtgtctcttc 1613tttcaactac atcttttaaa acaaaaaaaa aaaaaaaaaa aa
16554247PRTMurinae gen. sp. 4Met Leu Leu Leu Leu Pro Ile Leu Asn
Leu Ser Leu Gln Leu His Pro1 5 10 15Val Ala Ala Leu Phe Thr Val Thr
Ala Pro Lys Glu Val Tyr Thr Val 20 25 30Asp Val Gly Ser Ser Val Ser
Leu Glu Cys Asp Phe Asp Arg Arg Glu 35 40 45Cys Thr Glu Leu Glu Gly
Ile Arg Ala Ser Leu Gln Lys Val Glu Asn 50 55 60Asp Thr Ser Leu Gln
Ser Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu65 70 75 80Pro Leu Gly
Lys Ala Leu Phe His Ile Pro Ser Val Gln Val Arg Asp 85 90 95Ser Gly
Gln Tyr Arg Cys Leu Val Ile Cys Gly Ala Ala Trp Asp Tyr 100 105
110Lys Tyr Leu Thr Val Lys Val Lys Ala Ser Tyr Met Arg Ile Asp Thr
115 120 125Arg Ile Leu Glu Val Pro Gly Thr Gly Glu Val Gln Leu Thr
Cys Gln 130 135 140Ala Arg Gly Tyr Pro Leu Ala Glu Val Ser Trp Gln
Asn Val Ser Val145 150 155 160Pro Ala Asn Thr Ser His Ile Arg Thr
Pro Glu Gly Leu Tyr Gln Val 165 170 175Thr Ser Val Leu Arg Leu Lys
Pro Gln Pro Ser Arg Asn Phe Ser Cys 180 185 190Met Phe Trp Asn Ala
His Met Lys Glu Leu Thr Ser Ala Ile Ile Asp 195 200 205Pro Leu Ser
Arg Met Glu Pro Lys Val Pro Arg Thr Trp Pro Leu His 210 215 220Val
Phe Ile Pro Ala Cys Thr Ile Ala Leu Ile Phe Leu Ala Ile Val225 230
235 240Ile Ile Gln Arg Lys Arg Ile 2455744DNAMurinae gen. sp.
5atgctgctcc tgctgccgat actgaacctg agcttacaac ttcatcctgt agcagcttta
60ttcaccgtga cagcccctaa agaagtgtac accgtagacg tcggcagcag tgtgagcctg
120gagtgcgatt ttgaccgcag agaatgcact gaactggaag ggataagagc
cagtttgcag 180aaggtagaaa atgatacgtc tctgcaaagt gaaagagcca
ccctgctgga ggagcagctg 240cccctgggaa aggctttgtt ccacatccct
agtgtccaag tgagagattc cgggcagtac 300cgttgcctgg tcatctgcgg
ggccgcctgg gactacaagt acctgacggt gaaagtcaaa 360gcttcttaca
tgaggataga cactaggatc ctggaggttc caggtacagg ggaggtgcag
420cttacctgcc aggctagagg ttatccccta gcagaagtgt cctggcaaaa
tgtcagtgtt 480cctgccaaca ccagccacat caggaccccc gaaggcctct
accaggtcac cagtgttctg 540cgcctcaagc ctcagcctag cagaaacttc
agctgcatgt tctggaatgc tcacatgaag 600gagctgactt cagccatcat
tgaccctctg agtcggatgg aacccaaagt ccccagaacg 660tggccacttc
atgttttcat cccggcctgc accatcgctt tgatcttcct ggccatagtg
720ataatccaga gaaagaggat ctag 744626DNAArtificial Sequenceprimer
6ggagctactg catgttgatt gttttg 26724DNAArtificial Sequenceprimer
7tgcaaactga ggcactgaaa agtc 24825DNAArtificial Sequenceprimer
8ttgttgtctc cttctgtctc ccaac 25924DNAArtificial Sequenceprimer
9acagttgctc cttgtatcag gttc 241021DNAArtificial Sequenceprimer
10gtaacggccg ccagtgtgct g 211123DNAArtificial Sequenceprimer
11cgccagtgtg atggatatct gca 231211PRTMurinae gen. sp. 12Ser Phe Glu
Arg Phe Glu Ile Phe Pro Lys Glu1 5 10139PRTUnknownbinding sequence
13Ser Gln Asp Xaa Xaa Xaa Glu Leu Tyr1 5148PRTUnknownbinding
sequence 14Xaa Xaa Xaa Tyr Xaa Xaa Arg Thr1 51510PRTMurinae gen.
sp. 15Phe Glu Arg Phe Glu Ile Phe Pro Lys Glu1 5 1016290PRTMurinae
gen. sp. 16Met Arg Ile Phe Ala Gly Ile Ile Phe Thr Ala Cys Cys His
Leu Leu1 5 10 15Arg Ala Phe Thr Ile Thr Ala Pro Lys Asp Leu Tyr Val
Val Glu Tyr 20 25 30Gly Ser Asn Val Thr Met Glu Cys Arg Phe Pro Val
Glu Arg Glu Leu 35 40 45Asp Leu Leu Ala Leu Val Val Tyr Trp Glu Lys
Glu Asp Glu Gln Val 50 55 60Ile Gln Phe Val Ala Gly Glu Glu Asp Leu
Lys Pro Gln His Ser Asn65 70 75 80Phe Arg Gly Arg Ala Ser Leu Pro
Lys Asp Gln Leu Leu Lys Gly Asn 85 90 95Ala Ala Leu Gln Ile Thr Asp
Val Lys Leu Gln Asp Ala Gly Val Tyr 100 105 110Cys Cys Ile Ile Ser
Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Leu 115 120 125Lys Val Asn
Ala Pro Tyr Arg Lys Ile Asn Gln Arg Ile Ser Val Asp 130 135 140Pro
Ala Thr Ser Glu His Glu Leu Ile Cys Gln Ala Glu Gly Tyr Pro145 150
155 160Glu Ala Glu Val Ile Trp Thr Asn Ser Asp His Gln Pro Val Ser
Gly 165 170 175Lys Arg Ser Val Thr Thr Ser Arg Thr Glu Gly Met Leu
Leu Asn Val 180 185 190Thr Ser Ser Leu Arg Val Asn Ala Thr Ala Asn
Asp Val Phe Tyr Cys 195 200 205Thr Phe Trp Arg Ser Gln Pro Gly Gln
Asn His Thr Ala Glu Leu Ile 210 215 220Ile Pro Glu Leu Pro Ala Thr
His Pro Pro Gln Asn Arg Thr His Trp225 230 235 240Val Leu Leu Gly
Ser Ile Leu Leu Phe Leu Ile Val Val Ser Thr Val 245 250 255Leu Leu
Phe Leu Arg Lys Gln Val Arg Met Leu Asp Val Glu Lys Cys 260 265
270Gly Val Glu Asp Thr Ser Ser Lys Asn Arg Asn Asp Thr Gln Phe Glu
275 280 285Glu Thr 290
* * * * *
References