U.S. patent application number 10/643768 was filed with the patent office on 2004-09-30 for b-7 domain-specific antibodies.
This patent application is currently assigned to BRIGHAM AND WOMENS HOSPITAL. Invention is credited to Borriello, Francescopaolo, Freeman, Gordon J., Nadler, Lee M., Sharpe, Arlene H..
Application Number | 20040192899 10/643768 |
Document ID | / |
Family ID | 22763271 |
Filed Date | 2004-09-30 |
United States Patent
Application |
20040192899 |
Kind Code |
A1 |
Sharpe, Arlene H. ; et
al. |
September 30, 2004 |
B-7 domain-specific antibodies
Abstract
Novel structural forms of T cell costimulatory molecules are
described. These structural forms comprise a novel structural
domain or have a structural domain deleted or added. The structural
forms correspond to naturally-occurring alternatively spliced forms
of T cell costimulatory molecules or variants thereof which can be
produced by standard recombinant DNA techniques. In one embodiment,
the T cell costimulatory molecule of the invention contains a novel
cytoplasmic domain. In another embodiment, the T cell costimulatory
molecule of the invention contains a novel signal peptide domain or
has an immunoglobulin variable region-like domain deleted. The
novel structural forms of T cell costimulatory molecules can be
used to identify agents which stimulate the expression of
alternative forms of costimulatory molecules and to identify
components of the signal transduction pathway which results in
costimulation of T cells.
Inventors: |
Sharpe, Arlene H.;
(Brookline, MA) ; Borriello, Francescopaolo;
(Brookline, MA) ; Freeman, Gordon J.; (Brookline,
MA) ; Nadler, Lee M.; (Newton, MA) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP.
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
BRIGHAM AND WOMENS HOSPITAL
Boston
MA
Dana-Farber Cancer Institute, Inc.
Boston
MA
|
Family ID: |
22763271 |
Appl. No.: |
10/643768 |
Filed: |
August 18, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10643768 |
Aug 18, 2003 |
|
|
|
09837867 |
Apr 17, 2001 |
|
|
|
6608180 |
|
|
|
|
09837867 |
Apr 17, 2001 |
|
|
|
08205697 |
Mar 2, 1994 |
|
|
|
6218510 |
|
|
|
|
Current U.S.
Class: |
530/388.22 ;
435/320.1; 435/334; 435/69.1; 536/23.53 |
Current CPC
Class: |
A01K 2217/05 20130101;
C07K 14/70532 20130101 |
Class at
Publication: |
530/388.22 ;
536/023.53; 435/069.1; 435/320.1; 435/334 |
International
Class: |
C12Q 001/68; C07H
021/04; C07K 016/28; C12N 005/06 |
Goverment Interests
[0002] Work described herein was supported under CA-40216 and
GM46883 awarded by the National Instiutes of Health. The U.S.
government therefore may have certain rights in this invention.
Claims
1. An isolated nucleic acid encoding a protein which binds CD28 or
CTLA4 comprising a contiguous nucleotide sequence derived from at
least one T cell costimulatory molecule gene, the nucleotide
sequence represented by a formula A-B-C-D-E, wherein A comprises a
nucleotide sequence of at least one first exon of a T cell
costimulatory molecule gene, wherein the at least one first exon
encodes a signal peptide domain, B comprises a nucleotide sequence
of at least one second exon of a T cell costimulatory molecule
gene, wherein the at least one second exon encodes an
immunoglobulin variable region-like domain, C comprises a
nucleotide sequence of at least one third exon of a T cell
costimulatory molecule gene, wherein the at least one third exon
encodes an immunoglobulin constant region-like domain, D comprises
a nucleotide sequence of at least one fourth exon of a T cell
costimulatory molecule gene, wherein the at least one fourth exon
encodes a transmembrane domain, and E comprises a nucleotide
sequence of at least one fifth exon of a T cell costimulatory
molecule gene, wherein the at least one fifth exon encodes a
cytoplasmic domain, with the proviso that E does not comprise a
nucleotide sequence selected from a group consisting of SEQ ID
NO:25, SEQ ID NO:27, SEQ ID NO:29 and SEQ ID NO:31.
2. The isolated nucleic acid of claim 1 which is a cDNA.
3. The isolated nucleic acid of claim 2 which comprises a coding
region of the cDNA.
4. The isolated nucleic acid of claim 1, wherein the nucleotide
sequence is derived from a T cell costimulatory molecule gene
encoding B7-1.
5. The isolated nucleic acid of claim 4, wherein B7-1 is
murine.
6. The isolated nucleic acid of claim 4, wherein B7-1 is human.
7. The isolated nucleic acid of claim 5, wherein E comprises a
nucleotide sequence shown in SEQ ID NO:4.
8. The isolated nucleic acid of claim 5, wherein E comprises a
nucleotide sequence encoding an amino acid sequence shown in SEQ ID
NO:5.
9. An isolated nucleic acid encoding a protein which binds CD28 or
CTLA4 and is encoded by a T cell costimulatory molecule gene having
at least one first exon encoding a first cytoplasmic domain
comprising a nucleotide sequence selected from the group consisting
of a nucleotide sequence of SEQ ID NO:25, SEQ ID NO:27, SEQ ID
NO:29 and SEQ ID NO:31, and at least one second exon encoding a
second cytoplasmic domain, wherein the isolated nucleic acid
comprises a nucleotide sequence encoding the second cytoplasmic
domain.
10. The isolated nucleic acid of claim 9 which comprises a coding
region of a cDNA.
11. The isolated nucleic acid of claim 9 which does not comprise a
nucleotide sequence encoding the first cytoplasmic domain.
12. The isolated nucleic acid of claim 9 wherein the T cell
costimulatory molecule gene is B7-1.
13. The isolated nucleic acid of claim 12 wherein B7-1 is
murine.
14. The isolated nucleic acid of claim 12 wherein B7-1 is
human.
15. An isolated nucleic acid encoding a protein which binds CD28 or
CTLA4 comprising a nucleotide sequence shown in SEQ ID NO: 1.
16. An isolated nucleic acid encoding a protein which binds CD28 or
CTLA4 comprising a nucleotide sequence shown in SEQ ID NO:3.
17. An isolated nucleic acid encoding a cytoplasmic domain derived
from a protein which binds CD28 or CTLA4, the nucleic acid
comprising a nucleotide sequence shown in SEQ ID NO:4.
18. A recombinant expression vector comprising the nucleic acid
molecule of claim 15.
19. A host cell which contains the recombinant expression vector of
claim 18.
20. An isolated nucleic acid encoding a protein which binds CD28 or
CTLA4 comprising a contiguous nucleotide sequence derived from at
least one T cell costimulatory molecule gene, the nucleotide
sequence represented by a formula A-B-C-D-E, wherein A comprises a
nucleotide sequence of at least one first exon of a T cell
costimulatory molecule gene, wherein the at least one first exon
encodes a signal peptide domain, B comprises a nucleotide sequence
of at least one second exon of a T cell costimulatory molecule
gene, wherein the at least one second exon encodes an
immunoglobulin variable region-like domain, C comprises a
nucleotide sequence of at least one third exon of a T cell
costimulatory molecule gene, wherein the at least one third exon
encodes an immunoglobulin constant region-like domain, D, which may
or may not be present, comprises a nucleotide sequence of at least
one fourth exon of a T cell costimulatory molecule gene, wherein
the at least one fourth exon encodes a transmembrane domain, and E,
which may or may not be present, comprises a nucleotide sequence of
at least one fifth exon of a T cell costimulatory molecule gene,
wherein the at least one fifth exon encodes a cytoplasmic domain,
with the proviso that A does not comprise a nucleotide sequence
selected from a group consisting of SEQ ID NO:33, SEQ ID NO:35, SEQ
ID NO:37, SEQ ID NO:39 and SEQ ID NO:41.
21. The isolated nucleic acid of claim 20 which is a cDNA.
22. The isolated nucleic acid of claim 21 which comprises a coding
region of the cDNA.
23. The isolated nucleic acid of claim 20, wherein the nucleotide
sequence is derived from a T cell costimulatory molecule gene
encoding B7-2.
24. The isolated nucleic acid of claim 23, wherein B7-2 is
murine.
25. The isolated nucleic acid of claim 23, wherein B7-2 is
human.
26. The isolated nucleic acid of claim 24, wherein A comprises a
nucleotide sequence selected from the group consisting of SEQ ID
NO: 14.
27. An isolated nucleic acid encoding a protein which binds CD28 or
CTLA4 and is encoded by a T cell costimulatory molecule gene having
at least one first exon encoding a first signal peptide domain
comprising a nucleotide sequence selected from the group consisting
of a nucleotide sequence of SEQ ID NO:33, SEQ ID NO:35, SEQ ID
NO:37 SEQ ID NO:39 and SEQ ID NO:41, and at least one second exon
encoding a second signal peptide domain, wherein the isolated
nucleic acid comprises a nucleotide sequence encoding the second
signal peptide domain.
28. The isolated nucleic acid of claim 27 which comprises a coding
region of a cDNA.
29. The isolated nucleic acid of claim 27 which does not comprise a
nucleotide sequence encoding the first signal peptide domain.
30. The isolated nucleic acid of claim 27 wherein the T cell
costimulatory molecule gene is B7-2.
31. The isolated nucleic acid of claim 30 wherein B7-2 is
murine.
32. The isolated nucleic acid of claim 30 wherein B7-2 is
human.
33. An isolated nucleic acid encoding a protein which binds CD28 or
CTLA4 comprising a nucleotide sequence shown in SEQ ID NO:12.
34. An isolated nucleic acid encoding a signal peptide domain
derived from a protein which binds CD28 or CTLA4, the nucleic acid
comprising a nucleotide sequence shown in SEQ ID NO:14.
35. A recombinant expression vector comprising the nucleic acid
molecule of claim 33.
36. A host cell which contains the recombinant expression vector of
claim 35.
37. An isolated nucleic acid encoding a protein comprising a
contiguous nucleotide sequence derived from at least one T cell
costimulatory molecule gene, the nucleotide sequence represented by
a formula A-B-C-D, wherein A comprises a nucleotide sequence of at
least one first exon of a T cell costimulatory molecule gene,
wherein the at least one first exon encodes a signal peptide
domain, B comprises a nucleotide sequence of at least one second
exon of a T cell costimulatory molecule gene, wherein the at least
one second exon encodes an immunoglobulin constant region-like
domain, C comprises a nucleotide sequence of at least one third
exon of a T cell costimulatory molecule gene, wherein the at least
one third exon encodes a transmembrane domain, and D comprises a
nucleotide sequence of at least one fourth exon of a T cell
costimulatory molecule gene, wherein the at least one fourth exon
encodes a cytoplasmic domain.
38. The isolated nucleic acid of claim 37 comprising a nucleotide
sequence shown in SEQ ID NO:8.
39. The isolated nucleic acid of claim 37 comprising a nucleotide
sequence shown in SEQ ID NO:10.
Description
RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 09/837,867, filed Apr. 17, 2001, entitled "B7
Domain-Specific Antibodies (As Amended)"; which is a divisional
application of U.S. application Ser. No. 08/205,697 filed on Mar.
2, 1994, issued as U.S. Pat. No. 6,218,510 on Apr. 17, 2001,
entitled "Novel Forms of T Cell Costimulatory Molecules and Uses
Therefor". The contents of all of the aforementioned applications
are hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0003] For CD4+ T lymphocyte activation to occur, two distinct
signals must be delivered by antigen presenting cells to resting T
lymphocytes (Schwartz, R. H. (1990) Science 248:1349-1356;
Williams, I. R. and Unanue, E. R. (1991) J. Immunol. 147:3752-3760;
Mueller, D. L. et al., (1989) J. Immunol. 142:2617-2628). The
first, or primary, activation signal is mediated physiologically by
the interaction of the T cell receptor/CD3 complex (TcR/CD3) with
MHC class II-associated antigenic peptide and gives specificity to
the immune response. The second signal, the costimulatory signal,
regulates the T cell proliferative response and induction of
effector functions. Costimulatory signals appear pivotal in
determining the functional outcome of T cell activation since
delivery of an antigen-specific signal to a T cell in the absence
of a costimulatory signal results in functional inactivation of
mature T cells, leading to a state of tolerance (Schwartz, R. H.
(1990) Science 248:1349-1356).
[0004] Molecules present on the surface of antigen presenting cells
which are involved in T cell costimulation have been identified.
These T cell costimulatory molecules include murine B7-1 (mB7-1;
Freeman, G. J. et al., (1991) J. Exp. Med. 174:625-631), and the
more recently identified murine B7-2 (mB7-2; Freeman, G. J. et al.,
(1993) J. Exp. Med. 178:2185-2192). Human counterparts to the
murine B7-1 and B7-2 molecules have also been described (human B7-1
(hB7-1) Freedman, A. S. et al., (1987) J. Immunol. 137:3260-3267;
Freeman, G. J. et al., (1989) J. Immunol. 143:2714-2722; and human
B7-2 (hB7-2); Freeman, G. J. et al., (1993) Science 262:909-911;
Azuma, M. et al. (1993) Nature 366:76-79). The B7-1 and B7-2 genes
are members of the immmunoglobulin gene superfamily.
[0005] B7-1 and B7-2 display a restricted pattern of cellular
expression, which correlates with accessory cell potency in
providing costimulation (Reiser, H. et al. (1992; Proc. Natl. Acad.
Sci. USA 89:271-275; Razi-Wolf Z. et al., (1992) Proc. Natl. Acad.
Sci. USA 89:4210-4214; Galvin, F. et al. (1992) J. Immunol.
149:3802-3808; Freeman, G. J. et al., (1993) J. Exp. Med.
178:2185-2192). For example, B7-1 has been observed to be expressed
on activated B cells, T cells and monocytes but not on resting B
cells, T cells or monocytes, and its expression can be regulated by
different extracellular stimuli (Linsley, P. S. et al., (1990)
Proc. Natl. Acad. Sci. USA 87:5031-5035; Linsley, P. S. et al.,
(1991) J. Exp. Med. 174:561-569; Reiser, H. et al. (1992); Proc.
Natl. Acad. Sci. USA 89:271-275; Gimmi, C. D. et al. (1991) Proc.
Natl. Acad. Sci. USA 88:6575-6579; Koulova, L. et al. (1991) J.
Exp. Med. 173:759-762; Azuma, M. et al. (1993) J. Exp. Med.
177:845-850; Sansom, D. M. et al. (1993) Eur. J. Immunol.
23:295-298)
[0006] Both B7-1 and B7-2 are counter-receptors for two ligands,
CD28 and CTLA4, expressed on T lymphocytes (Linsley, P. S. et al.,
(1990) Proc. Natl. Acad. Sci. USA 87:5031-5035; Linsley, P. S. et
al., (1991) J. Exp. Med. 174:561-569). CD28 is constitutively
expressed on T cells and, after ligation by a costimulatory
molecule, induces IL-2 secretion and T cell proliferation (June, C.
H. et al. (1990) Immunol. Today 11:211-216). CTLA4 is homologous to
CD28 and appears on T cells after activation (Freeman, G. J. et al.
(1992) J. Immunol. 149:3795-3801). Although CTLA4 has a
significantly higher affinity for B7-1 than does CD28, its role in
T cell activation remains to be determined. It has been shown that
antigen presentation to T cells in the absence of the B7-1/CD28
costimulatory signal results in T cell anergy (Gimmi, C. D. et al.
(1993) Proc. Natl. Acad Sci. USA 90:6586-6590; Boussiotis, V. A. et
al. (1993) J. Exp. Med. 178:1753). The ability of T cell
costimulatory molecules such as B7-1 and B7-2 to bind to CD28
and/or CTLA4 on T cells and trigger a costimulatory signal in the T
cells provides a functional role for these molecules in T cell
activation.
SUMMARY OF THE INVENTION
[0007] This invention pertains to novel forms of T cell
costimulatory molecules. In particular, the invention pertains to
isolated proteins encoded by T cell costimulatory molecule genes
which contain amino acid sequences encoded by novel exons of these
genes. The isolated proteins of the invention correspond to
alternative forms of T cell costimulatory molecules. Preferably,
these alternative forms correspond to naturally-occurring,
alternatively spliced forms of T cell costimulatory molecules or
are variants of alternatively spliced forms which are produced by
recombinant DNA techniques. The novel forms of T cell costimulatory
molecules of the invention contain an alternative structural domain
(i.e., a structural domain having an amino acid sequence which
differs from a known amino acid sequence) or have a structural
domain deleted or added. The occurrence in nature of alternative
structural forms of T cell costimulatory molecules supports
additional functional roles for T cell costimulatory molecules.
[0008] The invention also provides isolated nucleic acid molecules
encoding alternative forms of proteins which bind to CD28 and/or
CTLA4 and isolated proteins encoded therein. Isolated nucleic acid
molecules encoding polypeptides corresponding to novel structural
domains of T cell costimulatory molecules, and isolated polypeptide
encoded therein are also within the scope of the invention. The
novel structural domains of the invention are encoded by exons of T
cell costimulatory molecule genes. In one embodiment of the
invention, the T cell costimulatory molecule gene encodes B7-1. In
another embodiment, the T cell costimulatory molecule gene encodes
B7-2.
[0009] Another aspect of the invention provides proteins which bind
CD28 and/or CTLA4 and contain a novel cytoplasmic domain. T cell
costimulatory molecule genes which contain exons encoding different
cytoplasmic domains which are used in an alternate manner have been
discovered. Alternative splicing of mRNA transcripts of a T cell
costimulatory molecule gene has been found to generate native T
cell costimulatory molecules with different cytoplasmic domains.
The existence of alternative cytoplasmic domain forms of T cell
costimulatory molecules supports a functional role for the
cytoplasmic domain in transmitting an intracellular signal within a
cell which expresses the costimulatory molecule on its surface.
This indicates that costimulatory molecules not only trigger an
intracellular signal in T cells, but may also deliver a signal to
the cell which expresses the costimulatory molecule. This is the
first evidence that the interaction between a costimulatory
molecule on one cell and its receptor on a T cell may involve
bidirectional signal transduction between the cells (rather than
only unidirectional signal transduction to the T cell).
[0010] In yet another aspect of the invention, proteins that bind
CD28 and/or CTLA4 and contain a novel signal peptide domain are
provided. T cell costimulatory molecule genes which contain exons
encoding different signal peptide domains which are used in an
alternate manner have been discovered. Alternative splicing of mRNA
transcripts of the gene can generate native T cell costimulatory
molecules with different signal peptide domains. The existence of
alternative signal peptide domain forms of T cell costimulatory
molecules also suggests a functional role for the signal peptide of
T cell costimulatory molecules.
[0011] An isolated nucleic acid molecule of the invention can be
incorporated into a recombinant expression vector and transfected
into a host cell to express a novel structural form of a T cell
costimulatory molecule. The isolated nucleic acids of the invention
can further be used to create transgenic and homologous recombinant
non-human animals. The novel T cell costimulatory molecules
provided by the invention can be used to trigger a costimulatory
signal in a T lymphocyte. These molecules can further be used to
raise antibodies against novel structural domains of costimulatory
molecules. The novel T cell costimulatory molecules of the
invention can also be used to identify agents which stimulate the
expression of alternative forms of costimulatory molecules and to
identify components of the signal transduction pathway induced in a
cell expressing a costimulatory molecule in response to an
interaction between the costimulatory molecule and its receptor on
a T lymphocyte.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a photograph of an agarose gel depicting the
presence of mB7-1 cytoplasmic domain II-encoding exon 6 in mB7-1
cDNA, determined by nested Reverse Transcriptase Polymerase Chain
Reaction (RT-PCR).
[0013] FIG. 2 is a schematic representation depicting three mB7-1
transcripts (A, B and C) detected by nested RT-PCR.
DETAILED DESCRIPTION OF THE INVENTION
[0014] This invention pertains to novel structural forms of T cell
costimulatory molecule which contain a structural domain encoded by
a novel exon of a T cell costimulatory molecule gene, or have a
structural domain deleted or added. Preferably, the isolated T cell
costimulatory molecule corresponds to a naturally-occurring
alternatively spliced form of a T cell costimulatory molecule, such
as B7-1 or B7-2. Alternatively, the isolated protein can be a
variant of a naturally-occurring alternatively spliced form of a T
cell costimulatory molecule which is produced by standard
recombinant DNA techniques.
[0015] Typically, a domain structure of a T cell costimulatory
molecule of the invention includes a signal peptide domain, an
immunoglobulin variable region-like domain (IgV-like), an
immunoglobulin constant region-like domain (IgC-like), a
transmembrane domain and a cytoplasmic domain. T cell costimulatory
molecule genes are members of the immunoglobulin gene superfamily.
The terms "immunogloublin variable region-like domain" and
"immunoglobulin constant region-like domain" are art-recognized and
refer to protein domains which are homologous in sequence to an
immunoglobulin variable region or an immunoglobulin constant
region, respectively. For a discussion of the immunoglobulin gene
superfamily and a description of IgV-like and IgC-like domains see
Hunkapiller, T. and Hood, L. (1989) Advances in Immunology 44:
1-63. Each structural domain of a protein is usually encoded in
genomic DNA by at least one exon. The invention is based, at least
in part, on the discovery of novel exons in T cell costimulatory
molecule genes which encode different forms of structural domains.
Moreover, it has been discovered that exons encoding different
forms of a structural domain of a T cell costimulatory molecule can
be used in an alternative manner by alternative splicing of primary
mRNA transcripts of a gene. Alternative splicing is an
art-recognized term referring to the mechanism by which primary
mRNA transcripts of a gene are processed to produce different
mature mRNA transcripts encoding different proteins. In this
mechanism different exonic sequences are excised from different
primary transcripts. This results in mature mRNA transcripts from
the same gene that contain different exonic sequences and thus
encode proteins having different amino acid sequences. The terms
"alternative forms" or "novel forms" of T cell costimulatory
molecules refer to gene products of the same gene which differ in
nucleotide or amino acid sequence from previously disclosed forms
of T cell costimulatory molecules, e.g., forms which result from
alternative splicing of a primary mRNA transcript of a gene
encoding a T cell costimulatory molecule.
[0016] Accordingly, one aspect of the invention relates to isolated
nucleic acids encoding T cell costimulatory molecules corresponding
to naturally-occurring alternatively spliced forms or variants
thereof, and uses therefor. Another aspect of the invention
pertains to novel structural forms of T cell costimulatory
molecules which are produced by translation of the nucleic acid
molecules of the invention, and uses therefor. This invention
further pertains to isolated nucleic acids encoding novel
structural domains of T cell costimulatory molecules, isolated
polypeptides encoded therein, and uses therefor.
[0017] The various aspects of this invention are described in
detail in the following subsections.
I. Isolated Nucleic Acid Molecules Encoding T Cell Costimulatory
Molecules
[0018] The invention provides an isolated nucleic acid molecule
encoding a novel structural form of a T cell costimulatory
molecule. As used herein, the term "T cell costimulatory molecule"
is intended to include proteins which bind to CD28 and/or CTLA4.
Preferred T cell costimulatory molecules are B7-1 and B7-2. The
term "isolated" as used herein refers to nucleic acid substantially
free of cellular material or culture medium when produced by
recombinant DNA techniques, or chemical precursors or other
chemicals when chemically synthesized. An "isolated" nucleic acid
is also free of sequences which naturally flank the nucleic acid
(i.e., sequences located at the 5' and 3' ends of the nucleic acid)
in the organism from which the nucleic acid is derived. The term
"nucleic acid" is intended to include DNA and RNA and can be either
double stranded or single stranded. Preferably, the isolated
nucleic acid molecule is a cDNA.
[0019] A. Nucleic Acids Encoding Novel Cytoplasmic Domains
[0020] One aspect of the invention pertains to isolated nucleic
acids that encode T cell costimulatory molecules, each containing a
novel cytoplasmic domain. It has been discovered that a gene
encoding a costimulatory molecule can contain multiple exons
encoding different cytoplasmic domains. In addition,
naturally-occurring mRNA transcripts have been discovered which
encode different cytoplasmic domain forms of T cell costimulatory
molecules. Thus, one embodiment of the invention provides an
isolated nucleic acid encoding a protein which binds CD28 or CTLA4
and comprises a contiguous nucleotide sequence derived from at
least one T cell costimulatory molecule gene. In this embodiment,
the nucleotide sequence can be represented by a formula A-B-C-D-E,
wherein
[0021] A comprises a nucleotide sequence of at least one first exon
encoding a signal peptide domain,
[0022] B comprises a nucleotide sequence of at least one second
exon of a T cell costimulatory molecule gene, wherein the at least
one second exon encodes an immunoglobulin variable region-like
domain,
[0023] C comprises a nucleotide sequence of at least one third exon
of a T cell costimulatory molecule gene, wherein the at least one
third exon encodes an immunoglobulin constant region-like
domain,
[0024] D comprises a nucleotide sequence of at least one fourth
exon of a T cell costimulatory molecule gene, wherein the at least
one fourth exon encodes a transmembrane domain, and
[0025] E comprises a nucleotide sequence of at least one fifth exon
of a T cell costimulatory molecule gene, wherein the at least one
fifth exon encodes a cytoplasmic domain,
[0026] with the proviso that E does not comprise a nucleotide
sequence encoding a cytoplasmic domain selected from the group
consisting of SEQ ID NO:28 (mB7-1), SEQ ID NO:30 (hB7-1), SEQ ID
NO:32 (mB7-2) and SEQ ID NO:34 (hB7-2).
[0027] In the formula, A, B, C, D, and E are contiguous nucleotide
sequences linked by phosphodiester bonds in a 5' to 3' orientation
from A to E. According to the formula, A can be a nucleotide
sequence of an exon which encodes a signal peptide domain of a
heterologous protein which efficiently expresses transmembrane or
secreted proteins, such as the oncostatin M signal peptide.
Preferably, A comprises a nucleotide sequence of at least one exon
which encodes a signal peptide domain of a T cell costimulatory
molecule gene. It is also preferred that A, B, C, D and E comprise
nucleotide sequences of exons of the B7-1 gene, such as the human
or murine B7-1 gene. As described in detail in Examples 1 and 2,
naturally-occurring murine B7-1 mRNA transcripts which contain a
nucleotide sequence encoding one of at least two different
cytoplasmic domains have been discovered. The alternative
cytoplasmic domains are encoded in genomic DNA by different exons
and the different mB7-1 mRNA transcripts are produced by
alternative splicing of the mRNA transcripts. The genomic structure
of mB7-1 has been reported to contain only a single exon encoding
cytoplasmic domain (Selvakumar, A. et al. (1993) Immunogenetics
38:292-295). The nucleotide sequence for the mB7-1 cDNA expressed
in B cells has been reported to correspond to usage of five exons,
1-2-3-4-5 (see Freeman, G. J. et al., (1991) J. Exp. Med.
174:625-631; the nucleotide sequence of which is shown in SEQ ID
NO: 16), including a single exon encoding cytoplasmic domain. As
described herein, the nucleotide sequence of a sixth exon for the
mB7-1 gene which encodes a cytoplasmic domain having a different
amino acid sequence than the cytoplasmic domain encoded by exon 5
has been discovered. The nucleotide sequence encoding the first
cytoplasmic domain of mB7-1 (i.e., exon 5) is shown in SEQ ID NO:
25 and the amino acid sequence of this cytoplasmic domain (referred
to herein as Cyt I) is shown in SEQ ID NO: 26. A nucleotide
sequence encoding a second, alternative cytoplasmic domain for
mB7-1 (i.e., exon 6) is shown in SEQ ID NO: 4. This alternative
cytoplasmic domain encoded by exon 6 (also referred to herein as
Cyt II) has an amino acid sequence shown in SEQ ID NO: 5.
[0028] The Cyt II domain of mB7-1 has several characteristic
properties. Of interest is the preferential expression of mRNA
containing the exon encoding Cyt II (i.e., exon 6) in thymus. In
contrast, mRNA containing exon 6 of mB7-1 is not detectable in
spleen. Accordingly, this invention encompasses alternative
cytoplasmic domain forms of T cell costimulatory molecules which
are expressed preferentially in thymus. As defined herein, the term
"expressed preferentially in the thymus" is intended to mean that
the mRNA is detectable by standard methods in greater abundence in
the thymus than in other tissues which express the T cell
costimulatory molecule, particularly the spleen. The Cyt II domain
of mB7-1 has also been found to contain several consensus
phosphorylation sites and, thus, alternative cytoplasmic domain
forms of T cell costimulatory molecules which contain at least one
consensus phosphorylation site are also within the scope of this
invention. As used herein, the term "consensus phosphorylation
site" describes an amino acid sequence motif which is recognized by
and phosphorylated by a protein kinase, for example protein kinase
C, casein kinase II etc. It has also been discovered that exon 6 is
encoded in genomic DNA approximately 7.5 kilobases downstream of
exon 5. This invention therefore includes alternative cytoplasmic
domain forms of T cell costimulatory molecules which are located in
genomic DNA less than approximately 10 kb downstream (i.e., 3' ) of
an exon encoding a first cytoplasmic domain of the T cell
costimulatory molecule. Additionally, a second, alternative
cytoplasmic domain of another T cell costimulatory molecule is
likely to be homologous to the Cyt II domain of mB7-1. For example,
the first cytoplasmic domains of mB7-1, hB7-1, mB7-2 and hB7-2
display between 4% and 26% amino acid identity (see Freeman, G. J.
et al. (1993) J. Exp. Med. 178:2185-2192). Accordingly, in one
embodiment, an alternative cytoplasmic domain of a T cell
costimulatory molecule has an amino acid sequence that is at least
about 5% to 25% identical in sequence with the amino acid sequence
of mB7-1 Cyt II (shown in SEQ ID NO: 5).
[0029] Another embodiment of the invention provides an isolated
nucleic acid encoding a protein which binds CD28 or CTLA4 and is
encoded by a T cell costimulatory molecule gene having at least one
first exon encoding a first cytoplasmic domain and at least one
second exon encoding a second cytoplasmic domain. The at least one
first exon comprises a nucleotide sequence selected from the group
consisting of a nucleotide sequence of SEQ ID NO:25 (mB7-1), SEQ ID
NO:27 (hB7-1), SEQ ID NO:29 (mB7-2) and SEQ ID NO:31 (hB7-2). In
this embodiment, the isolated nucleic acid includes a nucleotide
sequence encoding at least one second cytoplasmic domain.
Preferably, the isolated nucleic acid does not comprise a
nucleotide sequence encoding a first cytoplasmic domain. Preferred
T cell costimulatory molecule genes from which nucleotide sequences
can be derived include B7-1 and B7-2.
[0030] In yet another embodiment, the isolated nucleic acid of the
invention encodes a protein which binds CD28 or CTLA4 and comprises
a nucleotide sequence shown in SEQ ID NO: 1. This nucleotide
sequence corresponds to a naturally-occurring alternatively spliced
form of mB7-1 which includes the nucleotide sequences of exons
1-2-3-4-6. Alternatively, the isolated nucleic acid comprises a
nucleotide sequence shown in SEQ ID NO: 3, which corresponds to a
naturally-occurring alternatively spliced form of mB7-1 comprising
the nucleotide sequences of exons 1-2-3-4-5-6.
[0031] B. Nucleic Acids Encoding Novel Signal Peptide Domains
[0032] Other aspects of this invention pertain to isolated nucleic
acids which encode T cell costimulatory molecules containing novel
signal peptide domains. It has been discovered that a gene encoding
a costimulatory molecule can contain multiple exons encoding
different signal peptide domains and that mRNA transcripts occur in
nature which encode different signal peptide domain forms of T cell
costimulatory molecules. Thus, isolated nucleic acids which encode
proteins which bind CD28 or CTLA4 and comprise contiguous
nucleotide sequences derived from at least one T cell costimulatory
molecule gene are within the scope of this invention. The
nucleotide sequence can be represented by a formula A-B-C-D-E,
wherein
[0033] A comprises a nucleotide sequence of at least one first exon
of a T cell costimulatory molecule gene, wherein the at least one
first exon encodes a signal peptide domain,
[0034] B comprises a nucleotide sequence of at least one second
exon of a T cell costimulatory molecule gene, wherein the at least
one second exon encodes an immunoglobulin variable region-like
domain,
[0035] C comprises a nucleotide sequence of at least one third exon
of a T cell costimulatory molecule gene, wherein the at least one
third exon encodes an immunoglobulin constant region-like
domain,
[0036] D, which may or may not be present, comprises a nucleotide
sequence of at least one fourth exon of a T cell costimulatory
molecule gene, wherein the at least one fourth exon encodes a
transmembrane domain, and
[0037] E, which may or may not be present, comprises a nucleotide
sequence of at least one fifth exon of a T cell costimulatory
molecule gene, wherein the at least one fifth exon encodes a
cytoplasmic domain, with the proviso that A does not comprise a
nucleotide sequence encoding a signal peptide domain selected from
the group consisting of SEQ ID NO:33 (mB7-1), SEQ ID NO:35 (hB7-1),
SEQ ID NO:37 (mB7-2), SEQ ID NO:39 (hB7-2) and SEQ ID NO:41
(hB7-2).
[0038] In the formula, A, B, C, D, and E are contiguous nucleotide
sequences linked by phosphodiester bonds in a 5' to 3' orientation
from A to E. To produce a soluble form of the T cell costimlatory
molecule D, which comprises nucleotide sequence of a transmembrane
domain and E, which comprises a nucleotide sequence of a
cytoplasmic domain may not be present in the molecule. In a
preferred embodiment, A, B, C, D and E comprise nucleotide
sequences of exons of the B7-2 gene, such as the human or murine
B7-2 gene. As described in detail in Example 6, naturally-occurring
murine B7-2 mRNA transcripts which contain a nucleotide sequence
encoding one of at least two different signal peptide domains have
been discovered. An mRNA transcript containing a nucleotide
sequence encoding a novel signal peptide domain represents an
alternatively spliced form of murine B7-2. A naturally-occurring
mB7-2 mRNA transcript encoding an alternative signal peptide domain
preferably comprises the nucleotide sequence shown in SEQ ID NO:
12. The amino acid sequence of the novel signal peptide domain
encoded by this transcript is shown in SEQ ID NO: 13. Accordingly,
in this embodiment, the isolated nucleic acid encodes a protein
which binds CD28 or CTLA4 and preferably comprises a nucleotide
sequence shown in SEQ ID NO: 12.
[0039] In yet another embodiment of the invention, the isolated
nucleic acid encodes a protein which binds CD28 or CTLA4 and is
encoded by a T cell costimulatory molecule gene having at least one
first exon encoding a first signal peptide domain and at least one
second exon encoding a second signal peptide domain. The at least
one first exon comprises a nucleotide sequence selected from the
group consisting of a nucleotide sequence of SEQ ID NO:33 (mB7-1),
SEQ ID NO:35 (hB7-1), SEQ ID NO:37 (mB7-2) and SEQ ID NO:39 (hB7-2)
and SEQ ID NO:41 (hB7-2). In this embodiment, the isolated nucleic
acid includes a nucleotide sequence encoding at least one second
signal peptide domain. Preferably, the isolated nucleic acid does
not comprise a nucleotide sequence encoding the first signal
peptide domain. Preferred T cell costimulatory molecule gene from
which nucleotide sequences can be derived include B7-1 and
B7-2.
[0040] C. Nucleic Acids Encoding Proteins With Domains Deleted or
Added
[0041] Another aspect of the invention pertains to isolated nucleic
acids encoding T cell costimulatory molecules having structural
domains which have been deleted or added. This aspect of the
invention is based, at least in part, on the discovery that
alternative splicing of mRNA transcripts encoding T cell
costimulatory molecules generates transcripts in which an exon
encoding a structural domain has been excised or in which at least
two exons encoding two forms of a structural domain are linked in
tandem. A preferred nucleic acid is one in which an exon encoding
an IgV-like domain has been deleted. Accordingly, in one
embodiment, the isolated nucleic acid encodes a protein comprising
a contiguous nucleotide sequence derived from at least one T cell
costimulatory molecule gene, the nucleotide sequence represented by
a formula A-B-C-D, wherein
[0042] A comprises a nucleotide sequence of at least one first exon
of a T cell costimulatory molecule gene, wherein the at least one
first exon encodes a signal peptide domain,
[0043] B comprises a nucleotide sequence of at least one second
exon of a T cell costimulatory molecule gene, wherein the at least
one second exon encodes an immunoglobulin constant region-like
domain,
[0044] C comprises a nucleotide sequence of at least one third exon
of a T cell costimulatory molecule gene, wherein the at least one
third exon encodes a transmembrane domain, and
[0045] D comprises a nucleotide sequence of at least one fourth
exon of a T cell costimulatory molecule gene, wherein the at least
one fourth exon encodes a cytoplasmic domain.
[0046] In the formula, A, B, C and D are contiguous nucleotide
sequences linked by phosphodiester bonds in a 5' to 3' orientation
from A to D.
[0047] Naturally-occurring mRNA transcripts encoding murine B7-1
have been detected in which the exon encoding the IgV-like domain
(i.e, exon 2) has been excised and the exon encoding the signal
peptide domain (i.e., exon 1) is spliced to the exon encoding the
IgC-like domain (i.e., exon 3) (see Example 7). In one embodiment,
an isolated nucleic acid encoding an alternatively spliced form of
murine B7-1 in which an IgV-like domain exon has been deleted
comprises a nucleotide sequence corresponding to usage of exons
1-3-4-5 (SEQ ID NO: 8). Alternatively, an alternatively spliced
form of murine B7-1 comprises a nucleotide sequence corresponding
to usage of exons 1-3-4-6 (SEQ ID NO: 10), which contains the
second, alternative cytoplasmic domain of mB7-1.
[0048] Another aspect of this invention features an isolated
nucleic acid encoding a T cell costimulatory molecule which
contains exons in addition to a known or previously identified form
of the T cell costimulatory molecule. For example, a
naturally-occurring murine B7-1 mRNA transcript has been identified
which contains two cytoplasmic domain-encoding exons in tandem,
i.e., the transcript contains exons 1-2-3-4-5-6 (the nucleotide
sequence of which is shown in SEQ ID NO: 3). Since there is an
in-frame termination codon within exon 5, translation of this
transcript produces a protein which contains only the Cyt I
cytoplasmic domain. However, if desired, this termination codon can
be mutated by standard site-directed mutagenesis techniques to
create a nucleotide sequence which encodes an mB7-1 protein
containing both a Cyt I and a Cyt II domain in tandem.
[0049] An isolated nucleic acid having a nucleotide sequence
disclosed herein can be obtained by standard molecular biology
techniques. For example, oligonucleotide primers suitable for use
in the polymerase chain reaction (PCR) can be prepared based upon
the nucleotide sequences disclosed herein and the nucleic acid
molecule can be amplified from cDNA and isolated. At least one
oligonucleotide primer should be complimentary to a nucleotide
sequence encoding an alternative structural domain. It is even more
preferable that at least one oligonucleotide primer span a novel
exon junction created by alternative splicing. For example, an
oligonucleotide primer which spans the junction of exon 4 and exon
6 can be used to preferentially amplify a murine B7-1 cDNA that
contains the second, alternative cytoplasmic domain (e.g., a cDNA
which contains exons 1-2-3-4-6; SEQ ID NO: 1). Alternatively, an
oligonucleotide primer complimentary to a nucleotide sequence
encoding a novel alternative structural domain can be used to
screen a cDNA library to isolate a nucleic acid of the
invention.
[0050] Isolated nucleic acid molecules having nucleotide sequences
other than those specifically disclosed herein are also encompassed
by the invention. For example, novel structural forms of B7-1 from
species other than mouse are within the scope of the invention
(e.g., alternatively spliced forms of human B7-1). Likewise, novel
structural forms of B7-2 from species other than mouse are also
within the scope of the invention (e.g., alternatively spliced
forms of human B7-2). Furthermore, additional alternatively spliced
forms for murine B7-1 and murine B7-2 can be identified using
techniques described herein. These alternatively spliced forms of
murine B7-1 and B7-2 are within the scope of the invention.
Isolated nucleic acid molecules encoding novel structural forms of
T cell costimulatory molecules can be obtained by conventional
techniques, such as by methods described below and in the
Examples.
[0051] An isolated nucleic acid encoding a novel structural form of
a T cell costimulatory molecule can be obtained by isolating and
analyzing cDNA clones encoding the T cell costimulatory molecule
(e.g., mB7-1; hB7-1; mB7-2; hB7-2 etc.) by standard techniques (see
for example Sambrook et al. Molecular Cloning: A Laboratory Manual,
2nd Edition, Cold Spring Harbor Laboratory press (1989) or other
laboratory handbook). For example, cDNAs encoding the costimulatory
molecule can be amplified by reverse transcriptase-polymerase chain
reaction (RT-PCR) using oligonucleotide primers specific for the
costimulatory molecule gene. The amplified cDNAs can then be
subcloned into a plasmid vector and sequenced by standard methods.
Oligonucleotide primers for RT-PCR can be designed based upon
previously disclosed nucleotide sequences of costimulatory
molecules (see Freeman, G. J. et al., (1991) J. Exp. Med.
174:625-631 for mB7-1; Freeman, G. J. et al., (1989) J. Immunol.
143:2714-2722 for hB7-1; Freeman, G. J. et al., (1993) J. Exp. Med.
178:2185-2192 for mB7-2; and Freeman, G. J. et al., (1993) Science
262:909-911 for hB7-2; nucleotide sequences are shown in SEQ ID
NOS: 16, 18, 20, 22 and 24). For analyzing the 5' or 3' ends of
mRNA transcripts, cDNA can be prepared using a 5' or 3' "RACE"
procedure ("rapid amplification of cDNA ends) as described in the
Examples. Alternative to amplifying specific cDNAs, a cDNA library
can be prepared from a cell line which expresses the costimulatory
molecule and screened with a probe containing all or a portion of
the nucleotide sequence encoding the costimulatory molecule.
[0052] Individual isolated cDNA clones encoding a T cell
costimulatory molecule can then be sequenced by standard techiques,
such as dideoxy sequencing or Maxam-Gilbert sequencing, to identify
a cDNA clone encoding a T cell costimulatory molecule having a
novel structural domain. A novel structural domain can be
identified by comparing the sequence of the cDNA clone to the
previously disclosed nucleotide sequences encoding T cell
costimulatory molecules (e.g., sequences shown in SEQ ID NO: 16,
18, 20, 22 and 24). Once a putative alternative structural domain
has been identified, the nucleotide sequence encoding the domain
can be mapped in genomic DNA to determine whether the domain is
encoded by a novel exon. This type of approach provides the most
extensive information about alternatively spliced forms of mRNAs
encoding the costimulatory molecule.
[0053] Alternatively, a novel structural domain for T cell
costimulatory molecules can be identified in genomic DNA by
identifying a novel exon in the gene encoding the T cell
costimulatory molecule. A novel exon can be identified as an open
reading frame flanked by splice acceptor and splice donor
sequences. Genomic clones encoding a T cell costimulatory molecule
can be isolated by screening a genomic DNA library with a probe
encompassing all or a portion of a nucleotide sequence encoding the
costimulatory molecule (e.g., having all or a portion of a
nucleotide sequence shown in SEQ ID NO: 16, 18, 20, 22 and 24). For
costimulatory molecules whose genes have been mapped to a
particular chromosome, a chromosome-specific library rather than a
total genomic DNA library can be used. For example, hB7-1 has been
mapped to human chromosome 3 (see Freeman, G. J. et al. (1992)
Blood 79:489-494; and Selvakumar, A. et al. (1992) Immunogenetics
36:175-181. Genomic clones can be sequenced by conventional
techniques and novel exons identified. A probe corresponding to a
novel exon can then be used to detect the nucleotide sequence of
this exon in mRNA transcripts encoding the costimulatory molecule
(e.g., by screening a cDNA library or by PCR).
[0054] A more preferred approach for identifying and isolating
nucleic acid encoding a novel structural domain of a T cell
costimulatory molecule is by "exon trapping". Exon trapping is a
technique that has been used successfully to identify and isolate
novel exons (see e.g. Duyk, G. M. et al. (1990) Proc. Natl. Acad
Sci. USA 87:8995-8999; Auch, D. and Reth, M. (1990) Nucleic Acids
Res. 18:6743-6744; Hamaguchi, M. et al. (1992) Proc. Natl. Acad.
Sci. USA 89:9779-9783; and Krizman, D. B and Berget, S. M. (1993)
Nucleic Acids Res. 21:5198-5202). The approach of exon trapping can
be applied to the isolation of exons encoding novel structural
domains of T cell costimulatory molecules, such as a novel
alternative cytoplasmic domain of human B7-1, as described in
Example 5.
[0055] In addition to the isolated nucleic acids encoding
naturally-occurring alternatively spliced forms of T cell
costimulatory molecules provided by the invention, it will be
appreciated by those skilled in the art that nucleic acids encoding
variant alternative forms, which may or may not occur naturally,
can be obtained used standard recombinant DNA techniques. The term
"variant alternative forms" is intended to include novel
combinations of exon sequences which can be created using
recombinant DNA techniques. That is, novel exons encoding
structural domains of T cell costimulatory molecules, either
provided by the invention or identified according to the teachings
of the invention, can be "spliced", using standard recombinant DNA
techniques, to other exons encoding other structural domains of the
costimulatory molecule, regardless of whether the particular
combination of exons has been observed in nature. Thus, novel
combinations of exons can be linked in vitro to create variant
alternative forms of T cell costimulatory molecules. For example,
the structural form of murine B7-1 which has the signal peptide
domain directly joined to the IgC-like domain (ie., which has the
IgV-like domain deleted) has been observed in nature in combination
with the cytoplasmic domain encoded by exon 5. However, using
conventional techniques, an alternative structural form can be
created in which the IgV-like domain is deleted and the alternative
cytoplasmic domain is encoded by exon 6. In another example, a
murine B7-1 cDNA containing exons 1-2-3-4-5-6 can be mutated by
site-directed mutagenesis to change a stop codon in exon 5 to an
amino acid encoding-codon such that an mB7-1 protein can be
produced which contains both a Cyt I domain and a Cyt II domain in
tandem. Additionally, an exon encoding a structural domain of one
costimulatory molecule can be transferred to another costimulatory
molecule by standard techniques. For example, the cytoplasmic
domain of mB7-2 can be replaced with the novel cytoplasmic domain
of mB7-1 provided by the invention (i.e., exon 6 of mB7-1 can be
"swapped" for the cytoplasmic domain exon of mB7-2).
[0056] It will also be appreciated by those skilled in the art that
changes can be made in the nucleotide sequences provided by the
invention without changing the encoded protein due to the
degeneracy of the genetic code. Additionally, nucleic acids which
have a nucleotide sequence different from those disclosed herein
due to degeneracy of the genetic code may be isolated from
biological sources. Such nucleic acids encode functionally
equivalent proteins (e.g., a protein having T cell costimulatory
activity) to those described herein. For example, a number of amino
acids are designated by more than one triplet codon. Codons that
specify the same amino acid, or synonyms (for example, CAU and CAC
are synonyms for histidine) may occur in isolated nucleic acids
from different biological sources or can be introduced into an
isolated nucleic acid by standard recombinant DNA techniques
without changing the protein encoded by the nucleic acid. Isolated
nucleic acids encoding alternatively spliced forms of T cell
costimulatory molecules having a nucleotide sequence which differs
from those provided herein due to degeneracy of the genetic code
are considered to be within the scope of the invention.
II. Additional Isolated Nucleic Acid Molecules of the Invention
[0057] In addition to isolated nucleic acids encoding alternative
forms of T cell costimulatory molecules, the invention also
discloses previously undescribed nucleotide sequences of the murine
B7-1 gene and mRNA transcripts. As described in detail in Example
3, it has now been discovered that murine B7-1 mRNA transcripts
contain additional 5' untranslated (UT) sequences which were not
previously reported. A 5' UT region of approximately 250 base pairs
has been reported for mB7-1 mRNA transcripts, determined by primer
extension analysis (see Selvakumar et al. (1993) Immunogenetics
38:292-295). As described herein, an additional 1500 nucleotides of
5' UT sequences have been discovered in mB7-1. These 5' UT
sequences are contiguous with known exon 1 sequences, thereby
extending the size of exon 1 by approximately 1500 base pairs. Thus
the novel 5' UT sequence of the invention corresponds to the 5'
region of mB7-1 exon 1 (i.e., exon 1 extends an additional
.about.1500 nucleotides at its 5' end than previously reported)
rather than corresponding to a new exon upstream of exon 1.
Computer analysis of the potential secondary structure of the 5' UT
region reveals that the most stable structure is comprised of
multiply folded palindromic sequences. This high degree of
secondary structure may explain the results of Selvakumar et al.
((1993) Immunogenetics 38:292-295) in that the secondary structure
could account for premature termination of the primer extension
reaction. The potential for excessive secondary structure in the 5'
UT region suggests that post-transcriptional mechanisms are
involved in controlling mB7-1 expression. Thus, inclusion of the
long 5' UT sequence in recombinant expression vectors encoding
mB7-1 may provide post-transcriptional regulation that is similar
to that of the endogenous gene. Accordingly, the 5' UT region of
mB7-1 provided by the invention can be incorporated by standard
recombinant DNA techniques at the 5' end of a cDNA encoding a mB7-1
protein. The nucleotide sequence of the 5' UT region of mB7-1 (i.e,
the full nucleotide sequence of exon 1) is shown in SEQ ID NO:
6.
[0058] The discovery of additional 5' UT sequences in mB7-1 cDNA
demonstrates that transcription of the mB7-1 gene initiates further
upstream (i.e., 5' ) in genomic DNA than previously reported in
Selvakumar et al. (Immunogenetics (1993) 38:292-295). Transcription
of a gene is typically regulated by sequences in genomic DNA
located immediately upstream of sequences corresponding to the 5'
UT region of the transcribed mRNA. Nucleotides located within
approximately 200 base pairs of the start site of transcription are
generally considered to encompass the promoter of the gene and
often include canonical CCAAT or TATA elements indicative of a
typical eukaryotic promoter. For a gene having a promoter which
contains a TATA box, transcription usually starts approximately 30
base pairs downstream of the TATA box. In addition to CCAAT and
TATA-containing promoters, it is now appreciated that many genes
have promoters which do not contain these elements. Examples of
such genes include many members of the immunoglobulin gene
superfamily (see for example Breathnach, R. and Chambon, P. (1981)
Ann. Rev. Biochem. 50:349-383; Fisher, R. C. and Thorley-Lawson, D.
A. (1991) Mol. Cell. Biol. 11:1614-1623; Hogarth, P. M. et al.
(1991) J. Immunol. 146:369-376; Schanberg, L. E. (1991) Proc. Natl.
Acad Sci. USA 88:603-607; Zhou, L. J. et al. (1991) J. Immunol.
147:1424-1432). In such TATA-less promoters, transcriptional
regulation is thought to be provided by other DNA elements which
bind transcription factors. Sequence analysis of .about.180 base
pairs of mB7-1 genomic DNA immediately upstream of the newly
identified 5' UT region revealed the presence of numerous consensus
sites for transcription factor binding, including AP-2, PU.1 and
NF.kappa.B. The nucleotide sequence of this region is shown in SEQ
ID NO: 7. The structure of this region (i.e, the DNA elements
contained therein) is consistent with it functioning as a promoter
for transcription of the mB7-1 gene. The ability of this region of
DNA to function as a promoter can be determined by standard
techniques routinely used in the art to identify transcriptional
regulatory elements. For example, this DNA region can be cloned
upstream of a reporter gene (e.g., encoding chloramphenicol acetyl
transferase, .beta.-galactosidase, luciferase etc.) in a
recombinant vector, the recombinant vector transfected into an
appropriate cell line and expression of the reporter gene detected
as an indication that the DNA region can function as a
transcriptional regulatory element. If it is determined that this
DNA region can function as a B7-1 promoter, it may be advantageous
to use this DNA region to regulate expression of a B7-1 cDNA in a
recombinant expression vector to mimic the endogenous expression of
B7-1.
III. Uses for the Isolated Nucleic Acid Molecules of the
Invention
[0059] A. Probes
[0060] The isolated nucleic acids of the invention are useful for
constructing nucleotide probes for use in detecting nucleotide
sequences in biological materials, such as cell extracts, or
directly in cells (e.g., by in situ hybridization). A nucleotide
probe can be labeled with a radioactive element which provides for
an adequate signal as a means for detection and has sufficient
half-life to be useful for detection, such as .sup.32P, .sup.3H,
.sup.14C or the like. Other materials which can be used to label
the probe include antigens that are recognized by a specific
labeled antibody, fluorescent compounds, enzymes and
chemiluminescent compounds. An appropriate label can be selected
with regard to the rate of hybridization and binding of the probe
to the nucleotide sequence to be detected and the amount of
nucleotide available for hybridization. The isolated nucleic acids
of the invention, or oligonucleotide fragments thereof, can be used
as suitable probes for a variety of hybridization procedures well
known to those skilled in the art. The isolated nucleic acids of
the invention enable one to determine whether a cell expresses an
alternatively spliced form of a T cell costimulatory molecule. For
example, mRNA can be prepared from a sample of cells to be examined
and the mRNA can be hybridized to an isolated nucleic acid
encompassing a nucleotide sequence encoding all or a portion of an
alternative cytoplasmic domain of a T cell costimulatory molecule
(e.g., SEQ ID NO: 1) to detect the expression of the alternative
cytoplasmic domain form of the costimulatory molecule in the cells.
Furthermore, the isolated nucleic acids of the invention can be
used to design oligonucleotide primers, e.g. PCR primers, which
allow one to detect the expression of an alternatively spliced form
of a T cell costimulatory molecule. Preferably, this
oligonucleotide primer spans a novel exon junction created by
alternative splicing and thus can only amplify cDNAs encoding this
alternatively spliced form. For example, an oligonucleotide primer
which spans exon 4 and exon 6 of murine B7-1 can be used to
distinguish between the expression of a first cytoplasmic domain
form of mB7-1 (i.e, encoded by exons 1-2-3-4-5) and expression of
an alternative second cytoplasmic domain form of a costimulatory
molecule (i.e., encoded by exons 1-2-3-4-6) (e.g., see Example
2).
[0061] The probes of the invention can be used to detect an
alteration in the expression of an alternatively splicedform of a T
cell costimulatory molecule, such as in a disease state. For
example, detection of a defect in the expression of an
alternatively splicedform of a T cell costimulatory molecule that
is associated with an immunodeficiency disorder can be used to
diagnose the disorder (i.e., the probes of the invention can be
used for diagnostic purposes). Many congenital immunodeficiency
diseases result from lack of expression of a cell-surface antigen
important for interactions between T cells and antigen presenting
cells. For example, the bare lymphocyte syndrome results from lack
of expression of MHC class II antigens (see e.g., Rijkers, G. T. et
al. (1987) J. Clin. Immunol. 7:98-106; Hume, C. R. et al. (1989)
Hum. Immunol. 25:1-11)) and X-linked hyperglobulinemia results from
defective expression of the ligand for CD40 (gp39) (see e.g.
Korthauer, U et al. (1993) Nature 361:541; Aruffo, A. et al. (1993)
Cell 72:291-300). An immunodeficiency disorder which results from
lack of expression of an alternatively spliced form of a T cell
costimulatory molecule can be diagnosed using a probe of the
invention. For example, a disorder resulting from the lack of
expression of the Cyt II form of B7-1 can be diagnosed in a patient
based upon the inability of a probe which detects this form of B7-1
(e.g., an oligonucleotide spanning the junction of exon 4 and exon
6) to hybridize to mRNA in cells from the patient (e.g., by RT-PCR
or by Northern blotting).
[0062] B. Recombinant Expression Vectors
[0063] An isolated nucleic acid of the invention can be
incorporated into an expression vector (i.e., a recombinant
expression vector) to direct expression of a novel structural form
of a T cell costimulatory molecule encoded by the nucleic acid. The
recombinant expression vectors are suitable for transformation of a
host cell, and include a nucleic acid (or fragment thereof) of the
invention and a regulatory sequence, selected on the basis of the
host cells to be used for expression, which is operatively linked
to the nucleic acid. Operatively linked is intended to mean that
the nucleic acid is linked to a regulatory sequence in a manner
which allows expression of the nucleic acid. Regulatory sequences
are art-recognized and 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 known to
those skilled in the art or are described in Goeddel, Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990). It should be understood that the design
of the expression vector may depend on such factors as the choice
of the host cell to be transfected and/or the type of protein
desired to be expressed. Such expression vectors can be used to
transfect cells to thereby produce proteins or peptides encoded by
nucleic acids as described herein.
[0064] The recombinant expression vectors of the invention can be
designed for expression of encoded proteins in prokaryotic or
eukaryotic cells. For example, proteins can be expressed in
bacterial cells such as E. coli, insect cells (using baculovirus),
yeast cells or mammalian cells. Other suitable host cells can be
found in Goeddel, Gene Expression Technology: Methods in Enzymology
185, Academic Press, San Diego, Calif. (1990). Expression in
prokaryotes is most often carried out in E. coli with vectors
containing constitutive or inducible promotors directing the
expression of either fusion or non-fusion proteins. Fusion vectors
add a number of amino acids usually to the amino terminus of the
expressed target gene. Such fusion vectors typically serve three
purposes: 1) to increase expression of recombinant protein; 2) to
increase the solubility of the target recombinant protein; and 3)
to aid in the purification of the target recombinant protein by
acting as a ligand in affinity purification. Often, in fusion
expression vectors, a proteolytic cleavage site is introduced at
the junction of the fusion moiety and the target recombinant
protein to enable separation of the target recombinant protein from
the fusion moiety subsequent to purification of the fusion protein.
Such enzymes, and their cognate recognition sequences, include
Factor Xa, thrombin and enterokinase. 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-tranferase, maltose E binding
protein, or protein A, respectively, to the target recombinant
protein.
[0065] Inducible non-fusion prokaryotic expression vectors include
pTrc (Amann et al., (1988) Gene 69:301-315) and pET11d (Studier et
al., Gene Expression Technology: Methods in Enzymology 185,
Academic Press, San Diego, Calif. (1990) 60-89). In pTrc, target
gene expression relies on host RNA polymerase transcription from a
hybrid trp-lac fusion promoter. In pET11d, expression of inserted
target genes relies on transcription from the T7 gn10-lac 0 fusion
promoter mediated by a coexpressed viral RNA polymerase (T7 gn1).
This viral polymerase is supplied by host strains BL21(DE3) or
HMS174(DE3) from a resident .lambda. prophage harboring a T7 gn1
under the transcriptional control of the lacUV 5 promoter.
[0066] One strategy to maximize recombinant protein expression in
E. coli is to express the protein in a host bacterial strain with
an impaired capacity to proteolytically cleave the recombinant
protein (Gottesman, S., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128).
Another strategy is to alter the nucleic acid sequence of the
nucleic acid to be inserted into an expression vector (e.g., a
nucleic acid of the invention) so that the individual codons for
each amino acid would be those preferentially utilized in highly
expressed E. coli proteins (Wada et al., (1992) Nuc. Acids Res.
20:2111-2118). Such alteration of nucleic acid sequences of the
invention can be carried out by standard DNA synthesis techniques
and are encompassed by the invention.
[0067] Examples of vectors for expression in yeast S. cerivisae
include pYepSec1 (Baldari. et al., (1987) Embo J. 6:229-234), pMFa
(Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et
a, (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).
[0068] Expression of alternatively spliced forms of T cell
costimulatory molecules in mammalian cells is accomplished using a
mammalian expression vector. Examples of mammalian expression
vectors include pCDM8 (Seed, B., (1987) Nature 329:840) and pMT2PC
(Kaufman et al. (1987), EMBO J. 6:187-195). When used in mammalian
cells, the expression vector's control functions are often provided
by viral material. For example, commonly used promoters are derived
from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
The recombinant expression vector can be designed such that
expression of the nucleic acid occurs preferentially in a
particular cell type. In this situation, the expression vector's
control functions are provided by regulatory sequences which allow
for preferential expression of a nucleic acid contained in the
vector in a particular cell type, thereby allowing for tissue or
cell specific expression of an encoded protein.
[0069] The recombinant expression vectors of the invention can be a
plasmid or virus, or viral portion which allows for expression of a
nucleic acid introduced into the viral nucleic acid. For example,
replication defective retroviruses, adenoviruses and
adeno-associated viruses can be used. The recombinant expression
vectors can be introduced into a host cell, e.g. in vitro or in
vivo. A host cell line can be used to express a protein of the
invention. Furthermore, introduction of a recombinant expression
vector of the invention into a host cell can be used for
therapeutic purposes when the host cell is defective in expressing
the novel structural form of the T cell costimulatory molecule. For
example, in a recombinant expression vector of the invention can be
used for gene therapy purposes in a patient with an
immunodeficiency disorder resulting from lack of expression of a
novel structural form of a T cell costimulatory molecule.
[0070] C. Host Cells
[0071] The invention further provides a host cell transfected with
a recombinant expression vector of the invention. The term "host
cell" is intended to include prokaryotic and eukaryotic cells into
which a recombinant expression vector of the invention can be
introduced. The terms "transformed with", "transfected with",
"transformation" and "transfection" are intended to encompass
introduction of nucleic acid (e.g., a vector) into a cell by one of
a number of possible techniques known in the art. Prokaryotic cells
can be transformed with nucleic acid by, for example,
electroporation or calcium-chloride mediated transformation.
Nucleic acid can be introduced into mammalian cells via
conventional techniques such as calcium phosphate co-precipitation,
DEAE-dextran-mediated transfection, lipofectin, electroporation or
microinjection. Suitable methods for transforming and transfecting
host cells can be found in Sambrook et al. (Molecular Cloning: A
Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press
(1989)), and other laboratory handbooks.
[0072] The number of host cells transfected with a recombinant
expression vector of the invention by techniques such as those
described above will depend upon the type of recombinant expression
vector used and the type of transfection technique used. Typically,
plasmid vectors introduced into mammalian cells are integrated into
host cell DNA at only a low frequency. In order to identify these
integrants, a gene that contains a selectable marker (i.e.,
resistance to antibiotics) can be introduced into the host cells
along with the gene of interest. Preferred selectable markers
include those which confer resistance to certain drugs, such as
G418 and hygromycin. Selectable markers can be introduced on a
separate vector (e.g., plasmid) from the nucleic acid of interest
or, preferably, are introduced on the same vector (e.g., plasmid).
Host cells transformed with one or more recombinant expression
vectors containing a nucleic acid of the invention and a gene for a
selectable marker can be identified by selecting for cells using
the selectable marker. For example, if the selectable marker
encoded a gene conferring neomycin resistance, transformant cells
can be selected with G418. Cells that have incorporated the
selectable marker gene will survive, while the other cells die.
[0073] Preferably, the novel cytoplasmic domain form of the T cell
costimulatory molecule is expressed on the surface of a host cell
(e.g., on the surface of a mammalian cell). This is accomplished by
using a recombinant expression vector encoding extracellular
domains (e.g., signal peptide, V-like and/or C-like domains),
transmembrane and cytoplasmic domains of the T cell costimulatory
molecule with appropriate regulatory sequences (e.g., a signal
sequence) to allow for surface expression of the translated
protein.
[0074] In one embodiment, a host cell is transfected with a
recombinant expression vector encoding a second, novel cytoplasmic
domain form of a T cell costimulatory molecule. In a preferred
embodiment, the host cell does not express the first (i.e.,
previously disclosed) cytoplasmic domain form of the costimulatory
molecule. For example, a host cell which does not express a form of
murine B7-1 containing Cyt I can be transfected with a recombinant
expression vector encoding a form of murine B7-1 containing Cyt II.
Such a host cell will thus excusively express the form of B7-1
containing Cyt II. This type of host cell is useful for studying
signaling events and/or immunological responses which are mediated
by the Cyt II domain rather than the Cyt I domain of B7-1. For
example, one type of cell which can be used to create a host cell
which exclusively expresses the Cyt II-form of murine B7-1 is a
non-murine cell, since the non-murine cell does not express murine
B7-1. Preferably, the non-murine cell also does not express other
costimulatory molecules (e.g., COS cells can be used).
Alternatively, a mouse cell which does not express the Cyt-I form
of murine B7-1 can be used. For example, a recombinant expression
vector of the invention can be introduced into NIH 3T3 fibroblast
cells (which are B7-1 negative) or into cells derived from a mutant
mouse in which the endogenous B7-1 gene has been disrupted and thus
which does not natively express any form of B7-1 molecule (i.e.,
into cells derived from a "B7-1 knock-out" mouse, such as that
described in Freeman, G. J. et al. (1993) Science 262:907-909).
[0075] In another embodiment, the host cell transfected with a
recombinant expression vector encoding a novel structural form of a
T cell costimulatory molecule is a tumor cell. Expression of the
Cyt-I form of murine B7-1 on the surface of B7-1 negative murine
tumor cells has been shown to induce T cell mediated specific
immunity against the tumor cells accompanied by tumor rejection and
prolonged protection to tumor challenge in mice (see Chen, L., et
al. (1992) Cell 71, 1093-1102; Townsend, S. E. and Allison, J. P.
(1993) Science 259, 368-370; Baskar, S., et al. (1993) Proc. Natl.
Acad. Sci. 90, 5687-5690). Similarly, expression of novel
structural forms of costimulatory molecules on the surface of a
tumor cell may be useful for increasing the immunogenicity of the
tumor cell. For example, tumor cells obtained from a patient can be
transfected ex vivo with a recombinant expression vector of the
invention, e.g., encoding an alternative cytoplasmic domain form of
a costimulatory molecule, and the transfected tumor cells can then
be returned to the patient. Alternatively, gene therapy techniques
can be used to target a tumor cell for transfection in vivo.
Additionally, the tumor cell can also be transfected with
recombinant expression vectors encoding other proteins to be
expressed on the tumor cell surface to increase the immunogenicity
of the tumor cell. For example, the Cyt-I form of B7-1, B7-2, MHC
molecules (e.g., class I and/or class II) and/or adhesion molecules
can be expressed on the tumor cells in conjunction with the Cyt-II
form of B7-1.
[0076] D. Anti-Sense Nucleic Acid Molecules
[0077] The isolated nucleic acid molecules of the invention can
also be used to design anti-sense nucleic acid molecules, or
oligonucleotide fragments thereof, that can be used to modulate the
expression of alternative forms of T cell costimulatory molecules.
An anti-sense nucleic acid comprises a nucleotide sequence which is
complementary to a coding strand of a nucleic acid, e.g.
complementary to an mRNA sequence, constructed according to the
rules of Watson and Crick base pairing, and can hydrogen bond to
the coding strand of the nucleic acid. The hydrogen bonding of an
antisense nucleic acid molecule to an mRNA transcript can prevent
translation of the mRNA transcript and thus inhibit the production
of the protein encoded therein. Accordingly, an anti-sense nucleic
acid molecule can be designed which is complementary to a
nucleotide sequence encoding a novel structural domain of a T cell
costimulatory molecule to inhibit production of that particular
structural form of the T cell costimulatory molecule. For example,
an anti-sense nucleic acid molecule can be designed which is
complementary to a nucleotide sequence encoding the Cyt-II form of
murine B7-1 and used to inhibit the expression of this form of the
costimulatory molecule.
[0078] An anti-sense nucleic acids molecule, or oligonucleotide
fragment thereof, can be constructed by chemical synthesis and
enzymatic ligation reactions using procedures known in the art. The
anti-sense nucleic acid or oligonucleotide can be chemically
synthesized using naturally-occurring nucleotides or variously
modified nucleotides designed to increase the biological stability
of the molecules or to increase the physical stability of the
duplex formed between the ant-isense and sense nucleic acids e.g.
phosphorothioate derivatives and acridine substituted nucleotides
can be used. Alternatively, the anti-sense nucleic acids and
oligonucleotides can be produced biologically using an expression
vector into which a nucleic acid has been subcloned in an
anti-sense orientation (i.e. nucleic acid transcribed from the
inserted nucleic acid will be of an anti-sense orientation to a
target nucleic acid of interest). The anti-sense expression vector
is introduced into cells in the form of a recombinant plasmid,
phagemid or attenuated virus in which anti-sense nucleic acids are
produced under the control of a high efficiency regulatory region,
the activity of which can be determined by the cell type into which
the vector is introduced. For a discussion of the regulation of
gene expression using anti-sense genes see Weintraub, H. et al.,
"Antisense RNA as a molecular tool for genetic analysis",
Reviews--Trends in Genetics, Vol. 1(1) 1986.
[0079] E. Non-Human Transgenic and Homologous Recombinant
Animals
[0080] The isolated nucleic acids of the invention can further be
used to create a non-human transgenic animal. A transgenic animal
is an animal having cells that contain a transgene, wherein the
transgene was introduced into the animal or an ancestor of the
animal at a prenatal, e.g., an embryonic, stage. A transgene is a
DNA molecule which is integrated into the genome of a cell from
which a transgenic animal develops and which remains in the genome
of the mature animal, thereby directing the expression of an
encoded gene product in one or more cell types or tissues of the
transgenic animal. Accordingly, the invention provides a non-human
transgenic animal which contains cells transfected to express an
alternative form of a T cell costimulatory molecule. Preferably,
the non-human animal is a mouse. A transgenic animal can be
created, for example, by introducing a nucleic acid encoding the
protein (typically linked to appropriate regulatory elements, such
as a tissue-specific enhancer) into the male pronuclei of a
fertilized oocyte, e.g., by microinjection, and allowing the oocyte
to develop in a pseudopregnant female foster animal. For example, a
transgenic animal (e.g., a mouse) which expresses an mB7-1 protein
containing a novel cytoplasmic domain (e.g. Cyt-II) can be made
using the isolated nucleic acid shown in SEQ ID NO: 1 or SEQ ID NO:
3. Alternatively, a transgenic animal (e.g., a mouse) which
expresses an mB7-2 protein containing an alternative signal peptide
domain can be made using the isolated nucleic acid shown in SEQ ID
NO: 12. Intronic sequences and polyadenylation signals can also be
included in the transgene to increase the efficiency of expression
of the transgene. These isolated nucleic acids can be linked to
regulatory sequences which direct the expression of the encoded
protein one or more particular cell types. Methods for generating
transgenic animals, particularly animals such as mice, have become
conventional in the art and are described, for example, in U.S.
Pat. Nos. 4,736,866 and 4,870,009 and Hogan, B. et al., (1986) A
Laboratory Manual, Cold Spring Harbor, N. Y., Cold Spring Harbor
Laboratory. A transgenic founder animal can be used to breed
additional animals carrying the transgene.
[0081] The isolated nucleic acids of the invention can further be
used to create a non-human homologous recombinant animal. The term
"homologous recombinant animal' as used herein is intended to
describe an animal containing a gene which has been modified by
homologous recombination. The homologous recombination event may
completely disrupt the gene such that a functional gene product can
no longer be produced (often referred to as a "knock-out" animal)
or the homologous recombination event may modify the gene such that
an altered, although still functional, gene product is produced.
Preferably, the non-human animal is a mouse. For example, an
isolated nucleic acid of the invention can be used to create a
homologous recombinant mouse in which a recombination event has
occurred in the B7-1 gene at an exon encoding a cytoplasmic domain
such that this exon is altered (e.g., exon 5 or exon 6 is altered).
Homologous recombinant mice can thus be created which express only
the Cyt I or Cyt II domain form of B7-1. Accordingly, the invention
provides a non-human knock-out animal which contains a gene
encoding a B7-1 protein wherein an exon encoding a novel
cytoplasmic domain is disrupted or altered.
[0082] To create an animal with homologously recombined nucleic
acid, a vector is prepared which contains the DNA sequences which
are to replace the endogenous DNA sequences, flanked by DNA
sequences homologous to flanking endogenous DNA sequences (see for
example Thomas, K. R. and Capecchi, M. R. (1987) Cell 51:503). The
vector is introduced into an embryonic stem cell line (e.g., by
electroporation) and cells in which the introduced DNA has
homologously recombined with the endogenous DNA are selected (see
for example Li, E. et al. (1992) Cell 69:915). The selected cells
are then injected into a blastocyst of an animal (e.g., a mouse) to
form aggregation chimeras (see for example Bradley, A. in
Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.
J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric
embryo can then be implanted into a suitable pseudopregnant female
foster animal and the embryo brought to term. Progeny harbouring
the homologously recombined DNA in their germ cells can be used to
breed animals in which all cells of the animal contain the
homologously recombined DNA.
IV. Isolated Novel Forms of Costimulatory Molecules
[0083] The invention further provides isolated T cell costimulatory
molecules encoded by the nucleic acids of the invention. These
molecules have a novel structural form, either containing a novel
structural domain or having a structural domain deleted or added.
The term "isolated" refers to a T cell costimulatory molecule,
e.g., a protein, substantially free of cellular material or culture
medium when produced by recombinant DNA techniques, or chemical
precursors or other chemicals-when chemically synthesized. In one
embodiment, the novel T cell costimulatory molecule is a B7-1
protein. In another embodiment, the novel T cell costimulatory
molecule is a B7-2 protein.
[0084] A. Proteins with a Novel Cytoplasmic Domain
[0085] One aspect of the invention pertains to a T cell
costimulatory molecule which includes at least one novel
cytoplasmic domain. In one embodiment, the invention provides a
protein which binds to CD28 and/or CTLA4 and has an amino acid
sequence derived from amino acid sequences encoded by at least one
T cell costimulatory molecule gene. In this embodiment, the protein
comprises a contiguous amino acid sequence represented by a formula
A-B-C-D-E, wherein
[0086] A, which may or may not be present, comprises an amino acid
sequence of a signal peptide domain,
[0087] B comprises an amino acid sequence of an immunoglobulin
variable region-like domain encoded by at least one exon of a T
cell costimulatory molecule gene,
[0088] C comprises an amino acid sequence of an immunoglobulin
constant region-like domain encoded by at least one exon of a T
cell costimulatory molecule gene,
[0089] D comprises an amino acid sequence of a transmembrane domain
encoded by at least one exon of a T cell costimulatory molecule
gene, and
[0090] E comprises an amino acid sequence of a cytoplasmic domain
encoded by at least one exon of a T cell costimulatory molecule
gene,
[0091] with the proviso that E does not comprise an amino acid
sequence of a cytoplasmic domain selected from the group consisting
of SEQ ID NO: 26 (mB7-1), SEQ ID NO: 28 (hB7-1), SEQ ID NO: 30
(mB7-2), and SEQ ID NO: 32 (hB7-2).
[0092] In the formula, A, B, C, D, and E are contiguous amino acid
residues linked by amide bonds from an N-terminus to a C-terminus.
According to the formula, A can be an amino acid sequence of a
signal peptide domain of a heterologous protein which efficiently
expresses transmembrane or secreted proteins, such as the
oncostatin M signal peptide. Preferably, A, if present, comprises
an amino acid sequence of a signal peptide domain encoded by at
least one exon of a T cell costimulatory molecule gene. In one
preferred embodiment, the isolated protein is a B7-1 or a B7-2
protein. E preferably comprises an amino acid sequence of a murine
B7-1 cytoplasmic domain having an amino acid sequence shown in SEQ
ID NO: 5 (i.e., the amino acid sequence of the cytoplasmic domain
encoded by the novel exon 6 of the invention).
[0093] Another embodiment of the invention provides an isolated
protein which binds CD28 or CTLA4 and is encoded by a T cell
costimulatory molecule gene having at least one first exon encoding
a first cytoplasmic domain and at least one second exon encoding a
second cytoplasmic domain. The at least one first cytoplamic domain
comprises an amino acid sequence selected from the group consisting
of amino acid sequence of SEQ ID NO:26 (mB7-1), SEQ ID NO:28
(hB7-1), SEQ ID NO:30 (mB7-2) and SEQ ID NO:32 (hB7-2). In this
embodiment, the protein includes an amino acid sequence comprising
at least one second cytoplasmic domain. Preferably, the protein
does not include an amino acid sequence comprising a first
cytoplasmic domain. Preferred proteins which bind CD28 and/or CTLA4
are derived from B7-1 and B7-2. In a particularly preferred
embodiment, the invention provides an isolated protein which binds
CD28 or CTLA4 and has a novel cytoplasmic domain comprising an
amino acid sequence shown in SEQ ID NO: 2.
[0094] A. Proteins with a Novel Signal Peptide Domain
[0095] In yet another aspect of the invention, T cell costimulatory
molecules which include at least one novel signal peptide domain
are provided. In one embodiment, the isolated protein binds to CD28
or CTLA4 and has an amino acid sequence derived from amino acid
sequences encoded by at least one T cell costimulatory molecule
gene. In this embodiment, the protein comprises a contiguous amino
acid sequence represented by a formula A-B-C-D-E, wherein
[0096] A comprises an amino acid sequence of a signal peptide
domain encoded by at least one exon of a T cell costimulatory
molecule gene,
[0097] B comprises an amino acid sequence of an immunoglobulin
variable region-like domain encoded by at least one exon of a T
cell costimulatory molecule gene,
[0098] C comprises an amino acid sequence of an immunoglobulin
constant region-like domain encoded by at least one exon of a T
cell costimulatory molecule gene,
[0099] D, which may or may not be present, comprises an amino acid
sequence of a transmembrane domain encoded by at least one exon of
a T cell costimulatory molecule gene, and
[0100] E, which may or may not be present, comprises an amino acid
sequence of a cytoplasmic domain encoded by at least one exon of a
T cell costimulatory molecule gene,
[0101] with the proviso that A not comprise an amino acid sequence
of a signal peptide domain selected from the group consisting of
SEQ ID NO: 34 (mB7-1), SEQ ID NO: 36 (hB7-1), SEQ ID NO: 38
(mB7-2), SEQ ID NO: 40 (hB7-2), SEQ ID NO: 42 (hB7-2).
[0102] In the formula, A, B, C, D, and E are contiguous amino acid
residues linked by amide bonds from an N-terminus to a C-terminus.
To produce a soluble form of the T cell costimlatory molecule D,
which comprises an amino acid sequence of a transmembrane domain
and E, which comprises an amino acid sequence of a cytoplasmic
domain may not be present in the molecule. Preferably, A comprises
an amino acid sequence of a novel signal peptide domain shown in
SEQ ID NO: 15.
[0103] In another embodiment of the invention, the isolated protein
which binds CD28 or CTLA4 is encoded by a T cell costimulatory
molecule gene having at least one first exon encoding a first
signal peptide domain and at least one second exon encoding a
second signal peptide domain. The at least one first signal peptide
domain comprises an amino acid sequence selected from the group
consisting of an amino acid sequence of SEQ ID NO:34 (mB7-1), SEQ
ID NO:36 (hB7-1), SEQ ID NO:38 (mB7-2) and SEQ ID NO:40 (hB7-2) and
SEQ ID NO:42 (hB7-2). In this embodiment, the protein includes an
amino acid sequence comprising at least one second signal peptide
domain. Preferably, the protein does not include an amino acid
sequence comprising a first signal peptide domain. Preferred
proteins which bind CD28 and/or CTLA4 are derived from B7-1 and
B7-2. In a particularly preferred embodiment, the invention
features a murine B7-2 protein having a comprising an amino acid
sequence shown in SEQ ID NO: 13.
[0104] C. Isolated Proteins with Structural Domains Deleted or
Added
[0105] This invention also features costimulatory molecules which
have at least one structural domain deleted. A preferred structural
form has at least one IgV-like domain deleted. In one embodiment,
the isolated protein has an amino acid sequence derived from amino
acid sequences encoded by at least one T cell costimulatory
molecule gene and comprises a contiguous amino acid sequence
represented by a formula A-B-C-D, wherein
[0106] A, which may or may not be present, comprises an amino acid
sequence of a signal peptide domain encoded by at least one exon of
a T cell costimulatory molecule gene,
[0107] B comprises an amino acid sequence of an immunoglobulin
constant region-like domain encoded by at least one exon of a T
cell costimulatory molecule gene, and
[0108] C comprises an amino acid sequence of a transmembrane domain
encoded by at least one exon of a T cell costimulatory molecule
gene, and
[0109] D comprises an amino acid sequence of a cytoplasmic domain
encoded by at least one exon of a T cell costimulatory molecule
gene.
[0110] In the formula, A, B, C and D are contiguous amino acid
residues linked by amide bonds from an N-terminus to a C-terminus.
In a preferred embodiment, an isolated murine B7-1 protein having
an IgV-like domain deleted comprises an amino acid sequence shown
in SEQ ID NO: 9. Alternatively, an isolated murine B7-1 protein
having an IgV-like domain deleted comprises an amino acid sequence
shown in SEQ ID NO: 11.
[0111] The proteins of the invention can be isolated by expression
of the molecules (e.g., proteins or peptide fragments thereof) in a
suitable host cell using techniques known in the art. Suitable host
cells include prokaryotic or eukaryotic organisms or cell lines,
for example, yeast, E. coli and insect cells. The recombinant
expression vectors of the invention, described above, can be used
to express a costimulatory molecule in a host cell in order to
isolate the protein. The invention provides a method of preparing
an isolated protein of the invention comprising introducing into a
host cell a recombinant expression vector encoding the protein,
allowing the protein to be expressed in the host cell and isolating
the protein. Proteins can be isolated from a host cell expressing
the protein according to standard procedures of the art, including
ammonium sulfate precipitation, fractionation column chromatography
(e.g. ion exchange, gel filtration, electrophoresis, affinity
chromatography, etc.) and ultimately, crystallization (see
generally, "Enzyme Purification and Related Techniques", Methods in
Enzymology, 22, 233-577 (1971)).
[0112] Alternatively, the costimulatory molecules of the invention
can be prepared by chemical synthesis using techniques well known
in the chemistry of proteins such as solid phase synthesis
(Merrifield, 1964, J. Am. Chem. Assoc. 85:2149-2154) or synthesis
in homogeneous solution (Houbenweyl, 1987, Methods of Organic
Chemistry, ed. E. Wansch, Vol. 15 I and II, Thieme, Stuttgart).
V. Uses For the Novel T Cell Costimulatory Molecules of the
Invention
[0113] A. Costimulation
[0114] The novel T cell costimulatory molecules of the invention
can be used to trigger a costimulatory signal in T cells. When
membrane-bound or in a multivalent form, a T cell costimulatory
molecule can trigger a costimulatory signal in a T cell by allowing
the costimulatory molecule to interact with its receptor (e.g.,
CD28) on T cells in the presence of a primary activation signal. A
novel T cell costimulatory molecule of the invention can be
obtained in membrane-bound form by expressing the molecule in a
host cell (e.g., by transfecting the host cell with a recombinant
expression vector encoding the molecule). To be expressed on the
surface of a host cell, the T cell costimulatory molecule should
include extracellular domains (i.e., signal peptide, which may or
may not be present in the mature protein, IgV-like and IgC-like
domains), a transmembrane domain and a cytoplasmic domain. To
trigger a costimulatory signal, T cells are contacted with the cell
expressing the costimulatory molecule, preferably together with a
primary activation signal (e.g., MHC-associated antigenic peptide,
anti-CD3 antibody, phorbol ester etc.). Activation of the T cell
can be assayed by standard procedures, for example by measuring T
cell proliferation or cytokine production.
[0115] The novel T cell costimulatory molecules of the invention
can also be used to inhibit or block a costimulatory signal in T
cells. A soluble form of a T cell costimulatory molecule can be
used to competitively inhibit the interaction of membrane-bound
costimulatory molecules with their receptor (e.g., CD28 and/or
CTLA4) on T cells. A soluble form of a T cell costimulatory
molecule can be expressed in host cell line such that it is
secreted by the host cell line and can then be purified. The
soluble costimulatory molecule contains extracellular domains
(i.e., signal peptide, which may or may not be present in the
mature protein, IgV-like and IgC-like domains) but does not contain
a transmembrane or cytoplasmic domain. The soluble form of the T
cell costimulatory molecule can also be in the form of a fusion
protein, e.g. an immunoglobulin fusion protein wherein the
extracellular portion of the costimulatory molecule is fused to an
immunoglobulin constant region. A soluble form of a T cell
costimulatory molecule can be used to inhibit a costimulatory
signal in T cells by contacting the T cells with the soluble
molecule.
[0116] B. Antibodies
[0117] A novel structural form of a T cell costimulatory molecule
of the invention can be used to produce antibodies directed against
the costimulatory molecule. Conventional methods can be used to
prepare the antibodies. For example, to produce polyclonal
antibodies, a mammal, (e.g., a mouse, hamster, or rabbit) can be
immunized with a costimulatory molecule, or an immunogenic portion
thereof, which elicits an antibody response in the mammal.
Techniques for conferring immunogenicity on a protein include
conjugation to carriers or other techniques well known in the art.
For example, the protein can be administered in the presence of
adjuvant. The progress of immunization can be monitored by
detection of antibody titers in plasma or serum. Standard ELISA or
other immunoassay can be used with the immunogen as antigen to
assess the levels of antibodies. Following immunization, antisera
can be obtained and, if desired, polyclonal antibodies isolated
from the sera.
[0118] In addition to polyclonal antisera, the novel costimulatory
molecules of the invention can be used to raise monoclonal
antibodies. To produce monoclonal antibodies, antibody producing
cells (lymphocytes) can be harvested from an immunized animal and
fused with myeloma cells by standard somatic cell fusion procedures
thus immortalizing these cells and yielding hybridoma cells. Such
techniques are well known in the art. For example, the hybridoma
technique originally developed by Kohler and Milstein (Nature 256,
495-497 (1975)) as well as other techniques such as the human
B-cell hybridoma technique (Kozbor et al., Immunol. Today 4, 72
(1983)), the EBV-hybridoma technique to produce human monoclonal
antibodies (Cole et al. Monoclonal Antibodies in Cancer Therapy
(1985) Allen R. Bliss, Inc., pages 77-96), and screening of
combinatorial antibody libraries (Huse et al., Science 246, 1275
(1989)). Hybridoma cells can be screened immunochemically for
production of antibodies specifically reactive with the protein or
portion thereof and monoclonal antibodies isolated.
[0119] The term antibody as used herein is intended to include
fragments thereof which are also specifically reactive with an
alternative cytoplasmic domain of a costimulatory molecule.
Antibodies can be fragmented using conventional techniques and the
fragments screened for utility in the same manner as described
above for whole antibodies. For example, F(ab').sub.2 fragments can
be generated by treating antibody with pepsin. The resulting
F(ab').sub.2 fragment can be treated to reduce disulfide bridges to
produce Fab' fragments.
[0120] Chimeric and humanized antibodies are also within the scope
of the invention. It is expected that chimeric and humanized
antibodies would be less immunogenic in a human subject than the
corresponding non-chimeric antibody. A variety of approaches for
making chimeric antibodies, comprising for example a non-human
variable region and a human constant region, have been described.
See, for example, Morrison et al., Proc. Natl. Acad. Sci. U.S.A.
81, 6851 (1985); Takeda et al., Nature 314, 452 (1985), Cabilly et
al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397;
Tanaguchi et al., European Patent Publication EP171496; European
Patent Publication 0173494, United Kingdom Patent GB 2177096B.
Additonally, a chimeric antibody can be further "humanized"
antibodies such that parts of the variable regions, especially the
conserved framework regions of the antigen-binding domain, are of
human origin and only the hypervariable regions are of non-human
origin. Such altered immunoglobulin molecules may be made by any of
several techniques known in the art, (e.g., Teng et al., Proc.
Natl. Acad. Sci. U.S.A., 80, 7308-7312 (1983); Kozbor et al.,
Immunology Today, 4, 7279 (1983); Olsson et al., Meth. Enzymol.,
92, 3-16 (1982)), and are preferably made according to the
teachings of PCT Publication WO92/06193 or EP 0239400. Humanized
antibodies can be commercially produced by, for example, Scotgen
Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain.
[0121] Another method of generating specific antibodies, or
antibody fragments, reactive against an alternative cytoplasmic
domain of the invention is to screen phage expression libraries
encoding immunoglobulin genes, or portions thereof, with proteins
produced from the nucleic acid molecules of the present invention
(e.g., with all or a portion of the amino acid sequence of SEQ ID
NO: 7). For example, complete Fab fragments, V.sub.H regions and
F.sub.V regions can be expressed in bacteria using phage expression
libraries. See for example Ward et al., Nature 341, 544-546:
(1989); Huse et al., Science 246, 1275-1281 (1989); and McCafferty
et al. Nature 348, 552-554 (1990).
[0122] In a preferred embodiment, the invention provides an
antibody which binds to a novel structural domain of a T cell
costimulatory molecule provided by the invention. Such antibodies,
and uses therefor, are described in greater detail below in
subsection VI, part B.
[0123] C. Screening Assays
[0124] A T cell costimulatory molecule of the invention containing
a novel cytoplasmic domain can be used in a screening assay to
identify components of the intracellular signal tranduction pathway
induced in antigen presenting cells upon binding of the T cell
costimulatory molecule to its receptor on a T cell. In addition to
triggering a costimulatory signal in T cells, engagement of the
costimulatory molecule with a receptor on T cells is likely to
deliver distinct signals to the antigen presenting cell (i.e., the
cell expressing the T cell costimulatory molecule), e.g. through
the cytoplasmic domain. Signals delivered through a novel
cytoplasmic domain of the invention may be of particular importance
in the thymus, e.g., during positive selection of T cells during
development, since structural forms of costimulatory molecules
comprising a novel cytoplasmic domain are preferentially expressed
in the thymus. A host cell which exclusively expresses a Cyt-II
form of a costimulatory molecule (e.g., mB7-1) is especially useful
for elucidating such intracellular signal transduction pathways.
For example, a host cell which expresses only a Cyt-II form of the
costimulatory molecule can be stimulated through the costimulatory
molecule, e.g., by crosslinking the costimulatory molecules on the
cell surface with an antibody, and intracellular signals and/or
other cellular changes (e.g., changes in surface expression of
proteins etc.) induced thereupon can be identified.
[0125] Additionally, an isolated T cell costimulatory molecule of
the invention comprising a novel cytoplasmic domain can be used in
methods of identifying other molecules (e.g., proteins) which
interact with (i.e., bind to) the costimulatory molecule using
standard in vitro assays (e.g., incubating the isolated
costimulatory molecule with a cellular extract and determining by
immunoprecipitation if any molecules within the cellular extract
bind to the costimulatory molecule). It is of particular interest
to identify molecules which can interact with the novel cytoplasmic
domain since such molecules may also be involved in intracellular
signaling. For example, it is known that the cytoplasmic domains of
many cell-surface receptors can interact intracellularly with other
members of the signal transduction machinery, e.g., tyrosine
kinases.
[0126] The invention further provides a method for screening agents
to identify an agent which upregulates or downregulates expression
of a novel structural domain form of a T cell costimulatory
molecule. The method involves contacting a cell which expresses or
can be induced to express a T cell costimulatory molecule with an
agent to be tested and determining expression of a novel structural
domain form of the T cell costimulatory molecule by the cell. The
term "upregulates" encompasses inducing the expression of a novel
form of a T cell costimulatory molecule by a cell which does not
constitutively express such a molecule or increasing the level of
expression of a novel form of a T cell costimulatory molecule by a
cell which already expresses such a molecule. The term
"downregulates" encompasses decreasing or eliminating expression of
an a novel form of a T cell costimulatory molecule by a cell which
already expresses such a molecule. The term "agent" is intended to
include molecules which trigger an upregulatory or downregulatory
response in a cell. For example, an agent can be a small organic
molecule, a biological response modifier (e.g., a cytokine) or a
molecule which can crosslink surface structures on the cell (e.g.,
an antibody). For example, expression of the a novel cytoplasmic
domain form of the T cell costimulatory molecule by the cell can be
determined by detecting an mRNA transcript encoding the novel
cytoplasmic domain form of the T cell costimulatory molecule in the
cell. For example, mRNA from the cell can be reverse transcribed
and used as a template in PCR reactions utilizing PCR primers which
can distinguish between a Cyt I cytoplasmic domain form and a novel
Cyt II cytoplasmic domain form of the T cell costimulatory molecule
(see e.g., Example 2). Alternatively, a novel cytoplasmic
domain-containing T cell costimulatory molecule can be detected in
the cell using an antibody directed against the novel cytoplasmic
domain (e.g., by immunoprecipitation or immunohistochemistry). A
preferred T cell costimulatory molecule for use in the method is
B7-1. Cell types which are known to express the Cyt-I form of B7-1,
or which can be induced to express the Cyt-I form of B7-1, include
B lymphocytes, T lymphocytes and monocytes. Such cell types can be
screened with agents according to the method of the invention to
identify an agent which upregulates or downregulates expression of
the Cyt-II form of B7-1.
VI. Isolated Novel Structural Domains of T Cell Costimulatory
Molecules and Uses Therefor
[0127] Another aspect of the invention pertains to isolated nucleic
acids encoding novel structural domains of T cell costimulatory
molecules provided by the invention. In one embodiment, the
structural domain encoded by the nucleic acid is a cytoplasmic
domain. A preferred nucleic acid encoding a novel cytoplasmic
domain comprises a nucleotide sequence shown in SEQ ID NO: 4. In
another embodiment, the structural domain encoded by the nucleic
acid is a signal peptide domain. A preferred nucleic acid encoding
a novel signal peptide domain comprises a nucleotide sequence shown
in SEQ ID NO: 14.
[0128] The invention also provides isolated polypeptides
corresponding to novel structural domains of T cell costimulatory
molecules, encoded by nucleic acids of the invention. In one
embodiment, the structural domain is a cytoplasmic domain. A
preferred novel cytoplasmic domain comprises an amino acid sequence
shown in SEQ ID NO: 5. In another embodiment, the structural domain
is a signal peptide domain. A preferred novel signal peptide domain
comprises an amino acid sequence shown in SEQ ID NO: 15.
[0129] The uses of the novel structural domains of the invention
include the creation of chimeric proteins. The domains can further
be used to raise antibodies specifically directed against the
domains.
[0130] A. Chimeric Proteins
[0131] The invention provides a fusion protein comprised of two
peptides, a first peptide and a second peptide, wherein the second
peptide is a novel structural domain of a T cell costimulatory
molecule provided by the invention. In one embodiment, the novel
structural domain is a cytoplasmic domain, preferably comprising an
amino acid sequence shown in SEQ ID NO: 5. In another embodiment,
the novel structural domain is a signal peptide domain, preferably
comprising an amino acid sequence shown in SEQ ID NO: 15. For
example, a fusion protein can be made which contains extracellular
and transmembrane portions from a protein other than murine B7-1
and which contains a novel cytoplasmic domain (e.g., Cyt-I1) of
murine B7-1. This type of fusion protein can be made using standard
recombinant DNA techniques in which a nucleic acid molecule
encoding the cytoplasmic domain (e.g., SEQ ID NO:4) is linked
in-frame to the 3' end of a nucleic acid molecule encoding the
extracellular and transmembrane domains of the protein. The
recombinant nucleic acid molecule can be incorporated into an
expression vector and the encoded fusion protein can be expressed
by standard techniques, e.g., by transfecting the recombinant
expression vector into an appropriate host cell and allowing
expression of the fusion protein.
[0132] A fusion protein of the invention, comprising a first
peptide fused to a second peptide comprising a novel cytoplasmic
domain of the invention, can be used to transfer the signal
transduction function of the novel cytoplasmic domain to another
protein. For example, a novel cytoplasmic domain of B7-1 (e.g.,
Cyt-I1) can be fused to the extracellular and transmembrane domains
of another protein (e.g., an immunoglobulin protein, a T cell
receptor protein, a growth factor receptor protein etc.) and the
fusion protein can be expressed in a host cell by standard
techniques. The extracellular domain of the fusion protein can be
crosslinked (e.g., by binding of a ligand or antibody to the
extracellular domain) to generate an intracellular signal(s)
mediated by the novel cytoplasmic domain.
[0133] Additionally, a fusion protein of the invention can be used
in methods of identifying and isolating other molecules (e.g.,
proteins) which can interact intracellularly (i.e., within the cell
cytoplasm) with a novel cytoplasmic domain of the invention. One
approach to identifying molecules which interact intracellularly
with the cytoplasmic domain of a cell-surface receptor is to
metabolically label cells which express the receptor,
immunoprecipitate the receptor, usually with an antibody against
the extracellular domain of the receptor, and identify molecules
which are co-immunoprecipitated along with the receptor. In the
case of mB7-1, however, the cells which have been found to express
the naturally-occurring Cyt-II form of B7-1 have also been found to
express the naturally-occurring Cyt-I form of B7-1 (e.g.,
thymocytes, see Example 2). Thus, immunoprecipitation with an
antibody against the extracellular domain of mB7-1 would
immunoprecipitate both forms of the protein since the extracellular
domain is common to both the Cyt-I and Cyt-II containing forms.
Thus, molecules which interact with either Cyt-I or Cyt-II would be
co-immunoprecipitate. A fusion protein comprising a non-B7-1
extracellular domain (to which an antibody can bind), a
transmembrane domain (derived either from the non-B7-1 molecule or
from B7-1) and a B7-1 alternative cytoplasmic domain (e.g., Cyt-II)
can be constructed and expressed in a host cell which naturally
expresses the Cyt-II form of B7-1. The antibody directed against
the "heterologous" extracellular domain of the fusion protein can
then be used to immunoprecipitate the fusion protein and to
co-immunoprecipitate any other proteins which interact
intracellularly with the novel cytoplasmic domain.
[0134] B. Antibodies
[0135] An antibody which binds to a novel structural domain of the
invention can be prepared by using the domain, or a portion
thereof, as an immunogen. Polyclonal antibodies or monoclonal
antibodies can be prepared by standard techniques described above.
In a preferred approach, peptides comprising amino acid sequences
of the domain are used as immunogens, e.g. overlapping peptides
encompassing the amino acid sequence of the domain. For example,
polyclonal antisera against a novel cytoplasmic domain (e.g., Cyt
II of mB7-1) can be made by preparing overlapping peptides
encompassing the amino acid sequence of the domain and immunizing
an animal (e.g., rabbit) with the peptides by standard
techniques.
[0136] An antibody of the invention can be used to detect novel
structural forms of T cell costimulatory molecules. Such an
antibody is thus useful for distinguishing between expression by a
cell of different forms of T cell costimulatory molecules. For
example, a cell which is known to express a costimulatory molecule,
such as B7-1, (for example, by the ability of an antibody directed
against the extracellular portion of the costimulatory molecule to
bind to the cell) can be examined to determine whether the
costimulatory molecule includes a novel cytoplasmic domain of the
invention. The cell can be reacted with an antibody of the
invention by standard immunohistochemical techniques. For example,
the antibody can be labeled with a detectable substance and the
cells can be permeabilized to allow entry of the antibody into the
cell cytoplasm. The antibody is then incubated with the cell and
unbound antibody washed away. The presence of the detectable
substance associated with the cell is detected as an indication of
the binding of the antibody to a novel cytoplasmic domain expressed
in the cell. Suitable detectable substances with which to label an
antibody include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials and radioactive materials.
Examples of suitable enzymes include horseradish peroxidase,
alkaline phosphatase, .beta.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidinibiotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; and examples of suitable radioactive material include
.sup.125I .sup.131I, .sup.35S or .sup.3H.
[0137] C. Kinase Substrates
[0138] A novel cytoplasmic domain of the invention which contains a
concensus phosphorylation site (i.e., Cyt-II of mB7-1) can be used
as a substrate for a protein kinases which phosphorylates the
phosphorylation site. Kinase reactions can be performed by standard
techniques in vitro, e.g., by incubating a polypeptide comprising
the cytoplasmic domain (or a T cell costimulatory molecule which
includes the novel cytoplasmic domain) with the kinase. The kinase
reactions can be performed in the presence of radiolabeled ATP
(e.g., .sup.32P-.gamma.-ATP) to radiolabel the novel cytoplasmic
domain.
[0139] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references and published patents and patent applications cited
throughout the application are hereby incorporated by
reference.
[0140] The following methodology was used in the Examples.
MATERIALS AND METHODS
[0141] Genomic Cloning
[0142] A mouse 129 lambda genomic library was kindly provided by
Drs. Hong Wu and Rudolf Jaenisch of the Whitehead Institute for
Biomedical Research, Cambridge, Mass. Genomic DNA was prepared from
the J1 embryonic stem cell line (derived from the 129/sv mouse
strain), partially digested with MboI, sized (17-21 kb), and
ligated into the BamHI site of lambda-DASH II arms (Stratagene, La
Jolla Calif.). The library was probed with the coding region of
mB7-1 cDNA to yield four clones (.lambda.4, .lambda.9, .lambda.15,
and .lambda.16). These lambda clones were subdloned into
Bluescript-pKS II (Stratagene, La Jolla Calif.) for subsequent
restriction mapping.
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
[0143] Total cellular RNA was prepared from SWR/J mouse spleen and
thymus using RNA-Stat-60 (Tel-Test "B", Inc, Friendswood, Tex.).
Random hexamer primed reverse transcription (RT) was performed with
Superscript-RT (Gibco BRL, Gaithersburg Md.) using 1-10 .mu.g total
RNA in a 20 .mu.l reaction. All PCR reactions were performed in 25
.mu.l volumes using a manual "hot start", wherein 10.times.
deoxynucleotide triphosphates (dNTPs) were added to the samples at
80.degree. C. Final reaction conditions were: 60 mM Tris-HCl, pH
8.5, 15 mM (NH.sub.4).sub.2SO.sub.4, 2.5 mM MgCl.sub.2, 200 .mu.M
dNTPs, and 2 .mu.g/ml each of the specific primers. Cycling
conditions for all amplifications were 94.degree. C., 4 minutes
prior to 35 cycles of 94.degree. C. for 45 seconds, 58.degree. C.
for 45 seconds, and 72.degree. C. for 3 minutes, followed by a
final extension at 72.degree. C. for 7 minutes. The template for
primary PCR was 2 .mu.l of the RT reaction product and the template
for secondary nested PCR was 1 .mu.l of the primary PCR reaction
product.
Oligonucleotides
[0144] All oligonucleotides were synthesized on an Applied
Biosystems 381 A DNA Synthesizer. The oligonucleotides used in this
study are listed in Table I and their uses for primary or secondary
PCR, as well as sense, also are indicated.
[0145] Rapid Amplification of cDNA Ends (RACE) Procedure
[0146] Polyadenylated RNA purifed by two cycles of oligo-dT
selection was obtained from CH1 B lymphoma cells, which express
high levels of mB7-1. Primers designed to the most 5' end of the
cDNA were employed with the 5' RACE Kit (Gibco BRL, Gaithersburg,
Md.) according to the manufacturer's instructions. In brief, RNA
was reverse transcribed with a gene-specific oligonucleotide, the
cDNA purified, and a poly-dCTP tail was added with terminal
deoxynucleotide transferase. PCR was performed using a nested
primer and an oligonucleotide complimentary to the poly-dCTP tail.
PCR bands were cloned, sequenced, and correlated with the genomic
sequences.
[0147] Oligonucleotide Hybridization
[0148] Oligonucleotide(s) were 5' end-labeled with polynucleotide
kinase and .gamma..sup.32P-ATP. Hybridizations were carried out in
5.times.SSC and 5% SDS at 55.degree. C. overnight and subsequently
washed 3 times for 15 minutes with 2.times.SSC at 55.degree. C.
Blots were exposed to Kodak XAR-5 film with an intensifying screen
at -80.degree. C.
[0149] The oligonucleotides used for the PCR studies in Examples
1-4 are shown in Table I:
1TABLE I Oligonucleotides used for PCR studies Desig- nation
Sequence (5' to 3') sense PCR B7.27 CCAACATAACTGAGTCTGGAAA +
second- (SEQ ary ID NO: 43) B7.36 CTGGATTCTGACTCACCTTCA - second-
(SEQ ary ID NO: 44) B7.37 AGGTTAAGAGTGGTAGAGCCA - primary (SEQ ID
NO: 45) B7.38 AATACCATGTATCCCACATGG - second- (SEQ ary ID NO: 46)
B7.42 CTGAAGCTATGGCTTGCAATT + primary (SEQ ID NO: 47) B7.44
TGGCTTCTCTTTCCTTACCTT + second- (SEQ ary ID NO: 48) B7.48
GCAAATGGTAGATGAGACTGT - second- (SEQ ary ID NO: 49) B7.62
CAACCGAGAAATCTACCAGTAA - probe (SEQ ID NO: 50) B7.68
GCCGGTAACAAGTCTCTTCA + primary (SEQ ID NO: 51) B7.71
AAAAGCTCTATAGCATTCTGTC + primary (SEQ ID NO: 52) B7.80
ACTGACTTGGACAGTTGTTCA + second- (SEQ ary ID NO: 53) B7.547
TTTGATGGACAACTTTACTA - primary (SEQ ID NO: 54)
Example 1
Characterization of the mB7-1 Genomic Locus
[0150] Lambda clones containing mB7-1 genomic DNA were isolated
using a probe consisting of the coding region of mB7-1. Four
representative lambda clones (designated clones .lambda.4,
.lambda.9, .lambda.15, and .lambda.16) were selected for further
analysis. These lambda clones were subcloned and subjected to
restriction mapping with HindIII and BamHI. Regions containing
exons were further characterized with XbaI and PstI. Fine mapping
studies indicate that the mB7-1 locus is comprised of 6 exons
arranged in the following 5' to 3' order: 5' UT plus signal peptide
domain, Ig-V-like domain, Ig-C-like domain, transmembrane domain,
cytoplasmic domain I, and the alternative cytoplasmic domain II, to
be discussed below. The 4 lambda clones spanned over 40 kb of the
mB7-1 locus, excluding a gap of undetermined size between exon 1
(signal exon) and exon 2 (Ig-V-like exon). The gap between clones
.lambda.15 (transmembrane domain exon) and .lambda.16 (cytoplasmic
domain exon) was determined to be less than 100 base pairs by PCR
using a sense primer (B7.71) designed to the 3' end of clone
.lambda.15 and an antisense primer (B7.38) located at the 5' end of
clone X16. Clones ?9 and .lambda.15 overlapped in a region spanning
exon 2.
EXAMPLE 2
Identification of mB7-1 Exon 6: An Alternately Spliced Exon
Encoding a Novel Second Cytoplasmic Domain
[0151] Analysis of mB7-1 cDNAs isolated from an A20 B cell cDNA
library showed that one cDNA contained additional sequence not
previously described for the mB7-1 cDNA. This sequence was mapped
to the mB7-1 locus approximately 7-kb downstream of exon 5. A
canonical splice site was present immediately upstream of this
sequence and a polyadenylation site was present downstream. Taken
together, these data suggested that this novel sequence represents
an additional exon, encoding 46 amino acids, which may be
alternatively spliced in place of exon 5. This alternative
cytoplasmic domain is notable for two casein kinase II
phosphorylation sites (amino acid positions 11-15 (SAKDF) and amino
acid positions 28-32 (SLGEA) of SEQ ID NO: 5) (for a description of
casein kinase II phosphorylation sites see Pinna (1990) Biochimica
et Biophysica Acta 1054:267-284) and one protein kinase C
phosphorylation site (amino acid positions 11-14 (SAKD) of SEQ ID
NO: 5)(for a description of protein kinase C phosphorylation sites
see Woodgett et al. (1986) Biochemistry 161:177-184; and Kishimoto
et al. (1985) J. Biol. Chem. 260:12492-12499).
[0152] In order to assess whether exon 6 also could be used in an
alternative fashion, an antisense primer (B7.48) was designed to
the predicted exon 4/6 splice junction such that only the
alternatively spliced product would give rise to an amplified
product. This primer overhangs the putative exon 4/6 junction by 3
bp at its 3' end. The 3 bp overhang is insufficient to permit
direct priming in exon 4 outside the context of an exon 4/6 splice
(FIG. 1, lane 9, negative control is a cDNA clone containing only
mB7-1 CytI). The expected amplified product for the alternately
spliced transcript (FIG. 1, transcript C) would be 399 bp.
Interestingly, this transcript was observed only in thymic, but not
splenic RNA.
[0153] [In FIG. 1, lanes 1, 2 and 3 represent nested PCR products
from murine splenic RNA using PCR primers B7.27-B7.36, B7.27-B7.38,
and B7.27-B7.48, respectively. Lanes 4, 5 and 6 represent nested
PCR products from murine thymic RNA using PCR primers B7.27-B7.36,
B7.27-B7.38 and B7.27-B7.48, respectively. Lane 7 represents a
negative control (no input RNA). Lane 8 represents a positive
control (mB7-1 cDNA clone). Lane 9 represents a negative control
for B7.27-B7.48 amplification comprised of the mB7-1 cDNA
containing cytoplasmic domain I, which does not have the correct
exon 4-6 splice junction. Lane M is a 100 bp ladder with the lower
bright band equal to 600 bp. Letters A, B and C refer to the
transcripts detected and are further illustrated in FIG. 1. Note
that exon 6 splicing as an alternative cytoplasmic domain is
present only in the thymus, but not in the spleen].
[0154] To further investigate the use of exon 6 in mB7-1 mRNA
transcripts, nested RT-PCR spanning exons 3 through 6 was performed
using spleen RNA (FIG. 1, PCR product A). A PCR product longer than
predicted from the use of exon 6 as an alternatively spliced exon
also was observed. Subsequent sequence analysis indicated that in
this transcript, exons 5 and 6 were spliced in tandem, rather than
in an alternative fashion (FIG. 1, transcript A), making use of a
previously unrecognized splice donor site downstream of the
termination codon in exon 5. Thus, this alternative transcript
would not change the encoded protein. Subsequent sequence analysis
of a larger than expected product observed from spleen RNA (FIG. 1,
lane 3) revealed an additional example of the tandem splicing of
exon 6 to exon 5 using an alternative noncanonical splice site.
Transcripts with tandem splicing of exon 6 to exon 5 were observed
in the spleen and the thymus.
[0155] FIG. 2 is a schematic diagram of the three mB7-1 transcripts
(A, B, and C) detected by nested RT-PCR. Exons are depicted in
different shades of gray and untranslated sequences are white.
Oligonucleotide primers used for the initial RT-PCR and subsequent
nested PCR are indicated above their respective locations in the
transcripts. Only B7.48 spans an exon-exon junction as indicated.
The scale bar above indicates the length in base pairs.
EXAMPLE 3
Identification of Additional mB7-15' Untranslated Sequences
[0156] Rapid amplification of cDNA ends (RACE) is a PCR-based
strategy to determine the 5' end of a transcript. Three distinct
rounds of 5' RACE were performed on polyadenylated RNA from CH1 B
lymphoma cells, which express high levels of mB7-1 RNA. The
resulting sequences extended the 5' UT of the known mB7-1 cDNA by
1505 bp, beyond the transcriptional start site reported by
Selvakumar et al. ((1993) Immunogenetics 38:292-295). In order to
confirm that this long 5' UT sequence was indeed in the mB7-1 mRNA
and not generated by PCR amplification of genomic DNA, a nested
RT-PCR amplification (B7.68-B7.547 followed by B7.44-B7.80) was
performed. This amplification spans exon 2 (primer B7.80) and the
novel 5' UT sequences in exon 1 (B7-44), and should yield an 840 bp
PCR product. It should be noted that exon 2 is separated from exon
1 by greater than 12 kb in genomic DNA, thus making a genomic
DNA-derived PCR product of almost 13kb. The predicted band of 840
bp, indeed, was observed when this nested PCR amplification was
performed. To further confirm the nature of the PCR product,
hybridization was performed with an oligonucleotide (B7.62) derived
from sequences in exon 1 located 5' of the transcriptional start
site reported by Selvakumar et al. ((1993) Immunogenetics
38:292-295). This probe hybridized to the PCR product. In addition,
sequencing of the RACE product revealed that it contained sequences
identical to the previously known genomic sequences immediately
upstream of the known exon 1 and was contiguous with exon 1. Thus,
it did not identify an additional exon.
EXAMPLE 4
Fine Mapping of mB7-1 Intron-exon Boundaries
[0157] In order to characterize intron-exon boundaries,
oligonucleotide primers were synthesized to mB7-1 cDNA sequences
(described in Freeman et al. (1991) J. Exp. Med. 174:625-631), as
well as to sequences determined from PCR products characterized
during amplifications from tissue RNA. Sequences for exons 1
through 5, as well as exon-intron junctions have been reported
previously (Selvakumar et al. (1993) Immunogenetics 38:292-295).
The coding region of the exon 1 signal peptide domain is 115 bp and
is flanked at the 3' end with a canonical splice site. Exons 2 (318
bp), 3 (282 bp), and 4 (114 bp), are separated by 6.0 and 3.8 kb,
respectively, and all 3 exons are flanked on both their 5' and 3'
ends with canonical splice sites. Exon 5 is located 4 kb downstream
of exon 4, and contains a termination codon after the first 97 bp.
An additional functional canonical splice site was observed 43 bp
downstream of the termination codon in exon 5, since this site was
used to generate the transcript outlined in FIG. 1 (transcript A).
Exon 6 is located 7.2 kb downstream of exon 5 and encodes an open
reading frame with a termination codon after 140 bp. Both exons 5
and 6 are followed by polyadenylation sequences, ATTAAA and AATAAA
respectively.
EXAMPLE 5
Identification of Additional Novel Cytoplasmic Domains by Exon
Trapping
[0158] In this example, an exon trapping approach is used to
identify a novel exon encoding an alternative cytoplasmic domain
for human B7-1. The basic strategy of exon trapping is to create an
expression vector encoding a recombinant protein, wherein the
encoded protein cannot be functionally expressed unless an
appropriate exon, with flanking intron sequences that allow proper
mRNA splicing, is cloned into the expression vector. A recombinant
expression vector is created comprising transcriptional regulatory
sequences (e.g., a strong promoter) linked to nucleic acid encoding
the human B7-1 signal peptide exon, IgV-like and IgC-like exons
followed by a transmembrane exon with flanking 3' intron donor
splice sequences. These splice sequences are immediately followed
by translational stop codons in all three frames. A polyadenylation
recognition site is not included in the recombinant expression
vector. Following the stop codons are restriction enzyme sites
which allow genomic DNA fragments to be cloned into the expression
vector to create a library of recombinant expression vectors.
[0159] As a negative control, the parental recombinant expression
vector is transfected into a host cell line which is hB7-1.sup.-
(e.g, COS cells) and the absence of surface expression of hB7-1 is
demonstrated, confirming that the parental expression vector alone
is unable to direct stable surface expression of hB7-1 in the
absence of a cytoplasmic domain encoding exon. As a positive
control, the known hB7-1 cytoplasmic domain with a flanking 5'
intron acceptor splice sequence is cloned into a restriction enzyme
site downstream of the transmembrane exon such that the
transmembrane domain exon can be spliced to the cytoplasmic domain
exon. This positive control vector is transfected into a host cell
(e.g., COS cells) and the surface expression of hB7-1 on the cells
is demonstrated, confirming that the cloning into the vector of a
cytoplasmic domain encoding exon with the proper splice sequences
produces an hB7-1 molecule that can be stably expressed on the cell
surface.
[0160] To identify an alternative hB7-1 cytoplasmic domain exon,
genomic DNA fragments for the hB7-1 gene are cloned into the
parental recombinant expression at the restiction enzyme sites
downstream of the transmembrane domain exon. Cloning of genomic
fragments into the vector will "trap" DNA fragments which encompass
a functional exon preceded by an intron splice acceptor site and
followed by a polyadenylation signal, since cloning of such
fragments into the vector allows for expression of a functional
recombinant protein on the surface of transfected host cells. The
diversity of the genomic DNA fragments cloned into the vector
directly impacts the variety of sequences "trapped". Were total
genomic DNA to be used in such an approach, a variety of exons
would be trapped, including cytoplasmic domains from proteins other
than T cell costimulatory molecules. However, instead of using
total genomic DNA for subcloning into the expression vector, only
genomic DNA fragments located in the vicinity of the exon encoding
a known cytoplasmic domain of the T cell costimulatory molecule of
interest are subcloned into the vector. For example, for human
B7-1, genomic DNA clones can be isolated by standard techniques
which contain DNA located within several kilobases 5' or 3' of the
hB7-1 exon which encodes the known cytoplasmic domain. These
fragments are cloned into the parental recombinant expression
vector to create a library of expression vectors. The library of
expression vectors is then transfected into a host cell (e.g., COS
cells) and the transfectants are screened for surface expression of
hB7-1. Cell clones which express a functional B7-1 molecule on
their surface are identified and affinity purified (e.g., by
reacting the cells with a molecule which binds to B7-1, such as an
anti-B7-1 monoclonal antibody (e.g., mAb 133 describe in Freedman,
A. S. et al. (1987) J. Immunol. 137:3260; and Freeman, G. J. et al.
(1989) J. Immunol. 143:2714) or a CTLA4Ig protein (described in
Linsley, P. S. et al., (1991) J. Exp. Med. 174:561-569). Cell
clones which express a B7-1 molecule on their surface will have
incorporated into the expression vector DNA encoding a functional
cytoplasmic domain (e.g., an alternative cytoplasmic domain encoded
by a different exon than the known cytoplasmic domain). DNA from
positive clones encoding the alternative cytoplasmic domain can
then be amplified by PCR using a sense primer corresponding to the
transmembrane domain and an antisense primer corresponding to
vector sequences.
[0161] This same approach can be adapted by the skilled artisan to
identify alternative cytoplasmic domains for other T cell
costimulatory molecules (e.g., B7-2) or to "trap" exons encoding
other alternative structural domains of T cell costimulatory
molecules.
EXAMPLE 6
Identification of a Novel B7-2 Signal Peptide Domain
[0162] cDNA fragments corresponding to the 5' ends of
naturally-occurring murine B7-2 mRNA transcripts were prepared by
5' RACE: polyadenylated RNA isolated from murine spleen cells was
reverse transcribed with a gene-specific oligonucleotide, the cDNA
was isolated, and a poly-dCT tail was added to the 5' end with
terminal deoxynucleotide transferase. PCR was perfomed using a
nested primer and an oligonucleotide primer complementary to the
poly-dCTP tail to amplify 5' cDNA fragments of mB7-2 transcripts.
The gene-specific oligonucleotide primers used for PCR were as
follows:
2 CAGCTCACTCAGGCTTATGT reverse transcrip- (SEQ ID tion, - sense NO:
55) AAACAGCATCTGAGATCAGCA primary PCR, - (SEQ ID sense NO: 56)
CTGAGATCAGCAAGACTGTC secondary PCR, - (SEQ ID sense NO: 57)
[0163] The amplified fragments were subcloned into a plasmid vector
and sequenced. Of approximately 100 individual clones examined,
.about.75% of the clones had a 5' nucleotide sequence corresponding
to that reported for the 5' end of an mB7-2 cDNA (see Freeman, G.
J. et al. (1993) J. Exp. Med. 178:2185-2192). Approximately 25% of
the clones had a 5' nucleotide sequence shown in SEQ ID NO:14,
which encodes a novel signal peptide domain having an amino acid
sequence shown in SEQ ID NO: 15.
EXAMPLE 7
Identification of an Alternatively Spliced Form of B7-1 Having a
Structural Domain Deleted
[0164] Reverse-transcriptase polymerase chain reaction was used to
amplify mB7-1 cDNA fragments derived from murine spleen cell RNA.
Oligonucleotide primers used for PCR were as follows:
3 CTGAAGCTATGGCTTGCAATT primary PCR, + (SEQ ID sense NO: 58)
ACAAGTGTCTTCAGATGTTGAT secondary PCR, + (SEQ ID sense NO: 59)
CTGGATTCTGACTCACCTTCA primary PCR, - (SEQ ID sense NO: 60)
CCAGGTGAAGTCCTCTGACA secondary PCR, - (SEQ ID sense NO: 61)
[0165] A cDNA fragment was detected which comprises a nucleotide
sequence (SEQ ID NO:8) encoding a murine B7-1 molecule in which the
signal peptide domain was spliced directly to the IgC-like domain
(i.e., the IgV-like domain was deleted). The amino acid sequence of
mB7-1 encoded by this cDNA is shown in SEQ ID NO:9.
Equivalents
[0166] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 0
0
* * * * *