# Patent

## Title
T cell costimulatory polypeptides containing alternative domains

## Abstract

Structural forms of T cell costimulatory polypeptides are described. These forms comprise 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 structural forms correspond to naturally-occurring alternatively spliced forms of T cell costimulatory polypeptides or variants thereof which can be produced by standard recombinant DNA techniques. In one embodiment, the T cell costimulatory polypeptide of the invention contains an alternative cytoplasmic domain. In another embodiment, the T cell costimulatory polypeptide of the invention contains an alternative signal peptide domain or has an immunoglobulin variable region-like domain deleted. The alternative structural forms of T cell costimulatory polypeptides can be used to identify agents which stimulate the expression of alternative forms of costimulatory polypeptides and to identify components of the signal transduction pathway which results in costimulation of T cells.

## Background
This application is a continuation application of U.S. Ser. No. 08/702,525 filed Feb. 7, 1997, now U.S. Pat. No. 6,294,660, titled “Nucleic Acids encoding B7-1 and B7-2 Molecules”, which is a U.S. national phase application of PCT/US95/02576 filed on Mar. 2, 1995, titled “Novel Forms of T Cell Costimulatory Molecules and Uses Therefor”, which is a continuation-in-part application of U.S. Ser. No. 08/205,697 filed on Mar. 2, 1994 now U.S. Pat. No. 6,218,510, titled “B7-1 and B7-2 Polyeptides”. The contents of all of the aforementioned application(s) are hereby incorporated by reference.


BACKGROUND OF THE INVENTION
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).
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.
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 extacellular 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).
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. 142:3795–3801). 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 (Ginnni, 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
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.
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.
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).
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.
Still another aspect of the invention pertains to isolated proteins that bind CD28 and/or CTLA4 in which a structural domain has been deleted or added, and isolated nucleic acids encoding such proteins. In a preferred embodiment, the protein (e.g., B7-1) has an immunoglobulin constant-like domain deleted (i.e., an immunoglobulin variable-like domain is linked directly to a transmembrane domain). In another embodiment, the protein has an immunoglobulin variable-like domain deleted (i.e., a signal peptide domain is linked directly to an immunoglobulin constant-like domain).
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
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).
FIG. 2 is a schematic representation depicting three mB7-1 transcripts (A, B and C) detected by nested RT-PCR.
FIG. 3 is a graphic representation of interleukin-2 production by T cells stimulated with either untransfected CHO cells (CHO), CHO cells transfected-to express full-length mouse B7-1 (CHO-B7-1) or CHO cells transfected to express the IgV-like isoform of mouse B7-1 (CHO-SV).



DETAILED DESCRIPTION OF THE INVENTION
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.
Typically, a domain structure of a T cell costimulatory molecule of the invention includes a signal peptide domain (e.g., exon 1), an immunoglobulin variable region-like domain (IgV-like) (e.g., exon 2), an immunoglobulin constant region-like domain (IgC-like) (e.g. exon 3), a transmembrane domain (e.g., exon 4) and a cytoplasmic domain (e.g., exon 5). 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.
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 transcription and 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.
The various aspects of this invention are described in detail in the following subsections. Forming part of the present disclosure is the appended Sequence Listing. The numerous nucleotide and amino acid sequences presented in the Sequence Listing are summarized below.

    SEQ ID NO: 1—nucleotide sequence of mouse B7-1 exons 1-2-3-4-6
    SEQ ID NO: 2—amino acid sequence of mouse B7-1 protein encoded by exons 1-2-3-4-6
    SEQ ID NO: 3—nucleotide sequence of mouse B7-1 exons 1-2-3-4-5-6
    SEQ ID NO: 4—nucleotide sequence of mouse B7-1 exon 6 (CytII)
    SEQ ID NO: 5—amino acid sequence of mouse B7-1 peptide encoded by exon 6 (CytII)
    SEQ ID NO: 6—nucleotide sequence of mouse B7-1 full-length exon 1
    SEQ ID NO: 7—nucleotide sequence of mouse B7-1 promoter
    SEQ ID NO: 8—nucleotide sequence of B7-1 exons 1-3-4-5
    SEQ ID NO: 9—amino acid sequence of mB7-1 protein encoded by exons 1-3-4-5
    SEQ ID NO: 10—nucleotide sequence of mouse B7-1 exons 1-3-4-6
    SEQ ID NO: 11—amino acid sequence of mouse B7-1 protein encoded by exons 1-3-4-6
    SEQ ID NO: 12—nucleotide sequence of mouse B7-2 exons m1B-2-3-4-5
    SEQ ID NO: 13—amino acid sequence of mouse B7-2 protein encoded by exons m1B-2-3-4-5
    SEQ ID NO: 14—nucleotide sequence of mouse B7-2 exon m1B
    SEQ ID NO: 15—amino acid sequence of mouse B7-2 peptide encoded by exon m1B
    SEQ ID NO: 16—nucleotide sequence of mouse B7-1 exons 1-2-3-4-5 (as disclosed in Freeman, G. J. et al. (1991) J. Exp. Med. 174:625–631)
    SEQ ID NO: 17—amino acid sequence of mouse B7-1 protein encoded by exons 1-2-3-4-5
    SEQ ID NO: 18—nucleotide sequence of human B7-1 exons 1-2-3-4-5 (as disclosed in Freeman, G. J. et al. (1989) J. Immunol. 143:2714–2722)
    SEQ ID NO: 19—amino acid sequence of human B7-1 protein encoded by exons 1-2-3-4-5
    SEQ ID NO: 20—nucleotide sequence of mouse B7-2 exons m1A-2-3-4-5 (as disclosed in Freeman, G. J. et al. (1993) J. Exp. Med. 178:2185–2192)
    SEQ ID NO: 21—amino acid sequence of mouse B7-2 protein encoded by exons m1A-2-3-4-5
    SEQ ID NO: 22—nucleotide sequence of human B7-2 exons h1A-2-3-4-5 (as disclosed in Freeman, G. J. et al. (1993) Science 262:909–911)
    SEQ ID NO: 23—amino acid sequence of human B7-2 protein encoded by exons h1A-2-3-4-5
    SEQ ID NO: 24—nucleotide sequence of human B7-2 exons h1B-2-3-4-5 (as disclosed in Azuma, M. et al. (1993) Nature 366:76–79)
    SEQ ID NO: 25—nucleotide sequence of mouse B7-1 exon 5 (Cyt I)
    SEQ ID NO: 26—amino acid sequence of mouse B7-1 peptide encoded by exon 5 (Cyt I)
    SEQ ID NO: 27—nucleotide sequence of human B7-1 exon 5 (Cyt I)
    SEQ ID NO: 28—amino acid sequence of human B7-1 peptide encoded by exon 5 (Cyt I)
    SEQ ID NO: 29—nucleotide sequence of mouse B7-2 exon 5 (Cyt I).
    SEQ ID NO: 30—amino acid sequence of mouse B7-2 peptide encoded by exon 5 (Cyt I)
    SEQ ID NO: 31—nucleotide sequence of human B7-2 exon 5 (Cyt I)
    SEQ ID NO: 32—amino acid sequence of human B7-2 peptide encoded by exon 5 (Cyt I)
    SEQ ID NO: 33—nucleotide sequence of mouse B7-1 truncated exon 1 (signal)
    SEQ ID NO: 34—amino acid sequence of mouse B7-1 peptide encoded by exon 1 (signal)
    SEQ ID NO: 35—nucleotide sequence of human B7-1 exon 1 (signal)
    SEQ ID NO: 36—amino acid sequence of human B7-1 peptide encoded by exon 1 (signal)
    SEQ ID NO: 37—nucleotide sequence of mouse B7-2 exon m1A (signal)
    SEQ ID NO: 38—amino acid sequence of mouse B7-2 peptide encoded by exon m1A (signal)
    SEQ ID NO: 39—nucleotide sequence of human B7-2 exon h1A (signal)
    SEQ ID NO: 40—amino acid sequence of human B7-2 peptide encoded by exon h1A (signal)
    SEQ ID NO: 41—nucleotide sequence of human B7-2 exon h1B (signal)
    SEQ ID NO: 42—amino acid sequence of human B7-2 peptide encoded by exon h1B (signal)
    SEQ ID NOs: 43–61: oligonucleotide primers for PCR
    SEQ ID NO: 62: nucleotide sequence of mouse B7-1 exons 1-2-4-5


SEQ ID NO: 63: amino acid sequence of mouse B7-1 protein encoded by exons 1-2-4-5

    SEQ ID NO: 64: nucleotide sequence of mouse B7-1 exons 1-2-4-6


SEQ ID NO: 65: amino acid sequence of mouse B7-1 protein encoded by exons 1-2-4-6
I. Isolated Nucleic Acid Molecules Encoding T Cell Costimulatory Molecules
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 B-7-2. 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.
A. Nucleic Acids Encoding Novel Cytoplasmic Domains
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

    
    
        A comprises a nucleotide sequence of at least one first exon encoding 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 encoding a cytoplasmic domain selected from the group consisting 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 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 (i.e., either exon 5 or exon 6) and the different mB7-1 mRNA transcripts are produced by alternative splicing of the mRNA transcripts. The genomnic structure of mB7-1 has been reported to contain only a single exon encoding cytoplasmic domain (i.e., exon 5; see 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 (the nucleotide sequence of which is shown in SEQ ID NO: 16) corresponding to signal, Ig-variable, Ig-constant, transmembrane and cytoplasmic domains (see Freeman, G. J. et al., (1991) J. Exp. Med. 174:625–631). This transcript includes a single exon encoding cytoplasmic domain, exon 5. 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.
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 abundance 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, m1B7-2 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).
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 cytoplasmic domain exon of the gene 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 (i.e., the nucleic acid comprises an alternative splice form of a transcript of the gene in which the exon encoding-the first cytoplasmic domain, e.g., exon 5, has been excised from the transcript). Preferred T cell costimulatory molecule genes from which nucleotide sequences can be derived include B7-1 and B7-2.
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.
B. Nucleic Acids Encoding Novel Signal Peptide Domains
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

    
    
        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 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).

    
    


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 costimulatory 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. One of these signal domains corresponds to the signal domain of murine B7-2 disclosed in Freeman et al. (1993) J. Exp. Med. 178:2185–2192 (this signal domain is referred to herein as exon m1A). However, the second signal domain corresponds to a novel nucleotide sequence (referred to herein as m1B). Accordingly, an mRNA transcript containing a nucleotide sequence encoding the novel signal peptide domain (m1B) represents an alternatively spliced form of murine B7-2. A naturally-occurring mB7-2 mRNA transcript comprising the alternative signal peptide domain (i.e., comprising exons m1B-2-3-4-5) preferably comprises the nucleotide sequence shown in SEQ ID NO: 12, and encodes a protein comprising the amino acid sequence shown in SEQ ID NO: 13. The nucleotide and amino acid sequences of the novel signal peptide domain (i.e., exon m1B) are shown in SEQ ID NOs: 14 and 15, respectively.
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 (m1B7-1), 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 (i.e., the nucleic acid comprises an alternative splice form of a transcript of the gene in which the exon encoding a first signal domain has been excised from the transcript). Preferred T cell costimulatory molecule gene from which nucleotide sequences can be derived include B7-1 and B7-2.
C. Nucleic Acids Encoding Proteins with Domains Deleted or Added
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 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. In one embodiment, the nucleic acid is one in which an exon encoding an IgV-like domain has been deleted (i.e., the signal peptide domain exon is lined directly to the IgC-like domain exon). 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

    
    
        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.

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.

    
    


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.
In another embodiment, nucleic acid is one in which an exon encoding an IgC-like domain has been deleted (i.e., the IgV-like domain exon is linked directly to the transmembrane domain exon). 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

    
    
        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 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.

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.

    
    


In one embodiment, an isolated nucleic acid encoding an alternatively spliced form of murine B7-1 in which an IgC-like domain exon has been-deleted comprises a nucleotide sequence corresponding to usage of exons 1-2-4-5 (shown in SEQ ID NO: 62). The amino acid sequence of the protein encoded by this nucleic acid is shown in SEQ ID NO: 63. Moreover, in another embodiment, an alternatively spliced form of murine B7-1 in which an IgC-like domain exon has been deleted can comprise a nucleotide sequence corresponding to usage of exons 1-2-4-6 (shown in SEQ ID NO: 64), which contains the second, alternative cytoplasmic domain of mB7-1. The amino acid sequence of the protein encoded by this nucleic acid is shown in SEQ ID NO: 65. Naturally-occurring mRNA transcripts encoding murine B7-1 have been detected in which the exon encoding the IgC-like domain (i.e, exon 3) has been excised and the exon encoding the IgV-like domain (i.e., exon 2) is spliced to the exon encoding the transmembrane domain (i.e., exon 4) (see Example 7). When expressed in a host cell, the IgV-like isoform of mB7-1 is capable of binding to both mouse CTLA4 and mouse CD28 and can trigger a costimulatory signal in a T cell such that the T cell proliferates and produces interleukin-2 (see Example 7).
Yet 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.
II. Isolation of Nucleic Acids of the Invention
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.
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.
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.
Individual isolated cDNA clones encoding a T cell costimulatory molecule can then be sequenced by standard techniques, 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.
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).
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.
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).
For the purposes of this invention, the amino acid residues encompassing the different “domains” or “exons” (i.e., signal (S), IgV-like (V), IgC-like (C), transmembrane (TM) and cytoplasmic (Cyt)) of mouse and human B7-1 and B7-2 proteins are defined as follows: mouse B7-1 (as shown in SEQ ID NO: 17):  ̃1–37 (S),  ̃38–142 (V),  ̃143–247 (C),  ̃248–274 (TM) and  ̃275–306 (Cyt); human B7-1 (as shown in SEQ ID NO: 19):  ̃1–33 (S),  ̃34–138 (V),  ̃139–242 (C),  ̃243–265 (TM) and  ̃266–288 (Cyt); mouse B7-2 (as shown in SEQ ID NO: 21):  ̃1–5 (S),  ̃6–133 (V),  ̃134–233 (C),  ̃234–264 (TM) and  ̃265–309 (Cyt); and human B7-2 (as shown in SEQ ID NO: 23):  ̃1–6–22 (S),  ̃23–132 (V),  ̃133–245 (C),  ̃246–268 (TM) and  ̃269–329 (Cyt). It will be appreciated by the skilled artisan that regions slightly longer or shorter than these amino acid domains (i.e., a few amino acid residues more or less at either the amino-terminal or carboxy-terminal end) may be equally suitable for use as signal, IgV-like, IgC-like, transmembrane and/or cytoplasmic domains in the proteins of the invention (i.e., there is some flexibility in the junctions between different domains within the proteins of the invention as compared to the domain junctions delineated above for B7-1 and B7-2 proteins). Accordingly, proteins comprising signal, IgV-like, IgC-like, transmembrane and/or cytoplasmic domains having essentially the same amino acid sequences as those regions delineated above but which differ from the above-delineated junctions merely be a few amino acid residues, either longer or shorter, at either the amino- or carboxy-terminal end of the domain are intended to be encompassed by the invention. Nucleic acid segments encoding any of the domains delineated above can be obtained by standard techniques, e.g., by PCR amplification using oligonucleotide primers based on the nucleotide sequences disclosed herein, and can be ligated together to create nucleic acid molecules encoding recombinant forms of the proteins of the invention.
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.
III. Additional Isolated Nucleic Acid Molecules of the Invention
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  ̃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.
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  ̃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 NFκB. 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, β-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.
IV. Uses for the Isolated Nucleic Acid Molecules of the Invention
A. Probes
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 32P, 3H, 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).
The probes of the invention can be used to detect an alteration in the expression of an alternatively spliced form of a T cell costimulatory molecule, such as in a disease state. For example, detection of a defect in the expression of an alternatively spliced form 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. 1:98–106; 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).
B. Recombinant Expression Vectors
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.
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 promoters 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-transferase, maltose E binding protein, or protein A, respectively, to the target recombinant protein.
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 (17 gn1). This viral polymerase is supplied by host strains BL21 (DE3) or HMS174(DE3) from a resident λ prophage harboring a T7 gn1 under the transcriptional control of the lacUV 5 promoter.
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.
Examples of vectors for expression in yeast S. cerivisae include pYepSec1 (Baldari. et al., (1987) Embo J. 6:229–234), pMFa (Kuan and Herskowitz, (1982) Cell 30:933–943), pJRY88 (Schultz et al., (1987) Gene 54:113–123), and pYES2 (Invitrogen Corporation, San Diego, Calif.). Baculovirus vectors available for expression of proteins in cultured insect cells (SF 9 cells) include the pAc series (Smith et al., (1983) Mol. Cell Biol. 3:2156–2165) and the pVL series (Lucklow, V. A., and Summers, M. D., (1989) Virology 170:31–39).
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.
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.
C. Host Cells
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.
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.
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.
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 exclusively 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).
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.
D. Anti-Sense Nucleic Acid Molecules
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.
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-sense 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.
E. Non-Human Transgenic and Homologous Recombinant Animals
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.
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.
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 harboring 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.
V. Isolated Novel Forms of Costimulatory Molecules
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.
A. Proteins with a Novel Cytoplasmic Domain
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

    
    
        A, which may or may not be present, comprises an amino acid sequence of a signal peptide domain,
        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,
        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,
        D comprises an amino acid sequence of a transmembrane domain encoded by at least one exon of a T cell costimulatory molecule gene, and
        E comprises an amino acid sequence of a cytoplasmic domain encoded by at least one exon of a T cell costimulatory molecule gene,

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).

    
    


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).
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 cytoplasmic 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.
A. Proteins with a Novel Signal Peptide Domain
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

    
    
        A comprises an amino acid sequence of a signal peptide domain encoded by at least one exon of a T cell costimulatory molecule gene,
        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,
        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,
        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
        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,

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 (B7-2), SEQ ID NO: 42 (hB7-2).

    
    


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 costimulatory 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.
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 comprising an amino acid sequence shown in SEQ ID NO: 13.
C. Isolated Proteins with Structural Domains Deleted or Added
This invention also features costimulatory molecules which have at least one structural domain deleted. In one embodiment, the structural form has at least one IgV-like domain deleted. Accordingly, 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

    
    
        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,
        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
        C comprises an amino acid sequence of a transmembrane domain encoded by at least one exon of a T cell costimulatory molecule gene, and
        D comprises an amino acid sequence of a cytoplasmic domain encoded by at least one exon of a T cell costimulatory molecule gene.

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 (utilizing Cyt I of mB7-1). Alternatively, an isolated murine B7-1 protein having an IgV-like domain deleted comprises an amino acid sequence shown in SEQ ID NO: 11 (utilizing Cyt II of mB7-1).

    
    


In another embodiment, the structural form of the T cell costimulatory molecule has at least one IgC-like domain deleted. Accordingly, 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

    
    
        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,
        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, and
        C comprises-an amino acid sequence of a transmembrane domain encoded by at least one exon of a T cell costimulatory molecule gene, and
        D comprises an amino acid sequence of a cytoplasmic domain encoded by at least one exon of a T cell costimulatory molecule gene.

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 IgC-like domain deleted comprises an amino acid sequence shown in SEQ ID NO: 63 (utilizing Cyt I of mB7-1). Alternatively, an isolated murine B7-1 protein having an IgC-like domain deleted comprises an amino acid sequence shown in SEQ ID NO: 65 (utilizing Cyt II of mB7-1).

    
    


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)).
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).
VI. Uses for the Novel T Cell Costimulatory Molecules of the Invention
A. Costimulation
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.
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.
B. Antibodies
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.
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.
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′)2 fragments can be generated by treating antibody with pepsin. The resulting F(ab′)2 fragment can be treated to reduce disulfide bridges to produce Fab′ fragments.
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, Monfison 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. Additionally, 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.
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, VH regions and FV 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).
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.
C. Screening Assays
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 transduction 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.
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.
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
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.
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.
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.
A. Chimeric Proteins
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-II) 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. Thee 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.
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-II) 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.
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.
B. Antibodies
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.
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, O-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin 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 125I, 131I, 35S or 3H.
C. Kinase Substrates
A novel cytoplasmic domain of the invention which contains a consensus 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., 32P-γ-ATP) to radiolabel the novel cytoplasmic domain.
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.
The following methodology was used in the Examples.
Genomic Cloning
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 (λ4, λ9, λ15, and λ16). These lambda clones were subcloned into Bluescript-pKS II (Stratagene, La Jolla Calif.) for subsequent restriction mapping.
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
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 μg total RNA in a 20 μl reaction. All PCR reactions were performed in 25 μl volumes using a manual “hot start”, wherein 10× deoxynucleotide triphosphates (dNTPs) were added to the samples at 80° C. Final reaction conditions were: 60 mM Tris-HCl, pH 8.5, 15 mM (NH4)2SO4, 2.5 mM MgCl2, 200 μM dNTPs, and 2 μg/ml each of the specific primers. Cycling conditions for all amplifications were 94° C., 4 minutes prior to 35 cycles of 94° C. for 45 seconds, 58° C. for 45 seconds, and 72° C. for 3 minutes, followed by a final extension at 72° C. for 7 minutes. The template for primary PCR was 2 μl of the RT reaction product and the template for secondary nested PCR was 1 μl of the primary PCR reaction product.
Oligonucleotides
All oligonucleotides were synthesized on an Applied Biosystems 381A 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.
Rapid Amplification of cDNA Ends (RACE) Procedure
Polyadenylated RNA purified 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.
Oligonucleotide Hybridization
Oligonucleotide(s) were 5′ end-labeled with polynucleotide kinase and γ32P-ATP. Hybridizations were carried out in 5×SSC and 0.5% SDS at 55° C. overnight and subsequently washed 3 times for 15 minutes with 2×SSC at 55° C. Blots were exposed to Kodak XAR-5 film with an intensifying screen at −80° C.
The oligonucleotides used for the PCR studies in Examples 1–4 are shown in Table I:








TABLE I







Oligonucleotides used for PCR studies












Designation
Sequence (5′ to 3′)
sense
PCR
















B7.27
CCAACATAACTGAGTCTGGAAA
+
secondary
(SEQ ID NO:43)



 


B7.36
CTGGATTCTGACTCACCTTCA
−
secondary
(SEQ ID NO:44)


 


B7.37
AGGTTAAGAGTGGTAGAGCCA
−
primary
(SEQ ID NO:45)


 


B7.38
AATACCATGTATCCCACATGG
−
secondary
(SEQ ID NO:46)


 


B7.42
CTGAAGCTATGGCTTGCAATT
+
primary
(SEQ ID NO:47)


 


B7.44
TGGCTTCTCTTTCCTTACCTT
+
secondary
(SEQ ID NO:48)


 


B7.48
GCAAATGGTAGATGAGACTGT
−
secondary
(SEQ 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
+
secondary
(SEQ ID NO:53)


 


B7.547
TTTGATGGACAACTTTACTA
−
primary
(SEQ ID NO:54)









EXAMPLE 1
Characterization of the mB7-1 Genomic Locus
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 λ4, λ9, λ15, and λ16) were selected for further analysis. These lambda clones were subcloned and subjected to restriction mapping with HindIII and BamHI. Regions containing exons were farther 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 λ15 (transmembrane domain exon) and λ16 (cytoplasmic domainexon) was determined to be less than 100 base pairs by PCR using a sense primer (B7.71) designed to the 3′ end of clone λ15 and an antisense primer (B7.38) located at the 5′ end of clone λ16. Clones λ9 and λ15 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
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 poly-adenylation 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. 2:12492–12499).
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.
[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].
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.
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 mB7-1 5′ Untranslated Sequences
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 B17.80) 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 13 kb. 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
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
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.
As a negative control, the parental recombinant expression vector is transfected into a host cell line which is hB7-1− (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.
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 restriction enzyme sites downstream of the transmembrane domain exon. Cloning of genomic fragments into the vector will “trap” DNA figments 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 3B7-1, 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.
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
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 performed 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:










CAGCTCACTCAGGCTTATGT
reverse transcription, − sense
(SEQ ID NO:55)



 


AAACAGCATCTGAGATCAGCA
primary PCR, − sense
(SEQ ID NO:56)


 


CTGAGATCAGCAAGACTGTC
secondary PCR, − sense
(SEQ ID NO:57)






The amplified fragments were subcloned into a plasmid vector and sequenced. Of approximately 100 individual clones examined,  ̃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 Alternatively Spliced Forms of B7-1 Having a Structural Domain Deleted
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:










CTGAAGCTATGGCTTGCAATT
primary PCR, + sense
(SEQ ID NO:58)



 


ACAAGTGTCTTCAGATGTTGAT
secondary PCR, + sense
(SEQ ID NO:59)


 


CTGGATTCTGACTCACCTTCA
primary PCR, − sense
(SEQ ID NO:60)


 


CCAGGTGAAGTCCTCTGACA
secondary PCR, − sense
(SEQ ID NO:61)






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.
Another cDNA fragment was detected with comprises a nucleotide sequence (SEQ ID NO: 62) encoding a murine B7-1 molecule in which the IgV-like domain was spliced directly to the transmembrane domain (i.e., the IgC-like domain was deleted). The amino acid sequence encoded by this cDNA is shown in SEQ ID NO: 63). This protein is referred to herein as an IgV-like isoform of mB7-1. To examine the functional activity of the IgV-like isoform of mB7-1, its cDNA was cloned into an expression vector, pBK-CMV, in which transcription of the cDNA is placed under the control of the CMV promoter. The expression vector was cotransfected into Chinese Hamster Ovary (CHO) cells, along with a puromycin resistance gene, and drug resistant clones were selected. The resultant clones expressing the IgV-like isoform of mB7-1 on their surface are referred to herein as CHO-sV clones.
Expression of the IgV-like isoform of mB7-1 on the surface of the CHO-sV cells was confirmed by FACS analysis using either murine CTLA4Ig, murine CD28Ig or anti-B7-1 antibody as the primary staining reagent. Each of these reagents stained the CHO-sV cells. Positive staining of CHO-sV with both mCTLA4Ig and mCD28Ig indicate that the IgV-like isoform of mB7-1 is capable of interacting with both CTLA4 and CD28. In contrast to the results with mouse CTLA4Ig, human CTLA4Ig failed to stain the CHO-sV cells, although this reagent was able to stain CHO cells expressing the full-length mouse B7-1 molecule (CHO-B7-1 cells). These data implicate the IgC domain of mB7-1 in the binding to human CTLA4Ig, whereas the IgC domain of mB7-1 is not required for binding to mouse CTLA4Ig. These results suggest species differences in the binding parameters for human and murine CTLA4.
The ability of the IgV-like isoform of mB7-1 on CHO-sV cells to deliver a costimulatory signal to T cells was tested in standard T cell proliferation and interleukin-2 (IL-2) production assays. T cells that received a primary activation signal were stimulated to produce IL-2 when incubated with either CHO-B7-1 cells or CHO-sV cells but not when incubated with untransfected CHO cells. The results of this experiment is illustrated graphically in FIG. 3, in which IL-2 production by T cells is expressed as a function of the number of CHO cells used to costimulate the T cells. The data demonstrate that CHO-sV cells can trigger a costimulatory signal in T cells, although the level of IL-2 production by cells stimulated with CHO-sV was approximately 25–50% of the level of IL-2 production by cells stimulated with CHO-B7-1. Similar results were observed when T cell proliferation was assayed as an indicator of T cell costimulation.
EQUIVALENTS
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.

## Claims

1. An isolated polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:63.

2. An isolated polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:65.

3. An isolated polypeptide which binds CD28 or CTLA4, comprising the amino acid sequence set forth in SEQ ID NO:63.

4. An isolated polypeptide which binds CD28 or CTLA4, comprising the amino acid sequence set forth in SEQ ID NO:65.