HK1160479A

HK1160479A - New dendritic cell co-stimulatory molecules

Info

Publication number: HK1160479A
Application number: HK12100909.6A
Authority: HK
Inventors: 德鲁‧M‧帕夕尔; 土屋东夫; 凯文‧S‧戈尔斯基; 苏-伊‧特森
Original assignee: 约翰霍普金斯大学
Priority date: 2000-04-28
Filing date: 2012-02-01
Publication date: 2012-08-17

Abstract

A novel costimulatory protein molecule, B7-DC, which is a member of the B7 family, is described as is DNA coding therefor and expression vectors comprising this DNA. B7-DC protein, fragments, fusion polypeptides/proteins and other functional derivatives, and transformed cells expressing B7-DC are useful in vaccine compositions and methods. Compositions and methods are disclosed for inducing potent T cell mediated responses that can be harnessed for anti-tumor and anti-viral immunity.; A costimulatory protein molecule, B7-DC, which is a member of the B7 family, is described as is DNA coding therefor and expression vectors comprising this DNA. B7-DC protein, fragments, fusion polypeptides/proteins and other functional derivatives, and transformed cells expressing B7-DC are useful in vaccine compositions and methods. Compositions and methods are disclosed for inducing potent T cell mediated responses that can be harnessed for anti-tumor and anti-viral immunity.

Description

novel dendritic cell costimulatory molecules

The application is a divisional application with the international application number of PCT/US01/13430, the international application date of 2001, 4, 27, the date of entering China national phase of 2002, 12, 30, the Chinese application number of 01812016.4 and the invented name of new dendritic cell co-stimulatory molecule.

Background

Technical Field

The present invention is in the fields of biochemistry and medicine and relates to novel proteins selectively expressed on the surface of dendritic cells which can be used as cell surface molecules or in soluble form in vaccine compositions to stimulate an immune response.

Description of the background Art

The generation of a T lymphocyte immune response is a complex process involving cell-cell interactions and the production of soluble mediators (cytokines or lymphokines) that is regulated by several T cell surface molecules that act as "receptors", including the T Cell Receptor (TCR) complex and other "helper" surface molecules, many of which are cell surface "differentiation antigens" first defined by monoclonal antibodies ("CD molecules").

Two signals are required to achieve optimal activation of all lymphocytes: one antigen-specific or clonal signal and a second signal that is antigen-non-specific (Janeway, C., Cold Spring Harbor Symp. Quant. biol. 54: 1-14 (1989)). Without co-stimulation, known as a co-stimulatory molecule (such as B7 described below), when lymphocytes encounter an antigen alone, clonal inactivation, also known as "anergy" (Schwartz, r.science 248: 1349(1990)) or apoptosis (programmed cell death) occurs; if a costimulatory signal is provided, clonal expansion specific for the stimulating antigen is produced. Without co-stimulation, no significant amplification of the immune response against a given antigen occurs (June et al (Immunology Today 15: 321: 331-1994); Chen et al (Immunology Today 14: 483486); Townsend, SE and Allison, JP (1993) Science 259: 368-370).

The nature and potential of the immune response is largely dependent on the type of Antigen Presenting Cell (APC) that processes and presents antigen to T cells. Upon T cell binding and activation, the density of peptide antigen/MHC ligands used to bind the TCR and the provision of soluble and/or membrane-bound co-stimulatory signals by the APC are important. For these reasons, immunotherapeutic strategies have begun to focus on providing (a) target antigens for appropriate APC species and (b) appropriate co-stimulatory molecules to enhance T cell activation.

APCs that provide the signals required to activate T cells include monocytes/macrophages, B lymphocytes and most importantly Dendritic Cells (DCs). In the past, activated macrophages were thought to be the key APC for initiating T cell immune responses in vivo. This concept is based on the ability of the cell to efficiently phagocytose and display and present antigens on the surface. In particular, recently, attention has been directed to DCs as the primary trigger of antigen-specific T cell immune responses in vivo. DCs have a different phenotype from activated macrophages and can be divided into different subtypes capable of eliciting different immune responses. One functional feature of DCs is that they have approximately 100-fold greater capacity to activate naive T cells in vitro than macrophages. To date, the explanation for this potential is based on the number differences of molecules known to be important for antigen presentation. The invention is based on the discovery of an important quantitative difference.

The first signal of antigen presentation is triggered by the interaction of the TCR with an antigen present on the APC in the context of a Major Histocompatibility Complex (MHC) class II molecule (Allen, Immunol. today 8: 270 (1987)). Costimulatory signals originate from other molecules, best characterized by the B7 family (i.e., B7.1, B7.2, and alternatively B7.3), which are also present on APCs.

Two proteins expressed on the surface of T cells are the best characterized ligands or counter receptors for co-stimulatory molecules such as B7. CD28 is a homodimeric glycoprotein of the immunoglobulin (Ig) superfamily (Aruffo and Seed, Proc. Natl. Acad. Sci.84: 8573-8577(1987)), which is present on most mature human T cells that play a role in T cell activation. CD28 is constitutively expressed on resting T cells and increases upon activation. Ligation of CD28 following signal through the T cell receptor induces T cell proliferation and secretion of IL-2(Linsley, PS, et al. (1991) J exp. Med.173, 721-. CD28 mediates cell-cell contact ("intercellular adhesion"), an antigen-independent intercellular interaction necessary for immune responses (Springer et al, Ann. Rev. Immunol.5: 223-252 (1987)).

CTLA4 is a T cell surface molecule that is highly homologous to CD28, but which is not expressed on resting T cells and appears upon T cell activation (Brunet, j.f., et al, (1987) Nature 328, 267-270). CTLA-4 was originally identified by differential screening of murine cytolytic T cell cDNA libraries, Brunet et al, supra. Linsley et al, (1991) j.exp.med.174: the role of CTLA-4 as a secondary receptor for B7 is discussed in 561-569 and also discloses that B7 has a higher affinity for CTLA4 than for CD 28. Freeman et al, (1993) Science 262: 907-. Ligands for CTLA-4 are described in Lenschow et al, (1993) proc.nat' l.acad.sci.90: 11054 and 11058.

Cytokines such as IL-2, IL-4 and IL-6, which are secreted by the cells to grow and induce differentiation, may be present in the form of aggregates in the Th-B cell contact zone, which ensures that only B cells presenting antigens to Th cells are activated, avoiding activation of other B cells.

CD28 and CTLA-4 interact with a costimulatory molecule, typically B7. B7 was originally described as a B cell activating antigen and was named B7/BB-1 because it was found on B cells (Linsley et al, Proc. Natl. Acad. Sci. USA 87: 5031-5035 (1990). hereinafter, this molecule is named B7, B7-1 or B7.1). B7 and in particular the recently described B7 homolog is also a member of the Ig superfamily, and B7 contains two extracellular Ig domains in comparison to CD28 and CTLA-4: an N-terminal variable-like (V) domain followed by a constant region-like (C) domain.

B7 family members are generally expressed on APCs and, as mentioned, they are important for the activation of resting T cells. These family members include B7-1(═ B7, also known as CD80) and B7-2 (also known as CD 86). References to the description of B7-1 include Schwartz, r.h.cell 71: 1065-1068, 1992; chen, L, et al. Cell 71: 1093-; freeman, G.J. et al J Immunol 143: 2714 2722, 1989; and Freeman, g.j. et al J exp.med.174: 625-631, 1991)). References to the description of B7-2 include (Freeman, G.J. et al Science 262: 909. sup. 911813. sup. 960. 1993). Heretofore, murine B7-1 and B7-2 and human B7-1 and B7-2 have been described (Freeman et al, 1989, supra; 1991, supra; and 1993, supra). Activated human B lymphocytes express CTLA4/CD28 binding to the counterreceptors B7-2 and B7-3, both of which can transmit costimulatory signals to T cells via CD28 or CTLA 4.

B cells expressed B7-2 after 24 hours of co-stimulation with mAb against Ig or against MHC class II. B7-2 induced detectable IL-2 secretion and T cell proliferation. Approximately 48-72 hours after activation, B cells expressed B7-1 and a third CTLA4 counter receptor identified by mAbBB-1, (Yokochi, T, et al (1982) J Immunol.128, 823. sub.827) designated B7-3. B7-3 was also expressed on B7-negatively activated B cells and was able to co-stimulate T cell proliferation without detectable IL-2 production, indicating that the B7-1 and B7-3 molecules are different. B7-3 is expressed on a wide variety of cells, including activated B cells, activated monocytes, dendritic cells, langerhans cells, and keratinocytes. Expression of B7-1 and B7-3 began to decrease 72 hours after B cell activation. The presence of these CTLA4/CD28 binding counter receptors on the surface of activated B lymphocytes suggests that co-stimulation of T cells is regulated, in part, by transient expression of these molecules after B cells are activated.

B7: the importance of the CD28/CTLA4 costimulatory pathway has been demonstrated in vitro and in vivo. There is a direct link between increased T cell activity and increased expression of B7 (Razi-Wolf et al, Proc. Natl. Acad. Sci. USA, 89: 4210-4214 (1992)). T cells become anergic when contacted with a peptide antigen on cells lacking a costimulatory ligand to bind CD 28. This blockade of the costimulatory pathway leads to the development of antigen-specific resistance in both murine and human systems (Harding et al, supra; Lenschow, D.J. et al (1992) science.257, 789-Bu792; Turka, LA et al (1992) Proc. Natl. Acad. Sci. USA.89, 11102-11105; Gimmi, CD et al (1993) Proc. Natl. Acad. Sci USA 90, 6586-Buchner 6590; Boussiositis, V.et al (1993) J exp. Med.178, 1753-Buchner 1763). In contrast, B7-negative murine tumor cells expressing B7 induced a T cell-mediated specific immune response with concomitant tumor rejection and long-term protection against tumor challenge. (Chen, L, et al. (1992) Cell 71: 1093-561102; Townsend et al., supra; Baskar, S, et al. (1993) Proc. Natl. Acad. Sci.90, 5687-5690.). Therefore, B7: modulation of the CD28/CTLA4 pathway can create great potential to stimulate or suppress immune responses in humans.

The interaction between CD28 and B7 has been identified by genetic fusion of the extracellular segment of B7 or CD28 with the Ig C.gamma.1 chain (Linsley et al, J.exp. Med.173: 721-730 (1991)). When the B7Ig fusion proteins were immobilized, or when B7 was expressed on the surface of cells, such as transfected CHO cells, they co-stimulated T cell proliferation. Stimulation of T cells by B7+ CHO cells also specifically stimulates an increase in IL-2 transcription levels.

U.S.5,521,288 describes a method for modulating the immune response by contacting a fragment encoded by the partial DNA encoding B7, which fragment predominantly corresponds to the extracellular domain (ECD) of B7, with CD28 positive T cells. Derivatives of B7, which are fusion protein constructs comprising at least a portion of the B7ECD and another protein, such as a human Ig C γ 1 domain that alters the solubility, binding affinity, and/or valency of B7, may also be used to modulate immune responses. For example, DNA encoding amino acid residues 1-125 of the B7ECD and DNA encoding amino acid residues corresponding to the hinge, CH2, and CH3 regions of human Ig C.gamma.1 are ligated to form a DNA fusion product encoding the B7Ig fusion protein. Also disclosed herein is a method of treating a T cell mediated immune system disease by binding the CD28 receptor to react with T cells by administering the B7 or B7Ig fusion protein. The binding of an immunosuppressive agent inhibits T cell proliferation in graft versus host disease by reacting CD28+ T cells with B7 antigen or B7Ig fusion protein.

U.S. patent 5,861,310 discloses modified tumor cells that express one or more T cell co-stimulatory molecules, including B7-2 and B7-3. One embodiment further comprises the expression B7. The modification may be transfection with a nucleic acid encoding a B7-2, B7-3, or B7 protein. Tumor cells may also be genetically modified in vivo. Such modified tumor cells are said to be useful for treating tumor patients to prevent or inhibit metastatic spread or to inhibit tumor recurrence. This article discloses a method of specifically inducing a CD4+ T cell response against tumors.

U.S. patent 5,942,607 discloses isolated nucleic acids encoding novel CTLA4/CD28 ligands that co-stimulate T cell activation. In a specific embodiment, the isolated nucleic acid encodes B7-2. Also disclosed is a nucleic acid comprising at least a portion of the disclosed full-length B7-2 sequence. According to this article, the nucleic acid sequence can be integrated into different expression vectors, wherein the carrier in a variety of host cells including mammalian and insect cells can direct the corresponding protein or peptide synthesis. Also disclosed are transformed host cells that produce the proteins or peptides encoded by these nucleic acid sequences and isolated proteins and peptides containing at least a portion of the B7-2 sequence.

Dong H et al, Nat Med 19995: 1365-1399 describes a third member of the B7 family, designated B7-H1, which does not bind to CD28, CTLA4 or ICOS (inducible costimulators). The ligation response of B7-H1 co-stimulates T cells to respond to polyclonal stimulators and alloantigens, preferably to stimulate the production of interleukin-10. IL-2 produced in small amounts is essential for the co-stimulatory effect of B7-H1. This study identified a previously unknown costimulatory molecule that may be associated with the down-regulation of cell-mediated immune responses. The same laboratory (Wang S et al, blood.2000; 96: 2808-2813) described a novel human B7-like gene, designated B7-H2, whose expression was detected on the surface of monocyte-derived immature DCs. Soluble fusion proteins of B7-H2 and Ig, B7-H2Ig bound activated but not resting T cells. The above binding was inhibited by the soluble form of icos (icosid) but not by CTLA4 Ig. ICOSIg stained CHO cells transfected with the B7-H2 gene. Using suboptimal cross-linked CD3 as a stimulator, it was found that B7-H2Ig co-stimulates T cell proliferation in a dose-dependent manner and is associated with IL-2 secretion, whereas the optimal CD3 ligation response preferentially stimulates IL-10 production. The authors suggested that B7-H2 was a potential ligand for ICOS T cell molecules.

Swallow MM et al, Immunity, 1999, 11: 423-432 reported the cloning of a novel gene B7h, a homologue of the B7 molecule expressed on APC. B7h co-stimulates the proliferation of purified T cells by acting on receptors other than CD28 or CTLA-4. Surprisingly, although B7h was expressed in unstimulated B cells, it was also induced in non-lymphoid cells (3T3 cells; embryonic fibroblasts) treated with TNF α, which was upregulated in murine non-lymphoid tissues treated with LPS, a potent TNFa activator. These studies define a novel T cell costimulatory ligand, indicating that induction of B7h with TNF α can directly increase self-recognition during inflammation.

Yoshinaga SK et al, Nature, 1999, 402: 827-832 describe a novel murine costimulatory receptor-ligand pair. The receptor is related to CD28, is a murine homologue of the human protein ICOS, and is expressed on activated T cells and resting memory T cells. The ligand, which is homologous to the B7 molecule, was named B7 related protein-1 (B7 RP-1). B7RP-1 is a type 1 transmembrane protein with 20% and 19% amino acid identity to murine B7.1(CD80) and B7.2(CD86), respectively. This homologue is effective since B7.1 and B7.2 share only 27% amino acid identity (Freeman, GJ et al, J.exp. Med.178: 2185-2192 (1993)). This homologue contains cysteines at conserved positions (residues 62, 138, 185 and 242 from the initial methionine) which are important for the formation of Ig loops. The full length of B7RP-1 and the relative position of the transmembrane region were similar to that of the B7 molecule (Greenfield, EA et al., Crit. Rev. Immunol.18: 389-418 (1998)). B7RP-1 is expressed on B cells and macrophages. ICOS and B7RP-1 did not interact with proteins in the CD28-B7 pathway, and B7RP-1 costimulated T cells independently of CD 28. Transgenic mice expressing a fusion protein of the Fc fragment of B7RP-1 and Ig ("B7-RP 1-Fc") developed lymphoproliferation in the spleen, lymph nodes and Peyer's patches. The enhanced delayed type hypersensitivity in antigen pre-sensitized mice treated with B7RP-1-Fc demonstrated the co-stimulatory activity of B7RP-1 in vivo upon antigen challenge. The authors concluded that ICOS and B7RP-1 are unique and novel receptor-ligand pairs structurally related to CD28-B7 and involved in adaptive immune responses.

Yoshinagaga SK et al, Int Immunol, 2000, 12/10: 1439-1447 reported co-stimulation of human T cells by the interaction of human B7RP-1 and ICOS. The KD value for this ligand-receptor interaction is approximately 33nM, t_(1/2)Off-rate > 10 min. TNF α differentially modulates the expression of human B7RP-1 on B cells, monocytes and DCs. TNF α enhanced the expression of B7RP-1 on B cells and monocytes but inhibited the expression of B7RP-1 on DCs. Human B7RP-1-Fc protein or expression membrane boundThe B7RP-1 cells co-stimulate T cell proliferation in vitro. Specific cytokines, such as IFN γ and IL-10, were induced by co-stimulation with B7 RP-1. Although there was no significant increase in IL-2 levels, the co-stimulation induced by B7RP-1 was dependent on IL-2. These studies identified the human ortholog (human ortholog) of murine B7RP-1 and characterized its interaction with human ICOS.

PD-1 is an immunosuppressive receptor expressed by activated T, B and bone marrow cells. PD-1 deficient mice exhibit multiple forms of autoimmunity due to loss of peripheral tolerance. Freeman, GJ et al, J.Exp.Med.192: 1027-1034(2000) reported a ligand for PD-1 (PD-L1), which is a member of the B7 gene family. Binding of PD-1 to PD-L1 results in inhibition of TCR-mediated lymphocyte activation (proliferation, cytokine secretion). In addition, PD-1 signaling inhibits suboptimal levels of CD 28-mediated co-stimulation. PD-L1 was expressed by APCs (human monocytes stimulated with TNF γ, activated human DCs). In addition, PD-L1 is also expressed in the heart and lung. The authors concluded that the relative magnitude of the PD-L1 signal inhibited on APC and the co-stimulated B7-1/B7-2 signal can determine the degree of T cell activation and a threshold between tolerance and autoimmunity. The presence of PD-L1 in non-lymphoid tissues may contribute to the magnitude of the immune response at the site of inflammation.

The citation of documents above does not imply that any of the documents identified above are prior art to the present disclosure. All statements as to the date and descriptions as to the contents of these documents are based on the information available to the present application and do not necessarily imply any correctness as to the dates or contents of these documents.

Summary of The Invention

To identify the genes encoding novel Dendritic Cell (DC) -specific costimulatory molecules for T cell activation, the inventors screened a subtractive cDNA library between DCs and activated macrophages. This cDNA subtraction method allows the determination of genes expressed by DCs but not by activated macrophages. Several novel DC-specific genes have been discovered using this approach, and these genes are useful in enhancing the potential of vaccines that rely on T cell activation. The present application relates to one such gene.

Based on the fact that it is present in the DC library but not in the activated macrophage library, a new coding sequence was identified, designated "B7-DC". The B7-DC gene is a member of the B7 family of genes that encode co-stimulatory molecules. B7-DC is the first B7 family member with DC-specific expression and different receptor specificity. The product of this gene plays an important role in mediating the unique ability of DCs to stimulate T cells. Functional analysis showed that B7-DC had higher activity than B7-1 in stimulating IFN γ production by T cells. Thus, B7-DC DNA and polypeptides are used in compositions and methods to improve the efficacy of cellular and molecular vaccine compositions, regardless of antigen specificity.

In a specific embodiment, the present invention provides an isolated nucleic acid molecule encoding a mammalian protein designated B7-DC that is selectively expressed on dendritic cells and not on activated macrophages. The nucleic acid molecule preferably comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 (human) or SEQ ID NO: 5 (murine). The invention also relates to an isolated nucleic acid which hybridizes with the above-mentioned nucleic acid molecule under stringent hybridization conditions. Preferred stringent conditions include: incubate at 45 ℃ in 6X sodium chloride/sodium citrate (SSC) followed by rinsing with about 0.2X SSC at about 50 ℃. Preferably, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 1. the preferred nucleic acid molecule as described above encodes a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and SEQ ID NO: 4 or a biologically active fragment, homologue or other functional derivative encoding the protein. Preferably, the nucleic acid molecule encodes a polypeptide having the sequence SEQ ID NO: 2 (B7-DC of human origin) or a protein encoding SEQ ID NO: 2. a biologically active fragment, homologue or other functional derivative of (a).

In a preferred embodiment, the nucleic acid molecule encodes the extracellular domain of the B7-DC protein, which comprises residues 26-221, which encodes a co-stimulatory homologue, fragment or other functional derivative thereof.

In another embodiment, the above-described nucleic acid molecule encoding a B7-DC fusion protein comprises:

(a) a first nucleic acid sequence encoding a first polypeptide which is the entire B7-DC protein or a portion thereof (preferably SEQ ID NO: 2 or SEQ ID NO: 4);

(b) optionally, fusing the first nucleic acid sequence in frame to a linker nucleic acid sequence encoding a linker peptide; and

(c) a second nucleic acid sequence linked in reading frame to the first nucleic acid sequence or to a linker nucleic acid sequence, the second nucleic acid sequence encoding a second polypeptide.

The second polypeptide preferably comprises one or more Ig heavy chain constant region domains, preferably the C domains of both of human IgG, preferably IgG 1.

The invention also provides an expression vector comprising any one of the nucleic acid molecules described above, wherein the nucleic acid molecule is operably linked to (a) a promoter and (b) optionally, additional regulatory sequences which regulate expression of the nucleic acid in eukaryotic cells.

The expression vector may be a plasmid or a viral vector. These vectors contain self-replicating RNA replicons (DNA-primed or RNA), suicide RNA vectors, DNA viruses (e.g., adenovirus, vaccinia virus, etc.), and RNA viral particles grown in packaging cell lines.

The vector DNA or RNA can be complexed with a gold particle for gene gun mediated introduction into a host, or can be complexed with another polymer that enhances delivery to the desired target cells and tissues, for example, in a controlled release formulation.

The present invention also comprises a carrier composition comprising:

(a) a first recombinant expression vector having integrated into its sequence a nucleotide sequence encoding an antigen of interest, wherein the antigen of interest induces an immune response; and

(b) a second recombinant expression vector comprising a nucleic acid sequence incorporating one or more nucleotide sequences encoding co-stimulatory polypeptides, wherein at least one of the polypeptides is B7-DC, or a biologically active fragment, homologue or other functional derivative thereof.

Wherein the expression vector is capable of co-infecting or co-transfecting a host cell to produce co-expression of the antigen and the co-stimulatory polypeptide, polypeptide fragment, homologue or derivative.

In a refinement of the above embodiment, the present invention provides a third nucleic acid sequence encoding a protein of interest which (i) promotes diffusion of the expression product (antigen) between cells, preferably APCs, (ii) enhances display of the antigen on the APCs expressing the nucleic acid, and/or (iii) promotes re-presentation (cross-priming) and display of the antigen in the host APCs into which the vector has been introduced. The nucleic acid encoding the protein of interest may be fused to a nucleic acid encoding an antigen or a co-stimulatory molecule, or both. The first or second vector (the first or the second vector) comprises a nucleic acid. In one embodiment, the vector composition combines nucleic acid encoding an antigen, nucleic acid encoding a co-stimulatory molecule (preferably B7-DC), and nucleic acid encoding a "protein of interest" in a single fusion construct.

The invention encompasses cells transformed or transfected with any of the nucleic acid molecules or expression vectors described above. The cell is preferably a eukaryotic cell, more preferably a mammalian cell, most preferably a human cell, which may be a dendritic cell or a precursor (progenitors) cell thereof. In another embodiment, the cell is a tumor cell, more preferably the tumor cell carries an antigen that is identical to or cross-reactive with an antigen in the host tumor, thereby eliciting an immune response.

A preferred embodiment is the transfection of an isolated mammalian tumor cell with an exogenous nucleic acid molecule encoding a mammalian B7-DC protein (preferably SEQ ID NO: 2 or SEQ ID NO: 4) or a biologically active fragment, homolog or other functional derivative thereof, when said protein, fragment, homolog or derivative thereof is expressed by said tumor cell and said tumor cell is contacted with a T cell

(i) B7-DC protein, fragment, homologue or derivative thereof, binds to T cells; and

(ii) tumor cells co-stimulate T cells to proliferate and/or produce and secrete cytokines.

The invention also relates to a polypeptide selectively expressed on dendritic cells and not on activated macrophages, the polypeptide having the following properties:

(a) binding to a binding partner on a T cell; and

(b) co-stimulating T cells to proliferate and/or produce and secrete cytokines.

The invention also encompasses biologically active fragments, homologs or other functional derivatives of the polypeptides.

The polypeptide, fragment, homologue or functional derivative is preferably represented by the sequence SEQ ID NO: 1 or SEQ ID NO: 5, or a fragment, homologue or equivalent of said nucleic acid molecule. Preferred polypeptides have the amino acid sequence SEQ ID NO: 2 or SEQ ID NO: 4.

the polypeptide or biologically active fragment, homologue or other functional derivative thereof may be produced by recombinant expression of one of the nucleic acids described above.

Preferred polypeptides contain the extracellular domain of the B7-DC protein, preferably

(a) SEQ ID NO: 2 (human) amino acid residues 26-221, or

(b) SEQ ID NO: 4 (murine) amino acid residues 26-221.

The polypeptide may consist essentially of the extracellular domain of B7-CD.

The invention also provides a B7-DC fusion polypeptide comprising a first fusion partner comprising all or part of the B7-DC protein

(ii) Directly fused to a second polypeptide, or

(ii) Optionally, a linker peptide sequence fused to the second polypeptide.

The B7-DC fusion protein described above can also be combined with a second polypeptide, preferably one or more Ig heavy chain constant region domains, preferably having a hinge region, C, corresponding to the C.gamma.1 chain of a human immunoglobulin_H2 and C_H3 region, fused to each other.

In one embodiment of the above fusion protein, the first fusion partner is the extracellular domain of the B7-DC protein, SEQ ID NO: 2 or SEQ ID NO: 4, and (b) 4.

The fusion protein preferably binds to a binding partner on a T cell and co-stimulates the T cell in the presence of a suitable stimulus for a T cell receptor.

The present invention also provides a dimeric or trimeric fusion protein which is a dimer or trimer of the above fusion protein. Preferably, the chains are linked to each other by disulfide bonds or other interchain covalent bonds.

In preferred dimeric fusion proteins, the dimer consists of the C of two Ig heavy chains_HThe cysteine residues in the regions are covalently linked, wherein the cysteine residues are identical to disulfide-linked cysteine residues in a dimeric normal Ig H chain.

The fusion proteins of the invention may comprise multimers of two or more repeats of a first fusion partner, wherein the first fusion partners are linked end-to-end to each other, or to a linker sequence between one or more monomers.

The invention also provides an antibody specific for an epitope of the B7-DC protein, wherein the epitope is not present in a known member of the B7 family of proteins. The epitope may be SEQ ID NO: 2 or SEQ ID NO: 4 linear or conformational epitopes of a polypeptide. The antibody is preferably a monoclonal antibody, more preferably a human or humanized (by genetic engineering) monoclonal antibody.

The invention also provides a method for identifying or quantifying cells in a cell population that express a B7-DC polypeptide on their surface using the above-described antibodies, the method comprising

(a) Contacting cells in the cell population with the antibody to allow the antibody to bind to the epitope-expressing cells;

(b) detecting the presence of antibody-bound cells or quantitatively detecting the number of antibody-bound cells.

The present invention also provides another method for isolating from a population of cells a cell expressing a B7-DC polypeptide on its surface, the method comprising:

(a) contacting a population of cells with the antibody to allow the antibody to bind to the epitope-expressing cells;

(b) cells that bound to the antibody were positively selected or cells that did not bind to the antibody were negatively selected.

The present invention also provides a method for detecting the presence of or quantifying the amount of a B7-DC polypeptide, fragment or homologue thereof in a sample, the method comprising the steps of:

(a) contacting the sample with the antibody of claim 43 such that the antibody binds to any polypeptide or fragment containing the epitope;

(b) detecting the presence of or quantitatively detecting the polypeptide or fragment bound to the antibody.

The invention also relates to a method of inducing or enhancing expression of a B7-DC polypeptide in an antigen presenting cell or a precursor thereof to enhance the ability of the cell to co-stimulate the T cell in vitro or in vivo in the presence of a suitable stimulus for a T cell receptor, the method comprising transforming or transfecting the antigen presenting cell or precursor with an expression vector as described above, thereby inducing or enhancing expression of the B7-DC polypeptide on the cell. The antigen presenting cell is preferably a dendritic cell, and the precursor is a dendritic cell precursor.

The present invention provides methods for stimulating an immune response using cell costimulatory compositions and polypeptide costimulators. A method for enhancing a T cell response to antigen stimulation in a mammalian subject comprises administering to the subject an effective amount of the above cells, preferably tumor cells, and an antigen stimulator, wherein the cells are effective to enhance the T cell response to antigen stimulation in the subject. The above method preferably injects the antigen and co-stimulatory composition simultaneously.

A method of using a tumor associated antigen to enhance a mammalian subject's T cell response to stimulation by the antigen comprises administering to the subject an effective amount of a tumor cell as described above, wherein the tumor cell expresses the antigen and the administered tumor cell is effective to enhance the subject's T cell response to stimulation by the tumor antigen.

A method of enhancing a T cell response to antigen stimulation in a mammalian subject, comprising administering to the subject an effective amount of a polypeptide, fragment, homolog, or functional derivative as described above, or a fusion polypeptide or protein as described above, and an antigen stimulator, wherein the administered polypeptide is effective to enhance the T cell response to antigen stimulation in the subject.

The invention also provides a method of inhibiting a T cell response to antigen stimulation in a mammalian subject comprising administering to the subject an effective amount of the above antibody, wherein the administered antibody is effective to block T cell stimulation or eliminate antigen-reactive T cells, thereby inhibiting the T cell response. These methods may be particularly useful for treating a subject with a tissue or organ transplant to inhibit transplant rejection and/or to promote transplantation, and for autoantigens, to block or reduce autoimmune reactions and their pathological sequelae.

The invention provides methods of treatment using T cells that have been stimulated within the compositions of the invention. A method of enhancing an immune response to antigen stimulation in a mammalian subject comprising:

(a) obtaining T cells from a subject, from an immunologically compatible donor of the subject, or from an immunologically acceptable cultured cell line;

(b) contacting ex vivo T cells with an effective amount of the above cells, wherein the contacting is effective to increase the T cell response to antigen stimulation; and

(c) administering the T cells of step (b) to a subject,

thereby enhancing the immune response of the subject.

In another embodiment, a method of enhancing an immune response to antigen stimulation in a mammalian subject comprises:

(b) contacting ex vivo T cells with an effective amount of (i) a polypeptide, fragment, homolog or functional derivative described above, or (ii) a fusion polypeptide described above, wherein the contacting is effective to increase the T cell response to antigen stimulation; and

(c) administering the T cells of step (b) to a subject,

thereby enhancing (or generating) the immune response in the subject.

The invention also provides a vaccine composition comprising

(a) (ii) a cell expressing the B7-DC construct as described above, (ii) a B7-DC polypeptide, fragment, homologue or functional derivative, (iii) a B7-DC fusion polypeptide or protein

(b) Typically, an antigen of another origin against which an immune response is desired-although not required in a cell-based vaccine, wherein the cells are capable of expressing the antigen themselves (as are tumor cells containing tumor antigens);

(c) optionally, a conventional immunostimulant or adjuvant; and

(d) a pharmaceutically and immunologically acceptable excipient or carrier for (a), (b) and (c).

A method of inducing or enhancing an immune response to an antigen in a mammalian subject, the method comprising administering to the subject an effective amount of the above vaccine composition.

The present invention also provides a co-stimulatory composition for use with an antigen or vaccine, the composition comprising:

(a) B7-DC polypeptide (preferably SEQ ID NO: 2 or SEQ ID NO: 4), a fragment, homologue or functional derivative thereof or B7-DC fusion polypeptide, and

(b) a pharmaceutically and immunologically acceptable excipient or carrier.

A method of boosting the immune response to an antigen or vaccine in a mammalian subject, the method comprising administering to the subject the above co-stimulatory composition in combination with the antigen or vaccine.

A method of stimulating a systemic immune response to a tumor in a subject, the method comprising administering to the subject genetically altered tumor cells

(a) Derived from a tumor in a subject, and

(b) genetically altered by introduction of the B7-DC nucleic acid as described above in vivo, expression of which provides a co-stimulatory signal in a subject,

wherein the administration results in stimulation of a systemic immune response directed against the tumor.

The tumor cells are preferably treated, preferably by irradiation, to prevent their growth after administration.

Prior to administration of the above-described therapeutic composition, the subject may be subjected to chemotherapy for tumor regression treatment, irradiation, or surgical resection.

The present invention also provides a method of inducing an anti-tumor response in a mammal containing an antigen-positive (anti-positive) tumor, the method comprising:

(a) providing a tumor or a cell of a tumor cell line, the cell

(i) Expressing an antigen common to mammalian tumors;

(ii) transfection with the nucleic acid vector encoding B7-DC described above, whereby, when expressed, the B7-DC molecule elicits a cellular co-stimulatory T cell response to a tumor antigen;

(ii) optionally, irradiating prior to step (b);

(b) administering to a mammal an effective amount of cells that express an antigen and a B7-DC molecule;

thereby inducing an anti-tumor response.

In the above method, the anti-tumor response is characterized by:

(A) the total number of maximum vertical diameter products of all measurable damage is reduced by at least 50%;

(B) no evidence of new damage, and

(C) any original damage is not worsened.

The present invention also provides a method of inducing regression or attenuation of primary growth or regeneration of a tumor in a mammal having the tumor, the method comprising:

(a) providing a tumor or a cell of a tumor cell line, the cell

(i) Expressing an antigen common to mammalian tumors;

(ii) transfecting the nucleic acid vector encoding B7-DC as described above, whereby the B7-DC molecule, when expressed, causes the cell to co-stimulate the T cell to respond to the tumor antigen;

(iii) optionally, irradiating prior to step (b);

thereby inducing a systemic immune response specific for the melanoma tumor antigen and thereby inducing regression or attenuation.

A method of inhibiting recurrent growth of an antigen-positive tumor in a mammal, the method comprising:

(a) providing a tumor or a cell of a tumor cell line, the cell

(i) Expressing an antigen common to mammalian tumors;

(ii) transfecting the nucleic acid vector encoding B7-DC as described above, such that when expressed, the cells co-stimulate the T cells to respond to the tumor antigen;

(iii) optionally, irradiating prior to step (b);

thereby inducing a systemic immune response in the mammal specific for the tumor antigen, which immune response inhibits recurrent growth of the tumor cells.

Another embodiment relates to a method of providing a co-stimulatory signal in the vicinity of a localized administration of an antigen in a mammalian subject to promote localized development of inflammatory and immune responses, thereby generating systemic immunity to the antigen, the method comprising the localized administration to the subject of an antigen

(a) The above cells expressing a costimulatory effective amount of a B7-DC polypeptide, fragment, homolog or functional derivative, and

(b) antigens

So that co-stimulation of adjacent parts of the body with the antigen promotes the site-specific generation of a response, resulting in systemic immunity.

In the above method, the antigen is preferably a tumor antigen, and in (b) the antigen is administered in the form of a tumor cell or subcellular antigenic material. The tumor cell may also be the cell of (a) expressing the B7-DC polypeptide, fragment, homolog or derivative.

Brief Description of Drawings

FIG. 1 is a map showing hB7-DC mapping located on human chromosome 9p 24. hB7-DC map of BAC clone RPCI-11.2.

FIG. 2 shows that B7-DC was differentially expressed between DC and macrophages. The distribution of B7-DC mRNA in bone marrow DC, spleen DC, macrophages, macrophage cell lines and tissues was examined by running 0.5. mu.g/lane of purified DNA on a 1% agarose gel for efficient Northern blot analysis. G3PDH was used as a control. J774A1, Raw264.7, Pu5-1.8 and WEHI cells are macrophage lineages. BM: bone marrow.

FIG. 3 shows a potent Northern blot analysis of B7-DC expression on human DCs. Lanes 1 show human DCs cultured with GM-CSF + Flt-3L, lanes 2 show human placenta and lanes 3 show human DCs cultured with GM-CSF + IL 4. Oligonucleotides from the 5 'and 3' UTRs of human B7-DC were used as PCR DNA probes to perform efficient Northern blot analysis of human DC total RNA. Beta-actin was used as a control to determine the quality of the mRNA.

FIG. 4 represents flow cytometric analysis showing surface expression of B7-DC on mature BM-DC. Murine BM-DCs grown for 9 days were Fc blocked and stained with control antibody or B7-DC antiserum. Specificity of binding was confirmed by adding B7-DC-Ig to compete for anti-B7-DC binding to the DC surface.

FIG. 5 shows that B7-DC binds to PD-1, but not to CILA-4 or CD 28. 293T cells were transiently transfected with pCAGGS-B7.1opCAGGS-B7-DC. Transfectants were stained with PD-1-Ig, 28-Ig, and CTLA-4-Ig fusion molecules, followed by staining with a PE-labeled secondary antibody. Staining of PCAGGS (empty vector) transfectants was negative (not shown).

FIG. 6 (left and right panels) shows co-stimulation of T cell proliferation with anti-CD 3 and B7-DC-Ig. Left panel: purified T cells (CD4+ CD8) were cultured in wells previously coated with increasing concentrations of anti-CD 3(mAb2C11) and fixed concentrations (0.1. mu.g/ml) of fixed B7.1-Ig (. diamond-solid.), B7-DC-Ig (●) or isotype controls (. tangle-solidup.). The results describe a typical test of the three. Culturing the cells for 72h, and using³H-thymidine CPM marker, counts per minute. Right panel: purified CD 8T cells were cultured in wells pre-coated with increasing concentrations of anti-CD 3 and fixed concentrations (0.1. mu.g/ml) of fixed B7.1-Ig (. diamond-solid.), B7-DC-Ig (●) or isotype control (. tangle-solidup.). The results are typical of either. Culturing the cells for 72h, and using³H-thymidine CPM marker, counts per minute.

FIG. 7 shows co-stimulation of antigen-specific T cell proliferative responses. RENCA cells were treated with IFN γ for 72 hours to induce MHC class II expression and cultured with 12.5. mu.g/ml HA110-120 polypeptide. Purified HA + I-Ed specific transgenic T cells were added to soluble forms of increasing concentrations of B7.1-Ig (. diamond-solid.), B7-DC-Ig (●) or isotype controls (. tangle-solidup.). Culturing the cells for 48h, and using³H-thymidine CPM marker, counts per minute. The results are typical of one of the three.

FIG. 8 shows cytokine secretion by T cells co-stimulated with B7-DC.

Upper part: purified T cells were cultured in wells previously coated with anti-CD 3 (0.12. mu.g/ml) and 0.1. mu.g/ml immobilized B7.1-Ig (. diamond-solid.), B7-DC-Ig (●) or isotype control (. tangle-solidup.) as in FIG. 6 (left). The results are typical of one of the three.

The lower part: with purified HA + I-E^dSpecific transgenic T cells with 12.5. mu.g/ml HA (110-120-) Gamma-IFN-treated RENCA cells of the polypeptide. The results are typical of either. Supernatants were collected after 24h and 48h incubation and assayed for indicated lymphokines by ELISA.

FIG. 9 shows that B7-DC-Ig greatly enhanced antigen-specific proliferation after in vivo co-stimulation. At a proper transfer of 2.5X 10⁶Following individual HA-specific TCR transgenic cells, three groups of mice were immunized in the mouse hindpaw pad with HA polypeptide alone (110-120), Incomplete Freund's Adjuvant (IFA) or with IFA and B7-DC-Ig + IFA in combination or an isotype control antibody. Draining lymph nodes were collected on day 7. Mixing 1.5X 10⁵LN cells were cultured with the HA peptide for 48 hours with 1. mu. Ci [, [ solution of ] A³H]Thymidine was pulsed and after 12h the bound radioactivity was detected.

Description of the preferred embodiments

The present inventors have identified novel proteins and nucleic acids that can serve as the basis for improved immunotherapeutic compositions and methods. A novel co-stimulatory protein in human and murine forms, designated B7-DC, has been discovered and disclosed herein.

The DNA encoding human B7-DC has the nucleotide sequence SEQ ID NO: 1, shown below:

1 atcttcctcctgctaatgttgagcctggaattgcagcttcaccagatagcagcttta

61 ttcacagtgacagtccctaaggaactgtacataatagagcatggcagcaatgtgaccctg

121 gaatgcaactttgacactggaagtcatgtgaaccttggagcaataacagccagtttgcaa

181 aaggtggaaaatgatacatccccacaccgtgaaagagccactttgctggaggagcagctg

241 cccctagggaaggcctcgttccacatacctcaagtccaagtgagggacgaaggacagtac

301 caatgcataatcatctatggggtcgcctgggactacaagtacctgactctgaaagtcaaa

361 gcttcctacaggaaaataaacactcacatcctaaaggttccagaaacagatgaggtagag

421 ctcacctgccaggctacaggttatcctctggcagaagtatcctggccaaacgtcagcgtt

481 cctgccaacaccagccactccaggacccctgaaggcctctaccaggtcaccagtgttctg

541 cgcctaaagccaccccctggcagaaacttcagctgtgtgttctggaatactcacgtgagg

601 gaacttactttggccagcattgaccttcaaagtcagatggaacccaggacccatccaact

661 tggctgcttcacattttcatcccctcctgcatcattgctttcattttcatagccacagtg

721 atagccctaagaaaacaactctgtcaaaagctgtattcttcaaaagacacaacaaaaaga

781 cctgtcaccacaacaaagagggaagtgaacagtgctatc 819

the human B7-DC protein has the amino acid sequence SEQ ID NO: 2, shown below (with annotations for leader sequence, transmembrane domain and cytoplasmic tail):

the extracellular domain of the protein is derived from residue P²⁶To residue W²²¹。

A DNA clone comprising the coding sequence encoding murine B7-DC has the nucleotide sequence SEQ ID NO: 3, shown below. The coding sequence (underlined, in triplet form) begins with the methionine codon at nucleotide 210(bold), terminate inStop codon (bold) (nucleotides 951-953).

SEQ ID NO: 5 is SEQ ID NO: 3, or a coding sequence portion thereof.

Consisting of SEQ ID NO: 3, (e.g., the murine B7-DC protein encoded by the coding region of SEQ ID NO: 5) has the amino acid sequence SEQ ID NO: 4, shown below (with annotations for leader sequence, transmembrane domain and cytoplasmic tail):

The DNA sequence of the intact murine B7-DC (originally designated as "a lactotrophy protein-like protein" or "Btdc") has the genbank accession number AF 142780.2.

Basic molecular methods

This method will be described in more detail in the examples. The present inventors utilized a PCR selection method (PCR Select) which has two important features. First, only a small amount of RNA is required for the initial PCR reaction prior to the hybridization step. This technique allows the use of highly purified mature DCs that have been substantially depleted of contaminating macrophages, precursor cells, or other potentially contaminating cells. Such highly purified DCs are known to be difficult to obtain in large quantities. Second, the PCR selection procedure allows for the cloning of low copy number, differentially expressed genes.

In order to identify the cell counterpart corresponding thereto,genes differentially expressed by DC of activated macrophages and identifying genes associated with DC specific function, the present inventors applied a cDNA subtraction method (cDNA subtraction approach). They used a modified PCR-based, binding-inhibited PCR (PCR Select)^TM) "typical difference display analysis (RDA)" (Diatchenko, l., et al, proc.natl.acad.sci USA 93: 66025-6030(1996)).

Conventional recombinant DNA methods

Basic versions disclose conventional methods of molecular biology, all of which are incorporated herein by reference, including: sambrook, J et al, Molecular Cloning: a Laboratory Manual, second edition, Cold Spring Harbor Press, Cold Spring Harbor, New York, 1989; current Protocols in Molecular Biology, Vol.2, Wiley-Interscience, New York, (Current edition); kriegler, Gene Transfer and Expression: a Laboratory Manual (1990); glover, DM, ed, DNA Cloning: a Practical Approach, Vol. I & II, IRL Press, 1985; albers, B. et al, molar Biology of the Cell, second edition, Garland Publishing, Inc., New York, NY (1989); watson, JD et al, Recombinant DNA, second edition, Scientific American Books, New York, 1992; and Old, RW, et al, Principles of Gene management: an Introduction to Genetic Engineering, second edition, University of California Press (University of California Press), Burkholderia, California (1981).

Unless otherwise indicated, a particular nucleic acid sequence is intended to encompass conservatively substituted variants thereof (e.g., substitutions of degenerate codons) and complementary sequences. The terms "nucleic acid" and "polynucleotide" are synonymous and are intended to include a gene, cDNA molecule, mRNA molecule, and fragments thereof such as oligonucleotides, and further include equivalents thereof (as explained more fully below). The size of nucleic acids is expressed in kilobases (kb) or base pairs (bp). This is determined by agarose or polyacrylamide gel electrophoresis (PAGE) and nucleic acid sequences determined or published by the user. The size of a protein is expressed in terms of molecular weight or length (number of amino acid residues) in kilodaltons (kDa). Protein size is determined according to PAGE, sequencing, deduced amino acid sequence based on the encoded nucleic acid sequence, or known amino acid sequence.

Specifically, cDNA molecules encoding the amino acid sequence corresponding to B7-DC or fragments or derivatives thereof can be synthesized by Polymerase Chain Reaction (PCR) using primers derived from the protein sequences disclosed herein (see, e.g., u.s.4,683,202). These cDNA sequences are then transferred into eukaryotic or prokaryotic expression vectors, and the resulting vectors can be used to direct the synthesis of B7-DC or fragments or derivatives thereof in a suitable host cell, such as COS or CHO cells.

The present invention encompasses isolated nucleic acids having a nucleotide sequence encoding novel B7-DC, fragments thereof or equivalents thereof. The term nucleic acid as used herein is intended to encompass such fragments or equivalents. The nucleic acid sequence of the invention may be DNA or RNA. Preferred nucleic acids are nucleic acids encoding a polypeptide having the sequence SEQ ID NO: 1, human B7-DC or an equivalent thereof.

Preferably, the nucleic acid of the invention is a cDNA molecule encoding at least a part of B7-DC. The cDNA can be prepared from mRNA extracted from mature DC or other cells that naturally express the protein. The nucleic acid sequence encoding B7-DC can be obtained from the genomic DNA of DC. Thus, DNA encoding B7-DC can be cloned from cDNA or genomic libraries according to well-known techniques.

The cDNA nucleotide sequence encoding B7-DC can be obtained by isolating total mRNA from an appropriate cell line. Double-stranded cDNA was prepared from total mRNA. The cDNA can be inserted into a suitable plasmid, phage or viral vector using any of a variety of well-known techniques.

With respect to nucleotide sequences, the term "equivalent" is intended to encompass structural analogs and/or coding sequences of functionally equivalent proteins. For example, a natural polymorphism in the nucleotide sequence of B7-DC (particularly the third base of the codon) may be evidence of a "silent" mutation that does not alter the amino acid sequence. However, polymorphisms involving amino acid sequence changes in B7-DC may exist in the human (or other mammalian) population, and those of ordinary skill in the art will appreciate that because of the presence of naturally occurring allelic variants, it is likely that these allelic variants will be found in the human population with one or more nucleotide changes (up to 3-4% of the entire coding sequence). All such allelic variants and resulting polymorphisms of nucleic acids and polypeptides in the DNA encoding B7-DC are included within the scope of the invention.

Still further, there may be one or more naturally occurring isoforms of the B7-DC protein described herein or related, immunologically cross-reactive family members. Such isoforms or family members are defined as proteins having an amino acid sequence functionally similar to B7-DC, even though they are encoded by genes at different loci.

Nucleic acid fragments

A nucleic acid sequence fragment is defined as a nucleotide sequence having fewer nucleotides than the nucleotide sequence encoding the full-length B7-DC protein. The invention includes nucleic acid fragments encoding polypeptides that retain (1) the ability of B7-DC to bind to its natural ligand on T cells and (2) enhance or inhibit (depending on how they are presented) the activated T cell-mediated immune response (as measured by cytokine production and/or T cell proliferation of T cells that receive a primary activation signal).

For example, the nucleic acid fragments herein encode B7-DC polypeptides that retain the ability to bind to T cell surface receptors that have not been identified (but are apparently not CD28 or CTLA-4) and deliver a costimulatory signal to T lymphocytes. According to another criterion, the nucleic acid fragment of the present invention is a nucleic acid fragment that hybridizes to a nucleic acid from another animal species, and therefore can be used in a screening assay to detect novel proteins that cross-react with B7-DC.

Typically, the nucleic acid sequence encoding the B7-DC polypeptide fragment contains nucleotides from the coding sequence of the mature protein. However, in some instances, all or part of the leader sequence of the nucleic acid may be present. The nucleic acid sequences of the invention may also contain linker sequences, natural or modified restriction endonuclease sites and other sequences useful for cloning, expression and purification procedures of the encoded protein or fragment. These and other modified nucleic acid sequences are described herein or are known in the art.

In one embodiment, the DNA encoding the amino acid sequence corresponding to the ECD of B7-DC is linked to the DNA encoding the hinge region corresponding to human Ig C.gamma.1, C_H2 and C_H3 region, wherein the ECD of B7-DC contains amino acids at about positions 26-221, thereby forming a construct expressing a B7-DC-Ig fusion protein.

A similar DNA molecule encoding the B7-Ig fusion protein is disclosed in U.S. Pat. No. 5,521,288 and deposited with the American Type Culture Collection (American Type Culture Collection) at Rockville.Md under accession number 68627.

Techniques for rearranging and expressing DNA encoding B7-DC and soluble B7-DC fusion proteins, such as oligonucleotide synthesis, PCR, transforming cells, constructing vectors, expression systems, and the like, are well established in the art, and one of ordinary skill in the art is familiar with standard sources of materials for specific conditions and procedures.

In another embodiment, DNA encoding one domain or fragment of B7-DC is fused to a nucleic acid encoding most or all of the remainder of another B7 family protein, e.g., B7.1, B7.2, or B7.3. The DNA sequence of the intact human B7.1(CD80) has gene bank (Genbank) accession number X60958; the murine sequence is accession number X60958; the rabbit sequence is accession number U05593. The complete cDNA sequence of human B7.2(CD86) has the genbank accession number L25259; the murine sequence is accession number L25606.

Expression vectors and host cells

The present invention includes expression vectors comprising a nucleic acid sequence encoding a B7-DC polypeptide operably linked to at least one regulatory sequence. "operably linked" means that the coding sequence is linked to the regulatory sequences in a manner that allows for expression of the coding sequence. Well-known regulatory sequences are selected to direct the expression of the protein of interest in a suitable host cell. Thus, the term "regulatory sequence" encompasses promoters, enhancers and other expression regulatory elements. Such regulatory sequences are described, for example, in Goeddel, Gene Expression technology, methods in Enzymology, Vol.185, Academic Press, san Diego, Calif. (1990).

It will be appreciated by those of ordinary skill in the art that the specific construction of the expression vectors of the invention will require consideration of, for example, the host cell used for transfection and/or the type of protein expressed.

The expression vectors of the invention contain a vector encoding the B7-DC: full-length proteins and functional derivatives thereof (as defined herein), including full-length nucleic acid molecules such as polypeptide fragments, variants, fusion proteins, and the like. Thus, in one embodiment, the expression vector comprises a nucleic acid encoding at least a portion of a B7-DC protein, such as ECD, alone or fused to another protein.

Such expression vectors are used to transfect host cells for expression of the DNA and production of the encoded protein comprising the fusion protein or peptide. It is understood that a genetically modified cell expressing a B7-DC polypeptide can transiently express exogenous DNA for a period of time sufficient for the cell to be used for an intended purpose. Thus, if the cell is an immunogen with increased co-stimulatory capacity both in vivo and in vitro, the length of time required for expression or cell survival is the time necessary for the cell to exert its immunogenic and/or co-stimulatory functions. For example, for B7-DC expressing transduced tumor cells of the invention, expression of B7-DC can be only 6 hours, preferably 24 hours, more preferably at least 2-4 days. Of course, expression may also be stable (e.g., for living cells). Suitable expression vectors and regulatory elements (e.g., promoters, inducible or constitutive) discussed below can be selected for stability required for expression.

The present invention provides methods for producing B7-DC proteins, fragments, and derivatives. For example, a host cell is transfected with a nucleic acid vector encoding at least a portion of the B7-DC protein, and the host cell is cultured under suitable conditions to express the B7-DC polypeptide.

The host cell may also be transfected with one or more expression vectors, which may comprise, alone or in combination, DNA encoding at least a portion of the B7-DC protein and DNA encoding at least a portion of the second protein, such that the host cell produces a fusion polypeptide comprised by both portions.

When the recombinant vector contains a DNA encoding a portion of B7-DC and a DNA encoding another protein, such as human Ig C γ 1, the resulting fusion protein may have altered solubility, binding affinity, and/or valency. For example, the B7-DC Ig fusion protein is preferably secreted by transfected host cells in culture and can therefore be isolated from the culture medium. Alternatively, if the protein is retained in the cytoplasm, the cells are harvested and lysed, and the protein is isolated from the lysate.

The culture typically includes the host cells, appropriate growth media, and other byproducts. Suitable media are well known in the art. The B7-DC protein can be isolated from culture media or cell lysates using conventional Techniques for purifying proteins and peptides, including ammonium sulfate precipitation, fractional column chromatography (e.g., ion exchange, gel filtration, affinity chromatography, etc.), and/or electrophoresis (see generally, "Enzyme Purification and Related technologies", Methods in Enzymology, 22: 233-. Once purified, partially or homogeneously, the recombinant B7-DC protein of the invention can be used in pharmaceutical compositions as will be described in more detail herein.

Eukaryotic or prokaryotic host cells transformed or transfected to express B7-DC or a homolog or functional derivative thereof are within the scope of the present invention. For example, B7-DC can be expressed in bacterial cells such as e.coli (e.coli), insect cells (baculovirus), yeast or mammalian cells such as chinese hamster ovary Cells (CHO) or human cells. Other suitable host cells may be found in Goeddel, (1990) (supra) or are known to those of ordinary skill in the art.

Expression in eukaryotic cells can result in partial or complete glycosylation and/or formation of the corresponding intra-or interchain disulfide bonds of the recombinant protein.

Examples of vectors for expression in Saccharomyces cerevisiae (S.cerevisiae) include pYepSecl (Baldari et al, (1987) EMBO J6: 229. sup. 234), pMFa (Kurjan et al (1982) Cell 30: 933. sup. 943), pJRY88(Schultz et al, (1987) Gene 54: 113. sup. 123), and pYES2(Invitrogen Corporation, san Diego, Calif.). Baculovirus vectors for expressing proteins in cultured insect cells (SF 9 cells) include the pAc series (Smith et al, (1983) mol. cell biol.3: 2156-. Typically, COS cells (Gluzman, Y., (1981) Cell 23: 175-. The NS0 myeloma cell line (a glutamate synthetase expression system) was purchased from Celltech, Inc.

Typically, in fusion expression vectors, a protease cleavage site is introduced at the junction of the reporter group and the protein of interest to enable separation of the protein of interest from the reporter group after purification of the fusion protein. Proteinases suitable for such cleavage and their recognition sequences include factor Xa, thrombin and enterokinase.

Typical fusion expression vectors include pGEX (Amrad, Melbourne, Australia), pMAL (New England Biolabs, Beverly, Mass.), and pRIT5 (pharmacia, Piscataway, N.J.), which fuse glutathione S-transferase, maltose E binding protein, protein A, respectively, with the recombinant protein of interest.

Inducible non-fusion Expression vectors include pTrc (Amann et al, (1988) Gene 69: 301-. Expression of the gene of interest was dependent on transcription by host RNA polymerase from the hybrid trp-lac fusion promoter in pTrc and expression of the gene of interest inserted into pET 11d was dependent on transcription from the T7gn10-lacO fusion promoter mediated by co-expressed viral RNA polymerase (T7gn 1). The viral polymerase is provided by host bacteria BL21(DE3) or HMS174(DE3) from resident gamma phage having T7gn1 under the transcriptional control of the lacUV 5 promoter.

One embodiment of the invention is a transfected cell that re-expresses novel B7-DC. For cells that have expressed B7-DC, such as mature DCs, transfected cells express increased amounts of B7-DC protein or fragments thereof on their surface.

For example, tumor cells such as sarcoma, melanoma, leukemia, lymphoma, carcinoma, or neuroblastoma are transfected with an expression vector that directs the expression of B7-DC on the surface of the tumor cells described below. Such transfected tumor cells can be used as immunogens to induce therapeutic anti-tumor immunity as described herein.

Construction of vectors

Construction of suitable vectors containing the coding and regulatory sequences of interest uses standard ligation and restriction techniques well known in the art. The isolated plasmid, DNA sequence or synthetic oligonucleotide is cleaved, tailed and religated as desired.

The DNA sequence forming the vector may be of many origins. Backbone vectors and regulatory systems are typically present in available "host" vectors that are used as containers of sequences in constructs. For related coding sequences, the starting construct may be, and often is, a presumably suitable sequence retrieved from a cDNA or genomic DNA library. However, once the sequence is disclosed, the entire gene sequence can be synthesized in vitro starting from individual nucleotide derivatives. The entire gene sequence of a gene of considerable length, for example, 500-1000bp, can be prepared by synthesizing individual overlapping complementary oligonucleotides and filling the non-overlapping regions of the single strands with DNA polymerase in the presence of deoxynucleotide triphosphates. This approach has successfully constructed several genes of known sequence. See, for example, Edge, m.d., Nature (1981) 292: 756; nambair, K.P., et al, Science (1984) 223: 1299; and Jay, e., J Biol Chem (1984) 259: 6311.

the phosphotriester method described in the above references or Beaucage, s.l., and carothers, m.h., Tet Lett (1981) 22: 1859; and Matteucci, m.d., and carothers, m.h., J Am Chem Soc (1981) 103: 3185 synthetic oligonucleotides are prepared by the phosphoramidite method described in, or can be prepared using a commercially available automated oligonucleotide synthesizer. In 50mM Tris, H7.6, 10mM MgCl₂5mM dithiothreitol, 1-2mMATP, 1.7pmoles gamma-³²Annealing or treatment with a kinase of the pre-labeled single strand is achieved by treating 1nmol of the substrate with an excess of, for example, about 10 units of polynucleotide kinase in the presence of p-ATP (2.9mCi/mmole), 0.1mM spermidine, 0.1mM EDTA.

Once the desired portions of the carrier have thus been obtained, they can be cut and ligated using standard restriction and ligation procedures. Cleavage of site-specific DNA can be accomplished by treatment with an appropriate restriction enzyme (or enzymes) under conditions generally known in the art, particularly as specified by the manufacturer of these commercially available restriction enzymes. See, e.g., New England Biolabs, Product Catalog. In general, 1mg of plasmid or DNA sequence is cleaved with 1 unit of enzyme in about 20ml of buffer solution; in the examples herein, typically, an excess of restriction enzyme is used to ensure complete digestion of the DNA substrate. Although the variation is allowed, but generally at about 37 degrees C temperature 1 to 2 hours incubation. After each incubation, the proteins were removed by phenol/chloroform extraction followed by extraction with ether and recovery of the nucleic acids from the aqueous phase by precipitation with ethanol. If desired, the different sized cleaved fragments can be separated by polyacrylamide gel or agarose gel electrophoresis using standard techniques. General description of fragment size separation is given in Methods in Enzymology (1980) 65: 499-560.

In the presence of four deoxynucleotide triphosphates (dNTPs) by reaction in 50mM Tris pH 7.6, 50mM NaCl, 6mM MgCl₂Restriction cleaved fragments were treated to blunt ends with E.coli DNA polymerase I large fragment (Klenow) in 6mM DTT and 0.1-1.0mM dNTP at 20-25 ℃ for 15-25 minutes. Even if four dNTPs are present, the Klenow fragment fills the 5 'single strand overhang, while the overhanging 3' single strand is cleaved off. If desired, selective repair can be carried out by providing only one or selectively providing dNTPs, within limits determined by the nature of the overhang. After treatment with Klenow, the mixture was extracted with phenol/chloroform and precipitated with ethanol. Any single-stranded portion is hydrolyzed by treatment with S1 nuclease or BAL-31 under appropriate conditions.

A typical ligation reaction was carried out in a volume of 15-50ml under the following standard conditions and temperatures: for example, 20mM Tris-HCl pH 7.5, 10mM MgCl₂10mM DTT, 33. mu.g/ml BSA, 10-50mM NaCl, and 40. mu.M ATP, 0.01-0.02(Weiss) units of T4DNA ligase, 0 ℃ ("sticky end" ligation) or 1mM ATP, 0.3-0.6(Weiss) units of T4DNA ligase, 14 ℃ ("blunt end" ligation). The total concentration of DNA to effect intermolecular "sticky-end" ligation was 33-100. mu.g/ml (final total concentration was 5-100 nM). The final total concentration of DNA for which intermolecular "blunt-ended" ligation was performed was 1 mM.

In vector construction, a "vector fragment" is used, which is typically treated with Bacterial Alkaline Phosphatase (BAP) or bovine intestinal alkaline phosphatase (CIAP) to remove the 5' phosphate and prevent self-ligation. BAP or CIAP was digested at 60 ℃ for about 1 hour in about 10mM Tris-HCl, 1mM EDTA, pH 8, in an amount of about 1 unit/mg vector. The product was extracted with phenol/chloroform and precipitated with ethanol. Alternatively, religation can be prevented in vectors that have been double digested with additional restriction enzymes, and unwanted fragments isolated.

Any method may be used to introduce mutations into a coding sequence to produce variants of the invention, including simple deletions or insertions, systematic deletions, insertions or substitutions of clustered bases or substitutions of individual bases.

For example, the B7-DC DNA sequence (cDNA or genomic DNA) is modified by site-directed mutagenesis, a well-known technique, the protocols and reagents of which are commercially available (Zoller, MJ et al, Nucleic Acids Res (1982) 10: 6487-. For example, the correct ligation of the plasmid construction is determined by first transforming E.coli strain MC1061(Casadaban, M., et al, J Mol Biol (1980) 138: 179. sup. 207) or other suitable host with the ligation mixture. Transformants are selected based on the presence of ampicillin-, tetracycline-, or other antibiotic resistance genes (or other selection markers) using conventional methods, depending on the manner in which the plasmid is constructed. Plasmids were prepared from transformants with optional chloramphenicol amplification, optionally followed by chloramphenicol amplification (Clewell, DB et al, Proc Natl Acad Sci USA (1969) 62: 1159; Clewell, D.B., J Bacteriol (1972) 110: 667). Several micro-DNA fragments (preps) are commonly used, see, e.g., "Holmes, DS, et al, Anal Biochem (1981) 114: 193-197; birmboim, HC et al, Nucleic Acids Res (1979) 7: 1513-1523. Sequencing by restriction and/or by Sanger's dideoxynucleoside method (Proc Natl Acad Sci USA (1977) 74: 5463), or by Maxam et al Methods in Enzymology (1980) 65: 499, in Messing et al, Nucleic Acids Res (1981) 9: 309, as further described.

Vector DNA is introduced into mammalian cells by conventional techniques, such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofection, or electroporation. Suitable methods for transforming host cells can be found in Sambrook et al, supra, and other articles.

Generally, in fusion expression vectors, a protease cleavage site is introduced at the junction of the reporter group and the protein of interest, thus allowing the protein of interest to be separated from the reporter group after purification of the fusion protein. Proteinases suitable for such cleavage and their recognition sequences include factor Xa, thrombin and enterokinase.

Typical fusion expression vectors include pGEX (Amrad, Melbourne, Australia), pMAL (New England Biolabs, Beverly, Mass.), and pRIT5 (pharmacia, Pistavir, N.J.), which fuse glutathione S-transferase, maltose E binding protein, protein A, respectively, to the recombinant protein of interest.

Promoters and enhancers

The promoter region of a DNA or RNA molecule binds RNA polymerase, initiating transcription of an "operably linked" nucleic acid sequence. As used herein, "promoter sequence" refers to a nucleotide sequence of a promoter that is located on a DNA or RNA strand and is transcribed by RNA polymerase. Two sequences of a nucleic acid molecule, such as a promoter and a coding sequence, are "operably linked" when they are linked to each other in a manner that allows both sequences to be transcribed into the same RNA transcript or that allows an RNA transcript in one sequence to begin to be extended into a second sequence. Thus, two sequences, e.g., a promoter sequence and a coding sequence of DNA or RNA, are operably linked if transcription initiated at the promoter sequence produces an operably linked RNA transcript of the coding sequence. In order to achieve "operative linkage", it is not necessary to have two sequences immediately adjacent to each other in a linear sequence.

Preferred promoter sequences of the present invention must be operable in mammalian cells and may be eukaryotic or viral promoters. Suitable promoters may be inducible, repressible or constitutive. An example of a constitutive promoter is the viral promoter MSV-LTR, which is efficient and active in various types of cells and which has equally enhanced activity in arrested and growing cells compared to most other promoters. Other preferred viral promoters include those present in CMV-LTR (from cytomegalovirus) (Bashart, M. et al, Cell 41: 521(1985)) or RSV-LTR (from Rous sarcoma virus) (Gorman, C.M., Proc.Natl.Acad.Sci.USA 79: 6777 (1982)). Other useful promoters are the promoter of the murine metallothionein I gene (Hamer, D., et al, J.mol.appl.Gen.1: 273-288 (1982)); the TK promoter of herpes virus (McKnight, S., Cell 31: 355-jar 365 (1982)); SV40 early promoter (Benoist, C., et al, Nature 290: 304-310(1981)) and the yeast gal4 gene promoter (Johnston, S.A., et al, Proc. Natl. Acad. Sci. (USA) 79: 6971-6975 (1982); Silver, P.A., et al, Proc. Natl. Acad. Sci. (USA) 81: 5951-5955 (1984)). Other exemplary descriptions of transcription factors and the respective activities associated with the promoter regions and DNA that binds to the transcription factors include: keegan et al, Nature (1986) 231: 699; fields et al, Nature (1989) 340: 245; jones, Cell (1990) 61: 9; lewis, Cell (1990) 61: 1161; ptashne et al, Nature (1990) 346: 329 of the formula (I); adams et al, Cell (1993) 72: 306. the relevant disclosures of all references listed above are incorporated herein by reference.

The promoter region may further comprise an octamer region that functions as a tissue-specific enhancer by interacting with a protein in a particular tissue. The enhancer region of the DNA construct of the present invention is an enhancer region which is specific for the transfected target cell or is highly activated by a cytokine of such target cell. Examples of vectors (plasmids or retroviruses) are disclosed in (Roy-Burman et al, U.S. Pat. No. 5,112,767). A general discussion of enhancers in transcription and their activity is found in Lewis, B.M., Genes IV, Oxford University Press, Oxford, (1990), page 552-576. Particularly useful enhancers are retroviral enhancers (e.g., viral LTRs). Enhancers are preferably located upstream of the promoter, interacting with the promoter to stimulate gene expression. If retroviral vectors are used, the endogenous viral LTR may be used as a weak enhancer and may be replaced by other desired enhancer sequences which confer tissue specificity or other desired properties, such as transcriptional availability, to the B7-DC encoding DNA molecule of the invention.

The nucleic acid sequences of the invention may also be chemically synthesized using standard techniques. Various methods for chemically synthesizing polydeoxyribonucleotides are known, including solid phase synthesis, such as peptide synthesis, which can already be fully automated using commercially available DNA synthesizers (see, e.g., Itakura et al, U.S. Pat. No. 4,598,049; Caruthers et al, U.S. Pat. No. 4,458,066; and Itakura, U.S. Pat. Nos. 4,401,796 and 4,373,071, incorporated herein by reference).

Proteins and polypeptides

The invention comprises a polypeptide having the sequence SEQ ID NO: 2 or SEQ ID NO: 4 "isolated" B7-DC protein. The present specification exemplifies full-length human and murine B7-DC proteins (and DNA), but it is understood that homologs of B7-DC and mutants thereof from other mammalian species having the characteristics disclosed herein are also within the scope of the present invention.

The present invention also includes "functional derivatives" of B7-DC, which refers to variants of amino acid substitutions, "fragments" or "chemical derivatives" of B7-DC, the terms "fragments" and "chemical derivatives" being explained below. One functional derivative retains detectable B7-DC activity, preferably capable of binding to a receptor on a T cell and co-stimulating T cell activity, which allows its utility in accordance with the present invention. "functional derivatives" include "variants" and "fragments" whether used simultaneously or individually in the present invention.

A functional homologue must have the above-mentioned biochemical and biological activities. In view of their functional characteristics, the homologous protein B7-DC from other species used comprises proteins not yet found, which are within the scope of the present invention if they have sequence similarity and the listed biochemical and biological activities.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps are introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences are removed for comparison). In a preferred comparison method, cysteine residues are compared.

In a preferred embodiment, the length of the sequences to be compared is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80% or 90% of the length of the reference sequence. For example, when the second sequence is compared to the amino acid sequence of the human B7-DC protein having 276 amino acid residues (SEQ ID NO: 2), at least 83, preferably at least 110, more preferably at least 138, even more preferably at least 166, and even more preferably at least 193, 221 or 248 amino acid residues are compared. The amino acid residues (or nucleotides) at the corresponding amino acid positions (or nucleotide positions) are then compared. Two molecules are identical at a position in a first sequence if the position has the same amino acid residue (or nucleotide) as the corresponding position in the second sequence (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity of two sequences is a parameter that characterizes the number of identical sites shared by the sequences, taking into account the number of gaps introduced and the length of each gap required to perform an optimal alignment of the two sequences.

Sequence comparison and percent identity determination between two sequences can be accomplished using mathematical algorithms. In a preferred embodiment, the percent identity of two amino acid sequences is determined using the Needleman and Wunsch (J.MoL.BioL.48: 444-453(1970)) algorithms, which have been included in the GAP program in the GCG package (in the GAP weight) of the GCG package, using the Blossom 62 and PAM250 matrices and the GAP value (GAP weight) of 16, 14, 12, 10, 8, 6 or 4 and the length value (length weight) of 1,2, 3,4, 5 or 6http://www.gcg.comAvailable). In another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (in the case ofhttp://www.gcg.comAvailable), determined using NWSgapdna, CMP matrices and gap weights of 40, 50, 60, 70, or 80 and length weights of 1,2, 3,4, 5, or 6. In another embodiment, the percent identity of two amino acid or nucleotide sequences is determined using the algorithms of E.Meyers and W.Miller (CABIOS, 4: 11-17(1989)), using a PAM120 weight residue table (weight residual table), a gap length penalty of 12 and a gap penalty of 4, which have been included in the ALIGN program (version 2.0).

The nucleic acids and proteins of the invention are also used as "query sequences" to perform public database searches, e.g., to identify other family members or related sequences. Such a search can be performed using Altschul et al, (1990) j.mol.biol.215: NBLAST and XBLAST programs from 403-10 (version 2.0). A BLAST nucleotide search with NBLAST can be performed with a score (score) of 100 and wordlength of 12 to obtain nucleotide sequences homologous to human or murine B7-DC nucleic acid molecules. BLAST protein searches using the XBLAST program with a score (score) of 50 and wordlength of 3 can also be performed to obtain amino acid sequences homologous to the human or murine B7-DC protein molecules of the present invention. To obtain gap alignments (alignment) for comparison, the alignment can be performed as described in Altschul et al (1997) Nucleic Acids Res.25: 3389 The descriptions of 3402 utilize Gapped BLAST. When using BALST and Gapped BLAST programs, the default parameters in the respective programs (e.g., XBLAST and NBLAST) are used. See http:// www.ncbi.nlm.nih.gov.

Thus, the homologues of the B7-DC protein described above have the properties (a) of the functional activity of native B7-DC, and (B) of a sequence similarity to a native B7-DC protein (e.g., SEQ ID NO: 2 or SEQ ID NO: 4) of at least about 30% (amino acid level), preferably at least about 50%, more preferably at least about 70%, even more preferably at least about 90%, when determined by the above methods.

It is common knowledge in the art to obtain and express this protein using DNA probes based on the published B7-DC sequence. The biochemical and biological activity of the protein is then assayed using art-recognized methods such as those described herein, e.g., standard T cell proliferation or cytokine secretion assays. Biological assays of T cell co-stimulation will show whether the homolog has the requisite activity as a "functional" homolog.

Preferred assays determine functional properties of B7-DC such as stimulation of cytokine synthesis by T cells, which depend on TCR binding or cross-linking ("primary activation signal") and the transmission of co-stimulatory signals. B7-DC on T cells in combination with its natural ligand send out a signal that causes cytokine, such as IL-2 production increase, which in turn stimulates proliferation, the proliferation can be measured by conventional methods.

A "variant" of B6-DC is a molecule substantially identical to its full-length protein or a fragment thereof, in which one or more amino acid residues have been substituted (substitution variant) or one or more residues have been deleted (deletion variant) or added (addition variant). "fragments" of B6-DC refer to any subset of the molecule, preferably ECD-containing molecules, i.e., shorter polypeptides of the full-length protein.

There are many methods available for preparing fragments, mutants and variants of isolated DNA sequences. A small sub-region or fragment of nucleic acid encoding the B7-DC protein, e.g., 1-30 bases in length, can be prepared by standard chemical synthesis methods. Antisense oligonucleotides and primers can be used to prepare larger synthetic fragments.

A preferred functional derivative is a fusion protein, a polypeptide containing a functional fragment of B7-DC. For example, one useful B7 derivative is a B7-DC-Ig fusion protein containing a polypeptide corresponding to the ECD of B7-DC and a C region of Ig.

The presence of a fusion partner may alter the solubility, affinity and/or valency (defined herein as the number of binding sites available per molecule) of the B7-DC protein. A soluble B7-DC fusion protein that, when bound to a receptor on a T cell, produces a different biological effect than the native protein expressed on APCs, i.e., inhibits stimulation of T cells by competitive binding rather than co-stimulation.

The extracellular domain (ECD) of B7-DC as used herein refers to the entire extracellular portion of the protein or any fragment thereof that recognizes and binds PD-1 or other receptors on T cells other than CD28 or CTLA-4. Preferably, the ECD of B7-DC is encoded by SEQ ID NO: 2 or SEQ ID NO: 4 from position 26 to position 221.

"soluble B7-DC" refers to the cell-free form of B7-DC, which can be shed (shed), secreted, or otherwise extracted from the producing cells. Soluble B7-DCs include, but are not limited to, soluble fusion proteins such as the free ECD of B7-DC-Ig, B7-DC or the B7-DC ECD fused (genetically or chemically) to a bioactive molecule.

As previously mentioned, the present invention also encompasses hybrid fusion proteins formed of a B7-DC domain and a domain or fragment of another B7 family protein, preferably expressed on the cell surface in a co-stimulatory manner.

A preferred group of B7-DC variants are those in which at least one amino acid residue, preferably only one residue, is substituted with a different residue. For a detailed description of Protein chemistry and Structure, see Schulz, GE. et al, Principles of Protein structures, Springer Verlag, new york, 1978, and Creighton, t.e., Proteins: structure and Molecular Properties, w.h.freeman & co, san francisco, 1983, the above references being incorporated herein by reference. The types of substitutions that can occur in a protein molecule can be based on the analysis of the frequency with which amino acids are changed between homologous proteins of different species, e.g., tables 1-2 of Schulz et al (supra) and FIGS. 3-9 of Creighton (supra). Based on this analysis, the present invention defines conservative substitutions as one of the following 5 groups in which an exchange occurs:

the three amino acid residues in parentheses above have a particular role in the structure of the protein. Glycine is the only residue without side chains, thus making the chain elastic. Proline, due to its unusual geometric configuration, tightly binds the chain. Cysteines can participate in the formation of disulfide bonds, which are important in the folding of proteins.

More substantial changes in biochemical, functional (or immunological) properties are made by selective substitutions that are less conservative, such as substitutions within the above-mentioned 5 groups, rather than within the 5 groups. Such changes are more clearly distinct from their effect on maintaining (a) the structure of the peptide backbone of the substituted region, e.g., a sheet or helical structure, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain. Examples of such substitutions are (i) the substitution or deletion of glycine and/or proline with another amino acid or the insertion of glycine or proline; (ii) substitution of a hydrophilic residue such as serine or threonine for a hydrophobic residue such as leucine, isoleucine, phenylalanine, valine, or alanine, (by the hydrophobic residue); (iii) substitution of cysteine residues for other residues (or by other residues); (iv) substitution (or substitution) of a residue with a positively charged side chain, such as lysine, arginine or histidine, for a negatively charged residue, such as glutamic acid or aspartic acid; or (v) substitution of a residue with a bulky side chain, such as phenylalanine, for a residue without such a side chain, such as glycine (or substitution of a residue without such a side chain).

The most acceptable deletions, insertions and substitutions according to the invention are those which do not produce a fundamental change in the properties of the B7-DC protein in its T cell co-stimulatory activity. However, where it is difficult to predict the effect accurately before making a substitution, deletion, insertion, one of ordinary skill in the art will appreciate that the effect can be assessed by conventional detection methods, such as those described herein, without the need for undue experimentation.

For the present invention, shorter chain variants may be prepared by chemical synthesis, preferred longer chain variants are typically prepared by site-specific mutagenesis of a nucleic acid encoding the B7-DC polypeptide, expression of the variant nucleic acid in cell culture, and optionally purification of the polypeptide from the cell culture, for example by immunoaffinity chromatography using specific antibodies immobilized on a column to adsorb the variant by binding to at least one epitope.

Chemical derivatives of B7-DC

A "chemical derivative" of B7-DC contains an additional chemical moiety that is not normally a part of a protein. Covalent modifications of polypeptides are also included within the scope of the invention. Such derivatized moieties may improve solubility, adsorption, biological half-life, and the like. Parts capable of taking such effects are disclosed in, for example, Remington's Pharmaceutical Sciences, 16 th edition, Mack Publishing Co., Easton, Pa (1980).

Such modifications are introduced into the molecule by reacting the target amino acid residue of the polypeptide with an organic derivatizing agent that is capable of reacting with the selected side chain or terminal residue. Another modification is cyclization of the protein.

Examples of chemical derivatives of polypeptides are as follows.

The lysine residues and the amino terminal residues are derivatized with succinic anhydride or other carboxylic acid anhydrides, and the derivatives produced with cyclic carboxylic acid anhydrides have the effect of altering the charge of the lysine residues. Other suitable reagents for derivatizing amino-containing residues include imidoesters such as methyl picolinate (methyl picolinate); pyridoxal phosphate; pyridoxal; a chlorohydrocarbon compound; trinitrobenzenesulfonic acid; o-methylisourea; 2, 4-pentanedione; and a aminotransferase-catalyzed reaction with glyoxylic acid.

The carboxy side chain group, aspartyl group or glutamyl group, is selectively modified by reaction with carbodiimide (R-N ═ C ═ N-R') 1-cyclohexyl-3- (2-morpholinyl- (4-ethyl) carbodiimide or 1-ethyl-3- (4-azonia-4, 4-dimethylpentyl) carbodiimide.

Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of serine or threonine residues, methylation of the amino group of lysine (Creighton, supra, pages 79-86), acetylation of the N-terminal amino group, and amidation of the C-terminal carboxyl group.

Also included are polypeptides in which one or more D-amino acids are substituted for one or more L-amino acids.

Polypeptides (Multimeric Peptides)

The invention also encompasses longer polypeptides in which the basic peptide sequence obtained from the B7-DC sequence is repeated from about 2 to about 100 times, with or without intervening gaps and linkers. It will be apparent that such multimers may be constructed from any of the peptide variants defined herein. Furthermore, a peptide multimer may contain different combinations of peptide monomers and substituted variants thereof as disclosed. Such oligo-or polypeptides may be prepared by chemical synthesis or recombinant DNA techniques as described herein. When produced chemically, the oligomer preferably contains 2-8 repeats of the base peptide sequence. When produced by recombinant means, the multimer may contain as many repeats as the expression system can allow, for example, from 2 to about 100 repeats.

In tandem multimers of B7-DC peptides or polypeptides, preferably dimers and trimers, the chains are joined by intrachain disulfide bonds or other "artificial" covalent bonds, so that the chains are "side-by-side" rather than "end-to-end". Preferred dimers and trimers are fusion proteins with B7-DC, such as the dimers and trimers formed between B7-DC-Ig as described herein.

Antibody specificity of B7-DC peptide

In the following description, various methodologies well known to those of ordinary skill in the art of immunology, cell biology and molecular biology are incorporated by reference. Publications and other materials setting forth these well-known methodologies are incorporated by reference herein in their entirety. General references that illustrate the basic principles of Immunology include a.k.abbas et al, Cellular and Molecular Immunology (fourth edition), w.b.saunders co., philadelphia, 2000; janeway et al, immunobiology, The Immune System in Health and Disease, fourth edition, Garland Publishing co, new york, 1999; roitt, i.e., et al, Immunology, (current ed.) c.v. mosby co, st.louis, MO (1999); klein, J., Immunology, Blackwell Scientific Publications, Inc., Cambridge, MA, (1990).

Monoclonal antibodies (mAbs) and methods of making and using them are described in Kohler and Milstein, Nature 256: 495-497 (1975); U.S. Pat. nos. 4,376,110; hartlow, e., et al, Antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988); monoclone Antibodies and hybrids: a New Dimension in Biological analytes, Plenum Press, New York (1980); zola et al, in Monoclonal Hybridoma Antibodies: techniques and Applications, CRC Press, 1982)).

Immunoassay methods are also described in Coligan, j.e., et al, Current Protocols in Immunology, Wiley-Interscience, new york 1991 (or Current edition); butt, W.R, (ed) Practical Immunoassay: the State of The Art, Dekker, New York, 1984; bizollon, ch.a., eds., Monoclonal Antibodies and New Trends in Immunoassays, Elsevier, New york, 1984; butler, j.e., ELISA (chapter 29), In: van Oss, c.j., et al, (eds.), immucohemistry, Marcel Dekker, inc., new york, 1994, 759-; butler, J.E. (eds.), biochemistry of Solid-Phase immunology, CRC Press, Boca Raton, 1991; weintraub, B., Principles of Radioimmunoassays, seven Training court on Radioligand Assay Techniques, The endogrine Society, March, 1986; word, T.S. et al, Laboratory Techniques and Biochemistry in Molecular Biology, North Holland Publishing Company, New York, (1978) (chapter "An Introduction to radio analysis and Related Techniques" written by Chard.T.).

Anti-idiotypic antibodies are also described, for example, by Idiotypy in Biology and Medicine, academic Press, New York, 1984; volume Immunological Reviews 79, 1984; volume Immunological Reviews 90, 1986, curr. top. microbiol, volume Immunological 119, 1985; bona, c, et al, CRC crit.rev.immunol., pages 33-81 (1981); jerne, NK, ann.immunol.125c: 373-389 (1974); jerne, NK, In: idiotypees-antibiotics on the instrument, Westen-Schnurr, I., eds., editions Roche, Basel, 1982, Urban, J et al., Ann. Immunol.133D: 179- (1982); rajewsky, K et al, ann.rev.immunol.1: 569-607(1983).

The present invention provides antibodies, both monoclonal and polyclonal, that react with a novel epitope of B7-DC that is not present in known proteins of the B7 family. The antibody may be heterologous, allogeneic, syngeneic or a modification thereofVeneered forms, such as humanized or chimeric antibodies. Also included are anti-idiotype antibodies specific for the idiotype of the anti-B7-DC antibody. The term "antibody" is also meant to encompass intact molecules and fragments of the molecule that contain an antigen binding site and are capable of binding to an epitope of B7-DC. These include Fab and F (ab') lacking the Fc fragment of an intact antibody₂Fragments that clear from circulation more rapidly than intact antibodies and have less non-specific tissue binding (Wahl et al, J.Nucl. Med.24: 316-325 (1983)). Also included are Fv fragments (Hochman, J. et al (1973) Biochemistry 12: 1130-1595; Sharon, J. et al (1976) Biochemistry 15: 1591-1594). These various fragments can be prepared using conventional techniques such as protease cleavage or chemical cleavage (see, e.g., Rousseaux et al, meth.enzymol., 121: 663-69 (1986)).

Polyclonal antibodies are obtained in serum form from immunized animals such as rabbits, goats, rodents, etc., and may be used without further treatment, or may be subjected to conventional concentration or purification procedures such as ammonium sulfate precipitation, ion exchange chromatography, and affinity chromatography (see Zola et al, supra).

The immunogen may comprise the entire B7-D6 protein, or a fragment or derivative thereof. Preferred immunogens comprise all or part of the ECD of human B7-DC (amino acid residues 26-221), and these residues are found to contain post-translational modifications such as glycosylation on native B7-DC. The immunogen containing the extracellular domain produced by various methods known in the art, such as a cloned gene expressed by conventional recombinant methods, is isolated from the original cells, a cell population expressing high levels of B7-DC, and the like.

mAbs can be produced by conventional hybridoma techniques, such as those described by Kohler and Milstein (Nature, 256: 495-97(1975)), and modified versions thereof (see references above). An animal, preferably a mouse, is immunized with an immunogen as described above to produce the desired antibodies in the immunized animal.

Typically, B lymphocytes from the lymph nodes, spleen or peripheral blood of an immunized animal are fused with myeloma cells in the presence of a fusion promoting agent such as polyethylene glycol (PEG). Any cell of the murine myeloma cell line can be used for fusion: P3-NS1/1-Ag4-1, P3-x63-k0Ag8.653, Sp2/0-Ag14, or HL1-653 myeloma cell lines (available from ATCC, Rockville, Md.). The next step involves growth in selective media to allow complete death of the unfused parental myeloma cells and donor lymphocytes, while only the hybridoma cells survive. These hybridoma cells are cloned and cultured, and the supernatants are screened for antibodies with the desired specificity, such as by immunoassay techniques using the B7-DC-Ig fusion protein. Positive clones are subcloned, for example, by limiting dilution, and mAbs isolated.

Hybridomas produced according to these methods can be propagated in vitro or in vivo (in ascites fluid) using techniques well known in the art (see generally Fink et al, prog. Generally, single cell lines are propagated in culture medium, and the medium containing high concentrations of single mabs is collected by decantation, filtration, or centrifugation.

The antibodies produced may be single chain antibodies or scfvs, rather than the normal multimeric structure. The single chain antibody comprises a hypervariable region of the Ig of interest and when the single chain antibody is part of the complete Ig, reconstructs the antigen binding site of the native Ig (Skerra, A. et al (1988) Science, 240: 1038-; bird et al, (1988) Science 242: 423; huston et al, (1988) Proc.Natl.Acad.Sci.USA 85: 5879; jost CR et al,. J.biol chem.1994269: 26267-26273; U.S. Pat. nos. 4,704,692, 4,853,871, 4,94,6778, 5,260,203, 5,455,0Kn are contacted with a solution containing an unknown amount of labeled antibody, which acts as a "reporter molecule". After the second incubation, the labeled antibody is bound to the antigen, which is bound to the solid support by the unlabeled antibody, and the solid support is washed again to remove the unreacted labeled antibody. This type of forward sandwich (forward sandwich) assay can be a simple yes/no assay to identify the presence of an antigen, or can be quantified by comparing the amount of labeled antibody to the amount of antibody obtained from a standard sample containing a known amount of antigen.

In another type of "sandwich" assay, so-called "simultaneous" and "reverse" assays are used. The simultaneous detection involves one incubation step, since both the solid support bound antibody and the labeled antibody are added simultaneously to the sample being tested. After incubation, the solid support is washed to remove residual liquid sample and unbound labeled antibody. The presence of labeled antibody bound to the solid support is then detected as in a conventional "forward" sandwich assay.

In a "reverse" assay, a solution of labeled antibody is added to a liquid sample stepwise, followed by addition of unlabeled antibody bound to a solid support after an appropriate incubation time. After the second incubation, the solid phase is washed in a conventional manner to remove the residual sample to be detected and the unreacted labeled antibody solution. The labeled antibody bound to the solid support is then detected as in the "simultaneous" and "forward" assays.

The antibodies described above are useful in methods of inhibiting T cell stimulation and treating diseases associated with unwanted T cell activation, such as transplant rejection and autoimmunity. The method involves administering to a subject in need of such treatment an effective amount of an antibody, preferably a mAb, more preferably a human or humanized mAb specific for a costimulatory epitope of B7-DC. The administered antibody must be effective in blocking stimulation of T cells or eliminating antigenically active T cells, thereby inhibiting the targeted T cell response. The relevant dosage ranges are described below.

Use of nucleic acids encoding B7-DC proteins

The nucleic acids of the invention can be used diagnostically to detect the progression of disease by detecting the expression of B7-DC in cells of a biological sample or by detecting the effect of an agent on the expression of B7-DC. This is preferably achieved by measuring the mRNA level of the cells. For use in such diagnostic methods, the nucleic acid sequence is detectably labeled, e.g., with a radioactive or fluorescent label or biotin label, and used in a conventional dot blot or Northern hybridization procedure to detect preparations of mRNA molecules present in, e.g., total kor poly (a +) RNA of a biological sample.

Therapeutic compositions and administration thereof

The B6-DC polypeptide or a cell expressing the polypeptide, such as a DC or a tumor cell, is administered to a mammalian subject, preferably a human. Polypeptides that bind to cells, are immobilized or otherwise aggregate to enhance T lymphocyte reactivity and resulting immunity. The B6-DC-Ig fusion protein forms a dimer and, as shown in the examples, co-stimulates T cells. The soluble monomeric form of the B6-DC polypeptide binds to receptors on T cells without producing stimulatory activity and is therefore considered to be a competitive inhibitor or antagonist of T cell co-stimulation by the stimulatory form of the molecule. This binding of the B6-DC antagonist may inhibit ongoing T cell activity or may interfere with the action of co-stimulatory signals presented by endogenous B6-DC or even by other B7 family members through their receptors (e.g., CD28 or CTLA-4).

The compositions described herein having B7-DC activity are administered in a biologically or therapeutically effective amount in a pharmaceutically acceptable carrier. The B7-DC polypeptide (or cells expressing the polypeptide) can be administered alone or in combination with other proteins or polypeptides such as an active protein or polypeptide having another member of the B7 family or another immunostimulatory molecule. Treatment may comprise administration of an adjuvant which in the broadest sense comprises any non-specific immunostimulatory compound, such as an interferon. Adjuvants contemplated by the present invention comprise resorcinol, a non-ionic surfactant such as polyoxyethylene oleyl ether and n-cetyl polyvinyl ether.

The following is the dose of the antibody of the present invention administered to the subject.

A therapeutically effective amount is an amount that will achieve the desired immunological or clinical effect when administered for a period of time effective.

The therapeutically active amount of a polypeptide having B7-DC activity (or an antibody against B7-DC) may vary depending on factors such as the state of the disease, age, sex and weight of the individual and the ability of the peptide to elicit a desired response in the individual. Dosage therapy can be adjusted to provide the best therapeutic response. For example, several divided doses may be administered daily or the dose may be reduced appropriately as dictated by the exigencies of the therapeutic situation. A therapeutically effective amount of a cell-associated form of a protein can be determined from the protein or cell equivalent.

Thus, an effective amount is from about 1ng to about 1g, more preferably from about 1 μ g to 100mg/kg, and even more preferably from about 100 μ g to about 100mg/kg per kilogram of body weight of the recipient. Dosage forms suitable for internal administration preferably contain (for the latter dosage range) from about 0.1mg to 500mg of active ingredient per unit. The active ingredient may vary from 0.5 to 95% by weight, based on the total weight of the composition. Alternatively, an effective dose of B7-DC expressing cells, such preferred transduced cells as DCs or inactive tumor cells is about 10 per subject⁴-10⁹A cell, more preferably about 10⁶-10⁸Individual cells, preferably in isolated dosage form. One of ordinary skill in the art of immunotherapy can adjust these dosages without undue experimentation.

The active compound (e.g., B6-DC polypeptide or cells transduced with B6-DC DNA) can be administered by injection in a conventional manner, e.g., by conventional and effective routes. Preferred routes include subcutaneous, intradermal, intravenous and intramuscular routes. Other possible routes include oral, intrathecal, inhalation, transdermal or rectal administration. Direct intratumoral injection is also possible for the treatment of tumors that have not been completely resected.

Depending on the route of administration, the active compound may be coated in a material to prevent the compound from being affected by enzymes, acids and other natural conditions that inactivate the compound. Thus, for the mode of administering a polypeptide or peptide having B7-DC activity by the enteral route, it is necessary to coat the composition with or co-administer the composition with a material that prevents its inactivation. For example, the peptide may be administered to the subject in the form of a suitable carrier, diluent or adjuvant, co-administered with an enzyme inhibitor, such as pancreatic insulin inhibitor, diisopropyl fluorophosphate (DEP) and aprotinin, or in the form of a suitable carrier, such as liposomes, comprising water-in-oil-in-water emulsions and conventional liposomes (Strejan et al, (1984) J Neuro imunol 7: 27).

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersants, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents as pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the therapeutic compositions is contemplated. Auxiliary active compounds may also be used in the compositions.

Preferred pharmaceutically acceptable diluents include saline and aqueous buffers. Pharmaceutical compositions suitable for injection comprise sterile aqueous solutions (soluble in water) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Isotonic agents such as sugars, polyalcohols such as mannitol, sorbitol, sodium chloride may be included in the pharmaceutical composition. In any case, the composition should be sterile and should be fluid. Should be stable under the conditions of manufacture and storage, must contain a preservative to prevent contamination by microorganisms such as bacteria and fungi. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

The carrier can be a solvent or dispersant comprising, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size of the dispersion and by the use of surfactants.

The action of microorganisms can be inhibited by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.

Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Parenteral compositions are preferably formulated in dosage unit form to facilitate administration and uniformity of dosage. Dosage unit form refers to physically discrete units suitable for administration as a single dose to a mammalian subject; each unit consisting of a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage units of the invention will depend upon, or depend directly upon, (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) limitations inherent in the art of admixing such active compounds to treat sensitivity in individuals.

For pulmonary instillation, nebulized solutions may be used, and in sprayable aerosol formulations, the active protein may be combined with a solid or liquid inert carrier material. It may also be packaged in a squeeze bottle or mixed with a pressurized volatile, typically a propellant gas. The aerosol formulation may contain, in addition to the protein of the present invention, solvents, buffers, surfactants and antioxidants.

For topical application, the proteins of the invention may be combined with a topically applied vehicle such as an ointment or salve which has a smoothing effect on the skin and which is capable of delivering the active ingredient directly to the affected area.

The carrier for the active ingredient may be in either a sprayable or a non-sprayable form. The non-sprayable form may be a semi-solid or solid form containing one carrier suitable for topical application and having a dynamic viscosity preferably greater than water. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like. These may be sterilized or mixed with auxiliaries such as preservatives, stabilizers, humectants, buffers, or salts which influence the osmotic pressure, etc., if desired. Examples of preferred excipients for non-sprayable topical formulations include ointment bases such as polyethylene glycol-1000 (PEG-1000), conventional creams such as HEB cream, gelatin, petrolatum gel, and the like.

Other pharmaceutically acceptable carriers for the B7-DC polypeptides of the invention are liposomes, pharmaceutical compositions containing the active protein, said compositions being dispersed or present in the form of distinct particles, wherein the particles consist of an aqueous layer bound to a lipid layer. The active protein is preferably present in the aqueous and lipid layers, internally or externally or anywhere, or in a non-homogeneous system known as a liposome suspension. A hydrophobic layer, or a lipidic layer, usually but not exclusively containing phospholipids, such as lecithin and sphingomyelin, steroids, such as cholesterol, more or less ionic surfactants, such as dicetyl phosphate, stearylamine or phosphatidic acid and/or other natural hydrophobic materials.

Modification of tumor cells to express B7-DC and multiple costimulatory molecules

Another aspect of the invention is a cell, preferably a tumor cell, modified to express a multiple co-stimulatory molecule. Transient expression of costimulatory molecules B7, B7-2 and B7-3 on activated B cells was different. For example, B7-2 is expressed early in B cell activation, while B7-3 is expressed late. Different co-stimulatory molecules therefore have different functions during the course of the immune response. An effective T cell response requires that the T cells receive costimulatory signals from multiple costimulatory molecules.

Thus, the invention encompasses tumor cells that are genetically modified or that express more than one co-stimulatory molecule, e.g., tumor cells can be modified to express B7-DC and one or more of B7, B7-2, and B7-3.

Before modification, cells, such as tumor cells, may not express any co-stimulatory molecules or may express certain co-stimulatory molecules without expressing other co-stimulatory molecules. As described herein, tumor cells can be modified by transfection with a nucleic acid encoding B7-DC alone or with different costimulatory molecules. For example, tumor cells transfected with a nucleic acid encoding B7-DC may be further transfected with a nucleic acid encoding B7. The sequences of the cDNA molecules encoding the human or murine B7-DC protein are SEQ ID NOs: 1 and SEQ ID NO: 3. Alternatively, more than one modification may be used. For example, tumor cells transfected with nucleic acids encoding B7-DC can be stimulated with agents that induce expression of B7-1, B7-2, or B7-3.

Pathogen-associated antigens

The main application of the present invention is the use of the composition of the present invention in a therapeutic vaccine capable of treating cancer and the major chronic viral infections worldwide leading to morbidity and mortality. Such vaccines are designed to eliminate infected cells, which requires that the T cells react when the antibodies fail. In addition to the antigenic epitopes themselves, the vaccine of the invention comprises:

(a) vectors such as naked DNA, naked RNA, self-replicating RNA replicons and viruses including vaccinia virus, adenovirus, adeno-associated virus (AAV), lentivirus and RNA alphavirus;

(b) antigenic localization (targeting) or processing signals such as HSP70, calretin, the extracellular domain of Flt-3 ligand, domain II of Pseudomonas exotoxin A (ETA), herpes simplex VP22 target protein, and the like (see commonly assigned U.S. patent application Ser. No. 09//421,608; 09/501,097; 09/693,450; 60/222,9002; 60,/222,985; 60/268,575; and Chang, W-F et al, J.Virol.75: 2368-; and

(c) a costimulatory signal, preferably the B7-DC protein of the invention or a fusion protein, fragment or functional derivative thereof (alone or in combination with other known costimulatory proteins such as B7.1, B7.2, soluble CD40, etc.).

Tumor cells or other types of host cells, including APCs, are transformed, transfected or otherwise transduced with a nucleic acid encoding an antigen that elicits an immune response. Such antigens are preferably epitopes of pathogenic microorganisms against which the host cells are protected by effector T cell responses, including Cytotoxic T Lymphocytes (CTL) and delayed hypersensitivity reactions. These pathogenic microorganisms typically comprise viruses, intracellular parasites such as plasmodium (malaria) and bacteria that grow intracellularly such as mycobacteria (mycobactria) and listeria (listeria). Thus, the type of antigen comprised in the vaccine combination of the present invention is any form associated with these pathogenic bacteria (and of course tumor specific antigens). It is noteworthy that some viral antigens are also tumor antigens in the case where the virus is a causative agent of cancer.

In fact, two of the most prevalent cancers worldwide: hepatocellular carcinoma and cervical cancer are both associated with viral infections. Hepatitis B Virus (HBV) (Beasley, R.P., et al, Lancet 2, 1129-1133(1981)) has been identified as a causative agent of hepatocellular carcinoma. 80-90% of cervical cancers express the E6 and E7 antigens from four "high risk" human papillomavirus types: one of HPV-16, HPV-18, HPV-31 and HPV-45 (Gissmann, L. et al., Ciba Found Symp.120, 190-. Due to their ubiquitous expression in cervical cancer, HPV E6 and E7 antigens are most likely target antigens for virus-associated cancers in immunocompetent individuals. In addition to their importance as target antigens for therapeutic cancer vaccines, virus-associated tumor antigens are ideal candidates for prophylactic vaccines. In fact, the introduction of prophylactic HBV vaccines in Asia has reduced the incidence of liver cancer (Chang, M.H., et al. New Engl. J.Med.336, 1855-1859(1997)), which has a great impact on cancer prevention.

The most important viruses in chronic human viral infection are Human Papilloma Virus (HPV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Human Immunodeficiency Virus (HIV), epstein-barr virus (EBV) and Herpes Simplex Virus (HSV).

In addition to use in human cancers and infectious diseases, the present invention can also be used in the veterinary field to treat animal diseases. Thus, the methods described herein can be readily used by one of ordinary skill in the art to treat herpes virus infections in livestock, including equine herpes virus, bovine herpes virus, marek's disease virus in chickens and other poultry; animal retrovirus disease, pseudorabies and rabies.

The following references, all incorporated herein by reference, give principles and current information in the areas of basic virology, medical virology and veterinary virology: fields Virology, Fields, BN et al, eds., Lippincott Williams & Wilkins, new york, 1996; principles of Virology: molecular Biology, Pathologenetics, and Control, Flint, S.J., et al, eds., Amer Society for Microbiology, Washington, 1999; principles and Practice of Clinical Virology, fourth edition, zuckerman.a.j. et al, eds, John Wiley & Sons, new york, 1999; the Hepatitis C Viruses, by Hagedorn, CH et al, eds., Springer Verlag, 1999; hepatitis B Virus: molecular mechanics in diseases and Novel Strategies for Therapy, Koshy, R. et al, eds., World Scientific Pub Co, 1998; veteriary Virology, Murphy, f.a. et al, eds., Academic Press, new york, 1999; avian Viruses: function and Control, Ritchie, B.W., Iowa State University Press, Ames, 2000; virus Taxolomy: classification and Nomenclature of Viruses: seven Report of the International Committee on Taxomy of Virus, by M.H.V.Van Regemortel, MHV et al, compiled by Academic Press; new york, 2000.

Target molecules

A wide variety of proteins with different modes of action have been used as "target" molecules for binding to antigens, preferably as fusion polypeptides, to localize the antigen into cellular and subcellular compartments, thereby facilitating antigen presentation on T cells in a more efficient and useful manner.

Linkage of antigens to Heat Shock Proteins (HSPs) represents a potential approach for improving the effectiveness of nucleic acid (and other) vaccines. HSPs are apparently natural biological adjuvants in cancer and viral vaccines. Both gp96HSP residing in the Endoplasmic Reticulum (ER) and cytoplasmic Hsp70 can act as immunological adjuvants (Srivastava, PK et al, Semin. Immunol.3, 57-64 (1991); Udono, H et al, Proc. Natl. Acad. Sci. USA 91, 3077-. These HSPs or chaperones bind a large number of peptides (Lambert, E., et al., Eur. J Immunol.27, 923-927 (1997)). Hsp70 is a chaperone protein that targets the protein of interest to a proteosome-major cellular protease complex that produces peptides for binding to MHC class I molecules. Thus, antigens linked directly to Hsp70 are more efficiently presented by MHC class I (to direct the generation of, in particular, CTL responses). There are two characteristics that are clearly associated with the adjuvanticity of HSPs: (1) in vitro, gp 96-conjugated peptides are effective in introducing antigen into the MHC class I processing pathway; (2) the binding of gp96 to macrophages induces the secretion of pro-inflammatory cytokines, thereby augmenting the function of the cell targeted by the peptide antigen.

Immunization with HSP complexes isolated from tumors or from virus-infected cells can induce effective anti-tumor immunity (Srivastava, PK et al, Int J cancer.33: 417-22, 1984; Srivastava, PK et al, Proc Natl Acad Sci USA.83: 3407-11, 1986; Udono, H et al, J Immunol.152: 5398. 5403, 1994; Blachere, NE et al, J Immunotherh.14: 352-6, 1993; Udono, H et al, supra; Tamura, Y et al, science.278: net-20, 1997; Jazki, S et al, J munotherh.21: 269-76, 1998)) or anti-viral immunity (Heikema, A et al, munol Lett.57: 69-74, 1997; suto, R et al, science.269: 1585-8, 1995). Mixing peptides with HSP in vitro produces immunogenic HSP-peptide complexes (Ciupitu, AM et al, J Exp Med.187: 685-91, 1998; Blachere, NE et al, J Exp Med.186: 1315-22, 1997). Some HSP-based protein vaccines involve fusion of antigen with HSP (Suzue, K et al, J Immunol.156: 873-9, 1996; Suzue, K. et al, Proc Natl Acad Sci USA 94: 13146-51, 1997). Recently, the present inventors and their colleagues (e.g., Chen, C-H et al, Canc. Res.60: 1035-1042(2000)) have used HSPs in chimeric forms of DNA or RNA replicon vaccines. They use HPV-16E7 as an antigen, fused with Mycobacterium tuberculosis (HSP 70), and show enhanced amplification and activation of E7-specific CD8+ T cells, which results in effective antitumor immunity against existing tumors (Lin, K. -Y., et al, Cancer Res.56: 21-26., 1996).

Another useful target molecule is the translocation domain of Pseudomonas exotoxin A (ETA), such as domain II (dII) of ETA (comprising residues 253-364). A transport domain is a polypeptide that induces the transport of a protein or polypeptide into the cytoplasm of a cell. For example, similarly useful polypeptides are derived from diphtheria, Clostridium (Clostridium), tetani, anthrax, Yersinia, Vibrio cholerae or Bordetella pertussis toxins. In the preparation of such a composition, the DNA encoding the toxin domain of the hormone is preferably mutated or deleted.

Calreticulin (CRT), an abundant 46kDa protein localized in the lumen of the Endoplasmic Reticulum (ER), exhibits phytoclusterin activity known to be involved in folding and assembly of nascent glycoproteins (Nash (1994) mol. Cell. biochem. 135: 71-78; Hebert (1997) J Cell biol. 139: 613-, but not antigen-specific immunity of tumors (Basu, see above). The DCs were further stimulated in vitro with CRT conjugated peptides, which were re-presented in the above DC type I molecules and stimulated for peptide-specific CTL (Nair, supra).

Flt-3 ligand stimulates the growth of DC precursors in vivo and may promote the production of large numbers of DCs (Maraskovsky, E. et al, J Exp Med.184: 1953-62, 1996; Shurin, MR. et al, Cell Immunol.179: 174-84, 1997). Flt3, a murine tyrosine kinase receptor (Rosnet, O. et al, Oncogene 6: 1641-50, 1991), is a member of the type III receptor kinase family (see Lyman, SD, Curr Opin Hematol.5: 192-6, 1998). In hematopoietic tissues, Flt3 expression was restricted to CD34 positive precursors. Flt3 was used to identify and subclone the corresponding ligand, Flt 3-ligand (Lyman, SD et al, Cell 75: 1157-67, 1993; Hannum, C et al, Nature 368: 643-8, 1994). The major form of Flt-3 ligand synthesized serves as a transmembrane protein from which functionally similar soluble ECD was prepared by proteolytic cleavage (Lyman et al, supra). These proteins bind to and activate a unique tyrosine kinase receptor. In hematopoietic cells, expression of the Flt-3 receptor is largely restricted to the most primitive precursor cells, including DC precursors. ECD of Flt-3 ligand exerts a strong antitumor effect against several murine models of tumors, including fibrosarcoma, breast Cancer, liver Cancer, lung Cancer, melanoma and lymphoma (Lynch, DH et al, Nat Med.3: 625 631, 1997; Chen, K et al, Cancer Res.57: 3511-3516, 1997; Braun, SE et al, Hum Gene ther.10: 2141-2151, 1999; Peron, JM et al, J Immunol.161: 6164-6170, 1998; Chakravarty, PK et al, Cancer Res.59: 6028-6032, 1999; Esche, C et al, Cancer Res.58: 380-383, 1998) (19). The present inventors' colleagues linked DNA encoding HPV Ek7 protein to DNA encoding Flt-3 ligand ECD. The construct is used for immunization, the amplification and the activity of E7 antigen-specific CD8+ T cells are greatly improved, and effective anti-tumor immunity is generated on the existing metastatic tumor expressing E7.

The HSV-1 protein VP22, which is a prototype protein that, due to its outstanding intracellular translocation properties, is capable of, inter alia, increasing antigen spreading, can be used (Elliott, G., and P.O' Hare.1997.cell 88: 223-33). For example, VP22, which binds to p53(Phelan, A. et al., 1998, Nat Biotechnol 16: 440-443) or thymidine kinase (Dilber, MS et al, 1999, Gene Ther 6: 12-21), promotes the diffusion of proximally-linked proteins in vitro into surrounding cells and treats model tumors. In the DNA vaccine above, VP22 linked to the HPV-16E7 antigen caused a large (approximately 50-fold) increase in the number of E7-specific CD8+ T cell precursors in immunized mice, transforming the less effective DNA vaccine into a vaccine that was significantly effective against E7-expressing tumors. The non-spreading VP22 mutant did not enhance the efficacy of the vaccine. VP22 and proteins with similar modes of action enhance the efficacy of the vaccine in several ways: (1) promoting diffusion of antigen from transfected cells to surrounding APCs, thereby increasing the number of APCs that present antigen via the MHC class I pathway; (2) more efficient presentation of antigen in transfected cells; (3) cross-priming is performed to release the VP 22/antigen fusion protein, resulting in uptake and processing of DCs (or other APCs) for presentation via the MHC-I restricted pathway (Huang, Ay et al, 1994, Science 264: 961-.

Those of ordinary skill in the art know how to identify suitable epitopes, such as CTL epitopes, for related proteins from pathogens for use in accordance with the present invention.

Transfer of B7-DC DNA to cells and animals

DNA transfer, for example to achieve what is commonly known as "gene therapy", involves the introduction of "foreign" DNA into cells and, ultimately, into living animals. Several conventional gene therapy techniques have been studied and extensively repeated (Yang, N-S., Crit.Rev.Biotechnol.12: 335-.

One method involves transferring the nucleic acid into cultured primary cells, followed by autologous transplantation of the transformed cells ex vivo into the host, and homologous transfer to transfer the transformed cells into the host, either systemically or into specific organs or tissues.

To achieve the object of the present invention, nucleic acid therapy is achieved by transferring functionally active DNA directly into a body tissue or organ of a mammal in vivo. The transfer of DNA can be accomplished in a number of ways as described below. These systems were tested for successful expression in vitro using selectable markers (e.g., G418 resistance) to select transfected clones expressing DNA, followed by detection of the presence of the expression product of B7-DC by appropriate immunoassay using antibodies to the product (after treatment of the inducible system with an inducer). The effectiveness of this method, including DNA uptake, integration of the plasmid and stability of the integrated plasmid can be enhanced by linearizing the plasmid in a known manner and co-transfecting it with high molecular weight mammalian DNA as a "vector".

Examples of reports of successful "gene transfer" in the prior art include: (a) plasmid DNA was injected directly into murine muscle tissue to cause expression of marker genes over an indefinite period (Wolff, J.A. et al, Science 247: 1465 (1990); Acsadi, G. et al, The New Biologist 3: 71 (1991)); (b) retroviral vectors are effective for infecting vascular tissue in vivo and in situ; (c) retroviral preparations are injected portal and directly into the liver to affect Gene transfer and expression in vivo (Horzaglou, M. et al, J biol. chem.265: 17285 (1990); Koleko, M. et al, Human Gene Therapy 2: 27 (1991); Ferry, N. et al, Proc. Natl. Acad. Sci. USA 88: 8387 (1991)); (d) intratracheal injection of recombinant adenovirus into lung tissue is effective for in vivo transfer and long-term expression of foreign genes in the lung respiratory epithelium (Rosenfeld, m.a. et al, Science 252: 431 (1991)); (e) herpes simplex virus vectors are capable of transferring genes into brain tissue in vivo (Ahmad, F. et al., eds., Miami Short Reports-Advances in Gene Technology: The Molecular Biology of Human Genetic Disease, Vol.1, Boerriger Mannheim Biochemicals, USA, 1991).

Retroviral-mediated Human Therapy employs an amphotropic, replication-defective retroviral system (Temin, h.m., Human Gene Therapy 1: 111 (1990); Temin et al, U.S. Pat. No. 4,980,289; Temin et al, U.S. Pat. No. 4,650,764; Temin et al, U.S. Pat. No. 5,124,263; Wills, j.w. U.S. Pat. No. 5,175,099; Miller, a.d., U.S. Pat. No. 4,861,719). Such vectors have been used to introduce functional DNA into human cells or tissues, for example, adenosine deaminase gene into lymphocytes, NPT-II gene and gene encoding tumor necrosis factor into tumor infiltrating lymphocytes. Retrovirus-mediated gene transfer generally requires proliferation of target cells for gene transfer (Miller, d.g., et al, mol.cell.biol.10: 4239 (1990)). This condition is met by assuring that the DNA molecule of the invention will be introduced into the preferred target cells, i.e., actively growing tumor cells. Gene therapy for cystic fibrosis by any means of transfection with plasmids and transfection with retroviral vectors has been disclosed by Collins et al, U.S. Pat. No. 5,240,846.

DNA molecules encoding B7-DC sequences can be packaged into retroviral vectors using packaging Cell lines that produce replication-defective retroviruses, as is well known in the art (see, e.g., Cone, R.D. et al, Proc. Natl. Acad. Sci. USA 81: 6349-6353 (1984); Mann R.F. et al, Cell 33: 153-159 (1983); Miller, A.D. et al, Molec. Cell. biol. 5: 431-437 (1985); Sorge, J. et al, Molec. Cell. biol. 4: 1730-lec 1737 (1984); Hock, R.A. et al, Nature 320: 257 (1986); Miller, A.D. et al, Molec. Cell. biol. 6: 2895-282 (1986)). Newer packaging cell lines capable of safe and effective gene transfer have also been disclosed (Bank et al, U.S.5,278,056).

This method can be used to transfer retroviral vectors to selected tissues or organs in a site-specific manner. Thus, for example, a catheter transfer system (Nabel, EG et al, Science 244: 1342(1989)) may be used. Such methods using retroviral vectors or liposomal vectors are particularly effective for transferring the expressed nucleic acid into the blood circulation of the vessel wall or tumor.

Other viral vectors may also be used, including recombinant adenoviruses for neuron-specific transfer and retention (Horowitz, M.S., In: Virology, Fields, BN et al, eds., Raven Press, New York, 1990, page 1679; Berkner, K.L., Biotechniques 6: 6169191988), Strauss, S.E., In: the Adenoviruses, Ginsberg, HS, eds., Plenum Press, New York, 1984, Chapter 11), Herpes Simplex Virus (HSV). The benefits of human gene therapy using adenoviral vectors include that very little recombination occurs, no human malignancy has been found with this type of virus, the genome of the adenovirus is double-stranded DNA, it can be processed to accept foreign genes as large as 7.5kb in size, and live adenovirus is a safe human vaccine organism. Adeno-associated viruses have also been used in human therapy according to the present invention (Samulski, R.J., et al, EMBO J.10: 3941 (1991)).

Another vector which is capable of expressing the DNA molecule of the invention and which is useful in the treatment of the invention, particularly in human treatment, is vaccinia virus, which can be made non-replicative (U.S. Pat. No. 5,225,336; 5,204,243; 5,155,020; 4,769,330; Sutter, G et al, Proc. Natl. Acad. Sci USA (1992) 89: 10847-10851; Fuerst, T.R. et al, Proc. Natl. Acad. Sci. USA (1989) 86: 2549-2553; Falkner F. G. et al; Nucl. acids Res (1987) 15: 7192; Chakrabarti, S et al, Mo. cell. biol. (1985) 5: 3403-3409). Recombinant vaccinia viruses and other viruses containing heterologous DNA and their use in immunization and DNA therapy are described in: moss, b., curr, opin, genet, dev, (1993) 3: 86-90; moss, b.biotechnology (1992) 20: 345-; moss, b., Curr Top Microbiol Immunol (1992) 158: 25-38; moss, b., Science (1991) 252: 1662-1667; piccini, a et al, adv. virus Res, (1988) 34: 43-64; moss, B, et al, Gene Amplif Anal (1983) 3: 201-213.

In addition to naked DNA or RNA, or viral vectors, engineered bacteria may also be used as vectors. A large number of bacterial strains include Salmonella (Salmonella), BCG and Listeria Monocytogenes (LM) (Hoiseth & Stocker, Nature 291, 238-.

In addition to virus-mediated In vivo gene transfer, physical methods well known In the art can also be used to transfer DNA directly, including administration of plasmid DNA (Wolff et al, 1990, supra) and particle bombardment-mediated gene transfer (Yang, N. -S., et al, Proc.Natl.Acad.Sci.USA 87: 9568(1990), Williams, R.S. et al, Proc.Natl.Acad.Sci.USA 88: 2726(1991), Zelenin, A.V. et al, FEBS Lett.280: 94(1991), Zelenin, A.V. et al, FEBS Lett.244: 65(1989), Johnson, S.A. et al, In Vitro.cell.biol.27: 11 (1991)). In addition, electroporation, a well-known method for transferring genes into cells in vitro, can also be used to transfer the DNA of the present invention into in vivo tissues (Titomirov, A.V., et al, Biochim. Biophys. acta 1088: 131 (1991)).

"vector-mediated gene transfer" has also been described (Wu, C.H. et al, J.biol.chem.264: 16985 (1989)), Wu, G.Y. et al, J biol.chem.263: 14621(1988), Soriano, P. et al, Proc. Natl.Acad.Sci.USA 80: 7128(1983), Wang, C-Y. et al, Proc. Natl.Acad.Sci.USA 84: 7851 (1982); son, J.M. et al, Willi.chem.267: 963 (1992)). Preferred vectors are targeted liposomes (Nicolau, C. et al, Proc. Natl. Acad. Sci. USA 80: 1068 (1983); Soriano et al, supra), such as immunoliposomes capable of binding an acylated mAb into a lipid bilayer (Wang et al, supra). Polycations such as asialoglycoprotein/polylysine (Wu et al, 1989, supra) can also be used, where the conjugate comprises a molecule that recognizes the target tissue (e.g., asialoglycomucoid in the liver) and a compound that binds DNA to bind the DNA to be transfected. Polylysine is an example of a DNA binding molecule that can bind DNA without damaging it. The conjugate is then conjugated to the plasmid DNA of the invention for transfer.

Plasmid DNA for transfection or microinjection can be prepared by methods well known in the art, such as the method using Quiagen (Quiagen), followed by DNA purification by known methods, such as those enumerated herein.

Furthermore, as mentioned above, the use of transduced B7-DC molecules according to the present invention does not require stable expression. Instead, transient expression of the polypeptide is sufficient for the transduced cell to exert its immunogenic and/or co-stimulatory function.

Having generally described the invention, a better understanding of the invention may be obtained by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the invention unless otherwise specified.

Example I

Materials and methods

Preparation and culture of cells

Female BALB/c mice, 6-12 weeks old, were purchased from NCI and used to prepare DCs and macrophages.

Bone marrow-derived DCs were cultured in RPMI-1640(Gibco BRL) medium supplemented with 5% Fetal Calf Serum (FCS) (Hyclone), penicillin/streptomycin ((JRH Biosciences), gentamicin (Sigma), non-essential amino acids (JRH Biosciences), L-glutamic acid (salt) (JRH Biosciences), sodium pyruvate (S-H Biosciences) as described previously (26)igma), 2-mercaptoethanol (Sigma) and 1000 units/ml of recombinant murine GM-CSF (Immunex). Bone marrow-derived DCs grown for 8 days were stained with monoclonal antibodies by conventional methods. Monoclonal antibodies, 14-4-4s, against MHC class II were purified from the supernatants of the hybridomas. The CTLA4-Ig fusion molecule was friendly provided by Philiam Baldwin, University of John Hopkins (Johns Hopkins University). MHC class I antibody (28-14-8), F4/80 antibody (Cl. A3-1), B7.1 antibody (1G10), B7.2 antibody (GL1), Fc_γAntibodies to RII/III (2.4G2) and Mac-1 (M1/70) were purchased from PharMingen. To prepare the cDNA for detection, cells grown for 8 days were purified by cell sorter using 14-4-4s and CTLA4-Ig by Johns Hopkins University tumor Center (Johns Hopkins University Oncology Center). After sorting II^hiMHC class B7^hiThe purity of the population was 93-98%.

Macrophages derived from bone marrow were cultured in RPMI-1640 medium supplemented with 10% FCS, penicillin/streptomycin, non-essential amino acids, sodium pyruvate, L-glutamic acid (salts), 2-mercaptoethanol, and 250 units/ml recombinant murine M-CSF, as described previously (27), treated with 500 units/ml γ -IFN (Pharmingen) and 5 μ g/ml LPS (Sigma). After stimulation, cell surface expression of MHC class II and B7 was confirmed by flow cytometry (cytometric) analysis after 10 days of culture.

The macrophage lines WEHI-3, RAW264.7, J774.A.1, PU5-1.8 were supplied by doctor Joshua Farber of the National institute of Health, NIAID. Culture was carried out using the culture medium recommended by ATCC.

Heterogeneously mixed lymphocyte reaction

8 days of growth, characterized by II^hiMHC class B7^hiThe BM-derived DCs of (a) were tested for their ability to stimulate allogeneic T cells in MLC. By adding increased amount of BALB/c-stimulated cells to 3X 10⁵MLC reactions were performed in 96-well flat-bottom microplates in allogeneic C57BL/6 lymphocytes. After 3 days of culture, 1. mu. Ci of [ mu ] Ci is added to each well³H]-methyl-thymidine (Amersham)m), culturing for 18h, and evaluating the proliferation of the T cells. Cells were then harvested and radioactivity bound determined using a beta counter (Packard 96).

cDNA subtractive hybridization

Total RNA from sorted DCs and activated macrophages was extracted with TRIZOL (Gibco BRL). Messenger RNA was purified using Oligotex mRNA purification kit (Qiagen). We used a PCR-based SMART cDNA synthesis system (Clonetech) to amplify the cDNA followed by PCR-based subtractive system PCR selection (Clonetech). The reduction was performed according to the manufacturer's protocol. After the last subtractive PCR, the DNA fragments were ligated into plasmid vectors pCR2.1(Invitrogen) or pCR Blunt (Invitrogen). After transformation, each clone was cultured for plasmid DNA amplification and miniprep DNA, and then digested with EcoRI to confirm the presence of the insert. Plasmid dot blots were then performed to confirm that the cDNA clone was dendritic cell specific. The alkali denatured, miniprep DNA was spotted onto a Hybond N + membrane (Amersham) for hybridization with SMART cDNA probes derived from sorted DC or activated macrophages. Random primer labeling (Stratagene Prime-It II) was used³²P labels these cDNA probes, and hybridization and washing are performed as described previously (28). The membrane was exposed to film (Amersham) for 1-2 days and developed.

Dot blot analysis of plasmids

Spotting of alkali denatured miniprep DNA samples onto Hybond N + membranes (Amersham) with SMART from sorted DC or activated macrophagesAnd (3) cDNA probe hybridization. These cDNA probes were labeled with 32P using the random primer labeling method (Stratagene Prime-It II). Hybridization and washing were performed as described previously (cites), and the membranes were exposed to film (Amersham) for 1-2 days and developed.

construction and screening of cDNA library-cloning of B7-DC

Bone marrow-derived DCs were collected after 8 days of growth without sorting. Approximately 20% -40% of these cells express high levels of MHC class II and B7. Extraction of total RNA and purification of polyA RNA were performed as described above. For oligo dT-directed DC library construction, we used the γ ZAP expression cDNA synthesis system (Stratagene). The PCR DNA fragment of B7-DC was labeled as a probe for screening. Membrane transfer, denaturation, renaturation were performed according to the Stratagene protocol. Radiolabelling, hybridization, washing and autoradiography of the probes were performed as above. Positive clones were isolated and screened twice. After secondary screening, plasmids were excised by in vivo excision and detected by dot blot and sequencing. Sequencing was performed by Core Facility at the Johns Hopkins University medical School (Johns Hopkins University School of Medicine). Homology searches of nucleic acid sequences to determine their similarity to previously reported genes were performed using the BLAST program in Genbank (NCBI). From the DC cDNA library, a full-length B7-DC cDNA clone was isolated. 5' -RACE was performed using SMART RACE cDNA amplification kit (Clontech). The 5' -RACE product was cloned into pCR2.1 vector and sequenced. Two more than full-length B7-DCs were obtained by RT-PCR and their sequences were compared to avoid sequence errors.

Human B7-DC was cloned as follows: normal peripheral blood mononuclear cells were cultured in GM-CSF + IL-4 or GM-CSF + Flt-3L as described previously (29), from which human DCs were obtained. RNA was extracted as above. BLAST searches identified overlapping EST clones with GenBank accession No. AK001879, which are homologous to murine B7-DC. 5' RACE was performed as above. We sequenced a 5 '-RACE PCR fragment and designed a primer corresponding to the 5' -UTR of human B7-DC. The following primers located in the 5 '-UTR and 3' -UTR of B7-DC were used to amplify full-length human B7-DC:

5'-GGAGCTACTGCATGTTGATTGTTTTG-3' [ SEQ ID NO: 6] and

5’-TGCAAACTGAGGCACTGAAAAGTC-3’[SEQ ID NO：7]

full-length cDNA sequences of human and murine B7-DC cDNAs have been deposited at EMBL/GenBank/DDBS under accession numbers AF329193 and AF142780.

Screening/genomic cloning and mapping of BAC (129SVJ) library

BAC library screening was performed according to the manufacturer's protocol (Genome Systems, Inc.). The primers used were:

5'-TTGTTGTCTCCTTCTGTCTCCCAAC-3' [ SEQ ID NO: 8] and

5’-ACAGTTGCTCCTTGTATCAGGTTC-3’[SEQ ID NO：9]

the BAC library was screened to obtain 3 positive clones. Chromosome mapping was performed by fluorescence in situ hybridization (Genome Systems Inc.). A total of 80 metaphase cells were analyzed, 79 showed specific markers. The human B7-DC gene was located by using commercially available bioinformatics tools, the BLAST program of NCBI and the International RH Mapping protocol (Mapping Consortium). The hB7-DC sequence was searched in htsg and found to map to two BAC clones RP11-574F11(AL162253) and Rp11-635N21(AL354744) located on chromosome 9.

Northern blotting of viruses

Female Balb/c mice, 4-6 weeks old, were purchased from NCI and used to prepare tissue RNA. RNA extraction and tissue SMART cDNA synthesis, sorting DCs and activated macrophages were performed as described above. SMART PCR cDNA was purified using a PCR purification kit (Qiagen). 0.5. mu.g/lane of purified DNA was run on a 1% agarose gel and transferred to Nytran nylon membranes (Schleier and Schuell). To prepare the radioactive probes, we performed amplification using plasmid DNA from the subtractive library as a template. We amplified the DNA by PCR using a primer pair adjacent to the cloning site of the plasmid DNA, and purified PCRDNA of each clone was used as a hybridization probe. The nucleotide sequences of these primers are as follows:

5'-GTAACGGCCGCCAGTGTGCTG-3' [ SEQ ID NO: 10] and

5’-CGCCAGTGTGATGGATATCTGCA-3’[SEQ ID NO：11]

viral Northern analysis of total RNA from human DCs and control placenta was also performed. The probes and RNA used were prepared as described above. Radiolabelling, hybridization, washing and autoradiography of the probes was also performed as described above.

Hamster anti-mB 7-DC Ab product

The stable B7-DC transfectant from DC2.4, RAW246.7 and RENCA cell lines was used to immunize U.S. hamsters. B7-DC was cloned into a modified pCAGGS vector (30). The hamster was boosted three times with a plasmid containing B7-DC (Rockland). The anti-B7-DC antibody used in this study was derived from serum from one of three immunized hamsters.

CD28-Ig, CTLA4-I and PD-1-Ig binding assays

293T cells were transfected with B7.1-pCAGGS, B7-DC-pCAGG, PD-1-pCAGGS or a separate vector using lipofectamine 2000(Gibco BRL). 24h later, in FACS buffer (1 XHBSS, 2% calf serum, 10mM HEPES and 0.1% NaN)₃) The cells were resuspended and spun at 1000rpm for 5 minutes at 4 ℃. The buffer was then decanted, the antibody added to the tube, incubated for 20 minutes at 4 ℃ and washed twice with FACS buffer, and the procedure repeated for secondary antibodies. Out-of-pattern on FACScan. The B7.1 antibody was diluted 1: 5, 10. mu.l/sample (Gal-Tag). Recombinant CD28-Ig, CTLA-4-Ig and PD-1-Ig chimeras were used at concentrations of 2. mu.g/ml and 10. mu.l/sample, respectively (R)&D systems, Inc). Sheep F (ab')₂Anti-human IgG-PE was used at a 1: 20 dilution (Southern Biotechnology Associates, Inc.).

Synthesis of B7-DC-Ig dimer

By culturing in pIg-Tail Plus vector (R)&D systems) fusion of the sequence encoding the N-terminal amino acid of B7-DC and the sequence encoding human IgG₁Fc C-terminal amino acid sequence A B7-DC-Ig construct was prepared in which the N-terminal amino acid of B7-DC did not contain an in-frame transmembrane domain. Lipofectamine 2000(Gibco BRL) or GENE JAMMER (Stratagene) was used,COS-7 cells were transiently transfected with pIg/B7-DC. The B7-DC-Ig fusion protein was purified from serum-free supernatant by saturated ammonium sulfate precipitation. SDS-PAGE and silver staining confirmed the purity > 90%.

T cell proliferation and cytokine assay

For co-stimulation assay against CD3, 96-well flat-bottom plates (Immulon 4 from Dynex) were treated with anti-CD 3 antibody (2C11, Pharmingen) and B7.1-Ig (R)&D System), and 100ng/ml B7-DC-Ig or isotype control (Sigma) diluted in 1 XPBS (Gibco) pH7.4 were pre-coated for 2 hours at 37 ℃. The plates were then washed 3 times with 1 × PBS, blocked with RPMI1640 medium supplemented with 10% FCS, penicillin/streptomycin, non-essential amino acids, sodium pyruvate, L-glutamic acid (salts), 2-mercaptoethanol for half an hour, and T cells added. Spleen and lymph nodes were obtained from BALB/c mice 6-10 weeks old. RBC were lysed with ACK buffer and T cells were purified using dynabeads M-280(Dynex) using an indirect method. The beads were washed twice with PBS pH7.4+ 1% FCS, and cells were added to the beads, which were washed with anti-IE^dAnd B220/CD45RO or CD8 α (Pharmingen) were added to the cells with two-way mixing and incubated at 4 ℃ for 30'. The tube was placed in Dynal MPC 5 'to separate the cells, centrifuged 5' at 1500rpm, and washed twice with 2 XPBS, pH7.4+ 1% FCS to remove unbound Ab. With incubation 15', at 2X 10⁵The same procedure was repeated with cells/well seeded with cells. After 72h incubation, 10. mu.l were added³H-thymidine (1. mu. Ci/well) was added to each well and incubated for 18H. Cells were harvested using a Packard Micromate cell harvester and the filtrate was read on a Packard Matrix 96 direct beta counter.

For costimulation experiments with the RENCA system presenting HA antigens, RENCA cells were cultured in RPMI-1640 medium supplemented with 10% FCS, penicillin/streptomycin, non-essential amino acids, sodium pyruvate. Cells were induced with IFN- γ (75U/ml) for 72hr to allow MHC class II expression. Then irradiated with 13,200 rads to 2X 10⁴Cells/well (96-well flat bottom plate) were seeded. Then, 2.5. mu.g/well of HA110-120 peptide and different concentrations of Ig fusion molecules were added to the wells. As aboveThe isolated transgene I-E^d+ HA-specific T cells (H.von Boehmer hefei, Harvard university) at 4X 10⁵Cell/well seeding. After 48hr incubation, 10. mu.l of the suspension was added³H-thymidine (1. mu. Ci/well) was added to each well and incubated for 18 hrs. Cells were harvested using a Packard Micromate cell harvester and the filtrate was read on a Packard Matrix 96 direct beta counter.

For analysis of cytokine products by ELISA, cultures were performed as described above and supernatants were collected at the indicated times. The concentrations of IL-2, IL-10 and IFN-. gamma.were determined using commercially available ELISA kits (Endogen) and IL-4 and IL-6(R & D systems).

In vivo co-stimulation

LNs derived from mixed axillary, inguinal, cervical and mesenteric membranes of TCR transgenic murine line 6.5 were dissociated in RPMI medium (GIBCOBRL), passed through a 100 μm nylon cell filter and washed in sterile Hank's buffer (GIBCO BRL), wherein transgenic murine line 6.5 expresses an identity I-E in B10.D2 genetic background^dEpitope (¹¹⁰SFERFEIFPKE¹²⁰[SEQ ID NO：12]) The TCR of (1). FACSStaining to determine the rate of CD4 cells of a typical clone, 2.5X 10 in 0.2ml sterile Hank's buffer⁶A cell preparation of representative clonal cells was injected intravenously (i.v.) into the tail vein of recipient b10.d2 mice. 3 days after this transfer, the animals were immunized by subcutaneous (s.c) injection into the hind paw (hind paw). Each mouse received a bilateral injection of one of three formulations:

(A) 10 μ g of synthetic HA (per footpad) (HA peptide (110-120)) conjugated to Incomplete Freund's Adjuvant (IFA) (Sigma) in a volume ratio of 1: 1,

(B) HA-IFA mixture containing 25 μ g B7-DC-Ig, or

(C) HA-IFA mixture containing 25. mu.g isotype control antibody. Harvesting shed LN nodes 7 days later; 1.5X 10⁵LN cells were cultured in round bottom 96-well tissue culture plates with the indicated concentrations of synthetic HA peptide. By using 1. mu. Ci [ alpha ], [ alpha ]³H]Cultures were pulsed for 48 hours with thymidine and harvested after 12 hours of culture and the amount of bound radioactivity was measured for proliferation assays.

Example II

Identification and characterization of B7-DC

B7-DC were isolated from a subtractive library between DC and activated macrophages. The two cell populations used for cDNA subtraction were bone marrow-derived GM-CSF-cultured DCs as the "test" population and adhesive bone marrow-derived M-CSF macrophages activated by gamma-interferon + LPS as the "driver" population. Growth for 8 days II^hiMHCB-like 7^hi"mature" DCs were sorted to > 93% purity for use as a source of test cDNA. DCs were characterized by flow cytometry as having approximately 50-fold higher MHC class II levels than macrophages. Both populations express B7.1 and B7.2, but the level of B7.2 is much higher in DCs. F4/80 and CD16 were expressed at higher levels in the macrophage population. A functional comparison of these two cell populations demonstrated that the DC population was approximately 100-fold more effective than the macrophage population in stimulating an allogeneic mixed lymphocyte response.

After extracting RNA from both populations, we used a PCR-based cDNA synthesis system followed by a PCR-based subtraction step, PCR screening. We named one of the differentially expressed clones, which encoded a novel immunoglobulin supergene family member, B7-DC. The mouse B7-DCcDNA was-1.7 kb in length, encoding a 247 amino acid (aa) precursor protein containing a 23aa N-terminal signal peptide, with a predicted molecular weight of-25 kd (Table 1). The deduced leader sequence and transmembrane domain were identified using the SOSUI program (31). Two charged amino acids were found in the 23aa transmembrane domain of mB7-DC, indicating that this is a binding partner. At the amino acid level, mouse mB7-DC has 70% homology with human B7-DC, indicating that they are orthologs (see Table below). hB7-DC was slightly different from mouse B7-DC because it had a longer cytoplasmic tail.

By homology search, B7-DC was found to have high homology with B7-H1 (34% identity, 48% similarity) (table 2), a lower content of cremophil protein (30% identity, 45% similarity), and < 20% identity with B7.1 and B7.2 (table 3). Phylogenetic studies have shown that the milk fat-like proteins may be linked to the B7 family by exon shuffling (32, 33). They each contain the typical IgV-IgC structure and the transmembrane domain. In contrast to other B7 family members, mouse B7-DC has a very short cytoplasmic tail (4 aa).

TABLE 1

Amino acid sequences of mouse B7-DC and human B7-DC were compared. The upper line marks the mB7-DC deduced leader and transmembrane domains. Comparison using Clustalw Gonnet Pam250 matrix, [ ] indicates identical amino acids, [: conservative substitutions are indicated, and cysteine residues important for disulfide bond formation in the V or C region of an immunoglobulin are in italics.

TABLE 2

Amino acid sequence comparison of mB7-DC and mB7-H1

TABLE 3

Amino acid sequence comparison of B7-DC and B7 family members

Comparison with NCBI blast2 search (matrix BLOSUM62)

1. Amino acids identical at the corresponding sites

2. Amino acids that are similar at the corresponding sites-are grouped as follows: (A, G); (S, T); (E, D); (R, K, H); (Q, N); (V, I, L, M); (Y, F); (W); (C) (ii) a (P)

3. No significant similarity was found with the matrix BLOSUM62

BT ═ butter fat protein

5.＝PDL-1

To determine the genomic structure of mB7-DC, the present invention isolated a genomic clone by screening a mixed Bacterial Artificial Chromosome (BAC) library with 5 'and 3' UTR probes. BAC clones were used for chromosome mapping. Chromosomal mapping of B7-DC was performed using Fluorescence In Situ Hybridization (FISH). Determination of 10 specifically labeled chromosomes 19 confirmed that mB7-DC was located at 47% of the distance between the heterochromatin-euchromatin boundary and the telomere of chromosome 19, the corresponding region at the band 19C2 and 19C3 interfaces. Hybridization slides were incubated with fluorescein-bearing anti-digoxigenin antibodies and the spots stained with DAPI were counted to detect specific hybridization signals. This locus corresponds to the region of human chromosome 9 where hB7-H1 is located.

hB7-DC was found to be located on two chromosome 9 BAC clones. In addition, both hB7-DC and hB7-H1 were found to be located on a single chromosome 9 BAC clone with an insert size of about 164kb (FIG. 1). The proximity of the B7-DC and B7-H1 genomes reminds the B7.1/B7.2 pairs, which are located within 1 megabase-pair of each other.

Example III

Selective expression of B7-DC in dendritic cells

To determine the expression pattern of B7-DC,viral Northern analysis was performed using RNA extracted from various tissues, macrophage lines, macrophage cultures, and dendritic cells from bone marrow and spleen. Immature (4, 6 days) and mature (8 days and sorted MHCII) using B7-DC probe^hiB7^hi) Strong hybridization was detected in bone marrow-derived DCs and splenic DCs, whereas no signal was detected in any of the 4 macrophage lines, activated BM macrophages or peritoneal macrophages (figure 2). Strong expression of hB7-DC was detected in human DCs grown from peripheral blood mononuclear cells cultured with GM-CSF supplemented with IL-4 or Flt-3L (FIG. 3). To examine cell surface expression of the B7-DC protein, DCs were stained with anti-mB 7-DC antibody. Blocking with soluble B7-DC-Ig resulted in staining being found on DC (FIG. 4).

B7-DC did not bind to CD28 or CTLA-4, but bound to PD-1

Although B7-DC has a structure and sequence homologous to family B7, it does not contain the putative CD28/CTLA-4 binding sequence, sqdxxely [ SEQ ID NO: 13] or XXXYXXRT [ SEQ ID NO: 14] (34) (wherein X ═ any amino acid). To directly assess binding, the ability of dimers CD28-Ig and CTLA-4-Ig to stain 293T cells transfected with B7-DC or B7.1 was investigated. The B7.1 transfectants found strong binding, whereas the B7-DC transfectants did not (FIG. 5). Based on homology (homlogy) and genomic proximity of B7-DC and B7-H1/PD-L1, experiments were performed to test PD-1 as a candidate binding partner for B7-DC. Indeed, PD-100IG binds to the B7-DC transfectant, but not to the B7.1 transfectant. BPD-1-Ig bound less well to the B7-DC transfectant than CTL-4-It and CD28-Ig bound to the B7.1 transfectant, although It was specific. Positive staining of the stable B7-DC-GFP transfectant with PD-1-Ig further confirmed that PD-1 was able to bind to B7-Dc. It can therefore be concluded that B7-DC does not have CD28 or CTLA-4 as receptors, as do B7-H1 and B7H/B7 RP-1. In contrast, PD-1 appears to be a receptor for B7-DC.

Example IV

Function of B7-DC as a T cell costimulatory molecule

Soluble B7-DC-Ig fusion proteins were prepared for addition to T cell stimulation assays to test whether B7-DC has co-stimulatory activity. The proliferative response of T cells to stimulation was measured by increasing the amount of immobilized anti-CD 3 in the presence of B7-DC-Ig, B7.1-Ig or an isotype control. Figure 6 (left panel) shows that B7-DC co-stimulated T cell proliferative responses greater than B7.1 in the presence of suboptimal concentrations of anti-CD 3. Moreover, the proliferative response of B7-DC co-stimulation was higher in CD4 cells than in CD8 cells (figure six right). In the absence of the TCR-focused stimulus, B7-DC failed to stimulate T cells, indicating that B7-DC provided a true costimulatory signal.

B7-DC also co-stimulates a proliferative response when the MHC peptide complex produces "Signal 1". RENCA cells (which do not express endogenous B7.1, B7.2 or B7-DC as analyzed by RT-PCR) were treated with γ -IFN to induce MHC class II expression. These cells carry I-E^dThe restriction HA110-120 peptide (FERFEIFPKE) (35) [ SEQ ID NO: 15]. Adding from I to E^d+ HA110-120 specific TCR transgenic mouse line in the presence of B7-DC-Ig, B7.1-Ig or isotype control in purified splenic T cells determination of proliferative response. FIG. 7 shows that B7-DC has greater co-stimulatory activity than B7.1.

B7-DC co-stimulation of lymphokine production

The best characterized T cell response to costimulation by B7 family molecules is the production of lymphokines. These lymphokines are important T cell effector mediators. Studies have analyzed the production of many different lymphokines by T cells that have been stimulated by anti-CD 3 or HA antigens (FIG. 8) and co-stimulated by B7-DC-Ig, B7.1-Ig, or isotype controls. The mode of lymphokine co-stimulation is quite consistent, whether anti-CD 3 or MHC peptide complex is used as "signal 1". Clearly, B7-DC co-stimulated higher levels of γ -IFN than B7.1. B7-DC also co-stimulated production of large amounts of IL-6, whereas B7.1 was virtually completely unproductive. When two molecules co-stimulate IL-2 production, B7.1 is more potent than B7-DC. Thus, the way in which B7-DC and B7.1 co-stimulate is different, B7-DC is more effective in co-stimulating the important pro-inflammatory lymphokines.

Example V

B7-DC enhances immune response in vivo

To determine whether B7-DC is biologically active in vivo, the inventors investigated whether B7-DC-Ig could enhance the immune response to peptide vaccines. B7-DC-Ig or an isotype control antibody was added to the immunogenic mixture of HA110-120 peptide and IFA. To count HA-specific CD4T cells in vivo, 2.5 × 10 cells were plated 3 days prior to immunization⁶anti-HA 6.5T cells were transferred to mice. 7 days after immunization, the shed LN cells were harvested and stimulated with varying amounts of HA110-120 peptide for 2 days in vitro. FIG. 9 shows that the addition of B7-DC-Ig actually dramatically increased the proliferative response to HA. The total number of HA-specific T cells in the shed LN increased approximately 2-fold in the group to which B7-DC-Ig was added, relative to isotype antibody control. Thus, it can be concluded that B7-DC has the ability to enhance antigen-specific responses, even on a per-cell basis.

Example VI

Discussion and conclusions

The present inventors have discovered and characterized a novel B7 family member, the expression of which is highly restricted in DCs and which has unique T cell co-stimulatory properties. The human ortholog of B7-DC was also expressed in DC.

Unlike previously described B7 family members, this restricted expression pattern suggests that B7-DC are involved in an immune response distinct from the known B7.1/2 pathway. When a weak B7-DC signal was detected in activated macrophages, preliminary real-time RT-PCR analysis performed showed that B7-DC mRNA was expressed in DC > 15 fold higher than in activated macrophages. Also, very low levels of B7-DC were detected on the surface of activated macrophages using antibody staining techniques. It is unclear whether this is sufficient for efficient T cell activation.

The unusual way in which B7-DC co-stimulate lymphokine production compared to other B7 family members suggests a unique biological effect. The traditional classification of cytokines is as follows: th1 cytokines include IL-2, gamma-IFN and lymphotoxin; th2 cytokines include IL-4, IL-5, IL-6 and IL-13 (36). B7-DC did not induce any kind of Th1 or Th2 lymphokine profile (profile). B7-DC induced very little IL-4 and no IL-10. However, IL-6 is considered to be a Th2 cytokine. The lower IL-2 and higher γ -IFN co-stimulation by B7-DC did not meet the standard Th1 pattern relative to B7.1. However, the high γ -IFN production indicates that B7-DC elicits important T cell effector functions.

The ability of B7-DC to co-stimulate IL-6 is noteworthy. The strong T cell proliferative response induced by B7-DC was due in part to its strong co-stimulation of IL-6 production, which was not observed with B7.1. IL-6 is a potent expansion agent of T cell proliferation (in combination with other proliferation stimulators) (37, 38). IL-6 is a multifunctional cytokine that regulates not only T cell function, but also pro-inflammatory responses, monocyte differentiation, B cell differentiation, thrombosis, bone resorption, and growth of certain hematopoietic tumors (39, 40). IL-6 cooperates with the soluble IL-6 receptor (sIL-6R) to induce chemokine and leukocyte recruitment (41). It can mediate potent anti-apoptotic effects through Stat-3 activation. It has been reported that Stat-3-dependent activation of IL-6 in T cells is an important pathway for activated T cell survival (42, 43), although other reports speculate that Stat-3 plays a role in resting T cells.

Although B7-DC does not bind to CD28 or CTLA-4, it binds to one receptor of PD-1, B7-H1/PD-L1 (22, 47, 48). It has not been determined whether it binds to one receptor (23-25, 44-46) -ICOS of B7h/B7 RP-1. The apparent homology between B7-DC and B7-H1/PDL-1 (higher than between B7.1 and B7.2), the similar physical linkage of hB7-H1/PD-L1 to hB7-DC and their binding to common receptors suggest that they are linked by a relatively recent gene replication event. This is similar to the relationship between B7.1 and B7.2, with both B7.1 and B7.2 mapping to within one megabase pair of murine chromosome 16 and human chromosome 3 (49).

It is clear that the relative biological effects of B7-DC on B7-H1/PD-L1 are important when PD-1 and other putative receptors mediate. PD-1 is expressed after T cell activation and appears to inhibit T cell activation. PD-1 caused apoptosis in T cells stimulated with high concentrations of anti-CD 3. PD-1 can knock out mice to develop autoimmune syndrome (22), which is characterized by the clinical manifestations of myocardial hypertrophy. In contrast, Dong et al (21) reported that B7-H1/PD-L1 co-stimulated T cell proliferation and cytokine release at lower concentrations of anti-CD 3. By analogy with the CD28/CTLA-4 relationship, PD-L1 may be a counter-receptor to the still unidentified active receptor. Despite the ability to bind PD-1, B7-DC and B7-H1 differ in their lymphokine co-stimulatory patterns; B7-H1 co-stimulated T cell IL-10 production, whereas B7-DC did not. The unique cellular expression pattern and costimulatory effect of B7-DC suggests a unique role in immune response.

The references cited above and below are incorporated herein by reference in their entirety, whether or not specifically incorporated.

Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without undue experimentation, without departing from the spirit and scope of the invention.

While the invention has been described in conjunction with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.

Article of referenceDocument (A)

Some documents are cited by numerals only (additional description), except for documents cited in their entirety in the text; the latter is listed below:

1.Hathcock，K.S.，G.Laszlo，C.Pucillo，P.Linsley，and R.J.Hodes.1994.Comparative analysis of B7-1 and B7-2 costimulatory ligands：expression and function.J Exp Med 180，no.2：631.

2.Razi-Wolf，Z.，G.J.Freeman，F.Galvin，B.Benacerraf，L.Nadler，and H.Reiser.1992.Expression and function of the murine B7 antigen，the major costimulatory molecule expressed by peritoneal exudate cells.Proc Natl Acad Sci USA 89，no.9：4210.

3.Steinman，R.M.1991.The dendritic cell system and its role in immunogenicity.Annu Rev Immunol 9：271.

4.Banchereau，J.，and R.M.Steinman.1998.Dendritic cells and the control of immunity.Nature 392，no.6673：245.

5.Patterson，S.2000.Flexibility and cooperation among dendritic cells.Nature Immunol.1，no.4：273.

6.Langenkamp，A.，M.Messi，A.Lanzavecchia，and S.Federica.2000.Kinetics of dendritic cell activation：impact on priming of Th1，Th2 and nonpolarized T cells.Nature Immunol.1，no.4：311.

7.Thompson，C.B.，T.Lindsten，J.A.Ledbetter，S.L.Kunkel，H.A.Young，S.G.Emerson，J.M.Leiden，and C.H.June.1989.CD28 activation pathway regulates the production of multiple T-cell-derived lymphokines/cytokines.Proc Natl Acad Sci USA 86，no.4：1333.

8.Harding，F.A.，J.G.McArthur，J.A.Gross，D.H.Raulet，and J.P.Allison.1992. CD28-mediated signalling co-stimulates murine T cells and prevents induction of anergy in T-cell clones.Nature 356，no.6370：607.

9.Linsley，P.S.，W.Brady，L.Grosmaire，A.Aruffo，N.K.Damle，and J.A.Ledbetter.1991.Binding of the B cell activation antigen B7 to CD28 costimulates T cell proliferation and interleukin 2mRNA accumulation.J Exp Med 173，no.3：721.

10.Fraser，J.D.，B.A.Irving，G.R.Crabtree，and A.Weiss.1991.Regulation of interleukin-2 gene enhancer activity by the T cell accessory molecule CD28.Science 251，no.4991：313.

11.Lindstein，T.，C.H.June，J.A.Ledbetter，G.Stella，and C.B.Thompson.1989.Regulation of lymphokine messenger RNA stability by a surface-mediated T cell activation pathway.Science 244，no.4902：339.

12.Linsley，P.S.，W.Brady，M.Urnes，L.S.Grosmaire，N.K.Damle，and J.A.Ledbetter.1991.CTLA-4 is a second receptor for the B cell activation antigen B7.J Exp Med 174，no.3：561.

13.Waterhouse，P.，L.E.Marengere，H.W.Mittticker，and T.W.Mak.1996.CTLA-4，a negative regulator of T lymphocyte activation.Immunol Rev 153：183.

14.Kuchroo，V.K.，M.P.Das，J.A.Brown，A.M.Ranger，S.S.Zamvil，R.A.Sobel，H.L.Weiner，N.Nabavi，and L.H.Glimcher.1995.B7-1 and B7-2 costimulatory molecules activate differentially the Th1/Th2 developmental pathways：application to autoimmune disease therapy.Cell 80，no.5：707.

15.Lanier，L.L.，S.O’Fallon，C.Somoza，J.H.Phillips，P.S.Linsley，K.Okumura，D.Ito，and M.Azuma.1995.CD80(B7)and CD86(B70)provide similar costimulatory signals for T cell proliferation，cytokine production，and generation of CTL.J Immunol 154，no.1：97.

16.Van Parijs，L.，M.P.Sethna，A.N.Schweitzer，F.Borriello，A.H.Sharpe，and A.K.Abbas.1997.Functional consequences of dysregulated B7-1(CD80)and B7-2(CD86)expression in B or T lymphocytes of transgenic mice.J Immunol 159，no.11：5336.

17.Abbas，A.K.，and A.H.Sharpe.1999.T-cell stimulation：an abundance of B7s[news；comment].Nat Med 5，no.12：1345.

18.Borriello，F.，M.P.Sethna，S.D.Boyd，A.N.Schweitzer，E.A.Tivol，D.Jacoby，T. B.Strom，E.M.Simpson，G.J.Freeman，and A.H.Sharpe.1997.B7-1 and B7-2 have overlapping，critical roles in immunoglobulin class switching and germinal center formation.Immunity 6，no.3：303.

19.Sharpe，A.H.1995.Analysis of lymphocyte costimulation in vivo using transgenic and ‘knockout’mice.Curr Opin Immunol 7，no.3：389.

20.Green，J.M.，P.J.Noel，A.I.Sperling，T.L.Walunas，G.S.Gray，J.A.Bluestone，and C.B.Thompson.1994.Absence of B7-dependent responses in CD28-deficient mice.Immunity 1，no.6：501.

21.Dong，H.，G.Zhu，K.Tamada，and L.Chen.1999.B7-H1，a third member of the B7 family，co-stimulates T cell proliferation and interleukin-10 secretion [see comments].Nat Med 5，no.12：1365.

22.Freeman，G.J.，A.J.Long，Y.Iwai，K.Bourque，T.Chernova，H.Nishimura，L.J.Fitz，N.Malenkovich，T.Okazaki，M.C.Byrne，H.F.Horton，L.Fouser，L.Carter，V.Ling，M.R.Bowman，B.M.Carreno，M.Collins，C.R.Wood，and T.Honjo.2000.Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation.J Exp Med 192，no.7：1027.

23.Swallow，M.M.，J.J.Wallin，and W.C.Sha.1999.B7h，a novel costimulatory homolog of B7.1 and B7.2，is induced by TNFalpha.Immunity 11，no.4：423.

24.Wang，S.，G.Zhu，A.I.Chapoval，H.Dong，K.Tamada，J.Ni，and L.Chen.2000.Costimulation of T cells by B7-H2，a B7-like molecule that binds ICOS.Blood 96，no.8：2808.

25.Yoshinaga，S.K.，J.S.Whoriskey，S.D.Khare，U.Sarmiento，J.Guo，T.Horan，G.Shih，M.Zhang，M.A.Coccia，T.Kohno，A.Tafuri-Bladt，D.Brankow，P.Campbell，D.Chang，L.Chiu，T.Dai，G.Duncan，G.S.Elliott，A.Hui，S.M.McCabe，S.Scully，A.Shahinian，C.L.Shaklee，G.Van，T.W.Mak，and et al.1999.T cell co-stimulation through B7RP-1 and ICOS.Nature 402，no.6763：827.

26.Inaba，K.，W.J.Swiggard，R.Steinman，N.Romani，and G.Schuler.1998.Isolation of dendritic cells.John Wiley & Sons，Inc.，，New York.

27.Fortier，A.H.，and L.A.Falk.1994.Isolation of murine macrophages.John Wiley & Sons，Inc.，New York.

28.Orimoto，K.，H.Tsuchiya，J.Sakurai，M.Nishizawa，and O.Hino.1998.Identification of cDNAs induced by the tumor suppressor Tsc2 gene using a conditional expression system in Tsc2 mutant (Eker)rat renal carcinoma cells.Biochem Biophys Res Commun 247，no.3：728.

29.Romani，N.，S.Gruner，D.Brang，E.Kampgen，A.Lenz，B.Trockenbacher，G.Konwalinka，P.O.Fritsch，R.M.Steinman，and G.Schuler.1994.Proliferating dendritic cell progenitors in human blood.J Exp Med 180，no.1：83.

30.Niwa，H.，K.Yamamura，and J.Miyazaki.1991.Efficient selection for high-expression transfectants with a novel eukaryotic vector.Gene 108，no.2：193.

31.Hirolawa，T.，S.Boon-Chieng，and S.Mitaku.1998.SOSUI：Classification and secondary structure prediction system for membrane proteins.Bioinformatics 14，no.4：378.

32.Linsley，P.S.，R.Peach，P.Gladstone，and J.Bajorath.1994.Extending the B7(CD80)gene family.Protein Sci 3，no.8：1341.

33.Henry，J.，M.Ribouchon，D.Depetris，M.Mattei，C.Offer，R.Tazi-Ahnini，and P.Pontarotti.1997.Cloning，structural analysis，and mapping of the B30 and B7 multigenic families to the major histocompatibility complex(MHC)and other chromosomal regions.Immunogenetics 46，no.5：383.

34.Fargeas，C.A.，A.Truneh，M.Reddy，M.Hurle，R.Sweet，and R.P.Sekaly.1995.Identification of residues in the V domain of CD80(B7-1)implicated in functional interactions with CD28 and CTLA4.J Exp Med 182，no.3：667.

35.Kirberg，J.，A.Baron，S.Jakob，A.Rolink，K.Karjalainen，and H.von Boehmer.1994.Thymic selection of CD8+ single positive cells with a class II major histocompatibility complex-restricted receptor.J Exp Med 180，no.1：25.

36.Mosmann，T.R.，and R.L.Coffman.1989.TH1 and TH2 cells：different patterns of lymphokine secretion lead to different functional properties.Annu Rev Immunol 7：145.

37.Suda，T.，A.O’Garra，I.MacNeil，M.Fischer，M.W.Bond，and A.Zlotnik.1990.Identification of a novel thymocyte growth-promoting factor derived from B cell lymphomas.Cell Immunol 129，no.1：228.

38.Suda，T.，R.Murray，C.Guidos，and A.Zlotnik.1990.Growth-promoting activity of IL-1 alpha，IL-6，and tumor necrosis factor-alpha in combination with IL-2，IL-4，or IL-7 on murine thymocytes.Differential effects on CD4/CD8 subsets and on CD3+/CD3-double-negative thymocytes.J Immunol 144，no.8：3039.

39.Taga，T.，and T.Kishimoto.1997.Gp130 and the interleukin-6 family of cytokines.Annu Rev Immunol 15：797.

40.Chomarat，P.，J.banchereau，J.Davoust，and A.K.Palucka.2000.IL-6 switches the differentiation of monocytes from dendritic cells to macrophages.Nature Immunol 1，no.6：510.

41.Romano，M.，M.Sironi，C.Toniatti，N.Polentarutti，P.Fruscella，P.Ghezzi，R.Faggioni，W.Luini，V.Van Hinsbergh，S.Sozzani，F.Bussolino，V.Poli，G.Ciliberto，and A.Mantovani.1997.Role of IL-6 and its soluble receptor in induction of chemokines and leukocyte recruitment.Immunity 6，no.3：315.

42.Takeda，K.，T.Kaisho，N.Yoshida，J.Takeda，T.Kishimoto，and S.Akira.1998.Stat3 activation is responsible for IL-6-dependent T cell proliferation through preventing apoptosis：generation and characterization of T cell-specific Stat3-deficient mice.J Immunol 161，no.9：4652.

43.Teague，T.K.，B.C.Schaefer，D.Hildeman，J.Bender，T.Mitchell，J.W.Kappler，and P.Marrack.2000.Activation-induced inhibition of interleukin 6-mediated T cell survival and signal transducer and activator of transcription 1 signaling.J Exp Med 191，no.6：915.

44.Coyle，A.J.，S.Lehar，C.Lloyd，J.Tian，T.Delaney，S.Manning，T.Nguyen，T.Burwell，H.Schneider，J.A.Gonzalo，M.Gosselin，L.R.Owen，C.E.Rudd，and J.C.Gutierrez-Ramos.2000.The CD28-related molecule ICOS is required for effective T cell-dependent immune responses.Immunity 13，no.1：95.

45.Hutloff，A.，A.M.Dittrich，K.C.Beier，B.Eljaschewitsch，R.Kraft，I.Anagnostopoulos，and R.A.Kroczek.1999.ICOS is an inducible T-cell co-stimulator structurally and functionally related to CD28.Nature 397，no.6716：263.

46.Yoshinaga，S.K.，M.Zhang，J.Pistillo，T.Horan，S.D.Khare，K.Miner，M.Sonnenberg，T.Boone，D.Brankow，T.Dai，J.Delaney，H.Han，A.Hui，T.Kohno，R.Manoukian，J.S.Whoriskey，and M.A.Coccia.2000.Characterization of a new human B7-related protein：B7RP-1 is the ligand to the co-stimulatory protein ICOS.Int Irnmunol 12，no.10：1439.

47.Ishida，Y.，Y.Agata，K.Shibahara，and T.Honjo.1992.Induced expression of PD-1，a novel member of the immunoglobulin gene superfamily，upon programmed cell death.Embo J 11，no.11：3887.

48.Shinohara，T.，M.Taniwaki，Y.Ishida，M.Kawaichi，and T.Honjo.1994.Structure and chromosomal localization of the human PD-1 gene (PDCD1).Genomics 23，no.3：704.

49.Reeves，R.H.，D.Patch，A.H.Sharpe，F.Borriello，G.J.Freeman，S.Edelhoff，and C.Disteche.1997.The costimulatory genes Cd80 and Cd86 are linked on mouse chromosome 16 and human chromosome 3.Mamm Genome 8，no.8：581。

Claims

1. A fusion protein comprising a first fusion partner comprising a polypeptide comprising the amino acid sequence of seq id NO: 2, the first fusion partner being (i) fused to a second polypeptide, or (ii) fused to a linker peptide sequence fused to the second polypeptide, or (iii) the B7-DC protein extracellular domain of all or part of the amino acids at about positions 20-221 of said first fusion partner.

2. A variant B7-DC fusion protein comprising a first fusion partner comprising a variant B7-DC extracellular domain, wherein the variant B7-DC extracellular domain comprises a polypeptide having the amino acid sequence of SEQ ID NO: 2, wherein the amino acid sequence of SEQ ID NO: 2 by a different conserved amino acid or by deletion, and said first fusion partner (i) fusing to a second polypeptide, or (ii) fusing to a linker peptide sequence fused to said second polypeptide.