AU772061B2 - B7-2:CTL A4/CD 28 counter receptor - Google Patents

Description

AUSTRALIA

Patents Act 1990 Dana-Farber Cancer Institute

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Invention Title: B7-2: CTL A4/CD 28 counter receptor The following statement is a full description of this invention including the best method of performing it known to us:- Iatc I t3Y I Astralia g"

D

ocumens Dcumt, s received on: 7 BEt 23jL 0 Batch No: Government Funding Work described herein was supported under CA-40216-08 awarded by the National Institutes of Health. The U.S. government therefore may have certain rights in this invention.

Background of the Invention To induce antigen-specific T cell activation and clonal expansion, two signals provided by antigen-presenting cells (APCs) must be delivered to the surface of resting T lymphocytes (Jenkins, M. and Schwartz, R. (1987) J. Exp. Med. 165, 302-319; Mueller, D.L., et al. (1990) J. Immunol. 144, 3701-3709; Williams, I.R. and Unanue, E.R. (1990) J.

Immunol. 145, 85-93). The first signal, which confers specificity to the immune response, is mediated via the T cell receptor (TCR) following recognition of foreign antigenie-peptide presented in the context of the major histocompatibility complex (MHC). The second signal, termed costimulation, induces T cells to proliferate and become functional (Schwartz, R.H.

(1990) Science 248, 1349-1356). Costimulation is neither antigen-specific, nor MHC restricted and is thought to be provided by one or more distinct cell surface molecules expressed by'APCs (Jenkins, et al. (1988) J. Immunol. 140, 3324-3330; Linsley, P.S., et al. (1991)J. Exp. Med. 1, 721-730; Gimmi, et al., (1991) Proc. Natl. Acad. Sci.

USA. 88, 6575-6579; Young, et al. (1992) J. Clin. Invest. 90, 229-237; Koulova, et *al. (1991) J. Exp. Med. 17, 759-762; Reiser, et al. (1992) Proc. Natl. Acad Sci. USA. 89, 271-275; van-Seventer, et al. (1990) J Immunol. 144, 4579-4586; LaSalle, et al., (1991) J. Immunol. 147, 774-80; Dustin, et al., (1989) J. Exp. Med. 169, 503; Armitage, et al. (1992) Nature 357, 80-82; Liu, et al. (1992) J. Exp. Med. 175, 437-445).

25 Considerable evidence suggests that the B7 protein, expressed on APCs, is one such critical costimulatory molecule (Linsley, et al., (1991) J. Exp. Med. 173, 721-730; Gimmi, et al., (1991) Proc. Nail. Acad. Sci. USA. 88, 6575-6579; Koulova, et al., (1991) J. Exp. Med. 173, 759-762; Reiser, et al. (1992) Proc. Natl. Acad. Sci. USA. 89, 271-275; Linsley, P.S. et al. (1990) Proc. Natl. Acad Sci. USA. 87, 5031-5035; Freeman, G.J.

et al. (1991) J. Exp. M.ed. 174,625-631.). B7 is the counter-receptor for two ligands expressed on T lymphocytes. The first ligand, termed CD28, is constitutively expressed on resting T cells and increases after activation. After signaling through the T cell receptor, ligation of CD28 induces T cells to proliferate and secrete IL-2 (Linsley, et al. (1991) J Exp. Med. 73, 721-730; Gimmi. et al. (1991) Proc. Natl. Acad. Sci. USA. 88, 6575- 6579; Thompson, et al. (1989) Proc. Natl. Acad. Sci. USA. 86, 1333-1337; June, C.H., et al. (1990) Immunol. Today. J.L, 211-6; Harding, et al. (1992) Nature. 356, 607-609.).

The second ligand. termed CTLA4 is homologous to CD28 but is not expressed on resting T cells and appears following T cell activation (Brunet, et al., (1987) Nature 32., 267- 270). DNA seauences encoding the human and murine CTLA4 orotein are described in -2- Dariavich, et al. (1988) fur. j Immunol. 1901-1905; Brunet, et al. (1987) supra: Brunet. J.F. et al. (1988) Immunol. Rev. 10:21-36; and Freeman, et al. (1992) J.

Immunol. 192, 3795-3801. Although B7 has a higher affinity for CTLA4 than for CD28 (Linsley, et al., (1991) J. Exp. Med. 174, 561-569), the function of CTLA4 is still unknown.

Te importance of the B7:CD28/CTLA4 costimulatory pathway has been demonstrated in virro and in several in vivo model systems. Blockade of this costimulatory pathway results in the development of antigen specific tolerance in murine and humans systems (Harding, et al. (1992) Nature. 3.6, 607-609; Lenschow, et al. (1992) Science. 257, 789-792; Turka, et al. (1992) Proc. Natl. Acad. Sci. USA. 11102- 11105; Gimmi, et al. 1993) Proc. Natl. Acad. Sci USA 20, 6586-6590; Boussiotis, V..

et al. (1993) J. Ero. Med 178, 1753-1763). Conversely, expression of B7 bv-B7 negative murine tumor cells induces T-cell mediated specific immunity accompanied 5y tmir6 rejection and long lasting protection to tumor challenge (Chen, et al. (1992) Cell 71 1093-1102; Townsend, S.E. and Allison, J.P. (1993) Science 259, 368-370; Baskar, et al.

(1993) Proc. Nat. Acad. Sci. 90, 5687-5690.). Therefore, manipulation of the B7:CD28/CTLA4 pathway offers great potential to stimulate or suppress immune responses in humans.

S 20 Summary of the Invention This invention pertains to isolated nucleic acids encoding novel molecules which costimulate T cell activation. Preferred costimulatory molecules include antigens on the surface of B lymphocytes, professional antigen presenting cells monocytes. dendritic cells. Langerhan cells) and other cells keratinocvtes, endothelial cells, astrocytes, fibroblasts, oiigodendrocytes) which present antigen to immune cells, and which bind either CTLA4, CD28. both CTLA4 and CD28 or other known or as yet undefined receptors on immune cells. Such costimulatory molecules are referred to herein as CTLA4/CD28 binding counter-receptors or B lymphocyte antigens, and are capable of providing costimulation to activated T cells to thereby induce T cell proliferation and/or cytokine secretion. Preferred B lymphocrte antigens include B7-2 and B7-3 and soluble fragments or derivatives thereof which bind CTLA4 and/or CD2 and have the ability to inhibit or induce costimulation of immune cells. In one emoooiment. an isolated nucleic acid which encodes a peptide having the activity of the human B- lymphocyte antigen is provided. Preferabiy, the nucleic acid is a cDNA molecule having a nuceotide seouence encoding human B7-2. as shown in Figure 3 (SEQ ID Ir. another embodiment, the nucleic acid is a cDNA molecule having a nucieotide sequence encoding murine B-2. as shown in Figure 14 (SEQ ID NO:2).

-a The invention also features nucleic acids which encode a peptide havin B7-2 activity and at least about 50%, more preferably at least about 60% and most preferably at least about homologous with an amino acid sequence shown in Figure 8 (SEQ ID NO:2) or an amino acid sequence shown in Figure 14 (SEQ ID NO:23). Nucleic acids which encode peptides having B7-2 activity and at least about 80%, more preferably at least about more preferably at least about 95% and most preferably at least about 98% or at least about 99% homologous with an amino acid sequence shown in Figure 8 (SEQ ID NO:2) or an amino acid sequence shown in Figure 14 (SEQ ID NO:23) are also within the scope of the invention. In another embodiment, the peptide having B7-2 activity is encoded by a nucleic acid which hybridizes under high or Iow stingency conditions to a nucleic acid which encodes a peptide having an amino acid sequence of Figure 8 (SEQ ID NO:2) or a peptide having an amino acid sequence shown in Figure 14 (SEQ ID NO:23).

The invention further pertains to an isolated nucleic acid comprising a nucleotide sequence encoding a peptide having B7-2 activity and having a length of at least 20 amino acid residues. Peptides having B7-2 activity and consisting of at least 40 amino acid residues in length, at least 60 amino acid residues in length, at least 80 amino acid residues in length, at least 100 amino acid residues in length or at least 200 or more amino acid residues in length are also within the scope of this invention. Particularly preferred nucleic acids encode a peptide having B7-2 activity, a length of at least 20 amino acid residues o, more and at least .i 20 50% or greater homology (preferably at least 70%) with a sequence shown in Figure 8 (SEQ ID NO:2).

In one preferred embodiment, the invention features an isolated DNA encoding a peptide having B7-2 activity and an amino acid sequence represented by a formula: Xn-Y-Zm In the formula, Y consists essentially of amino acid residues 24-245 of the sequence shown in Figure 8 (SEQ ID NO:2). X n and Zm are additional amino acid residue(s) linked to Y bv an anide bond. X, and Zm are anino acid residues selected from amino acid residues contiguous to Y in the amino acid sequence shown in Figure 8 (SEQ ID NO:2).

X

n is amino acid residue(s) selected from amino acids contiguous to the amino terminus of Y in the sequenc shown in Figure 8 (SEQ ID NO:2), selected from amino acid residue 23 to 1.

Zm is amino acid residue(s) selected from amino acids contiguous to the carboxv terminus or Y in the sequence shown in Figure 8 (SEQ ID NO:2). sel:ee ':om amino cid rsidue 246 to 329. According to the formula. n is a number from 0 to 23 (n=0-23 and 7m is a number from 0 to 84 A particularly preferred DNA encodes a peptide having an -4amino acid sequence represented by the formula Xn-Y-Zm, where Y is amino acid residues 24-245 of the sequence shown in Figure 8 (SEQ ID NO:2) and n=0 and m=0.

Tne invention also features an isolated DNA encoding a B7-2 fusion protein which includes a nucleotide sequence encoding a first peptide having B7-2 activity and a nucleotide sequence encoding a second peptide corresponding to a moiety that alters the solubility, binding affinity, stability or valency of the first peptide. Preferably, the first peptide having B7-2 activity includes an extracellular domain portion of the B7-2 protein about amino acid residues 24-245 of the sequence shown in Figure 8 (SEQ ID NO:2)) and the second peptide is an immunoglobulin constant region, for example, a human Cyl or Cy4 domain, including the hinge, CH2 and CH3 region, to produce a B7-2 immunoglobulin fusion protein (B7-2Ig)(see Capon et al. (1989) Nature 337, 525-531 and Capon U.S. 5,116,964).

The nucleic acids obtained in accordance with the present invention can be inserted into various expression vectors, which in turn direct the synthesis of the corresp-oding protein or peptides in a variety of hosts, particularly eucaryotic cells, such as mammalian and insect cell culture, and procaryotic cells such as E. coli. Expression vectors within the scope of the invention comprise a nucleic acid encoding at least one peptide having the activity of a novel B lymphocyte antigen as described herein, and a promoter operably linked to the nucleic acid sequence. In one embodiment, the expression vector contains a DNA encoding a peptide having the activity of the B7-2 antigen and a DNA encoding a peptide having the 20 activity of another B lymphocyte antigen, such as the previously characterized B7 activation antigen, referred to herein as B7-1. Such expression vectors can be used to transfect host cells to thereby produce proteins and peptides, including fusion proteins, encoded by nucleic acids as described herein.

Nucleic acid probes useful for assaying a biological sample for the presence of B cells 25 expressing the B lymphocyte antigens B7-2 and B7-3 are also within the scope of the invention.

The invention further pertains to isolated peptides having the activity of a novel B lymphocyte antigen, including the B7-2 and B7-3 protein antigens. A preferred peptide having B7-2 activity is produced by recombinant expression and comprises an amino acid sequence shown in Figure S (SEQ ID NO: Another preferred peptide having B7-2 activity comprises an amino acid sequence shown in Figure 14 (SEQ ID NO:23). A particularly preferred peptide having the activity of the B7-2 antigen includes at least a portion of the mature form of the protein. such as an extracellular domain porion about amino acid residues 2-245 of SEQ ID NO:2) which can be used to enhance or suppress T-ceii mediated immune responses in a subject. Other prefe red peptides having B7-2 activity include pepiies mavin an amiino acid seue:.c rep resented by a formula: Xn-Y-Zm In the formula, Y is amino acid residues selected from the group consisting of: amino acid residues 55-68 of the sequence shown in Figure 8 (SEQ ID NO:2); amino acid residues 81-89 of the sequence shown in Figure 8 (SEQ ID NO:2); amino acid residues 128-142 of the sequence shown in Figure 8 (SEQ ID NO:2); amino acid residues 160-169 of the sequence shown in Figure 8 (SEQ ID NO:2); amino acid residues 188-200 of the sequence shown in Figure 8 (SEQ ID NO:2); and amino acid residues 269-282 of the sequence shown in Figure 8 (SEQ ID NO:2). In the formula Xn and Zm are additional amino acid residue(s) linked to Y by an amide bond and are selected from amino acid residues contiguous to Y in the amino acid sequence shown in Fiigde 8 (SEQ ID NO:2). Xn is amino acid residue(s) selected from amino acids contiguous to the amino terminus of Y in the sequence shown in Figure 8 (SEQ ID NO:2). Zm is amino acid residue(s) selected from amino acids contiguous-to the carboxy terminus of Y in the sequence shown in Figure 8 (SEQ ID NO:2). According to the formula, n is a number from 0 to 30 (n=0-30) and m is a number from 0 to 30 (m=0-30).

Fusion proteins or hybrid fusion proteins including a peptide having the activity of a novel B lymphocyte antigen B7-2, B7-3) are also featured. For example, a fusion protein comprising a first peptide which includes an extracellular domain portion of a novel B lymphocyte antigen fused to second peptide, such as an immunoglobulin constant region, that .i'0 alters the solubility, binding affinity, stability and/or valency of the first pepti'de are provided.

In one embodiment, a fusion protein is produced comprising a first peptide which includes amino acid residues of an extracellular domain portion of the B7-2 protein joined to a second pepide which includes amino acid residues of a sequence corresponding to the hinge. CH2 and CH3 regions of Cyl or Cy4 to form a B7-2Ig fusion protein. In another embodiment, a 5 hybrid fusion protein is produced comprising a first peptide which includes an extracellular domain portion of the B7-1 antigen and an extracellular domain portion of the B7-2 antigen and a second peptide which includes amino acid residues corresponding to.the hinge, CH2 and CH3 of Cyl (see Linsley et al. (1991) J Exp. Med. 1783:721-730: Capon et al.

(1989) Vature 525-53 I; and Capon U.S. 5,116.964).

i Isolated peptides and fusion proteins of the invention can be administered to a subject to either upregulate or inhibit the expression of one or more B lymphocyre antigens or block the ligation of one or more B iymphocyTe antigens to their natura! liLand on immune cei!s.

such as T cells, to thereby provide enhancement or suppression of cell-mediated immune responses in vivo.

Another embodiment of the invention provides antibodies. preferably monoc!onal antioocies. specifically reactive with a peptide of a novel B iymphocyte antigen or fusion protein as described herein. Preerred antiboodies are anti-human B7-2 monoclonal antibodies -6produced by hybridoma cells HF2.3D1, HA5.2B7 and HA3.1F9. These hybridoma cells have been deposited with the American Type Culture Collection at ATCC Accession No. (HF2.3D ATCC Accession No. (HA5.2B7), and ATCC Accession No.

(HA3. 1F9).

A still further aspect of the invention involves the use of the nucleic acids of the invention, especially the cDNAs, to enhance the immunogenicity of a mammalian cell. In preferred embodiments, the mammalian cell is a turnor cell, such as a sarcoma, a lymphoma, a melanoma, a neuroblastoma, a leukemia or a carcinoma, or an antigen presenting cell, such as a macrophage, which is transfected to allow expression of a peptide having the activity of a novel B lymphocyte antigen of the invention on the surface of the cell. Macrophages that express a peptide having the activity ofa B lymphocyte antigen, such as the B7-2 antigen, can be used as antigen presenting cells, which, when pulsed with an appropriate pathogenrelated antigen or tumor antigen, enhance T cell activation and immune stimulatin.'- Mammalian cells can be transfected with a suitable expression vector containing a nucleic acid encoding a peptide having the activity of a novel B lymphocyte antigen, such as the B7-2 antigen, ex vivo and then introduced into the host mammal, or alternatively, cells can be transfected with the gene in vivo via gene therapy techniques. For example, the nucleic acid encoding a peptide having B7-2 activity can be transfected alone, or in combination with nucleic acids encoding other costimulatory molecules. In enhancing the immunogenicity of tumors which do not express Class I or Class II MHC molecules, it may be beneficial to additionally transfect appropriate class I or II genes into the mammalian cells to be transfected with a nucleic acid encoding a peptide having the activity of a B lymphocyte antigen, as described herein.

The invention also provides methods for inducing both general immunosuppression 25 and antigen-specific tolerance in a subject by, for example, blocking the functional interaction of the novel B lymphocyte antigens of the invention, B7-2 and B7-3, to their natural ligand(s) on T cells or other immune system cells, to thereby block co-stimulation through the receptor-ligand pair. In one embodiment, inhibitor, molecules that can be used to block the interaction of the natural human B7-2 antigen to its natural ligands CTLA4 and CD28) include a soluble peptide having B7-2 binding activity but lacking the ability to cosimulate immune cells, antibodies that block the binding of B7-2 to its ligands and fail to deliver a co-stimulatory signal (so called "blocking antibodies", such as blocking anti-B7-2 antibodies). B7-2-Ig fusion proteins. which can be produced in accordance with the teachings of the present invention. as well as soluble forms of B7-2 recectors, such as CTLA4Ig or CD2Slg. Such biocking agents can be used alone or in combination with agents which block interaction of other cosiri.uiator olecu es with their natural ligands anti-B 7 antibody). iihibition o T ceil responses and induction ofT cell toierance according to the 25/02 '04 12:01 FAX 61 3 9663 3099 FB RICE CO, @oo_0 7 methods described herein may be useful prophylactically, in preventing transplantation rejection (solid organ, skin and bone marrow) and grft versus host disease, especially in allogenic bone marrow trnplantation. The methods of the invention may also be useful therapeutically, in the treatment of autoimmune diseases, allergy and allergic reactions, transplantation rejection, and established graft versus host disease on a subject.

Another aspect of the invention features methods for upregulating immune responses by delively of a costimulatory signal to T cells through use of a stimulatory form of 37-2 antigen, which include soluble, multivalent forms of B7-2 protein, such as a peptide having B7-2 activity and 87-2 fusion proteins. Delivery of a stimulatory form of B7-2 in conjunction with antigen may be useful prophylactically to enhance the efficacy of vaccination against a variety of pathogens and may also be useful therapeutically to upregulate an immune response against a particular pathogen during Is an infection or against a tumor in a tumior-bearing host.

The invention also features methods of identifying molecules which can inhibit either the interaction of B lymphocyte antigens, B7-2, B7-3, with their receptors or interfere with intracellular signalling through their receptors. Methods for identifying molecules which can modulate the expression of B lymphocyte antigens on cells are also provided. In addition, methods for identifying cytokines produced in response to costirnulation of T cells by novel B lymphocyte antigens are within the scope of the invention, 25 In a first aspect, the present invention provides an isolated nucleic acid molecule comprsing a nucleotide sequence encoding a peptide having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2), said peptide having the ability to costimulate T cell 0 proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4.

In a second aspect, the present invention provides an isolated nucleic acid molecule comprising the coding region of a nucleotide sequence shown in Figure 8 (SEQ

ID

NO:) and encoding peptide having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4.

00 COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:01 FAX 61 3 9663 3099 FB RICE CO, B 00i 7a In a third aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence shown in Figure 14 (SEQ ID NO:22) and encoding a peptide having the ability to costirnulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4.

In a further aspect, the present invention provides an isolated nucleic acid molecule encoding apeptide comprising an amino acid sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4.

In a further aspect, the present invention provides an isolated nucleic acid molecule encoding a peptide comprising an amino acid sequence shown in Figure 14 (SEQ ID NO:.23) said peptide having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4.

In a further aspect, the present invention provides an isolated nucleic acid molecule encoding a B7-2 peptide, wherein the peptide is encoded by a nucleic acid molecule which hybridises under high or low stringency conditions to a nucleic acid molecule which encodes a peptide comprising an amino acid sequence shown in Figure 8 (SEQ ID NO:2), said peptide having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4, In a further aspect, the present invention provides an isolated nucleic acid molecule Swhich hybridises under high or low stringe n c y conditions to a nucleic acid molecule 25 which encodes a peptide comprising an amino acid sequence shown in Figure 14 (SEQ ID NO:23), said peptide having the ability to costimulate T cell proliferation or T cell Se cytokine production or the ability to bind CD28 or CTLA4.

9 30 In a further aspect, the present invention provides an isolated DNA molecule 30 comprising a nucleotide sequence encoding a peptide having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2), said peptide having the ability to costimulate T ceCUll proliferation or T cell cyto ld kine production or the ability to bind CD28 or CTLA4, the peptide having an amino acid sequence represented by formnnula XnY-Zm, wherein

Y

5 comprises amino acid residues 24-245 of the sequence shown in Figure 8 (SEQ ID NO:2), wherein X, is amino acid residues selected from amino acid residues a .9 COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:02 FAX 61 3 9663 3099 FB RICE CO.

[a009 7b contiguous to the amino terminus of Y in the sequence shown in Figure 8 (SEQ ID wherein Zm is amino acid residues selected from amino acid residues contiguous, to the carboxy terminus ofY in the sequence shown in Figure 8 (SEQ ID NO:2), wherein n=0-23 and wherein m= 0-84.

In a further aspect, the present invention provides an isolated DNA molecule comprising a nucleotide sequence encoding a peptide of at least 20 amino acid residues or more in length having at least about 50% homology with a human B7-2 peptide comprising an amino acid sequence shown in Figure 8 (SEQ ID NO:2), said peptide having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4.

In a further aspect, the present invention provides an isolated nucleic acid molecule encoding a B7-2 fusion protein comprising a nucleotide sequence encoding a first peptide having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2), said peptide having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4 and a nucleotide sequence encoding a second peptide.

In a further aspect, the present invention provides an isolated polypeptide comprising an amino acid sequence having at least 50% amino acid sequence identity with the extracellular domain of a human 37-2 peptide shown in Figure 8 (SEQ ID NO:2) and the ability to bind CD28 or CTLA4.

25 In a fiurther aspect, the present invention provides a cell transfected with a nucleic acid molecule encoding a peptide having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2), said peptide having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4, said nucleic acid molecule 30 being in a fonn suitable for expression of the peptide on the cell surface.

In a further aspect, the present invention provides a tumor cell which is modified to express a T cell costimulatory molecule having at least 50% amino acid sequence 99ok identity with a human B7-2 peptide comprising the amino acid sequence shown in o• COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:02 FAX 61 3 9663 3099 FB RICE CO.

7c Figure 8 (SEQ ID NO0:2), said peptide having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4.

In a further aspect, the present invention provides .a tumor cell transfected with a nucleic acid molecule encoding a T cell costimulatory molecule having at least amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to costimnulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4, said nucleic acid molecule being in a form suitable for expression of B7-2.

In a further aspect, the present invention provides a method of treating a subject with a tumor, the method including: obtaining tumor cells from the subject; transfecting the tumor cells with a nucleic acid molecule encoding a molecule having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2), said peptide having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4 and said nucleic adcid molecule being in a form suitable for expression of B7-2; and administering the tumor cells to the subject, In a further aspect, the present invention provides a method of inducing an antitumor response by CD4 T lymphocytes in a subject with a tumor, the method including: obtaining tumor cells from the subject; transfecting the tumor cells with at least one nucleic acid molecule o: comprising DNA encoding: a molecule having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in 30 Figure 8 (SEQ ID NO:2), said peptide having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4, an MvfHC class II a chain protein, and (iii) an MHC class H P chain protein, wherein the nucleic acid molecule is in a 35 form suitable for expression of B7-2, the MHC class II a chain protein and the MHC class Ilf chain protein; and COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:02 FAX 61 3 9663 3099 FB RICE CO.

[a0Oi 7d administering the tumor cells to the subject.

In a further aspect, the present invention provides a method of treating a subject with a tumor, the method including modifying tumor cells in vivo via introduction of a nucleic acid molecule comprising a nucleotide sequence encoding a peptide having at least amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2), said peptide having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4.

In a further aspect the present invention provides a method of treating a subject with a tumor, the method including: obtaining tumor cells and T lymphocytes from the subject; culturing the T lymphocytes from the subject In vitro with the tumor cells from the subject and with a molecule having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2), said peptide having the ability to costimulate T cell proliferation or T cell cytoldkine production or the ability to bind CD28 or CTLA4; and administering the T lymphocytes to the subject.

In a further aspect, the present invention provides a substantially pure preparation of a peptide having at least 50% amino acid sequence identity with a human B7-2 peptide 2comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2), said peptide 25 having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4.

0 *000 In a further aspect, the present invention provides an isolated, recombinant or purified peptide having at least 50% amino adid sequence identity with a human B7-2 peptide 30 comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2), said peptide having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4 and having an amino acid sequence represented by a *formula Xna-Y-Zm, wherein Y is amino acid residues selected from the group consisting of: 35 amino acid residues 55-68 of the sequence shown in Figure 8 (SEQ ID NO:2); amino acid residues 81-89 of the sequence shown in Figure 8 (SEQ ID NO:2); 00 O go 000 00 00 COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:03 FAI 61 3 9663 3099 FB RICE CO. 012 7e amino acid residues 128-142 of the sequence shown in Figure 8 (SEQ ID NO:2); amino acid residues 160-169 of the sequence shown in Figure 8 (SEQ ID NO:2); amino acid residues 188-200 of the sequence shown in Figure 8 (SEQ ID NO:2); and amino acid residues 269-282 of the sequence shown in Figure 8 (SEQ ID NO:2), wherein X, is amino acid residues selected from amino acid residues contiguous to the amino terminus of Y in the sequence shown in Figure 8 (SEQ ID NO:2). wherein Z is amino acid residues selected from amino acid residues contiguous to the carboxy terminus of Y in the sequence shown in Figure 8 (SEQ ID NO:2), wherein n---0-30 and wherein m=0-30.

In a further aspect, the present invention provides a composition comprising an antibody that recognises a human B7-2 polypeptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) and an antibody that recognises a B7-1 polypeptide as shown in (SEQ ID NO:29).

In a further aspect, the present invention provides a nonhuman, transgenic animal which contains cells transfected to express a peptide having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to costimulate T cell proliferation or T cell cytoldkine production or the ability to bind CD28 or CTLA4.

In a further aspect, the present invention provides a nonhuman knockout animal which o 25 contains cells having an altered gene encoding a B lymphocyte antigen, B7-2.

9 9 $0 In a further aspect the present invention provides a composition suitable for pharmaceutical administration comprising a peptide having at least 50% amino acid 05 sequence identify with a human B7-2 peptide comprising the amino acid sequence 30 shown in Figure 8 (SEQ ID NO:2), said peptide having the ability to costimulate T cell proliferation or T cell cytoldkine production or the ability to bind CD28 or CTLA4, and a pharmaceutically acceptable carrier.

ose.

$$0 •In a further aspect, the present invention provides a method of inhibiting an interaction 35 of a B lymphocyte antigen, B7-2, with its natural ligand(s) on the surface of immune *e0@ 9 0099 @9 9 9 9.* 9.

COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:03 FAX 61 3 9663 3099 FB RICE CO. [013 7f cells, the method including, contacting an immune cell with an agent selected from the group consisting of: a peptide having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to bind CD28 or CTLA4 but being incapable of costimulatmng T cell proliferation or T cell cytokine production, and an antibody against B7-2 which inhibits B7-2 binding with its natural ligand(s), to thereby inhibit costimulation of the immune cell through B7-2 ligand interaction.

In a further aspect, the present invention provides a method of downregulating T cell mediated immune responses in a subject, the method including administering to the subject an agent selected from the group consisting of: a peptide having at least amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to bind CD28 or CTLA4 but being incapable of costimulating T cell proliferation or T cell cytokine production and an antibody against B137-2 which inhibits B7-2 binding with its natural ligand(s), in an amount effective to inhibit T cell proliferation and/or cytokine secretion in 'the subject In a further aspect, the present invention provides a method of treating an autoimmune disease in a subject mediated by interaction of a B Lymphocyte antigen, B7-2, with its natural ligand(s) on the surface of immune cells, the method including administering to the subject an agent selected from the group consisting of: a peptide having at least amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to bind 26 CD28 or CTLA4 but being incapable of costimulating T cell proliferation or T cell 9.

cytokine production and an antibody against B7-2 which inhibits B7-2 binding with its natural ligand(s), such that autoimmune disease is treated In a further aspect, the present invention provides a method of treating allergy in a *30 subject mediated by interaction of a B lymphocyte antigen, B7-2, with its natural S9.9 ligand(s) on the surface of immune cells, the method including administering to the subject an agent selected from the group consisting of: a peptide having at least amino acid sequence identity with a human B7-2 peptide comprising the amino acid "sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to bind 9 9 o 35 CD28 or CTLA4 but being incapable of costimulating T cell proliferation or T cell cytokine production and an antibody against B7-2 which inhibits B7-2 binding with its 9 99 9 9 9 oo COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:03 FAX 61 3 9663 3099 FB RICE CO.

7g natural ligand(s), to thereby inhibit costimulation of the immune cells through the B7-2 -ligand interaction.

In a further aspect, the present invention provides a method of inhibiting donor T cell S proliferation and/or cytokine secretion in a transplant recipient to thereby prevent graft-versus-host disease (GVIHD) in the recipient, the method including contacting donor T cells to be transplanted with an agent selected from the group consisting of a peptide having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to bind CD28 or CTLA4 but being incapable of costimulating T cell proliferation or T cell cytokine production and an antibody against B7-2 which inhibits B7-2 binding with its natural ligand(s), such that donor T cell proliferation and/or cytokine secretion is inhibited in a transplant recipient.

In a further aspect, the present invention provides a method of inhibiting transplantation rejection in a recipient of a tissue or organ transplant, the method including administering to the recipient an agent selected from the group consisting of a peptide having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to bind CD28 or CTLA4 but being incapable of costimulating T cell proliferation or T cell cytokine production and an antibody against B7-2.

In a further aspect, the present invention provides a method of upregulating T cell mediated imnmune responses in a subject, the method including administering to the S. 25 subject a B7-2 peptide having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) having the ability to costimulate T cell proliferation or T cell cytoldkine production or the ability to bind CD28 or CTLA4 in an amount effective to stimulate T cell proliferation and/or cytokine secretion in the subject In a further aspect, the present invention provides a method of identifying molecules which modulate expression of a B7-2 antigen, the method including: a) contacting a cell which expresses a peptide having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to costimulate T cell proliferation or T cell cytokidne production or the ability to COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:04 FAX 61 3 9663 3099 FB RICE CO. Q01-j-__ 7h bind CD28 or CTLA4 with a molecule to be tested, under conditions appropriate for interaction of the molecule with the cell; and b) determining the effect of the molecule on cell expression of the peptide.

In a further aspect, the present invention provides a method of identifying a cytokine produced by an immune cell in response to costirnulation with a Bl7-2 antigen, the method including: a) contacting an activated immune cell and a cell modified to include a nucleic acid molecule comprising DNA which encodes'a peptide having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4, in an appropriate cell culture medium under conditions appropriate for expression of the peptide in the cell; and b) determining the presence of a cytokine in the cell culture medium.

In a further aspect, the present invention provides a method of identifying molecules which inhibit costimulation of immune cells by a B7-2 antigen, the method including: contacting an immune cell which has received a primary induction signal with a protein having at least 50% amino acid sequence identity with a human B 7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) said protein having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4 and a molecule to be tested, under conditions appropriate for :11:25 interaction of the molecule with the immune cell and the protein; and b) determining the effect of the molecule on intracellular signalling by the immune cell in response to the protein.

e.00 .0 fi m l e u e In a farther aspect, the present invention provides a method of identifying molecules 0 30 which inhibit binding of a B17-2 antigen to a ligand on the surface of immune cells, the eoo method including: a) contacting a labelled B7-2 ligand and a molecule to be tested with a peptide having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4; 0: o• 0. C. C C COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:04 FAX 61 3 9663 3099 FB RICE CO.

Q016 7i b) removing unbound labelled B7-2 ligand; and c) determining the amount of labelled B7-2 ligand bound to the peptide having B7-2 activity, as an indication ofthe ability of the molecule to inhibit binding of the B7-2 ligand to a B7-2 antigen.

In a further aspect, the present invention provides a method of identifying molecules which inhibit intracellular signalling by an immune cell in response to a recombinant protein having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2), said peptide having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4, the method including: a) contacting an immune cell which has received a primary activation signal and which expresses a B7-2 ligand on the cell surface with the protein and a molecule to be tested, under conditions appropriate for interaction of the molecule with the immune cell and the protein; and b) determining the effect of the molecule on intracellular signalling by the immune cell in response to the protein.

In a further aspect, the present invention provides use of a peptide having at least amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to costimulate T cell proliferation or T cell cytolkine production or the ability to bind CD28 or CTLA4 in the manufacture of a medicament for the treatment of disease in a 25 subject.

In a furnther aspect, the present invention provides an isolated polypeptide consisting of .a B7-2 extracellular domain as shown in Figure 8 (SEQ ID NO:2).

0* 0 30 In a further aspect, the present invention provides an isolated polypeptide consisting of amino acid residues 24-243 shown in Figure 8 (SEQ ID NO:2).

In a further aspect, the present invention provides an isolated nucleic acid molecule consisting of a nucleotide sequence encoding the extracellular domain of the B7-2 35 molecule shown in Figure 8 (SEQ ID NO:2).

0* 0* 0* 00 COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:04 FAX 61 3 9663 3099 FB RICE CO, 017 7j In a further aspect, the present invention provides an isolated nucleic acid molecule consisting of a nucleotide sequence encoding amino acids 24-243 of Figure 8 (SEQ ID NO:2).

In a further aspect, the present invention provides a method of modulating an immune response, comprising administering to a subject a molecule selected from the group consisting of; the polypeptide shown in Figure 8 (SEQ ID NO:2); a polypeptide comprising a variable region domain shown in Figure 8 (SEQ ID NO:2); a polypeptide consisting of a variable region domain shown in Figure 8 (SEQ ID NO:2); a polypeptide comprising the extracellular domain shown in Figure 8 (SEQ ID NO:2); a polypeptide consisting of the extracellular domain shown in Figure 8 (SEQ ID NO:2); an antibody reactive with the B7-2 polypeptide shown in SEQ ID NO:2; a nucleic acid molecule encoding a polypeptide comprising the extracellular domain shown in Figure 8 (SEQ ID NO:2); a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide shown in Figure 8 (SEQ ID NO:2) and a nucleic acid molecule comprising the coding region of the nucleotide sequence shown SEQ ID NO:1, such that the immune response is modulated.

In a further aspect, the present invention provides an isolated variable region form of the B cell activation antigen, B7-2, which comprises a B7-2 immunoglobulin-like 11 variable region domain but does not comprise a B7-2 immunoglobulin-like constant region domain.

25 In a further aspect, the present invention provides an isolated B7-2 fusion protein comprising a human B7-2 immunoglobulin-like variable region domain operatively linked to a heterologous polypeptide, wherein the B7-2 fusion protein does not comprise a B7-2 immunoglobulin-like constant region domain.

*9 9 *oo o*o COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:05 FAX 61 3 9663 3099 FB RICE CO.

@018 7k In a further aspect, the present invention provides an isolated nucleic acid molecule encoding a variable region form of a B7-2 fusion protein, the B7-2 fusion protein comprising a human B7-2 immunoglobulin-like variable region domain operatively linked to a heterologous polypeptide, wherein the B7-2 fusion protein does not comprise a B7-2 inmmnunoglobulin-like constant region domain.

In a further aspect, the present invention-provides a method for stimulating a response by an activated T cell, comprising contacting the activated T cell with a variable region form of the B cell activation antigen B7-2, the variable region form of B7-2 comprising a B7-2 inmmunoglobulin-like variable region domain but not comprising a B7-2 immunoglobulin-like constant region domain, such that a response by the activated T cell is stimulated.

In a further aspect, the present invention provides a method for upregulating T cell mediated immune responses in a subject, the method including administering to the subject a variable region form of the B cell activation antigen, B7-2, the variable region form of B7-2 comprising a B7-2 immunoglobulin-like variable region domain but not comprising a B7-2 immunoglobulin-like constant region domain, in an amount effective to stimulate T cell proliferation and/or cytokine secretion in the subject S. In a further aspect, the present invention provides an antibody or fragment thereof .specifically reactive with a peptide of any one of claims 93-96, wherein the antibody is :i a fully human monoclonal antibody.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

S

COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:05 FAX 61 3 9663 3099 FB RICE CO, o019 71 Throughout this specification the word "comprise", or variations such as "comprises" or '"comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Brief Description of the Drawineg Figure IA-B are graphic representations of the responses ofCD28 T cells, as assessed by 3 H-thymidine incorporation or IL-2 secretion, to costimulation provided by either B7 (B7-1) transfected CHO cells (panel a) or syngeneic activated B lymphocytes (panel b) cultured in media, anti-CD3 alone, or anti-CD3 in the presence of the following monoclonal antibodies or recombinant proteins: aB7 (133, anti-B7-1); CTLAIg; Fab a CD28; control Ig fusion protein (isotype control for CTLA4IG); or aB5 (anti-BS, the isotype control for anti-B7-1).

Figure 2A-C are graphs of log fluorescence intensity of cell surface expression of B7-1 on splenic B cells activated with surface inmunoglobulin (sIg) crosslinking. The total (panel B7-1 positive (B7-1 panel b) and B7-1 negative panel c) activated B cells were stained with anti-B7-1 monoclonal antibody (133) and fluoroscein isothiocyanate (FITC) labeled goat anti-mouse immunoglobulin and analyzed by flow cytometry.

Figure 3A-B are graphic representations of the responses of CD28+ T cells, as assessed by 3 H-thymidine incorporation and IL-2 secretion, to costimulation provided by B7-1 (panel a) or B7-1r (panel b) activated syngenic B lymphocytes cultured in media, anti-

C

D3 alone, or anti-CD3 in the presence of the following monoclonal antibodies or COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 recombinant proteins: aBB-1 (133, anti-B7-1 and anti-B7-3); aB7 (anti-B7-1); CTLA4Ig; Fab aCD28; control Ig fusion protein or aB5 (anti-BS).

Figure 4 is a graphic representation of the cell surface expression of B7-1, B7-3 and total CTLA4 counter-receptors on fractionated B7-1I and B7-1- activated B lymphocytes.

Figure 5 is a graphic representation of temporal surface expression of B7-1 (CTLA4Ig and mAbs BB-1 and 133), B7-3 (CTLA4Ig and mAb BBI) and B7-2 (CTLA4Ig) counter-receptors on splenic B cells activated by sIg crosslinking.

Figure 6 is a graphic representation of temporal surface expression of B7-1 (CTLA4Ig and mAbs BB-I and 133), B7-3 (CTLA4Ig and mAb BB1) and B7-2 (CTLA4Ig) counter-receptors on splenic B cells activated by MHC class II crosslinking.

Figure 7A-B are graphic representations of the response of CD28- T cdells, as assessed by 3 H-thymidine incorporation and IL-2 secretion, to costimulation providedby syngeneic B lymphocytes activated by sIg crosslinking for 24 hours (panel a) or 48 hoursfpael and cultured in media, anti-CD3 alone, or anti-CD3 in the presence of the following monoclonal antibodies or recombinant protein: aB7(133, anti-B7-1); aBB I (anti-B7-1, anti-B7-3) CTLA4Ig; Fab aCCD28; and Figure 8 is the nucleotide and deduced amino acid sequence of the human B lymphocyte antigen B7-2 (hB7-2-clone29).

Figure 9 is a graphic representation of COS cells transfected with control piasmid (pCDNAI), plasmid expressing B7-1 or plasmid expressing B7-2 stained with either control mAb (IgM), anti-B7- (rnAbs 133 and BB-1), recombinant protein CTLA4Ig, or isotype matched control Ig protein followed by the appropriate second FITC labelled immunoglobulin and analyzed by flow cytometry.

Figure 10A-B show R NA blot analyses of B7-2 expression in unstimulated and anti- :25 Ig activated human spenic B cells and cell lines (panel a) and human myelomas (panel b).

Figure 11 is a graphic representation of the proliferation of CD28+ T cells, as assessed by 3 H-thymidine incorporation or IL-2 secretion, to submitogenic stimulation with phorbol myristic acid (PNIA) and COS cells transfected with vector alone or vectors directing the exporession of either B7-1 or B7-2.

Figure 12 is a graphic representation of the inhibition by mAbs and recombinant proteins of the proliferation of CD2S+ T cells, as assessed by 3 H-thymidine incorporation and IL-2 secretion. to stimulation by PMA and COS cells transfected with vector alone (vector), or with a vector expressing BT-1 (B7-1) or B7-2 Inhibition studies were performed with the addition of either no antibody (no mAb), anti-B7 m-b 133 (133), anti-B7 mA BB-l (BB anti-85 m.Ab Fab fragment of anti-CD2 (CD2S Fab). CTLA4Ig (CTLA4g). or Ig control protein (control Ig) to the PM>A stimulated COS cell admixed CD'S- T cells.

-9- Figure 13 shows the sequence homology between the human B7-2 protein (h 137-2) deduced amnino acid sequence (SEQ ID NO: 2) anid the amino acid sequence of both the human B7-1 protein (h B7-1) (SEQ ID NO: 28 and 29) and the wuine B7-1 protein (M B7) (SEQ ID NO: 30 and 3 1).

Figure 14 is the nucleotide and deduced amnino acid sequence of the murine B7-2 antigen (rnB7-2) (SEQ ID NO: 22 and 23).

Figure 15 is a graphic represenitation of the competitive inhibition of binding of biotinylatedCTLA4'rg to immobilized B37-2 Ig by B7 famnily-Ig fusion proteins. The Ig fusion proteins examined as competitors were: ful-length B7-2 (hB7.2), full-length B7-l (hB7. the variable region-like domain of B7-2 (hB7.2V) or the constant region-like domain of B7-2 (hB7.2C).

Figure IdA-B are graphic representations of the competitive inhibition of binding of biotinviated-B71-1 (panel A) or B7-2-lg (panel B) toI imobilized CTLA-4-lq by increasing concentrations of unlabelled B7-1l-g (panel A) or B7-2-Ig (panel T-he experimentally dete-rnined

IC

5 0 values are indicated in the upper right corner of the panels.

Figure 17 depicts flow cytomerric profiles of cells stained with an anti-hB7-2 gu

I

monoclonal antibody ',HA3. IF9. Cells stained with the antibody were CHO cells transfected to express human B37-2 (CHO-hB7.2), NIH 3T3 cells transfected to exporess human B7-2 20(3)T3-h37.2) and control transfected NTH 3T3 cells (3)T3-neo). The anti-hB 17.2 anti body was used as a positive control.

Figure 18 depicts flow cytromeu-ic profiles of cells stained with an anti-hB7-2 monoclonal antibody, HAS-.2B7. Cells stained with the antibody were. CHO cells transfected to express human 137-2 (CHO-hB7.2), NIH 3T3 cells transfected to express human B7-2 (3T3-hB7.2) and control transfected NIH 3T3 cells (3T3-nieo). The anti-hB7.2 antibody was used as a positive control.

Figure 19 depicts flow cytomerric profiles of cells stained with an anti-hB7-2 monoclonal antibody. HF'2.3D1. Cells stained with the antibody were C140 cellIs transfected V16600 to express human B37-2 (CHO-hB7.2), NIH 3T3 cells transfected to excress human B37-2 and control transf-ected NIH4 3T3 cells (T-e'.The ani-h37. antibody 0 was used as a positive control.

is a zraphic representation of tum or cell qrov.-th (as measured by tumnor Size) in mice CollowinLg transpiantation of J558 plasmac.'tomra c2lls or i558 plasrnac% coma Cells tra-nsf-eczed to xrS I7I(5S3.)o 72(:S3.) t eailed Descripion of the Invention In addition to the pre-viously charactzerized B iym hocyte activatiion antiaQen B7 (reerrd t heeinas 7-I huan lmphoc'ytes exp::ress othe-r rov.-i molecules which costimulate T cell activation. These costimulatory molecules include antigens on the surface of B lymphocytes, professional antigen presenting cells monocytes, dendritic cells, Langerhan cells) and other cells keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes) which present antigen to immune cells, and which bind either CTLA4, CD28, both CTLA4 and CD28 or other known or as yet undefined receptors on immune cells. Costimulatory molecules within the scope of the invention are referred to herein as CTLA4/CD28 ligands (counter-receptors) or B lymphocyte antigens. Novel B lymphocyte antigens which provide cotimulation to activated T cells to thereby induce T cell proliferation and/or cytokine secretion include the B7-2 (human and murine) and the B7-3 antigens described and characterized herein.

The B lymphocyte antigen B7-2 is expressed by human B cells at about 24 hours following stimulation with either anti-immunoglobulin or anti-MHC class IIjnonoclonal antibody. The B7-2 antigen induces detectable IL-2 secretion and T cell prolifertion. At about 48 to 72 hours post activation, human B cells express both B7-1 and a third CTLA4 counter-receptor. B7-3, identified by a monoclonal antibody BB-1, which also binds B7-1 (Yokochi, et al. (1982) J Immunol. 128, 823-827). The B7-3 antigen is also expressed on B7-1 negative activated B cells and can costimulate T cell proliferation without detectable IL-2 production, indicating that the B7-1 and B7-3 molecules are distinct. B7-3 is expressed on a wide variety of cells including activated B cells, activated monocytes, dendritic cells, 20 Langerhan cells and keratinocytes. At 72 hours post B cell activation, theexpression of B7-1 and B7-3 begins to decline. The presence of these costimulatory molecules on the surface of activated B lymphocites indicates that T cell costimulation is regulated, in part, by the temporal expression of these molecules following B cell activation.

Accordingly, one aspect of this invention pertains to isolated nucleic acids comprising 25 a nucleotide sequence encoding a novel costimulatory molecule, such as the B lymphocyte antigen, B7-2, fragments of such nucleic acids, or equivalents thereof. The term "nucleic acid" as used herein is intended to include such fragments or equivalents. The term "equivalent" is intended to include nucleotide sequences encoding functionally equivalent B l. Ivlmphocyte antigens or fiunctionallv equivalent peptides having an activity of a novel B lymphocyte antigen. the ability to bind to the natural iigand(s) of the B lymphocyte antigen on immune cells, such as CTLA4 and/or CD28 on T cells. and inhibit block) or stimulate enhance) immune cell costimulation. Such nucleic acids are considered equivalents of the human B7-2 nucieotide sequence provided in Figure 8 (SEQ ID NO:1) and the murine B7-.2 nucieotide sequence provided in Figure 14 (SEQ ID NO:22) and are within the scope of this inventior..

In one embcodiment. the nucieic acid is a cDNA encoding a peptide having an activity or the B7-2 B ilymphoc-te antigen. Preferably, the nucleic acid is a cDNA molecule -11consisting of at least a portion of a nucleotide sequence encoding human B7-2. as shown in Figure 8 (SEQ ID NO: 1) or at least a portion of a nucleotide sequence encoding murine B7-2.

as shown in Figure 14 (SEQ ID NO:22). A preferred portion of the cDNA molecule of Figure 8 (SEQ ID NO:1) or Figure 14 (SEQ ID NO:22) includes the coding region of the molecule.

In another embodiment, the nucleic acid of the invention encodes a peptide having an activity of B7-2 and comprising an amino acid sequence shown in Fieure 8 (SEQ ID NO:2) or Figure 14 (SEQ ID NO:23). Preferred nucleic acids encode a peptide having B7-2 activity and at least about 50% homology, more preferably at least about 60% homology and most preferably at least about 70% homology with an amino acid sequence shown in Figure 8 (SEQ ID NO:2). Nucleic acids which encode peptides having B7-2 activity and at least about more preferably at least about 95%, and most preferably at least about 9.8-99% homologous with a sequence set forth in Figure 8 (SEQ ID NO:2) are also wihin he scope of the invention. Homology refers to sequence similarity between two peptides having the activity of a novel B lymphocyte antigen, such as B7-2, or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequences is occupied by the same nucleotide base or amino acid, then the molecules are homologous at S* that position. A degree (or percentage) of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.

Another aspect of the invention provides a nucleic acid which hybridizes under high or low stringency conditions to a nucleic acid which encodes a peptide having all or a portion of an amino acid sequence shown in Figure 8 (SEQ ID NO:2) or a peptide having all or a portion of an.amino acid sequence shown in Figure 14 (SEQ ID NO:23). Appropriate stingency conditions which promote DNA hybridization, for example. 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50'C are known to those skilled in the art or can be found in Current Protocols in Mfolecular Biology John Wiley Sons. N.Y. (1989), 6.3.1-6.3.6. For example, the salt concentration in the wash step can be seiected from a low sr-ingencv of about 2.0 x SSC at 50°C to a hich st-rngency of about 0.2 x SSC at 50 0 C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about to high stringency conditions, at about Isolated nucleic acids encoding a peptide having an activity of a nove! B Ivmohocvte antigen, as described herein, and having a sequence which differs from nuc!eotide secuence shown in Figure 8 (SEQ ID NO: 1) or Figure 14 (SEQ ID NO:22) due to degeneracy in the genetic code are also within the scope of the invention. Such nucleic acids encode Functionally equivalent peptides ie.g.. a peptide having B7-2 activity) bu: differ in sequence -12from the sequence of Figure 8 or Figure 14 due to degeneracy in the genetic code. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) may occur due to degeneracy in the genetic code. As one example, DNA sequence polymorphisms within the nucleotide sequence of a B7-2 (especially those within the third base of a codon) may result in "silent" mutations in the DNA which do not affect the amino acid encoded. However, it is expected that DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the B7-2 antigen will exist within a population. It will be appreciated by one skilled in the art that these variations in one or more nucleotides (up to about 3-4% of the nucleotides) of the nucleic acids encoding peptides having the activity of a novel B lympho'yte antigen may exist among individuals within a population due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of the invention. Furthermore. there-'na be one or more isoforms or related, cross-reacting family members of the novel B lymphocyte antigens described herein. Such isoforms or family members are defined as proteins related in function and amino acid sequence to a B lymphocyte antigen the B7-2 antigen), but encoded by genes at different loci.

A "fragment" of a nucleic acid encoding a novel B lymphocyte antigen is defined as a nucleotide sequence having fewer nucleotides than the nucleotide sequence encoding the 20 entire amino acid sequence of the B lymphocyte antigen and which encodes a peptide having an activity of the B lymphocyve antigen the ability to bind to the natural ligand(s) of the B lymphocyte antigen on immune cells, such as CTLA4 and/or CD28 on T cells and either stimulate or inhibit immune cell costimulation). Thus, a peptide having B7-2 activity binds CTLA4 and/or CD28 and stimulates or inhibits a T cell mediated immune response, as 5 evidenced by, for example, cytokine production and/or T cell proliferation by T cells that have received a primary activation signal. In one embodiment, the nucleic acid fragment encodes a peptide of the B7-2 antigen which retains the ability of the antigen to bind CTLA4 and/or CD28 and deliver a costimulatory signal to T lymphocytes. In another embodiment.

the nucleic acid fragment encodes a peptide including an extracellular portion of the human ?0 B7-2 antigen approximate!y amino acid residues 24-245 of the sequence provided in Figure 8 (SEQ ID NO:2)) which can be used to bind CTLA4 and/or CD2S and, in monovalent form. irnhibi coszimulation. or in multivaleni form. induce or enhance costimulation.

Preferred nucleic acid fragments encode peotides of at least 20 amino acid residues in length. preferably at least 40 amino acid residues and length, and more preferably at least amino acid residues in length. Nucleic acid fragments which encode peptides of at least SO amino acid residues in length. at least 100 amino acid residues in length, and at least 200 or -13more amino acids in length are also within the scope of the invention. Particularly preferred nucleic acid fragments encode a peptide having the activity of human B7-2 and an amino acid sequence represented by a formula: Xn-Y-Zm In the fomula, Y comprises amino acid residues 24-245 of the sequence shown in Figure 8 (SEQ ID NO:2). Xn and Zm are additional amino acid residue(s) linked to Y by an amide bond. Xn and Zm are selected from amino acid residues contiguous to Y in the amino acid sequence shown in Figure 8 (SEQ ID NO:2). In the formula, Xn is amino acid residue(s) selected from amino acids contiguous to the amino terminus of Y in the sequence shown in Figure 8 (SEQ ID NO:2), from amino acid residue 23 to 1. Zm is amino ecid residue(s) selected from amino acids contiguous to the carboxy terminus of Y in the sequence shown in Figure 8 (SEQ ID NO:2), from amino acid residue 246 to 329. In addition, in the formula, n is a number from 0 to 23 (n=0-23) and m is a number from 0 to 84

A

i particularly preferred peptide has an amino acid sequence represented by the formula Xn-Y- Zm as above, where n=0 and m=0.

Nucleic acid fragments within the scope of the invention include those caoable of hybridizing with nucleic acid from other animal species for use in screening protocols to 20 detect novel proteins that are cross-reactive with the B lymphocyte antigens described herein.

These and other fragments are described in detail herein. Generally. the nucleic acid encoding a fragment ofa B lymphocvte antigen will be selected from the bases coding for the mature protein, however, in some instances it may be desirable to select all or part of a fragment or fragments from the leader sequence or non-coding portion of a nucleotide :5 sequence. Nucleic acids within the scope of the invention may also contain linker sequences, S modified restriction endonuclease sites and other sequences useful for molecular cloning, expression or purification of recombinant protein or fragments thereo.t These and other modifications of nucleic acid sequences are described in further detail herein.

A nucleic acid encoding a peptide having an activity of a novel B lymphocyte antigen.

0 such as the B7-2 antigen, may be obtained from mRUNA present in activated B Ivmohocvtes.

It should also be possible to obtain nucleic acid sequences encoding B lymphocyte antigens from B cell genomic DNA. For examp!e, the gene encoding the B7-2 atigen can be cloned from either a cDNA or a genomic library in accordance with protocols herein described. A cDNA encoding the B7-2 antigen can be obtained by isolating total mRNA from an appropriate cell line. Doubie stranded cDNAs can then prepared from the totair mrRNA.

Subsequently, the cDNAs can be insered into a suitable piasmid or vira! bac:eriophage) vector using any one of a number of known techniques. Genes encodin, nove- B lympohocvte -14antigens can also be cloned using established polymerase chain reaction techniques in accordance with the nucleotide sequence information provided by the invention. The nucleic acids of the invention can be DNA or RNA. A preferred nucleic acid is a cDNA encoding the human B7-2 antigen having the sequence depicted in Figure 8 (SEQ ID NO:1). Another preferred nucleic acid is a cDNA encoding the murine B7-2 antigen having the sequence shown on Figure 14 (SEQ ID NO:22).

This invention further pertains to expression vectors containing a nucleic acid encoding at least one peptide having the activity of a novel B lymphocyte antigen, as described herein, operably linked to at least one regulatory sequence. "Operably linked" is intended to mean that the nucleotide acid sequence is linked to a regulatory sequence in a manner which allows expression of the nucleotide sequence in cis or trans). Regulatory sequences are art-recognized and are selected to direct expression of the desired protein in an appropriate host cell. Accordingly, the term regulatory sequence includes pr-moters, enhancers and other expression control elements. Such regulatory sequences are known to those skilled in the art or one described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transfected and/or the type of protein desired to be expressed. In one embodiment, the expression vector includes a nucleic acid encoding at least a portion of the B7-2 protein, such V 20 as an extracellular domain portion. In another embodiment, the expression Vector includes a DNA encoding a peptide having an activity of the B7-2 antigen and a DNA encoding a peptide having an activity of another B lymphocyte antigen, such as B7-1. cDNAs encoding the human B7-1 and mouse B7-1 antigens are shown in SEQ ID NO:28 and SEQ ID respectively. The deduced amino acid sequences of these antigens are also shown in SEQ ID NO:29 and SEQ ID NO:3 1, respectively. Such expression vectors can be used to transfect cells to thereby produce proteins or peptides, including fusion proteins or peptides encoded *o by nucleic acid secuences as described herein. These and other embodiments are described in further detail herein.

The invention also features methods of producing peptides having an activity of a novei B lymphocyte antigen. For exampie, a host cell transfected with a nucieic acid vector directing expression of a nucleotide sequence encoding a peptide having an ac:ivitv of the B7-2 protein can be cultured in a medium under appropriate conditions to allow expression of the peptide to occur. In addition, one or more expression vectors containing DNA encoding a peptide having an activity of B-2 and DNA encoding another peptide. such as a pepide having an activity ofa second B Ivmphocyte antigen B7-1, B7-3 can be used to transfec: a host ceil to coexcress these :pepides or produce fusion proteins or peptides. In one embodiment, a recombinant expression vector containing DNA encoding a B7-2 fusion protein is produced. A B7-2 fusion protein can be produced by recombinant expression of a nucleotide sequence encoding a first peptide having B7-2 activity and a nucleotide sequence encoding second peptide corresponding to a moiety that alters the solubility, affinity, stability or valency of the first peptide, for example, an immunoglobulin constant region. Preferably, the first peptide consists of a portion of the etr-acellular domain of the human B7-2 antigen approximately amino acid residues 24-245 of the sequence shown in Figure 8 (SEQ ID The second peptide can include an immunoglobulin constant region, for example, a human C 1 y domain or Cy4 domain the hinge, CH2 and CH3 regions of human IgCyl, or human IgCy4, see Capon et al. US 5,116,964, incorporated herein by reference). A resulting B7-2Ig fusion protein may have altered B7-2 solubility, binding affinity, stability and/or valency the number of binding sites available per molecule) and miay increase the efficiency of protein purification. Fusion proteins and peptides produced by recombinant technique may be secreted and isolated from a mixture of cells and mediumontaining the protein or peptide. Alternatively, the protein or peptide may be retained cytoplasmically and the cells harvested, lysed and the protein isolated. A cell culture typically includes host cells.

media and other byproducts. Suitable mediums for cell culture are well known in the art.

Protein and peptides can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins and peptides. Techniques for transfecting host cells and purifying proteins and peptides are described in further detail herein.

20 Particularly preferred human B7-2Ig fusion proteins include the extracellular domain portion or variable region-like domain of human B7-2 coupled to an immunoglobulin constant region. The immunoglobulin constant region may contain genetic modifications which reduce or eliminate effector activity inherent in the immunoglobulin structure. For example, DNA encoding the extracellular portion of human B7-2 (hB7-2), as well as DNA 25 encoding the variable region-like domain of human B7-2 (hB7.2V) or the constant regionlike domain of human B7-2 (hB7.2C) can be joined to DNA encoding the hinge. CH2 and CH3 regions of human IgCyl and/or IgCy4 modified by site directed mutagenesis. The preparation and characterization of these fusion proteins is described in detail in Example 7.

Transfected cells which express peptides having an activity of one or more B lymphocyte antigens B7-2. B7-3 on the surface of the cell are also within the scope of this invention. In one embodiment. a host cell such as a COS cell is transfected with an expression vector directing the expression of a peptide having B7-2 activity on the surface of the cell. Such a transfected host cell can be used in methods ofidentifying mnolecules which inhibit binding ofB7-2 to its counter-receptor on T cells or which interfere with intracellular signaiing of costimulation to T cells in response to B7-2 interaction. In another embodiment.

a tumor cell such as a sarcoma, a melanoma, a leukemia. a Ivmohoma. a carcinoma or a neurobiastoma is transfected with an expression vector directing the expression of at least one -16peptide having the activity of a novel B lymphocyte antigen on the surface of the tumor cell.

In some instances, it may be beneficial to transfect a tumor cell to coexpress major histocompatibiliry complex (MHC) proteins, for example MHC class II ca and P chain proteins or an MIIC class I c chain protein, and, if necessary, a 32 microglobulin protein.

Such transfected tumor cells can be used to induce tumor immunity in a subject. These and other embodiments are described in further detail herein.

The nucleic acid sequences of the invention can also be chemically synthesized using standard techniques. Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See Itakura et al. U.S. Patent No. 4,598,049; Caruthers sa U.S. Patent No. 4,458,066; and Itakura U.S. Patent Nos.

4,401,796 and 4,373,071, incorporated by reference herein).

Another aspect of the invention pertains to isolated peptides having an activity of a novel B lymphocyte antigen B7-2, B7-3). A peptide having an activity ofa B lymphocyte antigen may differ in amino acid sequence from the B lymphocyte antigen, such as the human B7-2 sequence depicted in Figure 8 (SEQ ID NO:2), or murine B7-2 sequence depicted in Figure 14 (SEQ ID NO:22), but such differences result in a peptide which functions in the same or similar manner as the B lymphocyte antigen or which has the same :or similar characteristics of the B lymphocyte antigen. For example, a peptide having an 20 activity of the B7-2 protein is defined herein as a peptide having the ability, to bind to the natural ligand(s) of the B7-2 protein on immune cells, such as CLTA4 and/or CD28 on T cells and either stimulate or inhibit immune cell costimulation. Thus, a peptide having B7-2 activity binds CTLA4 and/or CD28 and stimulates or inhibits a T cell mediated immune response (as evidenced by, for example, cvtokine production and/or proliferation by T cells 25 that have received a primary activation signal). One embodiment provides a peptide having B7-2 binding activity, but lacking the ability to deliver a costimularory signal to T cells.

Such a peptide can be used to inhibit or block T cell proliferation and/or cvtokine secretion in a subject. Alternatively, a peptide having both B7-2 binding activity and the ability to deliver a costimulatory signal to T cells is used to stimulate or enhance T cell oroliferation and/or cytokine secretion in a subject. Varous modifications of the B7-2 protein to produce these and other functionally equivalent peptides are described in detail herein. The term "peptide" as used herein. refers to peptides. proteins and poiypeptides.

A peptide car, be produced by modification of the amino acid sequence of the human B7-2 protein shown in Figure 8 (SEQ ID NO:2) or the murine B7-2 orotein shown in Figure 14 (SEQ ID NO:23). such as a substitution. addition or deietion oian amino acid residue which is not directiv involved in the function of B7-2 the ability of B7-2 to bind CTLA4 anador CD2 and.or stimulate or irhibit T cell costimulation). Pe tdes of the invention are -17typically at least 20 amino acid residues in length, preferably at least 40 amino acid residues in length, and most preferably 60 amino acid residues in length. Peptides having B7-2 activitd and including at least 80 amino acid residues in length, at least 100 amino acid residues in length, or at least 200 or more amino acid residues in length are also within the scope of the invention. A preferred peptide includes an extracellular domain portion of the human B7-2 antigen about amino acid residues 24-245 of the sequence shown in Figure 8 (SEQ ID NO:2). Other preferred peptides have an amino acid sequence represented by a formula: xn-Y-z m X-4 where Y is amino acid residues selected from the group consisting of: amino acid residues 55-68 of the sequence shown in Figure 8 (SEQ ID NO:2); amino acid residueS 81 -89 of the sequence shown in Figure 8 (SEQ ID NO:2); amino acid residues 128-142 of the sequence shown in Figure 8 (SEQ ID NO:2); amino acid residues 160-169 of the sequence shown in Figure 8 (SEQ ID NO:2); amino acid residues 188-200 of the sequence shown in Figure 8 *(SEQ ID NO:2); and amino acid residues 269-282 of the sequence shown in Figure 8 (SEQ ID NO:2). In the formula, Xn and Zm are additional amino acid residues linked to Y b an amiide bond. X and Zm are amino acid residues selected from amino acids contiguous to Y 0 in the amino acid sequence shown in Figure 8 (SEQ ID NO:2). Xn is amino acid residues selected from amino acids contiguous to the amino terminus of Y in the sequence shown in Figure 8 (SEQ D NO:2). Zm is amino acid residues selected from amino acids contiguous to the carboxy terminus of Yin the sequence shown in Figure 8 (SEQ ID NO:2). According to the formula, n is a number from 0 to 30 (n=0-30) and m is a number from 0 to 30 (m=0-30).

A particularly preferred peptide has an amino acid sequence represented b the formula

X

n Y-Zm, where n=0 and m=0.

Another embodiment of the invention provides a substantially pure preparation of a peptide having an activity of a novel B lymphocyte antigen such as B7-2 or B7-3. Such a preparation is subs nialiy free of proteins and peptides with which the peptide naurailv occurs in a cell or with which it naturally occurs when secreted by a ceil.

The term "isolated" as used throughout this application refers to a nucleic acid prote:n or peticde having an activity or a novel B lymphocyte antigen, such as B7-2.

substantiaily free ofcelluiar material or culture medium when produced by recombinant DNA techniques. or chemical precursors or other chemicals when chemically synthesized.

An isolated nucleic acid is also free ofsequences which naturally flani the nucleic acid (i.e sequences located at the 5' ad 3' ends of the nucleic acid) in the organism from which h e nucleic acid is deived.

-18- These and other aspects of this invention are described in detail in the following subsections.

I. Isolation of Nucleic Acid From Cell Lines Suitable cells for use in isolating nucleic acids encoding peptides having an activity of a novel B lymphocyte antigen include cells capable of producing mRPNA coding for B lymphocyte antigens B7-1, B7-2, B7-3) and appropriately translating the mRNA into the corresponding protein. One source of mRNA is normal human splenic B cells, either resting or activated by treatment with an anti-immunoglobulin antibody or an anti-MHC class II antibody, or from subsets of neoplastic B cells. Expression of the human B7-2 antigen is detectable in resting B cells and in activated B cells, with mRNA levels increasing 4-fold from resting levels following stimulation. Total cellular RNA can be obtained using standard techniques from resting or activated B cells during these intervals and utilized"in The construction of a cDNA library.

In addition, various subsets of neoplastic B cells may express B7-2 and B7-3 and can alternatively serve as a source of the mRNA for construction of a cDNA library. For example, tumor cells isolated from patients with non-Hodgkins lymphoma express B7-1 mRNA. B cells from nodular, poorly differentiated lymphoma (NPDL). diffuse large cell lymphoma (LCL) and Burkit's lymphoma cell lines are also suitable sources of human B7-1 20 mRNA and, potentially B7-2 and B7-3 mRNA. Nyeiomas generally exprss B7-2, but not B7-1 mRNA, and, thus can provide a source of B7-2 mRNA. The Burkitt's Ivmohoma cell line Raji is one source of B lymphoc-te antigen mRNA. Preferably, B7-2 mRNA is obtained trom a population of both resting and activated normal human B cells. Activated B cells can be obtained by stimulation over a broad spectrum of time from minutes to days) with, 25 for example an anti-immunoglobulin antibody or an anti-MCH class II antibody.

0 0 II. Isolation of mR§NA and Construction ofcDNA Library Total cellular mRNA can be isolated by a variety of technicues. by using the guanicinmum-thiocvanate extraction procedure of Chirgwin et al.. Biochemisrr, i 5294- 5299 (1979). According to this method, Poly mRNA is prepared and purified for use in a cDNA librar construction using oligo (dT) cellulose selection. cDNA is then synthesized from the olyv(A-) RNA using oliso(dT) priming and reverse transcriptase. Nolonev MLV reverse transcriptase (available from Gibco/BRL. Be:hesda, MD) or AMV reverse transcriptase (available rom Seikas2Ku AmeIca. Inc.. St. Petersburg. FL) are preferabiy enioved.

Following reverse transCn-iption. the mRNkA DNA hybrid molecue is convened to double stranded DNA using conventional t-echnicues and incororated into a suitable vector.

19- The expeijments herein employed F. ccli DNA POlymerase I and ribonuclease Hi in the Convera ion, to double stranded cDNA.

Cloning of the cDNAs can be accomplished using any of the conventionaltehqs for joining double stande d DNA with an appropriate vector. The use of synthetic adaptors is Paticlarl prferrd, ince it alleviates the possibility, of cleavage of the cDNA with restriction enzyime prior to cloning. Using this method. no-efcmlmnay kinased adaptors are added to the DNA prior to ligation with the vector. Virtually any adaptor can, be emlyd AsstIot in more detail in the examples below, non-self complementary Bst~a adaptors are preferably added to the cDNA for cloning, for ligation into a pCDM8 vector prepared for cloning by digestion with BstXI.

Eucaryotic cDNA ca~n be expressed when placed in the sense orientatIo'n in a vector that supplies an apiropriate eucaryotic promoter and orignorelctnan.thrlmns including enhancers, splice acceptors and/or donor sequences and poivadenri The cDNAS of the present invention are placed in suitable vectors containing a eucaryotic promtoter,, an origin of reulication functional in E. colt, an SvO oiiofrlction which allows growth~ in COS cells. and a cDNA insertion site. Suitable vectors include jTtHJ (Seed *9and Aruffo, Proc. lVai. 4cad Sci., 841:3365-3369 (1987)), ,zH~m (Aniffo and Seed, Proc.

atl Acad Sc., 8,4:8573..8577 (1987)), pCDNf7 and pCDM8 (Seed. Naue32:4-l 2 0 1 9 8 7 w i t h t e D M S v co'e n p a r t i c u l a r l y p r e f e r r e d a v a i l a b l e c o m n n e c a l l f r o m *.20 lvitrge~ an iego, CA).

0044 Transfection of Uost ells and Sceenin for Novel B vice 4Activation ntiens The thus prepared cDNA library is then used to clone e zne ofinterest b 00.0 expDression cioning technique 5 A basic expeso coing techniqehsbe dsrbdb 251 Seed and Aruffo, Proc. iNad cad Sc. USA, 84:3 365-3369 (18)and AruffoanSed 0 :rc a.AadSi S 8":S573-8577 (1987), although modif-ications to this technique 0:006: may be necessary.

According to one emnbodiment. piasmid DNA is inodcditasmanCSel o line (Giuzriri CeZ 23:175 (198 by knowvn mthods of tranfeion (eg.DE.AE-Dex~rn) *00 and allowed to replicate and express the cDNA inserts. The trans rect anis expressing 87-1 arngen are depleted with an anti-B7-l monoclonal antibod.' e.

munne IG and 1gM'Y coated irnriuornagneric beads Tr-ad 11) rd ni c badsTras recranrs exrssn human B7-1 antigenl. ca be p st ve _l c~ed by re actin th :r n f c a it' the ftsion proteins CTL.-k4Lg andc CD2SIg. followed by pa--ning with anti-human ant:ibo0dy Coated plates.

Although human CTLA412 and CD28Tg fusion protinrs were used, in the- -xamoles descr-ibed hereirn. given the cross-species reactiVip, berween: .37-!1 an or eamcie rnurine 137-1. it can be expected that other- ftision proteins reactive with anoth.-r cross-r_-aCtIVe scecles cou idbe used. After panning, episomal DNA is recovered from the panned cells and transformed into a competent bacterial host, preferably E. coli. Plasmid DNA is subsequently reintroduced into COS cells and the cycle of expression and panning repeated at least two times. After the 'final cycle, plasmid DNA is prepared from individual colonies, transfected into COS cells and analyzed for expression of novel B lymphocyte antigens by indirect immunofluorescence with, for example, CTLA4Ig and CD28Ig.

IV. Sequencing of Novel B Lvmphocvte Antigens Plasmids are prepared from those clones which are strongly reactive with the CTLA4Ig and/or CD28Ig. These plasmids are then sequenced. Any of the conventional sequencing techniques suitable for sequencing tracts of DNA about 1.0 kb or larger can be employed.

As described in Example 4, a human B7-2 clone (clone29) was obtaifedRcontaining an insert of 1,120 base pairs with a single long open reading frame of 987 nucleotides and approximately 27 nucleotides of 3' noncoding sequences (Figure 8, SEQ ID NO:1). The predicted amino acid sequence encoded by the open reading frame of the protein is shown below the nucleotide sequence in Figure 8. The encoded human B7-2 protein, is predicted to be 329 amino acid residues in length (SEQ ID NO:2). This protein sequence exhibits many features common to other type I Ig superfamily membrane proteins. Protein translation is 20 predicted to begin at the methionine codon (ATG, nucleotides 107 to 109) based on the DNA homology in this region with the consensus eucarotic translation initiation site (see Kozak, M. (1987) Nuc!. Acids Res. 15:8125-8148). The amino terminus of the B7-2 protein (amino acids 1 to 23) has the characteristics of a secretory signal peptide with a predicted cleavage between the alanines at positions 23 and 24 (von Heijne (1987) Nuc!. Acids Res. 14:4683).

25 Processing at this site would result in a B7-2 membrane bound protein of 306 amino acids having an unmodified molecular weight of approximately 34 kDa. This protein would consist of an approximate extracellular Ig superfamily V and C like domains of from about amino acid residue 24 to 245. a hydrophobic transmembrane domain offrom about amino acid residue 246 to 268, and a long cytoplasmic domain of from about amino acid residue 269 to 329. The homologies to the Ig superfamily are due to the two contiguous Ig-like domains in the extracellular region bound by the cysteines at cositions 40 to 110 and 157 to 218. The extracellular domain also contains eight potential N-linked givcosvlation sites and.

like B7-1, is probably giycosylated. Glvcosvlaiion of the human BT-2 orotein may increase the molecular weight to about 50-70 kDa. The cytoDiasmic domain of human B7-2. while somewhat longer than B7-1. contains a common region of multiple cvsteines followed by positively charged amino acids which presumably function as signaling or regulator.

domains within an ancigen-presenctin cell (APC). Comoarison or both the nucleotide and -21amino acid sequences of the human B7-2 with the GenBank and EMBL databases yielded significant homology (about 26% amino acid sequence identity) with human B7-1. Since human B7-1, human B7-2 and murine B7-1 all bind to human CTLA4 and CD28, the homologous amino acids probably represent those necessary to comprise a CTLA4 or CD28 binding sequence. E. coli transfected with a vector containing a cDNA insert encoding human B7-2 (clone 29) was deposited with the American Type Culture Collection

(ATCC)

on July 26, 1993 as Accession No. 69357.

V, Cloning Novel B T vmphocte Antigens from Other Mammalian Species .0 The present invention is not limited to human nucleic acid molecules and contemplates that novel B lyrmphocyte antigen homologues from other mammalian species that express B lymphocyte antigens can be cloned and sequenced using the tedhniques described herein. B lymphocyte antigens isolated for one species humans' which exhibit cross-species reactivity may be used to modify T cell mediated immune responses in a different species mice). Isolation of cDNA clones from other species can also be accomplished using human cDNA inserts, such as human B7-2 cDNA. as hybridization probes.

As described in Example 6, a murine B7-2 clone (mB7-2, clone 4) was obtained containing an insert of 1,163 base pairs with a single long open reading frame of 927 nucleotides and approximately 126 nucleotides of 3' noncoding sequences (Figure 14, SEQ S ID NO:22). The predicted amino acid sequence encoded by the open reading frame of the protein is shown below the nucleotide sequence in Figure 14. The encoded murine B7-2 protein, is predicted to be 309 amino acid residues in length (SEQ ID NO:23). This protein sequence exhibits many features common to other type I Ig superfamily membrane proteins.

Protein translation is predicted to begin at the methionine codon (ATG. nucleotides 111 to 1. 1I3) based on the DNA homology in this region with the consensus eucarvotic translation S. initiation site (see Kozak. M. (1987) Nuc!. Acids Res. 15:8125-8148). The amino terminus of the murine B7-2 protein (amino acids 1 to 23) has the characteristics of a secretory signal peptide with a predicted cleavage between the alanine at position 23 and the valine at position 24 (von Heijne (1987) Vuc!. Acids Res. 14:4683). Processing at this site would result in a murine B7-2 membrane bound protein of 286 amino acids having an unmodified molecular we ght or approximate 32 kDa. This orotein would consist of an apcroximate extracellular Ig supenamily V and C like domains of from about amino acid residue 24 to 246. a hyarophocic transmembrane domain of from about amino acid residue 247 to 265. and a long cytoplasmic domain of from about amino acid residue 266 to 309. The homologies to the Ig super:amiy are due to the two contiguous Ig-like domains in the exiracelular region bound by the cysteines at positions 40 to I 10 and 157 to 2 i 6. The extrace!!u!ar domain also -22contains nine potential N-linked glycosylation sites and, like murine B7-1, is probably glycosylated. Glycosylation of the murine B7-2 protein may increase the molecular weight to about 50-70 kDa. The cytoplasmic domain of murine B7-2 contains a common region which has a cysteine followed by positively charged amino acids which presumably functions as signaling or regulatory domain within an APC. Comparison of both the nucleotide and amino acid sequences of murine B7-2 with the GenBank and EMBL databases yielded significant homology (about 26% amino acid sequence identity) with human and murine B7- 1. Murine B7-2 exhibits about 50% identity and 67% similarity with its human homologue, hB7-2. E. coli (DH106/p3) transfected with a vector (plasmid pmBx4) containing a cDNA insert encoding murine B7-2 (clone 4) was deposited with the American Type Culture Collection (ATCC) on August 18, 1993 as Accession No. 69388.

Nucleic acids which encode novel B lymphocyte antigens from other species, such as the murine B7-2. can be used to generate either transgenic animals or "knock out"animals which, in turn, are useful in the development and screening of therapeutically useful reagents.

A transgenic animal a mouse) is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, an embryonic stage. A transgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops. In one embodiment, murine B7-2 cDNA or an appropriate sequence thereof can be used to clone genomic B7-2 in accordance with 20 established techniques and the genomic sequences used to generate transgeni'c animals that contain cells which express B7-2 protein. Methods for generating transgenic animals particularly animals such as mice, have become conventional in the ar and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009. Typically, particular cells would be targeted for B7-2 transgene incornoration with tissue specific enhancers, which could result 25 in T cell costimulation and enhanced T cell proliferation and autoimmunitv. Transgenic animals that include a copy of a B7-2 transgene introduced into the germ line of the animal at an embryonic stage can be used to examine the effect of increased B7 expression. Such animals can be used as tester animals for reagents thought to confer protection from, for example. autoimmune disease. In accordance with this facet of the invention, an animal is 0 treated with the reagent and a reduced incidence of the disease, compared to untreated animals bearing the transgene--. would indicate a potential therapeutic intervention for the disease.

Altenativei. the non-human homologues of B7-2 can be used to construct a B7-2 "nock out" animal which has a defective or altered B7-2 gene as a result of homologous recombination beteen the endogenous B7-2 gene and altered B7- c-enomic DNA introduced into an emnbronic cell of the animal. For example. murine 3B-2 cDNA can be used to clone genomic B7-2 in accordance with established Lec iniques. A portion of the -23genomic B7-2 DNA such as an exon which encodes an extracellular domain ca b deleted or replaced with another gene, such as a gene encoding a selectable marker which ca be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector (see Thomas, K.R and Canecchi, M. R (1987) Cell i:503 for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line by electroporation) and cells in which the introducd DNA has homologously recombined with the endogenous DNA are selected (see Li, E. et al. (1992) Cell :915). The selected cells are then injected into a blastocyst of an animal a mouse) to form aggregation chimeras (see Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed.

(IRL, Oxford, 1987) pp. 11-152). A chimeric embryo can then be implanted-into a suitable pseudopregnant female foster animal and the embryo brought to termn to create a "knock out" animal. Progeny harbouring the homologously recombined DNA in their gerfn BIls can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homoiogously recombined DNA. Knockout animals can be characterized for their ability to accept grafts, reject tumors and defend against infectious diseases and can be used in the study of basic immunobiology.

V Expression of B Lvmphocvre Anens Host cells transfected to express peptides having the activity of a novel B lymphocyte .00. antigen are also within the scope of the invention. The host cell may be any procarvotic or eucaryotic cell. For example, a peptide having B7-2 activity may be expressed in bacterial cells such as E. coi, insect cells (baculovirus), yeast, or mammalian cells such as Chinese hamster ovary cells (CHO) and NSO cels. Other suitable host cels may be found in Goeddel, (1990) supra or are known to those skilled in the ar.

For example, expression in eucarvotic cells such as mammalian, yeast, or insect cells can lead to partial or complete glycosylation and/or formation of re!evant-inter- or intra-chain S disulfide bonds of recombinant protein. Examples of vectors for expression in veast S cerivisae include DYepSecl (Baldan. et al., (1987) Embo J 2293) pMFa (Kujan an S0 Herskowitz (1982) Ce! :,_.933-943) pJRY88 (Schultz e al, (1987) Gene 54:113-123). and pYES2 (Invitrogen Cororaion. San Diego, CA). Baculovius vectors available for exrssion of is in cultured isec cells (SF 9 cells) include the c series (Smith e al., (1983) M o. Cell Bio. 3 :21 56-2 165) and the pVL series (Lucklow, and Summers.

(1989) Viroio 1 70:3 Generally. COS cells (Gluzman. (1981) Ce!/ 2:175- 12) are used in conjunction with such vectors as pCDM8 (Seed. (1987) Nture 39:8 0) for transient a m p lifcation/expression in mammaiian cells. while CHO (dhfr- Chinese Hamste Ovary) cells are used with vectors such as pMT2PC (Kaufman e al (1987).

-24- EMBO J 6:187-195) for stable amplification/expression in mammalian cells. A preferred cell line for production of recombinant protein is the NSO myeloma cell line available from the ECACC (catalog #85110503) and described in Galfre, G. and Milstein, C. ((1981) Methods in Enzymology 72(13):3-46; and Preparation of Monoclonal Antibodies: Strategies and Procedures, Academic Press, Vector DNA can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofectin, or electroporation.

Suitable methods for transforming host cells can be found'in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks. When used in mammalian cells, the expression vector's control functions are often provided by viral material. For example. commonly used promoters are derived from polyoma. Adenovirus 2, cytomegalovirus and mast frequently, Simian Virus It is known that a small faction of cells (about 1 out of 10 typically integrate DNA into their genomes. In order to identify these integrants, a gene that contains a selectable marker resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418. hygromycin and methotrexate. Selectable markers may be introduced on the same plasmid as the gene of interest or may be introduced on a separate plasmid. Cells S 20 containing the gene of interest can be identified by drug selection; cells that have incorporated the selectable marker gene will survive, while the other cells die. The surviving cells can then be screened for production of novel B lymphocyte antigens by cell surface staining with ligands to the B cell antigens CTLA4Ig and CD2SIg). Alternatively, the protein can be metabolically radiolabeled with a labeled amino acid and immunoprecipitated from cell supernatant with an anti-B lymphocyte antigen monoclonal antibody or a fusion protein such as CTLA4Ig or CD28Ig.

Expression in procaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promotors directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids usually to the amino terminus of the S 30 expressed target gene. Such fusion vectors typically serve three purooses: 1) to increase expression of recombinant protein: 2) to increase the solubilitv of the target recombinant protein: and 3) to aid in the purification of the target recombinant protein by acting as a ligand in affinity purification. Ofen. in fusion expression vectors, a proteolvtic cleavage site is introduced at the junction of the fusion moiety and the target recombinant protein to enable separation of the taret recombinant crotein from the fusion moiety subsequent to ourification of the fusion protein. Such enzymes. and their cognate recognition sequences. include Factor Xa. thrombin and enterokinase. Tvrical fusion expression vectors include oGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, MA) and (Pharmacia, Piscataway, NJ) which fuse glutathione S-tranferase, maltose E binding protein, or protein A, respectively, to the target recombinant protein.

E. coli expression systems include the inducible expression vectors pTrc (Amann et aL, (1988) Gene 6:301-315) and pET 11 (Studier et Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89; commercially available from Novagen). In the pTrc vector system, the inserted gene is expressed with a pelB signal sequence by host RNA polymerase transcription from a hybrid trp-lac fusion promoter. After induction, the recombinant protein can be purified from the periplasmic fraction. In the pET 11 vector system, the target gene is expressed as non-fusion protein by transcription frondthe T7 gnlO-lac 0 fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host E. coli strains BL21(DE3) or HMS174(DE3) from a resident X prophage harboring a T7 gnl- unfer the transcriptional control of the lacUV 5 promoter. In this system, the recombinant protein can be purified from inclusion bodies in a denatured form and, if desired, renatured by step gradient dialysis to remove denaturants.

One strategy to maximize recombinant B7-2 expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gortesman, Gene Expression Technology: Methods in Enmolog 135., 0 Academic Press, San Diego. California (1990) 119-128). Another strategy would be to alter the nucleic acid sequence of the B7-2 gene or other DNA to be inserted into an exoression Svector so that the individual codons for each amino acid would be those preferentially utilized in highly expressed E. coli proteins (Wada al., (1992) Nuc. Acids Res. 20:2 111-2118).

Such alteration of nucleic acid sequences of the invention could be carried out by standard .5 DNA synthesis techniques.

Novel B lymphocyte antigens and portions thereof, expressed in mammalian cells or S otherwise, can be purified according to standard procedures of the art, including ammonium sulfate precipitation, fractionation column chromatography ion exchange, gel filtration, elec:rophoresis. affinity chromatography. etc.) and ultimately, crystallization (see generally.

0 "Enzme Purification and Related Techniques". Methods in En-vmoioag. 22:233-577 Once purified, partially or to homogeneity. the recombinantly produced B lymphocyte antigens or portions thereof can be utiiized in comcositions suitable for pharmaceutical administration as described in detail herein.

-26- VII. Modifications of Nucleic Acid and Amino Acid Sequence of the Invention and Assays for B7 Lvmphocyte Antigen Activitv It will be appreciated by those skilled in the art that other nucleic acids encoding peptides having the activity of a novel B lymphocyte antigen can be isolated by the above process. Different cell lines can be expected to yield DNA molecules having different sequences of bases. Additionally, variations may exist due to genetic polymorphisms or cellmediated modifications of the genetic material. Furthermore, the DNA sequence of a B lymphocyte antigen can be modified by genetic techniques to produce proteins or peptides with altered amino acid sequences. Such sequences are considered within the scope of the present invention, where the expressed peptide is capable of either inducing or inhibiting activated T cell mediated irimune responses and immune function.

A number of processes can be used to generate equivalents or fragments of an isolated DNA sequence. Small subregions or fragments of the nucleic acid encoding-the-B7-2 protein, for example 1-30 bases in length, can be prepared by standard, synthetic organic chemical means. The technique is also useful for preparation of anrisense oligonucleotides and primers for use in the generation of larger synthetic fragments ofB7-2 DNA.

Larger subregions or fragments of the genes encoding B lymphocyte antigens can be .expressed as peptides by synthesizing the relevant piece of DNA using the polymerase chain reaction (PCR) (Sambrook, Fritsch and Maniatis, 2 Molecular Cloning; A Laboratory 20 Manual, Cold Spring Harbor, (1989)), and ligating the thus obtained, DNA into an appropriate expression vector. Using PCR, specific sequences of the cloned double stranded DNA are generated. cloned into an expression vector, and then assayed for CTLA4/CD28 binding activity. For example, to express a secreted (soluble) form of the human B7-2 protein, using PCR, a DNA can be synthesized which does not encode the transmembrane S 25 and cytopiasmic regions of the protein. This DNA molecule can be iigated into an appropriate expression vector and introduced into a host cell such as CHO, where the B7-2 protein fragment is synthesized and secreted. The B7-2 protein fragment can then readily be obtained from the culture media.

In another embodiment, mutations can be introduced into a DNA by any one of a :°oo 30 number of methods. including those for producing simple deletions or insertions, systematic deletions. insertions or substitutions of clusters of bases or substitutions of single bases, to generate variants or modilfed ceuivaients of B ivmohocyte antigen DNA. For example.

cnanges in the human B7-: cDNA sequence show.vn in Figure 8 (SEQ ID NO:1) or murine B7-2 cDNA sequence shown in Figure i4 (SEQ [D NO :22 such as amino acid substitutions or deletions are :referablv obtained by site-direc:ed mutagenesis. Sice directed mutagenesis systems are we!i :.nown in the art. Protocols and reagents car be obtained commercially trom .Amersham intenmaional PLC. Amersham. L.K.

-27- Peptides having an activity of a novel B lymphocyte antigen, the ability to bind to the natual ligand(s) ofa B lymphocyte antigen on T cells and either stimulate (amplify) or inhibit (block) activated T cell mediated immune responses, as evidenced by, for example, cytokine production and/or T cell proliferation by T cells that have received a primary activation signal are considered within the scope ofthe invention. More specifically, peptides that bind to T lymphocytes, for example CD28 cells, may be capable of deliverin a costimulatory signal to the T lymphocytes, which, when transmitted in the presence of antigen and class II MHC, or other material capable of transmitting a primary signal to the T cell, results in activation of cytokine genes within the T cell. Alternatively, such a peptide can be used in conjunction with class I MHC to thereby activate CD8- cytolytic T cells. In addition, soluble, monomerib forms of the B7-2 protein, may retain the abilirv to bind to their natural ligand(s) on CD28 T cells but, perhaps because of insufficient crossinking with the liand, fail to deliver the secondary signal essential for enhanced cytokine producib and cell division. Such peptides, which provide a means to induce a state of anergy or tolerance in the cells, are also considered within the scope of the invention.

Screening the peptides for those which retain a characteristic B lymphocyte antigen activity as described herein can be accomplished using one or more of several different assays. For example, the peptides can be screened for specific reactivity with an anti-B7-2 .monoclonal antibody reactive with cell surface B7-2 or with a fusion protein, such as CTLA4g or CD2 8 1 Specifically, appropriate cells, such as COS cells, ca' be transfected with a B7-2 DNA encoding a peptide and then analyzed for cell surface phenotype by indirect immunofluorescence and flow cytometry to determine whether the peptide has B7-2 activity Cell surface expression of the transfected cells is evaluated using a monoclonal antibody specifically reactive with cell surface B7-2 or with a CTLA4Ig or CD28Ig fusion proten.

S roduction of secreed forms of B7-2 is evaluated using anti-B7-2 monoclonal antibody or CTLA4Ig or CD28 fusion protein for immunoprecipitation.

Other. more p referr e d assays take advantage of the functional characteristics of the B7-2 antigen. As previously set forth, the ability of T cells to svnthesize cvokines depends not only on occupancy or cross-linking of the T cell receptor for antigen (the "primary activation signal" provided by. for example anti-CD3. or phorbol ester to produce an "activated T cell"). but also on the induction ofa costimulatory sianal, in this case. by interaction with a B lvmphocyte antigen, such as B7-2. BT-! or B7-3. The binding of B7-2 to its natural ligand(s) on. for exampie. CD28 T ce!!s. has the effect of transmittina a signal to the T ce!l that induces the production of increased levels of cytokines. particularlv of miterieukin- which in turn si m uiates the proliferation o the T ivmphocvtes. Other assays for B -2 function thus involve assaying for the synthesis of c:~okines. such as interieukin-2 -28interleukin-4 or other known or unknown novel cytokines, and/or assaying for T cell proliferation by CD28' T cells which have received a primary activation signal.

In vitro, T cells can be provided with a first or primary activation signal by anti-T3 monoclonal antibody anti-CD3) or phorbol ester or, more preferably, by antigen in association with class II MHC. T cells which have received a primary activation signal are referred to herein as activated T cells. B7-2 function is assayed by adding a source of B7-2 cells expressing a peptide having B7-2 activity or a secreted form of B7-2) and a primary activation signal such as antigen in association with Class II MHC to a T cell culture and assaying the culture supernatant for interieukin-2, gamma interferon, or other known or unknown cytokine. For example, any one of several conventional assays for interleukin-2 can be employed, such as the assay described in Proc. Natl. Acad. Sci. USA, 86-1333 (1989) the pertinent portions of which are incorporated herein by reference. A kit for an assay for the production of interferon is also available from Genzyme Corporation (Cainbidge, MA.).

T cell proliferation can also be measured as described in the Examples below. Peptides that retain the characteristics of the B7-2 antigen as described herein may result in increased per cell production of cytokines, such as IL-2, by T cells and may also result in enhanced T cell proliferation when compared to a negative control in which a costimulatory signal is lacking.

The same basic functional assays can also be used to screen for peptides having B7-2 activity, but which lack the ability to deliver a costimulatory signal, but in the case of such peptides, addition of the B7-2 protein will not result in a marked increase in proliferation or cytokine secretion by the T cells. The ability of such proteins to inhibit or completely block the normal B7-2 costimulatory signal and induce a state of anergy can be determined using subsequent attempts at stimulation of the T cells with antigen presenting cells that express cell surface B7-2 and present antigen. If the T cells are unresponsive to the subsequent activation attempts. as determined by IL-2 synthesis and T cell proliferation, a state of anergy has been induced. See. Gimmi, C.D. et al. (1993) Proc. Natl. Acad. Sci. USA 90, 6586- 6. 6590; and Schwanz (1990) Science, 248, 1349-1356, for assay systems that can used as the basis for an assay in accordance with the present invention.

It is possibie to modify the structure of a peptide having the activity of a novel B 30 lymohocvte antigen for such purposes as increasing solubility, enhancing therapeutic or prophylactic efficacy. or stability she!f life ex vivo and resistance to proteolytic degradation in viIo. Such modified peptides are considered functional equivalents of the B lymphocyte antigens as defined herein. For example, a peptide having B7-2 activity can be modified so that it maiainains the abiiitv to co-stimulate T cell proiiferation and/or produce cytokines. Those residues shown to be essential to interact with the CTLA4'CD28 receptors on T cells can be modified by repiacing the essential amino acid with another. preferably similar arnino acid residue ia conser,.ative substitution) whose oresence is shown to enhance.

-29diminish, but not eliminate, or not effect receptor interaction. In addition, those amino acid residues which are not essential for receptor interaction can be modified by being replaced by another amino acid whose incorporation may enhance, diminish, or not effect reactivity.

Another example of modification of a peptide having the activity of a novel B lymphocyte antigen is substitution of cysteine residues preferably with alanine, serine, threonine, leucine or glutamic acid residues to minimize dimerization via disulfide linkages.

In addition, amino acid side chains of a peptide having B7-2 activity can be chemically modified. Another modification is cyclization of the peptide.

In order to enhance stability and/or reactivity, peptides having B7-2 activity can be .0 modified to incorporate one or more polymorphisms in the amino acid sequence of the antigen resulting from any nitural allelic variation. Additionally, D-amino acids, non-natural amino acids, or non-amino acid analogs can be substituted or added to produce a modified protein within the scope of this invention. Furthermore, the peptides can be modified using polyethylene glycol (PEG) according to the method of A. Sehon and co-workers (Wie et al supra) to produce a peptide conjugated with PEG. In addition, PEG can be added during chemical synthesis of the peptide. Other modifications of the peptides include reduction/alkylation (Tarr in: Methods of Protein Microcharacterizarion, J. E. Silver ed., S Humana Press, Clifton NJ 155-194 (1986)); acylation (Tarr, supra); chemical coupling to an appropriate carrier (Mishell and Shiigi, eds, Selected Methods in Cellular immunology,

WH

i Freeman, San Francisco, CA (1980), U.S. Patent 4,939,239; or mild formalin treatment (Marsh (1971), Int. Arch. ofAllerg and.4Dpl. Immunol. 41:199-215).

To facilitate purification and potentially increase solubility ofa peptide, it is possible to add an amino acid fusion moiety to the protein backbone. For example. hexa-histidine can be added to the peptide for purification by immobilized metal ion affinity chromatographv (Hochuli. E. et aL, (1988) Bio/TechnoloD :1321-1325). In addition, to facilitate isolation of a B lymphocyte antigen free of irrelevant sequences, specific endoprotease cleavage sites can be introduced between the sequences of a fusion moiety and the peptide. It-may be necessary to increase the solubility of a peptide by adding functional groups to the peptide, or by omitting hydrophobic regions of the peptide.

VTT. Uses nfNucleic Acid Secuences Encoding B Lv'mphocvte Anatiens and Perides Having B7-2 Acivit- A. Molecuar Probes The nucleic acids of this invention are useful diagnosically. for trackine the crocress of disease, by measuring the ac:ivation status of B lymphocytes in biological sampies or for assaving the effect ofa molecule on the expresssion of a B ivmphoc:,te antigen detecting cellular m.RNA levels). In accordance with these diagnostic assays, the nucleic acid sequences are labeled with a detectable marker, a radioactive, fluorescent, or biotinylated marker and used in a conventional dot blot or Northern hybridization procedure to probe mRNA molecules of total or poly(A+) RNAs from a biological sample.

B. Antibody Production The peptides and fusion proteins produced from the nucleic acid molecules of the present invention can also be used to produce antibodies specifically reactive with B lymphocyte antigens. For example, by using a full-length B7-2 protein, or a peptide fragment thereof, having an amino acid sequence based on the predicted amino acid sequence of B7-2, anti-protein/anti-peptide po~yclonal antisera or monoclonal antibodies can be made using standard methods. A mammal, a mouse, hamster, or rabbit) can be immunized with an immunogenic form of the protein or peptide which elicits an antibody response ifr the mammal. The immunogen can be, for example, a recombinant B7-2 protein, or fragment thereof, a synthetic peptide fragment or a cell that expresses a B lymphocyte antigen on its surface. The cell can be for example, a splenic B cell or a cell transfected with a nucleic acid encoding a B lymphocyte antigen of the invention a B7-2 cDNA) such that the B lymphocyte antigen is expressed on the cell surface. The immunogen can be modified to increase its immunogenicity. For example, techniques for conferring immunogenicity on a 20 peptide include conjugation to carriers or other techniques well known in the art. For example, the peptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassay can be used with the immunogen as antigen to assess the levels of antibodies.

Following immunization, antisera can be obtained and, if desired, polycional antibodies isolated from the sera. To produce monoclonal antibodies, antibody producing cells (lymphocytes) can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells. Such techniques are well known in the art. For example, the hybridoma 30 technique originaily deveioped by Kohler and Milstein (Nature (1975) 256:495-497) as well as other technicues such as the human B-cell hybridoma technique (Kozbar et al., Immunol.

Todav (19S3) the EBV-hvbridon a technique to produce human monoclonal antibodies (Cole et al. nor:ocion a :.i:nbo des "i Ccncer Theraoy (1985) (Allen R. Bliss. Inc.. pages 77- 96). and screenin: of combinatora! antibody libraries (Huse et ai.. Science (1989) 246:1275).

Hvbridoma cells can be sc rened imnmunochemically for production of antibodies specifically reactive with the pencide ,and monocional antibodies isolated.

The term a. .ody as used herein is intended to include fr~nents thereof which, ar also sprecifically reactive with a peptide having the activity of a novel B lyphoyt atge, or fusion protein as described herein. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab' fr-agents can be generated by treating antibody with pepsin. The resulting F(ab), frapient can be treated to reduce disulfide bridges to produce Fab' fiagents. The antibody of the present invention is frther intended to include bispecific and chimenic: molecules having an anti-B lymphocyte antigen, B7-2, B7-3) portion.

.Particularly preferred antibodies are anti.-human B7-2 monoclonal antibodies produced by hybri'domas HA3.1F9, HA5.2B7 and HF2.3D1. The preparationi and characterization of these antibodies is described in detail in Example 8. Monoclonal antibody HA3.1F9 was determined to be of the IgGi isotype; monoclonal antibody HA5.2B7 was d etermined to be of the IgG2b isotype; and monodlonal antibody HF2.3D1 was determined to be of the IgG2a isotype. Hybridoma cells were deposited with the American Type Culture Collection, which meets the requirements of the Budapest Treaty, on July 19, 1994 as ATCC Accession No. HB11688 (hybridoma HA3.1F9, ATCC Accession No. HB11087 (HAS.2B7) and ATCC Accession No. HB11686 (HF2.3DlJ.

When antibodies produced in non-human subjects are used thenreutically in humans, to 20 they are recognized to varying degrees as foreign and an immune response may be generated toL geea0~luoupes~l st rdc hiei nioydrvtvs in the patient One aprahfor minimni.ing or eliminating this problemn, which is preferable .0.molecules that cobn o-ua nmlvariable region and a humnan constant region.

to ~Chimeri4c antibodv molecules can include, for examnple, the antigen binding domain from an antibody of a mouse, rat, or other- species, with human constant regions. A variety of approaches for making chin eric antibodies have been descrbeancnbeuetom e chimeric antibodies containinz the immiunoglobuln variable region which recognizes the gene product of thie novel B lymphocyte antigens of the invention. Sez. for example, Morrison et al., Proc. iXarL Acad S'ci. U.S.A. 81:6851 (1985); Takeda et al., N~ature 314:452 30(1985), Cabilly et al, U.S. Parent No. 4,8 16,567; Boss et al., U.S. Paten; No. 4,816,3 97; Tanaguchi et al., European Patent Publication Elm171496; European Patent Publication 0173494, United KindomPatent rCE 21 77096B. It is ex:ec:ed that Such chimerlic antibodies would be less irm-unioenic in a human subject than the corresponding nion-chimneric antibody.

For humran zherapeutiic purposes, the monoclorai or chimeric arittbodies Specificaly reactive with a centide havi2 the ac:ivlr; of a B lvmophoc,/te antigen as describedi herein can.

be ftirher hurnanizted by croducina 'hu.n v ari'able r-2'iorl Cznirre:s. %vrt c wncn s of the variable regions, especially the conserved framnework regions of the ant~igen-binding domain, are of human on1m1 and only the hypervariable rgosaeo o-ua rgn eea reviews of "humnanize!d" chimeric antibodies are provided by Mlvorris on, S. L. (1985) Science 222:1202-1207 and by 0i et al. (1986) BioTechniaues 4:214. Such altered iimunoglobulin S molecules may be made by any of several techniques known in the art, (egITnge ,Proc. Nat!. Ica,! Sci. US.A., 30:7308-73 12 (1983); Kozbor et al., Immunology Today, 4:779 198);Olsson et al.,Met/i. En-7mol., 92:3-16 (1982)), and are preferab ly mad according to the teacainzs of PCT Publication W092/06 193 or EP 0239400. Humanized antibodies can be conmmercially produced by, for example. Scotgen Limited, 2Holly Road, Twickernam, Middlesex, Great Britain. Suitable "humanized" anibodies can be alternatively produced by CD)R or CEA substitution (see U.S. Patent 5.225,539 to Winter;, Jones et al. (1986) Nature 31:552- '125; Verhoevan er. al. (1988) Science 239:1534; and Beidler et al. (1983)] I.mmuo!. h1- :4053-4060). Humanized antibodies whi'ch -lfave reduced immunogenicity are orefer-red for imimunotherapy in human subjets Imtmunotherapy with a humanized antibody will likely reduce- the necessity for any concomitant iminunosuppress o n and may result in increased long term effectiveniess for te treatment of chronic disease situations or situations requiring repeated antibody treatments.

an alterntive to hiLmanimi a monoclonal antibody from a mouse or other species, human monoclon-al antibody direc.ed azains: a human protein can be aenerated. Transaenic mice carrying humian antibody rezneroires have been created which can. be linmunized with a human B lymohocy-te antizen. such as B7-2. Sclenoc-tes from these imunilzed traisgenilc mice can then be used to create hvbri1dornas that secrete hum an monoclo nal antibodies srecifically reactive with a human B lyvmphocyte antigeni (see, Wood e- al. PCT publication WO 9 1/00906. Kucheriacati er al. PCT publication WO 9 1/10741: Lonberg et al.

25 PCT publication WO 9120'0I S; Kay tt al. PCT publicati on 92/03 917: L onrbera. N. et al.

(1994) Narure 3L: S55- S59: Greenq. L et, al. 19 94) Nature Gene. 7:13-21; 1:Mvo rriso n. S.-L.

et 1 9 9 4) P r oc. .,Va tL. Sc. USA 4 31:6 85i1- 68 5 5 ;B r- 1 aem-an e: a. (-19 9 3) Ye7 Im mu ro 17: 3 3-140: T u a Io n a (19) .VS 90: 3 7'2037: an 3r a 7t -1.(191 Eur Jmmunol 61231t).

~vooioni antibd comcos zions of the invention can also be crod uced by other C e'thods well known to tcse .kiler, the ar- of recombinant DNA iec.1110ozv. An alte~mat:ve rtrhod.reere as tr"combirlatorli antibody disciay' rreth: od. has been :Z invent ion (for descniions o mc-ina~oriai anibody display seetc. Sast,-; ea!.(S9 PXSjS2; ueea.~jS~Sfrc -K7:and Oriandi e, 1989) P-V-.S' 8 ~3 S 3 3 A fte 2rmnzn na~n!wt B Ivnichoc%7t antice-n teacio -te-roire -33of the resulting B-cel pool is cloned. Methods are generally known for directly obtaining the DNA sequence of the variable regions of a diverse population of immunoglobulin molecules by using a mixture of oligomer primers and PCR. For instance, mixed oligonucleotide primers corresponding to the 5' leader (signal peptide) sequences and/or framework I (FRI) sequences, as well as primer to a conserved 3' constant region primer can be used for PCR amplification of the heavy and light chain variable regions from a number of murine antibodies (Larrick et al. (1991) Biotechniques 11:152-156). A similar strategy can also been used to amplify human heavy and light chain variable regions from human antibodies (Larrick et al. (1991) Methodsr: Companion to Methods in Enzvmology 2:106-110).

In an illustrative embodiment, RNA is isolated from activated B cells of, for example, peripheral blood cells, bond marrow, or spleen preparations, using standard protocols U.S. Patent No. 4,683,202; Orlandi, et al. PNAS (1989) 8:3833-3837; Sastrv et al., PNAS (1989) 86:5728-5732; and Huse et al. (1989) Science 246:1275-1281.) First-straind cDNA is synthesized using primers specific for the constant region of the heavy chain(s) and each of the K and X light chains, as well as primers for the signal sequence. Using variable region PCR primers, the variable regions of both heavy and light chains are amplified, each alone or in combinantion, and ligated into appropriate vectors for further manipulation in generating the display packages. Oligonucleotide primers useful in amplification protocols may be unique or degenerate or incorporate inosine at degenerate positions. Restriction endonuclease 20 recognition sequences may also be incorporated into the primers to allow fot the cloning of the amplified fragment into a vector in a predetermined reading frame for expression.

The V-gene library cloned from the immunization-derived antibody repertoire can be S expressed by a population of display packages, preferably derived from filamentous phage, to form an antibody display library. Ideally, the display package comprises a system that allows the sampling of very large diverse antibody display libraries, rapid sorting after each affinity separation round, and easy isolation of the antibody gene from purified display packages. In addition to commercially available kits for generating phage display libraries the Pharmacia Recombinant Phage .nribody System, catalog no. 27-9400-01; and the Stratagene SurrZ4P-TM v phage display kit. catalog no. 240612), examples of methods and reagents ;0 particularly amenable for use in generating a diverse antibody disolay library can be found in.

for example, Ladner et al. U.S. Patent No. 5,223.409; Kang et al. International Publication No. WO 92/18619: Dower e: al. International Publication No. WO 91/17271; Winter et a!.

International Publication WO 92/2079 1: Markland et al. International Publication No. WO 92/15079: Breit!ing et al. International Publication WO 93/01288: McCafferrv et a!.

International Publication No. WO 92'01047; Garrard et al. International Publication No. WO 92'09690: Ladner et al. International Publication No. WO 90/02809: Fuchs et al. (19 1) B3o/.1ech.nology 1370-1372: Hay et ai. 992) Hum A.qnibod HybridomCas 1:8 -85: Huse et -34al. (1989) Science 246:1275-1281; Griffths et al. (1993) EMBO J12:725-734; Hawkins et al.

(1992) JMol Biol 226:889-896; Clackson et al. (1991) Nature 3.2:624-628; Gram et al.

(1992) PNAS 39:3576-3580; Garrad et al. (1991) Bio/Technology 2:1373-1377; Hoogenboom et al. (1 9 9 1) NucAcid Res19:4133-4137; and Barbas et al. (1991) PNAS S:7978-7982.

In certain embodiments, the V region domains of heavy and light chains can be expressed on the same polypeptide, joined by a flexible linker to form a single-chain Fv fragment, and the scFV gene subsequently cloned into the desired expression vector or phage genome. As generally described in McCafferty et al., Nature (1990) 34.:552-554, complete VH and VL domains of an antibody, joined by a flexible (Gly 4 -Ser)3 linker can be used to produce a single chain antibody which can render the display package separable based on antigen affinity. Isolated scfV antibodies immunoreactive with a peptide having activity of a B lymphocyte antigen can subsequently be formulated into a pharmaceutica'preparation for use in the subject method. Once displayed on the surface of a display package filamentous phage), the antibody library is screened with a B lymphocyte antigen protein, or peptide fragment thereof, to identify and isolate packages that express an antibody having specificity for the B lymphocyte antigen. Nucleic acid encoding the selected antibody can be recovered from the display package from the phage genome) and subcloned into other expression vectors by standard recombinant DNA technicues.

The antibodies of the current invention can be used therapeutically tq inhibit T cell activation through blocking receptor:igand interactions necessary for costimulation of the T cell. Tnese so-called "biocking antibodies" can be identified by their ability to inhibit T cell proliferation and/or cytokine production when added to an in vitro costimulation assay as described herein. The ability of blocking antibodies to inhibit T cell functions may result in immunosuppression and/or tolerance when these antibodies are administered in vivo.

C. Protein Purification The polyclonal or monoclonal antibodies of the current invention, such as an antibody specifically reactive with a recombinant or synthetic peptide having B7-2 activity or B7-3 30 activity can also be used to isolate the native B lymphocyte antigen from cells. For example.

antibodies reactive with the peptide can be used to isolate the naturally-occurring or native tfoi of B7-2 from activated B lymphocytes by immunoaffinity chromatography. In addition, the native fom ofr B-3 can be isolated from B cells by immunoaffinity chromatography with monoclonai antibody BB-i.

D Other TherapticuRa nts The nucleic acid sequences and novel B lymphocyte anti described herein can be used in the deveopment of therapeutic reagents having the ability to either upregulate amplify) or downregulate suppress or tolerize) T cell mediated immune responses. For example, peptides having B7-2 activity, including soluble, monomeric forms of the B7-2 antigen or a B7-2 fusion protein, B7-2Ig, and anti-B7-2 antibodies that fail to deliver a costimulatory signal to T cells that have received a primary activation signal, can be used to block the B7-2 ligand(s) on T cells and thereby provide a specific means by which to cause immunosuppression and/or induce tolerance in a subject. Such blocking or inhibitory forms of B lymphocyte antigens and fusion proteins and blocking antibodies can be identified by their ability to inhibit T cell proliferation and/or cytokine production when added to an in vitro costimulation assay as previously described herein. In contrast to the rmonomeric form, stimulatory forms of B7-2, such as an intact cell surface B7-2, retain the ability to 6 transmit the costimulatory signal to the T cells, resulting in an increased secretion ofcytokines when compared to activated T cells that have not received the secondary signal.

In addition. fusion proteins comprising a first peptide having an activity of B7-2 fused to a second peptide having an activity of another B lymphocyte antigen B7-I) can be used to modify Tcell mediated immune responses. Alternatively, two separate peptides S* having an activity of B lymphocyte antigens, for example, B7-2 and B7-I or a combination 20 of blocking antibodies anti-B7-2 and anti-B 7- monoclonal antioodies' cn be combined as a single composition or administered separately (simultaneously or Ssequentially), to upregulate or downregulate T cell mediated immune responses in a subject.

Furthermore, a therapeutically active amount of one or more peptides havinE B7-2 activity and or B7-1 activity can be used in conjunction with other iimunomodulating reagents to Fmun e edsponses influence immune responses. Examples of other immunomodulating reagents include blocking antibodies, against CD28 or CTLA4, aeainst other T cell markers or against cvIoknes fu s 1 mre or agains1 cytokines, fusion proteins, CTLA4tg. or immunosuppressive drugs. cyclosporine

A

or FK506. cclosporine

A

The peptides produced from the nucleic acid molecules of the present also be usela thm e eoe present mvention may also be useful in the construction of therapeutic agents which block Tcel!! function by destruction of the T cell. For example. as described. secreted forms of a B lymohocvte rtigen can be constructed by standard genetic engneering technioues. By linking a soluble form ofB7-I, B7-2 or B7-3 to a toxin such as ricin. an agent capable ofCpreventin, T ceil acvaion can be mad. Infusion of one or a combination of immunotoxins, B7-2-ricin.

B7-l-ricin. into a parie-nt may result in th- death ofT ceils. m iaicurly o ac vaed T cels that express higher amounts of CD2S and CTLA4. Soluble forms of in a monovalent -36form alone may be useful in blocking B7-2 function, as described above, in which case a carrier molecule may also be employed.

Another method of preventing the function of a B lymphocyte antigen is through the use of an antisense or triplex oligonucleotide. For example, an oligonucleotide complementary to the area around the B7-1, B7-2 or B7-3 translation initiation site, for B7-1, TGGCCCATGGCTTCAGA, (SEQ ID NO:20) nucleotides 326-309 and for B7-2, GCCAAAATGGATCCCCA (SEQ ID NO:21)), can be synthesized. One or more antisense oligonucleotides can be added to cell media, typically at 200 .g/ml, or administered to a patient to prevent the synthesis of B7-1, B7-2 and/or B7-3. The antisense oligonucleotide is taken up by cells and hybridizes to the appropriate B lymphocyte antigen mRNA to prevent translation. Alternatively, da oligonucleotide which binds double-stranded DNA to form a triplex construct to prevent DNA unwinding and transcription can be used. As a result of either, synthesis of one or more B lymphocyte antigens is blocked. E. Therapeutic ULses by Downreguiation of Immune Responses Given the structure and function of the novel B lymphocyte antigens disclosed herein, it is possible to downregulate the function of a B lymphocyte antigen, and thereby downregulate immune responses, in a number of ways. Downregulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing 20 the induction of an immune response. The functions of activated T cells may be inhibited by o9 suppressing T cell responses or by inducing specific tolerance in T cells, or both.

Immunosuppression ofT cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent. Tolerance, which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased. Operationally. tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.

Downregulating or preventing one or more B lymphocvte antigen functions, e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations 30 of tissue. skin and organ transplantation and in graft-versus-host disease (GVHD). For example, blockage ofT cell function should result in reduced tissue destruction in tissue transplantation. Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells. followed by an immune reaction that destroys the transolant. The administration of a molecuie which inhibits or blocks interaction of a B7 [ymphocyte antigen with its natural ligand(s) on immune cells (such as a soluble, monomeric form of a peptide having B7-2 activity alone or in conjunction with a monomeric form of a pentide having n a ctivity ofanother B lvmchocvte antigen B7-1. B7-3) or blocking -37antibody), prior to transplantation can lead to the binding of the molecule to the natural ligand(s) on the immune cells without transmitting the corresponding costimulatory signal.

Blocking B lymphocyte antigen function in this manner prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject. Induction of long-term tolerance by B lymphocyte antigen-biocking reagents may avoid the necessity of repeated administration of these blocking reagents. To acheive suficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination ofB lymphocyte antigens. For example, it may be desirable to block the function of B7-2 and B7-1, B7-2 and B7-3, B7-1 and B7-3 or B7-2, B7-1 and B7-3 by administering a soluble fdn of a combination of peptides having an activiti of each of these antigens or a blocking antibody (separately or together in a single composition) prior to transplantation. Alternatively, inhibitory forms of B lymphocyte antigens can-oe ued with other suppressive agents such as blocking antibodies against other T cell markers or against cytokines, other fusion proteins, CTLA4Ig, or immunosuppressive drugs.

The efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans. The Sfunctionally important aspects of B7-1 are conserved structurally bevween species and it is therefore likely that other B lymphocyte antigens can function across species, thereby 2 0 allowing use of reagents composed of human proteins in animal systems. Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al., Science, 27: 789-792 (1992) and Turka et al., Proc. Nat. Acad Sci. USA, 9: 11102- 11105 (1992). In addition. murine models of GVHD (see Paul ed.. Fundamental Immunoloy, Raven Press, New York. 1989, pp. 846-847) can be used to determine the effect o r blocking B lymphocyte antigen function in vivo on the development of that disease.

Blocking B iymphocyte antigen function, by use ofa peptide having B7-2 activity alone or in combination with a peptide having 87-1 activity and/or a peptide having 0 B7-3 activity. may also be therapeutically useful for treating autoimmune diseases. Many autoimmune disorders are the result of inappropriate activation ofT ce!ls that are reactive against se!f tissue and which promote the production ofcytokines and autoantibodies involved in the pathoiogy of the diseases. Preventing the activation o autoreactive T cells may reauce or eliminate disease symptoms. Administration of reaents which block S cosimulation of T cells by disruoting receopor:iigand interactions of B Ivmohocvte antigens can be used to inhibit T cell ac:ivation and prevent production ofautoantibodies or T ce!Iderived cvtokines which may be involved in the disease process. Additionally, blocking -38reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease. The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized 'animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/pr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamenral Immunology, Raven Press, New York, 1989, pp. 840-856).

The IgE antibody response in atopic allergy is highly T cell dependent and, thus, inhibition of B lymphocyte antigen induced T cell activation may be useful therapeutically in the treatment of allergy and allergic reactions. An inhibitory form of B7-2 protein, such as a peptide having B7-2 activity alone or in combination with a peptide having the activity of another B lymphocyte antigen, such as B7-1, can b administered to an allergic subject to inhibit T cell mediated allergic responses in the subject. Inhibition ofB lymphocyte antigen costimulation ofT cells may be accompagnied by exposure to allergen in conjunction with appropriate MHC molecules.

Allergic reactions may be systemic or local in nature, depending on the route of entry of the allergen and the pattern of deposition of IgE on mast cells or basophils. Thus, it may be necessary to inhibit T ceil mediated allergic responses locally or systemically by proper administration of an inhibitory form of B7-2 protein.

Inhibition of T cell activation through blockage of B lymphocte antigen function may also be important therapeutically in viral infections ofT cells. For example, in the acquired immune deficiency syndrome (AIDS), viral replication is stimulated by T cell activation. Blocking B7-2 function could lead to a lower level of viral replication and 25 thereby ameliorate the course of AIDS. In addition, it may also be necessary to block the function of a combination ofB lymphocyte antigens B7-1, B7-2 and B7-3. Surprisingly, HTLV-I infected T cells express B7-1 and B7-2. This expression may be important in the growth of HTLV-I infected T cells and the blockage of B7-1 function to-geher with the function of B7-2 and/or B7-3 may slow the growth of HTLV-I induced leukemias.

30 Alternatively, stimulation of viral replication by T cell activation may be induced by contact with a stimulator form of 37-2 protein. for such purposes as generating retroviruses various HIV isolates) in sufficient cuantities for isolatation and use.

F. Therapeutic f:ses hv i7:reguiation of mmune Responses Upreguiation o a B lymphocyre antigen function. as a means of upregulating immune responses. may also be usefli in therapy. Upreguiation or immune resoonses may be in the forn or ennancing an existing immune response or eliciting an initial immune response. For -39example, enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection. Viral infections are cleared primarily by cytolytic T cells. In accordance with the present invention, it is believed that B7-2 and thus, B7-1 and B7-3 with their natural ligand(s) on T cells may result in an increase in the cytolytic activity of at least some T cells. It is also believed that B7-2, B7-1, and B7-3 are involved in the initial activation and generation of CD8+ cytotoxic T cells. The addition of a soluble peptide having B7-2 activity, alone, or in combination with a peptide having the activity of another B lymphocyte antigen, in a multi-valent form, to stimulate T cell activity through the costimulation pathway would thus be therapeutically useful in situations where more rapid or thorough clearance of virus would be beneficial. These would include viral skin diseases such as Herpes or shingles, in which cases the multi-valent soluble peptide haviing B7-2 activity or combination of such peptide and/or a peptide having B7-1 activityand/or a peptide having B7-3 activity is delivered topically to the skin. In addition, systemic viral-diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of stimulatory forms of B lymphocyte antigens systemically.

Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide having B7-2 activity (alone or in combination with a peptide having B7-1 activity and/or a peptide having B7-3 activity) or together with a 0 stimulatory form of a soluble peptide having B7-2 activity (alone or in combination with a peptide having B7-1 activity and/or a peptide having B7-3 activity) and reintroducing the in vitro activated T cells into the patient. Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a peptide having the activity of a B lymphocyte antigen as described herein such .5 that the cells express all or a portion ofa B lymphocyte antigen on their surface, B7-2 or B7-3. and reintroduce the transfected ce!Is into the patient. The infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.

Stimulatory forms of B lymphocyte antigens may also be used prophylactically in vaccines against various pathogens. Immunity against a pathogen. a virus, could be induced by vaccinating with a viral protein along with a stimulatory form ofa peptide having B7-2 activity or another peptide having the activity ofB lymphocyte antigen in an appropriate adiuvant. Altemrately. an expression vector which encodes genes for both a pathogenic antigen and a peptide having the activity ofa B lymphocyte antigen. a vaccinia virus expression vector engineered to express a nucleic acid encoding a viral protein and a nucleic acid encoding a peptide having B7-2 activity as described herein. can be used for vaccination. Presentation of B7-2 with class I MHC proteins by. for example, a ce!l transre=:ed to coexpress a peptide having B7-2 activity and MHC class i a chain rotein and P2 microglobulin may also result in activation of cytolytic CD8+ T cells and provide immunity from viral infection. Pathogens for which vaccines may be useful include hepatitis B, hepatitis C, Epstein-Barr virus, cytomegalovirus, HIV-1, HIV-2, tuberculosis, malaria and schistosomiasis.

In another aspect, a stimulatory form of one or more soluble peptides having an activity of a B lymphocyte antigen can be administered to a tumor-bearing patient to provide a costimulatory signal to T cells in order to induce anti-tumor immunity.

G. Modification of a Tumor Cell to Express a Costimulatorv Molecule The inability of a tumor cell to trigger a costimulatory signal in T cells may be due to a lack of expression of a co'timulatory molecule, failure to express a costimulatory molecule even though the tumor cell is capable of expressing such a molecule, insufficient expression of a costimulatory molecule on the tumor cell surface or lack of expression of a"-appropriate costimulatory molecule expression of B7 but not B7-2 and/or B7-3). Thus, according to one aspect of the invention, a tumor cell is modified to express B7-2 and/or B7-3 by transfection of the tumor cell with a nucleic acid encoding B7-2 and/or B7-3 in a form suitable for expression of B7-2 and/or B7-3 on the tumor cell surface. Alternatively, the tumor cell is modified by contact with an agent which induces or increases expression ofB7- 2 and/or B7-3 on the tumor cell surface. In yet another embodiment, B7-2 and/or B7-3 is 20 coupled to the surface of the tumor cell to produce a modified tumor cell. These and other emodiments are described in further detail in the following subsections.

0 (1M. Transfection of a Tumor Cell with a Nucleic Acid Encoding a Costimulatory Molecule 25 Tumor cells can be modified ex vivo to express B7-2 or B7-3, alone or in combination or in combination with B7-1 by transfection of isolated tumor cells with a nucleic acid Sencoding B7-2 and/or B7-3 and B7-1 in a form suitable for expression of the molecule on the S surface of the tumor cell. The terms "transfection" or "transfected with" refers to the introduction of exogenous nucleic acid into a mammalian cell and encompass a variety of 30 techniques useful for introduction of nucleic acids into mammalian cells including electroporation, calcium-phosphate precipitation, DEAE-dextran treatment, lipofection, microinjection and infection with viral vectors. Suitable methods for transfecting mammalian cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual.

2nd Edition. Cold Spring Harbor Laboratory press (1989)) and other laboratory textbooks.

The nucleic acid to be introduced may be. for example. DNA encompassing the gene(s) encoding B7-2 and/or B7-3. sense strand RNA encoding B7-2 andor B7-3 or a recombinant -41expression vector containing a cDNA encoding B7-2 and/or B7-3. The nucleotide sequence of a cDNA encoding human B7-2 is shown in the Sequence Listing.

A preferred approach for introducing nucleic acid encoding B7-2 and/or B7-3 into tumor cells is by use of a viral vector containing nucleic acid, e.g. a cDNA, encoding B7-2 and/or B7-3. Examples of viral vectors which can be used include retroviral vectors (Eglitis, et al., Science 230, 1395-1398 (1985); Danos, 0. and Mulligan, Proc. Natl. Acad.

Sci. USA 85, 6460-6464 (1988); Markowitz, et al., J. Virol. 62, 1120-1124 (1988)), adenoviral vectors (Rosenfeld, et al., Cell 68, 143-155 (1992)) and adeno-associated viral vectors (Tratschin, et al., Mol. Cell. Biol. 5, 3251-3260 (1985)). Infection of tumor cells with a viral vector has the advantage that a large proportion of cells will receive nucleic acid, thereby obviating a need for selection of cells which have received nucleic acid, and molecules encoded within the viral vector, e.g. by a cDNA contained in the viral vector, are expressed efficiently in cells which have taken up viral vector nucleic acid.

Alternatively, B7-2 and/or B7-3 can be expressed on a tumor cell using a plasmid expression vector which contains nucleic acid, e.g. a cDNA, encoding B7-2 and/or B7-3.

Suitable plasmid expression vectors include CDM8 (Seed, Nature 329, 840 (1987)) and pMT2PC (Kaufman. et al., EMBO J. 6, 187-195 (1987)). Suitable vectors and methods for expressing nucleic acids in host cells, such as tumor cells are described in further detail herein.

'20 When transfection of tumor cells leads to modification of a large proportion of the S t umor cells and efficient expression of B7-2 and/or B7-3 on the surface of tumor cells, e.g.

when using a viral expression vector, tumor cells may be used without further isolation or subcloning. Alternatively, a homogenous population of transfected tumor cells can be prepared by isolating a single transfected tumor cell by limiting dilution cloning followed by 5 expansion of the single tumor cell into a clonal population of cells by standard techniques.

Induction or Increased Expression of a Costimulatorv Molecule on a Tumor Cell Surface A tumor cell can be modified to trigger a costimulatory signal in T cells by inducing or increasing the level of expression of B7-2 and/or B7-3 on a tumor cell which is capable of expressing B7-2 and/or B7-3 but fails to do so or which expresses insufficient amounts of B7-2 and/or B7-3 to activate T cells. An agent which stimulates expression of B7-2 and/or B7-3 can be used in order to induce or increase expression of B7-2 and/or B7-3 on the tumor cell surface. For example, tumor cells can be contacted with the agent in vitro in a culture medium. The agent which stimulates expression of B7-2 and/or B7-3 may act. for instance, by increasing transcription of B7-2 and/or B7-3 gene, by increasing translation of B7-2 and/or B7-3 mRNA or by increasing stability or transport of B7-2 and/or B7-3 to the cell -42surface. For example, it is known that expression of B7 can be upregulated in a cell by a second messenger pathway involving cAMP. Nabavi, et al. Nature 360, 266-268 (1992).

B7-2 and B7-3 may likewise be inducible by cAMP. Thus, a tumor cell can be contacted with an agent, which increases intracellular cAMP levels or which mimics cAMP, such as a cAMP analogue, e.g. dibutyryl cAMP, to stimulate expression of B7-2 and/or B7-3 on the tumor cell surface. It is also known that expression of B7 can be induced on normal resting B cells by crosslinking cell-surface MHC class II molecules on the B cells with an antibody against the MHC class II molecules. Kuolova, et al., J. Exp. Med 173, 759-762 (1991).

Similarly, B7-2 and B7-3 can be induced on resting B cells by crosslinking cell-surface MHC class II molecules on the B cells. Accordingly, a tumor cell which expresses MHC class II molecules on its surface car be treated with anti-MHC class II antibodies to induce or increase B7-2 and or B7-3 expression on the tumor cell surface. In addition, interleukin-4 (IL-4) which has been found to induce expression of B7-2 on B cells, may be.used:to upregulate expression of B7-2 on tumor cells (Stack et al., J. Cell. Biochem. Suppl 1(18):434 (1994).

Another agent which can be used to induce or increase expression of B7-2 and/or B7- 3 on a tumor cell surface is a nucleic acid encoding a transcription factor which upregulates transcription of the gene encoding the costimulatory molecule. This nucleic acid can be transfected into the tumor cell to cause increased transcription of the costimulatory molecule 20 gene, resulting in increased cell-surface levels of the costimulatory molecule.

Coupling of a Costimulatorv Molecule to the Surface of a Tumor Cell In another embodiment, a tumor cell is modified to be capable of triggering a costimulatory signal in T cells by coupling B7-2 and/or B7-3 to the surface of the tumor cell.

For example, B7-2 and/or B7-3 molecules can be obtained using standard recombinant DNA technology and expression systems which allow for production and isolation of the costimulatory molecule(s). Alternatively, B7-2 and/or B7-3 can be isolated from cells which S express the costimulatory molecule(s) using standard protein purification techniques. For example, B7-3 protein can be isolated from activated B cells by immunoprecipitation with an anti-B7-3 antibody such as the BB 1 monoclonal antibody. The isolated costimulatory molecule is then coupled to the tumor cell. The terms "coupled" or "coupling" refer to a chemical, enzymatic or other means antibody) by which B7-2 and/or B7-3 is linked to a tumor cell such that the costimulatory molecule is present on the surface of the tumor cell and is capable of triggering a costimulatory signal in T cells. For example. B7-2 and/or B7-3 can be chemically crosslinked to the tumor ceil surface using commercially available crosslinking reagents (Pierce, Rockford IL). Another approach to coupling B7-2 and/or B7-3 to a tumor cell is to use a bisnecific antibody which binds both the costimulatory molecuie and a cell- -43surface molecule on the tumor cell. Fragments, mutants or variants of B7-2 and/or B7-3 which retain the ability to trigger a costimulatory signal in T cells when coupled to the surface of a tumor cell can also be used.

Modification of Tumor Cells to Express Multiple Costimulatorv Molecules Another aspect of the invention is a tumor cell modified to express multiple costimulatory molecules. The temporal expression of costimulatory molecules on activated B cells is different for B7, B7-2 and B7-3. For example, B7-2 is expressed early following

B

cell activation, whereas B7-3 is expressed later. The different costimulatory molecules may thus serve distinct functions during the course of an immune response. An effective T cell response may require that the T cell receive costimulatory signals from multiple costimulatory molecules. Accordingly, the invention encompasses a tumor.cll which is modified to express more than one costimulatory molecule. For example, a tumo- cell can be modified to express both B7-2 and B7-3. Alternatively, a tumor cell modified to express B7- 2 can be further modified to express B7-1. Similarly, a tumor cell modified to express B7-3 can be further modified to express B7-1. A tumor cell can also be modified to express B7-1, B7-2 and B7-3. A tumor cell can be modified to express multiple costimulatory molecules B7-1 and B7-2) by any of the techniques described herein.

Before modification, a tumor cell may not express any costimulatory molecules, or may express certain costimulatory molecules but not others. As described herein, tumor cells 0 can be modified by transfecting the tumor cell with nucleic acid encoding a costimulatory *0 molecule(s), by inducing the expression of a costimulatory molecule(s) or by coupling a 0 costimulatory molecule(s) to the tumor cell. For example, a tumor cell transfected with nucleic acid encoding B7-2 can be further transfected with nucleic acid encoding B7-1. The 25 cDNA sequence and deduced amino acid sequence of human B7-1 is shown in the Sequence Listing. Alternatively, more than one type of modification can be used. For example, a tumor cell transfected with a nucleic acid encoding B7-2 can be stimulated with an agent which induces expression of B7-1.

30 Additional Modification of a Tumor Cell to Express MHC Molecules o.e. Another aspect of this invention features modified tumor cells which express a costimulatory molecule and which express one or more MHC molecules on their surface to tngger both a costimulatory signal and a primary, antigen-specific, signal in T cells. Before modification, tumor cells may be unable to express MHC molecules. may fail to express MHC molecules although they are capable of expressing such molecules, or may express insufficient amounts of MHC molecules on the tumor cell surface to cause T cell activation.

Tumor cells can be modified to express either MHC class I or MHC class II molecules, or both. One approach to modifying tumor cells to express MHC molecules is to transfect the tumor cell with one or more nucleic acids encoding one or more MHC molecules.

Alternatively, an agent which induces or increases expression of one or more MHC molecules on tumor cells can be used to modify tumor cells. Inducing or increasing expression of MHC class II molecules on a tumor cell can be particularly beneficial for activating CD4 T cells against the tumor since the ability of MHC class II tumor cells to directly present tumor peptides to CD4 T cells bypasses the need for professional MHC class II+ APCs. This can improve tumor immunogenicity because soluble tumor antigen (in the form of tumor cell debris or secreted protein) may not be available for uptake by professional MHC class II APCs.

One embodiment of the invention is a modified tumor cell which expresses B7-2 and/or B7-3 and one or more MHC class II molecules on their cell surface. MHC class II molecules are cell-surface a/P heterodimers which structurally contain a cleft irnto-which antigenic peptides bind and which function to present bound peptides to the antigen-specific TcR. Multiple, different MHC class II proteins are expressed on professional APCs and different MHC class II proteins bind different antigenic peptides. Expression of multiple MHC class II molecules, therefore, increases the spectrum of antigenic peptides that can be presented by an APC or by a modified tumor cell. The ct and P chains of MHC class II molecules are encoded. by different genes. For instance, the human MHC class II protein HLA-DR is encoded by the HLA-DRca and HLA-DRP genes. Additionally,, many polymorphic alleles of MHC class II genes exist in human and other species. T cells of a particular individual respond to stimulation by antigenic peptides in conjunction with self MHC molecules, a phenomenon termed MHC restriction. In addition, certain T cells can also respond to stimulation by polymorphic alleles of MHC molecules found on the cells of other 25 individuals, a phenomenon termed allogenicity. For a review of MHC class II structure and function, see Germain and Margulies, Ann. Rev. Immunol. 11: 403-450, 1993.

Another embodiment of the invention is a modified tumor cell which expresses B7-2 S and/or B7-3 and one or more MHC class I molecules on the cell surface: Similar to MHC class II genes, there are multiple MHC class I genes and many polymorphic alleles of these 30 genes are fournd in human and other species. Like MHC class II proteins, class I proteins bind peptide fragments of antigens for presentation to T cells. A functional cell-surface class I molecule is composed of an MHC class I ca chain protein associated with a 12microglobulin protein.

Trnsfection of a Tumor Cell with Nucleic Acid Encoding MHC Molecules Tumor cells can be modified ex vivo to express one or more MHC class II molecules by transfection of isolated tumor cells with one or more nucleic acids encoding one or more MHC class II a chains and one or more MHC class II 3 chains in a form suitable for expression of the MHC class II molecules(s) on the surface of the tumor cell. Both an a and a p chain protein must be present in the tumor cell to form a surface heterodimer and neither chain will be expressed on the cell surface alone. The nucleic acid sequences of many murine and human class I genes are known. For examples see Hood, et al. Ann. Rev. Immunol. 1, 529-568 (1983) and Auffray, C. and Strominger, Advances in Human Genetics 15, 197- 247 (1987). Preferably, the introduced MHC class II molecule is a self MHC class II molecule. Alternatively, the MHC class II molecule could be a foreign, allogeneic, MHC class II molecule. A particular foreign MHC class II molecule to be introduced into tumor cells can be selected by its ability to induce T cells from a tumor-bearing subject to proliferate and/or secrete cyt6kines when stimulated by cells expressing the foreign MHC class II molecule by its ability to induce an allogeneic response). The tumor cells to be transfected may not express MHC class II molecules on their surface prior to transfection or may express amounts insufficient to stimulate a T cell response. Alternatively, tumor cells which express MHC class II molecules prior to transfection can be further transfected with additional, different MHC class II genes or with other polymorphic alleles of MHC class II genes to increase the spectrum of antigenic fragments that the tumor cells can present to T cells.

Fragments, mutants or variants of MHC class II molecules that retain the ability to 20 bind peptide antigens and activate T cell responses, as evidenced by prolifertion and/or lymphokine production by T cells, are considered within the scope of the invention. A preferred variant is an MHC class II molecule in which the cytoplasmic domain of either one or both of the a and P chains is truncated. It is known that truncation of the cytoplasmic domains allows peptide binding by and cell surface expression of MHC class II molecules but 25 prevents the induction of endogenous B7 expression, which is triggered by an intracellular signal generated by the cytoplasmic domains of the MHC class II protein chains upon crosslinking of cell surface MHC class II molecules. Kuolova. et al., J. Exp. Med. 173, 759-762 (1991); Nabavi, et al. Nature 360, 266-268 (1992). Expression of B7-2 and B7-3 is also induced by crosslinking surface MHC class II molecules, and thus truncation of MHC S 30 class II molecules may also prevent induction of B7-2 and/or B7-3. In tumor cells transfected 'es" to constitutively express B7-2 and/or B7-3, it may be desirable to inhibit the expression of endogenous costimulatory molecules, for instance to restrain potential downregulatory feedback mechanisms. Transfection of a tumor cell with a nucleic acid(s) encoding a cytoplasmic domain-truncated form of MHC class II ac and P chain proteins would inhibit endogenous B7-1 expression and possibly also endogenous B7-2 and B7-3 expression. Such variants can be produced by. for example, introducing a stop codon in the MHC class II chain gene(s) after the nucleotides encoding the transmembrane spanning region. The cytoplasmic -46domain of either the a chain or the P chain protein can be truncated, or, for more complete inhibition of B7 (and possibly B7-2 and/or B7-3) induction, both the a and P chains can be truncated. See e.g. Griffith et al., Proc. Natl. Acad. Sci. USA 85: 4847-4852, (1988), Nabavi et al., J. Immunol. 142: 1444-1447, (1989).

Tumor cells can be modified to express an MHC class I molecule by transfection with a nucleic acid encoding an MHC class I a chain protein. For examples of nucleic acids see Hood, et al. Ann. Rev. Immunol. 1, 529-568 (1983) and Auffray, C. and Strominger, J.L., Advances in Human Genetics 15, 197-247 (1987). Optionally, if the tumor cell does not.

express P-2 microglobulin, it can also be transfected with a nucleic acid encoding the p-2 microglobulin protein. For examples of nucleic acids see Gussow, et al., J. Immunol. 139, 3132-3138 (1987) and Parns, et al., Proc. Natl. Acad. Sci. USA 78, 2253-2257 (1981).

As for MHC class II molecules, increasing the number of different MHC class I genes or polymorphic alleles of MHC class I genes expressed in a tumor cell can increisethe.spectrum of antigenic fragments that the tumor cells can present to T cells.

When a tumor cell is transfected with nucleic acid which encodes more than one molecule, for example a B7-2 and/or B7-3 molecule(s), an MHC class II a chain protein and an MHC class II P chain protein, the transfections can be performed simultaneously or sequentially. If the transfections are performed simultaneously, the molecules can be introduced on the same nucleic acid, so long as the encoded sequences do not exceed a 20 carrying capacity for a particular vector used. Alternatively, the molecules cqn be encoded by separate nucleic acids. If the transfections are conducted sequentially and tumor cells are selected using a selectable marker, one selectable marker can be used in conjunction with the ffirst introduced nucleic acid while a different selectable marker can be used in conjunction with the next introduced nucleic acid.

S 25 The expression of MHC molecules (class I or class II) on the cell surface of a tumor cell can be determined, for example, by immunoflourescence of tumor cells using Sfluorescently labeled monoclonal antibodies directed against different MHC molecules.

Monoclonal antibodies which recognize either non-polymorphic regions of a particular MHC molecule (non-allele specific) or polymorphic regions of a particular MHC molecule (allelespecific) can be used and are known to those skilled in the art.

Induction or Increased Expression of MHC Molecules on a Tumor Cell Another approach to modifying a tumor cell ex vivo to express MHC molecules on the surface of a tumor cell is to use an agent which stimulates expression of MHC molecules in order to induce or increase expression of MHC molecules on the tumor cell surface. For example, tumor cells can be contacted with the agent in vitro in a culture medium. An agent which stimulates expression of MHC molecules may act. for instance, by increasing -47transcription of MHC class I and/or class II genes, by increasing translation of MHC class I and/or class II mRNAs or by increasing stability or transport of MHC class I and/or class II proteins to the cell surface. A number of agents have been shown to increase the level of cell-surface expression of MHC class Ii molecules. See for example Cockfield, S.M. et al., j Immunol. 144,2967-2974 (1990); Noelle, R.J. et al. J. Immunol. 137, 1718-1723 (1986); Mond, et al., J Immunol. 127, 881-888 (1981); Willman, et al. J Exp. Med., 170, 1559-1567 (1989); Celada, A.and Maki, R. J Immunol 146, 114-120 (1991) and Gimcher, L.H. and Kara, C.J. An Rev. Immunol. 0, 13-49 (1992) and references therein. These agents include cytokines, antibodies to other cell surface molecules and phorbol esters. One agent which upregulates MHC class I and class II molecules on a wide variety of cell types is the cytokine interferon-y. Tus, for example, tumor cells modified to express B7-2 and/or B7-3 and B7-1 can be further modified to increase expression ofMHC molecules by contact with interferon-y.

Another agent which can be used to induce or increase expression of an MHC molecule on a tumor cell surface is a nucleic acid encoding a transcription factor which upregulates transcription of MHC class I or class II genes. Such a nucleic acid can be transfected into the tumor cell to cause increased transcription of MHC genes, resulting in Sncreased cell-surface levels of MHC proteins. MHC class I and class II genes are regulated *by different transcription factors. However, the multiple MHC class I genes are regulated coordinately, as are the multiple MHC class II genes. Therefore, transfectioh of a tumor cell with a nucleic acid encoding a transcription factor which regulates MHC gene expression may increase expression of several different MHC molecules on the tumor cell surface.

S everal transcription factors which regulate the expression of MHC genes have been Sidentified, cloned and characterized. For example, see Reith, W. et al., Genes Dev. 4, 1528- 1540, (1990); Liou. et al., Science 247, 1581-1584 (1988); Didier, et al., Proc.

Natl. Acad Sci. USA 85, 7322-7326 (1988).

Inhiition of Tnvariant hain presion in Tumor ell Another embodiment of the invention provides a tumor cell modified to express a T cell costimulatory molecule B7-2 and/or B7-3 and B7-1) and in which expression of an MHC class I-associated protein, the invariant chain, is inhibited. Invariant chain expression is inhibited to promote association of endogenously-derived TAA peptides with MHC class II molecules to create an antigen-MHC compiex. This complex can trigger an antigen-specific signal in T cells to induce activation of T cells in conjunction with a costimulatory signal.

MHC class II molecules have been shown to be capable of presenting endogenouslv-derived peptides. Nuchtem. et al. Nature 343, 74-76 (1990): Weiss. S. and Bogen. B. Cell 767- 776 (1991). However. in cells which naturally express MHC class II molecules, the c and 0 -48chain proteins are associated with the invariant chain (hereafter li) during intracellular transport of the proteins from the endoplasmic reticulum. It is believed that li functions in part by preventing the association of endogenously-derived peptides with MHC class II molecules. Elliott, et al. J Immunol. 138, 2949-2952 (1987); Stockinger, et al. Cell 56, 683-689 (1989); Guagliardi, et al. Nature (London) 343, 133-139 (1990); Bakke, O., et al. Cell 63, 707-716 (1990); Lottreau, et al. Nature 348,600-605 (1990); Peters, et al. Nature 349, 669-676 (1991); Roche, et al.Nature 345, 615-618 (1990); Teyton, et al. Nature 348, 39-44 (1990). Since TAAs are synthesized endogenously in tumor cells, peptides derived from them are likely to be available intracellularly. Accordingly, inhibiting the expression of Ii in tumor cells which express Ii may increase the likelihood that TAA peptides will associate with MIHC class II molecules. Consistent with this mechanism, it was shown that supertransfection of an MHC class II+, Ii- tumor cell with the Ii gene prevented stimulation of tumor-specific immunity by the tumor cell. Clements, et-l. -lmmunol.

149, 2391-2396 (1992).

Prior to modification, the expression of Ii in a tumor cell can be assessed by detecting the presence or absence of Ii mRNA by Northern blotting or by detecting the presence or absence of Ii protein by immunoprecipitation. A preferred approach for inhibiting expression of Ii is by introducing into the tumor cells a nucleic acid which is antisense to a coding or regulatory region of the Ii gene, which have been previously described. Koch, et al., 20 EMBOJ. 6, 1677-1683, (1987). For example, an oligonucleotide complementary to nucleotides near the translation initiation site of the Ii mRNA can be synthesized. One or more antisense oligonucleotides can be added to media containing tumor cells, typically at a \ee concentration of oligonucleotides of 200 g/ml. The antisense oligonucleotide is taken up by tumor cells and hybridizes to li mRNA to prevent translation. In another embodiment, a 25 recombinant expression vector is used in which a nucleic acid encoding sequences of the li gene in an orientation such that mRNA which is antisense to a coding or regulatory region of the Ii gene is produced. Tumor cells transfected with this recombinant expression vector thus contain a continuous source of Ii antisense nucleic acid to prevent production of Ii protein.

Alternatively, li expression in a tumor cell can be inhibited by treating the tumor cell with an agent which interferes with Ii expression. For example, a pharmaceutical agent which inhibits ii gene expression. Ii mRNA translation or Ii protein stability or intracellular transport can be used.

Tvpes of Tumor Cells to be Modified The tumor cells to be modified as described herein include tumor cells which can be transfected or treated by one or more of the approaches encompassed by the present invention to express B7-2 and or B7-3. alone or in combination with B7-1. If necessary. the tumor -49cells can be further modified to express MHC molecules or an inhibior of Ii expression.

A

tumor from which tumor cells are obtained can be one that has arisen spontaneously, e.g in a human subject, or may be experimentally derived or induced, e.g. in an animal subject. The tumor cells can be obtained, for example, from a solid tumor of an organ, such as a tumor of the lung, liver, breast, colon, bone etc. Malignancies of solid organs include carcinomas, sarcomas, melanomas and neuroblastomas. The tumor cells can also be obtained from a blood-borne (ie. dispersed) malignancy such as a lymphoma, a myeloma or a leukemia.

The tumor cells to be modified include those that express MHC molecules on their cell surface prior to transfection ahd those that express no or low levels of MHC class I and/or class II molecules. A minority of normal cell types express MHC class II molecules.

It is therefore expected that Aany tumor cells will not express MHC class II molecules naturally. These tumors can be modified to express B7-2 and/or B7-3 and MHC class II molecules. Several types of tumors have been found to naturally express surficeMHC class II molecules, such as melanomas (van Duinen et al., Cancer Res. 48, 1019-1025, 1988), diffuse large cell lymphomas (O'Keane et al., Cancer 66, 1147-1153 1990), suaous cell carcinomas of the head and neck (Mattijssen et al., Int. J Cancer 6, 95-100, 1991) and colorectal carcinomas (Moller et al., Int. J Cancer6, 155-162, 1991). Tumor cells which S. naturally express class II molecules can be modified to express B7-2 and/or B7-3, and, in addition, other class II molecules which can increase the spectrum of TA peptides which :20 can be presented by the tumor cell. Most non-malignant cell types express MNHC class

I

molecules. However, malignant transformation is often accompanied by downregulation of expression of MHC class I molecules on the surface of tumor cells. Csiba, et al., Brit. J Cancer 50, 699-709 (1984). Importantly, loss of expression of MHC class I antigens by tumor cells is associated with a greater aggressiveness and/or metastatic potential of the tumor cells. Schrier, et al. Nature 305, 771-775 (1983); Holden, et al. Am. Acad S Dermatol. 867-871 (1983); Baniyash, et al. J Immunol. 129, 318-1323 (1982).

Types of tumors in which M H C class I expression has been shown to be inhibited include melanomas, colorectal carcinomas and squamous cell carcinomas van Duinen et al., Cancer Res. 48, i019-1025, (1988); oller et nt. Cancer 6, 155-162 (1991); Csiba. et al., Brit. J Cancer 50, 699-709 (1984): Holden. et al. J Am. Acad. Dermatol. 867-871 (1983). A tumor cell which fails to express class I molecules or which expresses only low levels of MHC class I molecules can be modified by one or more of the techniques described herein to induce or increase expression of MHC class I molecules on the tumor cell surface to enhance tumor cell immunogenicity.

Modification of Tumor Cells In Vivo Another aspect of the invention provides methods for increasing the immunogenicity of a tumor cell by modification of the tumor cell in vivo to express B7-2 and/or B7-3 and B7- 1 to trigger a costimulatory signal in T cells. In addition, tumor cells can be further modified in vivo to express MHC molecules to trigger a primary, antigen-specific, signal in T cells.

Tumor cells can be modified in vivo by introducing a nucleic acid encoding B7-2 and/or B7-3 and B7-1 into the tumor cells in a form suitable for expression of the costimulatory molecule(s) on the surface of the tumor cells. Likewise, nucleic acids encoding MHC class I or class II molecules or an antisense sequence of the Ii gene can be introduced into tumor cells in vivo. In one embodiment, a recombinant expression vector is used to deliver nucleic acid encoding B7-2 and/or B7-3 and B7-1 to tumor cells in vivo as a form of gene therapy.

Vectors useful for in vivo gene therapy have been previously described and include retroviral vectors, adenoviral vectors and adeno-associated viral vectors. See e.g. Rosenfkid M.A., Cell 68, 143-155 (1992); Anderson, Science 226, 401-409 (1984); Friedman, T., Science 244, 1275-1281 (1989). Alternatively, nucleic acid can be delivered to tumor cells in vivo by direct injection of naked nucleic acid into tumor cells. See e.g. Acsadi, et al., Nature 332, 815-818 (1991). A delivery apparatus is commercially available (BioRad).

Optionally, to be suitable for injection, the nucleic acid can be complexed with a carrier such as a liposome. Nucleic acid encoding an MHC class I molecule complexed with a liposome 20 has been directly injected into tumors of melanoma patients. Hoffman, M.,,Science 256, 305- 309 (1992).

Tumor cells can also be modified in vivo by use of an agent which induces or increases expression of B7-2 and/or B7-3 and B7-1 (and, if necessary, MHC molecules) as described herein. The agent may be administered systemically, e.g. by intravenous injection, or, preferably, locally to the tumor cells.

The Effector Phase of the Anti-Tumor T Cell-Mediated Immune Response The modified tumor cells of the invention are useful for stimulating an anti-tumor T cell-mediated immune response by triggering an antigen-specific signal and a costimulatory signal in tumor-specific T cells. Following this inductive, or afferent. phase of an immune response, effector populations ofT cells are generated. These effector T cell populations can include both CD4-, T cells and CDS8 T cell. The effector populations are responsible for elimination of umnors cell. by, for example, cytolysis of the tumor cells. Once T cells are activated, expression of a costimulatory molecule is not required on a target cell for recognition of the target cell by effector T cells or for the effector functions of the T cells.

Harding. F.A. and Allison. J.P. J. Ex. Med. 177. 1791-1796 (1993). Therefore. the antitumor T cell-mediated immune response induced by the modified tumor cells of the invention -51is effective against both the modified tumor cells and unmodified tumor cells which do not express a costimulatory molecule.

Additionally, the density and/or type of MIHC molecules on the cell surface required for the afferent and efferent phases of a T cell-mediated immune response can differ. Fewer MuGC molecules, or only certain types of MI-C molecules MIHC class I but not MHC class II) may be needed on a tumor cell for recognition by effector T cells than is needed for the initial activation of T cells. Therefore, tumor cells which naturally express low amounts of MHC molecules but are modified to express increased amounts of MT{C molecules can induce a T cell-mediated immune response which is effective against the unmodified tumor cells. Alternatively, tumor cells which naturally express MHC class I molecules but not MHC class II molecules whichi are then modified to express MHC class II molecules can induce a T cell-mediated immune response which includes effector T cell populations which can elim-inate the parental MHC class class 11- tumor cells.

Therapeut ic Compositions of Tumor Cells Another aspect of the invention is a composition of modified tumor cells in a biologically compatible form suitable for pharmaceutical administration to a subject in vivo.

This composition comprises an amount of modified tumor cells and a physiologically CCC.***acceptable carrier. The amount of modified tumor cells is selected to be therapeutically effective. The term "biologically compatible form suitable for pharmnaceutical administration in vivo" means that any toxic effects of the tumor cells are outweighed by the therapeutic effects of the tumor cells. A "physiologically acceptable carrier" is one which is biologically compatible with the subject. Examples of acceptable carriers include saline and aqueous C buffer solutions. In all cases, the compositions must be sterile and must be fluid to the extent that easy syringability exists. The term "subject" is intended to include living organi sms in which tumors can arise or be experimentally induced. Examples of subjects include humans, CCC CC dogs, cats, mice, rats. and transgenic species thereof.

Administration of the therapeutic compositions of the present invention can be carried C u sn nw rcdrs at dosages and for periods of time effective to achieve the *4030 desired result. For example, a therapeutically effective dose of modified tumor cells may vary according to such factors as age. sex and weight of the individual, the type of tumor cell and degree of tumor burden, and the immunological competency of the subject. Dosage regimens may be adi,. sted to provide optimum therapeutic responses. For instance, a single dose of modified tumor cells may be administered or several doses may be administered over time. Administration may be by injection. irncluding intravenous, intramuscular.

intraperi toneal and subcutaneous injections.

-52- (1 Activation of Tumor-specific T Lymphocvtes In Vitro Another approach to inducing or enhancing an anti-tumor T cell-mediated immune response by triggering a costimulatory signal in T cells is to obtain T lymphocytes from a tumor-bearing subject and activate them in vitro by stimulating them with tumor cells and a stimulatory form of B7-2 and/or B7-3, alone or in combination with B7-1. T cells can be obtained from a subject, for example, from peripheral blood. Peripheral blood can be further fractionated to remove red blood cells and enrich for or isolate T lymophocytes or T lymphocyte subpopulations. T cells can be activated in vitro by culturing the T cells with tumor cells obtained from the subject from a biopsy or from peripheral blood in the case of blood-borne malignancies) together with a stimulatory form of B7-2 and/or B7-3 or, alternatively, by exposure to!a modified tumor cell as described herein. The term "stimulatory form" means that the costimulatory molecule is capable of crosslinking its receptor on a T cell and triggering a costimulatory signal in T cells. The stimulatory.form of the costimulatory molecule can be, for example, a soluble multivalent molecule or an immobilized form of the costimulatory molecule, for instance coupled to a solid support.

Fragments, mutants or variants fusion proteins) of B7-2 and/or B7-3 which retain the ability to trigger a costimulatory signal in T cells can also be used. In a preferred embodiment, a soluble extracellular portion of B7-2 and/or B7-3 is used to provide costimulation to the T cells. Following culturing of the T cells in vitro with tumor cells and 20 B7-2 and/or B7-3, or a modified tumor cell, to activate tumor-specific T cells, the T cells can be administered to the subject, for example by intravenous injection.

o(14). Therapeutic Uses of Modified Tumor Cells The modified tumor cells of the present invention can be used to increase tumor 25 immunogenicity, and therefore can be used therapeutically for inducing or enhancing T lymphocyte-mediated anti-tumor immunity in a subject with a tumor or at risk of developing a tumor. A method for treating a subject with a tumor involves obtaining tumor cells from S the subject, modifying the tumor cells ex vivo to express a T cell costimulatory molecule, for Sexample by transfecting them with an appropriate nucleic acid, and administering a 30 therapeutically effective dose of the modified tumor cells to the subject. Appropriate nucleic acids to be introduced into a tumor cell include nucleic acids encoding B7-2 and/or B7-3, alone or together with nucleic acids encoding B7-1, MHC molecules (class I or class II) or Ii antisense sequences as described herein. Alternatively, after tumor cells are obtained from a subject, they can be modified ex vivo using an agent which induces or increases expression of B7-2 and/or B7-3 (and possibly also using agent(s) which induce or increase B7-1 or MHC molecules).

Tumor cells can be obtained from a subject by, for example, surgical removal of tumor cells, e.g. a biopsy of the tumor, or from a blood sample from the subject in cases of blood-borne malignancies. In the case of an experimentally induced tumor, the cells used to induce the tumor can be used, e.g. cells of a tumor cell line. Samples of solid tumors may be treated prior to modification to produce a single-cell suspension of tumor cells for maximal efficiency of transfection. Possible treatments include manual dispersion of cells or enzymatic digestion of connective tissue fibers, e.g. by collagenase.

Tumor cells can be transfected immediately after being obtained from the subject or can be cultured in vitro prior to transfection to allow for further characterization of the tumor cells determination of the expression of cell surface molecules). The nucleic acids chosen for transfection can b6 determined following characterization of the proteins expressed by the tumor cell. For instance, expression of MHC proteins on the cell surface of the tumor cells and/or expression of the Ii protein in the tumor cell can be assessed. Tumors which express no, or limited amounts of or types of MHC molecules (class I or class II) can be transfected with nucleic acids encoding MHC proteins; tumors which express Ii protein can be transfected with Ii antisense sequences. If necessary, following transfection, tumor cells can be screened for introduction of the nucleic acid by using a selectable marker (e.g.

drug resistance) which is introduced into the tumor cells together with the nucleic acid of interest.

Prior to administration to the subject, the modified tumor cells can be'treated to render them incapable of further proliferation in the subject, thereby preventing any possible S outgrowth of the modified tumor cells. Possible treatments include irradiation or mitomycin C treatment, which abrogate the proliferative capacity of the tumor cells while maintaining the ability of the tumor cells to trigger antigen-specific and costimulatorv signals in T cells and thus to stimulate an immune response.

The modified tumor cells can be administered to the subject by injection of the tumor cells into the subject. The route of injection can be, for example, intravenous, intramuscular, intraperitoneal or subcutaneous. Administration of the modified tumor cells at the site of the original tumor may be beneficial for inducing local T cell-mediated immune responses 0 against the original tumor. Administration of the modified tumor cells in a disseminated manner, e.g. by intravenous injection, may provide systemic anti-tumor immunity and, furthermore, may protect against metastatic spread of tumor cells from the original site. The modified tumor cells can be administered to a subject prior to or in conjunction with other forms of therapy or can be administered after other treatments such as chemotherapy or surgical intervention.

Additionally. more than one type of modified tumor cell can be administered to a subject. For example. an effective T ce!I response may require exposure of the T cell to more -54than one type of costimulatory molecule. Furthermore, the temporal sequence of exposure of the T cell to different costimulatory mocules may be important for generating an effective response. For example, it is known that upon activation, a B cell expresses B7-2 early in its response (about 24 hours after stimulation). Subsequently, B7-1 and B7-3 are expressed by the B cell (about 48-72 hours after stimulation). Thus, a T cell may require exposure to B7-2 early in the induction of an immune response by exposure to B7-1 and/or B7-3 in the immune response. Accordingly, different types of modified tumor cells can be administered at different times to a subject to generate an effective immune response against the tumor cells.

For example, tumor cells modified to express B7-2 can be administered to a subject.

Following this administration, a tumor cell from the same tumor but modified to express B7- 3 (alone or in conjunction with B7-1) can be administered to the subject. Another method for treating a subject with a tumor is to modify tumor cells in vivo to express B7-2 and/or B7-3, alone or in conjunction with B7-1, MHC molecules and/or an inhibitor of li expression. This method can involve modifying tumor cells in vivo by providing nucleic acid encoding the protein(s) to be expressed using vectors and delivery methods effective for in vivo gene therapy as described in a previous section herein.

Alternatively, one or more agents which induce or increase expression of B7-2 and/or B7-3, and possibly B7-1 or MHC molecules, can be administered to a subject with a tumor.

The modified tumor cells of the current invention may also be used in a method for 20 preventing or treating metastatic spread of a tumor or preventing or treating,recurrence of a tumor. As demonstrated in detail in one of the following examples, anti-tumor immunity induced by B7-l-expressing tumor cells is effective against subsequent challenge by tumor cells, regardless of whether the tumor cells of the re-exposure express B7-1 or not. Thus, administration of modified tumor cells or modification of tumor cells in vivo as described 25 herein can provide tumor immunity against cells of the original, unmodified tumor as well as metastases of the original tumor or possible regrowth of the original tumor.

The current invention also provides a composition and a method for specifically inducing an anti-tumor response in CD4 T cells. CD4' T cells are activated by antigen in conjunction with MHC class II molecules. Association of peptidic fragments of TAAs with 30 MHC class II molecules results in recognition of these antigenic peptides by CD4 T cells.

Providing a subject with tumor cells which have been modified to express MHC class II molecules along with B7-2 and/or B7-3, or modified in vivo to express MHC class II molecules along with B7-2 and/or B7-3. can be useful for directing tumor antigen presentation to the MHC class II pathway and thereby result in antigen recognition by and activation ofCD4+ T cells specific for the tumor cells. Depletion of either CD4" or CD8+ T cells in vivo, by administration of anti-CD4 or anti-CDS antibodies, can be used to demonstrate that specific anti-tumor immunity is mediated by a particular CD4 T cell subpopulation.

Subjects initially exposed to modified tumor cells develop an anti-tumor specific T cell response which is effective against subsequent exposure to unmodified tumor cells. Thus the subject develops anti-tumor specific immunity. The generalized use of modified tumor cells of the invention from one human subject as an immunogen to induce anti-tumor immunity in another human subject is prohibited by histocompatibility differences between unrelated humans. However, use of modified tumor cells from one individual to induce antitumor immunity in another individual to protect against possible future occurrence of a tumor may be useful in cases of familial malignancies. In this situation, the tumor-bearing donor of tumor cells to be modified id closely related to the (non-tumor bearing) recipient of the modified tumor cells and therefore the donor and recipient share MHC antigens. A strong hereditary component has been identified for certain types of malignancies, fdr e*-ample certain breast and colon cancers. In families with a known susceptibility to a particular malignancy and in which one individual presently has a tumor, tumor cells from that individual could be modified to express B7-2 and/or B7-3, alone or in combination with B7-1 and administered to susceptible, histocompatible family members to induce an anti-tumor response in the recipient against the type of tumor to which the family is susceptible. This anti-tumor response could provide protective immunity to subsequent development of a 20 tumor in the immunized recipient. Tumor-Specific T Cell Tolerance In the case of an experimentally induced tumor, a subject a mouse) can be exposed to the modified tumor cells of the invention before being challenged with 25 unmodified tumor cells. Thus, the subject is initially exposed to TAA peptides on tumor cells together with B7-2 and/or B7-3, and B7-1 which activates TAA-specific T cells. The activated T cells are then effective against subsequent challenge with unmodified tumor cells.

In the case of a spontaneously arising tumor, as is the case with human subjects, the subject's immune system will be exposed to unmodified tumor cells before exposure to the modified tumor cells of the invention. Thus the subject is initially exposed to TAA peptides on tumor cells in the absence of a costimulatory signal. This situation is likely to induce TAA-specific T cell tolerance in those T cells which are exposed to and are in contact with the unmodified tumor cells. Secondary exposure of the subject to modified tumor cells which can trigger a costimulatory signal may not be sufficient to overcome tolerance in TAA-specific T cells which were anergized by primary exposure to the tumor. Use of modified tumor cells to induce anti-tumor immunity in a subject already exposed to unmodified tumor cells may therefore be most effective in early diagnosed patients with small tumor burdens, for instance -56a small localized tumor which has not metastasized. In this situation, the tumor cells are confined to a limited area of the body and thus only a portion of the T cell repertoire may be exposed to tumor antigens and become anergized. Administration of modified tumor cells in a systemic manner, for instance after surgical removal of the localized tumor and modification of isolated tumor cells, may expose non-anergized T cells to tumor antigens together with B7-2 and/or B7-3 alone, or in combination with B7-1 thereby inducing an antitumor response in the non-anergized T cells. The anti-tumor response may be effective against possible regrowth of the tumor or against micrometastases of the original tumor which may not have been detected. To overcome widespread peripheral T cell tolerance to tumor cells in a subject, additional signals, such as a cytokine, may need to be provided to the subject together with the modified tumor cells. A cytokine which functions as a T cell growth factor, such as IL-2, could be provided to the subject together with the modified tumor cells. IL-2 has been shown to be capable of restorinig the alloantigen-specific.

responses of previously anergized T cells in an in vitro system when exogenous IL-2 is added at the time of secondary alloantigenic stimulation. Tan, et al. J. Exp. Med. 177, 165-173 (1993).

Another approach to generating an anti-tumor T cell response in a subject despite tolerance of the subject's T cells to the tumor is to stimulate an anti-tumor response in T cells 2 from another subject who has not been exposed to the tumor (referred to as a naive donor) and transfer the stimulated T cells from the naive donor back into the tumor-bearing subject so that the transferred T cells can mount an immune response against the tumor cells. An anti-tumor response is induced in the T cells from the naive donor by stimulating the T cells in vitro with the modified tumor cells of the invention. Such an adoptive transfer approach is generally prohibited in outbred populations because of histocompatibity differences between 25 the transferred T cells and the tumor-bearing recipient. However, advances in allogeneic bone marrow transplantation can be applied to this situation to allow for acceptance by the recipient of the adoptively transferred cells and prevention of graft versus host disease. First, a tumor-bearing subject (referred to as the host) is prepared for and receives an allogeneic bone marrow transplant from a naive donor by a known procedure. Preparation of the host 30 involves whole body irradiation, which destroys the host's immune system, including T cells tolerized to the tumor, as well as the tumor cells themselves. Bone marrow transplantation is accompanied by treatment(s) to prevent graft versus host disease such as depletion of mature T cells from the bone marrow graft, treatment of the host with immunosuppressive drugs or treatment of the host with an agent, such as CTLA4Ig, to induce donor T cell tolerance to host tissues. Next, to provide anti-tumor specific T cells to the host which can respond against residual tumor cells in the host or regrowth or metastases of the original tumor in the host, T cells from the naive donor are stimulated in vitro with tumor cells from the host -57which have been modified, as described herein, to express B7-2 and/or B7-3. Thus, the donor T cells are initially exposed to tumor cells together with a costimulatory signal and therefore are activated to respond to the tumor cells. These activated anti-tumor specific T cells are then transferred to the host where they are reactive against unmodified tumor cells.

Since the host has been reconstituted with the donor's immune system, the host will not reject the transferred T cells and, additionally, the treatment of the host to prevent graft versus host disease will prevent reactivity of the transferred T cells with normal host tissues.

H. Administration of Therapeutic Forms of B Lymphocvte Antigens The peptides of the invention are administered to subjects in a biologically compatible form suitable for pharmaceutical administration in vivo to either enhance or suppress T cell mediated immune response. By "biologically compatible form suitable for administration in vivo" is meant a form of the protein to be administered in which any toxic effects are outweighed by the therapeutic effects of the protein. The term subject is intended to include living organisms in which an immune response can be elicited, mammals. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.

Administration of a peptide having the activity of a novel B lymphocyte antigen as described herein can be in any pharmacological form including a therapeutically active amount of peptide alone or in combination with a peptide having the activity of another B lymphocyte 0 antigen and a pharmaceutically acceptable carrier. Administration of a therape'utically active amount of the therapeutic compositions of the present invention is defined as an amount effective, at dosages and for periods of time necessay to achieve the desired result. For example, a therapeutically active amount of a peptide having B7-2 activity may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of peptide to elicit a desired response in the individual. Dosage regima may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

The active compound peptide) may be administered in a convenient manner 0. such as by injection (subcutaneous. intravenous, etc.), oral administration, inhalation.

transdermal application, or rectal administration. Depending on the route of administration, the active compound may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound.

To administer a peptide having B7-2 activity by other than parenteral administration, it may be necessary to coat the peptide with. or co-administer the peptide with. a material to prevent its inactivation. For example, a peptide hving B7-2 activity may be administered to an individual in an appropriate carrier. diluent or adjuvant. co-administered with enzyme -58inhibitors or in an appropriate carrier such as liposomes. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Adjuvant is used in its broadest sense and includes any immune stimulating compound such as interferon. Adjuvants contemplated herein include resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether and nhexadecyl polyethylene ether. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol. Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes (Strejan et al., (1984) J. Neuroimmunol 2:27).

The active compound may also be administered parenterally or intraperitoneally.

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.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases, the composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures 20 thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, asorbic Sacid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, 25 for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. 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.

Sterile injectable solutions can be prepared by incorporating active compound 30 peptide having B7-2 activity) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.

Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freezedr.i.n; which yields a powder of the active ingreient peptide) plus any additional desired ingredient from a previously sterile-filtered solution thereof.

-59- When the active compound is suitably protected, as described above, the protein may be orally administered, for example, with an inert diluent or an assimilable edible carrier. As used herein "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the therapeutic compositions is contemplated.

Supplementary active compounds can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units stited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharma6eufifal carrier.

The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

I. Identification ofCvtokines Induced by Costimulation P .0 The nucleic acid sequences encoding peptides having the activity of novel B lymphocyte antigens as described herein can be used to identify cytokines which are produced by T cells in response to stimulation by a form ofB lymphocyte antigen, B7-2.

T cells can be suboptimally stimulated in vitro with a primary activation signal, such as phorbol ester, anti-CD3 antibody or preferably antigen in association with an MHC class II *5 molecule, and given a costimulatory signal by a stimulatory form of B7-2 antigen, for instance by a cell transfected with nucleic acid encoding a peptide having B7-2 activity and .i expressing the peptide on its surface or by a soluble, stimulatory form of the peptide. Known cytokines released into the media can be identified by ELISA or by the ability of an antibody which blocks the cytokine to inhibit T cell proliferation or proliferation of other cell types 3 0 that is induced by the cytokine. An IL-4 ELISA kit is available from Genzyme (Cambridge MA), as is an IL-7 blocking antibody. Blocking antibodies against IL-9 and IL-12 are available from Genetics Institute (Cambridge. MA).

An in vitro T cell costimulation assay as described above can also be used in a method for identifying novel cytokines which may be induced by costimulation. If a particular activity induced upon costimulation, T cell proliferation, cannot be inhibited by addition ofbiocking antibodies to known cvtokines. the activity may result from the action of an unkown cytokine. Following costimulation, this cytokine could be purified from the media by conventional methods and its activity measured by its ability to induce T cell proliferation.

To identify cytokines which prevent the induction of tolerance, an in vitro T cell costimulation assay as described above can be used. In this case, T cells would be given the primary activation signal and contacted with a selected cytokine, but would not be given the costimulatory signal. After washing and resting the T cells, the cells would be rechallenged with both a primary activation signal and a costimulatory signal. If the T cells do not respond proliferate or produce IL-2) they have become tolerized and the cytokine has not prevented the induction of tolerance. However, if the T cells respond, induction of tolerance has been prevented by the cytokine. Those cytokines which are capable of preventing the induction of tolerance can bedtargeted for blockage in vivo in conjunction with reagents which block B lymphocyte antigens as a more efficient means to induce tolerance in transplant recipients or subjects with autoimmune diseases. For example, one could administer a B7-2 blocking reagent together with a cytokine blocking antibody to a subject.

J. Identification of Molecules which Inhibit Costimulation Another application of the peptide having the activity of a novel B lymphocyte antigen of the invention B7-2 and B7-3) is the use of one or more of these peptides in screening assays to discover as yet undefined molecules which are inhibitors of costimulatory ligand binding and/or of intracellular signaling through T cells following costimulation. For example, a solid-phase binding assay using a peptide having the activity of aB lymphocyte antigen, such as B7-2, could be used to identify molecules which inhibit binding of the antigen with the appropriate T cell ligand CTLA4, CD28). In addition, an in vitro T cell costimulation assay as described above could be used to identify molecules which 25 interfere with intracellular signaling through the T cells following costimulation as determined by the ability of these molecules to inhibit T cell proliferation and/or cytokine S production (yet which do not prevent binding ofB lymphocyte antigens to their receptors).

For example, the compound cyclosporine A inhibits T cell activation through stimulation via the T cell receptor pathway but not via the CD28/CTLA4 pathway. Therefore, a different 30 intracellular signaling pathway is involved in costimulation. Molecules which interfere with intracellular signaling via the CD28/CTLA4 pathway may be effective as immunosuppressive agents in vivo (similar to the effects of cyclosporine A).

K. Identification of Molecules which Modulate B Lymphocvte Anticen Expression The monoclonal antibodies produced using the proteins and peptides of the current invention can be used in a screening assay for molecules which modulate the expression of B lymphocyte antigens on cells. For example, molecules which effect intracelluiar signaling which leads to induction ofB lymphocyte antigens, e.g. B7-2 or B7-3, can be identified by assaying expression of one or more B lymphocyte antigens on the cell surface. Reduced immunofluorescent staining by an anti-B7-2 antibody in the presence of the molecule would indicate that the molecule inhibits intracellular signals. Molecules which upregulate

B

lymphocyte antigen expression result in an increased immunofluorescent staining.

Alternatively, the effect of a molecule on expression of a B lymphocyte antigen, such as B7- 2, can be determined by detecting cellular B7-2 mRNA levels using a B7-2 cDNA as a probe.

For example, a cell which expresses a peptide having B7-2 activity can be contacted with a molecule to be tested, and an increase or decrease in B7-2 mRNA levels in the cell detected by standard technique, such as Northern hybridization analysis or conventional dot blot of mRNA or total poly(A+)RNAs using a B7-2 cDNA probe labeled with a detectable marker.

Molecules which modulate B lymphocyte antigen expression may be useful therapeutically for either upregulating or downregulating immune responses alone or in conjunction with soluble blocking or stimulating reagents. For instance, a molecule which inhibits expression of B7-2 could be administered together with a B7-2 blocking reagent for immunosuppressive purposes. Molecules which can be tested in the above-described assays include cytokines such as IL-4, yINF, IL-10, IL-12, GM-CSF and prostagladins.

This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references and published patent applications cited .0-20 throughout this application are hereby incorporated by reference.

The following methodology was used in Examples 1, 2 and 3.

METHODS AND MATERIALS Mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation from single cell suspensions of normal human spleens and were separated into E- and E+ fractions by rosetting with sheep red blood cells (Boyd. et al. (1985) Immunol. 134, 1516). B cells were purified from the E- fraction by adherence of monocvtes on plastic and depletion of residual T. natural killer cells (NK) and residual monocytes by two treatments with anti-MsIgG and anti-MsIgM coated magnetic beads (Advanced Magnetics, Cambridge, MA). using monoclonal antibodies: anti-CD4, -CD8. -CDI Ib. -CD14 and -CD16. CD4- T cells were isolated from the E- fraction of the same spleens after adherence on plastic and depletion ofNK, B cells and residual monocytes with magnetic beads and monoclonal 3 antibodies: anti-CD20. -CDI Ib. -CDS and -CD16. CD28+ T cells were identically isolated from the E- fraction using anti-CD20, -CD Ib, -CD 14 and -CD 16 monoclonal antibodies.

The efficiency of the purification was analyzed by indirect immunofluorescence and flow -62cytometry using an EPICS flow cytometer (Coulter). B cell preparations were >95% CD3)+, CD 14+. CD4+ T cell preparations were >98% CD3'+, >98% CD4+.<1% CD8-, CD2O-, CD 14+. CD28+ T cell preparations were >98% CD3+, >98% CD28 <1I% CD20-:, CD 14-.

B. Monoclonal Antibodies and Fusion Proteins Monoclonal antibodies were used as purified Ig unless indicated otherwise: anti- B7: 133, IgM is a blocking antibody and has been previously described (Freedman, A.S. et al.

(1987) Immunol 13.7,3260-3267); anti-B7:B 1.1, IgGi (RepliGen Corp., Cambridge, MA) (Nickoloff, et al (1993 Pathol 14 2, 1029-1040) is a non-blocking monoclonal antibody; BB-1: IgM is a blocking antibody (Dr. E. Clark, University of Washington, Seattle, WA) (Yokochi, et al. (1982)]. ImmunoL 128, 823 -827); anti-CD2O: B1,'IgG2a (Stashenko, et al.(1 980) J. ImmunoL 125, 1678-1685); anti-B5: IggM (Freedm~an,. et al.

(1985)]J ImmunoL 13 4, 2228-223 anti-CD8: 7PT 3F9, JgG2a; anti-CD4: l9Thy5D7, IgG1_a; anti-CD I11 b: Mo 1, 16M and anti-CD 14: Mo2, IgM (Todd, R, et al. (198 1)]J ImmunoL 126, 1435-1442); anti-MHC class IL: 9-49, IgG2a (Dr R. Todd, University of 'Michigan, Ann Arbor) (Todd, et al. (1984) Hum Immunol. ILO 2-40 anjC2:93 g~ D.C June, Naval Research Institute, Bethesda) (Hansen, et al. (1980) Immunogenetics. 247-260); anti-CD 16: 3G8, IgG I (used as ascites) (Dr. J. Ritz, Dana-Farber Cancer Institute, Boston); anti-CD3: OKT3, IgG2a hybridoma was obtained from the American Type Culture Collection and the purified monoclonal antibody was adhered on plastic plates at-a concentration of lug/ml; anti-CD28 Fab fragments were generated from the 9.3 monoclonal antibody, by papain digestion and purification on a protein A column. according to the manufacturer's Instructions (Pierce, Rockford, IL). Human CTLA4 fusion protein .25 (CTLA41cg) and control fusion protein (control-lig) were prepared as previously described (Gimmi. et al. (1993) Proc. Nail. Acad. Sci USA 90l:6586-6590,), Boussiotis, et allJ Exp. M1ed. (accepted for publication)).

C. CH-O Cell Transfection transfectants (CHO-B7) were prepared from the B7-1 negative chinese hamster ovary (CHO) cell line, fixed_ with paraforrnaldehvde and used as previously described (Gimmi. et al. Proc. Acad Sci USA 88. 6575-6579).

D. h7 Vitro B Cell Activation and Selection of B7+ and B7- Cells Splenic B cells were- cultured at -x0 clsm incmlttutuemda RM 164'0 with 10%/l heat inactivated fetal caffserum (FCS). 2miv 2iutamnen. I mM1 sodium pyruvate. penicillin (100 umis/mIl). stt~mcnsulfate (I100y.±g/ml and gentanivcinl sulfate -63in tissue culture flasks and were activated by crosslinking of sig with affinity purified rabbit anti-human IgM coupled to Affi-Gel 702 beads (Bio-Rad), Richmond.

CA)

(Boyd, et al., (1985) J Immunol. 13,1516) or by crosslinking of MHC class II with 9- 49 antibody coupled to Affi-Gel 702 beads. B cells activated for 72 hours, were used as total activated B cell populations or were indirectly stained with anti-B7 (B 1.1) monoclonal antibody and fluorscein isothiocyanate (FITC) labeled goat anti-mouse immunoglobulin (Fisher, Pittsburgh, PA), and fractionated into B7-1+ and B7-1- populations by flow cytometric cell sorting (EPICS Elite flow cytometer, Coulter).

E. Immnflouorescence and Flow Cvtometr For surface phenotype analysis populations of B cells activated by either-sIg or MHC class II crosslinking for 6, 12, 24, 48, 72 and 96 hours were stained with either anti-B7 (133), BB-1 monoclonal antibodies, control IgM antibody, CTLA4Ig or control-Ig. Celfsuspensions were stained by two step indirect membrane staining with 1Oig/ml of primary monoclonal antibody followed by the appropriate secondary reagents. Specifically, immunoreactivity with anti-B7 (133) and BB-1 monoclonal antibodies was studied by indirect staining using goat anti-mouse Ig or immunoglobulin FITC (Fisher) as secondary reagent and immunoreactivity with fusion proteins was studied using biotinylated CTLA4Ig or biotinylated control-Ig and streptavidin-phycoerythrin as secondary reagent.

PBS

*20 containing 10% AB serum was used as diluent and wash media. Cells werefixed with 0.1% paraformaldehyde and analyzed on a flow cytometer (EPICS Elite Coulter).

F. Proliferation Assay T cells were cultured at a concentration of lxl05 cells per well in 96-well flat bottom 25 microtiter plate at 37'C for 3 days in 5% CO 2 Syngeneic activated B cells (total B cell population or B7+ and B7- fractions) were irradiated (2500 rad) and added into the cultures at a concentration of ixl05 cells per well. Factors under study were added t6 the required concentration for a total final volume of 200 pl per well. When indicated. T cells were incubated with anti-CD28 Fab (final concentration of 10 for 30 minutes at 4'C, prior to addition in experimental plates. Similarly, CHO-B7 or B cells were incubated with CTLA4Ig or control-Ig (I O.igml) for 30 minutes at 4°C. Thvmidine incorporation as an index ofmitogenic activity. was assessed after incubation with luCi (37kBq) of {methvi- 3

H

thymidine (Du Pont. Boston. MA) for the last 15 hours of the culture. The cells were harvested onto filters and the radioactivity on the dried filters was measured in a Pharmacia beta plate liquid scintilation counter.

-64- G. IL-2 and IL-4 Assay IL-2 and IL-4 concentrations were assayed by ELISA (R&D Systems, Minneapolis, MN and BioSource, Camarillo, CA) in culture supernatants collected at 24 hours after initiation of the culture.

EXAMPLE 1 Expression of a Novel CTLA4 Ligand on Activated B Cells Which Induces T Cell Proliferation Since crosslinking surface Ig induces human resting B cells to express B7-1 maximally (50-80%) at 72 hours, the ability of activated human B lymphocytes to induce submitogenically activated T cells to proliferate and secrete IL-2 was determired- Figure 1 depicts the costimulatory response of human splenic CD28+ T cells, submitogenically activated with anti-CD3 monoclonal antibody, to either B7 (B7-1) transfected CHO cells (CHO-B7) or syngeneic splenic B cells activated with anti-Ig for 72 hours. 3 H-Thymidine incorporation was assessed for the last 15 hours of a 72 hours culture. IL-2 was assessed by ELISA in supernatants after 24 hours of culture (Detection limits of the assay: 31-2000 S. pg/ml). Figure 1 is representative of seventeen experiments.

20 Submitogenically activated CD28+ T cells proliferated and secreted-high levels of IL- 2 in response to B7-1 costimulation provided by CHO-B7 (Figure 1, panel Both proliferation and IL-2 secretion were totally inhibited by blocking the B7-1 molecule on CHO cells with either anti-B7-1 monoclonal antibody or by a fusion protein for its high affinity receptor, CTLA4. Similarly, proliferation and IL-2 secretion were abrogated by 25 blocking B7-1 signalling via CD28 with Fab anti-CD28 monoclonal antibody. Control monoclonal antibody or control fusion protein had no effect. Nearly identical costimulation of proliferation and IL-2 secretion was provided by splenic B cells activated with anti-Ig for 72 hours (panel Though anti-B7-l monoclonal antibody could completely abrogate both proliferation and IL-2 secretion delivered by CHO-B7, anti-B7-l monoclonal antibody 30 consistently inhibited proliferation induced by activated B cells by only 50% whereas IL-2 secretion was totally inhibited. In contrast to the partial blockage of proliferation induced by anti-B7-l monoclonal antibody, both CTLA4Ig and Fab anti-CD28 monoclonal antibody completely blocked proliferation and IL-2 secretion. These results are consistent with the hypothesis that activated human B ce!is express one or more additional CTLA4/CD2S ligands which can induce T cell proliferation and IL-2 secretion.

EXAMPLE 2 Activated Human Splenic B Cells Express CTLA4 Ligand(s) Distinct from B7-1 In light of the above observations, whether other CTLA4 binding counter-receptors were expressed on activated B cells was determined. To this end, human splenic B cells were activated for 72 hours with anti-Ig and then stained with an anti-B7-l monoclonal antibody (B1.1) which does not inhibit B7-1 mediated costimulation. Fluoroscein isothiocyanate (FITC) and mAb BI.1 were used with flow cytometric cell sorting to isolate B7-1 and B7-1fractions. The resulting post-sort positive population was 99% B7-1+ and the post-sort negative population was 98% B7-1- (Figure 2).

To examine the costifnulatory potential of each population, human splenic CD28+ T cells were submitogenically stimulated with anti-CD3 monoclonal antibody in the presence of irradiated B7-1+ or B7-1- anti-Ig activated (72 hours) splenic B cells. 3 H-Thyrnidine incorporation was assessed for the last 15 hours of a 72 hours culture. IL-2 was assessed by ELISA in supernatants after 24 hours of culture (Detection limits of the assay: 31-2000 pg/ml). The results of Figure 3 are representative often experiments. B7-1+ B cells induced anti-CD3 activated T cells to proliferate and secrete IL-2 (Figure 3a) but not IL-4. As was observed with the unfractionated activated B cell population, anti-B7-l monoclonal antibody (133) inhibited proliferation only 50% but consistently abrogated IL-2 secretion. As above, CTLA4Ig binding or blockade of CD28 with Fab anti-CD28 monoclonal antibody completely inhibited both proliferation and IL-2 secretion. Control monoclonal antibody and control-Ig were not inhibitory. In an attempt to identify other potential CTLA4/CD28 binding costimulatory ligand(s) which might account for the residual, non-B7 mediated proliferation delivered by B7+ B cells, the effect ofBB-1 monoclonal antibody on proliferation and IL-2 ::05 secretion was examined. As seen. BB-1 monoclonal antibody completely inhibited both proliferation and IL-2 secretion (Figure 3a). Figure 3b displays the costimulatory potential of B7-1- activated human splenic B cells. Irradiated B7-1- activated (72 hr) B cells could also a" deliver a significant costimulatory signal to submitogenically activated CD4+ l mphocytes.

This costimulation was not accompanied by detectable IL-2 (Figure 3b) or IL-4 accumulation S 30 and anti-B7-l monoclonal antibody did not inhibit proliferation. However. CTLA4Ig. Fab anti-CD28 monoclonal antibody, and BB-1 monoclonal antibody all completely inhibited proliferation.

Phenotypic analysis of the B7-1- and B7-1- activated splenic B cells confirmed the above finciional results. Figure 4 shows the cell surface expression ofB7-,l B7-2 and B7-3 on fractionated B7-l and B7-1- activated B cell. As seen in Figure 4. B7-1- activated splenic B cells stained with anti-B7-1 (133) monoclonal antibody. BB-1 monoclonal antibody, and bound CTLA4-g. In contrast. B7- activated splenic B cells did not stain with -66anti-B7-l (133) monoclonal antibody but did stain with BB-1 monoclonal antibody and CTLA4Ig. These phenotypic and functional results demonstrate that both B7-l1 and B7-1activated (72 hours) human B lymphocytes express CTLA4 binding counter-receptor(s) which: 1) can induce submitogenically activated T cells to proliferate without detectable IL- 2 secretion; and 2) are identified by the BB-1 monoclonal antibody but not anti-B7-1 monoclonal antibody. Thus, these CTLA4/CD28 ligands can be distinguished on the basis of their temporal expression after B cell activation and their reactivity with CTLA4Ig and anti- B7 monoclonal antibodies. The results of Figure 4 are representative of five experiments.

EXAMPLE 3 Three Distinct CTLA4/CD28 Ligands Are Expressed Following Human B CellActivation To determine the sequential expression of CTLA4 binding counter-receptors following activation, human splenic B cells were activated by crosslinking of either surface Ig or MHC class II and the expression of B7-1, B7-3 and B7-2 binding proteins were examined by flow cyvometric analysis. Ig or MHC class II crosslinking induced a similar pattern of CTLA4Ig binding (Figures 5 and Figure 5 is representative of the results of experiments for anti-B7-1 and BB-I binding and 5 experiments for CTLA4Ig binding.

20 Figure 6 is representative of 25 experiments for anti-B7-l binding and 5 expgriments for CTLA4Ig binding. The results of these experiments indictes that prior to 24 hours, none of these molecules are expressed. At 24 hours post-activation, the majority of cells express a protein that binds CTLA4Ig however, fewer than 20% express either B7-1 or B7-3.

Crosslinking of MHC class II induces maximal expression and intensity of B7-1 and B7-3 at 25 48 hours whereas crosslinking of Ig induces maximal expression at 72 hours and expression declines thereafter. These results suggest that an additional CTLA4 binding counter-receptor is expressed by 24 hours and that the temporal expression of the distinct B7-1 and B7-3 proteins appears to coincide.

A series of experiments was conducted to determine whether the temporal expression of CTLA4 binding counter-receptors differentially correlated with their ability to costimulate T cell proliferation and/or IL-2 secretion. Human splenic CD28+ T cells submitogenically stimulated with anti-CD3 were cultured for 72 hours in the presence of irradiated human splenic B cells that had been previously activated in vitro by sig crossiinking for 24. 48, or 72 hours. IL-2 secretion was assessed by ELISA in supematants after 24 hours and T cell proliferation as assessed by -H-thymidine incorporation for the last 15 hours of a 72"hour culture. The results of Figure 7 are represenative of 5 experiments. As seen in Figure 7a, 24 hour activated B cells provided a costimulatory signal which was accompanied by modest -67levels of IL-2 production, although the magnitude of proliferaion was significantly less than observed with 48 and 72 hours activated human B cells (note differences in scale for 3 H-Thyidine incorporation) Neither proliferation nor IL-2 accumulation was inhibited by anti-b7 (133) or BB-1. In contrast, with CTLA4Ig and anti-CD28 Fab monoclonal antibody totally abrogated proliferation and IL-2 accumulation. B cells activated for 48 hours, provided costimulation which resulted in nearly maximal proliferation and IL-2 secretion (Figure 7b). Here, anti-B7-1 (133) monoclonal antibody, inhibited proliferation approximately 50% but totally blocked IL-2 accumulation. BB-1 monoclonal antibody totally inhibited both proliferation and IL-2 secretion. As above, CTLA4Ig and Fab anti-CD28 also totally blocked proliferation and IL-2 production. Finally, 72 hour activated B cells induced T cell respoilse identical to that induced by 48 hour activated B cells. Similar results are observed if the submitogenic signal is delivered by phorbol myristic acid (PMA) and if the human splenic B cells are activated by MHC class II rather than Ig erogslinking.

These results indicate that there are hree CTLA4 binding molecules that are temporarily expressed on activated B cells and each can induce submitogenically stimulated T cells to proliferate. Two of these molecules, the early CTLA4 binding counter-receptor (87-2) and 7- (133 i o) induce IL-2 production whereas 87-3 induces proliferation without detectable IL-2 production.

Previous studies provided conflicting evidence whether the anti-B7 monoclonal 20 antibody, l 33 and monoclonal antibody BB-I identified the same molecule (Freedman,

A.S.

et al. (1987) Immunol. j31, 3260-3267; Yokochi, et al. (1982)Jmmunol. 1, 823-827; *FreemanoG., et al. (1989) Immunol. 14, 2714-2722.). Although both monoclonal antibodies identified molecules expressed 48 hours following human B-cell activation, several reports suggested that B7 (87-1) and the molecule identified by monoclonal antibody BB-1 were distinct since they were differentially expressed on cell lines and B cell neoplasms (Freedman, A.S. et al. (1987) Immunol. 1 7, 3260-3267; Yokochi, et al. (1982) Immunol. 128, 823-827; Freeman, G.J. et al. (1989) Immunol. 1, 2714-2722; Clark,

E

and Yokochi, T. (1984) Leukocyte Tpving, 1st International References Workshov. 339-346; S* Clark. E. et al. (1984) Leukocyre Typing, 1st International References Workshop. 740). In eaddition immunoprecipitation and Western Blotting with these IM monoclonal antibodies suggested that they identified different molecules (Clark. E and Yokochi. T. (1984) Leukocvte 7 yping, Ist International References Workshop. 339-346; Clark, et al. (1984) Leukocyte Tvping is International References Workshop. 740). The original anti-B7 mionoclonal antibody 133. was generated bv immunization with anti-immunoglobulin activated human B i.mphoc es whereas the BB-I monoclonal antibody was generated by immunization wit. baboon cell line. Thus. the BB-! monoclonal antibody must identify an epitope on human that is conser,.ed between baboons and humans.

-68- Following the molecular cloning and expression of the human B7 gene B7 transfected COS cells were found to be identically stained with the anti-B7 (133) and BB-1 monoclonal antibodies and that they both precipitated the identical broad molecular band (44-54kD) strongly suggesting that they identified the same molecule (Freeman, et al.

(1989) J. Immunol. 143, 2714-2722). This observation was unexpected since the gene encoding the molecule identified by the BB-1 monoclonal antibody had been previously mapped to chromosome 12 (Katz, et al. (1985) Eur. J. Immunol. 103-6), whereas the B7 gene was located by two groups on chromosome 3 (Freeman, et al. (1992) Blood. 29, 489-494; Selvakumar, et al. (1992) Immunogenetics. 36, 175-181.). Subsequently, additional discrepancies between the phenotypic expression of B7 (B7-1) and the molecule identified by the BB-1 monclonal antibody were noted. BB-1 monoclonal antibody stained thymic epithelial cells (Turka, et al. (1991) J. Immunol. 146, 1428-36; Munro, et al. Blood submitted.) and keratinocytes (Nickoloff, et al (1993) Am. J. Pathol.'-i2, 1029- 1040; Augustin, et al. (1993) J. Invest. Dermatol. 100, 275-281.) whereas anti-B7 did not. Recently, Nickoloff et al. (1993) Am. J. Pathol. 142, 1029-1040, reported discordant expression of the molecule identified by the BB-1 monoclonal antibody and B7 on keratinocytes using a BB-1 and anti-B7 (B 1. 1 and 133) monoclonal antibodies. Nickoloff et al. also demonstrated that these BB-I positive cells did not express B7 mRNA yet bound CD28 transfected COS cells providing further support for the existence of a distinct protein 20 which binds monoclonal antibody BB-1.

.The present findings confirm that there is an additional CTLA4 counter-receptor identified by the BB-1 monoclonal antibody, B7-3, and that this protein appears to be functionally distinct from B7-1 (133). Although the expression of B7-1 and B7-3 following B cell activation appears to be concordant on B7 positive B cells, these studies demonstrate 25 that the B7-3 molecule is also expressed on B7 negative activated B cells. More importantly, the B7-3 molecule appears to be capable of inducing T cell proliferation without detectable IL-2 or IL-4 production. This result is similar to the previous observation that ICAM-1 could costimulate T cell proliferation without detectable IL-2 or IL-4 production (Boussiotis, et al J. Exp. Med. (accepted for publication)). These data indicate that the BB-1 monoclonal antibody recognizes an epitope on the B7-1 protein and that this epitope is also found on a distinct B7-3 protein, which also has costimulatory function. Phenotypic and blocking studies demonstrate that the BB-1 monoclonal antibody could detect one (on B7 negative cells) or both (on B7 positive cells) of these proteins. In contrast, the anti-B7 monoclonal antibodies, 133 and B1.1 detect only the B7-1 protein. Taken together. these results suggest that by 48 hours post B-cell activation by crosslinking of surface immunoglobulin or MHC class II. B cells express at least two distinct CTLA4 binding counter-receptors. one identified -69by both anti-B7 and BB-1 monclonal antibodies and the other identified only by BB-1 monoclonal antibody.

The B7-2 antigen is not detectable on activated B cells after 12 hours, but by 24 hours it is strongly expressed and functional. This molecule appears to signal via CD28 since proliferation and IL-2 production are completely blocked by Fab anti-CD28 monoclonal antibody. At 48 hours post activation, IL-2 secretion seems to be accounted for by B7-1 costimulation, since anti-B7 monoclonal antibody completely inhibits IL-2 production.

Previous studies and results presented here demonstrate that B7 (B7-1) is neither expressed (Freedman, A.S. et al. (1987) Immunol. 112, 3260-3267; Freedman, et al.

(1991) Cell. Immunol. 132, 429-437) nor capable of costimulating T cell proliferation or IL-2 secretion until 48 hours postB-cell activation. Previous studies have shown that-activation of T cells via the TCR in the absence of costimulation (Gimmi, et al. (1993) Proc. Natl.

Acad. Sci USA 90:6586-6590, Schwartz, et al. (1989) Cold Spring Harb7 Simp: Quant.

Biol 54, 605-10; Beverly, et al. (1992) Int. Immunol. 4 661-671.) and lack of IL-2 (Boussiotis, et al J Exp. Med. (submitted); Beverly, et al. (1992) Int. Immuno. 4, 661- 671; Wood, et al. (1993) J. Exp. Med. 177, 597-603) results in anergy. If B7-1 were the only costimulatory molecule capable of inducing IL-2 secretion, T cells would be anergized within the first 24 hours following activation since there is no B7-I present to costimulate IL-2 production. Therefore, the existence of another, early inducible costimulatory molecule, which can costimulate IL-2 secretion during the first 24 hours would be necessary to induce an effective immune response rather than anergy. The appearance of the early CTLA4 binding counter-receptor, B7-2, between 12 and 24 hours post B cell activation, fulfills this function.

Two observations shed light on the biologic and potential clinical significance of these two additional CTLA4 binding counter-receptors. First, B7 (B7-1) deficient mouse has been developed and its antigen presenting cells were found to still bind CTLA4Ig (Freeman and Sharpe manuscript in preparation). This mouse is viable and isolated mononuclear cells induce detectable levels of IL-2 when cultured with T cells in virro. Therefore, an alternative CD2S costimulatory counter-receptor or an alternative IL-2 producing pathway must be functional. Second, thus far the most effective reagents to induce antigen specific anergy in murine and human systems are CTLA4Ig and Fab anti-CD28, whereas anti-B7 monoclonal antibodies have been much less effective (Harding, et al. (1992).Vaure. 356, 607-609; Lenschow. et al. (1992) Science. 22, 789-792; Chen, et al. (1992) Cell 71, 1093- 1102: Tan. et al. (1993)J Exp. Med. 177, 165-173.). These observations are also consistent with the hypothesis that alternative CTLA4/CD28 ligands capable of inducing IL-2 exist, and taken together with the results presented herein. suggest that all three CTLA4 binding counter-receptors may be critical for the induction ofT cell immunity. Furthermore.

their blockade will likely be required for the induction of T cell anergy. Identical results have been observed in the murine system with the identification of two CTLA4 binding ligands, corresponding to the human B7-1 and B7-2 molecules. APCs in the B7 deficient mouse bind to the CTLA4 and can induce IL-2 secretion. Taken together, these observations indicate that multiple CTLA-4 binding counter-receptors exist and sequentially costimulate T cell activation in the murine system.

EXAMPLE 4 Cloning. Sequencing and Expression of the B7-2 Antigen A. Construction ofcDNA library A cDNA library was constructed in the pCDM8 vector (Seed, Nature, 329:840 (1987)) using poly (A) RNA from the human anti-IgM activated B cells as descibed (Aruffo et al, Proc. Natl. Acad. Sci. USA, 84:3365 (1987)). Splenic B cells were cultured at 2x10 6 cells/ml in complete culture media, {RPMI 1640 with 10% heat inactivated fetal calf serum (FCS), 2mM glutamine, 1 mM sodium pyruvate, penicillin (100 units/ml), streptomycin sulfate (100.g/ml) and gentamycin sulfate in tissue culture flasks and were activated by crosslinking ofsIg with affinity purified rabbit anti-human IgM coupled to Affi-Gel 702 beads (Bio-Rad), Richmond, CA) (Boyd, et al., (1985) J.

20 Immunol. 134,1516). Activated B cells were harvested after 1/6, 1/2, 4, 8 12, 24, 48, 72 and 96 hours.

RNA was prepared by homogenizing activated B cells in a solution of 4M guanidine thiocvanate. 0.5% sarkosyl, 25mM EDTA, pH 7.5, 0.13% Sigma anti-foam A, and 0.7% mercaptoethanol. RNA was purified from the homogenate by centrifugation for 24 hour at 25 32.000 rpm through a solution of 5.7M CsCI, 10mM EDTA, 25mM Na acetate, pH 7. The pellet of RNA was dissolved in 5% sarkosyl, ImM EDTA, 10mM Tris, pH 7.5 and extracted with two volumes of 50% phenol, 49% chloroform, 1% isoamyl alcohol. RNA was ethanol precipitated twice. Poly RNA used in cDNA library construction was purified by two cycles ofoligo (dT)-cellulose selection.

Complementary DNA was synthesized from 5.5,ug of anti-IgM activated human B cell poly(A)~ RNA in a reaction containing 50mM Tris, pH 8.3, 75mM KC1. 3mM MgCI2, dithiothreitol. 500uM dATP, dCTP, dGTP, dTTP. 50uigml oligo(dT)12-18, 180 units/ml RNasin. and 10.000 units/ml Moloney-MLV reverse transcriptase in a total volume of 55ul at 37 for 1 hr. Following reverse transcription, the cDNA was converted to doublestranded DNA by adjusting the solution to 25mM Tris. pH 8.3. 100mM KC1. 5mM MgCl, 250auM each dATP. dCTP, dGTP. dTTP, 5mM dithiothreitol. 250 unitsml DNA polymerase I, 8.5 units/ml ribonuclease H and incubating at 160 for 2 hr. EDTA was added to 18mM and -71the solution was extacted with an equal volume of 50% phenol, 49% chloroform. 1% isoamyl alcohol. DNA was precipitated with two volumes of ethanol in the presence of ammonium acetate and with 4 micrograms of linear polyacrylamide as carrier. In addition, cDNA was synthesized from 4.g of anti-IgM activated human B cell poIy(A) RNA in a reaction containing 50mM Tris, pH 8.8, 50pg/ml oligo(dT)12-1 8 327 units/mi RNasin, and 952 units/ml AMV reverse transcriptase in a total volume of 100pl at 42° for 0.67 hr.

Following reverse transcription, the reverse transcriptase was inactivated by heating at for 10 min. The cDNA was converted to double-stranded DNA by adding 320 1l H20 and 801l of a solution of 0.1M Tris, pH 7.5, 25mM MgCl2, 0.5M KCI, 250p.g/ml bovine serum albumin, and 50mM dithiothreitol, and adjusting the solution to 2 00pM each dATP, dCTP, dGTP, dTTP, 50 units/ml DA polymerase I, 8 units/ml ribonuclease H and incubating at 16 C for 2 hours. EDTA was added to 18 mM and the solution was extracted with an equal volume of 50 phenol, 49 chloroform, 1 isoamyl alcohol. DNA was precipitated with two volumes of ethanol in the presence of 2.5M ammonium acetate and with 4 micrograms of linear polyacrylamide as carrier.

The DNA from 4pg of AMV reverse transcription and 2.g of Moloney MLV reverse transcription was combined. Non-selfcomplementary BstXI adaptors were added to the DNA as follows: The double-stranded cDNA from 6pg of poly(A) RNA was incubated with 3.6i g of a kinased oligonucleotide of the sequence CTTTAGAGCACA (SEQ ID NO:15) and 2.4 g of a kinased oligonucleotide of the sequence CTCTAAAG (SEQ ID NO:1-6) in a solution containing 6mM Tris, pH 7.5, 6mM MgC12, 5mM NaCI, 350pg/ml bovine serum albumin, 7mM mercaptoethanol, 0.1rmM ATP, 2mM dithiothreitol, ImM spermidine, and 600 units T4 DNA ligase in a total volume of 0.45ml at 15 C for 16 hours. EDTA was added to 34mM and the solution was extracted with an equal volume of 50% phenol, 49% chloroform, 1% "25 isoamyl alcohol. DNA was precipitated with two volumes of ethanol in the presence of ammonium acetate.

DNA larger than 600bp was selected as follows: The adaptored DN-A was redissolved in 10mM Tris, pH 8. ImM EDTA, 600mM NaCI, 0.1% sarkosyl and chroiatographed on a Sepharose CL-4B column in the same buffer. DNA in the void volume of the column (containing DNA greater than 600bp) was pooled and ethanol precipitated.

The pCDMS vector was prepared for cDNA cloning by digestion with BstXI and purification on an agarose gel. Adaptored DNA from 6ug ofpoly(A)tRNA was ligated to 2.25p.g of BstXI cut pCDMS in a solution containing 6mM Tris, pH 7.5. 6mM MgCl2, NaCI. 3 50ug/ml bovine serum albumin. 7mM mercaptoethanol, 0. mM ATP. 2mM dithiothreitol. ImM spemnidine. and 600 units T4 DNA ligase in a total volume of 1.5ml at 150 for 24 hr. The ligation reaction mixture was transformed into competent E.coli MC106 /P3 and a total of 4.290.000 independent cDNA clones were obtained.

-72- Plasmid DNA was prepared from a 500 ml culture of the original transformation of the cDNA library. Plasmid DNA was purified by the alkaline lysis procedure followed by twice banding in CsCI equilibrium gradients (Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1987)).

B. Cloning Procedure In the first round of screening, thirty 100 mm dishes of 50% confluent COS cells were transfected with 0.05 g/ml anti-IgM activated human B cells library DNA using the DEAE- Dextran method (Seed et al, Proc. Natl. Acad. Sci. USA, 84:3365 (1987)). The cells were trypsinized and re-plated after 24 hours. After 47 hours, the cells were detached by incubation in PBS/0.5 mMv EDTA, pH 7.4/0.02% Na azide at 37 0 C for 30 min. The detached cells were treated with 10 pg/ml/CTLA4Ig and CD28Ig for 45 minutes at 4 0 C. Cells were washed and distributed into panning dishes coated with affinity-purified Goat'ani-human IgG antibody and allowed to attach at room temperature. After 3 hours, the plates were gently washed twice with PBS/0.5mM EDTA, pH 7.4/0.02% Na azide, 5% FCS and once with 0.15M NaCI, 0.01 M Hepes, pH 7.4, 5% FCS. Episomal DNA was recovered from the panned cells and transformed into E. coli DH10B/P3. The plasmid DNA was re-introduced into COS cells via spheroplast fusion as described (Seed et al, Proc. Natl. Acad. Sci. USA, S84:3365 (1987)) and the cycle of expression and panning was repeated twice. In the second 20 and third rounds of selection, after 47 hours, the detached COS cells were first incubated with a-B7-1 mAbs (133 and B1.1, 10 tg!ml), and COS cells expressing B7-1 were removed by amouse IgG and IgM coated magnetic beads. COS cells were then treated with 10 4g/ml of human CTLA4Ig (hCTLA4Ig) and human CD28Ig (hCD28Ig) and human B7-2 expressing COS cells were selected by panning on dishes with goat anti-human IgG antibody plates.

25 After the third round, plasmid DNA was prepared from individual colonies and transfected into COS cells by the DEAE-Dextran method. Expression of B7-2 on transfected COS cells was analyzed by indirect immunofluorescence with CTLA4Ig.

After the final round of selection, plasmid DNA was prepared from individual colonies. A total of 4 of 48 candidate clones contained a cDNA insert of approximately 1.2 kb. Plasmid DNA from these four clones was transfected into COS cells. All four clones were strongly positive for B7-2 expression by indirect immunofluorescence using CTLA4Ig and flow cytometric analysis.

C. Sequencing The B7-2 cDNA insert in cione29 was sequenced in the pCDM8 expression vector employing the following strategy. Initial sequencing was performed using sequencing primers T7. CDMSR (Invitrogen) homologous to pCDvMS vector sequences adjacent to the -73cloned B7-2 cDNA (see Table Sequencing was performed using dye terminator chemistry and an ABI automated DNA sequencer. (ABI, Foster City, CA). DNA sequence obtained using these primers was used to design additional sequencing primers (see Table This cycle of sequencing and selection of additional primers was continued until the B7-2 cDNA was completely sequenced on both strands.

TABLE

I

T7(F) (SEQ ID NO:3) CDM8(R) (SEQ ID NO:4) CDM8 RGV(2) (SEQ ID NO:5) HBX29-5P (2R) (SEQ ID NO:6) HBX29-5P (2F) (SEQ ID NO:7) HBX29-5P (SEQ ID NO:8) 5PA (SEQ ID NO:9) (3FA) (SEQ ID NO:10) HBX29-5P(1R) (SEQ ID NO:11) HBX29-3P(1R) (SEQ ID NO:12) HBX29-5P(3R) (SEQ ID NO:13) 20 HBX29-3P(P) (SEQ ID NO:14) 5'd[TAATACGACTCACTATAGGGJ 3 5'd[TAAGGTTCCTTCACAAAG3' 5'd[ACTGGTAGGTATGGAAGATCC]3' 5s'd[ATGCGAATCATTCCTGTGGGC]3 5'd[AAAGCCCACAGGATATGATTCG]3' 5'd[CTCTCAAAACCAAABCCTGAG] 3 5'd[TTAGGTCACAGCAGAAGCAGC]3' 5'd[TCTGGAAACTGACAAGACGCG]3' S'd[CTCAGGCTTTGGTTTTGAGAG]3' S'd[CACTCTCTTCCCTCTCCATTGJJ 5'd[GACAAGCTGATGGAAACGTCGI]3' The human B7-2 clone 29 contained an insert of 1,120 base pairs with a single long open reading frame of 987 nucleotides and approximately 27 nucleotides of 3' noncoding sequences (Figure 8 (SEQ ID NO: The predicted amino acid sequence encoded by the open reading frame of the protein is shown below the nucleotide sequence in Figure 8. The encoded protein, human B7-2, is predicted to be 329 amino acids in length-(SEQ ID NO:2).

This protein sequence exhibits many features common to other type 1 Ig superfamily embrane proteins. Protein translation is predicted to begin at the ATG codon (nucleotide 107-109) based on DNA homology in this region with the consensus eukarvotic translation Initiation site (Kozk. M. (1987) NucZ. Acids Res. LfL:8125-8148). The amino terminus of the human B7-2 protein (amino acids I to 23) has the characteristics of a secretory signal peptide with a predicted cleavage between the alanines at positions 23 and 24 (von Heijne (1986) Ncl. Acids Res. 14:4683). Processing at this site would result in a human B7-2 membrane bound protein of 306 amino acid with an unmodified molecular weight of approximately 34 lkDa. This protein would consist of an extracellular Ig superfamily V and C like domains, of from about amino acid residue 24-245. a hydrophobic transmembrane domain of from about -74amino acid residue 246-268 and a long cytoplasmic domain of from about amino acid residue 269-329. The homologies to the Ig superfamily are due to the two contiguous Ig-like domains in the extracellular region bound by the cysteines at positions 40 to 110 and 157 to 218. The extracellular domain also contains eight potential N-linked glycosylation sites. E.

coli transfected with a vector containing the cDNA insert of clone 29, encoding the human B7-2 protein, was deposited with the American Type Culture Collection (ATCC) on July 26, 1993 as Accession No. 69357.

Comparison of both the nucleotide and amino acid sequences of human B7-2 with the GenBank and EMBL databases showed that only the human and murine B7-1 proteins are related. Alignment of the three B7 protein sequences (see Figure 13) shows that human B7-2 has approximately 26% amirio acid identity with human B7-1. Figure 13 represents the comparison of the amino acid sequences for human B7-2 (hB7-2) (SEQ ID NO:2), human B7-1 (hB7-1) (SEQ ID NO: 28 and 29) and murine B7 (mB7) (SEQ ID NO: 30 afrd 31). The amino acid sequences for the human B7-1 and murine B7 (referred to herein as murine B7-1) can be found in Genbank at Accession #M27533 and X60958 respectively. Vertical lines in Figure 13 show identical amino acids between the hB7-2 and hB7-1 or mB7. Identical amino acids between hB7-1 and mB7 are not shown. The hB7-2 protein exhibits the same general structure as hB7-1 as defined by the common cysteines (positions 40 and 110, IgV domains; positions 157 and 217, IgC domain) which the Ig superfamily domains and by many other common amino acids. Since both hB7-1 and mB7 have been shown to bind to both human CTLA4 and human CD28, the amino acids in common between these two related proteins will be those necessary to comprise a CTLA4 or CD28 binding sequence. An example of such a sequence would be the KYMGRTSFD (position 81-89, hB7-2) (SEQ ID NO:17) or KSQDNVTELYDVS (position 188-200. hB7-2) (SEQ ID NO: 18). Additional related 25 sequences are evident from the sequence comparison and others can be inferred by considering homologous related amino acids such as aspartic acid and glutamic acid, alanine and glycine and other recognized functionally related amino acids. The B7 sequences share a highly positive charged domain with the cytoplasmic portion WKWKKKKRPRNSYKC (position 269-282. hB7-2) (SEQ ID NO:19) which is probably involved in intracellular signaling.

EXAMPLE Characterization of the Recombinant B7-2 Antigen A. B7-2 Binds CTL.A4Ig and Not Anti-B7-1 and Ani-B7-3 NMonoclonal Antibodies COS cells transfected with either vector DNA (pCDNAI). or an expression plasmid containing B7-1 (BT-1) or B7-2 (B7-2) were prepared. After 72 hours. the transfected COS cells were detached by incubation in PBS containing 0.5 mM EDTA and 0.02% Na azide for min. at 37 0 C. Cells were analyzed for cell surface expression by indirect immunofluorescence and flow cytometric analysis using fluoroscein isothiocyanate conjugated (FITC) goat-anti-mouse Ig or goat-anti-human IgG FITC (Figure Cell surface expression of B7-l was detected with mAbs 133 (anti-B7-1) and BB-1 (anti-B7-1 and anti- B7-3) and with CTLA4Ig, whereas B7-2 reacted only with CTLA4Ig. Neither of the B7 transfectants showed any staining with the isotype controls (IgM or control Ig). The vector transfected COS cells showed no staining with any of the detection reagents. In addition, none of the cells showed any staining with the FITC labeled detection reagents and alone.

This demonstrates that B7-2 encodes a protein that is a CTLA4 counter-receptor but is distinct from B7-1 and B7-3g.

B. RNA Blot Analysis of B7-2 Expression in Unstimulated and Activated Humafr B Cells.

Cell Lines, and Mvelomas Human splenic B cells were isolated by removing T cells and monocvtes as previously described (Freedman, Freeman, Horowitz, Daley, Nadler, L.M., J. Immunol. (1987) 132:3260-3267). Splenic B cells were activated using anti-Ig beads and cells were harvested at the indicated times (Freedman et al., (1987), cited supra). Human myelomas from bone marrow specimens were enriched by removing T cells and monocytes using E rosettes and adherence as previously described (Freeman, et Immunol.

(1989) 43.:2714-2722). RNA was prepared by guanidine thiocyanate homogenization and cesium chloride centrifugation. Equal amounts of RNA 2 04g) were electrophoresed on an agarose gel, blotted, and hybridized to 32P-labelled B7-2 cDNA. Figure 10, panel a, shows RNA blot analysis of unstimulated and anti-Ig activated human splenic B cells and of cell lines including Raji (B cell Burkitts lymphoma), Daudi (B cell Burkitt's lymphoma), RPMI 8226 (myeloma), K562 (erythroleukemia), and REX (T cell acute lymphoblastic leukemia).

Figure 10, panel b shows RNA blot analysis of human myeloma specimens.

Three mRNA transcripts of 1.35. 1.65 and 3.0 kb were identified by hybridization to the B7-2 cDNA (Figure 10, panel RNA blot analysis demonstrated that B7-2 mRNA is expressed in unstimulated human splenic B cells and increases 4-fold following activation (Figure 10, panel B7-2 mRNA was expressed in B cell neoplastic lines (Raji. Daudi) and a myeloma (RPMI 8226) but not in the erythroleukemia K562 and the T cell line REX. In contrast, we have previously shown that B7-1 mRNA is not expressed in resting B cells and is transiently expressed following activation Freeman et al. (1989) supra). Examination of mRNA isolated from human myelomas demonstrates that B7-2 mRNA is expressed in 6 of 6 patients, whereas B7-1 was found in only 1 of these 6 Freeman et al. (1989) supra).

Thus. B7-1 and B7-2 expression appears to be independently regulated.

C. Costimulation Human CD28 T cells were isolated by immunomagnetic bead depletion using monoclonal antibodies directed against B cells, natural killer cells and macrophages as previously described (Gimmi, et al. (1993) Proc. Natl. Acad Sci. USA 92, 6586-6590).

B7-1, B7-2 and vector transfected COS cells were harvested 72 hours after transfection, incubated with 25jag/ml of mitomycin-C for 1 hour, and then extensively washed. 105 CD28 and T cells were incubated with 1 ng/ml ofphorbol myristic acid (PMA) and the indicated number of COS transfectants (Figure 11). As shown in Figure 11, panel a, T cell proliferation was measured by 3H-thymidine (1 aCi) incorporated for the last 12 hours of a 72 hour incubation. Panel !of Figure 11 shows IL-2 production by T cells as measured by ELISA (Biosource, CA) using supematants harvested 24 hours after the initiation of culture.

D. B7-2 Costimulation is not Blocked by Anti-B7-l and Anti-B7-3 mAbs but is Blocked by CTLA4-Ig and Anti-CD28 Fab Human CD28+ T cells were isolated by immunomagnetic bead depletion using mAbs directed against B cells, natural killer cells, and macrophages as previously described (Gimmi, Freeman, Gribben, Gray, Nadler, L.M. (1993) Proc. Natl. Acad.

Sci USA 90, 6586-6590). B7-1, B7-2, and vector transfected COS cells were harvested 72 20 hours after transfection, incubated with 25.g/ml of mitomycin-C for 1 hour, and then extensively washed. 105 CD28 T cells were incubated with I ng/ml ofphorbol myristic acetate (PMA) and 2 x 104 COS transfectants. Blocking agents (10g/!ml) are indicated on the left side of Figure 12 and include: 1) no monoclonal antibody (no blocking agents), 2) mAb 133 (anti-B7-1 mAb), 3) mAb BBI (anti-B7-1 and anti-B7-3 mAb), 4) mAb (control IgM mAb), 5) anti-CD28 Fab (mAb 6) CTLA-Ig, and 7) control Ig. Panel a of Figure 12 shows proliferation measured by 3 H-thymidine (1 Ci) incorporation for the last 12 hours of a 72 hour incubation. Figure 12, panel b, shows IL-2 production as measured by ELISA (Biosource, CA) using supernatants harvested 24 hours after the initiation of culture.

B7- 1 and B7-2 transfected COS cells costimulated equivalent levels of T cell 0 proliferation when tested at various stimulator to responder ratios (Figure 11). Like B7-1, B7-2 transfected COS cell costimulation resulted in the production of IL-2 over a wide range of stimulator to responder cell ratios (Figure 11). In contrast, vector transfected COS cells did not costimulate T cell proliferation or IL-2 production.

-77- E. B7-2 Costimulaton is not Blocked by Anti-B7-1 and Anti-B7-3 mAbs but is Blocked by CTLA4-g and Anti-CD28 Fab Human CD28 T cells were isolated by immunomagnetic bead depletion using mAbs directed against B cells, natural killer cells, and macrophages as previously described (Gimmi, Freeman, Gribben, Gray, Nadler, L.M. (1993) Proc. Natl. Acad.

Sci USA 90, 6586-6590). B7-1, B7-2, and vector transfected COS cells were harvested 72 hours after transfection, incubated with 25pg/ml of mitomycin-C for 1 hour, and then extensively washed. 105 CD28- T cells were incubated with 1 ng/ml ofphorbol myristic acetate (PMA) and 2 x 104 COS transfectants. Blocking agents (0.ag/ml) are indicated on the left side of Figure 12 and include: 1) no monoclonal antibody (no blocking agents), 2) mAb 133 (anti-B7-l mAb), 3- mAb BB1 (anti-B7-1 and anti-B7-3 mAb), 4) mAb (control IgM mAb), 5) anti-CD28 Fab (mAb 6) CTLA-Ig, and 7) controlIg. Panel a of Figure 12 shows proliferation measured by 3 H-thymidine (l Ci) incorporation fo? the last 12 hours of a 72 hour incubation. Figure 12, panel b, shows IL-2 production as measured by ELISA (Biosource, CA) using supernatants harvested 24 hours after the initiation of culture.

To distinguish B7-2 from B7-1 and B7-3, mAbs directed against B7-1 and B7-3 were used to inhibit proliferation and IL-2 production of submitogenically activated human CD28 T cells. Both B7-1 and B7-2 COS transfectants costimulated T cell proliferation and IL-2 production (Figure 12). MAbs 133 (Freedman, A.S. etal. (1987) supra) (anti-B7-1) and BBI (Boussiotis, et al., (in review) Proc. Natl. Acad Sci. USA; Yokochi, T.'Holly, R.D., S Clark, E.A. (1982) J Immunol. 2 1, 823-827) (anti-B7-l and anti-B7-3) completely inhibited proliferation and IL-2 secretion induced by B7-1 but had no effect upon costimulation by B7- 2 transfected COS cells. Isotype matched control B5 mAb had no effect. To determine whether B7-2 signals via the CD28/CTLA4 pathway, anti-CD28 Fab and CTLA4-Ig fusion protein were tested to determine whether they inhibited B7-2 costimulation. Both anti-CD28 Fab and CTLA4-Ig inhibited proliferation and IL-2 production induced by either B7-1 or B7- 2 COS transfectants whereas control Ig fusion protein had no effect (Figure 12). While CTLA4-Ig inhibited B7-2 costimulation of proliferation by only 90%. in other experiments inhibition was more pronounced (98-100%). None of the blocking agents inhibited T cell proliferation or IL-2 production induced by the combination of PMA and phytohemagglutinin.

0* Like B7-1. B7-2 is a counter-receptor for the CD2S and CTLA4 T cell surface molecules. Both proteins are similar in that they are: 1) expressed on the surface of APCs: 2) structurally related to the Ig supergene family with an IgV and IgC domain which share 26% amino acid identity, and 3) capable of costimulating T cells to produce IL-2 and proliferate. However. B7-1 and B7-2 differ in several fundamental ways. First. B7-2 mRNA is constitutively expressed in unstimulated B cells, whereas B7-I mRNA does not appear -78until 4 hours and cell surface protein is not detected until 24 hours (Freedman, et al.

(1987) supra; Freeman, et al. (1989) supra). Unstimulated human B cells do not express CTLA4 counter-receptors on the cell surface and do not costimulate T cell proliferation (Boussiotis, et al. supra). Therefore, expression of B7-2 mRNA in unstimulated B cells would allow rapid expression of B7-2 protein on the cell surface following activation, presumably from stored mRNA or protein. Costimulation by B7-2 transfectants is partially sensitive to paraformaldehyde fixation, whereas B7-2 costimulation is resistant (Gimmi, et al. (1991) Proc. Natl. Acad. Sci. USA 88, 6575-6579). Second, expression of B7-1 and B7-2 in cell lines and human B cell neoplasms substantially differs. Third, B7-2 protein contains a longer cytoplasmic domain than B7-1 and this could play a role in signaling B-cell differentiation. These phenitypic and functional differences suggest that these homologous molecules may have biologically distinct functions.

EXAMPLE 6 Cloning and Sequencing of the Murine B7-2 Antigen A. Construction of cDNA Library A cDNA library was constructed in the pCDM8 vector (Seed, Nature, 329:840 (1987)) using poly (A) RNA from dibutryl cyclic AMP (cAMP) activated M12 cells (a murine B cell tumor line) as described (Aruffo et al, Proc. Natl. Acad. Sci. USA, 84:3365 (1987)).

M12 cells were cultured at lx 106 cells/ml in complete culture media, {RPMI 1640 with 10% heat inactivated fetal calf serum (FCS), 2mM glutamine, 1 mM sodium pyruvate, penicillin (100 units/ml), streptomycin sulfate (100g/ml) and gentamycin sulfate 25 in tissue culture flasks and were activated by 300p.g/ml dibutryl cAMiP (Nabavi, et al.

(1992) Nature 360, 266-268). Activated M12 cells were harvested after 0, 6, 12, 18, 24 and hours.

RNA was prepared by homogenizing activated M12 cells in a solution of 4M guanidine thiocyanate, 0.5% sarkosyl, 25mM EDTA, pH 7.5, 0.13%, Sigma anti-foam A, and 0.7% mercaptoethanol. RNA was purified from the homogenate by centrifugation for 24 hour at 32.000 rpm through a solution of 5.7M CsC1. 10mM EDTA. 25mM Na acetate, pH 7.

S The pellet of RNA was dissolved in 5% sarkosyl, ImM EDTA, 10mM Tris. pH 7.5 and extracted with two volumes of 50/o phenol, 49% chloroform, 1% isoamvl alcohol. RNA was ethanol precipitated twice. Poly RNA used in cDNA library construction was purified by two cycles of oligo (dT)-ciuiose seiection Complementary DNA was synthesized from 5.5pg of dibutrvi cAMP activated murine MI12 cell poly(A)~ RNA in a reaction containing 50mM Tns. pH 8.3. 75mM KC1, -79- 3mM MgC12, I10MM dithiothreitol, 500pLiM dATP, dCTP, dGTP, dTTP, oligo(dT)1 2 1 8 180 units/ml RNasin, anid 10,000 units/ml Moloney-VMV reverse transcniptase in a total volume of 55gjl at 37"C for I hr. Following reverse transcription, the cDNA was converted to double-standed DNA by adjusting the solution to 25MM Tris, pH- 8.3, 1l00mM~ KCI, 5mM MgCI2, 250jiM each dATP, dCTP, dGTP, dTTP, dithiothreitol, 250 units/mI DNA polymerase 1, 8.5 units/ml ribonuclease H and incubating at 16"C for 2 hr. EDTA was added to 18mM and the solution was extracted with an equal volume of 50% phenol, 49% chloroform, 1% isoamnyl alcohol. DNA was precipitated with two volumes of ethanol in the presence of 2.5M ammonium acetate and with 4 micrograms of linear polyacrylamide as carrier. Following reverse transcription, the reverse transcriptase was inactivated by heating at 70 0 C for 10 min. The cDNA was converted to double-.stranded DNA by adding 320 H20O and 8Ojal of a solution of 0.I1M Tris, pH 7.5, 25mM MgCl2, KCI, 2 S5jig/ml bovine serum albumin, and 50mM dithiothreitol, and adjusiing the solution to 200pL.M each dATP, dCTP, dGTP, dTTP, 50 units/mI DNA polymerase 1, 8 units/mI ribonuclease H and incubating at 160C for 2 hours. EDTA was added to 18 mM and the solution was extracted with an equal volume of 50% phenol, 49% chloroform,

I%

isoanmyl alcohol. DNA was precipitated with two volumes of ethanol in the presence of ammonium acetate and with 4 micrograms of linear polyacrylamide as carrier.

2,ug of non-selfcomplementary BsX dposwere ade to the DJNA~~ as follows: 20 The double-stranded cDNA from 5.Sug of poly(A)+ RNA was incubated w#t 3 .6jg, of a kinased oligonucleotide of the seuneCTTTAGAGCACA (SEQ ID NO 5 and 2.4 ofra kinased oligonucleotide of the sequence CTCTAAAG (SEQ ID NO: 16) in a solution containing 6mM Tris, PH 7.5, 6mM MgCla), 5mM NaCI, 3 50jig/ml bovine serum albumin, 7mIM mercaptoethanol, 0.1mIM ATP, 2mM dithiothreitol I1mM spermidine, and 600 units T4 DNA ligase in a total volume of O.

4 5mi at 15. for 16 hours. EDTA was added to 34mMlv and the solution was extracted with an equal volume of 50911 phenol, 49% chloroform, 1% isoanmil alcohol. DNA was precipitated with two volumes of ethanol in the presence of ammonium acetate.

DNA larger- than 600bp was selected as follows: The adaptored DNA was redissolved Z.o. in 10mIM Tnis., pH S. Ilm.M E DTA, 600rnM NaCI1, 0. sarkosyl and chromato graphed on a Sepharose CL-4B column in the same buffer. DNA in the void volume of the column (containin2 DNA greater than 600bp) wvas pooled and ethanol precipitated.

T-he pCDMS vector was prepared for CDNA Cloningy by digestion with BstXI and purification on an agarose gel. Adaptored DNA from 5.5ug of polv(AY':R.NA was ligyated to 33 2 2 5ua o fBstXI cut pCD!MS in a solution, containim rng mM Tris, PH 7.5. 6mIM ,\gfCI/, NaCI. 3 _50Lg'nl bov ine serum albumin. /7mv rnercao~toethanol. 0. 1 mM ATP. 2mMV dithiothreitol. M scermidine. and 600 units T4 DNA ligase in a total volume of I .5m1 at 150 for 24 hr. The ligation reaction mixture was transformed into competent E.coli MC1061/P3 and a total of 200 x 106 independent cDNA clones were obtained.

Plasmid DNA was prepared from a 500 ml culture of the original transformation of the cDNA library. Plasmid DNA was purified by the alkaline lysis procedure followed by twice banding in CsCI equilibrium gradients (Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1987)).

B. Cloning Procedure In the first round of screening, thirty 100 mm dishes of 50% confluent COS cells were transfected with 0.05pg/ml activated M12 murine B cell library DNA using the DEAE- Dextran method (Seed et al, Proc. Nat!. Acad. Sci. USA, 84:3365 (1987)). The cells were trypsinized and re-plated after 24 hours. After 47 hours, the cells were detachted by incubation in PBS/0.5 mM EDTA, pH 7.4/0.02% Na azide at 37 0 C for 30 min. The detached cells were treated with 10 4g/ml/human CTLA4Ig and murine CD28Ig for 45 minutes at 4°C.

Cells were washed and distributed into panning dishes coated with affinity-purified Goat antihuman IgG antibody and allowed to attach at room temperature. After 3 hours, the plates were gently washed twice with PBS/0.5mM EDTA, pH 7.4/0.02% Na azide, 5% FCS and once with 0.15M NaC, 0.01 M Hepes, pH 7.4, 5% FCS. Episomal DNA was recovered from the panned cells and transformed into E. coli DH10B/P3. The plasmid DNA was reintroduced into COS cells via spheroplast fusion as described (Seed et al, Proc. Natl. Acad.

Sci. USA, 84:3365 (1987)) and the cycle of expression and panning was repeated twice. In the second and third rounds of selection, after 47 hours, the detached COS cells were first incubated with ca-murine B7-1 mAb (16-10AI, 10 g/ml), and COS cells expressing B7-1 were removed by a-mouse IgG and IgM coated magnetic beads. COS cells were then treated with 104g/ml of human CTLA4Ig and murine CD28Ig and murine B7-2 expressing COS cells were selected by panning on dishes coated with goat anti-human IgG antibody. After the third round, plasmid DNA was prepared from individual colonies and transfected into COS cells by the DEAE-Dextran method. Expression of B7-2 on transfected COS cells was analyzed by indirect immunofluorescence with CTLA4Ig.

30 After the final round of selection, plasmid DNA was prepared from individual colonies. A total of 6 of 8 candidate clones contained a cDNA insert of approximately 1.2 kb. Plasmid DNA from these eight clones was transfected into COS cells. All six clones with the 1.2 Kb cDNA insert were strongly positive for B7-2 expression by indirect immunofluorescence using.CTLA4Ig and flow cvtometric analysis.

-81- The B7-2 cDNA insert in clone4 was sequenced in the pCDM8 expression vector employing the following strategy. Initial sequencing was performed using sequencing primers T7, CDM8R (Invitrogen) homologous to pCDM8 vector sequences adjacent to the cloned B7-2 cDNA (see Table Sequencing was performed using dye terminator chemistry and an ABI automated DNA sequencer. (ABI, Foster City, CA). DNA sequence obtained using these primers was used to design additional sequencing primers (see Table II).

This cycle of sequencing and selection of additional primers was continued until the muiine B7-2 cDNA was completely sequenced on both strands.

TABLE

II

T7(F) (SEQ ID NO:3) 5'd[TAATACGACTCACTATAGGGJ3, CDM8(R) (SEQ ID NO:4) MBX4-IF (SEQ ID NO:24) 5'd[ACATAAGCCTGAGTGAGCTGG]3' MBX4-2R (SEQ ID NO:25) 5'd[ATGATGAGCAGCATCACAAGG]3 MBX4-14 (SEQ ID NO:26) 5'd[TGGTCGAGTGAGTCCGAATACJ3' MBX4-2F (SEQ ID NO:27) 5'd[GACGAGTAGTAACATACAGTGJ3' A murine B7-2 clone (mB7-2, clone 4) was obtained containing an insert of 1,163 base pairs with a single long open reading frame of 927 nucleotides and approximately 126 nucleotides of 3' noncoding sequences (Figure 14, SEQ ID NO:22). The predicted amino acid sequence encoded by the open reading frame of the protein is shown below the nucleotide sequence in Figure 14. The encoded murine B7-2 protein, is predicted to be 309 25 amino acid residues in length (SEQ ID NO:23). This protein sequence exhibits many features common to other type I Ig superfamily membrane proteins. Protein translation is predicted to begin at the methionine codon (ATG, nucleotides II to 113) based on the DNA homology in this region with the consensus eucaryotic translation initiation site (see Kozak, (1987) uc. Acids Res. 15:8125-8148) The amino terinus of the murine 7- protein 30 (amino acids I to 23) has the characteristics of a secretory signal peptide with a predicted cleavage between the alanine at position 23 and the valine at position 2- (von Heijne (1987) ouc. Acids Res. :468 3 Processing at this site would result in a murine B7-2 membrane bound protein of 286 amino acids having an unmodified molecular weight of approximately 32 kDa. This protein would consist of an approximate etracelular i superfamily V and C like domains of from about amino acid residue 24 to 246. a hvdrophobic ransmembrane domain of from about amino acid residue 247 to 265. and a lone cytoplasmic domain of from about amino acid residue 266 to 309. The homologies to the Ig suerfamilv are due to the -82two contiguous Ig-like domains in the extracellular region bound by the cysteines at positions to 110 and 157 to 216. The extracellular domain also contains nine potential N-linked glycosylation sites and, like murine B7-1, is probably glycosylated. Glycosylation of the murine B7-2 protein may increase the molecular weight to about 50-70 kDa. The cytoplasmic domain of murine B7-2 contains a common region which has a cysteine followed by positively charged amino acids which presumably functions as signaling or regulatory domain within an APC. Comparison of both the nucleotide and amino acid sequences of murine B7-2 with the GenBank and EMBL databases yielded significant homology (about 26% amino acid sequence identity) with human and murine B7-1. Murine B7-2 exhibits about 50% identity and 67% similarity with its human homologue, hB7-2. E.

coli (DH106/p3) transfected with a vector (plasmid pmBx4) containing a cDNA insert encoding murine B7-2 (clone 4) was deposited with the American Type Culture Collection (ATCC) on August 18, 1993 as Accession No. 69388.

D. Costimulation CD4 murine T cells were purified by first depleting red blood cells by treatment with Tris-NH 4 C1. T cells were enriched by passage over a nylon wool column. CD4' T cells were purified by two-fold treatment with a mixture of anti-MHC class II and anti-CD28 mAbs and rabbit complement. Murine B7-1 (obtained from Dr. Gordon Freeman, Dana- 20 Farber Cancer Institute, Boston, MA; see also, Freeman, G.J. et al (1991) J Exp. Med. 174, 625-631) murine B7-2, and vector transfected COS cells were harvested 72 hours after tmasfection, incubated with 25pg/ml mitomycin-C for one hour, and then extensively washed. 105 murine CD4 T cells were incubated with Ing/ml of phorbol myristic acid (PMA) and 2 x 104 COS transfectants (Table III). T cell proliferation was measured by 3

H-

thymidine (luCi) incorporated for the last 12 hours of a 72 hour incubation.

TABLE III 3H-Thvmidine Incorporation (cpm) 30 CD4 T cells 175 CD4 T cells ing/ml PMA 49 CD4- T cells COS-vector 1750 CD4- T cells COS-B7-1 4400 CD4- T cells COS-B7-2 2236 CD4 T cells Ing/ml PMA COS-vector 2354 CD4 T cells Ing. ml PMA COS-B7-1 67935 CD4- T cells In./ml PMA COS-B7-2 43847 -83- EXAMPLE 7 C-ostuti dCacterization of Hum anB7-2 mmunoobnFusn A. Prearation OfHuman B7-2rg Fusion Proteins The extrace!lular portion of human B7-2 was prepared as a fusion protein coupled to an immunoglobulin constant region. The immunoglobulin constant region may contain genetic modifications including those which reduce or eliminate effector activity inherent in the immunoglobulin structure. Briefly, DNA encoding the extracellular portion ofhB7-2 was joined to DNA encoding thehinge, CH2 and CH3 regions of human IgCyl or IgCy4 modified by directed mutagenesis. This was accomplished as described in the following subsections.

B. Preparation of ene Fusions DNA fragments corresponding to the DNA sequences of interest were prepared by polymerase chain reaction (PCR) using primer pairs described below. In general,

PCR

reactions were prepared in 100

T

l final volume composed of Taq, polymerase buffer (Gene Amp PCR Kit, Perkin-Elmer/Cetus, Norwalk, CT) containing primers (I M each), dNTPs (200 .M each) 1 ng of template DNA, and Taq, polymerase (Saiki, et al. (1988) S Science 239:487-491) PCR DNA amplifications were run on a thermocycler (Ericomp, San Diego, CA) for 25 to 30 cycles each composed of a denaturation step (1 minute at 94°C), a renaturation step (30 seconds at 54 0 and a chain elongation step (1 minute at 72 0 The structure of each hB7-2 Ig genetic fusion consisted of a signal sequence to facilitate secretion coupled to the extracellular domain of B7-2 and the hinge, CH2 and CH3 domains of human IgCy or IgCy4. The IgC gamma 1 and IgC gamma 4 sequences contained nucleotide changes within the hinge region to replace cysteine residues available for disulfide bond S formation with serine residues and may contain nucleotide changes to replace amino acids S within the CH2 domain thought to be required for IgC binding to Fc recep.tors and complement activation.

Sequence analysis confirmed structures of both m/ 4 and clones. and each construct was used to transfect 293 cells to test transient expression. higG ELISA measured/confirmed transient expression levels approximately equal to 100 ng proteinrml ce!l supemnant for both constructs. NSO cell lines were transfected for permanent expression the the fusion proteins.

C. G eneic Construction ofhB-2J Fusion Prein.

Prearation of Signal Seaence PCR amplification was used to generate an immunogobulin signal sequence suitable for secretion of the BT-2Ig fusion protein from mammalian cells. The Ig signal seauence was -84prepared from a plasmid containing the murine IgG heavy chain gene (Orlandi. R. et al.

(1989) Proc. Natl. Acad. Sci. USA. 86:38333837) using the oligonucleotide GGCACTAGGTCTCCAGCTTGAGATCACAGTTCTCTCTAC-3' (SEQ ID NO: as the forward primer and the oligonucleotide GCTTGAATCTTCAGAGGAGCGGAGTGGACACCTGTGG-3' (SEQ ID NO: as the reverse PCR primer. The forward PCR primer (SEQ ID NO: contains recognition sequences for restriction enzymes BsaI and is homologous to sequences 5' to the initiating methionine of the Ig signal sequence. The reverse PCR primer (SEQ ID NO:) is composed of sequences derived from the 5' end of the extracellular domain of hB7-2 and the 3' end of the Ig signal sequence. PCR amplification of the murine Ig signal template DNA using these primers resulted in a 224 bp product which is composed of BsaI restriction sites followed by the sequence of the Ig signal region fused to the first 20 nucleotides of the coding sequence of the extracellular domain of hB7-2. The junction between the signal sequence anIhB7-2 is such that protein translation beginning at the signal sequence will continue into and through hB7-2 in the correct reading frame.

Preparation of the hB7-2 Gene Segment The extracellular domain of the hB7.2 gene was prepared by PCR amplification of plasmid containing the hB7-2 cDNA inserted into expression vector pCDNAI (Freeman et 20 al., Science 262:909-11 (1994)): The extracellular domain of hB7-2 was prepared by PCR amplification using oligonucleotide 5'-GCTCCTCTGAAGATTCAAGC-3' (SEQ ID NO: as the forward primer and oligonucleotide 5'-GGCACTATGATCAGGGGGAGGCTGAGGTCC-3' (#04) (SEQ ID NO: as the reverse primer. The forward PCR primer contained sequences 25 corresponding to the first 20 nucleotides of the B7-2 extracellular domain and the reverse PCR primer contained sequences corresponding to the last 22 nucleotides of the B7-2 extracellular domain followed by a Bcl I restriction site and 7 noncoding nucleotides. PCR amplification with primer #03 and #04 yields a 673 bp product corresponding to the extracellular IgV and IgC like domains ofhB7-2 followed by a unique Bcl I restriction site.

30 The signal sequence was attached to the extracellular portion ofhB7-2 by PCR as follows. DNA-PCR products obtained above corresponding to the signal sequence and the hB7-2 extracellular domain were mixed in equimolar amounts, denatured by heating to 100°C. held at 54 0 C for 30°C to allow the complementary ends to anneal and the strands were filled in using dNTPs and Toq polymerase. PCR primers #01 and #04 were added and the entire fragment produced by PCR amplification to yield a -880 fragment composed of a Bsai restriction site followed by the signal sequence fused to the extracellular domain of hB7- 2. followed by a Bc! I restriction site.

Cloning and Modification of Immunoglobulin Fusion Domain Plasmid pSP721gGI was prepared by cloning the 2000 bp segment of human IgGI heavy chain genomic DNA (Ellison, et al. (1982) Nucl. Acids. Res. 10:4071-4079) into the multiple cloning site of cloning vector pSP72 (Promega, Madison, WI). Plasmid pSP721gGl contained genomic DNA encoding the CHI, hinge, CH2 and CH3 domains of the heavy chain human IgCyl gene. PCR primers designed to amplify the hinge-CH2-CH3 portion of the heavy chain along with the intervening DNA were prepared as follows. The forward PCR primer GACAAAACTCACACATCTCCACCGTCTCCAGGTAAGCC-3 (SEQ ID NO: contained HindIII and Bcl irestriction sites and was homologous to the hinge domain sequence except for five nucleotide substitutions which would change the three cysteine residues to serines. The reverse PCR primer 5'TAATACGACTCACTATAGGG3' (SEQ ID NO: was identical to the commercially available T7 primer (Promega, Madison, WI).

Amplification with these primers yielded a 1050 bp fragment bounded on the 5' end by HindIII and BclI restriction sites and on the 3' end by BamHl, Smal, Kpnl, Sad, EcoR1, Clal, EcoR5 and Begll restriction sites. This fragment contained the IgC hinge domain in which the three cysteine codons had been replaced by serine codons followed by an intron, S the CH2 domain, an intron, the CH3 domain and additional 3' sequences. After PCR amplification, the DNA fragment was digested with Hindlll and EcoR1 and cloned into expression vector pNRDSH digested with the same restriction enzymes. This created plasmid S pNRDSH/IgGl.

A similar PCR based strategy was used to clone the hinge-CH2-CH3 domains of human IgCgamma4 constant regions. A plasmid, p428D (Medical Research Council, London, England) containing the complete IgCgamma4 heavy chain genomic sequence (Ellison, J. Buxbaum, J. and Hood, L.E. (1981) DNA 1: 11 -18) was used as a template for PCR amplification using oligonucleotide GTCCAAATATGGTCCCCCATCCCATCATCCCCAGGTAAGCCACCC-3' (SEQ ID NO: as the forward PCR primer and oligonucleotide 30 CCGCTCTGCCTCCC-3' (SEQ ID NO: as the reverse PCR primer. The forward PCR primer (SEQ ID NO: contains a Bc 11 restriction site followed by the coding sequence for the hinge domain ofIgCgamma4. Nucleotide substitutions have been made in the hinge region to replace the cysteines residues with serines. The reverse PCR primer (SEQ ID NO. contains a PspAI restriction site (5'CCCGGG-3'). PCR amplification with these primers results in a 1179 bp DNA fragment. The PCR product was digested with Bcll and PspAI and ligated to pNRDSH/IgGI digested with the same restriction enzymes to yield plasmid -86pNRDSH/IgG4. In this reaction, the IgCy 4 domain replaced the IgCyl domain present in pNRDSH/IgGl.

Modification of the CH2 domain in IgC to replace amino acids thought to be involved in binding to Fc receptor was accomplished as follows. Plasmid pNRDSH/IgGl served as template for modifications of the IgCyl CH2 domain and plasmid pNRDSH/IgG4 served as template for modifications of the IgCy 4 CH2 domain. Plasmid pNRDSH/IgGI was PCR amplified using a forward PCR primer (SEQ ID NO: and oligonucleotide GGGGGGAAGAGGAAGACTGACGGTGCCCCC TCGGCTTCAGGTGCTGAGGAAG-3' (SEQ ID NO: as the reverse PCR primer. The forward PCR primer (SEQ ID NO: has been previously described and the reverse PCR primer (SEQ ID NO: was homologous to the amino terminal portion bf the CH2 domain of IgG1 except for five nucleotide substitutions designed to change amino acids 234, 235, and 237 (Canfield, S.M. and Morrison, S. L. (1991) J. Exp. Med. 173: 1483-1491.) from Leu to Ala, Leu to Glu, and Gly to Ala, respectively. Amplification with these PCR primers will yield a 239 bp DNA fragment consisting of a modified hinge domain, an intron and modified portion of the CH2 domain. Plasmid pNRDSH/IgG was also PCR amplified with the oligonucleotide

CATCTCTTCCTCAGCACCTGAAGCCGAGGGGGCACCGTCAGTCTTCCTCTTCCC

CC-3' (SEQ ID NO:) as the forward primer and oligonucleotide (SEQ ID NO: as the reverse PCR primer. The forward PCR primer (SEQ ID NO: is complementary to primer S 20 (SEQ ID NO: and contains the five complementary nucleotide changes neaessary for the S: CH2 amino acid replacements. The reverse PCR primer (SEQ ID NO: has been previously described. Amplification with these primes yields a 875 bp fragment consisting of the modified portion of the CH2 domain, an intron, the CH3 domain, and 3' additional sequences.

The complete IgCyI segment consisting of modified hinge domain, modified CH2 domain 25 and CH3 domain was prepared by an additional PCR reaction. The purified products of the two PCR reactions above were mixed, denatured (95 0 C,l minute) and then renatured (54 0

C,

30 seconds) to allow complementary ends of the two fragments to anneal-. The strands were filled in using dNTP and Taq polymerase and the entire fragment amplified using forward PCR primer (SEQ ID NO: and reverse PCR primer (SEQ ID NO: The resulting fragment 30 of 1050 bp was purified, digested with HindIII and EcoRl and ligated to pNRDSH previously digested with the same restriction enzymes to yield plasmid pNRDSHIgGI m.

Two amino acids at immunoglobulin positions 235 and 237 were changed from Leu to Glu and Gly to Ala, respectively, within the IgCy4 CH2 domain to eliminate Fc receptor binding. Plasmid pNRDSI-'IgG4 was PCR amplified using the forward primer (SEQ ID NO: and the oligonucleotide

CGCACGTGACCTCAGGGGTCCGGGAGATCATGAGAGTGTCCTTGGGTTTTGGGG

GG.AACAGGAAGACTGATGGTGCCCCCTCGAACTCAGGTGCTGAGG-3 '(SEQ ID -87- NO: as the reverse primer. The forward primer has been previously described and the reverse primer was homologous to the amino terminal portion of the CH2 domain, except for three nucleotide substitutions designed to replace the amino acids described above. This primer also contained a Pmll restriction site for subsequent cloning. Amplification with these primers yields a 265 bp fragment composed of the modified hinge region, and intron, and the modified 5' portion of the CH2 domain.

Plasmid pNRDSH/IgG4 was also PCR amplified with the oligonucleotide

-CCTCAGCACCTGAGTTCGAGGGGGCACCATCAGTCTCCTGTTCCCCCC

AAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCG-3 (SEQ ID NO: as the forward primer and oligonucleotide (SEQ ID NO: as the reverse

PCR

primer. The forward PCR primer (SEQ ID NO:) is complementary to primer (SEQ ID NO:) and contains the three complementary nucleotide changes necessary for the GH2.amino acid replacements. The reverse PCR primer (SEQ ID NO: has been previously described.

Amplification with these primes yields a 1012 bp fragment consisting of the modified portion of the CH2 domain, an intron, the CH3 domain, and 3' additional sequences. The complete IgCy4 segment consisting of modified hinge domain, modified CH2 domain and CH3 domain was prepared by an additional PCR reaction. The purified products of the two PCR reactions above were mixed, denatured (95 0 C,1 minute) and then renatured (54'C, 30 seconds) to allow complementary ends of the two fragments to anneal. The strands were filled in using dNTP and Taq polymerase and the entire fragment amplified using forward PCR primer (SEQ ID S. NO: and reverse PCR primer (SEQ ID NO: The resulting fragment of 1179 bp was purified, digested with Bcll and PspAi and ligated to pNRDSH previously digested with the same restriction enzymes to yield plasmid pNRDSH/IgG4m.

25 Assembly of Final hB7-2Ig Genes The PCR fragment corresponding to the Ig signal-hB7-2 gene fusion prepared above was digested with Bsal and Bl I restriction enzymes and ligated to pNRDSH/IGI, pNRDSH/IgGlm, pNRDSH/IgG4, and pNRDSH/IgG4m previously digested with Hind III and BclI. The ligated plasmids were transformed into E. coil JMI09 using CaC12 competent cells and transformants were selected on L-agar containing ampicillin (50 qg/ml: Molecular Cloning: A Laboratory Manual (1982) Eds. Maniatis. Fritsch. E. and Sambrook.

J.

Cold Spring Harbor Laboratory). Plasmids isolated from the transformed 5. coli were anaiyzed by restriction enzyme digestion. Plasmids with the expected restriction plasmid were sequenced to verify all portions of the signal-hB7-2-IgG gene ftsion se-ments.

-88- D. Expression Cloning of hB7-2V-'IGl and hB7-2C IgGI The variable and constant domains of human B7-2 were separately cloned into pNRDSH/IgGl. Tese clonings were accomplished using PCR. The portions of hB7-2 corresponding to the variable and constant regions were determined from intron/exon mapping and previously published gene structure analysis.

Human B7-2 Variable Domain GAACTGTCAGTGCTT3' (SEQ ID NO:) A P L K I E L S V L (SEQ ID NO:) Human B7-2 Constant Domain CCTTTCTCTATAGAG3' (SEQ ID NO:) A N F S Q P F S I E (SEQ1D NO:) Assembly ofhB7-2VIg The hB7-2V domain Ig sequence was assembled using a PCR strategy similar to that shown above. The signal sequence was derived from the onco M gene by PCR amplification of a plasmid containing the onco M gene using oligonucleotide GCAACCGGAAGCTTGCCACCATGGGGGTACTGCTCACACAGAGGACG3' 20 (SEQ ID NO: as the forward PCR primer and

AGTCTCATTGALTAAGCTTGAATCTTCAGAGGAGCCATGCTGGCCATGCTTGGA

AACAGGAG-3' (SWQ ID NO: as the reverse primer. The forward PCR primer contains a Hind III restriction site and the amino terminal portion of the onco M signal sequence. The reverse PCR contains the sequence corresponding to the 3' portion of 25 the onco Mt signal sequence fused to the 5' end of the hB7-2 IgV like domain.

The hB7-2 IgV like domain was obtained by PCR amplification of the hB7-2 cDNA using oligonucleotide GATTCAGGCTTT TTCAATGAGAC-3' (SEQ ID NO: as the forward and oligonucleotide TGTGTGTGGAkTTCTCATTACTGATCAAGCACTGACAGTTCAGAATTCATC3 (SEQ ID NO as the reverse PCR primer. PCR amplification with these primers yields the hB7-2 IgV domain with a portion of the 3' end of the onco tM signal sequence on the 5' end and a Bcl I restriction site on the 3 end. The signal and IgV domain were linked together in a PCR reaction in which equimolar amounts of the onco M signal and IQV domain DNA fragments were mixed. denatured. annealed, and the strands filled in. Subsequent PCR amplification using forward primer #05 and reverse primer #08 yie!ded a DNA fragment containinu a Hind III restriction site, followed by the onco 'M signal fused to the B7-2 lZV -89domain followed by a Bcl I restriction site. This PCR fragment was digested with Hind I and Bcl I and cloned into expression vector pNRDSH/IgG digested with the same restiction enzymes to yield pNRDSH/B7-2CIg.

12). Assembly ofhB7-2CIg The expression plasmid for h.B7-2IgC domain was prepared as described above for the IgV domain except for using PCR primers specific for the IgC domain. The onco M signal sequence was prepared using oligonucleotide #05 as the forward PCR primer and oligonucleotide 5'- AGAAATTGGTACTA T CAGGTTGACTGAAGTTAGCCATGCTGGCCATGCTTGGA AACAGGAG-3' (SEQ ID NO: as the reverse PCR primer. The hB7-2 igC domain was prepared using oligonucleotide

CTCCTGTTTCCAAGCATGGCCAGCATGGCTAACTTCAGTC

AACCTGAATAGTACCAATTTC-3' (SEQ ID NO: as the reverse PCR primer.

The two PCR products were mixed and amplified with primers #05 and #I to assemble the onco M signal sequence with the hB7-2IgC domain. The PCR product was subsequently digested with Hind III and BclI and ligated to pNRDSH/IgGI digested with similar restriction enzymes to yield the final expression plasmid PNRDSH/hB7-2CIgGI.

20 Cm on b int ar s With H n 7-2T Fuio n Prte The ability of various B7 family-Ig fusion proteins to competitively inhibit the binding of biotinylated-CTLA4Ig to immobilized

B

7 2 Ig was determined. Competition binding assays were done as follows and analysed according to McPherson (McPherson, G.A. 985) J. Pharmacol. Methods 14:213-228). Soluble hCTLA4Ig was labelled with 1251 to a specific activity of approximately 2 x 106 cpm/pmol. hB7-2-Ig fusion protein was coated overnight onto microtiter plates at 10pOg/ml in 10 mM Tris-HCl, pH8.0, 50 1 /well.

The wells were blocked with binding buffer (DMEM containing 10% heat-inactivated

FBS,

:i BSA and 50 m BES, pH 6.8) for 2 h at room temperature. The labeled CTLA4-Ig (4nM) was added to each well in the presence or absence of unlabeled competing Ig fusion 0 proteins, including full-length B7-2 (hB7-2Ig), full-length B7-1 (hB7-Ig), the variable region-like domain of B7-2 (hB7-2VIg) and the constant region-like domain of B7-2 (hB7- 2C I) and allowed to bind for 2.5 h at room temperature. The wells were washed once with ice-cold binding buffer and then four times with ice-cold PBS. Bound radioactivity was recovered by treatment of the wells with 0.5 N NaOH ftr 5 mi and the solubi!i- d material removed and counted in a gamma counter.

The results of these assays are shown in Figure 15 in which both hB7-2Ig (10-20 nM) and hB 7-2Vg (30-40 m MI) competitively inhibit the binding ofCTLA41g to immobilized B7- 2 protein. hB7-2CIg is unable to compete with soluble CTLA4, indicating that the B7-2 binding region is in found in the variable-region like domain.

F. Competitive binding Assays for B7-1 and B7-2 fusion proteins The ability of the various recombinant CTLA4 forms to bind to hB7-1 or hB7-2 was assessed in a competitive binding ELISA assay as follows. Purified recombinant hB7-Ig pg/ml in PBS) was bound to a Costar EIA/RIA 96 well microtiter dish (Costar Corp, Cambridge MA, USA) in 50 pLL overnight at room temperature. The wells were washed three times with 200 pL of PBS and the unbound sites blocked by the addition of 1 BSA in PBS (200/well) for 1 hour at room temperature. The wells were washed as above. Biotinylated hCTLA4IgGI (ref, MFGR;1 ag/ml serially diluted in twofold steps to 15.6 ng/mL; 50 pL) was added to each well and incubated for 2.5 hours at room temperature. The wells were washed as above. The bound biotinylated CTLA4Ig was detected by the additio" of 50 1/1 of a 1:2000 dilution of streptavidin-HRP (Pierce Chemical Co., Rockford, IL) for 30 minutes at room temperature. The wells were washed as above and 50 uL ofABTS (Zymed, California) added and the developing blue color monitored at 405 nm after 30 min. A graphic representation of a typical binding assay is shown in Figure 16. The ability of the various forms of CTLA4 to compete with biotinylated CTLA4IgG was assessed by mixing varying amounts of the competing protein with a quantity of biotinylated CTLA4IgGI shown to be *20 non-saturating 70 ng/mL; 1.5nM) and performing the binding assays ass described above (Figure 15). A reduction in the signal (Abs 405 nm) expected for biotinylated CTLA4IgGI indicated a competition for binding to hB7-1.

Considering the previous evidence that CTLA4 was the high affinity receptor for B7- 1, the avidity of binding of CTLA4 and CD28 to B7-1 and B7-2 was compared. B7-1-Ig or 25 B7-2-Ig was labelled with biotin and bound to immobilized CTLA4-Ig in the presence or absence of increasing concentrations of unlabeled B7-l-Ig or B7-2-Ig. The experiment was repeated with 125-I-labeled B7-l-Ig or B7-2-Ig. Using this solid phase binding assay, the avidity of B7-2 (2.7 rvM) for CTLA4 was determined to be approximately two-fold greater than that observed for B7-1 (4.6 nMI). The experimentally determined IC50 values are indicated in the upper right comer of the panels. The affinity of both B7-I and B7-2 for CD28 was lower and was difficult to confidently determine.

-91- EXAMPLE 8 dutio and Characterization ofMonclonal Antibodies to u B7- A. mmuniations ad Cell Fusion Balb/c female mice (obtained from Taconic Labs, Germantown, NY) were immunized intraperitoneally with 50 g human B7.2-Ig emulsified in complete Freund's adjuvant (Sigma Chemical Co., St. Louis, MO) or 106 CHO-human B7.2 cells per mouse. The mice were given two booster immunizations with 10-25 g human B7.2-Ig emulsified in incomplere Freund's adjuvant (Sigma Chemical Co., St. Louis, MO) or CHO-human B7.2 cels at 0 fourtee day intervals following the initial immunization for the next two months. The mice were ubled by retro-orbital bled and the sera assayed for the presence ofantibodies reactive to the immunogen by ELISA against human B7.2-Ig. ELISA against hCTLA4 was also used to control for Ig tail directed antibody responses. Mice showing a strong seroogi-cal response were boosted intravenously via the tail vein with 25 sg human hB7.2-Ig diluted in phosphatebuffered saline (PBS), pH 7.2 (GIBCO, Grand Island, NY). Three to four days following this boost, the spleens from these mice were fused 5:1 with SP 2/0 myeloma cells (American Type Culture Collection, Rockville, MD, No. CRL8006), which are incapable of secreting both heavy and light immunogobulin chains (Kearney et al. (1979)J Immunol. 123:1548).

Standard methods based upon those developed by Kohler and Milstein (Nature (1975) '20 2,L:495) were used.

B. Antibodvy Screnoin After 10-21 days, supernatants from wells containing hybridoma colonies from the fusion were screened for the presence of antibodies reactive to human B7.2 as follows: Each well of a 96 well at bottomed plate (Costar Corp., Cat. 3590) was coated with 50 l per well of a I jg/mi human B7.2-Ig solution or 5 x 104 3T3-hB7.2 cells on lysine coated plates in Phosphate-buffered saline nH 7 x 2 ce l l s o n e coated plates in phosphate-buffered saline, pH 7.2, overnight at 4 oC. The human B7.2-g solution was aspirated off, or the cells were cross-linked to the plates with glutaraldehvde, and the wells were washed three times with PBS. then blocked with 10% BSA solution (in PBS) (i00g I/well) for one ho A soluton (in PBS) (100p washed three imes with PBS and 0 o hybridoma supernatan was added per well and incubated for 1.5 hours at room temperature. Following this incubation, the wells were washed three times with PBS and then incubated for 1.5 hours at room temperature with jl per weil of a 1:4000 dilution of horseradish peroxidase-conjugated. arrnit purified. aoat anti-mouse G or inl he-avv and ligh chain-specific antibodies (HRP: Zymed Laboratories, San Francisco. CA). The wells were then washed three times with PBS. followed by a minute incubation in l per we! o I M 22 -azino-bis-3-ehlbenhizoline-6-sufon -92acid (ABTS) in 0.1 M Na-Citrate, pH 4.2 to which a 1:1000 dilution of 30 hydrogen peroxide had been added as a substrate for HRP to detect bound antibody. The absorbence was then determined at OD 4 10 on a spectrophotometric autoreader (Dynatech, Virginia).

Three hybridomas, HA3.1F9, HA5.2B7 and HF2.3D1, were identified that produced antibodies to human B7.2-Ig. HA3.1F9 was determined to be of the IgGI isotype, HA5.2B7 was determined to be of the IgG2b isotype and HF2.3D1 as determined to be of the IgG2a isotype. Each of these hybridomas were subcloned two additional times to insure that they were monoclonal. Hybidoma cells were deposited with the American Type Culture Collection, which meets the requirements of the Budapest Treaty, on July 19, 1994 as ATCC Accession No. (hybridoma HA3.1F9), ATCC Accession No. (HA5.2B7) and ATCC Accession No. _(HF2.3D 1).

C. Competitive ELISA Supernatants from the hybridomas HA3.1F9, HA5.2B7 and HF2.3DI were further characterized by competitive ELISA, in which the ability of the monoclonal antibodies to inhibit the binding of biotinylated hCTLA4Ig to immobilized hB7-2 immunoglobulin fusion proteins was examined. Biotinylation of hCTLA4Ig was performed using Pierce Immunopure NHS-LC Biotin (Cat. No. 21335). B7-2 immunoglobulin fusion proteins used were: hB7.2-Ig (full-length hB7-2), hB7.2-VIg (hB7-2 variable domain only) and hB7.2-CIg 20 (B7-2 constant domain only). A hB7. I-Ig fusion protein was used as a control. For the ELISA, 96 well plates were coated with the Ig fusion protein (50 Ll/well of a 20 .g/ml solution) overnight at room temperature. The wells were washed three times with PBS, blocked with 10 fetal bovine serum (FBS), 0.1 bovine serum albumin (BSA) in PBS for S 1 hour at room temperature, and washed again three times with PBS. To each well was 25 added 50 u.l of Bio-hCTLA4-Ig (70 ng/ml) and 50 tl of competitor monoclonal antibody supernatant. Control antibodies were an anti-B7.1 mAb (EW3.5D12) and the anti-hB7-2 mAb B70 (IgG2bK, obtained from Pharmingen). The wells were washed-again and streptavidin-conjugated horse radish peroxidase (from Pierce. Cat. No. 21126; 1:2000 dilution. 50 ul/well) was added and incubated for 30 minutes at room temoerature. The wells I* 30 were washed again, followed by a 30 minute incubation in 50 ul per well of ABTS in 0.1 M Na-Citrate, pH 4.2 to which a 1:1000 dilution of 30 hydrogen peroxide had been added as a substrate for HRP to detect bound antibody. The absorbence was then determined at

OD

4 10 on a spectrophotometric autoreader (Dynatech, Virginia). The results. shown in Table IV below, demonstrate that each of the mAbs produced by the hybridomas HA3.1F9.

HA5.2B7 and HF2.3D I are able to competitively inhibit the binding o hCLTA4Ig to fulllength hB7.2-Ig or hB7.2-VIg (hCTLA41g does not bind to hB7.2CIl).

-93-

IALEJY

Blockinz ofBinding hB7.-1g hB7.2-T hB7.2-VIg h EW3.5D12 (anti-hB7.1 mAb) Yes No No No (anti-hB7-2) No Yes Yes No HA3.1F9 (anti-hB7-2) No Yes Yes No HA5.2B7 (anti-hB7-2) No Yes Yes No HF2.3D1 (anti-hB7-2) No Yes Yes No D. Flow Cvtometrv Supernatants from the hybridomas HA3. F9, HA5.2B7 and HF2.3D1 were also characterized by flow cytometry. Supernatants collected from the clones were screened by flow cytometry on CHO and 3T3 cells transfected to express hB7.2 (CHO-hB7.2 and 3T3hB7.2, respectively) or control transfected 3T3 cells (3T3-Neo). Flow cytometry was 10 performed as follows: 1 x 106 cells were washed three times in 1 BSA in PBS, then the S cells were incubated in 50 l.1 hybridoma supernatant or culture media per I x 106 cells for minutes at 4 OC. Following the incubation, the cells were washed three times with 1 BSA in PBS, then incubated in 50 gl fluorescein-conjugated goat anti-mouse IgG'or IgM antibodies (Zymed Laboratories, San Francisco, CA) at 1:50 dilution per 1 x 106 cells for minutes at 4 The cells were then washed three times in I BSA in PBS and fixed with 1 paraformaldehyde solution. The cell samples were then analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose CA). The results, shown in Figures 17, 18 and 19 S demonstrate the monoclonal antibodies produced by the hybridomas HA3. IF9, HA5.2B7 and HF2.3D each bind to hB7-2 on the surface of cells.

Inhibirinof Proliferation of Human T Cells b Anti-hB7-- mAbs Hybridoma supernatants containing anti-human B7-2 mAbs were tested for their ability to inhibit hB7-2 costimulation of human T cells. In this assay, purified CD28 human T cells were treated with submitogenic amounts of PMA (Ing/ml) to deliver the primary signal and with CHO cells expressing hB7-2 on their surface to deliver the costimulatorv signal. Proliferation of the T cells was measured after three days in culture by the addition of H-thymidine for the remaining 18 hours. As shown in Table V. resting T ce!!s show little proiieration as measured by ]H-thymidine incorporation (510 pm). Deliver of signal I by PMA results in some proliferation (3800 pm) and T cells receiving both the primar.

(PIA)

and costimulatory (CHO/hB7-2) signais proliferate maximally (9020 cpm). All three anti- -94hB7-2 mAbs tested reduce the costimulatory signal induced proliferation to that found for PMA treated cells alone showing that these mAbs can inhibit T cell proliferation by blocking the B7/CD28 costimulatory pathway.

TABLEV

Addition to CD28 T Cells hB7-2 mAb CPM S- 510 +PMA 3800 +PMA CHO/hB7-2 9020 +PMA CHO/hB7-2 HF2.301 3.030 HA5.2B7 1460 HA3.1F9. 2980 EXAMPLE 9 10 Regression of Implanted Tumor Cells Transfected to Express B7-2 In this example, untransfected or B7-2 transfected J558 plasmacytorna cells were used in tumor regression studies to examine the effect of expression of B7-2 on the surface of tumor cells on the growth of the tumor cells when transplanted into animals.

J558 plasmacytoma cells (obtained from the American Type Culture Collection, Rockville, MD; TIB 6) were transfected with an expression vector containing cDNA encoding either mouse B7-2 (pAWNEO3) or B7-1 (pNRDSH or pAWNE03) and a neomycinresistance gene. Stable transfectants were selected based upon their neomycin resistance and cell surface expression of B7-2 or B7-1 on the tumor cells was confirmed.by FACS analysis 20 using either an anti-B7-2 or anti-B7-1 antibody.

Syngeneic Balb/c mice, in groups of 5-10 mice/set, were used in experiments designed to determine whether cell-surface expression of B7-2 on tumor cells would result in regression of the implanted tumor cells. Untransfected and transfected J558 cells were cultured in vitro, collected, washed and resuspended in Hank's buffered salt solution (GIBCO, Grand Island, New York) at a concentration of 108 cells/ml. A patch of skin on the right flank of each mouse was removed of hair with a depilatory and. 24 hours later, 5 x 106 tumor cells/mouse were implanted intradermally or subdermallv. Measurements of tumor volume (by linear measurements in three perpendicular directions) were made every two to three days using calipers and a ruler. A typical experiment lasted 18-21 days, after which time the tumor size exceeded 10 of the body mass of mice transplanted with untransfected control J558 cells. As shown in Figure 20, J558 cells transfected to express B7-2 on their surface were rejected by the mice. No tumor growth was observed even after three weeks.

Similar results were observed with J558 cells transfected to express B7-1 on their surface. In contrast, the untransfected (wild-type) J558 cells produced massive tumors in as little as 12 days, requiring the animal to be euthanized. This example demonstrates that cell-surface expression of B7-2 on tumor cells, such as by transfection of the tumor cells with a B7-2 cDNA, induces an anti-tumor response in naive animals that is sufficient to cause rejection of the tumor cells.

EOTUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

o -96- SEQUENCE LISTING GENERAL INFORMAT ION: Ci) APPLICANT: NAME: DANA- FARBER CANCER INS TITUTE STREET: 44 BINNEY STREET CITY: BOSTON STATE: MASSACHUSETTS CE) COUNTRY: USA POSTAL CODE (ZIP): 02115 TELEPHONE: (617) 632-4016 TELEFAX: (617) 632-4012 CA) NAME: REPLIGEN CORPORATION STREET: F= KNALL SQUARE, BLDG 700 CITY: CAMBRIDGE STATE: MASSACHUSETTS CE) COUNTRY:- USA POSTAL CODE (ZIP): 02139 TELEPHONE: (617) 225-6000 TELEFAX; (617) 494-1975 TITLE OF INVE-_NTION: Novel CTLA4/CD28 Ligands and Uses Therefor (iii) NUMBER OF SEQUENCES: 31 (iv) CORRESPONDENCE ADDRESS: CA) ADDRE7SSEE: LAHIVE COCKFIELD STREET: 60 State Street, Suite 510 CITY: Boston D) STATE: Massachusetts CE) COUNTRY: USA 35 ZI-P: 02109 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS CD) SOFTWARE: Patentln Release 0, Version 12 o CURRENT APPLIjCA'TION DATA: APPLICAMTION NUNMER: FILING DATE:.

CL.ASSIFICATION:

(V44) PP2OR A PPL7CAT:0N DATA: APLICAT:O0N NUMBER: US08/201,62d; USO8/1.09,393; USOS/147,772 F:LI-NG DATE: 26-JUL-1993; 19-AUG-1993; 03-NOV-1993 (vi)ATTORNEY/AG CENT INFORM-ATION: zqm:!Ez* mandragccuras, Amy E T'TONNUMBER: 36,207 5 RZFEREC,'DOCKET NUIMBER: RP7-004CP2?C -97- (iJx) TELECOMMUN~ICATION

INFORMATION:

TzETHON-.: (617) 227-7400 TE-LEFAX: (617) 227-5941 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 1120 base pairs TYPE-: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE

TYPE:

1 cDNA (ix) FEATURE: NAME/KEY: CI)S LOCATION: 107. .1093 (xiJ) SEQUENCE DESCRIPTION: SEQ ID NO:l: 0: *00.

CACAGGGTGA AAGCTTTGCT TCTCTGCTGC TGTAACAGGG ACTAGCACAG ACACA CGGAT GAGTGGGGTC ATTTCCAGAT ATTAGGTCAC AGCAGAAGCA GCCAAA ATG GAT CCC Met Asm Pro CAG TGC ACT ATG Gln Cys Thr met 115 163 211 GGA CTG ACT AAC ATT CTC TTT GTG ATG GCC TTC CTG Gly Leu Ser Asn Ile Leu Phe Val Met Ala Phe Leu

CTC

Leu TCT GGT GCT Ser Gly Ala GCT CCT Ala Pro 25 CTG AAC ATT CAA Leu Lys Ile Gin 4 40 GCT TAT TTC AAT Ala Tyr Phe Asn 30 CA.A AAC CAA AGC Gin Asn. Gin Ser- CCA GAC CTG CCA Ala Asp Leu Pro GAG ACT Glu Thr CTG AGT Leu Ser

TGC

C,/s 40 CAA TTT CCA AAC Gin Phe Ala Asn

TCT

Ser 4S GAG CTA CTA GTA TTT7 TGG CAG CAC CAG CAA AAC TTG GTT Clii Lau Val Val Phe T= Gin Asp Gi:: Clu Asn Lau Val CTG AAT GAG Leu As:: Gl1, AAG TAT ATC Lvs T-vr Met: 307 355 GTA TAC TTA Val TyrLv CGC AAA GAG AAA Cl', Lys Clu Lvs TTT CAC Phe AspD ACT GTT CAT Ser Val His

TCC

Ser GGC CGC ACA AGT1 7T7T GAT TCG GAC ACT TCC ACC C7C Gly Arg Thm Ser Phe Aso ser Asp Sr T rz ThIn Le,- 90 AGA CTT CAC AAT -98- CTT C-AG ATC AAG GAC AAG GGC TTG TAT CAA TGT ATC ATC CAT C-AC AAA Leu Gin 100 Ile Lys Asp Lys Giy 105 Leu Tyr Gin Cys 110 Ile Ile His His Lys 115 AAG C-C-C ACA GGA ATG ATT C-GC ATC C-AC C-AG ATG Lys Pro Thr Giy Met Ile Ar-g AAT TCT GAA Asn Ser Glu Ile His Gin Met 125 C-TG TCA Leu Ser 130 499 547 GTG C-TT GCT Val Leu Ala AAC TTC Asn Phe 135 AGT CALA CC-T GAA ATA GTA Ser Gin Pro Giu Ile Val 140 CC-A ATT TC-T AAT ATA Pro Ile Ser Asn Ile 145 ACA GAA AAT GTG TAC Thr Giu Asn Val Tyr 150 ATA AAT TTG Ile Asn Leu 155 ACC TGC TCA TC-T Thr Cys Ser Ser

ATA

Ile 160 CAC GGT TAC His Gly Tyr 595 CCA GAA Pro Glu 165 CC-T AAG AAG ATG Pro Lys Lys Met

AGT

Ser 170 GT-T TTG C-TA AGA Val Leu Leu Arg ACC AAG AAT TCA ACT Thr Lys Asn Ser Thr- 175

ATC

Ile 180 GAG TAT GAT GGT Glu Tyr Asp Gly Ile 185 ATG CAG AAA TC-T Met Gin Lys Ser

C-AA

Gin 190 GAT AAT GTC AC-A Asp Asn Val Thr

GAA

Giu 195 35 .00.

0:* V6000 0 C-TG TAC GAC GTT Leu Tyr Asp Val

TCC

Ser 200 ATC AGC TTG TC-T Ile Ser Leu Ser

GTT

Val 205 TCA TTC C-CT GAT Ser Phe Pro Asp GTT AC-G Val Thr 210 AGC AAT ATG Ser Asn Met TTA TCT TCA Leu Ser Ser 230

ACC

Thr 215 ATC TTC TGT ATT Ile Phe C-vs Ile

C-TG

Leu 220 GAA ACT GAC AAG Glu Thr Asp Lys AC-G CGG C-TT Thr Arg Le'u 225 C-CT C-CC C-CA Pro Pro Pro 787 835 C-CT TTC TC-T ATA Pro Phe Ser Ile

GAG

Giu 235 C-TT GAG GAC C-CT Leu Giu Asp Pro

CAG

Gin 240 GAC C-AC Asp His 245 ATT C-CT TGG ATT Ile Pro T -m Ile

AC-A

Thr 250 GC-T GTA CTT C-CA Ala Val Leu Pro

AC-A

Thr 255 GTT ATT ATA TGT Val Ile Ile Cys

GTG

Val1 260 ATG GTT TTC TGT Me:- Val Phe Cys

C-TA

Leu 265 ATT C-T A TGG AAA Ile Leu Trp Lys

TGG

Trp 270 AAG AAG AAG Lys Lys Lvs AAG- -C-GG Lys Arg 275 GAAz GAG Glu Giu 290 C-CT C-CC AAC TCT Pro Arc Asn Ser

TAT

Tyr 290 AAA TGT GGA AC-C Lys Cys Giv Thr

AAC

As r' 285 AC-A ATG GAG AGG Thin Met Glu Ara 979 AGT GA C-AG Ser C-lu Gin C-AT C-"A GC Asz Glu Al a 310

AC-C

C hr 295 AAG AAA AC-A G;A Lvs Lvs Ara C-lu Lys 300 ATC C-AT A 7TA C-CT 1-1 H is lI I- Pro C-AA AC-A TC-T C-lu Arc Ser 305 T-C-A 7-C G.AC Ser C-vs Asp 1027 2.075 C-AC C-CT GTT TTT C-in Arc Val. Phe

A.A

Lvs 315.

AC-C CG AAG ACA Ser Ser Lvs Thr

T-C?

Ser 320

C

C

OC

C*'

C(

C

C.

M

A.

25 Se 30 Le 6 Ly Let 0 40 His Glu Ser 145 His Asn Val -99- AAA AGT GAT ACA TGT TTT TAATTAAAGA GTAAAGCCCA

AAAAAAA

Lys Ser Asp Thr Cys Phe 325 INFORMATION FOR SEQ ID NO:2: SEQUENCE

CHARACTERISTICS:

LENGTH: 329 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DE(SCRIPTION: SEQ ID NO:2: et Asp Pro Gin Cys Thr Met Gly Leu Ser Asn Ile Leu Ph 1 S 10 la Phe Leu Leu Ser Gly Ala Ala Pro Leu Lys Ile Gin Ala 25 in Glu Thr Ala Asp Leu Pro Cys Gin Phe Aia Asn Ser Gin 35 40 Leu Ser Gl' Leu Val Val Phe T= Gin Asp Gin Giu Asn 55 u Asn Glu Val Tyr Leu Gly Lys Glu Lys Phe Asp Ser Val 5 70 75 s Tyr Met Gly Arg Thr Ser Phe Asp Ser Asp Ser Tmp Thr 90 i His Asn Leu Gin lie Lys Asp Lys Gly Leu Tyr Gin Cys 100 105 110 His Lys Lys Pro Thr Gly Met Ile Arg Ile His Gin Met 115 120 125 Lau Ser Val Leu Ala Asn Phe Ser Gin Pro Glu Ile Val 130 135 140 Asn Ile Thr Glu Asn Val Tvr Ile Asn Leu Thr Cys Ser S ISO 155 G% Tyr Pro Glu Pro Lys Lvs Met Ser Val Leu Lau Aro i i6S 170 Ser Thr Ile Glu Tvr Asp Gly 1le Me C>

T

v1s Sa C> A 180 195 Thr Glu Leu Tyr Asp Val Ser Ile Ser Leu Ser Val Ser 195 200 205

V

AT

Le Hi Le 9!

~.E

Asn ?ro e r .rT al Met yr Phe ;n Gin u Val s Ser u Arg lie Ile lie Lvs Asn 1120 -100- Asp Val Thr Ser Asn Met Thr Ile Phe cys Ile Leu Glu Thr Asp Lys 210 215 220 Thr Arg Leu Leu Ser Ser Pro Pile Ser Ile Giu Leu Glu Aspo Pro Gin 225 230 235 240 Pro Pro Pro Asp His Ile Pro Tp Ile Thr Ala Val Leu Pro Thr Val 245 250 255 Ile le Cys Val. Met Val. Phe Cys Leu. Ile Leti Trp Lys T= Lys-Lys 260 265 270 Lys Lys Arg Pro Arg Asn Ser Tyr Lys Cys Gly Thr Asn Thr Met Giu 275 280 285 Arg Giu Giu Ser Giu Gin Thr Lys Lys Arg Giu Lys Ile His Ile Pro 290 295 300 Giu Arg Ser Asp Giu Ala Gin Ara Val Pile Lys Ser Ser Lys Thr Ser 305 310 315 320 Ser Cvs Asm Lvs Ser Asr Thr Cvs Phe 325 INFORMATION FOR SEQ ID NO:3: Ci SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYfPE: nucleic acid STRANDEDNES S: single (ID) TOPOLOGY: linear MOLECULE TYPE: oligoruclectide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TAATACGACT CACTATAGGG INFORMATION FOR SEQ1 ID NO:-4: LENGTH-: 1-3 base pairs TYP: uclei4c acid SPAEDE Si sngle TOPOLOGY--: linear O (ii) MOLETCULE" TY=E: clcculo-d (xi) SEQUENCE DES CF ?T!C SEQ 1D NO:4: TAAGGTTCCT TCACAAG i INFORMATION FOR SEQ ID C)SEQUENCE

CHARACTERISTICS:

LENGT H: 21 *base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: oliccnucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID No:S: ACTGGTAGGT ATGGAAGATC C 21 INFORMVATION FOR SEQ ID NO:6: Wi SEQUENCE

CHARACTERISTICS:

CA) LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS. single TOPOLOGY: linear (ii) MOLECULE TYPE: oligonuclectide SEQUENCE DESCRIPTION: SEQ ID NO:6: *ATGCGAATCA TTCCTGTGGG C 2 INFORMATION FOR SEQ ID NO:7: Wi SEQUENCE

CHARACTERISTICS:

CA) LENGTH: 21- base pairs TYPE: nucleic acid 40 STRANDEDNESS: sincle TOPOLOGY: linear (iJi) MOLEC ULE TYPE: oligonucleotide (xi) SEQUENCE IDESCRIPTION: SE7Q ID NO: 7: A.AAGC-CACA GGAATG.ATTC G 2 INFORMATION FO0R SEQ ITD NO:S: C:4) SEQUENCE

C-AACTERISTICS:

LENGTH: 2! base zairs TYPE: nucleic acid -102- STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECIULE TYPE: oligonuclectide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: CTCTCAAAAC CAAAGCCTGA G 21 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHAR.ACTERISTICS: LENGTH: 21 base pairs TYPE: nucileic acid ST.R=NEDNEzSS: single TOPOLOGY: linear (ii) MOLECUJLE TYPE: oligonucleotide, (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: TTAGGTCACA GCAGAAGCAG C 21 0 .0 INFORMATION FOR SEQ ID Wi SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STR7ADEDNESS: single TOPOLOGY: linear Uii) MOLECULE TYPE: oliaonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID TCTGGAXACT GAC:A'AGACGC G 21 INFORMATION FOR SEQ ID NO:11: (iJ) SEQUENCE CILA CTERISTTCS: LE7NGT-T: 2:1 base pairs TP:nucleic acid NMEDYESS: sincle TCO=L0GY: 1-Ljnear MOLECULE7 TYPE-: cliconuclectide -103- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l1: CTCAGGCTTT GG-TTTGAGA G 21 INFORMATION FOR SEQ ID NO:12: i) SEQUENCE

CHARACTERISTICS:

LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: CACTCTCTTC CCTCTCCATT G 21 2 INFORMATION FOR SEQ ID NO:13: SEQUENCE

CHARACTERISTICS:

LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GACAAGCTGA TGGAAACGTC G 21 INFORMATION FOR SEQ ID NO:14: SEQUENCE

CHARACTERISTICS:

LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: sincle TOPOLOG1: linear J(ii) MOLECULE TYPE: oliqcnucleotide (xi) SEQUNCE ESCRTION: SEQ ID NO:14: CAATGGAGAG GGAAGAGCT

G

INFORN~zATIN F3, S: Q ID NC' SEQUENCE CzHARA

CR:ST:CS.

-104- LENGTH: 12 base pairs TYPE: nucleic acid STP.AIDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE:.oligonucleotide (XiJ) SEQUENCE DESCRIPTION: SEQ ID CTTTAGAGCA CA INFORMATION FOR SEQ ID NO:16: Wi SEQUENCE CHARACTERISTICS: LENGTH: 8 base pairs TYPE: nucleic acid STRJANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA 30 CxiJ) SEQUENCE DESCRIPTION: SEQ ID NO:16:

CTCTAAAG

INFORMATION FOR SEQ ID NO:17: Ci) SEQUENCE CHAR.ACTERISTICS: CA) LENGTH: 9 amino acids TYP-'E: amino acid CD) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide SEQUENCE DESCR:2TION: SE-Q ID NO:17: Lys Tvr Met Gly Ara Thr Ser Phe Asp INFORMATION FOR SEQ ID NO:lS: C)SEQUENCE CHARAL c"7EISTICS: LENGTI- 13 aminc acids .amnz acid 7D) ?OPLOGY: linear Ci4) MGL-ECT--- TYPE: cent ide -105- (xi) SEQUIENC- DESCRIPTION: SEQ ID NO:18: Lys Ser Gin Aspo Asn Val Thr Glu Lys Tyr A-sp Val. Ser INFORMATION FOR SEQ ID) NO:19: i)SEQUENCE

CHARACTERISTICS:

LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear ii MOLECULtE TYPEa peptide (xi) SEQUENCE DESCRIPTION: SEQ ID) yO:19: Trp Lys Trp Lys Lys Lys Lys Arg Pro Arg Asn Ser Tyr Lys Cys s 10 1 INFORMATION FOR SEQ ID C)SEQUJENCE

CHARACTERISTICS:

LENGTH: 17 base ipairs TYPE: nucleic acid STR-NDEDNESS. single :30 TOPOLOGY: linear (ii) MOLECULE TypE: oligonuclectide (xi) SEQUENCE DESCRIPTION: SE7Q ID TGGCCCATGG CTTCAGA 1 o INFORMATION -7OR SEQ ID NO2i Ci) SEQUENCE CH.ARA1CTERISTICS: CA) LE.NGTH: 17 base paz.rs CB) TYPE-: nucleic acid CC) STR..NEDNEESS: sirgie TCP0L0Gv,' li4near (ii) MOLECUjE TYPE: cligonucilectid (xi4) S'EQUENCE DESCP:P-TON. SEQ 110 NO:21: GCC-AAAAT.GG A7TCCCCA7 -106- INFORMATION FOPR SEQ ID NO:22: Wi SEQUENCE CHARACTERISTICS: LENGTH: 1163 base pairs TYPE: nucleic acid sTR=NDNss: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/=-Y: CDS LOCATIONI: i1i.. 1040 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: CCCACGCGTC CGAGCAAG CAGACGCGTA AGAGTGGCTC CTGTAGGCAG CACGGACTTG AACAACCAGA. CTCCTGTAOA CGTGTTCCAG AACTTACGGA AGCACCCACG ATG GAC Met Aso CCC AGA TGC ACC ATG GOT TTO Pro Arg Cys Thr Met Giv Leu

GC-:

Al a ATC CTT ATC TTT Ile Leu Ile Phe

GTG

Val is ACA GTC TTG Thr Val Leu 164 CTG ATC Leu Ile TCA GAT OCT GTT Ser As-o Ala Val

TCC

Ser 25 GTG GAG ACO CAA Val Giu Thr Gin

OCT

Ala TAT TTC AAT 000 Tyr Phe Asn Gly

ACT

Thr OCA TAT CTG CCO Ala Tyr Leu Pro

TOC

Cys 40 CCA 77TT AC.A AAO Pro Phe Thr Lys

OCT

A-1a 45 CAA AAC ATA AGC Gin Asn Ile Ser

CTO

Leu AOT GAG CTO OTA Ser Olu Leu Val TTT TOO CAG GAC Phe Trm Gin Asp CAA AAO TTO GTT Gin Lys Leu Val CTO TAC Leu Tyr GAG CAC TAT 0Th His Tyr CTG 0CC CC Lau Olv Arg Le 70 CCC AC" GAO A Cly Thr Giu Lvs

CTT

Lau 75 OAT AOT OTO AAT Asp 5cr Val Asn GC- AAC TAC Ala Lvs Tvr CC-A CTT CAC Ar= Lau His 1 56 ACG AOC TTT GAC Ttr Ser ?he ASP AAC AAC TOO ACT Asn Asn ToThr

CTA

Leu 404 AAT OTT- 100 CAC ATC AAC GCT ATG M e s CCC TCC TAT OAT TOT C, s 110 T777 AT.A: C.A AA-A =Iie G1- Lys 452

AAG

Lys 115 CCA CCC ACA GGA Pro Pro Thr Gly TCA ATT Ser Ile 120 -107- ATC CTC CAA CAG le Leu Gin Gin 125 ACA TTA ACA GAA Thr Leu Thr Giu

CTG

Leu 130 TCA GTG ATC GCC AAC TTC AGT GAA CCT GAA ATA AAA CTG Ser Val Ile Al~a Asn Phe.Ser Giu Pro Glu 140 le Lys Leu GTA ACA GGA AAT TCT GGC ATA AAT Val Thr Giy Asn Ser IS0 Gly Ile Asn

TTG

Leu 155 ACC TGC ACG TCT Thr Cys Thr Ser GCT CAG AAT Aia Gin Asn 145 AAG CAA GGT Lys Gin Giy 160 TCA ACT AAT Ser Thr Asn 548 596 CAC CCG AAA His Pro Lys 165 CCT AAG AAG ATG Pro Lys Lys Met

TAT

Tyr 170 TTT CTG ATA ACT Phe Leu Ile Thr

AAT

Asn 175 644 GAG TAT Giu Tyr GGT GAT'AAC ATG Gly Asp Asn Met

CAG

Gin 185 ATA TCA CAA GAT Ile Ser Gin Asm

AAT

Asn 190 GTC ACA GAA Vai Thr Glu

CTG

TGG

Trp 210

TTC

Phe 195 AGT ATC TCC AAC Ser Ile Ser Asr.

AGC

Ser 200 CTC TCT CTT TCA Leu Ser Leu Ser CCG GAT GGT GTG Pro Asp Giy Val 740 788 .930 0. CAT ATG ACC'GTT His Met Thr Val

GTG

Val 215 TGT GTT CTG GAA Cys Val Leu Giu

ACG

Thr 220 GAG TCA ATG AAG Giu Ser Met Lys ATT TCC Ile Ser 225 TCC AAA CCT Ser Lvs Pro TGG AAG GAG Trp Lys Glu 24S

CTC

Leu 230 AAT TTC ACT CAA Asn Phe Thr G in

GAG

Giu 235 TTT CCA TCT CCT Phe Pro 5cr Pro CAA ACG TAT Gin Thr Tyr 240 CTT GTG ATG Leu Vai Mec 836 884 ATT ACA GCT TCA Ile Thr Ala Ser

GTT

Val1 250 ACT GTG GCC CTC Thr Vai Ala Leu

CTC

Leu 255 CTG CTC Leu Lau 260 ATC ATT GTA TGT Ile Ile Val Cys

CAC

His 265 AAG AAG CCG AAT Lys Lys Pro Asn

CAG

Gin 270 CCT AGC AGG CCC Pro Ser Ara Pro 932 980

AGC

Ser 275 AAC ACA GCC TCT Asn Thr Ala Ser

AAG

Lys 280 TTA GAG CGG GAT Lau Glu Arg Asp

AGT

Ser 285 AAC GCT GAC AG.X Asn Ala Asp Ara

GAG

Glu 290 ACT ATC AAC CTG AAG GXA. CTT G.X, CCC Thr 7'e Asn Lau Lvs Glu Lau Gl-' Pro

CAA

Gin 3~00 ATT G CT T CA GCA li Ala Ser Ala AAA CCA Lvs Pro- AAT GCA GAG TG.kGGC-T GAGA1G-CTA GGAAGAGTT- AA-TG1-CT Asn Ala Glu TTGCC?-GA TAAGA:AGTGcACCTC? ACATC A TGTTCT7CA =TATT AT-TCTA.CAGT TGAATAA-TTA AGAA-C 1028 1077 !1127 1163 -108- INFORMATION FOR SEQ ID NO:23: Ci) SEQUENCE CHARACTERISTICS: LENG-TE:..309 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Met Asp Pro Arg Cys Thr Met Giy Leu Ala Ile Leu Ile Phe Val Thr r 1 Val Asn Ser Leu 6q Lys Leu Gln Glu Gin 145 Gin hr c- Leu Gly Leu Tyr Tyr His Lvs Leu 130 As-, Asr-, Leu Leu Thr Ser Glu Leu Asn Lys 115 Ser Val Glu ?he Ile Ala Glu His Glv Val 100 Pro Val Thr Pr:) Tvr 190 Ser 5 Ser Tyr Leu Tyr Arg 85 Gin Pro lie Gly Lvs 165 Gly l1e Ala Pro Val 55 Gly Ser Lvs Giy Asn 135 Ser Lys Asn As val s5 Val Cvs 40 Phe Thr Phe Asp Ser .20 Phe Gly Lvs Met 5er z00 Ser Pro Trp Glu Asp Met 105 Ile Ser Ile Met Gin 185 Leu 10 Val Phe Gin Lys Arg 90 Gly Ile Glu Asn Tvr 170 Ile Ser Glu Thr Asp Leu Asn Ser Leu Pro Leu 155 Phe Ser Leu Thr Lys Gin Asn Asn Tyr Gin Glu 140 Thr Leu Gin Ser Gin Ala Gin Ser Trp Asp Gin 125 Ile Cys lie Aso Phe 205 Tyr Asn Leu Asn Leu Phe Leu Leu S er Asn 175 Val Asz Val 22.0 :s Lc: Th-r Cvs VaI Le e Tr Gu S %r Met Lvs 220 Ile 225 Thr Val Arg Arg Lys I 305

I

*3 0 3

ACATAA

IN

*5 (ii (xi

ATGATGA

Ser Ser Tyr Trp Met' Leu Pro Ser 275 Giu Thr 290 ?ro Asn *1 Lys Lys Leu 260 Asn ri la Pro Leu 230 Glu Ile 245 Ile Ile Thr Ala Asn Leu Glu Asn Phe Thr Ala Val Cys Ser Lys 280 Lys Glu 295 Thr Ser His 265 Leu Leu -109 Gin Val 250 Lys Glu Glu Glu Phe 235 Thr Val Lys Pro Arg Asp Pro Gin 300 Pro Ser Pro Gin 240 Ala Leu Leu Leu 255 Asn Gin Pro Ser 270 Ser Asn Ala Asp 285 Ile Ala Ser Ala NFORMATION FOR SEQ ID NO:24: SEQUENCE CHA,-UCTERISTICS: LENGTH: 2i base pairs TYPE: nucleic acid STRAXNDEDNESS: single TOPOLOGY: linear i) MOLECLE TYPE: oligonucieotide SEQUENCE DESCRIPTION: SEQ ID NO:24: GCCT GAGTGAGCTG 0 FORMATION FOR SEQ ID i) SEQUENCE CHARACTERISTICS: LENGTH: 21 base cairs TYPE: nucleic acid S RAED ESS* sniae TOPOLOGY: i-4ear MOLECE TYPE: cliconuclecide SEQUENCE DESCR SE ID NO:2S: GCA GCATC-z,--Ac

G

INF0RTON FOR. SEQ -n No:6: -110- Wi SEQUENCE CtLARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY:. linear (ii) MOLECUJLE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ.ID NO:26: TGGTCGAGTG AGTCCGAAT.A C 21 154 INFORMATION FOR SEQ ID NO:27: Wi SEQUENCE CH.ARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (xi4) SEQUENCE DESCRIPTION: SEQ ID NO:27: GACGAGTAGT AACATACAGT G 21 INFORMATION FOR SEQ ID NO:29: Wi SEQUENCE C:.-RXACTERISTICs: LENGTH: 1491 base pairs TYPE: nualeic acid STIRAkNMEDNESS: double T0POLCG-,':4~ (i4i) MOLECULE TY-PE: cDNA to mRNA (iii) H 7POTrHv'IC: -no (iJv) AN7I-SENSZ: no (Vi) 0RIG,NT. SCUP.CE: CRGCANS=!: Hornc sajjen' CELL L:"77: Ra~i -111- (vii) IMME~IATE SOURCE: LIBRARY: cDNA in pC~m8 vector CLONE: B7, Raji clone #13 (viii) POSITION IN GZNOME: CHOMOSOME/SEGMENT 3 (ix) FEATUR.E: NAME/KEY: Open reading frame (translated region) LOCATION: 318 to 1181 bp IDENTIFICATION METHOD: similarity to other pattern (ix) FEATURE: NAME/KEY: Alternate polyadenylation signal LOCATION: 1474 to 1479 bp IDENTIFICATION METHOD: similarity to other pattern (x PUBLICATION INFORMATION: AUTHORS: FREEMAN, GORDON J.

SEGIL, JEFFREY M.

LEE, GRACE WHITMAN, JAMES F.

30 NADLER, LEE M.

TITLE: B7, A New Member Of The Ig Superfamily With U~nique Expression On Activated And Neoulastic B Calls *35 JOURNAL: The Journal of Immunology VOLUME: 143 ISSUE: 8 PAGES: 2714-2722 DATE: 15-OCT-1989 RELVAN RESIDUES In S'EQ ID NO:28: FROM 1 TO 1491 :*,Goes SEQUENCE DESCR:7PTION: SEQ ID) No:28: 4: CCAAAGAAAA AGTGAT.TTGT CATTGCT"?A7 TAGACTGTAA GAAGAGAAC-A TCT-CAGAAGT GGAGTC77TAC CICTGAAATCA AAGGATTT7 AGAAAc-TG GATT-TTTCT TCAGCAAGCT 120 GTGACT-rA. ATCCACALACC ~G C CAGGAACA'CC CT-CCAATCT.C T G 7G7GTTT7T 180 GTA~ATA AT~TGTCATC.'cC CCTGCCTG-T 240 TTGCACrCTGG GA AGT1GCCCT C-GCTTAzC77 GGGTCCAA:AT TG'-TGGC7TTT C-%CTTTTG C 300 CCTAAGCATC TGAAGCC ATG GGC CAC ACA CGG AGG CAG GGA ACA TCA CCA TCC 353 Met Gly His Thr Arg Arg Gin Gly Thr Ser Pro Ser AAG TGT CCA TAC CTG AAT. TTC =TT CAG CTC TTG GTG CTG GCT GGT CTT Lys Cys Pro Tyr Leu Asn Phe Phe Gin Leu Leu Val Leu Ala Gly Leu 401 449 TCT CAC Ser His TTC TGT TCA GG T GTT ATC C.AC GTG ACC AAG GAA GTG AAA GAA Phe Cys Ser Giy Val. Ile His Val. Thr Lys Giu Val. Lys Giu GTG GCA ACG CTG Val. Ala Thr Leu

TCC

Ser

TGT

Cys GGT CAC AAT Gly His Asn

GTT

Val.

20 TCT GTT GAA GAG Ser Val Giu Glu CTG GCA Leu Ala CAA ACT CGC Gin Thr Arg ATG TCT GGG Met Ser Gly 45

ATC

Ile TAC TGG CAA AAG Tyr Tro Gin Lys

GAG

Giu 35 AAG AAA ATG GTG Lys Lys Met Vai CTG ACT ATG Leu Thr Met CGG ACC ATC Arc- Thr Ile 545

J'

GAC ATG AAT ATA Aso Met Asn Ile

TGG

Trp so CCC GAG TAC AAG Pro Giu Tyr Lys

A.AC

Asn 593 641 TTT GAT Phe Asp ATC ACT A-AT AAC Ile Thr Asn Asn

CTC

Leu 65 TCC ATT GTG ATC Ser Ile Vai Ile

CTG

Leu GCT CTG CGC CCA Ala Leu Ara Pro T CT Ser 75 GAC GAG GGC AC.A Aso Giu Giy Thr

TAC

Tyr 80 GAG TGT GTT GTT Giu Cys Vai Val

CTG

Leias AAG TAT GAA Lys Tyr Giu AAA GAC Lys -Aso AAA GCT Lvs Aia i GCT TTC AAG CGC Aia Phe Lys Arc7

GAA

Glu CAC CTG GCT GAA His Leu Aia Glu

GTG

Val 100 ACG TTA TCA'- GTC Thr Leu Ser Val GAC TTC CCT Asp Phe Pro

ACA

Thr lio- CCT AG? ATA TCT Pro 5cr le Ser

GAC

Asp 11.5 TTT GAA ATT CC.A Phe G2lu 1-ie Pro ACT AAT Thr Ser Asn 120 G AG C?7 CA C Pro H~is AT? AGA ACC lie Arc: Arg ATA A??7 TC-'C TCA ACC TOTC7CA CCM GG7Tlie lie Cvs Ser Thr 5cr Civ Civ, Phe 12 C C1 Pro CTC TCC TGG

TTG

Leu Ser Trp Leu 140 -113-* GAA AAT GGA GA GAA TTA AAT Giu A-Sn Gly Glu Glu Leu Asn 145

GCC

Ala

ISO

ATC AAC ACA ACA Ile Asn Thr Thr 881 929

GTT

Val 155 TCC CAA

GAT

Ser Gin As-o CCT GAA.ACT GAG CTC TAT Pro Glu Thr Giu Leu Tyr 160

GCT

Al a 165 GTT AGC AGC AAA Val Ser Ser Lys

CTG

Leu GAT TTC AAT ATG ACA ACC AAC CAC AGC Asp Phe Asn Met Thr Thr A-sn His Ser 175

TTC

Phe 180 ATG TGT CTC ATC Met Cys Leu Ile AAG TAT Lys Tyr GIGA CAT TTA AGA GTG AA I Gly His Leu Ara Val A-sn 190 CAG ACC TTC Gin Thr Phe 195 AAC TGG AAT ACA Asn Asn Thr

ACC

Thr 200 AAG CA Lys. Gin 1025 GAG CAT TTT Giu His Phe 205 CCT GAT AAC CTG Pro Asp Asn Leu

CTC

Leu 210 CCA TCC TGG GCC Pro Ser Trp Ala

ATT

Ile 215 ACC TTA ATC Thr Leu Ile 1073 ease 35 4* TCA GTA Ser Val 220 AAT GGA ATT TTT_ Asn Gly Ile Phe

GTG

Val 225 ATA TGC TGC CTG Ile Cys Cys Leu

ACC

Thir TAC TGC TTT GCC Tyr Cys Phe Ala 1121 1169

CCA

Pro 235 AGA TGC AGA AZrg Cys Ara GAG AGA AGG Glu Arg Arg 240 AGG AAT GAG Arg A-sn Giu

AGA

Arg TTG AGA AGG GAA Leu Arg Ara- Giu

AGT

Ser 250 GTA CGC CCT GTA TAACAGTGTC CGCAGAAGCA AGGGGCTGAA

AAGAT-!CT-GAA

Val Arg Pro Val. 1221 GGTAGCCTCC GTCATCTCTT CTGGGATACA TGGATCGTGG GGATCATGAG GaTTCTTCC 1281 *CTTAAC-kAT- TTAAC-CTGTT TTACCCACTA CCTCACCTTC TTAAA:AACCT C-TTTCAGATT 1341 AAGCTGAACA GTTACX AGAT GC--GGCATC CCTCTCCTTT CTCCCCAT-r GZAATTTGCT 1401 TAATG7-'kCC TCT7TTTG CCA'7 T T T CC ATTCTGCC;-CT TT C77GTCA CC 1461 AATTCA:,TTAT CTAT-!7AAACA C77A;ATTTGACG 1491.

iNF'ORPATION 7OR 7D NO: 29: -114- SEQUENCE CARACTERISTICS: LENGTH: 288 amino acids TYPE: amin-'o acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein DESCRIPTION: B cell activation antigen; natural ligand for CD28 T cell surface antigen; transmembrane protein (ix) FEATURE: NAME/KEY: signal sequence LOCATION: -34 'to -1 IDENTIFICATION METHOD: amino terminal sequencing of soluble prote.ni OTHER INFORMATION: hydrophobic (ix) FEATURE: NAME/KEY: extracellular domain LOCATION: 1 to 208 IDENTIF ICATION MNETHOD: similarity with known sequence (ix) FEATURE: NAIME/KEY: transmiembrane domain LOCATION: 209 to 235 IDEINTIFICA-TION METHOD: similarity with known sequence (ix)1 FEATURE: 4 NAME/KEY: intracellular domain :40 LOCATION: 236 to 254 IDENTIF7C-ATION MEITHOD: similarity with known s equenc e NAME/KZ*Y-: N-lin'..ed alvccsylationi LOCATION: 19 to 21 IDE.NT7:F:c.TIoN ME THOD: similaritv withi known sequ e nc e NAMJ E/KEIY: N-cr; ±vrcosvlar-ion LOCATION: 55 to 57 IDENTIFICATION METHOD: similarity with known sequence UiX) FEATURE: NAME/KE~y: N-linked glycosylation LOCATION: 64 to 66 IDENTIFICTION METOD: similarity with known seqruence (ix) FEATURE: NAME/KEY: N-linkfd lycosylation LOCATION: 152 to 154 IDETIFICATION M~THD: similarity with known sequence (ix) FEATUR17: NAME/KZ-Y: N-linked glycosylation LOCATION: 173 to 175 IDENTIFBICTION METOD: similarity with known sequence (ix) FEATURE: NAMr-/I-rY N-linked glyccsylation LOCTION: 177 to 179 IDENTTIFICXricN METHiOD: similarity with known ~sequence (i)FEATURE: !CO (A NAE/=Y N-linked qlvccs-vation (B1) LOCATION: 192 to 194 IDENIPiFICATIIN ME77HOD: similarity with known seuence N-Iiri.'Ked glcosvation _rOC.: _77CN 18 to 700 ON METHOD: sim-4laity with knoum se-quenc-2 -116- (ix) FEATURE: NAME/KEY: Ig V-set domain LOCATION: 1 to 104 IDENTIFICATION METHOD: similarity with known sequence (ix) FEATURE: 0 NAME/KEY: Ig C-set domain LOCATION: 105 to 202 IDENTIFICATION METHOD: similarity with known sequence PUBLICATION INFORATION: AUTHORS: FREEMAN, GORDON J.

FREEDMAN, ARNOLD S.

SEGIL, JEFFREY M.

LEE, GRACE WHITMAN, JAMES F.

NADLER, LEE M.

*s S. S 0000 0O 0 S. S

SS

S.

OSSO

25 TITLE: B7, A New Member Of The Ig Superfamily With Unique Exnression On Activated And Neoplastic B Cells JOURNAL: The Journal of Immunology VOLUME: 143 ISSUE: 8 30 PAGES: 2714-2722 DATE: 15-OCT-1989 RELEVANT RESIDUES IN SEQUENCE ID NO:29: From -26 to 262 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: Met Gly His Thr Arg Gin Gly Thr Ser Pro Ser Lys Cys Pro Tyr 45 9*6* 45 Leu Asn Phe Ser Glv Val -1 1 Phe Gin Leu Leu Val Leu Ala Gly Leu Ser His Phe Cys Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cvs Gly His Ash Val 20 Ser Val Glu Glu Leu Ala Gin Thr Arc Tvr Tr- Gin Lvs Lys Lvs Mec Val Leu Thr Met Met Ser Glv Aso Met Asn lie Tr P?ro G'1 Tvr Lvs Asn Arc Thr Ile Phe 55 Aso lie Thr -117- Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly Thr Glu Pro Ile Glu Pro Thr 175 Val 2 Asp 30 Ile P Ty7 80 His Ser Cys Asn Glu 160 Thr Glu Leu Ile Ser Gly 145 Thr Asn Cys Ala Ser Thr 130 Glu Glu His Va Gii AsE 115 Ser Glu Leu Ser 1.

Val Val 100 Phe Leu 85 Thr Glu Lys Leu Ile Tyr Ser Pro Glu Val Thr Lys Lys 105 Ser Asp Ala Asn Ala Asp Ile Phe PhE Arg 120 Gly Leu" Tyr Phe 180 Asn Ser Cys Gly Asn Ala 165 Met Trp Tro Leu Phe Ala 150 Val Cys Asn Ala Thr' Pro 135 Ile Ser Leu Thr Ile 215 I'yr Glu Asn Ser Ile Thr 200 Thr Cys Pro Thr Lys Lys 185 Lys Leu Phe His Thr Leu 170 Tyr Gin Ile Ala Leu Val 155 Asp Glv Glu Ser Pro 2 Ser 140 Ser Phe E s His VTal 220 kra Ly.

Pro Arg 125 Tr-m Gin Asn Leu Phe 205 Asn Cvs Arg Thr 110 Ile Leu Asp MeE Arg 190 Pro GJy Ara 0 .0 a 0 000.0 0.

oooo o .0.0 000.

0 ksn sn ,he Gin Leu Val Thr Leu 210 Ile Phe 195 Pro Cvs

I

225 230 235 Val Ara Pro Val Glu Arcr 240 Arg Arg Asn Glu Arg 245 Leu Arg Arg Glu Ser 250 14U INFORMATION FOR SEQ ID SEQUENCE CHAR-CTERISTICS: LENGTH: 1716 base pairs TYPE: nucleic acid SPil IMEDYESS: double TOPOLOGY: l (ii) MCLECrLJ-L 7YP: oDNA t\ mN (iii) HYPCPOT1-TICAL: nc (vi) OR7GINAL SOUR:

ORGANISI:

-118- DEVELOPMENTAL STAGE: germ line TISSUE TYPE: lymnphoid CELL TYPE: B lymphocyte CELL LINE: 70Z and (vii) IMME~DIATE SOURCE: LIBRARY: cDNA in pCDM8 vector CLONE: B7 #Is 1 and 29 (ix) FEATURE: NAME/T.EY: translated region LOCATION: 243 to 1166 bpo IDENTI-FICAT;ON METHOD: similarity to other pattern (ix) FEATURE: NAME/KEY: Alternate ATG initiation codons LOCATION: 225 to 227 and 270 to 272 IDENTIFICATION METHOD: similarity to other pattern 25(xi) SEQUENCE DESCRIPTION: SEQ ID GAGTTTTATA CCTC-AATAGA CTCTTACTAG TTTCTCTTTT TCAGGTTGTG AAACTCAACC TTCAAAGACA CTCTGTTCCA TTTCTGTGGA CTAATAGGAT CATCTTTAGC ATCTGCCGGG 120 TGGATGCC.AT CCAGGCTTCT TLTTTCTACAmT CTCTGTTTCT CGATTTTTGT GAGCCTAGGA 180 GGTGCCTAAG CTCCATTGGC TCTAGATTCC TGGCTTTCCC C-ATCAT-GTTC TCCAAAGCAT 240 CTGAAGCT ATG GCT TGC AAT TGT CAkG TTG ATG C-AG GAT ACA CCA CTC CTC 290 :Met Ala Cvs Asn Cys Gin Leu Met Gln Asp Thr Pro Lau Lau -30 AAG TTT CCA TGT CCA AGG CTC AAT CTT CTC TTT GTG CTG CTG AT? CGT 338 40 Lys Phe Pro Cvs Pro Ara Lau lie Leu Leu Phe Val Lau Leu Ile-Ara -15 CT? TCA CA GTG TCT TCA GAT G? C AT GAA CA CTG TCC AAG TCA GTG 386 Leu Ser Gin Val Ser 5cr Asp Val Asp Glu Gln Leu Ser Lys Ser Val 4 5 -s 1 GAT AAG GA TTG CTG CC ?-GC CC? TAC AAC TC?- CCT CAT G GAT 434 Lys Asp Lys Val Lau Leu Pro Cys Ara Ty/r Asn Ser Pro His Glu Aso :z20 2S GAG TCT GAA GAC CGA AT C TAC TGG CA-L A~A CAT GAC AAA GTG GG C?-G 482 Glu Ser Glu Aso Ar-= Ile Tvr Trz Gin Lvs His Aso Lvs Val Val Lau 35 TCT GTC ATT GCT GGG AAA CTA AAA GTG TO CCGG A AGx- G Ser Val Ile Ala Gly Lys Leu Lys Val Trp Pro Giu Tyr Lys Asn Arg 50 530 578 ACT TTA TAT GAC AAC ACT ACC TAC Thr Leu Tyr Asp Asn Thr.Thr Tyr 65 TCT CT-T ATC ATO CTG GGC CTG GTC Ser Leu Ile Ile Leu Gly Leu Val CTT TCA Leu Ser GAC CGO GGC ACA Asp Arg Gly Thr

TAC

Tyr so AGC TGT GTC GTT Ser Cys Val Val.

CAA

Gin AAG AAG GAA AGA Lys Lys Giu Arg 626

GGA

Gly go ACO TAT GAA GTT Thr Tyr Giu Val

AAA

Lys 95 CAC TTG GCT TTA His Leu Ala Leu

GTA

Val 100 AAG TTG TCC ATC Lys Leu Ser Ile

AA

Lys 674 722 OCT GAC TTC TCT ACC CCC AAC ATA ACT Ala Asp Phe Ser Thr Pro Asn Ie- Thr 110

GAG

Giu 115 TCT GGA AAC CC.A Ser Gly Asn Pro TCT GCA 05 .30 GAC ACT AAA Asp Thr Lys CGC TTC TCT Arg Phe Ser 140

AGG

Arg 125 ATT ACC TOC TTT Ile Thr Cys Phe

OCT

Ala 130 TCC GGG GGT TTC Ser Oly Oly Phe CCA AAO CCT Pro Lys Pro 135 ATC AAT ACO Ile Asn Thr TOG TTG GAA AAT Trp Leu Gilu Asn

GGA

Glv 145 AGA GAA TTA CCT Arg Oiu Leu Pro

GC

Oly ACA ATT Thr Ile TCC CAG OAT CCT Ser Gin Asp) Pro

GAA

Glu 160 TCT GAA TTG TAC Ser Glu Leu Tyr

ACC

Thr 165 ATT AGT AGC C-AA Ile Ser Ser Gin 770 818 866 914 962

CTA

Leu 170 OAT TTC AAT ACG Asp Phe Asn Thr

ACT

Thr 175 COO AAC C-PC ACC Arg Asn His Thr ATT AAG Ile Lys 180 TOT CTC ATT Cys Leu Ile

AAA

Lys TAT OGA OAT GCT CAC GTG TCA GAG GAC T'yr Gly Asp Ala His Val Ser Glu Asp 190

TTC

Phe 195 ACC TOG GAA A.AA: Thr Trp Glu Lys CCC CCA Pro Pro 200 GCA. GGA Ala Glv GAA GAC CCT Glu Asp Pro TTC GGC GCA Phe Gly Ala 220

CCT

Pro 205 GAT AGO AAG AAC Asp Ser Lvs Asn'

ACA

Thr 210 CTT GTG CTC TTT Leu Val Leu Phe 1010 GTA ATA ACA G00 Val T 7e Thr Val

GTC

Vali GT0 ATC O TT GTC Val 71i- Val Val

ATC

ile ATC AAA TGO Lvs CY/S 1058 1106 TT -TG Phe Cys 235 AAG CAC ACA AGO Lys Hiis Ara Ser Cvs TTC AGA AGA AAT Phe Ara Ara Asn G:A G Glu GCA AC-C AGA Ala Ser Ara GTh ACA AA Ac AGO 7 k CT C TO C0 OTTCC GA Thr Asm Asn Ser Leu Thr Phe Glv Pro 0G u Z3 50 253 GA TC TA 0-00 GAA Glu AlIa Lau Ala 0 u 2GO

CAG

Gin 265 1154 -120- ACC GTC TTC CTT TAGTTCTTCT CTGTCCATGT GGGATACATG GTATTATGTG Thr Val Phe Leu GCT1CATGAGG TACAATCTTT CT-TTCAGCAC CGTGCTAGCT GATCTTTCGG ACAACTTGAC ACAAGATAGA GTTAACTGGG AAGAGAAAGC CTGAATGAG GATTTCTTTC CATCAGGAAG CTACGGGCA). GTTTGCTGGG CCTTTGATTG CTTGATGACT GAAGTGGAAA GGCTGAGCCC .ACTGTGGGTG GTGCTAGCCC TGGGCAGGGG CAGGTGACCC TGGGTGGTAT AAGAAAAAGA GCTGTCACTA AAAGGAGAGG TGCCTAGTCT TACTGCAACT TGATATGTCA TGTTTGGTTG GTGTCTGTGG GAGGCCTGCC CTTTTCTGAA GAGA.AGTGGT GGGAGAGTGG ATGGGGTGGG GGCAGAGGAA AAGTGGGGGA GAGGGCCTGG GAGGAGAGGA GGGAGGGGGA CGGGGTGGGG GTGGGGAAAA CTATGG-TTGG GATGTAAAAA CGGATAATAA TATAAATATT AAATAAAAAU AGAGTATTGA GCAAAAAAAA AAAAAA INFORMATION FOR SEQ ID NO:3l: 2 SEQUENCE CH.ARACTERISTICS: LENGTH: 306 amino acids TYPE: amino acid 3 0 TOPOLOGY: linear (ii) MOLEC1JhE TYPE: protein DESCRIPTION: B lym hocyte activation antigen; Ig superfamily member; T cell costimulatory signal via activation of CD28 pathways, binds to CD28 T cells, transmembrane protein (ix) FEATUJRE: NAME/KEY: signal sequence LOCATION: -37 to -1 IDENTIFICAT:_ON METHOD: similarity with known s equenc e OTHER INFORMAL'TION: hydrophobic 1206 1266 1326 1386 1446 1506 1566 1626 1,686 1716 NAY.E/KEY: excracellular domain LOCATION: 1 tz: 210 IDENT:?:7CATICN METHOD: si.-ilari.nv wit-h known s eqauenc e -121- (ix) FEATURE: NAME/.KY: transmembrane domain LOCATION: 211 to 235 IDENTIFICATION METHOD: similarity with known sequence (ix) FEATURE: intracellular (cytoplasmic) domain LOCATION: 236 to 269 IDENTIFICATION METHOD: similarity with known sequence (ix) FEATURE: NAME/KEy: Ig V-set domain LOCATION: 1 to 105 IDENTIFICATION METHOD: similarity with known sequence (ix) FEATURE: NAME/KEY: IgC-set domain LOCATION: 106 to 199 IDENTIFICATION METHOD: similarity with known sequence PUBLICATION INFORMATION: AUTHORS: FREEMAN, GORDON J.

GRAY, GARY S.

GIMMI, CLAUDE D.

LOMBARD, DAVID B.

ZHOU, LIANG-JI WHITE, MICHAEL FINGEROTH, JOYCE D.

GRIBBEN, JOH G.

NADLER, LEE M.

TITLE: Structure, Expression, and T Cell Costimulatory Activity Of The Murine Homologue Of The Human B Lymphocyte Activazion Anciaen B7 JOUThNAL: Journal of Eer imenal Medicine

VOLUME:

ISSUE:

PAGES:

DATE: IN PRESS RELEVANT RE.SIDUES IN SEQUENCE ID NO:31: From -37 to 269 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: -122- Met Ala Cys Asn Cys Gin Leu Met Gin Asp Thr Pro Leu Leu Lys Phe -30 Pro Cys Pro Arg Leu I-le.Leu Leu Phe Val Leu Leu Ile Arg Leu Ser -15 Gin Val Ser Ser Asp Val Asp Glu Gin Leu Ser Lys Ser Vai Lys Asp -5 -l 1 5 Lys Val Leu Leu Pro Cys Arg Tyr Asn Ser Pro His Giu Asp Giu Ser 20 Giu Asp Arg Ile Tyr Trp Gin Lys His Asp Lys Val Val Leu Ser Val 9 35 Ile Ala GJy Lys Lei Lys Val Trp Pro GJu Tyr Lys Asn Arg Thr Lru 50 Tyr Asp Asn Thr Thr Tyr Ser Leu Ile Ile Leu Gly Leu Vai Leu Ser 65 70 Asp Arg Gly Thr Tyr Ser Cys Vai Val Gin Lys Lys Giu Arg Gly Thr 80 as Tyr Gly Vai Lys His Leu Ala Leu Val Lys Leu Ser Ile Lys Ala Asp 95 100 105 S 30 Phe Ser Thr Pro Asn Ile Thr Glu Ser Gly Asn Pro Ser Ala Asp TAr 110 115 120 Lys Arg Ile Thr Cys Phe Ala Ser Gly Gly Phe Pro Lys Pro Arg Phe 125 130 135 Ser Tro Leu Glu Asn Gly Arg Giu Leu Pro Gly Ile Asn Thr Thr Ile 140 145 ISO .155 Ser Gin Asp Pro Giu Ser Giu Leu Tyr Thr Ile Ser Ser Gin Leu Asp 160 165 170 Phe Asn Thr Thr Arg Asn His Thr Ile Lys Cys Leu Ile Lys Tyr Gly 175 180 185 Aso Ala His Val Ser Glu Asp Phe Thr Trp Glu Lys Pro Pro Glu Asp 190 195 200 Pro Pro Aso Ser Lvs As. Thr Leu Val Leu Phe Glv Ala G1v Phe Gly 205 210 215 Ala Val lie Thr Val Val Val Tle Val Val Ile lie Lvs Cys Phe Cys 220 225 230 235 Lys is Ara Ser Cys Phe Arg Ara Asn Glu Ala Ser Arg Glu Thr Asr 240 245 253 -123- Asn Ser Leu Thir Phe Gly Pro Glu Glu Ala Leu Ala Glu Gin Thr Val 255 260 265 Leu

Claims (249)

1. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a peptide having al least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID N0:2), said peptide Shaving the ability to costirnulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4.

2. The isolated nucleic acid molecule of claim 1 which is a cDNA sequence.

3. The isolated nucleic acid molecule of claim 2, wherein the cDNA is of human origin.

4. An isolated nucleic acid molecule of claim 3, wherein said nucleic acid molecule encodes a peptide having the ability to costimulate T cell proliferation or T cell cytoldkiune production or the ability to bind CD28 or CTLA4 and comprising a nucleotide sequence shown in Figure 8 (SEQ ID NO:I). An isolated nucleic acid molecule comprising the coding region of a nucleotide sequence shown in Figure 8 (SEQ ID NO:l) and encoding peptide having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4.

6. The isolated nucleic acid molecule of claim 2, wherein the cDNA is of muine origin. 25

7. An isolated nucleic acid molecule comprising a nucleotide sequence shown in Figure 14 (SEQ ID NO:22) and encoding a peptide having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4. *0 30 8. An isolated nucleic acid molecule of claim 6, wherein the cDNA comprises the coding region of a nucleotide sequence shown in Figure 14 (SEQ ID NO:22) and encoding a peptide having the ability to costimulate T cell proliferation or T cell cytoldine production or the ability to bind CD28 or CTLA4. oe@o 0e*o

9. An isolated nucleic acid molecule encoding a peptide comprising an amino acid sequence shown in Figure 8 (SEQ ID N0:2) said peptide having the ability to 0 000$ 0 *O *0 COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:06 FAX 61 3 9663 3099 FB RICE CO. 021 124 costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4. An isolated nucleic acid molecule encoding a peptide comprising an amino acid s sequence shown in Figure 14 (SEQ ID NO:23) said peptide having the ability to costimulate T cell proliferation or T cell cytoldne production or the ability to bind CD28 or CTLA4.

11. The isolated nucleic acid molecule of claim 1, wherein the peptide is at least 70% homologous with a sequence comprising an amino acid sequence of Figure 8 (SEQ ID NO:21).

12. An isolated nucleic acid molecule encoding a B7-2 peptide, wherein the peptide is encoded by a nucleic acid molecule which hybridises under high or low stringency conditions to a nucleic acid molecule which encodes a peptide comprising an amino acid sequence shown in Figure 8 (SEQ ID NO:2), said peptide having the ability to costimulate T cell proliferation or T cell cytoldine production or the ability to bind CD28 or CTLA4.

13. The isolated nucleic acid molecule of claim 1, wherein the peptide is at least amino acid residues in length.

14. The isolated nucleic acid molecule of claim 1, wherein the peptide is at least homologous with a sequence comprising an amino acid sequence of Figure 14 25 (SEQ ID NO:23). C15. An isolated nucleic acid molecule which hybridises under high or low Sstringency conditions to a nucleic acid molecule which encodes a peptide comprising an amino acid sequence shown in Figure 14 (SEQ ID NO:23), said peptide having the 30 ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4.

16. The isolated nucleic acid molecule of claim 15 wherein the peptide is at least ""amino acid residues in length. C C. o• *oooo COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:06 FAX 61 3 9663 3099 FB RICE CO. 0022 125

17. The isolated nucleic acid molecule of claim 1, wherein the peptide comprises amino acid residues 24-245 of the sequence shown in Figure 8 (SEQ ID NO:2).

18. An isolated DNA molecule comprising a nucleotide sequence encoding a peptide having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino adcid sequence shown in Figure 8 (SEQ ID N0:2), said peptide having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 .or CTLA4, the peptide having an amino acid sequence represented by formula wherein Y comprises amino acid residues 24-245 of the sequence shown in Figure 8 (SEQ ID NO:2), wherein Xa is amino adcid residues selected from amino acid residues contiguous to the amino terminus of Y in the sequence shown in Figure 8 (SEQ ID NO:2), wherein Z. is amino acid residues selected from amino acid residues contiguous to the carboxy terminus of Y in the sequence shown in Figure 8 (SEQ ID NO:2). wherein n=--0-23 and wherein 0-84.

19. The isolated DNA molecule of claim 18, wherein n=0 and m-0. An isolated DNA molecule comprising a nucleotide sequence encoding a peptide of at least 20 amino acid residues or more in length having at least about homology with a human B7-2 peptide comprising an amino adcid sequence shown in Figure 8 (SEQ ID NO:2), said peptide having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4.

21. An isolated nucleic acid molecule encoding a B7-2 fusion protein comprising a 25 nucleotide sequence encoding a first peptide having at least 50% amino acid sequence identity with a human B7-2 peptide comiprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2), said peptide having the ability to costitnulate T cell proliferation or T cell cytoldine production or the ability to bind CD28 or CTLA4 and a nucleotide sequence encoding a second peptide. e S22. The isolated nucleic acid molecule of claim 21 which is a DNA.

23. The isolated nucleic acid molecule of claim 22, wherein the first peptide ocomprises an extracellular domain of a human B7-2 protein. 4 00 04 04 44 4 *4 @4 4. COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:06 FAX 61 3 9663 3099 FB RICE CO. 2023 126

24. The isolated nucleic acid molecule of claim 23, wherein the first peptide comprises amino acid residues 24-245 of the sequence shown in Figure 8 (SEQ ID NO:2).

25. The isolated nucleic acid molecule of claim 23, wherein the first peptide comprises a variable region-like domain of a human B7-2 protein.

26. The isolated nucleic acid molecule of claim 23, wherein the first peptide comprises a constant region-like domain of a human B7-2 protein.

27. The isolated nucleic acid molecule of claim 22 wherein the second peptide comprises an immunoglobulin constant region.

28. The isolated nucleic acid molecule of claim 27, wherein the immunoglobulin constant region is a Cyl domain, including the hinge, CH2 and CH3 region.

29. The isolated nucleic acid molecule of claim 27, wherein the immunoglobulin constant region is modified to reduce constant region-mediated biological effector functions. The isolated nucleic acid molecule of claim 29, wherein the biological effecter function is selected from the group consisting of complement activation and Fc receptor interaction. S". 25 31. The isolated nucleic acid molecule of claim 30, wherein the immunoglobulin constant region is a Cy4 domain, including the hinge, CH2 and CH3 region. oo S S. '32. The isolated nucleic acid molecule of claim 31, wherein at least one amino acid *residue of the CH2 domain is modified by substitution, addition or deletion.

33. An isolated polypeptide comprising an amino acid sequence having at least amino acid sequence identity with the extracellular domain of a human B7-2 peptide ~shown in Figure 8 (SEQ ID NO:2) and the ability to bind CD28 or CTLA4, S S :0:0 35 34. The polypeptide of claim 33 wherein the polypeptide comprises the extracellular '.600domain of the human B7-2 protein shown in Figure 8 (SEQ ID NO:2). 55* S S COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:06 FAX 61 3 9663 3099 FB RICE CO. 11024 127 The polypeptide of claim 34, wherein the polypeptide comprises amino acid residues 24-245 of the sequence shown in Figure 8 (SEQ ID NO:2).

36. The polypeptide of claim 34, wherein the polypeptide comprises a variable region-like domain of the human B7-2 protein shown in Figure 8 (SEQ ID NO:2).

37. The. polypeptide of claim 33, wherein the polypeptide is fused to a second peptide,

38. The polypeptide of claim 37, wherein the second polypeptide is an immunoglobulin constant region comprising a Cyl domain including the hinge, CH2 and CH3 region.

39. The polypeptide of claim 37, wherein the immunoglobulin constant region is modified to reduce constant region-mediated biological effector functions. The polypeptide of claim 39, wherein the biological effector function is selected from the group consisting of complement activation and Fc receptor interaction.

41. The polypeptide of claim 40, wherein the immunoglobulin constant region is a Cy4 domain including the hinge, CH2 and CH3 region.

42. The polypeptide of claim 41, wherein at least one amino acid residue of the CH2 domain is modified by substitution, addition or deletion.

43. A composition suitable for pharmaceutical administration comprising a S* polypeptide of claim 33 or 34 and a pharmaceutically acceptable carrier.

44. A composition suitable for pharmaceutical administration comprising a polypeptide of claim 36 or 37 and a pharmaceutically acceptable carrier.

45. A recombinant expression vector including a nucleic acid molecule of claim 1. S: 46, The recombinant expression vector of claim 45, wherein the nucleic acid 35 molecule is a cDNA molecule. COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:07 FAX 61 3 9663 3099 FB RICE CO. [i025 128

47. The recombinant expression vector of claim 46, wherein the cDNA is of human origin and comprises a nucleotide sequence shown in Figure 8 (SEQ ID NO:1).

48. The recombinant expression vector of claim 46 which is a plasmid.

49. A recombinant expression vector including a nucleic acid molecule of claim 7. A composition suitable for pharmaceutical administration comprising a recombinant expression vector of claim 45 or 49 and a pharmnnaceutically acceptable carrier.

51. A host cell transfected with the expression vector of claim 45 capable of directing the expression of a peptide having an activity of a B-lymphocyte antigen B7- 2.

52. A host cell transfected with the expression vector of claim 47 capable of directing the expression of a peptide having an activity of a B lymphocyte antigen, B7- 2.

53. A host cell transfected ,with the expression vector of claim 49 capable of directing the expression of a peptide having an activity of a B lymphocyte antigen. B7- 2.

54. A composition suitable for pharmaceutical administration comprising a host cell 25 of claim 51 or 53 and a pharmaceutically acceptable carrier. p. p S* 55. An isolated, recombinant peptide having an activity of a B lymphocyte antigen, V B7-2. expressed by a host cell of claim 52.

56. A cell transfected with a nucleic acid molecule encoding a peptide having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2), said peptide having the ability "to costimulate T cell proliferation or T cell cytokine production or the ability to bind S. CD28 or CTLA4, said nucleic acid molecule being in a form suitable for expression of *35 the peptide on the cell surface. p. *p a a COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:07 FAX 61 3 9663 3099 FB RICE CO. [i026 129

57. The cell of claim 56, wherein the nucleic acid molecule is a eDNA comprising a nucleotide sequence shown in Figure 8 (SEQ ID NO:1) in a recombinant expression vector.

58. A tumor cell which is modified to express a T cell costimulatory molecule having at least 50% amino acid sequence identity with a human B7-2 peptide comprising, the amino acid sequence shown in Figure 8 (SEQ ID NO:2), said peptide having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4.

59. The tumor cell of claim 58 which is transfected with a nucleic acid molecule encoding human B7-2 in a form suitable for expression of B7-2. The tumor cell of claim 58 which is stimulated to express B7-2.

61. The tumor cell of claim 58 which has a human B7-2 antigen coupled to the tumor cell.

62. The tumor cell of claim 58 which expresses a T cell costimulatory molecule, B7-1.

63. The tumor cell of claim 58 which expresses an MHC class I molecule.

64. The tumor cell of claim 58 which expresses an MHC class H molecule. S :65. The tumor cell of claim 58 which normally expresses an MHC class H associated protein, the invariant chain, and wherein expression of the invariant chain is V. inhibited.

66. A tumor cell transfected with a nucleic acid molecule encoding a T cell costimulatory molecule having at least 70% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to costimulate T cell proliferation-or T cell cytokine production or the ability to bind CD28 or CTLA4, said nucleic acid molecule being in a 35 form suitable for expression of B7-2. oo o* o S *5 COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:07 FAX 61 3 9663 3099 FB RICE CO. 1027 130

67. The tumor cell of claim 66, wherein the nucleic acid molecule is a eDNA in a recombinant expression vector.

68. The tumor cell of claim 66, frnther transfected with a nucleic acid molecule encoding a T cell costimulatory molecule, B7-1, said nucleic acid molecule being in a form suitable for expression of B 7-1,

69. The tumor cell of claim 66, further transfected with at least one nucleic acid molecule comprising DNA encoding: at least one MHC class II a chain protein; and at least one MHC class II 3 chain protein, wherein the nucleic acid molecule is in a form suitable for expression of the MHC class II a chain protein(s) and the MHC class II P chain protein(s). 16 70, The tumor cell of claim 69 which does not express MHC class II molecules prior to transfection of the tumor cell. 71, The tumor cell of claim 66, further transfected with at least one nucleic acid molecule encoding at least one MHC class I a chain protein in a form suitable for expression of the MHC class I a chain protein(s).

72. The tumor cell of claim 71, further transfected with a nucleic acid molecule encoding a p-2 microglobulin protein in a form suitable for expression of the -2 *microglobulin protein. 2

73. The tumor cell of claim 66 which normally expresses an MHC class II associated protein, the invariant chain, and wherein expression of the invariant chain is "inhibited.

74. The tumor cell of claim 73, wherein expression of the invariant chain is inhibited by transfection of the tumor cell with a nucleic adcid molecule which is antisense to a regulatory or a coding region of the invariant chain gene.

75. The tumor cell of claim 66 which is a sarcoma. 7 Ter

76. The tumor cell of claim 66 which is a lymphoma. COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:07 FAX 61 3 9663 3099 FB RICE CO. Z028 131

77. The tumor cell of clam 66 which is selected from a group consisting of a melanoma, a neuroblastoma, a leukemia and a carcinoma.

78. A method of treating a subject with a tumor, the method including: obtaining tumor cells from the subject; transfecting the tumor cells with a nucleic acid molecule encoding a molecule having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2), said peptide having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4 and said nucleic acid molecule being in a form suitable for expression of B7-2; and administering the tumor cells to the subject.

79. The method of claim 78, wherein the tumor cells are further transfected with a nucleic acid molecule encoding B7-1. The method of claim 78, wherein the tumor cells are further transfected with at least one nucleic acid molecule encoding at least one MUC class II a chain protein and at least one MHC class I 13 chain protein in a form suitable for expression of the MHC class II a chain protein(s) and the MHC class II 3 chain protein(s). S.

81. The method of claim 78, wherein the tumor cells are further transfected with at ~least one nucleic acid molecule encoding at least one MHC class II a chain protein in a 25 form suitable for expression of the MHC class I a protein(s).

82. The method of claim 81, wherein the tumor cells are further transfected with a nucleic acid molecule encoding a 13-2 microglobulin protein in a form suitable for expression of the P-2 microglobulin protein.

83. The method of claim 78, wherein expression of an MHC class II associated protein, the invariant chain, is inhibited in the tumor cells.

84. The method of claim 83, wherein expression of the invariant chain is inhibited in the tumor cells by transfection of the tumor cell with a nucleic acid molecule which is antisense to a regulatory or a coding region of the invariant chain gene. S COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:08 FAX 61 3 9663 3099 FB RICE CO. [a 02 132 The m-thod of claim 78, wherein the tumor is a sarcoma.

86. The method of claim 78, wherein the tumor is a lymphoma,

87. The method of claim 78, wherein the tumor is selected from a group consisting of a melanoma, a neuroblastoma, a leukemia and a carcinoma.

88. A method of inducing an antitumor response by CD4+ T lymphocytes in a subject with a tumor, the method including: obtaining tumor cells from the subject; transfecting the tumor cells with at least one nucleic acid molecule comprising DNA encoding: a molecule having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2), said peptide having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4, (ii) an MHC class II a chain protein, and (iii) an MIHC class II P chain protein, wherein the nucleic acid molecule is in a form suitable for expression of B7-2, the MHC class H a chain protein and the MHC class U 13 chain protein; and administering the tumor cells to the subject.

89. A method of treating a subject with a tumor, the method including modifying tumor cells in vivo via introduction of a nucleic acid molecule comprising a nucleotide o sequence encoding a peptide having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2), said peptide having the ability to costimulate T cell proliferation or T cell cytoldne production or the ability to bind CD28 or CTLA4. *e.

90. The method of claim 89, wherein tumor cells are modified in vivo by delivering to the subject in vivo a nucleic acid molecule encoding B7-2 in a form suitable for expression of B7-2.

91. The method of claim 90, wherein the nucleic acid molecule is delivered to the subject in viva by injection of the nucleic acid molecule in an appropriate vehicle into the tumor. COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:08 FAX 61 3 9663 3099 FB RICE CO. [it03 133

92. A method of treating a subject with a tumor, the method including: obtaining tumor cells and T lymphocytes from the subject; culturing the T lymphocytes from the subject in vitro with the tumor cells from the subject and with a molecule having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2), said peptide having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4; and administering the T lymphocytes to the subject.

93. A peptide produced by recombinant expression of a nucleic acid molecule of claim 1.

94. A peptide produced by recombinant expression of a nucleic acid molecule of claim 4. is A peptide produced by recombinant expression of a nucleic acid molecule of claim

96. A peptide of claia 95 comprising an amino acid sequence set forth in Figure 8 (SEQ ID NO: 2).

97. A peptide produced by recombinant expression of a DNA of claim 18. :98. A peptide produced by recombinant expression of a DNA of claim

99. A substantially pure preparation of a peptide having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence •shown in Figure 8 (SEQ ID NO:2), said peptide having the ability to costimulate T cell "'"proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4.

100. An isolated, recombinant or purified peptide having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2), said peptide having the ability to costimulate T cell proliferation or T cell cytoline production or the ability to bind CD28 or CTLA4 and having an amino acid sequence represented by a formula Xn-Y-Z, wherein Y is amino acid residues selected from the group consisting of. amino acid residues 55-68 of the 9o1". 99 -9 o COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:09 FAX 61 3 9663 3099 FB RICE CO. [a 031 134 sequence shown in Figure 8 (SEQ ID NO:2); amino acid residues 81-89 of the sequence shown in Figure 8 (SEQ ID NO:2); amino acid residues 128-142 of the sequence shown in Figure 8 (SEQ ID NO:2); amino acid residues 160-169 of the sequence shown in Figure 8 (SEQ ID NO:2); amino acid residues 188-200 of the sequence shown in Figure 8 (SEQ ID NO:2); and amino acid residues 269-282 of the sequence shown in Figure 8 (SEQ ID NO:2), wherein X is amino acid residues selected from amino acid residues contiguous to the amino terminus of Y in the sequence shown in Figure 8 (SEQ ID NO:2), wherein is amino acid residues selected from amino acid residues contiguous to the carboxy terminus of Y in the sequence shown in Figure 8 (SEQ ID NO:2), wherein n--=0-30 and wherein m=--0-30.

101. The isolated, recombinant or purified peptide of claim 100, wherein n=0 and m=0.

102. A composition comprising an antibody that recognises a human B7-2 polypeptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) and an antibody that recognises a B7-1 polypeptide as shown in (SEQ ID NO:29).

103. The composition of claim 102, further comprising a pharmaceutical carrier.

104. The composition of claim 102, wherein the antibody that recognises B7-2 and the antibody that recognises B7-1 are monoclonal antibodies.

105. The composition of claim 102, wherein the antibody that recognises B7-2 and the antibody that recognises B7-1 are humanised antibodies. S106. The composition of claim 102 wherein at least one of the antibody that recognises B7-2 and the antibody that recognises B7-1 is an IgGI or an IgG2a antibody.

107. A nonhuman, transgenic animal which contains cells transfected to express a peptide having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) said peptide "o having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4. COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:09 FAX 61 3 9663 3099 FB RICE CO. Q032 135

108. The nonhuman, transgenic animal of claim 107 which is a mouse.

109. A nonhuman, knockout animal which contains cells having an altered gene encoding a B lymphocyte antigen, B7-2.

110. The nonhuman, knockout animal of claim 109 which is a mouse. I 11. A composition suitable for pharmaceutical administration comprising a peptide having at least 50% amino acid sequence identify with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2), said poptide having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4, and a pharmaceutically acceptable carrier.

112. The composition of claim 111 further comprising a B7-1 peptide.

113. The composition of claim 11, wherein the peptide comprises an amino acid sequence set forth in Figure 8 (SEQ ID NO: 2).

114. The composition of claim 113, wherein the peptide comprises amino acid residues 24-245 of the sequence shown in Figure 8 (SEQ ID NO:2).

115. A method of producing a peptide having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2), said peptide having the ability to costimulate T cell 25 proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4, the :o method including: culturing a host cell of claim 52 in a medium to express the peptide; and 9 isolating the peptide from the medium. a0 116. A method of producing a peptide having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SIEQ ID NO:2), said peptide having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4, the method including: culturing a host cell of claim 53 in a medium to express the peptide; and isolating the peptide from the medium. 9. 9o 99 9 COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:09 FAX 61 3 9663 3089 FB RICE CO. 1]033 136

117. A method of inhibiting an interaction of a B lymphocyte antigen, B7-2, with its natural ligand(s) on the surface of immune cells, the method including, contacting an immune cell with an agent selected from the group consisting of: a peptide having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to bind CD28 or CTLA4 but being incapable of costimulating T cell proliferation or T cell cytokihe production, and an antibody against B7-2 which inhibits B7-2 binding with its natural ligand(s), to thereby inhibit costimulation of the immune cell through B7-2-ligand interaction,

118. The method of claim 117, wherein the agent is a peptide having at least amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to bind CD28 or CTLA4 but being incapable of costimulating T cell proliferation or T cell cytolkine production.

119. The method of claim 118, wherein the peptide is a soluble, monomeric peptide.

120. The method of claim 119, wherein the peptide comprises amino acid residues 24-245 of the sequence shown in Figure 8 (SEQ ID NO:2).

121. The method of claim 119, wherein the agent is a B7-2 fusion protein comprising a first peptide which binds to CTLA4 or CD28 without delivering a costimulatory signal to a T cell and a second peptide comprising a moiety that alters the solubility, 25 binding affinity or valency of the first peptide. 0* 0*

122. The method of claim 121, wherein the first peptide comprises an extracellular *domain of the human B7-2 protein,

123. The method of claim 121, wherein the first peptide comprises amino acid residues 24-245 of the sequence shown in Figure 8 (SEQ ID NO:2).

124. The method of claim 121, wherein the second pepitide comprises an 00. immunoglobulin constant region. Io0* to.. 00 0 *0 0 0 0 *00 COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:09 FAX 61 3 9663 3099 FB RICE CO. [034 137

125. The method of claim 124, wherein the immunoglobulin constant region is a Cyl domain, including the hinge, CH2 and CH3 region.

126. The method of claim 118, wherein the agent is an antibody reactive with B7-2.

127. The method of claim 126, wherein the antibody is a monoclonal antibody.

128. A method ofdownregulating T cell mediated immune responses in a subject, the method including administering to the subject an agent selected from the group consisting of: a peptide having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to bind CD28 or CTLA4 but being incapable of costimulating T cell proliferation or T cell cytokine production and an antibody against B7-2 which inhibits B7-2 binding with its natural ligand(s), in an amount effective to inhibit T cell proliferation and/or cytokine secretion in the subject.

129. The method of claim 128, wherein the agent is a peptide having at least amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to bind CD28 or CTLA4 but being incapable of costimnulating T cell proliferation or T cell cytokine production.

130. The method of claim 128, wherein the agent is an antibody reactive with human l B7-2.

131. The method of claim 130, wherein the antibody is a monoclonal antibody. V.

132. The method of claim 128, further comprising administering to the subject an agent that inhibits B7-1 mediated costimulation.

133. The method of claim 132, wherein the agent is analog of a B7-1 peptide lacking the ability to deliver a costimulatory signal to T cells. S

134. The method of claim 132, wherein the agent is an antibodyreactive with B7-1. 555e 35 T.

135. The method of claim 134, wherein the antibody is a monoelonal antibody. COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:10 FAX 61 3 9663 3099 FB RICE CO. [035 138

136. The method of claim 128, further including administering to the subject an immunomodulating reagent selected from the group consisting of an antibody reactive with CD28, an antibody reactive with CTLA4, an antibody reactive with a cytoldkine, a CTLA4Ig fusion protein, a CD28Ig fusion protein, and an immunosuppressive drug.

137. A method of treating an autoimmune disease in a subject mediated by interaction of a B Lymphocyte antigen, B7-2, with its natural ligand(s) on the surface of immune cells, the method including administering to the subject an agent selected from the group consisting of: a peptide having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to bind CD28 or CTLA4 but being incapable of costimulating T cell proliferation or T cell cytoldkine production and an antibody against B7-2 which inhibits B7-2 binding with its natural ligand(s), such that autoimmune disease is treated.

138. The method of claim 137, wherein the autoimnmune disease is selected from the group consisting of diabetes mellitus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, systemic lupus erythematosus, and autoimnmune thyroiditis.

139. The method of claim 137, wherein the agent is a peptide having at least amino acid sequence identity with a human B7-2 peptide comprising the amino acid S. sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to bind CD28 or CTLA4 but being incapable of costinulating T cell proliferation or T cell cytokine production.

140. The method of claim 139, wherein the peptide is a soluble, monomeric peptide. 0

141. The method of claim 140, wherein the peptide comprises amino acid residues 24-245 of the sequence shown in Figure 8 (SEQ ID NO:2).

142. The method of claim 137, wherein the agent is a B7-2 immunoglobulin fusion protein (B7-2Ig) comprising a first peptide comprising an extracellular domain of the 0. B7-2 protein and a second peptide comprising an immunoglobulin constant domain. 9* S 9* S. COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:10 FAX 61 3 9663 3099 FB RICE CO. 139

143. The method of claim 142, wherein the extracellular domain of the B7-2 protein comprises amino acid residues 24-245 of the sequence shown in Figure 8 (SEQ ID NO:2).

144. The method of claim 137, wherein the agent is an antibody reactive with B7-2.

145. The method of claim 144, wherein the antibody is a monoclonal antibody.

146. The method of claim 139 further including administering to the subject a peptide having at least 50% amino acid sequence identity with a human B7-2 peptidc comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to bind CD28 or CTLA4 but being incapable of costimulating T cell proliferation or T cell cytokine production.

147. The method of claim 137, further including administering to the subject an immunomodulating reagent selected from the group consisting of: an antibody reactive with B7-1, an antibody reactive with CD28, an antibody reactive with CTLA4, an antibody reactive with a cytolkine, a CTLA4Ig fusion protein, a CD28Ig fusion protein, and an immunosuppressive drug.

148. A method of treating allergy in a subject mediated by interaction of a B lymphocyte antigen, B7-2, with its natural ligand(s) on the surface of immune cells, the method including administering to the subject an agent selected from the group •'••.•consisting of a peptide having at least 50% amino acid sequence identity with a human 25 B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to bind CD28 or CTLA4 but being incapable of o costimulating T cell proliferation or T cell cytokine production and an antibody against B7-2 which inhibits B7-2 binding with its natural ligand(s), to thereby inhibit costimulation of the immune cells through the B7-2 -ligand interaction.

149. The method of claim 148, wherein the agent is a peptide having B7-2 binding activity, but lacking the ability to deliver a costimulatory signal to immune cells. o

150. A method of inhibiting donor T cell proliferation and/or cytokine secretion in a transplant recipient to thereby prevent graft-versus-host disease (GVHD) in the recipient, the method including contacting donor T cells to be transplanted with an *0 COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:10 FAX 61 3 9663 3099 FB RICE CO. (t037 140 agent selected from the group consisting of: a peptide having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to bind CD28 or CTLA4 but being incapable of costimulating T cell proliferation or T cell cytokine production and an antibody against B7-2 which inhibits B7-2 binding with its natural ligand(s), such that donor T cell proliferation and/or cytokine secretion is inhibited in a transplant recipient,

151. The method of claim 150, wherein the agent is a peptide having at least amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to bind CD28 or CTLA4 but being incapable of costimulating T cell proliferation or T cell cytokine production.

152. The method of claim 151, wherein the peptide comprises amino acid residues 24-245 of the sequence shown in Figure 8 (SEQ ID NO:2).

153. The method of claim 150, wherein the agent is an antibody reactive with B7-2

154. The method of claim 153, wherein the antibody is a monoclonal antibody.

155. A method of inhibiting transplantation rejection in a recipient of a tissue or organ transplant, the method including administering to the recipient an agent selected from the group consisting of a peptide having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to bind CD28 or CTLA4 but being incapable of costimulating T cell proliferation or T cell cytokine production and S"an antibody against B7-2.

156. The method of claim 155, wherein the agent is a peptide having at least .amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to bind .*CD28 or CTLA4 but being incapable of costimulating T cell proliferation or T cell cytokine production. COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:11 FAX 61 3 9663 3099 FB RICE CO. [in 0s 141

157. The method of claim 156, wherein the peptide comprises amino acid residues 24-245 of the sequence shown in Figure 8 (SEQ ID NO:2).

158. The method of claim 155, wherein the agent is an antibody reactive with B7-2.

159. The method of claim 158, wherein the antibody is a monoclonal antibody.

160. A method of upregulating T cell mediated immune responses in a subject, the method including administering to the subject a B7-2 peptide having at least amino acid sequence identity with a human B7-2 pcptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) and having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4 in, an amount effective to stimulate T cell proliferation and/or cytokine secretion in the subject.

161. The method of claim 160, further comprising administering to the subject a B7-1 peptide.

162. The method of claim 160, further comprising administering to the subject a pathogen or portion thereof to thereby induce an anti-pathogen immune response in the subject.

163. The method of claim 162, wherein the pathogen is a virus. 25 164. A method of identifying molecules which modulate expression of a B7-2 antigen, the method including: a) contacting a cell which expresses a peptide having at least 50% amino acid *o sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to biud CD28 or CTLA4 with a molecule to be tested, under conditions appropriate for interaction of the molecule with the cell; and b) determining the effect of the molecule on cell expression of the peptide.

165. The method of claim 164, wherein the effect of the molecule on cell expression of the peptide is detemined by detecting the presence of the peptide on the cell surface. 0 COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:11 FAX 61 3 9663 3099 FB RICE CO. @039 142

166. The method of claim 165, wherein the presence of the peptide on te cell surface is detected by immunofluorescence with an antibody reactive with the peptide or with a CTLA4Ig or CD28Ig fusion protein.

167. The method of claim 164, wherein the effect of the molecule on cell expression of the peptide having B7-2 activity is determined by detecting the presence of mRNA encoding the peptide in the cell.

168. The method of claim 167, wherein the presence of mRNA is detected by hybridisation with B7-2 cDNA.

169. A method of identifying a cytokine produced by an immune cell in response to costimulation with a B7-2 antigen, the method including: a) contacting an activated immune cell and a cell modified to include a nucleic acid molecule comprising DNA which encodes a peptide having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to costimnulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4, in an appropriate cell culture medium under conditions appropriate for expression of the peptide in the cell; and b) determining the presence of a cytokine in the cell culture medium.

170. The method of claim 169, wherein the immune cell is a T cell. 9 9 .9* 25 171. The method of claim 169, wherein the presence of a cytolkine in the cell culture i medium is determined by contacting the medium with an antibody reactive with the 0: cytokine. *9*

172. A method of identifying molecules which inhibit costimulation of immune cells by a B7-2 antigen, the method including: contacting an immune cell which has received a primary induction signal with a protein having at least 50% amino acid sequence identity with a human B7-2 pptide comprising the amino acid sequence shown in Figure 8 (SEQ ID N0:2) said protein -having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4 and a molecule to be tested, under conditions appropriate for interaction of the molecule with the immune cell and the protein; and 99* COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:11 FAX 61 3 9663 3099 FB RICE CO. 0040 143 b) determining the effect of the molecule on costimulation of the immune cell by the protein.

173. The method of claim 172, wherein the immune cell is a T cell.

174. The method of claim 172, wherein the effect of the molecule on costimulation of the T cell is determined by detecting T cell proliferation and/or cytokine production.

175. The method of claim 172, wherein the protein is expressed on the surface of a cell.

176. A method of identifying molecules which inhibit binding of a B7-2 antigen to a ligand on the surface of immune cells, the method including: contacting a labelled B7-2 ligand and a molecule to be tested with a peptide having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to costimulate T cell proliferation or T cell cytoldkine production or the ability to bind CD28 or CTLA4; removing unbound labelled B7-2 ligand; and c) determining the amount of labelled B7-2 ligand bound to the peptide having B7- 2 activity, as an indication of the ability of the molecule to inhibit binding of the B7-2 ligand to a B7-2 antigen.

177. The method of claim 176, wherein the immune cell is a T cell and the B7-2 ligand is CTLA4 or CD28,

178. The method of claim 176, wherein the peptide is immobilised on a solid phase support.

179. A method of identifying molecules which inhibit intracellular signalling by an immune cell in response to a recombinant protein having at least 50% amino acid 0sequence identity with a human B7-2 peptide comprising the amino acid sequence S• shown in Figure 8 (SEQ ID NO:2), said peptide having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4, the method including: S *5 S S COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:12 FAX 61 3 9663 3099 FB RICE CO. 0J041 144 a) contacting an imrmune cell which has received a primary activation signal and which expresses a B7-2 ligand on the cell surface with the protein and a molecule to be tested, under conditions appropriate for interaction of the molecule with the immune cell and the protein; and b) determining the effect of the molecule on intracellular signalling by the immune cell in response to the protein.

180. The method of claim 179, wherein the immune cell is a T cell.

181. The method of claim 180, wherein the effect of the molecule on intracellular signalling by the immune cell is determined by detecting T cell proliferation and/or cytokine production.

182. The method of claim 179, wherein the protein is expressed on the surface of a cell.

183. Use of a peptide having at least 50% amino acid sequence identity with a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO:2) said peptide having the ability to costimulate T cell proliferation or T cell cytokine production or the ability to bind CD28 or CTLA4 in the manufacture of a medicament for the treatment of disease in a subject.

184. The use of claim 183, wherein the peptide comprises an amino acid sequence set forth in Figure 8 (SEQ ID NO: 2). e*eo

185. The use of claim 183, wherein the peptide comprises amino acid residues 24- 245 of the sequence shown in Figure 8 (SEQ ID NO:2).

186. The isolated nucleic acid of claim 1, wherein the peptide has at least identity to a sequence comprising an amino acid sequence of Figure 8 (SEQ ID NO:2). 9* .187. The isolated nucleic acid of claim 1, wherein the peptide has at least S* identity to a sequence comprising an amino acid sequence of Figure 8 (SEQ ID NO:2). COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:12 FAX 61 3 9663 3099 FB RICE CO. [042 145

188. The isolated nucleic acid of claim 1, wherein the peptide has at least identity to a sequence comprising an amino acid sequence of Figure 14 (SEQ ID N0:23),

189. The isolated nucleic acid of claim 1, wherein the peptide has at least identity to a sequence comprising an amino acid sequence of Figure 14 (SEQ ID N0:23).

190. The isolated nucleic acid molecule of claim 21, wherein said nucleic acid molecule comprises a nucleotide sequence encoding a first peptide having at least about amino acid sequence identity with the extracellular domain of a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO: 2).

191. The isolated nucleic acid molecule of claim 21, wherein said nucleic acid molecule comprises a nuclcotide sequence encoding a first peptide having at least about amino acid sequence identity with the extracellular domain of a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ED NO: 2).

192. The polypeptide of claim 33, wherein the polypeptide has at least about 20 amino acid sequence identity with the extracellular domain of a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO: 2). *oo•

193. The polypeptide of claim 33, wherein the polypeptide has at least about *0 amino acid sequence identity with the extracellular domain of a human B7-2 peptide comprising the amino acid sequence shown in Figure 8 (SEQ ID NO: 2).

194. The isolated polypeptide of claim 33, wherein the polypeptide has an amino acid sequence which differs from the amino acid sequence of the extracellular domain shown in Figure 8 (SEQ ID NO:2) due to substitution, addition, or deletion of at least 30 one amino acid residue. 00 0e* 00 COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:12 FAX 61 3 9663 3099 FB RICE CO. [a043 146

195. An isolated polypeptide consisting of a B7-2 extracellular domain as shown in Figure 8 (SEQ ID NO:2).

196. An isolated polypeptide consisting of amino acid residues 24-243 shown in Figure 8 (SEQ ID NO:2).

197. An isolated nucleic acid molecule consisting of a nucleotide sequence encoding the extracellular domain of the B7-2 molecule shown in Figure 8 (SEQ ID NO:2).

198. An isolated nucleic acid molecule consisting of a nucleotide sequence encoding amino acids 24-243 of Figure 8 (SEQ ID NO:2).

199. A method of modulating an immune response, comprising administering to a subject a molecule selected from the group consisting of: the polypeptide shown in Figure 8 (SEQ ID NO:2); a polypeptide comprising a variable region domain shown in Figure 8 (SEQ ID NO:2); a polypeptide consisting of a variable region domain shown in Figure 8 (SEQ ID NO:2); a polypeptide comprising the extracellular domain shown in Figure 8 (SEQ ID NO:2); a polypeptide consisting of the extracellular domain shown in Figure 8 (SEQ ID NO:2); an antibody reactive with the B7-2 polypeptide shown in 20 SEQ ID NO:2; a nucleic acid molecule encoding a polypeptide comprising the extracellular domain shown in Figure 8 (SEQ ID NO:2); a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide shown in Figure 8 (SEQ ID NO:2) and a nucleic acid molecule comprising the coding region of the nucleotide sequence shown SEQ ID NO:1, such that the immune response is modulated.

200. The composition of claim 111, wherein the composition modulates T cell proliferation.

201. The composition of claim 111, wherein the composition downregulates an 30 immune response. S. COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:12 FAX 61 3 9663 3099 FB RICE CO. U044 147

202. The composition of claim 111, wherein the composition upregulates an immune response.

203. The composition of claim 111, wherein the composition modulates at least one S of: autoimmune disease, allergy, and transplant rejection.

204. The composition of claim 111, wherein the composition modulates infection with a pathogen.

205. The composition of claim 111 wherein the pathogen is a virus.

206. The composition of claim 111, wherein the virus is HIV.

207. The method of claim 117, wherein the molecule modulates T cell proliferation.

208. The method of claim 117, wherein the molecule downregulates an immune response.

209. The method of claim 117, wherein the molecule upregulates an immune :20 response. @0oo

210. The method of claim 117, wherein the molecule modulates at least one of: :0 autoimmune disease, allergy, and transplant rejection.

211. The method of claim 117, wherein the molecule modulates infection with a pathogen.

212. The method of claim 117, wherein the pathogen is a virs.

213. Themethod of claim 117, wherein the virus is HIV. 0o00 00 000 COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:13 FAX 61 3 9663 3099 FB RICE CO. [045 148

214. The method of claim 199, wherein the molecule modulates T cell proliferation.

215. The method of claim 199, wherein the molecule downregulates an immune response.

216. The method of claim 199, wherein the molecule upregulates an immune response.

217. The method of claim 199, wherein the molecule modulates at least one of: autoimmune disease, allergy, and transplant rejection.

218. The method of claim 199, wherein the molecule modulates infection with a pathogen. 16 219. The method of claim 199, wherein the pathogen is avirus.

220. The method of claim 199, wherein the virus is HIV.

221. An isolated variable region form of the B cell activation antigen, B7-2, which *20 comprises a B7-2 finmunoglobulin-like variable region domain but does not comprise a B7-2 immunoglobulin-like constant region domain.

222. The B7-2 variable region form of claim 221, which is human. *e

223. The B7-2 variable region form of claim 221, which is a fusion protein comprising a B7-2 variable region polypeptide operatively linked to a heterologous ••polypeptide.

224. The B7-2 variable region formn of claim 223, wherein the fusion protein is a 30 soluble, monomeric polypeptide. o a. a a. COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:13 FAX 61 3 9663 3099 FB RICE CO. Q046 149

225. The B7-2 variable region form of claim 223, wherein the B7-2 variable region polypeptide is a human B7-2 variable region polypeptide.

226. The B7-2 variable region form of claim 225, wherein the human B7-2 variable region polypeptide comprises an amino acid sequence of about positions 24 to 133 of SEQ ID NO: 2.

227. The B7-2 variable region form of claim 223, wherein the heterologous polypeptide is an immunoglobulin constant region.

228. The B7-2 variable region form of claim 227,'wherein the immunoglobulin constant region comprises a Cyl domain, including the hinge, CH2 and CH3 region.

229. The B7-2 variable region form of claim 223, wherein the immunoglobulin constant region is modified to reduce constant region-mediated biological effector functions.

230. The B7-2 variable region form of claim 229, wherein the biological effector function is selected from the group consisting of complement activation and Fc receptor interaction.

231. The B7-2 variable region form of claim 230, wherein the immunoglobulin constant region is a Cy4 domain, including the hinge, CH2 and CH3 region.

232. The B7-2 variable region form of claim 231, wherein at least one amino acid residue of the CH2 domain is modified by substitution, addition or deletion.

233. An isolated B7-2 fusion protein comprising a human B7-2 immunoglobulin-like 9. variable region domain operatively linked to a heterologous polypeptide, wherein the B7-2 fusion protein does not comprise a B7-2 immunoglobulin-like constant region domain. COMS ID No: SMBI-00634744 Received by IPlAustralia: Time 12:21 Date 2004-02-25 25/02 '04 12:13 FAX 61 3 9663 3099 FB RICE CO. [a047 150

234. The B7-2 fusion protein of claim 233, wherein the human B7-2 immunoglobulin-like variable region domain comprises an amino acid sequence from about position 24 to position 133 of SEQ ID NO: 2.

235. The B7-2 fusion protein of claim 233, wherein the heterologous polypeptide comprises an immunoglobulin constant region polypeptide.

236. An isolated nucleic acid molecule encoding a variable region form of a B7-2 fusion protein, the B7-2 fusion protein comprising a human B7-2 immunoglobulin-like variable region domain operatively linked to a heterologous polypeptide, wherein the B7-2 fusion protein does not comprise a B7-2 immunoglobulin-like constant region domain.

237. The isolated nucleic acid molecule of claim 236, wherein the B7-2 variable region region polypeptide is a human B7-2 variable region polypeptide.

238. The isolated nucleic molecule of claim 237, wherein the human B7-2 variable :region polypeptide comprises an amino acid sequence of about positions 24-133 of SEQ ID NO:2.

239. The nucleic acid of claim 236, wherein the heterologous polypeptide is an immunoglobulin constant region polypeptide.

240. The nucleic acid of claim 239, wherein the immunoglobulin constant region comprises a Cyl domain, including the hinge, CH2 and CH3 region.

241. The nucleic acid of claim 239, wherein the immunoglobulin constant region is modified to reduce constant region-mediated biological effector functions. *0"9 COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:13 FAX 61 3 9663 3099 FB RICE CO. Z048 151

242. The nucleic acid of claim 241, wherein the biological effector function is selected from the group consisting of complement activation and Fc receptor interaction.

243. The'nucleic acid of claim 242, wherein the immunoglobulin constant region is a C 7 4 domain, including the hinge, CH2 and CH3 region. 244 The nucleic acid of claim 243, wherein at least one amino acid residue of the CR2 domain is modified by substitution, addition or deletion.

245. A recombinant expression vector comprising the nucleic acid molecule of any one of claims 236-244.

246. A host cell containing the recombinant expression vector of claim 245.

247. A composition suitable for pharmaceutical administration comprising an isolated variable region form of the B cell activation antigen, B7-2, of any one Of claims 221-232 and a pharmaceutically acceptable carrier. 20 248. The composition of claim 247, wherein the composition modulates T cell proliferation.

249. The composition of claim 247, wherein the composition upregulates an immune response.

250. The composition of claim 247, wherein the composition modulates infection with a pathogen. "251. The composition of claim 250, wherein the pathogen is a virus. 99

252. The composition of claim 251, whemin the virus is HIV. 99 COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:14 FAX 61 3 9663 3099 FB RICE CO. [a049 152

253. A method for stimulating a response by an activated T cell, comprising contacting the activated T cell with a variable region forn of the B cell activation antigen B7-2, the variable region form of B7-2 comprising a 87-2 immunoglobulin-like variable region domain but not comprising a B7-2 innunoglobulin-like constant region domain, such that a response by the activated T cell is stimulated.

254. The method of claim 253, wherein a Thelper-Type 2 (TH 2 response is preferentially stimulated.

255. A method for upregulating T cell mediated immune responses in a subject the method including administering to the subject a variable region form of the B cell activation antigen, B7-2, the variable region form of B7-2 comprising a B7-2 immunoglobulin-like variable region domain but not comprising a B7-2 immunoglobulin-like constant region domain, in an amount effective to stimulate T cell proliferation and/or cytokine secretion in the subject.

256. An antibody or fragment thereof specifically reactive with a peptide of any one of claims 93-96, wherein the antibody is a humanized antibody.

257. The antibody or fragment thereof of claim 256, which comprises a human constant region and a non-human variable region.

258. The antibody or fragment thereof of claim 256, which comprises a human constant region and a human variable, region chimera-

259. An anti-body or fragment thereof specifically reactive with a peptide of any one of claims 93-96, wherein the antibody is a fully human monoclonal antibody.

260. A composition suitable for pharmaceutical administration comprising the o 30 antibody or fragment thereof of any one of claims 256-259. COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:14 FAX 61 3 9663 3099 FB RICE C.O. 153

261. The composition of claim 260, wherein the composition modulates T cell proliferation.

262. The composition of claim 260, wherein the composition dowaregulates a T-cell mediated immune response.

263. The composition of claim 260, wherein the composition modulates at least one of: autoimmune disease, allergy, and transplant rejectior

264. A method of inhibiting an interaction of a B lymphocyte antigen, B7-2, with its natural ligand(s) on the surface of immune cells, the method including contacting an immune cell with the antibody or fragment thereof of any one of claims 256-259, to thereby inhibit costimulation of the immune cell through B7-2-ligand interaction.

265. A method of downregulating T cell mediated immune responses in a subject, the method including administering to the subject the antibody or fragment thereof of any one of claims 256-259, in an amount effective to inhibit T cell proliferation and/or cytokine secretion in the subject. :20 266. A method oftreating an autoimnmune disease in a subject mediated by interaction of a B Lymphocyte antigen, B7-2, with its natural ligand(s) on the surface of immune cells, the method including administering to the subject the antibody or fragment thereof of any one of claims 256-259, such that the autoimmune disease is treated.

267. An isolated nucleic acid molecule according to any one of claims I to 17, 21 to 32, 186 to 1'91, 197, 198 or 236 to 244 substantially as hereinbefore described with particular reference to the examples and/or the preferred embodiments.

268. An isolated DNA molecule according to any one of claims 18 to 20 substantially as hereinbefore described with particular reference to the examples and/or the preferred embodiments. °4 COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:14 FAX 61 3 9663 3099 FB RICE CO, o051 154

269. An isolated polypeptide according to any one of claims 33 to 42, 192 to 196 substantially as hereinbefore described with particular reference to the examples and/or the preferred embodiments.

270. A composition according to any one of claims 43, 44, 50, 54, 102 to 106, 111 to 114, 200 to 206, 247 to 252 or 260 to 263 substantially as hereinbefore described with particular reference to the examples and/or the preferred embodiments.

271. A recombinant expression vector according to any one of claims 45 to 49 or 245 substantially as hereinbefore described with particular reference to the examples and/or the preferred embodiments.

272. A host cell according to any one of claims 51 to 53 or 246 substantially as hereinbefore described with particular reference to the examples and/or the preferred embodiments.

273. Au isolated, recombinant peptide according to claim 55 substantially as hereinbefore described with particular reference to the examples and/or the preferred embodiments.

274. A cell according to claims 56 or 57 substantially as hereinbefore described with particular reference to the examples and/or the preferred embodiments.

275. A tumor cell according to any one of claims 58 to 77 substantially as S 0. 25 hereinbefore described with particular reference to the examples and/or the preferred embodiments.

276. A method according to any one of claims 78 to 92, 115 to 182, 199, 207 to 220, 253 to 255 or 264 to 266 substantially as hereinbefore described with particular reference to the examples and/or the preferred embodiments.

277. A peptide according to any one of claims 93 to 98, 100 or 101 substantially as hereinbefore'dscibed with particular reference to the examples and/or the preferred erribodiments, 9D *o 9. COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25 25/02 '04 12:14 FAX 61 3 9663 3099 FB RICE CO. o052 155

278- A substantially pure preparation according to claim 99 substantially as hereinbefore described with particular reference to the examples and/or the preferred embodiments.

279. A nonhuman transgenic animal according to claims 107 or 108 substantially as hereinbefore described with particular reference to the examples and/or the preferred embodiments.

280. A nonhuman knockout animal according to claims 109 or 110 substantially as hereinbefore described with particular reference to the examples and/or the preferred embodiments.

281. Use of a peptide according to any one of claims 183 to 185 substantially as hereinbefore described with particular reference to the examples and/or the preferred embodiments.

282. An isolated variable region form of the B-cell activation antigen, B7-2 according to any one of claims 221 to 232 substantially as hereinbefore described with particular reference to the examples and/or the preferred embodiments.

283. An isolated B7-2 fusion protein according to any one of claims 233 to 235 substantially as hereinbefore described with particular referenCe to the examples and/or the preferred embodiments. 999* 0.0. 25 284. An antibody or fragment thereof according to any one of claims 256 to 259 9@ substantially as hereinbefore described with particular reference to the examples and/or "the preferred embodiments. DATED this 25th day of February 2004 DANA-FARBER CANCER INSTITUTE, REPLIGEN CORPORATION Patent Attorneys for the Applicant: o99 9 .o F.B. RICE CO. 0 9 09 COMS ID No: SMBI-00634744 Received by IP Australia: Time 12:21 Date 2004-02-25