JP2009060905A - B7-2: CTLA4 / CD28 counter receptor - Google Patents

Abstract

The present invention provides a nucleic acid encoding a novel CTLA4 / CD28 ligand that co-stimulates activation of T cells.
In one embodiment, the nucleic acid has a sequence encoding B lymphocyte antigen B7-2. Preferably, the nucleic acid is a DNA molecule comprising at least a part of a nucleotide sequence consisting of a specific sequence. The nucleic acid sequence is integrated into various expression vectors and the corresponding protein or peptide is synthesized in various hosts, particularly eukaryotic cells such as mammalian and insect cell cultures.
[Selection figure] None

Description

  The present invention relates to an isolated nucleic acid comprising a nucleotide sequence encoding a peptide having the activity of a B lymphocyte antigen, B7-2.

  In order to induce antigen-specific T cell activation and clonal expansion, two signals provided by antigen-presenting cells (APC) must be transmitted to the surface of resting T lymphocytes (Jenkins, M. and Schwartz, R. (1987), J. Exp. Med., 165, 302-319; , ER (1990) J. Immunol., 145, 85-93). The first signal that gives specificity to the immune response is mediated by the T cell receptor (TCR) after recognition of the foreign antigenic peptide shown in the major histocompatibility complex (MHC). A second signal called costimulation induces T cells to proliferate and become functional (Schwartz, RH, (1990) Science, 248, 1349-1356). Costimulation is neither antigen-specific nor MHC-restricted and is provided by one or more other cell surface molecules expressed by APC (Jenkins, MK et al. (1988), J. Immunol., 140, 3324). Linsley, PS et al. (1991), J. Exp. Med., 173, 721-730; Gimmi, CD et al. (1991), Proc. Natl. Acad. Sci. Young, J.W. et al. (1992), J.Clin.Invest., 90, 229-237; Koulova, L. et al. (1991), J.Exp. Med., 173, 759-762; Reiser, H. et al. (1992), Proc. Natl. Acad. Sci. USA, 89, 271-275; van-Seventer, GA et al. (1990), J. Immunol., 144, 4579-4586; J. M. et al. (1991), J. Immunol., 147, 774-8. Dustin, M. I. et al. (1989), J. Exp. Med., 169, 503; Armitage, R. J. et al. (1992), Nature, 357, 80-82; Liu, Y. et al. J. Exp. Med., 175, 437-445).

  Considerable evidence suggests that the B7 protein expressed on APC is one such important costimulatory molecule (Linsley, PS et al. (1991), J. Exp. Med., 173). 721-730; Gimmi, CD, et al. (1991), Proc. Natl. Acad. Sci. USA, 88, 6575-6579, Koulova, L. et al. 759-762; Reiser, H. et al. (1992), Proc. Natl. Acad. Sci. USA, 89, 271-275; Linsley, PS et al. (1990), Proc. 87, 5031-5035; Freeman, GJ et al. (1991), J. Exp. Med., 174, 625-631). B7 is a counter-receptor for the two ligands expressed on T lymphocytes. A first ligand, named CD28, is constitutively expressed on quiescent 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, PS et al. (1991), J. Exp. Med., Gimmi, CD et al. (1991), Proc. Natl. Acad. Sci. USA, 88, 6575-6579; Thompson, C. B. et al. (1989), Proc. Natl. Acad. Sci. USA, 86, 1333-1337; June, C. H. et al. (1990), Immunol. Today, 11, 211-6; Harding, FA, et al. (1992), Nature, 356, 607-609 . A second ligand named CTLA4 is homologous to CD28 but is not expressed on resting T cells and occurs after T cell activation (Brunet, JF et al. (1987) Nature, 328, 267- 270). DNA sequences encoding human and mouse CTLA4 proteins are described in Daravich et al. (1988), Eur. J. et al. Immunol. 18 (12), 1901-1905; Brunet, J.F. Et al. (1987), supra; Brunet, J.F. Et al. (1988), Immunol. Rev. 103, 21-36; and Freeman, GJ. Et al. (1992), J. et al. Immunol. 149, 3795-3801. B7 has a greater affinity for CTLA4 than for CD28 (Linsley, PS et al. (1991), J. Exp. Med. 174, 561-569), but the function of CTLA4 is still Is unknown.

  The importance of the B7: CD28 / CTLA4 costimulatory pathway has been demonstrated in vitro and in several in vivo model systems. Blocking this costimulatory pathway results in the development of antigen-specific tolerance in mouse and human systems (Harding, FA et al. (1992), Nature, 356, 607-609; Lenschow, DJ et al. (1992). ), Science, 257, 789-792; Turka, LA, et al. (1992), Proc. Natl. Acad. Sci. USA, 89, 11102-11105, Gimmi, CD, et al. Natl.Acad.Sci.USA, 90, 6586-6590; Boussiotis, V. et al. (1993), J. Exp. Med., 178, 1753-1763). Conversely, B7 expression by B7-negative mouse tumor cells induces long-lasting protection against T cell-mediated specific immunity and tumor challenge associated with tumor rejection (Chen, L. et al. (1992), Cell Townsend, SE and Allison, JP (1993), Scienco, 259, 368-370; Baskar, S. et al. (1993), Proc. Natl. Acad. Sci. 90, 5687-5690). Thus, manipulation of the B7: CD28 / CTLA4 pathway offers great potential to stimulate or suppress immune responses in humans.

Cell, 71, 1093-1102 (1992) Scienco, 259, 368-370 (1993) Proc. Natl. Acad. Sci. USA, 90, 5687-5690 (1993)

  The present invention relates to isolated nucleic acids encoding novel molecules that costimulate T cell activation. Preferred costimulatory molecules present antigens to B lymphocytes, specialized antigen presenting cells (eg monocytes, dendritic cells, Langerhans cells), and immune cells, CTLA4, CD28, both CTLA4 and CD28, or others Antigens on the surface of other cells (eg, keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes) that bind to receptors on known or undefined immune cells. Such costimulatory molecules, referred to herein as CTLA4 / CD28 binding counterreceptors or B lymphocyte antigens, costimulate activated T cells, thereby causing T cell proliferation and / or cytokine secretion. Can be induced. Preferred B lymphocyte antigens include B7-2 and B7-3, and soluble fragments or derivatives thereof that have the ability to bind CTLA4 and / or CD28 and to inhibit or induce immune cell costimulation. In one aspect, an isolated nucleic acid encoding a peptide having human B7-2B lymphocyte antigen activity is provided. Preferably the nucleic acid is shown in FIG. 8 (SEQ ID NO: 1). A cDNA molecule having a nucleotide sequence encoding human B7-2. In another embodiment, the nucleic acid is a cDNA molecule having a nucleotide sequence encoding murine B7-2, shown in FIG. 14 (SEQ ID NO: 22).

  The present invention has B7-2 activity and is at least about 50%, more preferably at least about 60% of the amino acid sequence shown in FIG. 8 (SEQ ID NO: 2), or the amino acid sequence shown in FIG. 14 (SEQ ID NO: 23), Most preferably, it relates to a nucleic acid encoding a peptide that is at least 70% homologous. It has B7-2 activity and is at least about 80%, more preferably at least about 90%, more preferably at least about the amino acid sequence shown in FIG. 8 (SEQ ID NO: 2) or the amino acid sequence shown in FIG. 14 (SEQ ID NO: 23). Nucleic acids encoding peptides that are about 95%, most preferably at least about 98% or at least about 99% homologous are also within the scope of the invention. In other embodiments, the peptide having B7-2 activity is high or low in nucleic acid encoding a peptide having the amino acid sequence of FIG. 8 (SEQ ID NO: 2) or a peptide having the amino acid of FIG. 14 (SEQ ID NO: 23). It is encoded by a nucleic acid that hybridizes under stringency conditions.

  The invention further relates to an isolated nucleic acid comprising a nucleic acid sequence encoding a peptide having B7-2 activity and having a length of at least 20 amino acid residues. A peptide having B7-2 activity and comprising a length of at least 40 amino acid residues, at least 60 amino acid residues, at least 80 amino acid residues, at least 100 amino acid residues, or at least 200 amino acid residues Are also within the scope of the present invention. Particularly preferred nucleic acids have B7-2 activity, have a length of at least 20 amino acid residues or more, and at least 50% or more homology (preferably at least 70) with the sequence shown in FIG. 8 (SEQ ID NO: 2). %).

In one preferred embodiment, the present invention has B7-2 activity and has the formula:
Xn-Y-Zm
An isolated DNA encoding a peptide having the amino acid sequence represented by

  In the formula, Y consists essentially of amino acid residues 24-245 of the sequence shown in FIG. 8 (SEQ ID NO: 2). Xn and Zm are amino acid residues selected from amino acid residues adjacent to Y in the amino acid sequence shown in FIG. 8 (SEQ ID NO: 2). Xn is an amino acid residue selected from amino acids adjacent to the amino terminus of Y in the sequence shown in FIG. 8 (SEQ ID NO: 2), that is, selected from amino acid residues 23 to 1. Zm is an amino acid residue selected from amino acids adjacent to the carboxy terminus of Y in the sequence shown in FIG. 8 (SEQ ID NO: 2), that is, selected from amino acid residues 246 to 329. In the formula, n is a number from 0 to 23 (n = 0 to 23), and m is a number from 0 to 84 (m = 0 to 84). Particularly preferred DNA has an amino acid sequence represented by the formula Xn-Y-Zm, wherein Y is amino acid residues 24-245 of the sequence shown in FIG. 8 (SEQ ID NO: 2), n = 0, m = 0. Encodes the peptide.

  The present invention provides a nucleotide sequence encoding a first peptide having B7-2 activity, and a second peptide corresponding to a portion that changes the solubility, binding affinity, stability, valence of the first peptide. It also relates to an isolated DNA encoding a B7-2 fusion protein comprising the encoding nucleotide sequence. Preferably, the first peptide having B7-2 activity comprises the extracellular domain portion of the B7-2 protein (eg, amino acid residues about 24-245 of the sequence shown in FIG. 8 (SEQ ID NO: 2)), and the second peptide Is a human Cγ1 or Cγ4 domain containing the constant region of an immunoglobulin, eg, the hinge, CH2 and CH3 regions, and forms a B7-2 immunoglobulin fusion protein (B7-2Ig) (Capon et al. (1989), Nature 337, 525-531 and Capon, US Pat. No. 5,116,964).

  The nucleic acids obtained according to the present invention are inserted into various expression vectors, various hosts, in particular eukaryotic cells such as mammalian and insect cell cultures, and E. coli. Corresponding proteins or peptides are synthesized in prokaryotic cells such as Coli. Expression vectors within the scope of the invention include a nucleic acid encoding at least one peptide having the activity of the novel B lymphocyte antigen described herein, and a promoter operably linked to the nucleic acid sequence. In one embodiment, the expression vector comprises DNA encoding a peptide having the activity of the B7-2 antigen, and other B lymphocytes such as a previously characterized B7 activation antigen (referred to herein as B7-1). DNA encoding a peptide having the activity of a sphere antigen is included. Such expression vectors can be used to transfect host cells, thereby producing a protein or peptide comprising a fusion protein encoded by the nucleic acids described herein.

  Nucleic acid probes useful for analyzing biological samples for the presence of B cells expressing B lymphocyte antigens B7-2 and B7-3 are also within the scope of the present invention.

The invention further relates to isolated peptides having the activity of novel B lymphocyte antigens, including B7-2 and B7-3 protein antigens. A preferred peptide having B7-2 activity is produced by recombinant expression and comprises the amino acid sequence shown in FIG. 8 (SEQ ID NO: 2). Another preferred peptide having B7-2 activity comprises the amino acid sequence shown in FIG. 14 (SEQ ID NO: 23). Particularly preferred peptides having the activity of the B7-2 antigen are extracellular domain portions (eg, about amino acid residues about 24-245 of SEQ ID NO: 2) that can be used to enhance or suppress a patient's T cell-mediated immune response, etc. At least a portion of the mature form of the protein. Other preferred peptides having B7-2 activity include peptides having the amino acid sequence represented by the following formula:
Xn-Y-Zm

In the formula, Y represents amino acid residues 55 to 68 of the sequence shown in FIG. 8 (SEQ ID NO: 2), amino acid residues 81 to 89 of the sequence shown in FIG. 8 (SEQ ID NO: 2), and FIG. 8 (SEQ ID NO: 2). Amino acid residues 128-142 of the sequence shown, amino acid residues 160-169 of the sequence shown in FIG. 8 (SEQ ID NO: 2), amino acid residues 188-200 of the sequence shown in FIG. 8 (SEQ ID NO: 2), FIG. It is an amino acid residue selected from the group consisting of amino acid residues 269 to 282 shown in No. 2). In the formula, Xn and Zm are additional amino acid residues bonded to Y by an amide bond, and are selected from amino acid residues adjacent to Y in the amino acid sequence shown in FIG. 8 (SEQ ID NO: 2). Xn is an amino acid residue selected from amino acids adjacent to the amino terminus of Y in the sequence shown in FIG. 8 (SEQ ID NO: 2). Zm is an amino acid residue selected from amino acids adjacent to the carboxy terminus of Y in the sequence shown in FIG. 8 (SEQ ID NO: 2). In the formula, n is a number from 0 to 30 (n = 0 to 30), and m is a number from 0 to 30 (m = 0 to 30).

  It also relates to a fusion protein or hybrid fusion protein comprising a peptide having the activity of a novel B lymphocyte antigen (eg B7-2, B7-3). For example, including the extracellular domain portion of a novel B lymphocyte antigen fused to a second peptide such as an immunoglobulin constant region that alters the solubility, binding affinity, stability and / or valency of the first peptide A fusion protein comprising a first peptide is provided. In one embodiment, a first peptide comprising amino acid residues of the extracellular domain portion of the B7-2 protein bound to a second peptide comprising amino acid residues of sequences corresponding to the Cγ1 or Cγ4 hinge, CH2 and CH3 regions. A fusion protein comprising is made to form a B7-2Ig fusion protein. In other embodiments, a first peptide comprising the extracellular domain portion of the B7-1 antigen and an extracellular domain portion of the B7-2 antigen, and a second peptide comprising amino acid residues corresponding to the hinge, CH2 and CH3 of Cγ1 (Eg Linsley et al. (1991), J. Exp. Med., 1783, 721-730; Capon et al. (1989), Nature, 337, 525-531; and Capon US Pat. No. 5,116). 964).

  Isolated peptides and fusion proteins of the present invention upregulate or inhibit the expression of one or more B lymphocyte antigens, or on one or more B lymphocyte antigens on immune cells such as T cells. Can be administered to patients to block ligation to their natural ligands, thereby enhancing or suppressing cell-mediated immune responses in vivo.

  Another aspect of the present invention provides an antibody, preferably a monoclonal antibody, that specifically reacts with a peptide of a novel B lymphocyte antigen or a fusion protein described herein. Preferred antibodies are anti-human B7-2 monoclonal antibodies produced by hybridoma cells HF2.3D1, HA5.2B7 and HA3.1F9. These hybridoma cells have been deposited with the American Type Culture Collection under ATCC Deposit Number HB11686 (HF2.3D1), ATCC Deposit Number HB11687 (HA5.2B7), and ATCC Deposit Number HB11688 (HA3.1F9).

  A further aspect of the present invention includes enhancing the immunogenicity of mammalian cells using the nucleic acids of the present invention, particularly cDNA. In a preferred embodiment, the mammalian cell is a tumor cell such as a sarcoma, lymphoma, melanoma, neuroblastoma, leukemia, or carcinoma, or an antigen-presenting cell such as a macrophage. A peptide having B lymphocyte antigen activity is expressed on the cell surface. Macrophages expressing peptides having B lymphocyte antigen activity, such as B7-2 antigen, enhance T cell activation and immune stimulation when pulsed with appropriate pathogen-related antigens or tumor antigens It can be used as a cell.

  Mammalian cells are transfected ex vivo with an appropriate expression vector containing a nucleic acid encoding a peptide having the activity of a novel B lymphocyte antigen such as the B7-2 antigen, and then the host mammal Or cells can be transfected in vivo with that gene by gene therapy techniques. For example, a nucleic acid encoding a peptide having B7-2 activity can be transfected alone or in combination with nucleic acids encoding other costimulatory molecules. In a mammalian cell to be transfected with a nucleic acid encoding a peptide having the activity of a B lymphocyte antigen described herein in enhancing the immunogenicity of a tumor that does not express a class I or class II MHC molecule. It may be beneficial to additionally transfect appropriate class I or II genes.

  The present invention, for example, blocks the functional interaction of the novel B lymphocyte antigens of the present invention, such as B7-2 and B7-3, with their natural ligands on the T cell, thereby allowing receptor ligand pairs Also provided are methods of inducing general immunosuppression and antigen-specific tolerance in patients by blocking co-stimulation via. In one aspect, an inhibitory molecule that can be used to block the interaction of a natural human B7-2 antigen with its natural ligands (eg, CTLA4 and CD28) has B7-2 binding activity, A soluble peptide that does not have the ability to costimulate immune cells, an antibody that blocks the binding of B7-2 to its ligand and cannot transmit a costimulatory signal (a so-called “blocking antibody” such as a blocking anti-B7-2 antibody), B7-2-Ig fusion protein, which can be prepared according to the teachings of, as well as soluble forms of B7-2 receptors such as CTLA4Ig or CD28Ig. Such blocking agents can be used alone or in combination with agents that block their natural ligands of other costimulatory molecules (eg, anti-B7 antibodies). Inhibition of T cell response and induction of T cell tolerance by the methods described herein is prophylactic in the prevention of transplant rejection (solid organs, skin and bone marrow) and graft-versus-host disease, particularly in allogeneic bone marrow transplantation. Can be useful to. The methods of the invention may also be useful therapeutically in the treatment of autoimmune diseases, allergies and allergic reactions, transplant rejection, and graft-versus-host disease.

Another aspect of the present invention is by using stimulatory forms of B7-2 antigens, including soluble, multivalent forms of B7-2 proteins such as peptides having B7-2 activity and B7-2 fusion proteins. It relates to a method of upregulating the immune response by transmitting a costimulatory signal to T cells. Delivery of a stimulatory form of B7-2 together with an antigen is prophylactically useful to enhance the effectiveness of vaccination against various pathogens, immunizing against individual pathogens during infection or against tumors in a host with a tumor It may also be useful therapeutically to upregulate the response.
The present invention identifies molecules capable of inhibiting the interaction of B lymphocyte antigens such as B7-2, B7-3 with their receptors or preventing intracellular signaling through those receptors. Also related to how to do. Also provided are methods for identifying molecules capable of modulating the expression of B lymphocyte antigens on cells. Furthermore, a method for identifying cytokines produced in response to costimulation of T cells by a novel B lymphocyte antigen is also within the scope of the present invention.

  In addition to the previously characterized B lymphocyte activation antigen B7 (referred to herein as B7-1), human B lymphocytes express other novel molecules that costimulate T cell activation. These costimulatory molecules present antigens to B lymphocytes, specialized antigen-presenting cells (eg monocytes, dendritic cells, Langerhans cells) and immune cells, CTLA4, CD28, both CTLA4 and CD28, Or on the surface of other cells that bind to receptors on other known or undefined immune cells (e.g. keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes) Contains antigen. Co-stimulatory molecules within the scope of the present invention are referred to herein as CTLA4 / CD28 ligand (counter receptor) or B lymphocyte antigen. A novel B lymphocyte antigen that co-stimulates activated T cells, thereby inducing T cell proliferation and / or cytokine secretion, is described and characterized in B7-2 ( Human and murine) and the B7-3 antigen.

  B lymphocyte antigen B7-2 is expressed by human B cells approximately 24 hours after stimulation with either anti-immunoglobulin or anti-MHC class II monoclonal antibodies. The B7-2 antigen induces detectable IL-2 secretion and T cell proliferation. Approximately 48-72 hours after activation, human B cells are both B7-1 and a third CTLA4 counter receptor, B7-3, identified by monoclonal antibody BB-1, which also binds B7-1. (Yokochi, T. et al. (1982), J. Immunol., 128, 823-827). The B7-3 antigen is also expressed on B7-1 negative activated B cells and costimulates T cell proliferation without the production of detectable IL-2, indicating that B7-1 and B7-3 Indicates that the molecule is different. B7-3 is expressed on a variety of cells including activated B cells, activated monocytes, dendritic cells, Langerhans cells, and keratinocytes. At 72 hours after B cell activation, B7-1 and B7-3 expression begins to decline. The presence of these costimulatory molecules on the surface of activated B lymphocytes indicates that T cell costimulation is partially controlled by transient expression of these molecules after B cell activation.

  Accordingly, one aspect of the present invention is an isolated comprising a nucleotide sequence encoding a novel costimulatory molecule such as a B lymphocyte antigen, B7-2, a fragment of such a nucleic acid, or an equivalent thereof. Relates to the nucleic acid. As used herein, the term “nucleic acid” is intended to include such fragments or equivalents. The term “equivalent” refers to the activity of a novel B lymphocyte antigen (ie, binding to the natural ligand of a B lymphocyte antigen on immune cells such as CTLA4 and / or CD28 on T cells and co-stimulating immune cells Is intended to include a nucleotide sequence encoding a functionally equivalent B lymphocyte antigen or a functionally equivalent peptide having the ability to inhibit (eg, block) or stimulate (eg, enhance). Such a nucleic acid is considered to be equivalent to the human B7-2 nucleotide sequence (SEQ ID NO: 1) shown in FIG. 8 and the B7-2 nucleotide sequence of Murine (SEQ ID NO: 22) shown in FIG. 14, and is within the scope of the present invention. Is in.

  In one embodiment, the nucleic acid is a cDNA encoding a peptide having the activity of a B7-2 B lymphocyte antigen. Preferably, the nucleic acid is at least a portion of the nucleotide sequence (SEQ ID NO: 1) encoding human B7-2 shown in FIG. 8, or the nucleotide sequence (SEQ ID NO: 22) encoding murin B7-2 shown in FIG. 14 (SEQ ID NO: 2). A cDNA molecule comprising at least a portion of A preferred portion of the cDNA molecule of FIG. 8 (SEQ ID NO: 1) or FIG. 14 (SEQ ID NO :) comprises the coding region of the molecule.

  In another embodiment, the nucleic acid of the invention encodes a peptide having B7-2 activity and comprising the amino acid sequence shown in FIG. 8 (SEQ ID NO: 2) or FIG. 14 (SEQ ID NO: 23). Preferred nucleic acids have B7-2 activity and have at least about 50% homology with the amino acid sequence shown in FIG. 8 (SEQ ID NO: 2), more preferably at least about 60% homology, and most preferably at least about 70. Encodes peptides with% homology. Encodes a peptide having B7-2 activity and having at least about 90%, more preferably at least about 95%, most preferably at least about 98-99% homology with the sequence shown in FIG. 8 (SEQ ID NO: 2) Nucleic acids are also within the scope of the present 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 the positions in each sequence aligned for comparison purposes. If a position in the compared sequences is occupied by the same nucleotide base or amino acid, then the molecules are homologous at that position. The degree of percent homology between sequences is a function of the number of matched or homologous positions shared by the sequences.

Another embodiment of the present invention relates to a peptide having all or part of the amino acid sequence (SEQ ID NO: 2) shown in FIG. 8, or a peptide having all or part of the amino acid sequence (SEQ ID NO: 23) shown in FIG. Nucleic acids that hybridize to the encoding nucleic acid under high or low stringency conditions are provided. Appropriate stringency conditions to facilitate DNA hybridization, such as 6.0 × sodium chloride / sodium citrate (SSC) at about 45 ° C. followed by 2.0 × SSS wash at 50 ° C.) Known and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, New York (1989), 6.3.1-6.3.6. For example, the salt concentration in the washing step can be selected from a low stringency of about 2.0 × SSC (50 ° C.) to a high stringency of about 0.2 × SSC (50 ° C.). Furthermore, the temperature in the washing step can be selected from room temperature, low stringency conditions of about 22 ° C. to high stringency conditions of about 65 ° C.
An isolated nucleic acid encoding a peptide having a novel B lymphocyte antigen and having a sequence different from the nucleotide sequence shown in FIG. 8 (SEQ ID NO: 1) or FIG. 14 (SEQ ID NO: 22) due to degeneracy in the genetic code Are also within the scope of the present invention. Such nucleic acids encode functionally equivalent peptides (eg, peptides having B7-2 activity) but differ in sequence from the sequence of FIG. 8 or FIG. 14 due to degeneracy in the genetic code. For example, many amino acids are specified by one or more triplets. Codons that represent the same amino acid, ie synonyms (eg CAU and CAC are synonyms for histidine) arise due to degeneracy in the genetic code. As an example, DNA sequence polymorphisms within the nucleotide sequence of B7-2 (especially within the third codon of the codon) may result in “silent” mutations in the DNA sequence that do not affect the encoded amino acid. However, it is expected that DNA sequence polymorphisms that would result in changes in the amino acid sequence of the B7-2 antigen will exist within the population. These mutations (up to about 3-4% of the nucleotides) in one or more nucleotides of a nucleic acid encoding a peptide having the activity of a novel B lymphocyte antigen are between natural ones within the population due to natural allelic variation. Those skilled in the art will recognize that Any and all such nucleotide variants and resulting amino acid polymorphs are within the scope of the invention. In addition, there may be one or more isoforms of the novel B lymphocyte antigens described herein, or related cross-reacting family members. Such isoforms or family members are defined as proteins encoded by genes in different locations that are related in function and amino acid sequence to B lymphocyte antigens (eg, B7-2 antigen).

  A “fragment” of a nucleic acid encoding a novel B lymphocyte antigen has fewer nucleotides than the nucleotide sequence encoding the entire amino acid sequence of the B lymphocyte antigen, and the activity of the B lymphocyte antigen (ie, CTLA4 on T cells). And / or nucleotide sequence encoding a peptide having the ability to bind to the natural ligand of a B lymphocyte antigen on immune cells and stimulate or inhibit immune cell costimulation, such as CD28. Thus, a peptide having B7-2 activity binds CTLA4 and / or CD28 and is T cell mediated, eg as demonstrated by cytokine secretion production and / or T cell proliferation by a T cell that has received a primary activation signal. Stimulate or inhibit the immune response. In one aspect, the nucleic acid fragment encodes a peptide of the B7-2 antigen that retains the ability of the antigen to bind CTLA4 and / or CD28 and carries a costimulatory signal to T lymphocytes. In other embodiments, the nucleic acid fragment comprises the extracellular portion of human B7-2 antigen (eg, amino acid residues about 24-245 of the sequence shown in FIG. 8 (SEQ ID NO: 2)) and binds CTLA4 and / or CD28 However, the monovalent form encodes a peptide that can be used to inhibit costimulation or in the multivalent form to induce or enhance costimulation.

Preferred nucleic acid fragments encode peptides consisting of at least 20 amino acid residues in length, preferably at least 40 amino acid residues, and more preferably at least 60 amino acid residues. Nucleic acid fragments encoding peptides consisting of at least 80 amino acid residues in length, at least 100 amino acid residues, and at least 200 or more amino acid residues are also within the scope of the present invention. Particularly preferred nucleic acid fragments have the formula:
Xn-Y-Zm
And a peptide having human B7-2 activity. Where Y comprises amino acid residues 24-245 of the sequence shown in FIG. 8 (SEQ ID NO: 2), and Xn and Zm are additional amino acid residues linked to Y by an amide bond. Xn and Zm are selected from amino acid residues adjacent to Y in the amino acid sequence shown in FIG. 8 (SEQ ID NO: 2). In the formula, Xn is an amino acid residue selected from amino acid residues 23 to 1 adjacent to the amino terminus of Y in the sequence shown in FIG. 8 (SEQ ID NO: 2). Zm is an amino acid residue selected from amino acid residues 246 to 329 adjacent to the carboxy terminus of Y in the sequence shown in FIG. 8 (SEQ ID NO: 2). Further, in the formula, n is a number from 0 to 23 (n = 0 to 23), and m is a number from 0 to 84 (m = 0 to 84). Particularly preferred peptides have the amino acid sequence represented by the above formula Xn-Y-Zm where n = 0 and m = 0.

  Nucleic acid fragments within the scope of the present invention include nucleic acids from other animal species for use in screening protocols to detect novel proteins that are cross-reacting with the B lymphocytes described herein. Including those that can hybridize with. These and other fragments are described in detail below. In general, the nucleic acid encoding a fragment of a B lymphocyte antigen is selected from the bases encoding the mature protein, but in some cases, selecting all or part of the fragment from the leader sequence or non-coding portion of the nucleotide sequence It is preferable to do this. Nucleic acids within the scope of the present invention may also include linker sequences, modified restriction endonuclease sites, and other sequences useful for molecular cloning, recombinant protein expression or construction, or fragments thereof. These and other modifications of the nucleic acid sequence are described in detail below.

  Nucleic acids encoding peptides having novel B lymphocyte antigen activity, such as B7-2 antigen, can be obtained from mRNA present in activated B lymphocytes. It would also be possible to obtain nucleic acid sequences encoding B lymphocyte antigens from B cell genomic DNA. For example, a gene encoding a B7-2 antigen can be cloned from a cDNA or genomic library according to the protocol described herein. CDNA encoding the B7-2 antigen can be obtained by isolating total mRNA from an appropriate cell line. Next, double stranded cDNA can be prepared from total mRNA. The cDNA can then be inserted into an appropriate plasmid or viral (eg, bacteriophage) vector using any one of a number of known techniques. A gene encoding a novel B lymphocyte antigen can also be cloned using established polymerase chain reaction techniques according to the nucleotide sequence information provided by the present 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 shown in FIG. 8 (SEQ ID NO: 1). Another preferred nucleic acid is a cDNA encoding the murin B7-2 antigen having the sequence shown in FIG. 14 (SEQ ID NO: 22).

  The invention further relates to an expression vector comprising a nucleic acid encoding at least one peptide having the activity of a novel B lymphocyte antigen described herein operably linked to at least one regulatory sequence. “Operably linked” is intended to mean that the nucleic acid sequence is linked to a regulatory sequence (eg, in cis or trans) in such a way as to permit its expression. Control sequences are known in the art and are selected to direct expression of the desired protein in a suitable host cell. Thus, the term regulatory sequence includes promoters, enhancers and other expression control elements. Such regulatory sequences are known to those skilled in the art and are described in Goeddel, Gene Expression Technology, Methods in Enzymology, Academic Press, San Diego, California (1990). It should be understood that the design of the expression vector depends on the choice of the host to be transfected and / or the type of protein desired to be expressed. In one aspect, the expression vector includes a nucleic acid encoding at least a portion of a B7-2 protein, such as an extracellular domain portion. In other embodiments, the expression vector comprises DNA encoding a peptide having the activity of the B7-2 antigen and DNA encoding a peptide having the activity of another B lymphocyte antigen such as B7-1. CDNAs encoding human B7-1 and mouse B7-1 antigens are shown in SEQ ID NO: 28 and SEQ ID NO: 30, respectively. The deduced amino acid sequences of these antigens are shown in SEQ ID NO: 29 and SEQ ID NO: 31, respectively. Such expression can be used to transfect cells, thereby producing proteins or peptides, including fusion proteins or peptides encoded by the nucleic acid sequences described herein. These and other aspects are described in further detail herein.

  The present invention also relates to a method for producing a peptide having a novel B lymphocyte antigen activity. For example, a host cell transfected with a nucleic acid vector directed to the expression of a nucleotide sequence encoding a peptide having the activity of the B7-2 protein can be cultured in a medium under suitable conditions to express the peptide. In addition, the DNA includes a DNA encoding a peptide having B7-2 activity, and a DNA encoding another peptide such as a peptide having a second B lymphocyte antigen (for example, B7-1, B7-3) activity. These expression vectors can be used to transfect host cells to co-express these peptides or to produce fusion proteins or peptides. In one aspect, a recombinant expression vector comprising DNA encoding a B7-2 fusion protein can be produced. The B7-2 fusion protein comprises a nucleotide sequence encoding a first peptide having B7-2 activity and a second corresponding to a moiety that alters the solubility, affinity, stability or valence of the first peptide. It can be produced by recombinant expression of a nucleotide sequence encoding a peptide, such as an immunoglobulin constant region. Preferably, the first peptide consists of a portion of the extracellular domain of human B7-2 antigen (for example, amino acid residues about 24-245 of the sequence shown in FIG. 8 (SEQ ID NO: 2)). The second peptide is an immunoglobulin constant region, such as a human Cγ1 domain or Cγ4 domain (eg, human IgCγ1 or human IgCγ4 hinge, CH2 and CH3 regions, see Capon et al. US Pat. No. 5,116,964, which Which constitutes a part of this specification. The resulting B7-2Ig fusion protein may have altered B7-2 solubility, binding affinity, stability, and / or valency (ie, number of binding sites available per molecule) to increase the efficiency of protein purification. Can be increased. Fusion proteins and peptides produced by recombinant techniques are secreted and can be isolated from a mixture of cells and media containing the protein or peptide. Alternatively, the protein or peptide can be retained in the cytoplasm and the cells can be harvested, lysed and the protein isolated. A cell culture typically includes host cells, media, and other byproducts. Suitable media for cell culture are well known in the art. Proteins and peptides can be isolated from cell culture media, host cells, or both using protein and peptide purification techniques known in the art. Techniques for transfecting host cells and purifying proteins and peptides are described in further detail herein.

Particularly preferred human B7-2Ig fusion proteins comprise the extracellular domain portion or variable region-like domain of human B7-2 bound to an immunoglobulin constant region. The immunoglobulin constant region may include genetic modifications that reduce or eliminate effector activity inherent in the immunoglobulin structure. For example, the DNA encoding the extracellular part of human B7-2 (hB7-2) is also the DNA encoding the variable region-like domain of human B7-2 (hB7.2V) or the constant region-like domain of human B7-2. Can also be bound to DNA encoding the hinge, CH2 and CH3 regions of human IgCγ1 and / or IgCγ4 modified by site directed mutagenesis. The production and characterization of these fusion proteins is described in detail in Example 7.

  Transfected cells that express peptides having the activity of one or more B lymphocyte antigens (eg, B7-2, B7-3) on the surface of the cells are also within the scope of the present invention. In one aspect, a host cell, such as a COS cell, is transfected with an expression vector that directs the expression of a peptide having B7-2 activity on the surface of the cell. Such transfected host cells inhibit B7-2 binding to its counter-receptor on T cells, or costimulatory intramolecular signaling to T cells in response to B7-2 interactions. Can be used in methods of identifying molecules that interfere with In other embodiments, tumor cells such as sarcomas, melanomas, leukemias, lymphomas, carcinomas or neuroblastomas are directed to the expression of at least one peptide having a novel B lymphocyte antigen activity on the tumor cell surface. Transfect with an expression vector. In some cases, tumor cells are transfected and co-expressed with major histocompatibility complex (MHC) proteins, such as MHC class II α and β chain proteins or MHC class I α chain proteins and, if necessary, β2 microglobulin protein Is also beneficial. Such transfected tumor cells can be used to induce tumor immunity in patients. These and other aspects are described in further detail herein.

  The nucleic acid sequences of the invention can also be chemically synthesized using standard techniques. Various methods for chemically synthesizing polydeoxynucleotides are known, including fully automated solid-phase synthesis on commercially available DNA synthesizers, as well as peptide synthesis (Itakura et al., US Pat. No. 4,598,049; Caruthers et al., 4,458,066; and Itakura et al., 4,401,796 and 4,373,071, these patents are incorporated herein by reference. Constitute.).

  Another aspect of the present invention relates to an isolated peptide having the activity of a novel B lymphocyte antigen (eg B7-2, B7-3). The peptide having the activity of the B lymphocyte antigen is an amino acid different from the B lymphocyte antigen such as the human B7-2 sequence shown in FIG. 8 (SEQ ID NO: 2) or the murine B7-2 sequence shown in FIG. Although the sequences may vary, such differences function in the same or similar manner as the B lymphocyte antigen, or result in a peptide having the same or similar characteristics as the B lymphocyte antigen. For example, peptides having the activity of the B7-2 protein bind to natural ligands of the B7-2 protein on immune cells, such as CLTA4 and / or CD28 on T cells, and stimulate or inhibit immune cell costimulation. A peptide having the ability to do so is defined herein. Thus, peptides having B7-2 activity bind to CTLA4 and / or CD28 and stimulate or inhibit T cell mediated immune responses (eg, by cytokine production and / or proliferation by T cells that have received a primary activation signal). Evidenced). One embodiment provides a peptide that has B7-2 binding activity but lacks the ability to transmit a costimulatory signal to T cells. Such peptides can be used to inhibit or block T cell proliferation and / or cytokine secretion in a patient. Alternatively, peptides having both B7-2 binding activity and the ability to transmit costimulatory signals to T cells are used to stimulate or enhance T cell proliferation and / or cytokine secretion in a patient. Various modifications of the B7-2 protein that produce these and other functionally equivalent peptides are described in detail herein. As used herein, the term “peptide” refers to peptides, proteins, and polypeptides.

Substitution, addition, or deletion of amino acid residues that are not directly involved in the function of B7-2 (ie, the ability of B7-2 to bind to CTLA4 and / or CD28 and / or stimulate or inhibit T cell costimulation) A peptide can be produced by modifying the amino acid sequence of the human B7-2 protein shown in FIG. 8 (SEQ ID NO: 2) or the murine B7-2 protein shown in FIG. 14 (SEQ ID NO: 23). The peptides of the present invention are typically at least 20 amino acid residues in length, preferably at least 40 amino acid residues, and most preferably 60 amino acid residues. Peptides having B7-2 activity and having a length of at least 80 amino acid residues, at least 100 amino acid residues, or at least 200 or more amino acid residues are also within the scope of the present invention. Preferred peptides comprise the extracellular domain portion of the human B7-2 antigen (eg, amino acid residues about 24-245 of the sequence shown in FIG. 8 (SEQ ID NO: 2)). Other preferred sequences have the formula:
Xn-Y-Zm
(Wherein Y represents amino acid residues 55 to 68 of the sequence shown in FIG. 8 (SEQ ID NO: 2), amino acid residues 81 to 89 of the sequence shown in FIG. 8 (SEQ ID NO: 2), and FIG. 8 (SEQ ID NO: 2). Amino acid residues 128-142 of the sequence, amino acid residues 160-169 of the sequence shown in FIG. 8 (SEQ ID NO: 2), amino acid residues 188-200 of the sequence shown in FIG. 8 (SEQ ID NO: 2), and FIG. It is an amino acid residue selected from the group consisting of amino acid residues 269 to 282 of the sequence shown in number 2).
It has the amino acid sequence shown by In the formula, Xn and Zm are additional amino acid residues bonded to Y by an amide bond. Xn and Zm are amino acid residues selected from amino acids adjacent to Y in the amino acid sequence shown in FIG. 8 (SEQ ID NO: 2). Xn is an amino acid residue selected from amino acids adjacent to the amino terminus of Y in the sequence shown in FIG. 8 (SEQ ID NO: 2). Zm is an amino acid residue selected from amino acids adjacent to the carboxy terminus of Y in the sequence shown in FIG. 8 (SEQ ID NO: 2). In the formula, n is a number from 0 to 30 (n = 0 to 30), and m is a number from 0 to 30 (m = 0 to 30). Particularly preferred peptides have the amino acid sequence represented by the formula Xn-Y-Zm, where n = 0 and m = 0.

  Another aspect of the invention provides a substantially pure product of a peptide having the activity of a novel B lymphocyte antigen such as B7-2 or B7-3. Such a product is substantially free of proteins and peptides that are naturally together in the cell or that are naturally together when secreted by the cell.

  As used throughout this specification, the term “isolated” refers to cellular material or culture medium when produced by recombinant DNA technology, chemical precursor or other chemistry when chemically synthesized. A nucleic acid, protein, or peptide having the activity of a novel B lymphocyte antigen such as B7-2 that is substantially free of substances. Isolated nucleic acid also does not include sequences that are naturally flanking of the nucleic acid in the organism from which it is derived (ie, sequences located at the 5 ′ and 3 ′ ends of the nucleic acid).

These and other aspects of the invention are described in detail below.
I. Isolation of nucleic acids from cell lines Suitable cells used to isolate nucleic acids encoding peptides having novel B lymphocyte activity include B lymphocyte antigens (eg, B7-1, B7-2, B7- A cell capable of producing an mRNA encoding 3) and capable of almost translating the mRNA into the corresponding protein. One source of mRNA is normal human splenic B cells from a subset of quiescent or activated by anti-immunoglobulin antibodies or anti-MHC class II antibodies or from neoplastic B cells. Human B7-2 antigen expression is detectable in resting B cells and in activated B cells, and mRNA levels increase four-fold from resting levels after stimulation. Total cellular RNA can be obtained from B cells activated at rest or during these intervals using standard techniques and can be used to construct a cDNA library.

In addition, various subsets of neoplastic B cells express B7-2 and B7-3, which serve instead as a source of mRNA for cDNA library construction. For example, tumor cells isolated from patients with non-Hodgkins lymphoma express B7-1 mRNA. B cells from nodular, less differentiated lymphoma (NPDL) diffuse large cell lymphoma (LCL) and Burkitt lymphoma cell lines are also human B7-1 mRNA, and possibly B7-2 and B7 A suitable source of -3 mRNA. Myeloma generally expresses B7-2 but does not express B7-1 mRNA, thus providing a source of B7-2 mRNA. The Burkitt lymphoma cell line Raji is one source of B lymphocyte antigen mRNA. Preferably B7-2 mRNA is obtained from both populations of quiescent and activated normal human B cells. Activated B cells are obtained by stimulation over a wide range of times (eg minutes to days) using, for example, anti-immunoglobulin antibodies, or anti-MCH class II antibodies.
II. Isolation of mRNA and construction of cDNA library

  Total cellular mRNA can be isolated by various techniques, for example, using the guanidinium-thiocyanate extraction method of Chirgwin et al., Biochemistry, 18, 5294-5299 (1979). According to this method, Poly (A +) mRNA is prepared and purified for use in cDNA library construction using oligo (dT) cellulose selection. cDNA is then synthesized from poly (A +) RNA using oligo (dT) priming and reverse transcriptase. Moloney MLV reverse transcriptase (available from Gibco / BRL, Bethesda, Maryland) or AMV reverse transcriptase (available from Seikagaku America, Inc., St. Petersburg, Florida) is preferably used.

  After reverse transcription, the mRNA / DNA hybrid molecule is converted to double stranded DNA using conventional techniques and introduced into a suitable vector. The experiment here is E. coli for conversion to double stranded cDNA. E. coli DNA polymerase I and ribonuclease H are used.

  cDNA cloning can be performed using any of the conventional techniques for joining double-stranded DNA to an appropriate vector. It is particularly preferred to use a synthetic adapter. This is because it mitigates the possibility of cleaving the cDNA by restriction enzymes before cloning. Using this method, non-self-complementary, kinased adapters are added to the DNA prior to ligation with the vector. Virtually any adapter can be used. As shown in more detail in the examples below, non-self-complementary BstXI adapters are preferably added to the cDNA for cloning for ligation into the pCDM8 vector prepared for cloning by BstXI digestion.

A eukaryotic cDNA is placed in sense orientation in a vector that provides the appropriate eukaryotic promoter, origin of replication, and enhancer, splice acceptor and / or donor sequence, and other elements including a polyadenylation signal. Can be expressed. The cDNA of the present invention is transformed into prokaryotic promoter, E. coli. Placed in a suitable vector with an origin of replication that functions in E. coli, an SV40 origin of replication that allows growth in COS cells, and a cDNA insertion site. Suitable vectors include πH3 (Seed and Aruffo, Proc. Natl. Acad. Sci., 84, 3365-3369 (1987)), πH3m (Aruffo and Seed, Proc. Natl. Acad. Sci., 84, 8573-8777. (1987)), pCDM7 and pCDM8 (See, Nature, 329, 840-841 (1987)), with the pCDM8 vector being particularly preferred (available from Invitrogen, San Diego, Calif.).
III. Transfection of host cells and screening for novel B lymphocyte activating antigens

  Using the cDNA library thus prepared, the gene in question is cloned by an expression cloning technique. The basic expression cloning technique is described in See and Aruffo, Proc. Natl. Acad. Sci. USA, 84, 3365-3369 (1987), and Aruffo and Seed, Proc. Natl. Acad. Sci. USA, 84, 8573-8777 (1987), although modifications of this technique may be necessary.

According to one embodiment, plasmid DNA is introduced into a monkey COS cell line (Gluzman, Cell), 23, 175 (1981)) using known transfection techniques (eg DEAE-dextran) and the cDNA insert is Replicate and express. Transfectants expressing the B7-1 antigen are depleted with immunomagnetic beads coated with anti-B7-1 monoclonal antibodies (eg 133 and B1.1) and anti-Muulin IgG and IgM. Transfectants expressing the human B7-2 antigen can be positively selected by reacting the transfectants with the fusion proteins CTLA4Ig and CD28Ig and then panning on plates coated with anti-human Ig antibodies. Human CTLA4Ig and CD28Ig fusion proteins were used, but if there is cross speciles reactivity between B7-1 and eg mouse B7-1, other fusions reactive with other cross-reactive species It can be expected that proteins could also be used. After panning, episomal DNA is recovered from the panned cells and competent bacterial host, preferably E. coli. coli. Transform to The plasmid DNA is then reintroduced into the COS cells and the expression and panning cycle is repeated at least twice. After the final cycle, plasmid DNA is prepared from individual colonies, transfected into COS cells and analyzed for expression of the novel B lymphocyte antigen by indirect immunofluorescence using, for example, CTLA4Ig and CD28Ig.
IV. Sequencing of a novel B lymphocyte antigen

  Plasmids are prepared from these colonies that are highly reactive with CDLA4Ig and CD28Ig. These plasmids are then sequenced. Any conventional sequencing technique suitable for sequencing tracts of DNA greater than about 1.0 kb can be used.

As described in Example 4, a human B7-2 clone (clone 29) having a single long open reading frame of 987 nucleotides and a 1,120 base pair insert with about 27 nucleotides of 3 ′ non-coding sequence is Obtained (FIG. 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 FIG. The encoded 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 predicted to begin with a methionine codon (ATG, nucleotides 107-109) based on DNA homology in the region with the consensus eukaryotic translation initiation site (Kozak, M. (1987), Nucl. Acids Res. 15, 8125-8148). The amino terminus (amino acids 1-23) of the B7-2 protein has the characteristics of a secretory signal peptide due to the expected cleavage between alanines at positions 23 and 24 (von Heijne (1987), Nucl. Acids Res., 14 4683). Processing at this site yields a 306 amino acid B7-2 membrane-bound protein with an unmodified molecular weight of approximately 34 kDa. This protein consists of an approximately extracellular Ig superfamily V and C-like domain consisting of about 24-245 amino acid residues, a hydrophobic transmembrane domain consisting of about 246-268 amino acid residues, and about 269-329 amino acid residues. Consisting of a long cytoplasmic domain. The homology to the Ig superfamily is due to two adjacent Ig-like domains in the extracellular region joined by cysteines at positions 40-110 and 157-218. The extracellular domain also has eight potential N-linked glycosylation sites and is probably glycosylated similar to B7-1. Glycosylation of human B7-2 protein can increase the molecular weight to about 50-70 kDa. The cytoplasmic domain of human B7-2 is somewhat longer than B7-1, but generally consists of a number of cysteines that act as signaling or regulatory domains within the antigen presenting cell (APC), followed by positively charged amino acids Include various areas. Comparison of both the nucleotide and amino sequences of human B7-2 with the GenBank and EMBL databases showed considerable homology with human B7-1 (approximately 26% amino sequence identity). Since human B7-1, human B7-2 and murin B7-1 all bind to human CTLA4 and CD28, homologous amino acids probably indicate the amino acids necessary to contain a CTLA4 or CD28 binding sequence. E. coli transfected with a vector having a cDNA insert encoding human B7-2. coli (clone 29) was deposited with the American Type Culture Collection (ATCC) on July 26, 1993 under accession number 69357.
V. Cloning of novel B lymphocyte antigens from other mammalian species

  The present invention is not limited to human nucleic acid molecules, and novel B lymphocyte antigen homologues from other mammalian species that express B lymphocyte antigens can be cloned and sequenced using the techniques described below. Intended. B lymphocyte antigens isolated for one species (eg, human) that exhibit cross-species reactivity can be used to modify T cell-mediated immune responses in other species (eg, mice). Isolation of cDNA clones from other species can also be performed using a human cDNA insert such as human B7-2 cDNA as a hybridization probe.

  As shown in Example 6, a Mullin B7-2 clone containing a single long open reading frame consisting of 927 nucleotides and an insert consisting of 1,163 base pairs with about 126 nucleotides of 3 ′ non-coding sequence ( mB7-2, clone 4) was obtained (FIG. 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 FIG. The encoded myulin B7-2 protein is predicted to be 309 amino acid residues in length (SEQ ID NO: 23). This protein has many features in common with other type I Ig superfamily membrane proteins. Protein translation is expected to begin at the methionine codon (ATG, nucleotides 111-113) based on DNA homology in the region with the consensus eukaryotic translation initiation site (Kozak, M (1987), Nucl. Acids Res. 15, 8125-8148). The amino terminus (amino acids 1-23) of the murine B7-2 protein is characterized by a secretory signal peptide with the expected cleavage between alanine at position 23 and valine at position 24 (von Heijne (1987) Nucl. Acids.Res., 14, 4683). Processing at this site will result in a 286 amino acid murin B7-2 membrane-bound protein with an unmodified molecular weight of approximately 32 kDa. This protein comprises an approximately extracellular Ig superfamily V and C-like region of amino acid residues about 24-246, a hydrophobic transmembrane domain of amino acid residues 247-265, and a long cytoplasmic domain of amino acid residues 266-309 Will be. The homology to the Ig superfamily is due to two adjacent Ig-like regions in the extracellular region joined by cysteines at positions 40-110 and 157-216. The extracellular domain also contains nine potential N-linked glycosylation regions and, like murin B7-1, is probably glycosylated. Glycosylation of the Murine B7-2 protein can increase the molecular weight up to about 50-70 kD. The cytoplasmic domain of Murine B7-2 contains a general region with positively charged amino acids following a cysteine that probably functions as a signal transduction or control domain within APC. Both the nucleotide and amino acid sequence of Murin B7-2 shows considerable homology (approximately 26% amino acid sequence identity) with human and Murin B7-1 when compared to the GenBank and EMBL databases. Murine B7-2 shows about 50% identity and 67% similarity to its human homologue, hB7-2. E. coli transfected with a vector (plasmid pmBx4) containing a cDNA insert encoding murine B7-2. E. coli (DH106 / P3) (clone 6) was deposited with the American Type Culture Collection (ATCC) on August 18, 1993 as accession number 69388.

  Nucleic acids encoding novel B lymphocyte antigens from other species, such as Mullin B7-2, can be used to create transgenic animals, or “knockout” animals, which are useful for therapeutically useful drugs. Useful for development and screening. A transgenic animal (eg, a mouse) is an animal that has cells that carry the transgene, and the transgene is introduced into the animal or the predecessor of a prenatal, eg, embryonic, animal. A transgene is DNA that is then bound to the genome of a cell from which a transgenic animal develops. In one embodiment, a transgenic animal having cells that express the B7-2 protein using the genomic sequence cloned from genomic B7-2 by established techniques using the murine B7-2 cDNA or appropriate sequence thereof. Can be produced. Methods for creating transgenic animals, particularly animals such as mice, have become common in the art and are described, for example, in US Pat. Nos. 4,736,866 and 4,870,009. Yes. Typically, special cells could be targeted for B7-2 transgene introduction using tissue specific enhancers, resulting in T cell costimulation and increased T cell proliferation and autoimmunity . Transgenic animals containing a copy of the B7-2 transgene introduced into the germ line of animals at the embryonic stage can be used to examine the effect of increased B7 expression. Such animals can be used, for example, as tester animals for drugs thought to confer protection from autoimmune diseases. In accordance with this aspect of the invention, a reduction in the incidence of a disease compared to an untreated animal that has been treated with a drug and has a transgene will indicate the potential for therapeutic involvement in that disease.

Alternatively, B7-2 having a defect or change in the B7-2 gene as a result of homologous recombination between the endogenous B7-2 gene and the altered B7-2 genomic DNA introduced into the embryonic cells of the animal A non-human homologue of B7-2 can be used to create “knockout” animals. For example, murine B7-2 cDNA can be used to clone genome B7-2 according to established techniques. A portion of the genomic B7-2 DNA (such as an exon encoding the extracellular domain) can be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5 'and 3' ends) are included in the vector (eg, Thomas, KR and Capecchi, MR (1987), (See description of homologous recombination vectors in Cell, 51, 503). The vector is introduced into an embryonic stem cell line (e.g., by electroporation), and cells in which the introduced DNA is homologously recombined with endogenous DNA (e.g., Li, E et al. (1992)) Cell, 69, 915). Selected cells are then injected into blastocysts of animals (eg, mice) to form aggregation chimeras (eg, Braley, Teratocarcinomas and Embryonic Stem Cell: A Practical Approach, E. J. Robertson (IRL, Oxford, 1987) 113-152). The chimeric embryo is transferred to a suitable pseudopregnant female Foster animal, and the embryo is folded to create a “knockout animal”. Progeny having DNA homologously recombined in those embryonic cells can be identified by standard techniques and can be used to breed animals that have DNA homologously recombined in all cells of the animal. Knockout animals can be characterized for their ability to accept grafts, reject tumors, and protect against infectious diseases and can be used for basic immunobiological studies.
VI. Expression of B lymphocyte antigen

  Host cells transfected to express a peptide having the activity of a novel B lymphocyte antigen are also within the scope of the present invention. The host cell can be a prokaryotic cell or a eukaryotic cell. For example, a peptide having B7-2 activity is obtained from E. It can be expressed in mammals such as bacterial cells such as E. coli, insect cells (baculovirus), yeast, Chinese Ovalley cells (CHO) and NSO cells. Other suitable host cells are found in Goeddel (1990), supra, and are known to those skilled in the art.

For example, expression in eukaryotic cells such as mammalian, yeast, or insect cells can result in partial or complete glycosylation of the recombinant protein and / or formation of related interchain or intrachain disulfide bonds. . Yeast S. Examples of vectors for expression in cerivisae are pYepSecl (Baldari et al. (1987), Embo J., 6, 229-234), pMFa (Kurjan and Herskowitz (1982), Cell, 30, 933-943), pJRY88 (Schultz). Et al. (1987), Gene, 54, 113-123) and pYES2 (Invitrogen Corporation, San Diego, California). Baculovirus vectors that can be used for protein expression in cultured insect cells (SF9 cells) include the pAc series (Smith et al. (1983), Mol. Cell Biol. 3, 2156-2165) and the pVL series (Lucklow, V. et al. A. and Summers, MD (1989), Virology, 170, 31-39). In general, COS cells (Gluzman, Y. (1981), Cell, 23, 175-182) are transformed into vectors such as pCDM8 for transient amplification / expression in mammals (See, B. (1987), Nature, 329, 840), CHO (dhfr ^- Chinese hamster berry) cells are used for vectors such as pMT2PC (Kaufman et al. (1987), EMBO J., 6, 187) for stable amplification / expression in mammalian cells. ~ 195). A preferred cell line for the production of recombinant proteins is available from ECACC (Catalog # 85110503) and is described by Galfre, G. et al. And Milstein, C (1981), Methods in Enzymology, 73 (13), 3-46, and Preparation of Monoclonal Antibodies: Strategies and Procedure, Academic Press, New York. Vector DNA can be introduced into mammalian cells by conventional techniques such as calcium phosphate or calcium chloride coprecipitation, 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, Volume 2, 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, cytomegaloyl, and most often simian virus 40.

A small portion of cells (about 1/10 ⁵ ) typically integrates DNA into their genome. In order to identify these integrants, a gene containing a selectable marker (ie, resistance to antibiotics) is generally introduced into the host cell along with the gene of interest. Preferred selectable markers include those that confer resistance to drugs such as G418, hygromycin and methotrexate. The selectable marker may be introduced on the same plasmid as the gene of interest or on a different plasmid. Cells with the gene of interest can be identified by drug selection. That is, cells into which a selectable marker gene has been introduced survive and other cells die. Surviving cells can then be screened for production of new B lymphocyte antigens by cell surface staining with ligands for B cell antigens (eg CTLA4Ig and CD28Ig). Alternatively, the protein can be metabolically labeled with a labeled amino acid and immunoprecipitated from the cell supernatant with an anti-B lymphocyte antigen monoclonal antibody or a fusion protein such as CTLA4Ig or CD28Ig.
Expression in prokaryotes is a vector that contains a vector containing a constitutive or inducible promoter that directs the expression of a fusion or non-fusion protein. Most often done in coli. A fusion vector adds many amino acids to the normal amino terminus of the target gene to be expressed. Such fusion vectors 1) increase the expression of the recombinant protein; 2) increase the solubility of the target recombinant protein, and 3) purify the target recombinant protein by acting as a ligand in affinity purification. Typically serves the three purposes of helping. Often in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the target recombinant protein, allowing the target recombinant protein to be separated from the fusion moiety after purification of the fusion protein. Such enzymes, and their homologous recognition sequences, include Factor Xa, thrombin, and enterokase. A typical fusion expression vector includes a target recombinant protein, glutathione S-transferase, maltose E-binding tamper.
Or pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, Mass.), And pRIT5 (Pharmacia, Piscataway, NJ).

  E. coli. Expression systems include inducible expression vectors pTrc (Amann et al. (1988), Gene, 69, 301-315) and pET11 (Studier et al., Gene Expression Technology: Methods in Enzymology, 185, Academic Press, San Diego, Calif. (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. Following induction, the recombinant protein can be purified from the periplasmic fraction. In the pET11 vector system, the target gene is expressed as a non-fusion protein by transcription from a T7gn10-lac 0 fusion promoter (T7gnl) mediated by a co-expressed viral RNA polymerase. This viral polymerase is derived from a resident λ prophage harboring T7 gnl under the transcriptional control of the lacUV5 promoter. Supplied by E. coli strains BL21 (DE3) and HMS174 (DE3). In this system, the recombinant protein can be purified from denatured inclusion bodies and, if desired, reconstituted by step gradient dialysis to remove denatured products.

  E. One strategy to maximize recombinant B7-2 expression in E. coli is to express the protein in a host bacterium that has an impaired ability to cleave the recombinant protein more proteolytically (Gottesman, S., et al. Gene Expression Technology; Methods in Enzymology, 185, Academic Press, San Diego, California (1990), 119-128). Another strategy is to replace the nucleic acid sequence of the B7-2 gene or other DNA to be inserted into the expression vector with E. coli where the individual codons of each amino acid are highly expressed. will be changed to be preferentially used in E. coli proteins (Wada et al. (1992), Nuc. Acids Res., 20, 2111-11118). Such alteration of the nucleic acid sequences of the present invention can be done by standard DNA synthesis techniques.

Novel B lymphocyte antigens and portions thereof expressed in mammalian cells or otherwise are expressed in ammonium sulfate precipitation, fractional column chromatography (eg, ion exchange, gel filtration, electrophoresis, affinity chromatography, etc.), and ultimately Including crystallization. It can be purified according to standard methods in the art (see generally "Enzyme Purification and Related Technologies", Methods in Enzymology, 22, 233-577 (1971)). Once purified to partial or homogeneous, the recombinantly produced B lymphocyte antigen or portion thereof can be used in a composition suitable for pharmaceutical administration as described in detail below.
VII. Modification of nucleic acid and amino acid sequences of the present invention and analysis of B7 lymphocyte antigen activity

  One skilled in the art will recognize that other nucleic acids encoding peptides having novel B lymphocyte antigen activity can be isolated by the above methods. It can be expected that different cell lines will produce DNA molecules having different base sequences. Furthermore, variants may exist due to genetic polymorphisms and cell-mediated modifications of genetic material. Furthermore, the DNA sequence of the B lymphocyte antigen can be modified using genetic techniques to produce a protein or peptide having an altered amino acid sequence. Such sequences are considered to be within the scope of the present invention, and the expressed peptides can induce or inhibit activated T cell mediated immune responses and immune functions.

  Many methods can be used to make an equivalent or fragment of an isolated DNA sequence. Small subregions or fragments of nucleic acids encoding the B7-2 protein, such as 1-30 bases in length, can be prepared by standard synthetic organic chemistry means. The technique is also useful for the preparation of antisense oligonucleotides and primers used to make larger synthetic fragments of B7-2 DNA.

  Larger subregions or fragments of the gene encoding the B lymphocyte antigen are related using the polymerase chain reaction (PCR) (Sambrook, Fritsh and Maniatis, Molecular Cloning; A Laboratory Manual, Cold Spring Harbor, New York (1989)). By synthesizing a piece of DNA to be synthesized and ligating the DNA thus obtained to an appropriate expression vector, it can be expressed as a peptide. Using PCR, a special sequence of cloned double-stranded DNA is created, cloned into an expression vector, and then analyzed for CTLA4 / CD28 binding activity. For example, to express a secreted (soluble) form of human B7-2 protein, PCR can be used to synthesize DNA that does not encode the transmembrane and cytoplasmic regions of the protein. This DNA molecule can be ligated into an appropriate expression vector and introduced into a host cell such as CHO, where a B7-2 protein fragment is synthesized and secreted. The B7-2 protein fragment can be easily obtained from the culture medium.

  In other embodiments, the DNA in any of a number of ways, including simple deletions or insertions, systematic deletions of base clusters, insertions or substitutions, or methods of making single-base substitutions Mutations can be introduced into the B lymphocyte antigen DNA variants or modified equivalents. For example, changes such as amino acid substitution or deletion in the human B7-2 cDNA sequence shown in FIG. 8 (SEQ ID NO: 1) and the murine B7-2 cDNA sequence shown in FIG. 14 (SEQ ID NO: 2) are preferably caused by site-directed mutagenesis. can get. Site-directed mutagenesis is well known in the art. Protocols and reagents are obtained commercially from Amersham International PLC, Amersham, UK.

Binds the natural ligand of the B lymphocyte antigen on the T cell, as evidenced by the activity of the novel B lymphocyte antigen, ie, cytokine production and / or T cell proliferation by, for example, T cells that have received the primary activation signal Thus, peptides having the ability to stimulate (amplify) or inhibit (block) an activated T cell-mediated immune response are considered within the scope of the present invention. More specifically, peptides that bind to T lymphocytes, such as CD28 ⁺ , can carry a costimulatory signal to the T lymphocyte, which sends antigen and class II MHC, or primary signals to T cells. When transmitted in the presence of other substances that can be transmitted, activation of cytokine genes within the T cell results. Alternatively, such peptides can be used with class I MHC, thereby activating CD8 ⁺ cytolytic T cells. Furthermore, the soluble, monomeric form of B7-2 protein retains the ability to bind to its natural ligand on CD28 ⁺ T cells, but presumably reduces cytokine production and cell division due to insufficient crosslinking with the ligand. Inability to transmit secondary signals essential for enhancement. Such peptides that provide a means of inducing anergy or tolerance in cells are also considered to be within the scope of the present invention.

  Peptide screening for those that retain the characteristic B lymphocyte antigen activity described herein can be performed using one or more of several different assays. For example, the peptides can be screened for specific reactivity with cell surface B7-2, or an anti-7-2 monoclonal antibody reactive with a fusion protein such as CTLA4Ig or CD28Ig. Specifically, appropriate cells such as COS cells are transfected with B7-2 DNA encoding the peptide and then analyzed for cell surface phenotype by indirect immunofluorescence and flow cytometry, and the peptide is B7- Determine if you have 2 activities. Cell surface expression of transfected cells is assessed using monoclonal antibodies that react specifically with cell surface B7-2 or CTLA4Ig or CD28Ig proteins. Production of the secreted form of B7-2 is assessed using an anti-B7-2 monoclonal antibody or CTLA4Ig or CD28 fusion protein for immunoprecipitation.

Another more preferred analysis takes advantage of the functional characteristics of the B7-2 antigen. As explained above, the ability of T cells to synthesize cytokines is not only due to the occupancy or cross-linking of T cell receptors for antigens (eg by anti-CD3 or phorbol esters to create “activated T cells”). "Primary activation signal" provided, in this case also by induction of costimulation by interaction with B lymphocyte antigens such as B7-2, B7-1 or B7-3. For example CD28 ⁺ T The binding of B7-2 to its natural ligand on the cell induces an increased level of production of cytokines, particularly interleukin-2, which in turn stimulates T lymphocyte proliferation, a signal to T cells. Other analyzes of B7-2 function include interleukin-2, interleukin-4, or other known or unknown new services. Analysis of synthesis of cycaines such as icaine and / or analysis of T cell proliferation by CD28 ⁺ T cells that have received a primary activation signal.

  In vivo, T cells are given a primary or primary activation signal by an antigen conjugated to an anti-T3 monoclonal antibody (eg, anti-CD3) or phorbol ester, more preferably a class II MHC. A T cell that has received a primary activation signal is referred to herein as an activated T cell. B7-2 function is the primary activation of T cell culture, such as antigen bound to a B7-2 source (eg, a cell expressing a peptide having B7-2 activity, or a secreted form of B7-2) and a class II MHC. Signals are added and the culture supernatant is analyzed by analyzing for interleukin-2, γ-interferon or other known or unknown cytokines. For example, some conventional analysis methods for interleukin-2, such as Proc. Natl. Acad. Sci. USA, 86, 1333 (1989), any of which can be used (the relevant part of which is part of this specification). Interferon production analysis kits are also available from Genzyme Corporation (Cambridge, Mass.). T cell proliferation can also be measured as described in the examples below. Peptides that retain the characteristics of the B7-2 antigen described herein result in increased per cell production of cytokines such as IL-2 by T cells, compared to negative controls lacking a costimulatory signal. It results in increased T cell proliferation.

  The same basic functional analysis can also be used to screen for peptides that have B7-2 activity but lack the ability to carry a costimulatory signal, but for such peptides, the addition of B7-2 protein is either proliferating or There will be no significant increase in cytokine secretion by T cells. The ability of these proteins to inhibit or completely block normal B7-2 costimulatory signals and induce an anergic state is the ability of T cells to express cell surface B7-2 and to present antigen presenting T cells. It can be measured using subsequent attempts to stimulate. If the T cells are unresponsive to subsequent activation attempts, as measured by IL-2 synthesis and T cell proliferation, an anergic state has been induced. For the analysis system used as the basis of the analysis according to the present invention, see Gimmi, C. et al. D. Et al. (1993) Proc. Natl. Acad. Sci. USA, 90, 6586-6590.

  Novel B-lymph for purposes such as increased solubility, increased therapeutic or prophylactic effects, or increased stability (eg, resistance to degradation by proteolytic enzymes in vivo and ex vivo storage periods) Modification of the structure of a peptide having the activity of a sphere antigen is also possible. Such modified peptides are considered to be functional equivalents of the B lymphocyte antigen as defined herein. For example, a peptide having B7-2 activity can be modified to maintain its ability to stimulate T cell proliferation and / or produce cytokines. These residues, which have been shown to be essential for interacting with the CTLA4 / CD28 receptor on T cells, do not lose their essential amino acids, whether their presence increases and decreases receptor interaction. Or can be modified by substitution with other preferably similar amino acid residues that do not affect (conservative substitution). Furthermore, those amino acid residues that are not essential for receptor interaction may be modified by substitution with other amino acids whose introduction increases, decreases or does not affect the reactivity.

  Another example of modification of a peptide having activity of a novel B lymphocyte antigen is to dimerize by disulfide linkage by replacing cysteine residues, preferably with alanine, serine, threonine, leucine, or glutamic acid residues. It is to minimize. Furthermore, the amino acid side chain of the peptide having B7-2 activity can be chemically modified. Another modification is cyclization of the peptide.

  In order to increase stability and / or reactivity, peptides having B7-2 activity can be modified to introduce one or more polymorphisms of the antigen's amino acid sequence resulting from any natural allelic variation. Furthermore, it can be substituted with a D-amino acid, an unnatural amino acid, or a non-amino acid analog, and added to produce a modified protein within the scope of the present invention. In addition, the peptides Modifications can be made using polyethylene glycol (PEG) according to Sehon and coworkers' method (Wie et al., Supra) to make peptides conjugated with PEG. Furthermore, PEG can be added during chemical synthesis of peptides. Other modifications of the peptide include reduction / alkylation (Tarr, Methods of Protein Microcharacterzation, edited by JE Silver, Human Press, Clifton, New Jersey, 155-1994 (1986)), acylation (Tarr, supra), Chemical coupling to a suitable carrier (Edited by Michel and Shiigi, Selected Methods in Cellular lmmunology, WH Freeman, San Francisco, California (1980), US Pat. No. 4,939,239), or weak formalin treatment (Marsh (1971)) Int. Arch. Of Allergy and Appl. Immunol., 41, 199-215).

It is possible to add amino acid fusion moieties to the protein bone nucleus to facilitate peptide purification and possibly increase solubility. For example, hexa-histidine can be added to peptides for purification by immobilized metal ion affinity chromatography (Hochuli, E et al. (1988), Bio / Technology, 6, 1321-1325). In addition, a specific endoprotease cleavage site can be introduced between the fusion moiety and the peptide sequence to facilitate isolation of B lymphocyte antigens that do not contain unrelated sequences. It may be necessary to increase the solubility of the peptide by adding functional groups to the peptide or by omitting the hydrophobic region of the peptide.
VII. Use of nucleic acid sequences encoding B lymphocyte antigens and peptides having B7-2 activity.

A. Molecular probe.
The nucleic acids according to the present invention track disease progression by measuring the activation state of B lymphocytes in a biological sample, or assay the effect of molecules on the expression of B lymphocyte antigens (eg, cells Is useful for diagnosis). In accordance with these diagnostic assays, the nucleic acid sequence is labeled with a detectable marker, such as a radioactive, fluorescent, or biotinylated marker, and biological using a conventional dot blot or Northern hybridization method. Probing total or poly (A +) RNA mRNA molecules from the sample.

B. Antibody production.
The peptides and fusion proteins produced from the nucleic acid molecules according to the present invention can also be used for the production of antibodies that react specifically 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, an anti-protein / anti-peptide polyclonal antiserum or a monoclonal antibody can be obtained using standard methods. Can be produced. A mammal (eg, a mouse, hamster, or rabbit) can be immunized with an immunogenic protein or peptide that elicits an antibody response in the mammal. The immunogen may be, for example, a recombinant B7-2 protein, or a fragment thereof, a synthetic peptide fragment, or a cell that expresses a B lymphocyte antigen on its surface. This cell was transfected with, for example, a splenic B cell or a nucleic acid encoding a B lymphocyte antigen according to the present invention (eg B7-2 cDNA) such that the B lymphocyte antigen is expressed on the cell surface. It can be a cell. The immunogen can be modified to increase its immunogenicity. For example, techniques for conferring immunogenicity to a peptide include conjugation to a carrier or other techniques well known in the art. For example, the peptide can be administered in the presence of an adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as an antigen to assess antibody levels.

  After immunization, antisera can be obtained and polyclonal antibodies can be isolated from this serum if desired. To produce monoclonal antibodies, antibody-producing cells (lymphocytes) are collected from the immunized animal and fused with myeloma cells by standard somatic cell fusion techniques to immortalize these cells and produce hybridoma cells. Can do. Such techniques are well known in the art. For example, the hybridoma technology originally developed by Kohler and Milstein (Nature (1975), 256, 495-497), and the human B cell hybridoma technology (Cosbar et al., Immunol. Today (1983), Vol. 72, p. 72). , EBV-hybridoma technology for generating human monoclonal antibodies (Cole et al., Monoclonal Antibodies in Cancer Therapy (1985) (Allen R. Bliss, Inc., pp. 77-96)) and combination antibody live Larry's screening (Hughes et al., Science (1989), 246, 1275) Hybridoma cells were screened immunochemically for the production of antibodies that specifically react with this peptide, and monoclonal It can be separated from the body.

The term antibody as used herein is intended to encompass peptides having the activity of the novel B lymphocyte antigens described herein or fragments thereof that specifically react with a fusion protein. Yes. Antibodies are fragmented using conventional techniques, and the fragments can be screened for use in the same manner as described above for whole antibodies. For example, F (ab ′) ₂ fragments can be generated by treating antibody with pepsin. The resulting F (ab ′) ₂ fragment can be treated to reduce disulfide bridges to produce Fab ′ fragments. The antibodies according to the invention are further intended to include bispecific and chimeric molecules having anti-B lymphocyte antigen (ie B7-2, B7-3) moieties.

  Particularly preferred antibodies are anti-human B7-2 monoclonal antibodies produced by hybridomas HA3.1F9, HA5.2B7 and HF2.3D1. The production and characterization of these antibodies is described in detail in Example 8. Monoclonal antibody HA3.1F9 was determined to be of the IgG isotype; monoclonal antibody HA5.2B7 was determined to be of the IgG2b isotype; monoclonal antibody HF2.3D1 was determined to be of the IgG2a isotype. The hybridoma cells were identified as ATCC Acceptance Number (Hybridoma HA3.1F9), ATCC Acceptance Number (HA5.2B7) and ATCC Acceptance Number (HF2.3D1) in the American Type Culture Collection that meets the requirements of the Budapest Treaty. Deposited on May 19th.

  When antibodies produced in subjects other than humans are used therapeutically by humans, they are perceived as extraneous to some extent and an immune response can occur in the patient. One approach over systemic immunosuppression that minimizes or eliminates this problem is to produce chimeric antibody derivatives, ie, antibody molecules that combine a variable region of a non-human animal with a human constant region. A chimeric antibody molecule can include, for example, an antigen binding domain from a mouse, rat, or other species of antibody with a human constant region. Various approaches for making chimeric antibodies have been described and can be used to make chimeric antibodies comprising immunoglobulin variable regions that recognize the gene product of the novel B lymphocyte antigen according to the present invention. For example, Morrison et al., Proc. Natl. Acad. Sci. U. S. A. 81, 6851 (1985); Takeda et al., Nature, 314, 452 (1985), Cavily et al., U.S. Pat. No. 4,816,567; Boss, et al., U.S. Pat. No. 4,816,397; See publication 0173494, British patent GB2177096B. Such chimeric antibodies are expected to be less immunogenic in human subjects than the corresponding non-chimeric antibodies.

  For human therapeutic purposes, further humanize monoclonal or chimeric antibodies that react specifically with peptides having the activity of the B lymphocyte antigen described herein by generating human variable region chimeras. However, in this case, part of the variable region, in particular the conserved framework region of the antigen binding domain, is of human origin and only the hypervariable region is of non-human origin. A review of “humanized” chimeric antibodies can be found in L. Morrison (1985), Science, 229, 1202-1207 and Oy et al. (1986), BioTechniques, 4, 214. Such altered immunoglobulin molecules may be any of several techniques known in the art (e.g., Teng et al., Proc. Natl. Acad. Sci. USA, 80, 7308-7312). (1983); Kozubo et al., Immunology Today, 4 pages 7279 (1983); Olson et al., Meth. Enzymol. Alternatively, it is created according to the teaching of EP0239400. Humanized antibodies can be commercially produced, for example, by Scottgen Limited, 2 Holy Road, Twickenham, Middlesex, UK. Suitable “humanized” antibodies can alternatively be generated by CDR or CEA substitution (US Pat. No. 5,225,539 to Winter; Jones et al. (1986), Nature 321 552-525; Verhoyan et al. (1988), Science, 239, 1534; and Baidler et al. (1988), J. Immunol. 141, 4053-4060). Humanized antibodies with reduced immunogenicity are preferred for immunotherapy in human subjects. Immunotherapy with humanized antibodies appears to reduce the need for concomitant immunosuppression and results in increased long-term efficacy for treatment of chronic disease situations or situations where antibody treatment needs to be repeated Can do.

  Instead of humanizing monoclonal antibodies from mice or other species, human monoclonal antibodies against human proteins can be generated. Transgenic mice with a human antibody repertoire have been created that can be immunized with human B lymphocyte antigens such as B7-2. The spleen cells of these immunized transgenic mice can then be used to create hybridomas that secrete human monoclonal antibodies that specifically react with human B lymphocyte antigens (eg, Wood et al., PCT Publication WO 91 / 00906, Kucharapati et al., PCT publication WO91 / 10741; Ronberg et al., PCT publication WO92 / 03918; Kei et al., PCT publication 92/03917; N. Ronberg et al. (1994), Nature, 368: 856-859; L. Green et al. (1994), Nature Genet., 7: 13-21; SL Morrison et al. (1994), Proc. Natl. Acad. Sci. USA, 81: 6851-6855; 1993), Year Immunol., 7: 33-40; see Tuiron et al. (1993), PNAS, 90: 3720-3724; and Bradgeman et al. (1991), Eur. J. Immunol., 21: 1323-1326).

  The monoclonal antibody composition according to the present invention can also be produced by other methods well known to those having ordinary knowledge in the field of recombinant DNA technology. Another method, referred to as the “combined antibody display” method, was developed to identify and isolate antibody fragments having specific antigen specificity, which can be used to generate monoclonal antibodies that bind the B lymphocyte antigens of the present invention. (For example, see Sustry et al. (1989), PNAS, 86, 5728; Hughes et al. (1989), Science, 246, 1275; and Aurandy et al. (1989). PNAS, 86, 3833). After immunizing the animal with B lymphocyte antigen, the antibody repertoire of the resulting B cell pool is cloned. Methods are generally known for directly obtaining the variable region DNA sequences of diverse populations of immunoglobulin molecules by using a mixture of oligomer primers and PCR. For example, a mixed oligonucleotide primer corresponding to a 5 ′ leader (signal peptide) sequence and / or a framework 1 (FR1) sequence, and a primer for a conserved 3 ′ constant region primer can be used as a heavy chain from several mouse antibodies. And can be used for PCR amplification of the light chain variable region (Larlic et al. (1991), Biotechniques, 11: 152-156). Similar strategies can be used to amplify human heavy and light chain variable regions derived from human antibodies (Larrick et al. (1991) Methods: Methods to Methods in Enzymology, Vols. 106-110).

  In exemplary embodiments, RNA is isolated using standard protocols, eg, from activated B cells of peripheral blood cells, bone marrow, or spleen samples (eg, US Pat. No. 4,683,202; Aurandi et al., PNAS (1989)). 86, 3833-3837; Sustry et al., PNAS (1989), 86, 5728-5732; and Hughes et al. (1989), Science, 246, 1275-1281). The first cDNA strand is synthesized using primers specific for the constant region of the heavy chain and each of the kappa and lambda light chains and a signal sequence. Using variable region PCR primers, the variable regions of both heavy and light chains are amplified alone or in combination and ligated into an appropriate vector for further manipulation in the creation of a display package. Oligonucleotide primers useful for amplification protocols can be unique, degenerate, or incorporate inosine at a degenerate position. A restriction endonuclease recognition sequence can also be incorporated into this primer so that the amplified fragment can be cloned into an open reading frame for predetermined expression in the vector.

  V gene libraries cloned from immunization-derived antibody repertoires can preferably be expressed by populations of display packages derived from filamentous phage to form antibody display libraries. Ideally, this display package is a system that allows for the sampling of a very large variety of antibody display libraries, rapid classification after each affinity separation step, and easy isolation of antibody genes from purified display packages. including. Available commercially available kits for the creation of phage display libraries (eg, Pharmacia's recombinant phage antibody system, catalog number 27-9400-01; and Stratagene's SurfZAP ™ phage display kit, catalog number 240612) In addition, examples of methods and reagents that are particularly amenable to use in generating diverse antibody display libraries include, for example, Radnor et al., US Pat. No. 5,223,409; Kang et al., International Publication No. WO 92/18619; International Publication No. WO91 / 17271; Winter et al., International Publication WO92 / 20791; Mark Land et al., International Publication No. WO92 / 15679; Breitling et al., International Publication WO93 / 01288; McCaffati et al., International Publication No. WO92 / 010 No. 7; Garrard et al., International Publication No. WO 92/09690; Radnor et al., International Publication No. WO 90/02809; Fach et al. (1991), Bio / Technology, Vol. 9, pages 1370-1372; Hay et al. (1992), Hum Antibodies Hybridomas, 3 pp. 81-85; Hughes et al. (1989), Science 246: 1275-1281; Glyphs et al. (1993, EMBO J 12: 725-734; Hawkins et al. (1992), J Mol Biol, 226 889-896; Crackson et al. (1991), Nature, 352, 624-628; Gram et al. (1992), PNAS, 89, 3576-3580; Garado et al. (1991), Bio / Technology, 91373. 1377; Hogenboom et al. (1991), Nuc Acid Res 19: 4133-4137; and Barbus et al. (1991), PNAS, 88: 7978-7882.

In some embodiments, the heavy and light chain V region domains are expressed on the same polypeptide, joined by a variable linker to form a single chain Fv fragment, and then the scFV gene is transferred to the desired expression vector or It can be cloned into the phage genome. The complete V _H and V _{L of} an antibody linked by a variable (Gly ₄ -Ser) ₃ linker, as generally described in McCafferty et al., Nature (1990), 348: 552-554. Domains can be used to generate single chain antibodies that can make display packages separable based on antigen affinity. Subsequently, isolated scFV antibodies that immunoreact with peptides having B lymphocyte antigen activity can be formulated into medicinal preparations for use in the methods of the invention.
If displayed on the surface of a display package (eg, filamentous phage), the antibody library is screened with a B lymphocyte antigen protein or peptide fragment thereof to express antibodies with specificity for B lymphocyte antigen. Identify and isolate the package. Nucleic acid encoding the selected antibody can be recovered from the display package (eg, from the phage genome) and subcloned into other expression vectors by standard recombinant DNA techniques.

The antibodies according to the present invention can be used therapeutically to inhibit T cell activation by blocking the receptor: ligand interaction required for T cell co-stimulation. These so-called “blocking antibodies” can be identified by their ability to inhibit T cell proliferation and / or cytokine production when added to the in vitro co-stimulation assays described herein. The ability of blocking antibodies to inhibit T cell function can result in immunosuppression and / or tolerance when these antibodies are administered in vivo.
C. Protein purification.

Polyclonal or monoclonal antibodies according to the present invention, for example antibodies that react specifically with recombinant or synthetic peptides having B7-2 activity or B7-3 activity, can further be used to isolate natural B lymphocyte antigens from cells. Can be used. For example, antibodies that react with this peptide can be used to isolate naturally occurring or native B7-2 from activated B lymphocytes by immunoaffinity chromatography. In addition, native B7-3 can be isolated from B cells by immunoaffinity chromatography using monoclonal antibody BB-1.
D. Other therapeutic reagents

  The nucleic acid sequences described herein and the novel B lymphocyte antigen have the ability to either up-regulate (eg, amplify) or down-regulate (eg, establish suppression or tolerance) a T cell-mediated immune response It can be used for the development of therapeutic reagents. For example, soluble, monomeric B7-2 antigens or B7-2 fusion proteins, such as peptides having B7-2 activity, including B7-2Ig, and costimulatory signals to T cells that receive the initial activation signal Providing a specific means of blocking B7-2 ligand on T cells, thereby inducing immunosuppression and / or inducing tolerance using anti-B7-2 antibodies that cannot deliver it can. Such blocking or inhibitory B lymphocyte antigens and fusion proteins and blocking antibodies are based on their ability to inhibit T cell proliferation and / or cytokine production when added to an in vitro co-stimulation assay as described herein above. Can be identified. In contrast to the monomeric form, the stimulatory form of B7-2, such as the intact cell surface B7-2, retains the ability to transmit costimulatory signals to T cells, so that it receives secondary signals. Increased secretion of cytokines compared to non-activated T cells.

  In addition, using a fusion protein consisting of a first peptide having the activity of B7-2 fused to a second peptide having the activity of another B lymphocyte antigen (eg B7-1), T cell-mediated immunity The reaction can be modified. Alternatively, a single B lymphocyte antigen, eg, two separate peptides having B7-2 and B7-1 activity, or a combination of blocking antibodies (eg, anti-B7-2 and anti-B7-1 monoclonal antibodies) Can be administered together or separately (simultaneously or sequentially) to upregulate or downregulate a T cell mediated immune response in a subject. In addition, therapeutically effective amounts of one or more peptides having B7-2 activity and / or B7-1 activity can be used in conjunction with other immunomodulatory reagents to affect the immune response. Examples of other immunomodulatory reagents include blocking antibodies such as for CD28 or CTLA4, for other T cell markers, or for cytokines, fusion proteins such as CTLA4Ig, or immunosuppressants such as cyclosporin A or FK506. Is included.

  Peptides generated from the nucleic acid molecules according to the present invention may further be useful in the assembly of therapeutic agents that block T cell function by T cell destruction. For example, as described, secreted forms of B lymphocyte antigens can be assembled by standard genetic engineering techniques. A substance capable of preventing T cell activation can be created by linking soluble B7-1, B7-2 or B7-3 to a toxin such as ricin. Injection of one or a combination of immunotoxins, such as B7-2-lysine, B7-1-lysine, into the patient's body results in death of T cells, particularly activated T cells that express higher amounts of CD28 and CTLA4. Can bring The monovalent form of soluble form of B7-2 alone is also useful for blocking B7-2 function as described above, in which case carrier molecules may also be used.

Another way to interfere with the function of B lymphocyte antigens is through the use of antisense or triplex oligonucleotides. For example, oligonucleotides complementary to the region near the B7-1, B7-2 or B7-3 translation start site (eg, TGGCCCCATGCTTCAGA (SEQ ID NO: 20) nucleotides 326-309 for B7-1, and B7-2 Can synthesize GCCAAAATGGATCCCCA (SEQ ID NO: 21) One or more antisense oligonucleotides, typically 200 μg / ml, added to the cell culture medium or administered to the patient to produce B7-1 , B7-2 and / or B7-3 synthesis, antisense oligonucleotides are taken up by cells and hybridize with the appropriate B lymphocyte antigen mRNA to prevent translation. Binds to strand DNA to form triplex assembly Oligonucleotides that prevent unwinding and transcription of DNA can be used Te. In either result, the synthesis of one or more of B lymphocyte antigens can be cut off.
E. Therapeutic use by down-regulating the immune response.

  Given the structure and function of the novel B lymphocyte antigens disclosed herein, it is possible to down-regulate B lymphocyte antigen function in several ways, thereby down-regulating the immune response. Is possible. Downregulation may be in the form of inhibiting or blocking an immune response that is already in progress, or may include preventing induction of an immune response. The function of activated T cells can be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both. Immunosuppression of T cell responses is generally an active non-antigen specific process that requires that T cells are constantly exposed to inhibitory drugs. Tolerance includes inducing refractory or anergy in T cells, which is generally antigen-specific and can be distinguished from immunosuppression by persisting after exposure to the tolerizing agent. . Operationally, tolerance can be demonstrated by the lack of a T cell response when re-exposed to a specific antigen in the absence of a tolerizing agent.

  Down-regulating or preventing one or more B lymphocyte antigen functions, such as prevention of high-level lymphokine synthesis by activated T cells, in the context of tissue, skin and organ transplantation and graft-versus-host disease (GVHD) Would be useful to. For example, blockage of T cell function must result in reduced tissue destruction during tissue transplantation. Typically, in tissue transplantation, graft rejection begins with recognition by the T cell that it is foreign, followed by an immune response that destroys the graft. A molecule that inhibits or blocks the interaction of a B7 lymphocyte antigen with its natural ligand on immune cells (eg, a soluble monomeric peptide having B7-2 activity alone or another B lymphocyte antigen ( E.g. in combination with a monomeric peptide or blocking antibody having the activity of B7-1, B7-3) prior to transplantation to the natural ligand on immune cells without the transmission of the corresponding costimulatory signal The molecule can be bound. Blocking B lymphocyte antigen function in this manner prevents cytokine synthesis by immune cells, eg, T cells, and thus acts as an immunosuppressive means. Moreover, the lack of costimulation is sufficient for T cell anergy, thereby inducing tolerance in the subject. Induction of long-term tolerance by B lymphocyte antigen blocking reagents may avoid the need for repeated administration of these blocking reagents. In order to achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of the combined B lymphocyte antigen. For example, by administering a combination of soluble forms of peptides having the activity of each of their antigens or blocking antibodies prior to transplantation (separately or together in a single composition), B7-2 and B7-1, It may be desirable to block the function of B7-2 and B7-3, B7-1 and B7-3 or B7-2, B7-1 and B7-3. Alternatively, inhibitory B lymphocyte antigens can be used with other inhibitors, such as blocking antibodies against other T cell markers or against cytokines, other fusion proteins such as CTLA4Ig, or immunosuppressants.

The effectiveness of a particular blocking reagent in preventing organ transplant rejection or GVHD can be assessed using animal models that are expected to be effective in humans. The functionally important aspects of B7-1 are structurally conserved between species, so other B lymphocyte antigens may also function across species, and thus reagents composed of human proteins Can be used in animal systems. Examples of suitable systems that can be used include allogeneic heart transplantation in rats and xenogeneic islet cell transplantation in mice, both of which are described in Renshaw et al., Science 257: 789-792 (1992) and Turka et al., Proc. Natl. Acad. Sci. USA, Vol. 89, pages 11102-11105 (1992) and was used to investigate the immunosuppressive effect of CTLA4Ig fusion protein in vivo. In addition, the mouse model of GVHD (see Paul, Fundamental Immunology, Raven Press, New York, 1989, pp. 847-847) was used to block the in vivo B lymphocyte antigen function blockage on the progression of this disease. The effect can be measured.
For example, blocking B lymphocyte antigen function by using 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 is It may also be therapeutically useful for the treatment of immune diseases. Many autoimmune diseases are the result of inappropriate activation of T cells that are reactive to self tissue, which promotes the production of cytokines and autoantibodies involved in the pathology of the disease. Prevention of activation of autoreactive T cells may reduce or eliminate the symptoms of the disease. Receptor of B lymphocyte antigen: autoantibodies or T cells that inhibit T cell activation using administration of a reagent that blocks T cell co-stimulation by disrupting ligand interactions and may be involved in the course of disease Can prevent cytokine production induced by. In addition, blocking reagents induce antigen-specific tolerance of autoreactive T cells, leading to long-term healing from the disease. The effectiveness of blocking reagents to prevent or alleviate autoimmune diseases can be measured using several well-characterized animal models of human autoimmune diseases. Examples include mouse experimental autoimmune encephalitis, systemic lupus erythematosus in MRL / lpr / lpr mice or NZB hybrid mice, mouse autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and experimental myasthenia gravis in mice Disease is included (see Paul, Fundamental Immunology, Raven Press, New York, 1989, pages 840-856).

  The response of IgE antibodies in atopic allergy is highly T cell dependent, and thus inhibition of T cell activation induced by B lymphocyte antigens may be therapeutically useful in the treatment of allergies and allergic reactions. An inhibitory B7-2 protein, such as a peptide having B7-2 activity, alone or in combination with another B lymphocyte antigen, such as a peptide having B7-1 activity, is administered to an allergic patient and the T It can inhibit cell-mediated allergic reactions. Inhibition of B cell lymphocyte antigen costimulation of T cells may be accompanied by exposure to allergens associated with appropriate MHC molecules. The nature of the allergic reaction can be systemic or local, depending on the allergen entry pathway and the pattern of IgE deposition on mast or basophil cells. Thus, it may be necessary to locally or systemically inhibit T cell mediated allergic reactions by appropriate administration of inhibitory B7-2 protein.

Inhibition of T cell activation by blocking B lymphocyte antigen function may also be therapeutically important during viral infection of T cells. For example, in acquired immune deficiency syndrome (AIDS), viral replication is stimulated by T cell activation. Blocking B7-2 function leads to a lower level of viral replication, thereby improving the course of AIDS. In addition, blocking the function of the combined B lymphocyte antigens, ie B7-1, B7-2 and B7-3 may also be necessary. Surprisingly, T cells infected with HTLV-I express B7-1 and B7-2. This expression is important in the growth of HTLV-I infected T cells, and blockade of B7-1 function along with B7-2 and / or B7-3 function may slow the progression of HTLV-I induced leukemia I don't know. Alternatively, stimulation of viral replication by T cell activation may be induced by contact with a stimulatory B7-2 protein for the purpose of generating a sufficient amount of retrovirus for isolation and use.
F. Therapeutic use by up-regulating the immune response.

  Upregulation of B lymphocyte antigen function as a means of upregulating the immune response can also be useful in therapy. Up-regulation of an immune response can be in the form of promoting an existing immune response or manifesting an initial immune response. For example, enhancing the immune response by stimulating B lymphocyte antigen function may be useful in the case of viral infections. Viral infection is largely eliminated by cytolytic T cells. According to the present invention, B7-2 with natural ligands on T cells, and hence B7-1 and B7-3, can result in at least some increase in cytolytic activity of T cells. It is further believed that B7-2, B7-1 and B7-3 are involved in the initial activation and generation of CD8 + cytotoxic T cells. Stimulating T cell activity via a costimulatory pathway by adding a soluble peptide with B7-2 activity, in multivalent form, alone or in combination with a peptide having the activity of another B lymphocyte antigen Thus, it would be therapeutically useful in situations where faster or complete elimination of the virus is advantageous. These include viral skin diseases such as herpes or shingles, in which case a multivalent soluble peptide having B7-2 activity, or such a peptide and / or a peptide having B7-1 activity and / or B7 A peptide combination with -3 activity is applied topically to the skin. In addition, systemic viral diseases such as influenza, colds, and encephalitis can be ameliorated by systemic administration of stimulated B lymphocyte antigens.

  Alternatively, a viral antigen that removes T cells from the patient and expresses 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) Pulsed with viral antigens by APC pulsed with or with a stimulated form of 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) APC can also enhance antiviral immune responses in infected patients by co-stimulating T cells in vitro and reintroducing the in vitro activated T cells into the patient. Another method of enhancing the antiviral immune response is to separate infected cells from the patient and transfect them with a nucleic acid encoding a peptide having the activity of a B lymphocyte antigen as described herein. The cell is allowed to express on its surface all or part of a B lymphocyte antigen, such as B7-2 or B7-3, and the transfected cell is reintroduced into the patient's body. is there. Then, the infected cells will be able to transduce costimulatory signals to the T cells in vivo and thus activate the T cells.

Stimulated B lymphocyte antigens can also be used prophylactically in vaccines against various pathogens. Immunity against pathogens such as viruses can be induced by vaccination with a viral protein with a stimulated form of a peptide having B7-2 activity or another peptide having B lymphocyte antigen activity in a suitable adjuvant. . Alternatively, an expression vector encoding a gene for both a pathogenic antigen and a peptide having B lymphocyte antigen activity, such as a nucleic acid encoding a viral protein, as described herein Vaccinia virus expression vectors assembled to express a nucleic acid encoding a peptide having B7-2 activity can also be used for vaccination. For example, presentation of B7-2 with class I MHC protein by cells transfected with co-expressing peptides with B7-2 activity and MHC class I alpha chain protein and beta ₂ microglobulin is also cytolytic CD8 + T cells Activation and provides immunization from viral infections. 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 embodiment, a stimulated form of one or more soluble peptides having B lymphocyte antigen activity is administered to a patient with a tumor to generate a costimulatory signal for T cells to induce anti-tumor immunity. Can be provided.
G. Modification of tumor cells to express costimulatory molecules.

Tumor cells are unable to elicit a costimulatory signal in T cells because of the lack of expression of costimulatory molecules, even if tumor cells have the ability to express such molecules, It may be due to insufficient expression of surface costimulatory molecules or lack of appropriate costimulatory molecule expression (eg B7 expression rather than B7-2 and / or B7-3). Thus, according to one aspect of the invention, the tumor cells encode B7-2 and / or B7-3 in a form suitable for expression of B7-2 and / or B7-3 on the tumor cell surface. The tumor cells are modified to express B7-2 and / or B7-3 by transfection with a nucleic acid. Alternatively, the tumor cells are modified by contacting with a substance that induces or enhances the expression of B7-2 and / or B7-3 on the tumor cell surface. In yet another embodiment, B7-2 and / or B7-3 are bound to the surface of the tumor cell to produce a modified tumor cell. These and other aspects are described in further detail in the following subsections.
(1) Transfection of tumor cells with a nucleic acid encoding a costimulatory molecule.

  By transfecting the isolated tumor cells with a nucleic acid encoding B7-2 and / or B7-3 and B7-1 in a form suitable for expressing the molecule on the surface of the tumor cell, The tumor cells can be modified ex vivo to express B7-2 or B7-3 alone or in combination with B7-1. The term “transfection” or “transfect” means the introduction of exogenous nucleic acid into mammalian cells, by electroporation, calcium phosphate precipitation, DEAE dextran treatment, lipofection, microinjection and viral vectors. A variety of techniques useful for introducing nucleic acids into mammalian cells, including infection, are included. Suitable methods for transfecting mammalian cells include Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press (1989)) and other laboratories. Can be found in textbooks. The nucleic acid to be introduced is, for example, DNA including a gene encoding B7-2 and / or B7-3, sense strand RNA encoding B7-2 and / or B7-3, or B7-2 And / or a recombinant expression vector comprising cDNA encoding B7-3. The nucleotide sequence of cDNA encoding human B7-2 is shown in the sequence listing.

  A preferred approach for introducing a nucleic acid encoding B7-2 and / or B7-3 into a tumor cell is a viral vector comprising a nucleic acid encoding B7-2 and / or B7-3, eg cDNA Is due to the use of Examples of viral vectors that can be used include retroviral vectors (MA Aeglitis et al., Science, 230, 1395-1398 (1985); O. Danos and R. Mulligan, Proc. Natl. Acad. Sci. USA, 85. Vol. 6460-6464 (1988); D. Markowitz et al., J. Virol., 62: 1120-1124 (1988)), adenovirus vector (MA Rosenfeld et al., Cell, 68: 143-155). (1992)) and adeno-associated virus vectors (JD Tratchon et al., Mol. Cell. Biol. 5: 3251-3260 (1985)). Infection of tumor cells with a viral vector allows the majority of cells to accept the nucleic acid, thereby avoiding the need for selection of cells that have received the nucleic acid, and can be introduced into the viral vector by, for example, cDNA contained in the viral vector. The encoded molecule has the advantage of being efficiently expressed in cells that have taken up the nucleic acid of the viral vector.

Alternatively, B7-2 and / or B7-3 can be expressed on tumor cells using a plasmid expression vector comprising a nucleic acid encoding B7-2 and / or B7-3, eg, cDNA. Suitable plasmid expression vectors include CDM8 (B. 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.
If transfection of a tumor cell leads to the most modification of that tumor cell and efficient expression of B7-2 and / or B7-3 on the surface of the tumor cell, eg when using a viral expression vector, the tumor The cells can be used without further separation or subcloning. Alternatively, a homogenous population of transfected tumor cells is a limiting dilution clone of one transfected tumor cell, which is then expanded into a clonal population of cells by standard techniques. Can be prepared.
(2) Induction or enhanced expression of costimulatory molecules on the tumor cell surface.

  On tumor cells that express the ability to express B7-2 and / or B7-3, but cannot do that, or express B7-2 and / or B7-3 in an amount insufficient to activate T cells Tumor cells can be modified to induce costimulatory signals in T cells by inducing or enhancing expression levels of B7-2 and / or B7-3. Agents that stimulate B7-2 and / or B7-3 expression can be used to induce or enhance B7-2 and / or B7-3 expression on tumor cells. For example, tumor cells can be contacted with this substance in vitro in culture medium. Substances that stimulate the expression of B7-2 and / or B7-3 enhance the translation of B7-2 and / or B7-3 mRNA, for example by enhancing transcription of the B7-2 and / or B7-3 gene Or by enhancing the stability or transport of B7-2 and / or B7-3 to the cell surface. For example, it is known that B7 expression can be upregulated intracellularly by a second messenger pathway involving cAMP. N. Navavi et al., Nature, 360, 266-268 (1992). B7-2 and B7-3 can also be induced by cAMP. That is, tumor cells are contacted with a substance that increases intracellular cAMP levels or approximates cAMP, such as cAMP analogs, such as dibutyryl cAMP, and B7-2 and / or B7-3 on the surface of this tumor cell. Expression can be stimulated. It is also known that the expression of B7 is induced on normal resting B cells by cross-linking cell surface MHC class II molecules on B cells with antibodies against MHC class II molecules. L. Quorova et al. Exp. Med. 173, 759-762 (1991). Similarly, B7-2 and B7-3 can be induced on resting B cells by cross-linking cell surface MHC class II molecules on B cells. Thus, tumor cells that express MHC class II molecules on their surface can be treated with anti-MHC class II antibodies to induce or enhance B7-2 and / or B7-3 expression on the tumor cell surface. In addition, interleukin 4 (IL-4), which has been found to induce B7-2 expression on B cells, can be used to upregulate B7-2 expression on tumor cells ( RM Stack et al., J. Cell.Biochem.Suppl., 1 (18) Volume 434 (1994).

Another substance that can be used to induce or enhance B7-2 and / or B7-3 expression on the tumor cell surface encodes a transcription factor that upregulates the transcription of the gene encoding the costimulatory molecule. Nucleic acid. This nucleic acid can be transfected into tumor cells to enhance transcription of the costimulatory molecule gene, resulting in increased cell surface levels of the costimulatory molecule.
(3) Co-stimulatory molecule binding to the surface of tumor cells.

In another embodiment, B7-2 and / or B7-3 is modified to allow a costimulatory signal in T cells to be triggered by binding to the surface of the tumor cells. For example, B7-2 and / or B7-3 molecules can be obtained using standard recombinant DNA techniques and expression systems that allow the production and isolation of costimulatory molecules. Alternatively, B7-2 and / or B7-3 can be isolated from cells expressing a costimulatory molecule using standard protein purification techniques. For example, B7-3 protein can be isolated from activated B cells by immunoprecipitation using an anti-B7-3 antibody such as the BB1 monoclonal antibody. The isolated costimulatory molecule is then bound to the tumor cells. The term “bound” or “bound” means that B7-2 and / or B7-3 are linked to tumor cells so that costimulatory molecules are present on the surface of the tumor cells and co-activate in T cells. By chemical, enzymatic or other means (eg antibodies) such that a stimulation signal can be induced. For example, B7-2 and / or B7-3 can be chemically cross-linked to the tumor cell surface using commercially available cross-linking reagents (Pierce, Rockford, IL). Another approach to bind B7-2 and / or B7-3 to tumor cells is to use bispecific antibodies that bind to both costimulatory molecules and cell surface molecules on tumor cells. B7-2 and / or B7-3 fragments, mutants or variants that retain the ability to induce a costimulatory signal in T cells when bound to the surface of tumor cells can also be used. .
(4) Modification of tumor cells to express multiple costimulatory molecules

  Another embodiment of the invention is a tumor cell that has been modified to express a plurality of costimulatory molecules. Transient expression of costimulatory molecules on activated B cells is different for B7, B7-2 and B7-3. For example, B7-2 is expressed early after B cell activation, while B7-3 is expressed later. That is, different costimulatory molecules can perform distinct functions during the course of the immune response. An effective T cell response may require the T cell to receive a costimulatory signal from multiple costimulatory molecules. Accordingly, the present invention encompasses tumor cells that have been modified to express one or more costimulatory molecules. For example, tumor cells can be modified to express both B7-2 and B7-3. Alternatively, tumor cells modified to express B7-2 can be further modified to express B7-1. Similarly, tumor cells modified to express B7-3 can be further modified to express B7-1. Tumor cells can also be modified to express B7-1, B7-2 and B7-3. Tumor cells can be modified to express multiple costimulatory molecules (eg, B7-1 and B7-2) by any technique described herein.

Prior to modification, the tumor cells may not express any costimulatory molecules, or may express one costimulatory molecule but not others. As described herein, a tumor cell is transfected by transfecting the tumor cell with a nucleic acid encoding a costimulatory molecule, by inducing expression of the costimulatory molecule, or by Modifications can be made by attaching costimulatory molecules. For example, tumor cells transfected with a nucleic acid encoding B7-2 can be further transfected with a nucleic acid encoding B7-1. The cDNA of human B7-1 and the derived amino acid sequence are shown in the sequence listing. Alternatively, one or more types of modifications can be applied. For example, tumor cells transfected with a nucleic acid encoding B7-2 can be stimulated with a substance that induces expression of B7-1.
(5) Further modification of tumor cells to express MHC molecules.

Yet another aspect of the present invention is to express a costimulatory molecule and express one or more MHC molecules on their surface so that both the costimulatory signal and the first antigen-specific signal are transmitted to T cells. Characterized by tumor cells modified to induce Prior to modification, tumor cells may not be able to express MHC molecules, may be capable of expressing MHC molecules but may not be able to express such molecules, or are sufficient to elicit T cell activation. A significant amount of MHC molecules may not be expressed on tumor cells. Tumor cells can be modified to express either MHC class I or MHC class II molecules, or both. One approach to modify tumor cells to express MHC molecules is to transfect the tumor cells with one or more nucleic acids encoding one or more MHC molecules. Alternatively, tumor cells can be modified with agents that induce or enhance expression of one or more MHC molecules on the tumor cells. The ability of MHC class II ⁺ tumor cells to present tumor peptides directly to CD4 ⁺ T cells can circumvent the need for professional MHC class II ⁺ APC, thus inducing or enhancing expression of MHC class II molecules on tumor cells Can be particularly beneficial for the activation of CD4 ⁺ T cells against tumors. This can improve tumor immunogenicity because soluble tumor antigens (tumor cell debris or secreted protein forms) cannot be utilized for uptake by professional MHC class II ⁺ APC.

  One aspect of the invention is a modified tumor cell that expresses B7-2 and / or B7-3 and one or more MHC class II molecules on the cell surface. MHC class II molecules structurally contain a cleft into which an antigenic peptide binds and have the function of presenting the bound peptide to an antigen-specific TcR, a cell surface α / β heterodimer It is. Multiple different MHC class II proteins are expressed on professional APCs, and different MHC class II proteins bind to different antigenic peptides. Thus, the expression of multiple MHC class II molecules increases the spectrum of antigenic peptides that can be presented by APC or by modified tumor cells. The α and β chains of MHC class II molecules are encoded by different genes. For example, human MHC class II protein HLA-DR is encoded by HLA-DRα and HLA-DRβ genes. In addition, many polymorphic alleles of MHC class II genes are present in humans and other species. Certain individuals' T cells respond to stimulation by antigenic peptides bound to their own MHC molecules, a phenomenon called MHC restriction. In addition, certain T cells can respond to stimulation by polymorphic alleles of MHC molecules found on cells of other individuals, a phenomenon called allogeneic. For a review of the structure and function of MHC class II, see Jermaine and Margulies, Ann. Rev. Immunol. 11 403-450 1993.

Another aspect of the invention is a modified tumor cell that expresses B7-2 and / or B7-3 and one or more MHC class I molecules on the cell surface. Like the MHC class II genes, there are multiple MHC class I genes, and many polymorphic alleles of these genes have been found in humans and other species. Like MHC class II proteins, class I proteins bind to peptide fragments of the antigen for presentation to T cells. Functional cell surface class I molecules are composed of MHC class I α chain proteins linked to β2 microglobulin protein.
(6) Transfection of tumor cells with nucleic acids encoding MHC molecules.

  By one or more nucleic acids encoding one or more MHC class II α chains and one or more MHC class II β chains in a form suitable for expression of MHC class II molecules on the surface of tumor cells, Tumor cells can be modified ex vivo to express one or more MHC class II molecules by transfecting the isolated tumor cells. In order to form a surface heterodimer, both α and β chain proteins must be present on the tumor cell and neither chain alone will be expressed on the cell surface. Nucleic acid sequences for many mouse and human class II genes are known. For example, L.M. Food, etc., Ann. Rev. Immunol. 1, pages 529-568 (1983) and C.I. Aufrey and J.W. L. See 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 may be a foreign allogeneic MHC class II molecule. Certain foreign MHC class II molecules to be introduced into tumor cells increase the cytokines when stimulated by cells expressing the foreign MHC class II molecule in T cells from a subject having a tumor. Alternatively, it can be selected by its ability to induce secretion (ie, by its ability to induce homologous reactions). Tumor cells to be transfected may not express MHC class II molecules on their surface prior to transfection, or may express insufficient amounts to stimulate a T cell response. Alternatively, tumor cells that express MHC class II molecules prior to transfection are further transfected with additional different MHC class II genes or with other polymorphic alleles of the MHC class II genes, and the tumor cells Can also increase the spectrum of antigenic fragments that can be presented to T cells.

  Fragments, mutants or variants of MHC class II molecules that retain the ability to bind peptide antigens and activate T cell responses as evidenced by proliferation and / or lymphokine production by T cells are It is considered to be within the scope of the invention. Preferred variants are MHC class II molecules in which one or both cytoplasmic domains of the α and β chains are truncated. The truncation of the cytoplasmic domain allows peptide binding by MHC class II molecules and expression of MHC class II molecules on the cell surface, but upon cross-linking of cell surface MHC class II molecules the cytoplasmic domain of the MHC class II protein chain It is known to prevent the induction of endogenous B7 expression induced by intracellular signals generated by. L. Quorova et al. Exp. Med. 173, 759-762 (1991); Navavi et al., Nature, 360, 266-268 (1992). The expression of B7-2 and B7-3 is also induced by cross-linked surface MHC class II molecules, and thus truncation of MHC class II molecules may also prevent the induction of B7-2 and / or B7-3. In tumor cells transfected to effect constitutive expression of B7-2 and / or B7-3, inhibiting the expression of endogenous costimulatory molecules, eg, suppressing possible downregulation feedback mechanisms May be desirable. Transfection of tumor cells with nucleic acid encoding cytoplasmic domain truncated MHC class II α and β chain proteins also inhibits endogenous B7-1 expression and possibly endogenous B7-2 and B7-3 expression Will do. Such variants can be generated, for example, by introducing a stop codon after the nucleotide encoding the transmembrane region in the MHC class II chain gene. Either the α or β chain protein is truncated, or both α and β chains are truncated for more complete inhibition of B7 (and possibly B7-2 and / or B7-3) induction. can do. For example, Griffith et al., Proc. Natl. Acad. Sci. USA, 85, 4847-4852 (1988), Nabavi et al. Immunol. 142, 1444-1447 (1989).

  Tumor cells can be modified to express MHC class I molecules by transfection with a nucleic acid encoding an MHC class I α chain protein. For examples of nucleic acids, see L.L. Food, etc., Ann. Rev. Immunol. 1, pages 529-568 (1983) and C.I. Aufrey and J.W. L. See Strominger, Advances in Human Genetics, 15: 197-247 (1987). If desired, if the tumor cells do not express β2 microglobulin, it can be transfected with a nucleic acid encoding a β2 microglobulin protein. For examples of nucleic acids, see D.C. Gousou et al. Immunol. 139, 3132-3138 (1987) and J. Am. R. Parns et al., Proc. Natl. Acad. Sci. USA, Vol. 78, pages 2253-2257 (1981). Similar to MHC class II molecules, by increasing the number of MHC class I genes or polymorphic alleles of MHC class I genes expressed in tumor cells, the antigen that the tumor cells can present to T cells Sex fragment vectors can be increased.

  When transfecting tumor cells with a nucleic acid encoding one or more molecules, such as B7-2 and / or B7-3 molecules, MHC class II α chain protein and MHC class II β chain protein, the transfection may be simultaneous or sequential. Can be implemented. If transfection is performed simultaneously, the molecules can be introduced on the same nucleic acid as long as the encoded sequence does not exceed the capacity of the particular vector used. Alternatively, the molecule can be encoded by separate nucleic acids. When transfection is performed sequentially and tumor cells are selected using a selectable marker, one selectable marker is used in conjunction with the first introduced nucleic acid, while a different selectable marker is then introduced. Can be used in connection with the nucleic acid to be produced.

Expression of MHC molecules (class I or class II) on the cell surface of tumor cells can be measured, for example, by immunofluorescence of tumor cells using fluorescently labeled monoclonal antibodies against different MHC molecules. Monoclonal antibodies that recognize either the non-polymorphic region of a particular MHC molecule (non-allelic specificity) or the polymorphic region of a particular MHC molecule (allele specificity) can be used, and these Are known to those skilled in the art.
(7) Induction or enhanced expression of MHC molecules on tumor cells.

  Another approach to ex vivo modify tumor cells to express MHC molecules on the surface of tumor cells uses substances that stimulate the expression of MHC molecules to induce or enhance the expression of MHC molecules on the surface of the tumor cells It is to be. For example, tumor cells can be contacted with the substance in vitro in a culture medium. Agents that stimulate the expression of MHC molecules are, for example, by enhancing transcription of MHC class I and / or class II genes, by enhancing translation of MHC class I and / or class II mRNA, or by MHC class I And / or may work by enhancing the stability or transport of class II proteins to the cell surface. Several substances have been shown to enhance the level of cell surface expression of MHC class II molecules. For example, S.M. M.M. Cockfield et al. Immunol. 144, 2967-2974 (1990); J. et al. Noel et al. Immunol. 137, 1718-1723 (1986); J. et al. Mondo et al. Immunol. 127, 881-888 (1981); L. William et al. Exp. Med. 170, pp. 1559-1567 (1989); Serada and R.C. Maki, J.M. Immunol. 146, 114-120 (1991) and L.L. H. Grimture and C.I. J. et al. Kara, Ann. Rev. Immunol. 10: 13-49 (1992) and references thereof. These substances include cytokines, antibodies to other cell surface molecules, and phorbol esters. One substance that upregulates MHC class I and class II molecules in a wide range of cell types is the cytokine interferon gamma. Thus, for example, tumor cells modified to express B7-2 and / or B7-3 and B7-1 can be further modified to enhance expression of MHC molecules upon contact with interferon γ.

Another substance that can be used to induce or enhance expression of MHC molecules on the surface of tumor cells is a nucleic acid encoding a transcription factor that upregulates transcription of MHC class I or class II genes. Such nucleic acids can be transfected into tumor cells to cause enhanced transcription of the MHC gene, resulting in increased cell surface levels of the MHC protein. MHC class I and class II genes are regulated by different transcription factors. However, as is the case with multiple MHC class II genes, multiple MHC class I genes are cooperatively regulated. Thus, transfection of tumor cells with a nucleic acid encoding a transcription factor that regulates expression of the MHC gene can enhance the expression of several different MHC molecules on the surface of the tumor cell. Several transcription factors that regulate MHC gene expression have been identified, cloned, and characterized. For example, W.W. Rice et al., Genes Dev. 4, pages 1528-1540 (1990); C. Liu et al., Science, 247, 1581-1584 (1988); K. Didia et al., Proc. Natl. Acad. Sci. USA, 85, 7322-7326 (1988).
(8) Inhibition of non-mutant chain expression in tumor cells.
Another aspect of the present invention is a tumor cell modified to express a T cell costimulatory molecule (eg, B7-2 and / or B7-3 and B7-1), wherein the protein binds to MHC class II The present invention provides a tumor cell in which the expression of a non-mutant chain is inhibited. Unmutated chain expression is inhibited, facilitating the binding of endogenous TAA peptides to MHC class II molecules, creating an antigen-MHC complex. This complex can elicit an antigen-specific signal in T cells to induce T cell activation in conjunction with costimulatory signals. MHC class II molecules have been shown to be capable of presenting endogenous peptides. J. et al. G. Nacturn et al., Nature, 343, 74-76 (1990); Weiss and B.B. Bogen, Cell, 767-776 (1991). However, in cells that naturally express MHC class II molecules, α and β chain proteins are associated with the non-mutated chain (hereinafter Ii) during intracellular transport of this protein from the endoplasmic reticulum. Ii is believed to function in part by preventing binding of endogenous peptides to MHC class II molecules. W. Elliott et al. Immunol. 138, 2949-2952 (1987); Stockinger et al., Cell, 56, 683-689 (1989); Guagriadi et al., Nature (London), 343, 133-139 (1990); Bucke et al., Cell, 63, 707-716 (1990); Lotru et al., Nature, 348, 600-605 (1990); Peters et al., Nature, 349, 669-676 (1991); Rocce et al., Nature, 345, 615-618 (1990); Teton et al., Nature, 348, 39-44 (1990). Since TAA is endogenously synthesized in tumor cells, peptides derived from these appear to be available intracellularly. Thus, inhibition of Ii expression in Ii-expressing tumor cells may increase the likelihood that a TAA peptide will bind to MHC class II molecules. Consistent with this mechanism, it has been shown that supertransfection of MHC class II ⁺ , Ii ⁻ tumor cells with the Ii gene prevents stimulation of tumor-specific immunity by the tumor cells. V. K. Clements et al. Immunol. 149, 2391-2396 (1992).

Prior to modification, the expression of Ii in tumor cells 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 the expression of Ii is by introducing into the tumor cells an antisense nucleic acid to the coding or regulatory region of the Ii gene, which has been described previously. N. Koch et al., EMBO J. et al. 6: 1677-1683 (1987). For example, an oligonucleotide complementary to the nucleotide near the translation start site of Ii mRNA can be synthesized. One or more antisense oligonucleotides can be added to the medium containing the tumor cells, typically at an oligonucleotide concentration of 200 μg / ml. This antisense oligonucleotide is taken up by tumor cells and hybridizes with Ii mRNA, preventing translation. In another embodiment, a recombinant expression vector is used, wherein the nucleic acid encodes a sequence of the Ii gene in an orientation such that mRNA is produced that is antisense to the coding or regulatory region of the Ii gene. Thus, tumor cells transfected with this recombinant expression vector contain a continuous source of Ii antisense nucleic acid to prevent production of Ii protein. Alternatively, Ii expression in tumor cells can be inhibited by treating the tumor cells with substances that interfere with Ii expression. For example, drugs that inhibit Ii gene expression, Ii mRNA translation, or Ii protein stability or intracellular transport can be used.
(9) The type of tumor cell to be modified.

  Tumor cells that undergo modification as described herein may be one or more of the ones encompassed by the present invention for expressing B7-2 and / or B7-3 alone or in combination with B7-1. Includes tumor cells that can be transfected or treated by the approach. If necessary, the tumor cells can be further modified to express MHC molecules or inhibitors of Ii expression. The tumor from which tumor cells are obtained may be naturally occurring, eg, in a human patient, or may be experimentally induced or induced, eg, in an animal subject. Tumor cells can be obtained, for example, from solid tumors of organs such as lung, liver, breast, colon, bone and other tumors. Malignant tumors of solid organs include carcinomas, sarcomas, melanomas and neuroblastomas. Tumor cells can also be obtained from blood-derived (ie, dispersed) malignant tumors such as lymphoma, myeloma or leukemia.

Tumor cells to be modified include those that express MHC molecules on their cell surface prior to transfection and those that express no or low levels of MHC class I and / or class II molecules. A few of the normal cell types express MHC class II molecules. Thus, it is expected that many tumor cells will not naturally express MHC class II molecules. These tumors can be modified to express B7-2 and / or B7-3 and MHC class II molecules. Several types of tumors, such as melanoma (Van Duinen et al., Cancer Res. 48: 1019-1025, 1988), and chronic large cell lymphoma (Okin et al., Cancer 66: 1147-1153, 1990) Squamous cell carcinoma of the head and neck (Matijusen et al., Int. J. Cancer, 6: 95-100, 1991) and colorectal cancer (Moller et al., Int. J. Cancer, 6: 155-162, 1991) It has been found that surface MHC class II molecules are naturally expressed. Tumor cells that naturally express class II molecules can be modified to express B7-2 and / or B7-3, and also other class II molecules, resulting in a spectrum of TAA peptides that can be presented by the tumor cells. Can be increased. Most non-malignant tumor cell types express MHC class I molecules. However, transformation to malignant tumors often involves down-regulation of MHC class I molecule expression on the surface of tumor cells. A. Shiba et al., Brit. J. et al. Cancer, 50, 699-709 (1984). Importantly, loss of expression of MHC class I antigens by tumor cells is associated with greater aggressiveness and / or metastatic potential of tumor cells. P. I. Schlier et al., Nature, 305, 771-775 (1983); A. Holden et al. Am. Acad. Dermatol. 9: 867-871 (1983); Baniyash et al. Immunol. 129, 1318-1323 (1982). Tumor types that have been shown to inhibit MHC class I expression include melanoma, colorectal cancer and squamous cell carcinoma. Van Duinen et al., Cancer Res. 48, 1019-1025 (1988); Moller et al., Int. J. et al. Cancer, pp. 155-162 (1991); Shiba et al., Brit. J. et al. Cancer, 50, 699-709 (1984); A. Holden et al. Am. Acad. Dermatol. 9: 867-871 (1983). Tumor cells that are unable to express class I molecules or that express only low levels of MHC class I molecules are modified by one or more techniques described herein to produce MHC class I on the tumor cell surface. The expression of the molecule can be induced or enhanced and the immunogenicity of the tumor cell can be enhanced.
(10) Modification of tumor cells in vivo.

Another aspect of the invention is to modify a tumor cell in vivo to express B7-2 and / or B7-3 and B7-1 to induce a costimulatory signal in T cells. A method is provided for increasing the immunogenicity of In addition, tumor cells can be further modified in vivo to express MHC molecules and trigger a first, antigen-specific signal in T cells. By introducing a nucleic acid encoding B7-2 and / or B7-3 and B7-1 into the tumor cell in a form suitable for expression of a costimulatory molecule on the surface of the tumor cell, the tumor cell Can be modified in vivo. Similarly, nucleic acids encoding MHC class I or class II molecules or antisense sequences of the Ii gene can be introduced into tumor cells in vivo. In one embodiment, as a form of gene therapy, recombinant expression vectors can be used to deliver nucleic acids encoding B7-2 and / or B7-3 and B7-1 into tumor cells in vivo. Vectors useful for in vivo gene therapy have been described previously and include retroviral vectors, adenoviral vectors and adeno-associated viral vectors. For example, M.M. A. Rosenfeld, Cell, 68, 143-155 (1992); F. Anderson, Science 226, 401-409 (1984); See Friedman, Science, 244, 1275-1281 (1989). Alternatively, the nucleic acid can be delivered to the tumor cell in vivo by direct injection of the naked nucleic acid into the tumor cell. For example, G. See Asadi et al., Nature, 332, 815-818 (1991). Commercial devices are available for delivery (BioRad). If desired, the nucleic acid can be complexed with a carrier such as a liposome to be suitable for injection. Nucleic acids encoding MHC class I molecules complexed with liposomes were injected directly into the tumors of melanoma patients. M.M. Hoffman, Science, 256, 305-309 (1992).
Tumor cells are modified in vivo by the use of substances that induce or enhance expression of B7-2 and / or B7-3 and B7-1 (and MHC molecules if necessary) as described herein. You can also. This substance can be administered systemically, eg intravenously, or preferably locally to tumor cells.
(11) Effector phase of anti-tumor T cell mediated immune response.

  The modified tumor cells according to the present invention are useful for stimulating anti-tumor T cell-mediated immune responses by inducing antigen-specific and costimulatory signals in tumor-specific T cells. Following this inductive or afferent phase of the immune response, an effector population of T cells is generated. These effector T cell populations can include both CD4 + T cells and CD8 + T cells. This effector population is responsible for the elimination of tumor cells, for example by cytolysis of tumor cells. Once the T cell is activated, the expression of costimulatory molecules is not required on the target cell for the recognition of the target cell by the effector T cell or the effector function of the T cell. F. A. Harding and J.H. P. Allison, J.M. Exp. Med. 177, 1791-1796 (1993). Therefore, the anti-tumor T cell-mediated immune response induced by the modified tumor cells according to the present invention is effective both for the modified tumor cells and for the unmodified tumor cells that do not express costimulatory molecules.

Furthermore, the density and / or type of cell surface MHC molecules required for the afferent and centrifugal phases of the T cell mediated immune response may vary. Less MHC molecules required for initial activation of T cells or only certain types of MHC molecules (eg MHC class I but not MHC class II) may be required on tumor cells for recognition by effector T cells. I don't know. Thus, tumor cells that naturally express small amounts of MHC molecules, but modified to express larger amounts of MHC molecules, can induce an effective T cell-mediated immune response against unmodified tumor cells. Alternatively, tumor cells that naturally express MHC class I molecules but not MHC class II molecules where they are modified to express MHC class II molecules are parental MHC class I +, class II-tumor cells. Can induce T cell-mediated immune responses, including effector T cell populations that can eliminate.
(12) A composition for treating tumor cells.

  Another aspect of the invention is a composition of modified tumor cells of a biologically compatible type suitable for in vivo pharmaceutical administration to a subject. The composition includes an amount of modified tumor cells and a physiologically acceptable carrier. The amount of modified tumor cells is selected to be therapeutically effective. The term “biologically compatible form suitable for pharmaceutical administration in vivo” means that the therapeutic effect of the tumor cell is superior to the toxic effect of the tumor cell. A “physiologically acceptable carrier” is a carrier that is biologically compatible with a subject. Examples of acceptable carriers include saline and aqueous buffer solutions. In all cases, the composition must be sterile and must be fluid to the extent that easy syringability exists. The term “subject” is intended to include living organisms in which tumors can be developed or experimentally induced. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.

Administration of therapeutic compositions according to the present invention can be carried out using known methods for a period and dose effective to achieve the desired result. For example, a therapeutically effective amount of a modified tumor cell can vary depending on factors such as age, gender and individual weight, tumor cell type and degree of tumor burden, and the subject's ability to respond immunologically. Dosage regimens can be adjusted to provide the best therapeutic response. For example, a single dose of modified tumor cells can be administered, or several doses can be administered in a batch. Administration may be by injection, including intravenous, intramuscular, intraperitoneal and subcutaneous injection.
(13) In vitro activation of tumor-specific T lymphocytes.

Another approach to induce or enhance an anti-tumor T cell mediated immune response by inducing co-stimulatory signals in T cells is to obtain T lymphocytes from a subject with a tumor and either tumor cells and alone or B7-1 It is to activate it in vitro by stimulating it with a combined B7-2 and / or B7-3 stimulatory form. 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 enriched or isolated for T lymphocytes or T lymphocyte subpopulations. T cells are cultured with tumor cells obtained from a subject (eg, from a biopsy or, in the case of blood-derived malignant tumors, from peripheral blood) combined with stimulated B7-2 and / or B7-3 Or alternatively, can be activated in vitro by exposure to a modified tumor cell as described herein. The term “stimulated” means that the costimulatory molecule can cross-link with its receptor on T cells to induce a costimulatory signal in T cells. The stimulatory form of the costimulatory molecule can be, for example, a soluble multivalent molecule of the costimulatory molecule, or a fixed form of the costimulatory molecule, eg, bound to a solid support. B7-2 and / or B7-3 fragments, mutants or variants (eg, fusion proteins) that retain the ability to elicit a costimulatory signal in T cells can also be used. In a preferred embodiment, the soluble extracellular portion of B7-2 and / or B7-3 is used to provide costimulation to T cells. In order to activate tumor-specific T cells, the T cells are cultured in vitro with tumor cells and B7-2 and / or B7-3, or with modified tumor cells, and the T cells are then injected, for example, by intravenous injection Can be administered to a subject.
(14) Therapeutic use of modified tumor cells.

  The modified tumor cells according to the present invention can be used to increase the immunogenicity of a tumor, and thus provide T lymphocyte-mediated anti-tumor immunity in a subject with a tumor or where a tumor may develop. It can be used therapeutically to induce or enhance. A method for treating a subject with a tumor obtains tumor cells from the subject and ex vivo expresses the T cell costimulatory molecule by, for example, transfecting the tumor cells with a suitable nucleic acid. And administering to the subject a therapeutically effective dose of the modified tumor cells. Appropriate nucleic acids to be introduced into the tumor cells are combined with the nucleic acids described herein, either alone or encoding a B7-1, MHC molecule (class I or class II) or Ii antisense sequence. , Nucleic acids encoding B7-2 and / or B7-3. Alternatively, after obtaining tumor cells from a subject, using a substance that induces or enhances expression of B7-2 and / or B7-3 (and possibly a substance that induces or enhances B7-1 or MHC molecules) These can also be modified ex vivo.

  Tumor cells can be obtained, for example, from a subject by surgical removal of tumor cells, eg, biopsy of the tumor, or in the case of a blood-derived malignant tumor from a blood sample of the subject. In the case of an experimentally derived tumor, the cells used to induce the tumor, such as cells of a tumor cell line, can be used. Solid tumor samples can be processed to produce a single cell suspension of tumor cells prior to modification for maximum transfection efficiency. Possible treatments include manual dispersion of cells or enzymatic digestion of connective tissue fibers, eg, with collagenase.

  Tumor cells can be transfected immediately after being obtained from a subject, or can be cultured in vitro prior to transfection to allow further characterization of the tumor cells (eg, measurement of expression of cell surface molecules). it can. The nucleic acid selected for transfection can be determined after characterization of the protein expressed by the tumor cell. For example, the expression of MHC protein on the cell surface of the tumor cell and / or the expression of Ii protein in the tumor cell can be evaluated. Tumors that express no or limited amounts or types of MHC molecules (class I or class II) can be transfected with nucleic acids encoding MHC proteins, and tumors that express Ii proteins It can be transfected with a sense sequence. If necessary, tumor cells can be screened for introduction of the nucleic acid after transfection by using a selectable marker (eg, drug resistance) introduced into the tumor cell along with the nucleic acid of interest.

  Prior to administration to a subject, treatment can be performed to prevent further modification of the modified tumor cells within the subject, thereby preventing possible growth of the modified tumor cells. Possible treatments are irradiation or mitomycin C treatment, which destroys the proliferative capacity of the tumor cells while inducing antigen-specific and costimulatory signals in T cells, thus stimulating the immune response Maintain the ability of tumor cells.

  The modified tumor cells can be administered to a subject by injecting the tumor cells into the subject. The route of injection can be, for example, intravenous, intramuscular, intraperitoneal or subcutaneous. Administration of modified tumor cells to the site of the original tumor can be beneficial for the induction of a local T cell-mediated immune response against the original tumor. Administration of modified tumor cells in a disseminated manner, such as intravenous injection, provides systemic anti-tumor immunity and can further protect against metastatic spread of tumor cells from the original site. The modified tumor cells can be administered before or in conjunction with other therapeutic forms, or can be administered after other treatments such as chemotherapy or surgical insult.

In addition, one or more types of modified tumor cells can be administered to a subject. For example, an effective T cell response may require exposure to one or more types of costimulatory molecules. Furthermore, continuous exposure of T cells over time to different costimulatory molecules may be important for the generation of effective responses. For example, upon activation, B cells are known to express B7-2 early in the response (about 24 hours after stimulation). Subsequently, B7-1 and B7-3 are expressed by B cells (approximately 48-72 hours after stimulation). That is, T cells may need to be exposed to B7-2 early in the induction of an immune response by exposure to B7-1 and / or B7-3 during the immune response. Thus, different types of modified tumor cells can be administered at different times in order 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. After this administration, it expresses B7-3 from the same tumor (alone or B7
Tumor cells that have been modified to (with -1) can be administered to the subject.

  Another method of treating a subject with a tumor modifies tumor cells in vivo to express B7-2 and / or B7-3 alone or in combination with inhibitors of B7-1, MHC molecules and / or Ii expression. It is to be. This method involves the use of vectors and delivery methods effective for in vivo gene therapy as described in the previous section of this specification to provide tumor cells by providing nucleic acids encoding the protein to be expressed. It may include modifying in vivo. Alternatively, one or more substances that induce or enhance the 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 according to the invention can also be used in methods for preventing or treating metastatic dispersion of tumors or for preventing or treating tumor recurrence. As demonstrated in detail in one of the examples below, anti-tumor immunity induced by B7-1 expressing tumor cells, regardless of whether the re-exposed tumor cells express B7-1 or not. Effective for subsequent challenge with tumor cells. That is, administration of a modified tumor cell as described herein or in vivo modification of a tumor cell is directed against cells of the original, unmodified tumor as well as metastasis of the original tumor or possible regrowth of the original tumor. Tumor immunity can be provided.

The present invention further provides compositions and methods for specifically inducing an anti-tumor response in CD4 ⁺ T cells. CD4 ⁺ T cells are activated by antigens associated with MHC class II molecules. Binding of peptide fragments of TAA to MHC class II molecules yields recognition of these antigenic peptides by CD4 ⁺ T cells. For tumor cells modified to express MHC class II molecules with B7-2 and / or B7-3 or modified in vivo to express MHC class II molecules with B7-2 and / or B7-3 Is useful for directing presentation of tumor antigens to the MHC class II pathway, which results in antigen recognition and activation by CD4 ⁺ T cells specific for tumor cells. In vivo depletion of either CD4 ⁺ or CD8 ⁺ T cells with anti-CD4 or anti-CD8 antibodies is used to demonstrate that specific anti-tumor immunity is mediated by specific (eg, CD4 ⁺ ) T cell subpopulations. be able to.

Subjects initially exposed to modified tumor cells exhibit an anti-tumor specific T cell response that is effective against subsequent exposure to unmodified tumor cells. That is, the subject exhibits anti-tumor specific immunity. The general use of the modified tumor cells according to the present invention from one human subject as an immunogen to induce anti-tumor immunity in another human subject is due to differences in histocompatibility between unrelated humans. It is blocked. However, in order to protect against possible future tumor appearance, using modified tumor cells from one individual to induce anti-tumor immunity in another is useful in the case of familial malignancies It may be. In this situation, the tumor-bearing donor of the tumor cell to be modified has a close relationship with the recipient of the modified tumor cell (no tumor), so the donor and recipient share the MHC antigen. To do. Strong genetic elements have been identified in certain types of malignant tumors, such as certain breast and rectal cancers. In a family member who is known to be susceptible to a particular malignancy and one person currently has a tumor, tumor cells from that person can be B7-2 and / or B7-3 alone or B7-1. It can be modified to express in combination and administered to susceptible histocompatibility family members to induce the recipient's anti-tumor immune response to the type of tumor that the family is susceptible to. This anti-tumor response may provide protective immunity against subsequent tumor appearance in the immunized recipient.
(15) Tumor-specific T cell tolerance.

  In the case of experimentally derived tumors, a subject (eg, a mouse) can be exposed to the modified tumor cells of the present invention before being challenged with unmodified tumor cells. That is, the subject is first exposed to the TAA peptide on the tumor cells along with B7-2 and / or B7-3 and B7-1 that activate TAA-specific T cells. The activated T cells are then effective for subsequent challenge with unmodified tumor cells. In the case of spontaneous tumors, as in human patients, the subject's immune system is exposed to unmodified tumor cells prior to exposure to the modified tumor cells of the present invention. That is, the subject is first exposed to the TAA peptide on the tumor cells in the absence of a costimulatory signal. This situation appears to induce TAA-specific T cell tolerance in T cells that have been exposed to and in contact with unmodified tumor cells. A subject's secondary exposure to a modified tumor cell capable of inducing a costimulatory signal may not be sufficient to overcome the tolerance of TAA-specific T cells anergized by primary exposure to this tumor. Thus, the use of modified tumor cells to induce anti-tumor immunity in subjects already exposed to unmodified tumor cells was diagnosed early with a small tumor burden, eg, a small localized tumor that has not metastasized Can be most effective in patients. In this situation, the tumor cells are confined to a limited area of the body and therefore only a portion of the T cell repertoire is exposed to the tumor antigen and anergized. For example, administration of modified tumor cells by systemic methods following surgical removal of localized tumors and modification of isolated tumor cells can result in non-anergic T cells alone or in combination with B7-1. -2 and / or B7-3 can be exposed to tumor antigens, thereby inducing an anti-tumor response in non-anergic T cells. This anti-tumor response may be effective against potential tumor regrowth or against micrometastases of the original tumor that may not have been detected. In order to overcome widespread peripheral T cell tolerance to tumor cells in a subject, additional signals such as cytokines may need to be provided to the subject along with the modified tumor cells. Cytokines that function as T cell growth factors, such as IL-2, can be provided to a subject along with modified tumor cells. In an in vitro system, it has been shown that IL-2 can restore allergen-specific responses of previously anergized T cells when exogenous IL-2 is added at the time of secondary alloantigen stimulation. P. Tan et al. Exp. Med. 177: 165-173 (1993).

Another approach for generating an anti-tumor T cell response in a subject despite tolerance of the subject's T cells to the tumor is an anti-tumor response in T cells from another subject that has not been exposed to the tumor (referred to as a naive donor). And transferring the stimulated T cells from this naïve donor to a subject with a tumor so that the transferred T cells can be equipped with an immune response against the tumor cells. By stimulating the T cells in vitro with the modified tumor cells according to the present invention, an antitumor response is induced in naive donor-derived T cells. Such adoptive transfer approaches are generally hampered in outbred populations due to differences in histocompatibility between transfected T cells and tumor recipients. However, advances in allogeneic bone marrow transplantation can be applied to this situation to allow recipients to accept adoptively transferred cells and prevent graft-versus-host disease. First, a tumor-bearing subject (referred to as a host) is prepared for allogeneic bone marrow transplantation from a naive donor by a known method, and transplantation is performed. Host preparation involves whole body irradiation, which destroys the host's immune system, including T cells that are well tolerated against the tumor and the tumor cells themselves. Bone marrow transplantation is treatment to prevent graft-versus-host disease such as depletion of mature T cells from bone marrow transplants, treatment of the host with immunosuppressive agents, or to induce tolerance of donor T cells to host tissues. With treatment of the host with a substance such as CTLA4Ig. In order to provide the host with tumor cells remaining in the host or anti-tumor specific T cells that can respond to regrowth or metastasis of the host's original tumor, T cells from naive donors are herein described Stimulation in vitro by tumor cells from this host modified to express B7-2 and / or B7-3 as described. That is, donor T cells are first exposed to a tumor cell with a costimulatory signal and are therefore activated to respond to the tumor cell. These activated anti-tumor specific T cells are then transferred to a host that is reactive against unmodified tumor cells. Since the host has been reconstituted by the donor's immune system, the host does not reject the transferred T cells, and further, the host treatment to prevent graft-versus-host disease can be performed with the transferred T cells. Will interfere with reactivity with normal host tissue.
H. Administration of B lymphocyte antigen pharmaceutical preparation

  The peptide of the present invention is administered to a subject as a biologically adaptive dosage form suitable for in vivo pharmaceutical administration for enhancing or suppressing an immune response mediated by T cells. “Biologically adaptable dosage form suitable for in vivo administration” means a formulation of a protein to be administered whose therapeutic effect outweighs the toxic effect. The term subject is intended to include organisms that elicit an immune response, such as mammals. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Administration of a peptide having the novel B lymphocyte antigen activity described herein includes a therapeutically active amount of the peptide alone or in combination with a peptide having the activity of another B lymphocyte antigen and a pharmaceutically acceptable carrier. Any pharmaceutical dosage form can be taken. Administration of a therapeutically effective amount of the therapeutic composition of the present invention is defined as an amount effective at the dose and time required to obtain the desired result. For example, a therapeutically effective amount of a peptide having B7-2 activity may also vary depending on factors such as the disease state, age, sex, and individual weight and the ability of the peptide to elicit the desired response in the individual. Dosage ranges may be adjusted to provide the optimum therapeutic response. For example, the dose may be administered in several divided doses every day, or the dose may be reduced in proportion to what the treatment status indicates.

The active compound (eg, peptide) can also be administered in a convenient manner such as injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal administration, or rectal administration. Depending on the route of administration, the active compound may be coated with a substance that protects the compound from the action of enzymes and acids and other natural conditions that may inactivate the compound.
In order to administer a peptide having B7-2 activity other than by parenteral administration, it may be necessary to coat with a substance that prevents inactivation of the peptide or to administer it at the same time. For example, a peptide having B7-2 activity is administered to an individual in a suitable carrier, diluent or adjuvant, is administered concurrently with an enzyme inhibitor, or is administered in a suitable carrier such as a liposome. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Adjuvant is understood in the broadest sense and includes any immunostimulatory compound such as interferon. The adjuvants here are also intended to include non-ionic surfactants such as resorcinols, polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzyme inhibitors also include pancreatic trypsin inhibitor, diisopropyl fluorophosphate (DEP) and trasilol. Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes (Strejan et al. (1984), J. Neuroimmunol., 7:27).

  The active compound can also be administered parenterally or intraperitoneally. Dispersants can also be prepared in glycerin, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations can also contain a preservative to prevent the growth of microorganisms.

  Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. 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 prevented from contamination by microorganisms such as bacteria and mold. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and mixtures thereof. The proper fluidity can be maintained, for example, by using a coating agent such as lecithin, maintaining the required particle size in the case of a dispersant, and using a surfactant. Prevention of the action of microorganisms can be achieved, for example, by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Long-term absorption of injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

  A sterile injectable solution is prepared by adding an appropriate amount of an active compound (for example, a peptide having B7-2 activity) to a suitable solvent containing one or a combination of the above components, if necessary, followed by sterile filtration. Can do. Generally, dispersions are prepared by adding the active compound to a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the production of sterile injectable solutions, the preferred process is vacuum drying and lyophilization, giving the active ingredient (eg peptide) plus the desired additional ingredient powder from a pre-sterilized filtered solution .

  When the active compound is suitably protected as described above, the protein can also be administered orally, 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. Unless the active compound is contraindicated in its usual vehicle or drug, it is intended for use in the therapeutic composition. Supplementary active compounds can also be added to the compositions.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. The unit dosage form used herein is suitable for uniform administration to the mammalian subject to be treated, with a predetermined amount of active compound calculated in advance and the necessary pharmaceuticals necessary for each unit to obtain the desired therapeutic effect. Physically distinct units that are included with the carrier. The unit dosage form formulations of the present invention are essential to the technology of mixing active compounds for (i) unique properties of the active compounds and the specific therapeutic effects to be achieved and (b) treatment for the sensitivity of each individual. It is determined by the restrictions and depends directly.
I. Recognition of cytokines induced by costimulation

  Cytokines produced by T cells in response to stimulation by a type of B lymphocyte antigen, such as B7-2, using a nucleic acid sequence encoding a peptide having novel B lymphocyte antigen activity described herein Can be recognized. T cells are subject to suboptimal stimulation by primary activation signals such as phorbol esters, anti-CD3 antibodies or preferably antigens and MHC class II molecules in vitro, eg peptides with B7-2 activity A costimulatory signal is received from a stimulated form of the B7-2 antigen, such as by a cell transfected with a nucleic acid encoding and expressing a peptide on the surface, or by a soluble stimulated form of the peptide. Known cytokines released into the medium can be recognized by ELISA and by the ability of antibodies to block cytokines that inhibit T cell proliferation and the ability of antibodies to inhibit the proliferation of other cell types induced by cytokines . IL-4 ELISA kits and IL-7 blocking antibodies are available from Genzyme (Cambridge, MA). Blocking antibodies against IL-9 and IL-12 are available from Genetics Institute (Cambridge, MA).

  The in vitro T cell costimulation assay can also be used in methods for recognizing novel cytokines that can also be induced by costimulation. If a specific activity induced by costimulation, such as T cell proliferation, cannot be blocked by the addition of blocking antibodies to known cytokines, the activity may be due to the action of unknown cytokines. After costimulation, this cytokine should be purified from the medium by conventional methods and its activity measured by its ability to induce T cell proliferation.

The in vitro T cell costimulation assay described above can be used to recognize cytokines that prevent induction of tolerance. In this case, the T cell is given a primary activation signal and subsequently contacted with the selected cytokine without giving a costimulatory signal. After the T cells are washed and rested, the cells are given both a primary activation signal and a costimulatory signal. If the T cell does not respond (eg, proliferate or produce IL-2), the cell has acquired resistance and the cytokine did not prevent tolerance induction. However, if T cells respond, the cytokines have prevented tolerance induction. Cytokines that can prevent tolerance induction are targeted for in vivo blockade associated with drugs that block B lymphocyte antigens as a more effective means to induce tolerance in transplant recipients or patients with autoimmune disease be able to. For example, a B7-2 blocking agent could be administered to a subject along with a cytokine blocking antibody.
J. et al. Recognition of molecules that prevent costimulation

Another application of peptides with the activity of the novel B lymphocyte antigens of the present invention (eg, B7-2 and B7-3) is to inhibit one or more of these peptides as costimulatory ligand binding inhibitors and / or costimulatory. It is to be used in screening assays to discover undiscovered molecules that are later inhibitors of intracellular signaling in T cells. For example, solid phase binding assays using peptides with B lymphocyte antigen activity, such as B7-2, recognize molecules that block the binding of that antigen to an appropriate T cell ligand (eg, CTLA4, CD28). Could be used to do. In addition, the in vitro T cell costimulation assay described above is determined by the ability of the molecule to inhibit T cell proliferation and / or cytokine production (but does not prevent binding of B lymphocyte antigen to its receptor). Could be used to find molecules that inhibit intracellular signaling through T cells after costimulation. For example, cyclosporin A compounds block T cell activation by stimulation via the T cell receptor pathway rather than the CD28 / CTLA4 pathway. Therefore, costimulation involves different intracellular signaling pathways. Molecules that inhibit intracellular signaling through the CD28 / CTLA4 pathway would be effective as immunosuppressants (similar to the effects of cyclosporin A) in vivo.
K. Recognition of molecules that regulate B lymphocyte antigen expression

Monoclonal antibodies produced using the proteins and peptides of the present invention can be used in screening assays for molecules that regulate B lymphocyte antigen expression on cells. For example, molecules that cause intracellular signaling leading to B lymphocyte antigen induction, such as B7-2 or B7-3, can be recognized by assaying the expression of one or more B lymphocyte antigens on the cell surface. . A reduction in immunofluorescence staining with anti-B7-2 antibody in the presence of a molecule appears to indicate that the molecule blocks intracellular signals. Molecules that enhance B lymphocyte antigen expression result in enhanced immunofluorescent staining. Alternatively, the effect of certain molecules on the expression of B lymphocyte antigens such as B7-2 can be determined by detecting cellular B7-2 mRNA levels using B7-2 cDNA as a probe. For example, a cell expressing a peptide having B7-2 activity is brought into contact with a test molecule, and a B7-2 cDNA probe labeled with a marker capable of detecting the increase or decrease of the B7-2 mRNA concentration in the cell by Northern hybridization analysis is detected. It can be detected by standard techniques such as a conventional dot blot assay of the mRNA or total poly (A ⁺ ) RNA used. Molecules that modulate B lymphocyte antigen expression may be useful therapeutically to enhance or attenuate the immune response, alone or in combination with soluble blockers or stimulants. For example, a molecule that blocks the expression of B7-2 could be administered with a B7-2 blocking agent for immunosuppression purposes. Molecules that can be tested in the above assay include cytokines such as IL-4, γINF, IL-10, IL-12, GM-CSF and prostaglandins.

The present invention is illustrated by the following examples, which are not intended to be limiting. The contents of all references and published patent applications cited herein are for reference only.
In Examples 1, 2 and 3, the following methodology was used.

Methods and materials
A. Cell mononuclear cells were isolated from normal human spleen free cell suspension by Ficoll-Hypaque density gradient centrifugation and separated into E- and E + fractions by rosette formation with sheep erythrocytes (Boyd, A. W. et al. (1985), J. Immunol., 134: 1516). B cells are attached to monocyte plastics from the E-fraction and anti-MsIgG and anti-MsIgM coated magnetic beads (Advanced Magnetics, using anti-CD4, -CD8, -CD11b, -CD14 and -CD16 monoclonal antibodies, Purification by removal of residual T, natural killer cells (NK) and residual monocytes by two treatments with Cambridge, MA). CD4 + T cells were isolated from the same splenic E + fraction after adherence to plastic and removal of NK, B cells and residual monocytes with magnetic beads and anti-CD20, -CD11b, -CD8 and CD16 monoclonal antibodies. CD28 + T cells were similarly isolated from this E + fraction using anti-CD20, CD11b, -CD14 and -CD16 monoclonal antibodies. The purification effect was analyzed by indirect immunofluorescence and flow cytometry using an EPICS flow cytometer (Coulter). B cell products were> 95% CD20 +, <2% CD3 +, <1% CD14 +. CD4 + T cell products were> 98% CD3 +,> 98% CD4 +, <1% CD8 +, <1% CD20 +, <1% CD14 +. CD28 + T cell products were> 98% CD3 +,> 98% CD28 +, <1% CD20 +, <1% CD14 +.
B. Monoclonal antibodies and fusion proteins

Unless stated otherwise, monoclonal antibodies were used as purified Ig: anti-B7: 133, IgM was a blocking antibody and has been previously reported (Freedman, AS et al. (1987), Immunol. 137: 3260-3267); anti-B7: B1.1, IgG1 (RepliGen, Cambridge, MA) (Nickoloff, B et al. (1993), Am. J. Pathol., 142: 1029-1040. Page) is a non-blocking monoclonal antibody; BB-1: IgM is a blocking antibody (Dr. E. Clark, University of Washington, Seattle, WA) (Yokochi, T et al. (1982), J. Immunol., 128: 823-827); anti-CD20: B1, IgG2a (Stash nko, P et al. (1980), J. Immunol., 125: 1678-1685); anti-B5, IgM (Freedman, A. et al. (1985), J. Immunol., 134: 2228-2235. Anti-CD8: 7PT3F9, IgG2a; anti-CD4: 19Thy5D7, IgG2a; anti-CD11b: Mo1, IgM and anti-CD14: Mo2, IgM (Todd, R et al. (1981), J. Immunol., 126. Anti-MHC class II: 9-49, IgG2a (Dr. R. Todd, University of Michigan, Ann Arbor), (Todd, RI, et al. (1984), Hum. Immunol. 10: 23-40); anti-CD28: 9.3, IgG2a (Dr C. June, Naval Research Institute, Bethesda), (Hansen, JA et al. (1980), Immunogenetics, 10: 247-260); anti-CD16: 3G8, IgG1 (used as ascites) (Dr. J. Ritz, Dana-Farber, Cancer, Institute, Boston); anti-CD3: OKT3, IgG2a hybridomas were obtained from the American Type Culture Collection and purified monoclonal antibodies were attached to plastic plates at a concentration of 1 μg / mL. It was. The anti-CD28 Fab fragment was generated from the 9.3 monoclonal antibody by papain digestion according to the manufacturer's instructions (Pierce, Rockford, IL) and purified on a protein A column. Human CTLA4 fusion protein (CTLA4Ig) and control fusion protein (control-Ig) have been previously reported (Gimmi, CD et al. (1993), Proc. Natl. Acad. Sci. USA, 90: 6586-6590. ) Produced as described above. Bouusiotis, V. J. et al. Exp. Med. (Accepted for printing).
C. Transfection of CHO cells

The B7-1 transfectant (CHO-B7) was prepared from a B7-1 negative Chinese hamster ovary (CHO) cell line, fixed with paraformaldehyde, and a previous report (Gimmi, CD et al. (1993). Proc. Natl. Acad. Sci. USA, 88: 6575-6579).
D. B cell activation and selection of B7 + and B7− cells in vitro

Spleen B cells were cultured in complete culture medium in tissue culture flasks {RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM glutamine, 1 mM sodium pyruvate, penicillin (100 units / mL), streptomycin sulfate (100 μg / mL) and gentamicin sulfate (5 μg / mL)} to 2 × 10 ⁶ cells / mL and sIg Affi-Gel702 beads (Bio-Rad, Richmond, Calif.) (Boyd, A. W. et al. ( 1985), J. Immunol., 134: 1516) by cross-linking with affinity purified rabbit anti-human IgM or cross-linking with MHC class II 9-49 antibody bound to Affi-Gel702 beads. Activated by binding. B cells are activated for 72 hours and used as an activated total B cell population or goat anti-mouse immunoglobulin (Fisher) labeled with anti-B7 (B1.1) monoclonal antibody and fluorescein isothiocyanate (FITC). (Inc., Pittsburg, Pa.) And indirectly fractionated into B7-1 + and B7-1− populations by flow cytometry cell sorting (EPICS, Elite, flow, cytometer, Coulter).
E. Immunofluorescence and flow cytometry

For surface phenotype analysis, populations of B cells activated by MHC class II or sIg cross-linked for 6, 12, 24, 48, 72 and 94 hours were treated with anti-B7 (133), BB-1 monoclonal antibody, control IgM. Stained with antibody, CTLA4Ig or control-Ig. The cell suspension was stained with a 10 μg / mL primary monoclonal antibody followed by a two-step indirect membrane staining method with an appropriate secondary reagent. Specifically, immunoreactivity with anti-B7 (133) and BB-1 monoclonal antibodies was examined by indirect staining using goat anti-mouse Ig or immunoglobulin FITC (Fisher) as a secondary reagent, and fused. Immunoreactivity with the protein was examined using biotinylated CTLA4Ig or biotinylated control-Ig and streptavidin-phycoerythrin as secondary reagents. For dilution and washing, PBS supplemented with 10% AB serum was used. Cells were fixed with 0.1% paraformaldehyde and analyzed with a flow cytometer (EPICS, Elite, Coulter).
F. Proliferation assay

T cells are 37 ° C. at a concentration of 1 × 10 ⁵ cells per 96-well flat bottom microtiter plate hole
And cultured in 5% CO ₂ for 3 days. Genotype activated B cells (total B cell population or B7 + and B7− fractions) were irradiated (2500 rads) and added to the medium at a concentration of 1 × 10 ⁵ cells per well. Study factors were added at the concentration required to give a final total volume of 200 μL per well. When indicated, T cells were incubated with anti-CD28 Fab (final concentration 10 μg / mL) for 30 minutes at 4 ° C. and subsequently added to the laboratory plates. Similarly, CHO-B7 or B cells were incubated with CTLA4Ig or control Ig (10 μg / mL) for 30 minutes at 4 ° C. The thymidine incorporation as an indication of mitogenic activity in the final 15 hours of culture {methyl - ^{3 H}} thymidine (Du
• Pont, Boston, MA) Evaluated after incubation with 1 μCi (37 kBq). Cells were harvested by filtration and the radioactivity on the dry filter plate was measured in a Pharmacia beta plate liquid scintillation counter.
G. IL-2 and IL-4 assays

  The concentrations of IL-2 and IL-4 were assayed in the supernatant collected 24 hours after the start of culture by ELISA (R & D Systems, Minapolis, MN and BioSource, Camarillo, CA).

Example 1 Activation of a novel CTLA4 ligand that induces T cell proliferation Expression on activated B cells Cross-linked surface Ig is such that human resting B cells express B7-1 maximally (50-80%) at 72 hours. Therefore, the ability of activated human B lymphocytes to proliferate submitogenically activated T cells and secrete IL-2 was measured. FIG. 1 shows spleenmitogenically activated human splenic CD28 + T cells B7 (B7-1) transfected with CHO cells (CHO-B7) or anti-Ig activated for 72 hours with anti-CD3 monoclonal antibody. 2 shows costimulatory responses in splenic B cells of the same genotype. ³ H-thymidine incorporation was assayed during the final 15 hours of 72 hour culture. IL-2 was assayed with the supernatant after 24 hours of culture by ELISA (detection limit: 31 to 2000 pg / mL). FIG. 1 shows the results of 17 experiments.

  Semi-mitogenically activated CD28 + T cells proliferated in response to B7-1 costimulation given by CHO-B7 (FIG. 1, a) and secreted high concentrations of IL-2. Both proliferation and IL-2 secretion were completely blocked by blocking the B7-1 molecule on CHO cells with anti-B7-1 monoclonal antibody or with the fusion protein of CTLA4, which is a high affinity receptor. Similarly, proliferation and IL-2 secretion were removed by blockade of B7-1 signaling via the Fab anti-CD28 monoclonal antibody CD28. Control monoclonal antibody or control fusion protein was ineffective. Almost the same costimulation of proliferation and IL-2 secretion was shown by splenic B cells activated with anti-Ig for 72 hours (FIG. 1, b). The anti-B7-1 monoclonal antibody was able to completely eliminate the proliferation and IL-2 secretion caused by CHO-B7, whereas the anti-B7-1 monoclonal antibody showed 50% proliferation induced by activated B cells. Only reliably blocked, while IL-2 secretion was completely blocked. In contrast to the partial block of proliferation induced by anti-B7-1 monoclonal antibody, both CTLA4Ig and Fab anti-CD28 monoclonal antibody completely blocked proliferation and IL-2 secretion. These results fit the hypothesis that activated human B cells express one or more additional CTLA4 / CD28 ligands capable of inducing T cell proliferation and IL-2 secretion.

Example 2 From the above observation that activated human splenic B cells express a CTLA4 ligand different from B7-1, it was decided whether another CTLA4-binding counter-receptor would be expressed on activated B cells. . For this, human splenic B cells were activated with anti-Ig for 72 hours and then stained with an anti-B7-1 monoclonal antibody (B1.1) that does not block costimulation via B7-1. With fluorescein isothiocyanate (FITC) and MAbB1.1, performs cell selection by flow cytometry B7-1 ⁺ fraction and B7-1 ^- to separate the fractions. The resulting post-separation positive population was 99% B7-1 ⁺ and the post-separation negative population was 98% B7-1 ⁻ (FIG. 2).

To examine the costimulatory capacity of each population, human splenic CD28 + T cells were irradiated with B7-1 + or B7-1-anti-Ig activated (72 hours) with anti-CD3 monoclonal antibody in the presence of splenic B cells. Stimulated. ³ H-thymidine incorporation was studied during the last 15 hours of 72 hour culture. IL-2 was evaluated for the supernatant after 24 hours of culture by ELISA (detection limit of assay: 31 to 2000 pg / mL). FIG. 3 shows the results of 10 experiments. B7-1 + B cells induced proliferation and secretion of IL-2 in anti-CD3 activated T cells (FIG. 3a), but not IL-4. As observed in the unfractionated activated B cell population, the anti-B7-1 monoclonal antibody (133) blocked growth by only 50%, but IL-2 secretion was well extinguished. As described above, CTLA4Ig binding or blockade of CD28 by Fab anti-CD28 monoclonal antibody completely blocked proliferation and IL-2 secretion. Control monoclonal antibody and control-Ig were not inhibitory. In an attempt to discover other potential CTLA4 / CD28 binding costimulatory ligands that may be responsible for residual non-B7 mediated proliferation caused by B7 + B cells, the BB-1 monoclonal antibody against proliferation and IL-2 secretion The effect was tested. As mentioned above, the BB-1 monoclonal antibody completely blocked proliferation and IL-2 secretion (FIG. 3a). FIG. 3b presents the costimulatory capacity of B7-1-activated human splenic B cells. Irradiated B7-1-activated (72 hours) B cells will transmit a clear costimulatory signal to quasi-mitogenic activated CD4 + lymphocytes. This costimulation was not accompanied by detectable IL-2 (FIG. 3b) or IL-4 accumulation, and the anti-B7-1 monoclonal antibody did not block proliferation. However, CTLA4Ig, Fab anti-CD28 monoclonal antibody and BB-1 monoclonal antibody all completely blocked proliferation.

B7-1 ⁺ and B7-1 ^- phenotype analysis of activated splenic B cells was confirmed the functional consequences. Figure 4 is fractionated B7-1 ⁺ and B7-1 ^- shows the B7-1, cell surface expression of B7-2 and B7-3 on activated B-cells. As shown in FIG. 4, B7-1 + activated splenic B cells were stained with anti-B7-1 (133) monoclonal antibody, BB-1 monoclonal antibody and conjugated CTLA4Ig. In contrast, B7-activated splenic B cells did not stain with anti-B7-1 (133) monoclonal antibody, but stained with BB-1 monoclonal antibody and CTLA4Ig. These phenotypes and functional outcomes are accompanied by detectable IL-2 secretion in B7-1 + and B7-1-1-activated (72 hours) human B lymphocytes 1) quasi-mitogenically activated T cells. 2) demonstrates that it expresses a CTLA4 binding counter-receptor that is recognized by the BB-1 monoclonal antibody but not the anti-B7-1 monoclonal antibody. Thus, these CTLA4 / CD28 ligands can be distinguished based on their transient expression after B cell activation and their reactivity with CTLA4Ig and anti-B7 monoclonal antibodies. FIG. 4 shows the results of five experiments.

Example 3 After human B cell activation , human spleen B cells were cross-linked with surface Ig or MHC class II to determine the sequential expression of post-activation CTLA4 binding counterreceptors expressed by three different CTLA4 / CD28 ligands. Activated by binding, the expression of B7-1, B7-3 and B7-2 binding proteins was examined by flow cytometric analysis. Ig or MHC class II cross-linking induced a similar pattern of CTLA4Ig binding (FIGS. 5 and 6). FIG. 5 represents the results of 25 experiments for anti-B7-1 and BB-1 binding and 5 experiments for CTLA4Ig binding. FIG. 6 shows the results of 25 experiments for anti-B7-1 binding and 5 experiments for CTLA4Ig binding. These experimental results indicate that none of these molecules are expressed before 24 hours. Many cells express a protein that binds to CTLA4Ig 24 hours after activation (B7-2), however, 20% or less express B7-1 or B7-3. MHC class II cross-linking induces maximal B7-1 and B7-3 at 48 hours, Ig cross-linking induces maximal expression at 72 hours, after which expression decreases. These results suggest that a new type of CTLA4 binding counterreceptor is expressed at 24 hours and appears to be temporally consistent with the temporal expression of different types of B7-1 and B7-3 proteins.

A series of experiments was performed to determine if the correlation between transient expression of this CTLA4 binding counter-receptor and the ability to costimulate T cell proliferation and / or IL-2 secretion was different. Human splenic CD28 + T cells stimulated quasimitically with anti-CD3 were cultured for 72 hours in the presence of irradiated human spleen B cells that had been activated by cross-linking with sIg in vitro for 24, 48 or 72 hours. . IL-2 secretion was assessed by ELISA in the supernatant after 24 hours, and T cell proliferation was assessed by ³ H-thymidine incorporation during the final 15 hours of 72 hours in culture. FIG. 7 shows the results of five experiments. As can be seen in FIG. 7a, 24 hour activated B cells gave a costimulatory signal with moderate levels of IL-2 production, but the extent of their proliferation was 48 and 72 hour activated human B cells. Significantly less than observed (note the scale of ³ H-thymidine incorporation). Neither anti-B7-1 (133) or BB-1 blocked growth or IL-2 accumulation. In contrast, CTLA4Ig and anti-CD28 Fab monoclonal antibody completely abolished proliferation and IL-2 accumulation. B cells activated for 48 hours caused costimulation, resulting in almost maximum proliferation and IL-2 secretion (FIG. 7b). Here, anti-B7-1 (133) monoclonal antibody blocked growth by approximately 50%, but completely blocked IL-2 accumulation. The BB-1 monoclonal antibody completely blocked proliferation and IL-2 secretion. As mentioned above, CTLA4Ig and Fab anti-CD28 also completely blocked proliferation and IL-2 production. Finally, 72 hour activated B cells induced the same T cell response as that induced by 48 hour activated B cells. Similar results are obtained if a quasi-mitogenic signal is transmitted from phorbol myristate acetate (PMA) and if human spleen B cells are not Ig cross-linked and activated by MHC class II. Observed. These results indicate that there are three types of CTLA4 binding molecules, which are transiently expressed on activated B cells and each can stimulate and induce quasi-mitogenic proliferation in T cells. Two of these molecules, early CTLA4 binding counter receptors (B7-2) and B7-1 (133) induce IL-2 production, while B7-3 has no detectable IL-2 production. Induces proliferation.

  Previous reports have provided conflicting evidence as to whether anti-B7 monoclonal antibody 133 and monoclonal antibody BB-1 recognize the same molecule (Freedman, AS et al. (1987), Immunol. 137: 3260-3267; Yokochi, T. et al. (1982), J. Immunol., 128: 823-827; Freeman, G. J. et al. (1989), J. Immunol., 143: 2714-2722). Both monoclonal antibodies recognize molecules expressed 48 hours after activation of human B cells, but some reports indicate that B7 (B7-1) and the molecule recognized by monoclonal antibody BB-1 depend on the cell line and B cells. It has been suggested that it is different because it is expressed differently on neoplasms (Freedman, AS et al. (1987), Immunol., 137: 3260-3267; Yokochi, T. et al. (1982). J. Immunol., 128: 823-827; Freeman, G. J. et al. (1989), J. Immunol., 143: 2714-2722; Clark, E. and Yokochi, T .; (1984), Leukocyte Typing, 1st International Reference · Workshop, pp. 339~346;. Clark, E, etc. (1984), Leukocyte · Typing, 1st · International · References · Workshop, 740 pages). In addition, immunoprecipitation and Western blotting of these IgM monoclonal antibodies suggested that they recognize different molecules (Clark, E. and Yokochi, T. (1984), Leukocyte Typing, 1st International References. Workshop, 339-346; Clark, E. (1984), Leukocyte Type, 1st International References, Workshop 740). The original anti-B7 monoclonal antibody 133 was generated by immunization with anti-immunoglobulin activated human B lymphocytes, whereas the BB-1 monoclonal antibody was generated by immunization in a baboon cell line. Therefore, the BB-1 monoclonal antibody should recognize human cell epitopes conserved between baboons and humans.

  After molecular cloning and expression of the human B7 gene (B7-1), B7 transfected COS cells were found to be similarly stained with anti-B7 (133) and BB-1 monoclonal antibodies, both of which are identical The broad molecular band (44-54 kD) was strongly precipitated, suggesting that they recognize the same molecule (Freeman, GJ et al. (1989), J. Immunol., 143: 2714-2722. ). In this discovery, the gene encoding the molecule recognized by the BB-1 monoclonal antibody was previously assigned to chromosome 12 (Katz, FE et al. (1985), Eur. J. Immunol., Pages 103-6). However, two groups placed the B7 gene on chromosome 3 (Freeman, GJ et al. (1992), Blood, 79: 489-494; Selvakumar, A. et al. (1992), Immunogenetics, 36: 175-181), which was unexpected. Subsequently, another difference was noted in phenotype expression between molecules recognized by B7 (B7-1) and the BB-1 monoclonal antibody. The BB-1 monoclonal antibody is derived from thymic epithelial cells (Turka, LA, et al. (1991), J. Immunol., 146: 1428-36; Munro, JM, et al., Blood, posted) (Nickoloff, B. et al. (1993), Am. J. Pathol. 142: 1029-1040; Augustin, M. et al. (1993), J. Invest. Dermatol. 100: 275-281. ), While anti-B7 did not stain it. Recently, Nickoloff et al. (1993), Am. J. et al. Pathol. 142: 1029-1040 reported incongruent expression of BB-1 monoclonal antibodies and the molecules recognized by B7 in keratinocytes using BB-1 and anti-B7 (B1.1 and 133) monoclonal antibodies. Nickoloff et al. Also demonstrated that these BB-1 positive cells do not express B7 mRNA but bind to CD28 transfected COS cells and there is additional evidence for the presence of a different protein that binds to monoclonal antibody BB-1.

  This discovery confirms that there is another CTLA4 counter receptor recognized by the BB-1 monoclonal antibody B7-3 and that this protein is functionally different from B7-1 (133). The expression of B7-1 and B7-3 after B cell activation appears to be temporally consistent in B7 positive B cells, but these studies indicate that B7-3 molecules are also expressed in B7 negative activated B cells. Prove that. More importantly, the B7-3 molecule appears to be able to induce T cell proliferation without detectable IL-2 or IL-4 production. This result is similar to the previous observation that ICAM-1 can costimulate T cell proliferation without producing detectable IL-2 or IL-4 (Boussiotis, V. et al., J. Exp. Med. In press). is doing. These data indicate that the BB-1 monoclonal antibody recognizes an epitope on the B7-1 protein and that this epitope has a costimulatory function but is also present on a different protein B7-3. Phenotype and blocking studies have demonstrated that the BB-1 monoclonal antibody can recognize one or both of these proteins (on B7 negative cells) (on B7 positive cells). In contrast, anti-B7 monoclonal antibodies 133 and B1.1 recognize only the B7-1 protein. Taken together, these results indicate that 48 hours after activation of B cells by surface immunoglobulin or MHC class II cross-linking, one is recognized by both anti-B7 and BB-1 monoclonal antibodies and the other. Suggests that it expresses at least two different CTLA4-binding counter-receptors, although only recognized by the BB-1 monoclonal antibody.

  The B7-2 antigen is not detected after 12 hours on activated B cells, but is strongly expressed and functions after 24 hours. This molecule appears to signal through CD28 because proliferation and IL-2 production are completely inhibited by the Fab anti-CD28 monoclonal. Since anti-B7 monoclonal antibody completely blocks IL-2 production, IL-2 secretion appears to be due to B7-1 costimulation 48 hours after activation.

  From the previous studies and the results described here, B7 (B7-1) is not expressed until 48 hours after B cell activation (Freeman, AS et al. (1987), Immunol., 137: 3260- 3267; Freedman, AS et al. (1991), Cell Immunol., 137: 429-437) It is demonstrated that T cell proliferation or IL-2 secretion cannot also be costimulated. Activation of T cells via TCR in the absence of costimulation from previous studies (Gimmi, CD et al. (1993), Proc. Natl. Acad. Sci. USA, 90: 6586-6590; Schwartz , RH et al. (1989), Cold Spring Harb. Symp. Quant. Biol., 54: 605-10; Beverly, B. et al. (1992), Int. 661-671) and the absence of IL-2 (Boussiotis, V. et al., J. Exp. Med., Published; Beverly, B. et al. (1992), Int. Immunol. 4: 661-671 Wood, M. et al. (1993), J. Exp. Med., 177: 597-603) It has been demonstrated that cause ghee. If B7-1 was the only costimulatory molecule capable of inducing IL-2 secretion, there was no B7-1 that costimulates IL-2 production, so the T cells were in the first 24 hours after activation. It seems that it will become anergy. Therefore, the presence of other early inducible costimulatory molecules that can costimulate IL-2 secretion during the first 24 hours is not anergy and may be necessary to induce an effective immune response. The appearance of an early CTLA4-binding counter-receptor, B7-2, between 12 and 24 hours of B cell activation serves this function.

  Two new findings have highlighted the biological and potential clinical implications of these two added CTLA4-binding counterreceptors. First, B7 (B7-1) deficient mice were developed, but their antigen-presenting cells were also found to bind to CTLA4Ig (Freeman and Sharpe, in preparation for submission). The mice are healthy and the isolated mononuclear cells induce detectable levels of IL-2 when cultured with T cells in vitro. Therefore, another CD28 costimulatory counter-receptor or another IL-2 production pathway should be functioning. Second, CTLA4Ig and Fab anti-CD28 are the most prominent reagents for inducing antigen-specific anergy in murine and human systems, whereas anti-B7 monoclonal antibodies are much weaker (Harding, FA; (1992), Nature, 356: 607-609; Lenschow, DJ et al. (1992), Science, 257: 789-792; Chen, L. et al. (1992), Cell, 71: 1093-1102; Tan, P. et al. (1993), J. Exp. Med., 177: 165-173). These observations are consistent with the hypothesis that there is another CTLA4 / CD28 ligand capable of inducing IL-2, and considering the results presented here, all three CTLA4-binding counter-receptors induce T cell immunity. Suggest that it will be essential. Furthermore, it seems necessary to block them for the induction of T cell anergy. Identical results were also observed in the murine system with the recognition of two CTLA4-binding ligands corresponding to human B7-1 and B7-2 molecules. APC of B7-deficient mice can bind to CTLA4 and induce IL-2 secretion. Taken together, these observations indicate that there are multiple CTLA4-binding counter receptors in the murine system, which in turn co-stimulate T cell activation.

Example 4 Cloning, sequencing and expression of B7-2 antigen
A. Construction of cDNA Library Using poly (A) ⁺ RNA from human anti-IgM activated B cells described in literature (Aruffo et al., Proc. Natl. Acad. Sci. USA, 84: 3365 (1987)) pCDM8 vector (Seed, Nature, 32
9: 840 (1987)). Spleen B cells were placed in a tissue culture flask in complete culture medium {RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM glutamine, 1 mM sodium pyruvate, penicillin (100 units / mL), streptomycin sulfate (100 μg / mL) and gentamicin sulfate (5 μg / mL)} to 2 × 10 ⁶ cells / mL and sIg and Affi-Gel · 702 beads (Bio-Rad, Richmond, Calif.) (Boyd, A.W. et al. (1985). Year), J. Immunol., 134: 1516) Binding affinity was activated by cross-linking with purified rabbit anti-human IgM. Activated B cells were harvested after 1/6, 1/2, 4, 8, 12, 24, 48, 72 and 96 hours.

Activated B cells were homogenized in a solution of 4M-guanidine thiocyanate, 0.5% sarcosyl, 25 mM EDTA, pH 7.5, 0.13% Sigma Antiform A and 0.7% mercaptoethanol to obtain RNA. Prepared. The RNA was purified by centrifuging this homogenate from 5.7 M-CsCl, 10 mM EDTA, 25 mM sodium acetate, pH 7 at 32,000 rpm for 24 hours. The RNA pellet was dissolved in 5% sarkosyl, 1 mM EDTA, 10 mM Tris, pH 7.5 and extracted with 2 volumes of 50% phenol, 49% chloroform, 1% isoamyl alcohol. RNA was precipitated twice from ethanol. The poly (A) ⁺ RNA used for construction of the cDNA library was purified by 2 cycles of oligo (dT) -cellulose selection.

Anti-IgM activated human B cell poly (A) ⁺ RNA 5.5 μg to 50 mM Tris, pH 8.3, 75 mM KCl, 3 mM MgCl ₂ , 10 mM dithiothreitol, 500 μM dATP, dCTP, dGTP, dTTP, Complementary DNA was synthesized at 37 ° C. for 1 hour in a reaction solution containing oligo (dT) _12-18 50 μg / mL, RNasin 180 units / mL, and Moloney-MLV reverse transcriptase 10000 units / mL in a total volume of 55 μL. After reverse transcription, the solution was treated with 25 mM Tris, pH 8.3, 100 mM KCl, 5 mM MgCl ₂ , 250 μM dATP, dCTP, dGTP, dTTP, 5 mM dithiothreitol, DNA polymerase I250 units / mL, ribonuclease H8 Adjusted to 5 units / mL and incubated at 16 ° C. for 2 hours to convert the cDNA to double stranded DNA. 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 2 volumes of ethanol in the presence of 2.5 M ammonium acetate and 4 μg of linear polyacrylamide as a carrier. Separately, 4 μg of anti-IgM activated human B cell poly (A) ⁺ RNA to 50 mM Tris, pH 8.8, oligo (dT) _12-18 50 μg / mL, RNasin 327 units / mL and AMV reverse transcriptase 952 units / mL CDNA was synthesized at 42 ° C. for 0.67 hours in the reaction solution contained in 100 μL. After reverse transcription, the reverse transcriptase was inactivated by heating to 70 ° C. for 10 minutes. 320 μL of water and 0.1 M Tris, pH 7.5, 25 mM MgCl ₂ , 0.5 M KCl, 250 μg / mL bovine serum albumin and 80 μL of 50 mM dithiothreitol were added, and the solutions were each 200 μM-dATP, The DNA was adjusted to dCTP, dGTP, dTTP, DNA polymerase I 50 units / mL, ribonuclease H8 units / mL, and incubated at 16 ° C. for 2 hours to convert cDNA into double-stranded DNA. 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 2 volumes of ethanol in the presence of 2.5 M ammonium acetate and 4 μg of linear polyacrylamide as support.

DNA from 4 μg AMV reverse transcription and 2 μg Moloney MLV reverse transcription was mixed. A non-self-complementary BstXI adapter was added to the DNA as follows. Polynucleotide (A) ⁺ RNA ⁶ μg of double-stranded cDNA kinased sequence CTTTAGAGCACA (SEQ ID NO: 15) oligonucleotide 3.6 μg and kinased sequence CTCTAAAG (SEQ ID NO: 16) oligonucleotide 2.4 μg 6 mM − Tris, pH 7.5, 6 mM MgCl ₂ , 5 mM NaCl, 350 μg / mL bovine serum albumin, 7 mM mercaptoethanol, 0.1 mM ATP, 2 mM dithiothreitol, 1 mM spermidine and a solution containing 600 units of T4 DNA ligase Incubated for 16 hours at 15 ° C. in a total volume of 0.45 mL. EDTA was added to 34 mM, and the solution was extracted with an equal volume of 50% phenol, 49% chloroform, 1% isoamyl alcohol. DNA was precipitated from 2 volumes of ethanol in the presence of 2.5M ammonium acetate.

  DNA greater than 600 bp was selected as follows: adapter DNA was redissolved in 10 mM Tris, pH 8, 1 mM EDTA, 600 mM NaCl, 0.1% sarkosyl, on Sepharose CL-4B column, same buffer Chromatography with DNA in the column void volume (containing DNA of 600 bp or more) was collected and ethanol precipitated.

The pCDM8 vector was prepared by BstXI digestion and agarose gel purification and used for cDNA cloning. Adapter DNA from ⁶ μg of poly (A) ⁺ RNA was added to 2.25 μg of BstXI-cut pCDM8 in 6 mM Tris, pH 7.5, 6 mM MgCl ₂ , 5 mM NaCl, 350 μg / mL bovine serum albumin, 7 mM mercaptoethanol, 0. The cells were ligated for 24 hours at 15 ° C. in a solution containing 600 units of 1 mM ATP, 2 mM dithiothreitol, 1 mM spermidine and T4 DNA ligase in a total volume of 1.5 mL. The ligated reaction mixture was transformed into sensitive E. coli MC1061 / P3 to obtain a total of 4290000 independent cDNA clones.
Plasmid DNA was prepared from 500 mL of a culture solution of a cDNA library transformant. Plasmid DNA was purified by alkaline degradation followed by banding separation with two CsCl equilibration gradients (Maniatis et al., Molecular Cloning: A Laboratory Laboratory, Cold Spring Harbor, NY (1987)).
B. Cloning procedure

  In the first stage of screening, 30 100 mm dishes of 50% confluent COS cells were treated with 0.05 μg / mL of anti-IgM activated human B cell library DNA using the DEAE-dextran method (Seed et al., Proc. Natl. Acad. Sci. USA, 84: 3365 (1987)). Cells were trypsinized and replated 24 hours later. After 47 hours, cells were detached by incubation in PBS / 0.5 mM EDTA, pH 7.4 / 0.02% sodium azide for 30 minutes at 37 ° C. The detached cells were treated with CTLA4Ig and CD28Ig 10 μg / mL for 45 minutes at 4 ° C. Cells were washed and spread onto culture dishes coated with affinity purified goat anti-human IgG antibody and allowed to attach at room temperature. After 3 hours, the plates were washed twice with PBS / 0.5 mM EDTA, pH 7.4 / 0.02% sodium azide, 5% FCS and 0.15 M NaCl, 0.01 M Hepes, pH 7.4, 5 Gently washed once with% FCS. Episomal DNA was collected from cultured (pan) cells and transformed into E. coli DH10B / P3. Plasmid DNA was reintroduced into COS cells by spheroplast fusion described in the literature (Seed et al., Proc. Natl. Acad. Sci. USA, 84: 3365 (1987)), and expression and culture were repeated twice. .

  In the second and third stages of selection, after 47 hours, detached COS cells were first incubated with α-B7-1 mAbs (133 and B1.1, 10 μg / mL) to express C7-1 cells expressing B7-1. Were removed with α-mouse IgG and IgM coated magnetic beads. COS cells were then treated with human CTLA4Ig (hCTLA4Ig) and human CD28Ig (hCD28Ig) 10 μg / mL and human B7-2 expressing COS cells were cultured and selected on goat anti-human IgG antibody plate dishes. After the third stage, plasmid DNA was prepared from each colony and transfected into COS cells by DEAE-dextran method. B7-2 expression on transfected COS cells was analyzed by indirect immunofluorescence with CTLA4Ig.

After the final stage of selection, plasmid DNA was prepared from each colony. A total of 4 out of 48 candidate clones contained a cDNA insert of about 1.2 kb. These four clones were transfected into COS cells. All four clones were strongly positive for B7-2 expression by indirect immunofluorescence using CTLA4Ig and flow cytometry analysis.
C. Sequencing

The B7-2 cDNA insert of clone 29 was sequenced in the pCDM8 expression vector using the following strategy. Initial sequencing was performed using primer T7, CDM8R (Invitrogen) homologous to the CDM8 vector sequence adjacent to the cloned B7-2 cDNA (see Table I). Sequencing was performed using a dye termination reaction and an ABI automated DNA sequencer (ABI, Foster City, Calif.). The following sequencing primers were designed using the DNA sequences obtained using these primers (see Table I). This cycle of sequencing and the next primer selection was continued until the B7-2 cDNA was fully sequenced for both strands.

  Human B7-2 clone 29 contained a 1120 base pair insert, a single long reading frame of 987 nucleotides and a 3 'non-coding sequence of approximately 27 nucleotides (Figure 8, SEQ ID NO: 1). The amino acid sequence predicted to be encoded by the open reading frame of this protein is shown below the nucleotide sequence in FIG. 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 membrane proteins. Protein translation is based on DNA homology in this region with the consensus eukaryotic translation initiation site (Kozak, M (1987), Nucl. Acids Res., 15: 8125-8148). Expected to start at (nucleotides 107-109). The amino terminal of human B7-2 protein (amino acids 1 to 23) is the predicted cleavage site between alanine at positions 23 and 24 (von Heijne (1986), Nucl. Acids Res., 14: 4683). It has the characteristics of a secretory signal peptide. Processing at this site appears to result in a 306 amino acid human B7-2 membrane-bound protein with an unmodified molecular weight of approximately 34 kDa. This protein consists of an extracellular Ig superfamily V and C-like domain of approximately amino acid residues 24-245, a hydrophilic transmembrane domain of approximately amino acid residues 246-268, and a long protoplasmic domain of approximately amino acid residues 269-329. Seem. The homology with the Ig superfamily is due to two adjacent Ig-like domains in the extracellular region bound by cysteines at positions 40-110 and 157-218. The extracellular domain also contains eight potential sites for N-linked glycosylation. E. coli transfected with a vector containing the cDNA insert of clone 29 encoding the human B7-2 protein was deposited on July 26, 1993 with the American Type Culture Collection (ATCC) under accession number 69357.

  Comparison of both the nucleotide and amino acid sequences of human B7-2 with the GenBank and EMBL databases revealed that only human and murine B7-1 proteins are relevant. When the three B7 protein sequences are arranged (FIG. 13), it can be seen that human B7-2 has about 26% amino acid identity with human B7-1. FIG. 13 shows the amino acid sequences of human B7-2 (hB7-2) (SEQ ID NO: 2), human B7-1 (hB7-1) (SEQ ID NOs: 28 and 29) and murine B7 (mB7) (SEQ ID NOs: 30 and 31). A comparison of is shown. The amino acid sequences of human B7-1 and murine B7 (hereinafter referred to as murine B7-1) can be found in Genbank under accession numbers # M27533 and X60958, respectively. The vertical line in FIG. 13 shows the same amino acid between hB7-2 and hB7-1 or mB7. The same amino acid between hB7-1 and mB7 is not shown. The hB7-2 protein has the same general structure as hB7-1 defined by the common cysteines (positions 40 and 110 of the IgV domain, positions 157 and 217 of the IgC domain) of the Ig superfamily and many other common amino acids. Indicates. Since hB7-1 and mB7 are both known to bind to human CTLA4 and human CD28, the amino acids common between these two related proteins are required to contain CTLA4 or CD28 binding sequences. Seem. Examples of this sequence are KYMGRTFSD (positions 81-89, hB7-2) (SEQ ID NO: 17) or KSQDNVTELYDVS (positions 188-200, hB7-2) (SEQ ID NO: 18). Other related sequences are apparent from sequence comparisons, others can be inferred by considering homologously related amino acids such as aspartic acid and glutamic acid, alanine and glycine, and other recognized function related amino acids. The B7 sequence has a highly positively charged domain in the protoplasmic part WKWKKKKRPRNSYKC (positions 269-282, hB7-2) (SEQ ID NO: 19), which is probably involved in intracellular signaling.

Example 5 Properties of recombinant B7-2 antigen
A. B7-2 binds to CTLA4Ig but does not bind to anti-B7-1 and anti-B7-3 monoclonal antibodies. Vector DNA (pCDNAI) or B7-1 (B7-1) or B7-2 (B7- Transfected with any of the expression plasmids containing 2). After 72 hours, transfected COS cells were detached by incubation in PBS containing 0.5 mM EDTA and 0.02% sodium azide for 30 minutes at 37 ° C. The cells were analyzed for cell surface expression by indirect immunofluorescence and flow cytometric analysis using isothiocyanate fluorescein-conjugated (FITC) goat anti-mouse Ig or goat anti-human IgG-FITC (FIG. 9). Cell surface expression of B7-1 was detected with mAbs 133 (anti-B7-1) and BB-1 (anti-B7-1 and anti-B7-3) and with CTLA4Ig, while B7-2 reacted with CTLA4Ig only I let you. None of the B7 transfections stained with the isotype control (IgM or control Ig). COS cells transfected with the vector did not stain with any detection reagent. Furthermore, none of the cells were stained with the FITC labeled detection reagent. This demonstrates that B7-2 encodes a protein that is a CTLA4 counter-receptor but is different from B7-1 and B7-3.
B. RNA blot analysis of B7-2 expression in unstimulated and activated human B cells, cell lines and myeloma

Human spleen B cells were described in the literature by removing T cells and mononuclear cells (Freedman, AS, Freeman, GJ, Hollowitz, JC, Daley, J, Nadler, L.M. , J. Immunol. (1987), 137: 3260-3267). Spleen B cells were activated using anti-Ig beads and cells were harvested at the indicated times (Freedman et al. (1987), supra). T cells and mononuclear cells were removed as reported using E rosetting and attachment of bone marrow specimens (Freeman, GJ, et al., J. Immunol. (1989), 143: 2714-2722). Human myeloma was purified. RNA was prepared by homogenization with guanidine thiocyanate and cesium chloride centrifugation. An equal amount of RNA (20 μg) was electrophoresed on an agarose gel, stained, and hybridized to ³² P-labeled B7-2 cDNA. FIG. 10a shows unstimulated and anti-Ig activated human splenic B cells and Raji (B cell Burkitt lymphoma), Daudi (B cell Burkitt lymphoma), RPMI 8226 (myeloma), K562 (erythroleukemia) and REX. Figure 2 shows RNA blot analysis of cell lines containing (T cell acute lymphoblastic leukemia). FIG. 10b shows an RNA blot analysis of human myeloma.

Three 1.35, 1.65, and 3.0 kb mRNA transcripts were detected by hybridization to B7-2 cDNA (FIG. 10b). RNA blot analysis demonstrated that B7-2 mRNA was expressed in unstimulated human splenic B cells and increased 4-fold after activation. B7-2 mRNA was expressed in B cell neoplastic lines (Raji, Daudi) and myeloma (RPMI8226), but not in erythroleukemia K562 and T cell line REX. In contrast, we have previously reported that B7-1 mRNA is not expressed in resting B cells and is transiently expressed after activation (Freeman, GJ et al. (1989), supra). . A study of mRNA isolated from human myeloma demonstrated that B7-2 mRNA was expressed in 6 of 6 patients, but B7-1 was found in only 1 of 6 (Freeman) , GJ et al. (1989), supra). Thus, it is clear that the expression of B7-1 and B7-2 is independently controlled.
C. Costimulation

Human CD28 ⁺ T cells were analyzed by immunomagnetic bead removal as described in a previous literature (Gimmi, CD et al. (1993), Proc. Natl. Acad. Sci. USA, 90: 6586-6590). Isolation using monoclonal antibodies against cells, natural killer cells and macrophages. C7-1 cells transfected with B7-1, B7-2 and vector were harvested 72 hours after transfection, incubated with mitomycin C 25 μg / mL for 1 hour, and then washed extensively. 10 ⁵ CD28 ⁺ and T cells were incubated with 1 ng / mL phorbol myristate acetate (PMA) and the indicated number of COS transfectants (FIG. 11). As shown in FIG. 11a, T cell proliferation was measured by 3H-thymidine (1 μCi) incorporated in the last 12 hours of the 72 hour incubation. FIG. 11b shows IL-2 production by T cells measured by ELISA (Biosource, CA) using supernatant harvested 24 hours after the start of culture.
D. B7-2 costimulation is not blocked by anti-B7-1 and anti-B7-3 mAbs but is blocked by CTLA4Ig and anti-CD28 Fab

Previous reports of human CD28 ⁺ T cells using mAbs on B cells, natural killer cells and macrophages (Gimmi, CD, Freeman, GJ, Gribben, JG, Gray, G., Nadler , LM (1993), Proc. Natl. Acad. Sci. USA, 90: 6586-6590). C7-1 cells transfected with B7-1, B7-2 and vector were harvested 72 hours after transfection, incubated with mitomycin C 25 μg / mL for 1 hour, and then washed extensively. CD28 ⁺ T cells 10 ⁵ cells were incubated with phorbol myristate acetate (PMA) 1ng / mL and COS transfectants 2 × 10 ⁴ cells together. The blocking agent (10 μg / mL) is shown at the left end of FIG. 12. 1) No monoclonal antibody (no blocking agent), 2) mAb 133 (anti-B7-1 mAb), 3) mAbBB-1 (anti-B7- 1) and anti-B7-3 mAb), 4) mAbB5 (control IgM mAb), 5) anti-CD28 Fab (mAb 9.3), 6) CTLA-Ig and 7) control Ig. FIG. 12a shows proliferation measured by ³ H-thymidine (1 μCi) uptake during the last 12 hours of the 72 hour incubation. FIG. 12b shows IL-2 production measured by ELISA (Biosource, CA) using the supernatant collected 24 hours after the start of culture.

COS cells transfected with B7-1 and B7-2 costimulated corresponding levels of T cell proliferation when examined at a wide range of stimulator / responder cell ratios (FIG. 11). Costimulation of COS cells transfected with B7-2 as well as B7-1 showed IL-2 production at a wide range of stimulator / responder cell ratios (FIG. 11). In contrast, vector-transfected COS cells did not costimulate T cell proliferation or IL-2 production.
E. B7-2 costimulation is not blocked by anti-B7-1 and anti-B7-3 mAbs but is blocked by CTLA4-Ig and anti-CD28 Fab

Previous reports of human CD28 ⁺ T cells using mAbs on B cells, natural killer cells and macrophages (Gimmi, CD, Freeman, GJ, Gribben, JJ, Gray, G., Nadler). , LM (1993), Proc. Natl. Acad. Sci. USA, 90: 6586-6590). C7-1 cells transfected with B7-1, B7-2 and vector were harvested 72 hours after transfection, incubated with mitomycin C 25 μg / mL for 1 hour, and then washed extensively. CD28 ⁺ T cells 10 ⁵ cells were incubated with phorbol myristate acetate (PMA) 1ng / mL and COS transfectants 2 × 10 ⁴ cells together. The blocking agent (10 μg / mL) is shown at the left end of FIG. 12. 1) No monoclonal antibody (no blocking agent), 2) mAb 133 (anti-B7-1 mAb), 3) mAbBB-1 (anti-B7- 1) and anti-B7-3 mAb), 4) mAbB5 (control IgM mAb), 5) anti-CD28 Fab (mAb 9.3), 6) CTLA-Ig and 7) control Ig. FIG. 12a shows proliferation measured by ³ H-thymidine (1 μCi) incorporated during the last 12 hours of 72 hour incubation. FIG. 12b shows IL-2 production measured by ELISA (Biosource, CA) using supernatant collected 24 hours after the start of culture.

In order to distinguish B7-2 from B7-1 and B7-3, the proliferation and IL-2 production of human CD28 ⁺ T cells quasimitically activated with mAbs against B7-1 and B7-3 were determined. I stopped it. B7-1 and B7-2 COS transfectants costimulated T cell proliferation and IL-2 production (FIG. 12). mAb 133 (Freedman, A. S. et al. (1987) supra) (anti-B7-1) and BB1 (Boussiotis, V. A. et al. (in review), Proc. Natl. Acad. Sci. USA; Yokochi, T., Holly, RD, Clark, EA (1982), J. Immunol., 128: 823-827) (anti-B7-1 and anti-B7-3) are B7- 1 completely blocked proliferation and IL-2 production induced by 1, but had no effect on costimulation by COS cells transfected with B7-2. The control B5 mAb matched to the isotype was ineffective. Anti-CD28 Fab and CTLA4-Ig fusion proteins were examined to determine if the B7-2 signal follows the CD28 / CTLA4 pathway to determine if they block B7-2 costimulation. Both anti-CD28 Fab and CTLA4-Ig blocked proliferation and IL-2 production induced by B7-1 or B7-2COS transfectants, whereas the control Ig fusion protein was ineffective (FIG. 12). . CTLA4-Ig blocked B7-2 costimulation of proliferation by 90%, but in another experiment the inhibition was even greater (98-100%). None of the blocking agents inhibited T cell proliferation or IL-2 production induced by the combination of PMA and phytohemagglutinin.

  Like B7-1, B7-2 is a counter-receptor for CD28 and CTLA4 • T cell surface molecules. Both proteins are similar in the following ways: 1) expressed on the APC surface, 2) structurally related to the Ig superfamily, with IgV and IgC domains, and 26% amino acid identity; 3) T Cells are costimulated to produce and proliferate IL-2. However, B7-1 and B7-2 are fundamentally different in several respects. First, B7-2 mRNA is structurally expressed in unstimulated B cells, whereas B7-1 mRNA does not appear until 4 hours and cell surface proteins are not detected until 24 hours (Freedman, A. et al. S. et al. (1987), supra; Freeman, GJ et al. (1989), supra). Unstimulated human B cells do not express CTLA4 counterreceptors on the cell surface and do not costimulate T cell proliferation (Boussiotis, VA, supra). Therefore, expression of B7-2 mRNA in unstimulated B cells appears to rapidly express the B7-2 protein, possibly from storage mRNA or protein, on the cell surface after activation. Costimulation with B7-2 transfectants is partially sensitive to paraformaldehyde immobilization, whereas B7-2 costimulation is resistant (Gimmi, CD et al. (1991), Proc Natl.Acad.Sci.USA, 88: 6575-6579). Second, the expression of B7-1 and B7-2 in cell lines and human B cell neoplasms is substantially different. Third, the B7-2 protein has a longer protoplasmic domain than B7-1, which will play a role in B cell differentiation. These phenotypes and functional differences suggest that these homologous molecules may have distinct biological functions.

Example 6 Cloning and sequencing of the murine B7-2 antigen
A. Construction of cDNA library Dibutyryl cyclic AMP (cAMP) activated M12 cells (murine B cell tumor line) as described in the literature (Aruffo et al., Proc. Natl. Acad. Sci. USA, 84: 3365 (1987)) ) From poly (A) ⁺ RNA was used to construct cDNA with the pCDM8 vector (Seed, Nature, 329: 840 (1987)).
M12 cells up to 1 × 10 ⁶ cells / mL in complete culture medium {RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM glutamine, 1 mM sodium pyruvate, penicillin (100 units / mL), streptomycin sulfate (100 μg / mL) and gentamicin sulfate (5 μg / mL)} and activated with dibutyryl cAMP 300 μg / mL (Nabavi, N. et al. (1992), Nature, 360: 266-268. page). Activated M12 cells were harvested after 0, 6, 12, 18, 24 and 30 hours.

RNA was prepared by homogenizing activated M12 cells in a mixture of 4M-guanidine thiocyanate, 0.5% sarkosyl, 25 mM EDTA, pH 7.5, 0.13% Sigma Antiform A and 0.7% mercaptoethanol. did. The homogenate was centrifuged at 32,000 rpm for 24 hours in a mixture of 5.7 M-CsCl, 10 mM-EDTA, 25 mM-sodium acetate, pH 7, and the RNA was purified. The RNA pellet was dissolved in 5% sarkosyl, 1 mM EDTA, 10 mM Tris, pH 7.5 and extracted with 2 volumes of 50% phenol, 49% chloroform, 1% isoamyl alcohol. RNA was precipitated twice from ethanol. The poly (A) ⁺ RNA used for the construction of the cDNA library was purified by 2 cycles of oligo (dT) cellulose selection.

Complementary DNA is 50 mM Tris, pH 8.3, 75 mM KCl, 3 mM MgCl ₂ , 10 mM dithiothreitol, 500 μM dATP, dCTP, dGTP, dTTP, oligo (dT) _12-18 50 μg / mL, RNasin 180 units / Synthesized from 5.5 μg poly (A) ⁺ RNA of murine M12 cells activated with dibutyryl cAMP in 55 μL total reaction volume of 10000 units / mL of mL and Moloney-MLV reverse transcriptase for 1 hour at 37 ° C. After reverse transcription, the solution 25mM- Tris, pH8.3,100mM-KCl, 5mM-MgCl 2, each 250μM-dATP, dCTP, dGTP, dTTP, 5mM- dithiothreitol, DNA polymerase I250 units / mL, ribonuclease H8. The cDNA was converted to double-stranded DNA by adjusting to 5 units / mL and incubating for 2 hours at 16 ° C. 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 2 volumes of ethanol in the presence of 2.5M ammonium acetate and 4 μg of linear polyacrylamide as a carrier. After reverse transcription, the reverse transcriptase was inactivated by heating to 70 ° C. for 10 minutes. 320 μL of water and 80 μL of a mixture of 0.1 M Tris, pH 7.5, 25 mM MgCl ₂ , 0.5 M KCl, 250 μg / mL bovine serum albumin and 50 mM dithiothreitol were added, and the solutions were each 200 μM-dATP, dCTP, dGTP, dTTP, DNA polymerase I 50 units / mL, ribonuclease H8 units / mL were prepared, and incubated at 16 ° C. for 2 hours to convert cDNA into double-stranded DNA. 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 2 volumes of ethanol in the presence of 2.5M ammonium acetate and 4 μg of linear polyacrylamide as a carrier.

To this DNA, 2 μg of non-self-complementary BstXI adapter was added as follows: 3.6 μg of double-stranded cDNA from poly (A) ⁺ RNA 5.5 μg and kinase-treated oligonucleotide of sequence CTTTAGAGCACA (SEQ ID NO: 15) and sequence CTCTAAAAG (SEQ ID NO: 16) kinase-treated oligonucleotide 2.4 μg and 6 mM Tris, pH 7.5, 6 mM MgCl ₂ , 5 mM NaCl, bovine serum albumin 350 μg / mL, 7 mM mercaptoethanol, 0.1 mM ATP, Incubation was carried out at 15 ° C. for 16 hours in a total volume of 0.45 mL containing 2 mM dithiothreitol, 1 mM spermidine and 600 units of T4 DNA ligase. EDTA was added to 34 mM and the solution was extracted with an equal volume of 50% phenol, 49% chloroform, 1% isoamyl alcohol. DNA was precipitated from 2 volumes of ethanol in the presence of 2.5M ammonium acetate.

DNA of 600 bp or more was selected as follows: adapter DNA was redissolved in 10 mM Tris, pH 8, 1 mM EDTA, 600 mM NaCl, 0.1% sarkosyl, and the same buffer on a Sepharose CL-4B column. Chromatography on DNA in the column void volume (containing DNA of 600 bp or more) was collected and precipitated with ethanol.
A pCDM8 vector for cDNA cloning was prepared by digestion with BstXI and agarose gel purification. Adapter DNA from 5.5 μg of poly (A) ⁺ RNA was converted to 6 mM-Tris, pH 7.5, 6 mM-MgCl ₂ , 5 mM-NaCl, bovine serum albumin 350 μg / mL, 7 mM-mercaptoethanol, 0.1 mM-ATP, 2 mM- Ligated to 2.25 μg of BstXI-cleaved pCDM8 at 1.5 ° C. in 1.5 mL total solution containing dithiothreitol, 1 mM spermidine and 600 units of T4 DNA ligase at 15 ° C. for 24 hours. The ligation reaction mixture was transformed into competent E. coli MC1061 / P3, yielding 200 × 10 ^{6 in} all independent cDNA clones.

Plasmid DNA was prepared from 500 mL of the original cDNA library transformant culture solution. Plasmid DNA was purified by alkaline digestion and subsequently banded twice in a CsCl equilibrium gradient (Maniatis et al., Molecular Cloning: A Laboratory Laboratory, Cold Spring Harbor, NY (1987)).
B. Cloning procedure

  In the first stage of the screening, 30 100 mm dishes of 50% confluent COS cells were activated with the DEAE-dextrin method (Seed et al., Proc. Natl. Acad. Sci. USA, 0.05 μg / mL of activated M12 murine B cell library DNA. 84: 3365 (1987)). Cells were trypsinized and replated 24 hours later. After 47 hours, cells were detached by incubation in PBS / 0.5 mM EDTA, pH 7.4 / 0.02% sodium azide at 37 ° C. for 30 minutes. The detached cells were treated with human CTLA4Ig and murine CD28Ig 10 μg / mL for 45 minutes at 4 ° C. Cells were washed and spread onto culture dishes coated with affinity purified goat anti-human IgG antibody and allowed to attach at room temperature. After 3 hours, the plates were washed twice with PBS / 0.5 mM EDTA, pH 7.4 / 0.02% sodium azide, 5% FCS and 0.15 M NaCl, 0.01 M Hepes, pH 7.4, 5 Gently washed once with% FCS. Episomal DNA was harvested from the cultured cells and transformed into E. coli DH10B / P3. Plasmid DNA was reintroduced into COS cells by spheroplast fusion described in the literature (Seed et al., Proc. Natl. Acad. Sci. USA, 84: 3365 (1987)), with two cycles of expression and culture. Repeated. In the second and third stages of selection, after 47 hours, detached COS cells were first incubated with α-murine B7-1 mAb (16-10A1, 10 μg / mL), and COS cells expressing B7-1 were -Removed with magnetic beads coated with mouse IgG and IgM. COS cells were then treated with 10 μg / mL of human CTLA4Ig and murine CD28Ig and murine B7-2 expressing COS cells were selected by culturing on goat anti-human IgG antibody coated dishes. After the third stage, plasmid DNA was prepared from each colony 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 stage of selection, plasmid DNA was prepared from each colony. A total of 6 of the 8 candidate clones contained a cDNA insert of approximately 1.2 kb. Clone cells were transfected with 8 clones of plasmid DNA. All six clones having a 1.2 Kb cDNA insert were strongly positive for B7-2 expression by indirect immunofluorescence using CTLA4Ig and flow cytometry analysis.
C. Sequencing

The B7-2 cDNA insert of clone 4 was sequenced in the pCDM8 expression vector using the following strategy. Initial sequencing was performed using sequencing primer T7, CDM8R (Invitrogen) homologous to the CDM8 vector sequence adjacent to the cloned B7-2 cDNA (see Table II). Sequencing was performed using a dye terminator reaction and an ABI automated DNA sequencer (ABI, Foster City, Calif.). The following sequencing primers were designed using the DNA sequences obtained with these primers (see Table II). This sequencing and subsequent primer selection cycle was continued until the murine B7-2 cDNA was fully sequenced on both strands.

One of the resulting murine B7-2 clones (mB7-2, clone 4) has a 1163 base pair insert, a single long open reading frame of 927 nucleotides and a 3 ′ non-coding sequence of approximately 126 nucleotides (FIG. 14). SEQ ID NO: 22). The amino acid sequence predicted to be encoded by the open reading frame of this protein is shown below the nucleotide sequence in FIG. 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 based on DNA homology in this region with the consensus eukaryotic translation initiation site (see Kozak, M (1987), Nucl. Acids Res. 15: 8125-8148). Expected to start with a codon (ATG, nucleotide 111-113). The amino terminal of the murine B7-2 protein (amino acids 1 to 23) is the predicted cleavage site between alanine at position 23 and valine at position 24 (von Heijne (1987), Nucl. Acids Res., 14: 4683) with a characteristic of a secretory signal peptide. Processing at this site appears to result in a 286 amino acid murine B7-2 membrane-bound protein with an unmodified molecular weight of approximately 32 kDa. This protein consists of an extracellular Ig superfamily V and C-like domain of approximately amino acid residues 24-246, a hydrophilic transmembrane domain of approximately amino acid residues 247-265 and a long protoplasmic domain of approximately amino acid residues 266-309 It seems to be done. The homology with the Ig superfamily is due to two adjacent Ig-like domains in the extracellular region to which cysteines at positions 40 to 110 and 157 to 216 bind. The extracellular domain also contains nine potential sites for N-linked glycosylation and, like murine B7-1, is probably glycosylated. Glycosylation of murine B7-2 protein increases the molecular weight to about 50-70 kDa. The protoplasmic domain of murine B7-2 contains a common region with cystene followed by a positively charged amino acid that probably functions as a signaling or regulatory domain within APC. Comparison of both the nucleotide and amino acid sequences of murine B7-2 with the GenBank and EMBL databases showed that human and murine B7-1 showed significant homology (amino acid sequence identity about 26%). Murine B7-2 showed 50% identity and 67% similarity to the human homolog 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 under accession number 69388. Deposited at.
D. Costimulation

CD4 ⁺ mouse T cells were first purified by treatment with Tris-NH ₄ Cl to remove erythrocytes. T cells were concentrated through a nylon wool column. CD4 ⁺ T cells were purified twice by treatment with anti-MHC class II and anti-CD28 mAb mixtures and rabbit complement. Murine B7-1 (special gift from Dr. Gordon Freeman of Dana-Farber Cancer Institute, Boston, MA; Freeman, GJ et al. (1991), J. Exp. Med., 174: 625-631. (See), COS cells transfected with murine B7-2 and vector were harvested 72 hours after transfection, incubated with 25 μg / mL mitomycin C for 1 hour and washed thoroughly. Murine CD4 ⁺ T cells 10 ⁵ phorbol myristate acetate (PMA) was 1 ng / mL and COS transfectants 2 × 10 ⁴ cells and incubated (Table III). T cell proliferation was measured by ³ H-thymidine (1 μCi) incorporated in the final 12 hours of the 72 hour incubation.

Example 7 Construction and properties of human B7-2 immunoglobulin fusion protein
A. Manufacture of human B7-2Ig fusion protein It was manufactured as a fusion protein in which the extracellular portion of human B7-2 was bound to an immunoglobulin constant region. The immunoglobulin constant region may contain genetic modifications, including modifications that attenuate or eliminate effector activity essential for the immunoglobulin structure. Overall, DNA encoding the extracellular portion of hB7-2 was ligated to DNA encoding the hinge, CH2 and CH3 regions of human IgCγ1 or human IgCγ4 modified by directed mutation. This was done as described in the subsection below.
B. Preparation of gene fusion

A DNA fragment corresponding to the target DNA sequence was prepared by polymerase chain reaction (PCR) using the following primer pair. In general, a PCR reaction contains a primer (1 μM each), dNTP (200 μM each), template DNA 1 ng, and Taq polymerase (Saiki et al. (1988) Science 239: 487-491). (Gene Amp PCR kit, Perkin-Elmer / Cetus, Norwalk, CT) Prepared in a final volume of 100 μL. DNA amplification by PCR was performed using a thermocycler (Ericomp, San Diego, Calif.), Each with a denaturation step (94 ° C. for 1 minute), a regeneration step (54 ° C. for 30 seconds) and a strand extension step (72 ° C. for 1 minute). 25 to 30 cycles consisting of The structure of each hB7-2-Ig gene fusion is composed of sequences of secretory promoting signals that bind to the extracellular domain of B7-2 and the hinge, CH2 and CH3 domains of human IgCγ1 or IgCγ4. The sequences of IgCγ1 and IgCγ4 include nucleotide conversion in which a cysteine residue capable of forming a disulfide bond in the hinge region is replaced with a serine residue, and the CH2 domain is necessary for IgC binding to Fc receptor and complement activation. It may include nucleotide conversions that replace the suspected amino acid. Sequence analysis confirmed the structure of mγ4 and mγ1 clones, and 293 cells were transfected with each construct to examine transient expression. A transient expression level approximately equal to 100 ng protein / mL cell supernatant was observed / confirmed for both constructs by hIgG ELISA. NSO cell lines were transfected for permanent expression of the fusion protein.
C. Construction of hB7-2Ig fusion protein gene

(1) Preparation of signal sequence An immunoglobulin signal sequence suitable for secretion of B7-2Ig fusion protein from mammalian cells was produced using PCR amplification. This Ig signal sequence is derived from a plasmid containing a murine IgG heavy chain gene (Orlandi, R. et al. (1989), Proc. Acad. Natl. Sci. USA, 86: 38333837). (# 01) (SEQ ID NO :) is a forward primer, and oligonucleotide 5'-GCTTGAATCTTCAGAGGAGGCGAGTGGAACCCTTGGG-3 '(# 02) (SEQ ID NO :) is a reverse PCR primer (reverse-PCR-primer) Prepared as above. The forward PCR primer (SEQ ID NO :) contains a recognition sequence for the restriction enzyme BsaI and is homologous to the sequence of the Ig signal sequence to the 5 ′ initiation methionine. The reverse PCR primer (SEQ ID NO :) is composed of sequences derived from the extracellular domain 5 ′ terminal and the Ig signal sequence 3 ′ terminal of hB7-2. PCR amplification of murine Ig signal template DNA using these primers gives a 224 bp product that is fused to the first 20 nucleotide sequence encoding the BsaI restriction site followed by the extracellular domain of hB7-2. It consists of a signal region sequence. The point of attachment between the signal sequence and hB7-2 is such that protein translation starting with the signal sequence continues through hB7-2 in the correct reading frame.

(2) Preparation of hB7.2 gene segment The extracellular domain of hB7-2 gene is a plasmid containing hB7-2 cDNA inserted into expression vector pCDNAI (Freeman et al., Science, 262: 909-11 (1994)). Prepared by PCR amplification:
The extracellular domain of hB7-2 has oligonucleotide 5′-GCTCCTCTGAAGATTCAAGC-3 ′ (# 03) (SEQ ID NO :) as a forward primer and oligonucleotide 5′-GGCACTATGATCAGGGGGAGGCTGAGGTCC-3 ′ (# 04) (SEQ ID NO :) Was prepared by PCR amplification using as a reverse primer. The forward PCR primer contains a sequence corresponding to the first 20 nucleotides of the B7-2 extracellular domain, and the reverse PCR primer is the last 22 nucleotides of the B7-2 extracellular domain followed by a BclI restriction site and non-coding. Contains a sequence corresponding to 7 nucleotides. PCR amplification with primers # 03 and # 04 gives a 673 bp product corresponding to the extracellular IgV and IgC-like domains of hB7-2 followed by a unique BclI restriction site.
The signal sequence was bound to the extracellular part of hB7-2 by PCR as follows. PCR product of DNA corresponding to the signal sequence obtained above and hB7-2 extracellular domain is mixed in an equimolar amount, denatured by heating to 100 ° C., and maintained at 54 ° C. for 30 ° C. Annealing was performed and both strands were satisfied using dNTP and Toq polymerase. PCR primers # 01 and # 04 were added, and PCR amplification was performed to produce a complete fragment, which was composed of a BsaI restriction site, followed by a signal sequence fused to the extracellular domain of hB7-2, followed by a BclI restriction site. ~ 880 fragments were obtained.

(3) Cloning and modification of immunoglobulin fusion domain Plasmid pSP721gG1 is a 2000 bp segment of human IgG1 heavy chain genomic DNA (Ellison, JW et al. (1982), Nucl. Acids Res., 10: 4071-4079). Was cloned into the multiple cloning site of the cloning vector pSP72 (Promega, Madison, WI). Plasmid pSP721gG1 contained genomic DNA encoding the CH1, hinge, CH2 and CH3 domains of the human heavy chain IgCγ1 gene. PCR primers designed to amplify the hinge-CH2-CH3 portion of the heavy chain with intermediate DNA were prepared as follows. The forward PCR primer 5′-GCATTTTAAGCTTTTTCCTGGATCAGGAGCCCCAAATTCTCTGACAAAACTCACACATCTCCACCGTCTCCAGGTAAGCC-3 ′ (SEQ ID NO :) contained HindIII and BclI restriction sites except for the 5 homologous nucleotide sequence that changed the cysteine residue to serine. The reverse PCR primer 5′TAATACGACTCACTATAGGGG ′ (SEQ ID NO :) was identical to the commercially available T7 primer (Promega, Madison, Wis.). Amplification with these primers yields a 1050 bp fragment that binds to the HindIII and BclI restriction sites at the 5′-end and binds to BamH1, Sma1, Kpn1, Sac1, EcoR1, Cla1, EcoR5 and Bg111 restriction sites at the 3′-end. It was. This fragment contained an IgC hinge domain where three cysteine codons were replaced with serine codons followed by an intron, CH2 domain, intron, CH3 domain and a subsequent 3 ′ sequence. After PCR amplification, the DNA fragment was digested with HindIII and EcoR1 and cloned into the expression vector pNRDSH digested with the same restriction enzymes. This resulted in the plasmid pNRDSH / IgG1.

  A similar PCR strategy was used to clone the hinge-CH2-CH3 domain of the human IgCγ4 constant region. Plasmid p428D (Medical Research Council), containing the complete IgCγ4 heavy chain genomic sequence (Ellison, J., Buxbaum, J. and Hood, LE (1981), DNA, 11-11) (London, UK) is the oligonucleotide 5′GAGCATTTTCCTGATCAGGAGGTCCCAAATATGGTCCCCCCCATCCATCATCCCCAGGTAAGCCCAACCC-3 ′ (SEQ. It was used for the mold. The forward PCR primer (SEQ ID NO :) contains a BclI restriction site followed by a sequence encoding the hinge domain of IgCγ4. Nucleotide substitutions were made to replace cysteine residues in the hinge region with serine residues. The reverse PCR primer (SEQ ID NO :) contains a PspAI restriction site (5'CCCGGG-3 '). A 1179 bp DNA fragment was obtained by PCR amplification with these primers. The PCR product was digested with BclI and PspAI and ligated to pNRDSH / IgG1 digested with the same restriction enzymes to give plasmid pNRDSH / IgG4. In this reaction, the IgCγ4 domain replaced the IgCγ1 domain present in pNRDSH / IgG1.

  Modifications in which the amino acid thought to be involved in binding to the Fc receptor in the CH2 domain in IgC were replaced as follows. Plasmid pNRDSH / IgG1 was used as a template for modifying IgCγ1CH2 domain, and plasmid pNRDSH / IgG4 was used as a template for modifying IgCγ4CH2 domain. Plasmid pNRDSH / IgG1 was PCR amplified using the forward PCR primer (SEQ ID NO :) and oligonucleotide 5'-GGGTTTTGGGGGGAAGAGGAAGACTGACGGGTGCCCCCTCGCTTCAGGGTCTGAGGAAG-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 :) is amino acids 234, 235 and 237 (Canfield, SM and Morrison, SL (1991). , J. Exp. Med., 173: 1483-1491) with the exception of 5 nucleotide substitutions designed to convert Leu to Ala, Leu to Glu, and Gly to Ala, respectively. Homologous to the terminal part. Amplification of these PCR primers yields a 239 bp DNA fragment composed of the modified hinge domain, intron and modified CH2 domain. The plasmid pNRDSH / IgG1 was also subjected to PCR amplification using the oligonucleotide 5'-CATCTCTTCCTCAGCACCCTGAAGCCGAGGGGGCACCGTCCAGTCTCTCCTTCTCCCCC-3 '(SEQ ID NO :) as the forward primer and the oligonucleotide (SEQ ID NO :) as the reverse PCR primer. The forward PCR primer (SEQ ID NO :) is complementary to the primer (SEQ ID NO :) and contains 5 complementary nucleotide changes necessary for CH2 amino acid substitution. The reverse PCR primer (SEQ ID NO :) has been previously described. Amplification with these primers gave a 875 bp fragment composed of a modified CH2 domain portion, intron, CH3 domain and 3 'additional sequence. A complete IgCγ1 segment composed of a modified hinge domain, a modified CH2 domain and a CH3 domain was further prepared by a single PCR reaction. The product of the two PCR reactions was purified, mixed, denatured (95 ° C., 1 minute) and then regenerated (54 ° C., 30 seconds) to anneal the complementary ends of the two fragments. Both strands were filled (fill in) using dNTP and Taq polymerase, and all fragments were amplified using forward PCR primers (SEQ ID NO :) and reverse PCR primers (SEQ ID NO :). The obtained 1050 bp fragment was purified, digested with HindIII and EcoRI, and ligated to pNRDSH previously digested with the same restriction enzymes to obtain plasmid pNRDSHIgG1m.

  Two amino acids at positions 235 and 237 of the immunoglobulin were changed from Leu to Glu and Gly to Ala, respectively, to remove the Fc receptor binding site in the IgCγ4CH2 domain. Plasmid pNRDSH / IgG4 was used as a forward primer (SEQ ID NO :) and oligonucleotide 5'-CGCACGTGACCCTCAGGGGTCCCGGGAGATCAGGAGGTGTCCTTGGGTTTTGGGGGGGAACAGGAAGACTGATGGGTGCCCCCTCGACTGG The forward primer is described above and the reverse PCR primer is homologous to the amino terminal portion of the CH2 domain except for the three nucleotide substitutions designed for the amino acid substitution. This primer also contains a PmII restriction site for subsequent cloning. Amplification with these primers yielded a 256 bp fragment composed of the modified hinge region, the intron and the modified 5 'portion of the CH2 domain.

Oligonucleotide 5′-CCTCAGCACCTGGAGTTCGAGGGGGCACCCATCAGTCTCCTGTTCCCCCCAAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCCTGAGGTCACGTGGCG-3 ′ (SEQ ID NO :) was used as a forward primer, and IgG (SEQ ID NO :) was used as a reverse primer. The forward PCR primer (SEQ ID NO :) is complementary to the primer (SEQ ID NO :) and contains three complementary nucleotide changes necessary for CH2 amino acid replacement. The reverse PCR primer (SEQ ID NO :) has been previously described. Amplification with these primers yielded a 1012 bp fragment composed of the modified CH2 domain portion, intron, CH3 domain and 3 'additional sequence. A complete IgCγ4 segment composed of a modified hinge domain, a modified CH2 domain, and a CH3 domain was further prepared in a single PCR reaction. The product of the two PCR reactions was purified, mixed, denatured (95 ° C., 1 minute) and then regenerated (54 ° C., 30 seconds) to anneal the complementary ends of the two fragments. Both strands were filled using dNTP and Taq polymerase, and all fragments were amplified using forward PCR primers (SEQ ID NO :) and reverse PCR primers (SEQ ID NO :). The obtained 1179 bp fragment was purified, digested with BcII and PspAI, and ligated to pNRDSH previously digested with the same restriction enzymes to obtain plasmid pNRDSH / IgG4m.
(4) Assembly of the final hB7-2Ig gene

The PCR fragment corresponding to the Ig signal-hB7-2 gene fusion prepared above was digested with BsaI and BclI restriction enzymes, and previously digested with HindIII and BclI, pNRDSH / IgG1, pNRDSH / IgG1m, pNRDSH / IgG4 and Ligated to pNRDSH / IgG4m. The ligated plasmid was transformed into CaCl ₂ capable cells E. coli JM109 and the transformants were ampicillin (50 μg / mL, Molecular Cloning: A Laboratory Manual (1982), Maniatis, T., Fitsch, EE, and Selected on L-agar, including Sambrook, J., Cold, Spring, Harbor, Laboratory). Plasmids isolated from transformed E. coli were analyzed by restriction enzyme digestion. The predicted restriction plasmid was sequenced to confirm all parts of the signal hB7-2-IgG gene fusion segment.
D. Expression cloning of hB7-2V-IgG1 and hB7-2C-IgG1

The variable and constant domains of human B7-2 were cloned separately into pNRDSH / IgG1. These clonings were performed using PCR. The portion of hB7-2 corresponding to the variable and constant regions was determined from intron / exon maps and published gene structure analysis.

(1) Assembly of hB7-2VIg The hB7-2V domain Ig sequence was assembled using the same PCR strategy as described above. The signal sequence was obtained by PCR amplification of a plasmid containing the oncoM gene, and the oligonucleotide 5'-GCAACCCGGAAGCTTGCCACCATGGGGGTACTGCTCACACAGAGGACGCG-3 '(# 05) (SEQ ID NO :) was used as a forward PCR primer. ) (SEQ ID NO :) was used as a reverse primer. The forward PCR primer (# 05) contains a HindIII restriction site and the amino terminal portion of the oncoM signal sequence. Reverse PCR (# 06) contains the sequence corresponding to the 3 ′ portion of the oncoM signal sequence fused to the 5 ′ end of the hB7-2IgV-like domain.

hB7-2IgV-like domain is oligonucleotide 5'-CTCCCTTTTCCAAGCATGGCCAGCATGGCTCCCTCTGAAGATTCAGGCTTTATTTCAATGAGAC-3 '(# 07) forward (SEQ ID NO :), oligonucleotide 5'-TGTGTGTGATGATGCTATCATGATG Obtained by PCR amplification of hB7-2 cDNA used as a primer. PCR amplification with these primers yielded an hB7-2IgV domain having the 3 'end portion of the oncoM signal sequence at the 5' end and the BclI restriction site at the 3 'end. Signal and IgV domain were combined in a PCR reaction. There, equimolar amounts of oncoM signal and IgV domain DNA fragment were mixed, denatured, annealed and the strands were satisfied. Subsequent PCR amplification using forward primer # 05 and reverse primer # 08 yielded a DNA fragment containing a HindIII restriction site, followed by an oncoM signal fused to the B7-2 IgV domain, followed by a BclI restriction site. This PCR fragment was digested with HindIII and BclI and cloned into the expression vector pNRDSH / IgG1 digested with the same restriction enzymes to obtain pNRDSH / B7-2CIg.
(2) Assembly of hB7-2CIg

An expression plasmid for the hB7-2 IgC domain was prepared as described above for IgV except that PCR primers specific for the IgC domain were used. The signal sequence oncoM was prepared using oligonucleotide # 05 as the forward PCR primer and oligonucleotide 5′-AGAAATTGGTACACTTTTCAGGTTGACTGAAGTTGCCATGCTGGCCATGCCTTGGAAACAGGAG-3 ′ (SEQ ID NO :) as the reverse PCR primer. The hB7-2 IgC domain was prepared using the oligonucleotide 5'-CTCCCTGTTCCAAGCATGGCCAGCATGGCTACTACTTCAGTCAACCCTGAAATAGTACCAATTTC-3 '(# 11) (SEQ ID NO :) as a reverse PCR primer. Both PCR products were mixed and amplified with primers # 05 and # 11 to assemble the signal sequence oncoM and the hB7-2IgC domain. This PCR product was then digested with HindIII and BclI and ligated into the expression vector pNRDSH / IgG1 digested with similar restriction enzymes to yield the final expression plasmid pNRDSH / hB7-2CIgG1.
E. Competitive binding assay with human B7-2Ig fusion protein

The ability of various B7-Ig fusion proteins to competitively inhibit the binding of biotinylated CTLA4Ig to immobilized B7-2-Ig was measured. Competitive binding assays were performed as follows and analyzed according to McPherson (McPherson, GA (1985), J. Pharmacol. Methods, 14: 213-228). Soluble hCTLA4Ig was labeled with ¹²⁵ I to a specific activity of about 2 × 10 ⁶ cpm / pmol. A microtiter plate was coated overnight at 50 μL / well with 10 μg / mL pH 8.0 of 10 mM-Tris solution of hB7-2-Ig fusion protein. Wells were blocked for 2 hours at room temperature with binding buffer (DMEM with 10% heat inactivated FBS, 0.1% BSA and 50 mM BES, pH 6.8). Full length B7-2 (hB7-2Ig), full length B7-1 (hB7-1Ig), B7-2 (hB7-2VIg) variable region-like domain and B7-2 (hB7-2CIg) constant region-like domain in each well Labeled CTLA4-Ig (4 nM) was added in the presence or absence of an unlabeled competitive Ig fusion protein containing and allowed to bind for 2.5 hours at room temperature. The wells were washed once with ice-cold binding buffer and then 4 times with ice-cold PBS. The wells were treated with 0.5N NaOH for 5 minutes to recover bound radioactivity, lysate was removed and counted with a gamma counter.

  The results of these assays are shown in FIG. 15, where both hB7-2Ig (10-20 nM) and hB7-2VIg (30-40 nM) inhibit CTLA4Ig binding to the immobilized B7-2 protein. hB7-2CIg did not compete with soluble CTLA4 and the B7-2 binding region was found to be in the variable region-like domain. F. Competitive binding assays for B7-1 and B7-2 fusion proteins

The ability of various types of recombinant CTLA4 to bind to hB7-1 or hB7-2 was assessed by competitive binding ELISA assay as follows. Binding to Costar EIA / RIA 96-well microtiter dishes (Costar, Cambridge, MA, USA) overnight at room temperature in 50 μL of purified recombinant hB7-Ig (20 μg / mL PBS). Each well was washed 3 times with 200 μL of PBS, and 1% BSA · PBS was added (200 / well) to block unbound portions for 1 hour at room temperature. Each well was washed as described above. Biotinylated hCTLA4IgG1 (ref, MFGR, 1 μg / mL, serial 2-fold dilution, up to 15.6 ng / mL, 50 μL) was added to each well and incubated at room temperature for 2.5 hours. Each well was washed as described above. Bound biotinylated CTLA4Ig was detected by adding 50 l / l of 1: 2000 dilution of streptavidin-HRP (Pierce Chemical, Rockford, IL) at room temperature for 30 minutes. The wells were washed as before, ABTS (Zymed, California) 50 μL was added, and after 30 minutes, a blue color was observed at 405 nm. A graph of typical binding test results is shown in FIG. Various amounts of competing proteins were mixed with a fixed amount of biotinylated CTLA4 IgG1 (ie, 70 ng / mL, 1.5 nM) that was proved to be desaturated, and binding assays were performed as described above. The ability to compete with activated CTLA4IgG1 was evaluated (FIG. 15). A decrease in the expected signal (405 nm absorbance) for biotinylated CTLA4IgG1 indicates competition in binding to hB7-1.
Considering the above evidence that CTLA4 is a receptor having high affinity for B7-1, the binding strength of CTLA4 and CD28 to B7-1 and B7-2 was compared. B7-1-Ig or B7-2-Ig was labeled with biotin and allowed to bind to immobilized CTLA4-Ig in the presence or absence of various concentrations of unlabeled B7-1-Ig or B7-2-Ig. The experiment was repeated with ¹²⁵ I-labeled B7-1-Ig or B7-2-Ig. Using this solid phase binding assay, the binding force of B7-2 to CTLA4 (2.7 nM) was determined to be about twice as large as that observed with B7-1 (4.6 nM). Experimentally determined IC ₅₀ values are shown in the upper right corner of the figure. The affinity of both B7-1 and B7-2 for CD28 was low and it was difficult to conclude reliably.

Example 8 Production and properties of monoclonal antibody against human B7-2
A. Immunized and cell-fused Balb / c female mice (obtained from Taconic Laboratories, Germany, NY) human B7.2-2 emulsified in complete Freund's adjuvant (Sigma Chemical, St. Louis, MO) per animal. Immunization was performed by intraperitoneal administration of 50 μg of Ig or 10 ⁶ CHO-human B7.2 cells. Mice were infused twice with human B7.2-Ig 10-25 μg emulsified in incomplete Freund's adjuvant (Sigma Chemical, St Louis, Mo.) or following the first immunization with CHO-human B7.2 cells. Booster immunization was performed at intervals of 14 days for 2 months. Blood was collected from the retroorbital area of the mouse, and the serum was assayed for the presence of antibodies reactive with this immunogen by ELISA against human B7.2-Ig. An ELISA against hCTLA4-Ig was also performed as a control for Ig tail-directed antibody response. Mice that showed a strong serum reaction were intravenously boosted by diluting 25 μg of human hB7.2-Ig into phosphate buffered saline (PBS), pH 7.2 (GIBCO, Grand Island, NY) from the tail vein. Three to four days after this booster immunization, neither heavy chain nor light chain of immunoglobulin can be secreted into the mouse spleen (Kearney et al. (1979), J. Immunol., 123: 1548) SP2 / 0 myeloma cells ( American Type Culture Collection, Rockville, MD, No. CRL8006) was injected at 5: 1. A standard method based on the method developed by Kohler and Milstein (Nature (1975), 256: 495) was used.
B. Antibody screening

Ten to 21 days later, the supernatant of wells containing hybridoma colonies from the infusion was screened for the presence of antibodies reactive against human B7.2 as follows: flat bottom 96-well plate (Costar) , Catalog number 3590) with 50 μL of human B7.2-Ig 1 μg / mL solution or lysine-coated plates at 4 ° C. in phosphate buffered saline, pH 7.2, 3T3-hB7.2 cells 5 × 10 Coated with ⁴ pieces. Aspirate human B7.2-Ig solution or bind cells to plate with glutaraldehyde, wash each well 3 times with PBS, then with 1% BSA (PBS) solution (100 μL / well) for 1 hour at room temperature Blocked. After this blocking incubation, the wells were washed three times with PBS, 50 μL of hybridoma supernatant was added per well, and incubated at room temperature for 1.5 hours. Following this incubation, the wells were washed 3 times with PBS and then at room temperature horseradish peroxidase-binding, affinity purification, goat anti-mouse IgG or IgM heavy and light chain-specific antibodies (HRP, Zymed Laboratories, (San Francisco, Calif.) Was incubated for 1.5 hours with 50 μL per well of a 1: 4000 dilution. The wells were then washed 3 times with PBS, followed by 1 mM-2,2-azinobis-3-ethylbenzthiazoline-added 1: 1000 dilution of 30% hydrogen peroxide as a substrate to HRP to detect bound antibody A solution of 6-sulfonic acid (ABTS) in 0.1 M sodium citrate, pH 4.2 was incubated in 50 μL per well for 30 minutes. Subsequently, the absorbance was measured at OD ₄₁₀ with an automatic reading spectrophotometer (Dynatech, VA).
Three species, HA3.1F9, HA5.2B7 and HF2.3D1, were confirmed to produce antibodies against human B7.2-Ig. HA3.1F9 was determined to be an IgG1 isotype, HA5.2B7 was an IgG2b isotype, and HF2.3D1 was an IgG2a isotype. These hybridomas were further subcloned twice to confirm that they were monoclonal. Each hybridoma cell is an American type culture collection that conforms to the provisions of the Budapest Treaty as ATCC accession number HB11688 (hybridoma HA3.1F9), ATCC accession number HB11687 (hybridoma HA5.2B7) and ATCC accession number HB11686 (hybridoma HF2.3D1). Deposited on July 19th.
C. Competitive ELISA

Ｄ．フローサイトメトリー Supernatants from hybridomas HA3.1F9, HA5.2B7 and HF2.3D1 were further analyzed by competitive ELISA to demonstrate the ability of the monoclonal antibody to block biotinylated hCTLA4Ig binding to immobilized hB7-2 immunoglobulin fusion protein. investigated. Biotinylation of hCTLA4Ig was performed using Pierce's Immunopure, NHS-LC, Biotin (catalog number 21335). The B7-2 immunoglobulin fusion proteins used were hB7.2-Ig (full length hB7-2), hB7.2-VIg (variable domain only hB7-2) and hB7.2-CIg (constant domain only hB7- 2). hB7.1-Ig fusion protein was used as a control. For ELISA, a 96-well plate was coated with Ig fusion protein (20 μg / mL solution 50 μL / well) overnight at room temperature. The wells were washed 3 times with PBS, blocked with 10% fetal bovine serum (FBS) and 0.1% bovine serum albumin (BSA) in PBS for 1 hour at room temperature, and further washed 3 times with PBS. To each well, 50 μL of Bio-hCTLA4-Ig (70 ng / mL) and 50 μL of the competitive monoclonal antibody supernatant were added. Control antibodies were anti-B7.1 mAb (EW3.5D12) and anti-hB7-2 mAbB70 (IgG2bκ, obtained from Pharmingen). Each well was washed again and streptavidin-conjugated horseradish peroxidase (from Pierce, catalog number 21126, 1: 2000 dilution, 50 μL / well) was added and incubated for 30 minutes at room temperature. Wash wells again, followed by ABTS in 0.1 M sodium citrate, pH 4.2 solution, added at a dilution of 1: 1000 with 30% hydrogen peroxide to detect antibody as a substrate for HRP in 50 μL per well Incubated for 30 minutes. Subsequently, the absorbance was measured at OD ₄₁₀ with an automatic reading spectrophotometer (Dynatech, VA). The results shown in Table IV below show that each mAb produced from hybridomas HA3.1F9, HA5.2B7 and HF2.3D1 can competitively block the binding of hCLTA4Ig to full length hB7.2-Ig or hB7.2-VIg. (HCLTA4Ig does not bind to hB7.2-CIg).

D. Flow cytometry

Supernatants from hybridomas HA3.1F9, HA5.2B7 and HF2.3D1 were also analyzed by flow cytometry. Supernatants collected from clones on CHO and 3T3 cells (CHO-hB7.2 and 3T3-hB7.2) transfected to express hB7.2 or controls on transfected 3T3 cells (3T3-Neo) And screened by flow cytometry. Flow cytometry was performed as follows: 1 × 10 ⁶ cells were washed 3 times with 1% BSA in PBS, then the cells were washed in 50 μL of hybridoma supernatant or culture per 1 × 10 ⁶ cells. Incubated for 30 minutes at 4 ° C. After incubation, the cells were washed 3 times with 1% BSA in PBS, then fluorescein-conjugated goat anti-mouse IgG or IgM antibody (Zymed Laboratories, San Francisco, Calif.) At 1:50 dilution. Incubation for 30 minutes at 4 ° C. with 50 μL per 10 ⁶ cells. Cells were then washed 3 times with 1% BSA in PBS and fixed with 1% paraformaldehyde solution. Cell samples were then analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA). The results shown in FIGS. 17, 18 and 19 demonstrate that the monoclonal antibodies produced by hybridomas HA3.1F9, HA5.2B7 and HF2.3D1 each bind to hB7-2 on the cell surface.
E. Inhibition of human T cell proliferation by anti-hB7-2 mAb

The ability of a hybridoma supernatant containing anti-human B7-2 mAb to block hB7-2 costimulation of human T cells was examined. In this assay, purified CD28 ⁺ human T cells were treated with a quasi-mitogenic amount of PMA (1 ng / mL) to obtain primary signals, and costimulatory signals were obtained with CHO cells expressing hB7-2 on the surface. T cell proliferation was measured in the remaining 18 hours after addition of ³ H-thymidine after 3 days in culture. As shown in Table V, resting T cells showed no proliferation as measured by the ³ H-thymidine (510 pm) uptake method. Release of signal 1 by PMA showed some proliferation (3800 pm), with T cells receiving both primary (PMA) and costimulatory (COS / hB7-2) signals maximally proliferating (9020 cpm). All three tested anti-hB7-2 mAbs reduced costimulatory signal-induced proliferation to that seen in PMA-treated cells, and these mAbs blocked T7 cell proliferation by blocking the B7 / CD28 costimulatory pathway. Showed that to do.

Example 9 Regression of transplanted tumor cells transfected to express B7-2
In this example, when J558 plasmacytoma cells that were not transfected or transfected with B7-2 were used for tumor regression studies and implanted into animals, tumor cell growth of B7-2 expressed on the surface of the tumor cells The effect on was examined.

  Expression vector containing cDNA encoding mouse B7-2 (pAWNE03) or B7-1 (pNRDSH or pAWNE03) and neomycin resistance gene in J558 plasmacytoma cells (obtained from American Type Culture Collection, Rockville, MD, # TIB6) Transfected. Stable transfectants were selected based on neomycin resistance, and cell surface expression of B7-2 or B7-1 on tumor cells was confirmed by FACS analysis using anti-B7-2 or anti-B7-1 antibodies. .

The same genotype Balb / c mice were used in an experiment designed to determine whether B7-2 cell surface expression on tumor cells would cause regression of transplanted tumor cells in groups of 5 to 10 mice. Untransfected and transfected J558 cells were cultured in vitro, harvested, washed with Hank's buffered salt solution (GIBCO, Grand Island, New York) and resuspended at a concentration of 10 ⁸ cells / mL. . A part of the skin on the right flank of each mouse was depilated with a depilatory agent, and 24 hours later, 5 × 10 ⁶ tumor cells per mouse were transplanted intradermally or subcutaneously. Tumor volume was measured (with 3 linear measurements) every 2 or 3 days using calipers and rulers. A typical experiment was continued for 18-21 days, but at the end, the tumor size exceeded 10% of the mouse body volume in mice transplanted with untransfected control J558 cells. As shown in FIG. 20, the mice excluded J558 cells transfected to express B7-2 on the surface. No tumor growth was observed even after 3 weeks. Similar results were observed with J558 cells transfected to express B7-1 on the surface. In contrast, untransfected (wild-type) J558 cells produced large tumors that were untreatable in animals on day 12. This example demonstrates that B7-2 cell surface expression of tumor cells, such as by transfection of tumor cells with B7-2 cDNA, induces an anti-tumor response sufficient to cause tumor cell rejection in susceptible animals. is there.
Equal

  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 equivalent ranges are intended to be encompassed by the present claims.

Medium, anti-CD3 alone or the following monoclonal antibody or recombinant protein, αB7 (133, anti-B7-1); CTLA4Ig; FabαCD28; control Ig fusion protein (isotype control for CTLA4Ig); or αB5 (anti-B5, anti-B5 B7 (B7-1) transfected CHO cells (panel a), or syngeneic activated B cultured in anti-CD-3 in the presence of (isotype control for B7-1) FIG. 4 is a graphical representation of the response of CD28 ⁺ T cells as assessed by ³ H thymidine transduction or IL-2 secretion to costimulation provided by lymphocytes (panel b).

FIG. 6 is a graph of log fluorescence intensity of cell surface expression of B7-1 on splenic B cells activated with surface immunoglobulin (sIg) cross-linking. All (panel a), B7-1-positive (B7-1 ^+, panel b) and B7-1 negative (B7-1 ^-, panel c) the activated B cells, an anti-B7-1 monoclonal antibody (133) and fluoro Stained with cein isothiocyanate (FITC) labeled goat anti-mouse immunoglobulin and analyzed by flow cytometry.

, Medium, anti-CD3 alone, or the following monoclonal antibodies or recombinant proteins, αBB-1 (133, anti-B7-1 and anti-B7-3); αB7 (anti-B7-1); CTLA4Ig; FabαCD28; control Ig fusion Against costimulation provided by B7-1 ⁺ (panel a) or B7-1 ⁻ (panel b) activated syngeneic B lymphocytes cultured in protein or anti-CD3 in the presence of αB5 (anti-B5) , Graphic representation of CD28 ⁺ T cell responses as assessed by ³ H-thymidine transduction and IL-2 secretion.

Fractionated B7-1 ⁺ and B7-1 ^- activated B lymphocytes on the of B7-1, a graphical representation of cell surface expression of B7-3 and all CTLA4 counter-receptor.

Temporal surface expression of B7-1 (CTLA4Ig and mAbs BB-1 and 133), B7-3 (CTLA4Ig and mAbBB1) and B7-2 (CTLA4Ig) counter receptors on splenic B cells activated by sIg cross-linking It is a graph display.

Temporal surface expression of B7-1 (CTLA4Ig and mAbs BB-1 and 133), B7-3 (CTLA4Ig and mAbBB1), and B72 (CTLA4Ig) counter receptors on splenic B cells activated by MHC class II crosslinking It is a graph display.

Vehicle, anti-CD3 alone or the following monoclonal antibody or recombinant protein, αB7 (133, anti-B7-1); αBB1 (anti-B7-1, anti-B7-3); CTLA4Ig; FabαCD28; and αB5 (anti-B5) ³ H-thymidine against stimuli provided by syngeneic B lymphocytes activated by sIg cross-linking, cultured in anti-CD3 in the presence of 24 h (panel a) or 48 h (panel b) FIG. 5 is a graphical representation of CD28 ⁺ T cell responses as assessed by transduction and IL-2 secretion.

The nucleotide sequence and deduced amino acid sequence of human B lymphocyte antigen B7-2 (hB7-2-clone 29).

The nucleotide sequence and deduced amino acid sequence of human B lymphocyte antigen B7-2 (hB7-2-clone 29).

The nucleotide sequence and deduced amino acid sequence of human B lymphocyte antigen B7-2 (hB7-2-clone 29).

Staining with control mAb (IgM), anti-B7-1 (mAbs133 and BB-1), recombinant protein CTLA4Ig, or an isotype-matched control Ig protein followed by an appropriate second FITC labeled immunoglobulin and flow cytometry FIG. 2 is a graphic representation of COS cells transfected with a control plasmid (pCDNAI, a plasmid expressing B7-1 (B7-1) or a plasmid expressing B7-2 (B7-2)) analyzed in (1).

Shows RNA blot analysis of B7-2 expression in unstimulated and anti-Ig activated human splenic B cells and cell lines (panel a) and human myeloma (panel B).

³ H-thymidine introduction or IL-- for submitogenic stimulation in phorbol myristic acid (PMA) and COS cells transfected with vectors alone or vectors directed to expression of B7-1 or B7-2 2 is a graphical representation of the proliferation of CD28 ⁺ T cells evaluated by secretion. Vector alone (vector), or B7-1 (B7-1) or B7-2 (B7-2) transfected COS cells with a vector expressing and to stimulation by ^PMA, 3 H- thymidine incorporation and IL- 2 is a graphical representation of the inhibition of proliferation of CD28 ⁺ T cells by recombinant protein and mAbs as assessed by 2 secretion. Inhibition studies were performed on PMA-stimulated COS cell mixed CD28 ⁺ T cells without antibody addition (no mAB), anti-B7 mAb 133 (133), anti-B7 mAb BB-1 (BB1), anti-B5 mAb (B5), anti-CD28 Fab fragment. (CD28 Fab), CTLA4Ig (CTLA4Ig), or Ig control protein (control Ig) was added.

Vector alone (vector), or B7-1 (B7-1) or B7-2 (B7-2) transfected COS cells with a vector expressing and to stimulation by ^PMA, 3 H- thymidine incorporation and IL- 2 is a graphical representation of the inhibition of proliferation of CD28 ⁺ T cells by recombinant protein and mAbs as assessed by 2 secretion. Inhibition studies were performed on PMA-stimulated COS cell mixed CD28 ⁺ T cells without antibody addition (no mAB), anti-B7 mAb 133 (133), anti-B7 mAb BB-1 (BB1), anti-B5 mAb (B5), anti-CD28 Fab fragment. (CD28 Fab), CTLA4Ig (CTLA4Ig), or Ig control protein (control Ig) was added.

Human B7-2 protein (hB7-2) deduced amino acid sequence (SEQ ID NO: 2) and human B7-1 protein (hB7-1) (SEQ ID NOs: 28 and 29) and murin B7-1 protein (mB7) (SEQ ID NO: 30 and 31) shows the sequence homology between both amino acid sequences.

Human B7-2 protein (hB7-2) deduced amino acid sequence (SEQ ID NO: 2) and human B7-1 protein (hB7-1) (SEQ ID NOs: 28 and 29) and murin B7-1 protein (mB7) (SEQ ID NO: 30 and 31) shows the sequence homology between both amino acid sequences.

It is the nucleotide sequence and the deduced amino acid sequence (SEQ ID NO: 22 and 23) of mouse B7-2 antigen (mB7-2).

It is the nucleotide sequence and the deduced amino acid sequence (SEQ ID NO: 22 and 23) of mouse B7-2 antigen (mB7-2).

It is the nucleotide sequence and the deduced amino acid sequence (SEQ ID NO: 22 and 23) of mouse B7-2 antigen (mB7-2).

It is the nucleotide sequence and the deduced amino acid sequence (SEQ ID NO: 22 and 23) of mouse B7-2 antigen (mB7-2).

FIG. 4 is a graphical representation of competitive inhibition of biotinylated CTLA4Ig binding to immobilized B7-2Ig by a B7 family-Ig fusion protein. Ig fusion proteins examined as competitors are full length B7-2 (hB7.2), full length B7-1 (hB7.1), variable region-like domain of B7-2 (hB7.2V), or constant of B7-2. Region-like domain (hB7.2C).

Biotinylated B7-1-Ig (Panel A) or B7-2 against immobilized CTLA4-Ig by increasing the concentration of unlabeled B7-1-Ig (Panel A) or B7-2-Ig (Panel B) -Graphic representation of competitive inhibition of binding of Ig (panel B). Experimentally determined IC ₅₀ values are shown in the upper right corner of the panel.

The flow cytometry profile of the cell dye | stained with the anti- hB7-2 monoclonal antibody, HA3.1F9 is shown. Cells stained with antibody (3T3-hB7.2) are CHO cells transfected to express human B7-2 (CHO-hB7.2), NIH3T3 cells transfected to express human B7-2. (3T3-hB7.2) and control transfected NIH 3T3 cells (3T3-neo). Anti-hB7.2 antibody B70 was used as a positive control.

The flow cytometry profile of cells stained with anti-hB7-2 monoclonal antibody, HA5.2B7 is shown. Cells stained with the antibody were CHO cells transfected to express human B7-2 (CHO-hB7.2), NIH3T3 cells transfected to express human B7-2 (3T3-hB7.2), And control transfected NIH3T3 cells (3T3-neo). Anti-hB7.2 antibody B70 was used as a positive control.

2 shows a flow cytometry profile of cells stained with an anti-hB7-2 monoclonal antibody, HF2.3D1. Cells stained with the antibody include CHO cells transfected to express human B7-2 (CHO-hB7.2), NIH3T3 cells transfected to express human B7-2 (3T3-hB7.2), And control transfected NIH3T3 cells (3T3-neo). Anti-hB7.2 antibody B70 was used as a positive control.

Of J558 plasmacytoma cells transfected to express B7-1 (J558-B7.1) or B7-2 (J558-B7.2), or tumor cells in mice after transplantation of J558 plasmacytoma cells It is a graphical representation of growth (measured by tumor size).

Claims (81)

  An isolated nucleic acid comprising a nucleotide sequence encoding a peptide having the ability to bind CD28 or CTLA4, wherein the nucleic acid is the complement of a nucleic acid molecule encoding a peptide comprising the amino acid sequence shown in FIG. 8 (SEQ ID NO: 2) Isolated nucleic acid that is hybridized with 6.0 × NaCl / sodium citrate (SSC) at about 45 ° C. and then washed with 2 × SSC at a temperature of about 50 ° C.   The isolated nucleic acid of claim 1, wherein the peptide comprises amino acid residues 24-133 of the human B7-2 protein shown in Figure 8 (SEQ ID NO: 2).   An isolated nucleic acid comprising a nucleotide sequence encoding a peptide having the ability to bind CD28 or CTLA4, wherein the nucleic acid is the complement of a nucleic acid molecule encoding a peptide comprising the amino acid sequence shown in FIG. 14 (SEQ ID NO: 23) Isolated nucleic acid that is hybridized with 6.0 × NaCl / sodium citrate (SSC) at about 45 ° C. and then washed with 2 × SSC at a temperature of about 50 ° C.   A first nucleotide sequence encoding a first peptide capable of binding CD28 or CTLA4 (the first peptide is at least amino acid residues 24-245 of the human B7-2 peptide shown in FIG. 8 (SEQ ID NO: 2)) 90% amino acid sequence identity), as well as an isolated nucleic acid encoding a B7-2 fusion protein comprising a second nucleotide encoding a second peptide, wherein the nucleic acid is DNA, An isolated nucleic acid, wherein the peptide comprises amino acid residues 24-133 of the human B7-2 protein shown in FIG. 8 (SEQ ID NO: 2).   The polypeptide according to claim 4, wherein the polypeptide comprises amino acid residues 24-133 of human B7-2 shown in Fig. 8 (SEQ ID NO: 2).   A polypeptide having at least 90% identity to the polypeptide of SEQ ID NO: 2 and capable of binding CD28 or CTLA4, in a form suitable for B7-2 expression, for use in treating a patient having a tumor. A composition comprising tumor cells transfected with a peptide-encoding nucleic acid and a pharmaceutically acceptable carrier, wherein the nucleic acid is delivered to a patient in vivo by injecting the nucleic acid in a suitable vehicle into the tumor .   A composition comprising an antibody that recognizes the B7-2 polypeptide shown in FIG. 8 (SEQ ID NO: 2) and an antibody that recognizes the B7-1 polypeptide shown in SEQ ID NO: 29.   8. The composition of claim 7, further comprising a pharmaceutically acceptable carrier.   The composition according to claim 7, wherein the antibody that recognizes B7-2 and the antibody that recognizes B7-1 are monoclonal antibodies.   The composition according to claim 7, wherein the antibody recognizing B7-2 and the antibody recognizing B7-1 are humanized antibodies.   The antibody according to claim 9, wherein at least one of the antibody recognizing B7-2 and the antibody recognizing B7-1 is an IgG2a antibody. The polypeptide shown in FIG. 8 (SEQ ID NO: 2);
A polypeptide comprising amino acid residues 24-133 shown in FIG. 8 (SEQ ID NO: 2);
A polypeptide comprising amino acid residues 24-133 shown in FIG. 8 (SEQ ID NO: 2);
A polypeptide comprising amino acid residues 24-245 shown in FIG. 8 (SEQ ID NO: 2);
A polypeptide comprising amino acid residues 24-245 shown in FIG. 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 amino acid residues 24-245 shown in FIG. 8 (SEQ ID NO: 2);
A nucleic acid molecule comprising a nucleic acid sequence encoding the polypeptide shown in FIG. 8 (SEQ ID NO: 2);
And a nucleic acid molecule comprising the coding region of the nucleotide sequence shown in SEQ ID NO: 1;
A composition suitable for pharmaceutical administration for use in modulating an immune response comprising a molecule selected from the group consisting of a pharmaceutically acceptable carrier.   The composition according to claim 12, further comprising a peptide having the activity of B lymphocyte antigen, B7-1. A pharmaceutical composition for modulating an immune response, wherein the composition is the polypeptide shown in FIG. 8 (SEQ ID NO: 2);
A polypeptide comprising amino acid residues 24-133 shown in FIG. 8 (SEQ ID NO: 2);
A polypeptide comprising amino acid residues 24-133 shown in FIG. 8 (SEQ ID NO: 2);
A polypeptide comprising amino acid residues 24-245 shown in FIG. 8 (SEQ ID NO: 2);
A polypeptide comprising amino acid residues 24-245 shown in FIG. 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 amino acid residues 24-245 shown in FIG. 8 (SEQ ID NO: 2);
A nucleic acid molecule comprising a nucleic acid sequence encoding the polypeptide shown in FIG. 8 (SEQ ID NO: 2);
A nucleic acid molecule comprising the coding region of the nucleotide sequence set forth in SEQ ID NO: 1;
A composition comprising a molecule selected from the group consisting of: contacting said immune cell, thereby preventing costimulation of the immune cell by B7-2-ligand interaction. The polypeptide shown in FIG. 8 (SEQ ID NO: 2);
A polypeptide comprising amino acid residues 24-133 shown in FIG. 8 (SEQ ID NO: 2);
A polypeptide comprising amino acid residues 24-133 shown in FIG. 8 (SEQ ID NO: 2);
A polypeptide comprising amino acid residues 24-245 shown in FIG. 8 (SEQ ID NO: 2);
A polypeptide comprising amino acid residues 24-245 shown in FIG. 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 amino acid residues 24-245 shown in FIG. 8 (SEQ ID NO: 2);
A nucleic acid molecule comprising a nucleotide encoding the polypeptide shown in FIG. 8 (SEQ ID NO: 2);
Use of a molecule selected from the group consisting of a nucleic acid molecule comprising the coding region of the nucleotide sequence shown in SEQ ID NO: 1 for the manufacture of a pharmaceutical composition for modulating an immune response.   13. The composition of claim 12, wherein the composition modulates T cell proliferation.   13. The composition of claim 12, wherein the composition down regulates the immune response.   13. A composition according to claim 12, wherein the composition upregulates an immune response.   13. The composition of claim 12, wherein the composition modulates at least one of autoimmune disease, allergy, and transplant rejection.   13. The composition of claim 12, wherein the composition modulates infection by a pathogen.   The composition according to claim 12, wherein the pathogen is a virus.   The composition according to claim 12, wherein the virus is HIV.   15. A pharmaceutical composition according to claim 14, wherein the molecule modulates T cell proliferation.   15. A pharmaceutical composition according to claim 14, wherein the molecule down regulates the immune response.   15. A pharmaceutical composition according to claim 14, wherein the molecule upregulates an immune response.   15. The pharmaceutical composition of claim 14, wherein the molecule modulates at least one of autoimmune disease, allergy, and transplant rejection.   15. A pharmaceutical composition according to claim 14, wherein the molecule modulates infection by a pathogen.   The pharmaceutical composition according to claim 14, wherein the pathogen is a virus.   The pharmaceutical composition according to claim 14, wherein the virus is HIV-1 or HIV-2.   16. Use according to claim 15, wherein the molecule modulates T cell proliferation.   16. Use according to claim 15, wherein the molecule down regulates the immune response.   16. Use according to claim 15, wherein the molecule upregulates an immune response.   16. The use according to claim 15, wherein the molecule modulates at least one of autoimmune disease, allergy, and transplant rejection.   16. Use according to claim 15, wherein the molecule modulates infection by a pathogen.   The use according to claim 15, wherein the pathogen is a virus.   16. Use according to claim 15, wherein the virus is HIV-1 or HIV-2.   An isolated variable region form of the B cell activating antigen B7-2 comprising amino acid residues 24-133 of the human B7-2 protein shown in FIG. 8 (SEQ ID NO: 2), but the B7-2 immunoglobulin A variable region that does not contain a constant domain.   38. The B7-2 variable region form of claim 37 that is human.   38. The B7-2 variable region form of claim 37, which is a fusion protein comprising a B7-2 variable region polypeptide operably linked to a heterologous polypeptide.   40. The B7-2 variable region form of claim 39, wherein the fusion protein is a soluble monomeric polypeptide.   40. The B7-2 variable region form of claim 39, wherein the B7-2 variable region polypeptide is a human B7-2 variable region polypeptide.   42. The B7-2 variable region form of claim 41, wherein the human B7-2 variable region polypeptide comprises the amino acid sequence of about 24-133 of SEQ ID NO: 2.   40. The B7-2 variable region form of claim 39, wherein the heterologous polypeptide is an immunoglobulin constant region.   44. The B7-2 variable region form of claim 43, wherein the immunoglobulin constant region comprises a Cγ1 domain comprising a hinge, CH2, and CH3.   40. The B7-2 variable region form of claim 39, wherein the immunoglobulin constant region has been modified to reduce constant region mediated biological effector function.   46. The B7-2 variable region form of claim 45, wherein the biological effector function is selected from the group consisting of complement activation and Fc receptor interaction.   47. The B7-2 variable region form of claim 46, wherein the immunoglobulin constant region is a Cγ4 domain comprising a hinge, CH2, and CH3.   48. The B7-2 variable region form of claim 47, wherein at least one amino acid residue of the CH2 domain is modified by substitution, addition or deletion.   An isolated B7-2 fusion protein comprising amino acid residues 24-133 of the human B7-2 protein shown in FIG. 8 (SEQ ID NO: 2) operably linked to a heterologous polypeptide, the B7-2 fusion protein Is a B7-2 fusion protein that does not contain a B7-2 immunoglobulin-like constant region domain.   50. The B7-2 fusion protein of claim 49, wherein the human B7-2 immunoglobulin-like variable region domain comprises the amino acid sequence at about positions 24-133 of SEQ ID NO: 2.   50. The B7-2 fusion protein of claim 49, wherein the heterologous polypeptide comprises an immunoglobulin-like constant region polypeptide.   An isolated nucleic acid molecule encoding the variable region form of a B7-2 fusion protein, wherein the B7-2 fusion protein is operably linked to a heterologous polypeptide as shown in FIG. 8 (SEQ ID NO: 2) human B7-2 An isolated nucleic acid molecule comprising amino acid residues 24-133 of the protein, wherein the B7-2 fusion protein does not comprise a B7-2 immunoglobulin-like constant region domain.   53. The isolated nucleic acid molecule of claim 52, wherein the B7-2 variable region polypeptide is a human B7-2 variable region polypeptide.   54. The isolated nucleic acid molecule of claim 53, wherein the human B7-2 variable region polypeptide comprises an amino acid sequence at about positions 24-133 of SEQ ID NO: 2.   53. The nucleic acid of claim 52, wherein the heterologous polypeptide is an immunoglobulin constant region polypeptide.   56. The nucleic acid of claim 55, wherein the immunoglobulin constant region comprises a C [gamma] 4 domain comprising a hinge, CH2 and CH3.   56. The nucleic acid of claim 55, wherein the immunoglobulin constant region is modified to reduce constant region mediated biological effector function.   58. The nucleic acid of claim 57, wherein the biological effector function is selected from the group consisting of complement activation and Fc receptor interaction.   59. The nucleic acid of claim 58, wherein the immunoglobulin constant region comprises a C [gamma] 4 domain comprising a hinge, CH2 and CH3.   60. The nucleic acid of claim 59, wherein at least one amino acid residue of the CH2 domain is modified by substitution, addition or deletion.   61. A recombinant expression vector comprising the nucleic acid molecule of any of claims 52-60.   62. A host cell comprising the recombinant expression vector of claim 61.   . 49. A composition suitable for pharmaceutical administration for use in modulating an immune response, the isolated variable region of a B cell activating antigen, B7-2 according to any of claims 37-48, And a composition comprising a pharmaceutically acceptable carrier.   62. An isolated nucleic acid encoding a variable region form of the B7-2 fusion protein of any of claims 52-61, wherein the composition is suitable for pharmaceutical administration for use in modulating an immune response. And a pharmaceutically acceptable carrier.   65. The composition of claim 63 or 64, wherein the composition modulates T cell proliferation.   65. The composition of claim 63 or 64, wherein the composition upregulates an immune response.   65. The composition of claim 63 or 64, wherein the composition modulates infection by a pathogen.   68. The composition of claim 67, wherein the pathogen is a virus.   69. The composition of claim 68, wherein the virus is HIV-1 or HIV-2.   A composition for stimulating a response by activated T cells, comprising a variable region form of B cell activation antigen B7-2, the variable region form of B7-2 being shown in FIG. 8 (SEQ ID NO: 2) The human B7-2 protein shown amino acid residues 24-133, but not containing the B7-2 immunoglobulin-like constant region domain, and contacting the B7-2 variable region form activated T cell, Results A composition that stimulates a response by activated T cells.   71. The composition of claim 70, wherein the T helper type 2 (TH2) response is preferentially stimulated.   Use of the variable region form of B cell activation antigen B7-2 for the manufacture of a medicament for the modulation of T cell proliferation and / or cytokine secretion in a patient, wherein the variable region of B7-2 is shown in FIG. Use comprising amino acid residues 24-133 of the human B7-2 protein shown in No. 2) but not containing the B7-2 immunoglobulin-like constant region domain.   An antibody or a fragment thereof that specifically reacts with a polypeptide capable of binding to CD28 or CTLA4 produced by the recombinant expression according to claim 1, 4, or 5, wherein the antibody is a humanized antibody An antibody or fragment thereof.   74. The antibody or fragment thereof of claim 73 comprising a human constant region and a non-human variable region.   74. The antibody or fragment thereof of claim 73 comprising a human constant region and a human variable region chimera.   An antibody that specifically reacts with a polypeptide having the ability to bind to CD28 or CTLA4, produced by recombinant expression according to claim 1, 4, or 5, wherein the antibody is a fully human monoclonal antibody, or a method thereof fragment.   77. A composition suitable for pharmaceutical administration for use in modulating an immune response comprising the antibody or fragment thereof of any of claims 73-76.   78. The composition of claim 77, wherein the composition modulates T cell proliferation.   78. The composition of claim 77, wherein the composition down regulates a T cell mediated immune response.   78. The composition of claim 77, wherein the composition modulates at least one of autoimmune disease, allergy, and transplant rejection.   77. A method of preventing interaction of a B lymphocyte antigen, B7-2, with its natural ligand on the surface of an immune cell, the method comprising: Contacting with it, thereby preventing costimulation of immune cells by B7-2 interaction.

Images