WO1996029348A1 - Monoclonal antibody against human receptor protein 4-1bb and methods of its use for treatment of diseases - Google Patents

Abstract

Disclosed herein is a monoclonal antibody against H4-1BB that can be used to enhance or suppress T-cell activation and proliferation by treating T-cells that have expressed receptor protein H4-1BB with the anti H4-1BB monoclonal antibody. Also disclosed herein is cDNA for the human receptor H4-1BB. The cDNA of the human receptor H4-1BB is about 65 % homologous to the mouse cDNA 4-1BB and was isolated by using probes derived from murine cDNA 4-1BB. A fusion protein for detecting cell membrane ligands to human receptor protein H4-1BB was developed. It comprises the extracellular portion of the receptor protein H4-1BB and a detection protein (alkaline phosphatase) bound to the portion of the receptor protein H4-1BB. B-cells that have expressed a ligand to receptor protein H4-1BB can be treated with cells that have expressed receptor protein H4-1BB and B-cell proliferation may be induced. The use of H4-1BB to block H4-1BB ligand binding has practical application in the suppression of the immune system during organ transplantation or against autoimmune diseases.

Description

MONOCLONAL ANTIBODY A-3AINST HUMAN RECEPTOR PROTEIN 4-1BB AND METHODS OF ITS USE FOR TREATMENT OF DISEASES.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of co-pending application Serial No. 08/122,796 filed September 16, 1993, which is a continuation-in-part of co-pending application Serial No. 08/012,269 filed February 1, 1993, which is a continuation-in-part of co-pending application Serial No. 07/922,996 filed July 30, 1992, which is a continuation-in-part of copending application Serial No. 07/267,577 filed November 7, 1988, now abandoned.

The subject matter described herein was in part a subject invention of NIH Grants Nos. IR23AI23058-03, RO1 AI28175 and P-60 KD20542 of which the present inventor was the Principal Investigator and either the Donald Guthrie Foundation for Medical Research Inc. of Guthrie Square, Sayre, Pennsylvania 18849-1669 or Indiana University School of Medicine of Indianapolis, Indiana 46202, was the Grantee.

FIELD OF THE PRESENT INVENTION

The present invention relates to a previously unknown human receptor protein, H4-

1BB, a monclonal antibody that specifically recognizes H4-1BB and a ligand protein to H4- 1BB. H4-1BB was isolated and identified by research on a homologous murine (mouse) receptor protein, 4- IBB, which was isolated and identified by specific expression of the T cell genes by the present inventor. BACKGROUND OF THE PRESENT INVENTION

The immune system of humans and other .species require that white blood cells, which include phagocytes, T lymphocytes and B cells, be made in the bone marrow. As presently understood, the phagocytes include macrophage cells which .scavenge unwanted materials such as vims proteins from the system. The lymphocytes include helper T cells and killer T cells and B cells as well as other cells, including those categorized as suppressor T cells. The B cells produce the antibodies. The killer T cells physically pierce the cell -and the helper T cells facilitate the whole process. The immune process is facilitated by lymphokines.

Lymphokines are the proteins by which the immune cells communicate with each other. Scientists produce them in sufficient quantities for therapeutic use against immunologic diseases. There are many known lymphokine proteins and they include the interferons, interleulάn- 1,2,3,4,5,6,7, colony-stimulating factors, lymphotoxin, tumor necrosis factor and erythropoietin, as well as others.

Interleukin 1 , secreted from macrσphages activate the helper T cells and raise the body temperature causing fever which enhances the activity of the immune cells. The activated helper T Cells produce Interleukin 2, which in turn stimulates the helper and killer T cells to grow and divide. The helper T cells also produce another lymphokine, B cell growth factor (BCGF), which causes B cells to multiply. As the number of B cells increases, the helper T cells produce another lymphokine known as the B cell differentiating factor (BCDF), which instructs some of the B cells to stop replicating and start producing antibodies. T cells also produce a lymphokine, gamma interferon (IF), which has multiple effects like Interleukin 2. Interferon helps activate killer T cells, enabling them to attack the invading organisms. Like BCGF, interferon increases the ability of the B cells to produce antibodies. Interferon also keeps the macrophages at the site of the infection and helps the macrophages digest the cells they have engulfed. Gathering momentum with each kind of lymphokine signal between the macrophages and the T cells, the lymphokines amplify the immune system response and the vims protein or other foreign matter on the infected cells is overwhelmed. There are many other lymphokines, maybe a hundred or more, which participate in the immune process. Many lymphokines are known and many are not. Lymphokine activities are produced when a certain lymphokine binds to its specific receptor. Among scientists there is widespread use of cloned cell lines for production of lymphokines and their receptors. The isolation of lymphokine and lymphokine receptor mRNA has become a common technique. The mouse receptor protein, 4- IBB, was isolated and identified based on specific expression of the T cell genes using a technique identified by the present inventor in a publication (Proc. Natl. Acad. Sci. USA. 84, 2896-2900, May 1987, Immunology). The protocol reported in this publication can be used by scientists to detect virtually all of the lymphokines. The method is designed to detect virtually all mRNA expressed differentially and the mRNA sequences of the immune cells are expressed differentially (as they relate to the T cells and the killer T cells) even though the level of expression is low and the quantity of the lymphokine and its receptor protein is low. The present inventor believes that the analysis described in the above identified publication can reveal biologically important molecules such as lymphokines and their receptors because there are many indications that biologically important or active molecules are coded by the most scarce messages.

Most T cell factors have been classically identified by recognizing biologic activities in assays, purifying the protein information. An alternative approach is to isolate putative T cell genes based upon specific expression and then demonstrate the function of the unknown molecule. Using the aforesaid modified differential screening procedure, the present inventor cloned a series of T cell subset-specific cDNAs from cloned helper T (HTL) L2 and cloned cytolytic T lymphocyte (CTL) L3.

T cells are critically important in acquired immunity, providing protection against viral, bacterial and parasitic infection. T cells are activated when they encounter peptide from the invading pathogen in context with self-MHC via the T cell's own T cell receptor (TCR) complex and other costimulatory molecule(s), such as CD28(1 ,2). Without the engagement of the other co-stimulatory molecule(s) the T cell is rendered anergic(3). To date the best characterized co-stimulatory molecule has been CD28(1,2). More recently, however, other cell surface molecules have been suggested to play a co-stimulatory role, such as the molecule 4-1 BB. 4-1 BB is a -55 kDa homodimeric molecule expressed on activated T cells in the mouse and is a member of the Nerve Growth Factor receptor (NGFR)/Tumor Necrosis Factor receptor (TNFR) gene family(4). This family is characterized by the presence of cysteine-rich motifs in the extracellular domains. Other members of this family include NGFR, B cell activation molecule CD40, the T cell activation molecule OX-40 in rat and CD27, the two receptors for TNF called TNFR-1 and TNFR-11, the apoptotic inducing molecule called Fas, and CD30 which has been suggested to play a role in the regulation of cellular growth and transformation (4, 5).

A series of T-cell subset-specific cDNAs were isolated from cloned murine T-cεlls by employing a modified differential screening procedure. The nucleotide sequence and expression properties of some of the cDNA species have been reported. One of the genes not previously characterized, that encodes mouse receptor protein 4- IBB, was studied further. These studies liave led to the isolation of the human homologue to 4- IBB, H4-1BB.

Specific immune responses are governed by the recognition of antibodies to foreign antigens. Antibodies form a family of structurally related glyco-proteins and confer the protective effect of cell-mediated immunity. Antibodies are produced by B lymphocytes and are bound to the cell membrane, functioning as B cell receptors for antigens. Antibodies are also secreted by B cell progeny which differentiate in response to stimulation by antigens. A specific antigen will trigger the complementary B lymphocyte to proliferate and differentiate into effector cells which then eliminate the antigen. Each lymphocyte produces an antibody of a particular specificity, and thus immune responses are very specific for distinct antigens. The portion of the antigen recognized by T and B lymphocytes are called epitopes or determinants.

The development of techniques to produce virtually unlimited amounts of a single (monoclonal) antibody for a specific antigenic epitope has had an enormous impact on clinical immunology. To produce a monoclonal antibody of known specificity, a mouse can be injected with a particular antigen, such as a receptor protein and the spleen B lymphocytes (that produce the antibody against the protein) can be fused via somatic cell hybridization to a myeloma (lymphocyte tumor) to produce an immortal cell line. This is done because normal B lymphocytes can not grow indefinitely, yet when fused with the myeloma, the resulting hybridoma does produce a virtually endless supply of a specific monoclonal antibody. "Selection" mediums have been developed to ensure that only the fused cells continue to grow. Each hybridoma cell is specific for only one antigenic determinant. If several hybridomas are produced that secrete antibodies that recognize and bind to the surface of a particular cell, each hybridoma clone will secrete an antibody for only one surface antigenic determinant. To determine which mAbs specifically bind to the protein receptor, the hybridomas can be screened with ELISA (enzyme-linked immunosorbent assay).

Monoclonal antibodies have numerous applications: 1) The hybridoma can produce large quantities of specific antibodies that are normally either unavailable in small quantities or not available at all; 2) the hybridoma can be directed to produce antibodies against a single antigen determinant which, for complex antigens, may be normally very difficult; 3) pure antibodies can be obtained against antigens that cannot be purified; 4) immunodiagnosis of infectious and systemic diseases by detecting specific antigens circulating in tissues or using monoclonal antibodies in immunoassays; 5) characterization of protein receptors and the role they play in the transition from a naive to a memory T cell; and 6) blocking or enhancing immune response.

SUMMARY OF THE PRESENT INVENTION

The present invention includes the human receptor protein H4-1BB and the cDNA gene encoding for human receptor protein H4-1BB. The nucleotide sequence of the isolated cDNA is disclosed herein along with the deduced amino acid sequence. The cDNA gene identified as pH4-lBB was deposited at the Agricultural Research Service Culture Collection and assigned the accession number: NRRL B21131

The cDNA, including its fragments and derivatives, can be used as a probe to isolate DNA sequences encoding for proteins similar to the receptor protein encoded by the cDNA. The cDNA of the human receptor, H4-1BB, is about 65% homologous to the mouse cDNA 4- 1BB and was isolated by using probes derived from cDNA 4-1BB. The cDNA gene identified as p4-lBB was deposited at the American Type Culture Collection at 12301 Parklawn Drive, Rockville, Maryland 20852 under ATCC No.: 67825.

The human receptor protein H4-1BB can be produced by: 1) inserting the cDNA of H4-1BB into an appropriate expression vector, 2) transfecting the expression vector into an appropriate transfection host, c) growing the transfected hosts in appropriate culture media and d) purifying the receptor protein from the culture media. The protein and fragments and derivatives can be used: 1) as a probe to isolate ligands to human receptor protein H4-1BB, 2) to stimulate proliferation of B-cells expressing H4-1BB ligands, or 3) to block H4-1BB ligand binding.

B-cell proliferation can be induced by treating B-cells that have expressed a ligand to receptor protein H4-1BB with cells that have expressed receptor protein H4-1BB. The use of H4-1BB protein, H4-1BB ligand protein, or fragments of the proteins, to block H4-1BB ligand binding has practical application in the suppression of the immune system during organ transplantation. A similar costimulatory immune system pathway is being analyzed for this type of application. See "Mounting a Targeted Strike on Unwanted Immune Responses", Jon Cohen, Science, Vol. 257, 8-7-92; "aLong Term Survival of Xenogeneic Pancreatic Islet Grafts Induced by CTLA4Ig", Lenschow et al, Science Vol. 257, 7-8-92; and "Immunosuppresion in Vivo by a Soluble Form of the CTLA-4 T Cell Activation Molecule", Linsley et al, Science Vol. 257 7-8-92.

A monoclonal antibody against H4-1BB can be used to enhance T-cεll proliferation by treating T-cells that have expressed receptor protein H4-1BB with the anti H4-1BB monoclonal antibody. Some tumors are potentially immunogenic but do not stimulate an effective anti- immune response in vivo. Tumors may be capable of delivering antigen-specific signals to T cells, but may not deliver the co-stimulatory signals necessary for full activation of T cells. Expression of the co-stimulatory ligand B7 on of melanoma cells was found to induce the rejection of a murine melanoma in vivo. ("Tumor Rejection After Direct Co-Stimulation of CD8⁺ T Cells by B7-Transfected Melanoma Cells", Sarah E. Townsend and James P. Allison, Science Vol. 259, 1-5-93.) A monoclonal antibody against H4-1BB may be capable of the same effect as it is now known to enducε T cell proliferation and activation.

The present invention includes a monoclonal antibody 4B4-1 that specifically recognizes an epitσpe on the extracellular domain of human receptor protein 4- IBB on peripheral blood T cells. The monoclonal antibody is produced from a hybridoma identified as 4B4-1 and deposited under the Budapest Treaty at the American Type Culture Collection at 12301 .Parklawn Drive, Rockville, Maryland 20852 under ATCC No.: HB-11860. The 4B4-1 mAb will have great utility in assessing the role of the 4-1BB receptor protein in the transition from naive to memory T-cells. In addition, by treating T-cεlls that have expressed receptor protein 4-1BB with 4B4-1 mAb T-cell proliferation and activation can be enhanced. Cross-linking of the 4- IBB with 4B4-1 mAb will produce the effects similar to the binding of the 4-1BB ligand to 4-1BB. The cross-liking effect makes the 4B4-1 mAb very useful for culturing T-cells because cross-Unking is necesarry for adequate proliferation. Therefore, the 4B4-1 mAb is a useful adjunct to T-cell culture medium. The mAb can also be used to enhance T-cell activation which is useful in the treatment of cancers that avoid normal immune responses.

A mAb aganist H4-1BB can also be used to interfere with H4-1BB and H4-1BB ligand binding. By interfering with the ligand binding, the immune responses will be suppressed. The mAb is usefiill as a tool to elucidate the mechanisms of auto-immune disease and can be used to control such problems. For example, the mAb described herein can be used to rheumatoid arthritis, systemic lupus erythematosis, and diabetis. The blocking effect can also be used to suppress the immune system during organ transplantation.

The monoclonal antibody against the murine 4- IBB ligand was produced by injecting Sprague-Dawley rats with purified recombinant protein of the 4- IBB ligand. The spleen cells were then fused to myeloma cell line SP/O. The positive clones were tested with H4-1BB expressing T-cell line. Each step pf these processes summarized here will be described in detail below.

The monoclonal antibody can be used enhance T-cell proliferation and activation. This can be done by treating T-cells that have expressed receptor protein 4-1BB with anti H41-BB monoclonal antibody.

A fusion protein can detect cell membrane ligands to human receptor protein, H4-1BB. A fusion protein of the present invention comprises the extracellular portion of the receptor protein H4-1BB and a detection protein (alkaline phosphatase) bound to the portion of the receptor protein H4-1BB. The portion of the receptor protein H4-1BB binds to the cell membrane ligands and binding can be detected by relative activity assays for the detection protein. The fusion protein is placed in the presence of a cell suspected to express the receptor protein H4-1BB. Then the cell is washed of any fusion protein not bound to the cell membrane ligands. Once the washed cells are placed in the presence of a substrate for the detection protein and the relative activity of the detection protein can be measured.

The primary object of the present invention is the identification of the new human receptor, H4-1BB as identified herein by its sequence.

Another object of the present invention is to teach a fusion protein comprising the extracellular portion of H4-1BB and a detection protein.

Still another object of the present invention is to teach methods of using the cDNA H4- 1BB, the receptor protein H4-1BB, the monoclonal antibody and the lig.and for H4-1BB.

BRIEF DESCRIPTIONS OF THE FIGURES

Figures la and 2b illustrate the molecules involved in T-cell activation.

Figures 2a, 2b, and 2c illustrate a normal T-cell activation pathway.

Figures 3a, 3b, and 3c illustrate CTLA4-lg alone, 4-1BB/AP and CTLA4-lg together and 4-1BB/AP alone respectively -being used to block steps in the T-cell activation pathway.

DETAILED DESCRIPTION

In the following detailed description references are made to known procedures and studies, as well as published work of the applicant. These publications are incorported herein by reference for clarity and listed in an Appendix A at the end of this detailed description. A list of common abbreviations is included in Appendix B.

The following description teaches the isolation of 4-1BB and its human homologue, H4-1BB, the preparation of the peripheral blood cells, including the antibodies and reagents used, the production of fusion protein, immunization and monoclonal antibody production, and immunoprecipitation studies. In short, the research shows that the monoclonal antibody 4B4-1 specifically binds to human receptor protein 4- IBB on peripheral blood T cells. The results .are coupled with the precise detail of the methods employed.

Isolation and characterization of mouse receptor 4- IBB

SEQ ID NO: 1 shows the nucleotide sequence and the deduced amino acid sequence of the mouse receptor 4- IBB. The predicted amino acid sequence is shown below the nucleotide sequence. The transcript of 4- IBB was inducible by concanavalin A in mouse splenocytes, T cell clones, and hybridomas. The expression of 4-1BB transcripts was inhibited by cyclosporin A. The 4-1BB mRNA was inducible by antigen receptor stimulation but was not inducible by n-2 stimulation in the cloned T-cells (1). The 4- IBB cDNA encodes a peptide of 256 amino acids containing a putative leader sequence, a potential membrane anchor segment, and other features of known receptor proteins. Therefore, the expression pattern of 4- IBB resembles those of lymphokine mRNAs while the sequence appeared consistent with those of receptor proteins.

The major species of 4- IBB on the cell surface appears to be a 55-kDa dimer. 4- IBB also appears to exist as a 30-kDa monomer and possibly as a 110-kDa tetramer. Since these 4- 1BB species were immunoprecipitated from a homogeneous population of cells (T-cell clone fl), all forms potentially co-exist on each cell. We will need to compare the peptide digests from the 4-1BB monomer and dimer to determine whether 4-1BB exists as a homodimer on the cell surface. A variety of cell surface receptors such as the insulin receptor(2), the B cell surface immunoglobin receptor (3), the T cell Ag receptor (4), the CD28 costimulatory receptor (5), .and the CD27 T-cell .antigen (6) .are composed of disulfide-bonded subunits. Receptor dimerization may be required for ligand binding and subsequent biochemical signalling.

4- IBB is not expressed on resting T cells but is inducible by activators which deliver a complete growth stimulus to the T cell. The combination of PMA and ionomycin is capable of mimicing those signals required for T cell proliferation. Although PMA or ionomycin alone induced 4- IBB mRNA, the combination of PMA and ionomycin resulted in optimal 4- IBB expression. Furthermore, the expression of 4-1BB was not transient. When purified splenic T- cells were stimulated with immobilized anti-CD3, 4- IBB mRNA was expressed and this expression was maintained for up to 96 hrs poststimulation. Cell cycle analysis will be required to confirm that 4- IBB is expressed throughout cell cycle progression.

4-1BB is structurally related to members of the nerve growth factor receptor super-family. Although these receptors possess structurally similar ligand-binding properties (cyst ne-rich regions), the cytoplasmic domains of these proteins are nonconserved which could allow for diversity in transmembrane signaling. Some members of this family are involved in the T or B cell activation process. There are in vitro functional data on the OX-40, CD4O and CD27 antigens. Antibodies against the OX-40 augment the T-cell response in a mixed lymphocyte reaction (7) and antibodies against CD4O enhance B-cell proliferation in the presence of a coactivator, such as PMA or CD2O antibodies, and synergize with JL-4 in vitro to induce B-cell differentiation and to generate long-term normal B cell lines (8). One monoclonal antibody, anti-lA4, which recognizes an epitope on the CD27 molecule inhibited calcium mobilization, IL-2 secretion, helper T cell function, and T-cell proliferation. On the other hand, CLB-CD27/1, another anti-CD27 mAb enhanced proliferation of human T cells stimulated with PHA or anti-CD3 mAb (6). These results indicate that the CD27 molecule plays an important role in T cell activation. Except for TNFRs, NCFR and CD4O, the ligands or cell surface molecules to which the members of the superfamily bind are not yet identified. Identification and characterization of the ligands to which the receptors bind will be helpful in better defining the physiologic role of 4- IBB.

To ascertain whether cell surface 4- IBB could contribute to T cell activation, the anti-4-lBB 53A2 was used as an antagonist to 4- IBB. These data suggested that 4- IBB does in fact have the potential to function as an accessory signaling molecule during T cell activation and proliferation. The addition of soluble 53.A2 to purified splenic T cells stimulated with immobilized anti-CD3 resulted in an amplification of ³H thymidine incorporation compared to T cells stimulated with anti-CD3 alone. This pattern of enhancement ranged from 2- to 10-fold in three independent experiments.

In the original two signal model of Bretcher and Cohn, they proposed that signal 1, the occupancy of the T cell antigen receptor (TCR), resulted in inactivation of the T cell in the abaSence of signal 2, which is provided by .accessory cells. This has since been confirmed by a variety of studies (9). The identification of the accessory cell CD28 as a potent costimulatory receptor on T cells was a significant contribution in beginning to charactize the accessory signal(s) required for optimal T cell proliferation (10). It is possible that other cell surface molecules may contribute to these costimulatory activation requirements (11).

The biochemical signals delivered through 4-1BB are not completely known. One possibility considered was the observation that 4- IBB contains a putative p56^k* tyrosine kinase binding domain in its cytoplasmic tail. It was later determined that p56^lck tyrosinase kinase binds to 4- IBB. It will also be worthwhile to determine if 4-lBB-mediated signaling can regulate genes such as IL-2 and IL-2 receptor, whose expression is required for T cell activation and subsequent proliferation.

The precise functions of members of the Nerve Growth Factor Receptor (NGFR) family appear to be diverse. An emerging theme of inquiry concerns the ability of these molecules to maintain the responsiveness or viability of the particular cell type in which they are expressed. For instance, NGF is absolutely required for viability of neurons in vitro and in vivo (12). The crosslinking of CD4O by soluble antiCD40 monoclonal antibody blocks germinal center centrocytes from undergoing apoptosis in vitro (13). Signals delivered through CD4O may also aid in maintenance of responsiveness to differentiation factors. The ligation of CD4O with anti-CD4O F(ab')₂ fragments in the presence of IL-4 induced large increases IgE synthesis (14). Also, anti-CD40 activated naive B cells treated with IL-10 -and transforming growth factor-β became committed to IgA secretion (15). In addition to sharing the molecular characteristics with the NGFR superfamily, it was noted that the 4- IBB contained a putative zinc finger stmcture of the yeast elF-2b protein (16). 4- IBB also shares a conserved region with the sina seven in absentia ofDmsophila, which is required for correct photoreceptor cell development (17). That particular region is also similar to the protein product of the DGI7 gene of Dictyostelium, whose expression is specifically induced during aggregation by cAMP (18).

This region forms the pattern of C-X₂-C-X -C-X₃-H-X₃-C-X-C; and the cysteines and histidine are conserved in a similar space in 4- IBB, sina, and DG17 proteins. Ten of 24 amino acids between the 4- IBB and sina proteins are identical; 3 of 24 are conservative substitutes. The conserved pattern suggests that these amino acids are functionally important. The sina protein is localiazed in the nucleus, suggesting that it has a regulatory function in cells. The fact that the amino acid sequence of 4-1BB contains features like a zinc finger motif, a nuclear protein, and a receptor domain suggests that 4- IBB may play diverse roles during cellular proliferation and differentiation.

In addition, 4-1BB may represent another cell-surface molecule involved in T cell-APC interactions. The 4-1BB-AP fusion protein specifically bound to mature B-cell lines, anti-μ-activated primary B cells, and mature macrophage-cell lines. 4-1BB-AP bound at low or insignificant levels to immature B- and macrophage-cell lines, T cell clones, T cell lines, primary culture T cells, and various nonlymphoid-cell lines. Since 4-1BB-AP binds to mature B cells and macrophages, it is possible that signals delivered upon 4- IBB binding may modulate APC functions in some way. This possibility remains to be explored.

Chalupny and colleagues (19) have proposed that 4- IBB Rg, a fusion protein consisting of the extracellular domain of 4- IBB and the Fc region of human IgG, bound to the extracellular matrix (ECM). The highest level of 4-1BB Rg binding was to human vitronectin. In data not shown, an ELISA was performed using 4-1BB-AP and human vitronectin (Yelios Pharmaceuticals GIBCO-BRL, Grand Island, NY) immobilized at 0.007 mg-lOmg per well on mi-crotiter plates. No binding of 4-1BB-AP based on AP activity was observed. To mle out the possibility that 4-1BB-AP was binding to proteins extrinsically attached to the cell surface (possible extracellular matrix components), B-cell lymphomas were washed in acid conditions prior to the binding assay. 4-1BB-AP still bound specifically to mature B-cell lymphomas. It is still to be determined whether a 4-lBB-ligand specifically expressed on B cells and macrophages exists, and whether 4-1BB-AP may bind to the ECM under particular binding conditions. It is possible that the ECM could facilitate the binding of 4- IBB to a specific cell-sur ace ligand.

B cells and helper T cells interact with each other through receptors on B cells binding to their specific counter-receptors on T cells. Researchers believe that this interaction results in a cascade of biochemical signaling relays between these two cell types (20). As this interaction proceeds, these cells become committed to enter the S phase of the cell cycle. Initial interactions between TCR and CD4 on T cells, and processed antigen-MHC II on B cells, do not result in B cells capable of -entering the cell cycle (21). However, studies from in vitro systems suggest that once T cells are stimulated, they express newly synthesized or modified cell-surface molecules capable of inducing B cells to enter the cell cycle (22, 23). This T cell function is not antigen-specific or MHC-restricted (24). In addition, soluble factors are not required for the activated Th induction of B-cell activation (25). Once B cells enter the cell cycle, IL-4 induces B cells to progress from Gj to S phase. The ability of activated T cells or T-cell membranes to promote the entry of B cells into the cell cycle can be blocked by either cycloheximide or cyclosporin A treatment (26, 27). These newly expressed membrane proteins appear to be "lymphokine-like" in their induction characteristics.

4-1BB has expression properties which meet the requirements of a B-cell costimulator. 4-1BB is inducible by anti-CD3 or TCR-mediated T-cell stimulation, and its expression is sensitive to cyclosporin A as well as cycloheximide treatment (28). Interestingly, paraformaldehyde-fixed SF21-4-1BB cells, synergized anti-μ and induced B-cell proliferation. The costimulation of splenic B cells by SF21-4-1BB occurred at optimal (10 μg/ml) and suboptimal (1.0-0.1 mg/ml) doses of anti-μ. The addition of SF21-4-1BB cells to resting B cells, did not result in significant B-cell proliferation. SF21-4-1BB cells did not synergize with TPA or ionomycin, or suboptimal concentrations of LPS in inducing B-cell proliferation.

Although the baculovirus system has been used to express large amounts of recombinant soluble proteins, this system may be utilized for the expression of recombinant cell-surface proteins. The baculovims infection provides a convenient means to express uniformity high levels of recombinant protein on a per cell basis. It is noteworthy, that the addition of SF21 cells alone did not result in significant levels of costimulation. This can be a potential problem when using cos- or L- cell lines which can exhibit strong costimulator activity on their own.

Another member of the NGFR superfamily, CD4O, is expressed on B cells .and interacts with gp39, a molecule expressed on activated T cells. The cDNAs encoding the murine (29) and human (30) gp39 proteins have been cloned; this cell surface molecule is a type II membrane protein with homology to tumor necrosis factor. Noelle et al. (31) found that a CD4O-immunoglobulin fusion protein, is capable of blocking T cell-induced B-cell proliferation and differentiation in a dose-dependent manner. Armitage et al. have isolated a cDNA for murine gp39 and showed that gp39 could induce B-cell proliferation in the absence of co-stimuli, and result in IgE production in the presence of IL-4-. Hollenbaugh et al. (32) have shown that COS cells transfected with human gp 39 can synergize with either TPA or anti-CD2O in inducing human B-cell proliferation and is able to stimulate B cells without a costimulator only at low levels. These data indicate that CD4O may be one of the B-cell-surface molecules that transmit signals during physical contact with T cells.

Cell-surface receptors communicate with their external milieu by interacting either with soluble factors or other cell surface molecules expressed on neighboring cells. The role of biochemical signals delivered by cell-cell contact versus those delivered by soluble factors interacting with cell surface receptors is not clear. The NGFR superfamily is unusual for the TNFR I and II as well as the NGFR bind to more than one ligand. The TNFRs I and II both bind to TNF-a and TNF-R (33). The NGFR binds to NGF, brain-derived neurotrophic fector, and neurotrophin-3 (34).

In addition, one ligand may function as both a cell surface and soluble ligand. Recent evidence on the CD40 ligand, gp39, suggests that this ligand can exist as a membrane bound as well as a soluble ligand (35). It may be possible that 4- IBB is secreted and interacts with B cells in a soluble form as well as a membrane bound form. A member of the NGFR receptor family, CD27, which is expressed on T cells, is secreted in addition to being expressed on the cell surface (36). It is also possible that more than one 1 ligand (soluble and cell surface) may bind to 4-lBB.

Isolation of the human homologue. H4-1BB

In order to isolate the human homologue (H4-1BB) of mouse 4- IBB two sets of polymerase chain reaction (PCR) primers were designed. To design the PCR primers, the amino acid sequence among the members of nerve growth factor receptor (NGFR) superfamily were compared because 4- IBB is a member of the superfamily (37). The amino acid sequences employed were mouse 4- IBB (38), human NGFR (39), human tumor necrosis factor receptors (33), human CD40 (40), and human CD27 (6). The areas of sequence conservation among the NGFR superfamily were chosen. (NOTE: WILL BE REVISED TO REFER TO SEQUENCE LISTINGS - STILL BEING PREPARED)

Forward primer I (H4-1BBFI) spans from amino acids 36 to 41 and forward primer π (HR-lBBFϋ) spans from amino acids 52 to 58 of the mouse 4-1BB. Reverse primer I (H4- lBBRI) spans from amino acids 116 to 121 and reverse primer π (H4-IBBRU) spans from amino acids 122 to 128 of mouse 4-1BB. The regions used as PCR primers in mouse 4- IBB are indicated if Fig. 1.

The degenerative oligonucleotide sequence of each primer are shown in the Sequence Listing as follows:

SEQ ID NO:3 H4-1BBFI

SEQ ID NO:4 H4-1BBFII

SEQ ID NO:5 H4-1BBRI

SEQ ID NO:6 H4-1BBRII

Peripheral blood lymphocytes from normal healthy individuals were isolated and activated with PMA (10 ng/ml) and ionomycin (1 mM). mRNA from the lymphocytes was isolated. Using reverse transcriptase the human lymphocyte mRNA was converted to single- stranded cDNA. The cDNA was then amplified with Taq polymerase with combination of the primers. The combination of primers was as follows: H4-1BBFI vs H4-1BBRI; H4-1BBFI vs H4-1BBRIJ; H4-1BBFII vs H4-1BBRI; and H4-1BBFII vs H4-1BBR1T.

The primer set of H4-1BBFII and H4-1BBRII produced a specific band of ^"240bp. The 240bp is an expected size of human 4- IBB if the human homologue protein is similar to mouse 4- IBB in size. The PCR product (240bp) was cloned in PGEM3 vector and sequenced. One open reading frame of the PCR product was ^"65% identical to mouse 4-1BB. Therefore, it was concluded that the 240 bp PCR product is the human homologue of mouse 4- IBB. The 240 bp PCR product was used to screen λgtl 1 cDNA library of activated human T lymphocytes. An ^"0.85 kb cDNA was isolated. The sequence of the cDNA is shown in SEQ ID NO: 7 and the predicted amino acid sequence is shown in SEQ ID NO: 8 An expression plasmid to produce H4-1BB-AP fusion protein was constructed.

The 5' portion of the H4-1BB cDNA including sequences encoding the signal sequence and the entire extracellular domain, was amplified by PCR. For correctly oriented cloning, a Hind m site on the 5' end of the forward primer and a Bgl JJ site on the 5' end of the reverse primer were created.

The Hind III - Bgl II H4-1BB fragment was inserted into the mammalian expression vector APtaq-1, upstream of the coding sequence for human placental alkaline phosphatase (AP). The oligonucleotides PCR primers used for the amplification of 5' portion of H4-1BB are shown in the Sequence Listing as SEQ ID NO:9 for the forward primer and SEQ ID NO: 10 for the reverse primer.

H4-1BB-AP will be used to identify cells and tissues that express ligand for human 4- 1BB (i.e. H4-1BBL). The studies with mouse 4- IBB indicated that the ligand for 4- IBB is on the cell suiace. B cells and macrophages were major cells that express 4-1BBL. It is expected that H4-1BBL also expresses on human B cells and macrophages.

A mammalian expression cDNA library will be generated from human cell lines that express H4-1BBL. The library will be screened by [^,25J Mabeled H4-1BB-AP. cDNA for H4- 1BBL will then be isolated and characterized. Soluble recombinant H4-1BBL will then be produced. Both H4-1BB-AP and H4-1BBL will be used to suppress or enhance immune responses as described below. Monoclonal antibody to H4-1BBL will be produced and monoclonal antibody to H4- IBB is discussed below.

According to studies with murine 4- IBB, 4- IBB acts as a costimulatory signal. It is expected that H4-1BB will act as a costimulatory signal for T cell activation. Mouse 4- IBB helped B cells with proliferation and differentiation. It is expected that H4-1BB will do the same. H4-1BB-AP, H4-1BBL and monoclonal antibody can be used to suppress or enhance human immune responses.

Figures la and lb illustrate the molecules involved in T-cell activation. During early T-cell activation (cognitive phase), resting T cells express the TCR/CD3 complex and other "accessory" molecules. Among these constitutively expressed molecules, CD4 (or CD8), LFA-1 and CD28 are probably the ones to receive costimulatory signals. Initial interaction with the TCR/CD3 complex in combination with these 'accessory' costimulatory signals leads to subsequent expression of additional receptor molecules such as CD28, CTLA4, and 4- IBB. These newly expressed molecules are probably going to receive additional important costimulatory signals at later stages of T-cell activation (clonal expansion).

Suppression of immune responses.

Figures 2a-c illustrate a normal T-cell .activation pathway. Figures 3a-c illustrate the blocking of immune responses with soluble chimera of 4-1BB. If 4-1BB plays a role in T-cell activation, blocking of the interaction to its ligand on antigen-presentihg cells should result in suppression of T-cell dependent immune responses. It is well documented that blocking of the interaction of CD28 to its counter-receptor B7 suppresses in varying degrees, both in vivo antibody production and cell-mediated immune responses. Blocking of both interactions should result in a more effective immunosuppression; since 4- IBB is induced during T-cell activation. Blocking of the interaction of 4- 1 BB to its ligand may be of importance at later stages of the .activation process where the CD28 B7 interaction may no longer be of relevance.

As illustrated with mouse receptor 4- IBB and mouse ligand 4-1BBL above, addition of H4-1BB-AP will coat the H4-1BBL expressing cells and block the normal interaction between H4-1BB and H4-1BBL. This will lead to immunosuppression. This type of immunosuppression is antigen-specific. Therefore it avoids the generalized immunosuppression produced by antiCD3 or cyclosporin A treatments. H4-1BB-AP treatment can be used to treat certain autoimmune diseases and to facilitate organ transplantation.

Immune enhancement. H4-1BB may function at the late stage of T cell activation and may be a critical molecule for completion of T cell activation. Most tumors display tumor-specific antigens. One reason, however, why immunogenic tumors can escape host immunity is that tumor- reactive T cells receive inadequate costimulation. The introduction of the costimulatory molecules, such as H4-1BB into the tumor, therefore, could enhance the antitumor immunity of cytotoxic T cells (CTL). H4-1BBL can be expressed in cell-specific fashion. For example, the H4-1BBL can be expressed in melanoma using melanocyte-specific promoter such as tyrosinase promoters. The H4-lBBL-expressing melanoma will stimulate cytotoxic T cells through H4- IBB and activate the melanoma-specific CTL. The activated melanoma-specific CTL can destroy melanoma.

Monoclonal Antibody to H4-BB

4-1 BB is expressed on activated but not resting murine T cells. Crosslinking of 1 AH2 mAb directed against murine 4-1 BB has been shown to enhance anti-CD3-induced T cell proliferation (41). Normal splenic cell antigen presentation and T cell activation can be blocked by inhibiting the binding of 4- IBB on T cells to its ligand on B cells and macrophages with 4-1 BB/AP, a fusion protein containing the extracellular domains of 4- 1BB and alkaline phosphatase. We developed and here describe the characterization of human 4- IBB specific mAbs: 4B4-1, 4B11 and 1G5. All mAb specifically bind human 4- 1BB expressing SF-21 cells.

Production of recombinant human 4-1 gp

PGEX-3 expression vector (Pharmacia) containing the full length cDNA sequence encoding 4-1 BB and the GST-binding domain of glutathionine S transferase (GST) was constructed and the fusion protein expressed in bacteria. Fusing H4-1BB with GST, allowed for efficient purification of rH4-lBB when isolated by GST-sepharose and a Sepharose 4B column chromatographies. The GST-binding domain was cleaved prior to immunization. The rh4-lBB fraction was purified by GST-sepharose column and Sepharose 4B column chromatographies and subsequently cleaved with factor Xa to release the h4-l BB portion prior to immunization.

BALB/c animals were immunized with rh4-l BB protein and their splenocytes were fused with the Sp2/o fusion partner. Eight week-old BALB/c mice were immunized with 50 μg of sh4-l BB emulsified in Titermax (Cytkx) or Complete Friends adjuvant. Three intraperitoneal (ip) injections were administered 2-weeks apart (42). Three days following the last injection, the mouse was sacrificed and their spleens were removed. Spleen cells were fused with Sp2/o myeloma cells. Spleen cells and Sp2/o cells were mixed at 5: 1 ratio and fused using 50% PEG. Cells were washed, resuspended in OptiMEM (Gibco), 10% FCS, 5 mM hypoxanthine, 1 % aminopterin, and 0.8 mM thymidine (HAT) and cultured in 96 well U-bottom plates (Corning). Resulting cell supematants were screened by ELISA for rh4-l BB reactivity. Clones were isolated and subcloned.

Antibodies and reagents used

FITC-labeled goat anti-mouse mAb with specificity for the heavy chain of IgG

.and IgM (GAMGM)(Jackson Laboratories, MA) was used at 1 : 150. Tricolorlabeled anti-CD4, CD8, CD45RO, PE-labeled anti-CD3 mabs and isotype controls were purchased from CALTAG (S. San Francisco, CA) and used according to manufacturer's recommendations. 4B4-1 (IgGl ) was purified over a protein-G sepharose,column and directly labeled by incubating the purified mAb in 0.4 mg/ml of FITC (Pierce) on ice for 3 hrs followed by filtration through Centricon-30 Microconcentrators (Amicon) to remove free-unbound FITC. FITC-labeled 4B4-1 was used at 3-4 μg per 5.0 x 10⁵ cells for staining.

The resulting hybridomas supematants were screened for binding to rh4-l BB protein by ELISA. Cell supematants scoring positive for binding to rh4-l BB by ELISA were subsequently assessed for staining of CEM (human T lymphoma) cells by FCM. CEM and 4B4-1 , IG5, and 4B11 hybridoma cells were maintained in the basic culturing media. CEM cells were stimulated overnight to 48 hrs with 10 ng/ml PMA and 1 μM ionomycin in culturing media. Only 4B4-1 mAb specifically bound CEM cells above the non-specific binding of the isotype control and only after the cells had been stimulated with 10 ng/ml PMA and 1 μM ionomycin for 24 hrs. All other mAb positive by ELISA for rh4-l BB did not appear to bind CEM cells.

To conduct the immuπoprecipitations, cell surface proteins were labeled with biotin as previously described (17). Briefly, 1 x 10⁷cells were washed in ice-cold PBS, resuspended in freshly prepared 0.5 mg/ml animohexanoyl-biotin-N-hydroxysuccinimide ester (AH-BNHS, Zymed Lab., Inc) in PBS for 30 min at 4 C with constant agitation. Cells were washed twice with 0.2 M glycine in PBS. Cells were lysed on ice for 15 min in 200 μl lysis buffer containing 1.0% NP-40, 20 mM Tris-HCl, 0.15 NaCl, μmM sodium orthovanadate, 5 μl/ml aprotinin and 1 μg/ml leupeptin, pH 7.5. Suspension was centrifuged at 14,000 rpm for 10 min, 4 C to remove debris. 20 μl of hybridoma culture supematants were added for 1 hr, 4 C followed by the addition of 30 μl of a 50% suspension of protein G-Sepharose 4B (Zymed Lab, Inc.) and incubated for 1 hr, 4 C. Suspension was centrifuge and resulting protein G-Sepharose pellet was washed. The immunoprecipitates were boiled 5 min in 20 μl SDS sample buffer and n on 10% SDS- PAGE. Proteins were transfered to Immobilon-P membrane (Millipore Corp., Bedford, MA) and blocked with 5% BSA in buffer containing 25 mM Tris-HCI, 0. 15 NaCl, 0.05% Tween 20 , pH 7.5 (TBST). The blot was probed with avidin-horse radish peroxidase conjugate and developed using chemilu inescence detection kit (ECL, Amersham Corp., Arlington Heights, IL).

To conduct the Flow Cytomertric studies (FCM) fresh cells were washed once and cultured cells were washed 3 times in staining media consisting of PBS, 1 % BSA and 0. 1 % glucose prior to staining. Approximately 2.5-5 x 10⁵ cells were resuspended in 50-100 μl of diluted mAb in staining media and incubated at 4 C for 30-60 min. Cells were washed once, resuspended in a 1:150 dilution of FITC-labeled GAMGM secondary mAb (Jackson Laboratories, MA) when needed, incubated 30 min and washed 3-4 times or simply washed 3-4 times if primary mAb were directly labeled. Cells were fixed with 1 % paraformaidehyde for overnight prior to analysis on the FACScan by Becton Dickinson using Consort 30 or FACScan software. Appropriate isotype controls were included for all primary antibodies used. Gates were set on live cells only, based on forward verses side scatter profiles. Five to 15000 events were collected for each sample.

To further address whether 4B4-1 mAb specifically recognized 4-1 BB and to assess the specificity of the other mAb picked-up by ELISA, SF-21 insect cells were infected with baclovims containing cDNA encoding h4-l BB or an irrelevant protein. SF-21 cells were grown in Grace Media (Gibco) supplemented with 10% FCS, 3.3 g/i yeastolate (Gibco), 3.3g/l lactoalbumin hydrolysate(Gibco), 2 mM glutamine (Sigma) and antibiotics at 27 C.

Infected SF-21 insect cells were then analyzed by FCM for binding of the various mAbs. Only cells infected with the baclovims containing and expressing products the h4- 1 BB construct on the cell surface, but not the construct encoding and expressing an irrelevant protein were found to bind 4B4-1, 4B11 and 1G5 mAbs. This data demonstrates that 4B4-1 mAb is specific for h4-l BB. By immunoprecipitation, only 4B4-1 mAb precipitated a ^"35 kDa protein when mn on SDS-PAGE under reducing conditions -and ^"70 kDa protein under non-reducing conditions from rh4-l BB expressing SF-21 cells, but not those cells expressing irrelevant protein. Similar results were seen using CEM cells with PMA and ionomycin stimulation. IG5 is of an IgM isotype, thus explaining its apparent inability to immunoprecipitate rh4-l BB. 4B4-1 and 4B11 mAb are IgGls.

To evaluate 4-1BB expression in human peripheral blood, mononuclear cells were isolated by passage over Ωcoll, washed and stained for with 4B4-1 mAb. Human peripheral blood mononuclear cells (PBMC) were isolated by Histopaque 1077 (Sigma) for 30 min at 400 g and the resulting interface cells were washed twice prior to use. Human T cells were isolated according to manufacturer's instmctions. Briefly, PBMCs were treated with Lympho-kwik (One Lambda, Ince. , Canoga Park, CA) T cell isolation solution for 20 min at 37 C followed by a 5 min centrifugation at 1500 g. The resulting cell pellet was washed twice prior to use. PBMC or freshly isolated T cells were cultured in basic culturing media consisting of aRPMI 1 640 (Gibco Laboratories), 10% FCS (Hyclone, Utah), 1 x MEM nonessential amino acids (Sigma), 1 mM sodium pymvate (Gibco), 2 mM glutamine(Sigma), and 50 U/ml penicillin and 50 μg/ml streptomycin (Gibco) supplemented with 5 Rg/mi PHA (Calbiochem) and 50 @ 2-ME (Sigma).

Of the lymphocyte sized fraction, <2% were positive for 4B4-1 mAb binding (data not shown) when compared to binding observed with an irrevelant-isotype control. By multi-color analyws, <2% of CD3+ (T) cells were found to expressed 4-1 BB. Isotype-matched irrelevant mAb were used for each fluorescence parameter to correct for non-specific binding of the abs. By 48 hrs of PHA-stimulation, 18.5-21.5% of purified T cells expressed 4-1 BB on its cell surface. Without PHA-stimulation <2% of T cells express 4-1 BB. On PHA-stimulated T cells, 4-1BB was expressed on both CD4+ and CD8+ cells with ^"50% of 4-1 BB+ cells expressing CD4 and 50% expressing CD8.

Binding of 4B4-1 FITC-labeled mAb was almost completely blocked when PHA- stimulated T cells were pre-incubated with μ/ml purified rh4-l BB protein, but not irrelevant control protein at similar concentration made in a similar manner.

PHA-stimulated T cells were incubated 30 min. on ice with staining media or varying concentrations of either rh4-l BB protein or irrelevant recombinant protein that was produced in a similar manner as rh4-l BB. Without washing 2.5 μg of 4B4/FITC- labeled mAb or isotype matched FITC-labeled mAb, a suboptimal concentration, was added and cells were further incubated on ice for 30 min. Cells were washed 3 times with staining media, fixed with 1 % paraformaldehyde, and analyzed by FCM as described above.

The competitive blocking of 4B4-1 /FITC mAb by rh4-l BB was dependent upon the concentration of rh4-l BB. Together this data demonstrates that 4B4-1 mAb specifically recognizes the human 4-1 BB on peripheral blood T cells.

Activated T cells co-express 4-1BB+ andCD45RA andCD45RO.

We have previously shown that murine 4-IBB is associate with p56^lck by a series of immunoprecipitation studies and peptide mapping study. During the course of these studies we observed that not only did anti-4-1 BB mAb coimmunoprecipitate a 56 kDa protein but it also immunoprecipitated a protein of 200 kDa. Conversely, anti-CD45 mAb co-immunoprecipitated a ^"30 kDa protein from ConA stimulated murine thymocytes. This data suggests that 4-1 BB may form a multi-peptide complex with CD45 -and p56^,c on activated murine T cells. To assess the association of 4-1 BB and CD45 in humans, PBMCs stimulated with PHA for 48 hrs were analy.zed for expression of CD45RA and CD45RO isoforms by multi-color FCM. Sixteen to 19% of cells expressed 4-1 BB, and nearly all (except 1 %) expressed CD45RA -and nearly all express CD45RO after correcting for non-specific binding of the antibodies.

Discussion of Examples of Uses for mAb 4B4-1.

The 4B4-1 mAb specifically stained the T cell line, CEM and peripheral blood T cells following activation, but did not bind resting cells. In this regard, 4- IBB expression in humans is similar to that observed in the mouse. 4B4-1 mAb immunoprecipitates a homodimeric protein of "35 IcDa under reducing conditions -and "70 kDa under non- reducing conditions from 4-1 BB expressing SF-21 cells and activated CEM cells. The molecular weights are similar to the expected values based on amino acid sequence and anticipated glycosylation and similar to molecular weight observed in the mouse. Furthermore, 10 μg of rh4-l BB protein, but not irrelevant protein, completely block the binding of FITC-labeled 4B4-1 mAb to PHA stimulated T cells. Collectively, these data conclusively show that 4B4-1 mAb is specific for human 4-1 BB.

Although 4B 11 and I G5 mAbs specifically recognize 4-1 BB expressed on SF-21 cells, they do not recognize 4-1 BB expressed on activated T cells. This is most likely due to the mAb having specificity for a cryptic or unique binding site(s) that is not exposed or present on T-cells but is accessible or present on SF-21 cells due to slight differences in glycosylation and processing between human T cells and insect cells (SF-21).

In mice, neither 4- IBB mRNA nor surface expression is detectable on resting splenocytes or unstimulated cloned T cells. But upon activation of T cells by anti-CD3 or antiTCRαβ, 4-1 BB mRNA is detected within 3 hrs of stimulation and is first detectable on the cell surface 2-3 days following stimulation. Maximum surface expression is reached about 6 days following stimulation. As in the mouse, 4-1 BB is not detected on the surface of freshly isolated peripheral blood T cells in man, but is readily detected following PHA-stimulation. Unlike in the mouse, 4-1BB is expressed much more rapidly in humans, reaching a peak expression level within 12-48 hrs. 4-1BB expression begins to decrease within 72 hrs poststimulation, as do the number of cells expressing 4-1 BB on their cell surface. In both mouse and humans, 4-1 BB is expressed on CD4+ and CDS+ T cell subsets) (19).

4-1 BB is associated with p56^kk. A 56 kDa protein is detected when "PO* was transferred from gamma-labeled ATP onto the p56 protein in ConA activated thymocytes that were subjected to immunoprecipitated with anti-4-1 BB mAb, 1AH2. By peptide mapping, this 56 kDa phosphoprotein was identified as p56^lck. p56^lc and 4-1 BB was also be found to co-immunoprecipitate from insect cells (SF-21) and HeLa cells transfected with 4-1BB and p56^k Furthermore, cross-linking of 4-1BB activated p56^lck. Cysteine residues critical for p56^,c -CD4/CD8 complex formation were also critical for p56^kk - 4- 1BB interaction. In prelimary results, it was noted that anti-4-lBB also immunoprecipitated a protein of ^"200 kDa from biotin-surface labeled ConA activated thymocytes. When anti- CD45 mAb was used for immunoprecipitation, a ^"30 kDa protein, of similar size to murine 4-1BB, was detected. Other have previously shown that CD45 mediates the dephosphorylation of certain proteins such as p56^lck (44). Perhaps 4-1 BB plays a role in bringing CD45 and p56^,ck together and facilitates the dephosphorylation of p56^lc by the CD45 phosphatase.

To further assess the association of 4-1 BB and CD45, PHA-stimulated PBMCs were analyzed by multicolor FCM. Approximately 16-19% of PBMCs cultured in PHA for 48 hrs express 4-1 BB. If all 4-1BB+ cells express CD45RA (Figure 7A) and express CD45RO, the ^"17.5% of 4-IBB+ cells must co-express both CD45RA and CD45RO on their cell surface. Of the PHA-stimulated CD45RA^hiRO^hi cells, approximately 50% express 4-1 BB. This data futher supports the hypothesis that CD45 and 4- IBB share an association. More importantly, it suggests that 4-1 BB may play a role in T cell transition from a naive phenotype (CD45RA^hiRO^ro) to a memory phenotype (CD45RA^,0RO^U).

Picker et al. previously demonstrated through multi-color FCM, that naive T cells undergo a "stepwise, unidirectional progression" from a naive (CD45RA °RO^h') to a memory (CD45RA^l0RO^hi) phenotype through a distinct CD45RA^hiRO^hi intermediate cell type (21 ). Peripheral blood few cells that express this intermediate phenotype are detectable. However, in secondary lymphoid tissue, such as tonsil, 2-10% of T cells were found to be CD45RA^h,RO^hl. Much is know about the naive and memory T cells, but little is known about the CD45RA^hlRO^hl in transitional cells. Nor is it known what events occur during this transition phase that result in memory T cell development. Therefore, it will be necessary to assess the role of 4-1 BB in the transition from a naive to that of a memory T cell and ϋie apparent association of 4-1 BB and CD45. In this regard, 4B4-1 mAb will be invaluable.

The mAb 4B4-1 can be used to enhance T-cell cross-linking and therefore induce T-cell activation against certain types of cancer cells (e.g. melanoma). By using the mAb in experiments with various cancer cells in the presence of T-cells dosages and proper formulations of initiating T-cell activation against the cancer cells can be determined. The formulations are tested in animal models with the same type of cancer and the formulations and dosages are refined for testing in humans.

Sinovial T-Iymphocytes in patients with rheumatoid arthritis express H4-1BB, but 4-1BB is not expressed in sinovial T-lymphocytes of patients without this disease. This disease involves an undesired immune response against the patient's own tissue.

Therefore, blocking the undesired immune response would provide relief for the arthritis sufferer. By injecting the patient with the mAb 4B1-4 or the fusion protein, the binding between H4-1BB and its ligand would be blocked. If the binding of the mAb and H4-1BB did not enhance activation of the immune system, then the mAb 4B1-4 interference with binding would have the desired effect, otherwise the fusion protein would be used for blocking binding. The fusion protein (monomeric) does not stimulate H4-1BB or its ligand but is a good ligand binding blocker because it binds to the H4-1BB ligand thereby preventing H4-1BB from binding and stimulating the ligand.

A similar method of blocking ligand binding would be useful for treating patients with systemic lupus erythematosus. For patients with Type I diabetis - T-cells attack their own insulin producing cells, pancreatic Beta cells. By injecting the mAb or fusion protein this destruction can be blocked.

Peripheral blood T cells in patients with AIDS or certain types of viral flu are expressing H4-1BB, whereas the same cells in normal patients are not expressing H4-1BB. Therefore, 4- IBB is important in this immune response. The enhancement or blocking of H4-1BB ligand binding or cross-linking will be important in regulating the T-cells in patients with these diseases.

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38. Kwon, B.S., and Weissman, S.M. 1989. cDNA sequences of two inducible T-cell genes. Proc. Naϋ. Acad. Sci. USA. 86: 1963-1967. 39. Johnson, D., Lanahan, A., Buck C.R., Sehgal, A., Morgan, C, Mercer, E., Bothwell, M., and Chao, M. 1986. Expression and stmcture of the human NGF receptor. Cell 47:545- 554.

40. Stamenkovic, I., Clark, E., and Seed, B. 1989. A B-lymphocyte -activation molecule related to ϋie nerve growϋi factor receptor and induced by cytokines in carcinomas. EMBO. J. 8:1403-1408. 41. Pollok KE, Y-J Kim, Z Zhou, J Hurtado, KK Kim, and BS Kwon. 1993. The inducible T cell antigen 4-1 BB: Analysis of expression and function. / Immunol 150:771.

42. Antibody Lab Manual. 1 988. Editors are E Harlow and D Lane. Cold Spring Harbor Lab.

43. Pelchen-Matthews, A, JE Armes, G Griffiths, and M Marsh. 1991. Differential endocytosis of CD4 in lymphocytic and nonlymphocytic cells. J Exp Med 173:575.

44.Biffeπ, M, D McMichael-Phillips, T Larson, A Venkitaraman, and D Alexander. 1 994. The CD45 tyrosine phosphatase regulates specific pools of -antigen receptor-, associated P59fyn andCD4-associated p561ck tyrosine kinases in human T-cells. EMBO J 13:1920.

APPENDIX B

COMMON ABBREVIATIONS

CTL cytolytic T lymphocyte

HTL helper T lymphocyte

LGL large granular lymphocytes

NK natural killer cells

ConA concanavalin A

DTT dithiothreitol mAb monoclonal antibody.

4- IBB protein expressed on activated T cells rh) 4-1BB recombinant human 4- IBB

4-1BB/L ligand to 4- IBB found on activatedmacropage and mature B cells

4-1BB/AP fusion protein between 4- IBB and alkaline phosphatase.

SDS sodium dodecysulfate

SSC 150 mM sodium chloride/15 mM sodium citrate, pH 7.0

TPA 12-0-tetradecanoylphorbol- 13-acetate

Th helper T lymphocytes

IL-2 interleukin 2

IL-3 interleukin 3 rIL-2 recombinant 11-2

CSF-GM granulocyte/macrophage colony-stimulating factors cRNA complementary RNA ss single-stranded ds double- stranded

TCR T-cell antigen receptor

PTA phorbol 12-tetradecanoate 13-acetate r recombinant mu murine hu human

BFU-E burst forming unit-erythroid, an erythroid progenitor cell

CFU-GEMM colony forming unit-eranulocvte ervthroid macropha e megakaryocyte, a multipotential progenitor cell

CFU-GM colony forming unit-granulocyte macrophage, a granulocyte- macrophage progenitor cell

CFU-S colony forming unit-Spleen, a multipotential stem cell

H-ferritin the heavy chain subunit form of ferritin

MGF mast cell growth factor, a c-kdt ligand

CSF colony stimulating factors

G granulocyte

M macrophage

Epo erythropoietin

IL interleukin

LD low density

NALDT non-adherent low density T-lymphocyte depleted

PMSF phenylmethylsulfonyl fluoride

PBS phosphate buffered saline

AcNPV Autographa californica nuclear polyhedrosis vims

SDS sodium dodecyl sulfate

LPS lipopolysaccharide

The foregoing description has been directed to particular embodiments of the invention in accordance with ϋie requirements of ϋie Patent Statutes for the purposes of illustration and explanation. It will be apparent, however, to those skilled in this art that many modifications and changes will be possible without departure from the scope and spirit of the invention. It is intended that the following claims be interpreted to embrace all such modifications.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Kwon, Byoung Se

Kang, Chang-Yuil

(ii) TITLE OF INVENTION: Monoclonal antibody against human receptor 4-1BB

(iii) NUMBER OF SEQUENCES: 10

(iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Barnard, Brown & Michaels

(B) STREET: 306 East State Street, Suite 220

(C) CITY: Ithaca

(D) STATE: NY

(E) COUNTRY: USA (F) ZIP: 14850

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/122,796 (B) FILING DATE: 16-SEP-1993

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/012,269

(B) FILING DATE: 01-FEB-1993

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/922,996

(B) FILING DATE: 30-JUL-1992 (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/267,577

(B) FILING DATE: 07-NOV-1988

(viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Michaels, Christopher A

(B) REGISTRATION NUMBER: 34,390

(C) REFERENCE/DOCKET NUMBER: KW05

(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 607-273-1711

(B) TELEFAX: 607-273-2609

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2347 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: murine 4-1BB

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 146.. 16 (ix) FEATUREi

(A) NAME/KEY: mat_pβptidβ

(B) LOCATION: 146.. 13

(x) PUBLICATION INFORMATION: (A) AUTHORS: Kwon, Byoung Se et al.,

(B) TITLE: cDNA sequences of two inducible T-cell genes

(C) JOURNAL: Proc. Natl. Acad. Sci. U.S.A.

(D) VOLUME: 86 (E) ISSUE: March

(F) PAGES: 1963-1967

(G) DATE: 1989

(K) RELEVANT RESIDUES IN SEQ ID NO:l: FROM 1 TO 2347

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

ATGTCCATGA ACTGCTGAGT GGATAAACAG CACGGGATAT CTCTGTCTAA AGGAATATTA 60 CTACACCAGG AAAAGGACAC ATTCGACAAC AGGAAAGGAG CCTGTCACAG AAAACCACAG 120

TGTCCTGTGC ATGTGACATT TCGCC ATG GGA AAC AAC TGT TAC AAC GTG GTG 172

Met Gly Aβn Asn Cys Tyr Aβn Val Val 1 5

GTC ATT GTG CTG CTG CTλ GTG GGC TGT GAG AAG GTG GGA GCC GTG CAG 220 Val lie Val Leu Leu Leu Val Gly Cys Glu Lys Val Gly Ala Val Gin 10 15 20 25 AAC TCC TGT GAT AAC TGT CAG CCT GGT ACT TTC TGC AGA AAA TAC AAT 268 Asn Ser Cys Asp Asn Cys Gin Pro Gly Thr Phe Cys Arg Lys Tyr Aβn 30 35 40

CCA GTC TGC AAG AGC TGC CCT CCA ACT ACC TTC TCC AGC ATA GGT GGA 316 Pro Val Cyβ Lys Ser Cyβ Pro Pro Ser Thr Phe Ser Ser lie Gly Gly 45 50 55

CAG CCG AAC TGT AAC ATC TGC AGA GTG TGT GCA GGC TAT TTC AGG TTC 364 Gin Pro Asn Cys Asn He Cys Arg Val Cys Ala Gly Tyr Phe Arg Phe 60 65 70

AAG AAG TTT TGC TCC TCT ACC CAC AAC GCG GAG TGT GAG TGC ATT GAA 412 Lys Lys Phe Cys Ser Ser Thr His Aβn Ala Glu Cyβ Glu Cyβ He Glu 75 80 85

GGA TTC CAT TGC TTG GGG CCA CAG TGC ACC AGA TGT GAA AAG GAC TGC 460 Gly Phe His Cyβ Leu Gly Pro Gin Cyβ Thr Arg Cyβ Glu Lye Aβp Cyβ 90 95 100 105 AGG CCT GGC CAG GAG CTA ACG AAG CAG GGT TGC AAA ACC TGT AGC TTG 508 Arg Pro Gly Gin Glu Leu Thr Lys Gin Gly Cyβ Lye Thr Cys Ser Leu 110 115 120

GGA ACA TTT AAT GAC CAG AAC GGT ACT GGC GTC TGT CGA CCC TGG ACG 556 Gly Thr Phe Aβn λβp Gin Aβn Gly Thr Gly Val Cyβ Arg Pro Trp Thr 125 130 135

AAC TGC TCT CTA GAC GGA AGG TCT GTG CTT AAG ACC GGG ACC ACG GAG 604 Aβn Cys Ser Leu Aβp Gly Arg Ser Val Leu Lye Thr Gly Thr Thr Glu 140 145 150

AAG GAC GTG GTG TGT GGA CCC CCT GTG GTG AGC TTC TCT CCC AGT ACC 652 Lys Aβp Val Val Cyβ Gly Pro Pro Val Val Ser Phe Ser Pro Ser Thr 155 160 165

ACC ATT TCT GTG ACT CCA GAG GGA GGA CCA GGA GGG CAC TCC TTG CAG 700 Thr He Ser Val Thr Pro Glu Gly Gly Pro Gly Gly His Ser Leu Gin 170 175 180 185

GTC CTT ACC TTG TTC CTG GCG CTG ACA TCG GCT TTG CTG CTG GCC CTG 748 Val Leu Thr Leu Phe Leu Ala Leu Thr Ser Ala Leu Leu Leu Ala Leu 190 195 200

ATC TTC ATT ACT CTC CTG TTC TCT GTG CTC AAA TGG ATC AGG AAA AAA 796 He Phe He Thr Leu Leu Phe Ser Val Leu Lys Trp He Arg Lys Lys 205 210 215

TTC CCC CAC ATA TTC AAG CAA CCA TTT AAG AAG ACC ACT GGA GCA GCT 844 Phe Pro Hie He Phe Lye Gin Pro Phe Lye Lye Thr Thr Gly Ala Ala 220 225 230

CAA GAG GAA GAT GCT TGT AGC TGC CGA TGT CCA CAG GAA GAA GAA GGA 892 Gin Glu Glu λβp Ala Cyβ Ser Cyβ Arg Cyβ Pro Gin Glu Glu Glu Gly 235 240 245

GGA GGA GGA GGC TAT GAG CTG TGA TGTACTATCC TAGGAGATGT GTGGGCCGAA 946 Gly Gly Gly Gly Tyr Glu Leu * 250 255

ACCGAGAAGC ACTAGGACCC CACCATCCTG TGGAACAGCA CAAGCAACCC CACCACCCTG 1006

TTCTTACACA TCATCCTAGA TGATGTGTGG GCGCGCACCT CATCCAAGTC TCTTCTAACG 1066

CTAACATATT TGTCTTTACC TTTTTTAAAT CTTTTTTTAA ATTTAAATTT TATGTGTGTG 1126

ACTGTTTTGC CTGCCTGTAT GCACACGTGT GTGTGTGTGT GTGTGTGACA CTCCTGATGC 1186

CTGAGGAGGT CAGAAGACAA AGGGTTGGTT CCATAAGAAC TGGAGTTATG GATGGCTGTG 1246

AGCCGGGATA GGTCGGGACG GAGACCTGTC TTCTTATTTT AACGTGACTG TATAATAAAA 1306

AAAAAATGAT ATTTCGGGAA TTGTAGAGAT TGTCCTGACA CCCTTCTAGT TAATGATCTA 1366

AGAGGAATTG TTGATACGTA GTATACTGTA TATGTGTATG TATATGTATA TGTATATATA 1426

AGACTCTTTT ACTGTCAAAG TCAACCTAGA GTGTCTGGTT ACCAGGTCAA TTTTATTGGA 1486

CATTTTACGT CACACACACA CACACACACA CACACACACG TTTATACTAC GTACTGTTAT 1546

CGGTATTCTA CGTCATATAA TGGGATAGGG TAAAAGGAAA CCAAAGAGTG AGTGATATTA 1606

TTGTGGAGGT GACAGACTAC CCCTTCTGGG TACGTAGGGA CAGACCTCCT TCGGACTGTC 1666

TAAAACTCCC CTTAGAAGTC TCGTCAAGTT CCCGGACGAA GAGGACAGAG GAGACACAGT 1726

CCGAAAAGTT ATTTTTCCGG CAAATCCTTT CCCTGTTTCG TGACACTCCA CCCCTTGTGG 1786

ACACTTGAGT GTCATCCTTG CGCCGGAAGG TCAGGTGGTA CCCGTCTGTA GGGGCGGGGA 1846

OACACAGCCC CCCCGCλCCT ACGAGAATCG ACTCACAGGG CGCCCCGGGC TTCGCAAATG 1906

AAACTTTTTT AATCTCACAA GTTTCGTCCG GGCTCGGCGG ACCTATGGCG TCGATCCTTA 1966

TTACCTTATC CTGGCGCCAA GATAAAACAA CCAAAAGCCT TGACTCCGGT ACTAATTCTC 2026

CCTGCCGGCC CCCGTAAGCA TAACGCGGCG ATCTCCACTT TAAGAACCTG GCCGCGTTCT 2086 GCCTOGTCTC GCTTTCGTAλ ACGGTTCTTA CAAAAGTAAT TAGTTCTTGC TTTCAGCCTC 2146

CAAGCTTCTG CTAGTCTATG GCAGCATCAA GGCTGGTATT TGCTACGGCT GACCGCTACG 2206

CCGCCGCAAT AAGGGTACTG GGCGGCCCGT CβAAGGCCCT TTGGTTTCAG AAACCCAAGG 2266

CCCCCCTCAT ACCAACGTTT CGACTTTGAT TCTTGCCGGT ACGTGGTGGT GGGTGCCTTA 2326 GCTCTTTCTC GATAGTTAGA C 2347

(2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 257 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Gly Aβn Aβn Cyβ Tyr Asn Val Val Val He Val Leu Leu Leu Val 1 5 10 15

Gly Cys Glu Lye Val Gly Ala Val Gin Aβn Ser Cyβ λβp Aβn Cyβ Gin 20 25 30 Pro Gly Thr Phe Cyβ Arg Lye Tyr Aβn Pro Val Cyβ Lye Ser Cyβ Pro 35 40 45

Pro Ser Thr Phe Ser Ser He Gly Gly Gin Pro Aβn Cyβ λβn He Cys 50 55 60 λrg Val Cyβ Ala Gly Tyr Phe Arg Phe Lye Lye Phe Cye Ser Ser Thr 65 70 75 80

Hie λen Ala Glu Cyβ Glu Cys He Glu Gly Phe Hie Cyβ Leu Gly Pro 85 90 95

Gin Cyβ Thr Arg Cyβ Glu Lye Aβp Cyβ Arg Pro Gly Gin Glu Leu Thr 100 105 110 Lys Gin Gly Cyβ Lye Thr Cyβ Ser Leu Gly Thr Phe Aβn Aβp Gin Aβn 115 120 125

Gly Thr Gly Val Cyβ Arg Pro Trp Thr λβn Cyβ Ser Leu λβp Gly λrg 130 135 140

Ser Val Leu Lye Thr Gly Thr Thr Glu Lye λβp Val Val Cyβ Gly Pro 145 150 155 160

Pro Val Val Ser Phe Ser Pro Ser Thr Thr He Ser Val Thr Pro Glu 165 170 175

Gly Gly Pro Gly Gly Hie Ser Leu Gin Val Leu Thr Leu Phe Leu λla 180 185 190 Leu Thr Ser λla Leu Leu Leu λla Leu He Phe He Thr Leu Leu Phe 195 200 205

Ser Val Leu Lye Trp He λrg Lye Lye Phe Pro Hie He Phe Lye Gin 210 215 220

Pro Phe Lye Lye Thr Thr Gly Ala Ala Gin Glu Glu Aep Ala Cyβ Ser 225 230 235 240 Cyβ λrg Cyβ Pro Gin Glu Glu Glu Gly Gly Gly Gly Gly Tyr Glu Leu 245 250 255

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARλCTERISTICS: (λ) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /dββc - "PCR Primer"

(iii) HYPOTHETICλL: NO (iv) λNTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

TTYTGYMGλλ λRTλYλλYCC 20

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 20 baβe pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(λ) DESCRIPTION: /dββc - "PCR Primer" (iϋ) HYPOTHETICλL: NO

(iv) λNTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: TTYTCSTSCλ HTGGTGGλCλ 20

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARλCTERISTICS: (λ) LENGTH: 20 baβe pairβ (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (λ) DESCRIPTION: /deβc « "PCR Primer'

(iii) HYPOTHETICλL: NO

(iv) λNTI-SENSE: NO ( i) SEQUENCE DESCRIPTION: SEQ ID NO:5:

CCCλRGSWRC AGGTYTTRCλ 20 (2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARλCTERISTICS: (λ) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc - "PCR Primer"

(iii) HYPOTHETICλL: NO

(iv) λNTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: TTYTGRTCRT TRλλTGTTCC 20

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS: (λ) LENGTH: 838 baβe paire

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNλ to mRNλ

(iii) HYPOTHETICλL: NO

(iv) λNTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(λ) ORGANISM: Homo sapiens

(C) INDIVIDUAL ISOLATE: H4-1BB #1

(D) DEVELOPMENTλL STλGE: Differentiated T-cell (G) CELL TYPE: Lymphocyte

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..805 (D) OTHER INFORMATION: /cσdon_βtart» 41

/product" "H4-1BB" /number* 1

(ix) FEATURE: (A) NAME/KEY: mat peptide

(B) LOCATION: 41..802

(D) OTHER INFORMATION:

41 /product" "H4-1BB" /number* 1

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7; λλTCλGCTTT GCTACTλTCλ TλCCTGTGCC λGλTTTCλTC ATG GGA AλC λGC TGT 55

TλC λλC λTλ GTλ GCC λCT CTG TTG CTG GTC CTC AAC TTT GAG AGG λCλ 103 Tyr λβn He Val λla Thr Leu Leu Leu Val Leu λβn Phe Glu λrg Thr 10 15 20 λGλ TCλ TTG CλG GλT CCT TGT λGT AλC TGC CCλ GCT GGT λCλ TTC TGT 151 λrg Ser Leu Gin λβp Pro Cyβ Ser λβn Cye Pro λla Gly Thr Phe Cyβ 25 30 35

GλT λλT AλC λGG λλT CλG λTT TGC λGT CCC TGT CCT CCλ λλT λGT TTC 199 λβp λsn λen λrg λβn Gin He Cyβ Ser Pro Cyβ Pro Pro λβn Ser Phe 40 45 50

TCC λGC GCλ GGT GGλ CAA AGG ACC TGT GAC ATA TGC AGG CAG TGT AAA 247 Ser Ser λla Gly Gly Gin λrg Thr Cyβ λsp lie Cyβ λrg Gin Cyβ Lys 55 60 65

GGT GTT TTC AGG ACC AGG AAG GAG TGT TCC TCC ACC AGC AAT GCA GAG 295

Gly Val Phe λrg Thr λrg Lys Glu Cyβ Ser Ser Thr Ser Asn λla Glu

70 75 80 85 TGT GλC TGC λCT CCλ GGG TTT CλC TGC CTG GGG GCλ GGλ TGC λGC λTG 343 Cyβ λβp Cyβ Thr Pro Gly Phe Hie Cyβ Leu Gly λla Gly Cyβ Ser Met 90 95 100

TGT Gλλ CλG GλT TGT λλλ Cλλ GGT CAA GAA CTG ACλ AAλ λλλ GGT TGT 391 Cyβ Glu Gin λβp Cyβ Lye Gin Gly Gin Glu Leu Thr Lye Lye Gly Cyβ 105 110 115 λλλ GλC TGT TGC TTT GGG λCλ TTT λλC GλT CλG λλλ CGT GGC λTC TGT 439 Lye λep Cyβ Cyβ Phe Gly Thr Phe λβn λβp Gin Lye λrg Gly He Cyβ 120 125 130

CGλ CCC TGG λCλ λλC TGT TCT TTG GλT GGλ λλG TCT GTG CTT GTG AλT 487 λrg Pro Trp Thr λβn Cyβ Ser Leu λβp Gly Lye Ser Val Leu Val λβn 135 140 145

GGG λCG λλG GλG λGG GλC GTG GTC TGT GGλ CCλ TCT CCλ GCT GAC CTC 535 Gly Thr Lye Glu Arg Aβp Val Val Cyβ Gly Pro Ser Pro λla λβp Leu 150 155 160 165 TCT CCG GGλ GCλ TCC TCT GTG λCC CCG CCT GCC CCT GCG λGA GAG CCA 583 Ser Pro Gly Ala Ser Ser Val Thr Pro Pro λla Pro λla λrg Glu Pro 170 175 180

GGλ CλC TCT CCG CλG λTC λTC TCC TTC TTT CTT GCG CTG λCG TCG λCT 631 Gly Hie Ser Pro Gin He He Ser Phe Phe Leu λla Leu Thr Ser Thr 185 190 195

GCG TTG CTC TTC CTG CTG TTC TTC CTC λCG CTC CGT TTC TCT GTT GTT 679 λla Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu λrg Phe Ser Val Val 200 205 210 λλλ CGG GGC λGλ λλG λλλ CTC CTG TλT λTλ TTC λλλ Cλλ CCλ TTT λTG 727 Lye λrg Gly λrg Lye Lye Leu Leu Tyr He Phe Lye Gin Pro Phe Met 215 220 225 λGλ CCA GTA Cλλ λCT λCT Cλλ GAG GAA GAT GGC TGT AGC TGC CGA TTT 775 Arg Pro Val Gin Thr Thr Gin Glu Glu Aβp Gly Cyβ Ser Cyβ Arg Phe 230 235 240 245 CCλ Gλλ Gλλ Gλλ Gλλ GGλ GGλ TGT Gλλ CTG TGλλλTGGλλ GTCλλTλGGG 825

Pro Glu Glu Glu Glu Gly Gly Cyβ Glu Leu 250 255

CTGTTCGGAC TTT 838

(2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHλRλCTERISTICS:

(λ) LENGTH: 255 .amino acidβ (B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Met Gly λβn Ser Cyβ Tyr λβn He Val λla Thr Leu Leu Leu Val Leu 1 5 10 15

Aβn Phe Glu λrg Thr λrg Ser Leu Gin λep Pro Cyβ Ser λen Cye Pro 20 25 30 λla Gly Thr Phe Cyβ λβp λβn λβn λrg λβn Gin He Cyβ Ser Pro Cys 35 40 45

Pro Pro λβn Ser Phe Ser Ser λla Gly Gly Gin λrg Thr Cyβ λβp He 50 55 60

Cyβ λrg Gin Cyβ Lye Gly Val Phe λrg Thr λrg Lye Glu Cyβ Ser Ser 65 70 75 80 Thr Ser λβn λla Glu Cyβ λβp Cye Thr Pro Gly Phe Hie Cye Leu Gly

85 90 95 λla Gly Cyβ Ser Met Cyβ Glu Gin λβp Cye Lye Gin Gly Gin Glu Leu 100 105 110

Thr Lye Lye Gly Cyβ Lye λβp Cyβ Cyβ Phe Gly Thr Phe Aβn λβp Gin 115 120 125

Lye λrg Gly He Cyβ λrg Pro Trp Thr Aβn Cye Ser Leu λβp Gly Lye 130 135 140

Ser Val Leu Val λen Gly Thr Lye Glu λrg λβp Val Val Cyβ Gly Pro 145 150 155 160 Ser Pro λla λβp Leu Ser Pro Gly λla Ser Ser Val Thr Pro Pro λla

165 170 175

Pro λla λrg Glu Pro Gly Hie Ser Pro Gin He He Ser Phe Phe Leu 180 185 190 λla Leu Thr Ser Thr λla Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu 195 200 205 λrg Phe Ser Val Val Lye λrg Gly λrg Lye Lye Leu Leu Tyr He Phe 210 215 220

Lye Gin Pro Phe Met λrg Pro Val Gin Thr Thr Gin Glu Glu λβp Gly 225 230 235 240 Cyβ Ser Cyβ λrg Phe Pro Glu Glu Glu Glu Gly Gly Cye Glu Leu

245 250 255

(2) INFORMλTION FOR SEQ ID NO: : (i) SEQUENCE CHARACTERISTICS: (λ) LENGTH: 25 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(λ) DESCRIPTION: /deβc - "PCR Primer" (iii) HYPOTHETICλL: NO (iv) λNTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: : AλTAAGCTTT GCTλGTλTCλ TACCT 25

(2) INFORMλTION FOR SEQ ID NO:10:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 30 baβe pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /dββc - "PCR Primer"

(iii) HYPOTHETICAL: NO (iv) λNTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

TTλλGλTCTC TGCGGλGλGT GTCCTGGCTC 30

BUDAPEST TR EΛ IΎ ON THF IN I ER NΛTIONΛL R Γ.COGNI I ION or

TH E DEPOSIT OF M ICROORGANISMS FOR TH F. PU R POSES OF PΛ TF NT PROCEDU R E

INTERNA TIONA L rORM

RECEIPT IN THF CASE OF ΛN ORIGINAL DEPOSIT ISSUED PURSUANT TO R U I.T 7 AND VIABILITY STATEMENT ISSUED PU RSUANT TO R U LE 10 2 ,

To: (Name and Address of Depositor or Attorney)

Indiana University Foundation

Attπ: Byoung Se Kwon

Indiana University School of Medicine

Department of Microbiology & Immunology

635 Barnhill Drive, MS-255

Indianapolis, IN 46202-5120

Deposited on Behalf of: Indiana University Foundation

Identification Reference by Depositor: ATCC Designation

Hybridoma that produces 4B4-1 mAb HB- 1 1 860

The deposit was accompanied by: _ a scientific description _ a proposed taxonomic description indicated above.

The deposit was received March 10. 1995 by this International Depository Authority and has been accepted.

AT YOUR REQUEST:

X We will not inform you of requests for the strain.

The strain will be made available if a patent office signatory to the Budapest Treaty certifies one's right to receive, or if a U.S. Patent is issued citing the strain and ATCC is instructed by the United States Patent & Trademark Office or the depositor to release said strain.

If the culture should die or be destroyed during the effective term of the deposit, it shall be your responsibility to replace it with living culture of the same.

The strain will be maintained for a period of at least 30 years after the date of deposit, and for a period of at least five years after the most recent request for a sample. The United States and many other countries are signatory to the Budapest Treaty.

The viability of the culture cited above was tested March 15. 1995. On that date, the culture was viable.

International Depository Authority: American Type Culture Collection, Rockville, Md. 20852 USA Signa attuurree / ooff ppeerrssoonn h haavviinng aauutth

e, Director ug ority to represent ATCC:

Date: March 1 7, 1995

Annette /L. Bad . Patent Depository v cc: Christopher A. Michaels

Claims

What is claimed is:

1. A monoclonal antibody against H4-1BB which preferentially binds to at least a portion of receptor protein H4-1BB.

2. A hybridoma capable of producing a monoclonal antibody of claim 1.

3. A method of using the monoclonal antibody of claim 1 to enhance T-cell proliferation comprising the step of treating T-cells that have expressed receptor protein H4-1BB with said monoclonal antibody.

4. The method of claim 3 further comprising the step of conducting said treatment in the presence of co-stimulatory molecule.

5. A method of using the monoclonal antibody of claim 1 to enhance T-cell activation comprising the step of treating T-cells that have expressed receptor protein H4-1BB with said monoclonal antibody.

6. The method of claim 5 further comprising the step of conducting said treatment in the presence of co-stimulatory molecule.

7. The method of using the monoclonal antibody of claim 1 to inhibit the interaction of H4-1BB and its ligand.

8. The method of using the monoclonal antibody of claim 1 to aid in killing cancer cells by using said monoclonal antibody to activate T-cells against said cancer cells.

9. The method of using the monoclonal antibody of claim 1 to treat autoimmune diseases by using the monoclonal antibody to inhibit the interaction of H4-1BB and its ligand.

10. The method of using the monoclonal antibody of claim 1 to block a patient's immune response during organ transplantation by using the monoclonal antibody to inhibit the interaction of H4-1BB and its ligand.

11. An antibody that is immuno reactive with a purified human 4- IBB polypeptide comprising the N-terminal amino acid sequence aLeu-Gln-Asp-Pro-Cys-Ser-Asn- Cys-Pro-Ala-Gly-Thr.

12. An -antibody of claim 1 1, wherein said antibody is a monoclonal antibody.