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Link:  Pharm/Biotech Resources


Title:  Methods of using antibodies to human receptor protein 4-1BB

United States Patent:  6,905,685

Issued:  June 14, 2005

Inventors:  Kwon; Byoung S. (812 Mountain Ash, Carmel, IN 46033)

Appl. No.:  877338

Filed:  June 8, 2001

Abstract

Disclosed herein are the methods of using the H4-1BB protein, ligands to this protein, and various mAbs either directed against H4-1BB or other molecules that can be used therapeutically. The nature and importance of the H4-1BB molecule provides the ligands and related co-stimulatory molecules the ability to enhance or suppress T-cell activation and proliferation. By treating T-cells that have expressed receptor protein H4-1BB with one of the four anti-H4-1BB monoclonal antibodies disclosed herein activation or inhibition of the immune response is seen. 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 including diabetes, rheumatoid arthritis, and lupus. Other applications of this technology include the development of therapeutic methods for the treatment of HIV-1 infected individuals, and the treatment of cancerous tumors.

Description of the Invention

FIELD OF THE PRESENT INVENTION

The present invention relates to the therapeutic and scientific uses for the human H4-1BB protein, its ligands, and the development of monoclonal antibodies that recognize and bind the H4-1BB receptor protein.

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. The phagocytes include macrophage cells which scavenge unwanted materials, such as virus proteins or bacterial cell walls from the system. The lymphocytes include helper T cells, killer T cells and B cells, as well as other types of cells, including those categorized as suppressor T cells. The B cells produce the antibodies. The killer T cells physically destroy target cells and the helper T cells facilitate the whole process. The complexities of the immune system and its function is facilitated, at least in part, by the lymphokines.

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

Interleukin 1 (IL-1), secreted from macrophages activates the helper T cells and acts to raise body temperature, causing fever, which enhances the activity of the immune cells. The activated helper T cells produce Interleukin 2 (IL-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 gamma interferon (IF), which is similar to Interleukin 2 in that it has multiple effects. Gamma interferon helps activate killer T cells, enabling them to attack the invading organisms. Like BCGF, gamma interferon increases the ability of the B cells to produce antibodies. IF 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 such that the virus protein, an infected cell, or other foreign matter is overwhelmed and removed from the system. There are many lymphokines, maybe a hundred or more, which participate in the complex web that is the immune process. Many lymphokines and their precise effects remain unknown.

Lymphokine activities are produced when a certain lymphokine binds to its specific receptor on the surface of a target cell. Among scientists there is widespread use of cloned cell lines for production of lymphokines and their receptors. The isolation of lymphokine and lymphokine receptor messenger ribonucleic acid (mRNA) has become a common technique. The mouse receptor protein, 4-1BB, was isolated and identified based on specific expression of the T cell genes using a technique identified by the present inventor in a prior 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. Importantly, the mRNA sequences of immune cells are expressed differentially as they relate to T cells generally, and to the killer T cells specifically. Even though the level of expression is low and the quantity of the lymphokine and its receptor protein is low, this expressed mRNA can be detected and isolated. 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 initiated by cellular signals induced by very scarce message molecules (i.e., IF, interleukins, Map Kinase Kinase, etc.).

Most T cell factors have been classically identified by recognizing biologic activities in assays, and thereafter purifying the protein information. An alternative approach is to isolate putative T cell genes based upon specific expression, insert them into an appropriate expression vector, and then demonstrate the function of the unknown isolated protein. Using the aforesaid modified differential screening procedure, the present inventor cloned a series of T cell subset-specific complementary deoxyribonucleic acid (cDNA) from cloned helper T lymphocyte (HTL) L2 cells, and cloned cytolytic T lymphocytes (CTL) L3.

T cells are critically important in long-term acquired immunity, providing protection against viral, bacterial and parasitic infection. T cells are activated when they encounter a peptide from the invading pathogen in context with self-MHC (Major Histocompatibility Complex) via the T cell's own T cell receptor (TCR) complex and other co-stimulatory molecule(s), such as CD-28, or CD-3. Without the engagement of the other co-stimulatory molecule(s), the T cell is rendered anergic (Vassali et al., PNAS, 1979). To date, the best-characterized co-stimulatory molecule has been CD-28. More recently, however, other cell-surface molecules have been suggested to play a co-stimulatory role, such as the molecule 4-1BB. The 4-1BB protein 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 super family (Haskins et al., J. Exp. Med., 1983). 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 tumor necrosis factor (TNF) called TNFR-1 and TNFR-11, the apoptotic-inducing protein Fas, and CD-30 which plays a role in the regulation of cellular growth and transformation.

Some of these members have been shown to play important roles in human immunodeficiency virus-1 (HIV-1) infection, including CD4+ T cell proliferation, apoptosis and virus replication. The presence of high serum levels of CD30 has become a predictor of progression to acquired immunodeficiency syndrome (AIDS), although no circulating CD30 cells have been found in HIV-1 seropositive individuals. The expression of HIV-1 was induced by triggering CD30 of HIV-1 infected CD4+ T cells through a nuclear factor-κβ (NF-κβ)-dependent pathway. In HIV-1 individuals, high levels of Fas expression were observed in peripheral blood lymphocytes. Fas production was found to trigger or induce marked apoptosis of T lymphocytes, which might contribute to the CD4+ T cell depletion by HIV-1 infection. The ability of CD4+ T cells to express the CD40 ligand after in vitro stimulation is not impaired because of HIV-1 infection, but CD40/CD40 ligand interaction regulates HIV-1 replication of B cells in vitro. CD27 signaling enhanced proliferative response of T cells to the normal extent in HIV-1-infected individuals.

In the experiments that led to the development of this invention, a series of T cell subset-specific cDNAs were isolated from cloned murine T-cells 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, which encodes mouse receptor protein 4-1BB, was studied further. These studies have led to the isolation of the human homologue to 4-1BB, H4-1BB, as well as to a series of monoclonal antibodies capable of binding the H4-1BB receptor protein and acting thereby as agonists or antagonists of H4-1BB.

T cells interact with components of the extracellular matrix (ECM) through members of the integrin family after transendothelial migration during homing to sites of inflammation. Integrin molecules are very late antigens (VLA's) in a family of cell surface receptors that mediate the adhesion of cells to ECM proteins as well as other cells. The heterodimeric integrins comprising of various alpha and beta subunits, act as a transducing mechanism of extracellular signals. Regulation of integrin function is utilized by T cells and other leukocytes for rapid adhesion following activation of the cells.

The major factors known so far to affect the differentiation of the T cells are the lymphokines (also referred to as cytokines), such as IL-2 and IL-4. In vitro and in vivo studies with transgenic mice have demonstrated that IL-2 induces the development of the Th1 subset of T cells by priming them for efficient IF production and preventing development of IL-4-producing cells. Previously however, it was unknown how their interactions worked to direct the amplification of the immune response and development of the Th1 or Th2 subset of T cells.

Specific immune responses are governed by the recognition of antibodies to foreign antigens. Antibodies form a family of structurally related glycoproteins and confer, generally to the organism producing them, 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 that differentiate in response to stimulation by antigens. A specific antigen will trigger the complementary B lymphocyte(s) 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 (mAb) 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 to create a hybridoma. This is done because normal B-lymphocytes cannot grow indefinitely, yet when fused with the myeloma, the resulting hybridoma produces a virtually endless supply of a specific monoclonal antibody. Selection techniques 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 different antibody-producing hybridomas are produced, each hybridoma clone of an individual B lymphocyte will secrete an antibody for only one surface antigenic determinant. To determine which mAbs specifically bind to the protein receptor, or which has a desired activity (e.g., the mAb acts as an agonist, antagonist, or has the most specific binding to a critical epitope), the hybridomas can be screened with an 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 or activation.

The invention below presents uses for the H4-1BB protein, its ligands, antibodies thereto and other co-stimulatory molecules that can be used therapeutically in the treatment of cancer and HIV-1.

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 pH 4-1BB 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. The cDNA of the human receptor, H4-1BB, was isolated by using probes derived from cDNA 4-1BB. The cDNA gene identified as p4-1BB was deposited at the American Type Tissue Culture Collection at 10801 University Boulevard, Manassas, Va. 20110-2209 under ATCC NO.: 67825. The present invention also provides an antibody specific for H4-1BB. One exemplary hybridoma which secretes such an antibody was deposited at the American Type Tissue Culture Collection on Mar. 10, 1995 under ATCC No. HB-11860.

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.

Monoclonal antibodies generated against H4-1BB can be used to enhance or suppress T-cell proliferation and activation by treating T-cells that have expressed receptor protein H4-1BB with an anti-H4-1BB monoclonal antibodies. To enhance immune reaction antibodies which act as agonists can be generated, to suppress T-cell proliferation and/or activation antibodies which act as antagonists can be generated. To this end, four monoclonal antibodies have been developed for use. The monoclonal antibodies BBK-1 and BBK-4 are agonists to receptor protein H4-1BB, while monoclonal antibodies BBK-2 and BBK-3 are antagonists to receptor protein H4-1BB, and can be used to either upregulate the immune system or suppress its activity. 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. A monoclonal antibody generated against H4-1BB (e.g. an agonist) is capable of eradicating tumors with low immunogenicity by providing for the full activation, enhancement, and/or proliferation of T-cells. Moreover, an anti-H4-1BB mAb agonist has great utility in assessing the role of the 4-1BB receptor protein in the transition from naive to memory T-cells. Cross-linking of the 4-1BB with an anti-H4-1BB mAb agonist, such as BBK-1 or BBK-4 will produce the effects similar to the binding of the 4-1BB ligand to 4-1BB.

A mAb agonist H4-1BB can also be used to interfere with H4-1BB and H4-1BB ligand binding. By interfering with ligand binding, as with the use of an anti-H4-1BB mAb antagonist BBK-2, and BBK-3, the immune responses will be suppressed. In this context, diseases that would benefit from the therapeutic use of such a mAb include rheumatoid arthritis, systemic lupus erythematosus, and diabetes. Alternatively, this type of molecule is useful in organ transplantation to suppress immune system mediated rejection of transplanted tissue.

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) or Fc portion of an IgG1 bound to the portion of the receptor protein H4-1BB. In addition, this disclosure demonstrates that co-engagement of CD28 with 4-1BB promoted type 1 effector T cell development. The 4-1BB signal regulated CD28 mediated cytokine production profiles in two ways, enhancing type 1 and, at the same time, suppressing type 2 cytokine (lymphokine) production. The 4-1BB-mediated co-stimulation also induced γ-interferon (IF) production in Th1 cells. The expression of 4-1BB was subset-specific, being detected predominantly on IF-producing, but not on IL-4-producing cells. Moreover, it was determined that 4-1BB and CD30 expression were mutually exclusive, representing type 1 and type 2 subsets, respectively. The co-engagement of 4-1BB with CD28 enhanced long-term cell survival for cells susceptible to apoptosis induced by repeated TCR activation. Therefore, it was demonstrated that 4-1BB and CD30 interplay to regulate the balance between type 1 and type 2 T cell subsets, and the polarization of the immune response. Therapeutically, they can be used to achieve opposite effects.

This invention presents data, which demonstrates that there is a functional correlation between 4-1BB and CD28 in T cell adhesive responses. The inventors disclose herein that 4-1BB can induce of cell adhesion. Enhanced cell adhesion through the presence 4-1BB signal or agonist results in a maximal T cell activation in response to repeated exposure to sub-optimal concentrations of anti-CD3 and anti-CD28. Thus, 4-1BB effects were attributed by its ability in reducing the threshold of anti-CD3 concentration needed to repeatedly activate primary T cells. Therefore the degree of cell adhesion in response to anti-4-1BB correlated with the 4-1BB expression levels, and correspondingly effects the therapeutic use of these molecules.

The level of 4-1BB-expressing and the percentage of 4-1BB-expressing T cells was higher in HIV-1 positive individuals than in the HIV-1 controls (P<0.01). 4-1BB signal cooperated with CD28 to induce HIV-1, and CD4+ T cell proliferation. In addition, cross-linking 4-1BB with agonistic monoclonal antibodies enhanced HIV-1 replication both in primary stimulation and secondary re-stimulation of CD4+ T cells from HIV-1 individuals. Thus, 4-1BB is involved in the activation of HIV-1 replication from latently infected CD4+ T cells, and the 4-1BB co-stimulatory pathway can be the target of therapeutic intervention. If the pathway of HIV-1 infection is disturbed at the early stages of the infection, through the use of H4-1BB antibodies a lower virus load will result. Moreover, the increased 4-1BB expression on CD8+ T cells is noteworthy because it is correlated with the degree of immunodeficiency in HIV-1 infection, cross-linking 4-1BB on CD8+ T cells will induce enhanced cytotoxic activity against HIV-1 infected CD4+ T cells.

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

Another object of the present invention is to teach the method of constructing a monoclonal antibody against H4-1BB.

DETAILED DESCRIPTION OF THE INVENTION

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 the production of monoclonal antibodies acting as either agonists or antagonists of H4-1BB. Also disclosed is the therapeutic use of: 4-1BB, antibodies to it, its ligands, and immunoprecipitation studies.

Isolation and Characterization of Mouse Receptor 4-1BB

U.S. application Ser. No. 08/012,796 discloses the nucleotide sequence and the deduced amino acid sequence of the mouse receptor 4-1BB. The transcript of 4-1BB 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 IL-2 stimulation in the cloned T-cells (1). The 4-1BB 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 4-1BB resembles those of lymphokine mRNAs while the sequence appeared consistent with those of receptor proteins.

The major species of 4-1BB on the cell surface appears to be a 55-kDa dimer. 4-1BB 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 f1), all forms potentially co-exist on each cell. Peptide digests from the 4-1BB monomer and dimer are needed to determine whether 4-1BB exists as a homodimer on the cell surface. A variety of cell surface receptors such as the insulin receptor (Ebina et al., 1985), the B cell surface immunoglobin receptor (Vassali et al.,), the T cell Ag receptor (Haskins et al., 1983), the CD-28 co-stimulatory receptor (Lesslaver et al., 1986), and the CD27 T-cell antigen (Van Lier et al., 1987) are composed of disulfide-bonded subunits. Receptor dimerization may be required for ligand binding and subsequent cell signal transduction.

4-1BB 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 phorbol myristate acetate (PMA) and ionomycin is capable of mimicking those signals required for T cell proliferation. Although PMA or ionomycin alone induced 4-1BB mRNA, the combination of PMA and ionomycin resulted in optimal 4-1BB expression. Furthermore, the expression of 4-1BB was not transient. When purified splenic T cells were stimulated with immobilized anti-CD3, 4-1BB mRNA was expressed and this expression was maintained for up to 96 hrs post-stimulation. Cell-cycle analysis will be required to confirm that 4-1BB 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 (cysteine-rich regions), the cytoplasmic domains of these proteins are non-conserved 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, CD40 and CD27 antigens. Antibodies against the OX-40 augment the T-cell response in a mixed lymphocyte reaction and antibodies against CD40 enhance B-cell proliferation in the presence of a co-activator, such as PMA or CD20 antibodies, and synergizes with IL-4 in vitro to induce B-cell differentiation and to generate long-term normal B cell lines. One monoclonal antibody, anti-1A4, 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 phytohemagglutinin (PHA) or anti-CD3 mAb. These results indicate that the CD27 molecule plays an important role in T cell activation. Except for TNFR's, NCFR and CD40, 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 4-1BB.

To ascertain whether cell surface 4-1BB could contribute to T cell activation, the anti-4-1BB 53A2 was used as an antagonist to 4-1BB. The resulting data suggest that 4-1BB does in fact have the potential to function as an accessory signaling molecule during T cell activation and proliferation. The addition of soluble 53A2 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 absence of signal 2, which is provided by accessory cells. This has since been confirmed by a variety of studies (Moeller et al., 1989). The identification of the accessory cell CD28 as a potent co-stimulatory receptor on T cells was a significant contribution in beginning to characterize the accessory signal(s) required for optimal T cell proliferation. It is possible that other cell surface molecules may contribute to these co-stimulatory activation requirements.

The biochemical signals delivered through 4-1BB indicate that there is a putative p56lck tyrosine kinase-binding domain in its cytoplasmic tail. It was later determined that p56lck tyrosinase kinase binds to 4-1BB. It will also be worthwhile to determine if 4-1BB-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 member of the Nerve Growth Factor Receptor (NGFR) superfamily appear to be diverse. An emerging them 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 (Yamori et al., 1992). The cross-linking of CD40 by soluble antiCD40 monoclonal antibody blocks germinal center centrocytes from undergoing apoptosis in vitro. Signals delivered through CD40 may also aid in maintenance of responsiveness to differentiation factors. The ligation of CD40 with anti-CD40 F(ab′)2 fragments in the presence of IL-4 induced large increases in IgE synthesis. Also, anti-CD40 activated naive B cells treated with IL-10 and transfonning growth factor-β became committed to IgA secretion (DeFrance et al., 1992). In addition to sharing the molecular characteristics with the NGFR superfamily, it was noted that the 4-1BB contained a putative zinc finger structure similar to that of the yeast elF-2b protein. 4-1BB also shares a conserved region with the sina seven in absentia of Drosophila Melanogaster, which is required for correct photoreceptor cell development (Carthew and Rubin, 1990). That particular region is also similar to the protein product of the DGl7 gene of Dictyosteliun, whose expression is specifically induced during aggregation by cyclic adenosine monophosphate (cAMP).

This region forms the pattern of CX2CX9CX3HX3CXC (SEQ ID NO:9); and the cysteines and histidine are conserved in a similar space in 4-1BB, sina, and DGl7 proteins. Ten of 24 amino acids between the 4-1BB 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 localized 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 4-1BB may play diverse roles during cellular proliferation and differentiation.

In addition, 4-1BB may represent another cell-surface molecule involved in T cell-antigen presenting cell (APC) interactions. The 4-1BB-alkaline phosphatase (4-1BB-AP) fusion protein specifically bound to mature B-cell lines, anti-IF-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 non-lymphoid-cell lines. Since 4-1BB-AP binds to mature B cells and macrophages, it is possible that signals delivered upon 4-1BB binding may modulate APC functions in some way. This possibility remains to be explored.

Chalupny et al., proposed that 4-1BB Rg, a fusion protein consisting of the extracellular domain of 4-1BB 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. The inventors performed an ELISA to test this possibility using 4-1BB-AP and human vitronectin (Yelios Pharmaceuticals/GIBCO-BRL, Grand Island, N.Y.) immobilized at 0.007 mg, with 10 mg per well on microtiter plates. No binding of 4-1BB-AP based on AP activity was observed. To rule 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 4-1BB-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 4-1BB to a specific cell-surface 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. This interaction results in a cascade of biochemical signaling relays between the two cell types. 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 (Noelle and Snow et al., 1990). 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. This T cell function is not antigen-specific or MHC-restricted. In addition, soluble factors are not required for the Th induction of B-cell activation. Once B cells enter the cell cycle, IL-4 induces B cells to progress from G1 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. 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 co-stimulator. 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. Interestingly, paraformaldehyde-fixed SF21-4-1BB cells, synergized anti-μ and induced B-cell proliferation. The co-stimulation of splenic B cells by SF21-4-1BB occurred at optimal (10 μg/ml) and sub-optimal (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 tetradecanoylphorbolacetate (TPA) or ionomycin, or sub-optimal 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 baculovirus 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 co-stimulation. This can be a potential problem when using COS- or L-cell lines which can exhibit strong co-stimulation activity on their own.

Another member of the NGFR superfamily, CD40, is expressed on B cells and interacts with gp39, a molecule expressed on activated T cells. The cDNAs encoding the murine and human gp39 proteins have been cloned; this cell surface molecule is a type II membrane protein with homology to tumor necrosis factor. Noelle et al., found that a CD40-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., have shown that COS cells transfected with human gp39 can synergize with either TPA or anti-CD20 in inducing human B-cell proliferation and is able to stimulate B cells without a co-stimulator only at low levels. These data indicate that CD40 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. The NGFR binds to NGF, brain-derived neurotrophic factor, and neurotrophin-3.

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. It may be possible that 4-1BB 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 (Hintzen et al., 1991). It is also possible that more than one 1 ligand, if soluble and on the cell surface, may bind to 4-1BB.

Isolation of the Human Homologue, H4-1BB

In order to isolate the human homologue (H4-1BB) of mouse 4-1BB 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 4-1BB is a member of the superfamily (Mallett and Barclay, 1991). The amino acid sequences employed were mouse 4-1BB, human NGFR, human tumor necrosis factor receptors, human CD40, and human CD27. The areas of sequence conservation among the NGFR superfamily were chosen.

Materials and Methods

Peripheral blood lymphocytes from normal healthy individuals were isolated and activated with PMA (10 ng/ml) and ionomycin (1 mM). Messenger RNA 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 (SEQ ID Nos. 3-6). A combination of primers was used and produced a specific band of about 240 bp. The 240 bp is an expected size of human 4-1BB if the human homologue protein is similar to mouse 4-1BB in size. The PCR product (240 bp) 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 4-1BB. The 240-bp PCR product was used to screen λgt11 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:1 and the predicted amino acid sequence is shown in SEQ ID NO:2.

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 III site on the 5′ end of the forward primer and a Bgl II 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).

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 4-1BB indicated that the ligand for 4-1BB is on the cell surface. B cells and macrophages were major cells that express 4-1BBL. It is expected that H4-1BBL be expressed on human B cells and macrophages.

Results

A mammalian expression cDNA library was generated from human cell lines that express H4-1BBL. The library was screened by Iodine-labeled H4-1BB-AP. cDNA for H4-1BBL was then isolated and characterized. Soluble recombinant H4-1BBL was then produced. The generated antibodies were then used to suppress or enhance immune responses as described below. Monoclonal antibodies to H4-1BBL were produced and are discussed below.

According to studies completed by the inventor, 4-1BB acts as a co-stimulatory signal. It is expected then that H4-1BB acts as a co-stimulatory signal for T cell activation. Mouse 4-1BB helped B cells with proliferation and differentiation. H4-1BB has been found to do the same. H4-1BB-AP, H4-1BBL and various monoclonal antibodies disclosed below can be used to suppress or enhance human immune responses.

Claim 1 of 8 Claims

1. A method of treating cancerous tumors such that said cancerous tumors are reduced, comprising the administration of a first effective amount of anti-H4-1BB antibody such that said antibody comes into contact with at least one T-cell, and the administration of a second stimulatory molecule such that the molecule also comes into contact with said T-cell.

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