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Lymphotoxin-beta, lymphotoxin-beta complexes, pharmaceutical preparations and therapeutic uses thereofRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, LymphokineLymphotoxin-beta, lymphotoxin-beta complexes, pharmaceutical preparations and therapeutic uses thereof description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070116668, Lymphotoxin-beta, lymphotoxin-beta complexes, pharmaceutical preparations and therapeutic uses thereof. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] The present application is a continuation-in-part of co-pending applications PCT/US91/04588, filed Jun. 27, 1991 and U.S. patent application Ser. No. 07/544,862, filed Jun. 27, 1990. TECHNICAL FIELD OF THE INVENTION [0003] This invention relates to lymphotoxin-.beta., a lymphocyte membrane-type polypeptide. Lymphotoxin-.beta., also referred to as p33, has been identified on the surface of T lymphocytes, T cell lines, B cell lines and lymphokine-activated killer cells. [0004] This invention also relates to complexes formed between lymphotoxin-.alpha. (LT-.beta.) and other lymphotoxin-type peptides such as lymphotoxin (which we refer to herein as "lymphotoxin-.alpha. (LT-.alpha.)" to distinguish it from LT-.beta.) and to complexes comprising multiple subunits of LT-.beta.. The LT-.beta. polypeptide of this invention is expected to be useful in holding LT-.alpha. formed within the cell on the cell surface where either LT-.beta. or the LT-.alpha./LT-.beta. complex may act as an inflammation regulating agent, a tumor growth inhibiting agent, a T cell inhibiting agent, a T cell activating agent, an immunomodulatory agent, an autoimmune disease regulating agent or an HIV regulating agent. Furthermore, the antitumor activity of the LT-.alpha./LT-.beta. complex may be delivered to tumor cells by tumor infiltrating lymphocytes (TILs) transfected with the gene for LT-.beta.. BACKGROUND OF THE INVENTION [0005] The initiation of the immune response involves a complex array of intercellular signals. These signals typically involve soluble cytokines coupled with a number of cell-cell contact dependent-sign signals. The contact dependent events, most notably activation of the T-cell receptor, lend specificity to the response whereas the soluble mediators are generally responsible for maintenance of cell differentiation and proliferation. Tumor Necrosis Factor (TNF) and LT-.alpha. are two polypeptides generally recognized for involvement with the initiation of the immune response. [0006] TNF and LT-.alpha. are soluble proteins noted originally for their ability to inhibit the growth of tumors. [L. Old, "Tumor Necrosis Factor," Science, 230:630 (1985)]. Further research demonstrated that both proteins exhibit a wide range of activities. TNF is synthesized in response to various inflammatory insults by a variety of cell types including both hematopoietic and nonhematopoietic cells, while LT-.alpha., in contrast, is made specifically by lymphocytes. The two known TNF receptors do not appear to discriminate between LT-.alpha. and TNF. [T. Schall et al., "Molecular Cloning and Expression of a Receptor for Human Tumor Necrosis Factor," Cell, 61:361-370 (1990); C. Smith et al., "A Receptor for Tumor Necrosis Factor Defines an Unusual Family of Cellular and Viral Proteins," Science 248:1019 (1990)]. In general, LT-.alpha. and TNF display similar spectra of activities in in vitro systems, although LT-.alpha. is often less potent. [J. Browning et al., Studies Of The Differing Affects Of Tumor Necrosis Factor And Lymphotoxin On The Growth Of Several Human Tumor Lines," J. Immunol., 143:1859 (1989)]. [0007] TNF appears to play a major role in specific aspects of metabolic control, in the response to endotoxin shock, and in the regulation of hematopoietic cell development. [B. Beutler et al., "The History, Properties, and Biological Effects of Cachectin," Biochemistry, 27, (1988); M. Akashi et al., "Lymphotoxin: Stimulation And Regulation of Colony Stimulating Factors in Fibroblasts," Blood, 74:2383 (1989); G. Roodman et al., "Tumor Necrosis Factor-alpha and Hematopoietic Progenitors: Effects Of Tumor Necrosis Factor On The Growth Of Erythroid Progenitors CFU-E And BFU-E And The Hematopoietic Cell Lines k562, HL60, And HEL Cells," Exp. Hematol., 15:928 (1987)]. [0008] Along with IL-1 and IL-6, TNF is also a major mediator of the inflammatory response. [D. Cavender et al., "Endothelial Cell Activation Induced By Tumor Necrosis Factor And Lymphotoxin," Amer. Jour. Path., 134:551 (1989); R. Cotran et al., "Endothelial Activation Its Role In Inflammatory And Immune Reactions," in Endothelial Cell Biology, (Plenum Press, Simonescu & Simonescu, eds., 1988) 335]. TNF also appears to be involved in T cell activation under certain conditions. [M. Shalaby et al., "The Involvement Of Human Tumor Necrosis Factors-.alpha. And -.beta. In The Mixed Lymphocyte Reaction," J. Immunol., 141:499 (1988); N. Damle et al., "Distinct Regulatory Effects of IL-4 and TNF-.alpha. During CD3-Dependent and CD3-Independent Initiation Of Human T-Cell Activation," Lymph. Res., 8:85 (1989); G. Ranges et al., "Tumor Necrosis Factor-.alpha. As A Proliferative Signal For An IL-2-Dependent T Cell Line: Strict Species Specificity of Action," Amer. Assoc. Immunol., 142:1203 (1989); G. Ranges et al., "Tumor Necrosis Factor .alpha./Cachectin Is A Growth Factor For Thymocytes," J. Exp. Med., 167:1472 (1988); P. Scheurich et al., "Immunoregulatory Activity Of Recombinant Human Tumor Necrosis Factor (TNF)-.alpha.: Induction Of TNF Receptors On Human T Cells And TNF-.alpha.-Mediated Enhancement Of T Cell Responses," J. Immunol., 138:1786 (1987)]. [0009] TNF is produced by several types of cells, including monocytes, fibroblasts, T cells and Natural Killer (NK) cells. [D. Goeddel et al., "Tumor Necrosis Factors: Gene Structure And Biological Activities," Cold Spring Harbor Symposium Quant. Biol., 51, 597 (1986); D. Spriggs et al., "Tumor Necrosis Factor Expression In Human Epithelial Tumor Cell Lines," J. Clin. Invest., 81:455:(1988); M. Turner et al., "Human T cells From Autoimmune and Normal Individuals Can Produce Tumor Necrosis Factor," Eur. J. Immunol., 17:1807 (1987)]. Investigators have also detected murine and human forms of TNF that are associated with the surface of various cells either as a transmembrane protein or a receptor-bound molecule. [B. Luettig et al., "Evidence For the Existence of Two Forms of Membrane Tumor Necrosis Factor: An Integral Protein and a Molecule Attached To Its Receptor," J. Immunol., 143:4034 (1989); M. Kriegler et al., "A Novel Form of TNF/Cachectin Is a Cell Surface Cytotoxic Transmembrane Protein: Ramifications For the Complex Physiology of TNF," Cell, 53, pp. 45-53 (1988); and M. Kinkhabwala et al. "A Novel Addition To the T Cell Repertory," J. Exp. Med., 171:941-946 (1990)]. [0010] LT-.alpha. also has many activities, generally similar, but not identical to those of TNF, including tumor necrosis, induction of an antiviral state, activation of polymorphonuclear leukocytes, induction of class I major histocompatibility complex antigens on endothelial cells, induction of adhesion molecules on endothelium and growth hormone stimulation. [N. Ruddle and R. Homer, "The Role of Lymphotoxin in Inflammation," Prog. Allergy, 40:162-182 (1988)]. Both LT-.alpha. and TNF are ligands to members of the nerve growth factor (NGF) receptor family. [S. Mallett and A. N. Barclay, "A New Superfamily Of Cell Surface Proteins Related To The Nerve Growth Factor Receptor," Immunology Today, 12:7:220-223 (1991).] [0011] In contrast to TNF, LT-.alpha. secretion appears to be a specific property of only activated T cells and certain B-lymphoblastoid tumors. [N. Paul et al., "Lymphotoxin," Ann. Rev. Immunol., 6:407 (1988)]. Some researchers have also indicated that a membrane-associated form of LT-.alpha. may be expressed on the surface of lymphocytes under certain circumstances [J. Hiserodt, et al., "Identification of Membrane-Associated Lymphotoxin (LT) On Mitogen-Activated Human Lymphocytes Using Heterologous Anti-LT Antisera In Vitro," Cell. Immunol., 34:326-339 (1977); C. Ware et al., "Mechanisms of Lymphocyte-Mediated Cytotoxicity," J. Immunol., 126:1927-1933 (1981); U. Anderson et al. J. Immunol. Methods, 123, 233 (1989); Y. Abe et al., Jpn. J. Canc. Res., 82:23 (1991); Y. Abe et al., "Studies of Membrane Associated and Soluble (Secreted) Lymphotoxin In Human Lymphokine-Activated T-Killer Cells In Vitro," Lymphokine and Cytokine Research, 11, 2: 15-121 (1992)]. [0012] In recent years genes for both TNF and LT-.alpha. have been isolated and cloned, leading to their complete characterization and to the availability of recombinant forms of both proteins. [P. Gray et al., "Cloning and Expression of cDNA For Human Lymphotoxin, A Lymphokine With Tumor Necrosis Activity," Nature, 312:121-124 (1984); D. Pennica et al., "Human Tumor Necrosis Factor: Precusor Structure, Expression And Homology To Lymphotoxin," Nature, 312:724 (1984)]. [0013] Other "cytokine-like" cell surface proteins including the CD40 protein have recently been shown to share certain similarities with TNF and LT-.alpha.. Like TNF and LT-.alpha., the CD40 protein is a ligand to members of the TNF/nerve growth factor (NGF) receptor family. [S. Mallett and N. Barclay, Immunology Today, 12:220-223 (1991)]. The CD40 protein is a 277-amino acid protein expressed on the surface of B lymphocytes, epithelial cells, and some carcinoma cell lines. [R. Armitage et al., Nature, 357:80-82 (1992); T. Farrah and C. Smith, "Emerging Cytokine Family," Nature, 358:26 (1992)]. [0014] We have now identified a novel surface protein, lymphotoxin-.beta. (LT-.beta.) or p33. LT-.beta. has been identified on the surface of several types of lymphocyte cells, including OKT3-stimulated primary T cells, antigen specific IL-2 dependent CTL clones, and a PMA-stimulated human T cell hybridoma II-23.D7. LT-.beta. targets LT-.alpha. produced in the cell to the cell membrane where LT-.beta. and LT-.alpha. appear as a complex (designated "LT-.alpha./LT-.beta." throughout this disclosure). The LT-.alpha./LT-.beta. complex is believed to be a novel mechanism for membrane expression of LT-.alpha. by activated T-cells. [Androlewicz et al., "Lymphotoxin Is Expressed As a Heteromeric complex With A Distinct 33 kDa Glycoprotein On The Surface Of An Activated Human T Cell Hybridoma," Journal Of Biological Chemistry, 267:2542-2547 (1992)]. The LT-.alpha./LT-.beta. complex may exhibit cytolytic and cell regulatory activity similar to the soluble LT-.alpha., TNF and CD40 proteins. The membrane-associated LT-.beta. complexed with LT-.alpha. may represent, as a complex, a novel ligand for T cell interactions with other cells and may also be useful in targeted cell lysis. SUMMARY OF THE INVENTION [0015] The novel protein of the present invention has been named lymphotoxin-.beta. (LT-.beta.). This protein is found on the surface of several types of lymphocyte cells, including OKT3-stimulated primary T cells, antigen-specific IL-2 dependent CTL clones, and a PMA-stimulated human T cell hybridoma, II-23.D7. It forms a novel complex with LT-.alpha. and forms complexes with other LT-.beta. subunits (e.g., (LT-.beta.).sub.2 LT-.alpha. complexes). [0016] LT-.beta. has a molecular weight of 31-35 kD as determined by immunoprecipitation and SDS-PAGE. LT-.beta. exhibits N-linked glycosylation. The amino acid sequence of lymphotoxin-.beta. is set forth in SEQ ID NO:2, and the amino acid sequences of several soluble lymphotoxin-.beta. peptides are set forth in SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8. The DNA sequence coding for lymphotoxin-.beta. is set forth in SEQ ID NO:1 and DNA sequences coding for several soluble lymphotoxin-.beta. peptides are set forth in SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7. [0017] LT-.beta. as a cell membrane protein binds LT-.alpha. during synthesis, thus "targeting" the LT-.alpha. to the cell membrane. In the absence of LT-.beta., LT-.alpha. is secreted into the extracellular medium. The LT-.alpha./LT-.beta. complex is recognized by polyclonal antisera raised against recombinant lymphotoxin-.alpha. (rLT-.alpha.) expressed in CHO cells or by monoclonal antibodies (mAbs) raised against natural LT-.alpha.. Furthermore, antisera that recognize the LT-.alpha./LT-.beta. complex and (LT-.alpha.).sub.3 block the mixed lymphocyte reaction (MLR), a standard immunological assay of the expected proliferative response of T lymphocytes to allogenic stimulation, i.e., the introduction of T lymphocytes from another individual, which are recognized as foreign (non-self) by the "responder" lymphocytes [See, e.g., M. Shalaby et al., J. Immunol., 141:499 (1988)]. [0018] The LT-.beta. protein was purified by affinity chromatography, partially sequenced and a specific oligonucleotide probe was designed. The cDNA encoding the LT-.beta. was isolated by probing a cDNA library from activated II-23.D7 cells, a human T-cell hybridoma that displays large amounts of surface lymphotoxin upon phorbol ester activation. The identified amino acid sequence encodes a 240-244 amino acid sequence (a molecular mass of the unmodified protein of about 25130-25390 kDa). See SEQ ID NO:2. The amino acid sequence and the placement of the transmembrane region are typical of a type II membrane protein. [0019] This sequence comprises a short 14-18 amino acid N-terminal "cytoplasmic" domain. Following this cytoplasmic domain there is an extensive stretch of 30 hydrophobic amino acids which presumably acts as a membrane anchoring domain. No identical sequences were found within available databases. There is one cysteine residue in the extracellular domain and two methionines within the last C-terminal 17 amino acids. This is consistent with the very limited cyanogen bromide cleavage pattern exhibited by this protein. [0020] Comparison of the LT-.beta. sequence with other proteins known to bind to members of the TNF/NGF receptor family reveals considerable structural similarity. Four of the ligands to members of the TNF/NGF receptor family (TNF, LT-.alpha., LT-.beta. and the CD40 ligand) resemble type II membrane proteins and share at least four regions of sequence conservation in the extracellular domain as indicated in FIG. 14. The conserved TNF and LT-.alpha. domains shared with LT-.beta. are likely to be involved in intersubunit interactions and .beta. sheet organization. These regions of conservation can account for the association between LT-.alpha. and LT-.beta.. The existence of these homology regions may facilitate engineering the polypeptide to form complexes with, for example, TNF or the CD40 ligand. Such a molecule would have mixed functions and could possibly be used as a custom designed drug. [See J. Fuh et al., "Rational Design Of Potent Antagonists To The Human Growth Hormone Receptor," Science, 256:1677 (1992)]. [0021] We believe that the polypeptide complexes of this invention are important in T cell activation events and are useful in compositions and methods for T cell activation or T cell suppression and as therapeutic agents in the treatment of inflammation or applications requiring cytolytic activities, such as inhibition of tumor cell or neoplasia growth. We also believe that the polypeptide complexes may be important in cellular immunotherapies, including enhancing the tumoricidal properties of tumor infiltrating lymphocytes in Tumor Infiltrating Lymphocyte ("TIL") therapy. TIL immunotherapy may be improved by gene transfer techniques. For example, a gene may be added to tumor cells for the purpose of inducing the body's immune system to mediate an effective tumor-directed immune response. [See, e.g., W. F. Anderson, "Human Gene Therapy." Science, 256:808-813 (1992)]. [0022] We also believe, based upon similarities between a molecule identified as "Fas" and members of the TNF/NGF receptor family, that the polypeptide complexes of this invention may be involved in the internal cell process known as programmed cell death or apoptosis, and may therefore be involved in mediating autoimmune disease. [See, e.g., N. Itoh et al., "The Polypeptide Encoded By The cDNA For Human Cell Surface Antigen Fas Can Mediate Apoptosis," Cell, 66:233-243 (1991); R. Watanabe-Fukunaga et al., "Lymphoproliferation Disorder In Mice Explained By Defects In Fas Antigen That Mediates Apoptosis," Nature 356:314 (1992)]. Continue reading about Lymphotoxin-beta, lymphotoxin-beta complexes, pharmaceutical preparations and therapeutic uses thereof... 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