Indiana University

Other

Advanced Research & Technology Institute

Donald Guthrie Foundation for Medical Research, Inc.

Advanced Research and Technology

Advanced Research and Technologyinc

Byoung S Kwon

Hal E Broxmeyer

Byung Soo Youn

Doo-Hong Park

Seung Jae Baek

Ju-Hyung Ahn

Soo-Il Chung

Eun-Kyoung Lee

Kong-Ju Lee

The human receptor H4-1BB has been isolated, sequenced and disclosed herein. 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. 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. A monoclonal antibody against H4-1BB can be used to enhance T-cell proliferation by treating T-cells that have expressed receptor protein H4-1BB with the anti H4-1BB monoclonal antibody. Tumors transfected with H4-1BBL may be capable of delivering antigen-specific signals as well as the co-stimulatory signals and can be killed by human cytotoxic T lymphocytes.

Nucleic acid molecules encoding human 4-1BB

The present invention includes the receptor protein 4-1BB and the cDNA gene encoding for receptor protein 4-1BB. The nucleotide sequence of the isolated cDNA is disclosed herein along with the deduced amino acid sequence. The 4-1BB protein and fragments and derivatives can be used: 1) as a probe to isolate ligands to receptor protein 4-1BB, 2) to stimulate proliferation of B-cell's expressing 4-1BB, or 3) to block 4-1BB ligand binding. A monoclonal antibody against 4-1BB was developed which specifically recognizes an epitope on the extracellular domain of receptor protein 4-1BB. The monoclonal antibody can be used enhance T-cell proliferation and activation by treating T-cells that have expressed receptor protein 4-1BB with the monoclonal antibody. The effectiveness of the treatment was enhanced when conducted in the presence of protein tyrosinase kinase. A fusion protein for detecting cell membrane ligands to receptor protein 4-1BB was developed. It comprises the extracellular portion of the receptor protein 4-1BB and a detection protein bound to the portion of the receptor protein 4-1BB.

Antibody for 4-1BB

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.

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

The present invention relates to a variant of Lkn-1(shLkn-1) with enhanced biological activity, which is a truncated form of Lkn-1, a process for preparing a recombinant shLkn-1 by employing expression vector therefor and pharmaceutical application of the said protein. shLkn-1 is generated by missing 26 amino acid residues from the amino terminus of Lkn-1 to contain 66 amino acids. Recombinant shLkn-1 inhibits colony formation and cell proliferation in vivo, which suggests that it can be used as a potential drug for the antibody production, the treatment during HIV-1 infection, the protection of bone marrow stem cells during chemotherapy or radiotherapy, and the inhibition of leukemia.

Variant of C6 &bgr;-chemokine leukotactin-1(shLkn-1) with enhanced biological activity

Antibody specific for H4-1BB

Murine 4-1BB gene

Full length cDNAs, L2G25B and 4-1BB, were isolated and sequenced. The cDNA L2G25B encodes for the lymphokine, macrophage inflammatory protein-1&agr; or MIP-1&agr;. The studies disclosed herein suggest that MIP-1&agr; and MIP-&bgr; can, through rapid action, modulate early myeloid progenitor cell proliferation. Recombinant proteins have been produced for the cytokine, L2G25BP (Macrophage Inflammatory Protein-1&agr;, MIP-1&agr;). By employing the recombinant protein (rMIP-1&agr;), receptors for MIP-1&agr; were identified on Con A-stimulated and unstimulated CTLL-R8, a T-cell line, and LPS-stimulated RAW 264.7, a macrophage-cell line. Purified recombinant murine macrophage inflammatory protein-1 alpha (rmuMIP-&agr;), was assessed for effects on proliferation of granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells. The results suggest that rmuMIP-1&agr; has myelosuppressive activity in vivo. The cDNA clone, called 4-1BB, is an inducible receptor-like sequence found in both cytolytic and helper T-cells.

Nucleic acid encoding MIP-1&agr; Lymphokine

Method of using human receptor protein 4-1BB

The present invention started when a .lambda.gt11 cDNA library of normal human melanocytes was screened with antibodies directed against purified hamster tyrosinase. Two important cDNA clones were isolated: cDNA .lambda.mel 34 and .lambda.mel 17-1. It is concluded that .lambda.mel 34 contained cDNA encoding human tyrosinase. Moreover, the cDNA gene for human tyrosinase was recovered from the .lambda.mel 34 and can be used for many purposes including the production of pure human tyrosinase. This gene and its promoter are characterized herein. Studies suggested that .lambda.mel 17-1 gene product act on melanin biosynthesis's pathway distal to tyrosinase. Melanocytes preferentially express an mRNA species, Pmel 17, whose protein product cross reacted with anti-tyrosinase antibodies and whose expression correlated with the melanin content. The deduced protein structure has been analyzed and its chromosomal location in mouse and man has been mapped. The human Pmel 17 gene, designated D12S53E, maps to Chromosome 12, region 12pter-q21; and the mouse homologue, designated D12S53Eh, maps to the distal region of mouse Chromosome 10, a region also known to carry the coat color locus si (silver).

CDNA clones for a regulatory protein in the melanin-production pathway

This discovery resulted when a .lambda. gt11 cDNA library of normal human melanocytes were screened with antibodies directed against purified hamster tyrosinase. Sixteen independent clones which gave a positive signal were isolated from 5.times.10.sup.5 independent plaques. cDNA inserts of 13 clones among the 16 candidates cross-hybridized with each other, indicating that they were from related mRNA species. mRNA homologous to a representative cDNA .lambda. mel 34 was expressed specifically in melanocytes, detecting an approximately 2.4 kb mRNA species of human melanocytes. The nucleotide sequence of the three overlapping cDNA inserts spanning 1.88 kb was determined and an amino acid sequence was deducted. The human tyrosinase is composed of 548 amino acids with a molecular weight of 62,160 excluding a hydrophobic signal peptide. Mouse genomic DNA blot analysis revealed that the gene for .lambda. mel 34 was deleted in albino mouse homozygous for the deletion at and around the albino locus on chromosome 7. It is concluded that .lambda. mel 34 contained cDNA encoding human tyrosinase. Moreover, the cDNA gene for human tyrosinase was recovered from the .lambda. mel 34 and can be used for many purposes including the production of pure human tyrosinase.

Growing community of inventors

Carmel, IN, United States of America

Byoung S Kwon