WO2011031835A1 - Polypeptides dr5 chimères et leurs utilisations - Google Patents

Polypeptides dr5 chimères et leurs utilisations Download PDF

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WO2011031835A1
WO2011031835A1 PCT/US2010/048237 US2010048237W WO2011031835A1 WO 2011031835 A1 WO2011031835 A1 WO 2011031835A1 US 2010048237 W US2010048237 W US 2010048237W WO 2011031835 A1 WO2011031835 A1 WO 2011031835A1
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chimeric
mouse
human
cell
cells
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John D. Mountz
Jun Li
Hui-Chen Hsu
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The Uab Research Foundation
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A01K2217/052—Animals comprising random inserted nucleic acids (transgenic) inducing gain of function
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/20—Animal model comprising regulated expression system
    • A01K2217/203—Animal model comprising inducible/conditional expression system, e.g. hormones, tet
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0331—Animal model for proliferative diseases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0368—Animal model for inflammation
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0387—Animal model for diseases of the immune system

Definitions

  • abnormal cells are deleted by programmed cell death referred to as apoptosis.
  • Insufficient apoptosis leads to the development of many diseases including cancer and autoimmune diseases.
  • Enhanced apoptosis by apoptosis-inducing ligands such as agonistic antibodies are considered potential therapeutic strategies for eliminating abnormal cells.
  • DR5 expression is up-regulated in tumor tissues compared to normal tissues. Cancer cells can be therapeutically targeted using, for example, DR5 antibodies that promote apoptosis. Such treatment ideally leads to apoptosis of these target cells without systemic toxicity (Yada et al, Ann. Oncol. 19: 1060-7 (2008)).
  • DR5 is also upregulated on rheumatoid arthritis synovial fibroblasts, which can also be therapeutically targeted using DR5 antibodies (Ichikawa et al., J. Immunol. 171 : 1061- 9 (2003)).
  • chimeric DR5 polypeptides and nucleic acids encoding the chimeric DR5 polypeptides are also provided. Also provided are expression vectors, transgenes, and cells comprising the chimeric DR5 nucleic acids or polypeptides. Transgenic animals with one or more cells comprising the chimeric DR5 nucleic acids are provided. Also provided are methods of screening for an agent or a combination of agents with DR5- mediated apoptosis activity using the provided polypeptides, nucleic acids, vectors, transgenes, cells, and transgenic animals.
  • Figure 1 is a histogram showing the expression levels of endogenous mouse DR5 mRNA in B6 and autoimmune BXD2 mouse tissues.
  • Figure 2 shows increased expression of DR5 on autoreactive B-cells in BXD2 mice.
  • the top, left panel is an image of an agarose gel showing reverse transcriptase polymerase chain reaction (RT-PCR) analysis of DR5 expression in CD4+ T-cells and B-cells in B6 and BXD2 mice.
  • the top, right panel is an image of an agarose gel showing RT-PCR analysis of DR5 and hTNFI (last lane), in mouse synovial fibroblasts (MSF) in B6, arthritis BXD2 and arthritis human TNFI transgenic mice.
  • the bottom, left panel is a histogram showing quantitation of DR5 expression relative to expression of 18S RNA.
  • the bottom, right panel is a histogram showing quantitation of DR5 and hTNFI expression relative to expression of 18S RNA.
  • Figure 3 is a histogram showing that the DR5 antibody, TRA-8, enhances apoptosis of human CD4 T-cells (left) and CD 19 B-cells (right) from human lupus patients.
  • Figure 4 is an alignment of the amino acid sequences of human DR5 (SEQ ID No:5) and mouse DR5 (SEQ ID NO:4). Figure 4 also shows the transmembrane and death domains of DR5 and the location of the junctions of the chimeric DR5 constructs.
  • FIG. 5 is a Western blot showing the expression of various chimeric DR5 polypeptides under control of the cytomegalovirus (CMV) promoter or the mouse DR5 1 kilobase (kb) promoter in transiently transfected HEK293 cells.
  • CMV cytomegalovirus
  • kb mouse DR5 1 kilobase
  • Figure 6 are photomicrographs showing the viability of mouse NIH-3T3 cells expressing various chimeric DR5 polypeptides.
  • Figures 7A and 7B are histograms showing the viability of mouse NIH-3T3 cells ( Figure 7A) or mouse B16 melanoma cells ( Figure 7B) transfected with a construct with a CMV promoter driven human DR5, mouse DR5 or chimeric DR5 with and without the addition of TRA-8. Data also includes results from NIH-3T3 cells transfected with promoterless mouse DR5 and green fluorescent protein under the control of the CMV promoter.
  • Figure 8 is a histogram showing the viability of human HEK293 cells expressing human DR5 or chimeric DR5 under control of the CMV promoter with and without the addition of TRA-8. Data also include the results from HEK293 cells treated with lipofectamine only ("Mock").
  • Figure 9 is a schematic showing various chimeric DR5 constructs with different regulatory elements.
  • Figure 10 is a histogram showing the percentage of cell surface expression of chimeric DR5 in mouse NIH-3T3 cells using various chimeric DR5 constructs.
  • Figure 11 is a histogram showing the effects of various chimeric DR5 constructs with different regulatory elements on the viability of mouse NIH-3T3 cells.
  • Figure 12 is a FACS analysis showing TRA-8 enhanced apoptosis of mouse NIH-3T3 cells expressing chimeric DR5.
  • Figure 13 is a schematic showing /oxed-STOP-chimeric DR5 constructs.
  • Figure 14 are FACS analyses showing Floxed-STOF prevents the expression of DR5 in transfected cells.
  • Figure 15 are photomicrographs showing the Floxed-STOF can be removed by expression of Cre.
  • Figure 16 is a histogram showing Floxed-STOV enhanced the viability of mouse B16 melanoma cells expressing chimeric DR5.
  • Figure 17 is a histogram showing the effects of Floxed-STOV chimeric DR5 on early embryonic development.
  • Figure 18 is a schematic showing Floxed-STOF chimeric DR5 constructs with anti-apoptotic elements.
  • Figure 19 is a graphic map of the 3kb mouse DR5 promoter- /oxe ⁇ i-c-FLIP- STOP-chimeric DR5 construct.
  • Figure 20 is a histogram showing the effects of the 3kb mouse DR5 promoter- /oxe -c-FLIP-STOP-chimeric DR5 on early embryonic development.
  • Figure 21 is an image of a gel showing integration of transgenic DNA in the pre-implantation embryos as determined by nested PCR.
  • Figure 22 shows the genotyping of five (5) 3kb/FLIP/STOP/Chimeric DR5 transgenic mice. Primers were targeted to the human portion of the chimeric DR5 nucleic acid sequence.
  • Figure 23 shows the genotyping of thirteen (13) 3kb/FLIP/STOP/Chimeric DR5 transgenic mice. Primers were targeted to the human portion of the chimeric DR5 nucleic acid sequence.
  • Figure 24 shows the comparison of human, mouse and chimeric DR5.
  • Figure 24A shows amino acid alignment of human and mouse DR5.
  • Figure 24B shows a schematic diagram of human, mouse and chimeric DR5.
  • Figure 24C shows the expression of cell surface human DR5 recognized by anti-human DR5 antibody in mouse NIH-3T3 cells after transiently transfection with the indicated constructs as determined by flow cytometry analysis.
  • Figure 24D shows the confirmation of human DR5 expression and its molecular mass as determined by Western blot analysis.
  • Figure 25 shows the DR5/TRA-8 mediated cell killing and approach to generate human/mouse chimeric DR5 transgenic mice.
  • Figures 25A and 25B show DR5/TRA-8 mediated killing in HEK293 (Fig. 25 A) and NIH-3T3 (Fig. 25B) cells transiently trans fected with human, mouse, and chimeric DR5.
  • Figure 25 C shows a schematic representing the elements of the construct used for generating chimeric DR5 transgenic mice.
  • the human mouse chimeric DR5 is driven by mouse 3kb death receptor putative promoter.
  • a Floxed- FLIP L STOP cassette is introduced between the promoter and the chimeric DR5, which can be removed by crossing the DR5 transgenic mouse strain to Cre-expressing mouse strain.
  • Figure 25D shows real-time PCR analysis of the chimeric DR5 expression on the indicated tissues from the Ubc.CreDR5 double positive transgenic mice after induction of Cre expression by Tamoxifen. Expression is represented as the ratio of copy numbers of chimeric DR5 to that of GAPDH.
  • Figure 26 shows that TRA-8 treatment prevents the development and attenuates the severity of arthritis in Ubc.CreDR5 mice.
  • Arthritis was induced on Ubc.CreDR5 mice via intradermal injection of chick type II collagen emulsified in CFA and an immunofluorescence assay (IFA) was performed on day 0 and day 32 (arrows).
  • IFA immunofluorescence assay
  • TRA-8 treatment was initiated on day 0 and day 30 for early and late treatment group respectively (arrow heads).
  • Figure 26B shows representative H&E and Mac-3 immunohistochemistry (IHC) staining of the knee joint sections from DR5 Tg+ or DR5- wild-type mice treated with TRA-8.
  • IHC immunohistochemistry
  • Figure 26C shows representative histograms showing the percentage of human DR5.
  • Figure 26D shows a histogram demonstrating the average of the indicated population of cells expressing human DR5.
  • Figures 26E and 26F show the Percentage of CDl lbhigh spleen macrophages from Ubc.CreDR5 mice that were untreated, treated with TRA-8 at early time point, or treated with TRA-8 at late time point as determined by FACS analysis.
  • Figure 26G shows a histogram demonstrating the proliferation of LPS-stimulated spleen cells from Ubc.CreDR5 and Ubc.Cre mice as determined by a [ 3 H] -thymidine incorporation assay.
  • Figure 27 shows that TRA-8 treatment ameliorates the collagen II-induced arthritis (CIA) condition in Lyz.CreDR5 mice.
  • CIA was induced on Lyz.CreDR5 mice via intradermal injection of chick type II collagen.
  • TRA-8 treatment was initiated on day 28.
  • Clinical score were followed daily until the mice were sacrificed (Figure 27 A).
  • Figure 27B is a representative image showing cathespin-B activity in joints measured by in vivo imaging using a NIRF probe, ProSense 750.
  • Figures 27C and 27D show FACS analysis of cell surface expression of chimeric DR5 on CDl lb+ spleen cells (Fig. 27C) and CDl lb+ cell (Fig. 27D) depleted by TRA-8.
  • Figure 27E shows histological assessment of representative knee joints from either DR5 Tg+ or control DR5- wild type mice injected with TRA-8.
  • Figure 28 shows that TRA-8 treatment induces apoptosis of macrophages in fresh synovial fragments isolated from human synovial fluid.
  • Figure 28A shows two major component cells of synovial lining layer, macrophages and fibroblasts ion human rheumatoid arthritis (RA) and osteoarthritis (OA), revealed by confocal microscope using anti-CD68 and anti-CD90 antibodies, respectively.
  • Figure 28B shows cell surface expression of DR5 on macrophages in synovial lining layer from RA patients recognized by TRA-8.
  • Figure 28C shows cell death of the synovial fragments before and after TRA-8 treatment analyzed by 7-AAD staining.
  • Figures 28D and 28E show apoptosis induced by TRA-8 on macrophages isolated from RA synovial fragments confirmed by cell morphology (Fig. 28D) and Annexin-V FACS analysis (Fig. 28E). DETAILED DESCRIPTION
  • chimeric DR5 polypeptides e.g., human/mouse chimeric DR5 polypeptides
  • fragments as well as, nucleic acids, vectors, transgenes, and animal models expressing chimeric DR5.
  • These polypeptides, fragments, nucleic acids, vectors, transgenes, and animal models can be used to evaluate the effect of DR5 agonists or antagonists, for example DR5 antibodies.
  • the animal models can also be used to develop models of cancer and inflammatory arthritis and other autoimmune diseases.
  • human/mouse (hu/mo) chimeric DR5 molecules have been produced that express the human DR5 extracellular domain with a mouse transmembrane and mouse intracellular domain of DR5.
  • Transient expression of hu/mo DR5 in both human and mouse cells has been verified and enables TRA-8 mediated apoptosis in transfected cells.
  • the effect of various lengths of mouse DR5 promoter, first intron and 3' UTR on the expression of chimeric DR5 has been determined.
  • /oxe -STOP-Chimeric DR5 constructs have been developed to enable spatial and temporal regulation of DR5 expression and to test the efficacy of the temporal expression of DR5 and the TRA-8 mediated apoptosis in transfected cells.
  • Two new /oxed-STOP-Chimeric DR5 constructs, in which the anti-apoptotic gene, cFLIP or cIAP2 was embedded upstream of the STOP element and between two LoxP sites have been generated. This approach confers the anti-apoptotic protective effect until the /oxe ⁇ i-(cFLIP/cIAP2) STOP is excised and the chimeric DR5 is turned on in the Tg mice. Additionally, transgenic mice containing the /oxe -cFLIP-STOP-Chimeric DR5 transgene have been made.
  • transgenic mice containing the /oxe ⁇ i-STOP hu/mo chimeric DR5 molecule have been made and crossed with tamoxifen inducible ubiquitin Cre (Ubc.Cre) transgenic mice to produce Ubc.Cre DR5 transgenic mice. These mice were induced to develop collagen II-induced arthritis (CIA), and treatment with TRA- 8 inhibited the development of CIA.
  • the /oxe ⁇ i-STOP hu/mo chimeric DR5 transgenic mice were also crossed with Lyz.Cre transgenic mice to produce DR5 Lyz.Cre transgenic mice to achieve restricted expression of the chimeric DR5 molecule in myeloid lineage cells.
  • Treatment with TRA-8 reduced the number of macrophages in the transgenic mice and inhibited the severity of arthritis in mice induced to develop arthritis.
  • chimeric DR5 polypeptide sequences contains (i) the extracellular domain of human DR5 and (ii) the intracellular domain of a non-human DR5.
  • the chimeric DR5 polypeptide sequence further contains the transmembrane domain of a non-human DR5.
  • the non-human DR5 is mouse DR5.
  • the transmembrane domain and the intracellular domain are from the same or different species.
  • the transmembrane domain and intracellular domain are from a mouse DR5.
  • the extracellular domain of human DR5 comprises SEQ ID NO: 15, the
  • transmembrane domain of mouse DR5 comprises SEQ ID NO: 16, and/or the intracellular domain of mouse DR5 comprises SEQ ID NO: 17.
  • the chimeric DR5 polypeptide contains linker sequences between the domains.
  • chimeric DR5 polypeptide sequences comprise SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO:3.
  • sequences for DR5 from various species that are disclosed on Genbank and these sequences and others are herein incorporated by reference in their entireties as well as for individual subsequences contained therein.
  • the amino acid sequence for rat DR5 can be found at Genbank Accession No.
  • ACL51000 and the nucleic acid sequence for rat DR5 can be found at Genbank Accession No. NM 001108873. Further, the amino acid sequences for mouse DR5 and human DR5 are provided in SEQ ID NOs:4 and 5, respectively.
  • sequences or subsequences including, for example, sequences with one or more insertions, deletions or substitutions including conservative and/or non-conservative
  • linker polypeptides can include sequences between the various domains (including, e.g., linker sequences).
  • linker polypeptides include alternating Serine and Glycine stretches exemplified by (GGGGS) 3 (SEQ ID NO: 19) (Freund et al, FEBS Lett. 320:97-100 (1993)), (GSGSGS) n (SEQ ID NO:20), and G(SGGG) 2 SGGT (SEQ ID NO:21); the flexible linker peptide of Trichoderma reesi cellobiohydrolase I (CBHI) (Takkinen et al, Prot. Eng.
  • CBHI Trichoderma reesi cellobiohydrolase I
  • elbow-like peptides such as SAKTTP (SEQ ID NO:22), RADAAP (SEQ ID NO:23), and derivatives thereof (see Le Gall et al, Prot. Eng. Des. Sel. 17:357-366 (2004), which is incorporated herein by reference in its entirety for all derivatives and methods of making same).
  • the chimeric DR5 polypeptide optionally contains sequences encoding a purification tag such as a GST tag or HIS tag.
  • peptide, polypeptide, protein or peptide portion is used broadly herein to mean two or more amino acids linked by a peptide bond. Protein, peptide and polypeptide are also used herein interchangeably to refer to amino acid sequences. It should be recognized that the term polypeptide is not used herein to suggest a particular size or number of amino acids comprising the molecule and that a peptide described herein can contain up to several amino acid residues or more.
  • chimeric peptide, polypeptide, protein or peptide portion refers to a peptide, polypeptide, protein or peptide portion containing two amino acid sequences or subsequences from two different species.
  • amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional or deletional variants. Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence.
  • no more than about from 2 to 6 residues are deleted at any one site within the protein molecule.
  • These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture.
  • Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example Ml 3 primer mutagenesis and PCR mutagenesis.
  • Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues.
  • Deletions or insertions optionally are made in adjacent pairs, i.e., a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct.
  • the mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mR A structure.
  • Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Table 1 and are referred to as conservative substitutions.
  • modification with reference to a polynucleotide or polypeptide refers to a naturally-occurring, synthetic, recombinant, or chemical change or difference to the primary, secondary, or tertiary structure of a polynucleotide or polypeptide, as compared to a reference polynucleotide or polypeptide, respectively (e.g., as compared to a wild-type polynucleotide or polypeptide). Modifications include such changes as, for example, deletions, insertions, or substitutions. Polynucleotides and polypeptides having such
  • Nucleic acids that encode the aforementioned amino acid sequences are also disclosed. These sequences include all degenerate sequences related to a specific protein sequence, i.e., all nucleic acids having a sequence that encodes one particular protein sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protein sequences. Thus, while each particular nucleic acid sequence may not be written out herein, it is understood that each and every sequence is in fact disclosed and described herein through the disclosed protein sequence. Thus, provided are nucleic acid sequences encoding a chimeric DR5 polypeptide. The nucleic acid sequence may comprise untranslated regions at either end or internally.
  • the chimeric DR5 polypeptide contains (i) the extracellular domain of human DR5 and (ii) the intracellular domain of non-human DR5.
  • the chimeric DR5 polypeptide further contains the transmembrane domain of a non-human DR5.
  • the non-human DR5 is mouse DR5.
  • the extracellular domain of human DR5 comprises SEQ ID NO: 15
  • the transmembrane domain of mouse DR5 comprises SEQ ID NO: 16
  • the intracellular domain of mouse DR5 comprises SEQ ID NO: 17.
  • the chimeric DR5 polypeptide includes linker sequences between the domains.
  • the chimeric DR5 polypeptide comprises SEQ ID NO: l, SEQ ID NO:2 or SEQ ID NO:3.
  • the nucleic acid sequence is SEQ ID NO:9.
  • the nucleic acid sequences provided herein are examples of the genus of nucleic acids and are not intended to be limiting.
  • nucleic acid sequences or subsequences including, for example, sequences with one or more insertions, deletions or substitutions. Also contemplated herein are modified versions of the nucleic acid sequences with 85, 90, 95, or 99% identity to the disclosed sequences and subsequences.
  • the chimeric polypeptides can include intervening sequences between the various domains, the intervening sequences encoding, for example, sequences encoding linker sequences or untranslated sequences.
  • the identity can be calculated after aligning the two sequences so that the identity is at its highest level.
  • Another way of calculating identity can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981); by the identity alignment algorithm of Needleman and Wunsch, J. Mol Biol. 48:443 (1970); by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988); by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI); or by inspection.
  • expression vectors such as transgenes, comprising these nucleic acids, wherein the nucleic acids are operably linked to an expression control sequence.
  • an expression vector containing a first nucleic acid sequence encoding a chimeric DR5 polypeptide.
  • the first nucleic acid sequence is SEQ ID NO:9.
  • the expression vector further includes a promoter sequence operably linked to the first nucleic acid sequence.
  • the promoter sequence comprises SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.
  • the expression vector contains a second nucleic acid sequence encoding the first intron of a mouse DR5 polypeptide.
  • the second nucleic acid sequence is located 3 ' to the promoter sequence and 5 ' to the first nucleic acid sequence.
  • the expression vector contains regulatory elements, for example, a Floxed-STOF element.
  • the expression vector comprises SEQ ID NO: 14.
  • the vector further contains sequences that encode anti- apoptotic elements, for example, FLICE inhibitory protein (FLIP) or cellular inhibitor of apoptosis (cIAP2).
  • nucleic acids, constructs, expression vectors and transgenes provided herein further comprise a sequence encoding a reporter molecule.
  • the reporter molecule is green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), chloramphenicol acetyl transferase (CAT), or luciferase.
  • GFP green fluorescent protein
  • YFP yellow fluorescent protein
  • CFP cyan fluorescent protein
  • RFP red fluorescent protein
  • CAT chloramphenicol acetyl transferase
  • cells comprising the provided expression vectors or nucleic acids.
  • the provided nucleic acid sequences are located in the genome of the cell.
  • Such expression vectors and cultured cells can be used to make the disclosed polypeptides.
  • the cells are embryonic stem cells, gametes (e.g., an egg), cancer cells, T-cells, B-cells, macrophages or fibroblasts.
  • Also provided are methods of making a cell expressing a chimeric DR5 polypeptide comprising transforming the cell with a vector or nucleic acid encoding chimeric DR5.
  • the cell is transformed with a vector or nucleic acid sequence comprising SEQ ID NO:9 or SEQ ID NO: 14.
  • the vector is an expression vector or an integration vector.
  • the nucleic acids can be delivered through a number of direct delivery systems such as, electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or by transfer of genetic material in cells or carriers such as cationic liposomes.
  • direct delivery systems such as, electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or by transfer of genetic material in cells or carriers such as cationic liposomes.
  • direct delivery systems such as, electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or by transfer of genetic material in cells or carriers such as cationic liposomes.
  • transgenic animals comprising one or more cells expressing a chimeric DR5 polypeptide.
  • the term animal refers to non-human animals, including, mammals, amphibians and birds. Specifically, examples include sheep, feline, bovines, ovines, pigs, horses, rabbits, guinea pigs, mice, hamsters, rats, non-human primates, and the like.
  • the chimeric DR5 polypeptide comprises (i) the extracellular domain of human DR5 and (ii) the intracellular domain of a non-human DR5.
  • the chimeric DR5 polypeptide further comprises a transmembrane domain of a non-human DR5.
  • the extracellular domain comprises SEQ ID NO: 15, the intracellular domain comprises SEQ ID NO: 17, and/or the
  • transmembrane domain comprises SEQ ID NO: 16.
  • the chimeric DR5 polypeptide comprises SEQ ID NO: l, SEQ ID NO:2 or SEQ ID NO:3.
  • the genome of the cells of the transgenic animal comprises SEQ ID NO: 14.
  • the cells of the transgenic animal expressing the chimeric DR5 polypeptide are cancer cells, T-cells, B cells, macrophages or fibroblasts. Such cells are optionally isolated from the transgenic animal.
  • the animal is a model for cancer, arthritis or autoimmune disease.
  • the arthritis is rheumatoid arthritis.
  • the intracellular domain and the transmembrane domain of the chimeric DR5 polypeptide are optionally from the same species as the transgenic animal. For example, if the transgenic animal is a mouse, the intracellular and transmembrane domains are from mouse DR5. If the transgenic animal is a rat, the intracellular and transmembrane domains are from rat DR5.
  • Animal models for cancer, arthritis, or autoimmune disease can, for example, include genetic knockouts, wherein the function of a gene or multiple genes is lost due to targeting the gene or multiple genes, and genetic knock-ins, wherein a gene or multiple genes of interest are targeted specifically or non-specifically into the genome of the animal to express the gene or genes of interest.
  • Examples of animal models of cancer that contain a genetic knockout or knock-in include, for example, knockouts or knock-ins of cell cycle regulators (e.g., cyclin dependent kinases, cyclins, and mitogen activated protein kinases); components of signaling pathways (e.g., growth factors, growth factor receptors, cytokines, chemokines, and adapter proteins); and regulators of gene expression (e.g., transcription factors, splicing factors, translation factors, post-transcriptional regulators (e.g., miRNAs), and post-translational regulators (e.g., kinases, phosphatases, and acetyl-transferases)).
  • cell cycle regulators e.g., cyclin dependent kinases, cyclins, and mitogen activated protein kinases
  • components of signaling pathways e.g., growth factors, growth factor receptors, cytokines, chemokines, and adapter proteins
  • Animal models of arthritis can, for example include, knockouts or knock-ins of genes involved in regulating inflammation (e.g., cytokines, chemokines, tumor necrosis factor alpha (TNFa), transforming growth factor beta (TGF ), and interferon gamma (IFNy)).
  • Animal models of autoimmune diseases can, for example, include knockouts or knock-ins of genes involved in immunity (e.g., immunoglobulins, chemokine and chemokine receptors, tumor necrosis factor receptors, co-stimulatory molecules, and cytokines and cytokine receptors).
  • the transgenic animal comprising one or more cells expressing a chimeric DR5 polypeptide can be crossed with an animal model of cancer, arthritis, or an autoimmune disease.
  • a progeny animal resulting from a cross between a transgenic animal comprising one or more cells expressing a chimeric DR5 polypeptide and a transgenic animal model of cancer, arthritis, or an autoimmune disease is the DBA/l-hDR5 human/mouse chimeric Tg mouse or the B6-hu/mo chimeric DR5 Tg mouse, which are described in Example 10 below.
  • the transgenic animal is FVB.Cg-Tg(MMTV-vHaras)SHlLed/J-hDR5 human/mouse chimeric Tg mouse, which is described in Example 11 below.
  • Animal models of cancer can, for example, be created by directly injecting tumor cells into the animal.
  • the tumor cells can be injected subcutaneously, intraperitoneally, or intravenously.
  • Tumor cells can be injected in a targeted manner to a specific organ or location within the animal, or, alternatively, tumor cells can be injected at random, in a non-targeted manner.
  • the transgenic animal comprising one or more cells expressing a chimeric DR5 polypeptide is injected directly with tumor cells to create an animal model of cancer.
  • Animal models of cancer, arthritis, and autoimmune disease can, for example, be created by exogenously administering an agent or agents that cause cancer, arthritis, or an autoimmune disease.
  • an agent or agents that causes cancer, arthritis, or an autoimmune disease can include chemical compounds, radiation, viral infection, or bacterial infection. See Calbrese and Blain, Toxicological Sciences 50: 169-85 (1999); Van Der Berg, Kelley's Textbook of Rheumatology, 8 th ed., W.B. Saunders Company, Chapter 25 (2008); and Lusdewig et al, Experimental Physiology 85(6):653-9 (2000).
  • the transgenic animal comprising one or more cells expressing a chimeric DR5 polypeptide is administered an agent or agents that cause cancer, arthritis, or an autoimmune disease.
  • the animal model is a DBA/1, B6 or FVB.Cg-Tg(MMTV-vHaras)SHlLed/J mouse. These animals are described in Examples 10 and 11 below.
  • transgenic animal refers to any animal, e.g., a mammal, bird, or an amphibian, in which one or more of the cells of the animal contain a
  • heterologous nucleic acid can be introduced using known transgenic techniques.
  • the nucleic acid is introduced into the cell, directly or indirectly.
  • the nucleic acid can be introduced into a precursor of the cell or by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • the nucleic acid may be integrated within a chromosome, or it may be an extrachromosomally replicating DNA.
  • transgene is used herein to describe genetic material that has been or is about to be artificially inserted into the genome of a mammalian cell, particularly a mammalian cell of a living animal.
  • the transgene is used to transform a cell.
  • Vectors for stable integration include, but are not limited to, plasmids, retroviruses and other animal viruses and YACS.
  • the transgenic animal can be created by introducing a transgene construct into, for example, an embryonic stem cell, an unfertilized egg, a fertilized egg, a spermatozoon or a germinal cell containing a primordial germinal cell thereof, preferably in the embryogenic stage in the development of a non-human mammal (more preferably in the single-cell or fertilized cell stage and generally before the 8-cell phase).
  • the transgene can be introduced by known means, including, for example, the calcium phosphate method, the electric pulse method, the lipofection method, the agglutination method, the microinjection method, the particle gun method, the DEAE-dextran method and other such method.
  • the transgene is introduced into a somatic cell, a living organ, a tissue cell or other cell by gene transformation methods.
  • Cells including the transgene may be fused with the above-described germinal cell by a commonly known cell fusion method to create a transgenic animal.
  • an ES cell line may be employed, or embryonic cells may be obtained freshly from a host, e.g., mouse, rat, guinea pig, and the like. Such cells are grown on an appropriate fibroblast- feeder layer or grown in the presence of appropriate growth factors, such as leukemia inhibiting factor (LIF).
  • LIF leukemia inhibiting factor
  • ES cells When ES cells have been transformed, they may be used to produce transgenic animals. After transformation, the cells are plated onto a feeder layer in an appropriate medium. Cells containing the construct may be detected by employing a selective medium. After sufficient time for colonies to grow, they are picked and analyzed for the occurrence of homologous recombination or integration of the construct.
  • Blastocysts are obtained from 4 to 6 week old superovulated females.
  • the ES cells are trypsinized, and the modified cells are injected into the blastocoel of the blastocyst. After injection, the blastocysts are returned to each uterine horn of pseudopregnant females. Females are then allowed to go to term and the resulting litters screened for mutant cells having the construct.
  • chimeric progeny can be readily detected.
  • the chimeric animals are screened for the presence of the modified gene, and males and females having the modification are mated to produce homozygous progeny transgenic animals.
  • the method comprises the steps of providing a transgenic animal containing one or more cells expressing a chimeric DR5 polypeptide; administering to the animal the agent or agents to be tested; and determining whether the agent or agents cause apoptosis of the cells expressing the chimeric DR5 polypeptide.
  • Apoptosis indicates the agent or agents have DR5- mediated apoptosis activity.
  • Methods of determining apoptosis are known in the art and are also described in the Examples below. For example, apoptosis can be determined using the ATPLiteTM assay or annexin-V staining.
  • the method comprises the steps of providing cells expressing a chimeric DR5 polypeptide;
  • the agent is an antibody or small molecule.
  • any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods of using the disclosed compositions or animals. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.
  • Example 1 Expression of Human and Mouse DR5 in Different Tissue
  • hDR5 transgenic mice Analysis of the hDR5 mRNA expression in the previously generated hDR5 transgenic (Tg) mice indicated that the highest levels of Tg could be detected in liver, lung, spleen, heart, kidney, and peripheral blood mononuclear cells. The distribution pattern mirrors that of the endogenous human DR5. However, the expression of the Tg at the protein levels on the cell surface was low unless the Tg mice were immunized with sheep red blood cells to stimulate an immune response in vivo. Live imaging biodistribution analysis of the whole mouse body further showed that there were modestly increased levels of hDR5 detected in the liver and kidney of hDR5 Tg vs control wild-type mice.
  • DR5 protein In mouse, the DR5 protein is also widely expressed and its transcript was previously reported to be expressed in the heart, lung, and kidney. qRT-PCR analysis was carried out to determine the expression of mouse DR5 (mDR5) at the mRNA levels from different tissues in the B6 and autoimmune BXD2 mice (Fig. 1). As in human, DR5 mRNA is present in the lung and intestines of normal and autoimmune BXD2 mice.
  • DR5 is unregulated in T and B cells from autoimmune BXD2 mice relative to B6 mice
  • transcriptional expression of mouse DR5 was compared in CD4 T cells from B6 and BXD2 mice, and purified B cells from B6 and BXD2 mice.
  • DR5 is increased expression of DR5 on autoreactive B cells compared to BXD2 mice (Fig 2).
  • BXD2 mice exhibit increased generation of autoreactive B cells with increased autoantibody production.
  • GCs germinal centers
  • AID activation-induced cytidine deaminase
  • Mouse synovial fibroblasts exhibit abnormal features including increased proliferation, production of stromelysin, collagenase, IL-6 and RANKL that lead to erosion and inflammation.
  • Two models of arthritis have been analyzed, the BXD2, which develops a spontaneous erosive arthritis with synovial hyperplasia and erosion of cartilage and bone (Wu et al., Arthritis Rheum. 52:3257-68 (2005)).
  • the human TNF-a Tg mouse develops synovial hyperplasia and bone and cartilage erosion (Keffer et al, EMBO J. 10:4025-31 (1991)).
  • Primary cell lines were generated from mouse synovial fibroblasts from B6, BXD2, or human TNF-a transgenic mouse.
  • DR5 antibody-mediated apoptosis in both CD4 and CD 19 B cells obtained from PBMCs of human lupus patients was analyzed.
  • PBMCs were isolated and cultured either in the presence of absence of TRA-8 (5 ⁇ g/ml) at 37°C overnight.
  • Cells were harvested and the percentage of CD4 and CD 19 cells undergoing apoptosis was determined by FACS analysis. The results are shown in Figure 3.
  • TRAIL-R2 TNF related apoptosis-inducing ligand
  • the molecular structure of mouse and human DR5 consists of an extracellular domain, a transmembrane domain, and an intracellular domain which contains a death domain.
  • the extracellular domain of human is longer than mouse (210 amino acids versus 177 amino acids), and there is 33% amino acid identity between the human and mouse forms.
  • the intracellular domain of human is also longer than mouse (202 amino acids compared to 175 amino acids), and there is 35% amino acid identity between the two (Fig. 4).
  • Chimeric protein 1 (SEQ ID NO: l) produced the precise joining of the human extracellular domain with the first amino acid of the mouse transmembrane domain. See Figure 4, junction 1. Since it was not known if amino acids around the transmembrane domain were essential for proper anchoring and orientation of the molecule, and if these are species-specific, chimeric 2 (SEQ ID NO:2) was produced such that it included 5 additional extracellular domain amino acids of the mouse, which was fused to the human extracellular domain. See Figure 4, junction 2.
  • Example 3 Expression of Chimeric DR5 Proteins in HEK293 Cells.
  • Example 2 The three constructs described in Example 2 were tested for expression and function by cloning them into a pCMV/SPORT6 vector under the regulation of a strong CMV promoter. Three additional constructs were also generated by replacing the CMV promoter with the mouse DR5 1 kilobase (kb) promoter. SEQ ID NO: 6 shows the sequence of the mouse DR5 1 kb promoter. The expression of these chimeric constructs at the protein levels was first tested in transiently transfected (Lipofectamine 2000) human HEK293 cells by Western blot. Chimeric construct 3 was not detected, probably due to disruption of the human extracellular domain, which is critical for the binding of anti-human DR5 antibody. Both human DR5 and chimeric construct 1 and 2 produced chimeric proteins that are of their predicted sizes (47.9, 45.5 and 45.4 kDa for human DR5, chimeric 1 and 2 respectively) (Fig 5).
  • the mouse DR5 1 kb promoter exhibited a low activity in human HEK293 cells (lanes 6 and 7, Fig 5).
  • the CMV and the 1 kb mouse DR5 promoter exhibit equivalent ability to drive expression of DR5 in mouse cell lines.
  • apoptotic function of the different chimeric DR5 proteins was tested in transiently transfected (Lipofectamine 2000) NIH 3T3 cells.
  • Mouse NIH 3T3 cells were chosen for these experiments since all chimeric proteins expressed the mouse death domain.
  • signaling with anti-human DR5 was not carried out since it was known that high expression of the DR5 death domain results in spontaneous apoptosis that does not require TRAIL or anti-DR5 signaling.
  • the transfection efficiencies were determined by parallel transfection of a CMV-GFP vector in the NIH 3T3 cells indicating approximately 35-40% transfection efficiency.
  • chimeric DR5-3 did not induce significant apoptosis. Therefore, chimeric 1 DR5 (SEQ ID NO: l), which consists of the full- length human extracellular domain fused precisely in frame and to the mouse transmembrane domain, was used for further experiments.
  • mice NIH3T3 cells and mouse B16 melanoma cells after transfection with full-length human DR5, full-length mouse DR5, or the chimeric human/mouse DR5, was tested (Fig 7).
  • Cell viability was determined using the ATPLiteTM assay, which was carried out in a 96-well plate on a Packard TopCount (GMI, Inc., Ramsey, Minnesota). Viability was determined with and without the addition of the DR5 antibody, TRA-8. In both cell lines, transfection of the full- length human DR5 directed by CMV promoter did not result in significant spontaneous apoptosis.
  • apoptosis was not enhanced significantly by addition of TRA-8, even though this TRA-8 could bind to the extracellular domain of this full- length human DR5.
  • expression of the full-length mouse DR5 induced relatively lower levels of apoptosis.
  • TRA-8 is not capable of interacting with the mouse extracellular domain and thus does not enhance the apoptosis.
  • HEK293 human cells were trans fected with the full-length human DR5 and the chimeric human/mouse DR5 driven by the CMV promoter. These cells enabled not only a direct comparison of the spontaneous apoptosis of the human and mouse death domains in a human cell line, but also a comparison of TRA-8 mediated apoptotic effect since both molecules include the human extracellular domain. There was equivalent viability of both the full-length human and the human mouse chimeric construct in the 293 cell lines (Fig. 8). Also, addition of TRA-8 greatly decreased the viability of both the full-length human DR5 as well as the human/mouse chimeric DR5. This result shows that the mouse death domain does have an equivalent functional effect as a human death domain in a human cell line although the human death domain does not function in mouse cell lines (Fig. 7).
  • Example 5 Effects of Various Lengths of the Mouse DR5 Promoter, First Intron and 3' UTR on Expression of Mouse DR5.
  • the regulatory regions of the human DR5 are not completely known, but both the promoter and the first intron are thought to contain critical regulatory elements (Yoshida et al., FEBS Lett. 507:381-5 (2001)). Important regulatory elements include 2 Spl sites in the 1 kb human promoter and several NF- ⁇ and p53 binding sites in the first intron of human DR5. However, there is little homology between the mouse and human promoter or the first intron. Specifically, the first intron of the human is large (25.6 kB), whereas the first intron in the mouse is much smaller (2.9 kB). The 3" UTR of human and mouse DR5 are thought to contain important regulatory elements including micro-RNA regulatory sites.
  • first intron and 3 ' untranslated region (UTR) of mouse DR5 on DR5 expression constructs were produced that contained either a 1 kb promoter, a 3 kb promoter, a 2.9 kb first intron, and the 3 " untranslated region (Fig. 9). These constructs were tested for both expression and for the cell killing effect using FACS analysis and ATPLiteTM assay. For cell surface expression studies, the anti-human DR5 antibody (clone DJR2-4, Biolegend) was used.
  • NIH 3T3 cells were transfected with the chimeric DR5 driven by CMV, the 1 kb promoter-DR5, the 3 kb promoter-DR5, and the 3 kb promoter plus the first intron with and without the 3' UTR. Twenty-four to 48 hours after transfection, surface expression of the human/mouse chimeric DR5 was determined by FACS analysis.
  • TRA-8 was added 24 hours after the transfection. At 48 hours, cell viability was estimated by the ATPLiteTM assay. The highest spontaneous cell death was seen with chimeric DR5 driven by the CMV promoter and it was further enhanced by the addition of TRA-8 (Fig. 11). Both the 1 kb and 3 kb DR5 promoter resulted in a lower degree of spontaneous cell death, but cell death was greatly enhanced by TRA-8.
  • Spontaneous cell death by the 3 kb promoter/2.9 kb first intron/DR5 or the 3 kb promoter/2.9 kb first intron/DR5/3' UTR was low, and this was not greatly enhanced by the addition of TRA-8.
  • Apoptosis in chimeric DR5 transfected mouse 3T3 cells was also analyzed by using 7-amino-actinomycin d (7-AAD) staining and FACS analysis (Fig. 12). The results indicated that spontaneous cell death was relatively low with the 3 kb promoter transfected 3T3 cells but was significantly enhanced by addition of TRA-8.
  • Example 6 Tissue-Specific and Spatial/Temporal Regulation of Human/Mouse Chimeric DR5 Transgene.
  • a Floxed-STOF element was engineered between the promoter region and the chimeric DR5 (Fig 13).
  • HEK293 cells were used since they could be transfected and transfected efficiently, and, in this case, GFP was used to estimate transcription level. Approximately 20% of the cells were GFP positive (GFP+) after transfection with the CMV-IRES-GFP. In the CMV-STOP-IRES-GFP transfected cells, there were no GFP+ cells (Fig 15). However, cells transfected with the CMV-STOP-IRES-GFP and also transfected with an Ad-Cre (48-72 hours) exhibited GFP+ cells. These results showed that the Floxed STOP can accentuate expression of downstream genes and that the Ad-Cre can reverse it.
  • mouse B16 melanoma cells were transfected with either the 3 kb chimeric-DR5, the 3 kb/STOP/chimeric DR5 or control CMV/GFP.
  • Cell viability was determined using the ATPLiteTM assay.
  • Cells transfected with a chimeric DR5 exhibited low viability (Fig 16).
  • the viability of cells was further suppressed by addition of TRA-8 (Fig 16).
  • cell viability was increased in cells transfected with the 3 kb promoter/' STOP/chimeric DR5.
  • Example 7 Analysis of DR5 Transgenic Embryos.
  • DR5 is a pro-apoptotic molecule.
  • the transgene is not embryonic lethal. Therefore, the effect of different DR5 transgenes after injection into single cell embryos (zygotes) was determined. This was analyzed on day 3 after zygote microinjections.
  • FLIP FLICE inhibitory protein
  • v-FLIP gammaherpesviruses and molluscipoxviruses
  • Example 8 Development of Mice Expressing Chimeric DR5.
  • mice were produced using a human/mouse chimeric DR5 in which the Floxed stop had been placed upstream of the ATG start site of the human/mouse chimeric molecule.
  • this Floxed STOP element produced in human/mouse DR5 chimeric constructs is driven by the 3 kb promoter, a 3 kb promoter and first intron, or the 3 kb promoter/first intron and the 3' UTR of mouse.
  • Mice were produced with the FLIP L gene embedded into the Floxed sequence. This helped ensure survival during embryonic development of the mouse. This FLIP L and the STOP sequence was removed by targeted expression of Cre.
  • Figure 22 shows the genotyping of five (5) 3 kb/FLIP/STOP/Chimeric DR5 mice.
  • Figure 23 shows the genotyping of thirteen (13) 3 kb/FLIP/STOP/Chimeric DR5 mice. Primers were targeted to the human portion of the chimeric DR5 polypeptide.
  • LAP lymphoid apoptosis
  • BIR Baculoviral IAP repeat
  • One of the important functions of IAPs is inhibition of apoptosis by binding to caspases and other BIR-interacting proteins.
  • This new construct is used for development of other chimeric DR5 Tg lines of mice to enable study of the effects of DR5 antibodies in mice that develop autoimmune disease due to a decrease in the threshold of autoimmune disease induction by the expression of IAP.
  • Example 10 A Mouse Model of Arthritis that Expresses Chimeric DR5.
  • the DBA/1 collagen mouse model of arthritis is used.
  • the DBA/1 (H2q) and B6 (H2b) mouse is used for studies of the effects of IL-17 on type II collagen (CII)- induced arthritis (CIA).
  • B6 mice are relatively resistant to CIA compared to DBA/1 mice.
  • cyropreserved H2q congenic B6 mice are available from the Jackson Laboratory (Stock Number: 000945). These mice are first acquired and used to backcross with the hu/mo chimeric DR5 Tg B6 mice (H2q hu/mo chimeric DR5 Tg mice) to induce CIA.
  • a second backcross strain is also produced in which the DBA/1 mouse model is crossed back to the DR5 Tg mice using a speed-congenic approach with 50 markers to select for the DBA/1 genotype.
  • the DR5 transgene and the DBA/1 MHC are selected.
  • the DBA/1 H2q MHC is the most important locus in conferring susceptibility to arthritis in the DBA/1 mouse.
  • the H2q-hu/mo chimeric DR5 Tg mouse or the DBA/1 -hDR5 human/mouse chimeric Tg mouse are used to induce CIA.
  • CII arthritis is induced by injection of CII and complete Freund's adjuvant (CFA) in 3 to 4 wk-old mice following by a second injection of CII and CFA 3 weeks later. Arthritis is observed 10 to 14 days after the boost.
  • CFA complete Freund's adjuvant
  • Ad-IL- 17 Ad-IL- 17 before the priming dose of CII and CFA to accelerate the development of arthritis in strains of mice that are not normally susceptible to arthritis (including the C57BL/6 mouse that has the resistant H2b MHC allele).
  • Example 11 Chimeric Mouse Model for Optimization of DR5 Antibody for Cancer Therapy.
  • the DR5 chimeric Tg mouse is also used in combination with mouse tumor models. Studies are carried out by crossing the DR5 Chimeric 1 Tg mouse with an appropriate mouse model of cancer from Jackson Laboratories. The studies enable study of the effect of DR5 agonist agents, for example, an anti-human DR5 antibody, in mouse models of cancer.
  • mice Numerous mouse models of cancer are available commercially, from the Jackson Laboratories, and non-commercially. These mice develop cancer due to defects in regulation of cell growth or growth factors, proliferation, DNA repair, or apoptosis. Mice are available with preferential development of adenomas of the lung, intestine, gonads or multiple tissues. Models of liver cancer have been developed. There are a large number of mouse models of breast cancer due to altered expression of genes under the control of the mouse mammary tumor virus (MMTV) promoter. The selected mouse model of cancer is crossed with the hu/mo DR5 chimeric Tg mouse for studies of TRA-8 in treatment of established local or metastatic cancer.
  • MMTV mouse mammary tumor virus
  • the FVB.Cg-Tg(MMTV-vHaras)SHlLed/J mouse (“MMTV-HaRas mouse”) is a mouse model of breast cancer maintained and available from the Jackson Laboratory.
  • These transgenic mice express the Harvey RAS (human) gene under the direction of the mouse mammary tumor virus promoter. The majority of expression of the transgene is detected in the mammary tissue and salivary glands. Both male and female transgenic mice develop malignant lymphoid tissue and mammary and salivary gland tumors as early as 5 weeks of age. Half of the female transgenic animals develop tumors by 6 months of age.
  • MMTV-HaRas oncogene develops an oncogenic phenotype on a mixed B6/FVB mouse background.
  • the MMTV-HaRas mouse is crossed with the chimeric DR5 Tg mouse to produce double Tg mice for studies of the effect of DR5 antibodies on mouse cancer development in vivo.
  • Example 12 Identification of anti-DR5 antibody as an anti-macrophage reagent for rheumatoid arthritis in a chimeric human/mouse DR5 mouse model.
  • Mouse NIH3T3 and human HEK293 cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA) and cultured in DMEM (Invitrogen; Carlsbad, CA) supplemented with 10% FBS, 100 units/ml penicillin, 100 ⁇ g/ml streptomycin (Invitrogen), and 2mM glutamine (Invitrogen) at 37°C, 5% C0 2 in a humidified incubator.
  • Single cell suspensions from spleen and inguinal lymph nodes were prepared as described before and cultured in RPMI-1640 (Invitrogen) supplemented with 10% FBS, lOmM HEPES and 0.1% 2-mercaptoethanol (Invitrogen).
  • Human and mouse DR5 cDNAs were obtained from Open Biosystems (Huntsville, AL). The extracellular domain of human DR5 was amplified by PCR using primer A 5'-actgtcgacgccccaagtcagcctggacacata-3' (SEQ ID NO:24) and primer B 5'-tcctatccagaggcctagcttatgccaagaacagggagaggcaggagtccctgg-3' (SEQ ID NO:25).
  • mouse DR5 transmembrane and intracellular domains of mouse DR5 were amplified by PCR using primer C 5'-ccagggactcctgcctctccctgttcttggcataagctaggcctct ggatagga-3' (SEQ ID NO:26) and primer D 5'-gatgcggccgctcaaacgcactgagatcctcctgg- 3' (SEQ ID NO:27). There are 49 base pairs overlapping between these two PCR products. The fused chimeric DR5 was then generated by PCR using the mixture of above 2 products as the template and primers A and D.
  • the purified PCR product was then digested by Sail and Notl and this SaR-Notl chimeric DR5 fragment was used to replace the human DR5 on the pCMV-SPORT6 vector.
  • a 3 kb putative mouse DR5 promoter was cloned from mouse BAC RP24-355K8. This regulatory genomic DNA sequence was then subcloned upstream of the chimeric DR5. All constructs were sequenced to confirm their identities.
  • Transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Forty-eight hours after transfection, cells were lysed and a 50 ⁇ g sample of the protein lysate was analyzed by Western blot as described. Cell viability was determined by an ATP luminescence assay kit
  • DR5 Tg mice Establishment of the hu/mo chimeric DR5 Tg mouse.
  • the DNA used for generating DR5 Tg mice was based on the 3 kb mouse promoter/Chimeric DR5 construct described above.
  • a Floxed-STOF cassette (Addgene; Cambridge, MA) was introduced in between the promoter and chimeric DR5. All constructs were sequenced to confirm their identities.
  • a purified 8.3 kb Drdl-Drdl fragment was then used to generate the Tg mice by single cell embryo microinjection. Genotyping was performed by using the primers that target the human DR5 extracellular domain.
  • chimeric DR5 Ubc Cre double Tg and chimeric DR5 Lyz.Cre double Tg mice were bred with two different Cre-expressing mice: (1) the UBC-cre/ESRl)lEjb/J (The Jackson Laboratory; Bar Harbor, ME), which has strong tamoxifen-inducible cre activity in all reported tissues, and (2) Lysozyme M Cre (B6. (The Jackson Laboratory), which expresses Cre in myeloid cells.
  • RNA concentration was assessed by Quantitative reverse transcription PCR (qRT-PCR).
  • qRT-PCR Quantitative reverse transcription PCR
  • RNA quality was also verified by Bio-Rad Experion system (Bio Rad Laboratories, Inc.; Hercules, CA). Two micrograms of total RNA was used to synthesize the first- strand cDNA by using random hexamer primers and RevertAidTMM-MuLV Reverse Transcriptase (Fermentas Life Science; Glen Burnie, MD). QRT-PCR was performed on an IQ5 multicolor real-time PCR detection system).
  • the PCR reaction was done in 25 ⁇ 1 solution containing 12.5 ⁇ 1 SYBR Green Supermix (Bio-Rad), ⁇ ⁇ cDNA and 400nM of each primer, with the following setting: 95°C for 3 minutes followed by 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds. Quantification of gene expression was analyzed using the IQ5 Optical System Software version 2.0.
  • CIA collagen II-induced arthritis
  • Mice were immunized by intradermal administration of chicken type II collagen (Chondrex Inc.; Redman, WA) emulsified in Complete Freund's Adjuvant (CFA) followed by a boost of CII in Incomplete Freund's Adjuvant (IFA) on day 30 after the primary injection as previously described.
  • CFA Complete Freund's Adjuvant
  • IFA Incomplete Freund's Adjuvant
  • an adenovirus expressing mouse IL-17 (2xl0 9 pfu / mouse) was administered to all mice 2 days prior to the CII primary immunization.
  • the severity of arthritis was measured daily based on the 0-3 scale system as follows: 0, normal; 1 , erythema and mild swelling confined to the ankle, tarsal or one digit; 2, erythema and moderate swelling extending from the ankle to tarsal, metatarsal or two or more digits; 3, erythema and severe swelling encompassing the ankle, foot and digits.
  • the maximal clinical score for each mouse was 12.
  • NIRF near- infrared fluorescence
  • RA Human rheumatoid arthritis
  • Synovial fragments were isolated by low speed centrifugation and mononuclear cells were isolated by using Ficoll-paque solution (Amersham Biosciences; Piscataway, NJ) following the manufacturer's manual. Fresh synovial fragments were treated with and without TRA-8 (2 ⁇ g/ml for 16 hours) followed by staining with 7-AAD (Enzo Life Science; Madison Meeting, PA). Synovial fragments fixed with 4%
  • Cellular antagonist reagents such as anti-CD20 and anti-CD64 that eliminate autoimmune pathogenic cells often act via an antibody- or complement-mediated pathway to induce cytolysis.
  • successful anti-DR5 -mediated apoptosis to eliminate pathogenic cells in a mouse model of RA requires not only expression of human DR5 on the surface of cells but also proper docking and activation of mouse death domain adaptor molecules to activate the apoptosis cascade.
  • a chimeric human/mouse DR5 molecule was generated that can confer TRA-8 mediated DR5 apoptosis.
  • apoptotic susceptibility to TRA-8 was next determined in human HEK293 cells and mouse NIH3T3 cells expressing the human DR5, the mouse DR5, and the chimeric DR5 driven by the CMV promoter.
  • HEK293 cells that were transfected with human and chimeric DR5 exhibited approximately 60% reduction of viability by TRA-8 treatment.
  • HEK293 cells transfected with mouse DR5 did not exhibit a significant killing effect by TRA-8 (Fig. 25 A). These suggested that extracellular and death domain of human and mouse DR5 function properly in human and mouse cells, but the mouse death domain was silent in human cells.
  • the 3 kb promoter plays the major regulation role after the Floxed-STOV was removed. After induction of Cre by TMX, various levels of chimeric DR5 Tg transcript can be detected in the examined tissues (Fig. 25D) and its expression pattern mimics that of the endogenous mouse MK.
  • TRA-8 treatment reduced the CD 1 lb high activated macrophages by -50-60% (Figs. 26E and 26F).
  • Cathepsin B is predominately produced by macrophages associated with the severity of arthritis.
  • CIA was induced in DR5 Tg and control mice treated with and without TRA-8.
  • increased activated cathepsin B was found in the Lyz.Cre single Tg mouse treated with and without TRA-8 and Lyz.Cre DR5 Tg mice treated with TRA-8.
  • the increased protease activity was apparent in the ankle, tarsal joints and digits.
  • TRA-8 can deplete macrophages in human RA synovium
  • fresh synovial fluid (SF) samples were obtained from 15 RA patients attending an outpatient rheumatology clinic. Synovial fragments predominantly from the synovial lining layers were isolated from the SF. Immunofluoresence staining using anti-CD90 and anti-CD68 was carried out for identification of fibroblasts and macrophages, respectively. The results indicated that RA synovial lining layer consists of 75% macrophages and 25% fibroblasts compared to 30% and 70% in OA synovium respectively (Fig. 28A). TRA-8 staining on the fixed synovial fragment indicated the high expression of DR5 on the surface of synovial tissue fragment macrophages (Fig.

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Abstract

Cette invention concerne des polypeptides DR5 chimères et des acides nucléiques codant les polypeptides DR5 chimères. L'invention concerne des cellules et des vecteurs d'expression comprenant les acides nucléiques ou les polypeptides DR5 chimères. L'invention concerne des animaux transgéniques comprenant une ou plusieurs cellules exprimant les polypeptides DR5 chimères. L'invention concerne des méthodes de criblage d'un agent ou d'une association d'agents ayant une activité apoptotique dans laquelle intervient le DR5.
PCT/US2010/048237 2009-09-09 2010-09-09 Polypeptides dr5 chimères et leurs utilisations WO2011031835A1 (fr)

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US9127070B2 (en) 2010-10-29 2015-09-08 Daiichi Sankyo Company, Limited Anti-DR5 antibodies, polynucleotides and methods
EP3409110A1 (fr) * 2013-11-19 2018-12-05 Regeneron Pharmaceuticals, Inc. Animaux non humains ayant un gène de facteur activant les cellules b humanisé
US11370818B2 (en) 2014-01-31 2022-06-28 Boehringer Ingelheim International Gmbh Anti-BAFF antibodies
CN116970562A (zh) * 2023-09-22 2023-10-31 北京翊博生物集团有限公司 一种抗原特异性t细胞的制备方法及其在免疫治疗中的应用
WO2024130148A3 (fr) * 2022-12-16 2024-08-02 Denali Therapeutics Inc. Modèles transgéniques de cd98 à chaîne lourde

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9127070B2 (en) 2010-10-29 2015-09-08 Daiichi Sankyo Company, Limited Anti-DR5 antibodies, polynucleotides and methods
US9856321B2 (en) 2010-10-29 2018-01-02 Daiichi Sankyo Company, Limited Anti-DR5 antibodies, polynucleotides and methods
EP3409110A1 (fr) * 2013-11-19 2018-12-05 Regeneron Pharmaceuticals, Inc. Animaux non humains ayant un gène de facteur activant les cellules b humanisé
US10219494B2 (en) 2013-11-19 2019-03-05 Regeneron Pharmaceuticals, Inc. Non-human animals having a humanized B-cell activating factor gene
US11000020B2 (en) 2013-11-19 2021-05-11 Regeneron Pharmaceuticals, Inc. Non-human animals having a humanized B-cell activating factor gene
US12058987B2 (en) 2013-11-19 2024-08-13 Regeneron Pharmaceuticals, Inc. Mouse having a humanized B-cell activating factor gene
US11370818B2 (en) 2014-01-31 2022-06-28 Boehringer Ingelheim International Gmbh Anti-BAFF antibodies
WO2024130148A3 (fr) * 2022-12-16 2024-08-02 Denali Therapeutics Inc. Modèles transgéniques de cd98 à chaîne lourde
CN116970562A (zh) * 2023-09-22 2023-10-31 北京翊博生物集团有限公司 一种抗原特异性t细胞的制备方法及其在免疫治疗中的应用
CN116970562B (zh) * 2023-09-22 2024-01-02 北京翊博生物集团有限公司 一种抗原特异性t细胞的制备方法及其在免疫治疗中的应用

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