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Definitions

• the present invention relates to a novel member of the TNF-Ligand superfamily. More specifically, isolated nucleic acid molecules are provided encoding a human Apoptosis Inducing Molecule II (AIM II). AIM II polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of AIM II activity. Also provided are therapeutic methods for treating lymphadenopathy, aberrant bone development, autoimmune and other immune system diseases, graft versus host disease, rheumatoid arthritis, osteoarthritis and to inhibit neoplasia, such as tumor cell growth.
• TNF- and ⁇ are related members of a broad class of polypeptide mediators, which includes the interferons, interleukins and growth factors, collectively called cytokines (Beutler, B. and Cerami, A., Armu. Ret. Immunol. 7:625-655 (1989)).
• Tumor necrosis factor (TNF- ⁇ and TNF- ⁇ ) was originally discovered as a result of its anti-tumor activity, however, now it is recognized as a pleiotropic cytokine capable of numerous biological activities including apoptosis of some transformed cell lines, mediation of cell activation and proliferation and also as playing important roles in immune regulation and inflammation.
• TNF-ligand superfamily known members ofthe TNF-ligand superfamily include TNF- ⁇ , TNF- ⁇ (lymphotoxin- ⁇ ), LT- ⁇ , OX40L, Fas ligand, CD30L, CD27L, CD40L and
• the ligands of the TNF ligand superfamily are acidic, TNF-like molecules with approximately 20% sequence homology in the extracellular domains (range, 12%-36%) and exist mainly as membrane-bound forms with the biologically active form being a trime ⁇ c/multimeric complex. Soluble forms ofthe TNF ligand superfamily have only been identified so far for TNF. LT ⁇ , and Fas ligand (for a general review, see Gruss, H. and Dower, S.K., Blood, #5(72,) .3378-3404 (1995)), which is hereby incorporated by reference in its entirety
• Fas/CD95 a cell surface antigen that mediates apoptosis and is involved in clonal deletion of T-cells. Fas is expressed in activated T-cells, B -cells, neutrophils and in thymus, liver, heart and lung and ovary in adult mice (Watanabe-Fukunaga et al, J. Immunology. 148: 1274 ( 1992)). In experiments where a monoclonal Ab to Fas is cross-linked to Fas, apoptosis is induced (Yonehara et al, J. Exp. Med.
• Fas antigen is a cell surface protein of relative MW of 45 Kd. Both human and murine genes for Fas have been cloned by Watanabe-Fukunaga et al, (J. Immunol. 148: 1214 (1992)) and Itoh et al. (Cell 66:233 (1991)).
• the proteins encoded by these genes are both transmembrane proteins with structural homology to the Nerve Growth Factor/Tumor Necrosis Factor receptor superfamily, which includes two TNF receptors, the low affinity Nerve Growth Factor receptor and the LT ( , receptor CD40, CD27, CD30. and OX40.
• Fas ligand has been described (Suda et al, Cell 75: 1 169 (1993)).
• the amino acid sequence indicates that Fas ligand is a type II transmembrane protein belonging to the TNF family. Fas ligand is expressed in splenocytes and thymocytes.
• the purified Fas ligand has a MW of 40 kd.
• Fas/Fas ligand interactions are required for apoptosis following the activation of T-cells (Ju et al, Nature 373:444 (1995); Brunner et al. Nature 373:441 (1995)).
• Activation of T-cells induces both proteins on the cell surface.
• Subsequent interaction between the ligand and receptor results in apoptosis ofthe cells. This supports the possible regulatory role for apoptosis induced by Fas/Fas ligand interaction during normal immune responses.
• polypeptide of the present invention has been identified as a novel member of the TNF ligand super-family based on structural and biological similarities.
• the present invention provides isolated nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide encoding the AIM II polypeptide having the amino acid sequence shown in Figures 1 A and IB (SEQ ID NO:2) or the amino acid sequence encoded by the cDNA deposited as ATCC Deposit Number 97689 on August 22, 1996.
• the present invention also provides isolated nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide encoding the AIM 11 polypeptide having the amino acid sequence shown in Figures 1 C and 1 D (SEQ ID NO: 39) or the amino acid sequence encoded by the cDNA deposited as ATCC Deposit Number 97483 on March 15, 1996.
• the present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of AIM II polypeptides or peptides by recombinant techniques.
• the invention further provides an isolated AIM II polypeptide having an amino acid sequence encoded by a polynucleotide described herein.
• AIM II polypeptide includes membrane-bound proteins (comprising, or alternatively consisting of, a cytoplasmic domain, a transmembrane domain, and an extracellular domain) as well as truncated proteins that retain the AIM II polypeptide activity.
• soluble AIM II polypeptides comprise, or alternatively consist of, all or part ofthe extracellular domain of an AIM II protein, but lack the transmembrane region that would cause retention ofthe polypeptide on a cell membrane. Soluble AIM II may also include part of the transmembrane region or part of the cytoplasmic domain or other sequences, provided that the soluble AIM II protein is capable of being secreted.
• a heterologous signal peptide can be fused to the N-terminus of the soluble AIM II polypeptide such that the soluble AIM II polypeptide is secreted upon expression.
• the invention also provides for AIM II polypeptides, particularly human AIM II polypeptides, which may be employed to treat afflictions such as lymphadenopathy, rheumatoid arthritis, autoimmune disease (e.g., inflammatory autoimmune diseases, myasthenia gravis), graft versus host disease, IgE-mediated allergic reactions, anaphylaxis, adult respiratory distress syndrome, Crohn's disease, allergic asthma, acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), and Graves' disease.
• autoimmune disease e.g., inflammatory autoimmune diseases, myasthenia gravis
• graft versus host disease IgE-mediated allergic reactions, anaphylaxis, adult respiratory distress syndrome, Crohn's disease, allergic asthma, acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), and Graves' disease.
• ALL acute lymphoblastic leukemia
• NHL
• compositions comprising, or alternatively consisting of, an AIM II polynucleotide or an AIM II polypeptide for administration to cells in vitro, to cells ex vivo and to cells in vivo, or to a multicellular organism.
• the compositions comprise, or alternatively consist of, an AIM II polynucleotide for expression of an AIM II polypeptide in a host organism for treatment of disease.
• Particularly preferred in this regard is expression in a human patient for treatment of a dysfunction associated with aberrant endogenous activity of an AIM II.
• the present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a cellular response induced by AIM II, which involves contacting cells which express AIM II with the candidate compound, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made in absence ofthe candidate compound; whereby, an increased cellular response over the standard indicates that the compound is an agonist and a decreased cellular response over the standard indicates that the compound is an antagonist.
• a screening assay for AIM II agonists and antagonists is provided.
• the antagonists may be employed to treat, prevent, diagnose, and/or prognose septic shock, inflammation, cerebral malaria, activation ofthe HIN virus, graft-host rejection, immunodeficiency, bone resorption, and cachexia (wasting or malnutrition).
• the AIM II antagonists ofthe invention may be employed to treat, prevent, diagnose, and/or prognose septic shock, inflammation, cerebral malaria, activation ofthe HIN virus, graft-host rejection, immunodeficiency, bone resorption, and cachexia (wasting or malnutrition).
• anti-AIM II antibodies are used to treat, prevent, diagnose, and/or prognose graft versus host disease.
• AIM II may be used to treat rheumatoid arthritis (RA) by inhibiting the increase in angiogenesis or increase in endothe al cell proliferation required to sustain an invading pannus in bone and cartilage as is often observed in RA
• AIM II may be used to inhibit or activate a cellular response mediated by a cellular receptor (e g LT- ⁇ -R, TR2, CD27, and TRANK) by either inhibiting the binding of a ligand to the receptor or by binding to the receptor and activating a receptor mediated cellular response
• a cellular receptor e g LT- ⁇ -R, TR2, CD27, and TRANK
• An additional aspect ofthe invention is related to a method for treating an individual in need of an increased level of AIM II activity in the body comprising administering to such an individual a composition comprising or alternatively consisting of, a therapeutically effective amount of an isolated AIM II polypeptide ofthe invention or an agonist thereof
• a still further aspect ofthe invention is related to a method for treating an individual in need of a decreased level of AIM II activity in the body comprising, administering to such an individual a composition comprising, or alternatively consisting of, a therapeutically effective amount of an AIM II antagonist
• Figures 1A and IB show the nucleotide (SEQ ID NO 1) and deduced amino acid (SEQ ID NO 2) sequences of AIM II
• the protein has a deduced molecular weight of about 26 4 kDa
• the predicted Transmembrane Domain of the AIM II protein is underlined
• Figures 1C and ID show the nucleotide (SEQ ID NO 38) and deduced amino acid (SEQ ID NO 39) sequences of a partial AIM II cDNA that was also obtained
• Figures 2A-2F show the regions of similarity between the amino acid sequences of the AIM II protein and human TNF- ⁇ (SEQ ID NO 3), human
• FIGS 3A-3F show an analysis of the AIM II amino acid sequence Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.
• “Antigenic Index - Jameson-Wolf graph about amino acid residues 13 -20, 23-36, 69-79, 85-94, 167-178, 184-196, 221-233 in Figures 1 A and IB (SEQ ID NO:2) correspond to the shown highly antigenic regions ofthe AIM II protein.
• Figures 4A and 4B show the effect of AIM II on the in vitro proliferation of MDA-MB-231 human breast cancer cells. 5.000 MDA-MB-231/WT (circle), MDA-MB-231/Neo (triangle) or MDA-MB-231 /AIM II (square) cells were plated in triplicate in 24-well plates with IMEM in the presence of either 10% FBS
• FIG. 4B shows colony formation of MDA-MB-231 /WT, MDA-MB-231/Neo and MDA-MB-231/AIM II cells in 0.33% agarose.
• Figures 5A-5C show increased apoptotic cells in MDA-MB-231/AIM II (Figure 5C) in 0.5% serum compared with that ofthe MDA-MB-231 WT ( Figure 5 A) or MDA-MB-231/Neo ( Figure 5B) cells with Annexin-N FACS analysis as described in Example 5 Material and Methods.
• Figure 6A shows an evaluation of the effects of AIM II on growth of xenograft human breast carcinoma MDA-231 in nude mice.
• FIG. 6B shows the effect of AIM II transduction on inhibition of growth of MC-38 murine colon cancer in syngeneic C57BL/6 mice.
• Figure 7 shows the pFlag-AIM II plasmid construct ( Figure 7A) and cytotoxicity of a recombinant soluble form of AIM II (sAIM II) in MDA-MB-231 cells in the presence or absence of IFN ⁇ ( Figure 7B) or with IFN ⁇ alone ( Figure 7A).
• Figures 8A-8M Cell surface expression of the LT ⁇ R or TR2 by the FACS analyses using LT ⁇ R ( Figures 8A-8D) or TR2 ( Figures 8E-8H) mAb.
• MDA-MB-231 Figures 8 A and 8E
• HT-29 Figures 8B and 8F
• MC-3 Figures
• FIG. 8C and 8G U93T
• Figures 8D and 8H FACS binding analyses of soluble AIM II protein alone ( Figure 81) and blocking of a soluble AIM II protein binding by preincubation with the LT ⁇ R-Fc fusion protein ( Figure 8J) or TR2-Fc fusion protein (Figure 8K) in MDA-MB-231 cells.
• Figure 8L summarizes the surface expression of LT ⁇ R and TR2 in various cell lines. Effects of LT ⁇ R-Fc or TR2-Fc fusion protein to block the sAIM Il-mediated cytotoxicity in HT-29 cells ( Figure 8M).
• sLT ⁇ R-Fc open circle with LT ⁇ R-Fc alone, filled circle LT ⁇ R-Fc, and IFN ⁇
• TR2-Fc fusion protein open triangle with TR-2Fc alone, filled triangle TR2-Fc with sLT ⁇ and IFN ⁇
• Figure 9 shows secretion of IFN- ⁇ by sAIM II treated human PBL cells.
• Human PBLs (5 x 10 5 cells per well in the 96 well plate) were treated with or without anti-CD3 mAb and IL-2 (20 U/ml) in the presence or absence of sAIM II for 5 days. The supernatants were then collected from the following groups of cells: PBLs in the presence (filled circle) or absence (open circle) of sAIM II, or the resting PBLs with (filled triangle) or without (open triangle) sAIM II. Human IFN ⁇ concentrations were determined by ELISA.
• Figure 10 shows a schematic representation of the pHE4-5 expression vector (SEQ ID NO 50) and the subcloned AIM II cDNA coding sequence The locations ofthe kanamycin resistance marker gene, the AIM II coding sequence, the oriC sequence, and the laclq coding sequence are indicated
• Figure 1 1 shows the nucleotide sequence ofthe regulatory elements ofthe pHE promoter (SEQ ID NO 51) The two lac operator sequences, the Shine-Delgarno sequence (S/D), and the terminal Htndlll and Ndel restriction sites (italicized) are indicated
• Figure 12 shows a sensorgram of specificity of binding of MCA-38 AIM II conditioned media to LT ⁇ R-Fc versus MCIF-Fc immobilized on BIAcore chip
• Conditioned media was analyzed on a BIAcore instrument flowcell derivatized with lymphotoxin beta receptor Fc fusion protein
• the conditioned media (100 ⁇ l) was flown over the chip at 5 ⁇ l/min and washed with HBS buffer also at 5 ⁇ l/min
• the shown data represents the net bound (off-rate) region ofthe plot after binding of AIM II to immobilized receptor and is measured in relative mass units
• LT ⁇ R-Fc and MCIF-Fc refer to binding data from LT ⁇ R-Fc or MCIF-Fc immobilized BIAcore chip surfaces, respectively
• Figure 13 shows the determination ofthe LT ⁇ R binding by AIM II eluted from LT ⁇ R-Fc column Binding conditions were as described in Figure 1 1
• LT ⁇ R and MCIF refer to binding data from LT ⁇ R-Fc or MCIF-Fc immobilized BIAcore chip surfaces, respectively
• Undiluted conditioned media from MCA38 cells was analyzed before (pre) and after passage through MCIF-Fc (post-MCIF) and LT ⁇ R-Fc (post-LT ⁇ R) affinity columns
• Fractions (1 ml) eluted from the LT ⁇ R (E4-6) and MCIF-Fc (El -3) affinity columns were diluted 3 -fold and tested for binding to LT ⁇ R BIAcore chip
• Figures 14A-14B show the sequence of TR6 and aligned amino acid sequence of cysteine-rich motif
• A Shows a deduced amino acid sequence of TR6 (SEQ ID NO.52) The signal sequence is underlined The potential N-glycosylation site (NCT, amino acid residues 173-175) is underlined with shadow.
• NCT potential N-glycosylation site
• the N-terminal amino acid sequence of recombinant TR6-(His) reads as VAETPT — , which indicates that the first 29 amino acids constitute a signal sequence.
• B Shows an aligned amino acid sequence of cysteine-rich motif of TR6 with other TNF receptor family members (SEQ ID NO:53-58).
• TR6 The amino acid sequence of TR6 (SEQ ID NOs:52 and 59) was aligned with those of TNFR-I, TNFR-II, 4- IBB, TR2 (HVEM), LT ⁇ R and TRl (OPG) on the basis of sequence homology and conserved cysteines.
• Figures 15A-15C show the identification of the membrane-bound TR6 ligand.
• FEEK293 EBN A cells were transfected with pCEP4 control vector (shaded area) or with pCEP4/encoding full-length AIM II (LIGHT) cDNA (solid line). Cells were incubated with (A) HVEM/TR2-Fc (0.34 (g), (B) LT ⁇ R-Fc (0.34 (g), (C) TR6-(His) (0.34 (g) or buffer control (same as vector). Cells were stained with anti-hlgG-FITC for detecting HVEM/TR2 and LT ⁇ R binding. For detecting TR6 binding, cells were stained with anti-poly(His) and anti-mlgG-FITC. They were analyzed for binding by FACS.
• Figures 16A and 16B show TR6 inhibits AIM II (LIGHT)-induced cell death in HT29 cells.
• HT29 cells were incubated in 96-well plates with control medium, 10 U/ml IFN- ⁇ alone, purified sAIM II (sLIGHT) protein (10 ng/ml) in the absence or presence of IFN- ⁇ (10 U/ml), purified sLT ⁇ R-Fc (200 ng/ml) or
• TR6-(His) (200 ng/ml) in the presence of IFN- ⁇ (10 U/ml) and sAIM II (sLIGHT) (10 ng/ml).
• B Cells were incubated with various dose of TR6-(His) and IFN- ⁇ (10 U/ml) with (open circle) or without (filled circle) sAIM II (sLIGHT) (10 ng/ml).
• cells were cultured for 4 days, and proliferation was detected during the last 6 h of culture by the addition of 1 ⁇ Ci of [ 3 H]thymidine. Cells were harvested, and thymidine incorporation was determined using a liquid scintillation counter.
• Figure 17 shows the amino acid sequence and expression of mouse AIM II (LIGHT).
• the putative amino acid sequence of mouse AIM II deduced from the cDNA sequence was aligned with that of human AIM II ( Figure 17A). To obtain -l i ⁇
• Figure 17B-1 through 17B-4 was generated using human 293 cells transfected with the pmAIM II or pcDNA3 and stained with LT ⁇ R-Ig or the anti-AIM II antibody, followed by FITC-conjugated goat anti-human IgG or anti-rabbit IgG, respectively (solid line). Staining with human IgGI or rabbit IgG were used as controls (shaded area). ( Figure 17B-1 through 17B-4.)
• Figure 18 shows the costimulatory activity of mouse AIM II.
• Purified T cells 1 x 10 6 cells/ml
• irradiated COS cells which were transfected with pcDNA3 or pmAIM II plasmid for 72 hours
• Figure 18 A indicated doses of immobilized AIM II.
• flag fusion proteins Figure 18B
• the concentration of anti-CD3 mAb was 2 ⁇ g/ml in Figure 18A and 0.2 ⁇ g/ml in Figure 18B.
• Purified T cells (1 x 10 6 cells/ml) of either CD28 'A or CD28 + + splenocytes were stimulated with immobilized AIM Il.flag fusion protein (4 ⁇ g/ml) or soluble anti-CD28 (1 ⁇ g/ml) in the presence of immobilized anti-CD3 (0.2 ⁇ g/ml) ( Figure 18C). In all assays, the cells were incubated for 72 hr and the proliferative responses ofthe T cells were monitored by 3 H-TdR incorporation during the last 15 hr.
• Figure 19 shows that AIM II DNA injection induces enhanced cytolytic T cell (CTL) responses and regression of established P815 tumor with memory tumor immunity.
• CTL cytolytic T cell
• DBA/2 mice were inoculated with 2 x 10 5 cells of P815 cells at day 0 and then intratumorally injected with medium, pcDNA3 or pmAIM II mixed with liposome at day 7, 9 and 11. Seven days after last injection, spleen cells were harvested and re-stimulated with irradiated P815 cells for 4 days The CTL activity was assessed by a standard 4 hr 51 Cr release assay against P815 and L1210 cells in indicated effector:target (E/T) ratios. The results are expressed as the means + SD of triplicate wells. Similar results were obtained in three independent experiments. To generate the data shown in Figure 19B, DBA/2 mice were inoculated s.c.
• mice with palpable tumor nodules were intratumorally injected with either medium, pcDNA3 or pmAIM II mixed with liposome. The injections were repeated on day 10, 14 and 17. Tumor sizes were assessed by measuring diameters, and the tumor with more than 2 mm in the longest diameter was considered as a palpable tumor. The results are expressed as percentage of mice with palpable tumor. Similar results were obtained in three independent experiments.
• mice which had regressed P815 tumor after pmAIM II treatment were s.c. challenged with 2 x 10 s P815 cells at the right back and the same number of L1210 cells at the left back 40 days after primary tumor inoculation.
• Naive DBA/2 mice receiving both P815 and L1210 challenges were used as controls. Tumor sizes were assessed by measuring perpendicular diameters. The results are expressed as average + SD of 5 mice in each group.
• Figure 20 shows the inhibition of GVHD by blockage of AIM II.
• Figures 20A and 20B Sub-lethally (4 Gy) irradiated BDF1 mice were injected i.v. with 7 x 10 7 cells of B6 splenocytes on day 0. The recipient mice were administered i.v. with either LT ⁇ R-Ig or control human IgGl at 100 ⁇ g per mouse on day -1, 2, 5, 8, 1 1, 14 and 17 (control Ig were administered until day 1 1 because all mice died in day 12).
• the survival ( Figure 20A) and body weight (Figure 20B) ofthe recipients were monitored daily. The results in Figure 20B are expressed as average grams ⁇ SEM of five mice in each group.
• Non-irradiated BDFI mice were injected i.v. with 7 x 10 7 cells of either B6 (B6-F1) or BDF 1 (FI - FI) splenocytes on day 0.
• the recipient mice were administered i.v. with either LT ⁇ R-Ig or control human IgGl at 100 ⁇ g/ mouse on day -1, 1, 3, 5, 7, 9.
• the splenocytes were prepared from the recipient mice and assayed for their CTL activity against P815 (H-2 d ) and EL-4 (H-2 b ) in a standard 51 Cr release assay without further stimulation in vitro. ( Figures 20C-1 and 20C-2.)
• Non-irradiated BDF1 mice were injected i.v. with 7 x 10 7 cells of B6
• Figure 21 shows cytokine production by modulation of AIM II costimulatory pathway.
• Purified T cells (1 x 10 6 cells/ml) were stimulated with immobilized AIM Il.flag fusion protein (3.2 ⁇ g/ml) in 96-well flat-bottomed microplates in the presence or absence of immobilized anti-CD3 (0.5 ⁇ g/ml). After 48 hours, the culture supernatants were collected and the cytokine production was assessed by sandwich ELISA.
• Figure 21 A-1 through 21A-4. The data shown in Figure 21 B- 1 through 21 B-4 was generated using T cells ( 1 x
• the present invention provides isolated nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide encoding an AIM II polypeptide having the amino acid sequence shown in Figures 1A and IB (SEQ ID NO:2), which was determined by sequencing a cDNA.
• the AIM II protein ofthe present invention shares sequence homology with human TNF- ⁇ (SEQ ID NOJ), human TNF- ⁇ (SEQ ID NOJ). human lymphotoxin (SEQ ID NO:5) and human Fas Ligand (SEQ ID NO: 6) ( Figures 2A-2F).
• the nucleotide sequence shown in Figures 1A and IB (SEQ ID NOJ) were obtained by sequencing the cDNA, which was deposited on August 22, 1996, at the American Type Culture
• nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90%) identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence ofthe sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art.
• nucleic acid molecule ofthe present invention encoding an AIM II polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material.
• standard cloning and screening procedures such as those for cloning cDNAs using mRNA as starting material.
• the determined nucleotide sequence of the AIM II cDNA of Figures 1A and IB contains an open reading frame encoding a protein of 240 amino acid residues, with an initiation codon at positions 49-51 ofthe nucleotide sequence in Figures 1 A and IB (SEQ ID NOJ), an extracellular domain comprising, or alternatively consisting of, amino acid residues from about 60 to about 240 in Figures 1A and IB (SEQ ID NO:2), a transmembrane domain comprising, or alternatively consisting of, amino acid residues from about 37 to about 59 in Figures 1A and IB (SEQ ID NO:2), a intracellular domain comprising, or alternatively consisting of, amino acid residues from about 1 to about 36
• TNF-ligand like molecules function as dimers, given that AIM II is homologous to TNF-ligand like molecules, it is likely that it also functions as a homodimer.
• the predicted AIM II polypeptide encoded by the deposited cDNA comprises about 240 amino acids, but may be anywhere in the range of 230-250 amino acids.
• the exact “address” of, the extracelluar, intracellular and transmembrane domains ofthe AIM II polypeptide differ slightly.
• the exact location ofthe AIM II extracellular domain in Figures 1 A and IB may vary slightly (e.g., the address may "shift" by about 1 to 5 residues) depending on the criteria used to define the domain.
• nucleic acid molecules ofthe present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically.
• the DNA may be double-stranded or single-stranded.
• Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.
• isolated nucleic acid molecule(s) is intended a nucleic acid molecule,
• DNA or RNA which has been removed from its native environment
• recombinant DNA molecules contained in a vector are considered isolated for the purposes ofthe present invention.
• Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
• Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules ofthe present invention.
• Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
• isolated nucleic acid molecules according to the present invention may be produced naturally, recombinantly, or synthetically.
• Isolated nucleic acid molecules of the present invention include DNA molecules comprising, or alternatively consisting of, an open reading frame (ORF) shown in Figures 1A and IB (SEQ ID NOJ) or Figures 1C and ID (SEQ ID NO: 38); DNA molecules comprising, or alternatively consisting of, the coding sequence for the AIM II protein shown in Figures 1 A and IB (SEQ ID NO:2) or Figures I C and ID (SEQ ID NOJ9), and DNA molecules which comprise, or alternatively consist of, a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the AIM II protein.
• ORF open reading frame
• DNA molecules comprising, or alternatively consisting of, the coding sequence for the AIM II protein shown in Figures 1 A and IB (SEQ ID NO:2) or Figures I C and ID (SEQ ID NOJ9)
• Nucleic acid molecules according to the present invention further include those encoding the full-length AIM II polypeptide lacking the N-terminal methionine.
• the invention provides a nucleic acid molecule having a nucleotide sequence related to a portion of SEQ ID NOJ which has been determined from the following related cDNA: HT4CC72R (SEQ ID NO:20).
• the invention provides isolated nucleic acid molecules encoding the AIM II polypeptide having an amino acid sequence encoded by the cDNA contained in the plasmid deposited as ATCC Deposit No. 97689 on August
• this nucleic acid molecule will encode the polypeptide encoded by one of the above-described deposited cDNAs.
• the invention further provides an isolated nucleic acid molecule having the nucleotide sequence shown in Figures 1A and IB (SEQ ID NOJ) or Figures IC and ID
• nucleic acid molecule having a sequence complementary to one ofthe above sequences are useful, for example, as probes for gene mapping, by in situ hybridization with chromosomes, and for detecting expression of the AIM II gene in human tissue, for instance, by Northern blot analysis.
• nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide having a nucleotide sequence at least 80% identical, and more preferably at least 85%>, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to (a) a nucleotide sequence encoding the AIM II polypeptide having the complete amino acid sequence in Figures 1 A and IB (SEQ ID NO:2) or Figures I C and ID (SEQ ID NO:39); (b) a nucleotide sequence encoding the AIM II polypeptide having the amino acid sequence in Figures 1A and IB (SEQ ID NO:2), but lacking the N-terminal methionine; (c) a nucleotide sequence encoding the AIM II polypeptide having the complete amino acid sequence encoded by the cDNA contained in ATCC Deposit No.
• nucleotide sequence encoding the AIM II polypeptide extracellular domain (e) a nucleotide sequence encoding the AIM II polypeptide transmembrane domain; (f) a nucleotide sequence encoding the AIM II polypeptide intracellular domain; (g) a nucleotide sequence encoding a soluble AIM II polypeptide having the extracellular and intracellular domains but lacking the transmembrane domain; and (h) a nucleotide sequence complementary to any ofthe nucleotide sequences in (a), (b), (c), (d), (e), (f) or (g) above.
• Additional embodiments of the invention include isolated nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide having a nucleotide sequence at least 80%. identical, and more preferably at least 85%>, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to (a) a nucleotide sequence encoding the AIM II polypeptide having the sequence of amino acids about 1 to about 208 in Figures IC and ID (SEQ ID NO: 39); (b) a nucleotide sequence encoding the AIM II polypeptide having the sequence of amino acids about 7 to about 208 in Figures IC and ID (SEQ ID NO:39); (c) a nucleotide sequence encoding the AIM II polypeptide having the sequence of amino acids about 34 to about 208 in Figures IC and ID (SEQ ID NO:39); and (d) a nucleotide sequence complementary to any ofthe nucleotide sequences in (a), (b),
• nucleic acid molecule is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the nucleotide sequence shown in Figures 1A and IB (SEQ ID NOJ) or Figures IC and ID (SEQ ID NO:38) or to the nucleotide sequence of the deposited cDNA can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive.
• nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the AIM II polypeptide.
• a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% ofthe nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% ofthe total nucleotides in the reference sequence may be inserted into the reference sequence.
• These mutations ofthe reference sequence may occur at the 5' or 3 ' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
• the query sequence may be an entire sequence shown in Figures 1A and IB or Figures IC and ID, the ORF (open reading frame), or any fragment specified as described herein.
• nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the presence invention can be determined conventionally using known computer programs.
• a preferred method for determining the best overall match between a query sequence (a sequence ofthe present invention) and a subject sequence, also referred to as a global sequence alignment can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Co p. App. Biosci. 6:231-245 (1990)).
• a sequence alignment the query and subject sequences are both DNA sequences.
• An RNA sequence can be compared by converting U's to T's.
• the result of said global sequence alignment is in percent identity.
• Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are:
• This corrected score is what is used for the purposes ofthe present invention. Only bases outside the 5' and 3' bases ofthe subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score. For example, a 90 base subject sequence is aligned to a 100 base query- sequence to determine percent identity The deletions occur at the 5' end ofthe subject sequence and therefore, the FASTDB alignment does not show a match/alignment of the first 10 bases at the 5' end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5' and 3' ends not matched/total number of bases in the query sequence) so 10%. is subtracted from the percent identity score calculated by the FASTDB program.
• the final percent identity would be 90%.
• a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5' or 3' of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5' and 3' ofthe subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to made for the purposes ofthe present invention.
• the present application is directed to nucleic acid molecules at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in Figures 1A and IB (SEQ ID NOJ) or Figures IC and ID (SEQ ID NO:38) or to the nucleic acid sequence of the deposited cDNAs, irrespective of whether they encode a polypeptide having AIM II activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having AIM II activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer.
• PCR polymerase chain reaction
• nucleic acid molecules of the present invention that do not encode a polypeptide having AIM II activity include, />/te/' alia, (1) isolating the AIM II gene or allelic variants thereof in a cDNA library; (2) in situ hybridization (e.g. , "FISH") to metaphase chromosomal spreads to provide precise chromosomal location ofthe AIM II gene, as described in Verma et al. , Human Chromosomes: A Manual of Basic Techniques, Pergamon Press. New York ( 1988). and (3) Northern Blot analysis for detecting AIM II mRNA expression in specific tissues
• nucleic acid molecules having sequences at least 80%, 85%. 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in Figures 1A and IB (SEQ ID NOJ) or Figures IC and ID
• polypeptide having AIM II activity polypeptides exhibiting activity similar, but not necessarily identical, to an activity of the AIM II protein ofthe invention, as measured in a particular biological assay.
• AIM II protein cytotoxic activity can be measured using propidium iodide staining to demonstrate apoptosis as described by Zarres et al, Cell 70: 31-46 (1992).
• AIM II induced apoptosis can also be measured using TUNEL staining as described by Gavierli et al, J. Cell. Biol. 119: 493-501 (1992).
• polypeptides encoded by these polynucleotides are examples of these polynucleotides.
• the propidium iodide staining is performed as follows. Cells either from tissue or culture are fixed in formaldehyde, cut into frozen sections and stained with propidium iodide. The cell nuclei are visualized by propidium iodide using confocal fluorescent microscopy. Cell death is indicated by pyknotic nuclei
• nucleic acid molecules having a sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of one ofthe deposited cDNAs or the nucleic acid sequence shown in Figures 1A and IB (SEQ ID NOJ) or Figures IC and ID (SEQ ID NOJ 8) will encode a polypeptide "having AIM II protein activity "
• degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay.
• nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having AIM II protein activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid).
• the polynucleotides and polynucleotide fragments of the invention encode a polypeptide which demonstrates an AIM II functional activity.
• a polypeptide demonstrating an AIM II "functional activity" is meant, a polypeptide capable of displaying one or more known functional activities associated with a full-length and/or secreted AIM II polypeptide.
• Such functional activities include, but are not limited to, biological activity, antigenicity, ability to bind (or compete with a polypeptide for binding) to an anti-AIM II antibody, immunogenicity (ability to generate antibody which binds to a polypeptide), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide (e.g. , TR2 (International Publication No. WO 96/34095), LT- ⁇ receptor, TR6 (International Publication No. WO 98/30694), and CD27)).
• TR2 International Publication No. WO 96/34095
• LT- ⁇ receptor LT- ⁇ receptor
• TR6 International Publication No. WO 98/30694
• CD27 a receptor or ligand for a polypeptide
• AIM II polypeptides and fragments, variants derivatives, and analogs thereof, can be assayed by various methods.
• various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” i munoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, m situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.
• competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” i munoas
• antibody binding is detected by detecting a label on the primary antibody
• the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
• the secondary antibody is labeled
• Many means are known in the art for detecting binding in an immunoassay and are within the scope ofthe present invention.
• a ligand e.g., DR5 (See, International Publication No. WO 98/41629), TRl 0 (See, International Publication No. WO 98/54202), 312C2 (See, International Publication No. WO 98/06842), and TR1 1, TRl l SVl, and TR1 1 SV2 (See, U.S. Application Serial No.
• binding can be assayed, e.g. , by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky, E. et al, 1995, Microbiol Rev. 59:94-123.
• physiological correlates of binding to its substrates can be assayed.
• the present invention is further directed to polynucleotides comprising, or alternatively consisting of, fragments of the isolated nucleic acid molecules described herein.
• a fragment of an isolated nucleic acid molecule having the nucleotide sequence of one ofthe deposited cDNAs or the nucleotide sequence shown in Figures 1A and IB (SEQ ID NOJ) or Figures IC and ID (SEQ ID NO:38) is intended fragments at least about 15 nt, and more preferably at least about 20 nt.
• Figures IC and ID By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of one of the deposited cDNAs or the nucleotide sequence as shown in Figures 1 A and IB (SEQ ID NOJ) or Figures IC and ID (SEQ ID NO:38).
• the present invention is further directed to polynucleotides comprising, or alternatively consisting of, fragments of isolated nucleic acid molecules which encode subportions of AIM II domains.
• the invention provides polynucleotides comprising, or alternatively consisting of, the nucleotide sequences of a member selected from the group consisting of nucleotides 49-108,
• the present invention is further directed to polynucleotides comprising, or alternatively consisting of, isolated nucleic acid molecules which encode domains of AIM II.
• the invention provides polynucleotides comprising, or alternatively consisting of, nucleic acid molecules which encode beta-sheet regions of AIM II set out in Table 2.
• polynucleotides comprise, or alternatively consist of, nucleic acid molecules which encode a polypeptide having an amino acid sequence selected from the group consisting of amino acid residues from about 7 to about 14, amino acid residues from about 18 to about 23, amino acid residues from about 17 to about 25, amino acid residues from about 33 to about 46, amino acid residues from about 35 to about 39, amino acid residues from about 57 to about 60, amino acid residues from about 67 to about 72, amino acid residues from about 102 to about 107, amino acid residues from about 121 to about 126, amino acid residues from about 131 to about 166, amino acid residues from about 141 to about 152, amino acid residues from about 158 to about 169, amino acid residues from about 213 to about 221, and amino acid residues from about 232 to about 240 of SEQ ID NO:2.
• the invention is further directed to isolated polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of amino acid residues from about 7 to about 14, amino acid residues from about 18 to about 23, amino acid residues from about 17 to about 25, amino acid residues from about 33 to about 46, amino acid residues from about 35 to about 39, amino acid residues from about 57 to about 60, amino acid residues from about 67 to about 72, amino acid residues from about 102 to about 107, amino acid residues from about 121 to about 126, amino acid residues from about 131 to about 166, amino acid residues from about 141 to about 152, amino acid residues from about 158 to about 169, amino acid residues from about 213 to about 221, and amino acid residues from about 232 to about 240 of SEQ ID NO:2.
• amino acid sequence selected from the group consisting of amino acid residues from about 7 to about 14, amino acid residues from about 18 to about 23, amino acid residues from about 17 to about 25, amino acid residues from about 33
• Nucleic acid fragments of the present invention include nucleic acid molecules encoding beta-sheet regions of the AIM II protein, as well as isolated nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide having a nucleotide sequence at least 80%. identical, and more preferably at least 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to nucleic acid molecules encoding beta-sheet regions of the AIM II protein.
• Polynucleotides encoding polypeptides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to beta-sheet regions are also with the scope ofthe invention, as are the polypeptides encoded by these polynucleotides.
• the present invention is also directed to polynucleotides comprising, or alternatively consisting of, isolated nucleic acid molecules which encode a polypeptide having an amino acid sequence selected from the group consisting of amino acid residues from about 94 to about 100, amino acid residues from about 121 to about 124, amino acid residues from about 127 to about 135, amino acid residues from about 139 to about 149, amino acid residues from about 160 to about 168, amino acid residues from about 175 to about 185, amino acid residues from about 197 to about 209, amino acid residues from about 213 to about 220, and amino acid residues from about 232 to about 240 of SEQ ID NO:2.
• the invention is further directed to isolated polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of amino acid residues from about 94 to about 100, amino acid residues from about 121 to about 124, amino acid residues from about 127 to about 135, amino acid residues from about 139 to about 149, amino acid residues from about 160 to about 168, amino acid residues from about 175 to about 185, amino acid residues from about
• nucleic acid fragments of the present invention include nucleic acid molecules encoding epitope-bearing portions of the AIM II protein.
• nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising, or alternatively consisting of, one, two, three, four, five, or more amino acid sequences selected from amino acid residues from about 13 to about 20 in Figures 1A and IB (SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 23 to about 36 in Figure 1 (SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 69 to about 79 in Figures 1A and IB (SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 85 to about 94 in Figures 1A and
• IB (SEQ ID NO: 2); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 167 to about 178 in Figures 1 A and IB (SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 184 to about 196 in Figures 1 A and IB (SEQ ID NO: 2); and a polypeptide comprising, or alternatively consisting of, amino acid residues from about 221 to about 233 in Figures 1A and IB (SEQ ID NO:2).
• “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) amino acids.
• polypeptide fragments are antigenic regions of the AIM II protein. Methods for determining other such epitope-bearing portions of the AIM II protein are described in detail below. Polypeptides encoded by these polynucleotides are also encompassed by the invention.
• AIM II polynucleotides may be used in accordance with the present invention for a variety of applications, particularly those that make use of the chemical and biological properties of the AIM II.
• the invention provides an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide which hybridizes under stringent hybridization conditions to a portion ofthe polynucleotide in a nucleic acid molecule ofthe invention described above, for instance, the complement of a polynucleotide fragment described herein, or the cDNA plasmid contained in ATCC Deposit 97689 or ATCC Deposit 97483.
• stringent hybridization conditions is intended overnight incubation at 42° C in a solution comprising, or alternatively consisting of 50% formamide, 5x SSC (750 mM NaCl, 75mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in OJx SSC at about 65 °C.
• a polynucleotide which hybridizes to a "portion" of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70 nt of the reference polynucleotide.
• nt nucleotides
• a polynucleotide which hybridizes only to a poly A sequence such as the 3 ' terminal poly(A) tract ofthe AIM II cDNA shown in Figures 1 A and IB (SEQ ID NOJ) or Figures IC and ID (SEQ ID NO:38)
• a polynucleotide ofthe invention used to hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g.
• nucleic acid molecules ofthe present invention which encode an AIM II polypeptide may include, but are not limited to those encoding the amino acid sequence of the polypeptide, by itself; the coding sequence for the polypeptide and additional sequences, such as those encoding a leader or secretory sequence, such as a pre-, or pro- or prepro- protein sequence; the coding sequence of the polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5 ' and 3 ' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals, for example - ribosome binding and stability of mRNA; an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.
• the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification ofthe fused polypeptide.
• the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al, Proc. Nail Acad. Sci. USA 56:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein.
• the "HA” tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al, Cell 37:161 (1984).
• other such fusion proteins include the AIM II fused to Fc at the N- or C-terminus.
• the present invention further relates to variants of the nucleic acid molecules ofthe present invention, which encode portions, analogs or derivatives of the AIM II protein. Variants may occur naturally, such as a natural allelic variant. By an "allelic variant” is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985).
• Non-naturally occurring variants may be produced using art-known mutagenesis techniques, which include, but are not limited to: ohgonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site-directed mutagenesis (see, e.g., Carter et al, Nucl Acids Res. 73:4331 (1986); and Zoller etal, Nucl. Acids Res. 70:6487 (1982)), cassette mutagenesis (see, e.g., Wells et al, Gene 34:315 (1985)), restriction selection mutagenesis (see, e.g., Wells etal, Philos. Trans. R. Soc. London Ser.
• art-known mutagenesis techniques include, but are not limited to: ohgonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site-directed mutagenesis (see, e.g., Carter et al, Nu
• variants include those produced by nucleotide substitutions, deletions or additions which may involve one or more nucleotides.
• the variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the AIM II protein or portions thereof. Also especially preferred in this regard are conservative substitutions.
• Vectors and Host Cells The present invention also relates to vectors which include the isolated
• DNA molecules of the present invention are vectors which are genetically engineered with the recombinant vectors, and the production of AIM II polypeptides or fragments thereof by recombinant techniques.
• the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host.
• a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
• the DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the
• the expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
• the coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end ofthe polypeptide to be translated.
• the expression vectors will preferably include at least one selectable marker.
• markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicilhn resistance genes for culturing in E. coli and other bacteria.
• Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli. Streptomyces and Salmonella lyphimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
• the present invention further includes novel expression vectors comprising operator and promoter elements operatively linked to nucleotide sequences encoding a protein of interest.
• novel expression vectors comprising operator and promoter elements operatively linked to nucleotide sequences encoding a protein of interest.
• pH ⁇ 4-5 which is described in detail below.
• components ofthe pHE4-5 vector include: 1) a neomycinphosphotransferase gene as a selection marker, 2) an E. coli origin of replication, 3) a T5 phage promoter sequence, 4) two lac operator sequences, 5) a Shine-Delgarno sequence, 6) the lactose operon repressor gene (laclq)
• the origin of replication (oriC) is derived from pUC19 (LTI, Gaithersburg, MD). The promoter sequence and operator sequences were made synthetically. Synthetic production of nucleic acid sequences is well known in the art.
• a nucleotide sequence encoding AIM II (SEQ ID NO: 1 ), is operatively linked to the promoter and operator by inserting the nucleotide sequence between the Ndel and Aspl 18 sites ofthe pHE4-5 vector.
• the pHE4-5 vector contains a laclq gene.
• Laclq is an allele ofthe lacl gene which confers tight regulation ofthe lac operator. Amann,
• the laclq gene encodes a repressor protein which binds to lac operator sequences and blocks transcription of down-stream (i.e., 3') sequences.
• the laclq gene product dissociates from the lac operator in the presence of either lactose or certain lactose analogs, e.g., isopropyl B-D-thiogalactopyranoside (IPTG).
• IPTG isopropyl B-D-thiogalactopyranoside
• the promoter/operator sequences ofthe pHE4-5 vector comprise a T5 phage promoter and two lac operator sequences. One operator is located 5' to the transcriptional start site and the other is located 3' to the same site. These operators, when present in combination with the laclq gene product, confer tight repression of down-stream sequences in the absence of a lac operon inducer, e.g., IPTG. Expression of operatively linked sequences located down-stream from the lac operators may be induced by the addition of a lac operon inducer, such as IPTG.
• a lac operon inducer such as IPTG.
• the pHE4 series of vectors contain all ofthe components ofthe pHE4-5 vector except for the AIM II coding sequence.
• Features of the pHE4 vectors include optimized synthetic T5 phage promoter, lac operator, and Shine-Delgarno sequences. Further, these sequences are also optimally spaced so that expression of an inserted gene may be tightly regulated and high level of expression occurs upon induction.
• bacterial promoters suitable for use in the production of proteins ofthe present invention include the E. coli lacl and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter.
• Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those ofthe Rous Sarcoma Virus (RS V), and metallothionein promoters, such as the mouse metallothionein-I promoter.
• the pHE4-'S vector also contains a Shine-Delgarno sequence 5' to the AUG initiation codon
• Shine-Delgarno sequences are short sequences generally located about 10 nucleotides up-stream (/ e , 5 1 ) from the AUG initiation codon
• the present invention is also directed to expression vector useful for the production of the proteins of the present invention
• This aspect of the invention is exemplified by the pHE4-5 vector (SEQ ID NO 50)
• vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen, pBS vectors, Phagesc ⁇ pt vectors, Bluesc ⁇ pt vectors, pNH8A, pNHl ⁇ a, pNH18A pN ⁇ 46A, available from Stratagene, and ptrc99a, pKK223-3, pKK233-3 pDR540, pRIT5 available from Pharmacia
• preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene, and pSVK3, pBPV, pMSG and pSVL available from Pharmacia
• Other suitable vectors will be readily apparent to the skilled artisan
• the polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage Also, peptide moieties may be added to the polypeptide to facilitate purification Such regions may be removed prior to final preparation of the polypeptide The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others are familiar and routine techniques in the art
• a preferred fusion protein comprises a heterologous region from immunoglobulin that is useful to solubi ze proteins
• EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another human protein or part thereof
• the Fc part in a fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232262)
• it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and purified in the advantageous manner desc ⁇ bed This is the case when Fc portion proves to be a hindrance to use in therapy and diagnosis, for example when the fusion protein is to be used as antigen for immunizations
• human proteins, such as, hIL-5-receptor has been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5 See,
• polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
• the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., AIM II coding sequence), and/or to include genetic material (e.g. , heterologous polynucleotide sequences) that is operably associated with AIM II polynucleotides ofthe invention, and which activates, alters, and/or amplifies endogenous AIM II polynucleotides.
• endogenous genetic material e.g., AIM II coding sequence
• genetic material e.g., heterologous polynucleotide sequences
• heterologous control regions e.g., promoter and/or enhancer
• endogenous AIM II polynucleotide sequences via homologous recombination
• heterologous control regions e.g., promoter and/or enhancer
• endogenous AIM II polynucleotide sequences via homologous recombination
• the invention further provides an isolated AIM II polypeptide having the amino acid sequence encoded by one ofthe deposited cDNAs, or the amino acid sequence in Figures 1 A and IB (SEQ ID NO: 2) or Figures IC and ID (SEQ ID NO: 2)
• polypeptides ofthe present invention include the polypeptide encoded by one ofthe deposited cDNAs, the polypeptide of Figures 1A and IB (SEQ ID NO:2) or Figures IC and ID (SEQ ID NO:39), the polypeptide of Figures lA and IB (SEQ ID NO:2) lacking the N-terminal methionine, the extracellular domain, the transmembrane domain, the intracellular domain, soluble polypeptides comprising, or alternatively consisting of, all or part of the extracellular and intracellular domains but lacking the transmembrane domain, as well as polypeptides which are at least 80% identical, more preferably at least 85%, 90%, 92% or 95% identical, still more preferably at least 96%.
• polypeptides of the present invention further include (a) the AIM II polypeptide having the sequence of amino acids about 1 to about 208 in Figures IC and ID (SEQ ID NO 39), (b) the AIM II polypeptide having the sequence of amino acids about 7 to about 208 in Figures IC and ID (SEQ ID NO 39), and (c) the AIM II polypeptide having the sequence of amino acids about 34 to about 208 in Figures 1 C and ID (SEQ ID NO 39), as well as polypeptides which are at least
• polypeptide having an amino acid sequence at least, for example, 95%o "identical" to a reference amino acid sequence of an AIM II polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids ofthe reference amino acid ofthe AIM II polypeptide.
• up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% ofthe total amino acid residues in the reference sequence may be inserted into the reference sequence.
• These alterations ofthe reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
• any particular polypeptide is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence shown in Figures 1 A and IB (SEQ ID NO:2) or Figures IC and ID (SEQ ID NO:39) or to the amino acid sequence encoded by one ofthe deposited cDNAs can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 5371 1. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance,
• the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% ofthe total number of amino acid residues in the reference sequence are allowed.
• polypeptide having an amino acid sequence at least, for example, 95% "identical" to a query amino acid sequence of the present invention it is intended that the amino acid sequence ofthe subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence.
• the amino acid sequence ofthe subject polypeptide may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence.
• up to 5% ofthe amino acid residues in the subject sequence may be inserted, deleted, (indels) or substituted with another amino acid.
• These alterations ofthe reference sequence may occur at the amino or carboxy terminal positions ofthe reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence
• any particular polypeptide is at least 80%, 85%, 90%, 92%, 95%, 96%, 97% 98% or 99% identical to, for instance, the ammo acid sequences shown in Table 1 or to the amino acid sequence encoded by the deposited DNA can be determined conventionally using known computer programs
• a preferred method for determining the best overall match between a query sequence (a sequence ofthe present invention) and a subject sequence, also referred to as a global sequence alignment can be determined using the FASTDB computer program based on the algorithm of Brutlag et ⁇ / (Comp App Biosci 6 237-245 (1990))
• the query and subject sequences are either both nucleotide sequences or both amino acid sequences
• the result of said global sequence alignment is in percent identity
• Penalty 1
• Joining Penalty 20
• Randomization Group Length 0
• Cutoff Score 1 ,
• the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score This final percent identity score is what is used for the purposes of the present invention Only residues of the (reference) sequence that extend past the N- or C-termini of the subject sequence
• AIM II The natural processed form of AIM II that was affinity purified on an LT- ⁇ receptor column from conditioned media of MCA-38 cells transformed with full length AIM II cDNA is Leu-83 to Val-240 in SEQ ID NO.2 (See Example 10).
• AIM II is processed differently in COS cells, producing an AIM II that is cleaved between Glu-67 and Met-68 to yield a polypeptide having amino acids 68-240 in SEQ ID NO 2.
• COS cells also cleave the AIM II between Met-68 and Val-69, resulting a polypeptide having amino acids 69-240 in SEQ ID NO.2.
• polypeptides of the present invention are preferably provided in an isolated form.
• isolated polypeptide is intended a polypeptide removed from its native environment.
• a polypeptide produced and/or contained within a recombinant host cell is considered isolated for purposes ofthe present invention
• isolated polypeptide are polypeptides that have been purified, partially or substantially, from a recombinant host.
• a recombinantly produced version of the AIM II polypeptide can be substantially purified by the one-step method described in Smith and Johnson, Ge/7e 67:31 -40
• the AIM II polypeptides of the invention may be in monomers or multimers (/. e. , dimers, trimers, tetramers and higher multimers). Accordingly, the present invention relates to monomers and multimers ofthe AIM II polypeptides ofthe invention, their preparation, and compositions (preferably, pharmaceutical compositions) containing them.
• the polypeptides ofthe invention are monomers, dimers, trimers or tetramers.
• the multimers of the invention are at least dimers, at least trimers, or at least tetramers. Multimers encompassed by the invention may be homomers or heteromers.
• homomer refers to a multimer containing only AIM II polypeptides of .the invention (including AIM II fragments, variants, splice variants, and fusion proteins, as described herein). These homomers may contain AIM II polypeptides having identical or different amino acid sequences.
• a homomer ofthe invention is a multimer containing only AIM II polypeptides of .the invention (including AIM II fragments, variants, splice variants, and fusion proteins, as described herein). These homomers may contain AIM II polypeptides having identical or different amino acid sequences.
• a homomer ofthe invention is a multimer containing only
• a homomer of the invention is a multimer containing AIM II polypeptides having different amino acid sequences.
• the multimer of the invention is a homodimer (e.g., containing AIM II polypeptides having identical or different amino acid sequences) or a homotrimer (e.g., containing AIM II polypeptides having identical and/or different amino acid sequences).
• the homomeric multimer ofthe invention is at least a homodimer, at least a homot ⁇ mer, or at least a homotetramer.
• heteromer refers to a multimer containing one or more heterologous polypeptides (i.e.. polypeptides of different proteins) in addition to the AIM II and AIM II polypeptides of the invention.
• the multimer ofthe invention is a heterodimer, a heterotrimer, or a heterotetramer.
• the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.
• Multimers ofthe invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation.
• multimers ofthe invention such as, for example, homodimers or homotrimers
• heteromultimers of the invention such as, for example, heterotrimers or heterotetramers, are formed when polypeptides ofthe invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution.
• multimers ofthe invention are formed by covalent associations with and/or between the AIM II polypeptides of the invention.
• covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in SEQ ID NO: 2 or SEQ ID NO: 39, or contained in the polypeptide encoded by a cDNA of the deposits designated as ATCC Accession 97689 or 97483).
• the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occurring) polypeptide.
• the covalent associations are the consequence of chemical or recombinant manipulation.
• covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in an AIM II fusion protein
• covalent associations are between the heterologous sequence contained in a fusion protein ofthe invention (.vee, e.g., US Patent Number 5,478.925).
• the covalent associations are between the heterologous sequence contained in an AIM II-Fc fusion protein ofthe invention (as described herein).
• covalent associations of fusion proteins ofthe invention are between heterologous polypeptide sequence from another TNF family ligand/receptor member that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (.vee, e.g., WO 98/49305, the contents of which are herein incorporated by reference in its entirety).
• oseteoprotegerin e.g., WO 98/49305
• the multimers of the invention may be generated using chemical techniques known in the art.
• polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g. , US Patent Number 5,478.925. which is herein incorporated by reference in its entirety).
• multimers ofthe invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
• polypeptides ofthe invention may be routinely modified by the addition of cysteine or biotin to the C terminus or N-terminus ofthe polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (.vee, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety). Additionally, techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
• multimers ofthe invention may be generated using genetic engineering techniques known in the art.
• polypeptides contained in multimers ofthe invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e g , US Patent Number 5,478,925, which is herein incorporated by reference in its entirety)
• polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide ofthe invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (vee, e g , US Patent Number 5,478,925, which is herein incorporated by reference in its entirety)
• recombinant techniques described herein or otherwise known in the art are applied to generate
• the present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions
• the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof
• the antibody portion fused to a polypeptide ofthe present invention may comprise the hinge region, CH 1 domain, CH2 domain and CH3 domain or any combination of whole domains or portions thereof
• the polypeptides ofthe present invention may be fused or conjugated to the above antibody portions to increase the in vivo half life ofthe polypeptides or for use in immunoassays using methods known in the art
• the polypeptides may also be fused or conjugated to the above antibody portions to form multimers
• Fc portions fused to the polypeptides ofthe present invention can form dimers through disulfide bonding between the Fc portions
• Higher multime ⁇ c forms can be made by fusing the polypeptides to portions of IgA and IgM Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g., U.S.
• AIM II polypeptide includes membrane-bound proteins (comprising, or alternatively consisting of, a cytoplasmic domain, a transmembrane domain, and an extracellular domain) as well as truncated proteins that retain the AIM II functional activity
• soluble AIM II polypeptides comprise, or alternatively consisting of, all or part ofthe extracellular domain of an AIM II protein, but lack the transmembrane region that would cause retention ofthe polypeptide on a cell membrane.
• Soluble AIM II may also include part of the transmembrane region or part of the cytoplasmic domain or other sequences, provided that the soluble AIM II protein is capable of being secreted.
• a heterologous signal peptide can be fused to the ⁇ -terminus of the soluble
• AIM II polypeptide such that the soluble AIM II polypeptide is secreted upon expression.
• polypeptide ofthe present invention have uses that include, but are not limited to, functioning as a molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art.
• AIM II polypeptide can be varied without significant effect of the structure or function ofthe protein. If such differences in sequence are contemplated, it should be remembered that there will be critical areas on the protein which determine activity.
• the invention further includes variations ofthe AIM II polypeptide which show substantial AIM II polypeptide activity or which include regions of AIM II protein such as the protein portions discussed below.
• Such mutants include deletions, insertions, inversions, repeats, and type substitutions.
• guidance concerning which amino acid changes are likely to be phenotypically silent can be found in Bowie, J.U., et al, "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 2- 7J306-1310 (1990). Polynucleotides encoding these fragments, derivatives or analogs are also encompassed by the invention.
• the fragment, derivative or analog ofthe polypeptide of Figures 1 A and IB (SEQ ID NO :2) or Figures IC and ID (SEQ ID NO:39), or that encoded by one ofthe deposited cDNAs may be (i) one in which one or more ofthe amino acid residues (e.g., 3, 5, 8, 10, 15 or 20) are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group (e.g., 3, 5, 8, 10, 15 or 20), or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as an IgG Fc fusion region
• the AIM II receptor of the present invention may include one or more (e.g , 3, 5, 8, 10, 15 or 20) amino acid substitutions, deletions or additions, either from natural mutations or human manipulation
• changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity ofthe protein (see Table 1)
• Ammo acids in the AIM II protein of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and
• amino terminal deletion mutants Such mutants include those comprising the amino acid sequence shown in SEQ ID NO 2 having a deletion of at least first N-terminal amino acid but not more than the first 114 N-terminal amino acid residues of SEQ ID NO 2 Alternatively, the deletion will include at least the first 35 N-terminal amino acid residues but not more than the first 114 N-terminal amino acid residues of SEQ ID NO 2 Alternatively, the deletion will include at least the first 59 N-terminal amino acid residues but not more than the first 114 N-teiminal amino acid residues of SEQ
• the deletion will include at least the first 67 N-terminal amino acid residues but not more than the first 114 N-terminal amino acid residues of SEQ ID NO 2 Alternatively, the deletion will include at least the first 68 N-terminal amino acid residues but not more than the first 1 14 N-terminal amino acid residues of SEQ ID NO 2 Alternatively, the deletion will include at least the first 73 N-terminal amino acid residues but not more than the first 114 N-terminal amino acid residues of SEQ ID NO 2 Alternatively, the deletion will include at least the first 82 N-terminal amino acid residues but not more than the first 114 N-terminal amino acid residues of SEQ ID NO 2 Alternatively, the deletion will include at least the first 100 N-terminal amino acid residues but not more than the first 1 14 N-terminal amino acid residues of SEQ ID NO:2.
• Polynucleotides encoding these deletion mutants are also encompassed by the invention, as are polynucleotides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to polynucleotides encoding the deletion mutants described above and polynucleotides encoding polypeptides at least 80%, 85%, 90%, 92%, 95%, 96%,
• the present invention also encompasses the above polynucleotides fused to a heterologous polynucleotides and polypeptide expression products of these polynucleotides.
• the present invention is also directed to all combinations of the above described ranges.
• polynucleotides encoding these deletion mutants are also encompassed by the invention, as are polynucleotides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to polynucleotides encoding the deletion mutants described above and polynucleotides encoding polypeptides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to these deletion mutants.
• the present invention also encompasses the above polynucleotides fused to a heterologous polynucleotides and polypeptide expression products of these polynucleotides.
• Preferred AIM II polypeptides are those having one or more of the sequences shown below (numbering starts with the first amino acid in the protein (Met):
• Trp(114)toVal(240) Particularly preferred embodiments include polypeptides comprising, or alternatively consisting of, one or more ofthe AIM II N-terminal deletions Gln-60 to Val-240 (AIM II (aa 60-240)), Met-68 to Val-240 (AIM II (aa 68-240)), Val-69 to Val-240 (AIM II (aa 69-240)), Asp-74 to Val-240 (AIM II (aa 74-240)). Leu-83 to Val-240 (AIM II (aa 83-240)), and Ala-101 to Val-240
• polynucleotides encoding these polypeptides are also encompassed by the invention, as are polynucleotides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to polynucleotides encoding the polypeptides described above and polynucleotides encoding polypeptides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides.
• the present invention also encompasses the above polynucleotides fused to a heterologous polynucleotides and polypeptide expression products of these polynucleotides.
• N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that an AIM II mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six AIM II amino acid residues may often evoke an immune response.
• the present invention further provides polypeptides having one or more residues deleted from the amino terminus ofthe AIM II amino acid sequence shown in Figures 1A and IB (SEQ ID NO:2 up to the phenylalanine residue at position number 235, and polynucleotides encoding such polypeptides.
• the present invention provides polypeptides comprising, or alternatively consisting of the amino acid sequence of residues n-314 of Figures 1A and IB (SEQ ID NO 2), where n is an integer in the range of 2 to 235, and 236 is the position ofthe first residue from the N-terminus ofthe complete AIM II polypeptide believed to be required for at least immunogenic activity of the
• the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of the amino acid sequence of a member selected from the group consisting of residues of E-2 to V-240, E-3 to V-240, S-4 to V-240, V-5 to V-240, V-6 to V-240, R-7 to V-240, P-8 to V-240, S-9 to V-240, V-10 to V-240, F-l 1 to V-240, V-12 to V-240, V-13 to V-240, D- 14 to V-240, G- 15 to V-240, Q- 16 to V-240, T- 17 to V-240, D- 18 to V-240, 1-19 to V-240, P-20 to V-240.
• V-240 R-29 to V-240, R-30 to V-240, Q-31 to V-240, S-32 to V-240, C-33 to V-240, S-34 to V-240, V-35 to V-240, A-36 to V-240, R-37 to V-240, V-38 to V-240.
• G-39 to V-240, L-40 to V-240 G-41 to V-240, L-42 to V-240, L-43 to V-240, L-44 to V-240, L-45 to V-240, L-46 to V-240, M-47 to V-240, G-48 to V-240, A-49 to V-240, G-50 to V-240, L-51 to V-240, A-52 to V-240, V-53 to
• V-240 V-240, S-79 to V-240, W-80 to V-240, E-81 to V-240, Q-82 to V-240, L-83 to V-240, 1-84 to V-240, Q-85 to V-240, E-86 to V-240, R-87 to V-240, R-88 to V-240, S-89 to V-240, H-90 to V-240, E-91 to V-240, V-92 to V-240, N-93 to V-240, P-94 to V-240, A-95 to V-240, A-96 to V-240, H-97 to V-240, L-98 to V-240, T-99 to V-240, G- 100 to V-240, A- 101 to V-240, N- 102 to V-240, S- 103 to V-240, S- 104 to V-240, L- 105 to V-240, T- 106 to V-240, G- 107 to V-240, S-108 to V-240, G-
• V-240 V-240, R-172 to V-240, Y-173 to V-240, P-l 74 to V-240, E-175 to V-240, E-176 to V-240, L-l 77 to V-240, E-178 to V-240, L-l 79 to V-240, L-l 80 to V-240, V-181 to V-240, S-182 to V-240, Q-183 to V-240, Q-184 to V-240, S-185 to V-240, P-l 86 to V-240, C-187 to V-240, G-188 to V-240, R-189 to V-240, A-190 to V-240, T-191 to V-240. S-192 to V-240, S-193 to V-240,
• V-240 V-240, V-217 to V-240, R-218 to V-240, V-219 to V-240, L-220 to V-240, D-221 to V-240, E-222 to V-240, R-223 to V-240, L-224 to V-240, V-225 to V-240, R-226 to V-240, L-227 to V-240, R-228 to V-240, D-229 to V-240, G-230 to V-240, T-231 to V-240, R-232 to V-240, S-233 to V-240, Y-234 to V-240, and F-235 to V-240 of the AIM II sequence shown in SEQ ID NO 2 (which is identical to the sequence shown as Figures 1A and IB, with the exception that the amino acid residues in SEQ ID NO 2 are numbered consecutively from 1 through 240 from the N-terminus to the C-termmus)
• the present invention is also directed to nucleic acid molecules comprising, or alternatively consisting of, a
• the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence ofthe AIM II polypeptide shown in Figures 1 A and IB (SEQ ID NO.2), up to the valine residue at position number 6, and polynucleotides encoding such polypeptides
• the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues 1 -m of Figures 1 A and IB (i.e., SEQ ID NO 2), where m is an integer in the range of 6 to 239, and 6 is the position of the first residue from the C-terminus of the complete AIM II polypeptide believed to be required for at least immunogenic activity ofthe AIM II polypeptide
• Polynucleotides encoding these polypeptides are also encompassed by the invention More in particular the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of a member selected from the group
• M-l to E-222 M-l to D-221, M-l to L-220, M-l to V-219, M-l to R-218, M-l to V-217, M-l to V-216, M-l to V-215, M-l to E-214, M-l to E-213, M-l to G-212, M-l to A-21 1 , M-l to E-210, M-l to L-209, M-l to H-208, M-l to V-207, M-l to V-206, M-l to G-205, M-l to G-204, M-l to L-203, M-l to F-202, M-l to S-201, M-l to S-200, M-l to D-199, M-l to
• V-147 M-l to K-146, M-l to S-145, M-l to Y-144, M-l to 1-143, M-l to Y-142, M-l to Y-141 , M-l to Y-140, M-l to G-139, M-l to A-138, M-l to K-137, M-l to T-136, M-l to V-135, M-l to V-134, M-l to L-133, M-l to A-132, M-l to G-131, M-l to D-130, M-l to H-129, M-l to Y-128, M-l to S-127, M-l to L-126, M-l to G-125, M-l to R-124, M-l to L-123, M-l to F-122, M-l to A-121 , M-l to L-120, M- l to G-l 19, M-l to L-l 18.
• the present invention is also directed to nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequences encoding the polypeptides described above
• the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence Polypeptides encoded by these polynucleotide sequences are also encompassed by the invention
• the invention also provides polypeptides having one or more ammo acids deleted from both the am o and the carboxyl termini of an AIM II polypeptide, which may be described generally as having residues n-m of Figures 1 A and IB
• the present invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues 83-m 1 of Figures 1 A and IB (/ e., SEQ ID NO 2), where m 1 is an integer from 89 to 239, corresponding to the position of the amino acid residue in Figures 1A and IB (SEQ ID NO 2)
• the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of a member selected from the group consisting of residues L-83 to M-239, L-83 to F-238, L-83 to A-237, L-83 to G-236, L-83 to F-235, L-83 to Y-234, L-83 to S-233, L-83 to R-232, L-83 to
• T-231 L-83 to G-230, L-83 to D-229, L-83 to R-228, L-83 to L-227, L-83 to R-226, L-83 to V-225, L-83 to L-224, L-83 to R-223, L-83 to E-222, L-83 to D-221, L-83 to L-220, L-83 to V-219, L-83 to R-218, L-83 to V-217, L-83 to V-216, L-83 to V-215, L-83 to E-214, L-83 to E-213, L-83 to G-212, L-83 to A-211, L-83 to E-210, L-83 to L-209, L-83 to H-208, L-83 to V-207, L-83 to
• V-206 L-83 to G-205, L-83 to G-204, L-83 to L-203, L-83 to F-202, L-83 to S-201 , L-83 to S-200, L-83 to D-199, L-83 to W-198, L-83 to W-197, L-83 to V-196, L-83 to R-195, L-83 to S-194, L-83 to S-193, L-83 to S-192, L-83 to T-191, L-83 to A-190, L-83 to R-189, L-83 to G-188, L-83 to C-187, L-83 to P-186, L-83 to S-185, L-83 to Q-184, L-83 to Q-183, L-83 to S-182, L-83 to
• the present application is also directed to nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding the AIM II polypeptides described above
• the present invention also encompasses
• the polynucleotides of the invention encode functional attributes of AIM II
• Preferred embodiments of the invention in this regard include fragments that comprise, or alternatively consist of, one, two, three, four or more of one or more of the following functional domains alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions"), turn and turn-forming regions ("turn-regions”), coil and coil-forming regions ("coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions of AIM 11.
• the data representing the structural or functional attributes of AIM II set forth in Figures 3A-3F and/or Table 2 was generated using the various identified modules and algorithms of the DNA*STAR set on default parameters.
• the data presented in columns VIII, IX, XIII, and XIV of Table 2 can be used to determine regions of AIM II which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII. IX, XIII, and/or IV by choosing values which represent regions ofthe polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
• Figures 3 A-3F Certain preferred regions in these regards are set out in Figures 3 A-3F, but may, as shown in Table 2, be represented or identified in tabular form.
• the DNA* STAR computer algorithm used to generate Figures 3A-3F (set on the original default parameters) was used to present the data in Figures 3A-3F in a tabular format (See Table 2).
• the tabular format ofthe data in Figure 3 A-3F may be used to easily determine specific boundaries of a preferred region.
• inventions set out in Figures 3A-3F and in Table 2 include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figures 1A and IB. As set out in Figures 3A-3F and in Table 2, such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions (columns I. Ill, V. and VII in Table 2).
• ca ca ca co ca ca ca o iii i-i i-i iQ i-i i-i ffl i-i i-i t-i
• the present invention is further directed to isolated polypeptides comprising, or alternatively consisting of, fragments of AIM II
• the invention provides isolated polypeptides comprising or alternatively consisting of, the amino acid sequences of a member selected from the group consisting of amino acids 1 -60, 1 1 -70, 21 -80, 31 -90, 41-100, 51-1 10, 61 - 120, 71-130, 81-140, 91 -150, 101 -160 1 1 1 -170, 121 -180, 131-190, 141-200 151-210 161-220, 171 -230, and 181 -240 of SEQ ID NO 2, as well as isolated polynucleotides which encode these polypeptides
• the present invention is also directed to isolated polypeptides comprising, or alternatively consisting of, domains of AIM II
• the invention provides polypeptides comprising, or alternatively consisting of, beta-sheet regions of AIM II set out in Table 2
• These polypeptides include polypeptides comprising, or alternatively consisting of ammo acid sequences of a member selected from the group consisting of amino acid residues from about 7 to about 14, amino acid residues from about 18 to about 23 amino acid residues from about 17 to about 25, amino acid residues from about 33 to about 46 ammo acid residues from about 35 to about 39, amino acid residues from about 57 to about 60, amino acid residues from about 67 to about 72, amino acid residues from about 102 to about 107, amino acid residues from about 121 to about 126, amino acid residues from about 131 to about 166 amino acid residues from about 141 to about 152, amino acid residues from about 158 to about 169, amino acid residues from about 213 to about 221 and amino acid
• the invention is further directed to isolated polynucleotides comprising, or alternatively consisting of, nucleic acid molecules which encode the beta-sheet regions set out in Table 2, and isolated polypeptides comprising, or alternatively consisting of, amino acid sequences at least 80% identical, and more preferably at least 85% 90%, 92%, 95% 96% 97%, 98% or 99% identical to nucleic acid molecules encoding beta-sheet regions ofthe AIM II protein
• the invention is also directed to isolated polypeptides comprising, or alternatively consisting of. fragments of AIM II which have one or more functional activity associated with AIM II polypeptides having the amino acid sequence of
• Antigenic epitope-bearmg peptides and polypeptides ofthe invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide ofthe invention See. for instance, Wilson et al. Cell 37:767-778 (1984) at 777
• the present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO 2 or SEQ ID NO 39, or an epitope ofthe polypeptide sequence encoded by a polynucleotide sequence contained in deposited clone identified as ATCC Accession No 97689 or 97483 or encoded by a polynucleotide that hybridizes to the complement of the sequence of SEQ ID NO 1 or SEQ ID NO 38 or contained in deposited clone identified as ATCC Accession No 97689 or 97483 under stringent hybridization conditions or lower stringency hybridization conditions as defined supra
• the present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO: 1 or SEQ ID NO:38), polynucleotide sequences ofthe complementary strand of a polynucleotide sequence en
• epitopes refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human
• the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide.
• An "immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art. for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al, Proc. Nail Acad. Sci.
• antigenic epitope is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross- reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic.
• Fragments that function as epitopes may be produced by any conventional means. (See, e.g., Houghten, Proc. Nail. Acad. Sci. USA 52:5131-5135 (1985), further described in U.S. Patent No. 4,631,21 1).
• antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, and, most preferably, between about 15 to about 30 amino acids.
• Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10. 15, 20, 25, 30, 35, 40, 45, 50, 55, 60. 65.
• Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope
• Antigenic epitopes can be used as the target molecules in immunoassays (See, for instance, Wilson el al, Cell 37 767-778
• immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art (See, for instance, Sutcliffe et al, supra, Wilson el al, supra. Chow et al, Proc Nati Acad Sci USA 82 910-914, and Bittle el a/ , J Gen Virol 66.2347-2354 (1985)
• a preferred immunogenic epitope includes the secreted protein
• the polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as, for example, rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier
• immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e
• Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, m vitro immunization, and phage display methods See. e.g., Sutcliffe et al, supra, Wilson et al, supra, and Bittle et al,
• mice may be immunized with free peptide, however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid
• KLH keyhole limpet hemacyanin
• peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde
• Animals such as, for example, rabbits, rats, and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 micrograms of peptide or carrier protein and Freund's adjuvant or
• polypeptides of the present invention comprising an immunogenic or antigenic epitope can be fused to other polypeptide sequences
• the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CHI, CH2, CH3, or any combination thereof and portions thereof) resulting in chimeric polypeptides
• immunoglobulins IgA, IgE, IgG, IgM
• CHI constant domain of immunoglobulins
• Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g. , the hemagglutinin ("HA") tag or flag tag) to aid in detection and purification ofthe expressed polypeptide
• an epitope tag e.g. , the hemagglutinin ("HA") tag or flag tag
• HA hemagglutinin
• a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al.
• DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides See, generally, U S Patent Nos 5,605,793. 5,81 1,238, 5,830,721 , 5,834.252.
• alteration of polynucleotides corresponding to SEQ ID NO 1 and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling
• DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence
• polynucleotides of the invention, or the encoded polypeptides may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination
• one or more components, motifs, sections, parts, domains, fragments, etc , of a polynucleotide coding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of
• Table 2 A list of exemplified amino acid sequences comprising immunogenic epitopes are shown in Table 2 It is pointed out that Table 2 only lists amino acid residues comprising epitopes predicted to have the highest degree of antigenicity using the algorithm of Jameson and Wolf, Comp. App Biosci. - J 81 -186 (1988) (said references incorporated by reference in their entireties).
• the Jameson-Wolf antigenic analysis was performed using the computer program PROTEAN, using default parameters (Version 3.1 1 for the Power Macintosh. DNASTAR, Inc.,
• Table 2 1228 South Park Street Madison, WI
• Table 2 portions of polypeptides not listed in Table 2 are not considered non-immunogenic.
• the immunogenic epitopes of Table 2 is an exemplified list, not an exhaustive list, because other immunogenic epitopes are merely not recognized as such by the particular algorithm used.
• Amino acid residues comprising other immunogenic epitopes may be routinely determined using algorithms similar to the Jameson-Wolf analysis or by in vivo testing for an antigenic response using methods known in the art. See, e.g., Geysen et al, supra; U.S.
• Table 2 may include additional N-terminal or C-terminal amino acid residues.
• the additional flanking amino acid residues may be contiguous flanking N-terminal and/or C-terminal sequences from the polypeptides of the present invention, heterologous polypeptide sequences, or may include both contiguous flanking sequences from the polypeptides of the present invention and heterologous polypeptide sequences.
• the immunogenic and antigenic epitope-bearing fragments may be specified by either the number of contiguous amino acid residues, as described above, or further specified by N-terminal and C-terminal positions of these fragments on the amino acid sequence of SEQ ID NO:2. Every combination of a N-terminal and C-terminal position that a fragment of, for example, at least 7 or at least 15 contiguous amino acid residues in length could occupy on the amino acid sequence of SEQ ID NO:2 is included in the invention.
• At least 7 contiguous amino acid residues in length means 7 amino acid residues in length or any integer between 7 amino acids and the number of amino acid residues of the full length polypeptide ofthe present invention- Specifically, each and every integer between 7 and the number of amino acid residues of the full length polypeptide are included in the present invention.
• Immunogenic and antigenic epitope-bearing polypeptides ofthe invention are useful, for example, to make antibodies which specifically bind the polypeptides ofthe invention, and in immunoassays to detect the polypeptides of the present invention.
• the antibodies are useful, for example, in affinity purification ofthe polypeptides ofthe present invention.
• the antibodies may also routinely be used in a variety of qualitative or quantitative immunoassays, specifically for the polypeptides ofthe present invention using methods known in the art. e.g., Harlow et al, .ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor Laboratory Press; 2nd Ed. 1988).
• epitope-bearing polypeptides of the present invention may be produced by any conventional means for making polypeptides including synthetic and recombinant methods known in the art.
• epitope-bearing peptides may be synthesized using known methods of chemical synthesis.
• Houghten has described a simple method for the synthesis of large numbers of peptides, such as 10-20 mgs of 248 individual and distinct 13 residue peptides representing single amino acid variants of a segment of the HA1 polypeptide, all of which were prepared and characterized (by ELISA-type binding studies) in less than four weeks (Houghten et al, Proc. Nati Acad. Sci. U.S.A. 52:5131-5135 (1985)).
• Epitope-bearing polypeptides ofthe present invention are used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al, supra; Wilson et al, supra, and Bittle et al, J. Gen. Virol. 66:2341-2354 (1985).
• animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling of the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid.
• KLH keyhole limpet hemacyanin
• peptides containing cysteine residues may be coupled to a carrier using a linker such as N-maleimidobenzoyl- N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde.
• Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 ⁇ g of peptide or carrier protein and Freund's adjuvant. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface.
• the titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
• polypeptides of the present invention comprising an immunogenic or antigenic epitope can be fused to heterologous polypeptide sequences.
• the polypeptides ofthe present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH 1 , CH2, CH3 , any combination thereof including both entire domains and portions thereof) resulting in chimeric polypeptides.
• immunoglobulins IgA, IgE, IgG, IgM
• CH 1 , CH2, CH3 any combination thereof including both entire domains and portions thereof
• proteins ofthe invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N Y., and Hunkapiller, M. et al,
• a peptide corresponding to a fragment ofthe AIM II polypeptides ofthe invention can be synthesized by use of a peptide synthesizer.
• nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the AIM II polypeptide sequence.
• Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, alpha- amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, alpha- Abu, alpha-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, alpha-alanine, fluoro-amino acids, designer amino acids such as alpha-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general.
• Non-naturally occurring variants may be produced using art-known mutagenesis techniques, which include, but are not limited to ohgonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see e g Carter et al Nucl Acids Res 13 4331 (1986), and Zoller et al , Nucl Acids Res 10 6487 (1982)), cassette mutagenesis (see, e g Wells et al , Gene 34 315 (1985)), restriction selection mutagenesis (vee e g Wells et / Philos Trans R Soc London eiA 317 415 (1986))
• the invention additionally encompasses AIM II polypeptides which are differentially modified during or after translation, e g , by glycosylation, acetylation, phosphorylation, amidation, de ⁇ vatization by known protecting/blocking groups proteolvtic cleavage linkage to an antibody molecule or other cellular ligand, etc Any of numerous chemical modifications may be carried out by known techniques including but not limited to, specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH 4 , acetylation, formylation oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc
• Additional post-translational modifications encompassed by the invention include, for example, e g , N- nked or O-hnked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-hnked or O-hnked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression
• the polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation ofthe protein
• a detectable label such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation ofthe protein
• the chemical moieties for de ⁇ vitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like
• the polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two. three or more attached chemical moieties.
• the polymer may be of any molecular weight, and may be branched or unbranched.
• the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing.
• Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects ofthe polyethylene glycol to a therapeutic protein or analog).
• the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 1 1,000, 1 1,500, 12,000, 12,500, 13,000, 13,500, 14,000,
• polyethylene glycol may have a branched structure.
• Branched polyethylene glycols are described, for example, in U.S. Patent No.
• polyethylene glycol may be covalently bound through amino acid residues via a reactive group such as, a free ammo or carboxyl group
• Reactive groups are those to which an activated polyethvlene glvcoi molecule may be bound
• the amino acid residues having a free ammo group may include lysine residues and the N-terminal amino acid residues, those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules Preferred for therapeutic purposes is attachment at an ammo group such as attachment at the N-terminus or lysine group
• polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues
• polyethylene glycol can be linked to a proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues
• the method of obtaining the N-terminally pegylated preparation may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules
• Selective proteins chemically modified at the N- terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for de ⁇ vatization in a particular protein Under the approp ⁇ ate reaction conditions substantially selective de ⁇
• pegylation of the proteins of the invention may be accomplished bv any number of means
• polyethylene glycol may be attached to the protein either directly or by an intervening linker
• Lmkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al , Cm Rev Iheia Di ug Camei Sys 9 249-304 (1992), Francis et al Intern J of Hematol 68 1 -18 (1998) U S Patent No 4,002,531 , U S Patent No 5,349 052, WO 95/06058 and WO 98/32466 the disclosures of each of which are incorporated herein by reference
• One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monmethoxy polyethylene glycol (MPEG) using tresylchlo ⁇ de (ClSO 2 CH 2 CF ) Upon reaction of protein with tresylated MPEG, polyethylene glycol is directly attached to amine groups ofthe protein
• MPEG monmethoxy polyethylene glycol
• tresylchlo ⁇ de ClSO 2 CH 2 CF
• the invention includes protein-polyethylene glycol conjugates produced by reacting proteins ofthe invention with a polyethylene glycol molecule having a 2,2,2-t ⁇ fluoreothane sulphonyl group
• Polyethylene glycol can also be attached to proteins using a number of different intervening linkers
• U S Patent No 5,612,460 discloses urethane linkers for connecting polyethylene glycol to proteins
• Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG- succimmidylsuccinate MPEG activated with lJ'-carbonyldnmidazole, MPEG-
• the number of polyethylene glycol moieties attached to each protein ofthe invention may also vary.
• the pegylated proteins ofthe invention may be linked, on average, to 1, 2, 3, 4, 5, 6.
• the present invention further relates to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a polypeptide, preferably an epitope, ofthe present invention (as determined by immunoassays well known in the art for assaying specific antibody-antigen binding).
• TCR T-cell antigen receptors
• Antibodies ofthe invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies ofthe invention), and epitope-binding fragments of any of the above.
• antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen.
• the immunoglobulin molecules ofthe invention can be of any type (e.g. , IgG, IgE, IgM, IgD, IgA and IgY), class (e.g. ,
• IgGl IgG2, IgG3, IgG4, IgAl and IgA2 or subclass of immunoglobulin molecule.
• the antibodies are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab' and F(ab')2.
• Fd single-chain Fvs (scFv). single-chain antibodies, disulfide- nked Fvs (sdFv) and fragments comprising either a VL or VH domain
• Antigen-binding antibody fragments, including single-chain antibodies may comprise the variable reg ⁇ on(s) alone or in combination with the entirety or a portion ofthe following hinge region, CHI , CH2.
• antigen-binding fragments also comprising any combination of variable reg ⁇ on(s) with a hinge region, CHI , CH2, and CH3 domains
• the antibodies of the invention may be from any animal origin including birds and mammals
• the antibodies are human, murine, donkey, ship rabbit, goat, guinea pig. camel, horse, or chicken
• "human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U S Patent No 5,939,598 by Kucherlapati et al.
• the antibodies ofthe present invention may be monospecific, bispecific, trispecific or of greater multispecificity Multispecific antibodies may be specific for different epitopes of a polypeptide ofthe present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material See, e.g.,
• Antibodies ofthe present invention may be described or specified in terms ofthe epitope(s) or portion(s) of a polypeptide ofthe present invention that they recognize or specifically bind
• the epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or listed in the Tables and Figures
• Antibodies that specifically bind any epitope or polypeptide ofthe present invention may also be excluded Therefore the present invention includes antibodies that specifically bind polypeptides of the present invention and allows for the exclusion of the same
• Antibodies ofthe present invention may also be described or specified in terms of their cross-reactivity
• Antibodies that do not bind any other analog, ortholog or homolog of a polypeptide of the present invention are included
• Antibodies that bind polypeptides with at least 95%, at least 90% at least 85%o, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide ofthe present invention are also included in the present invention
• Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%o, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention
• Further included in the present invention are antibodies that bind polypeptid
• the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides ofthe invention either partially or fully.
• the invention features both receptor-specific antibodies and ligand-specific antibodies.
• the invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation.
• Receptor activation i.e., signaling
• receptor activation can be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting the phosphorylation (e-g . tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra).
• antibodies are provided that inhibit ligand or receptor activity by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%) ofthe activity in absence ofthe antibody.
• the invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand.
• receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand.
• neutralizing antibodies which bind the ligand and prevent binding ofthe ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor.
• antibodies which activate the receptor are also act as receptor agonists, / ' .
• the antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities ofthe peptides of the invention disclosed herein.
• the invention further relates to antibodies which act as agonists or antagonists ofthe polypeptides ofthe present invention.
• the above antibody agonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281 ; U.S. Patent No. 5,81 1,097; Deng et al, Blood 2 ⁇ : 1981- 1988 (1998), Chen el al , Cancer Res. 58(16) .
• Antibodies of the present invention may be used, for example, but not limited to, to purify, detect, and target the polypeptides ofthe present invention, including both in vitro and in vivo diagnostic and therapeutic methods.
• the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g. , Harlow et al . Antibodies: A Laboratory Manual,
• the antibodies ofthe present invention may be used either alone or in combination with other compositions.
• the antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions.
• antibodies ofthe present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, or toxins. See, e.g., PCT publications WO
• the antibodies of the invention include derivatives that are modified, i.e, by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response
• the antibody derivatives include antibodies that have been modified, e.g. , by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
• the antibodies ofthe present invention may be generated by any suitable method known in the art.
• Polyclonal antibodies to an antigen of interest can be produced by various procedures well known in the art.
• a polypeptide ofthe invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen.
• Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to.
• Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
• monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught,, for example, in Harlow et al. Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al, in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties).
• the term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technologv
• the term “monoclonal antibody' refers to an antibody that is derived from a single clone including any eukaryotic, prokaryotic or phage clone, and not the method by which it is produced Thus, the term “monoclonal antibody” is not limited to antibodies produced through hybridoma technology
• Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hyb ⁇ doma and recombinant and phage display technology
• mice can be immunized with a polypeptide of the invention or a cell expressing such peptide
• an immune response is detected e g
• antibodies specific for the antigen are detected in the mouse serum
• the mouse spleen is harvested and splenocytes isolated
• the splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC Hybridomas are selected and cloned by limited dilution
• the hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide ofthe invention Ascites fluid, which generally contains high levels of antibodies can be generated by immunizing mice with positive hybridoma clones Accordingly, the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising cult
• Antibody fragments that recognize specific epitopes may be generated by known techniques
• Fab and F(ab')2 fragments ofthe invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments)
• F(ab')2 fragments contain the variable region, the light chain constant region and the CHI domain of the heavy chain
• the antibodies ofthe present invention can also be generated using various phage display methods known in the art
• phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them
• phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine)
• Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e g.
• Phage used in these methods are typically filamentous phage including fd and Ml 3 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein
• phage display methods that can be used to make the antibodies ofthe present invention include those disclosed in
• the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below
• techniques to recombinantly produce Fab, Fab 1 and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324.
• a chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art.
• Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule.
• CDRs complementarity determining regions
• framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
• These framework substitutions are identified by methods well known in the art, e.g. , by modeling ofthe interactions ofthe CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al. , U.S. Patent No. 5,585,089; Riechmann et al, Nature 332:323 (1988), which are incorporated herein by reference in their entireties )
• Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-gratting (EP 239,400.
• Human antibodies are particularly desirable for therapeutic treatment of human patients
• Human antibodies can be made b ⁇ a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences See also, U S Patent Nos 4,444,887 and 4,716, 1 1 1, and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654.
• Human antibodies can also be produced using transge ic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes
• the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells
• the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes
• the mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination
• homozygous deletion of the JH region prevents endogenous antibody production
• the modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice
• the chimeric mice are then bred to produce homozygous offspring that express human antibodies
• the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all antigen, e.g
• IgA, IgM and IgE antibodies For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int Rev Immunol 13 65-93) For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies see, e g , PCT publications WO 98/24893, WO 96/34096, WO 96/33735, U S Patent Nos 5,413,923, 5,625, 126,
• Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection"
• a selected non-human monoclonal antibody e g , a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope (Jespers et al . Bio, 'technology 12 899-903 (1988))
• antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" polypeptides of the invention using techniques well known to those skilled in the art (See, e , Greenspan & Bona, FASEB J 7(5) 437-444 (1989) and Nissinoff, J Immunol 147(8) 2429-2438 (1991))
• antibodies which bind to and competitively inhibit polypeptide multime ⁇ zation and/or binding of a polypeptide ofthe invention to a ligand can be used to generate anti-idiotypes that "mimic" the polypeptide multime ⁇ zation and/or binding domain and.
• anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its gands/receptors and thereby block its biological act ⁇ v ⁇ t ⁇
• the invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof
• the invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e g as defined supia, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide of the invention preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO 2
• the polynucleotides may be obtained, and the nucleotide sequence ofthe polynucleotides determined, by any method known in the art
• a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e g , as described in Kutmeier et al BioTechniques 17 242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions ofthe sequence encoding the antibody, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR
• a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source If a clone containing a nucleic acid encoding a particular antibody is not available but the sequence ofthe antibody molecule is known, a nucle
• nucleotide sequence and corresponding amino acid sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al, 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al, eds., 1998,
• the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability.
• CDRs complementarity determining regions
• one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra.
• the framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (,vee, e.g., Chothia et al. , J. Mol. Biol. 278:451-419 (1998) for a listing of human framework regions).
• the polynucleotide generated by the combination ofthe framework regions and CDRs encodes an antibody that specifically binds a polypeptide ofthe invention.
• one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding ofthe antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intracham disulfide bond to generate antibody molecules lacking one or more intracham disulfide bonds Other alterations to the polynucleotide are encompassed by the present invention and within the skill ofthe art In addition, techniques developed foi the production of "chimeric antibodies" (Morrison et al , 1984, Proc Nati Acad Sci 81 851 -855, Neuberger et al . 1984, Nature 312 604-608, Takeda et al , 1985.
• a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g. , humanized antibodies
• the antibodies ofthe invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques
• an antibody of the invention or fragment, derivative or analog thereof, e.g., a heavy or light chain of an antibody of the invention, requires construction of an expression vector containing a polynucleotide that encodes the antibody
• the vector for the production ofthe antibody molecule may be produced by recombinant DNA technology using techniques well known in the art
• methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals
• These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and m vivo genetic recombination
• the invention thus provides rephcable vectors comprising a polynucleotide that encodes the antibody
• variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain
• the expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody ofthe invention
• the invention includes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof operably linked to a heterologous promoter
• vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression ofthe entire immunoglobulin molecule, as detailed below
• host-expression vector systems may be utilized to express the antibody molecules of the invention.
• Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ
• microorganisms such as bacteria (e.g., E. colt, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces,
• Pichia transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g. , baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus.
• insect cell systems infected with recombinant virus expression vectors e.g. , baculovirus
• plant cell systems infected with recombinant virus expression vectors e.g., cauliflower mosaic virus, CaMV
• tobacco mosaic virus e.g., cauliflower mosaic virus, CaMV
• telomeres e.g., TMV
• recombinant plasmid expression vectors e.g., Ti plasmid
• mammalian cell systems e.g., COS, CHO, BHK, 293, 3T3 cells
• promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
• bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule.
• mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al, 1986, Gene 45: 101 ; Cockett et al, 1990, Bio/Technology 8:2).
• a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E.
• coli expression vector pUR278 (Ruther et al , 1983, EMBO J 2 1791 ) in which the antibody coding sequence ma ⁇ be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced
• plN vectors Inouye & Inouye, 1985 Nucleic Acids Res 13 3101 -3109, Van Heeke & Schuster 1989 J Biol Chem 24 5503-5509
• pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST)
• GST glutathione S-transferase
• fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione
• the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites
• Aulographa ca fornica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes
• the virus grows in Spodoptera frugipeida cells
• the antibody coding sequence may be cloned individually into non-essential regions (for example the polyhed ⁇ n gene) of the virus and placed under control of an AcNPV promoter (for example the polyhed ⁇ n promoter)
• the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e g the late promoter and tripartite leader sequence
• This chimeric gene may then be inserted in the adenovirus genome by in vitio or in vivo recombination Insertion in a non- essential region ofthe viral genome (e g , region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (e g , see Logan & Shenk, 1984, Proc Nati Acad Sci USA 81 355-359)
• Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences These signals include the ATG initiation codon and adjacent sequences Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire
• exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc (see Bittner et al, 1987. Methods in Enzymol 153 51 -544)
• a host cell strain may be chosen which modulates the expression ofthe inserted sequences, or modifies and processes the gene product in the specific fashion desired Such modifications (e.g. , glycosylation) and processing (e.g. , cleavage) of protein products may be important for the function ofthe protein
• Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products
• Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed
• eukaryotic host cells which possess the cellular machinery for proper processing ofthe primary transcript, glycosylation, and phosphorylation ofthe gene product may be used
• mammalian host cells include but are not limited to CHO, VERY, BHK.
• breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.
• cell lines which stably express the antibody molecule may be engineered Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc ), and a selectable marker Following the introduction ofthe foreign DNA, engineered cells may be allowed to grow for 1 -2 days in an enriched media, and then are switched to a selective media
• the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines
• This method av advantageously be used to engineer cell lines which express the antibody molecule
• Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule
• a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al, 1977, Cell 1 1 223), hypoxanthme-guanine phospho ⁇ bosyltransferase (Szybalska & Szybalski.
• genes can be employed in tk-, hgprt- or aprt- cells, respectively
• antimetabohte resistance can be used as the basis of selection for the following genes dhfr, which confers resistance to methotrexate (Wigler et al, 1980, Nati Acad Sci USA 77 357, O'Hare et al , 1981, Proc Nati Acad Sci USA 78 1527), gpt.
• the expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells m DNA cloning, Vol 3 (Academic Press New York, 1987))
• vector amplification for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells m DNA cloning, Vol 3 (Academic Press New York, 1987)
• a marker in the vector system expressing antibody is amplifiable
• increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene Since the amplified region is associated with the antibody gene production ofthe antibody will also increase (Crouse et al 1983 Mol Cell Biol
• the host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide
• the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides
• a single vector may be used which encodes both heavy and light chain polypeptides
• the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot 1986 Nature 322 52, Kohler 1980, Pioc Nati Acad Sci USA 77 2197)
• the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA
• an antibody molecule of the invention may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e g , ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), cent ⁇ fugation, differential solubility, or by any other standard technique for the purification of proteins
• the present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20 or 50 amino acids of the polypeptide) of the present invention to generate fusion proteins
• the fusion does not necessarily need to be direct, but may occur through linker sequences
• the antibodies may be specific for antigens other than polypeptides (or portion thereof, preferably at least 10, 20 or 50 amino acids of the polypeptide) of the present invention
• antibodies may be used to target the polypeptides ofthe present invention to particular cell types, either m vitro or m vivo, by fusing or conjugating the polypeptides ofthe present invention to antibodies specific for particular cell surface receptors
• Antibodies fused or conjugated to the polypeptides of the present invention may also be used in /// vitro immunoassays and purification methods using methods known in the art See e.g .
• the present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions
• the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof
• the antibody portion fused to a polypeptide ofthe present invention may comprise the constant region, hinge region. CHI domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof
• the polypeptides may also be fused or conjugated to the above antibody portions to form multimers
• Fc portions fused to the polypeptides ofthe present invention can form dimers through disulfide bonding between the Fc portions
• the polypeptides of the present invention may be fused or conjugated to the above antibody portions to increase the m vivo half life of the polypeptides or for use in immunoassays using methods known in the art Further, the polypeptides ofthe present invention may be fused or conjugated to the above antibody portions to facilitate purification
• chimeric proteins consisting of the first two domains of the human CD4-polypept ⁇ de and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EP 394,827, Traunecker et al , Nature 331 84-86 (1988)
• the polypeptides of the present invention fused or conjugated to an antibody having disulfide- linked dime ⁇ c structures (due to the
• IgG mav also be more efficient in binding and neutralizing other molecules, than the monome ⁇ c secreted protein or protein fragment alone (Fountoulakis et al J Biochem 270 3958-3964 (1995))
• the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties (EP A 232,262)
• deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired
• the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations
• human proteins, such as hIL-5 receptor have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5 (See, D Bennett et al , J Molecular Recognition 8 52-58 (1995), K Johanson et al , J Biol Chem 270 9459-9471 (1995)
• the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitates their purification
• the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc , 9259 Eton Avenue, Chatsworth, CA, 9131 1), among others many of which are commercially available As described in Gentz et al , Proc Nati Acad Sci USA 86 821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein
• Other peptide tags useful for purification include, but are not limited to, the "HA" tag. which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al, Cell 37:767 (1984)) and the "flag" tag.
• the present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent.
• the antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. See, for example, U.S. Patent No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention.
• suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
• suitable prosthetic group complexes include streptavidin/biotin and avidin biotin;
• suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
• an example of a luminescent material includes luminol;
• examples of bioluminescent materials include luciferase, luciferin, and aequorin;
• suitable radioactive material include I25 I, 13I I, l ⁇ In or "Tc.
• an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion.
• a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.
• Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof
• Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine.
• 6-thioguan ⁇ ne. 6-thioguan ⁇ ne. cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cv.v-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g.. dactinomycin (formerly actinomycin), bleomycin. mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).
• alkylating agents e
• the conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents.
• the drug moiety may be a protein or polypeptide possessing a desired biological activity.
• Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as.
• a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin
• a protein such as tumor necrosis factor, a-interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin
• angiostatin or endostatin e
• lymphokines interleukin- 1
• IL-2 interleukin-2
• IL-6 interleukin-6
• GM-CSF granulocyte macrophase colony stimulating factor
• G-CSF granulocyte colony stimulating factor
• Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification ofthe target antigen.
• solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
• an antibody can be conjugated to a second antibody to form an antibody heteroconiugate as described by Segal in U S Patent No 4,676,980, which is incorporated herein by reference in its entirety
• An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytok ⁇ ne(s) can be used as a therapeutic
• the antibodies ofthe invention may be assayed for lmmunospecific binding by any method known in the art
• the immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion prec ⁇ itin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, lmmunoradiomet ⁇ c assays, fluorescent immunoassays, protein A immunoassays, to name but a few Such assays are routine and well known in the art (see, e g , Ausubel et al , eds, 1994,
• lmmunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as R1PA buffer ( 1 % NP-40 or Triton X- 100, 1 % sodium deoxycholate, 0.1 % SDS, 0J 5 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g.
• R1PA buffer 1 % NP-40 or Triton X- 100, 1 % sodium deoxycholate, 0.1 % SDS, 0J 5 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol
• protein phosphatase and/or protease inhibitors e.g.
• the antibody of interest to the cell lysate, incubating for a period of time (e.g. , 1 -4 hours) at 4° C, adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C, washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer.
• a period of time e.g. 1 -4 hours
• protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C, washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer.
• the ability ofthe antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g. western blot analysis.
• Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g. , 8%- 20% SDS-PAGE depending on the molecular weight ofthe antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose,
• a polyacrylamide gel e.g. , 8%- 20% SDS-PAGE depending on the molecular weight ofthe antigen
• blocking the membrane in blocking solution e.g., PBS with 3% BSA or non-fat milk
• washing buffer e.g. , PBS-Tween 20
• primary antibody the antibody of interest
• secondary antibody which recognizes the primary antibody, e.g., an anti-human antibody
• an enzymatic substrate e.g., horseradish peroxidase or alkaline phosphatase
• radioactive molecule e.g. , 32 P or 125 I
• ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e g , horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence ofthe antigen
• a detectable compound such as an enzymatic substrate (e g , horseradish peroxidase or alkaline phosphatase)
• a detectable compound such as an enzymatic substrate (e g , horseradish peroxidase or alkaline phosphatase)
• a detectable compound such as an enzymatic substrate (e g , horseradish peroxidase or alkaline phosphatase)
• the binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays
• a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e g , 3 H or 125 I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen
• the affinity ofthe antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis Competition with a second antibody can also be determined using radioimmunoassays
• the antigen is incubated with antibody of interest is conjugated to a labeled compound (e g , 3 H or 125 I) in the presence of increasing amounts of an unlabeled second
• the present invention is further directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the disorders or conditions described herein.
• Therapeutic compounds ofthe invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies ofthe invention (including fragments, analogs and derivatives thereof as described herein.
• the antibodies ofthe invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide ofthe invention, including, but not limited to, any one or more ofthe diseases, disorders, or conditions described herein such as, for example, graft versus host disease and autoimmune diseases, disorders, or conditions associated with such diseases or disorders (including, but not limited to, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (e.g., IgA nephropathy), Multiple Sclerosis, Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Purpura (e.g., Henloch-Scoenle
• Myasthenia Gravis and (c) insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, rheumatoid arthritis, schleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis/dermatomyositis, pernicious anemia, idiopathic Addison's disease, infertility, glomerulonephritis such as primary glomerulonephritis and IgA nephropathy, bullous pemphigoid, Sjogren's syndrome, diabetes millitus, and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis), chronic active hepatitis, primary biliary cirrhosis, other endocrine gland failure, vitiligo, vasculitis, post-MI, cardiotomy syndrome, urticaria, atopic dermatitis, asthma, inflammatory myopathies, and other inflammatory, granul
• X-linked infantile agammaglobulinemia acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia. transient hypogammaglobulinemia of infancy, unspecified hypogammaglobulinemia, agammaglobulinemia, common variable immunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS),
• CVID common variable immunodeficiency
• WAS Wiskott-Aldrich Syndrome
• X-linked immunodeficiency with hyper IgM non X-linked immunodeficiency with hyper IgM, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), antibody deficiency with normal or elevated Igs, immunodeficiency with thymoma, Ig heavy chain deletions, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), selective IgM immunodeficiency, recessive agammaglobulinemia (Swiss type), reticular dysgenesis, neonatal neutropenia, severe congenital leukopenia, thymic alymphoplasia-aplasia or dysplasia with immunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezelof syndrome-combined immunodeficiency with Igs, purine nucle
• antibodies ofthe invention are be used to treat, inhibit, prognose diagnose or prevent graft versus host disease and autoimmune disease
• antibodies ofthe invention are be used to treat, inhibit, prognose, diagnose or prevent rheumatoid arthritis
• antibodies of the invention are used to treat, inhibit, prognose diagnose or prevent systemic lupus erythematosis
• the treatment and/or prevention of diseases and disorders associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases and disorders
• Antibodies of the invention mav be provided in pharmaceutically acceptable compositions as known in the art or as described herein
• a summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e g as mediated by complement (CDC) or by effector cells (ADCC) Some of these approaches are described in more detail below Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation
• the antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e g . IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies
• the antibodies of the invention may be administered alone or in combination with other types of treatments (e g radiation therapy chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents) Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that ofthe patient is preferred.
• human antibodies, fragments derivatives, analogs, or nucleic acids are administered to a human patient for therapy or prophylaxis
• binding affinities include those with a dissociation constant orKd less than 5X10 "6 M, 10- 6 M, 5X10 "7 M, 10 "7 M, 5X10-*M.
• the invention also provides a method of screening compounds to identify those which enhance or block the action of AIM II on cells, such as its interaction with AIM Il-binding molecules such as receptor molecules.
• An agonist is a compound which increases the natural biological functions of AIM II or which functions in a manner similar to AIM II, while antagonists decrease or eliminate such functions.
• a cellular compartment such as a membrane preparation, may be prepared from a cell that expresses a molecule that binds AIM II, such as a molecule of a signaling or regulatory pathway modulated by AIM II. The preparation is incubated with labeled AIM II in the absence or the presence of a candidate molecule which may be an AIM II agonist or antagonist.
• AIM II-like effects of potential agonists and antagonists may by measured, for instance, by determining activity of a second messenger system following interaction of the candidate molecule with a cell or appropriate cell preparation, and comparing the effect with that of AIM II or molecules that elicit the same effects as AIM II.
• Second messenger systems that may be useful in this regard include but are not limited to AMP guanylate cyclase. ion channel or phosphoinositide hydrolysis second messenger systems.
• an assay for AIM II antagonists is a competitive assay that combines AIM II and a potential antagonist with membrane-bound AIM II receptor molecules or recombinant AIM II receptor molecules under appropriate conditions for a competitive inhibition assay.
• AIM II can be labeled, such as by radioactivity, such that the number of AIM II molecules bound to a receptor molecule can be determined accurately to assess the effectiveness ofthe potential antagonist.
• Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to a polypeptide of the invention, and thereby inhibit or extinguish its activity.
• Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a receptor molecule, without inducing AIM Il-induced activities, thereby preventing the action of AIM II by excluding AIM II from binding.
• Antagonists ofthe invention include fragments of the AIM II polypeptide having the amino acid sequence shown in SEQ ID NO: 2.
• Antisense technology can be used to control gene expression through antisense DNA or
• RNA or through triple-helix formation are discussed, for example, in Okano, J. Neuroche . 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988).
• RNA ohgonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into AIM II polypeptide
• the oligonucleotides described above can also be delivered to cells such that the antisense
• TNF Tumor Necrosis Factor
• the Tumor Necrosis Factor (TNF) family ligands are known to be among the most pleiotropic cytokines, inducing a large number of cellular responses, including cytotoxicity, anti-viral activity, immunoregulatory activities, and the transcriptional regulation of several genes (Goeddel, D V etal, "Tumor Necrosis Factors Gene Structure and Biological Activities," Symp Quant. Biol. 51 597-609 (1986), Cold Spring Harbor, Beutler, B , and Cerami, A , Aram. Rev. Biochem. 57 505-518 (1988), Old, L J . Set. Am. 258 59-75 (1988), Fiers, W , FEBS Lett. 285 199-224 (1991))
• the TNF-family ligands induce such various cellular responses by binding to TNF-family receptors
• AIM II polynucleotides, polypeptides, agonists or antagonists of the invention may be used in developing treatments for any disorder mediated (directly or indirectly) by defective, or insufficient amounts of AIM II AIM II polypeptides, agonists or antagonists may be administered to a patient (e.g., mammal, preferably human) afflicted with such a disorder Alternatively, a gene therapy approach may be applied to treat such disorders Disclosure herein of AIM II nucleotide sequences permits the detection of defective AIM II genes, and the replacement thereof with normal AIM Il-encoding genes Defective genes may be detected in m vitro diagnostic assays, and by comparison of the AIM II nucleotide sequence disclosed herein with that of a AIM II gene derived from a patient suspected of harboring a defect in this gene
• the AIM II polypeptide ofthe present invention may be employed to treat lymphoproliferative disease which results in lymphadenopathy, the AIM II mediates apoptosis by stimulating clonal deletion of T-cells and may therefore, be employed to treat autoimmune disease, to stimulate peripheral tolerance and cytotoxic T-cell mediated apoptosis
• the AIM II may also be employed as a research tool in elucidating the biology of autoimmune disorders including systemic lupus erythematosus (SLE), Graves' disease, immunoproliferative disease lymphadenopathy (IPL), angioimmunoproliferative lymphadenopathy (AIL), immunoblastive lymphadenopathy (IBL), rheumatoid arthritis, diabetes, and multiple sclerosis, allergies and to treat graft versus host disease.
• SLE systemic lupus erythematosus
• Graves' disease immunoproliferative disease lymphadenopathy
• AIL angioimmunopro
• the AIM II polynucleotides, polypeptides and/or agonists or antagonists of the invention may also be used to treat, prevent, diagnose and/or prognose diseases which include, but are not limited to, autoimmune disorders, immunodeficiency disorders, and graft versus host disease.
• the AIM II polypeptide ofthe present invention may also be employed to inhibit neoplasia, such as tumor cell growth.
• the AIM II polypeptide may be responsible for tumor destruction through apoptosis and cytotoxicity to certain cells.
• AIM II may also be employed to treat diseases which require growth promotion activity, for example, restenosis, since AIM II has proliferation effects on cells of endothelial origin.
• AIM II may, therefore, also be employed to regulate hematopoiesis in endothelial cell development.
• Diseases associated with increased cell survival, or the inhibition of apoptosis, that may be treated, prevented, diagnosed and/or prognosed with the AIM II polynucleotides, polypeptides and/or agonists or antagonists of the invention include, but are not limited to, cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma. endothelioma, osteoblastoma.
• cancers such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma,
• osteoclastoma osteosarcoma. chondrosarcoma. adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer
• autoimmune disorders such as, multiple sclerosis, Sjogren's syndrome. Grave's disease
• Hashimoto's thyroiditis autoimmune diabetes, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis, autoimmune gastritis, autoimmune thrombocytopenic purpura, and rheumatoid arthritis
• viral infections such as herpes viruses, pox viruses and adenoviruses
• inflammation graft vs. host disease (acute and/or chronic), acute graft rejection, and chronic graft rejection.
• AIM II polynucleotides, polypeptides, agonists, or antagonists ofthe invention are used to inhibit growth, progression, and/or metastasis of cancers, in particular those listed above or in the paragraph that follows.
• Additional diseases or conditions associated with increased cell survival, that may be treated, prevented, diagnosed and/or prognosed with the AIM II polynucleotides, polypeptides and/or agonists or antagonists of the invention include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic.
• leukemia including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, my
• sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pan
• Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma Diseases associated with increased apoptosis, that may be treated, prevented, diagnosed and/or prognosed with the AIM II polynucleotides, polypeptides and/or agonists or antagonists ofthe invention include, but are not limited to, AIDS; neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and brain tumor or prior associated disease); autoimmune disorders
• myelodysplastic syndromes such as aplastic anemia
• AIM II polynucleotides, polypeptides, agonists, and/or antagonists are used to treat the diseases and disorders listed above.
• AIM II polynucleotides, polypeptides, agonists or antagonists of the invention are used to treat AIDS and pathologies associated with AIDS.
• Another embodiment of the present invention is directed to the use of AIM II polynucleotides, polypeptides, or antagonists to reduce AIM Il-mediated death of T cells in HIV-infected patients
• AIM II polynucleotides, polypeptides, or antagonists to reduce AIM Il-mediated death of T cells in HIV-infected patients
• the role of T cell apoptosis in the development of AIDS has been the subject of a number of studies (see, for example, Meyaard et al. , Science 257:217-219 (1992); Groux et al , J. Exp. Med. , 775:331 (1992); and Oyaizu et al, in Cell Activation and Apoptosis in HIV Infection, Andrieu and Lu, Eds., Plenum Press, New York, pp. 101-1 14 (1995)).
• Fas-mediated apoptosis has been implicated in the loss of T cells in HIV individuals (Katsikis et al, J. Exp. Med. 181:2029-2036 (1995). It is also likely that T cell apoptosis occurs through multiple mechanisms. For example, at least some ofthe T cell death seen in HIV patients is likely to be mediated by AIM II.
• AICD activated-induced cell death
• the patient is asymptomatic when treatment with AIM II polynucleotides, polypeptides, or antagonists commences.
• peripheral blood T cells may be extracted from an HIV patient, and tested for susceptibility to AIM Il-mediated cell death by procedures known in the art.
• a patient's blood or plasma is contacted with AIM II antagonists (e.g., anti-AIM II antibodies) ofthe invention ex vivo.
• AIM II antagonists may be bound to a suitable chromatography matrix by procedures known in the art.
• the patient's blood or plasma flows through a chromatography column containing AIM II antagonist bound to the mat ⁇ x, before being returned to the patient The immobilized AIM II antagonist binds AIM II, thus removing AIM II protein from the patient's blood
• an AIM II polynucleotide, polypeptide, or antagonist ofthe invention is administered in combination with other inhibitors of T cell apoptosis
• Fas-mediated apoptosis also has been implicated in loss of T cells in HIV individuals (Katsikis et al , 1 Exp Med 181 2029-2036 (1995))
• a patient susceptible to both Fas ligand mediated and AIM Il-mediated T cell death may be treated with both an agent that blocks AIM II/AIM II receptor interactions and an agent that blocks Fas-hgand/Fas interactions Suitable agents for blocking binding of Fas-ligand to
• Fas include but are not limited to, soluble Fas polypeptides, multime ⁇ c forms of soluble Fas polypeptides (e g , dimers of sFas/Fc), anti-Fas antibodies that bind Fas without transducing the biological signal that results in apoptosis, anti-Fas-hgand antibodies that block binding of Fas-hgand to Fas, and muteins of Fas- gand that bind Fas but do not transduce the biological signal that results in apoptosis
• the antibodies employed according to this method are monoclonal antibodies
• suitable agents for blocking Fas-hgand/Fas interactions including blocking anti-Fas monoclonal antibodies, are described in WO 95/10540, hereby incorporated by reference
• agents which block binding of TRAIL to a TRAIL receptor are administered with the AIM II polynucleotides, polypeptides, or antagonists ofthe invention
• Hematopoiesis is a multi-step cell proliferation and differentiation process which begins with a pool of multipotent stem cells. These cells can proliferate and differentiate into hematopoietic progenitors in reply to different stimuli.
• the AIM II polypeptides ofthe invention, as well as agonists and antagonists thereof, may be used to either stimulate or inhibit development of hematopoietic cells and, in particular, erythropoietic precursor cells.
• the AIM II polypeptides used to regulate development of hematopoietic cells may be contacted with hematopoietic target cells in a number of forms, e.g., as full-length or mature polypeptides, AIM II polypeptide fragments, or as hybrid fusion proteins composed of AIM II and a non- AIM II polypeptide (e.g., colony stimulating factors, erythropoietin, interferons, interleukins, etc.). Methods for producing such fusion proteins are described, for example, in Mele et al., U.S. Patent No. 5,916,773.
• hematopoietic cells refers cells of hematopoietic origin such as erythrocytes, thrombocytes, lymphocytes, eosinophils, basophils, macrophages, and monocytes.
• erythropoietic precursor cells refers cells of erythropoietic origin, such as erythrocytes.
• AIM II polypeptides e.g., a polypeptide comprising, or alternatively consisting of amino acid residues 69 to 240 or 83 to 240 of SEQ ID NO:2
• agonists thereof e.g., anti-AIM II antibodies, soluble forms of AIM II having amino acids sequences contained in the extracellular domain of AIM II
• cytokines e.g., GM-CSF, G-CSF, IL-2, IL-3, IL-5, IL-6, IL-7, IL-12, IL-13, IL-15
• cytokines e.g., GM-CSF, G-CSF, IL-2, IL-3, IL-5, IL-6, IL-7, IL-12, IL-13, IL-15
• VEGF vascular endothelial growth factor
• AIM II polypeptides as well as agonists thereof, can be used to modulate T cell immune responses (e.g., to inhibit antigen induced T cell proliferation).
• AIM II polypeptides, as well as agonists thereof, can also be used to modulate cell mediated immune responses (e g , activate cytolytic T cells)
• AIM II polypeptides as well as agonists thereof, can be used to modulate apoptosis Further, AIM II polypeptides, as well as agonists thereof, can be used to induce cytolytic T cell responses and facilitate the formation of antigen-specific memory
• AIM II polynucleotides or polypeptides of the invention may also find application as the following A vaccine adjuvant that enhances immune responsiveness to specific antigen
• compositions of the invention include virus and virus associated diseases or symptoms described herein or otherwise known in the art
• the compositions ofthe invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of AIDS, meningitis, Dengue, EBV, and hepatitis (e.g., Hepatitis B)
• the compositions ofthe invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of HIV/AIDS, Respiratory syncytial virus.
• compositions of the invention include bacteria or fungus and bacteria or fungus associated diseases or symptoms described herein or otherwise known in the art
• the compositions ofthe invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: tetanus. Diphtheria, botulism, and meningitis type B
• the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: Vibrio cholerae, Mycobacterium leprae, Salmonella typhi,
• Salmonella paratyphi Meisseria menwgitidis. Streptococcus pneumoniae. Group B streptococcus, Shigella spp., Enterotoxigenic Escherichia coli, Enterohemorrhagic E. coli, Borrelia burgdorferi, and Plasmodium (malaria).
• compositions ofthe invention An adjuvant to enhance anti-parasitic immune responses.
• Anti-parasitic immune responses that may be enhanced using the compositions ofthe invention as an adjuvant, include parasite and parasite associated diseases or symptoms described herein or otherwise known in the art.
• the compositions of the invention are used as an adjuvant to enhance an immune response to a parasite
• the compositions ofthe invention are used as an adjuvant to enhance an immune response to Plasmodium
• B cell immunodeficiencies that may be ameliorated or treated by administering the AIM II polypeptides or polynucleotides ofthe invention, or agonists thereof, include, but are not limited to, severe combined immunodeficiency (SCID)-X linked, SCID-autosomal, adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton's disease, congenital agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired agammaglobulinemia.
• SCID severe combined immunodeficiency
• ADA deficiency adenosine deaminase deficiency
• XLA X-linked agammaglobulinemia
• Bruton's disease congenital agammaglobulinemia
• X-linked infantile agammaglobulinemia acquired agammaglobulinemia
• BLPD B cell lymphoproliferative disorder
• IgM immunodeficiency recessive agammaglobulinemia (Swiss type), reticular dysgenesis. neonatal neutropenia, severe congenital leukopenia, thymic alymphoplasia-aplasia or dysplasia with immunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezelof syndrome-combined immunodeficiency with Igs, purine nucleoside phosphorylase deficiency (PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severe combined immunodeficiency T cell immunodeficiencies that may be ameliorated or treated by administering the AIM II polypeptides or polynucleotides ofthe invention, or agonists thereof, include, but are not limited to, DiGeorge anomaly, thymic hypoplasia, chronic mucocutaneous candidiasis, natural killer cell defici
• Conditions resulting in an acquired loss of B or T cell function that may be ameliorated or treated by administering the AIM II polypeptides or polynucleotides ofthe invention, or agonists thereof, include, but are not limited to, HIV Infection, AIDS, bone marrow transplant, and B cell chronic lymphocytic leukemia (CLL).
• HIV Infection HIV Infection
• AIDS bone marrow transplant
• CLL B cell chronic lymphocytic leukemia
• Conditions resulting in a temporary immune deficiency that may be ameliorated or treated by administering the AIM II polypeptides or polynucleotides ofthe invention, or agonists thereof, include, but are not limited to, recovery from viral infections (e.g., influenza), conditions associated with malnutrition, recovery from infectious mononucleosis, or conditions associated with stress, recovery from measles, recovery from blood transfusion, recovery from surgery.
• viral infections e.g., influenza
• AIM II polynucleotides or polypeptides of the invention may be used to diagnose, prognose, treat or prevent one or more of the following diseases or disorders, or conditions associated therewith ' primary immuodeficiencies, immune-mediated thrombocytopenia, Kawasaki syndrome, bone marrow transplant (e.g. , recent bone marrow transplant in adults or children), chronic B-cell lymphocytic leukemia, HIV infection (e.g., adult or pediatric HIV infection), chronic inflammatory demyelinating polyneuropathy, and post-transfusion purpura.
• diseases or disorders, or conditions associated therewith may be used to diagnose, prognose, treat or prevent one or more of the following diseases or disorders, or conditions associated therewith ' primary immuodeficiencies, immune-mediated thrombocytopenia, Kawasaki syndrome, bone marrow transplant (e.g. , recent bone marrow transplant in adults or children), chronic B-cell lymphocytic leukemia, HIV infection (e.g.
• AIM II polynucleotides or polypeptides ofthe invention may be used to diagnose, prognose, treat or prevent one or more ofthe following diseases, disorders, or conditions associated therewith, Guillain-Barre syndrome, anemia (e.g., anemia associated with parvovirus B19, patients with stable multiple myeloma who are at high risk for infection (e.g., recurrent infection), autoimmune hemolytic anemia (e.g., warm- type autoimmune hemolytic anemia), thrombocytopenia (e.g., neonatal thrombocytopenia), and immune-mediated neutropenia), transplantation (e.g., cytomegalovirus (CMV)-negative recipients of CMV-positive organs), hypogammaglobulinemia (e.g.
• CMV cytomegalovirus
• hypogammaglobulinemic neonates with risk factor for infection or morbidity e.g., hypogammaglobulinemic neonates with risk factor for infection or morbidity
• epilepsy e.g. , intractable epilepsy
• systemic vasculitic syndromes e.g., myasthenia gravis (e.g., decompensation in myasthenia gravis), dermatomyositis, and polymyositis.
• Autoimmune disorders and conditions associated with these disorders that may be treated, prevented, and/or diagnosed with the AIM II polynucleotides, polypeptides, and/or antagonist of the invention (e.g., anti-AIM II antibodies), include, but are not limited to, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (e.g., IgA nephropathy), Multiple Sclerosis, Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura), Reiter's Disease, Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation
• autoimmune disorders that are highly probable
• autoimmune thyroiditis hypothyroidism (t ' .e., Hashimoto's thyroiditis) (often characterized, e.g., by cell-mediated and humoral thyroid cytotoxicity), systemic lupus erhythematosus (often characterized, e.g., by circulating and locally generated immune complexes), Goodpasture's syndrome (often characterized, e.g., by anti-basement membrane antibodies), Pemphigus
• receptor autoimmunities such as, for example, (a) Graves' Disease (often characterized, e.g., by TSH receptor antibodies), (b) Myasthenia Gravis (often characterized, e.g., by acetylcholine receptor antibodies), and (c) insulin resistance (often characterized, e.g. , by insulin receptor antibodies), autoimmune hemolytic anemia
• autoimmune thro bocytopenic purpura (often characterized, e.g. , by phagocytosis of antibody-sensitized platelets.
• Additional autoimmune disorders that are probable that may be treated, prevented, and/or diagnosed with the compositions ofthe invention include, but are not limited to, rheumatoid arthritis (often characterized, e.g., by immune complexes in joints), schleroderma with anti-collagen antibodies (often characterized, e.g., by nucleolar and other nuclear antibodies), mixed connective tissue disease (often characterized, e.g., by antibodies to extractable nuclear antigens (e.g., ribonucleoprotein)), polymyositis/dermatomyositis (often characterized, e.g., by nonhistone ANA), pernicious anemia (often characterized, e.g., by antiparietal cell, microsomes, and intrinsic factor antibodies), idiopathic Addison's disease (often characterized, e.g.
• infertility often characterized, e.g. , by antispermatozoal antibodies
• glomerulonephritis often characterized, e.g. , by glomerular basement membrane antibodies or immune complexes
• IgA nephropathy bullous pemphigoid
• Sjogren's syndrome often characterized e g , by multiple tissue antibodies, and/or a specific nonhistone ANA (SS-B)) diabetes millitus (often characterized, e g by cell-mediated and humoral islet cell antibodies)
• adrenergic drug resistance including adrenergic drug resistance with asthma or cystic fibrosis
• Additional autoimmune disorders that are possible) that may be treated prevented, and/or diagnosed with the compositions ofthe invention include, but are not limited to chronic active hepatitis (often characterized, e g by smooth muscle antibodies), primary biliary cirrhosis (often characterized, e g , by mitchond ⁇ al antibodies), other endocrine gland failure (often characterized, e g , by specific tissue antibodies in some cases), viti go (often characterized, e g , by melanocyte antibodies), vascuhtis (often characterized, e g , by Ig and complement in vessel walls and/or low serum complement), post-MI (often characterized, e g , by myocardial antibodies) cardiotomy syndrome (often characterized, e g , by myocardial antibodies), urticaria (often characterized, e g , by IgG and IgM antibodies to IgE), atopic dermatitis (often characterized,
• the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are treated, prevented, and/or diagnosed using anti-AIM II antibodies
• rheumatoid arthritis is treated, prevented, and/or diagnosed using anti-AIM II antibodies and/or other antagonist ofthe invention
• lupus is treated, prevented, and/or diagnosed using anti-AIM II antibodies and/or other antagonist ofthe invention
• nephritis associated with lupus is treated, prevented, and/or diagnosed using anti-AIM II antibodies and/or other antagonist ofthe invention.
• AIM II polynucleotides or polypeptides, or antagonists thereof are used to treat or prevent systemic lupus erythematosus and/or diseases, disorders or conditions associated therewith.
• Lupus-associated diseases, disorders, or conditions that may be treated or prevented with AIM II polynucleotides or polypeptides, or antagonists ofthe invention include, but are not limited to, hematologic disorders (e.g. , hemolytic anemia, leukopenia, lymphopenia, and thrombocytopenia), immunologic disorders
• the AIM II polynucleotides or polypeptides, or antagonists thereof are used to treat or prevent renal disorders associated with systemic lupus erythematosus.
• AIM II polynucleotides or polypeptides, or antagonists thereof are used to treat or prevent nephritis associated with systemic lupus erythematosus.
• soluble AIM II polypeptides ofthe invention are administered, to treat, prevent, prognose and/or diagnose an immunodeficiency (e.g., severe combined immunodeficiency (SCID)-X linked, SCID-autosomal, adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton's disease, congenital agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, transient hypogammaglobulinemia of infancy
• an immunodeficiency e.g., severe combined immunodeficiency (SCID)-X linked, SCID-autosomal, adenosine deaminase deficiency (
• a number of soluble forms ofthe polypeptides ofthe present invention can be used to treat, prevent, prognose and/or diagnose disease states These soluble forms will generally lack the AIM II transmembrane domain
• Examples of soluble AIM II polypeptides include those where the extracellular and intracellular domains are present, but the transmembrane domain has been deleted Further examples comprising, or alternatively consisting of, amino acids 60 to 240, 69 to 240, or 83 to 240 of SEQ ID NO 2
• AIM II polypeptides or polynucleotides ofthe invention, or agonists thereof is administered to treat, prevent, prognose and/or diagnose common variable immunodeficiency
• AIM II polypeptides or polynucleotides ofthe invention, or agonists thereof is administered to treat, prevent, prognose and/or diagnose X-hnked agammaglobulinemia
• AIM II polypeptides or polynucleotides ofthe invention, or agonists thereof is administered to treat, prevent, prognose and/or diagnose severe combined immunodeficiency (SCID).
• SCID severe combined immunodeficiency
• .AIM II polypeptides or polynucleotides of the invention, or agonists thereof is administered to treat, prevent, prognose and/or diagnose Wiskott-Aldrich syndrome
• AIM II polypeptides or polynucleotides of the invention, or agonists thereof is administered to treat, prevent, prognose and/or diagnose X-linked Ig deficiency with hyper IgM.
• AIM II polypeptides or polynucleotides of the invention, or agonists thereof is administered to treat, prevent, prognose and/or diagnose DiGeorge anomaly
• AIM II antagonists also may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinafter described.
• the antagonists may further be employed for instance to treat cachexia which is a lipid clearing defect resulting from a systemic deficiency of lipoprotein lipase. which is believed to be suppressed by AIM II.
• the AIM II antagonists may also be employed to treat cerebral malaria in which AIM II may play a pathogenic role.
• the AIM II antagonists may also be employed to prevent graft-host rejection (e.g. , by preventing the stimulation ofthe immune system in the presence of a graft).
• the AIM II antagonists may also be employed to prevent graft-host disease.
• anti-AIM II antibody is employed to prevent graft-host disease.
• the AIM II antagonists may also be employed to inhibit bone resorption and, therefore, to treat and/or prevent osteoporosis.
• the antagonists may also be employed as anti-inflammatory agents, and to treat endotoxic shock. This critical condition results from an exaggerated response to bacterial and other types of infection.
• AIM II antagonists also find application as the following
• nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a disease or disorder associated with aberrant expression and/or activity of a polypeptide ofthe invention, by way of gene therapy
• Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid
• the nucleic acids produce their encoded protein that mediates a therapeutic effect
• the compound comprises nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host
• nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue- specific
• nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression ofthe antibody nucleic acids (Koller and Smithies, 1989, Proc Nati Acad Sci USA 86 8932-8935, Zijlstra et al, 1989, Nature 342 435-438)
• the expressed antibody molecule is a single chain antibody
• the nucleic acid sequences include sequences encoding both the heavy and light chains
• nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid- carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient
• direct in which case the patient is directly exposed to the nucleic acid or nucleic acid- carrying vectors
• indirect in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient
• the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product.
• This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g.
• nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
• the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180 dated April 16. 1992 (Wu et al ); WO 92/22635 dated December 23, 1992 (Wilson et al); WO92/20316 dated November 26, 1992
• the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, 1989, Proc. Nati. Acad. Sci. USA 86:8932-8935; Zijlstra et ⁇ /., 1989, Nature 342:435-438).
• viral vectors that contains nucleic acid sequences encoding an antibody ofthe invention are used.
• a retroviral vector can be used (see Miller et al, 1993, Meth. Enzymol. 217:581-599). These retroviral vectors have been to delete retroviral sequences that are not necessary for packaging ofthe viral genome and integration into host cell DNA.
• the nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery ofthe gene into a patient.
• retroviral vectors More detail about retroviral vectors can be found in Boesen et al, 1994, Biotherapy 6:291-302, which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy.
• Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al, 1994, J. Clin. Invest. 93:644-651; Kiem et al, 1994, Blood 83 : 1467- 1473 ; Salmons and Gunzberg, 1993, Human Gene Therapy 4: 129- 141; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3: 110- 114.
• Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells Kozarsky and Wilson. 1993, Current Opinion in Genetics and Development 3 499-503 present a review of adenovirus-based gene therapy Bout et al .
• adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys
• adenovirus vectors are used Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh etal. 1993, Proc Soc Exp Biol Med 204 289-300, U S Patent No 5,436, 146)
• Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection Usually, the method of transfer includes the transfer of a selectable marker to the cells The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene Those cells are then delivered to a patient
• the nucleic acid is introduced into a cell prior to administration in vivo ofthe resulting recombinant cell
• introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, micro cell-mediated gene transfer, spheroplast fusion, etc
• Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, 1993, Meth Enzymol 217 599-618, Cohen et al, 1993, Meth Enzymol 217 618-644, Cline, 1985, Pharmac Ther 29 69-92) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted
• the technique should provide for the stable transfer ofthe nucleic acid to the cell, so that the nucleic acid is express
• Recombinant blood cells e.g., hematopoietic stem or progenitor cells
• Recombinant blood cells are preferably administered intravenously.
• the amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.
• Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g. , as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
• the cell used for gene therapy is autologous to the patient.
• nucleic acid sequences encoding an antibody are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered t>? vivo for therapeutic effect.
• stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment ofthe present invention (see, e.g., PCT Publication WO 94/08598, dated April 28, 1994; Stemple and Anderson, 1992, Cell 71 :973-985; Rheinwald,
• the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence ofthe appropriate inducer of transcription
• antagonists and agonists ofthe invention include AIM II polypeptides These polypeptides can modulate their effect by, for example, binding to cellular proteins such as receptors.
• Methods for identifying peptides which interact with a specific protein are known in the art for example, Phizicky and Fields, "Protem-protem interactions methods for detection and analysis” Microhiol Rev 59 94-123 (1995), describe methods for screening peptides to identify those having binding affinity for a second polypeptide
• Phizicky and Fields discuss methods such as protein affinity chromatography, affinity blotting, coimmunoprecipitation, and cross-linking
• Additional molecular biological methods suitable for use with the present invention include protein probing of expression libraries, the two-hybrid system, cell panning, and phage display
• Another method for identifying AIM II polypeptides of the invention which bind to a cell surface receptor involves transfecting eukaryotic cells with
• DNA encoding the receptor such that the cells expresses the receptor on their surfaces, followed by contacting the cells with a labeled (e g , radioactive label, biotm, etc ) AIM II polypeptide
• a labeled e g , radioactive label, biotm, etc
• the amount of labeled AIM II polypeptide bound to the cells is measured and compared to that bound to control cells
• the control cells will generally be cells which do not express the surface receptor
• the detection of an increased amount of label bound to the cells which express the receptor as compared to the control cells indicates that the cells which expresses the receptor bind the AIM II polypeptide
• cells which express and retain AIM II polypeptides can be used to identify AIM II ligands
• cells which express AIM II would be contacted with potential ligands which have been detectably labeled
• ligands may be polypeptides which are expressed as part of a library of sequences on the surface of a phage (e g , a phage display library)
• assays can be performed to determine whether the AIM II polypeptide induces or inhibits a receptor-mediated cellular response normally elicited by the particular receptor.
• Whether an AIM II polypeptide activates a receptor-mediated cellular response may be determined by measuring a cellular response known to be elicited by the receptor in the presence of the AIM II polypeptide or another ligand Further, whether an AIM II polypeptide inhibits a receptor-mediated cellular response may be determined by measuring a cellular response known to be elicited by the receptor in the presence of both a molecule which is known to induce the cellular response and the AIM II polypeptide.
• Soluble forms ofthe polypeptides ofthe present invention may be utilized in the ligand binding and receptor activation/inhibition assay described above.
• AIM II has also been shown to inhibit the differentiation and survival of photoreceptor cells. Further, AIM II has been shown to inhibit the production of rhodopsin by retinal cells. Thus, AIM II polypeptides and AIM II agonists are useful for inhibiting the differentiation and survival of photoreceptor cells (e.g., rods and cones) and inhibiting rhodopsin production by retinal cells (e.g., rods).
• AIM II polypeptides, agonists or antagonists ofthe invention may be used to treat cardiovascular disorders, including peripheral artery disease, such as limb ischemia.
• Cardiovascular disorders include cardiovascular abnormalities, such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia and Scimitar Syndrome.
• Congenital heart defects include aortic coarctation, cor triatriatum, coronary vessel anomalies, crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of fallot, transposition of great vessels, double outlet right ventricle, tricuspid atresia, persistent truncus arteriosus, and heart septal defects, such as aortopulmonary septal defect, endocardial cushion defects, Lutembacher's Syndrome, trilogy of Fallot, and ventricular heart septal defects.
• Cardiovascular disorders also include heart disease, such as arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), heart aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction heart rupture, ventricular septal rupture, heart valve diseases, myocardial diseases, myocardial ischemia, pericardial effusion, pericarditis (including constrictive and tuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.
• heart disease such as arrhythmias, carcinoid heart disease, high cardiac output, low cardiac
• Arrhythmias include sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams- Stokes Syndrome, bundle-branch block, sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, and ventricular fibrillation.
• Tachycardias include paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.
• Heart valve disease include aortic valve insufficiency, aortic valve stenosis, heart murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency, and tricuspid valve stenosis.
• Myocardial diseases include alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy. endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury, and myocarditis.
• Myocardial ischemias include coronary disease, such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning.
• Cardiovascular diseases also include vascular diseases such as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis.
• vascular diseases such as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis.
• aortitis Leriche's Syndrome, arterial occlusive diseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabetic angiopathies, diabetic retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids, hepatic veno-occlusive disease, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary veno- occlusive disease, Raynaud's disease, CREST syndrome, retinal vein occlusion, Scimitar syndrome, superior vena cava syndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagic telangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis, and venous insufficiency.
• Aneurysms include dissecting aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart aneurysms, and iliac aneurysms.
• Arterial occlusive diseases include arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya disease, renal artery obstruction, retinal artery occlusion, and thromboangiitis obliterans.
• Cerebrovascular disorders include carotid artery diseases, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery diseases, cerebral embolism and thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural ematoma, subdural hematoma, subarachnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient), subclavian steal syndrome, periventricularleukomalacia, vascular headache, cluster headache, migraine, and vertebrobasilar insufficiency Embolisms include air embolisms, amniotic fluid embolisms, cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and thromboembohsms.
• Thrombosis include coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, and thrombophlebitis.
• Ischemia includes cerebral ischemia, ischemic colitis, compartment syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion injuries, and peripheral limb ischemia.
• Vasculitis includes aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph node syndrome, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein- Henoch purpura, allergic cutaneous vasculitis, and Wegener's granulomatosis.
• an AIM II polynucleotide, polypeptide, agonist, or antagonist of the invention is used to treat thrombotic microangiopathies.
• One such disorder is thrombotic thrombocytopenic purpura (TTP) (Kwaan, H.C.,
• Plasma from patients afflicted with TTP induces apoptosis of human endothelial cells of dermal microvascular origin, but not large vessel origin (Laurence et al, Blood 57:3245 (1996)). Plasma of TTP patients thus is thought to contain one or more factors that directly or indirectly induce apoptosis..
• HUS hemolytic-uremic syndrome
• the invention is directed to use of AIM II to treat the condition that is often referred to as "adult HUS" (even though it can strike children as well).
• a disorder known as childhood/diarrhea-associated HUS differs in etiology from adult HUS.
• conditions characterized by clotting of small blood vessels may be treated using AIM II. Such conditions include, but are not limited to, those described herein.
• cardiac problems seen in about 5- 10% of pediatric AIDS patients are believed to involve clotting of small blood vessels. Breakdown of the microvasculature in the heart has been reported in multiple sclerosis patients.
• SLE systemic lupus erythematosus
• a patient's blood or plasma is contacted with AIM II polypeptides of the invention ex vivo.
• the AIM II polypeptides of the invention may be bound to a suitable chromatography matrix by procedures known in the art.
• the patient's blood or plasma flows through a chromatography column containing AIM II polynucleotides and/or polypeptides ofthe invention bound to the matrix, before being returned to the patient.
• the immobilized AIM II binds AIM II, thus removing AIM II protein from the patient's blood.
• AIM II polynucleotides, polypeptides, agonists or antagonists ofthe invention may be administered in vivo to a patient afflicted with a thrombotic microangiopathy.
• a soluble form of AIM II polypeptide ofthe invention is administered to the patient.
• the present invention provides a method for treating a thrombotic microangiopathy, involving use of an effective amount of AIM II polynucleotide, polypeptide, agonist or antagonist.
• An AIM II polypeptide may be employed in in vivo or ex vivo procedures, to inhibit AIM Il-mediated damage to (e.g., apoptosis of) microvascular endothelial cells.
• AIM II polynucleotides, polypeptides, agonists or antagonists of the invention may be employed in combination with other agents useful in treating a particular disorder.
• agents useful in treating a particular disorder For example, in an in vitro study reported by Laurence et al. , Blood 87:3245 (1996), some reduction of TTP plasma-mediated apoptosis of microvascular endothelial cells was achieved by using an anti-Fas blocking antibody, aurintricarboxylic acid, or normal plasma depleted of cryoprecipitate.
• a patient may be treated with a polynucleotide and/or polypeptide of the invention in combination with an agent that inhibits Fas-ligand-mediated apoptosis of endothelial cells, such as.
• an AIM II polynucleotide, polypeptide, agonist or antagonist, and an anti-FAS blocking antibody are both administered to a patient afflicted with a disorder characterized by thrombotic microangiopathy, such as TTP or HUS.
• angiogenesis is stringently regulated and spatially and temporally delimited. Under conditions of pathological angiogenesis such as that characterizing solid tumor growth, these regulatory controls fail. Unregulated angiogenesis becomes pathologic and sustains progression of many neoplastic and non-neoplastic diseases.
• a number of serious diseases are dominated by abnormal neovascularization including solid tumor growth and metastases, arthritis, some types of eye disorders, and psoriasis. See, e.g., reviews by Moses et al, Biotech. 9:630-634 (1991); Folkman et al, N. Engl. J. Med. 333: 1757-1763 (1995);
• the present invention provides for treatment of diseases or disorders associated with neovascularization by administration of the AIM II polynucleotides and/or polypeptides ofthe invention (including AIM II agonists and/or antagonists)
• AIM II polynucleotides and/or polypeptides ofthe invention including AIM II agonists and/or antagonists
• Malignant and metastatic conditions which can be treated with the polynucleotides and polypeptides of the invention include, but are not limited to those malignancies, solid tumors, and cancers described herein and otherwise known in the art (for a review of such disorders, see Fishman et al. , Medicine, 2d Ed., J. B. Lippincott Co , Philadelphia (1985)).
• ocular disorders associated with neovascularization which can be treated with the AIM II polynucleotides and polypeptides of the present invention (including AIM II agonists and AIM II antagonists) include, but are not limited to: neovascular glaucoma, diabetic retinopathy, retinoblastoma, retrolental fibroplasia, uveitis. retinopathy of prematurity, macular degeneration, corneal graft neovascularization, as well as other eye inflammatory diseases, ocular tumors and diseases associated with choroidal or iris neovascularization. See, e.g., reviews by Waltman et al, Am. J.
• disorders which can be treated with the AIM II polynucleotides and polypeptides of the present invention include, but are not limited to, hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing, granulations, hemophilic joints, hypertrophic scars, nonunion fractures, Osler- Weber syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.
• Polynucleotides and/or polypeptides of the invention, and/or agonists and/or antagonists thereof, are useful in the diagnosis and treatment or prevention of a wide range of diseases and/or conditions.
• diseases and conditions include, but are not limited to, cancer (e.g., immune cell related cancers, breast cancer, prostate cancer, ovarian cancer, follicular lymphoma, cancer associated with mutation or alteration of p53, brain tumor, bladder cancer, uterocervical cancer, colon cancer, colorectal cancer, non-small cell carcinoma of the lung, small cell carcinoma of the lung, stomach cancer, etc.), lymphoproliferative disorders (e.g., lymphadenopathy), microbial (e.g., viral, bacterial, etc.) infection (e g , HIV-1 infection HIV-2 infection herpesvirus infection (including but not limited to, HSV-1 , HSV-2, CMV, NZN, HHV-6, HHV-7, EBV), adenovirus infection, poxvirus
• cardiomyopathy e g , dilated cardiomyopathy
• diabetes e g , diabetic complications
• diabetic complications e g , diabetic nephropathy, diabetic neuropathy, diabetic retinopathy
• influenza e g , asthma, psoriasis, glomerulonephritis, septic shock, and ulcerative colitis
• Polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are useful in promoting angiogenesis, wound healing (e g , wounds, burns, and bone fractures)
• Polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are also useful as an adjuvant to enhance immune responsiveness to specific antigen and/or anti-viral immune responses
• polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are useful in regulating (/ e , elevating or reducing) immune response
• polynucleotides and/or polypeptides ofthe invention may be useful in preparation or recovery from surgery, trauma, radiation therapy, chemotherapy, and transplantation, or may be used to boost immune response and/or recovery in the elderly and immunocompromised individuals.
• polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are useful as immunosuppressive agents, for example in the treatment or prevention of autoimmune disorders.
• polynucleotides and/or polypeptides of the invention are used to treat or prevent chronic inflammatory, allergic or autoimmune conditions, such as those described herein or are otherwise known in the art.
• AIM II polypeptides particularly human AIM II polypeptides
• the uses of the AIM II polypeptides include but are not limited to, the treatment or prevention of viral hepatitis, Herpes viral infections, allergic reactions, adult respiratory distress syndrome, neoplasia, anaphylaxis, allergic asthma, allergen rhinitis, drug allergies (e.g., to penicillin, cephalosporins), primary central nervous system lymphoma (PCNSL), glioblastoma, chronic lymphocytic leukemia (CLL), lymphadenopathy, autoimmune disease, graft versus host disease, rheumatoid arthritis, osteoarthritis,
• PCNSL primary central nervous system lymphoma
• CLL chronic lymphocytic leukemia
• AIM II polypeptide ofthe present invention may be employed to inhibit neoplasia, such as tumor cell growth.
• immunotherapeutic agent such as IL-2 or IL-12 may result in synergistic or additive effects that would be useful for the treatment of established cancers.
• the AIM II polypeptide may also be useful for tumor therapy.
• AIM II may further be employed to treat diseases which require growth promotion activity, for example, restenosis, since AIM II has proliferative effects on cells of endothelial origin.
• AIM II may, therefore, also be employed to regulate hematopoiesis in endothelial cell development.
• the AIM II polypeptides ofthe invention may also be employed to inhibit the differentiation and proliferation of T cells and B cells.
• AIM II induced mhibition of T and B cell activation, differentiation and/or proliferation may be employed to treat a number of immunological based diseases, several of which are referred to above
• the AIM II polypeptides of the invention may also be employed to stimulate activation, differentiation and/or proliferation of T cells and B cells
• AIM II may act as a cytokine adjuvant or costimulatory molecule
• the following experiments are performed to assess the in vivo AIM II protein on the host immune system
• the spleens or the lymph nodes are used for the following in vitro analyses well known to those skilled in the art FACS analyses Expression of surface markers for T cells, B cells, NK cells, Monocytes, Dendritic cells, costimulatory and adhesion molecules Cytokine production assays T cell proliferation or cytotoxicity assay AIM II protein and tumor antigen may result in the induction of protective immunity, which could lead to protecting mice from subsequent tumor challenge
• the following experiment can be performed using syngeneic C57BL/6 mice to test the effect of AIM II on induction of tumor or Ag-specific protective immunity MC-38 tumor-free mice treated with AIM II protein will be challenged with MC-
• mice will be treated with various mAb which recognize either CD4+ or CD8+ T cells, NK cells, granulocyte (Grl+), or specific cytokine such as IFN ⁇ using techniques well known to those skilled in the art. Tumor growth in these antibody-treated mice is measured
• AIM II may also be used to treat rheumatoid arthritis (RA) by inhibiting the increase in angiogenesis or the increase in endothelial cell proliferation required to sustain an invading pannus in bone and cartilage as is often observed in RA Endothelial cell proliferation is increased in the synovia of RA patients as compared to patients with osteoarthritis (OA) or unaffected individuals.
• RA rheumatoid arthritis
• Neovascularization is needed to sustain the increased mass ofthe invading pannus into bone and cartilage
• Inhibition of angiogenesis is associated with a significant decrease in the severity of both early and chronic arthritis in animal models.
• the AIM II polypeptide is believed to possess binding activities for a number of proteins, including several human cellular receptors. These receptors include the lymphotoxin- ⁇ -receptor (LT- ⁇ -R), TR2 (also referred to as the Herpes virus entry mediator (HVEM) and ATAR), CD27, TR6 (also referred to as DcR3), TR9 (DR6) and TRANK (also referred to as receptor activator of nuclear factor-kappa B (RANK)).
• LT- ⁇ -R lymphotoxin- ⁇ -receptor
• TR2 also referred to as the Herpes virus entry mediator (HVEM) and ATAR
• CD27 CD27
• TR6 also referred to as DcR3
• TR9 TR9
• TRANK also referred to as receptor activator of nuclear factor-kappa B (RANK)
• polypeptides ofthe invention can be used to stimulate or block the action of ligands which bind cellular receptors having AIM II binding activity (e.g., LT- ⁇ -R, TR2, CD27, TR6, TR9 and TRANK).
• ligands which bind cellular receptors having AIM II binding activity e.g., LT- ⁇ -R, TR2, CD27, TR6, TR9 and TRANK.
• LT- ⁇ which binds to the LT- ⁇ -R
• LT- ⁇ -R has been implicated in the development of secondary lymphoid tissues and the maintenance of organized lymphoid tissues in adults LT- ⁇ -R may, in some instances, function in conjunction with TR2 to mediate cellular responses and has been shown to be expressed in a number of tissues in the lung including a subpopulation of T-lymphocytes LT- ⁇ -R has also been implicated in the formation of germinal centers and thus appears to be involved in humoral immune responses Rennert et al , Int Immunol 9 1627-1639 (1997)
• the AIM II polypeptides ofthe invention may be employed to inhibit the formation of germinal centers and LT- ⁇ -R mediated humoral responses by blocking access of cellular ligands to LT- ⁇ -R Further, polypeptides of the invention may stimulate the formation of germinal centers and LT- ⁇ -R mediated humoral responses by activating LT- ⁇ -R
• AIM II polypeptides of the invention may have different effects on LT- ⁇ -R
• the effect that the AIM II polypeptides ofthe invention would have on LT- ⁇ -R would vary with the individual peptide and the effect it has when bound to LT- ⁇ -R
• HCV Hepatitis C virus
• the core protein of Hepatitis C virus (HCV) has also been shown to associate with LT- ⁇ -R and enhance signaling mediated by this receptor Chen et al , J Virol 71 9417-9426 (1997) Further, the interaction of this protein with LT- ⁇ -R may contribute to the chronically activated, persistent state of HCV- mfected cells
• the AIM II polypeptides of the invention may be employed to block HVC stimulation of LT- ⁇ -R and the pathology associated with this virus
• TR2 is a member ofthe tumor necrosis factor (TNF) receptor family which is expressed in a number of human tissues and cell lines This protein is expressed constitutively and in relatively high levels in peripheral blood T cells, B cells, and monocytes Kwon et al , J Biol Chem 272 14272-14276 (1997) TR2 serves a number of functions in vivo, including the mediation of Herpes viral entry into cells during infection Further a TR2-Fc fusion protein has been demonstrated to inhibit mixed lymphocyte reaction-mediated proliferation These data suggest that the TR2 and its ligand play a role in T cell stimulation It has been shown along these lines that overexpression of TR2 activates NF- ⁇ B and AP-1 This activation appears to occur through a TNF receptor-associated factor (TRAF)- mediated mechanism
• TNF tumor necrosis factor
• the AIM II polypeptides of the invention may be employed to inhibit T cell activation, and thus T cell mediated immune responses, by blocking access to TR2 by cellular ligands which activate this receptor Similarly, polypeptides of the invention may stimulate T cell activation by activating TR2 As noted above for LT- ⁇ -R, one skilled in the art would recognize that different portions of the AIM II polypeptide may either inhibit or stimulate TR2 mediated cellular responses
• the AIM II polypeptides ofthe invention may also be employed to prevent or treat Herpes viral infections
• CD27 as well as its ligand CD70, is predominantly confined to lymphocytes Further, CD27 has been shown to interact with CD70 and to be involved in the induction of IgE synthesis in B cells Nagumo et al , J Immunol 161 6496-6502 (1998) In addition activation of CD27 may enhance IgE synthesis Inhibition ofthe interaction between CD27 and CD70 thus may inhibit
• AIM II polypeptides of the invention may be used for modulating immune responses
• AIM II polypeptides may be used to regulate the function of B cells by inhibiting the interaction between CD27 and CD70
• AIM II polypeptides may thus bind to CD27 and inhibit B cell differentiation and proliferation, as well as the secretion of proteins (e g , IgE) by these cells Therefore, AIM II polypeptides may be employed to suppress IgE antibody formation in the treatment of IgE-induced immediate hypersensitivity reactions, such as allergic rhinitis (also know as hay fever), bronchial asthma, allergic asthma, anaphylaxis, atopic dermatitis and gastrointestinal food allergy CD27 is also believed to be the receptor for a pro-apoptotic protein commonly known as Siva Pandanilam et al Kidney Int 54 1967-1975 (1998)
• AIM II polypeptides ofthe invention may be employed to inhibit the interaction between Siva and CD27 and thus prevent S ⁇ va/CD27 mediated induction of a
• AIM II polypeptides of the invention may also be employed to enhance activation ofthe S ⁇ va/CD27 apoptotic pathway and thus facilitate the induction of apoptosis Diseases associated with increased cell survival, or the inhibition of apoptosis, include cancers (such as folhcular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors), autoimmune disorders (such as systemic lupus erythematosus, immune-related glomerulonephritis, and rheumatoid arthritis) and viral infections (such as herpes viruses, pox viruses and adenoviruses), information graft v host disease, acute graft rejection, and chronic graft rejection
• cancers such as folhcular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors
• autoimmune disorders such as systemic lupus erythematosus, immune-related glomerulonephritis, and rheumatoid arthritis
• CD27 may be membrane-bound, a soluble form of CD27 is produced in the course ofthe immune response Soluble CD27 (sCD27) is found in a number of body fluids and may be measured to monitor local and systemic immune activation Further, CD27 is expressed on human malignant B cells and high levels of sCD27 are present in the sera of patients with va ⁇ ous B-cell malignancies Kersten et al , Blood 87 1985-1989 (1996) These elevated levels of sCD27 have been shown to strongly correlate with tumor load sCD27 has also been shown to be elevated in patients with a variety lymphoid malignancies and solid tumors of the central nervous system These afflictions include primary central nervous system lymphoma (PCNSL) and lymphoid malignancies located in the meninges (e.g. , acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL)).
• PCNSL primary central nervous system lymphoma
• ALL acute lymphoblastic leuk
• Soluble CD27 has also been found to be elevated in patients with a number of non-hyperproliferative diseases.
• sCD27 has been shown to be elevated in patients with untreated Graves' hyperthyroidism. Kallio et al, J. Lab.
• sCD27 may prevent leukemic B cells from stimulating T cells via CD70, and thus may impair the ability of B cells to function as antigen-presenting cells.
• Polypeptides of the invention may be employed to inhibit interactions between sCD27 and CD70, and, thus, to enhance the ability of B cells to act as antigen- presenting cells.
• AIM II polypeptides of the invention may also be employed to treat diseases and afflictions associated with increased levels of sCD27. While not wishing to be bound to a mechanistic theory, AIM II polypeptides may be useful in treatment regimens for these conditions since AIM II binds sCD27 and prevents it from interacting with cellular ligands.
• TR6 (See, Example 13).
• TR6 (DcR3) is a novel soluble member ofthe TNFR superfamily, which is constitutively expressed in lung tissue, tumor cells and in endothelial cells. TR6 binds specifically to AIM II and FasL and inhibits their activities. Like DcRl, DcR2 and another soluble member of TNFR superfamily, OPG, TR6 may act as an inhibitor of signaling through TNF family members, FasL and AIM II. TR6 appears to have important roles in the inhibition of apoptosis and tumor modulation.
• TR6 may act as an mhibitor of AIM II interaction and may also act in distinct roles depending on cell types TR6 (DcR3) may act as a decoy receptor and cont ⁇ bute to immune evasion both in slow and rapid tumor cell death, that are mediated bv AIM II or FasL mediated apoptosis pathway respectively Indeed, TR6 (DcR3) has been shown to bind to FasL and might contribute to immune evasion by certain tumors
• TR6 and its ligands are important for interactions between activated T lymphocytes and endothehum TR6 may be involved in activated T cell trafficking as well as endothelial cell survival
• TR6 may act as a cytokine to trigger membrane-bound FasL or AIM II and directly transduce signals through FasL or AIM II
• FasL could itself transduce signals, leading to cell-cycle arrest and cell death in CD4 + T cells but cell proliferation in CD8 + T cells (Desbarats, J , et al , Natui e Medicine 4 1377-1382 (1998), Suzuki, I , and Fink, P J , .7 Exp Med 757 123-128
• TR6 may act as a cytokine for signaling through FasL and AIM II
• AIM II polypeptides of the invention may also be employed to treat diseases and afflictions associated with increased levels of TR6, or conditions where increased apoptosis would be desirable While not wishing to be bound to a mechanistic theory AIM II polypeptides may be useful in treatment regimens for these conditions since AIM II binds TR6 and prevents TR6 from interacting with cellular ligands to block apoptosis AIM II polypeptides are also believed to bind to RANK (See Anderson etal , Nature 390 175-179 (1997) ) RANK is a protein which has been implicated in osteoclast differentiation and regulation of interactions between T cells and dendritic cells RANK apparently mediates its cellular effects via interaction with RANKL (also referred to as osteoprotege ⁇ n ligand (OPGL), TRANCE and ODF) Mice having a disrupted RANKL gene show severe osteoporos
• bone cells cells which make bone, osteoblasts, and cells which resorb bone, osteoclasts
• These cells each have very precise functions and the balance between their activities is critical to the maintenance ofthe skeletal system For example, in human adults, between 10 to
• the AIM II polypeptides of the invention may be employed to regulate osteoclast differentiation and proliferation (bone formation), as well as bone development and degradation
• Polypeptides of the invention may, for example, be employed to inhibit osteoclast differentiation and proliferation and, thus, may be employed to decease the rate of bone degradation
• Inhibition of osteoclast differentiation and proliferation and bone degradation may be useful in the treatment of conditions such as osteoporosis, skeletal and dental abnormalities, bone cancers, osteoarthritis, osteogenesis lmperfecta, and Hurler and Marfan syndromes
• Polypeptides ofthe invention may also be employed in processes for reshaping bone and teeth and in pe ⁇ odontal reconstructions where lost bone replacement or bone augmentation is required, such as in a jaw bone and supplementing alveolar bone loss resulting from pe ⁇ odontal disease to delay or prevent tooth loss (see, e g , S ⁇ gurdsson et / , ,7 Penodontol 66 511-21 (1995))
• the AIM II polypeptides ofthe invention may further be used to regulate T and B lymphocyte differentiation and proliferation AIM II polypeptides may thus bind to RANK and inhibit the differentiation and proliferation of T and B lymphocyte as well as the secretion of proteins (e g immunoglobins) from these cells AIM II polypeptides may therefore be employed to suppress lymphocyte-mediated immune responses, for example to prevent graft rejection AIM II polypeptides may also be used to inhibit osteoclast differentiation and proliferation AIM II polypeptides may thus be employed to treat diseases such as bone cancers
• the present invention also provides AIM II polypeptides which mimic one or more ofthe natural ligands of RANK and stimulate RANK-mediated cellular responses These cellular responses include the activation of T and B lymphocyte differentiation and proliferation and induction of osteoclast differentiation
• AIM II polypeptides may thus be employed to treat diseases such as infections (e g , bacterial, viral and protozoal infections)
• AIM II polypeptides may also be employed to enhance immune responses (e g , in the treatment of AIDS and AIDS related complexes) and to increase bone degradation rates
• TR9 TR9 also referred to as DR6
• TR9 TR9 is a novel member ofthe tumor necrosis factor family of receptors which possesses a cytoplasmic death domain (See, WO 98/56892)
• TR9 induces apoptosis in mammalian cells and is capable of engaging the NF- ⁇ B and JNK pathways TR9 is believed to play a role in inflammatory responses and immune regulation
• the AIM II polypeptides may be used to modulate inflammatory responses and immune regulation via TR9
• AIM II polypeptides may also be used to induce apoptosis via TR9 via the NF- ⁇ B and JNK pathways
• AIM II polypeptides may thus be employed to treat diseases such as infections
• AIM II polypeptides may also be employed to enhance immune responses
• the AIM II polypeptide may be cleaved in vivo to form a soluble form of the molecule
• a cleavage site appears to be located between amino acid residues 82 and 83 ofthe sequence shown in SEQ ID NO 2
• Cleavage of the AIM II polypeptide at this location is believed to result in the production of a soluble form of the molecule which comprises, or alternatively consisting of, amino acids 83-240 in SEQ ID NO 2
• Soluble forms of AIM II are especially useful for the treatment of diseases where systemic administration of these peptides is preferred
• soluble forms of AIM II are also useful for topical administration
• the complete and mature AIM II polypeptides of the invention, as well as subfragments of these polypeptides may be employed to treat afflictions associated with receptors and other ligands to which these molecules bind
• the invention provides a method of delivering compositions containing the polypeptides of the invention (e.g., compositions containing AIM II polypeptides or anti-AIM II antibodies associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs) to targeted cells, expressing the membrane-bound form of AIM II on their surface, or
• an AIM II receptor e.g , TR2, LTbeta receptor, and CD27
• AIM II polypeptides or anti-AIM II antibodies ofthe invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions
• the invention provides a method for the specific delivery of compositions ofthe invention to cells by administering polypeptides ofthe invention (e.g., AIM II or anti-AIM II antibodies) that are associated with heterologous polypeptides or nucleic acids
• polypeptides ofthe invention e.g., AIM II or anti-AIM II antibodies
• the invention provides a method for delivering a therapeutic protein into the targeted cell.
• the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell
• the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention (e.g., AIM II polypeptides or anti-AIM II antibodies) in association with toxins or cytotoxic prodrugs.
• polypeptides of the invention e.g., AIM II polypeptides or anti-AIM II antibodies
• the invention provides a method for the specific destruction of cells expressing TR2, LTbeta receptor, and/or CD27 on their surface (e.g., activated T cells, and/or T cell and/or B cell related leukemias or lymphomas) by administering AIM II polypeptides in association with toxins or cytotoxic prodrugs.
• the invention provides a method for the specific destruction of cells expressing the membrane-bound form of AIM II on their surface (e.g., spleen, bone marrow, kidney and PBLs) by administering anti- AIM II antibodies in association with toxins or cytotoxic prodrugs.
• toxins is meant compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, cytotoxins (cytotoxic agents), or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death.
• Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin.
• radioisotopes known in the art
• compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseu
• Toxin also includes a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g. , alpha-emitters such as, for example, 2I3 Bi, or other radioisotopes such as, for example, 103 Pd, I33 Xe, 131 L 68 Ge, 57 Co, 65 Zn, 85 Sr, 32 P, 35 S, 90 Y, 153 Sm, 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se, 113 Sn, 90 Yttrium, U7 Tin, 186 Rhenium, 166 Holmium, and 188 Rhenium; luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
• alpha-emitters such as, for example, 2I3 Bi
• radioisotopes such as, for example, 103 Pd, I33 Xe, 131 L 68 Ge
• a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.
• Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide. tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 - dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
• Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6- thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g. , mechlorethamine, thioepa chlorambucil, melphalan.
• antimetabolites e.g., methotrexate, 6-mercaptopurine, 6- thioguanine, cytarabine, 5-fluorouracil decarbazine
• alkylating agents e.g. , mechlorethamine, thioepa chlorambucil, melphalan.
• carmustine BSNU and lomustine (CCNU)
• cyclothosphamide busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin
• anthracyclines e.g., daunorubicin (formerly daunomycin) and doxorubicin
• antibiotics e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)
• anti-mitotic agents e.g., vincristine and vinblastine.
• cytotoxic prodrug is meant a non-toxic compound that is converted by an enzyme, normally present in the cell, into a cytotoxic compound.
• Cytotoxic prodrugs that may be used according to the methods ofthe invention include, but are not limited to, glutamyl derivatives of benzoic acid mustard alkylating agent, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubisin, and phenoxyacetamide derivatives of doxorubicin.
• the invention further relates to the use ofthe AIM II polynucleotides to detect complementary polynucleotides such as, for example, as a diagnostic reagent. Detection of a mutated form of an AIM II associated with a dysfunction will provide a diagnostic tool that can add or define a diagnosis of a disease or susceptibility to disease which results from under-expression, over-expression or altered expression of AIM II, such as. for example, autoimmune diseases, malignancies and/or immundeficiencies.
• the polynucleotide encoding the AIM II may also be employed as a diagnostic marker for expression ofthe polypeptide of the present invention.
• AIM II is expressed in spleen, thymus and bone marrow tissue.
• disorders such as septic shock, inflammation, cerebral malaria, activation ofthe HIV virus, graft-host rejection, bone resorption, rheumatoid arthritis and cachexia
• bodily fluids e.g., serum, plasma, urine, synovial fluid or spinal fluid
• the invention provides a diagnostic method useful during diagnosis of a disorder, which involves: (a) assaying AIM II gene expression level in cells or body fluid of an individual; (b) comparing the AIM II gene expression level with a standard AIM II gene expression level, whereby an increase or decrease in the assayed AIM II gene expression level compared to the standard expression level is indicative of a disorder. It is also believed that certain tissues in mammals with cancer express significantly reduced levels ofthe AIM II protein and mRNA encoding the AIM II protein when compared to a corresponding "standard" mammal, i.e., a mammal of the same species not having the cancer.
• the invention provides a diagnostic method useful during tumor diagnosis, which involves assaying the expression level of the gene encoding the AIM II protein in mammalian cells or body fluid and comparing the gene expression level with a standard AIM 11 gene expression level, whereby an decrease in the gene expression level over the standard is indicative of certain tumors.
• the present invention is useful as a prognostic indicator, whereby patients exhibiting enhanced AIM II gene expression may experience a better clinical outcome relative to patients expressing the gene at a lower level.
• saying the expression level ofthe gene encoding the AIM II protein is intended qualitatively or quantitatively measuring or estimating the level ofthe
• AIM II protein or the level of the mRNA encoding the AIM II protein in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the AIM II protein level or mRNA level in a second biological sample).
• the AIM II protein level or mRNA level in the first biological sample is measured or estimated and compared to a standard AIM II protein level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the cancer.
• a standard AIM II protein level or mRNA level is known, it can be used repeatedly as a standard for comparison.
• biological sample any biological sample obtained from an individual, cell line, tissue culture, or other source which contains AIM II protein or mRNA.
• biological samples include mammalian body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) which contain secreted mature
• the present invention is useful for detecting cancer in mammals.
• the invention is useful during diagnosis ofthe following types of cancers in mammals: breast, ovarian, prostate, bone, liver, lung, pancreatic and spleenic.
• Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans. Particularly preferred are humans.
• Labeled antibodies and derivatives and analogs thereof, which specifically bind to a polypeptide of interest can be used for diagnostic purposes to detect, diagnose, or monitor diseases and/or disorders associated with the aberrant expression and/or activity of a polypeptide of the invention
• the invention provides for the detection of aberrant expression of a polypeptide of interest, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of aberrant expression
• the invention provides a diagnostic assay for diagnosing a disorder, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder
• the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms
• a more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression ofthe cancer
• the diagnostic assays of the invention can be used for the diagnosis and prognosis of any disease related to the altered expression or production of AIM II These assays are believed to be particularly useful for the diagnosis and prognosis of graft versus host disease,
• Antibodies of the invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e g see Jalkanen, M et al J Cell Biol 101 976-985 (1985) Jalkanen, M et a/ , ] Cell Biol 105 3087-3096 (1987))
• Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA)
• Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase radioisotopes, such as iodine ( 125 I 121 I), carbon ( 14 C), sulfur ( 5 S), tritium ( H), indium ( I 12 In), and technetium ( 99 Tc) luminescent labels, such as luminol, and fluorescent labels, such as fluorescein and rhodamine, and biotin
• monitoring ofthe disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc
• Presence of the labeled molecule can be detected in the patient using methods known in the art for m vivo scanning These methods depend upon the type of label used Skilled artisans will be able to determine the appropriate method for detecting a particular label
• Methods and devices that may be used in the diagnostic methods ofthe invention include but are not limited to computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography
• the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al , U S Patent No 5,441,050)
• the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument
• the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography
• the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI)
• MRI magnetic resonance imaging
• mRNA encoding the AIM II protein is then assayed using any appropriate method. These include Northern blot analysis (Harada et ⁇ /., Cell 63:303-312 (1990)), SI nuclease mapping (Fujita et al, Cell 49:351-361 (1987)), the polymerase chain reaction (PCR), reverse transcription in combination with the polymerase chain reaction (RT-PCR) (Makino et al. Technique 2:295-301 (1990)), and reverse transcription in combination with the ligase chain reaction (RT-LCR). Assaying AIM II protein levels in a biological sample can occur using antibody-based techniques.
• AIM II protein expression in tissues can be studied with classical immunohistological methods (Jalkanen, M., et al, J. Cell. Biol 101:916-985 (1985); Jalkanen, M., et al, J. Cell Biol. 105:3081-3096 (1987)).
• Other antibody-based methods useful for detecting AIM II protein expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
• Suitable labels are known in the art and include enzyme labels, such as, Glucose oxidase, and radioisotopes, such as iodine ( 125 1, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( I I2 In), and technetium ( 99m Tc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
• enzyme labels such as, Glucose oxidase, and radioisotopes, such as iodine ( 125 1, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( I I2 In), and technetium ( 99m Tc)
• fluorescent labels such as fluorescein and rhodamine, and biotin.
• Endogenous gene expression can also be reduced by inactivating or "knocking out” the gene and/or its promoter using targeted homologous recombination, (e.g., see Smithies etal, Nature 317:230-234 (1985); Thomas &
• a mutant, non-functional polynucleotide ofthe invention flanked by DN A homologous to the endogenous polynucleotide sequence (either the coding regions or regulatory regions ofthe gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express polypeptides of the invention in vivo
• techniques known in the art are used to generate knockouts in cells that contain, but do not express the gene of interest Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the targeted gene
• Such approaches are particularly suited in research and agricultural fields where modifications to embryonic stem cells can be used to generate animal offspring with an inactive targeted gene (see, e g , Thomas & Capec
• cells that are genetically engineered to express the polypeptides ofthe invention, or alternatively, that are genetically engineered not to express the polypeptides of the invention are administered to a patient in vivo
• Such cells may be obtained from the patient (; e , animal, including human) or an MHC compatible donor and can include, but are not limited to fibroblasts, bone marrow cells, blood cells (e g , lymphocytes), adipocytes, muscle cells, endothelial cells etc
• the cells are genetically engineered in vitro using recombinant DNA techniques to introduce the coding sequence of polypeptides of the invention into the cells, or alternatively, to disrupt the coding sequence and/or endogenous regulatory sequence associated with the polypeptides ofthe invention, e g , by transduction (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to, the use of plasmid
• the cells to be administered are non-autologous or non-MHC compatible cells they can be administered using well known techniques which prevent the development of a host immune response against the introduced cells
• the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system
• polypeptides of the invention can also be expressed in transgenic animals
• Animals of any species including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e g , baboons, monkeys, and chimpanzees may be used to generate transgenic animals
• techniques desc ⁇ bed herein or otherwise known in the art are used to express polypeptides ofthe invention in humans, as part of a gene therapy protocol
• transgene / e , polynucleotides ofthe invention
• transgene / e , polynucleotides ofthe invention
• Such techniques include, but are not limited to, pronuclear m ⁇ cro ⁇ nject ⁇ on (Paterson et ⁇ / ,J ?p/ Microhiol Biotechnol 40 691-698 (1994), Carver el al. Biotechnology (NY) 77" 1263-1270 (1993): Wright et al. Biotechnology (NY) 9 830-834 (1991 ); and Hoppe et ⁇ /., U.S. Pat.
• U.S. Patent No. 5,631, 153 Capecchi et al, Cells and Non-Human Organisms Containing Predetermined Genomic Modifications and Positive- Negative Selection Methods and Vectors for Making Same
• U.S. Patent No. 4,736,866 Leder et al, Transgenic Non-Human Animals
• U.S. Patent No. 4,873,191 Wild-Wet al. Genetic Transformation of Zygotes
• transgenic clones containing polynucleotides of the invention for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (Campell et al, Nature 380:64-66 (1996); Wilmut et al, Nature
• the present invention provides for transgenic animals that carry the transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, t ' .e., mosaic animals or chimeric.
• the transgene may be integrated as a single transgene or as multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems.
• the transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al (Lasko ei al, Proc. Nati Acad. Sci. USA 59:6232-6236 (1992)).
• the regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
• gene targeting is preferred.
• vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene.
• the transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type, by following, for example, the teaching of Gu et al. (Gu et al. ,
• the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration ofthe transgene has taken place. The level of mRNA expression ofthe transgene in the tissues ofthe transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenic gene-expressing tissue may also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.
• founder animals may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal.
• breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the transgene on a distinct background that is appropriate for an experimental model of interest.
• Transgenic and "knock-out" animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of AIM II polypeptides, studying conditions and/or disorders associated with aberrant AIM II expression, and in screening for compounds effective in ameliorating such conditions and/or disorders.
• the present invention also relates to methods for separating cells into subpopulations based on whether these cells bind either the AIM II polypeptides of the invention or antibodies having specificity for these polypeptides.
• separation methods will generally be based on the principle that cells which either express a surface receptor which binds AIM II polypeptides or have an AIM II polypeptide on their surface can be identified using labeled AIM II polypeptides or AIM II specific antibodies. Such cells can then be separated from other cells in a population which do not bind these polypeptides or antibodies.
• Methods for separating cells commonly known as "cell sorting", are known in the art and are discussed in Crane, U.S. Patent No. 5,489,506.
• the invention provides methods for separating cells which bind either AIM II polypeptides or antibodies having specificity for AIM II polypeptides comprising contacting a population of cells with either an AIM II polypeptide or an antibody having specificity for the AIM II polypeptide, wherein the AIM II polypeptide or antibody is labeled with a detectable label and separating cells which bind either the AIM II polypeptide or anti-AIM II polypeptide antibody from cells which do not bind these molecules.
• Cells which bind AIM II polypeptides are believed to include those which express the lymphotoxin- ⁇ -receptor (LT- ⁇ -R). TR2, CD27, and TRANK.
• polypeptides ofthe present invention are used as a research tool for studying the biological effects that result from inhibiting LT ⁇ receptor/ AIM II, TR6/AIM II. and/or TRANK/AIM II interactions on different cell types.
• AIM II polypeptides also may be employed in in vitro assays for detecting LT ⁇ receptor, TR6, and/or TRANK or AIM II or the interactions thereof.
• This invention also provides a method for identification of molecules, such as receptor molecules, that bind AIM II.
• Genes encoding proteins that bind AIM II, such as receptor proteins can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting. Such methods are described in many laboratory manuals such as, for instance, Coligan et al, Current Protocols in Immunology, 1 (2): Chapter 5 (1991).
• expression cloning may be employed for this purpose.
• polyadenylated RNA is prepared from a cell responsive to AIM II
• a cDNA library is created from this RNA, the library is divided into pools and the pools are transfected individually into cells that are not responsive to AIM II.
• the transfected cells then are exposed to labeled AIM II.
• AIM II can be labeled by a variety of well-known techniques including standard methods of radio-iodination or inclusion of a recognition site for a site-specific protein kinase.
• the cells are fixed and binding of AIM II is determined. These procedures conveniently are carried out on glass slides.
• a labeled ligand can be photo affinity linked to a cell extract, such as a membrane or a membrane extract, and prepared from cells that express a molecule that it binds, such as a receptor molecule
• Cross-linked material is resolved by polyacrylamide gel electrophoresis ("PAGE") and exposed to X-ray film
• PAGE polyacrylamide gel electrophoresis
• the labeled complex containing the hgand-receptor can be excised, resolved into peptide fragments, and subjected to protein microsequencing
• the amino acid sequence obtained from microsequencing can be used to design unique or degenerate ohgonucleotide probes to screen cDNA libraries to identify genes encoding the putative receptor molecule
• Polypeptides ofthe invention also can be used to assess AIM II binding capacity of AIM II binding molecules, such as receptor molecules, in cells or in cell-free preparations
• the nucleic acid molecules ofthe present invention are also valuable for chromosome identification
• the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome
• the mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease
• one ofthe cDNAs herein disclosed is used to clone genomic DNA of an AIM II protein gene This can be accomplished using a variety of well known techniques and libraries, which generally are available commercially The genomic DNA then is used for in situ chromosome mapping using well known techniques for this purpose
• sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA Computer analysis of the 3 ' untranslated region ofthe gene is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes Fluorescence in situ hybridization ("FISH") of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step This technique can be used with probes from the cDNA as short as 50 or 60 bp For a review of this technique, see Verma et al, Human Chromosomes- A Manual Of Basic Techniques, Pergamon Press, New York (1988)
• in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample
• the effect ofthe compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays.
• in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.
• kits that can be used in the above methods.
• a kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers.
• the kits ofthe present invention contain a substantially isolated polypeptide comprising an epitope which is specifically immunoreactive with an antibody included in the kit.
• the kits ofthe present invention further comprise a control antibody which does not" react with the polypeptide of interest.
• kits of the present invention contain a means for detecting the binding of an antibody to a polypeptide of interest (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate).
• a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate.
• the kit is a diagnostic kit for use in screening serum containing antibodies specific against proliferative and/or cancerous polynucleotides and polypeptides.
• a kit may include a control antibody that does not react with the polypeptide of interest.
• a kit may include a substantially isolated polypeptide antigen comprising an epitope which is specifically immunoreactive with at least one anti-polypeptide antigen antibody.
• a kit includes means for detecting the binding of said antibody to the antigen (e.g.
• the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine which can be detected by flow cytometry
• the kit may include a recombinantly produced or chemically synthesized polypeptide antigen
• the polypeptide antigen ofthe kit may also be attached to a solid support
• the detecting means ofthe above-described kit includes a solid support to which said polypeptide antigen is attached Such a kit may also include a non-attached reporter-labeled anti-human antibody In this embodiment, binding ofthe antibody to the polypeptide antigen can be detected by binding ofthe said reporter-labeled antibody.
• the invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide ofthe invention.
• the diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding ofthe polynucleotide or polypeptide antigen to the antibody
• the antibody is attached to a solid support.
• the antibody may be a monoclonal antibody.
• the detecting means ofthe kit may include a second, labeled monoclonal antibody. Alternatively, or in addition, the detecting means may include a labeled, competing antigen.
• test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention.
• the reagent After binding with specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support.
• the reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined.
• the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, MO).
• the solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).
• the invention provides an assay system or kit for carrying out this diagnostic method.
• the kit generally includes a support with surface-bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.
• the invention further provides a method of treating an individual in need of an increased level of AIM II activity comprising administering to such an individual a pharmaceutical composition comprising, or alternatively consisting of, an effective amount of an isolated AIM II polypeptide of the invention, effective to increase the AIM II activity level in such an individual.
• a pharmaceutical composition comprising, or alternatively consisting of, an effective amount of an isolated AIM II polypeptide of the invention, effective to increase the AIM II activity level in such an individual.
• the invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition ofthe invention, preferably an antibody ofthe invention.
• the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects).
• the subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.
• Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above; additional appropriate formulations and routes of administration can be selected from among those described herein below.
• Va ⁇ ous delivery systems are known and can be used to administer a compound of the invention e g , encapsulation in liposomes microparticles, microcapsules recombinant cells capable of expressing the compound receptor- mediated endocytosis (see, e g Wu and Wu, 1987, J Biol Chem 262 4429- 4432), construction of a nucleic acid as part of a retroviral or other vector, etc
• compositions containing the AIM II of the invention may be administered orally, rectally, parenterally, intracistemally, intravaginally, lntrape ⁇ toneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray
• parenteral refers to modes of administration which include intravenous, intramuscular intraperitoneal, intrasternal, subcutaneous and lntra-articular injection and infusion
• Administration can be systemic or local
• lntravent ⁇ cular injection may be facilitated by an mtravent ⁇ cular catheter for example, attached to a reservoir, such as an Ommaya reservoir
• Pulmonary administration can also be employed, e g , by use of an inhaler or nebulizer, and formulation with an aerosolizing agent
• care must be taken to use materials to which the protein does not absorb
• the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, 1990, Science 249 1527-1533, Treat et al , in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds ), Liss, New York, pp 353- 365 (1989), Lopez- Berestein, ibid , pp 317-327, see generally ibid )
• a liposome see Langer, 1990, Science 249 1527-1533, Treat et al , in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds ), Liss, New York, pp 353- 365 (1989), Lopez- Berestein, ibid , pp 317-327, see generally ibid )
• the compound or composition can be delivered in a controlled release system
• a pump may be used (see Langer, supra, Sefton, 1987, CRC C ⁇ t Ref Biomed Eng 14 201 , Buchwald et al , 1980, Surgery 88 507 Saudek et al , 1989, N Engl J Med 321 574)
• polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds ), CRC Pres , Boca Raton, Florida (1974), Controlled Drug Bioavailabi ty, Drug Product Design and Performance, Smolen and Ball (eds ), Wiley, New York (1984), Ranger and Peppas, J , 1983, Macromol Sci Rev Macromol Chem 23 61 , see also Levy et al , 1985, Science 228 190, During et al , 1989, Ann Neurol 25 351, Howard et al , 1989,
• a controlled release system can be placed in proximity of the therapeutic target, i e , the brain, thus requiring only a fraction of the systemic dose (see, e g , Goodson, in Medical Applications of Controlled Release, supra, vol 2, pp 1 15-138 (1984)) Other controlled release systems are discussed in the review by Langer
• the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g , by use of a retroviral vector (see U S Patent No 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun, Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox- like peptide which is known to enter the nucleus (see, e.g , Johot et al, 1991, Proc Nati Acad Sci USA 88 1864-
• a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination
• particularly preferred modes of administration of AIM II polypeptides of the present invention include, intradermal, subcutaneous and intra-articular injection and infusion
• intradermal, subcutaneous and intra-articular injection and infusion Preferably,
• AIM II polypeptide administered intra-articularly or intra-dermally per dose will be in the range of about 0 1 to about 1 0 mg/kg of patient body weight
• compositions of the invention may be administered alone or in combination with other therapeutic agents (e.g., a costimulatory molecule)
• therapeutic agents that may be administered in combination with the compositions ofthe invention, include but are not limited to, other members ofthe TNF family, chemotherapeutic agents, antibiotics, steroidal and non-steroidal anti- inflammatories, conventional immunotherapeutic agents, cytokines and/or growth factors Combinations may be administered either concomitantly, e.g , as an admixture, separately but simultaneously or concurrently, or sequentially
• the compositions ofthe invention are administered in combination with other members ofthe TNF family TNF, TNF-related or TNF- like molecules that may be administered with the compositions ofthe invention include, but are not limited to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer
• LT-alpha2-beta OPGL. FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No WO 96/14328), AIMJ (International Publication No WO 97/33899), APRIL (J Exp Med 188(6) 1 185- 1 190), endokine- ⁇ (International Publication No WO 98/07880), TR6 (International Publication No WO 98/30694), OPG, and neutrokine- ⁇
• compositions of the invention are administered in combination with CD40 ligand (CD40L), a soluble form of CD40L (e.g.
• compositions of the invention are administered in combination with one, two, three, four, five, or more of the following compositions tacrolimus (Fujisawa), thalidomide (e.g., Celgene), anti- Tac(Fv)-PE40 (e.g., Protein Design Labs), inolimomab (Biotest), MAK-195F (Knoll), ASM-981 (Novartis), interleukin- 1 receptor (e.g.
• Jmmunex interleukin- 4 receptor
• ICM3 ICM3
• BMS-188667 Bristol-Myers Squibb
• anti-TNF Ab e.g.. Therapeutic antibodies
• CG-1088 Celgene
• anti-B7 Mab e.g., Innogetics
• MEDI-507 BioTransplant
• ABX-CBL Abgenix
• compositions ofthe invention are administered in combination with antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors.
• Nucleoside reverse transcriptase inhibitors that may be administered in combination with the compositions ofthe invention, include, but are not limited to, RETROVIRTM (zidovudine/AZT), VIDEXTM (didanosine/ddl), HIVIDTM (zalcitabine/ddC), ZERITTM (stavudine/d4T), EPIVIRTM (lamivudine/3TC), and COMBIVIRTM (zidovudine/lamivudine).
• RETROVIRTM zidovudine/AZT
• VIDEXTM didanosine/ddl
• HIVIDTM zalcitabine/ddC
• ZERITTM stavudine/d4T
• EPIVIRTM lamvudine/3TC
• COMBIVIRTM zidovudine/lamivudine
• Non-nucleoside reverse transcriptase inhibitors that may be administered in combination with the compositions ofthe invention, include, but are not limited to, VIRAMUNETM (nevirapine), RESCRIPTORTM (delavirdine), and SUSTIVATM (efavirenz).
• Protease inhibitors that may be administered in combination with the compositions ofthe invention, include, but are not limited to, CRIXIVANTM (indinavir), NORVIRTM (ritonavir),
• antiretroviral agents nucleoside reverse transcriptase inhibitors, non- nucleoside reverse transcriptase inhibitors, and/or protease inhibitors may be used in any combination with compositions of the invention to treat, prevent, and/or diagnose AIDS and/or to treat, prevent, and/or diagnose HIV infection.
• compositions ofthe invention may be administered in combination with anti-opportunistic infection agents.
• Anti-opportunistic agents that may be administered in combination with the compositions ofthe invention include, but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLETM DAPSONETM, PENTAMIDINETM, ATOVAQUONETM, ISONIT ⁇ ZIDTM
• compositions of the invention are used in any combination with TRI METH O P RI M - S ULF AMETHOXAZ OLETM, DAP S ONETM , PENTAMIDINETM, and/or ATOVAQUONETM to prophylactically treat, prevent, and/or diagnose an opportunistic Pneumocystis carinii pneumonia infection.
• compositions of the invention are used in any combination with ISONIAZIDTM, RIFAMPINTM, PYRAZIN AMIDETM, and/or ETHAMBUTOLTM to prophylactically treat, prevent, and/or diagnose an opportunistic Mycobacterium avium complex infection.
• compositions of the invention are used in any combination with
• compositions of the invention are used in any combination with GANCICLOVIRTM, FOSCARNETTM, and/or CIDOFOVIRTM to prophylactically treat, prevent, and/or diagnose an opportunistic cytomegalovirus infection.
• compositions of the invention are used in any combination with FLUCONAZOLETM, ITRACONAZOLETM, and/or KETOCONAZOLETM to prophylactically treat, prevent, and/or diagnose an opportunistic fungal infection.
• compositions of the invention are used in any combination with ACYCLOVIRTM and/or FAMCICOLVIRTM to prophylactically treat, prevent, and/or diagnose an opportunistic herpes simplex virus type I and/or type II infection.
• compositions ofthe invention are used in any combination with P YPJMETHAMINETM and/or LEUCO VORINTM to prophylactically treat, prevent, and/or diagnose an opportunistic Toxoplasma gondii infection.
• compositions ofthe invention are used in any combination with LEUCOVORINTM and/or NEUPOGENTM to prophylactically treat, prevent, and/or diagnose an opportunistic bacterial infection.
• compositions of the invention are administered in combination with an antiviral agent
• Antiviral agents that may be administered with the compositions ofthe invention include, but are not limited to, acyclovir, ⁇ bavi ⁇ n, amantadine, and remantidine
• compositions of the invention are administered in combination with an antibiotic agent
• Antibiotic agents that may be administered with the compositions of the invention include, but are not limited to, amoxicil n, aminoglycosides, beta-lactam (glycopeptide), beta- lactamases, Chndamycin, chloramphenicol, cephalospo ⁇ ns, ciprofloxacin, erythromycin, fluoroquinolones, macrohdes, metronidazole, penicillins, quinolones, ⁇ fampin, streptomycin sulfonamide, tetracychnes, t ⁇ methop ⁇ m t ⁇ methop ⁇ m-sulfamthoxazole, and vancomycin
• nonspecific immunosuppressive agents that may be administered in combination with the compositions ofthe invention include, but are not limited to, steroids, cyclospo ⁇ ne, cyclospo ⁇ ne analogs cyclophosphamide, cyclophosphamide IV, methylprednisolone, prednisolone, azathiop ⁇ ne, FK-506, 15-deoxysperguahn and other immunosuppressive agents that act by suppressing the function of responding T cells
• compositions ofthe invention are administered in combination with immunosuppressants
• Immunosuppressants preparations that may be administered with the compositions ofthe invention include, but are not limited to , ORTHOCLONETM (OKT3 ), SANDIMMUNETM/ NEORALTM/SANGDYATM (cyclospo ⁇ n), PROGRAFTM (tacrohmus), CELLCEPTTM (mycophenolate), Azathiop ⁇ ne, glucorticosteroids, and RAPAMUNETM (sirohmus)
• immunosuppressants may be used to prevent rejection of organ or bone marrow transplantation
• compositions of the invention are administered in combination with steroid therapy
• Steroids that may be administered in combination with the compositions ofthe invention, include, but are not limited to, oral corticosteroids, prednisone, and methylprednisolone (e g , IV methylprednisolone)
• compositions ofthe invention are administered m combination with prednisone
• the compositions of the invention are administered in combination with prednisone and an immunosuppressive agent
• Immunosuppressive agents that may be administered with the compositions ofthe invention and prednisone are those described herein, and include, but are not limited to, azathiop ⁇ ne, cylophosphamide, and cyclophosphamide IV
• compositions of the invention are administered in combination with methylprednisolone
• the compositions of the invention are administered in combination with methylprednisolone and an
• compositions of the invention are administered in combination with an NSAID
• compositions of the invention are administered in combination with one, two, three, four, five, ten, or more ofthe following drugs NRD-101 (Hoechst Marion Roussel), diclofenac (Dimethaid), oxaprozin potassium (Monsanto), mecasermin (Chiron), T-614 (Toyama), pemetrexed disodium (Eh Lilly), atreleuton (Abbott), valdecoxib (Monsanto), kornac (Byk Gulden), campath, AGM-1470 (Takeda), CDP-571 (Celltech Chiroscience), CM-101 (CarboMed), ML-3000 (Merckle), CB-2431 (KS Biomedix), CBF-BS2 (KS Biomedix), IL-lRa gene therapy (Valentis), JTE-522 (Japan Tobacco), pachtaxel (Angiotech), DW-166HC (Dong Wha), darbufelone mesy
• NRD-101 Ho
• compositions of the invention are administered in combination with one, two, three, four, five or more of the following drugs: methotrexate, sulfasalazine, sodium aurothiomalate, auranofin, cyclosporine, penicillamine, azathioprine, an antimalarial drug (e.g. , as described herein), cyclophosphamide, chlorambucil, gold, ENBRELTM (Etanercept), anti- TNF antibody, and prednisolone.
• drugs methotrexate, sulfasalazine, sodium aurothiomalate, auranofin, cyclosporine, penicillamine, azathioprine, an antimalarial drug (e.g. , as described herein), cyclophosphamide, chlorambucil, gold, ENBRELTM (Etanercept), anti- TNF antibody, and prednisolone.
• compositions of the invention are administered in combination with an antimalarial, methotrexate, anti-TNF antibody, ENBRELTM and/or suflasalazine.
• the compositions of the invention are administered in combination with methotrexate.
• the compositions ofthe invention are administered in combination with anti-TNF antibody.
• the compositions ofthe invention are administered in combination with methotrexate and anti-TNF antibody.
• the compositions ofthe invention are administered in combination with suflasalazine.
• the compositions of the invention are administered in combination with methotrexate, anti-TNF antibody, and suflasalazine.
• compositions of the invention are administered in combination ENBRELTM. In another embodiment, the compositions ofthe invention are administered in combination with ENBRELTM and methotrexate. In another embodiment, the compositions ofthe invention are administered in combination with ENBRELTM, methotrexate and suflasalazine. In another embodiment, the compositions of the invention are administered in combination with ENBRELTM, methotrexate and suflasalazine. In other embodiments, one or more antimalarials is combined with one ofthe above-recited combinations.
• compositions of the invention are administered in combination with an antimalarial (e.g., hydroxychloroquine), ENBRELTM, methotrexate and suflasalazine-
• an antimalarial e.g., hydroxychloroquine
• sulfasalazine e.g., anti-TNF antibody
• methotrexate e.g., hydroxychloroquine
• compositions ofthe invention include, but are not limited to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide methylprednisone, prednisone, azathioprine, FK-506, 15-deoxyspergualin, and other immunosuppressive agents that act by suppressing the function of responding T cells.
• the compositions of the invention are administered in combination with an antibiotic agent.
• Antibiotic agents that may be administered with the compositions ofthe invention include, but are not limited to, tetracycline, metronidazole, amoxicillin, beta-lactamases, aminoglycosides, macrolides, quinolones, fluoroquinolones, cephalosporins, erythromycin, ciprofloxacin, and streptomycin.
• compositions of the invention are administered alone or in combination with an anti-inflammatory agent.
• Anti- inflammatory agents that may be administered with the compositions of the invention include, but are not limited to, glucocorticoids and the nonsteroidal anti- inflammatories, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e- acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, parany
• compositions ofthe invention are administered in combination with a chemotherapeutic agent.
• Chemotherapeutic agents that may be administered with the compositions ofthe invention include, but are not limited to, antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactmomycin), antiestrogens (e g , tamoxifen), antimetabolites (e.g , fluorouracil, 5-FU. methotrexate, floxu ⁇ dine. interferon alpha-2b.
• antibiotic derivatives e.g., doxorubicin, bleomycin, daunorubicin, and dactmomycin
• antiestrogens e.g , tamoxifen
• antimetabolites e.g , fluorouracil, 5-FU. methotrexate, floxu ⁇ dine. interferon alpha-2b.
• cytotoxic agents e g , carmustine, BCNU, lomustme, CCNU. cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine. mitomycin. busulfan, cis-platin, and vincristine sulfate
• hormones e g , medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol, estradiol.
• megestrol acetate methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and testolactone
• nitrogen mustard derivatives e.g , mephalen, chorambucil, mechlorethamine (nitrogen mustard) and thiotepa
• steroids and combinations e g , bethamethasone sodium phosphate
• compositions of the invention are administered in combination with cytokines
• Cytokines that may be administered with the compositions ofthe invention include, but are not limited to, IL-2, IL-3,
• IL-4 IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, IFN- ⁇ and TNF- ⁇
• compositions of the invention are administered in combination with angiogenic proteins
• Angiogenic proteins include, but are not limited to, Glioma Derived Growth Factor (GDGF), as disclosed in European Patent Number EP-399816, Platelet Derived Growth Factor-A (PDGF-A), as disclosed in European Patent Number EP-682110, Platelet Derived Growth Factor-B (PDGF-B), as disclosed in European Patent Number EP-282317; Placental Growth Factor (P1GF). as disclosed in WO 92/06194, Placental Growth Factor
• Vascular Endothelial Growth Factor-2 (P1GF-2), as disclosed in Hauser et al. Growth Factors -7 259-268 (1993), Vascular Endothelial Growth Factor (VEGF), as disclosed in WO 90/13649, Vascular Endothelial Growth Factor-A (VEGF- A), as disclosed in European Patent Number EP-506477, Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosed in WO 96/39515, Vascular Endothelial Growth Factor B- 186 (VEGF-B 186), as disclosed in WO 96/26736, Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in WO 98/02543, Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in WO 98/07832, and Vascular Endothelial Growth Factor-E (VEGF-E), as disclosed in German Patent Number DEI 9639601 The above mentioned references are incorporated by reference herein
• compositions of the invention are administered in combination with Fibroblast Growth Factors
• Fibroblast Growth Factors include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4. FGF-5. FGF-6, FGF-7,
• compositions ofthe invention are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy
• compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier
• pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U S Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans
• a "pharmaceutically acceptable carrier” will generally be a non- toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type
• carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered
• Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like Water is a preferred carrier when the pharmaceutical composition is administered intravenously Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
• Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
• the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates.
• Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned.
• These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
• the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
• Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
• compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
• suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin.
• Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
• the formulation should suit the mode of administration.
• the parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
• AIM II polypeptides containing the transmembrane region can also be used when appropriately solubihzed by including detergents, such as triton X-100, with buffer.
• the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
• compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
• the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site ofthe injection
• the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent
• an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration
• the compounds of the invention can be formulated as neutral or salt forms Pharmaceutically acceptable salts include those formed with anions such
• the amount ofthe compound ofthe invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques
• in vitro assays may optionally be employed to help identify optimal dosage ranges
• the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness ofthe disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems
• the total pharmaceutically effective amount of AIM II polypeptide administered parenterally per dose will be in the range of about 1 ⁇ g/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion More preferably, this dose is at least 0 01 mg/kg/day, and most preferably for humans between about 0 01 and 1 mg/kg/day for the hormone
• the AIM II polypeptide is typically administered at a dose rate of about 1 ⁇ g/kg/hour to about 50 ⁇ g/kg/hour, either by 1 -4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump
• An intravenous bag solution may also be employed
• the dosage administered to a patient is typically 0 1 mg/kg to 100 mg/kg ofthe patient's body weight
• the dosage administered to a patient is between 0 1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg ofthe patient's body weight
• human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides
• the dosage and frequency of administration of antibodies ofthe invention may be reduced by enhancing uptake and tissue penetration (e g , into the brain) ofthe antibodies by modifications such as. for example, pidation
• the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more ofthe ingredients ofthe pharmaceutical compositions ofthe invention
• Optionally associated with such conta ⁇ ner(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration
• the DNA sequence encoding the AIM II protein in the cDNA assigned ATCC Accession No 97689 is amplified using PCR ohgonucleotide primers specific to the amino terminal sequences of the AIM II protein and to vector sequences 3 ' to the gene Additional nucleotides containing restriction sites to facilitate cloning are added to the 5 ' and 3 ' sequences respectively
• a 22 kDa AIM II protein fragment (lacking the N-terminus and transmembrane region) is expressed using the following primers
• the 5 ' ohgonucleotide primer has the sequence 5 ' GCGGGATCCGGAGAGATGGTCACC 3' (SEQ ID NO 7) containing the underlined BamHl restriction site, which includes nucleotides 244-258 of the AIM II protein coding sequence in Figure 1A (SEQ ID NO 1)
• the 3 ' primer has the sequence
• the entire AIM II protein can be expressed using the following primers
• the 5 ' ohgonucleotide primer has the sequence
• the 3 ' primer has the sequence
• restriction sites are convenient to restriction enzyme sites in the bacterial expression vector pQE9, which are used for bacterial expression in these examples (Qiagen Inc 9259 Eton Avenue Chatsworth, CA 9131 1 )
• pQE9 encodes ampicilhn antibiotic resistance (" Amp r ”) and contains a bacterial origin of replication ("on"), an IPTG inducible promoter, a ⁇ bosome binding site ("RBS”) a 6-H ⁇ s tag and restriction enzyme sites
• Amp r ampicilhn antibiotic resistance
• RBS ⁇ bosome binding site
• Insertion of the AIM II protein DNA into the restricted pQE9 vector places the AIM II protein coding region downstream of and operably linked to the vector's IPTG- mducible promoter and in-frame with an initiating AUG appropriately positioned for translation of AIM II protein
• pQE60 is used for bacterial expression in this example (QIAGEN, Inc , 9259 Eton Avenue, Chatsworth, CA, 91311)
• pQE60 encodes ampicilhn antibiotic resistance ("Amp r ”) and contains a bacterial origin of replication ("on”), an IPTG inducible promoter, a ⁇ bosome binding site
• RBS nickel-nit ⁇ lo-t ⁇ -acetic acid
• Ni-NTA nickel-nit ⁇ lo-t ⁇ -acetic acid
• suitable single restriction enzyme cleavage sites These elements are arranged such that an inserted DNA fragment encoding a polypeptide expresses that polypeptide with the six His residues (/ e , a "6 X His tag”) covalently linked to the carboxyl terminus of that polypeptide
• the DNA sequence encoding the desired portion ofthe AIM II protein is amplified from the deposited cDNA using PCR ohgonucleotide primers which anneal to the amino terminal sequences of the desired portion of the AIM II protein and to sequences in the deposited construct 3' to the cDNA coding sequence Additional nucleotides containing restriction sites to facilitate cloning in the pQE60 vector are added to the 5' and 3' sequences, respectively
• the 5' primer has the sequence 5' GACGC CCATGG AG GAG GAG AGT GTC GTA CGG C 3' (SEQ ID NOJ 7) containing the underlined NcoX restriction site followed by nucleotides complementary to the amino terminal coding sequence ofthe AIM II sequence in Figure 1A
• SEQ ID NOJ 7 the sequence 5' GACGC CCATGG AG GAG GAG AGT GTC GTA CGG C 3' (SEQ ID NOJ 7) containing the underlined NcoX restriction site followed by nucleotides complementary to the amino terminal coding sequence ofthe AIM II sequence in Figure 1A
• the 3' primer has the sequence:
• the amplified AIM II DNA fragment and the vector pQE60 are digested with BamHl and Ncol and the digested DNAs are then ligated together. Insertion ofthe AIM II DNA into the restricted pQE60 vector places the AIM II protein coding region downstream from the IPTG-inducible promoter and in-frame with an initiating AUG and the six histidine codons.
• AIM II deletion mutant with an N-terminal 6-His tag
• the DNA sequence encoding the AIM II protein in the deposited cDNA was amplified using PCR ohgonucleotide primers specific to sequences of the AIM II protein and to vector sequences 3 ' to the gene. Additional nucleotides containing restriction sites to facilitate cloning were added to the 5 ' and 3 ' sequences respectively.
• an N-terminal deletion AIM II mutant (Met(68) to Val(240) in SEQ ID NO:2) was constructed using the following primers:
• the 5' ohgonucleotide primer has the sequence: 5'-GGG GGA TCC ATG GTC ACC CGC CTG CC-3 1 (SEQ ID NO:21) containing the underlined BamHl restriction site, and includes 17 nucleotides of the AIM II protein coding sequence in Figure 1 A (SEQ ID NOJ).
• the 3 ' primer has the sequence: 5'-GGG AAGCTT CAC CAT GAA AGC CCC G-3 1 (SEQ IDNO:22) containing the underlined HindXXX restriction site followed by nucleotides complementary to nucleotides 753-768 as shown in Figure 1 B (SEQ ID NO: 1 ).
• restriction sites are convenient to restriction enzyme sites in the bacterial expression vector pQE9, which are used for bacterial expression in this example. (Qiagen, Inc. 9259 Eton Avenue, Chatsworth, CA, 9131 1). pQE9 encodes ampicilhn antibiotic resistance (“Amp 1 ”) and contains a bacterial origin of replication ("ori”), an IPTG inducible promoter, a ribosome binding site (“RBS”), a 6-His tag and restriction enzyme sites.
• Amp 1 ampicilhn antibiotic resistance
• ori ampicilhn antibiotic resistance
• RBS ribosome binding site
• 6-His tag 6-His tag
• E. coli strain M15/rep4 containing multiple copies of the plasmid pREP4, which expresses lac repressor and confers kanamycin resistance ("Kan r "), is used in carrying out the illustrative example described herein.
• This strain which is only one of many that are suitable for expressing AIM II protein, is available commercially from Qiagen.
• Transformants are identified by their ability to grow on LB plates in the presence of ampicilhn and kanamycin. Plasmid D ⁇ A is isolated from resistant colomes and the identity of the cloned DNA confirmed by restriction analysis
• Clones containing the desired constructs are grown overnight ("O/N") in liquid culture in LB media supplemented with both ampicilhn (100 ⁇ g/ml) and kanamycin (25 ⁇ g/ml)
• O/N culture is used to inoculate a large culture, at a dilution of approximately 1 100 to 1 250
• the cells are grown to an optical density at 600nm ("OD600") of between 0 4 and 0 6 Isopropyl-B-D-thiogalactopyranoside (“IPTG”) is then added to a final concentration of 1 mM to induce transcription from lac repressor sensitive promoters, by inactivating the lacX repressor Cells subsequently are incubated further for 3 to 4 hours
• Cells then are harvested by centrifugation and disrupted, by standard methods
• Inclusion bodies are purified from the disrupted cells using routine collection techniques, and protein is solubihzed from the inclusion bodies into 8M urea The 8M urea solution
• pQE60 is used for bacterial expression in this example (QIAGEN, Inc , 9259 Eton Avenue, Chatsworth, CA, 91311)
• pQE60 encodes ampicilhn antibiotic resistance ("Amp r ”) and contains a bacterial origin of replication ("ori"), an IPTG inducible promoter, a ribosome binding site (“RBS”), six codons encoding histidine residues that allow affinity purification using nickel-nitrilo-t ⁇ -acetic acid (“Ni-NTA”) affinity resin sold by QIAGEN,
• a DNA fragment encoding a polypeptide may be inserted in such as way as to produce that polypeptide with the six His residues (i.e., a "6 X His tag") covalently linked to the carboxyl terminus of that polypeptide
• the polypeptide coding sequence is inserted such that translation of the six His codons is prevented and. therefore, the polypeptide is produced with no 6 X His tag.
• the DNA sequence encoding the desired portion ofthe AIM II protein is amplified from the deposited cDNA using PCR ohgonucleotide primers which anneal to the amino terminal sequences of the desired portion of the AIM II protein and to sequences in the deposited construct 3' to the cDNA coding sequence. Additional nucleotides containing restriction sites to facilitate cloning in the pQE60 vector are added to the 5' and 3' sequences, respectively.
• the 5' primer has the sequence 5'GACGC
• the 3' primer has the sequence 5' CGC AAGCTT CCTT CAC ACC ATG AAA GC 3' (SEQ ID NO: 19) containing the underlined Hindlll restriction site followed by nucleotides complementary to the 3' end ofthe non-coding sequence in the AIM II DNA sequence in Figure IB (SEQ ID NOJ).
• the amplified AIM II DNA fragments and the vector pQE60 are digested with Ncol and Hindlll and the digested D ⁇ As are then ligated together. Insertion ofthe AIM II D ⁇ A into the restricted pQE60 vector places the AIM II protein coding region including its associated stop codon downstream from the IPTG- inducible promoter and in-frame with an initiating AUG. The associated stop codon prevents translation ofthe six histidine codons downstream ofthe insertion point.
• the 5' primer has the sequence 5'-GGG CCA TGG ATG GTC ACC CGC CTG CC-3' (SEQ ID NO:23) containing the underlined Ncol restriction site, and includes followed by 17 nucleotides of the AIM II protein coding sequence in Figure 1 A.
• the 3' primer has the sequence 5'-GGG AAG CTT CAC CAT GAA AGC CCC G-3' (SEQ ID ⁇ O:22) containing the underlined Hindlll restriction site followed by nucleotides complementary to nucleotides 753 to 768 in Figure IB (SEQ ID NOJ).
• the amplified AIM II (aa 68-240) DNA fragments and the vector pQE60 were digested with Ncol and Hindlll and the digested D ⁇ As were then ligated together. Insertion of the AIM II (aa 68-240) D ⁇ A into the restricted pQE60 vector places the AIM II (aa 68-240) protein coding region downstream from the IPTG-inducible promoter and in-frame with an initiating AUG.
• the Hindlll digestion removes the six histidine codons downstream ofthe insertion point.
• the 5' primer has the sequence 5'-GGG CCA TGG GCC AAC TCC AGC TTG ACC-3' (SEQ ID NO: 24) containing the underlined Ncol restriction site including nucleotides 349-366 in the AIM II protein coding sequence in Figure 1A.
• SEQ ID NO: 24 sequence 5'-GGG CCA TGG GCC AAC TCC AGC TTG ACC-3' (SEQ ID NO: 24) containing the underlined Ncol restriction site including nucleotides 349-366 in the AIM II protein coding sequence in Figure 1A.
• the point in the protein coding sequence where the 5' primer begins may be varied to amplify a desired portion ofthe complete protein (i.e., shorter or longer).
• the 3' primer has the sequence 5'-GGG AAG CTT CAC CAT GAA AGC CCC G-3 1 (SEQ ID ⁇ O:22) containing the underlined Hindlll restriction site followed by nucleotides complementary nucleotides 755-768 ofthe AIM II DNA sequence in Figure IB.
• the amplified AIM II (aa 101 -240) DNA fragments and the vector pQE60 were digested with Ncol and Hindlll and the digested D ⁇ As are then ligated together.
• Insertion ofthe AIM II (aa 101-240) D ⁇ A into the restricted pQE60 vector places the AIM II (aa 101 -240) protein coding region downstream from the IPTG-inducible promoter and in-frame with an initiating AUG.
• the Hindlll digestion removes the six histidine codons downstream ofthe insertion point
• a polynucleotide sequence encoding a soluble fragment of AIM II (corresponding to amino acid residues L83-V240 of SEQ ID NO 2) was cloned into the HGS E coli expression vector pHE4 The resulted plasmid DNA
• Tris-HCl pH7 4, 2 mM CaCl 2 was lysed by passing twice through a microfluidizer (Microfluidics, Newton, MA) at 6000-8000 psi
• the lysed sample was mixed with NaCl to a final concentration of 0 5M and then centrifuged at 7000 x g for 20 minutes
• the resulting pellet was washed again with the same buffer plus 0 5M NaCl and then centrifuged at 7000x g again for 20 minutes
• the partially purified inclusion bodies were then resuspended for 2-4 hours at 20-25 ⁇ C in 2 0 M guanidine hydrochloride containing 100 mM Tris pH 7 4, 2mM CaC12, 5 mM Cysteine and centrifuged The resulting pellet was then resuspended for 48-72 hours at 4 ⁇ C in 3 0-3 5 M guanidine hydrochloride containing 100 mM Tris pH 7 4, 2mM CaC12, with or without 5 mM Cysteine
• the 3M guanidine hydrochloride extract was quickly diluted with 20-30 volumes of a buffer containing 50 mM Tris-HCl pH8, 150 mM sodium chloride Detergents such as Tween-20, CHAPS can be added to increase the refold efficacy. Afterwards the mixture was placed at 4 ⁇ C without mixing for 2 to 7 days prior to the chromatographic purification steps described below H. Liquid Chromaiographic Purification of AIM II The diluted AIM II sample was clarified using a 0 45 ⁇ m sterile filter. The AIM II protein was then adjusted to pH6-6.8 with 0.5M MES and chromatographed over a strong cation exchange (POROS HS-50) column.
• POROS HS-50 strong cation exchange
• the HS column was washed first with 6-10 column volume of a buffer containing 50 mM MES-NaOH pH 6.6 and 150 mM sodium chloride.
• the bound protein was eluted using 3 to 5 column volume of a stepwise gradient of 300 mM, 700 mM, 1500 mM sodium chloride in 50 mM MES at pH 6.6.
• the HS fraction eluted with 0 7 M sodium chloride was diluted 3 -fold with water.
• the ligation mixture from expression constructs made in D, E or F, above were transformed into competent E. coli cells using standard procedures such as those described in Sambrook et al. Molecular Cloning: a Laboratory Manual, 2nd Ed; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).
• This strain which was only one of many that are suitable for expressing AIM II protein, was available commercially from QIAGEN, Inc., supra.
• Transformants were identified by their ability to grow on LB plates in the presence of ampicilhn and kanamycin. Plasmid DNA was isolated from resistant colonies and the identity of the cloned DNA confirmed by restriction analysis, PCR and DNA sequencing.
• Clones containing the desired constructs are grown overnight ("O/N") in liquid culture in LB media supplemented with both ampicilhn (100 ⁇ g/ml) and kanamycin (25 ⁇ g/ml).
• O/N culture was used to inoculate a large culture, at a dilution of approximately 1 :25 to 1 :250.
• the cells were grown to an optical density at 600 nm ("OD600”) of between 0J and 0.6.
• IPTG Isopropyl-b-D- thiogalactopyranoside
• the cells were then stirred for 3-4 hours at 4°C in 6M gaanidine-HCl pH8
• the protein can be successfully refolded by dialyzing it against 500 mM NaCl, 20% glycerol, 25 mM T ⁇ s/HCl pH7 4, containing protease inhibitors
• the protein can be purified by ion exchange, hydrophobic interaction and size exclusion chromatography
• an affinity chromatography step such as an antibody column can be used to obtain pure AIM II protein
• the purified protein is stored at 4°C or frozen at -80 °C
• the cDNA sequence encoding the full length AIM II protein in the deposited clone assigned ATCC Accession No 97689 is amplified using PCR ohgonucleotide primers corresponding to the 5' and 3 ' sequences ofthe gene
• the 5 ' primer has the sequence 5 'GCT CCA GGA TCC GCC ATC ATG
• GAG GAG AGT GTC GTA CGG C 3 ' (SEQ ID NO 1 1) containing the underlined BamHl restriction enzyme site followed by 22 bases (/ e , nucleotides
• the 5 ' end of the amplified fragment encoding AIM II provides an efficient signal peptide
• An efficient signal for initiation of translation in eukaryotic cells, as desc ⁇ bed by Kozak, M , J Mol Biol 196 947-950 (1987) is appropriately located in the vector portion of the construct
• the 3 ' primer has the sequence 5 'GA CGC GGT ACC GTC CAA TGC ACC ACG CTC CTT CCT TC 3 ' (SEQ ID NO 12) containing the underlined J.s/ 718 restriction site followed by nucleotides complementary to 770-795 nucleotides ofthe AIM II set out in Figure 1A
• the amplified fragment is isolated from a 1% agarose gel using a commercially available kit ("Geneclean," BIO 101 Inc . La Jolla, Ca ) The fragment then is digested with BamHl an ⁇ A.spl ⁇ 8 and again is purified on a 1% agarose gel This fragment is designated herein F2
• the vector pA2-GP is used to express the AIM II protein in the baculovirus expression system, using standard methods, as described in Summers et /., A Manual of Methods for Baculovirus Vectors and Insect Cell Culture
• This expression vector contains the strong polyhedrin promoter ofthe Autogr ⁇ ph ⁇ c ⁇ liformc ⁇ nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites
• the signal peptide of AcMNPV gp67, including the N-terminal methionine, is located just upstream of aB ⁇ mHl site
• the polyadenylation site of the simian virus 40 ("SV40") is used for efficient polyadenylation For an easy selection of recombinant virus the beta-galactosidase gene fromE.
• polyhedrin sequences are flanked at both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate viable virus that express the cloned polynucleotide
• baculovirus vectors could be used in place of pA2-GP, such as pAc373, pVL941 and pAcIMl provided, as those of skill readily will appreciate, that construction provides appropriately located signals for transcription, translation, trafficking and the like, such as an in-frame AUG and a signal peptide, as required
• pA2-GP such as pAc373, pVL941 and pAcIMl
• the plasmid is digested with the restriction enzyme B ⁇ mHl and AsplX8 and then is dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art.
• the DNA is then isolated from a 1% agarose gel using a commercially available kit ("Geneclean" BIO 101 Inc . LaJolla, Ca.). This vector DNA is designated herein "V”.
• Fragment F2 and the dephosphorylated plasmid V2 are ligated together with T4 DNA ligase.
• E. coli HB 101 cells are transformed with ligation mix and spread on culture plates.
• Bacteria are identified that contain the plasmid with the human AIM II gene by digesting DNA from individual colonies using Xbal and then analyzing the digestion product by gel electrophoresis. The sequence ofthe cloned fragment is confirmed by DNA sequencing. This plasmid is designated herein pBacAIM II.
• the cDNA sequence encoding the human AIM II protein in the deposited clone identified as ATCC Accession No. 97483 is amplified using PCR ohgonucleotide primers corresponding to the 5' and 3' sequences ofthe gene.
• the 5' primer has the sequence 5' CGC GGA TCC CGG AGA GAT GGT
• CAC C 3' (SEQ ID NO: 67) containing the underlined BamHl restriction enzyme site followed by 15 bases ofthe sequence of AIM II of SEQ ID NO:38. Inserted into an expression vector, as described below, the 5' end ofthe amplified fragment encoding human AIM II provides an efficient signal peptide.
• Biol, 196: 947-950 (1987) is appropriately located in the vector portion ofthe construct.
• the 3' primer has the sequence 5' CGC TCT AGA CCT TCA CAC CAT GAA AGC 3' (SEQ ID NO:68) containing the underlined Xbal restriction followed by nucleotides complementary to the last 18 nucleotides ofthe AIM II coding sequence set out in SEQ ID NO:38, including the stop codon.
• the amplified fragment is isolated from a 1% agarose gel using a commercially available kit ("Geneclean,” BIO 101 Inc., La Jolla, Ca.), digested with BamHl and Aspl X 8 and again is purified on a 1 % agarose gel.
• Geneclean BIO 101 Inc., La Jolla, Ca.
• the vector is then constructed essentially as described above in section A of this example, but using a pA vector
• the plasmid shuttle vector pA2 GP was used to insert the cloned DNA encoding the an N-terminal deletion of the AIM II protein into a baculovirus to express an AIM II mutant (Gln(60) to Val(240)) and AIM II mutant (Ser(79) to Val(240)) in SEQ ID NO:2, using a baculovirus leader and standard methods as described in Summers et al, A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experimental Station Bulletin No. 1555 (1987).
• This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by the secretory signal peptide (leader) ofthe baculovirus gp67 protein and convenient restriction sites such as BamHl, Xbal and AsplX8.
• the polyadenylation site of the simian virus 40 ("SV40") is used for efficient polyadenylation.
• the plasmid contains the beta-galactosidase gene fromE. coli under control of a weak Drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhedrin gene.
• the inserted genes are flanked on both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate viable virus that expresses the cloned polynucleotide.
• baculovirus vectors could be used in place ofthe vector above, such as pAc373, pVL941 and pAcIMl, as one skilled in the art would readily appreciate, as long as the construct provides appropriately located signals for transcription, translation, secretion and the like, including a signal peptide and an in-frame AUG as required.
• Such vectors are described, for instance, in Luckow et al, Virology 170: X -39.
• the cDNA sequence encoding the AIM II (Gln(60)to Val(240), Figure 1 A (SEQ ID NO:2), was amplified using PCR ohgonucleotide primers corresponding to the 5' and 3' sequences ofthe gene.
• the 5' primer has the sequence 5'-GGG GGA TCC CGCA GCT GCA CTG GCG TCT AGG-3 1 (SEQ ID NO 25) containing the underlined BamHX restriction enzyme site followed by 20 nucleotides (/ e , nucleotides 225-245) encoding the AIM II protein shown in Figures 1 A and IB, beginning with amino acid 60 ofthe protein
• the 3' primer has the sequence 5'-GGG TCT AGA CAC CAT GAA AGC CCC G-3 1 (SEQ ID NO 26) containing the underlined Xbal restriction site followed by nucleotides complementary to nucleotides 753-768 in Figure IB (SEQ ID NO 1)
• the amplified fragment was isolated from a 1% agarose gel using a commercially available kit ("Geneclean,” BIO 101 Inc , La Jolla, Ca ) The fragment then was digested with BamHX and XbaX and again was purified on a 1 % agarose gel This fragment was designated herein "FI "
• the plasmid was digested with the restriction enzymes BamHl and Nb l and optionally, can be dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art
• the DNA was then isolated from a 1% agarose gel using a commercially available kit ("Geneclean” BIO 101 Inc , La Jolla, Ca ) This vector DNA was designated herein "VI"
• E. coli HB 101 or other suitable E. coli hosts such as XL- 1 Blue (Stratagene Cloning Systems, La Jolla, CA) cells were transformed with the ligation mixture and spread on culture plates Bacteria were identified that contain the plasmid with the human AIM II gene using the PCR method, in which one of the primers that was used to amplify the gene and the second primer was from well within the vector so that only those bacterial colonies containing the AIM II gene fragment will show amplification ofthe DNA The sequence ofthe cloned fragment was confirmed by DNA sequencing This plasmid was designated herein pBacAIM II (aa 60-240)
• the 5' primer has the sequence 5' cgc GGATCC C TCCTGGGAGCAGCTGATAC 3' (SEQ ID NO 27) containing the underlined BamHl restriction enzyme site followed by nucleotides 283-301 encoding the AIM II protein shown in Figures lAand 1 B, beginning with amino acid 79 of the protein
• the 3' primer has the sequence
• the amplified fragment was isolated from a 1% agarose gel using a commercially available kit ("Geneclean,” BIO 101 Inc , La Jolla, Ca ) The fragment then was digested with BamHl and again was purified on a 1% agarose gel This fragment was designated herein "FI "
• the plasmid was digested with the restriction enzymes BamHl and optionally, can be dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art
• the DNA was then isolated from a 1%> agarose gel using a commercially available kit ("Geneclean” BIO 101 Inc., La Jolla, Ca ) This vector DNA was designated herein "Nl"
• Fragment FI and the dephosphorylated plasmid VI were ligated together with T4 D ⁇ A ligase E. coli HB101 or other suitable E. coli hosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, CA) cells were transformed with the ligation mixture and spread on culture plates Bacteria were identified that contain the plasmid with the mutant AIM II gene using the PCR method, in which one of the primers that was used to amplify the gene and the second primer was from well within the vector so that only those bacterial colonies containing the AIM II gene fragment will show amplification ofthe D ⁇ A The sequence ofthe cloned fragment was confirmed by D ⁇ A sequencing This plasmid was designated herein pBacAIM II (aa 79-240)
• the plate was rocked back and forth to mix the newly added solution The plate was then incubated for 5 hours at 27°C After 5 hours the transfection solution was removed from the plate and 1 ml of Grace's insect medium supplemented with 10%o fetal calf serum is added The plate was put back into an incubator and cultivation was continued at 27°C for four days
• plaque assay was performed, as described by Summers and Smith, cited above An agarose gel with "Blue Gal” (Life Technologies Inc , Gaithersburg) was used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques (A detailed description of a "plaque assay” of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc , Gaithersburg, page 9-10)
• V-AIM II V-AIM II (aa 60-240)
• Sf9 cells were grown in Grace's medium supplemented with 10% heat- inactivated FBS The cells were infected with the recombinant baculovirus V- AIM II or V-AIM II (aa60-240) at a multiplicity of infection ("MOI") of about 2
• Most ofthe vectors used for the transient expression ofthe AIM II protein gene sequence in mammalian cells should carry the SV40 origin of replication. This allows the replication ofthe vector to high copy numbers in cells (e.g. , COS cells) which express the T antigen required for the initiation of viral DNA synthesis. Any other mammalian cell line can also be utilized for this purpose
• a typical mammalian expression vector contains the promoter element, which mediates the initiation of transcription of mRNA, the protein coding sequence, and signals required for the termination of transcription and polyadenylation ofthe transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).
• LTRs long terminal repeats
• cellular signals can also be used (e.g., human actin promoter).
• Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152).
• pSV2dhfr ATCC 37146
• pBC12MI ATCC 67109
• Mammalian host cells that could be used include, human HeLa, 283, H9 and Jurkart cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1 , African green monkey cells, quail QC1-3 cells, mouse L cells and Chinese hamster ovary cells
• the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome
• a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transfected cells
• the transfected gene can also be amplified to express large amounts ofthe encoded protein
• the DFIFR dihydrofolate reductase
• Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al. Biochem J. 227:211-219 (1991), Bebbington et al, Bio/Technology 70.
• the expression vectors pCl and pC4 contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al, Molecular and Cellular Biology, 438-447 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al, Cell 41:521-530 (1985)) Multiple cloning sites, e.g., with the restriction enzyme cleavage sites BamHl, Xbal and AsplX8, facilitate the cloning of the gene of interest.
• the vectors contain in addition the 3 ' intron, the polyadenylation and termination signal ofthe rat preproinsulin gene Example 3(a): Cloning and Expression in COS
• the expression plasmid, pAIM II HA is made by cloning a cDNA encoding AIM II into the expression vector pcDNAI/Amp (which can be obtained from Invitrogen, Inc.).
• the expression vector pcDNAl/amp contains: (1) an E. coli origin of replication effective for propagation in E.
• coli and other prokaryotic cells (2) an ampicilhn resistance gene for selection of plasmid-containing prokaryotic cells; (3) an SV40 origin of replication for propagation in eukaryotic cells; (4) a CMV promoter, a polylinker, an SV40 intron, and a polyadenylation signal arranged so that a cDNA conveniently can be placed under expression control of the CMV promoter and operably linked to the SV40 intron and the polyadenylation signal by means of restriction sites in the polyline.
• a DNA fragment encoding the AIM II protein and an HA tag fused in frame to its 3 ' end is cloned into the polyline region of the vector so that recombinant protein expression is directed by the CMV promoter.
• the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein described by Wilson et al, Cell 37: 767 (1984).
• the fusion ofthe HA tag to the target protein allows easy detection ofthe recombinant protein with an antibody that recognizes the HA epitope.
• the plasmid construction strategy for full length AIM II is as follows. The
• AIM II cDNA of the deposited clone is amplified using primers that contain convenient restriction sites, much as described above regarding the construction of expression vectors for expression of AIM II in E. coli.
• primers that contain convenient restriction sites, much as described above regarding the construction of expression vectors for expression of AIM II in E. coli.
• one of the primers contains a hemagglutinin tag ("HA tag") as described above.
• Suitable primers include the following, which are used in this example.
• the 5 ' primer, containing the underlined BamHl site, and an AUG start codon has the following sequence: 5' GAG CTC GGA TCC GCC ATC ATG GAG GAG AGT GTC GTA CGGC 3' (SEQ 1D NOJ 3).
• the 3' primer containing the underlined Xbal site, a stop codon. 9 codons thereafter forming the hemagglutinin HA tag, and 33 bp of 3' coding sequence (at the 3' end) has the following sequence: 5'GAT GTT CTA GAA AGC GTA GTC TGG GAC GTC GTA TGG GTA CAC

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