CA2321186A1 - Apoptosis inducing molecule ii and methods of use - Google Patents

Abstract

The present invention relates to a 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.

Description

Apoptosis Inducing Molecule II and Methods of Use Background of tire Invention Field of the Invention 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.
Related Art Human tumor necrosis factors a (TNF-a) and ~i (TNF-(3, or lymphotoxin) 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., Annu. Ret.. Immunol., 7:625-655 (1989)).
Tumor necrosis factor (TNF-a and TNF-Vii) 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.
To date, known members of the TNF-ligand superfamily include TNF-a, TNF-~i (lymphotoxin-a), LT-Vii, OX40L, Fas ligand, CD30L, CD27L, CD40L and 4-IBBL. The ligands of the TNF ligand superfamily are acidic, TNF-like molecules with approximately 20% sequence homalogy in the extracellular domains (range, 12%-36%) and exist mainly as membrane-bound forms with the biological lv active form being a trimeric/multimeric complex. Soluble forms of the _7-TNF ligand superfamily have only been identified so far for TNF, LTa, and Fas ligand (for a general review, see Gruss, H. and Dower, S.K., Blood, 8.5(I2~:3378-3404 (1990), which is hereby incorporated by reference in its entirety.
These proteins are involved in regulation of cell proliferation, activation, and differentiation. including control of cell survival or death by apoptosis or cytotoxicity (Arn~itage, R.J., Curr. Opin. Immz~nol. 6:407 ( 1994) and Smith, C.A., Cell 75:959 ( 1994)).
Mammalian development is dependent on both the proliferation and differentiation of cells as well as programmed cell death which occurs through apoptosis (Walker, el ul., MelhodsAchiev. Exp. Palhol. 13:18 (1988). Apoptosis plays a critical role in the destruction of immune thymocytes that recognize self antigens. Failure of this normal elimination process may play a role in autoimmune diseases (Gammon el ul., Immunology Today 12:193 ( 1991 )).
Itoh et al. (Cell 66:233 ( 1991 )) described a cell surface antigen, Fas/CD95 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 e/ ul.. J. Immunolo. 148:1274 (1992)) in addition to activated T-cells, B-cells, neutorophils. In experiments where a monoclonal Ab to Fas is cross-linked to Fas, apoptosis is induced (Yonehara et ul.. .l. Exp. Med. 169:1747 (1989); Trauth et al., Science 2:15:301 (1989)). In addition, there is an example where binding of a monoclonal Ab to Fas may stimulate T-cells under certain conditions (Alderson et al., J. Exp.
Med.
178:2231 ( 1993)).
Fas antigen is a cell surface protein of relative M W of 45 Kd. Both human and murine genes for Fas have been cloned by Watanabe-Fukunaga et al., (J.
Immunvl. 148:1274 ( 1992)) and Itoh et ul. (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 LTa receptor CD40, CD27, CD30, and OX40.

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Rece~atly the Fas Iigand has been described (Suds er al., Cetl 15:1169 (1993}}. The amino acid sequence indicates that Fas ligand is a typc Ii transmembrane protzin i~lcnging to the TNf family. Fas ligand is expressed in splenocytes and thymocvtes. The purified Fas ligand has a MAN of 40 kd.
Recently, it has been demonstrated that FasIFas ligand interactions are required for apoptosis following the activation of T-cells (Ju et a?., Nsrrere 3'3:4=14 (1995}; $tt~ner et al., IJature 373=~1 (I995)). Activation o?' T-cells induces 'noth proteins on the cell surface. Subsequent interaction bettveefi the iigand sad receptor results in apc~ptosis of the cells. This supports the possible regulatory role for apoptosis induced by Fas.~as ligand interaction dou~r~g normal immune responses.
The polypeptide of the present invention has been iderti~ed as a novel rz~ember of the TIv'F ligand super-family based on structural and biological sirrilaritiss.
Clearly, there is a need for factors that regulate activation, and differenr:ation of normal and abnormal cells. There is a need, therefore, for identification and characterizatiozt of such factors that modulate activation and differentiation of cells, both r~orrnatly and in disease states. In particular, there is s need to isolate and eharacteri~e additional Fas Iigands that control apoptosis 2~ for. tl~e treattr~ent of autoimzn~:ue disease, graft versus host disease, rl;eumaaoid arthritis and lymphadenopathy.
Sufn~rrar,~ ~~''tlae Invention The present invertio:~ provides isrrlated nucleic acid malec;ules cvmprisiag a polj~nucleotide encoding the AIM II polr~peptide having the amino acid sequence. shown in Figures lA acrd B ($EQ Il7 N0:3) or tr~e atnii:o acid sequence encoded by the clJrdA clone deposited as ATCC Deposit Number 9'689 on August 22, 1996. 'fhe present invention also provides isolated nucleic acid r~~:oiecules comprising a polyz~ucleotide encoding the AhVI II
polypeptide having the ~arnioo acid sequen,~,e shown in Figures 1 C and ):7 (SEQ ID ND.39}
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~ilE3 7131:)-~ +v:3 t35 y;c)c~ii.~p"r,: # i <t LVy. :L IJ:11: JItvJ lire the amino acid sequence encod:d by the cDNA clone deposited as ATCC Deposit l~~umber 9783 on March 15, 1996.
'fhe present invention also relates to recombinant vectors, ~whieh include the isolated nucleic acid molecules of the present invention, and to host cells containing the recounbinant vectors, as wel l as to methods cfm,akiz~g such vectors and host cells and for using them for production of AIM II polypeptides or peptides by recombinant techniques.
'file invention further provides an isolated AIM II polypeptide having an amino acid sequence encoded by a paiynucleotide described herein.
IQ As used herein the term "AIM II" polypeptide includes membrane-bound proteins tcomprisin.g a cytoplasmic domain, a transmJ.embrarle domain, and an eatracellular domain) as well as truncated proteins that zetain the AIM II
palypeptide activity. In one embodiment, soluble AIM iI polypeptides comprise all or part of tt!e e~ctracellular domain of an ATM II protein, but lack the 15 ttansme~nbrane region that would cause retention of the ,polypeptide on a cell membrane. Soluble A1M II may also include part of the transtnembrane region cr part c.~f the cytoplasmic dotnair>, c~z ether sequences, provided that the soluble AIM II protein is capable of being secreted. A heterologous signal peptide can be fused tmt're N-terminus of the soluble AIM II poiypeptide such that the 20 soluble t3tM 1I Foly~peptide is secreted upon expression.
The invention also provides for AIIL: II polypeptides, particularly human AIM-L polypeptides, which may be employed to teat a~ictions such as i~~rtphade:~lopathy, rheumatoid arthritis, autoiznmune disease, graft versus host disease, IgE-mediated allergic reactions, anaphylaxis, adult respiratory distress 25 syndrcrne, Crohn's disease, allergic asthma, acute lymphoblastic leulcen~ia tALLI, non-i~odgkin's lvmghoyna(1~'HLj, and Gra4~es' disease. These polypeptides ofthe invention may also be used to Stimulate peripheral tolerance, destroy Borne transformed cell lines, mediate cell activation and proliferation and are functionally linked as primary mediators of immune regulation and inflammatory 34 response.
AMEiVDED SHEET

_5_ The invention further provides compositions comprising an AIM I1 polynucleotide or an AlM II polypeptide for administration to cells in vimo.
to cells ex viva and to cells in vivo, or to a multicellular organism. In certain particularly preferred embodiments of this aspect of the invention. the compositions comprise 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 Il, which involves contacting cells which express AIM I1 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 of the 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.
In another aspect, a screening assay for AIM II agonists and antagonists is provided. The antagonists may be employed to prevent septic shock, inflammation, cerebral malaria, activation of the HIV virus, graft-host rejection, bone resorption, and cachexia (wasting or malnutrition).
In a further aspect of the invention, AIM II may be used to treat rheumatoid arthritis (RA) by inhibiting the increase in angiogenesis or increase in endothelial cell proliferation required to sustain an invading pannus in bone and cartilage as is often observed in RA.
In an additional aspect of the invention, AIM I1 may be used to inhibit or activate a cellular response mediated by a cellular receptor (e.g., LT-~3-R, TR?.
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.
An additional aspect of the invention is related to a method for treating an individual in need of an increased level of AIM II activity in the body comprising kG\ y» F F' 1 'iC t:\CHt~.i\ c)~~ : 17- ~-,cA 02321186 2000 08 2p:~.X18 ~ 813 +4-;~ 89 '?:3~~4~~ FW : I~ t o administering to such an individual a composition comprising a therapeutically effective atxxount of an isolated AIM II polypeptide of the invention or as ago~ost thereof.
A still further aspect of the 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 a therapeutically effective amount of an AIM II antagonist.
brief .~~scription of the Figures Figures IA and B show the nucleotide (SEQ ID NO:1) and deduced 1~ amino 2cid (SEQ ID N(~:2) sequences of AIM II. The protein has a deduced molecular ~Neight ref about 2;;.4 kDa. The predicted ~'ransmembrsne Domain of the At~~ Ii protein is underlined.
Figures IC and D show the nucle,~tide (SEQ ID N0:38) and deduced amino acid {SEQ ID N0:39) sequences of a partial AIM II cDNA that w-as also obtained.
Figures 2A-F show the regiars of similarity behveen the amino acid sequences of the AIM II protein and human TNF-a (SEQ ID NO: 3). human TNF-~3 (SEQ ID h'G:4), human lymphotoxin (SEQ ID NO:~) and human Fas Ligand (SE's ID ht0:6).
Figures 3A-F show an analysis of the AIM II amino acic? sequence.
Alpha, beta, turn and coil regions; hyarophilieity a,~.d hydrophobieity;
acnphipathi~c regions; flexible regions; antigenic index and surface probability are shown. In the "Antigenic Index - 3ameson-Virolf' graph, about amino acid residues I3-?0, 23-35, 69-?9, 85-94, I67-178, 184-196, 221-233 in Figures lA
and B (SEA ID NG:2) correspond to the shown highly antigenic reeiens of the :ATM II protein.
Figures 4A and B show tha effect of AIPrf TI on the in zdtro proliferation of MDA-MB-231 human br;,ast cancer cells. S,QQO MDA-MB-23IlWT (circle), AMENDED SHcET

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w7-MDA-11~IS-~3llNeo (triangle) or MDA-MB-2311AIM II (square) cells were plated in triplicate in 24-well plates with I'~A~l in the presence of either 10%
FB S (filled circle, sqe~are or triangle) or 1 °f° FBS {open circle, square or triauglel.
The number of live cell s were determined by trypan blue exclusion method at day 3, day S or day 7. Cells were fed with fresh mediunn every two days during this time course. Figure :H shows colony formation of MDA-Nl>3-2311WT, MDA-MI3-231,~I~o and MDA-MB-?31lAIM II cdls in 0.33% agarose.
Figures .5A-C sho;v increased Apoptotic cells in MDA-hiB-2311AIM II
(Figur: SC; in 0. S % serum compared mith That of the MDA-~fB-~ 311WT {Figgie lU 5~.) or MDA-MB-?311Neo (h'igure SB) cells with Annexin-V FAGS arjalysis as desct~il;ed in Example 5 Material and Methods.
Figure 6 (A) shows an evaluation of the effects of AIh: II on growth of xeztogr~c hu.~~an breast carcinoma, hIDA-231 in nude trice. Female athyrnic nude micz were irijeeted s.e. with lc3' cells of parental MDA-23 1 (IvtDA-23 1 -WT), ar hlI3A-2 3 1 stably transfected with AII~i II, or vector control neo (n=10).
Mice wore Then ear tagged and randomized. Tumor growth was assessed twice weekly with a caliper in the blinded fashio~a. This panel repzesents three experirnents each with ten mice per group. (8) shows the effect of ,~lM II
transduetion on inhibition of erow~tiz of MC-3$ marine colon cancer in symgeneic C57BL16 mice. Female CS7131rit mice were injected s.c. vriih I0~ cells of parentFl MC-38 (MC-38-WT), cr MC-38 stabl~r transfected with AIM II, or sector control r~eo (n=10). Mice were then ear tagged and randomized. Tuzr~oz grovrth was aasessed twice weekly with a caliper in a coded, blinded fashion 'i his pan~i representv four experiments each with ten mice per broup.
Figu,~~ 7 shows the pFlag-AIM II plasmid construct (Figwre 7A).
CYtoto.~cicity of a, recombinant soluble form of AIM II (sAIM II) in MDA-MB-231 cells in the presence or absence of II':~iy (Figure ?B) or with IFNy alone ( Figure 7C). Experiments were ca.~ied out as described in Example S Materials and Methods.
AMENDED SHEET
°. .

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:..?: ..::..: ; ;.::;.; :::..:. . .'::'.: :::.::
-g-Figures 8A-M. Cell surface expression of the LT~iR or TRby tb~e FRCS
analyses using LT~iR (Fi6ures 8A-D) or TR2 (Figures 8E-I-i) mAb. MDA-'vI8-231 (Figures 8A and E), HT-29 (Figures 8B and F), MC-3 (Figures SC and G).
L'93T (Figures 8D and 1~. FACS binding analyses of soluble AII~1 II protein alone {Figure 8I) and blocking of a soluble AIM II protein binding by pxeincubatron with the LT~3R-Fc fusion protein (Figure 8~ or TR2-Fc fusion protein (Figure 8K) in MDA-MB-2 31 cells. Figure 8L sumrrzarizes the surface expr>Lssion of LT(3R and TR2 in various cell lines. (Figure 8M) Effects of Fc ox TR?-Fc fusion protein to block the sAIM II-mediated eytotoxiciry in HT-lIl cells. Cells were plated irzta 96-well plates and sAIlM II (1 (3nglml) was added fizz the presence of 5 Ulml ofIFNy wYth various amounts of sLT(3R-Fc (open circle with LTpR-Fe alone, filled circle LT~iR-Fc, and IFNy) or T''.t'c2-Fe fusion protein {open triangle with '1'Tt-2Fc' alone, filled triangle TR.2-1Fc with sLTy and IFN ~).
Cells were incubated for eve days and the viability of cells was deteunined by XTT assays, Figure 9 shows secretion of IfN-'y by sAIM II treated human PBL cells.
Hluman PBLs (S x ;0= cells per well in the 46 well plate) were treated with or without anti-CD3 mAb and IL-2(20 LTlml) in the presence or absent of s:~,IIvI
lI
fox 5 days. The supernatants were then collected frozr~ the following groups of ZO cells: YBLs in the presence (iiiled circle) or absence (opez7. circle) or sAIM II, or the resting Pl3Ls with (filled triangle) or without (open triangle) sAZM II.
Fluman IFNy concentration were determined by ELISA.
Figure 10 shows a schematic representation of the pHE~4-5 expression vector (SEQ ID N0:54) and the subcaoned ~.IM II cDNA coding sequence. The locations of the kanarnycin resistance marker gene, the A)Zvi IT coding sequence.
~dae oriC sequence, and the lacIq coding sedaence are indicated.
r figure 11 shows the nucleotide sequence of the regulatoryr elements ofthe pHE promoter (SEQ ID 7~'4:51 ). The two dac operator sequences, the Shine-AMENDED SHEET

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Figure 12 shows a sensorgxant of specificity of binding of MCA-"s8 AiM II conditioned media :,o LT~R-F c versus ?'~ICIF-Fc immobilized on BIAcore chip. Ceziditioned media was analy zed or a BIAcore ins'~tument flowcell derivatized with iyrr~phot~oxin beta receptar Fc f;~sion protein. T he conditioned media (l4lp p,L) was flown over the chip at ~ ~L/trin and washed with IBS
bui~er aiso at 5 wLlmin, The shown data represents the net bound (otf rate) region of the plot after binding of AIM II to irnxnobilized receptor and is IG measured in relative mass ~~rni~.s (RU) versus tithe. The binding conditions were performed at high receptor chip densities under diffusion-limited conditions.
Legend: LT~iR-Fa and MCIF-Fc :efer to binding data front LT~SR-Fc or MCIF-Fc ir:~mobilized BIAcors e1_zip star~F«ces, respectively.
Figure l 3 shows the determination of the LT(3R binding by AIM II eluted frozzz LT(3R-rc column. Binding conditions were as described in Figure 11' LegEnd: LTpR and MCIf refer to biztding data from LTpR-Fc or MCIF-Fc immobiii~ed BIALore chip suffaces, respectively. Undiluted Conditioned media from 1'viCA3 $ yells was analyzed before (pxe) and after passage through MCXF'-1~ c (post-~.iCIF) and L T pR-Fc (post-LT~R) afrinity columns. Fractions (1 rnL) eluted from the LTpR (E4-~) and MCIF-Fc (El-'~) affinity columns were diluted 3-fold and teJted for binding to LTpR BIAcore chip.
DeBaided ~7~esetipnon The present invention pro~.~ides isolated nucleic acid molecules comprising a polynucleotrde e~ZCOding an C~IM II polypeptide having the amino 2~ acid sequencz sho~~n in Fcg rues LA axed B ; SF.C~ lI5 N0:2), whick~ was determined by sequez~.cing a cloned cDIV'A. The AIM I! protein of the present invention shares sequence homology with human TIvF-a (SEQ ID IV~: 3), human TNF-~i (SEQ ID N~'~:4), human lymphotaxin (SEQ ID 1\70:0 and human Fas Ligand (SEQ ID N0:6) (Figures 2A-F). The nucleotide sequence shown in Figures lA
A~1ENDED SHEET
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and B (SEQ ID NO:I) urere obtained by sequencing the a cl7NA clone, which was deposited on August 22, 1896 at the American Type Culture Collection, 10801 University Blvd., ?ldanassas, VA 201 IO-2209, LISA, and given accession number 97689. The deposited c:la»e is contained in the pBluescript SK(-) plasmid (Stratagene, L3 Jolla, CA). The nucleotide sequence shov~~n in Figures C and D was obtained by sequencing the a cDNA clone, which was deposited ore ;vlarch 1 S, 1996 at the American Type Culture Collection, 10801 University blvd., Manassas, VA 20110-2209, USA, and given accession number 97483.
lYucleic ~l rirf i~lo!ecules XO Unless otlaercvzse indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA
sequenaer (such as the l~Zodel 373 from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides enc5ded by DNA molecules determined herein were predicted by translation of a DNA sequence dete:mined as above.
1S Therefore, as is known in the art for any DNA sequence ~~etermined bz this aatam~ated approach, any nucleotide sequence deterrlined herein may contain some errors, N»cleotide sequences determined by automation are ty-picaliy at least about 9U°~o identical, more typically at least about 95% to at least about 99.9oro identical to the actual nucleotide sequence of the sequenced DNA
20 molecule. Ths actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well kno~.vzx in the art.
As is also known in the art, a single insertion or deletion in a deteralined nucleotide sequence competed to the actual sequence will cause a frame shift in translation of the nucleotide sequence such t>aat tl~e predicted amino acid 25 sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA
~rmlecule, beginning at the point of such an inser~.ion or deletion.
Using the information provided herein, such as the nucleotide sequence in Fig~.ues : A and H, a nucleic acid molecule of the present invention encoding >>.:::-:,:.::.>:::,>.:'<:::_.<, ,.q f~tt ~ted~» ~ ~ ~ ~: MENDEp g~4EET

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~ I . . v . ~ . r _ : .'1 J i: V ~ ' :l y L J J r .. . . . ..:~. . . :.:. . . .::. . ..::.... .>, ..:
an A .IM II lxrlypeptide may be obtained using standard cloning and screening procedures, such as thane for cloning cI7NAs using mRNA as starting material.
Illustrative of the invention, the nucleic acid molecule described in Figures lA
and B (SEQ ID NC~:1) was discovered in a cDNA library derived from human macrophage ox LDL (HMCCB54). 'fhe gene was also identified in c.DNA
libraries from activated T-cells (I-IT4CC72). 'fhe determined nucleotide sequence of r"he A.IM II cDNA of Figwres l A and B (SIrQ ID NU: l ) contains an open reading frame encoding a protein of 240 amino acid residues, with an initiation codon at positions 49-51 ofthe nucleoside sequence in Fi~.ires lA and B (SEQ
1D ID NO: l), an extracellular domain comprising amino acid residues from about (i0 to about 2~0 in Figures lA and $ (SEQ ID NO:Z), a transmembrane domain comprising amino acid residues from about 37 to about 59 in Figures 1 A and B
(SEQ ID ~r0:2), a intracellular domain comprising amino acid residues from about 1 to about 3o in Figures l A and B (SEQ iD N0:2) and a deduced I5 molcz°alar weight of about ?6.4 kDa The AIM II protein shown in Figures d A
and $ (SEQ ID N0:2) is about 27J/° identical and about S i % similar to the amino acid sequence of hamar.~ Fan Ligand (Figures 2A-F) and is about 26% identical and about a''°/a similar to the amino acid sequence of human TNF-a ~T'igvres ~A-F). TNF-Iigand like molecules function as dimers, given that AIM II is 20 homologous to T Nl~-ligand like molecules, it is likely that it also functions as a homodimer_ As one of ordinary skill yvould appreciate, due to the possibilities of sequencing ezrors discussed abcwe, the predicted AIM II golvpeptide encoded by the deposited crNA comprises about X40 amino acids, but may be anywhere in 25 the range of 23G-250 amino acid. It sill further be appreciated that, depending on the criteria used, concerning the exact "address" of the extracelluar, intracellular and transmembrane domaira of the AI~I II polypeptidc ditTer slightly. For example, the exact location of the AIM II extraceliular domain in Figures 1 A and B ( S:EQ ID N0:2) may vary slightly (c.g., the address may "shin"
30 by about 1 to 5 residues;) depending on the criteria used to define the domain.
As indicated, nucleic acid molecules of the present invention may be in the form of RTE A, such as mRl'dA, or in the form of DNA. including, for instance, ,.;; .....:::. -..: .,-::;v:..:: :. ;:;a A
lri~ec~.~~~~::
MEN~~~ SHED

_1~_ 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.
By "isolated" nucleic acid molecules) is intended a nucleic acid molecule.
DNA or RNA, which has been removed from its native environment For example.
recombinant DNA molecules contained in a vector are considered isolated for the purposes of the 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 of the DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
Isolated nucleic acid molecules of the present invention include DNA
molecules comprising an open reading frame (ORF) shown in Figures 1 A and B
(SEQ ID NO:1 ) or Figures 1 C and D (SEQ ID N0:38); DNA molecules comprising the coding sequence for the AIM II protein shown in Figures IA and B (SEQ ID N0:2) or Figures 1C and D (SEQ ID N0:39); and DNA molecules which comprise a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the AIM II
protein.
Of course, the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate such degenerate variants.
In addition, the invention provides a nucleic acid molecule having a nucleotide sequence related to a portion of SEQ ID NO:1 which has been determined from the following related cDNA clone: HT4CC72R (SEQ ID
N0:20).
In another aspect, the invention provides isolated nucleic acid molecules encoding the AIM II polypeptide having an amino acid sequence encoded by the cDNA clone contained in the plasmid deposited as ATCC Deposit No. 97689 on August 22, 1996 or by the cDNA clone contained in the plasmid deposited as ATCC Deposit No. 97483 on March 15, 1996. Preferably, this nucleic acid molecule will encode the polypeptide encoded by the above-described deposited cDNA clone. The invention further provides an isolated nucleic acid molecule having the nucleotide sequence shown in Figures 1 A and B (SEQ ID NO: l ) or Figures 1 C and D (SEQ ID N0:38) or the nucleotide sequence of the AIM II
cDNA contained in the above-described deposited clones, or a nucleic acid molecule having a sequence complementary to one of the above sequences. Such isolated molecules, particularly DNA molecules, are useful 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.
The present invention is further directed to fragments of the isolated nucleic acid molecules described herein. By a fragment of an isolated nucleic acid molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in Figures I A and B (SEQ ID NO:1 ) is intended fragments at least about 1 S nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325. 350, 375, 400, 425, 450, 475, 500, 525. 550, 575. 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925. 950, 975. 1000, 1025, 1050, 1075, 1100, I 125 or 11 SO nt in length are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in Figures 1 A and B (SEQ ID NO: l ). By a fragment at least 20 nt in length, for example, is intended fragments which include 20 ormore contiguous bases from the nucleotide sequence ofthe deposited cDNA
or the nucleotide sequence as shown in Figures 1 A and B (SEQ ID NO:1 ).
Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding epitope-bearing portions of the AIM II protein. In particular, such nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising amino acid residues from about 13 to about 20 in Figures 1 A and B (SEQ ID N0:2); a polypeptide comprising amino acid residues from about 23 to about 36 in Figure 1 (SEQ ID
N0:2); a polypeptide comprising amino acid residues from about 69 to about 79 in Figures 1 A and B (SEQ ID N0:2); a polypeptide comprising amino acid residues from about 85 to about 94 in Figures 1 A and B (SEQ ID N0:2);a polypeptide comprising amino acid residues from about 167 to about 178 in Figures 1 A and B (SEQ ID N0:2);a polypeptide comprising amino acid residues from about 184 to about I 96 in Figures 1 A and B (SEQ ID N0:2); and a polypeptide comprising amino acid residues from about 221 to about 233 in Figures 1 A and B (SEQ ID N0:2). The inventors have determined that the above 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.
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. Among these applications in autoimmune disease and aberrant cellular proliferation. Additional applications relate to diagnosis and to treatment of disorders of cells, tissues, and organisms.
This invention is also related to the use of the 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. The polynucleotide encoding the AIM II may also be employed as a diagnostic marker for expression of the polypeptide of the present invention.
In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above. for instance, the cDNA clone contained in ATCC

_1J_ Deposit 97689. By "stringent hybridization conditions" is intended overnight incubation at 42 °C in a solution comprising: 50% formamide, Sx SSC
(750 mM
NaCI. 75mM trisodium citrate), 50 mM sodium phosphate (pH 7.6}, Sx Denhardt's solution.10% dextran sulfate, and 20 pg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 x SSC at about 65 °C.
By a polynucleotide which hybridizes to a "portion" of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 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 10 polynucleotide. These are useful as diagnostic probes and primers as discussed above and in more detail below.
By a portion of a polynucleotide of "at least 20 nt in length," for example, is intended 20 or more contiguous nucleotides from the nucleotide sequence of the reference polynucleotide (e.g. , the deposited cDNA or the nucleotide sequence as 15 shown in Figures 1 A and B (SEQ ID NO:1 )).
Of course, a polynucleotide which hybridizes only to a poly A sequence (such as the 3' terminal poly(A) tract of the AIM II cDNA shown in Figures 1 A
and B (SEQ ID NO:1 )), or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide of the 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., practically any double-stranded eDNA clone).
As indicated, nucleic acid molecules ofthe present invention which encode an AIM I1 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 splicin~~ and polyadenylation signals, for e~arnple -ribosome binding and stability of mRNA: an additional coding sequence which codes for additional amino acids. such as those which provide additional functionalities.
Thus. the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the fused polypeptide. In certain preferred embodiments of this aspect of the invention, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, lnc.), among others, many of which are commercially available. As described in Gentz et ul.. Proc. Nml. Acud Sci. USA 86:821-824 (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 ul., Cell 37: 767 (1984). As discussed below, other such fusion proteins include the AIM II fused to Fc at the N- or C-terminus.
Nucleic acid molecules according to the present invention further include those encoding the full-length AIM-II polypeptide lacking the N-terminal methionine.
The present invention further relates to variants of the nucleic acid molecules of the 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 ( i 985). Non-natural ly occurring variants may be produced using art-known mutagenesis techniques.
Such 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, _17_ 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.
Further embodiments of the invention include isolated nucleic acid S molecules comprising a polynucleotide having a nucleotide sequence at least 90%
identical, and more preferably at least 95%, 96%. 97%. 98% or 99% identical to (a) a nucleotide sequence encoding the AIM I1 polypeptide having the complete amino acid sequence in Figures 1 A and B (SEQ ID N0:2); (b) a nucleotide sequence encoding the AIM II polypeptide having the amino acid sequence in Figures 1 A and B (SEQ ID N0:2), but lacking the N-terminal methionine; (c) a nucleotide sequence encoding the AIM I I polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No.
97689; (d) a 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 I1 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 of the nucleotide sequences in (a), (b), (c), (d), (e), (f) or (g) above.
By a polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence encoding an AIM II
polypeptide is intended that the 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. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the _18_ S' 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.
As a practical matter. whether any particular nucleic acid molecule is at least 90%, 95%, 96%, 97%. 98% or 99% identical to, for instance. the nucleotide sequence shown in Figures 1 A and B or to the nucleotides sequence of the deposited cDNA clone 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. Madison, WI 53711. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 ( 1981 ), to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
By a polynucleotide having a nucleotide sequence at least. for example, 95% "identical" to a reference nucleotide sequence of the present invention, it is intended that the 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. In other words, to obtain a poymucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to S% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to ~% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be an entire sequence shown in Figures lA

and B. the ORF topen reading frame). or any fragment specified as described herein.
As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 90%, 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 overal l match bem~een a query sequence (a sequence of the 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 er ul. (C'onrp. App. l3io,rci. 6:237-245 ( 1990)). In 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: Matrix=Unitary, k-tuple=4, Mismatch Penalty=l, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=l, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter.
1f the subject sequence is shorter than the query sequence because of 5' or 3' deletions. not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for 5' and 3' truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5' or 3' ends. relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5' and 3' ofthe subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide 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 corrected score is what is used for the purposes of the present invention. Only bases outside the 5' and 3' bases of the 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 ~' end of the subject sequence and therefore, the FASTDB alignment does not show a match/alignment of the first 10 bases at the ~' end. The 10 unpaired bases represent 10% of the sequence (number of bases at the S' and i' 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. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, 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 S' and 3' of the 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 of the present invention.
The present application is directed to nucleic acid molecules at least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in Figures 1 A and B (SEQ ID NO: I ) or to the nucleic acid sequence of the deposited cDNA. 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. Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having AIM II activity include, inter 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 of the AIM II gene, as described in Verma el ul.. Huma» Chromoso»~e.s: A Ma»ual o f Basic Tc~cITni ytres.
Pergamon Press, New York ( 1988): and (3) Northern Blot analysis for detecting AIM II
mRNA expression in specific tissues.
Preferred, however. are nucleic acid molecules having sequences at least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in Figures I A and B (SEQ ID NO: I ) or to the nucleic acid sequence of the deposited cDNA which do. in fact, encode a polypeptide having AIM II protein activity. By "a polypeptide having AIM II activity" is intended polypeptides exhibiting activity similar. but not necessarily identical. to an activity ofthe AIM II
protein of the invention, as measured in a particular biological assay. For example, 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). Alternatively. AIM II induced apoptosis can also be measured using TUNEL staining as described by Gavierli et al., J. Cell. Biol. 119: 493-501 ( 1992).
Briefly, 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 (chromosome clumping, shrinking and/or fragmentation of nuclei).
Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%, 98%, or 99%
identical to the nucleic acid sequence of the deposited cDNA or the nucleic acid sequence shown in Figures 1 A and B (SEQ ID NO: I ) will encode a polypeptide "having AIM II protein activity." In fact, since 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.
It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide WO 99/42584 PCT/US99/037p3 _77_ having AIM I1 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).
For example. guidance concerning how to make phenotypically silent amino acid substitutions is provided in ,Bowie, J. U. et al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 2-/7:1306-1310 ( 1990), wherein the authors indicate that proteins are surprisingly tolerant of amino acid substitutions.
AIM ll "Knock-Orrts" and Homologous Recon:bination 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 et al., Nature 317:230-234 (1985); Thomas & Capecchi, Cell ~ 1:503-512 ( 1987); Thompson et al., Cell 5:313-321 ( 1989);
each of which is incorporated by reference herein in its entirety). For example, a mutant, non-functional polynucleotide of the invention (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous polynucleotide sequence (either the coding regions or regulatory regions of the 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. In another embodiment, 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 & Capecchi 1987 and Thompson 1989, supra). However this approach can be routinely adapted for use in humans provided the recombinant DNA constructs are directly administered or _73_ targeted to the required site in nioo using appropriate viral vectors that will be apparent to those of skill in the art. The contents of each of the documents recited in this paragraph is herein incorporated by reference in its entirety.
In further embodiments of the invention. cells that are genetically engineered to express the polypeptides of the invention: or alternatively, that are genetically engineered not to express the polypeptides of the invention (e.g., knockouts) are administered to a patient in vivo. Such cells may be obtained from the patient (i. 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 ir7 vines 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 plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. The coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention.
The engineered cells which express and preferably secrete the polypeptides ofthe invention can be introduced into the patient systemically, e.g., in the circulation, or intraperitoneally. Alternatively, the cells can be incorporated into a matrix and implanted in the body, e.g., genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft. (See, e.~., Anderson et al. U.S. Patent No.

5,399,349; and Mulligan & Wilson, U.S. Patent No. 5.460,959, each of which is incorporated by reference herein in its entirety).
When 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.
For example. 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.
Vectors nml Host Cells The present invention also relates to vectors which include the isolated DN A molecules of the present invention, host cells 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. Generally, 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 luc, nn and me promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
Other suitable promoters will be known to the skilled artisan. 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 of the polypeptide to be translated.
As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. cnli and other bacteria. Representative examples of appropriate -2 j-hosts include, but are not limited to, bacterial cells, such as E. coli, ,S~repronryce.s and Salmonella lyphimuriunr cells, fungal cells, such as yeast cells: insect cells such as Drosophila S2 and Spodop~ercr Sf~ cells; animal cells such as CHO. COS
and Bowes melanoma cells: and plant cells. Appropriate culture mediums and S conditions for the above-described host cells are known in the art.
In addition to the use of expression vectors in the practice of the present invention, the present invention further includes novel expression vectors comprising operator and promoter elements operatively linked to nucleotide sequences encoding a protein of interest. One example of such a vector is pHE4-which is described in detail below.
As summarized in Fi~~ures I 0 and 11. components of the pHE4-~ vector (SEQ ID NO:50) include: I ) a neomycinphosphotransferase gene as a selection marker, 2) an E. coli origin of replication, 3) a TS phage promoter sequence, 4) two lac operator sequences. 5) a Shine-Delgarno sequence, 6) the lactose operon repressor gene (IacIq). The origin of replication (oriC) is derived from pUC I 9 (LTI, Gaithersburg. MD). 'the promoter sequence and operator sequences were made synthetically. Synthetic production of nucleic acid sequences is well known in the art. CLONTECH 95/96 Catalog, pages 215-216, CLONTECH, 1020 East Meadow Circle, Palo Alto. CA 94303. A nucleotide sequence encoding AIM II (SEQ ID NO: I ), is operatively linked to the promoter and operator by inserting the nucleotide sequence between the NdeI and Asp718 sites of the pHE4-5 vector.
As noted above, the pHE4-5 vector contains a IacIq gene. LacIq is an allele of the lacl gene which confers tight regulation of the lac operator.
Amann, E. el al., Gene 69: 301-315 ( 1988); Stark, M., Gene 51:255-267 ( 1987). The laclq gene encodes a repressor protein which binds to lac operator sequences and blocks transcription of down-stream (i. e. , 3') sequences. However, the IacIq gene product dissociates from the lac operator in the presence of either lactose or certain lactose analogs, e. l;. , isopropyl B-D-thiogalactopyranoside (IPTG).
AIM II
thus is not produced in appreciable quantities in uninduced host cells containing the pHE4-~ vector. Induction of these host cells by tile addition of an agent such as IPTG. however. results in the expression of the AIM Il coding sequence.
The promoter/operator sequences of the pHE4-~ vector {SEQ ID NO:S 1 ) comprise a TS 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 lacIq gene product.
confer tight repression of down-stream sequences in the absence of a lac operon inducer, e.l,~., 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. Binding of a lac inducer to the lacIq proteins results in their release from the lac operator sequences and the initiation of transcription of operatively linked sequences. Lac operon regulation of gene expression is reviewed in Devlin, T., TEXTBOOK OF BIOCHEMISTRY WITH CLINICAL
CORRELATIONS, 4th Edition ( I 997), pages 802-807.
IS The pHE4 series of vectors contain all of the components of the pHE4-5 vector except for the AIM I1 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.
Among known bacterial promoters suitable for use in the production of proteins of the 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 of the Rous Sarcoma Virus (RSV), and metallothionein promoters, such as the mouse metallothionein-I
promoter.
The pHE4-5 vector also contains a Shine-Delgarno sequence 5' to the AUG initiation codon. Shine-Delgarno sequences are short sequences generally _77_ located about 10 nucleotides up-stream (i.e., S') from the AUG initiation codon.
These sequences essentially direct prokaryotic ribosomes to the AUG initiation codon.
Thus, the present invention is also directed to expression vector useful for S 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).
Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors. Bluescript vectors. pNHBA, pNH 16a, pNH 18A, pNH46A, available from Stratagene: and ptrc99a, pKK223-3, pKK233-3, pDR540, pRITS available from Pharmacia.
Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTI
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.
Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals. such as Davis el ul., Basic Methods In Molecular- Biolo~ry (1986).
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 solubilize proteins. For example. EP-A-O 464 X33 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another human protein or part thereof. In many cases, 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 0232 262). On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed. detected and purifed in the advantageous manner described. 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. In drug discovery. for example, human proteins, such as, hILS-receptor has been fused with Fe portions for the purpose of high-throughput screening assays to identify antagonists of hIL-~. See, D.
Bennett et al., Journal of Molecular Recognition, Vol. 8:52-58 (1995) and K.
Johanson e! al., The Journal of Biological Chemisly~, Vol. 270, No.
16:9459-9471 (1995).
The AIM II protein can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or canon exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification. 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 of the invention may also include an initial modified methionine residue. in some cases as a result of host-mediated processes.
In addition to encompassing host cells containing the vector constructs discussed herein, 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
polvnucleotides of the invention, and which activates, alters, and/or amplifies endogenous AIM

polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous AIM II polynucleotide sequences via homologous recombination (see, e. g., U.S. Patent No. 5,641,670, issued June 24, 1997; International Publication No. WO 96/29411, published September 26, 1996; International Publication No. WO 94/12650, published August 4, 1994; Koller et ul., Proc.
Natl. Acud Sci. USA 86:8932-8935 (1989): and Zijlstra et al., Nature 3=12:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).
Tra»sge»ic Non-H»ma» Animals The 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. In a specific embodiment, techniques described herein or otherwise known in the art, are used to express polypeptides of the invention in humans, as part of a gene therapy protocol.
Any technique known in the art may be used to introduce the transgene (i.v., polynucleotides of the invention) into animals to produce the founder lines WO 99/425$4 PCT/US99/03703 of tr~nsgenic animals. Such techniques include, but are not limited to, pronuclear microinjection (Paterson et al. , Appl. Microbiol. Biotechnol. -10:691-698 ( 1994);
Carver et al. , Biotecl2no7og~y (A~l') 11:1263-1270 ( 1993 ); Wright et al., Biotechnolo~~ (N3') 9:830-834 ( I 991 ); and Hoppe et al., U.S. Pat. No.
4.873.191 S ( 1989)): retrovirus mediated gene transfer into germ lines (Van der Putten et al., Prvc. Natl. Acad Sci.. U.SA 82:6148-6152 ( 1985)), blastocysts or embryos:
gene targeting in embryonic stem cells (Thompson et al., Cell 36:313-321 (1989));
electroporation of cells or embryos (Lo, Mol Cell. Biol. 3:1803-1814 (1983));
introduction of the polynucleotides of the invention using a gene gun (.see.
e.g., Ulmer et al., Science 239:1745 (1993); introducing nucleic acid constructs into embryonic pleuripotent stem cells and transferring the stem cells back into the blastocyst; and sperm-mediated gene transfer (Lavitrano et al., Cell37:717-723 ( 1989); etc. For a review of such techniques, see Gordon, "Transgenic Animals,"
Irztl. Ren. C'ytol. 113:171-229 (19$9), which is incorporated by reference herein in its entirety. Further, the contents of each of the documents recited in this paragraph is herein incorporated by reference in its entirety.
Any technique known in the art may be used to produce 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 385:810-813 (1997)). each of which is herein incorporated by reference in its entirety).
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, i.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 et al., Prnc. Natl. Acad. .Sci.
USA
89:6232-6236 (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. When it is desired that the polynucleotide transgene be integrated into the chromosomal site of the endogenous gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, 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. , Science 26:103-IOG (1994)). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. The contents of each of the documents recited in this paragraph is herein incorporated by reference in its entirety.
Once transgenic animals have been generated, 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 of the transgene in the tissues of the 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.
Once the founder animals are produced, they may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal. Examples of such 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 -j7_ 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.
AIM 11 Polypeptides and Fragments The invention further provides an isolated AIM II polypeptide having the amino acid sequence encoded by the deposited cDNA, or the amino acid sequence in Figures lA and B (SEQ ID N0:2), or a peptide or polypeptide comprising a portion of the above polypeptides.
It will be recognized in the art that some amino acid sequences of the AIM II polypeptide can be varied without significant effect of the structure or function of the protein. If such differences in sequence are contemplated, it should be remembered that there will be critical areas on the protein which determine activity.
Thus, the invention further includes variations of the AIM II polypeptide which show substantial AIM II polypeptide activity or which include regions of AIM I1 protein such as the protein portions discussed below. Such mutants include deletions, insertions, inversions. repeats, and type substitutions. As indicated above, guidance concerning which amino acid changes are likely to be phenotypically silent can be found in Bowie, J.U., et ul., "Deciphering the _JJ-Message in Protein Sequences: Tolerance to Amino Acid Substitutions." Science 2-17:1306-1310 ( 1990).
Thus, the fragment, derivative or analog of the polypeptide of Figures 1 A
and B (SEQ ID N0:2), or that encoded by the deposited cDNA. may be (i) one in which one or more of the amino acid residues (e.g., 3, 5, 8, 10, 1 ~ 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. S, 8, 10, 1 ~
or 20), or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half life of the 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 peptide or leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments. derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
Of particular interest are substitutions of charged amino acids with another charged amino acid and with neutral or negatively charged amino acids. The latter results in proteins with reduced positive charge to improve the characteristics of the AIM II protein. The prevention of aggregation is highly desirable.
Aggregation of proteins not only results in a loss of activity but can also be problematic when preparing pharmaceutical formulations, because they can be immunogenic. (Pinckard el al., Clin Exp. Immunnl. 2:331-340 ( 1967): Robbins et al., Diabetes 36:838-845 (1987): Cleland et al. C.'ril. Ren. Therapeutic Drag Carrier Systems 10:307-377 (1993)).
The replacement of amino acids can also change the selectivity of binding to cell surface receptors. Ostade et al., Nature 361:266-268 (1993) describes certain mutations resulting in selective binding of TNF-a to only one of the two known types of TNF receptors. Thus, the AIM 1I receptor of the present invention may include one or more (e.~~.. 3. 5. 8. 10. 1 ~ or 20) amino acid substitutions, deletions or additions, either from natural mutations or human manipulation.
As indicated, changes are preferably of a minor nature. such as S conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table 1 ).
TABLE 1. Conservative Amino Acid Substitutions.
Aromatic Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine ~ Polar ~ Glutamine Asparagine Basic Arginine Lysine Histidine Acidic ~ Aspartic Acid Glutamic Acid Small Alanine Serine Threonine Methionine Glvcine Of course, the number of amino acid substitutions a skilled artisan would make depends on many factors, including those described above. Generally speaking, the number of substitutions for any given AIM-II polypeptide will not be more than S0, 40, 30, 25, 20, 15, 10, 5 or 3.
Amino 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 Wells, Science 2=/-x:1081-1085 (19$9)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as receptor binding or in nimo. or in vitro proliferative activity. Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallization. nuclear magnetic resonance or photoaffinity labeling (Smith et ul.. J. Mol. Biol. ??-1:899-904 ( 1992) and de Vos et ul. Science 2.5.5:306-312 (1992)).
Amino a» d CarboxyTermi»nl Deletions Also included in the present invention are amino terminal deletion mutants.
Such mutants include those comprising the amino acid sequence shown in SEQ
ID N0:2 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 N0:2.
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 N0:2.
Alternatively, the deletion will include at least the first 59 N-terminal amino acid residues but not more than the first 114 N-terminal amino acid residues of SEQ
ID N0:2. Alternatively, 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 N0:2. Alternatively, the deletion will include at least the first 68 N-terminal amino acid residues but not more than the first 114 N-terminal amino acid residues of SEQ ID N0:2. 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 N0:2. Alternatively, the deletion will include at least the first 82 N-terminal amino acid residues but not more than the first N-terminal amino acid residues of SEQ ID N0:2. Alternatively, the deletion will include at least the first 100 N-terminal amino acid residues but not more than the first 114 N-terminal amino acid residues of SEQ ID N0:2.
In addition to the ranges of N-terminal deletion mutants described above, the present invention is also directed to all combinations of the above described ranges. For example. the deletions of at least the first 59 N-terminal amino acid residues but not more than the first 67 N-terminal amino acid residues of SEQ
ID
N0:2; deletions of at least the first 59 N-terminal amino acid residues but not more than the first 68 N-terminal amino acid residues of SEQ ID N0:2;
deletions of at least the first 59 N-terminal amino acid residues but not more than the first 73 N-terminal amino acid residues of SEQ ID N0:2; deletions of at least the first 59 N-terminal amino acid residues but not more than the first 82 N-terminal amino acid residues of SEQ ID N0:2; deletions of at least the first 59 N-terminal amino acid residues but not more than the first 100 N-terminal amino acid residues of SEQ ID N0:2; deletions of at least the first 67 N-terminal amino acid residues but not more than the first 73 N-terminal amino acid residues of SEQ ID N0:2;
deletions of at least the first 67 N-terminal amino acid residues but not more than the first 82 N-terminal amino acid residues of SEQ ID N0:2; deletions of at least the first 67 N-terminal amino acid residues but not more than the first 100 N-terminal amino acid residues of SEQ ID N0:2; deletions of at least the first terminal amino acid residues but not more than the first 73 N-terminal amino acid residues of SEQ ID N0:2; deletions of at least the first 68 N-terminal amino acid residues but not more than the first 82 N-terminal amino acid residues of SEQ
ID
N0:2; deletions of at least the first 68 N-terminal amino acid residues but not more than the first 100 N-terminal amino acid residues of SEQ ID N0:2;
deletions of at least the first 73 N-terminal amino acid residues but not more than the first 82 N-terminal amino acid residues of SEQ ID N0:2; deletions of at least the first 73 N-terminal amino acid residues but not more than the first 100 N-terminal amino acid residues of SEQ ID N0:2; deletions of at least the first terminal amino acid residues but not more than the first I 00 N-terminal amino acid residues of SEQ ID N0:2; etc. etc. etc. . . .
Preferred AIM II polypeptides are shown below (numbering starts with the first amino acid in the protein (Met):
Gln(residue 60) to Val(residue 240) Leu(61 ) to Val(240) His(62) to Val(240) Val(92) to Val(240) Trp(63) to Val(240) Asn(93) to Val{240) Ar~(64) to Val(240) Pro(94) to Val(240) Leu(65) to Val(240) Ala(95) to Val(240) Gly(66) to Val(240) Ala(96) to Val(240) Glu(67) to Val(240) His(97) to Val(240) Met(68) to Val(240) Leu(98) to Val(240) Val(69) to Val(240) Thr(99) to Val(240) Thr(70) to Val(240) Gly(100) to Val(240) Ar~(71) to Val(240) Ala(101) to Val(240) Leu(72) to Val (240) Asn(102) to Val(240) Pro(73) to Val(240) Ser(103) to Val(240) Asp(74) to Val(240) Ser(104) to Val(240) Gly(75) to Val(240) Leu(105) to Val(240) Pro(76) to Val(240) Thr(106) to Val(240) Ala(77) to Val(240) Gly(I07) to Val(240) Gly(78) to Val(240) Ser(108) to Val{240) Ser(79) to Val(240) Gly(109) to Val(240) Trp(80) to Val(240) Gly(110) to Val(240) Glu(81 ) to Val(240) Pro( 111 ) to Val(240) Gln(82) to Val(240) Leu(112) to Val(240) Leu(83) to Val(240) Leu(113) to Val(240) Ile(84) to Val(240) Trp(114) to Val(240) Gln(85) to Val(240) Glu(86) to Val(240) Arg(87) to Val(240) Arg(88) to Val(240) Ser(89) to Val(240) His(90) to Val(240) Glu(91 ) to Val(240) Particularly preferred embodiments include the AIM II N-terminal deletions Gln-60 to Val-240 (AIM II (aa 60-240)), Met-68 to Val-240 (AIM I1 {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 (AIM II (aa 101-240)).
Even if deletion of one or more amino acids from the N-terminus of a protein results in modification or loss of one or more biological functions of the protein, other biological activities may still be retained. Thus, the ability of shortened AIM II muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally wil l be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking 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.
Accordingly, the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the AIM II amino acid sequence shown in Figures 1 A and B (i. e., SEQ ID N0:2) up to the phenylalanine residue at position number 235, and polynucleotides encoding such polypeptides.
In particular, the present invention provides polypeptides comprising the amino acid sequence of residues n-314 of Figures I A and B (SEQ ID N0:2), where n is an integer in the range of 2 to 235, and 236 is the position of the first residue from the N-terminus of the complete AIM II polypeptide believed to be required for at least immunogenic activity of the AIM II polypeptide.
More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence 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-I1 to V-240: V-12 to V-240; V-13 to V-240: D-14 to V-240; G-I S to V-240; Q-16 to V-240: T-17 to V-240; D-18 to V-240; I-19 to V-240; P-20 to V-240: F-21 to V-240: T-22 to V-240; R-23 to V-240: L-24 to V-240; G-25 to V-240, R-26 to V-240: S-27 to V-240; H-28 to 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; Q-54 to V-240; G-55 to V-240; W-56 to V-240; F-57 to V-240; L-58 to V-240; L-~9 to V-240; Q-60 to V-240; L-61 to V-240; H-62 to V-240; W-63 to V-240; R-64 to V-240; L-65 to V-240; G-66 to V-240; E-67 to V-240; M-68 to V-240; V-69 to V-240; T-70 to V-240; R-71 to V-240; L-72 to V-240; P-73 to V-240; D-74 to V-240; G-75 to V-240; P-76 to V-240; A-77 to V-240; G-78 to 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; I-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-109 to V-240; G-110 to V-240; P-111 to V-240; L-112 to V-240; L-113 to V-240; W-114 to V-240;
E-115 to V-240; T-116 to V-240; Q-I 17 to V-240; L-118 to V-240; G-119 to V-240; L-120 to V-240; A-121 to V-240; F-122 to V-240; L-123 to V-240;
R-124 to V-240; G-125 to V-240; L-126 to V-240; S-127 to V-240; Y-128 to V-240: H-129 to V-240; D-130 to V-240; G-131 to~ V-240; A-132 to V-240;
L-133 to V-240; V-134 to V-240; V-135 to V-240; T-136 to V-240; K-137 to V-240; A-138 to V-240; G-139 to V-240; Y-140 to V-240; Y-141 to V-240:
Y-142 to V-240; I-143 to V-240; Y-144 to V-240; S-145 to V-240; K-146 to V-240; V-147 to V-240; Q-148 to V-240: L-149 to V-240; G-150 to V-240:

G-151 to V-240; V-152 to V-240; G-153 to V-240, C-154 to V-240; P-155 to V-240: L-156 to V-240: G-157 to V-240; L-158 to V-240: A-159 to V-240;
S-160 to V-240; T-161 to V-240: I-162 to V-240; T-163 to V-240; H-164 to V-240: G-165 to V-240; L-166 to V-240; Y-167 to V-240; K-168 to V-240;
R-169 to V-240; T-170 to V-240; P-171 to V-240: R-172 to V-240: Y-173 to V-240; P-174 to V-240: E-175 to V-240; E-176 to V-240; L-177 to V-240;
E-178 to V-240; L-179 to V-240; L-180 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-186 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; S-194 to V-240; R-195 to V-240;
V-196 to V-240; W-197 to V-240; W-198 to V-240; D-199 to V-240; S-200 to V-240; S-201 to V-240; F-202 to V-240; L-203 to V-240: G-204 to V-240;
G-205 to V-240; V-206 to V-240; V-207 to V-240; H-208 to V-240; L-209 to V-240; E-210 to V-240; A-211 to V-240; G-212 to V-240; E-213 to V-240;
E-214 to V-240; V-215 to V-240; V-216 to 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 N0:2 (which is identical to the sequence shown as Figure 1 A-B, with the exception that the amino acid residues in SEQ
ID
N0:2 are numbered consecutively from 1 through 240 from the N-terminus to the C-terminus): Polynucleotides encoding these polypeptides are also encompassed by the invention.
As mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification or loss of one or more biological functions of the protein, other biological activities may still be retained.
Thus, the ability of the shortened AIM II mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise know-n in the art. It is not unlikely that an AIM II mutein with a large number of deleted C-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.
Accordingly. the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the AIM I1 polypeptide shown in Figures 1 A and B (SEQ ID N0:2).
up to the valine residue at position number 6, and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising the amino acid sequence of residues 1-m of Figures IA and B (i.e., SEQ ID N0:2). 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 of the AIM
II polypeptide.
More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues M-1 to M-239;
M-1 to F-238;
M-1 to A-237;
M-1 to G-236;
M-1 to F-235; M-1 to Y-234; to S-233; to R-232; M-1 to T-231;
M-1 M-1 M-1 to G-230; M-1 to D-229; to R-228; to L-227; M-1 to R-226;
M-1 M-1 M-1 to V-225; M-1 to L-224; to R-223; to E-222; M-1 to D-221;
M-1 M-1 M-1 to L-220; M-1 to V-219; to R-218; to V-217; M-1 to V-216;
M-1 M-1 M-1 to V-215; M-1 to E-214; to E-213; to G-212; M-1 to A-211;
M-1 M-1 M-1 to E-210; M-1 to L-209; to H-208; to V-207; to V-206;
M-1 M-1 M-1 M-1 to G-205; M-1 to G-204; to L-203; to F-202; to S-201;
M-1 M-1 M-1 M-1 to S-200; M-1 to D-199; to W-198; to W-197; to V-196;
M-1 M-1 M-1 M-1 to R-195; M-1 to S-194; to S-193; to S-192; to T-191;
M-1 M-I M-1 M-1 to A-190; M-1 to R-189; to G-188; to C-187; to P-186;
M-1 M-1 M-1 M-I to S-185; M-1 to Q-184: to Q-183: to S-182: to V-181;
M-1 M-1 M-1 M-1 to L-180; M-1 to L-179; to E-178; to L-177; to E-176;
M-1 M-1 M-1 M-1 to E-175; M-I to P-174; to Y-173; to R-172; to P-171;
M-1 M-1 M-1 M-1 to T-170; M-1 to R-169; to K-168; to Y-167; to L-166;
M-1 M-1 M-1 M-1 to G-165; M-1 to H-164: 161: M-1 M-I to to S-160;
T-163;
M-1 to I-162;
M-1 to T-M-1 to A-159; M-1 to L-158; M-1 to G-157; M-1 to L-156; M-1 to P-155; M-1 to C-154; M-1 to G-153; M-1 to V-152; M-1 to G-151; M-1 to G-150; M-1 to L-149; M-1 to Q-148; M-I to V-147; M-1 to K-146; M-I to S-145; M-1 to Y-144; M-1 to I-143; M-1 to Y-142; M-1 to Y-141; M-1 to Y-140; M-1 to G-139; M-1 to A-138; M-1 to K-137; M-1 to T-136; M-1 to V-135; M-1 to V-134: M-1 to L-133; to A-132: to G-131; to D-130:
M-1 M-1 M-1 M-1 to H-129; M-1 to Y-128; to S-127; to L-126; to G-125;
M-1 M-1 M-1 M-1 to R-124; M-1 to L-123; to F-122; to A-121; to L-120;
M-1 M-1 M-I M-1 to G-119; M-1 to L-118; to Q-117; to T-116; to E-115;
M-1 M-1 M-1 M-1 to W-114; M-1 to L-113; to L-112; to P-I11; to G-110;
M-1 M-1 M-1 M-1 to G-109; M-1 to S-108; M-1 to G-107; M-1 to T-106; M-1 to L-105; M-1 to S-104; M-1 to S-103; M-1 to N-102; M-1 to A-101; M-1 to G-100, M-1 to T-99;
M-1 to L-98; M-1 to H-97; M-1 to A-96; M-1 to A-95; M-1 to P-94; M-1 to N-93; M-1 to V-92; M-1 to E-91; M-1 to H-90; M-I to S-89; M-1 to R-88; M-1 to R-87; M-1 to E-86; M-1 to Q-85; M-1 to I-84; M-1 to L-83; M-1 to Q-82;
M-1 to E-81; M-1 to W-80; M-1 to S-79; M-1 to G-78; M-1 to A-77; M-I to P-76; M-1 to G-75; M-I to D-74; M-1 to P-73; M-1 to L-72; M-1 to R-71; M-1 to T-70; M-1 to V-69; M-1 to M-68; M-1 to E-67; M-1 to G-66; M-1 to L-65;
M-1 to R-64; M-1 to W-63; M-1 to H-62; M-1 to L-61; M-1 to Q-60; M-1 to L-59; M-1 to L-58; M-I to F-57; M-1 to W-56; M-1 to G-55; M-I to Q-54; M-1 to V-53; M-1 to A-52; M-1 to L-51; M-1 to G-50; M-1 to A-49; M-1 to G-48;
M-1 to M-47; M-1 to L-46; M-1 to L-45; M-1 to L-44; M-1 to L-43; M-I to L-42: M-1 to G-41; M-1 to L-40; M-1 to G-39; M-1 to V-38; M-1 to R-37; M-1 to A-36; M-1 to V-35; M-1 to S-34; M-1 to C-33; M-1 to S-32, M-1 to Q-31;
M-1 to R-30; M-1 to R-29; M-1 to H-28; M-I to S-27; M-1 to R-26; M-1 to WO 99/425$4 PCT/US99/03703 G-''~: M-1 to L-24: M-1 to R-23; M-1 to T-22; M-1 to F-21; M-1 to P-20: M-1 to I-19; M-1 to D-18: M-1 to T-17; M-1 to Q-16: M-1 to G-15; M-1 to D-14:
M-1 to V-13: M-1 to V-12: M-1 to F-I 1; M-1 to V-10: M-1 to S-9; M-1 to P-8:
M-1 to R-7; M-I to V-6 ofthe sequence ofthe AIM II sequence shown in Figures 1 A and B (which is identical to the sequence shown as SEQ 1D NO:?, with the exception that the amino acid residues in SEQ ID N0:2 are numbered consecutively from 1 through 240 from the N-terminus to the C-terminus).
Polynucleotides encoding these polypeptides also are provided.
The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of an AIM II polypeptide.
which may be described generally as having residues n-m of Figures 1 A and B
(i. c'.. SEQ ID N0:2 ). where n and m are integers as described above.
The natural processed form of AIM II that was affinity purified on an LT-(3 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 N0:2. (See Example 10). However, it appears that 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 N0:2. In addition, COS cells also cleave the AIM II between Met-68 and Val-69. resulting a polypeptide having amino acids 69-240 in SEQ ID N0:2.
The polypeptides of the present invention are preferably provided in an isolated form. By "isolated polypeptide" is intended a polypeptide removed from its native environment. Thus, a polypeptide produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention.
Also intended as an "isolated poiypeptide" are polypeptides that have been purified, partially or substantially, from a recombinant host. For example, a recombinantly produced version of the AIM II polypeptide can be substantially purified by the one-step method described in Smith and .Tohnson, Gene 67: 31-( 1988).

The polypeptides of the present invention include the polypeptide encoded by the deposited cDNA, the polypeptide of Figures 1 A and B (SEQ ID Iv0:2).
the polypeptide of Figures lA and B (SEQ ID N0:2) lacking the N-terminal methionine, the extracellular domain, the transmembrane domain, the intracellular domain. soluble polypeptides comprising 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 90% or 95% identical, still more preferably at least 96%, 97%, 98% or 99% identical to the polypeptide encoded by the deposited cDNA, to the polypeptide of Figures 1 A
and B (SEQ ID N0:2), and also include portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids.
By a polypeptide having an amino acid sequence at least, for example, 95% "identical" to a reference amino acid sequence of an AIM II poivpeptide 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 of the reference amino acid of the AIM II polypeptide. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, 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% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the 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.
As a practical matter, whether any particular polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence shown in Figures I A and B (SEQ ID N0:2) or to the amino acid sequence encoded by deposited eDNA clone can be determined conventionally using known -4~-computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group. University Research Park. X75 Science Drive. Madison, WI 53711. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, 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% of the total number of amino acid residues in the reference sequence are allowed.
By a 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. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, (indels) or substituted with another amino acid. These alterations of the 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.
As a practical matter, whether any particular polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequences shown in Table 1 or to the amino acid sequence encoded by deposited DNA clone can be determined conventionally using known computer programs. A preferred method far determining the best overall match between a query sequence (a sequence of the 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. (Comp. App. Biosci. 6:237-245 ( 1990)). In a sequence alignment the quey and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence ali~~nment is in percent identity . Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2. Mismatch Penalty=I ..loining Penalty=20, Randomization Group Lengtlz=0. Cutoff Score=1.
Window Size=sequence length, Gap Penalty=5. Gap Size Penalty=0.05. Window Size=500 or the length of the subject amino acid sequence, whichever is shorter.
If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, 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 quen~ 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 query (reference) sequence that extend past the N- or C-termini of the subject sequence are considered for the purposes of manually adjusting the percent identity score.
That is, only residues which are not matched/aligned with the N- or C-termini of the query sequence are counted when manually adjusting the percent identity score.
For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a match/alignment of the first 10 residues at the N-terminus.
The 10 unpaired residues represent 10% of the sequence (number of residues at the N-and C- termini not matched/total number of residues in the quern sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB
program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini 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 residue positions outside the N-and C-terminal ends ofthe subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to made for the purposes of the present invention.
As used herein the term "AIM II" polypeptide includes membrane-bound proteins (comprising a cytoplasmic domain, a transmembrane domain, and an extracellular domain) as well as truncated proteins that retain the AIM II
polypeptide activity. In one embodiment, soluble AIM II polypeptides comprise all or part of the extracellular domain of an A1M II protein, but lack the transmembrane region that would cause retention of the 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 polypeptide of the present invention could be used 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.
In another aspect, the invention provides peptides and polypeptides comprising epitope-bearing portions of the polypeptides of the present invention.
These epitopes are immunogenic or antigenic epitopes of the polypeptides of the present invention. An "immunogenic epitope" is defined as a part of a protein that WO 99/~t25$4 PCT/US99/03703 elicits an antibody response in vivo when the whole polypeptide of the present invention. or fragment thereof. is the immunogen. On the other hand. a region of a polypeptide to which an antibody can bind is defined as an "antigenic detern~inant" or "antigenic epitope." The number of in rivo immunogenic epitopes S of a protein generally is less than the number of antigenic epitopes. ,See, e.~,~., Geysen, e~ crl.. Pnoc. Natl. Acad. Sci. USA 81:3998-4002 (1983). However, antibodies can be made to any antigenic epitope, regardless of whether it is an immunogenic epitope, by using methods such as phage display. See. e.R., Petersen G. el crl.. Mol. Gc~n. Gener. 249:425-431 ( 199 ). Therefore, included in the present invention are both immunogenic epitopes and antigenic epitopes.
As to the selection of peptides or polypeptides bearing an antigenic epitope (i.e., that contain a region of a protein molecule to which an antibody can bind), it is well known in that art that relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M., Green, N. and Learner, R.A. (1983) Antibodies that react with predetermined sites on proteins. Science ZI9:660-666. Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein. can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i. e. , immunogenic epitopes) nor to the amino or carboxyl terminals.
Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention. See, for instance, Wilson et al. Cell 37.~ 767-778 (1984) at 777.
Antigenic epitope-bearing peptides and polypeptides of the invention preferably contain a sequence of at least seven. more preferably at least nine and most preferably between about at least about 1 ~ to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention.

Non-limiting examples of antigenic polypeptides or peptides that can be used to generate AIM II-specific antibodies include: a polypeptide comprising amino acid residues from about 13 to about 20 in Figures 1 A and B (SEQ ID
NO:?): a polypeptide comprising amino acid residues from about 23 to about 36 in Figure 1 (SEQ ID N0:2); a polypeptide comprising amino acid residues from about 69 to about 79 in Figures 1A and B (SEQ ID N0:2): a polypeptide comprising amino acid residues from about 85 to about 94 in Figures 1 A and B
(SEQ ID N0:2); a polypeptide comprising amino acid residues from about 167 to about 178 in Figures 1 A and B (SEQ ID N0:2); a polypeptide comprising amino acid residues from about 184 to about 196 in Figures 1 A and B (SEQ ID N0:2);
and a polypeptide comprising amino acid residues from about 221 to about 233 in Figures IA and B (SEQ ID N0:2). As indicated above, the inventors have determined that the above polypeptide fragments are antigenic regions of the AIM II protein.
The AIM II polypeptides of the invention may be in monomers or multimers (i. e. , dimers, trimers, tetramers and higher multimers).
Accordingly, the present invention relates to monomers and multimers of the AIM II polypeptides of the invention, their preparation. and compositions (preferably.
pharmaceutical compositions) containing them. In specific embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In additional embodiments, 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.
As used herein, the term 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. In a specific embodiment, a homomer of the invention is a multimer containing only AIM II polypeptides having an identical amino acid sequence. In another specific embodiment, a homomer of the invention is a multimer containing AIM II

polypeptides having different amino acid sequences. In specific embodiments, the multimer of the invention is a homodimer (e.y.. 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). In additional embodiments, the homomeric multimer of the invention is at least a homodimer. at least a homotrimer, or at least a homotetramer.
As used herein, the term 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 I1 polypeptides of the invention. In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments, the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.
Multimers of the invention may be the result of hydrophobic, hydrophilic.
ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation. Thus, in one embodiment, multimers of the invention, such as, for example. homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution. In another embodiment.
heteromultimers of the invention, such as, for example. heterotrimers or heterotetramers, are formed when poiypeptides of the 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. In other embodiments, multimers of the invention are formed by covalent associations with and/or between the AIM II polypeptides of the invention. Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in SEQ ID N0:2 or SEQ ID N0:39, or contained in the polypeptide encoded by the clones designated as ATCC
Accession 97689 and 97483). In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occurring) polypeptide. In another WO 99/4258a PCT/US99/03703 _j1_ instance, the covalent associations are the consequence of chemical or recombinant manipulation. Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polvpeptide sequence in an AIM II fusion protein. In one example, covalent associations are between the heterologous sequence contained in a fusion protein of the invention (sec-. c~.,~r., LJS Patent Number 5.478,925). In a specific example, the covalent associations are between the heterologous sequence contained in an AIM II-Fc fusion protein of the invention (as described herein). In another specific example.
covalent associations of fusion proteins of the 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 (.sec, e.g., International Publication No. WO 98/49305, the contents of which are herein incorporated by reference in its entirety).
The multimers of the invention may be generated using chemical techniques known in the art. For example, 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.~~.. US Patent Number 5,478.925, which is herein incorporated by reference in its entirety). Additionally, multimers of the 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, c~.~,~., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety). Further, polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (.see, 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 (.sec'. c~.~>.. US
Patent Number 5.478.92, which is herein incorporated by reference in its entirety).
Alternatively. multimers of the invention may be generated using genetic engineering techniques known in the art. In one embodiment, polypeptides contained in multimers of the invention are produced recombinantIy using fusion protein technology described herein or otherwise known in the art (.sec, e.g., US
Patent Number 5,478,9?,. which is herein incorporated by reference in its entirety). In a specific embodiment, 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) (see, e.g., I1S Patent Number 5,478,925, which is herein incorporated by reference in its entirety). In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain (or hyrophobic or signal peptide) and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g. , US
Patent Number 5.478,92. which is herein incorporated by reference in its entirety).
The invention encompasses AIM II polypeptides which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic 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, NaBI-I4; acetylation. formylation, oxidation. reduction; metabolic synthesis in the presence of tunicamycin; etc.
Additional post-translational modifications encompassed by the invention include, for example, e.~,>.. N-linked or O-linked carbohydrate chains, processing WO 99/425$4 PCTNS99/03703 of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone. chemical modifications of N-linked or O-linked carbohydrate chains. and addition ofan N-terminal methionine residue as a result ofprocaryotic 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 of the protein.
Also provided by the invention are chemically modified derivatives of AIM I1 which may provide additional advantages such as increased solubility, stability and circulating time ofthe polypeptide. or decreased immunogenicity (see U. S. Patent No. 4,179,337). The chemical moieties for derivitization 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. For polyethylene glycol, 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 of the polyethylene glycol to a therapeutic protein or analog).
The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of the protein. There are a number of attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al.. Exp. Hematol. 20:1028-103 (1992) (reporting pegylation of GM-CSF using tresyl chloride). For example.

polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as. a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino 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 amino group, such as attachment at the N-terminus or lysine group.
One may specifically desire proteins chemically modified at the N-terminus. Using polyethylene glycol as an illustration of the present composition, one rnay select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (or peptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) 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 derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
The epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means. Houghten, R. A. ( 1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Pnoc.
Natl.
Acad. Sci. USA 82:5131-S 135. This "Simultaneous Multiple Peptide Synthesis WO 99/4258.1 PCT/US99/03703 _si_ (SMPS)" process is further described in L!.S. Patent No. 4.631,21 1 to Houzhten et «l. ( 1980.
The present invention further relates to antibodies and T-cell anti~~en receptors ('hC R) which specifically bind the polypeptides ofthe presern invention.
S The antibodies of the present invention include I~~G ( including IgG 1.
IgG2. I~~G3.
and IgCi~4), IgA (including IgAI and IgA2). IgD, lgE. IgM. and IgY. As used herein. the term "antibody" (Ab) is meant to include whole antibodies.
including single-chain whole antibodies, and antigen-binding fragments thereof. '~-lost preferably the antibodies are human antigen binding antibody fragments of the present invention that include, but are not limited to. Fab, Fab' and F(ab')'_'. Fd.
single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFW
and fragments comprising either a V L or VH domain. The antibodies may be tiom any animal origin including birds and mammals. Preferably, the antibodies are human, murine, rabbit, goat, guinea pig, camel, horse, or chicken.
Antigen-binding antibody fragments, including single-chain antibodies. may comprise the variable regions) alone or in combination with the entire or partial of the following: hinge region, CH 1. CI-I2. and CH 3 domains. Also included in the invention are am~ combinations of variable regions) and hinge region. CH
1, C1-i2. and C1I3 domains. The present invention further includes chimeric, 2O ht1ma111L(',d. and human monoclonal and polyclonal antibodies which specifically bind the polypeptides of the present invention. The present invention further includes antibodies which are anti-idiotypic to the antibodies of the present invention.
The antibodies of the present invention may be monospecifte, bispecific.
trispccific orofgreatermultispecificim. Multispecific antibodies may be specific for different epitopes of a polypepiide of the present invention or may be specific for both a polypeptide of the present invention as well as for heterolotous compositions, such as a heterologous polypeptide or solid support material.
Sec~, e.y., WO 93/17715: WO 92/08802: Vv'O 91 /00360; WO 92/05793; Tutt, A. et crl.
, .I. lmmunol. I -l: :60-6~) ( 1991 ): US Patents 5.573.920. ~l.-17=1.893.
5.601.819.
4.714.681, 4,925.648: Kostelm~. S.A. eJ ul.. .l. Imrr7uool. 1.1<f:1 i-17-1553 (1992).
Antibodies of the present invention may be described or specified in terms of the epitope(s) or portions) of a polypeptide of the present im~ention which are recognized or specifically bound by the antibody. The epitope(s) or polypeptide portions) may be specified as described herein, c~.y., by N-terminal and C-terminal positions, by size in contiguous amino acid residues. or as listed in the Tables and Figures. Antibodies which specifically bind any epitope or polvpeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides ofthe present invention. and allows for the exclusion of the same.
Antibodies of the 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 the polypeptides of the present invention are included.
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%, 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 which only bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein). Antibodies of the present invention may also be described or specified in terms of their binding affinity.
Preferred binding affinities include those with a dissociation constant or Kd less than 5X10'"M. 10-~M,5X10-'M. 10-'M,5X10- M, 10-~M.5X10~''M. l0wM.5X10-'°M.
10-"'M. 5X10-"M. 10~"M. 5X10-''-M, 10-''-M, 5X10-''M, 10~''M, 5X10~'~M, 10-'~M, 5X10-''M, and 10-''M.
Antibodies of the present invention have uses that include. but are not limited to, methods known in the art to purify, detect, and target the polypeptides of the present invention including both in vitro and ire nirn diagnostic and -~7-therapeutic methods. For example. the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. ,See, c~.g.. Harlow el al.. ANTIBODIES: :1 LABORATORY MANUAL, (Cold Spring Harbor Laboraton~ Press. 2nd ed.
1988) (incorporated by reference in the entirety).
The antibodies of the present invention may be used either alone or in combination with other compositions. The antibodies may further be recombinantiv fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polvpeptides or other compositions. For example, antibodies of the 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. Sec, e.y., WO 92/08495; WO 91/14438; WO 89/12624; L'S
Patent 5,314.995; and EP 0 396 387.
The antibodies of the present invention may be prepared by any suitable method known in the art. For example, a polypeptide of the present invention or an antigenic fragment thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies. Monoclonal antibodies can be prepared using a wide of techniques known in the art including the use of hybridomaandrecombinanttechnology.,See,e.g.,Harloweral.,ANT.IBODIES:
A LABORATORY MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed.
1988): Hammerling, et ul., in: MONOCLONAL ANTIBODIES AND T-CELL
HYBRIDOMAS 563-681 (Elsevier. N.Y., 1981 ) (said references incorporated by reference in their entireties).
Fab and F(ab')2 fragments may be produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce Flab' j'_' fragments).
Alternatively. antibodies of the present invention can be produced through the application of recombinant DNA technolol;y or through synthetic chemistw using methods known in the art. For example. the antibodies of the present _S8_ invention can be prepared L1S111~~ various phage display methods knovyn in the art.
In phage display methods. functional antibody domains are displayed on the surface of a phage particle which carries polynucleotide sequences encoding them.
Phage with a desired binding property are selected from a repertoire or combinatorial antibody library (c'. ~. human or murine) by selecting directly with antigen, typically antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M 13 with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman U. ei crl...l Immunvl. A-~elhocts 182:41-SO ( 1995);
Ames, R.S. c'! ul., .I. Imn~unvl. Mclhvds l ~-t:177-I 86 ( 1995); Kettleborough.
C.A. el al., Eur. .l. Immunvl. 2:952-9S8 (1994); Persic, L. e! al., Gene 187:9-18 (1997);
Burton, D.R. e! al., Advances in Inamunolv~,ry X7:191-280 (1994);
1S PCT/GB91 /0l 134: WO 90/02809; WO 91 /10737; WO 92/01047; WO 92/18619;

6; WO 9S/15982; WO 95/20401; and US Patents 5.698,426, 5,223,409, 5,403,484. S.S80.717, 5.427,908. S,7S0,7S3, 5,821,047. S,S71,698.
5.427.908, S.S16,637. 5.780.225. 5.658,727 and 5,733,743 (said references incorporated by reference in their entireties).
As described in the above references, after phage selection. 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. For example, techniques to recombinantly produce Fab. Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in WO 92/22324; Mullinax. R.L. et al., BivTcchniques 12:864-869 ( I 992); and Sawai, H. e! ul.. AJRI 3-1:26-34 ( 199S);
and Better, M. e! al.. Science ?-1(1:1041-1043 ( 1988) (said references incorporated by reference in their entireties).

Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Patents 4.9=16.778 and ~.2~8.498;
Huston cn crl. (1991 ) :l~cthvds in Era~yr~~ologv 203:46-88: Shu. L. et ul.
(1993) P11:~.5 90:7995-7999: and Skerra, A. e! ul.. Scier~cc~ 2-10:1038-1040 ( 1988).
For some uses, includin~~ in vivo use of antibodies in humans and in vrtro detection assays. it may he preferable to use chimeric. humanized. or human antibodies.
Methods for producing chimeric antibodies are known in the art. See e.R..
Morrison, .Science ?2:1202 (1985); Oi en crl., BioTechnigares -1:214 (1986);
Dillies. S.D. et crl. ( 1989) .I. Irnmrrnvl. NlethcWs 12:191-202; and US
Patent 5,807,71 s. Antibodies can be humanized using a variety of techniques including CDR-grafting (EP 0 239 400; WO 91 /09967; US Patent 5,530,1 O 1; and 5,585,089). veneering or resurfacing (EP 0 592 106; EP 0 ~ 19 596; Padlan E.A., ( 1991 ) Molecular Immunology 28{4/5):489-498: Studnicka G.M. et ul. (1994) I'roicin Enl,~inec~ring 7(6):805-814; Roguska M.A. et ul. { 1994) PNA.S
91:969-973), and chain shuffling (US Patent 5.565,332). Human antibodies can be made by a variety of methods known in the art including phage display methods described above. See also, US Patents 4.444.887, 4,716.111, 5,545,806. and 5,814,318; and WO 98/46645 (said references incorporated by reference in their entireties ).
Further included in the present invention are antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide of the present invention. The antibodies may be specific for antigens other than polypeptides of the present invention. For example, antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the 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 in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor er ul.
sarpru and WO 93/21232; EP 0 439 095; Naramura, M. ct ul.. Immunol. Left.

39:91-99 ( 1994): US Patent 5.474.981: Gillies, S.O. et crl. ( 199? ) P~\'.9S
<f >:l-1?8-143?: Fell. H.P. e! ul. ( 1991 ) J. Inrmt.mvl. l-1:2446-24~? (said references incorporated by reference in their entireties).
The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions. For example. 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, CI-I2 domain. and CI-I3 domain or any combination of whole domains or portions thereof. The polypeptides of the present invention may be fused or conjugated to the above antibody portions to increase the in viva half life of the 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. For example. Fc portions fused to the polypeptides of the present invention can form diners through disulfide bonding between the Fc portions. Higher multimeric 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.y.. US
Patents 5.336,603, 5,622,929, 5,359,046, 5.349,053. 5.447.851. 5.112.946; EI' 0 307 434, EP 0 367 166; WO 96/04388. WO 91 /06570; Ashkenazi. A. e! ul.
(1991) PNAS 88:10535-10539; Zheng. X.X, e! ul. (1995) ,I. Immunnl.
l~-1:5590-5600; and Vil, H. e! ul. (1992) P~'AS89:11337-11341 (said references incorporated by reference in their entireties).
The invention further relates to antibodies which act as agonists or antagonists of the polypeptides of the present invention. For example. the present invention includes antibodies which disrupt the receptor/ligand interactions with the polvpeptides of the invention either partially or fully. Included are both receptor-specific antibodies and ligand-specific antibodies. Included are receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise know 'n in the art. Also include are receptor-specific antibodies which both prevent ligand binding and receptor activation. Likewise. included are neutralizing antibodies which bind the ligand and prevent binding of the 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. Further included are antibodies which activate the receptor. These antibodies may act as agonists for either all or less than all of the biological activities al~iected by ligand-mediated receptor activation. The antibodies may be specified as agonists or antagonists for biological activities comprising; specific activities disclosed herein. The above antibody agonists can be made using methods known in the art. See e.ll.. WO 96/40281: US Patent 5.811,097; Deng. B. en crl. (1998) Blood 92(6}:1981-1988; Chen. Z. er ul.
(1998) Cancer Res. 58( 16):3668-3678: Harrop, J.A. et ul. ( 1998) J. Immunol.
161(4):1786-1794; Zhu, Z. el ul. (1998) Cancer Res. 58(15):3209-3214; Yoon, D.Y. el ul. (1998) J. lmmunol. 160(7):3170-3179: Prat, M. et crl. (1998) J.
Cell.
Sci. 111 (Pt2):237-247; Pitard, V. el al. ( 1997) .1. Immunol. Methods 205(2):177-190; Liautard, J. c~ ul. ( 1997) Cytokinde 9(4):233-241; Carlson.
N.G.
cu ul. (1997) J. Biol. Chem. 272(17):11295-11301: Taryman. R.E. er ul. (1995) Neuron 14(4):765-762: Muller, Y.A. cn crl. (1998) Structure 6(9):1153-1167;
Bartunek. P. m crl. ( 1996) Cytokine 8( 1 ):14-20 (said references incorporated by reference in their entireties).
In additional embodiments, the polynucleotides of the invention encode functional attributes of AIM II. Preferred embodiments of the invention in this regard include fragments that comprise 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 II.

The data representing the structural or functional attributes of AIM II set forth in Figures 3A-F and/or Table 2 was generated using the various modules and algorithms of the DNA*STAR set on default parameters. In a preferred embodiment. the data presented in columns VIII. IX, XIII, and XI~' of Table 2 can be used to determine regions of AIM Il which exhibit a hi~~h degree of potential for antigenicity. Regions of high an tigenicity are determined from the data presented in columns VIII, IX, XII1, and/or IV by choosing values which represent regions of the 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.
C.'ertain preferred regions in these regards are set out in Figures 3A-F, but may, as shown in Table 2. be represented or identified by using tabular representations of the data presented in Figures 3A-F. The DNA*STAR
computer algorithm used to generate Figures 3A-F (set on the original default parameters) was used to present the data in Figures 3A-F in a tabular format (See Table ?). The tabular format of the data in Figure 3A-F may be used to easily determine specific boundaries of a preferred region.
The above-mentioned preferred regions set out in Figures 3.A-F 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 1 A and B. As set out in Figures 3A-F and in Table 2. such preferred regions include Gamier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions.
Choir-Fasman alpha-regions, beta-regions, and coil-regions, Kvte-Doolittle hydrophilic regions and hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Emini surface-forming regions and .lameson-Wolf regions of high antigenic index.

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Among highly preferred fi~a~ments in this regard are those that comprise re~~ions ofAIM II that combine several structural features, such as several of the features set out above in Table ?.
'l~he AIM II polypeptide ofthe present invention may be employed to treat lymphoproliferatiye disease which results in lymphadenopathy. the AIM ll 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 Il may also be employed as a research tool in elucidatin~~ the biology of autoimmune disorders including systemic lupus erythematosus (SLE). Graves' disease, innnunoproliferatiye disease lymphadenopathy (IPI_). an~~ioimmunoproliferatiye lymphadenopathy (AIL).
immunoblastive lymphadenopathy (IBL). rheumatoid arthritis. diabetes, and multiple sclerosis, allergies and to treat 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. AlM 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.
This invention also provides a method for identification of molecules. such as receptor molecules. that bind AIM II. Genes encoding proteins that bind AIM i1, such as receptor proteins. can be identified by numerous methods known to those of skill in the art. for example. ligand panning and FRCS sorting.
Such methods are described in many laboratory manuals such as. for instance.
Coligan c~~ crl.. C'm-rem I'rmocols in Irnmurruloy. 1~2):Chapter 5 ( 1991 ).
For instance, expression cloning may be employed for this purpose. To this end polyadenyiated 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 I
I. The _'~ J _ transfected cells then are exposed to labeled AIM II. (AIM II can be labeled by a variety ofvyell-known techniques includin<, standard methods ofradio-iodination or inclusion of a recognition site for a site-specific protein kinase.l Followin~~
exposure. the cells are fixed and binding of A1M II is determined. These procedures conveniently are carried out on glass slides.
Pools are identified of cDNA that produced AIM II-binding cells.
Sub-pools are prepared from these positives. transfected into host cells and screened as described above. Using an iterative sub-pooling and re-screening process, one or more single clones that encode the putative binding molecule.
such as a receptor molecule. can be isolated.
Alternatively a labeled ligand can be photo affinity linked to a cell extract.
such as a membrane or a membrane extract, 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. The labeled complex containing the ligand-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 oligonucleotide probes to screen cDNA libraries to identify genes encoding the putative receptor molecule.
Polypeptides of the 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.
A list of exemplified amino acid sequences comprising immunogenic epitopes are shown in Table 2 above. 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. C.'nmp. .4ppl. Biosci.
-x:181-186 (1988) (said references incorporated by reference in their entireties).
The .Iameson-Wolfantigenic analysis was performed using the computer program PROTEAN. using default parameters (Version 3.1 I for the Power Macintosh, DNASTAR. Inc.. 1228 South Park Street Madison, WI). Table 2 and portions _74_ of polvpcptides not listed in Table ? are not considered non-immunogenic. The immuno~~cnic epitopes of Table ? 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 epitopcs may be routinely determined usin~~ algorithms similar to the .lameson-Vv'olf analysis or by ll7 l'll'() testing for an antigenic response using methods known in the art. .Seo, o.y.. Geysen et crl., .s~tyrcr: U.S. Patents 4.708.781;
5,194.39?: 4.433.09?; and 5.4$0.971 (said references incorporated by reference in their entireties).
(t is particularly pointed out that the amino acid sequences of Table comprise immuno~~enic epitopes. Table ? lists only the critical residues of immuno~~enic epitopes determined by the.lameson-Wolfanalysis. Thus. additional flanking residues on either the N-terminal. C-terminal, or both N- and C-terminal ends may be added to the sequences of Table 2 to generate an epitope-bearing polypeptide of the present invention. Therefore, the immunogenic epitopes of Table 2 may include additional N-terminal or C-terminal amino acid residues.
The additional Ranking amino acid residues may be contiguous flanking N-terminal and/or C-terminal sequences ti-om the polypeptides of the present invention, heterologous polypeptide sequences, or may include both contiguous flankin~~
sequences from the polypeptides of the present invention and heterologous polypeptide sequences.
Polypeptides of the present invention comprising immunogenic or antigenic epitopes are at least 7 amino acids residues in length. "At least"
means that a polypeptide of the present invention comprising an immunogenic or antigenic epitope may be 7 amino acid residues in length or any integer between 7 amino acids and the number of amino acid residues of the full len~.th polypeptides of the invention. Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10. 15. 20. ?~. 30. 35. 40. 45. 50. ~~. G0, 6~, 7U, 75. 80, 8s. 90. 9~. or 100 amino acid residues in length. However. it is pointed _7i_ out that each and every integer between 7 and the number of amino acid residues of the full length polypeptide are included in the present invention.
'fhe immunogenic and antigenic epitope-bearin~~ fi~a~~ments 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:?. Every cOmblllatl011 Of a N-terminal and C-terminal position that a fragment of. for example. at least 7 or at :east 1 ~ contiguous amino acid residues in length could occupy on the amino acid sequence of SEQ ID N0:2 is included in the invention. Again, "at least 7 conti~~uous 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 of the 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 of the invention. and in immunoassays to detect the polypeptides of the present invention. The antibodies are useful, for example. in affinity purification of the polypeptides of the present invention. The antibodies may also routinely be used in a variety of qualitative or quantitative immunoassays, specifically for the polypeptides of the present invention using methods known in the art. See, e.~,T., Harlow e! al., ANTIBODIES: A LABORATORY MANUAL, {Cold Spring Harbor Laboratory Press: 2nd Ed. 1988).
The 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. For instance. epitope-bearing peptides may be synthesized using known methods of chemical synthesis. For instance. 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 HAI

polypeptide. all of which were prepared and characterized (bv ELI SA-type bindin~~
studies) in less than four weeks (Houghten eml.. I'r-nc. ~1'a~l. Accrcf. Sci.
L:S-1 H?:51 31-~ 1 s~ ( 1985)). This "Simultaneous Multiple Peptide Synthesis (SMPS)"
process is further described in U.S. Patent No. 4.631.211 to Hou~~hten and coworkers ( 1986). In this procedure the individual resins for the solid-phase synthesis of various peptides are contained in separate solvent-permeable packets.
enabling the optimal use of the many identical repetitive steps involved in solid-phase methods. A completely manual procedure allows 500-1000 or more syntheses to be conducted simultaneously (Houghten c~t crl., I'rnc. Ncnl. .-1 ccrcl. .Sci.
USA 82:5131-5 I 35 ( 1985) at 5134).
Epitope-bearing polypeptides of the present invention are used to induce antibodies according to methods well known in the art including. but not limited to, in u~~ru immunization, in vitro immunization, and phage display methods.
~Seo.
e.g., Sutcliffe el crl.. supra; Wilson el ul., .supra. and Bittle e~ ul. J.
Gon. Y'irol.
66:2347-2354 ( 1985). If in vivo immunization is used. 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 (KLI-I ) or tetanus toxoid. For instance. 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 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 pg 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 EL.ISA 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 accordin<~ to methods well known in the art.
As one of skill in the art will appreciate, and discussed above, the polvpeptides of the present in~~ention comprising an immunogenic or anti~~enic epitope can be fused to heterolo4~ous polypeptide seduences. For example, the polypeptides of the present invention may be fused with the constant dOlllalll Of immunoglobulins (IgA. IgE. I4~G. IgM ), or portions thereof (CH 1. CI-I_'. CI-13. any combination thereof including both entire domains and portions thereof) resulting in chimeric polypeptides. These fusion proteins facilitate purification. and show an increased half life in oiw~. This has been shown, v.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant res~ions of the heavy or light chains of mammalian immunoglobulins. See, e.y., EPA 0, 394,827; Trauneckere/ul.. Nulzrrc~ 331:84-( 1988). Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion can also be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g..
Fountoulakis eI ul.. .I. Biochem. 2?0:39~8-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag to aid in detection and purification of the expressed polypeptide.
The present inventors have discovered that AIM I1 is expressed in spleen, thymus and bone marrow tissue. For a number of disorders, such as septic shock.
inflammation, cerebral malaria. activation ofthe H1V virus, graft-host rejection, bone resorption. rheumatoid arthritis and cachexia, it is believed that significantly higher or lower levels of AIM II gene expression can be detected in certain tissues (o.y., spleen, thymus and bone marrow tissue) or bodily fluids (c~.~,~., serum, plasma, urine, svnovial fluid or spinal fluid) taken from an individual having such a disorder. relative to a "standard" AIM II gene expression level, i.e., the AIM II
expression level in tissue or bodily fluids from an individual not having the disorder. Thus, 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 I7uid 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.
Cell Sortiy 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.
These 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 I1 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.
Thus. in one aspect. the invention provides methods for separating cells which bind either AI M I I polypeptides or antibodies having specificity for AIM II
polypeptides comprising contacting a population of cells with either an AIM I1 polypeptide or an antibody having specificity for the AIM 11 polypeptide, wherein the AIM II polypeptide or antibody is labelled 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-~3-receptor (LT-~i-R), TR2. CD27. and TRANK.

WO 99/4258.1 PCT/US99/03703 AI:'iI Il.-lgnJllSls QJIII AIIIlIb01)lSIS
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 A111 II-binding molecules such as receptor molecules. An a~~onist is a S COnlpOlllld which increases the natural biological functions of AIM 11 or which functions in a manner similar to AIM I1. while antagonists decrease or eliminate such functions.
For example, 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 I1 agonist or antagonist. The ability of the candidate molecule to bind the binding molecule or AIM II itself is reflected in decreased binding of the labeled ligand. Molecules which bind gratuitously, i. e., without inducing the effects of AIM II when bound to the AIM II binding molecule. are most likely to be good antagonists. Molecules that bind well and elicit effects that are the same as or closely related to AIM I1, are good agonists.
A1 M 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 11 or molecules that elicit the same effects as AIM I1. 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.
Another example ofan assay for AIM II antagonists is a competitive assay that C0117b111eS AIM II and a potential antagonist with membrane-bound AIM ll receptor molecules or recombinant AIM I I 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 of the potential antagonist.
Potential antagonists include small or~~anic molecules. peptides.
polypeptides and antibodies that bind to a polypeptide of the im~ention. and S 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 blndlng molecule. such as a receptor molecule, without 111dL1C111~~ AlM II-induced activities. thereby preventing the action ofAIM II by excluding AIM II from binding. Antagonists ofthe invention include fragments of the AIM II polypeptide having tile amino acid sequence shown in SEQ ID NO:?.
Other potential antagonists include antisense molecules. Antisense technology can be used to control gene expression through antisense DNA or RNA or through triple-helix formation. Antisense techniques are discussed, for example, in Okano, .I. Neurochenr. X6:560 ( 1991 ); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression. CRC Press, Boca Raton. FL (1988).
Triple helix formation is discussed in. for instance, Lee ce crl.. ;'~~ucleic Acids Re.seurch 6:3073 ( 1979): Cooney cu al.. ,Science 2-11:456 ( 1988), and Dervan e~t ul.. Science 2~ ! :1360 ( 1991 j. The methods are based on binding of a polynucleotide to a complementary DNA or RNA. For example. the S' coding portion of a polynucleotide that encodes the mature polypeptide of the present invention may be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription thereby 2S preventing transcription and the production of AIM Il. The antisense RNA
oligonucleotide hybridizes to the mRNA in vrv and blocks translation of the mRNA molecule into AIM l l polypeptide. The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in oioo to inhibit production of AIM II.

WO 99/4258.1 PCT/US99/037U3 -~1_ The antagonists may be employed in a composition with a pharmaceutically acceptable carrier. e. ~~.. as hereinafter described.
The antagonists may be employed for instance to treat cachexia which is a lipid clearing defect resulting from a systemic deficiency of lipoprotein lipase.
w-hich is believed to be suppressed by AIM II. The AIM II antagonists may also be employed to treat cerebral malaria in which A1 M II may play a pathogenic role.
The AIM II antagonists may also be employed to prevent graft-host rejection by preventing the stimulation of the immune system in the presence of a grant.
The AIM II antagonists may also be employed to inhibit bone resorption and. therefbre. 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.
As noted above. antagonists and agonists of the 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 know in the art. For example.
Phizickv and Fields. "Protein-protein interactions: methods for detection and analysis"
~~icrohiol. ller. X9:94-123 (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 Il polypeptides of the invention which bind to a cell surface receptor involves transfecting eukaryotic cells with DNA eIlCOdlng the receptor, such that the cells expresses the receptor on their surfaces. followed by contacting the cells with a labeled (e.g., radioactive label.
biotin. etc.) AIM II polypeptide. The amount of labeled AIM II polypeptide -g2_ 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.
Further. as one skilled in the art would recognize, cells which express and retain AIM Il polypeptides can be used to identify AIM II ligands. In one such an embodiment, cells which express AIM II would be contacted with potential ligands which have been detectably labeled. Further. such ligands may be polypeptides which are expressed as part of a library of seduences on the surface of a phage (e.~=., a phage display library).
Once an AIM II polypeptide has been identified which binds to the cell surface receptor of interest, 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 11 polypeptide or another ligand. Further, whether an AIM 11 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 of the polypeptides of the present invention (e.g.. an AIM
polypeptide comprising amino acid 83-240 of SEQ ID N0:2). for example, may be utilized in the ligand binding and receptor activation/inhibition assay described above.

WO 99/425$4 PCT/US99/03703 _8;_ Cancer Prn~;nosis~
It is 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.c~., a mammal of the same species not having the cancer. Further. it is believed that reduced levels of the AlM II protein can be detected in certain body fluids (e.y., sera, plasma. urine, and spinal fluid) Irom mammals with cancer when compared to sera from mammals of the same species not having the cancer. Thus, the invention provides a diagnostic method useful during tumor diagnosis, which involves assayin~~ the axpl'eSS1011 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.
Where a tumor diagnosis has already been made according to conventional methods, 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.
By "assaying the expression level of the gene encoding the AIM 1I protein"
is intended qualitatively or quantitatively measuring or estimating the level of the AIM II protein or the level of the mRNA encoding the AIM II protein in a first biological sample either directly (c~.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.~~., by comparing to the AIM II
protein level or mRNA level in a second biological sample).
Preferably, 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. As will be appreciated in the art, once a standard AlM Ii protein level or mRNA level is known, it can be used repeatedly as a standard for comparison.

-8~1_ By "biolo~.:ical sample" is intended any biological sample obtained from an individual. cell line. tissue culture. or other source which contains AI~1 I1 protein or mRNA. Biological samples include mammalian body fluids (such as sera.
plasma. urine, synovial fluid and spinal fluid) which contain secreted mature AIM II protein. and ovarian. prostate, heart. placenta. pancreas liver.
spleen. lung, breast and umbilical tissue.
The present invention is useful for detectin~~ cancer in mammals. In particular the invention is useful during diagnosis of the of followin~~ 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. Particularl~~ preferred are humans.
Total cellular RNA can be isolated from a biological sample using the single-step guanidinium-thiocyanate-phenol-chloroform method described in Chomczynski and Sacchi, Anul. Biochem. 162:156-159 ( 1987). Levels of mRNA
encoding the AIM II protein are then assayed using any appropriate method.
These include Northern blot analysis (Harada el ul., C.'ell 63:303-3 I? ( 1990)), S 1 nuclease mapping (Fujita et ul.. C.'el! -ll: 357-367 ( 1987)) , the polymerase chain reaction (PCR), reverse transcription in combination with the polymerase chain reaction (RT-PCR) (Makino el crl.. Te~chnidt~e 2:295-301 ( 1990)) , and reverse transcription in combination with the lipase chain reaction (RT-LCR).
Assaying AIM II protein levels in a biological sample can occur using antibody-based techniques. For example, AIM II protein expression in tissues can be studied with classical immunohistological methods (Jalkanen. !~ i.. en ul.. .I.
C'cll. l3inl. 101: 976-985 ( 1980: .Ialkanen. M.. et ul.. .I. Cell. Binl. Ill:

(1987)).
Other antibody-based methods useful for detecting AIM II protein gene 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 ('''I,'''I). carbon ("C).
sulfur _8j_ ("S). tritium ('H), llldllllll ("=In), and technetium (''''"'Tc). and fluorescent labels.
such as fluoreseein and rhodamine. and biotin.
Tlrerape~itics The uses of the AIM II polypeptides, particularly human AIM II
polypeptides, include but are not limited to the treatment viral hepatitis.
Herpes viral infections. aller~~ic reactions. adult respiratory distress syndrome, neoplasia.
anaphylaxis. aller~~ic asthma. allergen rhinitis. drug allergies (e.~,~.. to penicillin.
cephalosporins), primary central nervous system lymphoma (PCNSL). chronic lylllpllC)C~'tlC leukemia (CLL). lymphadenopathy. autoimmunc disease. graft versus host disease. rheumatoid arthritis, osteoarthritis. Graves' disease, acute lymphoblastic leukemia (ALL). non-Hodgkin's lymphoma (NHL).
ophthalmopathy. uveoretinitis, the autoimmune phase of Type 1 diabetes.
myasthenia gravis. glomerulonephritis, autoimmune hepatological disorder.
autoimmune inflammatory bowel disease. and Crohn's disease. In addition, the AIM I I polypeptide of the present invention may be employed to inhibit neoplasia.
such as tumor cell ~~rowth. The combination of AIM 1I protein with immunotherapeutic a~~ent 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 reduire growth promotion activity, for example.
restenosis, since AlM II has proliferative effects on cells of endothelial origin.
AIM 11 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 inhibition of T and B cell activation. differentiation andior proliferation may be employed to treat a numberofimmunological based diseases. several ofwhich are referred to above. Further. depending on the particular AIM II polypeptide _8~_ employed. the AIM ll 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 adjuyant or costimulatory molecule. The followin~~ experiments are performed to assess the in oioo AIM II protein on the host immune system.
Tumor or non-tumor bearing mice are treated with AIM II protein at three different doses (0. I mg/kg, l mg/kg and 10 mg/kg. i.p., QD, 10-14 days, N=5 per group) before or after immunization with tumor antigen or superantigen, the mice are sacrificed weekly post treatment after blood collection. The spleens or the lymph nodes are used for the following in rilro analyses well known to those skilled in the art:
FRCS 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 ofprotective immunity. which could lead to protecting mice from subsequent tumor challenge.
In order to examine possibility 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-i8 tumor-free mice treated with AIM II protein will be challenged with MC-38 or irrelevant murine sarcoma MCA-102 using techniques well known to those skilled in the art. Three possible results could be observed:
Result ~ I Result #!? Result # i MC-38.\~fT: tumor (-) tumor 1-1 tumor (+) MCA-10'_': tumor (+1 tumor 1-) tumor (+) Indication from II I : Evidence of tumor-specific protective immunity Indication from tt'?: Evidence of non-tumor specific immunity Indication from #t_p: Lack of protective immunity _87_ Ifgeneration oftumor-specific protective immunity upon AIM II treatment is demonstrated. the followin~~ depletion experiment are performed to identify which leukocyte sub-population is responsible for the tumor rejection. The mice ~°ill be treated with various mAb which recognize either CD~4t or CD8+
T cells.
NK cells, granulocvte (Gr1+), or specific cvtokine such as IFNy 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 otter observed in RA. Endothelial cell proliferation is increased in the synovia of RA
patients as compared to patients with osteoarthritis (OA) or unaffected individuals.
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-~3-receptor (LT-(3-R), TR2 (also referred to as the Herpes virus entry mediator (HVEM) and ATAR). CD27. and TRANK (also referred to as receptor activator of nuclear factor-kappa B (RANK)).
Each of the receptors listed immediately above is involved in various physiological processes which may be modulated by the AIM II polypeptides of the invention. More specifically. the polypeptides of the invention can be used to stimulate or block the action of ligands which bind cellular receptors having AIM II binding activity (e.g.. LT-~3-R, TR2, CD?7. and TRANK).
LT-~. which binds to the LT-~3-R, has been implicated in the development of secondary lymphoid tissues and the maintenance of organized lymphoid tissues in adults. LT-(3-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 -gs-tissues in the lung including a subpopulation ohT-lymphocytes. LT-~3-R has also been implicated in the formation of germinal centers and thus appears to be involved in humoral immune responses. Rennert et crl.. Irrt. Innrrur7ol.

9:1627-1669 ( 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-(3-R. Further, polypeptides of the invention ma~~ stimulate the formation of germinal centers and LT-~3-R
mediated humoral responses by activating LT-(3-R.
One skilled in the art would recognize that different portions ofthe AIM II
polypeptide may have different effects on I_T-~i-R. One skilled in the art would also recognize that the effect that the AIM II polypeptides of the invention would have on LT-~3-R would vary with the individual peptide and the effect it has when bound to LT-(3-R. Methods for screening molecules having agonistic and antagonistic activities of cellular receptor are described above.
The core protein of hepatitis C virus (HCV) has also been shown to associate with LT-~3-R and enhance signaling mediated by this receptor. Chen et crl.. .l. G'irol. ?l :9417-9426 ( 1997). Further. the interaction of this protein with LT-(3-R may contribute to the chronically activated. persistent state of HCV-infected cells. The AIM II polypeptides of the invention may be employed to block HVC stimulation of LT-~3-R and the pathology associated with this virus.
TR2 is a member of the 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. Chenz 2??:14272-14276 (1997). TR2 serves a number of functions ira oioo. 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-xB and AP-1. This activation appears to occur through a TNF receptor-associated factor (TRt'1F)-mediated mechanism.
The AIM II polypeptides of the invention may be employed to inhibit T eel I activation, and thus T cell mediated immune responses. by blockin~~
access to TR2 by cellular ligands which activate this receptor. Similarly.
polypeptides of the invention may stimulate T cell activation by activatin~~ TR?. As noted above for LT-~3-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 of the invention may also be employed to prevent or treat Herpes viral infections.
Expression of 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 e! ul.. .l.
Imrrrrrnol.
l <1:6496-6502 ( 1998). In addition. activation of CD27 may enhance IgE
synthesis. Inhibition of the interaction between CD27 and CD70 thus may inhibit IgE production and allergic responses.
'fhe AIM II polypeptides of the invention may be used for modulating immune responses. For example, AI1~~1 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 CD?7 and inhibit B cell differentiation and proliferation, as well as the secretion of proteins (e.~>.. IgE) by these cells.
Therefore, AIM II polypeptides may be employed to suppress IgE antibody formation in the treatment of 1gE-induced immediate hypersensitivity reactions, such as allergic rhinitis (also know as hay fever), bronchial asthma. allergic asthma, anaphylaxis. atopic dermatitis and gastrointestinal food aller~~y.
CD27 is also believed to be the receptor for a pro-apoptotic protein commonly known as Siva. Pandanilam e~t ul.. Kidney Im. ~-1:1967-197 ( 1998).
AIM II polypeptides of the invention may be employed to inhibit the interaction between Siva and CD27 and thus prevent Siva/CD27 mediated induction of apoptosis. Diseases associated with decreased cell survival. or increased apoptosis. include AIDS: neurodegenerative disorders (such as Alzheimer's disease. Parkinson's disease. Amyotrophic lateral sclerosis, Retinitis pigmentosa.
Cerebellar degeneration): mvelodysplastic syndromes (such as apiastic anemia), isehemic injury (such as that caused by myocardial infarction. stroke and reperfusion injury 1, toxin-induced liver disease (such as that caused by alcohol), septic shock. cachexia and anorexia.
AIM II polvpeptides of the invention may also be employed to enhanced activation of the Siva/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 follicular 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.
While CD?7 may be membrane-bound, a soluble form of CD27 is produced in the course of the immune response. Soluble CD27 (sCD?7) 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 sCD?7 are present in the sera of patients with various B-cell malignancies. Kersten el crl.. Blood 8?:1985-1989 ( 199G). 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.~,=.. acute lymphoblastie leukemia (ALL) and non-Hodgkin's lymphoma (NHL)).
Soluble CD27 has also been found to be elevated in patients with a number of non-hyperproliferative diseases. For example, sCD?7 has been shown to be elevated in patients with untreated Graves' hyperthyroidism. Kallio cu crl..
J. Gcrh.
('lin .llecl 132:478-48? (1998). Further. increases in sCD?7 serum levels have been found in patients with systemic lupus erythematosus (SLE) and this increase has been shown to be associated with the activity of the disease. Font eJ
crl., t'lin.
Imnrunul. ImnrtnxrhcrJl7ol. 81:?39-213 ( 1996): Swaak eJ ul.. Clip.
Rheuntcrtol.
l-l:?9;-300 ( 1990. Also, B cells tr0117 1170St patients with chronic l1'111p110CVt1C
leukemia (CLL) have been shown to co-express both membrane-bound and soluble CD?7, as well as CD70. Ranheim of crl.. l3lnod ~.5:3356-3566 (1995).
It has been postulated that sCD?7 may prevent leukemic B cells from stimulatin~~ T cells via CD70. and thus may impair the ability of B cells to function as antigen-presenting cells. Ranheim er ul.. l3loocl H.5:3556-3566 ( 1995).
Polypeptides of the invention may be employed to inhibit interactions between sCD?7 and CD70, and, thus, to enhance the ability of B cells to act as antigen-presenting cells.
AIM Il polypeptides of the invention may also be employed to treat diseases and afflictions associated with increase levels of sCD27. While not wishing to be bound to a mechanistic theory. AIM 1I polypeptides may be useful in treatment regimens for these conditions since it binding sCD27 and prevents it from interacting with cellular ligands.
A1M II polypeptides are also believed to bind to RANK. (See Anderson eJ crl.. N'mure 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 osteoprotegerin ligand (OPGL), TRANCE and ODF).
Mice having a disrupted RANKL gene show severe osteoporosis. exhibit defective tooth eruption, and lack osteoclasts. These mice also exhibit defects in T and B lymphocyte differentiation. Additionally. RANKL-deficient mice lack lymph nodes but exhibit normal splenic structure and Peyer's patches. These data indicate that RANKL mediated pathways regulate lymph node organogenesis, lymphocyte development. and osteoclast differentiation and proliferation.
There are two main classes of 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 of the skeletal system. For example. in human adults. between 10 to 1 S% of trabecular bone surfaces are covered with osteoid (new unmineralized bone made by osteoblasts) w-hile about 4% have active resorptive surfaces. The dynamic nature ofthe continuing flux ofbone cell activity is illustrated by the fact that approximately 18% of total skeletal calcium is often removed and deposited over a period of one year.
The AIM polypeptides of the invention may be employed to modulate osteoclast differentiation and proliferation, 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 imperfecta, and Hurler and Marfan syndromes.
Polypeptides of the invention may also be employed in processes for reshaping bone and teeth and in periodontal reconstructions where lost bone replacement or bone augmentation is required. such as in a jaw bone and supplementing alveolar bone loss resulting from periodontal disease to delay or prevent tooth loss (see.
e.g., Sigurdsson et al.. J. Peniodontol. 6b:511-21 (1995)).
The AIM II polypeptides of the 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 ofproteins {e.~., 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 of the 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 Il 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.
The AIM II polypeptide may be cleaved in viw to form a soluble form of the molecule. As noted in Example 10, a cleavage site appears to be located between amino acid residues 82 and 83 of the sequence shown in SEQ ID N0:2.
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 amino acids 83-in SEQ ID N0:2. Soluble forms ofAIM II are especially useful for the treatment of diseases where systemic administration of these peptides is preferred.
Further, 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.
Modes of adntinistration It will be appreciated that conditions, such as those discussed above, can be treated by administration of AIM II protein. Thus, the invention further provides a method of treating an individual in need of an increased level of AIM I I
activity comprising administering to such an individual a pharmaceutical WO 99/425$4 PCT/US99/03703 composition comprising an effective amount of an isolated AIM II polypeptide of the invention, effective to increase the AIM II activity level in such an individual.
As a general proposition, the total pharmaceutically effective amount of AIM II polypeptide administered parenterally per dose will be in the range of S about 1 pg/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. If given continuously, the AIM II polypeptide is typically administered at a dose rate of about 1 ug/kg/hour to about 50 pg/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.
Pharmaceutical compositions containing the AIM II of the invention may be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray. By "pharmaceutically acceptable carrier" is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term "parenteral" as used herein refers to modes of administration which include intravenous. intramuscular, intraperitoneal, intrasternal, subcutaneous and intra-articular injection and infusion.
In the treatment of rheumatoid arthritis, particularly preferred modes of administration of AIM II polypeptides of the present invention include, intradermal, subcutaneous and intra-articular injection and infusion.
Preferably, AIM I1 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.
Particularly preferred excipients include In addition to soluble AIM II polypeptides (i.e., AIM II polypeptides missing all or part of the transmembrane domain), AIM II polypeptides containing -9~-the transmembrane region can also be used when appropriately solubilized by including detergents, such as triton X-100, w°ith buffer.
Chromosome Assnl~s The nucleic acid molecules of the 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.
In certain preferred embodiments in this regard, the cDNA 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.
In addition, in some cases, sequences can be mapped to chromosomes by preparing PCR primers (preferably I 5-25 bp) from the cDNA. Computer analysis of the 3' untranslated region of the 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 e! al., Human C.'hnomosomev: A Manual OrBasic Techrriyues. Pergamon Press, New York ( 1988 ).
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found. for example, in V. MeKusick.
:l~endelian Inherilunce In ~l~crn, available on-line through Johns Hopkins University, Welch Medical Library. The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).
Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.
Examples Example l: Expression and Purification of AIM ll in E. coli A. Expression of AIM ll ~~ith an N-terminal 6-His tag The DNA sequence encoding the AIM II protein in the deposited cDNA
clone is amplified using PCR oligonucleotide 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' oligonucleotide primer has the sequence S' GCGGGATCCGGAGAGATGGTCACC 3' (SEQ ID N0:7) containing the underlined BamHI restriction site, which includes nucleotides 244-258 of the A1M II protein coding sequence in Figure 1 A (SEQ ID NO: l ).
The 3' primer has the sequence:

5' CGCAAGCTTCCTTCACACCATGAAAGC 3' (SEQ ID N0:8) containing the underlined Hind III restriction site followed by nucleotides complementary to nucleotides 7~7-774 as shown in Figure 1 B (SEQ ID NO: I ).
The entire AIM II protein can be expressed using the following primers:
The 5' oligonucleotide primer has the sequence:
5' GACC GGATCC ATG GAG GAG AGT GTC GTA CGG C 3' (SEQ ID
N0:9) containing the underlined BumHl restriction site. which includes nucleotides 49-70 of the AIM II protein coding sequence in Figure I A (SEQ ID
NO: I ).
The 3' primer has the sequence:
5' CGC AAGCTT CCT TCA CAC CAT GAA AGC 3' (SEQ ID N0:10) containing the underlined HindIIl restriction site followed by nucleotides complementary to nucleotides 756-783 as shown in Figure 1 B (SEQ ID NO:1 ).
The 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. 91311 ). pQE9 encodes ampicillin antibiotic resistance ("Amp"') 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.
The amplified AIM II DNA and the vector pQE9 both are digested with BamHI and Hind III and the digested DNAs are then ligated together. 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
inducible promoter and in-frame with an initiating AUG appropriately positioned for translation of AIM I1 protein.
B. Expression orAlM II with a C-terminal 6-His tcr~,~
The bacterial expression vector pQE60 is used for bacterial expression in this example. (QIAGEN. Inc., 9259 Eton Avenue, Chatsworth, CA, 9131 I ).
pQE60 encodes ampicillin antibiotic resistance ("Amp"') 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-tri-acetic acid ("Ni-NTA") affinity resin sold by QIAGEN.
Inc.. supra, and 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 (i.e., a "6 X His tag") covalently linked to the carboxyl terminus of that polypeptide.
The DNA sequence encoding the desired portion of the AIM II protein is amplified from the deposited cDNA clone using PCR oligonucleotide 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.
For cloning the protein, the 5' primer has the sequence:
S' GACGC CCATGG AG GAG GAG AGT GTC GTA CGG C 3' (SEQ ID NO:
17) containing the underlined NcoI restriction site followed by nucleotides complementary to the amino terminal coding sequence of the AIM II sequence in Figure I A. One of ordinary skill in the art would appreciate, of course, that the point in the protein coding sequence where the 5' primer begins may be varied to amplify a DNA segment encoding any desired portion of the complete protein (shorter or longer). The 3' primer has the sequence:
5' GACC GGATCC CAC CAT GAA AGC CCC GAA GTA AG 3' (SEQ ID NO:
18) containing the underlined BamHI restriction site followed by nucleotides complementary to the 3' end of the coding sequence immediately before the stop codon in the AIM II DNA sequence in Figure 1 B, with the coding sequence aligned with the restriction site so as to maintain its reading frame with that of the six His codons in the pQE60 vector.
The amplified AIM II DNA fragment and the vector pQE60 are digested with BamHl and NcoI and the digested DNAs are then ligated together. Insertion of the 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.
C'. Ex~n~ession of.-IL1~111 deletion mutant i1'1/h an J~'-terminal 6-His I ag The DNA sequence encoding the AIM II protein in the deposited eDNA
clone was amplified using PCR oligonucleotide 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.
In particular, an N-terminal deletion AIM II mutant (Met(68) to Val(240j in SEQ ID N0:2) was constructed using the following primers:
The 5' oligonucleotide primer has the sequence:
5'-GGG GGA TCC ATG GTC ACC CGC CTG CC-3' (SEQ ID N0:21 ) containing the underlined l3umHI restriction site, and includes 17 nucleotides of the AIM II protein coding sequence in Figure lA (SEQ ID NO:1 ).
The 3' primer has the sequence:
S'-GGG AAG CTT CAC CAT GAA AGC CCC G-3' (SEQ ID N0:22) containing the underlined Hind III restriction site followed by nucleotides complementary to nucleotides 753-768 as shown in Figure 1 B (SEQ ID NO:1 ).
These 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, 91311 ). pQE9 encodes ampicillin antibiotic resistance ("Amp"') 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.
The amplified AIM II taa 68-240) DNA and the vector pQE9 both were digested with BamHI and Hind III and the digested DNAs w.~ere then ligated together. Insertion of the AIM II (aa 68-240) protein DNA into the restricted pQE9 vector places the AIM II protein coding region downstream of and WO 99/42584 PCT/US99/03'703 operably linked to the vector's IPTG-inducible promoter and in-frame with an initiating AUG appropriately positioned for translation of AIM II deletion protein.
Transformation of the Bacteria:
The ligation mixture from the 6-His tagged expression constructs made in A, B or C. above. is transformed into competent E. colt cells using standard procedures. Such procedures are described in Sambrook et al.. Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). E. colt strain MI~/rep4, containing multiple copies of the plasmid pREP4, which expresses lac repressor and confers kanamycin resistance ("Kan"'), 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 ampicillin and kanamycin. Plasmid DNA is isolated from resistant colonies 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 ampicillin (100 pg/ml) and kanamycin (25 ug/ml).
The O/N culture is used to inoculate a large culture, at a dilution of approximately I : I 00 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 I mM to induce transcription from luc repressor sensitive promoters, by inactivating the lacI 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 solubilized from the inclusion bodies into 8M urea. The 8M urea solution containing the solubilized protein is passed over a PD-10 column in 2X
phosphate-buffered saline ("PBS"), thereby removing the urea, exchanging the buffer and refolding the protein. The protein is purified by a further step of chromatography to remove endotoxin. Then, it is sterile filtered. The sterile filtered protein preparation is stored in ?X PBS at a concentration of 9~
pg/ml.
D. Expression and Purificalion of full length AIM II aa~ilhoirW 6-His tag The bacterial expression vector pQE60 is used for bacterial expression in this example. (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311 ).
pQE60 encodes ampicillin antibiotic resistance ("Amp"') and contains a bacterial origin ofreplication ("ori"). an IPTG inducible promoter, a ribosome binding site ("RBS"), six codons encoding histidine residues that allow affinity purification using nickel-nitrilo-tri-acetic acid ("Ni-NTA") affinity resin sold by QIAGEN, Inc., supra, and suitable single restriction enzyme cleavage sites. These elements are arranged such that a DNA fragment encoding a polypeptide may be inserted in such as way as to produce that polypeptide with the six His residues (i.
c'.. a "6 X His tag") covalently linked to the carboxyl terminus of that polypeptide.
However, in this example. 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 of the AIM II protein is amplified from the deposited cDNA clone using PCR oligonucleotide 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.
For cloning the protein, the 5' primer has the sequence 5'GACGC
CCATGG AG GAG GAG AGT GTC GTA CGG C 3' (SEQ ID NO: 17) containing the underlined NcoI restriction site including nucleotides of the amino terminal coding region of the A1M II sequence in Figure 1 A. One of ordinary skill in the art would appreciate, of course, that the point in the protein coding sequence where the 5' primer begins may be varied to amplify a desired portion of the complete protein (i.e., shorter or longer). The 3' primer has the sequence 5' CGC AAGCTT CCTT CAC ACC ATG AAA GC 3' (SEQ ID NO: 19) containing the underlined Hind III restriction site followed by nucleotides complementary to the 3' end of the non-coding sequence in the AIM II DNA
sequence in Figure 1 B (SEQ ID NO: l ).
The amplified AIM II DNA fragments and the vector pQE60 are digested with Ncol and Hind III and the digested DNAs are then ligated together.
Insertion of the AIM II DNA 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 of the six histidine codons downstream of the insertion point.
E. Corrstruclion of an N-terminal AIM ll Deletion Mutant For cloning an AIM II deletion mutant (Met(68) to Val(240) in SEQ ID
N0:2), the 5' primer has the sequence 5'-GGG CCA TGG ATG GTC ACC CGC
CTG CC-3' (SEQ ID N0:23) 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 S'-GGG AAG CTT CAC CAT GAA
AGC CCC G-3' (SEQ ID N0:22) containing the underlined Hind III restriction site followed by nucleotides complementary to nucleotides 753 to 768 in Figure 1 B (SEQ ID NO:1 ).
The amplified AIM II (aa 68-240) DNA fragments and the vector pQE60 were digested with NcoI and Hind III and the digested DNAs were then ligated together. Insertion of the AIM II (aa 68-240) DNA 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 HindIII
digestion removes the six histidine codons downstream of the insertion point.

-lO3-F. Conrtruction of ~crn A%-terminal Alhl ll Deletion Mutant For cloning an AIM II deletion mutant (Ala( I O 1 ) to Val(240) in SEQ ID
NO:?). the 5' primer has the sequence 5'-GGG CCA TGG GCC AAC TCC AGC
TTG ACC-3' (SEQ ID N0:24) containing the underlined NcoI restriction site including nucleotides 349-366 in the AIM II protein coding sequence in Figure I A. One of ordinary skill in the art would appreciate, of course, that the point in the protein coding sequence where the 5' primer begins may be varied to amplify a desired portion of the complete protein (i.e., shorter or longer). The 3' primer has the sequence 5'-GGG AAG CTT CAC CAT GAA AGC CCC G-3' (SEQ ID
N0:22) containing the underlined Hind 1II restriction site followed by nucleotides complementary nucleotides 75~-768 of the AIM II DNA sequence in Figure 1 B.
The amplified AIM II (aa 10 I -240) DNA fragments and the vector pQE60 were digested with NcoI and Hind III and the digested DNAs are then ligated together, Insertion of the AIM II (aa 101-240) DNA into the restricted pQE60 vector places the AIM II (aa 1 O 1-240) protein coding region downstream from the IPTG- inducible promoter and in-frame with an initiating AUG. The HindII1 digestion removes the six histidine codons downstream of the insertion point.
G. Purification ofAlMlI,frnm E. coli A polynucleotide sequence encoding a soluble fragment of AIM II
(corresponding to amino acid residues L83-V240 of SEQ ID N0:2) was cloned into the HGS E. coli expression vector pHE4. The resulted plasmid DNA
(pHE4:AIMII.L83-V240) was used to transform SG13009 E. coli host cells. The bacterial transformants were grown in LB medium containing kanamycin. Upon IPTG induction, recombinant AIM II was expressed in E. coli as an insoluble protein deposited in inclusion bodies The E. coli cell paste was resuspended in a buffer containing 0.1 M
Tris-HCI pH7.4, 2 mM CaCl2 and was lysed by passing twice through a microfluidizer (Microfluidics, Newton, MA) at 6000-8000 psi. The lysed sample was mixed with NaCI to a final concentration of O.SM and then centrifuged at 7000x g for 20 minutes. The resulting pellet was washed again with the same buffer plus O.SM NaCI and then centrifuged at 7000x g again for 20 minutes.
The partial ly purified inclusion bodies were then resuspended for 2-4 hours at 20-25 pC in 2.0 M guanidine hydrochloride containing 100 mM Tris pH 7.4, 2mM CaCl2, ~ mM Cvsteine and centrifuged. The resulting pellet was then resuspended for 48-72 hours at 4 pC in 3.0-3.5 M guanidine hydrochloride containing 100 mM Tris pI-I 7.4, 2mM CaCl2, with or without ~ mM Cysteine.
At this time, a portion of AIM II was solublized and remained in the soluble phase after 7,OOOx g centrifugation.
The 3M guanidine hydrochloride extract was quickly diluted with 20-30 volumes of a buffer containing SO mM Tris-I-ICl pHB, 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.
Liquid Chromatographic Purification of AIM II
The diluted AIM II sample was clarified using a 0.45 pm sterile filter. The AIM II protein was then adjusted to pH6-6.8 with O.SM MES and chromatographed over a strong canon exchange (POROS HS-50) column. 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, I 500 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.
Transformation of the Bacteria:
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, _lOj_ Zncl EcL ; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY ( 1989).
E. coli strain Ml~/rep4, containing multiple copies of the plasmid pREP4, which expresses the lac repressor and confers kanamvcin resistance ("Kan"'), was used in carrying out the illustrative example described herein. 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 ampicillin 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 ampicillin (100 pg/ml) and kanamycin (25 pg/ml). 'The O/N culture was used to inoculate a large culture, at a dilution of approximately I :25 to 1:250. The cells were grown to an optical density at 600 nm ("OD600") of between 0.4 and 0.6. Isopropyl-b-D-thiogalactopyranoside ("IPTG") was then added to a final concentration of 1 mM
to induce transcription from the lac repressor sensitive promoter, by inactivating the IacI repressor. Cells subsequently were incubated further for 3 to 4 hours.
Cells then were harvested by centrifugation.
The cells were then stirred for 3-4 hours at 4°C in 6M guanidine-pHB. The cell debris was removed by centrifugation, and the supernatant containing the AIM II was dialyzed against 50 mM Na-acetate buffer pHb, supplemented with 200 mM NaCI. Alternatively, the protein can be successfully refolded by dialyzing it against 500 mM NaCI, 20% glycerol, 25 mM Tris/HCI
pH7.4, containing protease inhibitors. After renaturation the protein can be purified by ion exchange, hydrophobic interaction and size exclusion chromatography. Alternatively, 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.

Example 2: Cloning and Expression of AIM II protein in a Baculovirus E.rpression System A. Construction of a full length AIM II protein:
The cDNA sequence encoding the full length AIM II protein in the deposited clone is amplified using PCR oligonucleotide primers corresponding to the ~' and 3' sequences of the 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: I 1 ) containing the underlined BamHI restriction enzyme site followed by 22 bases (i. e, , nucleotides 49-70) of the coding region for the AIM II protein in Figure I A. Inserted into an expression vector, as described below, 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 described 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 S'GA CGC GGT ACC GTC CAA TGC
ACC ACG CTC CTT CCT TC 3' (SEQ ID N0:12) containing the underlined Asp718 restriction site followed by nucleotides complementary to 770-795 nucleotides of the AIM 1I set out in Figure lA.
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 BamHI and Asp718 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 al., A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experimental Station Bulletin No. 1 X55 (1987).
This expression vector contains the strong polyhedrin promoter of the Amographa californica 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 a BamHI site. The polyadenylation site of the simian virus 40 ("SV40") is used for efficient polyadenylation. For an easy selection of recombinant virus the beta-gaiactosidase gene from E. coli is inserted in the same orientation as the polyhedrin promoter and is followed by the polyadenylation signal of the polyhedrin gene. The 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.
Many other baculovirus vectors could be used in place of pA2-GP, such as pAc373, pVL941 and pAcIMI 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. Such vectors are described in Luckow et al., Virology 17U: 31-39, among others.
The plasmid is digested with the restriction enzyme BamHI and Asp718 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 1 O 1 Inc., La Jolla, Ca.). This vector DNA is designated herein "V".
Fragment F2 and the dephosphorylated plasmid V2 are ligated together with T4 DNA ligase. E. toll HB 1 O1 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 usingXbaI and then analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing. This plasmid is designated herein pBacAIM II.
B. Construction of an N-terminal AIM II deletion mutants:
In this illustrative example, the plasmid shuttle vector pA2 GP was used to insert the cloned DNA encoding the an N-terminal deletion of the AIM I1 WO 99/425$4 PCT/US99/03703 protein into a baculovirus to express an AIM II mutant (Gln(60) to Val(240)) and AIM II mutant (Ser(79) to Vai(240)) in SEQ ID N0:2, using a baculovirus leader and standard methods as described in Summers et crl., A Manual o f Methods.
for Baculvvirar.s Vectors and Insect Cell C'ulmre Procedures, Texas Agricultural S Experimental Station Bulletin No. 1 X55 ( 1987). This expression vector contains the strong polyhedrin promoter of the Aulographa californicu nuclear polyhedrosis virus (AcMNPV) followed by the secretory signal peptide (leader) of the baculovirus gp67 protein and convenient restriction sites such as BcrmHI, Xbal and Asp718. The polyadenylation site of the simian virus 40 ("SV40") is used for efficient polyadenylation. For easy selection of recombinant virus.
the plasmid contains the beta-galactosidase gene from E. 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.
Many other baculovirus vectors could be used in place of the vector above, such as pAc373, pVL941 and pAcIMI, 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 170:31-39.
The cDNA sequence encoding the AIM II (Gln(60)to Val(240), Figure 1 A
(SEQ ID N0:2), was amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene.
The 5' primer has the sequence:
5'-GGG GGA TCC CGCA GCT GCA CTG GCG TCT AGG-3' (SEQ ID N0:25) containing the underlined BamHI restriction enzyme site followed by 20 nucleotides (i.e., nucleotides 225-245) encoding the AIM II protein shown in Figure 1 A and B, beginning with amino acid 60 of the protein. The 3' primer has the sequence S'-GGG TCT AGA CAC CAT GAA AGC CCC G-3' (SEQ ID NO:

WO 99/425$4 PCT/US99/03703 26) containing the underlined .~hcrI restriction site followed by nucleotides complementary to nucleotides 7>s-768 in Figure 1 B (SEQ ID NO:1 ~.
The amplified fra~~ment was isolated from a 1 % agarose <~el using a commercially available kit ("Geneclean." BIO 101 Inc.. La ,Tolla. Ca.). The fragment then was di~~ested with l3aml-II and.lhcrl and again was puritied on a 1 agarose gel. This fragment was designated herein "Fl ".
The plasmid was digested with the restriction enzymes l3umHI and Xhcrl and optionally, can be dephosph orylated using calf intestinal phosphatase.
using routine procedures known in the art. The DNA was then isolated ti~om a 1%
agarose gel using a commercially available kit ("Geneclean" BIO 101 Inc., La ,Tolla. Ca.). This vector DNA was designated herein "V 1 ".
Fragment F 1 and the dephosphorylated plasmid V 1 were ligated together with T4 DNA ligase. C. colt HB 1 O1 or other suitable E. colt hosts such as XL-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. II1 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 of the DNA. Tl2e sequence of the cloned fragment was confirmed by DNA sequencing. This plasmid was designated herein pBacAIM II (aa 60-240).
The cDNA sequence encoding the AIM II (Ser(79)to Val(240). Figure 1 A
(SEQ ID N0:2), was amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene.
The 5' primer has the sequence:
~' cgc GGATCC C TCCTGC~GAGCAGCTGATAC 3' (SEQ ID N0:27) containing the underlined l3umH1 restriction enzyme site followed by nucleotides 283-301 encoding the AIM II protein shown in Figure 1 A and B. be~~inning with amino acid 79 of the protein. The >' primer has the sequence:

~'-cgc ti(i;1'ht'C TC'.1 C:ACCATGAAAGC s' fSFQ ID N0:29) containin<T the underlined l3crnrI-II restriction site followed by nucleotides complementary to nucleotides 7~7-771 in Figure IB (SEQ ID NO:1 ).
The amplified fragment was isolated from a 1% agarose gel using a S commercially available kit ("Geneclean." BIO 1 O1 Inc., La ,lolla. Ca. ).
The fragment then was di~~ested with Buml-ll and again was purified on a 1 %
a~=arose gel. This fragment was designated herein "F1 ".
The piasmid was digested with the restriction enzymes BanrI-II and optionally, can be dephosphoryiated using calf intestinal phosphatase. using routine procedures known in the art. The DNA was then isolated from a 1 agarose ~~el usin~~ a commercially available kit ("C~eneclean" B10 101 Inc..
La Jolla, Ca.). This vector DNA was designated herein "V I ".
Fragment F 1 and the dephosphorylated plasmid V 1 were ligated together with T4 DNA lipase. 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 wi I1 show amplification of the DNA. The sequence of the cloned fragment was confirmed by DNA sequencing,. This plasmid was designated herein pBacAlM II (aa 79-240).
C. Transfection of the Baculovirus vectors containing AIM II sequences:
~ ug of the plasmid either pBac A1M II or pl3acAIM II (aa 60-240) was co-transfected with 1.0 pg ofa commercially available linearized baculovirus DNA
("BaculoGoldT"~ baculovirus DNA", Pharmingen, San Diego, CA.). usin~~ the lipofcction method described by Felgner el ul.. Proc. Natl. Acad. Sci. USA 84:
741 s-7417 ( 1987 ). 1 yg of BaculoGoldT"" virus DNA and 5 ~tg of the plasmid pBac A1M II or pBacAIM 11 (aa 60-240) was mixed in a sterile well of a WO 99/d2584 PCT/US99/03703 microtiter plate containing: ~0 fll of serum-free Grace's medium (Life Technologies Inc.. Gaithersburg. MD). Afterwards 10 pl Lipofectin plus 90 fll Grace's medium were added, mixed and incubated for 1 > minutes at CO0111 te111peraTllt'e.
~rI1C11 the transfection mixture was added drop-wise to Sf~ insect cells (ATCC CRL 1711 ) seeded in a 35 lllnl tissue culture plate with 1 ml Grace's medium without serum.
The plate was rocked back and forth t0 17111 the newly added solution. The plate w-as then incubated for S hours at ?7°C. After ~ hours the transfection solution was removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. The plate was put back into an incubator and cultivation was continued at 27°C for four days.
After four days the supernatant was collected and a 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).
Four days after serial dilution, the virus was added to the cells. After appropriate incubation. blue stained plaques are picked with the tip of an Eppendorf pipette. The agar containing the recombinant viruses was then resuspended in an Eppendorf tube containing 200 X11 of Grace's medium. The agar was removed by a brief centrifugation and the supernatant containing the recombinant baculovirus is used to infect Sf~ cells seeded in 3~ mm dishes.
Four days later the supernatants of these culture dishes were harvested and then they were stored at 4°C. A clone containing properly inserted hESSB I, II
and III was identified by DNA analysis including restriction mapping and sequencin~~. This was designated herein as V-AIM II or V-AIM II (aa 60-240).
Sf~ 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 (about 1 to about 3 ). Six hours later the medium was removed and was replaced with SF900 II medium minus methionine and cvsteine (available from Life Technologies Inc.. Gaithersburg). 42 hours later. ~ pCi of~-'''S-methionine and ~
pCi ''S-cysteine (available from Amersham) were added. The cells were further incubated for 16 hours and then they were harvested by centrifugation, lysed and the labeled proteins were visualized by SDS-PAGE and autoradiography.
E.aanrple 3: Clnnin~ anrl Expression irr Manrmnlian Cells Most of the vectors used for the transient expression of the AIM II protein gene sequence in mammalian cells should carry the SV40 origin of replication.
This allows the replication of the vector to high copy numbers in cells (c~.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 of the 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, HTLV1, H1V1 and the early promoter of the cytomegalovirus (CMV).
However, cellular signals can also be used (e.~,>., human actin promoter).
Suitable expression vectors for use in practicing the present im~ention include, for example.
vectors such as pSVL and pMSG (Pharmacia. Uppsala. Sweden), pRSVcat (ATCC 37152), pSV2dhIr (ATCC 37146) and pBC12M1 (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 CVI. African green monkey cells, quail QC 1-3 cells. mouse L cells and Chinese hamster ovary cells.

Alternatively. the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome. The co-transfection with a selectable marker such as dhii-. gpt. neomycin. hygromvcin allows the identification and isolation of the transfected cells.
The transfected gene can also be amplified to express large amounts ofthe encoded protein. T'he DHFR (dihydrofolate reductase) is a useful marker to develop cel l lines that carry several hundred or even several thousand copies of the gene of interest. Another useful selection marker is the enzyme glutamine swthase (GS) (Murphy cl al., l3inclac~m .l. 22,':277-279 (1991); Bebbington cn al..
l3iulTecJmvlo~~~ Ill: 169-17~ ( 1992)). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified genes) integrated into a chromosome. Chinese hamster ovary (CHO) cells are often used for the production of proteins.
The expression vectors pC 1 and pC4 contain the strong promoter {I,TR) of the Rous Sarcoma Virus (Cullen et crl.. Molecular and Cellarlcrr Bioloy, 438-447 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart el al., Cell -II:521-530 ( 1985)). Multiple cloning sites, e.y. , with the restriction enzyme cleavage sites l3amHI. ~~al and Asp718, facilitate the cloning of the gene of interest. The vectors contain in addition the 3' intron, the polyadenvlation and termination signal of the rat preproinsulin gene.
Example 3(a): Clonir:g acrd Expression in COS Cells The expression plasmid, pAIM II HA. is made by clonin~~ a cDNA
encoding AIM II into the expression vector pcDNAI/Amp (which can be obtained from lnvitrogen, Inc.).
The expression vector peDNAI/amp contains: ( 1 ) an E. coli ori~~in of replication effective for propagation in E. coli and other prokaryotic cells:
(2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3 ) an SV40 origin of replication for propagation in cukarvotic cells: (4) a CMV
promoter. a polvlinker. 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 tra~~ment encoding the AIM II protein and an HA tag fused in frame to its s' 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 cpitope derived from the influenza hemagglutinin protein described by Wilson el crl., ('ell 3'~ 767 ( 1984). The fusion of the HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
The plasmid construction strategy is as follows. The AIM II cDNA ofthe 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 11 in E. cvli. To facilitate detection, purification and characterization of the expressed AIM II, 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 ~' primer, containing the underlined I3amHI 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 ID N0:13).
The 3' primer, containing the underlined h?~aI site. a stop codon, 9 codons thereafter forming the hemagglutinin HA tag, and 33 by of 3' coding sequence (at the 3' end) has the following sequence:
5'GAT GTT CT'A GAA AGC GTA GTC TGG GAC GTC GTA TGG GTA CAC
CAT GAA AGC CCC GAA GTA AGA CCG GGT AC 3' (SEQ ID N0:14).
The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digested with I-IindIll and Xhol and then ligated. The ligation mixture is _11;_ transformed into E. coli strain SURE (available from Stratagene Clonin~~
Systems, 1 1099 North Torrey Pines Road. La .Iolla. CA 92037). and the transformed culture is plated on ampicillin media plates which then are incubated to allow growth of ampicillin resistant colonies. Plasmid DNA is isolated ti~om resistant colonies and examined by restriction analysis and gel sizin~~ for the presence of the AIM II-encoding fragment.
For expression of recombinant AIM I1. COS cells are transfected with an expression vector. as described above. using DEAE-DEXTRAN. as described, for instance. in Sambrook el crl., Molecular Clonin;~: a Laboratory Manual. Cold Sprin~~ Laboratory Press. Cold Spring Harbor, New York ( 1989). Cells are incubated under conditions for expression of AIM II by the vector.
Expression of the AIM 11 HA fusion protein is detected by radiolabelling and immunoprccipitation. using methods described in, for example Harlow el ul.
, Antibodies: A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press. Cold Spring Harbor. New York ( 1988). To this end. two days after transfection, the cells are labeled by incubation in media containing 'SS-cysteine for 8 hours. The cells and the media are collected. and the cells are washed and the lysed with detergent-containing RIPA buffer: 1 ~0 mM NaCI. 1 % NP-40. 0.1 SDS. i % NP-40. 0.5% DOC. 50 mM TRIS. pI-I 7.~. as described by Wilson cn crl.
cited above. Proteins are precipitated from the cell lysate and from the culture media using an I-IA-specific monoclonal antibody. The precipitated proteins then are analyzed by SDS-PAGE gels and autoradiography. An expression product of the expected size is seen in the cell lysate. which is not seen in negative controls.
Exantple~ 3(b): Cloning and Expressive in CHO Cells The vectorpC4 is used for the expression ofAIM II protein. Plasmid pC.' 1 is a derivative of the plasmid pSV2-dhlr [ATCC Accession No. 37146]. Both piasmids contain the mouse DHFR gene under control of the SV40 early promoter. Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transtected with these plasmids can be selected by growing the cells in a selective medium (alpha minus MEM. Life Technologies) supplemented w-ith the chemotherapeutic agent methotrcxate. The amplification of the DI-IFR genes in cells resistant to methotrexate (MTX) has been well documented (.~~ee. c.~..
Alt.
F.Vf., Kellems. R.M.. Bertino. .1.R.. and Schimke, R.T.. 1978, .1. Biol. Chem.
'?~3:13~7-1370, Hamlin, ,1.L. and Ma, C. 1990, Biochem. et Biophys. Acta.
1097:107-143, Page, M..1. and Sydenham. M.A. 1991. Biotechnolow Vol. 9:64-68). Cells grown in increasing concentrations of MTX develop resistance to the drug by overproducing the target enzyme. DHFR, as a result of amplification of the DHFR gene. If a second gene is linked to the DHFR gene it is usually co-amplilled and over-expressed. It is state of the art to develop cell lines carryin~~
more than 1.000 copies of the genes. Subsequently, when the methotrexate is withdrawn. cell lines contain the amplified gene integrated into the i chromosome(s).
Plasmid pC4 contains for expressing the gene of interest the strong promoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus (Cullen.
er al.. Molecular' and C'elhrlcrr Biolo~,rJ~. March 1985:438-447) plus a fragment i isolated from the enhancer of the immediate early gene of human cytomegalovirus l (CMV) (Boshart el al.. C'cll -11:521-530 (1985)). Downstream of the promoter are BcrmHI, Xficrl. and .Asp718 restriction enzyme cleavage sites that allow integration of the genes. Behind these cloning sites the plasmid contains the 3' intron and polyadenylation site ofthe rat preproinsulin gene. Other high efficiency promoters can also be used for the expression, c~.g., the human ~3-actin promoter.
t the SV40 early or late promoters or the long terminal repeats from other retroviruses. e.~~., HIV and I-ITLVI. Clontech's Tet-Off and Tet-On gene G 25 expression systems and similar systems can be used to express the AIM II
in a regulated way in mammalian cells (Gossen, M.. & Bujard. H. 1992. Proc. Natl.
Accrd Sci. LISA <fJ: ~~47-~s~ 1 ). For the polyadenylation of the mRNA other signals, c~.~,~., from the human growth hormone orglobin genes can be used as well.
Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co-transfection with a selectable marker such as gpt.

or hygromvcin. It is advantageous to use more than one selectable marker in the beginnin~~. c. fir.. G-118 plus methotrexate.
The plasmid pC4 is digested with the restriction enzymes BamHl and Asp718 and then dephosphorylated using calf intestinal phosphatase by procedures known in the art. The vector is then isolated trom a 1 % agarose gel.
The DNA sequence encoding the complete AIM II protein is amplified using PC R oligonucleotide primers corresponding to the ~' and 3' sequences of the gene. The 5' primer has the sequence:
5' GCT CCA G(~A TCC GCC ATC ATG GAG GAG AGT GTC GTA CGG C3' (SEQ ID NO:I ~) containing the underlined BcrmHl restriction enzyme site followed by an efficient signal for initiation of translation in eukaryotes, as described by Kozak. M., J. Mvl. Biol. l >6:947-9~0 (1987), and 22 bases (i.e., nucleotides 49-70) of the coding region of the AIM II protein shown in Figure (SEQ ID NO:I ). The 3' primer has the sequence:
5'GA CGC GGT ACC GTC CAA TGC ACC ACG CTC CTT CCT TC 3' (SEQ
ID N0:16) containing the underlined Asp718 restriction site followed by nucleotides complementary to nucleotides 770-795 of the AIM II gene shown in Figure 1 B (SEQ ID NO:1 ).
The amplified fragment is digested with the endonucleases BanrHl and A.sP718 and then purified again on a 1 % agarose gel. The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA lipase. E. colt HB

or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC4 using, for instance, restriction enzyme analysis.
Example 3(c): Cloning ar:d Expression of n» AIM ll N-tern:ina! deletion in CHO Cells The vector pC4 was used for the expression of AIM II mutant (Met{68) -Val(240) in SEQ ID N0:2) protein. The plasmid pC.'4 was digested with the restriction enzymes BamHI and then dephosphorylated using calf intestinal WO 99/4258. PCT/US99/03703 phosphatase by procedures known in the art. The vector was then isolated from a 1 °ro a~~arose gel.
The DN.a sequence encodin~_ the AIM I1 (aa 68-240) protein was amplified usin~~ PCR oligonucleotide primers corresponding to the ~' and 3' sequences of the gene. The following ~' primer was used:
5' GAC AGT GGA TCC GCC ACC ATG GTC ACC CGC CTG CCT GAC GGA
C 3' (SEQ ID NO: ~0) containing the underlined BamHl restriction enzyme site followed by an efficient signal for initiation of translation in eukaryotes, as described by Kozak, M.,.I. Mnl. Biol. l9<:947-950 (1987), and nucleotides 202-?~6 in the codin~~ region for the AIM Il polypeptide shown in Figure 1 A (SEQ
ID
NO: l ). 'l~he following 3' primer was used:
(Baml-II + stop codon (italics)) 5'-GGG GGA TCC TGA CAC CAT' GAA AGC
CCC G- 3' (SEQ ID N0:28) containing the underlined BamHI restriction site followed by nucleotides complementary nt 753-768 shown in Figure 1 B (SEQ ID
NO:1 ).
The amplified fragment was digested with the endonucleases BarnHI and then purified again on a 1% agarose gel. The isolated fragment and the dephosphorylated vector were then ligated with T4 DNA ligase. E. cvli HB101 or XL-1 Blue eel is were then transformed and bacteria were identified that contain the ti~agment inserted into plasmid pC4 using, for instance, restriction enzyme analysis.
The vector pC4/Ck~i8 (a pC4 construct wherein the Ck(38 signal peptide was first cloned into the pC4 vector with a Baml-II site at the 3' end of Ck~38 signal sequence) was used for the expression of AIM II mutant (Trp(80) -Val(240) in SEQ ID NO:2) protein. The plasmid pC4/Ck~38 was digested with the restriction enzymes BamHl and then dephosphorylated using calf intestinal phosphatase by procedures knowm in the art. The vector was then isolated from a 1 % agarose gel.

The DNA sequence encoding the AIM I1 (aa 80-240) protein was amplified using PC R oligonucleotide primers corresponding to the ~' and 3' sequences of the gene. The following .s' primer was used:
S' cgc GGATCC ~fGGGAGCAGCTGATAC 3' (SEQ ID N0:41 ) containing the underlined Buml-II restriction enzyme site followed by nucleotides 286-301 in the coding region for the AIM II polypeptide shown in Figure 1 A (SEQ ID NO:1 ).
The following s' primer was used:
5' egc GGATCC TCA CACCATGAAAGC 3' (SEQ ID NO:29) containing the underlined Bun21-11 restriction site followed by nucleotides complementary nt 771 shown in Figure 1 B (SEQ ID NO:1 ).
The amplified fragment was digested with the endonucleases l3uml-II and then purified again on a 1 % agarose gel. The isolated fragment and the dephosphorylated vector were then ligated with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells were then transformed and bacteria were identified that contain the fragment inserted into plasmid pC4/Ck~i8 using, for instance. restriction enzyme analysis.
CHO Cell Transfection:
Chinese hamster ovary cells lacking an active DI-IFR gene are used for transfection. S pg of the expression pC4 vectors described above are cotransfected with 0.~ pg of the plasmid pSV2-neo using lipofectin (Felgner et al., .supra). The plasmid pSV2neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including 6418. The cells are seeded in alpha minus MEM
supplemented with 1 mg/ml 6418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner. Germany) in alpha minus MEM
supplemented with I 0. 35. or ~0 nglml of methotrexate plus 1 mg/ml 6418.
After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations ofmethotrexate (~0 nM,100 nM. 200 nM, 400 nM. 800 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containin~~ even hi~~her concentrations of methotrexate ( 1 ~tM. 2 yM. ~ ~tM. 10 ~tM. 20 ~tlvl >. The same procedure is repeated until clones are obtained which grow at a concentration of 100 - 200 pM. Expression of the desired gene product is analyzed, for instance.
by SDS-PAGE and Western blot or by reverse phase HPLC analysis.
E.ranrple 3(d): Cloning and Expression of nn AlM ll N-terminal deletion in CHD Cells The vector pC4 was used for the expression of AIM II mutant (Met(68 )-Val(240) in SEQ ID N0:2) protein that includes a C-terminal Fc immunoglobulin region. In this construct. the Ck~i8 signal peptide was first cloned into pC4 with a Burral--II site at the 3' end of Ck~i8. The Fc fragment flanked by BunrHI
and Xhul sites was cloned into the vector resulting in pC4/Ck~38/Fc. The AIM-II
fragment was then cloned between the CK-X38 leader and the Fc fragment in the BcrmHI
site.
The plasmid pC4 was digested with the restriction enzymes BcrmHl and then dephosphorylated using calf intestinal phosphatase by procedures known tn the art. The vector was then isolated from a I % agarose gel.
The DNA sequence encoding the complete AIM Il (aa 68-2-IO) protein was amplified using PCR oligonucleotide primers corresponding to the S' and ~' sequences of the gene. The following ~' primer was used: 5' GAC AGT GGA
TCC GCC ACC ATG GTC ACC CGC CTG CCT GAC GGA C 3' (SEQ ID NO:
40) containing the underlined BamHI restriction enzyme site followed by an efficient signal for initiation of translation in eukaryotes, as described by Kozak.
M., .I. Mr>l. Biol. l X6:947-950 ( 1987), and nucleotides 202-226 in the coding.:
region for the AIM II polypeptide shown in Figure 1 A (SEQ ID 1v'O:1 ). The following 3' primer was used: (BumHl ) 5'-CJGG GGA TCC CAC CAT GAA
AGC CCC G-s' (SEQ ID N0:30) containing the underlined BcrmHI restriction site followed by nucleotides complementary to nt 7~3-768 shown in Figures I A and B (SEQ ID NO: I ) followed by the Fc immuno~~lobulin fragment having the following sequence:

-1?1-s'-GGGATCCGGAGCCC:~AATCTTCTGACAAAACTCACACATGCCCAC
CGTGCCCAGCACCTGA.-~TTCGAGGGTGCACCGTCAGTCTTCCTCTTC
CCCCCAAAACCCAAGG.4CACCCTCATGATCTCCCGGACTCCTG.AGG
TCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAA
GTTCAACTGGTACGTGGACGGCGTGGAGGTGCA'hAATGCCAAGACA
AAGCCGCGGG.AGGAGCAGTACAACAGCACGTACCGTGTGGTCAGC
GTCCTCACCGTCCTGC.aCCAGGACTGGCTGAATGGCAAGGfIGTACA
AGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAAC
CATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACC
C'fGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGA
CCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTG
GGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC
CGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCG
TGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGT
GATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCC
CTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT-3' (SEQ ID N0:31 ).
The amplified fragment was digested with the endonucleases I3umHl and then purified again on a 1 % agarose gel. The isolated fragment and the dephosphorylated vector were then ligated with T4 DNA lipase. E. coli HB 101 or XL-1 Blue cells were then transformed and bacteria are identified that contain the fragment inserted into plasmid pC4 using. for instance, restriction enzyme analysis.
CHO cell transfection:
Chinese hamster ovary (CHO/dhfc -DG44) cells were transfected with the expression vector (pC4/spCK~38/Fc/AIM II) using lipofectin. Recombinant clones were isolated by growing the cells in MEM alpha selective medium with S%
dialyzed fetal bovine serum (DiFBS). 1 % penicillin/streptomycin (PS). 1 mg/mL
geneticin (G418) and 10nM methotrexate (MTX). High expressing clones. which _ 1 ~'7 -were confirmed by screening recombinant clones usin~~ a BIAcore method (see, below for more details). were then individually amplified by increasing stepwise the concentration of MTX to a final concentration of 100uM. The high e~pressin~ clones were used for the production of AIM II-igG l fusion protein in a microcarrier CHO perfusion bioreactor.
CIIO.AIi\-I Il-IgGI cells were grown on Cvtode~ 1 microcarriers (Pharrnacia Biotech. Upsala, Sweden) in HGS-CI-10- 3 medium containing 1 ultra-low IgG FBS. The cells grown in multiple microcarrierspinners were scaled up to a lOL microcarrier perfusion bioreactor. The perfusion bioreactor was operated cominuously for 27 days and during that period of time, 90 liters of microcarrier-flee supernatants containing AIM II-IgGI fusion protein were harvested. The supernatants were clarified through a filtration process using 0.2 pm sterile filters and stabilized by adding SmM EDTA. The clarified supernatants were loaded onto an affinity column to capture AIM II-IgG 1 fusion protein.
Purification of AIM II-IgGI fusion protein The AIM II-IgGI fusion protein was purified from 15I_ of CHO
conditioned media. The conditioned media was loaded onto a Protein A HyperD
(54mL bed volume. BioSepra) affinity column at a flow rate of 30mL/min at 10 °C
on a BioCad 60 ( I'erSeptives Biosystems). The column was preequilibrated with ?SmM sodium acetate. pI-18 and 0.1 M NaCI. After loading, the column was washed with 3 column volumes each of0.1 M sodium citrate, pH~ and 0.1 M NaCI
and 0.1 M sodium citrate, pI-I 2.8 and 0.1 M NaCI. The peak fractions containing AIM II-IgG fusion protein were determined by SDS-PAGE analysis and pooled.
The identity of the purified protein was confirmed by N-terminal sequence analysis. The final protein yield was about 9 mg/L condition media.

_l~;_ E_wmple 4: AIA~ II E.rpression Coustrr~cts Full-length constructs:
(a) pCMVsport: TheeukaryoticexpressionvectorpCMVsportcontains nucleotides encoding the AIM-II ORF from Met( 1 ) to Val(240). The plasmid construction strategy is as follows. The :AIM II cDNA of the deposited clone is amplilicd using primers that contain convenient restriction sites. Suitable primers 111Clllde the following which are used in this example. The s' primer, containing the underlined ,SuII site, an AUG start codon, nucleotides ~ 1-69 in the coding re~~ion ofthe AIM II poiypeptide (SEQ ID NO:1 ) and has the followine seduence:
5'-GGG GTC GAC GC'C'.-1 TC' ATG GAG GAG AGT GTC GTA CGG-3' (SEQ ID N0:32).
The 3' primer, containing the underlined No~I site. nucleotides complementary to nucleotides 753-767 in SEQ ID NO: l and a stop codon and has the following sequence:
5'-GGG GCG GCC GCG C'CT TC:,9 C'AC CAT GAA AGC CCCG-3' (SEQ ID N0:33).
The PCR amplified DNA ti-agment is digested with Sell and ,\'o~I and then gel purified. The isolated fragment was then ligated into the .Scrll and l~'otI
digested vector pCMVsport. The ligation mixture is transformed into E. coli and the transformed culture is plated on antibiotic media plates which are then incubated to allow growth of the antibiotic resistant colonies. Plasmid DNA is isolated from resistant colonies and examined by restriction analysis and gel sizing for the presence of the AIM II encoding fragment.
For expression of the recombinant AIM II, eukaryotic cells such as COS
or CHO are transfected with the expression vector, as described above. using DEAE-DEXTRAN as described above in Example 3. Expression of the AIM II
recombinant protein is detected by the methods described above in Example 3.
(b) pG 1 SamEN: The retroviral expression vector pG 1 SamEN encodes the AIM-II ORF from Met( 1 ) to Val(240). The pG 1 vector is described in Morgan. R. A., cu ul.. :\~rrcl. :9cicls Re.r. Z0~<):1293-1?99 ( 1992) and is similar to the LN vector (Miller. t1. D. and Rosman. G. ,1.. Bimc~chr7iguc~.v :980-990 ( 1989)), but has additional cloning sites. The plasmid construction strategy is as follows. The AIM II cDNA of the deposited clone is amplified using primers that contain com~enient restriction sites. Suitable primers include the following which are used in this example. The ~' primer, containing the underlined I~~cuI
site. and an AUG start codon. nucleotides 51-69 in the coding region for the AIM II
polypeptide (SFQ ID NO:1 ) has the following sequence:
~'-GGG GCCi GC'C GCG C'C;9 TCt9 TGG AGG AGA GTG TCG TAC
GG-3' (SEQ ID N0:34).
The 3' primer. containing the underlined ScrII site. nucleotides complementary to nucleotides 75 3-768 in SEQ ID NO:1 and a stop codon has the following sequence:
5'-GGG GTC GAC GC'C.' TTC:9 CAC CAT GAA AGC CCC G- 3' (SEQ ID
N0:35).
The PCR amplified DNA fragment is digested with Sul1 and ~'mI and then gel purified. The isolated fragment was then ligated into the Sul1 and NoU
digested vector. The ligation mixture is transformed into E. coli and the transformed culture is plated on antibiotic media plates which are then incubated to allow growth of the antibiotic resistant colonies. Plasmid DNA is isolated from resistant colonies and examined by restriction analysis and gel sizing for the presence of the AIM II encoding fragment.
Por expression of the recombinant AIM II. eukaryotic cells such as COS
or CHO are transfected with the expression vector, as described above, using DEAF-DEXTRAN as described above in Example 3. Expression of the AIM II
recombinant protein is detected by the methods described ahove in Example 3.

WO 99/x2584 PCT/US99/03703 -l~~_ 2. h-terminal deletion constructs:
(a) p(:1/c1c~38: The eukaryotic expression vector encodes the AIM-II mutant (Gln(60) to Val(240) in SEQ ID N0:2)(AIM-2 (aa60-240)) and was secreted under the direction of the human Ck-X38 signal peptide. The pG 1 vector is S described in Morgan. R. A., cn crl., Nticl. .-lciclo Rc~.s. 20(6):1293-1?99 ( 199?) and is similar to the LN vector (Miller. A. D. and Rosman. G. .l.
~l(Jlc'C'I7I7lc~LlL'.S' ?:980-990 ( 1989)). but has additional C1o11117g sites. 'The plasmid construction strategy is as follows. The AIM II cDNA of the deposited clone is amplified using primers that contain convenient restriction sites. Suitable primers include the following which are used in this example. The S' primer. containing the underlined Nml site, nucleotides in the coding region for the AIM II polypeptide (SEQ ID
NO: l ) and an AUG start colon has the following sequence:
S'-GGG G CG GCC GCG C'C:9 TC'A TGA AGG TCT CCG TGG CTG CCC TCT
CCT GCC TCA TGC TTG TTA CTG CCC TTG GAT CGC AGG CAG CTG
CAC TGG CGT-3' (NoJI + Kozak + CK-~38 leader (double underline)) (SEQ ID
N0:36).
The 3' primer, containing the underlined .SuII site. nucleotides complementary to nucleotides 7S3-768 in SEQ ID NO: l and a stop colon has the following sequence:
S'-GGG GTC GAC TCA CAC CAT GAA AGC CCC G-3' (SEQ ID N0:37).
The PCR amplified DNA fragment is digested with SuII and NolI and then gel purified. The isolated fragment was then ligated into the SaII and Noil digested vector pG 1. The ligation mixture is transformed into E. coli and the transformed culture is plated on antibiotic media plates which are then incubated 2S to allow growth ofthe antibiotic resistant colonies. Plasmid DNA is isolated from resistant colonies and examined by restriction analysis and gel sizing for the presence of the AIM I1 encoding fragment.
For expression of the recombinant AIM II, eukaryotic cells such as COS
or CHO are transfected with the expression vector. as described above. using _ ] 76_ DEAF-DEXTRAN as described above in Example 3. Expression of the AIM II
reCOlllblnallt protein is detected by the methods described above in Example 3.
(b) pHE-t: Plasmid pHE4 is a bacterial expression vector containing a strong synthetic promoter with two lac operators. Expression from this promoter is regulated by the presence of a lac repressor. and is induced using IPTG or lactose.
The plasmid also contains an efficient ribosomal binding site and a synthetic transcriptional terminator downstream of the AIM II mutant gene. The vector also contains the replication region of pUC plasmids and the kanamvcin resistance gene.
The AIM-11 N-terminal deletion mutants were constructed according to the following scheme. The AIM II cDNA of the deposited clone is amplified using primers that contain convenient restriction sites. Suitable primers include the following which are used in this example.
For the AIM II (Thr(70) to Val(240j) polypeptide in SEQ ID NO:?, the 5' primer, containing the underlined Ndol site, and an AUCJ start codon, nucleotides 256-271 in the coding region for the AIM II polvpeptide (SEQ ID
NO:1 ) has the following sequence:
5'-cgc CATATG A CCC,'GCCTGCCTGACG-3' (SEQ ID N0:42).
For the AIM II (Ser(79j to Val(240)) polypeptide in SEQ ID N0:2. the 5' primer, containing the underlined Ndel site, and an AUG start codon, nucleotides 283-310 in the coding region for the AIM I1 polvpeptide (SEQ ID
NO:1 ) has the following sequence:
5'-cgc CATATG A GC TGGGAGCAGCTGATAC-3' (SEQ ID N0:43).
For the AIM II (Ser(103) to Val(240)) polypeptide in SEQ ID N0:2, the 5' primer, containing the underlined Ndel site, and an AUG start codon, nucleotides 355-373 in the coding region for the AIM II polypeptide (SEQ ID
NO: l ) has the following sequence:
5'-cgc CATATG A GC AGCTTGACCGGCAGCG-3' (SEQ ID N0:44).

_ 1 ?'7-The follovvin~~ 3' primers can be used to construct the aforementioned I~-terminal deletions:
The s' primer. containing the underlined As7~718 site. nucleotides complementary to nucleotides 7~3-768 in SEQ ID NO:1 and a stop codon has the following sequence:
5'-c~~c GG'hACC TTA CACCATGAAAGCCCCG-~' (SEQ ID N0:4~).
The PCR amplified DNA fragment is digested with .~'deI and AsJ~718 and then gel purified. The isolated fragment was then ligated into the appropriately digested p(-IE4 vector. The ligation mixture is transformed into E. ruli and the IO transformed culture is plated on antibiotic media plates which are then incubated to allow growh of the antibiotic resistant colonies. Plasmid DNA is isolated from resistant colonies and examined by restriction analysis and gel sizing for the presence of the AIM II encoding fragment.
For expression of the recombinant AIM LI N-terminal deletion. bacterial cells are transfected with the expression vector, as described above in Example 1.
Expression of the AIM I1 recombinant protein is detected by the methods described above in Example 1.
Exn»rple S: Biological Cltnracterizatio» of the AIM ll Polypeptide The following set of experiments provides the biological characterization of the AIM II protein and demonstrates that AIM II has potent anti-tumor activity in vivo and 111 hlll'O.
A. AIM II is Itighly expressed irr activated lymphocytes 6»t »ot i»
ca»eer cells Northern blot analyses demonstrated that the AIM II mRNA is approximately 1.9 kb in length and is expressed predominantly in spleen, brain and peripheral blood cells. AIM 1I is also detectable to some extent in prostate.
testis, ovary. small intestine. placenta. liver, skeletal muscle and lung. AIM
Il _I7g_ message was not detected in fetal tissues. many endocrine glands and tumor lines of non-hematopoietic and 111 elOld origin.
RT-PCR assays were performed to investigate expression of .AIM II in activated vs. resting PBMC. Fresh PBMC including mixture of T cells, B
lymphocytes. NK cells. monocytes and granulocytes express the AI11 II mRNA
which is consistent with Northern blot analysis. No expression was found in resting PBLs as mixture of T. B and NK cells. Jurkat cells (restin~~ or activated) or K~62 cells. Increased expression of AIM II was found in activated PBLs.
CD 3+, CD4+ T-cells, CD8+ Tumor infiltrating lymphocytes (TIL). ~~ranulocytes.
and monocytes. Additional RT-PCR analyses demonstrated the presence of AIM II mRNA in LPS-activated neutrophils and PMA-stimulated L'937 cells.
Interestingly, expression of AIM II was not detectable in various cancer cell lines derived from breast, prostate or ovary, except in one human breast epithelial-derived, non-tumorigenic cell line MCA-1 OA cells. In addition, no expression of AIM I1 was found ii-om three breast cancer samples examined.
B. Constitutive e_apressiou ofAIMlI resulted in growth inhibition under serum starvation or treatment witlt IFNy To investigate the biological function of AIM II, the AIM II gene was stably transduced into human breast carcinoma cell line MDA-MB-231 using a retroviral vector. Expression of the AIM I1 gene in these cells was confirmed by Northern blot analyses. In addition, MDA-MB-231 cells expressin~~ the drug resistance gene Neo were used as control in this study. No difference in the growth rate in vilnn was observed within AIM II transfectants (MDA-MB-231 /AIM II) compared with that of the parental cells or vector control transfected cells (MDA-MB-23 l/Neo), when these cells were cultured in medium containing 10% FBS. However. when the serum concentration was reduced to I %, there was 80% growth inhibition (Fig. 4A) for the MDA-MB-231 /AIM 11 cells, but not for the parental or vector control MDA-MB-231 cells. A dose-dependent growth inhibition with a different amount of serum has also been observed.

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Wild type MDA-MB-?31 cells grew to a very high den.siry with typical pile-up feariues in either lU% or 1 % senun (Fig. 4A). Morphological changes were noticed in the MDA-MB-2311AIM 11 cells, ~~ith most cells floating into the medium and keeping a single layer growth pattern throughout the culture. No chanEes of morphology were found in the vector control MDA-MB-231 cells.
Growth izablibition of AIA9 II expressing ivIDA-MB-231 cells was further exazx~.ined with in soft agar colony assay. As shov~r. in Fig. 4B, 84%
reduction of colony formation was found In the MDA-MH-231lAIM II cells as compared e~~ith that of the pa.-ental or nectar control cells. Tretltment with 25 u/ml of IFNy can also cause 8tJ'/o growth inhibitiozr of AIM II expressing'vIDA-MB-231 cells, whereas in the parental or vector control cells, there is only 24-30°,'o inhibition.
Thus, AfI~I II expressing cells demonstrated enhanced sensitivity towards cytotoxicity mediated by cytokine IFN~y.
1 S C. Enhanced apor~taxir ire AflyII1 ex~rressin~ cells.
.Annexizi-V FACS analyses were performed to investigate underlying mechanisms of grow-th inhibition of AIM II expressing cells. In the presence of 10% sen:ri, there are less than 2% apoptotic cells in all three cell lines.
After 48 hou.~ incubation in reduced serum (0.5% FBS)~ the apoptotic population of the 2p MDA-MH-231 cells showed a three-fold increase, up to 8°~°.
There is little or:~o increase of apoptosis in the parental or vector control MDA-MB-231 cells (Figures 5A-C). Induction of apoptosis was further confirmed by DIvA
fragmentation assay afMDA-MB ~31i'W'T, MDA-'~18-431/rTeo andN~A-MB-2311AIM II cells :n 1~°/a and 0.5% serum, with cr withet:t I'aclitaxel (taxol).
ZS Fragmented DlvlA was only seen in the AIM II expressing MDA-M>3-23I cells, especially in 1% serum. Vfhen AIM II expressing cells v~ere treated v~zth Pacditaxei (taxol), there was much mare fragmented DNA observed than seen in parental o; vector control cells. Thus, the data suggest that AII~I lI
expression can trigger apoptosis of NIDA-lVlB-23I cells under serum starvation or with the 3C addition of IFNy or taxol.
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-13a-D. Potent in vivo anti-tumor activities of ALY11I.
We have evaluated the effects of AIM II transduction on the t':mor growth in vivo. When MDA-N1B-231 cells were inoculated into the mammary fat pads, AIM II expression sigruificantly inhibited tumor formation of h~A-YIB-231 in nude mice, mltereas the vector coatrol MDA-MB-231,~eo cells showed no change iz: tumor Growth as compared »~ith that of the parental MI3A-MB-231 cells dig. 6th). Similar tuzzaor suppression in the MDA-f~IB-?311AIM II cells was also demonstrated in SCID mice. A histological examination ofthe tumors from AIM il; expressing MAA-MB-231 cells or those from parental or vector J 4 control cells was performed. Parenial or vector control MDA-MB-X31 cells formed a large solid tumor zz~ass filled with predominantly tumor :ells with little or nu cell~,tlar infiltrates. In contrast, there was extensive necrosis observed even in small residual tuzzaors formed by the MD A-MB-231!AIM II cells in nude mice.
Furthermore; in A IM II expressing tumors, there is an significant increase in ~tumber of infiltratir_g neutropiiil cells, The average num'oer of neutrophiis (mean + S. ~.) per mmz tuzr~or size in wild type. Neo control, and AIM II transduced MDA-MB-231 tumors were 101= 2f,' i t 16 Goad 226 +38, respectively, based on the immunohistological staining using Gr-1 rnAb (lSharMingen, San Diego, CA).
?0 The in~hzbitar~~ ~2ffect of AIM II on tumor suppression was further validated in the syngeneic marine tumor r;~odel. Loral expression of AIM II in MC-38 marine colon cancer ceps resulted in complete suppression of tumor faz~atic~n in ~ out of 10 C57BL!6 mice (Fig. 6B), Local production of AI'VI II
was also dramatically prolona~ed the survival ofmice bearing MC-38 tumors, All animal experiments were repeated three times and similar results were obtaizted.
injection of A.IM iI-expressing tumor cells did not cause gross abnormalities inthe nude mice, SCID mice or C57BL!6 mice, such as weight loss or hepatic iniury, during the experimental period. This indicates that locally produced AIM II e~certs a potent anti-tumor effect without inducing systemic toxicity.
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E Expression and Cytatoxicily of a sotuble.4~~l.ll protein In order to study the activities of the ATil~t II protein, a recombinant soluble form of AIM II protein (sgl:N II1 ulnas produced by transient transfecting 293T cells with a construct pFlag :AIIvi II. This construct encodes the extracel lular domain of AIM II, but Lacking, the tsansmembranieportion of AIM
II.
(FiB. 7A) A single ZO kDa pol3~peptide (sAIM iI) carp be purified from the conditioned medium of pFlae-AIM II transduced 293T cells with anti-Flag monoclonal antibody. The proliferation of breast cancer :1~_sDA-MB-231 cells were inhibited in response to the treatment of this soluble AIM II protein, at a dose dependent manner Figures ?B-C. Addition of IfNy, at 10 uhux or 50 ulmh dramatically er~t:anced cytoto~.icity of the soluble AIM ti protein. IIrNy alone showed little activity ontlie MDA-MB-231 cells Figwes 7B-C. 'Fhi~ is consistent with previous report that MDA-IvIB-231 cells iJ resistant to single cytokine such as T?<'F or IFNy treatment.
I S A series of nor.'nal at:d cancer r~11 lines were tested for their sensitivity to the cy~tc~ta7cic effects of soluble Ah1-I II protein at sub-optimal concentration (SG
ngtrnl j in the presence of 10 ulml of INFy. As shown in Fig. 8C; cells from h~A-130, MCF-'~, I-IT-29 are se;~.sitivs to t~'~e cytotoxic effects of AIM II, whereas cells from LT9;iT, MC3-1, SW480, MCF-LOA are resistant to A1M II-r:~ediated cell killing. Anion: all the cell Lines tested, colon adenocarcinomm cell line FiT-''9 is the most sensitize; w;~un 1C" less than lnglml_ It has been shown that I3T'-29 is very sensi~.ve to 7 i~lF, Fas or lymphotoxin a receptor mediated killing in the presence of TFNy.
F. Boih L~',fiR and T~2 are req~sired for,41M11 induced growth inkibition of carfcer cells.
AIM II was originally identified from an activated T-cell cDNA librtuy but does not induct apoptosis in iyrnphocyte cell lines. Lfsinp; the RT-1'CR
analyses. all lyzxaphopoiet:c cells examined showed no expression of LT~3R, but TR2 expression was found in all these cells, especially in acti~~ated lurkat cells or PBLs. This is consistent with the previous rcpor~~.e that peripheral lvmphorytes AMENDED SHEET
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do not expz~ss the L'T~3R, while TR2 expression is associated with T-cell aCtlv atron.
Cell suzface expression ofthc LT/iR and TRZ in a series ofhuman cancer ails was examined using monoclonal antibodies against the LT~3R or TR2 by S FRCS analysis- As shown in Figures $A-H, high levels of both receptors were found on the MDA-MB-231, and HT-29 cells, whereas MC3-1 cells do not express 'fI?,2 and Jurkat cells do rot express LT~i~R. Figure SL summarizes surface expression of both receptors in all the cell lines examined. CeII
lines that express only one of the receptors, such as 3urkat or MC3~-1 are resistant to the c5~totoxicity of AL~Vl II. Takan together, these data suggest that AIM II-mediated growth inhibition in tumor cells may require both L'f ~R and TR2 receptors.
while cells expressing only one of the receptors is not sufficient to mediate cell kihing.
To ftuttter demonstrate that the AIM II is a relev ant ligand fox both LT~iR
and ~'.'R.?, receptors and the importance of both receptors in AIM II mediated tumor cell growth inhibition, the Flag-tagged AIM II protein was incubated with MDA-MB-Z31 or FIT-29 ctlls. then FRCS analyses were carried out using anti-Flag mAb. As shown in Figures 8I-K, there is a positive shift in binding ef MDA-MB-231 or I-IT-29 ells with Flag-tagged soluble AIM TI protein, The specificity of binding was further confi-med by pry-insrubation of LT~3R-Fc or TR2-Fc fusion p: otein with a soluble.AIM II-flag protein in ttie same cells, which effectively blocked binding of both receptors (Figures $I-K), The importance of the inwolvemert of both LTpR and Tfi? in the AIM II-rn.ediatc,~d cytoto:cic.ity toward tumor calls was ftu-ther supported by the data obtainEd from the in vitro growth assays: sAIh~i II-mediated cytotoxiviy of HT-29 was abolished by the addition of LT~3R-Fc or TR2-Fc f~ision protein in a dose-depended mannsr whereas the i,T~R-Fc or ?R2-Fc fusion protein itself showed no e~iect on cell gov~rth (Figure 8M). Ir. addition, in a similar assay, sAIM 11 was unable to bind to othe: members of TNFR, such as TriFRi, Fas, DR3 or DR14.
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In addition, co-culture of MDA-MB-231IWt or HT-29 cells with MD A-M$-2311?~1~I 1,I cells resulted in killing of the MDA-VIH-231,'Wt or wild type HT-29 cells. However, conditioned media collected from the co-culttued NLDA-biB-231/AIM II er MC-38iAIM IJ cells showed ro inhibitory effect on the in vitro proliferation of HT-29 calls. The results indicated that the natural AIM
II
protein may not be cleaved and secreted into the medium. Thus, the mernbrane-bound AIM II is functional in cells which express appropriate surface receptors such as MDA-MB-231 ar I-iT-29. Taken together, this data suggests that the.
Alhi II- mediated grorwttt inhibition of tumor cells may require both LTpR and la TI~2 receptors, while cells expressing only one of the receptors is not suff-icient to Irediate cell h-illin~.
G. Ejfe~cts vf.4lM.II on the lymphocytes AIIvI Ii was oziginally identified from an activated T-cell cDNA library 1.5 but does not induce apoptosis in Lymphocyte cell Lines. L,rs'tng RT-PCR
analyses, all lympltopoietic cells examined showed no expression of LT~iR, but TR2 was positive in all tl-~ese cells, especially in actwated .Turkat cells or PBLs.
This is -consistent with previous reports that peripheral lyznphocytcs do not express the LT~3R while TR2 expression was associated wiab T-;,ell activation.
20 To im~estigate whether the membrane-bound AIM II exerts different activities on the lymphocytes, co-culture experiments of TILI200 calls with ~iDA-MB-231;'t,.iM II cells was carried out. TILi20Q is a CDT (995; tumor in.~'iltraring Iyrnphocyte line expressing a high level of Fas. The membrame-bound AIM II did not induce apoptosis of TIL 1200, whereas the addition of Fns 25 antibody t;i~gered 90% of TIL12QU undezgone apoptvsis. Similar results were obtained ~~ith fresh TIL, cells or Juri:at cells.
Furthetzzlore, several Iyrnphoid cell limes and pBLs were screenedfortheir responsiveness to the soluble AIM II protein. No cytotoxicity of AIM II was shown in Jacket cells (either resting or CD3 mAb activated}, K552 cells, or 30 TILI20f3 (tumor in~~iltxating Lymphocytes)- PBMC (fresh or IL2lCD3 mAb activated) (Figure 8L). In contrast, tc~atment of PBLs with sAIM II, resulted in activation of TR2 oxpressin~ T cells as demonstrated by release of IFNy(Fig.
9).
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WO 99/4258. PCT/US99/03703 Discussion In the lore~~oing experiments. the biolo~~ical functions of AIM II and its possible mechanisms of action as a novel ligands of LT~3R and TR2 have been characterized. The results demonstrate that the AIM II protein exhibits potent cytotoxicity primarily in transformed tumor cells both ira rilro and in giro.
while at the same time. activating lymphocytes. The biological activities of AIM II
in oilro and in riw clearly distinguish AIM Il from other known members of the TNF/Fasl. family in several ways including binding to two distinct signaling pathways: LT(3R and TR2. Since the ability ofAIM ll expression to inhibit tumor growth vyas demonstrated in both xenographic (immunodeficient) and syngeneic (immunocompetent) models, the results suggest that the T-cell mediated tumor specific response may not be an essential factor for the primary tumor rejection in this study.
Activation of the TNF receptors family can directly induce cell proliferation, or differentiation or death. The foregoing experiments show that AIM Il expression resulted in grovvrth inhibition and apoptosis in the human breast carcinoma cell line MDA-MB-231 in conjunction with serum starvation, or addition of IFN~y. Induction of apoptosis appears to be the primary cause for the growth inhibition in vitro as shown in Annexin-V FACS analysis and DNA
fragmentation. The morphology and growth pattern of MDA-MB-231 /LT-y cells suggest involvement of some loss of cells adhesion. Browning el ul. have shown that Fas activation led to rapid cell death ( 12-24 h), TNF effects requires 24 h and LTcx 102 heterotrimers were slowest (2-3 days) in induction of apoptosis for HT-29 cells. Lysis of the LTyR and TR2 expressing MDA-MB-231 and I-IT-29 cells in response to the treatment with the soluble AIM II protein showed similar slow effect. i.e. at least 3-5 days. Substantial cell lysis does not occur even after 3-4 days for some cell lines. The dynamics ofaction of AIM II are more similar to LTaI ~i2 heterotrimers.

_I3~_ AIM II was originally identified from a human activated T cell library by screening of seduence hOlllolOg~' with cvsteine-rich motif of the T\F:'Fas Iigand and receptor superfamily. Like other TNF-related ligands. AIM II is a type-II
transmembrane protein with C-terII1111115 011 the exterior cell surface. a single S transmembrane domain, and a short cvtoplasmic tail. As predicted.
transduction of a full-length cDNA of AIM II gene resulted in cell surface expression of a protein which binds to two receptors as demonstrated in FACS analyses. A
soluble AlM II protein is sufficient to bind to both receptors and trig~~er cytotoxic effects on the target cells. However in the transwell co-culture experiment, where two type of cells shared the culture medium but are physically separated, cytotoxicity from the AIM II expressing MDA-MB-?3 i cells towards the wild type MDA-MB-231 or I-I'r-?9 cells was not observed. In the direct co-culture assay, membrane-bound AIM II effectively mediated killing from close contact.
Thus, it seems that natural AIM II protein may not be a secreted protein.
Fluorescence in situ hybridization (FISH) localized AIM II gene to human chromosome I G. band p 1 I .2. The AIM II position is in close proximity with Core binding protein. sulfotransferase, syntaxin 1 B, retinoblastoma-bindin~~
protein 6, zinc finger protein 44, cell adhesion regulator and Wilms tumor-3 gene. Genes encoding other known TNF ligands such as TNF, LTa, and LT~3 are tightly linked on human chromosome 6 within the major histocompatibilitv complex {MHC) sandwiched between the class iil and HLA-B locus.
Both LT~iR and TR2 lack the death domain. Thus, the demonstration of AIM I1 binding to both LT~3R and TR2 is intriguing. Although LT~iR and TR2 could activate common signaling pathways via association with TNFR-associated factors (TRAFs). AIM II-LT~3R and AIM II-TR2 interactions may trigger the distinct biological events. As shown in this Example, expression of AIM Il leads to the death of cells expressin~~ both LT(3R and TR? while activate lymphocytes which expressing only the TR2 receptor. Signaling through the LT~3R activates a TRAF3-dependent pathway. In contrast, AIM II-TR2 interaction probably elicits stimulatory responses of host immune system through TRAFs (TRAFI, TRAF?. TRAF3 and TRAH'~). This AIM II dual si~nalin'~ hypothesis is further supported by the distinct tissue and cell expression patterns of LT(3R and TR?.
LT~iR is prominent in tumor and other epithelial cells. but is absent on the T
and B cells. In contrast. TR? is abundantly expressed in comparable levels in resting and activated T cells, B cells and monocytes and granulocyte. Hence, AIM II
probably plays critical roles such as induction of apoptosis and immune activation and, therefore, may have an therapeutic application for cancer.
The LT(3R was originally described as a transcribed sequence encoded on human chromosome 1?p. a member of the TNFR superfamily. The LT~3R is implicated as a critical element in controlling lymph node development and cellular immune reactions. It has been showed that LT~3R is expressed in a variety of tissues and cell lines including tumor lines. Unlike other members of the TNFR
family, LT~iR is not expressed by T nor B lymphocytes. Activation of LT~iR by using recombinant LTa 1 X32 heterotrimers or by cross-linking with immobilized antibodies, induces the death of adenocarcinoma cell lines and production of chemokine IL-8 and RANTES. even though LT(3R does not contain the death domain in its cytoplasmic region.
TR2 is expressed in multiple human tissues and shows a constitutive and relatively high expression in hemopoietic lineage cells including resting and activated CD4+ and CD8-+ T cells. B cells, monocytes and neutrophils. The TR2 cytoplasmic tail does not contain the death domain seen in the Fas and TNFR-1 intracellular domains. and appears to be more related to those of CD40 and 4-1 BB. Signals through 4-1 BB and CD40 have been shown to be co-stimulatory to T cells and B cells, respectively. A TR2-Fc fusion protein inhibited a mixed lymphocyte reaction-mediated proliferation. in contrast to Fast and TNF, which trigger apoptosis. All the hemopoietic derived cells tested expresses the TR?
receptor but are resistant to AIM II mediated killing obsen~ed in the tumor cells.
This indicates that TR2 alone does not mediate death signal. However. since all cancer cells examined expressed both LT~3R and TR?, it remains to be elucidated whether both AIM Il-LT(3R and AIM II-TR2 signaling contributes equally for the AIM 11 mediated cvtotoxicity in tumor cells. Vve also can not exclude the possibility that AIM II interacts with other known or unknown death receptors such as DR3. DR~t and DRS, although soluble AIM II does not bind to DR3. DR4 and DRS in an in rilrv binding assay.
The dose-limiting toxicity of TNF and cytotoxicity of Fast for T-cells limits their clinical application. Treatment with AIM II could be alternatively attractive approach since AIM II trigger the stimulatory signal rather than the death signal to the host immune cells which expressing the TR2 but lacking the LT(3R. AIM II has the ability to selectively induce death of tumor cells probably through LT~iR and TR2 and at the same time can trigger secretion of IFNy from lymphocytes apparently through the TR2 signaling pathway. This model thus demonstrates that AIM II is not only an attractive candidate for the future development an anti-cancer agent, but more importantly, it provides an novel system, distinct from the previously defined TNF or Fas system, for the further understanding of the signaling pathway of members of TNF ligand-receptor interactions.
Methorls Molecular cloning of AIM II full lengtlt gene.
A database containing more than one million ESTs (expression sequence tags) obtained from over 500 different cDNA libraries has been generated through the combined efforts of Human Genome Science Inc. and The Institute for Genomic Research using high throughput automated DNA sequence analysis of randomly selected human eDNA clones. Sequence homology comparisons of each EST were performed against the GenBank database using the blastn and blastn algorithms. ESTs having homology to previously identified sequences (probability equal or less than 0.01 ) were given a tentative name based on the name of the sequence to which it was homologous. A specific homolo~~v and motif~search using the conserved amino acid sequence, GLYLIYSQVLF (SEQ ID
N0:46). of the TNF/Fas ligand family against this human EST database revealed several EST having >~U% homology. One clone containing GYYYIYSKVQL
(SEQ ID N0:47) from human activated T cell library was selected. This EST was sequenced on both strands to the 3' end. Its homology was confirmed. The initial clone lacks the 5' portion of the gene in comparison to other members of TNF
family. To obtain the full len;~th sequence. a nested PCR reaction was carried out using two gene specific oli~~onucleotides and two vector-specific primers. An additional 72 nucleotides at the ~' end was obtained. The full length sequence was then cloned into the vector pCMVsport 2.0 (Life Technologies Inc.. Rockville, MD).
Northern blot nrralysis.
Human multiple tissue Northern blots (Clontech. MTN blots, #7759-1 and #7760-1 ) were probed with a ''P-labelled AIM II full length cDNA according to the vendor's instructions. The blots were hybridized overnight in Hybrisol solution (Oncor). preheated to 42 °C before use. followed by two subsequent washes in 2X
SSC/0.1 % SDS and 0.2X SSC/0.1 % SDS at 42 °C and visualized using a PhospholmagerT~~ (Molecular Dynamics Co.).
In situ Irybrirlizrrtion arrd FISH detection.
To determine the precise chromosomal location of the AIM II gene, single-copy gene fluorescence ira .situ hybridization (FISH) to normal human metaphase chromosome spreads was attempted (Lawrence et crl., 1988). A 2 Kb cDNA was nick-translated using Digoxigenin-11-dUTP (Boehringer Mannheim) and FISH was carried out as detailed in Johnson et ul.. 1991 b. Individual chromosomes were counterstained with DAPI and color digital images.
containing both DAPI and gene signal detected with Rhodamine. were recorded using a triple-band pass filter set (Chroma Technology, Inc.. Brattleburo, VT) in combination with a cooled charge coupled-device camera (Photometrics, Inc..
Tucson. AZ) and variable excitation wave length filters (,lohnson el ul.. 1991 a).

Images were analyzed using the ISEE sottware package (Inovision Corp..
Durham. NC).
Cells and Reagents The human breast carcinoma MDA-MB-?31. subclone 2LMP. obtained from ij~ vivo passage of MDA-MB-23 I cells in athvmic nude mice, was used in all the experiments. MC-38 is a 1.2-dimethvlhvdrazine induced murine colon adenocarcinoma which is of H-2b origin. Human T lymphoma line Jurkat and CHO lines were obtained from the American Type Culture Collection (ATCC.
Rockville. MD). A human melanoma antigen gp100 reactive CD8+ T-cell line TIL1200 was kindly provided by Dr. Yutaka Kawakami (National Cancer Institute. Bethesda. MD). All tumor cell lines were grown and maintained in RPM1 1 G40 medium containing 10% FCS. except MDA-MB-231, which used Dulbecco's modified Eagle's medium as basal medium. HLA-A2 restricted TIL
1200 was grown in Aim-V medium containing 10% human serum and 1000 U of IL-2. The apoptosis inducing anti-Fas Mab CH-1 1 was obtained from Upstate Biotechnology. Interfreon was obtained from Biosource International (CA).
Production of soluble AIM IL
The sequence encoding amino acids 74-240 of AIM II, i.e.. the putative extracellulardomain, was subcloned into the vectorpFLAG.CMV-1 in frame with sequences encoding the preprotrypsin signal peptide and the FLAG peptide tag.
The resulting construct, pFLAG-sAIM II. was transfected into 293T cells to generate recombinant sAIM II. Culture media from cells transfected pFLAG.CMV-1 or pFLAG-sAIM II were passed through anti-FLAG mAb (Eastman Kodak Co.) affinity columns. The column eluents were fractionated by SDS-PAGE and sAIM II was detected by western blot analysis, using the anti-FLAG mAb and ECL detection reagents (Amersham International).

- l 40-Ge»erafiorr of reco»rbirra»t receptor-Fc f»siorr proteins A cDNA encoding extracellular domain of human LT~3R was amplified from a HepG2 cells by RT-PCR technique. The sequences of oligonucleotide primers are as following:
Forward S' CGGGATCCATGCTCCTGCCTTGGGCCAC >' (SEQ ID N0:48):
and Reverse: s' GCGGATCCTGGGGGCAG'fGGCTCTAATGG s' (SEQ ID
N0:49) and contained l3crrnI-ll restriction sites on each end to facilitate the cloning of PCR product into the pSK+ vector (Stratagene). The amplified sequence was subjected to l3unrHI digestion and ligated to BcrmHI cut pSK+ vector for sequencing. The fidelity of amplified cDNA fragment was confirmed by dideoxy DNA sequencing. To obtain human LT~3R-Fc fusion protein. excracellular domain of LT~3R was excised from pSK+ vector with l3umHI restriction endonuclease and ligated to BglI1 cut pUCl9-IgGI-Fc vector to allow in frame ligation. To generate recombinant baculovirus. fusion gene was firstly excised with HpaI/HindIII from pUC 19-IgG-Fc vector, followed by ligation with Sma1 cut pBacPAK9 vector (Clontech Co.) after fill-in, then co-transfected with linearized BacPAK6 DNA (Clontech Co.) into Std cells. To obtain recombinant soluble LT~iR fusion protein, five days culture supernatants from recombinant virus infected insect Sf?1 cells was filtered and trapped onto protein A Sepharose beads. the bound sLT~3R protein was then eluted with glycine buffer (pH 3.0) and followed by dialysis in PBS. Production of TR2-Fc fusion protein has been described.
Generation of LT,QR and TR2 antibodies Balb/cJ mice {The Jackson Laboratory, Bar Harbor, ME) were immunized with LT(3R-Fc fusion proteins in Freund's adjuvant. Mice were boosted three times then the spleen cells were fused with the marine myeloma NS-1 cells in the presence of 50% polyethylene glycol in HEPES (PEG 1500, Boehringer Mannheim), followed by culture in RPM/ l 640/HAT and RPMI 1640/HT selective media (Boehringer Co.). Supernatant from positive wells were tested for the ability to bind L.T~iR-Fc fusion protein. but not human IgGI by ELISA.
Hybridomas producinf: antibodies against LT~iR-Fc fusion protein were cloned by limiting dilution three times. To produce large amount of mAbs, 10' hybridoma cells were injected into pristane treated peritoneal cavim ohBalb/c mice. and mAbs was subsequent) purified from ascites by affinity chromatography. Similarly.
using TR2-GST fusion protein, monoclonal antibodies against TR? were produced and screened by ELISA assay.
in vitro growth assays Cells (5,000 cells per well) were plated in triplicate in 24-multiwell tissue culture plates with IMEM in the presence of either 10% FBS or I% FBS. The number of live cells were determined by trypan blue exclusion method at day 3, day 5 or day 7. Cells were refed with fresh medium every two days during this time course.
A soluble tetrazolium/formazan (XTT) assay for cell growth in a 96-well plate was performed. Cells (2,000-4,000 cells/well) were grown in IMEM
medium with 10% FBS or 1 % FBS. After four to five days culture, XTT ( 1.0 mg/ml plus PMS at 1.53 mg/ml) was added to each well and incubated for four hours at 37°C. Absorbance at 450 nm was measured with the Dvnatech Model MR700.
FRCS analysis Cells were collected by trypsinization or aspiration, and centrifuged at 1500-2000 rpm for 5 min. The cell pellets were resuspended and washed in 5 ml ice-cold PBS twice. And then, the cells were incubated with Flag-tagged AIM II
protein or Abs at I 0pg/ml in the binding buffer (HBSS containing 10% BSA, ?0 mM HEPES, pI-I 7.?, 0.02% NaN,. and 2~ pg/ml normal rat Ig) for 30 min at 4°C. Cells were then washed and stained with phycoerythrin (PE) conjugated to goat anti-mouse IgG at 20 pg/ml as described. 'l~o compete for cell surface binding, soluble LT(3R-Fc fusion protein, TR2-Fc at I O~g/ml was preincubated _1.1~_ with :11M II for 30 min before adding; to cells. Fluorescence was analyzed by a F:1C'scan flow cvtometer (Becton Dickinson, Mountain View. CA).
For apoptosis assay. cell pellets were resuspended in 1 X bindings buffer ( I

m~~t HEPES p1-1 7.4, 0.1 ~ M NaCI. ~ mM KC1, 1 mM MgCI,, 1.8 mM CaCI=) S COllta117117!! 1: I 00 dilution of Annexin V-F1TC (Trevigen. Gaithersburg~.
MD) and l.t!?!1111 of propidium iodide and incubated at 4°C for I J 111117. The fluorescence of :lnnexin V-FITC and propidium iodide of individual cells were analyzed by filow cWOmetry (Coulter).
Retroviral transdrrctiou of turrror cells 10 A retroviral vector was used to stably transduce tumor cells with AIM II
gene. ~I~o construct a plasmid encoding the AIM II, a 1.9 kb ~1'olIlScrlI
fragment containing the AIM II eDNA was insel-ted into a parental plasmid pG 1 SamEN.
This retroviral backbone was derived from the Moloney murine leukemia virus and the AIM II gene was under.the transcription control of the long-terminal repeat from the Moline murine leukemia virus. Generation of the retroviral packaging line was described previously (Markowitz el ul. ). Briefly, 30 pg of pG 1 SamEN-AIM II DNA were used to transfect a mixture of 2x10' PA317 amphotropic packaging line and 3x105 GP+E86 ecotropic packaging line. After 2 week of selection. high-titer 6418-resistant PA317 clones were then selected to recreate the packaging line PA-AIM II and used for gene transfer into tumor cells. A
control retrovirus producing line PA-neo was also used. These packaging lines were grown for 20 h and the retrovirai supernatants were harvested, added to a 75% confluent flask of wild type MDA-MB-231 or MC-38 respectively.
Fol lowing transduction with a recombinant retrovirus encoding the human AI M
I I.
AIM II expressing MDA-MB-231 or MC-38 cells were selected with the neomycin analogue 6418 and designated MDA-MB-231/AIM I1 or MC-38/AI M II respectively. AIM II expression in these tumor cells was confirmed by Northern blot analyses. All stable transfectants including MDA-MB-231 /AIM II.
vector control line MDA-MB-231 /neo. MC-38/AIM II and the vector control line MC-38/neo were grown and maintained in the presence of 6418 at I .J mg/ml and 0.375 mg/ml. respectively.
Coculture Assrrns of Jurkat Cells The MDA-MB-231 cells were plated in 6-well tissue culture plates and S allowed to grow to confluence. Following removal of media and washing of the monolayers with 1 X PBS. 1 x 10~ Jurkat cells (nonadherant) were plated in 1 ml of RPMI medium over a monolaveror an empty wells. Wells with MDA-MB-231 cells alone (without overlaying Jurkat cells) were maintained as additional control.
After 24 or 48 hours of culture. the nonadherant phase of the mixed culture was collected from the (-well plated after gentle rocking of the plate and assayed for viability using trypan blue exclusion. For detection of apoptosis. 20,000 cells were measured per sample using Annexin V-FITC FACScan flow cytometer.
Lynrphokine release ass~~~
The lymphokine release assays were performed to detect human PBL
1S reactivity with AIM II as previously described. (Zhai el crl.) Briefly, human PBL
cells were incubated for 5 days in the presence of anti -CD3 mAb (0. I pg/ml) and r1L-2 20 U/m1 plus AIM lI protein at various concentrations, the supernatants were collected and the secretion of IFNy were determined using ELISA kits purchased from R&D Systems (Minneapolis, MN).
Tumorigerricity studies Female athymic Ncr-nu nude mice, 6 week old, were obtained from the Frederick Cancer Research and Development Center, National Institute of Health (Frederick. MD) and Charles River Laboratories (Raleigh, NC). Female CS7BL/( mice, 6-7 wk old, were purchased from Harlan Sprague Dawley (Indianapolis.
2S IN). MDA-MB-231 cells ( 1 x 10~ ) were injected on day 0 into the mammary fat pad ofthe female athymic nude mice and similarly. MC-38 cells were injected s.c.
into the t7ank region of CSBBL/6 mice. Mice were then ear tagged and randomized. Tumor size was assessed by measuring perpendicular diameters with WO 99/4258: PCT/US99/03703 a caliper mice weekly in a blinded fashion. Each treatment group consisted of ten animals and experiments were repeated three times. Tumor histalogical examination was carried out with H/E staining.
E.rample 6: Detectio» oJAIM II expressio» bn BIAcore Analysis CI-IO cells were transfected with either an AIM II-Flag tag expression vector or an BAP-Flag (negative control). Three days after transfection. AIM

expression was determined using the BIAcore instrument (BIAcore, Inc.) which permits real-time measurements ofprotein binding events to immobilized AIM II
receptor, Iy111phOtOX111-~ receptor (BIAcore sensorgram detects binding by changes in refractive index at the surface of the flow cell). A lymphotoxin-(3 receptor-Fc fusion protein was covalently inunobilized to the BIAcore flow cell via amine groups using N-ethyl-N'-(dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide chemistry. Various dilutions of AIM II-Flag and the negative control (BAP-Flag) conditioned serum-free media were applied to the lymphotoxin-(3-receptor-derivatized flow cell at Spl/min for a total volume of SOpI. The amount of bound protein was determined after washing the flow cell with I-IBS buffer ( 10mM HEPES, pH 7.4, 150 mM NaCI, 3.4 mM EDTA, 0.005%
Surfactant P20). The flow cell surface was regenerated by displacing bound protein by washing with 20 pl of 10 mM HC1.
The specific binding to the lymphotoxin-~3-receptor was detected at up to 10-fold dilution ofthe conditioned media from A1M II-Flag cultures, whereas, no significant binding was observed for the negative control (BAP-Flag) conditioned media. This demonstrates that AI M II-Flag binding is specific to lymphotoxin-~3-receptor and not to the Fc portion of the fusion protein. Moreover, specific receptor binding by AIM II-Flag protein indicates that it exhibits a native structure as secreted by the cells. Thus, this BIAcore-based assay can be used to detect expression ofAlM I1 from conditioned media and other biological fluids.
Further.

-14~-by using known amounts of pure AIM II protein this assay can be developed into a quantitative assay for determining= AIM II concentrations.
Exarrrple 7: Activation-induced Apoptosis Assay Activation-induced apoptosis is assayed using SupT-1 > T leukemia cells and is measured by cell cycle analysis. The assay is performed as follows.
SupT-1 3 cells are maintained in RPMI containing 10% FCS in logarithmic growth (about 1 ~ 10''). Sup-T13 cells are seeded in wells of a 24 well plate at 0.5 x 106/ml, 1 ml/well. AIM II protein (0.01, 0.1, l, 10, 100, 1000 ng/ml) or buffer control is added to the wells and the cells are incubated at 37 °C for 24 hours. The wells of another 24 well plate were prepared with or without anti-CD3 antibody by incubating purified BC3 mAb at a concentration of 10 pg/ml in sterile-filtered O.OSM bicarbonate buffer, pH 9.5 or buffer alone in wells at 0.~ ml/well. The plate is incubated at 4°C overnight. The wells of antibody coated plates are washed 3 times with sterile PBS, at 4°C. The AIM II treated Sup-Tl 3 cells are transferred to the antibody coated wells and incubated for 18 hr., at 37°C.
Apoptosis is measured by cell cycle analysis using propidium iodide and flow cytometry. Proliferation of treated cells is measured by taking a total of 300 ~l of each treatment well and delivering in to triplicate wells (100 pl!well) of 96 well plates. To each well add 20 pl/well 3H thymidine (0.5 pCi/20 pl. ? Ci/mM) and incubate 18 hr., at 37°C. Harvest and count'H-thyrnidine uptake by the cells.
This measurement is used to confirm an effect on apoptosis if observed by other methods. The positive control for the assay is Anti-CD3 crosslinking alone. In addition, profound and reproducible apoptosis in this line using anti-fas monoclonal antibody (S00 11g/1111 in soluble form-IgM mAb) has been demonstrated. The negative control for the assay is medium or buffer alone.
Also, crosslinking with another anti-CD3 mAB (OKT3) has been shown to have no effect.

If an effect is obser,~ed by cell cycle analysis the cells will be further stained for the TLJN>rL assay for flow cytometrv or with Annexin V, techniques well k170W'll to those skilled in the art.
E.ra»rple 8: CD3-induced Proliferatio» AssaO
A CD 3-induced proliferation assay is performed on PBMCs and is measured by the uptake of 'H-thymidine. The assay is performed as follows.
Ninety-six well plates are coated with 100 pl/well of mAb to CD3 (HIT3a, Pharmingen) or isotype-matched control mAb (B33.1 ) overnight at 4 °C ( 1 pg/ml in .OSM bicarbonate buffer, pli 9.~). then washed three times with PBS. PBMC
are isolated by F/1-I gradient centrifugation ii-om human peripheral blood and added to quadruplicate wells (5 x IOa/well) of mAb coated plates in RPMI
containing 10% FCS and P/S in the presence of varying concentrations of AIM II
protein (total volume 200 pl). Relevant protein buffer and medium alone are controls. After 48 hr. culture at 37 °C, plates are spun for 2 min. at 1000 rpm and 100 pl of supernatant is removed and stored -20°C for measurement of IL-2 (or other cytokines) if effect on proliferation is observed. Wells are supplemented with 100 ul of medium containing 0.~ pCi of 'I-I-thymidine and cultured at 37 °C for 18-24 hr. Wells are harvested and incorporation of 'H-thymidine used as a measure of proliferation. Anti-CD3 alone is the positive control for proliferation. IL-(100 U/ml) is also used as a control which enhances proliferation. Control antibody which does not induce proliferation of T cells is used as the negative control for CD3-induced proliferation and medium or buffer are used as negative controls for the effects of AIM II proteins.
Example 9: Effect of AIM II o» the Expression of MHC Class II, Costi»rulatorv acrd Adhesion Molecules and Cell Differe»tiatio rr of ll~o»ocyte Derived Hrr»ra» De»dritic Cells WO 99/425$4 PCT/US99/03703 -1 ~7-Dendritic cells are generated by the expansion of proliferatin~~ precursors found in the peripheral blood: adherent PBMC or elutriated monocvtic li-actions are cultured for 7-10 days with GM-CSF (50 ng/ml) and IL-4 (20 I7~~; ml).
These dendritic cells have the characteristic phenotype of immature cells (expression of CDI. CD80. CD86, CD40 and MHC class II antigens). Treatment with activating factors, such as TNF-a. causes a rapid change in surface phenotype ( increased expression of MHC class 1 and II. costimulatory and adhesion molecules, downregulation of FcyRII, upregulation of CD83). These changes correlate with increased antigen-presenting capacity and with functional maturation of the dendritic cells.
FAGS analysis of surface antigens is performed as follows. Cells are treated 1-3 days with various concentrations of AIM-I I (0.1. 1, 10. I 00.

ng/ml) or LPS as positive control, washed with PBS containing 1 % BS.-~ and 0.02 mM sodium azide, and then incubated with 1:20 dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at 4°C. After an additional wash, the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).
Effect on the production of cvtokines Cytokines generated by dendritic cells, in particular IL-12, are important in the initiation of T-cell dependent immune responses. IL-12 strongly influences the development of Thl helper T-cell immune response, and induces cvtotoxic T
and NK cell function. An ELISA will be used to measure the IL- 12 release as follows. Dendritic cells ( 1 O6/ml) are treated with AIM-II (0.1. 1, 10. 100, I1~T.I1111) for 24 hours. LPS (100 ng/ml) is added to the cell culture as positive control. Supernatants from the cell cultures are then collected and analyzed for IL-12 content using commercial ELISA kit. The standard protocols provided with the kits are used.
Effect on the expression of MI-IC Class l I, costimulatorv and adhesion molecules Three major families of cell surface antigens can be identified on monocytes: adhesion molecules. molecules involved in antigen presentation. and Fc receptor. Modulation of the expression of MHC.' class II antigens and other costimulatorv molecules, such as B7 and ICAM-1. may result in changes in the antigen presentin~~ capacity of monocytes and ability to induce T cell activation.
Increase expression of Fc receptors may correlate with improved monocyte cytotoxic activim. cvtokine release and phagocytosis.
FACS analysis will be used to examine the surface antigens as follows.
Monocytes are treated 1-5 days with various concentrations of AIM-II (0.1. 1, 10, 100. 1000 ng/ml ) or LPS (positive control). washed with PBS containing 1 %
BSA
and 0.02 mM sodium azide, and then incubated with 1:20 dilution of appropriate F1TC- or PE-labeled monoclonal antibodies for s0 minutes at 4°C.
After an additional wash. the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).
Effect on monocvte survival Human peripheral blood monocytes progressively lose viability when cultured in absence oi'serum or other stimuli. Their death results from internally regulated processes (apoptosis). Addition to the culture of activ sting factors, such as TNF-a, dramatically improves cell survival and prevents DNA fragmentation.
Propidium iodide staining will be used to measure apoptosis as follows.
Monocytes ( I 0'/ml ) are cultured in suspension in polypropylene tubes in DMEM
for two days in presence or absence of TNF-a ( 100 ng/ml, positive control) or AIM-II (0.1. 1, 10. 100, 1000 ng/ml). Cell viability is assessed by propidium iodide (PI) staining. Cells are suspended at a concentration of 2 x 10~/ml in PBS
containing PI at a final concentration of ~ ltg/ml. and then incubated at room temperature for ~ minutes before FACScan analysis. PI uptake has been demonstrated to correlate with DNA fragmentation in this experimental paradigm.
Effect on cvtokine release WO 99/42584 PCT/tJS99/03703 - l 4n-An important function of monocvtes/macrophages is their regulatory activity on other cellular populations of the immune system through the release of cvtokines after stimulation. An ELISA to measure the IL- 1 (3 release is performed as follows. Human monocytes are added at 10''/ml in 48-well plates and various concentrations of AIM-II are added (0.1. I . 10, 100. 1000 ng/ml) in presence or absence of 100 ng/ml LPS. Atter 24 hour incubation, the supernatants are collected and assayed for the presence ofcwokines by ELISA kits. The standard protocols provided with the kits are used.
E_vantple !0: Afftnitn Purifrcatiort ofSolnbleAIMIIforN terminal Segtrertce Analysis Previous data indicated that a BIAcore chip derivatized with the lymphotoxin beta receptor (LT(3R)-Fc fusion protein was able to specifically bind AIM II (a.a. 74-240)-Flag fusion protein (See Example 5, section E and Figure 7A). The LT(3R BIAcore chip was then used to detect expression of soluble AIM II protein from conditioned media of non-Flag tagged AIM II stable transfectants in order to determine which cell lines) should be used for further purification for N-terminal sequence analysis.
CHO cells were transfected with an expression construct (pC4 vector) consisting of the extracellular region of AIM II (amino acids 60-240) fused to the ck-beta 8 signal peptide. Clones were selected for high expression by groWh in media containing methotrexate. The clones with the highest amount of binding to LT~3R BIAcore chip were further amplified. Conditioned media (20 mL) from CHO 11. a high level AIM II producing clone, was obtained. A second AIM II
construct encoding the complete full length cDNA was transfected into murine MCA-38 carcinoma cells and subject to selection with 6418. Conditioned media was obtained from these transfected MCA-38 cells.
Conditioned media from the stable transfectants, CHO 11 or MCA-s8 cells, were filtered, centrifuged at 10,000 x ~~ and then passed over an MCIF-Fc affinity column (control column) followed by the LTpR-Fc affinity column (0.2m1.

bed volume). The columns were washed with several bed column volumes I-IEPES buffered saline containing 0.00% Surfactant P-'?0. Bound protein was eluted with 10 mM HCl (3 X 0.~ mL fractions) and immediately neutralized with TRIS buffer. The fractions eluted from the LT(iR column retained binding to LT(3R BIAcore chip, whereas. fractions eluted from the control MCIF-Fc column were negative for binding. The eluted fractions were dried in Spedvac then resuspended in 20 yL water. An aliquot of the eluted protein was analyzed by reducing SDS-PAGE gels and detected by silver staining. A band of approximately -25 kDa and ~? 1 kDa was detected specifically bound to the L1'~iR
column from CHO-11 and MCA-38 cell lines. The remaining eluted material was subject to SDS-PAGE and blotted onto PVDF membrane forty-terminal sequence analysis.
The N-terminus of the AIM II molecule purified from MCA-38 cells started at residue 83 within the predicted extracellular region of the molecule (Table 3). The results of the AIM I1 from CHO-11 also confirmed that this protein correspond to AIM II protein; the N-terminus contained two sequences starting three residues apart which start within the ck-beta 8 signal peptide followed by the extracellular region of AIM II starting at residue 60 (Table 3).
Thus, the natural processed form of AIM II should correspond to residues 83-and have a molecular mass of 17,284 daltons. The apparent electrophoretic mobility of ~21 kDa is consistent with glycosylation as evident by presence of several electrophoretic species. Similarly, the --25kDa apparent molecular mass of the CHO-11 expressed ck-beta8/AIM II fusion protein was larger than that predicted from its sequence (20,361 ). Again this might also be due to glycosylation ofthe protein (there is one N-glycosvlation site at residue 104 of full length AIM II).

WO 99/42584 PCT/US99l03703 -I~l-Table 3. N-terminus of AIM2 purified from MCA-38 or CHO-11 clone conditioned media.
N-terminus MCA-381 LIQER . . . .
$ N-terminus C1-10I I 1 ( 4 o o ) SQAGS . . . . . . . . . . . . . . . . . . .
. . . . . . , . . .
N-terminusCHO-I11 (400) GSQLH. . . . . . . . . . . . . . . . . . . . . . . . .
. . .
ck-beta-8-AIMS sequence S AGSQLHWRLGEMVTRLPDGPAGSWEQLIQERN
1= Affinity purified AIM II from MCA-38 or CHO-1 I conditioned media.
2=Amino acid sequence at _ju.nction of ek-beta-8 and estracellular region of AIM 11.
Uouble underlined sequence corresponds to ck beta 8 signal sequence {SQA), and in the case of the GS residues sequence introduced during cloning. AIM 11 sequence starts at the 6th residue, Q.
Values in parenthesis represent percentage of each sequence found in AIM II
sample.
The Sensorgram of specificity of binding of MCA-38 AIM II conditioned media to LT(3R-lc versus MCIF-Fc immobilized on BIAcore chip is shown in Figure l 2. The conditioned media was analyzed on a BIAcore instrument flowcell derivatized with tymphotoxin beta receptor Fc fusion protein. The conditioned media ( I 00 ~tL ) was flown over the chip at ~ ~tL/min and washed with HBS
buffer also at 5 ~tL/min. The shown data represents the net bound (off rate) region of the plot after binding of AIM II to immobilized receptor and is measured in relative mass units (RU) versus time. The binding conditions were performed at high receptor chip densities under diffusion-limited conditions. Legend: LT(3R-Fc and MCIF-Fc refer to binding data from LT(3R-Fc or MCIF-Fc immobilized BIAcore chip surfaces. respectively.
Determination of the LTpR binding by AIM II eluted from LT(3R-Fc column is shown in Figure 13. LT(3R and MC1F refer to binding data from LT(iR-F c or MCIF-Fc immobilized BIAcore chip surfaces, respectively.
Undiluted Conditioned media from MCA38 cells was analyzed before (pre) and after -1 j?_ passage through MCIF-Fc (post-MCIF) and LTpR-Fc (post-LTpR) affinity columns. Fractions (I mL) eluted from the LT(3R (E4-6) and MCIF-Fc (El-3) affinity columns were diluted 3-fold and tested for binding to LT(3R BIAcore chip.
Example 11: Effect of AIM II irr Treating Arljrrvarrt-Irt~luce~l Arthritis in Rats An analysis of the use of A1M II to treat rheumatoid arthritis (RA) is performed through the use of an adjuvant-induced arthritis (AIA) model in rats.
AIA is a well-characterized and reproducible animal model of rheumatoid arthritis which is well-known to one of ordinary skill in the art (Pearson, Arm.
RJ?etIIIT. Di.s.
l ~: 379 ( 1956); Pearson eml.. .Arlhrins Rheum. 2: 440 ( 1959)). AIM I1 is expected to inhibit the increase in angiogenesis or the increase in endothelial cell proliferation reduired to sustain the invading pannus in bone and cartilage observed in this animal model of RA. Lewis and BB rats (available from Charles River Lab, Raleigh, N.C. and the University of Massachusetts Medical Center.
Worcester, MA) are used as the common and responsive strains for adjuvant-induced arthritis in these experiments.
Initiation of the arthritic condition is induced by the intradermal injection of 0.1 ml adjuvant (S mg/ml) into the base of the tail. Groups of 5 to 6 rats receive either 0.1 to 1.0 mg/kg AIM I I or vehicle intra-articularly 20 days after the injection of adjuvant. At this time point acute inflammation reaches a maximal level and chronic pannus formation will have just begun. The effect of AIM II
on pannus formation is analyzed radiologically once each week after day 15 following adjuvant challenge essentially as described by Taurog and colleagues (J. Exp.
Mecl 162: 962 ( 1985)). Briefly. rats are anesthetized with ether or chloral hydrate and positioned so that both hind limbs are X-rayed together. The X-ray films are examined blindly using a scoring system of 0-3 for periosteal reaction, bony erosions, joint space narrowing and destruction. When there is a significant amount of joint damage in vehicle-treated rats, the animals are sacrificed. At this WO 99/4258. PCT/US99/03703 point. the pava~s are evaluated histologicallv for the relative degree of tissue damage and for the therapeutic effect AIM II has elicited on these joints.
Finally. AIM II- and vehicle-treated animals undergo a clinical evaluation t~°ice per week to assess hind paw volume using a plethysmometer system and body weight.
Example 12: Effect of AIM II in Treating Collagen-lnclnced Arthritis in Mice An analysis ofthe use ofAIM II to treat rheumatoid arthritis (RA) may be performed through the use of a collagen-induced autoimmune arthritis (CIA) model in mice. CIA is another well-characterized and reproducible animal model of rheumatoid arthritis which is well-knowm to one of ordinary skill in the art (Courtenay el ul., Nmure 2~3: 666 ( 1980); Wooley el ul.. ,I. Exp. Med. I ~-t:

(1981 ): Holmdahl et ul.. Immunol. Reaie~~.s I18: 193 (1990)). AIM II is expected to induce apoptosis and inhibit the synovial cell proliferation required to form the invading pannus in bone and cartilage observed in both rheumatoid arthritis and this autoimmune animal model of RA.
DBA/1 Lac .1 mice, available from Jackson Lab (Bar Harbor. ME) are used as the most universally susceptible strain for collagen-induced arthritis in these experiments.
Initiation of the arthritic condition is induced by the intradermal injection of 0.1 ml of 1 mg/ml of bovine type II collagen in Complete Freund"s Adjuvant into the base of the tail. Three weeks later, the animal are injected with 40 pg of LPS to accelerate the development of arthritis. Groups of 10 mice will receive either 0.1 - 1 mg/kg AIM I I or vehicle intradermally or intra-articularly 7-15 days after the injection of LPS. At this time point. acute inflammation is expected to reached a maximal level and chronic pannus formation will have just begun. The effect of AIM II on arthritis is monitored and analyzed clinically using the following score: 0 = normal, 0.5 = swollen digits. 1 = entire paw swollen, 2 =
deformity and 3 = ankylosis. When it is determined that a significant amount of _1;:1_ ankylosis has occurred in the paws of vehicle-treated rats, the animals will be sacrificed and the paws are evaluated histologically for the relative decree of pannus formation. cartilage and bone destruction and for what effect AIM II
has elicited on these_joints.
It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples.
Numerous modifications and variations of the present invention are possible in light of the above teachin~~s and. therefore, are within the scope of the appended claims.
The entire disclosure of all publications (including patents, patent applications. journal articles, laboratory manuals, books, or other documents) cited herein are hereby incorporated by reference.

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_7_ (A) LENGTH: 1169 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (cDNA) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 49..768 (xi)SEQUENCE
DESCRIPTION:
SEQ
ID
NO:1:

GACCCAGGCG GCTCCAGAGA
TG

Met GluGlu SerValVal ArgPro SerValPhe ValValAsp GlyGlnThr AspIle ProPheThr ArgLeu GlyArgSer HisArgArg GlnSerCys SerVal AlaArgVal GlyLeu GlyLeuLeu LeuLeuLeu MetGlyAla GlyLeu AlaValGln GlyTrp PheLeuLeu GlnLeuHis TrpArgLeu GlyGlu MetValThr ArgLeu ProAspGly ProAlaGly SerTrpGlu GlnLeu IleGlnGlu ArgArg SerHisGlu ValAsnPro AlaAlaHis LeuThr GlyAlaAsn SerSer LeuThrGly SerGlyGly ProLeuLeu TrpGlu ThrGlnLeu GlyLeu AlaPheLeu ArgGlyLeu SerTyrHis AspGly AlaLeuVal ValThr LysAlaGly TyrTyrTyr IleTyrSer LysVal GlnLeuGly GlyVal GlyCysPro LeuGlyLeu AlaSerThr IleThr HisGlyLeu TyrLys ArgThrPro ArgTyrPro GluGluLeu GluLeu _J_ AGC CGG GCC AGC

Leu Val Gln Gln Ser Pro Cys Gly Thr Ser Ser Arg Ser Arg Ala Ser TGG GGT GTG CTG

Val Trp Asp Ser Ser Phe Leu Gly Val His Glu Ala Trp Gly Val Leu GAG GAT GAA GTT

Gly Glu Val Val Val Arg Val Leu Arg Leu Arg Leu Glu Asp Glu Val GGT GCT TTC TGAAGGAAGG

Arg Asp Thr Arg Ser Tyr Phe Gly Met Val Gly Ala Phe TCAAi~AAAAAF~aAAAAAAAA AAAAAAAAAA A 116 (2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 240 amino acids (B} TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Glu Glu Ser Val Val Arg Pro Ser Val Phe Val Val Asp Gly Gln Thr Asp Ile Pro Phe Thr Arg Leu Gly Arg Ser His Arg Arg Gln Ser Cys Ser Val Ala Arg Val Gly Leu Gly Leu Leu Leu Leu Leu Met Gly Ala Gly Leu Ala Val Gln Gly Trp Phe Leu Leu Gln Leu His Trp Arg Leu Gly Glu Met Val Thr Arg Leu Pro Asp Gly Pro Ala Gly Ser Trp Glu Gln Leu Ile Gln Glu Arg Arg Ser His Glu Val Asn Pro Ala Ala His Leu Thr Gly Ala Asn Ser Ser Leu Thr Gly Ser Gly Gly Pro Leu WO 99/42584 PCT/US99/03'103 Leu Trp Glu Thr Gln Leu Gly Leu Ala Phe Leu Arg Gly Leu Ser Tyr His Asp Gly Ala Leu Val Val Thr Lys Ala Gly Tyr Tyr Tyr Ile Tyr Ser Lys Val Gln Leu Gly Gly Val Gly Cys Pro Leu Gly Leu Ala Ser Thr Ile Thr His Gly Leu Tyr Lys Arg Thr Pro Arg Tyr Pro Glu Glu Leu Glu Leu Leu Val Ser Gln Gln Ser Pro Cys Gly Arg Ala Thr Ser Ser Ser Arg Val Trp Trp Asp Ser Ser Phe Leu Gly Gly Val Val His Leu Glu Ala Gly Glu Glu Val Val Val Arg Val Leu Asp Glu Arg Leu Val Arg Leu Arg Asp Gly Thr Arg Ser Tyr Phe Gly Ala Phe Met Val (2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 455 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Met Gly Leu Ser Thr Val Pro Asp Leu Leu Leu Pro Leu Val Leu Leu Glu Leu Leu Val Gly Ile Tyr Pro Ser Gly Val Ile Gly Leu Val Pro His Leu Gly Asp Arg Glu Lys Arg Asp Ser Val Cys Pro Gln Gly Lys Tyr Ile His Pro Gln Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly Pro Gly Gln Asp Thr Asp Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys Glu Met Gly Gln Val Glu Ile Ser Ser Cys Thr Val Asp Arg Asp Thr Val Cys Gly Cys Arg _5_ Lys Asn Gln Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe Gln Cys Phe Asn Cys Ser Leu Cys Leu Asn Gly Thr Val His Leu Ser Cys Gln Glu Lys Gln Asn Thr Val Cys Thr Cys His Ala Gly Phe Phe Leu Arg Glu Asn Glu Cys Val Ser Cys Ser Asn Cys Lys Lys Ser Leu Glu Cys Thr Lys Leu Cys Leu Pro Gln Ile Glu Asn Val Lys Gly Thr Glu Asp Ser Gly Thr Thr Val Leu Leu Pro Leu Val Ile Phe Phe Gly Leu Cys Leu Leu Ser Leu Leu Phe Ile Gly Leu Met Tyr Arg Tyr Gln Arg Trp Lys Ser Lys Leu Tyr Ser Ile Val Cys Gly Lys Ser Thr Pro Glu Lys Glu Gly Glu Leu Glu Gly Thr Thr Thr Lys Pro Leu Ala Pro Asn Pro Ser Phe Ser Pro Thr Pro Gly Phe Thr Pro Thr Leu Gly Phe Ser Pro Val Pro Ser Ser Thr Phe Thr Ser Ser Ser Thr Tyr Thr Pro Gly Asp Cys Pro Asn Phe Ala Ala Pro Arg Arg Glu Val Ala Pro Pro Tyr Gln Gly Ala Asp Pro Ile Leu Ala Thr Ala Leu Ala Ser Asp Pro Ile Pro Asn Pro Leu Gln Lys Trp Glu Asp Ser Ala His Lys Pro Gln Ser Leu Asp Thr Asp Asp Pro Ala Thr Leu Tyr Ala Val Val Glu Asn Val Pro Pro Leu Arg Trp Lys Glu Phe Val Arg Arg Leu Gly Leu Ser Asp His Glu Ile Asp Arg Leu Glu Leu Gln Asn Gly Arg Cys Leu Arg Glu Ala Gln Tyr Ser Met Leu Ala Thr Trp Arg Arg Arg Thr Pro Arg Arg Glu Ala Thr Leu Glu Leu Leu Gly Arg Val Leu Arg Asp Met Asp Leu Leu Gly Cys Leu Glu Asp Ile Glu Glu Ala Leu Cys Gly Pro Ala Ala Leu Pro Pro Ala Pro Ser Leu Leu Arg (2) INFORMATION FOR SEQ ID N0:4:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 205 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Met Thr Pro Pro Glu Arg Leu Phe Leu Pro Arg Val Cys Gly Thr Thr Leu His Leu Leu Leu Leu Gly Leu Leu Leu Val Leu Leu Pro Gly Ala Gln Gly Leu Pro Gly Val Gly Leu Thr Pro Ser Ala Ala Gln Thr Ala Arg Gln His Pro Lys Met His Leu Ala His Ser Thr Leu Lys Pro Ala Ala His Leu Ile Gly Asp Pro Ser Lys Gln Asn Ser Leu Leu Trp Arg Ala Asn Thr Asp Arg Ala Phe Leu Gln Asp Gly Phe Ser Leu Ser Asn Asn Ser Leu Leu Val Pro Thr Ser Gly Ile Tyr Phe Val Tyr Ser Gln Val Val Phe Ser Gly Lys Ala Tyr Ser Pro Lys Ala Thr Ser Ser Pro Leu Tyr Leu Ala His Glu Val Gln Leu Phe Ser Ser Gln Tyr Pro Phe His Val Pro Leu Leu Ser Ser Gln Lys Met Val Tyr Pro Gly Leu Gln Glu Pro Trp Leu His Ser Met Tyr His Gly Ala Ala Phe Gln Leu Thr Gln Gly Asp Gln Leu Ser Thr His Thr Asp Gly Ile Pro His Leu Val Leu Ser Pro Ser Thr Val Phe Phe Gly Ala Phe Ala Leu (2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 205 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein _'7 _ (xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Met Thr Pro Pro Glu Arg Leu Phe Leu Pro Arg Val Cys Gly Thr Thr Leu His Leu Leu Leu Leu Gly Leu Leu Leu Val Leu Leu Pro Gly Ala Gln Gly Leu Pro Gly Val Gly Leu Thr Pro Ser Ala Ala Gln Thr Ala Arg Gln His Pro Lys Met His Leu Ala His Ser Thr Leu Lys Pro Ala Ala His Leu Ile Gly Asp Pro Ser Lys Gln Asn Ser Leu Leu Trp Arg Ala Asn Thr Asp Arg Ala Phe Leu Gln Asp Gly Phe Ser Leu Ser Asn Asn Ser Leu Leu Val Pro Thr Ser Gly Ile Tyr Phe Val Tyr Ser Gln Val Val Phe Ser Gly Lys Ala Tyr Ser Pro Lys Ala Thr Ser Ser Pro Leu Tyr Leu Ala His Glu Val Gln Leu Phe Ser Ser Gln Tyr Pro Phe His Val Pro Leu Leu Ser Ser Gln Lys Met Val Tyr Pro Gly Leu Gln Glu Pro Trp Leu His Ser Met Tyr His Gly Ala Ala Phe Gln Leu Thr Gln Gly Asp Gln Leu Ser Thr His Thr Asp Gly Ile Pro His Leu Val Leu Ser Pro Ser Thr Val Phe Phe Gly Ala Phe Ala Leu (2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 281 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Met Gln Gln Pro Phe Asn Tyr Pro Tyr Pro Gln Ile Tyr Trp Val Asp Ser Ser Ala Ser Ser Pro Trp Ala Pro Pro Gly Thr Val Leu Pro Cys _g_ Pro Thr Ser Val Pro Arg Arg Pro Gly Gln Arg Arg Pro Pro Pro Pro Pro Pro Pro Pro Pro Leu Pro Pro Pro Pro Pro Pro Pro Pro Leu Pro Pro Leu Pro Leu Pro Pro Leu Lys Lys Arg Gly Asn His Ser Thr Gly Leu Cys Leu Leu Val Met Phe Phe Met Val Leu Val Ala Leu Val Gly Leu Gly Leu Gly Met Phe Gln Leu Phe His Leu Gln Lys Glu Leu Ala Glu Leu Arg Glu Ser Thr Ser Gln Met His Thr Ala Ser Ser Leu Glu Lys Gln Ile Gly His Pro Ser Pro Pro Pro Glu Lys Lys Glu Leu Arg Lys Val Ala His Leu Thr Gly Lys Ser Asn Ser Arg Ser Met Pro Leu Glu Trp Glu Asp Thr Tyr Gly Ile Val Leu Leu Ser Gly Val Lys Tyr Lys Lys Gly Gly Leu Val Ile Asn Glu Thr Gly Leu Tyr Phe Val Tyr Ser Lys Val Tyr Phe Arg Gly Gln Ser Cys Asn Asn Leu Pro Leu Ser His Lys Val Tyr Met Arg Asn Ser Lys Tyr Pro Gln Asp Leu Val Met Met Glu Gly Lys Met Met Ser Tyr Cys Thr Thr Gly Gln Met Trp Ala Arg Ser Ser Tyr Leu Gly Ala Val Phe Asn Leu Thr Ser Ala Asp His Leu Tyr Val Asn Val Ser Glu Leu Ser Leu Val Asn Phe Glu Glu Ser Gln Thr Phe Phe Gly Leu Tyr Lys Leu (2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs {B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear {ii) MOLECULE TYPE: cDNA

WO 99!42584 PCT/US99/03703 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:

(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:

(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:

(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear {ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:

(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:

(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:

(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:

(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:

(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:

(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
Pro Thr Ser Val Pro Arg Arg Pro -I?-(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:

(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 503 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (Dy TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION:
SEQ ID N0:20:

(2) INFORMATION
FOR SEQ ID N0:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:

(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:

(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:

(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:

WO 99/42584 PCTlUS99/03703 (2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:

{2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:

(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:

(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

-1 ~-(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:

(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:

(2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:

(2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 733 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:

(2) INFORMATION
FOR SEQ
ID N0:32:

(i) SEQUENCE
CHARACTERISTICS:

(A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE
TYPE:
cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:

(2) INFORMATION FOR SEQ ID N0:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:

(2) INFORMATION FOR SEQ ID N0:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:

(2) INFORMATION FOR SEQ ID N0:35:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:35:

(2) INFORMATION FOR SEQ ID N0:36:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 93 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:36:

(2) INFORMATION FOR SEQ ID N0:37:
(i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:37:

(2) INFORMATION FOR SEQ ID N0:38:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1017 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..625 (xi)SEQUENCE
DESCRIPTION:
SEQ
ID
N0:38:

IleProArgAla ArgValGly LeuGlyLeu LeuLeuLeu LeuMetGly AlaGlyLeuAla ValGlnGly TrpPheLeu LeuGlnLeu HisTrpArg LeuGlyGluMet ValThrArg LeuProAsp GlyProAla GlySerTrp GluGinLeuIle GlnGluArg ArgSerHis GluValAsn ProAlaAla HisLeuThrGly AlaAsnSer SerLeuThr GlySerGly GlyProLeu LeuTrpGluThr GlnLeuGly LeuAlaPhe LeuArgGly LeuSerTyr HisAspGlyAla LeuValVal ThrLysAla GlyTyrTyr TyrIleTyr SerLysValGln LeuGlyGly ValGlyCys ProLeuGly LeuAlaSer ThrIleThrHis GlyLeuTyr LysArgThr ProArgTyr ProGluGlu LeuGluLeuLeu ValSerGln GlnSerPro CysGlyArg AlaThrSer SerSerArgVal TrpTrpAsp SerSerPhe LeuGlyGly Va1ValHis LeuGluAlaGly GluGluVal ValValArg ValLeuAsp GluArgLeu CTG GGT ACC TTC GTG T
CGG

Val Arg Arg Asp Ser Tyr Gly Ala Phe Met Leu Gly Thr Phe Val Arg TCAGGGGAAAGAP.AACTCACGAAGCAGAGGCTGGGCGTGGTGGCTCTCGC CTGTAATCCC745 (2) INFORMATION FOR SEQ ID N0:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 208 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:39:
Ile Pro Arg Ala Arg Val Gly Leu Gly Leu Leu Leu Leu Leu Met Gly Ala Gly Leu Ala Val Gln Gly Trp Phe Leu Leu Gln Leu His Trp Arg Leu Gly Glu Met Val Thr Arg Leu Pro Asp Gly Pro Ala Gly Ser Trp Glu Gln Leu Ile Gln Glu Arg Arg Ser His Glu Val Asn Pro Ala Ala His Leu Thr Gly Ala Asn Ser Ser Leu Thr Gly Ser Gly Gly Pro Leu Leu Trp Glu Thr Gln Leu Gly Leu Ala Phe Leu Arg Gly Leu Ser Tyr His Asp Gly Ala Leu Val Val Thr Lys Ala Gly Tyr Tyr Tyr Ile Tyr Ser Lys Val Gln Leu Gly Gly Val Gly Cys Pro Leu Gly Leu Ala Ser Thr Ile Thr His Gly Leu Tyr Lys Arg Thr Pro Arg Tyr Pro Glu Glu Leu Glu Leu Leu Val Ser Gln Gln Ser Pro Cys Gly Arg Ala Thr Ser Ser Ser Arg Val Trp Trp Asp Ser Ser Phe Leu Gly Gly Val Val His Leu Glu Ala Gly Glu Glu Val Val Val Arg Val Leu Asp Glu Arg Leu Val Arg Leu Arg Asp Gly Thr Arg Ser Tyr Phe Gly Ala Phe Met Val (2) INFORMATION FOR SEQ ID N0:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:40:

(2) INFORMATION FOR SEQ ID N0:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:41:

(2) INFORMATION FOR SEQ ID N0:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ TD N0:42:

(2) INFORMATION FOR SEQ ID N0:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:43:
CGCCATATGA GCTGGGAGCA GCTGATAC 2g (2) INFORMATION FOR SEQ ID N0:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:44:

(2) INFORMATION FOR SEQ ID N0:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:45:

(2) INFORMATION FOR SEQ ID N0:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:46:

_77_ Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe (2) INFORMATION FOR SEQ ID N0:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:47:
Gly Tyr Tyr Tyr Ile Tyr Ser Lys Val Gln Leu (2) INFORMATION FOR SEQ ID N0:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:48:

(2) INFORMATION FOR SEQ ID N0:49:
(i) SEQ~NCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:49:

(2) INFORMATION FOR SEQ ID N0:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3974 base pairs (B) TYPE: nucleic acid (C) STR.ANDEDNESS: both (D) TOPOLOGY: both (ii) MOLECULE TYPE: cDNA

_73_ (xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:50:

ACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCA7$0 _7 j_ (2) INFORMATION FOR SEQ ID N0:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 112 base pairs (B) TYPE: nucleic acid (C) STRANpEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:51:

Claims (31)

1. What Is Claimed Is:
   I. An isolated nucleic acid molecule consisting of a polynucleotide having a nucleotide sequence at least 95% identical to a nucleotide sequence selected from the group consisting of:
   (a) a nucleotide sequence encoding a polypeptide having an amino acid selected from the group consisting of E-1115 to V-240: T-116 to V-240; Q-117 to V-240: L-118 to V-240: G-119 to V-240; L-120 to V-240;
   A-121 to V-240; F-122 to V-240; L-123 to V-240; R-124 to V-240: G-125 to V-240; L-126 to V-240, S-127 to V-240; Y-128 to V-240; H-129 to V-240;
   D-130 to V-240; G-131 to V-240; A-132 to V-240; L-133 to V-240; V-134 to V-240; V-135 to V-240; T-136 to V-240: K-137 to V-240; A-138 to V-240;
   G-139 to V-240; Y-140 to V-240; Y-141 to V-240; Y-142 to V-240; I-143 to V-240; Y-144 to V-240; S-145 to V-240: K-146 to V-240; V-147 to V-240;
   Q-148 to V-240; L-149 to V-240; G-150 to V-240; G-151 to V-240: V-152 to V-240; G-153 to V-240; C-154 to V-240; P-155 to V-240; L-156 to V-240;
   G-157 to V-240; L-158 to V-240; A-159 to V-240; S-160 to V-240; T-161 to V-240; I-162 to V-240; T-163 to V-240; H-164 to V-240: G-165 to V-240;
   L-166 to V-240; Y-167 to V-240; K-168 to V-240; R-169 to V-240; T-170 to V-240; P-171 to V-240: R-172 to V-240; Y-173 to V-240; P-174 to V-240;
   E-175 to V-240; E-176 to V-240; L-177 to V-240; E-178 to V-240: L-179 to V-240; L-180 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-186 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; S-194 to V-240; R-195 to V-240; V-196 to V-240; W-197 to V-240; W-198 to V-240: D-199 to V-240: S-200 to V-240: S-201 to V-240;
   F-202 to V-240: L-203 to V-240; G-204 to V-240; G-205 to V-240; V-206 to V-240; V-207 to V-240: H-208 to V-240: L-209 to V-240: E-210 to V-240;
   A-211 to V-240; G-212 to V-240: E-213 to V-240; E-214 to V-240; V-215 to V-240; V-216 to 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 in SEQ ID NO:2;
   (h) a nucleotide sequence encoding a polypeptide having an amino acid selected from the group consisting of M-1 to M-239: M-1 to F-238;
   
   M-1 to A-237: M-1 to G-236:M-1 to F-235:M-1 to Y-234: M-1 to S-233; M-1 to R-232; M-1 to T-231:M-1 to G-230;M-1 to D-229; M-1 to R-228; M-1 to L-227; M-1 to R-226; M-1 to V-225; M-1 to L-224; M-1 to R-223: M-1 to E-222: M-1 to D-221; M-1 to L-220; M-1 to V-219: M-1 to R-218; M-1 to V-217: M-1 to V-216; M-1 to V-215; M-1 to E-214: M-1 to E-213; M-1 to G-212; M-1 to A-211 M-1 to E-210: M-1 to L-209; M-1 to H-208: M-1 to V-207; M-1 to V-206; M-1 to G-205; M-1 to G-204; M-1 to L-203; M-1 to F-202: M-1 to S-201; M-1 to S-200; M-1 to D-199; M-1 to W-198: M-1 to W-197: M-1 to V-196; M-1 to R-195; M-1 to S-194; M-1 to S-193; M-1 to S-192; M-1 to T-191; M-1 to A-190; M-1 to R-189; M-1 to G-188; M-1 to C-187; M-1 to P-186; M-1 to S-185; M-1 to Q-184; M-1 to Q-183; M-1 to S-182; M-1 to V-181; M-1 to L-180; M-1 to L-179; M-1 to E-178; M-1 to L-177: M-1 to E-176: M-1 to E-175; M-1 to P-174: M-1 to Y-173: M-1 to R-172: M-1 to P-171; M-1 to T-170; M-1 to R-169; M-1 to K-168: M-1 to Y-167; M-1 to L-166; M-1 to G-165; M-1 to H-164; M-1 to T-163; M-1 to I-162: M-1 to T-161; M-1 to S-160; M-1 to A-159; M-1 to L-158; M-1 to G-157;
   
   M-1 to L-156; M-1 to P-155; M-1 to C-154; M-1 to G-153; M-1 to V-152; M-1 to G-151; M-1 to G-150; M-1 to L-149; M-1 to Q-148: M-1 to V-147; M-1 to K-146; M-1 to S-145; M-1 to Y-144; M-1 to I-143; M-1 to Y-142; M-1 to Y-141: M-1 to Y-140: M-1 to G-139; M-1 to A-138; M-1 to K-137: M-1 to T-136: M-1 to V-135; M-1 to V-134; M-1 to L-133; M-1 to A-132: M-1 to G-131: M-1 to D-130; M-1 to H-129; M-1 to Y-128: M-1 to S-127; M-1 to L-126; M-1 to G-125: M-1 to R-124; M-1 to L-123; M-1 to F-122; M-1 to A-121; M-1 to L-120: M-1 to G-119; M-1 to L-118; M-1 to Q-117; M-1 to T-116: M-1 to E-115; M-1 to W-114: M-1 to L-113: M-1 to L-112; M-1 to P-111: M-1 to G-110: M-1 to G-109; M-1 to S-108, M-1 to G-107: M-1 to T-106: M-1 to L-105: M-1 to S-104; M-1 to S-103; M-1 to N-102: M-1 to A-101: M-1 to G-100: M-1 to T-99: M-1 to L-98; M-1 to H-97; M-1 to A-96;
   M-1 to A-95; M-1 to P-94; M-1 to N-93; M-1 to V-92: M-1 to E-91: M-1 to H-90; M-1 to S-89; M-1 to R-88; M-1 to R-87; M-1 to E-86; M-1 to Q-85; M-1 to I-84: M-1 to L-83; M-1 to Q-82; M-1 to E-81; M-1 to W-80: M-1 to S-79;
   M-1 to G-78; M-1 to A-77: M-1 to P-76; M-1 to G-75: M-1 to D-74: M-1 to P-73; M-1 to L-72; M-1 to R-71; M-1 to T-70; M-1 to V-69; M-1 to M-68: M-1 to E-67; M-1 to G-66; M-1 to L-65: M-1 to R-64; M-1 to W-63; M-1 to H-62;
   M-1 to L-61; M-1 to Q-60: M-1 to L-59; M-1 to L-58; M-1 to F-57: M-1 to W-56; M-1 to G-55; M-1 to Q-54; M-1 to V-53; M-1 to A-52; M-1 to L-51; M-1 to G-50; M-1 to A-49; M-1 to G-48; M-1 to M-47; M-1 to L-46; M-1 to L-45;
   M-1 to L-44; M-1 to L-43; M-1 to L-42; M-1 to G-41; M-1 to L-40: M-1 to G-39; M-1 to V-38; M-1 to R-37: M-1 to A-36; M-1 to V-35; M-1 to S-34; M-1 to C-33; M-1 to S-32; M-1 to Q-31; M-1 to R-30; M-1 to R-29; M-1 to H-28;
   M-1 to S-27; M-1 to R-26; M-1 to G-25; M-1 to L-24; M-1 to R-23: M-1 to T-22; M-1 to F-21; M-1 to P-20; M-1 to I-19; M-1 to D-18; M-1 to T-17; M-1 to Q-16: M-1 to G-15; M-1 to D-14; M-1 to V-13: M-1 to V-12: M-1 to F-11;
   M-1 to V-10; M-1 to S-9; M-1 to P-8; M-1 to R-7; and M-1 to V-6 in SEQ ID
   N0:2; and (c) a nucleotide sequence complementary to any of the nucleotide sequences in (a) or (b).
2. 2. The isolated nucleic acid molecule of claim 1, which is (a).
3. 3. The isolated nucleic acid molecule of claim 1, which is (b).
4. 4. The isolated nucleic acid molecule of claim 1, which is DNA.
5. 5. The isolated nucleic acid molecule of claim 1. which is fused to a heterologous polynucleotide.
6. 6. The isolated nucleic acid molecule of claim 5, wherein said heterologous polynucleotide encodes a heterologous polypeptide.
7. 7. An isolated nucleic acid molecule of claim 1 comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of the AIM II polypeptide of SEQ ID NO:2.
8. 8. A method for making a vector comprising inserting an isolated nucleic acid molecule of claim 1 into a vector.
9. 9. A vector produced by the method of claim 8.
10. 10. A method of making a host cell comprising introducing the vector of claim 9 into a host cell.
11. 11. A host cell produced by the method of claim 10.
12. 12. A method for producing an AIM II polypeptide, comprising culturing the host cell of claim 9 under conditions such that said polypeptide is expressed and recovering said polypeptide.
13. 13. An isolated AIM polypeptide N-terminal or C-terminal deletion mutant consisting of a polypeptide selected from the group consisting of:
    (a) a polypeptide having an amino acid sequence selected from the group consisting of E-115 to V-240; T-116 to V-240; Q-117 to V-240: L-118 to V-240; G-119 to V-240: L-120 to V-240: A-121 to V-240: F-122 to V-240:
    L-123 to V-240: R-124 to V-240; G-125 to V-240; L-126 to V-240; S-127 to V-240: Y-128 to V-240; H-129 to V-240: D-130 to V-240: G-131 to V-240:
    A-132 to V-240; L-133 to V-240: V-134 to V-240; V-135 to V-240: T-136 to V-240: K-137 to V-240; A-138 to V-240: G-139 to V-240; Y-140 to V-240:
    Y-141 to V-240; Y-142 to V-240: I-143 to V-240; Y-144 to V-210: S-145 to V-240: K-146 to V-240; V-147 to V-240: Q-148 to V-240; L-1:19 to V-240;
    G-150 to V-240; G-151 to V-240; V-152 to V-240; G-153 to V-240: C-154 to V-240; P-155 to V-240; L-156 to V-240; G-157 to V-240: L-158 to V-240:
    A-159 to V-240; S-160 to V-240; T-161 to V-240: I-162 to V-240; T-163 to V-240; H-164 to V-240; G-165 to V-240: L-166 to V-240; Y-167 to V-240;
    K-168 to V-240: R-169 to V-240; T-170 to V-240: P-171 to V-240. R-172 to V-240: Y-173 to V-240; P-174 to V-240; E-175 to V-240: E-176 to V-240:
    L-177 to V-240; E-178 to V-240; L-179 to V-240; L-180 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-186 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; S-194 to V-240;
    R-195 to V-240; V-196 to V-240: W-197 to V-240; W-198 to V-240; D-199 to V-240; S-200 to V-240; S-201 to V-240; F-202 to V-240; L-203 to V-240:
    G-204 to V-240; G-205 to V-240; V-206 to V-240; V-207 to V-240: H-208 to V-240; L-209 to V-240; E-210 to V-240; A-211 to V-240; G-212 to V-240:
    E-213 to V-240; E-214 to V-240; V-215 to V-240; V-216 to 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 in SEQ ID NO:2;
    (b) a polypeptide having an amino acid sequence selected from the group consisting of M-1 to M-239; M-1 to F-238; M-1 to A-237; M-1 to G-236: M-1 to F-235; M-1 to Y-234; M-1 to S-233: M-1 to R-232; M-1 to T-231: M-1 to G-230: M-1 to D-229: M-1 to R-228; M-1 to L-227; M-1 to R-226: M-1 to V-225: M-1 to L-224: M-1 to R-223: M-1 to E-222: M-1 to D-221; M-1 to L-220: M-1 to V-219; M-1 to R-218: M-1 to V-217: M-1 to V-216: M-1 to V-215; M-1 to E-214: M-1 to E-213; M-1 to G-212: M-1 to A-211; M-1 to E-210: M-1 to L-209: M-1 to H-208; M-1 to V-207: M-1 to V-206; M-1 to G-205; M-1 to G-204; M-1 to L-203: M-1 to F-202: M-1 to S-201: M-1 to S-200; M-1 to D-199; M-1 to W-198; M-1 to W-197; M-1 to V-196; M-1 to R-195; M-1 to S-194; M-1 to S-193; M-1 to S-192; M-1 to T-191; M-1 to A-190; M-1 to R-189; M-1 to G-188; M-1 to C-187: M-1 to P-186; M-1 to S-185; M-1 to Q-184; M-1 to Q-183; M-1 to S-182; M-1 to V-181; M-1 to L-180; M-1 to L-179; M-1 to E-178; M-1 to L-177: M-1 to E-176; M-1 to E-175; M-1 to P-174; M-1 to Y-173; M-1 to R-172: M-1 to P-171; M-1 to T-170; M-1 to R-169; M-1 to K-168; M-1 to Y-167: M-1 to L-166; M-1 to G-165; M-1 to H-164; M-1 to T-163; M-1 to I-162; M-1 to M-1 to S-160; M-1 to A-159; M-1 to L-158; M-1 to G-157; M-1 to L-156; M-1 to P-I55; M-1 to C-154; M-1 to G-153; M-1 to V-152; M-1 to G-151: M-1 to G-150; M-1 to L-149; M-1 to Q-148; M-1 to V-147; M-1 to K-146; M-1 to S-145; M-1 to Y-144; M-1 to I-143; M-1 to Y-142; M-1 to Y-141: M-1 to Y-140; M-1 to G-139, M-1 to A-138; M-1 to K-137; M-1 to T-136: M-1 to V-135; M-1 to V-134; M-1 to L-133: M-1 to A-132; M-1 to G-131: M-1 to D-130: M-1 to H-129; M-1 to Y-128; M-1 to S-127; M-1 to L-126; M-1 to G-125; M-1 to R-124; M-1 to L-123; M-1 to F-122; M-1 to A-121; M-1 to L-120; M-1 to G-119, M-1 to L-118; M-1 to Q-117; M-1 to T-116: M-1 to E-115; M-1 to W-114; M-1 to L-113; M-1 to L-112; M-1 to P-111; M-1 to G-110: M-1 to G-109; M-1 to S-108: M-1 to G-107; M-1 to T-106: M-1 to L-105; M-1 to S-104: M-1 to S-103: M-1 to N-102; M-1 to A-101: M-1 to G-100; M-1 to T-99: M-1 to L-98, M-1 to H-97; M-1 to A-96; M-1 to A-95; M-1 to P-94; M-1 to N-93; M-1 to V-92; M-1 to E-91; M-1 to H-90; M-1 to S-89;
    M-1 to R-88; M-1 to R-87; M-1 to E-86; M-1 to Q-85; M-1 to I-84; M-1 to L-83; M-1 to Q-82; M-1 to E-81; M-1 to W-80; M-1 to S-79; M-1 to G-78; M-1 to A-77: M-1 to P-76; M-1 to G-75; M-1 to D-74: M-1 to P-73; M-I to L-72;
    M-1 to R-71: M-1 to T-70; M-1 to V-69; M-1 to M-68; M-I to E-67; h-1-1 to G-66: M- I to L-65: M-1 to R-64, M-1 to W -63: M-1 to H-62; M-1 to L-6 I : M-1 to Q-60: M-1 to I_-59: M-1 to L-58, M-1 to F-57; M-1 to W-56; M-I to G-55:
    M-1 to Q-54: M-I to V-53; M-1 to A-52: M-1 to I_-51; M-I to G-S0; M-1 to A-49; M-1 to G-48: M-1 to M-47; M-1 to L-46: M-1 to L-45; M-1 to L-44: M-1 to L-43; M-1 to L-42; M-1 to G-41; M-I to L-40; M-1 to G-39; M-1 to V-38;
    M-1 to R-37; M-1 to A-36: M-1 to V-35; M-1 to S-34: M-1 to C-33: M-1 to S-32; M-1 to Q-31; M-1 to R-30: M-1 to R-29; M-1 to H-28; M-1 to S-27; M-I
    to R-26; M-1 to G-25; M-1 to L-24; M-1 to R-23: M-1 to T-22; M-1 to F-21:
    M-1 to P-20: M-I to I-19; M-1 to D-18; M-1 to T-i7; M-1 to Q-16; M-1 to G-15: M-1 to D-14: M-1 to V-13; M-1 to V-12; M-I to F-1 1; M-1 to V-10; M-1 to S-9; M-1 to P-8; M-1 to R-7; and M-1 to V-6 in SEQ ID N0:2;
    (c) a polypeptide having an amino acid sequence of (a) plus a methionine residue at the N-terminus;
    (d) a polypeptide having an amino acid sequence of (b) plus a methionine residue at the N-terminus;
    (e) a substitution variant having an amino acid sequence of (a) except for one or more amino acid substitutions; wherein the amino acid sequence of said variant is at least 95% identical to said amino acid sequence of (a);
    and (f) a substitution variant having an amino acid sequence of(b) except for one or more amino acid substitutions: wherein the amino acid sequence of said variant is at least 95% identical to said amino acid sequence of (b).
14. 14. The deletion mutant of claim 13. which is (a).
15. 15. The deletion mutant of claim 13. which is (b).
16. 16. The deletion mutant of claim 13, which is (c).
17. 17. The deletion mutant of claim 13. which is (d).
18. 18. The deletion mutant of claim 13, which is (e).
19. 19. The deletion mutant of claim 13, which is (f).
20. 20. The isolated polypeptide of claim 13, wherein said AIM-II
    polypeptide N-terminal or C-terminal deletion mutant is fused to an Fc immunoglobulin polypeptide.
21. 21. The isolated polypeptide of claim 20. wherein said Fc immunoglobulin is fused at the C-terminal of said deletion mutant.
22. 22. The isolated polypeptide of claim 13, which is produced or contained in a host cell.
23. 23. The isolated polypeptide of claim 13. wherein said host cell is insect, mammalian, or bacterial.
24. 24. The isolated polypeptide of claim 13. together with a pharmaceutically acceptable carrier or excipient.
25. 25. The isolated polypeptide of claim 13, produced by a method comprising:
    (a) introducing a vector comprising a polynucleotide encoding said polypeptide into a host cell;
    (b) culturing said host cell; and (c) recovering said polypeptide.
26. 26. A method for producing a polypeptide comprising:
    (a) culturing the host cell of claim 25 under conditions that said vector is expressed: and (b) recovering said polypeptide.
27. 27. An isolated antibody that binds specifically to an AIM II
    polypeptide of claim 13.
28. 28. A method for the treatment of a disease state in a patient selected from the group consisting of:
    (a) rheumatoid arthritis;
    (b) osteoarthritis;
    (c) allergic reaction;
    (d) viral infection;
    (e) Graves' disease;
    (f) autoimmune disease; and (g) cancer;
    wherein said method comprises administering to the patient a therapeutically effective amount of the apoptosis inducing molecule II (AIM
    II) polypeptide selected from the group consisting of:
    (a) amino acids from about 1 to about 240 in SEQ ID NO:2;
    (b) amino acids from about 2 to about 240 in SEQ ID NO:2;
    (c) the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97689;
    (d) the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97483;
    (e) the amino acid sequence of the extracellular domain of the AIM II polypeptide:
    
    (f) the amino acid sequence of the transmembrane domain of the AIM II polypeptide;
    (g) the amino acid sequence of the intracellular domain of the AIM II polypeptide;
    (h) the amino acid sequence of a soluble AIM II polypeptide having the all or part of the extracellular and intracellular domain but lacking the transmembrane domain wherein the extracellular domain;
    (i) a polypeptide comprises amino acid 83 to 240 in SEQ ID
    NO:2; and (j) the amino acid sequence of an epitope-bearing portion of any one of the polypeptides of (a), (b), (c), (d), (e), (f), (g), (h) or (i).
29. 29. The method of claim 28, wherein said polypeptide is administered together with a pharmaceutically acceptable carrier or excipient.
30. 30. A method for separating eukaryotic cells which bind an apoptosis inducing molecule II (AIM II) polypeptide of claim 1 from eukaryotic cells which do not bind said AIM II polypeptide comprising:
    (a) contacting a mixed population of eukaryotic cells comprising cells which bind said AIM II polypeptide and cells which do not bind said AIM II polypeptide with said AIM II polypeptide;
    (b) identifying cells which bind aid AIM II polypeptide; and (c) separating cells which bind said AIM II polypeptide from the cells which do not bind said AIM II polypeptide.
31. 31. A method for diagnosis of a disorder in an individual, wherein said disorder is related to the over expression or underexpression of an AIM II
    gene, said method comprising:
    
    (a) measuring AIM II gene expression level in a biological sample of said individual; and (b) comparing the AIM II gene expression level of said individual with a standard AIM II gene expression level;
    wherein an increase or decrease in the AIM II gene expression level over said standard is indicative of an AIM II-related disorder.