JP2003528030A - Apoptosis-inducing molecule II - Google Patents

Abstract

  (57) [Summary] The present invention relates to novel members of the TNF ligand superfamily. More specifically, provided is an isolated nucleic acid molecule encoding human apoptosis-inducing molecule II (AIM II). AIM II polypeptides are also provided. Vectors, host cells and recombinant methods for producing AIM II are also provided. Further, the present invention relates to screening methods for identifying agonists and antagonists of AIM II activity. Therapeutic methods for treating lymphadenopathy, abnormal bone development, autoimmune and other immune system disorders, graft-versus-host disease, rheumatoid arthritis, osteoarthritis, and new treatments such as tumor cell proliferation Methods for inhibiting formation are also provided.

Description

Detailed Description of the Invention

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to novel members of the TNF ligand superfamily. More specifically, an isolated nucleic acid molecule encoding a human apoptosis inducing molecule II (AIM II) is provided. AIM II polypeptides are also provided, as are vectors, host cells and recombinant methods for producing AIM II polypeptides. The invention further relates to screening methods for identifying agonists and antagonists of AIM II activity. Therapeutic methods for treating lymphadenopathy, autoimmune diseases, graft versus host disease, and for inhibiting neoplasia (eg, tumor cell growth) are also provided.

(Related field) Human tumor necrosis factor α (TNF-α) and human tumor necrosis factor β (TNF-β,
Or lymphotoxin) is a member associated with a broad class of polypeptide mediators, including interferons, interleukins, and growth factors (collectively called cytokines) (Beutler, B. and Cera).
mi, A. , Annu. Ret ,. Immunol. , 7: 625-655 (1
989)).

Tumor necrosis factor (TNF-α and TNF-β) was originally discovered as a result of its antitumor activity, but is now the mediator of apoptosis, cell activation and cell proliferation in several transformed cell lines. It is recognized to play an important role as a pleiotropic cytokine with numerous biological activities including, as well as in immune regulation and inflammation.

To date, a known member of the TNF ligand superfamily has been TNF.
-Α, TNF-β (lymphotoxin α), LT-β, OX40L, Fas ligand, CD30L, CD27L, CD40L and 4-IBBL. The ligands of the TNF ligand superfamily are acidic TNF-like molecules with approximately 20% (range, 12% to 36%) sequence homology in the extracellular domain, and biologically active forms of trimers / It exists mainly as a membrane-bound form that is a complex of multimers. To date, soluble forms of the TNF ligand superfamily have been identified as TNF and LTα.
, And Fas ligand only (for a general review, see
Gruss, H .; And Dower, S .; K. , Blood, 85 (12): 3.
378-3404 (1995)), which is incorporated herein by reference in its entirety.

These proteins are involved in the regulation of cell proliferation, cell activation, and cell differentiation, including regulation of cell survival or death by apoptosis or cytotoxicity (A
rmitage, R .; J. Curr. Opin. Immunol. 6; 407
(1994) and Smith, C.I. A. , Cell 75: 959 (1994).
)).

Mammalian development depends on both cell proliferation and differentiation as well as programmed cell death, which occurs via apoptosis (Walker et al., Metho).
ds Achiev. Exp. Pathol. 13:18 (1988)). Apoptosis plays an important role in the destruction of immune thymocytes that recognize self-antigens. This failure of the normal clearance process may play a role in autoimmune disease (Gam
mon et al., Immunology Today 12: 193 (1991)).

[0007] Itoh et al. (Cell 66: 233 (1991)) described that the cell surface antigen Fas / CD95 mediates apoptosis and is involved in clonal clearance of T cells. Fas is a combination of activated T cells, B cells, neutrophils, and in thymus, liver, heart and lung, and in adult mice, activated T cells, B cells, neutrophils, and ovary (Watanabe-Fukunaga et al. , J. Immu
nolo. 148: 1274 (1992)). Apoptosis is induced in an experiment in which a monoclonal antibody against Fas is cross-linked to Fas (Yonehara et al., J. Exp. Med. 169: 1747 (1989);
Traauth et al., Science 245: 301 (1989)). Furthermore, F
There are examples where binding of a monoclonal antibody to as may stimulate T cells under certain conditions (Alderson et al., J. Exp. Med. 178: 2231 (
1993)).

Fas antigen is a cell surface protein with a relative molecular weight of 45 kd. Both human and mouse genes for Fas have been found in Watanabe-Fukuu.
naga et al. (J. Immunol. 148: 1274 (1992)) and It.
cloned by Oh et al. (Cell 66: 233 (1991)). The proteins encoded by these genes are both transmembrane proteins with structural homology to the nerve growth factor / tumor necrosis factor receptor superfamily, which contains two TNF receptors, a low-affinity nerve growth receptor. Factor receptor and LTβ receptor CD40, CD27, CD30
, And OX40.

Recently, Fas ligands have been described (Suda et al., Cell 75: 1).
169 (1993)). This amino acid sequence shows that Fas ligand is a type II transmembrane protein belonging to the TNF family. Fas ligand is expressed on spleen cells and thymocytes. This purified Fas ligand has a molecular weight of 40 kd.

Recently, it has been demonstrated that Fas / Fas ligand interaction is required for apoptosis following T cell activation (Ju et al., Nature 373: 444).
(1995): Bruner et al., Nature 373: 441 (1995).
). Activation of T cells induces both proteins on the cell surface. Subsequent interaction between the ligand and receptor results in cell apoptosis. This supports a potential regulatory role for Fas / Fas ligand interaction-induced apoptosis during the normal immune response.

The polypeptides of the present invention, based on structural and biological similarities,
It was identified as a new member of the NF ligand superfamily.

Clearly, there is a need for factors that regulate activation and differentiation of normal and abnormal cells. Therefore, there is a need for the identification and characterization of such factors that regulate activation and differentiation of both normal and diseased cells. In particular, there is a need to isolate and characterize additional Fas ligands that control apoptosis for the treatment of autoimmune diseases, graft-versus-host disease, rheumatoid arthritis and lymphadenopathy. SUMMARY OF THE INVENTION The present invention is encoded by the amino acid sequence shown in FIGS. 1A and 1B (SEQ ID NO: 2), or a cDNA clone inserted into a bacterial host on August 22, 1996 as ATCC Deposit No. 97689. Having an amino acid sequence
Provided is an isolated nucleic acid molecule that includes a polynucleotide that encodes a polypeptide. The present invention also provides the amino acid sequence shown in Figure 1C and Figure 1D (SEQ ID NO: 40), or the amino acid sequence encoded by the cDNA clone inserted into the bacterial host as ATCC Deposit No. 97483 on May 15, 1996. Have AI
Provided is an isolated nucleic acid molecule comprising a polynucleotide encoding a M II polypeptide.

The invention also provides a recombinant vector comprising the isolated nucleic acid molecule of the invention, and a host cell comprising this recombinant vector, as well as making such vectors and host cells, and by recombinant techniques. Methods for using these to produce AIM II polypeptides or peptides.

The present invention further provides an isolated AIM II polypeptide having an amino acid sequence encoded by the polynucleotides described herein.

The term “AIM II” polypeptide, as used herein, refers to membrane-bound proteins (including cytoplasmic, transmembrane, and extracellular domains) as well as truncated proteins that retain AIM II polypeptide activity. including. In one embodiment, the soluble AIM II polypeptide comprises all or part of the extracellular domain of the AIM II protein, but lacks the transmembrane region that results in retention of the polypeptide on the cell membrane. Soluble AIM II is also soluble AIM II
If the protein can be secreted, it can include parts of the transmembrane region or parts of the cytoplasmic domain or other sequences. The 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 when expressed.

The present invention also relates to lymphadenopathy, rheumatoid arthritis, autoimmune diseases (eg, inflammatory autoimmune disease, myasthenia gravis), graft-versus-host disease, IgE-mediated allergic reaction, anaphylaxis, adulthood. AIM II polypeptides, especially human AIM II poly, that can be used to treat distress such as respiratory distress syndrome, Crohn's disease, allergic asthma, acute lymphocytic leukemia (ALL), non-Hodgkin's lymphatic disease, and Graves' disease. Provide a peptide. These polypeptides can be used to stimulate peripheral tolerance, destroy some transformed cell lines, mediate cell activation and cell proliferation, and as the first mediator of immune regulation and inflammatory responses. Can be functionally related.

The invention further provides compositions comprising an AIM II polynucleotide or AIM II polypeptide for administration to cells in vitro, cells ex vivo, and cells in vivo, or to a multicellular organism. In certain preferred embodiments of this aspect of the invention, the composition comprises or consists of an AIM II polynucleotide for expressing the AIM II polypeptide in a host organism for treatment of a disease. . What I especially like about this is that
Expression in human patients for the treatment of dysfunction associated with aberrant endogenous activity of AIM II.

The present invention also provides screening methods for identifying compounds that can enhance or inhibit the cellular response induced by AIM II. The method comprises contacting cells expressing AIM II with a candidate compound, assaying the cellular response, and comparing the cellular response to a standard cellular response (the standard is Assayed when contact is made in the absence)
. An elevated cellular response to the standard thereby indicates that the compound is an agonist, and a reduced cellular response to the standard indicates that the compound is an antagonist.

In another aspect, screening assays for AIM II agonists and antagonists are provided. Use this antagonist to treat, diagnose, and / or prognose septic shock, inflammation, cerebral malaria, HIV virus activation, graft-host rejection, bone resorption, and cachexia (waxing or malnutrition). You can In a preferred embodiment, AIM II antagonists of the invention (eg, anti-AIM II antibodies) are used to treat graft versus host disease.
It may be treated, prevented, diagnosed, and / or prognostic.

In a further aspect of the invention, AIM II is used to inhibit increased angiogenesis or increased endothelial cell proliferation to invade bone and cartilage, as is often observed in RA. Can be used to treat rheumatoid arthritis (RA).

In a further aspect of the invention, AIM II is used to either inhibit the binding of a ligand to a receptor or to bind a receptor and activate a receptor-mediated cellular response. Cell receptors (eg L
It can inhibit or activate cellular responses mediated by T-β-R, TR2, CD27, and TRANS).

A further aspect of the invention is the step of treating an individual in need of elevated levels of AIM II activity in the body, a therapeutically effective amount of the isolated AIM of the invention.
A method comprising administering to such an individual a composition comprising a II polypeptide or an agonist thereof.

Yet a further aspect of the invention is the reduced levels of AIM II in the body.
A method comprising treating an individual in need of activity, administering to such an individual a composition comprising a therapeutically effective amount of an AIM II antagonist.

DETAILED DESCRIPTION The present invention encodes an AIM II polypeptide having the amino acid sequence shown in FIGS. 1A and 1B (SEQ ID NO: 2), which was determined by sequencing the cDNA. Provided is an isolated nucleic acid molecule that comprises, or alternatively consists of, a polynucleotide. The AIM II protein of the present invention is a human TN.
F-α (SEQ ID NO: 3), human TNF-β (SEQ ID NO: 4), human lymphotoxin (
Shares sequence homology with SEQ ID NO: 5), and the human Fas ligand (SEQ ID NO: 6) (Figures 2A-2F). The nucleotide sequence shown in FIGS. 1A and 1B (SEQ ID NO: 1) is the American Type Culture on August 22, 1996.
Collection, 10801, University Blvd. , M
anassas, VA 20110-2209, USA, deposit number 9
7689 was obtained by sequencing the cDNA given. The deposited cDNA clone is pBluescript SK (-) plasmid (Stra
Tagene, La Jolla, CA). The nucleotide sequences shown in FIGS. 1C and 1D are set forth in American Type March 15, 1996.
Culture Collection, 10801, University
Blvd. , Manassas, VA 20110-2209, USA, and was obtained by sequencing the cDNA given accession number 97483.

Nucleic Acid Molecules Unless otherwise indicated, all of the nucleotide sequences herein that have been determined by sequencing DNA molecules include an automated DNA sequencing device (eg, A
applied Biosystems, Inc. All of the amino acid sequences of the polypeptides encoded by the DNA molecules determined herein using the model 373), and predicted by translation of the DNA sequences as determined above. Was done. Therefore, as is known in the art for all DNA sequences determined by this automated approach, any nucleotide sequence determined herein may contain some errors. A nucleotide sequence determined by automated manipulation is typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. Is. The actual sequence can be more accurately determined by other efforts, including manual DNA sequencing methods well known in the art. Also, as is known in the art, one insertion or one deletion compared to the actual sequence in the determined nucleotide sequence causes a frameshift in the translation of the nucleotide sequence, so that it was determined. The deduced amino acid sequence encoded by the nucleotide sequence is completely different from the amino acid sequence actually encoded by the sequenced DNA molecule. This frameshift begins at the point of such insertion or deletion.

Using the information provided herein, eg, the nucleotide sequences of FIGS. 1A and 1B, nucleic acid molecules of the invention encoding AIM II polypeptides can be labeled using standard cloning and screening procedures (eg, starting Procedure for cloning cDNA using mRNA as the substance). As an illustration of the invention, the nucleic acid molecule described in Figures 1A and 1B (SEQ ID NO: 1) was found in a cDNA library derived from human macrophage ox LDL (HMCCB64). The same gene was also identified in a cDNA library derived from activated T cells (HT4CC72). The determined nucleotide sequence of the AIM II cDNA of FIGS. 1A and 1B (SEQ ID NO: 1) contains an open reading frame encoding a protein of 240 amino acid residues, which is shown in FIG.
And the extracellular sequence comprising the start codon at positions 49-51 of the nucleotide sequence of 1B (SEQ ID NO: 1), about 60 to about 240 amino residues of Figures 1A and 1B (SEQ ID NO: 2), or alternatively consisting thereof. A domain, a transmembrane domain comprising, or alternatively consisting of, amino acids from about positions 37 to about 59 of Figures 1A and 1B (SEQ ID NO: 2), from about positions 1 to about of Figures 1A and 1B (SEQ ID NO: 2). It has an intracellular domain comprising, or alternatively consisting of, the amino acid at position 36, as well as a predicted molecular weight of approximately 26.4 kDa. Figures 1A and 1B (SEQ ID NO: 2)
The AIM II protein shown in Figure 2 is about 27% identical and about 51% similar to the amino acid sequence of human Fas ligand (Figures 2A-2F), and the amino acid sequence of human TNF-α (Figures 2A-2F). About 26% identical and about 47% similar to the sequence. The TNF ligand-like molecule functions as a dimer, and AIM I
If I is homologous to a TNF ligand-like molecule, this molecule also appears to function as a homodimer.

As the skilled artisan will appreciate, due to the potential sequencing errors discussed above,
The putative AIM II polypeptide encoded by the deposited cDNA is approximately 2
It contains 40 amino acids, but can be anywhere in the range 230-250 amino acids. It is further understood that, depending on the criteria used, the relative precise "positioning" of the extracellular, intracellular, and transmembrane domains of the AIM II polypeptide is slightly different. For example, the exact location of the AIM II extracellular domain in FIGS. 1A and 1B (SEQ ID NO: 2) may vary slightly (eg, location (addr) depending on the criteria used to define the domain.
ess) may "shift" about 1-5 residues).

As shown, the nucleic acid molecules of the invention are in the form of RNA (eg, mRNA),
Or it may be in the form of DNA, including, for example, cDNA and genomic DNA obtained by cloning or produced synthetically. This DNA can be double-stranded or single-stranded. Single-stranded DNA or RNA can be the coding strand, also known as the sense strand, or it can be the non-coding strand, also referred to as the antisense strand.

By an “isolated” nucleic acid molecule, a nucleic acid molecule (D
NA or RNA) is intended. For example, recombinant DNA contained in a vector
The molecule is considered isolated for the purposes of the present invention. Isolated DNA
Further examples of 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 as produced synthetically.

However, a library (eg, a genomic library or a cDNA library) that has not been isolated from other members of the library (eg, in the form of a homogenous solution containing clones and other members of the library) Nucleic acid contained in a clone that is a member of the) or a chromosome that has not been isolated or removed from the cell or cell lysate (eg, "chromosomal spread" (as in karyotype)
chromosome spread)) is not "isolated" for the purposes of the present invention. As discussed further herein, the nucleic acids isolated according to the present invention can be produced naturally, recombinantly, or synthetically.

The isolated nucleic acid molecule of the present invention can be prepared as shown in FIGS. 1A and 1B (SEQ ID NO: 1) or FIG.
C and 1D (SEQ ID NO: 38) containing, or alternatively consisting of, an open reading frame (ORF); a DNA molecule shown in FIGS. 1A and 1B (
SEQ ID NO: 2) or AIM II shown in Figures 1C and 1D (SEQ ID NO: 39)
A DN comprising, or alternatively consisting of, a coding sequence for a protein; and comprising, or alternatively consisting of, a sequence substantially different from the above sequences
The A molecule, however, still encodes the AIM II protein due to the degeneracy of the genetic code. Of course, the genetic code is well known in the art. Therefore, it is routine for those skilled in the art to produce such degenerate variants.

The nucleic acid molecule according to the present invention further comprises a nucleic acid molecule encoding a full length AIM polypeptide lacking the N-terminal methionine.

Further, the invention provides a nucleic acid molecule having a nucleotide sequence related to a portion of SEQ ID NO: 1. It was determined from the following related cDNAs: HT4C
C72R (SEQ ID NO: 20).

In another aspect, the invention provides a cDNA contained in the plasmid deposited under ATCC Deposit No. 97689 deposited on August 22, 1996,
Or an isolated nucleic acid molecule encoding an AIM II polypeptide having the amino acid sequence encoded by the cDNA contained in the plasmid deposited under ATCC Deposit No. 97483 deposited on Mar. 15, 1996. . Preferably, the nucleic acid molecule encodes a polypeptide encoded by one of the deposited cDNAs above. The invention further provides an isolated nucleic acid molecule having the nucleotide sequence shown in Figures 1A and 1B (SEQ ID NO: 1) or Figures 1C and 1D (SEQ ID NO: 38), or the AIM contained in the deposited plasmid above. II. An isolated nucleic acid molecule having one nucleotide sequence of the cDNA or a nucleic acid molecule having a sequence complementary to one of the above sequences is provided. Such isolated molecules, especially DNA molecules, are used, for example, as probes for genetic mapping by in situ hybridization with chromosomes and for detecting expression of the AIM II gene in human tissues (eg, Northern light). It is useful as a probe for blotting analysis).

A further embodiment of the invention is at least 80% identical, and more preferably at least 85%, 90%, 92%, 95 to the following nucleotide sequences:
An isolated nucleic acid molecule comprising, or consisting of, a polynucleotide having a nucleotide sequence that is%, 96%, 97%, 98%, or 99% identical: (a) Figures 1A and 1B. (SEQ ID NO: 2) or a nucleotide sequence encoding an AIM II polypeptide having the complete amino acid sequence in Figures 1C and 1D (SEQ ID NO: 39); (b) Figures 1A and 1B (SEQ ID NO: 2).
A nucleotide sequence encoding an AIM II polypeptide having the amino acid sequence of, but lacking the N-terminal methionine; A nucleotide sequence encoding a peptide;
(D) a nucleotide sequence encoding the extracellular domain of AIM II polypeptide; (e) a nucleotide sequence encoding the transmembrane domain of AIM II polypeptide; (f) a nucleotide sequence encoding the intracellular domain of AIM II polypeptide; (g) ) A nucleotide sequence encoding a soluble AIM II polypeptide having an extracellular domain and an intracellular domain but lacking a transmembrane domain; and (h) above (a), (b), (c), (d), ( e), (f), or (g)
A nucleotide sequence complementary to any of the nucleotide sequences in.

A further embodiment of the invention is at least 80% identical, and more preferably at least 85%, 90%, 92%, 95 to the following nucleotide sequences:
An isolated nucleic acid molecule comprising, or consisting of, a polynucleotide having a nucleotide sequence that is%, 96%, 97%, 98%, or 99% identical: (a) Figures 1C and 1D. (SEQ ID NO: 39) a nucleotide sequence encoding an AIM II polypeptide having a sequence of about amino acids 1 to about 208; (b) about amino acids 7 to 7 in FIGS. 1C and 1D (SEQ ID NO: 39).
A nucleotide sequence encoding an AIM II polypeptide having a sequence of about 208; (c) amino acids about 34 to about 20 in Figures 1C and 1D (SEQ ID NO: 39).
A nucleotide sequence encoding an AIM II polypeptide having the sequence of 8; and (d) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), or (c) above.

As a practical matter, any particular nucleic acid molecule can be used, for example, against the nucleotide sequence shown in FIGS. 1A and 1B (SEQ ID NO: 1) or FIGS. 1C and 1D (SEQ ID NO: 38), or deposited cDNA At least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 9 relative to the nucleotide sequence of
Whether or not they are 9% identical is determined by known computer programs (eg, Best
fit program (Wisconsin Sequence Analysis)
sis Package, Version 8 for Unix (registered trademark)
, Genetics Computer Group, University
Research Park, 575 Science Drive, Madi
Son, WI53711) can be determined conventionally. Bestfit
, Smith and Waterman, Advances in Appli.
The local homology algorithm of ed Mathematics 2: 482-489 (1981) is used to find the segment of best homology between two sequences. Of course, if Bestfit or any other sequence alignment program is used to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the invention, the identity will be the same. Is calculated over the entire length of the reference nucleotide sequence and the parameters are set such that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.

A nucleic acid having a nucleotide sequence that is “identical” to the reference nucleotide sequence of the present invention, for example at least 95%, is a polynucleotide sequence whose AIM II
It is intended that the nucleotide sequence of the polynucleotide be identical to the reference sequence, except that it may contain up to 5 point mutations for each 100 nucleotides of the reference nucleotide sequence encoding the polypeptide. In other words, up to 5% of the nucleotides in the reference sequence may be deleted or replaced by another nucleotide in order to obtain a polynucleotide having a nucleotide sequence at least 95% identical to the reference nucleotide sequence. Alternatively, up to 5% of the total nucleotides of the reference sequence can be inserted into the reference sequence. These variations in the reference sequence may be interspersed, either individually between the nucleotides in the reference sequence or in one or more contiguous groups in the reference sequence, at the 5'terminal position or 3'of the reference nucleotide sequence. It can occur at the terminal positions or somewhere between these terminal positions. The query sequence can be the entire sequence shown in FIGS. 1A and 1B or FIGS. 1C and 1D, the ORF (open reading frame), or any particular fragment as described herein.

As a practical matter, any particular nucleic acid molecule or polypeptide will be at least 80%, 85%, 90%, 92%, 95%, 9% of the nucleotide sequences of the invention.
Whether 6%, 97%, 98%, or 99% identical can be conventionally determined using known computer programs. A preferred method for determining the best overall compatibility (also referred to as the overall sequence alignment) between a query sequence (the sequence of the invention) and a subject sequence is described by Brutlag et al. (Comp.
． App. Biosci. 6: 237-245 (1990)) based on the FASTDB computer program. In a sequence alignment, both the query and subject sequences are DNA sequences. RNA sequences can be compared by converting U to T. The results of this global sequence alignment are expressed as percent identity (%). The preferred parameters used in the FASTDB alignment of DNA sequences to calculate percent identity are: Matrix = Unitary, k-tuple = 4, Mismat.
ch Penalty = 1, Joining Penalty = 30, Rand
animation Group Length = 0, Cutoff Scor
e = 1, Gap Penalty = 5, Gap Size Penalty 0
． 05, Window Size = 500 or the length of the nucleotide sequence of interest (
Whichever is shorter).

If the subject sequence is shorter than the query sequence due to the 5'or 3'deletions (and not due to internal deletions), manual corrections must be made to the results. This is because the FASTDB program does not consider 5'and 3'truncations of the subject sequence when calculating percent identity. For a subject sequence that is cleaved at the 5'or 3'end, the percent identity to the query sequence is the 5'and 3'of the subject sequence that is not matched / aligned as a percentage of the total bases of the query sequence. Corrected by calculating the number of bases in. Whether the nucleotides are matched / aligned is determined by the results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity and calculated by the FASTDB program above using the specified parameters to arrive at the final percent identity score. This corrected score is what is used for the purposes of the present invention. Only the bases outside the 5'and 3'bases of the subject sequence that are not matched / aligned with the query sequence, as indicated by the FASTDB alignment, are calculated for the purpose of manually adjusting the percent identity score.

For example, a 90 base subject sequence is aligned with a 100 base query sequence to determine percent identity. The deletion occurs at the 5'end of the subject sequence, so the FASTDB alignment shows no match / alignment at the first 10 bases at the 5'end. 1
0 unpaired bases represent 10% of the sequence (number of bases at the 5'and 3'ends that do not match / total number of bases in the query sequence), so 10% is FASTD
Subtracted from the percent identity score calculated by the B program.
If the remaining 90 bases were an exact match, the final percent identity would be 90%. In another example, a 90 base subject sequence is compared to a 100 base query sequence. In this case, the deletion is an internal deletion, so that there are no bases at the 5'or 3'of the subject sequence that are not matched / aligned with the query. In this case, FASTD
The percent identity calculated by B is not manually corrected. Again, only the 5'or 3'bases of the subject sequence that do not match / align with the query sequence are manually corrected. No other manual correction is made for the purposes of the present invention.

The present application is directed to the nucleic acid sequences shown in FIGS. 1A and 1B (SEQ ID NO: 1), or FIGS. 1C and 1D (SEQ ID NO: 38), or to the nucleic acid sequence of the deposited cDNA, respectively. At least 80%, 85%, 90%, 92%, 95%, 9 with or without encoding a polypeptide having AIM II activity.
A nucleic acid molecule that is 6%, 97%, 98%, or 99% identical. Because, this is how one of skill in the art would use this nucleic acid molecule even if the particular nucleic acid molecule does not encode a polypeptide having AIM II activity (eg as a hybridization probe or the polymerase chain reaction ( (As PCR) as a primer). The use of the nucleic acid molecule of the present invention that does not encode a polypeptide having AIM II activity includes, in particular, (1) cDNA
Isolating the AIM II gene or allelic variants thereof in the library; (2) Verma et al., Human Chromosomes: A Manu.
al of Basic Technologies, Pergamon Pres
s, New York (1988) to provide the correct chromosomal location of the AIM II gene, metaphase chromosomal spread (metaph).
in situ hybridization (e.g., FISH) to an asceromosome spread; and (3) AI in specific tissues.
Northern blot analysis to detect M II mRNA expression is included.

However, at least 80 relative to the nucleic acid sequence shown in FIGS. 1A and 1B (SEQ ID NO: 1) and FIGS. 1C and 1D (SEQ ID NO: 38) or to one of the deposited cDNAs. %, 85%, 90%, 92%, 95%, 96%, 9
Nucleic acid molecules having sequences that are 7%, 98%, or 99% identical are preferred, which actually encode a polypeptide having the protein activity of AIM II. By a "polypeptide having AIM II activity", a polypeptide exhibiting similar, but not necessarily identical, activity to that of the AIM II protein of the invention as measured in a particular biological assay. Is intended.
For example, the AIM II protein cytotoxic activity is described in Zarres et al., Cell.
70: 31-46 (1992) and can be measured using propidium iodide staining to demonstrate apoptosis. Alternatively, AIM II that induces apoptosis is also described by Gavierli et al. Cell. Biol.
119: 493-501 (1992), can be measured using TUNEL staining. Polypeptides encoded by these polynucleotides are also within the scope of the invention.

Briefly, propidium iodide staining is performed as follows. Cells from either tissues or cultures are fixed in formaldehyde, cut into frozen sections and stained with propidium iodide. Cell nuclei are visualized with propidium iodide using a confocal fluorescence microscope. Cell death is due to the nuclear condensation nucleus (
Chromosomal agglomeration, shrinkage, and / or nuclear fragmentation).

Of course, due to the degeneracy of the genetic code, one of skill in the art would appreciate for one nucleic acid sequence in the deposited cDNA, or in FIGS. 1A-B (SEQ ID NO: 1), or FIG.
And at least 80% relative to the nucleic acid sequence set forth in 1D (SEQ ID NO: 38),
Many nucleic acid molecules having sequences that are 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical "have AIMII protein activity."
Immediately recognize that it encodes a polypeptide. In fact, all degenerate variants of these nucleotide sequences encode the same polypeptide, and this will be apparent to those skilled in the art without the comparison assay described above. It is further recognized in the art that a suitable number also encodes a polypeptide having AIM II protein activity for nucleic acid molecules that are not such degenerate variants. This is because the substitution of amino acids by which one of ordinary skill in the art either appears to be sufficiently unlikely or not likely to significantly affect the function of the protein, as further described below ( For example, substituting one aliphatic amino acid with a second aliphatic amino acid).

For example, guidance on how to make phenotypically silent amino acid substitutions can be found in Bowie, J. et al. U. Et al., "Deciphering the Message"
e in Protein Sequences: Tolerance to
Amino Acid Substitutions ", Science 24
7: 1306-1310 (1990), where the authors point out that the protein is surprisingly tolerant of amino acid substitutions.

Preferred polypeptides and polynucleotide fragments of the invention are AI
M II encodes a polypeptide exhibiting functional activity. AIM II "functionally active" polypeptides include full-length polypeptides and / or secreted AIM I
By I polypeptide is meant a polypeptide that may exhibit one or more known functional activities associated with it. These functional activities include, but are not limited to, biological activity, antigenicity, ability to bind anti-AIM II antibody (or ability to compete with polypeptide for binding). Immunogenicity (ability to produce antibodies that bind to the polypeptide), ability to form multimers with the polypeptides of the invention,
And the ability of the polypeptide to bind a receptor or ligand (eg, T
R2 (International Publication WO96 / 34095), LT-β receptor, TR6 (International Publication WO98 / 30694), and CD27).

The functional activity of AIM II polypeptides, and fragments, variants, derivatives and analogs thereof, can be assayed by a variety of methods.

For example, in one embodiment various immunoassays known in the art are assayed for the ability to bind to, or compete with, the full-length polypeptide of the invention for binding to anti-AIM II antibody. Can be used. Such assays include radioimmunoassay, ELISA (enzyme-linked immunosorbent assay),
"Sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (eg using colloidal gold, enzymes or radioisotope labels), Western blots, precipitation reactions, agglutination assays (eg gels) Competitive and non-competitive assay systems using techniques such as agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, immunoelectrophoresis assays and the like, but are not limited thereto.
In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment,
This secondary antibody is labeled. Many means are known in the art for detecting binding in immunoassays and are within the scope of the invention.

In another embodiment, the identified ligand (eg, DR5 (International Publication WO98
/ 41629), TR10 (see International Publication WO98 / 54202), 312C2 (see International Publication WO98 / 06842), and TR11, TR11SV1, and TR11SV2 (US patent application 09/17).
6,200))), or a polypeptide fragment, variant, or derivative of the invention is assessed for its ability to multimerize, binding can be achieved, for example, by reducing and non-reducing gel chromatography, protein affinity. Assays can be performed by means well known in the art such as chromatography, as well as affinity blotting. Generally, Phizicky, E .; Et al., 1995
, Microbiol. Rev. 59: 94-123. In another embodiment, the physiological correlates of binding to its substrate (signalling) can be assayed.

[0051]   Other methods are known to those of skill in the art and are within the scope of this invention.

The present invention further relates to polynucleotides comprising fragments of the isolated nucleic acid molecules described herein. The nucleotide sequence of the deposited cDNA, or the nucleotide sequence shown in Figures 1A and 1B (SEQ ID NO: 1) or Figure 1C.
And a fragment of the isolated nucleic acid molecule having the nucleotide sequence set forth in 1D (SEQ ID NO: 38), as discussed herein, is useful as a diagnostic probe and primer, at least about 15 nt. , And more preferably at least about 20 nt, even more preferably at least 30 nt, and even more preferably at least 40 nt in length. In this context, "about" includes the values specifically stated, or a few or more (5, 4, 3, 2, or 1) nucleotides larger or smaller. Naturally, 50, 75, 100, 125, 150, 175, 200, 22
5, 250, 275, 300, 325, 350, 375, 400, 425, 45
0, 475, 500, 525, 550, 575, 600, 625, 650, 67
5, 700, 725, 750, 775, 800, 825, 850, 875, 90
0, 925, 950, 975, 1000, 1025, 1050, 1075, 11
The longer fragments of 00, 1125, or 1150 nt in length are also nucleotide sequences of the deposited cDNA, or the nucleotide sequence shown in FIGS. 1A and B (SEQ ID NO: 1) or the nucleotide sequences shown in FIGS. 1C and 1D ( Useful according to the invention as a fragment corresponding to most, if not all, of SEQ ID NO: 38). A fragment with a length of at least 20 nt means
For example, containing 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in FIGS. 1A and B (SEQ ID NO: 1) or the nucleotide sequence shown in FIGS. 1C and 1D (SEQ ID NO: 38). A fragment.

The present invention further provides a subportion of the AIM II domain.
Ion) is directed to a polynucleotide containing a fragment of the isolated nucleic acid molecule. In particular, the present invention provides a polynucleotide comprising a nucleotide sequence of a member selected from the group consisting of the following nucleotides consisting of SEQ ID NO: 1: 49-108, 59-118, 69-128, 79-138, 89- 148,
99-156, 109-158, 109-168, 119-178, 129-1
88, 139-198, 149-208, 157-216, 157-225, 1
68-227, 178-237, 188-247, 198-257, 208-2
67, 218 to 277, 226 to 285, 236 to 295, 246 to 305, 2
56-315, 266-325, 276-335, 286-345, 296-3
55, 306 to 365, 316 to 375, 326 to 385, 336 to 395, 3
46-405, 356-415, 366-425, 376-435, 386-4
45, 396-455, 409-469, 456-515, 476-536, 4
96-556, 516-575, 536-595, 576-635, 596-6
55, 636-695, 656-715, 696-755, 706-765 and 716-768. The present invention is further directed to a polynucleotide comprising an isolated nucleic acid molecule encoding a domain of AIM II. In one aspect, the invention provides a polynucleotide comprising a nucleic acid molecule that encodes the AIM II β-sheet region described in Table 2. Representative examples of such polynucleotides include nucleic acid molecules that encode a polypeptide having an amino acid sequence selected from the group consisting of the following amino acid residues of SEQ ID NO: 2: about 7 to about 14 amino acid residues. A group, about 18 to about 23 amino acid residues, about 17 to about 25 amino acid residues, about 33 to about 46 amino acid residues,
About 35 to about 39 amino acid residues, about 57 to about 60 amino acid residues, about 67 to about 7
2 amino acid residues, about 102 to about 107 amino acid residues, about 121 to about 126 amino acid residues, about 131 to about 166 amino acid residues, about 141 to about 152 amino acid residues, about 158 to about 169 amino acid residues, about 213 to about 221 amino acid residues, about 232 to about 240 amino acid residues. The invention is further directed to an isolated polypeptide comprising an amino acid sequence selected from the group consisting of the following amino acid residues of SEQ ID NO: 2: about 7 to about 14 amino acid residues, about 18 to about. 23 amino acid residues, about 17 to about 25 amino acid residues, about 33 to about 46 amino acid residues, about 3
5 to about 39 amino acid residues, about 57 to about 60 amino acid residues, about 67 to about 72 amino acid residues, about 102 to about 107 amino acid residues, about 121 to about 126 amino acid residues, about 131 to about 166 amino acid residues, about 141 to about 152 amino acid residues, about 158 to about 169 amino acid residues, about 213 to about 221 amino acid residues, about 232 to about 240 amino acid residues. In this context, "about" includes the values specifically stated, or a few or more (5, 4, 3, 2, or 1) nucleotides larger or smaller.

A nucleic acid fragment of the invention is at least 80% identical to, and more preferably at least 85% identical to, the nucleic acid molecule encoding the β-sheet region of the AIM II protein, as well as the nucleic acid molecule encoding the β-sheet region of the AIM II protein. %
, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence. Beta sheet area and at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%
, Or 99% polynucleotide-encoding polypeptides, as well as polypeptides encoded by these polynucleotides are within the scope of the invention.

The present invention is also directed to a polynucleotide comprising an isolated nucleic acid molecule encoding a polypeptide having an amino acid sequence selected from the group consisting of the following amino acid residues of SEQ ID NO: 2: about 94. ~ About 100 amino acid residues, about 121 to about 12
4 amino acid residues, about 127 to about 135 amino acid residues, about 139 to about 149 amino acid residues, about 160 to about 168 amino acid residues, about 175 to about 185 amino acid residues, about 197 to 209. Amino acid residues, about 213 to about 220 amino acid residues, and about 232 to about 240 amino acid residues. The invention is further directed to an isolated polypeptide comprising an amino acid sequence selected from the group consisting of the following amino acid residues of SEQ ID NO: 2: about 94 to about 100 amino acid residues, about 121 to about. 12
4 amino acid residues, about 127 to about 135 amino acid residues, about 139 to about 149 amino acid residues, about 160 to about 168 amino acid residues, about 175 to about 185 amino acid residues, about 197 to 209. Amino acid residues, about 213 to about 220 amino acid residues, and about 232 to about 240 amino acid residues. In this context, "about" includes the values specifically stated, or a few or more (5, 4, 3, 2, or 1) nucleotides larger or smaller.

Preferred nucleic acid fragments of the invention include nucleic acid molecules that encode the epitope-containing portion of the AIM II protein. In particular, such nucleic acid fragments of the invention include nucleic acid molecules that encode: 1, 2, 3, selected from about 13 to about 20 amino acid residues of Figures 1 and 1B (SEQ ID NO: 2). A polypeptide comprising 4, 5 or more amino acid sequences; a polypeptide comprising about 23 to about 36 amino acid residues of Figure 1 (SEQ ID NO: 2); about 69 to about 79 of Figures 1A and 1B (SEQ ID NO: 2). A polypeptide comprising amino acid residues; about 85 to about 9 of Figures 1A and 1B (SEQ ID NO: 2).
A polypeptide comprising 4 amino acid residues; about 1 in FIGS. 1A and 1B (SEQ ID NO: 2).
A polypeptide comprising amino acid residues from 67 to about 178; a polypeptide comprising amino acid residues from about 184 to about 196 of FIGS. 1A and 1B (SEQ ID NO: 2); and FIG.
A polypeptide comprising about 221 to about 233 amino acid residues of A and 1B (SEQ ID NO: 2). In this context, "about" includes the values specifically stated, or a few or more (5, 4, 3, 2, or 1) nucleotides larger or smaller. The present inventors have found that the above-mentioned polypeptide fragment is AIM I
It was determined to be the antigenic region of the I protein. Methods for determining other such epitope-containing portions of the AIM II protein are described below. Polypeptides encoded by these polynucleotides are also included in the invention.

As described in more detail below, AIM II polynucleotides can be used in accordance with the present invention in a variety of applications, particularly those that make use of the chemical and biological properties of AIM II. Among these applications are autoimmune diseases, immune deficiencies and abnormal cell proliferation. A further application relates to the diagnosis and treatment of disorders of cells, tissues and organs.

In another aspect, the invention provides for the nucleic acid molecule of the invention described above (eg, the complement of a polynucleotide fragment described herein, or ATCC acceptance under stringent hybridization conditions). No. 97689 or ATCC Accession No. 97483). An isolated nucleic acid molecule comprising a polynucleotide that hybridizes to a portion of the polynucleotide in the plasmid. By “stringent hybridization conditions” is intended an overnight incubation at 42 ° C. in a solution containing the following, followed by washing the filters in 0.1 × SSC at about 65 ° C .: 50% formamide. 5 × SSC (750 mM NaCl, 75 mM trisodium citrate), 5
0 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, and 20 g / ml denatured, sheared salmon sperm DNA.

A polynucleotide that hybridizes to a “portion” of a polynucleotide is
At least about 15 nucleotides (nt) of the reference polynucleotide, and more preferably at least about 20 nt, even more preferably at least 30 nt.
, And even more preferably, a polynucleotide (either DNA or RNA) that hybridizes to about 30-70 nt. In this context, "about" means a value that is specifically stated, or a few (5, 4,
3, 2, or 1) nucleotides larger or smaller. These are useful as diagnostic probes and primers, as discussed above and in further detail below.

A portion of a “at least 20 nt long” polynucleotide refers to, for example, a reference polynucleotide (eg, as shown in FIGS. 1A and 1B (SEQ ID NO: 1) or FIGS. 1C and 1D (SEQ ID NO: 38)). 20 or more contiguous nucleotides from the nucleotide sequence of one of the deposited cDNAs or nucleic acid sequences) is intended.

Of course, polynucleotides that hybridize only to the poly A sequence (eg, FIG.
A and 1B (SEQ ID NO: 1) or the 3'terminal poly (A) region of the AIM II cDNA shown in Figures 1C and 1D (SEQ ID NO: 38), or a complementary stretch of T (or U) residues. Soybean polynucleotides are not included in the polynucleotides of the invention used to hybridize to a portion of the nucleic acids of the invention. This is because such a polynucleotide hybridizes to any nucleic acid molecule comprising a poly (A) stretch, or its complement (eg, in particular, any of the oligo dT-sensitized cDNA libraries produced,). Double-stranded cDNA).

As indicated, the nucleic acid molecule of the invention encoding the AIM II polypeptide may include, but is not limited to: a nucleic acid molecule encoding the amino acid sequence of the polypeptide alone; Coding sequences and additional sequences (
A sequence encoding a leader or secretory sequence, eg a pre-protein sequence or a pro-protein sequence or a pre-pro-protein sequence); with or without an additional non-coding sequence, said polypeptide Coding sequences (additional non-coding sequences include, for example, introns and non-coding 5 ′ and 3 ′ sequences (eg, including splicing and polyadenylation signals, roles in transcription, mRNA processing (eg, ribosome binding and mRNA stability ), Which contains transcribed non-translated sequences,
(Not limited to these))); additional coding sequences encoding additional amino acids to provide additional functionality. Thus, the sequence encoding the polypeptide can be fused to a marker sequence, such as a peptide encoding sequence that facilitates purification of the fused polypeptide. In certain preferred embodiments of this aspect of the invention, the marker amino acid sequence is, inter alia, a pQE vector (Qage).
n, Inc. ) Is a hexahistidine peptide, such as the tag provided in. Many of these are commercially available. For example, Gentz et al., Proc. Natl
． Acad. Sci. Hexahistidine provides convenient purification of fusion proteins, as described in USA 86: 821-824 (1989).
The "HA" tag is another peptide useful for purification that corresponds to an epitope derived from the influenza hemagglutinin protein, which is described by Wilson et al., C.
37: 767 (1984). As discussed below, other such fusion proteins include AIM II fused to Fc at the N-terminus or C-terminus.

The present invention further relates to variants of the nucleic acid molecules of the invention which encode part, analog or derivative of the protein of AIM II. Variants such as natural allelic variants can occur naturally. By "allelic variant" is intended one of several alternative forms of a gene that occupy a given locus on the chromosome of an organism. Genes II, Lewin, B.M. Edited by John Wiley
& Sons, New York (1985).

Non-naturally occurring variants can be produced using mutagenesis techniques known in the art including, but not limited to: oligonucleotide-mediated mutagenesis, alanine. Scanning, PCR mutagenesis, site-directed mutagenesis (eg Carter et al., Nucl. Acids Res. 13:
4331 (1986); and Zoller et al., Nucl. Acids Res
． 10: 6487 (1982)), cassette mutagenesis (eg, W
ells et al., Gene 34: 315 (1985)), restriction selective mutagenesis (see, eg, Wells et al., Philos. Trans. R. Soc. Lo).
ndon Ser. A 317: 415 (1986)).

Such variants include variants produced by nucleotide substitutions, deletions, or additions. The substitution, deletion, or addition can include one or more nucleotides. Variants can be altered in coding regions, non-coding regions, or both. Changes in the coding regions may result in conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions, and deletions, which do not alter the properties and activities of the AIM II protein, or portions thereof. Also particularly preferred in this regard are conservative substitutions.

Vectors and Host Cells The present invention also includes vectors containing the isolated DNA molecules of the present invention, host cells genetically engineered with recombinant vectors, and recombinantly engineered AIM II polypeptides or fragments thereof. Regarding the production of.

The polynucleotide may be ligated into a vector containing a selectable marker for propagation in the host. Generally, the plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in complex with a charged lipid. If the vector is a virus, the vector can be packaged in vitro using a suitable packaging cell line and then transduced into host cells.

The DNA insert may be labeled with a suitable promoter (eg, λ, to name a few).
PL promoter of phage, E. E. coli lac, trp, and tac promoters, SV40 early and late promoters, and retrovirus L
TR promoter). Other suitable promoters are known to those of skill in the art. Expression constructs include sites for transcription initiation, termination,
And in the transcribed region it further comprises a ribosome binding site for translation. The coding portion of the mature transcript expressed by this construct preferably comprises a translation codon that begins at the beginning of the translated polypeptide and a stop codon appropriately placed at the end of the translated polypeptide (UAA, UGA, or UAG) is included.

As indicated, the expression vector preferably contains at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture as well as E. coli. It contains a tetracycline or ampicillin resistance gene for cultivation in E. coli and other bacteria. Representative examples of suitable hosts include bacterial cells (eg E. coli, Streptomyces and Salmonella typhimurium cells); fungal cells (eg yeast cells); insect cells (eg Drosophila S2 and Spod).
optera Sf9 cells); animal cells (eg, CHO, COS, and Bo)
wes melanoma cells); and plant cells, but are not limited thereto. Appropriate culture mediums and 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 that include operator and promoter elements operably linked to the nucleotide sequence encoding the protein of interest. One example of such a vector is pHE4-5, which is described in detail below.

The pHE4-5 vector (SEQ ID NO: 5, as summarized in FIGS. 10 and 11)
The components of 0) include: 1) the neomycin phosphotransferase gene as a selectable marker, 2) E. E. coli replication origin, 3) T5 phage promoter sequence, 4) two lac operator sequences, 5) Shine-Dalgarno sequence, 6) lactose operon repressor gene (lacIq). The origin of replication (OriC) is derived from pUC19 (LTI, Gaithersburg, MD). The promoter and operator sequences were synthetically produced.
Synthetic preparation 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. AI
The nucleotide sequence encoding MII (SEQ ID NO: 1) is operably linked to these promoters and operators by inserting the nucleotide sequence between the NdeI and Asp718 sites of the pHE4-5 vector.

As mentioned above, the pHE4-5 vector contains the lacIq gene. lacI
q is an allele of the lacI gene, conferring tight regulation of the lac operator. Amann, E .; Et al., Gene 69: 30-315 (1988).
Stark, M .; Gene 51: 255-267 (1987). lacI
The q gene binds to the lac operator sequence and is downstream (ie, 3 ')
Encodes a repressor protein that blocks transcription of the sequence. But lac
The Iq gene product is separated from the lac operator in the presence of either lactose or certain lactose analogs such as isopropyl β-D-thiogalactopyranoside (IPTG). Therefore, AIM II has pHE4-5
It is not produced in appreciable amounts in uninduced host cells containing the vector. However, induction of these host cells by the addition of agents such as IPTG has been shown by AIM II
It results in the expression of the coding sequence.

The promoter / operator sequence of the pHE4-5 vector (SEQ ID NO: 51) contains the T5 phage promoter and two lac operator sequences. One operator is located 5'to the transcription 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 downstream sequences in the absence of the lac operon inducer (eg IPTG). Expression of the operably linked sequence located downstream from this lac operator results in expression of the lac operon inducer (
For example, it can be induced by the addition of IPTG). la to lacIq protein
Binding of c inducers results in their release from the lac operator sequence and initiation of transcription of the operably linked sequence. Regulation of the lac operon of gene expression is described by Devlin, T .; , Textbook of Biochemis
try with Clinical Correlations, 4th Edition (1
997), pp. 802-807.

The pHE4 series of vectors include pHE4-5 excluding the AIM II coding sequence.
Contains all the components of the vector. Features of the pHE4 vector include an optimized synthetic T5 phage promoter, a lac operator, and Shine-Dalgarno sequences. Furthermore, these sequences are also optimally spaced so that expression of the inserted gene can be tightly regulated and high levels of expression occur upon induction.

Among the known bacterial promoters suitable for use in producing the proteins of the present invention are E. coli. E. coli lacI and lacZ promoters, T3 and T7
Includes promoters, gpt promoters, λ PR and PL promoters, and trp promoters. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the retroviral LTR promoter (eg, Rous sarcoma virus (RSV)), and the metallothionein promoter (eg, mouse metallothionein-I). Promoter).

The pHE4-5 vector also contains a Shine-Dalgarno sequence 5 ′ to the AUG start codon. The Shine-Dalgarno sequence is generally a short sequence located approximately 10 nucleotides upstream (ie, 5 ') from the AUG start codon. These sequences essentially direct the prokaryotic ribosome to the AUG start codon.

Accordingly, the present invention also relates to expression vectors useful for producing the proteins of the present invention. This aspect of the invention is exemplified by the pHE4-5 vector (SEQ ID NO: 50).

Among the preferred vectors for use in bacteria are pQE70, pQE60 and pQE-9 (available from Qiagen); pBS vector, Phagescr.
ipt vector, Bluescript vector, pNH8A, pNH16a,
pNH18A, pNH46A (available from Stratagene); and ptrc99a, pKK223-3, pKK233-3, pDR540, pRI.
T5 (available from Pharmacia). Among the preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and p.
SG (available from Stratagene); and pSVK3, pBPV,
There are pMSG and pSVL (available from Pharmacia). Other suitable vectors will be readily apparent to those of skill in the art.

Introduction of the construct into the host cell may be carried out by calcium phosphate transfection, DE
It can be provided by AE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals (eg, Davis et al., Basic Methods In Mole).
circular Biology (1986)).

The polypeptide may be expressed in a modified form (eg a fusion protein) and may include not only secretory signals, but also additional heterologous functional regions. For example, additional amino acids, particularly regions of charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in host cells during purification or 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. Among other things, the addition of peptide moieties that cause secretion or excretion to polypeptides to improve stability and facilitate purification is well known and routine in the art.

A preferred fusion protein comprises a heterologous region from immunoglobulin that is useful for solubilizing proteins. For example, EP-A-0 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of the constant region of immunoglobulin molecules together with another human protein or portions thereof. In many cases, the Fc portion of the fusion protein will be of considerable benefit for use in therapy and diagnosis, thus resulting in, for example, improved pharmacokinetic properties (
EP-A 0232262). On the other hand, for some uses it is desirable to be able to delete the Fc portion after the fusion protein has been expressed, detected and purified in the advantageous manner described. This is the case when the Fc part proves to be an obstacle to its use in therapy and diagnosis, for example when the fusion protein is used as an antigen for immunization. For example, in drug discovery, hI
Human proteins such as the L-5 receptor have been fused to the Fc portion for the purpose of a Heisle Put screening assay to identify antagonists of hIL-5 (D. Bennett et al., Journal of Molec.
ural Recognition Volume 8: 52-58 (1995) and K.
． Johnson et al., The Journal of Biological
Chemistry. 270, 16: 9459-9471 (1995)).

AIM II protein can be recovered and purified from recombinant cell culture by well known methods. This method includes ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. To be Most preferably, high performance liquid chromatography (“HPLC”) is used for purification. As the polypeptide of the present invention,
Naturally purified products, products of chemical synthetic procedures, and recombinant techniques from prokaryotic or eukaryotic hosts, including, for example, bacterial cells, yeast cells, higher plant cells, insect cells, and mammalian cells. The products produced are included. Depending on the host used in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or non-glycosylated. further,
The polypeptides of the present invention may also include initiating modified methionine residues, in some cases as a result of host-mediated processes.

In addition to including host cells containing the vector constructs discussed herein, the invention also deletes or replaces endogenous genetic material (eg, AIM II coding sequences). And / or to include genetic material (eg, a heterologous polynucleotide sequence) that is operably linked to the AIM II polynucleotide of the invention and that activates, alters, and / or amplifies the endogenous AIM II polynucleotide. An engineered, primary host cell of vertebrate origin, especially of mammalian origin,
Includes secondary host cells, and immortalized host cells. For example, techniques known in the art include heterologous regulatory regions (eg, promoters and / or enhancers).
And endogenous AIM II polynucleotide sequences can be used to operably link via homologous recombination (eg, US Pat. No. 5,641,670).
Issue (Published June 24, 1996); WO96 / 29411 (September 26, 1996)
Published); WO94 / 12650 (published August 4, 1994); Koller et al., Proc. Natl. Acad. Sci. USA 86: 8932-8935.
(1989); and Zijlstra et al., Nature 342: 435-4.
38 (1989), the disclosure of each of which is incorporated by reference in their entirety).

AIM II Polypeptides and Fragments The invention further provides an amino acid sequence encoded by one of the deposited cDNAs, or the amino acid sequence of FIG. 1A and FIG. 1B (SEQ ID NO: 2) or FIG.
Isolated AIM I having the amino acid sequence of C and FIG. 1D (SEQ ID NO: 39).
There is provided a peptide or polypeptide comprising or consisting of I polypeptide, or a portion of the above polypeptides.

The polypeptide of the present invention may be a polypeptide encoded by one of the deposited cDNAs, or the polypeptide of FIGS. 1A and 1B (SEQ ID NO: 2) or the polypeptides of FIGS. 1C and 1D ( SEQ ID NO: 39), FIGS. 1A and 1B
A polypeptide consisting of (SEQ ID NO: 2) but lacking the N-terminal methionine, extracellular domain, transmembrane domain, intracellular domain, soluble polypeptide (including or all of the extracellular and intracellular domains) , But lacking the transmembrane domain), and a polypeptide encoded by one of the deposited cDNAs, or the polypeptide of FIGS. 1A and 1B (SEQ ID NO: 2) or the polypeptides of FIGS. 1C and 1D. A polypeptide having at least 80% identity to (SEQ ID NO: 39), more preferably at least 85%, 90%, 92% or 95% identical, even more preferably at least 96%, 97%, 98% or 99% identical. , And also at least 30 amino acids, and more preferably at least 50 Including a portion of such a polypeptide having an amino acid.

The polypeptides of the present invention are further (a) FIG. 1C and FIG. 1D (SEQ ID NO: 39).
An AIM II polypeptide having an amino acid sequence from about 1 to about 208; (b
) AIM II polypeptide having about 7 to about 208 amino acid sequences of Figures 1C and 1D (SEQ ID NO: 39); and (c) about 34 to about 208 of Figures 1C and 1D (SEQ ID NO: 39). AIM II polypeptide having a single amino acid sequence and a polypeptide encoded by one of the deposited cDNAs, FIGS. 1A and 1B (SEQ ID NO: 2) or FIGS. 1C and 1D (SEQ ID NO: 3).
9) polypeptide, at least 80% identical, more preferably at least 85
%, 90%, 92% or 95% identical, even more preferably at least 96%
, 97%, 98% or 99% identical polypeptides, and also includes portions of such polypeptides having at least 30 amino acids, and preferably at least 50 amino acids.

The reference amino acid sequence of an AIM II polypeptide contains, for example, at least 95%
A polypeptide having the "identical" amino acid sequence is
It is intended that the amino acid sequence of the polypeptide is identical to the reference sequence, except that it may contain up to 5 amino acid changes for every 100 amino acids of the reference amino acid of the IM II polypeptide. In other words, up to 5% of the amino acid residues of the reference sequence may be deleted or replaced by another amino acid in order to obtain a polypeptide having an amino acid sequence which is at least 95% identical to the reference amino acid sequence. , Or multiple amino acids up to 5% of all amino acid residues of the reference sequence can be inserted into the reference sequence. These changes in the reference sequence can occur at the amino-terminal or carboxy-terminal positions of the reference amino acid sequence, or somewhere between these terminal positions, either individually between the residues of the reference sequence or the reference sequence. Interspersed with either one or more as a contiguous group.

As a practical matter, any particular polypeptide may be labeled, for example, with the amino acid sequence shown in FIGS. 1A and 1B (SEQ ID NO: 2) or the amino acid sequence shown in FIGS. 1C and 1D (SEQ ID NO: 39). , Or at least 80%, 85%, 90%, 92% to the amino acid sequence encoded by one of the deposited cDNAs.
, 95%, 96%, 97%, 98% or 99% identical, Best
fit program (Wisconsin Sequence Analysis)
Package, Version 8 for Unix (registered trademark), Ge
netics Computer Group, University Res
rch Park, 575 Science Drive, Madison,
It can be determined conventionally using known computer programs such as WI 53711). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present invention, of course, there will be no homology over the entire length of the reference amino acid sequence. As a percentage is calculated, and 5 of the total number of amino acid residues in the reference sequence.
The parameters are set so that gaps of homology up to% are allowed.

By virtue of a polypeptide having an amino acid sequence which is at least 95% “identical” to a query amino acid sequence of the invention, the subject polypeptide sequence will contain up to 5 amino acid changes for every 100 amino acids of the query amino acid. Except to be obtained, the amino acid sequence of the subject polypeptide is intended to be identical to the query sequence. In other words, in order to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the query amino acid sequence, 5% of the amino acid residues of the target sequence are
Up to can be inserted, deleted, or replaced with another amino acid. These changes in the reference sequence can occur at the amino-terminal or carboxy-terminal positions of the reference amino acid sequence, or somewhere between these terminal positions, either individually between the residues of the reference sequence or the reference sequence. Scattered either within one or more consecutive groups within.

As a practical matter, any particular polypeptide may be represented by, for example, at least 80%, 85%, 90%, 92% of the amino acid sequence shown in Table 1 or the amino acid sequence encoded by the deposited DNA, 95%, 96%, 97%, 98% or 9
Whether 9% identical or not can be determined using conventional known computer programs. A preferred method for determining the best overall match (also referred to as an overall sequence alignment) between a query sequence (the sequence of the invention) and a subject sequence is described by Brutlag et al. (Comp. App. Biosci). . 6: 237-245 (
(1990)) based on the FASTDB computer program. In array alignment, the query and target sequences are
Either both are nucleotide sequences or both are amino acid sequences. The result of the overall sequence alignment above is in percent identity. Preferred parameters used for FASTDB amino acid alignment are: Matri
x = PAM 0, k-tuple = 2, Mismatch Penalty = 1
, Joining Penalty = 20, Randomization Gr
up Length = 0, Cutoff Score = 1, Windows S
size = length of sequence, Gap Penalty = 5, Gap Size Pen
arty = 0.05, Window Size = 500 or the length of the target amino acid sequence (whichever is shorter).

If the sequence of interest is shorter than the query sequence due to N-terminal or C-terminal deletions (and not due to internal deletions), manual corrections are made to the results. This is F
This is because the ASTDB program does not consider N-terminal and C-terminal truncations of the subject sequence when calculating the overall percent identity. For a subject sequence that is truncated at the N- and C-termini to the query sequence, the percent identity is the N-terminus of the subject sequence that does not match / align with the corresponding subject residue as a percentage of the total bases of the query sequence. Corrected by calculating the number of residues in the query sequence that are C-terminal. The determination of whether residues are matched / aligned is determined by F
Determined by the results of ASTDB sequence alignment. This percentage is then subtracted from the percent identity and calculated by the FASTDB program described above using specific parameters to arrive at the final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues in the query (reference) sequence that extend beyond the N-terminus or C-terminus of the subject sequence are considered for the purpose of manually adjusting the percent identity score. That is, only residues that are not matched / aligned with the N- or C-termini of the query sequence are calculated 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. Deletions occur 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 (mismatched N-terminus and C
Represents the number of residues at the end / total number of residues in the query sequence, and the result is FAST
10% is subtracted from the percent identity score calculated by the DB 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 to a 100 residue query sequence. In this case, the deletion is an internal deletion, so there are no residues at the N-terminus or C-terminus of the subject sequence that are not matched / aligned with the query sequence. In this case, the percent identity calculated by FASTDB is not manually corrected. Again, only residue positions outside the N- and C-termini of the subject sequence that do not match / align with the query sequence, as shown in the FASTDB alignment, are manually corrected.
No other manual correction is made for the purposes of the present invention.

A naturally processed form of AIM II, which has a full length AIM II cD
Affinity-purified on the LT-β receptor column from the conditioned medium of NA-transformed MCA-38 cells) was obtained from Leu-83 to Val of SEQ ID NO: 2.
-240 (see, eg, Example 10). However, AIM II is processed differently in COS cells to produce AIM II that is cleaved between Glu-67 and Met-68 to produce a polypeptide having amino acids 68-240 of SEQ ID NO: 2. Seems to occur. In addition, COS cells also cleave AIM II between Met-68 and Val-69, resulting in amino acids 69-2 of SEQ ID NO: 2.
Yields a polypeptide having 40.

The polypeptides of the present invention are preferably provided in isolated form. By "isolated polypeptide" is intended a polypeptide removed from its native environment. Thus, a polypeptide produced within a recombinant host and / or contained within a recombinant host cell is considered isolated for the purposes of the present invention. Also intended as an "isolated polypeptide" is a polypeptide that has been partially or substantially purified from a recombinant host. For example,
Recombinantly produced versions of AIM II polypeptides can be substantially purified by the one-step method described by Smith and Jhonson (Gene 67: 31-40 (1988)).

The AIM II polypeptides of the present invention can be monomeric or multimeric (ie, dimers, trimers, tetramers and higher multimers). Therefore, the present invention provides
It relates to monomers and multimers of the AIM II polypeptides of the invention, their preparation and compositions (preferably pharmaceutical compositions) containing them. In particular embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers.
In a further embodiment, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.

The multimers encompassed by the present invention can be homomers or heteromers. As used herein, the term homomer refers to the AIM II polypeptides of the invention (AIM described herein.
II fragments, variants, splice variants and fusion proteins)
A multimer containing only. These homomers can include AIM II polypeptides having the same or different amino acid sequences. In certain embodiments, homomers of the invention are multimers that include only AIM II polypeptides having the same amino acid sequence. In another particular embodiment, the homomers of the invention are multimers that include AIM II polypeptides with different amino acid sequences. In certain embodiments, a multimer of the invention comprises a homodimer (eg, containing an AIM II polypeptide having the same or different amino acid sequence) or a homotrimer (eg, the same and / or different amino acid sequence). Including AIM II polypeptides). In a further embodiment, the homomeric multimers of the invention are at least homodimers, at least homotrimers or at least homotetramers.

The term heteromer, as used herein, refers to a multimer that comprises one or more heterologous polypeptides (ie, polypeptides of different proteins) in addition to the AIM II polypeptide of the invention. In a particular embodiment, the multimers of the invention are
It is a heterodimer, a heterotrimer or a heterotetramer. In a further embodiment, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer or at least a heterotetramer.

The multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and / or covalent attachment and / or may be indirectly linked, for example by liposome formation. Thus, in one embodiment, multimers of the invention (eg, homodimers or homotrimers, etc.) are formed when the polypeptides of the invention contact each other in solution. In another embodiment, a heteromultimer of the invention (eg, a heterotrimer or heterotetramer, etc.) is a polypeptide of the invention in solution in which the antibody to the polypeptide of the invention (of the invention (Including antibodies to the heterologous polypeptide sequence in the fusion protein). In other embodiments, the multimers of the invention are formed by covalent attachment to and / or between polypeptides of the invention. Such covalent bonds are included in the polypeptide sequence (eg, the polypeptide sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 39, or in the polypeptide encoded by the cDNA of the deposit designated ATCC Accession No. 97689 or 97483). Polypeptide sequence). In one example, the covalent bond is a bridge between cysteine residues that are present within the polypeptide sequence that interact in the native (ie, naturally occurring) polypeptide. In another example, this covalent bond is the result of chemical or recombinant manipulation. Alternatively, such a covalent bond may comprise one or more amino acid residues contained in the heterologous polypeptide sequence in the AIM II fusion protein. In one example, the covalent bond is between the heterologous sequences contained in the fusion protein of the invention (see, eg, US Pat. No. 5,478,925). In a particular example, this covalent bond is between the heterologous sequences contained in the AIM II-Fc fusion protein of the invention (as described herein).
In another particular example, the covalent attachment of a fusion protein of the invention is carried out by another TNF family ligand / receptor member capable of forming covalently linked multimers (eg,
Between heterologous polypeptide sequences derived from osteoprotegerin and the like (eg International Publication No. WO 98/49305 (
The contents of which are incorporated herein by reference in their entirety)).

The multimers of the present invention can be produced using chemical techniques known in the art. For example, the polypeptides desired to be included in the multimers of the invention can be chemically crosslinked using linker molecules and linker molecule length optimization techniques known in the art (eg, herein). US Pat. No. 5,47, which is incorporated by reference in its entirety
See No. 8,925). Further, the multimers of the present invention can be generated using techniques known in the art to generate one or more intermolecular cysteine residues between cysteine residues present within the sequence of the polypeptide desired to be included in the multimer. Crosslinks can be formed (see, eg, US Pat. No. 5,478,925, incorporated herein by reference in its entirety). Furthermore, the polypeptides of the present invention can 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 are known to include one or more of these modified polypeptides. It can be applied to generate multimers containing peptides (see, eg, US Pat. No. 5,478,925, incorporated herein by reference in its entirety). In addition, techniques known in the art can be applied to produce liposomes containing the polypeptide components that are desired to be included in the multimers of the invention (see, eg, herein in its entirety). See US Pat. No. 5,478,925).

Alternatively, the multimers of the present invention can be produced using genetic engineering techniques known in the art. In one embodiment, the polypeptides included in the multimers of the invention are recombinantly produced using fusion protein technology described herein or otherwise known in the art (eg, the present The specification is incorporated by reference in its entirety,
See U.S. Pat. No. 5,478,925). In a particular embodiment, a polynucleotide encoding a homodimer of the invention comprises a polynucleotide sequence encoding a polypeptide of the invention linked to a sequence encoding a linker polypeptide and then further to the original C-terminus. To the N-terminus in the opposite direction to a synthetic polynucleotide encoding the translation product of the polypeptide (lacking the leader sequence) (eg, incorporated herein by reference in its entirety). The
See U.S. Pat. No. 5,478,925). In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to include a transmembrane domain (or hydrophobic peptide or signal peptide) and to reconstitute the membrane. A recombinant polypeptide of the invention is produced that can be incorporated into liposomes by techniques (see, eg, US Pat. No. 5,478,925, which is incorporated herein by reference in its entirety).

The invention further includes compositions comprising a polypeptide of the invention fused or linked to a domain other than the variable region of an antibody. For example, the polypeptides of the invention can be fused or conjugated to the Fc region of an antibody, or a portion thereof. The portion of this antibody fused to a polypeptide of the invention may comprise the hinge region, CH1 domain, CH2 domain, and CH3 domain, or any combination of all or portions thereof. The polypeptides of the invention are fused or conjugated to the antibody moieties described above using methods known in the art to increase the in vivo half-life of the polypeptide or for use in immunoassays. obtain. These polypeptides can also be fused or conjugated to portions of the above antibodies to form multimers. For example, an Fc portion fused to a polypeptide of the invention can form a dimer through a disulfide bond between the Fc portions. Higher multimeric forms can be made by fusing the polypeptide to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the invention to antibody moieties are known in the art. For example, US Pat. No. 5,336,603
No. 5,622,929; No. 5,359,046; No. 5,349.
No. 053; No. 5,447, 851; No. 5,112, 946; EP 0.
307 434; EP 0 367 166; WO96 / 04388; WO91.
/ 06570; Ashkenazi et al., PNAS 88: 10535-1053.
9 (1991); Zheng, X .; X. Et al., J. Immunol. 154: 55
90-5600 (1995); and Vil, H .; Et al., PNAS 89: 113.
37-11341 (1992) (the above references are incorporated by reference in their entireties).

The term “AIM II” polypeptide as used herein refers to a membrane-bound protein (comprising or consisting of a cytoplasmic domain, a transmembrane domain, and an extracellular domain) and AIM II functional activity. Contains the truncated protein that it retains. In one embodiment, the soluble AIM II polypeptide is AI
It contains or consists of all or part of the extracellular domain of the M II protein, but lacks a transmembrane region that results in retention of the polypeptide on the cell membrane. Soluble AIM II may also include parts of the transmembrane region or parts of the cytoplasmic domain or other sequences if the soluble AIM II protein can be secreted. The 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 when expressed.

The polypeptides of the present invention can be labeled with SDS-PAGE using methods well known to those of skill in the art.
It has uses in gel or molecular sieve gel filtration columns including, but not limited to, the purpose as molecular weight markers.

It is recognized in the art that some amino acid sequences of AIM II polypeptides can be altered without significant effect on protein structure or function.
When such differences in sequence are intended, it should be considered that there are critical regions on the protein that determine activity.

Accordingly, the present invention further provides substantial AIM II polypeptide activity,
Or variations of the AIM II polypeptide, including regions of the AIM II protein, such as the protein portions discussed below. Such variants include deletions, insertions, inversions, repeats, and type substitutions. As mentioned above, guidance on which amino acid changes may be phenotypically silent is given by Bowi
e, J. U. Et al., "Deciphering the Message in
Protein Sequences: Tolerance to Amino
Acid Substitutions ", Science 247: 130.
6-1310 (1990). Polynucleotides encoding these fragments, derivatives, or analogs are also encompassed by the present invention.

Accordingly, a fragment, derivative or analog of the polypeptide of FIGS. 1A and 1B (SEQ ID NO: 2) or FIG. 1C or 1D (SEQ ID NO: 39), or a fragment of the polypeptide encoded by one of the deposited cDNAs. , A derivative or analog can be: (i) one or more amino acid residues (eg 3, 5, 8, 10, 15 or 20) are conserved or non-conserved amino acid residues ( Preferably conserved amino acid residues), such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) one or more amino acid residues The group is a substituent (eg 3, 5,
8, 10, 15 or 20), or (iii) a mature polypeptide fused to another compound (eg, polyethylene glycol), such as a compound that increases the half-life of the polypeptide, or (Iv) Those in which additional amino acids are fused to a mature polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence used for purification of the mature polypeptide or proprotein sequence. Such fragments, derivatives and analogs are considered to be within the skill of those in the art given the teachings herein.
Polynucleotides encoding these fragments, derivatives, or analogs are also encompassed by the present invention.

A particular object is the replacement of a charged amino acid with another charged amino acid and a neutral or negatively charged amino acid. The latter results in proteins with a reduced positive charge, improving the characteristics of the AIM II protein. Preventing agglomeration
Highly desirable. Protein aggregation not only results in loss of activity, but can be problematic when preparing pharmaceutical formulations. Because aggregation can be immunogenic. (Pinckard et al., Clin Exp. Immunol. 2
: 331-340 (1967); Robbins et al., Diabetes 36:
838-845 (1987); Cleland et al., Crit. Rev. Ther
apeutic Drug Carrier Systems 10: 307-
377 (1993)).

Amino acid substitutions may also alter the selectivity of binding to cell surface receptors.
Ostade et al., Nature 361: 266-268 (1993) describe specific mutations that result in the selective binding of TNF-α to only one of the two known types of TNF receptors. Accordingly, the AIM II receptors of the present invention may be derived from one or more (eg, 3, 5, 8, 10,
15 or 20) amino acid substitutions, deletions or additions.

As shown, the alterations are preferably those of less significant nature, such as conservative amino acid substitutions that do not significantly affect protein folding or activity (see Table 1).

[0110]

[Table 1] Of course, the number of amino acid substitutions will be made by those skilled in the art depending on many factors, including those mentioned above. Generally, the number of substitutions for any given AIM-II polypeptide will not exceed 50, 40, 30, 25, 20, 15, 10, 5 or 3.

Amino acids essential for function in the AIM II proteins of the invention can be identified by methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Scie).
nce 244: 1081-1085 (1989)). The latter procedure introduces a single alanine mutation at every residue in the molecule. The mutant molecule obtained is then
Tested for biological activity such as receptor binding, or in vitro or in vitro proliferative activity. Sites of interest for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance, or photoaffinity labeling (Smith et al., J. Mol. Biol. 224: 899-904 (.
1992) and de Vos et al., Science 255: 306-312 (
1992)).

In the present invention, amino-terminal deletion mutants are also included. Such variants include deletion of at least the first amino acid at the N-terminus of SEQ ID NO: 2, but N-
Those comprising the amino acid sequence set forth in SEQ ID NO: 2 with a deletion of no more than the first 114 amino acid residues at the terminus. Alternatively, this deletion is
It comprises at least the first 35 amino acid residues at the N-terminus of SEQ ID NO: 2, but not more than the first 114 residues at the N-terminus. Alternatively, the deletion comprises at least the first 59 amino acid residues at the N-terminus of SEQ ID NO: 2, but not more than the first 114 residues at the N-terminus. Alternatively, this deletion is made by at least the first 67 amino acid residues at the N-terminus but not the first 1 at the N-terminus of SEQ ID NO: 2.
Includes amino acid residues not exceeding 14 residues. Alternatively, the deletion is made by at least the first 68 amino acid residues at the N-terminus of SEQ ID NO: 2, but the first 114 amino acids at the N-terminus.
Includes no more than amino acid residues. Alternatively, the deletion comprises at least the first 73 amino acid residues at the N-terminus of SEQ ID NO: 2, but not more than the first 114 residues at the N-terminus. Alternatively, this deletion comprises at least the first 82 amino acid residues at the N-terminus of SEQ ID NO: 2, but not more than the first 114 residues at the N-terminus. Alternatively, the deletion is at least N of SEQ ID NO: 2.
-Comprising the first 100 amino acid residues at the terminal, but not exceeding the first 114 residues at the N-terminal. Polynucleotides encoding these deletion variants are also encompassed by the invention and are at least 80%, 85%, 90%, 92%, 95% relative to the polynucleotides encoding the above deletion variants. , 96%, 9
At least 80%, 85%, 90%, 92%, 95%, 96% for polynucleotides that are 7%, 98%, or 99% identical, and deletion mutants thereof.
, 97%, 98%, or 99% identical. The invention also includes the above-described polynucleotides fused to heterologous polynucleotides and the polypeptide expression products of these polynucleotides.

In addition to the above N-terminal deletion mutant ranges, the present invention also relates to all combinations of the above ranges. For example, the first 59 of at least the N-terminus of SEQ ID NO: 2.
Amino acid residue, but no more than the first 67 amino acid residues at the N-terminus; more than at least the first 59 amino acid residues at the N-terminus but more than the first 68 amino acid residues at the N-terminus of SEQ ID NO: 2 No deletion; a deletion of at least the first 59 amino acid residues at the N-terminus of SEQ ID NO: 2, but not more than the first 73 amino acid residues at the N-terminus;
A deletion of no more than the first 59 amino acid residues at the N-terminus of SEQ ID NO: 2, but no more than the first 82 amino acid residues of the N-terminus; at least the first 59 amino acid residues of the N-terminus of SEQ ID NO: 2, but Deletion no more than the first 100 amino acid residues at the N-terminus; at least the first 67 amino acid residues at the N-terminus of SEQ ID NO: 2, but N-
Deletion no more than the first 73 amino acid residues at the terminal; SEQ ID NO: 2 at least the first 67 amino acid residues at the N-terminal but not more than the first 82 amino acid residues at the N-terminal; SEQ ID NO: 2 Of at least the first 67 amino acid residues at the N-terminus, but not more than the first 100 amino acid residues at the N-terminus; SEQ ID NO: 2, the first 68 amino acid residues at the N-terminus, but the N-terminus No more than the first 73 amino acid residues of SEQ ID NO: 2; at least the first 68 amino acid residues at the N-terminus of SEQ ID NO: 2;
However, the deletion does not exceed the first 82 amino acid residues at the N-terminus; the deletion does not exceed the first 68 amino acid residues at the N-terminus of SEQ ID NO: 2, but not the first 100 amino acid residues at the N-terminus; A deletion of no more than the first 73 amino acid residues at the N-terminus of SEQ ID NO: 2, but not the first 82 amino acid residues of the N-terminus; at least the first 73 amino acid residues of the N-terminus of SEQ ID NO: 2, but The first 100 at the N-terminus
A deletion of no more than amino acid residues; a deletion of at least the first 82 amino acid residues at the N-terminus of SEQ ID NO: 2, but no more than the first 100 amino acid residues of the N-terminus; Polynucleotides encoding these deletion variants are also encompassed by the invention and are at least 80%, 85%, 90%, 92%, 95% relative to the polynucleotides encoding the above deletion variants. , 96%, 97%, 98%, or 9
At least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to a polynucleotide that is 9% identical and deletion mutants thereof. A polynucleotide that encodes a polypeptide. The invention also includes the above-described polynucleotides fused to heterologous polynucleotides and the polypeptide expression products of these polynucleotides.

Preferred AIM II polypeptides are polypeptides having one or more of the sequences shown below (starting counting from the first amino acid (Met) in the protein):

[0115]

[Chemical 1] Particularly preferred embodiments include polypeptides that do not comprise or consist of one or more of the following AIM IIN terminal deletions: Gln-60-Val.
-240 (AIM II (amino acids 60-240)), Met-68 to Val-
240 (AIM II (amino acids 68-240)), Val-69 to Val-2.
40 (AIM II (amino acids 69-240)), Asp-74 to Val-24.
0 (AIM II (amino acids 74-240)), Leu-83 to Val-240.
(AIM II (amino acids 83-240)), and Ala-101 to Val-.
240 (AIM II (amino acids 101-240)). Polynucleotides encoding these polypeptides are also encompassed by the invention and are at least 80%, 85%, 90% of the polynucleotides encoding the above polypeptides.
%, 92%, 95%, 96%, 97%, 98%, or 99% identical polynucleotides, and at least 80%, 85% to these polypeptides
, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical. The invention also includes the above-described polynucleotides fused to heterologous polynucleotides and the polypeptide expression products of these polynucleotides.

Even if the deletion of one or more amino acids from the N-terminus of the protein results in an alteration or deletion of one or more biological functions of the protein, other biological activity is still retained. obtain. Thus, the ability of a truncated AIM II mutein to induce and / or bind an antibody that recognizes the intact or mature form of the polypeptide is
Generally, less than the majority of the full or mature polypeptides are retained when removed from the N-terminus. Whether a particular polypeptide lacking the N-terminal residue of the complete polypeptide retains such immunogenic activity will depend on the conventional methods described herein, and so on. If not, it can be easily determined by a conventional method known in the art. AIM II muteins with multiple deleted N-terminal amino acid residues appear to be able to retain some biological or immunogenic activity. In fact, peptides consisting of as few as six AIM II amino acid residues can often elicit an immune response.

Accordingly, the invention further provides a polypeptide having one or more residues deleted from the amino terminus of the AIM II amino acid sequence shown in FIGS. 1A and 1B (sequence up to phenylalanine at position 235). No. 2, and a polynucleotide encoding such a polypeptide). In particular, the present invention relates to FIGS. 1A and 1B.
(SEQ ID NO: 2) having a amino acid sequence of residues n-314, or a polypeptide thereof, wherein n is an integer in the range of 2-235,
And 236 is the position of the first residue from the N-terminus of the complete AIM II polypeptide that is considered necessary for at least the immunogenic activity of the AIM II polypeptide. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

More particularly, the present invention provides that the AIM II sequence set forth in SEQ ID NO: 2, in which the amino acid residues of SEQ ID NO: 2 are numbered consecutively 1 to 240, N-terminal to C-terminal. Except that that is identical to the sequences shown in FIGS. 1A and 1B), a polynucleotide encoding a polypeptide comprising or consisting of an amino acid sequence of a member selected from the group consisting of the following residues: provide:

[0119]

[Chemical 2] The present invention also relates to a polynucleotide sequence encoding the above polypeptide, which is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98.
%, Or 99% identical polynucleotide sequences. The present invention also includes the above polynucleotides fused to a heterologous polynucleotide. The polypeptides encoded by these polynucleotide sequences are also encompassed by the present invention.

As mentioned above, deletion of one or more amino acids from the C-terminus of a protein may result in modification or deletion of one or more biological functions of the protein, but not other biological functions. Activity can still be retained. Thus, a truncated AIM II mutein's ability to induce and / or bind an antibody that recognizes the intact or mature form of the polypeptide is generally less than the majority of the intact or mature polypeptide. Are retained when removed from the C-terminus. Whether a particular polypeptide lacking the C-terminal residue of the complete polypeptide retains such immunogenic activity depends on conventional methods described herein, and so on. If not, it can be easily determined by a conventional method known in the art. AIM II muteins with multiple deleted C-terminal amino acid residues appear to be able to retain some biological or immunogenic activity. In fact, peptides consisting of as few as six AIM II amino acid residues can often elicit an immune response.

Therefore, the present invention further includes FIGS. 1A and 1B up to the valine residue at position number 6.
Provided are polypeptides having one or more residues deleted from the carboxy terminus of the AIM II amino acid sequence set forth in (SEQ ID NO: 2), as well as polynucleotides encoding such polypeptides. In particular, the present invention relates to FIGS. 1A and 1B (
That is, there is provided a polypeptide comprising or consisting of the amino acid sequence of residues 1-m of SEQ ID NO: 2), where m is an integer in the range 2-239 and 6 is the AIM. The position of the first residue from the C-terminus of the complete AIM II polypeptide that is considered necessary for at least the immunogenic activity of the II polypeptide. Polynucleotides encoding these polypeptides are also
Included by the present invention.

More particularly, the present invention relates to the sequence of the AIM II sequence shown in FIGS. 1A and 1B (
This is because amino acid residues of SEQ ID NO: 2 are consecutively 1 to 2 from the N terminus to the C terminus.
(Identical to the sequence shown in SEQ ID NO: 2 except numbered 40)
A polynucleotide encoding a polypeptide comprising or consisting of an amino acid sequence of a member selected from the group consisting of the following residues is provided:

[0123]

[Chemical 3] The present invention also relates to a polynucleotide sequence encoding the above polypeptide, which is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98.
%, Or 99% identical, to a nucleic acid molecule comprising or consisting of a polynucleotide sequence. The present invention also includes the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotide sequences are also included by the invention.

The invention also provides a polypeptide having one or more amino acids deleted from both the amino and carboxyl termini of the AIM II polypeptide, wherein A
IM II polypeptides can be generally described as having residues nm of FIGS. 1A and 1B (ie, SEQ ID NO: 2), where n and m are the integers above.

In a further embodiment, the invention provides a polynucleotide encoding a polypeptide comprising or consisting of the amino acid sequence of residues 83-m ^{1 of} FIGS. 1A and 1B (ie, SEQ ID NO: 2). Where m ¹ is 89-239
And corresponds to the position of the amino acid residue in FIGS. 1A and 1B (SEQ ID NO: 2). For example, the present invention provides AIM I shown in Figures 1A and 1B (SEQ ID NO: 2).
The following residues of the sequence I sequence;

[0126]

[Chemical 4] A polynucleotide encoding a polypeptide comprising or consisting of an amino acid sequence of a member selected from the group consisting of: This application also includes a polynucleotide sequence encoding the above AIM II polypeptide,
At least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%
Or a nucleic acid molecule comprising or consisting of polynucleotide sequences that are 99% identical. The present invention also includes the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotide sequences are also included according to the invention.

In a further embodiment, the polynucleotide of the invention encodes a functional property of AIM II. In this regard, the preferred embodiment of the present invention is 1, 2
3, 4 or more one or more of the following functional domains: α-helix and α-helix forming region of AIM II (“α-region”), β-sheet and β-sheet forming region (“β- Area "), turns and turn-forming areas (" turn-area ")
, Coils and coil-forming regions (“coil-regions”), hydrophilic regions, hydrophobic regions, α-amphipathic regions, β-amphipathic regions, flexible regions, surface-forming regions and highly antigenic indicator regions. Or a fragment consisting of them.

Data representing structural or functional properties of AIM II as shown in FIGS. 3A-3F and / or Table 2 are set to default parameters DNA ^* STA.
Made using various identified modules and algorithms of R. In a preferred embodiment, the data shown in columns VIII, IX, XIII, and XIV of Table 2 can be used to determine regions of AIM II that show a high likelihood of antigenicity. Regions of high antigenicity are identified in columns VIII, IX, XIII by selecting values that represent regions of the polypeptide that are likely exposed to the surface of the polypeptide in an environment where antigen recognition may occur in the process of initiation of the immune response. , And / or IV.

Certain preferred regions for these are shown in FIGS. 3A-3F, but may be represented in tabular format or identified, as shown in Table 2. Figure 3A-
The data is shown in FIGS. 3A-3F in tabular form (see Table 2) using the DNA ^* STAR computer algorithm used to generate 3F (set to initial default parameters). It was The tabular format of the data in FIGS. 3A-3F may be used to easily determine the particular boundaries of the preferred region.

The above preferred regions shown in FIGS. 3A-3F and Table 2 are shown in FIGS. 1A and 1B.
Include, but are not limited to, regions of the above type identified by analysis of the amino acid sequence shown in. As shown in FIGS. 3A-3F and Table 2, such preferred regions are Garnier-Robson α-regions, β-regions, turn-regions.
Regions, and coil-regions (columns I, III, V, and VII in Table 2),
Chou-Fasman α-region, β-region, and turn-region (columns II, IV, and VI in Table 2), Kyte-Doolittle hydrophilic region (
Table 2, column VIII), Hopp-Woods hydrophobic region (Table 2, column IX), Ei.
senberg α-amphipathic region and Eisenberg β-amphipathic region (columns X and XI in Table 2), Karplus-Schulz flexible region (column XII in Table 2), high-antigenic index Jameson-Wolf region ( Included in Table 2 column XIII) and Emini surface forming areas (Table 2 column XIV).

[0131]

[Table 2] In this regard, highly preferred fragments include AIM I that combine several structural features, such as some of the features listed in Table 2 above.
There is a fragment containing the region of I.

The invention further relates to an isolated polypeptide comprising or consisting of a fragment of AIM II. In particular, the invention relates to amino acids 1-60, 11-70, 21-80, 31-90, 41-100, 51-110 of SEQ ID NO: 2.
, 61-120, 71-130, 81-140, 91-150, 101-160
, 111-170, 121-180, 131-190, 141-200, 151
-210, 161-220, 171-230, and 181-240, or an isolated polypeptide comprising or consisting of an amino acid sequence of a member selected from the group consisting of: Provide a separated polynucleotide.

The present invention also relates to an isolated polypeptide comprising or consisting of a domain of AIM II. In particular, the invention relates to the AIM I described in Table 2.
Polypeptides comprising or consisting of the β-sheet region of I are provided.
These polypeptides are from about 7 to about 14 amino acid residues, about 18 to about 23 amino acid residues, about 17 to about 25 amino acid residues, about 33 to about 46 amino acid residues, about 35 to about 35 of SEQ ID NO: 2. 39 amino acid residues, about 57 to about 60 amino acid residues, about 67 to about 72
Amino acid residues, about 102 to about 107 amino acid residues, about 121 to about 126 amino acid residues, about 131 to about 166 amino acid residues, about 141 to about 152 amino acid residues, about 158 to about 169 amino acid residues, about 213 to about 221 amino acid residues, about 232 to
It comprises a polypeptide comprising or consisting of an amino acid sequence of a member selected from the group consisting of about 240 amino acid residues. The invention further provides isolated polynucleotides comprising or consisting of the nucleic acid molecules encoding the β-sheet region listed in Table 2 and nucleic acid molecules encoding the β-sheet region of the AIM II protein, An isolated poly that comprises or consists of an amino acid sequence that is at least 80% identical, and more preferably at least 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical. Regarding peptides.

The present invention also provides an AIM having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 39.
Comprises a AIM II having one or more activities associated with a II polypeptide,
Alternatively, it relates to isolated polypeptides consisting thereof, as well as isolated polynucleotides encoding these polypeptides.

For selection of peptides or polypeptides that carry an antigenic epitope (ie, including the region of the protein molecule to which an antibody can bind), relatively short synthetic peptides that mimic a portion of the protein sequence are partially It is well known in the art that antisera that react with mimicked proteins can be routinely elicited. For example, Su
tcliffe, J .; G. Shinnick, T .; M. Green, N .; And Learner, R .; A. (1983) Antibodies that
react with preterminated sites on pr
oteins. See Science 219: 660-666. Peptides capable of eliciting protein-reactive sera are often shown in the primary sequence of proteins, can be characterized by a set of simple chemistries, and are immunodominant regions of intact proteins (ie, immunogenic epitopes). Nor is it limited to either the amino terminus or the carboxy terminus.

Thus, the antigenic epitope-bearing peptides and polypeptides of the invention are useful for raising antibodies (including monoclonal antibodies) that specifically bind to the polypeptides of the invention. . For example, Wilson et al.
See Cell 37: 767-778 (1984), 777.

The present invention is directed to an epitope of a polypeptide having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 39, or the polynucleotide sequence contained in the deposited clone identified as ATCC Deposit No. 97689 or 97483, or SEQ ID NO: 1 or an epitope of a polypeptide sequence encoded by a polynucleotide that hybridizes to the complement of the sequence of SEQ ID NO: 38, or stringent or lower stringency hybridization conditions as defined above. ATCC deposit number 9 below
It includes a polypeptide that comprises or consists of an epitope of the polypeptide sequence encoded by the polynucleotide sequence contained in the deposited clone identified as 7689 or 97483. The present invention further provides a polynucleotide sequence encoding an epitope of the polypeptide sequence of the present invention (eg, the sequence disclosed in SEQ ID NO: 1 or SEQ ID NO: 38), a complementary strand of the polynucleotide sequence encoding the epitope of the present invention. And polynucleotide sequences that hybridize to complementary strands under stringent hybridization conditions as defined above or under lower stringency hybridization conditions.

The term "epitope", as used herein, refers to that portion of a polypeptide that has antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably a human. Say. In a preferred embodiment,
The invention includes polypeptides that include an epitope, and polynucleotides that encode the polypeptides. An "immunogenic epitope", as used herein, is as determined by any method known in the art (eg, by the methods for producing the antibodies described below). , As part of a protein that elicits an antibody response in animals (eg, Geysen et al., Proc).
． Natl. Acad. Sci. USA 81: 3998-4002 (1983).
)checking). The term “antigenic epitope” as used herein refers to an antibody as determined by any method known in the art (eg, by the immunoassays described herein). Is defined as the part of the protein that is capable of immunospecifically binding to its antigen. Immunospecific binding excludes non-specific binding, but need not exclude cross-reactivity with other antigens. Antigenic epitopes need not be immunogenic.

Fragments that function as epitopes can be produced by any conventional method. (For example, Houghten, Proc. Natl. Acad. Sci.
． USA 82: 5131-5135 (1985). This is further described in U.S. Pat. No. 4,631,211).

In the present invention, an antigenic epitope preferably comprises at least 4, at least 5, at least 6, at least 7 amino acid sequences, more preferably at least 8, at least 9, at least 10, at least 15, at least 20.
, At least 25, and most preferably between about 15 and about 30 amino acids. Preferred polypeptides containing immunogenic or antigenic epitopes are at least 10,15,20,25,30,35,40,4.
5, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 10
It is 0 amino acid residues long. Antigenic epitopes are useful (eg, to raise antibodies (including monoclonal antibodies) that specifically bind to the epitope)
. Antigenic epitopes can be used as target molecules in immunoassays. (For example, Wilson et al., Cell 37: 767-778 (1984);
Sutcliffe et al., Science 219: 660-666 (1983).
checking).

Similarly, immunogenic epitopes can be used to induce antibodies, eg, according to methods well known in the art. (For example, Sutcliffe et al., Supra; Wilson et al., Supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:
910-914; and Bittle et al., J. Am. Gen. Virol. 66:23
47-2354 (1985)). Preferred immunogenic epitopes are
Contains secreted proteins. A polypeptide comprising one or more immunogenic epitopes is
If a carrier protein (eg albumin) can be presented to elicit an antibody response against an animal system (eg rabbit or mouse), or if the polypeptide is sufficiently long (at least about 25 amino acids), the polypeptide is Can be presented without a carrier. However, immunogenic epitopes containing as few as 8-10 amino acids have been shown to be sufficient to elicit at least antibodies capable of binding to linear epitopes in denatured polypeptides ( For example, in Western blotting).

Non-limiting examples of antigenic polypeptides or peptides that can be used to produce AIM II-specific antibodies include: amino acid residue about 13 in FIGS. 1A and B (SEQ ID NO: 2). To about 20 or a polypeptide consisting thereof; a polypeptide comprising about 23 to about 36 amino acid residues in FIGS. 1A and 1B (SEQ ID NO: 2), or a polypeptide consisting thereof; FIGS. 1A and B ( A polypeptide comprising about 69 to about 79 amino acid residues in SEQ ID NO: 2), or a polypeptide consisting thereof; a polypeptide comprising about 85 to about 94 amino acid residues in FIGS. 1A and B (SEQ ID NO: 2), or them. Consisting of about 167 amino acid residues in Figures 1A and B (SEQ ID NO: 2). Polypeptide comprising the 178 or polypeptide consisting thereof;
1A and B (SEQ ID NO: 2), a polypeptide comprising amino acid residues from about 184 to about 196, or a polypeptide consisting thereof; and FIGS. 1A and B.
A polypeptide comprising about 221 to about 233 amino acid residues in (SEQ ID NO: 2),
Alternatively, a polypeptide consisting of them. In this context, "about" means specifically a few or more (5, 4, 3, 2, or 1) amino acids greater than or less than the stated range at either or both termini. range)
including. As mentioned above, we have found that the above polypeptide fragment is AI
It was determined to be the antigenic region of the M II protein. Polynucleotides encoding these polypeptides are also included in the invention.

Epitope-bearing peptides and polypeptides of the invention can be produced by any conventional means. Houghten, R .; A. General method
d for the rapid solid-phase synthesi
s of large numbers of peptides: speci
ficit of antigen-antibody interacti
on at the level of individual amino
acids. Proc. Natl. Acad. Sci. USA 82: 5131.
~ 5135 (1985). This "Simultaneous Multiple
The Peptide Synthesis (SMPS) process is
Further described in US Pat. No. 4,631,221 to ten et al. (1986).

The epitope-bearing polypeptides of the present invention can be used to induce antibodies according to methods well known in the art. These well-known methods include in vivo immunization,
In vitro immunization, and phage display methods are included, but are not limited to. See, for example, Sutcliffe et al., Supra; Wilson et al., Supra, and Bittle et al., J. Chem. Gen. Virol. , 66: 2347-2354 (1
985). When using in vivo immunization, animals can be immunized with free peptide; however, anti-peptide antibody titers will bind the peptide to macromolecular carriers such as keyhole limpet hemocyanin (KLH) or tetanus toxoid. Can be boosted by For example, a peptide containing a cysteine residue is a maleimidobenzoyl-N-hydroxysuccinimide ester (M
It can be attached to the carrier using a linker such as BS). On the other hand, other peptides can be bound to the carrier using more common binding agents such as glutaraldehyde. Animals such as rabbits, rats, and mice are known to stimulate, for example, about 100 μg of peptide or carrier protein and Freund's adjuvant or immune response with either free peptide or carrier-bound peptide. Immunization is carried out by intraperitoneal and / or intradermal injection of an emulsion containing the adjuvant. Several booster injections may be required to provide a useful titer of anti-peptide antibody, eg, at intervals of about 2 weeks. This titer can be detected, for example, by an ELISA assay using free peptide adsorbed on the solid surface. The titer of anti-peptide antibody in the serum from the immunized animal depends on the choice of anti-peptide antibody (eg by adsorption to the peptide on a solid support and elution of the selected antibody according to methods well known in the art). Can rise.

As will be appreciated by those in the art, and as discussed above, the polypeptides of the present invention comprising immunogenic or antigenic epitopes can be fused to other polypeptide sequences. For example, the polypeptides of the present invention are immunoglobulin (Ig
A, IgE, IgG, IgM) constant domains or parts thereof (CH1, C)
H2, CH3, or any combination thereof, and portions thereof, which results in a chimeric polypeptide. Such fusion proteins can facilitate purification and increase half-life in vivo. This has been shown for a chimeric protein consisting of the first two domains of the human CD4 polypeptide and various domains of the constant region of the heavy or light chain of mammalian immunoglobulins. For example, EP 394,827; Traunecker et al., Natu.
Re, 331: 84-86 (1988). An IgG fusion protein having a disulfide-bridged dimeric structure is also more effective at binding and neutralizing other molecules than monomeric polypeptides or their fragments alone due to their IgG partial disulfide bonds. It was found to be. For example, Fo
untoulakis et al. Biochem. 270: 3958-3964
See (1995). Nucleic acids encoding the above epitopes may also be epitope tags (eg, hemagglutinin (“HA”) tags or flag tags).
Tag)) may be recombined with the gene of interest to aid in the detection and purification of the expressed polypeptide. For example, the system described by Janknecht et al. Allows for rapid purification of native fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sc.
i. USA 88: 8972-897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of 6 histidine residues. This tag serves as the matrix binding domain for the fusion protein. Extracts from cells infected with recombinant vaccinia virus can be loaded onto a Ni ²⁺ nitrilotriacetic acid agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffer.

Further fusion proteins of the invention can be produced through the techniques of gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). D
NA shuffling can be used to modulate the activity of the polypeptides of the invention, and such methods can be used to produce polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. Generally, US Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, And Pa
Tten et al., Curr. Opinion Biotechnol. 8: 724-
33 (1997); Harayama, Trends Biotechnol.
16 (2): 76-82 (1998); Hansson et al., J. Am. Mol. Bio
l. 287: 265-76 (1999); and Lorenzo and Bla.
sco, Biotechniques 24 (2): 308-13 (1998).
(Each of these patents and publications are hereby incorporated by reference in their entirety). In one embodiment, modification of the polynucleotides corresponding to SEQ ID NO: 1 and the polypeptides encoded by these polynucleotides can be accomplished by DNA shuffling. DNA shuffling involves assembling two or more DNA segments by homologous or site-specific recombination to produce changes in a polynucleotide sequence.
In another embodiment, the polynucleotides or encoded polypeptides of the present invention may be modified by subjecting them to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, portions, domains, fragments, etc., of a polynucleotide encoding a polypeptide of the invention are one or more components of one or more heterologous molecules. , Motifs, sections, parts, domains, fragments and the like.

A listing of examples of amino acid sequences that include immunogenic epitopes is shown in Table 2. This is shown in Table 2 by Jameson and Wolf, Comp. Appl. Biosci. , 4
: 181-186 (1988), which is incorporated by reference in its entirety, to list only the amino acid residues that contain the epitope predicted to have the highest antigenicity. It has been pointed out. Jameson
-Wolf antigenicity analysis, using the computer program PROTEAN (Power MacIntosh, Version, using default parameters)
3.11 DNASTAR, Inc. , 1228 South Park St
Reet Madison, WI). Table 2 and some of the polypeptides not listed in Table 2 are not considered non-immunogenic. The immunogenic epitopes in Table 2 are exemplary enumerations, not exclusive enumerations. Because other immunogenic epitopes are not recognized by the particular algorithm used. Amino acid residues containing other immunogenic epitopes are described by Jameso
It can be routinely determined using an algorithm similar to n-Wolf analysis, or by in vivo testing for antigenic reactions using methods known in the art. See, for example, Geysen et al., Supra; U.S. Patent Nos. 4,708,781; 5,194.
, 392; 4,433,092; and 5,480,971 (these references are incorporated by reference in their entireties).

It has been specifically shown that the amino acid sequences in Table 2 include immunogenic epitopes. Table 2 lists only the key residues of immunogenic epitopes determined by Jameson-Wolf analysis. Thus, additional flanking residues at either the N-terminus, the C-terminus, or both the N-terminus and the C-terminus may be added to the sequence to produce an epitope-bearing polypeptide of the invention. Thus, the immunogenic epitopes of Table 2 may include additional N-terminal amino acid residues or C-terminal amino acid residues. The additional flanking amino acid residues may be contiguous flanking N-terminal and / or C-terminal sequences from a polypeptide of the invention, a heterologous polypeptide sequence, or a polypeptide of the invention and a heterologous polypeptide. Both contiguous sequences from the sequence may be included.

Immunogenic and antigenic epitope-bearing fragments are further defined by the number of consecutive amino acid residues as described above, or the N-terminal and C-terminal positions of these fragments of the amino acid sequence of SEQ ID NO: 2. Can be identified. For example, an N-terminal position and C, where a fragment of at least 7 or at least 15 contiguous amino acid residue lengths may be occupied on the amino acid sequence of SEQ ID NO: 2.
Any combination of terminal positions is included in the present invention. Again, "at least 7 contiguous amino acid residue lengths" means 7 amino acid residue lengths, or any integer between 7 amino acids and the number of amino acid residues of the full length polypeptide of the invention. In detail,
Each and every integer between 7 and the number of amino acid residues in the full length polypeptide is included in the invention.

Immunogenic epitope-bearing polypeptides and antigenic epitope-bearing polypeptides of the invention can be used, for example, to raise antibodies that specifically bind to the polypeptides of the invention and to detect the polypeptides of the invention. Are useful in immunoassays for This antibody is useful for, for example, affinity purification of the polypeptide of the present invention. The antibody may also be conventionally used in various qualitative or quantitative immunoassays, particularly for the polypeptides of the invention using methods known in the art. For example, Harlow et al., ANTIBODIE
S: A LABORATORY MANUAL, (Cold Spring H
Arbor Laboratory Press; Second Edition, 1988).

The epitope-bearing polypeptides of the invention can be produced by any conventional means for making polypeptides, including synthetic and recombinant methods known in the art. For example, epitope-bearing peptides can be synthesized using known methods of chemical synthesis. For example, Houghten has a large number of peptides, eg 10-20 m.
g of 248 individual peptides and another 13-residue peptide representing a single amino acid variant of a segment of the HA1 polypeptide, all of which were prepared and characterized in less than 4 weeks (by ELISA-type binding studies. )) For the synthesis of (Houghten, Proc. Natl. Acad. Sc).
i. USA, 82: 5131-5135 (1985)). This "simultaneous multiple peptide synthesis (Simultaneous Multiple Peptide Sy
N. Thesis (SMPS) process is further described in US Pat. No. 4,631,211 by Houghten and Coworkers (1986). In this procedure, individual resins for solid phase synthesis of various peptides are included in separate solvent permeable packets. This allows the optimal use of a number of identical repetitive steps involved in solid phase methods. The complete manual procedure is carried out at the same time 500 ~
Allows more than 1000 syntheses (Hughten et al., Proc. Natl.
Acad. Sci. , U. S. A. 82: 5131-5135, 5134 (19
85)).

The 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 vivo immunization, in vitro immunization, and phage display methods. For example, Sutcli
ffe et al., supra; Wilson et al., supra and Bittle et al. J. Gen.
Virol. 66: 2347-2354 (1985). When using in vivo immunization, the animal can be immunized with free peptide;
KLH) or tetanus toxin) can be boosted by binding of the peptide.
For example, peptide-containing cysteine residues can be attached to the carrier using a linker such as N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides are more common binders. (Eg glutaraldehyde) may be used to bind to the carrier. Animals such as rabbits, rats, and mice are injected with either free peptide or carrier-bound peptide, for example, by intraperitoneal injection of an emulsion (containing about 100 μg of peptide or carrier protein and Freund's adjuvant) and / or Immunized by intradermal injection. Several booster injections may be required to provide a useful titer of anti-peptide antibody, eg, at intervals of about 2 weeks. This titer can be detected, for example, by an ELISA assay using free peptide adsorbed on the solid surface. The titer of anti-peptide antibodies in the serum from immunized animals is dependent on the choice of anti-peptide antibody (eg, by adsorption of the peptide to a solid support and eluate of the antibody selected according to methods well known in the art). Can rise.

As will be appreciated by those in the art, and as discussed above, the polypeptides of the present invention comprising immunogenic or antigenic epitopes can be fused to a heterologous polypeptide sequence. For example, the polypeptides of the present invention are immunoglobulin (Ig
A, IgE, IgG, IgM) constant domains or parts thereof (CH1, C)
H2, CH3, any combination thereof, including both entire domains and portions thereof, which results in a chimeric polypeptide. These fusion proteins facilitate purification and exhibit increased half-life in vivo.
It shows, for example, a chimeric protein consisting of the first two domains of the human CD4 polypeptide, as well as various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. For example, EPA 0,394,827; Traunec.
See ker et al., Nature, 331: 84-86 (1988). Ig
A fusion protein having a disulfide bridged dimer structure due to the G moiety also
It may be more effective in binding and neutralizing other molecules than the monomeric polypeptides or fragments thereof alone. For example, Fountoulakis
Et al. J. Biochem. , 270: 3958-3964 (1995). Nucleic acids encoding the above epitopes may also be recombined with the gene of interest as epitope tags to aid in the detection and purification of the expressed polypeptide.

In addition, the proteins of the present invention can be chemically synthesized using techniques known in the art (eg Creighton, 1983, Proteins: St).
rututures and Molecular Principles, W.R.
H. Freeman & Co. , N .; Y. And Hunkapiller, M et al.,
Nature, 310: 105-111 (1984)). For example,
Polypeptides corresponding to fragments of the AIM II polypeptides of the invention can be synthesized by the use of peptide synthesizers. Furthermore, if desired,
Non-classical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence of AIM II. Non-classical amino acids include D-isomers of common amino acids, 2,4-diaminobutyric acid, a-aminoisobutyric acid, 4-
Aminobutyric acid, Abu, 2-aminobutyric acid, α-Abu, α-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline,
Homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, α-alanine, fluoroamino acid,
Designer amino acids (eg, α-methyl amino acids), Ca-methyl amino acids,
Examples include, but are not limited to, Na-methyl amino acids, and common amino acid analogs. Furthermore, the amino acids can be D (dextrorotatory) or L (levorotatory).

Non-naturally occurring variants can be generated by mutagenesis techniques known in the art (oligonucleotide-mediated mutagenesis, alanine scanning, PCR mutagenesis, site-directed mutagenesis (eg Carter et al., Nucl. Acids Res. 13). : 4331 (
1986); and Zoller et al., Nucl. Acids Res. 10: 6
487 (1982)), cassette mutagenesis (see, eg, Wells et al., Gene 34: 315 (1985)), restriction selective mutagenesis (eg, Wells et al., Philos. Trans. R. Soc. London
SerA 317: 415 (1986)), but is not limited thereto).

The present invention may be used during or after translation, for example, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, antibody molecules or other cellular agents. Further included are AIM II polypeptides that are differentially modified, such as by binding to a ligand. Any of numerous chemical modifications include the following may be carried out by known, but not limited to techniques: cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH ₄
Specific chemical cleavage by acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin;

Further post-translational modifications included according to the invention include, for example: N-linked or O-linked carbohydrate chains (N-terminal or C-terminal processing), attachment of chemical moieties to the amino acid backbone. Attachment, chemical modification of N-linked or O-linked carbohydrate chains, and addition or deletion of N-terminal methionine residues as a result of prokaryotic host cell expression. The polypeptide may also be modified with a detectable label such as an enzymatic label, a fluorescent label, an isotope label or an affinity label to allow detection and isolation of the protein.

The present invention may also provide additional benefits such as increased solubility, stability and circulation time of the polypeptide, or decreased immunogenicity of AIM I.
Chemically modified derivatives of the polypeptides of I are provided (US Pat. No. 4,179,33).
(See No. 7). The chemical moieties for derivatization include water-soluble polymers (eg,
Polyethylene glycol, ethylene glycol / propylene glycol copolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol, etc.)
Can be selected from The polypeptide may be modified at random positions within the molecule or at predetermined positions within the molecule and may contain one, two, three or more attached chemical moieties.

The polymer may be a polymer of any molecular weight and may be branched or unbranched. For polyethylene glycol, a preferred molecular weight is between about 1 kDa and about 100 kDa for ease of handling and manufacturing (the term "about" means that in the preparation of polyethylene glycol, some molecules are Heavy, and some indicate lighter than the indicated molecular weight). The desired therapeutic profile (eg, desired duration of sustained release, effect on biological activity in some cases, ease of handling, degree of antigenicity or lack of antigenicity, and therapeutic protein or analog). Other sizes may be used, depending on other known effects of polyethylene glycol). For example, polyethylene glycol is about 200, 500, 1000, 1500, 2000,
2500, 3000, 3500, 4000, 4500, 5000, 5500, 6
000, 6500, 7000, 7500, 8000, 8500, 9000, 95
00, 10,000, 10,500, 11,000, 11,500, 12:00
0, 12,500, 13,000, 13,500, 14,000, 14,500
, 15,000, 15,000, 16,000, 16,500, 17,000,
17,500, 18,000, 18,500, 19,000, 19,500, 2
50,000, 25,000, 30,000, 35,000, 40,000, 5
50,000, 55,000, 60,000, 65,000, 70,000, 75
It can have an average molecular weight of, 000,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

As described above, the polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in: US Pat. No. 5,64
3,575; Morpurgo et al., Appl. Biochem. Biotec
hnol. 56: 59-72 (1996); Vorovjev et al., Nucleo.
sides Nucleotides 18: 2745-2750 (1999)
And Caliceti et al., Bioconjug. Chem. 10: 638-
646 (1999), the disclosure of each of which is incorporated herein by reference.

The polyethylene glycol molecule (or other chemical moiety) should be attached to the protein in view of its effect on the functional or antigenic domains of this protein. There are numerous coupling methods available to those of skill in the art (eg, EP 0 401 384 (G-CSF to P, incorporated herein by reference).
EG)), Malik et al., Exp. Hematol. 20: 1028-
1035 (1992) (pegylation of GM-CSF with tresyl chloride).
See also report)))). For example, polyethylene glycol can be covalently attached via a amino acid residue by a reactive group such as a free amino or carboxyl group. Reactive groups are groups to which activated polyethylene glycol molecules can be attached. The amino acid residue having a free amino group may include a lysine residue and an N-terminal amino acid residue; the amino acid residue having a free carboxyl group may include an aspartic acid residue, a glutamic acid residue and a C-terminal amino acid. Residues may be mentioned. Sulfhydryl residues can also be used as a reactive group to attach the polyethylene glycol molecule. Preferred for therapeutic purposes is a bond at the amino group, eg at the N-terminus or at the lysine group.

As alluded to above, polyethylene glycol can be attached to proteins via attachment to any of a number of amino acid residues. For example, polyethylene glycol can be linked to proteins via covalent bonds to lysine residues, histidine residues, aspartic acid residues, glutamic acid residues, or cysteine residues.
Using one or more reaction chemistries, a particular amino acid residue of a protein (eg, lysine, histidine, aspartic acid, glutamic acid or cysteine) or more than one type of amino acid residue of a protein (eg, lysine, Polyethylene glycol may be attached to histidine, aspartic acid, glutamic acid, cysteine and combinations thereof.

Proteins that are chemically modified at the N-terminus may be specifically desired. Using polyethylene glycol as an illustration of the composition of the present invention, the ratio of polyethylene glycol molecules to protein (or peptide) molecules in the reaction mixture from various polyethylene glycol molecules (by molecular weight, branching, etc.), pegs to be performed. The type of oxidization reaction and the method of obtaining the selected N-terminal pegylated protein can be selected. The method of obtaining the N-terminal PEGylated preparation (ie, separating this moiety from other mono-PEGylated moieties, if necessary) is performed by purification of the N-terminal PEGylated material from a population of PEGylated protein molecules. obtain. Selective proteins chemically modified with N-terminal modifications have different types of primary amino groups (
This can be achieved by reductive alkylation, which takes advantage of the differential reactivity of lysine to N-terminus). Under appropriate reaction conditions, substantially selective derivatization of proteins at the N-terminus with a carbonyl group-containing polymer is achieved.

As mentioned above, pegylation of the proteins of the invention can be accomplished by any number of means. For example, polyethylene glycol is
It may be connected to the protein either directly or by an intervening linker. Linkerless (for connecting polyethylene glycol to protein
linkless) system is described in Delgado et al., Crit. Rev. T
hera. Drug Carr Sys. 9: 249-304 (1992).
Francis et al., Intern. J. of Hematol. 68: 1-1
8 (1998); U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,0.
52; WO95 / 06058; and WO98 / 32466. The disclosure of each of these is incorporated herein by reference.

One system for connecting polyethylene glycol directly to amino acid residues of proteins without an intervening linker is tresylated.
A) MPEG is used. Tresylated MPEG is tresyl chloride (C
Is produced by modification of monomethoxypolyethylene glycol (MPEG) using 1SO ₂ CH ₂ CF ₃ . Tresylated M
During the reaction of proteins with PEG, polyethylene glycol is directly attached to the amine groups of the protein. Accordingly, the invention includes protein polyethylene glycol conjugates produced by reacting a protein of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoroethanesulfonyl group.

Polyethylene glycol can also be linked to proteins using a number of different intervening linkers. For example, US Pat. No. 5,612,460, the entire disclosure of which is incorporated herein by reference, discloses urethane linkers for linking proteins and polyethylene glycol. Protein-polyethylene glycol conjugates, where polyethylene glycol is connected to the protein by a linker, are also activated with compounds such as MPEG-succinimidyl succinate, 1,1′-carbonyldiimidazole. MPE
G, MPEG-2,4,5-trichloropenyl carbonate, MPEG-p-nitrophenol carbonate, and various MPEG succinate derivatives) and proteins. A number of additional polyethylene glycol derivatives and reaction chemistries for connecting polyethylene glycol to proteins are described in WO 98/32466, the entire disclosure of which is incorporated herein by reference. PEGylated protein products produced using the reaction chemistries set out herein are included within the scope of the invention.

The number of polyethylene glycol moieties attached to each protein of the invention (ie, the degree of substitution) can also vary. For example, the pegylated protein of the invention is
On average 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 2,
It can be linked to zero or more polyethylene glycol molecules. Similarly, the average degree of substitution per protein molecule is, for example, 1-3, 2-4, 3-5, 4-6, 5-
7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-1
4, 13-15, 14-16, 15-17, 16-18, 17-19, or 1
Within the polyethylene glycol portion of 8-20. Methods for determining the degree of substitution are described, for example, in Delgado et al., Crit. Rev. Thera. D
rug Carrier Sys. 9: 249-304 (1992).

Antibodies In addition, the invention provides immunospecificity to a polypeptide of the invention (as determined by immunoassays well known in the art for assaying specific antibody-antigen binding), preferably an epitope. Binding antibody and T cell antigen receptor (
TCR). The antibodies of the present invention include polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies or chimeric antibodies, single chain antibodies, Fabs.
Fragments, F (ab ') fragments, fragments produced by Fab expression libraries, anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies against antibodies of the invention), and epitope binding of any of the above. Including but not limited to fragments. As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, ie, molecules that contain an antigen binding site that immunospecifically binds an antigen. Say. The immunoglobulin molecules of the invention can be of any type of immunoglobulin molecule (eg, IgG, IgE,
IgM, IgD, IgA, and IgY), any class (eg, IgG1,
IgG2, IgG3, IgG4, IgA1 and IgA2) or any subclass.

Most preferably, the antibody is a human antigen-binding antibody fragment of the invention, which comprises Fab, Fab 'and F (ab') 2, Fd, single chain Fvs (sc).
Fv), single chain antibodies, disulfide-bonded Fvs (sdFv) and VL or V
Examples include, but are not limited to, fragments that include any of the H domains. Antigen-binding antibody fragments, including single chain antibodies, may include variable regions alone or in combination with all or part of the following: hinge region, CH1 domain, C
H2 domain and CH3 domain. Also included in the invention are antigen binding fragments that also include any combination of variable and hinge regions, CH1 domains, CH2 domains and CH3 domains. Antibodies of the invention may be derived from any animal origin including birds and mammals. Preferably, the antibody is a human, murine, donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken. As used herein, "human" antibody includes antibodies having the amino acid sequence of human immunoglobulin,
And an antibody isolated from an animal that is transgenic for a human immunoglobulin library or one or more human immunoglobulins and does not express endogenous immunoglobulins (as described below and, for example, Kucherlapa).
(as described in US Pat. No. 5,939,598 to Ti et al.).

The antibodies of the invention can be monospecific, bispecific, trispecific or of greater multispecificity. A multispecific antibody may be specific for different epitopes of a polypeptide of the invention, or both a polypeptide of the invention and a heterologous epitope (eg, a heterologous polypeptide or a solid support material). Can be specific to. For example, PCT Publication WO 93/17715; WO 9
2/08802; WO 91/00360; WO 92/05793; Tu
tt et al. Immunol. 147: 60-69 (1991); U.S. Pat. No. 4
, 474,893, 4,714,681, 4,925,648,
No. 5,573,920, No. 5,601,819; Kostelny et al.
J. Immunol. 148: 1547-1553 (1992).

The antibodies of the invention may be described or specified with respect to the epitopes or parts of the polypeptides of the invention to which they recognize or specifically bind. Portions of this epitope or polypeptide can be identified, for example, by N-terminal and C-terminal positions, by size at contiguous amino acid residues, or as listed herein in tables and figures. Antibodies that specifically bind any epitope or polypeptide of the invention can also be excluded. Accordingly, the present invention includes antibodies that specifically bind the polypeptides of the present invention and allow for the exclusion of the polypeptides of the present invention.

The antibodies of the invention may also be described or specified for their cross-reactivity. Antibodies that do not bind any other analog, ortholog or homolog of a polypeptide of the invention are included. At least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55% and at least 50% identity to the polypeptides of the present invention. Also included in the invention are antibodies that bind a polypeptide having (as calculated using methods known in the art and described herein). Less than 95%, less than 90%, relative to the polypeptide of the invention,
Less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%,
Antibodies that do not bind a polypeptide with less than 55% and less than 50% identity (as calculated using methods known in the art and methods described herein) are also within the scope of the invention. included. Further included in the invention are antibodies that bind the polypeptide encoded by the polynucleotide that hybridizes to the polynucleotide of the invention under stringent hybridization conditions (as described herein). . Antibodies of the invention may also be described or specified for their binding affinity for the polypeptides of the invention. Preferred binding affinity is less than 5 × 10 ⁻² M, less than 10 ⁻² M, less than 5 × 10 ⁻³ M, 10 ⁻³ M
Less than 5 × 10 ⁻⁴ M, less than 10 ⁻⁴ M, less than 5 × 10 ⁻⁵ M, less than 10 ⁻⁵ M, 5
Less than × 10 ^-6 M, less than 10 ^-6 M, less than 5 × 10 ^-7 M, less than 10 ^-7 M, less than 5 × 10 ^-8 M, less than 10 ^-8 M, less than 5 × 10 ^-9 M, 10 Less than ^-9 M, less than 5 × 10 ^-10 M, less than 10 ^-10 M, less than 5 × 10 ^-11 M, less than 10 ^-11 M, less than 5 × 10 ^-12 M, 1
0 less than ^-12 M, less than 5 × 10 ^-13 M, less than 10 ^-13 M, less than ^{5 × 10 -14 M, 10 -1} 4 below M, 5 × 10 ^-15 M and less than less than 10 ^-15 M dissociation Affinities with a constant or Kd are mentioned.

The invention also provides antibodies to the epitopes of the invention, as determined by any method known in the art for determining competitive binding, such as the immunoassays described herein. Antibodies that competitively inhibit the binding of In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

The antibodies of the present invention can act as agonists or antagonists of the polypeptides of the present invention. For example, the invention includes antibodies that disrupt the receptor / ligand interaction with a polypeptide of the invention either partially or completely. The invention also features receptor-specific antibodies that do not interfere with ligand binding but interfere with receptor activation. Receptor activation (ie signal transduction) can be determined by the techniques described herein, or otherwise known in the art. For example, receptor activation can be determined by phosphorylation of the receptor (eg, tyrosine or serine / threonine), or immunoprecipitation, followed by detection of its substrate by Western blot analysis (eg, as described above). . In certain embodiments, an antibody is provided that inhibits ligand or receptor activity by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50% of its activity in the absence of the antibody. It

The present invention also recognizes receptor-specific antibodies that interfere with both ligand binding and receptor activation, as well as receptor-ligand complexes, and preferably specific unbound receptors or unbound ligands. It has the characteristics of a receptor-specific antibody that does not recognize. Similarly, the present invention binds ligands and neutralizing antibodies that prevent the binding of ligands to receptors, and ligands, thereby preventing receptor activation, but not ligand binding to receptors. Contains antibodies. Further, the invention includes antibodies that activate the receptor. These antibodies may act as receptor agonists, ie, enhance or activate either all or a subset of the biological activations of ligand-mediated receptor activation. The antibody is an agonist for biological activity, including the specific biological activity of the peptides of the invention disclosed herein,
It can be specified as an antagonist or an inverse agonist. Thus, the invention further relates to antibodies that act as agonists or antagonists of the polypeptides of the invention. The antibody agonist can be produced using a method known in the art. For example, PCT Publication WO 96/40281; US Pat. No. 5,811.
1,097; Deng et al., Blood 92 (6): 1981-1988 (1.
998); Chen et al., Cancer Res. 58 (16): 3668-36.
78 (1998); Harrop et al., J. Am. Immunol. 161 (4): 17
86-1794 (1998); Zhu et al., Cancer Res: 58 (15).
: 3209-3214 (1998); Yoon et al., J. Am. Immunol. 160
(7): 3170-3179 (1998); Prat et al. Cell. Sci
． 111 (Pt2): 237-247 (1998); Pittard et al., J. Am. Im
munol. Methods 205 (2): 177-190 (1997); L
iautard et al., Cytokine 9 (4): 233-241 (1997).
Carlson et al., J .; Biol. Chem. 272 (17): 11295-
11301 (1997); Taryman et al., Neuron 14 (4): 75.
5-762 (1995); Muller et al., Structure 6 (9): 1.
153-1167 (1998); Bartunek et al., Cytokine 8 (
1): 14-20 (1996) (all of the above references are incorporated herein by reference in their entirety).

Antibodies of the invention can be used, for example, but not limited to, to purify, detect, and target the polypeptides of the invention. These include both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibody has use in immunoassays for qualitatively and quantitatively measuring the levels of polypeptides of the invention in biological samples. For example, Harl
ow et al., Antibodies: A Laboratory Manual, (
Cold Spring Harbor Laboratory Press,
2nd edition, 1988), which is hereby incorporated by reference in its entirety.

As discussed in further detail below, the antibodies of the invention can be used either alone or in combination with other compounds. The antibody can further be recombinantly fused to the heterologous polypeptide at the N-terminus or C-terminus to the polypeptide or other compound, or can be chemically linked (including covalent and non-covalent). For example, the antibodies of the invention can be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, or toxins. For example, PCT publication WO92 / 08495; WO91
/ 14438; WO89 / 12624; U.S. Pat. No. 5,314,995; and European Patent 396,387.

The antibodies of the invention are modified (ie, covalently attached).
by a covalent attachment of any type of molecule to the antibody so that it does not prevent the antibody from producing an anti-idiotypic response. For example, without limitation, antibody derivatives include, for example, glycosylation,
Acetylation, pegylation, phosphorylation (phosphyla)
cation), amidation, known blocking / blocking groups
antibodies modified by derivatization with up), proteolytic cleavage, ligation to cellular ligands or other proteins, and the like. Any of a number of chemical modifications can be performed by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the derivative may contain one or more non-classical amino acids.

The antibodies of the present invention may be produced by any suitable method known in the art.
Polyclonal antibodies to the antigen of interest can be produced by various procedures well known in the art. For example, the polypeptides of the present invention can be administered to a variety of host animals, including but not limited to rabbits, mice, rats, etc., to induce the production of serum containing polyclonal antibodies specific for the antigen. . Various adjuvants can be used to increase the immunological response, depending on the host species, and Freund's (complete and incomplete) mineral gels such as aluminum hydroxide (
mineral gel), surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,
Keyhole limpet hemocyanin, dinitrophenol, and BCG (bacillus Calmette-Guerin) and Corynebacterium parvum
Include, but are not limited to, potentially useful human adjuvants such as Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display techniques, or a combination thereof. For example, monoclonal antibodies can be produced using the hybridoma technology known in the art, including: Har
Low et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press
, 2nd edition, 1988); Hammerling et al., Monoclonal An.
tibodies and T-Cell Hybridomas 563-6
81 (Elsevier, NY 1981), which is incorporated by reference in its entirety. The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Thus, the term "monoclonal antibody"
Are not limited to antibodies produced through hybridoma technology. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma and recombinant and phage display techniques.

Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art, and are discussed in detail in Example 23.
Briefly, mice can be immunized with the polypeptides of the invention or cells expressing such peptides. Once an immune response is detected (eg, antibodies specific for the antigen are detected in mouse serum), the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells (eg cells from cell line SP20 available from the ATCC).
Hybridomas are selected and cloned by limiting dilution. The hybridoma clones are then assayed for cells secreting antibodies capable of binding the polypeptides of the invention by methods known in the art. Ascites fluid, which generally contains high levels of antibodies, can be produced by immunizing mice with positive hybridoma clones.

Accordingly, the invention provides a method of producing an antibody produced by a method comprising culturing a monoclonal antibody and a hybridoma cell secreting the antibody of the invention, wherein, preferably, this A hybridoma is a hybridoma clone that fuses myeloma cells with splenocytes isolated from a mouse immunized with the antigen of the present invention and then secretes an antibody capable of binding to the polypeptide of the present invention.
Produced by screening the hybridomas resulting from the fusion.

Antibody fragments that recognize a particular epitope can be generated by known techniques. For example, Fab and F (ab ′) 2 fragments of the invention are (Fa
It can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce the b fragment) or pepsin (to produce the F (ab ') 2 fragment). F (ab ′) 2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.

For example, the antibodies of the present invention can also be produced using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In particular, such phage can be utilized to display antigen binding domains expressed from repertoires or combinatorial antibody libraries (eg human or mouse). Phage expressing an antigen binding domain that binds to an antigen of interest can be selected or identified with the antigen (eg, using labeled antigen or antigen bound or supplemented to a solid surface or bead). Phage used in these methods are typically phage having the antibody domain of Fab, Fv or disulfide-stabilized Fv recombinantly fused to either the phage gene III protein or the phage gene VIII protein. Is a filamentous phage containing the fd and M13 binding domains expressed from E. coli. Examples of phage display methods that can be used to make the antibodies of the invention include those disclosed below: Brinkman et al. Immunol. Meth
ods 182: 41-50 (1995); Ames et al., J. Am. Immunol.
Methods 184: 177-186 (1995); Kettleboro.
Ugh et al., Eur. J. Immunol. 24: 952-958 (1994);
Persic et al., Gene 187 9-18 (1997); Burton et al.,
Advances in Immunology 57: 191-280 (19)
94); PCT application number PCT / GB91 / 01134; PCT publication WO 90.
/ 0202809; WO 91/10737; WO 92/01047; WO 92
/ 18619; WO 93/11236; WO 95/15982; WO 95
/ 20401; and U.S. Patent Nos. 5,698,426; 5,223,4.
No. 09; No. 5,403,484; No. 5,580,717; No. 5,42.
No. 7,908; No. 5,750,753; No. 5,821,047; No. 5
No. 5,571,698; No. 5,427,908; No. 5,516,637;
No. 5,780,225; No. 5,658,727; No. 5,733,74.
3 and 5,969,108 (the above references are incorporated herein by reference in their entirety).

As described in the above references, after phage selection, the region encoding the antibody derived from the phage can be used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment. It can be separated and used and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria (eg, as described in detail below). For example, techniques for recombinantly producing Fab, Fab 'and F (ab') 2 fragments can also be used using methods known in the art. Such methods are disclosed, for example, in: PCT Publication WO 92/22324; Mullinax et al., BioT.
techniques 12 (6): 864-869 (1992); and Saw.
ai et al., AJRI 34: 26-34 (1995); and Better et al., S.
science 240: 1041-1043 (1998) (these references are incorporated by reference in their entirety).

Examples of techniques that can be used to generate single chain Fvs and antibodies include US Pat. Nos. 4,946,778 and 5,258,498; Huston.
Et al., Methods in Enzymology 203: 46-88 (19.
91); Shu et al., PNAS 90: 7995-7999 (1993); and Skerra et al., Science 240: 1038-1040 (1988). For some uses, including in vivo use of antibodies in humans and in vitro detection assays, the use of chimeric, humanized or human antibodies may be preferred. A chimeric antibody is a molecule in which different parts of the antibody are derived from different animal species (for example, an antibody having a variable region derived from a mouse monoclonal antibody and a human immunoglobulin constant region). Methods for making chimeric antibodies are known in the art. For example, Morris
on, Science 229: 1202 (1985); Oi et al., BioTec.
hnies 4: 214 (1986); Gillies et al., (1989) J.
． Immunol. Methods 125: 191-202; US Pat. No. 5,
807,715; 4,816,567; and 4,816,397.
No., which are hereby incorporated by reference in their entireties. A humanized antibody is an antibody molecule from a non-human species antibody that binds to a desired antibody having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. . Often, framework residues in the human framework regions will be replaced with the corresponding residue from the CDR donor antibody to alter (preferably improve) antigen binding. Substitutions of these frameworks can be made by methods well known in the art, for example, modeling interactions between CDRs and framework residues to identify framework residues important for antigen binding, and aberrant sequences at particular positions. It can be identified by sequence comparison to identify framework residues.
(For example, Queen et al., US Pat. No. 5,585,089; Riechman.
n. Et al., Nature 332: 323 (1988). These are incorporated herein by reference in their entirety). Antibodies can be humanized using a variety of techniques known in the art including: For example, CDR-grafting (European Patent 239,400; PCT Publication WO 91/09967; US Patent 5,2).
No. 25,539; No. 5,530,101 and No. 5,585,089).
, Veneering or resurfacing
Facing) (European Patent Nos. 592,106; 519,596; Pad)
lan, Molecular Immunology 28 (4/5): 489.
-498 (1991); Studnicka et al., Protein Engine.
eering 7 (6): 805-814 (1994); Roguska et al., P.
NAS 91: 969-973 (1994)), and chain shuffling (US Pat. No. 5,565,332).

Fully human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. U.S. Pat. Nos. 4,444,887, 4,716,111; and PCT Publication W.
O 98/46645, WO 98/50433, WO 98/24893, W
See also O 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins but which are capable of expressing human immunoglobulin genes. For example, a complex of human heavy and light chain immunoglobulin genes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable regions, constant regions and diversity regions (diversity)
region) can be introduced into mouse embryonic stem cells in addition to the human heavy chain gene and human light chain gene. The mouse heavy and immunoglobulin light chain immunoglobulin genes can be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletions in the JH region interfere with endogenous antibody production. The modified embryonic stem cells are developed and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. Transgenic mice are immunized in the normal fashion with a selected antigen (eg, all or part of a polypeptide of the invention). Monoclonal antibodies directed against the antigen are obtained from the immunized, transgenic mice using conventional hybridoma technology. Human immunoglobulin transgenes are masked during B cell differentiation by rearrangement of transgenic mice and are followed by class switching and somatic mutations. Thus, the use of such techniques may result in therapeutically useful Ig
It is possible to produce G antibodies, IgA antibodies, IgM antibodies and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Husz.
ar, Int. Rev. Immunol. 13: 65-93 (1995). For a detailed discussion of this technique for producing human antibodies and human monoclonal antibodies, as well as the protocol for producing such antibodies, see
For example, PCT publication WO98 / 24893; WO96 / 34096; WO96 /
33735; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016.
Nos. 5,545,806; 5,814,318; and 5,9.
39,598, which are hereby incorporated by reference in their entireties. In addition, Abgenix, Inc. Companies such as (Freemont, CA) and Genpharm (San Jose, CA) can engage in providing human antibodies to selected antigens using techniques similar to those described above.

Human antibodies that fully recognize the selected epitope were "guided (gui).
ed) selection ”. In this approach,
Selected non-human monoclonal antibodies (eg, murine antibodies) are used to guide the selection of human antibodies that fully recognize the same epitope (Jespers et al., Bio / technology 12: 899-903 (1988)).

In addition, antibodies to the polypeptides of the present invention can then be utilized to produce anti-idiotypic antibodies that "mimic" the polypeptides of the present invention using techniques well known to those of skill in the art. (Eg Greenspan and Bona, FASE
BJ. 7 (5): 437-444; (1989) and Nisinoff,
J. Immunol. 147 (8): 2429-2438 (1991). ) For example, multimerization of polypeptides
n) and / or binding of a polypeptide of the invention to a ligand,
The antibody that competitively inhibits can then be used to produce an anti-idiotype that "mimics" the multimerization and / or binding domain of the polypeptide and, as a result, binds the polypeptide and / or its ligand, And neutralize. Neutralization of such anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligands. For example, such anti-idiotypic antibodies can be used to bind to a polypeptide of the invention and / or to bind its ligand / receptor, thereby blocking its biological activity.

A. Polynucleotides Encoding Antibodies The present invention further provides polynucleotides comprising nucleotide sequences encoding the antibodies of the present invention and fragments thereof. The invention also provides for stringent or low stringency hybridization conditions (eg,
Hybridizing to a polynucleotide encoding an antibody (as defined above), preferably an antibody that specifically binds to a polypeptide of the invention, preferably that binds to a polypeptide having the amino acid sequence of SEQ ID NO: 2. Contains soybean polynucleotides.

The polynucleotide may be obtained by any method known in the art and the nucleotide sequence of the polynucleotide determined. For example, if the nucleotide sequence of the antibody is known, the polynucleotide encoding the antibody may be a chemically synthesized oligonucleotide (eg, Kutmeier et al., BioTechn.
issues 17: 242 (1994)).
Briefly, this involves the following steps: synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of these oligonucleotides, followed by PCR of the ligated oligonucleotides. amplification.

Alternatively, antibody-encoding polynucleotides can be made from nucleic acids from suitable sources. No clone containing a nucleic acid encoding a particular antibody is available, but if the sequence of the antibody molecule is known, the nucleic acid encoding the immunoglobulin is
A cDNA library made from a suitable source (eg, an antibody cDNA library, or any tissue or cell expressing an antibody (eg, a hybridoma cell selected for expression of an antibody of the invention), or therefrom. From the isolated nucleic acid (preferably poly A + RNA), for example, a cDNA encoding an antibody
To identify cDNA clones from the library, by PCR amplification using synthetic primers hybridizable to the 3'and 5'ends of the sequence, or using oligonucleotide probes specific for a particular gene sequence. It can be obtained by cloning. The amplified nucleic acid produced by PCR is
It can then be cloned into a replicable cloning vector using any method known in the art.

Once the antibody nucleotide sequence and corresponding amino acid sequence are determined,
Nucleotide sequences of antibodies can be prepared by methods well known in the art for manipulation of nucleotide sequences (eg, recombinant DNA technology, site-directed mutagenesis, PCR, etc. (eg,
Sambrook et al., 1990, Molecular Cloning, AL.
Laboratory Manual, 2nd Edition, Cold Spring Har
bor Laboratory, Cold Spring Harbor, NY
And Ausubel et al., 1998, Current Protocols.
in Molecular Biology, John Wiley & Sons
, NY. Both are incorporated herein by reference in their entirety. )) Can be engineered to produce antibodies with different amino acid sequences to produce, for example, amino acid substitutions, deletions, and / or insertions.

In certain embodiments, the amino acid sequences of the variable domains of the heavy and / or light chains are identified by methods well known in the art for identification of sequences of complementarity determining regions (CDRs) (eg, sequence Other heavy chains to determine hypervariable regions,
And by comparing with the known amino acid sequence of the variable region of the light chain). Using conventional recombinant DNA techniques, one or more CDRs may be inserted within the framework regions as described above (eg, into the human framework regions for humanizing non-human antibodies). . This framework region can be naturally occurring or can be a consensus framework region, and preferably a human framework region (eg, for the listed human framework regions, Ch
othia et al. Mol. Biol. 278: 457-479 (1998)). Preferably, the polynucleotide produced by the combination of the framework regions and CDRs encodes an antibody that specifically binds to a polypeptide of the invention. Preferably, as discussed above, one or more amino acid substitutions may be made within the framework regions, and preferably the amino acid substitutions improve the binding of the antibody to its antigen. Further, such methods provide for amino acid substitutions or deletions of cysteine residues in one or more variable regions involved in intrachain disulfide bonds, so as to produce antibody molecules lacking one or more intrachain disulfide bonds. It can be used to create losses. Other changes to the polynucleotide are encompassed by the present invention and in the art.

In addition, it was developed to produce a "chimeric antibody" by splicing a gene from a mouse antibody molecule of suitable antigen specificity with a gene from a human antibody molecule of suitable biological activity. Technology (Morrison et al., 1984,
Proc. Natl. Acad. Sci. 81: 851-855; Neuber.
Ger et al., 1984, Nature 312: 604-608; Takeda et al., 1985, Nature 314: 452-454). As mentioned above, chimeric antibodies are molecules in which different parts are derived from different animal species,
Such molecules have variable regions derived from mouse mAbs and human immunoglobulin constant regions (eg, humanized antibodies).

Alternatively, techniques described for the production of single chain antibodies (US Pat. No. 4,694,
778; Bird, Science, 1988, 242: 423-42; Hu.
Ston et al., 1988, Proc. Natl. Acad. Sci. USA 85
: 5879-5883; and Ward et al., 1989, Nature 334:
544-54) may be adapted for the production of single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region through amino acid bridges, resulting in a single chain polypeptide. E. Techniques for the assembly of functional Fv fragments in E. coli can also be used (Skerr
a et al., Science 242: 1038-1041 (1988)).

B. Methods of Producing Antibodies The antibodies of the invention may be produced by any method known in the art for synthesizing antibodies, in particular by chemical synthesis or, preferably, recombinant expression technology. Can be produced by

Antibodies of the invention, or fragments, derivatives or analogs thereof (eg,
Recombinant expression of the heavy or light chain of the antibody of the invention or the single chain antibody of the invention)
Construction of an expression vector containing a polynucleotide encoding the antibody is required. Once the polynucleotide encoding the antibody molecule of the invention or the heavy or light chain of the antibody, or a portion thereof (preferably containing the variable domain of the heavy or light chain) is obtained, production of the antibody molecule Vectors for can be produced by recombinant DNA technology using techniques well known in the art. Accordingly, methods for preparing a protein by expression of a polynucleotide containing an antibody encoding a nucleotide sequence are described herein. Methods well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example:
In vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination are included. Accordingly, the invention provides a replicable vector comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. To do. Such vectors include constant regions of antibody molecules (eg, PCT Publication WO 86/0580).
7; PCT Publication WO 89/01036; and US Pat. No. 5,122,464) and antibodies that can be cloned into such vectors for full expression of heavy or light chains. Variable domains of

The expression vector is transferred into a host cell by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce the antibody of the invention. Thus, the invention includes a host cell containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, operably linked to a heterologous promoter. In a preferred embodiment for the expression of double chain antibodies, the vector encoding both the heavy and light chains is as detailed below.
It can be co-expressed in a host cell for total expression of immunoglobulin molecules.

A variety of host expression vector systems may be utilized to express the antibody molecule of the present invention. Such host expression systems represent vehicles in which the coding sequence of interest may be produced and subsequently purified, but also in situ when transformed or transfected with sequences encoding the appropriate nucleotides. 2 represents a cell capable of expressing an antibody molecule of the invention. These include bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody-encoding sequences (eg E. coli, B. subtilis (sub).
microorganisms such as yeasts transformed with a recombinant yeast expression vector containing a sequence encoding an antibody (eg, Saccharomyces, Pich).
ia); an insect cell line infected with a recombinant viral expression vector (eg, baculovirus) containing a sequence encoding an antibody; a recombinant viral expression vector (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV)
) Infected plant cell line or a plant cell line transformed with a recombinant plasmid expression vector containing a sequence encoding an antibody (eg Ti plasmid); or a promoter derived from the genome of a mammalian cell (eg metallothionein). Promoter) or a promoter derived from a mammalian virus (eg, adenovirus late promoter; vaccinia virus 7.5K promoter) and a mammalian cell line (eg, COS, CHO) carrying a recombinant expression construct.
, BHK, 293, 3T3 cells). Preferably bacterial cells such as Escherichia coli, and more preferably eukaryotic cells, especially for the expression of whole recombinant antibody molecules, are used for the expression of the recombinant antibody molecules. For example, in combination with a vector such as the major immediate early gene promoter element from human cytomegalovirus,
Mammalian cells, such as Chinese hamster ovary cells (CHO), are effective expression systems for antibodies (Föcking et al., Gene, 1986, 45).
: 101; Cocktt et al., 1990, Bio / Technology 8:
2).

In bacterial systems, a number of expression vectors may be advantageously selected depending on the use intended for the expression of the antibody molecule. For example, if large quantities of such proteins are to be produced, vectors may be desired that direct high levels of expression of fusion protein products that are easily purified for the production of pharmaceutical compositions of antibody molecules. In such a vector, the antibody-encoding region may be separately ligated in frame to the vector along with the lacZ-encoding region, such that a fusion protein is produced.
coli expression vector pUR278 (Ruther et al., 1983, EMBO J
． 2: 1791); pIN vector (Inouye & Inouye, 1985,
Nucleic Acids Res. 13: 3101-3109; Van H
Eeek & Schuster, 1989, J. Am. Biol. Chem. 24:55
03-5509) and the like, but are not limited thereto. The pGEX vector can also be used to express foreign polypeptides as a fusion protein with glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be readily purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. This pGEX vector was designed to contain thrombin or factor Xa protease cleavage sites, so that
This cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus (Aut
ographa California nuclear polyhedr
osis virus) (AcNPV) is used as a vector to express foreign genes. The virus is Spodoptera frugi
Proliferates in perda cells. The antibody coding sequence may be individually cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of an AcNPV promoter, such as the polyhedrin promoter.

Many viral-based expression systems can be utilized in mammalian host cells. When an adenovirus is used as an expression vector, the sequence of interest encoding the antibody can be linked to an adenovirus transcription / translation control complex (eg, a late promoter and a tripartite leader sequence). This chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion into a nonessential region of the viral genome (eg region E1 or E3) results in a recombinant virus that survives in the infected host and is capable of expressing antibody molecules. (For example, Logan & Shenk, 1984, Proc. Na.
tl. Acad. Sci. USA 81: 355-359). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, this start codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be increased by including appropriate transcription enhancer elements, transcription terminators, etc. (Bittner et al., 1987).
Methods in Enzymol. 153: 51-544).
.

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products can be important for protein function. Different host cells have characteristic and unique mechanisms for post-translational processing and modification of protein and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells may be used that have the cellular machinery for proper processing of the primary transcript, glycosylation of the gene product, and phosphorylation. Such mammalian host cells include CHO, VERY, BHK, Hela, COS, M
DCK, 293, 3T3, WI38 and, in particular, breast cancer cell lines (eg BT
483, Hs578T, HTB2, BT20 and T47D) as well as normal mammary gland cell lines such as CRL7030 and Hs578Bst.

Stable expression is preferred for long-term, high-yield production of recombinant proteins.
For example, cell lines that stably express the antibody molecule can be engineered. Rather than the use of expression vectors containing viral origins of replication, the host cell is controlled by appropriate expression control elements (eg, promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectable markers. It can be transformed with DNA. Following the introduction of exogenous DNA, engineered cells can be grown for 1-2 days in concentrated medium and then switched to selective medium. A selectable marker in the recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosomes and proliferate, forming a lesion that can then be cloned and propagated in the cell line. . This method can be advantageously used to engineer cell lines expressing antibody molecules. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.

Many selection systems may be used, including tk-cells, hgprt-, respectively.
Herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11: 223), hypoxanthine-guanine phosphoribosyl transferase (Szybalska and Szybalski, 1992, Proc. Natl. Acad. Sci.
USA 48: 202), and adenine phosphoribosyl transferase (
Lowy et al., 1980, Cell 22: 817) gene, but is not limited thereto. Antimetabolite resistance can also be used as a basis for selection for the following genes: dhfr (Wigl confers resistance to methotrexate)
er et al., 1980, Natl. Acad. Sci. USA 77: 357); O
'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 7
8: 1527; gpt conferring resistance to mycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 7
8: 2072); conferring resistance to the aminoglycoside G-418 neo (Cli
nical Pharmacy 12: 488-505; Wu and Wu, 19
91, Biotherapy 3: 87-95; Tolstoshev, 199.
3, Ann. Rev. Pharmacol. Toxicol. 32: 573-5
96; Mulligan, 1993, Science 260: 926-932.
And Morgan and Anderson, 1993, Ann. Rev.
Biochem. 62: 191-217; May 1993, TIB TECH.
11 (5): 155-215); and h conferring resistance to hygromycin
ygro (Santerre et al., 1984, Gene 30: 147). Recombinant DNA technology methods generally known in the art can be routinely applied to the selection of desired recombinant clones, and such methods are described, for example, in Ausubel et al.
Ed.), 1993, Current Protocols in Molecule.
ar Biology, John Wiley & Sons, NY; Kriegl
er, Gene Transfer and Expression, A La
laboratory Manual, Stockton Press, NY (19
90); and Dracopoli et al. (Eds.), 1994, Current P.
rotocols in Human Genetics, John Wile
y & Sons, NY, Chapters 12 and 13; Colberre-Garapin et al., 1981, J. Am. Mol. Biol. 150: 1, which are incorporated herein by reference in their entirety.

Expression levels of antibody molecules can be increased by vector amplification (for a review, Bebbington and Hentschel, The use of).
vectors based on gene amplification
for the expression of cloned genes i
n mammalian cells in DNA cloning, volume 3 (Academic Press, New York, 1987). If the marker can be amplified in the vector system expressing the antibody, increasing the level of inhibitor present in the culture medium of the host cell will increase the number of copies of the marker gene. Production of this antibody is also increased due to the binding of this amplified region to the antibody gene (Crouse et al., Mol. Cell. Biol. 3:
257 (1983)).

The host cell is co-transfected with two expression vectors of the invention, a first vector encoding a polypeptide derived from the heavy chain and a second vector encoding a polypeptide derived from the light chain. Can be done. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes both heavy and light chain polypeptides and allows for expression thereof. In such a situation, the light chain should be placed before the heavy chain to avoid excess toxic free heavy chain (Proudfoot, 1986, Nature 322: 52; Kohle.
r, Proc. Natl. Acad. Sci. USA 77: 2197 (198).
0)). The heavy and light chain coding sequences may include cDNA or genomic DNA.

Once the antibody molecule of the present invention is produced by animal, chemical synthesis, or recombinant expression, the antibody molecule may be produced by any method known in the art for the purification of immunoglobulin molecules (eg, Chromatography (eg, ion exchange chromatography, affinity (particularly by Protein A followed by affinity for a specific antigen) chromatography, and sizing (size).
ng) column chromatography), centrifugation, differential solubility, or by any other standard technique for purification of proteins).

C. Antibody Conjugates The present invention provides that the polypeptides of the present invention (or portions thereof, preferably at least 10, 20, or 50 of the polypeptides) are used to produce fusion proteins.
Individual amino acids) or chemically bound (including both covalent and non-covalent) antibodies. This fusion need not necessarily be direct, but can occur through linker sequences. The antibody is a polypeptide of the invention (or a portion thereof, preferably at least one of the polypeptides).
Can be specific for antigens other than 0, 20, or 50 amino acids). For example,
By fusing or binding a polypeptide of the invention to an antibody specific for a particular cell surface receptor, the antibody targets the polypeptide of the invention to a particular cell type, either in vitro or in vivo. Can be used to
Antibodies fused or conjugated to the polypeptides of the invention can also be used in in vitro immunoassays and purification methods using methods known in the art. See, eg, Harbor et al., Supra, and PCT Publication WO 93/21232.
EP 439,095; Naramura et al., Immunol. Lett. Three
9: 91-99 (1994); US Pat. No. 5,474,981; Gillie.
S. et al., PNAS 89: 1428-1432 (1992); Fell et al. I
mmunol. 146: 2446-2452 (1991), which are incorporated by reference in their entirety.

The invention further includes compositions comprising a polypeptide of the invention fused or bound to a domain of an antibody other than the variable region. For example, the polypeptides of the invention can be fused or conjugated to the Fc region of an antibody, or a portion thereof. The antibody portion fused to the polypeptide of the present invention comprises a constant region, a hinge region, a CH1 domain,
It may comprise the CH2 domain, and the CH3 domain, or any combination of all or part of those domains. The polypeptide can also be fused or conjugated to the antibody moieties described above to form multimers. For example, an Fc portion fused to a polypeptide of the invention may form a dimer via a disulfide bond between the Fc portions. Higher order multimeric forms can be made by fusing the polypeptide to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody moieties are known in the art. For example, US Pat. Nos. 5,336,603; 5,622,929; 5,359,04.
No. 6, No. 5,349,053; No. 5,447,851; No. 5,112.
, 946; EP 307,434; EP 367,166; PCT publication WO
96/04388; WO 91/06570; Ashkenazi et al., Proc.
． Natl. Acad. Sci. USA 88: 10535-10539 (19).
91); Zheng et al. Immunol. 154: 5590-5600 (1
995); and Vil et al., Proc. Natl. Acad. Sci. USA
89: 11337-11341 (1992) (the above references are incorporated by reference in their entireties).

As discussed above, the polypeptides of the present invention can be fused or conjugated to the antibody moieties described above to increase the in vivo half-life of the polypeptide or by methods known in the art. Used in the immunoassay used. Furthermore, the polypeptides of the present invention can be fused or conjugated to the antibody moieties described above to facilitate purification. One reported example is the first two of the human CD4 polypeptides.
A chimeric protein consisting of one domain and various domains of the constant region of the heavy or light chain of a mammalian immunoglobulin is described. (EP 394,827;
Traunecker et al., Nature 331: 84-86 (1988)).
Polypeptides of the invention fused or linked to an antibody having a disulfide-linked dimeric structure (due to IgG) may also bind to and bind to other molecules more than monomeric secreted proteins or protein fragments alone. May be more efficient in summation. (Funtoulakis et al., J. Biochem. 270: 3.
958-3964 (1995)). In many cases, the Fc of the fusion protein
The moieties are advantageous in therapy and diagnosis and thus may, for example, yield improved pharmacokinetic properties. (EP A 232, 262). Alternatively, deletion of the Fc portion after the fusion protein has been expressed, detected and purified is preferred. For example, if the fusion protein is used as an antigen for immunization, the F
The c protein can interfere with treatment and diagnosis. In drug discovery, for example, human proteins (eg, hIL-5 receptor) were fused with Fc proteins for the purpose of high throughput screening assays to identify antagonists of hIL-5. (Bennett et al., J. Molecular Recog.
region 8: 52-58 (1995); Johnson et al., J. Am. Bio
l. Chem. 270: 9459-9471 (1995). 3.) Furthermore, the antibodies of the invention or their fragments may be fused to a marker sequence, such as a peptide that facilitates purification. In a preferred embodiment, the marker amino acid sequence is, inter alia, the pQE vector (QIAGEN, Inc.
, 9259 Eton Avenue, Chatsworth, CA, 9131.
Hexa-histidine peptides such as the tags provided in 1), many of which are commercially available. For example, Gentz et al., Proc. Natl. Ac
ad. Sci. Hexa-histidine provides convenient purification of the fusion protein, as described in USA 86: 821-824 (1989). Other peptide tags useful for purification include the "HA" tag corresponding to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37: 767 (19).
84)) and "flag" tags.

[0214]   The invention further provides an antibody or fragment thereof conjugated to a diagnostic or therapeutic agent.
Including This antibody can be used, for example, as part of a clinical trial procedure (eg
Monitor the development or progression of tumors (as to determine the efficacy of dimene)
Can be used diagnostically to. Detection involves coupling the antibody to a detectable substance
Can be facilitated by doing. Examples of detectable substances are various enzymes, prosthetic groups.
Molecular groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, various proton emission faults
Positron-emitting metals using radiography, and non-radioactive paramagnetic metal ions are included.
It The detectable substance can be the antibody (or the antibody) using techniques known in the art.
Directly to these fragments) or intermediates (eg known in the art)
Coupled or bound, either indirectly via a linker)
obtain. Metal ions which can be bound to antibodies for use as diagnostics according to the invention
See, eg, US Pat. No. 4,741,900. Proper fermentation
Examples of primes include horseradish peroxidase, alkaline phosphatase, β-gala.
Suitable for prosthesis molecules include: succinase, or acetylcholinesterase;
Examples of group complexes include streptavidin / biotin and avidin / biotin.
Examples of suitable fluorophores include umbelliferone, fluorescein, fluor
Olecein isothiocyanate, rhodamine, dichlorotriazinylamine ful
Includes olecein, dansyl chloride or phycoerythrin; luminescent materials
Examples include luminol; examples of bioluminescent materials are luciferase,
Cypherins, and aequorins; and examples of suitable radioactive substances include ¹²⁵ I,¹³¹I,¹¹¹In or⁹⁹Tc is mentioned.

In addition, the antibody or fragment thereof can be conjugated to a therapeutic moiety such as a cytotoxin (eg, cytostatic or cytocidal agent), therapeutic agent or radioactive metal ion. Cytotoxic or cytotoxic agents include any agent that is harmful to cells. Examples include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide.
de), vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitozantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid,
Included are procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogs or homologs. The therapeutic agents include antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (eg, mechlorethamine), thioepa (thioepa).
pa) chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide
, Busulfan, dibromomannitol, streptozotocin (strepto
zotocin), mitomycin C, and cis-dichlorodiamine platinum (I
I) (DDP) cisplatin), anthracyclines
ne) (eg, daunorubicin (formerly daunomycin), and doxorubicin), antibiotics (eg, dactinomycin (formerly actinomycin),
Bleomycin, mithramycin, and anthramycin
cin) (AMC)), and anti-mitotic agents such as vincristine and vinblastine, but are not limited thereto.

The conjugates of the invention can be used for the modification of a given biological response and the therapeutic agent or drug moiety should not be construed as limited to classical chemotherapeutic agents. . For example, the drug moiety can be a protein or polypeptide that possesses the desired biological activity. Such proteins include, for example, toxins such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminose. Gen-activators, thrombotic agents or anti-angiogenic agents (eg angiostatin or endostatin (en
dostatin)); or biological response modifiers (eg, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 ( "IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor (
"G-CSF"), or other growth factors, etc.).

Antibodies can also be attached to a solid support that is particularly useful for immunoassay or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Techniques for coupling such therapeutic moieties to the antibody are well known, eg Arnon.
Et al., "Monoclonal Antibodies For Immuno.
targeting Of Drugs In Cancer Therapy "
Monoclonal Antibodies And Cancer The
rapid, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Lis.
s, Inc. 1985); Hellstrom et al., "Antibodies F."
or Drug Delivery ”, Controlled Drug De
livery (2nd edition), Robinson et al. (eds.), pp. 623-53 (Mar.
cel Dekker, Inc. 1987); Thorpe, "Antibod
y Carriers Of Cytotoxic Agents In Ca
ncer Therapy: A Review ”, Monoclonal An
tibodies'84: Biological And Clinical
Applications, Pinchera et al. (Eds.), Pp. 475-506 (1
985); "Analysis, Results, And Future Pr.
Descendant Of The Therapeutic Use Of
Radiolabeled Antibody In Cener Ther
apy ”, Monoclonal Antibodies For Cancel
r Detection And Therapy, Baldwin et al. (ed.),
Pp. 303-16 (Academic Press 1985), and Thor
pe et al., “The Preparation And Cytotoxic P
properties of Antibody-Toxin Conjugat
es ", Immunol. Rev. 62: 119-58 (1982).

Alternatively, the antibody is conjugated to a secondary antibody and the antibody heteroconjugate as described by Segal in US Pat. No. 4,676,980, which is incorporated herein by reference in its entirety. A body (heteroconjugate) may be formed.

Antibodies, with or without a therapeutic moiety attached to the antibody, administered alone or in combination with cytotoxic factors and / or cytokines, can be used as therapeutic agents.

D. Assays for Antibody Binding The antibodies of the invention can be assayed for immunospecific binding by any method known in the art. Immunoassays that may be used include (Western blot, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitation, to name just a few). Competitive assay systems and non-competitive assay systems using techniques such as, but not limited to, reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays. Such assays are routine and well known in the art (eg, Ausubel et al., Ed., 1994, Current Pro, which is incorporated herein by reference in its entirety).
tocols in Molecular Biology, Volume 1, John
Wiley & Sons, Inc. , New York). Exemplary immunoassays are briefly described below (but these are not intended to be limiting).

Immunoprecipitation protocols generally involve RIPA buffer (1% NP-40 or Trito) supplemented with protein phosphatase and / or protease inhibitors (eg, EDTA, PMSF, aprotinin, sodium vanadate).
nX-100, 1% sodium deoxycholate, 0.1% SDS, 0.
0.01 M sodium phosphate, 15% NaCl, pH 7.2, 1% Tr
lysing a population of cells in a lysis buffer such as asylol), adding the antibody of interest to the cell lysate, incubating at 4 ° C. for a period of time (eg 1-4 hours), protein A and / Or adding Protein G Sepharose beads to the cell lysate, incubating at 4 ° C for about 1 hour or more, washing the beads in lysis buffer, and resuspending the beads in SDS / sample buffer Including the step of The ability of the antibody of interest to immunoprecipitate a particular antigen is
It can be assayed, for example, by Western blot analysis. Those of skill in the art are familiar with parameters that can be modified to increase antibody binding to antigen and reduce background (eg, preclearing cell lysates with Sepharose beads). . For further discussion on immunoprecipitation protocols, see, eg, Ausubel et al., Eds., 1994, Current P.
rotocols in Molecular Biology, Volume 1, Jo
hn Wiley & Sons, Inc. , New York, 10.16.
See 1.

Western blot analysis generally involves the steps of preparing a protein sample, in which the protein sample is polyacrylamide gel (eg, 8 depending on the molecular weight of the antigen).
% -20% SDS-PAGE), transfer of protein sample from its polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking solution (eg 3% BSA or skim milk powder) Blocking the membrane in PBS), washing the membrane in wash buffer (eg, PBS-Tween 20), the membrane with primary antibody (antibody of interest) diluted in blocking buffer Blocking the membrane, washing the membrane in wash buffer, diluting enzyme substrate (eg horseradish peroxidase or alkaline phosphatase) or radioactive molecule (eg ³² P or ¹²⁵ I) in blocking buffer. Bound secondary antibody (recognizes primary antibody, eg anti-human Blocking the membrane with an antibody), washing the membrane in a wash buffer, and detecting the presence of the antigen. Those skilled in the art are familiar with parameters that can be modified to increase the signal detected and reduce background noise. For further discussion on Western blot protocols see, eg, Ausubel.
Et al., 1994, Current Protocols in Molecule.
ar Biology, Volume 1, John Wiley & Sons, Inc
． , New York, 10.8.1.

ELISA is for the purpose of preparing an antigen, coating the wells of a 96-well microtiter plate with the antigen, binding to a detectable compound such as an enzyme substrate (eg horseradish peroxidase or alkaline phosphatase). Antibody to the well and incubating for a period of time, and detecting the presence of antigen. In the ELISA, the antibody of interest need not be conjugated to a detectable compound; instead, a secondary antibody conjugated to the detectable compound (which recognizes the antibody of interest) can be added to the wells. Further, instead of coating the well with the antigen, the antibody can be coated to the well. In this case, a secondary antibody conjugated to a detectable compound can be added following addition of the antigen of interest to the coated wells. Those of skill in the art are familiar with parameters that can be modified to increase the signal detected, and other variations of the ELISA known in the art. For further discussion on ELISA, see, eg, Ausubel et al., Ed., 1994, Cu.
current Protocols in Molecular Biology
, Vol. 1, John Wiley & Sons, Inc. , New York
, 11.2.1.

The binding affinity of an antibody for its antigen and the off-rate of antibody-antigen interactions can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay, which comprises incubating a labeled antigen (eg, ³ H or ¹²⁵ I) with an antibody of interest in the presence of increasing amounts of unlabeled antigen, and Includes detection of antibody bound to labeled antigen. The affinity of the antibody of interest for a particular antigen, and the binding off-rate can be determined from the data by Scatchard plot analysis. Competition with secondary antibodies can also be determined using radioimmunoassay. In this case, the antigen is incubated with the antibody of interest bound to a labeled compound (eg, ³ H or ¹²⁵ I) in the presence of increasing amounts of unlabeled secondary antibody.

E. Antibody-Based Therapy The present invention further relates to antibody-based therapies, wherein the antibody of the invention is administered to one or more of the disorders described herein or Animals to treat the condition,
Preferably it comprises the step of administering to a mammal, and most preferably a human patient.
Therapeutic compounds of the invention include antibodies of the invention (including fragments, analogs and derivatives thereof, as described herein) as well as nucleic acids encoding the antibodies of the invention (described herein). (Including fragments, analogs and derivatives thereof). Abnormalities of a polypeptide of the invention, including, but not limited to, any one of the diseases, disorders, or conditions described herein, using the antibodies of the invention, such as, for example: Treatment, suppression of a disease, disorder, or condition associated with specific expression and / or activity,
Or preventable: Graft-versus-host disease and autoimmune diseases, disorders, or conditions associated with such diseases or disorders (autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenic purpura ( idiopathic thrombo
cytopenia purpura), autoimmune cytopenia, hemolytic anemia,
Antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (eg IgA nephropathy), multiple sclerosis, neuritis, uveal conjunctivitis , Multiple endocrine disorders, purpura (eg Henl
och-Scoenlein purpura), Reiter's disease, Stiffman syndrome, autoimmune pneumonia, Guyan-Barre syndrome, insulin-dependent diabetes mellitus, and autoimmune inflammatory eye disease, autoimmune thyroiditis, thyroid dysfunction (ie, Hashimoto thyroiditis), systemic lupus erythematosus, Goodpasture's syndrome, pemphigus, for example,
(A) Graves' disease, (b) myasthenia gravis, and (c) receptor autoimmunity such as insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, rheumatoid arthritis, anti-collagen Sclerodes with antibodies
rma), mixed connective tissue disease, polymyositis / dermatomyositis, pernicious anemia, idiopathic Addison's disease, infertility, glomerulonephritis (eg, primary glomerulonephritis and IgA nephropathy), bullous pemphigoid, Sjogren's syndrome. , Diabetes mellitus, and adrenergic drug resistance (including adrenergic drug resistance associated with asthma or fibrosis)
, Chronic active liver disease, primary biliary cirrhosis, other endocrine insufficiency, vitiligo, vasculitis,
Late myocardial infarction (post-MI), cardiotomy syndrome, urticaria, atopic dermatitis, asthma, inflammatory myopathy, and other inflammatory, granulomatic (granulamat)
, degenerative, and atrophic disorders, and immunodeficiency, or conditions associated with such diseases or disorders (severe combined immunodeficiency (SCID) -X-linked, SCID-autosomal). , Adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton's disease, congenital agammaglobulinemia, X-linked infant agammaglobulinemia, acquired agammaglobulinemia , Adult-onset agammaglobulinemia, late-onset agammaglobulinemia, hypogammaglobulinemia, hypergammaglobulinemia, neonatal transient gammaglobulinemia, unspecified hypergammaglobulinemia, Gamma globulinemia, unclassified immunodeficiency (CIVD) (acquired), Viscott-Aldrich syndrome ( AS), X-linked immunodeficiency with high IgM (hyper IgM), non-X-linked immunodeficiency with high IgM (hyper IgM), selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency) Absent)
, Antibody deficiency with normal or elevated Igs, immunodeficiency with thymoma, I
g heavy chain deficiency, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), selective Ig
M immunodeficiency, recessive agammaglobulinemia (Swiss type), reticulo-hypothyroidism (re
thyroid dysgenesis), neonatal neutropenia, severe congenital leukopenia, dysplasia with thymic lymphaplasia or immunodeficiency, ataxia-telangiectasia, dwarfism, X-linked lymphocyte proliferation Syndrome (XLP), immunodeficiency with Nezerov syndrome complex Igs, purine nucleoside phosphorylase deficiency (PNP), MHC class II deficiency (deficient lymphocyte syndrome), severe combined immunodeficiency, DiGeorge abnormality, thymic dysgenesis, chronic mucosa (Including but not limited to candidiasis, natural killer cell deficiency, idiopathic CD4 + T lymphopenia, and immunodeficiency with dominant T cell deficiency).

In certain embodiments, the antibodies of the invention may be used to treat, suppress, prognose, diagnose or prevent graft versus host disease and autoimmune disease.

In another specific embodiment, the antibodies of the present invention are used to treat rheumatoid arthritis,
Suppress, prognose, diagnose or prevent.

In another specific embodiment, the antibodies of the invention are used to treat, suppress, prognose, diagnose or prevent systemic lupus erythematosus. Treatment and / or prophylaxis of diseases and disorders associated with aberrant expression and / or activity of the polypeptides of the invention include, but are not limited to, ameliorating the symptoms associated with these diseases and disorders. The antibodies of the invention are provided in pharmaceutically acceptable compositions as known in the art or as described herein.

One summary of the ways in which the antibodies of the invention may be used therapeutically is locally or systemically in the body, or (eg, by complement (CDC), or by effector cells (ADCC)). Conjugating a polynucleotide or polypeptide of the invention by direct cytotoxicity of the antibody (as mediated). Some of these approaches are described in more detail below. Given the teachings provided herein, one of ordinary skill in the art would be able, without undue experimentation, to determine how to use the antibodies of the invention for diagnostic, monitoring, or therapeutic purposes. to understand.

Antibodies of the invention may be, for example, other monoclonal or chimeric antibodies, or lymphokines or hematopoietic growth factors (eg, IL-2) that act to increase the number or activity of effector cells that interact with the antibody. , IL-3 and I
L-7 and the like) can be used advantageously.

The antibodies of the invention can be administered alone or in combination with other types of treatments such as radiation therapy, chemotherapy, hormone therapy, immunotherapy and anti-tumor agents.
In general, it is preferred to administer a species-sourced or species-reactive product that is the same species as the patient's species (in the case of an antibody). Thus, in a preferred embodiment, human antibodies,
The fragment derivative, analog, or nucleic acid is administered to a human patient for treatment or prevention.

[0233]   Polynucleotides or polypeptides of the invention, including fragments thereof
) Immunoassays, and for the treatment of disorders associated therewith
, With high affinity and / or affinity for a polypeptide or polynucleotide of the invention.
Or potent in vivo inhibitory and / or neutralizing antibodies, fragments thereof
, Or that region is preferably used. Such antibodies, fragments,
Alternatively, the region is preferably a polynucleotide or polypeptide of the invention.
(Including these fragments). With a preferred binding affinity
5 × 10^-6M, 10^-6M, 5 x 10^-7M, 10^-7M, 5 x 10^-8M, 10 ^-8 M, 5 x 10^-9M, 10^-9M, 5 x 10^-TenM, 10^-TenM, 5 x 10^-11M,
10^-11M, 5 x 10^-12M, 10^-12M, 5 x 10^-13M, 10^-13M, 5 x 10^{- 14} M, 10^-14M, 5 x 10^-15M, 10^-11M, 5 x 10^-12M, 10^-12M, 5
× 10^-13M, 10^-13M, 5 x 10^-14M, 10^-14M, and 10^-15Less than M
The binding affinity has a dissociation constant, Kd.

Identification of AIM II Agonists and Antagonists The present invention is also for identifying compounds that enhance or block the action of AIM II on cells (eg, interaction with AIM II binding molecules such as receptor molecules). To provide a method of screening compounds. The agonist is
AIM is a compound that increases the natural biological function of II, or AIM
Compounds that function in a manner similar to II. On the other hand, antagonists reduce or eliminate such functions.

For example, a cell compartment (eg, a membrane preparation) may be a molecule that binds AIM II (
(Eg, AIM II regulated signaling or regulatory pathway molecules)
Can be prepared from cells expressing. The preparation is incubated with labeled AIM II in the absence or presence of a candidate molecule, which can be an AIM II agonist or AIM II antagonist. The ability of the binding molecule or the candidate molecule to bind to AIM II itself is reflected in a decrease in labeled ligand binding. Molecules that bind gratuitously, that is, without inducing the effects of AIM II when bound to AIM II binding molecules, are most likely to be good antagonists. Bind well, and AIM I
Molecules that elicit the same or a closely related effect as I are good agonists.

AIM II-like effects of potential agonists and antagonists have been demonstrated, for example:
By determining the activity of the second messenger system following interaction of the candidate molecule with cells or a suitable cell preparation, and its effect with the effect of AIM II or of a molecule that induces an effect similar to AIM II. It can be measured by comparing. Second messenger systems that may be useful in this regard include, but are not limited to, AMP guanylate cyclase, ion channels, or phosphoinositide hydrolyzing second messenger systems.

Another example of an assay for AIM II antagonists is the competition assay. This assay was performed under the appropriate conditions for competitive inhibition assays under the AIM
II and potential antagonists are combined with membrane-bound AIM II receptor molecules or recombinant AIM II receptor molecules. AIM II is, for example,
AIM I that can be labeled with a radioactive label and is thus bound to the receptor molecule
The number of I molecules can be accurately determined to assess the efficacy of potential antagonists.

Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to a polypeptide of the invention and thereby inhibit or abrogate its activity. Potential antagonists also interfere with the action of AIM II by binding to the same site on a binding molecule (eg, a receptor molecule) without inducing AIM II-inducing activity, thereby eliminating AIM II from binding. , Small organic molecules such as closely related proteins or antibodies, peptides, polypeptides. SEQ ID NO: 2 is an antagonist of the present invention.
And a fragment of the AIM II polypeptide having the amino acid sequence shown in.

Other potential antagonists include antisense molecules. Antisense technology can be used to control gene expression through antisense DNA or RNA, or through triplex helix formation. Antisense technology is described, for example, in Okano, J. et al. Neurochem. 56: 560 (1991)
); Oligodeoxynucleotides as Antisense
Inhibitors of Gene Expression, CRC P
less, Boca Rotan, FL (1988). Triplex helix formation is described, for example, by Lee et al., Nucleic Acids Res.
search 6: 3073 (1979); Cooney et al., Science 2
41: 456 (1988); and Dervan et al., Science 251:
1360 (1991). This method uses complementary DNA or R
Based on the binding of the polynucleotide to NA. For example, the 5'coding portion of the polynucleotide encoding the mature polypeptide of the invention can be used to design an antisense RNA oligonucleotide of about 10-40 base pairs in length. DN
The A oligonucleotide is designed to be complementary to a region of the gene involved in transcription, thereby preventing transcription and production of AIM II. Antisense R
NA oligonucleotides hybridize to mRNA in vivo and
Blocks translation of RNA molecules into AIM II polypeptides. The oligonucleotides described above can also be delivered to cells so that antisense RNA or DNA can be expressed in vivo to inhibit production of AIM II.

Therapeutic Uses of AIM II, AIM II Agonists and AIM II Antagonists Tumor necrosis factor (TNF) family ligands are found in many cellular responses (cytotoxicity, antiviral activity, immunomodulatory activity, and some of them). (Including gene transcriptional regulation)
, Is known to be one of the most pleiotropic cytokines (Goeddel,
D. V. Et al. "Tumor Necrosis Factors: Gene St
“Ructure and Biological Activities”, S
ymp. Quant. Biol. 51: 597-609 (1986), Cold.
Spring Harbor; Beutler, B .; And Cerami, A
． , Annu. Rev. Biochem. 57: 505-518 (1988);
Old, L.D. J. , Sci. Am. 258: 59-75 (1988); Fier.
S.W. FEBS Lett. 285: 199-224 (1991)). TNF
Family ligands, by binding to TNF family receptors,
It induces such various cellular responses.

The AIM II polynucleotides, polypeptides, agonists or antagonists of the present invention may be used for treatment of defective AIM II or any disorder mediated (either directly or indirectly) by insufficient amounts of AIM II. It can be used in development. AIM II polypeptides, agonists or antagonists can be administered to patients (eg, mammals (preferably humans)) suffering from such disorders. Alternatively, gene therapy approaches can be applied to treat such disorders. Disclosure herein of AIM II nucleotide sequences includes detection of defective AIM II genes, and normal AI of defective AIM II genes.
Allows replacement with the M II coding gene. Defective genes are detected in in vitro diagnostic assays and by comparison of the AIM II nucleotide sequences disclosed herein with the nucleotide sequences of AIM II genes from patients suspected of having a defect in this gene. Can be done.

The AIM II polypeptides of the present invention can be used to treat lymphoproliferative disorders that result in lymphadenopathy, AIM II mediates apoptosis by stimulating clonal clearance of T cells, thus It can be used to stimulate peripheral tolerant and cytotoxic T cell-mediated apoptosis to treat autoimmune diseases. AIM II also has systemic lupus erythematosus (SLE), Graves' disease, immunoproliferative disease lymphadenopathy (IPL), angioimmunoproliferative lymphadenopathy (
AIL), immunoblastic lymphadenopathy (IBL), rheumatoid arthritis, diabetes, and as a research tool in elucidating the biology of autoimmune disorders including multiple sclerosis, allergies, and graft-versus-host It can be used to treat a disease.

AIM II polynucleotides, polypeptides and / or agonists or antagonists of the present invention treat, prevent, but are not limited to, autoimmune disorders, immunodeficiency disorders, and graft-versus-host disease. It can be used for diagnosis and / or prognosis.

AIM II polypeptides of the present invention may also be neoplastic (eg, tumor cell growth).
Can be used to inhibit AIM II polypeptides may be responsible for tumor destruction by apoptosis and cytotoxicity to specific cells. AIM
Since II has a proliferative effect on cells of endothelial origin, AIM II can also be used to treat diseases that require pro-proliferative activity, such as restenosis. Therefore, AIM II can also be used to regulate hematopoiesis in endothelial cell development.

Diseases associated with increased cell survival or inhibition of apoptosis that can be treated, prevented, diagnosed and / or prognosed with the AIM II polynucleotides, polypeptides, and / or antagonists or agonists of the invention include: Cancer (eg, follicular lymphoma, carcinoma with p53 mutation, and hormone-dependent tumor, which are: colon cancer, heart tumor, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestine Cancer, testicular cancer, gastric cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelial tumor, osteoblastoma, giant cell tumor of the bone, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovary Including, but not limited to cancer; autoimmune disorders (eg, multiple sclerosis, Sjogren's syndrome, Graves' disease, Hashimoto thyroiditis) Autoimmune diabetes, biliary cirrhosis, Behcet's disease (Behcet's
disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) and viral infections (eg,
Herpesviruses, poxviruses and adenoviruses), inflammation, graft-versus-host disease (acute and / or chronic), acute graft rejection, and chronic graft rejection. In a preferred embodiment, the AIM polynucleotides, polypeptides, antagonists or antagonists of the invention are used to inhibit cancer growth, progression, and / or metastasis, especially those listed above or in the following paragraphs. To be done.

Additional diseases or conditions associated with increased cell survival that may be treated, prevented, diagnosed and / or prognosed with the AIM II polynucleotides, polypeptides, and / or antagonists or agonists of the invention include malignant Diseases and related disorders including but not limited to the progression and / or metastasis of: leukemia (acute leukemia (eg, acute lymphocytic leukemia, acute myelogenous leukemia (myeloblastic, promyelocytic, promyelocytic , Myelomonocytic, including monocytic and erythroleukemia) and chronic leukemia (eg, chronic myelogenous (granulocytic))
Leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (eg Hodgkin's and non-Hodgkin's disease), multiple myeloma, Waldenstrom macroglobulinemia, heavy chain disease, and solid tumors (sarcoma). And carcinomas (eg fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphatic endothelium,
Periosteoma, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat adenocarcinoma, sebaceous adenocarcinoma, Papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial progenitor carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, testicular tumor , Lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineocytoma, hemangioblastoma, acoustic nerve Tumors, oligodendroglioma, meningiomas, melanomas, neuroblastomas, and retinoblastomas).

Diseases associated with increased apoptosis that may be treated, prevented, diagnosed and / or prognosed with the AIM II polynucleotides, polypeptides, and / or antagonists or agonists of the invention include: , But not limited to: AIDS; neurodegenerative disorders (eg Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and brain tumors or previously associated disorders); autoimmune disorders (eg , Multiple sclerosis, Sjogren's syndrome, Graves' disease, Hashimoto's thyroiditis, autoimmune diabetes, biliary cirrhosis,
Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis, myelodysplastic syndrome (eg, aplastic anemia), graft-versus-host disease (Acute and / or chronic), ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury or disease (eg, hepatitis-related liver injury, cirrhosis, ischemia / reperfusion injury, cholestasis). Cholestosis (bile duct injury) and liver cancer); toxic-induced liver disease (such as alcohol-induced),
Septic shock, ulcerative colitis, cachexia and anorexia. In a preferred embodiment, AIM II polynucleotides, polypeptides, agonists and / or antagonists are used to treat the diseases and disorders described above.

Many pathologies associated with HIV are mediated by apoptosis, including HIV-induced nephropathy and HIV encephalitis. Thus, in a preferred embodiment, the AIM II polynucleotides, polypeptides, agonists or antagonists of the invention are used to treat AIDS and AIDS-related pathologies.

Another embodiment of the invention relates to the use of AIM II polynucleotides, polypeptides or antagonists to reduce AIM II mediated death of T cells in HIV infected patients. The role of T cell apoptosis in the development of AIDS has been the subject of many studies (eg, Meyaard et al., Science).
e 257: 217-219 (1992); Groux et al., J. Am. Exp. Med
． , 175: 331 (1992); and Cell Activation a.
nd Apothesis in HIV infection, Andrieu
And Lu, Plenum Press, New York, 101-114.
See page (1995) Oyaizu et al.). Fas-mediated apoptosis was associated with T cell loss in HIV individuals (Katsiki
S., et al. Exp. Med. 181: 2029-2036 (1995)). Also, T cell apoptosis appears to occur through multiple mechanisms. For example, HI
At least some of the T cell deaths found in V patients appear to be mediated by AIM II.

Activated human T cells undergo programmed cell death (apoptosis) (activation-induced cell death (apoptosis) upon induction via the CD3 / T cell receptor complex.
Induced to undergo a process called AICD). AICD of CD4 T cells isolated from HIV infected asymptomatic individuals has been reported (Grou.
x et al., supra). Therefore, AICD may play a role in depletion of CD4 + T cells and progression of AIDS in HIV-infected individuals. Therefore, the present invention
Provided is a method of inhibiting AIM II-mediated T cell death in an IV patient, the method comprising administering to the patient an AIM II polynucleotide, polypeptide or antagonist. In one embodiment, when treatment with the AIM II polynucleotide, polypeptide or antagonist is initiated,
The patient is asymptomatic. If desired, prior to treatment, peripheral blood T cells can be extracted from HIV patients and tested for susceptibility to AIM II-mediated cell death by means known in the art. In one embodiment, the blood or plasma of a patient has an AIM II antagonist of the invention (eg, an anti-AIM II antibody).
Are contacted ex vivo. The AIM II antagonist can be attached to a suitable chromatographic matrix by means known in the art. The patient's blood or plasma flows through a chromatography column containing the AIM II antagonist bound to the matrix and then returned to the patient. The immobilized AIM II antagonist binds AIM II and therefore clears the AIM II protein from the blood of the patient.

In a further embodiment, the AIM II polynucleotides, polypeptides, or antagonists of the invention are administered in combination with other inhibitors of T cell apoptosis. For example, as described above, Fas-mediated apoptosis was also associated with T cell loss in HIV individuals (Katsikis et al., J. Am.
Exp. Med. 181: 2029-2036 (1995)). Therefore, Fas
Patients susceptible to both ligand-mediated and AIM II-mediated T cell death can be treated with both agents that block AIM II / AIM II receptor interactions and agents that block Fas ligand / Fas interactions. . F
Suitable agents for blocking the binding of asi ligands to Fas include soluble Fas polypeptides; multimeric forms of soluble Fas polypeptides (eg, sFas / Fc dimers); transmission of biological signals that result in apoptosis. An anti-Fas antibody that binds Fas without; an anti-Fas ligand antibody that blocks the binding of Fas ligand to Fas; and a mutein of the Fas ligand that binds Fas but does not transduce a biological signal that causes apoptosis. But is not limited to these. Preferably, the antibody used according to this method is a monoclonal antibody. Examples of suitable agents for blocking Fas ligand / Fas interactions, including blocking anti-Fas monoclonal antibodies, are described in WO 95/10540, hereby incorporated by reference.

In another example, an agent that blocks TRAIL binding to the TRAIL receptor is administered with an AIM II polynucleotide, polypeptide, or antagonist of the invention. Such agents include, but are not limited to, soluble TRAIL receptor polypeptides (eg, OP
Solubilized forms of G, DR4 (WO98 / 32856); TR5 (WO98 / 306
93); DR5 (WO98 / 41629); and TR10 (WO98 / 542).
02)); a multimeric form of the soluble TRAIL receptor polypeptide; as well as a TRAIL receptor antibody that binds the TRAIL receptor without transmission of a biological signal that causes apoptosis, blocking the binding of TRAIL to one or more TRAIL receptors , An anti-TRAIL antibody, and a TRAIL receptor, but does not transduce a biological signal that results in apoptosis, TR
AIL mutein. Preferably, the antibody used in this method is a monoclonal antibody.

The AIM II polypeptides of the invention can also be used to regulate hematopoiesis, and especially erythropoiesis. Hematopoiesis is a multi-step cell proliferation and differentiation process that begins with a pool of pluripotent stem cells. These cells can proliferate and differentiate into hematopoietic progenitors in response to different stimuli. AIM I of the present invention
I polypeptides, and agonists and antagonists thereof,
And, in particular, can be used to either stimulate or inhibit the development of erythropoietic progenitor cells.

AIM II polypeptides used to regulate the development of hematopoietic cells can be in many forms (eg, full length or mature polypeptides, AIM II polypeptide fragments, or AIM II and non-AIM II polypeptides). (
For example, as a hybrid fusion protein composed of colony stimulating factor, erythropoietin, interferon, interleukin, etc.) can be contacted with hematopoietic target cells. Methods for producing such fusion proteins are described, for example, in Mele et al., US Pat. No. 5,916,773.

As described herein, the phrase “hematopoietic cells” refers to cells of hematopoietic origin (eg, red blood cells, platelets, lymphocytes, eosinophils, basophils, macrophages and monocytes).
Say. Further, the phrase "erythropoietic progenitor cells" refers to cells of erythropoietic origin (eg, red blood cells).

As discussed in Examples 6, 9 and 22 below, AIM II polypeptides (eg, containing or including amino acid residues 69-240 or 83-240 of SEQ ID NO: 2). And a antagonist thereof (eg, an anti-AIM II antibody, a solubilized form of AIM II having an amino acid sequence contained in the extracellular domain of AIM II) of various cell types (eg, T cells ), A cytokine (eg, GM-CSF, G-CS)
F, IL-2, IL-3, IL-5, IL-6, IL-7, IL-12, IL-
13, IL-15, anti-CD40, CD40L, IFN-γ, TNF-α, vascular endothelial growth factor (VEGF) can be used to stimulate production.

AIM II polypeptides, as well as their antagonists, can be used to modulate the T cell immune response (eg, to inhibit antigen-induced T cell proliferation).

AIM II polypeptides, as well as their agonists, can also be used to modulate cell-mediated immune responses (eg, activate cytotoxic T cells).

AIM II polypeptides, as well as their agonists, can also be used to regulate apoptosis.

In addition, AIM II polypeptides, as well as their agonists, can be used to induce a cytotoxic T cell response and promote the formation of antigen-specific memory.

The AIM II polynucleotides or polypeptides of the invention, or their agonists or antagonists, may also find applications as follows: A vaccine adjuvant that enhances immune responsiveness to specific antigens.

[0262]   Adjuvants for enhancing tumor-specific immune responses.

Adjuvants for Enhancing Antiviral Immune Responses: Antiviral immune responses that can be enhanced using the compositions of the invention as adjuvants are disclosed herein or otherwise known in the art. And viruses and virus-related diseases or conditions known in. In a particular embodiment, the composition of the invention is used as an adjuvant for enhancing the immune response against a virus, disease or condition selected from the group consisting of: AIDS, meningitis, dengue, EBV, and hepatitis (eg, hepatitis B). In another specific embodiment, the composition of the invention is used as an adjuvant for enhancing the immune response against a virus, disease or condition selected from the group consisting of: HIV
/ AIDS, RS virus, Dengue fever (Dengue), rotavirus, Japanese encephalitis type B, influenza type A and type B (Influenza A and B)
), Parainfluenza, Measles (Measle
s), Cytomegalovirus, Rabies, Junin
, Chikungunya, Rift Valley Fever, Herpes Simplex (
Herpes simplex), and yellow fever.

Adjuvants to Enhance Antibacterial or Antifungal Immune Responses: Disclosed herein are antibacterial or antifungal immune responses that can be enhanced using the compositions of the invention as adjuvants. And bacterial or fungal, and bacterial or fungal-related diseases or conditions known or known in the art. In a particular embodiment, the composition of the invention is used as an adjuvant for enhancing the immune response to a bacterium or fungus, disease or condition selected from the group consisting of: tetanus, Diphtheria, Botulism and type B meningitis. In another specific embodiment, the composition of the invention is used as an adjuvant for enhancing the immune response to a bacterium or fungus, disease or condition selected from the group consisting of: Vibrio cholera
e, Mycobacterium leprae, Salmonella ty
phi, Salmonella paratyphi, Meisseria m
eningitidis, Streptococcus pneumoniae
, Group B Streptococcus, Shigella sp., Enterotoxic Escherichia col
i, enterohemorrhagic E. E. coli, Borrelia burgdorferi, and Plasmodium (malaria).

Adjuvants for Enhancing Antiparasitic Immune Responses: Antiparasitic immune responses that can be enhanced using the compositions of the invention as adjuvants are disclosed herein or Examples include parasites and parasite-related diseases or conditions that are otherwise known in the art. In a particular embodiment, the compositions of the invention are used as an adjuvant to enhance the immune response against parasites. In another particular embodiment, the composition of the invention is used as an adjuvant for enhancing the immune response against Plasmodium (malaria).

As an agent for boosting immune responsiveness in a B cell and / or T cell immunodeficient individual, such as an individual who has undergone partial or complete splenectomy. B cell immunodeficiencies that may be ameliorated or treated by administering an AIM II polypeptide or polynucleotide of the invention, or agonists thereof, may include, but are not limited to, the following: severe combined immunodeficiency (SCI).
D) -X-linked, SCID-autosomal, adenosine deaminase deficient (ADA deficient), X-linked agammaglobulinemia (XLA), Bruton's disease (Bruton ')
s disease), congenital agammaglobulinemia, X-linked infant agammaglobulinemia, acquired agammaglobulinemia, adult-onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia , Hypogammaglobulinemia, transient infant hypogammaglobulinemia, unspecified hypogammaglobulinemia, agammaglobulinemia, unclassified immunodeficiency (CVID) (acquired),
Viscott-Aldrich syndrome (WAS), X-linked immunodeficiency with high IgM, non-X-linked immunodeficiency with high IgM, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), normal or Antibody deficiency with elevated Ig, immunodeficiency with thymoma, Ig heavy chain deletion, κ chain deficiency, B cell lymphoproliferative disorder (BLPD), selective IgM immunodeficiency, recessive agammaglobulinemia (Swiss type) , Retinal hypoplasia, neonatal neutropenia, severe congenital leukopenia, hypothyroidism or dysplasia with immunodeficiency, ataxia-telangiectasia, short limb dwarfism, X Linked lymphoproliferative syndrome (XLP), Nezerov's syndrome combined immunodeficiency with Ig, purine nucleoside phosphorylase deficiency ( PNP), MHC class II deficiency (deficient lymphocyte syndrome) and severe combined immunodeficiency. T cell immunodeficiencies that may be ameliorated or treated by administering an AIM II polypeptide or polynucleotide of the invention, or agonists thereof, may include, but are not limited to, DiGeorge abnormalities, thymic development. Immunodeficiency with insufficiency, chronic mucocutaneous candidiasis, natural killer cell deficiency, idiopathic CD4 + T lymphopenia, and predominant T cell deficiency.

As an agent to boost immune responsiveness in individuals with an acquired loss of B cell function. An AIM II polypeptide or polynucleotide of the invention,
Or conditions that result in an acquired loss of B or T cell function that may be ameliorated or treated by administering their agonists include, but are not limited to: HIV infection, AIDS, bone marrow transplant, And B-cell chronic lymphocytic leukemia (CLL).

As an agent to boost immune responsiveness in an individual with a temporary immune deficiency. Conditions that result in temporary immunodeficiency that may be ameliorated or treated by administering an AIM II polypeptide or polynucleotide of the invention, or agonists thereof, include, but are not limited to: viral infections (eg, , Influenza), malnutrition-related conditions, infectious mononucleosis recovery, or stress-related conditions, measles recovery, transfusion recovery, surgery recovery.

The AIM II polynucleotides or polypeptides of the invention, or agonists or antagonists thereof, can be used to diagnose, prognose, treat or prevent one or more of the following diseases or disorders, or conditions associated therewith. May: primary immunodeficiency, immune-mediated thrombocytopenia, Kawasaki disease, bone marrow transplant (eg, recent bone marrow transplant in adult or child), chronic B-cell lymphocytic leukemia, HIV infection (eg, adult or child HIV infection). ), Chronic inflammatory demyelinating polyneuropathy and post-transfusion purpura.

In addition, the AIM II polynucleotides or polypeptides of the invention, or agonists or antagonists thereof, are used to diagnose, prognose, treat one or more of the following diseases, disorders, or conditions associated therewith. Or may be prevented: Guyan-Barre syndrome, anemia (eg, parvovirus B19-related anemia, patients with stable multiple myeloma at risk of infection (eg, recurrent infection), autoimmune hemolytic) Anemia (eg, warm autoimmune hemolytic anemia), thrombocytopenia (eg, neonatal thrombocytopenia), and immune-mediated neutropenia), transplant (eg, CMV-positive organ, cytomegalovirus (CMV) -negative recipient) ), Hypogammaglobulinemia (eg, low gamma with risk factors for infection or morbidity) Roblin hypertriglyceridemia newborns), epilepsy (e.g., intractable epilepsy), systemic vasculitis syndrome, myasthenia gravis (e.g., decompensation in myasthenia gravis), dermatomyositis,
And polymyositis.

As autoimmune disorders and conditions associated with these disorders that can be treated, prevented and / or diagnosed with the AIM II polynucleotides, polypeptides, and / or antagonists of the invention (eg, anti-AIM II antibodies). Include, but are not limited to: autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenic purpura, autoimmune cytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergies. Encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (eg IgA nephropathy), multiple sclerosis, neuritis, uveitis ophthalmitis, multiple endocrine glands, purpura (For example, Henloch-Scoenlein purpura), Reiter's disease, Stiffman's syndrome. Groups, autoimmune pulmonary inflammation, Guyan-Barre syndrome, insulin-dependent diabetes mellitus, and autoimmune inflammatory eye disease.

Additional (highly suspicious) autoimmune disorders that may be treated, prevented and / or diagnosed with the compositions of the present invention include, but are not limited to:
Autoimmune thyroiditis, hypothyroidism (ie, Hashimoto thyroiditis) (often characterized by, for example, cell-mediated and humoral thyroid cytotoxicity),
Systemic lupus erhythema
tosus) (often characterized by, for example, circulating and locally produced immune complexes), Goodpasture's syndrome (often characterized by, eg, anti-basement membrane antibodies), pemphigus (often, eg, epidermis). Characterized by epithelial acantholytic antibodies, receptor autoimmunity (eg, (a) Graves' disease (often, eg,
TSH receptor antibodies, (b) myasthenia gravis (often characterized by, for example, acetylcholine receptor antibody), and (c) insulin resistance (often characterized by, for example, insulin receptor antibody). , Autoimmune hemolytic anemia (often, eg, antibody-sensitized RBC
, And autoimmune thrombocytopenic purpura (often characterized, for example, by the phagocytosis of antibody-sensitized platelets).

Additional (possible) additional autoimmune disorders that can be treated, prevented and / or diagnosed with the compositions of the present invention include, but are not limited to: rheumatoid arthritis (often, eg, , Characterized by immune complexes in joints), scleroderma with anti-collagen antibodies
(Often characterized, eg, by nucleoli and other nuclear antibodies), mixed connective tissue disease (often, eg, characterized by antibodies to extractable nuclear antigens (eg, ribonucleoproteins)), multiple Myositis / dermatomyositis (often
Eg, characterized by non-histone ANA), pernicious anemia (often characterized by, for example, anti-parietal cells, microsomes and intrinsic factor antibodies),
Idiopathic Addison's disease (often characterized, for example, by humoral and cell-mediated adrenal cytotoxicity), infertility (often, for example, by anti-sperm antibodies), glomerulonephritis (often, for example, Characterized by glomerular basement membrane antibodies or immune complexes), eg primary glomerulonephritis and IgA
Nephropathy, bullous pemphigoid (often characterized, for example, by IgG and complement in basement membrane), Sjogren's syndrome (often, for example, characterized by multiple tissue antibodies and / or specific non-histone ANA (SS-B)) Attached), diabetes (often characterized by, for example, cell-mediated and humoral islet cell antibodies), and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis) (often) , For example, β-
Characterized by adrenergic receptor antibodies).

Additional (possible) additional autoimmune disorders that may be treated, prevented and / or diagnosed with the compositions of the present invention include, but are not limited to: chronic active hepatitis (often Characterized by smooth muscle antibodies),
Primary biliary cirrhosis (often characterized by, for example, mitochondrial antibodies), other endocrine gland defects (often in some cases, for example, by specific tissue antibodies), vitiligo (often, for example, Vasculitis (often characterized by melanocyte antibodies), vasculitis (often characterized, for example, by Ig and complement in the vessel wall and / or low serum complement), after MI (po
st-MI) (often characterized by, eg, myocardial antibodies), open heart syndrome (often characterized by, eg, myocardial antibodies), urticaria (often characterized by, eg, IgG and IgM antibodies to IgE). ), Atopic dermatitis (often IgG and Ig to IgE, for example)
Characterized by M antibodies, asthma (often I for IgE, for example)
characterized by gG and IgM antibodies), inflammatory myopathy, and many other inflammatory disorders, granulomatous, degenerative and atrophic disorders.

In a preferred embodiment, autoimmune diseases and disorders and / or conditions associated with the above diseases and disorders are treated, prevented and / or diagnosed with anti-AIM II antibodies.

In certain preferred embodiments, rheumatoid arthritis is treated with anti-AIM II of the present invention.
Treated, prevented and / or diagnosed with antibodies and / or other antagonists.

In certain preferred embodiments, lupus is an anti-AIM II antibody of the invention and / or
Alternatively, it is treated, prevented and / or diagnosed with other antagonists.

In certain preferred embodiments, the lupus-associated nephritis is an anti-AIM I of the invention.
Treated, prevented and / or diagnosed with an I antibody and / or other antagonist.

In certain embodiments, AIM II polynucleotides or polypeptides,
Alternatively, their antagonists (eg, anti-AIM II antibodies) are used to treat or prevent systemic lupus erythematosus and / or diseases, disorders or conditions associated therewith. Diseases, disorders or conditions associated with lupus that may be treated or prevented with the AIM II polynucleotides or polypeptides of the invention, or antagonists include, but are not limited to: hematological disorders (eg, , Hemolytic anemia, leukopenia, lymphopenia and thrombocytopenia), immunological disorders (eg anti-DNA and anti-Sm antibodies), rash, photophobia, oral ulcer, arthritis, fever, fatigue, weight loss, serosa Inflammation (eg, peritonitis (ple
uritus) (pleurisy)), nephropathy (eg nephritis),
Neurological disorders (eg seizure, peripheral neuropathy, CNS)
Related disorders), gastrointestinal disorders, Raynaud's phenomenon, and pericarditis. In a preferred embodiment, AIM II polynucleotides or polypeptides, or antagonists thereof (eg, anti-AIM II antibodies) are used to treat or prevent renal disorders associated with systemic lupus erythematosus.
In the most preferred embodiment, AIM II polynucleotides or polypeptides, or antagonists thereof (eg, anti-AIM II antibodies) are used to
Treat or prevent nephritis associated with systemic lupus erythematosus.

In certain embodiments, soluble AIM II polypeptides of the invention (eg, polypeptides comprising or consisting of amino acids 69-240 or amino acids 83-240 of SEQ ID NO: 2) or agonists thereof are administered. To treat, prevent, prognose and / or diagnose: immunodeficiency (eg, severe combined immunodeficiency (SCID) -X-linked, SCID-autosomal, adenosine deaminase deficient (ADA deficient), X-linked agamma). Globulinemia (XLA), Bruton's disease,
Congenital agammaglobulinemia, X-linked infant agammaglobulinemia, acquired agammaglobulinemia, adult-onset agammaglobulinemia, late-onset agammaglobulinemia, abnormal gammaglobulinemia, hypogammaglobulinemia , Transient infant hypogammaglobulinemia, unspecified hypogammaglobulinemia, agammaglobulinemia, unclassified immunodeficiency (CVID) (acquired), Viscott-Aldrich syndrome (WAS), X-linked immunodeficiency with high IgM, non-X with high IgM
Linked immunodeficiency, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), antibody deficiency with normal or elevated Ig, immunodeficiency with thymoma, Ig heavy chain deletion, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), selective I
gM immunodeficiency, recessive agammaglobulinemia (Swiss type), retinal dysgenesis, neonatal neutropenia, severe congenital leukopenia, thymic lymphaplasia-hypoplastic or dysplastic with immunodeficiency, telangiectasia. Ataxia, dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezerov syndrome with Ig-complex immunodeficiency, purine nucleoside phosphorylase deficiency (PNP), MHC class II deficiency (deficient lymphocyte syndrome) and severe complex Immunodeficiency, DiGeorge abnormalities, thymic dysgenesis, chronic mucocutaneous candidiasis, natural killer cell deficiency, immunodeficiency with idiopathic CD4 + T lymphocytosis, and predominant T cell failure) or disorders associated with immunodeficiency.

A number of soluble forms of the polypeptides of the invention are used to treat, prevent, treat disease states,
Prognosis and / or diagnosis may be made. These soluble forms are commonly referred to as AIM I
It lacks the I transmembrane domain. Examples of soluble AIM II polypeptides include those that have an extracellular domain and an intracellular domain but lack the transmembrane domain. Further examples include amino acids 60-240, 69- of SEQ ID NO: 2.
240 or 83-240 are included or consisted of these.

In certain embodiments, AIM II polypeptides or polynucleotides of the invention, or agonists thereof, are administered to treat, prevent, prognose and / or diagnose non-typeable immunodeficiency.

In certain embodiments, AIM II polypeptides or polynucleotides of the invention, or agonists thereof, are administered to treat, prevent, prognose and / or diagnose X-linked agammaglobulinemia.

In another specific embodiment, AIM II polypeptides or polynucleotides of the invention, or agonists thereof, are administered to treat severe combined immunodeficiency (SC).
ID) is treated, prevented, prognosed and / or diagnosed.

In another particular embodiment, the AIM II polypeptides or polynucleotides of the invention, or agonists thereof, are administered to treat, prevent, prognose and / or diagnose Viscott-Aldrich syndrome.

In another specific embodiment, an AIM II polypeptide or polynucleotide of the invention, or agonist thereof, is administered to X-linked Ig with high IgM.
Treat, prevent, prognose and / or diagnose defects.

In another particular embodiment, the AIM II polypeptides or polynucleotides of the invention, or agonists thereof, are administered to treat, prevent, prognose and / or diagnose Di George abnormality.

AIM II antagonists can also be used in compositions with pharmaceutically acceptable carriers, for example, as described herein below.

Antagonists can further be used to treat cachexia, a lipid depletion deficiency resulting from a systemic deficiency of lipoprotein lipase, which is believed to be suppressed by AIM II, for example. AIM II antagonists may also be used to treat cerebral malaria. In cerebral malaria, AIM II may play a pathogenic role.

AIM II antagonists may also be used to prevent graft-host rejection (eg, by preventing stimulation of the immune system in the presence of the graft).

AIM II antagonists may also be used to prevent graft-host disease.
In a preferred embodiment, anti-AIM II antibodies are used to prevent graft-host disease.

AIM II antagonists may also be used to inhibit bone resorption and therefore treat and / or prevent osteoporosis.

Antagonists can also be used as anti-inflammatory agents and to treat endotoxin shock. This important condition results from an exacerbated response to bacterial infections and other types of infections.

[0294]   AIM II antagonists may also find applications as follows:   As an inhibitor of the production of one or more cytokines.

[0295]   As an inhibitor of cytolytic T cell responses and formation of antigen-specific memory.

As a modulator of a T cell immune response (eg, an inhibitor of antigen-induced T cell proliferation).

As a modulator of a cell-mediated immune response (eg, an inhibitor of cytolytic T cell activation).

In certain embodiments, a nucleic acid comprising a sequence encoding an antibody or functional derivative thereof is administered to gene therapy for diseases or disorders associated with aberrant expression and / or activity of the polypeptides of the invention. To treat, inhibit or prevent. Gene therapy refers to the treatment performed by administering an expressed or expressible nucleic acid to a subject. In this embodiment of the invention, the nucleic acids produce their encoded protein that mediates a therapeutic effect.

Any method available in the art for gene therapy can be used in accordance with the present invention. An exemplary method is described below.

For a general review of methods of gene therapy, see Goldspiel et al., 1993.
, Clinical Pharmacy 12: 488-505; Wu and W.
U, 1991, Biotherapy 3: 87-95; Tolstoshev.
, 1993, Ann. Rev. Pharmacol. Toxicol. 32: 5
73-596; Mulligan, 1993, Science 260: 926.
-932; and Morgan and Anderson, 1993, Ann.
Rev. Biochem. 62: 191-217; May 1993, TIBT.
See ECH 11 (5): 155-215. Methods that are commonly known and can be used in the field of recombinant DNA technology are described in Ausubel et al. (Eds.), 1993, Cur.
rent Protocols in Molecular Biology,
John Wiley & Sons, NY; and Kriegler, 199.
0, Gene Transfer and Expression, A Lab
Oratory Manual, Stockton Press, NY.

In a preferred aspect, the compound comprises a nucleic acid sequence encoding an antibody, the nucleic acid sequence being part of an expression vector expressing the antibody or fragment or chimeric protein or heavy or light chain thereof in a suitable host. Is. In particular, such nucleic acid sequences have a promoter operably linked to the antibody coding region, the promoter being inducible or constitutive, and optionally tissue-specific. In another specific embodiment, the antibody coding sequence and any other desired sequence are flanked by regions that promote homologous recombination at the desired site in the genome, thus providing for intrachromosomal expression of the antibody nucleic acid. A nucleic acid molecule that provides
er and Smithies, 1989, Proc. Natl. Acad. Sc
i. USA 86: 8932-8935; Zijlstra et al., 1989, Na.
true 342: 435-438). In certain embodiments, the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequence comprises sequences encoding both the heavy and light chains of the antibody, or fragments thereof.

Delivery of the nucleic acid to the patient can be direct (where the patient is directly exposed to the nucleic acid or nucleic acid-bearing vector) or indirect (where the cell is first transfected with the nucleic acid in vitro). Converted and then transplanted into the patient). These two approaches are known as in vivo gene therapy or ex vivo gene therapy, respectively.

In certain embodiments, nucleic acid sequences are directly administered in vivo, where they are expressed to produce the encoded product. This is accomplished by a number of methods known in the art, such as constructing them as part of a suitable nucleic acid expression vector and constructing them (eg, defective or attenuated retrovirus or other viral vectors (US Pat. No. 4,980,286), by intracellular administration), or by direct injection of naked DNA, or by microparticle bombardment (eg, gene gun; Biolist).
ic, Dupont) or by coating them with lipids or cell surface receptors or transfectants, or encapsulating them in liposomes, microparticles or microcapsules, or peptides known to enter the nucleus By administering to a ligand that undergoes receptor-mediated endocytosis, which can be used to target a cell type that specifically expresses the receptor. (For example,
Wu and Wu, 1987, J. Am. Biol. Chem. 262: 4429-44
32)) and the like. In another embodiment, a nucleic acid-ligand complex can be formed where the ligand comprises a fusogenic viral peptide that disrupts endosomes, allowing the nucleic acid to avoid lysosomal degradation. . In yet another embodiment, the nucleic acids can be targeted in vivo for cell-specific uptake and expression by targeting specific receptors (eg April 16, 1992).
PCT Publication No. WO 92/06180 (Wu et al.) Dated 23 December 1992
Dated WO 92/22635 (Wilson et al.); November 26, 1992.
Dated WO 92/20316 (Findeis et al.); July 22, 1993.
Dated WO 93/14188 (Clarke et al.), October 14, 1993.
See dated WO 93/20221 (Young et al.)). Alternatively, the nucleic acid can be introduced inside the cell and incorporated into the host cell DNA for expression by homologous recombination (Koller and Smithies, 198).
9, Proc. Natl. Acad. Sci. USA 86: 8932-893.
5; Zijlstra et al., 1989, Nature 342: 435-438).
.

In a specific embodiment, viral vectors that contain nucleic acid sequences that encode the antibodies of the invention are used. For example, a retroviral vector can be used (Mi
ller et al., 1993, Meth. Enzymol. 217: 581-599). These retroviral vectors lack retroviral sequences that are not required for correct packaging of the viral genome and integration into host cell DNA. The nucleic acid sequence encoding the antibody used in gene therapy is cloned into one or more vectors, which facilitates delivery of the gene into patients. For more details on retroviral vectors, see Boe.
sen et al., 1994, Biotherapy 6: 291-302, which describes the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells to make the stem cells more resistant to chemotherapy. Can be found in. Other references describing the use of retroviral vectors in gene therapy are: Clowes et al., 1994, J. MoI. Clin. Invest. 93
: 644-651; Kiem et al., 1994, Blood 83: 1467-14.
73; Salmons and Gunzberg, 1993, Human Gen.
e Therapy 4: 129-141; and Grossman and W.
Ilson, 1993, Curr. Opin. in Genetics and
Devel. 3: 110-114.

Adenovirus is another viral vector that can be used in gene therapy. Adenoviruses are particularly attractive vehicles for delivering genes to respiratory epithelia. Adenovirus naturally infects respiratory epithelia where it causes mild disease. Other targets of adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenovirus has the advantage that it can infect non-dividing cells. Kozarsky and Wilson, 1993.
, Current Opinion in Genetics and Dev
element 3: 499-503 provides an overview of adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy.
5: 3-10 demonstrated the use of adenovirus vectors to transfer genes to the airway epithelium of rhesus monkeys. Other examples of the use of adenoviruses in gene therapy are described by Rosenfeld et al., 1991, Science 252: 431-434.
Rosenfeld et al., 1992, Cell 68: 143-155; Mas.
transgeli et al., 1993, J. Am. Clin. Invest. 91: 225-
234; PCT Publication No. WO94 / 12649; and Wang et al., 1995,
Gene Therapy 2: 775-783. In a preferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med.
． 204: 289-300; U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer involves transfer of the selectable marker to the cells. The cells are then placed under selection to isolate those cells that take up and express the transferred gene.
The cells are then delivered to the patient.

In this embodiment, the nucleic acid is introduced into the cell prior to in vivo administration of the resulting recombinant cell. Such introduction can be performed by any method known in the art, including, but not limited to, transfection, electroporation, microinjection, viruses containing nucleic acid sequences thereof. Infection with vector or bacteriophage vector, cell fusion, chromosome-mediated gene transfer, microce
11) Mediated gene transfer, spheroplast fusion, etc. Many techniques are known in the art for introducing foreign genes into cells (eg, Loeff.
ler and Behr, 1983, Meth. Enzymol. 217: 599
-618; Cohen et al., 1983, Meth. Enzymol. 217: 61
8-644 (1993); Cline, 1985, Pharmac. Ther.
29: 69-92m), and if the necessary developmental and physiological functions of the recipient cells are not disrupted, can be used in accordance with the present invention. This technique should provide for stable transfer of the nucleic acid to the cell, such that the nucleic acid is expressible by the cell, and preferably heritable and expressible by the progeny of the cell. is there.

The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (eg, hematopoietic stem cells or hematopoietic progenitor cells) are preferably administered intravenously. The amount of cells contemplated for use depends on the desired effect, patient condition, etc. and can be determined by one of ordinary skill in the art.

Cells into which nucleic acids can be introduced for purposes of gene therapy include any desired available cell type, and include, but are not limited to: epithelial cells, endothelial cells, keratinocytes, fibers. Blast cells, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes;
Various stem or progenitor cells, particularly hematopoietic stem cells or hematopoietic progenitor cells (eg,
Cells such as those obtained from bone marrow, cord blood, peripheral blood, fetal liver, etc.).

In a preferred embodiment, the cells used for gene therapy are autologous to the patient.

In embodiments in which recombinant cells are used in gene therapy, the nucleic acid sequence encoding the antibody is introduced into the cell such that the nucleic acid sequence is expressible by the cell or its progeny, and then recombinant. The cells are administered in vivo for therapeutic effect. In certain embodiments, stem cells or progenitor cells are used.
Any stem and / or progenitor cells that can be isolated and maintained in vitro can potentially be used according to this embodiment of the invention (eg, PCT Publication WO 94/08598 (April 28, 1994): Temple and And
Erson, 1992, Cell 71: 973-985; Rheinwald.
1980, Meth. Cell Bio. 21A: 229; and Pitt
elkow and Scott, 1980, Mayo Clinic Proc.
61: 771).

In certain embodiments, the nucleic acid introduced for gene therapy purposes contains an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controlled by the appropriate transcription inducer. Can be controlled by controlling the presence or absence of.

As mentioned above, the antagonists and agonists of the present invention include AIM II polypeptides. These polypeptides may modulate their effect, for example, by binding to cellular proteins such as receptors. Methods for identifying peptides that interact with a particular protein are known in the art. See, for example, Phizicky and Fields, “Protein-protei.
n interactions: methods for detection
and analysis "Microbiol. Rev. 59: 94-12
3 (1995) describe methods for screening peptides to identify those that have binding affinity for a second polypeptide. Phiz
Icky and Fields discuss methods such as protein affinity chromatography, affinity blotting, co-immunoprecipitation, and cross-linking. Additional molecular biology methods suitable for use with the present invention include:
Protein probing of expression libraries, two-hybrid systems, cell panning, and phage display are included.

Another method for identifying an AIM II polypeptide of the invention that binds to a cell surface receptor is to transfect a eukaryotic cell with DNA encoding the receptor such that the cell is at its surface. A step of expressing the receptor on the cell, followed by labeling the cells (eg, radiolabel, biotin, etc.) AIM I
Contacting with the I polypeptide. Labeled AIM bound to the cell
The amount of II polypeptide is measured and compared to the amount bound to control cells. The control cells are generally cells that do not express their surface receptors. Detecting an increase in the amount of label bound to cells expressing the receptor as compared to the control cells indicates that cells expressing the receptor bind to the AIM II polypeptide.

Moreover, one of skill in the art will appreciate that cells expressing and retaining the AIM II polypeptide are
It will be appreciated that it can be used to identify AIM II ligands. In one such embodiment, cells expressing AIM II are contacted with a detectably labeled potential ligand. Further, such a ligand can be a polypeptide that is expressed as part of a library of sequences on the surface of phage, such as a phage display library.

Once an AIM II polypeptide that binds to a cell surface receptor of interest has been identified, whether that AIM II polypeptide induces or inhibits a receptor-mediated cellular response normally elicited by that particular receptor. In order to determine the,
The assay can be performed. Whether an AIM II polypeptide activates a receptor-mediated cellular response measures the cellular response known to be elicited by that receptor in the presence of that AIM II polypeptide or another ligand. Can be determined by Furthermore, whether an AIM II polypeptide inhibits a receptor-mediated cellular response is elicited by its receptor in the presence of both a molecule known to induce that cellular response and that AIM II polypeptide. Can be determined by measuring the cellular response known to be exerted.

A polypeptide of the invention (eg, amino acids 69-240 or 8 of SEQ ID NO: 2).
Soluble forms of AIM polypeptides comprising or consisting of 3-240 can be utilized, for example, in the ligand binding and receptor activation / inhibition assays described above.

AIM II has also been shown to inhibit photoreceptor cell differentiation and survival. Furthermore, AIM II has been shown to inhibit the production of rhodopsin by retinal cells. Thus, AIM II polypeptides and AIM II agonists inhibit the differentiation and survival of photoreceptor cells (eg, rods and cones) and inhibit rhodopsin production by retinal cells (eg, rods). It is useful.

The AIM II polypeptides, agonists or antagonists of the invention can be used to treat cardiovascular disorders, including peripheral arterial disease (eg, limb ischemia).

Cardiovascular disorders include the following cardiovascular abnormalities: arterial fistula (ar
terio-arterial fistula), arteriovenous fistula, cerebral arteriovenous malformation,
Congenital heart defect, pulmonary valve atresia, and Scimitar syndrome. Congenital heart defects include aortic coarctation, tricentric heart, coronary vessel malformation,
Cross heart, right thoracic heart, patent ductus arteriosus, Ebstein malformation, Eisenmenger complex,
Left ventricular underdevelopment syndrome, left thoracic heart, tetralogy of Fallot, translocation of the great arteries, bilateral right ventricular origin, tricuspid atresia, residual arterial canal, and septal defect (eg in aortopulmonary artery) Septal defect, endocardial bed defect, Luthambache syndrome, trilogy of Fallot, and ventricular septal defect).

Cardiovascular disorders also include heart diseases such as: arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (bacterial endocardium). (Including membranous), cardiovascular aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiomegaly, cardiac hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction Cardiac rupture, ventricular septal rupture, heart valve disease, myocardial disease, myocardial ischemia, pericardial effusion, pericarditis (including infarcted pericarditis and tuberculous pericarditis), pericarditis, pericardium Post-incision syndrome, right heart disease, rheumatic heart disease, ventricular failure, hyperemia, cardiovascular pregnancy complications, scimitar syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.

Arrhythmias include: sinus arrhythmias, atrial fibrillation, atrial flutter, bradycardia, premature contractions, Adams-Stokes syndrome, leg block, sinoatrial block, QT prolongation (lon).
g QT) syndrome, paraconstriction, Lone-Ganning-Levine syndrome, early-excitation Mahmeh's syndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardia, and ventricular fibrillation. Tachycardia includes the following: paroxysmal tachycardia,
Supraventricular tachycardia, accelerated ventricular rhythm, atrioventricular nodal reentrant tachycardia, ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrial nodal reentrant tachycardia, sinus tachycardia Beats, torsades de pointes, and ventricular tachycardia.

Heart valve diseases include aortic valve insufficiency, aortic valve stenosis, murmur, aortic valve prolapse,
Mitral valve prolapse, tricuspid valve prolapse, mitral valve failure, mitral valve stenosis, pulmonary valve closure, pulmonary valve failure, pulmonary valve stenosis, tricuspid valve closure, tricuspid valve failure, and tricuspid valve stenosis, To be

Cardiomyopathy includes alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, subvalvular aortic stenosis, subvalvular pulmonary stenosis, restricted cardiomyopathy, Chagas cardiomyopathy,
Endocardial fibroelasticity, endocardial myocardial fibrosis, Keens syndrome, myocardial reperfusion injury, and myocarditis are included.

Myocardial ischemia includes coronary artery disease (eg, angina, coronary aneurysm, coronary atherosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and stunning).
)) Is mentioned.

Cardiovascular disease also includes aneurysms, dysplasias, hemangiomatosis, bacterial hemangiopathies, Hippel-Lindau disease, Kliper-Tornone-Weber syndrome, Sturge-Weber syndrome, angioneurotic edema, aortic disease. , Takayasu arteritis, aortitis, Leurisch's syndrome, arterial occlusive disease, arteritis, arteritis
, Polyarteritis nodosa, cerebrovascular disease, diabetic angiopathy, diabetic retinopathy, embolism, thrombosis, erythromelalgia, hemorrhoids, hepatic vein occlusion disease, hypertension, hypotension, ischemia, peripheral vascular disease , Phlebitis, pulmonary vein occlusion, Raynaud's disease, CREST syndrome, retinal vein closure,
Scimitar syndrome, superior vena cava syndrome, telangiectasia, ataxia-telangiectasia (
atacia telangiectasia), hereditary hemorrhagic telangiectasia,
Such as varicocele, dilated tortuous vein, varicose ulcers, vasculitis, and venous insufficiency,
Includes vascular disease.

Aneurysms include dissecting aneurysms, pseudoaneurysms, infectious aneurysms, ruptured aortic aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, cardiac aneurysms, and iliac aneurysms.

Arterial atresia: arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasia, mesenteric vascular closure, Moyamoya disease, renal artery occlusion, retinal artery occlusion, and obstructive thrombotic vasculitis. including.

Cerebrovascular disorders include carotid artery disease, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery disease, cerebral embolism, and cerebral thrombosis, Carotid thrombosis, sinus thrombosis, Wallenberg syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subarachnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient), subclavian artery stealing Syndrome, periventricular leukomalacia (periventricular leuk)
omalacia), vascular headache, cluster headache, migraine, and vertebral basilar artery circulatory insufficiency.

Embolisms include air embolisms, amniotic fluid embolisms, cholesterol embolisms, toe cyanosis syndrome, fat embolisms, pulmonary embolisms, and thromboembolisms. Thromboses include coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid thrombosis, sinus thrombosis, Wallenberg syndrome, and thrombophlebitis.

Ischemia is cerebral ischemia, ischemic colitis, compartment syndrome, anterior
Partition syndrome, myocardial ischemia, reperfusion injury, and peripheral limb ischemia (periphe)
ral limb ischemia). Vasculitis is aortitis, arteritis,
Behcet's syndrome, Churg-Strauss syndrome, mucocutaneous lymph node syndrome,
Includes thromboangiitis obliterans, hypersensitivity vasculitis, Schoenline-Henoch purpura, allergic cutaneous vasculitis, and Wegener's granulomatosis.

In one embodiment, the AIMII polynucleotides, polypeptides, agonists or antagonists of the invention are used to treat thrombotic microangiopathy. One such disorder is thrombotic thrombocytopenic purpura (TTP) (Kwaan, HC, Semin. Hematol. 241: 71 (1
987); Thompson et al., Blood 80: 1890 (1992)).
Increasing TTP-related mortality has been reported in U.S.P. S. Centers for Disease
Se Control (Torok et al., Am. J. Hem).
atol. 50:84 (1995)). Plasma from patients with TTP, including HIV + and HIV-, induces apoptosis of human endothelial cells of microvascular origin in the skin but not human endothelial cells of large vascular origin (Laurence). Et al., Blood 87: 3245 (1996)). Thus, plasma of TTP patients is believed to contain one or more factors that induce apoptosis, either directly or indirectly. Another thrombotic microangiopathy is the hemolytic uremic syndrome (HUS) (Moake, JK, Lancet 343: 39).
3 (1994); Melnyk et al., Arch. Intern. Med. 155:
2077 (1995); Thompson et al., Supra). Accordingly, in one embodiment, the present invention relates to the use of AIM II to treat a condition often referred to as "adult HUS" (although it can also affect children). Childhood / diarrhea-related HU
The disorder known as S differs in etiology from adult HUS. In another embodiment,
Conditions characterized by the coagulation of small blood vessels can be treated using AIM II. Such conditions include, but are not limited to, those described herein. For example, heart problems observed in about 5-10% of pediatric AIDS patients are thought to be associated with the coagulation of small blood vessels. Destruction of the cardiac microvasculature has been reported in multiple sclerosis patients. As a further example,
Treatment of systemic lupus erythematosus (SLE) is contemplated. In one embodiment, a patient's blood or plasma is contacted with an AIM II polypeptide of the invention ex vivo. AIM II polypeptides of the invention can be attached to a suitable chromatographic matrix by procedures known in the art. According to this embodiment, the patient's blood or plasma flows through a chromatography column containing matrix-bound AIM II polynucleotides and / or polypeptides of the invention prior to being returned to the patient. The fixed AIM II is AIM I
It binds I and thus removes the AIM II protein from the patient's blood. Alternatively, the AIM II polynucleotides, polypeptides, agonists or antagonists of the invention can be administered in vivo to patients suffering from thrombotic microangiopathy. In one embodiment, soluble forms of the AIM II polypeptides of the invention are administered to a patient. Accordingly, the present invention provides a method for treating thrombotic microangiopathy, which method comprises the use of an effective amount of an AIM II polynucleotide, polypeptide, agonist or antagonist. AIM II polypeptides can be used to inhibit AIM II-mediated damage to (eg, apoptosis) microvascular endothelial cells in in vivo or ex vivo procedures.

The AIM II polynucleotides, polypeptides, agonists or antagonists of the invention can be used with other agents useful in treating a particular disorder. For example, in an in vitro study reported by Laurence et al., Blood 87: 3245 (1996), some reduction in TTP plasma-mediated apoptosis of microvascular endothelial cells resulted in anti-Fas blocking antibodies, aurintricarboxylic acid, or cryoprecipitates. This was achieved by using normal plasma without it. Accordingly, a patient may combine a polynucleotide of the invention and / or an agent that inhibits Fas ligand-mediated apoptosis of endothelial cells in combination with agents such as those described above.
Alternatively it can be treated with a polypeptide. In one embodiment, AIM II polynucleotides, polypeptides, agonists or antagonists, and anti-F
Both as-blocking antibodies are characterized by thrombotic microangiopathy and disorders (eg,
TTP or HUS). An example of a monoclonal blocking antibody against Fas antigen (CD59) is described in WO95 / 10540, which application is incorporated herein by reference.

The naturally occurring balance between endogenous stimulators and inhibitors of angiogenesis is the balance in which the effects of inhibition dominate. Rastinejad et al., Cell 5
6: 345-355 (1989). Rare examples of neovascularization under normal physiological conditions (eg, wound healing, organ regeneration, embryogenesis, and female reproductive processes)
In, angiogenesis is tightly regulated and spatially and temporally delimited. Under conditions of pathological angiogenesis, such as those characterized by solid tumor growth, these regulatory controls fail. Unregulated angiogenesis becomes pathological and maintains the progression of many neoplastic and non-neoplastic diseases. Many serious diseases are dominated by abnormal neovascularization. Such diseases include solid tumor growth and metastases, arthritis, some types of eye disorders, and psoriasis. See, eg, Moses et al., Biotech. 9: 630-634 (1991); Folk
man et al. Engl. J. Med. 333: 1757-1763 (1995
); Auerbach et al. Microvasc. Res. 29: 401-4
11 (1985); Folkman, Advances in Cancer.
Research, Klein and Weinhouse, Academic
Press, New York, pp. 175-203 (1985); Patz,
Am. J. Opthalmol. 94: 715-743 (1982); and the review by Folkman et al., Science 221: 719-725 (1983). In many pathological conditions, the process of angiogenesis is
Contribute to the condition of the disease. For example, a considerable amount of data has been accumulated that suggests that solid tumor growth depends on angiogenesis. Folkman and Kl
agsbrun, Science 235: 442-447 (1987).

The present invention provides for the treatment of diseases or disorders associated with neovascularization by the administration of AIM II polynucleotides and / or polypeptides of the present invention, including AIM II agonists and / or antagonists. Malignant and metastatic conditions that can be treated with the polynucleotides and polypeptides of the present invention include, but are not limited to, the following: malignant diseases described herein or otherwise known in the art. , Solid Tumors, and Cancer (For a review of such disorders, see Fishman et al., Medicine, 2nd Edition, J. B. Lipp.
incott Co. , Philadelphis (1985)).
.

Further, the AIM II polynucleotides and polypeptides of the present invention (AIM
Ocular disorders associated with neovascularization that may be treated with (including II agonists and AIM II antagonists) include, but are not limited to, neovascular glaucoma, diabetic retinopathy, retinoblastoma, Post lens fibroplasia,
Other eye inflammatory diseases, eye tumors and diseases associated with uveitis, retinopathy of prematurity, macular degeneration, corneal transplantation, neovascularization, and choroid or iris neovascularization. For example, Waltman et al., Am. J. Ophthal. 85: 704-7
10 (1978) and Gartner et al., Surv. Ophthal. 22.
See review by 291-312 (1978).

Further, the AIM II polynucleotides and polypeptides of the present invention (AIM
Disorders that may be treated with (including II agonists and AIM II antagonists) include, but are not limited to: hemangiomas, arthritis, psoriasis, angiofibromas, atherosclerotic plaques, delayed wound healing, granulation. , Hemophilic joints, hypertrophic scars, pseudoarticular fractures, Austler-Weber syndrome, purulent granulomas,
Scleroderma, trachoma, and vascular adhesions.

The polynucleotides and / or polypeptides of the invention, and / or agonists and / or antagonists thereof, are useful in the diagnosis and treatment or prevention of a wide range of diseases and / or conditions. Such diseases and conditions include, but are not limited to, cancers (eg, cancers associated with immune cells, breast cancer, prostate cancer, ovarian cancer, follicular lymphoma, p53 mutations or alterations). Cancer, brain tumor, bladder cancer, cervical cancer, colon cancer, colorectal cancer, non-small cell lung cancer, small cell lung cancer, gastric cancer, etc., lymphoproliferative (lymphoproli)
inflammatory (eg, lymphadenopathy), microbial (eg, viral, bacterial, etc.) infection (eg, HIV-1 infection, HIV-2 infection, herpesvirus infection (HSV-1, HSV-2, CMV, VZV, HHV-6, HHV
-7, including but not limited to EBV), adenovirus infection, poxvirus infection, human papillomavirus infection, hepatitis infection (eg HAV, HB
V, HCV, etc.), Helicobacter pylori infection, non-invasive S
taphylococcia, etc.), parasitic infections, nephritis, bone diseases (eg, osteoporosis), atherosclerosis, pain, cardiovascular disorders (eg, neovascularization, hypovascularization) or decreased circulation (eg, Ischemic disease (eg, myocardial infarction, stroke, etc.), AIDS, allergy, inflammation, neurodegenerative disease (eg, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, pigmentary retinopathy, cerebellar degeneration), Graft rejection (acute and chronic)
, Graft-versus-host disease, diseases caused by myelodysplasia (eg, aplastic anemia), joint tissue destruction in rheumatism, liver disease (eg, acute and chronic hepatitis, liver injury, and cirrhosis), Autoimmune diseases (eg, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, immune complex glomerulonephritis, autoimmune diabetes, autoimmune thrombocytopenic purpura, Graves' disease, Hashimoto thyroiditis, etc.),
Cardiomyopathy (eg, dilated cardiomyopathy), diabetes, diabetic complications (eg, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy), influenza, asthma, psoriasis, glomerulonephritis, septic shock, and Ulcerative colitis.

Polynucleotides and / or polypeptides of the invention, and / or agonists and / or antagonists thereof, are useful in promoting angiogenesis, wound healing (eg, wounds, burns, and fractures). .

The polynucleotides and / or polypeptides of the present invention, and / or agonists and / or antagonists thereof, are useful as adjuvants to enhance the immune and / or antiviral immune response to a particular antigen. .

More generally, the polynucleotides and / or polypeptides of the invention, and / or agonists and / or antagonists thereof, are useful in modulating (ie, increasing or decreasing) an immune response. is there. For example, the polynucleotides and / or polypeptides of the present invention may be useful in preparing for or recovering from surgery, trauma, radiation therapy, chemotherapy and transplantation, or immune response in older and immunocompromised individuals. Can be used to boost and / or accelerate recovery. Alternatively, the polynucleotides and / or polypeptides of the invention, and / or agonists and / or antagonists thereof, are useful as immunosuppressive agents, eg, in treating or preventing autoimmune disorders. In certain embodiments, the polynucleotides and / or polypeptides of the invention are chronic inflammatory, allergic or autoimmune conditions (e.g., as described herein or otherwise known in the art, Condition) is used to treat or prevent.

Uses of AIM II polypeptides, particularly human AIM II polypeptides, include, but are not limited to, the following treatments or preventions: viral hepatitis, herpes virus infection, allergic reactions, adult respiratory distress. Syndrome, neoplasia, anaphylaxis, allergic asthma, allergen
Rhinitis, drug allergies (eg to penicillin, cephalosporins), primary central nervous system lymphoma (PCNSL), glioblastoma, chronic lymphocytic leukemia (CL
L), lymphadenopathy, autoimmune disease, graft-versus-host disease, rheumatoid arthritis, osteoarthritis, Graves' disease, acute lymphoblastic leukemia (ALL), lymphoma (Hodgkin's disease and non-Hodgkin's lymphoma (NHL) )), Eye disorders, uveoretinitis (u
veoretinitis), the autoimmune phase of type I diabetes, myasthenia gravis, glomerulonephritis, autoimmune hepatic disorders, autoimmune inflammatory bowel disease, and Crohn's disease. In addition, the AIM II polypeptides of the invention can be used to inhibit neoplasia, such as tumor cell growth. The combination of the AIM II protein with immunotherapeutic agents such as IL-2 or IL-12 can produce synergistic or additive effects useful in the treatment of established cancers. The AIM II polypeptide may also be useful in tumor therapy. AIM II can further be used to treat diseases that require proliferative activity, such as restenosis. This is because AIM II has a proliferative effect on cells of endothelial origin. Therefore, AIM II can also be used to regulate hematopoiesis in the development of endothelial cells.

The AIM II polypeptides of the invention can also be used to inhibit T cell and B cell differentiation and proliferation. AIM II-induced T cells and B
Inhibition of cell activation, differentiation and / or proliferation can be used to treat many immunologically-based diseases. Some of these diseases are mentioned above. Furthermore, depending on the particular AIM II polypeptide used, the AIM II polypeptides of the invention can also be used to stimulate activation, differentiation and / or proliferation of T and B cells.

AIM II may act as a cytokine adjuvant or costimulatory molecule. The following experiments are performed to evaluate AIM II protein in vivo against the host immune system.

Tumor-bearing or tumor-bearing mice were administered at three different doses (0.1 mg / kg, 1 mg) before or after immunization with tumor antigens or superantigens.
/ Kg, and 10 mg / kg, i. p. , QD, 10-14 days, N per group
= 5) with AIM II protein, and mice are sacrificed after blood collection weekly after treatment. Spleens or lymph nodes are used for the following in vitro analyzes well known to those skilled in the art: FACS analysis: T cells, B cells, NK cells, monocytes, dendritic cells, costimulatory molecules,
And expression of surface markers for adhesion molecules. Cytokine production assay. T cell proliferation or cytotoxicity assay.

AIM II proteins and tumor antigens can result in the induction of protective immunity, which can lead to protection of mice from subsequent tumor challenge. To test this possibility, the following experiment can be performed using syngeneic C57BL / 6 mice to test the effect of AIM II on the induction of tumor or Ag-specific protective immunity.

MC-38 tumor-free mice treated with AIM II protein are challenged with MC-38 or an irrelevant murine sarcoma MCA-102 using techniques well known to those of skill in the art. Three possible outcomes can be observed:

[0349]

[Chemical 5] # 1 point: Evidence of tumor-specific protective immunity. # 2 point: evidence of non-tumor specific immunity. # 3 point: lack of protective immunity.

When the development of tumor-specific protective immunity upon AIM II treatment is demonstrated, the following depletion experiments are performed to identify which leukocyte subpopulation is responsible for tumor rejection. Mice are treated with CD4 ⁺ T cells or CD8 ⁺ T cells, N using techniques well known to those skilled in the art.
Treatment with various mAbs that recognize either K cells, granulocytes (Grl +), or specific cytokines such as IFNγ. Tumor growth in these antibody treated mice is measured.

AIM II also has increased angiogenesis or increased endothelial cell proliferation required to maintain the invading Pannus in bone and cartilage, which is often observed in rheumatoid arthritis (RA). It can be used to treat RA by inhibiting. Endothelial cell proliferation is increased in synovial fluid of RA patients when compared to patients with osteoarthritis (OA) or unaffected individuals. Neovascularization is required to maintain the mass increase of pannus invading bone and ointment. Inhibition of angiogenesis is associated with a significant reduction in the severity of both early and chronic arthritis in animal models.

AIM II polypeptides are believed to have binding activity for many proteins, including some human cell receptors. These receptors include lymphotoxin-β-receptor (LT-β-R), TR2 (also called herpesvirus entry mediator (HVEM) and ATAR), CD
27, TR6 (also called DcR3), TR9 (DR6) and TRANS (
Nuclear factor κB (RANK) receptor activator).

Each of the receptors just listed is involved in a variety of physiological processes.
This process can be regulated by the AIM II polypeptides of the invention. More specifically, using the polypeptide of the present invention, the action of a ligand that binds to a cell receptor having AIM II binding activity (for example, LT-β-R, TR2, CD27, TR6, TR9 and TRANS) is exerted. It may stimulate or block this effect.

LT-β (which binds to LT-β-R) was associated with the development of secondary lymphoid tissue and the maintenance of organized lymphoid tissue in the adult. LT-β-R can, in some instances, function in concert with TR2 to mediate cellular responses, and T-β-R
It has been shown to be expressed in many tissues in the lung, including a subpopulation of lymphocytes. LT-β-R is also associated with the formation of germinal centers and therefore appears to be involved in the humoral immune response. Rennert et al., Int. Immunol. 9
: 1627-1639 (1997).

AIM II polypeptides of the present invention can be used to inhibit germinal center formation and LT-β-R mediated humoral responses by blocking the access of cellular ligands to LT-β-R. . Furthermore, the polypeptide of the present invention is LT-β.
By activating -R, germinal center formation and LT-β-R mediated humoral response can be stimulated.

One of ordinary skill in the art will recognize that different portions of the AIM II polypeptide may have different effects on LT-β-R. One of ordinary skill in the art will also appreciate that the effects of the AIM II polypeptides of the invention on LT-β-R may be dependent on the individual peptide and LT-
It is recognized that when bound to β-R, it changes with the effect that the AIM II polypeptide has. Methods of screening for molecules with agonistic and antagonistic activities of cellular responses are described above.

The hepatitis C virus (HCV) core protein has also been shown to associate with LT-β-R and enhance signaling mediated by this receptor. Chen et al. Virol. 71: 9417-9426 (1997).
Furthermore, the interaction of this protein with LT-β-R may contribute to chronically activated, persistently HCV-infected cells. The AIM II polypeptides of the invention can be used to block HCV stimulation of LT-β-R and pathologies associated with this virus.

TR2 is a member of the tumor necrosis factor (TNF) receptor family,
This family is expressed in many human tissues and cell lines. This protein is expressed constitutively and at relatively high levels in peripheral blood T cells, B cells, and monocytes. Kwon et al. Biol. Chem. 272: 1427
2-141476 (1997). TR2 provides many functions in vivo, including mediating herpesvirus entry into cells during infection. Furthermore, the TR2-Fc fusion protein was demonstrated to inhibit mixed lymphocyte reaction-mediated proliferation. These data show that TR2 and its ligand are T
Suggests a role in cell stimulation. This was shown with these strains in which TR2 overexpression activates NF-κB and AP-I. This activation appears to occur through a TNF receptor associated factor (TRAF) mediated mechanism.

The AIM II polypeptides of the present invention activate T cells, and thus, by blocking access to TR2 by cellular ligands that activate this receptor.
It can be used to inhibit a T cell mediated immune response. Similarly, the polypeptides of the present invention may stimulate T cell activation by activating TR2. LT-β-
As described above for R, one of skill in the art will recognize that different portions of the AIM II polypeptide may either inhibit or stimulate a TR2-mediated cellular response.

The AIM II polypeptides of the invention can also be used to prevent or treat herpesvirus infections.

Expression of CD27 and its ligand, CD70, is primarily restricted to lymphocytes. In addition, CD27 interacts with CD70 and IgE in B cells
It was shown to be involved in the induction of synthesis. Nagamo et al. Imrnunol
． 161: 6496-6502 (1998). Furthermore, activation of CD27 results in I
It can enhance gE synthesis. Thus, inhibition of the interaction between CD27 and CD70 may inhibit IgE production and allergic response.

The AIM II polypeptides of the present invention can be used to modulate an immune response. For example, AIM II polypeptides can be used to regulate B cell function by inhibiting the interaction between CD27 and CD70. Therefore, AIM
II polypeptides can bind to CD27 and inhibit B cell differentiation and proliferation, as well as secretion of proteins (eg, IgE) by these cells. Thus, AIM II polypeptides have been used to detect IgE-induced immediate hypersensitivity reactions (eg, allergic rhinitis (also known as hay fever), bronchial asthma, allergic asthma, anaphylaxis, atopic dermatitis and gastrointestinal foods. IgE antibody formation in the treatment of (allergies).

CD27 is also considered to be a receptor for pro-apoptotic proteins commonly known as Siva. Pandanilam et al., Kidn
ey Int. 54: 1967-1975 (1998). AIM II of the present invention
The polypeptide may be used 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 the following:
AIDS; neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, pigmentary retinopathy, cerebellar degeneration); spinal dysplasia syndrome (myelod)
yplastic syndrome (eg, aplastic anemia); ischemic injury (eg, caused by myocardial infarction, stroke and reperfusion injury),
Toxin-induced liver disease (eg caused by alcohol), septic shock, cachexia and anorexia.

The AIM II polypeptides of the present invention may also be used to enhance activation of the Siva / CD27 apoptotic pathway and thus promote induction of apoptosis. Diseases associated with increased cell survival or inhibition of apoptosis include: cancer (eg, follicular lymphoma, carcinoma with p53 mutations, and hormone-dependent tumors), autoimmune disorder (eg, systemic). Lupus erythematosus, immune-related glomerulonephritis, and rheumatoid arthritis) and viral infections (eg, herpesvirus, poxvirus and adenovirus), graft-versus-host disease,
Acute graft rejection and chronic graft rejection information.

Although CD27 may be membrane bound, the soluble form of CD27 is produced in the course of the immune response. Soluble CD27 (sCD27) is found in many body fluids and can be measured to monitor local and systemic immune activation. In addition, CD27 is expressed on human malignant B cells and high levels of sCD2
7 is present in the sera of patients with various B cell malignancies. Kersten
Et al., Blood 87: 1985-1989 (1996). These elevated levels of sCD27 have been shown to be strongly correlated with tumor burden.

SCD27 has also been shown to be elevated in patients with a variety of lymphocyte malignancies and solid tumors of the central nervous system. What causes these pains (
Affliction includes primary central nervous system lymphoma (primary)
central nervous system lymphoma (PCN
SL)) and lymphoid malignancies located in the meninges, such as acute lymphocytic leukemia (ALL) and non-Hodgkin's lymphoma (NHL).

Soluble CD27 was also found to be elevated in patients with many non-hyperproliferative disorders. For example, sCD27 has been shown to be elevated in patients with untreated Graves hyperthyroidism. Kallio et al., J
． Lab. Clin Med. 132: 478-482 (1998). further,
An increase in sCD27 serum levels was found in patients with systemic lupus erythematosus (SLE), and this increase was found to be associated with disease activity. Font et al., Clin. Immunol. Immunopathol. 81:
239-243 (1996); Swaak et al., Clin. Rheumatol.
14: 293-300 (1995). Also, B cells derived from the majority of patients with chronic lymphocytic leukemia (CLL) have been shown to express both membrane-bound CD27 and soluble CD27, as well as CD70. Ranheim et al., Blood 85: 3556-3565 (1995).

It is hypothesized that sCD27 may prevent CD70-mediated T cell stimulation of leukemic B cells and thus impair the ability of B cells to function as antigen presenting cells. R
anheim et al., Blood 85: 3556-3565 (1995). The polypeptides of the present invention inhibit the interaction between sCD27 and CD70, thus
It can be used to enhance the ability of B cells to act as antigen presenting cells.

The AIM II polypeptides of the invention can also be used to treat those that result in diseases and distress associated with increased levels of CD27. Although not wishing to be bound by opportunistic theory, AIM II polypeptides
Binds to sCD27 and prevents sCD27 from interacting with cellular ligands, and thus may be useful in treatment regimens for these conditions.

AIM II polypeptides bind TR6 (see Example 13).
. TR6 (DcR3) is a novel soluble member of the TNFR superfamily. This member is constitutively expressed in lung tissue, tumor cells and endothelial cells. TR6 specifically binds to AIM II and FasL and inhibits their activity. Like DcR1, DcR2 and another soluble member OPG of the TNFR superfamily, TR6 is a TNF family member,
It can act as an inhibitor of signal transduction through FasL and AIM II. TR6 appears to have an important role in inhibiting apoptosis and tumor regulation. TR6 may act as an inhibitor of AIM II interactions and may also act in different roles depending on the cell type. TR6 (DcR3
) May act as a decoy receptor and may contribute to immune evasion in both slow and rapid tumor cell death, which are mediated by AIM II or FasL-mediated apoptotic pathways, respectively. In fact, TR6 (DcR3
Has been shown to bind to FasL and may contribute to immune evasion by certain tumors (Pitti, RM et al., Nature 396: 699-703 (199).
8)). AIM II and FasL are known to be expressed in activated T cells. Therefore, TR6 and its ligands are thought to be important for the interaction between activated T lymphocytes and endothelial cells. TR6 may be involved in activated T cell trafficking and endothelial cell survival. Another possibility is that TR6 may act as a cytokine to induce membrane bound FasL or AIM II, and Fas
The ability to signal directly through L or AIM II. Recently, De
group and Suzuki groups sbarats is, FasL is obtained transmits itself a signal, it leads to arrest and cell death of the cell cycle in CD4 ⁺ T cells, in CD8 ⁺ T cells was reported to lead to cell proliferation ( Des
barats, J .; Et al., Nature Medicine 4: 1377-13.
82 (1998); Suzuki, I .; And Fink, P .; J. J. Exp
． Med. 187: 123-128 (1998)). Therefore, TR6 is Fas
It can act as a cytokine for signal transduction through L and AIm II.

Since TR6 appears to have an important role in inhibiting apoptosis and tumor regulation, the AIM II polypeptides of the present invention also cause disease and distress associated with increased levels of TR6, or apoptosis. Can be used to treat a condition where an increase in While not wishing to be bound by opportunistic theory, the AIM II polypeptide blocks these states because AIM II binds to TR6 and interferes with the interaction of TR6 with cellular ligands, blocking apoptosis. Can be useful in a treatment regimen.

AIM II polypeptides are also believed to bind to RANK (And
Erson et al., Nature 390: 175-179 (1997)). RANK is a protein involved in the regulation of osteoclast differentiation and the interaction between T cells and dendritic cells. RANK is RANKL (osteoprotegerin ligand (OPGL), TRAN
It also mediates its cellular effects through its interaction with CE and ODF).

Mice with a disrupted RANKL gene show severe osteoporosis, the appearance of defective teeth, and lack of osteoclasts. These mice also have T
And shows defects in B lymphocyte differentiation. In addition, RANKL-deficient mice lack lymph nodes but show normal splenic structure and Peyer's patches. These data are
We show that the RANKL-mediated pathway regulates lymph node organogenesis, lymphopoiesis, and osteoclast differentiation and proliferation.

There are two major classes of bone cells: the cells that make up the bone, the osteoblasts, and the cells that absorb the bone, the osteoclasts. Each of these cells has a very precise function, and the balance between their activities is important for the maintenance of the skeletal system. For example, in human adults, 10-15% of the cancellous surface is covered with osteoids (new unmineralized bone made by osteoblasts), but about 4% has active resorption. Having a surface. The continuous flux kinetic nature of bone cell activity is demonstrated by the fact that about 18% of total skeletal calcium is often removed and deposited in one year.

The AIM II polypeptides of the present invention have the effect of osteoclast differentiation and proliferation (osteogenesis),
And can be used to regulate bone development and degradation. The polypeptides of the invention can be used, for example, to inhibit osteoclast differentiation and proliferation, and thus reduce the rate of bone degradation. Inhibition of osteoclast differentiation and proliferation and bone degradation are useful in the treatment of osteoporosis, skeletal and dental disorders, bone cancer, osteoarthritis, osteogenesis imperfections, and conditions such as Fuller's syndrome and Marfan syndrome Can be. The polypeptides of the present invention may also be used in processes for reshaping bones and teeth, and in periodontal reconstructions that require replacement or augmentation of lost bone (eg, in the mandible), and periodontal. It can be used to supplement alveolar bone loss resulting from disease to delay or prevent tooth loss (eg, Sigurdsson et al., J. Periodontol. 6).
6: 511-21 (1995)).

The AIM II polypeptides of the present invention can further be used to regulate the differentiation and proliferation of T and B lymphocytes. Thus, AIM II polypeptides bind to RANK and inhibit T and B lymphocyte differentiation and proliferation, as well as secretion of proteins (eg, immunoglobulins) from these cells. Thus, AIM II polypeptides can be used to suppress lymphocyte-mediated immune responses, eg, to prevent graft rejection. AIM II
The polypeptides can also be used to inhibit osteoclast differentiation and proliferation. Thus, AIM II polypeptides can be used to treat diseases such as bone cancer.

The present invention also mimics one or more natural ligands of RANK, and RAN
Provided are AIM II polypeptides that stimulate K-mediated cellular responses. These cellular responses include activation of T and B lymphocyte differentiation and proliferation, and induction of osteoclast differentiation. Accordingly, AIM II polypeptides can be used to treat diseases such as infections (eg, bacterial, viral, and protozoal infections). AIM II polypeptides can also be used to enhance the immune response (eg, in treating AIDS and AIDS-related complications) or to increase the rate of bone degradation.

AIM II polypeptides also bind TR9. TR9 (also called DR6) is a novel member of the tumor necrosis factor family of receptors with cytoplasmic death domain (see WO98 / 56892). TR9 can induce apoptosis in mammalian cells and engage the NF-κB and JNK pathways. TR9 is thought to play a role in inflammatory responses and immune regulation.

AIM II polypeptides can be used to modulate TR9-mediated inflammatory responses and immunomodulation. AIM II polypeptides can also be used to induce TR9-mediated apoptosis via the NF-κB and JNK pathways. Thus, AIM II polypeptides can be used to treat diseases such as infections. AIM II polypeptides can also be used to enhance the immune response.

AIM II polypeptides can be cleaved in vivo to form soluble forms of the molecule. As described in Example 10, the cleavage site appears to be located between amino acid residues 82 and 83 of the sequence shown in SEQ ID NO: 2. Cleavage of the AIM II polypeptide at this position is believed to result in the production of a soluble form of the molecule comprising or consisting of amino acids 83-240 of SEQ ID NO: 2. Soluble forms of AIM II are particularly useful for the treatment of diseases in which systemic administration of these peptides is preferred. In addition, the soluble form of AIM II is also useful for topical administration. The complete and mature AIM II polypeptides of the present invention, as well as subfragments of these polypeptides, can be used to treat those causing pain associated with the receptors and other ligands to which these molecules bind.

In one embodiment, the invention provides a composition comprising a polypeptide of the invention (eg, AIM associated with a heterologous polypeptide, a heterologous nucleic acid, a toxin or a prodrug.
II polypeptide or a composition comprising an anti-AIM II antibody) which expresses a membrane-bound form of AIM II on their surface or which has an AIM II receptor (eg TR2, LTβ A method of delivering the receptor and CD27) is provided. AIM II of the present invention
The polypeptide or anti-AIM II antibody binds to hydrophobic interactions, hydrophilic interactions,
A heterologous polypeptide by ionic and / or covalent interactions,
It may be associated with a heterologous nucleic acid, toxin or prodrug.

In one embodiment, the invention provides a composition of the invention to cells by administering a polypeptide of the invention associated with a heterologous polypeptide or nucleic acid (eg, AIM II or anti-AIM II antibody). Methods are provided for specific delivery. In one example, the invention provides methods for delivering therapeutic proteins to targeted cells. In another example, the invention provides a single standard nucleic acid (
For example, antisense or ribozymes) or dual target nucleic acids (eg, DNA that can be integrated into the cell's genome or replicated episomally and transcribed) to a targeted cell. .

In another embodiment, the invention provides a polypeptide of the invention (eg, an AIM II polypeptide or anti-AIM) associated with a toxin or a cytotoxic prodrug.
II antibody) provides a method for specific destruction of cells (eg, destruction of tumor cells).

In a specific embodiment, the invention provides that cells that express TR2, LTβ receptor, and / or CD27 on their surface by administering an AIM II polypeptide associated with a toxin or a cytotoxic prodrug (eg, cells). , Activated T cells, and / or specific destruction of leukemia or lymphoma-associated T cells and / or B cells).

In another particular embodiment, the invention provides that membrane-bound AIM I by administering an anti-AIM II antibody associated with a toxin or a cytotoxic prodrug.
Methods are provided for the specific destruction of cells that express I on their surface (eg, spleen, bone marrow, kidney and PBL).

[0386]   "Toxin" binds to and activates the endogenous cytotoxic effector system
The compound is meant, and such toxins include, for example, radioisotopes, holo
Toxins, modified toxins, catalytic subunits of toxins,
Totoxin (cytotoxic drug), or cells under defined conditions that cause cell death
Any molecule or enzyme that is not normally present in and on the surface of. Book
Toxins that may be used in accordance with the methods of the invention include, but are not limited to:
Without limitation: radioisotopes known in the art, eg native or derived
That bind to the endogenous cytotoxic effector system (or its complement-containing moiety)
Min), thymidine kinase, endonuclease, RNase, α
Toxin, ricin, abrin, pseudomonas exotoxin A, diphtheria toxin, sapo
Phosphorus (saporin), momordin (momordin), gelonin (gel)
onin), pokeweed antiviral protein, alpha sarcin (sarc)
in) and cholera toxin. "Toxin" also refers to cytostatic (cytos)
static) or cytocidal, therapeutic or radiation
Active metal ions (eg,²¹³An alpha emitter such as Bi), or, for example,¹⁰³ Pd,¹³³Xe,¹³¹I,⁶⁸Ge,⁵⁷Co,⁶⁵Zn,⁸⁵Sr,³²P,³⁵S,⁹⁰Y, ¹⁵³ Sm,¹⁵³Gd,¹⁶⁹Yb,⁵¹Cr,⁵⁴Mn,⁷⁵Se,¹¹³Sn,⁹⁰It Liu
Mu,¹¹⁷Tin,¹⁸⁶rhenium,¹⁶⁶Holmium, and¹⁸⁸Other releases such as rhenium
Radioisotopes; luminescent labels (eg luminol); and fluorescent labels (eg full)
Orosein and rhodamine) and biotin.

[0387] The protein (including antibody) of the present invention can be applied by applying the techniques known in the art.
Can be labeled. Such techniques include, but are not limited to, the use of bifunctional conjugation agents (eg, US Pat. Nos. 5,756,065; 5,7,5).
No. 14,631; No. 5,696,239; No. 5,652,361; No. 5,505,931; No. 5,489,425; No. 5,435,990; No. 5,428,139; No. 5,342,604; No. 5,274,1.
19; 4,994,560; and 5,808,003; the contents of each of which is incorporated herein by reference in its entirety).
. Cytotoxic or cytotoxic agents include any agent that is harmful to cells.
Examples include paclitaxel (paclitaxol), cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide (tenoposide), vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, Actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. As therapeutic agents, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (eg, mechlorethamine, thioepa chlorambucil, melphalan, carmustine ( BSNU) and lomustine (C
CNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracycline (eg, daunorubicin (formerly daunomycin). ) And doxorubicin), antibiotics (eg, dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (eg, vincristine and vinblastine). However, it is not limited to these.

“Cytotoxic prodrug” means a non-toxic compound that is converted into a cytotoxic compound by the enzymes normally present in cells. Cytotoxic prodrugs that may be used in accordance with the methods of the present invention include glutamyl derivatives of benzoic acid mustard alkylating agents, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubicin, and phenoxyacetamide derivatives of doxorubicin. However, the present invention is not limited to these.

Diagnostic and Imaging The present invention further relates to the use of AIM II polynucleotides for detecting complementary polynucleotides (eg, as diagnostic reagents). Detection of mutated forms of AIM II associated with dysfunction may result in susceptibility to a disease (eg, autoimmune disease, malignant disease and / or immunodeficiency, etc.) or disease resulting from underexpression, overexpression or altered expression of AIM II. Provides diagnostic tools that can add or define diagnostics for. The polynucleotide encoding AIM II also includes
It can be used as a diagnostic marker for the expression of the polypeptides of the invention.

The inventors have found that AIM II is expressed in spleen, thymus and bone marrow tissues. "Normal" AIM II gene expression levels for a number of disorders (eg, septic shock, inflammation, cerebral malaria, HIV virus activation, graft-host rejection, bone resorption, rheumatoid arthritis and cachexia) Significantly higher or lower levels of AIM II gene expression compared to (ie, AIM II expression levels in tissues or fluids from individuals without the disorder) were collected from individuals with such disorders. It is believed that it can be detected in certain tissues (eg spleen, thymus and bone marrow tissues) or body fluids (eg serum, plasma, urine, synovial fluid or spinal fluid). Accordingly, the present invention provides a diagnostic method that is useful during diagnosing disorders, which method comprises: (a) assaying AIM II gene expression levels in cells or body fluids of an individual; (B) comparing this AIM II gene expression level to a standard AIM II gene expression level, whereby increasing or decreasing the assayed AIM II gene expression level relative to the standard expression level. Is a process that is an indicator of failure.

Certain tissues in mammals with cancer have been identified by the corresponding “standard” mammals (
That is, m that encodes significantly reduced levels of AIM II protein and AIM II protein when compared to the same mammal that does not have cancer).
It is also conceivable to express RNA. Furthermore, reduced levels of AIM II protein when compared to serum from a mammal of the same species that does not have cancer may result in certain body fluids (eg, serum, plasma, urine, and It can be detected in the cerebrospinal fluid). Accordingly, the present invention provides a diagnostic method that is useful during tumor diagnosis, the method comprising assaying the expression level of a gene encoding an AIM II protein in mammalian cells or body fluids, and this gene. Comparing the expression level to a standard AIM II gene expression level, whereby a decrease in gene expression level above the standard is indicative of a particular tumor.

If the 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 will express this gene at lower levels. Better clinical results may be experienced as compared to patients who do.

“Assaying the expression level of the gene encoding the AIM II protein”
By directly measuring the level of AIM II protein or the level of mRNA encoding AIM II protein in a first biological sample (eg, by assaying or estimating absolute protein or mRNA levels). Or relatively (eg, AIM I in the second biological sample)
It is intended to be measured or estimated, either qualitatively or quantitatively, either (by comparison with I protein levels or mRNA levels).

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 a cancer From a second biological sample obtained from an individual who does not have. As will be appreciated in the art, once the standard AIM II protein or mRNA level is known, it can be used repeatedly as a standard for comparison.

By “biological sample” is meant an individual, cell line, tissue culture, or AIM.
Any biological sample obtained from other sources containing II protein or mRNA is contemplated. As biological sample, secreted mature AIM
Mammalian body fluids containing the II protein (eg, serum, plasma, urine, synovial fluid and spinal fluid), as well as ovary, prostate, heart, placenta, pancreas, liver, spleen, lung, breast and umbilical cord tissue.

The present invention is useful for detecting cancer in mammals. In particular, the invention is useful during diagnosing the following types of cancer in mammals: breast cancer, ovarian cancer, prostate cancer, bone cancer, liver cancer, lung cancer, pancreatic cancer and spleen cancer. Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits, and humans. Especially preferred is human.

Labeled antibodies, and derivatives and analogs thereof, that specifically bind to a polypeptide of interest, are for detecting, diagnosing diseases and / or disorders associated with aberrant expression and / or activity of the polypeptides of the invention Or it may be used for diagnostic purposes to monitor. The present invention provides for the detection of aberrant expression of a polypeptide of interest, which detection includes: (a) using one or more antibodies specific for the polypeptide of interest, Assaying the expression of the polypeptide of interest in the cells or body fluids of the individual, and (b) the level of expression of this gene,
Comparing to a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level relative to the standard expression level is an indicator of aberrant expression.

The present invention provides a diagnostic assay for diagnosing a disorder, which assay comprises (
a) assaying the expression of the polypeptide of interest in cells or fluids of an individual using one or more antibodies specific for the polypeptide of interest, and (b) this gene expression level and standard Comparing the level of gene expression, whereby an increase or decrease in the assayed polypeptide gene expression level compared to its standard expression level is indicative of a particular disorder. With respect to cancer, the presence of relatively high amounts of transcripts in biopsied tissue from an individual may indicate a predisposition for the development of the disease or may provide a means for detecting the disease prior to the onset of actual clinical symptoms. Can be provided. A more definitive diagnosis of this type may allow health professionals to use preventative measures or early aggressive treatment, thereby preventing the development or further progression of cancer.

The diagnostic assays of the invention can be used for the diagnosis and prognosis of any disease associated with altered expression or production of AIM II. These assays are believed to be particularly useful for the diagnosis and prognosis of graft-versus-host disease, autoimmune diseases and immune deficiencies.

Classical immunohistological methods known to those of skill in the art using the antibodies of the invention (eg, Jalkanen, M. et al., J. Cell. Biol. 101: 976-98).
5 (1985); Jalkanen, M .; Et al., J. Cell. Biol. 105
: 3087-3096 (1987)) can be used to assay protein levels in biological samples. Other antibody-based methods useful for detecting protein gene expression include immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA).
)). Suitable antibody assay labels are known in the art,
And enzyme labels (eg glucose oxidase); radioisotopes (eg
Iodine ( ¹²⁵ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H),
Indium ( ¹¹² In), and technetium ( ⁹⁹ Tc); luminescent labels (eg,
Luminol); and fluorescent labels (eg fluorescein and rhodamine)
, And biotin.

One aspect of the invention is the detection and diagnosis of a disease or disorder associated with aberrant expression of a polypeptide of interest in an animal, preferably a mammal, most preferably a human. In one embodiment, diagnosis includes: a) administering (eg, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule that specifically binds to a polypeptide of interest. Step; b) following this administration,
The time in the subject to allow the labeled molecule to be preferentially concentrated (and to remove unbound labeled molecule to background levels) at the site where the polypeptide is expressed. Waiting an interval; c) determining background levels; and d) detecting labeled molecules in the subject, such that the detection of labeled molecules above this background level is , Show that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest. Background level can be determined by a variety of methods, including comparing the amount of labeled molecule detected to standard values previously determined for a particular system.

It is understood in the art that the size of the subject and the imaging system used will determine the amount of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally be 99mTc, in the range of about 5-20 millicuries. The labeled antibody or labeled antibody fragment will then preferentially accumulate at the location of cells which contain the particular protein. In vivo tumor imaging is described by S. et al. W. Burchiel et al.
"Immunopharmacokinetics of Radiolabe
"led Antibodies and Their Fragments" (
Tumor Imaging, Chapter 13: The Radiochemical
Detection of Cancer, S.M. W. Burchiel and B.I. A. Rhodes, Masson Publishing Inc. (1
982).

Depending on some variables, including the type of label used and the mode of administration, the labeled molecule is allowed to preferentially concentrate at a site in the subject and is bound to The time interval after administration for removal of unlabeled molecules to background levels is 6-48 hours or 6-24 hours or 6-12 hours. In another embodiment, the time interval after administration is 5-20 days or 5-10 days.

In one embodiment, monitoring diseases and disorders is performed by repeating the method for diagnosing a disease or disorder (eg, one month after initial diagnosis, first 6 months after diagnosis, 1 year after initial diagnosis, etc.).

The presence of labeled molecule can be detected in a patient using methods known in the art for in vivo scanning. These methods depend on the type of label used. One of ordinary skill in the art can determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the present invention include computer tomography (CT), whole body scans (eg, protons).
n) emission tomography (PET)), magnetic resonance imaging (MRI), and ultrasound diagnostics, but not limited to these.

In a particular embodiment, the molecule is labeled with a radioisotope and a radiation responsive surgical instrument.
(Thurston et al., US Pat. No. 5,441,050)
To detect in patients using. In another embodiment, the molecule is labeled with a fluorescent compound and detected in a patient using a fluorescence response scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and detected in the patient using positron emission tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and detected in the patient using magnetic resonance imaging (MRI).

Total cellular RNA was prepared as described in Chomczynski and Sacchi, Anal. B
iochem. 162: 156-159 (1987), can be isolated from biological samples using the single step guanidinium-thiocyanate-phenol-chloroform method. The level of mRNA encoding the AIM II protein is then assayed using any suitable method. These include Northern blot analysis (Harada et al., Cell 63: 303-3.
12 (1990)), S1 nuclease mapping (Fujita et al., Cell
49: 357-367 (1987)), polymerase chain reaction (PCR) reverse transcription (RT-PCR) in combination with polymerase chain reaction (Makino et al., T.
technique 2: 295-301 (1990)), and reverse transcription combined with ligase chain reaction (RT-LCR).

Assaying AIM II protein levels in a biological sample comprises:
It can occur using antibody-based techniques. For example, AIM II protein expression in tissues can be studied using classical immunohistological methods (Jalkan.
en, M. Et al., J. Cell. Biol. 101: 976-985 (1985).
Jalkanen, M .; Et al., J. Cell. Biol. 105: 3087-3
096 (1987)).

Other antibody-based methods useful for detecting AIM II protein expression include immunoassays (eg, enzyme-linked immunosorbent assay (ELI).
SA) and radioimmunoassay (RIA)).

[0410]   Suitable labels are known in the art and include: glucose
Enzyme labels such as oxidase, as well as iodine (¹²⁵I,¹²¹I), carbon (¹⁴C)
,sulfur(³⁵S), tritium (³H), indium (¹¹²In), technetium ( ^99m Radioisotopes such as Tc) and fluorescein and rhodamine
Such as a fluorescent label, as well as biotin.

AIM II “Knockout” and Homologous Recombination Endogenous gene expression can also be targeted to homologous recombination (eg Smithies).
Et al., Nature 317: 230-234 (1985); Thomas and Capecchi, Cell 51: 503-512 (1987); Thomp.
Son et al., Cell 5: 313-321 (1989), each of which is incorporated herein by reference in its entirety), for its gene and / or its promoter. It can be reduced by inactivating or “knocking out”. For example, a variant, non-functional polynucleotide (or completely unrelated DNA) of the invention flanked by DNA homologous to an endogenous polynucleotide sequence (either the coding or regulatory region of this gene). Sequence) can be used with or without a selectable marker and / or a negative selectable marker to transfect cells expressing a polypeptide of the invention in vivo. In another embodiment, techniques known in the art are used to make knockouts in cells that contain the gene of interest but do not express it. Insertion of this DNA construct, via targeted homologous recombination, results in inactivation of the targeted gene. Such an approach is particularly suited to the research and agricultural fields where modifications to embryonic stem cells can be used to produce animal progeny that have inactive targeted genes (eg, Thomas and Capecchi 198).
7 and Thompson 1989, supra). However, this approach uses recombinant viral vectors, which will be apparent to those of skill in the art, using recombinant DNA.
If the construct is administered directly or targeted to the required site in vivo, it may be routinely adapted for use in humans. The content of each of the documents cited in this paragraph is incorporated herein by reference in its entirety.

In a further embodiment of the invention, a cell that has been genetically engineered to express a polypeptide of the invention, or a cell that has been genetically engineered not to express a polypeptide of the invention (eg, a knockout), Administer to patients in vivo. Such cells can be obtained from patients (ie, animals, including humans) or MHC compatible donors, and can be fibroblasts, bone marrow cells, blood cells (eg lymphocytes), adipocytes, muscle cells, endothelial cells, etc. Can be included, but is not limited to. The cells can be transformed, for example, by transduction (using a viral vector and preferably a vector that integrates the transgene into the cell genome) or transfection procedures (plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Endogenous regulation to introduce a coding sequence of a polypeptide of the invention into a cell or to bind to this coding sequence and / or a polypeptide of the invention. To destroy the array
Genetically engineered in vitro using recombinant DNA technology. The coding sequence of the polypeptide of the invention may be placed under the control of a strong constitutive or inducible promoter or promoter / enhancer to achieve expression and preferably secretion of the polypeptide of the invention. Engineered cells that express and preferably secrete a polypeptide of the invention can be introduced systemically into the patient, eg, in circulation or intraperitoneally. Alternatively, the cells may be incorporated into a matrix and transplanted into the body (eg, genetically engineered fibroblasts may be transplanted as part of a skin graft); genetically engineered endothelial cells may be transplanted into a lymphatic graft or vessel. Can be transplanted as part of an implant (see, eg, Anderson et al., US Pat. No. 5,399,3).
49 and Mulligan and Wilson, US Pat. No. 5,460,
See No. 959. Each of these is hereby incorporated by reference in its entirety).

If the cells to be administered are non-self or non-MHC compatible cells, they may be administered using well known techniques that interfere with the development of a host immune response against the introduced cells. For example, the cells can be introduced in a capsular form, which does not allow the introduced cells to be recognized by the host immune system, while permitting exchange of components with the immediate extracellular environment.

Transgenic Non-Human Animals The polypeptides of the present invention can also be expressed in transgenic animals. Mouse, rat, rabbit, hamster, guinea pig, pig, miniature pig, goat,
Animals of any species, including but not limited to sheep, cows and non-human primates such as baboons, monkeys and chimpanzees, can be used to produce transgenic animals. In certain embodiments, techniques described herein or otherwise known in the art are used for expression of a polypeptide of the invention in humans as part of a gene therapy protocol.

Any technique known in the art can be used to introduce a transgene (ie, a polynucleotide of the invention) into an animal to establish the foo of the transgenic animal.
nder line) can be generated. Such techniques are described in Pronuclear Microinjection (Patterson et al., Appl. Microbiol. Biotec.
hnol. 40: 691-698 (1994); Carver et al., Biotec.
hology (NY) 11: 1263-1270 (1993); Wright
Et al., Biotechnology (NY) 9: 830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); Germline retrovirus-mediated gene transfer (Van der Putten et al., Pr.
oc. Natl. Acad. Sci. USA 82: 6148-6152 (19).
85)), blastocysts or embryos; gene targeting in embryonic stem cells (Thompso)
n et al., Cell 56: 313-321 (1989)); electroporation of cells or embryos (Lo, Mol. Cell. Biol. 3: 1803-181).
4 (1983)); introduction of the polynucleotide of the present invention using a gene gun (see, for example, Ulmer et al., Science 259: 1745 (1993)); nucleic acid constructs into embryonic pluripotent stem cells. And transfer of this stem cell into the blastocyst; and sperm-mediated gene transfer (Lavitra
no et al., Cell 57: 717-723 (1989)); and the like. For a review of such techniques, see Gordon, "Transgenic Animals," which is incorporated herein by reference in its entirety.
, Intl. Rev. Cytol. 115: 171-229 (1989). Further, the content of each of the documents cited in this paragraph is incorporated herein by reference in its entirety. Also, US Pat. No. 5,464,764 (Ca
pecchi et al., Positive-Negative Selection
Methods and Vectors; US Pat. No. 5,631,153 (Capecchi et al., Cells and Non-Human Organ).
isms Contining Predetermined Genomi
c Modifications and Positive-Negative
e Selection Methods and Vectors for
Making Same); U.S. Pat. No. 4,736,866 (Leder et al.,
Transgenic Non-Humnan Animals; and US Pat. No. 4,873,191 (Wagner et al., Genetic Transf).
Ormation of Zygotes), each of which is hereby incorporated by reference in its entirety.

Any technique known in the art can be used to generate transgenic clones containing a polynucleotide of the invention (eg, cultured, quiescently induced embryonic cells, fetal cells or adult cells). Nuclear Transfer of Cell-Derived Enucleated Oocytes to the Nucleus (Campell et al., Nature 380: 64-66 (1996); Wil
mut et al., Nature 385: 810-813 (1997)); each of which is hereby incorporated by reference in its entirety.

The present invention provides transgenic animals that carry the transgene in all their cells, as well as animals that carry the transgene in some (but not all) of the cells (ie, mosaic animals or chimeras). To do. The transgene may be as a single transgene or in a concatemer (eg head-to-head tandem or head-to-head).
Multiple copies, such as tail tandem type). This transgene is also described, for example, in the teaching of Lasko et al. (Lasko et al., Proc. Natl. Ac.
ad. Sci. USA 89: 6232-6236 (1992)) and can be selectively introduced into and activated by specific cell types. The regulatory sequences required for such cell type-specific activation depend on the particular cell type of interest, and they will be apparent to those of skill in the art. Targeting of the gene is preferred when it is desired that the polynucleotide transgene be integrated into the chromosomal site of the endogenous gene. Briefly, if such a technique is utilized, a vector containing several nucleotide sequences homologous to the endogenous gene may be used to bind the nucleotide sequence of the endogenous gene via homologous recombination with the chromosomal sequence. And is designed for the purpose of destroying the function of the nucleotide sequence. Transgenes can also be selectively introduced into specific cell types, and thus, for example, Gu et al. (Gu et al. Science 26
5: 103-106 (1994)), the endogenous gene is inactivated only in the introduced cell type. The regulatory sequences required for such cell type-specific inactivation depend on the particular cell type of interest, and they will be apparent to those of skill in the art. The content of each of the documents cited in this paragraph is incorporated herein by reference in its entirety.

Once a transgenic animal is produced, expression of its recombinant gene can be assayed using standard techniques. Initial screening can be accomplished by Southern blot analysis or PCR techniques to confirm that transgene integration has occurred by analysis of animal tissue. The level of transgene mRNA expression in the tissues of transgenic animals was also determined by Northern blot analysis, in situ hybridization analysis and reverse transcriptase P of tissue samples obtained from the animals.
It can be assessed using techniques including, but not limited to CR (rt-PCR). Samples of transgenic gene expressing tissues can also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.

Once founder animals are produced, they can be crossed, inbred, outbred or crossed to produce colonies of particular animals. Examples of such mating strategies include, but are not limited to: founder outcrosses with more than one integration site to establish another lineage; additive of each transgene. Expression effects allow for the inbreeding of separate strains to produce composite transgenics that express transgenes at higher levels; a predetermined for both enhancing expression and eliminating the need for screening animals by DNA analysis. Of heterozygous transgenic animals that produce homozygous animals for the integration site of; heterozygous heterozygous or individual homozygous strains for producing heterozygous lines; and suitable for experimental model of interest For placing transgenes on different backgrounds.

The transgenic and "knockout" animals of the present invention are detailed in the biological function of AIM II polypeptides, studies of conditions and / or disorders associated with aberrant AIM II expression, and such conditions and It has uses, including but not limited to, animal model systems useful in screening compounds that are effective in ameliorating disorders.

Other Uses The present invention is also based on whether cells bind to either the AIM II polypeptides of the invention or antibodies with specificity for these polypeptides.
It relates to a method of separating these cells into subpopulations. These separation methods generally involve cells that either express a surface receptor that binds AIM II polypeptide at the cell surface or have AIM II polypeptide bound to labeled AIM II polypeptide or It is based on the principle that it can be identified using AIM II specific antibodies. Such cells can then be separated from other cells in the population that do not bind these polypeptides or antibodies. Methods for separating cells (generally known as "cell sorting") are known in the art and discussed in Crane, US Pat. No. 5,489,506.

Accordingly, in one aspect, the invention features an AIM II polypeptide or AI.
Methods for separating cells that bind to any of the antibodies with specificity for M II polypeptides are provided. This method involves contacting a population of cells with either an AIM II polypeptide or an antibody having specificity for an AIM II polypeptide, wherein the AIM II polypeptide or antibody is labeled with a detectable label. And AIM II polypeptide or anti-AIM I
Separating cells that bind to any of the I polypeptide antibodies from cells that do not bind to these molecules. Cells that bind the AIM II polypeptide are believed to include cells that express the lymphotoxin-β-receptor (LT-β-R), TR2, CD27, and TRANS.

In another embodiment, the polypeptides of the invention inhibit the LTβ receptor / AIM II interaction, TR6 / AIM II interaction, and / or TRANK / AIM II interaction on different cell types. Used as a research tool to study the biological effects that result from. The AIM II polypeptide may also be the LTβ receptor, TR6, and / or TRANS, or AIM
II, or in vitro assays for detecting their interaction.

The present invention also provides methods for identifying molecules (eg, receptor molecules) that bind AIM II. Genes encoding proteins that bind to AIM II (eg, receptor proteins) can be identified by a number of methods known to those of skill in the art, such as ligand panning and FACS sorting. , Coligan et al., Curr
ent Protocols in Immunology, 1 (2): Chapter 5 (1991), described in many laboratory manuals.

For example, expression cloning can be used for this purpose. To this end, polyadenylated RNA was prepared from cells responsive to AIM II and cD
A NA library is made from the RNA, the library is divided into pools, and the pools are individually transfected into cells that do not respond to AIM II. The transfected cells were then labeled with labeled AIM I.
Exposed to I (AIM II can be labeled by a variety of well-known techniques, including standard methods of radioiodination or introduction of recognition sites for site-specific protein kinases). After exposure, cells are fixed and AIM II binding is determined. These procedures are conveniently performed on glass slides.

A pool of cDNAs that produce AIM II binding cells is identified. Sub-pools are prepared from these positives, transfected into host cells and screened as described above. Using a repetitive subpool and rescreening process, one or more single clones encoding putative binding molecules (eg, receptor molecules) can be isolated.

Alternatively, the labeled ligand can be photoaffinity linked to a cell extract (eg, membrane or membrane extract) prepared from cells expressing a molecule to which the ligand binds (eg, a receptor molecule). . Cross-linked material is separated by polyacrylamide gel electrophoresis ("PAGE") and exposed to X-ray film.
The labeled complex containing the ligand-receptor can be excited, separated into peptide fragments, and subjected to protein microsequencing. The amino acid sequences obtained from microsequencing were used to design unique or degenerate oligonucleotide probes for screening cDNA libraries to identify genes encoding putative receptor molecules. Can be used.

The polypeptides of the present invention can also be used to assess the AIM II binding capacity of AIM II binding molecules (eg, receptor molecules) in cells or in cell-free preparations.

The nucleic acid molecules of the invention are also useful for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. The mapping of DNA to chromosomes according to the present invention is an important first step in correlating disease-associated genes with their sequences.

In certain preferred embodiments in this regard, one of the cDNAs disclosed herein is used to genomic DNA of the AIM II protein gene.
Clone. This can be accomplished using a variety of well known techniques and libraries that are generally commercially available. The genomic DNA is then used for in situ chromosome mapping, using well known techniques for this purpose.

Furthermore, in some cases, sequences can be mapped to the chromosome by preparing PCR primers (preferably 15-25 bp) from the cDNA. Using computer analysis of the 3'untranslated region of the gene, genomic DN
Rapidly select primers that do not span more than one exon in A, thereby 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 cDNA clones to metaphase chromosome spreads can be used to provide the correct chromosomal location in one step. This technique can be used with probes derived from cDNA as short as 50 or 60 bp. For a review of this technology, see Verma et al., Human.
Chromosomes: A Manual Of Basic Techni
ques, Pergamon Press, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, the physical location of that sequence on the chromosome can be correlated to genetic map data. Such data can be found, for example, in V.I. McKusick, Mendelian Inheritance I
n Man (Johns Hopkins University, Welch
(Available online from Medical Library). The relationships between genes and diseases that map to the same chromosomal region are then identified through linkage analysis (co-inheritance of physically adjacent genes).

[0434] It is then necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If the mutation is observed in some or all of the affected individuals but not in normal individuals, the mutation may be the causative agent of the disease.

Demonstration of Therapeutic or Prophylactic Activity The compounds or pharmaceutical compositions of the invention are preferably tested in vitro prior to use in humans and then in vivo for the desired therapeutic or prophylactic activity. To be done. For example, in vitro assays to demonstrate therapeutic or prophylactic utility of a compound or pharmaceutical composition include the effect of the compound on cell lines or patient tissue samples. The effect of a compound or composition on cell lines and / or tissue samples can be determined utilizing techniques known to those of skill in the art, including but not limited to rosette formation assays and cytolytic assays. In vitro assays that can be used in accordance with the present invention to determine whether administration of a particular compound is directed include in vitro cell culture assays, in which a patient tissue sample is grown in culture,
The compound is then exposed or otherwise administered and the effect of such compound on the tissue sample is observed.

(Kit) The present invention provides a kit that can be used in the above method. In one embodiment, the kit comprises an antibody of the invention (preferably a purified antibody) in one or more containers. In certain embodiments, the kits of the invention comprise a substantially isolated polypeptide comprising an epitope that is specifically immunoreactive with the antibodies included in the kit. Preferably, the kit of the present invention further comprises a control antibody that does not react with the polypeptide of interest. In another particular embodiment, the kit of the invention comprises means for detecting binding of the antibody to a polypeptide of interest (e.g., the antibody is a detectable substrate (e.g., a fluorescent compound,
Enzyme substrate, a radioactive compound or a luminescent compound) or a secondary antibody that recognizes the primary antibody can be conjugated to a detectable substrate).

In another specific embodiment of the invention, the kit is for diagnostic use in screening serum containing antibodies specific for proliferative and / or cancerous polynucleotides and polypeptides. It's a kit. Such a kit may include a control antibody that does not react with the polypeptide of interest. Such a kit may comprise a substantially isolated polypeptide antigen that comprises an epitope that is specifically immunoreactive with at least one anti-polypeptide antigen antibody. Further, such kits are for detecting the binding of the antibody to an antigen (eg, the antibody can be conjugated with a fluorescent compound such as fluorescein or rhodamine, which can be detected by flow cytometry). Means are provided. In certain embodiments, the kit may comprise a recombinantly produced polypeptide antigen or a chemically synthesized polypeptide antigen. The polypeptide antigens of this kit can also be attached to a solid support.

In a more particular embodiment, the detection means of the above kit comprises a solid support to which the polypeptide antigen is attached. Such a kit also comprises a non-attached reporter-labeled anti-human antibody. In this embodiment, binding of the antibody to the polypeptide antigen can be detected by binding of the reporter-labeled antibody.

In a further embodiment, the invention comprises a diagnostic kit for use in screening serum containing antigens of the polypeptides of the invention. The diagnostic kit comprises a substantially isolated antibody that is specifically immunoreactive with a polypeptide or polynucleotide antigen, and means for detecting binding of the polynucleotide or polypeptide antigen to the antibody. . In one embodiment, the antibody is
Attached to a solid support. In a particular embodiment, the antibody may be a monoclonal antibody. The detection means of this kit may include a second labeled monoclonal antibody. Alternatively, or additionally, the detection means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solid phase reagent having surface bound antigen obtained by the method of the invention. After binding the specific antigen antibody with this reagent and removing the unbound serum components by washing, in order to bind the reporter with this reagent, depending on the amount of anti-antigen antibody bound on the solid support, This reagent is reacted with a reporter-labeled anti-human antibody. The reagent is washed again to remove unbound labeled antibody and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme detected by incubating this solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, MO).

The solid surface reagents in the above assay are used to convert protein material to solid support material (
For example, prepared by known techniques for attachment to polymeric beads, dipsticks, 96 well plates or filter materials). These methods of attachment generally include non-specific adsorption of proteins to the support or chemically active groups (eg, activated carboxyl groups, hydroxyl groups, or aldehyde groups) on the solid support. Covalent attachment of proteins
ment) (typically via a free amine group). Alternatively, streptavidin-coated plates can be used with biotinylated antigen.

The invention thus provides an assay system or kit for carrying out this diagnostic method. The kit generally comprises a support having surface-bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.

Therapeutic / Prophylactic Administration and Compositions It is understood that conditions as discussed above can be treated by administration of AIM II protein. As a result, the invention further provides methods of treating an individual in need of increasing levels of AIM II activity. The method comprises an effective amount of an isolated AIM II polypeptide of the invention effective to increase the activity level of AIM II in such an individual, or an effective amount of the isolated AIM II polypeptide of the invention. Administering to the individual a pharmaceutical composition comprising the AIM II polypeptide. Accordingly, the present invention provides methods of treatment, inhibition and prevention by administering to a subject an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody of the invention. In a preferred aspect, the compound is substantially purified (eg, substantially free of substances that limit its effect or produce undesired side effects). The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, and preferably mammals, and most preferably humans.

The formulations and methods of administration that can be used when the compound comprises nucleic acids or immunoglobulins are described above; additional suitable formulations and routes of administration are selected from among those described herein below. Can be done.

Various delivery systems are known and can be used to administer the compounds of the invention (eg, liposomes, microparticles, microcapsules, encapsulation in recombinant cells capable of expressing the compound). , Receptor-mediated endocytosis (eg, Wu and Wu, 1987, J. Biol. Chem. 262: 4429-4.
432), construction of nucleic acids as part of retroviruses or other vectors). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compound or composition may be administered by any convenient route, such as by infusion or bolus injection, by absorption through the epithelial or mucosal lining, such as the oral, rectal and intestinal mucosa, and other It can be administered with a biologically active agent. Therefore, a pharmaceutical composition comprising AIM II of the present invention may be administered orally, rectally,
Parenteral, intracisternally, vaginal, intraperitoneal, topical (
By powder, ointment, drop, or transdermal patch), bucally
) Or as an oral or nasal spray.

The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

Administration can be systemic or local. Furthermore, the pharmaceutical compounds or compositions of the invention may be administered by any suitable route, including intraventricular and intrathecal injections; Introduction to the central nervous system by means of a catheter). Pulmonary administration may also be employed, eg, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

In certain embodiments, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this is for example, but not limited to, surgery. By local injection, topical application (eg in combination with post-surgical wound dressing), injection, by catheter, by suppository, or by an implant (the implant being a membrane such as a sialastic membrane or Porous, non-porous, or glue-like materials, including fibers). Preferably, when administering proteins of the invention, including antibodies, care must be taken to use materials that do not absorb proteins.

In another embodiment, the compound or composition can be delivered into vesicles, particularly liposomes (Langer, 1990, Science 249: 1527-.
1533; Treat et al., Liposomes in the Therapy.
of Infectious Disease and Cancer, Lo
pez-Berstein and Fidler (ed.), Liss, New Y
Ork, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see ibid.).

In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (Langer, (supra); Sefton, 1987, CRC Crit. Ref. Biomed. En.
g. 14: 201; Buchwald et al., 1980, Surgery 88: 5.
07; Saudek et al., 1989, N. et al. Engl. J. Med. 321: 574
checking). In another embodiment, polymeric materials can be used (Medi
cal Applications of Controlled Release
Se, Langer and Wise (eds.), CRC Pres. , Boca R
Aton, Florida (1974); Controlled Drug B
ioavailability, Drug Product Design a
nd Performance, Smolen and Ball (ed.), Wile
y, New York (1984); Ranger and Peppas, J .; ,
1983, Macromol. Sci. Rev. Macromol. Chem.
23:61; Levy et al., 1985, Science 228: 1.
90; During et al., 1989, Ann. Neurol. 25: 351; Ho
ward et al., 1989, J. Am. Neurosurg. 71: 105). In yet another embodiment, the controlled release system can be placed at the therapeutic target, ie proximal to the brain, thus requiring only a portion of the systemic dose (eg, Go.
Odson, Medical Applications of Contro
Led Release, (supra), Volume 2, pages 115-138 (1984).
checking).

Other controlled release systems are discussed in the review by Langer (19
90, Science 249: 1527-1533).

In certain embodiments, where the compound of the invention is a protein-encoding nucleic acid, the nucleic acid constitutes it as part of a suitable nucleic acid expression vector and is administered so that it is intracellular. (Eg, by using a retroviral vector (see US Pat. No. 4,980,286), or by direct injection, or microparticle bombardment (eg, gene gun; Biolist).
ic, Dupont), or by coating with lipids or cell surface receptors or transfectants, or in combination with homeobox-like peptides known to enter the nucleus (eg, J.
oliot et al., 1991, Proc. Natl. Acad. Sci. USA 8
8: 1864-1868) and the like, so as to enhance the expression of the encoded protein in vivo. Alternatively, the nucleic acid can be introduced intracellularly and incorporated into host cell DNA for homologous recombination for expression.

In the treatment of rheumatoid arthritis, particularly preferred modes of administration of AIM II polypeptides of the present invention include intradermal, subcutaneous, and intraarticular injection and infusion. Preferably, AIM I administered intra-articularly or intradermally per dose
The I polypeptide ranges from about 0.1 to about 1.0 mg / kg body weight of the patient.

The compositions of the invention may be used alone or with other therapeutic agents (eg, costimulatory molecules).
Can be administered in combination with. Therapeutic agents that may be administered with the compositions of the present invention include, but are not limited to, other members of the TNF family, chemotherapeutic agents, antibiotics, steroidal and non-steroidal anti-inflammatory agents. , Conventional immunotherapeutic agents, cytokines and / or growth factors. The combination can be administered either, for example, simultaneously as a mixture, separately but simultaneously or in parallel; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also in procedures where the combined agents are administered separately but simultaneously (eg, when to the same individual via separate intravenous lines). Also includes. "Combination" administration further comprises the separate administration of one of the compounds or agents given first, followed by the second. Thus, in practice, therapeutic agents may be administered to an individual either simultaneously or at different times.
In most cases where the therapeutic agents are administered to the individual at different times, the therapeutic agents are generally administered in a manner such that the therapeutic effects of these agents overlap over time.

In one embodiment, the compositions of this invention are administered in combination with other members of the TNF family. TNF molecules, TNF-related molecules, or TNF-like molecules that may be administered with the compositions of the present invention include, but are not limited to, soluble forms of TNF-α, lymphotoxin-α, (LT-α,
Also known as TNF-β), LT-β (found in complex heterotrimer LT-α2-β), OPGL, FasL, CD27L, CD30L, CD40L, 4-
1BBL, DcR3, OX40L, TNF-γ (International Publication Number WO96 / 143
28), AIM-I (International Publication No. WO97 / 33899), APRIL (J.
Exp. Med. 188 (6): 1185-1190), endokine (end)
kine) -α (international publication number WO98 / 07880), TR6 (international publication number WO98 / 30694), OPG, and neutrokine (neutrok).
ine) -α (International Publication No. WO 98/18921), OX40, and nerve growth factor (NGF), and soluble forms of Fas, soluble forms of CD30, soluble forms of CD27, soluble forms of CD40 and soluble forms of 4-IBB. , TR
2 (International Publication Number WO96 / 34095), DR3 (International Publication Number WO97 / 3)
3904), DR4 (International Publication Number WO98 / 32856), TR5 (International Publication Number WO98 / 30693), TR6 (International Publication Number WO98 / 30694),
TR7 (International Publication Number WO98 / 41629), TRANS, TR9 (International Publication Number WO98 / 56892), TR10 (International Publication Number WO98 / 54202)
312C2 (International Publication No. WO98 / 06842), and TR12, and soluble forms of CD154, soluble forms of CD70, and soluble forms of CD1.
53.

In a preferred embodiment, the compositions of the invention are administered in combination with: CD40 ligand (CD40L); a soluble form of CD40L (eg AV.
REND ^™ ); a biologically active fragment, variant or derivative of CD40L;
An anti-CD40L antibody (eg, agonist or antagonist antibody); and / or an anti-CD40 antibody (eg, agonist or antagonist antibody).

In a preferred embodiment, the composition of the invention is administered in combination with 1, 2, 3, 4, 5 or more of the following compositions: tacrolimus (Fuj
isawa), thalidomide (eg, Celgene), anti-Tac
(Fv) -PE40 (e.g. Protein Design Labs), i
nolimomab (Biotest), MAK-195F (Knoll), A
SM-981 (Novartis), interleukin-1 receptor (eg, Immunex), interleukin-4 receptor (eg, Immune)
x), ICM3 (ICOS), BMS-188667 (Bristol-Mye)
rs Squibb), anti-TNF Ab (for example, Therapeutic antibody), CG-1088 (Celgene), anti-B7 Mab (for example, Innog).
etics), MEDI-507 (Bio Transplant), ABX-
CBL (Abgenix).

In certain embodiments, the compositions of the invention are administered in combination with antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors and / or protease inhibitors. Examples of nucleoside reverse transcriptase inhibitors that can be administered in combination with the composition of the present invention include:
Not limited to: RETROVIR ^™ (Zidovudine / AZT), VIDE
Z ^™ (Didanocin / ddI), HIVID ^™ (Zalcitabine / ddC), ZER
IT ^™ (stavudine / d4T), EPIVIR ^™ (lamivudine / 3TC), and COMBIVIR ^™ (zidovudine / lamivudine). Non-nucleoside reverse transcriptase inhibitors that may be administered in combination with the compositions of the present invention include, but are not limited to, VIRAMUNE ^™ (Nevirapine), RESCRI.
PTOR ^™ (delavirdine), and SUSTIVA ^™ (efavirenz). Protease inhibitors that can be administered in combination with the compositions of the present invention include:
Including but not limited to: CRIXIVAN ^TM (indinavir), NORVIR ^TM (ritonavir), INVIRASE ^TM (saquinavir))
, And VIRACCEPT ^™ (Nelfinavir). In certain embodiments, the antiretroviral agent, nucleoside reverse transcriptase inhibitor, non-nucleoside reverse transcriptase inhibitor, and / or protease inhibitor is for treating and / or diagnosing AIDS and / or HIV infection. Treatment, prevention and / or
Alternatively, it may be used in any combination with the compositions of the invention to diagnose.

In another embodiment, the compositions of this invention may be administered in combination with anti-opportunistic infection agents. Anti-opportunistic infection agents that may be administered in combination with the compositions of the present invention include, but are not limited to, TRIMETHOP.
RIM-SULFAMETHOXAZOLE ^™ , DAPSONE ^™ , PENTA
MIDINE ^™ , ATOVAQONE ^™ , ISONIAZID ^™ , RIFAM
PIN ^™ , PYRAZINAMIDE ^™ , ETHAMBUTOL ^™ , RIFAB
UTIN ^™ , CLARITHROMYCIN ^™ , AZITHROMYCIN ^™ ,
GANCICLOVIR ^™ , FOSCARNET ^™ , CIDOFOVIR ^™ , F
LUCONAZOLE ^™ , ITRACONAZOLE ^™ , KETOCONAZO
LE ^™ , ACYCLOVIR ^™ , FAMCICOLVIR ^™ , PYRIMETH
AMINE ^™ , LEUCOVORIN ^™ , NEUPOGEN ^™ (filgrastim / G-CSF), and LEUKINE ^™ (sargramostin (sargra
mostim) / GM-CSF). In certain embodiments, the compositions of the invention prophylactically treat an opportunistic Pneumocystis carinii pneumonia infection,
TRIMETHOPRIM-SULFAM for prevention and / or diagnosis
Used in any combination with ETHOXAZOLE ^™ , DAPSONE ^™ , PENTAMIDINE ^™ , and / or ATOVAQUONE ^™ . In another particular embodiment, the compositions of the invention are opportunistic Mycobacterium
In order to prophylactically treat, prevent and / or diagnose an avium complex infection, I
Used in any combination with SONAZID ^™ , RIFAMPIN ^™ , PYRAZINAMIDE ^™ , and / or ETHAMBUTOL ^™ . In another particular embodiment, the compositions of the invention are opportunistic Mycobacterium
Used in any combination with RIFABUTIN ^™ , CLARITHROMYCIN ^™ , and / or AZITHROMYCIN ^™ to prophylactically treat, prevent and / or diagnose tuberculosis infections. In another particular embodiment, the composition of the invention provides a GANCICLOVIR ^™ for the prophylactic treatment, prevention and / or diagnosis of opportunistic cytomegalovirus infections.
Used in any combination with FOSCARNET ^™ , and / or CIDOFOVIR ^™ . In another particular embodiment, the composition of the invention provides a FLUCONAZ for the prophylactic treatment, prevention and / or diagnosis of opportunistic fungal infections.
OLE ^™ , ITRACONAZOLE ^™ , and / or KETOCONAZO
Used in any combination with LE ^™ . In another particular embodiment, the composition of the invention comprises ACYCLOVIR ^™ and / or FAM to prophylactically treat or prevent an opportunistic herpes simplex virus type I and / or type II infection.
Used in any combination with CICOLVIR ^™ . In another particular embodiment, the compositions of the invention are used in any combination with PYRIMETHAMINE ^™ and / or LEUCOVORIN ^™ to prophylactically treat or prevent opportunistic Toxoplasma gondii infections. In another particular embodiment, the compositions of the invention are used in any combination with LEUCOVORIN ^™ and / or NEUPOGEN ^™ to prophylactically treat or prevent opportunistic bacterial infections.

In a further embodiment, the compositions of this invention are administered in combination with an antiviral agent. Antiviral agents that may be administered with the compositions of the invention include acyclovir, ribavirin, amantadine, and remantidine (remantidine).
Tidine), but is not limited thereto.

In a further embodiment, the compositions of this invention are administered with an antibiotic. Antibiotics that can be administered with the compositions of the present invention include amoxicillin, aminoglycosides, β-lactams (glycopeptides), β-lactamase, clindamycin, chloramphenicol, cephalosporins, ciprofloxacin. , Erythromycin, fluoroquinolones, macrolide antibiotics, metronidazole, penicillin, quinolones, rifampin, streptomycin, sulfonamide, tetracycline, trimethoprim, trimethoprim-sulfamethoxazole, and vancomycin. Not limited to.

Conventional non-specific immunosuppressive agents that may be administered with the compositions of the present invention include steroids, cyclosporine, cyclosporine analogs, cyclophosphamide,
Cyclophosphamide IV, methylprednisolone, prednisolone, azathioprine, FK-506, 15-deoxyspergualin (15-deoxyspe
rgualin), and other immunosuppressive agents that act by suppressing the function of responding T cells, but are not limited thereto.

In a particular embodiment, the compositions of this invention are administered in combination with an immunosuppressive agent. Immunosuppressant preparations that can be administered with the compositions of the invention include ORT
HOCLONE ^TM (OKT3), SANDIMMUNE ^TM / NEORAL ^TM / S
ANGDYA ^™ (cyclosporine), PROGRAF ^™ (tacrolimus), CE
Examples include, but are not limited to, LLCCEPT ^™ (mycophenolate salt), azathioprine, glucocorticosteroids, and RAPAMUNE ^™ (sirolimus). In certain embodiments, immunosuppressive agents may be used to prevent rejection of organ or bone marrow transplants.

In a preferred embodiment, the compositions of this invention are administered in combination with steroid therapy. Steroids that may be administered in combination with the compositions of the present invention include oral corticosteroids, prednisone, and methylprednisolone (methyl).
prednisolone (eg, IV methylprednisolone), but is not limited thereto. In certain embodiments, inventive compositions are administered in combination with prednisone. In a further specific embodiment, the compositions of the invention are administered in combination with prednisone and an immunosuppressive agent. Immunosuppressive agents that may be administered with the compositions of the invention and prednisone include, but are not limited to, the immunosuppressive agents described herein and azathioprine, cyclophosphamide, and cyclophosphamide IV. Not limited. In another particular embodiment, the composition of the invention is administered in combination with methylprednisolone. In a further specific embodiment, the compositions of the invention are administered in combination with methylprednisolone and an immunosuppressive agent. Immunosuppressive agents that may be administered with the compositions of the present invention and methylprednisolone are immunosuppressive agents described herein and include, but are not limited to, azathioprine, cyclophosphamide and cyclophosphamide IV. Not done.

In a preferred embodiment, the compositions of this invention are administered in combination with an antimalarial drug. Antimalarial drugs that may be administered with the compositions of the present invention include, but are not limited to, hydroxychloroquine, chloroquine, and / or quinacrine.

In a preferred embodiment, the compositions of this invention are administered in combination with an NSAID.

In a non-limiting embodiment, the composition of the invention comprises 1, 2, 3, 4, 5, 10
Administered in combination with one or more of the following drugs: NRD-101
(Hoechst Marion Roussel), Diclofenac (Dim
Ethaid), potassium oxaprozin (Monsanto), mecase
rmin (Chiron), T-614 (Toyama), permetrexe
d disodium (Eli Lilly), atreleon (Abbo
tt), valdecoxib (Monsanto), Ertenak (Byk G
ulden), campath, AGM-1470 (Takeda), CDP-
571 (Celltech Chiroscience), CM-101 (Ca
rboMed), ML-3000 (Merckle), CB-2431 (KS
Biomedix), CBF-BS2 (KS Biomedix), IL-1R
a gene therapy (Valentis), JTE-522 (Japan Tobac)
co), paclitaxel (Angiotech), DW-166HC (Dong
Wha), darbuferone mesylate (Warner-La
mvert), soluble TNF receptor 1 (synergen; Amgen),
IPR-6001 (Institut for Pharmaceutical)
l Research), trocade (Hoffman-La Roche
), EF-5 (Scotia Pharmaceuticals), BIIL-
284 (Boehringer Ingelheim), BIIF-1149 (
Boehringer Ingelheim), Leuko Vax (Infl
amtics), MK-663 (Merck), ST-1482 (Sigma)
a-Tau), and butixocot propionate (Warn
erLambert).

In a preferred embodiment, the compositions of the invention are administered in combination with 1, 2, 3, 4, 5 or more of the following drugs: methotrexate, sulfasalazine, sodium gold thiomalate, aura. Nofin, cyclosporine, penicillamine, azathioprine, antimalarial drugs (eg as described herein), cyclophosphamide, chlorambucil, gold, ENBREL ^™ (Etanerc
ept), anti-TNF antibody, and prednisolone. In a more preferred embodiment, the composition of the invention comprises an antimalarial drug, methotrexate, anti-TNF antibody, EN
Administered in combination with BREL ^™ and / or sulfasalazine. In one embodiment, the compositions of this invention are administered in combination with methotrexate. In another embodiment, the composition of the invention is administered in combination with an anti-TNF antibody.
In another embodiment, the composition of the invention is administered in combination with methotrexate and an anti-TNF antibody. In another embodiment, the composition of the invention is administered in combination with sulfasalazine. In another particular embodiment, the compositions of the invention are administered in combination with methotrexate, an anti-TNF antibody, and sulfasalazine. In another embodiment, the compositions of the invention are administered in combination with ENBREL ^™ . In another embodiment, the compositions of the invention are administered in combination with ENBREL ^™ and methotrexate. In another embodiment, the composition of the invention comprises ENBRE
It is administered in combination with L ^™ , methotrexate and sulfasalazine. In another embodiment, the compositions of the invention are administered in combination with ENBREL ^™ , methotrexate and sulfasalazine. In other embodiments, one or more antimalarial agents are administered in combination with one of the above listed combinations. In certain embodiments, inventive compositions are administered in combination with an antimalarial drug (eg, hydroxychloroquine), ENBREL ^™ , methotrexate and sulfasalazine. In another particular embodiment, the compositions of the invention are administered in combination with an antimalarial drug (eg hydroxychloroquine), sulfasalazine, anti-TNF antibody and methotrexate.

Conventional non-specific immunosuppressive agents that may be administered in combination with the compositions of the present invention include, but are not limited to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide, methylprednisone. , Prednisone, azathioprine, FK-506, 15-deoxyspergualin, and other immunosuppressive agents that act by suppressing the function of responding T cells.

In a further embodiment, the compositions of this invention are administered in combination with an antibiotic drug. Antibiotics that may be administered with the compositions of the present invention include, but are not limited to, tetracycline, metronidazole, amoxicillin, β-lactamase, aminoglycosides, macrolides, quinolones,
Fluoroquinolone, cephalosporins, erythromycin, ciprofloxacin, streptomycin.

In a further embodiment, the compositions of this invention are administered alone or in combination with anti-inflammatory agents. Anti-inflammatory agents that can be administered with the compositions of the present invention include glucocorticoid and non-steroidal anti-inflammatory agents, aminoaryl carboxylic acid derivatives, aryl acetic acid derivatives, aryl butyric acid derivatives, aryl carboxylic acids, aryl propionic acid derivatives, pyrazoles, Pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrin
e), bendazac, benzydamine, bucolome, diphenpyramide, ditazole, emorfazone, guaiazulene, nabumetone, nimesulide, orgothein, oxaseprol, paranyline, perisoxal,
Examples include pifoxime, procazone, proxazole, and tenidap,
It is not limited to these.

In another embodiment, the compositions of this invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered with the compositions of the invention include antibiotic derivatives (eg, doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens (eg, tamoxifen); antimetabolites (eg, fluorouracil, 5). -FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid, plicamycin
, Mercaptopurine, and 6-thioguanine); cytotoxic agents (eg, carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cis-platin). , And vincristine sulfate); hormones (eg, medroxyprogesterone, estramustine sodium phosphate, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and test lactone); Nitrogen mustard derivatives (eg mephalen, chlorambucil, mechlorethamine Roger emissions mustard) and thiotepa); steroids and combinations (e.g., betamethasone sodium); and others (
Examples include, but are not limited to, dicarbazine, asparaginase, mitoten, vincristine sulfate, vinblastine sulfate, and etoposide.

In a further embodiment, the compositions of this invention are administered in combination with a cytokine. Cytokines that can be administered with the compositions of the invention include IL
-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL
-12, IL-13, IL-15, anti-CD40, CD40L, IFN-γ and TNF-α, but are not limited thereto.

In a further embodiment, the compositions of this invention are administered in combination with angiogenic proteins. Angiogenic proteins that can be administered with the compositions of the present invention include glioma derived growth factor (GDGF) as disclosed in European Patent No. EP-399816; platelet derived as disclosed in European Patent No. EP-682110. Growth factor A (PDGF-A); Platelet-derived growth factor B (PDGF-B) as disclosed in European Patent No. EP-228317; International Publication No. WO92 / 0.
Placental Growth Factor (PIGF) as disclosed in 6194; Hauser et al., G.
placental growth factor 2 (PIGF-2) as disclosed in orwt Factors, 4: 259-268 (1993); vascular endothelial growth factor (VEGF) as disclosed in International Publication No. WO90 / 13649; European Patent No. EP-5064
Vascular Endothelial Growth Factor A (VEGF-A) as disclosed in 77; International Publication Number W
Vascular Endothelial Growth Factor 2 (VEGF-2) as disclosed in O96 / 39515
Vascular endothelial growth factor B (VEGF-3); vascular endothelial growth factor B-186 (VEGF-B186) as disclosed in International Publication No. WO 96/26736; as disclosed in International Publication No. WO 98/02543. Vascular endothelial growth factor D (VE
GF-D); vascular endothelial growth factor D (VEGF-D) as disclosed in WO98 / 07832; and vascular endothelial growth factor E (VEGF-E) as disclosed in German Patent No. DE19639601. ), But is not limited thereto. The above references are incorporated herein by reference.

In a further embodiment, the compositions of the invention are administered in combination with fibroblast growth factor. Fibroblast growth factors that can be administered with the composition of the present invention include FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FG.
F-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11,
FGF-12, FGF-13, FGF-14, and FGF-15 include, but are not limited to.

In a further embodiment, the compositions of the invention are administered in combination with other therapeutic or prophylactic regimens (eg, radiation treatment).

The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier. In certain embodiments,
The term "pharmaceutically acceptable" is recognized by the Federal Regulatory Agency or the U.S. Government for use in animals, and more particularly in humans, or to the United States Pharmacopeia or other generally accepted pharmacopoeia. Means listed. Furthermore, a "pharmaceutically acceptable carrier" is generally a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

The term “carrier” refers to a diluent, adjuvant, with which the composition is administered.
Excipient, or vehicle. Such pharmaceutical carriers include water and oil (
Oils of petroleum, animal, vegetable or synthetic origin (eg peanut oil,
(Eg soybean oil, mineral oil, sesame oil, etc.)). Water is
It is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, grain, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene,
Examples include glycol, water, ethanol and the like. The composition can, if desired, also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates, phosphates. Antibacterial properties (eg benzyl alcohol or methylparaben); antioxidants (eg ascorbic acid or sodium bisulfite); chelating agents (eg ethylenediaminetetraacetic acid); and agents for adjusting tonicity (eg sodium chloride or Dextrose) is also contemplated. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. Examples of suitable pharmaceutical carriers are E.I. W. "Rem" by Martin
Inton's Pharmaceutical Sciences ". Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration. The original preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

In addition to soluble AIM II polypeptides (ie, AIM II polypeptides lacking all or part of the transmembrane domain) AIM II polypeptides, AIM II polypeptides containing a transmembrane region are also surface-active. It can be used if it is suitably dissolved in a buffer by including an agent (eg Triton X-100).

In a preferred embodiment, the composition is formulated according to conventional procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If desired, the composition can also include a solubilizer and a local anesthesia such as lignocaine to relieve pain at the site of injection. Generally, the components are either dried separately or mixed together in a unit dosage form, for example, in a dry frozen form in an airtight container such as an ampoule or sachet indicating the amount of active agent. Supplied as a powder or water free concentrate. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The compounds of the present invention may be formulated as neutral or salt forms. Pharmaceutically acceptable salts include salts formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and sodium, potassium, ammonium,
Mention may be made of salts formed with cations such as those derived from calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine and the like.

The amount of a compound of the invention that is effective in treating, inhibiting and preventing a disease or disorder associated with aberrant expression and / or activity of a polypeptide of the invention can be determined by standard clinical techniques. . In addition, in vitro assays can optionally be used to help identify optimal dosage ranges. Precise dosages to be used in the formulation will also depend on the route of administration, and the severity of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

As a general proposition, the total pharmaceutically effective amount of AIM II polypeptides administered parenterally per dose is about 1 μg / kg / day to 10 mg / kg / patient body weight.
Although in the day range, this is at therapeutic discretion, as described above. More preferably, for the hormone, the dose is at least 0.01 mg / kg / day, most preferably between about 0.01 mg / kg / day and about 1 mg / kg / day for humans. When administered continuously, the therapeutic agent is typically injected one to four times daily or continuously subcutaneously (eg, using a minipump) at a dosage rate of about 1 μg / kg / hour to about 50 μg / kg / hour. It is administered by either. Intravenous bag solutions may also be used.

For antibodies, the dosage administered to a patient is typically 0.1 mg to 100 mg per kg patient body weight. Preferably, the dosage administered to a patient is between 0.1 mg / kg and 20 mg / kg of the patient's body weight, more preferably 1 mg to 10 mg / kg of the patient's body weight. In general, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, low dosages and infrequent administrations of human antibodies are often possible. Further, the dosage and frequency of administration of the antibody of the present invention may vary depending on, for example, antibody uptake and tissue penetration (eg, by modification such as lipidation).
, To the brain).

The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more ingredients of the pharmaceutical compositions of the invention. Notifications in the form prescribed by governmental agencies regulating the manufacture, use or sale of pharmaceutical or biological products may optionally be accompanied by such containers. This notice
It reflects the agency's approval of manufacture, use or sale for human administration.

Having described the invention in general, the invention will be more readily understood with reference to the following examples. The following examples are provided for purposes of illustration and are not intended to be limiting.

Examples Example 1: Expression and purification of AIM II in E. coli A. Expression of AIM II with N-terminal 6-His tag ATCC Deposit No. 97689 in the deposited cDNA. The DNA sequence encoding the AIM II protein is amplified using PCR oligonucleotide primers specific for the amino terminal sequence of the AIM II protein and the vector sequence 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-terminal and transmembrane regions) is expressed using the following primers: The 5 ′ oligonucleotide primer is the AIM II of FIG. 1A (SEQ ID NO: 1).
Underlined Ba containing nucleotides 244-258 of the protein coding sequence
Sequence containing the mHI restriction site:

[0489]

[Chemical 6] Have.

The 3'primer was sequenced with an underlined HindIII restriction site followed by nucleotides complementary to nucleotides 757-774 shown in Figure IB (SEQ ID NO: 1):

[0491]

[Chemical 7] Have.

All AIM II proteins can be expressed using the following primers: The 5'oligonucleotide primer is AIM II of Figure 1A (SEQ ID NO: 1).
Underlined BamH containing nucleotides 49 to 70 of the protein coding sequence
Sequence containing the I restriction site:

[0493]

[Chemical 8] Have.

[0494] The 3'primer has the sequence:

[0495]

[Chemical 9] Have.

These restriction sites are used for bacterial expression in these examples,
It is convenient for the restriction enzyme site in the bacterial expression vector pQE9. (Qia
gen, Inc. 9259 Eton Avenue, Chatsworth,
CA, 91311). pQE9 is resistant to ampicillin antibiotics ("Amp ^r ")
And includes a bacterial origin of replication (“ori”), an IPTG-inducible promoter, a ribosome binding site (“RBS”), a 6-His tag, and a restriction enzyme site.

Both the amplified AIM II DNA and the vector pQE9 were digested with BamH.
Digest with I and HindIII and then ligate the digested DNA together. By inserting the AIM II protein DNA into the restricted pQE9 vector,
Placing the AIM II protein coding region downstream of the vector's IPTG inducible promoter so that it is operably linked thereto, and in frame with respect to the starting AUG appropriately positioned for translation of the AIM II protein. To do.

B. Expression of AIM II with C-Terminal 6-His Tag The bacterial expression vector pQE60 is used for bacterial expression in this example. (QIAGEN, Inc., 9259 Eton Avenue, Chat.
SWTH, CA, 91311). pQE60 encodes ampicillin antibiotic resistance ("Amp ^r ") and contains a bacterial origin of replication ("ori"), an IPTG inducible promoter, a ribosome binding site ("RBS"), QIAGEN, Inc.
． Nickel-Nitrilotriacetic Acid ("Ni-NTA") sold by (above)
) Contains 6 codons encoding a histidine residue, which allows affinity purification using an affinity resin, and a suitable single restriction enzyme cleavage site. These elements consist of six His residues (ie, the following: an insert DNA fragment encoding a polypeptide covalently linked to the carboxyl terminus of the polypeptide).
It is arranged to express a polypeptide having a "6xHis tag").

The DNA sequence encoding the desired portion of the AIM II protein is amplified from the deposited cDNA using PCR oligonucleotide primers. This primer contains the amino terminal sequence and c of the desired portion of the AIM II protein.
Anneal to the sequence in the deposited construct 3'to the DNA coding sequence. pQE
Additional nucleotides containing restriction sites to facilitate cloning in the 60 vector are added to the 5'and 3'sequences, respectively.

[0500]   For protein cloning, the 5'primer has the sequence:

[0501]

[Chemical 10] Have. This is the underlined NcoI restriction site, followed by AIM in FIG. 1A.
It contains a nucleotide complementary to the amino terminal coding sequence of the II sequence. Of course,
Those skilled in the art will appreciate that the point in the protein coding sequence where the 5'primer starts can be varied to amplify a DNA segment (shorter or longer) encoding any desired portion of the complete protein. To do. The 3'primer has the sequence:

[0502]

[Chemical 11] Have. This is the underlined BamHI restriction site followed by AI in FIG. 1B.
It contains a nucleotide complementary to the 3'end of the coding sequence immediately preceding the stop codon in the M II DNA sequence so that the coding sequence maintains its reading frame with that of the six His codons in the pQE60 vector. Aligned with the restriction site.

The amplified AIM II DNA fragment and the vector pQE60 were
Digest with BamHI and NcoI and then ligate the digested DNA together. Insertion of AIM II DNA into the restricted pQE60 vector
The AIM II protein coding region is located downstream from the PTG inducible promoter and in frame with the initiation AUG and the six histidine codons.

C. Expression of AIM II Deletion Mutants with N-Terminal 6-His Tag The DNA sequence encoding the AIM II protein in the deposited cDNA was
It was amplified using PCR oligonucleotide primers specific for the sequence of the AIM II protein and the vector sequence 3'to the gene. Additional nucleotides containing restriction sites to facilitate cloning were added to the 5'and 3'sequences, respectively.

In particular, the N-terminal deleted AIM II variant (Met (68) to V in SEQ ID NO: 2
al (240)) was constructed using the following primers: The 5'oligonucleotide primer has the sequence:

[0506]

[Chemical 12] Have. It contains an underlined BamHI restriction site and contains 17 nucleotides of the AIM II protein coding sequence in Figure 1A (SEQ ID NO: 1).

[0507]   The 3'primer has the sequence:

[0508]

[Chemical 13] Have. It contains an underlined HindIII restriction site followed by nucleotides complementary to nucleotides 753-768 as shown in Figure 1B (SEQ ID NO: 1).

[0509]   These restriction sites correspond to the restriction enzyme sites in the bacterial expression vector pQE9.
It is convenient. These are used for bacterial expression in this example. (
Qiagen, Inc. 9259 Eton Avenue, Chatsworth
th, CA, 91311). pQE9 is ampicillin antibiotic resistance ("Amp ^r )), And an origin of bacterial replication (“ori”), an IPTG-inducible promoter.
, A ribosome binding site (“RBS”), a 6-His tag, and a restriction enzyme
Contains parts.

Amplified AIM II (aa68-240) DNA and vector pQE9
Both were digested with BamHI and HindIII and the digested DNA was then ligated together. The insertion of the AIM II (aa68-240) protein DNA into the restricted pQE9 vector is for the translation of the AIM II deleted protein downstream from and operably linked to the vector's IPTG inducible promoter. The AIM I in the start AUG and in-frame properly located in
Place the I protein coding region.

Bacterial Transformation The ligation mixture from the 6-His tagged expression construct made in A, B, or C above was prepared using standard procedures using competent E. coli. E. coli cells. Such procedures are described in Sambrook et al., Molecular.
Cloning: a Laboratory Manual, 2nd Edition; Col
d Spring Harbor Laboratory Press, Col
d Spring Harbor, N.D. Y. (1989). E
． coli M15 / rep4 strain (contains multicopy plasmid pREP4. It expresses the lac repressor and is resistant to kanamycin (“Kan ^r
))) Is used in carrying out the exemplary embodiments described herein. This strain is the only one of many suitable for expression of the AIM II protein and is commercially available 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 is confirmed by restriction analysis.

Clones containing the desired construct are grown overnight (“O / N”) in liquid culture in LB medium supplemented with both ampicillin (100 μg / ml) and kanamycin (25 μg / ml). .

This O / N culture is used to inoculate a large culture at a dilution of about 1: 100 to 1: 250. The cells have an optical density at 600 nm (“OD600”) of 0.
． Grow to between 4 and 0.6. Isopropyl-β-D-thiogalactopyranoside (“IPTG”) was then added to a final concentration of 1 mM and lacI was added.
Inactivating the repressor induces transcription from the lac repressor sensitive promoter. The cells are subsequently incubated for a further 3-4 hours.
The cells are then harvested by centrifugation and disrupted using standard methods. Inclusion bodies are purified from disrupted cells using conventional harvesting techniques and proteins are solubilized from inclusion bodies in 8M urea. An 8M urea solution containing solubilized protein was passed through a PD-10 column in 2x phosphate buffered saline ("PBS") to remove urea, exchange buffer and replace protein. Refold. The protein is purified by a further step of chromatography to remove endotoxin. It is then sterile filtered. The sterile filtered protein preparation is stored in 2 × PBS at a concentration of 95 μg / ml.

D. Expression and Purification of Full-Length AIM II Without a His Tag [0551] The bacterial expression vector pQE60 is used for bacterial expression in this example (QIAGEN, Inc. 9259 Eton Avenue, Chatsw).
orth, CA, 91311). pQE60 is resistant to ampicillin antibiotics (“
Amp ^r ") and a bacterial origin of replication (" ori "), IPTG inducible promoter, ribosome binding site (" RBS "), QIAGEN, Inc. (
A codon encoding a histidine residue that allows affinity purification using a nickel-nitrilotriacetic acid (“Ni-NTA”) affinity resin sold by K., and a suitable single restriction enzyme cleavage site. contains. These elements consist of six His residues (ie, “6 × H”) to which a DNA fragment encoding the polypeptide is covalently linked to the carboxyl terminus of the polypeptide.
is arranged so that it can be inserted to produce a polypeptide having an "is tag"). However, in this example, the polypeptide coding sequence is inserted such that translation of the 6His codon is disturbed, and thus the polypeptide is produced without the 6xHis tag.

The DNA sequence encoding the desired portion of the AIM II protein is amplified from the deposited cDNA clone using PCR oligonucleotide primers. This primer anneals to the amino terminal sequence of the desired portion of the AIM II protein and the sequence 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.

[0517]   For protein cloning, the 5'primer has the sequence:

[0518]

[Chemical 14] Have. It contains an underlined NcoI restriction site and is shown in FIG.
Contains the nucleotides of the amino terminal coding region of the II sequence. Of course, one of skill in the art will appreciate that the position in the protein coding sequence where the 5'primer begins can be varied to amplify the desired portion (ie, shorter or longer) of the complete protein. The 3'primer has the sequence:

[0519]

[Chemical 15] Have. It contains an underlined HindIII restriction site, followed by a nucleotide complementary to the 3'end of the non-coding sequence in the AIM II DNA sequence in Figure 1B (SEQ ID NO: 1).

The amplified AIM II DNA fragment and the vector pQE60 were
Digest with NcoI and HindIII, and then ligate the digested DNA together. Insertion of AIM II DNA into the restricted pQE60 vector resulted in downstream from the IPTG inducible promoter and in frame with the initiation AUG.
Position the AIM II protein coding region, including its associated stop codon. The relevant stop codon blocks the translation of the 6 histidine codons downstream of the insertion point.

(E. Construction of N-terminal AIM II deletion mutant) AIM II deletion mutant (Met (68) to Val (24 in SEQ ID NO: 2)
For cloning 0)), the 5'primer has the sequence:

[0522]

[Chemical 16] Have. It contains an underlined NcoI restriction enzyme recognition site, followed by FIG.
Contains 17 nucleotides of the AIM II protein coding sequence. The 3'primer has the sequence:

[0523]

[Chemical 17] (Comprising an underlined Hind III restriction site followed by nucleotides complementary to nucleotides 753-768 in Figure 1B (SEQ ID NO: 1)).

The amplified AIM II (amino acids 68-240) DNA fragment and vector pQE60 were digested with NcoI and Hind III, then the digested DNA was ligated together. Of AIM II (amino acids 68-240) DNA,
Insertion into the restriction enzyme-digested pQE60 vector resulted in AIM II (so that it had the initiation AUG downstream from the IPTG-inducible promoter and in frame.
Amino acids 68-240) position the protein coding region. HindIII digestion removes the six histidine codons downstream of this insertion site.

(F. Construction of N-terminal AIM II Deletion Mutant) AIM II deletion mutant (Ala (101) to Val (240 in SEQ ID NO: 2)
)) For cloning, the 5'primer has the sequence:

[0526]

[Chemical 18] (Including the underlined NcoI restriction enzyme recognition site and including nucleotides 349-366 in the AIM II protein coding sequence in FIG. 1A). Of course, one of ordinary skill in the art will appreciate that the start of the 5'primer in the protein coding sequence may be varied to amplify (ie, shorter or longer) the desired portion of the complete protein. The 3'primer has the sequence:

[0527]

[Chemical 19] (Comprising an underlined Hind III restriction site followed by nucleotides complementary to nucleotides 755-768 of the AIM II DNA sequence of FIG. 1B).

The amplified AIM II (amino acids 101-240) DNA fragment and vector pQE60 were digested with NcoI and Hind III, then the digested DNA was ligated together. AIM II (amino acids 101-240) DN
Insertion of A into the restricted pQE60 vector positions the AIM II (amino acids 101-240) protein coding region downstream from the IPTG-inducible promoter and with the initiating AUG in frame. Hind III digestion removes the six histidine codons downstream of the insertion site.

Purification of AIM II from G. E. coli AIM II solubilized fragment (amino acid residues L83-V2 of SEQ ID NO: 2)
(Corresponding to 40) and the polynucleotide sequence encoding HGS E. coli
It was cloned into the expression vector pHE4. The obtained plasmid DNA (pHE4
: AIMII. 83-V240), SG13009 E. E. coli host cells were transformed. Bacterial transformants were grown in LB medium containing kanamycin. Upon induction of IPTG, recombinant AIM II was expressed as an insoluble protein attached to inclusion bodies in E. coli. expressed in E. coli.

E. coli cell paste with 0.1 M Tris-HCl (pH 7.4),
Resuspend in a buffer containing ₂ mM CaCl ₂ and microfluidized bed (Micro
Fluidics, Newton, MA) was lysed by two passes at 6000-8000 psi. The lysed sample was mixed with NaCl to a final concentration of 0.5 M and then centrifuged at 7000 xg for 20 minutes. The resulting pellet is washed again with the same buffer with 0.5 M NaCl, then 700
Centrifuged again at 0xg for 20 minutes.

The partially purified inclusion bodies were then treated with 100 mM Tris (pH 7.4),
Resuspended in 2.0 M guanidine hydrochloride containing ₂ mM CaCl ₂ , 5 mM cysteine at 20-25 μC for 2-4 hours and centrifuged. Then, the obtained pellet was treated with 100 mM Tris (pH 7.4), 2 mM CaC.
3.0~3.5M guanidine hydrochloride containing l ₂ (Yes 5mM cysteine, or 5mM no cysteine) during and resuspended over 48-72 hours at 4MyuC. At this time, part of AIM II was solubilized and remained in the soluble phase after centrifugation at 7,000 xg.

3M guanidine hydrochloride extract was added to 50 mM Tris-HCl (pH 8), 1 mM.
It was quickly diluted with 20-30 volumes of buffer containing 50 mM sodium chloride.
Surfactants (eg Tween-20, CHAPS) may be added to increase the folding effect. This mixture was then left unmixed at 4 μC for 2-7 days prior to the chromatographic purification steps described below.

Liquid Chromatographic Purification of H.AIM II The diluted AIM II sample was clarified using a 0.45 μm sterile filter. AIM II protein was then added to 0.5M MES at pH 6-6.
Adjusted to 8 and chromatographed on a strong cation exchange resin (POROS HS-50) column. This HS column is first loaded with 50 mM MES-NaOH (p
H6.6) and 150 mM sodium chloride and washed with 6-10 column volumes of buffer. Bound proteins were loaded with 3-5 column volumes in a stepwise gradient (300
Sodium chloride (p, mM, 700 mM, 1500 mM) in 50 mM MES (p
Elute with H6.6).

The HS fraction, which was eluted with 0.7M sodium chloride, was diluted 3-fold with water.

(Bacterial transformation) The ligation mixture from the expression construct produced in D, E or F above was treated with Sa
Mbrook et al., Molecular Cloning: a Laborato.
ry Manual, 2nd edition; Cold Spring Harbor Lab
oratory Press, Cold Spring Harbor, NY (
1989) and using standard procedures such as those described in Competent E.C. E. coli cells were transformed. expresses the lac repressor and is resistant to kanamycin (“Ka
E. giving the n ^r ") E. coli strain MI5 / rep4 (comprising multiple copies of plasmid pREP4) was used in performing the illustrative examples described herein. Of the many strains that are suitable for expressing the AIM II protein, only one is this strain, QIAGEN, Inc. It was commercially available from (above). 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 identification of cloned DNA was performed by restriction enzyme analysis, PCR and D
Confirmed by NA sequencing.

Clones containing the desired construct are grown overnight (“O / N”) in liquid culture in LB medium supplemented with both ampicillin (100 μg / ml) and kanamycin (25 μg / ml). The O / N culture was used to inoculate a large culture at a dilution of about 1:25 to about 1: 250. The cells were transferred to 600 nm (“OD
600 ") was grown to an optical density of between 0.4 and 0.6. Isopropyl-b-D-thiogalactopyranoside ("IPTG") was then added to inactivate the lacI repressor to induce transcription from the lac receptor-sensitive promoter to a final concentration of 1 mM. Was added to. The cells were subsequently incubated for a further 3-4 hours. The cells were then harvested by centrifugation.

The cells were then placed in 6M guanidine HCl, pH 8 at 4 ° C. for 3-4 hours.
It was stirred. The cell debris was removed by centrifugation and the supernatant containing AIM II was added to 50 mM sodium acetate buffer pH supplemented with 200 mM NaCl.
Dialyzed against 6. Alternatively, the protein is 500 mM NaCl, 20% glycerol, 25 mM Tris / HC containing protease inhibitors.
Can be successfully refolded by dialysis against pH 7.4. After re-denaturation, the protein can be purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography.
Alternatively, an affinity chromatography step such as an antibody column can be used to obtain pure AIM II protein. Purified protein was stored at 4 ° C or frozen at -80 ° C.

Example 2: Cloning and Expression of AIM II Protein in Baculovirus Expression System (A. Cloning and Expression of Full Length AIM II Protein :) Full Length A in the Deposited Clone Assigned ATCC Deposit No. 97689
The cDNA sequence encoding the IM II protein is amplified using PCR oligonucleotide primers corresponding to the 5'and 3'sequences of the gene: This 5'primer has the sequence:

[0539]

[Chemical 20] (Has an underlined Bam HI restriction site followed by 22 bases (ie, nucleotides 49-70) of the coding region for the AIM II protein in FIG. 1A). The 5'end of the amplified fragment, inserted into an expression vector and encoding AIM II, provides an effective signal peptide as follows. K
ozak, M .; J. Mol. Biol. 196: 947-950 (1987).
Efficient signals for translation initiation in eukaryotic cells, as described by, are properly located in the vector portion of the construct.

[0540]   The 3'primer has the sequence:

[0541]

[Chemical 21] (Underlined Asp718 restriction enzyme recognition site followed by AIM II shown in FIG. 1A.
(Comprising nucleotides complementary to the 770-795 nucleotides).

The amplified fragment was transferred to a commercially available kit (“Geneclean”, BIO
101 Inc. , La Jolla, Ca. ) Is used to isolate from a 1% agarose gel. This fragment is then cloned into Bam HI and Asp71.
Digest with 8 and again purify on a 1% agarose gel. This fragment is designated herein as F2.

Vector pA2-GP was added to Summers in a baculovirus expression system.
Et al., A Manual of Methods for Baculovirus.
s Vectors and Insect Cell Culture Pr
ocedures, Texas Agricultural Experime
nal Station Bulletin No. Used to express AIM II protein using standard techniques, as described in 1555 (1987). This expression vector is an Autographa californ
nica nuclear polyhedrosis virus (AcMNPV) strong polyhedrosis promoter,
It then contains convenient restriction enzyme sites. The signal peptide of AcMNPV gp67 (including the N-terminal methionine) is located just upstream of the BamHI site.
The simian virus 40 (“SV40”) polyadenylation site is used for efficient polyadenylation. As an easy selection of recombinant virus, E. col
The β-galactosidase gene from i is inserted in the same orientation as the polyhedrin promoter and is followed by the polyadenylation signal of the polyhedrin gene.
This polyhedrin sequence is flanked on both ends by viral sequences for cell-mediated homologous recombination with wild-type viral DNA, in order to produce a viable virus expressing the cloned polynucleotide.

Many other baculovirus vectors (eg pAc373, pVL941)
, And pAcIM1) are well-positioned signals for constructs for transcription, translation, transport, etc. (if required, eg,
So long as it provides an in-frame AUG and signal peptide) it can be used in place of pA2-GP. Such a vector is, for example, Lu
ckow et al., Virology 170: 31-39 (1989).

The plasmid is digested with the restriction enzymes BamHI and Asp718 and then dephosphorylated with calf intestinal phosphatase using conventional procedures known in the art. The DNA is then placed in a commercially available kit ("Geneclean" BIO 101
Inc. , La Jolla, Ca), and is isolated from a 1% agarose gel. This vector DNA is referred to herein as "V".

Fragment F2 and dephosphorylated plasmid V2 are ligated together using T4 DNA ligase. E. E. coli HB101 is transformed with the ligation mixture and spread on culture plates. Bacteria containing plasmids containing the human AIM II gene are identified by digesting DNA from individual colonies using Xba I and analyzing the digestion products by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing. This plasmid is referred to herein as pBacAIM II.

B. Cloning and Expression of an Alternative Form of AIM II Protein The cDNA sequence encoding the human AIM II protein in the deposited clone identified as ATCC Deposit No. 97483 was replaced with the 5 ′ and 3 ′ sequences of this gene. Amplify using the corresponding PCR oligonucleotide primers.

[0548]   The 5'primer has the following sequence:

[0549]

[Chemical formula 22] Which has an underlined Bam HI restriction site followed by A of SEQ ID NO: 38.
It contains 15 bases of the IM II sequence. 5'amplified fragment (encoding human AIM II) inserted into an expression vector as described below.
Provides an effective signal peptide. Effective signal peptides for initiation of translation in eukaryotic cells have been described by Kozak, M .; J. Mol. Bil
o. 196: 947-950 (1987), appropriately located in the vector portion of the construct.

[0550]   The 3'primer has the following sequence:

[0551]

[Chemical formula 23] This sequence has an underlined XbaI restriction followed by a nucleotide complementary to the last 18 nucleotides of AIMI and encodes the sequence shown in SEQ ID NO: 38, including the stop codon.

Amplified fragments are isolated from a 1% agarose gel using a commercial kit ("Geneclean", BIO 101 Inc., La Jolla, CA). This fragment is then cloned into Bam HI and Asp71.
Digest with 8 and again purify on a 1% agarose gel. This vector was then constructed essentially with the pA vector as described above in Section A of this Example.

C. Construction of N-Terminal AIM II Deletion Mutants In this illustrative example, plasmid shuttle vector pA2GP was used to clone a cloned DN encoding an N-terminal deletion of the AIM II protein.
A is an AIM II mutant of SEQ ID NO: 2 (Gln (60) to Val (240))
And a baculovirus expressing AIM II variants (Ser (79) -Val (240)), Summer et al., A Manual of Methods.
for Baculovirus Vectors and Insect
Cell Culture Procedures, Texas Agricu
lural Experimental Station Bulletin
Insert using standard methods such as those described in No. 1555 (1987). This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV), followed by the secretion signal peptide (leader) of the baculovirus gp67 protein, Ba
Includes convenient restriction sites such as mHI, XbaI, and Asp718. The polyadenylation site of simian virus 40 (“SV40”) is used for efficient polyadenylation. For easy selection of recombinant virus, the plasmid was constructed in the same orientation, under the control of the weak Drosophila promoter, in E. coli. β derived from E. coli
It contains the galactosidase gene, followed by the polyadenylation signal of the polyhedrin gene. The inserted gene flanks the viral sequence on both sides due to cell-mediated homologous recombination with the DNA of the wild-type virus, producing a viable virus expressing the cloned polynucleotide.

As will be appreciated by those in the art, the construct will provide appropriately positioned signals for transcription, translation, secretion, etc., optionally including, for example, a signal peptide and an in-frame AUG. As long as you do many other baculovirus vectors (
For example, pAc373, pVL941 and pAcIM1) can be used in place of the above vector. Such vectors are described by Luckow et al.
Virology 170: 31-39.

AIM II (Gln (60) -Val (240)) (FIG. 1A (SEQ ID NO: 2)
) To the P sequence corresponding to the 5'and 3'sequences of this gene.
Amplification was performed using CR oligonucleotide primers.

[0556]   This 5'primer has the following sequence:

[0557]

[Chemical formula 24] This sequence has an underlined BamHI restriction site followed by FIGS. 1A and 1B.
It contains 20 nucleotides (ie, 225 to 245) encoding the AIM II protein shown in and begins at amino acid 60 of the protein. This 3'primer has the following sequence:

[0558]

[Chemical 25] And has an underlined XbaI restriction site followed by nucleotides complementary to nucleotides 753-768 of Figure 1B (SEQ ID NO: 1).

The amplified fragment was transferred to a commercial kit (“Geneclean” BIO).
101 Inc. , La Jolla, Ca) was used to isolate from a 1% agarose gel. This fragment was then digested with BamHI and XbaI,
Then, it was purified again on a 1% agarose gel. This fragment is referred to herein as "
It is called "F1".

The plasmid may be digested with the restriction enzymes Bam HI and Xba I and optionally dephosphorylated with calf intestinal phosphatase using conventional procedures known in the art. Then, this DNA was put into a commercially available kit ("Gene
clean ”BIO 101 Inc. , La Jolla, Ca. ) Is used to isolate from a 1% agarose gel. This vector DNA is referred to herein as "V1".

Fragment F1 and dephosphorylated plasmid V1 were both ligated using T4 DNA ligase. E. coli HB101 or other suitable E. coli. co
Li host (eg, XL-1 Blue (Stratagene Clonin
g Systems, La Jolla, CA)) cells were transformed with the ligation mixture and spread on culture plates. Human AIM using PCR method
Bacteria containing a plasmid carrying the II gene were identified. In the PCR method, one of the primers used was to amplify this gene, and the second primer was well inside the vector, so that only bacterial colonies containing the AIM II gene fragment contained DNA. Amplification of The sequence of the cloned fragment was confirmed by DNA sequencing. This plasmid
It was designated herein as pBacAIM II (aa 60-240).

AIM II (Ser (79) -Val (240)) (FIG. 1A (SEQ ID NO: 2).
) To the P sequence corresponding to the 5'and 3'sequences of this gene.
Amplification was performed using CR oligonucleotide primers.

[0563]   This 5'primer has the following sequence:

[0564]

[Chemical formula 26] This sequence has an underlined BamHI restriction site followed by FIGS. 1A and 1B.
It contains 283 to 301 nucleotides encoding the AIM II protein shown in and begins at amino acid 79 of the protein. This 3'primer has the following sequence:

[0565]

[Chemical 27] This sequence has an underlined BamHI restriction site, followed by FIG. 1B (SEQ ID NO: 1).
Of nucleotides 757 to 771.

The amplified fragment was transferred to a commercial kit (“Geneclean” BIO).
101 Inc. , La Jolla, Ca) was used to isolate from a 1% agarose gel. This fragment was then digested with BamHI and again 1%
Purified on agarose gel. This fragment was designated herein as "F1".

The plasmid may be digested with the restriction enzyme Bam HI and optionally dephosphorylated with calf intestinal phosphatase using conventional procedures known in the art. This DNA is then placed in a commercially available kit ("Geneclean" B
IO 101 Inc. , La Jolla, Ca. ) Is used to isolate from a 1% agarose gel. This vector DNA was designated herein as "V1".

Fragment F1 and dephosphorylated plasmid V1 were both ligated using T4 DNA ligase. E. coli HB101 or other suitable E. coli. co
Li host (eg, XL-1 Blue (Stratagene Clonin
g Systems, La Jolla, CA)) cells were transformed with the ligation mixture and spread on culture plates. Mutant AI using PCR method
Bacteria containing a plasmid carrying the M II gene were identified. In the PCR method,
One of the primers used was to amplify this gene, and the second was well from inside the vector so that only bacterial colonies containing the AIM II gene fragment show amplification of DNA. . The sequence of the cloned fragment was confirmed by DNA sequencing. This plasmid was designated herein as pBacAIM II (aa79-240).

(D. Transfection of Baculovirus Vector Containing AIM II Sequence) 5 μg of either pBacAIM II or pBacAIM II (aa60-240) plasmid was added to commercially available linear baculovirus DNA (“Bac
uroGold ^™ Baculovirus DNA ”, Pharmingen, San
(Diego, CA) 1.0 μg with Felgner et al., Proc. Natl
． Acad. Sci. Co-transfection using the lipofection method described in USA, 84: 7413-7417 (1987). 1 μg
BaculoGold ^™ viral DNA and 5 μg of plasmid pBacA
IM II or pBacAIM II (aa60-240) was added to 50 μl of serum-free Grace's medium (Life Techno).
logs Inc. , Gaithersburg, MD) in a sterile microtiter plate well. Then 10 μl of Li
Pofectin and 90 μl Grace's medium were added, mixed and incubated for 15 minutes at room temperature. Then add 1 ml of transfection mixture
Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with serum-free Grace's medium were added dropwise. The plate was rocked back and forth to mix the newly added solution. The plate was then incubated at 27 ° C for 5 hours. After 5 hours, remove the transfection solution from the plate,
Then, 1 ml of Grace's insect medium supplemented with 10% fetal bovine serum was added. The plate was returned to incubation and incubation at 27 ° C continued for 4 days.

After 4 days, the supernatant is collected and plaque assayed with Summers and S.
It was carried out as described by mith (cited above). "Blue Gal
"(Life Technologies Inc., Gaithersburg
gal producing plaques that stain blue using an agarose gel with g)
Allowed easy identification and isolation of expression clones. (A detailed description of this type of "plaque assay" is also available at Life Technologies Inc.
． , Insect Cell Culture and Baculovirology User Guide distributed by Gaithersburg (pages 9-10).

Four days after serial dilution, the virus was added to the cells. After appropriate incubation, blue-stained plaques were picked up with the tip of an Eppendorf pipette. The agar containing the recombinant virus was then resuspended in an Eppendorf tube containing 200 μl Grace's medium. The agar was removed by brief centrifugation and the supernatant containing the recombinant baculovirus was used on insect Sf9 cells seeded in a 35 mm dish. After 4 days, the supernatants of these culture dishes were harvested and then stored at 4 ° C. Properly inserted hESSB
Clones containing I, II and III were identified by DNA analysis including restriction mapping and sequencing. This clone is referred to herein as V-AIM.
II or V-AIM II (aa 60-240).

Sf9 cells were grown in Grace's medium supplemented with 10% heat-inactivated FBS.
The cells were infected with recombinant baculovirus V-AIM II or V-AIM II (aa 60-240) at a multiplicity of infection (“MOI”) of about 2 (about 1 to about 3). After 6 hours, the medium was removed and SF900II medium without methionine and cysteine (Life Technologies In) was removed.
c. , Available from Gaithersburg). 42 hours later, 5
μCi ³⁵ S methionine and 5 μCi ³⁵ S cysteine (available from Amersham) were added. The cells were incubated for an additional 16 hours, then harvested by centrifugation, lysed and labeled protein was added to SDS-PAG.
Visualized by E and autoradiography.

Example 3: Cloning and Expression in Mammalian Cells In mammalian cells, most vectors used for transient expression of the AIM II protein gene sequence contain an SV40 origin of replication. Will. This allows replication of the vector up to high copy numbers in cells that express the T antigen required for initiation of viral DNA synthesis (eg, COS cells). Any other mammalian cell line can also be used for this purpose.

A typical mammalian expression vector contains promoter elements that mediate the initiation of mRNA transcription, protein coding sequences, signals required for termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences, and intervening sequences flanking the donor and acceptor sites of RNA splicing. Highly efficient transcription is achieved by the SV40 early and late promoters, retroviruses (eg RSV, HTLVI, HIVI).
Long terminal repeats (LTR), as well as the cytomegalovirus (CMV) early promoter. However, cellular signals such as the human actin promoter can also be used. Suitable expression vectors for use in practicing the present invention include, for example, pSVL and pMSG (Pharm).
acia, Uppsala, Sweden), pRSVcat (ATCC 37
152), pSV2dhfr (ATCC 37146) and pBC12MI (
Vectors such as ATCC 67109). Mammalian host cells that can be used include human HeLa, 283, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos1, Cos7 and CV1, African green monkey cells, quail QC1-3 cells, mouse L cells and Chinese hamster ovary. Cells.

Alternatively, the gene can be expressed in stable cell lines containing the gene integrated into a chromosome. Co-transfection with selectable markers such as dhfr, gpt, neomycin, hygromycin allows identification and isolation of transfected cells.

The transfected gene can also be amplified to express large amounts of the encoded protein. DHFR (dihydrofolate reductase) is a useful marker for the development of cell lines that carry hundreds or even thousands of copies of the gene of interest. Another useful selectable marker is the glutamine synthase enzyme (GS) (Mu
rphy et al., Biochem J. et al. 227: 277-279 (1991); Be.
Bbington et al., Bio / Technology 10: 169-175 (
1992)). These markers are used to grow mammalian cells in selective medium and select the cells with the highest degree of resistance. These cell lines contain a propagated gene integrated into the chromosome. Chinese hamster ovary (
CHO) cells are often used for protein production.

The expression vectors pC1 and pC4 represent the Rous sarcoma virus strong promoter (LTR) (Cullen et al., Molecular and Cellula).
r Biology, 438-447 (March, 1985)), and CM.
A fragment of the V enhancer (Boshart et al., Cell 41: 521-
530 (1985)). Multiple cloning sites (eg BamHI, X
with restriction sites for baI and Asp718) facilitates cloning of the gene of interest. This vector contains, in addition to the 3'intron, the polyadenylation and termination signals of the rat preproinsulin gene.

Example 3 (a): Cloning and Expression in COS Cells The expression plasmid pAIM II HA was cloned into cDNA encoding AIM II.
Is made by cloning into the expression vector pcDNAI / Amp (available from Invitrogen, Inc.).

The expression vector pcDNAI / amp contains: (1) E. E. coli effective for growth in E. coli and other prokaryotic cells. origin of replication of E. coli; (2) Ampicillin resistance gene for selection of prokaryotic cells carrying the plasmid; (3
) SV40 origin of replication for growth in eukaryotic cells; (4) Sequenced CM
V promoter, polylinker, SV40 intron, and polyadenylation signal (so that the cDNA can be conveniently placed under the expression control of the CMV promoter due to restriction sites in polyline, and SV40
Can be operably linked to introns and polyadenylation signals).

A DNA fragment encoding the AIM II protein and an HA tag fused in frame to its 3 ′ end were added to the polyline region of the vector so that expression of the recombinant protein was directed by the CMV promoter. To clone. HA tags are described by Wilson et al., Cell 37: 767 (
1984), corresponding to an epitope derived from the influenza hemagglutinin protein. Fusion of the HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes its HA epitope.

The strategy for plasmid construction for full length AIM II is as follows. The AIM II cDNA of the deposited clone was cloned into E. Amplification is performed using primers containing convenient restriction sites, much as described above for construction of expression vectors for AIM II expression in E. coli. Expressed AIM II
In order to facilitate the detection, purification and characterization of H. aureus, one of the primers contains a hemagglutinin tag ("HA tag") as described above.

Suitable primers include the following, which were used in this example. The 5'primer (including the underlined BamHI site and AUG start codon) has the following sequence:

[0583]

[Chemical 28] 3'primer (underlined XbaI site, stop codon, and later haemagglutinin HA
The 9 codons that form the tag, and the 33 'of the 3'coding sequence (at the 3'end) include the following sequences:

[0584]

[Chemical 29] The PCR amplified DNA fragment and the vector pcDNAI / Amp were digested with HindIII and XhoI and then ligated.

The strategy for plasmid construction for the alternative form of AIM II is as follows. The AIM II cDNA of the deposited clone (identified as ATCC Deposit No. 97483) was cloned into E. E. coli and S. furgiperd
Amplification is done using primers containing convenient restriction sites, much as described above for the construction of expression vectors for AIM II expression in a. Expressed A
To facilitate the detection, purification and characterization of IM II, one of the primers contains the hemagglutinin tag (“HA tag”) described above.

Suitable primers include the following, which were used in this example. The 5'primer (including the underlined BamHI site, the AUG start codon and the five codons with the following sequences) has the following sequence:

[0587]

[Chemical 30] The 3'primer (including the underlined XbaI site) has the following sequence:

[0588]

[Chemical 31] The PCR amplified DNA fragment and the vector pcDNAI / Amp were digested with the appropriate restriction enzymes and then ligated.

In each of the above cases, the ligation mixture was transformed into E. coli SURE strain (Stra
tagene Cloning Systems, 11099 North T
orrey Pines Road, La Jolla, CA 92037) and the transformed cultures can be plated on ampicillin medium plates and then the plates can be incubated to grow ampicillin resistant colonies. Plasmid DNA was isolated from resistant colonies and AIM
The presence of the II coding fragment was tested by restriction analysis and gel electrophoresis.

For expression of recombinant AIM II, COS cells were treated with DEAE-DEXTRA.
N (eg Sambrook et al., Molecular Cloning: a
Laboratory Manual, Cold Spring Labora
tory Press, Cold Spring Harbor, New Yo
rk (as described in 1989)) and transfected with the expression vector described above. The cells were incubated under conditions for expression of AIM II by the vector.

Expression of AIM II HA fusion proteins is described in, eg, Harlow et al., An.
tibodies: A Laboratory Manual, 2nd edition; Col
d Spring Harbor Laboratory Press, Col
Detection by radiolabeling and immunoprecipitation, using the method described in d Spring Harbor, New York (1988). For this purpose, 2 days after transfection, the cells were labeled by incubation for 8 hours in medium containing ³⁵ S cysteine. Cells and medium were collected and cells were washed and detergent containing RIPA buffer: 150 mM NaCl, 1
% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM
Dissolved in TRIS, pH 7.5 (as described by Wilson et al. Cited above). Proteins were precipitated from cell lysates and from culture medium using HA-specific monoclonal antibodies. The precipitated protein was then analyzed by SDS-PAGE gel and autoradiography. An expression product of the expected size was found in the cell lysate, which was not found in the negative control.

Example 3 (b): Cloning and Expression in CHO Cells Vector pC4 was used for expression of AIM II protein. Plasmid pC
1 is a derivative of plasmid pSV2-dhfr (ATCC Accession No. 37146). Both plasmids contain mouse DHFR under the control of the SV40 early promoter.
Contains genes. Chinese hamster ovary cells or other cells deficient in dihydrofolate activity that are transfected with these plasmids are selected media (α minus MEM, Life Techn) supplemented with the chemotherapeutic agent methotrexate.
can be selected by growing the cells in Amplification of the DHFR gene in cells that are resistant to methotrexate (MTX) has been well documented (eg, Alt, FW, Kellems, RM, Berti.
no, J. R. , And Skimke, R .; T. , 1978, J .; Biol.
Chem. 253: 1357-1370, Hamlin, J .; L. And Ma,
C. 1990, Biochem. et Biophys. Acta, 1097:
107-143, Page, M .; J. And Sydenham, M .; A. 199
1, Biotechnology Volume 9: 64-68). Cells grown at increasing concentrations of MTX develop resistance to the drug by overproducing the target enzyme DHFR as a result of amplification of the DHFR gene. When the second gene is linked to the DHFR gene, it is usually co-amplified and overexpressed. It is common knowledge in the art to generate cell lines that carry more than 1000 copies of a gene. Subsequently, when methotrexate is recovered, the cell line carries the amplified gene integrated into the chromosome.

Plasmid pC4 is a strong promoter of the Rous sarcoma virus long terminal repeat (LTR) for expression of the gene of interest (Cullen et al., Molec).
ural and Cellular Biology, March 1985
: 438-447) and human cytomegalovirus (CMV) immediate-early gene enhancers (Boshart et al., Cell 4).
1: 521-530 (1985)). Downstream of the promoter are BamHI, XbaI, and Asp718 restriction enzyme cleavage sites that allow integration of the gene. This plasmid contains these cloning sites followed by the 3'intron and polyadenylation site of the rat preproinsulin gene.
Other high efficiency promoters can also be used for expression (eg, human β-actin promoter, SV40 early or late promoters, or long terminal repeats from other retroviruses (eg, HIV and HTLVI)).
Clontech's Tet-Off and Tet-On gene expression systems and similar systems can be used to express AIM II by the method of regulating mammalian cells (Gossen, M. and Bujard, H. 1992, Proc.
． Natl. Acad. Sci. USA 89: 5547-5551). mRN
Other signals, such as those from the human growth hormone or globin genes, may also be used for polyadenylation of A as well. Stable cell lines that carry the gene of interest integrated into the chromosome also have selectable markers (eg, gpt).
, G418 or hygromycin). It is advantageous to initially use more than one selectable marker (eg G418 and methotrexate).

The plasmid pC4 is digested with the restriction enzymes BamHI and Asp718 and then dephosphorylated using calf intestinal phosphatase by procedures known in the art. The vector is then isolated from a 1% agarose gel.

The DNA sequence encoding the complete AIM II protein is amplified using PCR oligonucleotide primers corresponding to the 5'and 3'sequences of this 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: 15), which is an underlined BamHI restriction enzyme site, followed by a potent signal for initiation of translation in eukaryotes (Kozak, M .;
J. Mol. Biol. 196: 947-950 (1987)), and 22 bases of the coding region of the AIM II protein shown in Figure 1A (SEQ ID NO: 1) (i.e., nucleotides 49-70). The 3'primer has the sequence 5'GA CGC GGT ACC GTC CAA TGC AC.
C ACG CTC CTT CCT TC 3 ′ (SEQ ID NO: 16), which is an underlined Asp718 restriction site, followed by FIG. 1B (SEQ ID NO: 1).
Containing nucleotides complementary to nucleotides 770-795 of the AIM II gene shown in.

The amplified fragment was digested with the endonucleases BamHI and Asp71.
Digested with 8, then purified again on a 1% agarose gel. The isolated fragment and the dephosphorylated vector were then ligated with T4 DNA ligase. Then E. E. coli HB101 or XL-1 Blue cells are transformed and bacteria containing the fragment inserted into plasmid pC4 are identified by, for example, restriction enzyme analysis.

Example 3 (c): Cloning and expression of N-terminal deleted AIM II in CHO cells Vector pC4 was transformed with AIM II mutants (Met (68) -V in SEQ ID NO: 2).
al (240)) was used for protein expression. Plasmid pC4 was digested with the restriction enzyme BamHI and then dephosphorylated using calf intestinal phosphatase by procedures known to those skilled in the art. This vector was then isolated from a 1% agarose gel.

The DNA sequence encoding the AIM II (aa68-240) protein was amplified using PCR oligonucleotide primers corresponding to the 5'and 3'sequences of this gene. The following 5'primers were used: 5'GAC AGT
GGA TCC GCC ACC ATG GTC ACC CGC CTG
CCT GAC GGA C 3 '(SEQ ID NO: 40). This is the underlined Ba
mHI restriction site followed by a competent signal for initiation of translation in eukaryotes (Kozak, M., J. Mol. Biol. 196: 947-950 (
1987)), and the AI shown in Figure 1A (SEQ ID NO: 1).
Includes nucleotides 202-226 in the coding region of the M II polypeptide. The following 3'primers were used: (BamHI and stop codon (italicized))

[0599]

[Chemical 32] It contains an underlined BamHI restriction site followed by nucleotides complementary to nt 753-768 shown in Figure 1B (SEQ ID NO: 1).

The amplified fragment is digested with the endonuclease BamHI and then
Purified again on a 1% agarose gel. The isolated fragment and the dephosphorylated vector were then ligated with T4 DNA ligase. Then
E. E. coli HB101 or XL-1 Blue cells are transformed and bacteria containing the fragment inserted into plasmid pC4 are identified by, for example, restriction enzyme analysis.

The vector pC4 / Ckβ8 (pC4 construct cloned into the pC4 vector at the BamHI site at the 3 ′ end of the Ckβ8 signal peptide, first with the Ckβ8 signal peptide) was transformed with the AIM II mutant (Trp of SEQ ID NO: 2 80) ~
Val (240)) was used for protein expression. Plasmid pC4 / C
kβ8 was digested with the restriction enzyme BamHI and then dephosphorylated using calf intestinal phosphatase by procedures known to those skilled in the art. This vector is then
Isolated from a% agarose gel.

The DNA sequence encoding the AIM II (aa80-240) protein was amplified using PCR oligonucleotide primers corresponding to the 5'and 3'sequences of this gene. The following 5'primers were used: 5'cgc GGA TCC TGGGAGCAGCTGATAC 3 '(SEQ ID NO: 41). It contains an underlined BamHI restriction enzyme site, followed by nucleotides 286-301 in the coding region of the AIM II polypeptide shown in Figure 1A (SEQ ID NO: 1). The following 3'primers were used: 5'cgc GGATCC TCA CA.
CCATGAAAGC 3 '(SEQ ID NO: 29). It contains an underlined BamHI restriction site followed by nucleotides complementary to nt 757-771 shown in FIG. 1B (SEQ ID NO: 1).

The amplified fragment was digested with the endonuclease BamHI and then 1
Repurified on a% agarose gel. The isolated fragment and the dephosphorylated vector were then ligated with T4 DNA ligase. Then E. col
iHB101 or XL-1 Blue cells were transformed and bacteria containing the fragment inserted into plasmid pC4 / Ckβ8 were identified using, for example, restriction enzyme analysis.

CHO Cell Transfections Chinese hamster ovary cells lacking the active DHFR gene are used for transfection. 5 μg of the above pC4 expression vector was cotransfected with 0.5 μg of the plasmid pSV2-neo using lipofectin (Felgner et al., Supra). The plasmid pSV2neo contains the dominant selectable marker, the neo gene, which is derived from Tn5 which encodes an enzyme that confers resistance to a group of antibodies including G418. 1 mg of the cells
Seed in α-minus MEM supplemented with 1 / ml G418. Two days later, cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in α-MEM supplemented with 10, 25, or 50 ng / ml methotrexate and 1 mg / ml G418. About 10
After -14 days, single clones were trypsinized and then treated with different concentrations (50n.
M, 100 nM, 200 nM, 400 nM, 800 nM) are used to seed 6-well petri dishes or 10 ml flasks. The clones that grow at the highest concentration of methotrexate are then transferred to higher concentrations (1 μM
2 μM, 5 μM, 10 μM, 20 μM) to a new 6-well plate containing methotrexate. The same procedure is repeated until clones are obtained that grow at a concentration of 100-200 μM. Expression of the desired gene product can be achieved, for example, by SDS-P
Analyze by AGE and Western blotting, or reverse phase HPLC analysis.

Example 3 (d): Cloning and Expression of AIM II N-Terminal Deletion in CHO Cells AIM II variant containing a C-terminal Fc immunoglobulin region (Met (68) -Val in SEQ ID NO: 2). (240)) The vector pC4 was used for protein expression. In this construct, the Ckβ8 signal peptide was added to Ckβ
It was first cloned into pC4 using the BamHI site at the 3'end of 8. Ba
The Fc fragment flanked by mHI and XbaI sites was cloned into the vector resulting in pC4 / Ckβ8 / Fc. Then AIM I
The I fragment was cloned between the CK-β8 leader and the Fc fragment at the BamHI site.

The plasmid pC4 was digested with the restriction enzyme BamHI and then dephosphorylated with calf intestinal phosphatase by procedures known in the art. This vector was then isolated from a 1% agarose gel.

The DNA sequence encoding the complete AIM II (aa68-240) protein was amplified using PCR oligonucleotide primers corresponding to the 5'and 3'sequences of this gene. The following 5'primers were used: 5'GAC AGT
GGA TCC GCC ACC ATG GTC ACC CGC CTG
CCT GAC GGA C3 '(SEQ ID NO: 40). This primer was constructed with an underlined BamHI restriction enzyme site, followed by Kozak, M. et al. J. Mol. B
iol. 196: 947-950 (1987), an effective signal for initiation of translation in eukaryotes, and nucleotide 202 in the coding region for the AIM II polypeptide shown in Figure 1A (SEQ ID NO: 1).
.About.226. The following 3'primers were used: (BamHI) 5'-GG
G GGA TCC CAC CAT GAA AGC CCC G-3 '(SEQ ID NO: 30). This primer contains an underlined BamHI restriction site followed by a nucleotide complementary to nt 753-768 shown in Figures 1A and 1B (SEQ ID NO: 1), followed by an Fc immunoglobulin fragment having the following sequence:

[0608]

[Chemical 33] The amplified fragment was digested with the endonuclease BamHI and then 1
Repurified on a% agarose gel. The isolated fragment and the dephosphorylated vector were then ligated with T4 DNA ligase. Then E. col
iHB101 or XL-1 Blue cells were transformed. Bacteria containing the fragment inserted into plasmid pC4 are then identified, for example using restriction enzyme analysis.

(CHO Cell Transfection) Chinese hamster ovary (CHO / dhfr-DG44) cells were transfected with expression vector (pC4 / spCKβ8 / Fc / AIM I) using lipofectin.
I) was used for transfection. Recombinant clones were 5% dialyzed fetal bovine serum (DiFBS), 1% penicillin / streptomycin (PS), 1 mg /
It was isolated by growing the cells in MEMα selection medium with mL geneticin (G418) and 10 nM methotrexate (MTX). Then
High expressing clones identified by screening the recombinant clones using the BIAcore method (see below for more details) were isolated by stepwise increasing the concentration of MTX to a final concentration of 100 μM. Were grown. This high-expressing clone was cloned into A in a microcarrier CHO perfusion bioreactor.
Used for production of IM II-IgG1 fusion protein.

CHO AIM II-IgG1 cells were loaded with 1% ultra-low IgG.
Growing on Cytodex 1 microcarriers (Pharmacia Biotech, Upsala, Sweden) in HGS-CHO-3 medium with FBS. 1 cell grown in a multiple microcarrier spinner
Scaled up to 0 L microcarrier perfusion bioreactor. The perfusion bioreactor was operated continuously for 27 days and during that time the AIM II
-A 90 liter supernatant containing IgGl fusion protein and no microcarriers was collected. The supernatant was clarified through a filtration process using a 0.2 μm sterile filter and stabilized by adding 5 mM EDTA. The clarified supernatant was loaded onto the affinity column and the AIM II-I
The gG1 fusion protein was captured.

Purification of AIM II-IgG1 Fusion Protein AIM II-IgG1 fusion protein was purified from 15 L CHO conditioned medium. This conditioned medium was passed through BioCad60 (PerSe) at a flow rate of 30 mL / min.
Protein A Type at 10 ° C. on ptives Biosystems).
Loaded on rD (54 mL total volume, BioSepra) affinity column. The column was loaded with 25 mM sodium acetate, pH 8 and 0.1 M NaCl.
Was equilibrated in advance with. After loading, the column was washed with 3 volumes of 0.1 M sodium citrate, pH 5 and 0.1 M NaCl, and 0.1 M sodium citrate, pH 2.8 and 0.1 M NaCl, respectively. AIM
The peak fractions containing II-IgG fusion protein were determined by SDS-PAGE analysis and pooled. The identity of this purified protein was confirmed by N-terminal sequence analysis. Final protein yield is approximately 9m / l conditioned medium
It was g.

Example 4: AIM II Expression Construct (Full Length Construct) (a) pCMVsport: Eukaryotic Expression Vector pCMVsport is M
Includes nucleotides encoding the AIM II ORF from et (1) to Val (240). The plasmid construction strategy is as follows. The AIM II cDNA of the deposited clone is amplified using primers containing convenient restriction sites. Suitable primers include the following used in this example: The 5'primer is an underlined SalI site, AUG start codon, nucleotides 51-6 in the coding region of the AIM II polypeptide (SEQ ID NO: 1).
Including 9 and having the following sequences:

[0613]

[Chemical 34] The 3'primer contains an underlined NotI site, contains nucleotides complementary to nucleotides 753 to 767 of SEQ ID NO: 1, and a stop codon, and has the following sequence:

[0614]

[Chemical 35] The PCR amplified DNA fragment is digested with SalI and NotI, then gel purified. The isolated fragment was then ligated into the SalI and NotI digested vector pCMVsport. The ligation mixture was transformed into E. E. coli and the transformed cultures are plated on antibiotic medium plates, which are then incubated to allow growth of antibiotic resistant colonies.
Plasmid DNA is isolated from resistant colonies and tested for the presence of the AIM II coding fragment by restriction analysis and gel sizing.

For expression of recombinant AIM II, eukaryotic cells (eg, COS cells or CHO cells) were prepared as described above using DEAE-DEXTRAN as described in Example 3 above. Transfected with the expression vector. AIM II
Recombinant protein expression is detected by the method described above in Example 3.

(B) pG1SamEN: The retroviral expression vector pG1SamEN is
It encodes the AIM II ORF of Met (1) -Val (240). pG1
The vector is described by Morgan, R. et al. A. Nucl. Acids Res. 20
(6): 1293-1299 (1992) and similar to the LN vector (Miller, AD and Rosman, GJ Bio).
techniques 7: 980-990 (1989)), with additional cloning sites. The plasmid construction strategy is as follows. The AIM II cDNA of the deposited clone is amplified using primers containing convenient restriction sites. Suitable primers include the following used in this example: The 5'primer contains an underlined NotI site, and AUG start codon, nucleotide 51 of the coding region of the AIM II polypeptide (SEQ ID NO: 1).
To 69 and has the following sequence:

[0617]

[Chemical 36] The 3'primer is an underlined SalI site, nucleotide 7 of SEQ ID NO: 1.
It contains nucleotides complementary to 53-768, and a stop codon, and has the following sequence:

[0618]

[Chemical 37] The PCR amplified DNA fragment is digested with SalI and NotI and then gel purified. The isolated fragment was then ligated into the SalI and NotI digested vector. The ligation mixture was transformed into E. E. coli and the transformed cultures are plated on antibiotic medium plates and then incubated to allow growth of antibiotic resistant colonies. Plasmid DNA is isolated from resistant colonies and tested for the presence of the AIM II coding fragment by restriction analysis and gel sizing.

For expression of recombinant AIM II, eukaryotic cells (eg, COS cells or CHO cells) were prepared as described above using DEAE-DEXTRAN as described in Example 3 above. Transfected with the expression vector. AIM II
Recombinant protein expression is detected by the method described above in Example 3.

2. N-Terminal Deletion Constructs (a) pG1 / ckβ8: This eukaryotic expression vector contains AIM-II mutants (Gln (60) to Val (240) in SEQ ID NO: 2) (AIM). -2 (
aa60-240) and is secreted under the direction of the human Ck-β8 signal peptide. This pG1 vector is described by Morgan, R. et al. A. Nucl
． Acids Res. 20 (6): 1293-1299 (1992) and is similar to the LN vector (Miller, AD and Ro.
sman, G .; J. Biotechniques 7: 980-990 (198).
9)), with additional cloning sites. This plasmid construction strategy is as follows. The AIM II cDNA of the deposited clone is amplified using primers containing convenient restriction sites. Suitable primers include the following primers used in this example. N underlined
otI site, a nucleotide in the coding region for the AIM II polypeptide (
SEQ ID NO: 1), and the 5'primer containing the AUG start codon has the following sequence:

[0621]

[Chemical 38] Underlined SalI site, nucleotides 753-768 in SEQ ID NO: 1
The 3'primer containing a nucleotide complementary to and a stop codon has the following sequence: 5'-GGG GTC GAC TCA CAC CAT GAA AGC C.
CC G-3 '(SEQ ID NO: 37).

The PCR amplified DNA fragment was digested with SalI and NotI,
It is then gel purified. The isolated fragment is then digested with SalI and No.
It was ligated into the tI digested vector pG1. The ligation mixture was transformed into E. E. coli and the transformed cultures are plated on antibiotic medium plates and then incubated to allow growth of antibiotic resistant colonies. Plasmid DNA is isolated from resistant colonies and tested for the presence of the AIM II encoding fragment by restriction analysis and gel sizing.

For expression of recombinant AIM II, eukaryotic cells such as COS or CHO are transfected with the above expression vector using DEAE-DEXTRAN as described in Example 3 above. Expression of AIM II recombinant protein is detected by the method described in Example 3 above.

(B) pHE4: 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 the lac repressor and is induced with IPTG or lactose. This plasmid also contains an efficient ribosome binding site, and a synthetic transcription terminator downstream of the AIM II mutant gene. This vector also contains the replication region of the pUC plasmid and the kanamycin resistance gene.

This AIM II N-terminal deletion mutant was constructed according to the following scheme.
The AIM II cDNA of the deposited clone is amplified with primers containing convenient restriction sites. Suitable primers used in this example are:
Including:

For the AIM II (Thr (70) to Val (240)) polypeptide in SEQ ID NO: 2, the underlined NdeI site and AUG start codon, AI
The 5'primer containing nucleotides 256-271 (SEQ ID NO: 1) in the coding region for the M II polypeptide has the following sequence: 5'-cgc CATATG A CCCCCCTGCCTGACG-3 '(SEQ ID NO: 42).

For the AIM II (Ser (79) to Val (240)) polypeptide in SEQ ID NO: 2, the underlined NdeI site and AUG start codon, AI
The 5'primer containing nucleotides 283 to 310 (SEQ ID NO: 1) in the coding region for the M II polypeptide has the following sequence: 5'-cgc CATAGG A GC TGGGAGCAGCTGATAC-
3 '(SEQ ID NO: 43).

AIM II in SEQ ID NO: 2 (Ser (103) to Val (240))
For polypeptides, the underlined NdeI site and AUG start codon, A
Nucleotides 355-373 (in the coding region for IM II polypeptides
The 5'primer comprising SEQ ID NO: 1) has the following sequence: 5'-cgc CATAGG AGC AGCTTGACCGGCAGCG-.
3 '(SEQ ID NO: 44).

The following 3'primers can be used to construct the N-terminal deletion described above: Underlined Asp718 site, nucleotides 753-7 in SEQ ID NO: 1.
The 3'primer containing a nucleotide complementary to 68 and a stop codon has the following sequence: 5'-cgc GGTACC TTA CACCATGAAAGCCCCG-3.
'(SEQ ID NO: 45).

This PCR amplified DNA fragment is digested with NdeI and Asp718 and then gel purified. This isolated fragment was then ligated into the appropriately digested pHE4 vector. This ligation mixture was transformed into E. E. coli and the transformed cultures are plated on antibiotic medium plates and then incubated to allow growth of antibiotic resistant colonies. Plasmid D
NA is isolated from resistant colonies and tested for the presence of the AIM II encoding fragment by restriction analysis and gel sizing.

For expression of N-terminal deleted recombinant AIM II, bacterial cells are transfected with an expression vector as described above in Example 1. AIM II
Recombinant protein expression is detected by the method as described above in Example 1.

Example 5 Biological Characterization of AIM II Polypeptides The following set of experiments provide biological characterization of AIM II proteins, and AIM II is potent in vivo and in vitro. It is demonstrated to have antitumor activity.

(A. AIM II is highly expressed in activated lymphocytes, but not highly in cancer cells.) Northern blot analysis showed that AIM II mRNA was approximately 1.9 kb in length. , And was mainly expressed in spleen, brain, and peripheral blood cells. AIM II is also partially detectable in prostate, testis, ovary, small intestine, placenta, liver, skeletal muscle, and lung. No AIM II message was detected in fetal tissue, many endocrine glands, and non-hematopoietic and bone marrow-derived tumor lines.

The RT-PCR assay was performed using AIM I in activated PBMC vs. resting PBMC.
It was done to investigate the expression of I. Fresh PBMCs containing a mixture of T cells, B lymphocytes, NK cells, monocytes, and granulocytes are consistent with Northern blot analysis.
Express IM II mRNA. Expression can be resting PBL, Jurkat cells (resting or activating) or K5 as a mixture of T cells, B cells, and NK cells.
It was not found in 62 cells. Increased expression of AIM II results in activated P
It was found in BL, CD3 +, CD4 + T cells, CD8 + tumor infiltrating lymphocytes (TIL), granulocytes, and monocytes. Further RT-PCR analysis showed that AIM II mRNA in LPS activated neutrophils and PMA stimulated U937 cells.
The presence of Interestingly, expression of AIM II was found in various cancer cell lines derived from breast, prostate or ovary (non-tumorigenic cell line MCA- derived from human mammary epithelium).
Was not detectable in (except for 1OA cells). Furthermore, AIM II expression was not found in the three breast cancer samples tested.

(B. Constitutive Expression of AIM II Caused Growth Inhibition Under Serum Starvation or IFNγ Treatment) To investigate the biological function of AIM II, the AIM II gene was cloned into the human breast carcinoma cell line MDA. -MB-231 was stably transduced with a retroviral vector. Expression of the AIM II gene in these cells was confirmed by Northern blot analysis. Furthermore, MDA-expressing the drug resistance gene Neo-
MB-231 cells were used as a control in this study. When these cells were cultured in medium containing 10% FBS, the AIM II transfectants (MDA-MB- compared to their parental or vector control transfected cells (MDA-MB-231 / Neo). 231 / AIM I
In I) no difference in growth rate in vitro was observed. However, when the serum concentration dropped to 1%, MDA-MB-231 / AIM II
There was 80% growth inhibition on cells (FIG. 4A), but not on parental or vector control MDA-MB-231 cells. Dose-dependent growth inhibition for different amounts of serum was also observed.

Wild-type MDA-MB-231 cells grew to very high densities in either 10% or 1% serum with typical pile-up characteristics (FIG. 4A). . Morphological changes were noted in MDA-MB-231 / AIM II cells, with most cells floating in the medium and maintaining a monolayer growth pattern throughout the culture. No morphological changes were found in vector control MDA-MB-231 cells. MD
Growth inhibition of AIM II expressing A-MB-231 cells was further tested in a soft agar colony assay. As shown in FIG. 4B, 80 colonies were formed.
A% reduction was found in MDA-MB-231 / AIM II cells compared to their parental or vector control cells. 25 μ / of IFNγ
Treatment with ml could also cause 80% growth inhibition of AIM II expressing MDA-MB-231 cells, whereas in parental or vector control cells there was only 20-30% inhibition. Therefore, cells expressing AIM II showed enhanced sensitivity to cytotoxicity mediated by the cytokine IFNγ.

C. Enhanced Apoptosis in AIM II Expressing Cells Annexin-V FACS analysis was performed to investigate the potential mechanism of growth inhibition of AIM II expressing cells. In the presence of 10% serum, there are less than 2% apoptotic cells in all three cell lines. Reduced serum (0.5% FBS)
After 48 hours of incubation in, the apoptotic population ratio of MDA-MB-231 cells showed a 3-fold (up to 8%) increase. There is a slight or no increase in apoptosis in parental or vector control MDA-MB-231 cells (FIGS. 5A-5C). The induction of apoptosis was further confirmed by DNA fragmentation. The fragmented DNA is AI
It was found only in MII expressing MDA-MB-231 cells, especially in 1% serum. When AIM II expressing cells were treated with Paclitaxel (taxol), there was an observation of more fragmented DNA than was seen in parental or vector control cells. Therefore, this data is AIM
II expression was MD in serum starvation or addition of IFNγ or taxol.
It suggests that it may trigger the apoptosis of A-MB-231 cells.

D. Potent In Vivo Anti-Tumor Activity of AIM II We evaluated the effect of AIM II transduction on tumor growth in vivo. AIM II expression significantly inhibited MDA-MB-231 tumorigenesis in nude mice when MDA-MB-231 cells were inoculated into a mammary fat pad, while vector control MDA-MB-231 / Neo cells. Showed no change in tumor growth compared to their parental MDA-MB-231 cells (Fig. 6A). Similar tumor suppression in MDA-MB-231 / AIM II cells was also shown in SCID mice. Histological examination of tumors from AIM II expressing MDA-MB-231 cells or from their parental or vector control cells was performed. Parental or vector control MDA-MB-231 cells formed large solid tumor masses predominantly filled with tumor cells with little or no cell infiltration. In contrast, extensive necrosis was observed even in small residual tumors formed by MDA-MB-231 / AIM II cells in nude mice. Furthermore, there is a significant increase in the number of neutrophil cell infiltrates in AIM II expressing tumors.
Based on immunohistological staining with Gr-1 mAb (PharMingen, San Diego, CA), wild type, Neo control, and AIM I.
The average number of neutrophils per mm ² tumor size (mean ± SD) in I-transduced MDA-MB-231 tumors was 101 ± 26, 77 ± 16 and 22 respectively.
It was 6 ± 38.

The inhibitory effect of AIM II on tumor suppression was further confirmed in a syngeneic mouse tumor model. Localized expression of AIM II in MC-38 mouse colon cancer cells resulted in complete suppression of tumorigenesis in 8 out of 10 C57BL / 6 mice (FIG. 6B). Local production of AIM II also dramatically prolonged survival of mice bearing MC-38 tumors. All animal experiments were repeated 3 times and similar results were obtained.

Nude mice (SC) were injected for the duration of the experiment by injection of AIM II expressing tumor cells.
No significant abnormalities (eg weight loss or liver damage) were caused in ID mice or C57BL / 6 mice). This is a locally generated AI
It is shown that M II exerted a strong antitumor effect without inducing systemic toxicity.

E. Soluble AIM II Protein Expression and Cytotoxicity To study the activity of the AIM II protein, a recombinant soluble form of the AIM II protein (sAIM II) was constructed 29 with the construct pFlag-AIM II.
Generated by transiently transfecting 3T cells. This construct encodes the extracellular domain of AIM II, but lacks the transmembrane portion of AIM II (FIG. 7A). Anti-Fla from a conditioned medium of 293T cells transduced with a single 20 kDa polypeptide (sAIM II) into pFlag-AIM II.
g monoclonal antibody. Breast cancer MDA-MB-231 cell proliferation is
It was inhibited in response to treatment of this soluble AIM II protein in a dose-dependent manner (FIGS. 7B-7C). Addition of INFγ (10 u / ml or 50 u / ml) dramatically increased the cytotoxicity of soluble AIM II protein. IFNγ alone showed little activity against MDA-MB-231 cells (Fig. 7B-
FIG. 7C). This is consistent with previous reports that MDA-MB-231 cells are resistant to single cytokines such as TNF or IFNγ treatment.

A series of normal and cancer cell lines were tested for their sensitivity to the cytotoxic effect of soluble AIM II protein at suboptimal concentrations (50 ng / ml) in the presence of 10 u / ml INFγ. . As shown in FIG. 8C, M
DA-130, MCF-7, HT-29 derived cells are susceptible to AIM II cytotoxicity, but U93T, MC3-1, SW480, MCF-10.
A-derived cells are resistant to AIM II-mediated cell killing. Of all the cell lines tested, the colon adenocarcinoma cell line HT-29 is the most sensitive with an IC ₅₀ of 1
It is less than ng / ml. HT-29 cells showed TNF, Fa in the presence of IFNγ.
It has been shown to be highly susceptible to killing s or lymphotoxin β receptors.

(F. Both LTβR and TR2 Require AIM II-Induced Growth Inhibition of Cancer Cells) AIM II was originally identified from an activated T cell cDNA library, but it was a lymphocyte cell line. Do not induce apoptosis in. RT-PCR analysis was used to show that all lymphocyte cells tested did not show expression of LTβR, whereas TR2 expression showed all these cells, especially activated Jurkat cells or PBLs.
Was found in. This is consistent with previous reports that peripheral lymphocytes do not express LTβR, but TR2 expression is associated with T cell activation.

Cell surface expression of LTβR and TR2 on a series of human cancer cells was demonstrated by LTβR
Alternatively, it was examined by FACS analysis using a monoclonal antibody against TR2. As shown in Figures 8A-8H, high levels of both receptors were found in MDA-MB-231 and HT-29 cells, while M
C3-1 cells do not express TR2 and Jurkat cells do not express LTβR. Figure 8L summarizes the surface expression of both receptors in all cell lines tested. Cell lines expressing only one of the receptors (eg Jurkat or MC3-1) are resistant to the cytotoxicity of AIM II. Taken together, these data show that AIM II-mediated growth inhibition in tumor cells is LTβR.
And the TR2 receptor may be required, while suggesting that cells expressing only one of the receptors are not sufficient to mediate cell killing.

To further demonstrate that AIM II is a related ligand for both LTβR and TR2 receptors, and is important for both receptors in AIM II-mediated tumor cell growth inhibition, Flag-tagged AIM II protein was Incubated with MDA-MB-231 or HT-29 cells, then FACS analysis was performed with anti-Flag mAb. 8I-8
Flag of MDA-MB-231 or HT-29 cells as shown in K.
Binding to the tagged soluble AIM II protein shifts positively. The specificity of binding was determined in the same cells as LTβR-Fc with soluble AIM II-flag protein.
It was further confirmed by preincubation of the fusion protein or TR2-Fc fusion protein, which effectively blocked the binding of both receptors (FIGS. 8I-8K).

LTβR and TR2 in AIM II-mediated cytotoxicity against tumor cells
The importance of both improvements was further supported by the data obtained from the in vitro proliferation assay: sAIM II-mediated cytotoxicity of HT-29 in a dose-dependent manner LTβR-Fc fusion protein or TR2-Fc fusion protein. Of the LTβR-Fc fusion protein or TR2-
The Fc fusion protein itself had no effect on cell proliferation (FIG. 8M). Furthermore, in a similar assay, sAIM II was found to
Could not bind to NFR (eg TNFRI, Fas, DR3 or DR14).

In addition, MDA-MB-231 / Wt or HT-29 cells and MDA-MB-
Co-culture with 231 / AIM II cells resulted in killing of MDA-MB-231 / Wt or wild type HT-29 cells. However, co-cultured MDA-MB-231
Conditioned medium harvested from / AIM II or MC-38 / AIM II cells showed no inhibitory effect on the growth of HT-29 cells in vitro. The results showed that the native AIM II protein could not be cleaved and secreted into the medium. Therefore, membrane-bound AIM II can be bound to MDA-MB-23
1 or functional in cells expressing appropriate surface receptors such as HT-29. Taken together, this data shows that AIM II-mediated growth inhibition of tumor cells is LTβ.
Both R and TR2 receptors may be required, suggesting that cells expressing only one of the receptors are not sufficient to mediate cell killing.

G. Effect of AIM II on Lymphocytes AIM II was originally identified from an activated T cell cDNA library but does not induce apoptosis in lymphocyte cell lines. RT-PCR analysis was used to show that all lymphocyte cells tested did not express LTβR, but T
R2 was positive on all these cells, especially activated Jurkat cells or PBLs. This is consistent with previous reports that peripheral lymphocytes do not express LTβR, but TR2 expression is associated with T cell activation.

To investigate whether membrane-bound AIM II exerts different activities on lymphocytes, a co-culture experiment of TIL1200 cells with MDA-MB-231 / AIM II cells was performed. TIL1200 expresses CD8 ⁺ (expressing high levels of Fas
995) Tumor infiltrating lymphocyte line. Membrane-bound AIM II did not induce TIL1200 apoptosis, but addition of Fas antibody resulted in 90% TIL
1200 were induced to undergo apoptosis. Similar results were obtained with fresh TIL cells or Jurkat cells.

In addition, several lymphoid cell lines and PBLs were screened for their responsiveness to soluble AIM II protein. AIM II cytotoxicity was demonstrated by Jurkat cells (either resting or CD3 mAb activation),
Not found in K562 cells, or TIL1200 (tumor infiltrating lymphocytes), PBMC (fresh or IL-2 / CD3 mAb activation) (FIG. 8L). In contrast, treatment of PBLs with sAIM II results in the activation of TR2-expressing T cells, as demonstrated by the release of IFNγ (FIG. 9).

Discussion The above experiments characterized the biological function of AIM II and its possible mechanism of action of LTβR and TR2 as novel ligands. This result demonstrates that the AIM II protein shows predominantly strong cytotoxicity in transformed tumor cells both in vitro and in vivo, but at the same time activates lymphocytes. The biological activity of AIM II in vivo and in vitro has been demonstrated by other known members of the TNF / FasL family in several ways, including binding to two distinct signaling pathways: LTβR and TR2, A
A clear distinction is made from IM II. This result indicates that the ability of AIM II expression to inhibit tumor growth was demonstrated in both xenographic (immunodeficient) and syngeneic (immune competent) models, and that T cell mediated tumor-specific responses We suggest that it may not be an essential factor for primary tumor rejection in this study.

Activation of the TNF receptor family can directly induce cell proliferation, or cell differentiation or cell death. The experiments described above show that AIM II expression resulted in growth inhibition and apoptosis in the human breast cancer cell line MDA-MB-231, associated with serum starvation or the addition of IFNγ. Induction of apoptosis appears to be the major cause for growth inhibition in vitro, as shown by Annexin-V FACS analysis and DNA fragmentation. MDA-MB-231 / LT-
The morphology and growth pattern of gamma cells suggests involvement of some loss of cell adhesion. Browning et al., Fas activation leads to rapid cell death (12-24 h), requires 24 h for the effect of TNF, and LTcX120 heterotrimer is the slowest inducer of apoptosis on HT-29 cells. (2-3
Days). In response to treatment with soluble AIM II protein, LTγ
Lysis of MDA-MB-231 and HT-29 cells expressing R and TR2 showed a similar slowing effect (ie at least 3-5 days). Substantial cell lysis does not occur with some cell lines even after 3-4 days. AI
The kinetics of action of M II is more similar to the LTαIβ2 heterotrimer.

AIM II was originally identified from a human activated T cell library by screening for sequence homology with TNF / Fas ligand and cysteine-rich motifs of the receptor superfamily. Like other TNF-related ligands, AIM II is a type II transmembrane protein with a C-terminus on the outer cell surface, a single transmembrane domain, and a short cytoplasmic tail. As expected,
Transduction of the full length cDNA of the AIM II gene results in cell surface expression of the protein, which binds to two receptors, as demonstrated by FACS analysis. Soluble AIM II protein binds to both receptors and is sufficient to induce a cytotoxic effect on target cells. However, in transwell co-culture experiments (where the two types of cells share a culture medium but are physically separate), against wild type MDA-MB-231 or HT-29 cells, AIM II expressing MDA
-No cytotoxicity derived from MB-231 cells was observed. In a direct co-culture assay, membrane-bound AIM II effectively mediated killing from close contact. Therefore, it is possible that the native AIM II protein may not be a secreted protein. By fluorescence in situ hybridization (FISH), the AIM II gene was identified as human chromosome 16, band p1.
Located at 1.2. The location of AIM II is very close to the core binding protein, sulfotransferase, syntaxin 1B, retinoblastoma binding protein 6, zinc finger protein 44, cell adhesion regulatory gene and Wilms tumor-3 gene. Other known TNF ligands (eg, TNF, LTα, and LT
The gene encoding β) is closely related on human chromosome 6 within the major histocompatibility complex (MHC), which is flanked by class III and HLA-B loci.

Both LTβR and TR2 lack the death domain. Therefore, LT
The demonstration of AIM II binding to both βR and TR2 is of interest. LTβ
R and TR2 can activate the general signaling pathway through association with the association factor for TNFR (TRAF), but AIM II-LTβR and AI
The MII-TR2 interaction can trigger different biological events. As shown in this example, expression of AIM II activates lymphocytes expressing only the TR2 receptor but induces death of cells expressing both LTβR and TR2. Signaling via LTβR activates the TRAF3-dependent pathway. In contrast, the AIM II-TR2 interaction probably resulted in TRAF (T
It excites the stimulatory response of the host immune system via RAF1, TRAF2, TRAF3 and TRAF5). This AIM II dual signaling hypothesis is further supported by the different tissue and cellular expression patterns of LTβR and TR2. LTβR is prominent in tumors and other epithelial cells, but T and B
Not present in cells. In contrast, TR2 is a resting and activated T cell, B
It is abundantly expressed at comparable levels in cells and in monocytes and granuocytes. Therefore, AIM II probably plays a crucial role such as inducing apoptosis and immune activation, and may therefore have therapeutic applications for cancer.

LTβR was originally a member of the TNFR superfamily, human 12th.
It was described as a transcriptional sequence encoded on the p chromosome. LTβR is implicated as an important element in regulating lymph node development and the cellular immune response. LTβR has been shown to be expressed in various tissues and cell lines, including tumor lines. Unlike other members of the TNFR family, LTβR
It is not expressed on either T or B lymphocytes. Recombinant LTα1
Activation of LTβR by using β2 heterotrimers or by cross-linking with immobilized antibodies leads to the death and death of adenocarcinoma cell lines even though LTβR does not contain a death domain within its cytoplasmic region. Chemokines IL-8 and RAN
Induces the production of TES.

TR2 is constitutively and relatively high in hematopoietic lineage cells expressed in multiple human tissues and including resting and activated CD4 + and CD8 + T cells, B cells, monocytes and neutrophils. Shows expression. TR2 cytoplasmic tail is Fa
It does not contain the death domain found in the s and TNFR-I intracellular domains and appears to be more related to the domains of CD40 and 4-1BB. 4-1B
Signals through B and CD40 have been shown to be costimulatory for T and B cells, respectively. The TR2-Fc fusion protein inhibited mixed lymphocyte reaction-mediated proliferation in contrast to FasL and TNF, which triggered apoptosis. All hematopoietic-derived cells tested express the TR2 receptor but are resistant to the AIM II-mediated killing observed in tumor cells. This indicates that TR2 alone does not mediate death signals. However, all the cancer cells tested expressed both LTβR and TR2, but AIM II-LT
It remains to be elucidated whether both βR signaling and AIM II-TR2 signaling contribute equally to AIM II-mediated cytotoxicity in tumor cells. We also found that soluble AIM II was DR3, DR
4 and DR5 do not bind in the in vitro binding assay, but AIM I
The possibility that I may interact with other known or unknown death receptors such as DR3, DR4 and DR5 cannot be ruled out.

The dose limiting toxicity of TNF and the cytotoxicity of FasL on T cells limits their clinical application. Treatment with AIM II could be an alternative attractive approach because AIM II triggers stimulatory rather than death signals for host immune cells that express TR2 but lack LTβR. . AI
M II presumably has the ability to selectively induce tumor cell death via LTβR and TR2, and at the same time can potentially trigger IFNγ secretion from lymphocytes via the TR2 signaling pathway. . Therefore, this model is
Not only to show that IM II is an attractive candidate for further development as an anti-cancer drug, but more importantly, to further understand the signaling pathways of members of the TNF ligand-receptor interaction. Previously defined TNF
Alternatively, a new system different from the Fas system is provided.

Methods Molecular Cloning of AIM II Full Length Genes A database containing more than 1,000,000 ESTs (expressed sequence tags) from over 500 different cDNA libraries was randomly selected for humans. Using high-throughput automated DNA sequence analysis of cDNA clones, Hum
an Genome Science Inc. And The Insitu
Produced through the combined efforts of te for Genomic Research. A sequence homology comparison of each EST was made with blastn and blastn.
The algorithm was used to run against the GenBank database, and ESTs with homology to previously identified sequences (approximately 0.01 or less) were given temporary names based on the name of the homologous sequences. Conserved amino acid sequence of the TNF / Fas ligand family against this human EST database (GLYL
A specific homology and motif search using IYSQVLF (SEQ ID NO: 46)) revealed several ESTs with> 50% homology. One clone containing GYYYYIYSKVQL (SEQ ID NO: 47) from a human activated T cell library was selected. The EST was sequenced on both strands to the 3'end. The homology was confirmed. The first clone lacks the 5'part of the gene as compared to other members of the TNF family. Nested PCR reactions were performed with two gene-specific oligonucleotides and two vector-specific primers to obtain the full length sequence. Additional 7 at the 5'end
2 nucleotides were obtained. The full length sequence is then cloned into vector pCMVport2.
0 (Life Technologies Inc., Rockville, M.
Cloned into D).

Northern Blot Analysis Human Multiple Tissue Northern Blot (Clontech, MTNblots, # 7)
759-1 and # 7760-1) were probed with ³² P-labeled AIM II full length cDNA according to the manufacturer's instructions. Blot before use 42
Hybridized overnight in hybridizol solution (Oncor) preheated at ° C, followed by 2xSSC / 0.1% SDS and 0.2xSSC at 42 ° C.
/ Washed twice in 0.1% SDS, Phosphormager ^™ (Molec
ural Dynamics Co. ) Was used for visualization.

In situ Hybridization and FISH Detection To determine the precise chromosomal location of the AIM II gene, fluorescent in situ hybridization (FISH) of single copy genes to standard human metaphase chromosome spreads was performed. , (Lawrence et al., 1988). Two
Kb cDNA was transferred to Digoxigenin-11-dUTP (Boehrin
nick translation using ger Mannheim) and FISH was performed as detailed in Johnson et al., 1991b. Individual chromosome is D
Those that were counterstained with API and color digital images and contained both gene signals detected by DAPI and Rhodamine were loaded with a triple-bandpass filter set (Chroma Technology, Inc., Brattle).
buro, VT, cooled charge coupled device camera (Photometrics, In)
c. , Tucson, AZ) and tunable excitation wavelength filters (variable)
excitement wave length filters) (Joh
Recorded using in combination with Nson et al., 1991a). Images are transferred to the ISEE software package (Invision Corp., Durham, NC).
) Was used for analysis.

Cells and Reagents Human breast cancer MDA-MB-231, subclone 2LMP was obtained from in vivo passage of MDA-MB-231 cells in athymic nude mice and used in all experiments.
MC-38 is a 1,2-dimethylhydrazine-induced mouse colon adenocarcinoma of H-2b origin. Human T lymphocyte strains Jurkat and CHO strains were obtained from American Type Culture Collection (ATCC, Rockville, MD). The human melanoma antigen gp100-reactive CD8 + T cell line TIL1200 was produced by Dr. Yu
Taka Kawakami (National Cancer Institute)
ute, Bethesda, MD). MDA-MB-23
All tumor cell lines except 1 were grown and maintained in RPMI 1640 medium containing 10% FCS. As the medium, Dulbecco's modified Eagle's medium was used as a basic medium. HLA-A-2 restricted TIL 1200 was grown in Aim-V medium containing 10% human serum and 1000 U IL-2. Apoptosis-inducing anti-Fas Mab CH-11 was added to Upstate Biotech.
It was obtained from the noology. Interferon, Biosource Inter
Obtained from national (CA).

Generation of Soluble AIM II The sequence encoding amino acids 74-240 of AIM II (ie the putative extracellular domain) was constructed in frame with the sequences encoding the preprotrypsin signal peptide and the FLAG peptide tag, vector pFLAG. Subcloned into CMV-1. The resulting construct, pFLAG-sAIM II, was transformed into recombinant s
293T cells were transfected to generate AIM II. pFLA
G. Culture medium from cells transfected with CMV-1 or pFLAG-sAIM II was treated with anti-FLAG mAb (Eastman Kodak Co.
． ) Passed through affinity column. The column eluent was fractionated by SDS-PAGE and sAIM II was detected by Western blot analysis with anti-FLAG mAb and ECL detection reagent (Amersham International).

Generation of Recombinant Receptor-Fc Fusion Protein cDNA encoding the extracellular domain of human LTβR was amplified from HepG2 cells by RT-PCR technology. The sequence of the oligonucleotide primer is: forward 5'CGGGATCCATGCTCCTGCCTTGGGGCC.
AC3 '(SEQ ID NO: 48); and reverse 5'GCGGATCCCTGGGGGC.
BTGHI restriction enzyme sites were included at each end to facilitate cloning of the PCR product into AGTGGCTCTAATGG 3 '(SEQ ID NO: 49), and the pSK + vector (Stratagene). The amplified sequences were subjected to BamHI digestion and ligated into BamHI cut pSK + vector for sequencing. The fidelity of the amplified cDNA fragment was confirmed by dideoxy DNA sequencing. To obtain the human LTβR-Fc fusion protein, the extracellular domain of LTβR was excised from the pSK + vector using BamHI restriction endonuclease and BglII cut pUC19-IgGl-Fc to be frame-ligated.
Ligated into the vector. To generate recombinant baculovirus, the fusion gene was first excised from the pUC19-IgG-Fc vector with Hpal / HindIII, followed by the SmaI cut pBacPAK9 vector (Clontech Co.
． ), And then linearized into Sf9 cells BacPAK6
Co-transfected with DNA (Clontech Co.). Insect Sf2 infected with recombinant virus to obtain recombinant soluble LTβR fusion protein
Supernatants cultured for 5 days from 1 cell were filtered and captured on Protein A Sepharose beads, then bound sLTβR protein was bound to glycine buffer (pH 3.
0) and subsequently dialyzed in PBS. The production of TR2-Fc fusion protein has been described.

(Production of LTβR Antibody and TR2 Antibody) Balb / cJ Mouse (The Jackson Laboratory, B
ar Harbor, ME) was immunized with the LTβR-Fc fusion protein in Freund's adjuvant. Mice were boosted 3 times, then splenocytes were
In the presence of 50% polyethylene glycol in EPES (PEG 1500, Boe
the human myeloma NS-1 cells at Hringer Mannheim), followed by PRMI1640 / HAT and RPMI1640 / HT selection medium (
Boehinger Co. ). Supernatants from positive wells were tested by ELISA for their ability to bind LTβR-Fc fusion protein but not human IgG. Hybridomas producing antibodies against the LTβR-Fc fusion protein were cloned by three limiting dilutions. To produce large amounts of mAb, 10 ⁷ hybridoma cells were injected intraperitoneally into pristane-treated Balb / c mice, and the mAb was subsequently purified from ascites by affinity chromatography. Similarly, the TR2-GST fusion protein was used to raise monoclonal antibodies against TR2 and screened by an ELISA assay.

In Vitro Proliferation Assay Cells (5,000 cells / well) were plated in triplicate in 24-multiwell tissue culture plates with IMEM in the presence of either 10% FBS or 1% FBS. . The number of viable cells on day 3, 5 or 7 was determined by trypan blue exclusion method. The cells were replaced with fresh medium every 2 days during this time course.

A soluble tetrazolium / formazan (XTT) assay for cell growth was performed in 96-well plates. Cells (2,000-4,000 cells / well)
Were grown in IMEM medium with 10% FBS or 1% FBS. 4-5
After the day of culture, XTT (1.0 mg / ml and 1.53 mg / ml PMS) was added to each well and incubated at 37 ° C. for 4 hours. Absorbance at 450 nm was measured with a Dynatech Model MR700.

FACS Analysis Cells were harvested by trypsinization or aspiration and centrifuged at 1500-2000 rpm for 5 minutes. The cell pellet was resuspended and washed twice with 5 ml ice cold PBS. The cells were then treated with binding buffer (10% BSA, 20 mM HEP).
ES (pH 7.2), 0.02% NaN ₃ and 25 μg / ml normal rat I
g of HBSS) for 30 minutes at 4 ° C. with 10 μg / ml Flag
-Incubated with tagged AIM II protein or Abs. Cells were then washed and stained with 20 μg / ml phycoerythrin (PE) conjugated to goat anti-mouse IgG as described. Soluble LTRβR-Fc fusion protein, TR2-Fc, to compete for cell surface binding
Was preincubated with AIM II for 30 minutes before addition to cells at 10 μg / ml. FAC scan flow cytometer (Becton Dic
Kinson, Mountain View, CA).

For the apoptosis assay, the cell pellet was diluted 1: 100 in Annex.
in V-FITC (Trevigen, Gaithersburg, MD) and 1 × binding buffer (10 mM H 2) containing 50 μg / ml propidium iodide.
EPES (pH 7.4), 0.15M NaCl, 5 mM KCl, 1 mM M
Resuspended in gCl ₂ , 1.8 mM CaCl ₂ ) and incubated at 4 ° C. for 15 minutes. Annexin V-FITC and propidium iodide fluorescence of each cell was analyzed by flow cytometry (Coulter).

Retroviral Transduction of Tumor Cells Retroviral vectors were used to stably transduce tumor cells with the AIM II gene. To construct a plasmid encoding AIM II, AI
The 1.9 kd Not I / Sal I fragment containing the M II cDNA was inserted into the parental plasmid pG1SamEN. This retroviral backbone is derived from the Moloney murine leukemia virus, and the AIM II gene is a Moloney (M
lane) was under the transcriptional control of long terminal repeats derived from murine leukemia virus. Generation of retroviral packaging strains has been previously described (Markowit.
z et al.). Briefly, 30 μg of pG1SamEN-AIM II DNA was added 2 ×.
Used to transfect a mixture of 10 ⁵ PA317 amphotropic packaging strain and 3 × 10 ⁵ GP + E86 ecotropic packaging strain.
After 2 weeks of selection, a high titer G418 resistant PA317 clone was then selected to regenerate the packaging strain PA-AIM II and used for gene transfer into tumor cells. A control retrovirus producing strain, PA-neo, was also used. These packaging strains were grown for 20 hours, retroviral supernatants were harvested and added to wild type, MDA-MB-231 or MC-38 75% confluent flasks, respectively. AIM II expressing MDA-MB-23 after transduction with a recombinant retrovirus encoding human AIM II
1 or MC-38 cells were selected with the neomycin analog, G418, and M
DA-MB-231 / AIM II or MC-38 / AIM II were designed respectively. AIM II expression in these tumor cells was confirmed by Northern blot analysis. MDA-MB-231 / AIM II, vector control strain, M
All stable transfectants, including DA-MB-231 / neo, MC-38 / AIM II and the vector control strain, MC-38 / neo, were grown and grown at 1.5 mg / ml and 0.375 mg / ml. Each G41
Maintained in the presence of 8.

Jurkat Cell Co-Culture Assay MDA-MB-231 cells were plated in 6-well tissue culture plates and grown to confluence. After removal of medium and washing of the monolayer with 1 × PBS, 1 × 10 ⁶ Jurkat cells (non-adherent) were placed on the monolayer or 1 m on empty wells.
Plated in 1 RPMI medium. Wells with MDA-MB-231 cells alone (without overlaying Jurkat cells) were maintained as a further control. After 24 or 48 hours of culture, the non-adherent phase of the mixed culture was collected from 6 wells plated after gentle rocking of the plate and assayed for viability using trypan blue exclusion. For detection of apoptosis, Anne
A xin V-FITC FACScan flow cytometer was used to measure 20,000 cells per sample.

Lymphokine Release Assay The lymphokine release assay was as described previously (Zhai et al.) In AIM I.
I was performed to detect human PBL reactivity. Briefly, human PBL cells were incubated for 5 days in the presence of anti-CD3 mAb (0.1 μg / ml) and rlL-2 20 U / ml and various concentrations of AIM II protein, and the supernatants were collected. , And IFNγ secretion by R & D Systems (Minneap
(Olis, MN) and determined using an ELISA kit.

(Tumor forming ability study) Female athymic Ncr-nu nude mice (6 weeks old) were subjected to Frederick C.
ancer Research and Development Center
, National Institute of Health (Freder
ich, MD) and Charles River Laboratories
(Raleigh, NC). Female C57BL / 6 mouse (6-7 weeks old)
Harlan Sprague Dawley (Indianapolis,
IN) and MDA-MB-231 cells (1 × 10 ⁶ ) were injected into the mammary fat pad of female athymic nude mice on day 0. Similarly, MC-38 cells from C58BL / 6 mice were injected. Subcutaneous (sc) injection in the adjacent area. Mice were then ear-tagged and randomized. Tumor size was assessed in a blinded fashion by measuring vertical diameter twice a week with calipers. Each treatment group consisted of 10 animals and the experiment was repeated 3 times. Histological examination of tumors was performed using H / E staining.

Example 6 Detection of AIM II Expression by BIAcore Analysis CHO cells were treated with AIM II-Flag tag expression vector or BAP-Fl.
Transfected with either ag (negative control). Three days after transfection, AIM II expression was analyzed by the BIAcore device (BIAcore,
Inc. ), Which allows a real-time measurement of the protein binding phenomenon for the immobilized AIM II receptor, lymphotoxin-β receptor (BIAcore sensorgram detects binding by a change in refractive index at the surface of the flow cell). To). The lymphotoxin-β receptor-Fc fusion protein was covalently immobilized to the BIAcore flow cell via the amine group using N-ethyl-N ′-(dimethylaminopropyl) carbodiimide / N-hydroxysuccinimide chemistry. Various dilutions of AIM II-Frag and negative control (BAP-Flag) conditioned serum-free medium were added to lymphotoxin-β.
-Receptor-derivatized flow cell was applied at 5 μl / min for a total dose of 50 μl. The amount of binding protein was adjusted to HBS buffer (10 mM HEPES (pH 7
． 4), 150 mM NaCl, 3.4 mM EDTA, 0.005% Sur
Determined after flow cell washing with factor P20). The surface of the flow cell was regenerated by displacement of bound protein by washing with 20 μl of 10 mM HCl.

Specific binding to the lymphotoxin-β-receptor was determined by AIM II-Fla.
No significant binding was observed for the negative control (BAP-Flag) conditioned medium, although detection was done with up to 10-fold conditioned medium dilutions from the g culture.
This demonstrates that AIM II-Flag binding is specific for the lymphotoxin-β-receptor and not the Fc portion of this fusion protein. Furthermore, specific receptor binding by the AIM II-Flag protein indicates that this binding represents a native construct such that it is secreted by the cell. Therefore, this BIAcore-based assay can be used to detect the expression of AIM II from conditioned media and other biological fluids. In addition, by using known amounts of pure AIM II protein, this assay can be developed into a quantitative assay for determining AIM II concentration.

Example 7 Activation-Induced Apoptosis Assay Activation-induced apoptosis is assayed using SupT-13 T leukemia cells and measured by cell cycle analysis. The assay is performed as follows. SupT-13 cells were treated with 10% F in logarithmic growth (approximately 1 × 10 ⁶ ).
Maintain in RPMI with CS. Sup-T13 cells at 0.5 × 10 ⁶ / m
1, seed wells of a 24-well plate at 1 ml / well. AIM II
Protein (0.01, 0.1, 1, 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 sterile filtered at 0.05 ml / well in the presence or absence of anti-CD3 antibody at 0.05 M / well.
Purified BC3 m at a concentration of 10 μg / ml in bicarbonate buffer (pH 9.5)
Prepared by incubating Ab, or buffer alone. The plate is incubated overnight at 4 ° C. The wells of the antibody-coated plate are washed 3 times with sterile PBS at 4 ° C. AIM II treated Sup-T13 cells are transferred to antibody coated wells and incubated at 37 ° C for 18 hours. Apoptosis is measured by cell cycle analysis using propidium iodide and flow cytometry. Proliferation of treated cells is measured by taking a total volume of 300 μl in each treated well and delivering to triplicate wells of a 96-well plate (100 μl / well). To each well, add 20 μl / well ³ H thymidine (0.5 μCi / 20 μl, 2 Ci / mM) and incubate at 37 ° C. for 18 hours. The cells are harvested and the ³ H-thymidine incorporation by the cells is counted. This measurement is used to confirm the effect on apoptosis if observed otherwise. The positive control for this assay is anti-CD3 crosslinking alone. In addition, anti-fas monoclonal antibody (soluble form-500 ng / ml in IgM mAb) has been used to demonstrate marked and reproducible apoptosis in this strain. The negative control for this assay is medium or buffer alone. In addition, another anti-CD3 mAB (OK
Crosslinking at T3) shows no effect.

If an effect is observed by cell cycle analysis, TUN the cells to flow cytometry or with Annexin V, a technique well known to those of skill in the art.
Further stain for EL assay.

Example 8: CD3-Induced Proliferation Assay The CD3-induced proliferation assay is performed on PBMC and measured by ³ H-thymidine incorporation. The assay is performed as follows. 96 well plates with 100 μl / well of mAb for CD3 (HIT3a, Phar
Mingen), or an isotype-matched control mAb (B33.1).
) Overnight at 4 ° C. (1 in 0.05M bicarbonate buffer, pH 9.5).
μg / ml), then washed 3 times with PBS. PBMC from human peripheral blood F
/ H gradient isolated and added to quadruplicate wells (5 x 10 ⁴ / well) of mAb coated plates in RPMI containing 10% FCS and P / S in the presence of different concentrations of AIM II protein. Add (total volume, 200 μl).
Buffers of related proteins alone and medium alone are controls. 48 hours,
After incubation at 37 ° C., the plates were spun down at 1000 rpm for 2 minutes, and if an effect on growth was observed, 100 μl of supernatant was removed and IL-2 (
Or other cytokines) were stored at -20 ° C. 0.5 well
Supplement with 100 μl of medium containing μCi ³ H-thymidine and incubate at 37 ° C.
Incubate for ~ 24 hours. Wells are harvested and ³ H-thymidine incorporation is used as a measure of proliferation. Anti-CD3 alone is a positive control for proliferation. IL-2 (100 U / ml) is also used as a control to enhance proliferation. A control antibody that does not induce T cell proliferation was used as a negative control for CD3-induced proliferation, and medium or buffer was used for AIM.
Used as a negative control for the effect of protein II.

Example 9: Effect of AIM II on MHC Class II, Co-stimulatory and Adhesion Molecule Expression, and Cell Differentiation of Human Dendritic Cell-Derived Monocytes, Dendritic Cells Seen in Peripheral Blood Produced by expansion of proliferating precursors: Adherent PBMC or eluted monocyte fractions are cultured with GM-CSF (50 ng / ml) and IL-4 (20 ng / ml) for 7-10 days. To do. These dendritic cells have the characteristic phenotype of immature cells (CD1, CD80, CD86,
Expression of CD40 and MHC class II antigens). Treatment with activators such as TNF-α results in rapid changes in surface phenotype (MHC class I and II, increased expression of costimulatory and adhesion molecules, downregulation of FcγRII, upregulation of CD83. ). These changes correlate with increased antigen presenting capacity of dendritic cells and functional maturation.

FACS analysis of surface antigens is performed as follows. Cells with various concentrations of AIM
-II (0.1, 1, 10, 100, 1000 ng / ml) or treated with LPS as a positive control for 1-3 days, washed with PBS containing 1% BSA and 0.02 mM sodium azide, then 4 Incubate for 30 minutes at 0 ° C. with a 1:20 dilution of the appropriate FITC- or PE-labeled monoclonal antibody. After further washing, labeled cells were washed with FACScan (Bec
Ton Dickinson) is analyzed by flow cytometry.

Effect on cytokine production Cytokines produced 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 Th1 helper T cell immune response and induces cytotoxic T cell function and NK cell function. The ELISA is used to measure the release of IL-12 as follows. Dendritic cells (10 ⁶ / ml) were added to AIM-II (0.1, 1, 10, ¹ ).
00, 1000 ng / ml) for 24 hours. LPS (100 ng / ml)
Is added to the cell culture as a positive control. Supernatants from cell cultures are then harvested and analyzed for IL-12 content using a commercially available ELISA kit. Use the standard protocol provided in the kit.

Effects on expression of MHC class II, costimulatory molecules and adhesion molecules Three major families of cell surface antigens can be identified on monocytes: adhesion molecules,
Molecules involved in antigen presentation, and Fc receptors. Modulation of expression of MHC class II antigens and other costimulatory molecules (eg, B7 and ICAM-1) can result in altered monocyte antigen presenting capacity and ability to induce T cell activation. Elevated Fc receptor expression may be correlated with improved monocyte cytotoxic activity, cytokine release and phagocytosis.

FACS analysis is used to test surface antigens as follows. Monocytes were treated with various concentrations of AIM-II (0.1, 1, 10, 100, 1000 ng / ml) or LPS (positive control) for 1-5 days with 1% BSA and 0.
． Wash with PBS containing 02 mM sodium azide, then incubate for 30 minutes at 4 ° C. with 1:20 dilution of the appropriate FITC- or PE-labeled monoclonal antibody. After further washing, the labeled cells are F
Analyze by flow cytometry on ACSScan (Becton Dickinson).

Effect on Monocyte Survival Human peripheral blood monocytes gradually diminish viability when cultured in the absence of serum or other stimulants. Their death is an internally regulated process (apoptosis)
caused by. Adding activators such as TNF-α to the culture dramatically improves cell survival and prevents DNA fragmentation.

Propidium iodide staining is used to measure apoptosis as follows. Monocytes (10 ⁷ / ml) in the presence or absence of TNF-α (100 ng / ml, positive control) or AIM-II (0.1, 1, 10, 100, 1000 ng / ml) for 2 days. , Suspend and culture in a polypropylene tube containing DMEM. Cell viability is assessed by propidium iodide (PI) staining. Cells are suspended in PBS containing PI at a final concentration of 5 μg / ml at a concentration of 2 × 10 ⁶ / ml and then incubated for 5 minutes at room temperature before FACScan analysis. PI uptake was demonstrated to correlate with DNA fragmentation in this experimental paradigm.

Effect on Cytokine Release An important function of monocytes / macrophages is their regulatory activity on other cell populations of the immune system through the release of cytokines after stimulation. E for measuring IL-1β release
Run LISA as follows. Human monocytes were plated at 10 ⁶ / m in a 48-well plate
1 and added at various concentrations of AIM-II (0.1, 1, 10, 100, 1,
000 ng / ml) in the presence or absence of 100 ng / ml LPS. After 24 hours of incubation, supernatants are collected and assayed for the presence of cytokines by an ELISA kit. Use the standard protocol provided in the kit.

Example 10: Affinity purification of soluble AIM II for N-terminal sequence analysis Previous data showed that BIAcore chips derivatized with lymphotoxin β receptor (LTβR) -Fc fusion protein were labeled with AIM II (a. 74-2
40) -Flag fusion protein was shown to be able to specifically bind (Example 5).
, Section E and Figure 7A). Then, to determine which cell line should be used for further purification for N-terminal sequence analysis, LTβR BIAc
The ore chip was used to detect the expression of soluble AIM II protein from the conditioned medium of non-Flag tagged AIM II stable transfectants.

CHO cells were transfected with an expression construct (pC4 vector) consisting of the extracellular domain of AIM II (amino acids 60-240) fused to the ck-β8 signal peptide. Clones were selected for high expression by growth in medium containing methotrexate. The clone with the highest amount of binding to the LTβR BIAcore chip was further amplified. Conditioned medium (20 mL) from CHO 11 (high level AIM II producing clone) was obtained. Second A encoding full-length cDNA
The IM II construct was transfected into mouse MCA-38 cancer cells and subjected to selection with G418. Conditioned medium was obtained from these transfected MCA-38 cells.

Conditioned medium from stable transfectants (CHO 11 or MCA-38 cells) was filtered and centrifuged at 10,000 × g, then loaded onto an MCIF-Fc affinity column (control column) followed by , LTβR-Fc affinity column (0.2 mL bed volume). 0.005 this column
Washed with several bed column volumes of HEPES buffered saline containing 10% surfactant P-20. Bound proteins were eluted with 10 mM HCl (3 x 0.5 mL fractions) and immediately neutralized with TRIS buffer. Fractions eluted from the LTβR column retained binding to the LTβR BIAcore chip, while fractions eluted from the control MCIF-Fc column were negative for binding. The eluted fractions were dried in the Speedvac and then resuspended in 20 μL water. Aliquots of the eluted protein were analyzed by reducing SDS-PAGE gel and detected by silver staining. Bands of approximately 25 kDa and 21 kDa specifically bound to the LTβR column derived from CHO-11 and MCA-38 cell lines were detected. The remaining eluate was subjected to SDS-PAGE and blotted onto PVDF membrane for analysis of N-terminal sequences.

The N-terminus of the AIM II molecule purified from MCA-38 cells started at residue 83 in the putative extracellular region of the molecule (Table 3). AIM I derived from CHO-11
The I results also confirmed that this protein corresponds to the AIM II protein; the N-terminus has two sequences starting 3 residues apart (AIM starting at residue 60).
II extracellular domain followed by ck-β8 signal peptide).
Table 3). Thus, the naturally processed form of AIM II should correspond to residues 83-240 and have a molecular weight of 17,284 daltons. The apparent electrophoretic mobility of approximately 21 kDa is consistent with glycosylation as evidenced by the presence of some electrophoretic species. Similarly, CK-11 expressed ck-β8 / AIM
The apparent molecular weight of the II fusion protein of approximately 25 kDa was higher than the molecular weight predicted from its sequence (20,361). Again, this may be due to glycosylation of the protein (there is one N-glycosylation site at residue 104 of full length AIM II).

[0690]

[Table 3] FIG. 1 shows a sensorgram of the specificity of binding of MCA-38 AIM II conditioned medium to LTβR-Fc versus MCIF-Fc immobilized on BIAcore chip.
2 shows. Conditioned media was analyzed on a BIAcore instrument flow cell derivatized with lymphotoxin β receptor Fc fusion protein. Conditioned medium (100 μL
) Was flushed onto the chip at 5 μL / min and washed with HBS buffer at 5 μL / min. The data shown represent the net off-rate area of the plot after binding of AIM II to the immobilized receptor and are measured in relative mass units (RU) against time. The binding conditions are diffusion limited conditions (diffusio
n-limited condition) and high receptor chip density. Legend: LTβR-Fc and MCIF-Fc are LTβR-F, respectively.
It refers to the binding data from the chip surface of BIAcore on which Fc and MCIF-Fc are immobilized.

The determination of LTβR binding by AIM II eluted from the LTβR-Fc column is shown in FIG. LTβR and MCIF refer to binding data from the chip surface of BIAcore on which LTβR-Fc or MCIF-Fc is immobilized, respectively. Undiluted conditioned medium from MCA38 cells was passed through the affinity column (pre: pre) as well as after passing through the MCIF-Fc affinity column (pre).
post-MCIF) and after passing through the LTβR-Fc affinity column (p
ost-LTβR). LTβR (E4-6) and MCIF-Fc (
Fractions (1 mL) eluted from the E1-3) affinity column were diluted 3-fold and tested for LTβR BIAcore binding to the chip.

Example 11: AIM II in the Treatment of Adjuvant-induced Arthritis in Rats
Effect of AIM II analysis of the use of AIM II to treat rheumatoid arthritis (RA) is performed through the use of an adjuvant-induced arthritis model (AIA) in rats. AIA is a well characterized and reproducible animal model of rheumatoid arthritis, which is well known to those of skill in the art (Pearson et al., Ann.
Rheum. Dis. 15: 379 (1956)); Pearson et al., Art.
christis Pheum. 2: 440, (1959)). AIM II is expected to inhibit an increase in angiogenesis or an increase in RA during endothelial cell proliferation that is required to maintain the invasive pannus of bone and cartilage observed in this animal model. Lewis and BB rats (Charles Live
r Lab, Raleigh, N .; C. And University of M
assachusetts Medical Center, Worceste
r, available from MA) is used as a general and responsive strain to adjuvant-induced arthritis in these experiments.

The onset of the arthritic condition was determined by the addition of 0.1 ml adjuvant (5 mg / ml in the base of the tail).
) Is intradermally injected. Groups of 5-6 rats received 0.1-10 mg / kg AIM II or vehicle intra-articularly 20 days after injection of adjuvant. At this point, chronic arthritis has reached its maximum level and chronic pannus formation has begun. The effect of AIM II on chronic arthritis
aurog and co-workers essentially as described (J. Exp.
Med. 162: 962, (1985)), radiologically analyzed 15 days after adjuvant challenge, once a week. Briefly, rats are anesthetized with either ether or chloral hydrate and placed so that both hindlimbs are X-radiated together. X-ray films are blindly tested using a 0-3 scoring system for periosteal reaction, bone erosion, joint space narrowing and destruction. If there is a significant amount of joint damage in vehicle-treated rats,
These animals are sacrificed. At this point, the paws are histologically evaluated for the relative degree of tissue damage and the therapeutic effect AIM II exerted on these joints.

Finally, AIM II-treated and vehicle-treated animals undergo clinical evaluation twice per week to assess hindpaw volume using the Plethysmometer system and body weight.

Example 12: AIM in treating collagen-induced arthritis in mice
Effect of II An analysis of the use of AIM II to treat rheumatoid arthritis (RA) is performed through the use of collagen-induced autoimmune arthritis (CIA) in mice. CIA is another well characterized and reproducible animal model of rheumatoid arthritis, which is well known to those of skill in the art (Courtenay et al.,
Nature 283: 666 (1980); Wooley et al. Exp. M
ed. 154: 688 (1981); Holmdahl et al., Immunol. R
views 118: 193 (1990)). AIM II is expected to induce apoptosis and inhibit synovial cell proliferation required to form invasive pannus in bone and cartilage observed in this autoimmune animal model of rheumatoid arthritis and RA.

DBA / 1 Lac J mice (Jackson Lab (Bar Harb
or ME,) is used as the most ubiquitous chain of collagen-induced arthritis in these experiments.

Onset of arthritic condition is induced by intradermal injection of bovine type II collagen (1 mg / ml) in 0.1 ml complete Freund's adjuvant in the base of the tail. After 3 weeks, animals are injected with 40 μg LPS to accelerate the progression of arthritis. Groups of 10 mice received 0.1-1 mg 7-15 days after injection of LPS.
/ Kg AIM II or vehicle received intradermally or intraarticularly. At this point, chronic inflammation was expected to reach maximal levels, and chronic pannus formation had just begun. The effect of AIM II on chronic arthritis was scored as follows: 0
= Normal, 0.5 = increased number, 1 = total paw increase, 2 = deformity and 3 = ankylosis, and monitored and analyzed. If it is determined that a specific amount of ankylosis occurs in the paws of vehicle-treated rats, the animals are sacrificed and the paws histologically examined for relative degrees of pannus formation, cartilage and bone destruction. It was evaluated and, for this reason, AIM II elicited these bindings.

Example 13 TR6 Suppresses AIM II (LIGHT, HVEM-L) -Mediated Apoptosis TR6 (DcR3) is a novel member of the tumor necrosis factor receptor (TNFR) family. The following studies show that AIM II is a ligand for TR6.

Background Tumor necrosis factor (TNF) family members are members of a wide variety of biological activity modulators (eg, cell proliferation, differentiation, cell survival, cell death, cytokine production, lymphocyte co-stimulation, immunoglobulins). Regulation of secretion and isotype conversion) (
Armitage, R.M. Curr. Opin. Immunol. 6: 407-
413 (1994); Tewari, M .; And Dixit, V .; M. , Cur
r. Opin. Genet. Dev. 6: 39-44 (1996). Receptors in this family share a common structural motif in their extracellular domain of multiple cysteine-rich repeats of about 30-40 amino acids (Gruss, HJ and Dower, SK. , Bloom
d 85: 3378-3404 (1995)). TNFR1, CD95 / Fas
/ APO-1, DR3 / TRAMP / APO-3, DR4 / TRAIL-R1 /
The APO-2, DR5 / TRAIL-R2 and DR6 receptors contain a conserved intracellular motif of 30-40 amino acids called the death domain and are involved in the activation of apoptotic signaling pathways and other members Containing the low sequence identity of NF-κB and stimulates the transcription factors NF-κB and AP-1 (Armita
ge, R.G. Curr. Opin. Immunol. 6: 407-413 (19
94); Tewari, M .; And Dixit, V .; M. Curr. Opin
． Genet. Dev. 6: 39-44 (1996); Gruss, H .; -J.
And Dower, S .; K. , Blood 85: 3378-3404 (199).
5)).

Most TNF receptors contain functional cytoplasmic domains and include TNFR1 (Loetsher, H. et al., Cell 61: 351-3.
56 (1990); Schall, T .; J. Et al., Cell 61: 361-37.
0 (1990)), TNFR2 (Smith, CA et al., Science).
248: 1019-1023 (1990)), lymphotoxin β receptor (L
TβR) (Baens, M. et al., Genomics 16: 214-218 (
1993)), 4-IBB (Kwon, BS and Weissman, SS.
M. , Proc. Natl. Acad. Sci. U. S. A. 86: 1963-
1967 (1989)), HVEM / TR2 / ATAR (Kwon, BS, et al., J. Biol. Chem. 272: 14272-14276 (1997); M.
ontgomery, R.A. I. Et al., Cell 87: 427-436 (1996).
); Hsu, H .; Et al., J. Biol. Chem. 272: 13471-1347
4 (1997)), NGFR (Johnson, D. et al., Cell 47: 545).
-554 (1986)), CD27 (Van Lier, RA, et al., J. Chem.
Immunol. 139: 1589-1596 (1987)), CD30 (Du
rkorp, H .; Et al., Cell 68: 421-427 (1992)), CD4.
0 (Banchereau, J. et al., Cell 68: 421-427 (199).
4)), OX40 (Mallett, S. et al., EMBO J. 9: 1063-1).
068 (1990)), Fas (Itoh, N. et al., Cell 66: 233-).
243 (1991)), DR3 / TRAMP (Chinnayan, AM.
Et al., Science 274: 990-992 (1996)), DR4 / TRA.
IL-RI (Chinnayan, AM et al., Science 274: 9).
90-992 (1996)), DR5 / TRAIL-R2 (Pan, G. et al.,
Science 277: 815-818 (1997)), and RANK (A
nderson, D.N. M. Et al., Nature 390: 175-179 (199
7)). Some members of the TNFR superfamily (eg, osteoprotegerin (OPG)) (S
immunonet, W.M. S. Et al., Cell 89: 309-319 (1997)).
Has no cytoplasmic domain and is not secreted or is a glycophospholipid tail (eg TRID / DcR1 / TRAIL-R3 (Degli-Epost
i, M. A. Et al., J. Exp. Med. 186: 1165-1170 (1997)
); Sheridan, J .; P. Et al., Science 277: 818-821.
(1997)) to the membrane. Soluble TNFR (eg SFV-T
2 (Smith, CA et al., Science 248: 1019-1023 (
1990)), Va53 (Howad, ST et al., Virology 180).
: 633-647 (1991) et al., G4RG (Hu, FQ. Et al., Virolo.
gy 204: 343-356 (1994)), and crmB (Gruss,
H. -J. & Dower, S.S. K. , Blood 85: 3378-3404 (
A viral open reading frame encoding (1995)) has also been identified.

By searching the expressed sequence tag (EST) database, TNFR
A novel member of the superfamily was identified, designated TR6, and characterized as a soluble cognate ligand for AIM II (also referred to as LIGHT) and FasL / CD95L. AIMII (LIGHT
) And FasL mediate apoptosis, which is the most common physiological form of cell death and occurs during embryogenesis, tissue remodeling, immune regulation and tumor regression.

AIM II (LiGHT) is highly induced in activated T lymphocytes and macrophages. AIM II (LIGHT) is HVEM / TR2 and LTβR (Mauri, DN et al., Immunity 8: 21-30 (1).
998)). HVEM / TR2 is a receptor for herpes simplex virus type I (HSV-1) entry into human T lymphoblasts. Soluble form of HVEM / TR2-Fc and HVEM / TR2
Antibodies against were shown to inhibit mixed lymphocyte responses, suggesting a role for this receptor or its ligand in T lymphocyte expansion (Kw).
on, B.I. S. Et al., J. Biol. Chem. 272: 14272-14276
(1997); Mauri, D .; N. Immunity 8: 21-30 (
1998); Harrop, J .; A. Et al., J. Immunol. 161: 178
6-1794 (1998)). The level of LTβR expression is prominent in epithelial cells but absent in T and B lymphocytes. Signaling via LTβR induces cell death in some line cancers (Browning, J. L.
Et al., J. Exp. Med. 183: 867-878 (1996)). AIM II (LIGHT) produced by activated lymphocytes can cause immunomodulation from hematopoietic cells that express only HVEM / TR2 and induce apoptosis of tumor cells, which is LTβR and HVEm /. Expresses both TR2 receptors (Zhai, Y. et al., J. Clin. Invest. 102: 114).
2-1152 (1998); Harrop, J. et al. A. Et al., J. Biol. Che
m. 273: 27548-27556 (1998)).

FasL is one of the major effectors of cytotoxic T lymphocytes and natural killer cells. It also relates to the establishment of peripheral tolerance in activation-induced cell death of lymphocytes. Furthermore, expression of FasL in non-lymphoid cells and tumor cells contributes to maintaining immune privilege of tissues by preventing invasion of Fas-sensitive lymphocytes (Nagata, S., Cell 88: 355-365 ( 1997
)). FasL is also promoted and reduced from the surface of human cells (Sc
hneider, P.H. Et al., J. Exp. Med. 187: 1205-1213 (
1998)).

The following experiments demonstrate that a novel member of the TNFR superfamily, TR6 (DcR3), binds AIM II (LIGHT) and FasL. Therefore, TR6 (DcR3) is expected to act as an inhibitor in AIM II (LIGHT) -induced tumor cell death by blocking the interaction of AIM II with its receptor.

Identification and Cloning of Novel Members of the TNFR Superfamily The EST cDNA database (obtained from more than 600 different cDNA libraries) was cloned using the BLASTN and TBLASTN algorithms.
We screened for sequence homology with cysteine-rich motifs of the NFR superfamily. Three EST clones whose amino acid sequences contained identical open reading frames showing specific homology to TNFR-II were identified from human normal prostate and pancreatic tumor cDNA libraries. A full length TR6 cDNA clone encoding an intact N-terminal signal peptide was obtained from a human normal prostate library.

RT-PCR Analysis For RT-PCR analysis, total RNA was treated with PMA / ionomycin or LP.
Before and after stimulation with S, Trizol (GIBCO) was used to isolate from various human cell lines. RNA is converted to cDNA by reverse transcription and P
35 cycles were amplified by CR. The primers used for amplification of TR6 fragment follow the sequence of TR6. β-actin was used as an internal control for RNA integrity. PCR products were run on a 2% agarose gel, stained with ethidium bromide and visualized by UV irradiation.

Recombinant Protein Production and Purification Recombinant TR6 protein was produced with hexahistidine at the C-terminus. TR6- (His), which encodes the entire TR6 protein, was amplified by PCR. For properly oriented cloning, the forward primer (5'-AGAC
CCAAGCTTCCCTGCTCCAGCAAGGACCATG-3 ′) (SEQ ID NO: 60) at the 5 ′ end with a HindIII site and a reverse primer (5
'-AGACGGGATCCTTAGTGGGTGGTGGTGCACAGGGA
BGAHi at the 5'end of GGAAGCGCTC-3 ') (SEQ ID NO: 61)
The site was created. The amplified fragment is cut with HindIII / BamHI and the mammalian expression vector (pCEP4) (Invitrogen)
Cloned into. The TR-6 (His) / pCEP4 plasmid was added to the recombinant T
HEK 293 EBNA cells were stably transfected to produce R-6 (His). Serum-free culture medium from cells transfected with TR6- (His) / pCEP4 was passed through a Ni column (Novagen).
The column eluate was fractionated by SDS-PAGE and TR6- (His)
Was detected by Western blot analysis with anti-poly (His) ₆ antibody (Sigma).

Production of HVEM / TR2-Fc, LTβR-Fc and Flag-tagged soluble AIM II (sAIM II, sLIGHT) fusion proteins has been previously described (Zhai, Y. et al., J. Clin. Invest. 102). : 1142-
1151 (1998)). The supernatant containing the Fc fusion protein was filtered and trapped on Protein-G Sepharose beads. Flag tagged sAIM I
The I (sLIGHT) protein was purified using an anti-flag mAb affinity column.

(Immunoprecipitation) TR6- (His) was bound to binding buffer (150 mM NaCl, 0.
1% NP-40, 0.25% gelatin, 50 mM HEPES, pH 7.4.
Flag-ligands and anti-Fl of the TNF superfamily in
Incubated overnight with ag agarose and then precipitated. The conjugated protein was lysed by 12.5% SDS-PAGE and detected by Western blot with HRP-conjugated anti-poly (His) ₆ or anti-human IgG1 antibody.

Cell Binding Assay For cell binding assay, HEK 293 EBNA cells were treated with AIM II.
(LIGHT) cDNA was stably transfected using the pCEP / full sequence or the calcium phosphate method using only the pCEP4 vector. Hygrom
After selection with ycinB, cells were harvested with 1 mM EDTA in PBS and TR6- (His), HVEM / TR2-Fc, or LTβR-.
Incubated with Fc for 20 minutes on ice. To detect Fc fusion proteins, cells were stained with FIFT-conjugated goat anti-human IgG. To detect TR6 binding, cells were serially stained with anti-poly (His) ₆ and FITC-conjugated goat anti-mouse IgG. FACscan (Becto
n Dickinson).

Cytotoxicity Assay A cytotoxicity assay using HT29 cells has been previously described (Browni).
ng, J.N. L. Et al., J. Exp. Med. 183: 867-878 (1996).
). Briefly, 5000 HT29 cells were treated with 1% FBS, DME.
96-well plates with M and sAIM II (sLIGHT
T) (10 ng / ml) and 10 units / ml of human recombinant interferon-γ (IFN-γ). Serial dilutions of TR6- (His) were added to microtiter wells in quadruplicate. IFN-γ and sAIM
Cells treated with II (sLIGHT) were incubated with various amounts of TR6- (His) for 4 days, after which [ ³ H] thymidine was added and cultured for 6 hours. Cells were harvested and thymidine incorporation was determined using a liquid scintillation counter.

Results and Discussion (TR6 is a novel member of the TNFR superfamily) TR6 was identified by searching the EST database. Three clones containing an identity open reading frame were identified from a human normal prostate and pancreatic tumor cDNA library. A full length TR6 cDNA encoding an intact N-terminal signal peptide was obtained from a human normal prostate library.
The TR6 open reading frame encodes 300 amino acids. To determine the N-terminal amino acid sequence of mature TR6, hexahistidine-tagged TR
6 was expressed in a mammalian cell expression system and the N-terminal amino acid sequence was determined by peptide sequencing. Processed mature TR6- (His)
The N-terminal sequence of A starts from amino acid 30 and shows that the first 29 amino acids make up the signal sequence (FIG. 14A). Therefore, the mature protein of TR6 was composed of 271 amino acids with no transmembrane region. There is one potential N-linked glycosylation site (Asn173) in TR6. (OPG) (Simone
t, W. S. Et al., Cell 89: 309-319 (1997)), this predicted protein is a soluble, secreted protein, and recombinant TR6 expressed in mammalian cells was expressed on polyacrylamide gels. It was about 40 kD as estimated. FIG. 14B shows TNFR-I, TNFR-II, 4-
1 shows potential cysteine-rich motifs aligned in 1BB, TR2 / HVEM, LTβR, TR1 / OPG and TR6. TR6 contains two complete cysteine-rich motifs and two incomplete cysteine-rich motifs,
And its amino acid sequence was remarkably similar to the TR1 / OPG amino acid sequence. TR6 has about 30% sequence homology with OPG and TNFR-II.

MRNA Expression The expression of TR6 mRNA in human multiple tissues was analyzed by Northern blot hybridization. Northern blot analysis shows that TR6 mRNA is approximately 1.3 kb long and is predominantly expressed in lung tissue and the colorectal cancer cell line SW480. RT-PCR analysis was performed on TR6 in various cell lines.
Was performed to determine the expression pattern of This expression was then induced on activation signals in stimulated Jurkat T leukemia cells. Interestingly, TR
6 mRNA was constitutively expressed at high levels in an endothelial cell line (HUVEC).

Identification of Ligands for TR6 To identify ligands for TR6, some Flag-tagged soluble proteins of TNF ligand family members were bound to recombinant TR6- (His) proteins by immunoprecipitation. Screened to embody. TR
6- (His) is AIM II (LIGHT) -Flag and tested Fl
The FasL-flag between the ag-tagged soluble TNF ligand members (ie T
RANCE, TL-3, TL-6, TL-7, 4-1BBL, AIM II and FasL). The results show that TR6 binds at least two ligands, AIM II (LIGHT) and FasL. AI
M II (LIGHT) shows specific sequence homology with the C-terminal receptor binding domain of FasL (31%), whereas sAIM II (sLIGHT) shows FasL (31%)
(Mauri, DN, et al., Immunity 8: 21-30.
(1998); Zhai, Y .; Et al., J. Clin. Invest. 102: 11
42-1151 (1998)). They may have similar binding epitopes for TR6 binding.

Previously, Zhai and Harrop (Zhai, Y. et al., J. Clin. I
nvest. 102: 1142-1151 (1998); Harrop, J .; A
． Et al., J. Biol. Chem. 273: 27548-27556 (1998).
), Has reported the biological function of AIM II (LIGHT) and its possible mechanism of action as a ligand for HVEM / TR2 and / or LTβR. AIM II (LIGHT) is expressed in activated T cells. In association with serum starvation or addition of IFN-γ, AIM II (LIGHT) shows cell proliferation in tumor cells, MDA-MB-231 and HT29.

TR6 may interact with HVEM / TR2 or LTβR with AIM II (
A) to determine whether it acts as an inhibitor for A.
TR6- (His) was used as a competitive inhibitor in the IM II (LIGHT) -HEVEN / TR2 interaction. AIM II (LIGHT) to T
When immunoprecipitated with HVEM / TR2-Fc in the presence of R6- (His), HVEM / TR2-Fc bound to AIM II (LIGHT) is TR6-.
TR6- (His) binding to AIM II (LIGHT) was competitively decreased by (His), but not changed by HVEM / TR2-Fc. Furthermore, conjugation of HVEM / TR2-Fc (6nM) or LTβR (6nM)
It was completely inhibited by 20 nM TR6- (His) protein in the immunoprecipitation assay. These results indicate that TR6 has HVEM / TR2 and LTβR
Supports the intention to act as a potent inhibitor of AIM II (LIGHT) function.

Binding of TR6- (His) to AIM II (LIGHT) Transfected Cells Determines whether TR6 binds to AIM II (LIGHT) expressed on cell surface membranes. For this purpose, the binding assay was performed by flow cytometry using HIM293EBNA cells transfected with AIM II (LIGHT). AIM II (LIGHT) HEK293EBNA cells
Stained significantly with R6- (His) as well as HVEM / TR2-Fc, LTβR-Fc. No binding was detected in HEK 293 EBNA cells transfected with the pCEP4 vector with any fusion protein (
15A-15C). Furthermore, the control isotope is AIM II (LIGHT
TEK-transfected HEK 293 EBNA cells did not bind, and none of the fusion proteins described above bound the vector-transfected cells, confirming the specificity of these bindings. These bonds are linked by TR6
We show that M II (LIGHT) can bind to both soluble and membrane bound forms.

TR6 Inhibits AIM II (LIGHT) Derivatized Cytotoxicity to HT29 Cells Browning et al., (J. Exp. Med. 183: 867-878 (19).
96)), while Fas activation leads to rapid cell death (12-24 hours),
It has been shown that LTβR takes 2-3 days in inducing apoptosis for a colorectal cancer cell line (HT29). Zhai, Y. Et al., J. Clin. In
vest. 102: 1142-1151 (1998) and Mauri, D .; N
． Et al., Immunity 8: 21-30 (1998) also lead to the death of cells expressing both LTβR and HVEM / TR2, although LTβR or HVEM.
It did not lead to the death of cells expressing only the / TR2 receptor. HVE
Both M / TR2 and LTβR are associated with AIM II in HT29 cells (LIGHT
) Cooperatively with vector killing (Zhai, Y. et al., Supra).

To determine whether TR6 conjugation inhibits AIM II (LIGHT) -mediated cytotoxicity, HT29 cells were treated with 200 ng / ml of LTβR-Fc or TR6- (His). 10 ng / ml of sAIM II (sLIG
HT) and IFN-γ (10 U / ml). As shown in Figure 16A, TR6- (His) blocked most of the total AIM II (LIGHT) mediated cell killing. Cells were also treated with different concentrations of TR6- (His)
Was incubated with sAIM II (sLIGHT) and / or IFN-γ in the presence of TR6- (His) dose-dependently blocked sAIM II (sLIGHT) -induced cell death (FIG. 16B). Taken together, TR6 may act as a natural inhibitor of AIM II (LIGHT) -induced tumor cell death and may contribute to immune evasion by the tumor.

Interaction of HVEM / TR2 and / or LTβR with AIM II (LIGHT) is associated with different biological events (eg, T cell proliferation, HVEM dependent HSV).
1 blocking of infection, and antitumor activity) (Mauri,
D. N. Et al., Immunity 8: 21-30 (1998); Zhai, Y. et al.
Et al., J. Clin. Invest. 102: 1142-1151 (1998);
Harrop, J .; A. Et al., J. Biol. Chem. 273: 27548-2
7556 (1998)). TR6 is thought to act as an inhibitor of AIM II (LIGHT) interaction and also plays a different role depending on the cell type. Indeed, TR6 (DcR3) has been shown to bind FasL and is believed to contribute to immune evasion by certain tumors (P
itti, R .; M. Et al., Nature 396: 699-703 (1998)).
. TR6 (DcR3) is thought to act as a decoy receptor, and is the AIM II (LIGHT) -mediated apoptotic pathway or FasL, respectively.
It is believed to contribute to immune evasion in both slow and rapid tumor cell death mediated by the mediated apoptotic pathway.

Another possibility is that TR6 is associated with membrane bound FasL or AIM II (LIG
HT) and may act as a cytokine to induce FasL or A
Signals can be directly converted via IM II (LIGHT). Recently, a group of Desbarats and Suzuki is, FasL is obtained by converting a signal by itself, CD4 ^- in T cells leads to arrest and cell death of the cell cycle but, CD8 ^- in T cells reported to lead to cell proliferation Did (Desba
rats, J. et al. Et al., Nature Medicine 4: 1377-1382.
(1998); Suzuki, I .; And Fink, P .; J. J. Exp. M
ed. 187: 123-128 (1998)). Therefore, TR6 may act as a cytokine for signaling via FasL and AIM II (LIGHT).

HUVEC cells constitutively expressed TR6 in RT-PCR analysis.
AIM II (LIGHT) and FasL are known to be expressed in activated T cells. Therefore, TR6 and its ligands are thought to be important for the interaction between activated T lymphocytes and endothelium. TR6 may be involved in activated T cell trafficking as well as endothelial cell survival.

Therefore, a new soluble member of the TNFR superfamily, TR6, was identified. TR6 is constitutively expressed in lung tissue, tumor cells and endothelial cells. TR6 ligands appear to be AIM II (LIGHT) and FasL, which are involved in the cell death pathway. TR6 is AIM II (LI
GHT) and FasL specifically, and AIM II (LIGHT
) And FasL activity. Similar to DcR1, DcR2 and another soluble member of the TNFR superfamily, OPG, TR6 is thought to act as an inhibitor of signaling through the TNF family members FasL and AIM II (LIGHT). Therefore, TR6
It is believed to have an important role in inhibiting apoptosis and tumor regulation. Example 14: Isolation of antibody fragments against a polypeptide of the invention from a library of scFvs The naturally occurring V-gene isolated from human PBLs, with or without donor exposure. Construction into large libraries of antibody fragments containing good reactivity to polypeptides of the invention (eg US Pat. No. 5,885,7).
No. 93, which is incorporated herein by reference in its entirety).

Library Rescue A scFv library is constructed from human PBL RNA as described in WO 92/01047. To rescue the phage displaying the antibody fragment, about 10 ⁹ E. coli carrying the phagemid were rescued. 50 ml using E. coli
Of 2 × TY (containing 1% glucose and 100 μg / ml ampicillin) (2 × TY-AMP-GLU) and 0.8 OD with shaking. D
． Grow to. Using 5 ml of this culture, 50 ml of 2xTY-AMP-
GLU was inoculated, 2 × 10 ⁸ TU of Δ gene 3 helper phage (M13Δ gene III, see WO92 / 01047) was added, and the culture was incubated for 45 minutes at 37 ° C. without shaking. , Then incubate at 37 ° C for 45 minutes with shaking. 400 this culture for 10 minutes
0r. p. m. Centrifuge at 37 ° C., and pellet the pellet with 2 liters of 2 × TY (100
ug / ml ampicillin and 50 ug / ml kanamycin)) and grow overnight. Phages are prepared as described in WO92 / 01047.

M13Δ gene III is prepared as follows: M13Δ gene III helper phage does not encode gene III protein. Therefore, phage displaying antibody fragments (phagemids) have greater binding avidity for antigen. During phage morphogenesis, infectious M13Δ gene III particles are made by growing helper phage in cells carrying the pUC19 derivative that supplies the wild-type gene III protein. The culture is incubated for 1 hour at 37 ° C without shaking and then for another hour at 37 ° C with shaking. Pellet cells (IEC-Centra 8,400
revs / min for 10 minutes) and 100 μg ampicillin and 1 / ml
2 x TY broth containing 25 μg kanamycin per ml (2 x TY-AM
P-KAN) in 300 ml and grown overnight at 37 ° C. with shaking. Phage particles were purified and concentrated from the culture medium by two PEG precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS, and 0.4.
A 5 μm filter (Minisart NML; Sartorius) was passed through to give a final concentration of about 10 ¹³ transducing units (ampicillin resistant clones) per ml.

(Paning of Library) 100 mg / ml or 10 mg / ml of Immunotubes (Nunc) was used.
Coat overnight in PBS with 4 ml of any of the polypeptides of the invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at 37 ° C and then washed 3 times in PBS. Approximately 10 ¹³ TU of phage are applied to the tube and incubated for 30 minutes at room temperature with rocking up and down and then left for a further 1.5 hours. Tube with PBS 0.1% Twee
Wash 10 times with n-20 and 10 times with PBS. The phage were eluted by adding 1 ml of 100 mM triethylamine and spinning up and down for 15 minutes on a rotating disc, then adding 0.5 ml of 1.0 M Tris-HCl, to the solution.
Immediately neutralize at pH 7.4. Next, the eluted phages were incubated with the bacteria at 37 ° C.
Phage was used to incubate 10 ml of mid-log phase E. E. coli TG1. Then E. E. coli is plated on TYE plates containing 1% glucose and 100 μg / ml ampicillin. The resulting bacterial library is then rescued with Δ gene 3 helper phage as described above to prepare phage for the next round of selection. This process is then repeated for all four rounds of affinity purification, increasing the tube wash to PBS, 0.1% Tween-20 20 times and 20 times to PBS 3 and 4 times.

Characterization of Binders Phage eluted from the third and fourth round of selection were used to transform E. coli. coli H
B 2151 is infected and soluble scFv are generated from a single colony for the assay (Marks et al., J. Mol. Biol. 222: 581-59).
7 (1991)). The ELISA is carried out using microtiter plates coated with either 10 pg / ml of the polypeptide of the invention in 50 mM bicarbonate, pH 9.6. Positive clones in the ELISA are further characterized by PCR fingerprinting (see, eg, WO92 / 01047) followed by sequencing.

Example 15: Method for Determining Changes in the AIM II Gene RNs from whole family or individual patients presenting with the phenotype of interest (eg, disease).
Isolate A. CDNA is then generated from these RNA samples using protocols known in the art (see Sambrook et al.). This cDNA is then used as a template for PCR with primers surrounding the region of interest in SEQ ID NO: 1. Suggested PCR conditions are Sidra
nsky, D.A. Et al., Science 252: 706 (1991) using 95 ° C for 30 seconds; 52-58 ° C for 60-120 seconds; and 7
It consists of 35 cycles of 60-120 seconds at 0 ° C.

The PCR product was then transferred to SequiTherm Polymerase (Ep
Sequencing is performed using a primer labeled with T4 polynucleotide kinase at the 5 ′ end. AIM II
Also determined intron-exon boundaries of selected exons of
The CR product is analyzed to confirm the result. The PCR product with the suspected mutation in AIM II is then cloned and sequenced to confirm the results of direct sequencing.

The AIM II PCR product was prepared as described in Holton, T .; A. And Graham,
M. W. , Nucleic Acids Research, 19: 1156 (
1991) and cloned into the T-tail vector and sequenced with T7 polymerase (United States Biochemical). Affected individuals are identified by mutations in AIM II that are not present in unaffected individuals.

Genomic rearrangement is also observed as a method to determine alterations in the AIM II gene. Genomic clones isolated using techniques known in the art were cloned into digoxigenin deoxy-uridine 5'-triphosphate (Boehringer Manhe).
im) and nick translation, and Johnson, C. et al. ,
Methods Cell Biol. FISH is performed as described in 35: 73-99 (1991). For specific hybridization to the AIM II locus, a large excess of human cot-1 DNA is used to hybridize with the labeled probe.

Chromosomes are counterstained with 4,6-diamino-2-phenylidole and propidium iodide, producing a combination of C and R bands. Aligned images for accurate mapping can be displayed on a cooled charge coupled device camera (Photometri).
cs, Tucson, AZ) and variable excitation wavelength filter combined triple band filter set (Chroma Technology, Brattle)
eboro, VT) (Johnson, Cv. et al., Genet. A).
nal. Tech. Appl. , 8:75 (1991)). ISee Grap
hikal Program System (Inovision Corp)
image acquisition, analysis, and chromosome segment length measurement using the S. Oration, Durham, NC). AIM I (hybridized by probe)
Chromosomal changes in the genomic region of I are identified as insertions, deletions and translocations. These AIM II changes are used as diagnostic markers for related diseases.

Example 16: Method of Detecting Aberrant Levels of AIM II in Biological Samples AIM II polypeptides can be detected in biological samples, and AIM
If elevated or decreased II levels are detected, then the polypeptide is a marker of a particular phenotype. It is understood that there are numerous detection methods, and therefore one of skill in the art can modify the following assays to suit their particular needs.

For example, an antibody sandwich ELISA is used to detect AIM II in a sample, preferably a biological sample. The wells of a microtiter plate are coated with a specific antibody against AIM II at a final concentration of 0.2-10 μg / ml. The antibody is either monoclonal or polyclonal and is produced using techniques known in the art. The wells are blocked so that non-specific binding of AIM II to the wells is reduced.

The coated wells are then RT washed with AIM II containing samples.
Incubate for> 2 hours. Preferably, serial dilutions of the sample should be used to confirm the results. The plate is then washed 3 times with deionized or distilled water to remove unbound AIM II.

Next, 50 μl of the specific antibody-alkaline phosphatase conjugate was added to 25-400 n.
Add at a concentration of g and incubate for 2 hours at room temperature. Plates are washed again three times with deionized or distilled water to remove unbound conjugate.

75 μl of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate (NPP) substrate solution was then added to each well and incubated at room temperature for 1 hour to allow cleavage of substrate and fluorescence. to enable. Fluorescence is measured with a microtiter plate reader. A calibration curve is generated using experimental results from serial dilutions of control samples, and sample concentration is plotted on the X-axis (log scale) and fluorescence or absorbance on the Y-axis (linear scale). Then, based on the measured fluorescence of this sample, an AI in the sample was prepared using a calibration curve.
Interpolate M II polypeptide concentration.

Example 17: Method of Treating Elevated Levels of AIM II The present invention relates to a method for treating an individual in need of reducing the level of biological activity of AIM II in the body, the method comprising: Is a therapeutically effective amount of AIM
The step of administering to such an individual a composition containing or consisting of the II antagonist. A preferred antagonist for use in the present invention is an AIM II-specific antibody.

It is further understood that conditions caused by reduced normal or normal expression levels of AIM II in an individual can be treated by administering AIM II, preferably in soluble and / or secreted form. Accordingly, the present invention also provides a method of treating an individual in need of increased levels of AIM II polypeptide. The method includes in such an individual an amount of AIM II that increases the level of biological activity of AIM II in such an individual, or this amount of AI.
Administering a pharmaceutical composition comprising M II.

For example, a patient with reduced levels of AIM II polypeptide receives the polypeptide at a daily dose of 0.1-100 μg / kg for 6 consecutive days. Preferably, the polypeptide is in soluble and / or secreted form.

Example 18: Method of Treating AIM II Level Decrease The present invention also relates to a method for treating an individual in need of increasing the level of AIM II biological activity in the body, comprising: The method comprises a therapeutically effective amount of AI.
Administering to such an individual a composition comprising or consisting of M II or an agonist thereof.

Antisense technology is used to inhibit the production of AIM II. This technique is one example of a method of reducing the level of AIM II polypeptides, preferably soluble and / or secreted forms of AIM II polypeptides, that are due to various etiologies such as cancer.

For example, in patients diagnosed with abnormally elevated levels of AIM II, antisense polynucleotides were administered at 0.5, 1.0, 1.5, 2.0 and 3.0 mg / kg per day. For 21 days. If well tolerated, the procedure is repeated after a 7 day washout period.

Example 19 Treatment Methods Using Gene Therapy—Ex Vivo One method of gene therapy is to implant fibroblasts into a patient that are capable of expressing soluble and / or mature AIM II polypeptides. . Generally, fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue culture medium and divided into small pieces. Place the nodule of tissue on the wet surface of the tissue culture flask,
Place approximately 10 pieces in each flask. The flask is inverted, closed tightly, and left at room temperature overnight. After 24 hours at room temperature, the flask is inverted, the tissue mass remains fixed at the bottom of the flask and fresh medium (eg Ham's F12 medium containing 10% FBS, penicillin and streptomycin) is added.
The flask is then incubated at 37 ° C for approximately 1 week.

At this point, fresh medium is added and then replaced every few days. After culturing for another two weeks, a monolayer of fibroblasts appears. Trypsinize the monolayer and scale up to a larger flask.

[0746] Moloney murine sarcoma virus is flanked by long terminal repeats of pMV-7 (Ki
rschmeier, P .; T. Et al., DNA, 7: 219-25 (1988)) is digested with EcoRI and HindIII and then treated with calf intestinal phosphatase. The linear vector is fractionated on an agarose gel and purified using glass beads.

The cDNA encoding AIM II can be amplified using PCR primers corresponding to the coding sequences at the 5'and 3'ends, respectively. Preferably, the 5'primer contains an EcoRI site and the 3'primer contains a HindIII site. Equal amounts of Moloney murine sarcoma virus linear backbone and amplified Ec
The oRI and HindIII fragments are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under suitable conditions for ligating the two fragments. The ligation mixture was then used to transform E. E. coli HB101 is transformed. It was then plated on agar containing kanamycin for the purpose of confirming that the vector had the AIM II inserted correctly.

Amphoteric pA317 or GP + am12 packaging cells were
Dul with 10% Calf Serum (CS), Penicillin and Streptomycin
Grow to confluent density in tissue culture in becco modified Eagles medium (DMEM). The MSV vector containing the AIM II gene is then added to the medium and the packaging cells are transduced with the vector. At this time, the packaging cells produce infectious virus particles containing the AIM II gene (here, the packaging cells are referred to as producer cells).

Fresh medium is added to the transduced producer cells and then the medium is added to 10c.
Harvest from m-plate confluent producer cells. Spent media containing infectious viral particles is filtered through a Millipore filter to remove detached producer cells, which is then used to infect fibroblasts. Media is removed from the subconfluent plates of fibroblasts and quickly replaced with media from producer cells. This medium is removed and replaced with fresh medium. If the virus titer is high, virtually all fibroblasts will be infected and no selection is required. If the titer is very low, neo or hi
It is necessary to use a retroviral vector with a selectable marker such as s. Once the fibroblasts were efficiently infected, the fibroblasts were analyzed and the AI
Determine if M II protein is being produced.

The engineered fibroblasts are then transplanted into the host either alone or after being grown to confluence on Cytodex 3 microcarrier beads.

Example 20: Method of Treatment Using Gene Therapy-In Vivo Another aspect of the invention is the use of in vivo gene therapy methods to treat disorders, diseases, and conditions. This gene therapy method is used to increase or decrease the expression of AIM II polypeptide in order to leave naked nucleic acid (DNA, RNA) in animals.
, And antisense DNA or RNA) AIM II sequences. AIM II polynucleotides are AIs derived from a promoter or target tissue.
It may be operably linked to any other genetic element required for the expression of M II polypeptide. Techniques and methods for such gene therapy and delivery are known in the art, eg, WO90 / 11092, WO98 / 11779; US Pat. Nos. 5,693,622, 5,705,151, 5,580,859; Ta.
bata H. Et al., Cardiovasc. Res. 35: 470-479 (1
997); Chao J. et al. Et al., Pharmacol. Res. 35: 517-5
22 (1997); Wolff J. et al. A. , Neuromuscul. Diso
rd. 7: 314-318 (1997); Schwartz B .; Et Gene
Ther. 3: 405-411 (1996); Tsurumi Y. et al. Et Ci
See recirculation 94: 3281-3290 (1996), which is incorporated herein by reference.

AIM II polynucleotide constructs can be delivered by any method that delivers an injectable substance to cells of an animal, such as injection into the interstitial space of a tissue (heart, muscle, skin, lung, liver, intestine, etc.). Can be delivered. The AIM II polynucleotide construct can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

The term “naked” polynucleotide, DNA or RNA refers to any delivery vehicle (viral sequence, viral particle, liposome formulation, lipofectin, or (Including a precipitating agent) is not included. However, AIM II polynucleotides can also be prepared in liposome formulations (eg, Felgne) that can be prepared by methods well known to those of skill in the art.
r P. L. Et al., Ann. NY Acad. Sci. 772: 126-139 (
1995) and Abdallah B. Et al., Biol. Cell 85: 1-
7 (1995).

The AIM II polynucleotide vector construct used in this method of gene therapy is preferably one that does not integrate into the host genome and does not contain sequences that allow replication. Any strong promoter known to those of skill in the art can be used to drive the expression of DNA. Unlike other gene therapy techniques, one major advantage of introducing a naked nucleic acid sequence into a target cell is the transient nature of polynucleotide synthesis in that cell. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide for the production of desired polypeptides for periods of up to 6 months.

The AIM II polynucleotide construct is used to determine the tissues in animals (muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney,
(Including the gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue). The interstitial space of this tissue is either the intercellular fluid, the mucopolysaccharide matrix (between the reticulum fibers of organ tissue, the elastic fibers in the walls of blood vessels or chambers, the collagen fibers of fibrous tissue), or the connective tissue sheath. Includes the same matrix within natural muscle cells or in the hiatus of bone. This is likewise the space occupied by circulating plasma and lymphatic fluid of the lymphatic channels. Delivery of muscle tissue to the interstitial space is preferred for the reasons discussed below. They can be conveniently delivered by injection into the tissue containing these cells. They are preferably delivered to and expressed in differentiated, persistent, non-dividing cells, which delivery and expression can be effected on undifferentiated cells or cells that are not fully differentiated (eg, blood stem cells). Or skin fibroblasts). In vivo, muscle cells are particularly qualified for their ability to take up and express polynucleotides.

For naked AIM II polynucleotide injections, an effective dosage of DNA or RNA is in the range of about 0.05 μg / kg body weight to about 50 mg / kg body weight.
Preferably, this dosage is about 0.005 mg / kg to about 20 mg / kg, and more preferably about 0.05 mg / kg to about 5 mg / kg.
Of course, as the skilled artisan will appreciate, this dosage will vary according to the tissue site of injection. Suitable and effective dosages of nucleic acid sequences can be readily determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration. The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used including, for example, inhalation of aerosol formulations, especially for delivery to the lungs or bronchial tissues, throat, or mucous membranes of the nose. In addition, naked AI
The M II polynucleotide construct can be delivered to the artery during angioplasty by the catheter used in this procedure.

The dose response effect of infused AIM II polynucleotides in muscle in vivo is determined as follows. MRN encoding AIM II polypeptide
Appropriate AIM II template DNA for the production of A is prepared according to standard recombinant DNA methodologies. This template DNA, which can be either circular or linear, is either used as naked DNA or complexed with liposomes. Mice are then injected into the quadriceps with various amounts of template DNA.

To 5-6 week old female and male Balb / C mice, 0.3 ml of 2.5%
Anesthetize by intraperitoneal injection of Avertin. A 1.5 cm incision is made in the anterior thigh and the quadriceps are visualized directly. AIM II template DNA was added to 0.
Inject into 1 ml of carrier through a 27 cc needle with a 1 cc syringe for 1 minute into the knee at a distance of about 0.5 cm from the distal insertion site of the muscle at a depth of about 0.2 cm. Sutures are made over the injection site for future localization and the skin is closed with stainless steel clips.

After a suitable incubation time (eg 7 days), muscle extracts are prepared by cutting out the entire quadriceps muscle. 15 μm sections of every 5 quadriceps muscles,
Histochemical staining for AIM II protein expression. The time course for AIM II protein expression can be done in a similar manner except that quadriceps from different mice are harvested at different times. AIM II in muscle after injection
DNA persistence can be determined by Southern blot analysis after preparation of total cellular DNA and HIRT supernatants from injected and control mice. The results of the above experiments in mice can be used to estimate appropriate doses and other treatment parameters in humans and other animals using AIM II naked DNA.

Example 21 Gene Therapy Using the Endogenous AIM II Gene Another method of gene therapy according to the present invention is the use of an endogenous AIM II sequence, eg
US Pat. No. 5,641,670 (filed June 24, 1997); International Publication No. W
O 96/29411 (published September 26, 1996); International Publication No. WO 94
/ 12650 (published August 4, 1994); Koller et al., Proc. Na
tl. Acad. Sci. U. S. A. 86: 8932-8935 (1989)
And Zijlstra et al., Nature, 342: 435-438 (198).
Operably linked to the promoter via homologous recombination, as described under 9). The method involves activation of a gene that is present in the target cell but is not expressed in the cell or is expressed at a lower level than desired. A polynucleotide construct containing a promoter and target sequence is made. This target sequence is homologous to the 5'non-coding sequence of the endogenous AIM II sequence, which is flanked by promoters. The target sequence is 5 of the AIM II sequence.
Close enough to the'end so that the promoter is operably linked to the endogenous sequence upon homologous recombination. PCR of this promoter and target sequence
Can be used to amplify. Preferably, the amplified promoter contains different restriction enzyme sites on the 5'and 3'ends. Preferably, 3 of the first target sequence
The'end contains the same restriction enzyme sites as the 5'end of the amplified promoter, and the 5'end of the second target sequence contains the same restriction sites as the 3'end of the amplified promoter.

The amplified promoter and amplified target sequence are digested with the appropriate restriction enzymes followed by treatment with calf intestinal phosphatase. The digested promoter and digested target sequence are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions suitable for ligation of these two fragments. The construct is size fractionated on an agarose gel, then purified by phenol extraction and ethanol precipitation.

In this example, the polynucleotide construct is administered as a naked polynucleotide via electroporation. However, the polynucleotide construct may also be administered with a transfection facilitating agent such as liposomes, viral sequences, viral particles, precipitating agents and the like. Such delivery methods are known in the art.

Once the cells have been transfected, homologous recombination will occur and this endogenous AI
This results in a promoter operably linked to the M II sequence. This results in the expression of AIM II in this cell. Expression can be detected by immunological staining or any other method known in the art.

Fibroblasts are obtained from a subject by skin biopsy. The obtained tissue is DMEM +
Place in 10% fetal bovine serum. Exponentially growing or early stationary phase fibroblasts are trypsinized and rinsed from the surface of the plastic with nutrient medium. An aliquot of cell suspension is removed for counting and the remaining cells are subjected to centrifugation. The supernatant is aspirated and the pellet is mixed with 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM KCl).
, 0.7 mM Na ₂ HPO ₄ , 6 mM dextrose). The cells are recentrifuged, the supernatant is aspirated, and the cells are acetylated bovine serum albumin 1
Resuspend in electroporation buffer containing mg / ml. This final cell suspension contains approximately 3 × 10 ⁶ cells / ml. Electroporation should be performed immediately after resuspension.

Plasmid DNA is prepared according to standard techniques. For example, to construct a plasmid for targeting to the AIM II locus, plasmid pUC1
8 (MBI Fermentas, Amherst, NY) is digested with HindIII. CMV promoter with XbaI site at 5'end and B at 3'end
Amplify by PCR with an amHI site. Two AIM II non-coding sequences are amplified via PCR: one AIM II non-coding sequence (AIM II fragment 1) is amplified with a HindIII site at the 5'end and an XbaI site at the 3'end; the other. The AIM II non-coding sequence of AIM II (AIM II fragment 2) is amplified with a BamHI site at the 5'end and a HindIII site at the 3'end. This CMV promoter and AIM II fragment (1
And 2) with the appropriate enzymes (CMV promoter-XbaI and BamHI;
AIM II fragment 1-XbaI; AIM II fragment 2-Bam
Digested with HI) and ligated together. The resulting ligation product is digested with HindIII and ligated with the HindIII digested pUC18 plasmid.

Plasmid DNA was added to a sterile cuvette (B
io-Rad). Final DNA concentration is generally at least 120μ
g / ml. Then 0.5 ml of the cell suspension (containing approximately 1.5 × 10 ⁶ cells) is added to the cuvette and the cell suspension and DNA solution are gently mixed. Electroporation is performed using a Gene-Pulser instrument (Bio-Rad). The capacitance and voltage are set to 960 μF and 250-300 V, respectively. Increasing the voltage reduces cell survival, but dramatically increases the proportion of viable cells that stably integrate the introduced DNA into their genome. Considering these parameters, the pulse time is about 14 ~
20mSec should be observed.

The electroporated cells are kept at room temperature for about 5 minutes, then the contents of the cuvette are gently removed using a sterile transfer pipette. The cells were placed in a 10 cm dish containing pre-warmed nutrient medium (D containing 15% bovine serum).
MEM) 10 ml directly and incubate at 37 ° C. The next day, the medium was aspirated and replaced with 10 ml of fresh medium and an additional 16-24
Incubate for hours.

The engineered fibroblasts are then injected either into the host, either alone or after being grown to confluence on cytodex 3 microcarrier beads. To do. here,
The fibroblasts produce the protein product. The fibroblasts can then be introduced into a patient as described above.

Example 22: Modification of T cell-mediated immunity in a tumor and host-versus-graft disease model via the AIM II costimulatory pathway [Introduction] T lymphocytes proliferate and further develop an immune response. Often two distinct signals are required for differentiation into effector cells that mediate (Schw
artz, R .; H. , Cell 71: 1065-1068 (1992)). T
In addition to antigen signals delivered by CR, costimulatory receptors on T cells and their ligands or counter-receptors (c) on professional antigen presenting cells (APCs)
The interaction with the interreceptor is essential for optimal activation and survival of T cells. Soluble CTLA4-Ig or anti-B7 monoclonal antibody (
Blockage of the B7-CD28 costimulatory pathway by mAb) inhibits autoimmune disease, rejection of transplanted organs and progression of graft versus host disease (GVHD) (Cross).
, A. H. Et al., J. Clin. Invest. 95: 2783-2789 (19
95); Lenschow, D .; J. Et al., Science 257: 789-7.
92 (1992); Blazzar, B .; R. Et al., Blood 83: 3815-.
3825 (1994)). On the other hand, B7 gene transfer or stimulation of costimulatory signals by the agonist mAb to 4-1BB can enhance the anti-tumor immune response (Chen, L. et al., Cell 71: 1093-1102 (1992); Me.
lero, I. Et al., Nat. Med. 3: 682-685 (1997)). These studies indicate that costimulatory interactions play an important role during the induction of immune responses. More importantly, the costimulatory pathway can be manipulated for therapeutic purposes.

AIM II, referred to as LIGHT, which shows homology to lymphotoxin, inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes, is also a member of the TNF cytokine family ( Mauri, DN et al., Immunity 8: 21-30 (
1998)), this mRNA is activated in the majority of peripheral blood mononuclear cells (PB).
MC) (Mauri, DN et al., Immunity).
8: 21-30 (1998); Zhai, Y .; Et al., J. Clin. Invest
． 102: 1142-1151 (1998)). Two cell receptors,
HVEM (ATAR, TR2) and LTβR are found to bind AIM II with high affinity (Mauri, DN et al. Immunity 8:
21-30 (1998)). HVEM expression can be detected in the majority of hematopoietic cells, including T cells, B cells and NK cells, and weakly in endothelial cells (Harrop, JA et al., J. Immunol. 161: 178).
6-1794 (1998); Kwon, B .; S. Et al., J. Bio. Chem. Two
72: 14272-14276 (1997)). In contrast, LTβR is not found in T and B cells, but is expressed at high levels in monocytes and stromal cells (Browning, JL et al., J. Immunol. 159:
3288-3298 (1997)). AIM II induces apoptosis in several tumor lines in vitro and in vivo, and its effect appears to require expression of both HVEM and LTβR on tumor cells (Zhai, Y.
． Et al., J. Clin. Invest. 102: 1142-1151 (1998).
). However, these receptors lack the typical death domain (Montgo).
merry, R.M. I. Et al., Cell 87: 427-436 (1996); Smi.
th, C.I. A. Et al., Cell 76: 959-962 (1994)). Recent reports indicate that AIM II can also bind to TR6, a decoy receptor (DcR3), which is expressed only in soluble form due to the lack of transmembrane sequences (Yu, KY). J. Biol. Chem. 274: 13733.
13736 (1999)).

Some recent studies suggest that AIM II may be involved in the regulation of cellular immune responses. AIM II stimulates the ternary mixed lymphocyte reaction (MLR) of peripheral blood mononuclear cells (PBMC) (Harrop, JA, et al., J. Biol. C).
hem. 273: 27548-27556 (1998)). This stimulus is HVE
It seems to mediate via M-linkage. This is because T cells do not express LTβR (Browning, JL et al., J. Immunol. 159:
3288-3298 (1997)). HVEM interacts with several TNF receptor-associated factors (TRAF), and overexpression of HVEM in 293 cells induces activation of nuclear factor (NF) -κB (Hsu, H. et al., J. Chem. Bi
ol. Chem. 272: 13471-13474 (1997); Marste.
rs, S. A. Et al., J. Biol. Chem. 272: 14029-14032
(1997)). Blocking HVEM with HVEM-Ig or specific mAbs
It can inhibit allogeneic MLR of PBMC (Harrop, JA, et al., J. Immu).
nol. 161: 1786-1794 (1998); Kwon, B .; S., et al.
Bio. Chem. 272: 14272-14276 (1997)). generally,
These results support that the interaction of AIM II and HVEM is involved in the generation of allogeneic T cell responses.

The mouse homologue of human AIM II has been cloned and characterized. Furthermore, AIM II has been shown to be a CD28-independent costimulatory molecule that is important for T cell proliferation and differentiation. It was also shown that T cell-mediated immune responses in tumors and GVDF can be modulated by in vivo manipulation of the AIM II costimulatory pathway.

(Molecular Cloning of Mouse AIM II) The full-length cDNA of the human homologue of human AIM II was prepared by combining ConACE-activated mouse T with RACE (rapid amplification of cDNA ends) and RT-PCR.
Isolated from cells, and pcDNA3 mammalian cell expression vector (pmAIM
II). The mouse AIM II cDNA encodes a putative 239-amino acid protein, which has the characteristics of a type II transmembrane protein and shows 77% amino acid homology with human AIM II (
FIG. 17A). The putative receptor binding region of mouse AIM II has significant sequence homology with the putative receptor binding regions of Fas ligand, LTβ, LTα, TNF, RANK ligand and TRAIL (Fas ligand (33%), LTβ (
30%), LTα (28%), TNF (27%), RANK ligand (26%)
And TRAIL (23%)) (data not shown). PmAIM to 293 cells
Upon transfection of II, surface expression of AIM II was stained with rabbit anti-AIM II antiserum or LTβR-Ig fusion protein by flow cytometric analysis (FIGS. 17B-1 to 17B-4).

AIM II Co-Stimulates CD28-Independent T Cell Responses It has been shown that human AIM II protein can enhance the proliferative response of human peripheral blood mononuclear cells to allogeneic antigens in in vitro culture. (Harrop, J.
A. Et al., J. Biol. Chem. 273: 27548-27556 (1998)
)). This suggests that AIM II may be involved in the T cell response. However, the nature of this enhancement is not clear. The possibility that AIM II could function as a stimulating molecule in the presence of T cell receptor signals was tested. To this end, an in vitro co-stimulation assay using transfected COS cells expressing AIM II to stimulate purified mature T cells in the presence of suboptimal fixed anti-CD3 mAb was performed. used. COS cells were transfected with pmAIM II, which induces a significant increase in T cell proliferation as compared to the increase in COS cells transfected with the control vector (FIG. 18A).
). In the absence of anti-CD3, COS cells transfected with pmAIM II did not stimulate T cell proliferation (Figure 18A). This result indicates that AIM II can co-stimulate T cell proliferation in the presence of antigen signals. To provide direct evidence for this conclusion, a fusion protein consisting of the extracellular domain of mouse AIM II fused to an 8-amino acid flag peptide (AIM I
I. Flag) was prepared. A fixed AIM, as shown in FIG. 18B.
II. Flag strongly stimulated proliferation of purified mouse T cells in the presence of suboptimal anti-CD3 in a dose-dependent manner. Similar to the results in FIG. 18A, the AIM
II. Flag did not stimulate T cells in the absence of anti-CD3 (Figure 18B).
. These results indicate that AIM II can co-stimulate T cell proliferation when TCR is involved.

AIM II co-stimulation was found in the B7-CD28 pathway (the most intensively studied co-stimulation pathway (Lenschow, DJ, et al., Annu. Rev. Immunol.
14: 233-258 (1996)) to determine whether T cells and anti-CD3 isolated from CD28-deficient (CD28 ^{− / −} ) mice.
A stimulates the proliferation of T cells isolated from normal littermates in the presence of mAb, A
IM II. The abilities of the flags were compared. As shown in FIG. 18C, the AIM
II. The flag fusion protein stimulated proliferation of CD28 ^{− / −} T cells at levels comparable to that of CD28 ^{+ / +} T cells. On the other hand, no proliferative response to anti-CD28 mAbs in CD28 ^{− / −} mice was observed. Taken together, these results indicate that AIM II can function independently of the CD28 costimulatory pathway.

[0776]   (Amplification of T cell-mediated tumor immunity by AIM II gene transfer in vivo)   Increased AIM II costimulation may increase cell-mediated immune response in vivo
To test whether or not pmAMI II plasmid was added to P815 tumor cells.
More provoked, injected directly into established tumor masses, and directed against tumor antigens
In the induction of cytolytic T cells (CTL) and in the regression of its tumor mass
The effect of AIM II expression was tested. Liposome-carried or naked, free
Plasms encoding epidemiotropic genes (eg, antigens (B7 and cytokines))
Administration of Sumid can express the molecule in mammalian tissues and stimulate T cell responses.
It was shown to be effective in violent conditions (Kim, JJ, et al., Nat. Bio).
thch. 15: 641-646 (1997); Templeton, N .; S.
Et al., Nat. Biotech. 15: 647-652 (1997); Plaut.
z, G. E. Et al., Proc. Natul. Acad. Sci. USA 90: 4
645-4649 (1993); Syrengelas, A .; D. Et al., Nat.
Med. 2: 1038-1041 (1996); Iwaski, A .; Et al., J. I
mmunol. 158: 4591-4601 (1997)). Furthermore, 2 × 10 ^Five Mice seeded subcutaneously (sc) with P815 cells showed an average diameter of 1 week.
Has previously been shown to produce clear tumors in the 3-5 mm size range
(Chen, L. et al., J. Exp. Med. 179: 523-532 (1994).
)).

Liposome-carried pmAIM II was repeatedly injected into the tumor mass starting 7 days after subcutaneous seeding of tumor cells. Transfected AIM I
Expression of I can be detected in the tumor mass by RT-PCR (data not shown).
One week after the last plasmid injection, splenocytes were stimulated in mixed lymphocyte-tumor cultures for 4 days and assayed for their CTL activity against P815 cells in a standard ⁵¹ Cr release assay (Chen). , L. et al., Cell
71: 1093-1102 (1992)). Mice treated with pmAIM II showed p81 compared to mice treated with medium or control vector.
It had a clearly increased CTL activity on 5 cells (FIG. 19A-1). CTL
Lysed p815 cells at high levels, while CTL showed L
It did not kill 1210 cells (Fig. 19A-2), indicating that CTLs are specific for the P185 tumor antigen. These results indicate that co-stimulation of AIM II in vivo can enhance the CTL response to the P815 tumor antigen.

Repeated injections of pm-AIM II further induced tumor regression in all treated mice within 3 weeks of the start of injection (FIG. 19B). On the other hand, mice in the control group treated with medium or control vector developed progressive growth tumors. In some mice, injection of the control vector caused a sAIM II delay in tumor growth, probably due to non-specific inflammation. In all cases, all tumors in the control group grew progressively and eventually killed the mice.

Mice with disappeared tumors after injection of pmAIM II plasmid did not relapse and remained tumor-free for more than 40 days (FIG. 19B). The mice were tested with a lethal dose of P815 cells to test if long-term tumor immunity had occurred. As shown in Figures 19C-1 these mice remained tumor free after this study, while all native mice developed tumors. This protection was specific to P815. This is because testing mice with L1210 (antigenically unrelated syngeneic lymphoma) cells induces tumor growth at a similar rate to that in native mice (FIG. 19C-2).

Restoration of Alloreactive T Cell-Mediated GVHD by Blocking AIM II In order to determine the role of endogenous AIM II in cell-mediated immune responses, L.
The effect of administering TβR-Ig, a soluble receptor for AIM II, and blocking the AIM II pathway in the mouse acute GVHD model was tested. In this model, lethally injected in B6 (H-2 ^b) derived T cell to radiation irradiated or not irradiated ^{BDF1 (H-2 b × d} ) recipient mouse may increase acute GVHD And is associated with rapid weight loss, alloreactive donor T cell expression, host splenocyte loss, thymus constriction and rapid mouse death (Via,
C. S. And Shearer, G .; M. , Immunol. Today 9:
207-213 (1988)). After intravenous (iv) injection with 7 × 10 ⁷ splenocytes from B6 mice, recipient mice received i.v. injections every 3 days starting 1 day before non-cell transfer. v. Received LTβR-Ig. All mice treated with LTβR-Ig survived up to 30 days (FIG. 20A), transiently lost mouse weight gain after GVHD induction (FIG. 20B), and 11 days after B6 splenocyte transfer. in assayed spleen decreased alloreactive (against H-2 ^d) CT
It had L activity (FIGS. 20C-1 and 20C-2). In contrast, all mice treated with control Ig died within 2 weeks after non-cell transfer with severe weight loss (FIGS. 20A and 20B). Higher levels of alloreactive CTL compared to control BDF1 mice that were transfected with splenocytes from BDF1 mice
Activity can be detected in mice that have undergone GVHD (FIGS. 20C-1 and 20C-2). Furthermore, the typical consequences of GVHD, the reduction of host splenic B lymphocytes and double positive thymocytes, were significantly inhibited by LTβR-Ig injection (data not shown). These results indicate that LTβR-Ig can restore alloreactive T cell-mediated GVHD.

These data implicate blockade of the AIM II costimulatory pathway in preventing GVHD, but it is possible that LTβ may play an additional role. This is because LTβR-Ig can neutralize endogenous LTβ production in mice with GVHD. To eliminate this possibility, splenocytes from LTα-deficient B6 mice lacking both the LTα3 complex and the LTα1β2 heterotrimer (LTβ) (Ware, CF, et al., Curr. Top. Microbiol. Im.
munol. 198: 175-218 (1995)) was transferred into non-irradiated BDF1 mice and tested for production of anti-host CTL activity. LTα
BDF1 Recipient mice transferred using splenocytes from deletion B6 mice showed an alloreactive CTL response similar to that of LTα ^{+ / +} littermate controls (FIGS. 20D-1 and 20D-). 2). This result indicates that LTβ from donor cells
We show that production is not required to generate an alloreactive CTL response in this GVHD model.

The effect of LTβR-Ig on the generation of CTL against the cognate antigen in vitro using LTα ^{− / −} B6 T cells was also tested. 20-1 and 20E-
As shown in 2, LTα ^{− / −} by BALB / c splenocytes irradiated for 5 days
Stimulation of B6T cells induce significant CTL activity against H-2 ^d target. Therefore, similar to the in vivo experiments shown in Figures 20D-1 and 20D-2, in vitro induction of alloreactive T cells is LT independent. Furthermore, cultures in which alloreactive CTL responses were significantly inhibited had LTβR- but not control Ig.
Ig is included (FIGS. 20E-1 and 20E-2). Taken together, these results negate LT's involvement in the generation of alloreactive T cells, and LTβR-Ig
Suggests that the blockade of AIM II by A. may be responsive to alloreactive CTL-mediated inhibition of GVHD.

Preferential induction of Th1-like cytokines by AIM II costimulation The effect of costimulation and inhibition of AIM II on T cell differentiation is studied. Purified T cells from B6 mice were stimulated with immobilized AIM II flag fusion protein in the presence of suboptimal amounts of anti-CD3 for 2 days, and culture supernatants were assayed for Th1- and Th2-type cytokines by sandwich ELISA. Was analyzed. As shown in FIGS. 21A-1 to 21A-4, the AIM II flag dramatically co-stimulated IFN-γ and GM-CSF production, while IL-4 and IL-10 secretion were altered. There wasn't. As shown in FIG. 21E, cytokine production in the culture supernatant of allogeneic MLR was tested. T cells purified from LTα-deficient splenocytes were used to eliminate the effects of LT. Culture supernatant of allogeneic MLR contains high levels of IFN
-Γ and GM-CSF, while IL-4 and IL-10 were either weak or undetectable. Inclusion of LTβR-Ig results in IFN-γ and G
It significantly inhibited M-CSF production (FIGS. 21B-1 to 21B-4). These results indicate that the AIM II costimulatory pathway preferentially elicits a Th1 type T cell response, while blockade of AIMII costimulation reduces Th1 type cytokine production.

Discussion A mouse homologue of human AIM II was cloned and its immunomodulatory function was studied. The data obtained show that AIM II is a key costimulatory molecule for CD28-independent T cell activation and preferentially induces Th1 type cytokine production. Two mouse models were used to test T cell-mediated immune responses against P815 tumors and against cognate antigens in acute GVHD. Tumor-specific CTL activity stimulated by AIM II gene transfer, which induces the recovery of established P815 tumors, and blockade of AIM II by LTβR-Ig resulted in GV
Can retreat HD. Therefore, these studies are based on a novel costimulatory molecule (AIM I
Establish I) as a key component in eliciting a cell-mediated immune response.

AIM II has potentially three receptors (HVEM, LTβR and Dc).
R3 / TR6) (Mauri, DN et al., Immunity 8: 2).
1-30 (1998); Yu, K .; Y. Et al., J. Biol. Chem. 274:
13733-13736 (1999)), transduces signals and interacts with other cells, but only HVEM appears to be able to respond to T cell costimulation, as described in this study. This indicates that LTβR was not found on T cells (Browning, JL et al., J, Immunol. 159: 3288-32).
98 (1997)), and the DcR3 / TR6 protein has no transmembrane domain (Yu, KY, et al., J. Biol. Chem. 274: 133733-1).
3736 (1999); Pitti, R .; M. Et al., Nature 396: 69.
9-703 (1998)). HV by AIM II
Engagement of EM clearly delivers costimulatory signals, while other receptors have AI
It may be involved in the regulation of M II mediated costimulation. LTβR is expressed by multiple non-lymphoid stromal cells of lymphoid tissue (Browning, JL, et al., J, Immuno).
l. 159: 3288-3298 (1997); Force, W .; R. Et al., J.
Immunol. 115: 5280-5288 (1995)) and binds to AIM II with high affinity (Mauri, DN et al., Nat. Med. 3: 6).
82-685 (1997)), the AI in lymphoid organs.
It can be speculated that MII's access to HVEM is blocked by LTβR. The results regarding the interaction of AIM II and LTβR are unknown, but cross-linking of LTβR by specific mAbs or LTβ induces secretion of the chemokines IL-8 and RANTES (Degli-Esposti, MA et al. ,
J. Immunol. 158: 1756-1762 (1997)) and deliver growth-inhibitory signals to several tumor lines in vitro (Degli-Esp.
osti, M .; A. Et al., J. Immunol. 158: 1756-1762 (1
997); Browing, J .; L. Et al., J. Exp. Med. 183: 867
-878 (1996)). Therefore, by binding to different receptors,
That AIM II may be involved in the regulation of innate and adaptive immune responses:
It is possible. Another receptor for AIM II, DcR3 / TR6, is overexpressed in many lung and colon cancers and also binds FasL (Pi
tti, R.A. M. Et al., Nature 396: 699-703 (1998)). These results show that Pittti and co-workers (Pittti, RM et al., Nature 396: 699-703 (19).
98)), in addition to preventing Fas-FasL-mediated apoptosis by DcR3 / TR6, some tumors secrete soluble DcR3 / TR6, neutralize AIM II and increase T cell activation. Suggest that you can. However, these studies show that AIM II gene transfer to the tumor mass enhanced A
It was shown that the IM II-HVEN interaction can induce regression of established tumor cells (FIG. 19), which can manipulate the AIM II-HVEM costimulatory pathway and enhance T cell response to tumors. Suggest that it induces. At the same time, A
It will be seen whether gene transfer by IM II could be effective on a broad spectrum of tumors in addition to P815.

AIM II can induce tumor cell death in the absence of other components of the immune system. It was shown that AIM II directly induces apoptosis of some tumor cells in cell culture, and that the AIM II-transfected human breast cancer line regresses in T cell-deficient thymic nude mice (Zhai, Y. et al., J. Cl
in, Invest. 102: 1142-1151 (1998)). Only cells expressing both LTβR and HVEM are sensitive to AIM II-mediated apoptosis (Zhai, Y. et al., J. Clin, Invest. 102: 1).
142-1151 (1998)) is interesting. Nevertheless, this mechanism of apoptosis can be ruled out in these studies. Because
This is because P815 was negative for both LTβR and HVEM by RT-PCR analysis (data not shown). Furthermore, the mechanism for regression of P815 tumors clearly correlates with amplified CTL responses specific for P815 (FIGS. 19A-1 and 19A2). Previous studies have shown that depletion of CD4 ⁺ , CD8 ⁺ , T cells or NK cells, at least in part, abrogates the effects of AIM II-induced tumor regression (data not shown), which indicates that It has been suggested that these subsets of lymphocytes are involved in AIM II-induced tumor immunity.

Intrinsic costimulatory pathways (eg B7-CD28 and CD40L-CD40)
Blockade induces GVHD remission in an animal model (Blazar, B
． R. Et al., Blood 83: 3815-3825 (1994); Durie,
F. H. Et al., J. Clin. Invest. 94: 1333-1338 (199
4)). AIM II can be detected in the presence of large amounts of activated PBMC (Harr
op, J. A. Et al., J. Immunol. 161: 1786-1794 (199
8)), and the role of these endogenously expressed AIM IIs in the immune response is poorly known. Recent studies have shown that mAbs to HVEM can significantly inhibit T cell proliferation in allogeneic MLRs in vitro (Har
rop, J .; A. Et al., J. Immunol. 161: 1786-1794 (19
98)), which suggested that the endogenous AIM II-HVEM interaction is required for the induction of allogeneic T cells. These studies develop this finding and that infusion of LTβR-Ig prevents the development of GVHD in F1 recipients and inhibits allogeneic CTL activity (FIG. 20) and donor T cells in F1 recipients. Inhibits expression (data not shown). These results are
Although it can be interpreted as a result of blockade of endogenous AIM II by LTβR-Ig,
Several alternative interpretations should be considered. LTα, LTβ or LTβ
Genetic disruption of R, or injection of LTβR-Ig into pregnant mice, inhibits lymph node development and interferes with the development of normal spleen architecture (Chaplin, DD and Fu, Y., Curr. Opin. Immunol. 10: 289-297.
(1998)). In adult mice, LTβR-Ig injection reduces follicular dendritic cells (FDC) and induces a humoral immune response (Mackay, F. and B.
rowing, J.M. L. , Nature 395: 26-27 (1998)).
However, it is clear that the reduction in FDC does not affect antigen presenting function in these mice. For example, GVHD can be induced in TNFRp55-deficient mice (Matsumoto, M. et al., Science 271: 1289).
-1291 (1996)), however, these mice disrupted the FDC network and impaired spleen architecture, similar to that observed in LTα knockout mice (Speiser, DE et al.). , J. Immunol.
158: 5185-190 (1997)). LTα ^{− / −} donor cells lacking both LTα and membrane-bound LTβ (Ware, CF et al., Curr. Top. Mi.
crobiol. Immunol. 198: 175-218 (1995)) still generated anti-host CTL responses in vivo (FIG. 20), which indicates that LT
It suggests that T cells in α-deficient mice are functionally normal, and that the donor cell's LTα or LTαβ complex is not required for the induction phase of GVDH.

In addition, these studies show that irradiated BALB / c spleen cells with L
Co-culture of purified B6 T cells from Tα-deficient mice was shown to normally induce allogeneic CTL, and donor cell LTα or LTαβ complexes are not required for induction of allogeneic CTL in this system Indicates that. Finally, LTβ
The inclusion of R-Ig is associated with allogeneic CTL from LTα-deficient T cells in the MLR assay.
Suppress generation completely. Recipient-derived LTβ is the G observed in this study.
It does not seem to play a role in VHD. This is because T cells, which are important responders to GVHD, do not express LTβR. In addition, T cells,
Donor T cells transferred to irradiated BDF1 mice that had been deprived of radiation-sensitive LTβ-producing cells, including B cells, NK cells, can still be sensitized to induce GVHD (FIGS. 20A, 20B). Taken together, these data deny a role for LT in the development of GVHD and support a significant contribution of AIM II in these responses. In summary, these studies show that AIM II is a key component in cell-mediated immune responses, and that AIM II costimulatory pathways can be engineered for therapeutic benefit in diseases such as cancer, transplantation and autoimmune diseases. Indicates.

(Method) (Mouse) Female C57BL / 6 (B6) mouse, BALB / c mouse, DBA
/ 2 and F1 (B6 x DBA / 2) (BDF1) mice
purchased from al Cancer Institute-Frederick, MD. CD28 ^{− / −} and LTα ^{− / −} mice in the B6 background have been previously described (Johnson, AJ et al., J. Virol. 73:
3702-3708 (1999); DeTogni, P .; Et al, Science
264: 703-707 (1994)).

(Cell line) COS, 293 human liver epithelial cells, P815 mouse mastocytoma, L
1210 mouse EL-4 and EL-4 mouse T cell lymphoma
Purchased from an Type Culture Collection (ATCC). The C26 mouse colorectal cancer strain was obtained from Jackson Laboratory, Ba.
r Harbor, ME, kindly provided by Dr. Robert Evans. Cell line is 10% FBS (HyClone, Logan, UT), 25 mM
RPMI-1640 (Gibco BRL, Gai) supplemented with HEPES, 100 U / ml penicillin G and 100 μg / ml streptomycin sulfate.
(Thersburg, MD).

Isolation of Mouse AIM II cDNA Mouse AIM II cDNA was isolated by PCR using degenerate primers for human AIM II. Sense primer (5'-TTTCGTIGATCGGGICA-3 '(SEQ ID NO: 62) (I
Represents inosine))) carries a sequence corresponding to human AIM II in 31-47. Antisense (5'AGGATGIACIACICCICC-3
'(SEQ ID NO: 63)) corresponds to the human AIM II sequence at 609 to 626. Poly from the spleen of BALB / c mice ^(A) - RNA was reverse transcribed and subjected to PCR using the above primers. The sequence of the PCR product is human AIM
II is highly homologous to the sequence of the cDNA and this product was used to
athon ^™ cDNA Amplification Kit (Clontec
The missing 5'and 3'ends were isolated by a RACE (rapid amplification of cDNA ends) procedure using h, Palo Alto, CA). Mouse AIM II
Full-length cDNA of pcDNA3 vector (Invitrogen, Carlsb
(Ad, CA). The homology between mouse AIM II and human AIM II was analyzed by the ClustrtalW program of Mac 6.5.

(Fusion protein and antibody) To prepare a mouse LTβR-Ig fusion protein, a cDNA encoding a mouse LTβR extracellular domain derived from mouse lung mRMA was used as a sense primer: (5′-AAAGGGCCGCCATGGGGCC).
T-3 '(SEQ ID NO: 64)) and antisense primer: (5'-TTAA
GCTTCAGTAGCATTGCTCCTGGCT-3 ′ (SEQ ID NO: 65))
Generated by RT-PCR. After digestion with NocI / HindIII, the PCR product was fused with the IL-3 leader sequence in the p30242 vector,
Then a p containing the IE-175 promoter and the Fc protein of human IgG1
Subcloned into X58 vector. This construct is then added to BHK / VP1
6 cells were transfected and the mouse LTβR-Ig fusion protein was purified from the conditioned medium by Sepharose Protein A affinity column. Fractions were then analyzed by SDS-PAGE through a 4-20% gradient gel to confirm the presence of the desired protein and further dialyzed against PBS. After purification, the resulting mouse LTβR-Ig was> 95% pure as determined by Coomassie blue staining of SDS-PAGE gels.

AIM II. To generate a flag fusion protein, mouse AIM I
The cDNA encoding the extracellular domain of I is fused to a flag peptide sequence, and E. Expressed in inclusion bodies in E. coli. After solubilization in 6M guanidine hydrochloric acid,
The protein was subsequently diluted and folded. The folded protein was then purified to near homogeneity by cation exchange column chromatography.
The final product was> 95% pure as determined from Coomassie blue staining of SDS-PAGE gels. Endotoxin concentration was determined by LAL assay (CA
<1 pg / mg of purified protein according to PE COD, Woods Hole, MA).

Rabbit anti-mouse AIM II peptide antibody is a synthetic peptide (CEAGEEVVVRVRPGG) of mouse AIM II conjugated to KLH at 209-232.
Immunization with NRLVRPRDGTRS (SEQ ID NO: 66)) gave
calico Biologicals, Inc. (Reamstown, PA
) Was prepared in. The antibody was affinity purified from serum using a peptide-conjugated column. Purified mAbs to mouse CD3, CD28, fluorescein isothiocyanate (FITC) conjugated CD4 and CD8
Purchased from Pharmingen (San Diego, CA). FITC-conjugated goat anti-human mAb and anti-rabbit mAb were added to Biosource I, respectively.
international (Camarillo, CA) and Souther
n Biotechnology Associates, Inc. (Birm
ingham, LA). Purified human IgG1 and rabbit Ig
G for Sigma (St. Louis, MO) and Rockland (G
ilbertsville, PA).

Flow Cytometric Analysis Human 293 cells (1 × 10 ⁶ cells) were transfected with either the mouse AIM II pcDNA3 vector (10 μg) or the mock vector (10 μg) by the calcium phosphate method. 30 hours after transfection, 293 cells were harvested and 5 min at 4 ° C for 5 min.
Stained with 1 μg mouse LTβR-Ig or anti-AIM II antibody in 50 μl PBS supplemented with% FBS and 0.02% azide. The cells were then washed and further incubated at 4 ° C. for 30 minutes with FITC-conjugated anti-human IgG or anti-rabbit IgG, respectively. Add Fluorescein to Cell Qu
FACSC with est software (Becton Dickinson)
alibur flow cytometry (Becton Dickinson, M
analyzed by the Outaintain View, CA).

(Tumor treatment model) DBA / 2 mice in a group of 5 to 10 were treated with 2 × 10 ⁵ P.
815 cells were seeded subcutaneously. One week later, this mouse with well-defined tumors with an average diameter of 3-5 mm was used for treatment. AIM II DN for in vivo injection
To prepare A, 10 μg of pmAIM II and 25 μg of cationic liposome, DMRIE-C (Gibco BRL) were added to 50 μl of Opti-.
Mixed in MEM I (Gibco BRL) and incubated in polystyrene tubes for 30 minutes at room temperature. This complex was administered 7 days, 10 days after tumor inoculation,
Injections were made intratumorally 14 and 17 days later. 50 μl for control
Opti-MEM I, or parental pc in 50 μl Opti-MEM I
A mixture of DNA3 (10 μg) and DMRIE-C (25 μg) was injected as above. Tumor size was assessed by measuring orthogonal diameters every 3 or 4 days.

DBA / 2 mice with P815 tumor regression after pmAIM II injection were treated with 2 × 10 ⁵ P815 cells on the right back and the same number of L1210 cells on the left back 40 days after the first tumor inoculation. The experiment was repeated subcutaneously. 2 × 10 ⁵ P815 and L
Naive DBA / 2 mice receiving 1210 were used as controls. Tumor size is then described (Chen, L et al., Cell 71: 1).
093-1102 (1992)).

For CTL assay, splenocytes of DBA / 2 mice subcutaneously inoculated with 2 × 10 ⁵ P815 cells at 7, 9 and 11 days and at 7, 9 and 11 days. DBA treated with either pmAIM II or control as described above
/ 2 mouse splenocytes were harvested on day 18. Splenocytes (5 × 10 ⁶ cells / ml) were cultured for 4 days in the presence of P815 cells irradiated with 150 Gy and then standard ⁵¹ Cr release assay (Chen, L. et al., Cell 71: 1093-).
1102 (1992)) for CTL activity. After 4 hours incubation, 100 μl of supernatant was collected and radioactivity was analyzed by MicroBe.
ta TriLux liquid scintillation counter (Wallac, Finl
and)).

(GVHD Model and In Vitro Allogeneic MLR) For incubation of acute GVHD, 7 × 10 ⁷ splenocytes from B6 mice were lethal irradiated (4 Gy) or non-irradiated BDF1. Recipient (B6-F1)
Intravenous injection in either Irradiated recipient mice were treated with 100 μg LTβR-Ig or control human IgG1 one day prior to splenocyte injection.
, And then every 6 days for 6 additional doses. The mice were monitored daily for survival and body weight. For CTL assay, non-irradiated recipients were treated with 100 μg of LTβR-Ig 1 day before splenocyte injection.
Alternatively, control Ig was given intravenously and repeated five more times daily. On day 11, recipient spleen cells were assayed for CTL activity by co-culturing with labeled P815 (H-2 ^d ) or EL-4 (H-2 ^b ) in a standard ⁵¹ Cr release assay. Splenocytes from BDF1 mice (F1-F1) that received BDF1 splenocyte transfer were used as a negative control. The same experiment was also performed using splenocytes from B6LTα ^{− / −} mice, and age-matched B6 mice were used as a control in this experiment.

[0800]   The in vitro introduction of CTL against the cognate antigen in the MLR is described (D
eTongi, P .; Et al., Science 264: 703-707 (1994).
) So went. Briefly, T cells were purchased from the manufacturer (Miltenyi Biot
ec Inc. Anti-FIT with magnetic fields, as taught by Auburn, CA)
FITC-conjugated anti-CD4 mAb and CD using C microbeads
Positive selection with 8 mAb. The purity of isolated T cells depends on anti-CD3
95% or higher when assessed by flow cytometry using mAb
It was. Purified B6LTα^-/-Mouse T cells were transfected with LTβR-Ig or control.
Of the same number of 30 Gy-irradiation in the presence or absence of human IgG1
1 x 10 with BALB / c splenocytes⁶Co-cultured at cells / ml. 5 days later
, C26 (H-2^d) And EL-4 (H-2^b) Activity against the standard ⁵¹ Tested in Cr release assay.

(T cell co-stimulation assay) 1 × 10 ⁶ cells / ml of magnetic beads-purified T cells derived from mouse spleen cells were exposed to 150 Gy-irradiated COS cells (5 × 10 ⁴ cells).
Cells / ml) and stimulated with plate-coated anti-CD3 mAb. The COS cells have been transfected with either pcDNA3 or pmAIM II by the DEAE-dextran method for 48 hours. Alternatively,
Plate the anti-CD3 coated plate for 4 hours at 37 ° C. with the desired amount of AIM II.
． Further coated with flag fusion protein. After washing, T cells purified from B6, CD28 ^{− / −} mice or any spleen of a litter (1 × 10 ⁶ cells / ml)
) Was added to the wells. Anti-CD28 (1 μg / ml) mAb was used as the soluble form. Proliferation of T cells was controlled by 1 μC during the last 15 hours of 3 day culture.
It was evaluated by the addition of i / well of ³ H-TdR. ³ H-TdR uptake
microBeta TriLux liquid scintillation counter (Walla
c, Finland).

(Cytokine Assay) 1 × 10 ⁶ cells / ml of purified B6 T cells were added to
Plate-coated AIM II.V. in the presence of anti-CD3 (0.5 μg / ml) as described above. The cells were stimulated with flags (3.2 μg / ml). Culture supernatants were collected at 48 hours and IFN-followed by the manufacturer's instructions (PharMingen).
Sandwich ELIS for γ, GM-CSF, IL-4 and IL-10
Assayed by A. To assay cytokine production in allogeneic MLR, 1 × 10 ⁶ cells / ml of purified B6LTα ^{− / −} T cells were added to LTβR-Ig.
Stimulation was performed with the same number of irradiated BALB / c splenocytes in the presence or absence of fusion protein and control human IgG. Culture supernatants were harvested at 72 hours and analyzed by IFN-γ, GM-CSF, IL-4 and IL.
It was assayed for -10 production.

Example 23: Production of Antibodies A. Hybridoma Technology The antibodies of the invention can be prepared by a variety of methods. (Ausbel et al., Ed.
1998, Current Protocols, Molecular Bio
LOGY, John Wiley & Sons, NY, Chapter 2). As one example of such a method, cells expressing AIM II are administered to an animal to induce the production of serum containing polyclonal antibodies. In a preferred method, a preparation of AIM II protein is prepared and purified,
Made substantially free of natural contaminants. Such preparations are then introduced into animals to produce greater specific activity of polyclonal antisera.

Monoclonal antibodies specific for the protein AIM II are prepared using hybridoma technology (Kohler et al. Nature 256: 49.
5 (1975); Kohler et al., Eur. J. Immunol. 6: 511 (
1976); Kohler et al., Eur. J. Immunol. 6: 292 (19
76); Hammerling et al .: Monoclonal Antibody.
s and T-Cell Hybridomas, Elsevier, N .; Y
． , Pp. 563-681 (1981)). Generally, an animal, preferably a mouse, is immunized with AIM II polypeptides, or more preferably secreted AIM II polypeptides expressing cells. Such polypeptide-expressing cells are cultured in any suitable tissue culture medium, preferably supplemented with 10% fetal bovine serum (inactivated at about 56 ° C.), and containing about 10 g / l of non-essential amino acids. , About 1,000U
Cultured in Earle's modified Eagle medium supplemented with 1 / ml penicillin and about 100 μg / ml streptomycin.

The splenocytes of such mice are extracted and fused with an appropriate myeloma cell line. Any suitable myeloma cell line may be used in accordance with the present invention; however, it is preferred to use the parental myeloma cell line (SP20) available from the ATCC. After fusion, the resulting hybridoma cells were selectively maintained in HAT medium and then Wands et al. (Gastroenterology 80:22).
Clone by limiting dilution as described by 5-232 (1981)). The hybridoma cells obtained by such selection are then AIM
II is assayed to identify clones secreting antibodies capable of binding the polypeptide.

Alternatively, additional antibodies capable of binding to AIM II polypeptides may be generated in a two step procedure using anti-idiotype antibodies. Such a method takes advantage of the fact that the antibody is itself an antigen and it is therefore possible to obtain an antibody which binds to the second antibody. According to this method, protein-specific antibodies are used to immunize animals, preferably mice. The splenocytes of such animals are then used to produce hybridoma cells, and the hybridoma cells identify antibodies-producing clones whose ability to bind AIM II protein-specific antibodies can be blocked by AIM II. To be screened. Such antibodies include anti-idiotypic antibodies directed against AIM II protein-specific antibodies and are used to immunize animals to induce the formation of additional protein-specific antibodies.

For use in vivo use of the antibody in humans, the antibody is "humanized." Such antibodies can be produced using genetic constructs derived from hybridoma cells which produce the monoclonal antibodies described above. Methods for producing chimeric and humanized antibodies are known in the art and discussed herein. (
For a review, see Morrison, Science 229: 1202 (19
85); Oi et al., BioTechniques 4: 214 (1986); Ca.
billy et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP
171496; Morrison et al., EP 173494; Neuberge.
r et al., WO 8601533; Robinson et al., WO 8702671; B.
aulianne et al., Nature 312: 643 (1984); Neube.
rger et al., Nature 314: 268 (1985)) Isolation of Antibody Fragments Against AIM II from a Library of B. scFv The naturally occurring V-gene isolated from human PBLs was Construct in a library of antibody fragments containing reactivity to AIM II that may or may not be exposed (eg, US Pat. No. 5,885,793, incorporated herein by reference in its entirety). Will be))).

Library Rescue From human PBL RNAs as described in PCT Publication WO92 / 01047.
Build a cFv library. To rescue the phage displaying the antibody fragment, about 10 ⁹ E. coli carrying the phagemid were rescued. With E. coli,
Inoculated with 50 ml 2 × TY (containing 1% glucose and 100 μg / ml ampicillin) (2 × TY-AMP-GLU),
Then, while shaking, the O. D. Grow to. 5 ml of this culture was used to inoculate 50 ml of 2xTY-AMP-GLU and 2x10 ⁸ TU of the delta gene 3 helper (M13 delta gene III, see PCT publication WO92 / 01047) was added, The culture is then incubated at 37 ° C. for 45 minutes without shaking and then at 37 ° C. for 45 minutes with shaking. 10 times this culture
4000r.min. p. m. Centrifuge at room temperature, and pellet the pellet with 2 liters of 2xT.
Resuspend in Y (containing 100 μg / ml ampicillin and 50 μg / ml kanamycin) and grow overnight. PCT release WO92 / 0
Prepare as described in 1047.

M13Δ gene III is prepared as follows: M13Δ gene III helper phage does not encode gene III protein. Therefore, phage displaying antibody fragments (phagemids) have greater binding avidity for antigen. During phage morphogenesis, infectious M13Δ gene III particles are made by growing helper phage in cells carrying the pUC19 derivative that supplies the wild-type gene III protein. The culture is incubated for 1 hour at 37 ° C without shaking and then for another hour at 37 ° C with shaking. Spin down cells (IEC-Centra 8, 40
0 r. p. m for 10 minutes), 100 μg / ml ampicillin and 25 μm
2 × TY broth containing 2 g / ml of kanamycin (2 × TY-AMP-KAN
) Resuspended in 300 ml and grown overnight at 37 ° C. with shaking. Phage particles were purified and concentrated from the culture medium by two rounds of PEG precipitation (Sambrook et al., 1990), resuspended in 2 ml PBS, and passed through a 0.45 μm filter (Minisart NML; Sartorius), about 1 μm.
A final concentration of 0 ¹³ transducing units / ml (ampicillin resistant clones) was obtained.

(Paning Library) Immunotubes (Nunc) were added at 100 μg / ml or 10 μg / ml.
4. Coat overnight in PBS with 4 ml of any of the polypeptides of the invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at 37 ° C and then washed 3 times in PBS. Approximately 10 ¹³ TU of phage are applied to this tube and incubated for 30 minutes at room temperature with tilting up and down on a turntable, then left to stand for a further 1.5 hours. Tube with PBS 0.1% Tween-2
Wash 10 times with 0 and 10 times with PBS. The phage were eluted by adding 1 ml of 100 mM triethylamine and spinning up and down for 15 minutes on a turntable, after which the solution was added to 0.5 ml of 1.0 M Tris-HCl, pH.
Neutralize immediately at 7.4. The eluted phage was then incubated with the bacteria at 37 ° C for 3
Phage was used to incubate 10 ml of mid-log phase E. coli by incubating for 0 min. E. coli TG1. Then E. E. coli is plated on TYE plates containing 1% glucose and 100 μg / ml ampicillin. The resulting bacterial library is then rescued with Δ gene 3 helper phage as described above to prepare phage for the next round of selection. This process is then repeated for all four rounds of affinity purification, increasing the tube wash to PBS, 0.1% Tween-20 20 times and 20 times to PBS 3 and 4 times.

Characterization of Binders The phage eluted from the third and fourth rounds of selection were used to transform E. coli. coli H
B 2151 is infected and soluble scFv are generated from a single colony for the assay (Marks et al., 1991). 50 mM bicarbonate, pH 9.6
The ELISA is performed with microtiter plates coated with either 10 pg / ml of the polypeptide of the invention in. P positive clones in ELISA
Further characterization by CR fingerprinting (see, eg, PCT Publication WO92 / 01047) followed by sequencing.

Example 24: Tissue distribution of AIM II expression Northern blot analysis was performed to test the level of expression of AIM II in human tissues using the method described by Sambrook et al. (Supra), among others. To be executed. RNAzol ^™ B system (B
iotecx Laboratories, Inc. , 6023 South
Loop East, Houston, Tex).

About 10 μg of total RNA is isolated from tissue samples. RNA is size separated by electrophoresis through a 1% agarose gel under highly denaturing conditions. RNA is blotted from the gel onto a nylon filter, then the filter is prepared for hybridization to a detectably labeled polynucleotide probe.

As a probe to detect mRNA encoding AIM II, the antisense strand of the coding region of the cDNA insert in the deposited clone identified as ATCC Deposit No. 97483 is labeled for high specific activity. . cDN
A uses the Prime-It kit from Stratagene to
Labeled by primer extension. This reaction is performed using 50 ng of cDNA according to the standard reaction protocol recommended by the supplier. The labeled polynucleotide is 5 Prime-3 Prime, Inc (560
3 Sel obtained from Arapahoe Road, Boulder, CO)
Purified from other labeled reaction components by column chromatography using an ect-G-50 column.

The labeled probe was hybridized to the filter overnight at 65 ° C. in a small volume of 7% SDS, 0.5 M NaPO ₄ , pH 7.4 at a concentration of 1,000,000 cpm / ml. It

Thereafter, the probe solution was drained and the filter was washed with 0.5 × SSC, 0.1
Wash twice with room temperature SDS and twice at 60 ° C. The filters are then dried and exposed to film with an intensifying screen at -70 ° C overnight.

Autoradiography showed that the mRNA for AIM II was spleen, bone marrow,
And abundant in the thymus.

It will be appreciated that the present invention may be practiced other than as described in detail in the foregoing description and examples.

Many modifications and variations of the present invention are possible in light of the above teachings, and are therefore within the scope of the appended claims.

Full disclosure of all publications cited herein (patents, patent applications, academic literature,
Experimental manuals, books or other documents) are incorporated herein by reference.

[0821]

[Table 4]

[0822]

[Table 5]

[Brief description of drawings]

FIG. 1A shows the nucleotide (SEQ ID NO: 1) and deduced amino acid of AIM II (SEQ ID NO: 1).
Sequence No. 2) shows the sequence. This protein has a predicted molecular weight of approximately 26.4 kDa. The putative transmembrane domain of the AIM II protein is underlined.

FIG. 1B shows AIM II nucleotides (SEQ ID NO: 1) and deduced amino acids (
Sequence No. 2) shows the sequence. This protein has a predicted molecular weight of approximately 26.4 kDa. The putative transmembrane domain of the AIM II protein is underlined.

FIG. 1C shows the nucleotide (SEQ ID NO: 38) and deduced amino acid (SEQ ID NO: 39) SEQ ID NOs of similarly obtained partial AIM II cDNAs.

FIG. 1D shows the nucleotide (SEQ ID NO: 38) and deduced amino acid (SEQ ID NO: 39) SEQ ID NOs of similarly obtained partial AIM II cDNAs.

FIG. 2A shows AIM II protein and human TNF-α (SEQ ID NO: 3), human TNF-β (SEQ ID NO: 4), human lymphotoxin (SEQ ID NO: 5), and human Fas ligand (SEQ ID NO: 6). The region of similarity between the amino acid sequences of

FIG. 2B shows AIM II protein and human TNF-α (SEQ ID NO: 3), human TNF-β (SEQ ID NO: 4), human lymphotoxin (SEQ ID NO: 5), and human Fas ligand (SEQ ID NO: 6). The region of similarity between the amino acid sequences of

FIG. 2C shows AIM II protein and human TNF-α (SEQ ID NO: 3), human TNF-β (SEQ ID NO: 4), human lymphotoxin (SEQ ID NO: 5), and human Fas ligand (SEQ ID NO: 6). The region of similarity between the amino acid sequences of

FIG. 2D shows AIM II protein and human TNF-α (SEQ ID NO: 3), human TNF-β (SEQ ID NO: 4), human lymphotoxin (SEQ ID NO: 5), and human Fas ligand (SEQ ID NO: 6). The region of similarity between the amino acid sequences of

FIG. 2E shows AIM II protein and human TNF-α (SEQ ID NO: 3), human TNF-β (SEQ ID NO: 4), human lymphotoxin (SEQ ID NO: 5), and human Fas ligand (SEQ ID NO: 6). The region of similarity between the amino acid sequences of

FIG. 2F shows AIM II protein and human TNF-α (SEQ ID NO: 3), human TNF-β (SEQ ID NO: 4), human lymphotoxin (SEQ ID NO: 5), and human Fas ligand (SEQ ID NO: 6). The region of similarity between the amino acid sequences of

FIG. 3A shows analysis of AIM II amino acid sequence. Alpha, beta, turn, and coil regions; hydrophilic and hydrophobic; amphipathic regions; flexible regions; antigenic index and surface probabilities are shown. In the "antigenicity index-Jameson-Wolf" graph, about amino acid residues 13 to 20 and 23 to those of FIGS. 1A and B (SEQ ID NO: 2).
36, 69-79, 85-94, 167-178, 184-196, 221-2
33 corresponds to the indicated highly antigenic region of the AIM II protein.

FIG. 3B shows analysis of the AIM II amino acid sequence. Alpha, beta, turn, and coil regions; hydrophilic and hydrophobic; amphipathic regions; flexible regions; antigenic index and surface probabilities are shown. In the "antigenicity index-Jameson-Wolf" graph, about amino acid residues 13 to 20 and 23 to those of FIGS. 1A and B (SEQ ID NO: 2).
36, 69-79, 85-94, 167-178, 184-196, 221-2
33 corresponds to the indicated highly antigenic region of the AIM II protein.

FIG. 3C shows analysis of the AIM II amino acid sequence. Alpha, beta, turn, and coil regions; hydrophilic and hydrophobic; amphipathic regions; flexible regions; antigenic index and surface probabilities are shown. In the "antigenicity index-Jameson-Wolf" graph, about amino acid residues 13 to 20 and 23 to those of FIGS. 1A and B (SEQ ID NO: 2).
36, 69-79, 85-94, 167-178, 184-196, 221-2
33 corresponds to the indicated highly antigenic region of the AIM II protein.

FIG. 3D shows analysis of AIM II amino acid sequence. Alpha, beta, turn, and coil regions; hydrophilic and hydrophobic; amphipathic regions; flexible regions; antigenic index and surface probabilities are shown. In the "antigenicity index-Jameson-Wolf" graph, about amino acid residues 13 to 20 and 23 to those of FIGS. 1A and B (SEQ ID NO: 2).
36, 69-79, 85-94, 167-178, 184-196, 221-2
33 corresponds to the indicated highly antigenic region of the AIM II protein.

FIG. 3E shows analysis of the AIM II amino acid sequence. Alpha, beta, turn, and coil regions; hydrophilic and hydrophobic; amphipathic regions; flexible regions; antigenic index and surface probabilities are shown. In the "antigenicity index-Jameson-Wolf" graph, about amino acid residues 13 to 20 and 23 to those of FIGS. 1A and B (SEQ ID NO: 2).
36, 69-79, 85-94, 167-178, 184-196, 221-2
33 corresponds to the indicated highly antigenic region of the AIM II protein.

FIG. 3F shows analysis of AIM II amino acid sequence. Alpha, beta, turn, and coil regions; hydrophilic and hydrophobic; amphipathic regions; flexible regions; antigenic index and surface probabilities are shown. In the "antigenicity index-Jameson-Wolf" graph, about amino acid residues 13 to 20 and 23 to those of FIGS. 1A and B (SEQ ID NO: 2).
36, 69-79, 85-94, 167-178, 184-196, 221-2
33 corresponds to the indicated highly antigenic region of the AIM II protein.

4A and B show the effect of AIMII on the in vitro proliferation of MDA-MB-231 human breast cancer cells. 5,000 MDA-MB-231 / WT (Mull), MDA-MB-231 / Neo (triangle) or MDA-MB-231 / AI
MII (squares) cells were plated in triplicate in 24-well plates with 10% F with IMEM.
BS (black circle, black square, black triangle) or 1% FBS (white circle, white square,
Plated in the presence of either of the (open triangles). The number of viable cells was determined by the trypan blue exclusion method on day 3, 5 or 7. During this time course, cells were replenished with fresh medium every two days. FIG. 4B shows colony formation of MDA-MB-231 / WT, MDA-MB-231 / Neo, and MDA-MB-231 / AIMII cells in 0.33% agarose.

5A-C are Annexin-V described in the materials and methods of Example 5.
MDA-MB-231 / WT (Fig. 5A) cells or MDA- by FACS analysis.
MDA in 0.5% serum compared to MB-231 / Neo (FIG. 5B) cells
-Represents an increase in apoptotic cells in MB-231 / AIMII (Fig. 5C).

FIG. 6A shows evaluation of the effect of AIMII on the growth of xenograft human breast cancer MDA-231 in nude mice. Female athymic nude mice were subcutaneously injected with parental MDA-23 1 (MDA-23 1-WT) or MIM-23 1 stably transfected with AIMII or vector control neo (n).
= 10) 10 ⁶ cells were injected. The mice were then earmarked and randomized. Tumor growth was assessed twice weekly with calipers in a blinded fashion. This panel
Shown are three experiments with 10 mice each per group.

FIG. 6B shows the effect of AIMII transduction on the inhibition of MC-38 mouse colon cancer growth in syngeneic C57BL / 6 mice. Female C57BL / 6 mice were subcutaneously injected with parental MC-38 (MC-38-WT) or MC-38 stably transfected with AimII, or vector control neo (n =
10 ^{6 of} 10) was injected. The mice were then earmarked and randomized. Tumor growth was assessed twice a week with calipers in a coded, blinded fashion. This panel represents 4 experiments with 10 mice each per group.

FIG. 7A: pFlag-AIMII plasmid construct (FIG. 7A), in the presence or absence of IFNγ (FIG. 7B) or IFNγ alone (FIG. 7C), MD.
Recombinant soluble form of AIM II (sAIMII in A-MB-231 cells
) Shows cytotoxicity. The experiment was performed as described in the materials and methods of Example 5.

FIG. 7B. PFlag-AIMII plasmid construct (FIG. 7A), in the presence or absence of IFNγ (FIG. 7B) or with IFNγ alone (FIG. 7C), MD.
Recombinant soluble form of AIM II (sAIMII in A-MB-231 cells
) Shows cytotoxicity. The experiment was performed as described in the materials and methods of Example 5.

7C shows the pFlag-AIMII plasmid construct (FIG. 7A), in the presence or absence of IFNγ (FIG. 7B) or with IFNγ alone (FIG. 7C), MD.
Recombinant soluble form of AIM II (sAIMII in A-MB-231 cells
) Shows cytotoxicity. The experiment was performed as described in the materials and methods of Example 5.

FIG. 8A is a cell surface expression of LTβR or TR2 by FACS analysis using LTβR (FIGS. 8A-8D) or TR2 (FIGS. 8E-8H) mAb. MD
A-MB-231 (FIGS. 8A and 8E), HT-29 (FIGS. 8B and 8F), M
C-3 (Figures 8C and 8G), U93T (Figures 8D and 8H). MDA-MB-
FACS binding analysis of soluble AIM II protein alone in 231 cells (
8I), and blocking of soluble AIM II protein binding by preincubation with LTβR-Fc fusion protein (FIG. 8J) or TR2-Fc fusion protein (FIG. 8K). FIG. 8L summarizes surface expression of LTβR and TR2 in various cell lines. The effect of LTβR-Fc or TR2-Fc fusion proteins on blocking sAIM II-mediated cytotoxicity in HT-29 cells. Cells were plated in 96 well plates and sAIM II (
10 ng / ml) in the presence of 5 U / ml IFNγ and varying amounts of sLTβR-F.
c (open circle LTβR-Fc alone, black circle LTβR-Fc and IFNγ
), Or a TR2-Fc fusion protein (open triangle TR-2Fc alone, black triangle TR2-Fc and sLTγ and IFNγ). Cells were incubated for 5 days, and cell viability was determined by XTT assay.

FIG. 8B is a cell surface expression of LTβR or TR2 by FACS analysis using LTβR (FIGS. 8A-8D) or TR2 (FIGS. 8E-8H) mAb. MD
A-MB-231 (FIGS. 8A and 8E), HT-29 (FIGS. 8B and 8F), M
C-3 (Figures 8C and 8G), U93T (Figures 8D and 8H). MDA-MB-
FACS binding analysis of soluble AIM II protein alone in 231 cells (
8I), and blocking of soluble AIM II protein binding by preincubation with LTβR-Fc fusion protein (FIG. 8J) or TR2-Fc fusion protein (FIG. 8K). FIG. 8L summarizes surface expression of LTβR and TR2 in various cell lines. The effect of LTβR-Fc or TR2-Fc fusion proteins on blocking sAIM II-mediated cytotoxicity in HT-29 cells. Cells were plated in 96 well plates and sAIM II (
10 ng / ml) in the presence of 5 U / ml IFNγ and varying amounts of sLTβR-F.
c (open circle LTβR-Fc alone, black circle LTβR-Fc and IFNγ
), Or a TR2-Fc fusion protein (open triangle TR-2Fc alone, black triangle TR2-Fc and sLTγ and IFNγ). Cells were incubated for 5 days, and cell viability was determined by XTT assay.

FIG. 8C is cell surface expression of LTβR or TR2 by FACS analysis using LTβR (FIGS. 8A-8D) or TR2 (FIGS. 8E-8H) mAb. MD
A-MB-231 (FIGS. 8A and 8E), HT-29 (FIGS. 8B and 8F), M
C-3 (Figures 8C and 8G), U93T (Figures 8D and 8H). MDA-MB-
FACS binding analysis of soluble AIM II protein alone in 231 cells (
8I), and blocking of soluble AIM II protein binding by preincubation with LTβR-Fc fusion protein (FIG. 8J) or TR2-Fc fusion protein (FIG. 8K). FIG. 8L summarizes surface expression of LTβR and TR2 in various cell lines. The effect of LTβR-Fc or TR2-Fc fusion proteins on blocking sAIM II-mediated cytotoxicity in HT-29 cells. Cells were plated in 96 well plates and sAIM II (
10 ng / ml) in the presence of 5 U / ml IFNγ and varying amounts of sLTβR-F.
c (open circle LTβR-Fc alone, black circle LTβR-Fc and IFNγ
), Or a TR2-Fc fusion protein (open triangle TR-2Fc alone, black triangle TR2-Fc and sLTγ and IFNγ). Cells were incubated for 5 days, and cell viability was determined by XTT assay.

FIG. 8D is cell surface expression of LTβR or TR2 by FACS analysis using LTβR (FIGS. 8A-8D) or TR2 (FIGS. 8E-8H) mAb. MD
A-MB-231 (FIGS. 8A and 8E), HT-29 (FIGS. 8B and 8F), M
C-3 (Figures 8C and 8G), U93T (Figures 8D and 8H). MDA-MB-
FACS binding analysis of soluble AIM II protein alone in 231 cells (
8I), and blocking of soluble AIM II protein binding by preincubation with LTβR-Fc fusion protein (FIG. 8J) or TR2-Fc fusion protein (FIG. 8K). FIG. 8L summarizes surface expression of LTβR and TR2 in various cell lines. The effect of LTβR-Fc or TR2-Fc fusion proteins on blocking sAIM II-mediated cytotoxicity in HT-29 cells. Cells were plated in 96 well plates and sAIM II (
10 ng / ml) in the presence of 5 U / ml IFNγ and varying amounts of sLTβR-F.
c (open circle LTβR-Fc alone, black circle LTβR-Fc and IFNγ
), Or a TR2-Fc fusion protein (open triangle TR-2Fc alone, black triangle TR2-Fc and sLTγ and IFNγ). Cells were incubated for 5 days, and cell viability was determined by XTT assay.

FIG. 8E is cell surface expression of LTβR or TR2 by FACS analysis using LTβR (FIGS. 8A-8D) or TR2 (FIGS. 8E-8H) mAb. MD
A-MB-231 (FIGS. 8A and 8E), HT-29 (FIGS. 8B and 8F), M
C-3 (Figures 8C and 8G), U93T (Figures 8D and 8H). MDA-MB-
FACS binding analysis of soluble AIM II protein alone in 231 cells (
8I), and blocking of soluble AIM II protein binding by preincubation with LTβR-Fc fusion protein (FIG. 8J) or TR2-Fc fusion protein (FIG. 8K). FIG. 8L summarizes surface expression of LTβR and TR2 in various cell lines. The effect of LTβR-Fc or TR2-Fc fusion proteins on blocking sAIM II-mediated cytotoxicity in HT-29 cells. Cells were plated in 96 well plates and sAIM II (
10 ng / ml) in the presence of 5 U / ml IFNγ and varying amounts of sLTβR-F.
c (open circle LTβR-Fc alone, black circle LTβR-Fc and IFNγ
), Or a TR2-Fc fusion protein (open triangle TR-2Fc alone, black triangle TR2-Fc and sLTγ and IFNγ). Cells were incubated for 5 days, and cell viability was determined by XTT assay.

FIG. 8F is cell surface expression of LTβR or TR2 by FACS analysis using LTβR (FIGS. 8A-8D) or TR2 (FIGS. 8E-8H) mAb. MD
A-MB-231 (FIGS. 8A and 8E), HT-29 (FIGS. 8B and 8F), M
C-3 (Figures 8C and 8G), U93T (Figures 8D and 8H). MDA-MB-
FACS binding analysis of soluble AIM II protein alone in 231 cells (
8I), and blocking of soluble AIM II protein binding by preincubation with LTβR-Fc fusion protein (FIG. 8J) or TR2-Fc fusion protein (FIG. 8K). FIG. 8L summarizes surface expression of LTβR and TR2 in various cell lines. The effect of LTβR-Fc or TR2-Fc fusion proteins on blocking sAIM II-mediated cytotoxicity in HT-29 cells. Cells were plated in 96 well plates and sAIM II (
10 ng / ml) in the presence of 5 U / ml IFNγ and varying amounts of sLTβR-F.
c (open circle LTβR-Fc alone, black circle LTβR-Fc and IFNγ
), Or a TR2-Fc fusion protein (open triangle TR-2Fc alone, black triangle TR2-Fc and sLTγ and IFNγ). Cells were incubated for 5 days, and cell viability was determined by XTT assay.

FIG. 8G is cell surface expression of LTβR or TR2 by FACS analysis using LTβR (FIGS. 8A-8D) or TR2 (FIGS. 8E-8H) mAb. MD
A-MB-231 (FIGS. 8A and 8E), HT-29 (FIGS. 8B and 8F), M
C-3 (Figures 8C and 8G), U93T (Figures 8D and 8H). MDA-MB-
FACS binding analysis of soluble AIM II protein alone in 231 cells (
8I), and blocking of soluble AIM II protein binding by preincubation with LTβR-Fc fusion protein (FIG. 8J) or TR2-Fc fusion protein (FIG. 8K). FIG. 8L summarizes surface expression of LTβR and TR2 in various cell lines. The effect of LTβR-Fc or TR2-Fc fusion proteins on blocking sAIM II-mediated cytotoxicity in HT-29 cells. Cells were plated in 96 well plates and sAIM II (
10 ng / ml) in the presence of 5 U / ml IFNγ and varying amounts of sLTβR-F.
c (open circle LTβR-Fc alone, black circle LTβR-Fc and IFNγ
), Or a TR2-Fc fusion protein (open triangle TR-2Fc alone, black triangle TR2-Fc and sLTγ and IFNγ). Cells were incubated for 5 days, and cell viability was determined by XTT assay.

FIG. 8H is cell surface expression of LTβR or TR2 by FACS analysis using LTβR (FIGS. 8A-8D) or TR2 (FIGS. 8E-8H) mAb. MD
A-MB-231 (FIGS. 8A and 8E), HT-29 (FIGS. 8B and 8F), M
C-3 (Figures 8C and 8G), U93T (Figures 8D and 8H). MDA-MB-
FACS binding analysis of soluble AIM II protein alone in 231 cells (
8I), and blocking of soluble AIM II protein binding by preincubation with LTβR-Fc fusion protein (FIG. 8J) or TR2-Fc fusion protein (FIG. 8K). FIG. 8L summarizes surface expression of LTβR and TR2 in various cell lines. The effect of LTβR-Fc or TR2-Fc fusion proteins on blocking sAIM II-mediated cytotoxicity in HT-29 cells. Cells were plated in 96 well plates and sAIM II (
10 ng / ml) in the presence of 5 U / ml IFNγ and varying amounts of sLTβR-F.
c (open circle LTβR-Fc alone, black circle LTβR-Fc and IFNγ
), Or a TR2-Fc fusion protein (open triangle TR-2Fc alone, black triangle TR2-Fc and sLTγ and IFNγ). Cells were incubated for 5 days, and cell viability was determined by XTT assay.

FIG. 8I is cell surface expression of LTβR or TR2 by FACS analysis using LTβR (FIGS. 8A-8D) or TR2 (FIGS. 8E-8H) mAb. MD
A-MB-231 (FIGS. 8A and 8E), HT-29 (FIGS. 8B and 8F), M
C-3 (Figures 8C and 8G), U93T (Figures 8D and 8H). MDA-MB-
FACS binding analysis of soluble AIM II protein alone in 231 cells (
8I), and blocking of soluble AIM II protein binding by preincubation with LTβR-Fc fusion protein (FIG. 8J) or TR2-Fc fusion protein (FIG. 8K). FIG. 8L summarizes surface expression of LTβR and TR2 in various cell lines. The effect of LTβR-Fc or TR2-Fc fusion proteins on blocking sAIM II-mediated cytotoxicity in HT-29 cells. Cells were plated in 96 well plates and sAIM II (
10 ng / ml) in the presence of 5 U / ml IFNγ and varying amounts of sLTβR-F.
c (open circle LTβR-Fc alone, black circle LTβR-Fc and IFNγ
), Or a TR2-Fc fusion protein (open triangle TR-2Fc alone, black triangle TR2-Fc and sLTγ and IFNγ). Cells were incubated for 5 days, and cell viability was determined by XTT assay.

FIG. 8J is cell surface expression of LTβR or TR2 by FACS analysis using LTβR (FIGS. 8A-8D) or TR2 (FIGS. 8E-8H) mAb. MD
A-MB-231 (FIGS. 8A and 8E), HT-29 (FIGS. 8B and 8F), M
C-3 (Figures 8C and 8G), U93T (Figures 8D and 8H). MDA-MB-
FACS binding analysis of soluble AIM II protein alone in 231 cells (
8I), and blocking of soluble AIM II protein binding by preincubation with LTβR-Fc fusion protein (FIG. 8J) or TR2-Fc fusion protein (FIG. 8K). FIG. 8L summarizes surface expression of LTβR and TR2 in various cell lines. The effect of LTβR-Fc or TR2-Fc fusion proteins on blocking sAIM II-mediated cytotoxicity in HT-29 cells. Cells were plated in 96 well plates and sAIM II (
10 ng / ml) in the presence of 5 U / ml IFNγ and varying amounts of sLTβR-F.
c (open circle LTβR-Fc alone, black circle LTβR-Fc and IFNγ
), Or a TR2-Fc fusion protein (open triangle TR-2Fc alone, black triangle TR2-Fc and sLTγ and IFNγ). Cells were incubated for 5 days, and cell viability was determined by XTT assay.

FIG. 8K is cell surface expression of LTβR or TR2 by FACS analysis using LTβR (FIGS. 8A-8D) or TR2 (FIGS. 8E-8H) mAb. MD
A-MB-231 (FIGS. 8A and 8E), HT-29 (FIGS. 8B and 8F), M
C-3 (Figures 8C and 8G), U93T (Figures 8D and 8H). MDA-MB-
FACS binding analysis of soluble AIM II protein alone in 231 cells (
8I), and blocking of soluble AIM II protein binding by preincubation with LTβR-Fc fusion protein (FIG. 8J) or TR2-Fc fusion protein (FIG. 8K). FIG. 8L summarizes surface expression of LTβR and TR2 in various cell lines. The effect of LTβR-Fc or TR2-Fc fusion proteins on blocking sAIM II-mediated cytotoxicity in HT-29 cells. Cells were plated in 96 well plates and sAIM II (
10 ng / ml) in the presence of 5 U / ml IFNγ and varying amounts of sLTβR-F.
c (open circle LTβR-Fc alone, black circle LTβR-Fc and IFNγ
), Or a TR2-Fc fusion protein (open triangle TR-2Fc alone, black triangle TR2-Fc and sLTγ and IFNγ). Cells were incubated for 5 days, and cell viability was determined by XTT assay.

FIG. 8L is cell surface expression of LTβR or TR2 by FACS analysis using LTβR (FIGS. 8A-8D) or TR2 (FIGS. 8E-8H) mAb. MD
A-MB-231 (FIGS. 8A and 8E), HT-29 (FIGS. 8B and 8F), M
C-3 (Figures 8C and 8G), U93T (Figures 8D and 8H). MDA-MB-
FACS binding analysis of soluble AIM II protein alone in 231 cells (
8I), and blocking of soluble AIM II protein binding by preincubation with LTβR-Fc fusion protein (FIG. 8J) or TR2-Fc fusion protein (FIG. 8K). FIG. 8L summarizes surface expression of LTβR and TR2 in various cell lines. The effect of LTβR-Fc or TR2-Fc fusion proteins on blocking sAIM II-mediated cytotoxicity in HT-29 cells. Cells were plated in 96 well plates and sAIM II (
10 ng / ml) in the presence of 5 U / ml IFNγ and varying amounts of sLTβR-F.
c (open circle LTβR-Fc alone, black circle LTβR-Fc and IFNγ
), Or a TR2-Fc fusion protein (open triangle TR-2Fc alone, black triangle TR2-Fc and sLTγ and IFNγ). Cells were incubated for 5 days, and cell viability was determined by XTT assay.

FIG. 8M is cell surface expression of LTβR or TR2 by FACS analysis using LTβR (FIGS. 8A-8D) or TR2 (FIGS. 8E-8H) mAb. MD
A-MB-231 (FIGS. 8A and 8E), HT-29 (FIGS. 8B and 8F), M
C-3 (Figures 8C and 8G), U93T (Figures 8D and 8H). MDA-MB-
FACS binding analysis of soluble AIM II protein alone in 231 cells (
8I), and blocking of soluble AIM II protein binding by preincubation with LTβR-Fc fusion protein (FIG. 8J) or TR2-Fc fusion protein (FIG. 8K). FIG. 8L summarizes surface expression of LTβR and TR2 in various cell lines. The effect of LTβR-Fc or TR2-Fc fusion proteins on blocking sAIM II-mediated cytotoxicity in HT-29 cells. Cells were plated in 96 well plates and sAIM II (
10 ng / ml) in the presence of 5 U / ml IFNγ and varying amounts of sLTβR-F.
c (open circle LTβR-Fc alone, black circle LTβR-Fc and IFNγ
), Or a TR2-Fc fusion protein (open triangle TR-2Fc alone, black triangle TR2-Fc and sLTγ and IFNγ). Cells were incubated for 5 days, and cell viability was determined by XTT assay.

FIG. 9 shows IFN-γ secretion by sAIM II-treated human PBL cells.
Human PBLs (5 × 10 ⁵ cells per well in 96-well plates) sAIM I with or without anti-CD3 mAb and IL-2 (20 U / ml).
Treated in the presence or absence of I for 5 days. Supernatants were then collected from the following groups of cells: PBL in the presence (black circles) or absence (black circles) of sAIMII, or with sAIMII (black triangles) or without (open triangles). Pause PBL. Human IFNγ concentration was determined by ELISA.

FIG. 10 shows a schematic diagram of the pHE4-5 expression vector (SEQ ID NO: 50) and the subcloned AIMII cDNA coding sequence. The position of the kanamycin resistance marker gene, AIMII coding sequence, oriC sequence, and lacIq coding sequence are indicated.

FIG. 11 shows the nucleotide sequence of the regulatory element of the pHE promoter (SEQ ID NO: 51). Two lac operator sequences, Shine-Delgarn
o sequence (S / D), and terminal HindIII and NdeI restriction sites (italic).

Figure 12 shows the MCA-38 AIM II conditioned medium, a sensorgram of specific binding to LTßR-Fc vs. MCIF-F _C immobilized on the BIAcore chip. Conditioned media was analyzed on a BIAcore instrument flow cell derivatized with lymphotoxin β receptor Fc fusion protein. Conditioned medium (100 μL)
Was flowed over the chip at 5 μL / min and was similarly washed with HBS buffer at 5 μL / min. The data shown represent the net bound (off-rate) area of the plot after binding of AIMII to the immobilized receptor and are measured in relative mass units (RU) vs. time. Binding conditions were performed at high receptor chip density under diffusion limited conditions. Description: LTβR-Fc and MCIF-Fc are LTβR, respectively.
-Refer to binding data from Fc or MCIF-Fc immobilized BIAcore chip surface.

FIG. 13 shows the determination of LTβR binding by AIM II elution from the LTβR-Fc column. The binding conditions were as described in Figure 11. Description: LTβR
And MCIF are LTβR-Fc or MCIF-Fc-immobilized BIA, respectively.
Reference binding data from core chip surface. Undiluted conditioned medium from MCA38 cells was treated with MCIF-Fc (post MCIF) and LTβR-Fc (L
Analysis was carried out before (pre) and after passing through the affinity column (after TβR). L
Fractions (1 mL) eluted from TβR (E4-6) and MCIF-Fc (E1-3) affinity columns were diluted 3-fold and LTβR BIAcore.
The binding to the chip was tested.

14A-14B show the sequence of TR6 and the aligned amino acid sequence of the cysteine rich motif. (A) shows the deduced amino acid sequence of TR6 (SEQ ID NO: 52). The signal sequence is underlined. Possible N-glycosylation sites (NCT
, Amino acid residues 173-175) are shaded and underlined. Recombinant TR
The N-terminal amino acid sequence of -6 (His) was read as VAETPT ---, indicating that the first 29 amino acids make up the signal sequence.

15A-C show the identity of membrane-bound TR6 ligands. HEK293EBN
A cells were transfected with pCEP4 control vector (hatched area) or pCEP4 / encoding full length AIM II (LIGHT) cDNA (solid line). The cells were treated with (A) HVEM / TR2-Fc (0.34 g), (B) LTβR-.
Incubated with Fc (0.34g), (C) TR6- (His) (0.34g) or buffer control (same as vector). Cells were stained with anti-hIgG-FITC to detect HVEM / TR2 and LTβR binding.
Cells were tested for anti-poly (His) and anti-mIgG-FI to detect TR6 binding.
Stained with TC. These were analyzed for binding by FACS.

16A and 16B show that in HT29 cells, TR6 was AIM II (L
(IGHT) is shown to inhibit cell death. HT2 in 96-well plate
9 cells with control medium, 10 U / ml IFN-γ alone, I
Purified sAIM II (sLI in the presence or absence of FN-γ (10 U / ml)
Purified sLT in the presence of IFN-γ (10 U / ml) and sAIM II (sLIGHT) (10 ng / ml) along with GHT) protein (10 ng / ml).
βR-Fc (200 ng / ml) or TR6- (His) (200 ng / ml)
) And incubated.

FIG. 17 shows the amino acid sequence and expression of mouse AIM II (LIGHT). The deduced amino acid sequence of mouse AIM II deduced from the cDNA sequence was aligned with the amino acid sequence of human AIM II (FIG. 17A). To obtain the optimal alignment, some gaps (-) were introduced using the ClustalW program using Macvector software (version 6.5). The region of homology was boxed and identical residues were shaded. The transmembrane region (TM) is underlined. An asterisk ( ^* ) indicates the position of the predicted N-glycosylation site. Amino acid numbers are shown to the side of the alignment. Using human 293 cells transfected with pmAIM II or pcDNA3 and stained with LTβR-Ig or anti-AIM II antibody, followed by FITC-conjugated goat anti-human IgG or anti-rabbit IgG, respectively (solid line), The data shown in FIGS. 17B-1 to 17B-4 were obtained. Staining with human IgGI or rabbit IgG was used as a control (hatched area (FIGS. 17B-1 to 17B-4)).

FIG. 17 shows the amino acid sequence and expression of mouse AIM II (LIGHT). The deduced amino acid sequence of mouse AIM II deduced from the cDNA sequence was aligned with the amino acid sequence of human AIM II (FIG. 17A). To obtain the optimal alignment, some gaps (-) were introduced using the ClustalW program using Macvector software (version 6.5). The region of homology was boxed and identical residues were shaded. The transmembrane region (TM) is underlined. An asterisk ( ^* ) indicates the position of the predicted N-glycosylation site. Amino acid numbers are shown to the side of the alignment. Using human 293 cells transfected with pmAIM II or pcDNA3 and stained with LTβR-Ig or anti-AIM II antibody, followed by FITC-conjugated goat anti-human IgG or anti-rabbit IgG, respectively (solid line), The data shown in FIGS. 17B-1 to 17B-4 were obtained. Staining with human IgGI or rabbit IgG was used as a control (hatched area (FIGS. 17B-1 to 17B-4)).

FIG. 18A shows the costimulatory activity of mouse AIM II. Purified T cells (1 x 1) in 96-well flat bottom microplates in the presence or absence of immobilized anti-CD3.
10 ⁶ cells / ml) and 5 × 10 ⁴ cells / ml of irradiated COS cells (this is pcD
Transfected with NA3 or pmAIM II plasmid for 72 hours) (
FIG. 18A), or the indicated dose of immobilized AIM II. Stimulated with either flag fusion protein (Figure 18B). The concentration of anti-CD3 mAb was 2 μg / ml in FIG. 18A and 0.2 μg / ml in FIG. 18B. C
Purified T cells (1 × 10 ⁶ cells / ml), either D28 ^{− / −} or CD28 ^{+ / +} spherical erythrocytes, were fixed AIM in the presence of immobilized anti-CD3 (0.2 μg / ml).
II. Flag fusion protein (4 μg / ml) or soluble anti-CD28 (
1 μg / ml) (FIG. 18C). In all assays, these cells were incubated for 72 hours and the proliferative response of T cells was monitored during the last 15 hours by incorporation of ³ H-TdR.

FIG. 18B shows the costimulatory activity of mouse AIM II. Purified T cells (1 x 1) in 96-well flat bottom microplates in the presence or absence of immobilized anti-CD3.
10 ⁶ cells / ml) and 5 × 10 ⁴ cells / ml of irradiated COS cells (this is pcD
Transfected with NA3 or pmAIM II plasmid for 72 hours) (
FIG. 18A), or the indicated dose of immobilized AIM II. Stimulated with either flag fusion protein (Figure 18B). The concentration of anti-CD3 mAb was 2 μg / ml in FIG. 18A and 0.2 μg / ml in FIG. 18B. C
Purified T cells (1 × 10 ⁶ cells / ml), either D28 ^{− / −} or CD28 ^{+ / +} spherical erythrocytes, were fixed AIM in the presence of immobilized anti-CD3 (0.2 μg / ml).
II. Flag fusion protein (4 μg / ml) or soluble anti-CD28 (
1 μg / ml) (FIG. 18C). In all assays, these cells were incubated for 72 hours and the proliferative response of T cells was monitored during the last 15 hours by incorporation of ³ H-TdR.

FIG. 18C shows costimulatory activity of mouse AIM II. Purified T cells (1 x 1) in 96-well flat bottom microplates in the presence or absence of immobilized anti-CD3.
10 ⁶ cells / ml) and 5 × 10 ⁴ cells / ml of irradiated COS cells (this is pcD
Transfected with NA3 or pmAIM II plasmid for 72 hours) (
FIG. 18A), or the indicated dose of immobilized AIM II. Stimulated with either flag fusion protein (Figure 18B). The concentration of anti-CD3 mAb was 2 μg / ml in FIG. 18A and 0.2 μg / ml in FIG. 18B. C
Purified T cells (1 × 10 ⁶ cells / ml), either D28 ^{− / −} or CD28 ^{+ / +} spherical erythrocytes, were fixed AIM in the presence of immobilized anti-CD3 (0.2 μg / ml).
II. Flag fusion protein (4 μg / ml) or soluble anti-CD28 (
1 μg / ml) (FIG. 18C). In all assays, these cells were incubated for 72 hours and the proliferative response of T cells was monitored during the last 15 hours by incorporation of ³ H-TdR.

FIG. 19A   FIG. 19A shows that injection of AIM II DNA resulted in increased cytolytic T cells (C
TL) response, and induce regression of established P815 tumors with memory tumor immunity.
And indicates. To obtain the data shown in Figures 19A-1 and 19A-2, DB
2 x 10 for A / 2 mouse^FiveCells P815 cells were inoculated on day 0 and then 7
, 9 and 11 days, pcDNA3 or pmA mixed with vehicle, liposomes
IM II was injected intratumorally. Seven days after the last injection, spleen cells were collected and
Restimulated with irradiated P815 cells for 4 days. CTL activity, effectors shown
-: Target (E / T) ratio of 4 hours standard for P815 and L1210 cells ⁵¹ It was evaluated by Cr release assay. Results are means ± S of triplicate wells
Shown as D. Similar results were obtained in three independent experiments.   To obtain the data shown in Figure 19B, DBA / 2 mice were treated with 2 x 10^FiveFine
Vesicles P815 cells were inoculated subcutaneously on day 0. 7 days later with palpable tumor mass
Mice, pcDNA3 or pmAIM II mixed with vehicle, liposomes
Were injected intratumorally. Injections were repeated on days 10, 14 and 17
. Tumor size was assessed by measuring the diameter and the longest diameter was 2 m.
Tumors larger than m were considered palpable tumors. Results have palpable tumor
It is shown as a percentage of the mice. Similar results were obtained in three independent experiments
It was   To obtain the data shown in Figures 19C-1 and 19C-2, pmAIM
2 × 10 in mice with regressed P815 tumors after II treatment^FiveP815 thin
Cells were intratumorally loaded on the right back and 40 days after the first tumor inoculation, an equal number of L1210 cells were
Vesicles were loaded into the left dorsal tumor. Nai receiving both P815 and L1210 loads
DBA / 2 mice in the control were used as controls. The size of the tumor
It was evaluated by measuring the straight diameter. Results are from 5 mice in each group
Shown as mean ± SD.

FIG. 19B   FIG. 19B shows that injection of AIM II DNA resulted in increased cytolytic T cells (C
TL) response, and induce regression of established P815 tumors with memory tumor immunity.
And indicates. To obtain the data shown in Figures 19A-1 and 19A-2, DB
2 x 10 for A / 2 mouse^FiveCells P815 cells were inoculated on day 0 and then 7
, 9 and 11 days, pcDNA3 or pmA mixed with vehicle, liposomes
IM II was injected intratumorally. Seven days after the last injection, spleen cells were collected and
Restimulated with irradiated P815 cells for 4 days. CTL activity, effectors shown
-: Target (E / T) ratio of 4 hours standard for P815 and L1210 cells ⁵¹ It was evaluated by Cr release assay. Results are means ± S of triplicate wells
Shown as D. Similar results were obtained in three independent experiments.   To obtain the data shown in Figure 19B, DBA / 2 mice were treated with 2 x 10^FiveFine
Vesicles P815 cells were inoculated subcutaneously on day 0. 7 days later with palpable tumor mass
Mice, pcDNA3 or pmAIM II mixed with vehicle, liposomes
Were injected intratumorally. Injections were repeated on days 10, 14 and 17
. Tumor size was assessed by measuring the diameter and the longest diameter was 2 m.
Tumors larger than m were considered palpable tumors. Results have palpable tumor
It is shown as a percentage of the mice. Similar results were obtained in three independent experiments
It was   To obtain the data shown in Figures 19C-1 and 19C-2, pmAIM
2 × 10 in mice with regressed P815 tumors after II treatment^FiveP815 thin
Cells were intratumorally loaded on the right back and 40 days after the first tumor inoculation, an equal number of L1210 cells were
Vesicles were loaded into the left dorsal tumor. Nai receiving both P815 and L1210 loads
DBA / 2 mice in the control were used as controls. The size of the tumor
It was evaluated by measuring the straight diameter. Results are from 5 mice in each group
Shown as mean ± SD.

FIG. 19C   FIG. 19C shows that injection of AIM II DNA resulted in increased cytolytic T cells (C
TL) response, and induce regression of established P815 tumors with memory tumor immunity.
And indicates. To obtain the data shown in Figures 19A-1 and 19A-2, DB
2 x 10 for A / 2 mouse^FiveCells P815 cells were inoculated on day 0 and then 7
, 9 and 11 days, pcDNA3 or pmA mixed with vehicle, liposomes
IM II was injected intratumorally. Seven days after the last injection, spleen cells were collected and
Restimulated with irradiated P815 cells for 4 days. CTL activity, effectors shown
-: Target (E / T) ratio of 4 hours standard for P815 and L1210 cells ⁵¹ It was evaluated by Cr release assay. Results are means ± S of triplicate wells
Shown as D. Similar results were obtained in three independent experiments.   To obtain the data shown in Figure 19B, DBA / 2 mice were treated with 2 x 10^FiveFine
Vesicles P815 cells were inoculated subcutaneously on day 0. 7 days later with palpable tumor mass
Mice, pcDNA3 or pmAIM II mixed with vehicle, liposomes
Were injected intratumorally. Injections were repeated on days 10, 14 and 17
. Tumor size was assessed by measuring the diameter and the longest diameter was 2 m.
Tumors larger than m were considered palpable tumors. Results have palpable tumor
It is shown as a percentage of the mice. Similar results were obtained in three independent experiments
It was   To obtain the data shown in Figures 19C-1 and 19C-2, pmAIM
2 × 10 in mice with regressed P815 tumors after II treatment^FiveP815 thin
Cells were intratumorally loaded on the right back and 40 days after the first tumor inoculation, an equal number of L1210 cells were
Vesicles were loaded into the left dorsal tumor. Nai receiving both P815 and L1210 loads
DBA / 2 mice in the control were used as controls. The size of the tumor
It was evaluated by measuring the straight diameter. Results are from 5 mice in each group
Shown as mean ± SD.

FIG. 20   FIG. 20 shows inhibition of GVHD by blockade of AIM II. (Fig. 20A and
And 20B) sub-lethal (4Gy) irradiated BDF1 mice were treated with 7 × 10 ⁷ Cells B6 splenocytes were injected intravenously. LTβR-Ig was added to recipient mice.
Alternatively, one of the control human IgG1s was added to -1, 2, 5, 8, 11, 14,
And on day 17 at 100 μg / mouse intravenously (control Ig
Was administered until day 11 because all mice died on day 12). Reshi
Pient survival (Figure 20A) and body weight (Figure 20B) were monitored daily.
. Results in FIG. 20B are shown as mean gram ± SEM of 5 mice in each group.
Express.   7x10 on day 0 in BDF1 mice not irradiated⁷B6 of cells (B6-F
1) Spleen cells or BDF1 (F1-F1) splenocytes were injected intravenously. Recipe en
Tomato mice were given -1 of either LTβR-Ig or control human IgG1.
On days 3, 5, 7, and 9, 100 μg / mouse was administered intravenously. On the 11th day
, Splenocytes were prepared from recipient mice, and P815 (H-2^d)and
EL-4 (H-2^b) For their CTL activity against⁵¹Cr release
In the output assay, assayed in vitro without further stimulation (FIG. 20).
C-1 and 20C-2).   7x10 on day 0 in BDF1 mice not irradiated⁷B6 LTα of cells^-/- Splenocytes or LT^{+ / +}Splenocytes were injected intravenously. BDF1 splenocytes (7 x 10⁷cell
) Was injected as a control (F1-F1).
On day 11, recipient spleen cells were evaluated for CTL activity as described above (FIG. 2).
0D-1 and 20D-2).   B6 LTα^-/-Purified T cells of splenocytes (1 x 10⁶Cells / ml) to 25 μ
Irradiated BA in the presence of g of LTβR-Ig or control human IgG1
LB / c splenocytes (1 x 10⁶Cells / ml). Five days later, C26 (
H-2^d) And EL-4 (H-2^b) Activity against⁵¹Cr release
It was tested in the output assay (FIGS. 20E-1 and 20E-2). The result is
Expressed as the mean ± SD of triplicate wells (FIGS. 20C-1 and 20C-2,
20D-1 and 20D-2, and 20E-1 and 20E-2).
.

FIG. 21 shows cytokine production by modulation of the AIM II costimulatory pathway.
Purified T cells (1 × 10 ⁶ cells / ml) were transferred to immobilized AIM II.I. Stimulation with flag fusion protein (3.2 μg / ml). After 48 hours, culture supernatants were collected and cytokine production was assessed by sandwich ELISA (FIGS. 21A-1 to 21A-4). 21B-1-2
The data shown in 1B-4 is for irradiated BALB / c in the presence of either 25 μg / ml of LTβR-Ig fusion protein or control human IgG1.
LTα cultured with spleen cells (1 × 10 ⁶ cells ^/ ml) ^- / ^- were generated using T cells purified from spleen cells (1 × 10 ⁶ cells / ml). After 72 hours, culture supernatants were collected and cytokine production was assessed by sandwich ELISA. IL-10 is undetectable (n, d.) (<0.3 ng) in all wells.
/ Ml) (Figs. 21B-1 to 21B-4).

[Sequence list]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 11/00 A61P 19/00 4H045 19/00 29/00 29/00 31/00 31/00 35/00 35/00 37/02 37/02 37/08 37/08 43/00 105 43/00 105 C07K 14/525 ZNA C07K 14/525 ZNA 16/24 16/24 19/00 19/00 C12P 21/02 C // C12N 15/09 C12R 1:91 C12P 21/02 A61K 37/02 (C12P 21/02 C12N 15/00 A C12R 1:91) (31) Priority claim number 60 / 142,657 (32) Priority date July 6, 1999 (July 1999) (33) Priority claiming country United States (US) (31) Priority claim number 60 / 148,326 (32) Priority date August 11, 1999 ( August 11, 1999) (33) Priority claiming country United States (US) (31) Priority claiming number 60/16 8,380 (32) Priority date December 2, 1999 (Dec. 2, 1999) (33) Priority claiming country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD) , RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ. , EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, K , KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, S I, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Ebner, Rainhard USA Maryland 20878, Gaithersburg, Shell Baan Terras Number 316 9906 (72) Inventor Yu, Guo Liang United States California 94705, Berkeley, Gravatt Drive 242 (72) Inventor Reuven, Stephen M. United States Maryland 20832, Ornay, Heritage Hills Driven 18528 (72) Inventor Tsai, Ifan United States Maryland 20879, Gaithersburg, Royal Woods Court 10330 (72) Inventor Ulrich, Stephen United States Maryland 20853, Rockville , Rams Head Coat 4713 F Term (Reference) 4B024 AA01 AA11 AA12 BA28 BA44 CA04 DA02 EA04 GA11 HA01 HA12 HA15 HA17 4B064 AG07 CA10 CA19 CC24 DA05 DA13 DA14 4C076 CC26 CC27 CC41 EE23 EE59 4C084 AA02 BA20 BA20 BA20 BA20 BA12 BA20 BA12 BA20 BA12 BA20 BA20 BAA17 BA01 ZB07 ZB26 4C085 AA14 BB11 BB42 CC02 CC23 CC32 4H045 AA10 AA11 AA30 BA10 BA41 BA57 CA40 DA14 DA76 DA86 EA22 EA28 EA29 FA50 FA74

Claims (29)

[Claims] 1. A method for treating graft-versus-host disease, immunodeficiency or autoimmunity, comprising a therapeutically effective amount of: (a) binding to a polypeptide consisting of amino acids 1-240 of SEQ ID NO: 2. A second therapeutic agent selected from the group consisting of: (b) the following: (1) a tumor necrosis factor; (ii) an immunosuppressive agent; (iii) an antibiotic. (Iv) anti-inflammatory agent; (v) chemotherapeutic agent; (vi) cytokine; (vii) angiogenic agent; and (viii) administering fibroblast growth factor to an individual. 2. The method of claim 1, wherein the first therapeutic agent and the second therapeutic agent are administered to the individual at the same time. 3. The method of claim 1, wherein the first therapeutic agent and the second therapeutic agent are administered to the individual at different times. 4. The method of claim 1, wherein the tumor necrosis factor is from the group consisting of: (a) TNF-α; (b) TNF-β; and (c) TNF-γ. The method chosen. 5. The method of claim 1, wherein the immunosuppressant is: (a) cyclosporine; (b) cyclophosphamide methylprednisone; (c) prednisone; (d) azathioprine. (E) FK-506; and (f) 15-deoxyspergualine. 6. The method of claim 1, wherein the cytokine is: (a) IL-2; (b) IL-3; (c) IL-4; (d) IL-. 5; (e) IL-6; (f) IL-7; (g) IL-10; (h) IL-12; (i) IL-13; (j) IL-15; and (k) IFN-. A method selected from the group consisting of γ. 7. A composition comprising: (a) a first therapeutic agent comprising an antibody that binds to a polypeptide consisting of amino acids 1-240 of SEQ ID NO: 2; and (b) a group consisting of: A second therapeutic agent selected from: (i) tumor necrosis factor; (ii) immunosuppressive agents; (iii) antibiotics; (iv) anti-inflammatory agents; (v) chemotherapeutic agents; (vi) cytokines; A composition comprising (vii) an angiogenic agent; and (viii) fibroblast growth factor. 8. The composition of claim 7, further comprising a pharmaceutically acceptable carrier or excipient. 9. A method for treating an individual having a disorder selected from the group consisting of: (a) graft versus host disease; (b) immunodeficiency; and (c) autoimmunity. Wherein the method comprises the step of administering to the individual a therapeutically effective amount of the composition of claim 7. 10. A method for treating an immune deficiency or cancer comprising: (a) an isolated polypeptide comprising amino acid residues at least 90% identical to amino acids 83-240 of SEQ ID NO: 2. A first therapeutic agent contained; and (b) a second therapeutic agent selected from the group consisting of: (i) an antibiotic; (ii) an anti-inflammatory agent; (iii) an angiogenic agent; and (Iv) a method comprising administering to an individual a therapeutically effective amount of fibroblast growth factor. 11. The method of claim 10, wherein the isolated polypeptide is fused to an Fc immunoglobulin polypeptide. 12. The method of claim 10, wherein the first therapeutic agent and the second therapeutic agent are administered to the individual at the same time. 13. The method of claim 10, wherein the first therapeutic agent and the second therapeutic agent are administered to the individual at different times. 14. The method of claim 10, wherein the antibiotic agent is: (a) amoxicillin; (b) clindamycin; (c) chloramphenicol; A method selected from the group consisting of: profloxacin; (e) erythromycin; (f) rifampin; (g) streptomycin; (h) tetracycline; and (i) vancomycin. 15. A method for treating an immune deficiency or cancer comprising: (a) an isolated polypeptide comprising amino acid residues at least 90% identical to amino acids 60-240 of SEQ ID NO: 2. A first therapeutic agent contained; and (b) a second therapeutic agent selected from the group consisting of: (i) an antibiotic; (ii) an anti-inflammatory agent; (iii) an angiogenic agent; and (Iv) a method comprising administering to an individual a therapeutically effective amount of fibroblast growth factor. 16. A composition, which comprises: (a) a first therapeutic agent comprising an isolated polypeptide comprising amino acid residues at least 90% identical to amino acids 83-240 of SEQ ID NO: 2. And (b) a second therapeutic agent selected from the group consisting of: (i) an antibiotic; (ii) an anti-inflammatory agent; (iii) an angiogenic agent; and (iv) a fibroblast growth factor. A composition comprising: 17. The composition of claim 16, further comprising a pharmaceutically acceptable carrier or excipient. 18. The composition of claim 16, wherein the isolated polypeptide is fused to an Fc immunoglobulin polypeptide. 19. A method for the treatment of an individual having a disorder selected from the group consisting of: (a) immunodeficiency; and (b) cancer, wherein the method comprises a therapeutically effective amount. 17. A method comprising the step of administering the composition of claim 16 to an individual. 20. An isolated polypeptide containing amino acid residues at least 90% identical to amino acids 83-240 of SEQ ID NO: 2; wherein said polypeptide is covalently linked to polyethylene glycol, Polyethylene glycol is 2000, 3000, 4000, 5000, 6000, 7
000, 8000, 9000, 10,000, 15,000 and 20,000
An isolated polypeptide having an average molecular weight selected from the group consisting of: 21. The isolated polypeptide of claim 20, wherein the polypeptide is polyethylene glycol and 1-3, 2-4, 3-5,
Isolated having an average degree of substitution classified within a range selected from the group consisting of 4-6, 5-7, 6-8, 7-9, 8-10, 9-11 and 10-12. Polypeptide. 22. The isolated polypeptide of claim 20, produced by a recombinant host cell. 23. The isolated polypeptide of claim 20, which contains a heterologous polypeptide. 24. A composition comprising the isolated polypeptide of claim 20 and a pharmaceutically acceptable carrier. 25. An isolated polypeptide containing an amino acid residue that is at least 95% identical to amino acids 83-240 of SEQ ID NO: 2, wherein the polypeptide is covalently linked to polyethylene glycol, The polyethylene glycol is 2000, 3000, 4000, 5000, 6000, 7
000, 8000, 9000, 10,000, 15,000 and 20,000
An isolated polypeptide having an average molecular weight selected from the group consisting of: 26. An isolated polypeptide comprising amino acids 83-240 of SEQ ID NO: 2; wherein the polypeptide is covalently linked to polyethylene glycol, wherein the polyethylene glycol is 2000, 3000, 4000. 5,000, 6000, 7
000, 8000, 9000, 10,000, 15,000 and 20,000
An isolated polypeptide having an average molecular weight selected from the group consisting of: 27. An isolated polypeptide containing an amino acid residue that is at least 90% identical to amino acids 60-240 of SEQ ID NO: 2, wherein the polypeptide is covalently linked to polyethylene glycol, The polyethylene glycol is 2000, 3000, 4000, 5000, 6000, 7
000, 8000, 9000, 10,000, 15,000 and 20,000
An isolated polypeptide having an average molecular weight selected from the group consisting of: 28. An isolated polypeptide containing an amino acid residue that is at least 95% identical to amino acids 60-240 of SEQ ID NO: 2, wherein the polypeptide is covalently linked to polyethylene glycol, The polyethylene glycol is 2000, 3000, 4000, 5000, 6000, 7
000, 8000, 9000, 10,000, 15,000 and 20,000
An isolated polypeptide having an average molecular weight selected from the group consisting of: 29. An isolated polypeptide containing amino acids 60-240 of SEQ ID NO: 2; wherein the polypeptide is covalently linked to polyethylene glycol, wherein the polyethylene glycol is 2000, 3000, 4000. 5,000, 6000, 7
000, 8000, 9000, 10,000, 15,000 and 20,000
An isolated polypeptide having an average molecular weight selected from the group consisting of: