WO2004017991A1 - Use of iap for the diagnosis and of iap-inhibitors for the treatment of hodgkin’s lymphomas - Google Patents

Abstract

A novel use of inhibitors of IAPs (inhibitors of apoptosis) in compositions for the treatment of Hodgkin’s lymphomas is described. In particular, XIAP-inhibitors are disclosed for methods of treatment of Hodgkin’s lymphomas. Furthermore, diagnostic methods for the detection of Hodgkin’s lymphomas using IAP specific agents are provided.

Description

Use of IAP for the diagnosis and of IAP-Inhibitors for the treatment of Hodgkin's

Lymphomas

The present invention is directed to the use of IAP (inhibitors of apoptosis)-inhibitors for the diagnosis or treatment of Hodgkin's lymphomas and in particular to the use of XIAP- inhibitors. The present invention is further directed to methods for the diagnosis of Hodgkin's lymphomas.

Apoptosis represents an efficient elimination of unwanted cells which is carried out by a family of aspartate-specific proteases, termed caspases. Suppression of caspases contributes to the pathogenesis of cancer by several mechanisms. Apoptosis is an active process of cellular self-destruction with distinctive morphological and biochemical features and is essential for the development and maintenance of multicellular organisms. The principle effectors of apoptotic signaling are caspases, aspartate-specific endoproteases, which are activated upon various apoptotic stimuli. This activation usually occurs via cleavage of the proenzyme at specific aspartate residues to generate the active enzyme '. As a consequence, the active enzymes cleave numerous cellular proteins necessary for cellular homeostasis, which results in the typical morphological hallmarks of apoptosis ². At least two distinct major apoptotic signaling pathways have been identified. The triggering of death domain containing cell surface receptors of the tumor necrosis factor (TNF) -super family results in the recruitment and activation of the initiator caspase, caspase-8, followed by a rapid cleavage of caspase-3 and -7, which in turn cleave vital substrates in the cell ³'⁴. The second apoptotic signaling pathway involves mitochondria and results in the release of pro-apoptotic factors from mitochondria, such as cytochrome c and Smac/DIABLO ^5"7. Subsequently, the released cytochrome c, the cytosolic Apaf-1 (apoptosis protease-activating factor), and procaspase-9 form the apoptosome, the mitochondrially controlled initiator complex of apoptosis ' . Activation of caspase-9 is induced by dimerization ¹⁰ driven by formation of the multimeric Apaf-1 complex ^π. Once activated, caspase-9 can directly process and activate caspase-3 and -7. Both pathways converge at the level of caspase-3, which is expressed in cells as an inactive 32-kDa precursor and is cleaved into 17-kDa and 12-kDa subunits of the mature caspase-3 during apoptosis ¹². The processing of caspase-3 occurs in two steps, beginning with a site specific cleavage by caspase-8 or -9 to form two subunits. Following this cleavage, caspase-3 then removes its own prodomain in two sequential steps generating the ultimate pl7 large subunit ^13"17. The activity of the caspases is modulated by another set of proteins, the IAPs (inhibitor of apoptosis) ¹⁸. One of these, XIAP, binds to and inhibits caspase-3, -7 and -9, but not caspase- 8¹⁹. In the presence of XIAP the first cut of procaspase-3 can occur but not the second autocatalytic cleavage, due to the inhibition of intrinsic caspase-3 activity ^14"17. Failure to remove the caspase-3 prodomain as a result of this partial cleavage serves as a "footprint" of XIAP action ²⁰'²¹.

Additional regulation is provided by Smac (second mitochondria-derived activator of caspases) or its murine homolog DIABLO (direct IAP-binding protein with low pi), which binds to IAPs and abrogates caspase inhibition

. The proform of Smac contains a N- terminal sequence, that targets this protein to the intermembrane space of mitochondria. Upon induction of apoptosis, Smac is released into the cytosol with similar kinetics to cytochrome c and modulates apoptosis ²²'²³. Like cytochrome c, the release of Smac is under the control of Bcl-2 family proteins, suggesting that both molecules may leave mitochondria via the same route ' . Smac promotes apoptosis by repressing the anti-caspase activity of XIAP, thereby promoting the enzymatic activity of mature caspase-3. This function depends on the ability of Smac to interact physically with IAPs. The N-terminal part of Smac shares significant homology with the conserved class of IAP-binding motifs ²⁴. It is likely that the N-terminus of the Smac protein simply displaces the bound caspase and releases the activated enzyme ²³'²⁵.

Apoptosis in the immune system is a fundamental process regulating lymphocyte maturation, receptor repertoire selection and homeostasis. Death by apoptosis is as essential for the function of lymphocytes as growth and differentiation. The ability to resist induction of

, apoptosis is one of the keys to cancer cell survival. The malignant Hodgkin and Reed- Steinberg (H-RS) cells of Hodgkin's lymphoma (HL) are germinal center B-cells with rearranged but nonproductive immunoglobulin genes ²⁶'²⁷. However, through an as yet unknown mechanism, H-RS cells resist the apoptotic fate characteristic of defective B-cells with crippled immunoglobulin genes. So, the malignant Hodgkin and Reed-Sternberg (H-RS) cells of Hodgkin's lymphoma (HL) were shown to be resistant to different apoptotic stimuli.

Therefore, it is an object of the present invention to provide a strategy to induce apoptotic cell death in Hodgkin's lymphomas. It is a further object of the present invention to provide an improved method for the diagnosis of the Hodgkin's lymphoma in vivo and ex vivo. These objects are solved by providing the embodiments characterized in the claims and described further below.

The present invention is based on the discovery that IAP's, for example X chromosome linked IAP (XIAP), are constitutively overexpressed, in both HL-derived B-cell lines and in primary HL tissues, and that in B-cell lines it is associated with and inhibits in particular caspase-3. Based on this finding, the present invention provides the basis for a new therapy of the Hodgkin's lymphoma, which is based on an inhibition of IAP's, in particular XIAP, in order to reduce or eliminate the caspase inhibiting activity thereof. As a result, this approach leads to the restoration of apoptotic mechanisms, wliich results in an increased number of cell death events in malignant HL cells. Since IAPs, in particular XIAP, are not overexpressed in normal tissues, the present invention provides a selective therapy for the HL.

Apart from the therapeutical aspect explained above, the discovery that IAP's, in particular XIAP, is constitutively overexpressed, in both HL-derived B-cell lines, and in primary HL tissues, provides a novel diagnostic approach for the in vivo or ex vivo diagnosis of tissues, which are suspected as being afflicted by HL.

Therefore, according to a general aspect, the present invention is directed to the use of IAP for the diagnosis of Hodgkin's lymphomas and to the use of IAP-inhibitors for the treatment of Hodgkin's lymphomas. Preferably, said IAP inhibitors are XIAP inhibitors.

IAP proteins initially were identified in baculovirus cells as proteins that inhibited apoptosis of insect cells infected with the virus (Crook et al., J. Virol. 61 (1993), 2168-2174). As an example, the sequence of XIAP is well known and has been published by Liston et al., Nature 379 (1996), 349-353, and the sequence data can be obtained from NCBI Genbank accession no. U45880; see also SEQ ID NO: 1 and 2.

IAP proteins have a caspase inhibitory activity. Specifically, IAP proteins such as XIAP reduce or prevent apoptosis by inhibiting activation of pro-caspases and by inhibiting caspase activity. As used herein, reference to an IAP protein as an "inhibitor of caspase activation" or "inhibitor of caspase activity" or as having "caspase inhibitory activity" means that the proteolytic activity of a caspase in the presence of the LAP or when bound to the IAP is less than it would be in the absence of the IAP or in the absence of IAP binding. This caspase inhibitory activity of an IAP can be due to a) inhibition of an upstream caspase required for proteolytic activation of a downstream caspase; b) inhibition of the completion of caspase processing by the IAP; or c) a direct inhibitory effect of the IAP on caspase proteolytic activity.

The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of partially or completely curing a disease and/or adverse effect attributed to the disease. The term "treatment" as used herein covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e. arresting its development; or (c) relieving the disease, i.e. causing regression of the disease.

Furthermore, the term "subject" as employed herein relates to animals in need of amelioration, treatment and/or prevention of Hodgkin lymphoma. Most preferably said subject is a human.

The terms "antagonist/inhibitor" in accordance with the present invention include chemical agents that modulate the action of IAP, either through altering its enzymatic or biological activity or through modulation of expression, e.g., by affecting transcription or translation. In some cases the antagonist/inhibitor may also be a substrate or ligand binding molecule.

The term "inhibitor" includes both substances which reduce the activity of the IAP and these which nullify it altogether. When more than one possible activity is defined herein for IAP, the inhibitor or activator may modulate any or all of IAP activities. An "antagonist" or "agonist" that modulates the activity of IAP and causes for example a response in a cell based assay refers to a compound that alters directly or indirectly the activity of IAP or the amount of active IAP. Typically, the effect of an antagonist is substantially the same as that of Smac or Smac-derived agonistic peptide described further below. Antagonists include competitive as well as non-competitive antagonists. A competitive antagonist (or competitive blocker) interacts with or near the site specific for agonist binding. A non-competitive antagonist or blocker inactivates the function of the receptor by interacting with a site other than the agonist interaction site. Preferably, the antagonist/inhibitor of IAP are small chemical agents which directly interact with IAP. Therefore, there will preferably be a direct relationship between the molar amount of compound required to inhibit or stimulate LAP activity and the molar amount of IAP present or lacking in the cell. IAP antagonists may be peptides, proteins, nucleic acids, antibodies, small organic compounds, peptide mimics, aptamers or PNAs (Milner, Nature Medicine 1 (1995), 879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Cell 79 (1994), 193-198; Gold, Ann. Rev. Biochem. 64 (1995), 736-797). For the preparation and application of such compounds, the person skilled in the art can use the methods known in the art, for example those referred to herein.

According to a preferred embodiment, said IAP-inhibitors are used in an amount so that caspase induced apoptotic signalling is restored in Hodgkin's lymphoma cells. Preferably, caspase-3, -7 and/or -9 are activated.

Caspases can be divided into two main classes: initiator and effector caspases. Initiator caspases (like caspase-9) are the upstream activators of the effector caspases (like caspase-3). Effector caspases cleave the proteins that actually induce apoptosis in the cell. These cleavages lead to morphological features like membrane blebbing, cytoplasmic and nuclear condensation, DNA fragmentation, and the formation of apoptotic bodies. They initially exist as immature pro-caspases. The pro-caspases lack caspase activity; caspase activation occurs due to proteolytic processing of the pro-caspase. For example, caspase-3 is a heterotetramer composed of approximately 17-20 kDa and 11 kDa polypeptides that are formed by proteolysis of a 32 kDa polypeptide precursor, pro-caspase-3. Cleavage of the pro- caspase-3 proceeds in two steps. The first cleavage results in production of a partially processed large subunit (22-24 kDa) that still contains the pro-domain, and a smaller, fully processed, subunit of about 11 kDa. In the second step, the pro-domain is cleaved from the partially processed large subunit, probably by an autocatalytic process, to produce the 17-20 kDa mature, fully processed large subunit of the active caspase-3 enzyme. Removal of the pro-domain, however, is not necessary for protease activation, as the partially processed caspase also has caspase activity.

The IAP-inhibitors of the present invention are preferably selected from the group consisting of a molecule that reduces the level of mRNA encoding IAP, a molecule that reduces the level of LAP, a molecule that inhibits the binding of an LAP to a caspase, and a molecule that reduces the biological activity of IAP. Said molecule preferably is selected from the group consisting of an antisense nucleic acid, siRNA, a ribozyme, an anti-IAP antibody, an anti-IAP aptamer, small molecules, a peptide and a peptidomimetic.

According to a preferred aspect, the antisense nucleic acid is a nucleic acid, which selectively hybridizes to transcriptional products of the nucleic acid encoding an IAP under moderate stringent conditions. According to the invention an antisense nucleic acid is used as an XIAP- inhibitor, which hybridizes to transcriptional products of SEQ ID NO: 1 or variants thereof. Said variants are each defined as having one or more substitutions, insertions and/or deletions as compared to the sequence of SEQ ID NO: 1, provided that said variants hybridize under moderately stringent conditions to a nucleic acid which comprises the sequence of SEQ ID NO: 1 and further provided that said variants code for XIAP or provided that said variants comprise nucleic acid changes due to the degeneracy of the genetic code, which code for the same or a functionally equivalent amino acid as the nucleic acid sequence of SEQ ID NO:l (i.e. the XIAP amino acid sequence presented in SEQ ID NO: 2). Said antisense nucleic acids of the present invention are used to hybridize to transcriptional products of SEQ ID NO: 1, e.g. mRNA, as well as to nucleic acids, which selectively hybridize to said transcriptional products of the nucleic acids under moderate stringent conditions. It is noted that SEQ ID NO: 1 provides the genomic sequence of the XIAP gene.

The term "nucleic acid sequence" refers to a heteropolymer of nucleotides or the sequence of these nucleotides. The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to a heteropolymer of nucleotides.

Stringency of hybridization, as used herein, refers to conditions under which polynucleotide duplexes are stable. As known to those of skill in the art, the stability of duplex is a function of sodium ion concentration and temperature (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 2^nd Ed. (Cold Spring Harbor Laboratory, (1989)). Stringency levels used to hybridize can be readily varied by those of skill in the art. The phrase "low stringency hybridization" refers to conditions equivalent to hybridization in 10% formamide, 5 x Denhart's solution, 6 x SSPE, 0.2% SDS at 42°C, followed by washing in 1 x SSPE, 0.2% SDS, at 50°C Denhart's solution and SSPE (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989) are well known to those of skill in the art as are other suitable hybridization buffers. As used herein, the phrase "moderately stringent hybridization" refers to conditions that permit DNA to bind a complementary nucleic acid that has about 60% identity, preferably about 75% identity, more preferably about 85% identity to the DNA; with greater than about 90%) identity to said DNA being especially preferred. Preferably, moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5 x Denhart's solution, 5 x SSPE, 0.2% SDS at 42°C, followed by washing in 0.2 x SSPE, 0.2% SDS, at 65°C.

The phrase "high stringency hybridization" refers to conditions that permit hybridization of only those nucleic acid sequences that form stable duplex in 0.018M NaCI at 65°C. (i.e., if a duplex is not stable in 0.018M NaCI at 65.degree°C, it will not be stable under high stringency conditions, as contemplated herein). High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5 x Denhart's solution, 5 x SSPE, 0.2% SDS at 42°C, followed by washing in 0.1 x SSPE, and 0.1% SDS at 65°C.

According to a further embodiment, the antisense nucleic acids of the present invention provide antisense DNA or RNA. Antisense DNA or R A molecules bind specifically with a targeted RNA message, interrupting the expression of the mRNA product. The antisense binds to the messenger RNA forming a double stranded molecule that cannot be translated by the cell. Typically, an antisense oligonucleotide is about 15-25 nucleotides in length. In addition, chemically reactive groups, such as iron-linked ethylenediaminetetraacetic acid (EDTA-Fe), can be attached to an antisense oligonucleotide, causing cleavage of the mRNA at the site of hybridization. These and other uses of antisense methods to inhibit the translation of nucleic acid are well known in the art (Marcus-Sakura, Anal. Biochem. 172 (1988), 289).

Nucleic acid molecules specifically hybridizing to LAP encoding genes and/or their regulatory sequences may be used for repression of expression of said gene, for example due to an antisense or triple helix effect or they may be used for the construction of appropriate ribozymes (see, e.g., EP-B1 0 291 533, EP-A1 0 321 201, EP-A2 0 360 257) which specifically cleave the (pre)-mRNA of a gene encoding IAP. The nucleic acid sequence encoding IAP is known in the art; see references supra. Selection of appropriate target sites and corresponding ribozymes can be done as described for example in Steinecke, Ribozymes, Methods in Cell Biology 50, Galbraith et al. eds Academic Press, Inc. (1995), 449-460. In another embodiment the dsRNA or RNAi approach is used for inhibiting expression of IAP. dsR A between 21 and 23 nucleotides in length is preferred. The dsRNA molecule can also contain a terminal 3 '-hydroxyl group and may represent an analogue of naturally occurring RNA, differing from the nucleotide sequence of said gene or gene product by addition, deletion, substitution or modification of one or more nucleotides. General processes of introducing an RNA into a living cell to inhibit gene expression of a target gene in that cell comprising RNA with double-stranded structure, i.e. dsRNA or RNAi are known to the person skilled in the art and are described, for in WO99/32619, WO01/68836, WO01/77350, WO00/44895, WO02/055692 and WO02/055693, the disclosure content of which is hereby incorporated by reference. Furthermore, the so-called "peptide nucleic acid" (PNA) technique can be used for the inhibition of the expression of a gene encoding an IAP .

According to a preferred embodiment, such a antisense nucleic acid can be used also for diagnostic purposes, and then, the antisense nucleic acid is used as a probe. Nucleic acid hybridization techniques can be used to identify transcriptional products of the nucleic acid sequence of XIAP (SEQ ID NO: 1). Since it has been found by the present inventors that XIAP is constitutively overexpressed in HL-cells, a probe hybridizing to transcriptional products of the XIAP gene can - preferably in vitro - serve as a valuable tool for the diagnosis of HL. Briefly, any nucleic acid having some homology to a transcriptional product set forth in this invention, or fragment thereof, can be used as a probe to identify a similar nucleic acid by hybridization under conditions of moderate to high stringency. Such similar nucleic acid then can be isolated, sequenced, and/or quantified in order to determine an overexpression in the tissues suspected to be HL tissues.

Hybridization can be done by Southern or Northern analysis to identify a DNA or RNA sequence, respectively, that hybridizes to a probe. The probe can be labeled with a radioisotope such as ³²P, an enzyme, digoxygenin, or by biotinylation. The DNA or RNA to be analyzed can be electrophoretically separated on an agarose or polyacrylamide gel, transferred to nitrocellulose, nylon, or other suitable membrane, and hybridized with the probe using standard techniques well known in the art such as those described in sections 7.39-7.52 of Sambrook et al., (1989) Molecular Cloning, 2^nd edition, Cold Spring Harbor Laboratory, Plainview, NY.

Typically, a probe is at least about 20 nucleotides in length. For example, a probe corresponding to a 20 nucleotide sequence set forth in this invention can be used to identify a nucleic acid identical to or similar to a nucleic acid sequence set forth in the group of nucleic acids of the present invention. In addition, probes longer or shorter than 20 nucleotides can be used.

According to a further preferred embodiment the present invention provides an antibody, which is directed against IAP or a mutant, variant or fragment of IAP, as disclosed above. The term "antibody" as used herein refers to intact antibodies as well as antibody fragments that retain some ability to selectively bind an epitope. Such fragments include, without limitation, Fab, F (ab') 2, and Fv antibody fragments. The term "epitope" refers to an antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules (e. g., amino acid or sugar residues) and usually have specific three dimensional structural characteristics as well as specific charge characteristics. Any antibody having specific binding affinity for an amino acid encoded by SEQ ID NO: 1 may be used for the detection of XIAP overexpression in tissues. Thus, any monoclonal or polyclonal antibody having specific binding affinity for a herein defined amino acid sequence, preferably SEQ ID NO: 2, may preferably be used.

Antibodies within the scope of the invention can be prepared using any method. For example, any substantially pure protein provided herein, or fragment thereof, can be used as an immunogen to elicit an immune response in an animal such that specific antibodies are produced. Thus, an intact full-length protein or fragments containing small peptides can be used as an immunizing antigen. In addition, the immunogen used to immunize an animal can be chemically synthesized or derived from translated cDNA. Further, the immunogen can be conjugated to a carrier polypeptide, if desired. Commonly used carriers that are chemically coupled to an immunizing polypeptide or protein include, without limitation, keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. The preparation of polyclonal antibodies is well-known to those skilled in the art. See, e. g., Green et al., Production of Polyclonal Antisera, in Immunochemical Protocolls (Manson, ed.), pages 1-5 (Humana Press 1992) and Coligan et al., Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters, in Current Protocolls In Immunology, section 2.4.1 (1992). In addition, those of skill in the art will know of various techniques common in the immunology arts for purification and concentration of polyclonal antibodies, as well as monoclonal antibodies (Coligan, et al, Unit 9, Current Protocols in Immunology, Wiley Interscience, 1994).

The preparation of monoclonal antibodies also is well-known to those skilled in the art; see, e.g., Kohler & Milstein, Nature 256 (1975), 495; Coligan et al., sections 2.5.1-2.6.7; and Harlow et al., Antibodies: A Laboratory Manual, page 726 (Cold Spring Harbor Pub. 1988). Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by analyzing a serum sample, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures. Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, e. g., Coligan et al., sections 2.7.1-2.7.12 and sections Immunoglobulin G (IgG), in Methods In Molecular Biology, Vol. 10, pages 79- 104 (Humana Press 1992).

In addition, methods of in vitro and in vivo multiplication of monoclonal antibodies are well- known to those skilled in the art. Multiplication in vitro can be carried out in suitable culture media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium, optionally replenished by mammalian serum such as fetal calf serum, or trace elements and growth- sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, and bone marrow macrophages. Production in vitro provides relatively pure antibody preparations and allows scale-up to yield large amounts of the desired antibodies. Large scale hybridoma cultivation can be carried out by homogenous suspension culture in an airlift reactor, in a continuous stirrer reactor, or in immobilized or entrapped cell culture. Multiplication in vivo may be carried out by injecting cell clones into mammals histocompatible with the parent cells (e. g., osyngeneic mice) to cause growth of antibody-producing tumors. Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. After one to three weeks, the desired monoclonal antibody is recovered from the body fluid of the animal.

The antibodies which are of use in the present invention also can be made using non-human primates. General techniques for raising therapeutically useful antibodies in baboons can be found, for example, in Goldenberg et al., International Patent Publication WO91/11465 (1991) and Losman et al, Int. J Cancer 46 (1990), 310. Alternatively, the antibodies can be "humanized" monoclonal antibodies. Humanized monoclonal antibodies are produced by transferring mouse complementarity determining regions (CDRs) from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then substituting human residues in the framework regions of the murine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions when treating humans. General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al., Proc. Natl. Acad. Sci. USA 86 (1989), 3833. Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al, Nature 321 (1986), 522; Riechmann et al, Nature 332 (1988), 323; Nerhoeyen et al, Science 239 (1988),:1534; Carter et al, Proc. Νatl. Acad. Sci. USA 89 (1992), 4285; Sandhu, Crit. Rev. Biotech. 12 (1992), 437 and Singer et al, J. Immunol. 150 (1993), 2844.

Antibodies of the present invention also may be derived from human antibody fragments isolated from a combinatorial immunoglobulin library; see, for example, Barbas et al, Methods: A Companion To Methods In Enzymology, Vol. 2 (1991), page 119 and Winter et al, Ann. Rev. Immunol. 12 (1994), 433. Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained, for example, from STRATAGEΝE Cloning Systems (La Jolla, CA, USA).

In addition, antibodies of the present invention may be derived from a human monoclonal antibody. Such antibodies are obtained from transgenic mice that have been "engineered" to produce specific human antibodies in response to antigenic challenge. In this technique, elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy and light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens and can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described by Green et al, Nature Genet. 7 (1994), 13; Lonberg et al, Nature 368 (1994), 856 and Taylor et al, Int. Immunol. 6 (1994), 579.

All mentioned antibodies may additionally be linked to a toxic agent and/or to a detectable agent. Alternatively, the present invention provides aptamers, which are directed against an LAP, preferably XIAP or a mutant, fragment or variant thereof. Aptamers are DNA or RNA molecules that have been selected from random pools based on their ability to bind other molecules. Aptamers have been selected which bind nucleic acid, proteins, small organic compounds, and even entire organisms. Aptamers in the form of so called Spiegelmers have been described by Nater and Klussmann, Cur. Opin. Drug Disc. Devel. 6 (2003), 253-261.

According to a particular preferred embodiment, the peptide used as an IAP-inhibitor is a second mitochondria-derived activator of caspase (Smac) protein. The Smac protein is an already well known inhibitor of IAP's and is also described in WO02/16418, which is incorporated herein in its entirety by this reference. The nucleotide and amino acid sequences of Smac and peptides thereof can be obtained from ΝCBI Genbank accession nos. ΝM_138930, NM 38929 and NM_019887. Furthermore, compositions and methods for regulating apoptosis such peptides and peptidomimetics capable of modulating apoptosis through their interaction with cellular IAPs (inhibitor of apoptosis proteins) are described in WO02/26775. Those peptides and mimetics are based on the N-terminal tetrapeptide of IAP- binding proteins, such as Smac/DIABLO, Hid, Grim and Reaper, which interact with a specific surface groove of LAP. Further IAP inhibitors based on small molecules mimicking the natural binding partners are described in Kipp et al, Biochemistry 41 (2002), 7344-7349. Peptides targeting the X-inhibitor of apoptosis protein (XIAP) can be identified by phage library screening using recombinant full-length human XIAP or a fragment containing only the baculovirus IAP repeat 2 (BIR2) domain; see, e.g., Tamm et al, J. Biol. Chem. 278 (2003), 14401-14405.

In this context it should be noted that in accordance with the present invention the IAP inhibitor may also be expressed in the target HL cells and/or be induced to be expressed. For example, it has recently been shown that overexpression of second mitochondria-derived activator of caspase/direct IAP-binding protein with low pi (Smac/DIABLO) sensitizes prostate cancer cells to Apo2L/TRAIL-mediated apoptosis; see Ng and Bonavida, Mol. Cancer Ther. 1 (2002), 1051-1058. Accordingly, the IAP inhibitor to be used in accordance with the present invention can also be the gene encoding said IAP inhibitor, i.e. use of a LAP inhibitor encoding cDNA operably linked to a promoter (see also Jia et al, Oncogene 22 (2003), 1589-1599), or a transcriptional activator of the endogenous gene encoding said IAP inhibitor. Accordingly, it is envisaged by the present invention that nucleic acid molecules encoding IAP inhibitors may be stably integrated into the genome of a target cell of subject or may be maintained in a form extrachromosomally, see, e.g., Calos, Trends Genet. 12 (1996), 463-466. On the other hand, viral vectors described in the prior art may be used for transfecting certain cells, tissues or organs. Furthermore, it is possible to use a pharmaceutical composition of the invention which comprises a nucleic acid molecule encoding a IAP in gene therapy. Suitable gene delivery systems may include liposomes, receptor-mediated delivery systems, naked DNA, and viral vectors such as herpes viruses, retroviruses, adenoviruses, and adeno- associated viruses, among others. Delivery of nucleic acid molecules to a specific site in the body for gene therapy may also be accomplished using a biolistic delivery system, such as that described by Williams (Proc. Natl. Acad. Sci. USA 88 (1991), 2726-2729). Standard methods for transfecting cells with nucleic acid molecules are well known to those skilled in the art of molecular biology, see, e.g., WO 94/29469. Gene therapy to prevent or decrease the development of diseases described herein may be carried out by directly administering the nucleic acid molecule encoding an IAP inhibitor to a patient or by transfecting cells with said nucleic acid molecule ex vivo and infusing the transfected cells into the patient. Furthermore, research pertaining to gene transfer into cells of the germ line is one of the fastest growing fields in reproductive biology. Gene therapy, which is based on introducing therapeutic genes into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer. Suitable vectors and methods for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Wang, Nature Medicine 2 (1996), 714-716; WO94/29469; WO 97/00957 or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640, and references cited therein. The nucleic acid molecules comprised in the pharmaceutical composition of the invention may be designed for direct introduction or for introduction via liposomes, or viral vectors (e.g. adenoviral, retroviral) containing said nucleic acid molecule into the cell. Preferably, said cell is a germ line cell, embryonic cell, or egg cell or derived therefrom.

Likewise, the IAP inhibitor can be compound that down-regulates the expression of IAP, for example actinomycin D; see, e.g., Ng et al, Prostate 53 (2002), 286-299. Furthermore, the experiments performed in accordance with the present invention suggest that nuclear factor- kappaB (NFkappaB) regulates expression of IAPs in Hodgkin's lymphoma cells. Accordingly, inhibitors of NFkappaB may also be used as LAP inhibitors in accordance with the present invention. For example, N50 (a cell permeable inhibitory peptide of NFkappaB translocation) may be used, which has been shown to suppress FSH-stimulated NFkappaB- DNA binding and XIAP expression in rat granulosa cells as well follicular growth; see Wang et al, Endocrinology 143 (2002), 2732-2740.

The present invention further provides compositions, comprising the IAP-inhibitors as mentioned above, optionally in combination with a pharmaceutically acceptable carrier and/or diluent, and/or other therapeutic agents.

The ingredients of the present invention are preferably used in form of a pharmaceutical composition where they are mixed with suitable carriers or excipients in doses to treat or ameliorate the disease. Such a composition may also contain (in addition to the ingredient and the carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers and other materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition may further contain other agents which either enhance the activity or use in treatment. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect or to minimize side-effects.

Techniques for formulation and administration of the compounds of the instant application may be found in "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, PA, latest edition. Whenever the compositions are to be used for medical purposes, they will contain a therapeutically effective dose of the respective ingredient. A therapeutically effective dose further refers to that amount of the compound/ingredient sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of such conditions. In the context of the present invention, a therapeutically effective dose is to be understood as an amount of the compound/ingredient, which results in a statistically significant reduction of HL or symptoms, which are related therewith. Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal or intranasal injections. Administration of the antibody of the present invention used in the pharmaceutical composition of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection. Intravenous administration to the patient is preferred.

A typical composition for intravenous infusion can be made up to contain 250 ml of sterile Ringer's solution, and 10 mg of antibody. See Remington's Pharmaceutical Science (15^th Ed., Mack Publishing Company, Easton, Ps., 1980).

As described in the examples, in cytosolic extracts of HL-derived B cells, inhibition of XIAP by the second mitochondria-derived activator of caspases (Smac)/DIABLO, or immunodepletion of XIAP restores cytochrome c-triggered processing and activation of caspase-3. Moreover, it could be shown in accordance with the present invention that Smac or a Smac-derived agonistic peptide also sensitized intact HL-derived B cells for the apoptotic action of staurosporine. This finding enables regimen to improve the effectiveness of chemotherapeutic agents which otherwise would be less effective or even not effective at all. Indeed, it has been shown that overexpression of XIAP and its family members suppresses apoptosis induced by a variety of stimuli, including TNF, Fas-L, menadione, staurosporine, etoposide, taxol, and growth factor withdrawal; see, e.g., Muzio et la., J. Biol. Chem. 272 (1997), 2952-2956; Martin et al, EMBO J. 15 (1996), 2407-2416 and Stennicke et al, J. Biol. Chem. 274 (1999), 8359-8362. In accordance with the present invention this suppressive effect can be reversed by sensitizing the target cells, i.e. HL and HL-derived cells, in particular B cells with an IAP inhibitor, preferably XIAP inhibitor such as one of those described hereinbefore.

Thus, in another embodiment the present invention relates to the use of the above described IAP-inhibitors for sensitizing lymphoma cells and lymphoma derived cells for the activity of cytotoxic approaches such as apoptosis induction by chemotherapeutic drugs, staurosporine, γ-irridiation, and triggering of death receptors such as the CD95; see also supra. In particular, the present invention relates to compositions, preferably pharmaceutical compositions, for sensitizing HL-derived cells, in particular B cells for the activity of chemotherapeutic agents. Chemotherapy for lymphoma is varied, because there are so many different forms of this disease. Treatment may rely on a single anticancer medication - that is, single agent chemotherapy - or it may involve combination chemotherapy with a number of different anticancer drugs. Such drugs destroy cancer cells by preventing them from growing and dividing rapidly. In a preferred embodiment of the invention, said cytotoxic approaches induce apoptosis. An overview about apoptosis, the cell's intrinsic death program and key regulator of tissue homeostasis, is given in, e.g., Fulda and Debatin, Curr. Med. Chem. Anti- Cane. Agents 3 (2003), 253-262.

Although many different chemotherapeutic plans are available for the treatment of Hodgkin's Disease (HD), two regimens are employed most frequently: MOPP, an abbreviation for mechlorethamine, vincristine (Oncovin®), procarbazine, and prednisone; and ABND, an abbreviation for doxorubicin (Adriamycin®), bleomycin, vinblastine, and dacarbazine. These therapies are used primarily for patients who have advanced disease or high-risk localized HD. Chemotherapy with chlorambucil (Leukeran®), vincristine sulfate (Oncovin®), procarbazine hydrohloride (Matulane®), and prednisone; also called "LOPP". Chemotherapy with chlorambucil (Leukeran®), vinblastine sulfate (Velban®), procarbazine hydrohloride (Matulane®), and prednisone; also called "ChlNPP". Chemotherapy with etoposide (NP-16; NePesid®), vinblastine sulfate (Velban®), doxorubicin (Adriamycin®), and prednisone; also called "ENAP". Alternative chemotherapies comprise the use of agents such as bleomycin, which is part of the combination drug plan ABND. Νon-bleomycin-based treatments include: ENA - etoposide, vinblastine, and doxorubicin; ENAP - etoposide, vinblastine, doxorubicin, and prednisone; NEEP - vincristine, epirubicin, etoposide, and prednisolone; and ΝONP - mitoxantrone (Νovantrone®), vincristine (Oncovin®), vinblastine, and prednisone. Furthermore, biological therapies are available. One of the biological therapies used for the treatment of lymphoma is interferon therapy. Interferons are a class of proteins that are released by virus-infected cells. They help normal cells to make antiviral proteins. Interferons also help the body to reduce tumor cell proliferation (growth and reproduction), while strengthening the body's immune response. Interferon-alpha (IΝFα) is a type of interferon that may be used to treat lymphoma.

Hence, in a preferred embodiment the present invention relates to the use of any one of the above described IAP inhibitors for the treatmetnof Hodgkin's lymphoma (HL) by sensitizing HL-derived cells for the cytotoxic activity of of staurosporine, mechlorethamine, vincristine, procarbazine, prednisone, doxorubicin, bleomycin, vinblastine, dacarbazine, chlorambucil, etoposide, mitoxantrone, genestein, phenoxodiol, interferons, drugs triggering death receptors such as the CD95, and pro-drugs and pharmaceutically acceptable salts of any one thereof. Those compounds can either be present in the same pharmaceutical composition as the IAP inhibitor or in a separately be prepared for administration. In the latter, the pharmaceutical compositions are preferably being applicable simultaneously or sequentially.

For example, antisense oligonucleotides targeting the LAP can be used to induce or enhance cytotoxic effects of doxorubicin, Taxol, vinorelbine, or etoposide. Appropriate doses are, for example, 5 to 20 mg/kg antisense oligonucleotides with 1 to 10 mg/kg of the chemotherapeutic agent. A similar approach has been described for antisense oligonucleotides targeting XIAP and inducing apoptosis and enhancing chemotherapeutic activity against human lung cancer cells in vitro and in vivo; see Hu et al., Clin. Cancer Res. 9 (2003), 2826- 2836.

The above mentioned compositions may also be used as diagnostic compositions including their use in screening methods. Hence, in a further embodiment the present invention relates to a method of diagnosis of a disease related to Hodgkin's lymphomas, which comprises a) assaying a sample from a subject for IAP transcriptional activity or IAP protein; and b) determining the level of IAP gene product or activity, wherein an altered level compared to a control indicates the presence of the disease. In a still further embodiment the present invention relates to a method of diagnosis of a disease related to Hodgkin's lymphomas, which comprises determining a mutation in the nucleic acid molecule encoding an IAP or an IAP inhibitor in a sample from a subject, wherein the presence of a mutation indicates presence of or predisposition for the disease. Methods of detection of the expression level of the transcriptional products of the nucleic acid of SEQ ID NO: 1 in a mammal can comprise obtaining a biological sample from said mammal; and contacting said biological sample with a reagent which detects said transcriptional products.

Preferably, this reagent is a nucleic acid and is even more preferably detectably labelled (for further information, see also chapter "probes", above). The nucleic acid may also be otherwise modified. Such modifications include, for-, example, spacer sequences for the separation of the nucleic acid as such and the labelling! Furthermore, such modifications include targeting sequences, such as those, which target the nucleic acid to specific tissues or sections in cells, such as mitochondria in the cytosol of a cell. If so, the expression level of the transcriptional products may be detected by detecting in a sample a mRNA transcript that encodes the IAP as disclosed herein, said method comprising the steps of contacting said sample under moderately stringent hybridizing conditions with the nucleic acid disclosed herein to form a duplex; and detecting the presence of said duplex.

Analogously, a method of detection of the expression level of XIAP is provided, said method comprising obtaining a biological sample from said mammal and contacting said biological sample with a reagent which detects the XIAP of the invention. According to a preferred embodiment, said reagent is an antibody as herein defined, wliich is directed against amino acid of SEQ ID NO: 2, and is more preferably detectably labelled, e.g. biotinylated or coupled to a fluorescent agent. A comparison of normal tissues, in which XIAP is not overexpressed and the biological sample, is indicative for HL being present in said sample or not.

As mentioned before, in the embodiments relating to diagnostic assays the IAP specific reagents are preferably labeled. A variety of techniques are available for labeling biomolecules, are well known to the person skilled in the art and are considered to be within the scope of the present invention. Such techniques are, e.g., described in Tijssen, "Practice and theory of enzyme immuno assays", Burden, RH and von Knippenburg (Eds), Volume 15 (1985), "Basic methods in molecular biology"; Davis LG, Dibmer MD; Battey Elsevier (1990), Mayer et al, (Eds) "Immunochemical methods in cell and molecular biology" Academic Press, London (1987), or in the series "Methods in Enzymology", Academic Press,

Inc. There are many different labels and methods of labeling known to those of ordinary skill in the art. Commonly used labels comprise, inter alia, fluorochromes (like fluorescein, rhodamine, Texas Red, etc.), enzymes (like horse radish peroxidase, b-galactosidase, alkaline phosphatase), radioactive isotopes (like 32P or 1251), biotin, digoxygenin, colloidal metals, chemi- or bioluminescent compounds (like dioxetanes, luminol or acridiniums). Labeling procedures, like covalent coupling of enzymes or biotinyl groups, iodinations, phosphorylations, biotinylations, random priming, nick-translations, tailing (using terminal transferases) are well known in the art. Detection methods comprise, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzymatic reactions, etc.

In addition, the above-described compounds etc. may be attached to a solid phase. Solid phases are known to those in the art and may comprise polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, animal red blood cells, or red blood cell ghosts, duracytes and the walls of wells of a reaction tray, plastic tubes or other test tubes. Suitable methods of immobilizing IAP nucleic acids, (poly)peptides, proteins, antibodies, etc. on solid phases include but are not limited to ionic, hydrophobic, covalent interactions and the like. The solid phase can retain one or more additional receptor(s) which has/have the ability to attract and immobilize the region as defined above. This receptor can comprise a charged substance that is oppositely charged with respect to the reagent itself or to a charged substance conjugated to the capture reagent or the receptor can be any specific binding partner which is immobilized upon (attached to) the solid phase and which is able to immobilize the reagent as defined above. Commonly used detection assays can comprise radioisotopic or non-radioisotopic methods. These comprise, inter alia, RIA (Radioisotopic Assay) and IRMA (Immune Radioimmunometric Assay), EIA (Enzym Immuno Assay), ELISA (Enzyme Linked Immuno Assay), FIA (Fluorescent Immuno Assay), and CLIA (Chemioluminescent Immune Assay). Other detection methods that are used in the art are those that do not utilize tracer molecules. One prototype of these methods is the agglutination assay, based on the property of a given molecule to bridge at least two particles.

In a preferred embodiment of the diagnostic assays of the present invention, immunohistochemistry is used for the LAP based diagnosis of Hodgkin's lymphomas (see also the examples), which is preferably performed with an antibody with is able to penetrate paraffin.

For diagnosis and quantification of (poly)peptides, polynucleotides, etc. in clinical and/or scientific specimens, a variety of immunological methods, as described above as well as molecular biological methods, like nucleic acid hybridization assays, PCR assays or DNA Enzyme Immunoassays (Mantero et al, Clinical Chemistry 37 (1991), 422-429) have been developed and are well known in the art. In this context, it should be noted that the IAP nucleic acid molecules may also comprise PNAs, modified DNA analogs containing amide backbone linkages. Such PNAs are useful, inter alia, as probes for DNA RNA hybridization.

The above-described compositions may be used for methods for detecting expression of a IAP polynucleotide by detecting the presence of mRNA coding for a IAP (poly)peptide wliich comprises, for example, obtaining mRNA from cells of a subject and contacting the mRNA so obtained with a probe/primer comprising a nucleic acid molecule capable of specifically hybridizing with a IAP polynucleotide under suitable hybridization conditions, and detecting the presence of mRNA hybridized to the probe/primer. Further diagnostic methods leading to the detection of nucleic acid molecules in a sample comprise, e.g., polymerase chain reaction (PCR), ligase chain reaction (LCR), Southern blotting in combination with nucleic acid hybridization, comparative genome hybridization (CGH) or representative difference analysis (RDA). These methods for assaying for the presence of nucleic acid molecules are known in the art and can be carried out without any undue experimentation.

In one embodiment of the screening method of the invention the expression of nucleic acids is analyzed with an expression array and/or realtime PCR. Chip and array technology are well known to the person skilled in the art. Advances in approaches to DNA-based diagnostics are reviewed, for example, by Whitcombe et al. in Curr. Opin. Biotechnol. 9 (1998), 602-608. Furthermore, DNA chips and microarray technology devices, systems, and applications are described by, e.g. Cuzin, Transfus. Clin. Biol. 8 (2001), 291-296 and Heller, Annu. Rev. Biomed. Eng. (2002), 129-153. Likewise, biomedical applications of protein chips is known and described in, e.g., Ng, J. Cell. Mol. Med. 6 (2002), 329-340.

The present invention also relates to a kit for use in any one of the above described methods, said kit comprising for example an anti-IAP antibody or IAP antisense nucleic acid molecule, or a derivative thereof. Likewise, the kit may contain a chip as described above. Such kits are used to detect RNA or DNA which hybridize to LAP DNA or to detect the presence of IAP protein or peptide fragments in a sample. Such characterization is useful for a variety of purposes including but not limited to forensic analyses, diagnostic applications, and epidemiological studies in accordance with the above-described methods of the present invention. The recombinant IAP proteins, DNA molecules, RNA molecules and antibodies lend themselves to the formulation of kits suitable for the detection and typing of IAP. Such a kit would typically comprise a compartmentalized carrier suitable to hold in close confinement at least one container. The carrier would further comprise reagents such as recombinant IAP protein or anti-IAP antibodies suitable for detecting IAP. The carrier may also contain a means for detection such as labeled antigen or enzyme substrates or the like.

To summarize, the present invention relates to the use of inhibitors of IAPs (inhibitors of apoptosis) in compositions for the treatment of Hodgkin's lymphomas. In particular, XIAP- inhibitors are disclosed for methods of treatment of Hodgkin's lymphomas. The present invention includes methods of treatment of diseases related to Hodgkin's lymphomas comprising administering to a subject in need thereof a therapeutically effective amount of an IAP inhibitor as defined above either alone or in combination with anti-cancer drugs. The use and methods of the invention can be used for the treatment of all kinds of Hodgkin's lymphomas or related diseases hitherto unknown as being related to or dependent on the modulation of LAP. The methods and uses of the present invention may be desirably employed in humans, although animal treatment is also encompassed by the methods and uses described herein.

These and other embodiments are disclosed and encompassed by the description and examples of the present invention. Further literature concerning any one of the materials, methods, uses and compounds to be employed in accordance with the present invention may be retrieved from public libraries and databases, using for example electronic devices. For example the public database "Medline" may be utilized, which is hosted by the National Center for Biotechnology Information and/or the National Library of Medicine at the National Institutes of Health. Further databases and web addresses, such as those of the European Bioinformatics Institute (EBI), which is part of the European Molecular Biology Laboratory (EMBL) are known to the person skilled in the art and can also be obtained using internet search engines. An overview of patent information in biotechnology and a survey of relevant sources of patent information useful for retrospective searching and for current awareness is given in Berks, TIBTECH 12 (1994), 352-364.

The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only and are not intended to limit the scope of the invention.

Several documents are cited throughout the text of this specification either by name or are referred to by numerals within parenthesis. Full bibliographic citations may be found at the end of the specification immediately preceding the claims. The contents of all cited references (including literature references, issued patents, published patent applications as cited throughout this application and manufacturer's specifications, instructions, etc) are hereby expressly incorporated by reference; however, there is no admission that any document cited is indeed prior art as to the present invention. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Methods in molecular genetics and genetic engineering are described generally in the current editions of Molecular Cloning: A Laboratory Manual, (Sambrook et al, (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Gene Transfer Vectors for Mammalian Cells (Miller & Calos, eds.); Current Protocols in Molecular Biology and Short Protocols in Molecular Biology, 3rd Edition (F. M. Ausubel et al, eds.); and Recombinant DNA Methodology (R. Wu ed., Academic Press). Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods Ln Enzymology (Academic Press, Inc., N.Y.); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986). Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial vendors such as BioRad, Stratagene, Invitrogen, and Clontech. General techniques in cell culture and media collection are outlined in Large Scale Mammalian Cell Culture (Hu et al, Curr. Opin. Biotechnol. 8 (1997), 148); Serum-free Media (Kitano, Biotechnology 17 (1991), 73); Large Scale Mammalian Cell Culture (Curr. Opin. Biotechnol. 2 (1991), 375); and Suspension Culture of Mammalian Cells (Birch et al, Bioprocess Technol. 19 (1990), 251); Extracting information from cDNA arrays, Herzel et al, CHAOS 11, (2001), 98-107.

In the following, the present invention is illustrated by the enclosed figures as well as the following examples which are directed to the inhibition of XIAP:

In the figures, the following is shown: Fig. 1. Failure of cytochrome c to induce caspase activation in cytosolic extracts of HL- derived B-cell lines. Cytosolic extracts of LI 236, L591, L428 and KMH2 cells, and of control B-cell LI 309 were prepared and equal amounts of protein were incubated with or without cytochrome c/dATP for lh at 30°C. Cytosolic extracts were resolved by SDS/PAGE and subjected to western blot analysis. A) Procaspase-9 (p46) and its fragments (p37, p35) were detected by polyclonal rabbit anti-caspase-9 antibody. B) Procaspase-3 (p32) and its fragments fragments (p20, pi 7) were detected by polyclonal anti-caspase-3 antibody. C) Measurement of relative caspase activity using DEVD-AFC. Samples were normalized for total cytosolic protein content. Asterisks (*) indicate nonspecific bands recognized by polyclonal antibodies .

Fig. 2. Expression of caspase activators and inhibitors in HL-derived B-cell lines. Equal amounts of proteins from total cell lysate, cytosolic and membrane fractions (including mitochondria) of L1236, L591, L428, and KMH2 cells and of control B-cell L1309 were subjected to SDS/PAGE and western blot analysis. Proteins were detected using antibodies against Apaf-1, XIAP, Smac and Cytochrome c, followed by secondary antibody and ECL as described in material and methods.

Fig. 3. XIAP co-immunoprecipitates with activated caspase-3. Cytosolic extracts of L1236, L591, L428 and KMH2 cells, and of control B-cell L1309 were prepared and equal amounts of protein (with same concentration) were incubated with or without cytochrome c/dATP for lh at 30°C. A) Samples were resolved by SDS/PAGE and subjected to western blot analysis. XIAP was detected by mouse anti-XIAP antibody. B) Caspase-3 was immunoprecipitated by mouse anti-caspase-3 antibody in cytosolic extracts and subjected to SDS/PAGE and western blotting. Co-precipitated XIAP was detected by mouse anti-XIAP antibody. Asterisks (*) indicate Mouse IgG.

Fig. 4. Caspase-3 activation by caspase-8 and granzyme B. Cytosolic extracts of LI 236 and KMH2 cells, and of control B-cell L1309 were prepared and equal amounts (with same concentration) of protein were left untreated or incubated for lh with A) 100 nM recombinant active caspase-8, B) granzyme B at 30°C. Cytosolic extracts were resolved by SDS/PAGE and subjected to western blot analysis. Caspase-3 was detected by polyclonal rabbit anti- caspase-3 antibody. Relative caspase activity was measured by using DEVD-AFC after incubation with C) recombinant active caspase-8 and D) Granzyme B. Samples were normalized for total cytosolic protein content.

Fig. 5. Degradation of XIAP by caspase-8. Cytosolic extracts of L1236 and KMH2 cells, and of control LI 309 cells were prepared and equal amounts of proteins were left untreated or incubated for lh at 30°C with A) 100 nM recombinant active caspase-8 or B) granzyme B. Cytosolic extracts were resolved by SDS/PAGE and subjected to western blot analysis. XIAP (p54) and XIAP fragments (p48, p30/34, p25) were detected by mouse anti-XIAP antibody.

Fig. 6. Depletion of XIAP restores caspase-3 processing and activity. Cytosolic extracts of L1236 and KMH2 cells, and of control B-cell L1309 were prepared and equal amounts of protein were incubated with or without cytochrome c/dATP in the absence and presence of Smac protein for lh at 30°C. A) Cytosolic extracts were resolved by SDS/PAGE and subjected to western blot analysis. Caspase-3 was detected by polyclonal rabbit anti-caspase-3 antibody. B) Relative caspase activity was measured by hydrolysis of DEVD-AFC. Samples were normalized for total cytosolic protein content. C) XIAP was immunodepleted by mouse anti-XIAP antibody. Cytosolic extracts of KMH2 cells with or without XIAP were incubated with or without cytochrome c/dATP for lh at 30°C. Samples were normalized for total cytosolic protein content and relative caspase-3 activity was measured by DEVDase activity.

Fig. 7. Smac restores caspase-9 processing in HL-derived B-cell line. Cytosolic extracts of LI 236 and KMH2 cells, and of control B-cell LI 309 were prepared and equal amounts of proteins were left untreated or incubated for lh at 30°C with cytochrome c/dATP and in the presence or absence of Smac protein. Cytosolic extracts were resolved by SDS/PAGE and subjected to western blot analysis. Caspase-9 was detected by polyclonal rabbit anti-caspase-3 antibody.

Fig. 8. Paraffin section immunohistochemistry of case #6 (table 1), HL, nodular sclerosis type using anti-hilp/XIAP specific monoclonal antibody. There is strong granular intracytoplasmic staining for XIAP in essentially all morphologically recognizable Hodgkin or Reed-Stemberg cells. Background lymphocytes are either negative for XIAP, or show variably weak to (focally) moderate positivity. (EnVision/diaminobenzidine). Fig. 9. XIAP mediated inhibition of apoptosis in HL-derived B-cell lines. First, upstream to mitochondria XIAP may prevent activation of Bax through induction of phosphrylation and activation of Akt. Since activated Bax is a potent trigger of cytochrome c release, the block of Bax activation also impairs the release of proapoptotic factors from mitochondria. Second, through binding to caspase-9 in the apoptosome, XIAP blocks the activation of caspase-3. Third, XIAP directly binds to caspase-3 and inhibits its proteolytic activity.

EXAMPLES

Materials and methods

Cell culture

The establishment and culturing of the Hodgkin and control B cell lines have been described previously (Kashkar et al, Cell Death Diff. 7 (2002), 750-757). The establishment of the Hodgkin B-cell lines L591, L428, L1236 and KMH2 has been described elsewhere ⁴³'⁴⁴. The LI 309 cell line was established by immortalization of human primary B-cells by EBV (Epstein-Barr virus). The cell lines were cultured in VLE RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 100 μg/ml streptomycin and 100 U/ml penicillin. All chemicals were purchased from Biochrom, Berlin, Germany. Cells were transfected with 0.5 μg Smac NH2-terminal peptide, H-AVPIAQK-OH, (Calbiochem), Smac protein, or β-galactosidase (119-kD subunit) as a control using the Chariot protein transfection kit according to the instructions of the manufacturer (Active Motif). Apoptosis was induced by lμM staurosporine (Qbiogene) and cell death was examined using trypan blue exclusion.

Sample Preparation and Immunoblotting

Whole cell extracts were prepared by lysing cells in CHAPS lysis buffer (10 mM HEPES, pH 7.4, 150 mM NaCI, 1% CHAPS, protease complete cocktail, Roche Diagnostics, Mannheim, Germany) on ice for 30 min. The crude lysate was then centrifuged at 14,000 g for 20 min at 4 °C and the resulting supernatant (total cell extract) was recovered. Protein concentration was determined by the bicinchroninic acid assay method (Pierce, Rockford, Illinois) using BSA as a standard and the lysates were adjusted to equal concentration. Equal amounts of protein of cytosol and whole cell extract were separated by SDS-PAGE and transferred to nitrocellulose membrane (Protran 0.2 μm; Schleicher and Schuell, Dassel, Germany). Rabbit polyclonal antisera specific for human caspase-3, rabbit polyclonal antisera specific for Apaf- 1 and monoclonal mouse anti-cytochrome c were obtained from Pharmingen (Heidelberg, Germany). Polyclonal anti-serum specific for caspase-9 was maid as described . Polyclonal anti-serum specific for human Smac/DIABLO was from Alexis (Grϋnberg, Germany). Monoclonal mouse anti-hilp/XIAP antibody and monoclonal mouse anti-caspase-3 antibody were from BD transduction laboratories (Heidelberg, Germany). Horseradish peroxidase conjugates of anti-rabbit and anti-mouse IgG (Biorad) were used as secondary antibodies and signals were detected by ECL (Amersham, Freiburg, Germany).

Preparation of cytosolic extracts and caspase activation For preparation of cell free lysates the procedure described by Ellerby et al, 1997, and Stennicke et al, 1998 was used with minor modifications. Cells were harvested, washed twice in phosphate-buffered saline at 4 °C, pelleted for 5 min at 1200 g, resuspended in buffer A (20 mM PIPES, pH 7.0, 50 mM KC1, 2 mM MgCl₂, 5 mM EGTA, 1 mM dithiothreitol) and allowed to swell on ice for 20 min. After addition of PMSF to 100 μM, cells were cracked by passing through a 27-gauge needle and pelleted at 14,000 g for 20 min at 4 °C. The resulting supernatant (cytosolic extract) was recovered. Protein concentration was determined by the bicinchroninic acid assay method (Pierce) using BSA as a standard and was adjusted to 20 mg/ml. For initiating caspase activation, either 10 μM horse heart cytochrome c together with 1 mM dATP, or 100 nM of purified recombinant active caspase-8 (ref. 47) or 60 μg granzyme B (Sigma) were added to 20 μl of cell extracts or buffer A as a control and incubated for 1 h at 30°C. To determine the effect of Smac on caspase activation, cytosolic extracts were incubated with 10 μM horse heart cytochrome c together with 1 mM dATP and recombinant Smac protein (1 μM) for 60 min. at 30 °C. 1 μl of resulting cytosolic extracts and buffer A were added to 99 μl caspase buffer (20 mM Pipes, 100 mM NaCI, 1 mM EDTA, 0.1% CHAPS, 10 % sucrose, 10 mM dithiothreitol) and reactions were initiated by addition of 100 μM Ac-DEVD-AFC (Ac-DEVD-7-amino-4-trifluoromethyl coumarin) ⁴⁷. Caspase activity was assayed by release of 7-amino-4-trifluoromethyl-coumarin (AFC) from DEVD containing synthetic peptides using a continuous-reading plate reader (Wallac victor TM multilabel counter 1420) thermostated at 30°C at 400 /505 nm excitation and emission respectively.

Immunoprecipitation

Equal amounts of cytosolic extracts were adjusted to a final volume of 500 μl with buffer A.

Samples were rotated for 6 h at 4 °C with 1 μg of monoclonal mouse anti-caspase-3 antibody. Antigen-antibody complexes were immobilized by rotation for 2 h at 4 °C with GammaBind G Sepharose (Pharmacia Biotech, Uppsala, Sweden). The complexes were pelleted (1 min, 14000 g) and the supernatant removed. After 3 washes with the same buffer used for the immunoprecipitation, samples were subjected to SDS-PAGE and immunoblotted as described above.

Immunodepletion of XIAP

20 μl (250 μg/ml) of anti-XIAP Mab (Transduction Laboratories) were added to 100 μl of

GammaBind G Sepharose (Pharmacia Biotech) in 500 μl of PBS and rotated at 4°C for 3 h. The beads were collected by centrifugation (1 min at 14000 g 4°C). After removal of the supernatant, the beads were washed once with 1 ml of buffer A and incubated with 300 μl of cytosolic extract (20 μg/μl) for 6 h in a rotator at 4°C. The beads were subsequently pelleted by centrifugation (1 min. at 14000 g 4°C). The resulting supernatant was the cytosolic extract immunodepleted of XIAP.

Expression and purification of recombinant Smac protein

The cDNA encoding the amino acids 56-239 of Smac was cloned from a human thymus cDNA library by PCR. The PCR product was cloned into the Ndel and Xhol sites of the pET- 23b vector (Novagen), resulting in a C-terminal His-6 tagged Smac. Expression of recombinant Smac was induced with 0.5 mM IPTG for 5 hours at 30°C. The protein was purified on Ni-chelate sepharose (Pharmacia) from the soluble fraction of sonicated cells.

Detection of XIAP in Hodgkin's lymphoma tissues

Primary tumor tissues from twelve cases of HL in Danish patients (two cases of lymphocytic predominance type; ten cases of classical type, comprising three mixed cellularity and seven nodular sclerosis type) were selected from the archives of the Institute of Pathology, Aarhus University Hospital (Table 1). Histological diagnosis and subtyping of HL was based on accepted morphological and immunophenotypical criteria ⁴⁹. All cases had been examined for the presence of Epstein-Barr virus sequences using EBV-encoded small RNA (EBER) in situ hybridization (detailed data not shown). In addition, two tonsils and three lymph nodes showing benign reactive lymphoid hyperplasia were included as controls for immunohistology. All tissues had been routinely fixed in formalin and embedded in paraffin. Immunohistolo g

Formalin-fixed and paraffin-embedded tissues were cut at 4 μm onto silanized slides. Antigen retrieval was performed by microwave superheating in TEG buffer (lOmM TRIS, 0.5mM EGTA) at pH 9.0. Sections were incubated with primary monoclonal anti-hilp/XIAP antibody (Transduction Laboratories) for 30 minutes (dilution 1:50). Reactions were detected using a standard, highly-sensitive EnVison™ horseradish peroxidase staining system (DAKO, Glostrup, Denmark) developed with diaminobenzidine.

Example 1: Failure of processing and activation of caspase-9 and -3 in HL-derived B- cells

HL-derived B-cell lines have been shown to be resistant to apoptosis induction by CD95 and staurosporine ²⁸'²⁹. In order to investigate if caspases are functional in these cells, a cell-free system was employed and exogenously added cytochrome c to induce proteolytic processing of procaspase-9 and -3 (ref.15, 30). First, the activation of caspase-9 and -3 was analysed after addition of cytochrome c to cytosolic extracts of four HL-derived B-cell lines, L1236, L591, L428, KMH2 and the control B-cell line L1309. In untreated cell extracts caspase-9 is detected as its 46-kDa proform. Addition of cytochrome c to the cytosolic extracts of L1309, leads to autocatalytical cleavage of procaspase-9, producing the characteristic p35 fragment. A second cleavage, mediated by subsequently activated caspase-3, results in the p37 fragment of caspase-9 (ref. 31) (Fig. 1A). Concomitantly, the initial p20 fragment and ultimate pl7 subunit of caspase-3 were detected (Fig. IB). In contrast to the control B-cell line, cytochrome c failed to induce any processing of caspase-9 in cytosolic extracts of HL-derived B-cell lines. Similarly, caspase-3 remained in its inactive form after addition of cytochrome c to the cytosolic extracts of all HL-derived B-cell lines (Fig. 1A, B). Addition of cytochrome c to cytosolic extracts induced caspase-3 enzymatic activity in the control B-cell line but not in the HL-derived B-cell lines indicating defective activation of both caspase-9 and caspase-3 (Fig. 1C).

The defective caspase activation in the HL-derived B-cell lines prompted the inventors to examine the expression of proteins involved in this process downstream of mitochondria. Apaf-1, cytochrome c and Smac/DIABLO are expressed in all cell lines at comparable levels. Notably, the lack of detection of cytochrome c and Smac/DIABLO in the cytosolic fractions excludes the possibility that contamination of cytosolic fractions by mitochondrial proteins could have caused caspase activation in the control B-cell line (Fig. 2A and B). In contrast, the anti-apoptotic protein XIAP appeared to be significantly overexpressed in the HL-derived B-cell lines L1236, L591, L428 and KMH2, compared to the control B-cell line L1309.

Example 2: XIAP association with caspase-3 in HL-derived B-cell lines

XIAP inhibits the catalytic activity of caspases through physical interaction ^15,24. The observation that XIAP is abundantly expressed in HL-derived B-cell lines prompted the inventors to examine whether XIAP is associated with caspase-3 in these cell lines. XIAP was detected in cytosolic extracts of control and HL-derived B-cell lines at about 54-kDa. Addition of cytochrome c appeared to induce partial degradation of XIAP in the control and in HL-derived B-cell cytosolic extracts into one detectable fragment at about 48-kDa. Immunoprecipitation of caspase-3 revealed a substantial interaction of the activated protease with XIAP in cysolic extracts of HL-derived B-cell lines but not in that of the control B-cell line (Fig. 3A, B). This indicated a physical interaction of XIAP with activated caspase-3, a hallmark for the aberrant antiapoptotic function of XIAP in HL-derived B-cells.

Example 3: Caspase-8 and granzyme B partially bypass the XIAP-mediated inhibition of caspase-3 in HL-derived B-cell lines

XIAP is known to suppress apoptosis induced by stimulation of both the death receptor and mitochondrial pathways ¹⁵'¹⁶. To detect the target of XIAP action, caspase-3 was activated by the other initiator proteases caspase-8 or granzyme B ¹³'³². Indeed, when recombinant active caspase-8 or granzyme B were added to the cytosolic extracts of HL-derived B-cells, these two proteases stimulated the cleavage of caspase-3 resulting in the p20 fragment, characteristic of the initial cleavage of caspase-3 (ref. 13, 15, 16) (Fig. 4A, B). Notably, the autocatalytic maturation generating the pl7 fragment proceeds to a lesser degree in the cytosolic extracts of HL-derived B-cell lines compared to the control B-cell line. We conclude that in HL-derived B-cells XIAP does not prevent the initial cleavage of caspase-3 induced by caspase-8 or granzyme B, but rather inhibits the subsequent autocatalytic processing to the mature pi 7 fragment. Incomplete caspase-3 processing corresponds with decreased caspase-3 activity in HL-derived B-cell extracts (Fig. 4C and D).

The observation that caspase-8 overcomes caspase-3 inhibition by XIAP to a greater extent than does granzyme B might be explained partly by its ability to cleave XIAP into different fragments, which results in the depletion of the full length XIAP (Fig. 5A, B) and is accompanied by the lose of its inhibitory activity ¹⁴. Taken together, the data in Fig. 4 and 5 suggest that caspase-3 in HL-derived B-cells can be processed by addition of activator proteases like caspase-8 and granzyme B, but that its activity is checked by XIAP levels. The competence of caspase-3 in HL-derived B-cell lines was confirmed by sequence analyses of caspase-3, that revealed a lack of mutations in this gene.

Example 4: Physical or functional depletion of XIAP restores cytochrome c-induced caspase-3 activity in HL-derived B-cells

So far the present data show that in HL-derived B-cell lines XIAP is overexpressed, inhibits initial cleavage of caspase-3 -mediated by the caspase-9-apoptosome, and thereby prevents caspase-3 activation. Moreover, XIAP may also act at the level of caspase-3 to shut down the caspase- 8 -mediated death receptor pathway and the granzyme B pathway. If XIAP was the key inhibitor of apoptosis in HL-derived B-cells, removal of XIAP should relieve the inhibition of caspase-3 activation. To provide further evidence for the effect of XIAP on the mitochondrial apoptotic pathway in HL-derived B-cells, XIAP was removed from cytosolic extracts by two independent methods. First, a mitochondrial inhibitor of XIAP, Smac, was added to cytosolic extracts, pretreated with cytochrome c. In the presence of recombinant Smac protein cytochrome c triggers processing and activation of caspase-3 in HL-derived B- cells (Fig. 6A). Corresponding to the processing of caspase-3 into the p20 and the ultimate pi 7 fragments, addition of Smac to cytosolic extracts of HL-derived B-cell lines restored the enzymatic activity of caspase-3 (Fig. 6B). As a second approach, XIAP was immunodepleted from cytosolic extracts of KMH2 cells using a mouse monoclonal anti-XIAP antibody. Immunodepletion of XIAP resulted in an enhancement of caspase-3 activity (Fig. 6C), providing further evidence for a central role of XIAP in inhibition of caspase-3 activation in HL-derived B-cells.

Furthermore, transfection of intact HL-derived B cells with Smac or a Smac agonistic peptide sensitized these cells for the proapoptotic activity of staurosporine confirming that high level expression of XIAP constitutes a state of apoptotic resistance in HL.

Addition of Smac protein to cytosolic extracts of L1236 and KMH2 cells also promoted caspase-9 processing into p35 and p37 fragments (Fig. 7). The additional inhibition of XIAP by Smac also restored the processing of caspase-9, suggesting that XIAP not only prevents maturation of caspase-3 but more likely inhibits caspase-9-mediated initial cleavage of caspase-3. Example 5: XIAP is expressed in primary Hodgkin' s lymphoma tumor tissues

Moderate or strong immunohistological expression of XIAP was seen in H-RS cells in all cases of classical HL examined. Staining was predominantly intracytoplasmic, with a striking granularity (Fig. 8). Staining was seen in both EBV-positive and EBV-negative HL cases. The two cases of lymphocytic predominance HL showed respectively, weak and very weak XIAP staining in their "L and H" Reed-Sternberg variant cells. Control tissues, and non-neoplastic lymphoid components within the HL tumors showed variable XIAP staining. In most cases this was absent; when present it was usually focal and weak. However, in some tumors, stronger staining was detected in background lymphocytes, both within germinal centers, and in the interfollicular regions (table 1).

Table 1: XIAP expression in primary Hodgkin's lymphoma tissues

No Age Sex Diagnosis EBV H-RS cells

1. 33yr Male HL-LP neg +

2. 37yr Male HL-LP neg ++

3. 20yr Male HL-MC pos +++

4. 88yr Male HL-MC pos +++

5. 67yr Female HL-MC pos ++++

6. 23yr Male HL-NS pos ++++

7. 78yr Male HL-NS pos ++++

8. 42yr Female HL-NS neg +++

9. 29yr Male HL-NS neg ++++

10. 20yr Female HL-NS neg

11. 40yr Male HL-NS neg ++++

12. 60yr Male HL-NS neg

Abbreviations: HL-LP: Hodgkin's lymphoma, lymphocytic predominance; HL-MC:

Hodgkin's lymphoma, mixed cellularity; HL-NS: Hodgkin's lymphoma, nodular sclerosis; neg: negative reaction in H-RS cells; pos: positive reaction in H-RS cells.

XIAP positivity in H-RS cells; + = very weak; ++ = weak; +++ = moderate; ++++ = strong staining. Primary H-RS cells and Hodgkin's lymphoma-derived B-cell lines established from patients with Hodgkin's disease have been shown to be resistant to different apoptotic stimuli '²⁹. In the present study, a novel defect in apoptotic signaling has been identified and localized in the caspase cascade downstream of mitochondria. The data indicate that the cytochrome c- induced activation of caspase-3 is severely impaired in HL-derived B-cell lines. The evidence provided suggests that XIAP is overexpressed in HL-derived B-cells, constitutively binds to any activated caspase-3 and plays a key role in inhibiting caspase-3 processing and activation by caspase-9. In HL-derived B-cell lines cytochrome c/dATP addition failed to activate caspase-9 and -3. Correspondingly, neither the initial cleavage of caspase-9 through autocatalysis nor any subsequent proteolytic cleavage mediated by caspase-3 was observed. When complexed with Apaf-1 and cytochrome c, caspase-9 is thought to be activated without cleavage , and ultimately processed by an autocatalytic mechanism ⁸. It has been suggested that the processing of caspase-9 to p37 is the signal for inhibition by XIAP, due to the production of a neo-epitope sequence in the small subunit ²⁴. Therefore, we would have expected to observe p37 fragments in cytochrome c-programmed extracts from all cells, which was not the case (Fig. 1). However, the presence of the characteristic p35 and p37 fragments following Smac addition to the cytosols (Fig. 7) indicates that caspase-9 is now active. Thus, XIAP may also inhibit the unprocessed form of caspase-9, as previously described ¹⁵. Regardless of the exact mechanism of XIAP inhibition of caspase-9, our data are fully consistent with a XIAP- mediated block on caspase activation in HL-derived B-cell lines.

It is important to note that Apaf-1, caspase-9 and -3 were expressed at the same levels (Fig. 1, 2) and caspase-3 did not reveal any mutations in the primary structure. It was thus inferred that one of the known caspase inhibitors might prevent caspase processing and activation in HL-derived B-cells. Indeed, XIAP was found to be overexpressed in all HL-derived B-cells (Fig. 1A). XIAP inhibits the catalytic activity of caspases by physically interacting with them. The finding that XIAP could be co-immunoprecipitated with caspase-3 in HL-derived B-cells but not control B-cells, supported the hypothesis that XIAP binds to caspase-3 as soon as it is activated. That the binding of XIAP to caspase-3 results in inhibition of this protease was demonstrated by functional removal of XIAP from cytosolic extracts.

In conclusion, four lines of evidence suggest that XIAP inhibits caspase-3 in HL-derived B- cell lines i) XIAP is overexpressed, ii) XIAP co-immunoprecipitates with activated caspase-3, iii) the XIAP inhibitor Smac restores caspase-3 activity and iv) physical removal of XIAP by immunodepletion promotes caspase-3 activation. As an executioner caspase, caspase-3 represents a target of converging caspase cascades including caspase-8, caspase-9 and granzyme B. When added to cytosolic extracts of HL- derived B-cells, caspase-8 and granzyme B induced protease activity of caspase-3, although at reduced levels. This is due to the initial cleavage into large and small subunits, while autocatalytic processing is still inhibited by XIAP (ref. 13, 15, 16). In contrast to caspase-8 and granzyme B, caspase-9 induced caspase-3 activation was completely abrogated. This is explained by the observation that XIAP inhibits the activity of caspase-9. Thus the inhibitory activity of XIAP is not restricted to caspase-3 but rather involves the level of the apoptosome. Thus, all major apoptotic pathways are blocked in the HL-derived B-cells lines overexpressing XIAP .

The XIAP gene can be upregulated by NF-κB (ref. 34, 35). Interestingly, H-RS cells strongly express CD30 and CD40, two members of the TNF receptor family that both can activate NFKB. Constitutive activation of NF-κB appears to be an unique feature of H-RS cells, which has previously been suggested to play an important role in the pathogenesis of H-RS cells ³⁹. The overexpression of XIAP, as a target gene for NF-κB is in line with these observations and provides a plausible link for NF-κB mediated resistance to apoptosis in HL-derived B- cells.

Hodgkin's lymphoma is unusual among neoplasms in that the malignant H-RS are typically rare in the affected tissues. This has hampered research into the pathogenesis of the disease for many years. The use of HL-derived B-cell lines has helped us to investigate the functional cross-communication between different molecules in these tumors. Our immunohistology experiments confirm that overexpression of XIAP is also consistently found in the H-RS cells in primary HL tumors, suggesting that our proposed model for XIAP's action is also relevant in vivo. In a previous report, it was shown that HL-derived B-cell lines have an defect at the level of

• 9R activation of Bax and therefore are resistant against staurosporine-induced apoptosis Recent reports indicate that XIAP can prevent apoptosis through a PI3-kinase/Akt dependent inhibition of the caspase cascade ⁴⁰. Apparently, XIAP can stimulate Akt phosphorylation and activation ⁴⁰. Akt/PI3-kinases in turn have been reported to regulate apoptotic events by modulating the function of Bcl-2 family members ⁴¹. Specifically, Bax conformational change can be inhibited by Akt activation ⁴². These observations can be reconciled in a model where overexpressed XIAP exerts its anti-apoptotic effects at three levels of mitochondrial apoptotic pathway (Fig. 9). References

I. Alnemri, E.S. Mammalian cell death proteases: A family of highly conserved aspartate specific cysteine proteases. J Cell Biochem. 64, 33-42 (1997). 2. Thornberry, N.A., & Lazebnik, Y. Caspases: enemies within. Science 281, 1312-1316 (1998).

3. Scaffidi, C. et al. Two CD95 (APO-1/Fas) signaling pathways. EMBO J. 17, 1675-1687 (1998).

4. Ashkenazi, A., & Dixit, V.M. Apoptosis control by death and decoy receptors. Curr. Opin. Cell Biol. 11, 255-260 (1999).

5. Jϋrgensmeier, J.M., Xie, Z., Deveraux, Q.L., Ellerby, L.M., Bredesen, D.E., & Reed, J.C. Bax directly induces release of cytochrome c from isolated mitochondria. Proc. Natl. Acad. Sci. USA 95, 4997-5002 (1998).

6. Du, C, Fang, M., Li, Y., Li, L., & Wang, X. Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 102, 33-42

(2000).

7. Reed, J.C, Jϋrgensmeier J.M, & Matsuyama, S. Bcl-2 family proteins and mitochondria. Biochim Biophys Acta. 1366,127-137 (1998).

8. Li, P. et al. Cytochrome c and dATP-dependent formation of Apaf-1 /caspase-9 complex initiates an apoptotic protease cascade. Cell 91, 479-489 (1997).

9. Saleh, A., Srinivasula, S.M., Acharya, S., Fishel, R., & Alnemri, E.S. Cytochrome c and dATP-mediated oligomerization of apaf-1 is a prerequisite for procaspase-9 activation. J. Biol. Chem. 274, 17941-17945 (1999).

10. Renatus M., Stennicke, H.R., Scott, F.L., Liddington, R.C., & Salvesen, G.S. Dimer formation drives the activation of the cell death protease caspase 9.

Proc. Natl. Acad. Sci. USA 98, 14250-14255 (2001).

I I. Acehan, D. et al. Three-dimensional structure of the apoptosome: implications for assembly, procaspase-9 binding, and activation. Mol. Cell 9, 423-32 (2002).

12. Han, Z., Hendrickson, E.A., Bremner, T.A., & Wyche, J.H. A sequential two-step mechamsm for the production of the mature pl7:pl2 form of caspase-3 in vitro. J. Biol.

Chem. 272, 13432-13436 (1997).

13. Martin, S.J. et al. The cytotoxic cell protease granzyme B initiates apoptosis in a cell-free system by proteolytic processing and activation of the ICE/CED-3 family protease, CPP32, via a novel two-step mechanism. EMBO J. 15, 2407-2416 (1996). 14. Deveraux, Q.L., Leo, E., Stennicke, H.R., Welsh, K., Salvesen, G.S., & Reed, JC. Cleavage of human inhibitor of apoptosis protein XIAP results in fragments with distinct specificities for caspases. EMBO J. 18, 5242-5251 (1999).

15. Deveraux, Q.L. et al. IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases. EMBO J. 17, 2215-2223 (1998).

16. Deveraux, Q.L., Takahashi, R., Salvesen, G.S., & Reed, J.C. X-linked IAP is a direct inhibitor of cell-death proteases. Nature. 388, 300-304 (1997).

17. Fernandes-Alnemri, T. et al. In vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains. Proc. Natl. Acad. Sci. USA 93, 7464-7469 (1996).

18. Deveraux, Q.L., & Reed, J.C. LAP family proteins—suppressors of apoptosis. Genes Dev. 13, 239-252 (1999).

19. Stennicke, H.R., Ryan, C.A., & Salvesen, G.S. Reprieval from execution: the molecular basis of caspase inhibition. Trends Biochem Sci. 2, 94-101 (2002). 20. Green D.R. Apoptotic Pathways: Paper Wraps Stone Blunts Scissors. Cell, 102, 1-4 (2000).

21. Deng, Y., Lin, Y., & Wu, X. TRAIL-induced apoptosis requires Bax-dependent mitochondrial release of Smac/DIABLO. Genes Dev. 16, 33-45 (2002).

22. Nerhagen, A.M. et al. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 102, 43-53 (2000).

23. Adrain, C, Creagh, E.M., & Martin, S.J. Apoptosis-associated release of Smac/DIABLO from mitochondria requires active caspases and is blocked by Bcl-2. EMBO J. 20, 6627-6636 (2001).

24. Srinivasula, S.M. et al. A conserved XIAP-interaction motif in caspase-9 and Smac/DIABLO regulates caspase activity and apoptosis. Nature 410, 112-116 (2001).

25. Chai, J. et al. Structural and biochemical basis of apoptotic activation by Smac/DIABLO. Nature 406, 855-862 (2000).

26. Kϋppers, R., Hansmann, M.L., & Rajewsky, K. Clonality and germinal centre B-cell derivation of Hodgkin/Reed-Sternberg cells in Hodgkin's disease. Ann Oncol. 9, 17-20 (1998).

27. Cossman, J. et al. Reed-Sternberg cell genome expression supports a B-cell lineage. Blood 94, 411-416 (1999). 28. Kashkar, H., Krδnke, M., & Jϋrgensmeier, JM. Defective Bax Activation in Hodgkin B- Cell Lines Confers Resistance to Staurosporine-induced Apoptosis. Cell Death Diff. 7, 750- 757 (2002).

29. Re, D., Hofmann, A., Wolf, J., Diehl, N., & Staratschek-Jox, A. Cultivated H-RS cells are resistant to CD95L-mediated apoptosis despite expression of wild-type CD95. Exp. Hematol.

28, 348 (2000).

30. Liu, X., Kim, C.Ν., Yang, J., Jemmerson, R., & Wang, X. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86, 147-157 (1996).

31. Srinivasula, S.M., Ahmad, M., Fernandes-Alnemri, T., & Alnemri, E.S. Autoactivation of procaspase-9 by Apaf-1 mediated oligomerization. Mol. Cell 1, 949-957 (1998).

32. Muzio, M., Salvesen, G.S., & Dixit, N.M. FLICE induced apoptosis in a cell-free system. Cleavage of caspase zymogens. J. Biol. Chem. 272, 2952-2956 (1997).

33. Zhang, X.D., Zhang, X.Y., Gray, C.P., Nguyen, T., & Hersey, P. Tumor necrosis factor- related apoptosis-inducing ligand-induced apoptosis of human melanoma is regulated by smac/DIABLO release from mitochondria. Cancer Res. 61, 7339-7348 (2001).

34. Stehlik, C. et al. Nuclear factor (NF)-kappaB-regulated X-chromosome-linked iap gene expression protects endothelial cells from tumor necrosis factor alpha-induced apoptosis. J. Exp. Med. 188, 211-216 (1998).

35. Lee, R. & Collins, T. Nuclear factor-kappaB and cell survival: IAPs call for support. Circ. Res. 88, 262-264 (2001).

36. Bargou, R.C. et al. Constitutive nuclear factor-kappa B-RelA activation is required for proliferation and survival of Hodgkin's disease tumor cells. J. Clin. Invest. 100, 2961-2969 (1997).

37. Hinz, M. et al. Constitutive NF-kappaB maintains high expression of a characteristic gene network, including CD40, CD86, and a set of antiapoptotic genes in Hodgkin/Reed-Sternberg cells. Blood 97, 2798-2807 (2001).

38. Izban, K.F. et al. Characterization of NF-kappaB expression in Hodgkin's disease: inhibition of constitutively expressed NF-kappaB results in spontaneous caspase-independent apoptosis in Hodgkin and Reed-Sternberg cells. Mod. Pathol. 14, 297-310 (2001). 39. Fiumara, P. et al. Functional expression of receptor activator of nuclear factor kappaB in Hodgkin disease cell lines. Blood 98, 2784-2790 (2001).

40. Asselin, E., Mills, G.B., & Tsang, B.K. XIAP regulates Akt activity and caspase-3- dependent cleavage during cisplatin-induced apoptosis in human ovarian epithelial cancer cells. Cancer Res. 61, 1862-1868 (2001). 41. Deng, X., Ruvolo, P., Carr, B., & May, W.S. Jr. Survival function of ERK1/2 as IL-3- activated, staurosporine-resistant Bcl2 kinases. Proc. Natl. Acad. Sci. USA 97, 1578-83 (2000).

42. Yamaguchi, H., & Wang, H.G. The protein kinase PKB/Akt regulates cell survival and apoptosis by inhibiting Bax conformational change. Oncogene 20, 7779-7786 (2001).

43. Drexler H.G., Recent results on the biology of Hodgkin and Reed-Sternberg cells. II. Continuous cell lines. Leukemia and Lymphoma 9, 1-25 (1993).

44. Wolf, J. et al. Peripheral blood mononuclear cells of a patient with advanced Hodgkin's lymphoma give rise to permanently growing Hodgkin-Reed Steinberg cells. Blood 87, 3418- 3428 (1996).

45. Stennicke, H.R. et al. Pro-caspase-3 is a major physiologic target of caspase-8. J. Biol. Chem. 273, 27084-27090 (1998).

46. Ellerby, H.M. et al. Establishment of a cell-free system of neuronal apoptosis: comparison of premitochondrial, mitochondrial, and postmitochondrial phases. J. Neurosci. 17, 6165- 6178 (1997).

47. Stennicke, H.R., & Salvesen, G.S. Biochemical Characteristics of Caspases-3, -6, -7, and - 8. J. Biol. Chem. 272, 25719-25723 (1997).

48. Stennicke, H.R. et al. Caspase-9 can be activated without proteolytic processing. J Biol Chem. 274, 8359-8362 (1999). 49. Jaffe, E.S., Harris, N.L., Stein, H., & Nardiman, J.W., eds. World Health Organization Classification of Tumours. Pathology and Genetics. Tumours of Haematopoietic and Lymphoid Tissue (IARC Press: Lyon, 2001).

Claims

Claims

1. Use of an IAP -inhibitor for the preparation of a pharmaceutical composition for the treatment of Hodgkin's lymphomas.

2. The use of claim 1 , wherein the IAP-Inhibitor is an XIAP-Inhibitor.

3. The use of claim 1 or 2, wherein said IAP-inhibitor in the composition is present sufficient in an amount to restore caspase-activation and caspase-dependent apoptotic signalling in Hodgkin' s lymphoma cells.

4. The use of claim 3, wherein caspase-3, -7 and/or -9 are activated.

5. The use of one any one of claims 1 to 3 claims, wherein said IAP-inhibitor is selected from the group consisting of a molecule that reduces the level of mRNA encoding said

IAP, a molecule that reduces the level of said IAP, a molecule that inhibits the binding of said IAP to a caspase and a molecule that reduces the biological activity of said IAP.

6. The use of claim 5, wherein said inhibitor is selected from the group consisting of an antisense nucleic acid, siRNA, a ribozyme, an anti-IAP antibody, an anti-IAP aptamer, small molecules, a peptide and a peptidomimetic.

7. The use of claim 6, wherein the antisense nucleic acid is a nucleic acid, which selectively hybridizes to the nucleic acid of SEQ ID NO: 1 or to a complementary strand thereof under moderate stringent conditions.

8. The use of claim 6 or 7, wherein the antisense nucleic acid is antisense DNA or RNA for the treatment of Hodgkin' s lymphomas.

9. The use of claim 6 or 7, wherein the antisense nucleic acid is a probe for the diagnosis of Hodgkin's lymphomas.

10. The use of claim 6, wherein said antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a humanized antibody, a fully human antibody, a chimeric antibody, and a synthetic antibody.

11. The use of claim 6 and 10, wherein the antibody specifically recognises a protein or peptide comprising the amino acid of SEQ ID NO: 2 or part thereof.

12. The use of claim 6, 10 or 11, wherein said antibody is linked to a toxic agent.

13. The use of claim 6, 10 or 11 , wherein said antibody is detectably labelled.

14. The use of claim 6, wherein the peptide is a second mitochondria-derived activator of caspases (Smac) or a Smac derived agonistic peptide.

15. Use of an IAP-inhibitor as defined in any one of claims 1 to 14 as a first agent for the preparation of a pharmaceutical composition for sensitizing Hodgkin's lymphoma (HL) or HL-derived cells for the activity of a cytotoxic second agent.

16. The use of claim 15, wherein said second agent is an apoptosis inducing agent.

17. The use of claim 15 or 16, wherein said second agent is a chemotherapeutic drug or γ- irridiation.

18. The use of any one of claims 15 to 17, wherein said second agent is selected form the group consisting of staurosporine, mechlorethamine, vincristine, procarbazine, prednisone, doxorubicin, bleomycin, vinblastine, dacarbazine, chlorambucil, etoposide, mitoxantrone, genestein, phenoxodiol, interferons, drugs triggering death receptors such as the CD95, or pro-drugs or pharmaceutically acceptable salts of any one thereof.

19. The use of any one of claims 15 to 18 comprising the use of a second agent as defined in a any one of claims 15 to 18 for the preparation of a pharmaceutical composition for the treatment of Hodgkin's lymphoma (HL) or HL-derived cells, wherein if said first agent and second agent are comprised in a first and second pharmaceutical compositions, said first and second pharmaceutical composition being applicable simultaneously or sequentially.

20. A method of diagnosis of a disease related to Hodgkin' s lymphomas, which comprises a) assaying a sample from a subject for LAP transcriptional activity or IAP protein; and b) determining the level of IAP gene product or activity, wherein an altered level compared to a control indicates the presence of the disease.

21 A method of diagnosis of a disease related to Hodgkin' s lymphomas, which comprises determining a mutation in the nucleic acid molecule encoding an IAP or an IAP inhibitor in a sample from a subject, wherein the presence of a mutation indicates presence of or predisposition for the disease.

22. The method of claim 20 or 21 , wherein the reagent is a nucleic acid.

23. The method of claim 22, wherein the nucleic acid is labelled or otherwise modified.

24. The method of claim 20, wherein the IAP gene product is determined by an antibody selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a humanized antibody, a chimeric antibody, and a synthetic antibody.

25. The method of claim 24, wherein the antibody is detectably labelled or otherwise modified.

26. A kit for use in a method of any one of claims 20 to 25, said kit comprising an anti- IAP antibody or IAP antisense nucleic acid molecule, or a derivative thereof.

27. A method of treatment of a disease related to Hodgkin's lymphomas comprising administering to a subject in need thereof a therapeutically effective amount of a composition as defined in any one of claims 1 to 19.