CA2155554A1 - Role of atp-ubiquitin-dependent proteolysis in mhc-1 restricted antigen presentation and inhibitors thereof - Google Patents

Abstract

Described herein are methods and drugs that inhibit the processing of antigens for presentation by major histocompatibility complex class I molecules. Specifically, inhibitors of the ATP-ubiquitin dependent proteolytic pathway are described, which can inhibit MHC-I
antigen presentation. These methods and drugs may be useful for the treatment of autoimmune diseases and for reducing rejection of organs and graft transplants.

Description

WO94tl7816 2 1~ PCT~S94/01183 ROLE OF ATP-UBIOUITIN-DEPENDENT PROTEOLYSIS IN
MHC-l RESTRICTED ANTIGEN PRESENTATION
AND INHIBITORS THEREOF

DESCRIPTION

Backqround There are many diseases that result from the immune system attacking and destroying tissues of the body.
These conditions can cause severe morbidity and in many cases can be fatal. Some of these diseases, such as systemic lupus erythematosus, result from an individual's immune system reacting against its own cells as if they were foreign or pathogenic.
Collectively these diseases are referred to as autoimmune diseases. Other diseases result from the immune system responding against molecules, such as therapeutic drugs, that are present in the body's tissues. Yet other diseases are caused when the immune system responds against transplanted foreign tissues in the body. The immune response against foreign tissues is the major cause of failure of transplanted organs or grafts in transplantation and is the principle barrier to this procedure.
In these diseases, the cells that cause the destructive immune response are lymphocytes, and in the vast majority of cases, T lymphocytes play an obligatory role in these responses. The immune response is initiated when T lymphocytes recognize fragments of antigens that are bound to major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells. The antigenic peptide bound to theMHC molecules stimulates the T cells and directs them to W094/17816 PCT~S94tO1183 ~
~15~55 ~

respond against the cells bearing the antigen-MHC
complex.
There are several different kinds of T lymphocytes.
The two major subsets of T lymphocytes, which are distinguished by the expression of certain cell surface molecules, differ in their specificity and function. T
cells that express the CD4 molecule are specific for antigens presented by MHC class II molecules and usually stimulate antibody or inflammatory responses. T cells that express CD8 molecules are specific for antigens presented by MHC class I molecules and function to kill host cells. The CD8+ T cells mediate tissue damage in several of the immune mediated diseases described above.
The therapeutic approaches to treating the immune-mediated diseases are directed at blocking the response of the lymphocytes. Some therapies, such as anti-lymphocyte globulin, seek to eliminate T cells. Other therapies, such as cyclosporin A and steroids, seek to block the response of lymphocytes by inhibiting lymphocyte activation. Still other therapies, such as treatment with monoclonal antibodies against cell interaction molecules, seek to block lymphocytes from interacting with other cells. All of these therapies have significant adverse side effects. Several of the drugs have associated toxicities; e.g., cyclosporin is associated with nephrotoxicity, and steroids cause Cushing's disease. Monoclonal antibodies are associated with immune responses against the antibodies, which makes continued therapy ineffective. In all cases, immunosuppression results in increased susceptibility to infection, which can be lethal. A better approach to controlling or altering T cell responses would be very beneficial therapeutically.

WO94/17816 PCT~S94/01183 215S5~4 Summary of the Invention Described herein are methods and drugs for blocking cytolytic immune responses, which are useful for the treatment of a variety of autoimmune diseases and for preventing rejection of transplanted organs or grafts.
These methods and drugs block the generation of cellular immunity by preventing the initial presentation of cellular and viral antigens on MHC-class I molecules to T cells. The methods comprise using inhibitors of the ATP-ubiquitin-dependent proteolytic pathway to prevent or reduce the processing of intracellular proteins into the antigenic peptides that bind to MHC-l molecules.
This invention results from Applicants' work, which shows that inhibition of an early step (ubiquitin activation) and a late step (cleavage of intracellular proteins into peptides) in the ATP-ubiquitin-dependent proteolytic pathway can inhibit MHC - 1 antigen presentation by blocking the generation of peptides binding to MHC-l molecules. This work provides strong evidence for the role of this cytosolic protein degradation system in the processing of MHC-l presented antigens for MHC-l presentation. Furthermore, Applicants have discovered new features of the function of the proteosome (multicatalytic protease complex), an essential component of the ATP-ubiquitin pathway. As a result of their work, methods and drugs are provided for reducing cytolytic immune responses without affecting antibody-mediated or inflammatory responses. This invention should thus provide therapeutics useful for autoimmune diseases and prevention of transplant rejection without the attendant disadvantages of generalized immune suppression.

21~a 4 ~4~ PCT11~'"94/01183 Specifically, Applicants have discovere~ that gammaAU~ l994 interferon (~-IFN), which has long been known to stimulate antigen presentation, alters the relative activities of the multiple peptidases of the proteasome, so as to promote the generation of peptides with the characteristic carboxyl-terminal residues found on MHC-l bound peptides. The trypsin-like peptidase, which cleaves on the carboxyl side of basic residues, and the chymotrypsin-like peptidase, which cleaves after hydrophobic residues, were found to have increased activity, while the peptidylglutamyl peptidase, which cleaves after acidic residues, had decreased activity.
Furthermore this effect of ~-IFN on the proteasomes is dependent on the expression of two genes at the MHC
locus, LMP 2 and LMP 7.
Secondly, Applicants have demonstrated that MHC-I
antigen presentation can be blocked by inactivation of ubiquitin conjugation, an early step in the ATP-ubiquitin-dependent proteolytic pathway. It was found that, at the nonpermissive temperature, class I-restricted antigen presentation was inhibited in a mutant cell line expressing a th~rmolabile ubiquitin activating enzyme (El).
Applicants have further ~mo~trated that chymostatin, an inhibitor of chymotrypsin-like proteases, inhibits MHC-I presentation by antigen presenting cells. Other inhibitors, particularly of the chymotrypsin-like and trypsin-like activities of proteasomes, are further described herein.

Brief Description of Drawings Figure la to lf shows the effect of gamma interferon (~-IFN) on peptidase activities of proteasomes from U937 215~;55~

cell9. Figure la to lc sho; the hydroly~ls of~thé S 3 7AUG ~54 indicated substrate~ by proteasome fractions from control cells and cells treated with y-IFN. Figure ld to lf shows kinetic analysis of rates of cleavage of the substrates by the same proteasome fractions.
Figure 2a to 2f shows the effect of ~-IFN on peptidase activities of purified 20S and 26S proteasomes.
Figure 3a to 3f shows the difference in activities of the peptidases of proteasomes from wild-type and MHC-deficient lymphoblastoid cells. Figure 3a to 3c shows the peptidase activities. Figure 3d to 3f shows kinetic analys is .
Figure 4a to 4b shows the frequency of amino acid residues preceding bonds cleaved to generate peptides presented on MHC-class I molecules. Figure 4a shows the frequency of amino acids at the carboxyl-termini. Figure 4b shows the frequency of amino acids preceding the amino-term; n~ 1 residues.
Figure 5a to 5d shows MHC class I-restricted presentation of ovalbumin (OVA) by mutant (ts20.10.2) and wild-type (E36.12.4) cells.
Figure 6a to 6b shows the effect of temperature on the synthesis and maturation of H-2Kb MHC heavy ch~' n~ .
Figure 7a to 7b shows MHC class I-restricted presentation of endogenously synthesized OVA
peptide.
Figure 8a to 8b shows inhibition of MHC class I-restricted presentation of ovalbumin (Figure 8a) and ovalbumin peptide (Figure 8b) by chymostatin.

Detailed De~cription This invention relate~ to an approach for inhibiting cytolytic mmllne responses that avoids A~ ~ S~

W O 9~/17816 PCTrUS94/01183 2i~55~ ~

generalized suppression of T lymphocyte function with its attendant risks of infection. The methods and drugs of this approach are useful for treating autoimmune diseases and preventing rejection of foreign tissues, such as transplanted organs or grafts. The strategy is to inhibit antigen presentation by major histocompatibility complex (MHC) I molecules rather than suppress T cell activity. This approach has the advantage that it selectively affects only class I MHC-restricted immune responses and not antibody or otherCD4+ T cell-mediated responses. Consequently, there should be less generalized immunosuppression and susceptibility to infection in the patient. Drugs that interfere with class I antigen presentation should also be useful in combination with other existing therapies and may lower the dose ùsed in these therapies, which would decrease toxicities.
Specifically, methods and drugs are described that inhibit the processing of internalized cellular or viral antigens into the kind of peptides, referred to as antigenic peptides, that bind to the MHC-I molecules.
MHC-I binding peptides have strict sequence and size requirements. In the absence of the antigenic peptides, the antigens are not presented at the cell surface and CD8+ T cells are not stimulated. This method should block both the initiation of immune response and stop ongoing immune responses.
This invention is based on Applicants' work (see Examples below) supporting the role of the ATP-ubiquitin dependent proteolytic pathway, and in particular, proteasomes (multicatalytic proteinase complexes), in the generation of MHC-I-associated antigenic peptides.
An initial step in the presentation of intracellular and WO94117816 PCT~S94/01183 1-- 215~54 viral proteins to the immune system is their proteolytic degradation in the cytosol to small peptides (Townsend and Bodmer, 1989). The antigenic fragments are then taken up via specific membrane transporters (Monaco, 1992; Powis, et al., 1991; Spies and DeMars, 1991) into the endoplasmic reticulum, where they associate with the MHC-I molecules. These peptide-protein complexes are then transported to the cell surface for presentation to cytotoxic T cells. The cleavage of proteins into antigenic peptides seems to be a regulated process, since there are strict size and sequence constraints on the peptides that can bind to MHC-class I molecules.
The identity of the cleavage enzymes and the nature and precise location(s) of the proteolytic steps that generate the 8-9 residue peptides presented on MHC-class I molecules have not been definitively established, although it is clear that the proteolysis does not occur in lysosomal or endosomal compartments (Morrison et al., 1986).
Recent studies have implicated the proteasomes, an essential component of the ATP-ubiquitin-dependent proteolytic pathway, in the generation of antigenic peptides. Several proteolytic systems exist in eukaryotic cells, and the ATP-ubiquitin pathway is the major cytosolic pathway of protein degradation (Finley and Chau, 1991; Hershko and Ciechanover, 1982;
Rechsteiner, 1987). In this multistep process, protein substrates are first modified by covalent conjugation to multiple ubiquitin moieties, which marks them for rapid degradation by a 26S (1,500 kD) ATP-dependent proteolytic complex, called the 26S proteosome or UCDEN
(Goldberg and Rock, 1992; Goldberg, 1992; Tanaka et al., 1992; Waxman et al., 1987; Hough et al., 1987). This WO94/17816 PCT~S94/01183 ~

21~i5~5 ~

large structure contains the 20S (about 700 kD) proteasome as its proteolytic core plus many additional regulatory and catalytic components (Goldberg and Rock, 1992; Orlowski, 1990; Rivett, 1989; Waxman et al., 1987;
Hough et al., 1987; Armon et al., 1990; Driscoll and Goldberg, 1990; Eytan et al., 1989). The precise intracellular function of the 20S proteosome is not clear; when isolated, this particle can degrade proteins and oligopeptides, but not ubiquitin-conjugated proteins (Goldberg, 1992; Tanaka et al., 1992; Armon, 1990;
Driscoll and Goldberg, 1990a; Eytan et al., 1989;
Driscoll and Goldberg, 1989).
The 20S proteasome is composed of about 15 distinct subunits of 20 - 30 kD. It contains three or four different neutral peptidases, which cleave specifically on the carboxyl side of hydrophobic, basic and acidic amino acids (Goldberg and Rock, 1992; Goldberg, 1992;
Tanaka et al., 1992; Orlowski, 1990; Rivett, 1989).
These peptidases are referred to as the chymotrypsin-like peptidase, the trypsin-like peptidase, and the peptidylglutamyl peptidase, respectively. Which subunits are responsible for these activities is unknown, although the cDNAs encoding several subunits have been cloned (Tanaka et al ., 1992).
Recent studies have found that the 20S proteasomes resemble in size and subunit composition the MHC-linked low molecular weight protein (LMP) particles (Driscoll and Finley, 1992; Goldberg and Rock, 1992; Monaco and McDevitt, 1986; Parham, 1990; Martinez and Monaco, 1991;
30 Oritz-Navarette et al., 1991; Glynne et al., 1991; Kelly et al., 1991; Monaco and McDevitt, 1982; Brown et al., 1991; Goldberg, 1992; Tanaka et al., 1992). The LMP
particles contain two polypeptides, LMP 2 and LMP 7, W094/17816 PCT~S94/01183 215 ~ 3 S ~

which are encoded in the MHC chromosomal region.
Treatment of cells with gamma interferon (y-IFN) stimulates antigen presentation (Townsend and Bodmer, 1989; Yewdell and Bennink, 1992) and causes the induction of LMP 2 and LMP 7, as well as other MHC genes (Glynne et al., 1991; Kelly et al., 1991; Monaco and McDevitt, 1982; Yang et al ., 1992). Immunochemical studies strongly suggest that LMP 2 and LMP 7 are two subunits of particles representing a small fraction of the 20S proteosome population. However, the importance of these subunits in the immune response is uncertain, since deletion of these genes does not prevent antigen presentation, in contrast to deletion of other MHC-genes (Arnold et al ., 1992; Mamburg et al ., 1992). Moreover, y-IFN can alter the polypeptide composition of 20S and 26S proteasomes (Yang et al., 1992) in mutants lacking the LMP 2 and 7 It has, thus, been a matter of uncertainty whether proteasomes play a role in antigen presentation (see discussions in Goldberg and ~ock, 1992; Driscoll and Finley, 1992; Yewdell and Bennin~, 1992, pp. 26-28).
Applicants' work described herein provides confirming evidence for a role for the 20S and 26S
proteasomes and the ATP-ubiquitin proteolytic pathway in the generation of antigenic peptides for MHC-1 antigen presentation. In addition, this work reveals new features of the pathway of proteolysis of antigens and of proteasome function, which led to the present invention. Firstly, they have shown that gamma interferon, which is known to stimulate antigen presentation, selectively enhances those peptidase activities, namely, the trypsin-like and chymotrypsin-like activities, of the proteasomes that produce WO94/17816 PCT~S94/01183 21~5~$~

peptides that can bind to MHC-l molecules, i.e. peptides with basic and hydrophobic carboxyl-terminal residues (Example l). Gamma interferon increased 2- to 6-fold the capacity of purified 20S and 26S proteasomes to cleave peptides after hydrophobic and basic residues, while reducing cleavage after acidic residues. They have further shown that this enhancement by gamma interferon is dependent on the expression of the MHC
gene products, LMP 2 and LMP 7. Their findings, thus, provide evidence to support the role of proteasomes in antigen presentation. Furthermore, they indicate that the three peptidases of proteasomes are distinctly regulated, that changes in the relative activities of the peptidases can alter the nature of peptides that are generated by the proteasome and which are available for MHC-l antigen presentation, and that the MHC-encoded subunits are involved in the regulation of the peptidase activities.
Secondly, Applicants have shown that a defect in ubiquitin conjugation, an early step in the ATP-ubiquitin-dependent proteolytic pathway, leads to reduced MHC-I-restricted antigen presentation (Example 2). Using cells exhibiting a temperature-sensitive defect in the ubiquitin activation enzyme, El, they found that the nonpermissive temperature inhibited class I-restricted presentation of ovalbumin (OVA) introduced into the cytosol, but did not affect presentation of an OVA peptide synthesized from a minigene.
These findings provide evidence for the role of the ATP-ubiquitin proteolytic system in MHC-I antigen presentation. They demonstrate that a block at either an early step (ubiquitin conjugation) or a late step (processing of carboxyl-termini of the peptides) in the ~ WO94/17816 PCT~S94/01183 2~iSS4 proteolysis of intracellular proteins by the ATP-ubiquitin system can inhibit class I restricted presentation. It is reasonable to expect that inhibition of other steps in this proteolytic pathway will also produce a similar effect.
Applicants' discoveries of these features of proteosome function and their demonstration that inhibition at a single step in the ATP-ubiquitin pathway can inhibit antigen presentation lead to the present invention. It is reasonable to expect that inhibitors that block single or multiple steps of this proteolytic pathway would inhibit MHC-I-restricted antigen presentation, and that such inhibitors would be useful in reducing CD8+ mediated cytolytic immune responses.
Such inhibitors should be useful for therapy or prevention of autoimmune diseases and for reducing rejection of transplanted and other foreign tissues.
Inhibitors of either or both the trypsin-like and chymotrypsin-like peptidases of proteasomes and inhibitors of ubiquitin conjugation are particularly expected to be useful for these purposes.
As described herein, Applicants have demonstrated the feasibility of this invention by showing that chymostatin, which selectively inhibits cleavage after hydrophobic residues, inhibits the MHC-I presentation of ovalbumin (Example 3). Antigen presentation of an OVA
peptide introduced into the cytosol was not inhibited, indicating that chymostatin is affecting the processing of the OVA protein into the peptide. This and other inhibitors are further described in the following section.

WO94/17816 PCT~S94/01183 ~

215~5~

Inhibitors of the ATP-Ubiquitin Dependent Proteolytic Pathway Various inhibitors of the peptidases of proteasomes have been reported (see, e.g., Tanaka et al., 1992;
Orlowski, 1990; Goldberg, 1992; Rivett et al., 1989a [Arch. Biochem. Biophys. 268:1-8] and 1989b [J. Biol.
Chem. 264:12,215-12219]; Dick et al., 1991). These include known inhibitors of chymotrypsin-like and trypsin-like proteases, as well as inhibitors of thiol (or cysteine) and serine proteases. In addition, some endogenous inhibitors of proteosome activities have been isolated. These include the 240 kD and the 200 kD
inhibitors isolated from human erythrocytes (Murakami and Etlinger, 1986; Li et al., 1991 and purified CF-2 (described in Example 4; Goldberg, 1992). As exemplified by these known inhibitors of proteasomes, other molecules, including known inhibitors of chymotrypsin-like, trypsin-like, thiol, and serine proteases, can be tested using assays for the proteosome peptidases (see Examples).
The inhibitors can be naturally isolated or synthetic and peptide or non-peptide molecules.
Preferably, the inhibitors would selectively inhibit the chymotrypsin-like and trypsin-like peptidases, while leaving the peptidylglutamyl peptidase relatively unaffected. A typical inhibitor would be a peptide aldehyde, like leupeptin, which inhibits primarily cleavage after basic residues, or chymostatin, which inhibits primarily after hydrophobic residues. In addition to these antibiotic inhibitors originally isolated from Actinomycetes (Aoyagi and Umezawa, 1975), a variety of peptide aldehydes have been synthesized, WO94/17816 PCT~S94/01183 .

such as the inhibitors of chymotrypsin-like proteases described by Siman et al. (WO 91/13904).
Novel molecules can also be obtained and tested for inhibitory activity. As illustrated by the above cited references, various strategies are known in the art for obtaining the inhibitors for a given protease. Compound or extract libraries can be screened for inhibitors using peptidase assays. Alternatively, peptide and peptidomimetic molecules can be designed based on knowledge of the substrates of the protease. For example, substrate analogs can be synthesized containing a reactive group likely to interact with the catalytic site of the protease (see, e.g., Siman et al., WO
91/13904; Powers et al., 1986). The inhibitors can be stable analogs of transition intermediates (transition state analogs), such as Cbz-Gly-Gly-leucinal, which inhibits the chymotrypsin-like activity of the proteosome (Orlowski, 1990; see also Kennedy and Schultz, 1979). Variants or analogs of known inhibitors, such as chymostatin, can be also be synthesized.
Various natural and chemical protease inhibitors reported in the literature, or molecules similar to them, are likely to inhibit the activity of the two proteasomal peptidases. These include peptides containing an ~-dicarbonyl unit, such as an ~-diketone or an ~-keto ester, peptide chloromethyl ketones, isocoumarins, peptide sulfonyl fluorides, peptidyl boronates, peptide epoxides and peptidyl diazomethanes (Angelastro et al., 1990; Bey et al., EPO 363,284; Bey et al., EPO 364,344; Grubb et al., WO 88/10266; Higuchi et al., EPO 393,457; Ewoldt et al., 1992; Hernandez et al., 1992; Vlasak et al., 1989; Hudig et al., 1991;

WO94/17816 PCT~S94/01183 ~i5S~5 ~

Odakc et al. 1881; Vijayalakshmi et al. 1991; Kam et al. 1990; Powers et al., 1989; Oweida et al. 1990;
Powers et al. 1990; Hudig et al. 1989; Orlowski et al. 1989; Zunino et al. 1988; Kam et al., 1988; Parkes et al . 1985; Green and Shaw, 1981; Angliker et al .
1987; Puri et al. 1989; Hanada et al . 1983; Kajiwara et al . 1987; Rao et al . 1987; Tsujinaka et al . 1988].
Various inhibitors of ubiquitin conjugation to proteins are also known (see Wilkinson et al. 1990).
Ubiquitin conjugation itself is a multistep process, involving the activities of an ubiquitin activating enzyme (El), a group of ubiquitin-carrier proteins (E2), which catalyze the transfer of ubiquitin to target proteins, and the ubiquitin-protein ligase, E3.
Molecules that block ubiquitin conjugation at any of these steps are also expected to be inhibitors of antigen presentation. Preferably, specific inhibitors of these enzymes will be used, such as substrate and transition state analogs. For example, adenosyl-phospho-ubiquitinol, an analog of the substrate ubiquitin adenylate, has been found to be a specific and effective inhibitor of E1 (Wilkinson et al., 1990).
The inhibitors can be used in vitro or in vivo to block MHC-I antigen presentation. They can be administered by any number of known routes, including orally, intravenously, intramuscularly, topically, and by infusion (see, e.g., Platt and Stracher, U.S. Patent 4,510,130; Badalamente et al., 1989; Staubli et al., 1988). Preferably, the inhibitors are low molecular weight molecules. Suitable vehicles for protein drug delivery, such as liposomes, may also be used. Vehicles than can target the drug to specific tissues can also be used.

~ W094/17816 PCT~S94/01183 21~5~

ExamPles The following Examples further describe Applicants' work in more detail and more specifically illustrate this invention. Tables are located at the end of each Example.

ExamPle l Requlation of the Peptidase Activities of Proteasomes ~y Y-IFN and MHC Genes The present studies were undertaken to clarify the role of y-IFN, proteasomes, and MHC-encoded proteins in the processing of cell antigens. The work described below demonstrates that the pattern of peptidase activities of the 20S and 26S proteasome complexes is altered following treatment of cells with y-IFN and upon expression of the MHC region that contains the LMP 2 and 7 genes. These changes in catalytic activities should favor the generation of the types of peptide sequences (Falk et al., l99l) that bind specifically to MHC-class I proteins.

y-Interferon Alters Proteosome Activities.
To learn whether y-IFN regulates the function of proteasomes, several hydrolytic activities of the proteasomes and of soluble extracts from control U937 cells (a human monocytic line) and U937 cells treated with y-IFN were examined. Although this treatment caused a large induction of MHC-class I genes and proteins encoded in the MHC region (i.e. LMP 2 or TAP
l), no significant difference was found in the ability of isolated proteasomes or cell extracts to degrade l25I-lactalbumin or ubiquitinated l25I-lactalbumin to WO94/17816 PCT~S94/01183 ~SSS 4 acid-soluble fragments. However, clear changes were found after y-IFN treatment in the ability of the proteasomes to hydrolyze different peptide substrates.
Fluorogenic peptide substrates were used to measure the hydrolytic activities of proteasomes: Suc-LLVY-MCA ~SEQ ID No: 3]
(hydrophobic) for the "chymotrypsin-like" peptidase, Boc-LR~-MCA (basic) for the "trypsin-like" peptidase, and Cbz-LLE-MNA (acidic) for the peptidylglutamyl peptidase.
Proteasome-enriched fractions, as well as crude extracts, from y-IFN-treated U937 cells hydrolyzed the hydrophobic peptide, Suc-LLVY-MCA, 2- to 3-fold faster and the basic peptide, Boc-LRR-MCA, 3- to 4-fold faster than similar preparations from control cells (Figure l).
These effects of y-IFN-treatment were seen in the absence of ATP. Kinetic analysis indicated that y-IFN
caused a 2-fold increase in the maximal capacity (Vmax) of the proteasome to hydrolyze the hydrophobic substrate and a 6-fold increase in VmaX with the basic substrate (Figure l). In addition, a small increase in the Kms for these substrates (averaging 50%) was found consistently, but such an effect cannot account for the accelerated peptide hydrolysis (Figure l). Very similar changes in VmaX were seen in analogous studies using purified 20S proteasome (see below~. The cleavage of a variety of other hydrophobic and basic peptides, which are degraded more slowly than Suc-LLVY-MCA or Boc-LRR-MCA (respectively), increased in a similar fashion after y-IFN treatment, as did hydrolysis of these standard substrates (Table l). Thus, proteasomes from y-IFN-treated cells are generally more efficient in cleaving after hydrophobic and basic residues, although the WO94/17816 PCT~S94/01183 2 1 5 ~

specificities of these active sites do not appear to change.
Under these conditions, the overall capacity of the cells to hydrolyze these basic and hydrophobic peptides increases. However, no change was found in total proteasome content. The ability of the crude extracts to hydrolyze these peptides changed in a similar fashion, as did proteasome fractions, which contain about 80% of the activities against these substrates in post-nuclear cell extracts. Most of the remaining peptidase activities (15% total) was due to proteasomes that were tightly associated with microsomal membranes, and y-IFN treatment also enhanced the cleavage of Suc-LLVY-MCA and Boc-LRR-MCA by proteasomes in the microsomal and nuclear fractions.
It is noteworthy that not all of the proteasomes's peptidase activities increase after y-IFN treatment.
Proteasomal fractions and the crude extracts from y-IFN-treated U937 cells cleaved the acidic substrate, Cbz-LLE-MNA (at lO0 mM), at roughly similar rates as controls. Further kinetic analysis, however, indicated that there was a reproducible decrease of approximately 30% in the VmaX value for this substrate (Figure l) (as well as a decrease in the Km)~ Thus, the several catalytic sites of the proteasome are regulated in distinct fashions by y-IFN.

Behavior of 20s ~nd 268 Complexes Within cells, proteasomes are found either as part of the 26S (1,500 kD) complexes, which degrade preferentially ubiquitin-conjugated proteins, or as 20S
particles, which by themselves cannot hydrolyze Ub-conjugated proteins, despite having multiple proteolytic W094/17816 PCT~S94/01183 ~5~5~ ~

activities (Goldberg and Rock, 1992; Goldberg, 1992;
Tanaka et al., 1992; Armon et al., 1990; Driscoll and Goldberg, 1992; Eytan et al., 1989; Driscoll and Goldberg, 1989; Matthews et al., 1989). To ~mine whether y-IFN alters the catalytic activities of both the 20S and 26S proteasomes, these structures were purified by FPLC using anion exchange and gel filtration columns (Figure 2). A good separation of the 20S and 26S forms of the proteasome was achieved. Native PAGE
lo of the 20S fraction showed the characteristic 700 kD
band, while the 20S samples contained a 1500 kD band plus some 700 kD material. As expected, these 26S
preparations degraded ubiquitinated-l25I-lactalbumin in the presence of ATP, but exhibited little or no activity against free l25I-lactalbumin. In contrast, the 20S
fraction degraded this protein, but not if it was ubiquitinated. Both 20S and 26S forms exhibited the three peptidase activities, which could be stimulated several-fold by ATP. ATP (2 mM) stimulated the degradation of Suc-LLVY-MCA by 20S proteasomes and the degradation of the same substrate 5-fold by 26S
proteasomes from control cells. In the presence of ATP, significant differences between control and ~-IFN-treated preparations were retained for 20S proteasomes Interferon-treatment caused similar changes in the peptidase activities of both 20S and 26S proteasomes, as were found with cruder preparations. With both forms, cleavages of hydrophobic and basic substrates increased significantly with y-IFN, although the percent increase tended to be somewhat smaller for the 26S fraction.
Interestingly, cleavage of the acid substrate by either particle did not increase, and its hydrolysis by the 26S
particle decreased by 50% after y-IFN treatment (Figure ~ W094/17816 215 5 ~ ~ 4 PCT~S94/01183 2). By contrast, after y-IFN treatment, no consistent differences were observed in the abilities of the 20S or 26S particles to degrade free 125I-lactalbumin or ubiquitinated l25I-lactalbumin either in the absence or presence of ATP. Taken together, these findings suggest that y-IFN alters the pattern of cleavages in polypeptides made by both 20S and 26S proteasomes, without affecting the overall rates of protein digestion.

Proteasome Activities in MHC-Deficient Cells Since two proteins encoded in the MHC region, LMP 2 and LMP 7, associate with proteasomes (Driscoll and Finley, 1992; Goldberg and Rock, 1992; Monaco and McDevitt, 1986; Parham,.l990), and since y-IFN
stimulates the expression of these components, Applicants investigated whether deletion of this part of the MHC-locus might influence the peptidase activities of proteasomes and their response of ~-IFN. For this purpose, they isolated the proteasomal fraction from 721 human B lymphoblastoid cells and from 721.174, a variant of these cells, that carries a homozygous deletion in the MHC region that includes the LMP 2 and LMP 7 genes DeMars et al., 1985). Proteasomes from the mutant - cells were found to degrade the basic substrate at approximately 40% of the rate and the hydrophobic substrate at 70% of the rate of the wild-type cells (Figure 3). VmaX values for the two substrates decreased by more than 50% for proteasomes from mutant cells (Figure 3). The total content of the proteasomes appeared similar in the two cell types, and no clear difference was seen in the ability of the proteasomes to degrade l25I-lysozyme or ubiquitinated-125I-lysozyme to WO94/17816 PCT~S94/01183 ~

2~5~5 ~

-20- .
acid-soluble products. Furthermore, the breakdown of several more slowly degraded hydrophobic and basic substrates (the same ones studied in Table 1) was also lower in the MHC-deleted mutant by 40% to 80. In contrast to the reduction in these peptidase activities, proteasomes from the MHC-deleted variant consistently cleaved the acidic substrate, Cbz-LLE-MNA, about 40%
faster than similar preparations from wild-type cells (Figure 3). A small, but consistent, increase in the lo Vmax was seen for this substrate (Figure 3).
Similar changes in the three peptidase activities were found in crude extracts from the mutant and wild-type cells and proteasome microsomal or "nuclear"
fractions. Thus, the peptidase capacity of proteasomes in all fractions of the cell appear to be regulated by MHC-encoded genes and by y-IFN. The findings that expression of MHC-encoded genes increases basic and hydrophobic cleavages by the proteasome, while suppressing acidic cleavages, parallels the results obtained when U937 cells were treated by y-IFN. These observations raise the possibility that the y-IFN-dependent changes in proteasome activities are mediated, at least in part, through the expression of MHC-encoded proteins, most likely the LMP 2 and LMP 7 proteasome subunits. To examine this hypothesis, Applicants used the 721 and 721.174 variant to investigate whether y-IFN
treatment could alter these peptidase activities in the absence of the LMP genes.
In wild-type cells treated with y-IFN, there was an increased rate of degradation of hydrophobic and basic substrates and a reduced capacity to cleave the acidic substrate. Thus, the proteasomes from wild-type lymphocytes responded in a manner similar to those of ~ W094/17816 PCT~S94/01l~
2~S~

U937 cells (although the relative changes appeared larger in the monocyte line). However, y-IFN treatment of the MHC-deleted cells did not alter at all the degradation of the basic substrate, Boc-LRR-MCA, in contrast to the large changes seen with proteasomes from wild-type cells. In the 721.174 mutant, y-IFN caused a much smaller increase in cleavage of the hydrophobic peptide, Suc-LLVY-MCA, than in the wild-type parent cells. These differences between the cells were evident with a number of different basic and hydrophobic peptides (Table 2). Surprisingly, in mutant lymphoblasts, y-IFN actually enhanced hydrolysis by proteasomes of the acidic substrate (Table 2), even though in the wild-type parent cells (and in the U937 line), y-IFN reduced reproducibly cleavage of this substrate. Clearly, the IFN-induced alterations in the activities of the three catalytic sites of the proteasome, either directly or indirectly, are MHC-dependent.

Protein Brea~down and Proteasome Function Two general new conclusions have emerged from these experiments: 1) that the catalytic functions of proteasomes are qualitatively regulated by gamma-interferon and these actions require the presence of MHC-encoded genes; and 2) that the different peptidase sites of these particles can be regulated in distinct fashions. After y-IFN treatment, proteasomes in monocytes and lymphoblasts, and presumably in many other cells, show a greater capacity to hydrolyze peptides following hydrophobic and basic residues, but a reduced capacity to cleave after acidic residues.

W094/17816 PCT~S94/01183 ~

2155~5~

Previous studies have demonstrated several cellular factors that can bind reversibly to the proteasome and stimulate (Dubiel et al., 1992; Ma et al., 1992b) or inhibit (Driscoll et al., 1992; Li et al., 1991) its three peptidase activities coordinately (Goldberg and Rock, 1992; Goldberg, 1992; Tanaka et al., 1992). In contrast, y-IFN affects each of these catalytic sites differently, presumably because it regulates gene expression and thereby alters the subunit composition of the proteasome (see below). Interestingly, y-IFN was not found to alter the rates of digestion of a protein substrate, 125I-lactalbumin or ubiquitin-conjugated l25I-lactalbumin to acid-soluble fragments by either the 20S
or 26S proteasome. These findings suggest that in vivo, the initial, presumably rate-limiting, cleavages of polypeptides or ubiquitinated proteins by proteasomes are not altered by y-IFN. Instead, this cytokine appears to affect the later steps in this pathway, i.e.
the subsequent cleavages of the acid-soluble protein fragments to short peptides. Thus, the changes in the relative activities of the three peptidases should alter the nature of the oligopeptides that are generated by the proteasome and are available for further proteolytic processing andtor delivery to MHC-class I molecules (see below).
It will be important to define the structural changes in the protease that lead to the increased rates of hydrolysis after basic and hydrophobic sequences and to the decreased cleavage after acidic residues. The major alterations in these activities induced by y-IFN
clearly require the presence of the portion of the MHC
region containing the LMP 2 and 7 genes. Deletion of this region by itself (without y-IFN treatment) caused WO94/17816 PCT~S94/01183 21555~

the reverse changes in peptidase activities of those induced by y-IFN. Furthermore, y-IFN is known to stimulate the expression of the two MHC-encoded subunits, LMP 2 and LMP 7, and to increase the abundance of "LMP particles" in cells (i.e. those proteasomes containing LMP subunits). The simplest explanation for these findings is that the y-IFN-induced alterations in catalytic properties result from incorporation into proteasomes of these MHC-encoded subunits. The LMP 2 and/or LMP 7 proteins may themselves contain "tryptic-like" or "chymotryptic-like" sites. If so, these sites may supplement the peptidase sites present in the absence of y-IFN or may replace them with sites of a higher catalytic efficiency. In this content, it is intriguing that, in yeast, the "chymotryptic-like"
activity of proteasomes is inactivated by point mutations in a proteasome-subunit gene very homologous to LMP 2 (Driscoll et al., 1992; Li et al., l99l; Ma et al., 1992). Alternatively, incorporation into proteasomes of LMP 2 or LMP 7 might enhance the activity of other subunits that contain the trypsin-like or chymotrysin-like catalytic sites. Similarly, incorporation of these MHC-encoded subunits may replace or reduce the activity of subunits that comprise the peptidylglutamyl catalytic site of the proteasomes.
Although these findings suggest functions for the LMP-encoded subunits, y-IFN clearly induce some changes in proteasome activity in the mutants lacking LMP 2 and LMP 7 genes. In these cells, y-IFN caused a small (but reproducible) increase in the chymotryptic activity. In addition, the peptidylglutamyl activity in the mutants increases upon y-IFN treatment, even though in wild-type cells, this activity decreases. These functional W094/17816 PCT~S94/01183 215~5 ~

changes must arise through other alterations in proteasome subunit composition, not involving LMP 2 and LMP 7. Presumably, ~-IFN can reduce or suppress the synthesis of subunits encoded outside of this portion of the MHC region or may otherwise modify proteasome structure. In fact, Peterson and coworkers (Yang et al., 1992) have reported that y-IFN alters the pattern of proteasome subunits in mutants lacking the LMP genes.
It is noteworthy that LMP 2 and LMP 7 proteins are present in only a subset of the cell's proteasomes (Brown et al., l99l), and within this subset, some particles may exist that contain only one of these subunits, while others may contain both. In either case, the LMP 2- and LMP 7-containing populations probably differ to a very large degree from the preexistent population in their hydrolytic activities, since the induction of these subsets or proteasomes by y-IFN were observed to cause large changes in the total peptidase activities in the cell within 3 days. Thus, the changes in catalytic activity found here probably underestimate the actual differences between the activities of LMP-containing and LMP-deficient subsets of proteasomes.
In the past, the proteasome has generally viewed as a single type of structure with characteristic enzymatic properties. This view is clearly simplistic or incorrect in light of the present findings, which emphasize the functional heterogeneity and plasticity of these particles. These findings suggest that in many cells, proteasome properties change in vivo during infections or sepsis or other conditions, in which y-IFN
levels rise and immune responses are activated. These alterations in proteasome properties must depend also on ~ WO94/17816 PCT~S94/01183 215S~ 4 ~ ~ ~

dosage and duration of exposure to this cytokine. The existence of functionally distinct subclasses of proteasomes in cells may also be important in many other biological contexts. In fact, there have been reports of alterations in proteasome-subunit composition during cell development (Ahn et al., l99l; Scherrer, l990), which presumably result in distinct changes in function and have different physiological consequences from those reported here.

Implication~ for Antigen Presentation As part of normal protein turnover, most of the peptides generated by the proteasomes in cytosol or nucleus must be rapidly digested to free amino acids by non-proteasomal peptidases. However, a fraction appears to be transported into the endoplasmit reticulum (ER) by the TAP 1-2 complex and to bind to MHC-class I
molecules, either directly or after further proteolytic processing (Townsend and Bodmer, 1989; Yewdell and Bennink, 1992; Monaco, 1992; Powis et al., l99l; Spies and DeMars, l99l). The changes in the proteasomal activities induced by y-IFN or resulting from the MHC-deletion should alter the peptides generated by the proteasome during degradation of cellular and viral - proteins, and thus change the repertoire of peptides available for presentation to T cells. These alterations in the relative rates of cleavage after basic, hydrophobic, and acidic residues should result in production of a different group of peptides or of the same peptides, but in very different proportions.
Specifically, the 2- to 6-fold increase in the "chymotryptic-like" and "tryptic-like" activities and the decrease in peptidylglutamyl activity should WO94/17816 PCT~S94/01183 21~

generate more short peptides ending with basic or hydrophobic carboxyl termini, and fewer peptides ending with acidic carboxyl termini.
These changes in catalytic properties with y-IFN or expression of MHC-encoded genes would thus appear to favor the generation of just those types of peptides that are known to bind to MHC-class I molecules. The C-terminal residue of antigenic peptides plays a key role in their binding to specific MHC-class I molecules (Falk 10 et al . , 1991; Guo et al ., 1992; Hunt et al ., 1992;
Jardetzky et al ., 1991; Matsumura et al ., 1992; Parham, 1992), as is also evident from the X-ray analysis of several MHC-protein-peptide complexes (Rotzschke and Falk, 1991; Fremont et al ., 1992; Silver et al., 1992;
Zhang et al ., 1992). It is indeed striking that the vast majority of naturally-processed peptides that have been found associated with class I molecules have hydrophobic or basic C-termini, and virtually none has an acidic C-terminus (Figure 4). This conclusion is based upon Applicants' survey of the published literature of MHC-associated peptides. Furthermore, an even stronger preference for hydrophobic C-termini is evident in the sequences of the average population of peptides that can be eluted from two specific subtypes of MHC-class I proteins. This predominance of basic and hydrophobic residues was not seen at the amino acids preceding the N-termini of MHC-associated peptides (Figure 4).
Several immunological roles have been postulated for LMP 2 and LMP 7 subunits. It has been proposed that the LMP subunits direct the proteasome particles to the endoplasmic reticulum (ER), where they would be in contiguity with the TAP-1/2 transporters. However, ~ WO94/17816 PCT~S94/01183 21~5~

Applicants and others (Yang et al., 1992) have found no evidence that the loss of LMP genes or y-IFN-stimulation of LMP expression affects the subcellular distribution of proteasomes. Instead, Applicants have found that the y-IFN-treatment and MHC-deletion change the peptidase activities of proteasomes in the soluble fraction of the c~ll, as well as the activities of the minor population of proteasomes associated with ER (10% total) and with the nuclear fraction. For transport into the ER, these antigenic peptides generated by soluble proteasomes must somehow withstand complete hydrolysis by cytosolic exopeptidases.
More recently, it has been argued that LMPs do not play an important role in antigen presentation (Arnold et al., 1992; Mamburg et al., 1992), because MHC-deleted cells (like 721.174) can present antigens if they are transfected only with the genes for the TAP-l and -2 transporters. This work shows, however, that the proteasomes isolated from these mutant cells still exhibit the tryptic- and chymotryptic-like activities.
These findings may explain why the MHC-deleted cells can still present antigens on class I molecules. The results lead to the prediction of a decrease in the efficiency of antigen processing and presentation by the MHC-deleted mutant cells. This was in fact reported by Hammerling and coworkers (Mamburg et al., 1992). On the other hand, the actions of y-IFN to increase expression of LMPs and thereby to change the pattern of peptides produced by the proteasome may contribute, along with the induction of the transporters and MHC-class I
proteins, to the enhancement of antigen presentation.

WO94/17816 PCT~S94/01183 2155~5~

Methods Treatment of Cells ~ith y-IFN
U937 cells (0.15 X l06/ml) were grown in 150 cm3 flasks, in RPMI 1640 medium containing 10% FCS and antibiotics at 37C for 72 hours in the presence or absence of 1000 U/ml of human recombinant y-IFN (kindly provided by Biogen, Inc. Cambridge, MA). In preliminary studies, this concentration of y-IFN was found to cause maximal changes of proteasomal peptidases activities in U937 cells. Mutant (721.174) and wild-type (721) cells were grown for three days in the presence or absence of 3,000 U/ml of y-IFN, which was found to cause maximal changes in peptidases activities.

Peptid~se Assays Hydrolysis of 125I-human lactalbumin or ubiquitin-conjugated-l25I-lactalbumin was assayed by measuring the production of radioactive peptides soluble in 10%
trichloracetic acid (Waxman et al., 1987). Proteasome fractions or crude extracts (0.1 mg/ml) and purified proteasomes (5 ~g/ml of 20S or 15 ~g/ml of 20S); were incubated in the Buffer B (see Detailed Description of Figure 1) with radioactive substrates at 37C for 60 or 120 minutes in the presence of 2 mM ATP or 5 U/ml of apyrase (to destroy residual ATP). Nonradioactive lactalbumin (20 ~g/ml) was added to reaction mixtures containing Ub-125I-lactalbumin.

Preparation of Crude Cell ~xtracts and Proteosome-~nriched Fractions To prepare extracts, cells were collected by centrifugation for 5 minutes at 700 x g, washed twice, and resuspended in Homogenization Buffer (50 mM Tris, 5 2~5 -29- ~ - 94/01 v mM MgCl2, 1 mM dithiothreitol (DTT), 2 mM ATP, 250 mM
sucrose, pH 7.4). Cell suspensions were homogenized by several passages through a Dounce homogenizer (Wheaton), followed by vortexing for 3 minutes with glass beads.
After centrifugation at 10,000 x g for 20 minutes, the supernatant ("crude extract") was centrifuged for 1 hour at 100,000 x g to obtain a "microsome pellet". The ~proteasome fraction" was obtained by spinning the post-microsomal supernatant for 5 hours at 100,000 x g.
Pellets were resuspended in about 10 volumes of Buffer A
(50 mM Tris, 5 mM MgCl2 1 mM DTT, 2 mM ATP, 20~
glycerol), homogenized and centrifuged at 14,000 rpm for 15 minutes. The supernatants, which contained 20~ of the protein in crude extracts, were adjusted to a concentration of 1 mg/ml (Bradford, 1976) prior to assay of proteolytic activities, as were the crude extracts (after dialysis against Buffer A). With each set of cultured cells, two sets of extracts were prepared and analyzed in parallel. The averages of results obtained with the two sets were taken as the result of a particular experiment.

Detailed Description of Fiqure 1 Figure la to lc shows the hydrolysis of the indicated substrates (100 ~M) by proteasome fractions from control cells and cells treated with ~-IFN (Mamburg et al., 1992). Peptidase activities were determined by the release of 7-amino-4-methylcoumarin (MCA) or methoxynaphthylamine (MNA) from fluorogenic peptide substrates (Ischiura et al., 1985). Proteasome fractions (final protein concentration: 50 ~g/ml in 0.1 ml of Buffer B: 50 mM Tris, 5 mM MgCl2, 5 mM DTT, pH 7.4, containing 5 U/ml apyrase) were incubated for 40 or ,." ~ 'r-~

215~4 PCTfIJ~ 94/ 01 ~ ~ -30 IPEA/Us 3 ' AU~ '?~

60 minutes at 37C. The reaction was terminated by adding 1 ml of 1~ sodium dodecyl sulphate. The increase in degradation of hydrophobic and basic substrates was highly significant tP~0.01 with n=6 different experiments). In the crude extracts, very similar changes (Pc0.02, n=3) were observed with suc-LLvy-Mca and Boc-LRR-MCA, while degradation of Cbz-LLE-MNA did not change ~ignificantly. Units = mM of substrate cleaved per mg of protein per hour.
Figure ld to lf shows kinetic analysis (Lineweaver-Burk method) of rates of cleavage of the substrates (v) by the same proteasome fraction~ used above. Incubations were carried out as in Figure la to lc, with varying substrate concentration (S). Vm~ for SucLLVY-MCA was 0.7 units and 1.6 with ~-IFN; for Boc-LRR-MCA, 0.3 units and 1.9 with ~-IFN; for Cbz-LLE-MNA, 0.3 units and 0.2 with ~-IFN. Km for Suc-LLVY-MCA was 0.2 mM and 0.3 mM with r-IFN; for ~oc-LRR-MCA, 0.4 mM and 0.5 mM with ~-IFN; for Cbz-LLE-MNA, 0.10 mM and 0.06 mM with ~-IFN.

Detailed De~cription of Figure 2 Figure 2 shows the effect of ~-IFN treatment on peptidase activities in 20S and 26S proteasomes purified from U937 cells. The results are representative of 3 different experiments. All differences between control preparations and those from ~-IFN-treated cells were statistically significant (P~0.01), but no significant difference was seen for Cbz-LLE-MNA cleavage by 20S.
Units and assay conditions are as in Figure 1, but the final concentrations of the proteasomes were 2.5-3.0 ~g/ml (26S) or 8-10 ~g/ml (20S). 20S and 26S
proteasomes were isolated from the proteasome fraction 21 S ~ 5~ ~ PCTI~,'' 9 4 / O 1 ~ '-7 3 IPEA/US 3, AU~, jJ~
by Q Sepharose anion exchange and Superose 6 gel filtration FPLC chromatographies (Pharmacia) (Driscoll and Goldberg, 1989; Matthews et al., 1989). The 26S-rich fraction was identified by its ability to support the ATP-dependent degradation of Ub-l2sI-lactalbumin.
Routinely, about 30 ~g of 26S and 90 ~g of 20S were obtained from 5 mg of proteasome fraction.

Detailed Description of Figure 3 Figure 3a to 3c shows the difference in activities of the peptidases of proteasomes from wild-type (721) and MHC-deficient (721.174) lymphoblastoid cells. The rates of hydrolysis of three substrates (100 ~M) were measured in proteasome fractions, as done for Figure 1.
Differences in all three activities were statistically significant (PcO.01, n=4 different preparations).
Figure 3d to 3f shows the kinetic analysis of cleavage for all the substrates at different concentrations (S) by the same proteasome fractions from wild-type and mutant cells. Vm~ f or Suc-LLVY-MCA was 6.5 units (wild-type) versus 2.4 (mutant); for Boc-LRR-MCA, 1.6 units vs 0.7; for Cbz-LLE-MNA, 0.7 units vs 1.2. Km for Suc-LLVY-MCA was 0.5 mM (wild-type) vs 0.3 mM (mutant); for Boc-LRR-MCA, 0.3 mM V8 0.5 mM; for Cbz-LLE-MNA, 0.4 mM (wild-type and mutant). Very similar results were obtained in two different experiments.

Detailed Description of Figure 4 Figure 4a shows the frequency of amino acids at the carboxyl termini of 44 peptides that bind MHC class I
molecules. The data were collected from published sequences of peptides (Falk et al., 1991;

~E~ S~EE~

~ 2 1 ~ 4 ~ 4 f~

-32- ~P ~ lus 3 Guo et al., 1992; Hunt et al., 1992; Jardetzky et al., 1991; Matsumura et al., 1992). These sequences were determined in different studies either by Edman degradation of individual peptides that were eluted and purified from MHC class I molecules or by functional analysis of synthetic antigenic peptides and alignment to known MHC-binding motifs. The MHC class I molecules included in this analysis fall into two groups: Kb, Db, Kd, Ld and HLA-A2.1, which bind peptides that generally have a hydrophobic C-termin~l residue, and HLA-B27 and HLA-AW68, which bind peptides that generally have a basic C-terminal residue.
Figure 4b shows the frequency of amino acids preceding the N-termini of MHC class I-bound peptides.
The protein sequences from which 39 of the peptides in Figure 4a were derived were available. Data were collected from the EMBL (Heidelberg, Germany) and National Biomedical Research Foundation (Bethesda, Md) databases.

~ S~

W094/17816 PCT~S94/01183 215~5~

TABLE l Stimulation of the Tr~sin-like and Chymotrysin-like PePtidases of Proteasomes by Y-IFN

ActivitY (uM cleaved/mq/h Substrate Control + ~-IFN% increase Hydrophobic Suc-LLVY-MCA 230 820+357 Suc-LY-MCA 90 210+133 Suc-AAF-MCA 60 l20+lO0 Basic Boc-LRR-MCA 57 250+439 Boc-GGR-MCA 3.4 5.2+53 Cbz-GKR-MCA 2.8 6.0+114 Degradation rates of substrates (lO0 uM) by proteasome fractions of U937 cells were analysed as in Figure l (upper panel). Presented are averages from 6 (Suc-LLVY-MCA and Boc-LRR-MCA) or 2 (all other substrates) different experiments.

WO94/17816 PCT~S94/01183 21~5554 Comparison of Y-IFN-Induced Chanqes in Peptidase Activities of Proteaso~es from Wild-type and MHC-deficient Cells y-IFN-induced Increase in Activity Substrates Wild-type MHC-deficient Mutant unitsl (% change) unitsl (% change) HYdrophob i c Suc-LLVY-MCA 340 (+33) 120 (+18)t Suc-LY-MCA 66 (+34)* 10 (+26) t Suc-AAF-MCA 62 (+42)* 8 (+15) t Basic Boc-LRR-MCA 180 (+48)* ns Boc-GKR-MCA 1.6 (+28)* ns Cbz-GGR-MCA 1.2 (+34)* ns Acidic Cbz-LLE-MNA -40 (-24)* +80 (+35) t Wild-type cells were 721 and mutant cells were 721.174. With all hydrophobic and basic substrates, y-IFN caused consistently (n=3) larger changes in the wild-type cells. Interferon consistently caused opposite effects with the acidic substrate in the wild-type and mutant cells. Data are differences between means for proteasome fractions. Activities were determined as in Figure 1.
1 Units = ~M of substrate cleaved per mg of protein per hour.
* P<0.02, t p<o,o5~ ns - not significant (T test).

~ W094/17816 21 S 5 ~ 5 4 PCT~S94/0l1~

ExamPle 2 Inhibition of MHC-I Antiqen Presentation bY a Defect in Ubiquitin Coniugation The rate-limiting step in the complete degradation of most cellular proteins is their covalent conjugation with ubiquitin (Hershko and Ciechanover, 1992;
Rechtsteiner, 1987). This step marks the proteins for rapid hydrolysis by a 26S (1500 kD) proteolytic complex that contains a 20S (700 kD) degradative particle called the proteasome (Goldberg, 1992; Goldberg and Rock, 1992). In addition to completely hydrolyzing cell proteins, some system in the cytosol generates antigenic peptides from endogenously synthesized cellular and viral proteins (Townsend et al ., 1985; Townsend et al ., 1986; Morrison et al ., 1986; Moore et al ., 1988; Spies and Demars, 1991; Powis et al., 1991). These peptides bind to newly synthesized class I major histocompatibility complex (MHC) molecules in the endoplasmic reticulum and peptide-class I complexes are then transported to the cell surface for presentation to cytotoxic T cells (Yewdell and Bennink, 1992; Monaco, 1992). Precisely how antigenic peptides are produced is unknown, although indirect evidence suggests a role for proteolytic particles (LMP) closely resembling and perhaps identical to the 20S (700 kD) proteasome (Goldberg and Rock, 1992; Monaco 1992; Parham, 1990;
Yang et al., 1992). Applicants therefore tested if the ATP-ubiquitin- dependent proteolytic system is involved in MHC class I-restricted antigen (Ag) presentation. Using cells that exhibit a temperature-sensitive defect in ubiquitin con~ugation, Applicants have found that the nonpermissive temperature inhibited WO94/17816 PCT~S94/01183 ~

~155~S ~

class I-restricted presentation of ovalbumin (OVA) introduced into the cytosol, but did not affect presentation of an OVA peptide synthesized from a minigene. These results implicate the ubiquitin-dependent proteolytic pathway in the production of antigenic peptides.
Ts20 cells, a chemically mutagenized variant of the Chinese hamster lung cell line E36, express a thermolabile ubi~uitin-conjugating enzyme El (Kulka et al., 1988). E1 catalyzes the ATP-dependent activation of ubiquitin, an essential first step in conjugation of ubiquitin to cellular proteins (Hershko and Ciechanover, 1992; Rechtsteiner, 1987). Ubiquitin-protein conjugation and protein degradation are reduced in ts20 cells at nonpermissive temperature (41C) (Kulka et al., 1988; Gropper et al., 1991). El is irreversibly inactivated in ts20 cells at 41C.
To determine whether blocking ubiquitin conjugation affected class I-restricted Ag presentation, ts20 and E36 cells were transfected with the cDNA genes for H-2Kb and ICAM-1 to enable the cells to present Ag to the OVA-specific, Kb-restricted murine T-cell hybridoma line RF33.70 (Rock et al., 1990). The transfected cell lines, ts20.10.2 (mutant) and E36.12.4 (wild-type), were incubated at 41C or 37C for 1 hour before introducing OVA into the cytosol by osmotic lysis of pinosomes (Townsend et al., 1986). Incubations during and after the introduction of OVA were performed at 37C; this eliminated temperature as a variable between the groups during and after their exposure to OVA. The cells were allowed to process the Ag for 1 hour before Ag processing was stopped by aldehyde fixation. Incubation WO94117816 PCT~S94/01183 ~155~

at 41C greatly decreased presentation of OVA by mutant (Figure 5A) but not wild-type (Figure 5B) cells.
Several hypotheses can be suggested to explain these results. These possibilities were examined. The antigen presenting cells (APCs) above acquired OVA by pinocytosis (Townsend et al., 1986). Therefore, presentation of OVA by mutant cells could have been inhibited if exposure to 41C blocked pinocytosis.
Mutant cells incubated at 41C or 37C did not differ in their uptake of extracellular fluid (0.94 + 0.27 nl/106 cells at 41C versus 0.96 + 0.47 nl/1O5 cells at 37C, mean + SD of four experiments) as determined by the uptake of poly(vinylpyrrolidone) (PVP), a commonly used marker of fluid-phase pinocytosis (Wiley and McKinley, 1987). The incubation conditions with PVP were identical to those described above for OVA. Thus, the failure of mutant cells to present OVA was not due to a defect in pinocytosis.
Alternatively, the inability of mutant cells incubated at 41C to present cytosolic OVA could have been due to a block in the generation or presentation of the antigenic OVA peptide-Kb complexes. Incubation at 41C did not affect the ability of mutant (Figure 5C) or wild-type (Figure 5D) cells to present exogenous 0VA257_264 peptide (amino acids 257-264 of ovalbumin) added to the medium. Under these conditions, the sYnthetic 0VA257-264 peptide (which represents the naturally processed epitope from ovalbumin) binds directly to Kb on the cell surface (Falk et al., 1991;
Rock et al., 1992). Therefore, presentation of OVA
peptide-Kb complexes at the APC surface was not significantly affected by blocking ubiquitin conjugation.

215~ ` 3 -38- IPEA/US 3 1 A~G 1`394 After antigenic peptides bind to newly synthesized class I molecules, the peptide-class I complexes are transported through the Golgi complex to the cell surface for presentation to T cells. Therefore, Applicants examined the effects of 41C on the synthesis and transport of H-2Kb in mutant cells. Following 1 hour incubation at 41C or 37C, cells were radiolabeled with [35S ] -methionine, and H-2Kb molecules were sequentially immunoprecipitated with a mAb (Y-3) (Townsend et al., 1990) that is specific for assembled Kb molecules. This was followed by precipitation with rabbit anti-heavy chain antisera (exon 8) (Smith and Barber, 1990). Similar amounts of assembled H-2Kb were immunoprecipitated from mutant cells incubated at the two temperatures (Figure 6a to 6b). Applicants also found at both temperatures similar amounts of free heavy chains (Figure 6a to 6b) and ~2 -microglobulin that were potentially available for assembly with peptide.
To analyze the transport of assembled Kb molecule~
through the Golgi complex, cells were incubated at 41C
or 37C for 1 hour and then pulse-radiolabeled and chased at 37C. H-2Kb molecules were immunoprecipitated and subjected to two-~mPn~ional IEF/SDS-PAGE.
Temperature did not affect the degree of charge or molecular mass heterogeneity of Kb molecules in either mutant or wild-type cells (Figure 6c to 6f). Since changes in charge and molecular mass during the intracellular maturation of class I molecules are associated with complex Yugar additions in the Golgi complex, these findings indicate that assembly and transport of H-2Kb occurred normally in the mutant cells following incubation at 41C. This suggested that inhibition of .., ~ i ~ . ; ~ -WO94/17816 PCT~S94/01183 2~ 55~4 Ag presentation was due to a selective defect in the production of antigenic OVA peptides.
To examine this point further, mutant cells, treated as described in Figure 5, were incubated with OVA or infected with a recombinant vaccinia virus (Ova257_264Vac) containing a minigene encoding the 0VA2S7_264 peptide preceded by an initiating Met.
Presentation of the endogenously-synthesized 0VA257_264 peptide was unaffected by incubation at 41C (Figure 7A), while, as shown earlier, the presentation of OVA
was inhibited (Figure 7B). After one hour of infection, the presentation of 0VA257_264 + Kb by the vaccinia infected cells was considerably less than the presentation by cells that were incubated with OVA
(compare the 37C groups in Figure 7A and 7B).
Therefore, it is unlikely that overproduction of the OVA257_264 peptide could account for the lack of effect of the 41C incubation on presentation by the vaccinia-infected mutant cells (also see Detailed Description of Figure 7). This finding indicates that the reduction in ubiquitin conjugation caused by incubating mutant cells at 41C did not affect either the transport of 0VA257_264 peptide from the cytosol into the endoplasmic reticulum, the subsequent assembly of the 0VA257_264 peptide and H-2Kb, or the transport of the class l-peptide complex to the cell surface.
The simplest interpretation of the present findings is that ubiquitin conjugation plays a critical role in the processing of OVA for class I restricted presentation. In related studies, Applicants found that OVA added to cell-free extracts was degraded by the ubiquitin-dependent pathway, since this process required W094/17816 PCT~S94/01183 . .
2155~S 4 ATP, ubiquitin, a proteasome fraction from rabbit reticulocytes, and free amino groups on the ovalbumin.
The presence of two MHC-encoded proteins in the proteasome has led to the hypothesis that the proteasome is involved in processing cellular and viral Ags (Parham, 1990; Yang e t al ., 1992). Recent data, however, indicate that these subunits are not essential for class I-restricted Ag presentation (personal communication from Hammerling et al . ) . Since ubiquitin-conjugated proteins are degraded by the 26S
proteasome complex (Goldberg, 1992; Goldberg and Rock, 1992), these findings directly implicate this structure in processing of an Ag for class I-restricted presentation. Thus, the immune system utilizes the cell's two primary and phylogenetically old degradative pathways to supply antigenic peptides: the ATP-ubiquitin-proteasome dependent pathway for class I
and the vacuolar pathway for class II-restricted presentation.

Detailed Description of Figure 5 Figure 5 shows MHC class I-restricted presentation of OVA by mutant (ts20.10.2) and wild-type (E36.12.4) cells. Ts20.10.2 (A and C) and E36.12.4 (B and D) cells were incubated at 41C (filled) or 37C (open) and then incubated with (A and B) or without (C and D) OVA. The indicated number of APCs incubated with OVA were cultured with RF33.70 (A and B), or control APCs (5 x 104) were cultured with RF33.70 and the indicated concentration of OVA257_264 peptide (C and D).
Ts20.10.2 (mutant) and E36. 12.4 (wild-type) cells (2 x 1o6 cells/ml) were incubated for 1 hour at 41C or 37C. For introducing OVA into the cytosol, the cells WO94/17816 PCT~S94/01183 ~15555~

were incubated for 10 minutes at 37~C with 20 mg/ml OVA
in hypertonic media, treated with hypotonic media, and then washed with ice-cold, serum-free RPMI 1640, as previously described (Townsend et al., 1986; Michalek et al., 1991). The washed APCs (2 x 106 cells/ml) were incubated for 1 hour at 37C to allow for the expression of processed Ag on the cell surface. During this 37C
incubation, some recovery of ubiquitin conjugating activity (16 + 10% mean + SD of three experiments) did occur in the mutant cells that had been previously incubated at 41C. APCs were fixed with 1%
paraiormaldehyde to prevent further recovery of ubiquitin conjugation and/or Ag processing and added to duplicate microcultures (200 ~l) with RF33.70 (105 cells), as previously described (Michalek et al., 1991).
Control APCs were as described above except OVA was omitted from the hypertonic media. RF33.70 is stimulated to produce IL-2 upon recognition of processed OVA + Kb on the surface of APCs (Rock et al., 1990b).
The microcultures were prepared, incubated, and assayed for IL-2 content as previously described (Michalek et al., 1991).
The H-2Kb cDNA, pBG367-Kb, was kindly provided by Dr. Gerald Waneck. The vector pcDL-SRc~296 was provided by Dr. Naoko Arai (Takebe et al., 1988). The H-2Kb cDNA
was subcloned into the Yhol~BamHl sites of the pcDL-SR~296 vector. Murine ICAM-I cDNA in the pHBAPr-l-neo vector was provided by Drs. Hedrick and Brian (Siu et al., 1989). E36 and ts20 cells were cotransfected with H-2Kb and ICAM-I using the Lipofectin reagent (BRL) and selection with G418 (GIBCO) were performed as previously described (Dang et al., 1990), e~cept ts20 cells were grown at 37C following transfection. G418-resistant ~ 215~55~ pCT,~rg4~0118 ~

-42- IP4/Vs 3 ~ jJ34 clones were screened for expression of ICAM-I and H2Kb with mAb YN1/1.7.4 (Dang et al., 1990) and B8-24-3 (Kohler et al., 1981), respectively. The transfected cell lines ts20.10.2 and E36.12.4 were passaged at 31C
and 37C, respectively.

Detailed De~cription of Figure 6 Figure 6 shows the effect of temperature on the synthesis and maturation of H-2Kb. In Figure 6a and 6b, mutant (ts20.10.2) and wild-type (E36.12.4) cells were incubated for 1 hour at 41C or 37C and then metabolically radiolabeled for 15 minutes at 37C.
H-2Kb was sequentially immunoprecipitated with Y-3 (Jones and Janeway, 1981) followed by precipitation with anti-exon 8 (Smith and Barber, 1990) (a kind gift from Dr. Brian Barber, University of Toronto) and analyzed by one-~lmon~ional SDS-PAGE. Y-3 recognizes a conformational epitope formed by the CY1 and ~2 domains of H-2Kb and binds only assembled H-2Kb molecules, whereas the anti-exon 8 antisera was raised against the C-terminal cytoplasmic ~lomA;n of H-2Kb (encoded by exon 8) and binds both assembled H2-2Kb molecules and free heavy chains. The position~ of H-2Kb heavy chA; n~ (H) and ,~2-microglobulin (L) and of the relative molecular mass stAn~lArds (Mr X 10-3) are indicated in Figure 6.
Forty million cells were incubated for 1 hour at 41C or 37C in methionine-free mP~ and then radiolabeled for 15 minutes at 37C with 0.7 mCi of [35S]-methionine (~1000 Ci/mmol, NEN). Detergent lysates, immunoprecipitations and SDS-PAGE were preformed essentially as described (Townsend et al., 1990), except that additional preclearing steps with ~ANDE~ S~EES

2155~5~
P~ J 94/OI l 8 IPEA/US 3 ~ ~(IG 1994 IgGsorb (5 times) and PA-sepharose (1 time) were included before and after the firRt immunoprecipitation, respectively.
In Figure 6c to 6f, ts20.10.2 and E36.12.4 cell~
were incubated for 1 hour at 41C or 37C, metabolically pulse-radiolabeled for 10 minutes at 37C, and then chased for 50 minutes at 37C. H-2Kb was immunoprecipitated with Y-3 and analyzed by two-~;mPn~ional IEF/SDS-PAGE. TmmllnoprecipitateR made at the 0 time point of the chase were analyzed and showed a single immature heavy chain spot, which chased into the more acidic and larger molecular mass forms ~hown above. The heavy ch~inR of H-2Kb are indicated by the H, and the ~2-microglobulin is indicated by the L.
Cells were incubated and pulsed-radiolabeled for 10 minutes with [35S]-methionine as described above. The cells were then chased in the presence of cold methionine (15 mg/ml) for 50 minutes at 37C. Cell lysates were prepared and immunoprecipitations with Y-3 were performed as described above. For two-~;mPnRional IEF/SDS-PAGE, the imml~noprecipitates were analyzed by using the Immobiline DryStrip Kit (Pharmacia). Gels were incubated in Autofluor (National Diagnostics), and radiolabeled proteins were visualized by autoradiography at -80C.

De~ailed Descri~tion of Figure 7 Figure 7 shows MHC class I-restricted presentation of endogenously-synthesized 0VA2572C~ peptide.
Ts20.10.2 cells were incubated at 41C (filled) or 37C
(open) for 1 hour and then either infected with Ova2s726~Vac for 1 hour at 37C (Figure 7a) or incubated AMc~~ ,H'~r WO94/17816 PCT~S94/01183 ~

~1S~i~5~

with OVA (Figure 7B). The indicated number of APCs were incubated with RF33.70.
Ts20.10.2 cells (7 x l05 cells/ml) were incubated for l hour at 41~C or 37C and then infected with recombinant vaccinia virus (l0 PFU/cell) for l hour at 37C (Figure 7A). Ts20.]0.2 cells were incubated at 41C and 37OC, treated with OVA, and th~n incubated at 37C as described above (see Detailed Description of Figure 5). The APCs infected with vaccinia virus or incubated with OVA were fixed with 1% paraformaldehyde and incubated in microcultures with RF33.70 (105 cells) as described in the Detailed Description of Figure 5.
The microcultures were prepared, incubated, and assayed for IL-2 content as also described therein. APCs infected with a recombinant vaccinia virus containing an influenza nucleoprotein gene did not stimulate RF33.70.
Time-course experiments with Ova257_264Vac showed that the amount of 0VA257_264 peptide-Kb complexes on the surface of cells infected for l hour was limiting because ceils infected for 3 hours were 64-fold more efficient at stimulating RF33.70.
Ova257_264Vac was constructed by inserting a synthetic oligonucleotide behind the vaccinia virus p7.5 early /late promoter in pScll (Chakrabati et al ., 1985), which was modified such that the restriction sites SalI
and NotI were substituted for the SmaI site. The oligonucleotide:
5~-TcGAccAccATGTcTATAATAAAcTTTGAGAAGTTATAGTGAccATGGGc-3r 3'-GGTGGTACAGATATTATTTGAAACTCTTCAATATCACTGGTACCCGCCGG-5', (Seq. ID ~l), consisted of a SalI site, Kozak's consensus sequence for efficient translation, an initiation codon, nucleotides encoding the peptide s I
F E ~ L (Seq. ID #2), two stop codons, and a NotI

~LWO94/17816 PCT~S94101183 -2 ~

site, which is not present in pSCll and therefore provides a simple method for determining the presence of the insert in the plasmid. After strand annealing, the synthetic oligonucleotide was phosphorylated with T4 polynucleotide kinase. pSCll was digested with Sal I and NotI, purified, and ligated with the oligonucleotide using T4 ligase. Homologous recombination of the plasmid with vaccinia virus, selection, and propagation of recombinants was then performed as described (Moss and Earl, l99l).

ExamPle 3 Inhibition of MHC-l Presentation BY Chymostatin ANT~GEN PRE~ENTING CEL~
The antigen-presenting cells (APCs) in this experiment were a mouse B lymphoblastoid cell line (LB27.4) that was passaged in media supplemented with normal mouse serum (1%) and lipopolysaccharide (l0 ~g/ml X 72 hours). Except for the inclusion of the lipopolysaccharide, this is described in Rock et al., l990b.

IKHIBITOR TREATMENT AND ANTIGEN LOADING
The LB27.4 cells were washed serum free and incubated in the presence or absence of chymostatin (200 ~g~ml; Boehringer Mannheim, Indianapolis, IN) for one hour at 37C. The cells were then resuspended in Electroporation buffer (phosphate buffered saline, 1 mM
Hepes, 0.4 M mannitol; chymostatin was also added to the appropriate groups) containing either the antigen ovalbumin (30 mg/ml) or a synthetic peptide corresponding to amino acids 257-25~ of ov2lbumin. The resuspended cells were then subjected to electroporation 2155554 ~ 01 ~ ~ 3 IPEA/US 3 1 AUG t99~
on the CELL-PORATOR from Gibco BRL (Gaithersburg, MD) at 4C with setting of high ohms, capacitance 1180. The cells were washed 4 times at 4C and incubated at 37C
for two hours in the continued presence or absence of chymostatin. After this two hour incubation, the cells were fixed with 1~ paraformaldehyde for 10 minutes at room temperature, followed by washing.

ANTIGEN PRESENTATION ASSAY
The indicated number of fixed antigen-presenting cells were incubated with 105 RF33.70 cells (an OVA + K~
specific T-T hybridoma) in duplicate 200 ~l microcultures. After 18 hours at 37C, an aliquot (100 ~1) of culture supernatant was harvested and assayed for IL-2 content using HT-2 cells, as described in Rock et al., l990a and l990b.

Detailed De~cription of Figure 8 Figure 8 shows MHC-I presentation of ovalbumin (OVA, Figure 8a) and an ovalbumin peptide (Figure 8b). In Figure 8a, APCs were treated with (open circles) or without (closed circles) chymostatin and electroporated with ovalbumin. In Figure 8b, APCs were treated with (open circles) or without (closed circles) chymostatin and electroporated with ovalbumin peptide.
Figure 8a ~monRtrates that chymostatin inhibits the presentation of ovalbumin with class I MXC molecules.
Figure 8b ~PmonRtrates that chymostatin does not inhibit the presentation of electroporated peptide. This result indicates that the inhibition of antigen presentation is occurring through chymostatin inhibition of the processing of the ovalbumin protein into the ovalbumin peptide.

Ji ' `~_ ~ J `~

~ WO94/17816 PCT~S94/01183 2 ~

Isolation of an Endoqenous Inhibitor of the Proteasome As described in Example 7, a 40 kDa polypeptide regulator of the proteasome, which inhibits the proteasome's proteolytic activities, has been purified from reticulocytes and shown to be an ATP-binding protein whose release appears to activate proteolysis.
The isolated inhibitor exists as a 250 kDa multimer and is quite labile (at 42C). It can be stabilized by the addition of ATP or a non-hydrolyzable ATP analog, although the purified inhibitor does not require ATP to inhibit proteasome function and lacks ATPase activity.
The inhibitor has been shown to correspond to an essential component of the 1500 kDa proteolytic complex.
If reticulocytes are depleted of ATP, the 1500 kDa UCDEN
is not found. Instead, Ganoth et al. identified three components, designated CF-l, CF-2 and CF-3, (J. Biol.
Chem. 263:12412-12419 (1988)). The inhibitor isolated as described herein appears identical to CF-2 by many criteria. These findings indicate the idea that the inhibitor plays a role in the ATP-dependent mechanism of the UCDEN complex. It is possible, for example, that during protein breakdown, within the 1500 kDa complex, ATP hydrolysis leads to functional release of the 40 kDa inhibitor, temporarily allowin~ proteasome activity, and that ubiquitinated proteins trigger this mechanism.
The purified factor has been shown to inhibit hydrolysis by the proteasome of both a fluorogenic tetrapeptide and protein substrates. When the inhibitor, the proteasome and partially purified CF-1 were mixed in the presence of ATP and Mg2 , the 1500 kDa WO94/17816 PCT~S94/01183 ~

2155~5~

complex was reconstituted and degradation of Ub-l25I-lysozyme occurred.
Isolation of this inhibitor of the multiple peptidase activities of the proteasome makes available an attractive site for pharmacological intervention. As described subsequently, this provides a natural inhibitor whose structural and functional features can be assessed to provide information useful in developing proteasome inhibitors.

Materials and Methods DEAE-cellulose (DE-52), CM-cellulose (CM-52), and phosphocellulose (Pll) were obtained from Whatman. Ub-conjugating enzymes (El, E2 and E3) were isolated using Ub-sepharose affinity column chromatography (Hershko et 15 al ., J. Biol . Chem. 258:8206-8215 (1983)), and were used to prepare Ub-125I-lysozyme conjugates (Hershko and Heller, Biochem. Biophys. Res. Comm. 128:1079-1086 (1985)). All other materials used were as described in the previous examples.

Purification Rabbit reticulocytes induced by phenylhydrazine injection were prepared (as described previously or purchased from Green Hectares (Oregon, WI). They were depleted of ATP by incubation with 2, 4-dir. tr~p:enol and 2-deoxyglucose as described (Ciechanover et al ., Biochem. Biophys. Res. Comm. 81:1100-1105 (1978)).
Lysates were then prepared and subjected to DE-52 chromatography. The protein eluted with 0.5M KCl (Hershko et al., J. Biol. Chem., 2~8:8206-8214 (1983)) was concentrated using am~,onium sulfate to 80%
saturation, centrifugated at 10,000 x g for 20 minutes, ~ WO94/17816 PCT~S94101183 2~5~5~

and suspended in 20 mM Tris-HCl (pH 7.6), 1 mM DTT
r (Buffer A). Following extensive dialysis against the same buffer, the protein (fraction II) was either stored at -80~C in 0.5 mM ATP or fractionated further.
Fraction II ~-200 mg) was applied to a Ub-sepharose column, and the Ub-conjugating enzymes were specifically eluted (Hershko et al., ~. Biol . Chem., 258:8206-8214 (1983)) and used in making TTb-lysozyme (Hershko and Heller, Bioche~. Biophys . Res. Comm. 128:1079-1086 10 (1985)). The unabsorbed fraction was brought to 38%
saturation using ammonium sulfate and mixed for 20 minutes, as described by Ganoth et al. (Ganoth et al., J. Biol. Chem. 263:12412-12419 (1988)). The precipitated proteins were collected by centrifugation 15 at 10,000 x g for 15 minutes. The pellet was resuspended in Buffer A and brought again to 38%
saturation with ammonium sulfate. The precipitated material was collected as above and then suspended in Buffer A containing 10% glycerol. After dialysis against this buffer, the 0-38% pellet was chromatographed on a Mono-Q anion exchange column equilibrated with Buffer A containing 10% glycerol. The protein was eluted using a 60 ml linear NaCl gradient from 20 to 400 mM. Fractions which inhibited the peptidase activity of the proteasome were pooled, concentrated, and then chromatographed on a Superose 6 (HR 10/30) gel filtration column equilibrated in Buffer A containing 100 mM NaC1 and 0.2 mM ATP. The column was run at a flow rate of 0.2 ml/minute, and 1 ml fractions were collected. Further purification of the inhibitor was achieved by a second more narrow Mono-Q
chromatographic gradient (from 50 to 300 mM NaCl), which yielded a sharp peak of inhibitor where only the 40 kDa W094/17816 PCT~S94tO1183 ~
2~555~ ~

band was visible after SDS-PAGE and Coo~asie staining.
Fractions with inhibitory activity against the proteasome were pooled and dialyzed against Buffer B
which contained 20 mM KH2P04 (pH 6.5), 10% glycerol, 1 mM DTT and 1 mM ATP. The sample was then applied to a 2 ml phosphocellulose column equilibrated in Buffer B.
The column was washed with 4 ml of this buffer, followed by 4 ml of this buffer, followed by 4 ml of Buffer B
containing either 20, 50, lOO, 400 or 600 mM NaCl.
To obtain partially pure CF-l, the Mono-Q fractions that eluted from lOO to 240 mM NaCl were pooled, concentrated to l ml and applied to a superose 6 column equilibrated in Buffer A containing lOO mM NaCl and 0.2 mM ATP. The fractions eluting at approximately 600 kDa were used as the CF-l containing fraction.
The proteasome was isolated from the supernatants of the two 38% ammonium sulfate precipitations. The supernatants were brought to 80% saturation with ammonium sulfate and mixed for 20 minutes. The precipitated protein was collected by centrifugation, resuspended in Buffer A, and dialyzed extensively against this buffer. The proteasome was isolated by Mono-Q anion exchange chromatography followed by gel filtration on superose 6 as described previously (Driscoll and Goldberg, Proc . Natl . Acad . Sci ., USA
86:789 791 (1989)).
T~e l,500 ~Da proteolytic complex was generated by incubating reticulocyte fraction II at 37C for 30 minutes in the presence of 2 mM ATP, 5 mM MgCl2 in 50 mM
Tris-HCl (pH 7.6). After precipitation with ammonium sulfate to 38% saturation, the pellet ~as collected at lO,OOO x g for lO minutes, suspended ir. 3uffer A, and WO94/17816 PCT~S94/01183 2~5~

isolated by Mono-Q anion exchange and superose 6 chromatography.

~ssaYs Inhibition of the proteasome was measured by preincubating individual column fractions with the proteasome in the presence of 1 mM ATP at 37C for 10 minutes. After preincubation, the reaction tubes were placed on ice, and either l25I-lysozyme or Suc-LLVY-MCA
was added. Reactions were carried out at 37C for 60 minutes with l25I-lysozyme or 10 minutes with Suc-LLVY-MCA. Protein hydrolysis was assayed by measuring production of radioactivity soluble in 10%
trichloroacetic acid, and peptide hydrolysis by the release of methylcoumaryl-7-amide (Driscoll and Goldberg, Proc. Natl. Acad. sci., USA ~6:789-791 (1989)). Degradation of Ub-conjugated l25-I-lysozyme was assayed at 27C for 60 minutes. Reactions contained either 5 mM EDTA or 2 mM ATP and 5 mM MgC12 and were terminated by adding 10% trichloroacetic acid.

RESULTS

Isolation of the Inhibitor To understand how the proteasome is regulated in vivo and how it functi.ons in the Ub-conjugate-degrading complex, Applicants attempted to isolate factors which influence its activity. Reticulocyte fraction II was separated using ammonium sulfate into fractions which precipitated with either 0-38% or 40-80%. The latter fraction was used to isolated proteasomes. The particles (obtained in this way from ATP-depleted reticuiocytes) showed appreciable activity against l25I-WO94/17816 PCT~S94/01183 ~
2~55~5 ~

lysozyme and Suc-LLVY-MCA which was independent of ATP
(Eytan et al., Proc. Natl. Acad. sci., USA 86:7751-7755 (1989); Driscoll and Goldberg, J. Biol. Chem. 265:4789-4792 (l990)). Neither the proteasome nor the 0-38%
fraction showed significant activity against Ub-conjugated l25I-lysozyme (Eytan et al., Proc. Natl.
Acad. sci., USA 86:7751-7755 (1989); Driscoll and Goldber~, J. Riol . Chem.265: 4789-4792 (l990)). However, as reported previously, ATP-dependent degradation of the ubiquitinated lysozyme was observed after the protease and the 0-38%fraction were preincubated together in the presence of ATP.
The 0-38% precipitated material was then separated using Mono-Q anion exchange and each fraction assayed for its ability to influence the proteasome activity against Suc-LLVY-MCA or l25I-lysozyme. Column fractions were preincubated with the proteasome for lO minutes and then either substrate was added. None of the column fractions by itself showed significant hydrolytic activity. A peak of inhibitory activity was eluted around 240 to 280 mM NaCl. It significantly decreased proteolytic activity against both substrates.
Hydrolysis of lysozyme and the peptide was inhibited to a similar extent.
To purify the inhibitory activity further, the active fractions were pooled and chromatographed by gel filtration. The inhibitor eluted as a sharp peak with an apparent molecular weight of about 100-150 kDa.
The active fractions were then pooled and assayed for their ability to inhibit the substrate hydrolyzing activities of the proteasomes. With increasing inhibitor concentration, proteasome activity decreased in a linear manner with both l25I-lysozyme and Suc-LLVY-W094/17816 PCT~S94tO1183 215~54 MCA as substrates, although the degree of the inhibition was highly variable between preparations.

The Inhibitor Is A Component of the 1,500kDa Proteolytic Complex Like the inhibitor, one component of the 1,500 kDa proteolytic complex (CF-2) has been reported to have a molecular weight of about 250 kDa. To test if the inhibitor corresponds to CF-2, the inhibitor obtained by gel filtration was subjected to phosphocellulose chromatography. Eytan et al. had noted that CF-2 has little affinity for phosphocellulose and elutes with less than 100 mM NaC1. Accordingly, Applicants found that the inhibitory activity was recovered in the f low through and 20 mM NaCl eluate (i.e., in the region where CF-2 activity was reported). Individual phosphocellulose fractions were then assayed for their ability to reconstitute degradation of ubiquitinated lysozyme. Individually or combined, the proteasome and CF-l containing fraction did not support rapid breakdown of ubiquitinated lysozyme. However, when this mixture was combined with the peak of the inhibitor activity, the rate of Ub-125I-lysozyme degradation increased sharply. No other phosphocellulose fractions stimulated this process.
These results sugqest strongly that the inkibi,Qr corresponds to CF-2 and thus is essential for hydrolysis of Ub-ligated proteins. One unusual property of CF-2 is that it is quite labile upon heating to 42C, but is stabilized by ATP (Ganoth et al., J. Biol. Chem.
263:12412-12419 (1988)). To test further if the inhibitor of the proteasome corresponds to CF-2, the purified inhibitor was preincubated at 42CC with or 2~s~s5 4 without ATP or the nonhydrolyzable analog, AMPPNP. The proteasome was added and after 10 minutes, peptidase activity was assayed. The degree of inhibition decreased rapidly during preincubation without 5 nucleotide added. The presence of either ATP or AMPPNP
prevented this loss of activity. Furthermore, the a~ility of this material to reconstitute degradation of Ub-conjugated lysozyme also decreased rapidly during incubation of 42C, and the addition of ATP or AMPPNP
10 (not shown) prevented this activation. Since the inhibition and reconstitution of Ub-conjugate degradation showed similar inactivation kinetics and were stabilized similarly by ATP, these two functions probably reside in a single molecule which appears to 15 bind ATP.
Although ATP stabilizes the inhibitory factor, it is not essential for inhibition of the proteasome.
After preincubation of the inhibitor with proteasome for up to 20 minutes with or without ATP, a similar degree 20 of inhibition was observed. Nevertheless, because of the stabilization by ATP, this nucleotide was routinely added to all incubations.
When analyzed by SDS-PAGE, the inhibitor preparations showed a major band of 40 kDa. To test 25 whether this 40 kDa subunit corresponded to any subunit of the 1,500 kDa complex, the 1,500 kDa complex was forI2ed by incubation of fraction II with Mg2+-ATP and isolated by anion exchange and gel filtration chromatography. SDS-PAGE of these active fractions 30 indicated many polypeptides similar to those previously reported for this complex (Hough et al., J. Biol. Chem.
262:8303-8313 (1987~; Ganoth et al., J. Biol. Chem.
263:12412-125419 (lg88); Eytan, E. et al ., Prc~. N~tl .

WO94117816 PCT~S94/01183 2155~ ~

Acad. Sci . USA 86: 7751-7755 (1989)). However, a readily apparent band of 40 kDa was evident in this fraction.
To further address the question of proteins associated with the proteasome, fraction II was immunoprecipitated using an anti-proteasome monoclonal antibody and analyzed by SDS-PAGE. Ub-conjugate degrading activity had previously been shown to be removed upon immunoprecipitation of fraction II (Matthews et al., Proc. Natl . Adac. sci. USA 86:2597-2601 (1989~). Upon SDS-PAGE of the immunoprecipitates, Applicants observed the characteristic set of proteasome subunits ranging from 20 to 34 kDa, along with other higher molecule weight bands. Importantly, a 40 kDa band, similar to that of the inhibitor and similar to that seen in the partially purified complex was detected in the immunoprecipitate.

W094/17816 PCT~S94/01183 2~5~5 ~

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~ WO94/17816 215 5 ~ ~ ~ PCT~S94/01183 Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentaiton, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

2 ~ 5 5 5 ~ 4 SEQUENCE LISTING

~1) GENERAL INFORMATIoN:
(i) APPLICANT: THE PRESIDENT AND FELLOWS OF HARVARD COLLEGE AND
DANA FARBER CANCER INSTITUTE
(ii) TITLE OF INVENTION: Role o~ ATP-Ubiquitin-Dependent Proteolysis in MHC-1 Restricted Antigen Presentation And Inhi~itors Thereof (iii) NUMBER OF SEQUENCES: 3 (iv) CORRESPONDENCE ADDRESS~
(A) ADDRESSEE: Sterne, Kessler, Goldstein & Fox (B) STREET: 1100 New York Avenue, Suite 600 (C) CITY: Washington (D) STATE: D.C.
(E) COUNTRY: USA
(F) ZIP: 20005-3934 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B~ COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: TO BE ASSIGNED
(B) FILING DATE: 27-JAN-1994 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Gold~tein, Jorge A.
(B) REGISTRATION NUMBER: 29,021 (C) REFERENCE/DOCÆT NUMBER: 1448.003PC00 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (202) 371-2600 (B) TELEFAX: (202) 371-2540 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: both (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acidG
(B) TYPE: amino acid (D) TOPOLOGY: both WO 94117816 215 5 ~ ~ 4 PCT/US94/01183 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Ser Ile Ile Asn Phe Glu Lys Leu (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acid~
(B) TYPE: amino acid (D) TOPOLOGY: both (ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 1..4 (D) OTHER INFORMATION: /note= "The sequence Leu Val Val Tyr is part of a modified peptide having the structure Suc-Leu Val Val Tyr-MCA."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Leu Leu Val Tyr

Claims (17)

The invention claimed is:

1. A method for inhibiting MHC-I antigen presentation in an antigen presenting cell, comprising contacting the cell with an inhibitor that inhibits proteolysis of intracellular proteins by the ATP-ubiquitin-dependent pathway, under conditions suitable for the inhibitor to enter the cell.

2. The method of Claim 1, wherein the inhibitor inhibits a peptidase of the proteasomes, said peptidase having an activity which is selected from the group consisting of:
a) cleavage after basic residues;
b) cleavage after hydrophobic residues; and c) a combination of a) and b).

3. The method of Claim 2, wherein the inhibitor is a peptide aldehyde.

4. The method of Claim 3, wherein the peptide aldehyde is selected from the group consisting of chymostatin and leupeptin.

5. The method of Claim 1, wherein the inhibitor inhibits ubiquitin conjugation of the intracellular proteins.

6. The method of Claim 5, wherein the inhibitor inhibits a protein selected from the group consisting of:
a) E1, also referred to as ubiquitin activating enzyme;
b) E2, also referred to as ubiquitin-carrier protein; and c) E3, also referred to as ubiquitin-protein ligase.

7. The method of Claim 6, wherein the inhibitor of E1 is a substrate analog of ubiquitin adenylate.

8. A method for inhibiting cytolytic immune responses in a mammalian tissue, comprising contacting the tissue with an inhibitor that inhibits proteolysis of intracellular proteins by the ATP-ubiquitin dependent pathway, under conditions suitable for the inhibitor to enter antigen-presenting cells in the tissue.

9. The method of Claim 8, wherein the inhibitor inhibits a step in the proteolysis selected from the group consisting of:
a) cleavage by a peptidase of the proteasomes, which is selected from the group consisting of cleavage after basic residues and cleavage after hydrophobic residues;
b) ubiquitin conjugation; and c) a combination of a) and b).

10. The method of Claim 9, wherein the inhibitor inhibits a protein selected from the group consisting of:
a) E1, also referred to as ubiquitin activating enzyme;
b) E2, also referred to as ubiquitin-carrier protein; and c) E3, also referred to as ubiquitin-protein ligase.

11. A method for inhibiting cytolytic immune responses in an individual, comprising administering to the individual an inhibitor that inhibits proteolysis of intracellular proteins by the ATP-ubiquitin dependent pathway, under conditions suitable for the inhibitor to enter antigen-presenting cells in the individual.

12. The method of Claim 11, wherein the inhibitor inhibits a step in the proteolysis selected from the group consisting of:
a) cleavage by a peptidase of the proteasomes, which is selected from the group consisting of cleavage after basic residues and cleavage after hydrophobic residues;
b) ubiquitin conjugation; and c) a combination of a) and b).

13. A method of therapy or prevention of an autoimmune disease in an individual, comprising administering to the individual an inhibitor that inhibits proteolysis of intracellular proteins by the ATP-ubiquitin-dependent pathway, under conditions suitable for the inhibitor to enter antigen-presenting cells in the individual.

14. The method of Claim 13, wherein the inhibitor inhibits a step in the proteolysis selected from the group consisting of:
a) cleavage by a peptidase of the proteasomes, which is selected from the group consisting of cleavage after basic residues and cleavage after hydrophobic residues;
b) ubiquitin conjugation; and c) a combination of a) and b).

15. A method for reducing rejection of foreign tissue by an individual, comprising administering to the individual an inhibitor that inhibits proteolysis of intracellular proteins by the ATP-ubiquitin-dependent pathway, under conditions suitable for the inhibitor to enter antigen-presenting cells in the individual.

16. The method of claim 15, wherein the inhibitor inhibits a step in the proteolysis selected from the group consisting of:
a) cleavage by a peptidase of the proteasomes, which is selected from the group consisting of cleavage after basic residues and cleavage after hydrophobic residues;
b) ubiquitin conjugation; and c) a combination of a) and b).

17. The method of Claim 15, wherein the foreign tissue is a transplanted organ or graft.