AU3022300A - Modified myelin protein molecules - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT Names of Applicants: *o

S

Actual Inventors: Address for Service: ALEXION PHARMACEUTICALS, INC.

UNITED STATES OF AMERICA, represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN

SERVICES

Steven H. NYE, Louis A. MATIS, Eileen Elliott MUELLER, John P.

MUELLER, Stephen P. SQUINTO, James A. WILKINS, Michael J.

LENARDO, Henry F. McFARLAND and Clara M. PELFREY CULLEN CO., Patent Trade Mark Attorneys, 239 George Street, Brisbane, Qld. 4000, Australia.

Invention Title: MODIFIED

MOLECULES

MYELIN PROTEIN The following statement is a full description of this invention, including the best method of performing it known to us: MODIFIED MYELIN PROTEIN MOLECULES FIELD OF THE INVENTION The present invention relates to the treatment of autoimmune diseases. In particular, the invention provides compositions and methods facilitating the diagnosis and treatment of Multiple Sclerosis More particularly, engineered human Myelin Basic Protein (MBP) molecules, MBP polypeptides and nucleic acid molecules encoding MBP polypeptides, and Proteolipid Protein (PLP) molecules, polypeptides comprising PLP sequences and nucleic acid molecules encoding such polypeptides, are provided, as well as methods for the use of such polyp&ptides for the diagnosis, 15 clinical assessment, and therapeutic treatment of multiple sclerosis.

BACKGROUND OF THE INVENTION The discussion in this section is not limited to subject matter that qualifies as "prior art" against the present invention. Therefore, no admission of such prior art status shall be implied or inferred by reason of inclusion of particular subject matter in this discussion, and no declaration against the present inventors' interests shall be implied by reason of such inclusion.

25 Autoimmune Diseases Autoimmune diseases result from the loss of tolerance to certain self antigens, resulting in an inappropriate attack by the immune system upon these antigens. Numerous mechanisms normally S"function to maintain immune self-tolerance in both the antibodymediated (humoral) and cellular aspects of the immune system. It is when these mechanisms malfunction that autoimmune diseases occur.

Illnesses resulting from such misdirected immune system activity affect more than 10 million patients in the U.S. alone.

Therapies that treat the causes, rather than the symptoms of these diseases have long been sought. While agents have been found that provide beneficial reductions in autoimmune activity, such treatments, in general, have the undesirable and dangerous effect of also compromising normal immune functions, and are thus considered sub optimal.

Multiple Sclerosis Multiple Sclerosis (MS) is a progressive neurodegenerative autoimmune disorder affecting about 350,000 Americans (see, for example, Hauser, 1994). Females are twice as likely as males to develop the disease. MS usually affects patients who are between the ages of 15 and 50 years, most commonly young women between the ages of 20 and 40. MS derives its name from the multiple scarred (sclerotic) areas of degeneration visible on macroscopic examination of the central nervous system (CNS) of affected individuals. The degeneration associated with MS includes demyelination, chronic inflammation, and gliosis (scarring) of affected areas of the brain, optic nerve, and spinal cord.

15 MS is characterized by different types and stages of disease progression. Patients are diagnosed as having relapsing and remitting MS when they experience periods of exacerbations and remissions. Rapidly progressive or chronically progressive MS is diagnosed depending upon the pace of disease progression. These 20 stages usually occur later in the course of the disease when the extent of recovery from individual attacks decreases and there are clinically stable periods between periods of deterioration.

Inactive MS typically occurs late in disease progression and is characterized by fixed neurologic deficits of variable magnitude.

25 MS is always debilitating and may sometimes lead to paralysis and death. Although the factors triggering the initial onset of MS remain unknown, evidence is persuasive that MS pathology results from an abnormal immune response against the myelin sheath. This immune response involves autoimmune actions of certain white blood cells. It is believed that neuroantigenspecific T cells are especially important in this regard.

Pathologically, MS is characterized by chronic inflammation, demyelination, and gliosis of white matter. The classic lesions of MS, termed plaques, are well-demarcated gray or pink areas easily distinguished from surrounding white matter. (The coloration of white matter is due to the high concentrations of myelin in this tissue.) The acute MS lesion is characterized by demyelination associated with tissue infiltration by mononuclear cells, predominantly T cells (both helper and cytotoxic) and macrophages, with B cells and plasma cells rarely being present. These inflammatory infiltrates appear to mediate the demyelination that is characteristic of the disease. Since activated T cells release cytokines that promote macrophage infiltration and activation, T cells are considered the primary mediators of pathogenic autoimmune attack in MS. More detailed discussions of T cells and myelin are found below under "T Cell Physiology," "T Cells and Autoimmune Pathogenesis," and "T Cells Target Defined Autoantigens in MS." Current treatments for MS vary. Depending on the severity of disease and the response to treatment, a variety of options for drug therapy are available. Drugs used to treat MS include steroids such as prednisone and methylprednisolone, hormones such as adrenocorticotropic hormone (ACTH), antimetabolites such as 15 azathioprine, alkylating agents such as cyclophosphamide, and Tcell inhibitory agents such as cyclosporine. The administration of any of these drugs is dangerous, as they all typically produce some level of generalized immunosuppression and leave the patient oo** more prone to infection. Patients may also experience side 20 effects such as nausea, hair loss, hypertension, and renal dysfunction when treated with such drugs. In addition, some of these drugs are carcinogenic.

New approaches to treating MS include interferon-beta therapy, which can lessen the frequency of MS attacks and may slow 25 disease progression. Other new approaches include administration of antigens involved in MS autoimmune responses, as discussed below.

Diaanosis of MS MS is typically diagnosed based on medical history and physical examinations. No clinical signs or diagnostic tests are unique to MS. Diagnosis of a patient with a single, initial symptom commonly associated with MS cannot be definitive, although symptoms of relapsing and remitting disease increases the likelihood of an MS diagnosis. Two or more episodes of worsening each lasting 24 hours or occurring at least a month apart, or slow stepwise progression of signs and symptoms over at least six months are considered strong indications of MS. MRI findings implicating involvement in two or more areas of CNS white matter and evidence of systemic disease are also indicative of MS.

Currently, various laboratory tests are performed to confirm the diagnosis and assess the progression of the disease. Such tests include analysis of human cerebrospinal fluid (CSF) and blood for chemical and cellular signs of MS pathology.

CSF abnormalities associated with MS consist of mononuclear cell pleocytosis and the presence of autoreactive (typically myelin reactive) T cells, an elevation in the level of total Ig, and the presence of oligoclonal Ig, typically seen as two or more oligoclonal bands. In approximately 80 percent of patients, the CSF content of IgG is increased in the presence of a normal concentration of total protein. This results from the selective Production of IgG within the CNS.

S. Oligoclonal banding of CSF IgG is detected by agarose gel electrophoresis techniques. Two or more oligoclonal bands are 15 found in 75 to 90 percent of MS patients. The presence of oligoclonal banding correlates with an elevated total IgG level in MS. Other Ig abnormalities in MS CSF include free kappa or lambda light chains and elevated levels of other Ig isotypes including IgA 20 Metabolites derived from myelin breakdown also may be detected in CSF. Elevated levels of PLP or its fragments may be detected, by radioimmunoassay, both in MS and in some patients with other neurologic diseases.

In addition to many of the pathologic signs described above S. 25 for CSF, blood of MS patients may show increased levels of IgG synthesis, polymorphonuclear leukocytes, decreased serum B 12 levels, elevated erythrocyte sedimentation rate, and presence of autoantibodies or autoreactive T cells. As discussed below, the areactive T cell index" is a particularly useful cellular finding for monitoring the clinical course of MS.

While these various indicators of MS disease are clinically useful, other means of following the course and extent of autoimmune activity in MS patients using relatively inexpensive and easily quantifiable tests, such as blood or cerebrospinal fluid tests (as opposed to expensive imaging techniques such as MRI) are needed.

T Cells. Antigen Presenting Cells, and T Cell Eritoes As mentioned above, MS pathogenesis is believed to be mediated by the inappropriate actions of white blood cells (leukocytes), most importantly T cells. T cells are mononuclear white blood cells that provide many essential immune functions.

The importance of T cells in human autoinmmune diseases has been increasingly appreciated in the past decade. Studies using treatments that result in generalized immunosuppression have defined a critical role for a subset of T cells, known as CD4+ or helper T cells, as primary regulators of all immune responses (both cellular and humoral) to protein or peptide antigens.

T cells mediate tissue injury by indirect and direct means.

T cells of both CD8+ (cytotoxic) and CD4+ (helper) subsets secrete a variety of inflammatory cytokines that can damage tissues indirectly by activating various other types of white blood cells.

Examples of such T cell effects include activation of antibody secreting B cells (stimulating humoral immune activity) and activation of macrophages, which can cause acute tissue damage and inflammation by releasing hydrolytic enzymes, reactive oxygen species, and additional pro-inflammatory cytokines. In addition to these indirect effects of T cell activity, direct tissue damage can- be mediated by CD81 cytotoxic T cells attacking cells displaying target antigens.

One unique aspect of the physiology of T cells is the presence of membrane bound antibody-like binding structures called T cell receptors (TCRs) on their cell surfaces. Like antibodies, TCRs bind with high specificity to particular antigens. Like antibody-producing cells, which develop as multitudinous clones of cells, each clone producing antibodies with unique specificities, T cells develop as a vast number of distinct clones, and any particular T cell clone expresses a single type of TCR with a defined binding specificity. T cell clones with TCRs that bind specifically to self antigens are responsible for the development of autoimmune diseases.

In addition to being cell surface, rather than soluble molecules, TCRs differ from antibodies in the way they recognize antigens. While antibodies bind to antigens in various contexts antigens that are native, denatured, soluble, or membrane bound), TCRs only bind to most antigens after the antigens have been broken down (processed) by certain cells known as antigen presenting cells (APCs) and the resulting peptides displayed (presented) on the cell surfaces of the APCs in association with class II or class I proteins of the major histocompatibility complex (MHC). In a human population, different individuals may display very different MHC molecules of these classes. Therefore, many different epitopes may be preferentially presented in such individuals.

The details of the mechanism by which antigen processing is carried out by APCs are poorly understood. There is consequently considerable uncertainty regarding the ability of APCs to process a given antigen in such a way as to produce and display a particular peptide unless that antigen has already been characterized in this respect.

One exception to the requirement that APCs process and present antigens in order for the antigens to stimulate T cells via their TCRs is the case of small peptide antigens. Such 15 peptides can bind directly to MHC class I molecules on cell oo 'surfaces without being processed by APCs, and may then be S"recognized" and bound by specific TCRs and thereby stimulate

T

cells.

Studies of the interactions of antibodies and TCRs with their 20 specific antigens have shown that a particular polypeptide antigen typically comprises numerous submolecular features, known as epitopes, that each can serve as a distinct binding site for a particular antibody or (subsequent to APC processing of the polypeptide and MHC display of a derived peptide comprising the T 25 cell epitope) a particular

TCR.

Thus, TCRs and antibodies are similar in that each only recognizes a small portion of a polypeptide antigen. They differ in that an antibody typically recognizes its specific epitope within the context of the intact polypeptide, while a TCR only recognizes a specific epitope as an MHC class II or class I associated peptide fragment of a processed polypeptide on the surface of an APC. Importantly, this TCR epitope recognition process can only occur if an APC can process the polypeptide antigen so as to generate and display the appropriate peptide.

Thus, even though a peptide that is recognized by a specific TCR may be present in a particular polypeptide antigen, it is uncertain whether peptides capable of stimulating T cells expressing that specific TCR will be derived from that polypeptide antigen in vivo. This is because it is uncertain whether APCs can generate the peptide recognized by the specific TCR by processing the particular polypeptide antigen.

This lack of certainty regarding the results of APC processing of a particular polypeptide antigen stems from several factors. One reason why an APC may not process a particular polypeptide antigen so as to display a specific peptide epitope contained within the polypeptide is that the APC efficiently cleaves the polypeptide at a site within the epitope and thereby destroys it. A second reason is that the polypeptide cannot enter into or be effectively broken down by the subcellular compartments of APCs responsible for polypeptide processing.

Certain aspects of the primary structure (linear amino acid sequence), secondary structure (3D structure resulting from interactions of amino acid residues that are close to one another C 15 in the linear amino acid sequence), or tertiary structure (3D structure resulting from interactions of amino acid residues that are far from one another in the linear amino acid sequence but come into proximity with each other as a result of folding of the polypeptide chain) can impact APC processing. The amino acid 20 sequence of a.polypeptide is clearly the most important factor in determining its potential to be processed and displayed by APCs so as to stimulate specific T cells. The peptide recognized by the specific T cell's TCRs must be contained within the amino acid sequence of the polypeptide. The amino acid sequence also 25 determines the potential secondary and tertiary structure the folding) of the polypeptide.

The folding of a polypeptide can also significantly impact APC processing. Both the first and second reasons given above for the uncertainty of the display by APCs of a specific epitope derived from a particular polypeptide can result from the way in which the polypeptide is folded. Proteolytic cleavage during processing within the APC can be influenced by the exposure or masking of a cleavage site due to folding. Entry of polypeptides into subcellular compartments is well known to be influenced by the 3D structure of the polypeptide, which structure is a function of folding.

T Cells and Autoimmune Diseases In autoimmune diseases, only a limited number of T cell clones, reactive with various epitopes of a small number of autoantigens, become activated and are involved in pathogenesis.

Various mechanisms have been postulated to play a role in this pathogenic activation of disease-causing autoreactive T cells.

Primary activation of antigen presenting cells (APCs) by infection or local inflammation is implicated in one such mechanism. APCs activated in this way can then provide powerful co-stimulation for hitherto unreactive T cells.

Other proposed mechanisms involve the polyclonal activation of previously quiescent autoreactive T cells by superantigens, such as bacterial toxins; or a coincidental molecular mimicry between foreign and self antigens (Abbas et. al. 1994). In this last case, the host immune system mounts a response to an epitope on a protein expressed by a pathogen, such as a virus, that resembles a homologous epitope on a host protein. Autoimmune 15 attack then results from the cross-reactive immune response that ensues.

In addition to external factors, underlying the emergence of all T cell-mediated autoimmune disease is a complex pattern of inherited susceptibility determined by multigenic factors. For further discussions of these various factors, Steinman, 1995, reviews current theories of autoimmunity.

n several autoimmune diseases, including MS (as discussed in detail immediately below under "T Cells Target Defined Autoantigens in some or all of the autoantigens targeted by 25 abnormal immune responses have been identified. Knowledge of these self antigens and the specific epitopes within these antigens that are targeted by autoreactive T cells in an autoimmune disorder such as MS provides an approach to therapy, as discussed in detail below under *Treatment of MS by Administration of Antigens" and "Therapeutic Induction of Apoptosis".

T Cells Target Defined Autoantiaens in MS Although, as discussed above, the precise etiology of MS remains unknown, autoimmune attack is clearly responsible for the destruction of central nervous system (CNS) myelin that is the hallmark of the disease. Myelin is the characteristic component of the myelin sheath that surrounds the axons of certain neurons, acts as an electrical insulator, and is essential for the proper signal transmission functions of these neurons. The demyelination associated with MS thus causes a loss of function in affected neurons, disrupting neuronal signaling and leading to paralysis and severe impairment of sensory functions.

The myelin sheath is made by oligodendrocytes (in the central nervous system) and Schwann cells (in the peripheral nervous system). Myelin is composed of regularly alternating layers of lipids cholesterol, phospholipids, and sphingolipids) and proteins.

The four major protein components of myelin, myelin basic protein (MBP), proteolipid protein (PLP), myelin associated glycoprotein (MAG) and myelin oligodendrocyte protein (MOG), are recognized by autoreactive T lymphocytes isolated from MS patients (Endoh et al. 1986; Martin et al. 1992; Kerlero de Rosbo et al.

1993; Amor et al. 1994; Johns et al. 1995).

Myelin basic protein (MBP) and proteolipid protein (PLP) are 15 major protein components of myelin, comprising approximately and 50% respectively of the total protein content of the myelin sheath. MBP and PLP have been shown to be major target autoantigens in MS, and T cells reactive with MBP and PLP play key 9*oo roles in its pathogenesis (see, for example, Schwartz, 1993; Brown 20 and McFarlin 1981. Lab Invest 45, pp. 278-284; Allegretta et al.

1990; Lehmann et al. 1992; Martin et al. 1992; Sprent 1994; Su and Sriram. 1991. J of Neuroimmunol 34, pp. 181-190; and Weimbs and Stoffel. 1992).

MBP-specific and PLP-specific T lymphocytes are found in the 25 blood of MS patients. While they can sometimes be found in the blood of healthy individuals, they are typically present in the cerebrospinal fluid (CSF) of patients with MS. Significantly, such T cells are not found in CSF from healthy individuals (Kerlero de Rosbo et al. 1993; Zhang et al. 1994).

The immune responses of MS patients towards MBP and PLP clearly differ from those of healthy individuals. MBP and PLP reactive T cells are preferentially activated in MS patients, as demonstrated by the observation that the frequency of MBP-specific and PLP-specific T cells expressing markers of activation IL-2 receptors) is elevated in MS patients (see, for example, Zhang, et al., 1994).

Gene mutation frequency analysis also provides evidence that MBP reactive T lymphocytes are specifically activated in MS patients. Since gene mutation is more frequent in dividing than in resting T cells, an increased mutation frequency in T cells of a particular specificity provides an indication of the specific activation of those cells in vivo (Allegretta et al. 1990).

T lymphocytes from MS patients were cultured in thioguanine to test the frequency of mutations in the hprt gene that would render them resistant to this purine analogue. A high frequency of thioguanine resistant T cell clones, up to 10 times the frequency of T cells from normal individuals, was found in MS patients, and a significant percentage of these mutant clones proliferated in response to brain MBP, although they had never been intentionally exposed to this antigen. In contrast, no resistant clones obtained from normal subjects recognized

MEP.

:MBP, PLP, and HOG are also considered to be primary :autoantigens in MS because of their ability to induce experimental allergic encephalomyelitis (EAE) in animals. EAE is an experimentally induced condition that closely resembles MS and is Sthe benchmark animal model of MS. In addition, transfer of T cells from an individual suffering from EAE (or MS) to a healthy *560 animal can produce EAE in the recipient, a method of disease 9 20 induction referred to as "adoptive transfer". For example, in a human to animal transfer study, CSF mononuclear cells (including

T

cells) from MS patients caused paralysis, ataxia, and inflanmmatory brain lesions when injected into the CSF in the brain ventricles of severe combined immunodeficiency (SCID) mice (Saeki et al.

1992). Also, immunization of animals with MBP and/or PLP and/or MOG can elicit the CNS inflammation, paralysis, and other signs and symptoms of EAE (see, for example, Alvord et al. 1984; Abbas et al. 1994; Amor et al. 1994; and Johns et al. 1995).

Although it is clear that MEP, PLP, and HOG are primary antigens targeted by the abnormal immune response in MS, studies have revealed a marked heterogeneity of MBP and PLP epitopes that can induce T cell proliferative responses. These studies have not consistently revealed a single epitope that is recognized with higher frequency by reactive T cells of MS patients than those of normal healthy individuals (Chou et al. 1989; Richert et al. 1989; Martin et al. 1990; Ota et al. 1990; Pette et al. 1990; Martin et al. 1992; Meini et al. 1993). This heterogeneity in antigen targeting may, in part, be a function of the variety of the MHC molecules and TCRs expressed by different individuals in a human population.

Different molecular forms (isoforms) of MBP are generated by differential splicing of MBP hnRNAs, resulting in the presence in the encoded protein of some or all of the seven exons of the single MBP gene. In healthy adults, MBP is found almost exclusively as an 18.5 kDa molecule which is produced from an mRNA comprising all exons of the MBP gene except exon 2 (Kamholtz et al. 1988). Other forms of MBP include a full length (all 7 exons) 21.5 kDa isoform, and two other minor isoforms (17.2 and 20.2 kDa). The expression of the two exon 2 containing isoforms (21.5 kDa and 20.2 kDa) appears to increase with myelin formation, during both early fetal development and remyelination of damaged tissue (Kamholtz et al. 1988; Roth et al. 1987). These two 15 isoforms are referred to in the art, and herein, as "fetal" isoforms, although they are also found in remyelinating damaged adult tissue.

MS plaques contain areas of remyelination and thus should contain higher levels of the 21.5 isoform of MBP than found in 20 healthy adult CNS tissue, suggesting that an immune response to an epitope within the common 26 amino acid region (corresponding to the sequence spanning amino acid residue 60 to amino acid residue 85 of SEQ ID NO:1) of each of the two fetal isoforms of MBP coded for by exon 2 (which regions are referred to as *X2MBP" or simply could exacerbate the clinical course of established disease (Prineas et al. 1993; Raine and Wu, 1993; Bruck et al. 1994).

9. Since remyelination may occur cyclically in the course of MS, each cycle of remyelination could theoretically serve to drive an ongoing immune response by activating resting X2MBP specific

T

cells in the CNS. Supporting this hypothesis, several lines of evidence suggest the involvement of an epitope encoded by exon 2 of the MBP gene an epitope within X2MBP) in MS pathogenesis.

Studies of the role of alternate isoforms of MBP in MS require the availability of quantities of purified myelin antigens in order to evaluate their immunological properties. Such studies have therefore generally been limited to utilizing syntheticallyderived peptides, peptides comprising X2MBP. Recently, CD4 MHC class II-restricted T cells reactive with peptides containing exon 2 encoded sequences of human MBP were isolated from peripheral blood of both MS patients and normal healthy controls (Voskuhl et al., 1993a; Voskuhl et al. 1994). In a family afflicted with MS, the frequency of T lymphocytes specific for an X2 comprising peptide was higher than the frequency of T cells specific for epitopes within the 18.5 kDa isoform of MBP that does not contain X2 (Voskuhl et al., 1993b). In addition to this data from human subjects, a murine X2 comprising peptide was recently found to be immunogenic in SJL/J mice, and severe EAE was induced by adoptive transfer of exon 2 peptide-sensitized lymphocytes (Segal et al., 1994; Fritz and Zhao, 1994).

Taken together, these human and animal findings demonstrate that an in vivo cellular immune response to the myelin derived antigen MBP causes at least some of the pathogenesis associated 15 with multiple sclerosis. It should be noted, however, that all of the studies regarding X2 epitopes used synthetic peptides as antigens and none of them used full length MBP 21.5 protein. In light of the uncertainty regarding processing and display of particular epitopes of untested proteins by APCs, it has been 20 questioned in the art whether these results are truly relevant to in vivo MS pathogenesis.

PLP is a highly hydrophobic integral myelin membrane protein whose physical and chemical properties render it difficult to isolate, study, or administer to a patient (see, for example, Sobel et al. 1994; Tuohy 1994; Van der Venn et al. 1989; Van der Venn et al. 1990; Van der Venn et al. 1992; van Noort et al.

1994). The primary amino acid sequence of PLP is highly conserved between species. Typically, the mature PLP polypeptide does not include the initiator methionine coded for by the PLP gene; this amino acid appears to be removed in mammalian cells by a posttranslational processing event. Accordingly, as used herein, the amino acid numbering of human PLP is that shown in SEQ ID NO:22, and is numbered starting with a glycine residue as amino acid number 1.

The 276 amino acid PLP polypeptide contains approximately hydrophobic residues and is described as being structured into five hydrophilic domains and four extremely hydrophobic domains, which are numbered one to four starting at the amino terminus of the protein. Protein domains may be defined as having different extents, depending upon the criteria used to define the domain boundaries. Thus, by the most stringent criteria, the hydrophobic domains of the human PLP molecule span amino acid residues 10 to 36 (hydrophobic domain 59 to 87 (hydrophobic domain 151 to 178 (hydrophobic domain and 238 to 267 (hydrophobic domain 4) of the amino acid sequence of human PLP (SEQ ID NO: 22). Less stringent criteria are also used to define these domains, so that the hydrophobic domains may alternatively be said to span amino acid residues 10 to 18 (hydrophobic domain 70 to (hydrophobic domain 162 to 170 (hydrophobic domain and 250 to 258 (hydrophobic domain 4) of the amino acid sequence of human PLP (SEQ ID NO: 22).

Accordingly, the hydrophilic domains of PLP may be defined as amino acid residues 1 to 9 (hydrophilic domain 37 to 58 15 (hydrophilic domain 88 to 150 (hydrophilic domain 179 to 237 (hydrophilic domain and 267 to 276 (hydrophilic domain of the amino acid sequence of human PLP (SEQ ID NO: 22).

PLP-reactive T cell lines react strongly to PLP peptides.

Synthetic peptides with sequences based on the PLP sequence have been used to identify murine and human encephalitogenic epitopes.

See, for example, Fritz et al. 1983. J Immunol 130, pp. 191-194; Endoh et al. 1986; Greer et al. 1992; Kuchroo et al. 1992; Kuchroo et al. 1994; McRae et al. 1992; Pelfrey et al. 1993; Pelfrey et al. 1994; Sobel et al. 1992; Tuohy et al. 1988; Tuohy et al. 1989; Tuohy et al'. 1992; Whitham et al. 1991. J Immunol 147, pp. 101- 107; Whitham et al. 1991. J Immunol 147, pp. 3803-3808; and Correale et al. 1995. The human peptide-defined epitopes are shown in table 1.

In accordance with a recently proposed structure of PLP (Weimbs and Stoffel. 1992), these encephalitogenic epitopes are found in the both intramembrane and extramembrane domains of PLP.

PLP peptides have been shown to be encephalitogenic, and can induce disease in rabbits, rats, guinea pigs, and a variety of mouse strains (see, for example, Trotter et al. 1987). Murine PLP is identical in sequence to human PLP (SEQ ID NO:22) Encephalitogenic epitopes in mouse models include those shown in Table 2. In at least some mouse strains, PLP represents the major encephalitogen within the CNS (Kennedy et al. 1990). In various rodent models, significantly more demyelination was observed with PLP-induced EAE compared to MBP-induced disease (Tabira 1988). In clinical studies, significant differences in the number of PLPpeptide-reactive T cells in MS patients versus normal healthy control individuals have been reported (Sun et al. 1991; Trotter et al. 1991; Chou et al. 1992; Zhang et al. 1994).

In addition to these observations, the importance of PLP in MS pathogenesis is suggested by the observation that PLP, unlike MBP, is found solely in the CNS and not in the peripheral nervous system, where relatively little damage occurs in MS (Lees and Mackin. 1988).

Treatment of MS by Administration of Antigens The ideal therapeutic treatment for any disease is one that specifically blocks pathogenesis without affecting normal physiology. In the case of autoimmune diseases, an approach to 15 such ideal therapy is a treatment that specifically induces immune tolerance to autoimmune disease-associated self antigens without affecting immune responses to foreign antigens. New therapeutic agents and treatment strategies are being sought that will allow the induction of tolerance to specific autoantigens, while leaving 20 all other aspects of immune function unaltered.

Attempts have been made to therapeutically modify T cell responses via the administration of antigens to suppress specific autoreactive lymphocytes, especially T cells, and thereby elicit tolerance to disease-associated autoantigens. A distinct advantage of such antigen-specific therapy is that it can achieve the therapeutic modulation of the activities of only those T cells that, by reacting with the self antigens, are responsible for the development of pathology. This specificity provides therapeutic benefits without altering the important immune activities of T cells reactive with other antigens.

MS antigens have been studied as tolerization inducers for the treatment of MS/EAE, since therapies that suppress autoreactive T cells may significantly alleviate nervous tissue demyelination and resulting symptoms (see, for example, Adronni et al. 1993; Critchfield et al. 1994; Miller and Karpus 1994; Racke et al. 1995). A number of treatment protocols and antigens have been used in these studies, with animal rather than human forms of the antigens predominantly being used. For example, Weiner et al.

1993 used MBP purified from bovine myelin and Miller et al. 1992 used guinea pig, rat, and mouse MBPs. In studies using human MBP antigen, MBP was purified from cadaveric human brain (See, for example, Zhang, et al. 1994).

Oral tolerance involves regulatory CD8+ T cells that suppress immune responses both in vitro and in vivo through the secretion of cytokines, including TGF-beta (Chen et al. 1994 Science 265:1237-1240). The down-regulation of the activity of T cells mediated by this mechanism is not specific to particular T cell clones, and does not involve antigen-specific immunosuppression, but acts on any T cells in close enough proximity to the suppressive T cells to be affected by their secreted cytokines.

This phenomenon has been termed "bystander suppression".

Recent studies have investigated the tolerizing effects of Soral administration of bovine myelin to MS patients (Weiner et al.

1993 Science 259:1321-1324; Yoon et al. 1993). While fewer of the patients treated with oral myelin developed exacerbations of their MS symptoms than the patients treated with placebo, the results of the study were inconclusive, as the patients were not properly randomized. In fact, the authors cautioned that "It must be strongly emphasized that this study does not demonstrate efficacy of oral myelin in the treatment of MS." Thus, while oral tolerization studies support the usefulness of myelin proteins as immunomodulatory agents for the treatment of MS, new, more effective antigens, and alternative modes of administration of such antigens for the immunomodulatory treatment of MS continue to be sought.

Clearly, for the treatment of human disease, human-derived antigens have advantages over animal-derived antigens, as they are the actual autoantigens targeted for autoimmune attack in human disease, and suppression of disease should be most effective when homologous protein is administered (Miller et al. 1992). This is because the human protein will have the same MHC binding specificity and be subject to the same antigen processing as the endogenous protein targeted by the autoimmune response.

In fact, it is known that immunodominant epitopes the antigenic regions of the protein most often recognized by CD4 autoreactive T cells) of important MS autoantigens differ depending on the species from which the antigen is derived, even though many myelin antigens exhibit high interspecies homology at the amino acid sequence level. For example, as determined by analysis of T cells obtained from MS patients, an immunodominant epitope of human MBP is contained with the region spanning amino acids 84-102 and another is found in the region spanning amino acids 143-168. In contrast, a major immunodominant eptiope of murine MBP is found in the region spanning amino acids 1-9 (Zamvil et al. Nature 324:258, 1986) and a major immunodominant epitope of rat MBP is found in the region spanning amino acids 68-88 (Burns, et al. J. Ex. Med. 169:27, 1989).

The use of antigens isolated from human CNS tissue as therapeutic agents is, however, undesirable. This is due not only to problems associated with purifying antigens from CNS tissue generally and the difficulty of obtaining human raw materials, but, more importantly, to the problem of eliminating the 15 possibility of pathogenic contamination. One example of potential contaminants in the purification of CNS-derived proteins are prion particles that transmit the spongiform encephalopathies Creutzfeldt-Jakob disease and kuru. The prion particles present a particularly intractable problem because they are resistant to any known means of sterilization that will not also destroy the proteins being purified.

A useful approach to obtaining human antigens that avoids these problems is the production of protein antigens using recombinant DNA technology, typically by preparing DNA molecules encoding the antigens and using the DNAs to drive expression of the antigens in non-human host cells. Oettinger et al. (1993) :e have prepared a recombinant DNA molecule comprising unmodified human sequences encoding the 18.5 kDa form of human MBP and used this DNA to express recombinant human 18.5 kDa MBP in Escherichia coll. The expression of PLP polypeptides in E coli, however, has proven an intractable problem until now, as at least some PLP sequences appear to have toxic effects upon bacteria.

In fact, the hydrophobicity of PLP severely limits aqueous solubility (Tuohy 1994), rendering native PLP from any source difficult to prepare and to administer intravenously.

T Cell Deletion Alterations in the T cell repertoire occur naturally during T cell development. Only a small fraction of thymocytes (immature T cells) survive the development and selection events in the thymus that result in emigration of developing T cells to the peripheral circulation where they complete their maturation (von Boehmer, 1988; Marrack and Kappler, 1987). Experimental evidence strongly suggests that a large number of thymocytes that bear receptors for autoantigens are initially present in the thymus. During T cell development in the thymus, those cells reactive with self antigens are deleted (killed) as part of the normal developmental pathway.

This intrathymic tolerization process is referred to as "thymic tolerance".

Developing T cells do not encounter certain autoantigens in the thymus, but may encounter them as mature peripheral T cells.

For example, it may be that neural antigens are never presented in the thymus. Tolerance to such autoantigens is normally produced outside the thymus, and is referred to as "peripheral tolerance".

15 Peripheral tolerance can occur by at least two mechanisms, one of which is a similar but distinct process to thymic tolerization that results in the deletion of those mature peripheral T cells that are specifically reactive with a previously unencountered autoantigen. In addition, T cells with certain specific 20 reactivities can be induced to become inactive (anergic).

Peripheral deletion and the induction of anergy are physiologic mechanisms that result in the development of "peripheral tolerance". As a result of thymic and peripheral tolerization, mature T cells are normally tolerant to most autoantigens, however, autoreactive T cells may persist because their antigen is not presented with the required costimulation or is found in an immunologically privileged site.

The mechanism by which tolerization via T cell deletion is generated has recently been shown to depend upon repeated exposure to an antigen under certain defined conditions. Specific T cell deletion can therefore be induced by the appropriate administration of exogenous compounds comprising the relevant epitopes. As only a limited number of autoantigens (notably comprising a much greater number of epitopes) are involved in the pathogenesis of any individual autoimmune disease, it is possible, when they are known, to administer the self epitopes targeted in a disease to sufferers in the form of one or more isolated autoantigen-derived compounds containing the epitopes involved in pathogenesis. To have an optimal clinical effect, it may be necessary to have a comprehensive mixture of MBP and PLP epitopes, perhaps together with MOG epitopes, because of the large degree of human MHC and TCR polymorphism, and because new epitope reactivities may appear during autoimmune disease progression (McCarron et al. 1990; Lehmann et al. 1992; Kaufman et al. 1993).

The deletion of autoreactive T cells is an example of programmed cell death, which represents an important process in the regulation of many biological systems (Singer et al. 1994).

Programmed cell death occurs by a mechanism referred to as apoptosis, in which cells respond to certain stimuli by undergoing a specific sequence of predetermined events that effectively constitute cellular suicide. Apoptosis clearly plays a large role in shaping and maintaining the T cell repertoire and contributes 15 to the establishment of self-tolerance by actively eliminating .cells expressing autoreactive TCRs.

has recently been discovered that T cells are sensitive to apoptotic cell death induced by a variety of stimuli at multiple points in their lifespan (see, for example, Lenardo 1991; Boehme and Lenardo 1993; Critchfield et al. 1994). Positive selection S'factors are also believed to play a role in regulating the survival of specific T cell clones. The reduction or expansion of the number of individual T cells Qf a particular clone in an organism by these and other mechanisms serve to modulate the responsiveness of the organism's immune system to a particular antigen. It is now firmly established in several" autoimmune disease models, as well as in certain viral infections, that apoptosis can be induced (upon exposure to antigen under certain defined conditions) in mature peripheral antigen-specific

T

lymphocytes as well as in immature thymocytes.

Apoptosis occurs in many biological systems (see, for example, Kerr et al. 1991; Lockshin and Zakeri, 1991; Cohen et al.

1992; Duvall and Wyllie, 1986; Cotter et al. 1990). A cell undergoing apoptosis undergoes a specific program of events cellular and biochemical processes that depend upon active metabolism and contribute to the cell's self-destruction. In apoptotic T cells, the nucleus shrinks, the chromatin condenses, the genetic material (DNA) progressively degrades into small (nucleosomal repeat sized) fragments, there is cytoplasmic compaction, the cell membrane forms blebs, and the cell eventually collapses (Kawabe and Ochi, 1991; Smith et al. 1989). Cells cannot recover from apoptosis, it results in irreversible cell death (Kawabe and Ochi, 1991; Smith et al. 1989).

Recent reports have indicated a role for the TNF-related cytokine known as the FAS ligand and its receptor, CD95 (the FAS receptor), in the induction of apoptosis in T cells (Crispe et al.

1994; Nagata and Suda, 1995; Strasser, 1995; Dhein et al., 1995; Brunner et al., 1995; and Ju et al., 1995).

T cells that do not undergo apoptosis, but which have become activated, will carry out their "effector" functions by causing cytolysis, or by secreting lymphokines that cause B cell responses or other immune effects (Paul, 1989). These effector functions are the cause of tissue damage in autoimmune and other diseases.

15 Therapeutic Induction of AnoDtosis A powerful approach to avoiding or treating autoimmune o* diseases is to permanently eliminate lymphocytes involved in the autoimmune response by apoptosis. For example, a therapeutic effect can be achieved by eliminating only those T cells reactive 20 with autoantigens targeted in the particular autoimmune disease being treated, while leaving the vast majority of the T cell repertoire intact. In vivo studies have demonstrated that EAE can be treated by administration of myelin antigens at a dose and interval effective to induce apoptosis of T cells reactive with the antigens (se, for example, Critchfield et al. 1994).

This approach is described in co-pending U.S. patent application No. 07/751,090, filed in the name of Michael J.

Lenardo, and entitled Interleukin-2 Stimulated T Lymphocyte Cell Death for the Treatment of Autoimmune Diseases, Allergic Disorders, and Graft Rejection and co-pending U.S. patent application No. 07/926,290, filed in the name of Michael J.

Lenardo, and entitled Interleukin-4 Stimulated T Lymphocyte Cell Death for the Treatment of Autoimmune Diseases, Allergic Disorders, and Graft Rejection.

The accompanying figures, which are incorporated in and constitute part of the specification, illustrate certain aspects of the invention, and together with the description, serve to explain the principles of the invention. It is to be understood, of course, that both the figures and the description are explanatory only and are not restrictive of the invention.

Brief Description of the Drawinas Fig. 1. Clinical course of active (antigen-induced) EAE in four individual SJL/J mice treated with ovalbumin (Fig. IA OVA), PLP peptide 139-151 (Fig. 1B a peptide with an amino acid sequence corresponding to amino acid residues 139-151 of SEQ IS NO:22), APLP4 (Fig. 1C PLP4), or MP4 (Fig. 1D) which are administered in CFA adjuvant. Disease was graded 0, no abnormality; 1, limp tail; 2, limp tail with inability to right upon being turned over; 3, hind limb weakness or dragging one hind limb; 4, paralysis of both hind limbs; 5, moribund; and 6, death.

Clinical score open circles; weight in grams closed circles.

Fig. 2. Prevention treatment of adoptive EAE by 15 intravenous APLP4 administration. PLP-specific lymph node cells from APLP4/CFA immunized mice were stimulated in vitro with PLP peptide 139-151 (described above for Fig. T cells from these animals were transferred by intravenous injection into naive recipients at 107 cells/mouse on day 0. The five mice in the 20 treated group (PLP4 Day 2, 4, 6) received two intravenous injections (separated by 6-8 h) of 125 gg of APLP4 on days 2, 4, and 6 post transfer. The five untreated mice (Control animals) received an equal volume (100 il) of sterile water. Mice were monitored daily and a mean clinical score for each group was determined (scored as in Fig. 1).

Fig. 3 Proliferative responses of T cell enriched lymph node cells from, as indicated on the x axis, naive mice (SJL/J) and mice immunized with PLP peptide 139-151, described above for Fig. 1, (PEPTIDE) or APLP4 (PLP4) in response to in vitro stimulation with synthetic PLP peptides (139-151 or 178-191 a peptide with an amino acid sequence corresponding to amino acid residues 178-191 of SEQ ID NO:22) or intact APLP4 (PLP4) at the concentrations indicated. T cells were incubated for 72 h in media alone or with antigens. Proliferation was measured by 3

H-

thymidine incorporation following an 18 h pulse. All assays were replicated with triplicate cultures.

Fig. 4 Prevention treatment of APLP4-induced active EAE by intravenous APLP4 administration. EAE was induced in SJL/J mice by subcutaneous injection on days 0 and 3 with 100 gg of APLP4 in CFA followed by 300 ng of pertussis toxin. The five experimental mice received two intravenous injections (separated by 6-8 h) of 125 jg of APLP4 on days 5, 7, and 9 post-immunization (PLP4 Day 5, 7, 9, closed circles). The five untreated mice (Control animals, open circles) were given an equal volume (100 gl) of sterile water on the same schedule. Mice were monitored daily and a mean clinical score (determined as in Fig. 1) was assigned for each group.

Fig. 5 PLP treatment eliminates T cell proliferation in response to PLP and MBP antigens. T cell proliferation assays were performed on lymph node cells obtained from mice immunized to induce EAE and treated with APLP4. Induction and treatment were as described for Fig. 4. Antigens used in the in vitro T cell proliferation assays at 50 pg/ml were APLP4 (PLP4) or MP4, as 15 indicated.

Fig. 6 Proliferation of human MBP-specific T cell lines in response to stimulation with recombinant MP4. MP4 was used at a concentration of 10 g/ml. Human T cell lines 2A2 (reactive with MBP peptide 31-50), 3H5 (reactive with MBP peptide 87-106) 20 and 5B2 (reactive with MBP peptide 151-170) were initially obtained from a healthy individual. These cell lines are specific for MBP epitopes indicated by the corresponding amino acid positions of adult human brain (18.5 kDa) MBP (SEQ ID NO:4) displayed in parentheses.

Fig. 7 MP4 stimulates murine T-cells after disease induction with PLP and PLP treatment eliminates T cell proliferation in response to MP4 antigens. T cell proliferation assays were performed on lymph node cells obtained from mice immunized with APLP4 to induce EAE and treated with APLP4. MP4 was used at 50 pg/ml in the in vitro T cell proliferation assays.

Fig. 8 Proliferative responses of T cell enriched lymph node cells from SJL/J mice immunized with PLP peptide 139-151 (described above for Fig. 1) in response to in vitro stimulation with synthetic PLP peptides (139-151, 43-64 a peptide with an amino acid sequence corresponding to amino acid residues 43-64 of SEQ ID NO:22, or 215-232 a peptide with an amino acid sequence corresponding to amino acid residues 215-232 of SEQ ID NO:22) or intact APLP4 (PLP4) at 10pg/ml. T cells were incubated for 72 h in media alone or with antigens. Proliferation was measured by 3 H-thymidine incorporation following an 18 h pulse. All assays were replicated with triplicate cultures.

Fig. 9 Proliferative responses of T cell enriched lymph node cells from SWR mice immunized with PLP peptide 103-116 in response to in vitro stimulation with synthetic PLP peptides (178-191 discussed above, 139-151 discussed above, or 103- 116 a peptide with an amino acid sequence corresponding to amino acid residues 103-116 of SEQ ID NO:22) or intact APLP4 (PLP4) at 10gg/ml. T cells were incubated for 72 h in media alone or with antigens. Proliferation was measured by 3 H-thymidine incorporation following an 18 h pulse. All assays were replicated with triplicate cultures.

Fig. 10 Proliferative responses of T cell enriched lymph node cells from PL/J mice immunized with PLP peptide 43-64 in 15 response to in vitro stimulation with synthetic PLP peptides (139-151, 43-64, or 178-191), discussed above, or intact APLP4 (PLP4) at 10g/ml. T cells were incubated for 72 h in media alone or with antigens. Proliferation was measured by 3 H-thymidine *see incorporation following an 18 h pulse. All assays were replicated &'se 20 with triplicate cultures.

Fig. 11 Treatment of EAE induced by the transfer of 30,000,000 T cells that were activated with guinea pig MBP.

Treatments were: 200gg MP4; 200lg guinea pig MBP (GP-MBP); 400g guinea pig MBP; or 400g ovalbumin (OVA, control); as indicated.

These treatments were administered twice daily (at 6 hour intervals) i.v. on days 6, 8, and 10 after adoptive transfer of encephalitogenic T cells. Each treatment group consisted of 3 to animals.

Fig. 12 Treatment of EAE induced by immunization of SJL mice with 100g PLP peptide 139-151, discussed above for Fig. 1.

Treatments were with 250pg MP4 or 250lg pigeon cytochrome c (control); as indicated. These treatments were administered twice daily (at 6 hour intervals) i.v. on days 5,7, and 9 after immunization. Each treatment group consisted of 3 animals.

Fig. 13. PCR strategy for construction of a synthetic MBP21.5 gene (cDNA). Indicated by bracket A is the alignment of overlapping oligonucleotides 1 through 6 (SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID that were used to construct the MBP+X2Cys81/Bact. gene. Three 22 subdomains of the gene II, and III as shown by the diagram indicated by bracket B) were initially synthesized. Larger domains (I+II, II+III) were formed by overlapping PCR using the appropriate outside oligonucleotides (oligonucleotides 1 and 4, and oligonucleotides 3 and 6, respectively) as shown by the diagram indicated by bracket C. The full-length molecule was completed by overlapping-PCR of domains I+II and II+III using outside oligonucleotides 1 and 6. A map of the final product is shown by the diagram indicated by bracket D. In this diagram, the hatched region in this map of the full-length molecule depicts the location of exon 2, with the cysteine at amino acid residue 81 (Cys 8 1 shown as altered to serine (Ser 8 1 The dark box at the 3' end of the gene (right hand side of the diagram) illustrates the addition of sequences encoding the histidine tag 15 that was added to facilitate purification.

Fig. 14. Recombinant MBP expression and subcellular localization in bacterial cells unfractionated whole cell lysates. Cell lysates were prepared from induced cultures of BL21(DE3) cells that were transformed with control pET22b vector without added insert pET22b/MBP18.5 hu m. or pET22b/MBP+X2Cys81/Bact. Whole cell lysates were separated by 16% SDS-PAGE under reducing conditions (note that under these conditions, no dimers are seen), then Coomassie stained (Coom) or immunoblotted with monoclonal antibodies that recognize either a 25 carboxy-terminal epitope ("C-term Ab') or an amino-terminal epitope ("N-term Ab") of human brain MBP. Asterisks highlight the position of two fragments of MBP+X2Cy s 8 1 that are recognized by only the "N-term Ab" mAb. Molecular weights in kilodaltons (as determined by electrophoreising marker proteins) appear on the left. The open and closed arrows mark the positions of MBP+X2Cys 81 and MBP18.5, respectively.

Fig. 15. Recombinant MBP expression and subcellular localization in bacterial cells soluble vs. insoluble fractions. Cell lysates were prepared from induced cultures of BL21(DE3) cells that were transformed with control pET22b vector without insert pET22b/MBP18.5 h um. or pET22b/MBP+X2Cys 8 1 B ac t Bacterial lysates were fractionated into soluble or insoluble pellet fractions using either neutral buffer ("Tris") or 0.1N HC1 ("Acid") conditions as described above. Shown are the Coomassie stained gels obtained by SDS-PAGE of the cell fractions under reducing conditions (note that under these conditions, no dimers are seen). The open and closed arrows mark the positions of MBP+X2Cys81 and MBP18.5, respectively. Note that the acid extraction (but not the neutral extraction) allowed recovery of the MBP+X2CYs 81 and the MBP18.5 polypeptides in the soluble fractions.

Fig. 16. Large scale acid extraction of recombinant MBP from bacterial cells. Shown is a Coomassie stained SDS/PAGE gel carried out under reducing conditions (note that under these conditions, no dimers are seen). Each group of three lanes shows whole cell lysate ("lysate") and insoluble ("insol") and soluble fractions obtained from simultaneous acid extraction and mechanical disruption. Cells were harvested from induced cultures of BL21(DE3) cells transformed with either pET22b vector without added insert pET22b/MBP18.5hum.

or pET22b/MBP+X2Cys 8 1/bact. The positions of MBP+X2Cys81 (open arrows) and MBP1 8 5 h um (closed arrows) are indicated. Note that this large scale acid extraction allowed recovery of almost all of the MBP+X2Cys 81 and the MBP18.5 polypeptides in the soluble fractions.

Fig. 17. Chromatograph showing reversed-phase chromatographic isolation of acid-extracted MBP+X2CY s81 The soluble fraction recovered from the experiment shown in Fig. 16 ("sol" lane was chromatographed over a VYDAC C4 reverse phase column and eluted via a 25-50% (CH 3 CN)/0.1%TFA gradient.

MBP+X2Cy s81 is found in pooled fractions that correspond to the large peak eluting between 17 and 20 minutes. A similar chromatograph was obtained for MBP18.5.

Fig. 18. Purification of MBP+X2CY s81 (top panel) and MBP18.5 (bottom panel) by metal chelation chromatography of acid extracts of bacterial cells. Shown are Coomassie stained gels of protein fractions collected during the affinity purification and subjected to SDS-PAGE. The positions of MBP+X2Cy s81 (open arrow) and MBP18.5 (closed arrow) are indicated. Lanes are labeled *load" (the lysate loaded onto the column), "unbound" (the column flow-through, "wash wash and "wash 3" (the column eluate from each wash), "elution elution and "elution 3" (the column eluate from each elution step), and resin (a sample of column resin taken after the final elution, boiled in sample buffer, and loaded on the gel).

Fig. 19. Yield of bacterially expressed MBP polypeptides in bacteria transfected with nucleic acid vectors comprising the nucleic acid sequences MBP 18 5 hum. (SEQ ID NO:4), MBP+X2Cy s81 hu m. (SEQ ID NO:1), MBP+X2S er 8l/ ba ct. (SEQ ID NO:3), and MBP+X2Cys81/ ba ct. (SEQ ID NO:2), as indicated.

Fig. 20. MBP antigens elicit proliferative responses from human T cell clones specific for adult, brain-derived MBP. T cell lines specific for adult brain MBP18.5 were stimulated with medium alone ("control") or medium containing 10mg of either purified adult human brain MBP ("Brain MBP"), bacterially produced MBP18.5 ("MBP18.5"), or bacterially produced MBP+X2Cys 8 1("MBP+X2Cys 81 Reported are total incorporated 3

H-

CPM from one representative proliferation assay done in triplicate as described in the Examples. "2A2" and "3H5" are human T cell lines obtained from normal individuals as described in the Examples.

Fig. 21. Proliferative responses of exon 2-specific human T cell lines to MBP antigens. Human T cell lines 1H7 and 1G1 were stimulated with medium alone ("control") or medium containing 10g of either purified adult human brain MBP ("Brain MBP"), bacterially produced MBP18.5 ("MBP18.5"), bacterially produced MBP+X2Cy s 8 1 ("MBP+X2Cys81"), or exon 2-encoded peptide corresponding to amino acids 59 to 84 of SEQ ID NO:1 ("X2 peptide"). Presented are the total 3 H-CPM incorporated during the proliferation assays, which were done in triplicate as described in the Examples. 1H7 and 1G1 are human T cell lines that are specific for the exon 2 encoded region of MBP and were obtained from the same MS patient as the 3All line used in the experiment set forth below in Fig. 22. Presented are the total 3H cpm incorporated during the proliferation assays, which were done in triplicate as described in the Examples.

Fig. 22. Proliferative responses of exon 2-specific human T cell lines to MBP+X2CYs 8 1 and MBP+X2Ser81. Human T cell line 3A11 was stimulated with varying doses of exon 2 peptide MBP+X2Cys81 MBP+X2Ser81 or medium alone 3A11 is a human T cell line that is specific for the exon 2 encoded region of MBP and was obtained from the same MS patient as the 1H7 and 1G1 lines used in the experiment described in Fig.

21. Presented are the total 3 H cpm incorporated during the proliferation assays, which were done in triplicate as described in the Examples.

Fig. 23. Sequence comparison of recombinant human MBP+X2Cys81/bact. (fetal form, SEQ ID NO:1) to that of adult brain-derived human MBP (adult form SEQ ID NO:4).

The adult brain-derived human MBP sequence (Genbank accession S 15 #M13577) is noted only in positions that deviate from the E. coli preferred codon sequence of MBP+X2Cys81/bact. The initiator (ATG) and stop codons (TAA) are indicated for both genes. Dashes in the adult brain-derived human MBP sequence reflect the positions of exon 2 (bp 177-255) and the histidine tag (bp 595- 612) additions to this version of MBP+X2Cys81/bact. pMBP+X2Cys81/bact. with 6 carboxy terminal histidine residues, also referred to as a histidine tag). Regions of overlap between synthetic oligonucleotides used for the construction of the MBP+X2Cys81/bact. gene are underlined. C to T bp mutations from the intended MBP+X2Cys81/bact. gene sequence are noted by asterisks above positions 462, 528 and 532. These changes conserve the MBP+X2Cys8 1 amino acid sequence. Sense oligonucleotide 1 (SEQ ID NO:5) includes the sequence GGAATTCCGT AAGGAGGTAT AG (not shown in this figure) located 5' to the NdeI cloning site, and extends through base 108. Oligonucleotide 6 (bp 516-622, SEQ ID NO:10) is an antisense oligonucleotide to the sequence shown and includes the tetranucleotide CCCC (not shown in this figure) located 3' to the HindIII site. Four other oligonucleotides used include sense oligonucleotides 3 (SEQ ID NO:7) and 5 (SEQ ID NO:9) and antisense oligonucleotides 2 (SEQ ID NO:6) and 4 (SEQ ID NO:8). The cysteine at amino acid 81 is noted in boldface type.

7 Fig. 24. Diagrammatic representation of location of MBP epitopes of recombinant human MBP 21.5 ("rhMBP21.5). numbers indicate amino acid residues of SEQ ID NO:1 corresponding to the known epitope specificity of the T cell lines tested (indicated by number letter number designations or "Gimer"). Each of the T cell lines shown gave a positive T cell response to the purified rhMBP21.5 molecules of the invention.

Fig. 25. Details of the specific molecules tested and results obtained with each T cell line shown in Fig. 24.

SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide compositions and methods for the diagnosis, clinical assessment, and therapeutic treatment of MS in human patients, and for the assessment of the potential responsiveness of MS patients to such therapeutic treatment.

The invention provides compositions comprising novel recombinant human PLP polypeptides. As used herein and in the claims, "PLP polypeptides" are polypeptides that contain at least one sequence corresponding to at least one hydrophilic domain of o 20 human PLP, as discussed above. In accordance with the invention, such PLP polypeptides may further comprise MBP, MOG, and/or MAG ooo.: polypeptide sequences, as well as other relevant polypeptide sequences. Also provided are DNA constructs which encode PLP polypeptides and which have been engineered to optimize the S.25 production and isolation of such molecules from bacterial cells.

More specifically, the molecules of the invention include immunoreactive polypeptides comprising PLP muteins that comprise a sequence of amino acids comprising the sequence of a native PLP polypeptide minus at least one hydrophobic peptide region, and preferably minus at least two hydrophobic regions. More preferably, the sequence of amino acids comprises the sequence of a native PLP polypeptide minus at least three hydrophobic peptide regions. Most preferred are immunoreactive polypeptides comprising PLP muteins that comprise a sequence of amino acids comprising the sequence of a native PLP polypeptide minus at least some of the amino acid residues making up each of all four hydrophobic domains of PLP.

The polypeptide and nucleic acid molecules of the invention further comprise MBP sequences, sequences corresponding to any span of at least 10 contiguous amino acid residues of SEQ ID NO: 1 or SEQ ID NO:3. As used herein and in the claims, an "MBP polypeptide" is a polypeptide comprising such an MBP sequence, and "an amino acid sequence encoded by at least part of exon 2 of the human MBP gene" is a sequence of at least 10 contiguous amino acids corresponding to at least 10 contiguous amino acids from the region spanning amino acids 60-85 of SEQ ID NO:1.

The invention provides compositions comprising novel recombinant human MBP 21.5 polypeptides MBP polypeptides that comprise an amino acid sequence encoded by at least part of exon 2 of the human MBP gene). Preferably, these MBP polypeptides include amino acid sequences encoded by all seven exons of the human MBP gene. In certain preferred embodiments, the sequence encoded by exon 2 is modified to facilitate large scale production 15 and purification of the polypeptide. Also provided are DNA constructs which encode MBP 21.5 polypeptides and which have been engineered to optimize the production and isolation of such molecules from bacterial cells.

The methods of the invention comprise the use of the compositions of the invention in the diagnosis and clinical assessment of MS, as well as in the therapeutic treatment of MS ooo and in the assessment of the potential responsiveness of MS o patients to such therapeutic treatment.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 25 As discussed above, the present invention relates to MBP and PLP polypeptides (proteins) for use in the treatment, diagnosis, and clinical assessment of MS, and to nucleic acid molecules useful in producing MBP and PLP polypeptides.

I. MBP polvetides As used in this specification and in the claims "MBP 21.5 polypeptides" refers to one or more of the following polypeptides: the polypeptide of SEQ ID NO:1 (human 21.5 kDa MBP, "MBP+X2"), the polypeptide of SEQ ID NO:1 with amino acid 81 being any standard amino acid ("MBP+X2XXx81"), the polypeptide of SEQ ID NO:1 with cysteine 81 replaced with any other standard amino acid ('MBP+X2Xaa81"), the polypeptide of SEQ ID NO:1 with cysteine 81 replaced with an uncharged amino acid an amino acid that is uncharged at a pH of between 6 and 7) having a molecular weight of cell lines) specifically reactive with X2MBP (X2MBP-specific T cells) to MBP+X2X a a 8 1 or, preferably, to a test peptide (the X2X aa81 peptide) comprising the exon 2 encoded region of MBP+X2X a a 8 1 as described in detail below. The test peptide is preferably a 26 amino acid peptide with a sequence corresponding to amino acid residues 59 to 84 of SEQ ID NO:1 with Cys 81 replaced with the other standard amino acid (the "X2Xaa8 1 26mer").

X2MBP-specific T cells can be obtained as T cell lines by conventional methods using a peptide containing the amino acid sequence encoded by exon 2 (hereinafter referred to as an "X2MBP peptide"). For example, the methods described by Voskuhl et al.

1993a may be used. See also Voskuhl et al., 1993b; Segal et al., 1994; Voskuhl et al., 1994; and Fritz and Zhao, 1994.

Preferably human T cell lines are obtained by such standard 15 methods following stimulation with an X2MBP peptide that has just :the 26 amino acids encoded by exon 2, an X2MBP peptide whose a sequence corresponds to amino acid residues 59 to 84 of SEQ ID NO:1 (the "X2 26mer"). In particular, stimulation with the X2 26mer is preferred to stimulation with the 40 amino acid X2MBP 20 peptide or the 18.5 kDa isoform of MBP described in the Voskuhl et o. al. 1993a publication.

In accordance with the present invention, X2MBP-specific T cell lines thus obtained are used, inter alia, to determine the epitope neutrality of a particular amino acid substitution at position 81. This is accomplished by assessing the reaction of the cells of the X2MBP-specific human T cell line to the X2X aa81 peptide. (MBP+X2X a a 8 1 can also be used to test epitope neutrality, but this is less preferred.) If the X2MBP-specific T cells respond to the X2X a a 8 1 peptide containing the particular amino acid substitution to an extent that satisfies the criterion for X2MBP-specificity set forth by Voskuhl et al. 1993a, i.e. if the particular X2X a a 8 1 peptide demonstrates a stimulation index of greater than 2, as compared to medium alone controls, then epitope neutrality of a particular replacement amino acid is confirmed.

Preferably the stimulation index is greater than 3.

In accordance with the present invention, such an epitope neutral replacement can generally be achieved using an uncharged less than about 150 ('MBP+X2Xaa81<150"), and the polypeptide of SEQ ID NO:1 with cysteine 81 replaced with serine ("MBP+X2Ser 81 "MBP 21.5 polypeptides" also comprise variations of the foregoing four sequences, provided that the sequence continues to include at least some of the sequence of amino acids encoded by exon 2 of the human MBP gene, and further provided that the polypeptide can induce a "T cell response" in a population of MBP reactive T cells isolated from an MS patient. The term "T cell response" is discussed below.

A preferred MBP 21.5 polypeptide of the invention is a bacterially expressed human recombinant MBP containing amino acids encoded by exon 2 of the human MBP gene and having a molecular weight of approximately 21.5 kDa in which Cys 81 has been replaced with another standard amino acid (this polypeptide is referred to S* 15 herein as "MBP+X2 X a a 8 1 and nucleic acid molecules encoding it are referred to as "MBP+X2 X a a 8 l h u m. or "MBP+X2X a a 8l b a c t with the superscript hum. or bact.indicating the codon usage in the coding region of the nucleic acid molecule, as discussed below).

As used in the art, a "standard" amino acid is one of the 20 amino S 20 acids commonly found in proteins.

As used herein, the amino acid sequence encoded by exon 2 oe will be referred to as X2MBP or simply X2. In accordance with the invention, the X2MBP sequence may be located at any position in the MBP+X2Xxx 8 1 polypeptide, although the naturally occurring 25 position of the native exon 2 encoded sequence (as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3) is preferred. Other S" polypeptides comprising X2MBP sequences are described below.

Preferably, the replacement amino acid does not cause epitope conversion, T cell recognition of the immunodominant epitope or epitopes of X2MBP is substantially unaltered by the replacement of Cys 81 with the particular replacement amino acid. Prior to the present invention it was unknown whether replacement of amino acid residue 81 with another standard amino acid would cause such epitope conversion whether such alterations would be epitope neutral).

Lack of epitope conversion by the substitution of any standard amino acid can be determined in accordance with the present invention by testing the responses of T cells T amino acid that has a molecular weight of less than about 150 and that preferably is not strongly hydrophobic.

Amino acids that satisfy these requirements include Ala, Asn, Gly, Pro, Thr, and Ser. Most preferably, the replacement is Ser, resulting in an MBP 21.5 polypeptide comprising an exon 2 encoded region in which Cys 81 has been changed to Ser 81 (hereinafter this polypeptide is referred to as "MBP+X2Ser81 and nucleic acid molecules encoding it are referred to as "MBP+X2Ser81/hum.. or "MBP+X2Ser81/bact.., with the superscripts hum. and bact.

indicating the codon usage in the coding region of the nucleic acid molecule, as discussed below).

Prior to the present invention, it was not known whether bacterially expressed MBP+X2 polypeptides would be recognized and responded to by T cells to the same extent as mammalian expressed S 15 MBP polypeptides human derived MBP-X2). This uncertainty was due, inter alia, to the differences in protein folding during the expression of proteins in bacteria or mammalian cells.

Bacterially expressed proteins are typically not folded into the native conformation of proteins expressed in mammalian cells. As discussed within the Background of the Invention section above under the heading "T Cells, Antigen Presenting Cells, and T Cell Epitopes', protein folding can determine whether a specific epitope is appropriately processed by APCs. For this reason, bacterially expressed proteins may not be processed and presented 25 by APCs in the same manner as native proteins, and may therefore not be recognized by T cells.

The exon 2 sequences in MBP+X2Cy s 8 1 were cause for additional uncertainty, as such sequences had only been shown to stimulate T cells when added to T cells as synthetic peptides, (which do not have to be processed by APCs in order to be recognized by TCRs and responded to by T cells). Prior to the present invention, it had never been shown that the 21.5 kDa isoform of MBP (regardless of source) could be correctly processed by APCs so as to stimulate encephalitogenic T cells, a question of particular interest with regard to the role of X2 epitopes in MS pathogenesis. The present invention has allowed the demonstration that this is the case, demonstrating the clinical relevance of the previously reported X2MBP peptide work.

II. PLP Polvnentides A preferred PLP polypeptide of the invention is a bacterially expressed human recombinant PLP containing hydrophilic domains 2, 3 and 4. Such PLP polypeptides may include one or more hydrophobic domains. More preferrably, PLP polypeptides comprise the PLP epitopes associated with MS shown in Table 1. Such preferred PLP polypeptides include APLP3 (SEQ ID NO:23) and APLP4 (SEQ ID NO:24). Particularly preferred molecules of the invention are PLP muteins comprising an amino acid sequence corresponding to the amino acid sequence set forth in SEQ ID NO:23 or SEQ ID NO:24, or, preferably, SEQ ID NO:25, SEQ ID NO:26 (preferably amino acid residues 1 to 368, inclusive, of SEQ ID NO:26), SEQ ID NO:27 (preferably amino acid residues 6 to 374, inclusive, of SEQ ID NO:27), or SEQ ID NO:28 (preferably amino acid residues 1 to 487, 15 inclusive, of SEQ ID NO:28).

In a particularly preferred embodiment, the immunoreactive polypeptides comprise at least 10 contiguous amino acids a linear polymer of amino acids sufficient in size to comprise an epitope), all but one target amino acid residue of which correspond to a region of the 21.5 kDa isoform of human MBP (SEQ ID NO:1) comprising amino acid residue 81 of SEQ ID NO:1. In this embodiment, the target amino acid residue is located in a position within the MBP amino acid sequence corresponding to the position of amino acid residue 81 of SEQ ID NO:1 and the target amino acid 25 residue is any standard amino acid other than cysteine.

Certain preferred immunoreactive polypeptides of the invention further comprise a myelin oligodendrocyte glycoprotein amino acid sequence corresponding to at least 10 contiguous amino acids of the amino acid sequence of human myelin oligodendrocyte glycoprotein (amino acid residues 199 to 319, inclusive, of SEQ ID NO:28).

Preferably, the immunoreactive polypeptides of the invention are expressed in bacteria at higher levels than the native PLP polypeptide and/or are more soluble in aqueous solution than the native PLP polypeptide.

PLP-specific T cells can be obtained as T cell lines by conventional methods using a peptide containing a PLP amino acid sequence. For example, the methods described by Voskuhl et al.

1993a may be used. See also Voskuhl et al., 1993b; Segal et al., 1994; Voskuhl et al., 1994; Fritz and Zhao, 1994; Pelfrey et al.

1993; Pelfrey et al. 1994; and Correale et al. 1995.

Prior to the present invention, it was not known whether bacterially expressed PLP polypeptides would be recognized and responded to by T cells in a manner that would allow their use as therapeutic agents. This uncertainty was due, inter alia, to the differences in protein folding during the expression of proteins in bacteria or mammalian cells. Bacterially expressed proteins are typically not folded into the native conformation of proteins expressed in mammalian cells. As discussed within the Background of the Invention section above under the heading "T Cells, Antigen Presenting Cells, and T Cell Epitopes", protein folding can determine whether a specific epitope is appropriately processed by APCs. For this reason, bacterially expressed proteins may not be 15 processed and presented by APCs in the same manner as native proteins. Therefore, some or all of the epitopes in such a bacterially expressed protein may not be recognized by T cells.

III

9III. Nucleic Acid Molecules Encodin MBP and PLP Polpeptides Nucleic acid molecules useful in the practice of the present invention can be prepared using a variety of techniques now known or subsequently developed in the art. For example, using techniques well known in the art they can be produced using cloned genes. The terms gene and genes, as used herein, encompass expressed protein-encoding) nucleic acid molecules, either 25 with intron-comprising sequences or without introns, e.g. cDNAs.

The cloned genes are manipulated by conventional techniques, e.g., PCR amplification and/or restriction digestion of nucleic acid molecules to generate restriction fragments encoding portions of the MBP or PLP polypeptides. These fragments can be assembled using, for example, PCR fusion (overlapping PCR) or enzymatic ligation of the restriction digestion products. The assembled constructions or fragments thereof can be modified by mutagenic techniques such as oligonucleotide mediated site-directed mutagenesis.

Numerous publications are available that teach these conventional methods, including Sambrook, et al. 1989; Ho et al.

Gene 1989; Farrell 1993; Ausubel et al. 1994; Griffin and Griffin 1994; Mullis et al. 1994; Harwood 1994; and Davis et al. 1994.

Alternatively, the nucleic acid molecules encoding the MBP or PLP polypeptides used in the practice of the invention or any or all of the nucleic acid fragments used to assemble such nucleic acid molecules can be synthesized by chemical means (see, for example, Talib et al. 1991 and Ausubel et al. 1994).

In accordance with the present invention, codons for various of the amino acids of the MBP and PLP polypeptides of the invention may be "bacterialyzed" to enhance the production of the protein in bacteria. As known in the art, bacteria tend to use certain codons for particular amino acids in preference to other possible codons which encode the same amino acid. Accordingly, it is believed that the protein synthetic machinery of the bacteria may work more effectively when processing the preferred codons.

Bacterialization and other alterations of myelin protein-encoding codons will now be discussed in greater detail as exemplified by 15 specific reference to the MBP molecules of the invention.

SEQ ID NO:1 sets forth the amino acid and nucleotide °O*o sequences for the native human 21.5 kDa fetal isoform of MBP. A nucleic acid molecule encoding MBP+X2Xaa81 can be produced by modifying at least one of nucleotides 241 through 243 codon 81) of SEQ ID NO:1 so that the codon corresponds to the desired replacement amino acid. Such modification can be achieved using a variety of nucleic acid manipulation techniques now known or subsequently developed in the art, including conventional recombinant DNA techniques such as oligonucleotide mediated site- 25 directed mutagenesis, PCR mutagenesis, or de novo synthesis of the desired polynucleotide, as discussed above.

For MBP+X2Ser81, the native TGC codon can be changed to any o. of AGC, AGT, TCA, TCC, TCG, and TCT. In general, the change is preferably to TCG, as this change results in the creation of a new TCGA restriction site at this location. The creation of a new restriction site at this location facilitates the identification and separation of a nucleic acid molecule comprising the desired modification from the mixture of modified and unmodified nucleic acid molecules that is typically obtained as an intermediate step in the overall process of producing a nucleic acid molecule encoding MBP+X2Xaa81, such as a nucleic acid molecule encoding MBP+X2Ser81. When considerations of optimization of protein production override considerations of ease of nucleic acid manipulation, and when MBP+X2Ser81 is to be produced in bacteria, E. coli (where the TCG codon is not a bacterially preferred codon) the change is preferably to TCC, TCT, or AGC, since these codons are preferred in bacteria.

SEQ ID NO:2 sets forth the amino acid sequence for the native human 21.5 kDa fetal isoform of MBP and a modified nucleotide sequence encoding this protein wherein the codons for various of the amino acids have been "bacterialyzed" to enhance the production of the protein in bacteria. As known in the art, bacteria tend to use certain codons for particular amino acids in preference to other possible codons which encode the same amino acid. Accordingly, it is believed that the protein synthetic machinery of the bacteria may work more effectively when processing the preferred codons. However, as also known in the 15 art, it is unpredictable whether substituting preferred codons for non-preferred codons will in fact result in a substantial enhancement in production of a particular protein in bacteria. As discussed in detail in the Examples, below, the bacterialization of SEQ ID NO:2 increased production of MBP in E. coli by at least 20 50 percent.

In SEQ ID NO:2, the bacterialization has been performed by substituting bacterially preferred codons for native human codons which did not already correspond to bacterially preferred codons (criterion In selecting which codons to change, particular 25 attention was paid to the following seven amino acids: Arg (17 of 21 codons changed); Gly (13 of 28 codons changed); Pro (10 of 17 codons changed); Lys (12 of 14 codons changed); Leu (3 of 11 codons changed); Thr (6 of 8 codons changed); and Val (3 of codons changed). These amino acids were emphasized because of a strong bias for the use of certain of their redundant codons in E.

coli. (Wada et al., 1992.). Of these seven, Arg, Pro, and Lys were considered the most important since they constitute 26% of the amino acid residues in MBP 21.5. As an alternate criterion, some codons were changed to a codon which is preferentially used in highly expressed bacterial genes (criterion 2, see Grosjean and Fiers, 1982). A complete listing of codon changes incorporated in the nucleic acid molecule corresponding to SEQ ID NO:3 (except for the native cysteine codon 81 being retained in this comparison instead of the Ser codon for amino acid number 81 found in SEQ ID NO:3) is given in Table 4, where the native (fetal) human MBP21.5 sequence data are indicated as "huMBP 21.5" and the bacterialized recombinant MBP (MBP+X2Cys 8 1/bact.) sequence data are indicated as "recMBP 21.5".

As used herein and in the claims, the expression "bacterially preferred codon" refers to a codon selected on the basis of either of the above two criteria, and the superscripts "hum.. and (2) obact.. designate MBP-encoding nucleic acid sequences with (1) native human codons and at least some codons that have been changed from native human codons to bacterially preferred codons.

More or less bacterialization can be performed if desired; the criterion being whether a desired level of production increase Sis achieved. Also, with regard to MBP, the bacterialyzed sequence can be further altered to produce MBP+X2Xaa8l/bact. preferably or preferably MBP+X2Ser81/bact. The bacterialization and the further alterations at codon 81 can be performed using the nucleic acid manipulation techniques discussed above and in the Examples.

As discussed above, SEQ ID NO:3 shows such a bacterialyzed 'o nucleotide sequence encoding MBP+X2Ser81, and further comprising an additional 18 nucleotide sequence at the 3' end (immediately preceding the termination codon, nucleotides 592-609 of SEQ ID NO:3) that encodes six histidine residues at the carboxy terminus of the encoded polypeptide (such a multiple histidine addition of at least four residues being referred to as a 25 histidine tag). This histidine tag is not found in the native MBP+X2Cys81/hum. protein, and has been added to facilitate purification of the polypeptide product of the expression of this MBP+X2Ser81/bact. gene.

Histidine tags are groups of at least five consecutive histidine residues that dct as metal chelators and allow the use of metal chelation chromatography or the like to rapidly and efficiently purify polypeptides containing such tags from mixtures of proteins. In accordance with the invention, such a histidine tag may be added to any of the polypeptides of the invention, or a sequence encoding such a tag may be added to any of the nucleic acid molecules of the invention so as to allow the ready purification of the polypeptides of the invention.

Preferred nucleic acid molecules of the invention are isolated nucleic acid molecules that comprise a nucleotide sequence (and/or a nucleotide sequence complementary thereto) which, when expressed in a suitable host, directs the expression of the MBP and/or PLP polypeptides of the invention.

The protein-encoding nucleic acid molecules of the invention can be inserted into an appropriate expression vector, a vector that contains the necessary elements for the transcription and translation of the inserted protein-encoding sequence, and then used to produce MBP and/or PLP polypeptides. A variety of host vector systems may be utilized to express the protein encoding sequence. These include, but are not limited to, mammalian cell systems infected with a virus such as vaccinia virus, adenovirus, a retrovirus, etc.; mammalian cell systems 15 transfected with plasmids; insect cell systems infected with a virus such as baculovirus; microorganisms such as yeast containing yeast expression vectors, or bacteria transformed with bacteriophage DNA, plasmid DNA, cosmid DNA, or the like.

Useful expression vectors for bacterial use can comprise a 20 selectable marker and bacterial origin of replication derived from commercially available plasmids including those comprising genetic elements of the well-known cloning vector pBR322 (American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, United States of America; ATCC Accession No. 37017). These pBR322 "backbone sections," or functionally equivalent sequences, are combined with an appropriate promoter and the structural gene to be expressed.

Preferred bacterial expression vectors include, but are not limited to, the phage T7 promoter plasmids pET14b, and pET22b (Novagen, Madison, WI). These vectors are preferably expressed in E. coli BL21(DE3) (Novagen, Madison, WI). This strain is lysogenic for a recombinant bacteriophage DE3 lysogen, which contains the gene for T7 polymerase behind the E. coli promoter (Studier et al., 1990). Other preferred bacterial expression vectors are Trc vectors including the pET Trc vector (SEQ ID NO:21) the pTrc 99A vector (Pharmacia) and the pSE vectors (Invitrogen, San Diego, CA).

Other promoters commonly used in recombinant microbial expression vectors include, but are not limited to, the lactose promoter system (Chang, et al., 1978), the tryptophan (trp) promoter (Goeddel, et al., 1980) and the tac promoter, or a fusion between the tac and trp promoters referred to as the trc promoter (see Sambrook, et al., 1989, and Maniatis, et al., 1982, particualrly page 412). Particularly preferred promoters are bacteriophage promoters, the T7 promoter discussed above, that can be used in conjunction with the expression of the corresponding bacteriophage RNA polymerase, T7 RNA polymerase, in the host cell.

Recombinant MBP and PLP polypeptides may also be expressed in fungal hosts, preferably yeast of the genus Saccharomyces such as S. cerevisiae. Fungi of other genera such as Aspergillus, Pichia or Kluyveromyces may also be employed. Fungal vectors will generally contain an origin of replication from the 2 pm yeast 15 plasmid or another autonomously replicating sequence (ARS), a promoter, DNA encoding the MBP and/or PLP polypeptide, sequences directing polyadenylation and transcription termination, and a selectable marker gene. Preferably, fungal vectors will include origins of replication and selectable markers permitting transformation of both E. coli and fungi.

Suitable promoter systems in fungi include the promoters for metallothionein, 3-phosphoglycerate kinase, or other glycolytic enzymes such as enolase, hexokinase, pyruvate kinase, and glucokinase, as well as the glucose-repressible alcohol dehydrogenase promoter (ADH2), the constitutive promoter from the alcohol dehydrogenase gene, ADH1, and others. See, for example, Schena, et al. 1991. Secretion signals, such as those directing the secretion of yeast alpha-factor or yeast invertase, can be incorporated into the fungal vector to promote secretion of the MBP and/or PLP polypeptide into the fungal growth medium. See Moir, et al., 1991.

Preferred fungal expression vectors can be constructed using DNA sequences from pBR322 for selection and replication in bacteria, and fungal DNA sequences, including the ADH1 promoter and the alcohol dehydrogenase ADH1 termination sequence, as found in vector pAAH5 (Ammerer, 1983).

Various mammalian or insect cell culture systems can be employed to express the recombinant MBP and/or PLP polypeptides of the invention. Suitable baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow, et al., 1988. Examples of suitable manmalian host cell lines include the COS cell of monkey kidney origin, mouse C127 mammary epithelial cells, mouse BALB/c-3T3 cells, mouse MOP8 cells, Chinese hamster ovary cells (CHO), human 293T cells, HeLa, myeloma, and baby hamster kidney (BHK) cells. Mammalian expression vectors may comprise non-transcribed elements such as an origin of replication, a suitable promoter and an enhancer linked to the MBP and/or PLP encoding sequence to be expressed, and other 5' or 3' flanking sequences such as ribosome binding sites, polyadenylation sequences, splice donor and acceptor sites, and transcriptional termination sequences.

The transcriptional and translational control sequences in mammalian expression vector systems to be used in transforming 15 vertebrate cells may be provided by viral sources. For example, commonly used promoters and enhancers are derived from Polyoma virus, Adenovirus, Simian Virus 40 (SV40), vaccinia, and human cytomegalovirus (CMV), including the cytomegalovirus immediateearly gene 1 promoter and enhancer.

20 Particularly preferred eukaryotic vectors for the expression of recombinant MBP and/or PLP polypeptides are pAPEX-1 (SEQ ID NO:11 and, more preferably, pAPEX-3p, SEQ ID NO:12. The vector pAPEX-1 is a derivative of the vector pcDNAI/Amp (Invitrogen) which was modified to increase protein expression levels. First, the 3'-untranslated SV40 small-t antigen intron was removed by deletion of a 601 base pair XbaI/HpaI fragment since this intron is susceptible to aberrant splicing into upstream coding regions (Evans and Scarpulla, 1989; Huang and Gorman, 1990). Second, a chimeric adenovirus-immunoglobulin hybrid intron was introduced into the 5'-untranslated region by replacing a 484 base pair NdeI- NotI fragment with a corresponding 845 base pair NdeI-NotI fragment from the vector pRc/CMV7SB (Sato et al., 1994, J. Biol.

Chem. 269:17267 et seq). Finally, to increase plasmid DNA yields from E. coli, the resulting CMV promoter expression cassette was shuttled into the vector pGEM-4Z (Promega Corp. Madison, WI).

The vector pAPEX-3 is a derivative of the vector pDR2 (Clontech Laboratories, Inc. Palo Alto, CA) in which the EBNA gene was first removed by deletion of a 2.4 kb ClaI/AccI fragment. The RSV promoter was then replaced with the CMV promoter and the adenovirus/immunoglobulin chimeric intron by exchanging a 450 bp MluI/BamHI fragment from pDR2 with a 1.0 kb MluI/BamHI fragment from the vector pAPEX-1. For construction of pAPEX-3P, a 1.7 kb BstBI/SwaI fragment containing the HSV tk promoter and hygromycin phosphotransferase (hyg) gene was removed from pAPEX-3 and replaced with a 1.1 kb SnaBI/NheI fragment containing the early promoter and puromycin acetyltransferase (pac) gene (Morgenstern and Land, 1990, Nucleic Acids Res. 18:3587-3596) plus a 137 bp XbaI/ClaI fragment containing an polyadenylation signal from the vector pAPEX-1.

A particularly preferred host cell for the expression of recombinant MBP- and/or PLP-encoding inserts in the pAPEX vectors is the human 293 EBNA cell line (Invitrogen, San Diego, CA).

Another preferred eukaryotic vector for the expression of 15 recombinant MBPs and/or PLPs is pcDNAI/Amp (Invitrogen Corporation, San Diego, California) The pcDNAI/Amp expression vector contains the human cytomegalovirus immediate-early gene I S: promoter and enhancer elements, the Simian Virus 40 consensus intron donor and acceptor splice sequences, and the 20 consensus polyadenylation signal. This vector also contains an SV40 origin of replication that allows for episomal amplification in cells COS cells, MOP8 cells, etc.) transformed with large T antigen, and an ampicillin resistance gene for propagation and selection in bacterial hosts.

III. Preparation of the PolvDetides of the Invention Purified recombinant MBPs and PLPs are prepared by culturing suitable host/vector systems (preferably bacterial systems) to express the recombinant MBP and/or PLP translation products of the nucleic acid molecules of the present invention, which are then purified from the culture media or cell extracts of the host system, the bacteria, insect cells, fungal, or mammalian cells. The invention thus provides a method for producing MBP and PLP polypeptides comprising growing a recombinant host containing a nucleic acid molecule of the invention, such that the nucleic acid molecule is expressed by the host, and isolating the expressed polypeptide.

Fermentation of cells that express recombinant MBP and/or PLP proteins containing one or more histidine tag sequences (a sequence comprising a stretch of at least 5 histidine residues) as a secreted product greatly simplifies purification. Such a histidine tag sequence enables binding under specific conditions to metals such as nickel, and thereby to nickel (or other metal) columns for purification.

In general terms, the purification is performed using a suitable set of concentration and fractionation chromatography) steps. For purification of MBP polypeptides, a particularly preferred purification step involves acid extraction, as described in the examples, below, under the heading "Purification and characterization of MBP Polypeptides".

The purified MBP and PLP polypeptides of the invention, however prepared, will in general be characterized by the presence of some impurities. These impurities may include proteins, carbohydrates, lipids, or other molecules in amounts and of a 15 character which depend on the production and purification processes used. These components will ordinarily be of viral, prokaryotic, eukaryotic, or synthetic origin, and preferably are non-pyrogenic and present in innocuous contaminant quantities, on the order of less than about 1% by weight.

20 IV. Clinical ADnlications As discussed above, the MBP and PLP polypeptides encoded by the MBP and/or PLP nucleic acid molecules of the invention can be used in the diagnosis, clinical assessment, and treatment of MS, and for the assessment of the potential responsiveness of MS patients to therapeutic treatment involving the administration of "the PLP polypeptides. Procedures for such diagnosis and assessment involve an assay entailing the incubation of replicate cultures of T cells in the presence and absence of one or more of the MBP and PLP polypeptides discussed herein, and the detection of T cell activation and/or T cell apoptosis (referred to in this specification and in the claims as a "T cell response") resulting from incubation in the presence, but not the absence, of the one or more polypeptides.

More specifically, such an assay preferably comprises isolating and partially purifying T cells from a patient, combining the isolated T cells with a PLP and/or MBP polypeptide such as a polypeptide selected from the group consisting of the polypeptide of SEQ ID NO:1, the polypeptide of SEQ ID NO:1 with cysteine 81 replaced with any other standard amino acid, the polypeptide of SEQ ID NO:1 with cysteine 81 replaced with an uncharged amino acid having a molecular weight of less than about 150, and the polypeptide of SEQ ID NO:1 with cysteine 81 replaced with serine; and/or with the polypeptide of SEQ ID NO:23, the polypeptide of SEQ ID NO:24, the polypeptide of SEQ ID NO:26, the polypeptide of SEQ ID NO:27, the polypeptide of SEQ ID NO:28, or one of the other preferred MBP or PLP polypeptides described above, and measuring the level of a T cell response induced by the polypeptide. Methods for measuring T cell responses are described below under the subheading "Detection of T Cell Responses." In accordance with the present invention, such an assay may be provided as a kit for the detection of MBP or PLP reactive T cells comprising an isolated PLP or MBP 21.5 polypeptide in close confinement and/or proximity with an agent for use in the S 15 detection of a T cell response, such as any of the agents described below under the subheading "Detection of T Cell Responses". In a preferred embodiment of such a kit, the kit further comprises a label indicating that the kit is for use in the diagnosis and/or clinical assessment of multiple sclerosis.

A finding of T cells in a patient's CSF that exhibit a T cell response when incubated with PLP or MBP 21.5 polypeptides in this fashion is taken as an indication that the patient is suffering from MS. A finding of such MBP or PLP responsive T cells in CSF and/or blood of an MS patient is an indication that the patient is an appropriate candidate for treatment with MBP and/or PLP polypeptides. The levels of such T cells in the blood or CSF may be monitored as an indication of disease progression and response to treatment.

The number of such reactive T cells in a patient's blood and/or CSF (the "precursor frequency" or "reactive T cell index") can be monitored over time, and can be used as an indicator of the clinical progression of the disease, with increasing numbers indicating exacerbation and decreasing numbers indicating improvement. The reactive T cell index also serves as a predictor of when a therapeutic treatment would be appropriate, a sudden increase in the index would suggest that therapeutic intervention should be commenced or intensified. If the index is monitored during a course of treatment, whether or not the treatment involves the administration of MBP and/or PLP polypeptides, a significant decline in the reactive T cell index is an indication of therapeutic success, while a significant rise in the index indicates therapeutic failure, and suggests that the therapeutic regimen should be adjusted.

The invention thus provides an assay comprising isolating and partially purifying T cells from a patient, combining the isolated T cells with an immunoreactive MBP 21.5 polypeptide or PLP polypeptide (the PLP polypeptide comprising a PLP mutein amino acid sequence having the amino acid sequence of a native PLP polypeptide minus at least one or two hydrophobic peptide regions, preferably minus at least three hydrophobic peptide regions) and measuring the level of a T cell response induced by the polypeptide.

The invention further provides a kit for the detection of MBP 15 reactive T cells comprising an immunoreactive PLP polypeptide (comprising a PLP mutein amino acid sequence having the amino acid sequence of a native PLP polypeptide minus at least one or two hydrophobic peptide regions, preferably minus at least three hydrophobic peptide regions) in close confinement and/or proximity 20 with an agent for use in the detection of a T cell response. In accordance with the invention, such a kit may further comprise a label indicating that the kit is for use in the clinical assessment of multiple sclerosis.

A. Detection of T Cell Responses 25 Assays of T cell activation and of apoptosis are well known to those of skill in the art. Detailed discussions of and protocols for such assays can be found in numerous publications including, Wier, 1978; Klaus, 1987; Voskuhl et al., 1993; and Ormerod, 1994. Such assays measure alterations of certain key indicators of T cell activation, and/or apoptosis.

For T cell activation, these indicators generally include reagents for the detection of T cell proliferation, cytokine release, and expression of cytokine receptors and other activation-associated cell surface markers. For apoptosis, these indicators generally include dyes, stains, and other reagents for the observation/detection of nuclear shrinkage and/or cell death; metabolic inhibitors capable of inhibiting apoptotic cell death; stains, enzymes, labeled nucleic acid precursors, and other indicators of DNA degradation.

All assays of T cell activation and of apoptosis involve the use of cell culture (tissue culture) supplies, typically including culture vessels such as multi-well plates, dishes, and flasks, as well as test tubes and centrifuge tubes, liquid measuring devices such as pipettes, droppers, and dropper bottles, cell culture media, and buffer solutions. Many of these assays also involve a readout that involves a labeled antibody, often a secondary antibody against a primary, unlabeled antibody that specifically binds to the indicator being measured. In addition, these assays involve numerous other reagents and instruments, as discussed below and in the Examples. As used in this specification, and in the claims, an "agent for use in the detection of a T cell response" is any of the reagents (including antibodies), supplies, media, and instruments discussed herein that can be used for such 15 detection.

Unless reagents specific for T cells are used as indicators, ;the measurements of T cell responses will generally involve the labeling and/or further purification of T cells from preparations of white blood cells, which are typically obtained partially purified) by centrifugation and/or filtration of the body fluid cerebrospinal fluid or decoagulated blood) in which they are isolated. As used hereinafter, and in the claims, "isolated T cells" are T cells that have been removed from the Sbody of a living subject, but not necessarily further purified by centrifugation to remove white blood cells from a body fluid or by separation of T cells from other blood cells). The isolation of T cells thus involves lancets, needles, syringes, evacuated blood collection tubes, and other blood and/or CSF collection supplies, and may further involve the use of filtration and centrifugation supplies.

Methods for specifically labeling T cells typically involve conventional immunohistochemical and/or FACS techniques involving antibodies to T cell specific markers, which are generally T cell receptors, subunits thereof, and associated molecules such as CD3.

Such antibodies are commercially available from numerous sources.

Methods for at least partially purifying T cells include cell sorting by FACS using the above-mentioned antibodies, various affinity purification methods, including passage over glass beads and/or nylon wool, the use of antibodies to markers for other white blood cell types to remove cells other than T cells from mixtures of white blood cells, and differential centrifugation, centrifugal elutriation and/or density gradient centrifugation using density gradient media such as polysucrose (FICOLL), albumin, colloidal silica, and the like.

Detection of T cell proliferation can be accomplished by labeling or partially purifying T cells as discussed above and applying methods used to detect cell proliferation generally One such method involves labeling newly synthesized DNA by culturing the T cells in the presence of detectable nucleic acid precursor molecules that can be incorporated into nascent DNA by living cells. Such precursors include 3 H thymidine and other radioactively labeled precursors, and BrdU and other conveniently :detectable non-radioactive precursors. When radioactively labeled 15 precursors are used, unincorporated precursors are washed away and Slevels of incorporated precursors are measured by autoradiography, Sscintillation counting, or other conventional methods of radiation quantification.

2 When BrdU and the like are used, unincorporated precursors are washed away and antibodies or other reagents capable of specifically binding to the precursor are used to detect precursor that has been incorporated into nuclear DNA. Additionally, reagents that label metabolically active cells can be used to follow increases in cell number. Such reagents include MTT, XTT, 25 MTS, and WST-1, which are cleaved by mitochondrial enzymes to Syield products that can be readily detected and measured "spectrophotometrically, with the level of cleavage products thus measured being proportional to the number of metabolically active cells in the sample being tested. Such reagents are commercially available from many sources.

Numerous cell surface markers of T cell activation are known in the art, and are generally detected by antibodies (which are commercially available from numerous sources) using conventional immunohistochemical and/or FACS techniques. These markers include CD25 (the IL-2 receptor), CD26, CD30, CD69, and CD71 (the transferrin receptor).

T cell activation can also be detected by measuring cytokine release into culture medium (see, for example, Correale et al.

1995). Inactive T cells do not release cytokines, while at least some active T cells release IL-2, IL-4, IL-5, IL-6, IL-10, IL-11, IL-12, IL-13, IL-14, gamma interferon, TNF alpha, and the TNFrelated cytokine known as the FAS ligand. In addition, T cell activation may be detected by T cell surface expression of activation-specific markers including CD95 (the FAS receptor).

Antibodies for detecting each of these cytokines and markers are well known in the art and are commercially available; assays using such antibodies to measure cytokines, in culture medium, are also well known in the art and are items of commerce.

A particularly sensitive assay for T cell activation is the recently developed enzyme-linked inmunospot (ELISPOT) assay, which typically detects cytokine release by single T cells as spots on an antibody coated substrate upon which the T cells are cultured.

Such assays are described in Taguchi et al., 1990, and Sun et al., 15 1991. Preferably the ELISPOT assay is used to detect the secretion of gamma interferon.

Materials and methods for determining whether cellular morbidity is a result of an ongoing process of apoptosis are also well known to workers in the art. In addition to conventional 20 histochemical stains, which allow the detection of apoptosisassociated ultrastructural changes, apoptosis detection o procedures, including assays and staining techniques, have been in use in the art for many years. These procedures typically determine if cell death depends upon active metabolism protein synthesis) or whether dying cells exhibit DNA degradation (fragmentation).

The former type of procedure involves growing replicate cultures containing dying cells in the presence or absence of a metabolic inhibitor, a protein synthesis inhibitor such as cycloheximide, an RNA synthesis inhibitor such as actinomycin

D,

or an immune-specific inhibitor such as cyclosporin, and determining whether such inhibition delays cell death; if it does then apoptosis is almost certainly involved. See, for example, Dhein et al., 1995, in which cell death is detected as the ability of the dye propidium iodide to enter the cell.

Procedures for the detection of DNA fragmentation may involve the isolation and size separation of DNA, typically by phenol extraction and gel electrophoresis. A newer technique involves the use of the enzyme terminal deoxynucleotidyl transferase ("TdT" or "terminal transferase'), an appropriate buffer cacodylate buffer containing a cobalt salt and a reducing agent such as DTT, DTE, or BME) and a labeled deoxynucleotide triphosphate (dNTP) or a labeled derivative or analog thereof BrdUTP, a biotynilated dNTP, a digoxigen labeled dNTP, or a radiolabeled dNTP, collectively referred to as a "labeled XTP').

TdT incorporates labeled XTPs onto free ends of DNA molecules. Since DNA degradation associated with apoptosis involves the generation of a great many free ends compared with a much smaller number in healthy cells, the incorporation of high levels of labeled XTPs relative to healthy cells indicates ongoing apoptosis. TdT methods for detecting apoptosis thus involve the detection of the incorporated labeled XTP (usually following washing of the cells to remove unincorporated labeled XTPs) typically using conventional techniques such as autoradiography or Simmunohistochemistry using antibodies against the labeled XTP either tagged, fluorescently or enzymatically tagged antibodies, or in conjunction with tagged secondary antibodies) S* A commercial kit for the practice of this method is available from ONCOR, Inc., Gaithersburg, MD, as the "APOPTAG" kit.

Another recently developed technique involves an ELISA using an anti-histone capture antibody and an anti-DNA detection antibody. This assay depends on the conventional separation of intact chromatin from fragmented chromatin, with the levels of 25 fragmented chromatin so separated being measured by the above mentioned ELISA. A commercial kit for the practice of this method is available from Boehringer Mannheim Corporation, Indianapolis, IN, as the "cell death detection" kit.

B. Treatment With regard to treatment using the MBP polypeptides of the invention, it should be noted that the MBP 21.5 polypeptides of the invention, MBP+X2Ser81) have various advantages in comparison to non-human-derived MBP antigens used in prior approaches for obtaining antigen tolerization in MS patients.

Such advantages include the inclusion of the full spectrum of MBP immunodominant regions, and the consequent ability of these polypeptides to induce tolerance in T cells reactive with any such MBP immunodominant regions.

Intra-antigenic and inter-antigenic spread of autoreactivity are related phenomena associated with autoimmune diseases in which additional epitopes within an antigen, or additional antigens within a target tissue, become targeted by autoreactive T cells during disease progression. Such antigen spreading has been observed during the course of the inflammatory autoimmune process in the murine models of experimental allergic encephalomyelitis (EAE) and insulin-dependent diabetes (Lehmann et al. 1992; McCarron et al. 1990; Kaufman et al. 1993; Tisch et al. 1993).

These findings of antigen spreading, as well as the demonstration of variability in the immunodominant epitopes recognized by MBP reactive activated T cells in MS patients, indicate that an effective MBP-specific therapy will need to target a heterogeneous population of MBP-specific autoreactive T 15 cells. Therefore, in order for parenteral MBP administration to be maximally effective in the treatment of MS, the complete repertoire of its immunodominant epitopes must be presented to T lymphocytes.

In accordance with certain aspects of the present invention, 20 a method for treating a patient suffering from multiple sclerosis comprises administering to the patient an MBP 21.5 polypeptide.

Preferably the MBP 21.5 polypeptide comprises the complete repertoire of MBP immunodominant epitopes. The MBP 21.5 polypeptide is administered in an amount sufficient to achieve a concentration of the polypeptide in a relevant compartment body fluid or tissue compartment) of the patient's body, the patient's blood, cerebrospinal fluid, lymph, reticuloendothelial system, liver, lymph nodes, spleen, thymus, and the like, sufficient to induce apoptosis of MBP reactive T cells.

Preferably the polypeptide is administered to the patient at least two times at an interval of at least twelve hours and not more than four days.

In accordance with certain aspects of the present invention, a method for treating a patient suffering from multiple sclerosis comprises administering to the patient a PLP polypeptide APLP3, APLP4, MP3, MP4, PM4, MMOGP4). Preferably the PLP polypeptide comprises the complete repertoire of known human PLP immunodominant epitopes. The PLP polypeptide is administered in an amount sufficient to achieve a concentration of the polypeptide in a relevant compartment body fluid or tissue compartment) of the patient's body, the patient's blood, cerebrospinal fluid, lymph, reticuloendothelial system, liver, lymph nodes, spleen, thymus, and the like, sufficient to induce apoptosis of PLP reactive T cells. Preferably the polypeptide is administered repeatedly to the patient at least two times at an interval of at least twelve hours and not more than four days between administrations. The polypeptide is preferably administered without the concomitant administration of an adjuvant, so that tolerance, rather than exacerbation of disease, will result.

In accordance with the present invention, the concentration of the MBP and/or PLP polypeptide in the patient's body fluid or tissue compartment that is sufficient to induce apoptosis of MBP and/or PLP reactive T cells is determined using the materials, 15 methods, and assays described above under "Clinical Applications" and "Detection of T Cell Responses". A concentration is considered sufficient to induce apoptosis of MBP or PLP reactive T cells when a substantial decrease in the number of T cells from peripheral blood exhibiting responses to MBP or PLP epitopes (the S 20 "precursor frequency" or "reactive T cell index") is seen following treatment (compared to T cells from blood samples taken before treatment) in response to the polypeptide, as compared to control assays, which are performed using irrelevant polypeptides albumin). An at least 25% reduction in reactive T cell index will, in general, comprise a "substantial reduction".

Smaller reductions are also considered "substantial" if they represent a statistically significant reduction, a reduction that, when analyzed by a standard statistical test, such as the student's T test, will give a probability value, p, less than or equal to 0.05 and, preferably, less than or equal to 0.015.

Alternatively, the concentration of the polypeptide in the patient's blood and/or cerebrospinal fluid that is sufficient to induce apoptosis of MBP or PLP reactive T cells may be determined by routine in vivo experimentation as the amount required to stabilize the clinical course or improve the clinical symptoms of EAE or MS.

In accordance with the invention, PLP and/or MPB 21.5 polypeptides may also be used to induce tolerization of PLP and/or MBP reactive T cells in an MS patient by administration on a schedule designed to induce tolerization without inducing apoptosis by inducing T cell anergy). Such schedules are typically used to tolerize patients to allergens, and generally involve administration of smaller doses (typically ranging from micrograms to hundreds of micrograms) of the tolerizing agent (in this case the MBP 21.5 preparation) on a weekly, biweekly, or monthly basis.

The amount of administered polypeptide that is sufficient to achieve a desired concentration of the polypeptide in a body fluid or tissue compartment of the patient can be readily determined from routine human and animal study data using standard pharmacokinetic calculations well known to those of skill in the art. Initial in vivo studies are done in mice that have been treated to induce EAE. Preferably the dose of polypeptide is "i 15 subsequently determined in a primate, a human patient or a marmoset (a monkey that is known to have MBP reactive T cells in its peripheral blood). Preferably the dosage is adjusted to achieve a clinical improvement (preferably in animals) or a substantial reduction in the number of T cells from peripheral 20 blood exhibiting responses to MBP or PLP epitopes.

The dose will also vary depending on the manner of administration, the particular symptoms of the patient being treated, the overall health, condition, size, and age of the patient, and the judgment of the prescribing physician.

25 Subject to the judgment of the physician, a typical therapeutic treatment includes a series of doses, which will usually be administered concurrently with the monitoring of clinical severity of disease and reactive T cell index.

Administration of the polypeptides will generally be performed by an intravascular route, via intravenous infusion by injection. Other routes of administration subcutaneous injection, intradermal injection, intramuscular injection, inhaled aerosol, oral, nasal, vaginal, rectal, and the like) may be used if desired as determined by the physician.

Formulations suitable for injection are found in Reminaton's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985). Such formulations must be sterile and nonpyrogenic, and generally will include a pharmaceutically effective carrier, such as saline, buffered phosphate buffered) saline, Hank's solution, Ringer's solution, dextrose/saline, glucose solutions, and the like. The formulations may contain pharmaceutically acceptable auxiliary substances as required, such as, tonicity adjusting agents, wetting agents, bactericidal agents, preservatives, stabilizers, and the like.

The formulations of the invention can be distributed as articles of manufacture comprising packaging material and the polypeptides. The packaging material will include a label which indicates that the formulation is for use in the treatment of neurologic disease and may specifically refer to multiple sclerosis.

Without intending to limit it in any manner, the present invention will be more fully described by the following examples.

EXAMPLES

15 Construction of bacterial vectors directing the expression of MBP 21.5 polveDptides and native MBP18.5 A full-length cDNA coding for the 18.5 kDa isoform of human MBP was obtained from the ATCC (#5748; ATCC, Rockville, MD).

Plasmid pHBP-1 was used as a template in a standard PCR reaction 20 using AmpliTaq (Perkin-Elmer, Norwalk, CT.) for 30 cycles with denaturation at 94 0 C for 1 min, annealing at 52 0 C for 1 min and extension at 72 0 C for 1 min. The sense oligonucleotide primer (5'-CATATGGCGT CACAGAAGAG AC-3', SEQ ID NO:13) encodes the Nterminus of hMBP18.5 (MASQKR) and contains an NdeI cloning site, whereas the antisense primer (5'-GGATCCTTAG CGTCTAGCCA TGGGTG-3', SEQ ID NO:14) encodes the C-terminal residues (PMARR) and contains a BamHI cloning site. Following an additional extension at 72 0

C

for 10 min, the resulting 526 base pair (bp) fragment was subcloned into pCRII (Invitrogen, San Diego, CA) as described by the supplier. Kanamycin-resistant E. coli DH10B (Gibco/BRL, Gaithersburg, MD) transformants were selected and the insert identified by restriction analysis and verified by dideoxy sequence analysis. The MBP coding region was subcloned into the NdeI and XhoI sites of the phage T7 promoter plasmid pET14b (Novagen, Madison, WI) and later recloned into pET22b (Novagen, Madison, WI). The resulting recombinant MBP18.5 gene contains only unmodified native codons, except for an additional 18 nucleotide sequence that encodes a histidine tag at the 3' end (immediately preceding the termination codon) that is not found in the native human MBP18.5 protein, and has been added to facilitate purification of the product of this MBP18.5 hum gene. The resulting recombinant vector (pET22b/MBP18.5hum.) was transformed into E. coli BL21(DE3) (Novagen, Madison, WI) where the DE3 lysogen contains the gene for T7 polymerase behind the E. coli promoter (Studier et al., 1990).

A synthetic recombinant gene encoding the 21.5 kDa isoform of human MBP was constructed in three rounds of overlapping PCR (Ho et al. 1989) (see Fig. 13). Each of three gene subdomains was synthesized in a 100l reaction using 5 pmole of each the appropriate pair of HPLC purified oligonucleotides and 0.5 units of Taq polymerase (Perkin-Elmer). Thirty cycles of denaturation for 1 minute at 95 0 C, annealing at 50 0 C for 1 minute and DNA 15 strand extension at 72 0 C for 1 minute were carried out. Five percent of each purified PCR fragment was then used as a template in a second round of PCR, where two subdomains were combined using flanking oligonucleotides. Purification of these DNA fragments and a third round of PCR resulted in amplification of a 648bp product.

20 The PCR product was digested with EcoRI and HindIII, subcloned into and transformed into E. coli XL-1 Blue (Stratagene, LaJolla, CA). Ampicillin-resistant transformants were selected and the desired constructions identified by restriction and sequence analysis. Restriction fragments from several independent clones were combined to remove undesired mutations that occurred during PCR cloning, and the resulting MBP+X2

C

ys 81 B a ct. gene was cloned into pET22b at the NdeI and HindIII sites.

An altered gene encoding a cysteine to serine substitution at amino acid residue 81 of the 21.5 kDa isoform of human MBP was constructed by the following steps. PCR amplification of an internal MBP fragment was carried out using pET22b/MBP21.5hum. as template along with the mutagenic antisense primer ATGTTCGACA GGCCCGGCTG GCTACG-3', SEQ ID NO:15, Ser 81 codon underlined, NspI site in italics) in combination with a sense oligonucleotide primer (5'-CAGCACCATG GACC-3', SEQ ID NO:16, NcoI site in italics). The NspI-NcoI restriction fragment in MBP+X2

C

ys 8 1/Bact. was then exchanged with the mutated fragment to create MBP+X2S er81 Bact By using the MBP18.5 hum gene as template in overlapping PCR, a version of MBP+X2Cys 81 was created with native human codons. A PCR fragment that includes human exon 2 sequence was generated from pET22b/rhMBP18.5 by utilizing sense oligonucleotide GGTGCGCCAA AGCGGGGCTC TGGCAAGGTA CCCTGGCTAA AGCCGGGCCG GAGCCCTCTG CCCTCTCATG CCCGCAGCCA GCCTGGGCTG TGCAACATGT ACAAGGACTC ACACCACCCG GCAAGAAC-3', SEQ ID NO:17, in combination with an antisense oligonucleotide (SEQ ID NO:18) that hybridizes to the T7 terminator of plasmid pET22b. A second PCR fragment was generated using the same template but with a T7 promoter oligonucleotide (SEQ ID NO:19) in combination with an antisense oligonucleotide AGGGTACCTT GCCAGAGCCC CGCTTTGGC SEQ ID that hybridized to the 5' end of exon 2. Fusion of both PCR products by amplification with T7 promoter and terminator 15 oligonucleotides in a second round of PCR completed the construction of a PCR product containing the MBP+X2Cys81/hum.

gene. A restriction fragment obtained from this PCR product was then subcloned into pET22b at the Ndel and HindIII sites and the selection of the desired clone was confirmed by sequence analysis.

20 Bacterial expression and identification of recombinant MBP For expression of recombinant MBP polypeptides, E. coli strain BL21(DE3) was transformed with the expression plasmids and ampicillin-resistant colonies selected and grown in Terrific Broth (TB) medium (Sambrook et al. 1989) to an OD 600 of 0.6. Protein expression was induced for 4 hours with ImM isopropylthiogalactoside (IPTG). Analytical characterization of recombinantly expressed MBP polypeptides was carried out by removing iml of induced cells at an OD 6 0 0 of 1.5. Cell pellets were lysed by boiling in 100il of 20 mM Tris-HCl, pH7.5 with of the lysate analyzed by 16% SDS-PAGE (Novex, San Diego, CA).

recombinantly expressed MBP polypeptides were identified by either Coomassie R-250 staining or immunoblotting with rat monoclonal antibodies specific to either the human MBP amino-terminal residues 36-50 corresponding to MBP exon 1 (MCA 408, SeroTec, Indianapolis, IN) or carboxy-terminal residues 129-138 corresponding to MBP exon 6 (MCA 70, SeroTec, Indianapolis, IN).

For fractionation of E. coli cells into soluble and insoluble fractions, cell pellets from two ml of each induced culture was collected at an OD 600 of 1.5 and resuspended in 400ml of Tris-HCl pH 8.0. To prepare a total cell lysate, the suspension was made 100mg/ml with lysozyme and ImM with phenylmethylsulfonyl fluoride, then incubated at 30 0 C for 15 minutes. This was followed by the addition of 10mM MgCl 2 and 200 mg/ml of DNase I (Sigma, St. Louis, MO) and incubation for 20 minutes at room temperature. The cell lysate was divided, one-half receiving additional Tris buffer and the other half made 0.1N HC1 and extracted at room temperature for 30 minutes. After centrifugation, the soluble supernatant was removed from the insoluble pellet and each fraction boiled for 5 minutes in SDScontaining loading dye. SDS-PAGE gels of 20% of each fraction were analyzed for recombinantly expressed MBP polypeptides as described above.

15 Purification and characterization of recombinant MBPs For purification of recombinantly expressed MBP polypeptides, 1L cultures of induced cells were harvested by centrifugation and pellets homogenized in 10 ml/g (10% w/v) of 0.1N HC1 using a TEKMAR homogenizer (The Tekmar Co., Cincinnati, OH). Cells were 20 mechanically disrupted by 3 passes (at 10,000 psi with nitrogen) through a MICROFLUIDIZER (Model M110-T, Microfluidics Corp., Newton, MA) with all manipulations performed on ice. The soluble fraction containing recombinantly expressed MBP was collected as the supernatant following centrifugation of the cell lysate at 10,000xg for 30 min at 4 0 C in a Beckman JA-10 rotor. The supernatant was filtered through a WHATMAN POLYCAP TF (0.45pm) i membrane (Whatman LabSales, Hillsboro, OR) and concentrated 5-10 fold using a PM-10 membrane in an AMICON stir cell apparatus (Amicon, Beverly, MA). Particulates were removed from the concentrated fraction by passing through a MILLEX GV (0.2mm) syringe filter (Millipore Corporation, Bedford, MA) and the filtered sample loaded onto a VYDAC C4 reverse phase column length, VYDAC, Hesperia, CA) at 4.1 ml/minute. Proteins were eluted using a linear 25-40% acetonitrile/0.1% trifluoroacetic acid (TFA) gradient for 30 minutes, then lyophilized.

For purification of recombinantly expressed MBP polypeptides, the lyophilized material was resuspended in binding buffer (8M urea, 10mM beta-mercaptoethanol, 0.1M NaH2PO 4 0.01 M Tris-HC1, pH and bound to Ni-NTA resin according to the manufacturers instructions (Qiagen Inc., Chadsworth, CA). The column was washed twice with the same binding buffer, and contaminating E. coli proteins were removed with binding buffer that was adjusted to pH 6.3 (wash rhMBP was eluted with a step gradient that included binding buffer at pH 5.9 (elution 1) and pH 4.5 (elution and finally 6M guanidine hydrochloride, 0.2M acetic acid (elution 3).

All fractions and a portion of the column resin were analyzed by 16% SDS-PAGE in the presence of reductant.

MBP polypeptides were quantified using a rapid analytical reversed-phase HPLC assay. A 4.6x50mm C18 column (C18 HYTACH, Glycotech, Branford, CT) was used and assays were performed at 0 C in a manner similar to the HPLC described by Kalghatgi and 15 Horvath, 1987. Recombinantly expressed MBP polypeptides were extracted from disrupted cells with 0.1N HC1 and fractionated on the C18 HYTACH reversed-phase column using a linear 10-30% S* acetonitrile/0.1% triflouroacetic acid (TFA) gradient over 1 minute. In the linear assay range, measurement of the MBP 20 polypeptide peak height is directly proportional to the quantity of MBP polypeptide. The concentration of an MBP+X2Cys81 standard was determined by amino acid composition. The molecular weight for MBP+X2Cys81 was determined by mass spectrophotometry to be 22,188 daltons. N-terminal sequencing of the purified MBP+X2Cy s 8 1 protein gave the amino acid sequence Ala Ser Gln Lys Arg Pro Ser Gln Arg His Gly Ser Lys Tyr Leu Ala Thr Ala Ser Thr Met Asp His Ala Arg, corresponding to the first 25 amino acids predicted from the nucleotide sequence of MBP+X2Cys 81 /hum. (SEQ ID NO:1).

Establishment of MBP18.5- and exon 2-secific T cell lines and Proliferation assays Native human MBP was prepared as described previously (Voskuhl et al. 1993a). MBP exon 2-encoded synthetic peptide was purchased from Synthecell Corp. (Rockville, MD) and was greater than 95% pure by HPLC analysis. Peripheral blood lymphocytes were isolated by leukapheresis and separation on FICOLL gradients.

Cells were then cryopreserved in RPMI 1640 (Whittaker Bioproducts, Walkersville, MD) with 10% DMSO and stored in liquid nitrogen until use. T cell lines were generated using a limiting cell concentration, as described previously (Voskuhl et al. 1993a).

2A2 and 3H5 are human T cell lines that were obtained from normal individuals. 1H7, 1G1 and 3A11 are human T cell lines obtained from MS patients and are specific for the exon 2-encoded region of MBP. T cell lines were rested for 10 days after the last restimulation, then used as responders at a concentration of 2x10 cells/ml. Autologous irradiated (3000 rad) peripheral blood lymphocytes (PBL) were used as stimulators at a concentration of 1x10 6 /ml. Fifty microliters of both responder and stimulator cells were mixed in each well of a round bottomed 96-well microtiter plate (Nunc, Roskilde, Denmark) with 1001 of the particular MBP antigen or medium alone. For the recombinant MBPs, lyophilized preparations from the reversed-phase HPLC purification were resuspended in PBS at a concentration of 8-10 mg/ml then diluted 15 with medium immediately prior to use. Assays were done in triplicate and carried out in Iscove's Modified Dulbecco's Medium (IMDM, Gibco, Grand Island, NY) containing 2mM L-glutamine, 100U/ml penicillin and 100mg/ml streptomycin (all Whittaker Bioproducts, Walkersville, MD) supplemented with 10% pooled human *20 serum (obtained from 4-7 normal AB NIH blood bank donors, heat inactivated and sterile filtered before use). Cultures were incubated for 72h at 37 0 C in 5% C0 2 During the last 18h of culture, cells were pulsed with ImCi/well 3 [H]-thymidine, harvested onto glass fiber filters, and thymidine incorporation measured by scintillation counting.

Construction and bacterial expression of recombinant human MBP 'genes A synthetic gene was constructed to encode the fetal isoform of adult human MBP (21.5 kDa isoform, MBP+X2Cys81) (see Figs. 1 and While others have typically constructed synthetic genes by ligating numerous oligonucleotides that encompass the complete sense and antisense strands of a particular coding region (Jayarman et al. 1991; Williams et al. 1988; Hernan et al. 1992; Wosnick et al. 1987), only six oligonucleotides (SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID were utilized here to synthesize the 644bp gene encoding recombinant human MBP+X2Cy s81 The HPLC-purified oligonucleotides ranged in size from 110 to 130 bp, with 20-25 bp overlapping regions designed for hybridization of sense and antisense strands during 3 rounds of PCR (Fig. 13). For optimal bacterial expression of the recombinant MBP gene, many of the human codons were converted to preferred bacterial codons based on codon bias tables created for all known (Wada et al. 1992) or highly expressed (Grosjean and Fiers, 1982) E. coli genes. Significant codon changes were employed, especially for those encoding arginine, proline and lysine, which comprise 26% of the amino acid residues in MBP21.5.

Several independent clones were sequenced and each had multiple nucleotide substitutions or deletions attributed to either rejection of the synthetic DNA by the bacterial cloning strain or PCR-based errors. All of these errors were corrected except for cytosine to thymine substitutions that were identified 15 at nucleotide positions 462, 528 and 532. These changes were not S*.i corrected, as they conserve the encoded MBP+X2Cys81 amino acid sequence and are not deleterious to the bacterial codon preference (Wada et al. 1992). For recombinant expression of the adult human brain derived (18.5 kDa) isoform of MBP, a cDNA clone with native 20 human codons encoding this isoform (MBP18.5/hum., encoding MBP18.5) was modified by PCR to include the appropriate restriction sites for cloning into the same expression vector.

The expression of recombinant MBP polypeptides in bacteria was initially characterized using small-scale shake flask cultures grown in rich TB medium. Following induction of 10ml cultures with IPTG, both recombinant forms of MBP were expressed to high levels in BL21(DE3) cells. MBP18.5 and MBP+X2Cys81 were the major proteins identified by Coomassie dye staining of total bacterial proteins separated by SDS-PAGE (Fig. 14, "Coom") and were recognized specifically by antibodies directed to either the carboxy- (Fig. 14, "C-term Ab") or amino-(Fig. 14, "N-term Ab") terminus of human MBP. Two smaller MBP-immunoreactive polypeptides (between 6-16 kDa) could be identified in the MBP+X2Cys81 lysate, but only by immunoblot analysis with the Nterminal antibody, indicating that premature termination of translation near the carboxy terminus, rather that proteolysis, was responsible for their presence. This was confirmed in pulsechase labeling experiments which showed that the smaller polypeptides were stable during the course of the experiment.

57 Although inclusion bodies were not evident in shake flask experiments, recombinant MBPs were observed in the insoluble fraction of lysed bacterial cells (Fig. 15, "Tris"). Previously, a homogeneous protein purified from bovine spinal cord was shown to have encephalitogenic activity and be soluble at pH 2-3 (Einstein et al. 1962). This encephalitogenic protein was subsequently identified as MBP, and consists almost exclusively of the 18.5 kDa isoform (Deibler et. al. 1972). Since MBP is acid soluble, we reasoned that it might be possible to streamline purification by direct acid extraction of bacterial lysates. We therefore attempted to solublize rhMBPs under acidic conditions.

Treatment of total cellular lysates with 0.1N HC1 (Fig. "Acid") released most of the rhMBPs into the soluble fraction The inability to extract all of the rhMBPs from the insoluble 15 pellet fraction may be due to incomplete lysis of cells during this particular sample preparation.

Purification and characterization of MBP PolvDetides For purification of recombinantly expressed MBP polypeptides, cells from 1L shake flask cultures were mechanically disrupted in 20 the acidic conditions described above. Following simultaneous cell disruption and acid extraction, all of the recombinantly expressed MBP polypeptides were found in the soluble fraction (Fig. 16, The soluble acid fraction was applied directly onto a VYDAC C4 reversed-phase column and rhMBPs eluted as a 25 single, sharp peak at 17-20 min with a 25-40% acetonitrile/0.1% TFA gradient (Fig. 17). N-terminal sequencing of the peak fraction verified the correct amino-terminal sequence for the MBP polypeptides, as described above. The predicted molecular weight of MBP+X2Cys 8 1 with an additional carboxy-terminal histidine tag agreed with the mass of 22,185 daltons obtained by mass spectrophotometric analysis of the peak fraction. Coomassie stained gels of the pooled peak fractions identified the recombinant MBP polypeptides, but also showed a heterogeneous mix of truncated MBP fragments apparently produced by limited acid hydrolysis of full-length MBP polypeptides (Fig. 18, "load"). By exploiting the C-terminal histidine tag, full-length MBP material was obtained by metal chelation chromatography using denaturing conditions and acidic pH elutions (Fig. 18). The majority of the full-length MBP polypeptides eluted with either elution 2 (8M urea, 10mM beta-mercaptoethanol, 0.1M NaH 2

PO

4 0.01 M Tris, pH or elution 3 (6M guanidine hydrochloride, 0.2M acetic acid), although contaminating E. coli proteins were observed in the eluate from the less stringent second elution (Fig. 18, "elution To quantitatively compare the expression of the MBP+X2

C

ys8l/bact. to that of MBP18.5/hum., soluble acid lysates were prepared from three sets of one liter bacterial cultures and analyzed using the rapid analytical reversed-phase HPLC assay described above. Using a standard amount of MBP+X2Cy s 8 1 as determined by amino acid analysis, and relating the peak height to protein concentration, we observed that 1.5 to 2.0-fold more MBP 21.5 polypeptide was expressed from the synthetic MBP+X2

C

ys81/bact. gene compared to the expression from the 15 MBP 1 8.

5 /hum gene. The average expression level of recombinant protein from MBP genes with bacterial codons was 50 mg/L compared to 30 mg/L from genes with human codons. This reflects bacterial codon bias and not an effect of exon 2-related sequences, as a strain that expressed the MBP+X2Cy s8 1 hum gene produced a similar 20 amount of MBP polypeptide as the strain expressing the MBP18.5/hum. (see Fig. 19 and Table Under physiological conditions, a fraction of MBP+X2Cy s 8 1 but not MBP18.5, formed an apparent dimeric molecule that was identified by Coomassie staining and Western blotting of 25 nonreduced samples on SDS-PAGE gels. Dimers are not observed under similar conditions with reduced samples. MBP dimers also have been observed after reversed-phase HPLC fractionation of myelin proteins from bovine CNS (van Noort et al. 1994).

Such dimers are particularly undesirable in a protein preparation that is to be formulated for pharmaceutical administration, as, for such use, such proteins are generally preferred as single molecular entities with defined characteristics, including a unique molecular weight. It was thus important to devise a means by which single, monomeric forms of MBP 21.5 polypeptides could be conveniently and efficiently prepared. In order to test whether dimer formation of MBP+X2Cy s 8 1 was mediated through the single cysteine residue at position 81 (Cys 81 of exon 2, the cysteine (Cys 8 1 was converted to a serine (Ser 81 by site-directed mutagenesis.

Reversed-phase HPLC showed that MBP+X2S e r 8 1 was expressed in bacteria at a level similar to MBP+X2

C

y s 8 1 (on average 50 mg/L, see Fig. 19) and remained monomeric in physiological solution, without reductant. As an alternative method of testing such an amino acid substitution for effective elimination of dimer formation, X2MPB peptides may be prepared and tested for dimer formation in physiological solution, without reductant.

MBP18.5- and MBP exon 2-snecific T cells recognize Recombinant Human (rh) MBPs To assess the biological activity of recombinant forms of MBP, we tested the in vitro proliferation response of human MBPspecific T cell lines when challenged with the recombinant 15 proteins. T cell lines were generated that respond to braino derived human MBP18.5 or a synthetic exon 2 peptide (amino acid i. residues 60-85 of MBP21.5, SEQ ID NO:1). Two MBP18.5-specific lines, 2A2 (recognizing residues 31-50) and 3H5 (recognizing residues 87-106), were stimulated by incubation for 72 hours in 20 vitro with either MBP18.5 or the MBP+X2 polypeptides. During the final 18 hours of this incubation the cells were pulsed with 3

H-

thymidine to allow measurement of cell proliferation. As shown in Fig. 20, both T cell lines responded equally well to MBP+X2Cy s 8 1 and MBP18.5, regardless of whether purified from human brain or 25 bacteria. We also analyzed the antigen recognition of additional human T cell lines that respond to MBP epitopes that hve been described in the art. As designated in the art, and described herein, these MBP 18.5 epitopes are contained within residues 106- 125, 136-155, 141-170, and 151-170 of MBP18.5, with the numbering being that used in the art, which is based on the amino acid sequence of the porcine MBP molecule. In each case, significant T cell proliferation was observed in response to native MBP18.5 and recombinant MBP+X2Cy s 81 The MBP+X2 molecules were engineered to include exon 2encoded peptide sequences. In addition to providing a means to prepare therapeutic agents containing X2, the molecules allowed the determination of whether or not APCs could display exon 2 epitopes derived from full length MPB 21.5 in a manner that allowed recognition by T cells. This was also important for the MBP+X2Ser81 polypeptide, as it was not known if the single cysteine residue in exon 2 was essential for T cell recognition.

Proliferation assays with two independent exon 2-peptidespecific human T cell lines clearly demonstrated that only synthetic exon 2 peptide, MBP+X2CYs 8 1 (Fig. 21) and MBP+X2Ser81 (Fig. 22) could elicit a T cell response. In addition, dose response assays (Fig. 22) revealed that both MBP+X2Cys81 and MBP+X2Ser81 were efficiently displayed to the T cells in vitro.

This indicates that Cys 8 1 is dispensable for presentation of the exon 2-encoded epitope recognized by the clones tested. T cell proliferation data are also summarized in Fig. 24 and Fig. These results demonstrate that human T cells can respond to processed X2 epitopes derived from full length MBP 21.5 molecules, 15 and that the bacterially expressed recombinant forms of MBP, including MBP18.5, MBP+X2Cys81, and MBP+X2Ser81, can be as effective in stimulating encephalitogenic T cells as the native MBP18.5 protein.

Synthesis. Expression and Purification of APLP4 and other PLP 20 Muteins A DNA template consisting of a 1.5 kb fragment containing full-length human PLP target sequence cloned in plasmid pUC8 (ATCC# 57466) was extensively modified to produce APLP4. Three polynucleotides, each encoding a peptide comprising hydrophilic 25 domain 2, 3, or 4 were synthesized independently by PCR and o subsequently fused by overlapping PCR. The sequence integrity of the DNA containing the entire APLP4 open reading frame was verified by dideoxy sequence analysis. The APLP4 coding region was subcloned into plasmid pET22b (Novagen) as an NdeI/HindIII fragment. The APLP4 protein contains five additional amino acids (Met-Leu-Glu-Asp-Pro) fused to the N terminus and five additional histidines (a histidine tag) fused to the C-terminal histidine.

Plasmid pAPLP4 was transformed into E. coli strain W3110 (DE3) comprising a lambda DE3 lysogen (Studier et al 1990) and the APLP4 polypeptide produced by the transformed bacteria was identified by Coomassie Blue staining, and by probing Western blots with rabbit polyclonal serum raised against a synthetic peptide (amino acids 118-130) of human PLP (Serotec, AHP261) followed by detection with horseradish perxoidase labeled goat anti-rabbit antibody and an enhanced chemiluminescence

(ECL)

detection system (Amersham). Following induction with 1 mM IPTG, APLP4 accounted for approximately 50% of the total cell protein and appeared to be exclusively located in inclusion bodies.

Selective extraction of the inclusion bodies is carried out with a buffer containing 6M guanidine HCL, or, preferably, 5M guanidine HCL, 20mM sodium citrate, pH 5.0. Such extraction resulted in a preparation with a purity of up to 90% as judged by SDS-PAGE and analytical reverse phase HPLC. N-Terminal amino acid sequence determination confirmed that the sequence of the isolated polypeptide contained the predicted amino terminal residues of APLP4.

Using similar conventional techniques, nucleic acid molecules 15 encoding other PLP mutein polypeptides were constructed and expressed in W3110 (DE3). These include APLP3 (SEQ ID NO:23), in which hydrophobic domains 1, 3, and 4 are absent, and a similar construct encoding a PLP mutein lacking only hydrophobic domains 1 and 4 (APLP2, SEQ ID NO:29). APLP2 also includes a His tag sequence attached to its amino terminus, which as linked to and ,separated from the amino terminus of the second hydrophilic domain of PLP by a linker containing a thrombin cleavage site (amino acid residues 14-19 of SEQ ID NO:29) Expression of the encoded PLP muteins revealed that APLP3 was expressed at levels comparable to 25 those for APLP4, discussed above, while APLP2 was expressed at levels so low that they could only be detected by pulse chase radiolabeling analysis. A native PLP construct tested in the same expression system did not yield any detectable PLP polypeptide, even when analyzed by pulse chase radiolabeling.

Construction, Expression. and Purification of MP4 and other Chimeric PLP Molecules An MBP21.5 APLP4 fusion protein, MP4 was constructed as follows. A synthetic DNA fragment encoding MBP21.5 (SEQ ID NO:1) was placed under the control of the T7 promoter in the expression vector pET22b as described above. Next, the DNA fragment containing an appropriately spaced ribosome binding site and the APLP4 gene was ligated downstream of the MBP21.5 gene, creating a dicistronic operon for independent expression of MBP21.5 and APLP4. The dicistronic construct was digested with AatII-XhoI and ligated to a synthetic AatII-XhoI linker/adapter (corresponding to the sequence spanning nucleotides 588 to 605 of SEQ ID NO:26) creating a gene fusion encoding the MBP21.5/APLP4 chimeric protein designated MP4 (SEQ ID NO:26). The sequence integrity of the resulting expression construct for the MP4 fusion protein was confirmed and the MP4-encoding plasmid- was used to transform E.

coli W3110 (DE3), referred to above harboring a lysogenic chromosomal copy of the bacteriophage T7 RNA polymerase.

Using similar conventional techniques, nucleic acid molecules encoding other chimeric PLP polypeptides were constructed and expressed in W3110 (DE3). These include MP3, (SEQ ID NO:25), PM4 (SEQ ID NO:27), and MMOGP4 (SEQ ID NO:28). MP3 was an analogous chimera to MP4 except that the PLP mutein moiety was the APLP3 chimera of SEQ ID NO:23. PM4 was analogous to MP4 except that a 15 different linker (corresponding to the sequence spanning nucleotides 508 to 519 of SEQ ID NO:27) was used in an overlapping PCR procedure to link MBP21.5 and APLP4 in the opposite orientation to that in MP4. MMOGP4 was constructed by inserting a sequence encoding the extracellular domain of human myelin oligodendrocyte glycoprotein (Pham-Dinh et al. J Neurochem 1994, 63:2353 et seq) into MP4 between the MBP and PLP derived sequences. Expression of the encoded chimeric PLP polypeptides revealed that they were expressed at levels comparable to those for MP4, as discussed below.

25 MP4 synthesis was induced in E. coli W3110 (DE3) carrying the MP4 plasmid by the addition of 1 mM IPTG, and the protein appeared to be exclusively located in an insoluble fraction, accounting for approximately 20% of the total cell protein. MP4 was isolated as follows. E. coli paste from a 20L IPTG induced fermentation was resuspended in 10 volumes (10mL/g wet weight) of lysis buffer Na Citrate, 1mM EDTA, pH The paste was uniformly suspended using an UltraTurrax T 50 homogenizer on ice. HCl was added to pH 5.0 and cells were lysed on ice using a Microfluidizer Model M-11OT homogenizer operated at a pressure of 15,000-20,000 psi at the interaction chamber. The resultant lysate was centrifuged at approx. 10,000x g to separate soluble and insoluble fractions. The soluble fraction was discarded and the insoluble fraction was resuspended in 10 volumes (10mL/gram wet wt.) of extraction buffer (6M Guanidine-HCl, 0.5M NaCl, 20mM sodium phosphate, pH 5.0) using a homogenizer (Tekmar TP 18/1051). The extract was allowed to incubate with stirring for 60 min. at 2-8 degrees C. The extract was then centrifuged at 10,000x g for min. The supernatant from the centrifugation was sonicated on ice with a Branson Sonifier 450 for 5 min to shear contaminating nucleic acids and filtered through a 0.45 micron filter (Whatman Polycap) to yield a filtered supernatant.

Column chromatography was performed in two steps. In the first step, metal chelate chromatography was employed as follows: A column with dimensions of 5 cm diameter x 20 cm length containing Chelating SEPHAROSE Fast Flow (Pharmacia Biotech) was packed in deionized water. Approximately the upper two thirds of the column was charged with Ni by loading 1 mL of 0.1M NiC12 per 7.8 mL of resin- The column was then washed with 5 CV of 15 deionized water. The column was equilibrated with 2 CV of Buffer A (6M Guanidine-HC1, 0.5M NaC1, 20 mM sodium phosphate, ImM 2mercaptoethanol, pH 7.2) and a baseline optical density at 280nm S was measured. The filtered supernatant was adjusted to pH 7.2 with NaOH and 2-mercaptoethanol was added to a final concentration of ImM. This reduced sample was warmed to room temperature. The reduced sample was loaded at a flow rate of 50mL/min. The flow rate was then adjusted to 100mL/min and the column was washed with Buffer A until the column outflow reached the baseline optical density at 280nm. The column was then washed three times successively with 6M Urea, 0.5M NaC1, 0.02M sodium phosphate, first at pH 7.2, then at pH 6.3, and finally at pH 5.5, with each wash being continued until the optical density returned to baseline.

MP4 was eluted from the column with 6M Urea, 0.5M NaCl, 0.02M sodium phosphate, pH 3.5, while monitoring optical density at 280nm. Protein containing fractions were pooled and MP4 was further purified as follows: An aliquot of pooled fractions containing approximately thirty-five mg of MP4 (as estimated by analytical HPLC and SDS PAGE) was fully reduced by adding dithiothreitol to a final concentration of 50mM, guanidine HCL to a final concentration of 6M, and adjusting the pH to 8.0. The sample was then incubated at 37 degrees C for 0.5 hr. The resulting reduced and denatured preparation was then filtered through a 0.45 micron filter and applied to a 1 cm diameter x cm length C4 VYDAC (Hesperia, CA) reversed phase HPLC column equilibrated in 55% solvent A (50% Formic Acid/50%

H

2 0) and solvent B (50% Acetonitrile/50% Formic Acid) at room temperature.

(A baseline optical density reading at 2 80nm is taken prior to loading the sample on the column.) After loading, the column effluent was monitored at 280nm until the reading returned to baseline. The column was then eluted with a linear gradient increasing solvent B concentration from 55% solvent A, 45% solvent B to 0% solvent A, 100% Solvent

B.

Pooled protein containing fractions were concentrated in a Rotavap concentrator (Buchi Corp.) until brought to a concentration of approximately 2-3 mg/mL. Deionized water was then added to the flask to bring the sample to approximately its 1 original volume and the sample was again concentrated to remove 15 residual formic acid. This process was repeated until approximately five to ten times the original volume of water was added and removed. The sample was then transferred to a stirred cell concentrator (Amicon) equipped with a 10,000 dalton cutoff membrane. Diafiltration was performed at 4 degrees C with 20 three additions of deionized water until a total of twelve diavolumes of deionized water was passed through the sample. (The final pH of the sample was The resulting concentrated material had a purity of up to MP4 as judged by SDS-PAGE and analytical reversed phase HPLC.

N-

Terminal amino acid sequence determination confirmed that the sequence of the isolated polypeptide contained the predicted amino terminal residues of SEQ ID NO:26).

Further purification of MP4 may be desired. Additional purification steps include gel filtration chromatography and ion exchange chromatography (preferably cation exchange chromatography). These steps are facilitated by the addition of a non-ionic detergent (preferably TWEEN 20) to a concentration of 0.1% to The non-ionic detergent may be added at any point in the purification subsequent to cell lysis, as it does not interfere with metal chelate chromatography.

As with any pharmaceutical preparation derived from bacteria, the MP4 preparation is tested for toxicity in animals before administration to humans, with any toxic preparations being further purified or discarded. Such toxicity testing is preferably done using mice, and most preferably using mice in which EAE has been induced as described below, either by injection of encephalitogenic proteins or, preferably, by adoptive transfer of T cells from animals suffering from EAE. Testing in this manner has the additional benefit of allowing efficacy of treatment to be assessed in the animal model system. For such testing, 300 pg doses of bacterially produced polypeptide are preferably administered according to the appropriate treatment schedule described below under the subheading "Treatment of Mice with EAE." T Cell Resnonses Induced by the PLP PolvoeDtides of the Invention The APLP4 and MP4 polypeptides of the invention, were tested in in vitro and in vivo systems for their ability to stimulate

T

cell responses and to prevent and treat EAE/MS. The results of 15 these studies are set forth in Figures 1-12 and Table 3. These results demonstrate that the PLP polypeptides of the invention can induce T cell responses and affect T cell reactivity to a variety of MBP and PLP epitopes, and can induce and prevent and treat EAE.

Induction of EAE by Active Immunization S 20 Female SJL/J mice were purchased from The Jackson Laboratories (Bar Harbor, ME). All mice were used between 8 and 12 weeks of age. All mice were maintained on standard laboratory food and water ad libitum. Feeding was adjusted to assure that paralyzed animals were afforded easier access to food and water.

Female SJL/J mice were immunized by subcutaneous injection with 150 pl of an incomplete Freund's adjuvant emulsion containing 150 g of Mycobacterium tuberculosis H37Ra (Difco; complete Freund's adjuvant) and antigen. Antigens included 100 ug ovalbumin, 100 pg recombinant APLP4, 300 gg MP4, or 150 Jg of PLP peptide 1CS (amino acid residues 139-151 of SEQ ID NO:22 with a serine substituted for the cysteine at position 140). Each 150 gl injection was distributed over three sites on the dorsal flank.

All mice also received subsequent injections of 300 ng of pertussis toxin (List Biologicals, Campbell, CA) on days 0 and 3, as a higher incidence of disease was obtained when pertussis toxin was coadministered in preliminary tests. The immunomodulating effect of pertussis toxin on EAE induction is well known, although the precise mode of action is unknown. Pertussis toxin is a vasoactive substance believed to produce blood-brain barrier permeability and thus facilitate the entry of encephalitogenic cells into the CNS.

Initial clinical signs of disease were usually observed between day 12 and 16 post-innmunization. Mice were monitored daily and a mean clinical score was assigned to each group. Mean day of onset was calculated based upon the initial appearance of clinical signs.

Adoptive Transfer of EAE Donor SJL/J mice were immunized subcutaneously with 100 gg of APLP4 as described above. Nine to eleven days later, draining lymph node cells were harvested and stimulated with 25 gg/ml of PLP peptide 1CS for 4 days in the presence of syngeneic

SJL/J

antigen presenting cells (APCs). The peptide activated T cells (1.6x10 7 in 0.1 ml PBS) were harvested, washed twice, and injected 15 intravenously into syngeneic naive recipients.

Treatment of Mice with EAE Mice were divided into treatment groups that included untreated mice and mice receiving intravenous injections of either 125 pg of APLP4 or pigeon cytochrome c (as a control) twice a day 20 (separated by 6-8 hours) on days 2, 4, and 6 in the adoptive transfer experiments or days 5, 7, and 9 in the active immunization experiments.

Throughout this application various publications and patent disclosures are referred to. The teachings and disclosures thereof, in their entireties, are hereby incorporated by reference into this application to more fully describe the state of the art to which the present invention pertains.

Although preferred and other embodiments of the invention have been described herein, further embodiments may be perceived and practiced by those skilled in the art without departing from the scope of the invention as defined by the following claims.

Table 1 Autoreactive Human PLP Peptides In MS Patients PLP Peptide Residues in PLP Peptide Name SEQ ID NO:24 Amino Acid Sequence PLP 38-49 9-20 ALTGTEKLIETYa PLP 40-60 11-31 TGTEKLIETYFSKNYQDYEYLb, c PLP 88-108 42-62 EGFYTTGAVRQIFGDYKTTICe PLP 89-106 43-60 GFYTTGAVRQIFGDYKTTb PLP 91-104 45-58 YTTGAVRQIFGDYKa PLP 95-116 49-70 AVRQIFGDYKTTICGKGLSATVd PLP 103-116 57-70 YKTTICGKGLSATVa,e PLP 104-117 58-71 KTTICGKGLSATVTf PLP 115-128 69-82 TVTGGQKGRGSRGQa PLP 117-150 71-104 TGGQKGRGSRGQHQAHSLERVCHCLGKWLGHPDKc .PLP 139-151 93-105 HCLGKWLGHDKFg, a PLP 139-154 93-108 HCLGKWLGHPDKFVGle PLP 142-153 96-107 GKWLGHPDKFVGf PLP 183-195 115-127 CQSIAFPSKTSASa PLP 195-206 127-138 SIGSLCADARMyc PLP 195-208 127-140 SIGSLCADARMYGVa PLP 220-234 152-166 GSNLLSICKTAEFQMa The numbers following the letters PLP in the PLP peptide names (left hand column) indicate that the sequence of that peptide spans and Corresponds to the amino acid residues of those numbers (inclusive) of SEQ ID N:22.

uperscript lower case letters at the ends of the peptide sequences indicate references discussing the peptides, as follows: a Kinkel et al., 1992. Neurology 42 (Suppl. 159 (abstr. 87P).

b Pelfrey et al., 1993. J Neuroimmunol 46: 33-42.

c Trotter et al., 1993. J Immunol 1 5 0:196A (abstr. 1117).

d Inobe et al., 1992. Neurology 42 (Suppl. 3) :159-160 (abstr. 87P).

e Trotter et al., 1991 J Neuroimmunol 33: 55-62.

f Correale et al., 1995. J Immunol 154: 2959-2968.

g Chou et al., 1992. J Neuroimmunol 38: 105-113.

Table 2 Encephalitogenic Epitopes of PLP in Inbred Mouse Strains Residues In PLP Peptide SEQ ID NO:24 Amino Acid Sequence Strain 43-64 14-35 EKLIETYFSKNYQDYEYLIVI PL/J (H-2d) 103-116 57-70 YKTTICGKGLSATV SWR (H-2q) 139-151 93-105 HCLGKWLGHPDIKY SJL/J (H-2s) :0.66.

:The numbers in the left hand column ("PLP Peptideff) indicate that the sequence of that peptide spans and corresponds to the amino acid residues of those numbers (inclusive) of SEQ ID NO:22.

Table 3. Induction of EAE in SJL/J Mice Group Antigen* Incidence Mean Day of Onsett Mean Clin. Score* A Ovalbumin 0/6 B APLP4 6/6 C 139-151 6/6 r a 12.6 (12-15) 14.2 (13-22) 12.8 (12-16) 3.7 (3-4) 4.6 2.3 (2-3) D MP4 5/5 Immunizations were performed on day 0 with CFA (150 jg H37Ra). Antigens used were either 100 gg ovalbumin (Sigma), 100 jg APLP4, 150 gg PLP peptide 139-151, or 300 jg MP4. All groups received 300 ng pertussis toxin injected i.v. on day 0 and 3.

t The mean number of days between immunization and the first signs of EAE is shown for each group of animals, with the range in brackets.

*The mean clinical grade at the height of disease severity is shown, with the range in brackets.

TABLE 4 huMBP recMBP IhuMBP recMBP' amino acid codon 21.5 21.5 jamino acid codon 21.5 21.5 Arg CGT 2 19 ISer TCT .1 7 Gly Lys Leu Pro Thr Phe Cys Met

CGC:

CGA

CGG

AGA'

AGG

GGT

GGC

GGA

GGG

AMA

MAG

CUT

CTC

CTA

CTG

TTA

UTG

CCT

CCC

CCA

CCG

ACT

ACC

ACA

ACG

rrr

TTC

TGT

TGC

ATG

Ala Val H is* Gin Asn Asp Glu lie Tyr Trp

TCC

TCA

TCG

AGT

AGC

GCT

GCC

GCA

GCG

GTT

GTC

GTA

GTG

CAT

CAC

CMA

CAG

MAT

MAC

GAT

GAC

GMA

GAG

AUT

ATC

ATA

TAT

TAG

TGG

recMBP2l .5 contains six additional Histidines at the C-terminus.

TABLE WET WT 0.1 N HCL PEAK HT LYSATE GENE 0D600 (g/mI) (cm) VOL (ml) MBp+X2Cys8I/bact. 2.70 8.0 0.080 4.3 126 MBp+X2Ser8l/bact. 1.89 8.8- 0.088 3.6 126 MBP18.5hum. 1 .96 8.0 0.080 2.8 126 MBp+X2Cys8l/hum. 1.76 6.0 0.060 1.6 126 References Abbas et. al. 1994. Cell and Mol Immunolocv, pp. 377-391.

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*00 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Mueller, John P.

Lenardo. Michael J.

McFarland, Henry F.

Matis, Louis A.

Mueller, Eileen Elliott Nye, Steven H.

Pelfrey, Clara M.

Squinto, Stephen P.

Wilkins, James A.

(ii) TITLE OF INVENTION: Modified Myel: (iii) NUMBER OF SEQUENCES: 29 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Maurice M. Klee STREET: 1951 Burr Street CITY: Fairfield STATE: Connecticut COUNTRY: USA ZIP: 06430 COMPUTER READABLE FORM: MEDIUM TYPE: 3.5 inch, 0.8 Mb storage COMPUTER: Macintosh Centris 610 OPERATING SYSTEM: System 7 SOFTWARE: Microsoft Word 6.0.1 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE: 02-MAY-1995

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: 08/431,6 FILING DATE: May 2, 1 APPLICATION NUMBER: 08/431,6 FILING DATE: May 2, 1 APPLICATION NUMBER: 08/482,1 FILING DATE: June 7, in Protein Molecules r 44 995 48 995 14 1995 (viii) ATTORNEY/AGENT INFORMATION: NAME: Klee, Maurice M.

REGISTRATION NUMBER: 30,399 REFERENCE/DOCKET NUMBER: ALX-129 (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: (203) 255 1400 TELEFAX: (203) 254 1101 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 594 base pairs TYPE: Nucleic acid STRANDEDNESS: Double TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA to mRNA DESCRIPTION: MBP+X2Cys81/hum. (Human 21.5 kD form o* of MBP) (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No PUBLICATION INFORMATION: AUTHORS: Roth, H. J.

Kronquist, K. E.

Kerlero de Rosbo, N.

Crandall, B. F.

Campagnoni, A. T.

TITLE: Evidence for the Expression of Four Myelin Basic Protein Variants in the Developing Human Spinal Cord Through cDNA Cloning JOURNAL: Journal of Neuroscience Research VOLUME: 17 PAGES: 312 328 DATE: 1987 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: ATG GCG TCA CAG AAG AGA CCC TCC CAG AGG CAC GGA TCC 39 Met Ala Ser Gln Lys Arg Pro Ser Gin Arg His Gly Ser 1 5 AAG TAC CTG GCC ACA GCA AGT ACC ATG GAC CAT GCC AGG CAT 81 Lys Tyr Leu Ala Thr Ala Ser Thr Met Asp His Ala Arg His 20 GGC TTC CTC CCA AGG CAC Gly Phe Leu Pro Arg His AGA GAC ACG GGC ATC CTT GAC TCC Arg Asp Thr Gly Ile Leu Asp Ser ATC GGG CGC TTC Ile Gly Arg Phe GGC TCT GGC AAG Gly Ser Gly Lys TTT GGC GGT GAC AGG GGT GCG CCC AAG CGG Phe Gly Gly Asp Arg Gly Ala Pro Lys Arg 50

GTA

Val CCC TGG CTA AAG CCG GGC CGG AGC CCT Pro Trp Leu Lys Pro Gly Arg Ser Pro

CTG

Leu CCC TCT CAT GCC Pro Ser His Ala

CGC

Arg 75 AGC CAG CCT GGG CTG TGC AAC ATG Ser Gin Pro Gly Leu Cys Asn Met CCG GCA AGA ACT GCT CAC TAT GGC Pro Ala Arg Thr Ala His Tyr Gly 90 TAC AAG GAC TCA CAC CAC Tyr Lys Asp Ser His His a.

a TCC CTG CCC CAG Ser Leu Pro Gin 100 CCC GTA GTC CAC Pro Val Val His 115 CCA CCC CCG TCG Pro Pro Pro Ser AAG TCA CAC GGC CGG ACC CAA GAT GAA AAC Lys Ser His Gly Arg Thr Gin Asp Glu Asn 105 110 TTC TTC AAG AAC ATT GTG ACG CCT CGC ACA Phe Phe Lys Asn Ile Val Thr Pro Arg Thr 120 125 123 165 207 249 291 333 375 417 459 501 543 585

CAG

Gin 130 GGA AAG GGG AGA GGA CTG TCC CTG AGC Gly Lys Gly Arg Gly Leu Ser Leu Ser 135

AGA

Arg 140 TTT AGC TGG GGG Phe Ser Trp Gly

GCC

Ala 145 GAA GGC CAG AGA CCA GGA TTT GGC Glu Gly Gin Arg Pro Gly Phe Gly 150 TAC GGA GGC AGA Tyr Gly Gly Arg 155 TTC AAG GGA GTC Phe Lys Gly Val 170 AAG CTG GGA GGA Lys Leu Gly Gly 185 GCG TCC GAC TAT AAA TCG GCT CAC AAG GGA Ala Ser Asp Tyr Lys Ser Ala His Lys Gly 160 165 GAT GCC CAG GGC ACG CTT TCC AAA ATT TTC Asp Ala Gln Gly Thr Leu Ser Lys Ile Phe 175 180 AGA GAT AGT CGC TCT GGA TCA CCC ATG GCT Arg Asp Ser Arg Ser Gly Ser Pro Met Ala 190 200 AGA CGC TGA Arg Arg INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 612 base pairs TYPE: Nucleic acid STRANDEDNESS: Double 594 TOPOLOGY: Linear (ii) MOLECULE TYPE: Other nucleic acid DESCRIPTION: MBP+X2Cys81/bact.

(iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: ATG GCG TCT CAG AAA CGT CCG TCC CAG Met Ala Ser Gin Lys Arg Pro Ser Gin 1 5 TAC CTG GCC ACC GCC AGC ACC ATG GAC Tyr Leu Ala Thr Ala Ser Thr Met Asp CGT CAC GGC TCC AAA Arg His Gly Ser Lys CAT GCC His Ala r r TTC CTG Phe Leu CCG CGT CAC CGT GAC Pro Arg His Arg Asp 35 ACC GGC ATC CTG Thr Gly Ile Leu CGT CAT GGC Arg His Gly GAC TCC ATC Asp Ser Ile AAA CGT GGC Lys Arg Gly GGC CGC TTC Gly Arg Phe TCT GGC AAA Ser Gly Lys TTC GGC GGT GAC Phe Gly Gly Asp

CGT

Arg 50 GGT GCG CCG Gly Ala Pro 126 168 210

GTG

Val 60 CCG TGG CTG AAA Pro Trp Leu Lys

CCG

Pro 65 GGC CGT AGC CCG CTG Gly Arg Ser Pro Leu CCG TCT CAT GCC Pro Ser His Ala

CGT

Arg AGC CAG CCG GGC Ser Gin Pro Gly CTG TGC Leu Cys GCG CAC Ala His AAC ATG TAC Asn Met Tyr TAT GGC TCC Tyr Gly Ser 252

AAA

Lys GAC TCC CAC CAC Asp Ser His His

CCG

Pro 90 GCT CGT ACC Ala Arg Thr 294 r CTG CCG Leu Pro 100 CAG AAA TCC CAC Gin Lys Ser His

GGC

Gly 105 CGT ACC CAG GAT GAA AAC CCG Arg Thr Gin Asp Glu Asn Pro 110 336 378 GTG GTG CAC TTC TTC AAA Val Val His Phe Phe Lys 115 CCG CCG TCT CAG GGC AAA Pro Pro Ser Gin Gly Lys 130 AAC ATT Asn Ile 120 GTG ACC CCG CGT ACC CCG Val Thr Pro Arg Thr Pro 125 GGC CGT GGC Gly Arg Gly 135 CTG TCC CTG AGC CGT Leu Ser Leu Ser Arg 140 420 462 TTC AGC TGG GGC GCC GAA GGC CAG Phe Ser Trp Gly Ala Glu Gly Gin 145 CGT CCG GGC TTC GGT TAC Arg Pro Gly Phe Gly Tyr 150 GGC GGC CGT GCG TCC GAC TAT AAA TCT GCT CAC AAA GGC TTC 504 Gly Gly Arg Ala Ser Asp Tyr Lys Ser Ala His Lys Gly Phe 155 160 165 AAA GGC GTG GAT GCC CAG GGT ACC TTG TCC AAA ATT TTC AAA 546 Lys Gly Val Asp Ala Gin Gly Thr Leu Ser Lys Ile Phe Lys 170 175 180 CTG GGC GGC CGT GAT AGC CGT TCT GGC TCT CCG ATG GCT AGA 588 Leu Gly Gly Arg Asp Ser Arg Ser Gly Ser Pro Met Ala Arg 185 190 200 CGT CAT CAC CAT CAC CAT CAC TAA 612 Arg His His His His His His 205 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 612 base pairs S* TYPE: Nucleic acid STRANDEDNESS: Double TOPOLOGY: Linear S* (ii) MOLECULE TYPE: cDNA to mRNA DESCRIPTION: MBP+X2Ser81/bact.

o (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ATG GCG TCT CAG AAA CGT CCG TCC CAG CGT CAC GGC TCC AAA 42 Met Ala Ser Gin Lys Arg Pro Ser Gin Arg His Gly Ser Lys 1 5 TAC CTG GCC ACC GCC AGC ACC ATG GAC CAT GCC CGT CAT GGC 84 Tyr Leu Ala Thr Ala Ser Thr Met Asp His Ala Arg His Gly 20 TTC CTG CCG CGT CAC CGT GAC ACC GGC ATC CTG GAC TCC ATC 126 Phe Leu Pro Arg His Arg Asp Thr Gly Ile Leu Asp Ser Ile 35 GGC CGC TTC TTC GGC GGT GAC CGT GGT GCG CCG AAA CGT GGC 168 Gly Arg Phe Phe Gly Gly Asp Arg Gly Ala Pro Lys Arg Gly 50 TCT GGC AAA GTG CCG TGG CTG AAA CCG GGC CGT AGC CCG CTG 210 Ser Gly Lys Val Pro Trp Leu Lys Pro Gly Arg Ser Pro Leu 65 CCG TCT CAT GCC Pro Ser His Ala

CGT

Arg AGC CAG CCG GGC Ser Gin Pro Gly

CTG

Leu TCG AAC ATG TAC Ser Asn Met Tyr

AAA

Lys GAC TCC CAC CAC Asp Ser His His

CCG

Pro 90 GCT CGT ACC GCG Ala Arg Thr Ala

CAC

His TAT GGC TCC Tyr Gly Ser GAA AAC CCG Glu Asn Pro 110 252 294 336 CTG CCG Leu Pro 100 CAG AAA TCC CAC Gin Lys Ser His

GGC

Gly 105 CGT ACC CAG GAT Arg Thr Gin Asp GTG GTG CAC Val Val His 115 CCG CCG TCT Pro Pro Ser TTC TTC AAA AAC Phe Phe Lys Asn

ATT

Ile 120 GTG ACC CCG CGT Val Thr Pro Arg ACC CCG Thr Pro 125

CAG

Gin 130 GGC AAA GGC CGT Gly Lys Gly Arg

GGC

Gly 135 CTG TCC CTG AGC Leu Ser Leu Ser

CGT

Arg 140 TTC AGC TGG GGC Phe Ser Trp Gly

GCC

Ala 145 GAA GGC CAG CGT Glu Gly Gin Arg

CCG

Pro 150 GGC TTC GGT TAC Gly Phe Gly Tyr 378 420 462 504 546

GGC

Gly 155 GGC CGT GCG TCC Gly Arg Ala Ser

GAC

Asp 160 TAT AAA TCT GCT Tyr Lys Ser Ala

CAC

His 165 AAA GGC TTC Lys Gly Phe ATT TTC AAA Ile Phe Lys 180 AAA GGC Lys Gly 170 GTG GAT GCC CAG Val Asp Ala Gin

GGT

Gly 175 ACC TTG TCC AAA Thr Leu Ser Lys CTG GGC GGC Leu Gly Gly 185 CGT CAT CAC Arg His His CGT GAT AGC CGT Arg Asp Ser Arg

TCT

Ser 190 GGC TCT CCG ATG Gly Ser Pro Met GCT AGA Ala Arg 195 588

CAT

His 200 CAC CAT CAC TAA His His His 612 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 519 base pairs TYPE: Nucleic acid STRANDEDNESS: Double TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA to mRNA DESCRIPTION: Human 18.5 (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No kDa form of MBP (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CAT ATG GCG His Met Ala 1 AAG TAC CTG Lys Tyr Leu GGC TTC CTC Gly Phe Leu ATC GGG CGC Ile Gly Arg GGC TCT GGC Gly Ser Gly TCA CAG AAG AGA CCC TCC CAG AGG CAC GGA TCC Ser Gin Lys Arg Pro Ser Gin Arg His Gly Ser 5 GCC ACA GCA AGT ACC ATG GAC CAT GCC AGG CAT Ala Thr Ala Ser Thr Met Asp His Ala Arg His 20 CCA AGG CAC AGA GAC ACG GGC ATC CTT GAC TCC Pro Arg His Arg Asp Thr Gly Ile Leu Asp Ser 35 TTC TTT Phe Phe AAG GAC Lys Asp GGC GGT GAC AGG GGT GCG CCA AAG CGG Gly Gly Asp Arg Gly Ala Pro Lys Arg 50 TCA CAC CAC CCG GCA AGA ACT GCT CAC Ser His His Pro Ala Arg Thr Ala His CAG AAG TCA CAC GGC CGG ACC CAA GAT Gin Lys Ser His Gly Arg Thr Gin Asp 75 CAC TTC TTC AAG AAC ATT GTG ACG CCT His Phe Phe Lys Asn Ile Val Thr Pro 90 r TAT GGC TCC CTG CCC Tyr Gly Ser Leu Pro GAA AAC CCC GTA GTC Glu Asn Pro Val Val 42 84 126 168 210 252 294 336 378 420 462 504 CGC ACA CCA Arg Thr Pro 100 CTG AGC AGA Leu Ser Arg TTT GGC TAC Phe Gly Tyr AAG GGA TTC Lys Gly Phe 140 ATT TTT AAG Ile Phe Lys 155 CCC CCG TCG CAG GGA AAG GGG AGA GGA CTG TCC Pro Pro Ser Gin Gly Lys Gly Arg Gly Leu Ser 105 110 TTT AGC Phe Ser 115 GGA GGC Gly Gly 130 TGG GGG GCC GAA GGC CAG AGA CCA GGA Trp Gly Ala Glu Gly Gin Arg Pro Gly 120 125 AGA GCG TCC GAC TAT AAA TCG GCT CAC Arg Ala Ser Asp Tyr Lys Ser Ala His 135 AAG GGA GTC GAT GCC CAG GGC ACG CTT TCC AAA Lys Gly Val Asp Ala Gin Gly Thr Leu Ser Lys 145 150 CTG GGA GGA AGA GAT AGT CGC TCT GGA TCA CCC Leu Gly Gly Arg Asp Ser Arg Ser Gly Ser Pro 160 165 ATG GCT AGA CGC TAA Met Ala Arg Arg 170 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 130 bases TYPE: Nucleic acid STRANDEDNESS: Single 519 TOPOLOGY: Linear (ii) MOLECULE TYPE: Other nucleic acid DESCRIPTION: PCR primer oligonucleotide 1 (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID GGAATTCCGT AAGGAGGTAT AGCATATGGC GTCTCAGAAA CGTCCGTCCC AGCGTCACGG CTCCAAATAC CTGGCCACCG CCAGCACCAT GGACCATGCC 100 CGTCATGGCT TCCTGCCGCG TCACCGTGAC 130 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 129 bases TYPE: Nucleic acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: Other nucleic acid DESCRIPTION: PCR primer oligonucleotide 2 (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: Yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GACGGCAGCG GGCTACGGCC CGGTTTCAGC CACGGCACTT TGCCAGAGCC •o ACGTTTCGGC GCACCACGGT CACCGCCGAA GAAGCGGCCG ATGGAGTCCA 100 GGATGCCGGT GTCACGGTGA CGCGGCAGG 129 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 133 bases TYPE: Nucleic acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: Other nucleic acid DESCRIPTION: PCR primer oligonucleotide 3 (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CCGGGCCGTA GCCCGCTGCC GTCTCATGCC CGTAGCCAGC CGGGCCTGTG CAACATGTAC AAAGACTCCC ACCACCCGGC TCGTACCGCG CACTATGGCT 100 CCCTGCCGCA GAAATCCCAC GGCCGTACCC AGG 133 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 131 bases TYPE: Nucleic acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: Other nucleic acid DESCRIPTION: PCR primer oligonucleotide 4 (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: Yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: CGGCGCCCCA GCTGAAACGG CTCAGGGACA GGCCACGGCC TTTGCCCTGA 9 GACGGCGGCG GGGTACGCGG GGTCACAATG TTTTTGAAGA AGTGCACCAC 100 CGGGTTTTCA TCCTGGGTAC GGCCGTGGGA T 131 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 119 bases TYPE: Nucleic acid o STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: Other nucleic acid DESCRIPTION: PCR primer oligonucleotide (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GCCGTTTCAG CTGGGGCGCC GAAGGCCAGC GTCCGGGCTT CGGCTACGGC GGCCGTGCGT CCGACTATAA ATCTGCTCAC AAAGGCTTCA AAGGCGTGGA 100 TGCCCAGGGC ACCCTGTCC 119 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 111 bases TYPE: Nucleic acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: Other nucleic acid DESCRIPTION: PCR primer oligonucleotide 6 (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: Yes (xi) SEQUENCE DESCRIPTION: SEQ ID CCCCAAGCTT ATTAGTGATG GTGATGGTGA TGACGTCTAG CCATCGGAGA GCCAGAACGG CTATCACGGC CGCCCAGTTT GAAAATTTTG GACAGGGTGC CCTGGGCATC C 0. 0.

*0 S 06 9 090 0 0000 a s*

S

0600 0. 0

S

9.

9 *0 0e U 0* 05 (xi)

ACGCG

GTCAT

TAAAT

AATAA

GTCAA

GTGTA

GCCCG

GGCAG

INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 4059 base pairs TYPE: Nucleic Acid STRANDEDNESS: Double TOPOLOGY: Circular (ii) MOLECULE TYPE: Other nucleic acid DESCRIPTION: Apex-I Eukaryotic Expression Vector SEQUENCE DESCRIPTION: SEQ ID NO:11: TTGAC ATTGATTATT GACTAGTTAT TAATAGTAAT CAATT TAGTT CATAGCCCAT ATATGGAGTT CCGCGTTACA TAACT GGCCC CGCCTGGCTG ACCGCCCAAC GACCCCCGCC CATTG TGACG TATGTTCCCA TAGTAACGCC AATAGGGACT TTCCA TGGGT GGACTATTTA CGGTAAACTG CCCACTTGGC AGTAC TCATA TGCCAAGTAC GCCCCCTATT GACGTCAATG ACGGT CCTGG CATTATGCCC AGTACATGAC CTTATGGGAC TTTCC TACAT CTACGTATTA GTCATCGCTA TTACCATGGT GATGC

ACGGG

TACGG

ACGTC

TTGAC

ATCAA

AAATG

TACTT

GGTTT

100 111 100 150 200 250 300 350 400 TGGCAGTACA TCAATGGGCG TGGATAGCGG TTTGACTCAC GGGGATTTCC45 450 a. a a a. a.

a

AAGTCTCCAC

AACGGGACTT

GGCGGTAGGC

GAACCGTCAG

TCTTTCCAGT

CGCCACCGAG

TCGACTGTTG

CTAAGATTGT

CGCGGTGATG

TCTTTTTGTT

ACTTGAGTGA

TCCCAGGTCC

TAGTAACGGC

CGGCCGCTCG

TACAAATAAA

ACTGCATTCT

TCTGGATCGA

GGACCTCTTC

GCACCTTGAA

AAACACAGGC

TAGTTGCTAG

GTAATTTCGC

TGCCGAGAGT

CTACTAGAAT

CAGCTTCAGA

CTCTTAAAAT

ACGCAGCTGG

CTCCAGAGGG

CCCATTGACG

TCCAAAATGT

GTGTACGGTG

AATTCTGTTG

ACTCTTGGAT

GGACCTGAGC

GGGTGAGTAC

CAGTTTCCAA

CCTTTGAGGG

GTCAAGCTTG

CAATGACATC

AACTGCAGGT

CGCCAGTGTG

AGCATGCATC

GCAATAGCAT

AGTTGTGGTT

TCCCGCCATG

GTTGTGTAGG

CTGTCTGCAT

ACAGTACTGA

GGCTGTCTCC

CATCAAGGGC

CCCGTAAGGG

AGTCAGTGCG

AGATGGCGGA

AGAAAATGTC

CCGTGCGACA

CGTGTGGTTT

TCAATGGGAG

CGTAACAACT

GGAGGTCTAT

GGCTCGCGGT

CGGAAACCCG

GAGTCCGCAT

TCCCTCTCAA

AAACGAGGAG

TGGCCGCGTC

AGGTGTGGCA

CACTTTGCCT

CGACCGGCTT

CTGGAATTCT

TAGAACTTGT

CACAAATTTC

TGTCCAAACT

GTATCAACGC

TACCGCTGTA

CAGCCATATA

CAAACCCATA

GAACTCATTA

AGCGAGGGCT

TAGACACTTC

GCTCCCATTT

GGGCCTCCAA

AAGTCAGTTA

TCCTCTTTTA

TGCAAGAGGA

TTTGTTTTGG

CCGCCCCATT

ATAAGCAGAG

TGATTACAAA

TCGGCCTCCG

CGACCGGATC

AAGCGGGCAT

GATTTGATAT

CATCTGGTCA

GGCTTGAGAT

TTCTCTCCAC

GGTACCGAGC

GCAGATATCC

TTATTGCAGC

ACAAATAAAG

CATCAATGTA

CATATTTCTA

TTCCTAGGGA

GCCCCCGCTG

CACCTCCTCT

CACCCTCCAA

TCTCCAGATA

AGCTAATCCC

TGAAAATTCA

CACAGTAATT

AGCAGGAAGT

ATTAGTTGCT

AGCAAAAGCC

CACCAAAATC

GACGCAAATG

CTCGTTTAGT

CTCTTCGCGG

AACGGTACTC

GGAAAACCTC

GACTTCTGCG

TCACCTGGCC

GAAAAGACAA

CTGGCCATAC

AGGTGTCCAC

TCGGATCCAC

ATCACACTGG

TTATAATGGT

CATTTTTTTC

TCTTATCATG

TTTACAGTAG

AATAGTAGAG

TTCGACTTAC

GAAATACCCA

AGTCAGAGCT

AAATAGCTTC

TCGATGAGGT

CTTACTTGAT

TTCCTCCCGA

GGACTAACTG

AGGCAACGCC

TCTCCACCCA

500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 a a. a a a. a.

GGCCTAGAAT

TAGTATTAAG

AAGAAGAGAG

CTGCTTCTAT

TCCATACCCC

GCCCATCCCG

GCTGACTAAT

GAGCTATTCC

CAAAAAGGAG

CTGGCGTTTT

ACGCTCAAGT

CGTTTCCCCC

CTTACCGGAT

TCAATGCTCA

AGCTGGGCTG

TCCGGTAACT

ACTGGCAGCA

GTGCTACAGA

ACAGTATTTG

AGTTGGTAGC

TTTTTGTTTG

GATCCTTTGA

ACGTTAAGGG

TCCTTTTAAA

TAAACTTGGT

AGCGATCTGT

AGATAACTAC

ATACCGCGAG

GCCAGCCGGA

CCATCCAGTC

GTTTCCACCC

CAGAGGCCGG

GCATTGTAGA

TTCTGTCACA

CTTTAATAAG

CCCCTAACTC

TTTTTTTATT

AGAAGTAGTG

CTCCCAGCAA

TCCATAGGCT

CAGAGGTGGC

TGGAAGCTCC

ACCTGTCCGC

CGCTGTAGGT

TGTGCACGAA

ATCGTCTTGA

GCCACTGGTA

GTTCTTGAAG

GTATCTGCGC

TCTTGATCCG

CAAGCAGCAG

TCTTTTCTAC

ATTTTGGTCA

TTAAAAATGA

CTGACAGTTA

CTATTTCGTT

GATACGGGAG

ACCCACGCTC

AGGGCCGAGC

TATTALATTGT

AATCATTACT

GGACCCCTGG

GGCTTCCAGA

CTGTCTGGCC

CAGTTTGGGA

CGCCCAGTTC

TATGCAGAGG

AGGAGGCTTT

AAGGCCAGGA

CCGCCCCCCT

GAAACCCGAC

CTCGTGCGCT

CTTTCTCCCT

ATCTCAGTTC

CCCCCCGTTC

GTCCAACCCG

ACAGGATTAG

TGGTGGCCTA

TCTGCTGAAG

GCAAACAAAC

ATTACGCGCA

GGGGTCTGAC

TGAGATTATC

AGTTTTAAAT

CCAATGCTTA

CATCCATAGT

GGCTTACCAT

ACCGGCTCCA

GCAGAAGTGG

TGCCGGGAAG

89

ATGACAACAG

GCCCGCTTAC

GGCAACTTGT

CTGTCACAAG

ACGGGTGCGG

CGCCCATTCT

CCGAGGCCGC

TTTGGAGGCC

ACCGTAAAAA

GACGAGCATC

AGGACTATAA

CTCCTGTTCC

TCGGGAAGCG

GGTGTAGGTC

AGCCCGACCG

GTAAGACACG

CAGAGCGAGG

ACTACGGCTA

CCAGTTACCT

CACCGCTGGT

GAAAAAAAGG

GCTCAGTGGA

AAAAAGGATC

CAATCTAAAG

ATCAGTGAGG

TGCCTGACTC

CTGGCCCCAG

GATTTATCAG

TCCTGCAACT

CTAGAGTAAG

CTGTTTTTTT

TCTGGAGAAA

CAAAACAGGA

GTCCAGCACC

GTCTTACTCC

CCGCCCCATG

CTCGGCCTCT

TAGGCTTTTG

GGCCGCGTTG

ACAAAAATCG

AGATACCAGG

GACCCTGCCG

TGGCGCTTTC

GTTCGCTCCA

CTGCGCCTTA

ACTTATCGCC

TATGTAGGCG

CACTAGAAGG

TCGGAAAAAG

AGCGGTGGTT

ATCTCAAGAA

ACGAAAACTC

TTCACCTAGA

TATATATGAG

CACCTATCTC

CCCGTCGTGT

TGCTGCAATG

CAATAAACCA

TTATCCGCCT

TAGTTCGCCA

1900 1950 2000 2050 2100 2150 2200 2250 2300 2350 2400 2450 2500 2550 2600 2650 2700 2750 2800 2850 2900 2950 3000 3050 3100 3150 3200 3250 3300 3350 GTTAATAGTT TGCGCAACGT TGTTGCCATT GCTACAGGCA TCGTGGTGTC

ACGCTCGTCG

GGCGAGTTAC

GGTCCTCCGA

GGTTATGGCA

GCTTTTCTGT

ATGCGGCGAC

GCCACATAGC

GGCGAAAACT

CCCACTCGTG

TTCTGGGTGA

GGGCGACACG

TGAAGCATTT

TATTTAGAAA

TGCCACCTG

TTTGGTATGG

ATGATCCCCC

TCGTTGTCAG

GCACTGCATA

GACTGGTGAG

CGAGTTGCTC

AGAACTTTAA

CTCAAGGATC

CACCCAACTG

GCAAAAACAG

GAAATGTTGA

ATCAGGGTTA

AATAAACAAA

CTTCATTCAG

ATGTTGTGCA

AAGTAAGTTG

ATTCTCTTAC

TACTCAACCA

TTGCCCGGCG

AAGTGCTCAT

TTACCGCTGT

ATCTTCAGCA

GAAGGCAAA

ATACTCATAC

TTGTCTCATG

TAGGGGTTCC

CTCCGGTTCC

AAAAAGCGGT

GCCGCAGTGT

TGTCATGCCA

AGTCATTCTG

TCAATACGGG

CATTGGAAAA

TGAGATCCAG

TCTTTTACTT

TGC!CGCAAAA

TCTTCCTTTT

AGCGGATACA

GCGCACATTT

CAACGATCAA

TAGCTCCTTC

TATCACTCAT

TCCGTAAGAT

AGAATAGTGT

ATAATACCGC

CGTTCTTCGG

TTCGATGTAA

TCACCAGCGT

AAGGGAATAA

TCAATATTAT

TATTTGAATG

CCCCGAAAAG

3400 3450 3500 3550 3600 3650 3700 3750 3800 3850 3900 3950 4000 4050 4059 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 8540 base pairs TYPE: Nucleic Acid STRANDEDNESS: Double TOPOLOGY: Circular (ii) MOLECULE TYPE: Other nucleic acid DESCRIPTION: Apex-3P Eukaryotic Expression Vector (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GTGACCAATA CAAAACAAAA GCGCCCCTCG TACCAGCGAA GAAGGGGCAG AGATOCCOTA GTCAGGTTTA GTTCGTCCGG CGGCGGGGGA TCTGTATGGT GCACTCTCAG TACAATCTGC TCTGATGCCG CATAGTTAAG CCAGTATCTG CTCCCTGCTT GTGTGTTGGA GGTCGCTGAG TAGTGCGCGA GCAAAATTTA AGCTACAACA AGGCAAGGCT TGACCGACAA TTGCATGAAG AATCTGCTTA 100 150 200 250 GGGTTAGGCG TTTTGCGCTG CTTCGCGATG TACGGGCCAG ATATACGCGT30 300 0 0*e* 0

TGACATTGAT

AGTTCATAGC

GCCCGCCTGG

ACGTATGTTC

GGTGGACTAT

ATATGCCAAG

TGGCATTATG

CATCTACGTA

ACATCAATGG

CACCCCATTG

CTTTCCAAAA

GGCGTGTACG

CAGAATTCTG

AGTACTCTTG

GAGGGACCTG

TTGGGGTGAG

TGTCAGTTTC

ATGCCTTTGA

GTTGTCAAGC

TGACAATGAC

TCCAACTGCA

GACCTGCAGC

ACTGGGAAAP,

CCTTTCGCCP

GATAAGATAC

AAAAATGCTI

ATTATAAGCI

GTTTCAGGT9I

TATTGACTAG

CCATATATGG

CTGACCGCCC

CCATAGTAAC

TTACGGTAAA

TACGCCCCCT

CCCAGTACAT

TTAGTCATCG

GCGTGGATAG

ACGTCAATGG

TGTCGTAACA

GTGGGAGGTC

TTGGGCTCGC

GATCGGAAAC

AGCGAGTCCG

TACTCCCTCT

CAAAAACGAG

GGGTGGCCGC

TTGAGGTGTG

ATCCACTTTG

*GGTCGACCGG

CATGCAAGCrl

CCCTGGCGTI

GCTGGCGTAP

ATTGATGAGI

TATTTGTGAJ

GCAATAAACI

1 CAGGGGGAGC

TTATTAATAG

AGTTCCGCGT

AACGACCCCC

GCCAATAGGG

CTGCCCACTT

ATTGACGTCA

GACCTTATGG

.CTATTACCAT

CGGTTTGACT

GAGTTTGTTT

ACTCCGCCCC

TATATAAGCA

GGTTGATTAC

CCGTCGGCCT

CATCGACCGG

CAAAAGCGGG

GAGGATTTGA

GTCCATCTGG

GCAGGCTTGA

CCTTTCTCTC

CTTGGTACCG

TGGCACTGGC

ACCCAACTTA

LTAGCGAAGAG

'TTGGACAAAC

SATTTGTGATG

SAGTTAACAAC

TGTGGGAGG'I

TAATCAATTA

TACATAACTT

GCCCATTGAC

ACTTTCCATT

GGCAGTACAT

ATGACGGTAA.

GACTTTCCTA

GGTGATGCGG

CACGGGGATT

TGGCACCAAA

ATTGACGCA-A

GAGCTCGTTT

AAACTCTTCG

CCGAACGGTA

ATCGGAAAAC

CATGACTTCT

TATTCACCTG

TCAGAAAAGA

GATCTGGCCA

CACAGGTGTC

AGCTCGGATC

CGTCGTTTTA

ATCGCCTTGC

GCCCGCACCG

CACAACTAGA

CTATTGCTTT

AACAATTGCA

TTTTTAAAGC

GGGGTCATT

ACGGTAAATG

GTCAATAATG

GACGTCAATG

CAAGTGTATC

ATGGCCCGCC

CTTGGCAGTA

TTTTGGCAGT

TCCAAGTCTC

ATCAACGGGA

ATGGGCGGTA

AGTGAACCGT

CGGTCTTTCC

CTCCGCCACC

CTCTCGACTG

GCGCTAAGAT

GCCCGCGGTG

CAATCTTTTT

TACACTTGAG

CACTCCCAGG

CTCTAGAGTC

CAACGTCGTG

AGCACATCCC

ATCCAGACAT

ATGCAGTGAA

ATTTGTAACC

TTCATTTTAT

AAGTAAAACC

350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700

TCTACAAATG

CCTCATAAAC

TGCCCATCCA

GGAAATTGGG

AATATCTGGG

AGCCCACAAA

GGGCCCAACA

TGTGCAAAAC

CTAGTGTTTT

AGCATTATCC

TGTCAACTGT

CTGTAGTTTG

AAAAAAATGA

GAGTTTTTTG

ACCTCAGTTT

TAAGTCCTCA

TGTGTCACTT

ATGCAAAGCA

AGGC TC C CCA

CAACCATAGT

AGTTCCGCCC

AGAGGCCGAG

GCTTTTTTGG

AGCCCACGGT

ACCCTCGCCG

CCCGGACCGC

CGCGCGTCGG

GCGGTGGCGG

CGCCGAGATC

CGCAGCAACA

TGGTATGGCT

CCTAACCTCC

CCCTCTGTGT

TAGGGGTTTT

AAGTCCCTTC

TGTCAACAGC

CCCTGCTCAT

AGGAGGCACA

CATTTTTACT

TTATCCAAAA

AGCATTTTTT

CTAACACACC

AAATTTGACC

TGTCCCTGAA

TAACAGTAAC

TTTGTAGAAT

AGGGTGTGGA

TGCATCTCAA

GCAGGCAGAA

CCCGCCCCTA

ATTCTCCGCC

GCCGCCTCGG

AGGCCTAGGC

GCGCCTCGCC

CCGCGTTCGC

CACATCGAGC

GCTCGACATC

TCTGGACCAC

GGCCCGCGCA

GATGGAAGGC

GATTATGATC

TCTACTTGAG

CCTCCTGTTA

TCACAGACCG

CACTGCTGTG

AGAAACATAC

CAAGAAGCAC

TTTTCCCCAC

TGGATCAGGA

CAGCCTTGTG

GGGGTTACAG

CTGCAGCTCC

CTTGAATGGG

TGCAAGTTTA

AGCTTCCCAC

TCGCCAGCAC

AAGTCCCCAG

TTAGTCAGCA

GTATGCAAAG

ACTCCGCCCA

CCATGGCTGA

CCTCTGAGCT

TTTTGCAAAA

ACCCGCGACG

CGACTACCCC

GGGTCACCGA

GGCAAGGTGT

GCCGGAGAGC

TGGCCGAGTT

CTCCTGGCGC

92

CCCAGGAAGC

AGGACATTCC

ATTAGGTCAC

CTTTCTAAGG

TTCCAGAAGT

AAGCTGTCAG

TGTGGTTGCT

CTGTGTAGGT

ACCCAGCACT

GTCAGTGTTC

TTTGAGCAGG

AAAGGTTCCC

TTTTCCAGCA

ACATAGCAGT

ATCAAAATAT

AGTGGTCGAC

GCTCCCCAGC

ACCAGGTGTG

CATGCATCTC

TCCCGCCCCT

CTAATTTTTT

ATTCCAGAAG

GCTTACCATG

ACGTCCCCCG

GCCACGCGCC

GCTGCAAGAA

GGGTCGCGGA

GTCGAAGCGG

GAGCGGTTCC

CGCACCGGCC

TCCTCTGTGT 1750 AATCATAGGC 1800 TTAACAAAAA 1850 GTAATTTTAA 1900 GTTGGTAAAC 1950 CTTTGCACAA 2000 GTGTTAGTAA 2050 TCCAAAATAT 2100 CCACTGGATA 2150 ATCTGCTGAC 2200 ATATTTGGTC 2250 CACCAACAGC 2300 CCATTTTCAT 2350 TACCCCAATA 2400 TTCCACAGGT 2450 CCTGTGGATG 2500 AGGCAGAAGT 2550 GAAAGTCCCC 2600 AATTAGTCAG 2650 AACTCCGCCC 2700 TTATTTATGC 2750 TAGTGAGGAG 2800 ACCGAGTACA 2850 GGCCGTACGC 2900 ACACCGTCGA 2950 CTCTTCCTCA 3000 CGACGGCGCC 3050 GGGCGGTGTT 3100 CGGCTGGCCG 3150 CAAGGAGCCC 3200 9 GCGTGGTTCC TGGCCACCGT CGGCGTCTCG TCTGGGCAGC GCCGTCGTGC TCCCCGGAGT GGGTGCCCGC CTTCCTGGAG ACCTCCGCGC GAGCGGCTCG GCTTCACCGT CACCGCCGAC CGCGACCTGG TGCATGACCC GCAAGCCCGG ACCCGCAGCG CCCGACCGAA AGGAGCGCAC TAAA.AGATTT TATTTAGTCT CCAGAAAAAG CTGTAGGTTT GGCAAGCTAG AACTTGTTTA

AAATAAAGCA

GCATTCTAGT

GGATCGATCC

CCTCTTCGTT

AAGACGCTGG

TATTTCTTCC

TGAAGCAGCC

GAGCCAATCA

CAGAACATAT

CTCGGGGCCG

CTGGATGGCC

TGCCCGCGTT

GGACAGCTTC

AGGAACCGTA

CCCTGACGAG

CGACAGGACT

CGCTCTCCTG

CCCTTCGGGA

GTTCGGTGTA

GTTCAGCCCG

CCCGGTAAGA

ATAGCATCAC AAATTTCACA TGTGGTTTGT CCAAACTCAT CGCCATGGTA TCAACGCCAT GTGTAGGTAC CCCGGGTTCG GCGGGGTTTG TGTCATCATA GGGGACACCG CCAGCAAACG CGGCGGCACC TCGCTAACGG ATTCTTGCGG AGAACTGTGA CCATCGCGTC CGCCATCTCC ACGCGCTGGG CTACGTCTTG TTCCCCATTA TGATTCTTCT GCAGGCCATG CTGTCCAGGC AAGGATCGCT CGCGGCTCTT AAAAGGCCGC GTTGCTGGCG CATCACAAAA ATCGACGCTC ATAAAGATAC CAGGCGTTTC TTCCGACCCT GCCGCTTACC AGCGTGGCGC TTTCTCATAG GGTCGTTCGC TCCAAGCTGG ACCGCTGCGC CTTATCCGGT CACGACTTAT CGCCACTGGC CCCGACCACC AGGGCAAGGG GGAGGCGGCC GAGCGCGCCG CCCGCAACCT CCCCTTCTAC GTCGAGTGCC CGAAGGACCG TGCCTGACGC CCGCCCCACG GACCCCATGC ATCGATAAAA GGGGGAATGA AAGACCCCAC TTGCAGCTTA TAATGGTTAC AATAAAGCAT TTTTTTCACT CAATGTATCT TATCATGTCT ATTTCTATTT ACAGTAGGGA AAATCGAATT CGCCAATGAC GAACTAAAGA CATGCAAATA CGAGCAACGG GCCACGGGGA ATTCACCACT CCAAGAATTG ATGCGCAAAC CAACCCTTGG AGCAGCCGCA CGCGGCGCAT CTGGCGTTCG CGACGCGAGG CGCTTCCGGC GGCATCGGGA AGGTAGATGA CGACCATCAG ACCAGCGCCA GCAAAAGGCC TTTTTCCATA GGCTCCGCCC AAGTCAGAGG TGGCGAAACC CCCCTGGAAG CTCCCTCGTG GGATACCTGT CCGCCTTTCT CTCACGCTGT AGGTATCTCA GCTGTGTGCA CGAACCCCCC AACTATCGTC TTGAGTCCAA AGCAGCCACT GGTAACAGGA 3250 3300 3350 3400 3450 3500 3550 3600 3650 3700 3750 3800 3850 3900 3950 4000 4050 4100 4150 4200 4250 4300 4350 4400 4450 4500 4550 4600 4650 TTAGCAGAGC GAGGTATGTA CCTAACTACG GCTACACTAG GAAGCCAGTT ACCTTCGGAA AAACCACCGC TGGTAGCGGT CGCAGAAAAA AAGGATCTCA TGACGCTCAG TGGAACGAAA TATCAAAAAG GATCTTCACC AAATCAATCT AAAGTATATA CTTAATCAGT GAGGCACCTA TAGTTGCCTG ACTCCCCGTC CCATCTGGCC CCAGTGCTGC TCCAGATTTA TCAGCAATAA GTGGTCCTGC AACTTTATCC GAAGCTAGAG TAAGTAGTTC CATTGCTGCA GGCATCGTGG TCAGCTCCGG TTCCCAACGA TGCAAAAAAG CGGTTAGCTC GTTGGCCGCA GTGTTATCAC TTACTGTCAT GCCATCCGTA ACCAAGTCAT TCTGAGAATA GGCGTCAACA CGGGATAATA TCATCATTGG AAAACGTTCT CTGTTGAGAT CCAGTTCGAT AGCATCTTTT ACTTTCACCA AAAATGCCGC AAAAAAGGGA ATACTCTTCC TTTTTCAATA CATGAGCGGA TACATATTTG TTCCGCGCAC ATTTCCCCGA ATTATCATGA CATTAACCTA TCTTCAAGAA TTCTCATGTT

GGCGGTGCTA

AAGGACAGTA

AAAGAGTTGG

GGTTTTTTTG

AGAAGATCCT

ACTCACGTTA

TAGATCCTTT

TGAGTAAACT

TCTCAGCGAT

GTGTAGATAA

AATGATACCG

ACCAGCCAGC

GCCTCCATCC

GCCAGTTAAT

TGTCACGCTC

TCAAGGCGAG

CTTCGGTCCT

TCATGGTTAT

AGATGCTTTT

GTGTATGCGG

CCGCGCCACA

TCGGGGCGAA

GTAACCCACT

GCGTTTCTGG

ATAAGGGCGA

TTATTGAAGC

AATGTATTTA

AAAGTGCCAC

TAAAAATAGG

TGACAGCTTA

94 CAGAGTTCTT GAAGTGGTGG TTTGGTATCT GCGCTCTGCT TAGCTCTTGA TCCGGCAAAC TTTGCAAGCA GCAGATTACG TTGATCTTTT CTACGGGGTC AGGGATTTTG GTCATGAGAT TAAATTAAAA ATGAAGTTTT TGGTCTGACA GTTACCAATG CTGTCTATTT CGTTCATCCA CTACGATACG GGAGGGCTTA CGAGACCCAC GCTCACCGGC CGGAAGGGCC GAGCGCAGAA AGTCTATTAA TTGTTGCCGG AGTTTGCGCA ACGTTGTTGC GTCGTTTGGT ATGGCTTCAT TTACATGATC CCCCATGTTG CCGATCGTTG TCAGAAGTAA GGCAGCACTG CATAATTCTC CTGTGACTGG TGAGTACTCA CGACCGAGTT GCTCTTGCCC TAGCAGAACT TTAAAAGTGC AACTCTCAAG GATCTTACCG CGTGCACCCA ACTGATCTTC GTGAGCAAAA ACAGGAAGGC CACGGAAATG TTGAATACTC ATTTATCAGG GTTATTGTCT GAAAAATAAA CAAATAGGGG CTGACGTCTA AGAAACCATT CGTATCACGA GGCCCTTTCG TCGTAGACAT CATGCGTGCT 4700 4750 4800 4850 4900 4950 5000 5050 5100 5150 5200 5250 5300 5350 5400 5450 5500 5550 5600 5650 5700 5750 5800 5850 5900 5950 6000 6050 6100 6150 GTTGGTGTAT TTCTGGCCAT CTGTCTTGTC ACCATTTTCG TCCTCCCAAC 6200

ATGGGGCAAT

AACACCTTCT

CAGACATGCG

GAGCAACCAG

AGGTGGCGGC

ATATGCTGAC

TTAGGATAGC

CAGATATAGA

ATATACTACC

TTAGGATAGC

CAGATATAGA

GCTACCCAGA

GATAGCATAT

TATAGATTAG

GCTACCCAGA

GATAGCATAT

TATAGATTAG

CCCATGGCAA

GACCAACAAC

CTCCAGATCG

GCAGGTATTC

CCGCAGTGGT

TACAGTCCAA

TCCACAATTT

ATTGGCGTGG

GAGTCCGCTG

TGTAACAACA

TAACCCCAGT

TGGGCATACC

CGCGTTGGAA

ACGGCTTTAG

CAGGAAAAGG

ATATGCAAAG

TGTATATGCA

ATATACTACC

TTAGGATAGC

CAGATATAGA

CTATGCTACC

TTAGGATAGC

TATAAATTAG

GCTACCCGGA

GATAGCATAT

TATAAATTAG

GCTACCCAGA

GATAGCATAT

CATTAGCCCA

CCTGTGCTTG

CAGCAATCGC

CCCGGGGTGC

TAGCGGGGTT

AACCGCAGGG

CAAAAAAAAG

AGCCCCGTTT

CTGTCGGCGT

TGGTTCACCT

ATCATATTGC

CATGTTGTCA

AACATTAGCG

CCTGGCCTCC

ACAAGCAGCG

GATAGCACTC

TGAGGATAGC

CAGATATAGA

CTATGCTACC

TTAGGATAGC

CAGATATAGA

ATATGCTATC

GATAGCATAT

TACAGATTAG

GCTACCCAGA

GATAGCATAT

TATAGATTAG

GCTATCCAGA

CCGTGCTCTC

GCGCTCAGGC

GCCCCTATCT

CATTAGTGGT

ACAATCAGCC

CGGCGTGTGG

AGTGGCCACT

AATTTTCGGG

CCACTCTCTT

GTCTTGGTCC

ACTAGGATTA

CGTCACTCAG

ACATTTACCT

TTAAATTCAC

AAA.ATTCACG

CCACTCTACT

ATATGCTACC

TTAGGATAGC

CAGATATAAA

ATATGCTACC

TTAGGATAGC

CAGATATTTG

ACTACCCTAA

GATAGCATAT

TATAGATTAG

ACTACCCAGA

GATAGCCTAT

TATTTGGGTA

AGCGACCTCG

GCAAGTGTGT

TGGCCCGCCC

TTTGTGGGCA

AAGTTATTAC

GGGCTGACGC

TGTCTTTGTT

GGTGTTAGAG

TCCCCTTGTT

CTGCCTGGGA

TGTGTTGCCC

CTCCGCGCTC 6250 GGTGAGCAAT 6300 CTAAGAATGG 6350 CCCCCTTGGG 6400 ACTGGGTATC 6450 CGGATACAGA 6500 ATATGCTACC 6550 TTAGGATAGC 6600 CAGATATAGA 6650 ATATGCTACC 6700 GGTAGTATAT 6750 TCTCTATTAG 6800 ACTACCCAGA 6850 GATAGCCTAT 6900 TATAGATTAG 6950 GCTACCCAGA 7000 GTATATGCTA 7050 TGAATATGAG 7100 GTAATTTGTC 7150 ACCTACTTAT 7200 AGTGGTTTGA 7250 ACCCTTATTT 7300 GTGCCCCCAC 7350 TATGGGCCCC 7400 ACAACCAGTG 7450 ACAAATAGAG 7500 CACATCTTAA 7550 ATAGCCATAA 7600

ATTCGTGTGA

ATTTCTATTG

ATTGCCCAAG

CCACTGAACC

TGTTTTC GAG

TCTATTAGCT

GTTCACTGCC

GTTCACTACC

CTCATGGGGT

AATTCGATAG

GTTA.GTCTGG

TAGGGTTAAC

GGTCCGCTTA

CCTCCCGTAG

GTGTAAGAGC

GTCCACAGAC

AAATGTGCAC

TTGTGTTTGG

AGTTGTACCA

GATGGACATC CAGTCTTTAC GGCTTGTCCC CACCCCATGG TTAAAGATAT TCAGAATGTT TCATTCCTAC ACTAGTATTT GGGTTTGTGA GGGTTATATT GGTGTCATAG CACAATGCCA CCCCGTCCAA ATTTTATTCT GGGGGCGTCA CCTGAAACCT CACCTCACAT ACACCTTACT GTTCACAACT CAGCAGTTAT AAACGAAGGA GAATGAAGAA GCAGGCGAAG ATTCAGGAGA CGCTCCTTGA TCTTCAGCCA CTGCCCTTGT GACTAAAATG CTCGTGGAAT CCTGACCCCA TGTAAATAAA ACCGTGACAG GGGAGATATC GCTGTTCCTT AGGACCCTTT TACTAACCCT CATATGCTTC CCGTTGGGTA ACATATGCTA TTGAATTAGG ATAGTATATA CTACTACCCG GGAAGCATAT GCTACCCGTT AAGGGGGCCT TATAAACACT ATTGCTAATG CCCTCTTGAG TCGGTAGCTA CACAGGCCCC TCTGATTGAC GTTGGTGTAG, TCTTCCTGGG CCCCTGGGAG GTACATGTCC CCCAGCATTG TTCAGCCAAG AGTTACACAT AAAGGCAATG TTGTGTTGCA TGCAAAGTCT GCTCCAGGAT GAAAGCCACT CAGTGTTGGC ATCCATTTAT AAGGATGTCA ACTACAGTCA GAGAACCCCT TCCCCCCCCG TGTCACATGT GGAACAGGGC CCAGTTGGCA ACCAACTGAA GGGATTACAT GCACTGCCCC 7650 7700 7750 7800 7850 7900 7950 8000 8050 8100 8150 8200 8250 8300 8350 8400 8450 8500 8540 9* INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 22 bases TYPE: Nucleic acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: PCR primer N-terminus of

(MASQKR)

(iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: CATATGGCGT CACAGAAGAG AC 22 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 26 bases TYPE: Nucleic acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: Other nucleic acid DESCRIPTION: MBP C-terminal (PMARR) PCR primer (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: Yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GGATCCTTAG CGTCTAGCCA TGGGTG 26 1.

e INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 36 bases TYPE: Nucleic acid STRANDEDNESS: Single S. TOPOLOGY: Linear (ii) MOLECULE TYPE: Other nucleic acid DESCRIPTION: PCR mutagenic Ser 81 primer (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: Yes (xi) SEQUENCE DESCRIPTION: SEQ ID GTCTTTGTAC ATGTTCGACA GGCCCGGCTG GCTACG 36 e INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 14 bases TYPE: Nucleic acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: Other nucleic acid DESCRIPTION: PCR primer for Ser mutagenesis (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: CAGCACCATG GACC 14 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 128 bases TYPE: Nucleic acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: Other nucleic acid DESCRIPTION: X2 PCR primer (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: GGTGCGCCAA AGCGGGGCTC TGGCAAGGTA CCCTGGCTAA AGCCGGGCCG GAGCCCTCTG CCCTCTCATG CCCGCAGCCA GCCTGGGCTG TGCAACATGT 100 ACAAGGACTC ACACCACCCG GCAAGAAC 128 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 19 bases TYPE: Nucleic acid STRANDEDNESS: Single S. TOPOLOGY: Linear S(ii) MOLECULE TYPE: Other nucleic acid DESCRIPTION: T7 terminator primer (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: Yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: GCTAGTTATT GCTCAGCGG 19 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 20 bases TYPE: Nucleic acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: Other nucleic acid DESCRIPTION: T7 promoter primer T7PP (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: TAATACGACT CACTATAGGG 0 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 39 bases TYPE: Nucleic acid STRANDEDNESS: Single TOPOLOGY: Linear (ii) MOLECULE TYPE: Other nucleic acid DESCRIPTION: 3' primer for X2 assembly (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID GGCTTTAGCC AGGGTACCTT GCCAGAGCCC CGCTTTGGC 39 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 5248 base pairs TYPE: Nucleic Acid STRANDEDNESS: Double TOPOLOGY: Circular (ii) MOLECULE TYPE: Other nucleic acid DESCRIPTION: pET Trc SOS/NI prokaryotic expression vector (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: TGGCGAATGG GACGCGCCCT GTAGCGGCGC ATTAAGCGCG GCGGGTGTGG TGGTTACGCG CAGCGTGACC GCTACACTTG CCAGCGCCCT AGCGCCCGCT 10 CCTTTCGCTT TCTTCCCTTC CTTTCTCGCC ACGTTCGCCG GCTTTCCCCG 15 TCAAGCTCTA AATCGGGGGC TCCCTTTAGG GTTCCGATTT AGTGCTTTAC 20 GGCACCTCGA CCCCAAAAAA CTTGATTAGG GTGATGGTTC ACGTAGTGGG 25 CCATCGCCCT GATAGACGGT TTTTCGCCCT TTGACGTTGG AGTCCACGTT 30 CTTTAATAGT GGACTCTTGT TCCAAACTGG AACAACACTC AACCCTATCT 35 CGGTCTATTC TTTTGATTTA TAAGGGATTT TGCCGATTTC GGCCTATTGG 40 TTAAAAAATG AGCTGATTTA ACAAAAATTT AACGCGAATT TTAACAAAAT 45 0 0 0 0 0 0 0 0 Is S S S. S S S

S*

S S

S

ATTAACGTTT ACAATTTCAG GTGGCACTTT CCCCTATTTG TTTATTTTTC

SS

55 5 S S 9 5*

AGACAATAAC

GAGTATTCAA

GCCTTCCTGT

GAAGATCAGT

CGGTAAGATC

GCACTTTTAA

GGGCAAGAGC

TGAGTACTCA

GAGAATTATG

TTACTTCTGA

CAACATGGGG

ATGAAGCCAT

CCTGATAAAT

CATTTCCGTG

TTTTGCTCAC

TGGGTGCACG

CTTGAGAGTT

AGTTCTGCTA

AACTCGGTCG

CCAGTCACAG

CAGTGCTGCC

CAACGATCGG

GATCATGTAA

ACCAAACGAC

TAAATACATT

GCTTCAATA.A

TCGCCCTTAT

CCAGAAACGC

AGTGGGTTAC

TTCGCCCCGA

TGTGGCGCGG

CCGCATACAC

AAAAGCATCT

ATAACCATGA

AGGACCGAAG

CTCGCCTTGA

GAGCGTGACA

TCGGGGAAAT GTGCGCGGAA CAAATATGTA TCCGCTCATG TATTGAAAAA GGAAGAGTAT TCCCTTTTTT GCGGCATTTT TGGTGAAAGT AA.AAGATGCT ATCGAACTGG ATCTCAACAG AGAACGTTTT CCAATGATGA TATTATCCCG TATTGACGCC TATTCTCAGA ATGACTTGGT TACGGATGGC ATGACAGTAA GTGATAACAC TGCGGCCAAC GAGCTAACCG CTTTTTTGCA TCGTTGGGAA CCGGAGCTGA CCACGATGCC TGCAGCAATG 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 100 S. OS 0 5

S

00 0 @5 05 @5 5

OS

55 *550 5

SSSS

00 @0 5 5

S

GCAACAACGT

CCGGCAACAA

TTCTGCGCTC

GCCGGTGAGC

TAAGCCCTCC

TGGATGAACG

CATTGGTAAC

AAAACTTCAT

ATCTCATGAC

GACCCCGTAG

-CGTAATCTGC

GTTTGCCGGA

AGCAGAGCGC

CCACCACTTC

TCCTGTTACC

TTGGACTCAA

GGGGGGTTCG

TGAGATACCT

AGAAAGGCGG

CACGAGGGAG

GGTTTCGCCA

GGGCGGAGCC

GGCCTTTTGC

ATTCTGTGGA

CGCAGCCGAA

GCGCCTGATG

GCATATATGG

GCCAGTATAC

GACACCCGCC

TGCGCAAACT

TTAATAGACT

GGCCCTTCCG

GTGGGTCTCG

CGTATCGTAG

AA.ATAGACAG

TGTCAGACCA

TTTTAATTTA

CAAAATCCCT

AAAAGATCAA

TGCTTGCAAA

TCAAGAGCTA

AGATACCAAA

AAGAACTCTG

AGTGGCTGCT

GACGATAGTT

TGCACACAGC

ACAGCGTGAG

ACAGGTATCC

CTTCCAGGGG

CCTCTGACTT

TATGGAAAAA

TGGCCTTTTG

TAACCGTATT

CGACCGAGCG

CGGTATTTTC

TGCACTCTCA

ACTCCGCTAT

AACACCCGCT

ATTAACTGGC

GGATGGAGGC

GCTGGCTGGT

CGGTATCATT

TTATCTACAC

ATCGCTGAGA

AGTTTACTCA

AAAGGATCTA

TAACGTGAGT

AGGATCTTCT

CAAAAAAACC

CCAACTCTTT

TACTGTCCTT

TAGCACCGCC

GCCAGTGGCG

ACCGGATAAG

CCAGCTTGGA

CTATGAGAAA

GGTAAGCGGC

GAAACGCCTG

GAGCGTCGAT

CGCCAGCAAC

CTCACATGTT

ACCGCCTTTG

CAGCGAGTCA

TCCTTACGCA

GTACAATCTG

CGCTACGTGA

GACGCGCCCT

GAACTACTTA

GGATAAAGTT

TTATTGCTGA

GCAGCACTGG

GACGGGGAGT

TAGGTGCCTC

TATATACTTT

GGTGAAGATC

TTTCGTTCCA

TGAGATCCTT

ACCGCTACCA

TTCCGAAGGT

CTAGTGTAGC

TACATACCTC

ATAAGTCGTG

GCGCAGCGGT

GCGAACGACC

GCGCCACGCT

AGGGTCGGAA

GTATCTTTAT

TTTTGTGATG

GCGGCCTTTT

CTTTCCTGCG

AGTGAGCTGA

GTGAGCGAGG

TCTGTGCGGT

CTCTGATGCC

CTGGGTCATG

GACGGGCTTG

CTCTAGCTTC

GCAGGACCAC

TAAATCTGGA

GGCCAGATGG

CAGGCAACTA

ACTGATTAAG

AGATTGATTT

CTTTTTGATA

CTGAGCGTCA

TTTTTCTGCG

GCGGTGGTTT

AACTGGCTTC

CGTAGTTAGG

GCTCTGCTAA

TCTTACCGGG

CGGGCTGAAC

TACACCGAAC

TCCCGAAGGG

CAGGAGAGCG

AGTCCTGTCG

CTCGTCAGGG

TACGGTTCCT

TTATCCCCTG

TACCGCTCGC

AAGCGGAAGA

ATTTCACACC

GCATAGTTAA

GCTGCGCCCC

TCTGCTCCCG

1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000 2050 2100 2150 2200 2250 2300 2350 2400 2450 2500 2550 2600 S @5 0 0 S 55 @0 0 0* 0 05 101 GCATCCGCTT ACAGACAAGC TGTGACCGTC TCCGGGAGCT GCATGTGTCA

GAGGTTTTCA

CATCAGCGTG

TCCAGCTCGT

GCGGGCCATG

TGTAAGGGGG

AGGATGCTCA

ACGTTGTGAG

AAATCACTCA

CCACAGGGTA

GCAGGGCGCT

AGACCATTCA

CTTCACGTTC

ACCCCGCCAG

CCCGTGGGGC

TTGGTGGCGG

GAATACCGCA

CCTCGCCGAA

ATGATAAAGA

CCACCGGAAG

ATCCCGGTGC

CTGCCCGCTT

CGGCCAACGC

TTTCTTTTCA

GCCCTGAGAG

GAAAATCCTG

TCGGTATCGT

GGACTCGGTA

CCAGCATCGC

TGAAAACCGG

CCGTCATCAC

GTCGTGAAGC

TGAGTTTCTC

TTAAGGGCGG

ATTTCTGTTC

CGATACGGGT

GGTAAACAAC

GGGTCAATGC

GCCAGCAGCA

GACTTCCGCG

TGTTGTTGCT

GCTCGCGTAT

CCTAGCCGGG

CGCCATGCCG

GACCAGTGAC

AGCGACAGGC

AATGACCCAG

AGACAGTCAT

GAGCTGACTG

CTAATGAGTG

TCCAGTCGGG

GCGGGGAGAG

CCAGTGAGAC

AGTTGCAGCA

TTTGATGGTG

CGTATCCCAC

ATGGCGCGCA

AGTGGGAACG

ACATGGCACT

CGAAACGCGC

GATTCACAGA

CAGAAGCGTT

TTTTTTCCTG

ATGGGGGTAA

TACTGATGAT

TGGCGGTATG

CAGCGCTTCG

TCCTGCGATG

TTTCCAGACT

CAGGTCGCAG

CGGTGATTCA

TCCTCAACGA

GCGATAATGG

GAAGGCTTGA

CGATCATCGT

AGCGCTGCCG

AAGTGCGGCG

GGTTGAAGGC

AGCTAACTTA

AAACCTGTCG

GCGGTTTGCG

GGGCAACAGC

AGCGGTCCAC

GTTAACGGCG

TACCGAGATA

TTGCGCCCAG

ATGCCCTCAT

CCAGTCGCCT

102

GAGGCAGCTG

TGTCTGCCTG

AATGTCTGGC

TTTGGTCACT

TGATACCGAT

GAACATGCCC

GATGCGGCGG

TTAATACAGA

CAGATCCGGA

TTACGAAACA

ACGTTTTGCA

TTCTGCTA.AC

CAGGAGCACG

CCTGCTTCTC

GCGAGGGCGT

CGCGCTCCAG

GCACCTGTCC

ACGATAGTCA

TCTCAAGGGC

CATTAATTGC

TGCCAGCTGC

TATTGGGCGC

TGATTGCCCT

GCTGGTTTGC

GGATATAACA

TCCGCACCAA

CGCCATCTGA

TCAGCATTTG

TCCCGTTCCG

CGGTAAAGCT

TTCATCCGCG

TTCTGATAAA

GATGCCTCCG

GAAACGAGAG

GGTTACTGGA

GACCAGAGAA

TGTAGGTGTT

ACATAATGGT

CGGAAACCGA

GCAGCAGTCG

CAGTAAGGCA

ATCATGCGCA

GCCGAAACGT

GCAAGATTCC

CGAAAGCGGT

TACGAGTTGC

TGCCCCGCGC

ATCGGTCGAG

GTTGCGCTCA

ATTAATGAAT

CAGGGTGGTT

TCACCGCCTG

CCCAGCAGGC

TGAGCTGTCT

CGCGCAGCCC

TCGTTGGCAA

CATGGTTTGT

CTATCGGCTG

2650 2700 2750 2800 2850 2900 2950 3000 3050 3100 3150 3200 3250 3300 3350 3400 3450 3500 3550 3600 3650 3700 3750 3800 3850 3900 3950 4000 4050 4100

AATTTGATTG

CCGAGACAGA

AATGCGACCA

AATAATACTG

GAACATTAGT

GGATAGTTAA

CGCCGCTTTA

CGCTGGCACC

GACGGCGCGT

CTGTTTGCCC

CCGCCATCGC

GCCTGGTTCA

TGCGACATCG

TCTCTTCCGG

ATGGTGTCCG

AGCAGCCCAG

GGTGCATGCG

GTACTGTGTG

TTTGTTTAAC

TGAATTCGAG

CACCACCACT

GGCTGCTGCC

AACGGGTCTT

CGAGTGAGAT

ACTTAATGGG

GATGCTCCAC

TTGATGGGTG

GCAGGCAGCT

TGATCAGCCC

CAGGCTTCGA

CAGTTGATCG

GCAGGGCCAG

GCCAGTTGTT

CGCTTCCACT

CCACGCGGGA

TATAACGTTA

GCGCTATCAT

GGATCTCGAC

TAGTAGGTTG

GTACCAGCTG

GAATTGTGAG

TTTAAGAAGG

CTCCGTCGAC

GAGATCCGGC

ACCGCTGAGC

GAGGGGTTTT

ATTTATGCCA

CCCGCTAACA

GCCCAGTCGC

TCTGGTC.AGA

TCCACAGCAA

ACTGACGCGT

CGCCGCTTCG

GCGCGAGATT

ACTGGAGGTG

GTGCCACGCG

TTTTCCCGCG

A.ACGGTCTGA

CTGGTTTCAC

GCCATACCGC

GCTCTCCCTT

AGGCCGTTGA

TTGACAATTA

CGCTCACAAT

AGATATACCA

AAGCTTGCGG

TGCTAACAAA

AATAACTAGC

TTGCTGAAAG

GCCAGCCAGA

GCGCGATTTG

GTACCGTCTT

GACATCAAGA

TGGCATCCTG

TGCGCGAGAA

TTCTACCATC

TAATCGCCGC

GCAACGCCAA

GTTGGGAATG

TTTTCGCAGA

TAAGAGACAC

ATTCACCACC

GAAAGGTTTT

ATGCGACTCC

GCACCGCCGC

ATCATCCGGC

TCCACACATC

TGGAGATCTG

CCGCACTCGA

GCCCGAAAGG

ATAACCCCTT

GAGGAACTAT

CGCAGACGCG

CTGGTGACCC

CATGGGAGAA

AATAACGCCG

GTCATCCAGC

GATTGTGCAC

GACACCACCA

GACAATTTGC

TCAGCAACGA

TAATTCAGCT

AACGTGGCTG

CGGCATACTC

CTGAATTGAC

GCGCCATTCG

TGCATTAGGA

CGCAAGGAAT

TCGTATAATA

TAGAAATAAT

GATCCATCGA

GCACCACCAC

AAGCTGAGTT

GGGGCCTCTA

ATCCGGAT

4150 4200 4250 4300 4350 4400 4450 4500 4550 4600 4650 4700 4750 4800 4850 4900 4950 5000 5050 5100 5150 5200 5248 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 277 amino acids TYPE: Amino Acid STRANDEDNESS: Single TOPOLOGY: Linear 103 (ii) MOLECULE TYPE: Other nucleic acid DESCRIPTION: native human PLP (iii) HYPOTHETICAL: No PUBLICATION

INFORMATION:

AUTHORS:

Kronquist, K. E.

Crandall, B. F.

Macklin, W.B.

Campagnoni, A. T.

TITLE: Expression of Myelin Proteins in the Developing Human Spinal Cord: Cloning and Sequencing of Human Proteolipid Protein cDNA JOURNAL: Journal of Neuroscience Research VOLUME: 18 PAGES: 395 401 DATE: 1987 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Met -1 Gly Leu Leu Glu Cys Cys Ala Arg Cys Leu Val Gly Ala Pro Phe 1 5 10 Ala Ser Leu Val Ala Thr Gly Leu Cys Phe Phe Gly Val Ala Leu 20 25 Phe Cys Gly Cys Gly His Glu Ala Leu Thr Gly Thr Glu Lys Leu 35 40 o Ile Glu Thr Tyr Phe Ser Lys Asn Tyr Gin Asp Tyr Glu Tyr Leu 55 Ile Asn Val Ile His Ala Phe Gin Tyr Val Ile Tyr Gly Thr Ala 65 70 Ser Phe Phe Phe Leu Tyr Gly Ala Leu Leu Leu Ala Glu Gly Phe 85 Tyr Thr Thr Gly Ala Val Arg Gin Ile Phe Gly Asp Tyr Lys Thr 100 105 Thr Ile Cys Gly Lys Gly Leu Ser Ala Thr Val Thr Gly Gly Gin 110 115 120 104 Lys Gly Arg Gly Ser Arg Gly 125 Gin His Gin Ala His Ser Leu Glu 130 Arg Val Phe Val Phe Ala Thr Thr Ile Gly Trp Asn Ile Cys Ala Ala Phe Met Gly Arg Cys His Gly Ile Cys Ser Cys Gin Ser Leu Ala Phe Lys Thr Phe Val Ile Ala Gly Thr Cys 140 Thr 155 Ala 170 Ser 185 Cys 200 Pro 215 Ala 230 Gly 245 Ala 260 Lys 275 Leu Gly Tyr Ala Val Pro Ile Ala Ala Asp Gly Lys Glu Phe Ala Ala Thr Tyr Phe Lys Leu Val Phe Ala Val Gin Ala Asn Trp Leu Gly 145 Thr Val Val 160 Tyr Ile Tyr 175 Pro Ser Lys 190 Arg Met Tyr 205 Cys Gly Ser 220 Met Thr Phe 235 Thr Leu Val 250 Phe Ala Val 265 His Trp Phe Thr Gly Asn His Ser Leu Pro Leu Asn Ser Val Leu Leu Leu Lys Asp Leu Thr Ala Leu Leu Phe Leu Leu 135 Lys 150 Val 165 Trp 180 Ser 195 Pro 210 Ser 225 Ile 240 Thr 255 Met 270 r INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 561 base pairs TYPE: Nucleic acid STRANDEDNESS: Double TOPOLOGY: Linear (ii) MOLECULE TYPE: Other nucleic acid DESCRIPTION: Delta PLP3 (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: ATG CTC Met Leu 1 105 GAG GAT CCG GGA CAT GAA Glu Asp Pro Gly His Glu ATT GAG ACC TAT TTC TCC Ile Glu Thr Tyr Phe Ser CTC ATC AAT GTG ATC CAT Leu Ile Asn Val Ile His ACT GCC Thr Ala GAG GGC Glu Gly TCT TTC TTC TTC Ser Phe Phe Phe 50 TTC TAC ACC ACC Phe Tyr Thr Thr r r r r GCC CTC ACT GGC Ala Leu Thr Gly 10 AAA AAC TAC CAA Lys Asn Tyr Gin 25 GCC TTC CAG TAT Ala Phe Gin Tyr CTT TAT GGG GCC Leu Tyr Gly Ala GGC GCA GTC AGG Gly Ala Val Arg 65 TGC GGC AAG GGC Cys Gly Lys Gly 80 GGG AGG GGT TCC Gly Arg Gly Ser 95 CGG GTG TGT CAT Arg Val Cys His 110 AAG TTT GTG GGC Lys Phe Val Gly ATT GCC TTC CCC Ile Ala Phe Pro 135 ACA GAA AAG CTA Thr Glu Lys Leu GAC TAT GAG TAT Asp Tyr Glu Tyr GTC ATC TAT GGA Val Ile Tyr Gly CTC CTG CTG GCT Leu Leu Leu Ala CAG ATC Gin Ile TTT GGC Phe Gly 48 132 174 216 258 300 GAC TAC AAG Asp Tyr Lys GTA ACA GGG Val Thr Gly CAA GCT CAT Gin Ala His ACC ACC ATC Thr Thr Ile GGC CAG AAG Gly Gin Lys TCT TTG GAG Ser Leu Glu 105 TGG CTA Trp Leu 115 TGG ACC Trp Thr 130 GGA CAT CCC GAC Gly His Pro Asp 120 ACC TGC CAG TCT Thr Cys Gin Ser CTG AGC GCA ACG Leu Ser Ala Thr AGA GGC CAA CAT Arg Gly Gin His 100 TGT TTG GGA AAA Cys Leu Gly Lys ATC TTC AAC ACC Ile Phe Asn Thr 125 AGC AAG ACC TCT Ser Lys Thr Ser 140 AGA ATG TAT GGT Arg Met Tyr Gly 155 GTT TGT GGC TCC Val Cys Gly Ser 170 342 384 426 GCC AGT ATA Ala Ser Ile 145 GTT CTC CCA Val Leu Pro AAC CTT CTG Asn Leu Leu TTC CAC TAA Phe His 185 GGC AGT CTC Gly Ser Leu TGG AAT GCT Trp Asn Ala 160 TCC ATC TGC Ser Ile Cys 175 TGT GCT GAC GCC Cys Ala Asp Ala 150 TTC CCT GGC AAG Phe Pro Gly Lys 165 468 510 AAA ACA GCT GAG TTC CAA ATG ACC Lys Thr Ala Glu Phe Gin Met Thr 180 552 561 106 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 525 base pairs TYPE: Nucleic acid STRANDEDNESS: Double TOPOLOGY: Linear (ii) MOLECULE TYPE: Other nucleic acid DESCRIPTION: Delta PLP4 (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: ATG CTC GAG GAT CCG GGA CAT Met Leu Glu Asp Pro Gly His 1 5 GAA AAG CTA ATT GAG ACC TAT TTC Glu Lys Leu Ile Glu Thr Tyr Phe 20 TAT GAG TAT CTC ATC AAT GTG ATC Tyr Glu Tyr Leu Ile Asn Val Ile 35 GAG GGC TTC TAC ACC ACC GGC GCA Glu Gly Phe Tyr Thr Thr Gly Ala GAC TAC AAG ACC ACC ATC TGC GGC Asp Tyr Lys Thr Thr Ile Cys Gly GTA ACA GGG GGC CAG AAG GGG AGG Val Thr Gly Gly Gin Lys Gly Arg 70 75 CAA GCT CAT TCT TTG GAG CGG GTG Gin Ala His Ser Leu Glu Arg Val 90 TGG CTA GGA CAT CCC GAC AAG TTT Trp Leu Gly His Pro Asp Lys Phe 100 105 TGG ACC ACC TGC CAG TCT ATT GCC Trp Thr Thr Cys Gin Ser Ile Ala 115 GCC AGT ATA GGC AGT CTC TGT GCT Ala Ser Ile Gly Ser Leu Cys Ala 130 GAA GCC CTC ACT GGC ACA Glu Ala Leu Thr Gly Thr TCC AAA AAC TAC CAA GAC Ser Lys Asn Tyr Gin Asp CAT GCC TTC CAG TAT GCT His Ala Phe Gin Tyr Ala GTC AGG CAG ATC TTT GGC Val Arg Gin Ile Phe Gly 50 AAG GGC CTG AGC GCA ACG Lys Gly Leu Ser Ala Thr GGT TCC AGA GGC CAA CAT Gly Ser Arg Gly Gin His TGT CAT TGT TTG GGA AAA Cys His Cys Leu Gly Lys GTG GGC ATC TTC AAC ACC Val Gly Ile Phe Asn Thr 110 TTC CCC AGC AAG ACC TCT Phe Pro Ser Lys Thr Ser 120 125 GAC GCC AGA ATG TAT GGT Asp Ala Arg Met Tyr Gly 135 39 81 123 165 207 249 291 333 375 417 107 GTT CTC CCA TGG AAT GCT TTC CCT GGC AAG GTT TGT GGC TCC 459 Val Leu Pro Trp Asn Ala Phe Pro Gly Lys Val Cys Gly Ser 140 145 150 AAC CTT CTG TCC ATC TGC AAA ACA GCT GAG TTC CAA ATG ACC 501 Asn Leu Leu Ser Ile Cys Lys Thr Ala Glu Phe Gin Met Thr 155 160 165 TTC CAC CAT CAC CAT CAC CAT TAA 525 Phe His His His His His His 170 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1155 base pairs TYPE: Nucleic acid STRANDEDNESS: Double TOPOLOGY: Linear (ii) MOLECULE TYPE: Other nucleic acid DESCRIPTION: MP3 chimera (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No *e (xi) SEQUENCE DESCRIPTION: SEQ ID ATG GCG TCT CAG AAA CGT CCG TCC CAG CGT CAC GGC TCC AAA 42 Met Ala Ser Gin Lys Arg Pro Ser Gin Arg His Gly Ser Lys 1 5 TAC CTG GCC ACC GCC AGC ACC ATG GAC CAT GCC CGT CAT GGC 84 Tyr Leu Ala Thr Ala Ser Thr Met Asp His Ala Arg His Gly 20 TTC CTG CCG CGT CAC CGT GAC ACC GGC ATC CTG GAC TCC ATC 126 Phe Leu Pro Arg His Arg Asp Thr Gly Ile Leu Asp Ser Ile 35

S

GGC CGC TTC TTC GGC GGT GAC CGT GGT GCG CCG AAA CGT GGC 168 Gly Arg Phe Phe Gly Gly Asp Arg Gly Ala Pro Lys Arg Gly 50 TCT GGC AAA GTG CCG TGG CTG AAA CCG GGC CGT AGC CCG CTG 210 Ser Gly Lys Val Pro Trp Leu Lys Pro Gly Arg Ser Pro Leu 65 CCG TCT CAT GCC CGT AGC CAG CCG GGC CTG TGC AAC ATG TAC 252 Pro Ser His Ala Arg Ser Gin Pro Gly Leu Cys Asn Met Tyr 108 AAA GAC TCC CAC CAC CCG GCT CGT ACC GCG Lys Asp Ser His His CTG CCG CAG AAA TCC Leu Pro Gin Lys Ser 100 GTG GTG CAC TTC TTC Val Val His Phe Phe 115 CCG CCG TCT CAG GGC Pro Pro Ser Gin Gly 130 TTC AGC TGG GGC GCC Phe Ser Trp Gly Ala 145 Pro Ala Arg Thr 90 CAC GGC CGT ACC His Gly Arg Thr 105 AAA AAC ATT GTG Lys Asn Ile Val 120

CAC

His CAG GAT GAA AAC CCG Gin Asp Glu Asn Pro 110 TAT GGC TCC Tyr Gly Ser ACC CCG CGT Thr Pro Arg CTG TCC CTG Leu Ser Leu ACC CCG Thr Pro 125 AGC CGT Ser Arg 140 AAA GGC CGT Lys Gly Arg

GGC

Gly 135 294 336 378 420 462 504 546 GAA GGC CAG CGT Glu Gly Gin Arg

CCG

Pro 150 GGC TTC GGT TAC Gly Phe Gly Tyr

GGC

Gly 155 GGC CGT GCG TCC Gly Arg Ala Ser GAC TAT AAA Asp Tyr Lys 160 CAG GGT ACC Gin Gly Thr 175 TCT GCT CAC Ser Ala His 165 TTG TCC AAA Leu Ser Lys AAA GGC TTC Lys Gly Phe ATT TTC AAA Ile Phe Lys 180 AAA GGC GTG GAT GCC Lys Gly Val Asp Ala 170 CTG GGC GGC CGT GAT Leu Gly Gly Arg Asp 185 CGT CTG GGA GGC CTC Arg Leu Gly Gly Leu 200 GGC ACA GAA AAG CTA Gly Thr Glu Lys Leu 215 CAA GAC TAT GAG TAT Gin Asp Tyr Glu Tyr 225 TAT GTC ATC TAT GGA Tyr Val Ile Tyr Gly 240 GCC CTC CTG CTG GCT Ala Leu Leu Leu Ala 255 AGG CAG ATC TTT GGC Arg Gin Ile Phe Gly 270 GGC CTG AGC GCA ACG Gly Leu Ser Ala Thr 285 AGC CGT TCT Ser Arg Ser 190 GAG GAT CCG Glu Asp Pro GGC TCT CCG ATG Gly Ser Pro Met

GGA

Gly 205 CAT GAA GCC His Glu Ala ATT GAG ACC TAT Ile Glu Thr Tyr CTC ATC AAT GTG Leu Ile Asn Val 230 ACT GCC TCT TTC Thr Ala Ser Phe 245 GAG GGC TTC TAC Glu Gly Phe Tyr 260 GAC TAC AAG ACC Asp Tyr Lys Thr 275

TTC

Phe 220 TCC AAA AAC TAC Ser Lys Asn Tyr GCT AGA Ala Arg 195 CTC ACT Leu Thr 210 ATC CAT GCC TTC CAG Ile His Ala Phe Gin 235 TTC TTC CTT TAT GGG Phe Phe Leu Tyr Gly 250 588 630 672 714 756 798 840 ACC ACC GGC Thr Thr Gly ACC ATC TGC Thr Ile Cys GCA GTC Ala Val 265 GGC AAG Gly Lys 280 AGG GGT Arg Gly GTA ACA GGG GGC CAG AAG GGG Val Thr Gly Gly Gin Lys Gly 290 882 109

TCC

Ser 295 AGA GGC CAA CAT CAA GCT CAT TCT Arg Gly Gin His Gin Ala His Ser 300 CAT TGT TTG GGA AAA TGG CTA GGA CAT His Cys Leu Gly Lys Trp Leu Gly His 310 315 GGC ATC TTC AAC ACC TGG ACC ACC TGC Gly Ile Phe Asn Thr Trp Thr Thr Cys 325 330 CCC AGC AAG ACC TCT GCC AGT ATA GGC Pro Ser Lys Thr Ser Ala Ser Ile Gly 340 345 GCC AGA ATG TAT GGT GTT CTC CCA TGG Ala Arg Met Tyr Gly Val Leu Pro Trp 355 TTG GAG CGG GTG TGT Leu Glu Arg Val Cys 305 CCC GAC AAG TTT GTG Pro Asp Lys Phe Val 320 CAG TCT ATT GCC TTC Gin Ser Ile Ala Phe 335 AGT CTC TGT GCT GAC Ser Leu Cys Ala Asp 350 AAT GCT TTC CCT GGC Asn Ala Phe Pro Gly 360 ATC TGC AAA ACA GCT Ile Cys Lys Thr Ala 375 924 966 1008 1050 1092 1134 1155

AAG

Lys 365 GTT TGT GGC TCC AAC CTT CTG TCC Val Cys Gly Ser Asn Leu Leu Ser 370 GAG TTC CAA ATG ACC TTC CAC Glu Phe Gin Met Thr Phe His 380 385 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 1122 base pairs TYPE: Nucleic acid STRANDEDNESS: Double TOPOLOGY: Linear (ii) MOLECULE TYPE: Other nucleic acid DESCRIPTION: MP4 chimera (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: ATG GCG TCT CAG AAA CGT CCG TCC CAG CGT CAC GGC Met Ala Ser Gin Lys Arg Pro Ser Gin Arg His Gly 1 5 9c 9

TCC

Ser AAA TAC CTG GCC ACC GCC AGC ACC ATG GAC CAT GCC CGT CAT Lys Tyr Leu Ala Thr Ala Ser Thr Met Asp His Ala Arg His 20 110 GGC TTC CTG CCG CGT CAC Gly Phe Leu Pro Arg His CGT GAC ACC GGC ATC CTG GAC TCC Arg Asp Thr Gly Ile Leu Asp Ser 35 123 ATC GGC CGC TTC TTC Ile Gly Arg GGC TCT GGC- Gly Ser Gly Phe Phe AAA GTG Lys Val GGC GGT GAC Gly Gly Asp CCG TGG CTG Pro Trp Leu CGT GGT GCG CCG AAA CGT Arg Gly Ala Pro Lys Arg 50 AAA CCG Lys Pro GGC CGT AGC CCG Gly Arg Ser Pro 165 207 249 291

CTG

Leu CCG TCT CAT GCC Pro Ser His Ala

CGT

Arg 75 AGC CAG CCG GGC Ser Gin Pro Gly

CTG

Leu TGC AAC ATG Cys Asn Met CAC TAT GGC His Tyr Gly TAC AAA Tyr Lys GAC TCC CAC CAC Asp Ser His His

CCG

Pro 90 GCT CGT ACC GCG Ala Arg Thr Ala @0 00 0 S 0 0* 0 e, 0 00 0* 0000

S

0*40 OS go 0

S

TCC CTG CCG Ser Leu Pro 100 CCG GTG GTG Pro Val Val CCG CCG CCG Pro Pro Pro CAG AAA TCC CAC Gin Lys Ser His

GGC

Gly 105 CGT ACC CAG GAT Arg Thr Gin Asp GAA AAC Glu Asn 110 CAC TTC His Phe 115 TCT CAG Ser Gin 130 TTC AAA AAC Phe Lys Asn GGC AAA GGC Gly Lys Gly ATT GTG Ile Val 120 CGT GGC Arg Gly 135 ACC CCG CGT ACC Thr Pro Arg Thr 125 CTG TCC CTG AGC Leu Ser Leu Ser 333 375 417 459 0 0000

CGT

Arg 140 TTC AGC TGG GGC Phe Ser Trp Gly

GCC

Ala 145 GAA GGC CAG CGT Giu Gly Gin Arg

CCG

Pro 150 GGC TTC GGT Gly Phe Gly CAC AAA GGC His Lys Gly 165 TAC GGC Tyr Gly 155 GGC CGT GCG TCC Gly Arg Ala Ser

GAC

Asp 160 TAT AAA TCT GCT Tyr Lys Ser Ala 0* 00 0 @0 TTC AAA GGC Phe Lys Gly 170 GTG GAT GCC CAG Val Asp Ala Gin

GGT

Gly 175 ACC TTG TCC AAA Thr Leu Ser Lys ATT TTC Ile Phe 180 AAA CTG GGC GGC CGT Lys Leu Gly Gly Arg 185 AGA CGT CTG GGA GGC Arg Arg Leu Gly Gly 200 GAT AGC CGT Asp Ser Arg CTC GAG GAT Leu Glu Asp TCT GGC Ser Gly 190 CCG GGA Pro Gly 205 TCT CCG ATG GCT Ser Pro Met Ala 195 CAT GAA GCC CTC His Glu Ala Leu TTC TCC AAA AAC Phe Ser Lys Asn 220 543 585 627 669

ACT

Thr 210 GGC ACA GAA AAG Gly Thr Glu Lys

CTA

Leu 215 ATT GAG ACC TAT Ile Giu Thr Tyr TAC CAA Tyr Gin 225 GAC TAT GAG TAT Asp Tyr Giu Tyr CTC ATC Leu Ile 230 AAT GTG ATC CAT GCC TTC Asn Val Ilie His Ala Phe 235 CAG TAT GCT GAG GGC TTC TAC ACC ACC GGC GCA GTC AGG CAG 753 Gin Tyr Ala Glu Gly Phe Tyr Thr Thr Gly Ala Val Arg Gin 240 245 250 ATC TTT GGC GAC TAC AAG ACC ACC ATC TGC GGC AAG GGC CTG 795 Ile Phe Gly Asp Tyr Lys Thr Thr Ile Cys Gly Lys Gly Leu 255 260 265 AGC GCA ACG GTA ACA GGG GGC CAG AAG GGG AGG GGT TCC AGA 837 Ser Ala Thr Val Thr Gly Gly Gin Lys Gly Arg Gly Ser Arg 270 275 GGC CAA CAT CAA GCT CAT TCT TTG GAG CGG GTG TGT CAT TGT 879 Gly Gin His Gin Ala His Ser Leu Glu Arg Val Cys His Cys 280 285 290 TTG GGA AAA TGG CTA GGA CAT CCC GAC AAG TTT GTG GGC ATC 921 Leu Gly Lys Trp Leu Gly His Pro Asp Lys Phe Val Gly Ile 295 300 305 TTC AAC ACC TGG ACC ACC TGC CAG TCT ATT GCC TTC CCC AGC 963 Phe Asn Thr Trp Thr Thr Cys Gin Ser Ile Ala Phe Pro Ser 310 315 320 SAAG ACC TCT GCC AGT ATA GGC AGT CTC TGT GCT GAC GCC AGA 1005 Lys Thr Ser Ala Ser Ile Gly Ser Leu Cys Ala Asp Ala Arg 325 330 335 ATG TAT GGT GTT CTC CCA TGG AAT GCT TTC CCT GGC AAG GTT 1047 Met Tyr Gly Val Leu Pro Trp Asn Ala Phe Pro Gly Lys Val 340 345 TGT GGC TCC AAC CTT CTG TCC ATC TGC AAA ACA GCT GAG TTC 1089 Cys Gly Ser Asn Leu Leu Ser Ile Cys Lys Thr Ala Glu Phe 350 355 360 S* CAA ATG ACC TTC CAC CAT CAC CAT CAC CAT TAA 1122 Gin Met Thr Phe His His His His His His 365 370 0 INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 1125 base pairs TYPE: Nucleic acid STRANDEDNESS: Double TOPOLOGY: Linear (ii) MOLECULE TYPE: Other nucleic acid DESCRIPTION: PM4 chimera (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No 112 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: ATG CTC GAG GAT CCG GGA CAT GAA GCC Met 1

AAG

Lys Leu Glu Asp CTA ATT GAG Leu Ile Glu Pro Gly His Glu Ala 5 CTC ACT GGC ACA GAA Leu Thr Gly Thr Glu ACC TAT TTC Thr Tyr Phe 20 TCC AAA AAC TAC CAA GAC TAT Ser Lys Asn Tyr Gin Asp Tyr GAG TAT CTC Glu Tyr Leu GGC TTC TAC Gly Phe Tyr TAC AAG ACC Tyr Lys Thr ATC AAT GTG ATC CAT GCC Ile Asn Val Ile His Ala 35 TTC CAG TAT GCT GAG Phe Gin Tyr Ala Glu CAG ATC TTT GGC GAC Gin Ile Phe Gly Asp ACC ACC GGC GCA Thr Thr Gly Ala GTC AGG Val Arg 50

ACC

Thr ACA GGG GGC CAG Thr Gly Gly Gin ATC TGC GGC Ile Cys Gly AAG GGG AGG Lys Gly Arg GAG CGG GTG Glu Arg Val 90 AAG GGC CTG AGC GCA ACG GTA Lys Gly Leu Ser Ala Thr Val 65 GGT TCC AGA GGC CAA CAT CAA Gly Ser Arg Gly Gin His Gin TGT CAT TGT TTG GGA AAA TGG Cys His Cys Leu Gly Lys Trp GTG GGC ATC TTC AAC ACC TGG Val Gly Ile Phe Asn Thr Trp 110

GCT

Ala CAT TCT TTG His Ser Leu 42 84 126 168 210 252 294 336 378 420 462 504 546 588 CTA GGA CAT Leu Gly His 100 ACC ACC TGC Thr Thr Cys 115 AGT ATA GGC Ser Ile Gly CTC CCA TGG Leu Pro Trp CTT CTG TCC Leu Leu Ser 155 CCC GAC AAG TTT Pro Asp Lys Phe 105 CAG TCT ATT GCC Gin Ser Ile Ala TTC CCC Phe Pro 120 AGC AAG ACC TCT GCC Ser Lys Thr Ser Ala 125 AGT CTC TGT GCT GAC Ser Leu Cys Ala Asp 130 AAT GCT TTC CCT GGC Asn Ala Phe Pro Gly 145 ATC TGC AAA ACA GCT Ile Cys Lys Thr Ala 160 GCC AGA ATG TAT GGT GTT Ala Arg Met Tyr Gly Val 135 140 AAG GTT TGT GGC TCC AAC Lys Val Cys Gly Ser Asn 150 GAG TTC CAA ATG ACC TTC Glu Phe Gin Met Thr Phe 165 CAC GGC GGT GGC GGT GCG TCT CAG AAA His Gly Gly Gly Gly Ala Ser Gin Lys 170 175 CAC GGC TCC AAA TAC CTG GCC ACC GCC His Gly Ser Lys Tyr Leu Ala Thr Ala 185 190 CGT CCG TCC CAG CGT Arg Pro Ser Gin Arg 180 AGC ACC ATG GAC CAT Ser Thr Met Asp His 195 113 GCC CGT CAT Ala Arg His CTG GAC TCC Leu Asp Ser GGC TTC CTG Gly Phe Leu 200 ATC GGC CGC Ile Gly Arg 215 CCG CGT CAC Pro Arg His 205 TTC TTC GGC Phe Phe Gly AAA GTG CCG Lys Val Pro CGT GAC ACC GGC ATC Arg Asp Thr Gly Ile 210 GGT GAC CGT GGT GCG Gly Asp Arg Gly Ala 220 TGG CTG AAA CCG GGC Trp Leu Lys Pro Gly 235 630 672

CCG

Pro 225 AAA CGT GGC TCT GGC Lys Arg Gly Ser Gly 230 714 CGT AGC CCG Arg Ser Pro 240 TGC AAC ATG Cys Asn Met 255 CAC TAT GGC His Tyr Gly GAT GAA AAC Asp Glu Asn CTG CCG TCT Leu Pro Ser TAC AAA GAC Tyr Lys Asp TCC CTG CCG Ser Leu Pro 270 CCG GTG GTG Pro Val Val 285 CAT GCC His Ala 245 TCC CAC Ser His 260 CAG AAA Gin Lys CGT AGC CAG CCG GGC CTG Arg Ser Gin Pro Gly Leu 250 CAC CCG GCT CGT ACC GCG His Pro Ala Arg Thr Ala 265 TCC CAC GGC CGT ACC CAG Ser His Gly Arg Thr Gin 275 280 756 798 840 882

CCG

Pro 295 CGT ACC CCG CCG CCG Arg Thr Pro Pro Pro 300 CAC TTC TTC His Phe Phe TCT CAG GGC Ser Gin Gly TGG GGC GCC Trp Gly Ala 315 AAA AAC ATT GTG ACC Lys Asn Ile Val Thr 290 AAA GGC CGT GGC CTG Lys Gly Arg Gly Leu 305 GAA GGC CAG CGT CCG Glu Gly Gin Arg Pro 320 924 966 TCC CTG AGC Ser Leu Ser 310 GGC TTC GGC Gly Phe Gly 325 CAC AAA GGC His Lys Gly CGT TTC AGC Arg Phe Ser TAC GGC GGC Tyr Gly Gly TTC AAA GGC Phe Lys Gly 340 CGT GCG Arg Ala 330 TCC GAC TAT AAA TCT GCT Ser Asp Tyr Lys Ser Ala 335 1008 1050 r GTG GAT GCC Val Asp Ala 345 GGC CGT GAT Gly Arg Asp CAG GGC ACC CTG TCC Gin Gly Thr Leu Ser 350 AGC CGT TCT GGC TCT Ser Arg Ser Gly Ser 365

AAA

Lys 355 ATT TTC AAA CTG GGC Ile Phe Lys Leu Gly 360 1092 CCG ATG GCT Pro Met Ala 370 AGA CGT CAT Arg Arg His CAC CAT His His 375 CAC CAT CAC His His His 1125 INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 1476 base pairs 114 TYPE: Nucleic acid STRANDEDNESS: Double TOPOLOGY: Linear (ii) MOLECULE TYPE: Other nucleic acid DESCRIPTION: MMOGP4 chimera (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: ATG GCG TCT CAG AAA CGT Met Ala Ser Gin Lys Arg GCC ACC GCC AGC ACC ATG Ala Thr Ala Ser Thr Met CAC CGT GAC ACC GGC ATC His Arg Asp Thr Gly Ile CCG TCC CAG CGT CAC GGC TCC AAA Pro Ser Gin Arg His Gly Ser Lys 10 TAC CTG Tyr Leu GAC CAT GCC CGT CAT GGC TTC CTG CCG CGT Asp His Ala Arg His Gly Phe Leu Pro Arg 25 r CTG GAC TCC ATC GGC CGC TTC TTC Leu Asp Ser Ile Gly Arg Phe Phe 40 GGC GGT Gly Gly GAC CGT Asp Arg 50 GGT GCG CCG AAA Gly Ala Pro Lys CGT GGC TCT GGC AAA GTG CCG TGG CTG AAA Arg Gly Ser Gly Lys Val Pro Trp Leu Lys 55 144 192 240 288

CCG

Pro GGC CGT AGC CCG CTG Gly Arg Ser Pro Leu 70 TGC AAC ATG TAC AAA GAC Cys Asn Met Tyr Lys Asp GGC TCC CTG CCG CAG AAA Gly Ser Leu Pro Gin Lys 100 GTG GTG CAC TTC TTC AAA Val Val His Phe Phe Lys 115 CCG TCT CAT GCC CGT AGC CAG CCG Pro Ser His Ala Arg Ser Gin Pro 75 TCC CAC CAC CCG GCT CGT ACC GCG Ser His His Pro Ala Arg Thr Ala 90 TCC CAC GGC CGT ACC CAG GAT GAA Ser His Gly Arg Thr Gin Asp Glu 105 110 GGC CTG Gly Leu CAC TAT His Tyr AAC CCG Asn Pro AAC ATT GTG ACC CCG CGT ACC CCG CCG CCG Asn Ile Val Thr Pro Arg Thr Pro Pro Pro 120 125 GGC CTG TCC CTG AGC CGT TTC AGC TGG GGC Gly Leu Ser Leu Ser Arg Phe Ser Trp Gly 135 140 TCT CAG Ser Gin 130 GCC GAA Ala Glu 145 GGC AAA GGC CGT Gly Lys Gly Arg GGC CAG CGT CCG Gly Gin Arg Pro 150 336 384 432 480 528 GGC TTC GGT TAC GGC GGC CGT GCG Gly Phe Gly Tyr Gly Gly Arg Ala 155 GGC TTC AAA GGC GTG GAT GCC CAG Gly Phe Lys Gly Val Asp Ala Gin 170 TCC GAC Ser Asp 160 GGT ACC Gly Thr 175 TAT AAA TCT GCT CAC AAA Tyr Lys Ser Ala His Lys 165 115 TTG TCC AAA ATT TTC AAA CTG GGC oooo oeoo o Leu Ser Lys CCG ATG GCT Pro Met Ala 195 CCT ATC CGG Pro Ile Arg 210 TCT CCT GGG Ser Pro Gly 225 CCC TTC TCT Pro Phe Ser GGA GAC CAG Gly Asp Gin GCT ATT GGT Ala Ile Gly 275 TCA GAT GAA Ser Asp Glu 290 GAG GAG GCA Glu Glu Ala 305 GAG GAT CCG Glu Asp Pro ACC TAT TTC Thr Tyr Phe ATC CAT GCC Ile His Ala 355 AGG CAG ATC Arg Gin Ile 370 AGC GCA ACG Ser Ala Thr 385 Ile Phe Lys Leu Gly 180 AGA CGT CCC GGG CAG Arg Arg Pro Gly Gin 200 GCT CTG GTC GGG GAT Ala Leu Val Gly Asp 215 AAG AAC GCT ACA GGC Lys Asn Ala Thr Gly 230 AGG GTG GTT CAT CTC Arg Val Val His Leu 245 GCA CCT GAA TAT CGG Ala Pro Glu Tyr Arg 260 GAG GGA AAG GTG ACT Glu Gly Lys Val Thr GGC CGT GAT AGC CGT TCT Gly Arg Asp Ser Arg Ser 185 190 TTC AGA GTG ATA GGA Phe Arg Val Ile Gly 205 GAA GTG GAA TTG CCA Glu Val Glu Leu Pro 220 ATG GAG GTG GGG TGG Met Glu Val Gly Trp 235 TAC AGA AAT GGC AAG Tyr Arg Asn Gly Lys 250 GGC CGG ACA GAG CTG Gly Arg Thr Glu Leu 265 CTC AGG ATC CGG AAT Leu Arg Ile Arg Asn 285 CCA AGA CAC Pro Arg His TGT CGC ATA Cys Arg Ile TAC CGC CCC Tyr Arg Pro 240 GAC CAA GAT Asp Gin Asp 255 GGC TCT Gly Ser 576 624 672 720 768 816 864

CTG

Leu 270 AAA GAT Lys Asp GGA GGT TTC ACC Gly Gly Phe Thr 295 GCA ATG GAA TTG Ala Met Glu Leu 310 GGA CAT GAA GCC Gly His Glu Ala 325 TCC AAA AAC TAC Ser Lys Asn Tyr 340 TTC CAG TAT GCT Phe Gin Tyr Ala TTT GGC GAC TAC Phe Gly Asp Tyr 375 GTA ACA GGG GGC Val Thr Gly Gly 390 TGC TTC TTC CGA GAT CAT Cys Phe Phe Arg Asp His 300 AAA GTA GAA GAT CCC TTC Lys Val Glu Asp Pro Phe 315 CTC ACT GGC ACA GAA AAG Leu Thr Gly Thr Glu Lys 330 CAA GAC TAT GAG TAT CTC Gin Asp Tyr Glu Tyr Leu 345 GTA AGG TTC Val Arg Phe TCT TAC CAA Ser Tyr Gin TAC TGG CTC Tyr Trp Leu 320 CTA ATT GAG Leu Ile Glu 335 ATC AAT GTG Ile Asn Val 350 GGC GCA GTC Gly Ala Val 1008 1056 912 960

GAG

Glu 360 GGC TTC TAC ACC ACC Gly Phe Tyr Thr Thr 365 1104 AAG ACC ACC ATC TGC GGC Lys Thr Thr Ile Cys Gly 380 CAG AAG GGG AGG GGT TCC Gin Lys Gly Arg Gly Ser 395 AAG GGC CTG 1152 Lys Gly Leu AGA GGC CAA 1200 Arg Gly Gin 400 116 CAT CAA GCT CAT TCT TTG GAG CGG GTG TGT CAT TGT TTG GGA AAA TGG 1248 His Gin Ala His Ser Leu Glu Arg Val Cys His Cys Leu Gly Lys Trp 405 410 415 CTA GGA CAT CCC GAC AAG TTT GTG GGC ATC TTC AAC ACC TGG ACC ACC 1296 Leu Gly His Pro Asp Lys Phe Val Gly Ile Phe Asn Thr Trp Thr Thr 420 425 430 TGC CAG TCT ATT GCC TTC CCC AGC AAG ACC TCT GCC AGT ATA GGC AGT 1344 Cys Gin Ser Ile Ala Phe Pro Ser Lys Thr Ser Ala Ser Ile Gly Ser 435 440 445 CTC TGT GCT GAC GCC AGA ATG TAT GGT GTT CTC CCA TGG AAT GCT TTC 1392 Leu Cys Ala Asp Ala Arg Met Tyr Gly Val Leu Pro Trp Asn Ala Phe 450 455 460 CCT GGC AAG GTT TGT GGC TCC AAC CTT CTG TCC ATC TGC AAA ACA GCT 1440 Pro Gly Lys Val Cys Gly Ser Asn Leu Leu Ser Ile Cys Lys Thr Ala 465 470 475 480 GAG TTC CAA ATG ACC TTC CAC CAT CAC CAT CAC CAT 1476 Glu Phe Gin Met Thr Phe His His His His His His 485 490 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 732 base pairs TYPE: Nucleic acid STRANDEDNESS: Double TOPOLOGY: Linear (ii) MOLECULE TYPE: Other nucleic acid DESCRIPTION: Delta PLP2 (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: ATG GGC AGC AGC CAT CAT CAT CAT CAT CAC Met Gly Ser Ser His His His His His His AGC AGC GGC CTG GTG CCG CGC GGC AGC CAT ATG CTC GAG GAT CCG Ser Ser Gly Leu Val Pro Arg Gly Ser His Met Leu Glu Asp Pro 20 GTG GCC ACT GGA TTG TGT TTC TTT GGG GTG GCA CTG TTC TGT GGC 120 Val Ala Thr Gly Leu Cys Phe Phe Gly Val Ala Leu Phe Cys Gly 35 TGT GGA CAT GAA GCC CTC ACT GGC ACA GAA AAG CTA ATT GAG ACC 165 Cys Gly His Glu Ala Leu Thr Gly Thr Glu Lys Leu Ile Glu Thr 50 117 TAT TTC TCC AAA AAC TAC CAA GAC TAT GAG TAT CTC ATC AAT GTG 210 Tyr Phe Ser Lys Asn Tyr Gin Asp Tyr Glu Tyr Leu Ile Asn Val 65 ATC CAT GCC TTC CAG TAT GTC ATC TAT GGA ACT GCC TCT TTC TTC 255 Ile His Ala Phe Gin Tyr Val Ile Tyr Gly Thr Ala Ser Phe Phe 80 TTC CTT TAT GGG GCC CTC CTG CTG GCT GAG GGC TTC TAC ACC ACC 300 Phe Leu Tyr Gly Ala Leu Leu Leu Ala Glu Gly Phe Tyr Thr Thr 95 100 GGC GCA GTC AGG CAG ATC TTT GGC GAC TAC AAG ACC ACC ATC TGC 345 Gly Ala Val Arg Gin Ile Phe Gly Asp Tyr Lys Thr Thr Ile Cys 105 110 115 GGC AAG GGC CTG AGC GCA ACG GTA ACA GGG GGC CAG AAG GGG AGG 390 Gly Lys Gly Leu Ser Ala Thr Val Thr Gly Gly Gin Lys Gly Arg 120 125 130 GGT TCC AGA GGC CAA CAT CAA GCT CAT TCT TTG GAG CGG GTG TGT 435 Gly Ser Arg Gly Gin His Gin Ala His Ser Leu Glu Arg Val Cys C135 140 145 CAT TGT TTG GGA AAA TGG CTA GGA CAT CCC GAC AAG TTT GTG GGC 480 His Cys Leu Gly Lys Trp Leu Gly His Pro Asp Lys Phe Val Gly 150 155 160 ATC ACC TAT GCC CTG ACC GTT GTG TGG CTC CTG GTG TTT GCC TGC 525 Ile Thr Tyr Ala Leu Thr Val Val Trp Leu Leu Val Phe Ala Cys 165 170 175 TCT GCT GTG CCT GTG TAC ATT TAC TTC AAC ACC TGG ACC ACC TGC 570 Ser Ala Val Pro Val Tyr Ile Tyr Phe Asn Thr Trp Thr Thr Cys S180 185 190 CAG TCT ATT GCC TTC CCC AGC AAG ACC TCT GCC AGT ATA GGC AGT 615 Gin Ser Ile Ala Phe Pro Ser Lys Thr Ser Ala Ser Ile Gly Ser 195 200 205 CTC TGT GCT GAC GCC AGA ATG TAT GGT GTT CTC CCA TGG AAT GCT 660 Leu Cys Ala Asp Ala Arg Met Tyr Gly Val Leu Pro Trp Asn Ala 210 215 220 TTC CCT GGC AAG GTT TGT GGC TCC AAC CTT CTG TCC ATC TGC AAA 705 Phe Pro Gly Lys Val Cys Gly Ser Asn Leu Leu Ser Ile Cys Lys 225 230 235 ACA GCT GAG TTC CAA ATG ACC TTC CAC 732 Thr Ala Glu Phe Gin Met Thr Phe His 240 118

Claims (51)

1. An isolated immunoreactive polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 except that amino acid 81 may be any standard amino acid.

2. The polypeptide of Claim 1 wherein the standard amino acid is not cysteine.

3. The polypeptide of Claim 1 wherein the standard amino acid is an uncharged amino acid having a molecular weight of less than about 150.

4. The polypeptide of Claim 3 wherein the other standard amino acid is serine. An isolated nucleic acid molecule comprising: a nucleotide sequence which when expressed in a suitable host directs the expression of the polypeptide of Claim 1; or a sequence complementary to or both and

6. An isolated nucleic acid molecule comprising: a nucleotide sequence which when expressed in a suitable host directs the expression of a polypeptide comprising ego• the amino acid sequence set forth in SEQ ID NO:1 except that amino acid 81 may be any standard amino acid; or a sequence complementary to or both and wherein the nucleotide sequence defines an altered set of codons, said altered set of codons differing from the native set of codons defined by the nucleotide sequence set forth in SEQ ID NO:1 in that at least one of the codons of the altered set of codons, other than the codon for amino acid 81, is a bacterially preferred codon such that higher levels of the polypeptide are produced when nucleic acid molecules having the altered set of codons are expressed in bacteria than produced when nucleic acid molecules having the native set of codons are expressed in bacteria.

7. The isolated nucleic acid molecule of Claim 6 wherein the amino acid at position 81 is not cysteine.

8. The isolated nucleic acid molecule of Claim 7 wherein the amino acid at position 81 is serine. 119

9. The isolated nucleic acid molecule of Claim 6 wherein the level of the polypeptide produced when nucleic acid molecules having the altered set of codons are expressed in bacteria is at least about 1.5 times the level of the polypeptide produced when nucleic acid molecules having the native set of codons are expressed in bacteria. A method for producing a myelin basic protein polypeptide comprising: growing a recombinant host containing the nucleic acid molecule of Claim 5, 6, 7, or 8 such that the nucleic acid molecule is expressed by the host; and isolating the expressed polypeptide.

11. The method of Claim 10 wherein the host is a bacterial host.

12. The method of Claim 10 wherein the isolation of the expressed polypeptide is accomplished by a method comprising disruption of the host to yield a disruptate followed by fractionation of the disruptate, said fractionation comprising a step involving acid extraction of the disruptate.

13. A method for treating a patient suffering from multiple sclerosis comprising administering to said patient an isolated immunoreactive polypeptide comprising a myelin basic protein amino acid sequence comprising an amino acid sequence encoded by at least part 'of exon 2 of the human MBP gene, in an amount sufficient to--achieve a concentration of the polypeptide in a compartment of the patient's body sufficient to induce tolerization of MBP reactive T cells.

14. The method of Claim 13 wherein the compartment is the patient's cerebrospinal fluid. The method of Claim 13 wherein the compartment is the patient's blood.

16. The method of Claim 13 wherein the compartment is a lymph node.

17. The method of Claim 13 wherein the polypeptide is administered to the patient according to a regimen comprising administration of the polypeptide to the patient at least two times at an interval of at least twelve hours and not more than four days. 120

18. The method of Claim 13 wherein the method further comprises administering interleukin-2 to the patient in an amount sufficient to achieve a concentration of interleukin-2 in the patient's blood or cerebrospinal fluid sufficient to stimulate T cell division.

19. A tolerance inducing composition comprising a purified myelin basic protein polypeptide and a pharmaceutically acceptable carrier, said myelin basic protein polypeptide comprising an amino acid sequence encoded by at least part of exon 2 of the human MBP gene and said composition being suitable for administration to a human patient. The composition of Claim 18 wherein the myelin basic protein polypeptide is the polypeptide of SEQ ID NO:1

21. The composition of Claim 18 wherein the myelin basic protein polypeptide is the polypeptide of Claim 4.

22. An article of manufacture comprising packaging material and a pharmaceutical formulation contained within said packaging material, wherein: said pharmaceutical formulation comprises a purified myelin basic protein polypeptide and a pharmaceutically acceptable carrier, said myelin basic protein polypeptide comprising an amino acid sequence encoded by at least part of exon 2 of the human MBP gene; said formulation is suitable for administration to a human patient; and said packaging material comprises a label which indicates that said pharmaceutical formulation is for use in the treatment of multiple sclerosis.

23. An assay comprising isolating and partially purifying T cells from a patient, combining the isolated T cells with a purified immunoreactive polypeptide comprising a myelin basic protein polypeptide comprising an amino acid sequence encoded by at least part of exon 2 of the human MBP gene, and measuring the level of a T cell response induced by the polypeptide.

24. A kit for the detection of MBP reactive T cells comprising a purified myelin basic protein polypeptide having a mass of approximately 21.5 kD, said myelin basic protein polypeptide comprising an amino acid sequence encoded by at least part of exon 2 of the human MBP gene, in close confinement and/or 121 proximity with an agent for use in the detection of a T cell mesponse. The kit of Claim 24 wherein the kit further comprises a label indicating that the kit is for use in the clinical assessment of multiple sclerosis.

26. An immunoreactive polypeptide comprising a PLP mutein, said mutein comprising a sequence of amino acids, said sequence comprising the sequence of a native PLP polypeptide minus at least three hydrophobic peptide regions.

27. The immunoreactive polypeptide of Claim 26, wherein the PLP mutein comprises an amino acid sequence corresponding to the amino acid sequence spanning amino acid residues 6 to 186, inclusive, of SEQ ID NO:23.

28. The immunoreactive polypeptide of Claim 26, wherein the PLP mutein is expressed in bacteria at higher levels than the native PLP polypeptide. *29. The immunoreactive polypeptide of Claim 26, wherein the PLP mutein is more soluble in aqueous solution than the native PLP polypeptide.

30. The immunoreactive polypeptide of Claim 26, wherein the PLP mutein comprises an amino acid sequence comprising an amino acid sequence corresponding to the amino acid sequence spanning amino acid residues 6 to 169, inclusive, of SEQ ID NO:24.

31. The immunoreactive polypeptide of Claim 26 further comprising an MBP amino acid sequence comprising at least contiguous amino acids of myelin basic protein, SEQ ID NO:1. 1

32. The immunoreactive polypeptide of Claim 26 further comprising an MBP amino acid sequence comprising at least contiguous amino acids, all but one target amino acid residue of which correspond to a region of SEQ ID NO:1 comprising amino acid residue 81 of SEQ ID NO:1, wherein the target amino acid residue is located in a position within the MBP amino acid sequence corresponding to the position of amino acid residue 81 of SEQ ID NO:1 and wherein the target amino acid residue is any standard amino acid other than cysteine, amino acid residue 81 of SEQ ID NO:1. 122 I. S S a s 5 *005 S a

33. The PLP mutein of Claim 26 comprising an amino acid sequence corresponding to the amino acid sequence set forth in SEQ ID

34. The PLP mutein of Claim 26 comprising an amino acid sequence corresponding to an amino acid sequence corresponding to the amino acid sequence spanning amino acid residues 1 to 368, inclusive, of SEQ ID NO:26. The PLP mutein of Claim 26 comprising an amino acid sequence corresponding to the amino acid sequence spanning amino acid residues 6 to 374, inclusive, of SEQ ID NO:27.

36. The PLP mutein of Claim 26 comprising an amino acid sequence corresponding to an amino acid sequence corresponding to the amino acid sequence spanning amino acid residues 1 to 487, inclusive, of SEQ ID NO:28.

37. The polypeptide of Claim 26 further comprising a myelin oligodendrocyte glycoprotein amino acid sequence corresponding to at least 10 contiguous amino acids of the amino acid sequence of human myelin oligodendrocyte glycoprotein, said amino acid sequence of human myelin oligodendrocyte glycoprotein corresponding to the amino acid sequence spanning amino acid residues 199 to 319, inclusive, of SEQ ID NO:28.

38. An isolated nucleic acid molecule comprising: a nucleotide sequence which when expressed in a suitable host directs the expression of the polypeptide of Claim 26; or a sequence complementary to or both and

39. An isolated nucleic acid molecule comprising: a nucleotide sequence which when expressed in a suitable host directs the expression of the polypeptide of Claim 27; or a sequence complementary to or both and An isolated nucleic acid molecule comprising: a nucleotide sequence-which when expressed in a suitable host directs the expression of the polypeptide of Claim 29; or a sequence complementary to or both and S. @0 0 0* S c S. S 55

41. An isolated nucleic acid molecule comprising: a nucleotide sequence which when expressed in a suitable host directs the expression of the polypeptide of Claim or a sequence complementary to or both and

42. An isolated nucleic acid molecule comprising: a nucleotide sequence which when expressed in a suitable host directs the expression of the polypeptide of Claim or a sequence complementary to or both and

43. An isolated nucleic acid molecule comprising: a nucleotide sequence which when expressed in a suitable host directs the expression of the polypeptide of Claim 31; or a sequence complementary to or both and

44. An isolated nucleic acid molecule comprising: a nucleotide sequence which when expressed in a suitable host directs the expression of the polypeptide of Claim 32; or a sequence complementary to or both and An isolated nucleic acid molecule comprising: a nucleotide sequence which when expressed in a suitable host directs the expression of the polypeptide of Claim 33; or a sequence complementary to or both and

46. An isolated nucleic acid molecule comprising: a nucleotide sequence which when expressed in a suitable host directs the expression of the polypeptide of Claim 34; or a sequence complementary to or both and o 124

47. An isolated nucleic acid molecule comprising: a nucleotide sequence which when expressed in a suitable host directs the expression of the polypeptide of Claim or a sequence complementary to or both and

48. An isolated nucleic acid molecule comprising: a nucleotide sequence which when expressed in a suitable host directs the expression of the polypeptide of Claim 36; or a sequence complementary to or both and

49. An isolated nucleic acid molecule comprising: a nucleotide sequence which when expressed in a suitable host directs the expression of the polypeptide of Claim 37; or a sequence complementary to or both and A method for producing a PLP polypeptide comprising growing a recombinant host containing the nucleic acid molecule of Claim 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39, such that the nucleic acid molecule is expressed by the host, and isolating the.expressed polypeptide.

51. The method of Claim 50 wherein the host is a bacterial host.

52. A method for treating a patient suffering from multiple sclerosis comprising administering to said patient an isolated immunoreactive polypeptide in an amount sufficient to achieve a concentration of the polypeptide in a compartment of the patient's body sufficient to induce tolerization of PLP reactive T cells, said polypeptide comprising a PLP mutein having an amino acid sequence comprising the amino acid sequence of a native PLP polypeptide minus at least three hydrophobic peptide regions.

53. The method of Claim 52 wherein the compartment is the patient's cerebrospinal fluid.

54. The method of Claim 52 wherein the compartment is the patient's blood. 125 The method of Claim 52 wherein the compartment is a lymph node.

56. The method of Claim 52 wherein the polypeptide is administered to the patient according to a regimen comprising repeated administration of the polypeptide to the patient at least two times at an interval of at least twelve hours and not more than four days between administrations.

57. The method of Claim 52 wherein the method further comprises administering interleukin-2 to the patient in an amount sufficient to achieve a concentration of interleukin-2 in the patient's blood or cerebrospinal fluid sufficient to stimulate T cell division.

58. A composition comprising: a purified PLP polypeptide comprising a PLP mutein having an amino acid sequence comprising the amino acid sequence of a native PLP polypeptide minus at least three hydrophobic peptide regions; and a pharmaceutically acceptable carrier; said composition being suitable for administration to a human patient. o.o 59. The composition of Claim 58 wherein the PLP mutein comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:23.

60. The composition of Claim 58 wherein the PLP mutein o comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:24.

61. A tolerance inducing composition which comprises a PLP mutein having an amino acid sequence comprising the amino acid sequence of a native PLP polypeptide minus at least three hydrophobic peptide regions, such that the PLP mutein is expressed in bacteria at higher levels than the native PLP polypeptide; and a pharmaceutically acceptable carrier; said composition being suitable for administration to a human patient.

62. An article of manufacture comprising packaging material and a pharmaceutical formulation contained within said packaging material, wherein: 126 said pharmaceutical formulation comprises a PLP polypeptide comprising a PLP mutein amino acid sequence having the amino acid sequence of a native PLP polypeptide minus at least three hydrophobic peptide regions, and a pharmaceutically acceptable carrier; said formulation is suitable for administration to a human patient; and said packaging material comprises .a label which indicates that said pharmaceutical formulation is for use in the treatment of multiple sclerosis.

63. An assay comprising isolating and partially purifying T cells from a patient, combining the isolated T cells with an immunoreactive polypeptide comprising a PLP mutein amino acid sequence having the amino acid sequence of a native PLP polypeptide minus at least three hydrophobic peptide regions, and measuring the level of a T cell response induced by the polypeptide. oo 64. A kit for the detection of PLP reactive T cells comprising a PLP polypeptide comprising a PLP mutein amino acid sequence having the amino acid sequence of a native PLP polypeptide minus at least three hydrophobic peptide regions in close confinement and/or proximity with an agent for use in the detection of a T cell response. S65. The kit of Claim 64 wherein the kit further comprises a label indicating that the kit is for use in the clinical assessment of multiple sclerosis. DATED this 2 8 t h day of April 2000 ALEXION PHARMACEUTICALS, INC. UNITED STATES OF AMERICA, represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES By their Patent Attorneys CULLEN CO. 127