AU781104B2 - Novel expression vectors containing accessory molecule ligand genes and their use for immunomodulation and treatment of malignancies and autoimmune disease - Google Patents

Description

S&F Ref: 465722D1

AUSTRALIA

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ORIGINAL

Name and Address of Applicant: Actual Inventor(s): Address for Service: Invention Title: University of California Technology Transfer Office Mail Code: 0093, 9500 Gilman Drive La Jolla California 92093-0093 United States of America Thomas J. Kipps, Sanjai Sharma, Mark Cantwell Spruson Ferguson St Martins Tower,Level 31 Market Street Sydney NSW 2000 (CCN 3710000177) Novel Expression Vectors Containing Accessory Molecule Ligand Genes and Their Use for Immunomodulation and treatment of Malignancies and Autoimmune Disease The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c i 1 Novel Expression Vectors Containing Accessory Molecule Ligand Genes and Their Use for Immunomodulation and treatment of Malignancies and Autoimmune Disease Technical Field of the Invention The present invention relates to novel expression vectors containing genes which encode an accessory molecule ligand and the use of those vectors for immunomodulation, improved vaccination protocols and the treatment of malignancies and autoimmune diseases. More particularly, this invention provides expression vectors and methods for treating various neoplastic or malignant cells, and expression vectors and methods for treating autoimmune disease. This invention also contemplates the production and expression of accessory molecule ligands with greater stability and enhanced function.

Background of the Invention Leukemias, lymphomas, carcinomas and other malignancies are well known and described in, eg., Harrison's Principles of Internal Medicine, Wilson et al., eds., McGraw-Hill, New York, pp. 1599- 15 1612. These malignancies appear to have somehow escaped the immune system surveillance mechanisms that eliminate rapidly and continuously proliferating cells. The exact mechanism by which these malignancies escape the immune system surveillance is not known.

*oo C06777 Some of these malignant immune system cells are malignant antigen presenting cells which do not function properly within the immune cascade. For example, neoplastic B cells cannot induce even weak allogeneic or autologous mixed lymphocyte reactions in vitro. Further evidence that malignancies survive due to the failure of the immune surveillance mechanism includes the increased frequency of such malignancies in immunocompromised individuals, such as allograft recipients and those receiving long-term immunosuppressant therapy. Further, the frequency of these malignancies is increased in patients having Acquired Immune Deficiency Syndrome (AIDS) and patients with primary immune deficiency syndromes, such as X-linked lymphoproliferative syndrome or Wiscott-Aldrich Syndrome (Thomas et al., Adv. Cancer Res. 57:329, 1991) The immune system normally functions to eliminate malignant cells by recognizing the malignant cells as foreign cells and clearing those cells from the body.

An immune reaction depends on both the immune system's antibody response and on the cellular immune response within a patient. More specifically, the cellular immune response which acts to recognize the malignant cells as foreign requires a number of different cells of the immune system and the interaction between those cells. An immune reaction begins with a T lymphocyte

(T

cell) which has on its cell surface the T cell receptor.

The T cell also has the ability to express on its surface various accessory molecules which interact with accessory molecules on the B lymphocyte (B cell). When the T cell receptor of the T cell specifically binds to a foreign antigen, such as a malignant cell, it becomes activated and expresses the accessory molecule ligand, ligand on its cell surface. The accessory cell molecule ligand is only present on the activated T cells for a short period of time and is rapidly removed from the cell surface. After the accessory cells molecule ligand is removed from the surface of the activated

T

cell, its ability to bind to B cells via the accessory molecule ligand is destroyed.

When present on the surface of an activated T cell, the accessory cell ligand can specifically bind to the accessory cell molecule present on the B cell. This specific T-B cell interaction causes the B and T cell to express costimulutory surface accessory molecule and cytokines which result in an immune activation which lead to cytolytic T cells which specifically kill and remove the malignant cell from the body.

SThe interaction with an activated T cell is not Ssolely limited to B cells but rather can be carried out by any cell which is able to present antigen to the T cell (an antigen presenting cell). These cells include B lymphocyte, macrophages, dendritic cells, monocytes Langerhans cells, interdigitating cells, follicular dendritic cells or Kupffer cells. These cells all are known to have various accessory molecules on the cell surface which allow them to interact with other cells of the immune system. For example, these antigen presenting cells all have the accessory molecule CD40 on t. their cell surface. The presence of these accessory molecules allows these antigen presenting cells to specifically bind to complimentary accessory molecule ligand and thus directly interact with other immune cells.

A large number of accessory molecule ligands are members of the tumor necrosis factor superfamily.

(Fanslow et al., Sem. Immun., 6:267-268 (1994). The genes for a number of these accessory molecule ligands have been cloned and identified. These accessory molecule ligand genes encode accessory molecules which all have the configuration of Type II membrane proteins and exhibit varying degrees of homology with other accessory molecule ligand genes. For example, the accessory molecule ligand genes encoding both murine ligand and human CD40 ligand have been isolated.

See, Armitage et al., Nature, 357:80-82 (1992) and Hollenbaugh et al., EMBO 11:4313-4321 (1992).

and its ligand, CD40 ligand are critical components of a normal immune response. CD40 mediated signals induce immune lymphocytes to proliferate and differentiate and become potent antigen presenting cells. Malignant or neoplastic B cells are poor antigen presenter cells and are unable to stimulate a vigorous allogeneic mixed lymphocyte reaction. Successful cross linking of CD40 molecules on immune cells results in a 'strong allogeneic mixed lymphocyte reaction suggesting a strong immune reaction. Various soluble CD40 ligands or 'antibodies specific for CD40 have been used to 15 potentially cross link CD40. These soluble CD40 ligands and CD40-specific antibodies are not optimal for cross linking the CD40 molecules on antigen presenting cells and do not work as effectively as CD40 ligand expressed on a cell membrane to produce strong stimulation of antigen presenting cells. These methods are also difficult to implement because large amounts of Sligand constructs or antibodies must be isolated which is difficult and time-consuming work. Other strategies to utilize CD40 ligand in solution or as a membrane bound molecule including transformation of fibroblasts with CD40 ligand to produce cultured cells which are then used to present antigen are not amenable to in vivo human clinical protocols.

(Fas) interaction with its ligand (Fas-ligand, or FasL) functions to limit the duration of the immune response and/or life-span of activated lymphocytes.

Apoptosis induced by Fas-FasL binding serves to clear activated self-reactive lymphocytes. Problems caused by altering this pathway have been demonstrated in animals with defects in Fas<->Fas-ligand interactions. Mice having mutations, which inactivate CD95 or FasL, develop numerous disorders including autoimmune pathologyresembling that seen in patients with rheumatoid arthritis (RA) or systemic lupus. Zhang, et al., in J.

Clin. Invest. 100:1951-1957 (1997) show that injection of FasL-expressing virus, into the joints of mice with collagen-induced-arthritis, results in apoptosis of synovial cells and relief of arthritis symptoms.

Expression of Fas ligand allows clearance of activated cells which play a role in the pathogenesis of autoimmune disease. Therefore, a gene therapy strategy for introducing FasL into the joints of rheumatoid arthritis patients could function to improve disease pathology by leading to destruction of the infiltrating mononuclear cells.

Administration of soluble accessory molecules and 15 accessory molecule ligands has been shown to trigger or So.. to be associated with adverse physiological effects.

For example, treatment of mice, having wild-type receptor expression, with soluble CD40L-CD8 fusion protein resulted in a pulmonary inflammatory response.

This was not observed in mice in which the gene for the S CD40 receptor had been knocked out. These experiments, described in Wiley, J.A. et al., Journal of Immunology 158:2932-2938 (1997), support in vitro data which suggest that CD40 ligation can result in inflammatory responses.

Direct administration of purified recombinant soluble Tumor Necrosis Factor (either a or 3) results in shock and tissue injury, as described in Tracey, K. J., and A. Cerami, Annu. Rev. Med. 45:491-503 (1994) Within minutes after acute intravenous or intra-arterial administration of TNF, a syndrome of shock, tissue injury, capillary leakage syndrome, hypoxia, pulmonary edema, and multiple organ failure associated with a high mortality ensues. Chronic low dose of TNF causes anorexia, weight loss, dehydration and depletion of whole-body protein and lipid.

Soluble Fas ligand and receptor have also been shown to be associated with tissue damage and other adverse effects. CD95, the Fas receptor, is a mediator of apoptosis. Fas ligand induces apoptosis by binding to Fas receptor. As shown in Galle, et al., J.

Exp. Med. 182:1223-1230 (1995) administering an agonistic anti-Fas antibody resulted in liver damage to mice. Mice injected intraperitoneally with the agonistic antibody died within several hours, and analyses revealed that severe liver damage by apoptosis was the most likely cause of death.

The role of soluble Fas ligand (FasL), in the pathogenesis of systemic tissue injury in aggressive lymphoma is described in Sato, K. et al., British 15 Journal of Haematoloqv, 94:379-382 (1996). The findings presented in this report indicate that soluble FasL is directly associated with the pathogenesis of liver injury and pancytopenia.

CD27, the receptor for the accessory molecule 20 ligand, CD70, was shown, in a report written by van Oers, et al., in Blood 82:3430-3436 (1993), to be associated with B cell malignancies.

The above findings all contraindicate the administration of soluble accessory molecule ligands, highlighting the need for therapies that increase the levels of these molecules without resulting in an elevation of their soluble forms.

Despite the wealth of information regarding accessory molecule ligand genes and their expression on the surface of various immune cells, the exact mechanism by which the accessory molecule ligand genes are regulated on antigen presenting cells is not yet known.

Without specific knowledge of the regulation of expression of accessory molecule ligand genes on these antigen presenting cells, altering the immune response by varying expression of an accessory molecule ligand gene has to date not been possible. Without any specific knowledge as to how to regulate the expression of an accessory molecule ligand gene on an antigen presenting cell, it is not possible to alter the immune response towards malignant cells. Thus, there was a need for a method of increasing the expression of an accessory molecule ligand gene on normal and malignant cells including antigen presenting cells.

Further, without the ability to regulate the expression of accessory molecule ligands, it is not possible to alter the immune clearance of these cells.

Summary of the Invention The present invention fills these needs by providing novel expression vectors containing accessory molecule ligand genes and methods for introducing those genes into normal and malignant antigen presenting cells thereby allowing the alteration of an immune response, the treatment of autoimmune diseases and the treatment of various neoplasias. This invention provides vectors, including gene therapy vectors which contain accessory molecule ligand genes. These vectors also contain the additional genetic elements, such as promoters, enhancers, polyadenylation signals ends), which allow that vector to be successfully placed within the cell and to direct the expression of the accessory molecule ligand gene in a cell. Such gene therapy vectors are capable of :transforming animal cells directly and thereby introducing the accessory molecule ligand gene into the cells of that animal in a form which can be utilized to produce accessory 20 molecule ligands within that cell.

According to a first aspect of the present invention there is provided a method for expressing a chimeric CD40 ligand in a CD40+ human cell, wherein the chimeric ligand includes one or more human domains derived from a human CD40 ligand gene and one or more murine domains derived from a murine CD40 ligand gene, and wherein 25 further the method comprises delivering a chimeric polynucleotide encoding said chimeric CD40 ligand into a CD40+ human cell.

According to a second aspect of the present invention there is provided a chimeric polynucleotide encoding a chimeric CD40 ligand comprising one or more human domains derived from a human CD40 ligand gene and one or more murine domains derived from a murine CD40 ligand gene.

According to a third aspect of the present invention there is provided a pharmaceutical composition for use in expressing a chimeric CD40 ligand in a human cell, wherein the chimeric CD40 ligand includes one or more human domain segments derived from a human CD40 ligand gene and one or more murine domain [R:\LIBVV]O3651 .doc:THR 7asegments derived from a murine CD40 ligand gene, and wherein further the composition comprises a chimeric polynucleotide encoding said chimeric CD40 ligand.

According to a fourth aspect of the present invention there is provided a method for expressing a chimeric Fas ligand in a human cell, wherein the chimeric ligand includes one or more human domain segments derived from a human Fas ligand gene and one or more murine domain segments derived from a murine Fas ligand gene, and wherein further the method comprises delivering a chimeric polynucleotide encoding said chimeric Fas ligand.

According to a fifth aspect of the present invention there is provided a chimeric polynucleotide encoding a chimeric Fas ligand comprising one or more human domain segments derived from a human Fas ligand gene and one or more murine domain segments derived from a murine Fas ligand gene.

According to a sixth aspect of the present invention there is provided a pharmaceutical composition for use in expressing a chimeric Fas ligand in a human cell, 15 wherein the chimeric Fas ligand includes one or more human domain segments derived from a human Fas ligand gene and one or more murine domain segments derived from a murine Fas ligand gene, and wherein further the composition comprises a chimeric polynucleotide encoding said chimeric Fas ligand.

According to a seventh aspect of the present invention there is provided a 20 pharmaceutical composition consisting of a mammalian, bacterial or insect cell transfected with the chimeric CD40 ligand polynucleotide of the second aspect.

According to an eighth aspect of the present invention there is provided a pharmaceutical composition consisting of a mammalian, bacterial or insect cell transfected with the chimeric Fas ligand polynucleotide of the fifth aspect.

25 As disclosed herein, the function of an accessory molecule ligand may be modified by altering the half life of the molecule on the cell surface or by changing the level of expression of that molecule on the cell surface. The present invention contemplates accessory molecule [R:\LIBVV]03651 .doc:THR ligands which are modified to improve the stability of such accessory molecule ligands on the cell surface.

Such increased stability may be accomplished using any of the disclosed methods of molecules described in this applicatioA, including chimeric molecules and molecules into which mutations have been introduced at least one location. The present invention also contemplates increasing the expression of such a molecule.

The present invention also provides gene therapy vectors containing the accessory molecule ligand genes which are chimeric in that portions of the gene are derived from two separate accessory molecule ligands which may or may not be from different species. The accessory molecule ligand genes of the present invention 15 include genes which encode molecules of the tumor necrosis factor (TNF) family. The molecules which make up the TNF family include TNF,, TNFe, CD40 ligand, Fas ligand, CD70, CD30 ligand, 41BB ligand (4-1BBL), nerve growth factor and TNF-related apoptosis inducing ligand 20 (TRAIL). In some embodiments of the present invention, the chimeric accessory molecule ligand genes of the present invention contain at least a portion of a murine accessory molecule ligand gene together with portions of accessory molecule ligand genes derived from either mouse, humans or other species. Some preferred embodiments of the present invention utilize murine ligand genes and chimeric CD40 ligand genes containing at least a segment of the murine CD40 ligand gene together with at least a segment of the human ligand gene. The present invention contemplates chimeric accessory molecule ligand genes wherein segments from the accessory molecule ligand gene of one species have been interchanged with segments from a second accessory molecule ligand gene which may optionally be from a different species. For example, in one preferred embodiment, the murine CD40 ligand gene transmembrane and cytoplasmic domains have been attached to the extracellular domains of human CD40 ligand gene.

The present invention contemplates gene therapy vectors which are capable of directly infecting the human, mammal, insect, or other cell. The use of such gene therapy vectors greatly simplifies inserting an accessory molecule ligand gene into those cells. The contemplated gene therapy vectors may be used in vivo or in vitro to infect the desired cell and are particularly useful for infecting malignant cells to effect sustained high-level expression of a physiologic ligand.

The present invention also contemplates animal, S. mammal, and human cells containing a gene therapy vector which includes an accessory molecule ligand gene and 15 sufficient genetic information to express that accessory molecule ligand within that cell. In preferred embodiments, the present invention also contemplates human neoplastic antigen presenting cells which contain the gene therapy vectors of the present invention or contain an accessory molecule ligand gene together with a promoter and 3' end region.

The present invention also contemplates human cells and human neoplastic cells containing a gene therapy vector which includes a chimeric accessory molecule ligand gene. The present invention also contemplates bacterial cells or animal cells containing accessory molecule ligand genes, chimeric accessory molecule ligand genes, murine accessory molecule ligand genes, human accessory molecule ligand genes, the gene therapy vectors of the present invention, the vectors of the present invention, and a chimeric accessory molecule ligand gene together with a heterologous promoter, enhancer or polyadenylation sequence.

The present invention also contemplates methods of altering immune response within a human patient or the immunoreactivity of human cells in vivo by introducing a gene which encodes an accessory molecule ligand gene into the human cells so that that accessory molecule ligand is expressed on the surface of those human cells.

This method includes the introduction of the accessory molecule ligand gene as part of a gene therapy vector or in association with a heterologous or native promoter, enhancer or polyadenylation signal. Some preferred embodiments of the present invention utilize introduction of Fas ligand genes and chimeric Fas ligand genes, constructed as contemplated above for CD40, into human cells to alter their immunoreactivity. The present invention also includes methods in which such accessory molecule ligand genes are inserted into cells which have the accessory molecule to which the accessory molecule ligand binds on the surface of the cell into 15 which the accessory molecule ligand gene.

The present methods of altering immunoreactivity are applicable to all types of human, animal, and murine cells including human neoplastic cells such as human lymphomas, leukemias and other malignancies. In 20 preferred embodiments, this method is used to introduce the gene encoding the accessory molecule ligand into potential antigen presenting cells of a human patient or cell which can stimulate bystanding antigen presenting cells. Such antigen presenting cells include monocytes, macrophages, B cells, Langerhans cells, interdigitating cells, follicular dendritic cells, Kupffer cells, and the like. The various antigen presenting cells may be present as part of a known malignancy in a human patient such as leukemias, lymphomas, acute monocytic leukemia (AML), chronic lymphocytic leukemia (CLL), acute myelomonocytic leukemia (AMML), chronic myelogenous or chronic myelomonocytic leukemia (CMML) and thus would include all tumors of any cell capable of presenting antigen to the human or animal immune system or are capable of stimulating bystanding antigen presenting cells. The present invention also contemplates modulating the immune system by introducing genes encoding an accessory molecule ligand gene of the present invention into any number of different cells found in a patient, including muscle cells, skin cells, stromal cells, connective tissue cells, fibroblasts and the like. The present invention also contemplates methods of treating neoplasias in either a human patient or an animal patient. In one preferred embodiment, the method comprises isolating the neoplastic cells from the human or animal patient and inserting into those isolated cells the gene which encodes the chimeric accessory .molecule ligand or the accessory molecule ligand so that that molecule is expressed on the cell surface of those neoplastic cells or other somatic cells. The neoplastic 15 cells are then infused back into the human or animal patient and may then participate in an enhanced immune response.

.The present invention also contemplates the coinfection or co-introduction of the accessory molecule 20 ligand gene together with a gene which encodes a tumor or carcinoma specific antigen. This combination of molecules are then expressed on the surface of the neoplastic cells and when those cells are introduced into the patient lead to the rapid immune response resulting in the destruction of those cells.

The present methods also include directly introducing the gene therapy vector or other vector carrying the accessory molecule ligand gene directly into the tumor or tumor bed of a patient. Upon entering the tumor bed of the patient, the gene therapy vector or other vector enter the cells present in the tumor or tumor bed and then express the accessory molecule ligand gene on the surface of those cells. These cells then are able to participate fully in the human immune or animal immune response.

The present invention also contemplates methods of augmenting an immune response to a vaccine. The present method of vaccinating an animal against a predetermined organism or antigen by administering to that animal a vaccine which has a genetic vector containing an accessory molecule ligand gene. Other embodiments of the present invention include vaccinating an animal by administering two separate genetic vectors, one containing the antigens from the organism to which immunity is desired by isolating the cells of the target animal and contacting with those cells a vector encoding at least one antigen from a predetermined organism so *.*.that the antigen is expressed by the cells and also contacting those cells with a different vector which expresses the accessory molecule ligand gene on the surface of the animal's antigen presenting cells.

S. 15 Together these two separate vectors produce a vaccination which is much stronger and of longer duration than is vaccination with antigen alone.

.The present methods of vaccination are applicable to vaccinations designed to produce immunity against a virus, a cell, a bacteria, any protein or a fungus. The present methods are also applicable to immunization against various carcinomas and neoplasias. In these embodiments, the tumor antigen against which immunity is desired is introduced into the animal together with the genetic vector containing the accessory molecule ligand gene.

The present invention also contemplates methods of treating arthritis utilizing a gene therapy vector encoding an accessory molecule ligand. Of particular interest for use with arthritis is the Fas ligand molecule in which the expression of Fas ligand activity has been increased in the joint and/or the stability of the Fas ligand activity on cells within the joint enhanced. In other embodiments, the present invention contemplated methods of treating arthritis utilizing chimeric accessory molecule ligands and chimeric accessory molecule ligand genes. The present invention 13 also contemplates both ex vivo therapy and in vivo therapy of arthritis utilizing the expression vectors of the present invention together with the Fas ligand and modified versions of that molecule including chimeric molecules.

Brief Description of the Drawings Figure 1. Figure 1 is a diagram showing a number of accessory molecule ligand genes and Domains I-IV of those genes as deduced from sequence data.

10 Figure 2. Figure 2 is a diagram showing example chimeric accessory molecule ligand genes. The domains derived from the murine accessory module are shown shaded.

Figure 3. Figure 3 shows the amount of either mouse or human CD40 ligand found on the surface of Hela or CLL cells infected with gene therapy vectors containing the genes encoding these molecules. Figure 3A shows uninfected Hela cells (shaded) and Hela cells infected with a gene therapy vector encoding murine ligand. Figure 3B shows uninfected Hela cells (shaded) and Hela cells infected with a gene therapy vector encoding human CD40 ligand. Figure 3C shows uninfected CLL cells (shaded) and CLL cells infected with a gene therapy vector encoding murine CD40 ligand. Figure 3D shows uninfected CLL cells (shaded) and CLL cells infected with a gene therapy vector encoding human ligand.

Figure 4. Figure 4 shows histograms of the increased expression of CD54 (Figure 4B) and (Figure 4D) on CLL cells into which a gene therapy vector containing the accessory molecule ligand gene (murine CD40 ligand gene) has been introduced. The shaded graph indicates control stain in FACS analysis and the open graph indicates staining with monoclonal antibodies immunospecific for either CD54 (Figures 4A and 4B) or CD80 (Figures 4C and 4D).

Figure 5. Figure 5 shows the cell proliferation as measured by 3 H-TdR incorporation of allogeneic T cells in response to various stimulation regimes. The CLL cells containing a gene therapy vector expressing an accessory molecule ligand gene (the murine CD40 ligand gene) were introduced, stimulating allogeneic T cells to proliferate.

Figure 6. Figure 6 shows the production of gamma interferon (IFNg) by allogeneic T cells stimulated with CLL cells containing an accessory molecule ligand gene.

Figure 7. Figure 7 shows the treatment of a S. neoplasia in an animal using a gene therapy vector containing an accessory molecule ligand gene of the present invention. The open squares show mice immunized 15 with neoplastic cell not expressing an accessory molecule ligand of the present invention. Mice immunized with neoplastic cells expressing an accessory molecule ligand of the present invention are shown as the horizontal line at the top of the Figure and show no 20 morbidity.

Figure 8. Figure 8 shows the production levels and stabilities of CD40 ligand and CD40 ligand transcript in CLL (upper graph) and normal blood mononuclear cells (lower graph).

Figure 9. Figure 9 shows the time course of transgene expression in CLL B cells infected with the accessory molecule ligand (CD40 ligand). The MFIR (mean fluorescence intensity ratio), comparing the fluorescence intensity of CD19' CLL cells stained with PE-labeled CD40 ligand versus the same stained with a PE-labeled isotype control mAb at each time point, are represented by the closed circles connected by solid lines according to the scale provided on the left-hand ordinate.

Figure 10. Figure 10 shows changes in surface antigen phenotype of CLL B cells infected with a gene therapy vector containing an accessory molecule ligand, ligand. Shaded histograms represent staining of uninfected CLL cells (thin lines) stained with nonspecific control antibody, open histograms drawn with thin lines represent uninfected CLL cells stained with FITC-conjugated specific mAb, and open histograms drawn with thick lines (labeled CD154-CLL) represent CLL cells infected with the accessory molecule ligand gene therapy vector and stained with FITC-conjugated specific mAb.

Figure 11. Figure 11 shows levels of CD27 produced in CLL cells infected with a gene therapy vector containing an accessory molecule ligand. Figure 11A shows that CD40L-infected CLL (CD154-CLL) cells express reduced levels of surface CD27. Open histograms represent staining of non-infected CLL cells (thin 15 lines) or infected CLL (thick lines) with FITCconjugated aCD27 mAb, respectively. Figure 11B shows production of soluble form of CD27 by CLL B cells.

Figure 12. Figure 12 shows allogeneic T cell responses induced by CLL cells infected with a gene 20 therapy vector containing an accessory molecule ligand ligand, also called CD154). Figure 12A indicates the concentration of IFNg in the supernatants after stimulation of allogeneic T cells with CLL cells containing the accessory molecule ligand. Figure 12B shows cell proliferation, as assessed by incorporation of 3 H-thymidine. Figures 12C and 12D show secondary allogeneic T cell responses induced by CLL containing the accessory molecule ligand.

Figure 13. Figure 13 depicts autologous T cell responses induced by CLL B cells containing the accessory molecule ligand, CD40 ligand or CD154, and controls. Figure 13A shows incorporation of 'H-thymidine by autologous T cells co-cultured with the CLL cells.

Figure 13B shows the levels of human IFNg produced by autologous T cells co-cultured with the CLL cells. In Figure 13C, the CTL activities of autologous T cells induced by CLL B cells containing the accessory molecule ligand are graphed.

Figure 14. Figure 14 shows specificity of CTL for autologous CLL B cells. IFNg concentration was assessed in the supernatants after 48 h of culture (Figure 14A), and cytolytic activity was assessed at 3 h of culture (Figure 14B). In Figure 14C, mAb were added to the autologous leukemia target cells prior to the CTL assay.

Figure 15. Figure 15 shows that intercellular stimulation plays a role in production of the phenotypic changes observed in CLL cells expressing the accessory molecule ligand. In Figure 15A, the effect of culture density on the induced expression of CD54 and following infection with a gene therapy vector 15 containing the accessory molecule ligand (CD40 ligand, CD154) is shown. Shaded histograms represent staining of leukemia B cells with a FITC-conjugated isotype control mAb. Open histograms represent CD154-CLL B cells, cultured at high or low density (indicated by 20 arrows), and stained with a FITC-conjugated mAb specific for CD54 or CD80. Figure 15B shows inhibition of CD154- CLL cell activation by anti-CD154 mAb. Figures 15C and 15D depict expression of immune accessory molecules on bystander non-infected CLL B cells induced by CLL cells expressing the accessory molecule ligand. Shaded histograms represent staining with PE-conjugated isotype control mAb.

Figure 16. Figure 16 shows that the vector encoding an accessory molecule ligand enhances immunization against G-gal in mice. Figure 16A shows that mice that received intramuscular injections of the vector produced significantly more antibodies to P-gal than did mice injected with either the nonmodified pcDNA3 vector or pCD40L. Figure 16B, ELISA analyses of serial dilutions of sera collected at d28, shows that mice co-injected with placZ and pCD40L had an eight-fold higher mean titer of anti-3-gal antibodies at d28 than mice treated with placZ pcDNA3.

Figure 17. Figure 17 shows analysis of the IgG, and IgG 2 immune responses to intramuscular plasmid DNA immunizations with and without a vector, encoding an accessory molecule ligand. IgG 2 a anti-i-gal antibodies predominated over IgG, subclass antibodies in the sera of mice injected with either placZ and pcDNA3 or placZ and pCD40L. In contrast, BALB/c mice injected with 3-gal protein developed predominantly IgG, anti-pgal antibodies, and no detectable IgG 2 anti-B-gal antibodies.

Figure 18. Figure 18 shows the comparison between injection of mice with a vector, pCD40L, encoding an 15 accessory molecule ligand, at the same and different sites as placZ. Adjuvant effect of pCD40L requires coinjection with placZ at the same site.

Figure 19. Figure 19 shows that co-injection into dermis of a vector encoding an accessory molecule 20 ligand, pCD40L, with placZ enhances the IgG anti-g-gal response in BALB/c mice.

Fiqure 20. Figure 20 shows that a vector encoding an accessory molecule ligand, pCD40L, enhances the ability of placZ to induce CTL specific for syngeneic bgal-expressing target cells. Splenocyte effector cells, taken from mice which had received injections of placZ and pCD40L, specifically lysed significantly more cells than did splenocytes from mice that received control injections.

Figure 21. Figure 21 shows downmodulation of human but not murine CD40L, in lung tumor cell lines that express Figure 22. Figure 22A shows that CD40 binding induces enhanced expression of the tumor cell surface markers CD95 (Fas), CD54 (ICAM-1), and MHC-I, in lung tumor cell lines. Figure 22B shows downmodulation of human CD40L by CD40-positive tumor cells.

Figure 23. Figure 23 shows the inhibition of Fas ligand expression by lymphocytes in the presence of RA synovial fluid.

Figure 24. Figure 24 shows an outline for a clinical trial of an accessory molecule ligand gene therapy treatment for B cell CLL.

Figure 25. Figure 25 shows a sequence line-up of human Fas ligand with human Fas ligand in which Domain III is replaced by Domain III of murine Fas ligand. The top protein sequence is native human Fas ligand. Domain III is underlined with the dotted line. The double S, underline indicates a putative MMP cleavage site. The bottom protein sequence is that of chimeric human-mouse Fas ligand. Domain III of the mouse Fas ligand 15 (underlined with dotted line) is substituted for Domain III of human Fas ligand. The numbers correspond to the amino acid sequence number using 1 for the start of the S. polypeptide sequence. The number of the first nucleotide base for the codon encoding the amino acid is 20 l+3x(n-1), where n is the amino acid sequence number.

Figure 26. Figure 26 shows a sequence line-up of human Fas ligand with human Fas ligand in which Domain III has been replaced with Domain III of human The top protein sequence is native human Fas ligand, and the bottom sequence is that of chimeric Fas ligand, in which Domain III of human CD70 has been substituted for Fas Domain III. Other markings are used similarly as in Figure Figure 27. Figure 27 shows a sequence line-up of human Fas ligand with human Fas ligand in which Domain I has been replaced with Domain III of human CD70. The top protein is native human Fas ligand, and the bottom protein sequence is that of chimeric Fas ligand, in which Domain III has been replaced with Domain I of human CD70. Other markings are used similarly as in Figure Figure 28. Figure 28 shows the amino acids around and at known matrix metalloproteinase (MMP) cleavage sites, as described in Smith, M.M. et al., Journal of Biol. Chem. 270:6440-6449 (95) and Nagase, and G.B.

Fields, Biopolymers (Peptide Science) 40:399-416 (96).

The cleavage site is indicated with an arrow.

Detailed Description of the Invention All references cited herein are hereby incorporated in their entirety by reference.

10 I. Definitions An "accessory molecule ligand gene" is a gene which encodes all or part of an accessory molecule ligand.

The gene comprises at least the nucleotide sequence required to encode the functional portion of an 15 accessory molecule ligand. The gene may optionally include such genetic elements as promoters, enhancers and 3' ends. The accessory molecule ligand gene is derived from a ligand which is a member of the tumor necrosis factor (TNF) family, including CD40 ligand, Fas 20 ligand, CD70, TNF,, TNF,, CD30 ligand, 4-1BB ligand (4- 1BBL), nerve growth factor and TNF-related apoptosis inducing ligand (TRAIL). As used herein, the term "accessory molecule ligand gene" includes chimeric accessory molecule ligand genes as defined below.

As used herein, the term "malignant cells or neoplastic cells," is defined to mean malignant or cancerous cells which are found in a human patient or an animal. Preferred types of malignant or neoplastic cells include any malignant antigen-presenting cell. In some preferred embodiments, these malignant antigen presenting cells have at least low levels of present on the cell surface.

As used herein, the term "neoplastic human cells" is defined to mean human cells which are neoplastic including but not limited to antigen presenting cells, any neoplastic cell which may function as an antigen presenting cell or function to facilitate antigen presentation, neoplastic monocytes, neoplastic macrophages, neoplastic B cells, neoplastic dendritic cells, neoplastic Langerhans cells, neoplastic interdigitating cells, neoplastic follicular dendritic cells, or neoplastic Kupffer cells and the like. The definition of neoplastic human cells includes those cells which are associated with neoplastic cells in the tumor bed of human patients. Typically, the neoplastic .g human cells are either leukemias, lymphomas, AML, ALL, AMML, CML, CMML, CLL other tumors of antigen presenting cells or breast, ovarian or lung neoplastic cells. It is also contemplated that the accessory molecule ligand 15 genes or chimeric accessory molecule ligand genes of the present invention may be inserted into somatic cells.

These somatic cells can be created by a genetic *ee e.peep engineering process which has introduced into those cells genes which encode molecules which render those 20 cells capable of presenting antigen to the immune system.

o As used herein, the term "chimeric gene" is defined to mean a gene in which part of the gene is derived from a second different gene and combined with the first gene so that at least a portion of each gene is present in the resulting chimeric gene. A gene may be chimeric if any portion of the sequence which encodes the resulting protein is derived from a second and different gene.

Typical chimeric genes include genes in which specific functional domains from one gene have been transferred to a second gene and replace the analogous domains of that second gene. For example, the resulting chimeric gene may have one domain derived from a murine gene and several domains derived from a human gene. These domains may range in size from 5 amino acids to several hundred amino acids. Other examples of chimeric accessory molecule ligand genes include genes which contain nucleotides encoding amino acids not found in any naturally occurring accessory molecule ligand gene.

Examples of chimeric genes and potential various combinations of domains are numerous and one of skill in the art will understand that no limit is placed on the amount of one gene that must be present in a second gene to render it chimeric.

As used herein, the term "murine CD40 ligand gene" is defined to mean an accessory molecule ligand gene 10 which is derived from a murine CD40 ligand gene.

Examples of such murine CD40 ligand genes include the gene isolated by Armitage et al., Nature, 357:80-82 (1992) and other genes derived from murine origin which hybridize to the gene described by Armitage et al. under low stringency hybridization conditions.

As used herein, the term "vector or genetic vector" is defined to mean a nucleic acid which is capable of replicating itself within an organism such as a bacterium or animal cell. Typical genetic vectors include the plasmids commonly used in recombinant

DNA

technology and various viruses capable of replicating within bacterial or animal cells. Preferred types of genetic vectors includes plasmids, phages, viruses, retroviruses, and the like.

As used herein, the term "gene therapy vector" is defined to mean a genetic vector which is capable of directly infecting cells within an animal, such as a human patient. A number of gene therapy vectors have been described in the literature, and include, the gene therapy vector described in Cantwell et al., Blood, In Press (1996) entitled "Adenovirus Vector Infection of Chronic Lymphocytic Leukemia B Cells." Such vectors have been described for example by Woll, P. J. and I. R.

Hart, Ann. Oncol., 6 Suppl 1:73 (1995); Smith, K. A.

J. Shepherd, J. E. Boyd, and G. M. Lees, Gene Ther., 3:190 (1996); Cooper, M. Semin. Oncol., 23:172 (1996); Shaughnessy, D. Lu, S. Chatterjee, and K. K.

Wong, Semin. Oncol., 23:159 (1996); Glorioso, J. N.

A. DeLuca, and D. J. Fink, Annu. Rev. Microbiol., 49:675 (1995); Flotte, T. R. and B. J. Carter, Gene Ther., 2:357 (1995); Randrianarison-Jewtoukoff, V. and M.

Perricaudet, Biologicals., 23:145 (1995); Kohn, D. B., Curr. Opin. Pediatr., 7:56 (1995); Vile, R. G. and S. J.

Russell, Br. Med. Bull., 51:12 (1995); Russell, S. J., Semin. Cancer Biol., 5:437 (1994); and Ali, N. R.

Lemoine, and C. J. Ring, Gene Ther., 1:367 (1994). All 10 references cited herein are hereby incorporated by reference.

II. Genetic Vectors and Constructs Containing an Accessory Molecule Ligand Gene A. Accessory Molecule Ligand Genes 15 In one embodiment of the present invention, preferred gene therapy vectors contain an accessory molecule ligand gene. This accessory molecule ligand gene may be derived from any source and may include molecules which are man-made and do not appear in 20 nature. The present invention contemplates accessory molecule ligand genes which are derived from the genes encoding molecules within the tumor necrosis family (TNF) which includes the genes encoding: murine ligand, human CD40 ligand, Fas ligand, TNF, TNFO, ligand, 4-1BB ligand, nerve growth factor, CD70, TNFrelated apoptosis inducing ligand (TRAIL) and chimeric accessory molecule ligands. The nucleotide sequence of one accessory molecule ligand, the sequence of at least one form of the murine CD40 ligand gene, has been determined and is listed as SEQ ID NO: 1. The present invention contemplates the use of any accessory molecule ligand gene which is homologous to the sequence present in SEQ ID NO: 1, and thus hybridizes to this sequence at low stringency hybridization conditions. One of skill in the art will understand that accessory molecule ligand genes, including murine CD40 ligand gene, useful in the present invention may be isolated from various different murine strains.

The nucleotide sequence of a human CD40 ligand gene has been determined and is shown as SEQ ID NO: 2. The present invention contemplates the use of any accessory molecule ligand gene which is homologous to SEQ ID NO: 2, and thus hybridizes to this sequence at low stringency conditions. One of ordinary skill in the art will understand that the accessory molecule ligand genes, including the human CD40 ligand genes, useful in the present invention, may vary depending on the individual from which the gene is isolated and such variations may prove useful in producing unique accessory molecule ligand genes. The present invention contemplates the use of the domains, sub-domains, amino acid or nucleotide sequence of the human CD40 ligand and/or human CD40 ligand gene as part of a chimeric accessory molecule ligand or chimeric accessory molecule ligand gene.

The nucleotide sequence of a bovine CD40 ligand gene has been determined and is shown as SEQ ID NO: 8.

:i The present invention contemplates the use of any accessory molecule ligand gene which is homologous to SEQ ID NO: 8, and thus hybridizes to the sequence at low stringency conditions. One of ordinary skill in the art will understand that the accessory molecule ligand genes, including the bovine CD40 ligand genes, may vary depending on the individual animal from which the gene is isolated and that such variations may prove useful in producing unique accessory molecule ligand genes.

The nucleotide sequence of human TNF, and human TNF, have been determined and are shown as SEQ ID NOS: 9 and respectively. The present invention contemplates the use of any accessory molecule ligand gene which is homologous to either human TNF, or human TNFO (SEQ ID NOS: 9 and 10, respectively), and thus hybridizes to these sequences at low stringency conditions. The accessory molecule ligand genes useful in the present invention, including the human TNF, and TNFp genes, may vary depending on the particular individual from which the gene has been isolated and these variations may prove useful in producing unique accessory molecule genes.

The nucleotide sequence of porcine TNF, and TNF, have been determined and are shown as SEQ ID NO: 11.

The present invention contemplates the use of any 10 accessory molecule ligand gene which is homologous to either SEQ ID NO: 11, and thus would hybridize to these sequences at low stringency conditions. One of ordinary skill in the art will understand that the accessory molecule ligand genes, including the porcine TNF, and TNF, genes, may vary depending on the particular animal from which the gene is isolated and that such variation may prove useful in producing unique accessory molecule genes.

The nucleotide sequence of a murine TNF, gene has been determined and is shown as SEQ ID NO: 12. The present invention contemplates the use of any accessory molecule ligand gene which is homologous to SEQ ID NO: 12, and thus hybridizes to the sequence at low stringency conditions. One of ordinary skill in the art will understand that the accessory molecule ligand genes, including the murine TNF, gene may vary depending on the individual from which the gene is isolated and that these variations may prove useful in producing unique accessory molecule genes.

The nucleotide sequence of human Fas ligand and murine (C57BL/6) Fas ligand have been determined and are shown as SEQ ID NOS: 13 and 14, respectively. The nucleotide sequence of murine Balb/c Fas ligand is shown as SEQ ID NO: 31. The present invention contemplates the use of any accessory molecule ligand gene which is homologous to any of SEQ ID NOS: 13, 14, and 31, and thus hybridizes to the sequences at low stringency conditions. One of ordinary skill in the art will understand that the accessory molecule ligand genes, including the human Fas ligand or murine Fas ligand genes may vary depending on the particular individual or animal from which the gene is isolated and that such variations may prove useful in producing any accessory molecule genes.

The nucleotide sequence of a human CD70 gene has been determined and is shown as SEQ ID NO: 15. The murine CD70 gene sequence has also been determined, and is shown as SEQ ID NO: 36 and was described by Tesselaar et. al, J. Immunol. 159:4959-65(1997). The present invention contemplates the use of any accessory molecule ligand gene which is homologous to SEQ ID NO: 15 or 36, and thus hybridizes to this sequence at low stringency conditions. One of ordinary skill in the art will .understand that the accessory molecule ligand genes, including the human CD70 gene may vary depending on the individual from which the gene is isolated and that these variations may prove useful in producing unique accessory molecule ligand genes.

The nucleotide sequence of human CD30 ligand gene has been determined and is shown as SEQ ID NO: 16. The present invention contemplates the use of any accessory molecule ligand gene which is homologous to SEQ ID NO: 16, and thus hybridizes to this sequence at low stringency conditions. One of ordinary skill in the art will understand that the accessory molecule ligand genes, including the human CD30 ligand gene, may vary depending on the individual from which the gene is isolated and that such variations may prove useful in producing unique accessory molecule ligand genes.

The present invention also contemplates variations and variants of the nucleotide sequences of the accessory molecule ligand genes provided herein which are caused by alternative splicing of the messenger

RNA.

This alternative splicing of the messenger RNA inserts additional nucleotide sequences which may encode one or more optional amino acid segments which in turn allows the accessory molecule ligand encoded to have additional properties or functions.

The .nucleotide sequence of a human and mouse 4-1BBL have been determined and are shown as SEQ ID NOS: 17 and 18, respectively. The present invention contemplates the use of any accessory molecule ligand gene which is homologous to either SEQ ID NOS: 17 or 18, and thus 10 hybridizes to these sequences at low stringency conditions. One of ordinary skill in the art will understand that accessory molecule ligand genes, including the human 4-1BBL gene may vary depending on the individual from which it is isolated and that such variations may prove useful in producing unique accessory molecule ligand genes.

The present invention also contemplates chimeric accessory molecules containing any domain, sub-domain portion, or amino acid sequence encoded by the following genes: bovine TNF-a (SEQ ID NO: 21), murine CD40 ligand (SEQ ID NO: 22), human nerve growth factor-3 (SEQ ID NO: 23), murine nerve growth factor (SEQ ID NO: 24), rat Fas ligand (SEQ ID NO: 25), human TNF-related apoptosis inducing ligand (TRAIL) (SEQ ID NO: 41, Genbank accession number U37518), murine TNF-related apoptosis inducing ligand (TRAIL) (SEQ ID NO: 42, Genbank accession number U37522), murine CD30-Ligand (SEQ ID NO: 43), human 4-1BBL (SEQ ID NO: 17), and murine 4-1BBL (SEQ ID NOS: 44 and 18). The present invention also contemplates chimeric accessory molecules which utilize genes encoding amino acid sequences homologous to these sequences.

The present invention contemplates chimeric accessory molecule ligand genes which are comprised of a nucleotide segment derived from one accessory molecule ligand gene operatively linked to a nucleotide sequence derived from a different accessory molecule ligand gene or other gene.

For example, chimeric accessory molecule ligand genes are contemplated which are comprised of a segment of the murine CD40 ligand gene which has been operatively linked to at least one other additional gene segment derived from a different accessory molecule ligand gene. The size of the particular segment derived from the different accessory molecule ligand gene may .1 0 vary from a nucleotide sequence encoding a few amino .acids, a sub-domain of the accessory molecule ligand, a domain of the accessory molecule ligand or more than a domain of an accessory molecule ligand. Other chimeric accessory molecules of the present invention are 15 comprised of an accessory molecule ligand gene into a. which nucleotides encoding an amino acid segment which is not found as part of a naturally occurring accessory molecule ligand have been inserted. This amino acid ~segment may be artificially created or derived from a protein found in nature. The chimeric accessory molecule ligand gene encodes a chimeric amino acid i sequence and thus a chimeric accessory molecule ligand encoded may possess unique properties in addition to the properties found on the individual segments derived from the different accessory molecule ligand genes. The chimeric accessory molecule ligand gene may encode an accessory molecule ligand which has properties derived from the accessory molecule ligand used to construct the chimeric gene.

Each of the accessory molecule ligand genes which are a member of the tumor necrosis factor family have a similar secondary structure consisting of a number of domains. This domain structure includes a first domain which is encoded by the 5' region of the accessory molecule ligand gene. The second domain (Domain II) is the domain which contains the amino acids which span the cell membrane and is thus called the transmembrane domain. The third domain (Domain III) is the proximal extracellular domain and these amino acids are the amino acids which are found proximal to the cellular membrane.

The fourth domain (Domain IV), is encoded by the 3' end of the accessory molecule ligand gene and has been called the distal extracellular domain. The distal extracellular domain (Domain IV) generally makes up the soluble form of the tumor necrosis factor family molecule. Based on the x-ray crystal structure of human 1. 0 TNF, the predicted secondary structure of the accessory molecule, CD40 ligand has been deduced together with the .domain structure of these molecules by M. Peitsch and C.

Jongeneel, International Immunology, 5:233-238 (1993) The secondary structures of the other members of the tumor necrosis factor family were deduced using computer S. analysis together with comparison to the human TNF and CD40 ligand domain structure. In Table I, the domain boundaries of a number of accessory molecule ligand genes is shown. A diagram of these domains for a number of these accessory cell molecule ligands is shown in Figure 1. The assignments of the domain boundaries are i approximate and one of ordinary skill in the art will understand that these boundaries may vary and yet still provide useful identification of domains.

*5 S S S* S S S

S.

*5 S S S. S S

S

S.

TAB3LE I DOMAIN STRUCTURE OF TUMOR NECROSIS FACTOR FAMILY MOLECULES* Domain I Domain 11 Domain III Domain IV (Cytoplasmic) (Transmembrane) (Proximal (Distal Extraceilular) Extracellular) Human CD40 1-42 43-135 136-330 331-786 Ligand Murine CD40 1-42 43-135 136-327 328-783 Ligand Bovine CD40 1-42 43-135 136-330 331-786 Ligand Human TNF-a 1-87 88-168 169-228 229-699 Murine TNF-a 1-87 88-168 169-237 238-705 Porcine TNF-a 1-87 88-168 169-228 229-696 Human TNF-pl 1-39 40-129 130-153 154-615 Porcine TNF-fl 1-39 40-126 127-150 151-612 Human Fas 1-237 238-315 316-390 391-843 Ligand Murine Fas 1-237 238-309 310-384 385-837 Ligand Human CD70 1-61 62-117 118-132 133-579 Murine CD70 1-73 74-123 124-138 139-585 Human CD30 1-117 118-186 187-240 241-702 Ligand Murine CD30 1-135 136-201 202-255 256-717 Ligand Human 4-1 BBL 1-69 70-174 175-210 211-762 Murine 4-1BBL 1-237 238-333 334-369 370-927 Human TRAIL 1-39 40-117 118-375 376-843 Murine TRAIL 1-51 52-111 112-387 388-873 The Domains above are identified by the nucleotide boundaries of each domain using the first nucleotide of the initial methionine of the cDNA as nucleotide number 1.

One of ordinary skill in the art will understand that typical chimeric accessory molecule genes would include genes produced by exchanging domains or subdomain segments between, for example, a mouse ligand gene and a human CD40 ligand gene. For example, chimeric accessory molecule gene may be constructed by operatively linking Domain I of the human CD40 ligand gene to Domains II-IV of the murine CD40 ligand gene.

One of ordinary skill in the art will understand the 1 0 variety of chimeric accessory molecule ligand genes which may be produced using the accessory molecules identified in Table I. The present invention also contemplates chimeric accessory molecules which are not shown in Table I but which are shown to have a similar domain structure. Other chimeric genes are also contemplated in which smaller segments (sub-domain segments) are exchanged between, for example, a murine ligand gene and a human CD40 ligand gene or a second murine CD40 ligand gene. One of skill in the art will understand that genes encoding accessory molecules will have at least gene segments which correspond to various functional segments of an accessory molecule ligand such as the murine CD40 ligand encoded by the murine CD40 ligand gene (SEQ ID NO: It will also be apparent to one of skill in the art that the nucleotide boundaries identified in Table I may vary considerably from those identified for the murine CD40 ligand gene (SEQ ID NO: 1) and still define domains which are useful in the present invention.

In one preferred embodiment, the chimeric accessory molecule ligand gene is comprised of the nucleotides encoding extracellular domains (Domains III and IV) of human CD40 ligand operatively linked to the nucleotides encoding transmembrane (Domain II) and the nucleotides encoding cytoplasmic domain (Domain I) of the murine ligand gene. Examples of such preferred chimeric accessory molecules are shown in Figure 2. An exemplary nucleotide sequence for such a gene is SEQ ID NO: 7. In other chimeric accessory molecule ligand genes of the present invention, the nucleotides encoding the extracellular domains (Domains III and IV) of the murine ligand gene may be operatively linked to nucleotides encoding the transmembrane (Domain II) and cytoplasmic domain (Domain I) of the human CD40 ligand gene. An exemplary nucleotide sequence for such a gene is SEQ ID NO: 3. In other preferred chimeric accessory molecule ligand genes of the present invention, the nucleotides encoding the extracellular domains (Domains III and IV) and transmembrane domain (Domain II) of human ligand are coupled to the nucleotides encoding cytoplasmic domain (Domain I) of murine CD40 ligand gene.

15 An exemplary nucleotide sequence for such a gene is SEQ ID NO: 6. Other chimeric accessory molecule genes contemplated by the present invention comprise the nucleotides encoding the extracellular domains (Domains III and IV) and transmembrane domain (Domain I) of the 20 murine CD40 ligand gene operatively linked to the nucleotides encoding cytoplasmic domain of the human CD40 ligand gene. An exemplary nucleotide sequence for .such a gene is SEQ ID NO: 5. Other chimeric accessory molecule ligand genes are contemplated by the present invention in which the human CD40 ligand gene extracellular domains (Domain III and IV) is operatively linked to the murine CD40 ligand gene transmembrane domain (Domain I) which is operatively linked to the human CD40 ligand gene cytoplasmic domain (Domain I).

An exemplary nucleotide sequence for such a gene is SEQ ID NO: 4.

One of ordinary skill in the art will understand that many more combinations which utilize domains or other selected segments of any of the accessory molecule ligand genes including the human CD40 ligand genes and the mouse CD40 ligand genes are possible. Such additional chimeric accessory molecule genes would include the following genes: chimeric accessory molecule genes in which the nucleotides encoding Domain I are selected from a particular accessory molecule ligand gene and operatively linked, either directly or by an additional nucleotide sequence to the nucleotides encoding Domain II from a particular accessory molecule ligand gene. These domains then would be operatively linked either directly or by an additional nucleotide sequence to the nucleotides encoding Domain III from a particular accessory molecule ligand gene. This molecule would then be operatively linked either directly or by an additional nucleotide sequence to the :.:.nucleotides encoding Domain IV of a particular accessory molecule ligand gene. The chimeric accessory molecule 15 ligand gene constructed in this manner may have additional nucleotides on either end or between domains which are useful to provide different amino acids in these positions. One of ordinary skill in the art will understand that these particular combinations are merely 20 illustrations and that numerous other combinations could be contemplated in which gene segments comprising nucleotides encoding less than the entire domain of an accessory molecule are exchanged between different accessory molecules.

The present invention also contemplates chimeric accessory molecule ligand genes which are comprised of gene segments of mouse or human CD40 ligand in combination with gene segments derived from Fas ligand, TNF,, TNF, CD70, CD30L, 4-1BBL, nerve growth factor or TNF-related apoptosis inducing ligand (TRAIL).

Particularly useful chimeric accessory molecule ligand genes comprise at least one gene segment which is derived from a murine CD40 ligand gene together with gene segments or a gene segments derived from a different accessory molecule ligand gene.

The present invention also contemplates chimeric accessory molecule ligand genes in which the accessory molecules produced have been modified to remove amino acids within the chimeric accessory molecule that are used by post-translational mechanisms to regulate the level of expression of the accessory molecule or accessory, molecule protein on a particular cell. The sites removed from the chimeric accessory molecules or chimeric molecule may include amino acids or sites which make up protease cleavage sites including metallothionine proteases, serine proteases and other proteases that recognize an amino acid sequence either specifically or nonspecifically. In particular preferred embodiments, amino acids in Domain III which make up potential or actual recognition site(s) used by post-translational regulatory mechanisms have been 15 modified or removed.

The present invention also contemplates chimeric accessory molecule ligand genes in which the domains, [.subdomain fragments or other amino acid residues have been taken from one accessory molecule ligand gene and 20 moved into a second accessory molecule ligand gene from the same species. For example, in this particular embodiment, the human Domain I, and the human Domain II from the CD40 ligand molecule may be operatively linked to the nucleotides encoding the human Domain III from, for example, the CD70 molecule which is in turn operatively linked to human Domain IV for the ligand molecule. This chimeric accessory molecule therefore contains human CD40L Domains I, II and IV and human CD70 Domain III. An exemplary nucleotide sequence for such a gene is SEQ ID NO: 19. One of ordinary skill in the art will understand that a number of such combinations using domains from the same species from different accessory molecule ligand genes may create a number of chimeric accessory molecule genes which may all have specific activities and properties.

The present invention contemplates chimericaccessory molecule ligand genes in which the Domain III of a particular accessory molecule ligand gene has been replaced with a Domain III from a different accessory molecule ligand gene. In one particularly preferred embodiment, the mouse Domain III has been used to replace the human Domain III in the CD40 ligand molecule. This chimeric accessory molecule therefore contains the human CD40L Domain I, the human Domain II, mouse CD40L Domain III, and human Domain IV. An exemplary nucleotide sequence for such a gene is SEQ ID NO: The present invention also contemplates the use of chimeric accessory molecules that contain man-made amino acid sequences inserted into or in place of a portion of a domain or other amino acid sequence of an accessory 15 molecule gene. These man-made amino acid segments may be created by selecting any amino acid sequence that may be used to give the accessory molecule a particular function or to remove another undesired function. These man-made amino acid segments are produced by inserting 20 into the accessory molecule ligand gene or chimeric accessory molecule ligand gene the nucleotide sequences required to encode those particular man-made amino acid segments in the desired positions. Further, the chimeric accessory molecule ligand genes may contain nucleotide segments which comprise sub-domain segments of other molecules or small segments in which amino acids have been changed for a desired purpose. The use of sub-domain nucleotide segments allows the introduction of short amino acid sequences derived from other molecules into chimeric accessory molecules of the present invention. The incorporation of such short subdomain segments or amino acid changes into the accessory molecule ligand allows the introduction of desired or the removal of undesired features of that molecule.

The identification of domain structures within accessory cell molecules is well known in the art and generally requires the identification of cysteineresidues within the accessory molecules and the subsequent mapping of disulfide bonds between various cysteine residues. The mapping of various sub-domain segments of an accessory molecule is well known in the art and involves analysis of the amino acid sequence of the accessory molecules and generally involves a comparison of the crystal structure of tissue necrosis factor with the use of predictive algorithms thereby producing a predicted structure of a chimeric accessory molecule or an accessory molecule. This predicted .structure of these molecules can then be used to select various sub-domain portions of the molecule to be used to construct further chimeric accessory molecules.

Examples of such mapping studies include the studies by 15 M. Pitsch and C. V. Jongeneel, International Immunoloqy, 5:233-238 (1993) and the analysis shown in Figure 1.

The present invention also contemplates accessory molecule ligand genes and chimeric accessory molecule ligand genes which are truncated and encode less than 20 the full length of the amino acid sequence found in the native accessory molecule ligand. These truncations may alter the properties of the accessory molecule ligand i gene but some identified activity is maintained. Such truncations may be made by removing a gene segment or gene segments from the accessory molecule gene and typically would be performed by removing nucleotides encoding domains which are not directly involved in the binding of the accessory molecule ligand with its accessory molecule. These truncated accessory molecule ligand genes or chimeric truncated accessory molecule ligand genes may contain further gene segments which encode amino acid segments or domains which replace the domains removed from that truncated accessory molecule gene. However, such replacement of the portions of the accessory molecule removed by truncation is not necessary.

The chimeric accessory molecule genes of the present invention may be constructed using standard genetic engineering methods to operatively link a particular nucleotide sequence from one accessory molecule Ligand gene to a different nucleotide sequence derived from the same or different accessory molecule ligand gene. In addition, standard genetic engineering methods may be used to insert man-made nucleotide sequences or sub-domain nucleotide sequences into the chimeric accessory molecule ligand gene. One of ordinary skill in the art will understand that various methods may be utilized to produce such chimeric accessory molecule genes. For example, a gene conversion method known as "SOEN" may be used to produce 15 a chimeric accessory molecule gene which contains nucleotide segments derived from different chimeric accessory molecules. The methods for using this gene conversion method are well known in the art and have been described for example in Horton, R. Mol.

20 Biotechnol., 3:93 (1995); Ali, S. A. and A.

Steinkasserer, Biotechniques, 18:746 (1995); Vilardaga, J. E. Di Paolo, and A. Bollen, Biotechnigues, 18:604 (1995); Majumder, F. A. Fattah, A. Selvapandiyan, and R. K. Bhatnagar, PCR. Methods Appl., 4:212 (1995); Boles, E. and T. Miosga, Curr. Genet. 28:197 (1995); Vallejo, A. R. J. Pogulis, and L. R. Pease, PCR.

Methods Appl., 4:S123 (1994); Henkel, T. and P. A.

Baeuerle, Anal. Biochem., 214:351 (1993); Tessier, D. C.

and D. Y. Thomas, Biotechniques, 15:498 (1993); Morrison, H. G. and R. C. Desrosiers, Biotechnicues, 14:454 (1993); Cadwell, R. C. and G. F. Joyce, PCR.

Methods Appl., 2:28 (1992); and, Stappert,

J.

Wirsching, and R. Kemler, Nucleic Acids Res., 20:624 (1992). Alternatively, one of ordinary skill in the art will understand that site-directed mutagenesis may be used to introduce changes into a particular nucleotide sequence to directly produce or indirectly be used to produce a chimeric accessory molecule gene of the present invention. For example, the mutagen kit provided by BioRad Laboratories may be used together with the methods and protocols described within that kit to producq the desired changes in the nucleotide sequence. These methods were originally described by Kunkel, Proc. Natl. Acad. Sci. USA, 82:488-492 (1985) and Kunkel et al., Meth. Enzol. Mol., 154:367-382 (1987). By using the site directed mutagenesis protocols described herein and known within the art, a skilled investigator may induce individual nucleotide changes which result in an altered amino acid sequence or which preserve an amino acid sequence but introduce a desired restriction enzyme recognition sequence into the gene.

15 This new restriction endonuclease recognition site may then be used to cut the gene at that particular point and use it to a gene or segment of another accessory molecule ligand gene. In addition to these methods, one of ordinary skill in the art will understand that an entire chimeric accessory molecule ligand gene may be synthesized using synthetic methods known in the art.

""This methodology only requires that the skilled artesian i generating nucleotide sequence of a chimeric accessory molecule ligand gene and provide that sequence to a company which is capable of synthesizing such a gene.

B. Genetic Constructs The present invention contemplates the use of accessory molecule ligand genes or chimeric accessory molecule ligand genes which are present in various types of genetic vectors. A genetic vector refers to a DNA molecule capable of autonomous replication in a cell into which another DNA segment can be inserted to cause the additional DNA segments to replicate. Vectors capable of expressing genes contained in that vector are referred to as "expression vectors." Thus, the genetic vectors and expression vectors of the present invention are recombinant DNA molecules which comprise at least two nucleotide sequences not normally found together in nature.

The genetic vectors useful in the present invention contain an accessory molecule ligand gene which encodes an accessory molecule ligand which is optionally operatively linked to a suitable transcriptional or translational regulatory nucleotide sequence, such as one derived from a mammalian, microbial, viral, or insect gene. Such regulatory sequences include .sequences having a regulatory role in gene expression, such as a transcriptional promoter or enhancer, an operator sequence to control transcription, a sequence encoding a ribosomal binding site within the messenger 15 RNA and appropriate sequences which control transcription, translation initiation or transcription termination.

S.Particularly useful regulatory sequences include the promoter regions from various mammalian, viral, 20 microbial, and insect genes. The promoter region directs an initiation of transcription of the gene and causes transcription of DNA through and including the accessory molecule ligand gene. Useful promoter regions include the promoter found in the Rous Sarcoma Virus (RSV) long terminal repeat (LTR), human cytomegalovirus (HCMV) enhancer/promoter region lac promoters, and promoters isolated from adenovirus, and any other promoter known by one of ordinary skill in the art would understand to be useful for gene expression in eukaryotes, prokaryotes, viruses, or microbial cells.

Other promoters that are particularly useful for expressing genes and proteins within eukaryotic cells include mammalian cell promoter sequences and enhancer sequences such as those derived from polyoma virus, adenovirus, simian virus 40 (SV40), and the human cytomegalovirus. Particularly useful are the viral early and late promoters which are typically found adjacent to the viral origin of replication in viruses such as the SV40. Examples of various promoters which have been used in expression vectors have been described by Okiama and Berg (Mol. Cell. Biol. 3:280, 1983), the pMLSVN SV40 described by Kossman et al., Nature 312:768 (1984). One of ordinary skill in the art will understand that the selection of a particular useful promoter depends on the exact cell lines and the other various parameters of the genetic construct to be used to express the accessory molecule ligand gene or the *.chimeric accessory molecule ligand gene within a particular cell line. In addition, one of ordinary skill in the art will select a promoter which is known to express genes in the target cell at a sufficiently 15 high level to be useful in the present invention.

The genetic vectors and expression vectors of the present invention optionally contain various additional regulatory sequences including ribosome binding sites which allow the efficient translation of the messenger 20 RNA produced from an expression vector into proteins, the DNA sequence encoding various signals peptides which may be operatively linked to the accessory molecule ligand gene or the chimeric accessory molecule ligand gene. The signal peptide, if present, is expressed as a precursor amino acid which enables improved extracellular secretion of translation fusion polypeptide.

The genetic constructs contemplated by the present invention therefore include various forms of accessory molecule ligand genes described above which are operatively linked to either a promoter sequence or a promoter and enhancer sequence and also operatively linked to a polyadenylation sequence which directs the termination and polyadenylation of messenger RNA. It is also contemplated that the genetic constructs of the present invention will contain other genetic sequences which allow for the efficient replication and expression of that construct within the desired cells. Suchsequence may include introns which are derived from native accessory molecule ligand genes or, for example, from a virus gene.

The present invention also contemplates gene therapy vectors which are able to directly infect mammalian cells so as to introduce the desired accessory molecule ligand gene or chimeric accessory molecule ligand gene into that cell. These gene therapy vectors are useful for directly infecting cells which have been isolated from an animal or patient, or can be directly introduced into an animal or patient and thereby directly infect the desired cell within that animal or patient.

Many types of gene therapy vectors which are able 15 to successfully transfer genes and cause the expression of desired foreign DNA sequences have been developed and described in the literature. For example, the article entitled "Gene Transfer Vectors for Mammalian Cells" in Current Comm. Mol. Biol., Cold Springs Harbor 20 Laboratory, New York (1987). Further, naked DNA can be physically introduced into eukaryotic cells including S: human cells by transvection using any number of o* techniques including calcium phosphase transfection (Berman et al., Proc. Natl. Acad. Sci. USA, 81:7176 (1984)), DEAE-Dextran Transfection, protoplast fusion (Deans et al., Proc. Natl. Acad. Sci. USA, 81:1292 (1984)), electroporation, liposome fusion, polybrene transfection and direct gene transfer by laser micropuncture of the cell membrane. In addition, one of ordinary skill in the art will understand that any technique which is able to successfully introduce the DNA into a cell in such a manner as to allow it to integrate into the genome of a cell and allow the expression of the desired gene would be useful in the present invention.

Specifically, gene therapy vectors which utilize recombinant infectious virus particles for gene delivery have been widely described. See, for example, Brody, S.

L. and R. G. Crystal, Ann. N. Y. Acad. Sci., 716:90 (1994); Srivastava, Blood. Cells, 20:531 (1994); Jolly, Cancer Gene Ther., 1:51 (1994); Russell, S.

Eur. J. Cancer, 30A:1165 (1994); Yee, J. T.

Friedmann, and J. C. Burns, Methods Cell Biol., 43 Pt A:99 (1994); Boris-Lawrie, K. A. and H. M. Temin, Curr.

Opin. Genet. Dev., 3:102 (1993); Tolstoshev, Annu.

Rev. Pharmacol. Toxicol., 33:573 (1993); and, Carter, B.

Curr. Opin. Biotechnol., 3:533 (1992). The present invention contemplates the use of gene therapy vectors *to carry out the desired methodology of the present invention by introducing a gene encoding an accessory molecule ligand gene or a chimeric accessory molecule 15 ligand gene into the cell. Many viral vectors have been defined and used as gene therapy vectors and include virus vectors derived from simian virus 40 adenoviruses, adeno-associated viruses, and retroviruses. One of ordinary skill in the art will 20 understand that useful gene therapy vectors are vectors which are able to directly introduce into the target cells the DNA which encodes the accessory molecule ligand and allow that DNA to persist in the cell so as to express the accessory molecule ligand in the desired manner within the cell.

The gene therapy vectors of the present invention are useful for introducing accessory molecule ligand genes into a variety of mammalian cells including human cells. The particular cells infected by the gene therapy vector will depend on the various specifics of the vector and such vectors can be used to introduce the accessory molecule ligand genes of the present invention into hematopoietic or lymphoid stem cells, antigen presenting cells, embryonic stem cells, and other cells which are capable of presenting antigen within the immune system including cells which have CD40 on their surface. Further, such gene therapy vectors are able to introduce a gene encoding an accessory molecule ligand gene into a human neoplastic cell such as a lymphoma, leukemia, AML, CLL, CML, AMML, CMML, breast cancer, lung cancer, ovarian cancer or any tumor capable of acting as antigen presenting cells or cells which can stimulate bystander antigen presenting cells. Further, the contemplated gene therapy vectors may be used to introduce the accessory molecule ligand genes of the present invention into cells which have been engineered to make those cells capable of presenting antigen to the immune system.

III. Cells Containing Genetic Constructs Encoding an Accessory Molecule Ligand or Chimeric Accessory Molecule Ligand 15 The present invention also contemplates various cells which contain the genetic constructs of the •present invention. These cells contain the constructs which encode the accessory molecule ligand gene and thus contain the various genetic elements described in a 20 Section II.B. above. These cells may be microbial cells, eukaryotic cells, insect cells, and various mammalian cells including human cells. In preferred embodiments of the present invention, these cells include various neoplastic cells including human neoplastic cells. These neoplastic cells may be of any cell type and include cells of the immune system, and other blood cells. Particularly preferred are any neoplastic cells which may function as an antigen presenting cells within the immune system or which may stimulate bystander antigen presenting cells by expression of a transgenic accessory cell molecule of the present invention. Typically these neoplastic which are able to function to present antigen to the immune system have or have had an accessory molecule, such as the CD40 molecule, on the cell surface. Generally, these cells are naturally capable of presenting antigen to the immune system, but the present invention also contemplates the introduction of accessory molecule ligand genes into a cell which is not naturally able to present antigen to the immune system but which has been genetically engineered to make that cell capable of presenting antigen to the immune system. Typically, these cells include various known cell types such as monocytes, macrophages, B cells, Langerhans cells, interdigitating cells, follicular dendritic cells or Kupffer cells and the like which have become neoplastic.

In addition, the present invention also contemplates *cells from various carcinomas, breast, ovarian and lung *cancers which contain the genetic constructs described herein. In other preferred embodiments, an accessory molecule ligand gene of the present invention is placed 15 into cells which may be injected into a treatment site such as a tumor bed or joint. For example, the accessory molecule ligand gene of the present invention S. may be inserted into a fibroblast cell and the accessory molecule ligand expressed on the surface of that cell.

20 The fibroblasts are then injected into the treatment site and cause the desired immuno effect due to the presence of the accessory molecule ligand on the surface i of those cells. These cells stimulate other immune cells present in that treatment site (bystander cells) This process then results in the desired effect on the immune system.

IV. Methods Utilizing Genetic Vectors and Constructs Containing an Accessory Molecule Ligand Gene The present invention contemplates methods of altering the immunoreactivity of human cells using a method which includes introducing a gene encoding an accessory molecule ligand gene into the human cells so that the accessory molecule ligand encoded by that gene is expressed on the surface of those cells. The present invention is useful for any human cells which participate in an immune reaction either as a target for the immune system or as part of the immune system which responds to the foreign target. A large variety of methods are contemplated in which the final result is that the accessory molecule ligand gene is introduced into the desired cells. These methods include ex vivo methods, in vivo methods and various other methods which involve injection of DNA, genetic vectors or gene therapy vectors into the animal or human, including injection directly into the tumor bed present in any animal or human.

Ex vivo methods are contemplated wherein the cells into which the accessory molecule ligand gene is to be introduced are isolated from the animal or patient and then the gene is introduced into those isolated cells 15 using suitable methods. Examples of useful ex vivo methods have been described for example by Raper, S. E., M. Grossman, D. J. Rader, J. G. Thoene, B. J. Clark, D.

M. Kolansky, D. W. Muller, and J. M. Wilson, Ann. Surg., 223:116 (1996); Lu, R. N. Shen, and H. E. Broxmeyer, Crit. Rev. Oncol. Hematol., 22:61 (1996); Koc, 0. J.

A. Allay, K. Lee, B. M. Davis, J. S. Reese, and S. L.

:Gerson, Semin. Oncol., 23:46 (1996); Fisher, L. J. and J. Ray, Curr. Opin. Neurobiol., 4:735 (1994); and, Goldspiel, B. L. Green, and K. A. Calis, Clin.

Pharm., 12:488 (1993). D. Dilloo et al., in Blood 90:1927-1933 (1997), describe a method, using activated cells, for treating B-acute lymphoblastic leukemia (ALL). They cocultured leukemia cells with fibroblasts infected with a retroviral vector encoding CD40L, then injected the cell mix into mice. Such an approach, if taken in humans, would differ from that contemplated here in that the therapeutic cells are stimulated in vitro, by another cell line expressing the accessory molecule ligand. Schultze, J.L. et al., in Blood 89: 3806-3816 (1997), describe a method for stimulating T-TILs (tumor-infiltrating T cells) cytotoxic for follicular lymphoma (FL) cells by exposing them, in vitro, to FL B cells which were previously cultured with CD40L-expressing fibroblasts. They propose an adoptive immunotherapy in which T-TILS stimulated in this manner are transfused into patients.

This method also requires in vitro stimulation, of the cells to be transfused, with another cell line expressing an accessory molecule.

Following the introduction of the gene, including any optional steps to assure that the accessory molecule ligand gene has been successfully introduced into those isolated cells, the isolated cells are introduced into the patient either at a specific site or directly into the circulation of the patient. In preferred embodiments of the present invention, cell surface 15 markers, including molecules such tumor markers or antigens identify the cells are used to specifically isolate these molecules from the patient. One of ordinary skill in the art will understand that such isolation methods are well known and include such 20 methodologies as fluorescence activated cell sorting (FACS), immunoselection involving a variety of formats including panning, columns and other similar methods.

o: The present invention also contemplates introducing the accessory molecule ligand gene into the desired cells within the body of an animal or human patient without first removing those cells from the patient.

Methods for introducing genes into specific cells in vivo, or within the patient's body are well known and include use of gene therapy vectors and direct injection of various genetic constructs into the animal or patient. Examples of useful methods have been described by Danko, I. and J. A. Wolff, Vaccine, 12:1499 (1994); Raz, A. Watanabe, S. M. Baird, R. A. Eisenberg,

T.

B. Parr, M. Lotz, T. J. Kipps, and D. A. Carson, Proc.

Natl. Acad. Sci. U. S. 90:4523 (1993); Davis, H. L., R. G. Whalen, and B. A. Demeneix, Hum. Gene Ther., 4:151 (1993); Sugaya, K. Fujita, A. Kikuchi, H. Ueda, K.

Takakuwa, S. Kodama, and K. Tanaka, Hum. Gene Ther., 7:223 (1996); Prentice, R. A. Kloner, Y. Li, L.

Newman, and L. Kedes, J. Mol. Cell Cardiol., 28:133 (1996); Soubrane, R. Mouawad, O. Rixe, V. Calvez, A.

Ghoumari, O0. Verola, M. Weil, and D. Khayat, Eur. J.

Cancer, 32A:691 (1996); Kass-Eisler, K. Li, and L.

A. Leinwand, Ann. N. Y. Acad. Sci., 772:232 (1995); DeMatteo, R. S. E. Raper, M. Ahn, K. J. Fisher, C.

Burke, A. Radu, G. Widera, B. R. Claytor, C. F. Barker, and J. F. Markmann, Ann. Surq., 222:229 (1995); Addison, C. T. Braciak, R. Ralston, W. J. Muller, J. Gauldie, and F. L. Graham, Proc. Natl. Acad. Sci. U. S. A., 92:8522 (1995); Hengge, U. P. S. Walker, and J. C.

Vogel, J. Clin. Invest., 97:2911 (1996); Felgner, P. L., 15 Y. J. Tsai, L. Sukhu, C. J. Wheeler, M. Manthorpe, J.

Marshall, and S. H. Cheng, Ann. N. Y. Acad. Sci., 772:126 (1995); and, Furth, P. A. Shamay, and L.

Hennighausen, Hybridoma, 14:149 (1995). In a typical application, a gene therapy vector containing an 20 accessory molecule ligand gene is introduced into the circulation or at a localized site of the patient to allow the gene therapy vector to specifically infect the desired cells. In other preferred embodiments the gene therapy vector is injected directly into the tumor bed present in an animal which contains at least some of the cells into which the accessory molecule ligand gene is to be introduced.

The present invention also contemplates the direct injection of DNA from a genetic construct which has a promoter and accessory molecule ligand gene followed by a polyadenylation sequence into a patient or animal.

Examples of such useful methods have been described by Vile, R. G. and I. R. Hart, Ann. Oncol., 5 Suppl 4:59 (1994). The genetic construct DNA is directly injected into the muscle or other sites of the animal or patient or directly into the tumor bed of the animal or patient.

Alternatively, DNA from a genetic construct containing at least an accessory molecule ligand gene is used and directly injected into the animal.

In preferred embodiments of the present invention, the immune reaction or response of a human patient or animal is .altered by introducing the accessory molecule ligand gene into cells, including human cells which have an accessory molecule present on the cell surface. Such cells include human cells, human antigen presenting cells and optionally these cells may be neoplastic antigen presenting cells which have the capacity to express the accessory molecule on the surface of the cell or cells which are capable of stimulating. In some embodiments, the amount of accessory molecule present on the surface of the cells into which the accessory 15 molecule ligand gene is to be introduced is very small and such small amounts of the accessory molecule may result from down-regulation of that accessory molecule on the surface of such cells. In some embodiments, the cells into which the accessory molecule ligand gene is 20 introduced have at least low levels of the CD40 molecule present on the cell surface or are derived from cells which did express the CD40 ligand molecule on the cell surface but have reduced or eliminated that expression.

The preferred methods of altering the immunoreactivity of a particular cell are applicable to mammalian cells including human cells. These human cells may include neoplastic human cells such as human lymphomas, leukemias, and other malignancies including breast, lung and ovarian cancers. In some preferred embodiments the cells are normal antigen presenting cells of a human patient such as monocytes, macrophages, B cells, Langerhans cells, interdigitating cells, follicular dendritic cells, Kupffer cells, and other similar cells. In preferred embodiments, the cells are lymphocytes which acquire altered immunoreactivity when the accessory molecules of the present invention-are introduced into those cells. In other preferred embodiments, the cells may be neoplastic or normal cells which are capable of stimulating bystander antigen presenting cells when the accessory molecule ligand genes of the present invention are introduced into these cells. The present invention also contemplates that cells which are not naturally capable of presenting antigen to the immune system may be genetically engineered to introduce the genes encoding the molecules required for antigen presentation, including genes encoding an accessory molecule, and thus allow these cells to act as artificial antigen presenting cells.

The accessory molecule ligand gene may then be introduced into these artificial antigen presenting cells. Various tests are well known in the literature 15 to determine whether a particular cell is able to function as an antigen presenting cell, such as cell proliferation or the production of lymphokines and therefore this aspect of the present invention may be easily determined.

In addition to the above normal human cells, the present invention also contemplates introducing the accessory molecule ligand gene into various neoplastic or malignant cells which optionally are antigen presenting cells. Such human neoplastic cells which are contemplated include leukemias, lymphomas, AML, AMML, or CMML, CML, CLL and any neoplastic cell which is capable of stimulating bystander antigen presenting cells when an accessory molecule ligand is introduced into that cell. Also contemplated are neoplastic cells such as a breast, ovarian or lung cancer cell which is capable of or is engineered to act as an antigen presenting cell.

However, the present immunomodulation also applicable to other malignancies not specifically identified and thus would include any tumor of any cell capable of presenting antigen within the animal or human immune system or any cell which is capable of acting asan antigen presenting cell or capable of stimulatingbystanding antigen presenting cells after an accessory molecule ligand gene has been introduced into those cells. Generally these antigen presenting cells have accessory molecules on the surface of the cells.

The present methods of altering the immunoreactivity of a human or animal cell contemplate the introduction of an accessory molecule ligand gene into the cells for which altered immunoreactivity is desired. The genes useful in the present invention include the wide range of accessory molecule ligand genes and chimeric accessory molecule ligand genes identified above and in preferred embodiments include at least a portion of the murine CD40 ligand gene. In particularly preferred embodiments, the accessory .15 molecule ligand gene introduced into the cells using the methods of the present invention is selected to correspond to the accessory molecule present on the surface of the cells for which altered immunoreactivity is desired. In one particular application of the present invention, the immunoreactivity of a cell which expresses the CD40 molecule on the cell surface would be accomplished by introducing the gene which encodes the CD40 ligand molecule and more preferably the murine ligand molecule.

The present invention also contemplates altering the immunoreactivity of human or animal cells by introducing an accessory molecule ligand gene which is a chimeric accessory molecule ligand gene into the cell.

The various useful chimeric accessory molecule ligand genes were identified above and could include a wide variety of molecules and allow the unique properties of those chimeric accessory molecule ligand genes to be utilized to alter the immunoreactivity of the target cells. In preferred embodiments, useful chimeric accessory molecule ligand genes are genes which encode at least a portion of the accessory molecule ligand which is capable of binding the accessory moleculepresent on the surface of the cells for which altered immunoreactivity is desired.

The methods of the present invention for altering the immunoreactivity contemplate the use of genetic vectors and genetic constructs including gene therapy vectors which encode an accessory molecule ligand and therefore contain an accessory molecule ligand gene.

Typically, the genetic vectors and genetic constructs including the gene therapy vectors of the present invention have a promoter which is operatively linked to the accessory molecule ligand gene followed by a polyadenylation sequence. In other embodiments, the only requirement is that the genetic vectors, genetic constructs, and gene therapy vectors of the present 15 invention contain the accessory molecule ligand gene or the chimeric accessory molecule ligand gene.

V. Methods of Treating Neoplasia The present invention also contemplates methods of treating human neoplasia comprising inserting into a human neoplastic cell a gene which encodes an accessory molecule ligand so that the accessory molecule ligand is expressed on the surface of the neoplastic cells. The present invention contemplates treating human neoplasia both in vivo, ex vivo and by directly injecting various DNA molecules containing a gene which encodes an accessory molecule ligand into the patient. However, at a minimum, the present methods for treating human neoplasia involve inserting the gene encoding the accessory molecule ligand into the neoplastic cells in such a way as to allow those neoplastic cells to express the accessory molecule ligand on the cell surface. The expression of the accessory molecule ligand gene in these neoplastic cells modulates the immune system to cause the neoplasia to be reduced or eliminated.

In a preferred method of treating human neoplasia, the method further comprises the steps of first obtaining the human neoplastic cells from a human patient and then inserting into the isolated human neoplastic cells a gene which encodes an accessory molecule ligand so that the accessory molecule ligand is expressedon the surface of the neoplastic cells. The human neoplastic cells having the accessory molecule ligand on the surface of that cell are then infused back into the human patient. One of ordinary skill in the art will understand that numerous methods are applicable for infusing the altered human neoplastic cells containing the gene encoding the accessory molecule ligand back into the patient and that these methods are well known in the art.

The contemplated methods of treating human 15 neoplasia are applicable to a wide variety of human neoplasias including lymphomas, leukemias, and other malignancies. In preferred embodiments the human e neoplasia is a neoplasia which involves the antigen presenting cells of the human immune system and includes 20 monocytes, macrophages, B cells, Langerhans cells, interdigitating cells, follicular dendritic cells, Kupffer cells, and the like. In other preferred embodiments, the human neoplasia is a leukemia, a lymphoma, AML, AMML, CMML, CML or CLL, lung cancer, breast cancer, ovarian cancer and other similar neoplasias.

The genetic vectors, genetic constructs and gene therapy vectors useful in the methods of treating human neoplasia of the present invention have been disclosed above and include constructs in which a promoter is operatively linked to the accessory molecule ligand gene or the chimeric accessory molecule ligand gene which is in turn operatively linked to a polyadenylation sequence. The methods of treating human neoplasia contemplate the use of genetic constructs, genetic vectors and gene therapy vectors as described in- this specification. In addition, the present invention contemplates the use of DNA which contains at least a gene encoding an accessory molecule ligand gene. This gene may or may not contain a promoter and other regulatory sequences.

In pieferred embodiments of the present invention, the cells comprising the human neoplasia are located in at least one defined site termed a tumor bed within the human patient. This tumor bed typically contains the tumor or neoplastic cell together with a number of other cells which are associated with the tumor or neoplastic cells. The present invention contemplates methods of treating such human neoplasia present in a tumor bed by injecting into the tumor bed of the patient, a gene which encodes an accessory molecule ligand so that the 15 accessory molecule ligand is expressed on the surface of the tumor cells thereby causing the cells to participate in an immune reaction. The gene which encodes the accessory molecule ligand may be present as part of a gene therapy vector, genetic construct or genetic 20 vector.

In preferred embodiments, the accessory molecule ligand gene is a chimeric accessory molecule ligand gene i which has at least a portion of the murine CD40 ligand gene is used. In other preferred embodiments, the accessory molecule ligand encoded is capable of binding an accessory molecule present on the human neoplasia to be treated.

The various gene therapy vectors used in the treatment methods of the present invention include vectors which are capable of directly infecting human cells. Such vectors have been described in the literature and are readily adaptable to the methods described in the present invention.

The present invention contemplates the use of any type of gene therapy including the methods of Raper, S.E. et al., Ann. Surq., 223:116 (1996); Lu, L. et al., Crit. Rev. Oncol. Hematol., 22:61 (1996); Koc, et al., Semin. Oncol., 23:46 (1996); Fisher, L. J. et al., Curr. Opin. Neurobiol., 4:735 (1994); Goldspiel, B. R.

et al., Clin. Pharm., 12:488 (1993); Danko, I. et al., Vaccine, 12:1499 (1994); Raz, E. et al., Proc. Natl.

Acad. Sci. 90:4523 (1993); Davis, H. L. et al., Hum. Gene Ther., 4:151 (1993); Sugaya, S. et al., Hum.

Gene Ther., 7:223 (1996); Prentice, H. et al., J. Mol.

Cell Cardiol., 28:133 (1996); Soubrane, C. et al., Eur.

J. Cancer, 32A:691 (1996); Kass-Eisler, A. et al., ann.

N. Y. Acad. Sci., 772:232 (1995); DeMatteo, R. P. et Ann. Surq., 222:229 (1995); Addison, C. L. et al., Proc. Natl. Acad. Sci. 92:8522 (1995); Hengge, U. R. et al., J. Clin. Invest., 97:2911 (1996); Felgner, P. L. et al., Ann. N. Y. Acad. Sci., 772:126 (1995); 15 Furth, Hybridoma, 14:149 (1995); Yovandich, J. et al., Hum. Gene Ther., 6:603 (1995); Evans, C.H. et al., Hum. Gene Ther., 7:1261.

VI. Methods of Vaccination The present invention contemplates methods of vaccinating an animal against a predetermined organism comprising administering to that animal a vaccine containing immunogenic animal antigens capable of causing an immune response in that animal against the desired organism together with a vector containing a gene encoding an accessory molecule ligand. The present invention also contemplates methods of vaccinating an animal which include administering the genes which encode the immunogenic antigen capable of causing a desired immune response or altering the immune response to a particular antigen together with a vector containing a gene including the accessory molecule ligand gene. In this particular embodiment, the vector or vectors introduced encode the immunogenic antigens desired and the desired accessory molecule ligand. The present invention also contemplates that the gene or genes encoding the immunogenic peptide or peptides may be present on the same vector as is the gene or genes encoding the accessory molecule ligand.

The vaccination methods of the present invention are general in that they may be used to produce a vaccinatiqn against any predetermined organism, such as a virus, a bacteria, a fungus or other organism. In addition, the present vaccination methods may be used to produce an immune response against a neoplastic cell.

In other preferred embodiments, the vaccination methods of the present invention utilize a genetic vector, a genetic construct or a gene therapy vector which contains an accessory molecule ligand gene which is a chimeric accessory molecule ligand gene. That chimeric accessory molecule ligand gene preferably 15 contains at least a portion of the murine CD40 ligand gene. In other preferred embodiments, the vaccination method utilizes a DNA molecule which encodes at the minimum the accessory molecule ligand gene or a chimeric accessory molecule ligand gene. This particular DNA may or may not include a promoter sequence which directs the expression of the accessory molecule ligand gene.

The present invention also contemplates that the vaccination method may utilize a genetic vector which is capable of expressing an accessory molecule ligand within a particular cell or organism together with a vector which is capable of expressing at least a single polypeptide from an andovirus. This andovirus polypeptide may be expressed from the same or different vector which expresses the accessory molecule ligand in that cell. In this particular embodiment, the andovirus polypeptide is also expressed in at least one cell type within the organism and serves to modulate the immune response found in response to this vaccination protocol.

The present invention also contemplates the introduction of an accessory molecule ligand gene into cells which are present in the joints of patients with rheumatoid arthritis. In preferred embodiments, the accessory molecule ligand gene introduced comprises at least a portion of the Fas ligand gene and upon expression the accessory ligand induces the cell death of cells expressing Fas on the cell surface. This process leads to the reduction of the destructive inflammatory process.

The following examples are provided to illustrate various aspects of the present invention and do not limit the scope of that invention.

VII. Methods of Treating Arthritis The present invention also contemplates methods of treating arthritis comprising inserting into a joint, cells which have been transformed with an accessory molecule, such as the Fas ligand. In preferred embodiments, the expression of that accessory molecule ligand or the stability of that molecule on the surface of the cells has been altered. In these preferred embodiments, the accessory molecule ligand functions in an enhanced manner to aid in the treatment of arthritis within the joint. The present invention contemplates treating human arthritis both in viv, ex vivo, and by directly injecting various DNA molecules containing genes which encode the useful accessory molecule ligand into the patients. Various useful protocols may be designed to rheumatoid arthritis including those described in the example section below.

The present invention contemplates the treatment of arthritis utilizing accessory molecule ligand genes which may be chimeric accessory molecule ligand genes comprised of portions of that gene being derived from two different accessory molecule ligand genes. In other embodiments, the chimeric accessory molecule ligands may be produced by utilizing domains from the same accessory molecule ligand gene. The resulting chimeric accessory molecule ligands have an altered stability on the surface of cells upon which they are expressed. This altered stability modulates the function of the immune system in the local environment around the cells in which these chimeric accessory molecule ligands are expressed. For example, in certain preferred embodiments, Fas ligand stability is altered on the surface of cells within a joint of a patient suffering from arthritis. This altered stability modulates the immune system and causes the cells to be targeted for apoptosis and thus reducing the immune response within the inflamed joint. In other embodiments, the accessory molecule ligand genes described within are altered such that the resulting accessory molecule ligand has an altered stability and causes an immunomodulatory effect which can be useful in the treatment of arthritis.

15 The present invention contemplates in preferred embodiments that chimeric accessory molecule ligands genes be utilized in the treatment of arthritis. These S. chimeric accessory molecule ligand genes preferably contain at least a portion of the Fas ligand gene Domain 20 IV, which carries the effect or function for Fas ligand.

In preferred embodiments, at least in the portion of that domain, is present which allows Fas ligand to have its biologic effects. In other preferred chimeric accessory molecule ligands, those ligands contain domains from other accessory molecule ligand genes of the present invention or from a different domain of the same accessory molecule ligand. Particularly preferred are Fas chimeric accessory molecule ligand genes made up on Domain IV of the human Fas ligand operatively linked with Domain III of the mouse Fas ligand. This particular combination results in more stable Fas ligand and thus, by replacing Domain III of human Fas ligand with Domain III of the mouse ligand, the activity of the human Fas ligand gene is altered.

Alternatively, in other preferred embodiments, the murine Fas ligand gene is used to encode the murine Fas ligand on the surface of cells in place of the human Fas ligand. The murine Fas ligand is more stable than the human Fas ligand and thus, alters the Fas ligand activity in the joint. The resulting alter Fas ligand activity is useful in the treatment of rheumatoid arthritis, Further preferred embodiments include embodiments in which the effect or function present on Domain IV of the humand Fas ligand is combined with other domains from other accessory molecule ligands. For example, CD70 Domain III is more stable than Domain III of the human Fas ligand and thus the chimeric accessory molecule ligand made up of Domain III from the human CD70 and Domain IV of the Fas ligand together with other supporting domains would be more stable. The increased 15 stability leads to increase Fas ligand activity. In other preferred embodiments, Domain III of the Fas ligand is replacd with multiple copies of a domain or domains. Such multiple copies of domains include domains made up of two or more copies of other domains 20 such as Domains III or I of the CD70 molecule.

In other preferred embodiments, the present invention contemplates accessory molecule ligand genes, such as Fas ligand genes, in which a cleavage site for matrix-metalloproteinase (MMP), have been removed from the accessory molecule ligand. MMP cleavage and recognition sites, charted in Figure 28, are discussed in Smith, M.M. et al., Journal of Biol. Chem. 270:6440- 6449 (95) and Nagase, and G.B. Fields, Biopolymers (Peptide Science) 40:399-416 In preferred embodiments, at least one MMP site has been removed from at least Domain III of the Fas ligand gene. The removal of the MMP site from the Fas ligand gene makes the Fas ligand more stable and thus, more effective in the treatment of arthritis.

In other preferred embodiments, chimeric accessory molecule ligand genes are comprised of portions of the human Fas ligand gene with other domains from other human accessory molecule ligands or domains from accessory molecules derived from other species. For example, the present invention contemplates the use of domains from CD40 ligand, CD70 ligand, CD30 ligand, TNFrelated apoptosis inducing ligand (TRAIL), TNF-a as well as mutants of human Fas ligand and murine Fas ligand.

Production of such chimeric accessory molecule ligands is easily accomplished by manipulating and producing accessory molecule ligand genes which are chimeric and thus has portions derived from at least two different accessory molecule ligand genes.

C C e

C..

EXAMPLES

1. Expression of Human and Mouse Accessory Molecule Ligand in Human CLL Cells a. Construction of a Genetic Construct and Gene .Therapy Vector Containing a Human and Mouse Accessory Molecule Ligand Gene Either the human accessory molecule ligand gene (human CD40 ligand) or the murine accessory molecule ligand gene (murine CD40 ligand) was constructed utilizing the respective human and murine genes. Each of these genes was cloned in the following manner.

i. Murine CD40-L cloning Total RNA was isolated using the RNA STAT-60 kit (Tel-Test Inc., Friendswood, TX) from 1 x 107 B6 15 mouse splenocytes that were previously activated for 8 hours with immobilized CD3-specific mAb. cDNA was then synthesized with the Superscript cDNA synthesis kit (Gibco BRL, Grand Island, NY) using oligo-dT primers.

The murine CD40 ligand (mCD40-L) gene was then amplified 20 from the cDNA by PCR using the following specific primers. 5'-GTTAAGCTTTTCAGTCAGCATGATAGAA

(SEQ

ID NO: 26), 5'-GTTTCTAGATCAGAGTTTGAGTAAGCC (SEQ ID NO: 27). The amplified mCD40-L PCR product was subcloned into the HindIII and Xbal sites of the eukaryotic expression vector pcDNA3 (Invitrogen, San Diego, CA). A DNA fragment encompassing the CMV promoter, gene, and polyadenylation signal was released from this plasmid construct after restriction digestion with BglII and XhoI enzymes. This DNA fragment was then subcloned into the shuttle plasmid MCS(SK)pXCX2 (Spessot R, 1989, Virology 168:378) that was designated mCD40-L pXCX2.

This plasmid was used for adenovirus production as described below.

ii. Human CD40-L Cloning A plasmid containing the gene for human CD40-L was used to produce the human CD40-L gene used herein.- The sequence of this gene is available and thus this source of the gene was used merely for convenience. See GenBank accession no. X67878. This plasmid was used for PCR amplification of the human CD40-L gene using the specific primers, sense primer CCAAGACTAGTTAACACAGCATGATCGAAA 3' (SEQ ID NO: 28) and antisense primer 5' CCAATGCGGCCGCACTCAGAATTCAACCTG 3' (SEQ ID NO: 29).

These primers contain flanking restriction enzyme sites for subcloning into the eukaryotic expression e plasmid pRc/CMV (Invitrogen). The PCR amplified fragment was subcloned into the Spel and NotI sites of pRc/CMV and designated hCD40-L pRc/CMV. A BglII and XhoI fragment encompassing the CMV promoter, 15 gene, and polyadenylation signal was then released from this plasmid and subcloned into the shuttle plasmid MCS(SK)pXCX2 as described above. This plasmid was designated hCD40-L pXCX2. This plasmid was used for adenovirus production as described below.

iii. Adenovirus Synthesis Either mCD40-L pXCX2 or hCD40-L pXCX2 plasmids were co-transfected with pJM17 (Graham and Prevec, 1991, Methods in Molecular Biology, Vol 7) into 293 cells (American Type Culture Collection, Rockville, MD) using the calcium phosphate method (Sambrook, Fritsch, and Maniatis, 1989, Molecular Cloning. A Laboratory Manual, 2nd edition, chapter 16:33-34). Isolated adenovirus plaques were picked and expanded by again infecting 293 cells. High titer adenovirus preparations were obtained as described (Graham and Prevec, 1991, Methods in Molecular Biology, Vol except for the following modifications. The cesium chloride gradient used for concentrating viral particles was a step gradient, with densities of 1.45 g/cm 3 and 1.2 g/cm 3 The samples were spun in a SW41 rotor (Beckman, Brea, CA) at 25,0-00 rpm at 4 0 C. The viral band was desalted using a Sephadex 61 DNA grade column (Pharmacia, Piscataway, NJ). The isolated virus was stored at 70 0 C in phosphate buffered saline with 10% glycerol. The virus titer was determined by infecting 293 cells with serial dilutions of the purified adenovirus and counting the number of plaques formed. Viral titers typically ranged from 1010 to 1012 plaque forming units/ml (PFU/ml).

b. Introduction of a Murine and Human Accessory Molecule Ligand Gene into CLL Cells and HeLa I* 1 0 Cells For adenovirus infection, 106 freshly thawed and washed CLL cells or HeLa cells were suspended in 0.5 to 1 mL of culture medium for culture at 37 0 C in a 5% CO 2 in-air incubator. Adenovirus was added to the cells at 15 varying multiplicity of infection (MOI), and the infected cells were cultured for 48 hours, unless otherwise stated, before being analyzed for transgene expression.

c. Expression of an Accessory Molecule Ligand 20 Gene in CLL Cells and HeLa Cells The CLL and HeLa cells which were infected with the adenovirus vector containing either mouse or human ligand genes prepared in Example lb. were then stained with commercially available monoclonal antibodies immunospecific for either human or mouse CD40 ligand (Pharmingen, San Diego, CA) using the manufacturer's directions. The CLL and HeLa cells were washed in staining media (SM) consisting of RPMI-1640, 3% fetal calf serum and 0.05% sodium azide and containing propidium iodide and then analyzed on a FACScan (Becton Dickinson, San Jose, CA). Dead cells and debris were excluded from analysis by characteristic forward and side light scatter profiles and propidium iodide staining. Surface antigen expression was measured as the mean fluorescence intensity ratio (MFIR). MFI-R equals the mean fluorescence intensity (MFI) of cells stained with a specific FITC-conjugated MoAb, divided by the MFI of cells stained with a control IgG-FITC. This method controls for the nonspecific increases in autofluorescence seen in larger, more activated cells.

The histograms, generated for the CLL cells and HeLa cells containing either a genetic vector containing the human CD40 ligand gene or the murine CD40 ligand gene and the appropriate controls, are shown in Figure 10 3A-3D. The expression of both the murine and human accessory molecule ligand gene (CD40 ligand) in HeLa cells is shown in Figures 3A and 3B, respectively. The expression of the murine and human accessory molecule ligand in CLL cells is shown in Figures 3C and 3D. The expression of an accessory molecule ligand gene in CLL cells and the expression of murine CD40 ligand on the surface of the CLL cells is shown in Figure 3C. The failure of the human accessory molecule ligand to be expressed on the surface of the CLL cells is shown in Figure 3D.

Figure 8 shows data from an experiment done to examine whether the CD4' T cells of CLL patients could be induced to express the accessory molecule ligand mRNA after CD3 ligation. An ELISA-based quantitative competitive RT-PCR was used to measure CD40 ligand transcript levels. In this experiment, CD40 ligand and RNA transcribed from the CD40 ligand gene in CLL cells are compared with levels of CD40 ligand and RNA made in normal donor cells, after induction by CD3 ligation.

For CD3 activation, plate coats of CD3 mAb were made and incubated with plated CLL or normal donor mononuclear cells for the indicated amount of time, after which cells were analyzed for expression of surface antigens or CD154 RNA message levels. CLL or normal donor serum was added to the cells at the beginning of the activation assay for examination of modulation of ligand surface expression.

For quantitative CD154 RT-PCR ELISA, total RNA was extracted and competitor RNA was generated from the insert containing CD40 ligand (CD154) cDNA. Varying amounts of competitor RNA were added to separate wells of isolated total RNA that subsequently were converted into cDNA. CD3 activation, ELISAs and PCR reactions were performed as described in Cantwell, M. et al., Nature Medicine 3:984-989 (1997). Biotinylated

PCR

products were captured onto microtiter plates (Becton Dickinson, Oxnard, CA) coated with streptavidin (Sigma), and incubated. The plate was treated with NaOH to remove the sense strands and subsequently washed. The DNA was then hybridized with either wild-type gene- .i specific or competitor-specific oligonucleotides. Using terminal transferase, each probe was labeled with a molecule of digoxigenin-ll-dideoxyUTP (Boehringer Mannheim). The plate was incubated and washed with HYBE buffer and blocking buffer, then peroxidase-conjugated anti-digoxigenin antibody (150 U/ml; Boehringer Mannheim) in blocking buffer was added.

TMB

(tetramethylbenzidine) and peroxidase (Kirkegaard and Perry Laboratories, Gaithersburg, MD) were added for color development, and optical densities were measured at 450 nm and Deltasoft II (Biometallics, Princeton,

NJ)

was used for data analysis.

Standard curves plotting the moles of RNA product versus the optical density were made for the standard cDNA reactions. The equations describing these standard curves were then used to calculate the moles of wildtype or competitor DNA present in the unknown

PCR

reactions based on the optical densities obtained in the ELISA readings. The ratio of the quantity of wild-type DNA to the amount of competitor DNA was then plotted against the known quantity of competitor RNA added in the initial samples. The ratio of 1 was taken for the extrapolation of the amount of unknown moles of target RNA in the sample (a ratio of 1 means the amount of target RNA versus competitor RNA are equal). The molecules of target RNA per CD4 cell was then calculated based on the following formula: [(moles target CD154 RNA) x (6 X 1023 molecules/mole) x (dilution factor of test of CD4 T cells in total cell population).

The upper graph in Figure 8 shows that T cells of patients with CLL do not express detectable CD40 ligand after CD3 ligation. CD40 ligand RNA is produced, but it is not stable. Although both CD40 ligand and ligand RNA are expressed in normal donor T cells (lower graph), the levels of neither the protein or RNA are stably maintained.

Figure 9 shows a time course for surface expression of CD40 ligand. Expression reached a peak level at 48 hours after infection and persisted at high levels for at least 6 days thereafter. In this experiment, CLL B cells were infected with a gene therapy vector containing an accessory molecule ligand, at a MOI of 1000 at time zero, and then assessed by flow cytometry at various times thereafter. At each time point listed on the abscissa, the proportions of viable CLL B cells that expressed detectable CD154 are indicated by the vertical bars corresponding to the percentage scale depicted on the right-hand ordinate.

d. Function of the Human and Murine Accessory Molecule Ligands i. Induction of CD80 and CD54 on Cells Containing a Gene Therapy Vector Encoding an Accessory Molecule The CLL cells infected with the murine accessory molecule ligand gene prepared in Example lb. were then cultured in tissue culture plates. The CLL cells were then analyzed using multiparameter FACS analysis to detect induction of CD80 and CD54 expression using fluroescein isothiocyanate-conjugated monoclonalantibodies immunospecific for each of these respective surface antigens. Non-infected CLL cells were used as a control. The cells were subjected to the appropriate FACS analysis and histograms were generated. CD80 mAb was obtained from Dr. Edward Clark and CD54 mAb was purchased.from CALTAG Inc. The CD80 was conjugated using standard methods which have been described in Kipps et al., Laboratory Immunology II, 12:237-275 (1992).

The results of this analysis are shown in Figure 1. 0 4A-4D. Figures 4A-4B compare the amount of CD54 expression in CLL cells which have not been transfected (Figure 4A) or CLL cells into which a gene therapy vector containing the murine CD40 ligand gene was introduced (Figure 4B). The shaded graph indicates the isotype control for FACS staining and the open graph indicates the cells stained with the anti-CD54 antibody.

These results show that the level of expression of CD54 is increased in CLL cells into which the gene therapy vector containing the murine CD40 ligand was introduced.

Figures 4C and 4D compare the amount of expression in CLL cells which have not been transfected (Figure 4C) or CLL cells into which a gene therapy vector containing the murine CD40 ligand gene was introduced (Figure 4D). The shaded graph indicates the isotype control for FACS staining and the open graph indicates the cells stained with the anti-CD80 antibody.

These results show that the level of expression of is increased in the CLL cells into which the gene therapy vector containing the murine CD40 ligand was introduced.

In an additional experiment, CLL cells infected with a gene therapy vector containing the murine accessory molecule ligand gene were evaluated by flow cytometry for induced expression of not only CD54 and CD80, but also CD86, CD58, CD70 and CD95. Fluoresceinconjugated mAb specific for human CD54 and CD70 were purchased from CALTAG. Fluorescein-conjugated mAb specific for human CD27, CD58, CD80, CD86, or CD95, and phycoerythrin-conjugated mAb specific for human or mouse ligand, were obtained from PharMingen. Shaded histograms represent staining of CLL B cells with FITCconjugated isotype nonspecific mAb. In contrast to uninfected CLL cells (Figure 10, thin-lined histograms), or Ad-lacZ-infected CLL cells (data similar to that obtained with uninfected cells, but not shown), CLL cells infected with the adenovirus vector encoding the 10 CD40 ligand (CD154) expressed high levels of CD54 (Figure 10, top left), CD80 (Figure 10, top middle), CD86 (Figure 10, top right), CD58 (Figure 10, bottom left), CD70 (Figure 10, bottom middle), and CD95 (Figure 10, bottom right). On the other hand, CD40 ligand-CLL (CD154 CLL) expressed significantly lower levels of both surface membrane CD27 (Figure 11A, thick-lined histogram) and soluble CD27 (Figure 11B) than uninfected (Figure 11A, thin-lined histogram)(P 0.01, Bonferroni t-test) or Ad-lacZ-infected CLL cells (data similar to that obtained with uninfected cells, but not shown). In the experiment shown in Figure 11A, the CLL B cells were examined for expression of CD27 via flow cytometry, three days after infection. Shaded histograms represent staining of CLL B cells with FITC-conjugated isotype control mAb. In Figure 11B, cell-free supernatants were collected, after the infection or stimulation of CLL B cells, for 72 hours and tested for the concentration of human CD27 by ELISA. The reduced expression of CD27 (Figure 11B) is similar to that noted for leukemia B cells stimulated via CD40 cross-linking with mAb G28-5 presented by CD32-expressing L cells, as described in Rassenti, L.Z. and T.J. Kipps, J. Exp. Med. 185:1435- 1445.

ii. Allogeneic T Cell Responses to CLL Cells Into Which a Genetic Therapy Vector Containing a Murine CD40 Liand Gene Has Been Introduced The ability of CLL cells which have been infected with a gene therapy vector containing the murine ligand gene to stimulate allogeneic T cells from another individual) was analyzed using cell proliferation assays. Briefly, the test cells were co-cultured 10 with the genetic therapy vector containing the lac-Z gene or the murine CD40 ligand gene at a multiplicity of infection of 1,000 in the presence of IL-4 at a concentration of 10 ng/ml. In other samples, the CLL cells were stimulated with MOPC21 (a control IgG) or G28-5 (an anti-CD40 monoclonal antibody) or were preincubated on CD32-L cells and at the same time treated with IL-4.

The preincubation with the CD32-L cells together with IL-4 treatment have been shown to be an efficient form of cross-linking the CD40 molecule other than direct gene transfection.

After three days of culture at 37 0 C, these cells iwere treated with mitomycin C to prevent their proliferation and then used to stimulate allogeneic

T

cells. Prior to this co-culture, the different aliquots of CLL cells had either been treated with the monoclonal antibody or had been infected with the gene therapy vector containing either the lac-Z or murine ligand gene at a stimulator ratio of 1:10. After two days of culture at 37 0 C, interferon gamma (IFNg) production was measured by ELISA assay. After five days of co-culture at 37 0 C, the incorporation of 3 H-thymidine into replicating cells was measured after an eight hour pulse label. The results of this assay are shown in Table II below and in Figure In another experiment, CLL B cells infected with the gene therapy vector containing the CD40 ligand gene were evaluated for their ability to act as stimulator cells in an allogeneic mixed lymphocyte T cell reaction (MLTR). In parallel, the stimulatory capacity of control lac-Z-vector-infected CLL cells and CLL B cells that had been cultured with CD32-L cells and an anti- CD40 mAb JG28-5) or an isotype control Ig, was also examined as described in Ranheim, E.A. and T.J. Kipps, J. Exp. Med., 177:925-935 (1993), Clark, E.A. and J.A.

Ledbetter, Proc. Natl. Acad. Sci. USA, 83:4494-4498 (1986), and Banchereau, J. et al., Science 251:70-72 1. 0 (1991). Effector T cells from a non-related donor were co-cultured with the CLL stimulator cells at an effector to target ratio of 4:1. After 18 h culture at 37'C, over 30% of the allogeneic CD3* cells were found to express the activation-associated antigen CD69 when cultured with CD154-CLL cells (data not shown). In contrast, less than 4% of the T cells expressed CD69 when co-cultured with uninfected or Ad-lacZ-infected

CLL

cells (data not shown).

Two days after the initiation of the MLTR, the concentrations of IFNg in the culture supernatants were assayed by ELISA. The supernatants of the MLTR stimulated with CLL cells infected with the accessory molecule ligand CD40L (Figure 12A, CD154-CLL) contained significantly higher levels of IFNg (306 5 ng/ml, m SE, n 3) than that of MLTR cultures stimulated with the anti-CD40 mAb (Figure 12A, aCD40-CLL) (23 3 ng/ml) (P 0.05, Bonferroni t-test). The latter was not significantly different from that of MLTR cultures stimulated with control Ad-lacZ-infected CLL cells (Figure 12A, lacZ-CLL) (43 10 ng/ml) (P 0.1, Bonferroni t-test). The supernatants of effector cells alone, or of MLTR cultures stimulated with uninfected CLL cells (Figure 12A, CLL) or control Ig treated CLL cells (Figure 12A, MOPC-CLL), did not contain detectable amounts of IFNg(<2 ng/ml). Similarly, none of the leukemia B cell populations produced detectable amounts 69 of IFNg when cultured alone, without added effector T cells (data not shown).

After 5 days, cell proliferation was assessed by incorporation of 'H-thymidine. Cultures with isotype control IgG-treated (Figure 12B, MOPC-CLL) or uninfected (Figure 12B, CLL) stimulator cells did not incorporate more 'H-thymidine than cultures without added leukemiastimulator cells (Figure 12B, None). Ad-lacZ-infected CLL B cells (Figure 12B, lacZ-CLL) also were unable to 1. 10 stimulate allogeneic T cells to incorporate amounts of H-thymidine that were much greater than that of control cultures. In contrast, anti-CD40-stimulated leukemia cells or CD154-CLL cells each induced significant effector cell proliferation (Figure 12B, aCD40-CLL or CD154-CLL) (P 0.05, Bonferroni t-test). Moreover, the amount of 'H-thymidine incorporated by cultures stimulated with CD154-CLL cells (41,004 761 cpm (m SE), n 3) was significantly greater than that of cultures stimulated with equal numbers of aCD40-CLL cells (22,935 1,892 cpm, n 3) (P 0.05, Bonferroni t test). However, neither of these mitomycin-C-treated leukemia cell populations incorporated 'H-thymidine when cultured without effector T cells (data not shown).

Also, as described for the MLTR between allogeneic

T

cells and CD40-stimulated CLL cells {6549, 7167, 7168), allogeneic T cell proliferation in response to CD154-CLL could be inhibited by CTLA-4-Ig or CD1la mAb when added at the initiation of the MLTR, indicating that respective interactions between CD80/CD86 and CD28, or CD54 and CDlla/CD18, contribute to the noted allogeneic T cell reaction (data not shown).

Table II Allogeneic T cell responses to CLL cells infected with mCD40-L adenovirus positive cells Alloqeneic response (mean+SEM) IFNy Human 3H-TdR uptake production Stimulators mCD40-L CD80 (cpm) (ng/ml) 5 None (t cells only) 3577 821 n.d.* CLL with: No activation 0 1.4 4577 1097 n.d.

MOPC21 0 1.0 5259 1788 n.d.

G28-5 0 26.7 22935 1892 22.3 1.6 10 lac-Z adeno 0 4.8 9037 1781 43.2 10.5 mCD40-L adeno 17.5 19.7 41004 761 305.7 n.d. not detectable Itl*t: iii. Stimulation of Gamma Interferon by CLL Cells Containing an Accessory Molecule Ligand Gene The function of CLL cells containing an accessory molecule ligand gene (mouse CD40 ligand) was analyzed by determining the ability of those cells to activate T lymphocytes. The procedure was performed as follows: allogeneic T lymphocytes from a healthy donor (greater than 90% CD3') were purified using magnetic beads and monoclonal antibodies specific for the CD14 and CD19 antigen. These allogeneic T lymphocytes then were cultured together with MMC-treated CLL cells which were infected with the accessory molecule ligand gene (murine ligand) or the lac-Z gene. This co-culture was performed in RPMI-1640 medium containing 10% fetal calf serum. After culture for 24 hours, the cells were collected and analyzed to determine the expression of the antigen CD69 on the T lymphocytes using a standard FACS sorting protocol. The cell culture supernatants were collected after two days in culture and tested to determine the concentration of human interferon gamma using an ELISA assay. A portion of the CLL cells containing an accessory molecule ligand gene (murine ligand) and a portion of the cells containing the 10 adenovirus expressing the lac-Z were cultured in the presence of human interleukin 4 IL-4 (5 ng/mL). The production of interferon gamma by allogeneic T lymphocytes in the presence of this amount of human interleukin 4 was also analyzed. The results from these analyses are shown in Figure 6.

As can be seen, the human CLL cells containing the accessory molecule ligand gene (murine CD40) produced substantially higher concentrations of interferon gamma in the cell culture supernatant when compared to CLL cells which contained the lac-Z gene. The increased production of interferon gamma (IFNg) by T lymphocytes exposed to CLL cells containing the accessory molecule ligand gene indicates that these CLL cells containing the accessory molecule ligand genes were effective in producing an enhanced immune response.

iv. Stimulation of Allogeneic T Cells Pre- Exposed to Non-Modified CLL B Cells Containing an Accessory Molecule Liand Gene Prior studies indicated that antigen presentation to T cells, in the absence of the signals derived from costimulatory molecules such as CD28, can lead to specific T cell clonal anergy. For this reason, allogeneic T cells that had previously been cultured, with non-modified CLL B cells lacking expression-of and other immune accessory molecules, were tested-for their ability to respond to CLL cells containing the ligand gene. Allogeneic effector cells did not incorporate more 3 H-thymidine in response to non-modified CLL cells (Figure 12C, CLL), or control CLL cells infected with Ad-lacZ (Figure 12C, lacZ-CLL), than when they were cultured alone (Figure 12C, None). In contrast, even after prior co-culture with non-modified CLL B cells, allogeneic effector cells could still be induced to proliferate (Figure 12C, CD154-CLL) or to 10 produce IFNg (Figure 12D, CD154 CLL) in response to cells expressing an accessory molecule ligand. Although modest amounts of IFNg were detected in the supernatants of such secondary cultures when Ad-lacZ-infected leukemia cells were used as stimulator cells (Figure 12D, lacZ-CLL), this level was significantly lower than that noted for secondary cultures with infected CLL cells (Figure 12D, CD154-CLL) (P 0.05, Bonferroni t-test). Similarly, the supernatants of the leukemia cells alone (data not shown), and the effector cells alone (Figure 12D, None), of the MLTR cultures stimulated with uninfected CLL cells (Figure 12D, CLL), contained negligible amounts of IFNg(<2 ng/ml). These results indicate that allogeneic effector cells cultured with nonmodified CLL B cells are not precluded from responding to CLL B cells infected with a gene therapy vector containing the accessory molecule ligand gene.

v. Autologous T Cell Responses to CLL Cells Into Which a Gene Therapy Vector Encoding a Murine Accessory Molecule Ligand Gene Has Been Introduced T cells isolated from the blood of CLL patients were examined for their ability to respond in vitro to autologous CLL B cells containing a gene therapy vector which encodes the murine accessory molecule, ligand. T cells were isolated to >95% purity, and then co-cultured with mitomycin-C-treated autologous leukemia cells in serum-free AIM-V medium supplemented with exogenous interleukin-2 at 25 U/ml. Modest 3 H-thymidine incorporation (sl0,000 cpm) was detected in cultures without added stimulator cells, secondary in part to the exogenous.IL-2 (Figure 13A, and data not shown). The level of T cell proliferation, however, did not increase in response to uninfected CLL cells (Figure 13A, CLL) or Ad-lacZ-infected CLL cells (Figure 13A, lacZ-CLL). In contrast, CLL cells infected with a gene therapy vector 10 containing the accessory molecule ligand (Figure 13A, CD154-CLL) induced autologous T cells to incorporate significantly more 3 H-thymidine (17, 368 1,093 cpm, n=3) than any of the control cultures (P 0.05, Bonferroni t-test). Furthermore, the MLTR stimulated with CLL cells infected with a vector encoding an accessory molecule ligand (CD40L) also generated significantly more IFNg (165 3 ng/ml, n=3) than any of the other cultures (Figure 13B)(P 0.05, Bonferroni ttest).

The T cells were harvested after 5 days from the autologous MLTR and assessed for CTL activity against autologous CLL B cells. T cells co-cultured with autologous CD40-ligand-CLL cells developed CTL activity for non-modified CLL B cells, effecting 40.1% lysis at an E:T ratio of 2:1 (Figure 13C, CD154).

However, such T cells did not develop detectable

CTL

activity for the same target cells in the control reactions, when co-cultured with uninfected or Ad-lacZinfected CLL cells (Figure 13C).

vi. Specificity of CTL Stimulated by Autoloqous CD40-Ligand-CLL B Cells for Allogeneic CLL B Cells Effector cells stimulated with autologous ligand-CLL were evaluated for their ability to secrete IFNg or manifest CTL activity against allogeneic CLL B cells (Figure 14). After 5 days of autologous MLTR with CD154-CLL or lacZ-CLL, T cells were isolated by Ficoll density gradient centrifugation, washed extensively, and then cultured in media for 24 h. Washed T cells were mixed with autologous ("Auto CLL", solid bar) or allogeneic ("Allo-1 CLL" or "Allo-2 CLL", shaded or hatched bars) target CLL B cells. T cells stimulated in the autologous MLTR with CD40-ligand-CLL cells, but not with lacZ-CLL cells, produced significantly more IFNg in response to secondary culture with non-modified 10 autologous CLL B cells than with allogeneic CLL B cells (Figure 14A) (P 0.05, Bonferroni t-test). Furthermore, T cells stimulated with CD40-ligand-CLL cells, but not with lacZ-CLL cells, were cytotoxic for autologous CLL cells, but not allogeneic CLL cells (Figure 14B) Similar results were obtained with the autologous MLTRactivated T cells of the allogeneic donor, again demonstrating specific cytotoxicity for autologous CLL B cells (data not shown). Finally, W6/32, a mAb to class I major histocompatibility complex (MHC I) antigens could significantly inhibit the cytotoxicity of T cells stimulated with CD40-ligand-CLL cells for autologous CLL B cells (Figure 14C, aHLA-class I) 0.05, Bonferroni t-test). Such inhibition was not observed with mAb specific for MHC class II antigen (Figure 14C, aHLA-DP), mAb specific for the Fas-ligand (Figure 14C, aFasL), or an isotype control mAb of irrelevant specificity (Figure 14C, MOPC-21). Collectively, these studies indicate that Ad-CD40-ligand-infected CLL cells can induce an autologous anti-leukemia cellular immune response in vitro, leading to the generation of MHCclass I-restricted CTL specific for autologous nonmodified leukemia B cells.

e. Transactivation of Non-Infected Bystander Leukemia B Cells by Ad-CD40L CLL Cells To address whether the changes in tumor marker expression (described in section Idi.) resulted from intracellular versus intercellular stimulation, the effect of culture density on the induced expression of CD54 and CD80 following infection with adenovirus gene therapy vector encoding the accessory molecule ligand (CD40L, or CD154) was examined. After infection,

CLL

cells were cultured at standard high density 1 x 106 cells/ml) or low density 2 x 10' cells/ml) for 3 days at 37. C. Cells plated at high density contained homotypic aggregates, whereas cells plated at low density remained evenly dispersed and without substantial cell-cell contact (data not shown). Despite expressing similar levels of heterologous CD154, CD154- CLL B cells cultured at high density were induced to express higher levels of CD54 and CD80 than CD154-CLL cells cultured at low density (Figure 15A). The stimulation achieved at high density could be inhibited by culturing the cells with a hamster anti-mouse CD154 mAb capable of blocking CD40<->CD154 interactions (Figure 15B, aCD154 Ab). Collectively, these studies indicate that CD154-CLL cells can activate each other in trans and that surface expression of CD154 is necessary for optimal leukemia cell stimulation.

In addition, Ad-CD154-infected, uninfected, AdlacZ-infected, or G 2 8-5-stimulated CLL cells were labeled with a green-fluorescence dye to examine whether CD154-CLL could stimulate non-infected bystander leukemia cells. Dye-labeled cells were used as stimulator cells for equal numbers of non-labeled syngeneic CLL B cells. After 2 days' culture, stimulator cells cultured by themselves retained the green-fluorescence dye, allowing such cells to be distinguished from non-labeled CLL cells by flow cytometry. Bystander (green-fluorescence-negative) CD19' CLL B cells were induced to express CD54 (Figure right histogram) or CD86 (Figure 15D, right histogram) when co-cultured with Ad-CD154-infected leukemia B cells, but not with mock infected CLL cells (Figures and 15D, left histograms), G28-5-stimulated CLL cells, or Ad-lacZ-infected CLL cells (data not shown). As expected, these bystander (green-fluorescence-negative) CLL cells also were negative for heterologous CD154.

f. Treatment of Leukemia with Gene Therapy Vectors Encodina an Accessory Molecule Liqand Figure 24 shows an outline for a clinical trial for testing treatment of B cell CLL with adenovirus gene therapy vectors encoding modified CD40 ligand. Leukemia 1 0 cells harvested by pheresis are infected with replication-defective vectors that encode the modified ligand. Following expression of this protein, the cells will be administered back to the patient for the purpose of stimulating a host anti-leukemia-cell immune 15 response. This strategy is far superior to one that uses gene therapy to affect expression of only one immune stimulatory molecule on the leukemia cell surface. Indded, this strategy results in the leukemia cells expressing an array of immune-stimulatory 20 accessory molecules and cytokines, as well as a molecule that can affect the same changes in leukemia cells of the patient that were never harvested.

2. Expression of Chimeric Accessory Molecule Ligand Genes The chimeric accessory molecule ligand genes described below are prepared using standard techniques as described herein.

a. Preparation of Chimeric Accessory Molecule Ligand Genes Utilizing Domains from Two Different Accessory Molecule Genes The human CD40 ligand gene was isolated from RNA prepared from T cells which had been activated by an anti-CD3 monoclonal antibody using 5' and 3' primers together with well known PCR methods. Chimeric accessory molecule genes of human CD40 ligand and-murine ligand are constructed from the newly cloned human ligand gene and mouse CD40 ligand gene described herein as SEQ ID NO: 2. The transmembrane and cytoplasmic domains of human CD40 ligand genes are exchanged.with those of the murine CD40 ligand gene and designated H(Ex)-M(Tm-Cy) CD40 ligand. These chimeric accessory molecule ligand genes are produced using the gene conversion technique described as SOEN which has been previously described by Horton, Mol. Biotechnol., 1. 1 0 3:93 (1995). A diagram depicting the chimeric accessory molecule ligand genes which are produced is shown in Figure 4. The nucleotide sequences of each of these respective chimeric accessory molecule ligand genes is designated SEQ ID NOS: 3-7 as indicated in the Table below.

e* Table III Chimeric Accessory Molecule Ligand Gene SEO ID NO: HuIC/HuTM/MuEX CD40-Ligand SEQ ID NO: 3 HuIC/MuTM/HuEX CD40-Ligand SEQ ID NO: 4 20 HuIC/MuTM/MuEX CD40-Ligand SEQ ID NO: MuIC/HuTM/HuEX CD40-Ligand SEQ ID NO: 6 MuIC/MuTM/HuEX CD40-Ligand SEQ ID NO: 7 Adenovirus vectors encoding each of the chimeric accessory molecules shown in Figure 2 are constructed using the methods described in Example 1. Each of these constructs are then transfected into either HeLa cells or CLL cells according to the methods of Example 1.

b. Expression of Chimeric Accessory Molecule Ligands on CLL and HeLa Cells The expression of each of the chimeric accessory molecule ligand genes constructed above is analyzed by using FACS analysis as specified in Example 1. The appropriate monoclonal antibody immunospecific for the external domain of either human or mouse CD40 ligand is selected and used to determine the level of expression of the chimeric accessory molecules on the surface of these cells. After appropriate analysis and preparation of appropriate histograms, the expression of chimeric accessory molecules containing at least a portion of the murine CD4.0 ligand gene is confirmed.

c. Function of Chimeric Accessory Molecule Liqands CLL cells are infected with various MOI of the mCD40L adenovirus and then cultured in 48 or 24 well 10 tissue culture plates for various times after infection (48, 72, and 96 hours). The CD19' B cells are then analyzed by multiparameter FACS analysis for induction of CD80 and CD54 expression using fluroescein isothiocyanate-conjugated mAb specific for each 15 respective surface antigen as described in Example 1.

Increased amounts of CD54 and CD80 are found on cells which have the chimeric accessory molecules containing the domain or domains derived from the mouse CD40 ligand gene.

20 Further analysis of the cells containing the chimeric accessory molecule genes is carried out according to Example The cells containing the chimeric accessory molecule genes which contain the domains derived from the murine CD40 ligand gene are able to stimulate the production of gamma interferon and T cell proliferation.

d. Expression of Chimeric Accessory Molecule Genes Which Contain Proximal Extracellular Domains from Two Different Accessory Molecules from the Same Species A chimeric accessory molecule ligand gene is prepared which contains the proximal extracellular domain from the human CD70 gene (Domain III) with the remainder of the domains derived from the human ligand gene. This gene is prepared using standard 79 biologic techniques as previously described herein.

This chimeric accessory molecule ligand gene has the DNA sequence shown as SEQ ID NO: 19. A different chimeric accessory molecule ligand gene is prepared which contains the proximal extracellular domain from the murine CD40 ligand gene with the remainder of the domains derived from the human CD40 ligand gene. This gene is prepared using standard techniques as previously described herein. This chimeric accessory molecule ligand gene has the DNA sequence shown as SEQ ID NO: The chimeric accessory molecule genes shown as SEQ ID NOS: 19 and 20 are inserted into the appropriate vectors as described in Example 1 and introduced into human neoplastic cells. The expression of that chimeric accessory molecule gene in the cells is determined as was described in Example 1.

The chimeric accessory molecule encoded by each of these chimeric accessory molecule genes is found on the surface of the human neoplastic cells using the FACS analysis described in Example 1. Increased amounts of CD54 and CD80 are found on the cells containing the i chimeric accessory molecule genes using the techniques described in Example 1. The cells containing the chimeric accessory molecule gene are able to stimulate the production of gamma interferon and T cell proliferation as described and assayed according to Example 1.

3. Augmentation of Vaccination Using Vectors Encoding Accessory Molecules The following procedures were used to demonstrate the augmentation of a vaccination protocol using a gene therapy vector encoding an accessory molecule.

a. Aucmentation of the Antibody Response in Mice Co- Injected with an Accessory Molecule Gene Therapy Vector and placZ Three different gene therapy constructs were prepared ising standard techniques including those techniques described herein. The first was a control gene therapy vector, pcDNA3, which did not contain any gene. The second, placZ, contained the Lac-Z gene which encoded 0-galactosidase (0-gal). The third, 10 contained the murine CD40 ligand gene described in Example 1.

Prior to any immunizations, serum was isolated from 6-8 week old BALB/c-mice to determine the amount of any :initial antibodies to 0-galactosidase. Each animal was injected i.m. with 100 micrograms of plasmid DNA per injection. Four separate injections were given at one week intervals.

Prior to the third injection, the animals were bled to monitor the early antibody response to 0-gal. One week after the final injection of plasmid DNA, the .:animals were bled to monitor the late antibody response .to beta-galactosidase. To test the sensitivity of the assay, known amounts of anti-3-gal antibodies isolated from an anti-p-gal antiserum were tested in parallel.

Serum dilutions of 1:40, 1:200, or 1:1000 were tested in an ELISA for anti-p-gal antibodies. For this, polystyrene microtiter ELISA plates were coated with 3gal at 10 microgram/ml in phosphate buffered saline.

The plates were washed thrice with blocking buffer containing 1% bovine serum albumin (BSA), 0.2% Tween in borate buffered saline (BBS) (0.1M borate, 0.2M NaC1, pH 50 microliters of diluted serum were added to separate wells. After at least 1 hour at room temperature, the plates were washed thrice with blocking buffer and then allowed to react with alkaline phosphatase-conjugated goat anti-mouse IgG antibody.

One hour later, the plates again were washed four-times with blocking buffer and incubated with 25 ml of TMB peroxidase substrate (Kirkegaard Perry, Gaithersburg, MD). The absorbance at 405 nm of each well was measured using a microplate reader (Molecular devices, Menlo Park, CA). The higher the O.D. reading, the greater the amount of specific antibody in the sample.

The data for each of two experiments are provided in Tables IV and V which follow on separate sheets. The results are summarized in Tables VI and VII collating 0 the data from the two experiments is provided as well.

n the summary page n stands for the number of animals in each of the four groups. S.D. stands for standard deviation and Avg. is the average O.D. reading for all the animals in a particular group.

The results of Group 4 demonstrate that the use of a gene therapy vector encoding an accessory molecule ligand (CD40L) enhances the immunization against 3-gal encoded by a genetic or gene therapy vector. The average O.D. reading of the 1:40 dilution of the sera from animals of this group is significantly higher than that of groups 1, 2, and 3 (P 0.05, Bonferroni t tests, see Table VII).

Data from an additional experiment further reinforce the finding that the gene therapy vector encoding an accessory molecule ligand enhances immunization against 3-gal (Figure 16). Here, and placZ were co-injected into skeletal muscle, to test for enhancement of the immune response to placZ, a pcDNA3-based vector encoding E. coli 0-galactosidase.

The relative anti-p-gal Ab activities weie determined via ELISA. As expected, mice injected with either the non-modified pcDNA3 vector or pCD40L alone did not produce detectable antibodies to b-gal (Figure 16A) Mice were injected with either 100 ig pcDNA3 (checkered bar), 50 ug pcDNA3 50 mg pCD40L (lined bar), 50 lg pcDNA3 50 ig placZ (striped bar), or 50 pg pCD40L pg placZ (solid bar). On the other hand, mice that received placZ and pcDNA3 developed detectable anti-bgal antibodies one week after the fourth and final injection, at d28. Mice that received placZ and developed higher titers of anti-?-gal antibodies than mice injected with placZ and pcDNA3. Figure 16B, ELISA analyses of serial dilutions of sera collected at d28, shows that mice co-injected with placZ and pCD40L had an eight-fold higher mean titer of anti-3-gal antibodies at d28 than mice treated with placZ pcDNA3.

i. Immunoqlobulin Subclass Production Stimulated by Accessory Molecule Vector Co-Injection Despite enhancing the titer of the anti-p-gal antibody response, the subclass of anti-P-gal IgG induced by injection of placZ was not altered by the coinjection of pCD40L. IgG2a anti-A-gal antibodies predominated over IgG, subclass antibodies in the sera of mice injected with either placZ and pcDNA3 or placZ and pCD40L (Figure 17). Also depicted are the ELISA O.D.

20 measurements of anti-a-gal IgG, and anti-A-gal IgG2a present in the pre-immune sera (striped bar) or postimmune sera (solid bar), collected at d28) of each group of mice, injected as indicated on the abscissa. In contrast, BALB/c mice injected with A-gal protein developed predominantly IgG, anti-A-gal antibodies, and no detectable IgG 2 anti-A-gal antibodies.

ii. Augmentation of Vaccination by Accessory Molecule Vector Requires Co-Injection with placZ at the Same Site The adjuvant effect of the pCD40L plasmid on the anti-A-gal antibody response was noted only when it was injected into the same site as placZ (Figure 18).

Groups of BALB/c mice received intramuscular injections of placZ and pCD40L together at the same site, or as simultaneous separate injections at distal sites (right and left hind leg quadriceps). A control group received intramuscular injections of placZ and pcDNA3 at the same site. Animals were bled at d28 and the sera tested for anti-/-gal Ab at different dilutions, as indicated on the abscissa. The graph illustrates a representative experiment depicting the mean O.D. at 405 nm of replicate wells of each of the serum samples for each group, at a 1:40, 1:200, or 1:1000 dilution. Animals injected simultaneously with 10 placZ and pCD40L, but at different sites, did not develop detectable anti-/-gal antibodies until d28.

Moreover, the anti-p-gal antibody titers of the sera from such animals at d28 were similar to that of mice that received placZ and pcDNA3, and significantly less than that of animals that received placZ and together at the same site.

S*

iii. Augmentation of Vaccination When Accessory Molecule Vector and placZ are Co-Iniected into Dermis 20 The pCD40L plasmid also enhanced the anti-b-gal antibody response to placZ when injected into the dermis. In the experiment shown in Figure 19, mice received intradermal injections, near the base of the tail, with either 50 pg pcDNA3 (checkered bar), 25 pg pcDNA3 25 pg pCD40L (lined bar), 25 pg pcDNA3 25 pg placZ (striped bar), or 25 pg pCD40L 25 pg placZ (solid bar). Injections, bleeds and ELISA analyses were performed as in Figure 16A. The checkered bar and lined bar groups each consisted of 8 mice while the striped bar and solid bar groups each consisted of 12 mice. The height of each bar represents the mean O.D. of sera at a 1:40 dilution of each group S.E. A statistical analysis of the data indicated that the striped bar and solid bar groups are independent (P As observed with intramuscular injection, mice co-injected with placZ and pCD40L developed detectable serum anti-9--gal antibodies one week following the second injection (d14), and two weeks earlier than mice injected with placZ and pcDNA3. Moreover, these animals also had an eight-fold higher mean titer of anti-3-gal antibodies than mice.of the placZ-injected group at d28. Mice injected with either the non-modified pcDNA3 vector or alone did not produce detectable antibodies to 3gal.

b. Augmentation of the CTL Response in Mice Co- 1 0 Injected with an Accessory Molecule Gene Therapy Vector and placZ The ability of pCD40L to enhance induction, by placZ, of CTL specific for syngeneic b-gal-expressing target cells was tested. BALB/c mice co-injected with 15 pCD40L and placZ into skeletal muscle (Figure 20A) or S dermis (Figure 20B) generated greater numbers of CTL specific for P13.2, a placZ transfected P815 cell line, S: than mice co-injected with placZ and pcDNA3. At a 5:1 effector:target ratio, the splenocyte effector cells 20 from mice that received intramuscular injections of placZ and pCD40L achieved greater than 20% specific lysis of P13.2. In contrast, when splenocytes of mice that received the control injection with placZ and pcDNA3 were used, a 9-fold greater ratio of effector to target cells was required to achieve this level of specific lysis. Similarly, the splenocyte effector cells from mice that received intradermal injections of placZ and pCD40L killed more than 50% of the P13.2 cells at effector:target ratios of 4:1. To achieve comparable levels of specific lysis required eight-fold higher effector:target ratios using splenocytes from mice that received intradermal injections of placZ and pcDNA3.

Nevertheless, the splenocytes of mice co-injected with and placZ did not have greater non-specific

CTL

activity for P815 cells than that of mice that received placZ along with pcDNA3 (Figure 20). As expected, the splenocytes from mice that received injections of pcDNA3 alone, or pcDNA3 and pCD4OL, did not mediate specific lysis of P13.2 or P815 cells.

000 06 00. 9** .600 so* 0 V ec Table

IV

Experiment #1 Injections of plasmid DNA im: 4/3196; 4/10/96; 4/17/96; 4/24/96 ELISA for anti-beta galactosiaase antibodies: Group Dilution of Pre-Bleed (4/3) 1/140 1/200 1/1000 Dilution of Bleed (4/17) Dilution of Bleed (5/1) Animal pcUNA3 (p-control, IU~ mcg) 1 (Control vector) 2 4- UU U1 0.09 0.11 0.09 0.09 0.12 0.11 0.10 0.11 0.10 0.10 1/140 1/200 1/1000 1/40 1/200 1/1000 U.U6 U.Ubi U.Ub U 1 u-77 0.07 0.07 0.07 0.10 0.09 0.08 0.09 0.09 0.10 0.12 0.08 0.08 0.08 0.11 0.07 0.11 0.07 0.08 0.11 0.11 0.11 0.11 0.11 0.11 0.01 0.02 0.02 0.01 0.04 0.01

I

w. 41 S. D. 0.01 u..I U.1 I 0.01 0.01 p-iacZ (50 mcg) 5

-F

p-C;ontrol (5U mcg)

T

0.13 0.10 0+10 0.10 0+11 0.10 0.19 0.10 0.18 0.10 0.09 0.10 Avg.

S.D.

U.13 A U .1D2 0.04 0.01 0.04 0.06 0.06 .0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.07 0.11 0.06 0.15 0.10 0.08 0.07 0.06 0.06 0+22 0.15 0.14 0.07 0.07 0.06 0.78 0.29 0.12 0.08 0.07 0.07 3.04 1.84 0.77 0.0 a.0 0.06_ 1.0 0703.

0.01 0.02 0.00 1.36 0.84 0.33 0.13 0+11 0.08 2.30 1.68 0.72 0.27 0.13 0.10 2.35 0.09 0.28 0.23 0.19 0.11 2.06 1.09 0.39 0.23 0.19 0.11 2.06 1.09 0.39 0.00 0.00 0.0 0.01.06 0.66 0.24 0.13 0.67 0.19 Table Experiment #2 Injections of plasmid DNA 6/5/96; 6/12/96; 6/19/96; 6/26/96 DiJlutions 6t zsera tar anti-66e galactosidase antibodies: r,;1 9- C Group Animal P-i-vF11101 k3u, mcg) 9 1U -Xv-9-t S. D.

U00 -e-ieed(65 Dilution of Bleed (7/19) Dilution of Bleed (8/3) 1/140 1/200 1/1000 1/140 1/200 1/1000 1/40 1/200 1/1000 L.e UU JU U.U4 U Ul -U Ul U.U A U U.U -ys 0.06 0.02 0.10 0.02 0.02 0.00 0.08 0.09 0.05 0.02 0.02 0.07 0.03 0.01 0.00 0.02 0.02 0.05 0.06 0.03 0.05 0.18 0.04 0.01 0.11 0.4 .5 0 .I 0 .4I T 0 0 4 40 0 0 0 4 0 0 0.02 0.01 0.02 0.07 0.01 0.01 0. 04 0.03 0.00 9JgI ciig, 5 0.02 0.03 0.02 0.06 0.04 0.04 0.39 0.11 0.03 00 0.03 0.02 0.03 0.14 0.03 0.04 2.85 1.58 0.41 7-oto 0.56 0.13 0.06 0.29 0.06 0.02 0.22 0.07 0.03 0.01 0.02 0.05 0.06 0.02 0.02 0.11 0.04 0.05 Avg. j 0.15 0.05 4.0..3.0.0410.04 1.59 0.45.1 S.D. 0.27 0.05 0.02 0.11 0.02 0.01 1.31 0.75 0.19 P-IacL (tau mcg) 13 0.23 0.06 0.05 0.28 0.07 0.02 2.37 0.73 0.18 S. D. 0.10 0.02 0.02 0.39 0.09 0.02 0.31 0.67 0.19 Table VI Summary P-re-Immfiune W~beta-gal Early eaa Late co bela-gal 1 p _4 _A v g 1 1 4 0 1 /2 0 0 1 /1 0 0 0 1 /1 4 0 1 /2 0 0 1 0 0 0 1 /4 0 1 /2 0 0 1 /1 0 0 0 1)p- onro (S.D)Av. 0.01 0.01 U.1 I.101 l 01 .101 D 0 0 10 10 0 1 0 0 1 0 0 2 0 0 2 0 0 1 0 0 4 0 0 1 Z) P-MUI4UL p-Uonrol (n Avg. 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.4.4 S Q 20 .0 1 0 .0 2 0 .0 7 0 .0 1 0 .0 1 0 .0 4 0 .0 3 0 .0 0 3) p-lacZ p-;ontrol (n =Avg. 0.11 0.04 0.04 0.11 0.03 0.03 0.61 0.1.9 SD 02 004 0.01 0.09 0.02 0.02 1.11 0.62 0.16 4) p-lacL P MCUU4UL (n =)Avg. 0.11 0.04 0.03 0.25 0.06 0.03 2.06 1.04 02 01 00 .10.32 0.07 0.02 0.97 0.69 0.18 Anti-beta-galactosidase standard: 67 ng 22ng 7.4ng 2.5ng .82ng .27ng O.D. 3.01 2.98 2+05 1.10 0.52 0.26 3.14 3.14 2.25 1.20 0.56 0.26 89 Table VII BONFERRONI t-TESTS Degrees of freedom: ONE WAY ANALYSIS OF VARIANCE ONE WAY ANALYSIS OF VARIANCE Source of Variation SS DF Variance Est (MS) Between Groups 18.29 6 3.05 Within Groups 18.39 29 0.63 Total 36.69 s2_bet F s2 wit MSbet Mswit 3.05 0.63 4.81 P 0.002 4. Treatment of Neoplasia Using a Gene Therapy Vector Containing an Accessory Molecule Gene or Chimeric Accessory Molecule Gene a. Treatment of Neoplasia in Mice The treatment of a neoplasia in a mouse model system has been demonstrated using the genes encoding accessory molecule ligands of the present invention.

Gene therapy vectors containing an accessory molecule ligand gene (murine CD40 ligand) were prepared as has been previously described in the above examples. These gene therapy vectors were used to introduce that accessory molecule ligand gene into neoplastic cells, Linel cells, from a tumor which originated in BALB/c mice. The accessory molecules were introduced into the 15 neoplastic cells according to the above examples. The expression of the accessory molecule ligand on the surface of these neoplastic cells was confirmed using flow cytometry as has been described in the above examples.

20 The effectiveness of the accessory molecule ligand genes for treating neoplasia was shown as follows.

SFemale BALB/c mice (6-8 weeks old) were injected i.p.

with 1.0 X 10 5 irradiated Linel neoplastic cells. The neoplastic Linel cells are derived from a spontaneous lung adenocarcinoma in a BALB/c mouse. This neoplastic cell has been described by Blieden et al., Int. J.

Cancer Supp., 6:82 (1991). Other female BALB/c mice were injected i.p. with 1.0 X 10 5 irradiated Linel tumor cells that had previously been transduced with the gene therapy vector encoding the accessory molecule ligand gene (murine CD40) as described above.

Each group of mice was allowed to generate an immune response for 10 days. After 10 days each mouse was challenged with 1.0 X 104 live, non-irradiated Linel neoplastic cells. These mice were then monitored for the formation of tumors and then sacrificed when the tumors grew to 2.0 cm because of morbidity. The-results of this monitoring are shown in Figure 7. As can be seen by Figure 7, the mice immunized with the neoplastic cell expressing the accessory molecule ligands of the present invention on the cell surface remained free of tumor thrqughout the experiment. Mice immunized with the neoplastic cells not having the accessory molecule ligand genes of the present invention succumbed to tumor days after challenge with the neoplastic cells.

Figure 21 demonstrates downmodulation of human CD40L, but not murine CD40L, in lung tumor cell lines that express CD40. Human cell lines HeLa cervical carcinoma, Figure 21A), A427 lung carcinoma, Figure 21B), NCI 460 (weakly positive lung large cell carcinoma, Figure 21C), and SK- 15 Mes-1 (strongly CD40-positive lung squamous cell tumor, Figure 21D) were infected with adenovirus encoding lac-Z oo-. (Ad-LacZ), murine CD40L (Ad-mCD40L), and human (Ad-hCD40L) at an MOI of 0 (Blank), 1, and 10. 48 hours after infection, murine CD40L and human CD40L surface 20 expression was determined. The percentage of cells that express ligand are plotted on the Y-axis. Human and mouse CD40L are expressed at equal levels in *negative cell lines. However, only murine expression is stable on cell lines that express In contrast to mCD40L, human CD40L is downmodulated on tumors.

The data graphed in Figure 22A show that binding induces expression of tumor surface markers.

Treating CD40-expressing lung cancer cell lines with cCD40 mAb resulted in enhanced expression of the tumor cell surface markers CD95 (Fas), CD54 (ICAM-1) and class I major histocompatability antigens (MHC NCI 460, a weakly CD40-positive lung large cell carcinoma, was incubated with a CD40-specific monoclonal antibody (thick line), or MOPC21, an isotype control mAb (thin line), on CD32-expressing mouse fibroblasts for 48 hours. Following the 48 hr incubation, the lung tumor cells were analyzed for CD95, CD54, and MHC-I expression by FACS.

Figure 22B again shows downmodulation of human by CD40-positive tumor cells. HeLa negative), CLL (CD40-positive), and SK-MES-1 positive) tumor cells were cocultured for 24 hours with CD3-activated normal donor T cells at a tumor cell:T cell ration of 2.5:1. Following coculture, CD2expressing T cells were analyzed for CD40L surface expression by FACS. Thin lines represent T cells stained with FITC-labeled isotype control antibody (MOPC21) and thick lines represent activated T cells stained with FITC-labeled aCD40L antibody (aCD154 antibody). The CD40-positive tumor cell lines, SK-MES- 15 1 and CLL, do not express CD40 ligand on their surfaces.

*e Expression of the Human and Mouse Accessory Molecule Liqand, Fas Liaand, in Human Blood Lymphocytes a. Construction of a Genetic Construct and Gene Therapy Vector Containing the Human and Mouse Fas Ligand Gene Either the human accessory molecule ligand gene (human Fas ligand) or the murine accessory molecule ligand gene (murine Fas ligand) was constructed utilizing the respective human and murine genes.

An altered accessory cell molecule, in which a putative MMP-cleavage site was removed, was made and designated AFasL-pcDNA3. The nucleotide sequence of AFasL-pcDNA3 is listed as SEQ ID NO: 40. Human Fas ligand nucleotides 325 to 342, encoding six amino acids, are missing from AFasL. The design of AFasL was based on reasoning that Domain III contains sites most accessible to MMPs, and could thus be the target on the molecule for cleavage from the surface of the cell. Sequences of the human Fas ligand gene have been determined and are listed as SEQ ID NOS: 13 and 30 (Genbank accession U11821). Sequences of mouse Fas ligand genes have been determined and are listed as SEQ ID NOS: 14 (C57BL/6, Genbank accession U10984) and 31 (Balb/c, Genbank accessionU58995). The sequence of the rat Fas ligand gene has been determined and is listed as SEQ ID NO: (Genbank accession U03470). Chimeric constructs are made, as described in Example 2 for CD40 ligand chimeric constructs, in which Domain III of human Fas ligand is replaced with Domains of other proteins, particularly proteins of the TNF family. Chimeric constructs include, but are not limited to, human Fas ligand with Domain III replaced by Domain III of murine Fas ligand (chimeric sequence listed as SEQ ID NO: 37, sequence 15 line-up shown in Figure 37), or replaced by Domain III of human CD70 (chimeric sequence listed as SEQ ID NO: 38, sequence line-up shown in Figure 38), or replaced with Domain I of human CD70 (chimeric sequence listed as SEQ ID NO: 39, sequence line-up shown in Figure 39) S* 20 Chimeric constructs in which multiple domains, for example, two copies of human CD70 Domain III, are inserted into human Fas ligand in place of Domain III, are also made using methods described in Example

I.

Chimeric constructs in which synthetic sequences are used to replace Domain III of human Fas ligand are also made.

i. Human Fas Ligand Cloning The cDNA encoding human Fas-ligand was subcloned in the eukaryotic expression vector pcDNA3. Normal donor blood lymphocytes were activated for 4 hours with 1 ng/ml PMA plus 0.5 uM ionomycin. Total RNA was isolated with the Qiagen Rneasy kit. cDNA was then synthesized from poly-A RNA with oligo-dT primers using the Gibco- BRL Superscript cDNA synthesis kit. The gene encoding human Fas-ligand was then PCR amplified with the-Fasligand-specific primers (sense primer, SEQ ID NO: -32, antisense primer, SEQ ID NO: 33). The Fas-ligand

PCR

product was then subcloned into pcDNA3 using standard molecular biology techniques. RT-PCR products, subcloned into pcDNA3, are designated hFasL-pcDNA3.

ii. Murine Fas Liqand Cloning The murine Fas-ligand geneS from Balb/c and C57/BL6 strains of mice were also amplified following activation of mouse splenocytes with PMA plus ionomycin as described above, and amplified from poly-A synthesized 10 cDNA as described above (sense primer, SEQ ID NO: 34, antisense primer, SEQ ID NO: 35). These genes were .subcloned in the pTARGET expression vector (Promega, Madison, WI). RT-PCR products, subcloned into pcDNA3, are designated mFasL-pcDNA3.

15 iii. Adenovirus Vector Construction For construction of adenovirus vectors encoding human Fas-ligand, murine Fas-ligand or AFas-ligand, the o *cloned cDNA insert is subcloned into the plasmid pRc/RSV (Invitrogen, San Diego, CA) at the HindIII-Xbal site. A 20 BglII-Xhol fragment with the RSV promoter-enhancer and o the bovine growth hormone poly-A signal sequence was subcloned into the BamHI-Xhol site of plasmid MCS(SK) pXCX2. The plasmid MCS(SK)pXCX2 is a modification of the plasmid pXCX2, in which the pBluescript polylinker sequence was cloned into the El region. The resulting plasmid then is co-transfected along with pJM17 into 293 cells using the calcium phosphate method. Isolated plaques of adenovirus vectors are picked and expanded by infecting 293 cells. High titer adenovirus preparations are obtained, as described above which uses a cesium chloride gradient for concentrating virus particles via a step gradient, with the densities of 1.45g/cm 3 and 1.20g/cm 3 in which samples are centrifuged for 2 hours in an SW41 rotor (Beckman, Brea, CA) at 25,000 rpm at C. The virus band is desalted using a Sephadex G-25 DNA grade column (Pharmacia, Piscataway, NJ), and the isolated virus is stored at -700 C in phosphate-buffered saline with 10% glycerol. The titer of the virus is determined by infecting permissive 293 cells at various dilutions and counting the number of plaques. Titers typically range from 1010 to 1012 plaque forming units/ml.

The adenovirus constructs are designated Ad-hFasL, AdmFasL and Ad-AFasL.

b. Introduction of the Murine and Human Fas Liqand 10 Genes into Human Cells The constructs hFasL-pcDNA3, mFasL-pcDNA3 and AFasLpcDNA3 are transfected into 293 via electroporation. The transfected cells are selected in medium containing G418.

Fas-ligand transfectants are screened for expression of the transgene using anti-Fas-ligand antibody and flow cytometry. The methods used are similar to those described for transfection of CD40L into CLL cells.

For FasL-adenovirus infection, 106 freshly thawed 99 C* and washed CLL cells or HeLa cells are suspended in to 1 mL of culture medium for culture at 37 0 C in a C0 2 -in-air incubator. Adenovirus are added to the cells at varying multiplicity of infection (MOI), and the infected cells are cultured for 48 hours, unless otherwise stated, before being analyzed for transgene expression.

c. Expression of the Fas Ligand Genes in Human Cells Mice with the lymphoproliferative or generalized lymphoproliferative disorder are unable to delete activated self-reactive cells outside of the thymus.

This is related to the fact that, in-these mice, interactions between the Fas receptor and an accessory molecule ligand, Fas ligand, are defective. These animals develop numerous disorders including lymphadenopathy, splenomegaly, nephritis, and systemic autoimmune pathology which resembles that seen in patients with systemic lupus erythematosus or rheumatoid arthritis It is conceivable that the normal interactions between the Fas receptor and the accessory molecule ligand that are responsible for clearance of activated lymphocytes from joints may be impaired in RA patients.

RA synovial lymphocytes express the Fas receptor at a higher proportion than that of matched RA blood lymphocytes to matched normal donor blood lymphocytes.

On the other hand, RA synovial lymphocytes express little or no accessory molecule ligand. Since the RA synovial lymphocytes are sensitive to Fas-induced apoptosis, it is feasible that local expression of Fas ligand in the RA 15 joint could serve to eliminate the synovial mononuclear cells that potentially mediate RA autoimmune pathology.

Figure 23 shows that Fas-ligand expression in lymphocytes is inhibited by exposure to RA synovial fluid. Normal donor blood T cells were activated for 20 hours with 1 ng/ml PMA plus 0.5 AM ionomycin. Cells were incubated in the presence of rheumatoid arthritis blood plasma (circles), RA synovial fluid (diamonds), or ::neither (squares). In addition, cells were incubated with increasing concentrations of the MMP inhibitor BB94.

Following activation, cells were analyzed for Fas-ligand surface expression by FACS. The percentage of cells expressing Fas ligand are plotted in Figure 23. This experiment demonstrates that there is a factor(s) present in RA synovial fluid and serum that prevents surface expression of Fas-ligand.

d. Function of Human, Murine and Chimeric Accessory Molecule Licand. Fas Liqand To determine the capacity of the AFasL constructs, the above-mentioned transfected cells are mixed with the Fas-ligand sensitive human T cell line, JURKAT.

Following 4 hours coculture, the nonadherant JURKAT cells are collected and evaluated for apoptosis. The fluorescent compound 3,3' dihexyloxacarbocyanine iodide (DiOC 6 is used to evaluate for apoptosis using a modification of a previously described protocol. For this, the cells are washed once at room temperature in phosphate buffered saline (PBS, pH Cells are placed into separate wells of a 96 well U-bottom plastic 5 microtiter plate at 10 5 x 10 cells/well in 50 ml total volume. If indicated, saturating amounts of PEconjugated antibodies are added followed by addition of DiOC 6 and propidium iodide DiOC 6 and PI are used at 40 nM and 10 ng/ml final concentrations, respectively.

The cells are then incubated 15 minutes in a 37 0 C, 5% CO 2 tissue culture incubator. The stained cells are then 15 washed twice in ice cold PBS and ultimately suspended in 200 ml SM and analyzed by FACS. Dead cells and debris with characteristic forward and light scatter profiles and PI staining are excluded from analysis.

The ability of cells expressing AFasL-pcDNA3 to 20 direct Fas-mediated apoptosis of cells expressing CD95 is compared with that of cells expressing FasL-pcDNA3.

Relative stability of the protein products encoded by .6FasL-pcDNA3 or FasL-pcDNA3 pre- and post- culture with RA synovial fluid, and with or without the metalloproteinase inhibitors, are assessed via flow cytometry of cells expressing either ligand.

6. Treatment of Arthritis with Gene Therapy Vectors Encoding an Accessory Molecule Liqand. Fas Liqand The heterologous Fas-ligand constructs, made as described above, that show the highest stability of expression in combination with the greatest ability to mediate Fas-induced apoptosis, are used in gene therapy for RA. Potential therapeutic constructs are tested in well-characterized mouse models of arthritis to assess efficacy and function in vivo.

a. Gene Therapy Treatment of Arthritis in Mice i. Mouse Models for Arthritis One mouse arthritis model is collagen-induced arthritis. It is known that injecting DBA/1 mice with type II cqllagen in complete Freund's adjuvant (CFA) induces an arthritis with synovitis and erosions that histologically resemble RA. For our studies, male DBA/I mice are immunized with bovine type II collagen in complete Freund's adjuvant on day 0 and boosted itraperitoneally on day 21. On day 28, animals are given an additional i.p. injection with lipopolysaccharide (LPS) and/or the same type collagen, or an injection of acetic acid alone. Swelling and/or redness of a fore or hind paw in animals immunized with 15 collagen typically is detected the third or fourth week following the second injection. The vertebrae are only rarely affected, and then only weeks after the initial peripheral joint swelling. Affected joints display initial histologic changes of synovial edema, followed by S: 20 synovial hyperplasia.

Another animal model, recently described by Kouskoff, V. et al., in Cell 87:811-822 (1997) was generated fortuitously, by crossing a T cell receptor (TCR) transgenic mouse line with the non-obese-diabetic (NOD) strain to produce the KRN x NOD mouse model of RA.

The offspring of such a mating universally develop a joint disease that is highly similar to that of patients with RA. Moreover, the disease in these animals has an early and reproducible time of onset and a highly reproducible course. The arthritis apparently is induced by chance recognition of an NOD-derived major histocompatibility complex (MHC) class II molecule by the transgenic TCR, leading to breakdown in the general mechanisms of self-tolerance and systemic selfreactivity.

ii. Relief of Arthritis Symptoms in Mice Treated with a Gene Therapy Vector Encoding an Accessory Molecule Liqand We have adapted and modified a protocol originally described.by Sawchuk and colleagues for micro-injecting adenovirus vectors into mouse joints. Using this procedure we can reproducibly inject a 5 1 volume into the articular space of the mouse knee. In this procedure, the mice are anesthetized with metofane. A small incision of approximately 2-3 mm is made with a #11 scalpel blade in the skin over the lateral aspect of the knee to visualize the patello-tibial ligament. We can inject up to 5 pl of fluid using a micro-100 4l-Hamilton syringe and a 30-gauge needle. After the injection, the 15 knee incision is closed with Nexabond (Veterinary Products Laboratory). Our adenovirus titers typically exceed 1010 plaque forming units (pfu) per ml, making it possible to deliver at least 5 x 108 pfu of virus in 5 ml into the knee joints, as outlined above. Control animals 20 are injected with control Ad-lacZ vector, a replicationdefective adenovirus vector lacking a transgene, or with S: the buffer used to suspend the virus (10 mM Tris, 1 mM MgCl, 10% glycerol) i In another method, splenocytes will be harvested from mice that are syngeneic to the host animal intended for adoptive transfer of transduced cells. Cell proliferation will be induced with exogenous IL-12 (100 units/ml) for 48 h. Cells are counted and then re-plated at densities of 5 x 105 or 1 x 106 cells per ml in a 12well dish with 1 ml complete culture medium per well.

Virus and ConA are added together at the time of replating in the presence of polybrene (8 yg/ml). The medium is changed 24 hours after infection with complete medium containing 100 units of recombinant IL-2 per ml.

Aliquots of the transduced cells are examined, for Fasligand expression, at 48 hours after infection via flow cytometry.

100 Animals will receive standardized numbers of cytokine-producing cells or control mock-transfected cells intraperitoneally. Concentrated cell suspensions are injected directly into the mouse synovium, as described.in section 4A above. In parallel, aliquots of the transferred cell populations are maintained in tissue culture supplemented with exogenous IL-2.

Mice are monitored in a blinded fashion for signs of arthritis. The date of disease onset is recorded and clinical severity of each joint or group of joints (toes, tarsus, ankle, wrist, knee) are graded as follows: 0 (normal), 1 (erythema), 2 (swelling), 3 (deformity), 4 (necrosis). The scores are summed to yield the arthritic score. The severity of arthritis is expressed both as 15 the mean score observed on a given day, and as the mean of the maximal arthritic score reached by each mouse during the clinical course of the disease. At the time of death, hind paws are dissected free and processed for histologic examination or for RT-PCR. The histologic 20 severity of the arthritis is scored on a scale of 0-3 for synovial proliferation and inflammatory cell infiltration, where a score of 0 normal and 3 severe.

For mice receiving intra-synovial injection of control of test adenovirus vector, the level of arthritis observed between contralateral sites is compared. In addition, the overall joint score minus that of the injected joint for the entire animal is compared with that observed in the joint injected with the control or test adenovirus vector.

Local administration of Fas-ligand adenovirus expression vectors will result in clearance of activated cells, as assessed by measuring the relative levels of mRNA by quantitative RT-PCR. This treatment also will lead to an enhanced level. Also, whether such level of apoptosis identified in affected mouse synovial tissue is assessed by the TUNEL assay ("Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP Nick End 101 Labeling"). TUNEL is performed by immersing the sections in TdT buffer (30 mM Tris-HC1, pH 7.2, 140 nM sodium cacodylate, 1 mM cobalt chloride), and then adding TdT (GIBCO BRL, Grand Island, NY) and biotinylated dUTP (Boehringpr Mannheim, Indianapolis, IN). The reaction is terminated by immersing the sections in TB buffer (300 mM sodium chloride, 30 mM sodium citrate). Subsequently, the samples are treated with peroxidase-labeled streptavidin and then visualized using the VECTASTAIN ABC kit (Vector Laboratories Inc., Burlingame, CA). For immunohistochemistry, the sections are blocked with 4% skim milk for 30 minutes at room temperature, then incubated with biotinylated mAbs specific for mouse CD3, B220, CD80, or CD95 (Fas). These antibodies are 15 available from Pharmingen (San Diego, CA).

b. Treatment of Rheumatoid Arthritis Patients with a Gene Therapy Vector Encoding an Accessory Molecule Ligand, Fas Ligand Candidate Fas-ligand constructs identified as having potential therapeutic benefit are used in human protocols to treat RA. Human protocols encompass either in vivo or ex vivo methods to deliver the Fas-ligand constructs.

Furthermore, the Fas-ligand constructs are potentially delivered by either viral or non-viral methods. Outlines of therapeutic strategies are described below.

An ex vivo therapy is similar to a protocol described for intra-articular transplantation of autologous synoviocytes retrovirally transduced to synthesize interleukin-1 receptor antagonist (Evan, Christopher et. al., Clinical Trial to Assess the Safety, Feasibility, and Efficacy of Transferring a Potentially Anti-Arthritic Cytokine Gene to Human Joints with Rheumatoid arhtritis, Human Gene Therapy, Vol. 7, 1261- 1280). In this procedure, after clinical diagnosis of RA, the synovium is harvested during total joint replacement. The synoviocytes re-isolated and expanded, 102 then transduced or transfected with heterologous Fasligand into synoviocytes (via retrovirus, adenovirus, naked DNA, etc.). The gene-modified synoviocytes are then reinjected into the patient, who is monitored and tested for amelioration of RA-associated symptoms, and for expression and function of the Fas-ligand in modified synoviocytes.

In another ex vivo protocol, an allogeneic immortalized cell line that stably expresses the heterologous Fas-ligand is administered to the RA patient. In this protocol, a stable immortalized cell line expressing Fas-ligand (introduced by transfection of the gene into the cell by nonviral methods, such as electroporation), or by viral transduction of the gene 15 into the cell) is constructed. The modified cell line is injected into the patient, who is monitored and tested for amelioration of RA associated symptoms, and for expression and function of the hFas-ligand in modified synoviocytes.

20 An in vivo based therapy will is similar in concept to the amelioration of collagen-induced-arthritis using a murine Fas-ligand adenovirus gene therapy vector, described in Zhang, et al., J. Clin. Invest. 100:1951- 1957 (1997). In our use of such an approach, delivery of the hFas-ligand construct or chimeric AfasL directly to the joints of RA patients is performed using either viral or non-viral methods. In this procedure, the Fas-ligand construct hFas-ligand adenovirus) is directly injected into the synovium. Patients are monitored and tested for amelioration of RA-associated symptoms as well as biological testing for expression and function of the hFas-ligand in modified synoviocytes.

Page(s) i2 6 -'are claims pages they appear after the sequence listing 103 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANTS:

Kipps, Thomas J.

Sharma, Sanjai Cantwell, Mark (ii) TITLE OF INVENTION: (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE

ADDRESS:

NOVEL EXPRESSION

VECTORS

CONTAINING

ACCESSORY

MOLECULE LIGAND GENES AND THEIR USE FOR IMMUNOMODULA- TION AND TREATMENT

OF

MALIGNANCIES AND AUTOIMMUNE

DISEASE

ADDRESSEE:

STREET:

(C)

(D)

(E)

(F)

CITY:

STATE:

COUNTRY:

ZIP:

Lyon Lyon 633 West Fifth Street Suite 4700 Los Angeles California

U.S.A.

90071-2066 .r COMPUTER READABLE FORM: MEDIUM TYPE:

COMPUTER:

OPERATING SYSTEM:

SOFTWARE:

3.5" Diskette, 1.44 Mb storage IBM Compatible IBM P.C. DOS FastSeq Version (vi) CURRENT APPLICATION

DATA:

APPLICATION NUMBER: To Be Assigned FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION

DATA:

APPLICATION

NUMBER:

FILING DATE: 60/132145 12/9/96 104 (Viii) ATTORNEY/AGENT

INFORMATION:

NAME: Guise, Jeffrey W.

REGISTRATION NUMBER: 34,613 REFERENCE/DOCKET NUMBER: 231/003 (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE:

TELEFAX:

TELEX:

(213) 489-1600 (2 13) 9 55 -044 0 67- 3510 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

786 base pairs nucleic acid single linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

ATGATCGAAA

ATGAAAATTT

CTTTTTGCTG

GAAGATTTTG

TTACTGAACT

AACAAAGAGG

CAAATTGCGG

GCTGAAAAAG

CTGACCGTTA

CGGGAAGCTT

TTCGAGAGAA

CAATCCATTC

GTGACTGATC

CTCTGA

CATACAACCA

TTATGTATTT

TGTATCTTCA

TATTCATGAA

GTGAGGAGAT

AGACGAAGAA

CACATGTCAT

GATACTACAC

AAAGACAAGG

CGAGTCAAGC

TCTTACTCAG

ACTTGGGAGG

CAAGCCAAGT

AACTTCTCCC

ACTTACTGTT

TAGAAGGTTG

AACGATACAG

TAAAAGCCAG

AGAAAAC-AGC

AAGTGAGGCC

CATGAGCAAC

ACTCTATTAT

TCCATTTATA

AGCTGCAAAT

AGTATTTGA-A

GAG CCATGGC

CGATCTGCGG

TTTCTTATCA

GACAAGATAG

AGATGCAACA

TTTGAAGGCT

TTTGAAATGC

AGCAGTAAAA

AACTTGGTAA

ATCTATG CCC

GCCAGCCTCT

ACCCACAGTT

TTGCAACCAG

ACTGGCTTCA

CCACTGGACT

CCCAGATGAT

AAGATGAAAG

CAGGAGAAAG

TTGTGAAGGA

AAAAAGGTGA

CAACATCTGT

CCCTGGAAAA

AAGTCACCTT

G CCTAAAGTC

CCGCCAAACC

GTGCTTCGGT

CGTCCTTTGG

GCCCATCAGC

TGGGTCAGCA

GAATCTTCAT

ATCCTTATCC

TATAATGTTA

TCAGAATCCT

GTTACAGTGG

TGGGAAACAG

CTGTTCCAAT

CCCCGGTAGA

TTGCGGGCAA

GTTTGTCAAT

CTTACTCAAA

120 180 240 300 360 420 480 540 600 660 720 780 786 INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

783 base pairs nucleic acid single linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

ATGATAGAAA

ATGAAGATTT

CTTTTTGCTG

GAAGATTTTG

TTGCTGAACT

AACAAAGAAG

ATTGCAGCAC

AAGAAAGGAT

ACGGTTAAAA

GAGCCTTCGA

CATACAGCCA

TTATGTATTT

TGTATCTTCA

TATTCATAAA

GTGAGGAGAT

AGAAAAAAGA

ACGTTGTAAG

ATTATACCAT

GAGAAGGACT

GTCAACG CC C

ACCTTCCCCC

ACTTACTGTT

TAGAAGATTG

AAAGCTAAAG

GAGAAGGCAA

AAACAGCTTT

CGAAGCCAAc

GAAAAGCAAC

CTATTATGTC

ATTCATCGTC

AGATCCGTGG

TTCCTTATCA

GATAAGGTCG

AGATGCAACA

TTTGAAGACC

GAAATGCAAA

AGTAATGCAG

TTGGTAATGC

TACACTCAAG

GGCCTCTGGC

CAACTGGACT

CCCAAATGAT

AAGAGGAAGT

AAGGAGAAGG

TTGTCAAGGA

GAGGTGATGA

CATCCGTTCT

TTGAAAATGG

TCACCTTCTG

TGAAGCCCAG

TCCAGCGAGC

TGGATCTGTG

AAACCTT CAT ATCTTTATcC

TATAACGTTA

GGATCCTCAA

ACAGTGGGCC

GAAACAGCTG

CTCTAATCGG

CATTGGATCT

105 GAGAGAATCT TACTCAAGGC GGCAAATACC CACAGTTCCT CCCAGCTTTG CGAGCAGCAG TCTGTTCACT TGGGCGGAGT GTTTGAATTA CAAGCTGGTG CTTCTGTGTT TGTCAACGTG ACTGAAGCAA GCCAAGTGAT CCACAGAGTT GGCTTCTCAT CTTTTGGCTT ACTCAAACTC

TGA

INFORMATION FOR SEQ ID NO: 3: Wi SEQUENCE CHARACTERISTICS: LENGTH: 783 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

S*

S S

*SSS

S

.5S* S

ATGATCGAAA

ATGAAAATTT

CTTTTTGCTG

GAAGATTTTG

TTGCTGAACT

AACAAAGAAG

ATTGCAGC-AC

AAGAAAGGAT

ACGGTTAAAA

GAGCCTTCGA

GAGAGAATCT

TCTGTTCACT

ACTGAAGCAA

TGA

CATACAACCA

TTATGTATTT

TGTATCTTCA

TATTCATAAA

GTGAGGAGAT

AGAAAAAAGA

ACGTTGTAAG

ATTATACCAT

GAGAAGGACT

GTCAACGCCC

TACTCAAGGC

TGGGCGGAGT

GCCAAGTGAT

AACTTCTCCC

ACTTACTGTT

TAGAAGATTG

AAAGCTAAAG

GAGAAGGCAA

AAACAGCTTT

CGAAGCCAAC

GAAAAGCAAC

CTATTATGTC

ATTCATCGTC

GGCAAATACC

GTTTGAATTA

CCACAGAGTT

CGATCTGCGG

TTTCTTATCA

GATAAGGTCG

AGATGCAACA

TTTGAAGACC

GAAATGCAAA

AGTAATGCAG

TTGGTAATGC

TACACTCAAG

GGCCTCTGGC

CACAGTTCCT

CAAGCTGGTG

GGCTTCTCAT

CCACTGGACT

CCCAGATGAT

AAGAGGAAGT

AAGGAGAAGG

TTGTCAAGGA

GAGGTGATGA

CATCCGTTCT

TTGAAAATGG

TCACCTTCTG

TGAAGCCC-AG

CCCAGCTTTG

CTTCTGTGTT

CTTTTGGCTT

GCCCATCAGC

TGGGTCAGCA

AAACCTTCAT

ATCTTTATCC

TATAACGTTA

GGATCCTCAA

ACAGTGGGCC

GAAACAGCTG

CTCTAATCGG

CATTGGATCT

CGAGCAGCAG

TGTCAACGTG

ACTCAAACTC

INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

786 base pairs nucleic acid single linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:

ATGATCGAAA

ATGAAAATTT

CTTTTTGCTG

GA6AGATTTTG

TTACTGAACT

AACAAAGAGG

CAAATTGCGG

GCTGAAAAAG

CTGACCGTTA

CGGGAAGCTT

TTCGAGAGAA

CAATCCATTC

GTGACTGATC

CTCTGA

CATACAACCA

TTATGTATTT

TGTATCTTCA

TATTCATGAA

GTGAGGAGAT

AGACGAAGAA

CACATGTCAT

GATACTACAC

AAAGACAAGG

CGAGTCAAGC

TCTTACTCAG

ACTTGGGAGG

CAAGCCAAGT

AACTTCTCCC

ACTTACTGTT

TAGAAGGTTG

AACGATACAG

TAAAAGCCAG

AGAAAACAGC

AAGTGAGGCC

CATGAGCAAC

ACTCTATTAT

TCCATTTATA

AGCTGCAAAT

AGTATTTGAA

GAGCCATGGC

CGATCTGCGG

TTCCTTATCA

GACAAGATAG

AGATGCAACA

TTTGAAGGCT

TTTGAAATGC

AGCAGTAAAA

AACTTGGTAA

ATCTATGCCC

GCCAGCCTCT

ACCCACAGTT

TTGCAACCAG

ACTGGCTTCA

CCACTGGACT

CCCAAATGAT

AAGATGAAAG

CAGGAGAAAG

TTGTGAAGGA

AAAAAGGTGA

CAACATCTGT

CCCTGGAAAA

AAGTCACCTT

GCCTAAAGTC

CCGCCAAACC

GTGCTTCGGT

CGTCCTTTGG

GCCCATCAGC

TGGATCTGTG

GAATCTTCAT

ATCCTTATCC

TATAATGTTA

TCAGAATCCT

GTTACAGTGG

TGGGAAACAG

CTGTTCCAAT

CCCCGGTAGA

TTGCGGGCAA

GTTTGTCAAT

CTTACTCAAA

120 180 240 300 360 420 480 540 600 660 720 780 786 106 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

783 base pairs nucleic acid single linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: *9 .4

ATGATCGAAA

ATGAAAATTT

CTTTTTGCTG

GAAGATTTTG

TTGCTGAACT

AACAAAGAAG

ATTGCAGCAC

AAGAAAGGAT

ACGGTTAAAA

GAGCCTTCGA

GAGAGAATCT

TCTGTTCACT

ACTGAAGCAA

TGA

TTATGTATTT

TGTATCTTCA

TATTCATAAA

GTGAGGAGAT

AGAAAAAAGA

ACGTTGTAAG

ATTATACCAT

GAGAAGGACT

GTCAACGCCC

TACTCAAGGC

TGGGCGGAGT

GCCAAGTGAT

ACTTACTGTT

TAGAAGATTG

AAAGCTAAAG

GAGAAGGCAA

AAACAGCTTT

CGAAGCCAAC

GAAAAGCAAC

CTATTATGTC

ATTCATCGTC

GGCAAATACC

GTTTGAATTA

CCACAGAGTT

CATACAACCA AACTTCTCCC

CGATCTGCGG

TTCCTTATCA

GATAAGGTCG

AGATGCA4CA

TTTGAAGACC

GAAATGCAAA

AGTAATGCAG

TTGGTAATGC

TACACTCAAG

GGCCTCTGGC

CACAGTTCCT

CAAGCTGGTG

GGCTTCTCAT

CCACTGGACT

CCCAAATGAT

AAGAGGAAGT

AAGGAGAAGG

TTGTCAAGGA

GAGGTGATGA

CATCCGTTCT

TTGAAAATGG

TCACCTTCTG

TGAAGCCCAG

CCCAGCTTTG

CTTCTGTGTT

CTTTTGGCTT

GCCCATCAGC

TGGATCTGTG

AAACCTT CAT

ATCTTTATCC

TATAACGTTA

GGATCCTCAA

ACAGTGGGCC

GAAACAGCTG

CTCTAATCGG

CATTGGATCT

CGAGCAGCAG

TGTCAACGTG

ACTCAAACTC

120 180 240 300 360 420 480 540 600 660 '720 780 783 .44 4 4**4 INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS:

(A)

(B)

(C)

(D)

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY!

786 base pairs nucleic acid single linear 4 4 .44 4 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

ATGATAGAAA

ATGAAGATTT

CTTTTTGCTG

GAAGATTTTG

TTACTGAACT

AACAAAGAGG

CAAATTGCGG

GCTGAAAAAG

CTGACCGTTA

CGGGAAGCTT

TTCGAGAGAA

CAATCCATTC

GTGACTGATC

CTCTGA

CATACAGCCA

TTATGTATTT

TGTATCTTCA

TATTCATGAA

GTGAGGAGAT

AGACGAAGAA

CACATGTCAT

GATACTACAC

AAAGACAAGG

CGAGTCAAGC

TCTTACTCAG

ACTTGGGAGG

CAAGCCAAGT

ACCTTCCCCC

ACTTACTGTT

TAGAAGGTTG

AACGATACAG

TAAAAGCC-AG

AGAAAACAGC

AAGTGAGGCC

CATGAGCAAC

ACTCTATTAT

TCCATTTATA

AGCTGCAAAT

AGTATTTGAA

GAGCCATGGC

AGATCCGTGG

TTTCTTATCA

GACAAGATAG

AGATGCAACA

TTTGAAGGCT

TTTGAAATGC

AGCAG;TAAAA

AACTTGGTAA

ATCTATGCCC

GCCAGCCTCT

ACCCACAGTT

TTGCAACCAG

ACTGGCTTCA

CAACTGGACT

CCCAGATGAT

AAGATGAAAG

CAGGAGAAAG

TTGTGAAGGA

AAAAAGGTGA

CAACATCTGT

CCCTGGAAAA

AAGTCACCTT

GCCTAAAGTC

CCGCCAAACC

GTGCTTCGGT

CGTCCTTTGG

TCCAGCGAGC

TGGGTCAGCA

GAATCTTCAT

ATCCTTATCC

TATAATGTTA

TCAGAATCCT

GTTACAGTGG

TGGGAAACAG

CTGTTCCAAT

CCCCGGTAGA

TTGCGGGCAA

GTTTGTCAAT

CTTACTCAAA

INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

786 base pairs nucleic acid single linear 107 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:

ATGATAGAAA

ATGAAGATTT

CTTTTTGCTG

GAAGATTTTG

TTACTGAACT

AACAAAGAGG

CAAATTGCGG

GCTGAAAAAG

CTGACCGTTA

CGGGAAGCTT

TTCGAGAGAA

CAATCCATTC

GTGACTGATC

CTCTGA

CATACAGCCA

TTATGTATTT

TGTATCTTCA

TATTCATGAA

GTGAGGAGAT

AGACGAAGAA

CAr-ATGTCAT

GATACTACAC

AAAGACAAGG

CGAGTCAAGC

TCTTACTCAG

ACTTGGGAGG

CAAGCCAAGT

ACCTTCCCCC

ACTTACtGTT

TAGAAGGTTG

AACGATACAG

TAAAAGCCAG

AGAAAACAGC

AAGTGAGGCC

CATGAGCAAC

ACTCTATTAT

TCCATTTATA

AGCTGCAAAT

AGTATTTGAA

GAGCCATGGC

AGATCCGTGG

TTCCTTATCA

GACAAGATAG

AGATGCAACA

TTTGAAGGCT

TTTGAAATGC

AGCAGTAAAA

AACTTGGTAA

ATCTATG CCC

GCCAGCCTCT

ACCCACAGTT

TTGCAACCAG

ACTGGCTTCA

CAACTGGACT

CCCAAATGAT

AAGATGAAAG

CAGGAGAAAG

TTGTGAAGGA

AAAAAGGTGA

CAACATCTGT

CCCTGGAAAA

AAGTCACCTT

GCCTAAAGTC

CCGCCAAACC

GTGCTTCGGT

CGTCCTTTGG

TCCAGCGAGC

TGGATCTGTG

GAATCTTCAT

ATCCTTATCC

TATAATGTTA

TCAGAATCCT

GTTACAGTGG

TGGGAAACAG

CTGTTCCAAT

CCCCGGTAGA

TTGCGGGCAA

GTTTGTCAAT

CTTACTCAAA

120 180 240 300 360 420 480 540 600 660 720 780 786 0 *0 000* 0* 0 a *000 0*00 00 0 0* 0.

00*0 000* 0e *0 0 0 0 0 0 0 0000*S 0 INFORMATION FOR SEQ ID NO: 8: Ci) SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

864 base pairs nucleic acid single linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

AACTCTAACG

GACCACCTGT

TGATTGGGTC

AAAGGAATCT

AGGGGTCCTT

AGGATATAAT

GTGATCAGGA

CTGTTCTCCA

AAAACGGGAA

CCTTCTGTTC

AGTCCCCAAG

AACCATGCGG

CGGTGTTTGT

TTGGCTTACT

GGTCTTCACA

CAGCATGATC

CAGTATGAAA

AGCGCTTTTT

TCATGAAGAT

ATCCTTACTG

GCAAAACAAA

GCCTCAGATA

GTGGGCCCCC

ACAGCTGGCC

CAATCGGGAA

TGGATCAGAG

GCAGCAATCC

CAATGTGACT

CAAACTCTGA

ATCCAGGAAA

GAAACATACA

ATTTTTATGT

GCTGTGTATC

TTTGTGTTCA

AACTGTGAGG

GAAGTAAAGA

GCGGCACATG

AAAGGATACT

GTGAAAAGAC

ACTTTGAGTC

AGAATCTTAC

ATTCACTTAG

GATCCAAGTC

ACGGTGTAAG

GCAG

GTCAACCTTC

ATTTACTTAC

TTCACAGACG

TGAAAACGAT

AAATTAGAAG

AGAAAGAAAA

TCATCAGTGA

ACACCCTAAG

AAGGATTCTA

AAGCTCCATT

TGAGAGCTGC

GAGGAGTCTT

AAGTGAGCCA

CCAGCAGGCT

TCCCCGCTCC

AGTTTTTCTT

ATTGGACAAG

ACAGAGATGC

CCGGTTTGAA

AAACTTTGAA

GGCCAGTAGT

CAACAACCTG

TTACATCTAC

TATAGCCAGC

AAACACCCAC

TGAATTGCAA

CGGGACGGGC

GCGGCTGGGC

GTGGCCACTG

ATCACCCAGA

ATAGAAGACG

AATAAAGGAG

GACTTGGTCA

ATGCACAAGG

AAAACAACCT

GTAACCCTCG

AC CCAAGT CA

CTCTGCCTGA

AGTTCTTCCA

TCGGGTGCTT

TTCACATCAT

TGATGCTGGT'

INFORMATION FOR SEQ ID NO: 9: Ci) SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

3634 base pairs nucleic acid single linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:

GAATTCCGGG

TCCTGAGGCC

CAGGCCTCAG

CAGCTTTCTG

TTAGAAGGAA

GGGCATGGGG

AGACCCCCCT

GTGTGTCCCC

TO CAGGO CCC

TGATTTCACT

TCAAGCCTGC

GACTCAACAC

AAGCCCCTCC

ACAGACCACA

ACGGGGTTCA

CGGAATCGGA

AACTTTCCAA

ACTACCGCTT

CCCGGCTGTC

CACCAAGCCC

AGCTTTTCCC

CAGTTCTAGT

GACCTGGTCC

GCCTCCAGGG

GCAGGGAGGA

ATCCCCGCCC

CCTCCAGATG

CAGGCTTGTC

CCAGCTCCTT

TCCAACCCGT

TCTATCTTTT

CCAAAAGAAA

TCCTACACAC

TGGGGAGTGT

CCGCGATGGA

AGCTCATGGG

CTGCTACCCC

CTCCCCGCAG

TTTCTCTCCC

TCCTGCATCC

TGGAGGCAAT

AAATCAGTCA

GAGGGGTATC

GAAGAAACCG

TTTCTCCA CC

ACCCAGCCTT

GACCCAAACA

TCAACGGACT

TGTCTGGAAG

AGGTTTTGAG

GTGGCCCAGA

CTTGATGCTT

AGACAGAAGG

AAGGAAGTTT 120 180 240 300 360 420 480 540 108

S.

6 @0 5@@O

S

OS

S Se..

0 005e *0 C S S

SS

6e S

S~.

S

S*

C

S

5 6 @6S 0

S

*00555 0

TCCGCTGGT,

TTGTTGGCA(

AGGGAGAGJ

CAAGCTGCC)

CCTGGAAAGC

GCGCTCCCC)

TTCTCCTTCC

ATCGGCCCCC

GGGGAAATG;

AGGGAGGGA~I

AAGAGATGGC

TGCTCACTA.Z

AATAAGATA'r

ATG'AATAAAC

GGAGATAAGG

TGTTGAATGC

GCTAAGAGCG

TGTTP.ACCAT

GGCCCAGGCA

GGTAGGGTTA

GGATGGATGG

TCTTTTCTCT

ATGTTGTAGG

TTGAAGCCCG

CAAGGGAAGG

GTGGGATGTG

AGGGCCAGGA

CTCCCTCCAG

GCCCTCCTGG

CTGTACCTCA

CTCCTCACCC

TCTGCCATCA

TATGAGCCCA

GAGATCAATC

ATTGCCCTGT

TTTATTACCC

CTTAGGGTCG

GATTCAGGAA

GGCCTCCAGA

AGGGAGCCTT

GACACAAGTG

GAGCCCAGCC

ATTATTTATT

TCCTGGGGGA

CTGAACAATA

TTTTA-AAATA

CTCATTGCTG

GGCGAGAAAT

ATCTGCCTCT

ATCCCTCGGA

AGCCCAACAG

CTCAGGGCAT

r GAATGATTC'

-ACCCAGCCA(

kGCAACTACAC kGGCAGGTTC'J

;ACACCATGAC

AGAAGACAGC

TGATCGTGGC

AGAGGGAAG;

GA.GACGCAAG

GAGAGAGAA-A

GAAGAGAGAG

GTGTGTATGG

GGAGACAGAT

ATGGTGAGAC

AGAGAAGAAG

TGGAAGGTGA

CAGGCCAGAC

TCTCCTTCTC

GTCAGTAAGT

GTACCGGTAT

AGGTGAAAGT

CTCCTCTTCA

TAAGAGC2CT

GCTGATGGTA

GTGGAGGAAC

GGATGAC-AGA

TGTGGAGAGT

CAAACCCTCA

CCAATGGCGT

TCTACTCCCA

ACACCATCAG

AGAGCCCCTG

TCTATCTGGG

GGCCCGACTA

GAGGAGGACG

CCTCCTTCAG

GAACCCAAGC

TOTGTGGCCT

ACTCACTGGG

TGGTTCTGGC

GACCTTAGGC

CTCCCCATGG

TATTATTTAT

CCCAATGTAG

GGCTGTTCCC

TTTATCTGAT

AGCCTCTGCT

AAAGTTTGCT

TCTTGTGGGT

CCCAGTCCCA

AATATTCCCC

GGGAATTTCC

r TTCCCCGCCC

;CAGACGCTCC

;ACCCCCCCTG

rCTTCCTCTCA

;CACTGAAAGC

;GGGGCCCCAG

'AGGCGCCACC

GGTGAGTGCC

AGAGGGAGAG

AAAACATGGA

AGAGAAAGAT

AGTGAATGAA

GTGGGGTGTG

AGAAAGAGCG

ATAGGGTGTC

ATACACAGAT

AGGCAGCCAG

CCCAACAGTT

GTCTCCAAAC

GGAAGCAGTG

AGGGGGGTAT

GGATCATCTT

GAGGATGTGT

GGCAGAACTT

AGCACAGGCC

CAGAGAGGAC

GAACCGACAT

AGCTGAGGGG

GGAGCTGAGA

GGTCCTCTTC

CCGCATCGCC

CCAGAGGGAG

AGGGGTCTTC

TCTCGACTTT C AACATCCAAC C ACACCCTCAA C

TTAGAACTTT

GCACAGTGAA G GCCTACAGCT TI

CAGAATGCTGC

CTTCCTCTCT C AGCCAGCTCC C TTATTTACAG A GAGCTGCCI'T G ATGTAGCCCC C

TAAGTTGTCTA

CCCCAGGGGA G TAGAAAAGAA A G4GGAAGAAGC T TCCTTAGACT C

ATCCCCCAGGA

AACTCTGGGA A

TCCTCTCGCC

CTCAGCAAGG

AAAACAACCC

CATACTGACC

ATGATCCGGG

GGCTCCAGGC

ACGCTCTTCT

TGGCC-AGCCT

AGATGGGATG

GAAAGACGGG

GGAGAGACAG

TGAATGAATG

AGAAGAGAGA

GGAAATATGA

TGGCAC-ACAG

GAATGGAGAG

CTGTTCCTCC

CCCCAGGGAC

CTCTTTCCTA

GGGGAAATTT

TTTCTAGGAA

CTCGAACCCC

CTTGGAACTT

GGAGACAATG

TTAGTGGGAT

ELGGAACCGGA

GGCCACACTG

CAGCTCCAGT

3ATAACCAGC

!LAGGGCCAAG

~TCTCCTACC3 kCCCCAGAGG C :AGCTGGAGA CCGAGTCTG C TTCCCAAAC G ~CTCTTCTGG C AGCAACAAG A~ ;TGCTGGCAA C ~TGATCCCTG A 'AGGACTTGA G CAGATGTTT C TCTATTTAT G .TGAATGTAT T GCTCAGACA T TGGCCTCTG T AACAATGCT G

TTGTGTCTGT

CATGGTCTC C CCCTAAGTC C CTAGGGCCC T AACAAGAGC C

TTC

CCAGGGACAT

ACAGCAGAGG

TCAGACGCCA

CACGGCTTCA

ACGTGGAGCT

GGTGCTTGTT

GCCTGCTGCA

TCATCCACTC

GGTGAAAGAT

GATGC-AGAAA

GATGTCTGGC

AATGAACAAG

TGGGGGAAGA

CAGCTAAGGA

AAGACACTCA

AGAAAACCAG

TTTAAGGGTG

CTCTCTCTAA

ATTCTGGGTT

AAAGTTTTGG

GTTTAAGGGT

GAGTGACAAG

GGAGGGCTAG

rGAGAAGGAC

ACTCAGAACG

1'GTGGGGTGG

!LCTCTCCTCT

3GCTGAACCG rGGTGGTGCC 3CTGCCCCTC kGACCAAGGT

;GGCTGAGGC

~GGGTGACCG

;GCAGGTCTA

;CCTCCCCTGC

TCAAAAAGA C LCCACCACTT C CACTAAGAA T ~CATCTGGAA T AAGACCTCA C CAGACTTCC TI :TTTGCACTT G TATTTGGGA G GTTTTCCGT G 'GCCTTCTTT T ATTTGGTGA C AATCGCCCT A TTCTTGGAA T TCTCTCCAC A GGAGACCCT A TGAACCTAA T

ATAAAGGCAG

ACCAGCTAAG

CATCCCCTGA

CCCTCTCTCC

GGCCGAGGAG

CCTCAGCCTC

CTTTGGAGTG

TCCCACCCAA

GTGCGCTGAT

GAGATGTGGC

ACATGGAAGG

CAGATATATA

AACAAGTGAT

GAGAGATGGG

GGGAAAGAGC

ACACCTCAGG

ACTCCCTCGA

TCAGCCCTCT

TGGGTTTGGG

TCTTGGGGGA

CTCAGCTTTT

CCTGTAGCCC

GATTTGGGGA

TCGCTGAGCT

TCATGGCCAG

GCAGAGCTCG

CCCTCTCTCC

CCGGGCCAT

%TCAGAGGGC

:ACCCATGTG

-AACCTCCTC

.AAGCCCTGG

~CTCAGCGCT

TTTGGGATC

~CCCAATCCC

AATTGGGGG

GAAACCTGG

TCAAACTGG

CTGGAGACC

CTAGAAATT

TGAGACACG

ITGATTATTT

ACCGGGGTA

AAAACGGAG

GATTATGTT

CAACTGTCA

CTATTCAGT

TAATTCTGC

GGCTTTAAG

CATAAACAA

TACCTCTCC

600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820* 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3634 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 1997 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:

GAGACAGAGT

CAGCCTCCGC

TGCAGATGCG

CCATGTTGGC

CAAAGTGCCA

CTTGCTCTGT CCCCCAGGCT GGAATACAGT GGTGCGATCT TGACTCACTG CTCcCAGGTT CAAATAATTC TcCAGCCTCA GCCTCCCGAG TAGCTGGGAC CACCAGCACG CCTGGcTAAT TTTTGTATTT ATTATAGAGA TGGGGTTTCA CAGCTGGTcT CAAAcTCCTG ACcTCAAGTA ATCCGccCAC CTCAGACTCC- GGATTACAGG TGTGAGCCAC TGC-ACCAGGC CTGGAACAAT TTTAAAATAA 109 TGTATTGGCT CTGCAAATGC AAGTCACCAC

CCTTAAGCCT

TGGAAGGTGT

CTGCCACATT

TTCTGTGGAA

GCCACCTGAA

GACCTCATCA TACTTGACTG TTCCAAACTA

GGACACTTTC

AGAGGCAAAC

ACCAGAATGT

AACCTGCCAG ATGGGCTGGA GACAAGGACA

GGGGTACAAG

GGGTGC-AGAG ATbTTATATA AGCTGCTCAA CCACCTCCTC CCCACTCCCT GGCCCTCCCA AGTCGCCTGA ACCCCCAGCC TGAAACAGCA

GTATCTTCTA

CCCGCCCGCT GCCTGCCACG GGGCCCAGGG GCTCCGCACA TGCTTCGTGC TTTGGACTAC TCCTGCACCT

GCTGCCTGGA

TGGTTTCCTT

CTCTGTCTCT

CTCTGTTTCT GCCATGATTC CACCTTGGGG

TTTCTCTGAC

TCGGGGGGTG CTCTCTCCCA *CACTGTCTCT

CTCTCTCTCT

*TCTCTTCCTC

CCAAGGGTGT

*GGTTCTGCTG

CCTGGGGCCC

AGCCAAACCT

TGAGCCCTAG

*GTTGGCCTCA

CACCTTCAGC

CACAGCACCC

TCAAACCTGC

ATGTCCCCAC

CAGCTCT

AGCTTCAGAA

CACCCATGTG

GATCCTGGAA

GTCAGGAAGA

TCCAGCATCA

AAGAGTGGAA

CCCCGATGAA

AAGTCCGTAT

AGAAGGAAAT

TGATTGCTCT

TCTGAATTGA

GCCCACGATT

TGTGGTTCTC

AGCCCTGGGG

CTGCCACTGC

GCAGGTGAGG

CGCCC-AGCAG

TCCCCGGCCT

GACTCTCCAT

CTCTCTGTTC

TGCATCTTGT

GGGCGGGAGG

CTCTTTCrCT

GTGGCACCAC

AGGTGAGGCA

AGCCCCCCTC

TGCCCAGACT

TGCTCACCTC

CAAGTCCCTT

GAATTCTGAA

TGTGTGTTTA

GATGGAGGGC

TCTCTGAGTA

GGGGGATCCA

GGGGATATAT

ACTGGGACAA

GGGCAAAGAG

TCAGGGAACC

CTGTCCCTTC

CCCCTGACCC

TCCTAGGCCT

GCTTCCCCGG

CGCTTCCTCT

CTCTCCTGCC

TGTCCTGCCC

GCCTGGGCCT

CTGTCAGTCT

CCTTCCTGTC

CCCCTTCTCT

TCTGTCTTCcC

GCAGGTTCTC

CCTACACCTC

GCAGGAGAATC

AACTCTGTTC

GCCCGTCAGC I %TTGGTAAAC

I

AGCTGTCCCC

ACTTCCTTTG

TTTGGGGTTA

ATCCTTCAGG

AGGGGACCAA

TTAATATTTT

AATGGACCTT

GTATGATTTG

AGAAGCCTGT

GGGCCTCCAG

TTTGGAACTC

GACTCCCTTT

CAGCCTTTCC

GCCCCAG CCC

ATAAAGGGAC

CCATCTCCTT

TCTGCCTGGG

GGGCTTGGTG

CATTGTCTCT

rTCTCTTGTC

GTCGATCTCT

GCCGCGTGCC

CCCATGACAC

:TCCTTCTGG

;GGGGCTGCT

rCCCCTAGGG

~CCCCAAGAT

~TCCACCTGA

ACCCCACCCT

TAGAAAACTT

TATAAATCTG

AGTGAGATGA

AAAATTTATC

CACCTGGACA

CTTGATGTGA

AGTTGTTTGG

ACTCAGCCAA

CTCACACCCC

TAGGCCTGAC

CCCAGAACTC

TGCCTTTGAC

CGACCTAGAA

CTGAGCGTcC

GGGCTGCCCG

CCTCGGTCCC

GGTTTGGTTT

GTCACACATT

TCCCTCTGCT

CTCTCGGGGG

CCGCCCCGCT

CACCTGAACG

GGCTGCTGCT

GGGGTGGCTC

GCTCCCTGGT

GCATCTTGCC

CCTCCCAGAC

360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 1997 INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

10240 base pairs nucleic acid single linear o, ooo 0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

GAATTCCCCG

TTTGAGTCTA

GCAGAGACCA

CCCCTCCCAC

TTTCACACAA

TCCAGCACAT

TCTGTCTCTC

CCCTTCTTCC

TAATAAAGCC

ACAGCAGGAC

ACCAAGAGAG

CCAAGATAGA

CATCTGGTCA

TGCTGCTGCT

ACGAATGCAG

CCACACTTCT

CC-ATGCCCAT

ACCGTGTCAG

TTTAAATATA

CATGATGAGA

CAGCTTACTT

CACACCATAT

TCACCATC-TT

TCTGTGGGCT

TCTTTTTAGG

TATCTGCCAG

GTCCTGTGGC

CCCTGCTTGA

GATCAAAGTC

ATGTCATTTC

CTGTAGAAAC

ACAGGGAAAG

CCTCCGAAAC

TGAGAATGTC

ATGTCTTCTC

TCTCTTCCTG

GAGGTC'rCCT

GGCAAAGGGA

AGGCCAACAG

GACCAAGCTA

GCCACCAAGA

GCTGCTGCTG

GCTCTTGTCA

CATTTCCCCT

GGCATGTGGC

ATCCTTAACC

CCAATTTGAG

AGGAATTCCA

CCCAAGCCTC

ACTCTTTTTT

TTTTGCTTTG

GCTTTAAGCC

GCCGCACCCA

CCTGCGCTGG

ACAGAAGTTA

GGCCCTGCAG

AGCATTAAAT

CCCAGTTTAG

AGCCTTGTTT

GGAGGACTCA

TGCATTTCCC

TGAGTTTTGG

CCCCAAACCA ACTGCTGGCC TAAGATTGAA

ACCAAGATTG

GCTGATGGAG

CCTCGGCCCA

ATCACTCCTT

CTCACCTTCC

GGGTTTTTCC

CTTTATTTCT

AGTCCATCAG

CTCCTACTGT

CTCCAGAGAA

AGAGACTCAG

TGACACAAGA

CACAGTGAGG

TTTAAAAGAG

CCATCTGTGG

TTTTGCCTAG

AAACGTTCCT

CTGCTGCTGC

TGCTGCTGCT

TCTCCTTGCT

GGGTTGTTGC

CCAGCCCCCC ATCTTCCATA AGTTCCAGGG

GCCAGGGATC

CACTGCACAc

AAGGCAACGC

GGGGTCCCTC TTTCACTTAA AACCATTTTT

CTTGTGTGCA

ATCCCTGCTC

CTTCTACGTG

ACCTCCATC

TTTGCACCTT

ATACTTAATG

CCTCTCCCTC

CACTGTCATG GAACTTATCA GC-ATATGGAG

ATTCCCAGGC

AGCCACAGCA

ACGTGGGATC

CATTCAGGCT

GTGCATGAAC

CTTTGCCTCT

CATGCCTTGC

GTTTTGAGGC

GAGATTTCAC

GCACAAGACT

TCCTCAAGAA

GCCCATCTCA

GCTCTCGAGC

CGTTTTTGTc

CACTGTACCA

TGGAGAGGAG

AGGAAAGCA

TTAAAAGAAA

CTACCCTTCT

CCTCTCCATT

GCTGCTGCTG

AAAATCCTCC

CTTCCATTTA

AAACCTGTGC

CCCTCGAGTC

AAATTTTGGC

AACCCTTCAG

TACCCATGTG

CTGTTCCCTC

AGGCCAGGTT

CATTTTATTT

TAGGGATCTA

CGAGCCTGAG

rATTTCTCCT

TGCTCTGACC

TAAGTTCAAG

TAGTTTCTCC

CCAGTCATCA

AGAAACCGAA

AGGCGCGTCC

TTCCTTCCTT

CTGCAATGCC

TTTTATATT

GCAGAAAGA

AGAAACAGGG

TAAGATGAGG

TCCGCCATCG

CTCCTGCTGC

CTGCCTTAAT

TAACTGGTCT

TTTATTTTGG

CAATGCAGTG

ATTCTCATTT

AGCTCCCTAT

C-ATGTGTCCT

TACATCTCc-A TCTCTGcccc

CAATGGCTTT

rATTTGACTT A.TCGGAGcTG

GGGTTTTGAT

GTTCTCCTCC rATGACTTCT.

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 110 TTTTGTTTGC ATTCCATCTC TTTAGTTTI TACATGAGGC ATTGAGGATA CGGATGGGP GACCAATTGT GGAAGACTGG TTGGTTATC TCATGGGTCA GCAGGTAATG AACCCAGTP GGCCTTGCTC AGCGGGTTAA GGATCCAGC GCGACTCAGA TCTTGCATTG CTGTGGCTG GAACCTCCAT ATGCCTCAGG TGCGGCCCT TCTTTTTCTT AATGTGTAAC CTACAAGCT TGTATCATTT T7:CATACTAG CCAAATACT GAAACAAATT CGACTAGGAA CCATGAGGT GTTAAGGATC CGGCGTTGCC GTGAGCTGT TGTTGCTGTG GCTCTGGTGT AGGCCGGCA AACCTCCATA AGCTGTGGCT GCGGCCC-rA TAGAAhCAAA CCAAATG CCC ATCAACAGA ACAATAG CAT CACACAATAA CATGCACAC TGACAGACAC AACCATATAG ATGAATTTC.

ACAGATGAGT TGTCTGTGTG GATTCATTTi CTATAGTGAC AGAGGTCAGA GAGCAGTTGI GCATGAGAGA ACTTTCTGGA GACCTAAAJ ACAAGAAGAT GGAAAAGTTC CTCAGGCTG' TAAGTTCTGC ATCAGCAGAG GGGGAAATCC GCTTTACCGA TGTATTTTCA ACACAATCC( ACCCTTAAGC TCCACCTGTG TGGAATTCTC GCCACGTTCT TTTGATGCAG GAACGTGTTC CCCACCCAAG GTTGGGACAT GGTGGGGGGC TAGGTGATTT CGTGGCCTCA TCCCTGAGTC CAAAAGGGGG CTCCCTGAAG ATTTCCTCC? GCAGGAGGGT ATAGAAGGGA ACTTGTTGA] ACAGACTGGG ACAAGTGTGA CTTGAGCCTC AACGTGCACA GAGAGAAGCC CGTAATCAGC TCTTCACGCA ACCGGGCAAG CAGCCCACGC TCACCGTCCC TTCTCTGGAA CTCCTAAGCC GGTTCCCCTG ACCCCACTCC CTTTCCCAGP CTCTCCTAGG CCTCAGCCTT TCCTGCCTTC GGCTTCCCCA GGCCCCAGCC CCGGCCTAGP CTGCCGGCTT CCTCTATAAA GGGACCCAGG CACAGCAGGT GAGACTCTCC CACCCCATCT CTCACTTCGG TCCCTCCGCC CGCTCCCTGG GCCTGGGCCT TGGTGGGTTT GGTTTTGGTT CTTTCTAGCT CTCACACACT CTGATCCCTC GGGTCTCCCC CTGCTCACCT CGGGATTTCC GCGCCCCGTC TCTTGTCTCT CGGGGTGGCT GGTGCCCCGC CCCGCTCACT GTCTCTCTCC GACGCCTCTA CCTCCGGAGG GTGTGCAGCA TGGCCCTGCC GCCCGAGGCC CAGGTGAGGC CGCCAACCTT GGGCCTCAGA

GCCTCTCTGA

CGGCCTCCCA CCCTCAGCTG CACAGCCTGC AGGCACCCTC AAACCTGCCG CTCACCTCGT GTAGCCCCCA GTCCTCCTCC TATGCCCCTG TCTCCCCCCA CCCCCTAAAT GGAGGCATCC GGAACTCAGT CCAGCACCTG CTTCCTCAGG CTCCCACCCC CTCTGGCTCT TCCTAGGAGA AGCGAACACG GATCGTGCCT TCCTCCGCCA GGTCCCCACC AGTGGCCTCT ACTTTGTCTA CTTCCCCAAG GCCACCCCCA CCCCTCTCTA CCAGTACCCC TTCCACGTGC CGCTCCTCAG GGGACCTTGG GTGCGCTCTG TGTACCAGGG GCTGTCCACA CACACAGACG GCACCCCCCA TGGAGCCTTC GCTCTATAGA AGAATCCAGA CCTTTTCACC TCCCTTATGA CCACTTCGGA AACAGTCTCA TCTTCCCCCA CGCTCAGCAC CCTCGGGGGA ACTGGAACCA CCCCGGATGC CCCTTCCCCT GTCCTGCCCG TCTAGGGGCC CATGAGGGAC TTTGAACGGA TGACCGGAGC ATTAAATTAT TTATTTATGG AGGATGGAGA TGTGACACAT GTGCCCAAGA GATAAAGTGA AGAGAGGGCA AAGTGGCTCA

GGAAGGGGCT

GACCCTCGAT GAAACCCAAT AAAGCTCTTT ACTCGGGAGG GGAGAATTCT CCAGATGTCT GGGGACGGGA GCTGTGGGTG TGTGATGAGG TCAGGGTGAG GGCAGCCAGA CCCCTGCAGG GGAAGAGATG CAGGGCCAAG GTCTTGAGAA CCACAGGTAG AGGGACAGAG GAATAGGGGT ACTGAGGGGT GTTCGGGATT CTGAGGGAGG C TCTCTGTTC LA AGACAGTCC A GATAATTAC A ACGATCCAT 'G TTCCCACAA T GGTGTAGGC 'A AAAGACAAA A AGTGAAAAC A GAAACAGGG T GCGGGTTCG G GTGTAGGTC G CAACAGCTC T AAAGACAAA A. GAATAGATA PL ATAACATCAi P. CAAACACAAN

CTATGAAGTTI

TTGTCTTTG(

k. TATATTGGAC r CCACTTGGGj

TTAATGATT'

TCTGCTGTC(

;AAGCCTCCCc

;TCTAGAGTT)

GGCCTTCTG(

7AGATCTTCC7

SGGACAGCAGC

ATGAAATCAC

TTGGGCCCAC

CAAGGCTGCP

CCCAGCTGCP

TGACCCCGC'I

LACTCAGTCAI

GCGTGAAACA

ACCCGCCCAG

GCGCCCAGAA

CCTAGGGCTG

CCTTCCTGCC

TGTTCTCTCT

TCTGTTCCCT

CTGAGTGCCT

GTCTCCGAGG

CCCCACAGGT

CCCCCATCCT

AGCAGGAGAG

CGCTCTTCTC

CCATCAGCAC

TGGTAAACAT

CTTCAGGGAC

CACTCCCGAC

GATTGAGACC

CCCCAGCACC

TGGCTTCTTG

CTCCCAGGTC

CCTGGCCCAC

CGCTCAGAAG

GGCTGTGT-TC

CCTGCTCCTC

AAGAAAAAAA

GGTCACCGCG

CTGGAGCTTC

TCTGCTGAGG

CTCGTCCAGG

GGTGTGGGGG

GAAGGGAATC

CAGAAGG CAT

GCTTGACTGG

TCTCTGAAAT

CTAAGGAGTG

CCTAAGGGGC

AGAAGCAGAT

CCGAGGTCGG

GACAGGAGGC

AGCACGGGGA

'C ACAAACATTT 'C CTGACCTCTG A ATGAAGTGTG 'G AGGATGCAGA .G CTGTGGTGTA 'T GGTGGCTACC A AAAAAAAAGA

GGCTCCTATT

A GTTCCCGTCG A TCCCTGGCCT G CAGATGTGGC T GATTAGACTC Xk AAAAAAAAAA h. GTTAATTGGG

AATGAAATAA

2AGCGAGAATA

AAGCGCAGGA

3CAGGTATGAA

TGGATGGTGG

k. GACGGGCTTC r~ GACAATTACA

CCACCCCACC

TGTAGAGAAC

k. GACACATCTG

AGTGAGATGA

SAATGAGGACA

;AACAAACCAG

;CCAGATGACC

GACAGGGGTA

LGAGGTGTTAT

LCTCCATCTCC

CCCTGGCCCT

CTGAGCCCCC

GCAGCATCTT

CCGACCTGCCC

,AGGGGCCCAC

TCCGGGTGCT

CCTCCTGCAT

GATTCTTTAT

TCCCATCTCT C CTCGTCCCCT I GCAGGAGGCC I] TTTCCCCATG CCTCCTCCTG G CGGGCCGTGG G CCCTAGGGCC T

CCCCCAAAGCA

CCACCTGGCC T TGAAGCATCC C TCCCTCCCAA C TCCGACCCCC A CCGGACTCAC T CTGAGCAACA A GTCTTCTCCG G GAGGTCCAGC T TCCGTGTGCC C CTGCTCACCC A AGCCCCAGTA G TTGGTTTCAA G CCTCTCCTCT G TGTAGAAGGA A ATCTGAATGC C ACGTGGAAGG G GGTTATTTAT G ACAGAGGGAT G GGGCTCCAGA T4 AGGCTCATGA GC GCTGTCTGCT CC GAGOGAGGAC A TCAGGTGAGA G~ GGTTCCTCTG AC GGGTCGCCTG G( TTCCCGGGAG A) CGCCCTGGGA GJ

ACTGAGCATC

GGACCTCAAA

GGAGTCCCTG

TTCAATCCCT

GGTCGCAGAT

CCTAGCCTGG

GAGAAACTTT

CCATAACGTT

TGGTGCAGCA

TGCTCAGTGG

TCGGATCTAG

CTAGCCTGAG

GGCCAAATAC

GTATATGCAC

AAATTACTAC

AAAGCCAAGC

ACAACTTAAT

CTGGGAGTGG

CAACATGGCT

TCACGGGACC

AAGTGTATTG

CTAGGTCACC

TTTAGCAGTT

ATCTGTGGGG

AACCTCATTG

CTTTGGAGAG

GATGTCCCAG

TGGAAAATAC

CAGAGGAGGA

PCATAATCGC

TCCTCTGAAC

:CCCAGCCCAC

b.GCCTGCGTT

CTAAGCCCTG

CACGCTGCCA

kGGGGTCCCG

"GACTCCCCC

:TTCACCCCG

:TGTCAGGCT

;TTTCTCTCT

rCTCTGTCTG

TCTTCCGCA

~CACCACCTG

;GGCTGCTGC

GGCAGCCTT

'CCCTGGCGT

LCTTGGCCAG

CCCAGACCT

TCCCCCCCA

CATCCCCCA

GGTCCTTGA

'GCGCTGGAC

*CTCCCTGCT

GGAAGGCTG

CTTCTCCTC

CGGGCCACA

GGGAGATCA

CGTCTTCTT

GCCTTCTCC

ACAATTTCC

TTCTAGGCA

CGCCTGGAG

PAGCTGACC

PAGGGGAAA

TCAGAAGAG

GACCCGGCC

GAGACGGCT

GTATCTGTC

3GAATCAGA kTGGCGGCC

GAAGACAAA

CAGATATGG

LGGGAACAC

k.CATGCCGT 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 ill CCAGGGCCAz

GAGGAGACP

GAAGCAGGA

CATGCAGAA

TTTGGTGAG

CAGCCTGGC

CCCGTGTAG

TGGGTCTCA

CTGAAGCTC

AAATAGAAG

CCAAAGAAA

GGTCCTACA

GGTAGGAAG'

GGAGATGAA

GGGTTTCTCi

TCACCCAGA(

AGGACACCA(

CCTCTCAGAj

CCCCGGGGCC

ACTGAGAGC)

GGCCCCCAG(

GGAGCCACCI

GTGAGCGCC'

GGCCAGAGAC

GAGAAAGATC

GAGAGAAATC

GTGAGTAAGC

AAAGATAGGC

GATAGGAAAC

CTCAATGAAC

AACTAGACA'r

GGGGACTTAT

CTTGAGCATC

TTCTGAGCTT

GTCTGGGTTT

GGGTCTCATC

CCCGTCG CC

GCAGGACAGA

GAGATAGTAT

AGGAGGATAC

GAACCGGATG

GGGTCACACT

GGGACAGCTC

GAAAGACAAC

CTTCAGGGGC

CGCCGTCTCC

GGAGACCCCC

CTTCCAGCTG

CTTTGCTGAA

CGTTCCCTCC

TGGTTTAGAA

AACAGCAACA

CCACCACCAA

CTGGATGCAA

ATACTTCAGA

CTTTCTTCCA

CAGCTCCCTC

ATTTACTAGT

GCTGCCTTGG

CTGGCCTCCT

ATCGAGTTGT

TGCTCCCCAG

GTGCTTAGAA

TGAGGC'rGCT

GAGCTTGGAC

CTCCCCTAAG

GTCTGGGAAT

GAGCCAGGGA

TGAGGCAACT

LT GAGGAGTGG ,C GGTCATGGC A TTCTTTGAG ,G TCAGGGCCT T TCCTGTCAG T TCCTCTCCC G GCCTCCAGC G CACCCAGCG C TCgCATTCC T CATCCCCCA T GGAAACAAT C ACAGAGCAG' r' ATCCCTGATi k CTAAGACAGQ

-ACCAAGGAA(

3 CCTATAAAT( G GGACCAGC( k. GACACACCC( 3CCACCATCTC k. TGATCCGAG)

'GCTCCAGGAC

k CGCTCTTCTC r GGCCAGCCTI

'GAGCTGGGC

;GGCAGGCAG;

CCGTGCCCCC

AGGGACACCC

AAAAAACAAC

AGAGATAAC

GAGTTGTTGA

CACGGCAGAG

TCAAATCTAA

AACCCTCTCG

GGACAGGGGT

GGCGGGGGGA

TTTTTCTTTC

CACGTTGTAG

GAGGGATAGG

CAGGAGGACT

TCGGAACCTC

TGGGGGGCGG

GACTCACCCC

CAATGGCACA

CACCTGGTCG

CAAGGCTCC

TACCAGACCA

GAGGGGGCCC

GAGAAGGATG

TCTGGGCACG

CCTGTCCATC

AGAGAATGAG

CTTAGAAATC

GAATTGGAAC

CCTGGGACAT

ACATTCCTTG

ATTTTCCAGA

TTATTTATAT

GAATGTATTT

TTCAGATGTG

AGCCTCTGTG

CTAAATAATG

GGGAGTTGTG

AAGAAATCTG

CCCCAAAATT

TGCCTGGCTC

AAAGGAACCT

TCCTATCCAG

GCGGCCAGAT

GGGCTCCAAA

;G ACACCCTC7I 'C ACGAGCCGC 'G ATACGTGGC 'C ACATTCCCT A GTGAAAGGA T CTGGCGCTG T GCCCTTCTC C GGTGTCGTC T AGTTCTACC A AGAAAAGGA

SGGAAATGGC.

T AACTGCCCC 3 CCTGGGTGT k. AGGTGCAGGI 3 TTTTCCGCT 3, CAGCTGTTT(

-AGCAAGAGA

CCAACAGCj

CCAGCTGGA(

k. CGTGGAGCT(

GTGCCTGTG(

CCTACTGCA(

GGCTCATTCJ

AAAGAAGTGI

AGGAGACGTC

CCCCTTGGA?

ATATAALAGAC

TGATCTGGAz

AGAGAAGAAG.

ATGGATGGGI

AGTACAAGC

TTAATCCTTC

CCCAAGGACT

GGGGTTAGTG

ATGCAGGTCA

CTCTTTCCTC

GTAAGAGTTC

ATTTGGCGGC

CGCTGACCTC

ATGGACAGAT

CCAGAACTCG

TCCCTCTTTG

GTGGGTATGC

TGCCGACAGA

CTTCCACCAA

AGGTCAACCT

AGGCCAAGCC

ATCGACTCAG

TCTATTTTGG

CCTTTATTAT

GGGCTGGGGA

AGGGATTCAG

TGGGGCTTCC

CTGGAATGTG

ACAAGATTTC

CTTCCCTGGG

TTGCACTTGG

ATTCAGGACG

TTTTCTGTGA

CCTCCTTTTG

CTGATTTGGT

TCTGTAACCG

GCCTCTTTCT

CTTTCTCCAC

AGGAGCCTCT

GAAGGTAATT

CTCGGGAAGT

GCTCAGGTAC

TAACCTGATC

'G AGGCTAGCGG .G AGGCCTCGA 'C ACACAAAGGG T' CGAAGCCGAC G AAGGCCCGCC T CCCAGGCCTG C CAGCTCCTCT T AAGTTTTCTC C CTACCTGAGC A .TTTGTCCCCC A GGCAGGGGCC A. CCAACCCCAC C CCCAACTTTC 3 CCCGCTACCG

GTTGAAAGAG

3 CACACCCAGC k. CAAGCCATCT

~GCCGGACGAC

CTGAGCCCCT

GCGGAGGACG

7CTCACC'CT

TTCGAGGTTA

CCCACCCCCA

GCTGATGGG

GAGAGAGATG

ATGCTCTCTA

AGATGAGTAC

AAACATAGTG

GAAGCGTCG

GGATGAGAAA

ACAGAAGCAG

TTCTTCTCCC

CAGTAAGTAT

CTGCCGTGGA

AAGTAGTGAG I CTCAGGATCA 'I TGAGGATGTG

I]

TGAAGCCAGG

C

CAGGCCAGCA

'I

GGGATGTCGC AGCCCCACCA

T

TCTCCTCCCT

C

CAATGCCCTC

C

TGGCCTGTAC

C

CGTTTTCCTC A CCTCTCTGCC A CTGGTACGAA C

TGCCGAGATCA

GATCATTGCC

C

TTTACTCCTTC

CTCGGCTCCA

A

GGATGTGTGG

C

AGACTCGCTG

G

GCTGCCAGGGA

ACCTCAATCT Ti

ATGGGGACCC

CATTATTATT

T~

GCGAGGTGTC

C,

AAACCGAGCT G CTTATGTTTT

T

GACTAACTTC T( CCCTACTGCT

C

GCGACTGAAT

T(

CGGGCTTAGG

A~

GCAAGAAACA A; ACCTCTCCCT

C

CTGCAGTGCA

GC

CTGAACCAGA

CC

CGGGAATTC

CTGGAGATAC

CAGTCTCTAG

AGGCTGAGGT

ACTGAAACCA

ATGGTGGGTT

TTCCTGCCGT

TCCCTCCAGG

TCCATTAAGA

CCTGTTCCGA

AAAGAAACAG

ACCTGCGGTC

CTCAGGATCC

CAAACCGCCG

CTTCCTCCAG

AGCCTCTCCC

CAGCAGAAGC

CCAGGACCCC

TCTCTCCCTC

CTGAAAAACA

CGCTCGCCAA

TCTCCTTCCT

TCGGCCCCCA

GACAAATGGC

AGTGTGGGGA

GGGGGAGAGA

A.ATATTTGTT

P.CAGACAAGG

AGACAGGAAG

TGTCTGGCAC

kTGGATGAGT 3TGGCTGTTT

CAACAGTTTC

TCTAAAACC

kGGAAGAAGG ~TATTTTCTG

C

'CGTCTCAAA C

CTGGGGGATC

TGAGGGTAG

C

GCGGGATAC I1

LAGACAGACCC

GTGCAGAGT

CAGCCAATG TI TGGCCAACC

G

TCATCTACT

C

CTCACACCA

T

.TCAAGAGCCC

CCATCTACC T ACCTGCCCC

A

TGTGAGGGG

G

AGACCCCCTC

GCTTAAAAC

T

CTGGACAACC

GGTCCTTGC

C

AGCTTGGGT

T

GATGACTTT

T

11.GCCCCAAA C ATTTATTTA T' rCGGAGAcCC.

kACTGTAGG

T'

'AAAACAAA Tj CGCTACATC

G(

kGCTGGCGAC

A

CTGCATCTC C' E'TCCCTGGG

C'

GCCCAGCC A) ~GGGTGTGG C ;TGAGACTT

CC

CAAGGGAC

T)

AGGGACATTT 6180 CAATCTCGAA 6240 GTGCGACTT 6300 GCAGCAGAGT 6360 TCTGAATTCC 6420 CCTCCCCCAG 6480 AGACGAAACA 6540 ACTCAGCTTT 6600 AATCAGAGAC 6660 AACTTGTCCC 6720 CAGCCTCCAG 6780 GGCAGGCAC 6840 CCCCCGCTAT 6900 ATGAGCTCAT 6960 CGCCCTCTTC 7020 TCCCAGAGTC 7080 CTAGAAATAA 7140 TCACACGCTG 7200 CACCATGAGC 7260 GAAGGCCGG 7320 CCTGGTCGCA 7380 GAAGGAAGAG 7440 GAAGAAACAC 7500 GGAAATCATG 7560 GAGAAGGATG 7620 GCACGAATGA 7680 GGTGTGGTAG 7740 bGGTAGAGCA 7800 GTGGAAGGCA 7860 GGAGAGAAAA 7920 rCCCTTCAGA 7980 ZAGCTGGCCC 8040 rGTCTCTCAG 8100 'AAATTTAGG 8160 ;GAAGCcTGA 8220 CTCACATAA 8280 ;AAGAAATAG 8340 CAGAGCTTG 8400 CAGAACTTG 8460 ,AGGGGACAG 8520 GAACTGACA 8580 CAAAGCCGA 8640 ICGTGAAGCT 8700 CCAGGTCCT 8760 'CAGCCGCAT 8820 'TTCCCAGAG 8880 GCGAGGGGT 8940 CTATCTGGA 9000 CAGGACATC 9060 ACGTCCTTC 9120 TTAAA.CAAC 9180 AGGCACTCA 9240 TTTGGATTC 9300 CCAATCGGA 9360 TAGGCTTCC 9420 CCCACAGGC 9480 rTATTATTT 9540 kGCATAAGG 9600 rGCTCCCAC 9660 TTTATCTC 9720 CTGAACCTC 9780 kATAAAAGC 9840 TTGGGGGCC 9900 rTCACTCCT 9960 ~iCAGGTCC 10020 ATTTCCAA 10080 GGCCTGAAA 10140 ~cCAGACAG 10200 10240 112 INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

1644 base pairs nucleic acid single linear SEQUENCE DESCRIPTION: SEQ ID NO: 12:

CCTCAGCGAG

GAAATAGCTC

GCCCAAGGCG

GACGTGGAAC

CGGTGCCTAT

TGTCTACTGA

CCTCTCATCA

GACAAGCCTG

AGCCAGCGCG

GTGCCAGCCG

CCCGACTACG

GTCAACCTCC

CTCAAACCCT

CAACTCAGCG

TACTTTGGAG

CACTCTGACC

TCCTTCTAAC

ACAACTACTC

CCACCATCAA

GAGTTCTGCA

CTCAGGCCTT

TCCTCACAGA

TATTTATTTA

AGTGTGGGAJ\

CACCTGGCCT

CTAACCCAAT

CTCTGCTCCC

ATCGCTTAAA

GACAGCAAGG

CCAGAAAAGC

CCAC-ATCTCC

TGGCAGAAGA

GTCTCAGCCT

ACTTCGGGGT

GTTCTATGGC

TAGCCCACGT

CCAACGCCCT

ATGGGTTGTA

TGCTCCTCAC

TCTCTGCCGT

GGTATGAGCC

CTGAGGTCAA

TCATTGCTCT

CCTTTACTCT

TTAGAAAGGG

AGAAACACAA

GGACTCAAAT

AAGGGAGAGT

CCTACCTTCA

GCCAGCCCCC

TTTGCTTATG

GCTGTCTTCA

CTCTACCTTG

TGTCTTAATA

CACGGGAGCC

ATAAAAAAC

GACTAGCCAG

AAGCAGCCAA

CTCCAGAAAA

GGCACTCCCC

CTTCTCATTC

GATCGGTCCC

CCAGACCCTC

CGTAGCAAAC

CCTGGCCAAC

CCTTGTCTAC

CCACACCGTC

CAAGAGCCCC

CATATACCTG

TCTGCCCAAG

GTGAAGGGAA

GACCCCTTTA

GATTATGGCT

GATGCTGGGA

GGGCTTTCCG

GGTCAGGTTG

GACCTTTCCA

CTCTATTTAT

AATGTATTTA

GACAGACATG

TTGCCTCCTC

ACGCTGATTT

GTGACTGTAA

CC=c

GAGGGAGAAC

CCAGGCAGGT

GACACCATGA

CAAAAGATGG

CTGCTTGTGG

CAAAGGGATG

ACACTCAGAT

CACCAAGTGG

GGCATGGATC

TCCCAGGTTC

AGCCGATTTG

TGCCCCAAGG

GGAGGAGTCT

TACTTAGACT

TGGGTGTTCA

TTGTCTACTC

CAGAGTCCAA

CAGTGACCTG

AATTCACTGG

CCTCTGTCTC

GACTCTTCCC

ATTTGCACTT

TTTGGAAGGC

TTTTCTGTGA

TTTTGCTTAT

GGTGACCAGG

TTGCCCTACA

AGAAACTCCA

TCTGTCCCTT

GCACAGAAAG

GGGGCTTCCA

CAGGGGCCAC

AGAAGTTCCC

CATCTTCTCA

AGGAGCAGCT

TCAAAGACAA

TCTTCAAGGG

CTATCTCATA

ACACCCCTGA

TCCAGCTGGA

TTGCGGAGTC

TCCATTCTCT

CTCAGAGCCC

CTCTGTGCTC

GACTGTGGGC

AGCCTCGAAT

AGAATGAGGC

TGAGGTGCAA

ATTATTTATT

CGGGGTGTCC

AAACGGAGCT

GTTTAAAACA

CTGTCGCTAC

GTCAATTGAG

GAACATCCTG

TCACTCACTG

CATGATCCGC

GAAC'rCCAGG

CACGCTCTTC

AAATGGCCTC

AAATTCGAGT

GGAGTGGCTG

CCAACTAGTG

ACAAGGCTGC

CCAGGAGAAA

GGGGGCTGAG

GAAGGGGGAC

CGGGCAGGTC

ACCCAGCCCC

CCAGTCTGTG

AGAGCTTTCA

CTCTCATGCA

GTCCATTCCT

TGGATAAGAT

TGCACAGCCT

ATTTATTTAT

TGGAGGACCC

GAGCTGTCCC

PAATATTTAT

ATCACTGAAC

ELGAAATAA.c 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1644 INFORMATION FOR SEQ ID NO: 13: Wi SEQUENCE

CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

1890 base pairs nucleic acid single linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

AAACAGAGAG

ATAAGAGAGA

CCTCAGCCTC

CAGCTGCCAT

GTGCCAGCTC

GAAGGCCTGG

CGCCGCCGCC

GCACAGGCCT

GCCTGGGGAT

CCAGCCAGAT

CTGAAAAAAA

TGCCTCTGGA

AGGGTGGCCT

GGGGTCAATC

AGATAGAGAA

TCCAGCTTGC

TACAGGACTG

GCAGCAGCCC

TCCCTGGGCC

TCAAAGGAGG

ACCACTGCCT

GTGTCTCCTT

GTTTCAGCTC

GCACACAGCA

GGAGCTGAGG

ATGGGAAGAC

TGTGATCAAT

TTGCAACAAC

AGAGAAAGAC

CTCCTCTTGA

AGAAGAAGTA

TTCAATTACC

CCTCCAGGCA

CCACCACCAC

CCACTACCGC

GTGATGTTTT

TTCCACCTAC

TCATCTTTGG

AAAGTGGCCC

ACCTATGGAA

GAAACTGGGC

CTGCCCCTGA

AGAGGTGTTT

GCAGTCAGCA

AAACCGTTTG

CATATCCCCA

CAGTTCTTCC

CACCGCCACC

TGCCACCCCT

TCATGGTTCT

AGAAGGAGCT

AGAAGCAAAT

ATTTAACAGG

TTGTCCTGCT

TGTACTTTGT

GCCACAAGGT

CCCTTAGCTA

ACAGGGTCCC

CTGGGGCTGG

GATCTACTGG

CTGTCCAACC

GCCACCACTA

GAAGAAGAGA

GGTTGCCTTG

GGCAGAACTC

AGGCCACCCC

CAAGTCCAAC

TTCTGGAGTG

ATATTCCAAA

CTACATGAGG

TGGAAACTCT

GTCCTTGACA

CCTGACTCAC

GTGGACAGCA

TCTGTGCCCA

CCACCTCCGC

GGGAACCACA

GTAGGATTGG

CGAGAGTCTA

AGTCCACCCC

TCAAGGTCCA

AAGTATAAGA

GTATACTTCa-

AACTCTAAGT

120 180 240 300 360 420 480 540 600 660 720 780 840 113

ATCCCCAGGA

TGTGGGCCCG

ATGTCAACGT

TATATAAGCT

ACCGAGAATG

CATGAACCAA

CTAATGTTTA

GCCATGTGAA

GTGCCTGAGA

GCCTTGCTAC

GGATGGAAAA

GCCCCACTGG

GATAAGCAAG

AGTGCCAATT

TGTAGATATT

TGCTTCCTGA

TGTTTTCCTA

GTAATAAAAA

TCTGGTGATG

CAGCAGCTAC

ATCTGAGCTC

CTAAGAGAAG

TTGTATTCAG

GTGGACCTTG

TGAGCCAGAC

GAGGGAGAAG

GTATTTAGGC

CTCAAGGGGG

GGAAGACTAG

CAGCATCTTC

AGAGATGTT

GTAGGGGTGT

GTGAAGTACA

CAATCAACTC

TAATATAATA

GTATATGTTA

ATGGAGGGGA

CTGGGGGCAG

TCTCTGGTCA

CACTTTGGGA

TGAGGGTCTT

AGACCACAGG

AAATGGAGGA

CATGAAAAAG

AGATTGAAAA

ACTGTCTTTC

AGGCTTGCAT

ACTTCTAAAT

TGGGGACTCA

GTGTGTGTGT

TATTAGGAAA

TAATAGTGCT

AATATTTATG

GGATACAAAT

AGATGATGAG

TGTTCAATCT

ATTTTGAGGA

TTCTTTCCAT

CTTACATGCA

GTTCAAAATG

ATATGACGGA

CAGCTACCCA

GGACACCTTT

AGATACATGG

AATAAGCTAA

GCATATCCTG

TTTCATTCCT

GTGTGTGTGT

ATATGGGTTG

TAAAAATCAT

TAGATGTGCA

CTACTGCACT

TACCAGTGCT

ATCTCAGACG

TATGATTCTT

TTTGAGGTCA

TCTGTAGCTC

AGAACATAGA

GGTGTTCTAC

TAACTCACCT

TTGTGACCTG.

AGAGGCTGAA

AGCCATCGGT

AACACAG CAT

GTGTATGACT

CATTTGGTCA

TGATTGTCAG

TTTTTGTGAA

ACTGGGCAGA

GATCATTTAT

TTTTTCGGCT

TGTTACAGGC

AGTAAGAAGA

CTCAACTCAC

ACTCTGGGCT

ACTCATCTTA

CTCAAGGTGG

AGGATTTAAG

AGAGGCCAAT

GAAACTAACA

GTGTATTTCC

AAAGAGAGAA

AGATTTTGAA

CTACTAATGA

ATGAAAACAT

900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1890 INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS:

C

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

1541 base pairs nucleic acid single linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: GGGTGTCTCA CAGAGAAGCA AAGAGAAGAG

GGAAACTTTA

GACACCTGAG

ATGCAGCAGC

TCATCTTGGG

GACCAAAGGA

CCACTCCCAC

CTACCGGTGG

CAGCTCTTCC

AAAGTATCAT

CCGAGGAGTG

GAAGACACAT

ATCAACGAAA

AACAACCAGC

GTGCTAATGG

AGCTACCTGG

CAACTCTCTC

AAGAAAAAGC

TGAATGAGAG

CTCATGACCG

GTCCTTAATG

CTTTGGGCTG

CCAAGAGAAT

TTACTACCTC

AAAGGAGGCT

CCATCTTTAC

TAAAGAAAAC

TCTCCTCCAC

CCATGAATTA

CTCCTCCAGG

GACCGCCACC

TGCCGCCACT

TATTTTTCAT

ACCTGCAGAA

CTTTTGAAAA

TGGCCCATTT

ATGGAACCGC

CTGGGTTGTA

CCCTAAACCA

AGGAGAAGAG

GGGCAGTATT

TGATCAATTT

ATTTTAAAAT

TCTTCTTAAG

CAAGGTCCAA

CCTGCATCAT

CTGTGTAGCA

TTTAACCATT

ACAGAGGCCG

CATTTGACTG

TGTTACCTGA

TTAGCTTCTC

AAGGCTGTGA

CCCATGTCCC

GTCAGTTTTT

TCCACCACCA

GACCCCTCTA

GGTTCTGGTG

GGAACTGGCA

GCAAATAGCC

AACAGGGAAC

TCTGATCTCT

CTTCGTGTAT

CAAGGTCTAT

GTTGAACTAC

CAATCTTACC

TGAGGAATCT

GATCTACTAT

ACCTATTGAG

CAGGTCAGCT

GAGCCAGATG

ATGCAGAGGC

GAAGAAGACA

TTTTTGAGAC

ATAAGCTAGA

TGTTTTCTGA

AACAGGAGAA

TGGAGCAGTC

GAAGGAAACC

CAGATCTTCT

CCCTGTCCAT

CCTGTGTCAC

AAGAAGAAGG

GCTCTGGTTG

GAACTCCGTG

AACCCCAGTA

CCCCACTCAA

GGAGTGAAGT

TCCAAAGTAT

ATGAGGAACT

TGCACTACTG

AGTGCTGAcC

AAGACCTTTT

TCTTTATC-AT

ATTAATTAAG

ATCCTTCATT

GAAGGAGGTC

ACAGAGAAAG

CCTTTACACT

ATAGTTGTGG

GACTGAAAAA

GCCGACCTTT

ATGGTGTTTC

AGCGTCAGAG

CTTTCCTGGG

GGGTAGACAG

CTTGTGGGCC

CACTACCACC

ACCACAACAC

GAATGGGATT

AGTTCACCAA

CACCCTCTGA

GGTCCATCCC

ATAAGAAAGG

ACTTCCGGGG

CTAAGTATcC

GCCAGATATG

ATTTATATGT

TCGGCTTGTA

GGGCACCAGG

ACTACATGAG

TTCTCGAGGT

TGTGACTGAG

AACTGTCTGA

CACTTCCAGG

TATGAATATA

AAGACAGTGT

G

CCTTGACTGC

TTCTGTCCTT

GCTGGGTGCC

CAGTGCCACT

TAGAGGGCCG

GCCATCACAA

AAATCTGTG

AGGAATGTAT

CCAAAGCCTT

AAAAAAAGAG

TCTGGAATGG

TGGCCTTGTG

TCAGTCTTGC

TGAGGATCTG

GGCCCACAGC

CAACATATCT

TAAGCTTTAA

AATATTGTCT

CCACAAAGAC

CCATGGAGTG

GGACATAAAG

TGTTAAATGG

GTGGGTCTAC

CAAGGGTGAG

CTCATTGGCA

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1541 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

888 base pairs nucleic acid single linear 114 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:

GGCTGGTCCC

GTTTCCTTCC

AGGTGATCGC

ATGGGTGCGT

TGGTGTGCAT

GGGACGTAGC

GGCAGGGGGG

AGCTACGTAT

GCTCCTCCAC

CCGCCTCCCG

CCCAGCGCCT

TTTTGCCTTC

CACTGCTGCT

AGTGTACACA

AGGACAAGAG

CTGACAGGTT

TTCCTTCTCG

CGCGGCGATG

CCTGCGGGCT

CCAGCGCTTC

TGAGCTGCAG

CCCAGC-ACTG

CCATCGTGAT

GACGGCCTCC

TAGCATCAGC

GACGCCCCTG

CCGAAACACT

GATTAGGGTT

CAGGGGCCAC

AAGGCATTGA

GAAGCAAGTA

GCAGCGCTCC

CCGGAGGAGG

GCTTTGGTCC

GCACAGGCTC

CTGAATCACA

GGCCGCTCCT

GGC-ATCTACA

AGGCACCACC

CTGCTGCGTC

GCCCGAGGGG

GATGAGACCT

TTTTAAATTT

CCGGGGTTGG

GCTTTTTCTT

GACGCCCAGG

GCGCCCCCAT

GTTCGGGCTG

CATTGGTCGC

AGCAGCAGCT

CAGGACCTCA

TCC'TGCATGG

TGGTACAC-AT

CCACCACCCT

TCAGCTTCCA

ACACACTCTG

TCTTTGGAGT

TATTTTATTT

GGTGGGAGTG

T.CATTTTCCT

AGCCCCGGGA

CGCCCCTCCT

CTCGGTGCGG

GGGCTTGGTG

GCCGCTCGAG

GC-AGGACCCC

ACCAGAGCTG

CCAGGTGACG

GGCCGTGGGA

CCAAGGTTGT

CACCAACCTC

GCAGTGGGTG

TATTTAAGTT

TGGTGGGGGG

ATTAAAAA

GGGGGCTGCA

GCGCTAGCGG

CGCAGGCCCT

ATCTGCCTCG

TCACTTGGGT

AGGCTATACT

GACAAGGGGC

CTGGCCATCT

ATCTGCTCTC

ACCATTGCCT

ACTGGGACAC

CGCCCCTGAC

CAAGAGAAAA

TAGTGGTGGC

120 180 240 300 360 420 480 540 600 660 720 780 840 888 INFORMATION FOR SEQ ID NO: 16: SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

1906 base pairs nucleic acid single linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

CCAAGTCACA

AACTGAATCA

CCAGGGCTGC

CCGGCGGGCT

ACAGCCACTC

CAGAGGACGG

TCAGAAGACC

CAAGTGGCAA

GGAGTCAGAT

TGCCAACTGC

CTCATCAACA

ACGAAACACG

ACCATATCAG

AATGTGTTGT

GAAGAAAGCG

AACTTTAGAC

TGGAAAGATA

AGGCAGGGAA

GGATGTCCCA

GAGCCTCCAG

AAATGAAGGG

TCTGCAGATG

AACTTCCCTA

ACTGTCACTC

CCCCAGGGAA

TACACCACCA

GGCATAATGA

GTCAGACAGA

TCAATTCACT

TCCAGTCTGC

TAGAACCAGA

TTGTATACAA

TGATTCAGGA

GATGAAGAGA

AGCAAGCACT

CCGTGGCCAG

TGGCTCTGTG

ACTCCATTCC

TCTTATGTAT

AGCATCTAAA

ATCAGGATGG

AGTTTCTTGT

AGCATATCAA

TATACCAGAA

TCAATGTGGA

CCATCTTCTT

CCTCTCTACC

CAAGACAAAC

CAGCTCCAGG

GCAATGTAGT

GCCCAACCAC

TTAAAATGGG

GAGCTGGGTG

GCCTAATGCT

TACTGGCAGA

TGGCACTAGG

AACCAGGCTC

CAATGGAAAA

GTCTGAAGGG

GACAGCAAGA

GGCAAATGC C

TAGGCAGGAA

AACCCATATC

AATGGTTGCC

TTCAGGGGGA GAATCCTTCT GATAAGGTGT GATGTGGGGA CAACGGAATG GCCCCTCCTG CCACCTGGGG ACCACGAGCC CCTTGTCTTC ACGGTGGCCA CAACTCACCT GACAACGTCC CCTGAAAAGA GCTCCATTCA CAAAACCAAG TTGTCTTGGA GAATCTGGTG ATCCAATTCC ACAATGCCCA AATAATTCTG AAAACAGGCC CTGGTGACAG TCTCTCTCAA TTCTTGCTGG TACATTCCAG TACATAGATA ATACAGTAAT TCAGACTGAA ATACAGTATT TCATCCCTCC TACACAOGGT ATTAAATAGT GTTAAAAAGA GAGTTTTTAG GTGGTGGGCA GAGCCCCACA TAATTCACTG TATGGTCTTG GCTTCAGTAC CAGAGCAGCT TGAGTGTTTA CACTGTGCCC TAGTTGTCCA AGTCGCGATC TACACTTGTG ACTGAACCAT AGGCTGATCT TGTACTCCAT GGACAGCCCC CTGTTCCTGA CAAGTTCAAA

GACTTTTACT

CAGTCCTCCT TCTCCAGGTT CAGTTAACAA CGTAGGTAAA TGAATGGTCT GTCTGAAGGA AGATGCCTCT AAGTTCTTGT AAGGGTGACT AAGCCCGGCT AAGGCAACAT AAAATTATAA

TGGAACAGAG

AGACTATATA

GAGACACAGC

GCAGCTATTT

CTATTATGGT

CCCTCAAAGG

AGAAGTCATG

ACAAAGATGG

CTGGTTTGTA

TCGATCTGAA

TGTGTGAGTC

ATTACCTGCA

CAAGCACCTT

CAGTTTCTCT

AAACACTTGG

ATACTTCTCC

TGAAGTATCT

CAGAATCAGA

ATCTATTTCT

AGCAACTCTG

TTCACGGGAT

AAGGACTCTC

GCCCAGTTTA

ATGACCCCAC

GATGGAAAGC

TACAGATCCT

ACATGAGGCA

GAAATAGGGT

PGCAACAGAG

CTCTGGCCAG

TCCGGTATGA

GAATTC

ATGGGCCCAG

AAGAATGGAC

CATGCATGTG

CTATTTGACC

GTTGGTCGTT

AGGAAATTGC

GGCCTACCTC

CATTCTCCAT

CTTCATCATT

GTTGGAGCTT

TGGAATGCAA

GGTCAACACC

TCCTCTTGAG

TGGCCTTCAG

GCAAAAAGAA

TTCTGTCTCT

TTCAGATAGC

AGGGATGAAT

TCTGTTTTGA

CCCTAATGGG

ACTTCTTTTA

TCACACAGGA

TGCCTGTCTG

CCCTAGGAAC

ACAAATTTAA

GGACAGAAAG

GGAATAAGAA

GTGGTCACTC

AAGTGGGGAA

AGGTGTGGTA

GAAATTAAAc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1906 115 INFORMATION FOR SEQ ID NO: 17: SEQUENCE

CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

1619 base pairs nucleic acid single linear SEQUENCE DESCRIPTION: SEQ ID NO: 17:

C

C

C C

C

GTCATGGAAT

CGCGCTCGCG

CTGCTCGCTG

GCCTCGCCCG

GATCCCGCCG

GTTCTGCTGA

CTGACGGGGG

GTCTACTATG

TCCGTTTCAC

GCTTTGACCG

CAGGGCCGCT

GCCAGGGCAC

GTGACCCCCG

GTGCAGCCCA

TGGGTCCCTG

CCCTTGAGGA

CCTGAGCTCA

GATCCTGAGT

ATTGCCGAGG

GCAACTGGGA

GAGCATCTGT

TAGACTGAAA

CCTCCACCTC

TGGTCTTGTT

CCAGCCTCGA

ACCACAGTTG

GCTATGTTGC

ACGCCTCTGA

CGCTTCACTG

CCTGCCGCGT

ACTGCCTTGG

CCGCCTGCGC

CGTCTTCCTC

GCTCCGCGGC CAGCCCGAGA GCCTCTTGGA CCTGCGGCAG TCGATGGGCC

CCTGAGCTGG

GCCTGAGCTA CAAAGAGGAC TCTTCTTTCA

ACTAGAGCTG

TTGCGCTGCA CCTGCAGCCA TGGACCTGCC ACCCGCCTCC TGCTGCACCT

GAGTGCCGGC

GCCATGCCTG GCAGCTTACC AAATCCCAGC CGGACTCCCT CCTGGACAGA GTCCGAATCC CTGCTTTCTC TACCTCAAGG CCCTCCTCAC

CCACTCCTTC

GATAATATAT

TATATATATT

TTGTGAATGG

ACTTTTTTAG

CTGGTCTTGA ACTCCTGGAC TTCATCCTTT CTATTAATTC kATGTGCCAG CATTGTGCCC GAAAATCTGA GTTATGGTAA CTGTGTGATA CTTGGGGGCT CTGTCAACCA GGCTAGAATG :CTCCCGAGG CTCAGGTGAT rGTGCCACCA CACTTGGCTA ZAAGGTTGTT TACATGCCAG

GACCCCGAAG

GCCCTGGTCG

GCCTGCCCCT

CTCCGCGAGG

GGCATGTTTG

TACAGTGACC

ACGAAGGAGC

CGGCGCGTGG

CTGCGCTCTG

TCCGAGGCTC

CAGCGCCTGG

CAGGGCGCCA

TCACCGAGGT

TACTCCATCC

GGCTTGGCAG

CCCAAGTTGG

ATATATATAT

AGGAGTTGTT

TTAGACGATC

ATTGTACTTA

AGGCTAGGGG

TACGTGAGGA

AGCTTTTTTC

CAGCGGTGCA

CCTCCCATCT

ACTTTTTAAT

TACAATTTAT

CCCCGTGGCC

CGGGGCTGCT

GGGCCGTGTC

GTCCCGAGCT

CGCAGCTGGT

CAGGCCTGGC

TGGTGGTGGC

TGGCCGGCGA

CTGCTGGGGC

GGAACTCGGC

GCGTCCATCT

CAGTCTTGGG

CGGAATAACG

TTCATGGAGA

GGGTCCCTGC

ACCTTGATAT

ATATATTTCT

TTGGGGGGGG

CTCCTGCCTC

TTTGCCTATT

GCTATAGAAA

ATTTAAAGAC

rTTCTTTCTT ATCATGAGTC2 CAGCCTCTCG2 1'TTTTTGCGG kATAAACACT

TCCCGCGCCC

GCTGCTGCTG

CGGGGCTCGC

TTCGCCCGAC

GGCCCAAAAT

AGGCGTGTCC

CAAGGCTGGA

GGGCTCAGGC

CGCCGCCCTG

CTTCGGTTTC

TCACACTGAG

ACTCTTCCGG

CCCAGCCTGG

CCCCTGGTGC

TGCTGACCTC

TTATTCTGAG

ATTTAAAGAG

GGTCTTCGAC

AGCCTCCCAA

TGTGTGTATT

CATCTAGAAA

rCATCCCCAG rTTTTTGAGA

CLATGCAGCCT

!LGTAGCTGGG

kGACGGTATT

'ATTTTTCC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1619 INFORMATION FOR SEQ ID NO: 18: SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

1239 base pairs nucleic acid single linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: AG CCTATAAA

GCACACACTT

CTCGGATGCG

TACTGTGCGC

TCGGGATCGC

CTATGGCCTA

CCGCACCGAG

GAATAATGCA

GGGCTCTCCT

TCTGAACTGG

CGAAGAAGAC

ACTGAAGCTC

TGTTTTGCAA

GTTCCCTTGC

GAAGGCTGGC

GCACGGGCAC

GATGTGGAGG

GCGCTCCTCA

CCCACAAATG

GAGGCCGCGT

GTCGCTTTGG

CCTCGGCCAG

GACCAGGTCA

GTGTTCGCCA

CACAGCCAAG

AAAAAGGAGT

AGTCCAACAT

GCAAAGCCTC

TCCATGGAGA

CACCGCCTCA

TGGCGGGAGA

CGTGCACTGA

ATACCGCGGA TGCCAGACAT GAGATACCGG

GCTCCTCGCG

CCGCGCTCCC

CACGGATGCT

GGCCGCCTGC ACTGAACT'rC TTTTGCTGCT

TCTGATCGCC

CGCTCACAAT

CACCACCTCG

CCCCTGTTTC

CCACATTGGC

AGCTACTGGC TAAAAACCAA ATGGAGCTGG GAGCTCATAC TGGTGGTAGA

CAGTCCCGGG

TCACAAACAC

AGGCCACAAG

AGGTAGATGA

CTTTGACAAC

ACAAGTTAGT GGACCGTTCC GTGTGGGTCT

GAGGGCTTAT

CCGACCGTGG

CCAGCAGGTA

GACGCTGCGC

GCCTACCCTG

TGTTCCCGCC

GCCTGTGTTC

CCCAACCTGG

TGCCCCAACA

GCATCGTTGT

CTATCTCAAG

CTCTACTACG

GTGCAGGGCT

TTGGCCCTGA

TGGAGTCAAC

CTGCATGGAG

TAATGGACCA

CTTCGTGCCC

TCCTCTCAGA

CGGTTAATGT

ACCCAAAGCT

CTATCTTCAC

GTACCCGAGA

CTACACAACA

GCAATACAAC

GTCTGAGGTA

TATTTTTGGA

GGGTCTCTCT

CAGTGGAACT

TGTTGCTCCT

CCCAGGATGC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 116

ATACAGAGAC

ACCCGACAAC

AAGCTGCTAT

AACTGGACTT

TAGCATTAGT

TATTTATATT

TGGGAGCTGT

CCATGGGAAT

GTTTTATGGG

GGTATTTATT

AAACTGCTGG

AATATATAAA

CTTATCCCAA

GAGAACTATC

GTCTGAGCAG

CTGAGCATAG

GCAGCTGCTA

AATAAATGTG

CACCACCAGC

CTTCTTGTGA

GGGTCCCTTC

CTC-AGACAAG

GATAAAAAAA

TTTGTAAAT

TTTGGACTCT

CTCCTAGTTG

CATGACTTTC

ACTTTATATA

AATTTCTAAA

TTCTTGTGAA

CTAAGTCCTC

TCTTGTCTTT

ATTCACTAGA

TCAAAGTTTA

960 1020 1080 1140 1200 1239 INFORMATION FOR SEQ ID NO: 19: SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

606 base pairs nucleic acid single linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

C

C

ATGATCGAAA

ATGAAAATTT

CTTTTTGCTG

CAAATTGCGG

GCTGAAAAAG

CTGACCGTTA

CGGGAAGCTT

TTCGAGAGAA

CAATCCATTC

GTGACTGATC

CTCTGA

CATACAACCA

TTATGTATTT

TGTATCGCTT

CACATGTCAT

GATACTACAC

AAAGACAAGG

CGAGTCAAGC

TCTTACTCAG

ACTTGGGAGG

CAAGCCAAGT

AACTTCTCCC

ACTTACTGTT

CGCACAGGCT

AAGTGAGGCC

CATGAGCAAC

ACTCTATTAT

TCCATTTATA

AGCTGCAAAT

AGTATTTGAA

GAG CCATGG C

CGATCTGCGG

TTTCTTATCA

TTTGAAATGC

AGCAGTAAAA

AACTTGGTAA

ATCTATGCCC

GCCAGCCTCT

ACCCACAGTT

TTGCAACCAG

ACTGGCTTCA

CCACTGGACT

CCCAGATGAT

AAAAAGGTGA

CAACATCTGT

CCCTGGAAAA

AAGTCACCTT

GCCTAAAGTC

CCGCCAAACC

GTGCTTCGGT

CGTCCTTTGG

GCCCATCAGC

TGGGTCAGCA

TCAGAATCCT

GTTACAGTGG

TGGGAAACAG

CTGTTCCAAT

CCCCGGTAGA

TTGCGGGCAA

GTTTGTCAAT

CTTACTCAAA

120 180 240 300 360 420 480 540 600 INFORMATION FOR SEQ ID NO: Wi SEQUENCE CHARACTERISTICS:

C

C

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

783 base pairs nucleic acid single linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:

ATGATCGAAA

ATGAAAATTT

CTTTTTGCTG

GAAGATTTTG

TTGCTGAACT

AACAAAGAAG

ATTGCGGCAC

GAAAAAGGAT

ACCGTTAAAA

GAAGCTTCGA

GAGAGAATCT

TCCATTCACT

ACTGATCCAA

TGA

CATACAACCA

TTATGTATTT

TGTATCTTCA

TATTCATAAA

GTGAGGAGAT

AGAAAAAAGA

ATGTCATAAG

ACTACACCAT

GACAAGGACT

GTCAAGCTCC

TACTCAGAGC

TGGGAGGAGT

GCCAAGTGAG

AACTTCTCCC

ACTTACTGTT

TAGAAGATTG

AAAGCTAAAG

GAGAAGGCAA

AAACAGCTTT

TGAGGCCAGC

GAGCAACAAC

CTATTATATC

ATTTATAGCC

TGCAAATACC

ATTTGAATTG

CCATGGCACT

CGATCTGCGG

TTTCTTATCA

GATAAGGTCG

AGATGCAACA

TTTGAAGACC

GAAATGCAAA

AGTAAAACAA

TTGGTAACCC

TATGCCCAAG

AGCCTCTGCC

CAC-AGTTCCG

CAACCAGGTG

GGCTTCACGT

CCACTGGACT

CCCAGATGAT

AAGAGGAAGT

AAGGAGAAGG

TTGTCAAGGA

AAGGTGATCA

CATCTGTGTT

TGGAAAATGG

TCACCTTCTG

TAAAGTCCCC

CCAAACCTTG

CTTCGGTGTT

CCTTTGGCTT

GCCCATCAGC

TGGGTCAGCA

AAACCTTCAT

ATCTTTATCC

TATAACGTTA

GAATCCTCAA

ACAGTGGGCT

GAAACAGCTG

TTCCAATCGG

CGGTAGATTC

CGGGCAACAA

TGTCAATGTG

ACTCAAACTC

120 180 240 300 360 420 480 540 600 660 720 780 INFORMATION FOR SEQ ID NO: 21: i)SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

558 base pairs nucleic acid single 117

TOPOLOGY:

linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

CTGCTGCACT

ATCAACAGCC

GTAGCCCACG

GCCAATGCCC

GACGGGCTTT

CCCTTGTTCC

ATCCTGTCTG

CCCTGGTACG

AGTGCTGAGA

GGGATCATTG

TCGGGGTAAT

CTCTGGTTCA

TTGTAGCCGA

TCATGGCCAA

ACCTCATCTA

TCACCCACAC

CCATCAAGAG

AACCCATCTA

TCAACCTGCC

CCCTGTGA

CGGCCCCCAG

AACACTCAGG

CATCAACTCT

CGGTGTGAAG

CTCACAGGTC

CATCAGCCGC

CCCTTGCCAC

CCAGGGAGGA

GGACTACCTG

AGGGAAGAGC

TCCTCTTCTC

CCGGGGCAGC

CTGGAAGACA

CTCTTCAGGG

ATTGCAGTCT

AGGGAGACCC

GTCTTCCAGC

GACTATGCCG

AGTCCCCAGG

AAGCCTCAAG

TCCGGTGGTG

ACCAGCTGGT

GCCA.AGGCTG

CCTACCAGAC

CAGAGTGGGC

TGGAGAAGGG

AGTCCGGGCA

TGGCCCCTCC

TAACAAGCCG

GGACTCGTAT

GGTGCCTGCT

CCCTTCCACC

CAAGGTCAAC

TGAGGCCAAG

AGATCGCCTC

GGTCTACTTT

120 180 240 300 360 420 480 540 558 INFORMATION FOR SEQ ID NO: 22: SEQUENCE

CHARACTERISTICS:

S.

0

S.

*5 S 0 LENGTH: 1783 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: S. Si

S

**bS

S.

S S 0ee 0 5*500S

S

CAAGTCACAT

CCGAATCAGA

AGGGCTGCAA

GCCCGGCTCG

CTACTTCTAC

GGTACTGGTA

AGGAGGAAAT

ATGGGCCTAC

TGGCACCATC

GTACTTCATC

GACATTGCAG

GTCTGGAGTT

ACAGGTCAAC

TTTCCCTC TT

TCTTAACCTT

AAAGAAAATA

CTTTGGAAAA

GAAAGAATGT

CAGTATAACC

CTGAATCTGG

AGACGAGACT

ATGATGGTAC

CTGCCTrGTT

GAGGGGAGTA

CATTCTGTGT

GTGGGAAGAG

AGCTTGGTCT

AAACCATTTG

GTTCTAGTGC

AAAGTTCCAG

GATCCAGGAT

TGAGGAGAGA

CAAGCAGGCA

GTAGCCAGCC

CTCAGCACCA

GTCCAGAAAA

TGCTCAGAGG

CTCCAAGTGT

CACGGACTCA

GTTTGCCAAC

CTCCTCATCA

CAGAGTAAGA

TCTACCATAT

GATAATGTGC

TATGAAAATG

TTAGGACAAC

ATACAAACCC

GATATGTGCA

ACTAATTCAC

AAAAGTAGGA

GTGGAGCTGA

CAGTAATGTC

GAAAGATACT

GCTGCTGCCA

GCAGTTTTTA

CTTCTGGACA

TAGACCCATC

AGTTGATGTC

TGTGTCAGAG(

AAAAAGGCTA

GCAGGGGAAJ

TAAGGTGTGI

GCTGTGGGGC

CCTGGAGAAC

CCGCACTGG]

AGGACTCCAC

ATCTCTTCTG

CAAAGCATCI

TATACCAGGA

TGCAGTTCCT

ATTCCAAGAx

ACATCTACCA

CAGTCAGGGT

TATCCGTCTT

CTGTCTACCA

TCAAACTAAG

AGGATTTAGA

CAGAGACCTA

TGACCTTGAG

GATGAGATTT

GCTCCCTACA

TGTCTGACTG

CTAACCTCTT

rCTCCTGAGA k.GTATTCCCC

.ICAAACATTA

kAGCTTGAGG rGGGCCCCTG 3ATGGAGTAA 2AAAATGTTT ~ATCCTTCTTG

GAACAGAGCT

~TGTGGGACAG

ACTATATAAA

TCCTTCCCCT GACCCAGCCA CACGAGGCCC

TGGAGAAGCA

GTGCCTTGTT

GTGGCAGTGG

TCCAAATACA

ACTGAGAAGG

TACCCTGAAA AGTACTCCAT CAACAATACC

AAACTGTCAT

CGGGAACCTG

ATAGTCCAAT

CGTGCAGTGC

TCAAATCATT

CAAAAAGCAG ACGTTGGTAA GAATCTCTCT

CAGTTTTTGC

GGATAATTTC

CAGTATGTGG

CTTATATAGT

AGCTCAGACT

TACAGTACTT

CATCTGTCCA

CATGTGAGTT

AGTGCACTTC

AAGTGGAGTC

TCCTTCAGAT

CTTGGGCACT

AGAAGGGGTG

CCATTTTTCC

TTCCCCCTGG

ACATTTCCCC

AATATTTTCT

CAGATGAAGG

CCTCCCATGG

TCATCTCAGC AAGTCCTAAG GTAATGGGCA

AAGTGATCCT

CATACATGGA GACATTTTCT CAAAAGTTCT

TGACAATGAG

ACAGCTTCTC

CTGACCAGAG

TTTCTACATT

GTGGGAGACA

GGAGTTCTCC

TTCAGTAAGG

AATAGACACT

TTTCTGAAGG

AAAAAGAAAA AAA

GGGTACAGAA~

GCATGGAGCC

TGCAGGTGCA

CAAGTCGCAG

CGATCATTCT

CCCCCCTTAA

CCAAGAAGTC

GGAACGAAGA

TCCCTGGCTT

CTGTGGACCT

CAGTGTGTGA

TGCATTACTT

ATACAAACAC

GAATAGTTGT

AACATGGGCC

TCTTTCTGTC

GCACAAACAG

TGAGTTGTCC

AACTTGGGGT

TCAACTCAGA

CATGAGGAAA

GACTTCCATG

GTCTCTCACT

GCCCAAATTC

AACTTTGAAT

ACACCATGCA

GACTTTTGAC

P.GAGCAAGCC

A.AAGGAGAAC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1783 INFORMATION FOR SEQ ID NO: 23: SEQUENCE

CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

1047 base pairs nucleic acidsingle linear 118 (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 23:

AGAGAGCGCT

AGGGGCTGGA

TACCAAGGGA

GTTCTACACT

CAATGTCCCT

TGACACTGCC

GGGGCAGACC

ACCCCGTGTG

CTTCGAGGTC

CCATCCCATC

GGA-TAAGACC

CATTAACAAC

CGTTGACAGC

TCACACCTTT

GATAGATACG

CGACACGCTC

GTAAATTATT

ATCATTATTT

GGGAGCCGGA

TGGCATGCTG

GCAGCTTTCT

CTGATCACAG

GCAGGACACA

CTTCGCAGAG

CGCAACATTA

CTGTTTAGCA

GGTGGTGCTG

TTCCACAGGG

ACCGCCACAG

AGTGTATTCA

GGGTGCCGGG

GTCAAGGCGC

GCCTGTGTGT

CCTCCCCCTG

TTAAATTATA

ATTAAATTTT

GGGGAGCGCA

GACCCAAGCT

ATCCTGGCCA

CTTTTCTGAT

CCATCCCCCA

CCCGCAGCGC

CTGTGGACCC

CCCAGCCTCC

CCCCCTTCAA

GCGAATTCTC

ACATCAAGGG

AACAGTACTT

GCATTGACTC

TGACCATGGA

GTGTGCTCAG

CCCCTTCTAC

AGGACTGCAT

TGGAAGC

GCGAGTTTTG

CAGCTCAGCG

CACTGAGGTG

CGGCATACAG

AGTCCACTGG

CCCGGCAGCG

CAGGCTGTTT

CCGTGAAGCT

CAGGACTCAC

GGTGTGTGAC

CAAGGAGGTG

TTTTGAGACC

AAAGCACTGG

TGGCAAGCAG

CAGGAAGGCT

ACTCTCCTGG

GGTAATTTAT

GCCAGTGGTC

TCCGGACCCA

CATAGCGTAA

GCGGAACCAC

ACTAAACTTC

GCGATAGCTG

AAAAAGCGGC

GCAGACACTC

AGGAGCAAGC

AGTGTCAGCG

ATGGTGTTGG

AAGTGCCGGG

AACTCATATT

GCTGCCTGGC

GTGAGAAGAG

GCCCCTCCCT

AGTTTATACA

GTGCAGTCCA

ATAACAGTTT

TGTCCATGTT

ACTCAGAGAG

AGCATTCCCT

CACGCGTGGC

GACTCCGTTC

AGGATCTGGA

GGTCATCATC

TGTGGGTTGG

GAGAGGTGAA

ACCCAAATCC

GTACCACGAC

GGTTTATCCG

CCTGACCTGC

ACCTCAACCT

GTTTTAAAGA

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1047 INFORMATION FOR SEQ ID NO: 24: SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

1176 base pairs nucleic acid single linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: GAGCGCCTGG AGCCGGAGGG GAGCGCATCG TCTCCCGGGC AGCTTTTTGG AAACTCCTAG TAGGCTCCC'r GGAGGTGGGA CACGGGCAGC TCCAGGGGGC TGGATGGCAT

GCTGGACCCA

GTTTTGCCAA GGACGCAGCT TTCTATACTG TGTTGTTCTA CACTCTGATC ACTGCGTTTT ATAGCAATGT CCCAGAAGGA GACTCTGTCC CCCTTGACAC AGCCCTCCGC

AGAGCCCGCA

TGACAGGGCA GACCCGCAAC

ATCACTGTAG

ACTCACCCCG TGTGCTGTTC

AGCACCCAGC

TAGACTTCCA GGCCCATGGT

ACAATCCCTT

CCACCCACCC AGTCTTCCAC ATGGGGGAGT TTGGAGATAA GACCACAGCC

ACAGACATCA

TGAACATTAA CAAC-AGTGTA

TTCAGACAGT

ATCCTGTTGA GAGTGGGTGC

CGGGGCATCG

CGACTCACAC CTTCGTcAAG

GCGTTGACAA

TCCGGATAGA CACAGCCTGT

GTGTGTGTGC

TTGCCTGCAG CCCCC'TCCC

CACCTGCCCC

CAGCCTGTAA ATTATTTTAA

ATTATAAGGA

TAAAGACATT ATTTATTAAA TTTTCAAAGC

AGTGACTTTG

TGAACATGCT

ATGGTGGAGT

AGCTCACCTC

GCCGCAGTGA

TGATCGGCGT

CTGAAGCCCA

GTGCCCCTAC

ACCCCAGACT

CTCCACCCAC

TCAACAGGAC

TCTCAGTGTG

AGGGCAAGGA

ACTTTTTTGA

ACTCCAAACA

CAGATGAGAA

TCAGCAGGAA

CTCCACACTC

CTGCATGATA

ATCCTG

GAG CTGGCCT

GTGCCTCAAG

TTTGGCCTGT

AGTGTCTGGG

GGTGCATAGC

ACAGGCAGAA

CTGGACTAAA

TGCACCAATA

GTTTAAGAAA

CTCTTCAGAC

TCACCGGAGC

TGACAGTGTC

GGTGACAGTG

GACCAAGTGC

CTGGAACTCA

GCAGGCTGCC

GGCTACAAGA

TCTTGGGCCC

ATTTATCGTT

TATATTTGGA

CCAGTGAAAT

GGTCGTGCAG

CCCAATAAAG

GTAATGTCCA

CCGTACACAG

CTTCAGCATT

GCTGCCCGAG

CGGAGACTCC

ACTCTGGATC

AAGCGCTCAT

AGTGTGTGGG

CTGGCCGAGG

CGAGCCTCCA

TACTGLJACCA

TGGAGGTTCA

AGAGGCTGAC

CTCCCTACCT

TATACAATTT

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1176 INFORMATION FOR SEQ ID NO: SEQUENCE

CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

1623 base pairs nucleic acid single linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 119 TCAGAGTCCT GTCCTTGACA

GGGGCTGGGT

C-AGCAGTGCC

GCCTAGAGGG

ACCGCCTTCC

AGAGCTGTGG

AGGAATGTAT

CCACAGCCTT

AACCAAAAAG

TCTGGAATGG

CGGCCTTGTG

TCAGTCTTGC

TGGGGATCTG

GGCCCACAGC

CAACATATCT

TAAGCTTTA-A

AATATTGTCT

AACAGGTCAG

ATGAGCCAGA

CAATGCAGAG

TTGAAGAAA.A

GGAGGCCGTC

AGGTTTCATG

TTTTAACTGA

GAGGCACAAG

TTTTTATGTA

GGAGTACATA

CTA

GCCATGCAGC

ACTTCTCCTT

CCAGGACAAA

CAACCACCCC

CTACCGGTGA

CAACTCTTTC

AGAGTATCAT

CCAAGGAGTG

GAAGACACAT

ATCAATGAGG

AACAGCCAGC

GTGCTAATGG

AGCTACCTAG

CAACTCTCTC

AGGAAAAAGC

TCAATGAGAG

CTACCCTTCA

TGGGAAGAAG

GCAAAGAGAA

AAAAAACCTT

TTTTAGACAC

GATAAGCTAG

TGTTTTCTGA

TCATTCTCTA

TGAGAGTAGA

TTAGAAACGT

CTTCAGTCTC

AGCCCGTGAA

GGGCTCCTCC

GGAGACCACC

CGCTGCCTCC

TATTTTTCAT

ATCTACAGAA

CTTTTGAAAA

TGGCCCACTT

ATGGAACTGC

CTGGGTTGTA

CCCTAAGCCA

AGGAGAAGAA

GGGC-AGTATT

TGATCAATTT

ATTTTAGAAT

TCTTCTTAAG

TTTTCTAGAG

ACTGTTCCTG

GGAACTGTCT

TAAACTCACC

ATGGTTGTGG3

AGACTGAAAA

GCCCACCTTT

C-ATAGCATGT

GCGATTCTAA~

ATGTGTTACA

*CACAAGACTG

*TTACCCATGT

AGGGTCAGTT

GCCTCCACCA

ACTAAGCCCT

GGTGCTGGTG

GGAACTGGCA

GCAAATAGCC

AACAGGGAAC

TTTGATCTCT

CTTCGTATAT

CAAGGTCTAT

GTTGAATTAC

TAATCTTACC

TGAGGAATCT

GATCTATTAT

ACCAATTGAG

GTCCATGGAG

AGGAACATAA

GATGTTAAAT

TTCCAGGGTG

TATGACTATA

AAGCCAGTGT

GATGCTAACA

GTACCTCCAG

AGAGTCACAT

TTTGATGCTA

AGAGGAGGAA

CCCCAGATCT

TTTTCTTGTC

CCACCTCCAT

CTAAAGAAGA

GCTCTGGTTG

GAACTCCGTG

AACCCCAGCA

CCCCGCTCAA

GGAGTGAAGT

TCCAAAGTAT

ATGAGGAACT

TGC-ACTACTG

GTTGCTGACC

AAGACCTTTT

TCTTTATCAT

CCACAAAGAC

TGGTCCTTAA

AGTTTTGGGC

GGCCAAGAGC

GGTCTACTTG

CAAGGGTGAG

CCCATTGGCA

GAGAAATAAG

rGCAATGATGI

GAGTACAACG

3AATATCTGA

ACCCTTTCCT

ACTGGGTAGA

CATCCTCTGG

C-ACCACTACC

AGGACAACAT

GAATGGGGTT

AGTTC-ACCAA

CACCCTCTGA

GGTCCATCcC

ATAAGAAAGG

ACTTCCGGGG

TTAAGTATCC

GCC-AGATArG

ATTTATATGT

TTGGCTTATA

GGATGCCAGG

CACAAGGTCC

rGCCTGCATC

TGCTGTGTGG

ATTTTAGCCA

CTACCTCACA

IAAGGATGCT

rCATCTTTAT kGGGGTGTTT rCTGTGTGTG

GTACATTAC

~TGTTTCTTG

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1623 INFORMATION FOR SEQ ID NO: 26: SEQUENCE

CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

28 base nucleic single linear pairs acid 2 6: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GTTAAGCTTT TcAGTCAGCA

TGATAGAA

INFORMATION FOR SEQ ID NO: 27: SEQUENCE

CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

27 base nucleic single linear pairs acid 27: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GTTTCTAGAT CAGAGTTTGA

GTAAGCC

INFORMATION FOR SEQ ID NO: 28: SEQUENCE

CHARACTERISTICS:

LENGTH:

LNGTH:30 base pairs 120

TYPE:

STRANDEDNESS:

TOPOLOGY:

nucleic acid single 1linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CCAAGACTAG TTAACACAGC ATGATCGAAA INFORMATION FOR SEQ ID NO: 29: SEQUENCE CHARACTERISTICS: 28:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

30 base nucleic single linear pairs acid 2 9: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CCAATGCGGC CGCACTCAGA ATTCAACCTG INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

972 base pairs nucleic acid single linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:

TCTAGACTCA

TGCCATGCAG

CAGCTCTCCC

GCCTGGTCAA

GCCGCCACCA

AGGCCTGTGT

GGGGATGTTT

CCAGATGCAC

AAAAAAGGAG

TCTGGAATGG

TGGCCTTGTG

TCAATCT'TGC

CCAGGATCTG

GGCCCGCAGC

CAACGTATCT

TAAGCTCTAA

AGATGTTCTA

GGACTGAGAA

CAGCCCTTCA

TGGGCCCCTC

AGGAGGCCAC

CTGCCTCCAC

CTCCTTGTGA

CAGCTCTTCC

ACAGCATCAT

CTGAGGAAAG

GAAGACACCT

ATCAATGAAA

AACAACCTGC

GTGATGATGG

AGCTACCTGG

GAGCTCTCTC

GAGAAGCACT

GA

GAAGTAAAAC

ATTACCCATA

CAGGCACAGT

CACCACCACC

TACCGCTGCC

TGTTTTTCAT

ACCTACAGAA

CTTTGGAGAA

TGGCCCATTT

ATGGAATTGT

CTGGGCTGTA

CCCTGAGCCA

AGGGGAAGAT

GGGCAGTGTT

TGGTCAATTT

TTGGGATTCT

CGTTTGCTGG

TCCCCAGATC

TCTTCCCTGT

GCCACCGCCA

ACCCCTGAAG

GGTTCTGGTT

GGAGCTGGCA

GCAAATAGGC

AACAGGCAAG

CCTGCTTTCT

CTTTGTATAT

CAAGGTCTAC

GATGAGCTAC

CAATCTTACC

TGAGGAATCT

TTCCATTATG

GGCTGGCCTG

TACTGGGTGG

CCAACCTCTG

CCACTACCAC

AAGAGAGGGA

GCCTTGGTAG

GAACTCCGAG

CACCCCAGTC

TCCAACTCAA

GGAGTGAAGT

TCCAAAGTAT

ATGAGGAACT

TGCACTACTG

AGTGCTGATC

CAGACGTTTT

ATTCTTTGTT

ACTCACCAGC

ACAGCAGTGC

TGCCCAGAAG

CTCCGCCGCC

ACCAcAGCAC

GATTGGGCCT

AGTCTACCAG

CACCCCCTGA

GGTCCATGCC

ATAAGAAGGG

ACTTCCGGGG

CTAAGTATCC

GGCAGATGTG

ATT'rATATGT

TCGGCTTATA

ACAGGCACCG

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 972 INFORMATION FOR SEQ ID NO: 31: Wi SEQUENCE CHARACTERISTICS: LENGTH: LNGTH:88S base pairs.

121

TYPE:

STRANDEDNESS:

TOPOLOGY:

nucleic acid single linear (xil) SEQUENCE DESCRIPTION: SEQ ID NO: 31: ATGCAGCAGC CCATGAATTA CCCATGTCCC TCATCTTGGG CTCCTCCAGG GTCAGTTTTT GACCAAAGGA GACCGCcACc TCCACCACCA CCACTCCCAC TGCCGCCACT GACCCCTCTA CTACCGGTGG TATTTTTCAT GGTTCTGGTG CAGCGTCTTCC -ACCTGCAGAA GGAACTGGCA AAAGTATCAT CTTTTGAAAA GCAAATAGCC CCGAGGAGTG TGGCCCATTT AACAGGGAAC GAAGACACAT ATGGAACCGC TCTGATCTCT ATCAACGAAG CTGGGTTGTA CTTCGTATAT AACAACcAGC CCCTAAACCA CAAGGTCTAT GTGCTAATGG AGGAGAAGAG GTTGAACTAC AGCTACCTGG GGGCAGTATT CAATCTTACC CAACTCTCTC TGATCAATTT TGAGGAATCT AAGAAAAAGC ATTTTAAAAT GATCTIACTAT

CAGATCTTCT

CCCTGTCCAT

CCTGTGTCAC

AAGAAGAAGG

GCTCTGGTTG

GAACTCCGTG

AACCCCAGTA

CCC CACTCAA

GGAGTGAAGT

TCCAAAG TAT

ATGAGGAACT

TGCACTACTG

AGTGCTGACC

AAGACCTTTT

TCTTTATCAT

GGGTAGACAG

CTTGTGGGCC

CACTACCACC

ACCACAACAC

GAATGGGATT

AGTTCACCAA

CACCCTCTGA

GGTCCATCCC

ATAAGAAAGG

ACTTCCGGGG

CTAAGTATCC

GACAGATATG

ATTTATATGT

TCGGCTTGTA

GGGCA

CAGTGCCACT

TAGAGGGCCG

GCCATCACAA

AAATCTGTGG

AGGAATGTAT

CCAAAGCCTT

AAAAAAAGAG

TCTGGAATGG

TGGCCTTGTG

TCAGTCTTGC

TGGGGATCTG

GGCCCACAGC

CAACATATCT

TAAGCT'rTAA 120 180 240 300 360 420 480 540 600 660 720 780 840 885 INFORMATION FOR SEQ ID NO: 32: SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

29 base pairs.

nucleic acid single linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CTTAAGCTTC TACAGGACTG AGAAGAAGT INFORMATION FOR SEQ ID NO: 33: Wi SEQUENCE CHARACTERISTICS: 32

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

30 base nucleic single linear pairs acid (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: CTTGAATTCC AACATTCTCG GTGCCTGTAA INFORMATION FOR SEQ ID NO: 34: SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

27 base pairs nucleic acid single linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: TCAGGATCCA CAAGGCTGTG AGAAGGA 122 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS:

(A)

(B)

C)

(D)

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

27 base pairs nucleic acid single linear SEQUENCE DESCRIPTION: SEQ ID NO: CTTGTCTAGA CCTGGTGCC CATGATA INFORMATION FOR SEQ ID NO: 36: Wi SEQUENCE CHARACTERISTICS: 35

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

680 base pairs nucleic acid single linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:

ATGCCGGAGG

CAATGGCCAT

AGCGGACTAC

GAGTTACAGC

CCAGCCTTGG

CATCAAGATG

GGCAGCACCC

GGCATCAGCT

ACATACCTGG

CGCAACGCTG

GATGACTTGT

CACATAAAAA

AAGGTCGCCC

GGCTGCTGCT

TCAGTAAGCA

TGAATCTCAC

GAAGGTCCTT

GCCTCTACAG

TGCAGCACAG

TGCTGCGTGG

TCCACGGAGA

ATGAGACCTT

GAATATTTTT

AAAAAAAAAA

TTGCCCCTGG

GGTGGTGTTT

GCAACAGAGG

AGTTCCTCGG

CACACACGGA

GCTGCATATC

GGCCACCCTG

GCGCTTTGGA

TGTCCTCTGT

CTTTGGAGTT

TTTCTTTTCA

GTTCGCTGGA

ATTACTGTGT

CTGCTGGAGC

AAGGACCCcA

CC-AGAGCTGG

CAGGTGACAC

GCTGTGGGCA

CAGGACTGTA

ACCAACCTCA

CAGTGGATAT

AGTTCTACGT

GCGGGACCGC

TTTGCTGTTG

ACCCTGAGCC

CACTGCGCTG

AGGAGGGCCA

TGGCCAACTG

TCTGCTCCCC

CAGTGGCATT

CCCTGCCTCT

GCCCTTGACC

ATTTATAAAT

GTTCCAGCGC

GTTTCATTGT

GCACACAGCT

GGGAGCAGGC

TCTGCGTATC

CTCTTCCCCA

CGCTGCGCAC

ACAGCGCCTG

GCTGCCGTCC

ACAACTCCAG

GTATATAGTA

120 180 240 300 360 420 480 540 600 660 680 INFORMATION FOR SEQ ID NO: 37: SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

846 base pairs nucleic acid single linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: ATGCAGCAGC CCTTCAATTA CCCATATCCC TCTCCCTGGG CCCCTCCAGG CACAGTTCTT GGTCAAAGGA GGCCACCACC ACCACCGCCA CCACCACTGC CTCCACTACC GCTGCCACCC CTGTGTCTCC TTGTGATGTT TTTCATGGTT ATGTTTCAGC TCTTCCACCT GCAGAAGGAA AGCCTTAAAG TATCATCTTT TGAAAAGCAA AAGGAGCTGA GGAAAGTGGC CCATTTAACA GAATGGGAAG ACACCTATGG AATTGTCCTG CTTGTGATCA ATGAAACTGG GCTGTACTTT TCTTGCAACA AcCTGCCCCT GAGCCACAAG GATCTGGTGA TGATGGAGGG GAAGATGATG CGCAGCAGCT ACCTGGGGGC AGTGTTCAAT

CAGATCTACT

CCCTGTCCAA

CCGCCACCAC

CTGAAGAAGA

CTGGTTGCCT

CTGGCAGAAC

ATAGGCCACC

GGCAAGTCCA

CTTTCTGGAG

GTATATTCCA

GTCTACATGA

AGCTACTGCA

CTTACCAGTG

GGGTGGACAG

CCTCTGTGCC

TACCACCTCC

GAGGGAACCA

TGGTAGGATT

TCCGTGAGTT

CCAGTCCACC

ACTCAAGGTC

TGAAGTATAA

AAGTATACTT

GGAACTCTAA

CTACTGGGCA

CTGATCATTT

CAGTGCCAGC

CAGAAGGCCT

GCCGCCGCCG

CAGCACAGGC

GGGCCTGGGG

CACCAACCAA

CCCTGAAAAA

CATGCCTCTG

GAAGGGTGGC

CCGGGGTCAA

GTATCCCCAG

GATGTGGGCO

ATATGTCAAC

123 GTATCTGAGC TCTCTCTGGT CAATTTTGAG GAATCTCAGA CGTTTTTCGG CTTATATAAG

CTCTAA

INFORMATION FOR SEQ ID NO: 38: SEQUENCE CHARACTERISTICS: 840 846

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

786 base pairs nucleic acid single linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:

ATGCAGCAGC

TCTCCCTGGG

GGTCAAAGGA

CCACCACTGC

CTGTGTCTCC

ATGTTTCAGC

AAGGAGCTGA

GAATGGGAAG

CTTGTGATCA

TCTTGCAACA

GATCTGGTGA

CGCAGCAGCT

GTATCTGAGC

CTCTAA

CCTTCAATTA

CCCCTCCAGG

GGCCACC-ACC

CTCCACTACC

TTGTGATGTT

TCTTCCGCTT

GGAAAGTGGC

ACACCTATGG

ATGAAACTGG

ACCTGCCCCT

TGATGGAGGG

ACCTGGGGGC

TCTCTCTGGT

CCCATATCCC

CACAGTTCTT

ACCACCGCCA

GCTGCCACCC

TTTCATGGTT

CGCACAGGCT

CCATTTAACA

AATTGTCCTG

GCTGTACTTT

GAGCCACAAG

GAAGATGATG

AGTGTTCAAT

CAATTTTGAG

CAGATCTACT

CCCTGTCCAA

CCGCCACCAC

CTGAAGAAGA

CTGGTTGCCT

ATAGGCCACC

GGCAAGTCCA

CTTTCTGGAG

GTATATTCCA

GTCTACATGA

AGCTACTGC-A

CTTACCAGTG

GAATCTCAGA

GGGTGGACAG

CCTCTGTGCC

TACCACCTCC

GAGGGAACCA

TGGTAGGATT

CCAGTCCACC

ACTCAAGGTC

TGAAGTATAA

AAGTATACTT

GGAACTCTAA

CTACTGGGCA

CTGATCATTT

CGTTTTTCGG

CAGTGCCAGC

CAGAAGGCCT

GCCGCCGCCG

CAGCACAGGC

GGGCCTGGGG

CCCTGAAAAA

CATGCCTCTG

GAAGGGTGGC

CCGGGGTCAA

GTATCCCCAG

GATGTGGGCC

ATATGTCAAC

CTTATATAAG

120 180 240 300 360 420 480 540 600 660 720 780 786 INFORMATION FOR SEQ ID NO: 39: SEQUENCE

CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

864 base pairs nucleic acid single linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: ATGCAGCAGC CCTTCAATTA

CCCATATCCC

TCTCCCTGGG CCCCTCCAGG

CACAGTTCTT

GGTCAAAGGA GGCCACCACC ACCACCGCCA CCACCACTGC CTCCACTACC

GCTGCCACCC

CTGTGTCTCC TTGTGATGTT

TTTCATGGTT

ATGTTTCAGC TCTTCCAATC

CTCCATCCTC

GGTGCGGCGC AGGCCCTATG

GGTGCGTCCT

AGTCCACCCC CTGAAAAAAA

GGAGCTGAGG

TCAAGGTCCA TGCCTCTGGA ATGGGAAGAC AAGTATAAGA AGGGTGGCCT

TGTGATCAAT

GTATACTTCC GGGGTCAATC

TTGCAA~CA

AACTCTAAGT ATCCCCAGGA

TCTGGTGATG

ACTGGGCAGA TGTGGGCCCG

CAGCAGCTAC

GATCATTTAT ATGTCAACGT

ATCTGAGCTC

TTTTTCGGCT TATATAAGCT

CTAA

864

CAGATCTACT

CCCTGTCCAA

CCGCCACCAC

CTGAAGAAGA

CTGGTTGCCT

CCCTATGCCG

GCGGCCATCC

AAAGTGGCCC

ACCTATGGAA

GAAACTGGGC

CTGCCCCTGA

ATGGAGGGGA

CTGGGGGCAG

TCTCTGGTCA

GGGTGGACAG

CCTCTGTGCC

TACCACCTCC

GAGGGAACCA

TGGTAGGATT

GAGGAGGGTT

TCAATCCTAT

ATTTAACAGG

TTGTCCTGCT

TGTACTTTGT

GCCACAAGGT

AGATGATGAG

TGTTCAATCT

ATTTTGAGGA

CAGTGCCAGC

CAGAAGGCCT

GCCGCCGCCG

CAGCACAGGC

GGGCCTGGGG

CGGGCTGCTC

AGGCCACcCCC

CAAGTCCAAC

TTCTGGAGTG

ATATTCCAAA

CTACATGAGG

CTACTGCACT

TACCAGTGCT

ATCTCAGACG

120 180 240 300 360 420 480 540 600 660 720 780 840 INFORMATION FOR SEQ ID NO: SEQUENCE

CHARACTERISTICS:

LENGTH:

LNGTH:828 base pairs 124

TYPE:

STRANDEDNESS:

TOPOLOGY:

nucleic acid single linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:

ATGCAGCAGC

TCTCCCTGGG

GGTCAAAGGA

CCACCACTGC

CTGTGTCTCC

ATGT'rTCAGC

TTGGAGAAGC

GCCCATTTAA

GGAATTGTCC

GGGCTGTACT

CTGAGCCACA

GGGAAGATGA

GCAGTGTTCA

GTCAATTTTG

CCTTCAATTA

CCC'CTCCAGG

GGCCACCACC

CTCCACTACC

TTGTGATGTT

'TCTTCCACCT

AAATAGGCCA

CAGGCAAGTC

TGCTTTCTGG

TTGTATATTC

AGGTCTACAT

TGAGCTACTG

ATCTTACC-AG

AGGAATCTCA

CCCATATCCC

CACAGTTCTT

ACCACCGCCA

GCTGCCACCC

TTTCATGGTT

ACAGCGAGAG

CCCCAGTCCA

CAACTCAAGG

AGTGAAGTAT

CAAAGTATAC

GAGGAACTCT

CACTACTGGG

TGCTGATCAT

GACGTTTTTC

CAGATCTACT

CCCTGTCCAA

CCGCCACCAC

CTGAAGAAGA

CTGGTTGCCT

TCTACCAGcC

CCCCCTGAAA~

TCCATGCCTC

AAGAAGGGTG

TTCCGGGGTC

AAGTATCCCC

CAGATGTGGG

TTATATGTCA

GGCTTATATA

GGGTGGACAG

CCTCTGTGCC

TACCACCTCC

GAGGGAACCA

TGGTAGGATT

AGATGCACAC

AAAAGGAGCT

TGGAATGGGA

GCCTTGTGAT

AATCTTGCAA

AGGATCTGGT

CCCGCAGCAG

ACGTATCTGA

AG CT CTAA

CAGTGCCAGC

CAGAAGGCCT

GCCGCCGCCG

CAGCACAGGC

GGGCCTGGGG

AGCATCATCT

GAGGAAAGTG

AGACACCTAT

CAATGAAACT

CAACCTGCCC

GATGATGGAG

CTACCTGGGG

GCTCTCTCTG

120 180 240 300 360 420 480 540 600 660 720 780 828 INFORMATION FOR SEQ ID NO: 41: SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

846 base pairs nucleic acid single linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:

ATGGCTATGA

ATCTTCACAG

GAGCTGAAGC

GATGACAGTT

AAGTGGCAAC

TCTACAGTTC

AGAGTAGCAG

TCCAAGAATG

CATTCATTCC

TTTTACTACA

AAGAACGACA

TTGTTGATGA

TCCATCTATC

ACAAATGAGC

GGCTAA

TGGAGGTCCA

TGCTCCTGCA

AGATGCAGGA

ATTGGGACCC

TCCGTCAGCT

AAGAAAAGCA

CTCACATAAC

AAAAGGCTCT

TGAGCAACTT

TCTATTCCCA

AACAAATGGT

AAAGTGCTAG

AAGGGGGAAT

ACTTGATAGA

GGGGGGACCC

GTCTCTCTGT

CAAGTACTCC

CAATGACGAA

CGTTAGAAAC

ACAAAATATT

TGGGACCAGA

GGGCCGCAAA

GCACTTGAGG

AACATACTTT

CCAATATATT

AAATAGTTGT

ATTTGAGCTT

CATGGACCAT

AGCCTGGGAC

GTGGCTGTAA

AAAAGTGGCA

GAGAGTATGA

ATGATTTTGA

TCTCCCCTAG

GGAAGAAGCA

ATAAACTCCT

AATGGTGAAC

CGATTTCAGG

TACAAATACA

TGGTCTAAAG

AAGGAAAATG

GAAGCCAGTT

AGACCTGCGT

CTTACGTGTA

TTGCTTGTTT

ACAGCCCCTG

GAACCTCTGA

TGAGAGAAAG

ACACATTGTC

GGGAATCATC

TGGTCATCCA

AGGAAATAAA

CAAGTTATCC

ATGCAGAATA

ACAGAATTTT

TTTTCGGGGC

GCTGATCGTG

CTTTACCAAc

CTTAAAAGAA

CTGGCAAGTC

GGAAACCATT

AGGTCCTCAG

TTCTCCAAAC

AAGGAGTGGG

TGAAAAAGGG

AGAAAACACA

TGACCCTATA

TGGACTCTAT

TGTTTCTGTA

CTTTTTAGTT

120 180 240 300 360 420 480 540 600 660 720 780 840 846 INFORMATION FOR SEQ ID NO: 42: Wi SEQUENCE

CHARACTERISTICS:

(A)

(B3)

(C)

(D)

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

876 base pairs nucleic acid single linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42: ATGCCTTCCT CAGGGGCCCT GAAGGACCTC AGCTTCAGTC AGCACTTCAG

GATGATGGTG

ATTTGCATAG TGCTCCTGCA GGTGCTCCTG CAGGCTGTGT CTGTGGCTGT GACTTACATG 125

TACTTCACCA

TTCTCAAAGA

TGCTTGCAGG

CAGGACACCA

GGAAGACCTC

TTAATTCCAA

TCTCGGAAAG

GAGCAGGAGG

GACGCTTCCA

TACAAGTACA

TGGTCCAGAG

AAAAAAAATG

GAAGCCAGCT

ACGAGATGAA

CGGATGAGGA

TTAAGAGGCA

TTTCTAC.AGT

AGAAAGTGGC

TCTCCAAGGA

GGCATTCATT

GCCTGTATTA

AGATGGTCTC

CCAGCTATCC

ATGCCGAGTA

ACAGGATTTT

TCTTTGGAGC

GCAGCTGCAG

TTTCTGGGAC

ACTGTATCAG

TCCAGAAAAG

AGCTCACATT

TGGAAAGACC

TCTCAACCAC

CATCTATTCC

AAAGGACAAG

GGATCCCATA

CGGACTGTAC

TGTTTCTGTG

CTTTTTAATT

GACAATTACT

TCCACTGATG

CTCATTGAAG

CAGCTAAGTA

ACTGGGATCA

TTAGGCCAGA

GTGCTCTTTA

CAAACATACT

GTGAGAACCA

GTGCTCATGA

TCCATCTATC

ACAAATGAAC

AACTAA

CCAAAATTGG

GAGAGATCTT

AGGTGACTTT

CTCCTCCCTT

CTCGGAGAAG

AGATTGAATC

GGAATGGAGA

TCCGATTTCA.

AACAGCTGGT

AGAGCGCCAG

AGGGAGGATT

ATTTGATGGA

ACTAGCTTGC

GAACAGACCC

GAGAACCTTT

GCCCAGAGGT

CAACTCAGCT

CTGGGAGTCC

GCTGGTCATC

GGAAGCTGAA

GCAGTACATC

AAAC.AGCTGT

GTTCGAGCTA

CCTGGATCAA

180 240 300 360 420 480 540 600 660 720 780 840 876 INFORMATION FOR SEQ ID NO: 43: SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

720 base pairs nucleic acid single linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43

ATGGAGCCAG

CAGGTGCAGC

AGTCGCAGCT

ATCATTCTGG

CCCCTTAAAG

AAGAAGTCAT

AACGAAGATG

CCTGGCTTGT

GTGGACCTGA

GTGTGTGAGT

CATTACTTAC

ACAAACACTT

GGCTGCAACA

CCGGCTCGGT

ACTTCTACCT

TACTGGTAGT

GAGGAAATTG

GGGCCTACCT

GCACCATCCA

ACTTCATCGT

CATTGCAGCT

CTGGAGTTCA

AGGTCAACTC

TCCCTCTTGA

AGCAGGCAGC

AGCCAGCCCC

CAGCACCACC

CCAGAAAAAG

CTCAGAGGAT

CCAAGTGTCA

CGGACTCATA

TTGCCAACTG

CCTCATCAAT

GAGTAAGAAC

TACCATATCA

TAATGTGCTA

TGTGGGGCTC

TGGAGAAGCA

GCACTGGTGT

GACTCCACTC

CTCTTCTGTA

AAGCATCTCA

TACCAGGACG

CAGTTCCTCG

TCCAAGATCA

ATCTACCAGA

GTCAGGGTGG

TCCGTCTTCT

CTTCCCCTGA

CGAGGCCCTG

GCCTTGTTGT

CAAATACAAC

CCCTGAAAAG

ACAATACCAA

GGAACCTGAT

TGCAGTGCTC

AAAAGCAGAC

ATCTCTCTCA

ATAATTTCCA

TATATAGTAG

CCCAGCCATG

GAGAAGCACA

GGCAGTGGCG

TGAGAAGGCC

TACTCCATCC

ACTGTCATGG

AGTCCAATTC

AAATCATTCT

GTTGGTAACA

GTTTTTGCTG

GTATGTGGAT

CTCAGACTGA

120 180 240 300 360 420 480 540 600 660 720 INFORMATION FOR SEQ ID NO: 44: SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

930 base pairs nucleic acid single linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:

ATGGACCAGC

TCGTGCCCCT

CTCTCAGATA

GTTAATGTTC

CCAAAGCTCT

ATCTTCACCC

ACCCGAGAGA

ACACAACAGG

AATACA.ACTC

CTGAGGTACG

TTTTTGGAAC

GTCTCTCTTG

GTGGAACTGT

TTGCTCCTGA

CAGGATGCAT

CTTGTGAAAC

ACACACTTGA

CGGATGCGGC

CTGTGCGCCC

GGGATCGCGA

ATGGCCTAGT

GCACCGAGCC

ATAATGCAGA

GCTCTCCTGT

TGAACTGGCA

AAGAAGACAA

TGAAGCTCAG

TTTTGCAAGC

TCCCTTGCTC

AGGCTGGCCA

ACAGAGACTG

CCGACAACCC

TGTGGAGGAT

GCTCCTCAGA

CACAAATGCC

GGCCGCGTGG

CGCTTTGGTT

TCGGCCAGCG

CCAGGTCACC

GTTCGCCAAG

CAGCCAAGAT

AAAGGAGTTG

TCCAACATTC

AAAGCCTCAG

CATGGAGAAC

CCGCCTCAGT

GGAGCTGTCT

ATGGGAATGA

ACCGCGGATG

GATACCGGGC

GCGCTCCCCA

CCGCCTGCAC

TTGCTGCTTC

CTCACAATCA

CCTGTTTCCC

CTACTGGCTA

GGAGCTGGGA

GTGGTAGACA

ACAAACACAG

GTAGATGACT

AAGTTAGTGG

GTGGGTCTGA

TATCCCAACA

CCAGACATCC

TCCTCGCGGA

CGGATGCTGC

TGAACTTCTG

TGATCGCCGC

CCACCTCGCC

ACATTGGCTG

AAAACCAAGC

GCTCATACCT

GTCCCGGGCT

GCCACAAGGT

TTGACAACTT

ACCGTTCCTG

GGGCTTATCT

CCACCAGCTT

AGCAGGTACT

CGCTGCGCTC

CTACCCTGCG

TTCCCGCCAC

CTGTGTTCCT

CAACCTGGGT

CCCCAACAC'r

ATCGTTGTGC

ATCTCAAGGT

CTACTACGTA

GCAGGGCTGG

GGCCCTGACA

GAGTCAACTG

GCATGGAGCC

TGGACTCTTT

120 180 240 300 360 420 480 540 600 660 720 780 840 900 930

Claims (12)

1. A method for expressing a chimeric CD40 ligand in a CD40+ human cell, wherein the chimeric CD40 ligand includes one or more human domain segments derived from a human CD40 ligand gene and onc or more murine domain segments derived from a murine CD40 ligand gene, and wherein further the method comprises delivering a chimeric polynucleotide encoding said chimeric CD40 ligand into a CD40+ human cell.

2. The method of claim I wherein the murine CD40 ligand domain segment comprises an extracellular murine CD40 ligand domain segment.

3. The method of claim 2 wherein the extracellular murine CD40 ligand domain segment comprises Domain III.

4. The method of claim 2 wherein the extracellular murine CD40 ligand domain segment comprises Domain IV.

5. The method of claim 1 wherein the murine CD40 ligand domain segment consists of Domain I. is 6. The method of claim 1 wherein the murine CD40 ligand domain segment consists of Domain II. o: 7. The method of claim 1 wherein the chimeric polynucleotide consists of the Snucleic acid sequence of SEQ ID NO. 3.

8. The method of claim 1 wherein the chimeric polynucleotide consists of the 20 nucleic acid sequence of SEQ ID NO.

9. The method of claim 1, wherein the method is active in human Sneoplastic cells.

10. The method of claim 9, wherein the neoplastic cells comprise neoplastic B do cells. 25 11. The method of claim 9 wherein the neoplastic cells comprise neoplastic T cells.

12. The method of claim 1 wherein the chimeric polynucleotide consists of the nucleic acid sequence of SEQ ID NO. 4.

13. The method of claim 1 wherein the chimeric polynucleotide consists of the nucleic acid sequence SEQ ID NO.

14. The method of claim 1 wherein the chimeric polynucleotide consists of the nucleic acid sequence SEQ ID NO. 6. The method of claim 1 wherein the chimeric polynucleotide consists of the nucleic acid sequence SEQ ID NO. 7. [R:\LIBVV]03651 .doc:THR

127- 16. A chimeric polynucleotide encoding a chimeric CD40 ligand comprising one or more human domain segments derived from a human CD40 ligand gene and one or more murine domain segments derived from a murine CD40 ligand gene. 17. The chimeric polynucleotide of claim 16 wherein the murine CD40 ligaid s domain segment comprises an extracellular murine CD40 ligand domain segment. 18. The chimeric polynucleotide of claim 17 wherein the extracellular murine ligand domain segment comprises Domain III. 19. The chimeric polynucleotide of claim 17 wherein the extracellular murine ligand domain segment comprises Domain IV. 20. The chimeric polynucleotide of claim 16 wherein the murind CD40 ligand domain segment consists of Domain I. 21. The chimeric polynucleotide of claim 16 wherein the murine CD40 ligand domain segment consists of Domain II. 22. The chimeric polynucleotide of claim 16 wherein the chimeric polynucleotide i. 15 consists of the nucleic acid sequence of SEQ ID NO. 3. 23. The chimeric polynucleotide of claim 16 wherein the chimeric polynucleotide consists of the nucleic acid sequence of SEQ ID NO. 24. The chimeric polynucleotide of claim 16 wherein the chimeric polynucleotide consists of the nucleic acid sequence of SEQ ID NO. 4. i: 20 25. The chimeric polynucleotide of claim 16 wherein the chimeric polynucleotide consists of the nucleic acid sequence of SEQ ID NO. 26. The chimeric polynucleotide of claim 16 wherein the chimeric polynucleotide consists of the nucleic acid sequence of SEQ ID NO. 6. 27. The chimeric polynucleotide of claim 16 wherein the chimeric polynucleotide 25 consists of the nucleic acid sequence of SEQ ID NO. 7. 28. A chimeric polynucleotide encoding a chimeric CD40 ligand comprising one or more human domain segments derived from a human CD40 ligand gene and one or more murine domain segments derived from a murine CD40 ligand gene, substantially as hereinbefore described with reference to any one of the examples. 29. A pharmaceutical composition for use in expressing a chimeric CD40 ligand in a CD40+ human cell, wherein the chimeric CD40 ligand includes one or more human domain segments derived from a human CD40 ligand gene and one or more murine domain segments derived from a murine CD40 ligand gene, and wherein further the composition comprises a chimeric polynucleotide encoding said chimeric CD40 ligand. 30. The composition of claim 29 wherein the murine CD40 ligand domain segment comprises an extracellular murine CD40 ligand domain segment. [R:\LIBVV]03651 .doc:THR -128- 31. The composition of claim 30 wherein the extracellular murine CD40 ligand domain segment comprises Domain III. 32. The composition of claim 30 wherein the extracellular murine CD40 ligand domain segment comprises Domain IV. 33. The composition of claim 29 wherein the murine CD40 ligand domain segment consists of Domain I. 34. The composition of claim 29 wherein the murine CD40 ligand domain segment consists of Domain II. The composition of claim 29 wherein the chimeric polynucleotide consists of to the nucleic acid sequence ofSEQ ID NO. 3. 36. The composition of claim 29 wherein the chimeric polynucleotide consists of the nucleic acid sequence of SEQ ID NO. 37. The composition of claim 29 wherein the composition is active in human CD40+ neoplastic cells. 5I 38. The composition of claim 37 wherein the neoplastic cells comprise neoplastic B cells. 39. The composition of claim 37 wherein the neoplastic cells comprise neoplastic T cells. The composition of claim 29 wherein the chimeric polynucleotide consists of 20 the nucleic acid sequence of SEQ ID NO. 4. 41. The composition of claim 29 wherein the chimeric polynucleotide consists of the nucleic acid sequence of SEQ ID NO. 42. The composition of claim 29 wherein the chimeric polynucleotide consists of the nucleic acid sequence of SEQ ID NO. 6. 25 43. The composition of claim 29 wherein the chimeric polynucleotide consists of the nucleic acid sequence of SEQ ID NO. 7. 44. A method for expressing a chimeric Fas ligand in a human cell, wherein the chimeric ligand includes one or more human domain segments derived from a human Fas ligand gene and one or more murine domain segments derived from a murine Fas ligand gene, and wherein further the method comprises delivering a chimeric polynucleotide encoding said chimeric Fas ligand. The method of claim 44 wherein the chimeric polynucleotide consists of the nucleic acid sequence of SEQ ID NO. 37. 46. The method of claim 44 wherein the chimeric polynucleotide consists of the nucleic acid sequence of SEQ ID NO. 38. [R:\LIBVV]03651 .doc:THR -129- 47. The method of claim 44 wherein the chimeric polynucleotide consists of the nucleic acid sequence of SEQ ID NO. 39. 48. A chimeric polynucleotide encoding a chimeric Fas ligand comprising one or more human domain segments derived from a human Fas ligand gene and one or more murine domain segments derived from a murine Fas ligand gene. 49. The chimeric polynucleotide of claim 48 consisting of the nucleic acid sequence of SEQ ID NO. 37. The chimeric polynucleotide of claim 48 consisting of the nucleic acid sequence of SEQ ID NO. 38. to 51. The chimeric polynucleotide of claim 48 consisting of the nucleic acid sequence of SEQ ID NO. 39. 52. A pharmaceutical composition for use in expressing a chimeric Fas ligand in a human cell, wherein the chimeric Fas ligand includes one or more human domain segments derived from a human Fas ligand gene and one or more murine domain 1s segments derived from a murine Fas ligand gene, and wherein further the composition comprises a chimeric polynucleotide encoding said chimeric Fas ligand. 53. The composition of claim 52 wherein the chimeric polynucleotide consists of p the nucleic acid sequence of SEQ ID NO. 37. 54. The composition of claim 52 wherein the chimeric polynucleotide consists of 20 the nucleic acid sequence of SEQ ID NO. 38. 55. The composition of claim 52 wherein the chimeric polynucleotide consists of S the nucleic acid sequence of SEQ ID NO. 39. 1 56. A pharmaceutical composition consisting of a mammalian, bacterial or insect cell transfected with the chimeric CD40 ligand polynucleotide of Claim 16. 25 57. A pharmaceutical composition consisting of a mammalian, bacterial or insect cell transfected with the chimeric Fas ligand polynucleotide of Claim 48. 58. A chimeric polynucleotide encoding a chimeric Fas ligand comprising one or more human domain segments derived from a human Fas ligand gene and one or more murine domain segments derived from a murine Fas ligand gene substantially as hereinbefore described with reference to any one of the Examples. Dated 7 March, 2005 University of California Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON (R:\LIBVV]03651 .doc:THR