SK3532000A3 - Dna sequence coding for kay ligand, method for the preparation of kay ligand, pharmaceutical composition containing said ligand and the use thereof - Google Patents

Abstract

Kay-ligand, a novel member of the tumor necrosis factor family (TNF), modified Kay-ligands and pharmaceutical compositions comprising them.

Description

Technical field

The present invention relates to a novel Kay ligand that is a member of the tumor necrosis factor family. This protein or its receptors have antitumor and / or immunoregulatory utility. In addition, cells transfected with genes for this novel ligand can be used in gene therapy to treat tumors, autoimmune and inflammatory diseases, or inherited genetic disorders, as well as antibodies blocking these proteins may have immunoregulatory utility.

BACKGROUND OF THE INVENTION

Tumor Necrosis Factor (TNF) -related cytokines are mediators of host defense and immune regulation. Members of this protein family exist in a cell membrane-anchored form where they act locally through cell-cell contact, or occur as secreted proteins that are capable of diffusing to more distant targets. A parallel family of receptors draws attention to the presence of these molecules causing the initiation of cell death or cell proliferation and differentiation in the target tissue. Currently, the TNF ligand and receptor family contains at least 11 known receptor-ligand pairs: TNF: TNF-R, LT-α: TNF-R, LT-α / β: Ι / Γ-β-R, FasL: Fas, CD40L: CD40, CD30L: CD30, CD27L: CD27, OX40L: OX40, and 4-1BBL: 4-1BB. The DNA sequences coding for these ligands have an identity of only 25 to 30%, even at the highest affinity, although the relative at the amino acid sequence level is about 50%.

The determining feature of this family of cytokine receptors is the extracellular cysteine-rich domain, which has been discovered in the molecular cloning of two different TNF receptors ⁽¹⁾ . This gene family encodes glycoproteins having properties of type I transmembrane proteins with an extracellular ligand binding domain, one transmembrane domain, and a cytoplasmic region that is involved in activating cellular functions. The cysteine-rich region, which is also a ligand-binding region, has a central domain of firmly linked disulfide bridges that repeats several times, depending on the particular family member. Most receptors have four domains, although the receptors have only three domains or up to six domains.

TNF ligand family proteins are characterized at their N-terminus by a short stretch of normally short hydrophilic amino acids, which often contains several lysine or arginine residues that appear to serve as stop-transfer sequences. They then have a transmembrane region and an extracellular region of variable length that separates the receptor-binding domain at the C-terminus from the cell membrane. This section is sometimes called a stem. The C-terminal binding region represents the major portion of the protein and often, although not always, contains glycosylation sites. These genes do not contain the classical signal sequences characteristic of type I membrane proteins, but rather correspond to type II membrane proteins having a C-terminal extending outside the cell and a short N-terminal extending into the cytoplasm. In some cases, such as TNF and LT-α, cleavage may occur during the early processing of the protein in the stalk region, and the ligand then mainly exists in secreted form. Most ligands occur in membrane form and thus mediate localized signal transduction.

The structure of these ligands was defined by crystallographic analysis of TNF, LT-α and CD40L. The structure of TNF and lymphotoxin alfa (LT-α) is "sandwiched", ie it consists of two antiparallel β-pleated leaves with a topology called "erythrocyte". meander (Greek key or jelly roll ⁽¹¹⁾ ). The deviation of the rms between the residues of the Ca and β chains is 0.61 C, suggesting a high degree of similarity in their molecular topography. A structural feature that apparently results from molecular studies of CD40L, TNF, and LT-α is the tendency of these ligands to form oligomeric complexes. Characteristic of the oligomeric structure is the generation of receptor binding sites at the junction site between adjacent subunits, thereby forming a multivalent ligand. By analyzing the crystal structure, the quaternary structure of CD40L, TNF and LT-α was examined and CD40L, TNF and LT-α appeared to exist as trimmers. Many amino acids conserved for these ligands occur precisely in the framework region of the β-sheet structure. It is likely that the basic sandwich structure is preserved among the different members of the whole family. Apparently, the quaternary structure is also maintained, since the conformation of the subunits is also likely to remain similar.

TNF family members are best described as the major switches in the immune system controlling cell survival and differentiation. Currently, secreted cytokines, TNF and LT-α, are known, unlike most other membrane-anchored TNF family members. While membrane forms of TNF have been well characterized and likely to have a unique biological function, the function of secreted TNF is the general signaling of "alarm to cells remote from the site of the event that triggered the event." TNF secretion can multiply the event leading to well-known changes in the lining of the vessels and in the cells at the site of inflammation. In contrast, membrane-bound TNF family members sell the signal via the TNF-receptor only to cells in direct contact. E.g. helper T cells provide "CD40 mediated assistance only to those B cells with which they come into direct contact through TCR interactions. Similar limitations to immediate cell contact relate to the ability to induce cell death in a well-investigated Fas system.

TNF ligands can be divided into three groups based on their ability to induce cell death. In the first group there are TNF, Fas and TRAIL ligands that can effectively induce cell death in many cell lines and their receptors have mostly confirmed deadly domains. Ligand DR-3 (TRAMP / WSL-1) would also probably belong to this category. The second group consists of ligands that trigger a weaker signal limited to only certain cell types. This second group includes e.g. TWEAK, ligand CD30 and LTalb2. An interesting question is how members of this group can trigger a mechanism leading to cell death when they do not have a confirmed "death domain." The absence of this domain suggests that there is another, weaker way of signaling in the cell death mechanism. The last group includes those members who are unable to transmit any signal leading to cell death. However, probably members of all groups may act antiproliferative to some cell types as a result of induction of differentiation, such as e.g. CD40 (Funakoshi et al., 1994).

The TNF family has grown dramatically lately, and now contains 11 different signaling pathways including immune system regulation. The widespread expression patterns of TWEAK and TRAIL show that there is considerable functional variability in this family, not yet known. This was notably due to the new discovery of two receptors that affect the replication ability of Rous sarcoma virus and Herpes simplex virus, as well as historically significant discoveries that TNF has antiviral activity and pox virus encodes decoy TNF receptors (Brojatsch et al., 1996, Montgomery et al., 1996, Smith 1994, Vassali 1992).

TNF is a mediator of septic shock and cachexia ¹¹¹¹¹ and is involved in the regulation of hematopoietic cell ^{(lv) development} . He seems to be playing

Λ a major role as a mediator in the inflammatory response and in defense against bacterial, viral and parasitic infections ^(v) ,

apparently anti-tumor activity ^(v1) . TNF has also been implicated in a variety of autoimmune diseases ^(VIII) . TNF is produced by various cell types, e.g. these are macrophages, fibroblasts, T-cells of cytotoxic NK-cells (natural killer) ^(V111) . TNF binds to two different receptors, each acting through different specific signaling molecules within the cell, resulting in different effects of TNF ^(1X) . TNF may exist both in a membrane-bound form and as a free secreted cytokine '*'.

LT-α has many common activities with TNF, such as. binding to TNF receptors ^1x11 , but unlike TNF, it is mainly secreted by activated T cells and some β-lymphoblastoid tumors ^(X11) . The heteromeric LT-α and LT-β complex is a membrane-bound complex where it binds to LT-β receptors ^(X111) . The LT system (ie LT with LT-R receptor) is involved in the development of peripheral lymphoid organs, since genetic disruption of LT-β causes T- and B-cell disorders in the spleen and disappearance of lymph nodes ^txlv) . The LT-β system is also involved in cell death in some adenocarcinoma cells ^(xv) .

Fas-L, another member of the TNF family, is expressed preferentially on activated T-cells ^(XV1) . It induces the death of the receptor-bearing cells, tumor cells and HIV-infected cells by a mechanism known as programmed cell death or apoptosis ^(xvll> . In addition, Fas or Fas-L deficiency is known to cause lymphoproliferative disorders, confirming the role Fas system in regulation of immune response ^(XV111) Fas system also participates in liver damage due to chronic viral hepatitis infection ^(X1X) and autoimmune response in HIV infected patients ^(XX) Fas also participates in Tb cell destruction in HIV infected patients ^(XX1> .

TRAIL, another member of the TNF family, also appears to be involved in the cell death of various transformed cell lines of diverse origin ^(XX11) .

CD40, another member of the TNF family, is expressed on the surface of T cells and induces regulation of B cells bearing CD40 ( ^xxxIII) . In addition, changes in the CD40-L gene are known to cause a disease known as X-linked hyper-IgM ^(xxlv) . The CD40 system is also associated with many different autoimmune diseases ^(xxv) and CD40-L also has antiviral properties ^(XXV1). Although the CD40 system is involved in the rescue of apoptotic B cells ^(xxvll) in other cells such as cells of the immune system induces apoptosis ^(XXV111>. Many other members of the TNF family of lymphocyte involved in costimulation ^(xxlxl.

In general, TNF family members play an essential regulatory role in controlling the immune system and activating the acute host defense system. Given the recent advances in influencing TNF family members for therapeutic benefit, it is likely that family members can provide unique remedies against disease. Some TNF family ligands can directly induce apoptotic death of many transformed cells, e.g. LT, TNF, Fas ligand and TRAIL (Nagata 1997). Activation of the Fas receptor and possibly TNF and CD30 can induce cell death of untransformed lymphocytes, which may have an important immunoregulatory function (Amakawa et al., 1996, Nagata 1997, Sytwu et al., 1996, Zheng et al., 1995). It is generally known that cell death is triggered upon aggregation of the "death domains" that are located on the cytoplasmic side of TNF receptors. These "lethal domains" organize the arrangement of the various components of signal transduction, ultimately causing the activation of the entire cascade (Nagata 1997). Some receptors do not contain confirmed "death domains", e.g. the LTb receptor and CD30 (Browning et al., 1996, Lee et al., 1996) and yet may induce cell death, albeit substantially weaker. These receptors are likely to function primarily by inducing cell differentiation and cell death is an aberrant consequence in some transformed cell lines, although the whole picture is still unclear, since a CD30 null mouse study suggests a lethal role in negative selection in the thymus ( Amakawa et al., 1996). In contrast, the transmission of signals by other paths, such as e.g. it is through CD40 that it is required to maintain and survive cells. Accordingly, there is a need to characterize other members of the TNF family and to provide additional means for treating diseases and affecting the immune system.

SUMMARY OF THE INVENTION

The present invention relates to a polypeptide called Kay ligand, which essentially solves a number of problems arising due to the limitations and drawbacks of the prior art solutions. The inventors discovered a new member of the TNF cytokine family and determined the amino acid sequence of both the murine and human proteins, including the respective DNA sequences encoding these proteins. The invention can be used to identify novel compositions for the diagnosis and treatment of a variety of diseases and conditions, as described in more detail below, as well as to obtain further information about the immune system and to self-influence the immune system and immune processes. In addition, the present invention also relates to the induction of cell death in cancers.

Other features and advantages of the present invention will be described in more detail in the following description, and in part will be apparent to those skilled in the art and in part will be recognized in the practice of the invention. The objects of the invention and its advantages can be achieved by means of the compositions and methods particularly pointed out in the description and claims, as well as in the accompanying drawings.

In accordance with the objective of the invention, in order to achieve the advantages of the invention as set forth in the description and examples of the invention, the invention includes DNA sequences encoding a Kay ligand. Specifically, the invention relates to a DNA sequence encoding a human Kay ligand (i.e., SEQ ID NO: 1). In addition, the invention also relates to the amino acid sequence of a novel ligand. The amino acid sequence of the human Kay ligand is shown herein as SEQ ID NO: 2. In addition, the murine Kay ligand sequence is shown partially as SEQ ID NO: 3, and the protein encoded by SEQ ID NO: 3 is shown herein as SEQ ID NO: 3. NO: 4. Another embodiment of the invention relates to sequences having at least 50% homology to a DNA sequence encoding a 5'-terminal receptor binding domain, while hybridizing to a sequence of the invention or a fragment thereof, and which encodes a Kay ligand with the sequence disclosed herein as SEQ ID NO: 1 or SEQ ID NO: 4.

Some embodiments of the invention pertain to DNA sequences encoding a Kay ligand, wherein the sequences are operably linked to an expression control sequence. Any expression control sequence is suitable for use in the invention and the appropriate sequence can be readily selected by those skilled in the art.

The present invention further relates to recombinant DNA which comprises a DNA sequence encoding a Kay ligand or a fragment thereof, as well as hosts having a Kay ligand sequence permanently integrated in the genome or containing it in the form of an episomal element. Any suitable host can be used according to the invention and can be readily selected by one skilled in the art without the need for undue experimentation.

A further embodiment of the present invention relates to a method for preparing substantially pure Kay ligands comprising the steps of culturing a transformed host and further relates to Kay ligand substantially free of all animal proteins normally associated with it.

The invention also relates to Kay ligands having the amino acid sequence set forth herein as SEQ ID NO: 2, or fragments or homologs thereof. In various embodiments of the invention, the amino acid sequences or DNA sequences may comprise conservative insertions, deletions and substitutions as shown below, or may comprise fragments of said sequences.

In another embodiment, the present invention relates to a soluble Kay ligand construct that can be used to directly trigger a Kay ligand-mediated pharmacological event. Such an event may have a therapeutic effect in stimulating growth, in treating cancer and tumors, or in manipulating the immune system to treat immunological diseases. The soluble forms of the ligand of the invention can be engineered to contain a readily recognizable label by means of genetic engineering, thereby allowing identification of the receptors for these ligands.

Further embodiments of the present invention pertain to antibodies directed against Kay ligand, which can be used e.g. for the treatment of cancer, tumors, or for influencing the immune system in the treatment of immunological diseases.

Yet another embodiment of the present invention relates to methods of gene therapy using the Kay ligand genes of the present invention.

The pharmaceutical composition of the present invention may optionally also contain a pharmaceutically acceptable carrier, adjuvant, filler or other pharmaceutical compositions, and may be administered by any method known in the art.

Both the foregoing summary and the following detailed description of the invention are to be understood as explanations of the invention and merely as examples of embodiments of the invention which serve to explain the present invention in more detail.

Also, the figures, which serve to better understand the invention and forming part of the description of the invention, are intended to illustrate some embodiments of the invention and together with the description serve to explain the principle of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1. A comparison of the amino acid sequence of human and mouse Kay ligand. The mouse sequence shown in the top row was obtained by direct cloning of cDNA. The human sequence was obtained by folding the partial cDNA with the 5'RACE assay. The third sequence, shown on the bottom line, shows the confirmed sequence.

Fig. 2. A human Kay ligand cDNA fragment (KayL cDNA) was used as a probe for Northern blot analysis of RNA from various human tissues. It can be seen that the 2.4 kb KayL RNA was mainly expressed in the spleen and peripheral blood lymphocytes, i.e. in the secondary lymphoid organs.

DETAILED DESCRIPTION OF THE INVENTION

The description will relate to a detailed description of preferred embodiments of the present invention. The present invention relates to sequences

DNAs that encode human or murine Uganda Kay, fragments or homologs thereof, and further relates to the expression of these DNAs in hosts transformed with the DNA. The invention further relates to the use of these DNA sequences and of the peptides encoded by these sequences. In addition, the invention relates to both the human and murine amino acid sequence of Kay ligand, or fragments or homologs thereof, and further relates to a pharmaceutical composition comprising or derived from these sequences.

A. Definitions

The term "homologous" herein describes the similarity between the sequences of the molecules to be compared. If a position in both sequences being compared is occupied by the same base or amino acid, e.g. if the same position in each of the two molecules is occupied by adenine, then these molecules are homologous at that position. The homology between two sequences, expressed as a percentage, is a function of the number of equal, i.e. homologous, positions having both sequences, divided by the number of all positions compared and multiplied by 100. for two sequences, 6 positions out of 10 are the same or homologous, then these sequences have 60% homology. Or eg. the two ATTGCC and TATGGC sequences have 50% homology. Generally, a comparison is made by sequencing the sequences to provide maximum homology.

The terms "purified preparation" or "substantially pure preparation of a polypeptide" are intended to refer to a polypeptide that has separated from other proteins, lipids, and nucleic acids with which it naturally occurs. Preferably, the purified polypeptide is also separated from other substances such as e.g. antibodies or matrix used for purification.

The term "transformed host" includes a host having a sequence, i. a ligand sequence integrated into the genome.

any Kay, permanent

The term "treatment or any therapeutic treatment" is used herein to e.g. administration of a therapeutic agent or agent, e.g. the drug.

The term "substantially pure nucleic acid, e.g. substantially pure DNA, means a nucleic acid which 1) is not directly linked to one or two sequences (one at the 5'-end and one at the 3'-end), e.g. coding sequences adjacent to the naturally occurring genome of the respective organism from which the nucleic acid originates, or 2) is substantially free of nucleic acid sequences that occur in the organism from which said nucleic acid originates. Thus, the term includes e.g. recombinant DNA inserted into the vector, e.g. into an autonomously replicating plasmid or virus, or into genomic DNA of a prokaryotic or eukaryotic organism, or which exists as a separate molecule (e.g., cDNA or genomic DNA fragment generated by PCR or by restriction endonuclease treatment) independent of other DNA sequences. Substantially pure DNA also includes recombinant DNA that is part of a hybrid gene encoding a Kay ligand.

The terms "peptides," "proteins" and "polypeptides" are used interchangeably.

The term "biologically active" is used to mean having an activity, either in vitro or in vivo, that is manifested directly or indirectly. E.g. a biologically active fragment of a Kay ligand may have e.g. 70% homology of the ligand active site amino acid sequence, more preferably at least 80% homology, and most preferably at least 90% homology. Identity or homology with respect to ligands is defined as the percentage of amino acid residues in the respective selected sequence that are identical to the amino acid residues of the Kay ligand sequence of SEQ ID NO: 2.

Embodiments of the present invention require, unless otherwise indicated, the use of techniques common in cell biology, cell and tissue cultures, molecular biology, transgenic biology, microbiology, recombinant DNA techniques, and immunological methods known in the art. These techniques are described in the relevant literature.

B. DNA Sequences of the Invention

One aspect of the present invention is a substantially pure (or recombinant) nucleic acid comprising a nucleotide sequence encoding a Kay ligand, such as e.g. the DNA sequence set forth herein as SEQ ID NO: 1 and / or equivalents of such nucleic acids. The term "nucleic acid" also includes fragments or equivalents, such as e.g. sequences encoding functionally equivalent polypeptides. Equivalent nucleotide sequences may comprise sequences that differ by one or more nucleotide substitutions, additions or deletions, such as e.g. allelic variants, mutations, etc., and may also contain nucleotide sequences that differ from the sequence encoding the Kay ligand set forth herein as SEQ ID NO: 1 due to the degeneracy of the genetic code.

The present invention is described generally with respect to the sequences of the human gene, although it will be apparent to one skilled in the art that it also relates to the murine sequence. Human protein appears to have all the characteristics of the TNF family, i. type II membrane protein alignment and conservative sequence motifs involved in protein alignment into the anti-parallel β-sheet structure that is typical of TNF.

The nucleotide sequence of Kay ligand is referred to herein as SEQ ID NO: 1 and the amino acid sequence of Kay ligand is referred to herein as SEQ ID NO: 2.

The sequences of the present invention can be used to prepare a series of DNA probes that are suitable for testing various sets of natural or synthetic DNA for the presence of Kay ligand-related sequences or fragments or derivatives thereof. The skilled artisan will appreciate that the term "Kay ligand" is used herein to include biologically active derivatives, fragments, and homologs thereof.

The Kay ligand coding sequence of the present invention can be used to express the peptide of the invention in various prokaryotic or eukaryotic hosts transformed with such sequences. The peptides can be used as anti-cancer or immunoregulatory agents. In general, this means that one step of the application is to cultivate a host transformed with a DNA molecule comprising a coding sequence for a Kay ligand operably linked to an expression control sequence.

The DNA sequence and recombinant DNA molecules of the present invention can be expressed using a wide variety of host / vector combinations. So eg. useful vectors may comprise segments of chromosome or synthetic DNA sequences. Expression vectors of the invention are characterized in that they comprise at least one expression control sequence that is operably linked to a sequence encoding a Kay ligand inserted into the vector to control or control the expression of the DNA sequence.

Within each expression vector, various Kay ligand insertion sequences of the present invention can be selected. The sites are usually designed according to the restriction endonucleases that cleave at the site, and these sites and endonucleases are well known to those skilled in the art. The skilled artisan will also appreciate that the vector for use in the present invention need not contain restriction endonuclease sites for insertion of the desired DNA fragment. Instead, the fragment can be cloned into the vector in an alternative manner. The choice of an expression vector, and in particular sites suitable for insertion of the selected DNA fragment and its association with the expression control sequence, depends on many factors. These factors include e.g. the size of the protein to be expressed, the sensitivity of the protein to proteolytic degradation by the host cell enzymes, the number of sites for the respective restriction enzyme, the contamination or binding of the expressed protein by host cell proteins that are difficult to remove during purification, etc. the expression characteristics such as e.g. the position of the start and stop codons relative to the vector sequences, and other factors that will be apparent to those skilled in the art. Thus, the selection of the vector and the appropriate DNA sequence insertion sites of the present invention is a result of consideration of all these factors, and not all solutions are equally effective for the desired applications. However, for a person skilled in the art, the analysis of all these factors is a routine matter and the choice of a suitable system then depends primarily on the particular application.

One skilled in the art is readily able to modify expression control sequences to obtain a higher level of protein expression, e.g. by substituting codons or by selecting codons of certain particular amino acids that are preferably used by a particular organism to reduce proteolysis or alter the glycosylation pattern. Similarly, cysteine residues may be replaced by other amino acids to facilitate protein production or assembly, or to avoid stability problems.

Thus, not all host / expression vector combinations function with the same efficiency in terms of expression of the DNA sequence of the present invention. However, the selection of a particular suitable host / expression factor combination can be readily made by one skilled in the art. Factors to be taken into account include, for example: host-vector compatibility, toxicity of proteins encoded by the vector's DNA sequences for the host, ease of isolation of the desired protein, expression characteristics of DNA sequences and expression control sequences operably linked thereto, biosecurity, energy requirement for protein structure alignment, protein form and protein modification, that occur after expression.

The Kay ligand and its homologues produced by the hosts transformed with the DNA sequences of the present invention, as well as the native Kay ligand purified by the method of the invention, or generated based on the amino acid sequence of the invention, are useful for a variety of anti-cancer and immunoregulatory applications. They are also useful in therapy and methods relating to other diseases.

The present invention also relates to the use of the DNA sequences of the invention for the expression of this ligand under special conditions, i. in gene therapy. In addition, the Kay ligand can be expressed in tumor cells, where expression is controlled by promoters suitable for this purpose. Such expression may enhance the anti-tumor immune response or directly affect tumor survival. The ligand of the invention may also affect the survival of organ grafts by altering the local immune response. In such a case, the graft or cells surrounding it would itself be modified by the APRIL gene modified by genetic engineering methods.

Another aspect of the present invention is the use of an isolated nucleic acid encoding a Kay ligand for the so-called " “Antisense therapy. The term "antisense therapy" refers to the administration or generation of in situ oligonucleotides or derivatives thereof that specifically hybridize under normal cellular conditions to cellular mRNA and / or DNA encoding the desired Kay ligand to inhibit expression of the encoded protein by inhibiting transcription and / or translation . Said binding may be conventional base pair complementarity or e.g. in the case of binding to DNA duplexes, it may be binding through specific interactions in the main (large) incision of double-stranded DNA. Generally, "antisense therapy" refers to a variety of techniques used in the art and includes essentially any therapy based on specific binding of the oligonucleotide sequence.

The antisense construct of the present invention can be administered e.g. in the form of an expression plasmid which, when transcribed in a cell, generates RNA which is complementary to the cell mRNA encoding the Kay ligand or at least a portion thereof. Alternatively, an oligonucleotide probe can also be prepared ex vivo by the antisense construct. Such oligonucleotide probes are preferably modified oligonucleotides that are resistant to endogenous nucleases and are therefore stable in vivo. Examples of such nucleic acid molecules suitable as "antisense oligonucleotides" are phosphoramidate, phosphothioate, and methylphosphonate analogs of DNA (see US patent applications 5,176,996, 5,264,564 and 5,256,775). In addition, a general procedure for constructing oligomers suitable for "antisense therapy" has been reviewed in Van Der Krol et al., 1988, Biotechniques 6: 958 = 976 and Stein et al., 1988, Cancer Res. 48: 2659-2668, which are incorporated herein by reference.

C. Ligand Kay and its amino acid sequence

As discussed above, Ligand Kay is a member of the TNF family. The protein itself, or fragments or homologs thereof, has a wide range of therapeutic and diagnostic uses, as discussed in more detail below.

Ligand Kay occurs mainly in the spleen and in peripheral blood lymphocytes, suggesting a significant regulatory role in the immune system. Comparison of the Kay ligand sequence of the invention with other members of the human TNF family shows considerable structural similarity. All of these proteins share several conserved sequences in the extracellular domain.

Although the three-dimensional structure of the ligand of the invention is not yet accurate, it can be assumed that, as a member of the TNF family, it has certain structural characteristics in common with other family members.

The novel polypeptide reacts with the receptor, peptides and methods of the present invention not specifically identified to date. However, according to the present invention, it is possible to identify receptors that specifically react with the Kay ligand or fragments thereof.

Certain embodiments of the present invention include peptides derived from a Kay ligand that have the ability to bind to its receptors. Ligand fragments can be prepared in various ways, e.g. by recombinant technology, by PCR, proteolytic cleavage, or chemical synthesis. Internal or terminal fragments of a polypeptide can be prepared by removing one or more nucleotides from one or both ends of a nucleic acid that encodes the polypeptide.

Polypeptide fragments can be prepared by expression of the mutant

DNA.

Polypeptide fragments can also be chemically synthesized using a conventional technique known as solid phase synthesis using the f-moc and t-boc biochemical procedure of Merriffield. So eg. the peptides and DNA sequences of the present invention can be basically arbitrarily divided into fragments of desired length that do not overlap, or overlapping fragments of desired length. The methods are described in more detail below.

D. Preparation of soluble forms of Kay ligand and tumor ligand

Soluble forms of Kay ligand can be very effective in signal transduction and can therefore be administered as a drug that mimics the natural membrane form. It is possible that the Kay ligand of the present invention is naturally secreted as a soluble cytokine, but if not, it is possible to adapt the gene by genetic engineering methods to enhance secretion. In order to prepare a soluble secreted form of APRIL, the N-terminal transmembrane region and some stalk segments should be removed at the DNA level and replaced with type I leader sequences, or alternatively, an type IZ leader sequence that allows efficient proteolytic cleavage in the appropriate host system. One of ordinary skill in the art is able to vary the range of "stem segments" retained in the expression construct to achieve optimal receptor binding properties and secretory efficacy. So eg. By shortening the N-terminus, it is possible to construct constructs containing all possible lengths of the stem to produce proteins that will start with amino acids 81 to 139. By this type of analysis it is possible to determine the optimal length of the stem sequence.

E. Preparation of Kay Ligand Reactive Antibodies

The present invention also relates to antibodies specifically reacting with the Kay ligand or its receptor. Anti-protein / anti-peptide or monoclonal antibodies may be prepared by standard methods (see, e.g., Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Press, 1988). A mammal such as e.g. a mouse, hamster or rabbit can be immunized with an immunogenic form of the peptide. Methods to make the protein immunogenic, e.g. by conjugating to a carrier or other methods are known in the art.

The immunogenic portion of Kay ligand or its receptor may be co-administered with an adjuvant. The progress of the immunization can be monitored by detecting antibody titer in plasma or serum. Standard ELISA or other immunoassays can be used with an immunogen as an antigen to assess antibody levels.

In a preferred embodiment of the present invention, the antibodies are immunospecific for antigenic determinants of Kay ligand or its receptor, i. antigenic determinants of the polypeptide of SEQ ID NO: 2, or for a closely related human, mammalian, non-human homologue (with a homology of 70, 80, or 90%, most preferably with a homology of at least 95%). In yet another preferred embodiment of the present invention, the anti-Kay ligand or Kay ligand receptor antibodies do not substantially cross-react (i.e., only react specifically) with a protein that is e.g. less than 80% homologous to SEQ ID NO: 2, preferably less than zz 95% homologous to SEQ ID NO: 2. By "substantially non-reactive" is meant that the antibody has a binding affinity for a non-homologous protein less more than 10%, more preferably less than 5% and even more preferably less than 1% of the protein binding affinity of SEQ ID NO: 2.

The term "antibody" is used herein to include antibody fragments that also specifically react with the Kay ligand or its receptors. Antibodies can be fragmented using conventional techniques known in the art, and the possibilities of using the fragments can then be tested in the same manner as described for whole antibodies. So eg. the F (ab ') ₂ fragment can be prepared by treatment with pepsin. The resulting fragment is then treated with the disulfide bridges of the present invention to reduce it to form a Fab 'fragment. Antibodies of the invention further include biospecific and chimeric molecules having activity directed against the Kay ligand or its receptor. Thus, both monoclonal and polyclonal antibodies (Ab) directed against Kay ligand, tumor ligand and their receptors, as well as antibody fragments such as e.g. Fab 'or F (ab') ₂ can be used to block a ligand and its receptor.

Various forms of antibodies can be prepared by standard recombinant DNA techniques (Winter and Milstein, Nature 349: 293-299, 1991). E.g. it is possible to construct such chimeric antibodies, wherein the antigen binding domain of the animal's antibody is linked to a human constant domain (Cabilly et al., U.S. Patent 4,816,567). Chimeric antibodies reduce the immune response elicited by animal antibodies when used in clinical applications for human patients.

In addition, recombinant " humanized antibodies recognizing Kay ligand or its receptor can be synthesized. Humanized antibodies are chimeric antibodies that contain mostly human IgG sequences into which sequences responsible for specific antigen binding have been inserted. The animals are immunized with the gene of interest, then the appropriate antibody is isolated and the portion of the variable region responsible for specific antigen binding is removed. The antigen binding region from the animal is then cloned into a suitable site of the human antibody gene from which the antigen binding region has been removed. Humanized antibodies minimize the use of heterologous (i.e., cross-species) sequences in human antibodies, thus reducing the likelihood of eliciting an immune response in the treated patient.

Different classes of recombinant antibodies can also be generated by preparing chimeric or humanized antibodies comprising variable domains, and human constant isolated from different classes can prepare recombinant domains (CH1, CH2 and CH3) of immunoglobulins. So eg. by an antibody with increased valency of antigen binding sites by cloning the antigen binding site into a vector carrying a human antibody constant region (Arulandam et al., J.Exp.Med. 177: 1439-1450, 1993) .

In addition, standard recombinant DNA techniques can be used to alter the binding affinity of recombinant antibodies to their antigens by altering the amino acid residues near the antigen binding site. The antigen binding affinity of a humanized antibody can be increased by molecular modeling mutagenesis (Quenn et al., Proc. Natl. Acad. Sci. USA 86: 10029-33, 1989).

F. Generation of peptide sequence analogs

Preparation of altered DNA sequences a

The Kay ligand analogs may be found to differ from the naturally occurring ligand in the amino acid sequence, or in a manner other than the amino acid sequence, or both. Modifications other than sequence modification include both in vitro and in vivo chemical derivatization of Kay ligand. These non-sequential modifications include, but are not limited to, changes in acetylation, methylation, phosphorylation, carboxylation, or glycosylation.

Preferred Kay ligand analogs include biologically active fragments thereof whose sequence differs from the sequence set forth herein as SEQ ID NO: 2 by one or more conservative amino acid substitutions, deletions, or insertions that do not interfere with the biological activity of the Kay ligand. Conservative substitutions typically include substitution of one amino acid with another amino acid with similar properties, e.g. substitution within the following groups valine - glycine, glycine - alanine, valine - isoleucine - leucine, aspartic acid - glutamic acid, asparagine - glutamine, serine - threonine, lysine arginine and phenylalanine - tyrosine.

Conservative amino acid changes are summarized in Table 1.

Table 1

Conservative amino acid substitutions

amino acid code can be replaced by any of: alanine A D-Ala, Gly, beta-Ala, L-Cys, and D-Cys arginine R D-Arg, Lys, D-Lys, homo-Arg, D-homo- -Arg, Met, D-Met, Clay, D-Ile, Orn, D-Orn asparagine A D-Asn, Asp, D-Asp, Glu D-Gln Aspartic acid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gin, D-Gln cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr, D- Thr glutamine Q D-Gln, D-Glu, Asp, D-Asp Glutamic acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gin, D-Gln glycine G Ala, D-Ala, Pro, D-Pro, -Ala, Acp isoleucine I D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met leucine L D-Leu, Val, D-Val, Leu, D-Leu, Met, D-Met lysine The D-Lys, Arg, D-Arg, H -mo-Arg, D-homo- Arg, Met, D-Met, White, D-Ile, Orn, D- -Crn methionine M D-Met, S-Me-Cys, White, D-Ile, Leu, D- -Leu, Val, D-Val

phenylalanine F D-Phe, Tyr, D-Thr, L-Dopa, His, D- -His, Trp, D-Trp, trans-3,4 or 5- -phenylproline, cis-3,4 or 5-phenyl- proline proline P D-Pro, L-1-thioazolidine-4-carboxylic acid acid, D- or L-1-0xazolidin-4- carboxylic acid serine WITH D-Ser, Thr, D-Thr, allo-Thr, Met, D- - Met, Met (O), D - Met (O), L - Cys, D - Cys threonine T D-Thr, Ser, D-Ser, allo-Thr, Met, D- -Met, Met (0), D-Met (0), Val, D-Val tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His, D- -His valine IN D-Val, Leu, D-Leu, Clay, D-lle, Met, D-Met

Useful methods of mutagenesis include mutagenesis by PCR and saturation mutagenesis, which will be described in detail in the following. It is also possible to create a library of random amino acid sequence variants by synthesizing a set of degenerate oligonucleotide sequences.

PCR mutagenesis

B

PCR mutagenesis uses a low precision Taq polymerase to introduce random mutations into the cloned DNA fragment (Leung et al., Technique 1: 11-15, 1989). It is a very efficient yet very fast method for introducing random mutations. The DNA portion to be mutated is amplified by polymerase chain reaction (PCR) under conditions that reduce the accuracy of Taq polymerase, e.g. if the dGTP / dATP ratio is approximately 5 and Mn ² * is added to the PCR reaction mixture. The entire set of amplified fragments is then cloned into suitable vectors to form a library of random mutants.

Saturation mutagenesis

Saturation mutagenesis allows the rapid introduction of a large number of single base substitution type mutations into a cloned DNA fragment (Mayers et al., Science 229: 242, 1985). This technique involves the generation of mutations, e.g. chemical treatment or irradiation of single-stranded DNA in vitro, and then synthesis of the complementary DNA strand. The frequency of mutations can be influenced by the potency of the effect, and basically substitutions of all possible bases can be achieved. Since this process does not involve any genetic selection of the mutated fragments, both neutral substitution fragments and randomly mutated DNA fragments are obtained from which proteins with altered function can be prepared. The distribution of point mutations is unaffected by conservative sequence elements.

Degenerated oligonucleotides

A homolog library can be prepared using a set of degenerate oligonucleotides. Chemical synthesis of degenerate sequences can be performed on an automatic DNA synthesis apparatus, and the synthetic genes are then ligated into a suitable expression vector. Synthesis of degenerate oligonucleotides is a method well known in the art ^(xxx) and has already been used for targeted development of other proteins ^(XXX1) .

Methods of non-random or targeted mutagenesis can be used to prepare specific sequences or mutations in certain specific regions. These methods can be used to generate variants that contain deletions, insertions, or substitutions of amino acid residues in a known amino acid sequence of a protein. The mutation sites may be modified individually or in series e.g. by 1) substituting the conserved amino acids first and then making more radical changes depending on the results obtained 2) deleting the target residues and 3) inserting residues of the same or a different class into the vicinity of the site, or combining all of the above methods 1 to third

Alanine replacement mutagenesis

Alanine scanning mutagenesis is a method suitable for identifying certain residues or stretches of proteins of interest that are suitable sites or domains for mutagenesis. (Cunningham and Wells, Science 244: 10811085, 1989). In this method, residues or groups of residues (e.g., charged amino acid residues such as Arg, Asp, His, Lys, Glu) are identified and replaced with a neutral or negative amino acid (most preferably alanine or polyalanine). These amino acid substitutions can affect the interaction of amino acids with the surrounding aqueous environment inside or outside the cell. Domains that exhibit functional sensitivity to substitutions can be further refined by introducing further changes or by altering already substituted sites. Thus, while the site for insertion of the amino acid sequence variation is predetermined, the nature of the mutation is not predetermined in itself. So eg. to optimize mutation efficiency at a particular site, alanine replacement mutagenesis or random mutagenesis at the target codon or region can be performed and the subunit variants of the protein of interest can be expressed and then tested for optimal combination and activity.

Oligonucleotide-mediated mutagenesis

Oligonucleotide-mediated mutagenesis is a method suitable for the preparation of DNA substitution, deletion and insertion variants (see, e.g., Adelman et al., DNA 2: 183, 1983).

The DNA of interest can be altered by hybridizing to an oligonucleotide that contains a mutation of the DNA template, wherein the template is a single-stranded form of a plasmid or bacteriophage containing an unchanged or native DNA sequence for the protein of interest. After hybridization, DNA polymerase is used to synthesize the entire complementary strand so that the template then contains the inserted oligonucleotide primer and thus encodes selected DNA changes for a given protein. In general, oligonucleotides of at least 25 nucleotides in size are used. The optimal oligonucleotide contains 12 to 15 nucleotides that are fully complementary to the template on either side of the nucleotide (s) encoding the mutation. This will ensure that the oligonucleotide will properly hybridize to the single-stranded template DNA. Oligonucleotides can be readily synthesized by methods known in the art (see, e.g., Crea et al., Proc. Natl. Acad. Sci. USA 75: 5765, 1978).

Cassette mutagenesis

Another way to prepare variants is by cassette mutagenesis, which is based on the technique described by Wells et al. (Gene 34: 315 (1985)). The starting material is a plasmid (or other vector) that contains DNA for the protein subunit to be mutated. First, it is necessary to identify the codon (s) to be mutated in the protein subunit. There must be a unique restriction endonuclease site on either side of the mutated site. In the absence of such a restriction site, it must be generated at the desired location by oligonucleotide-mediated mutagenesis as described above. After the restriction sites have been introduced into the plasmid, the plasmid is cleaved at these sites and thus linearized. A double stranded oligonucleotide is then synthesized in a standard manner, which encodes a DNA sequence between two restriction sites and contains the desired mutation. Each oligonucleotide strand is synthesized separately and then hybridized together again using standard methods known in the art. This double-stranded oligonucleotide is called a cassette. The cassette is designed to contain a 3'-end and a 5'-end compatible with the ends of the linearized plasmid so that it can be ligated directly into the plasmid. The plasmid then contains the mutated DNA sequence of the protein of interest.

Combination mutagenesis

Combination mutagenesis can also be used to generate mutations. E.g. the amino acid sequences of a family of homologous or otherwise related protein sequences are aligned (preferably) so as to achieve maximum homology. All sequences that appear at a given position for all aligned sequences are selected to form a degenerate set of combination sequences. Combination mutagenesis creates a very diverse library of variants at the nucleic acid level. So eg. the mixture of synthetic oligonucleotides is enzymatically ligated into gene sequences such that a degenerate set of potential sequences can be expressed as individual peptides, or alternatively, as a set of other fusion proteins comprising a set of degenerate sequences.

Various techniques are known in the art for screening products produced by mutated genes. Techniques for screening large gene libraries often include cloning the library into replicable expression vectors, transforming the appropriate cells with the resulting vector library, and expressing the genes under conditions where detection of the desired quality, i. in this case, binding to the Kay ligand or its receptor allows relatively easy isolation of the vector encoding the gene whose product has been detected. Each of the techniques described in the following paragraphs is suitable for highly efficient and high-throughput screening of a large number of sequences generated e.g. by random mutagenesis techniques.

The invention further provides the possibility of preparing mimetics to reduce the protein binding domains of a polypeptide of the invention or its receptor, e.g. peptide agents or agents other than peptides. Peptide mimetics are capable of disrupting the binding of Kay ligand and its receptor. Critical residues of Kay ligand involved in the molecular recognition process of the receptor polypeptide or the corresponding downstream intracellular protein in the signaling pathway can be determined and used to prepare peptide ligands for Kay ligand or its receptor that competitively or incompetently inhibit the binding of Kay ligand and its receptor (see e.g. "Peptide inhibitors of human papilloma virus protein binding to retinoblastoma gene protein, patent applications EP 412 762 A and EP 831 080 A, which are incorporated by reference).

G. Pharmaceutical preparations

By providing purified and recombinant Kay ligands, the present invention also provides a test method that can be used to test drug candidates that are functionally or agonists or antagonists of normal cellular function, in this case Kay ligand or its receptor. In one embodiment of the present invention, the ability of a compound to modulate binding between a Kay ligand and its receptor is assessed using such an assay. It will be apparent to those skilled in the art that various assay variants of the present invention may be made.

Many drug screening programs that are used to test an entire library of compounds or natural extracts require methods with high efficacy and processed sample capacity to maximize the number of compounds tested per unit of time. Tests that are performed on a cell-free system, such as e.g. purified or semi-purified proteins are often preferred as tests for so-called " primary screening, which allows rapid development of these compounds, since it allows a relatively easy detection of changes in the molecular target that occur by the test compound. However, the toxic effect on the cells and / or the bioavailability of the test compound may remain unnoticed in such an in vitro system, since the assay is primarily directed to the drug's action on the molecular target where it manifests by altering the binding affinity to other proteins or altering the enzymatic properties of the molecular target.

The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of Kay ligand or its receptor, or fragments or mimetics thereof, and optionally comprise pharmaceutically acceptable carriers and excipients.

Thus, the invention also relates to a method of treating cancer and a method of stimulating, or, in certain cases, inhibiting, the immune system, or portions thereof, comprising administering a pharmaceutically effective amount of a compound of the present invention or a pharmaceutically active salt or derivative thereof. The skilled artisan will appreciate that the compounds and methods of the invention may be combined with a variety of other therapies.

The compositions of the present invention can be formulated for a variety of routes of administration, including systemic, topical or topical administration. For systemic administration, administration by injection, including intramuscular, intravenous, intraperitoneal and subcutaneous injection, is preferred, wherein the formulation according to

present of the invention can formulate than aqueous solution, preferably within saline buffer such as e.g. Hank or Ringer solution. Except for that agent by present invention the can formulate in solid a to any

dissolution or resuspension immediately prior to use. The present invention also relates to lyophilized pharmaceutical forms.

The composition of the present invention may also be administered by the oral, transmucotic or transdermal route. In the formulation for transmucotic or transdermal administration, suitable penetration enhancers (penetrants), based on the barrier to be overcome, are used. Such penetrants are known in the art and include e.g. for transmucotic administration of bile salt, fusidic acid derivatives and detergents. Transmucosal administration may be by either nasal spray (spray) or suppositories. For oral administration, the formulation is formulated in a conventional dosage form suitable for oral administration such as capsules, tablets and tonicas. For topical administration, the composition may be in the form of ointments, ointments, gels or creams as is generally known in the art.

The compositions of the invention are preferably in unit dosage form and are administered once or more daily. The amount of active ingredient administered either in a single dose or during treatment is dependent on many different factors. So eg. the age and size of the subject, the severity and course of the disease being treated, the mode and form of administration, and of course the judgment of the attending physician. However, an effective dose is in the range of 0.005 to 5 mg / kg / day, preferably 0.05 to 0.5 mg / kg / day. One skilled in the art will appreciate when lower or higher doses may be beneficial.

The gene constructs of the present invention can also be used as part of a gene therapy protocol to transfer nucleic acids encoding either agonist or antagonist forms of the Kay ligand polypeptide.

The Kay ligand expression constructs of the invention can be administered via any biologically active carrier, e.g. any pharmaceutical composition capable of efficiently transferring in vivo the Kay ligand gene of the invention into a cell. This approach involves inserting a gene into a viral vector that can directly transfect a cell or deliver plasmid DNA, e.g. liposomes, or some intracellular carrier, or may be a direct injection of the gene construct. A preferred method is transmission using a viral vector.

The pharmaceutical composition with the gene construct for gene therapy consists essentially of a gene transfer system and a suitable diluent, or it may be a slow release matrix into which the gene transfer system is embedded. Alternatively, if the entire gene transfer system can be prepared intact from recombinant cells, e.g. as a retroviral vector, the pharmaceutical composition then comprises one or more cells that produce a gene transfer system.

In addition to use in therapy, the oligomers of the present invention can be used as diagnostic reagents to detect the presence or absence of target DNA, RNA, or amino acid sequences to which they specifically bind. Another aspect of the present invention is the use for evaluating a chemical compound for its ability to react, i. e.g. bind or otherwise physically associate with the Kay ligand or fragments thereof. The method comprises contacting the chemical with a Kay ligand and evaluating the ability of the substance to react with a Kay ligand. In addition, the Kay ligand of the present invention can be used in methods for assessing naturally occurring Kay ligands or Kay receptors, as well as for evaluating chemicals that associate with or bind to Kay ligand receptors.

This method of Kay ligand

Another aspect of the present invention provides a method for assessing a chemical for its ability to modulate the interaction between a Kay ligand and its receptor.

comprising mixing the Kay ligand and the receptor under conditions where such a pair is capable of interacting, then adding the chemical to be tested and detecting the formation or disintegration of the complex. Such modulating agents may be further evaluated by in vitro assays e.g. testing their activity in the cell-free system, optionally treating the cells with these agents, or administering the agents to animals and then evaluating the effects.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Isolation of the Kay ligand binding receptor

TNF family ligands were used for identification and cation. Using the described Kay ligand sequences, it was possible to fuse the 5'-end of the extracellular domain that forms the receptor binding sequence to a marker or marker sequence and then to add a leader sequence that promotes ligand secretion in any of a number of possible expression systems. One example of such a procedure has been described in Browning et al. (JBC 271: 86188626, 1996), where the LT-β ligand was secreted in such form. The VCAM leader sequence was joined to the short marker sequence of myc with the LT-β extracellular domain. The VCAM sequence was used to aid secretion of the LT-β molecule that is normally membrane bound. The secreted protein retained the myc tag at its N-terminus, which did not impair its ability to bind to the receptor. Such a secreted protein is then expressed in either transiently transfected COS cells or a similar system, e.g. EBNA-derived vectors, insect cell and baculovirus systems, or Pichia sp. and the like. An unpurified cell supernatant was used as a source of labeled ligand.

Cells expressing the receptor could be identified by exposure to the labeled ligand. Ligand-bound cells were then identified by FACS flow cytometry when myc was labeled with an anti-myc peptide (anti-myc, 9E10) and then phycoerythrin (or a similar label) labeled with anti-mouse immunoglobulin. FACS-positive cells were readily identified and served as a source of RNA encoding the receptor. An expression library was then prepared from this RNA by standard procedures and divided into pools. These subgroups of clones were then transfected into suitable host cells and the binding of the labeled ligand to the transfected receptor cells was identified by microscopic examination after the bound myc peptide was labeled with an anti-mouse immunoglobulin labeled enzyme, e.g. galactosidase, alkaline phosphatase or luciferase. Once a positive subset of clones was identified, the size of the subgroups decreased until the cDNA encoding the receptor was identified. The whole procedure can be performed with either the mouse or human Kay ligand, one of which will more readily lead to the receptor.

It will be apparent to those skilled in the art that modifications of the APRIL formulation of the present invention may be made within the inventive spirit of the invention. Therefore, the present invention also includes such modifications and variations as are within the scope of the invention as defined by the following claims, or as an equivalent solution.

LIST OF SEQUENCES

SEQ ID NO: 1

TGCCAAGCCC TGCCATGTAG TGCACGCAGG ACATCAACAA ACACAGATAA 51 CAGGAAATGA TCCATTCCCT GTGGTCACTT ATTCTAAAGG CCCCAACCTT 101 CAAAGTTCAA GTAGTGATAT GGATGACTCC ACAGAAAGGG AGCAGTCACG 151 CCTTACTTCT TGCCTTAAGA AAAGAGAAGA AATGAAACTG AAGGAGTGTG 201 TTTCCATCCT CCCACGGAAG GAAAGCCCCT CTGTCCGATC CTCCAAAGAC 251 GGAAAGCTGC TGGCTGCAAC CTTGCTGCTG GCACTGCTGT CTTGCTGCCT 301 CACGGTGGTG TCTTTCTACC AGGTGGCCGC CCTGCAAGGG GACCTGGCCA 351 GCCTCCGGGC AGAGCTGCAG GGCCACCACG CGGAGAAGCT GCCAGCAGGA 401 GCAGGAGCCC CCAAGGCCGG CCTGGAGGAA GCTCCAGCTG TCACCGCGGG 451 ACTGAAAATC TTTGAACCAC CAGCTCCAGG AGAAGGCAAC TCCAGTCAGA 501 ACAGCAGAAA TAAGCGTGCC GTTCAGGGTC CAGAAGAAAC AGTCACTCAA 551 GACTGCTTGC AACTGATTGC AGACAGTGAA ACACCAACTA TACAAAAAGG 601 ATCTTACACA TTTGTTCCAT GGCTTCTCAG CTTTAAAAGG GGAAGTGCCC 651 TAGAAGAAAA AGAGAATAAA ATATTGGTCA AAGAAACTGG TTACTTTTTT 701 ATATATGGTC AGGTTTTATA TACTGATAAG ACCTACGCCA TGGGACATCT 751 AATTCAGAGG AAGAAGGTCC ATGTCTTTGG GGATGAATTG AGTCTGGTGA 801 CTTTGTTTCG ATGTATTCAA AATATGCCTG AAÄCACTACC CAATAATTCC 8 51 TGCTATTCAG CTGGCATTGC AAAACTGGAA GAAGGAGATG AACTCCAACT 901 TGCAATACCA AGAGAAAATG CACAAATATC ACTGGATGGA GATGTCACAT 951 TTTTTGGTGC ATTGAAACTG

1051 TA

SEQ ID NO: 2

MDDSTEREQS RLTSCLKKRE EMKLKECVSI LPRKESPSVR SSKDGKLLAA 51 TLLLALLSCC LTWSFYQVA ALQGDLASLR AELQGHHAEK LPAGAGAPKA 101 GLEEAPAVTA GLKIFEPPAP GEGNSSQNSR NKRAVQGPEE TVTQDCLQLI 151 ADSETPTIQK GSYTFVPWLL SFKRGSALEE KENKILVKET GYFFIYGQVL 201 YTDKTYAMGH LIQRKKVHVF GDELSLVTLF RCIQNMPETL PNNSCYSAGI

251 AKLEEGDELQ LAIPRENAQI SLDGDVTFFG ALKLL

SEQ ID NO: 3

GTGGTCACTT ACTCCAAAGG CCTAGACCTT CAAAGTGCTC CTCGTGGAAT 52 GGATGAGTCT GCAAAGACCC TGCCACCACC GTGCCTCTGT TTTTGCTCCG

101 AGAAAGGAGA AGATATGAAA GTGGGATATG ATCCCATCAC TCCGCAGAAG 151 GAGGAGGGTG CCTGGTTTGG GATCTGCAGG GATGGAAGGC TGCTGGCTGC 201 TACCCTCCTG CTGGCCCTGT TGTCCAGCAG TTTCACAGCG ATGTCCTTGT 251 ACCAGTTGGC TGCCTTGCAA GCAGACCTGA TGAACCTGCG CATGGAGCTG 301 CAGAGCTACC GAGGTTCAGC AACACCAGCC GCCGCGGGTG CTCCAGAGTT 351 GACCGCTGGA GTCAAACTCC TGACACCGGC AGCTCCTCGA CCCCACAACT 401 CCAGCCGCGG CCACAGGAAC AGACGCGCTT TCCAGGGACC AGAGGAAACA 451 GAACAAGATG TAGACCTCTC AGCTCCTCCT GCACCATGCC TGCCTGGATG 501 CCGCCATTCT CAACATGATG ATAATGGAAT GAACCTCAGA AACAGAACTT 551 ACACATTTGT TCCATGGCTT CTCAGCTTTA AAAGAGGAAA TGCCTTGGAG 601 GAGAAAGAGA ACAAAATAGT GGTGAGGCAA ACAGGCTATT TCTTCATCTA 651 CAGCCAGGTT CTATACACGG ACCCCATCTT TGCTATGGGT CATGTCATCC 701 AGAGGAAGAA AGTACACGTC TTTGGGGACG AGCTGAGCCT GGTGACCCTG 751 TTCCGATGTA TTCAGAATAT GCCCAAAACA CTGCCCAACA ATTCCTGCTA 801 CTCGGCTGGC ATCGCGAGGC TGGAAGAAGG AGATGAGATT CAGCTTGCAA 851 TTCCTCGGGA GAATGCACAG ATTTCACGCA ACGGAGACGA CACCTTCTTT 901 GGTGCCCTAA AACTGCTGTA ACTCACTTGC TGGAGTGCGT GATCCCCT TC 951 CCTCGTCTTC TCTGTACCTC CGAGGGAGAA ACAGACGACT GGAAAAACTA

1001 AAAGATGGGG AAAGCCGTCA GCGAAAGTTT TCTCGTGACC CGTTGAATCT 1051 GATCCAAACC AGGAAATATA ACAGACAGCC ACAACCGAAG TGTGCCATGT 1101 GAGTTATGAG AAACGGAGCC CGCGCTCAGA AAGACCGGAT GAGGAAGACC 1151 GTTTTCTCCA GTCCTTTGCC AACACGCACC GCAACCTTGC TTTTTGCCTT 1201 GGGTGACACA TGTTCAGAAT GCAGGGAGAT TTCCTTGTTT TGCGATTTGC 1251 CATGAGAAGA GGGCCCACAA CTGCAGGTCA CTGAAGCATT CACGCTAAGT 1301 CTCAGGATTT ACTCTCCCTT CTCATGCTAA GTACACACAC GCTCTTTTCC 1351 AGGTAACTAC TATGGGATAC TATGGAAAGG TTGTTTGTTT TTAAATCTAG 1401 AAGTCTTGAA CTGGCAATAG ACAAAAATCC TTATAAATTC AAGTGTAAAA 1451 TAAACTTAAT TAAAAAGGTT TAAGTGTG

sequence SEQ ID N 1 MDESAKTLPP 51 ATLLLALLSS 101 LTAGVKLLTP 151 CRHSQHDDNG 201 YSQVLYTDPI 251 YSAGIARLEE

D: 4

PCLCFCSEKG EDMKVGYDPI SFTAMSLYQL AALQADLMNL AAPRPHNSSR GHRNRRAFQG MNLRNRTYTF VPWLLSFKRG FAMGHVIQRK KVHVFGDELS GDEIQLAIPR ENAQISRNGD

TPQKEEGAWF GICRDGRLLA RMELQSYRGS ATPAAAGAPE PEETEQDVDL SAPPAPCLPG NALEEKENKI WRQTGYFFI LVTLFRCIQN MPKTLPNNSC DTFFGALKLL

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Claims (35)

PATENT CLAIMS A DNA sequence encoding a Kay ligand or fragment thereof. 2. A DNA sequence encoding a Kay ligand consisting essentially of SEQ ID NO: 1 or SEQ ID NO: 3. 3. A DNA sequence consisting essentially of SEQ ID NO: 1 or SEQ ID NO: 3 which encodes a polypeptide consisting essentially of SEQ ID NO: 2 or SEQ ID NO: 4. A DNA sequence that hybridizes to at least a fragment of SEQ ID NO: 1 or SEQ ID NO: 3, wherein the fragment comprises a contiguous stretch of at least 20 consecutive bases, and the DNA sequence encodes the polypeptide at least 30% homologous to the Kay ligand active site. DNA sequence according to claim 2, which consists essentially of SEQ ID NO: 1 or SEQ ID NO: 3 with conservative substitutions, alterations or deletions. 6. A recombinant DNA molecule comprising a sequence encoding a Kay ligand that is operably linked to an expression control sequence. The molecule of claim 6, comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 3. 8. One-cell host! transformed with a recombinant DNA molecule according to claim 6 or 7. A DNA sequence encoding a Kay ligand having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. 10. A method for preparing a substantially pure Kay ligand, comprising the step of culturing a single cell host according to claim 8. 11. Kay Ligand substantially free of the animal proteins with which it is normally associated. The Kay ligand of claim 11 consisting essentially of SEQ ID NO: 2 or SEQ ID NO: 4. * t 13. A pharmaceutical composition comprising a therapeutically effective amount of a Kay ligand or active fragment thereof and a pharmaceutically acceptable carrier. 14. A method of preventing or reducing the severity of an autoimmune disease, comprising the step of administering a therapeutically effective amount of the pharmaceutical composition of claim 13. The pharmaceutical composition of claim 13, wherein the Kay ligand or active fragment thereof comprises the sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or a biologically active fragment thereof. 16. A method of preventing or reducing the rate of an autoimmune response to a tissue graft, comprising the step of administering a therapeutically effective amount of the pharmaceutical composition of claim 13. 17. A method of stimulating the immune system comprising the step of administering the composition of claim 13. i • 18. A method of suppressing the immune system comprising the step of administering an effective amount of a pharmaceutical composition according to claim 13. 19. A method of treating cancer comprising the step of administering an effective amount of a pharmaceutical composition according to claim 13. 20. A method for identifying a Kay ligand receptor comprising the steps of: (a) a Kay ligand or fragment thereof is provided; (b) the Kay ligand or fragment thereof is labeled with a detectable label; c) screening the compounds to detect receptors that bind to the detectably labeled Kay ligand of step b). The soluble biologically active fragment of Kay ligand according to claim 11. 22. A polypeptide comprising an amino acid sequence that is encoded by a DNA selected from the group consisting of: (a) a DNA sequence comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 3 b) a DNA sequence that hybridizes to the DNA defined in a) and which encodes the polypeptide at least 40% homologous to the Kay ligand of claim 12. An antibody preparation that is reactive with a Kay ligand or its receptor or biologically active fragments thereof. The antibody preparation of claim 23, which comprises a monoclonal antibody. 25. A method for preparing an antibody preparation reactive with a Kay ligand or receptor thereof, comprising the step of immunizing an organism with a Kay ligand or receptor or antigenic fragment thereof. 26. An anti-Kay ligand antisense nucleic acid comprising a nucleic acid sequence hybridizing to at least a portion of SEQ ID NO: 1 or SEQ ID NO: 3. 27. A pharmaceutical composition comprising the antibody preparation of claim 24. 28. A method of expressing a gene in a mammalian cell, the method comprising the steps of: a) the gene encoding the Kay ligand is introduced into the cell b) the cell is allowed to live under conditions such that the gene is expressed in the mammal. 29. A method for treating diseases associated with Kay ligand in a mammal comprising: a) introducing into the cell a therapeutically effective amount of a vector comprising a gene encoding a Kay ligand, and b) the gene is expressed in a mammalian cell. 30. The method of claim 29, wherein the mammal is a human. The method of claim 29, wherein the vector is a virus. 32. A method of inducing cell death comprising administering an agent capable of disrupting the binding of a Kay ligand to its receptor. 33. The method of claim 32, further comprising administering interferon-γ. 34. A method of treating, suppressing or altering an immune response comprising a signaling pathway between a Kay ligand and its receptor, comprising the step of administering an effective amount of an agent capable of disrupting the binding between the Kay ligand and its receptor. 35. The method of claim 34, wherein the immune response comprises human adenocarcinoma cells.