JP2002534964A - Mammalian α-helix protein - Google Patents

Abstract

  (57) [Summary] The present invention relates to polynucleotides and polypeptides for mammalian α-helix-12 (Zalpha12). The polypeptides and polynucleotides that encode them are hormonal and can be used to modulate the function of the immune system. The invention also includes antibodies to the Zalpha 12 polypeptide. Antagonists are used to treat inflammation and inflammation-related diseases.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION Inflammation is usually a localized response to harmful agents and damaged tissue that destroys, dilutes, or wallo-offs, trauma or microbial invasion. is there. It is characterized by its conventional signs of pain, fever, redness, swelling and loss of function in the acute form. Microbiologically, it is a complex series of phenomena,
This includes, for example, dilatation of arterioles, capillaries and venules, and enhanced permeability and blood flow, exudation of fluids, such as plasma proteins, and leukocyte migration into areas of inflammation.

[0002] Diseases characterized by inflammation are a significant cause of morbidity and mortality in humans. Normally, inflammation occurs as a protective response to exogenous, especially microbial, invasion of the host. Responses to mechanical trauma, toxins and neoplasia can also result in an inflammatory response. Leukocyte accumulation and subsequent activation is a central phenomenon in the pathogenesis of most forms of inflammation. Loss of inflammation puts the host at risk. Hyperinflammation caused by abnormal recognition of the host tissue as an exogenous substance or prolonged inflammatory processes may include diabetes, arteriosclerosis, cataract, reperfusion injury and cancer, post-infection syndromes such as infectious meningitis, rheumatism, It can lead to fever and various inflammatory diseases such as rheumatic diseases such as systemic lupus erythematosus and rheumatoid arthritis.

[0003] The afferentness of the inflammatory response in these various disease processes makes its regulation a major factor in the prophylactic control or cure of human disease. Thus, there is a need to discover cytokines that contribute to inflammation and inflammation-related diseases, so that antagonists, eg, antibodies, can be administered to down regulate cytokines to improve inflammation.

SUMMARY OF THE INVENTION The present invention addresses this need by providing novel polypeptides and related compositions and methods, and antagonists thereof. In one aspect, the invention provides an isolated polynucleotide encoding a mammalian cytokine designated Zalpha12. The data indicate that cytokines are involved in the inflammatory response. Thus, antagonists of Zalpha 12 may be used to reduce inflammation, especially coronary heart disease, arteriosclerosis, Crohn's disease and inflammatory bowel disease.

[0005] Three variants have been discovered as shown in SEQ ID NOs: 1, 2, 3, 4, 5 and 6.
Each Zalpha 12 polypeptide is derived from amino acid residue 1, ie, methionine,
SEQ ID NOs: 2, 4 and 6 extending to amino acid residue 34, ie, serine (inclusive)
Has a signal sequence of Accordingly, the Zalpha 12 polypeptide represented by SEQ ID NO: 2 extends from amino acid residue 35, ie, alanine, to amino acid residue 202, ie, asparagine (inclusive), and has the mature sequence represented by SEQ ID NO: 8. .

[0006] The Zalpha 12 polypeptide represented by SEQ ID NO: 4 extends from amino acid residue 35, ie, alanine, to amino acid residue 288, ie, asparagine (inclusive), and has the mature sequence represented by SEQ ID NO: 9. Have. The Zalpha 12 polypeptide represented by SEQ ID NO: 6 extends from amino acid residue 35, ie, alanine, to amino acid residue 158, ie, asparagine (inclusive), and SEQ ID NO: 1.
It has the mature sequence represented by 0.

In a second aspect of the present invention, (a) a transcription promoter; (b) Zalpha 12
An expression vector comprising a DNA segment encoding a polypeptide and (c) a transcription terminator, wherein the promoter, DNA segment and terminator are operably linked is provided. In a third aspect of the present invention there is provided a cultured prokaryotic or eukaryotic cell expressing a protein polypeptide encoded by said DNA segment, wherein said expression vector is integrated as disclosed above. .

[0008] In a further aspect of the invention, there is provided a chimeric polypeptide consisting essentially of a first portion and a second portion linked by a peptide bond. The first portion of the chimeric polypeptide is (a) a Zalpha12 polypeptide; (b) an allelic variant of Zalpha12; and (c) at least 80% identical to (a) or (b). Consists essentially of the protein potypeptide. The second portion of the chimeric polypeptide consists essentially of another polypeptide, for example, an affinity tag. In one aspect, the affinity label is an immunoglobulin Fc polypeptide. The invention also provides an expression vector encoding the chimeric polypeptide, and a host cell transfected to produce the chimeric polypeptide.

In a further aspect of the present invention, an antibody that specifically binds to a Zalpha 12 polypeptide as disclosed above, and also an anti-idiotype antibody that neutralizes an antibody to a Zalpha 12 polypeptide Is provided.

[0010] A further aspect of the present invention relates to a peptide or polypeptide having the amino acid sequence of the epitope-bearing portion of a Zalpha 12 polypeptide having the above amino acid sequence. The peptide or polypeptide having the amino acid sequence of the epitope-bearing portion of the Zalpha12 polypeptide of the present invention has at least 9, preferably at least 9,
Includes portions of such polypeptides having 15 and more preferably at least 30-50 amino acids, provided that the epitope-bearing polypeptide has any length up to the entire amino acid sequence of the polypeptide of the invention. Peptides are also included in the present invention. Examples of such epitope-bearing regions include SEQ ID NOs: 18, 19, 20, 25, 26, 27
, 28, 29, 30, 35, 36, 37, 38, 39, 40, 41, 42, 43 and 44. Also provided are another polypeptide or any of those polypeptides fused to a carrier molecule.

[0011] These and other aspects of the invention will become apparent based on the following specific description of the invention.

Specific Description of the Invention: The teachings of all references cited herein are hereby incorporated by reference in their entirety. Before describing the present invention in detail, the following terms may be helpful in understanding the present invention:

An “affinity label” is attached to a second polypeptide to provide for purification or detection of the second polypeptide, or to provide a site for binding of the second polypeptide to a substrate. Used herein to indicate the resulting polypeptide segment. Primarily, antibodies or any peptide or protein for which other specific binding agents are available can be used as an affinity label.

Affinity labels are poly-histidine-based, ie, protein A (Nilsson et al.,
(EMBO J. 4: 1075, 1985; Nilsson et al., Methods Enzymol. 198: 3, 1991).
, Glutathione S transferase (Smits and Johnson, Gene 67; 31, 19
88), Glu-Glu affinity label (Grussenmeyer, etc., Proc. Natl. Acad. Sci. USA
82: 7952-4, 1985), substance P, ie Flag ™ peptide (Hopp et al., B
iotechnology 6: 1204-1210, 1988), streptavidin binding peptides, or other antigenic epitopes or binding domains. Generally, Ford etc., Prot
See ein Expression and Purification 2: 95-107, 1991. Affinity labels on DNA codes can be obtained from commercial suppliers (eg, Pharmacia Biotech, Piscataway, NJ; Eas
tman Kodak, New Heven, CT; New England Biolabs, Beverly, MA).

The term “allelic variant” is used herein to indicate any of a plurality of alternative forms of a gene occupying the same chromosomal locus. Allelic variation occurs naturally through mutation, and can result in phenotypic polymorphisms within a population. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence. The term allelic variant is also used herein to indicate the protein encoded by an allelic variant of a gene.

The terms “amino-terminal” and “carboxyl-terminal” are used herein to indicate a position within a polypeptide. Where the context allows, those terms are used in reference to a particular sequence or portion of a polypeptide to indicate accessibility or relative position. For example, certain sequences located at the carboxyl terminus of a subject sequence in a polypeptide are located adjacent to the carboxyl terminus of the subject sequence, but are not required at the carboxyl terminus of the complete polypeptide.

The term “complement / anti-complement pair” refers to non-identical components that form, under appropriate conditions, non-covalently associated stable pairs. For example, biotin and avidin (or streptavidin) are the prototype members of a complement / anti-complement pair. Other exemplary complement / anti-complement pairs include receptor / ligand pairs, antibody / antigen (or hapten or epitope) pairs, sense / antisense polynucleotide pairs, and the like. If complement pair followed dissociation is desirable, the complement / anti - - complement / anti-complement pair preferably has a binding affinity of <10 ⁹ M ^-1.

The term “complement of a polynucleotide molecule” is a polypeptide molecule that has a complementary base sequence and an opposite orientation as compared to a reference sequence. For example, the sequence 5 'ATGCAC
GGG 3 'is complementary to 5' CCCGTGCAT 3 '. The term "contig" refers to a polynucleotide having a contiguous sequence of identical or complementary sequences to another polynucleotide. A contiguous sequence is said to "overlap" a length of a polynucleotide sequence, in whole or along a portion of the polynucleotide. For example, a polynucleotide sequence
Representative contigs for 5'-ATGGCTTAGCTT-3 'are 5'-TAGCTTgagtct-3' and 3'-gtcgacTACCGA-5 '.

The term “degenerate nucleotide sequence” refers to a sequence of nucleotides (as compared to a control polynucleotide that encodes a polypeptide) that includes one or more degenerate codons. Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (ie, GAU and GAC triplets are each Asp
Code).

The term “expression vector” is used to indicate a linear or circular DNA molecule comprising a segment encoding a polypeptide of interest operably linked to an additional segment that provides for its transcription. You. Such additional segments include promoter and terminator sequences and one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors generally can be derived from plasmid or viral DNA, or can include elements of both.

The term “isolated” when applied to a polynucleotide, removes the polynucleotide from its natural genetic environment and, therefore, has no other extraneous or unwanted coding sequences. And exist in a form suitable for use in a genetically engineered protein production system. Such isolated molecules are those that have been separated from their natural environment and include cDNA and genomic clones. An isolated DNA molecule of the present invention does not contain other genes which are not normally involved, but can contain naturally occurring 5 'and 3' untranslated regions, such as promoters and terminators. Identification of relevant regions will be apparent to those of skill in the art (see, for example, Dynan and Tijan, Nature 316: 774-78, 1985).

An “isolated” polypeptide or protein may have conditions other than its native environment,
For example, polypeptides or proteins found under conditions that are separate from blood and animal tissues. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, especially other polypeptides of animal origin. Highly refined form,
That is, it is preferable to supply the polypeptide with a purity of 95% abnormal, more preferably a purity of 99% or more. As used in this context, the term "isolated"
It does not exclude the presence of the same polypeptide in other physical forms, such as a dimeric form or other glycosylated or derivatized form.

“Operably linked” when applied to a DNA segment, said segments function cooperatively for their intended purpose, eg, transcription is initiated at a promoter, and Indicates that it is arranged to proceed to the terminator through the code segment. The term "orthology" refers to a polypeptide or protein from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences between ortho-forms are the result of specification.

“Paralogs” are different but structurally related proteins produced by an organism. Para bodies appear to occur through gene duplication. For example, α-globin, β-globin and myoglobin are paraboloids of each other.

“Polynucleotide” is a single or double stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5 ′ end to the 3 ′ end. Polynucleotides include RNA and DNA, and can be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. Polynucleotide sizes are expressed as base pairs (abbreviation "bp"), nucleotides ("nt"), or kilobases ("kb").

Here, the latter two terms describe polynucleotides that are single-stranded or double-stranded. When the term is applied to double-stranded molecules, it will be used to indicate the overall length and will be understood to be equivalent to the term "base pairs". It is understood by those skilled in the art that the two strands of a double-stranded polynucleotide differ slightly in length, and that their ends differ as a result of enzymatic degradation;
Not all nucleotides within a double-stranded polynucleotide molecule can be paired. Such unpaired ends do not exceed 20 nt in length.

A “polypeptide” is a polymer of amino acid residues linked by peptide bonds, whether produced naturally or synthetically. Polypeptides of up to about 10 amino acid residues are commonly referred to as "polypeptides." The term "promoter" is used herein to indicate a portion of a gene that contains a DNA sequence that provides for the binding of RNA polymerase and initiation of transcription. A promoter sequence is usually, but not necessarily, found in the 5 'non-coding region of a gene.

A “protein” is a macromolecule that comprises one or more polypeptide chains.
Proteins can also contain non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptide substituents will be added by the cell in which the protein is produced, and will vary with the cell type. Proteins are defined herein by their amino acid backbone; substituents, such as carbohydrate groups, are generally not specified, but can nevertheless be present.

The term “receptor” refers to a cell-associated protein that binds a bioactive molecule (ie, “ligand”) and mediates the effect of the ligand on a cell. Membrane-bound receptors
It is characterized by a multi-peptide structure comprising an extracellular ligand binding domain and, typically, an intracellular effector domain involved in signal transduction. Binding of the ligand to the receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecules in the cell. This interaction induces a change in the metabolism of the cell.

Metabolic phenomena linked to receptor-ligand interactions include gene transcription, phosphorylation,
Includes dephosphorylation, increased AMP production, cellular calcium metabolism, membrane lipid metabolism, cell attachment, inositol lipid hydrolysis, and phospholipid hydrolysis. Generally, receptors are membrane-bound, cytosolic or nuclear; monomers (e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor), or multimers (e.g.,
PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor).

The term “secretory signal sequence” refers to a DNA encoding a polypeptide (“secretory peptide”) that directs a larger polypeptide as a component of a larger polypeptide through the secretory pathway of the cell in which it is synthesized. Shows the sequence. The larger polypeptide is usually degraded during transit through the secretory tract to remove the secreted peptide.

The term “splice variant” is used herein to indicate an alternative form of RNA transcribed from a gene. Splice mutations occur in transcribed RNA
Through the use of alternative splicing sites between intramolecular or normally low, but separately transcribed RNA molecules, some mRNA may occur naturally and be transcribed from the same gene. Splice variants can encode polypeptides having an altered amino acid sequence. The term splice variant is also used herein to indicate the protein encoded by the splice variant of mRNA transcribed from the gene.

It will be appreciated that the molecular weight and length of the polymer as determined by inaccurate analytical methods (eg, gel electrophoresis) are approximate. Where such a value is expressed as “about” X or “approximately” X, it will be understood that the stated value of X is exactly ± 10%.

Introduction: The Zalpha 12 protein is an α-helical protein. SEQ ID NO: 2 is 4
It has species α-helices A, B, C and D. Helix A of SEQ ID NO: 2 extends from amino acid residue 56, ie, leucine, to amino acid residue 70, ie, isoleucine (including), also defined by SEQ ID NO: 21. Helix B of SEQ ID NO: 2 extends from amino acid residue 96, ie, leucine, to amino acid residue 110, ie, tyrosine (inclusive), and is also defined by SEQ ID NO: 22.

Helix C of SEQ ID NO: 2 extends from amino acid residue 130, ie, leucine, to amino acid residue 144, ie, leucine (inclusive), and is also defined by SEQ ID NO: 23. Helix D of SEQ ID NO: 2 extends from amino acid residue 162, ie, methionine, to amino acid residue 176, ie, serine (inclusive), and is also defined by SEQ ID NO: 24. SEQ ID NO: 25 contains helix A + B + C; SEQ ID NO: 26 contains helix A + B + C + D; SEQ ID NO: 28 contains helix B + C + D; SEQ ID NO: 29 contains helix B + C; and SEQ ID NO: 30 Contains helix C + D of SEQ ID NO: 2.

The polypeptide of SEQ ID NO: 4 also includes four helices A, B, C and D. Helix A of SEQ ID NO: 4 extends from amino acid residue 45, histidine, to amino acid residue 59, leucine (inclusive), and is also defined by SEQ ID NO: 31. Helix B of SEQ ID NO: 4 extends from amino acid residue 116, valine, to amino acid residue 130, lysine (inclusive), and is also defined by SEQ ID NO: 32. Helix C of SEQ ID NO: 4 extends from amino acid residue 142, ie, leucine, to amino acid residue 156, ie, isoleucine (includes), SEQ ID NO: 33
Is also defined by

Helix D of SEQ ID NO: 4 extends from amino acid residue 182, leucine, to amino acid residue 196, tyrosine (inclusive), and is defined by SEQ ID NO: 34. SEQ ID NO: 35 comprises helix A + B of SEQ ID NO: 4. SEQ ID NO: 36 includes helices A + B + C of SEQ ID NO: 4. SEQ ID NO: 37 is the helix A of SEQ ID NO: 4
+ B + C + D. SEQ ID NO: 38 comprises helix B + C + D of SEQ ID NO: 4. SEQ ID NO: 39 comprises helix B + C of SEQ ID NO: 4. SEQ ID NO: 4 contains the helix C + D of SEQ ID NO: 4.

[0038] Expression of the Zalph 12 gene can be detected in many different tissues, such as spleen, thymus, testis,
It is found in the small intestine, peripheral blood lymphocytes (PBL), stomach, trachea, T-cells such as CD4 ⁺ and CD8 ⁺ cells, and bone marrow. This pattern of expression indicates that Zalpha 12 plays a general role in growth and exerts key regulatory controls on testicular differentiation, hypothalamus-pituitary-gonad axis, and gonadal steroidogenesis and spermatogenesis. Suggest.

The progression of testicular hormone formation is divided into early and late stages, and the latter relies on the activation of functionally determined Leydig cell precursors by luteinizing hormone (LH). However, the factors controlling the early stages of this process remain unknown, and Huhtaniemi, Reprod. Fertil. Dev. 7: 1025-1035 (1995)
This suggests that pha 12 can be responsible for non-steroidogenic, non-LH responsive precursor cell activation.

When Leydig cell differentiation occurs, the production of steroid hormones in the testis is
It depends on the secretion of gonadotropin, LH and follicle-stimulating hormone (FSH) by the pituitary gland. LH stimulates the production of testosterone by Leydig cells, and spermatogenesis depends on both FSH and high testicular testosterone concentrations. LH and FSH secretion is under the control of gonadotropin releasing hormone (GnRH) produced in the hypothalamus. Kaufman, The neuro endocrine regulation of male reproduction.In:
Male Infertility.Clinical Investigation, Cause Evaluation and Treatment
., FH Comhaire, ed., Pp. 29-54 (Chapman and Hall, London, 1996). Since testis products have been shown to control LH and FSH production,
This suggests that alpha 12 regulates hormone production by the hypothalamus.

It is well known that steroidogenesis and spermatogenesis occur in two different cellular compartments of the testis, and Leydig and Sertoli cells are responsible for the former and the latter, respectively. See Saez, Endocrin. Rev. 15: 574-626 (1994). The individual activities of those cell types appear to be regulated by other secreted products. Sertoli cell-derived tumor necrosis factor-a, fibroblast growth factor, interleukin-1, transforming growth factor-B, epidermal growth factor / transforming growth factor-a, activin, inhibin, insulin-like growth factor-1 , Platelet-derived growth factors, endothelin and arginine-vasopressin have all been shown to regulate Leydig cell function. See Seaz, Endocrin. Rev. 15: 574-626 (1994). Thus, Zalpha 12 can control or regulate the activity of one or more of those genes.

In men, aging is associated with a progressive decline in testicular function. These changes are clinically apparent due to reduced fertility, fertility and libido, indicating relative testicular deficiency. Vermeulen, Ann.Med. 25: 531-534 (1993); Pugea
t et al., Horm. Res. 43: 104-110 (1995). Hormone replacement therapy in elderly men is not currently recommended, but suggests that new therapies for male menopause are very valuable.

Polynucleotides: The present invention also provides polynucleotide molecules, such as DNA and RNA molecules, that encode a Zalpha 12 polypeptide disclosed herein. One skilled in the art will readily recognize that, in light of the degeneracy of the genetic code, considerable sequence variation is possible between those polynucleotide molecules. A polynucleotide, generally a cDNA sequence, of the present invention encodes a polypeptide according to the present invention. The cDNA sequence encoding a polypeptide of the invention is composed of a series of codons, each amino acid residue of the polypeptide is encoded by a codon, and each codon is composed of three nucleotides. Amino acid residues are encoded by their respective codons, as follows.

Alanine (Ala) is encoded by GCA, GCC, GCG or GCT; Cysteine (Cys) is encoded by TGC or TGT; Aspartic acid (Asp) is encoded by GAC or GAT; Glutamic acid (Glu ) Is encoded by GAA or GAG; Phenylalanine (Phe) is encoded by TTC or TTT; Glycine (Gly) is encoded by GGA, GGC, GGG or GGT; Histidine (His) is encoded by CAC or CAT Isoleucine (Ile) is encoded by ATA, ATC or ATT; Lysine (Lys) is encoded by AAA or AAG;

Leucine (Leu) is encoded by TTA, TTG, CTA, CTC, CTG or CTT; Methionine (Met) is encoded by ATG; Asparagine (Asn) is encoded by AAC or AAT; Proline ( Pro) is encoded by CCA, CCC, CCG or CCT; Glutamine (Gln) is encoded by CAA or CAG; Arginine (Arg) is encoded by AGA, AGG, CGA, CGC, CGG or CGT; (Ser) is encoded by AGC, AGT, TCA, TCC, TCG or TCT; Threonine (Thr) is encoded by ACA, ACC, ACG or ACT; Valine (Val) is GTA, GTC, GTG or GTT Tryptophan (Trp) is encoded by TGG; and tyrosine (Tyr) is encoded by TAC or TAT.

According to the present invention, where the polynucleotides are claimed as described herein, the claimed is annealed together by sense strands, antisense strands, and their respective hydrogen bonds. It should be understood that the DNA is double stranded with the sense and antisense strands. Messenger RNA encoding a polypeptide of the invention and encoded by a cDNA described herein
(MRNA) is also claimed. mRNA encodes a polypeptide using the same codons as those defined herein, except that each thymine nucleotide (T) is replaced by a uracil nucleotide (U).

Those skilled in the art will also appreciate that different species exhibit "alternative codon usage." Generally, Grantham, et al., Nuc. Acids Res. 8: 1893-912, 1980;
Haas, et al., Curr. Biol. 6: 315-24, 1996; Wain-Hobson, et al., Gene 13.
: 355-64, 1981; Grosjean and Fiera, Gene 18: 199-209, 1982; Holm, Nu.
c. Acids Res. 14: 3075-87, 1986; Mol. Biol. 158: 573-97
1982. As used herein, the term "alternative codon usage" or "alternative codon" refers to the possible codons that are used most frequently in certain types of cells and thus encode individual amino acids. Technical term referring to protein translation codons that favor one or a few representatives (see Table 2).

For example, the amino acid threonine (Thr) is encoded by ACA, ACC, ACG, or ACT, but in mammalian cells, ACC is the most commonly used codon; in other species, For example, in insect cells, yeast, viruses or bacteria, different Thr codons are preferred. Alternative codons for a particular species can be introduced into a polynucleotide of the invention by various methods known in the art. For example, the introduction of alternative codon sequences into recombinant DNA enhances the production of the protein by rendering it more efficient in a particular cell type or species. Sequences containing selection codons can be tested for expression in various species and tested for functionality disclosed herein.

In a preferred embodiment of the present invention, the isolated polypeptide comprises SEQ ID NO: 1,2
Alternatively, it will hybridize under stringent conditions to a similarly sized region of 5, or a sequence complementary thereto. In general, the stringency conditions are selected to be about 5 ° C. below the defined ionic strength and thermal melting point (Tm) for the particular sequence. Tm is the temperature (under defined ionic strength pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typical stringent conditions are those wherein the salt concentration is up to about 0.03 at pH 7 and the temperature is at least about 60 ° C.

[0050] As indicated above, the isolated polynucleotides of the present invention include DNA and RNA. Methods for preparing DNA and RNA are well known in the art. Generally, RNA is isolated from tissues or cells that produce large amounts of Zalpha 12 RNA. Such tissues and cells are obtained from Northern blots (Thomas, Proc.
Natl. Acad. Sci. USA 77: 5201, 1980) and is discussed below. Total RNA can be prepared by guanidinium Hcl extraction, followed by isolation by centrifugation on a CsCl gradient (Chirgwin et al., Biochemistry 18: 52-94, 1979).
).

Poly (A) ⁺ RNA is obtained from Aviv and Leder (Proc. Natl. Acad. Sci. USA 69: 1408-
1412, 1972). Complementary DNA (cDNA) is prepared from poly (A) ⁺ RNA using known methods. On the other hand, genomic DNA can be isolated. Next, a polynucleotide encoding a Zalpha 12 polypeptide is
Identified and isolated, for example, by hybridization or polymerase chain reaction.

[0052] Full-length clones encoding Zalpha12 polypeptides can be obtained by conventional cloning methods. Complementary DNA (cDNA) clones are preferred, provided that
For some uses (eg, expression in transgenic animals)
Use of genomic clones or cDs containing at least one genomic intron
It is preferred to modify the NA clone. Methods for preparing cDNA and genomic clones are well-known and within the level of ordinary skill in the art, and the sequences disclosed herein for probing or sensitizing libraries. Or a partial use thereof. Expression libraries can be probed with an antibody to Zalpha12, a receptor fragment, or other specific binding partner.

The polynucleotide of the present invention can also be synthesized using a DNA synthesizer. Currently, the method of choice is the phosphoramidite method. Chemically synthesized double-stranded DNA
Is required for the synthesis of a gene or gene fragment, the individual complementary strands are produced separately. The generation of short genes (60-80 bp) is technically straightforward and can be achieved by synthesis of complementary strands and subsequent annealing thereof. However, for the production of longer genes (> 300 bp)
Certain methods are desirable because the coupling efficiency of individual cycles during NA synthesis is rarely 100%. To overcome this problem, synthetic genes (double-stranded) are assembled in a controlled fashion from single-stranded fragments that are 20 to 100 nucleotides in length.

Glick and Pasternak, Molecular Biotechnology, Principles & Application
s of Recombinant DNA, (ASM Press, Washington, DC 1994); Itakura et al.,
Annu. Rev. Biochem. 53: 323-56, 1984 and Climie et al., Proc. Natl. Acad.
Sa. USA 87: 633-7, 1990.

The present invention further provides counterpart ligands and polynucleotides from other species (ortho-forms). These species include, but are not limited to, mammals, birds, amphibians, reptiles, fish, insects and other vertebrate and invertebrate species.
Of particular interest are Zalpha 12 polypeptides from other mammalian species, for example, mice, pigs, sheep, cows, dogs, cats, horses, and other primate ligands. Human Zalpha
Orthoforms of the 12 polypeptides can be cloned using the information and compositions provided by the present invention in combination with conventional cloning techniques.

For example, cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses Zalpha 12 as disclosed herein. A suitable source of mRNA is
It can be identified by probing a Northern blot with a probe designed from the sequences disclosed herein. Next, a library is prepared from mRNA of positive tissues or cell lines. The Zalpha12-encoding cDNA can then be transformed in various ways, such as by complete or partial human cDNA, or based on the disclosed sequences above.
Alternatively, it can be isolated by probing with a plurality of denaturing probes.

The cDNA can also be prepared using the polymerase chain reaction (PCR) (Mulli) using primers designed from the representative human Zalpha 12 polynucleotide sequences disclosed herein.
s, U.S. Pat. No. 4,683,202). In a further method, a cDNA library is used to transform or transfect host cells, and expression of the cDNA of interest can be detected by an antibody against the Zalpha12 polypeptide. Similar techniques can also be applied to isolate genomic clones.

One of skill in the art will recognize that the sequences disclosed in SEQ ID NOs: 1, 3 or 5 represent particular alleles of human Zalpha 12 and are expected to result in allelic variation and alternating splicing. Will. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard methods. Allelic variants of the DNA sequence shown in SEQ ID NO: 1, such as those containing a silent mutation and those in which the mutation results in an amino acid sequence alteration, are allelic variants of SEQ ID NO: 2, 4, or 6. As with the body protein, it is within the scope of the present invention.

Another spliced mRNA that retains the properties of a Zalpha 12 polypeptide.
CDNAs, as well as polypeptides encoded by such cDNAs and mRNAs, are included within the scope of the present invention. Allelic and splice variants of those sequences can be prepared according to standard methods known in the art.
It can be cloned by probing cDNA or genomic libraries from different individuals or tissues.

The present invention also provides polypeptides of SEQ ID NOs: 2, 4, 6, 8, 9 or 10, and isolated Zalpha 12 polypeptides that are substantially identical to their ortho forms. . The term "substantially identical" refers to 50%, 60%, 70%, 80%, and most preferably 50%, 60%, 70%, 80%, or Is used herein to indicate a polypeptide having at least 90%, 95% or 99% sequence identity. % Sequence identity is determined by conventional methods.

For example, Altschul et al., Bull. Math. Bio. 48: 603-616, 1986 and henikof.
f and Henikoff, Pruc. Natl. Acad. Sci. USA 89: 10915-10919, 1992. Briefly, two amino acid sequences have a gap opening penalty of 10, a gap extension penalty of 1, and a Henikoff and Henikoff (as described in Table 1 (amino acids are indicated by the standard one letter code)). ) 'S “blosum
Using the 62 "scoring matrix, it is matched to optimize its matching score. The percent identity is then calculated as follows:

[0062]

(Equation 1)

[0063]

[Table 1]

The skilled person understands that there are many established algorithms for aligning two amino acid sequences. Pearson and Lipman's “FASTA” similarity search algorithm uses an appropriate protein alignment method to test the level of identity shared by the amino acid sequences disclosed herein and the amino acid sequence of the putative variant. is there. The FASTA algorithm is based on Pearson and Lipman, Proc.
Acad. Sci. USA 85: 2444 (1988), and Pearson, Meth. Enzymol. 183: 63 (199).
0).

Briefly, FASTA first determines the highest density of identity (when the ktup variable is 1) without considering the sequence in question (eg, SEQ ID NO: 2) and conservative amino acid substitutions, insertions or deletions. Alternatively, characterize the sequence by identifying regions shared by test sequences having either pairwise identity (if ktup = 2). Next, the ten regions with the highest density of identity are reevaluated by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions It is "trimmed" to include only those residues that contribute to the score.

If there are some regions with a score higher than the “cutoff” value (calculated by a formula that is based on the length of the sequence and the ktup value), the trimmed initial region will be , Are tested to determine if the region can be joined to form an approximate alignment with the gap. Finally, Needleman-Wunsch, where the highest scoring regions of the two amino acid sequences allow for amino acid insertions and deletions.
Algorithm (Needleman and winsch, J. Mol. Biol. 48: 444, 1970; Sellers
, SIAM J. Appl. Math. 26: 787, 1974).

Exemplary parameters for FASTA analysis are: ktup = 1, gap start penalty = 10, gap extension penalty = 1 and substitution matrix = BLOSUM62. These parameters are described in Appendix 2 of Pearson, 1990 (supra).
Can be introduced into the FASTA program by adjusting the rating matrix, as described in

[0068] FASTA can also be used to determine the sequence identity of nucleic acid molecules, using ratios as disclosed above. For nucleotide sequence comparisons, ktup values range from 1-6, preferably 4-6, with other parameters set as errors.
Can be

The present invention includes a nucleic acid molecule that encodes a polypeptide that has one or more conservative amino acid changes compared to the amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 9 or 10. The BLOSUM62 table is an amino acid substitution matrix derived from approximately 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of over 500 groups of related proteins [Henikoff and Henikoff, Proc. Nat'l Acad
Sci. USA 89: 10915 (1992)]. Thus, the BLOSUM62 substitution frequency can be used to define conservative amino acid substitutions that can be introduced into the amino acid sequences of the invention.

As used herein, the term “conservative amino acid substitution” refers to a substitution represented by a BLOSUM62 value greater than −1. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3. Preferred conservative amino acid substitutions are at least 1 (eg, 1, 2, or 3
), But more preferably the conservative substitutions are characterized by a BLOSUM62 value of at least 2 (eg, 2 or 3). The sequence identity of the polynucleotide molecules may be determined using ratios as disclosed above,
Determined in a similar manner.

[0071] Variant Zalpha12 polypeptides and substantially homologous Zalpha12 polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably conservative amino acid substitutions (see Table 2) and other substitutions that do not substantially affect the folding or activity of the protein and polypeptide; small deletions, typically 1 ~ 30 amino acid deletions; and small amino- or carboxyl-terminal extensions such as amino-terminal methionine residues, small linker peptides of up to about 20-25 residues, or affinity labeling. It is an extension.

Thus, the present invention provides for at least 90%, preferably at least 95%, and more preferably at least 99% or more of its corresponding region of SEQ ID NO: 2, 4, 6, 8, 9 or 10 A polypeptide of 20 to 30 amino acid residues comprising a sequence that is identical to Polypeptides comprising an affinity tag further comprise a proteolytic site between the Zalpha 12 polypeptide and the affinity tag. Preferred such sites include thrombin degradation sites and factor Xa degradation sites.

[0073]

[Table 2]

The present invention further provides various other polypeptide fusions (and related multimeric proteins comprising one or more polypeptide fusions). For example,
Zalpha 12 polypeptides can be prepared as fusions to dimeric proteins as disclosed in US Pat. Nos. 5,155,027 and 5,567,584. Preferred dimer proteins therefor include an immunoglobulin constant region domain. The immunoglobulin-Zalpha12 polypeptide fusions can be expressed in various multimeric zfsta2
To produce analogs, they can be expressed in genetically engineered cells. Auxiliary domains can be fused to Zalpha12 polypeptides to target them to particular cells, tissues or macromolecules (eg, collagen).

For example, a Zalpha 12 polypeptide or protein can be targeted to a predetermined cell type by fusing the Zalpha 12 polypeptide to a ligand that specifically binds to a receptor on the surface of a target cell. In this case, the polypeptides and proteins can be targeted for therapeutic or diagnostic purposes. A Zalpha 12 polypeptide can be fused to multiple components, for example, an affinity tag for purification and a targeting domain. Polypeptide fusions can also include one or more cleavage sites, particularly between domains. See Tuan et al., Connective Tissue Research 34: 1-9, 1996.

[0076] The proteins of the invention also comprise non-naturally occurring amino acid residues. Non-naturally occurring amino acids include trans-3-methylproline, 2,4-metaproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N
-Methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidinecarboxylic acid, dehydroproline, 3- and 4-
Methylproline, 3,3-dimethylproline, tert-leucine, norvaline, 2
-Azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine and 4-fluorophenylalanine. Several methods are known in the art for introducing non-naturally occurring amino acid residues into proteins. For example, a suppressor in which a nonsense mutation is chemically aminoacylated.
In vitro systems that are suppressed using tRNA can be used.

[0077] Methods for synthesizing amino acids and aminoacylating tRNA are known to those skilled in the art. Transcription and translation of the plasmid containing the nonsense mutation is performed in a cell-free system comprising E. coli S30 extract and commercially available enzymes and other reagents. The protein is purified by chromatography.
For example, Rovertson et al., J. Am. Chem. Soc. 113: 2722, 1991; Ellman et al.,
Meth. Enzymol. 202: 301, 1991; Chung et al., Science 259: 806-09, 1993;
And Chung et al., Proc. Natl. Acad. Sci. USA 90: 10145-49, 1993.

In a second method, translation is performed in Xenopus oocytes by microinjection of mutagenized mRNA and chemically aminoamylated suppressor-tRNA (Turcatti et al., J. Biol. Chem. 271:
1991-98, 1996). In a third method, E. coli cells are used in the absence of the natural amino acid to be replaced (eg, phenylalanine) and the desired non-naturally occurring amino acid (eg, 2-azaphenylalanine, 3-aza). Phenylalanine, 4-azaphenylalanine or 4-fluorophenylalanine).

[0079] Non-naturally occurring amino acids are introduced into proteins in place of their natural counterparts. See Koide et al., Biochem. 33: 7470-46, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modifications can be combined with site-directed mutagenesis to further extend the scope of the substitution (Wynn and Richards, Protein Sci. 2: 395-403,
1993). A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids can be replaced by Zalpha 12 amino acids.

Essential amino acids in the polypeptides of the present invention can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081- 1085, 1989; Bass et al., Proc. Natl. Scad. Sci. USA 88: 4498-502, 1991). In the latter technique, a single alanine mutation is introduced at every residue in the molecule, and the resulting mutant molecule is used to identify amino acid residues that are critical for the activity of the molecule. , Tested for biological activity as disclosed below.

See also, Hilton et al., J. Biol. Chem. 271: 4699-5708, 1996.
The site of ligand-receptor interaction can also be determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling for mutations of putative contact site amino acids. For example, de Vos, etc., Science 255: 306-312, 1992; Sm
ith et al., J. Mol. Biol. 224: 899-904, 1992; Wlodaver et al., FEBS Lett.
309: 59-64, 1992.

Multiple amino acid substitutions can be made by known methods of mutagenesis and screening, eg, Re
idhaar-Olson and Sauer (science 241: 53-57, 1988) or Bowie and Sauer
(Proc. Natl. Acad. Sci. USA 86: 2152-2156, 1989). Briefly, the authors found that co-randomizing multiple positions in a polypeptide, screening for functional polypeptides, and then suddenly determining the range of possible substitutions at individual positions. Disclosed are methods for sequencing a mutagenized polypeptide. Other methods that can be used include phage display (eg, Lowman et al., Biochem. 30: 10832-10837).
Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/062.
No. 04), and region-directed mutagenesis (Derbyshire et al., Gene 46:14).
5, 1986; Ner et al., DNA 7: 127, 1988).

Variants of the disclosed Zalpha 12 DNA and polypeptide sequences are described in Stemmer, Nature
370: 389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91: 10747-51,
It can be generated through DNA shuffling as disclosed by 1994 and WIPO publication WI97 / 20078. Briefly, mutant DNA is produced by random fragmentation of the parent DNA, resulting in randomly introduced point mutations, followed by in vitro homologous recombination by assembly using PCR. This technique can be improved with families of parent DNA, eg, allelic variants or DNA from different species, to introduce additional variability during the process. Selection or screening of the desired activity, followed by further interaction of the mutagenesis and assay, provides rapid "evolution" of the sequence by selecting for the desired mutation while simultaneously selecting for deleterious changes. I do.

The mutagenesis method as disclosed herein is combined with a high-throughput automated screening method to detect the activity of the cloned mutagenized polypeptide in a host cell. Can be done. Mutagenized DNA molecules encoding the active polypeptide can be recovered from the host cells and immediately sequenced using modern equipment. These methods allow for the rapid determination of the importance of individual amino acid residues in a polypeptide of interest and can be applied to polypeptides of unknown structure.

Using the methods discussed herein, one of skill in the art has identified various polypeptide fragments of SEQ ID NO: 2, 4 or 6 that retain the properties of the wild-type Zalpha 12 protein and / or Can be prepared. For any Zalpha12 polypeptide, including variants and fusion proteins, one of skill in the art can use the information provided in Tables 1 and 2 above to prepare a sufficient degenerate polynucleotide sequence encoding the variant. Can be generated.

Protein Production: Zalpha12 polypeptides of the present invention, eg, full-length polypeptides, biologically active fragments and fusion polypeptides, can be produced in genetically constructed host cells according to conventional techniques. Can be generated. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, especially cultured cells of multicellular organisms, are preferred. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into various host cells are disclosed in Sambrool et al., Molecular Cloning: A Laborato
^{ry Manual, 2 nd ed.,} Cold Spring Harbor Laboratory Press, Cold Spring Har
bor, NY, 1989, and Ausubel et al., eds., Current Protocol in Molecular Bi.
ology, John Wiley and Sons, Ins., NY, 1987.

In general, a DNA sequence encoding a zfsta2 polypeptide of the present invention acts on other genetic elements required for its expression, such as, in general, transcription promoters and terminators in expression vectors. Linked as possible. The vector will also usually include one or more selectable markers and one or more origins of replication,
However, one of skill in the art will recognize that, within certain systems, the selectable marker can be provided on a separate vector, and replication of the exogenous DNA can be provided by integration into the host cell genome. Promoters, terminators, selectable markers,
The choice of vector and element is routine within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.

To direct a Zalpha 12 polypeptide into the secretory tract of a host cell, a secretory signal sequence (or also known as a signal sequence, leader sequence, prepro sequence or pre sequence) is added to the expression vector. Supplied. The secretory signal sequence is
Zalpha 12 or another secreted protein (eg, t-P
It can be derived from A) or newly synthesized.

The secretory signal sequence is operably linked to the Zalpha 12 DNA sequence, ie,
The two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polynucleotide into the secretory pathway of the host cell. Secretory signal sequences are usually located 5 'to the DNA sequence encoding the polypeptide of interest, although certain secretory signal sequences can be located elsewhere in the DNA sequence of interest (eg, Welch No. 5,037,743; Holland et al.
No. 5,143,830).

On the other hand, a secretory signal sequence contained in a polypeptide of the present invention is used to direct another polypeptide into the secretory tract. The present invention provides such fusion polypeptides. The secretory signal sequence contained in a fusion polypeptide of the invention is preferably fused amino-terminally to the additional peptide in order to direct the additional peptide in the secretory tract. Such structures have many uses known in the art. For example, these novel secretory signal sequence fusion constructs can direct the secretion of proteins that are not normally secreted, eg, the active component of the receptor. Such fusions can be used in vivo or in vitro to direct the peptide through the secretory tract.

A cultured mammalian cell or a suitable host within the present invention. Exogenous DNA,
Methods for introduction into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14: 725, 1978; Corsaro and Pearson, Som.
atic Cell Genetics 7: 603, 1981; Graham et al., Virology 52; 456, 1973),
Electroporation (Neumann et al., EMBO J. 1: 841-845, 1982); DEAE
Dextran-mediated transfection (Ausubel et al., Supra), and liposome-mediated transfection (Hawley-Nelson et al., Focus 15: 73, 1).
993; Ciccarone et al., Focus 15:80, 1993).

The production of recombinant polypeptides in cultured mammalian cells has been described, for example, by Levinson
U.S. Patent No. 4,713,339; Hagen et al., U.S. Patent No. 4,784,950
No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cells are COS-
1 (ATCC No. CRL 165), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 16
32), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham
J. Gen. Viro. 36: 59-72, 1977), and the Chinese hamster ovary (eg, CHO-K1; ATCC No. CCL61) cell line.

[0093] Additional suitable cell lines are known in the art and are available from public depositories, such as the American Type Culture Collection, Rockville, Maryland. Generally, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus. See, for example, U.S. Pat. No. 4,956,288. Other suitable promoters include those from the metallothionein gene (U.S. Pat. Nos. 4,579,821 and 4,601,978), the adenovirus major late promoter.

[0094] Drug selection is generally used to select for cultured mammalian cells into which exogenous DNA has been inserted. Such cells are usually "transfectants"
Is referred to as Cells that are cultured in the presence of the selection agent and are capable of transmitting the gene of interest to their progeny are referred to as "suitable transfectants." A preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. The selection is performed in the presence of a neomycin-type drug, such as G-418 or the like.

The selection system, a method referred to as “amplification,” is also used to increase the level of expression of the gene of interest. Amplification involves culturing the transfectant in the presence of low levels of the selection agent, and then increasing the amount of the selection agent to select for cells that produce high levels of the introduced gene product. Will be implemented.
A preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate.

[0096] Other drug resistance genes (eg, hygromycin resistance, multiple drug resistance, puromycin acetyltransferase) can also be used. Other markers that introduce an altered phenotype, such as green fluorescent protein, or cell surface proteins, such as CD4, CD8, class I MHC, placental alkaline phosphatase, can be trans-transformed by means such as FACS classification or magnetic bead separation techniques. It can be used to classify untransfected and transfected cells.

[0097] Other higher eukaryotic cells, such as insect cells, plant cells and bird cells, can also be used as hosts. The use of Agrobacterium rhizogenes as a vector to express genes in plant cells is described by Sinka
r, et al., J. Biosci. (Bangalore) 11: 47-58, 1987.
Transformation of insect cells, and the production of foreign polypeptides therein, has been described by Guarino
No. 5,162,222; and WIPO Publication WO 94/06463. Insect cells are Autographa californica
It can be infected by a recombinant baculovirus normally derived from nuclear polyhedrosis virus (AcNPV).

[0098] DNA encoding the Zalpha 12 polypeptide can be obtained from AcNPV by one of two methods.
It is inserted into the baculovirus genome in place of the polyhedrin gene coding sequence. The first method is a conventional method of homologous DNA recombination between a wild-type AcNPV and a transfer vector containing zfsta2 having an AcNPV sequence at its end. Appropriate insect cells, eg, SF9 cells, are infected with wild-type AcNPV and operably linked to the AcNPV polyhedrin gene promoter, terminator and flanking sequences.
Transfected with a transfer vector comprising a Zalpha 12 polynucleotide.

King, LA and Possee, RD, The Baculovirus Exprossion System: A Lab
oratory Guide, London, Chapman &Hall;O'Reilly, DR., Baculovirus E
xpression Vector: A Laboratory Manual, New York, Oxford University Press
., 1994; and Richardson, CD, Ed., Baculovirus Expression Protocols. M
See ethods in Molecular Biology, Totowa, NJ, Humana Press, 1995. Natural recombination in insect cells is achieved by Zalp driven by the polyhedrin promoter.
would result in a recombinant baculovirus containing ha 12. Recombinant virus stocks are produced by methods commonly used in the art.

A second method for producing recombinant baculovirus is Luckow (Luckow, V
A, et al., J. Virol 67: 4566-79, 1993). This system is based on the Bac-to-Bac ™ kit (Lif
e Technologies, Rockville, MD). This system is
To transfer the DNA encoding the Zalpha12 polypeptide into the baculovirus genome maintained in E. coli as a large plasmid called the "bacmid", a transfer vector containing the Tn7 transposon, pFastBacI (TM) (
Life Technologies).

The PFastBacl ™ transfer vector contains the gene of interest, in this case, Z
The AcNPV polyhydrin promoter is used to drive the expression of alpha12.
However, pFastBacl ™ can be modified to a considerable extent. The polyhydrin promoter is removed and expressed early in baculovirus infection, and the baculovirus basic protein promoter known to be advantageous for expressing secreted proteins (also Pcor, p6.9
Or also known as the MP promoter). Hill-Perkin
s, MS and Possee, RD, J. Gen. Virol. 71: 971-6, 1990; Bonning, BC
J. Gen. Virol. 75: 1551-6, 1994; and Chazenbalk, GD, and Ra.
See poport, B., J. Biol Chem. 270: 1543-9,1995.

[0102] In such transfer vector constructs, short or long versions of the basic protein promoter may be used. In addition, transfer vectors can be constructed in which the secretory signal sequence from an insect protein has replaced the native Zalpha 12 secretory signal sequence. For example, ecdysteroid glucosyltransferase (EGT), honeybee Melittin (Invitrogen, Carlsba
d, CA) or baculovirus gp67 (PharMingem, San Diego, CA)
It can be used in constructs to replace the pha12 secretion signal sequence. further,
Transfer vectors can include an epitope tag at the C- or N-terminus of the expressed Zalpha12 polypeptide, for example, an in-frame fusion with DNA encoding a Glu-Glu epitope tag (Grussenmeyer, T. et al. , Peoc. Natl.
Acad. Sci. 82: 7952-6, 1985).

Using techniques known in the art, E. coli is transformed with a transfer vector containing Zalpha 12 and screened for a bacmida containing the intermittent lacZ gene, a representation of a recombinant baculovirus. You. A bacmid DNA containing the recombinant baculovirus genome is isolated using conventional techniques, and Spodoptera frugiperda cells, eg, Sf9
Used to transfect cells. A recombinant virus expressing Zalpha 12 is produced. Recombinant virus stocks are produced by methods commonly used in the art.

The recombinant virus is used to infect host cells, typically cell lines derived from the armyworm larva, Spodoptera frugiperda. In general, Glick and Pasternak, Molecular Biotechnology: Principles and Applicati
See on of Recombinant DNA, ASM Prss, Washington, DC, 1994. Another suitable cell line is H from Trichoplusia ni.
igh FiveO ^™ cell line (Invitrogen) (US Pat. No. 5,300,435). Commercial serum-free media is used to grow and maintain the cells.

Suitable media are SF900II ™ (Life Technologies) for Sf9 cells, or
Is EST 921 ™ (Expression Systems); and for T. ni cells, Ex-C
ellO405 ™ (JRH Biosciences, Lenza, KS) or Express FiveO ™ (L
ife Technologies). Cells are about 2-5 x 10⁵Individual cells ~ 1-2 x 10 ⁶ From the seeding density of individual cells, at which point the recombinant virus stock is
It is added at a multiplicity of infection (MCI) of 0.1 to 10, more typically around 3. Recombinant c
Virus-infected cells are typically 12-72 hours post-infection with recombinant Zalpha 12
Produces the polypeptide and secretes it into the medium with various efficiencies.

Cultures are usually harvested 48 hours after infection. Centrifugation is used to separate cells from the medium (supernatant). The supernatant containing the Zalpha 12 polypeptide is filtered through a filter with a pore size of usually 0.45 μm. The methods used are generally described in available laboratory manuals (King, LA ann.
d Possee, RD, supra; O'Reilly, DR et al., supra; Richardson, CD,
Above). Subsequent purification of the Zalpha 12 polypeptide from the supernatant can be achieved using the methods described herein.

[0107] Fungal cells, such as yeast cells, can also be used within the present invention. In this regard,
A yeast species of particular interest is Saccharomyces cereviae.
siae), Pichia pastoris and Pichia methanolica (pich)
ia methanolica). Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are described, for example, in Kawa.
saki, U.S. Patent No. 4,599,311; Kawasaki et al., U.S. Patent No. 4,931,373.
Brake, U.S. Patent No. 4,870,008; Welch et al., U.S. Patent No. 5,037,74.
And Murray et al., U.S. Patent No. 4,845,075. Transformed cells are selected by phenotype determined by the selectable marker, usually drug resistance, or the ability to grow in the absence of a particular nutrient (eg, leucine).

A preferred vector system for use in Saccharomyces cerevisiae is the POT1 disclosed by Kawasaki et al. (US Pat. No. 4,931,373), which allows for the selection of cells transformed by growth in a glucose-containing medium. It is a vector system. Suitable promoters and terminators for use in yeast are glycolytic enzyme genes (eg, Kawasaki, US Pat. No. 4,599,311; Kin.
gsman et al., U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No. 4,977,0.
No. 92) and from the alcohol dehydrogenase gene. See also U.S. Patent Nos. 4,990,446; 5,063,154; 5,139,936; and 4,661,454.

Other enzymes, such as Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis
veromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolic
Transformation systems for a), Pichia guillermondii, and Candida maltosa are known in the art.

For example, Gleeson et al., J. Gen. Microbiol. 132: 3459-3465, 1986 and Cr
See egg, U.S. Patent No. 4,882,279. Aspergillus cells, Mckni
ght et al., US Pat. No. 4,935,349. A method for transforming Acremonium chrysogenum is disclosed by Sumino., US Patent No. 5,162,228. Neurospora (N
Eurospora) is described in Lambowitz, U.S. Pat. No. 4,486,53.
No. 3 disclosed.

The use of Pichia methanolica as a host for the production of recombinant proteins
It is disclosed in WIPO publications WO97 / 17450, WO97 / 17451, WO98 / 02536 and WO98 / 02565. DNA molecules for use in transforming P. methanolica will usually be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation. P.
For polypeptide production in methanolica, the promoter and terminator in the plasmid are preferably those of the P. methanolica gene, for example, the P. methanolica alcohol utilization gene (AUG1 or AUG2). Other useful promoters include those of the dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes.

To facilitate integration of the DNA into the host chromosome, it is preferable to have a complete expression segment of the plasmid with the host DNA sequence at both ends. A preferred selectable marker for use in Pichia methanolica is P. methanolica encoding phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC. 4.1.1.21), which allows the growth of ade2 host cells in the absence of adenine. ADE2 gene. For large-scale industrial processes where it is desired to minimize the use of methanol, it is preferable to use host cells in which both methanol utilization genes (AUG1 and AUG2) have been deleted.

For the production of secreted proteins, host cells that lack the vacuolar protease genes (PEP4 and PRB1) are preferred. Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding the polypeptide of interest into P. methanolica cells. Having an electric field strength of 2.5 to 4.5 kV / cm, preferably about 3.75 kV / cm, and a time constant (r) of 1 to 40 ms, most preferably about 20 ms;
Electroporation using a pulsed electric field that decays exponentially
Preferably, P. methanolica cells are transformed.

Prokaryotic host cells, such as strains of the bacteria E. coli, Bacillus and other genera, are also useful host cells in the present invention. Techniques for transforming those hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, eg, Sambrook et al., Supra). When expressing a Zalpha 12 polypeptide in a bacterium, such as E. coli, the polypeptide can be retained in the cytoplasm, typically as insoluble granules, or can be directed to the periplasmic space by bacterial secretory sequences.

In the former case, the cells are lysed and the granules are collected and denatured using, for example, guanidine isothiocyanate or urea. Next, the denatured polypeptide is regenerated and dimerized by diluting the denatured polypeptide, for example, by dialysis against a solution of a combination of urea and reduced and oxidized glutathione, followed by dialysis against a buffer solution. Can be In the latter case, the polypeptide is soluble and functional from the periplasmic space by disrupting the cells to release the contents of the periplasmic space (eg, by sonication or osmotic shock) and recovering the protein. Recovered in form, thereby avoiding the need for denaturation and regeneration.

[0116] The transformed or transfected host cells are cultured according to conventional methods in a culture medium containing the nutrients and other components required for growth of the selected host cells. A variety of suitable media, such as defined and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. The medium can also include components such as growth factors or serum, if needed.

The growth medium generally comprises cells containing exogenously added DNA, for example, in an essential nutrient supplemented with a selectable marker carried on an expression vector or co-transfected into a host cell. Will choose by drug choice or nutritional deficiency. P. methanolica cells are cultured at a temperature of about 25 ° C. to 35 ° C. in a medium comprising a suitable carbon source, nitrogen source and micronutrients. The liquid culture is provided with sufficient aeration by conventional means, such as shaking a small flask or sparging a fermenter. A preferred culture medium for P. methanolica is YEPD (2% D
-Glucose, 2% Bacto ^™ peptone (Difco Laboratories, Detroit, MI),
1% Bacto ^™ yeast extract (Difco Laboratories), 0.004% adenine and 0.006
% L-leucine).

Another embodiment of the present invention is directed to an epitope of a Zalpha 12 polypeptide of the present invention-
A peptide or polypeptide comprising a carrier moiety is provided. The epiop of the polypeptide portion is an immunogenic or antigenic epitope of the invention. The region of the protein to which an antibody can bind is defined as an "antigenic epitope." For example, Geysen, HM, etc., Proc. Natl. Acad. Sci. USA 81: 3998-4002 (1984)
checking ...

With respect to the selection of peptides or polypeptides that carry an antigenic epitope (ie, including the region of the protein molecule to which an antibody can bind), a relatively short synthetic peptide that mimics a portion of the protein sequence will It is well known in the art that antisera that normally react with proteins mimicked by can be induced. Su
tcliffe, JG, etc. See Science 219: 660-666 (1983).

Peptides capable of inducing protein-reactive sera are occasionally represented in the major sequence of a protein, can be characterized by a set of simple chemical rules, and can be characterized by the immunodominant region of an intact protein (ie, ) Or at the amino or carboxyl terminus. Extremely hydrophobic peptides and those peptides of 6 or fewer residues are generally ineffective at eliciting antibodies that bind to the mimicked protein; longer soluble peptides,
In particular, those peptides containing a proline residue are usually effective.

The antigenic epitope-bearing peptides and polypeptides of the invention can also be used to raise antibodies, eg, monoclonal antibodies, that specifically bind to a polypeptide of the invention. The antigenic epitope-bearing peptides and polypeptides of the invention comprise at least 9 and preferably 15 to about 30 amino acids contained within the amino acids of the polypeptide of the invention. However, a large portion of the amino acid sequence of the present invention, i.e., 30 to 50 amino acids of the amino acid sequence of the polypeptide of the present invention, or any length up to and including the entire length of the amino acid sequence of the polypeptide of the present invention is included. The resulting peptide or polypeptide is also useful for inducing antibodies that react with the protein.

[0122] Preferably, the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (ie, the sequence comprises relatively hydrophilic residues and Residues are preferably avoided); and sequences containing proline residues are particularly preferred. All of the polypeptides shown in the sequence listing are
Includes antigenic epitopes used in accordance with the present invention. The invention also provides a polypeptide fragment or peptide comprising an epitope-bearing portion of a Zalpha 12 polypeptide described herein.

Such fragments or peptides comprise an “immunogenic epitope that is part of a protein that elicits an antibody response when the entire protein is used as an immunogen. Can be identified using standard methods (see, eg, Geysen et al., Supra) U.S. Patent No. 4,708,
No. 781 (1987) further describes how to identify a peptide bearing an immunogenic epitope of a desired protein.

Protein Isolation: It is preferred that the polypeptides of the present invention be purified to a purity of 80% or more, more preferably 90% or more, even more preferably 95% or more, and contaminating macromolecules, especially Particularly preferred is a pharmaceutically pure state that is at least 99.9% pure with respect to other proteins and nucleic acids and free of infectious and pyrogenic agents. Preferably, the purified polypeptide is substantially free of other polypeptides, especially other polypeptides of animal origin.

[0125] The expressed recombinant Zalpha12 polypeptide (or chimeric Zalpha12 polypeptide) can be purified using fractionation and / or conventional purification methods and media. Ammonium sulfate precipitation and acid or chaotropic agent extraction are used for sample fractionation. Typical purification steps include hydroxyapatite, size exclusion, FPLC and reverse phase high performance liquid chromatography. Suitable chromatographic media include derivatized dextran, agarose, cellulose, polyacrylamide, special silica, and the like. PEI, DEAE, QAE and Q derivatives are preferred.

Typical chromatographic media are those derivatized with a phenyl, butyl, or octyl group, such as phenyl-Sepharose FF (pharmacia), Toyopearl
Butyl 650 (Toso Haas, Montgomeryville, PA), Octyl-Sepharrose (Pharm
acia) and the like; or polyacrylic resins such as Amberchrom CG71 (Toso H
aas) and the like. Suitable solid supports include glass beads, silica-based resins, cellulose resins, agarose beads, cross-linked agarose beads,
Includes polystyrene beads, cross-linked polyacrylamide resins and the like which are insoluble under the conditions in which they are used.

The supports can be modified with amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and / or reactive groups that allow the attachment of proteins by carbohydrate moieties. Examples of coupling chemistries include carboxyl and amino derivatives for cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation and carbodiimide coupling chemistries. These and other solid media are well known in the art and are widely used, and are available from commercial suppliers.

[0128] Methods for coupling ligand or receptor polypeptides to a support medium are well known in the art. The choice of a particular method is conventional and is determined in part by the nature of the support chosen. For example, Affinity Chromatograpy: Pr
See inciples & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.

The polypeptides of the present invention can be isolated by exploiting their properties. For example,
Immobilized metal ion adsorption (IMAC) chromatography can be used to purify histidine-rich proteins and those proteins comprising a polyhistidine tag. Briefly, the gel is first charged by divalent metal ions and a chelate is formed (Sulkowski, Trends in Biochem. 3: 1-
7, 1985). Histidine-rich proteins are adsorbed to this matrix with different affinities, depending on the metal ions used, and competitive elution,
It may be eluted by lowering the pH or by using a strong chelating agent.

[0130] Other purification methods include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography (Methods in
Enzymol., Vol. 182, “Guide to Protein Purification”, M. Deutscher, (
ed.), Acad. Press, San Diego, 1990, pp. 529-39). In a further aspect of the invention, a fusion of the polypeptide of interest and an affinity tag (eg, a maltose-binding protein, an immunoglobulin domain) may be configured to facilitate purification.

Further, according to methods described in the art, polypeptide fusion or hybrid Zalpha 12 proteins are constructed using Zalpha 12 regions or domains of the invention (Sambrook et al., Supra; Altschul et al., Picard, Cur.
Opin. Biology, 5: 511-5, 1994). These methods allow for the determination of the biological significance of a large domain or region in a polypeptide of interest. Such hybrids alter reaction kinetics, binding, suppress or extend substrate specificity, or alter the tissue and cellular localization of the polypeptide, and are applied to polypeptides of unknown structure. You.

A fusion protein can be prepared by methods known to those skilled in the art by preparing the individual components of the fusion protein and chemically conjugating them. On the other hand, polynucleotides encoding both components of the fusion protein, in correct reading frame alignment, can be produced using known techniques and expressed by the methods described herein. For example, some or all of the domains that confer a biological function may be exchanged between a Zalpha12 of the invention and its functionally equivalent domain from another family member.

Such domains include, but are not limited to: secretory signal sequences, and significant domains or regions in this family.
Such fusion proteins are expected to have the same or similar biological functional profile as the polypeptides of the invention or other known families of proteins, depending on the fusion being constructed. Further, such fusion proteins can also exhibit other properties, as disclosed herein.

[0134] Zalpha12 polypeptides or fragments thereof can also be prepared by chemical synthesis. The Zalpha 12 polypeptide is a monomer or multimer; may be glycosylated or unglycosylated; may be pegylated or unpegylated; and may include the starting methionine amino acid residue. It is not necessary to do it.

Chemical Synthesis of Polypeptides: Polypeptides, particularly polypeptides of the present invention, can also be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation, or conventional solution methods. Polypeptides can preferably be prepared by solid phase peptide synthesis as described by Merrifield, J. Am. Chem. Soc. 85: 2149, 1963.

Assays: The activity of the molecules of the invention can be measured using various assays. Of particular interest are steroidogenesis, spermatogenesis, LH and FSH production in sperm, and changes in GnRH in the hypothalamus. Such assays are well-known in the art.

The proteins of the present invention are useful for enhancing sperm production. Zalpha 12 is
It can be measured in vitro using cultured cells or in vivo by administering a molecule of the invention to a suitable animal model. For example, Zalpha12 transfected (or co-transfected) expression host cells can be embedded in an alginate environment and injected (implanted) into a recipient animal. Alginate-poly-L-lysine microencapsulation, permeable membrane encapsulation and diffusion chambers have been described as a means for capturing transfected or primary mammalian cells.

[0138] Those types of non-immunogenic "encapsulation" or microenvironment allow nutrient transfer to the microenvironment and / or proteins and other macromolecules secreted or released by the captured cells. Allows for its spread to recipient animals through environmental barriers. Most importantly, the capsule or microenvironment masks and blocks foreign embedded cells from the immune response of the recipient animal. Such a microenvironment can extend the life span of injected cells from hours or days (naked cells) to weeks (embedded cells).

[0139] Alginate threads provide a simple and rapid means for generating embedded cells. The materials needed to produce alginate threads are readily available and relatively inexpensive. When manufactured, the alginate thread:
Relatively strong and durable in vivo, and based on data obtained with the yarn, in vivo. The alginate thread is easy to operate and its methodology can be evaluated for many preparations. In a typical method, 3% alginate is prepared in sterile water and sterile filtered. Immediately before the preparation of the alginate thread, the alginate solution is filtered again. About 50% cell suspension (1
about 5 × 10 ⁵ to about 5 × 10 ⁷ cells per ml) are mixed with a 3% alginate solution.

1 ml of alginate / cell suspension is extruded into a 100 mM sterile filtered Cacl ₂ solution for about 15 minutes to form a “Thread”. next,
The extruded yarn is transferred to a solution of 50 mM Cacl ₂ and then to a solution of 25 mM Cacl ₂ . Next, the yarn is rinsed with deionized water, after which the poly-L-
The threads are coated by incubating in a 0.01% solution of lysine. Finally, the thread is rinsed with a lactated Ringer's solution and withdrawn from the solution in a syringe (without a needle). A large bore needle is then attached to the syringe, and the thread is injected intraperitoneally into the recipient in a minimal amount of lactated Ringer's solution.

[0141] Another in vivo approach to assay the proteins of the invention involves a virus delivery system. Typical viruses for this purpose are adenovirus, herpes virus, vaccinia virus and adeno-associated virus (AAV)
Is included.

Adenovirus, a double-stranded DNA virus, is currently the most studied gene transfer vector for the provision of heterologous spread (TC Becker et al., Meth. Cell Bio. 43: 161-89, 1994; And JT Douglas and DT Curie
1, Science & Medicine 4: 44-53, 1997). The adenovirus system offers several advantages: (i) adenovirus has a relatively large D;
NA inserts can be adapted; (ii) can be grown to high titers; (iii
) Infects a wide range of mammalian cell types; and (iv) can be used with a number of available vectors containing different promoters. Also, because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection.

By deleting a part of the adenovirus genome, larger inserts of heterologous DNA (up to 7 kb) can be accommodated. The inserts are constructed by direct ligation or by homologous recombination with a co-transfected plasmid.
A can be incorporated into A. In a typical system, the essential E1 gene is deleted from the viral vector, and the virus will not replicate unless the E1 gene is supplied by a host cell (a human 293 cell line is typical).

When administered intravenously to an intact animal, adenovirus primarily targets the liver. If the adenovirus delivery system has an E1 gene deletion, the virus cannot replicate in the host cell. However, the host tissue (eg, liver) will express and process the heterologous protein (and secrete if a secretory signal sequence is present). Secreted proteins enter the circulation in the highly vascularized liver and the effect on infected animals can be determined.

The adenovirus system can also be used for protein production in vitro. By culturing adenovirus-infected non-293 cells under conditions such that the cells do not divide rapidly, the cells can produce proteins for extended periods of time. For example, BHK cells are grown to confluence in a cell factory and then exposed to an adenovirus vector encoding a secreted protein of interest.

The cells are then grown under serum-free conditions that allow the infected cells to survive for several weeks without significant cell division. On the other hand, 293S cells infected with adenovirus vector can be grown at relatively high cell densities, either as adherent cells or in suspension culture, to produce significant amounts of protein (Garnier et al., Cytotechnol. : 145-55, 1994). By either protocol, the expressed and secreted heterologous protein can be repeatedly isolated from the supernatant of the cell culture. In the protocol for generating infected 293S cells, non-secreted proteins are effectively obtained.

Agonists and antagonists: In view of the tissue distribution observed for Zalpha 12, agonists (eg, natural ligands / substrates / cofactors / etc.) And antagonists have enormous potential for in vitro and in vivo applications. Compounds identified as Zalpha 12 agonists are useful for stimulating the immune system or spermatogenesis. For example, Zalpha 1
2 and agonist compounds are useful as components of defined cell culture media,
And can be used alone or in combination with other cytokines and hormones to replace the serum commonly used in cell culture.

Antagonists: Antagonists are also useful as research reagents for characterizing sites of ligand-receptor interaction. Antagonists of Zalpha 12 can also be used to down-regulate inflammation as discussed in more detail below. Inhibitors of Zalpha 12 activity (Zalpha 12 antagonists) are anti-Zalpha 12
12 antibodies and soluble Zalpha 12 receptors and other peptide and non-peptide agents (
Ribozymes).

Zalpha 12 can also be used to identify inhibitors (antagonists) of its activity. Test compounds are added to the assays disclosed herein to identify compounds that inhibit the activity of Zalpha12. In addition to those assays disclosed herein, samples can be used to measure Zalpha1 by various assays designed to measure receptor binding or stimulation / inhibition of Zalpha12-dependent cellular responses.
2 Can be tested for inhibition of activity. For example, the Zalpha12-responsive cell line is Zalp
ha 12 -can be transfected by a reporter gene construct responsive to the stimulated cellular pathway.

[0150] Reporter gene constructs of this type are known in the art and generally comprise a Zalpha12-DNA response element operably linked to a protein that can be assayed, such as a gene encoding luciferase. Will. DNA response elements include cyclic AMP response element (CRE), hormone response element (HRE), insulin response element (IRE) (Nasrin et al., Proc. Natl. Acad. Sci. USA 87: 5273-
7, 1990) and serum response element (SRE) (Shaw et al., Cell 56: 563-72, 1989), but are not limited thereto.

The cyclic AMP response element is described in Roestler et al., J. Biol. Chem. 263 (19): 90
63-6, 1988 and Haener, Molec. Endocrinol. 4 (8): 1087-94, 1990. Hormone response elements are reviewed in Beato, Cell 56: 335-441, 1989. Candidate compounds, solutions, mixtures or extracts are tested for their ability to inhibit the activity of Zalpha12 on target cells, as evidenced by a reduction in Zalpha12 stimulation of reporter gene expression. This type of assay binds to cell surface receptors
Compounds that block Zalpha 12 directly and those that block steps in the cellular pathway following reporter ligand binding will be detected.

On the other hand, if the compound or other sample has a detectable label (eg, ¹²⁵ I,
Zalpha12 labeled with biotin, horseradish peroxidase, FITC, and the like) can be used to test for direct blocking of Zalpha12 binding to the receptor. In this type of assay, the ability of the test sample to inhibit the binding of labeled Zalpha 12 to the receptor is an indication of inhibitory activity that can be ascertained through a secondary assay. The receptor used in the binding assay can be a cellular receptor, or an isolated, immobilized receptor.

[0153] Zalpha 12 polypeptides can be expressed as a fusion with an immunoglobulin heavy chain constant region, typically an Fc fragment, which contains two region domains and lacks the variable regions. Methods for preparing such fusions are disclosed in U.S. Patent Nos. 5,155,027 and 5,567,584. Such fusions are typically secreted as multimeric molecules, wherein the Fc portions are disulfide bonded to each other and the two non-Ig polypeptides are arranged in close proximity to each other. This type of fusion can be used to affinity purify the ligand. For use in assays, the chimeras are bound to a support through the Fc region and used in an ELISA format.

[0154] Zalpha12 ligand-binding polypeptides can also be used for purification of ligand. The polypeptide may be present on a solid support, such as beads of agarose, cross-linked agarose, glass, cellulose resin, silica-based resin, polystyrene, cross-linked polyacrylamide, or similar materials that are stable under the conditions of use. Identified.

Methods for linking polypeptides to solid supports are known in the art and include amine chemistry, cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation and Hydrazide activation. The resulting medium is generally formed in the form of a column, and a fluid containing the ligand is passed through the column one or more times to allow binding of the ligand to the receptor polypeptide. Next, the ligand is used for salt concentration, chaotropic (guanidine).
HCl), or a change in pH, disrupting ligand-receptor binding.

The ligand-bound receptor (or antibody, one member of a complement / anti-complement pair), or a binding fragment thereof, and a commercially available biosensor device (BIAcore, Pharmacia Bio)
Assay systems using sensors, Piscataway, NJ) may be conveniently used. Such receptors, antibodies, members of complement / anti-complement pairs, or fragments are immobilized on the surface of the receptor chip. Use of this device is described in Karlsson, J. Imm
unol.Methods 145: 229-40, 1991 and Cunningham and Wells, J. Mol. Biol.
234: 554-63,1993.

[0157] Receptors, antibodies, members or fragments are covalently attached to dextran fibers that are attached to gold film in flowing cells using amine or sulfhydryl chemistry. A test sample is passed through the cells. If a ligand, epitope or opposing member of a complement / anti-complement pair is present in the sample, it binds to the immobilized receptor, antibody or member, respectively, and is detected as a change in plasmon resonance on the surface of the gold film. Cause a change in the refractive index of the medium. This system allows for the determination of on- and off-rates, from which the binding affinity is calculated and the stoichiometry of the binding is allowed.

[0158] Ligand-bound receptor polypeptides can also be used in other assay systems known in the art. Such a system is described in Scatchard analysis for the determination of binding affinity (Scatchard, Ann. NY. Acad. Sci. 51: 660-72, 194).
9) and calorimetric assay (Cunningham et al., Science 253: 545-48, 1991; Cu
nningham et al., Science 245: 821-25, 1991).

[0159] Zalpha12 polypeptides can also be used to prepare antibodies that specifically bind to a Zalpha12 epitope, peptide or polypeptide. Zalpha 12 polypeptide or its fragment acts as an antigen (immunogen) to inoculate animals and elicit an immune response. Suitable antigens are SEQ ID NOs: 2, 4, 6,
Zalpha12 polypeptides encoded by 8-10, 18-20, 25-30 and 35-44. Antibodies generated from this immune response can be isolated as described herein.

[0160] Methods for preparing and isolating polyclonal and monoclonal antibodies are well-known in the art. For example, Current Protocols in Immunol
ogy, Cooligan, etc., (eds.), National Institutes of Health, John Wiley
and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: A Laboratory Ma
nual, Second Edition, Cold Spring Harbor, NY, 1989; and Hurrell, JGR,
Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC
See Press, Inc., Boca Raton, FL, 1982.

As will be apparent to those of skill in the art, polyclonal antibodies can be used to conjugate a variety of warm-blooded animals, such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats, with Zalpha12 polypeptides or fragments thereof. It can be produced by inoculation. Zalpha 1
2 The immunity of the polypeptide can be increased by the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant. Polypeptides useful for immunization also include fusion polypeptides with an immunoglobulin polypeptide or a maltose binding protein, such as a fusion of Zalpha12 or a portion thereof.

A polypeptide immunogen can be a full-length molecule or a portion thereof. If the polypeptide moiety is "hapten-like", such a moiety will, for immunization,
It can be conveniently linked or conjugated to a polymeric carrier such as limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid.

As used herein, the term “antibody” refers to polyclonal antibodies, affinity purified polyclonal antibodies, monoclonal antibodies and antigen binding fragments, such as F (ab ′) ₂ and Fab proteolytic fragments Is included. Also encompassed are genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigen binding peptides and polypeptides.

Non-human antibodies can be prepared by grafting only non-human CDRs onto the human backbone and constant regions, or by incorporating the entire non-human variable domain (optionally by substituting exposed residues for human). -Cover these domains with a like surface (cl
oaking) "; where the result is a" laminated "antibody), which can be humanized. In many cases, a humanized antibody will have a human variable region scaffold to enhance the correct binding properties. Non-human residues can be retained within the domain.
Through the use of humanized antibodies, the biological half-life is increased and the potential for adverse immune reactions upon administration to humans is reduced.

Another technique for generating or selecting antibodies useful herein is Zalpha.
12 in vitro exposure of lymphocytes to 12 proteins or peptides, and selection of antigen display libraries in phage or similar vectors (eg, through the action of immobilized or labeled Zalpha 12 proteins or peptides).
Genes encoding polypeptides with potential Zalpha 12 polypeptide binding domains can be phage (phage display) or bacteria, such as E. coli. It is obtained by screening a random peptide library displayed on E. coli. The nucleotide sequence encoding the polypeptide can be obtained through a number of means, such as random mutagenesis and random polynucleotide synthesis.

These random peptide display libraries screen for peptides that interact with known targets, which can be proteins or polypeptides, such as ligands or receptors, biological or synthetic macromolecules, or organic or inorganic substances. Can be used to Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al., US Pat. No. 5,223,409; Ladner et al., US Pat. No. 4,946,778).
Ladner et al., U.S. Pat. Nos. 5,403,484 and Ladner et al., U.S. Pat. No. 5,571,698), and random peptide display libraries and kits for screening such libraries are described, for example, in Clontech (Palo Alt.
o, CA), Invitrogen Inc. (San Diego, CA), New England Biolabs, Inc. (Beve
rly, MA) and Pharmacia LKB Biotechnology Inc. (Piccataway, MJ).

A random peptide display library can be screened using the Zalpha 12 sequences disclosed herein to identify proteins that bind to Zalpha 12. Those "binding proteins" that interact with Zalpha12 polypeptides can be used to label cells; to isolate homologous polypeptides by affinity purification; they can be used as drugs, toxins, radionuclides and the like. Directly or indirectly.

The binding proteins can also be used in analytical methods, for example, to screen expression libraries and neutralize activity. Bound tampon protein can also be used in diagnostic assays to determine circulating levels of the polypeptide; to detect or quantify soluble polypeptide as a marker of underlying pathology or disease. These binding proteins also inhibit Zalpha12 binding and signal transduction in vitro and in vivo.
Can be used as "antagonis".

Antibodies were determined to specifically bind 1) if they exhibited a marginal level of binding activity, and 2) if they did not significantly cross-react with related polypeptide molecules. First, the antibodies herein may be those which are 10 ⁶ M ⁻¹ or higher;
Zalpha12 polypeptides, peptides preferably having a binding affinity (Ka) of 10 ⁷ M ^-1 or higher, more preferably 10 ⁸ M ^-1 or higher, and most preferably 10 ⁹ M ^-1 or higher Or, when it binds to an epitope, it binds specifically. The binding affinity of the antibody can be determined, for example, by Scatchard analysis (Scatchard, G., Ann. NY Acad. Sci. 51:66).
0-672, 1949) can be readily determined by those skilled in the art.

Second, antibodies are determined to bind significantly if they do not significantly cross-react with related polypeptide molecules. Antibodies detect Zalpha12 using standard Western blot analysis, but do not significantly cross-react with related polypeptide molecules if they are not known related polypeptides (Au
subel etc., supra). Examples of known related polypeptides include orthoforms, proteins from the same species that are members of the protein family (eg, IL-16), Z
alpha12 polypeptide and non-human Zalpha12.

In addition, antibodies can be "screened" against known related polypeptides to isolate populations that specifically bind to a polypeptide of the invention. For example, antibodies raised against Zalpha 12 are adsorbed to relevant polypeptides attached to an insoluble matrix; antibodies specific for Zalpha 12 flow through the matrix under appropriate buffer conditions. Will.

Such screening allows the isolation of polyclonal and monoclonal antibodies that do not cross-react with known closely related polypeptides (Anti
bodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor
Laboratory Press, 1988; Current Protocols in Immunology, Cooligan, etc.
(eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995.
).

Screening and isolation of specific antibodies is well known in the art. Fundamental Immunology, Paul (eds.), Raven Press, 1993; Getz
off et al., Adv. in Immunol. 43: 1-98, 1988; Monoclonal Antibodies: Princip
les and Practice, Goding, JW (eds.), Academic Press Ltd., 1996; Benjam
See Ann. Rev. Immunol. 2: 67-101, 1984.

A variety of assays known to those of skill in the art can be used to detect antibodies that specifically bind to a Zalpha 12 protein or peptide. A typical assay is
Antibodies: A Laboratory Manual, Harlow and lane (Eds.), Cold Speing Har
bor Laboratory Press, 1988. Representative examples of such assays include: co-immunoelectrophoresis, radioimmunoassay,
Radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), dot or western blot assay, inhibition or competition assay. And sandwich assays. In addition, antibodies that bind to wild type versus mutant Zalpha12 protein or peptide can be screened.

Antibodies against Zalpha 12 can be used to label cells that express Zalpha 12; to isolate Zalpha 12 by affinity purification; and for diagnostic assays to determine circulating levels of Zalpha 12 polypeptide. To detect or quantify soluble Zalpha 12 as a marker of underlying pathology; to screen expression libraries in analytical methods using FACS; to generate anti-indiotype antibodies; And can be used as a neutralizing antibody or antagonis to block Zalpha 12 -mediated activity in vitro.

Suitable direct labels or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect labels or labels include intermediates It features the use of biotin-avidin or other complement / anti-complement pairs as a body. The antibodies and binding proteins herein can also be conjugated directly or indirectly to drugs, toxins, radionuclides, and the like, and the conjugates are used for in vivo diagnostic or therapeutic applications. obtain. In addition, antibodies to Zalpha 12 or fragments thereof can be used in assays, such as Western blots or other assays known in the art.
It can be used in vitro to detect denatured Zalpha12 or fragments thereof.

Bioactive conjugates: The antibodies or polypeptides described herein can also be conjugated directly or indirectly to drugs, toxins, radionuclides, and the like, and the conjugates can be It can be used for in vivo diagnostic or therapeutic applications. For example, a polypeptide or antibody of the invention may comprise a corresponding anti-complementary molecule (eg, a receptor or antigen, respectively).
Can be used to identify or treat tissues or organs that express. More particularly, a Zalpha12 polypeptide or an anti-Zalpha12 antibody, or a biologically active fragment or portion thereof, is bound to a detectable or cytotoxic molecule and expresses an anti-complementary molecule, cell, tissue or It can be supplied to a mammal having an organ.

Suitable detectable molecules can be directly or indirectly linked to the polypeptide or antibody, and can include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers,
Includes chemiluminescent markers, magnetic particles, and the like. Suitable cytotoxic molecules can be conjugated directly or indirectly to the polypeptide or antibody, and can be used for bacterial or phytotoxic (eg, diphtheria toxin, pseudomonas endotoxin, ricin, abrin and the like), and therapeutic Includes radionuclides such as I-131, rhenium-188 or yttrium-90, which are bound directly to a polypeptide or antibody or indirectly by a chelating moiety.

A polypeptide or antibody can also be conjugated to a cytotoxic drug, for example, adriamycin. For indirect binding of a detectable or cytotoxic molecule, the detectable or cytotoxic molecule can be bound by a member of a complementary / anti-complementary pair, where the other member is bound to the polypeptide or antibody moiety. . For those purposes, biotin / streptavidin is a typical complementary / anti-complementary pair.

In another embodiment, the polypeptide-toxin fusion protein or antibody-toxin fusion protein is used for targeted cell or tissue inhibition or removal (eg, to treat cancer cells or tissues). Can be used. On the other hand, if the polypeptide has multiple functional domains (ie, an activation or ligand binding domain, and a targeting domain), a fusion protein that includes only the targeting domain will have a detectable, cytotoxic, or complementary molecule. Suitable for directing a target molecule to a cell or tissue type of interest. If the domain-only fusion protein comprises a complementary molecule, the anti-complementary molecule can be conjugated to a detectable or cytotoxic molecule. Thus, such domain-complementary molecular fusion proteins are generic anti-complementary-detectable
/ Represents a generic targeting vehicle for cytotoxic molecular conjugates.

In another embodiment, the Zalpha12-cytokine fusion protein or antibody-cytokine fusion protein is a Zalpha12 polypeptide or an anti-Zalpha1
2 If the antibody targets hyperproliferative blood and bone marrow cells, it can be used to enhance in vivo killing of target tissues (eg, blood and bone marrow cancer) (generally, Ho
rnick et al., Blood 89: 4437-4447, 1997). The described fusion proteins allow targeting of the cytokine to the desired site of action, thereby providing an increased local concentration of the cytokine.

Suitable Zalpha12 polypeptides or anti-Zalpha12 antibodies target unwanted cells or tissues (eg, tumors or leukemias) and the fused cytokines mediate improved target cell lysis by effector cells. I do. For example, suitable cytokines for this purpose include interleukin-2 and granulocyte-macrophage colony stimulating factor (GM-CSM).

In yet another embodiment, the Zalpha12 potipeptide or anti-Zalpha1
2 If the antibody targets vascular cells or tissues, such polypeptides or antibodies can be conjugated with a radionuclide, and particularly a beta-emitting radionuclide, to reduce restenosis. Such a treatment approach poses little risk to the clinician managing the radiotherapy.

For example, an iridium-192 impregnated ribbon placed into a patient's stented blood vessel until the required radiation dose is delivered can result in reduced tissue growth in the blood vessel and placebo. It showed a larger lumen than the control group that received the ribbon. In addition, revascularization and stent thrombosis were significantly lower in the treatment group. Similar results are expected from targeting a bioactive conjugate that includes a radionuclide, as described herein. The bioactive polypeptides or antibody conjugates described herein can be supplied intravenously, intraarterially or intravenously, or can be introduced locally at the intended site of action.

Use of Polynucleotides / Polypeptides: Molecules of the invention may be receptors involved in spermatogenesis, steroidogenesis, testicular differentiation, and hypothalamic-pituitary-gonadal axis regulation or receptors in the immune system Can be used to identify and isolate For example, the proteins and peptides of the invention are immobilized on a column, and the membrane preparation is passed over the column (Immobilized Affini
ty Ligand Techniques, Hermanson et al., eds., Academic Press, San Diego,
CA, 1992).

Proteins and peptides are also radiolabeled (Methods in Enzymol., V
ol. 182, “Guide to Protein Purification”, M. Deutscher, ed., Acad. Pre
ss, San Diego, 1990, 721-737) or photoaffinity labeled (Brunner et al., A
nn. Rev. Biochem. 62: 483-514, 1993 and Fedan et al., Biochem. Pharnacol.
33: 1176-1180, 1984), and specific cell-surface proteins can be identified. The molecules of the present invention will be useful for testing diseases of the reproductive and immunological systems.

Gene Therapy: Polynucleotides encoding Zalpha 12 polypeptides are useful in gene therapy applications where it is desired to increase or inhibit Zalpha 12 activity. If the mammal has or lacks the mutated Zalpha 12 gene,
The Zalpha 12 gene can be introduced into mammalian cells. In one embodiment, Zal
A gene encoding a pha 12 polypeptide is introduced in vivo in a viral vector. Such vectors include attenuated or defective DNA viruses such as herpes simplex virus (HSV), papillary virus, Epstein-Barr virus (EBV), adenovirus, adeno-associated virus (AAV) and the like. However, it is not limited only to them.

Defective viruses that completely or almost completely lack viral genes are preferred. The defective virus is not infectious after introduction into the cell. The use of defective viral vectors allows for administration to cells in specific localized areas without having to worry about the vector infecting other cells. Examples of particular vectors include, but are not limited to: defective herpesvirus 1
(HSV1) vector (Kaplitt et al., Molc. Cell. Neurosci. 2: 320-30, 1991),
Attenuated adenovirus vectors, such as Stratford-Perricaudat.
(J. Clin. Invest. 90: 626-30, 1992) and defective adeno-associated virus vectors (Samulski et al., J. Virol. 61: 3096-101, 1987).
Samulski et al., J. Virol. 63: 3822-28,1989).

[0189] In another embodiment, the zdint1 gene is as described in
Can be introduced into retroviral vectors: Anderson et al., US Patent 5,39.
No. 9,346; Mann et al., Cell 33: 153, 1983; Temin et al., U.S. Pat.
No. 0,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J. Vir
ol. 62: 1120, 1988; Temin et al., U.S. Patent No. 5,124,263; Dougherty.
, WIPO Publication WO95 / 07358; and kuo, etc., Blood 82: 845-52,
1993.

On the other hand, vectors can be introduced by in vivo lipofection using liposomes. Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of genes encoding markers (Felg
ner et al., Proc. Natl. Acad. Sci. USA 84: 7413-17, 1987; and Mackey etc.
Natl. Acad. Sci. USA 85: 8027-31, 1988). The use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages.

[0191] Molecular targeting of liposomes to specific cells represents one area of benefit. It is clear that directing transfection to specific cells represents an advantage in one area. It is clear that directing transfection to specific cell types is particularly advantageous in tissues with cellular heterogeneity, such as pancreas, liver, kidney and brain. Lipids are scientifically derived into other molecules for targeting. Targeted peptides, such as hormones or neurotransmitters, and proteins, such as antibodies or non-peptide molecules, can be chemically attached to liposomes.

It is possible to remove cells from the body and introduce the vector as a naked DNA plasmid, and then re-transplant the transformed cells into the body. Naked DNA vectors for gene therapy can be introduced into the desired host cells by any method known in the art, such as transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, It can be introduced by the use of a cancer or the use of a DNA vector transporter (eg, Wu et al., J. Biol. Chem. 267: 963-7, 1992; W
u et al., J. Biol. Chem. 263: 14621-24, 1988).

Antisense methods can be used to inhibit Zalpha 12 gene transcription, for example, to inhibit cell growth in vivo. A polynucleotide that is complementary to a segment of a Zalpha 12-encoding polynucleotide (eg, a polynucleotide as set forth in SEQ ID NO: 1) binds to a Zalpha 12-encoding mRNA and is capable of binding such mRNA. It is designed to inhibit translation. Such antisense polynucleotides are used to inhibit the expression of a Zalpha12 polypeptide-encoding gene in a cell culture or subject.

The present invention also provides reagents that could be used for diagnosis. For example, Zalpha 1
2 The probe comprising the gene, Zalpha 12 DNA or RNA, or a subsequence thereof is Za
It can be used to determine if the lpha 12 gene is dependent on chromosome 2q33.1 or if a mutation has occurred. Detectable chromosomal aberrations at the Zalpha 12 locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes, and translocations. Such abnormalities can be determined by molecular genetic techniques such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis using PCR techniques, and other genes known in the art. The polynucleotides of the present invention can be detected by using linkage analysis techniques (Sambrook et al., Supra; Ausubel et al., Supra; Marian, Chest 108:
255-65, 1995).

Transgenic mice constructed to express the Zalpha12 gene, and mice exhibiting a complete absence of Zalpha12 gene function, referred to as "knockout mice" (Snouwaert et al., Science 257: 1083, 1992). Can also be produced by those skilled in the art (Lowell et al., Nature 366: 740-42, 1993). These mice are used to study the Zalpha12 gene and the protein encoded thereby in an in vivo system.

Chromosome localization: Zalpha 12 has been mapped to chromosome 22q13.1. Radial gland hybrid mapping is a somatic genetic technique that has been developed to construct a high-resolution serial map of mammalian chromosomes (Cox et al., Science 250; 245-50, 1990). Partial or complete knowledge of the gene sequence allows the design of appropriate PCR primers for use by the chromosomal radiation hybrid mapping panel. Radiation hybrid mapping panels covering the entire human genome, such as the Stanford G3 RH panel and the GeneBridge 4 RH panel, are commercially available (Research Genetics, Inc.,
Huntsville, AL).

[0197] These panels demonstrate rapid, PCR-based chromosomal localization and alignment of genes, sequence-labeled sites (STS), and other non-polymorphic and polymorphic markers within the region of interest. enable. This involves establishing a directly proportional physical distance between the newly discovered gene of interest and the previously mapped marker. Accurate knowledge of gene location is useful for many purposes, including: 1)
Determining whether a sequence is part of an existing contig and various forms, such as YA
Acquisition of additional surrounding gene sequences in C, BAC or cDNA clones; 2) Provision of possible candidate genes for genetic diseases showing binding to the same chromosomal region; and 3
A) Cross reference of model organisms, such as mice, to help determine the function of a particular gene.

The sequence labeled sites (STS) are also used independently for chromosome localization. An STS is a DNA sequence that is unique in the human genome and can be used as a reference point for a particular chromosome or region of chromosome. STS is defined by a pair of oligonucleotide primers used in the polymerase chain reaction to specifically detect this site in the presence of all other genomic sequences. Because STSs are solely based on DNA sequences, they can be used in electronic databases, such as Dat.
abase of Sequence Tagged Sites (dbSTS), GenBank (National Center for Bio
logical Information, National Institutes of Health, Bethesda, MD, http: /
/www.ncbi.nlm.nih.gov) and the mapping data contained within their short genomic landmark STS sequences allows one to interrogate gene sequences of interest.

For use in medicine, the proteins of the present invention may be prepared according to conventional methods, parenterally,
Particularly formulated for intravenous or subcutaneous delivery. Intravenous administration will be by bolus injection or infusion for a typical period of one to several hours. Generally, the pharmaceutical formulation will comprise a Zalpha12 protein in combination with a pharmaceutically acceptable vehicle, such as a salt solution, a buffer solution, 5% dextrose in water, or the like. The formulation can further include one or more excipients, preservatives, solubilizers, buffers, albumin to prevent protein loss on the vial surface, and the like.

Methods of compounding are well known in the art and are described, for example, in Remington: T
he Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co.,
^{Easton, PA, 19 th ed.} , Disclosed in 1995. Therapeutic doses are generally 0.1-100 μ
g / kg body weight / day of the patient, preferably in the range of 0.5-20 mg / kg / day, and the exact dose is acceptable taking into account the nature and severity of the condition being treated, patient characteristics, etc. Determined by the clinician according to standards. Determination of the dosage is within the level of ordinary skill in the art. The protein may be administered for one week or less, often for one to three days, for acute treatment, or may be used for months to years for chronic treatment. The administration of the protein depends on the disease to be treated,
It can be subcutaneous, intraperitoneal or rectal.

Tissue Expression and Use : Zalpha 12 provides a novel polypeptide with a putative signal peptide leader sequence and an α-helical structure. Several putative isoforms have been identified. Thus, this gene can encode a secreted polypeptide having a secondary structure that indicates it is a member of the four helix bundle cytokine family. On the other hand, the polypeptide may have other activities associated with other biological functions, such as enzymatic activity, binding to cell membranes, or function as a carrier protein.

[0202] Northern blot analysis detects transcripts for Zalpha12 in the spleen, thymus, testis, small intestine, PBL, stomach, lymph nodes, trachea and spinal cord. Many of these organs contain cells that have important immunological functions or play a role in the immune system.
Zalpha12 is also expressed on CD4 ⁺ -rich peripheral T cells and to a lesser extent on CD8 ⁺ T cells. There is very weak expression in CD19 ⁺ B cells, suggesting that Zalpha 12 is expressed in T lymphocyte lineage cells and little if any in B lymphocyte lineage cells. In RNA from the human mixed lymphocyte reaction on day 7, Zalpha 12
There is an enhanced expression of This indicates that Zalpha 12 expression is regulated and increases following T cell activation.

Use of Zalpha 12: Zalpha 12 can be administered to immunocompromised mammals, preferably humans, eg, cancer patients who have undergone chemotherapy, AIDS patients and elderly people. This will stimulate their immune system. Zalpha 12 is also available prior to vaccine administration.
It can be used as a vaccine adjuvant to be administered with or after administration. Zalpha 12 can also be administered to stimulate the immune system to attack tumors.

Use of Antagonists of Zalpha12: Antagonists to Zalpha12, such as antibodies, soluble receptors or small molecule antagonists, can be administered to a mammal, preferably a human, to mitigate the inflammatory response. Antagonists, such as antibodies, against Zalpha 12 can be used to treat inflammation-related diseases,
For example, atherosclerotic heart disease (Paulsson, G. etc., Arterioscler Thromb. Vasc. B
iol., 20: 10-17 (2000)), can be used to treat patients with inflammatory bowel disease, Crohn's disease, rheumatoid arthritis and pancreatitis. The present invention is further illustrated by the following non-limiting examples.

[0205] Example Example 1. Cloning of Zalpha 12 Zalpha 12 was discovered in the spleen cDNA library by using the expressed sequence tag of SEQ ID NO: 7 as a probe. The library was prepared using polyA RN isolated from spleen tissue of a 2-year-old Spanish male boy who died of cerebral anoxia.
A1 μg was used. cDNA synthesis was initiated using a NotI-oligo (dT) primer. The double-stranded cDNA is blunt-ended, size-selected, and the vector N
Cloned into otI and EcoRI sites. The sequenced clones yielded full length DNA and the polypeptide sequences of sequencing 1 and 2.

The EST of SEQ ID NO: 17 resulted in the expression of a second clone. The clone was sequenced and it was found to have an unspliced intron. Oligonucleotide primers SEQ ID NOS: 11 and 12 were designed to amplify full length sequences without intron sequences. PCR was performed using lymph node MARATHON READY ™ cDNA (Clontech, Palo Alto, CA) as a template. The 823 bp were excised and the gel was run with the QIAQUICK ™ gel extraction kit (Qiagen, S
anta Clarita, CA). The excised PCR fragment was sequenced and did not contain any intron sequences. The next step was to find out if the clone of SEQ ID NO: 17 had another start codon upstream.

A RACE ™ reaction was performed using the lymph node cDNA to obtain a 5 ′ sequence. Oligonucleotide primers SEQ ID NOs: 13 and 14 were designed for the 5 'RACE ™ reaction and nested (immobilized) RACE ™. The RACE ™ reaction represented an additional start codon upstream. Actual full length was obtained through PCR using primers SEQ ID NOS: 15 and 16 flanking the coding sequence. Six bands were amplified and each was gel purified using the QIAQUICK ™ gel extraction kit and plasmid pCR2.1 TOPO vector using the TOPO TA ™ cloning kit (Invitrogen, Carlsbad, CA). Subcloned into Clones were sequenced and resulted in the full length sequences of SEQ ID NOs: 3, 4, 5 and 6. Therefore, three variants were expressed, namely SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, and SEQ ID NOs: 5 and 6.

Example 2 Northern blot analysis of Zalpha 12 Northern blot analysis was performed using the polynucleotides of SEQ ID NOs: 1 and 3 by standard techniques. The results showed that Zalpha 12 was not found in lymph nodes, activated mixed lymphocytes, spleen, thymus, testis, small intestine, human aortic endothelial cells, smooth muscle, kidney,
Mast cells, eosinophils, tonsils, pancreas, colon, peripheral serum lymphocytes (PBL), stomach, trachea, T
-Indicates expression in cells, such as CD4 ⁺ and CD8 ⁺ cells and bone marrow.

Zalpha 12 is also expressed by the following cell lines: HL60 (ATCC45500),
Acute promyelocytic leukemia cell line; Jurkat cells (ATCC TIB-152), T lymphocytes from acute T cell leukemia; MOLT-4 cells (ATCC CRL-1582), ie, acute lymphoblastic leukemia. T lymphoblasts; and HuT78 cells (ATCC TIB-161)
, Ie cutaneous T lymphocytes from lymphoma. From those data, it can be concluded that Zalpha 12 is a cytokine involved in the inflammatory cascade. Antagonists of Zalpha 12 can be used to reduce inflammation associated with a number of diseases, such as atherosclerotic heart disease, cardiovascular disease, rheumatoid arthritis, inflammatory bowel disease and Crohn's disease. Example 3 Zalpha 12 anti-peptide antibody Polyclonal anti-peptide antibody, the peptide huzalpha12X1-1: AQQHKGSLQKDP
LLSQACVGCLEALLDYLDAR (SEQ ID NO: 41), peptide PLPATKDTVLAPL from SEQ ID NO: 4
RMSQVRSLVIGLQNLLVC (SEQ ID NO: 42), peptide CEGLPPSTSSGQPPL from SEQ ID NO: 4
QDMLCLGGVAVSLSHIRN (SEQ ID NO: 43) or peptide FMRYRSSSVLS from SEQ ID NO: 4
Prepared by immunizing two New Zealand white rabbits with HEEC (SEQ ID NO: 44). Peptides were synthesized using an Applied Biosystems Model 431A peptide synthesizer (Applied Biosystems, Inc., foster City, CA) according to the manufacturer's instructions.

Next, the peptide was conjugated to the carrier protein maleimide-activated limpet (keyhole limpet) (KLH) (Pierce, Rockford, IL) via a cysteine residue according to the manufacturer's instructions. Rabbits were given an initial intraperitoneal (IP) injection of 200 μg of the conjugated peptide in complete Freund's adjuvant (Pierce, Rockford, IL), followed by a boost IP of 100 μg of the conjugated peptide in incomplete Freund's adjuvant. Infusions were given every three weeks. Seven to ten days after the third booster injection, animals were bled and serum was collected. The rabbits were then boosted and bled every three weeks.

[0211] Zalpha 12 peptide-specific rabbit sera were characterized by ELISA titration using 1 μg / ml peptide used to produce antibodies as antibody targets. Two rabbit sera against the SEQ ID NO: 41 peptide were 1: 5E5 (1: 100,000
) With titers against their specific peptide. The two rabbit sera against the SEQ ID NO: 42 peptide had titers against their specific peptide at a dilution of 1: 5E4 (1: 10,000). The two rabbit sera against the SEQ ID NO: 43 peptide had titers to their specific peptide at a dilution of 1: 5E4.
The two rabbit sera against the SEQ ID NO: 44 peptide had titers to their specific peptide at a dilution of 1: 5E5.

While the foregoing has described embodiments of the features of the present invention by way of example, it will be understood that various modifications may be made within the scope of the present invention. Accordingly, the invention is not limited except as by the appended claims.

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 19/02 A61P 29/00 29/00 C07K 14/52 C07K 14/52 16/24 16/24 C12N 15 / 00 ZNAA (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA ( BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR , BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO , RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventor Cooper, Emily United States of America, Washington 98117, Seattle, 9th Avenue Northwest 7745 (72) Inventor Jaspers, Stephen R. United States, Washington 98026, Edmonds, One Hundred Fifty Place Southwest 5829 (72) Inventor Renner, Joyce M. USA, Washington 98103, Seattle, Finney Avenue North 6522, # 201 F-term (reference) 4B024 AA01 BA21 BA43 CA04 CA11 DA01 DA02 DA05 DA11 EA01 EA02 EA03 EA04 FA02 GA01 GA11 HA01 4C084 AA17 ZA36 ZA45 ZA68 ZA96 ZB11 ZB15 BB15 BB15 4H045 AA10 AA11 AA20 AA30 BA10 CA40 DA01 DA76 EA20 FA72 FA74

Claims (12)

[Claims] [Claim 1] SEQ ID NOs: 2, 4, 6, 8, 9, 10, 18, 19, 20, 25, 26, 27, 28
, 29, 30, 35, 36, 37, 38, 39, 40, 41, 42, 43, and 44, or an isolated polypeptide comprising an amino acid sequence selected from the group consisting of: A polypeptide that is at least 80% identical to the polypeptide. 2. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 9, and 10, or at least 70% identical to said polypeptide. A polypeptide. 3. SEQ ID NOs: 2, 4, 6, 8, 9, 10, 18, 19, 20, 25, 26, 27, 28
Encodes a polypeptide consisting of an amino acid sequence selected from the group consisting of:, 29, 30, 35, 36, 37, 38, 39, and 40, or a polypeptide that is at least 80% identical to said polypeptide An isolated polynucleotide. 4. A polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 9, and 10, or a polypeptide at least 70% identical to said polypeptide. An isolated polynucleotide encoding. 5. SEQ ID NOs: 2, 4, 6, 8, 9, 10, 18, 19, 20, 25, 26, 27, 28
, 29, 30, 35, 36, 37, 38, 39 and 40, an antibody which specifically binds to a polypeptide consisting of an amino acid sequence selected from the group consisting of: 6. The antibody according to claim 5, which specifically binds to a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 9, and 10. 7. SEQ ID NO: 2, 4, 6, 8, 9, 10, 18, 19, 20, 25, 26, 27, 28
That specifically binds to an antibody that specifically binds to a polypeptide consisting of an amino acid sequence selected from the group consisting of:, 29, 30, 35, 36, 37, 38, 39, and 40 Idiotype antibody. 8. The anti-idiotype antibody according to claim 7, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 9 and 10. 9. Use of an antagonist against a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 9, and 10 for the treatment of inflammation. 10. The method according to claim 9, wherein the antagonist is an antibody that binds to a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 9, and 10. use. 11. Use of an antagonist against a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 9, and 10 for the manufacture of a medicament for the treatment of inflammation. . 12. The antibody according to claim 11, wherein the antagonist is an antibody that binds to a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 9, and 10. use.