CA2190679A1 - Lymphocyte tachykinin - Google Patents

Abstract

Abstract Not Yet Available

Description

LYMPHOCYTE TACHYKININ
Field of the Invention _ _ This invention relates to the biologically active peptides known as tachykinins. More particularly, the invention rela~es to a new tachykinin gene and to a new tachykinin peptide.

Background of the Invention _ ~
In the description which folIows, references are made to certain literature citations which are listed at the end of the specification.
A large number of biologically active peptides have been identified in higher animals One family of biologically active peptides is the tachykinin family.
Previously identified mammalian tachykinins are produced in the nervous system and brain and have therefore been called neurokinins. Three have been identified, namely, Substance P (SP), Neurokinin A (NKA; also called Substance K, neurokinin a, and neuromedin L), and Neurokinin B (NKB;
also called neuroklnin ~, and neuromedin K). Other tachykinins, founcl in non-r l;~n species,-include Kassinin, Eledoisin, and Physalaemin (1, 2).
All tachykinin peptides contain the characteristic carboxy-terminal amino acid motif Phe-X-Gly-Leu-Met-NH2 or F-X-G-L-M-NH2 Genes which encode the three r 1; ~n neurokinins have been described (1, 2). One gene, PPT-A, by alternate splicing encodes three different mRNA transcripts. The first of fhese, aPPT mRNA, encodes the precursor protein, preprotachykinln (PPT), from which SP is released by cleavage.
The other two, ~-PPT and r-PPT, encode a precursor which yields both SP and an additional amino acid sequence of 36 or 21 amino acids~ respectively. These peptides are long forms of NKA which has 10 amiro acids. Both long forms of NKA are also neurokinins. A second gene, PPT-B, encodes NKB only.
The previously described neurokinin genes are expressed=primarily in the nervous system and brain. The -- 2 -- =
neuro~inins ara released from nerve en~ings and act on the immune system. Release of neurokinins is associated with pain and antagonists or inhibitors cf the neurokinins act as analgesics (1,2).
A wide variety of additional bioactlvities have been attributed to the neurokinins, including decrease of blood pressure, plasma extravasation, smooth muscle contraction, release of neu~otransmitters, modulation of neuronal activities, a variety of haematopoietic effects and release of histamine, prostaglandins and other inflammatory mediators. These effects are mediate~d by one of three cellular receptors ~NK-l, NK2, and NK3) which have been identified on the plasma membrane of a variety of target cells (l,2).
:
Summary of Drawings _ ~
Certain embodiments of the invention are described, reference being made to the accompanying drawings, wherein:
Figure 1 shows a Northern blot of poly (A) RNA from the following cell lines and tissues:
70Z/3:~Pre=B cell line; 70Z/3y RAG=positive variant of 70Z~3; IIB4 CB17 I.l and 5.1, scid 2.1 and 4.1, RAG1-14 and -17 and RAG2-5 and -21: fetal liver cell lines transformed with Abelson murine leukemia virus;
WE~I 231: immature B cell line; J558: myeloma cell line; RBL 5 and EL4: T lymphoma cells; P338~1 myeloid lineage cell line; CB 5: erythroid lineage cell line;
NIH 3Y3 and L929: fibroblast cell lines; BMS 2.2:
stromal cell line.
Tissues were normal mouse tissues.
Figure 2 shows the effect of Substance Z on murine mast cell degranulation. X axis is concentration of substance Z(,uM) ar,d Y axis is mast cell degranulation expres~sed as % labelled serotonin release.
Figure 3 shows the activity of Substance Z on human fibroblasts in a cartilage degradation assay. X axis:
Sample numbers.~ Bars indicate % labelled glycosaminoglycans released from cartilage disc.
Figure 4 shows the effect of various concentrations of _, , .. ~

~ 21 90679 - 3 - ~ ~
Substance Z on the proliferation of murine leukemia cells, proliferation expressed as colonies per 500 plated cells.

Detailed Description of the Invention The inventors have identified a new mammalian gene, designated PP~-C, which encodes a pr-acur-sor protein ior a previously undescribed tachykinin peptide.
The cDNA sequence of the mouse PPT-C gene (Sequence ID
No:l) is shown in Table 1. It compri~es a sequence of 1249 nucleotides including an open reading frame encoding a sequence of 128 amino acids. The start codon is underlined in Table 1.
The deduced amino acid sequence (Sequence ID No:2) of :
the new precursor protein, designated substance Z precursor protein, is shown in Table 2. Its structure shows it to be a typical tachykinin precursor protein.
The SP precursor protein preprotachykinin, ior example, includes the amino acid sequence of Substance P, flanked by cleavage sites which are acted on by proteolytic enzymes known as convertases=to~release Substance P. The processing pathway has been described by Harris and Steiner et al.(3,4). Cleavage takes place at a doublet of basic amino acids, such as Lys-Arg or Arg-Arg.
Harris has proposed two basic types of recognition sequence for endoproteolysis: 1) a monobasic amino acid in close proximity to a cleavage doublet nf basic amino acids;
or 2) a strongly polar amino acid (Glu or Asp) in close proximity to a cleavage doublet of basic amLno acids. The neurokinin Substance P is an example of the first type, with an Arg in front of the cleavage doublet. For this type of recognition sequence, cleavage may occur either between the amino acids of the doublet (like Substance P) or àfter the doublet.
Neurokinin A is an example of the second type, with a Glu residue in front of the doublet. For the second type, cleavage usually occurs after the doublet.
Substance Z precursor protein has cleavage sites at amino acids 55~56 and amino acids 68169 (underlined in Table 2); these::cleavage sites flank a putative peptide ~ 21 9067q having the carboxy terminal motif FXGLM-NH2 characteristic of all known tachykinins.
The recognition sequence N terminal to the putative tachykinin could be classified as either type described by Harris.
By analogy with the other tachykinin precursor proteins, it is predicted that substance Z precursor prDtein is cleaved either within the amino acid doublet 55/56 ~R), to give the 11 amino acid=.peptide RSRTRQFYGLM-0 NH2 (Sequence ID No.:3 and designated herein substance Z) orC terminal to the second basic amino acid of the doublet, to give the 10 amino acid peptide SRTRQFYGLM-NH2 (designated substance 2-short form).
The characteristic tachykinin carboxy terminal motL~
of Substance Z is shown above in bold type. The remainder of its amino acid sequence differs from previously described tachykinins, as see in Table 3. Substance Z
precursor protein alsD differs considerably from the precursors of the previously described r ~ n tachykinIns.
Also included in the scope of the invention are fragments or analogues of the 11 amino acid peptide substance Z, including the lO amino a d d peptide, substance Z-short form, which are agonists retaining the biological activity of substance Z or act as antagonists of substance Z.
By analogy with studies on Substance P, it is predicted that up to about four N terminal amino acids may be deleted from substance Z while retaining full or partial agonist activity. The amino acid sequence comprising the tachykinin carboxy terminal motif is likely to be useful as an antagonist of substance Z activity.
Substance Z or fragments or.analogues thereof may be prepared by any suitable peptide synthetic method.
Chemical synthesis may be employed, for example standard solid-phase peptide synthetic techniques may be used. In standard solid phase peptide synthesis, peptides of varying length can be prepared using commercially available equipment. This equipment can be obtained from ~ 21 90679 Applied Biosystems (Foster City, CA.). The reaction conditions in peptide synthesi3 are optimized to. prevent isomerization of stereochemical centres, to prevent side reactions and to obtain high yields. The peptides are synthesized using standard autPmated protocols, using t-butoxycarbonyl-alpha-amino acids, and ~ollowing the manufacturer's instructions for blocking interfering groups, protecting the amino acid to be reacted, coupling, deprotecting and capping of unreacted residues. The solid support is generally based on a polystyrene resin, the resln acting both as a support for the growing peptide chain, and as a protective group for the carboxy terminus.
Cleavage from the resin yields the free carboxylic acid~
Peptides are purified by HPLC techniques, for example on a preparative Cl~ reverse phase column, using acetonitrile gradients in 0.1% trifIuoroacetic acid, followed by vacuum drying.
The peptides of the invention may also be produced by recombinant synthesis. A DNA sequence encoding the desired peptide is prepared, for example by cloning the required fragment from the DNA sequence encoding the complete precursor protein, and subcloning into an expression plasmid DNA. Suitable mammalian express-ion plasmids include pRC/CMV from Invitrogen Inc. The gene construct is expressad in a suitable cell line, such as a Cos or CHO
cell line and the expressed peptide is extracted and purified by conventional methods. Suitable methods for I
recombinant synthesis of peptides are readily available (5).
Analogues of substance Z may be prepared by similar synthetic methods. The term "analogue" extends to any functional and/or chemical equivalent of substance Z and includes peptides having one or more conservative amino acid substitutions, peptides incorporating unnatural amino acids and peptides having modified side chains.
Examples of side chain modifications contemplated by the present inventlon include modification of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidation with ~ 21 qO67q methylacetimidate; acetylation with acetic anhydride;
carbamylation of amino groups with cyanate;
trinitrobenzylation of amino groups with 2, 4, 6, trinitrobenzene sulfonic acid (TNBS); alkylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5'-phosphate followed by reduction with NaBH4.
The guanidino group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2, 3-butanedione, phenylglyoxal and glyoxal.
The carboxyl group may be modified by carbodiimide activation via -acylisourea formati'on fol'lowed by subsequent derivatisation, for example, to a corresponding amide.
Tyrosine residues may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid-, t-butylglycine, norvaline, phenylglycine, ornithine, sarc-osin-e, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers or amino acids.
Examples of conservative amino acid substitutions are substitutions within the folIowing five groups of amino acids:
Group 1: F Y W
Group 2: V L I
30 Group 3:' H K R
Group= 4: ' M S T P A G
Group 5: D E
For creation of antagonist compounds, C terminal residues are fre'quently susceptible targets. For example, in substance Z, Phe and/or Gly may be substituted with D-Trp and/or Met may be substituted with Leu.
Additionally, metabolically stable, non-peptide small molecules may be useful substance Z antagonists, by analogy with the previous described non-peptide antagonists of ~ 21 90679 Substance P (1).
Fragments or analogues of substance Z may be conveniently screened for their effectiveness as agonists or antagonists. For example, agonlst activity may be assessed in the cartilage degradation assay described herein. Identification of antagonists is discussed further below.
The gene PPT-~ appears to be expressed in haematopoietic cells, for example in pre-B cells, but not in tissues such as brain, lung, heart, liver, spleen, tymus and kidney, as can be seen in Figure 1. PPT-C expression hag also been fsund in isolated fetal liver cells, where expression was increased by administration of IL-7.
The following embodiments of the present invention are pro~ided.
1. IsolAted Nucleic Acids In accordance with one series of embodiments, the pres:ent invention provides isolated nucleic acids corresponding to, or related to, the PTT-C nucleic acid sequence disclosed herein.
In accordance with a first embodiment, an isolated nucleic acid sequence is provided which encodes substance Z
precursor pro:tein. The invention includes degeneracy equivalents of the disclosed nucleic acid sequences ~and sequences which hybridize to the disclosed sequences under stringent conditions~.
In addition to the disclosed nucleic acid sequences, one of ordinary~skilI in the art is now enabled to identify and isolate nucleic acids representinq PPT-C genes or cDNAs allelic to~the disclosed sequ-ence-s or which are_homologues of the disclosed se;~uences. One of ordinary skill in the art may now screen preparations of genomic or cDNA from any selected organism, including humans, other mammals, bacteria, viruses or yeasts or from genomic or -cDNA
librarles, using-probes or PCR primers to identify allelic or homologous sequences. In particular, the pre5ent invention enables the identification of the human homologue of the murine gene identified herein. ~It is also contemplated that additional PPT-C nucleic acid sequences _ _ _ _ _ _ _ _ . _ _ . , . . . _ , ~ 21 9067q will be isolated from human subjects suffering from a variety of disorders, enabling the identification of gene mutations which may contribute to these disorders.
Additionally, homologues of the mammalian PPT-C gene identified in lower organisms such as yeast, invertebrates or insects, may~provide suitable means for drug screening.
As will be understood by those in the art, allelic or homologous nucleic acid sequ-ences ~ay be identified and isolated using standard hybridization screening or=PCR
techniques, using short portions of~the nucleic acid sequences disclosed herein.
The present invention further provides portions of the disclosed nucleic acids sequences which are useful as probes and PCR primers, for example for identification of homologous ganes.

2. Substantially Pure Proteins The present invention further provides for substantially pure preparations of substance Z precursor 20' protein or frag'ments thereof. ~-Substance Z precursor protein may be produced by recombinant methods, as will be understood by those skilled in the art.

3. Antibodies The present invention also provides antibodies, and methods of making antibodies, which selectively bind to substance Z.
The antibodies of the invention may be polyclonal or monoclonal, or may be antibody fragments, including Eab fragmen-ts'an'd sIngle chain antibody fragments. In addition, recombinant antibodies may be generated, as well as humanized antibodies based upon non=human antibodies to substance Z. Antibody preparation techniques are generally described in references such as Antibody Engineering: A
Practical Guide, (6), or Antibody Engineering, (7).
In order to prepare polyclonal antibodies to substance Z, substance Z peptide may be conjugated to a carrier protein or m~y-be expressed recombinantly as a fusion protein which contains the peptide sequence of substance Z.

~ 2~ qO679 Preferably, the carrier protei~ or fused protein is conjugated to~ the carboxy terminal end of substance Z
peptide.
E. coli expression systems such as lacZ fusions using the pU~ series of vectors and trpE fusions using the pATH
vectors may, for example, be used to produce substance Z
fusion proteins. The expressed protein can then be purified, coupled to a carrier protein ~f desired, and mixed with Freund's adjuvant and injected into rabbits or other appropriate laboratory animals. Following booster injections at weekly intervals, the rabbits or other laboratory animals are then bled and the sera isolated.
The sera can be used directly or purified by conventional methods, such as affinity chromato~graphy. The sera can be used as probes to ldentify substance Z on gels of protein extracts from cells and tissues.
Monoclonal antibodles (MAbs) against substance Z may be raised in a number of animals, such as mice. Methods for making MAbs are well known and are described in publications such as that by Harlow and Lane(8). As an example, one such method is described.
Peptide Z is first coupled with the carrier protein, keyhole limpet haemocyanin, using carbodimide. The peptide-protein conjugate is then separated from free peptide by dialysis. The conjugate is injected into mice (typically 2-6 mice) at a dose~of 50~g per mouse with complete Freund's adjuvant. At two-week intervals, the mice receive a second and third booster. Test bleeds are performed 10 days after each booster to assess the development of the antibody response to substance Z. Antibody capture enzyme immunoassay may be used to determine the anti-Substance Z
antibody titer. In one embodiment of this assay, polyvinylchloride wells are coated with 50~1 of synthetic substance Z at a concentration of 2mg/ml. The wells are ~then blocked with 8% BSA/PBS. Serum obtained from the immunized mice:is serially diluted and 50~1 of samples of each dilution are added to the wells. Unbound antibodies are removed by washing and the presence of mouse anti-Substance Z antiboclies is then detected using horseradish ~ 21 9067q peroxidase-labelled rabbit anti-mouse lmmunoglobulin antibody. The antibody titer is determined by the highest dilution of the serum which shows the presence of anti-Substance Z antibo1ies.
A mouse with a high anti-Substance Z titer is selected for hybridoma production. After a final booster, spleen cells are obtained from the mouse and fused with myeloma cells such as sp2/0. Following fusion, the resultant hybridoma cells are diluted and plated in multi-well culture dishes. Supernatants of the cultures are screened for the presence of anti-Substance Z antibodies. Cells from positive wells are single-cell cloned. MAbs produced by these cloned hybridoma lines may be harvested from tissue culture supernatants or ascitic fluid.
Production of monoclonal antibodies as starting materials for obtaining humanised antibodies is well known (9). There are se~eral examples of humanisation of MAbs in the literature (10). Typically, a humanised antibody contains a binding portion obtained from non-human cells ~.
20 . (e.g., mouse cells) and one or more human portions, particularly framework portions of antibody obtained from human sources Particular examples are offered in the patent literature; United States Patent No. 5,558,864, issued September 24, 1996 to Bendig et al. described humanised and chimeric anti-epidermal growth factor receptor monoclonal antibodies; and United States Patent ~o. 5,482,85~, issued January 9, 1996 to Fell, Jr. et al.
described production of chimeric antibodies by homologous recombination. The specifications of both of these patent references and references mentioned therein are incorporated herein by reference.
Once a humanised antibody is obtaired, it should be tested in one or more anlmal models (ll-lb). Testing for toxic effects should also be conducted. For example, a single dosage (between 0.1 and 1 mg per kilogram of body weight) of antibody is administered intraperitoneally to mice and~or guinea pigs. The animals are observed for a week or so for adverse effects such as weight change and other obvious signs of toxicity. Immunohistological ~ 21 qO679 studies involving human tissues can be carri~ed out. For example, the reactivity of an antibody is evaluated using immunoperoxida5e staining on a varièty of normal human tissues. High dosage pharmacology/toxicology studies in adult chimpanze~es can be carri~d out. Analysis of blood chemistry, hematology, and urinalysis:is conducted.
Further, an assay for immunocompetence is conducted.
Animals are challenged with different strengths of dinitrochlorobenzene in acetone and the extent of response to DNCB is evaLuated.
A dosage r-egimen used for treating a patient will be determined by the attending physician considering various factors which affect drug action, e.g., the condition, body weight, sex and diet of the patient, the severity of the disease, time of administration and other clinical factors.
For example, for treatment of rheumatoid arthritis, a r~ n~d dosage is likely to be in the range of. 10 to 100 mg, over a period of a week or 50 (17). Such recommendatian must be based on an objective study, and particularly a stu~y which measures the level of agent in patient serum over time. Development of recommended dosages would likely be preceded by analysis of plasma levels of MAb in chimpanzees.
The antibadies of the invention may be labelled or conjugated for diagnostic and/or therapeutic uses. ~For example, they may be coupled to radionuclides, fluorescent compounds or enzymes for imaging or therapy or may be incorporated into liposomes for targeting to a specific tissue site.
The antibodies of the invention have utility as laboratory reagents, for example, for Western blotting to identify cells:or tissues~cxpressing~the PPT-C gene or immunocytochemistry or immunofluoresence techniques to identify the subcellular location of the precursor protein.
~ Additionally, the antibodies of the invention may be used as therapeutic agents to selectively bind and inhibit the activity of suastance Z for treatment of disorders associated with excess or inappropriate production of substance Z. For example, cancer cells such as leukemia .

21 ~067~

cells, whose growth is stimulated by substance Z, may be inhibited or suppres3ed by administration of antibodies against substan:ce Z.
~imilarly, any cell type which is dependent for growth or activation on stimulation by substance Z may be controlled by administration of anti-substance Z
antibodies.

4. Trnnsgenic Animnl Models The present invention also provides for the production of transgenic~non-human animal models for the study of the effects of over expression of the PPT-C gene and over-production of substance Z, for the screening of candidate compounds as potential antagonists of substance Z and for the evaluation of potential therapeutic interventions.
The transgenic ani~als of the invention also provide models of disease conditions associated with abnormalities of substance Z production. For example, the transgenic animals of the invention may provide an animal model of at least some aspects of rheumatoid arthritis.
Animal species suitable for use in the animal models of the inventlon include mice, ra-ts, rabbits, dogs, cats, goats, sheep, pigs and non-human primates.
Animal models may be produced by inserting a selected nucleic acid sequence into a germ line cell~or a stem cell using previously described techniques such as oocyte microinjection o-r transfection or microinjection lnto embryonic stem cells. Alternatively, an endogenous PPT-C
gene may be inactivated or replaced by homologous recombination within embryonic stem cells to produce "knock-out" or "knock-in" animal models. Techniques for obtaining transgenic an-imals are widely available in the literature. For example, laboratory techniques for=the production of transgenic mice is described in Hogan et al.
(18).
In accordance with one embodiment of the invention, transgenic- animals generated by the introduction of a PPT-C
transgene into a fertili~ed animal oocyte, with subsequent growth of the embryo to birth as a live animal. The PPT-C
transgene is a transcription~unit which directs the ~ 21 qo67q expression of PPT-C gene in eukaryotic cells. To create the transgene, PPT-C gene is ligated with an eukaryotic expression module. The basic eukaryotic expression module contains a promoter element to mediate transcription of PPT-C sequences and signals required for efficlent for termination and polyadenylation of the transcript.
Additional elements of the module may include enhancers which stimulate transcription of PPT-C sequences. The most frequently utilized termination and polyadenylation signals are those ~erived from SV40 (5). The choice of promoter and enhancer elements to be incorporated into the PPT-C
transgene is determined by the cell types in which PPT-C
gene is to be expressed. To achieve expression in a broad range of cells, promoter and enhancer elements derived from viruses may be utilized, such as the herpes simplex virus thymidine promoter and polyoma enhancer (19). To achieve exclusive expression in a particular cell type, such as B
cells, specific~promoter and enhancer:elements could be used, such as the promoter of the mb-1 gene and the intronic enhanc~er of the immunoglobuiin heavy chain gene (20).
The PPT-C transgene is inserted into a plasmid vector, such as pBR322 for amplification. The entire PPT-C
transgene is then released from the plasmid by enzyme digestion, purlfied and injected into an oocyte. The oocyte is subsequently implanted into a psèudopregnant female animal. Southern blot analysis or other approaches are used to determined the genotype of the ~ounder animals and animals generated in the subsequent backcross and intercross. ~
The PPT-C nucleic acid sequences of the invention may also be utilized in the creation of transgenic mice deficient in the production of Substance Z by homologous recombination.
Methods of disrupting the genes of an animal are well established and are described in publications such as Kitamura et al. (21). As the first step, the genomic sequence which gives rise to the PPT-C mRNA is pulled out by screening a mouse genomic library wi~h the PPT-C cDNA

21 ~0679 molecule as a probe. A fragment containing the coding sequence for Substance Z peptide and some flanking sequences is cloned into a plasmid vector such as pBR322. A
lkb pMClneo fragment contalning the neomycin resistant gene is then insert~ed into the sequences encoding Substance Z
peptide. This resultant construct is linearized, and introduced into D3 embryonic stem (ES) cells by electroporation. Neomycin-resistant colonies are selected and expanded. Homologous recombination events are identified by PCR and Southern blotting. ES cell clones carrying the disrupted PPT-C genes are in~ected into blastocysts of C57BL/6 mice, and the resulting male chimeras are m~ted to C57BLj6 females. Agouti offspring are analyzed by Southern blotting for the presence of the mutant PPT-C gene. Heterozygous mice are intercrossed, and homozygous PPT-C-mutant mice are identified by Southern blotting.
These mice will provide a model for study of the effects of substance Z deficiency and the inter-relationship between substance Z and other factors inmaintenance Q~ health, including the maintenance of a normal immune response. These animals will also provide tools for scree-ning candidate compounds for their interaction with substance Z or the signalling pathway activated by substance Z.

5 . .5~- e~n; nq for Druqs Which Affect Expression o~ the PPT-C Gene The present invention also enables the analysis of factors affectIng the expression of the PPT-C gene in humans or in animal models. The invention further provides a system for screening caffdidate compounds for their ability to turn on or turn off expression of the PPT-C
gene.
~ For example, pre-B cells may be isolated from a mammal and grown in culture in the presence of IL-7 (43-45). Such a cell culture system can be used to-Ldentify compounds which activate productiDn of substance Z or, once substance z production has been activated in the cells, they can be .. . _ _ ~ .. . . .

- 15 - 2 1 9 0 67q used to rdentify compounds which lead to suppression or switching off o~ substance~Z production.
Compounds thus identified are useful as therapeutics in conditions where substance Z production is deficient or is excessive. 5~

6. Identirication o~ Antagoni3ts The present invention enables also a screening method ior compounds of therapeutlc utility as antagonists of the biological activity of substance Z. Such antagonist compounds are useful, for example, to reduce or prevent tissue damage resulting from activatlon of synovial ~.
fibroblasts by substance Z, for example in conditions such as rheumatoid arthritis, and to reduce or prevent symptoms or tissue damage resulting from mast cell activation by substance Z, for example in conditions such as acute allergy.
Those skiLled in the art will be able to devise a number of possible scIeening methods for screening candidate compounds for substance Z antagonism.
~ For example, candidate compounds may be screened for biological activity and for antagonist activity in the cartilage degradation assay described herein.
A screening method may also be based on binding to the substance 2 receptor. Such competitive binding assays are well known to those skilled in the art. Once binding has been established for a particular compound, a biological activity assay is employed to determine agonist or antagonist potential.

7. Diagnostic Methods . The present invention enabies the identification of disorders associated with overproduction or underproduction of substance Z by assay of substance Z in appropriate tissue samples.
Substance Z may be assayed by a variety of methods, immunoassay being ~referred. Many types of immunoassay are described in the literature. For example, radioimmunoassay may be employed (22).
In accordance with one embodiment, substance Z is labelled with 12sI by chloramine T, as described in Example ~ 21 ~067q -~16 -1, for use in radLoimmunoassay.
A 100~1 al'iquot of serially diluted substance Z
standard or sample is mixed with 100~1 of l2sI-labelled substance Z and 100~1 of a dilution of anti-substance Z MAb that gives approximately 50% binding in the absence of unlabelled peptide. One ml. o~ a mixture of 6mg/ml Norit A
~Amend Drug and Chemical, Irvington, NJ) and 0.75mg/ml Dextran 70 in 0.25~ BSA-Dulbecco's ~BS buffer is added_ The tubes are vortexed and centrifuged. A lml aliquot is counted in a gamma counter. Standard concentration is plotted against (cpm bound in the pres-ence of standard)/(cpm bound in the absence of standard).
~oncent'ra~ion of substance Z in tissue samples is determined by reference to the standard curve. A normal range of subs~tance Z levels is obtained by assay of a numoer of normal subject tissue samples, as is understood by those skilled in the art. ~ -8. ~h~r~r~l-tic Methods The present invention further enables therapeutic intervention in disorders ass-ociated with an inappropriate level or location of substance Z.
Such interYentiOnS include .=
~ a) in conditions associated with undesired biological activity of substance Z, inhibition of its activity by administration of antagonist compounds or anti-substance Z antibodies.
For example, tumour cells whose growth is responsive to substance Z may be inhibited or suppressed by administration of substance Z antagonists.
In condltions such as rheumatoid arthritis or acute allergy, where cells are activated to produce substance Z
leading to tissue damage, therapeutic intervention may be achieved by administration of substance Z antagonists to reduce substance Z activity.
Alternatively, substance Z-producing cells may be targeted and destroyed to reduce undesired substance Z
production.
~b) conditions associated with a deficiency of substance Z may be treated by administration of - 17 - 2~qO67q pharmaceutical compositions lncluding substance Z.
Substance:2 or therapeutically effective analogues or fragments thereof may be be administered therapeutically by injection or by oral, nasal, buccal, rectal, vaginal, transdermal or ocular routes in a variety of formulations, as is known to those in the art.
For oral administration of peptides, various techniques can be used to improve stability, based for example on chemical modification, formulation and use of protease inhibitors. Stability can be improved if synthetic amino acids are used, such as peptoids or betidamino acids, or if metabolically stable analogues are prepared.
Formulation may be, for example, in water/oil emulsion or in liposomes for improved stability. Oral administration of peptides may be accompanied by protease inhibitors such as aprotinin, soybean trypsin inhibitor or FK-448, to provide protection for the peptide. Suitable methods for preparation of oral formulations of peptide drugs have been described, for example, by Saffran et al., (23~ (use~of trasyloI protease inhibitor); Lundin et al.
(24) and Vilhardt et al., (25).
Due to the high surface area and extensive vascular network, the nasal cavity provides a good site for absorption of both lipophilic and hydrophilic drugs, especially when coadministered with absorption enhancers.
The nasal absorption of peptide-based drugs can be improved by using aminoboronic acid derivatiYes, amastatin, and other enzyme inhibitors as absorption enhancers and by using surfactan~s such as sodium glycolate, as described in Amidon et al., (26).
~ he transdermal route provides good control of delivery and maintenance of the therapeutic level of drug over a prolonged period of time. A means of increasing skin permeability is desirable, to provi-de for systemic access of peptides. For example, iontophoresis can be used as an active driving force for. charged peptides or chemical enhancers such as the nonionic surfactant n-decylmethyl sulfoxide (NDMS) can be used. --~ 2l ~067~

Transdermal delivery of peptides is described in Amidon et al. (26) and Choi et aI. (2i).
Peptides may also be conjugated with water soluble polymers such as polyethylene glycol, dextran or albumin or incorporated into drug delivery systems such as polymeric matrices to increase plasma half-liie. ~
More generally, formulations suitable fer particular .
modes of administration of peptides are descrlbed, for example, in Remington's Pharmaceutical Sciences (28).
As an alte~native therapy in substance Z deficiency, gene theraFy may be carried out, comprising administration of a PPT-C gene to a substance Z deficient subject.
Appropriate techniques may be employed to target the introduced gene to a desired target tissue. Gene therapy has the potential to avoid life lo~g administration of exogenous peptides and may provide for a more physiologically-appropriate level of substance Z than exogenous ~administration.
EXAMPLES
The examples are described for the purposes of illustration and are not intended to~limit the scope of the invention.
Methods of molecular genetics, protein and peptide biochemistry and immunology referred~to but not explicitly described in this disclosure and exampies are reported in the scientific literature and are well known to those skilled in the art.

Example 1: Isolation Qf PPT~-C cDNA ,_ RNA prep~ration and Northern analysis Total RNA was isolated from CDl mouse tissues and cultured cell lines as described by Bergman et al. (29) and Sambrook et al.(5). Poly (A) RNA was selected by passage over oligo(dT)-cellulose (Pharmacia). For Northern anaIysis, 5~g o~ poly(A)+RNA was separated on 1% agarose gels containing 20mM NaHPO4, and lM formaldehyde, transferred onto Hybond-N nyion membranes (A~ersham), UV-immobilized, and hybridized with 32P-labelled probes prepared by a random hexamer-primed method (30).

2 1 90(~7~

Hybridization was at 42 C in 5X SSPE (750mM NaCl, 5mM EDTA, 50mM NaH2PO4, pH 7.4), 2% SDS, 5X Denhart's solution, 100~g of sheared/boiled salmon sperm DNA, 100~g of poly A, and 50% formamide. washIng was in 0.1X SSC~(i5mM NaCl, 1.5mM
sodium citrate, pH 7.0), 0.1% SDS at 65 .
~;ff~ntial displ~y PCR
Differential display PCR was performed following the method described by Liang and Pardee t31) with GenHunter Kit ~Brookline, MA). Poly (A)+RNA (0.2~g) from IIB4 and 70Z/3 cells (both transformed murine pre-B cell lines) was used for first strand cDNA synthesis with each of the four modified oligo(dT) primers (T12MN). The synthesized first strand cDNA was used as a template in the subsequent PCR
- reaction. In a 0.2ml PCR tube the following were added:
2~1 of 10X PCR buffer (500mM KCl, i5mM MgCi2, 100mM Tris-HCl at pH 8.3), 5'-arbitrary 10mer ¦2~M), T12 MN (10,uM, same as used in cDNA synthesis), cDNA synthesisl, cDNA template, 1.6~1 of dNTP mix (25~M), 12.5~Ci 2sS-dA~P (100 Ci/mmole), 1 U of Taq DNA polymerase (Perkin Elmer), and 9-2~1 of dH2O.
PCR was performed as follows: 94 C, 30s; 40 C, 2min; 72 C, 30s for 40 ~ycles. Four microliters of the PCR products from the two starting cells were run side by side on a 6%
urea:acrylamide sequencing gel. ~The dried gel was exposed to an X-ray film and the autoradiogram was analyzed for differentialiy displayed bands. These bands were cut out from the gel, and the DNA was eluted by soaking the gel slices in 100~1 of TE buffer for 10mIn and then boiling for 10min. The eluted DNA was precipitated using glycogen and ethanol, air-dried, and redissolved in 10~1 of dH2O. This DNA was reamplified with the same comoination of primers used in the first PCR. The reamplified DNA was gel-purifLed and used as a probe in Northern analysis. Once the differential expression was confirmed, the DNA was cloned using the TA Cloning Kit (Invitrogen, CA).
I cDNA library construction ~nd screening A 70~/3 cDNA library was constructed using standard procedures es~entially as described by Sambrook et al., (5). Reverse transcription was carried out on 5~g of _ _ . _ _ _ _ _ _ . .. .. . . .. .

~ 21 qO67q poly(A) RNA to qenerate first strand cDNA using an oligo~dT)12-1~ primer. The mRNA-cDNA hybrid was treated - with Rnase H. Remnants of mRNA served as primers for the synthesis of second strand cDNA. The double strand cDNA
was cleaved with Klenow to create blunt-ends, and then ligated to an EcoR I/Not I adapter. This adapter-tailed cDNA was purifled to remove the unligated adapters, and then inserted into lambda ZAPII vectors ~Stratagene, CA).
The constructs were packaged into infectious phage partLcles, amplified in E. coli strain XL1-Blue. The ratio of recombinants in the library was over 85~. The total yield of the recombinants was -4x106. The size of cDNA
inserts from 12 randomly picked up clones ranged from 0.8-4.5kb with an average of 1.4kb. Using the 440bp differentIal display PCR fragment as a probe, 2X106 plaques were screened. Up to 20 positive clones were isolated by three rounds of screening. The in vivo excision procedure was followed to release pBluescript plasmid from the lambda ZAPII vector. The insert size of the ten clones varied from 0.5-1 1 kb. Nucleotide sequence of each clone from both strands was determined by the dideoxynucleotide chain termination method ~6).
5' RACE of n~A
The largest cDNA clone which was isolated from the above mentioned ~library still lacked the 5' end of the gene. Therefore the 5' end of PPT-C cDNA was amplified using the method of 5' RACE ~rapid amplification of complementary DNA ends) using standard protocols included with a reagent kit from Clontech iPalo Alto, CA). The PPT-C gene specific primer sequence used was5'GGACAGAGAG~CA~ lC-3'. The amplified PCR product was cloned using the TA Cloning kit ~Invitrogen, CA).
Nucleotide sequences were determined by dideoxynucleotide chain reaction termination method.
~ The nucleotide sequence of the cDNA is shown in Table 1 and the amino acld sequencé deduced ~rom the open reading frame is shown in Table 2.

Exnmple 2: Expression of PPT-C gene _ _ _ _ , _ _ _ _ _ _ _ _ ~1 2' 90679 RNA was extracted from cells and tissues, and poly (A)~ RNA was isolated by passage over cligo (dT) -cellulose. Northern blot analysis was performed as described in Example l. 7G9 was a probe derived from PPT-C
cDNA. L32, a ribosomal protein-coding gene, was used as a loading control. II4B, CB17 l.1 and 5.1, scid 2.1 and 4.1, RAGl 14 and 17, RAG2 5 and 21 are fetal liver cell lines transformed with Abelson Murine Leukemia virus. 70Z/3 is a pre-B cell line. 70Z/3y is a RAG-positive variant of 70Z/3.
WEHI 231 is an immature B cell line. J558 is a myeloma cell line. RBL5 and EL4 are T lymphoma cells. P33BD1 and CB5 are cell lines of myeloid and erythroid lineage respectively. NIH3T3 and L929 are fibrobLasts. BMS2.2 is a 5 stromal cell line. Mouse tissues included in the scheme were brain, lung, heart, liver, spleen, thymus and kidney.
The results are shown in Figure 1. Expresslon of PPT-C
mRNA was restricted to cells o~ B lineage at its early developmental stage.
To test PPT-C expresslon in primary cells representing pre-B cells, RT-PCR was used to amplify PPT-C
mRNA (5) from fresh mouse.fetal liver celis and from mouse fetal liver cells expanded with IL-7 for~enrichment of B
lineage cells. The primers used were 5'-TAACCACCAGCAACGAGA-3' and 5'-ATGGCTGAGGAAGCTACCT-3'. PCR
products were blotted on a nylon membrane, and probed with 7G9.
PPT-C was expressed in fetal liver cells and culturing of the cells in the presence of IL-7 further increased PPT-C expression ~data not shown).
Ex~mple 3: prepAration of Subst~nce Z
Peptides~RSRTRQFYGLM (substance Z) and SRTRQFYGLM
(substance Z - short form) were synthesised using conventional solid phase peptide synthesis (33).
Analytical and preparative high-performance liquid chromatography were utilized to characterize and isolate the final compounds. Fast bombardment mass spectrometry was used to confirm the molecular weight.
Synthetic substance Z was used for the studies of ~ 21 90679 biological activity described in the following examples.

Ex~le 4: Efl~ect of Substance Z on cartilage degradation by human fibroblasts Generation of Synovial Fibroblast Lines The ability of 3 human fibroblast cell lines, synovial fibroblast lines RA1 and RA2 and skin fibroblast cell line CCD-967, to degrade cartilage discs~ was tested.
RA1 and RA2 were synovial fibroblast lines derived from synovium obtained from Rheumatoid Arthritis patients ~according to ACR criteria ~34)) undergoing knee replacement. They were established by placing finely minced synovial tissue into a 25cm tissue culture flask with medium, consisting of OPTI-MEM ~Gibco, Grand Island, NY., USA) supplemented with 10% FCS ~Gibco, Grand Island, NY, USA), antibiotic-antimycotic mix ~penicillin G sodium [1000 units]/streptomycin sulfate [1000 unitsl/amphotericin B [2.5 mg~ml]) ¦Gibco, Grand Island, NY, USA), 5.5 x 10 sM
b-mercaptoethanol ~Sigma, St. Louse, MO, USA) and 2.4 g/L
.sodaum carbonate ~Mallinckrodt Inc., Point-Claire, Quebec, Canada) . Minced synovial tissue was left in the culture flask for a period of 1 week to allow the fibroblasts to grow out of the tissue and onto the s rface of the culture flask, at which point the tissue was rerrLoved. Although 25: there was variation from line to line, in general, cells were passaged every 2 weeks and media was replenished every 3 days. The distinct morphology of fibroblasts along with their unique ability to survive multiple passages in the absence of added growth factors in vitro was used to assign 30 a lineage to these cells.
Skin fibroblast cell line CCD-967 ~Skin 1) was obtained from ATCC and cultured exactly as described for the synovial fibroblast c~ll lines.
The macrophage cell line U937 was used to generate a 35 monditioned medium. To prepare U93i-conditioned medium, U937 cells were ~grown to a concentration of approximately lxl05/ml of OPTI-MEM. This medium was subsequently centrifuged ~30a0rpm for 15rn n at 4 C) and filtered ~0.2mm millex-G~I filter, I~ pore, Bedford, MA, USA) prior to use ~ 21 9n67q in the assay. If the conditioned medium was not used immediately, it was stored immediately at -70 C.
Cartilagc degr~dAtion ~ss~y The cartilage degradation assay originally described by Steinberg et al. (35), and modified by Janusz and Hare (36) was~used with human cell lines and human normal cartilage. In brief, measurement of degradation of cartilage was performed by culturing of fibroblasts in the presence of radiolabelled human cartilage discs. Cartilage discs (qmm x lmm~ were prepared from normal human femoral cartilage using a 4mm cork bore. Femoral cartilage was obtained from normal appearing cartilage in patients with osteoarthritis-undergoing ~oint arthroplasty. Discs were incubated overnight with OPTI-MEM containing S33 Na~S04 (~OmCi/ml~ (Amersham, Oakville, ON, Canada~. Label was incorporated into the glycosaminoglycan side chains of the proteoglycan within the cartilage. The discs were then washed (x5) with sterile PBS to remove unincorporated r~diosotDpe. Discs were then freeze-thawed 5 times and heated at 65 C for 15 min. to inactivate endogenous enzymes and cytokine activity. The discs were store~ at -20 C prior to use. Inco~poration of radionuclide was normally found to be between 50,000 to 100,000 dpms/disc.
Adherent fibroblasts to be cocultured were trypsinized from culture flasks with 0.05% trypsiniO.53 mM EDTA 4Na (Gibco, Grand Island, NY, USA). 1 x 104 fibroblasts were cultured together with a radioactive cartilage disc, either in U937-conditionea medium or with l nM substance Z for=7 days in 96 well Nunclon~plates (Nunc, Roskilde, Denmark).
On day 3, t~e original medium was removed and replaced with 200mI fresh medium, supplemented as before. In some experiments, cells were:cultured with U937-conditioned medium or with substance Z in transwell tissue culture inserts for 96 well tissue culture piates with a 0.2mm anapore membrane (Nunc, Roskilde, Denmark) between disc and fibroblasts.
On day 7, 200 ml of medium was removed and added to 3ml scintillation fluid (Beckman Instruments Inc., Fullerton, CA, USA) and counted in a scintillation counter - 24 _ 2 1 q 0 6 7 9 (Beckman Instruments InC., Fullerton, CA, USA 151071). The remaining isotope in the cartilage di5c was measured by completely digesting the disc in 0.5ml of tissue solubilizer (Beckman Instruments Inc., Fullerton, CA, USA).
Solubili~ed discs were counted in scintiilation counter using 3ml scintillation fIuid (Beckman Instruments Inc., Fullerton,~CA, USA). Data were expressed~as % of S35 released into the supernatant using the equation: %= (dpm in supernatant)/(dpm in supernatant + disc) X 100.
In all experiments, culture of the radio-labeled disc alone was performed as a control. All experiments reported were ~arried out in quadruplicate and the % release of S35 calculated for each individuai replicate.
The results are shown in Figure 3.
The results show that synovial fibroblast lines degLaded cartilage if both contact with cartilage discs (Contact) and U937-conditioned medium (U937) were present.
In the absence of either i-actor, degradation did not occur. Substance Z, however, stimulated the degradation of cartilage by synovial fibroblast3 when neither contact nor conditioned medium was present.
Skin fibroblasts did not degrade cartilage when provided with contact and U937-conditioned medium or when challenged with Substance Z.
: ~ ~
Ex~m~le 5: Effect of S~L~ L~n~e Z on growth of 1 enk~mi A
C~
Growth of 70Z/~3 T.~~1k~mi J- Cell~
70Z/3 murine leukemia cells were maintained in liquid culture using the supplemented OPTI-~EM medlum described in Example 4, except that 5% FCS was used instead of 10%. To examine the effects of Substance Z on the growth of 70Z~3, cells were cloned in medium containing 0.3% melted agar (Bacto Agar, Gibco) following standard procedures described by Sauter and Paige (37). Briefly this consisted of pouring lml of medium contalning 0.3% melted agar into a 35mm tissue culture:plate. This layer was allowed to gel for 2~min at room temperature after which a second lmi layer, containing 70Z/3 cells (300 - I0~D/plate) in medium .... . . . .. . _ . . . . _ _ . .... . . .

2 1 qO67q - 25 - - ~
supplemented with 0.3% agar was poured. After 7 days of culture, the colonies tFoci containlng at least 50 cells) were enumerated visually with the aid of a stereomicroscope. ~ ~
The results are 5hown in Figure .4 and show that leukemia -cell growth was augmented by Substance Z.

XxAmple 6: ~nhAn~ Ma~t Cell D~ An~lAtion by Sub~t~nce Z
Primary mast cells were obtained by culturing bone ~marrow cells in IL-3-containing WEHI-3 conditioned medium as described by Berger (38~. By 6 weeks, >99% of the cells in the culture were mast cells. Experiments were carried out generally as described by Berger (38), except that degranulation wàs measured by checking the release of 3H-serotonin lnstead of that of b-hrxos~m~n;dase.
Mast cells were cultured overnight in Opti-MEM (Gibco) + 5~FCS + b-mercaptoethanol + WEHI-3 conditioned medium plus 3.5mCi/ml of 3H-serotonin. 3H-serotonin would be preferentially incorporated into granules of mast cells during the cuiture. The cells were then washed to remove excess serotonin, and cultured in fresh medium for 15 min.
Subsequently, cells were washed and resuspended in Tyrode's buffer (lOmM Hepes" pH7.4, 130mM NaCl, 5mM KCl, 1.4mM CaCl2, lmM MgCl2, 5.6mM glucose, 0.1% BSA). Aliquots of 2~105 cells were incubated with 5mg/ml SPE-7 anti-DNP monoclonal IgE
antibodies ( Sigma Chemicals Co.) for 30-~0 min, washed and treated with DNP-HSA antigen (Sigma Chemicals Co.) at a concentration of 0, 1 and 5ng/ml for=30-6Q min at 37 C.
Substance Z was included in the finai incubation at a :concentratior Eanging from Q-50mM. Cells were pelleted at 3000rpm for 5 min. An aliquot of supernatant was removed and placed in scintillation vials with appropriate scintillation fluid. ~emaining supernatant was discarded, cell pellet was lysed ln an equlvalent volume of Tyrode's +
0.5% Triton X-100 and 1/5th of the iysate was transferred to a fresh scintillation vial with scintillation fluid Samples were counted in scintillation counter.
~ Degranulation = counts in super~atant x 100 ,_ . . = i, . , _ ~ .

~ 21 9067q counts in (Supernatant + pellet) The results are shown in Figure 2.
Mast cells express high affinity receptors for IgE
antibodies ~rass-linking of surface-baund IgE by antigen, typicaLly at high concentrations, leads to the activation of these cells and the release of granules containing inflammatory mediators. This process of degranulation is also regulated by other signals~. Substance P, for example, has been shown to increase the sensitivity of mast cells to degranulation stimulated by exposure to antigen (39).
As demonstrated here, substance Z also enhanced IgE-mediated degranulation of mast cells Example 7: Isolation of ~_C~yLOI ~or S~L~...ce Z
For identification and isolation of the receptor for substance Z, substance Z is labelled with an easily detectable marker or label. For example, substance Z is labelled with a radio-label such as I by a conventional method such as those described by Harlow and Lane (8). One such method employs~chloramine T. 10mg of synthetic Substance Z in 25ml of 0.5M sodium phosphate (pH 7.5) ls mixed with 500mci of Na12sI and 25ml of 2mg/ml chloramine T.
After a incubation of Z0sec, 50ml of stop solution (2.4mg/ml sodium metabisulfite, 10mg/ml tyrosine, 10 glyceroL, 0.1% xylene cylanol in PBS) ~s added. The iodinated Substance Z is subsequently separated from the iodotyrosine on gel filtration column.
Labeled Substance Z is utilized to determine the distribution of its receptor in various cells and tissues, and to elucida~te the binding affinity and kinetics of Substance Z with its receptor (1).
Various approaches may be employed for the isolation of the receptor gere for Substance Z. Expression cloning is one of the most frequently applied strategies in receptor cloning (41, 42). For example, RNA is extracted from the cells or tissues which express Substance Z receptor as determined by using l2sI -labeled Substance Z. Poly(A)+ RNA
is isolated using cligo-dT cellulose, cDNA is synthesized and ligated into the mammalian expression vector pMET7 ~ ~1 9067q - 27 ~
(42). Ligated DNA is EtOH precipitated and resuspended in dH~O at 25ng/ml. DNA (lml) is used to transform each of 40ml of competent DHlOB E. coli cells by electroporation. Based on the titers of the cDNA transformations, 96-well plates .
are inoculated~with 150cfu per well in lml of LB-Amp.
Cultures are~grawn for 15-16hr at 37 C. 100ml of each culture is:removed and added to 100ml of 50% Glycerol, mixed, and stored at -80 C. Plasmid DNA is prepared from the rest of the culture.:The library is thus produced as pools of 150 clones.
To screen the library, plasmid DNA is transiently transfected into a 10cm dish of COS cells with lipofectamine. DNA from eight pools is used for each transfection. After 48hr, the cells, just at or before conf~uence, are transferred onto culture slides, and then incubated with 0.SnM l2sI -labeled Substance Z. After two washes , the slides are exposed to film. Positive pools are subsequently broken down to subpools of 15Q clones each. The positive subpools are further divided until a single clone encoding a Substance Z-binding activity is identified.
The nucleotide sequences of the isolated clones are determined, and deduced amino acld sequences are obtained.
In order to confirm that the isolated clones encode receptor specific for Substance= Z, COS cells are transfected with each of the individual clones. Substance Z
binding characteListics conferred by these clones should be comparable with that observed in receptor-bearing cells.
In addition, the value of the dissoc~ation constant (KD) as determined by Scatchard analysis should be similar, and the binding of l~sId-labeled Substance Z to the transfected cell should be ,able to be completely blocked by cold Substance Z, but not by other tachykinins.

The present invention is not limited to the features of the embodiments described herein, but includes all variations and modifications within the scope of the claims.

~ 2l 90679 Re~erences All of the listed references ~e inco~porated herein by reference.
1. Regoli, D. Bodon, A. And Fauchere J-L. (1994) Pharmacological Reviews v. 46, pp. 551-599.
2. Nakanishi, S., (1987), Physiological Reviews v. 117, p. 1142.
3. HarLis Arch, (1989), Biochem. Biophys. v. 275, pp.
315-333.~
4. Steiner et al. (1992), J. BioI. Chem. v. 267, pp.
23435-23438.
5. Sambronk, J., Fritsch, E.F., and Maniatis, T. (198) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
6. Antibody Enqineering: A Practical ~Gyide, (1992~, _ Borrebaek, Ed., W.H. Freeman & Company, N.Y.
7. Antibody Engineering, (1995), 2nd Ed., Borrebaek, Ed., Oxfo~d University Press, Oxford.
8. Harlow and Lane, Antibodies, a Laboratory Manual, (1988) Col~ Spring Harbor Laboratory, New York.

9. Tami J.A., Parr, M.D., Brown, J.S., (1986), Am. J. Hosp.
Pharm., v. 43, p. 2816-25.

10. Gorman, S.D., Clark, M.R. (1990), Semin Immunol., v. 2, pp. 457-466.

11. Halloran, M.M., Szekanecz, Z., Barquin, N., Haines, G.K., Koch, A. (1996), Arthritis Rheum., v. 39, pp. 81-19 .

12. Joosten, L.A.B., Nelsen, M.M.A., van de Loo, F.A.S., van den Berg, W.B., (1996), Arthrltis Rheum., v. 39, pp.
3Q 797-8~:g;

13. Jasin, H.E., Lightfoor, E., David. L.S., Rothlien, R., Foanes, R.B., Lipsky, P., (1992),~~Arthritis Rheum, v. 35, pp. 541-549.

14. Geiler, T., Kriegsmann, J., Keyszer, G.M., Gay, R., Gay, S., (1994), v. 37, pp. 1664-1671.

15. Yoshina, S., Quattrochhi, E., Weiner, H.L., (1995), Arthritis Rheum., v. 38, pp. 1091-1~96.

16. Caccesse, R.G., Zimmerman, J.L., Carlson, R.P., ~I992), Mediators ~lamm., v. 1, pp. 273-279.

~ 21 9067q 17. Dëla~uente, ~.C., Resman-Targo~, B.H., ~1999), Annals of Pharmacotherapy, v. 28, pp. 650-654.

18. Hogan et al. (1986) Manipulating the Mouse Embryoc Coldspring Harbour Laboratory Press, Coldspring Harbour, ~ N.Y.
l9. Thomas and Capecchi, Cell, (1987), v. 51, pp. 503-512.
20. Sun Cell, ~19g4), v. 79, pp. 853-900.'' 21. Kitamura et al. Cell (1992), v. 69,'pp. 823-831.
22. ~einstock et al., J. Immunol. ~1588J, v. 141, pp. 961-23. Saffran et'''al., (1979) (use of'trasylol proteaseinhibitor~;
24. 1undin et al. (1986);
25. Vilhardt et al., (17) (1986), Gen. Pharmacol., v. 17, ~ pp. 481-483.: -26. Amidon et al., tl994), Ann. Rev. Pharmacol.~Toxicol., v. 34, pp. 321-341.
27. Choi et al.', (l990),.Pharm. ~es., v. 7, pp. 1099-1106.
28. Remington's Pharmaceutical Sciences.
29. Bergman; Y., Stewart, S.J., Levy, S., and Levy, R.
(1983) J. Immunol. v. 131, pp. 1876-1881.
30. Feinberg, A.P. and Vogelstein, B. (1983), Anal, Biochem. v. 192, pp. 6-13.
31. 1iang, P. and Pardee, A.B. 1992) Science v. 257, pp.
967:-970.~
32. Sanger, F., Nicklen, S., and Coulson, A.R. (1977) Proc. =
Natl. Acad. Sci USA v. 74, pp. 5463-5467.
33. Barany and Merrifield, In the Peptides~ Analysis, Synthesis,=Biolo.7y, ed. by Gross and Meienhofer (1979), v. 2, pp. 1-~34, Acadëmic Press, New York.
34. Arnett F.C., Edworth, S.M., Bloch, D.A., (1988), Arthritis ~heum. v. 31, pp. 315-324_ 35~ Steinberg, J., Tsukamoto, S., Sledge, C.B. (I979) Arth.
Rheum.
35 36. Janusz, M.J., Hare, M. (1993) J. Immunoi., v. 150(5), pp. 1922-1931. _ __ 37. Sauter, H. and Paige, C.J. (i98~c), Proc. Natl. Acad.
Sci USA, v. 84, pp. 4989-4993.

~ 21 90679 38. Berger et al. J. Exp. Med. (1994), v. 180, pp. 471-476.
39. Johnson A.R., Erdcs~ E.G., (1973), Proc. Soc. Exp.
Biol. Med., v. 142, pp. 1252-1256.
40. Kluxen et al. Proc. Natl. Acad. Sci USA, (1992), v.
89, pp. 4618-4622.
41. Rose et aL_ (1995), ~. Biol. Chem. v. 270, pp. 22661-22664. ~~ ~ :
42. Takebe et al. Mol. Cell Biol. (1988~, v. 8, pp. 466-472.
43. Kee, B., Cumano, A., Iscove, N., and Paige, C.J., -(1994), Internat. Immunog, v. 6, pp. 4~f-407.
44. Ray, R.J., Eurlonger, C., Williams, D.E., Paige, C.J., (1996), Eur; J. Immunol. , v, 26, pp. 1~-16.
45. Marshall, A.J., Wu, G.E., Paige, C.J., (1996), J.
Immunol., v. 156, pp. 2077-284.

. .

~ 21 90679 ~ 31 -TABLE 1 ~

1 ggagaggctg tcccatgaag cagtgcaaag gtggggtgag gcataggaag gaagcagaga 61 gtctaggaca catctcagcc ac4tacaagt ggccccgtgg ggaccggagc ctgctggagg 121 aggtggcatc aggctgagtg gggctccctg gacagagaag cacctgctca ccatqttgcc 181 tctccttgcc ctgcttct-c tgatcgggcc atcagtgtgc,actacagcag gagacaqaga 241 ggaactggct tt~ggtgcag aggcagagtc ctgggtgacc gtg~acctga agggaatccc 301 cgtccccagc attgaactta agcttcagga gttgaagaga agcaggactc gccagttcta 361 tggtctgatg gggaagcggg tgggagggta tcagctggga cgt.atagtgc aggatctcct 421 tggcacgaga ggtttgtcca tagaagggac ctgcagacag gcggcgagtc aacagagggc 481 acgacccgga gcagtgacca gagaaagcct ccagagtcga gaggaagatg aggctcccct 541 aaccaccagc aacgtgtagc actctgccac ccatctctcc ccagaacatc acagctgagg 601 agcctcagcc aa~aqtcctg tcttttgtcc tcaacttggg tatttcctgc caggccctcg 661 tcacactctg cat~tttctcc caqgactcac tcttggcatc tggtagcaac gacacacaaa 721 gcacggctgc ctcctiacaa gctggactga cccagctctc ccttgtccct acagcaaagc 781 ttcacactct gtatcccagg cctagcacac agtagggctc aatcaacgac gagttagca~
841 taataggtag cttcctcagc catgcagggt agagggtggg atgtcagaaa agc:agagacc 901 aacattacac agcccaggtc taatgtctaa tccttgggtg ggaagagagc ttggggcctg 961 gctaaaacgg caatagaaac aagaaaaaga ccctggtgga~gaagacgctg catgtattgt 1021 atttgggggc ggggtcagga gccttcttcc ctttagattc ctgatgttgc taccaacaga 1081 catctccctc tgtgctgagg ctatggctca gtgggctaag gcgattgtgg ccaagcctga 1141 caacttgagt tcaatcccca ggacccacaa agtggaagaa aaaaattgtc cttttacctc 1201 tgcatgtgtc atggcatat.g ttacacaaaa taaaaagaca gtttggaaa ~ 21 ~067~

ULPLLALLLL :IGPS~CTTAG DREELAFGAE AESWVTVNLK GIPVPSIELK

I,QF.T,~K~ ~h~G _ V GGYQLGRIVQ DLLGTRGLSI: EGTCRQAASQ
60 :~0 80 90 100 QRARPGAVTR F..qT.Q.5RFF.nF APLTTSNV
11~ 120 128 Substance Z peptide is shown in ~old.

~ 21 9067~

~ABLL 3 j An tachykininS
NAME
Substance PR P K P Q Q F F G L M-NH~
Neurokinin AH K T D S F V G L M-NH2 Neurokinin 8D M H D F F V G L M-NH2 Substance ZR S R T R Q F Y G L M-NHz Non~1iAn tachykinins NAME
Physalaemin E A D P N K F Y G L M-NH2 Eledoisin E P s K A F I G L M-NH2 Kassinin D V P K s D G F V G L M-NH2

Claims

Claims Not Yet Available