CA2204995A1 - Antibiotic cryptdin peptides and methods of their use - Google Patents

Abstract

The present invention provides substantially purified cryptdin peptides and nucleic acid molecules encoding cryptdin peptides. The invention further provides methods for detecting inflammatory pathologies in a subject and methods for treating an inflammatory pathology in a subject by administering a pharmaceutical composition containing a cryptdin peptide. Representative cryptdin peptides are presented in the figure.

Description

CA 0220499~ 1997-0~-09 P~ 2 8 ~V V ~ U~
ANTIBIOTIC ~ ~IN PEPTIDES AND MET~ODS OF THEIR USE
This application is a continuation-in-part of United States Serial No. 07/930,649, filed August 14, 1992, which is a continuation-in-part of U.S. Serial No.
07/889,020, filed May 26, 1992, each of which is incorporated herein by reference.
This invention was made with government support under grant numbers AI22931, AI31696, DR08851, DK44632 and DK33506, awarded by National Institutes of Health.
The Government has certain rights in the invention.

B~R~ROUND OF TU~ INV~NTION
FT~Tn OF T~ lNv~lION
This invention relates generally to antimicrobial peptides and more specifically to cryptdin peptides, nucleic acid molecules encoding cryptdins, and their uses.

BACRGROUND INFORMATION
Survival in a world teaming with microorganisms depends on a network of host defense mechanisms. Among these mechanisms are phagocytosis by cells are resident in tissues or that circulate in the blood system and ingest, kill and digest potentially harmful microbes.
Although pathogenic microbes may vary considerably, phagocytes are able to destroy the vast majority by sequestering them in intracytoplasmic vacuoles and exposing them to a lethal mixture of organic and inorganic toxins.
Perhaps the most remarkable ultrastructural feature of phagocytes are their several thousand cytoplasmic granules, which are membrane-bound organelles typically about 0.3 ~m in diameter. During phagocytosis, some of these granules fuse to phagocytic vesicles thus enabling the contents of the granule to enter the lumen of the vesicle. Early observers surmised correctly that the granules contained factors which were responsible for J 'J~

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intraphagosomal killing in digestion of microbes. These granules contain a mixture of antimicrobial molecules including various peptides such as the so-called defensins.
Defensins are abundant antimicrobial peptide components of vertebrate neutrophil and macrophage granules. Members of the defensin family have been identified previously in human, rabbit, guinea pig and rat phagocytes, primarily those phagocytes termed phagocytic granulocytes. Defensins are cationic peptides that have molecular weights between about 3 and 4 kiloDaltons (kDa) and that exhibit broad-range antimicrobial activities against gram negative and gram positive bacteria, many fungi and some enveloped viruses.
The peptides are characterized by eight invariant amino acids, including six invariant cysteine residues that constitute a unique disulfide motif. The three disulfide bonds stabilize a tertiary conformation consisting predominantly of ~-sheet. The highly ordered structure and the absence of a helix make defensins unique among known antimicrobial peptides. It appears that defensins exert their antibacterial effect by permeabilizing the cytoplasmic membrane of the target microorganism by a mechAnism that may involve the formation of ion channels or transmembrane pores.
Until recently, defensins had been identified only in circulating or tissue phagocytes of myeloid origin. However, based on the presence of a particular mRNA, it has been surmised that similar peptides might be present in the epithelial cells of the small intestine.
Such intestinal peptides may prevent access of microorganisms through the small intestine into the systemic circulation and, therefore, can be useful as a therapeutic or prophylactic agent. Thus, a need exists to identify peptides that have antimicrobial activity within the mucosal epithelium or in the intestinal lumen.

CA 0 2 2 0 4 9 9 ~ 19 9 7 - 0 ~ - 0 9 ~ ) i j 2 8 ~ J ~ J~6 The present invention satisfies this need and provides additional benefits as well.

SUM~DY OF TU~ INV~NTION
The present invention provides a substantially purified cryptdin peptide having a consensus amino acid sequence:

Xl-c-x2-c-R-x3-c-x4-E-xs-c-x6-c-c-x7 wherein X1 is 3 to 9 amino acids; X2 is 1 amino acid, preferably Y, H or R; X3 is 2 or 3 amino acids; X4 is 3 amino acids; Xs is 5 amino acids; X6 is 6 to 10 amino acids; and X~ is O to 9 amino acids.
The invention also provides a substantially purified mouse cryptdin having a consensus Pmino acid sequence:

Xl-L-X2-C-Y-C-R-X3-C-K-X4-E-Xs-G-T-C-X6-C-C-X, wherein X1 is 3 or 4 amino acids, preferably LRD, LSKK
(SEQ ID NO: 1) or LRG;
X2 is 1 amino acid, preferably V, L or I;
X3 is 3 amino acids, preferably KGH or *RG, where * is S, T, K, I or A;
X4 is 2 amino acids, preferably GR, RR or RG;
Xs is 3 amino acids, preferably RMN, RVR, RVF
HMN or HIN;
X6 is 6 to 9 amino acids, preferably GIRFLY
(SEQ ID NO: 2) or RNLFLTFVF (SEQ ID NO: 3), RRGHLMYTL
(SEQ ID NO: 4) or RKGHL*YT* (SEQ ID NO: 5), where *
independently is L or M; and X, is 0 to 3 amino acids, preferably R, S or PRR.
For example, the invention provides various mouse, rat or human cryptdins having the sequence:

CA 0220499~ 1997-0~-09 - '2 8 t~96 1) LRDLVCYCRSRGCKGRERMN61C~KGHLLYTLCCR (SEQ ID N0: 6);

2) LRDLVCYCRTRGCKRRERMNGTCRKGHLMYTLCCR (SEQ ID N0: 7);

3) LRDLVCYCRKRGCKRRERMNGTCRKGHLMYTLCCR (SEQ ID N0: 8);

4) GLLCYCRKGHCKRGERVRGTCGIRFLYCCPR (SEQ ID N0: 9);

5) LSKKLICYCRIRGCKR~v~GlC~N~FLTFVFCC (SEQ ID N0: 10);

6) LKQCHCRKFCRPYEKAEGSCRPGLFIKRKICCIQQWTPG (SEQ ID
N0: 11);

7) GLLCYCRKGHCKRGERVRGTCGIRFLYCCPRR (SEQ ID N0: 12);

8) LSKKLICYCRIR~KK~v~lCKN~FLTFVFCCS (SEQ ID N0: 13);

9) LRDLVCYCRARGCKGRERMNGTCRKGHLLYMLCCR (SEQ ID N0: 14);

10) LKQCHCRKFCRPYEKAEGSCRPGLFIKRKICCIQQWTPGRT (SEQ ID
N0: 15);

11) IGRPVRRCRCRANCGPKEYATAFCAQGPFKQFKFCCT (SEQ ID
N0: 16);

12) l~w~w~nCHCRSFCRPYENATSFCAQGLFKQHKFCCLDTWPPRMK (SEQ
ID N0: 17);

13) TSGSQARATCYCRTGRCATRESLSGVCEISGRLYRLCCR (SEQ ID
N0: 18); and 14) AFTCHCRRSCYSl~YSY~Lcl~MGINHRFCCL (SEQ ID N0: 19).
Cryptdins are typ;c~lly characterized by being naturally found in the epithelial cells of the small intestine, being cationic, being about 30 to about 45 amino acid~ in length, having at least three and, preferably, three to nine, amino acids to the N-terminal of the first cysteine residue, exhibiting specific antimicrobial activity against intestinal pathogens and opportunistic pathogens and being relatively non-toxic to cells of the host organi~m. However, there may be diversity in the~e structural and functional characteristics. The invention also provides cryptdin analogs, which are devoid of one or more amino acids N-terminal to the fir~t cysteine. In addition, the invention also provides nucleic acid molecules encoding cryptdin peptide~. For example, the invention provide~
genomic DNA sequences and cDNA sequences encoding mouse and rat cryptdins.

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CA 0220499~ 1997-0~-09 '2 a --~y~ JT 19g6 The invention further provides a method for detecting an inflammatory pathology in a subject by determining the amount of cryptdin in a biological sample from the subject and comparing that amount to the amount present in a normal subject. Such a method can be used to determine the presence of an inflammatory pathology such as inflammatory bowel disease, pancreatitis, malignancy, infection or ileitis.
The invention also provides a method for treating an inflammatory pathology in a subject by a~ministering a cryptdin to the subject. Such treatment is particularly advantageous in patients who are immunocompromised due, for example, to malnutrition, radiation burns, immunosuppressive infections, autoimmune disease, neonatality, bone marrow transplantation or chemotherapy. A cryptdin can be administered orally, by nasogastric intubation, by transabdominal catheter, intravenously or by aerosol inhalation. When administered orally, it is preferably in a delayed release formulation designed to permit release in the small intestine. The cryptdin can be administered as a composition with a physiologically acceptable medium, and more than one cryptdin can be a~ministered simultaneously or sequentially.
P~T~F D~-~CRIPTION OF ~ DRAWINGS
Figure 1 provides the structures of mouse cryptdins 1-5 (SEQ ID NOS: 6 to 10, respectively) and rat cryptdin 1 (SEQ ID NO: 11). Amino acid residues are indicated by single letter code. Dashed lines are included in mouse cryptdin 4 (SEQ ID NO: 9) and rat cryptdin 1 (SEQ ID NO: 11) in order to preserve the consensus sequence where these peptides are shorter than other cryptdins. Invariant residues in the enteric cryptdin peptides are boxed. Disulfide bonding motifs are depicted by connecting double lines.

CA 0220499~ 1997-0~-09 n~ t9~

Figures 2.A. to 2.C. show chromatograms representing the purification of enteric cryptdins. Acid extract of jejunum and ileum was chromatographed in 30%
acetic acid on a P-60 column. Fractions indicated by the bracket (Figure 2.A.) were pooled and rechromatographed on the P-60 column (Figure 2.B.). Cryptdin cont~ining fractions (bracket, panel B) were pooled and further purified by reversed-phase high performance liquid chromatography (RP-HPLC) on 0.46 x 25 cm Vydac C-18 column. Water-acetonitrile gradient elution (--) using 0.13% (vol/vol) HFBA as modifier was used to purify cryptdins 1-5 (SEQ ID NOS: 6 to 10, respectively). The brackets in Figure 2.C. in~icAte the peptide contAine~ in each peak, and the portion of each which was subjected to a second round of RP-HPLC.
Figure 3 shows acid-urea PAGE of purified enteric cryptdins. Samples of low molecular weight enteric peptides obtAine~ by P-60 gel filtration (Figure 2, panel B) and purified cryptdins were electrophoresed on a 12.5% acid-urea gel and stained with formalin-contAi n ing Coomassie Blue. Lane A: approximately 20 ~g P-60 low molecular weight peptide fractions; lanes B-F:
1 ~g each of cryptdins 1-5 (SEQ ID NOS: 6 to 10), respectively.
Figures 4.A. and 4.B. compare mouse cryptdins 1-5 (SEQ ID NOS: 6 to 10, respectively) and partially purified luminal peptides.
Figure 4.A. Lyophilized luminal lavage of small intestine from 12 mice and 20 ~g protein was fractionated by P-60 gel filtration and electrophoresed on an acid-urea acrylamide gel (lane 2) along ~ide a similarly prepared sample of bowel tissue (lane 1). The positions of cryptdins 1-5 (SEQ ID NOS: 6 to 10, respectively) are indicated.
Figure 4.B. Partially purified luminal peptides (20 ~g; as for Figure 4.A., lane 2) were electrophoresed in a second acid-urea gel (lane 3) along & , ~, CA 0220499~ 1997-0~-09 r~

with an identical sample previously treated with performic acid (lane 4). In lane 4, rapidly migrating, cyst(e)ine-cont~ining peptides are absent due to the increased net negative charge resulting from the conversion of cyst(e)ines to cyqteic acid re~idues.
Figure 5 shows the identification of mouse cryptdins 1-5 (SEQ ID NOS: 6 to 10, respectively) in small intestine epithelium. Acid extracts of intact, whole small intestine (W) or epithelial sheets (E) were lyophilized, dis~olved in sample solution and resolved on a 12.5% acid-urea acrylamide gel. Cryptdins 1-5 (SEQ ID
NOS: 6 to 10) are identified numerically.
Figures 6.A. to 6.F. show the immunohistochemical localization of cryptdin 1 (SEQ ID
NO: 6) in small intestine. Full thickne~s sections of adult mouse jejunem were incubated with preimmune (Figures 6.A., 6.C. and 6.E.) or anti-cryptdin C rabbit IgG (Figures 6.B., 6.D. and 6.F.) and developed using peroxidase anti-peroxidase secondary antibody magnifications: Figures 6.A. and 6.B., 40X;
Figures 6.C. and 6.D., 250X; Figures 6.E. and 6.F., 640X.
Figures 7.A. and 7.B. depict the antimicrobial activity of mouse cryptdin 1 (SEQ ID NO: 6). Samples of purified natural mouse cryptdin 1 (Figure 7.A.) or rabbit NP-l (Figure 7.B.) were dissolved in 0.01% acetic acid and pipetted into wells produced in a 0.6% agarose/0.3%
tryptone plate cont~ining 1 X 106 log phase bacterial cells. After incubation at 37~C for 18 hr, ant;microbial activity was evaluated by measuring the diameters of the clear zones. Closed circles denote wild type S. typhimurium; open circles denote the phoP~ mutant.
Figure 8 shows the amino acid sequences for rat cryptdins 1-3 (SEQ ID NOS: 15-17, respectively), human cryptdins 5 and 6 (SEQ ID NOS: 18 and 19; HD-5 and HD-6) and a consensus sequence (Def consensus). Also shown are the amino acids sequences for rat prepro-cryptdins 1-3 CA 0220499~ 1997-0~-09 PCT/US'.7 ~ 3 2. ~

(SEQ ID NOS: 20-22) as deduced from cDNA or genomic DNA
sequences as indicated.
Figures 9.A. and 9.B. show the amino acid sequences of mouse cryptdins 1-17 (SEQ ID NOS: 23 to 39, respectively) as determined from the cDNA sequences encoding the specific cryptdin.
Figure 9.A. shows the entire amino acid sequence of the mouse cryptdins. The amino acid sequences of cryptdins 1-6 (SEQ ID NOS: 23 to 28) were determined by sequencing the purified peptides. The amino acid sequences of cryptdins 7-17(SEQ ID NOS: 29 to 39) were deduced from the cDNA sequences (see Figure 10).
The amino acids encoded by Exon 1, which encodes the signal peptide and propiece, and Exon 2, which encodes the mature cryptdin peptide, are indicated. A dot ;n~icAtes the sequence wa~ not encoded by the cDNA clone.
"*" indicates a space, which preserves the homology of the sequences.
Figure 9.B. in~icAtes the degree of relate~ness of the mouse cryptdins. Amino acids that are identical to the amino acid shown for cryptdin 1 (SEQ ID NO: 23) are indicated by a dot.
Figure 10 shows the nucleic acid sequences for the cDNA seqllen~es e~co~ing mouse cryptdins 1-17 (SEQ ID
NOS: 40 to 56, respectively). A consensus nucleotide sequence also is shown (SEQ ID NO: 57). A dot indicates the nucleotide is the same as shown for cryptdin 1. The amino acid sequence for cryptdin 1 (SEQ ID NO: 23) is shown above the nucleic acid sequence. Numbers below the nucleotide sequence indicate the nucleotide position relative to the methionine start codon (+1). Numbers above the amino acid sequence indicate the amino acid position. Italics indicate the mature cryptdin peptide ~equence. Nucleotides in lower case letters indicate non-coding sequences. "***" indicates a stop codon.
"(A)~" indicates poly-A tail. "*" indicates a space and ~ ET

CA 0220499~ 1997-0~-09 ., ' ' ~ ~
g ~r~ S 0 9 OCT 1996 "-" indicates the particular nucleotide could not be determined unambiguously.
Figure 11 shows the genomic DNA sequences for mouse cryptdins 1, 2, 3, 5 and 6 (SEQ ID NOS: 58 to 62, respectively) and the genomic sequence for the apparently inactivated mouse cryptdin i gene (Crypi; SEQ ID NO: 63), in which a stop codon (~Ç~) is substituted for a cysteine residue. Numbering is as described in the legend to Figure 11. The upper sequence represents a consensus cryptdin gene sequence (SEQ ID NO: 64). "X" indicates positions at which at least two sequences contAi ni ng nucleotide changes. The TATAAA box is shown in lowercase italics; exons are shown in capital letters; "**"
indicates intron DNA; "n" represents approximately 500 base pairs that were not sequenced. "~" indicates the start of the cryptdin peptide coding region at codon 59.
Coding sequences are indicated in bold print. Prepro-regions are coded by nucleotides 1-172; cryptdin peptides are coded by nucleotidase 173-279. The stop codon is underlined.
Gen~nk accession numbers for these sequences are 002994 (cryptdin 1, exon 1); 002995 (cryptdin 1, exon 2); 002996 (cryptdin 2, exon 1); 002997 (cryptdin 2, exon 2); 002998 (cryptdin 3, exon 1); 002999 (cryptdin 3, exon 2); 003000 (cryptdin 5, exon 1); 003001 (cryptdin 5, exon 2); 003002 (cryptdin 6, exon 1); 003003 (cryptdin 6, exon 2); 003004 (cryptdin i, exon 1); and 003005 (cryptdin i, exon 2).
Figures 12.A. to 12.C. demonstrate the effectiveness of mouse cryptdins (a~ indicated) in inhibiting the growth of E. coli ML35 cells in an agar diffusion assay.
Figures 13.A. to 13.C. demonstrate the effectiveness of mouse cryptdins (as indicated) in killing E. coli cells in suspension.
Figures 14.A. to 14.C. show the cDNA sequences encoding rat cryptdin 1 (Figure 14.A.), rat cryptdin 2 CA 0220499~ 1997-0~-09 ,~ r ~ r ~ .J 1~ 7~

(Figure 14.B.) and rat cryptdin 3 (Figure 14.C.).
Nucleotide numbers are indicated.
Figures lS.A. to 15.C. show the genomic DNA
sequences encoding rat cryptdin 1 (Figure 15.A.), rat cryptdin 2 (Figure 15.B.) and rat cryptdin 3 (Figure 15.C.). Nucleotide numbers are indicated.

D~TATT~n D~t~CRIPTION OF TR~ INV~NTION
The invention provides small peptide molecules, termed cryptdins, which express a broad range of antimicrobial activity, particularly against intestinal pathogen~, and for this reason are useful antimicrobial agents. For example, cryptdins have antimicrobial activity AgAin~t gram negative and gram positive bacteria and againqt protozoan pathogens (see Example III).
Cryptdin peptides and nucleic acid sequence~ encoding cryptdins were isolated from the small intestine and are active within the epithelial lining of the small intestine and within the lumen of the inte~tine. Because it is in~icAtive of inflammatory processes, the presence of cryptdins can be utilized in the diagnosi~ of a wide range of infla~matory conditions.
As used herein, the term ~'cryptdin" or "enteric defensins' refers to peptides having generally between about 30 and 45 amino acids. Cryptdins are characterized, in part, by a consensus sequence contAining six cysteine residue~. Illustrative sequences are provided in Figure 1, which shows invariant residues and the disulfide bonding motif. In addition, those re~idues which are preferably invariant are identified (see, also, Figures 8 and 9).
Cryptdins are further characterized by their cationic charge and their broad range of antimicrobial activity. While related to leukocyte-derived defensins, cryptdins are distinguished from these other molecules by the presence of 3 to 9 amino acids N-terminal to the first cysteine molecule. Cryptdins may have C-terminal CA 0220499~ 1997-0~-09 J ~ 3 2 8 .

extensions as well. In addition, they exhibit antimicrobial activity against enteric microorganisms, which can become blood-borne pathogens if the intestinal barrier is breached. Since cryptdins are associated with the secretory granules of Paneth cells in the small intestine, they can be secreted from the cells in which they are produced (Satoh, Cell T;ss. Res. 251:87-93 (1988); Satoh et al., AC~A Ui~tochem 83:185-188 (1988)).
Unlike leukocyte-derived defensins, cryptdins are not toxic to mammalian cells.
It should be appreciated that various modifications can be made to the cryptdin amino acid sequence without diminishing the antimicrobial activity of the peptide. It i9 intended that peptides exhibiting such modifications, including amino acid additions, deletions or substitutions are within the meaning of the term "cryptdin" and, therefore, within the scope of the invention. For example, cryptdin analogs, which are devoid of one or more ~;no acids N-terminal to the first cysteine residue, are within the present invention. Such cryptdin analogs can be synthesized using well known methods (see Example VI) or can be purified from the intestine where they may occur naturally due, for example, to partial proteolysis of a cryptdin peptide in the intestinal lumen.
Use of the phrase "substantially pure" in the present specification and claims as a modifier of peptide, protein or nucleic acid means that the peptide, protein or nucleic acid BO designated has been separated from its in vivo cellular environment. As a result of the separation and purification, the substantially pure peptides, proteins and nucleic acids are useful in ways that the non-separated impure peptides, proteins and nucleic acids are not.
The cryptdin peptides of the present invention preferably contain between about 30 and 45 amino acids (see Figures 1, 8 and 9). Cryptdins can be synthesized ~*r ~' CA 0220499~ 1997-0~-09 V_"

12 IF~J O ~ ~CT 1 by methods well known in the art, such as through the use of automatic peptide synthesizers or by well-known manual methods of peptide synthesis (see Example VI). In addition, they can be purified from natural sources such as small intestinal epithelium of vertebrate, preferably mammalian, origin (see Example I). Such epithelium can be obtained, for example, from rats, mice or humans using means well known to those skilled in the art.
As disclosed herein, various cryptdin peptides were isolated from intestinal epithelium, purified by chromatographic methods and characterized by electrophoresis and amino acid sequencing. Cryptdins were identified by their rapid migration on acid-urea PAGE and by their apparent molecular weight of about 4 kDa (see Examples I and II).
Anti-cryptdin antiho~;es were made using methods conventional in the art. For example, polyclonal antiserum can raised in appropriate animals, such as rabbits, mice or rats. Cryptdin peptides, either synthetic or obtained from natural sources, can be used to immunize the animal. As described in Example IV, a cryptdin analog, cryptdin C, which corresponds to residues 4-35 of mouse cryptdin 1 (SEQ ID N0: 6) as shown in Figure 1, was used to immunize rabbits using well known methods. Serum samples were collected until the anti-cryptdin titer was appropriate. Various fractions of the antiserum, such as IgG, can be isolated by means well known in the art. Cryptdin immunogens also can be used to obtain monoclonal antibodies using methods well known in the art (see, for example, Harlow and Lane, Antihod;es: A TIAhoratory Man~ (Cold Spring Harbor Laboratory Press 1988), which is incorporated herein by reference).
The antimicrobial activity of a cryptdin can be measured against various pathogens. As disclosed in Example III, various microorganisms were grown to an appropriate concentration, mixed with an appropriate . 0 ~

CA 0220499~ 1997-0~-09 . B

medium such as an agarose-trypticase soy medium and contacted with a cryptdin. Antimicrobial activity was apparent, for example, from the clear zones that surrounded the cryptdins in an agar diffusion assay. The area of the clear zone~ was concentration dependent (see Figure 12).
Anti-cryptdin antibodies can be used to determine the presence of cryptdin in a biological sample such as a histological sample. For example, sections of small intestine are fixed by means well known to those skilled in the art and incubated with anti-cryptdin antibodies such as an IgG fraction of antiserum. If desired, the anti-cryptdin antibody can be detectably labelled or an appropriate detectable second antibody can be used to identify the presence of the primary antibody attached to the cryptdin. Means of detection include the use of radioactive protein A or enzyme substrates such as peroxidase (see Harlow and Lane, supra, 1988).
Alternative methods of determining the presence of cryptdin in a biological sample obtAine~, for example, by intestinal lavage or by disrupting cells or tissues can be useful to determine the presence of inflammatory processes. In the presence of inflammatory processes, the concentration of cryptdins is ~ignificantly altered from that found in the normal cell. In particular, a deviation from the normal level of cryptdins by one to two st~n~rd deviations is indicative of an inflammatory process. Such an inflammatory process can include, for example, inflammatory bowel disease, pancreatitis, malignancy, infection or ileitis.
Because of their broad range of antimicrobial activity and their ability to function within the intestinal epithelium or lumen, cryptdins are potent therapeutic agents for infections of the intestine. In particular, cryptdins are useful where the subject is immunocompromised due, for example, to malignancy, malnutrition, chemotherapy, radiation, immunosuppressive &~' '' ~

CA 0220499~ 1997-0~-09 IS ~ ~ 1 1 3 3 2 8 5 ~ ~ . L ~ ~

viruses, autoimmune disease or neonatality. In addition, cryptdins are uqeful in surgical prophylaxis, for example, by functioning to help sterilize the small bowel. Thus, cryptdins can be useful as medicaments for S treating a subject having a pathology characterized, in part, by an inflammatory process.
A cryptdin, either purified from natural sources or synthetic, can be a~inistered to a subject in need of such therapy by various means, including orally, preferably in a slow-release type formulation, which will avoid release within the stomach. Alternatively, cryptdins can be administered through nasogastric intubation, transabdominal catheter, intravenously or aerosol administration. Individual species of cryptdin can be administered alone or in combination. Cryptdins administered in combination can be administered simultaneously or sequentially and can be repeated as necessary.
Prior to the characterization of a mouse intestinal defensin cDNA, expression of defensins was thought to be limited to profe~sional phagocytes, i.e., neutrophils and macrophages. The presence of high levels of cryptdin mRNA in Paneth cells led to the hypothesis that defensins synthesized in intestinal epithelium may contribute to antimicrobial barrier function in the small bowel (Ouellette et al., J. Cell R;ol. 108:1687-1695 (1989a), which is incorporated herein by reference).
Isolation and characterization of six mouse cryptdin peptides, two rat cryptdin peptides and 2 human cryptdin peptides, and the demonstration of antimicrobial activity of various cryptdin peptides i n~ i CAtes that the cryptdins have an antimicrobial role in the small intestine. The immunohistochemical localization of cryptdin(s) to Paneth cells is consistent with previous in situ hybridization analysis and suggests that defensins produced by these cells may contribute to restricting the colonization and invasion of the small bowel by bacteria.

~ s CA 0220499~ 1997-0~-09 PCT/US '~ i~ J'' ~ -~ 'J 2 8 Initial efforts to purify intestinal defensins focused on the isolation of mouse cryptdin 1 (SEQ ID NO:
6), the peptide predicted from the cryptdin cDNA
sequence. Since the deduced structure of the peptide is highly cationic, intestinal peptides were solubilized by homogenizing intact mouse jejunum and ileum in 30% formic acid. Acid-urea PAGE of the crude extract revealed several bands with Rf values similar to those of rabbit defensin NP-1 and cryptdin C, a folded synthetic defensin congener corresponding to residues 4 to 35 in cryptdin 1 (SEQ ID NO: 6). Peptides corresponding to these bands were purified approximately 200-fold by sequential gel filtration chromatography on Bio-Gel P-60 (Figures 2.A.
and 2.B.). Electrophoresis of P-60 column fractions on acid-urea gels showed that five fractions eluting between two prominent peaks (Figures 2.A. and 2.B., brackets) contA;ne~ putative cryptdin peptides (Figure 3, lane a).
Peptides in these P-60 fractions migrated with an apparent molecular mas~ of approximately 4 kDa on SDS-PAGE (not shown), consistent with the molecular weight of defensins. Furthermore, treatment of P-60 fraction samples with performic acid reduced the electrophoretic mobility of the five putative mou~e cryptdins in acid-urea gels, behavior that i~ characteristic of defensins and polypeptides that contain multiple cysteine residues.
Defensins in pooled P-60 fractions were purified further using sequential rounds of RP-HPLC
utilizing different ion-pair agents. Initial HPLC
fractionation utilized water-acetonitrile gradients containing 0.13% heptafluorobutyric acid (HFBA) as the ion-pairing agent, whereby each of the five peptides contained in the pooled P-60 fractions was resolved to near purity in a single run (Figure 2.C.). Complete purification of five peptides, mouse cryptdins 1-5 (SEQ
ID NOS: 6 to 10, respectively), was achieved by subsequent RP-HPLC using 0.1% trifluoroacetic acid (TFA) (Figure 3, lanes B-F). Assuming extraction of individual . . .

CA 0 2 2 0 4 9 9 ~ 1 9 9 7 - 0 ~ - 0 9 , r ~
t~ ~ ;?~ i peptides is equally efficient, both acid-urea gel electrophoresis and RP-HPLC of the P-60 fractions containing putative cryptdins showed that the relative abundance of the peptide~ is cryptdin 1 > cryptdin 2 >
5 cryptdin 5 > cryptdin 3 > cryptdin 4. The relative amounts of cryptdins 1-5 (SEQ ID NO: 6 to 10, respectively) have been qualitatively reproducible in every preparation of acid-extracted protein from mouse small intestine.
Using a modification of the method described above, mouse cryptdin 6, rat cryptdin 2, and human cryptdins 5 and 6 also were isolated (see Examples I and II; see, also, Figures 8 and 9). In addition, longer forms of mouse cryptdins 4 and 5 (compare SEQ ID NOS: 9 and 12; 10 and 13) and rat cryptdin 1 (compare SEQ ID
NOS: 11 and 15) were obt~;ne~l. This result suggests that the initial method of purifying cryptdin peptides resulted in partial degradation of the C-termini of some peptides. Significantly, both forms of the purified cryptdin peptides have antimicrobial activity.
Biochemical characterization of the isolated cryptdins demonstrated that these peptides are defensins.
Amino acid analysis of each peptide showed their compositions (cationic peptides of about 30 to 45 amino acid re~idues, including 6 half-cysteines) are compatible with defensin-like molecules. The complete sequences of mouse cryptdins 1-6 (SEQ ID NOS: 23 to 28), rat cryptdins and 2 (SEQ ID NOS: 11, 15 and 16) and human cryptdins 5 and 6 (SEQ ID NOS: 18 and 19) were determined by automated Edman degradation and, in some cases, by amino acid analysis of carboxyl terminal chymotryptic peptides (see Figures 1, 8 and 9). The primary structures of the cryptdins contain the distinctive structural features of human, rabbit, rat and guinea pig neutrophil defensins (Lehrer et al., Cell 64:229-230 (199la), which is incorporated herein by reference), i.e., the six invariant cysteine residues and the glycine and glutamic .~ r,,~
V ~

CA 0 2 2 0 4 9 9 ~ 19 9 7 - 0 ~ - 0 9 ~ r ~ ~ 2 'J~ 96 acid in positions that are highly conserved in myeloid defensins.
The cryptdin peptides disclosed herein contain features that are unique and distinct from defensins of myeloid origin. For example, mouse cryptdins 1, 2, 3 and 6 (SEQ ID NOS: 23 to 25 and 28, respectively) are almost identical, differing only at two or three positions (see Figure 9.A.). Analysis of codons from which these amino acid differences could arise shows that the conversion, for example, of Serl~ to Lysl~ in cryptdin 1 (SEQ ID NO:
23) and cryptdin 3 (SEQ ID NO: 25), respectively, requires two nucleotide substitutions. On the other hand, single nucleotide changes in the codon encoding Thrl~ in cryptdin 2 (SEQ ID NO: 24) could give rise to cryptdins 1, 3 and 6, suggesting that the cryptdin 2 gene may be an intermediate or progenitor of the cryptdin 1, 3 and 6 genes. Si~ilarly, a single nucleotide change in the codon for Thrl~ of cryptdin 2 can account for the deduced amino acid at position 10 in cryptdins 7-17 (see Figure 10, nucleotides 203-205; SEQ ID NOS: 46 to 56).
~y homology with the structures of known myeloid defensins, the cryptdin 1 N-terminus was predicted to begin at Leu4 or Val5, which is 1-2 residues prior to the first ro~erved cysteine. However, compared to myeloid defensins, cryptdins have variably extended N-termini that contain from three (mouse cryptdin 4, SEQ
ID NO: 26; rat cryptdin 1, SEQ ID NO: 11) to nine (human cryptdin 5, SEQ ID NO: 18) amino acids preceding the first cysteine. In mouse cryptdins 1-3 and 6-17 (SEQ ID
NOS: 23 to 25 and 28 to 39, respectively), the N-peptidyl extensions consist of two charged internal residues flanked by amino acids with hydrophobic sidechains.
Since natural variation in defensin amino termini correlates with relative antimicrobial potency in vitro (Ganz et al., J. Cl;n. Invest. 76:1427-1435 (1985), which is incorporated herein by reference), the extended s~' ,_ ~

CA 0220499~ 1997-0~-09 i~T/US ,;! ~ f ~

N-termini of enteric defensins may have evolved for a unique role in the bowel.
Mouse cryptdin 4 (SEQ ID NO: 9), the most cathodal and, apparently, least abundant mouse enteric defensin, was the first defensin found to contain a chain length variation between the fourth and fifth cysteine residue~. Unlike the majority of previously known defensins, in which nine amino acids separate the fourth and fifth cysteines (Lehrer et al., Supra~ l991a), mouse cryptdin 4 (SEQ ID NO: 9) contains only 8iX residues between the same two amino acids (Figure 1). In addition, rat cryptdins 1-3 (SEQ ID NOS: 11 and 15-17) contain ten amino acid residues between the fourth and fifth cysteines. These findings indicate the defensin fold involving this stretch of the peptide chain can accommodate significant variability in the size of the loop, as compared to the invariant loop size defined by crystal and NMR structures, respectively, of human and rabbit neutrophil defensins. Also, rat cryptdins 1-3 (SEQ ID NOS: 11 and 15-17) are the only cryptdins cont~ining three, instead of four, amino acid residues between the second and third cysteine residues.
Since cryptdin mRNA levels increase during postnatal development of mouse small bowel (Ouellette et al., supra, 1989a), it was investigated whether accumulation of enteric defensins was regulated similarly. Analysis of intestinal acid extracts from male and female mice showed that mouse cryptdins 1-3 and 5 ( SEQ ID NOS: 6 to 8 and 10, respectively) are present in adult mice, regardless of gender. On the other hand, extracts from 9 day-old mice lack the peptides, consistent with postnatal accumulation of cryptdin MRNA.
Mouse cryptdins 1-5 ( SEQ ID NOS: 6 to 10) were purified from intestinal epithelial cells. In the presence of EDTA, the intestinal epithelium no longer adheres to the underlying basement membrane and floats free of the lamina propria upon gentle agitation ~ 0499 99 -~ -09PCT/liS ' / 1Zi ' 2 8 , ~,, J ~J

(Bjerknes and Cheng, Am. J. ~n~t. 160:51-63 (1981), which is incorporated herein by reference). Preparations of epithelial sheets isolated in this manner were concentrated by low speed centrifugation and extracted with 30% formic acid. Peptides extracted from isolated epithelial sheets comigrate with cryptdins 1-5 (SEQ ID
NOS: 6 to 10) when analyzed by acid-urea PAGE (Figure 5), demonstrating their epithelial origin.
Immunoperoxidase stAining of full-thickness sections of small intestine with an anti-cryptdin antibody demonstrate cryptdin antigen in Paneth cells, consistent with localization of cryptdin mRNA by in situ hybridization (Ouellette et al., supra, ( 1989a)).
Incubation of sections of adult mou~e jejunum and ileum with a polyclonal anti-cryptdin IgG produced by rabbits immunized with the synthetic congener cryptdin C
localized the immunoperoxidase reaction to granulated cells, morphologically defined as Paneth cells, at the base of every crypt (Figure 6). The st~in;ng pattern accentuates the granular appearance of the cytoplasm in these cells and the immunoreactivity appears particularly strong over Paneth cell granules. The antibody is specific for mouse cryptdin(s), since it is negative both for rat and human Paneth cells (data not shown).
Leukocytes in the lamina propria of the villi also were negative, suggesting that related enteric defensins are not expressed by phagocytes or lymphocytes. Because of the extensive similarity of mouse cryptdins 1-3 (Figure 1; SEQ ID NOS: 6 to 8), the polyclonal antibody produced against cryptdin C probably recognizes the three peptides. Conversely, because mouse cryptdin 4 (SEQ ID
NO: 9) and cryptdin 5 (SEQ ID NO: 10) differ markedly from the other mouse cryptdins, the anti-cryptdin C
antibody is unlikely to react with cryptdin 4 (SEQ ID NO:
9) and cryptdin 5 (SEQ ID NO: 10), leaving their origin in Paneth cells somewhat unresolved.

CA 0 2 2 0 4 9 9 ~ 19 9 7 - 0 ~ - 0 9 ~ J ~ 2 Immunohistoch~micAl data suggest cryptdins are secreted into the intestinal lumen. Material in the small intestinal lumen is strongly positive for the antibody but negative for pre-immune sera or IgG (Figures 6.A. and 6.B.). Although the agonist for Paneth cell defen~in secretion is unknown, ly~ozyme, another protein constituent of Paneth cell granules, is secreted into the lumen under cholinergic regulation. Consistent with immunochemical detection of anti-cryptdin C positive material in the intestinal lumen, acid-urea PAGE of saline washes of adult jejunum and ileum contain peptides with mobilities very similar to but distinct from the mobility of cryptdins (Figure 4). Nevertheless, the peptides are not identical to cryptdins 1-5 (SEQ ID NOS:
6 to 10, respectively) by either migration in acid-urea PAGE or by HPLC analysis, suggesting they may correspond to cryptdins that have been processed further.
Conceivably, luminal cryptdin or cryptdin-like material could derive from exfoliated Paneth cells in the lumen, but the low rate of Paneth cell turnover suggests this is nl; kely. The release of cryptdin~ or processed variants into the small bowel by Paneth cells contrasts with the apparent lack of defensin secretion by leukocytes, and it is inferred that a secretory pathway may exist for the constitutive delivery of defensins into the intestinal lumen by Paneth cells.
The antibacterial activity of purified mouse cryptdins 1-5 (SEQ ID NOS: 6-10) was tested against wild type and phoP mutant S. typhimurium using a modified plate diffusion assay (Lehrer et al., J. Immunol. Methods 137:167-173 (199lb), which is incorporated herein by reference). phoP is a two-component regulatory locus that is essential to S. typhimurium virulence and survival within macrophages (Fields et al., Science 243:1059-1062 (1989); Miller et al., Proc. Natl. Acad.
Sci., USA 86:5054-5058 (1989), each of which is incorporated herein by reference). Mutants in the phoP

CA 0220499~ 1997-0~-09 ,~

locus are particularly sensitive to rabbit defensins NP-l and NP-2 when compared to wild type parent strains (Fields et al., supra, 1989; Miller et al., Infect.
Immun. 58:37 06-3710, (1990), which is incorporated herein by reference).
Under assay condition~ using a pho~phate buffer as described in Example III, the antimicrobial activity of rabbit defensin NP-l against wild type and the phoP
mutant organisms was quite similar (Figure 7.B.). On the other hand, at concentrations of mouse cryptdin 1 (SEQ ID
NO: 6) that are effective again~t the attenuated mutant, wild type S. typhimurium is completely resistant to the effects of the peptide (Figure 7.A.).
The differential activity of cryptdin 1 (SEQ ID
NO: 6) again~t avirulent S. typhimurium suggests that resi~tance to muco~al defensins may be important for the evolution of virulence in enteric pathogens. However, in experiment~ u~ing HEPES or PIPES a~ buffer~ as de~cribed in Example III, concentrations of 100 yg/ml or 300 yg/ml cryptdin 1 were as effective as NP-l in inhibiting the growth of wild type S. typhimurium. Furthermore, at these concentration~, cryptdins 4 and 5 were more effective than NP-l in preventing the growth of mutant and wild type S. typhimurium (not shown).
The present invention also provides substantially purified nucleic acid sequence~ encoding cryptdins. For example, the cDNA sequences for mouse cryptdins 1-17 (SEQ ID NOS: 40-56) are shown in Figure 10 and the cDNA sequences for rat cryptdins 1-3 ( SEQ ID NOS:
65-67) are shown in Figures 14 .A. to 14.C. In addition, the genomic DNA sequences for mouse cryptdins 1, 2, 3, 5 and 6 ( SEQ ID NOS: 58-62) and for an apparently inactivated cryptdin gene, cryptdin i (SEQ ID NO: 63) are shown in Figure 11 and the genomic DNA sequences for rat cryptdins 1-3 ( SEQ ID NOS: 68-70) are shown in Figures 15.A. to 15.C.

~ 'J ~ f CA o22o499~ 1997-0~-09 PCT/US ~ 3 2 8 : -...,~... .,: ai~ oC~1~396 The skilled artisan would recognize that various nucleotide substitutions could be made in the nucleic acid sequences shown in Figures 10, 11 14 and 15 without altering the amino acid sequence of the encoded cryptdin peptide due to degeneracy of the genetic code.
Such nucleotide sequences, which are equivalent to the sequences shown in Figures 10, 11, 14 and 15 are encompassed within the claimed invention.
The invention also provides nucleotide sequences that con~ist of a portion of a nucleic acid sequence as shown in Figures 10, 11, 14 and 15. Such a nucleotide sequence can be useful, for example, as a probe, which can hybridize under relatively stringent conditions to a nucleic acid molecule enco~ing a cryptdin peptide. For hybridization, such a nucleotide sequence should be at least about 10 nucleotide~ in length. One skilled in the art would know that appropriate condition~
for hybridization can be determined empirically or can be calculated based, for example, on the G:C content of the nucleotide sequence, the length of the sequence and the number of mismatches, if any, between the probe and the target sequence (see, for example, Sambrook et al., Molecl~l Ar cl oni ng A lAh~rAtory manual (Cold Spring Harbor Laboratory Press 1989), which is incorporated herein by reference).
A nucleotide sequence as described above can be detectably labelled by attaching, for example, a radioactive label or biotin, or can be unlabelled. A
labelled or unlabelled sequence also can be used as a primer for the polymerase chain reaction (PCR; see, for example, Erlich, PCR Technology: Principles ~n~
appl;~Ations for DNA Am~lifi~A~io~ (Stockton Press 1989), which is incorporated herein by reference). Such a sequence can be useful, for example, to identify a nucleic acid sequence encoding a cryptdin in a cell.
A nucleic acid molecule as shown in Figures 10, 11, 14 and 15 or a nucleotide sequence derived therefrom CA 0220499~ 1997-0~-09 ~ ~ ,~r also can be useful, for example, for preparing a cryptdin peptide or a portion of a cryptdin peptide using well known methods of recombinant DNA technology. For example, the nucleic acid sequence can be cloned into an expression vector such as a baculovirus vector or a viral vector, which can infect a mammalian cell and express an encoded cryptdin peptide in the cell. Expression from such a vector can be useful for producing large amounts of a cryptdin, which can be used to treat a subject having an inflammatory pathology as described herein, or for producing a cryptdin directly in a subject. Thus, the invention provides vectors ContA i n i ng a nucleic acid molecule as shown in Figures 10, 11, 14 and 15 as well as specific host cells, in which the vector can propagate or can express a cryptdin.
The following examples are intended to illustrate but not limit the invention.

EXAMPLE I
PurificAtio~ of ~ntrriC Defensins Outbred Swiss mice, (Crl:CD-l)(ICR)BR, 45 day old males (30~35 g) or timed-pregnant dams, were obtained from Charles River Breeding Laboratories, Inc. (North Wilmington MA). In studies of newborn mice, litters were 25 culled to 8 pups within 12 hr of delivery. Mice were housed under 12 hr cycles of light and darkness and had free access to food and water.
Cryptdins were isolated by a modification of the method described by Selsted et al., J. Cell. Biol.
118: 929-936 (1992); Ouellette et al., Infect. Im 62: 5040~5057 (1994) ~ each of which is incorporated herein by reference. Jejunal and ileal intestinal segments were excised from 60 mice immediately after carbon dioxide euthanasia. The tissue was washed and the lumen was 35 flushed with ice cold water prior to homogenization in 500 ml ice cold 30 % acetic acid. The homogenate was clarified by centrifugation, lyophilized, dissolved in CA 0220499~ 1997-0~-09 ' 200 ml 30% acetic acid, clarified by filtration through Whatman 541 filter paper and applied to a 10 x 60 cm Bio-Gel P-60 column equilibrated with 30% acetic acid. The elution rate was 100 ml/hr. Fractions contA;ning cryptdins were identified by electrophoresis in acid-urea polyacrylamide gels (Selsted and Harwig, Infect. Tmmun.
55:2281-2285 (1987), which is incorporated herein by reference).
Cryptdin-cont~ining fractions were pooled and lyophilized, then purification was completed by RP-HPLC.
Initial separation of mouse cryptdins 2-5 was achieved by HPLC on a 1 x 25 cm Vydac C-18 column using a gradient of water and acetonitrile contAining 0.13% HFBA. Solvents were delivered at 3 ml/min to generate the following acetonitrile gradient: 0-28% (10 min); 28-34% (20 min);
and 34-40% (60 min). Cryptdins 1 and 6, which coeluted under these conditions, were resolved by C-18 RP-HPLC
using 0.1% TFA as the ion pair and a 16-21% acetonitrile gradient delivered in 35 min at 3 ml/min. To eliminate traces of residual HFBA, preparations of cryptdins 2-5 were subjected to an addition RP-HPLC step using 0.1%
TFA. All peptides were lyophilized and quantitated by amino acid analysis prior to antimicrobial testing.
Essentially identical method~ were used to purify rat and human cryptdin peptides, except that rat cryptdins were isolated from the small intestine of adult Sprague-Dawley rats and human cryptdins were isolated from a surgically resected normal adult human male small intestine.

EXAMPLE II
Peptide ChArActerizAtion Amino acid analyses were performed on 6 N HCl hydrolysates (150 ~C, 2 hr) of unmodified or performic acid-oxidized peptides. Hydrolysates were derivatized with phenylisothiocyanate and the re~ulting phenylthiocarbamyl amino acids were quantitated as described previously (Selsted and Harwig, supra, 1987;

I f ~ J ~

CA 0 2 2 0 4 9 9 ~ l 9 9 7 - 0 ~ - 0 9, .~ . 7 ~ 2 ~ Q~

Selsted et al., supra, 1992; Ouellette et al., F~R~ T~tt.
304:146-148 (1992), which is incorporated herein by reference). Peptide samples were reduced with dithiothreitol ( DTT ) and pyridylethylated with 4-vinyl pyridine for sequencing (Henschen, In AdVAn~ed Meth~ i n Protein ~i~rosequence Analysi B (Wittmann-Liebold et al., pages 244-255 (1986), which is incorporated herein by reference). Sequence determinations were performed by automated Edman degradation on an ABI model 477 system (Applied Biosystems, Inc.; Foster City CA) with on-line PTH amino acid analysis. In some cases, the C-terminus of a cryptdin peptide was confirmed by ~m; no acid analysis of chymotryptic peptides. Cryptdins 4 and 5 also were analyzed by positive-ion fast atom bombardment mass spectrometry on a VG 7070E-HF instrument (Ouellette et al., supra, 1994).

EXA~PLE III
Antimicrobi~l Assays Antibacterial activity was measured in an agar radial diffusion assay (Lehrer et al., supra, 1991b) using wild type 5. typhimurium (ATCC 10428) or an isogenic phoP mutant of S. typhimurium ( strain CS015 phoP102::TnlOd-Cam, Miller et al., supra, 1989). Cells were grown to log phase in trypticase soy broth at 37 ~C, harvested by centrifugation and resuspended to 1 x 107 colony forming units (CFU) per ml in 10 mM sodium phosphate buffer (pH 7.4).
A 100 yl aliquot of each organism was mixed with 10 ml 1% agarose in 0.03% (w/v) trypticase soy medium, 10 mM sodium phosphate (pH 7.4) at 42 ~C.
Five yl samples of peptide solution were pipetted into 3 mm diameter wells formed in the agarose with a sterile punch. After 3 hr at 37 ~C, the inoculated agarose plate was overlayed with 1% agarose cont~in;ng 6% trypticase soy medium. After 12-16 hr, antimicrobial activity was apparent as clear zones surrounding wells loaded with CA 0220499~ 1997-0~-09 P~IJUS J' iJ J~ 2 8 antibacterial samples; the size~ of the clear zones were concentration-dependent.
Cryptdin antimicrobial activity in vitro was substantially enhanced in piperazine-N,N~-bis (2-ethane sulfonic acid) (PIPES) or in N-2-hydroxyethylpiperazine-N -2-ethanesulfonic acid (HEPES) as compared to the activity in ~odium phosphate. Purified cryptdin peptides were dissolved at 3 to 300 ~g/ml in 0.01% acetic acid and activity was examined against E. coli ML35 (ATCC). In the radial diffusion assay, 5 ~1 peptide solution was transferred into wells formed in plates of 1% agaro~e buffered with 10 mM PIPES (pH 7.4) and contAining 1 x 106 log-phase bacteria grown in trypticase soy broth. After 3 hr at 37 ~C, the plates were overlayed with 0.8%
agarose contAining 2X trypticase soy broth and incubated overnight. The antibacterial activities of cryptdin peptides was compared with the activity of rabbit neutrophil defensin NP-l, which was purified from peritoneal exudates as described by Selsted et al. (J.
Biol. Chem. 260:4579-4584 (1985), which is incorporated herein by reference). Antibacterial activity was determined by measuring the diameter of clearing around each well and plotted as a function of peptide concentration.
As shown in Figure 12, each cryptdin peptide produced a dose-dependent zone of clearing, which indicates that E. coli growth was inhibited. The potencies of the cryptdins varied, with cryptdins 1, 3 and 6 showing similar activity, which wa~ about 3-5x greater than the activity of cryptdin 2. Cryptdin 5 was approximately 5x more active than rabbit NP-1 at concentration above 100 yg/ml (Figure 12.C.) and cryptdin 4 was at least 50x more active than NP-l when compared at 100 ~g/ml and 300 ~g/ml (Figure 12.B.).
These higher concentrations of cryptdins 4 and 5 also were more effective than the same concentration of NP-l at inhibiting the growth of S. aureus and of wild type ~D ~FT

CA 0220499~ 1997-0~-09 ,t~ ~ ~
O ~ O~T ~3S~

and mutant strains of S. typhimurium ( not shown). These results demonstrate that various cryptdin peptides can inhibit bacterial growth.
In order to determine whether the effect of the cryptdin peptides against E. coli is bacteriostatic or bacteriocidal, bacterial killing was quantitated as a function of time. Bactericidal assays were performed by incubating 1-2 x 106 log-phase bacteria in 10 mM PIPES
cont~ining 10 ~g peptide/ml. After incubation for 15 or 30 min at 37 ~C, aliquots were removed, serially diluted and plated on tryptic~e 90y agar. Bactericidal activity was quantitated by counting colonies after overnight incubation at 37 ~C.
As shown in Figure 13, cryptdins 1 and 3-6 rapidly killed the E. coli Cella. In each of these case~, survival was re~n~eA to less than 1% after only 15 min incubation. Cryptdin 2 was the only peptide tested that was not bactericidal under the assay condition.
Cryptdin~ 2 and 3 differ only at Pm;no acid po~ition 10 (threonine and lysine, respectively).
The bactericidal activity of rat cryptdin 1 al~o was examined. E. coli ML35 cells, S. aureus 502A
cells or mutant or wild type S. typhimurium cells were incubated with various concentrations of rat cryptdin 1 or rabbit NP-l. Ten ~g/ml rat cryptdin 1 killed about 90% of the S. aureus cells and greater than 99% of the E. coli and mutant S. typhimurium cells, but was relatively ineffective in killing wild type S. typhimurium ( not shown). Rat cryptdin 1 was more effective than NP-l in killing the E. coli and mutant S. tyrhi~l~rium cells, whereas NP-l wa~ more effective in killing S. aureus.
The effect of mouse cryptdins 1-3 and 6 at inhibiting the growth of the protozoan, Giardia lamblia, which is the most common cause of protozoan disease in the human small intestine, also wa~ e~A~ined. Briefly, trophozoites of the C6 clone of Giardia lamblia WB (ATCC

CA 0220499~ 1997-0~-09 2B i~ i J~ ~Jul 1~36 30957) were grown to late log phase in TYI-S-33 medium contA; ni ng bovine bile. Free-swimming trophozoites were discarded and tubes with attached trophozoites were refilled with warm Dulbecco~ 8 PBS. Trophozoites were detached by chilling 10 min on ice, then harvested by centrifugation, resuspended at 2 x 107/ml in 25 mM HEPES
(pH 7.5) contA;n;ng 9% (isotonic) sucrose and incubated for 2 hr at 37 ~C with various concentrations of mouse cryptdins 1-3 or 6. Following incubation, trophozoite viability was determined by trypan blue exclusion.
The cryptdin peptides killed Giardia trophozoites in a dose-dependent manner (not shown). In particular, 20 ~g/ml of cryptdin 2 or cryptdin 3 reduced Giardia growth by greater than 2 orders of magnitude (not shown). These results ;n~ic~te that cryptdins are active against a variety of microorganisms.

E~AMPLE IV
Ant;-cryp~;n ~nt;ho~y A polyclonal rabbit antibody was prepared to a synthetic analogue of cryptdin 1. The peptide, termed cryptdin C, correspo~; n~ to residues 4-35 in cryptdin 1 (SEQ ID NO: 6; Figure 1) was synthesized by solid phase chemistry using Na-butoxycarbonyl protection (Kent, Ann.
Rev. B;ochem~ 57:957-989 (1988), which is incorporated herein by reference). Following cleavage/deprotection of synthetic cryptdin C with TFA-trifluoromethanesulfonic acid, the peptide was precipitated in ethyl ether and dried in vacuo. A 100 mg sample was dissolved in 10 ml 6.0 M guanidine-HCl, 0.2 M Tris-HCl, pH 8.2, cont~; n; ng 20 mg DTT. The sample was purged with nitrogen, heated to 50 ~C for 4 hr and diluted 100-fold with deionized water, then was dialyzed exhaustively, first against 0.1 M sodium phosphate (pH 8.2), 20 mM guanidine-HCl, 100 mM NaCl, then against 5% acetic acid. The sample was lyophilized, dissolved in 10 ml 5% acetic acid and subjected to RP-HPLC on a 1 x 25 cm Vydac C-18 column.

D ~E~

CA 0220499~ 1997-0~-09 29 . . ~ r The earliest eluting peak, representing about 0.5% of the crude peptide, was determined by A~i no acid analysis to have the desired composition.
A sample (1.5 mg) of cryptdin C was supplied, without conjugation to carrier, to Berkeley Antibody Company (Berkeley, CA) for immunization of 2 New Zealand White rabbits. Serum samples were collected for 12 weeks, until the anti-cryptdin C titer, determined by ELISA, reached about 1:10,000 for each rabbit. IgG was isolated from antiserum u~ing DEAE Econo-Pac chromatography (Bio-Rad; Richmond CA) as described by the manufacturer.

E~A~PLE V
Imml~nohistochemistry Paraffin sections of formalin-fixed mouse mid-small bowel were deparaffinized, treated with 1.1%
hydrogen peroxide for 40 min, then washed extensively with water followed by PBS. Slides were treated for 20 min at 37 ~C with 500 ~g/ml tryp~in in PBS, washed twice with PBS, and blocked by incubation for 20 min with 5%
porcine serum. Slides were incubated for 20 min in rabbit anti ~ly~din IgG (1:10 dilution relative to serum IgG concentration), then washed with blocking serum.
Porcine anti-rabbit IgG was used as linking reagent between the primary antibody and rabbit antiper~ Ase-peroxidase conjugate (Dako; Carpinteria CA).
Diaminobenzidine was used as peroxidase substrate and parallel incubations were performed using equivalent dilutions of rabbit preimmune IgG as the primary antibody.

EXAMPLE VI
Pre~r~t;on of Sy~thet;c Crypt~; n 1 This example provides a method for synthesizing, purifying and characterizing synthetic cryptdin 1.

r~

~ CA 0220499~ 1997-0~-09 iJ ~ 2 8 ~r~ v ~

A. Sy~thesis Synthesis was initiated at the 0.13 mmole scale using Wang resin coupled to flourenylmethoxycarbonyl (Fmoc)-arginine using an acid labile linker. Synthesis was carried out in dimethylformamide (DMF) using (relative to resin substitution) a 3-fold excess of Fmoc-amino acids activated in situ with a 3-fold excess of BOP
(benzotriazol-1-yl-oxy-tri~ (dimethylamino) phosphonium hexafluorophosphate) and HOBt (hydroxybenzotriazole) and a 6-fold molar excess of N-methylmorpholine (Nmm). Fmoc removal during ~ynthetic cycles was achieved using cycles of 50% and 25% piperidine in DMF. The side-chain protection scheme utilized the following Fmoc-~m;no acids: OtBut-aspartic acid, Pmc-arginine, tBut-tyrosine, tBut-serine, Trt-cysteine, tBoc-lysine, OtBut-glutamic acid, Trt-asparagine, tBut-threonine and Trt-histidine.
The peptide chain was assembled in a Synostat batch synthesizer using single coll~lings at all addition~
except at leucine and valine which were double coupled.
The cycle sequence is as follows:
1. Wash with DMF 4X for 2 min;
2. Deblock: 50% piperidine lX for 5 min;
3. Deblock: 25% piperidine lX for 15 min;
4. Wash with DMF 4X for 2 min;
5. Dissolve amino acids + BOP + HOBt in DMF
and transfer to reaction vessel;
6. Add Nmm to RV and mix for 60 min; and 7. Wash with DMF lX for 2 min.

After coupling of the amino terminal residue, the terminal Fmoc group was removed using the following cycle:
1. Wash with DMF 4X for 2 min:
2. Deblock: 50% piperidine lX for 5 min;
3. Deblock: 25% piperidine lX for 15 min;
4. Wash with DMF 4X for 2 min;
5. Wash with dichloromethane lX for 5 min;

CA 0220499~ 1997-0~-09 .~ ;
t r, ~ r r; ~ r~ ~Q~

6. Wash with isopropanol 4X for 5 min;
7. Dry under stream of Nz lX for 10-20 min;
and 8. Dry under vacuum lX for 12 hr.
The peptide-resin was weighed to determine mass increase. To cleave and deprotect the peptide-resin, it was first reswelled in dichloromethane, then cleaved and deprotected by addition of reagent R ( 90% trifluoroacetic acid, 5% thioanisole, 3% ethanedithiol, 2% anisole) at a ratio of 10 ml/g peptide-resin. Cleavage/deprotection was carried out under nitrogen for 18 hr at RT.

B. PurificAtion The cleavage mixture was separated from resin by filtration through a scintered glass funnel, washed with 1-2 ml fresh reagent R and diluted 5-fold with 50%
acetic acid. Glacial acetic acid was added to a final acetic acid concentration of 50%. The resulting solution was extracted 3x with 0.33 vol methylene chloride and the aqueous phase was lyophilized to dryness, then dissolved in 50% acetic acid and relyophil;zed. The extraction and lyophilization steps were repeated 3-4 times, then the dry peptide was dissolved in 30% acetic acid at a concentration of 20 mg/ml and passed over an 800 ml Sephadex G-10 column equilibrated in 30% acetic acid.
Peptide-cont~ining fractions were pooled, lyophilized, dissolved in 5% acetic acid, then diluted ten-fold with water to a final protein concentration of about 1 mg/ml.
The solution was adjusted to pH 8.0 with ammonium hydroxide and mixed rapidly with a magnetic stirrer at RT
in a beaker open to room air. The pH wa~ adju~ted periodically to pH 8.0 over a period of 4 days. The solution was then acidified with acetic acid to pH 3.5 and lyophilized.
C-18 RP-HPLC using 0.1% TFA-water/acetonitrile gradients was used to purify the folded peptide.
Fractions were analyzed on acid-urea gels and compared to ~ CA 0220499~ 1997-0~-09 . 8 ~ ,~

natural cryptdin 1. The yield from an initial crude peptide preparation of 500 mg was approximately 30 mg.

C. CharacteriZAti011 Synthetic cryptdin 1 was compared to natural peptide on analytical RP-HPLC, SDS-PAGE and under three different conditions on acid-urea PAGE. For analysis on acid-urea PAGE, peptide was electrophore~ed either without modification, after reduction with DTT or after performic acid oxidation. Under all conditions described, native and synthetic cryptdin 1 behaved identically. The amino acid compositions of natural and synthetic cryptdin 1 were indistinguishable.

E~A~PLE VII
Cl o~i ng of Nucleic Aci~ Molecllles ~nco~i ng Cryp~Ai n8 Individual crypt~ were isolated using a modification of the EDTA elution method of Bjerknes and Cheng, supra , 1981, as described by Cano-Gauci et al., ~Y~t. Cell Res. 208:344-349 (1993), which is incorporated herein by reference. Briefly, the central 10 cm of small intestine from an adult C3H/HeJ mouse was everted on a Buchler gradient-making apparatus, then intact crypt~
were dislodged by vibration in ice cold 30 mM EDTA in calcium-free, magnesium-free PBS. Isolated crypts were disrupted in a sonicating water bath prior to cDNA
synthesis.
The crypt library was con~tructed by mRNA-directed PCR amplification (Cano-Gauci et al., supra , 1992). Phage were screened at a density of approximately 300 PFU/dish using the partial cDNA clone, asb4/134, as a probe (Ouellette et al., supra, 1989a). Positive phage were collected and denatured plasmid cDNA was sequenced by the dideoxynucleotide termination method using SequenaseTM (U.S. Biochemical Corp.; Cleveland OH).
Sequencing primers included T3 and T7 promoter primers and Defcrpl30, which i~ a 16-mer that corresponds to ~ CA 0220499~ 1997-0~-09 ~ifu~ . ~ ; v ~. 2 8 ci~ 9~, 1996 nucleotides 90-105 in cryptdin 1 mRNA (Huttner et al., G~nnmi cs 19: 448-453 (1994), which is incorporated herein by reference). Reaction mixtures were separated by electrophoresi~ in gel~ consisting of 5% Long RangerTM (AT
Biochem, Inc.; Malvern PA) and DNA sequence data were analyzed (Ouellette et al., supra, 1994). Computations for similarity searche~ of DNA sequences in nonredundant nucleic acid and protein sequence databases were performed at the National Center for Biotechnology Information with the BLAST network service (Ouellette et al., supra, 1994).
A cDNA library also was prepared by amplification of cryptdin mRNA (Huttner et al., supra , 1994). Total RNA was isolated from the small intestine of a male 129/SVJ mouse using RNazolT~ (Biotecx Lab;
Houston TX). First strand cDNA synthesis was performed using the cDNA Cycle Kit (Invitrogen; San Diego CA).
Amplification of 5' ends was performed using the 5' RACE
method (Frohman et al., proc. NAtl. ACA~. Sci.~ USA
85:8998-9002 (1988), which is incorporated herein by reference) with a reverse primer that was specific for a conserved region of the cryptdin 3~-untranslated sequence (UTS).
Blot hybridization of the PCR products using an oligonucleotide probe specific for the cryptdin prepro-coding region detected a ~ingle band. DNA from the band was isolated using the Geneclean IIT~ kit (BiolOl; La Jolla CA), subcloned into the Bluescript II plasmid using the pCR-Script SK(+) cloning kit (Stratagene) and transfected into competent XL-l Blue cells (Stratagene).
Colonies contAining cryptdin-related sequences were identified by hybridization to a labelled asb4/134 probe.
DNA sequence analysis of the positive clones was performed as described above, except that internal primers were utilized as required.
Using these methods, cDNA sequences encoding 17 distinct mouse cryptdin peptides were idontified (Figure ~ .~t~~

CA 0220499~ 1997-0~-09 J .' ~''.J'~
'9 ~ ~ V iJ . ,~9 10; SEQ ID NOS: 40-56). The variou~ mouse cryptdin cDNA
sequences share 93-100% nucleotide sequence identity with cryptdin 1, except cryptdin 5 and cryptdin 4 share 73%
and 69~ sequence identity, respectively, with cryptdin 1.
The amino acid sequences were deduced from the cDNA sequences for the 17 mouse cryptdins (see Figure 9.A.; SEQ ID NOS: 23 to 39). As shown in Figure 9.A., the cDNA sequences encode prepro-cryptdin peptides consisting of a Qignal peptide, a propiece and the cryptdin peptide. The prepro-cryptdins, including the mature cryptdin peptide, share significant amino acid sequence identity with cryptdin 1, although cryptdins 4 and 5 are less homologous (Figure 9.B.). Amino acid variability was most striking at position 10 of the mature cryptdin peptide, where either serine, threonine, alanine, i~oleucine or lysine can be found.
Interestingly, a single nucleotide change in the sequence of cryptdin 2 can account for each of these Amino acids.
In addition, position 15 can contain arginine or lysine.
The amino acid variability among cryptdin peptides can be involved in conferring different antimicrobial properties to the cryptdins.
Mouse cryptdin genomic clones also were obtained and ~equenced (Huttner et al., supra, 1994).
Asb4/134 was used as a probe to screen a custom-made 129/SVJ mouse genomic library constructed in lambda DASH
II (Stratagene Cloning Systems, Inc.; La Jolla CA).
Approximately 1 x 10C phage were screened in duplicate and 25 positive phage were identified. Ten clones were purified and phage DNA was isolated using Qiagen 100 columns (Qiagen, Inc.; Chatsworth CA). Southern blots of Eco RI-digested DNA from individual phage were hybridized to asb4/134 and hybridizing fragments were subcloned into Bluescript II SK+TM (Stratagene) or pUC18 (BRL;
Gaithersburg MD) for sequencing.
Sequencing was performed as described above, except that primers were selected based on the cryptdin ;, !~

CA 0220499~ 1997-0~-09 P~ US ;~ 3 ~ 2 8 ~r' ~ ~ G r;~

1 cDNA sequence and with the expectation that mouse cryptdin genes would be structurally homologous to the rabbit MCP-1 and MCP-2 defensin genes (see Huttner et al., supra , 1994). DNA sequence data were analyzed using the programs of Staden (Ri ochem. Soc. Trans. 12:1005-1008 (1984) and the University of Wisconsin Genetics Computer Group (Devereux et al., Nucl. Acid~ Res. 12:387-395 (1985)). Searches for homology were performed as described above.
As shown in Figure 11, screening of the genomic library produced nucleic acid sequences that contained the complete coding sequences for mouse cryptdins 1, 2, 3, 5 and 6 (SEQ ID NOS: 58-62). In addition, a homologous gene, designated cryptdin i (Crypi; SEQ ID
NO: 63), which apparently was inactivated due to a point mutation that changed a cysteine codon to an in-frame stop codon, was isolated. Examination of the nucleic acid sequences revealed that the cryptdin genes contain two exons, the first of which codes for the 5~-UTS and the prepro-coding region and the second of which encodes the mature cryptdin peptide and the 3~-UTS (not shown;
but see Figure ll.A.). A simllar structure has been described for the human cryptdin genes (Jones and Bevins, J. Biol. Chem~ 267:23216-23225 (1992)).
Similar methods as described above were used to obtain the cDNA sequences encoding rat cryptdins 1-3 (Figures 14.A. to 14.C.; SEQ ID NOS: 65-67, respectively), except that RNA was obtained from the small intestine of Sprague-Dawley rats. In addition, genomic DNA sequence~ encoding rat cryptdins 1-3 (Figures 15.A. to 15.C.; SEQ ID NOS: 68-70, respectively) were obtained using methods as described above, except that a genomic library cont~i n ing Sprague-Dawley DNA cloned in EMBL3 was purchased from Clontech (Palo Alto CA).
Although the invention has been described with reference to the disclosed embodiments, it should be - understood that various modifications can be made without CA 02204995 1997 05 0 iJCï/Us J J / i 3 3 ~ ~1 ~r~ ,3~

departing from the spirit of the invention. Accordingly, the invention i-~ limited only by the following claim~.

W O96/16075 PCTrUS95/13328 SEQUENCE LISTING

(1) GENERAL INFORMATION:
(i) APPLICANT: THE REGENTS OF THE UNIVERSITY OF
CALIFORNIA
(ii) TITLE OF INVENTION: Antibiotic Cryptdin Peptides and Methods of Their Use (iii) NUMBER OF SEOUENCES: 70 (iv) CORRESPONDENCE ADDRESS:
(A) ArD~ : Robbins, Ber~iner ~ Carson (B) STREET: 201 N. Figueroa Street, 5th F~oor (C) CITY: Los Ange~es (D) STATE: Ca~ifornia (E) CWNTRY: USA
(F) ZIP: 9001Z-2628 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatib~e (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFT~ARE: Patentln Re~ease #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/930,649 (B) FILING DATE: 14-AUG-1992 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/889,020 (B) FILING DATE: 26-MAY-1992 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Ber~iner, Robert (B) REGISTRATION NUMBER: 20,121 (C) REFEREUCE/DOCKET UUMBER: 5555-339C1-XPC
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (213) 977-1001 (B) TELEFAX: (213) 977-1003 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEOUENCE CHARACTERISTICS:
~A) LENGTH: 4 amino acids ~B) TYPE: amino acid ~D) TOPOLOGY: linear ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Leu Ser Lys Lys ~2) INFORMATION FOR SEQ ID NO:Z:

~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 6 amino acids ~B) TYPE: amino acid ~D) TOPOLOGY: ~inear CA 02204995 1997-0~-09 (xi) SEQUENCE DESCRIPTION: SEO ID No:2:
Gly lle Arg Phe Leu Tyr (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEO ID NO:3:
Arg Asn Leu Phe Leu Thr Phe Val Phe (2) IUFORMATION FOR SEQ ID Nû:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Arg Arg Gly His Leu Met Tyr Thr Leu (2) INFORMATION FOR SEQ ID No:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ix) FEATURE:
(A) NAME/KEY: Peptide ~B) LOCATION: 6 (D) OTHER I~FORMATION: /note= "Xaa = Amino acid is ind~r~.~t,.~ly L or M."
(ix) FEATURE:
(A) NAME/KEY: Peptide (B) LOCATION: 9 (D) OTHER INFORMATION: /note= "Xaa = Amino acid is ir~e~ t"Lly L or M"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Arg Lys Gly His Leu Xaa Tyr Thr Xaa (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Leu Arg Asp Leu Val Cys Tyr Cys Arg Ser Arg Gly Cys Lys Gly Arg CA 0220499~ l997-0~-09 Glu Arg Met Asn Gly Thr Cys Arg Lys Gly His Leu Leu Tyr Thr Leu zo 25 30 Cys Cys Arg ~Z) INFORMATION FOR SEO ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 35 amino acids ~B) TYPE: amino acid ~D) TOPOLOGY: linear ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Leu Arg Asp Leu Val Cys Tyr Cys Arg Thr Arg Gly Cys Lys Arg Arg Glu Arg Met Asn Gly Thr Cys Arg Lys Gly His Leu Met Tyr Thr Leu Cys Cys Arg 2 ) INFORMATION FOR SEQ ID NO:8:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 35 amino acids ~B) TYPE: amino acid (D) TOPOLOGY: linear ~xi) SEQUENCE DESCRIPTION: SEQ ID No:8:
Leu Arg Asp Leu Val Cys Tyr Cys Arg Lys Arg Gly Cys Lys Arg Arg _ _ ==~}-~ .. . Glu Arg Met Asn Gly Thr Cys Arg Lys Gly His Leu Met Tyr Thr Leu Cys Cys Arg ~2) INFORMATION FOR SEQ ID NO:9:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 31 amino acids ~B) TYPE: amino acid ~D) TOPOLOGY: linear ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Gly Leu Leu Cys Tyr Cys Arg Lys Gly His Cys Lys Arg Gly Glu Arg Val Arg Gly Thr Cys Gly lle Arg Phe Leu Tyr Cys Cys Pro Arg CA 0220499~ 1997-0~-09 (2) INFORMATION FOR SEQ ID NO:10:
ti) SEOUENCE CHARACTERISTICS:
(A) LENGTH: 35 amino acids ~B) TYPE: amino acid ~D) TOPOLOGY: linear ~xi) SEQUENCE DESCRIPTION: SEO ID NO:10:
Leu Ser Lys Lys Leu lle Cys Tyr Cys Arg lle Arg Gly Cys Lys Arg Arg Glu Arg Val Phe Gly Thr Cys Arg Asn Leu Phe Leu Thr Phe Val Phe Cys Cys ~2) INFORMATION FOR SEQ ID NO:11:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 39 amino acids ~B) TYPE: amino acid ~D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Leu Lys Gln Cys His Cys Arg Lys Phe Cys Arg Pro Tyr Glu Lys Ala Glu Gly Ser Cys Arg Pro Gly Leu Phe lle Lys Arg Lys lle Cys Cys lle Gln Gln Trp Thr Pro Gly _ ~ - 35 ... . (Z) INFORMATION FOR SEO ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1Z:
Gly Leu Leu Cys Tyr Cys Arg Lys Gly His Cys Lys Arg Gly Glu Arg Val Arg Gly Thr Cys Gly lle Arg Phe Leu Tyr Cys Cys Pro Arg Arg (2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUEUCE CHARACTERISTICS:
(A) LENGTH: 36 amino acids ~B) TYPE: amino acid ~D) TOPOLOGY: linear (xi~ SEQUENCE DESCRIPTION: SEQ ID NO:13:
Leu Ser Lys Lys Leu lle Cys Tyr Cys Arg lle Arg Gly Cys Lys Arg Arg Glu Arg Val Phe Gly Thr Cys Arg Asn Leu Phe Leu Thr Phe Val Phe Cys Cys Ser ~2) INFORMATION FOR SEQ ID NO:14:
ti) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 amino acids ~B) TYPE: amino acid tD) TOPOL0GY: linear ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Leu Arg Asp Leu Val Cys Tyr Cys Arg Ala Arg Gly Cys Lys Gly Arg Glu Arg Met Asn Gly Thr Cys Arg Lys Gly His Leu Leu Tyr Met Leu Cys Cys Arg (2) INFORMATION FOR SEQ ID NO:15:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 41 amino acids ~B) TYPE: amino acid ~D) TOPOLOGY: linear ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Leu Lys Gln Cys His Cys Arg Lys Phe Cys Arg Pro Tyr Glu Lys Ala Glu Gly Ser Cys Arg Pro Gly Leu Phe lle Lys Arg Lys lle Cys Cys lle Gln Gln Trp Thr Pro Gly Arg Thr ~2) INFORMATION FOR SEQ ID NO:16:

~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
lle Gly Arg Pro Val Arg Arg Cys Arg Cys Arg Ala Asn Cys Gly Pro Lys Glu Tyr Ala Thr Ala Phe Cys Ala Gln Gly Pro Phe Lys Gln Phe CA 0220499~ 1997-0~-09 Lys Phe Cys Cys Thr ~Z) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 45 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
lle Arg Trp Pro Trp Lys Arg Cys His Cys Arg Ser Phe Cys Arg Pro Tyr Glu Asn Ala Thr Ser Phe Cys Ala Gln Gly Leu Phe Lys Gln His Lys Phe Cys Cys Leu Asp Thr Trp Pro Pro Arg Met Lys (2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Thr Ser Gly Ser Gln Ala Arg Ala Thr Cys Tyr Cys Arg Thr Gly Arg Cys Ala Thr Arg Glu Ser Leu Ser Gly Val Cys Glu lle Ser Gly Arg Leu Tyr Arg Leu Cys Cys Arg (2) INFORMATION FOR SEQ ID NO:19:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 32 amino acids ~B) TYPE: amino acid ~D) TOPOLOGY: linear ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

Ala Phe Thr Cys His Cys Arg Arg Ser Cys Tyr Ser Thr Glu Tyr Ser Tyr Gly Thr Cys Thr Val Met Gly lle Asn His Arg Phe Cys Cys Leu ~2) INFORMATION FOR SEQ ID NO:20:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 103 amino acids ~B) TYPE: amino acid ~D) TOPOLOGY: linear CA 0220499~ 1997-0~-09 ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Met Lys Thr Leu Val Leu Leu Ser Ala Leu Val Leu Leu Ala Phe G~n Val Gln Ala Asp Pro lle Gln Glu Ala Glu Glu Glu Thr Lys Thr G~u Glu Gln Pro Ala Asp Glu Asp Gln Asp Val Ser Val Ser Phe Glu G~y Pro Glu Pro Ser Ala Leu G~n Asn Leu G~u lle Gly Trp Pro Leu Lys G~n Cys His Cys Arg Lys Phe Cys Arg Pro Tyr Glu Lys A~a G~u Gly Ser Cys Arg Pro Gly Leu Phe lle Lys Arg Lys lle Cys Cys lle Gln Gln Trp Thr Pro Gly Arg Thr (2) INFORMATION FOR SEO ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 96 amino acids (B) TYPE: amino acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Met Lys Thr Leu Val Leu Leu Ser Ala Leu Va~ Leu Va~ Ala ~yr Gln Val G~n Ala Asp Pro lle G~n G~y Ala Glu G~u Glu Thr Lys Thr Glu Glu Gln Pro Ser Asp Glu Asp Gln Asp Val Ser Val Ser Phe Glu Gly Pro Glu Ala Ser Ala Leu Gln Asp Phe Glu l~e Gly Arg Pro Val Arg Arg Cys Arg Cys Arg Ala Asn Cys Gly Pro Lys Glu Tyr Ala Thr Ala Phe Cys A~a Gln Gly Pro Phe Lys G~n Phe Lys Arg Phe Cys Cys Thr (2) INFORMATION FOR SEQ ID No:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 103 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear CA 0220499~ 1997-0~-09 (xi~ SEQUENCE DESCRIPTION: SEQ ID NO:22:
Met Lys Thr Leu Val Leu Leu Ser Ala Leu Val Leu Leu Ala Phe Gln lle Gln Ala Asp Pro lle Gln Glu Ala Glu Glu Glu Thr Lys Thr Glu Glu Gln Pro Ala Asp Glu Asp Gln Asp Val Ser Val Ser Phe Glu Gly Pro Glu Pro Ser Ala Leu Gln Asn Leu Glu lle Arg Trp Pro Trp Lys Arg Cys His Cys Arg Ser Phe Cys Arg Pro Tyr Glu Asn Ala Thr Ser Phe Cys Ala Gln Gly Leu Phe Lys Gln His Lys Phe Cys Cys Leu Asp Thr Trp Pro Pro Arg Met Lys (2) INFORMATION FOR SEO ID No:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 93 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Met Lys Thr Leu Val Leu Leu Ser Ala Leu Val Leu Leu Ala Phe Gln Val Gln Ala Asp Pro lle Gln Asn Thr Asp Glu Glu Thr Lys Thr Glu Glu Gln Pro Gly Glu Asp Asp Gln Ala Val Ser Val Ser Phe Gly Asp Pro Glu Gly Thr Ser Leu Gln Glu Glu Ser Leu Arg Asp Leu Val Cys Tyr Cys Arg Ser Arg Gly Cys Lys Gly Arg Glu Arg Met Asn Gly Thr Cys Arg Lys Gly His Leu Leu Tyr Thr Leu Cys Cys Arg (2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 93 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEa ID NO:24:
Met Lys Thr Leu lle Leu Leu Ser Ala Leu Val Leu Leu Ala Phe Gln Val Gln Ala Asp Pro lle Gln Asn Thr Asp Glu Glu Thr Lys Thr Glu Lys Gln Pro Gly Glu Glu Asp Gln Ala Val Ser Val Ser Phe Gly Asp Pro Glu Gly Ser Ser Leu Gln Glu Glu Ser Leu Arg Asp Leu Val Cys Tyr Cys Arg Thr Arg Gly Cys Lys Arg Arg Glu Arg Met Asn Gly Thr Cys Arg Lys Gly His Leu Met Tyr Thr Leu Cys Cys Arg ~2~ INFORMATION FOR SEO ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 93 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEOUENCE DESCRIPTION: SEO ID No:25:
Met Lys Thr Leu Val Leu Leu Ser Ala Leu Val Leu Leu Ala Phe Gln Val Gln Ala Asp Pro lle Gln Asn Thr Asp Glu Glu Thr Lys Thr Glu Glu Gln Pro Gly Glu Asp Asp Gln Ala Val Ser Val Ser Phe Gly Asp Pro Glu Gly Ser Ser Leu Gln Glu Glu Ser Leu Arg Asp Leu Val Cys Tyr Cys Arg Lys Arg Gly Cys Lys Arg Arg Glu Arg Met Asn Gly Thr Cys Arg Lys Gly His Leu Met Tyr Thr Leu Cys Cys Arg (2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 92 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ix) FEATURE:
~A) NAME/KEY: Peptide _ - ~B~ LOCATION: 79 (D) OTHER INFORMATION: /note= "Xaa = Amino acid is ir~ e,.~ly L or M."
(ix) FEATURE:

CA 0220499~ 1997-0~-09 (A) NAME/KEY: Peptide (B) LOCATION: 80 (D) OTHER INFORMATION: /note= "Xaa = Amino acid is il~t~ .,lly L or M."
(ix) FEATURE:
(A) NAME/KEY: Peptide (B) LOCATION: 82 (D) OTHER INFORMATION: /note= "Xaa = Amino acid is iI~tp~ ..tly L or M."

(xi) SEOUENCE DESCRIPTION: SEQ ID NO:26:
Leu Val Leu Leu Ser Ala Leu Val Leu Leu Ala Phe Gln Val Gln Ala Asp Pro lle Gln Asn Thr Asp Glu Glu Thr Lys Thr Glu Glu Gln Pro Gly Glu Glu Asp Gln Ala Val Ser lle Ser Phe Gly Gly Gln Glu Gly Ser Ala Leu His Glu Lys Ser Leu Arg Gly Leu Leu Cys Tyr Cys Arg Lys Gly His Cys Lys Arg Gly Glu Arg Val Arg Gly Thr Cys Xaa Xaa Gly Xaa lle Arg Phe Leu Tyr Cys Cys Pro Arg Arg (2) INFORMATION FOR SEQ ID No:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 93 amino acids (B) T~PE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID No:27:
Met Lys Thr Phe Val Leu Leu Ser Ala Leu Val Leu Leu Ala Phe Gln Val Gln Ala Asp Pro lle His Lys Thr Asp Glu Glu Thr Asn Thr Glu Glu Gln Pro Gly Glu Glu Asp Gln Ala Val Ser lle Ser Phe Gly Gly Gln Glu Gly Ser Ala Leu His Glu Glu Leu Ser Lys Lys Leu lle Cys Tyr Cys Arg lle Arg Gly Cys Lys Arg Arg Glu Arg Val Phe Gly Thr Cys Arg Asn Leu Phe Leu Thr Phe Val Phe Cys Cys Ser CA 0220499~ 1997-0~-09 (2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 93 amino acids (B) TYPE: amino acid ~D) TOPOLOGY: linear ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
Met Lys Thr Leu Val Leu Leu Ser Ala Leu Val Leu Leu Ala Phe Gln Val Gln Ala Asp Pro lle Gln Asn Thr Asp Glu Glu Thr Lys Thr Glu Glu Gln Pro Gly Glu Glu Asp Gln Ala Val Ser Val Ser Phe Gly Asp Pro Glu Gly Thr Ser Leu Gln Glu Glu Ser Leu Arg Asp Leu Val Cys Tyr Cys Arg Ala Arg Gly Cys Lys Gly Arg Glu Arg Met Asn Gly Thr Cys Arg Lys Gly His Leu Leu Tyr Met Leu Cys Cys Arg (2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 93 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
Met Lys Thr Leu lle Leu Leu Ser Ala Leu Val Leu Leu Ala Phe Gln Val Gln Ala Asp Pro lle Gln Asn Thr Asp Glu Glu Thr Lys Thr Glu Glu Gln Pro Gly Glu Asp Asp Gln Ala Val Ser Val Ser Phe Gly Asp Pro Glu Gly Ser Ser Leu Gln Glu Glu Ser Leu Arg Asp Leu Val Cys Tyr Cys Arg Thr Arg Gly Cys Lys Arg Arg Glu His Met Asn Gly Thr Cys Arg Lys Gly His Leu Met Tyr Thr Leu Cys Cys Arg (2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 93 amino acids ~B) TYPE: amino acid ~D) TOPOLOGY: linear CA 0220499~ 1997-0~-09 ~xi) SEOUENCE DESCRIPTION: SEQ ID NO:30:
Met Lys Thr Leu Val Leu Leu Ser Ala Leu Val Leu Leu Ala Phe Gln Val Gln Ala Asp Pro lle Gln Asn Thr Asp Glu Glu Thr Lys Thr Glu Glu Gln Pro Gly Glu Glu Asp Gln Ala Val Ser Val Ser Phe Gly Asp Pro Glu Gly Ser Ser Leu Gln Glu Glu Ser Leu Arg Asp Leu Val Cys Tyr Cys Arg Lys Arg Gly Cys Lys Arg Arg Glu His Met Asn Gly Thr Cys Arg Lys Gly His Leu Leu Tyr Met Leu Cys Cys Arg (2) INFORMATION FOR SEO ID NO:31:
(i) SEOUENCE CHARACTERISTICS:
(A) LENGTH: 81 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
Leu Ala Phe Gln Val Gln Ala Asp Pro lle Gln Asn Thr Asp Glu Glu Thr Lys Thr Glu Glu Gln Pro Gly Glu Asp Asp Gln Ala Val Ser Val Ser Phe Gly Asp Pro Glu Gly Ser Ser Leu Gln Glu Glu Ser Leu Arg Asp Leu Val Cys Tyr Cys Arg Lys Arg Gly Cys Lys Arg Arg Glu His Met Asn Gly Thr Cys Arg Lys Gly His Leu Met Tyr Thr Leu Cys Cys Arg (2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 92 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
Lys Thr Leu Val Leu Leu Ser Ala Leu Val Leu Leu Ala Phe Gln Val CA 0220499~ 1997-0~-09 Gln Ala Asp Pro Ile Gln Asn Thr Asp Glu Glu Thr Lys Thr Glu Glu Gln Pro Gly Glu Asp Asp Gln Ala Val Ser Val Ser Phe Gly Asp Pro Glu Gly Ser Ser Leu Gln Glu Glu Ser Leu Arg Asp Leu Val Cys Tyr Cys Arg Lys Arg Gly Cys Lys Gly Arg Glu Arg Met Asn Gly Thr Cys Arg Lys Gly His Leu Leu Tyr Thr Leu Cys Cys Arg ~2) INFORMATION FOR SEû ID No:33:
(i) SEOUENCE CHARACTERISTICS:
~A) LENGTH: 85 amino acids ~B) TYPE: amino acid ~D) TOPOLOGY: linear ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
Ala Leu Val Leu Leu Ala Phe Gln Val Gln Ala Asp Pro Ile Gln Asn Thr Asp Glu Glu Thr Lys Thr Glu Glu Gln Pro Gly Glu Glu Asp Gln Ala Val Ser Val Ser Phe Gly Asp Pro Glu Gly Thr Ser Leu Gln Glu ,=................... Glu Ser Leu Arg Asp Leu Val Cys Tyr Cys Arg Ser Arg Gly Cys Lys ' 50 55 60 Gly Arg Glu Arg Met Asn Gly Thr Cys Arg Lys Gly His Leu Leu Tyr Met Leu Cys Cys Arg ~2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 93 amino acids ~B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID No:34:
Met Lys Thr Leu Ile Leu Leu Ser Ala Leu Val Leu Leu Ala Phe Gln Val Gln Ala Asp Pro Ile Gln Asn Thr Asp Glu Glu Thr Lys Thr Glu -CA 0220499~ 1997-0~-09 Glu Gln Pro Gly Glu Glu Asp Gln Ala Val Ser Val Ser Phe Gly Asp Pro Glu Gly Thr Ser Leu Gln Glu Glu Ser Leu Arg Asp Leu Val Cys Tyr Cys Arg Ala Arg Gly Cys Lys Gly Arg Glu Arg Met Asn Gly Thr Cys Arg Lys Gly His Leu Met Tyr Thr Leu Cys Cys Arg (2) INFORMATION FOR SEO ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 93 amino acids (B) TrPE: amino acid (D) TOPOLOGr: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
Met Lys Thr Leu Val Leu Leu Ser Ala Leu Val Leu Leu Ala Phe Gln Val Gln Ala Asp Pro lle Gln Asn Thr Asp Glu Glu Thr Lys Thr Glu Glu Gln Pro Gly Glu Glu Asp Gln Ala Val Ser Val Ser Phe Gly Asp Pro Glu Gly Thr Ser Leu Gln Glu Glu Ser Leu Arg Asp Leu Val Cys Tyr Cys Arg Lys Arg Gly Cys Lys Arg Arg Glu His Met Asn Gly Thr . ~--... ...
Cys Arg Arg Gly His Leu Met Tyr Thr Leu Cys Cys Arg (2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 85 amino acids (B) TYPE: amino acid (D) TOPOLOGr: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
Ala Leu Val Leu Leu ALa Phe Gln Val Gln Ala Asp Pro lle Gln Asn Thr Asp Glu Glu Thr Lys Thr Glu Glu Gln Pro Gly Glu Glu Asp Gln Ala Val Ser Val Ser Phe Gly Asp Pro Glu Gly Ser Ser Leu Gln Glu Glu Ser Leu Arg Asp Leu Val Cys Tyr Cys Arg Thr Arg Gly Cys Lys CA 0220499~ 1997-0~-09 Arg Arg Glu Arg Met Asn Gly Thr Cys Arg Lys Gly His Leu Met His Thr Leu Cys Cys Arg (2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 93 amino acids (B) TYPE: amino acid (D) TOPOLOGr: linear (xi) SEQUENCE DESCRIPTION: SEO ID No:37:
, Met Lys Thr Leu Val Leu Leu Ser Ala Leu Val Leu Leu Ala Phe Gln Val Gln Ala Asp Pro lle Gln Asn Thr Asp Glu Glu Thr Lys Thr Glu Glu Gln Pro Gly Glu Asp Asp Gln Ala Val Ser Val Ser Phe Gly Asp Pro Glu Gly Ser Ser Leu Gln Glu Glu Ser Leu Arg Asp Leu Val Cys Tyr Cys Arg Lys Arg Gly Cys Lys Arg Arg Glu His lle Asn Gly Thr Cys Arg Lys Gly His Leu Leu Tyr Met Leu Cys Cys Arg (2) INFORMATION FOR SEQ ID No:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 93 amino acids (B) TYPE: amino acid (D) TOPOLOGr: linear (xi) SEOUENCE DESCRIPTION: SEQ ID NO:38:
Met Lys Thr Leu lle Leu Leu Ser Ala Leu Val Leu Leu Ala Phe Gln Val Gln Ala Asp Pro lle Gln Asn Thr Asp Glu Glu Thr Lys Thr Glu Glu Gln Pro Gly Glu Glu Asp Gln Ala Val Ser Val Ser Phe Gly Asp Pro Glu Gly Thr Ser Leu Gln Glu Glu Ser Leu Arg Asp Leu Val Cys Tyr Cys Arg Ser Arg Gly Cys Lys Gly Arg Glu Arg Met Asn Gly Thr CA 0220499~ 1997-0~-09 Cys Arg Lys Gly His Leu Met Tyr Thr Leu Cys Cys Arg ~2) INFORMATION FOR SEQ ID NO:39:
i ) SEQUENCE CHARACTER I ST I CS:
~A) LENGTH: 82 amino acids ~B) TYPE: amino acid ~D) TOPOLOGY: linear ~xi ) SEQUENCE DESCRIPTION: SEQ ID NO:39:
Leu Leu Ala Phe Gln Val Gln~Asp Pro Ile Gln Asn Thr Asp Glu Glu Thr Lys Thr Glu Glu Gln Pro Gly Glu Glu Asp Gln Ala Val Ser Val Ser Phe Gly Asp Pro Glu Gly Thr Ser Leu Gln Glu Glu Ser Leu Arg Asp Leu Val Cys Tyr Cys Arg Lys Arg Gly Cys Lys Arg Arg Glu His Met Asn Gly Thr Cys Arg Lys Gly His Leu Leu Tyr Thr Leu Cys Cys Arg 2) I N FORMAT I ON FOR SEQ ID NO:40:
i ) SEQUENCE CHARACTER I ST I CS:
~A) LENGTH: 422 base pairs ~B) TYPE: nucleic acid C ) STRANDEDNE SS: doub l e ~D) TOPOLOGY: linear ~xi ) SEQUENCE DESCRIPTION: SEQ ID NO:40:
ACACATTGAG u I C~ cA CCMTCCTCC AGGTGACTCC CAGCCATGM GACACTAGTC 60 c I UL I c I L I G CCCTTGTCCT GCTGGCCTTC CAGGTCCAGG CTGATCCTAT CC4AAAr4r4 120 GATGMGAGA CTMMCTGA cr~lrr-~crr4 rrrr~Ar~lrr. Arr4rrrrGT ATCTGTCTCC 180 TTTGGAGACC C.4r4Arrr4r TTCTCTTCM GAGGMTCGT TGAGAGATCT GGTATGCTAT 240 TGTAGATCM GAGGCTGCM ArCMr4r4A CGCATGMTG GGACCTGCAG MAGGGTCAT 300 TTATTGTACA CGL ~ ,u I G TCGCTGMCA TCr'\r'\rr~r A-~\rr~\r4Ar ACGMCATGA 360 2) I N FORMAT I ON FOR SEQ I D NO : 41:
i ) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 422 base pairs ~B) TYPE: nucleic acid C ) STRANDEDNESS: doubl e ~D) TOPOLOGY: linear CA 0220499~ 1997-0~-09 (xi ) SEQUENCE DESCRIPTION: SEQ ID NO:41:

L I LL 11,1 C I G CCL I Cl, 1 LL I GCTGGCCTTC CAGGTCCAGG CTGATCCTAT CCAAMTACA 120 GATGMGAGA CTAAMCTGA ~cr45rr4 rrrr~\Ar~rr AC~4rrrr5T A~l.;l~jl~;lLC 180 TGTAGMCM GAGGCTGCM Mr'\Ar'\r'~A CGCATGMTG GGACCTGCAG MMGGGTCAT 300 TTAATGTACA CGLI~ .LlG TCGCTGMCA TG5Ar~~rrl~r A-~rr'~r~A- ATGACCATGA 360 (2) I N FORMAT I ON FOR SEQ I D NO: 42:
i ) SEQUENCE CHARACTER I ST I CS:
(A) LENGTH: 422 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: double (D) TOPOLOGY: linear (xi ) SEQUENCE DESCRIPTION: SEQ ID NO:42:

CTCCTCTCTG CCL I LL I LL I LL I GCcL I I C CAGGTCCAGG CTGATCCTAT CCAl\Mr4r4 120 GATGAAGAGA CTMMCTGA Crl\rr~rrr4 crrr~\Ar~r5 ArcArrccGT AT~ .C 180 TGTAGAMMA GAGGCTGCM Mr'\Arl-'\A CGCATGMTG GGACCTGCAG MMGGGTCAT 300 TTAATGTACA CACTCTGCTG TCGCTGMCA TC5Ar'\rr4r A-'\-r'\r4Ar ACGMCATGA 360 (2) INFORMATION FOR SEQ ID NO:43:
( i ) SEQUENCE CHARACTER I ST I CS:
(A) LENGTH: 365 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double ( D ) TOPOLOGY: l i near (xi ) SEQUENCE DESCRIPTION: SEQ ID No:43:

AAATACAGAT GAAGAGACTA MACTGAGGA crl~rrC4rrr ~;Mr~rr.\rC AGGCCGTATC 120 TGTCTCCTTT cr~lr~\rcr4r MGGCTCTGC TCTTCATGM MMTCTTTGA GAGGTTTGTT 180 ATGCTATTGT Ar'\AMrr'\r ACTGCAAMG Arr îr'~ACCA GTTCGTGGGA CTTGTGGAAT 240 TTGTCTCTGA GGCCGCTGAT GCCGGGGr~ I GATGACCACT TCTCAAGMA TGTTTGCMT 360 CA 0220499~ 1997-0~-09 WO 96/1607~ PCT/US95/13328 (2) I N FORMAT I ON FOR SEq I D NO: 44:
( j ) SEOUENCE CHARACTERISTICS:
(A) LENGTH: 421 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (X; ) SEQUENCE DESCRIPTION: SEQ ID NO:44:

CTCCTCTCTG CCCTTGTCCT 6CIG6CI,I IC CAGGTCCAGG CTGATCCTAT CC4~A/\MCA 120 GATGAAGAGA CTMTACTGA rr~rr~~rCA CCCCMr~rr ArC4rrrrGT ATCTATCTCC 180 TTTGGAGGCC MGAAGGGTC TGCTCTTCAT GAGGMTTGT CAMAAArrT GATATGCTAT 240 TGTAGMTM GAGGCTGCM MCMr-r~\A CCCI~I I I I I G GGACCTGCAG MMTCTTTTT 300 (2) INFORMATION FOR SEO ID NO:45:
( j ) SEOUENCE CHARACTER I STICS:
(A) LENGTH: 422 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: double (D) TOPOLOGY: I inear ( X j ) SEaUE N CE D E SCR I PT I ON: SEQ I D NO: 45:

CTCLILILIG CLI,~ IC~.I GLI~C~UI IC CAGGTCCAGG CTGATCCTAT CCMMTACA 120 GATGMGAGA CTMAACTGA ~ rr4rrCA cr~rro~A~I\CC Arr4rrr~rGT ATCIGlLICC 180 TTTGGAGACC ~r4r~lA~r~r TTCTCTTCM GAGGMTCAT TGAGATATCT GGTATGCTAT 240 TGTAGAGCM GAGGCTGCM Arr'lAr'~r'~A CGCATGMTG GGACCTGCAG MMGGGTCAT 300 TTATTGTACA TGCTCTGCTG TCGCTGMCA TGGAGACCTC AG~Ir~Ar4Ar ACGACCATGA 360 2) I N FORMAT I ON FOR SEQ I D NO: 46:
( j ) SEQUENCE CHARACTERI STICS:
(A) LENGTH: 420 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: doubLe (D) TOPOLOGY: linear (X;) SEQUENCE DESCRIPTION: SEQ ID NO:46:

CCTCTCTGCC CTCGTCCTGC TGGCCTTCCA GGTCCAGGCT GATCCTATCC AI~Mr4CAr'\ 120 TGAAGAGACT MMCTGAGG Arr4r,rC4rr CCMr~rr~C CAGGCCGTAT LII~ICI~ 1 180 CA 0220499~ 1997-0~-09 TCCAr~Cr4 GMGGCTCTT CTCTTCMGA GGMTCGTTG AGAGATCTGG TATGCTATTG 240 TAr~lAr~GA GGCTGCAAM CMC~\rJAAr4 CATGMTGGG ACCTGCAGM AGGGTCATTT 300 AATGTACACG CTCTGCTGTC GCTGMCATG GAGACCTCAG Ar~\Ar4ACAr GACCATGAGT 360 (2) INFORMATlON FOR SEQ ID NO:47:
j ) SEqUENCE CHARACTERISTICS:
(A) LENGTH: 342 base pairs tB) TYPE: nucleic acid (C) STRANDEDNESS: double ~D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEO ID NO:47:
GCTGGCCTTC CAGGTCCAGG CTGATCCTAT CCMMr4r4 GATGMGAGA CTMMCTGA 60 CG~rr4-rCA GGr5Mr~Cr. Arr~-rrCGT ATCIG1t;IGL TTTGGAGACC CAGMGGCTC 120 AAr'\Ar'\-'\A CACATGMTG GGACCTGCAG MMGGGTCAT TTMTGTACA CCL~ GLIG 240 TCGCTGMCA T5GAC~rrtlr Ar~rr~-4Ar ACMGCATGA GTACTGAGGC CACTGATGCT 300 ~2) INFORMATION FOR SEQ ID NO:48:
( j ) SEWENCE CHARACTERISTICS:
(A) LENGTH: 377 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (X; ) SEWENCE DESCRIPTION: SEQ ID NO:48:

CCTATCCMA ACACAGATGA AGAGACTMM ACTGAGGAGC ArrC4rrrGA Ar~_r~rr4_ 120 GCCGTATCTG I G I GL I I I GG Ar'lrCC4r'~A GGL I G I I G I C TTCMGAGGA ATCGTTGAGA 180 GATCTGGTAT GCTATTGTAG AAMAr'~rrr Trr4MAr~A C~ ~Ar4r4T GMTGGGACC 240 TGC4r'~AArr GTCATTTATT GTACATGCTC TGLII.TC6LI GMCATGGAG Arr4r4r'~rr 300 2) I N FORMAT I ON FOR SEq I D NO: 49:
( j ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 375 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (X; ) SEQUENCE DESCRIPTION: SEQ 10 NO:49:
GMGACACTA G I CL I GL ~ CT CTGCCCTCGT CL I GL I GGCC TTCCAGGTCC AGGCTGAT CC 60 TATCCMMAC ACAGATGMG AGACTAMAC TGAGGAGCAG Cr~rrrCMr Arr~\rr,l\rrr 120 CA 0220499~ 1997-0~-09 CGTATCTGTC TCCTTTGGAG ACCr4rAA~rC CICI ILICI I CAAr~~r~AT CGTTGAGAGA 180 TCTGGTATGC TATTGTAGM MM GAGGCTG CAM rr~ArA GMCGCATGA ATGGMCCTG 240 C~5AM rrrT CATTTATTGT ACACGCTCTG CTGTCGCTGA ACATGGAGAC CArAr~rr,Ar 300 M CArr~AC4 TGAGTACTGA GGCCACTGAT GCTGGTGCCT GATGACCACT TCGCM TMM 360 ~2) INFORMATION FOR SEO ID No:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 352 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEO ID NO:50:
CC~ I L~ I L~ I G~I~CCL I I C CAGGTCCAGG CTGATCCTAT CCAA~r4r4 GATGMGAGA 60 CTMMACTGA cr~rr~rrr4 CGrCM r~GG ArrArrrCGT ATCTGTCTCC TTTGGAGACC 120 CACM r~CAr TTCTCTTCM GAGGM TCGT TGAGAGATCT GGTATGCTAT TGTAGATCM 180 GAGGCTGCM Ar5M r~r~A CGCATGMTG GMCCTGCAG MM GGGTCAT TTATTGTACA 240 TGCTCTGCTG TCGCTGMCA TCr~r~rrAr Ar~r~Ar~Ar ACGACCATGA GTACTGAGGC 300 (2) INFORMATION FOR SEQ ID NO:51:
(i) SE WENCE CHARACTERISTICS:
(A) LENGTH: 422 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (xi) SEqUENCE DESCRIPTION: SEq ID NO:51: --CTCCTCTCTG CCLIC~I L~T GLIGGLLllc CAGGTCCAGG CTGATCCTAT CCAAAArAr~ 120 GATGAAGAGA CT M M CTGA CGArCAr,rCA Crrr~Ar~rr ACCACrrCGT ATcI~l~lLC 180 TTTGGAGACC CAr~Arrr4r lIcTLlL~-AA GAGGMTAGT TGAGAGATCT GGTATGCTAT 240 TGTAGAGCM GAGGCTGCM Arr~ArArAA CGCATGMTG GGACCTGCAG MM GGGTCAT 300 TTAATGTACA CGCTCTGCTG TCGCTGM CA TGGAGACCTC Ar~r~Ar4AC ACGACCATGA 360 (2) INFORMATION FOR SEQ ID No:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 388 base pairs (B) TrPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: ~inear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:

CA 0220499~ 1997-0~-09 GACT CCCAGC CATGMGACA CTAGTCCTCC T L I L I CLLL I Tlj I CL I 6L I G GCCTTCCAGG 60 TCCAGGCTGA TCCTATCCM MCACAGATG MGAGACTM MCTGAGGAG r4rrr~\cr~- 120 AAr'~rr~rrA GGCCGTATCT GICICLI I IG r'\-'\rrr4rA AGGCACTTCT CTTCMGAGG 180 MTCGTTGAG AGATCTGGTA TGCTATTGTA CAAAM-~rr CTGCMAAGA A-'\-'~Ar4r4 Z40 C îrr4r4r~lr CA-AAr'\rrlA ACATGAGTAC TGAGGCCACT GATGCTGGTG CCTGATGACC 360 (2) INFORMATION FOR SEq ID No:53:
( i ) SEQUENCE CHARACTER I ST I CS:
(A) LENGTH: 352 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D ) TOPOLOGY: I i near (xi) SEqUENCE DESCRIPTION: SEQ ID NO:53:

CTMMCTGA rr~rr4rrr4 ,r~rrrl\Ar~\Gr~ ArCArr~rCGT ATt. I ~i I C I LC TTTGGAGACC 120 GAGGCTGCAA AAr'\Ar~r'\A CGCATGMTG GGACCTGCAG MMGGGTCAT TTAATGCACA 240 CGCTCTGCTG TCGCTGMCA Tcc~lr~\rr4r Ar'lrr~CMC ACGAGCATGA GTACTGAGGC 300 (2) INFORMATION FOR SEq ID NO:54:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 401 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (xi ) SEOUENCE DESCRIPTION: SEO ID NO:54:
CCMTCCTCC CAGTGACTCC CAGCCATGM GACACTAGTC L I ~.L I C I c I G CCCTTGTCCT 60 GCTGGCCTTC CAGGTCCAGG CTGATCCTAT Cr~4r4r4 GATGAAGAGA CTMAACTGA 120 Gr~\rr4r~rA rrrr~ArACG ACC4rr,rrGT ATCll.lLll,L TTTGGAGACC CAGMGGCTC 180 Mr~A~~r~A CACATMMTG GGACCTGCAG MMGGGTCAT TTATTGTACA CTCTCTGCTG 300 TCGCTGMCA Tr~A'"\rCAr A~rqÇr'\r'~AC ATGACCATGA GTACTGAGGC CACTGATGCT 360 (2) INFORMATION FOR SEQ ID No:55:
( i ) SEqUENCE CHARACTER I ST I CS:
(A) LENGTH: 391 base pairs ~B) TYPE: nucleic acid ~C) STRANDEDNESS: double ~D ) TOPOLOGY: I i near CA 0220499~ 1997-0~-09 ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:
GGTGACTCCC AGCCATGMG ACACTMTCC TCCTCTCTGC Cull,CrCLlG l.;lGGCul ICC 60 AGGTCCAGGC TGATCCTATC CANL'\r4r4'' ATGMGAGAC TMMCTGAG C~\rr4rrr4r 120 C'\'''\Ar'~~';A CCAGGCCGTA T~;ltill.ll,L.I TTGGAGACCC A''U9rr4rT TCTCTTCMG 180 AGGMTCGTT GAGAGATCTG GTATGCTATT GTAGATCMG AGGCTGCMM ('CUr~r'\Ar 240 GCATGMTGG GACCTGCAGA MGGGTCATT TMTGTACAC 61, I L I CL 11,1 CGCTGMCAT 300 GGAGACCTCA C'\''Ur4A''~ CGACCATGAG TACTGAGGCC ACTGATGCTG GTGCCTGATG 360 (2) I N FORMAT I ON FOR SEQ I D NO: 56:
i ) SEOUENCE CHARACTER I ST I CS:
(A) LENGTH: 342 base pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (xi ) SEQUENCE DESCRIPTION: SEQ ID NO:56:

rrt~ 4~rr4 ,rr~ Ar~\rr A~r4rrrrGT Ar~ .,ICC TTTGGAGACC C4'''\A~rr4r 120 M'''lAr'\'''\A CACATGMTG GGACCTGCAG MMGGGTCAT TTATTGTACA CGCTCTGCTG 240 TCGCTGAACA Tt'r'~r~"r4r A'"\'"''\~4Ar ATGACCATGA GTACTGAGGC CACTGATGCT 300 (2) INFORMATION FOR SEQ ID No:57:
( i ) SEQUENCE CHARACTER I ST I CS:
(A) LENGTH: 403 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (xi ) SEQUENCE DESCRIPTION: SEQ ID NO:57:

GAGACTMMA CTGAGGAGCA Grr4rrrr'\A r~rl\rr4rrr T6T(~Tu I 1~1 C TCTTTTGGAG 180 ArCr4r~A~C CLI ILI-;I IC MGAGGMTC GTTGAGAGAT CTGGTATGCT ATTGTAGMA 240 GAGGCTGCAA Ar'\AG'lr~Ar CATGMTGGG ACCTGCAGM AGGGTCATTT ATGTACAGCT 300 CTGCTGTCGC TGMCATGGA C~rCC4~ \ CMGMCATG AGTACTGAGG CCACTGATGC 360 (2) I N FORMAT I ON FOR SEQ I D NO: 58:
( i ) SEQUENCE CHARACTER I ST I CS:
(A) LENGTH: 419 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: s i ngle (D) TOPOLOGY: linear -CA 0220499~ 1997-0~-09 (ix) FEATURE:
~A) NAME/KEY: misc feature (B) LOCATION: Z79..286 (D) OTHER INFORMATION: /note= "N r~.sellt8 DNA that ~as not sequenced.~I

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:

GGTGACTCCC AGCCATGM G ACACTAGTCC TCCTCTCTGC CuII~lCulG CTGGCCTTCC 120 AGGTCCAGGC TGATCCTATC C4AAArA~A~ ATGM GAGAC TM MCTGAG GAGCAGCCAG 180 C~~4A~~~GA CCAGGCCGTA ILIGIcTLLI TTGGAGACCC A~~A~r~CT TCTCTTCM G 240 CGTTGAGAGA TCTGGTATGC TATTGTAGAT CM GAGGCTG CAM r~AÇA GM CGCATGA 360 ATGGAACCTG CACA~ T CATTTATTGT ACACGCTCTG uIGlC6ClGA ACATGGAGA 419 , (2) 1NFORMATION FOR SEQ ID NO:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 419 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: sing~e (D) TOPOLOGY: linear ~ix) FEATURE:
(A) NAME/KEY: misc feature (B) LOCATION: 279..286 (D) OTHER INFORMATION: /note= "N r~,ese.,t~ DNA that ~as not sequenced."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:

AGTGACTCCC AGCCATGM G CCACTTGTCC TCCTTTCTGC CuIlGTCulA ~lGTLull IL 120 AGGTCCAGGC TGATCCTATC r~AACAr~~ ATGM GAGAC T M AACTGAG GAGCAGTCAG 180 GTGM GAGGA CCAGGCTGTG TCTGTCTCCT TTGGAGACCG Ar4AGr,rGCT TCTCTTCM G 240 CGTTGAGAGA TCTGGTATGC TATTGTAGAA CMGAGGTTG r4~AAr4Ar~ GMCGCATGA 360 ATGGGACCTG C~ T CATTTM TGT ACACGCTCTG CTGCCGCTGA ACATGGAGA 419 (2) INFORMATION FOR SEO ID NO:60:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 419 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: ~inear (ix) FEATURE:
(A) NAME/KEY: misc feature (B) LOCATION: 279..286 (D) OTHER INFORMATION: /note= "N re~ t~ DNA that ~as not sequenced."

(xi) SEOUENCE DESCRIPTTON: SEQ ID NO:60:

GGTGACTCCC AGCCATGMG ACACTAGTCC TCL I L I C I GC C~,T L~. I CL I G CTGGCCTTCC 120 AGGTCCAGGC TGATCCTATC CAMArACAr ATGMGAGAC TMAACTGAG C~rr~lrrCAr 180 GCCMr~r5A CCAGGCTGTG TLI~CICI I TTGGAGACCC AGMGGCTCT TCTCTTCMG 240 CGTTGAGAGA TCTGGTATGC TATTGTAGM MMGAGGCTG CAAM~'~Ar'~ GMCGCATGA 360 ATGGGACCTG CAr'~MrCrT CATTTMTGT ACACACTCTG CTGTCGCTGA ACATGGAGA 419 ~2) INFORMATION FOR SEq ID NO:61:
~; ) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 419 base pairs ~B) TYPE: nucleic acid C ) STRANDEDNESS: S; ngle ~D) TOPOLOGr: linear j X ) FEATURE:
~A) NAME/KEY: misc_feature B ) LOCAT I ON: 279. .286 ~D) OTHER INFORMATION: /note= "N re~res_.,ls DNA that ~as not sequenced."

~X; ) SEQUENCE DESCRIPTION: SEQ ID NO:61:

AGGTCCAGGC TGATCCTATC CAr~MMr4r ATGAAGAGAC TMTACTGAG C~rr~rCCAr 180 rCCAAr~rr~ CCAGGCTGTG TCAGTCTCCT TTGGAGGCCA AGMGGGTCT GCTCTTCATG 240 TGTCMMM GCTGATATGC TATTGTAGM TMGAGGCTG CAAM~~Ar~l GMCGCGTTT 360 2) I N FORMAT I ON FOR SEQ I D NO: 62:
~; ) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 419 base pairs ~B) TrPE: nucleic acid ~C) STRANDEDNESS: single ~D) TOPOLOGr: linear j X ) FEATURE:
~A) NAME/KEr: misc feature B ) LOCAT I ON: 279. .286 ~D) OTHER INFORMATION: /note= "N re~,,ese.,~s DNA that ~as not sequenced."

(X;) SEQUENCE DESCRIPTION: SEQ ID NO:62:

GGTGACTCCC AGCCATGMG ACACTMTCC TCCTCTCTGC CL I LL~ I CL I G CTGGCCTTCC 120 AGGTCCAGGC TGATCCTATC CMMTACAG ATGMGAGAC TMMCTGAG ,C~rrArrC4G 180 Crr~A~~GCA CCAGGCTGTG TCI6T~;ILI I TTGGAGACCC Ar~A~rCACT TCTCTTCMG 240 CATTGAGAGA TCTGGTATGC TATTGTAGAG CMGAGGCTG CAAA~r~Ar'\ GMCGCATGA 360 -CA 0220499~ 1997-0~-09 ATGGGACCTG CArAJUlCCCT CATTTATTGT ACATGCTCTG CTGTCGCTGA ACATGGAGA 419 (2) INFORMATION FOR SEa ID NO:63:
i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 419 base pairs ~B) TYPE: nucleic acid ~C) STRANDEDNESS: single ~D) TOPOLOGY: linear ( i x ) FEATURE:
(A) NAME/KEY: misc feature ( B ) LOCAT I ON: 279. .286 (D) OTHER INFORMATI : ~ te- "N re~n ci~ DNA that ~las not sequenced."

(xi ) SEQUENCE DESCRIPTION: SEQ ID No:63:

GGTGACTCCC AGCCATGMG ACACTAGTCC )Gl,ILILI6L. C~ GCC(.lG LIGGCL.I ICC 120 MGTCCAGGC TGATCCTATC CAAAAr-A~r-A~r ATGMGAGAC TMMCTGAG c~rr-A~rrc4r 180 CrA~M'-MrA CCMGCTGTT TCIGI1;11.~.1 TTGGAGACCC AGMGGCTCT TCTCTTCMG 240 AGGMTGTGA GTACTGGTGC CCAGTGTGAT GGATGCTTNN NNNNNNTTTT lil~ 1GICCAG 300 CGTTGAGAGA TCTGATATGA TATTGTAGM CMGAGGCTG C'AAM~,AA"'~ GMCGCCTGA 360 ATGGGACCTG MrAAArr,rT CATTTATTGT ACATGCTCTG CTGCTGCTGA ACATGGAGA 419 ~2) INFORMATION FOR SEQ ID NO:64:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 411 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (xi ) SEQUENCE DESCRIPTION: SEQ ID NO:64:
TATAMTGCA RRulG6rllhl TCACTNTCCA CACATTGRGC TCCTGCTCAC CMTCCTCCA 60 GGTGACTCCC AGCCATGMG ACACTUGTCC TCCTCTCTGC C~ G I CI, I G CTGGCCTTCC 120 AGGTCCAGGC TGATCCTATC rAAMU~r~" ATGMGAGAC TAMMCTGAG GAGCAGCCAG 180 rnC~\Ar~rGA CCAGGCTGTD TCTGTCTCYT TTGGAGACCV ArA~ArrrnCT TCTCTTCMG 240 GATCTGRTAT GCTATTGTAG AD:'AAC~rrr TGCAMrCM GAGMCGCVT GMTGGGACC 360 Tr~rAGAMrr GTCATTTAUT GTACANNCTC TGCTGYRGCT GMCATGGAG A 411 (2) I N FORMAT I ON FOR SEQ I D NO: 65:
( i ) SEQUENCE CHARACTER I ST I CS:
(A) LENGTH: 445 base pairs (B) TYPE: nucleic acid (C) ST~A' _' : doubLe ~D) TOPOLOGY: linear (xi ) SEQUENCE DESCRIPTION: SEQ ID NO:65:

-CA 0220499~ 1997-0~-09 WO 96/16075 PCTrUS95113328 CCTCTCTGCC CTTGTCCTGC TGGCATTCCA GGTCCAGGCT GATCCCATTC Mr'~GCC~-'\ 120 A~ r~\~T MMCTGAGG ArJr,A~ CA~r AGATGAGGAC CAGGATGTGT Ll1-1LIL~.I I 180 TCMr''rCCA GAACCCTCTG CTCTTCMM TTTAGAGATA GGATGGCCAT TMMGCAGTG 240 CCATTGCCGA MGTTCTGCA GACCTTATGA MM_rCCG~r GC~, I Ll, I G I C GTCCAGGTCT 300 ATTTATMAA CGCMMTCT GCTGCATACA ACMTGGACA rC~rrr~,G0A CATMCCACG 360 TGMCTGGGA CCTCACMTC TGTCATTCTT GGGCTTCMC TCGACTGCTT I ILI,I ILICl, 420 ( 2 ) I N FORMAT I ON FOR SEQ I D NO: 66 ( j ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 445 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear -(X; ) SEQUENCE DESCRIPTION: SEQ ID NO:66:
ACACTGGTCT CCAGCTCACC MTCCTCCAG GTGACTTCCA GCCATGMGA L I L I I 1. I LL 1 60 CLILILI6CC Ll lI.rCl,l66 TGGCCTACCA GGTCCAGGCT GATCCCATTC M_rr~ \ 120 A~'~Ar~C'~rT MMCTGAAG AGCAACCATC AGATGAGGAC CAGGATGTGT CTGTCTCCTT 180 TCM''~;CCCA GMGCCTCTG CTCTTCMGA TTTTGAGATA CC~ArrCC4r TGAGGAGGTG 240 TGCTCTGGGA CCTCACMTC TGTCATTATT GTGCTTGACC TCMCTGCTT I IL~ LICC 420 MTMMCTCC TGr~rA-~-4A MMA 445 (2) I N FORMAT I ON FOR SEQ I D NO: 67:
( j ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 445 base pairs (B) TYPE: nucleic acid ~C) STRANDEDNESS: double ~D) TOPOLOGY: linear (X; ) SEQUENCE DESCRIPTION: SEO ID NO:67:

CCTCTCTGCC Ll ILILI,I6C TGGCATTCCA GATCCAGGCT GATCCCATTC Mr~'''C~C'\ 120 Ar,~Ar~r~rT MMCTGAGG A~rA~rC~B~r AGATGAGGAC CAGGATGTGT C I G I L I L~ I 1 180 TGCTCTGGGA CCTCACMTC TGTCATCATT GTGCTTGGCC TCMCTTCTT I IC~I ILILL 420 CA 0220499~ 1997-0~-09 (2) INFORMATION FOR SEQ ID NO:68:

( i ) SEQUENCE CHARACTER I ST I CS:
(A) LENGTH: 2457 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (xi ) SEQUENCE DESCRIPTIOU: SEQ ID NO:68:

AGACATGGGA TAAAr4r~r4 CTGMGAGTG TTCCTGMTG ACATGGCGGC ACTTCTCGAG 420 ACCGGGTAGC AGCTTCTGAG CCTCTCTACA TTGTGGATGT ~ ILl.llil AGGTCAGGTC 480 TCATTGTCTA MMGTMMG CATTGCAGCA TCTCAGACCT Crr4AAr,Arr CCATGGCTTG 540 -TGGLI lCl lC A~4Ar4Arrr ACTACAGCTT CACTCTGACA MTCCTAGAA ACTTGMCTC 660 AATTCACTAG Arrrr4rrAT MMGCCATCA TACCTTATAA TGr~rrCCAAA GGAGGTGATT 720 CTCTMCTTG AAAAr4r4AA MTTGTTTGT TCTATGAGCT CATTMMTTA GGCAGTGTTC 900 AGCTATTTTC TTTCCTGACC ACTGAGAGGT MMTACTCM GCAGATGGGA A~~~~rrr~r 960 GACAGTMMG CCTGTTCATC ATTATCAGTG GGAGTGTGCA TC~~rrr4rr GGTGTCAGTG 1020 AAr4r4r4r~ GCATCAGGM GGMGCCTTG AG~r,Ar4r~rr MCATCMMG GGATCCTGAG 1080 C~r4Ar~\rrT GGGAGCAGTT GCCATCAATG AGTGCCTTCT CTMGTATGG GGCATGTTCT 1140 TTGCCCTATA MTGCAGGCT G~ I C TCCACACACT GGTCTCCAGC TCACCMTCC 1200 TCCAGGTGAC TTCCAGCCAT GMGACTCTT GTCCTCCTCT LIG~ CCTGCTGGCA 1260 TTCCAGGTCC AGGCTGATCC CATTCMGAG r~r4r~ A~ AGACTMMC TC~rr~~r'lr 1320 CCAGCAGATG ArGArC4rrJA T~ rLll.lC TCCTTTGMG G~r~rrl\r4Arr LILIl.l,ILI I 1380 CAAMTTTAG L~G~ GTGCACAGM TGATGGAGGC TTGGAGTCTC CTGATGGAGG 1440 TCCTTTCAGA ALI ILCCTI;I CTTATTCATA GMMTMCAG TC4r~r~~4A GCCATTGGGC 1560 TTGACTTTTT CCTTTTMGA ~ ,IcIA ACMTTTATC TGTGMAMC CTTTMMTA 1620 CGTTTTACCC TMAMTTAG ATTGGTACCC MATGCCAGT GTATGMGGT 1~l l6GI-ILM 1740 C4AAAr4r4A MAMCTGTT AGMTATGGT GTAGATGAM ATTCCTATAT GTGATTMCA 1800 CA 0220499~ 1997-0~-09 WO 96/1607~ PCT/US9S/13328 CCC4r4r4rr AMCCTACTC TCTACCATGC ACAGGACATC TTCATGGGGT AGTTCACTGT 1920 TACACACTAC T6GCLIL~I I ACTTCATGCC TGATGCTTTC I IGI I ll.LIC AGAGATAGGA 1980 TGGCCATTAA AGCAGTGCCA TTGCCGMAG TTCTGCAGAC CTTATGAAM GGCCCArrrr 2040 CrC~rGAr4T MCCACGTGA ACTGGGACCT CACMTCTGT CATTCTTGGG CTTCAACTCG 2160 A~ l l l lc CTTCTCCMT AMCCCCTTG CAGACAAATA ACCTGTTTAT GTTTTTTTGA 2220 AGATCCAMG AGACMGATG [j ~ C ~ GC6CCA GCTGCTTCTG TGTCTATCM ~.11;1 11.1 G I C 2400 2) I N FORMAT I ON FOR SEQ I D NO: 69:
( j ) SEOUENCE CHARACTERI STICS:
(A) LENGTH: 2408 base pairs (B) TrPE: nucleic acid ( C ) STRANDEDNESS: S j ng I e (D) TOPOLOGr: linear (X;) SEQUENCE DESCRIPTION: SEq ID NO:69:
TATTACGMT TCGAGCTCGG TACCGGTATA TCMrqrrGA CCACTGCCAG GACGMMGTG 60 TC~ 666~.~ CGGTTATAAA TTCTGATTAG CCAGGTMCA CAGTGTTATG Ar4rrCCrrr 240 GGMCCGGTG 66~1111116 TGGGGTGMT ATGGCAGTM AGATTTCAGG AGTCCT6AM 300 CArr.r.r4rAr GAAAArrGGT ACAGMCTGC ACCATTCAGC T,GAMrrr,Ar ACGTMCAGC 360 ACCACGGTGG TGGT6MCAC 6~.16~6~,1 CA GAGMTCCGG ATGMGCCTG CTTTTTTATA 420 CTMGTTGGC ATTATAMM AGCATTGCTT ATCMTTTGT TErMCrMC AGGTCACTAT 480 CAGTCAAAAT MMATCATTA TTTGATTTCA Al l I IGILI~C ACI~CLI6~ TCTGTCATCA 540 CGATACTGTG ATGCCATGGT GTCCGACTTA Tr~rrCG~CAA GATGTTGAGC MMCTTATCG 600 ICT66blC6A CTCTAGGCCT CACTGGCCTA ATACGACTCA CTATAGGGAG CTCGAGGATC 720 TE'~Ar~r AGGCTCCMC TTCTMCCAC MAMCTATT TTTTCCATGC GCTCCTTAM 840 TTAGGCAGCG CCCAGCTATT I ICI ~ ILLIG ACCACTGAGA GGTMMTACT CMGCAGATG 900 Cr ~ r4rrE GMGATAGCA A66CLICI IC ATCATTATCA CTGGGTGTGT GCGTGAGGGG 960 AGGGGTGTCA TTGCATACAC ArrrrAAr4T CAGGATGGM GCCTTGAGGA C4ÇACEMr4 1020 GTGTGGGGCC 1 1 I L ~ cC ACATAAATGC A61.~ 16~ 1 C L I L I C I ~CAC ACACTGGTCT 1140 Cl IGTLLIGG TGGCCTACCA GGTCCAGGCT GATCCCATTC Mrrrrr4r4 AGMr~C4rT 1260 -CA 0220499~ 1997-0~-09 MMCTGMG AGCMCCATC AGATGAGGAC CAGGATGTGT 1.; I G I G I G~. I I TCMrrrcr~ 1320 GTAGCTGTCT CTTGGTCCCT TTACATTCCT TGIGI II;I IC ATAGMGTM CGr~r~r.~r~ 1500 TATMGMGA MTATTCGTT CTACTTTMM MTTAGATTT rrr'~rCCMA TGCCAGTGTA 1680 TGAAGCTGTT GGGTMGGAA AAArr~AAAA TGGTGATAGA ATGTTGTGTA GATGACMTT 1740 GTGCTGACTG TGATGTCACC CAr~r~5rAA ACCTACTCTC TACCATGCAC AGGACATCTT 1860 GTTTCCTCAG AGATAGGMG GCCAGTGAGG AG~ .CI,I~I I GC~r~\rCMA CTGCGGACCT 1980 TTATTGTGCT TGACCTCMC TC~,I I I IGLI TCTCCMTM ACTCCTGGCA GACMMTAAT 2160 ~2) INFORMATION FOR SEQ ID NO:70:
j ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2551 base pairs (a) TYPE: r~cleic acid ( C ) STRANDEDNESS: S; ng I e (D) TOPOLOGY: linear (X; ) SEQUENCE DESCRIPTION: SEQ ID NO:70:

CTTCTTGACT GATAGATCTA ACCCTACAGA GAGATTCMG IGClLl IGTc CCATTGMCA 120 TCTCAGACCT rrr~AAr~rC CCATGGCTTG AG6~, I GCCGC AGGTGMGAG CCACCTGGAG 660 CA 0220499~ l997-0~-09 WO 96/16075 PCTrUS95/13328 CTCACTCTTG GCAGATGTGT TCCATGACTT TGI~LI ICI IC Ar~ArC4rCC ACTACAGCTT 720 CACTCTGACA MTCTTAGM ACTTGMCTC MTTCACTGG ACCr~Ar~AT MMGCCATCT 780 TACTTTCTCT MMATGGCCC rAAArr~rrr GATTCACMM GTTTGCCTTG ATGAGGACCA 840 TTGCTMTAC CCCMMCTT Gr~AAAAAAA TTGAGTGTCC AGTCMCCTG GTCMGGACT 900 GGTCCTGGAT CCACAGTTTC TC~r'\AAAr'~ AGGCTCCMC TTCMMCAC MMCCACTCC 960 GGTMMTACT CMGCAGATG ,rr~AAr~rr~ C~rr~rA~rA MGCCTGTTC ATCATTATCA 1080 GTGGGAGTGT GCGTGAGGGG AGGGGTGTCA GTGMCACAC AGAGCATCAG GMrGMr~'r 1140 TTGAGGACAG AGGMCATCA MGGGATCCT r~rr~r~Ar~ GCTGGGAGCA GTTGGCATCA 1200 CTGAGTGCCG TCTCTMGTG TGGG6CLI I I CTCTGCCACA TMMTGCAGG ~ J6Cl~Llc 1260 I IGTCLI~CI CTCTGCCCTT l;l~LII~LI~.G CATTCCAGAT CCAGGCTGAT CCCATTCMG 1380 Arrr/~r~Ar~ AGAGACTMM ACTGAGGAGC Ar~rl~rrA~ TC~rr~rr4r GATGTGTCTG 1440 TCTCCTTTGA Ar~CC~rAC~A CCLI~ LIC TTCMMTTT AGGTGCGTGC TTGTGCACAG 1500 CTGATTGATA ATTTTGTTAT Arr~AA~AAT MCTGTTCTA CTTTMAMT TAGATTTGGT 1800 ACCTMATGC CAGTGTATTA AGGTGTTGGG Tr.~rr,AAAAr ACMTMTGC TGATAGMTG 1860 GTTTGGGGTT GATCATGGTG CTGGCTGTGA TGTCACCCAC Ar~rr,AAArr TACTTTCTAC 1980 CATGCACAGG ACATCTTCAT~ AGGGTAGTTC ACTGTCACAC ACTGCTGGCC TCCTTACTTC 2040 ATGCCTGATG ~ TCCTCAGAGA TCAGATGGCC ATGGAAGAGG TGCCATTGCA 2100 AACACMMTT CTGCTGCCTA GMMCATGGC CCCr~ArGAT GAMTAACCA CGTGCTCTGG 2220 GACCTCACAA TCTGTCATCA I IGII L~ 6 CCTCMCTTC ~ LLI IGI CCAATMMCT 2280 ATTTCMCAG TCATATCMG GATGAGCAGG AGGTTAGATC CAAAr'\r'~ AGATGCTCTG 2460

Claims (14)

WE CLAIM:

1. A substantially purified nucleic acid molecule encoding a cryptdin.

2. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule is a cryptdin gene or a portion thereof.

3. A substantially purified nucleic acid molecule, comprising a gene selected from the group consisting of the mouse cryptdins 1, 2, 3, 5, 6 and i genes as shown in Figure 11 (SEQ ID NOS: 58-63).

4. A substantially purified nucleic acid molecule, comprising a gene selected from the group consisting of the rat cryptdins 1, 2 and 3 genes as shown in Figures 15.A. to 15.C. (SEQ ID NOS: 68-70).

5. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule is a cryptdin cDNA
sequence or a portion thereof.

6. A substantially purified nucleic acid molecule, comprising a cDNA sequence selected from the group consisting of the mouse cryptdins 1-17 cDNA
sequences as shown in Figure 10 (SEQ ID NOS: 40-56).

7. A substantially purified nucleic acid molecule, comprising a cDNA sequence selected from the group consisting of the mouse cryptdins 2-17 cDNA
sequences as shown in Figure 10 (SEQ ID NOS: 41-56).

8. A substantially purified nucleic acid molecule, comprising a cDNA sequence selected from the group consisting of the rat cryptdins 1, 2 and 3 cDNA
sequences as shown in Figures 14.A. to 14.C. (SEQ ID
NOS: 65-67).

9. A nucleotide sequence that can hybridize under relatively stringent conditions to a nucleic acid molecule encoding a cryptdin.

10. The nucleotide sequence of claim 9, comprising a portion of a nucleic acid molecule selected from the group consisting of the mouse cryptdins 2-17 cDNA sequences as shown in Figure 10 (SEQ ID NOS: 40-56);
the mouse cryptdins 1, 2, 3, 5, 6 and i genes as shown in Figure 11 (SEQ ID NOS: 58-63); the rat cryptdins 1, 2 and 3 genes as shown in Figures 15.A. to 15.C. (SEQ ID NOS:
68-70) and the rat cryptdins 1, 2 and 3 CDNA sequences as shown in Figures 14 .A. to 14.C. ( SEQ ID NOS: 65-67).

11. A method of detecting the presence of a nucleic acid molecule encoding a cryptdin in a biological sample, comprising the steps of:

a. contacting the biological sample with the nucleotide sequence of claim 9 under relatively stringent hybridization conditions;
and b. detecting hybridization of said nucleotide sequence to a nucleic acid molecule present in said sample, wherein said hybridization indicates the presence of a nucleic acid molecule encoding a cryptdin.

12. A substantially purified cryptdin peptide of enteric origin having an amino acid sequence selected from the group consisting of:

13. A method for treating inflammation of the intestine in a subject, comprising topical admininstration of a cryptdin peptide to the subject.

14. A substantially purified mouse cryptdin peptide of enteric origin having an amino acid sequence as follows:

C-Y-C-R-X3-C-K-X4-E-X5-G-T-C-X6-C-C-X, wherein X3 is 3 amino acids, preferably KGH or *RG, where * is S, T, K, I or A;
X4 is 2 amino acids, preferably GR, RR or RG;
X5 is 3 amino acids, preferably RMN, RVR, RVF
HMN or HIN;
X6 is 6 to 9 amino acids, preferably GIRFLY
(SEQ ID NO: 2), RRGHLMYTL (SEQ ID NO: 4) or RNLFLTFVF
(SEQ ID NO: 3) or RKGHL*YT* (SEQ ID NO: 5), where *
independently is L or M; and X7 is 0 to 3 amino acids, preferably R, S or PRR.