WO2000029435A1

WO2000029435A1 - 12 human secreted proteins

Info

Publication number: WO2000029435A1
Application number: PCT/US1999/025031
Authority: WO
Inventors: Jian Ni; Steven M. Ruben; Henrik S. Olsen; Paul E. Young; Joseph J. Kenny; Paul A. Moore; Ying-Fei Wei; John M. Greene
Original assignee: Human Genome Sciences, Inc.
Priority date: 1998-10-28
Filing date: 1999-10-27
Publication date: 2000-05-25
Also published as: CA2348824A1; EP1124850A1; EP1124850A4; AU1231400A; JP2002530062A

Abstract

The present invention relates to 12 novel human secreted proteins and isolated nucleic acids containing the coding regions of the genes encoding such proteins. Also provided are vectors, host cells, antibodies, and recombinant methods for producing human secreted proteins. The invention further relates to diagnostic and therapeutic methods useful for diagnosing and treating disorders related to these novel human secreted proteins.

Description

12 Human Secreted Proteins

Field of the Invention

This invention relates to newly identified polynucleotides and the polypeptides encoded by these polynucleotides, uses of such polynucleotides and polypeptides, and their production.

Background of the Invention

Unlike bacterium, which exist as a single compartment surrounded by a membrane, human cells and other eucaryotes are subdivided by membranes into many functionally distinct compartments. Each membrane-bounded compartment, or organelle, contains different proteins essential for the function of the organelle. The cell uses "sorting signals," which are amino acid motifs located within the protein, to target proteins to particular cellular organelles.

One type of sorting signal, called a signal sequence, a signal peptide, or a leader sequence, directs a class of proteins to an organelle called the endoplasmic reticulum (ER). The ER separates the membrane-bounded proteins from all other types of proteins. Once localized to the ER, both groups of proteins can be further directed to another organelle called the Golgi apparatus. Here, the Golgi distributes the proteins to vesicles, including secretory vesicles, the cell membrane, lysosomes, and the other organelles. Proteins targeted to the ER by a signal sequence can be released into the extracellular space as a secreted protein. For example, vesicles containing secreted proteins can fuse with the cell membrane and release their contents into the extracellular space - a process called exocytosis. Exocytosis can occur constitutively or after receipt of a triggering signal. In the latter case, the proteins are stored in secretory vesicles (or secretory granules) until exocytosis is triggered. Similarly, proteins residing on the cell membrane can also be secreted into the extracellular space by proteolytic cleavage of a "linker" holding the protein to the membrane.

Despite the great progress made in recent years, only a small number of genes encoding human secreted proteins have been identified. These secreted proteins include the commercially valuable human insulin, interferon, Factor VIII, human growth hormone, tissue plasminogen activator, and erythropoeitin. Thus, in light of the pervasive role of secreted proteins in human physiology, a need exists for identifying and characterizing novel human secreted proteins and the genes that encode them. This knowledge will allow one to detect, to treat, and to prevent medical disorders by using secreted proteins or the genes that encode them.

Summary of the Invention

The present invention relates to novel polynucleotides and the encoded polypeptides. Moreover, the present invention relates to vectors, host cells, antibodies, and recombinant and synthetic methods for producing the polypeptides and polynucleotides. Also provided are diagnostic methods for detecting disorders and conditions related to the polypeptides and polynucleotides, and therapeutic methods for treating such disorders and conditions. The invention further relates to screening methods for identifying binding partners of the polypeptides. Detailed Description Definitions

The following definitions are provided to facilitate understanding of certain terms used throughout this specification.

In the present invention, "isolated" refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered "by the hand of man" from its natural state. For example, an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be "isolated" because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide. The term "isolated" does not refer to genomic or cDNA libraries, whole cell total or mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots), sheared whole cell genomic DNA preparations or other compositions where the art demonstrates no distinguishing features of the polynucleotide/sequences of the present invention.

In the present invention, a "secreted" protein refers to those proteins capable of being directed to the ER, secretory vesicles, or the extracellular space as a result of a signal sequence, as well as those proteins released into the extracellular space without necessarily containing a signal sequence. If the secreted protein is released into the extracellular space, the secreted protein can undergo extracellular processing to produce a "mature" protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.

In specific embodiments, the polynucleotides of the invention are at least 15, at least 30, at least 50, at least 100, at least 125, at least 500, or at least 1000 continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides of the invention comprise a portion of the coding sequences, as disclosed herein, but do not comprise all or a portion of any intron. In another embodiment, the polynucleotides comprising coding sequences do not contain coding sequences of a genomic flanking gene (i.e., 5' or 3' to the gene of interest in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).

As used herein, a "polynucleotide" refers to a molecule having a nucleic acid sequence contained in SEQ ID NO:X or the cDNA contained within the clone deposited with the ATCC. For example, the polynucleotide can contain the nucleotide sequence of the full length cDNA sequence, including the 5' and 3' untranslated sequences, the coding region, with or without the signal sequence, the secreted protein coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence. Moreover, as used herein, a "polypeptide" refers to a molecule having the translated amino acid sequence generated from the polynucleotide as broadly defined.

In the present invention, the full length sequence identified as SEQ ID NO:X was often generated by overlapping sequences contained in multiple clones (contig analysis). A representative clone containing all or most of the sequence for SEQ ID NO:X was deposited with the American Type Culture Collection ("ATCC"). As shown in Table XIII, each clone is identified by a cDNA Clone ID (Identifier) and the ATCC Deposit Number. The ATCC is located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA. The ATCC deposit was made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure.

A "polynucleotide" of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NO:X, the complement thereof, or the cDNA within the clone deposited with the ATCC. "Stringent hybridization conditions" refers to an overnight incubation at 42 degree C in a solution comprising 50% formamide, 5x SSC (750 mM NaCI, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in OJx SSC at about 65 degree C.

Also contemplated are nucleic acid molecules that hybridize to the polynucleotides of the present invention at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, lower stringency conditions include an overnight incubation at 37 degree C in a solution comprising 6X SSPE (20X SSPE = 3M NaCI; 0.2M NaH₂PO₄; 0.02M EDTA, pH 74), 0.5% SDS, 30% formamide, 100 ug/ml salmon sperm blocking DNA; followed by washes at 50 degree C with IXSSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5X SSC).

Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility. Of course, a polynucleotide which hybridizes only to polyA+ sequences (such as any 3' terminal polyA+ tract of a cDNA shown in the sequence listing), or to a complementary stretch of T (or U) residues, would not be included in the definition of "polynucleotide," since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone generated using oligo dT as a primer). The polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that may be single- stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms. The polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications Polypeptides may be branched , for example, as a result of ubiquitmation, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-πbosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide oi nucleotide derivative, covalent attachment of a hpid oi hpid derivative, covalent attachment of phosphotidyhnositol, cross-linking, cychzation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchoi formation, hydroxylation, lodmation, methylation, myπstoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, lacemization, selenoylation, sulfation, transfei- RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitmation (See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983), Seifter et al , Meth Enzymol 182:626-646 (1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992) ) "SEQ ID NO:X" refers to a polynucleotide sequence while "SEQ ID NO:Y" refers to a polypeptide sequence, both sequences identified by an integer specified in Table XIII

"A polypeptide having biological activity" refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose- dependence a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25- fold less and, preferably, not more than about tenfold less activity, and most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention.)

Polynucleotides and Polypeptides of the Invention

FEATURES OF PROTEIN ENCODED BY GENE NO: 1

The translation product of this gene shares sequence homology with a protein from Xenopus laevis that is descnbed as upregulated in response to thyroid hormone in tadpoles, and is thought to be important in the tail resorption process during Xenopus laevis metamorphosis (See Proc Natl. Acad. Sci. USA (1996 Mar. 5):93(5): 1924-9, which is herein incorporated by reference). In addition, translation product of this gene shares sequence homology with a recently descπbed group of proteins, called hedgehog interacting proteins (HIPs) (See International Publication No. WO98/12326, which is herein incorporated by reference). These proteins bind to hedgehog polypeptides such as Shh and Dhh with high affinity (Kd approx. 1 nM). HIPs exhibit spatiallyand temporally restricted expression domains indicative of important roles hedgehog-mediated induction. They regulate differentiation of neuronal cells, regulate survival of differentiated neuronal cells, proliferation of chondrocytes, proliferation of testicular germ line cells and/or expression of patched or hedgehog genes. The biological activity of this polypeptide is assayed by techniques known in the art, otherwise disclosed herein and as descπbed International Publication No. WO98/12326, which is herein incorporated by reference.

Preferred polypeptides of the invention comprise the following amino acid sequence: MLRTSTPNLCGGLHCRAPWLSSGILCLCLIFLLGQVGLLQGHPQCLDYGPPFQPP LHLEFCSDYESFGCCDQHKDRRIAARYWDIMEYFDLKRHELCGDYIKDILCQEC SPYAAHLYDAENTQTPLRNLPGLCSDYCSAFHSNCHSAISLLTNDRGLQESHGRD GTRFCHLLDLPDKDYCFPNVLRNDYLNRHLGMVAQDPQGCLQLCLSEVANGLR NPVSMVHAGDGTHRFFVAEQVGVVWVYLPDGSRLEQPFLDLKNIVLTTPWIGD ERGFLGLAFHPKFRHNRKFYIYYSCLDKKKVEKIRISEMKVSRADPNKADLKSER VILEIEEPASNHNGGQLLFGLDGYMYIFTGDGGQAGDPFGLFGNAQNKSSLLGK VLRIDVNRAGSHGKRYRVPSDNPFVSEPGAHPAIYAYGIRNMWRCAVDRGDPIT RQGRGRIFCGDVGQNRFEEVDLILKGGNYGWRAKEGFACYDKKLCHNASLDDV LPIYA YGHA VGKS VTGGYVYRGCESPNLNGLYIFGDFMSGRLMALQEDRKNKK WKKQDLCLGSTTSCAFPGLISTHSKFIISFAEDEAGELYFLATSYPSAYAPRGSIYK FVDPSRRAPPGKCKYKPVPVRTKSKRIPFRPLAKTVLDLLKEQSEKAARKSSSAT LASGPAQGLSEKGSSKKLASPTSSKNTLRGPGTKKKARVGPHVRQGKRRKSLKS HSGRMRPSAEQKRAGRSLP (SEQ ID NO: 47). Also preferred are polypeptides comprising the mature polypeptide which is predicted to consist of residues 42-724 of the foregoing sequence, and biologically active fragments of the mature polypeptide.

Figures 1A-C show the nucleotide (SEQ ID NO: 11) and deduced amino acid sequence (SEQ ID NO:29) of this protein.

Figure 2 shows the regions of similarity between the amino acid sequences of SEQ ID NO:29, the Xenopus laevis tail resorption protein (gi|1234787) (SEQ ID NO:48), and the Hedgehog Interacting Protein ("HIP"; gi|AAD31172.1) (SEQ ID NO:49).

Figure 3 shows an analysis of the amino acid sequence of SEQ ID NO: 29. Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.

Northern analysis indicates that a 2.5-3.0 kb transcript of this gene is expressed primarily in testes tissue and A549 lung carcinoma tissue, but interestingly is absent from normal lung tissue. This gene is also expressed in osteoarthritis tissue and human fetal tissues.

The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the polypeptide having the amino acid sequence shown in Figures 1A-C (SEQ ID NO:29), which was determined by sequencing a cloned cDNA. The nucleotide sequence shown in Figures 1A-C (SEQ ID NOJ 1) was obtained by sequencing a cloned cDNA, which was deposited on Nov. 17, 1998 at the American Type Culture Collection, and given Accession Number 203484. The deposited gene is inserted in the pSport plasmid (Life Technologies, Rockville, MD) using the Sall/Notl restriction endonuclease cleavage sites.

The present invention is further directed to fragments of the isolated nucleic acid molecules described herein. By a fragment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ ID NOJ 1 is intended DNA fragments at least about 15nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments 50-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in SEQ ID NOJ 1. By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NOJ 1. In this context "about" includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Representative examples of polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, and from about 501 to about 550, and from about 551 to about 570 of SEQ ID NO 11, or the complementary strand thereto, or the cDNA contained in the deposited gene. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, oi 1) nucleotides, at either terminus or at both termini In additional embodiments, the polynucleotides of the invention encode functional attπbutes of the conesponding piotein

Preferred embodiments of the invention in this regard include fragments that compπse alpha-helix and alpha-helix forming legions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions"), turn and turn-forming regions ("turn- regions"), coil and coil-forming regions ("coil-iegions"), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions The data representing the structural or functional attπbutes of the protein set forth Figure 3 and/oi Table I, as descnbed above, was generated using the various modules and algoπthms of the

DNA ""STAR set on default parameters. In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table I can be used to determine regions of the protein which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur m the process of initiation of an immune response.

Certain preferred regions in these regards are set out in Figure 3, but may, as shown Table I, be represented or identified by using tabular representations of the data presented in Figure 3. The DNA*STAR computer algoπthm used to generate Figure 3 (set on the oπginal default parameters) was used to present the data in Figure 3 in a tabular format (See Table I). The tabular format of the data m Figure 3 is used to easily determine specific boundaries of a preferred region. The above-mentioned preferred regions set out m Figure 3 and in Table I include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figures 1A-C (SEQ ID NO:29). As set out in Figure 3 and in Table I, such preferred regions include Garni er-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-iegions, beta-iegions, and turn-iegions, Kyte-Doohttle hydiophi c regions and Hopp-Woods hydiophobic regions, Eisenbeig alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenic index and Emini surface-forming legions. Even if deletion of one or more amino acids from the N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g , biological activities, ability to multimeπze, etc.) may still be retained Foi example, the ability of shortened muteins to induce and oi bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be leta ed when less than the majoπty of the residues of the complete or mature polypeptide are removed from the N-termmus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods descπbed herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence shown in Figures 1A-C, up to the alanine residue at position number 524 and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides compnsing the amino acid sequence of residues n 1-524 of Figures 1A-C, where nl is an integer from 1 to 524 corresponding to the position of the amino acid residue in Figures 1A-C (which is identical to the sequence shown as SEQ ID NO:29). N-terminal deletions of the polypeptide of the invention shown as SEQ ID NO:29 include polypeptides comprising the amino acid sequence of residues: V-2 to P-529; A-3 to P-529; Q-4 to P- 529; D-5 to P-529; P-6 to P-529; Q-7 to P-529; G-8 to P-529; C-9 to P-529; L-10 to P- 529; Q-l l to P-529; L-12 to P-529; C-13 to P-529; L-14 to P-529; S-15 to P-529; E-16 to P-529; V-17 to P-529; A-18 to P-529; N-19 to P-529; G-20 to P-529; L-21 to P-529; R- 22 to P-529; N-23 to P-529; P-24 to P-529; V-25 to P-529; S-26 to P-529; M-27 to P- 529; V-28 to P-529; H-29 to P-529; A-30 to P-529; G-31 to P-529; D-32 to P-529; G-33 to P-529; T-34 to P-529; H-35 to P-529; R-36 to P-529; F-37 to P-529; F-38 to P-529; V- 39 to P-529; A-40 to P-529; E-41 to P-529; Q-42 to P-529; V-43 to P-529; G-44 to P- 529; V-45 to P-529; V-46 to P-529; W-47 to P-529; V-48 to P-529; Y-49 to P-529; L-50 to P-529; P-51 to P-529; D-52 to P-529; G-53 to P-529; S-54 to P-529; R-55 to P-529; L- 56 to P-529; E-57 to P-529; Q-58 to P-529; P-59 to P-529; F-60 to P-529; L-61 to P-529; D-62 to P-529; L-63 to P-529; K-64 to P-529; N-65 to P-529; 1-66 to P-529; V-67 to P- 529; L-68 to P-529; T-69 to P-529; T-70 to P-529; P-71 to P-529; W-72 to P-529; 1-73 to P-529; G-74 to P-529; D-75 to P-529; E-76 to P-529; R-77 to P-529; G-78 to P-529; F- 79 to P-529; L-80 to P-529; G-81 to P-529; L-82 to P-529; A-83 to P-529; F-84 to P-529; H-85 to P-529; P-86 to P-529; K-87 to P-529; F-88 to P-529; R-89 to P-529; H-90 to P- 529; N-91 to P-529; R-92 to P-529; K-93 to P-529; F-94 to P-529; Y-95 to P-529; 1-96 to P-529; Y-97 to P-529; Y-98 to P-529; S-99 to P-529; C-100 to P-529; L-101 to P-529; D-102 to P-529; K-103 to P-529; K-104 to P-529; K-105 to P-529; V-106 to P-529; E- 107 to P-529; K-108 to P-529; 1-109 to P-529; R-l 10 to P-529; 1-111 to P-529; S-112 to P-529; E-113 to P-529; M-114 to P-529; K-115 to P-529; V-116 to P-529; S-117 to P- 529; R-l 18 to P-529; A-l 19 to P-529; D-120 to P-529; P-121 to P-529; N-122 to P-529; K-123 to P-529; A- 124 to P-529; D-125 to P-529; L-126 to P-529; K-127 to P-529; S-

128 to P-529; E-129 to P-529; R-130 to P-529; V-131 to P-529; 1-132 to P-529; L-133 to P-529; E-134 to P-529; 1-135 to P-529; E-136 to P-529; E-137 to P-529; P-138 to P-529; A-139 to P-529; S-140 to P-529; N-141 to P-529; H-142 to P-529; N-143 to P-529; G- 144 to P-529; G-145 to P-529; Q-146 to P-529; L-147 to P-529; L-148 to P-529; F-149 to P-529; G-150 to P-529; L-151 to P-529; D-152 to P-529; G-153 to P-529; Y-154 to P- 529; M-155 to P-529; Y-156 to P-529; 1-157 to P-529; F-158 to P-529; T-159 to P-529; G-160 to P-529; D-161 to P-529; G-162 to P-529; G-163 to P-529; Q-164 to P-529; A- 165 to P-529; G-166 to P-529; D-167 to P-529; P-168 to P-529; F-169 to P-529; G-170 to P-529; L-171 to P-529; F-172 to P-529; G-173 to P-529; N-174 to P-529; A-175 to P- 529; Q-176 to P-529; N-177 to P-529; K-178 to P-529; S-179 to P-529; S-180 to P-529; L-181 to P-529; L-182 to P-529; G-183 to P-529: K-184 to P-529; V-185 to P-529; L- 186 to P-529; R-l 87 to P-529; 1-188 to P-529; D-189 to P-529; V-190 to P-529; N-191 to P-529; R-192 to P-529; A-193 to P-529; G-194 to P-529; S-195 to P-529; H-196 to P- 529; G-197 to P-529; K-198 to P-529; R-199 to P-529; Y-200 to P-529; R-201 to P-529; V-202 to P-529; P-203 to P-529; S-204 to P-529; D-205 to P-529; N-206 to P-529; P-207 to P-529; F-208 to P-529; V-209 to P-529; S-210 to P-529; E-211 to P-529; P-212 to P- 529; G-213 to P-529; A-214 to P-529; H-215 to P-529; P-216 to P-529; A-217 to P-529; 1-218 to P-529; Y-219 to P-529; A-220 to P-529; Y-221 to P-529; G-222 to P-529; 1-223 to P-529; R-224 to P-529; N-225 to P-529; M-226 to P-529; W-227 to P-529; R-228 to P-529; C-229 to P-529; A-230 to P-529; V-231 to P-529; D-232 to P-529; R-233 to P- 529; G-234 to P-529; D-235 to P-529; P-236 to P-529; 1-237 to P-529; T-238 to P-529; R-239 to P-529; Q-240 to P-529; G-241 to P-529; R-242 to P-529; G-243 to P-529; R- 244 to P-529; 1-245 to P-529; F-246 to P-529; C-247 to P-529; G-248 to P-529; D-249 to P-529; V-250 to P-529; G-251 to P-529; Q-252 to P-529; N-253 to P-529; R-254 to P- 529; F-255 to P-529; E-256 to P-529; E-257 to P-529; V-258 to P-529; D-259 to P-529; L-260 to P-529; 1-261 to P-529; L-262 to P-529; K-263 to P-529; G-264 to P-529; G-265 to P-529; N-266 to P-529; Y-267 to P-529; G-268 to P-529; W-269 to P-529; R-270 to P- 529; A-271 to P-529; K-272 to P-529; E-273 to P-529; G-274 to P-529; F-275 to P-529; A-276 to P-529; C-277 to P-529; Y-278 to P-529; D-279 to P-529; K-280 to P-529; K- 281 to P-529; L-282 to P-529; C-283 to P-529; H-284 to P-529; N-285 to P-529; A-286 to P-529; S-287 to P-529; L-288 to P-529; D-289 to P-529; D-290 to P-529; V-291 to P- 529; L-292 to P-529; P-293 to P-529; 1-294 to P-529; Y-295 to P-529; A-296 to P-529; Y-297 to P-529; G-298 to P-529; H-299 to P-529; A-300 to P-529; V-301 to P-529; G- 302 to P-529; K-303 to P-529; S-304 to P-529; V-305 to P-529; T-306 to P-529; G-307 to P-529; G-308 to P-529; Y-309 to P-529; V-310 to P-529; Y-311 to P-529; R-312 to P- 529; G-313 to P-529; C-314 to P-529; E-315 to P-529; S-316 to P-529; P-317 to P-529; N-318 to P-529; L-319 to P-529; N-320 to P-529; G-321 to P-529; L-322 to P-529; Y- 323 to P-529; 1-324 to P-529; F-325 to P-529; G-326 to P-529; D-327 to P-529; F-328 to P-529; M-329 to P-529; S-330 to P-529; G-331 to P-529; R-332 to P-529; L-333 to P- 529; M-334 to P-529; A-335 to P-529; L-336 to P-529; Q-337 to P-529; E-338 to P-529; D-339 to P-529; R-340 to P-529; K-341 to P-529; N-342 to P-529; K-343 to P-529; K- 344 to P-529; W-345 to P-529; K-346 to P-529; K-347 to P-529; Q-348 to P-529; D-349 to P-529; L-350 to P-529; C-351 to P-529; L-352 to P-529; G-353 to P-529; S-354 to P- 529; T-355 to P-529; T-356 to P-529; S-357 to P-529; C-358 to P-529; A-359 to P-529; F-360 to P-529; P-361 to P-529; G-362 to P-529; L-363 to P-529; 1-364 to P-529; S-365 to P-529; T-366 to P-529; H-367 to P-529; S-368 to P-529; K-369 to P-529; F-370 to P- 529; 1-371 to P-529; 1-372 to P-529; S-373 to P-529; F-374 to P-529; A-375 to P-529; E- 376 to P-529; D-377 to P-529; E-378 to P-529; A-379 to P-529; G-380 to P-529; E-381 to P-529; L-382 to P-529; Y-383 to P-529; F-384 to P-529; L-385 to P-529; A-386 to P- 529; T-387 to P-529; S-388 to P-529; Y-389 to P-529; P-390 to P-529; S-391 to P-529; A-392 to P-529; Y-393 to P-529; A-394 to P-529; P-395 to P-529; R-396 to P-529; G- 397 to P-529; S-398 to P-529; 1-399 to P-529; Y-400 to P-529; K-401 to P-529; F-402 to P-529; V-403 to P-529; D-404 to P-529; P-405 to P-529; S-406 to P-529; R-407 to P- 529; R-408 to P-529; A-409 to P-529; P-410 to P-529; P-411 to P-529; G-412 to P-529; K-413 to P-529; C-414 to P-529; K-415 to P-529; Y-416 to P-529; K-417 to P-529; P- 418 to P-529; V-419 to P-529; P-420 to P-529; V-421 to P-529; R-422 to P-529; T-423 to P-529; K-424 to P-529; S-425 to P-529; K-426 to P-529; R-427 to P-529; 1-428 to P- 529; P-429 to P-529; F-430 to P-529; R-431 to P-529; P-432 to P-529; L-433 to P-529; A-434 to P-529; K-435 to P-529; T-436 to P-529; V-437 to P-529; L-438 to P-529; D- 439 to P-529; L-440 to P-529; L-441 to P-529; K-442 to P-529; E-443 to P-529; Q-444 to P-529; S-445 to P-529; E-446 to P-529; K-447 to P-529; A-448 to P-529; A-449 to P- 529; R-450 to P-529; K-4 1 to P-529; S-452 to P-529; S-453 to P-529; S-454 to P-529; A-455 to P-529; T-456 to P-529; L-457 to P-529; A-458 to P-529; S-459 to P-529; G- 460 to P-529; P-461 to P-529; A-462 to P-529; Q-463 to P-529; G-464 to P-529; L-465 to P-529; S-466 to P-529; E-467 to P-529; K-468 to P-529; G-469 to P-529; S-470 to P- 529; S-471 to P-529; K-472 to P-529; K-473 to P-529; L-474 to P-529; A-475 to P-529: S-476 to P-529; P-477 to P-529; T-478 to P-529; S-479 to P-529; S-480 to P-529; K-481 to P-529; N-482 to P-529; T-483 to P-529; L-484 to P-529; R-485 to P-529; G-486 to P- 529; P-487 to P-529; G-488 to P-529; T-489 to P-529; K-490 to P-529; K-491 to P-529; K-492 to P-529; A-493 to P-529; R-494 to P-529; V-495 to P-529; G-496 to P-529; P- 497 to P-529; H-498 to P-529; V-499 to P-529; R-500 to P-529; Q-501 to P-529; G-502 to P-529; K-503 to P-529; R-504 to P-529; R-505 to P-529; K-506 to P-529; S-507 to P- 529; L-508 to P-529; K-509 to P-529; S-510 to P-529; H-511 to P-529; S-512 to P-529; G-513 to P-529; R-514 to P-529; M-515 to P-529; R-516 to P-529; P-517 to P-529; S- 518 to P-529; A-519 to P-529; E-520 to P-529; Q-521 to P-529; K-522 to P-529; R-523 to P-529; A-524 to P-529; of SEQ ID NO:29. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification or loss of one or more biological functions of the protein, other functional activities (e.g., biological activities (e.g., ability to illicit mitogenic activity, induce differentiation of normal or malignant cells, bind to EGF receptors, etc.)), may still be retained. For example the ability to induce and or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majoπty of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods descnbed herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted C-terminal amino acid residues may retain some biological oi immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the polypeptide shown in Figures 1A-C. up to the glutamine lesidue at position number 7, and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides compπsing the amino acid sequence of residues 1-ml of Figures 1 A-C, where ml is an integer from 7 to 528 corresponding to the position of the amino acid residue in Figures lA-C. Moreover, the invention provides polynucleotides encoding polypeptides compπsing, or alternatively consisting of, the ammo acid sequence of C-terminal deletions of the polypeptide of the invention shown as SEQ ID NO:29 include polypeptides compπsing the ammo acid sequence of residues: M-1 to L- 528; M-1 to S-527; M-1 to R-526; M-1 to G-525; M-1 to A-524; M-1 to R-523; M-1 to K-522; M-1 to Q-521; M-1 to E-520; M-1 to A-519; M-1 to S-518; M-1 to P-517; M-1 to R-516; M-1 to M-515; M-1 to R-514; M-1 to G-513; M-1 to S-512; M-1 to H-511; M-1 to S-510; M-1 to K-509; M-1 to L-508; M-1 to S-507; M-1 to K-506; M-1 to R-505; M-1 to R-504; M-1 to K-503; M-1 to G-502; M-1 to Q-501; M-1 to R-500; M-1 to V-499; M- 1 to H-498; M-1 to P-497; M-1 to G-496; M-1 to V-495; M-1 to R-494; M-1 to A-493; M-1 to K-492; M-1 to K-491; M-1 to K-490; M-1 to T-489; M-1 to G-488; M-1 to P- 487; M-1 to G-486; M-1 to R-485; M-1 to L-484; M-1 to T-483; M-1 to N-482; M-1 to K-481; M-1 to S-480; M-1 to S-479; M-1 to T-478; M-1 to P-477; M-1 to S-476; M-1 to A-475; M-1 to L-474; M-1 to K-473; M-1 to K-472; M-1 to S-471; M-1 to S-470; M-1 to G-469; M-1 to K-468; M-1 to E-467; M-1 to S-466; M-1 to L-465; M-1 to G-464; M-1 to Q-463; M-1 to A-462; M-1 to P-461; M-1 to G-460; M-1 to S-459; M-1 to A-458; M- 1 to L-457; M-1 to T-456; M-1 to A-455; M-1 to S-454; M-1 to S-453; M-1 to S-452; M- 1 to K-451; M-1 to R-450; M-1 to A-449; M-1 to A-448; M-1 to K-447; M-1 to E-446; M-1 to S-445; M-1 to Q-444; M-1 to E-443; M-1 to K-442; M-1 to L-441; M-1 to L-440; M-1 to D-439; M-1 to L-438; M-1 to V-437; M-1 to T-436; M-1 to K-435; M-1 to A- 434; M-1 to L-433; M-1 to P-432; M-1 to R-431; M-1 to F-430; M-1 to P-429; M-1 to I- 428; M-1 to R-427; M-1 to K-426; M-1 to S-425; M-1 to K-424; M-1 to T-423; M-1 to R-422; M-1 to V-421; M-1 to P-420; M-1 to V-419; M-1 to P-418; M-1 to K-417; M-1 to Y-416;M-1 toK-415;M-l to C-414; M-1 toK-413;M-l to G-412; M-1 to P-411; M- 1 to P-410; M-1 to A-409; M-1 to R-408; M-1 to R-407; M-1 to S-406; M-1 to P-405; M-1 to D-404; M-1 to V-403; M-1 to F-402; M-1 to K-401; M-1 to Y-400; M-1 to 1-399; M-1 to S-398; M-1 to G-397; M-1 to R-396; M-1 to P-395; M-1 to A-394; M-1 to Y- 393; M-1 to A-392; M-1 to S-391; M-1 to P-390; M-1 to Y-389; M-1 to S-388; M-1 to T-387; M-1 to A-386; M-1 to L-385; M-1 to F-384; M-1 to Y-383; M-1 to L-382; M-1 to E-381; M-1 to G-380; M-1 to A-379; M-1 to E-378; M-1 to D-377; M-1 to E-376; M-1 to A-375; M-1 to F-374; M-1 to S-373; M-1 to 1-372; M-1 to 1-371; M-1 to F-370; M-1 to K-369; M-1 to S-368; M-1 to H-367; M-1 to T-366; M-1 to S-365; M-1 to 1-364; M-1 to L-363; M-1 to G-362; M-1 to P-361; M-1 to F-360; M-1 to A-359; M-1 to C-358; M-1 to S-357; M-1 to T-356; M-1 to T-355; M-1 to S-354; M-1 to G-353; M-1 to L-352; M-1 to C-351; M-1 to L-350; M-1 to D-349; M-1 to Q-348; M-1 to K-347; M-1 to K-346; M- 1 to W-345; M-1 to K-344; M-1 to K-343; M-1 to N-342; M-1 to K-341; M-1 to R-340; M-1 to D-339; M-1 to E-338; M-1 to Q-337; M-1 to L-336; M-1 to A-335; M-1 to M- 334; M-1 to L-333; M-1 to R-332; M-1 to G-331; M-1 to S-330; M-1 to M-329; M-1 to F-328; M-1 to D-327; M-1 to G-326; M-1 to F-325; M-1 to 1-324; M-1 to Y-323; M-1 to L-322; M-1 to G-321; M-1 to N-320; M-1 to L-319; M-1 to N-318; M-1 to P-317; M-1 toS-316;M-l toE-315;M-l toC-314;M-l toG-313;M-l toR-312;M-l toY-311;M- 1 to V-310; M-1 to Y-309; M-1 to G-308; M-1 to G-307; M-1 to T-306; M-1 to V-305; M-1 to S-304; M-1 to K-303; M-1 to G-302; M-1 to V-301; M-1 to A-300; M-1 to H- 299; M-1 to G-298; M-1 to Y-297; M-1 to A-296; M-1 to Y-295; M-1 to 1-294; M-1 to P-293; M-1 to L-292; M-1 to V-291; M-1 to D-290; M-1 to D-289; M-1 to L-288; M-1 to S-287; M-1 to A-286; M-1 to N-285; M-1 to H-284; M-1 to C-283; M-1 to L-282; M- 1 to K-281; M-1 to K-280; M-1 to D-279; M-1 to Y-278; M-1 to C-277; M-1 to A-276; M-1 to F-275; M-1 to G-274; M-1 to E-273; M-1 to K-272; M-1 to A-271; M-1 to R- 270; M-1 to W-269; M-1 to G-268; M-1 to Y-267; M-1 to N-266; M-1 to G-265; M-1 to G-264; M-1 to K-263; M-1 to L-262; M-1 to 1-261; M-1 to L-260; M-1 to D-259; M-1 to V-258; M-1 to E-257; M-1 to E-256; M-1 to F-255; M-1 to R-254; M-1 to N-253; M-1 to Q-252; M-1 to G-251; M-1 to V-250; M-1 to D-249; M-1 to G-248; M-1 to C-247; M-1 to F-246; M-1 to 1-245; M-1 to R-244; M-1 to G-243; M-1 to R-242; M-1 to G-241; M-1 to Q-240; M-1 to R-239; M-1 to T-238; M-1 to 1-237; M-1 to P-236; M-1 to D-235; M-1 to G-234; M-1 to R-233; M-1 to D-232; M-1 to V-231; M-1 to A-230; M-1 to C-229; M- 1 to R-228; M-1 to W-227; M-1 to M-226; M-1 to N-225; M-1 to R-224; M-1 to 1-223; M-1 to G-222; M-1 to Y-221; M-1 to A-220; M-1 to Y-219; M-1 to 1-218; M-1 to A- 217; M-1 to P-216; M-1 to H-215; M-1 to A-214; M-1 to G-213; M-1 to P-212; M-1 to E-211; M-1 to S-210; M-1 to V-209; M-1 to F-208; M-1 to P-207; M-1 to N-206; M-1 to D-205; M-1 to S-204; M-1 to P-203; M-1 to V-202; M-1 to R-201; M-1 to Y-200; M-1 to R-199; M-1 to K-198; M-1 to G-197; M-1 to H-196; M-1 to S-195; M-1 to G-194; M- 1 to A-193; M-1 to R-192; M-1 to N-191; M-1 to V-190; M-1 to D-189; M-1 to 1-188; M-1 to R-187; M-1 to L-186; M-1 to V-185; M-1 to K-184; M-1 to G-183; M-1 to L- 182; M-1 to L-181; M-1 to S-180; M-1 to S-179; M-1 to K-178; M-1 to N-177; M-1 to Q-176; M-1 to A-175; M-1 to N-174; M-1 to G-173; M-1 to F-172; M-1 to L-171; M-1 to G-170; M-1 to F-169; M-1 to P-168; M-1 to D-167; M-1 to G-166; M-1 to A-165; M- 1 to Q-164; M-1 to G-163; M-1 to G-162; M-1 to D-161; M-1 to G-160; M-1 to T-159; M-1 to F-158; M-1 to 1-157; M-1 to Y-156; M-1 to M-155; M-1 to Y-154; M-1 to G- 153; M-1 to D-152; M-1 to L-151; M-1 to G-150; M-1 to F-149; M-1 to L-148; M-1 to L-147; M-1 to Q-146; M-1 to G-145; M-1 to G-144; M-1 to N-143; M-1 to H-142; M-1 to N-141; M-1 to S-140; M-1 to A-139; M-1 to P-138; M-1 to E-137; M-1 to E-136; M-1 to 1-135; M-1 to E-134; M-1 to L-133; M-1 to 1-132; M-1 to V-131; M-1 to R-130; M-1 to E-129; M-1 to S-128; M-1 to K-127; M-1 to L-126; M-1 to D-125; M-1 to A-124; M- 1 to K-123; M-l to N-122: M-l to P-121; M-l to D-120; M-l to A-1 19; M-l to R-l 18; M-1 to S-117; M-l to V- 116; M-l to K-115; M-l to M-114; M-l to E-113; M-l to S- 112; M-1 to I-l l l ; M-l to RJ 10; MJ to 1-109; M-1 to K-108; M-1 to E-107; M-l to V- 106; M-1 to K-105; M-1 to K-104: M-1 to K-103; M-1 to D-102; M-1 to L-101; M-1 to C-100; M-1 to S-99; M-1 to Y-98; M-1 to Y-97; M-1 to 1-96; M-1 to Y-95; M-1 to F-94; M-1 to K-93; M-1 to R-92; M-1 to N-91 ; M-1 to H-90; M-1 to R-89; M-1 to F-88; M-1 to K-87; M-1 to P-86; M-1 to H-85; M-1 to F-84; M-1 to A-83; M-1 to L-82; M-1 to G- 81 ; M-1 to L-80; M-1 to F-79; M-1 to G-78; M-1 to R-77; M-1 to E-76; M-1 to D-75; M- 1 to G-74; M-1 to 1-73; M-1 to W-72; M-1 to P-71 ; M-1 to T-70; M-1 to T-69; M-1 to L- 68; M-1 to V-67; M-1 to 1-66; M-1 to N-65; M-1 to K-64; M-1 to L-63; M-1 to D-62; M- 1 to L-61; M-1 to F-60; M-1 to P-59; M-1 to Q-58; M-1 to E-57; M-1 to L-56; M-1 to R- 55; M-1 to S-54; M-1 to G-53; M-1 to D-52; M-1 to P-51; M-1 to L-50; M-1 to Y-49; M- 1 to V-48; M-1 to W-47; M-1 to V-46; M-1 to V-45; M-1 to G-44; M-1 to V-43; M-1 to Q-42; M-1 to E-41; M-1 to A-40; M-1 to V-39; M-1 to F-38; M-1 to F-37; M-1 to R-36; M-1 to H-35; M-1 to T-34; M-1 to G-33; M-1 to D-32; M-1 to G-31; M-1 to A-30; M-1 to H-29; M-1 to V-28; M-1 to M-27; M-1 to S-26; M-1 to V-25; M-1 to P-24; M-1 to N- 23; M-1 to R-22; M-1 to L-21; M-1 to G-20; M-1 to N-19; M-1 to A-18; M-1 to V-17; M-1 to E-16; M-1 to S-15; M-1 to L-14; M-1 to C-13; M-1 to L-12; M-1 to Q-11; M-1 to L-10; M-1 to C-9; M-1 to G-8; M-1 to Q-7; of SEQ ID NO:29. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, developmental disorders, and degenerative disorders; osteoarthritis, and lung cancer. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of developing tissues, cartilage, and bone, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g. bone, lung, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e.. the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 29 as residues: Asp-52 to Glu-57, Arg-89 to Tyr-95, Asp- 102 to Glu-107, Ser-117 to Ser-128, Glu-137 to Gly-145, Arg-192 to Arg-199, Val-231 to Gly- 243, Val-250 to Glu-256, Arg-312 to Asn-318, Glu-338 to Asp-349, Pro-405 to Lys-417, Thr-423 to Ue-428, Lys-442 to Ser-453, Glu-467 to Ala-475, Thr-478 to Arg-494, Pro- 497 to Arg-526. Polynucleotides encoding said polypeptides are also provided.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 11 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a- b, where a is any integer between 1 to 2595 of SEQ ID NOJ 1, b is an integer of 15 to 2609, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 11, and where b is greater than or equal to a + 14. FEATURES OF PROTEIN ENCODED BY GENE NO: 2

The translation product of this gene, sometimes referred to herein as TIDE (for Ten Integrin Domains with EGF homology), shares sequence homology with integrins, which are a superfamily of dimeric ab cell-surface glycoproteins that mediate the adhesive functions of many cell types, enabling cells to interact with one another and with the extracellular matrix (See Genomics 56, 169-178 (1999); all information and references contained within this publication are hereby incorporated herein by reference). Eight human integrin b subunits have been described to date, and in combination with the 12 known a subunits form a large family of heterodimeric cell surface receptors that mediate cell adhesion to counter-receptors on neighboring cells, and to ECM proteins (reviewed by Hynes, 1992). Integrin-ligand interactions are crucial for fundamental biological processes such as cell migration and motility, and lymphocyte extravasation. In another embodiment, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention comprise the following amino acid sequence: TSTPPRAVPLPKSSQAAHQRNCNSGWSPGPASLGVRGSVCPAICWWHLS LLPPPSVNPTLQKCSSPGAAQELSMRPPGFRNFLLLASSLLFAGLSAVPQSFSPSLR SWPGAACRLSRAESERRCRAPGQPPGAALCHGRGRCDCGVCICHVTEPGMFFGP LCECHEWVCETYDGSTCAGHGKCDCGKCKCDQGWYGDACQYPTNCDLTKKK SNQMCKNSQDIICSNAGTCHCGRCKCDNSDGSGLVYGKFCECDDRECIDDETEEI CGGHGKCYCGNCYCKAGWHGDKCEFQCDITPWESKRRCTSPDGKICSNRGTCV CGECTCHDVDPTGDWGDIHGDTCECDERDCRAVYDRYSDDFCSGHGQCNCGR CDCKAGWYGKKCEHPQSCTLSAEESIRKCQGSSDLPCSGRGKCECGKCTCYPPG DRRVYGKTCECDDRRCEDLDGVVCGGHGTCSCGRCVCERGWFGKLCQHPRKC NMTEEQSKNLCESADGILCSGKGSCHCGKCICSAEEWYISGEFCDCDDRDCDKH DGLICTGNGICSCGNCECWDGWNGNACEI WLGSEYP (SEQ ID NO:50). Polynucleotides encoding these polypeptides are also provided.

Included in this invention as preferred domains are EGF-like domain signature 1 and 2 domains, which were identified using the ProSite analysis tool (Swiss Institute of Bioinformatics). A sequence of about thirty to forty amino-acid residues long found in the sequence of epidermal growth factor (EGF) has been shown [1 to 6] to be present, in a more or less conserved form, in a large number of other, mostly animal proteins. The functional significance of EGF domains in what appear to be unrelated proteins is not yet clear. However, a common feature is that these repeats are found in the extracellular domain of membrane-bound proteins or in proteins known to be secreted (exception: prostaglandin G/H synthase). The EGF domain includes six cysteine residues which have been shown (in EGF) to be involved in disulfide bonds. The main structure is a two- stranded beta-sheet followed by a loop to a C-terminal short two-stranded sheet. Subdomains between the conserved cysteines strongly vary in length as shown in the following schematic representation of the EGF-like domain:

I I I I x (4) -C-x( 0 , 48 ) -C-x(3 , 12 ) -C-x( l , 70 ) -C-x( 1. 6) -C-x(2 ) -G-a-x( 0. 21 ) -G-x(2 ) -C-x

I *********_********_******_****_*********

'C: conserved cysteine involved in a disulfide bond. 'G': often conserved glycine 'a': often conserved aromatic amino acid '*': position of both patterns. Y: any residue The region between the 5th and 6th cysteine contains two conserved glycines of which at least one is present in most EGF-like domains. The concensus pattern is as follows: C-x-

C-x(5)-G-x(2)-C [The 3 C's are involved in disulfide bonds].

Preferred polypeptides of the invention comprise the following amino acid sequence: GKCDCGKCKCDQGWYGDACQYPTNCDLTK (SEQ ID NO: 51), GGHGKCYCGNCYCKAGWHGDKCEFQCDIT (SEQ ID NO'52), HGQCNCGRCDCKAGWYGKKCEHPQSCTLS (SEQ ID NO 53), HGTCSCGRCVCERGWFGKLCQHPRKCNMT (SEQ ID NO: 54), GNGICSCGNCECWDGWNGNACEIWLGSEY (SEQ ID NO 55), and ICGGHGKCYCGNCYCKAGWHGDKCEFQCDITPWESK (SEQ ID NO 73) Polynucleotides encoding these polypeptides aie also piovided

Further preferred are polypeptides comprising the EGF-hke domain signature 1 and 2 domains of the sequence referenced in Table I for this gene, and at least 5, 10, 15, 20, 25, 30, 50, or 75 additional contiguous amino acid residues of this referenced sequence The additional contiguous ammo acid lesidues is N-teimmal oi C- terminal to the EGF-hke domain signature 1 and 2 domains

Alternatively, the additional contiguous amino acid residues is both N-termmal and C-termmal to the EGF- ke domain signature 1 and 2 domains, wherein the total N- and C-termmal contiguous am o acid residues equal the specified number The above preferred polypeptide domain is characteristic of a signature specific to EGF- ke domain 1 and 2 containing proteins Based on the sequence similarity, the translation product of this gene is expected to share at least some biological activities with EGF-hke containing proteins Such activities are known in the art, some of which are descnbed elsewhere herein. Included in this invention as preferred domains are integrins beta chain cysteine- πch domains, which were identified using the ProSite analysis tool (Swiss Institute of Bio formatics). Integnns [7,8] are a large family of cell surface receptors that mediate cell to cell as well as cell to matrix adhesion. Some mtegπns recognize the R-G-D sequence m their extracellular matrix protein ligand. Structurally, integnns consist of a dimer of an alpha and a beta chain. Each subunit has a large N-terminal extracellular domain followed by a transmembrane domain and a short C-terminal cytoplasmic region Some receptors share a common beta chain while having different alpha chains All the integrin beta chains contain four repeats of a forty amino acid region in the C-terminal extremity of their extracellular domain. Each of the repeats contains eight cysteines. The concensus pattern is as follows: C-x-[GNQ]-x(l,3)-G-x-C-x-C-x(2)-C-x-C [The five C's are probably involved in disulfide bonds]. Preferred polypeptides of the invention comprise the following amino acid sequence: GQPPGAALCHGRGRCDCGVCICHVTEPGMFFGPLC (SEQ ID NO: 74), ETYDGSTCAGHGKCDCGKCKCDQGWYGDACQYP (SEQ ID NO:58), MCKNSQDIICSNAGTCHCGRCKCDNSDGSGLVYG (SEQ ID NO:59), IDDETEEICGGHGKCYCGNCYCKAGWHGDKC (SEQ ID NO:60), KRRCTSPDGKICSNRGTCVCGECTCHDVDPTGDW (SEQ ID NO:61), DRYSDDFCSGHGQCNCGRCDCKAGWYGKKCEHPQ (SEQ ID NO:62), CQGSSDLPCSGRGKCECGKCTCYPPGDRRVYGK (SEQ ID NO:63), CEDLDGVVCGGHGTCSCGRCVCERGWFGKLC (SEQ ID NO:64), SADGILCSGKGSCHCGKCICSAEEWYISGEFC (SEQ ID NO:65), and CDKHDGLICTGNGICSCGNCECWDGWNGNACEI (SEQ ID NO: 66). Polynucleotides encoding these polypeptides are also provided.

Further preferred are polypeptides comprising the integrins beta chain cysteine- rich domain of the sequence referenced in Table XIII for this gene, and at least 5, 10, 15, 20, 25, 30, 50, or 75 additional contiguous amino acid residues of this referenced sequence. The additional contiguous amino acid residues is N-terminal or C- terminal to the integrins beta chain cysteine-rich domain.

Alternatively, the additional contiguous amino acid residues is both N-terminal and C-terminal to the integrins beta chain cysteine-rich domain, wherein the total N- and C-terminal contiguous amino acid residues equal the specified number. The above preferred polypeptide domain is characteristic of a signature specific to integrin proteins. Based on the sequence similarity, the translation product of this gene is expected to share at least some biological activities with integrin proteins, and specifically those containing an integrins beta chain cysteine-rich domain. Such activities are known in the art, some of which are described elsewhere herein. The following publications were referenced above and are hereby incorporated herein by reference: [ 1] Davis C.G., New Biol. 2:410-419(1990); [ 2] Blomquist M.C., Hunt L.T., Barker W.C, Proc. Natl. Acad. Sci. U.S.A. 81 :7363-7367(1984); [ 3] Barker W.C, Johnson G.C., Hunt L.T., George D.G., Protein Nucl. Acid Enz. 29:54-68(1986); [ 4] Doolittle R.F., Feng D.F., Johnson M.S., Nature 307:558-560(1984); [ 5] Appella E., Weber I.T., Blasi F., FEBS Lett. 231:1- 4(1988); [ 6] Campbell I.D., Bork P., Cuιτ. Opin. Struct. Biol. 3:385-392(1993); [ 7] Hynes R.O., Cell 48:549-554(1987); and [ 8] Albelda S.M., Buck C.A., FASEB J. 4:2868-2880(1990).

The polypeptide of the present invention has been putatively identified as a member of the integrin family and has been termed Ten Integrin Domains with EGF homology ("TIDE"). This identification has been made as a result of amino acid sequence homology to the human integrin beta-8 subunit (See Genbank Accession No. gi| 184521).

Figures 4A-C shows the nucleotide (SEQ ID NO: 12) and deduced amino acid sequence (SEQ ID NO:30) of TIDE. Predicted amino acids from about 1 to about 23 constitute the predicted signal peptide (amino acid residues from about 1 to about 23 in SEQ ID NO:30) and are represented by the underlined amino acid regions; amino acids from about 108 to about 136, from about 195 to about 223, from about 291 to about 319, from about 379 to about 407, and/or from about 465 to about 493 constitute the predicted EGF-like domain signature 1 and 2 domains (amino acids from about 108 to about 136, from about 195 to about 223, from about 291 to about 319, from about 379 to about 407, and/or from about 465 to about 493 in SEQ ID NO:30) and are represented by the double underlined amino acids; and amino acids from about 55 to about 89, from about 97 to about 129, from about 142 to about 175, from about 186 to about 216, from about 228 to about 261, from about 281 to about 314, from about 327 to about 359, from about 368 to about 398, from about 417 to about 448, and/or from about 455 to about 487 constitute the predicted integrins beta chain cysteine-rich domains (amino acids from about 55 to about 89, from about 97 to about 129, from about 142 to about 175, from about 186 to about 216, from about 228 to about 261, from about 281 to about 314, from about 327 to about 359, from about 368 to about 398, from about 417 to about 448, and/or from about 455 to about 487 in SEQ ID NO:30) and are represented by the shaded amino acids.

Figure 5 shows the regions of similarity between the amino acid sequences of the Ten Integrin Domains with EGF homology (TIDE) protein (SEQ ID NO:30) and the human integrin beta-8 subunit (SEQ ID NO: 67). Figure 6 shows an analysis of the Ten Integrin Domains with EGF homology

(TIDE) amino acid sequence. Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.

A polynucleotide encoding a polypeptide of the present invention is obtained from human osteoblasts, synovial hypoxia tissue, osteoblast and osteoclast, bone marrow stromal cells, umbilical vein, smooth muscle, placenta, and fetal lung. The polynucleotide of this invention was discovered in a human osteoblast II cDNA library. Its translation product has homology to the characteristic integrins beta chain cysteine- rich domains of integrin family members. The polynucleotide contains an open reading frame encoding the TIDE polypeptide of 494 amino acids. TIDE exhibits a high degree of homology at the amino acid level to the human integrin beta-8 subunit (as shown in Figure 5).

The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the TIDE polypeptide having the amino acid sequence shown in Figures 4A-C (SEQ ID NO:30). The nucleotide sequence shown in Figures 4A-C (SEQ ID NOJ 2) was obtained by sequencing a cloned cDNA (HOHCH55), which was deposited on November 17 at the American Type Culture Collection, and given Accession Number 203484. The present invention is further directed to fragments of the isolated nucleic acid molecules described herein. By a fragment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ ID NO: 12 is intended DNA fragments at least about 15nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments 50-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in SEQ ID NO: 12. By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NO: 12. In this context "about" includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Representative examples of TIDE polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, from about 501 to about 550, from about 551 to about 600, from about 601 to about 650, from about 651 to about 700, from about 701 to about 750, from about 751 to about 800, from about 801 to about 850, from about 851 to about 900, from about 901 to about 950, from about 951 to about 1000, from about 1001 to about 1050, from about 1051 to about 1100, from about 1101 to about 1150, from about 1151 to about 1200, from about 1201 to about 1250, from about 1251 to about 1300, from about 1301 to about 1350, from about 1351 to about 1400, from about 1401 to about 1450, from about 1451 to about 1500, from about 1501 to about 1550, from about 1551 to about 1600, from about 1601 to about 1650, from about 1651 to about 1700, from about 1701 to about 1750, from about 1751 to about 1800, from about 1801 to about 1850, from about 1851 to about 1900, from about 1901 to about 1950, from about 1951 to about 2000, from about 2001 to about 2050, from about 2051 to about 2100, from about 2101 to about 2150, from about 2151 to about 2200, from about 2201 to about 2250, from about 2251 to about 2300, from about 2301 to about 2350. from about 2351 to about 2400, from about 2401 to about 2450, from about 2451 to about 2499, from about 289 to about 1705, and/or from about 221 to about 1705 of SEQ ID NO: 12, or the complementary strand thereto, or the cDNA contained in the deposited gene. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.

Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding a member selected from the group: a polypeptide comprising or alternatively, consisting of, the mature TIDE protein (amino acid residues from about 221 to about 1705 in Figures 4A-C (amino acids from about 221 to about 1705 in SEQ ID NO:30). Since the location of these domains have been predicted by computer analysis, one of ordinary skill would appreciate that the amino acid residues constituting these domains may vary slightly (e.g., by about 1 to 15 amino acid residues) depending on the criteria used to define each domain. In additional embodiments, the polynucleotides of the invention encode functional attributes of TIDE.

Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions"), turn and turn-forming regions ("turn- regions"), coil and coil-forming regions ("coil-regions"), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions of TIDE. The data representing the structural or functional attributes of TIDE set forth in Figure 6 and/or Table II, as described above, was generated using the various modules and algorithms of the DNA*STAR set on default parameters. In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table II can be used to determine regions of TIDE which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.

Certain preferred regions in these regards are set out in Figure 6, but may, as shown in Table II. be represented or identified by using tabular representations of the data presented in Figure 6. The DNA*STAR computer algorithm used to generate Figure 6 (set on the original default parameters) was used to present the data in Figure 6 in a tabular format (See Table II). The tabular format of the data in Figure 6 is used to easily determine specific boundaries of a preferred region. The above-mentioned preferred regions set out in Figure 6 and in Table II include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figures 4A-C As set out in Figure 6 and in Table II, such preferred regions include Garnier- Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha- regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions and Hopp- Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus- Schulz flexible regions, Jameson-Wolf regions of high antigenic index and Emini surface-forming regions. Even if deletion of one or more amino acids from the N- terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, etc.) may still be retained. For example, the ability of shortened TIDE muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-termmus. Whether a particular polypeptide lacking N-termmal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods descπbed herein and otherwise known in the art. It is not unlikely that an TIDE mutein with a large number of deleted N-termmal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six TIDE amino acid residues may often evoke an immune response

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the TIDE ammo acid sequence shown in Figures 4A-C, up to the leucme residue at position number 489 and polynucleotides encoding such polypeptides In particular, the present invention provides polypeptides comprising the am o acid sequence of residues n 1-494 of Figures 4A-C, where nl is an integer from 2 to 489 corresponding to the position of the amino acid residue in Figures 4A-C (which is identical to the sequence shown as SEQ ID NO:30). In another embodiment, N-termmal deletions of the TIDE polypeptide can be descπbed by the general formula n2-494, where n2 is a number from 2 to 489, corresponding to the position of ammo acid identified in Figures 4A-C N-terminal deletions of the TIDE polypeptide of the invention shown as SEQ ID NO:30 include polypeptides comprising the amino acid sequence of residues: N-terminal deletions of the TIDE polypeptide of the invention shown as SEQ ID NO:30 include polypeptides compπsing the amino acid sequence of residues: R-2 to P-494; P-3 to P-494; P-4 to P-494; G-5 to P-494; F-6 to P- 494; R-7 to P-494; N-8 to P-494; F-9 toP-494; L-10 to P-494; L-l 1 to P-494; L-12 to P- 494; A-13 to P-494; S-14 to P-494; S-15 to P-494; L-16 to P-494; L-17 to P-494 ;F-18 to P-494; A- 19 to P-494; G-20 to P-494; L-21 to P-494; S-22 to P-494; A-23 to P-494; V- 24 to P-494; P-25 to P-494; Q-26to P-494; S-27 to P-494; F-28 to P-494; S-29 to P-494; P-30 to P-494; S-31 to P-494; L-32 to P-494; R-33 to P-494; S-34 toP-494; W-35 to P- 494, P-36 to P-494; G-37 to P-494; A-38 to P-494, A-39 to P-494, C-40 to P-494; R-41 to P-494; L-42 toP-494; S-43 to P-494; R-44 to P-494; A-45 to P-494; E-46 to P-494; S- 47 to P-494; E-48 to P-494; R-49 to P-494; R-50 toP-494; C-51 to P-494; R-52 to P-494; A-53 to P-494; P-54 to P-494; G-55 to P-494; Q-56 to P-494; P-57 to P-494; P-58 toP- 494; G-59 to P-494; A-60 to P-494; A-61 to P-494; L-62 to P-494; C-63 to P-494; H-64 to P-494; G-65 to P-494; R-66 toP-494; G-67 to P-494; R-68 to P-494; C-69 to P-494; D- 70 to P-494; C-71 to P-494; G-72 to P-494; V-73 to P-494; C-74 toP-494; 1-75 to P-494; C-76 to P-494; H-77 to P-494; V-78 to P-494; T-79 to P-494; E-80 to P-494; P-81 to P- 494; G-82 to P-494;M-83 to P-494; F-84 to P-494; F-85 to P-494; G-86 to P-494; P-87 to P-494; L-88 to P-494; C-89 to P-494; E-90 to P-494; C-91to P-494; H-92 to P-494; E-93 to P-494: W-94 to P-494; V-95 to P-494; C-96 to P-494; E-97 to P-494; T-98 to P-494; Y-99 toP-494; D-100 to P-494; G-101 to P-494; S-102 to P-494; T-103 to P-494; C-104 to P-494; A- 105 to P-494; G-106 to P-494;H-107 to P-494; G-108 to P-494; K-109 to P- 494; C-110 to P-494; D-l 11 to P-494; C-112 to P-494; G-l 13 to P-494; K-114 toP-494; C-115 to P-494; K-116 to P-494; C-117 to P-494; D-l 18 to P-494; Q-l 19 to P-494; G- 120 to P-494; W- 121 to P-494; YJ 22 to P-494; G-l 23 to P-494; D-l 24 to P-494; A- 125 to P-494; C-126 to P-494; Q-l 27 to P-494; Y-128 to P-494; P-129 toP-494; T-130 to P- 494; N-131 to P-494; C-132 to P-494; D-133 to P-494; L-134 to P-494; T-135 to P-494; K-136 to P-494;K-137 to P-494; K-138 to P-494; S-139 to P-494; N-140 to P-494; Q- 141 to P-494; M-142 to P-494; C-143 to P-494; K-144 toP-494; N-145 to P-494; S-146 to P-494; Q-147 to P-494; D-148 to P-494; 1-149 to P-494; 1-150 to P-494; C-151 to P- 494; S-152to P-494; N-153 to P-494; A- 154 to P-494; G-155 to P-494; T-156 to P-494; C-157 to P-494; H-158 to P-494; C-159 to P-494;G-160 to P-494; R-161 to P-494; C-162 to P-494; K-163 to P-494; C-164 to P-494; D-165 to P-494; N-166 to P-494; S-167 toP- 494; D-168 to P-494; G-169 to P-494; S-170 to P-494; G-171 to P-494; L-172 to P-494; V-173 to P-494; Y-174 to P-494;G-175 to P-494; K-176 to P-494; F-177 to P-494; C-178 to P-494; E-179 to P-494; C-180 to P-494; D-181 to P-494; D-182 toP-494; R-183 to P- 494; E-184 to P-494; C-185 to P-494; 1-186 to P-494; D-187 to P-494; D-188 to P-494; E-189 to P-494; T-190to P-494; E-191 to P-494; E-192 to P-494; 1-193 to P-494; C-194 to P-494; G-195 to P-494; G-196 to P-494; H-197 to P-494;G-198 to P-494; K-199 to P- 494; C-200 to P-494; Y-201 to P-494; C-202 to P-494; G-203 to P-494; N-204 to P-494; C-205 toP-494; Y-206 to P-494; C-207 to P-494; K-208 to P-494; A-209 to P-494; G- 210 to P-494; W-211 to P-494; H-212 to P-494;G-213 to P-494; D-214 to P-494; K-215 to P-494; C-216 to P-494; E-217 to P-494; F-218 to P-494; Q-219 to P-494; C-220 toP- 494; D-221 to P-494; 1-222 to P-494; T-223 to P-494; P-224 to P-494; W-225 to P-494; E-226 to P-494; S-227 to P-494;K-228 to P-494; R-229 to P-494; R-230 to P-494; C-231 to P-494; T-232 to P-494; S-233 to P-494; P-234 to P-494; D-235 toP-494; G-236 to P- 494; K-237 to P-494; 1-238 to P-494; C-239 to P-494; S-240 to P-494; N-241 to P-494; R-242 to P-494;G-243 to P-494; T-244 to P-494; C-245 to P-494; V-246 to P-494; C-247 to P-494; G-248 to P-494; E-249 to P-494; C-250 toP-494; T-251 to P-494; C-252 to P- 494; H-253 to P-494; D-254 to P-494; V-255 to P-494; D-256 to P-494; P-257 to P- 494;T-258 to P-494; G-259 to P-494; D-260 to P-494; W-261 to P-494; G-262 to P-494; D-263 to P-494; 1-264 to P-494; H-265 toP-494; G-266 to P-494; D-267 to P-494; T-268 to P-494; C-269 to P-494; E-270 to P-494; C-271 to P-494; D-272 to P-494 ;E-273 to P- 494; R-274 to P-494; D-275 to P-494; C-276 to P-494; R-277 to P-494; A-278 to P-494; V-279 to P-494; Y-280 toP-494; D-281 to P-494; R-282 to P-494; Y-283 to P-494; S-284 to P-494; D-285 to P-494; D-286 to P-494; F-287 to P-494;C-288 to P-494; S-289 to P- 494; G-290 to P-494; H-291 to P-494; G-292 to P-494; Q-293 to P-494; C-294 to P-494; N-295 toP-494; C-296 to P-494; G-297 to P-494; R-298 to P-494; C-299 to P-494; D- 300 to P-494; C-301 to P-494; K-302 to P-494;A-303 to P-494; G-304 to P-494; W-305 to P-494; Y-306 to P-494; G-307 to P-494; K-308 to P-494; K-309 to P-494; C-310 toP- 494; E-311 to P-494; H-312 to P-494; P-313 to P-494; Q-314 to P-494; S-315 to P-494; C-316 to P-494; T-317 to P-494;L-318 to P-494; S-319 to P-494; A-320 to P-494; E-321 to P-494; E-322 to P-494; S-323 to P-494; 1-324 to P-494; R-325 toP-494; K-326 to P- 494; C-327 to P-494; Q-328 to P-494; G-329 to P-494; S-330 to P-494; S-331 to P-494; D-332 to P-494;L-333 to P-494; P-334 to P-494; C-335 to P-494; S-336 to P-494; G-337 to P-494; R-338 to P-494; G-339 to P-494; K-340 toP-494; C-341 to P-494; E-342 to P- 494; C-343 to P-494; G-344 to P-494; K-345 to P-494; C-346 to P-494; T-347 to P- 494;C-348 to P-494; Y-349 to P-494; P-350 to P-494; P-351 to P-494; G-352 to P-494; D-353 to P-494; R-354 to P-494; R-355 toP-494; V-356 to P-494; Y-357 to P-494; G- 358 to P-494; K-359 to P-494; T-360 to P-494; C-361 to P-494; E-362 to P-494;C-363 to P-494; D-364 to P-494; D-365 to P-494; R-366 to P-494; R-367 to P-494; C-368 to P- 494; E-369 to P-494; D-370 toP-494; L-371 to P-494; D-372 to P-494; G-373 to P-494; V-374 to P-494; V-375 to P-494; C-376 to P-494; G-377 to P-494;G-378 to P-494; H- 379 to P-494; G-380 to P-494; T-381 to P-494; C-382 to P-494; S-383 to P-494; C-384 to P-494; G-385 toP-494; R-386 to P-494; C-387 to P-494; V-388 to P-494; C-389 to P- 494; E-390 to P-494; R-391 to P-494; G-392 to P-494;W-393 to P-494; F-394 to P-494; G-395 to P-494; K-396 to P-494; L-397 to P-494; C-398 to P-494; Q-399 to P-494; H- 400 toP-494; P-401 to P-494; R-402 to P-494; K-403 to P-494; C-404 to P-494; N-405 to P-494; M-406 to P-494; T-407 to P-494;E-408 to P-494; E-409 to P-494; Q-410 to P- 494; S-41 1 to P-494; K-412 to P-494; N-413 to P-494; L-414 to P-494; C-415 toP-494; E-416 to P-494; S-417 to P-494; A-418 to P-494; D-419 to P-494; G-420 to P-494; 1-421 to P-494; L-422 to P-494; C-423to P-494; S-424 to P-494; G-425 to P-494; K-426 to P- 494; G-427 to P-494; S-428 to P-494; C-429 to P-494; H-430 to P-494;C-431 to P-494; G-432 to P-494; K-433 to P-494; C-434 to P-494; 1-435 to P-494; C-436 to P-494; S-437 to P-494; A-438 toP-494; E-439 to P-494; E-440 to P-494; W-441 to P-494; Y-442 to P- 494; 1-443 to P-494; S-444 to P-494; G-445 to P-494;E-446 to P-494; F-447 to P-494; C- 448 to P-494; D-449 to P-494; C-450 to P-494; D-451 to P-494; D-452 to P-494; R-453 toP-494; D-454 to P-494; C-455 to P-494; D-456 to P-494; K-457 to P-494; H-458 to P- 494; D-459 to P-494; G-460 to P-494;L-461 to P-494; 1-462 to P-494; C-463 to P-494; T-464 to P-494; G-465 to P-494; N-466 to P-494; G-467 to P-494; 1-468 toP-494; C-469 to P-494; S-470 to P-494; C-471 to P-494; G-472 to P-494; N-473 to P-494; C-474 to P- 494; E-475 to P-494;C-476 to P-494; W-477 to P-494; D-478 to P-494; G-479 to P-494; W-480 to P-494; N-481 to P-494; G-482 to P-494; N-483 toP-494; A-484 to P-494; C- 485 to P-494; E-486 to P-494; 1-487 to P-494; W-488 to P-494; L-489 to P-494; of SEQ ID NO:30. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification or loss of one or more biological functions of the protein, other functional activities may still be retained. For example the ability of the shortened TIDE mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that an TIDE mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six TIDE amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the TIDE polypeptide shown in Figures 4A-C, up to the phenylalanine residue at position number 6, and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising the amino acid sequence of residues 1-ml of Figure 1, where ml is an integer from 6 to 494 corresponding to the position of the amino acid residue in Figures 4A-C Moreover, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of C- terminal deletions of the TIDE polypeptide of the invention shown as SEQ ID NO:30 include polypeptides comprising the amino acid sequence of residues: M-1 to Y-493; M- 1 to E-492; M-1 to S-491; M-1 to G-490; M-1 to L-489; M-1 to W-488; M-1 toI-487; M- 1 to E-486; M-1 to C-485; M-1 to A-484; M-1 to N-483; M-1 to G-482; M-1 to N-481; M-1 to W-480; M-1 to G-479;M-1 to D-478; M-1 to W-477; M-1 to C-476; M-1 to E- 475; M-1 to C-474; M-1 to N-473; M-1 to G-472; M-1 to C-471; M-1 toS-470; M-1 to C-469; M-1 to 1-468; M-1 to G-467; M-1 to N-466; M-1 to G-465; M-1 to T-464; M-1 to C-463; M-1 to 1-462; M-lto L-461; M-1 to G-460; M-1 to D-459; M-1 to H-458; M-1 to K-457; M-1 to D-456; M-1 to C-455; M-1 to D-454; M-1 to R-453;MJ to D-452; M-1 to D-451; M-1 to C-450; M-1 to D-449; M-1 to C-448; M-1 to F-447; M-1 to E-446; M- 1 to G-445; M-1 toS-444; M-1 to 1-443; M-1 to Y-442; M-1 to W-441; M-1 to E-440; M- 1 to E-439; M-1 to A-438; M-1 to S-437; M-1 to C-436; M-lto 1-435; M-1 to C-434; M- 1 to K-433; M-1 to G-432; M-1 to C-431; M-1 to H-430; M-1 to C-429; M-1 to S-428; M-1 to G-427;M-1 to K-426; M-1 to G-425; M-1 to S-424; M-1 to C-423; M-1 to L-422; M-1 to 1-421; M-1 to G-420; M-1 to D-419; M-1 toA-418; M-1 to S-417; M-1 to E-416; M-1 toC-415;M-l toL-414;M-l toN-413;M-l to K-412; M-1 toS-411;M-l to Q- 410;M-1 to E-409; M-1 to E-408; M-1 to T-407; M-1 to M-406; M-1 to N-405; M-1 to C-404; M-1 to K-403; M-1 to R-402; M-1 toP-401; M-1 to H-400; M-1 to Q-399; M-1 to C-398; M-1 to L-397; M-1 to K-396; M-1 to G-395; M-1 to F-394; M-1 to W-393;MJ to G-392; M-1 to R-391; M-1 to E-390; M-1 to C-389; M-1 to V-388; M-1 to C-387; M- 1 to R-386; M-1 to G-385; M-1 toC-384; M-1 to S-383; M-1 to C-382; M-1 to T-381; M- 1 to G-380; M-1 to H-379; M-1 to G-378; M-1 to G-377; M-1 to C-376;M-1 to V-375; M-1 to V-374; M-1 to G-373; M-1 to D-372; M-1 to L-371; M-1 to D-370; M-1 to E- 369; M-1 to C-368; M-1 toR-367; M-1 to R-366; M-1 to D-365; M-1 to D-364; M-1 to C-363; M-1 to E-362; M-1 to C-361; M-1 to T-360; M-1 to K-359;M-1 to G-358; M-1 to Y-357; M-1 to V-356; M-1 to R-355; M-1 to R-354; M-1 to D-353; M-1 to G-352; M-1 to P-351; M-1 toP-350; M-1 to Y-349; M-1 to C-348; M-1 to T-347; M-1 to C-346; M-1 to K-345; M-1 to G-344; M-1 to C-343; M-1 to E-342;M-1 to C-341; M-1 to K-340; M-1 to G-339; M-1 to R-338; M-1 to G-337; M-1 to S-336; M-1 to C-335; M-1 to P-334; M-1 toL-333; M-1 to D-332; M-1 to S-331 ; M-1 to S-330; M-1 to G-329; M-1 to Q-328; M-1 to C-327; M-1 to K-326; M-1 to R-325;M-1 to 1-324; M-1 to S-323; M-1 to E-322; M-1 to E-321; M-1 to A-320; M-1 to S-319; M-1 to L-318; M-1 to T-317; M-1 toC-316; M-1 to S-315; M-1 to Q-314; M-1 to P-313; M-1 to H-312; M-1 to E-311; M-1 to C-310; M-1 to K-309; M-1 to K-308;M-1 to G-307; M-1 to Y-306; M-1 to W-305; M-1 to G-304; M- 1 to A-303; M-1 to K-302; M-1 to C-301 ; M-1 to D-300; M-1 toC-299; M-1 to R-298; M-1 to G-297; M-1 to C-296; M-1 to N-295; M-1 to C-294; M-1 to Q-293; M-1 to G- 292; M-1 to H-291 ;M-1 to G-290; M-1 to S-289; M-1 to C-288; M-1 to F-287; M-1 to D-286; M-1 to D-285; M-1 to S-284; M-1 to Y-283; M-1 toR-282; M-1 to D-281; M-1 to Y-280; M- 1 to V-279; M-1 to A-278; M-1 to R-277; M-1 to C-276; M-1 to D-275; M-1 to R-274;M-1 to E-273; M-1 to D-272; M-1 to C-271; M-1 to E-270; M-1 to C-269; M-1 to T-268; M-1 to D-267; M-1 to G-266; M-1 toH-265; M-1 to 1-264; M-1 to D-263; M-1 to G-262; M-1 to W-261; M-1 to D-260; M-1 to G-259; M-1 to T-258; M-1 to P-257;M- 1 to D-256; M-1 to V-255; M-1 to D-254; M-1 to H-253; M-1 to C-252; M-1 to T-251 ; M-1 to C-250; M-1 to E-249; M-1 toG-248; M-1 to C-247; M-1 to V-246; M-1 to C-245 M-1 to T-244; M-1 to G-243; M-1 to R-242; M-1 to N-241; M-1 to S-240;M-1 to C-239 M-1 to 1-238; M-1 to K-237; M-1 to G-236; M-1 to D-235; M-1 to P-234; M-1 to S-233 M-1 to T-232; M-1 toC-231; M-1 to R-230; M-1 to R-229; M-1 to K-228; M-1 to S-227 M-1 to E-226; M-1 to W-225; M-1 to P-224; M-1 to T-223;M-1 to 1-222; M-1 to D-221 M-1 to C-220; M-1 to Q-219; M-1 to F-218; M-1 to E-217; M-1 to C-216; M-1 to K- 215; M-1 toD-214; M-1 to G-213; M-1 to H-212; M-1 to W-211; M-1 to G-210; M-1 to A-209; M-1 to K-208; M-1 to C-207; M-1 to Y-206;M-1 to C-205; M-1 to N-204; M-1 to G-203; M-1 to C-202; M-1 to Y-201; M-1 to C-200; M-1 to K-199; M-1 to G-198; M- 1 toH-197; M-1 to G-196; M-1 to G-195; M-1 to C-194; M-1 to 1-193; M-1 to E-192; M- 1 to E-191; M-1 to T-190; M-1 to E-189; M-lto D-188; M-1 to D-187; M-1 to 1-186; M- 1 to C-185; M-1 to E-184; M-1 to R-183; M-1 to D-182; M-1 to D-181; M-1 to C- 180;M-1 to E-179; M-1 to C-178; M-1 to F-177; M-1 to K-176; M-1 to G-175; M-1 to Y-174; M-1 to V-173; M-1 to L-172; M-1 toG-171; M-1 to S-170; M-1 to G-169; M-1 to D-168; M-1 to S-167; M-1 to N-166; M-1 to D-165; M-1 to C-164; M-1 to K-163;M-1 to C-162; M-1 to R-161; M-1 to G-160; M-1 to C-159; M-1 to H-158; M-1 to C-157; M-1 to T-156; M-1 to G-155; M-1 toA-154; M-1 to N-153; M-1 to S-152; M-1 to C-151; M-1 to 1-150; M-1 to 1-149; M-1 to D-148; M-1 to Q-147; M-1 to S-146; M-lto N-145; M-1 to K-144; M-1 to C-143; M-1 to M-142; M-1 to Q-141; M-1 to N-140; M-1 to S-139; M- 1 to K-138; M-1 to K-137;M-1 to K-136; M-1 to T-135; M-1 to L-134; M-1 to D-133; M-1 to C-132; M-1 to N-131; M-1 to T-130; M-1 to P-129; M-1 toY-128; M-1 to Q-127; M-1 to C-126; M-1 to A-125: M-1 to D-124; M-1 to G-123; M-1 to Y-122; M-1 to W- 121; M-1 to G-120;M-1 to Q-119; M-1 toD-118:M-l toC-117;M-l toK-116;M-l to C-115;M-1 toK-114;M-l toG-113;M-l toC-112;M-l toD-lll;M-l to CJ 10; MJ to K-109; M-1 to G-108; M-1 to H-107; M-1 to G-106; M-1 to A-105; M-1 to C-104; M-1 to T-103;M-1 to S-102; M-1 to G-101; M-1 to D-100; M-1 to Y-99; M-1 to T-98; M-1 to E-97; M-1 to C-96; M-1 to V-95; M-1 to W-94;M-1 to E-93; M-1 to H-92; M-1 to C-91; M-1 to E-90; M-1 to C-89; M-1 to L-88; M-1 to P-87; M-1 to G-86; M-1 to F-85; M-1 toF-84; M-1 to M-83; M-1 to G-82; M-1 to P-81; M-1 to E-80; M-1 to T-79; M-1 to V- 78; M-1 to H-77; M-1 to C-76; M-1 to I-75;M-1 to C-74; M-1 to V-73; M-1 to G-72; M- 1 to C-71; M-1 to D-70; M-1 to C-69; M-1 to R-68; M-1 to G-67; M-1 to R-66; M-1 toG-65; M-1 to H-64; M-1 to C-63; M-1 to L-62; M-1 to A-61; M-1 to A-60; M-1 to G- 59; M-1 to P-58; M-1 to P-57; M-1 to Q-56;M-1 to G-55; M-1 to P-54; M-1 to A-53; M- 1 to R-52; M-1 to C-51; M-1 to R-50; M-1 to R-49; M-1 to E-48; M-1 to S-47; M-1 toE- 46; M-1 to A-45; M-1 to R-44; M-1 to S-43; M-1 to L-42; M-1 to R-41; M-1 to C-40; M- 1 to A-39; M-1 to A-38; M-1 to G-37;M-1 to P-36; M-1 to W-35; M-1 to S-34; M-1 to R-33; M-1 to L-32; M-1 to S-31; M-1 to P-30; M-1 to S-29; M-1 to F-28; M-1 toS-27; M-1 to Q-26; M-1 to P-25; M-1 to V-24; M-1 to A-23; M-1 to S-22; M-1 to L-21; M-1 to G-20; M-1 to A-19; M-1 to F-18;M-1 to L-17; M-1 to L-16; M-1 to S-15; M-1 to S-14; M-1 to A-13; M-1 to L-12; M-1 to L-l 1; M-1 to L-10; M-1 to F-9; M-1 toN-8; M-1 to R- 7; M-1 to F-6; of SEQ ID NO:30. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

In addition, the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NOJ2 which have been determined from the following related cDNA genes: HLHFV34R (SEQ ID NO:68), HSRDA85R (SEQ ID NO:69), HSRAZ62R (SEQ ID NO:70), HSRDA17R (SEQ ID NO:71), and HSLEC45R (SEQ ID NO:72).

Based on the sequence similarity to the human integrin beta-8 subunit, translation product of this gene is expected to share at least some biological activities with integrin proteins, and specifically the human integrin beta-8 subunit. Such activities are known in the art, some of which are described elsewhere herein.

Specifically, polynucleotides and polypeptides of the invention are also useful for modulating the differentiation of normal and malignant cells, modulating the proliferation and/or differentiation of cancer and neoplastic cells, and modulating the immune response. Polynucleotides and polypeptides of the invention may represent a diagnostic marker for hematopoietic and immune diseases and/or disorders. The full- length protein should be a secreted protein, based upon homology to the integrin family. Therefore, it is secreted into serum, urine, or feces and thus the levels is assayable from patient samples. Assuming specific expression levels are reflective of the presence of immune disorders, this protein would provide a convenient diagnostic for early detection. In addition, expression of this gene product may also be linked to the progression of immune diseases, and therefore may itself actually represent a therapeutic or therapeutic target for the treatment of cancer. Polynucleotides and polypeptides of the invention may play an important role in the pathogenesis of human cancers and cellular transformation, particularly those of the immune and hematopoietic systems. Polynucleotides and polypeptides of the invention may also be involved in the pathogenesis of developmental abnormalities based upon its potential effects on proliferation and differentiation of cells and tissue cell types. Due to the potential proliferating and differentiating activity of said polynucleotides and polypeptides, the invention is useful as a therapeutic agent in inducing tissue regeneration, for treating inflammatory conditions (e.g., inflammatory bowel syndrome, diverticulitis, etc.). Moreover, the invention is useful in modulating the immune response to aberrant polypeptides, as may exist in rapidly proliferating cells and tissue cell types, particularly in adenocarcinoma cells, and other cancers.

Alternatively, the expression within cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis, treatment, and/or prevention of developmental diseases and disorders, including cancer, and other proliferative conditions. Representative uses are described in the "Hyperproliferative Disorders" and "Regeneration" sections below and elsewhere herein. Briefly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation.

Dysregulation of apoptosis can result in inappropriate suppression of cell death, as occurs in the development of some cancers, or in failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain neurodegenerative disorders, such as spinal muscular atrophy (SMA).

Alternatively, this gene product is involved in the pattern of cellular proliferation that accompanies early embryogenesis. Thus, aberrant expression of this gene product in tissues - particularly adult tissues - may correlate with patterns of abnormal cellular proliferation, such as found in various cancers. Because of potential roles in proliferation and differentiation, this gene product may have applications in the adult for tissue regeneration and the treatment of cancers. It may also act as a morphogen to control cell and tissue type specification. Therefore, the polynucleotides and polypeptides of the present invention are useful in treating, detecting, and/or preventing said disorders and conditions, in addition to other types of degenerative conditions. Thus this protein may modulate apoptosis or tissue differentiation and is useful in the detection, treatment, and/or prevention of degenerative or proliferative conditions and diseases. The protein is useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues. The protein can also be used to gain new insight into the regulation of cellular growth and proliferation. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues. The translation product of this gene, sometimes refeπ'ed to herein as TIDE (for

Ten Integrin Domains with EGF homology), shares sequence homology with integrins, which are a superfamily of dimeric ab cell-surface glycoproteins that mediate the adhesive functions of many cell types, enabling cells to interact with one another and with the extracellular matrix (See Genomics 56, 169-178 (1999); all information and references contained within this publication are hereby incorporated herein by reference). The gene encoding the disclosed cDNA is believed to reside on chromosome 13, at locus 13q33. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 13, generally, and particularly at locus 13q33. This gene is expressed primarily in synovial hypoxia tissue, osteoblast and osteoclast, bone marrow stromal cells, and to a lesser extent in umbilical vein, smooth muscle, placenta, and fetal lung cDNA libraries. Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, disorders of bone and connective tissues, immune and hematopoietic diseases and/or disorders, vascular disorders, and other disorders involving aberrations in cell-surface interactions. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the connective tissue and skeletal system, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g. cartilage, bone, vascular, hypoxic tissue, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 30 as residues: Met-1 to Phe-6, Arg-44 to Arg-52, His-64 to Cys- 69, Tyr-99 to Gln-147, His-158 to Gly-169, Phe-177 to Asp-182, Cys-194 to Cys-202, Gly-213 to Phe-218, Pro-224 to Gly-236, Asp-254 to Trp-261, Asp-263 to Ala-303, Trp- 305 to Cys-316, Lys-326 to Asp-332, Pro-334 to Cys-343, Pro-350 to Asp-370, Thr-407 to Asn-413, Gly-425 to Cys-431, Asp-449 to Asp-459, Gly-472 to Asn-483. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution and homology to the human integrin beta-8 subunit indicates polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the "Immune Activity" and "infectious disease" sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein.

Briefly, the expression indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. Involvement in the regulation of cytokine production, antigen presentation, or other processes indicates a usefulness for treatment of cancer (e.g. by boosting immune responses). Expression in cells of lymphoid origin, indicates the natural gene product is involved in immune functions. Therefore it would also be useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous Disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T- cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's Disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Based upon the tissue distribution of this protein, antagonists directed against this protein is useful in blocking the activity of this protein. Accordingly, preferred are antibodies which specifically bind a portion of the translation product of this gene.

Also provided is a kit for detecting tumors in which expression of this protein occurs. Such a kit comprises in one embodiment an antibody specific for the translation product of this gene bound to a solid support. Also provided is a method of detecting these tumors in an individual which comprises a step of contacting an antibody specific for the translation product of this gene to a bodily fluid from the individual, preferably serum, and ascertaining whether antibody binds to an antigen found in the bodily fluid. Preferably the antibody is bound to a solid support and the bodily fluid is serum. The above embodiments, as well as other treatments and diagnostic tests (kits and methods), are more particularly described elsewhere herein. Furthermore, the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 12 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a- b, where a is any integer between 1 to 2485 of SEQ ID NOJ2, b is an integer of 15 to 2499, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 12, and where b is greater than or equal to a + 14.

FEATURES OFPROTEIN ENCODED BY GENE NO: 3

The translation product of this gene shares sequence homology with RAMP3 (receptor-activity-modifying proteins), which another group has recently published, which is thought to be important in the transport of the calcitonin-receptor-like receptor (CRLR) to the plasma membrane. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like-receptor. There are two other related receptor-activity- modifying proteins, known as RAMP1 and RAMP2 (Nature 1998 May 28;393(6683):333-9). RAMP1 is thought to present the receptor at the cell surface as a mature glycoprotein and a Calcitonin-gene-related peptide (CGRP) receptor. Alternatively, RAMP2-tran sported receptors are core-glycosylated and are adrenomedullin receptors. CGRP (a 37-amino-acid neuropeptide) and its receptors are widely distributed in the body, and it is the most potent endogenous vasodilatory peptide discovered so far (Crit Rev Neurobiol 1997J 1(2-3): 167-239). Specific binding sites for adrenomedullin were present in every region of human brain (cerebral cortex, cerebellum, thalamus, hypothalamus, pons and medulla oblongata), suggesting that a novel neurotransmitter/neuromodulator role may exist for adrenomedullin in human brain (Peptides 1997; 18(8): 1125-9).

Figures 7A-B show the nucleotide (SEQ ID NO: 13) and deduced amino acid sequence (SEQ ID NO:31 ) of the Intestine derived extracellular protein. Predicted amino acids from about 1 to about 27 constitute the predicted signal peptide (amino acid residues from about 1 to about 27 in SEQ ID NO:31) and are represented by the underlined amino acid regions; and amino acids from about 122 to about 138 constitute the predicted transmembrane domain (amino acid residues from about 122 to about 138 in SEQ ID NO:31) and are represented by the double-underlined amino acids.

Figure 8 shows the regions of similarity between the amino acid sequences of the Intestine derived extracellular protein SEQ ID NO:31, and the RAMP3 protein (gi|4587099) (SEQ ID NO: 75).

Figure 9 shows an analysis of the amino acid sequence of SEQ ID NO: 31.

Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.

Northern analysis indicates that a 1.4kb transcript of this gene is primarily expressed in small intestine tissue, and to a lesser extent in colon and prostate tissue.

The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the polypeptide having the amino acid sequence shown in Figure 1 (SEQ ID NO:31), which was determined by sequencing a cloned cDNA (HTLEW81). The nucleotide sequence shown in Figures 7A-B (SEQ ID NO: 13) was obtained by sequencing a cloned cDNA (HTLEW81), which was deposited on Nov. 17, 1998 at the American Type Culture Collection, and given Accession Number 203484. The deposited gene is inserted in the pSport plasmid (Life Technologies, Rockville, MD) using the Sall/Notl restπction endonuclease cleavage sites. The present invention is further directed to fragments of the isolated nucleic acid molecules descπbed herein. By a fragment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ ID NO: 13 is intended DNA fragments at least about 15nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and pπmeis as discussed heiein Of couise, larger fragments 50-1500 nt in length are also useful according to the present invention, as are fragments conesponding to most, if not all, of the nucleotide sequence of the deposited cDNA oi as shown in SEQ ID NOJ3 By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or moie contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NO: 13. In this context "about" includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Representative examples of polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, and from about 501 to about 550, and from about 551 to about 600, from about 601 to about 650, from about 651 to about 700, from about 701 to about 750, from about 751 to about 800, from about 801 to about 850, from about 851 to about 900, from about 901 to about 950, from about 951 to about 1000, from about 1001 to about 1050, from about 1051 to about 1100, from about 1101 to about 1150, from about 1151 to about 1200, from about 1201 to about 1250, from about 1251 to about 1300, from about 1301 to about 1339 of SEQ ID NOJ3, or the complementary strand thereto, or the cDNA contained in the deposited gene. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. In additional embodiments, the polynucleotides of the invention encode functional attributes of the corresponding protein.

Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions"), turn and turn-forming regions ("turn- regions"), coil and coil-forming regions ("coil-regions"), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions. The data representing the structural or functional attributes of the protein set forth in Figure 9 and/or Table III, as described above, was generated using the various modules and algorithms of the DNA*STAR set on default parameters. In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table IIII can be used to determine regions of the protein which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.

Certain preferred regions in these regards are set out in Figure 9, but may, as shown in Table III, be represented or identified by using tabular representations of the data presented in Figure 9. The DNA*STAR computer algorithm used to generate Figure 9 (set on the original default parameters) was used to present the data in Figure 9 in a tabular format (See Table III). The tabular format of the data in Figure 9 is used to easily determine specific boundaries of a preferred region. The above-mentioned preferred regions set out in Figure 9 and in Table III include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figures 7A-B. As set out in Figure 9 and in Table III, such preferred regions include Garnier- Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha- regions, beta-regions, and turn-regions, Kyte-Doohttle hydrophilic regions and Hopp- Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus- Schulz flexible regions, Jameson-Wolf regions of high antigenic index and Emini surface-forming regions. Even if deletion of one or more ammo acids from the N- terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g , biological activities, ability to multimeπze, etc.) may still be retained. For example, the ability of shortened muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the lesidues of the complete or mature polypeptide are removed from the N-termmus Whether a particulai polypeptide lacking N-termmal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods descnbed herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted N-termmal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides having one oi more residues deleted from the amino terminus of the ammo acid sequence shown in Figures 7A-B, up to the argmine residue at position number 143 and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides compπsing the ammo acid sequence of residues nl-148 of Figures 7A-B, where nl is an integer from 2 to 143 corresponding to the position of the amino acid residue in Figures 7A-B (which is identical to the sequence shown as SEQ ID NO:31). N-terminal deletions of the polypeptide of the invention shown as SEQ ID NO:31 include polypeptides compπsing the amino acid sequence of residues: E-2 to L-148; T-3 to L-148; G-4 toL- 148; A-5 to L-148; L-6 to L-148; R-7 to L-148; R-8 to L-148;P-9 to L-148; Q-10 to L- 148; L-l 1 to L-148; L-12 to L-148; P-13to L-148; L-14 to L-148; L-15 to L-148; L-16 to L-148; L-17 toL-148; L-18 to L-148; C-19 to L-148; G-20 to L-148; G-21 toL-148; C-22 to L-148; P-23 to L-148; R-24 to L-148; A-25 toL-148; G-26 to L-148; G-27 to L-148; C-28 to L-148; N-29 toL-148; E-30 to L-148; T-31 to L-148; G-32 to L-148; M-33 toL- 148; L-34 to L-148; E-35 to L-148; R-36 to L-148; L-37 toL-148; P-38 to L-148; L-39 to L- 148; C-40 to L-148; G-41 toL-148; K-42 to L-148; A-43 to L-148; F-44 to L-148; A- 45 toL-148; D-46 to L-148; M-47 to L-148; M-48 to L-148; G-49 toL- 148; K-50 to L- 148; V-51 to L-148; D-52 to L-148; V-53 toL-148; W-54 to L-148; K-55 to L-148; W-56 to L-148; C-57 toL-148; N-58 to L-148; L-59 to L-148; S-60 to L-148; E-61 toL-148; F- 62 to L-148; 1-63 to L-148; V-64 to L-148; Y-65 toL-148; Y-66 to L-148; E-67 to L-148; S-68 to L-148; F-69 toL-148; T-70 to L-148; N-71 to L-148; C-72 to L-148; T-73 toL- 148; E-74 to L-148; M-75 to L-148; E-76 to L-148; A-77 toL-148; N-78 to L-148; V-79 to L-148; V-80 to L-148; G-81 toL-148; C-82 to L-148; Y-83 to L-148; W-84 to L-148; P-85 toL-148; N-86 to L-148; P-87 to L-148; L-88 to L-148; A-89 toL-148; Q-90 to L- 148; G-91 to L-148; F-92 to L-148; 1-93 toL-148; T-94 to L-148; G-95 to L-148; 1-96 to L-148; H-97 toL-148; R-98 to L-148; Q-99 to L-148; F-100 to L-148; F-101 toL-148; S- 102 to L-148; N-103 to L-148; C-104 to L-148; T-105 toL-148; V-106 to L-148; D-107 to L-148; R-108 to L-148; V-109 toL-148; H-l 10 to L-148; L-l 11 to L-148; E-l 12 to L- 148; D-l 13 toL-148; P-l 14 to L-148; P-l 15 to L-148; D-l 16 to L-148; E-l 17 toL-148; V-l 18 to L-148; L-l 19 to L-148; 1-120 to L-148; P-121 toL-148; L-l 22 to L-148; 1-123 to L-148; V-124 to L-148; 1-125 toL-148; P-l 26 to L-148; V-127 to L-148; V-128 to L- 148; L-l 29 toL-148; T-130 to L-148; V-131 to L-148; A-132 to L-148; M-133 toL-148; A- 134 to L-148; G-135 to L-148; L-l 36 to L-148; V-137 toL-148; V-138 to L-148; W- 139 to L-148; R-140 to L-148; S-141 toL-148; K-142 to L-148; R-143 to L-148; of SEQ ID NO:31. Polypeptides encoded by these polynucleotides are also encompassed by the invention. Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification or loss of one or more biological functions of the protein, other functional activities (e.g., biological activities (e.g., ability to illicit mitogenic activity, induce differentiation of normal or malignant cells, bind to EGF receptors, etc.)), may still be retained. For example the ability to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or matuie polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted C-terminal amino acid residues may retain some biological oi immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response. Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the ammo acid sequence of the polypeptide shown in Figures 7A-B, up to the argmine residue at position number 7, and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising the ammo acid sequence of residues 1-ml of Figures 7A-B, where ml is an integer from 7 to 147 corresponding to the position of the amino acid residue in Figures 7A-B. Moreover, the invention provides polynucleotides encoding polypeptides compnsing, or alternatively consisting of, the ammo acid sequence of C- terminal deletions of the polypeptide of the invention shown as SEQ ID NO:31 include polypeptides compπsing the amino acid sequence of residues: M-1 to L-147; M-1 to T- 146;M-1 to D-145; M-1 to T-144; M-1 to R-143; M-1 to K-142; M-1 toS-141; M-1 to R- 140; M-1 to W-139; M-1 to V-138; M-1 to V-137;M-1 to L-136; M-1 to G-135; M-1 to A-134; M-1 to M-133; M-1 toA-132; M-1 to V-131; M-1 to T-130; M-1 to L-129; M-1 to VJ28;MJ to V-127; M-1 to P-126; M-1 to 1-125; M-1 to V-124; M-1 toI-123; M-1 to L-122; M-1 to P-121; M-1 to 1-120; M-1 to L-l 19;M-1 to V-l 18; M-1 to E-l 17; M-1 to D-l 16; M-1 to P-l 15; M-1 toP-114; M-l to D-l 13; M-1 to E-l 12; M-1 to L-l 11; M-1 to H-110;M-1 to V-109; M-1 to R-108; M-1 to D-107; M-1 to V-106; M-1 toT-105; M-1 to C-104; M-1 to N-103; M-1 to S-102; M-1 to F-101;M-1 to F-100; M-1 to Q-99; M-1 to R-98; M-1 to H-97; M-1 toI-96; M-1 to G-95; M-1 to T-94; M-1 to 1-93; M-1 to F-92; M-1 toG-91 ; M-1 to Q-90; M-1 to A-89; M-1 to L-88; M-1 to P-87; M-lto N-86; M-1 to P-85; M-1 to W-84; M-1 to Y-83; M-1 to C-82;M-1 to G-81 ; M-1 to V-80; M-1 to V-79; M-1 to N-78; M-1 toA-77; M-1 to E-76; M-1 to M-75; M-1 to E-74; M-1 to T-73; M-lto C-72; M-1 to N-71; M-1 to T-70; M-1 to F-69; M-1 to S-68;M-1 to E-67; M-1 to Y-66; M-1 to Y-65; M-1 to V-64; M-1 to 1-63 :M-1 to F-62; M-1 to E-61; M-1 to S-60; M-1 to L-59; M-1 to N-58; M-1 to C-57; M-1 to W-56; M-1 to K-55; M-1 to W-54; M-1 toV- 53; M-1 to D-52; M-1 to V-51 ; M-1 to K-50; MJ to G-49; M-lto M-48; M-1 to M-47; M-1 to D-46; M-1 to A-45; M-1 to F-44;M-1 to A-43; M-1 to K-42; M-1 to G-41; M-1 to C-40; M-1 toL-39; M-1 to P-38; M-1 to L-37; M-1 to R-36; M-1 to E-35; M-1 toL-34; M-1 to M-33; M-1 to G-32; M-1 to T-31; M-1 to E-30; M-lto N-29; M-1 to C-28; M-1 to G-27; M-1 to G-26; M-1 to A-25;M-1 to R-24; M-1 to P-23; M-1 to C-22; M-1 to G- 21; M-1 toG-20; M-1 to C-19; M-1 to L-18; M-1 to L-17; M-1 to L-16; M-lto L-15; M-1 to L-14; M-1 to P-13; M-1 to L-12; M-1 to L-l 1; M-lto Q-10; M-1 to P-9; M-1 to R-8; M-1 to R-7; of SEQ ID NO:31. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

In addition, the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO:31 which have been determined from the following related cDNA genes: HLHCH17RA (SEQ ID NO:76), HTOAT51R (SEQ ID NO:77), and/or HBNBO41R (SEQ ID NO:78). The polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 122 - 138 of the amino acid sequence referenced in Table XIII for this gene. Moreover, a cytoplasmic tail encompassing amino acids 139 to 149 of this protein has also been determined. Based upon these characteπstics, it is believed that the protein product of this gene shares structural features to type la membrane proteins.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tιssue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, gastrointestinal and neurodegenerative diseases and disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tιssue(s) or cell type(s). For a numbei of disorders of the above tissues or cells, particularly of the central nervous and gastrointestinal systems, expression of this gene at significantly highei or lower levels is routinely detected in certain tissues or cell types (e.g. brain, CNS, gastrointestinal, cancel ous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, uπne, synovial fluid and spmal fluid) oi another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 31 as residues. Ala-5 to Gin- 10, Pro-23 to Cys-28, Arg- 140 to Asp- 145. Polynucleotides encoding said polypeptides are also provided. The tissue distπbution and homology to RAMP3 suggest that the translation product of this gene is useful for the detection/treatment of neurodegenerative disease states and behavioural disorders such as Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, the gene or gene product may also play a role in the treatment and/or detection of developmental disorders associated with the developing embryo. Alternatively, the tissue distπbution in small intestine and colon tissues indicates that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and/or treatment of disorders involving the small intestine. This may include diseases associated with digestion and food absorption, as well as hematopoietic disorders involving the Peyer's patches of the small intestine, or other hematopoietic cells and tissues within the body. Similarly, expression of this gene product in colon tissue indicates again involvement in digestion, piocessing, and elimination of food, as well as a potential lole for this gene as a diagnostic marker or causative agent in the development of colon cancer, and cancer in general Protein, as well as. antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 13 and may have been publicly available pnor to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence descπbed by the general formula of a- b, where a is any integer between 1 to 1325 of SEQ ID NOJ3, b is an integer of 15 to 1339, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 13, and where b is greater than or equal to a + 14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 4

The translation product of this gene shares sequence homology with a proteoglycan from Gallus gallus, and this proteoglycan is believed to participate in the osteogenic processes of cartilage ossification (See Genbank Accession No. gι|222847) Based on the sequence similaπty. The translation product of this gene is expected to share biological activities with the Gallus gallus proteoglycan polypeptide.

Figures 10A-B shows the nucleotide (SEQ ID NOJ4) and deduced amino acid sequence (SEQ ID NO.32) of the retinal specific protein Predicted ammo acids from about 1 to about 21 constitute the predicted signal peptide (amino acid residues from about 1 to about 21 SEQ ID NO 32) and are repiesented by the undei lined amino acid iegions

Figure 1 1 shows the regions of similarity between the amino acid sequences of the letinal specific protein SEQ ID NO 32, and the Gallus gallus pioteoglycan (SEQ ID NO 79)

Figuie 12 shows an analysis of the ammo acid sequence of SEQ ID NO: 32.

Alpha, beta, turn and coil regions, hydrophilicity and hydrophobicity: amphipathic regions; flexible regions, antigenic index and surface probability are shown Northern analysis indicates that this gene is expressed in adrenal cortex and adrenal medulla tissues. This gene is also expressed in retinal tissue

The present invention provides isolated nucleic acid molecules compπsing a polynucleotide encoding the polypeptide having the amino acid sequence shown in Figures 10A-B (SEQ ID NO:32), which was determined by sequencing a cloned cDNA (HARAO44). The nucleotide sequence shown in Figures 10A-B (SEQ ID NOJ4) was obtained by sequencing a cloned cDNA (HARAO44), which was deposited on Nov. 17, 1998 at the Ameπcan Type Culture Collection, and given Accession Number 203484 The deposited gene is inserted in the pSport plasmid (Life Technologies, Rockville, MD) using the Sall/Notl restπction endonuclease cleavage sites. The present invention is further directed to fragments of the isolated nucleic acid molecules descπbed herein. By a fragment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ ID NOJ4 is intended DNA fragments at least about 15nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt length which are useful as diagnostic probes and pπmers as discussed herein. Of course, larger fragments 50-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA oi as shown in SEQ ID NO: 14. By a fragment at least 20 nt in length, tor example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NOJ4. In this context "about" includes the particularly recited size, larger or smallei by several (5, 4, 3, 2, oi 1) nucleotides, at either terminus oi at both termini

Representative examples of polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, and from about 501 to about 550, and from about 551 to about 600, from about 601 to about 650, from about 651 to about 700, from about 701 to about 750, from about 751 to about 800, from about 801 to about 850, from about 851 to about 900, from about 901 to about 950, from about 951 to about 1000, from about 1001 to about 1050, from about 1051 to about 1100, from about 1101 to about 1150, from about 1151 to about 1200, from about 1201 to about 1250, from about 1251 to about 1300, from about 1301 to about 1350, from about 1351 to about 1389, and from about 187 to about 1119 of SEQ ID NO: 14, or the complementary strand thereto, or the cDNA contained in the deposited gene. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. In additional embodiments, the polynucleotides of the invention encode functional attπbutes of the corresponding protein.

Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions"), turn and turn-forming regions ("turn- regions"), coil and coil -forming iegions ("coil-regions"), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions. The data representing the structural or functional attnbutes of the protein set forth in Figure 12 and/or Table IV, as descπbed above, was generated using the various modules and algorithms of the

DNA'STAR set on default parameters In a preten^ded embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table IV can be used to determine regions of the protein which exhibit a high degiee of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.

Certain preferred regions in these regards are set out in Figure 12, but may, as shown in Table IV, be represented or identified by using tabular representations of the data presented in Figure 12. The DNA STAR computer algorithm used to generate

Figure 12 (set on the oπginal default parameters) was used to present the data in Figure 12 in a tabular format (See Table IV). The tabular format of the data in Figure 12 is used to easily determine specific boundaries of a preferred region. The above-mentioned preferred regions set out in Figure 12 and m Table IV include, but are not limited to, regions of the aforementioned types identified by analysis of the ammo acid sequence set out in Figures 10A-B. As set out in Figure 12 and in Table IV, such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doohttle hydrophilic regions and Hopp- Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenic index and Emini surface-forming regions. Even if deletion of one or more amino acids from the N-terminus of a protein results modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimeπze, etc.) may still be retained Foi example, the ability of shortened muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed fiom the N-termmus Whether a paiticulai polypeptide lacking N-terminal lesidues of a complete polypeptide retains such immunologic activities can readily be determined by loutine methods described herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted N-termmal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the ammo acid sequence shown in Figures 10A-B, up to the proline residue at position number 327 and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides composing the ammo acid sequence of residues nl-332 of Figures 10A-B, where nl is an integer from 2 to 327 corresponding to the position of the ammo acid residue in Figures 10A-B (which is identical to the sequence shown as SEQ ID NO:32). N-terminal deletions of the polypeptide of the invention shown as SEQ ID NO:32 include polypeptides comprising the amino acid sequence of residues: R-2 to T-332; L-3 to T-

332; L-4 to T-332; A-5 to T-332;F-6 to T-332; L-7 to T-332; S-8 to T-332; L-9 to T-332; L-10 to T-332; A-l 1 to T-332; L-12 to T-332; V-13 to T-332;L-14 to T-332; Q-15 to T- 332; E-16 to T-332; T-17 to T-332; G-18 to T-332; T-19 to T-332; A-20 to T-332; S-21 toT-332; L-22 to T-332; P-23 to T-332; R-24 to T-332; K-25 to T-332; E-26 to T-332; R- 27 to T-332; K-28 to T-332;R-29 to T-332; R-30 to T-332; E-31 to T-332; E-32 to T- 332; Q-33 to T-332; M-34 to T-332; P-35 to T-332; R-36 toT-332; E-37 to T-332; G-38 to T-332; D-39 to T-332; S-40 to T-332; F-41 to T-332; E-42 to T-332; V-43 to T-332;L- 44 to T-332; P-45 to T-332; L-46 to T-332; R-47 to T-332; N-48 to T-332; D-49 to T- 332; V-50 to T-332: L-51 toT-332; N-52 to T-332; P-53 to T-332; D-54 to T-332; N-55 to T-332; Y-56 to T-332; G-57 to T-332; E-58 to T-332;V-59 to T-332; 1-60 to T-332; D- 61 to T-332; L-62 to T-332; S-63 to T-332; N-64 to T-332; Y-65 to T-332; E-66 toT- 332; E-67 to T-332; L-68 to T-332; T-69 to T-332; D-70 to T-332; Y-71 to T-332; G-72 to T-332; D-73 to T-332;Q-74 to T-332; L-75 to T-332; P-76 to T-332; E-77 to T-332; V-78 to T-332; K-79 to T-332; V-80 to T-332; T-81 toT-332; S-82 to T-332; L-83 to T- 332; A-84 to T-332; P-85 to T-332; A-86 to T-332; T-87 to T-332; S-88 to T-332;I-89 to T-332; S-90 to T-332; P-91 to T-332; A-92 to T-332; K-93 to T-332; S-94 to T-332; T- 95 to T-332; T-96 toT-332; A-97 to T-332; P-98 to T-332; G-99 to T-332; T-100 to T- 332; P-101 to T-332; S-102 to T-332; S-103 toT-332; N-104 to T-332; P-105 to T-332; T-106 to T-332; M-107 to T-332; T-108 to T-332; R-109 to T-332; P-l 10 toT-332; T- 111 to T-332; T-l 12 to T-332; A-l 13 to T-332; G-l 14 to T-332; L-l 15 to T-332; L-l 16 to T-332; L-l 17 toT-332; S-l 18 to T-332; S-119 to T-332; Q-120 to T-332; P-121 to T- 332; N-122 to T-332; H-123 to T-332; G-124 toT-332; L-125 to T-332; P-126 to T-332; T-l 27 to T-332; C-128 to T-332; L-129 to T-332; V-130 to T-332; C-131 toT-332; V- 132 to T-332; C-133 to T-332; L-134 to T-332; G-135 to T-332; S-136 to T-332; S-137 to T-332; V-138 toT-332; Y-139 to T-332; C-140 to T-332; D-141 to T-332; D-142 to T- 332; 1-143 to T-332; D-144 to T-332; L-145 toT-332; E-146 to T-332; D-147 to T-332; 1-148 to T-332; P-149 to T-332; P-150 to T-332; L-151 to T-332; P-152 toT-332; R-153 to T-332; R-154 to T-332; T-155 to T-332; A-156 to T-332; Y-157 to T-332; L-158 to T- 332; Y-159 toT-332; A-160 to T-332; R-161 to T-332; F-162 to T-332; N-163 to T-332; R-164 to T-332; 1-165 to T-332; S-166 toT-332; R-167 to T-332; 1-168 to T-332; R-169 to T-332; A- 170 to T-332; E-171 to T-332; D-172 to T-332; F-173 toT-332; K-174 to T- 332; G-175 to T-332; L-176 to T-332; T-177 to T-332; K-178 to T-332; L-179 to T-332; K-180 toT-332; R-181 to T-332; 1-182 to T-332; D-183 to T-332; L-184 to T-332; S-185 to T-332; N-186 to T-332; N-187 toT-332; L-188 to T-332; 1-189 to T-332; S-190 to T- 332; S-191 to T-332; 1-192 to T-332; D-193 to T-332; N-194 toT-332; D-195 to T-332; A- 196 to T-332; F-197 to T-332; R-198 to T-332; L-l 99 to T-332; L-200 to T-332; H- 201 toT-332; A-202 to T-332; L-203 to T-332; Q-204 to T-332; D-205 to T-332; L-206 to T-332; 1-207 to T-332; L-208 toT-332; P-209 to T-332; E-210 to T-332; N-211 to T- 332; Q-212 to T-332; L-213 to T-332; E-214 to T-332; A-215 toT-332; L-216 to T-332; P-217 to T-332; V-218 to T-332; L-219 to T-332; P-220 to T-332; S-221 to T-332; G- 222 toT-332; 1-223 to T-332; E-224 to T-332; F-225 to T-332; L-226 to T-332; D-227 to T-332; V-228 to T-332; R-229 toT-332; L-230 to T-332; N-231 to T-332; R-232 to T- 332; L-233 to T-332; Q-234 to T-332; S-235 to T-332; S-236 toT-332; G-237 to T-332; 1-238 to T-332; Q-239 to T-332; P-240 to T-332; A-241 to T-332; A-242 to T-332; F- 243 toT-332; R-244 to T-332; A-245 to T-332: M-246 to T-332; E-247 to T-332; K-248 to T-332; L-249 to T-332; Q-250 toT-332; F-251 to T-332; L-252 to T-332; Y-253 to T- 332; L-254 to T-332; S-255 to T-332; D-256 to T-332; N-257 toT-332; L-258 to T-332; L-259 to T-332; D-260 to T-332; S-261 to T-332; 1-262 to T-332; P-263 to T-332; G-264 toT-332; P-265 to T-332; L-266 to T-332; P-267 to T-332; P-268 to T-332; S-269 to T- 332; L-270 to T-332; R-271 toT-332; S-272 to T-332; V-273 to T-332; H-274 to T-332; L-275 to T-332; Q-276 to T-332; N-277 to T-332; N-278 toT-332; L-279 to T-332; 1-280 to T-332; E-281 to T-332; T-282 to T-332; M-283 to T-332; Q-284 to T-332; R-285 toT- 332; D-286 to T-332; V-287 to T-332; F-288 to T-332; C-289 to T-332; D-290 to T-332; P-291 to T-332; E-292 toT-332; E-293 to T-332; H-294 to T-332; K-295 to T-332; H- 296 to T-332; T-297 to T-332; R-298 to T-332; R-299 toT-332; Q-300 to T-332; L-301 to T-332; E-302 to T-332; D-303 to T-332; 1-304 to T-332; R-305 to T-332; L-306 toT- 332; D-307 to T-332; G-308 to T-332; N-309 to T-332; P-310 to T-332; 1-311 to T-332; N-312 to T-332; L-313 toT-332; S-314 to T-332; L-315 to T-332; F-316 to T-332; P-317 to T-332; S-318 to T-332; A-319 to T-332; Y-320 toT-332; F-321 to T-332; C-322 to T- 332; L-323 to T-332; P-324 to T-332; R-325 to T-332; L-326 to T-332; P-327 toT-332; of SEQ ID NO:32. Polypeptides encoded by these polynucleotides are also encompassed by the invention

Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification oi loss of one or more biological functions of the protein, other functional activities (e g., biological activities (e.g., ability to illicit mitogenic activity, induce difteientiation ot normal or malignant cells, bind to EGF receptors, etc.)), may still be retained For example the ability to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majoπty of the lesidues of the complete or mature polypeptide are removed from the C-terminus Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods descπbed herein and otherwise known m the art. It is not unlikely that a mutein with a large number of deleted C-termmal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the polypeptide shown in Figures 10A-B, up to the glutamine residue at position number 7, and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides compπsing the ammo acid sequence of residues 1-ml of Figures 10A-B, where ml is an integer from 7 to 331 corresponding to the position of the amino acid residue in Figures 10A-B. Moreover, the invention provides polynucleotides encoding polypeptides compπsing, or alternatively consisting of, the ammo acid sequence of C-terminal deletions of the polypeptide of the invention shown as SEQ ID NO:32 include polypeptides comprising the amino acid sequence of residues: M-1 to F- 331; M-1 to R-330; M-1 to G-329; M-1 to 1-328; M-1 to P-327; M-1 to L-326; M-1 to R- 325; M-1 to P-324; M-1 to L-323; M-1 to C-322; M-1 to F-321; M-lto Y-320; M-1 to A- 319; M-1 to S-318; M-1 to P-317; M-1 to F-316; M-1 to L-315; MJ to S-314; M-l to L- 313; M-lto N-312; M-1 to 1-311; M-1 to P-310; M-l to N-309; M-l to G-308; M-1 to D- 307; M-1 to L-306; M-1 to R-305; M-lto 1-304; M-1 to D-303; M-1 to E-302; M-1 to L- 301 ; M-1 to Q-300; M-1 to R-299; M-1 to R-298; M-1 to T-297; M-lto H-296; M-1 to K-295; M-1 to H-294; M-1 to E-293; M-1 to E-292; M-1 to P-291; M-1 to D-290; M-1 to C-289; M-lto F-288; M-1 to V-287: M-1 to D-286; M-1 to R-285; M-1 to Q-284; M-1 to M-283; M-1 to T-282; M-1 to E-281; M-lto 1-280; M-1 to L-279; M-1 to N-278; M-1 to N-277; M-1 to Q-276; M-1 to L-275; M-1 to H-274; M-1 to V-273; M-lto S-272; M-1 to R-271; M-1 to L-270; M-1 to S-269; M-1 to P-268; M-1 to P-267; M-1 to L-266; M-1 to P-265; M-lto G-264; M-1 to P-263; M-1 to 1-262; M-1 to S-261; M-1 to D-260; M-1 to L-259; M-1 to L-258; M-1 to N-257; M-lto D-256; M-1 to S-255; M-1 to L-254; M-1 to Y-253; M-1 to L-252; M-1 to F-251; M-1 to Q-250; M-1 to L-249; M-lto K-248; M-1 to E-247; M-1 to M-246; M-1 to A-245; M-1 to R-244; M-1 to F-243; M-1 to A-242; M- 1 to A-241 ;M-1 to P-240; M-1 to Q-239; M-1 to 1-238; M-1 to G-237; M-1 to S-236; M- 1 to S-235; M-1 to Q-234; M-1 to L-233;M-1 to R-232; M-1 to N-231; M-1 to L-230; M- 1 to R-229; M-1 to V-228; M-1 to D-227; M-1 to L-226; M-1 to F-225;M-1 to E-224; M- 1 to 1-223; M-1 to G-222; M-1 to S-221 ; M-1 to P-220; M-1 to L-219; M-1 to V-218; M- 1 to P-217;M-1 to L-216; M-1 to A-215; M-1 to E-214; M-1 to L-213; M-1 to Q-212; M- 1 to N-211; M-1 to E-210; M-1 to P-209;M-1 to L-208; M-1 to 1-207; M-1 to L-206; M- 1 to D-205; M-1 to Q-204; M-1 to L-203; M-1 to A-202; M-1 to H-20LM-1 to L-200; M-1 to L-199; M-1 to R-198; M-1 to F-197; M-1 to A-196; M-1 to D-195; M-1 to N- 194; M-1 to D-193;M-l to 1-192; M-1 to S-191; M-l to S-190; M-l to 1-189; M-1 to L- 188; M-1 to N-187; M-1 to N-186; M-1 to S-185;M-1 to L-184; M-1 to D-183; M-1 to I- 182; M-1 to R-181; M-1 to K-180; M-1 to L-179; M-1 to K-178; M-1 to T-177;M-1 to L-176; M-1 to G-175; M-1 to K-174; M-1 to F-173; M-1 to D-172; M-1 to E-171; M-1 to A-170; M-1 to R-169;M-1 to 1-168; M-1 to R-167; M-1 to S-166; M-1 to 1-165; M-1 to R-164; M-1 to N-163; M-1 to F-162; M-1 to R-161;M-1 to A-160; M-1 to Y-159; M-1 to L-158; M-1 to Y-157; M-1 to A-156; M-1 to T-155; M-1 to R-154; M-1 to R-153;M-1 toP-152;M-l toL-151;M-l toP-150;M-l toP-149;M-l to 1-148; M-1 toD-147;M-l to E-146; M-1 to L-145;M-1 to D-144; M-1 to 1-143; M-1 to D-142; M-1 to D-141: M-1 to C-140; M-1 to Y-139; M-1 to V-138; M-1 to S-137;M-1 to S-136; M-1 to G-135; M-1 to L-134; M-1 to C-133; M-1 to V-132; M-1 to C-131; M-1 to V-130; M-1 to LJ29;MJ to C-128; M-1 to T-127; M-1 to P-126; M-1 to L-125; M-1 to G-124; M-1 to H-123; M-1 toN-122;M-l toP-121;M-l to QJ20; MJ toS-119;M-l toS-118;M-l to L-l 17: M-1 to L-l 16; M-1 toL-115;M-l to G-l 14; M-1 toA-113;M-l toT-112;M-l to T-l 11; M-1 to P-l 10; M-1 toR-109;M-l toT-108;M-l to MJ07; MJ toT-106;M-l toP-105;M-l toN-104;M-l to S-103; M-1 to S-102; M-1 toP-101;M-l toT-100;M-l toG-99:M-l to P-98; M-1 to A-97;M-1 to T-96; M-1 to T-95; M-1 to S-94; M-1 to K-93; M-1 to A- 92; M-1 to P-91; M-1 to S-90; M-1 to 1-89; M-1 toS-88; M-1 to T-87; M-1 to A-86; M-1 to P-85; M-1 to A-84; M-1 to L-83; M-1 to S-82; M-1 to T-81; M-1 to V-80; M-lto K- 79; M-1 to V-78; M-1 to E-77; M-1 to P-76; M-1 to L-75; M-1 to Q-74; M-1 to D-73; M-1 to G-72; M-1 to Y-71;M-1 to D-70; M-1 to T-69; M-1 to L-68; M-1 to E-67; M-1 to E-66; M-1 to Y-65; M-1 to N-64; M-1 to S-63; M-1 toL-62; M-1 to D-61; M-1 to 1-60; M-1 to V-59; M-1 to E-58; M-1 to G-57; M-1 to Y-56; M-1 to N-55; M-1 to D-54;M-1 to P-53; M-1 to N-52; M-1 to L-51; M-1 to V-50; M-1 to D-49; M-1 to N-48; M-1 to R- 47; M-1 to L-46; M-1 toP-45; M-1 to L-44; M-1 to V-43; M-1 to E-42; M-1 to F-41; M-1 to S-40; M-1 to D-39; M-1 to G-38; M-1 to E-37; M-lto R-36; M-1 to P-35; M-1 to M- 34; M-1 to Q-33; M-1 to E-32; M-1 to E-31; M-1 to R-30; M-1 to R-29; M-1 to K-28;M- 1 to R-27; M-1 to E-26; M-1 to K-25; M-1 to R-24; M-1 to P-23; M-1 to L-22; M-1 to S- 21; M-1 toA-20;M-l toT-19;M-l to G-18; M-1 to T-17; M-1 to E-16; M-1 toQJ5;M- 1 to L-14; M-1 to V-13; M-1 to L-12; M-1 to A-l 1;M-1 to L-10; M-1 to L-9; M-1 to S-8; M-1 to L-7; of SEQ ID NO:32. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

In addition, the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO: 14 which have been determined from the following related cDNA genes: HARAY79R (SEQ ID NO:80), HARAO44R (SEQ ID NO:81), HARAJ74R (SEQ ID NO:82), HARAO66R (SEQ ID NO:83), HARAN19R (SEQ ID NO:84), and HARAT78R (SEQ ID NO:85).

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to. retinal disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the retina, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g. retinal, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 32 as residues: Leu-22 to Asp-39, Asn-64 to Pro-76, Pro-98 to Thr-111, Pro-291 to Glu-302. Polynucleotides encoding said polypeptides are also provided. The tissue distribution in retinal tissue, and the homology to a Gallus gallus proteoglycan involved in the ossification process indicates that polynucleotides and polypeptides corresponding to this gene are useful for the treatment of disorders of the retina which involve the adhesion of tissues, or the binding of certain proteins to the cell surface. The translation products of this gene are useful for the treatment of retinal disorders such as retinal detachment in individuals suffeπng from myopia, or in the treatment of macular degeneration. Furthermore, this gene may serve as a tumor marker for retinoblastomas, or related tumors. Moie generally, the tissue distribution in retinal tissue indicates that The translation pioduct ot this gene is useful for the diagnosis, detection and/or treatment of eye disordeis including blindness, color blindness, impaired vision, short and long sightedness, retinitis pigmentosa, retinitis prohferans, and retinoblastoma, retinochoroiditis, retmopathy and retinoschisis. Based upon the tissue distribution of this protein, antagonists dπected against this protein is useful in blocking the activity of this protein Accordingly, prefen-ed are antibodies which specifically bind a portion of the translation product of this gene.

Also provided is a kit for detecting tumors in which expression of this protein occurs. Such a kit comprises one embodiment an antibody specific for the translation product of this gene bound to a solid support. Also provided is a method of detecting these tumors in an individual which compπses a step of contacting an antibody specific for the translation product of this gene to a bodily fluid from the individual, preferably serum, and ascertaining whether antibody binds to an antigen found in the bodily fluid. Preferably the antibody is bound to a solid support and the bodily fluid is serum. The above embodiments, as well as other treatments and diagnostic tests (kits and methods), are more particularly described elsewhere herein. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate hgands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues. Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NOJ4 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a- b, where a is any integer between 1 to 1375 of SEQ ID NOJ4, b is an integer of 15 to 1389, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 14, and where b is greater than or equal to a + 14.

FEATURES OFPROTEIN ENCODED BY GENE NO: 5

The translation product of this gene shares sequence homology with the CD33 protein (See Genbank Accession No. gi|2913995). The expression pattern of CD33 within the hematopoietic system indicates a potential role in the regulation of myeloid cell differentiation. However, this expression is absent from hematopoietic stem cells. CD33 is expressed in clonogenic leukemia cells in about 90% of patients suffering from acute myeloid leukemia (AML). While about 60-70% of adults suffering from AML experience complete remission due to chemotherapy application, most of these patients will ultimately die of relapsed leukemia. It is believed that, like CD33, the CD33-like protein of the present invention is also expressed by clonogenic leukemia cells from the vast majority of patients with AML. Thus, there is a clear need to identify and isolate nucleic acid molecules encoding additional polypeptides having

CD33-like protein activity. It is believed that cancerous tissue contains significantly greater amounts of CD33-like protein gene copy number and expresses significantly enhanced levels of CD33-like protein and mRNA encoding the CD33-like protein when compared to a "standard" mammal, i.e.-a mammal of the same species not having the cancer or inflammatory disease. Thus, enhanced levels of the CD33-like protein will be detected in certain bodily fluids (e.g., serum, plasma, urine, synovial fluid and spinal fluid) from mammals when compared to sera from mammals of the same species not having the cancer or inflammatory disease.

Two related cDNA genes, HDPIB36 and HEOMH10, have been isolated. These cDNA genes appear to encode splice variants of this gene. Preferred polynucleotides comprise the following sequences: CGACCCACGCGTCCGCCGCCTTCGGCTTCCCCTTCTGCCAA

GAGCCCTGAGCCACTCACAGCACGACCAGAGA (SEQ ID NO: 86), GTATGGAATGGGGTGGGAACCCCTGCCTCTCACACTGGGGAGGGACCCTGGG GACAGCCTATGGGCTGAGCAGAGAGGGCTCTCAGGGACCCCTGCAGCACAA GAATCTCCCACCACGGTCTCTGTCCCAGCCCTGACTCAGAAGCCTGATGTCTA CATCCCCGAGACCCTGGAGCCCGGGCAGCCGGTGACGGTCATCTGTGTGTTT AACTGGGCCTTTGAGGAATGTCCACCCCCTTCTTTCTCCTGGACGGGGGCTGC CCTCTCCTCCCAAGGAACCAAACCAACGACCTCCCACTTCTCAG (SEQ ID NO: 87), ATCCTCCAGAGAACCTGAGAGTGATGGTTTCCCAAGCAAACAGGACAGGTA GGAAAGGGGACAGAGGAGCCAAGGCCTCTCAGTGCCGAATTGGGGGCCCAG GAGTCTGGAGGGTCCCCACGCAGGAGGGTCCCTGAGCCCTGAGCTGCTCATC GATTCTGCCTCTTCCTTCCCT (SEQ ID NO: 88),

GTGAGTGGGGGAAAGGGGACACCTGGGTCCCAGGAAGGGGACCCTGCTGAG TCCTGTCCTCCCTCCCCTCAG (SEQ ID NO: 89), CTGGCCCCCTGGCTCAGAAGCGGAATCAGAAAGCCACACCAAACAGTCCTCG GACCCCTCTTCCACCAGGTGCTCCCTCCCCAGAATCAAAGAAGAACCAGAAA AAGCAGTATCAGTTGCCCAGTTTCCCAGAACCCAAATCATCCACTCAAGCCC CAGAATCCCAGGAGAGCCAAGAGGAGCTCCATTATGCCACGCTCAACTTCCC AGGCGTCAGACCCAGGCCTGAGGCCCGGATGCCCAAGGGCACCCAGGCGGA TTATGCAGAAGTCAAGTTCCAATGAGGGTCTCTTAGGCTTTAGGACTGGGAC TTCGGCTAGGGAGGAAGGTAGAGTAAGAGGTTGAAGATAACAGAGTGCAAA GTTTCCTTCTCTCCCTCTCTCTCTCTCTTTCTCTCTCTCTCTCTCTTTCTCTCTCT TTT (SEQ ID NO: 90), and/or

AAAAAAACATCTGGCCAGGGCACAGTGGCTCACGCCTGTAATCCCAGCACTT TGGGAGGTTGAGGTGGGCAGATCGCCTGAGGTCGGGAGTTCGAGACCAGCC TGGCCAACTTGGTGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCTGGG CATGGTGGCAGGCGCCTGTA ATCCTACTACTTGGGAAGCTGAGGCAGG AG AA TCACTTGAACCTGGGAGACGGAGGTTGCAGTGAGCCAAGATCACACCATTGC ACGCCAGCTTGGGCAACAAAGCGAGACTCCATCTCAAAAAAAAAATCCTCC AAATGGGTTGGGTGTCTGTAATCCCAGCACTTTGGGAGGCTAAGGTGGGTGG ATTGCTTGAGCCCAGGAGTTCGAGACCAGCCTGGGCAACATGGTGAAACCCC ATCTCTAC AAAAAATACA AAAC ATAGCTGGGCTTGGTGGTGTGTGCCTGTAG TCCCAGCTGTCAGACATTTAAACCAGAGCAACTCCCATCTGGAATGGGAGCT GAATAAAATGAGGCTGAGACCTACTGGGCTGCCATTCTCAGACAGTGGAGGC CATTCTAAGTCACAGGATGAGACAGGAGGTCCGTACAAGATACAGGTCATA AAGACTTTGCTGATAAAACAGATTGCAGTAAAGAAGCCAACCAAATCCCACC AAAACCAAGTTGGCCACGAGAGTGACCTCTGGTCGTCCTCACTGCTACACTC CTGACAGCACCATGACAGTTTACAAATGCCATGGCAACATCAGGAAGTTACC CGATATGTCCCAAAAGGGGGAGGAATGAATAATCCACCCCTTGTTTAGCAAA TAAGCAAGAAATAACCATAAAAGTGGGCAACCAGCAGCTCTAGGCGCTGCT CTTGTCTATGGAGTAGCCATTCTTTTGTTCCTTTACTTTCTTAATAAACTTGCT TTCACCTTAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:91). Also preferred are the polypeptides encoded by these polynucleotides. Figures 13A-C shows the nucleotide (SEQ ID NOJ5) and deduced amino acid sequence (SEQ ID NO:33) of the CD33-like protein. Predicted amino acids from about 1 to about 16 constitute the predicted signal peptide (amino acid residues from about 1 to about 16 in SEQ ID NO:33) and are represented by the underlined amino acid regions; and amino acids from about 496 to about 512 constitute the predicted transmembrane domain (amino acid residues from about 496 to about 512 in SEQ ID NO:33) and are represented by the double-underlined amino acid regions.

Figure 14 shows the regions of similarity between the amino acid sequences of the CD33-like protein SEQ ID NO:33, and the CD33L1 protein (gi|88178) (SEQ ID NO: 92).

Figure 15 shows an analysis of the amino acid sequence of SEQ ID NO:33.

Northern analysis indicates that this gene is expressed highest in spleen tissue and peripheral blood leukocytes, and to a lesser extent in ovary and lung tissue.

The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the polypeptide having the amino acid sequence shown in Figures 13A-C (SEQ ID NO:33), which was determined by sequencing a cloned cDNA (HDPCL05). The nucleotide sequence shown in Figures 13A-C (SEQ ID NO: 15) was obtained by sequencing a cloned cDNA (HDPCL05), which was deposited on Nov. 17, 1998 at the American Type Culture Collection, and given Accession Number 203484. The deposited gene is inserted in the pSport plasmid (Life Technologies, Rockville, MD) using the Sall/Notl restriction endonuclease cleavage sites.

The present invention is further directed to fragments of the isolated nucleic acid molecules described herein. By a fragment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ ID NO: 15 is intended DNA fragments at least about 15nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments 50-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in SEQ ID NO: 15. By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NO: 15. In this context "about" includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Representative examples of polynucleotide fragments of the invention include. for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150. from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, from about 501 to about 550, from about 551 to about 600. from about 601 to about 650, from about 651 to about 700, from about 701 to about 750, from about 751 to about 800, from about 801 to about 850, from about 851 to about 900. from about 901 to about 950, from about 951 to about 1000, from about 1001 to about 1050, from about 1051 to about 1100, from about 1101 to about 1150, from about 1151 to about 1200, from about 1201 to about 1250, from about 1251 to about 1300, from about 1301 to about 1350, from about 1351 to about 1400, from about 1401 to about 1450, from about 1451 to about 1500, from about 1501 to about 1550, from about 1551 to about 1600, from about 1601 to about 1650, from about 1651 to about 1700, from about 1701 to about 1750, from about 1751 to about 1800, from about 1801 to about 1850, from about 1851 to about 1900, from about 1901 to about 1950, from about 1951 to about 2000, from about 2001 to about 2050, from about 2051 to about 2100, from about 2101 to about 2150, from about 2151 to about 2200, from about 2201 to about 2250, from about 2251 to about 2295, from about 307 to about 1977, and from about 106 to about 1977, of SEQ ID NOJ5, or the complementary strand thereto, or the cDNA contained in the deposited gene. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini In additional embodiments, the polynucleotides of the invention encode functional attnbutes of the corresponding piotein

Preferred embodiments of the invention in this regaid include ftagments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet forming iegions ("beta-regions"), turn and turn-forming regions ("turn- regions"), coil and coil-forming regions ("coil-iegions"), hydrophilic regions, hydrophobic regions, alpha amphipathic iegions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index iegions The data representing the structural or functional attπbutes of the protein set forth in Figure 15 and/oi Table V, as descnbed above, was generated using the vaπous modules and algorithms of the DNA^STAR set on default parameters In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table V can be used to determine regions of the protein which exhibit a high degree of potential for antigenicity Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response

Certain preferred regions m these regards are set out Figure 15, but may, as shown in Table V, be represented or identified by using tabular representations of the data presented in Figure 15. The DNA*STAR computer algoπthm used to generate Figure 15 (set on the oπgmal default parameters) was used to present the data m Figure 15 in a tabular format (See Table V). The tabular format of the data in Figure 15 is used to easily determine specific boundaries of a preferred region The above-mentioned preferred regions set out in Figure 15 and m Table V include, but are not limited to, regions of the aforementioned types identified by analysis of the ammo acid sequence set out in Figures 13A-C As set out in Figure 15 and in Table V, such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic iegions and Hopp-Woods hydiophobic iegions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible iegions, Jameson-Wolf iegions of high antigenic index and Emini surface-forming iegions Even if deletion of one oi more ammo acids fiom the N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e g , biological activities, ability to multimeπze, etc ) may still be retained For example, the ability of shortened muteins to induce and/or bind to antibodies which recognize the complete or matuie forms of the polypeptides generally will be retained when less than the majority of the lesidues of the complete or mature polypeptide are removed from the N-termmus Whether a paiticulai polypeptide lacking N-termmal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods descπbed herein and otherwise known in the art It is not unlikely that a mutein with a large number of deleted N-termmal amino acid residues may retain some biological or immunogenic activities In fact, peptides composed of as few as six amino acid lesidues may often evoke an immune response

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence shown in Figures 13A-C, up to the alanme residue at position number 634 and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising the ammo acid sequence of residues nl-639 of Figures 13A-C, where nl is an integer from 2 to 634 corresponding to the position of the amino acid residue in Figures 13A-C (which is identical to the sequence shown as SEQ ID NO 33) N-terminal deletions of the polypeptide of the invention shown as SEQ ID NO:33 include polypeptides comprising the amino acid sequence of residues: L-2 to Q-639; L-3 to Q- 639; P-4 to Q-639;L-5 to Q-639; L-6 to Q-639; L-7 to Q-639; S-8 to Q-639; S-9 toQ- 639; L-10 to Q-639; L-l 1 to Q-639; G-12 to Q-639; G-13 toQ-639; S-14 to Q-639; Q-15 to Q-639; A-16 to Q-639; M-17 toQ-639; D-18 to Q-639; G-19 to Q-639; R-20 to Q-639; F-21 toQ-639; W-22 to Q-639; 1-23 to Q-639; R-24 to Q-639; V-25 toQ-639; Q-26 to Q- 639; E-27 to Q-639; S-28 to Q-639; V-29 toQ-639; M-30 to Q-639; V-31 to Q-639: P-32 to Q-639; E-33 toQ-639; A-34 to Q-639; C-35 to Q-639; D-36 to Q-639; 1-37 toQ-639; S-38 to Q-639; V-39 to Q-639; P-40 to Q-639; C-41 toQ-639; S-42 to Q-639; F-43 to Q- 639; S-44 to Q-639; Y-45 toQ-639; P-46 to Q-639; R-47 to Q-639; Q-48 to Q-639; D-49 toQ-639; W-50 to Q-639; T-51 to Q-639; G-52 to Q-639; S-53 toQ-639; T-54 to Q-639; P-55 to Q-639; A-56 to Q-639; Y-57 toQ-639; G-58 to Q-639; Y-59 to Q-639; W-60 to Q-639; F-61 toQ-639; K-62 to Q-639; A-63 to Q-639; V-64 to Q-639; T-65 toQ-639; E- 66 to Q-639; T-67 to Q-639; T-68 to Q-639; K-69 toQ-639; G-70 to Q-639; A-71 to Q- 639; P-72 to Q-639; V-73 toQ-639; A-74 to Q-639; T-75 to Q-639; N-76 to Q-639; H-77 toQ-639; Q-78 to Q-639; S-79 to Q-639; R-80 to Q-639; E-81 toQ-639; V-82 to Q-639; E-83 to Q-639; M-84 to Q-639; S-85 toQ-639; T-86 to Q-639; R-87 to Q-639; G-88 to Q-639; R-89 toQ-639; F-90 to Q-639; Q-91 to Q-639; L-92 to Q-639; T-93 toQ-639; G- 94 to Q-639; D-95 to Q-639; P-96 to Q-639; A-97 toQ-639; K-98 to Q-639; G-99 to Q- 639; N-100 to Q-639; C-101 toQ-639; S-102 to Q-639; L-103 to Q-639; V-104 to Q-639; 1-105 toQ-639; R-106 to Q-639; D-107 to Q-639; A-108 to Q-639; Q-109to Q-639; M- 110 to Q-639; Q-l 11 to Q-639; D-l 12 to Q-639;E-113 to Q-639; S-114 to Q-639; Q-l 15 to Q-639; Y-116 to Q-639;F-117 to Q-639; F-118 to Q-639; R-l 19 to Q-639; V-120 to Q-639;E-121 to Q-639; R-122 to Q-639; G-123 to Q-639; S-124 toQ-639; Y-125 to Q- 639; V-126 to Q-639; R-127 to Q-639; Y-128to Q-639; N-129 to Q-639; F-130 to Q- 639; M-131 to Q-639;N-132 to Q-639; D-133 to Q-639; G-134 to Q-639; F-135 toQ- 639; F-136 to Q-639; L-137 to Q-639; K-138 to Q-639; V-139to Q-639; T-140 to Q-639; V-141 to Q-639; L-142 to Q-639; S-143to Q-639; F-144 to Q-639; T-145 to Q-639; P- 146 to Q-639; R-147to Q-639; P-148 to Q-639; Q-149 to Q-639; D-150 to Q-639;H-151 to Q-639; N-152 to Q-639; T-153 to Q-639; D-154 toQ-639; L-155 to Q-639; T-l 56 to Q-639; C-157 to Q-639; H-158to Q-639; V-159 to Q-639; D-l 60 to Q-639; F-161 to Q- 639; S-162to Q-639; R-163 to Q-639; K-164 to Q-639; G-165 to Q-639;V-166 to Q-639; S-167 to Q-639; A- 168 to Q-639; Q-l 69 toQ-639; R-170 to Q-639; T-171 to Q-639; V- 172 to Q-639; R-173to Q-639: L-174 to Q-639; R-175 to Q-639; V-176 to Q-639;A-177 to Q-639; Y-178 to Q-639; A-179 to Q-639; P-l 80 toQ-639; R-181 to Q-639; D-182 to Q-639; L-183 to Q-639; V-184to Q-639; 1-185 to Q-639; S-186 to Q-639; 1-187 to Q- 639; S-188to Q-639; R-189 to Q-639; D-190 to Q-639: N-191 to Q-639;T-192 to Q-639; P-193 to Q-639; A-194 to Q-639; L-195 to Q-639;E-196 to Q-639; P-197 to Q-639; Q- 198 to Q-639; P-199 to Q-639;Q-200 to Q-639; G-201 to Q-639; N-202 to Q-639; V-203 toQ-639; P-204 to Q-639; Y-205 to Q-639; L-206 to Q-639; E-207 toQ-639; A-208 to Q- 639; Q-209 to Q-639; K-210 to Q-639; G-21 lto Q-639; Q-212 to Q-639; F-213 to Q- 639; L-214 to Q-639; R-215to Q-639; L-216 to Q-639; L-217 to Q-639; C-218 to Q-639; A-219to Q-639; A-220 to Q-639; D-221 to Q-639; S-222 to Q-639;Q-223 to Q-639; P- 224 to Q-639; P-225 to Q-639; A-226 to Q-639;T-227 to Q-639; L-228 to Q-639; S-229 to Q-639; W-230 toQ-639; V-231 to Q-639; L-232 to Q-639; Q-233 to Q-639; N-234to Q-639; R-235 to Q-639; V-236 to Q-639; L-237 to Q-639; S-238to Q-639; S-239 to Q- 639; S-240 to Q-639; H-241 to Q-639; P-242to Q-639; W-243 to Q-639; G-244 to Q- 639; P-245 to Q-639;R-246 to Q-639; P-247 to Q-639; L-248 to Q-639; G-249 toQ-639; L-250 to Q-639; E-251 to Q-639; L-252 to Q-639; P-253 toQ-639; G-254 to Q-639; V- 255 to Q-639; K-256 to Q-639; A-257to Q-639; G-258 to Q-639; D-259 to Q-639; S-260 to Q-639;G-261 to Q-639; R-262 to Q-639; Y-263 to Q-639; T-264 toQ-639; C-265 to Q-639; R-266 to Q-639; A-267 to Q-639; E-268to Q-639; N-269 to Q-639; R-270 to Q- 639; L-271 to Q-639;G-272 to Q-639; S-273 to Q-639; Q-274 to Q-639; Q-275 toQ-639; R-276 to Q-639; A-277 to Q-639; L-278 to Q-639; D-279to Q-639; L-280 to Q-639; S- 281 to Q-639; V-282 to Q-639; Q-283to Q-639; Y-284 to Q-639; P-285 to Q-639; P-286 to Q-639; E-287to Q-639; N-288 to Q-639; L-289 to Q-639; R-290 to Q-639;V-291 to Q-639; M-292 to Q-639; V-293 to Q-639; S-294 toQ-639; Q-295 to Q-639; A-296 to Q- 639; N-297 to Q-639; R-298to Q-639; T-299 to Q-639; V-300 to Q-639; L-301 to Q-639; E-302to Q-639; N-303 to Q-639; L-304 to Q-639; G-305 to Q-639;N-306 to Q-639; G- 307 to Q-639; T-308 to Q-639; S-309 toQ-639; L-310 to Q-639; P-31 1 to Q-639; V-312 to Q-639; L-313 toQ-639; E-314 to Q-639; G-315 to Q-639; Q-316 to Q-639; S-317to Q- 639; L-318 to Q-639; C-319 to Q-639; L-320 to Q-639; V-321to Q-639; C-322 to Q-639; V-323 to Q-639; T-324 to Q-639;H-325 to Q-639; S-326 to Q-639; S-327 to Q-639; P- 328 to Q-639;P-329 to Q-639; A-330 to Q-639; R-331 to Q-639; L-332 to Q-639;S-333 to Q-639; W-334 to Q-639; T-335 to Q-639; Q-336 toQ-639; R-337 to Q-639; G-338 to Q-639; Q-339 to Q-639; V-340to Q-639; L-341 to Q-639; S-342 to Q-639; P-343 to Q- 639; S-344to Q-639; Q-345 to Q-639; P-346 to Q-639; S-347 to Q-639; D-348to Q-639; P-349 to Q-639; G-350 to Q-639; V-351 to Q-639;L-352 to Q-639; E-353 to Q-639; L- 354 to Q-639; P-355 to Q-639;R-356 to Q-639; V-357 to Q-639; Q-358 to Q-639; V-359 toQ-639; E-360 to Q-639; H-361 to Q-639; E-362 to Q-639; G-363to Q-639; E-364 to Q- 639; F-365 to Q-639; T-366 to Q-639; C-367to Q-639; H-368 to Q-639; A-369 to Q-639; R-370 to Q-639;H-371 to Q-639; P-372 to Q-639; L-373 to Q-639; G-374 toQ-639; S- 375 to Q-639; Q-376 to Q-639; H-377 to Q-639; V-378to Q-639; S-379 to Q-639; L-380 to Q-639; S-381 to Q-639; L-382to Q-639; S-383 to Q-639; V-384 to Q-639; H-385 to Q-639;Y-386 to Q-639; S-387 to Q-639; P-388 to Q-639; K-389 toQ-639; L-390 to Q- 639; L-391 to Q-639; G-392 to Q-639; P-393 toQ-639; S-394 to Q-639; C-395 to Q-639; S-396 to Q-639; W-397to Q-639; E-398 to Q-639; A-399 to Q-639; E-400 to Q-639;G- 401 to Q-639; L-402 to Q-639; H-403 to Q-639; C-404 toQ-639; S-405 to Q-639; C-406 to Q-639; S-407 to Q-639; S-408 toQ-639; Q-409 to Q-639; A-410 to Q-639; S-411 to Q-639; P-412 toQ-639; A-413 to Q-639; P-414 to Q-639; S-415 to Q-639; L-416 toQ- 639; R-417 to Q-639; W-418 to Q-639; W-419 to Q-639; L-420to Q-639; G-421 to Q- 639; E-422 to Q-639; E-423 to Q-639; L-424to Q-639; L-425 to Q-639; E-426 to Q-639; G-427 to Q-639;N-428 to Q-639; S-429 to Q-639; S-430 to Q-639; Q-431 toQ-639; D- 432 to Q-639; S-433 to Q-639; F-434 to Q-639; E-435 toQ-639; V-436 to Q-639; T-437 to Q-639; P-438 to Q-639; S-439 toQ-639; S-440 to Q-639; A-441 to Q-639; G-442 to Q-639; P-443to Q-639; W-444 to Q-639; A-445 to Q-639; N-446 to Q-639;S-447 to Q- 639; S-448 to Q-639; L-449 to Q-639; S-450 to Q-639;L-451 to Q-639; H-452 to Q-639; G-453 to Q-639; G-454 toQ-639; L-455 to Q-639; S-456 to Q-639; S-457 to Q-639; G- 458 toQ-639; L-459 to Q-639; R-460 to Q-639; L-461 to Q-639; R-462 toQ-639; C-463 to Q-639; E-464 to Q-639; A-465 to Q-639; W-466to Q-639; N-467 to Q-639; V-468 to Q-639; H-469 to Q-639;G-470 to Q-639; A-471 to Q-639; Q-472 to Q-639; S-473 toQ- 639; G-474 to Q-639; S-475 to Q-639; 1-476 to Q-639; L-477 toQ-639; Q-478 to Q-639; L-479 to Q-639; P-480 to Q-639; D-481 toQ-639; K-482 to Q-639; K-483 to Q-639; G- 484 to Q-639; L-485to Q-639; 1-486 to Q-639; S-487 to Q-639; T-488 to Q-639; A-489to Q-639; F-490 to Q-639; S-491 to Q-639; N-492 to Q-639;G-493 to Q-639; A-494 to Q- 639; F-495 to Q-639; L-496 toQ-639; G-497 to Q-639; 1-498 to Q-639; G-499 to Q-639; 1-500 toQ-639; T-501 to Q-639; A-502 to Q-639; L-503 to Q-639; L-504 toQ-639; F-505 to Q-639; L-506 to Q-639; C-507 to Q-639; L-508 toQ-639; A-509 to Q-639; L-510 to Q-639; 1-511 to Q-639; 1-512 toQ-639; M-513 to Q-639; K-514 to Q-639; 1-515 to Q- 639; L-516 toQ-639; P-517 to Q-639; K-518 to Q-639; R-519 to Q-639; R-520to Q-639; T-521 to Q-639; Q-522 to Q-639; T-523 to Q-639; E-524to Q-639; T-525 to Q-639; P- 526 to Q-639; R-527 to Q-639; P-528to Q-639; R-529 to Q-639; F-530 to Q-639; S-531 to Q-639; R-532to Q-639; H-533 to Q-639; S-534 to Q-639; T-535 to Q-639; I-536to Q- 639; L-537 to Q-639; D-538 to Q-639; Y-539 to Q-639; I-540to Q-639; N-541 to Q-639; V-542 to Q-639; V-543 to Q-639;P-544 to Q-639; T-545 to Q-639; A-546 to Q-639; G- 547 toQ-639; P-548 to Q-639; L-549 to Q-639; A-550 to Q-639; Q-551 toQ-639; K-552 to Q-639; R-553 to Q-639; N-554 to Q-639; Q-555to Q-639; K-556 to Q-639; A-557 to Q-639; T-558 to Q-639;P-559 to Q-639; N-560 to Q-639; S-561 to Q-639; P-562 to Q- 639;R-563 to Q-639; T-564 to Q-639; P-565 to Q-639; L-566 to Q-639;P-567 to Q-639; P-568 to Q-639; G-569 to Q-639; A-570 toQ-639; P-571 to Q-639; S-572 to Q-639; P- 573 to Q-639; E-574 toQ-639; S-575 to Q-639; K-576 to Q-639; K-577 to Q-639; N- 578to Q-639; Q-579 to Q-639; K-580 to Q-639; K-581 to Q-639;Q-582 to Q-639; Y-583 to Q-639; Q-584 to Q-639; L-585 toQ-639; P-586 to Q-639; S-587 to Q-639; F-588 to Q- 639; P-589 toQ-639; E-590 to Q-639; P-591 to Q-639; K-592 to Q-639; S-593 toQ-639; S-594 to Q-639; T-595 to Q-639; Q-596 to Q-639; A-597 toQ-639; P-598 to Q-639; E- 599 to Q-639; S-600 to Q-639; Q-60i toQ-639; E-602 to Q-639; S-603 to Q-639; Q-604 to Q-639; E-605 toQ-639: E-606 to Q-639; L-607 to Q-639; H-608 to Q-639; Y-609to Q- 639: A-610 to Q-639; T-61 1 to Q-639; L-612 to Q-639;N-613 to Q-639; F-614 to Q-639; P-615 to Q-639; G-616 toQ-639; V-617 to Q-639; R-618 to Q-639; P-619 to Q-639; R- 620to Q-639; P-621 to Q-639; E-622 to Q-639; A-623 to Q-639; R-624to Q-639; M-625 to Q-639; P-626 to Q-639; K-627 to Q-639;G-628 to Q-639; T-629 to Q-639; Q-630 to Q-639; A-631 toQ-639; D-632 to Q-639; Y-633 to Q-639; A-634 to Q-639; of SEQID NO:33. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification or loss of one or more biological functions of the protein, other functional activities (e.g., biological activities (e.g., ability to illicit mitogenic activity, induce differentiation of normal or malignant cells, bind to EGF receptors, etc.)), may still be retained. For example the ability to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response. Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the polypeptide shown in Figures 13A-C, up to the leucine residue at position number 7, and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising the amino acid sequence of residues 1-ml of Figures 13A-C, where ml is an integer from 7 to 638 coιτesponding to the position of the amino acid residue in Figures 13A-C Moreover, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of C-terminal deletions of the polypeptide of the invention shown as SEQ ID NO:33 include polypeptides comprising the amino acid sequence of residues: M-1 to F- 638; M-1 to K-637;MJ to V-636; M-1 to E-635; M-1 to A-634; M-1 to Y-633; M-1 toD-632; M-1 to A-631; M-1 to Q-630; M-1 to T-629; M-1 to G-628;M-1 to K-627; M-1 to P-626; M-1 to M-625; M-1 to R-624; M-1 toA-623; M-1 to E-622; M-1 to P-621; M-1 to R-620; M-1 to P-619;MJ to R-618; M-1 to V-617; M-1 to G-616; M-1 to P-615; M-1 toF-614; M-1 to N-613; M-1 to L-612; M-1 to T-611; M-1 to A-610;M-1 to Y-609; M-1 to H-608; M-1 to L-607; M-1 to E-606; M-1 toE-605; M-1 to Q-604; M-1 to S-603; M-1 to E-602; M-1 to Q-601;M-1 to S-600; M-1 to E-599; M-1 to P-598; M-1 to A-597; M-1 toQ-596; M-1 to T-595; M-1 to S-594; M-1 to S-593; M-1 to K-592;M-1 to P-591; M-1 to E-590; M-1 to P-589; M-1 to F-588; M-1 toS-587; M-1 to P-586; M-1 to L-585; M-1 to Q-584; M-1 to Y-583;M-1 to Q-582; M-1 to K-581; M-1 to K-580; M-1 to Q-579; M- 1 toN-578; M-1 to K-577; M-1 to K-576; M-1 to S-575; M-1 to E-574;M-1 to P-573; M- 1 to S-572; M-1 to P-571; M-1 to A-570; M-1 toG-569; M-1 to P-568; M-1 to P-567; M- 1 to L-566; M-1 to P-565;M-1 to T-564; M-1 to R-563; M-1 to P-562; M-1 to S-561 ; M- 1 toN-560; M-1 to P-559; M-1 to T-558; M-1 to A-557; M-1 to K-556;M-1 to Q-555; M- 1 to N-554; M-1 to R-553; M-1 to K-552; M-1 toQ-551; M-1 to A-550; M-1 to L-549; M-1 to P-548; M-1 to G-547;M-1 to A-546; M-1 to T-545; M-1 to P-544; M-1 to V-543;

M-1 toV-542; M-1 to N-541; M-1 to 1-540; M-1 to Y-539; M-1 to D-538;M-1 to L-537;

M-1 to 1-536; M-1 to T-535; M-1 to S-534; M-1 toH-533; M-1 to R-532; M-1 to S-531;

M-1 to F-530; M-1 to R-529;M-1 to P-528; M-1 to R-527; M-1 to P-526; M-1 to T-525; M-1 toE-524; M-1 to T-523; M-1 to Q-522; M-1 to T-521; M-1 to R-520;M-1 to R-519;

M-1 to K-518; M-1 to P-517; M-1 to L-516; M-1 toI-515; M-1 to K-514; M-1 to M-513;

M-1 to 1-512; M-1 to I-511 ;M-l to L-510; M-l to A-509; M-l to L-508; M-l to C-507;

M-1 toL-506; M-1 to F-505; M-1 to L-504; M-1 to L-503; M-1 to A-502;M-1 to T-501;

M-1 to 1-500; M-1 to G-499; M-1 to 1-498; M-1 toG-497; M-1 to L-496; M-1 to F-495; M-1 to A-494; M-1 to G-493;M-1 to N-492; M-1 to S-491 ; M-1 to F-490; M-1 to A-489;

M-1 toT-488; M-1 to S-487; M-1 to 1-486; M-1 to L-485; M-1 to G-484;M-1 to K-483;

M-1 to K-482; M-1 to D-481; M-1 to P-480; M-1 toL-479; M-1 to Q-478; M-1 to L-477;

M-1 to 1-476; M-1 to S-475;MJ to G-474; M-1 to S-473; M-1 to Q-472; M-1 to A-471;

M-1 toG-470; M-1 to H-469; M-1 to V-468; M-1 to N-467; M-1 toW-466; M-1 to A- 465; M-1 to E-464; M-1 to C-463; M-1 to R-462;M-1 to L-461; M-1 to R-460; M-1 to L-

459; M-1 to G-458; M-1 toS-457; M-1 to S-456; M-1 to L-455; M-1 to G-454; M-1 to G-

453;M-1 to H-452; M-1 to L-451; M-1 to S-450; M-1 to L-449; M-1 toS-448; M-1 to S-

447; M-1 to N-446; M-1 to A-445; M-1 to W-444;M-1 to P-443; M-1 to G-442; M-1 to

A-441 ; M-1 to S-440; M-1 toS-439; M-1 to P-438; M-1 to T-437; M-1 to V-436; M-1 to E-435;M-1 to F-434; M-1 to S-433; M-1 to D-432; M-1 to Q-431; M-1 toS-430; M-1 to

S-429; M-1 to N-428; M-1 to G-427; M-1 to E-426;M-1 to L-425; M-1 to L-424; M-1 to

E-423; M-1 to E-422; M-1 toG-421; M-1 to L-420; M-1 to W-419; M-1 to W-418; M-1 toR-417; M-1 to L-416; M-1 to S-415; M-1 to P-414; M-1 to A-413;M-1 to P-412; M-1 to S-411 ; M-1 to A-410; M-1 to Q-409; M-1 toS-408; M-1 to S-407; M-1 to C-406; M-1 to S-405 ; M- 1 to C-404;M- 1 to H-403 ; M- 1 to L-402; M- 1 to G-401 ; M- 1 to E-400; M- 1 toA-399; M-1 to E-398; M-1 to W-397; M-1 to S-396; M-1 to C-395;M-1 to S-394; M-1 to P-393; M-1 to G-392; M-1 to L-391; M-1 toL-390; M-1 to K-389; M-1 to P-388; M-1 to S-387; M-1 to Y-386;M-1 to H-385; M-1 to V-384; M-1 to S-383; M-1 to L-382; M-1 toS-381; M-1 to L-380; M-1 to S-379; M-1 to V-378; M-1 to H-377;M-1 to Q-376; M-1 to S-375; M-1 to G-374; M-1 to L-373; M-1 toP-372; M-1 to H-371; M-1 to R-370; M-1 to A-369; M-1 to H-368;M-1 to C-367; M-1 to T-366; M-1 to F-365; M-1 to E-364; M-1 toG-363; M-1 to E-362; M-1 to H-361; M-1 to E-360; M-1 to V-359;M-1 to Q-358; M-1 to V-357; M-1 to R-356; M-1 to P-355; M-1 toL-354; M-1 to E-353; M-1 to L-352; M-1 to V-351 ; M-1 to G-350;M-1 to P-349; M-1 to D-348; M-1 to S-347; M-1 to P-346; M-1 toQ-345; M-1 to S-344; M-1 to P-343; M-1 to S-342; M-1 to L-341 ;M-1 to V-340; M-1 to Q-339; M-1 to G-338; M-1 to R-337; M-1 toQ-336; M-1 to T-335; M-1 to W-334; M- 1 to S-333; M-1 to L-332;M-1 to R-331; M-1 to A-330; M-1 to P-329: M-1 to P-328; M- 1 toS-327; M- 1 to S-326; M-1 to H-325; M-1 to T-324; M-1 to V-323;M-1 to C-322; M- 1 to V-321; M-l to L-320; M-l to C-319; M-l toL-318; MJ to S-317; M-l to Q-316; M- 1 to G-315; M-l to E-314;M-1 to L-313; M-1 to V-312; M-1 to P-311; MJ to L-310; M- 1 toS-309; M-1 to T-308; M-1 to G-307; M-1 to N-306; M-1 to G-305;M-1 to L-304; M- 1 to N-303; M-1 to E-302; M-1 to L-301; M-1 toV-300; M-1 to T-299; M-1 to R-298; M-1 to N-297; M-1 to A-296;M-1 to Q-295; M-1 to S-294; M-1 to V-293; M-1 to M- 292; M-1 toV-291; M-1 to R-290; M-1 to L-289; M-1 to N-288; M-1 to E-287.MJ to P- 286; M-1 to P-285; M-1 to Y-284; M-1 to Q-283; M-1 toV-282; M-1 to S-281; M-1 to L- 280; M-1 to D-279; M-1 to L-278;M-1 to A-277; M-1 to R-276; M-1 to Q-275; M-1 to Q-274; M-1 toS-273; M-1 to G-272; M-1 to L-271; M-1 to R-270; M-1 to N-269;M-1 to E-268; M-1 to A-267; M-1 to R-266; M-1 to C-265; M-1 toT-264; M-1 to Y-263; M-1 to R-262; M-1 to G-261; M-1 to S-260;M-1 to D-259; M-1 to G-258; M-1 to A-257; M-1 to K-256; M-1 toV-255; M-1 to G-254; M-1 to P-253; M-1 to L-252; M-1 to E-251;M-1 to L-250; M-1 to G-249; M-1 to L-248; M-1 to P-247; M-1 toR-246; M-1 to P-245; M-1 to G-244; M-1 to W-243; M-1 to P-242;M-1 to H-241; M-1 to S-240; M-1 to S-239; M-1 to S-238; M-1 toL-237; M-1 to V-236; M-1 to R-235; M-1 to N-234; M-1 to Q-233;M-1 to L-232; M-1 to V-231; M-1 to W-230; M-1 to S-229; M-1 toL-228; M-1 to T-227; M-1 to A-226; M-1 to P-225; M-1 to P-224;M-1 to Q-223; M-1 to S-222; M-1 to D-221; M-1 to A-220; M-1 toA-219; M-1 to C-218; M-1 to L-217; M-1 to L-216; M-1 to R-215;M-1 to L-214; M-1 to F-213; M-1 to Q-212; M-1 to G-211; M-1 toK-210; M-1 to Q-209; M-1 to A-208; M-1 to E-207; M-1 to L-206;M-1 to Y-205; M-1 to P-204; M-1 to V-203; M-1 to N-202; M-1 toG-201; M-1 to Q-200; M-1 to P-199; M-1 to Q-198; M-1 to P-197;M-1 to E-196; M-1 to L-195; M-1 to A-194; M-1 to P-193; M-1 toT-192; M-1 to N-191; M-1 to D-190; M-1 to R-189; M-1 to S-188;M-1 to 1-187; M-1 to S-186; M-1 to 1-185; M-1 to V-184; M-1 toL-183; M-1 to D-182; M-1 to R-181; M-1 to P-180; M-1 to AJ79.MJ to Y-178; M-1 to A-177; M-1 to V-176; M-1 to R-175; M-1 toL-174; M-1 to R-173; M-1 to V-172; M-1 to T-171; M-1 to R-170;M-1 to Q-169; M-1 to A-168; M-1 to S-167; M-1 to V-166; M-1 toG-165; M-1 to K-164; M-1 to R-163; M-1 to S-162; M-1 to F-161;M-1 to D-160; M-1 to V-159; M-1 to H-158; M-1 to C-157; M-1 toT-156; M-1 to L-155: M-1 to D-154; M-1 to T-153; M-1 to N-152;M-1 to H-151; M-1 to D-150; M-1 to Q-149; M-1 to P-148; M-1 toR-147; M-1 to P-146; M-1 to T-145; M-1 to F-144; M-1 to S-143;M-1 to L-142; M-1 to V-141; M-1 to T-140; M-1 to V-139; M-1 toK-138; M-1 to L-137; M-1 to F-136; M-1 to F-135; M-1 to G-134;M-1 to D-133; M-1 to N-132; M-1 to M-131; M-1 to F-130; M-1 toN-129; M-1 to Y-128; M-1 to R-127; M-1 to V-126; M-1 to Y-125;M-1 to S-124; M-1 to G-123; M-1 to R-122; M-1 to E-121; M-1 toV-120; M-1 to R-119; M-1 toF-118;M-l toF-117;M-l toY-116;M-l toQ-115;M-l to SJ 14; MJ toE-113;M-l to D-l 12; M-1 toQ-111; M-1 to M-110; M-1 to Q-109; M-1 to A-108; M-1 to D-107;M- 1 to R-106; M-1 to 1-105; M-1 to V-104; M-1 to L-103; M-1 toS-102; M-1 to C-101; M- 1 to N-100; M-1 to G-99; M-1 to K-98;M-1 to A-97; M-1 to P-96; M-1 to D-95; M-1 to G-94; M-1 toT-93; M-1 to L-92; M-1 to Q-91; M-1 to F-90; M-1 to R-89; M-1 toG-88; M-1 to R-87; M-1 to T-86; M-1 to S-85; M-1 to M-84; M-lto E-83; M-1 to V-82; M-1 to E-81; M-1 to R-80; M-1 to S-79; M-lto Q-78; M-1 to H-77; M-1 to N-76; M-1 to T-75; M-1 to A-74;M-1 to V-73; M-1 to P-72; M-1 to A-71; M-1 to G-70; M-1 toK-69; M-1 to T-68; M-1 to T-67; M-1 to E-66; M-1 to T-65; M-1 toV-64; M-1 to A-63; M-1 to K-62; M-1 to F-61; M-1 to W-60; M-lto Y-59; M-1 to G-58; M-1 to Y-57; M-1 to A-56; M-1 to P-55;M-1 to T-54; M-1 to S-53; M-1 to G-52; M-1 to T-51; M-1 toW-50; M-1 to D- 49; M-1 to Q-48; M-1 to R-47; M-1 to P-46; M-lto Y-45; M-1 to S-44; M-1 to F-43; M- 1 to S-42; M-1 to C-41; M-lto P-40; M-1 to V-39; M-1 to S-38; M-1 to 1-37; M-1 to D- 36; M-lto C-35; M-1 to A-34; M-1 to E-33; M-1 to P-32; M-1 to V-31 ;M-1 to M-30; M- 1 to V-29; M-1 to S-28; M-1 to E-27; M-1 toQ-26; M-1 to V-25; M-1 to R-24; M-1 to I- 23; M-1 to W-22; M-lto F-21; M-1 to R-20; M-1 to G-19; M-1 to D-18; M-1 to M- 17;M-1 to A-16; M-l to Q-15; M-l to S-14; M-l to G-13; M-l toG-12; M-l to L-l l; M- 1 to L-10; M-1 to S-9; M-1 to S-8; M-1 toL-7; of SEQ ID NO:33. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

In addition, the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO: 15 which have been determined from the following related cDNA genes: HTOFA26R (SEQ ID NO:93), HWAEM43R (SEQ ID NO:94), HDPMQ69R (SEQ ID NO:95), HDPGA09RA (SEQ ID NO:96), HEOMH10R (SEQ ID NO:97), and HFKCT73F (SEQ ID NO:98).

The polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 496 - 512 of the amino acid sequence referenced in Table XIII for this gene. Moreover, a cytoplasmic tail encompassing amino acids 513 to 639 of this protein has also been determined. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type la membrane proteins.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of the following diseases and conditions which include, but are not limited to, disorders of the immune system, in particular the immunodiagnosis of acute leukemias. Similarly, polypeptides and antibodies directed to these polypeptides are useful to provide immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels is detected in certain tissues or cell types (e.g., immune, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid or spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue from an individual not having the disorder.

Preferred epitopes include those comprising a sequence shown in SEQ ID NO: 33 as residues: Pro-46 to Gly-52, Asn-76 to Val-82, Ser-85 to Phe-90, Gly-94 to Asn-100, Gln-1 11 to Tyr-116, Pro-146 to Leu-155, Ser-188 to Asn-202, Ser-240 to Arg-246. Gly- 258 to Tyr-263, Ala-267 to Arg-276, Ser-326 to Arg-331, Ser-333 to Gln-339, Pro-343 to Asp-348, Glu-426 to Asp-432, Pro-517 to His-533, Ala-550 to Pro-565, Gly-569 to Gln-582, Pro-589 to Glu-606, Gly-616 to Ala-623, Met-625 to Ala-631.

CD33 monoclonal antibodies (MoAB) are important in the immunodiagnosis of AML. CD33 MoABs have been used in preliminary therapeutic trials for purging bone marrow of AML patients, either before transplantation or for diseases resistant to chemotherapy. To prevent AML patients in remission from suffering relapse, or due to the lack of an appropriate allogenic bone marrow donor, a method is necessary for purging leukemia cells from the autografts of patients with advanced AML. By the invention, this method is provided by which bone marrow from an AML patient is obtained by, for example, percutaneous aspirations from the posterior iliac crest, isolating bone marrow mononuclear by Ficoll-hypaque density gradient centrifugation, and incubating with an anti-CD33-like protein MoAB, for example, 3-5 times for 15-30 min. at 4-6 degrees C, followed by incubation with rabbit complement at about 37 degrees C for 30 minutes. The patient is then subject to myeloablative chemotherapy, followed by reinfusion of the treated autologous bone marrow according to standard techniques. By the invention, clonogenic tumor cells are depleted from the bone marrow while sparing hematopoietic cells necessary for engraftment. In a further embodiment,the invention provides an in vivo method for selectively killing or inhibiting growth of tumor cells expressing CD33-like protein antigen of the present invention. The method involves administering to the patient an effective amount of an antagonist to inhibit the CD33-like protein receptor signaling pathway. By the invention, administering such antagonist of the CD33-like protein to a patient may also be useful for treating inflammatory diseases including arthritis and colitis. Antagonists for use in the present invention include polyclonal and monoclonal antibodies raised aginst the CD33-like protein or a fragment thereof, antisense molecules which control gene expression through antisense DNA or RNA or through triple-helix formation, proteins or other compounds which bind the CD33-like protein domains, or soluble forms of the CD33-like protein, such as protein fragments including the extracellular region from the full length receptor, which antagonize CD33-like protein mediated signaling by competing with the cell surface CD33-like protein for binding to CD33 receptor ligands.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 15 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2281 of SEQ ID NOJ5, b is an integer of 15 to 2295, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 15, and where b is greater than or equal to a + 14. FEATURES OFPROTEIN ENCODED BY GENE NO: 6

This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. The polypeptide of the present invention has been putatively identified as a CD33 homolog derived from a human primary dendritic cells cDNA library. More particularly, the polypeptide of the present invention has been putatively identified as a human siglec homolog, sometimes hereafter referred to as "CD33-like 3" and/or "siglec 7". The invention also relates to inhibiting the action of such polypeptides.

The siglecs (sialic acid binding Ig-like lectins) are type 1 membrane proteins that constitute a distinct subset of the Ig superfamily, characterised by their sequence similarities and abilities to bind sialic acids in glycoproteins and glycolipid (Crocker, P.R., et al., Glycobiology:8 (1998)). Members of the Ig Superfamily of proteins are defined as molecules that share domains of sequence similarity with the variable or constant domains of antibodies.

Many Ig superfamily proteins consist of multiple tandem Ig-like domains connected to other domains, such as Fn-III repeat domains (Vaughn, D.E., and P.J. Bjorkman, Neuron, 16:261-73 (1996)). At the primary structural level, traditional Ig-like domains can be identified by the presence of two cysteine residues separated by approximately 55-75 amino acid residues, and an "invariant" tryptophan residue located 10-15 residues C-terminal to the first of the two conserved cysteine residues. The two conserved cysteine residues are thought to be involved in disulfide bonding to form the folded Ig structures (Vaughn, D.E., (1996)).

Ig-like domains further share a common folding pattern, that of a sandwich or fold structure of two b-sheets consisting of antiparallel b-strands containing 5-10 amino acids (Huang, Z., et al., Biopolymers, 43:367-82 (1997)). Ig-hke domains are divided, based upon sequence and structural similarities, into four classifications known as Cl, C2, 1 and V-hke domains.

The functional determinants of the Ig-like domains are presented on the faces of b-sheets or the loop regions of the Ig-fold Accordingly, protem-protem interactions can occur either between the faces of the b-sheets, or the loop regions of the Ig-fold (Huang, Z., ( 1998)). These Ig-like domains are involved in mediating a diversity of biological functions such as mtermolecular binding and protem-protem homophihc oi heterophihc interactions. Thus, Ig-like domains play an integial role in facilitating the activities of proteins of the Ig superfamily. In mammals, the group curcently comprises sιaloadhesιn/sιglec-1, CD22/sιglec-2, CD33/sιglec-3, myehn associated glycoprotein (MAG/sιglec-4), siglecs- 5, -6 and -7 (Crocker, P.R., et al., EMBO J., 13.4490-503 (1994); Sgroi, D., et al., J Biol. Chem., 268:7011-18 (1993); Freeman, D.S., et al., Blood, 85:2005-12 (1995); Kelm, S., et al., Curr Biol., 4:965-72 (1994); Cornish, A.L., et al., Blood, 92:2123-132 (1998); Patel, N., et al., J Biol. Chem, 274:22729-738 (1999); Nicol, G., et al., J Biol. Chem, In Press (1999)). Sιglec-7 has also recently been characteπsed independently as the NK receptor p75/AIRMl (Falco, M., et al., J. Exp. Med., 190:793-802 (1999)). In addition, the gene encoding another siglec-like sequence, OBBP-like protein has been reported but there is no information on its binding activity (Yousef, G.M., et al., Biochem. Biophys. Res. Commun., In Press (1999)).

Each of these proteins has an extracellular region made up of a membrane distal V-set domain followed by varying numbers of C2 set domains which range from 16 in sialoadhesin to 1 in CD33. In the cases of sialoadhesin, CD22, MAG and CD33, the sialic acid binding site has been mapped to the V-set domain and for sialoadhesin it has been further characteπsed at the molecular level by X-ray crystallography 11 (Nath, D., et al., J Biol. Chem., 270:26184-91 (1995); van der Merwe, P.A., et al., J. Biol. Chem., 271:9273-80 (1996); Tang, S., et al., J. Cell Biol., 138:1355-66 (1997); Taylor, V.C, et al., J. Biol. Chem., 274:11505-12 (1999); May, A.P., et al., Molecular Cell, 1:719-28 (1998)). Apart from MAG and SMP that are found exclusively in the nervous system, all siglecs characterised to date are expressed on discrete subsets of hemopoietic cells and can provide useful lineage-restricted markers. Thus, CD22 is present only on mature B cells, sialoadhesin is on macrophage subsets, CD33 is a marker of early committed myeloid progenitor cells, siglec-5 is expressed by monocytes and mature neutrophils, siglec-6 is on B cells and siglec-7 is expressed by NK cells and monocytes (Dorken, B., et al., J. Immunology, 136:4470-79 (1986); Crocker, P.R., et al., J. Exp. Med., 164:1862- 75 (1986); Peiper, S.C., et al., In Leukocyte Typing IV. Oxford University Press, Oxford. 814-16 (1989); Cornish, A.L., et al., Blood, 92:2123-132 (1998); Patel, N., et al., J Biol. Chem, 274:22729-738 (1999); Nicol, G., et al., J Biol. Chem, In Press (1999)). These expression patterns indicate discrete functions amongst hemopoietic cell subsets, but apart from CD22, a well-characterised negative regulator of B cell activation (reviewed in Cyster, J.G. and CC Goodnow, Immunity, 6:509-17 (1997)), the biological functions of siglecs expressed in the hemopoietic system are unknown. Proposed functions include cell-cell interactions through recognition of sialylated glycoconjugates on other cells. However, a number of studies have also shown that cell-cell adhesion mediated by siglecs can be modulated by cis-interactions with sialic acids present in the host plasma membrane. This is particularly striking for CD22, CD33 and siglec-5, whose binding activities can be greatly increased if host cells are pretreated with sialidase to remove the cis-competing sialic acids (Freeman, D.S., et al., Blood, 85:2005-12 (1995); Cornish, A.L., et al., Blood, 92:2123-132 (1998); Sgroi, D., et al., P.N.A.S., 92:4026-30 (1995)). Besides potential roles in cellular interactions, there is growing evidence that, similar to CD22, the more recently characterised siglecs are involved in signalling functions. The cytoplasmic tails of CD33 and siglecs-5, -6 and -7 have two well- conserved tyrosine-based motifs that are similar to well-characterised signaling motifs in other leukocyte receptors (Gergely, J., et al., Immun. Lett., 68:3-15 (1999)). Where studied, both tyrosine residues can be phosphorylated by src-like kinase(s) and, in the case of the membrane proximal tyrosine, this leads to subsequent recruitment of the tyrosine phosphatases, SHP-1 and SHP-2 (Falco, M., et al., J. Exp. Med., 190:793-802 (1999); Taylor, V.C, et al., J. Biol. Chem., 274: 11505-12 (1999)). Thus there exists a clear need for identifying and exploiting novel members of the siglec family of immunoglobulin proteins. Although structurally related, such proteins may possess diverse and multifaceted functions in a variety of cell and tissue types. The inventive purified siglec proteins are research tools useful for the identification, characterization and purification of cell signaling molecules. Furthermore, the identification of new siglecs permits the development of a range of derivatives, agonists and antagonists at the nucleic acid and protein levels which in turn have applications in the treatment and diagnosis of a range of conditions such as cancer, inflammation, neurological disorders and immunological disorders, amongst many other conditions. The polypeptide of the present invention has been putatively identified as a member of the siglec family and has been termed CD33-like 3. This identification has been made as a result of amino acid sequence homology to the human cd3311 (See Genbank Accession No. gi|2913995).

Figures 16A-B show the nucleotide (SEQ ID NO: 16) and deduced amino acid sequence (SEQ ID NO:34) of CD33-like 3. Predicted amino acids from about 1 to about 18 constitute the predicted signal peptide (amino acid residues from about 1 to about 18 in SEQ ID NO:34) and are represented by the underlined amino acid regions; and amino acids from about 360 to about 376 constitute the predicted transmembrane domain

(amino acids from about 360 to about 376 in SEQ ID NO:34) and are represented by the double underlined amino acids. Figure 17 shows the regions of similarity between the amino acid sequences of the CD33-like 3 protein (SEQ ID NO:34) and the human CD33L1 protein (SEQ ID NO:99).

Figure 18 shows an analysis of the CD33-like 3 amino acid sequence. Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.

A polynucleotide encoding a polypeptide of the present invention is obtained from human NK cells, T-cells, primary dendritic cells, placenta, spleen, primary breast cancer, gall bladder, apoptotic t-cells, macrophage. and chronic lymphocytic leukemia spleen. The polynucleotide of this invention was discovered in a human primary dendritic cell cDNA library.

As shown in Figures 16A-B, CD33-like 3 has a transmembrane domain (the transmembrane domains comprise amino acids from about 360 to about 376 of SEQ ID NO:34; which correspond to amino acids from about 360 to about 376 of Figures 16A-B ). The polynucleotide contains an open reading frame encoding the CD33-like 3 polypeptide of 467 amino acids. CD33-like 3 exhibits a high degree of homology at the amino acid level to the human CD33L1 (as shown in Figure 18).

The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the CD33-like 3 polypeptide having the amino acid sequence shown in Figures 16A-B (SEQ ID NO:34). The nucleotide sequence shown in Figures 16A-B (SEQ ID NO: 16) was obtained by sequencing a cloned cDNA (HDPUW68), which was deposited on November 17 at the American Type Culture Collection, and given Accession Number 203484.

The present invention is further directed to fragments of the isolated nucleic acid molecules described herein. By a fragment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ ID NO: 16 is intended DNA fragments at least about 15nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments 50-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in SEQ ID NO: 16. By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NO: 16. In this context "about" includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Representative examples of CD33-like 3 polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, from about 501 to about 550, from about 551 to about 600, from about 601 to about 650, from about 651 to about 700, from about 701 to about 750, from about 751 to about 800, from about 801 to about 850, from about 851 to about 900, from about 901 to about 950, from about 951 to about 1000, from about 1001 to about 1050, from about 1051 to about 1100, from about 1101 to about 1150, from about 1151 to about 1200, from about 1201 to about 1250, from about 1251 to about 1300, from about 1301 to about 1350, from about 1351 to about 1400, from about 1401 to about 1450, from about 1451 to about 1500, from about 1501 to about 1550, from about 1551 to about 1600, from about 1601 to about 1650, from about 1651 to about 1700, from about 1701 to about 1748 of SEQ ID NO: 16, or the complementary strand thereto, or the cDNA contained in the deposited gene. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding a member selected from the group: a polypeptide compnsing or alternatively, consisting of, the transmembrane domain (amino acid residues from about 360 to about 376 in Figures 16A-B (ammo acids from about 360 to about 376 in SEQ ID NO:34). Since the location of these domains have been predicted by computei analysis, one of ordinary skill would appreciate that the amino acid lesidues constituting these domains may vary slightly (e g , by about 1 to 15 amino acid residues) depending on the criteπa used to define each domain In additional embodiments, the polynucleotides of the invention encode functional attπbutes of CD33-hke 3

Preferred embodiments of the invention in this regaid include fiagments that compnse alpha-helix and alpha-helix forming iegions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions"), turn and turn-forming regions ("turn- regions"), coil and coil-forming iegions ("coil-iegions"), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions of CD33-hke 3 The data representing the structural or functional attributes of CD33-hke 3 set forth in Figuie 18 and/or Table VI, as descπbed above, was generated using the vaπous modules and algoπthms of the DNA^J STAR set on default parameters In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table VI can be used to determine regions of CD33-lιke 3 which exhibit a high degree of potential for antigenicity Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.

Certain preferred regions in these regards are set out in Figure 18 , but may, as shown in Table VI, be represented or identified by using tabular representations of the data presented m Figure 18 The DNA*STAR computer algoπthm used to generate Figure 18 (set on the oπgmal default parameters) was used to present the data in Figure 18 in a tabular format (See Table VI). The tabular format of the data in Figure 18 is used to easily determine specific boundaries of a preferred region. The above-mentioned preferred regions set out in Figure 18 and Table VI include, but aie not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figures 16A-B As set out in Figure 18 and in Table VI, such preferred iegions include Garniei-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-iegions, Kyte-Doolittle hydrophilic iegions and Hopp-Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic iegions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenic index and Emmi surface-forming regions Even if deletion of one or more amino acids from the N-termmus of a protein results m modification of loss of one or more biological functions of the protein, othei functional activities (e.g., biological activities, ability to multimerize, modulate cellular interaction, or signalling pathways, etc ) may still be retained For example, the ability of shortened CD33-hke 3 muteins to induce and/or bind to antibodies which recognize the complete or matuie forms of the polypeptides generally will be retained when less than the majoπty of the residues of the complete oi mature polypeptide are removed from the N-terminus Whether a particular polypeptide lacking N-termmal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods descπbed herein and otherwise known in the art. It is not unlikely that an CD33-hke 3 mutein with a large number of deleted N-termmal amino acid residues may retain some biological or immunogenic activities In fact, peptides composed of as few as six CD33-lιke 3 ammo acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the CD33-hke 3 amino acid sequence shown in Figures 16A-B , up to the glutamic acid residue at position number 462 and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising the amino acid sequence of residues nl-467 of Figures 16A-B , where nl is an integer from 2 to 462 corresponding to the position of the amino acid residue in Figures 16A-B (which is identical to the sequence shown as SEQ ID NO:34). In another embodiment, N-terminal deletions of the CD33-like 3 polypeptide can be described by the general formula n2-467, where n2 is a number from 2 to 462, coπ'esponding to the position of amino acid identified in Figures 16A-B . N-terminal deletions of the CD33-like 3 polypeptide of the invention shown as SEQ ID NO:34 include polypeptides comprising the amino acid sequence of residues: N-terminal deletions of the CD33-like 3 polypeptide of the invention shown as SEQ ID NO:34 include polypeptides comprising the amino acid sequence of residues: L-2 to K-467; L-3 to K-467; L-4 to K-467; L-5 to K-467; L-6 to K-467; L-7 to K-467; P-8 to K-467; L-9 to K-467; L-10 toK-467; W-l 1 to K-467; G-l 2 to K-467; R-l 3 to K-467; E-l 4 to K-467; R-15 to K-467; V-16 to K-467; E-17 to K-467; G-18 to K-467;Q-19 to K-467; K-20 to K-467; S-21 to K-467; N-22 to K-467; R-23 to K-467; K-24 to K-467; D-25 to K-467; Y-26 to K-467; S-27 toK-467; L-28 to K-467; T-29 to K-467; M-30 to K-467; Q-31 to K-467; S-32 to K-467; S-33 to K-467; V-34 to K-467; T-35 to K-467;V-36 to K-467; Q- 37 to K-467; E-38 to K-467; G-39 to K-467; M-40 to K-467; C-41 to K-467; V-42 to K- 467; H-43 to K-467; V-44 toK-467; R-45 to K-467; C-46 to K-467; S-47 to K-467; F-48 to K-467; S-49 to K-467; Y-50 to K-467; P-51 to K-467; V-52 to K-467 ;D-53 to K-467; S-54 to K-467; Q-55 to K-467; T-56 to K-467; D-57 to K-467; S-58 to K-467; D-59 to K-467; P-60 to K-467; V-61 toK-467; H-62 to K-467; G-63 to K-467; Y-64 to K-467; W-65 to K-467; F-66 to K-467; R-67 to K-467; A-68 to K-467; G-69 to K-467;N-70 to K-467; D-71 to K-467; 1-72 to K-467; S-73 to K-467: W-74 to K-467; K-75 to K-467; A-76 to K-467; P-77 to K-467; V-78 toK-467; A-79 to K-467; T-80 to K-467; N-81 to K-467; N-82 to K-467; P-83 to K-467; A-84 to K-467; W-85 to K-467; A-86 to K- 467;V-87 to K-467; Q-88 to K-467; E-89 to K-467; E-90 to K-467; T-91 to K-467; R-92 to K-467; D-93 to K-467; R-94 to K-467; F-95 toK-467; H-96 to K-467; L-97 to K-467; L-98 to K-467; G-99 to K-467; D-100 to K-467; P-101 to K-467; Q-102 to K-467; T-103 toK-467; K-104 to K-467; N-105 to K-467; C-106 to K-467; T-107 to K-467; L-108 to K-467; S-109 to K-467; 1-110 to K-467; R-l 11 toK-467; D-112 to K-467; A-113 to K- 467; R-l 14 to K-467; M-115 to K-467; S-116 to K-467; D-l 17 to K-467; A-l 18 to K- 467; G-l 19 toK-467; R-l 20 to K-467; Y-121 to K-467; F-122 to K-467; F-123 to K-467; R-l 24 to K-467; M-125 to K-467; E-l 26 to K-467; K-127 toK-467; G-l 28 to K-467; N- 129 to K-467; 1-130 to K-467; K-131 to K-467; W-132 to K-467; N-133 to K-467; Y- 134 to K-467; K-135to K-467; Y-136 to K-467; D-137 to K-467; Q-138 to K-467; L-139 to K-467; S-140 to K-467; V-141 to K-467; N-142 to K-467; VJ43to K-467; T-144 to K-467; A- 145 to K-467; L-l 46 to K-467; T-l 47 to K-467; H- 148 to K-467; R-149 to K- 467; P-150 to K-467; N-151to K-467; 1-152 to K-467; L-153 to K-467; 1-154 to K-467; P-155 to K-467; G-156 to K-467; T-l 57 to K-467; L-158 to K-467; E-159 toK-467; S- 160 to K-467; G-161 to K-467; C-162 to K-467; F-163 to K-467; Q-164 to K-467; N- 165 to K-467; L-l 66 to K-467; T-l 67 toK-467; C-168 to K-467; S-169 to K-467; V-170 to K-467; P- 171 to K-467; W-172 to K-467; A- 173 to K-467; C-174 to K-467; E-l 75 toK-467; Q-176 to K-467; G-177 to K-467; T-178 to K-467; P-179 to K-467; P-180 to K-467; M-181 to K-467; 1-182 to K-467; S-183 toK-467; W-184 to K-467; M-185 to K- 467; G-186 to K-467; T-187 to K-467; S-188 to K-467; V-189 to K-467; S-190 to K-

467; P-191 toK-467; L-192 to K-467; H-193 to K-467; P-194 to K-467; S-195 to K-467; T-196 to K-467; T-197 to K-467; R-198 to K-467; S-199 toK-467; S-200 to K-467; V- 201 to K-467; L-202 to K-467; T-203 to K-467; L-204 to K-467; 1-205 to K-467; P-206 to K-467; Q-207 toK-467; P-208 to K-467; Q-209 to K-467; H-210 to K-467; H-211 to K-467; G-212 to K-467; T-213 to K-467; S-214 to K-467; L-215 toK-467; T-216 to K- 467; C-217 to K-467; Q-218 to K-467; V-219 to K-467; T-220 to K-467; L-221 to K- 467; P-222 to K-467; G-223 toK-467; A-224 to K-467; G-225 to K-467; V-226 to K- 467; T-227 to K-467; T-228 to K-467; N-229 to K-467; R-230 to K-467; T-231 toK-467; 1-232 to K-467; Q-233 to K-467; L-234 to K-467; N-235 to K-467; V-236 to K-467; S- 237 to K-467; Y-238 to K-467; P-239 toK-467; P-240 to K-467; Q-241 to K-467; N-242 to K-467; L-243 to K-467; T-244 to K-467; V-245 to K-467; T-246 to K-467; V-247 toK-467; F-248 to K-467; Q-249 to K-467; G-250 to K-467; E-251 to K-467; G-252 to K-467; T-253 to K-467; A-254 to K-467; S-255 toK-467; T-256 to K-467; A-257 to K- 467; L-258 to K-467; G-259 to K-467; N-260 to K-467; S-261 to K-467; S-262 to K- 467; S-263 toK-467; L-264 to K-467; S-265 to K-467; V-266 to K-467; L-267 to K-467; E-268 to K-467: G-269 to K-467; Q-270 to K-467; S-271 toK-467; L-272 to K-467; R- 273 to K-467; L-274 to K-467; V-275 to K-467; C-276 to K-467; A-277 to K-467; V- 278 to K-467; D-279 toK-467; S-280 to K-467; N-281 to K-467; P-282 to K-467; P-283 to K-467; A-284 to K-467; R-285 to K-467; L-286 to K-467; S-287 toK-467; W-288 to K-467; T-289 to K-467; W-290 to K-467; R-291 to K-467; S-292 to K-467; L-293 to K- 467; T-294 to K-467; L-295 toK-467; Y-296 to K-467; P-297 to K-467; S-298 to K-467; Q-299 to K-467; P-300 to K-467; S-301 to K-467; N-302 to K-467; P-303 toK-467; L- 304 to K-467; V-305 to K-467; L-306 to K-467; E-307 to K-467; L-308 to K-467; Q-309 to K-467; V-310 to K-467; H-311 toK-467; L-312 to K-467; G-313 to K-467; D-314 to K-467; E-315 to K-467; G-316 to K-467; E-317 to K-467; F-318 to K-467; T-319 toK- 467; C-320 to K-467; R-321 to K-467; A-322 to K-467; Q-323 to K-467; N-324 to K- 467; S-325 to K-467; L-326 to K-467; G-327 toK-467; S-328 to K-467; Q-329 to K-467; H-330 to K-467; V-331 to K-467; S-332 to K-467; L-333 to K-467; N-334 to K-467; L- 335 toK-467; S-336 to K-467; L-337 to K-467; Q-338 to K-467; Q-339 to K-467; E-340 to K-467; Y-341 to K-467; T-342 to K-467; G-343 toK-467; K-344 to K-467; M-345 to K-467; R-346 to K-467; P-347 to K-467; V-348 to K-467; S-349 to K-467; G-350 to K- 467; V-351 toK-467; L-352 to K-467; L-353 to K-467; G-354 to K-467; A-355 to K- 467; V-356 to K-467; G-357 to K-467; G-358 to K-467; A-359 toK-467; G-360 to K- 467; A-361 to K-467; T-362 to K-467; A-363 to K-467; L-364 to K-467; V-365 to K- 467; F-366 to K-467; L-367 toK-467; S-368 to K-467; F-369 to K-467; C-370 to K-467; V-371 to K-467; 1-372 to K-467; F-373 to K-467; 1-374 to K-467; V-375 toK-467; V- 376 to K-467; R-377 to K-467; S-378 to K-467; C-379 to K-467; R-380 to K-467; K-381 to K-467; K-382 to K-467; S-383 toK-467; A-384 to K-467; R-385 to K-467; P-386 to K-467; A-387 to K-467; A-388 to K-467; D-389 to K-467; V-390 to K-467; G-391 toK- 467; D-392 to K-467; 1-393 to K-467; G-394 to K-467; M-395 to K-467; K-396 to K- 467; D-397 to K-467; A-398 to K-467; N-399 toK-467; T-400 to K-467; 1-401 to K-467: R-402 to K-467; G-403 to K-467; S-404 to K-467; A-405 to K-467; S-406 to K-467; Q- 407 toK-467; G-408 to K-467; N-409 to K-467; L-410 to K-467; T-411 to K-467; E-412 to K-467; S-413 to K-467; W-414 to K-467; A-415 toK-467; D-416 to K-467; D-417 to K-467; N-418 to K-467; P-419 to K-467; R-420 to K-467; H-421 to K-467; H-422 to K- 467; G-423to K-467; L-424 to K-467; A-425 to K-467; A-426 to K-467; H-427 to K- 467; S-428 to K-467; S-429 to K-467; G-430 to K-467; E-431to K-467; E-432 to K-467; R-433 to K-467; E-434 to K-467; 1-435 to K-467; Q-436 to K-467; Y-437 to K-467; A- 438 to K-467; P-439to K-467; L-440 to K-467; S-441 to K-467; F-442 to K-467; H-443 to K-467; K-444 to K-467; G-445 to K-467; E-446 to K-467; P-447to K-467; Q-448 to K-467; D-449 to K-467; L-450 to K-467; S-451 to K-467; G-452 to K-467; Q-453 to K- 467; E-454 to K-467; A-455to K-467; T-456 to K-467; N-457 to K-467; N-458 to K- 467; E-459 to K-467; Y-460 to K-467; S-461 to K-467; E-462 to K-467; of SEQ ID NO:34. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification or loss of one or more biological functions of the protein, other functional activities may still be retained. For example the ability of the shortened CD33-like 3 mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a CD33-like 3 mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six CD33-like 3 amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the CD33-like 3 polypeptide shown in Figures 16A-B , up to the leucine residue at position number 6, and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising the amino acid sequence of residues 1-ml of Figure 1. where ml is an integer from 6 to 467 corresponding to the position of the amino acid residue in Figures 16A-B . Moreover, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of C-terminal deletions of the CD33- like 3 polypeptide of the invention shown as SEQ ID NO:34 include polypeptides comprising the amino acid sequence of residues: M-1 to P-466; M-1 to 1-465; M-1 to K- 464; M-1 to 1-463; M-1 to E-462; M-1 to S-461; M-1 to Y-460; M-1 toE-459; M-1 to N- 458; M-1 to N-457; M-1 to T-456; M-1 to A-455; M-1 to E-454; M-1 to Q-453; M-1 to G-452; M-1 to S-451; M-1 toL-450; M-1 to D-449; M-1 to Q-448; M-1 to P-447; M-1 to E-446; M-1 to G-445; M-1 to K-444; M-1 to H-443; M-1 to F-442; M-1 toS-441; M-1 to L-440; M-1 to P-439; M-1 to A-438; M-1 to Y-437; M-1 to Q-436; M-1 to 1-435; M-1 to E-434; M-1 to R-433; M-1 toE-432; M-1 to E-431 ; M-1 to G-430; M-1 to S-429; M-1 to S-428; M-1 to H-427; M-1 to A-426; M-1 to A-425; M-1 to L-424; M-1 toG-423; M-1 to H-422; M-1 to H-421 ; M-l to R-420; MJ to P-419; M-1 to N-418; M-l to D-417; M-l to D-416; M-l to A-415; M-l toW-414; M-1 to S-413; M-l to E-412; M-l to T-411; M-l to L-410; M-1 to N-409; M-1 to G-408; M-1 to Q-407; M-1 to S-406; M-1 toA-405; M-1 to S-404; M-1 to G-403; M-1 to R-402; M-1 to 1-401; M-1 to T-400; M-1 to N-399; M-1 to A-398; M-1 to D-397; M-1 toK-396; M-1 to M-395; M-1 to G-394; M-1 to 1-393; M-1 to D-392; M-1 to G-391; M-1 to V-390; M-1 to D-389; M-1 to A-388; M-1 toA-387; M- 1 to P-386; M-1 to R-385; M-1 to A-384; M-1 to S-383; M-1 to K-382; M-1 to K-381; M-1 to R-380; M-1 to C-379; M-1 toS-378; M-1 to R-377; M-1 to V-376; M-1 to V-375; M-1 to 1-374; M-1 to F-373; M-1 to 1-372; M-1 to V-371; M-1 to C-370; M-1 toF-369; M-1 to S-368; M-1 to L-367; M-1 to F-366; M-1 to V-365; M-1 to L-364; M-1 to A-363; M-1 to T-362; M-1 to A-361; M-1 toG-360; M-1 to A-359; M-1 to G-358; M-1 to G- 357; M-1 to V-356; M-1 to A-355; M-1 to G-354; M-1 to L-353; M-1 to L-352; M-1 toV-351; M-1 to G-350; M-1 to S-349; M-1 to V-348; M-1 to P-347; M-1 to R-346; M-1 to M-345; M-1 to K-344; M-1 to G-343; M-1 toT-342; M-1 to Y-341; M-1 to E-340; M- 1 to Q-339; M-1 to Q-338; M-1 to L-337; M-1 to S-336; M-1 to L-335; M-1 to N-334; M-1 toL-333; M-1 to S-332; M-1 to V-331; M-1 to H-330; M-1 to Q-329; M-1 to S-328; M-1 to G-327; M-1 to L-326; M-1 to S-325; M-1 toN-324; M-1 to Q-323; M-1 to A-322; M-1 to R-321; M-1 to C-320; M-1 to T-319; M-1 to F-318; M-1 to E-317; M-1 to G-316; M-1 toE-315; M-1 to D-314; M-1 to G-313; M-1 to L-312; M-1 to H-311; M-1 to V-310; M-1 to Q-309; M-1 to L-308; M-1 to E-307; M-1 toL-306; M-1 to V-305; M-1 to L-304; M-1 to P-303; M-1 to N-302; M-1 to S-301; M-1 to P-300; M-1 to Q-299; M-1 to S-298; M-1 toP-297; M-1 to Y-296; M-1 to L-295; M-1 to T-294; M-1 to L-293; M-1 to S-292; M-1 to R-291; M-1 to W-290; M-1 to T-289; M-1 toW-288; M-1 to S-287; M-1 to L- 286; M-1 to R-285; M-1 to A-284; M-1 to P-283; M-1 to P-282; M-1 to N-281; M-1 to S-280; M-1 toD-279; M-1 to V-278; M-1 to A-277; M-1 to C-276; M-1 to V-275; M-1 to L-274; M-1 to R-273; M-1 to L-272; M-1 to S-271; M-1 toQ-270; M-1 to G-269; M-1 to E-268; M-1 to L-267; M-1 to V-266; M-1 to S-265; M-1 to L-264; M-1 to S-263; M-1 to S-262; M-1 toS-261; M-1 to N-260; M-1 to G-259; M-1 to L-258; M-1 to A-257; M-1 to T-256; M-1 to S-255; M-1 to A-254; M-1 to T-253; M-1 toG-252; M-1 to E-251; M-1 to G-250; M-1 to Q-249; M-1 to F-248; M-1 to V-247; M-1 to T-246; M-1 to V-245; M-1 to T-244; M-1 toL-243; M-1 to N-242; M-1 to Q-241; M-1 to P-240; M-1 to P-239; M-1 to Y-238; M-1 to S-237; M-1 to V-236; M-1 to N-235; M-1 toL-234; M-1 to Q-233; M-1 to 1-232; M-1 to T-231; M-1 to R-230; M-1 to N-229; M-1 to T-228; M-1 to T-227; M-1 to V-226; M-1 toG-225; M-1 to A-224; M-1 to G-223; M-1 to P-222; M-1 to L-221; M-1 to T-220; M-1 to V-219; M-1 to Q-218; M-1 to C-217; M-1 toT-216; M-1 to L-215; M-1 to S-214; M-l to T-213; MJ to G-212; MJ to H-211; M-l to H-210; M-l to Q-209; M- 1 to P-208; M-1 toQ-207; M-1 to P-206; M-1 to 1-205; M-1 to L-204; M-1 to T-203; M-1 to L-202; M- 1 to V-201; M-1 to S-200; M-1 to S-199; M-1 toR-198; M-1 to T-197; M-1 to T-196; M-1 to S-195; M-1 to P-194; M-1 to H-193; M-1 to L-192; M-1 to P-191; M-1 to S-190; M-l toV-189; M-l to S-188; M-l to T-187; M-1 to G-186; M-1 to M-185; M-l to W-184; M-1 to S-183; M-1 to 1-182; M-1 to M-181; M-1 toP-180; M-1 to P-179; M-1 to T-178; M-1 to G-177; M-1 to Q-176; M-1 to E-175; M-1 to C-174; M-I to A-173; M- 1 to W-172; M-1 toP-171; M-1 to V-170; M-1 to S-169; M-1 to C-168; M-1 to T-167; M-1 to L-166; M-1 to N-165; M-1 to Q-164; M-1 to F-163; M-1 toC-162; M-1 to G-161 ; M-1 to S-160; M-1 to E-159; M-1 to L-158; M-1 to T-157; M-1 to G-156; M-1 to P-155; M-1 to 1-154; M-1 toL-153; M-1 to 1-152; M-1 to N-151; M-1 to P-150; M-1 to R-149; M-1 to H-148; M-1 to T-147; M-1 to L-146; M-1 to A-145; M-1 toT-144; M-1 to V-143; M-1 to N-142; M-1 to V-141; M-1 to S-140; M-1 to L-139; M-1 to Q-138; M-1 to D- 137; M-1 to Y-136; M-1 toK-135; M-1 to Y-134; M-1 to N-133; M-1 to W-132; M-1 to K-131 ; M-1 to 1-130; M-1 to N-129; M-1 to G-128; M-1 to K-127; M-1 toE-126; M-1 to M-125; M-1 to R-124; M-1 to F-123; M-1 to F-122; M-1 to Y-121; M-1 to R-120; M-1 to G-l 19; M-1 to A-118; M-1 toD-117; M-1 to S-116; M-1 to M-115; M-1 to R-l 14; M- 1 to A-l 13; M-1 to D-l 12; M-1 to R-l 11; M-1 to 1-110; M-1 to S-109; M-1 toL-108; M- 1 to T-107; M-1 to C-106; M-1 to N-105; M-1 to K-104; M-1 to T-103; M-1 to Q-102; M-1 to P-101; M-1 to D-100; M-1 toG-99; M-1 to L-98; M-1 to L-97; M-1 to H-96; M-1 to F-95; M-1 to R-94; M-1 to D-93; M-1 to R-92; M-1 to T-91; M-1 to E-90; M-1 toE- 89; M-1 to Q-88; M-1 to V-87; M-1 to A-86; M-1 to W-85; M-1 to A-84; M-1 to P-83; M-1 to N-82; M-1 to N-81; M-1 to T-80; M-1 toA-79; M-1 to V-78; M-1 to P-77; M-1 to A-76; M-1 to K-75; M-1 to W-74; M-1 to S-73; M-1 to 1-72; M-1 to D-71; M-1 to N-70; M-1 toG-69; M-1 to A-68; M-1 to R-67; M-1 to F-66; M-1 to W-65; M-1 to Y-64; M-1 to G-63; M-1 to H-62; M-1 to V-61; M-1 to P-60; M-1 toD-59; M-1 to S-58; M-1 to D- 57; M-1 to T-56; M-1 to Q-55; M-1 to S-54; M-1 to D-53; M-1 to V-52; M-1 to P-51; M- 1 to Y-50; M-1 toS-49; M-1 to F-48; M-1 to S-47; M-1 to C-46; M-1 to R-45; M-1 to V- 44; M-1 to H-43; M-1 to V-42; M-1 to C-41; M-1 to M-40; M-1 toG-39; M-1 to E-38; M-1 to Q-37; M-1 to V-36; M-1 to T-35; M-1 to V-34; M-1 to S-33; M-1 to S-32; M-1 to Q-31 ; M-i to M-30; M-1 toT-29; M-1 to L-28; M-1 to S-27: M-1 to Y-26: M-1 to D-25: M-1 to K-24; M-1 to R-23; M-1 to N-22; M-1 to S-21; M- 1 to K-20; M-1 toQ-19; M-1 to G-18; M-1 to E-17; M-1 to V-16; M-1 to R-15; M-1 to E-14; M-1 to R-13; M-1 to G-12; M-1 to W-l l; M-l to L-10; M-l toL-9; M-1 to P-8; M-1 to L-7; M-1 to L-6; of SEQ ID NO:34. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

In addition, the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO: 16 which have been determined from the following related cDNA genes: HGBAY02R (SEQ ID NO: 100) and HLYBY62R (SEQ ID NOJ01). Based on the sequence similarity to the human CD33L1, translation product of this gene is expected to share at least some biological activities with CD33 proteins, and specifically myeloid modulatory proteins and/or siglec proteins. Such activities are known in the art, some of which are described elsewhere herein.

Specifically, polynucleotides and polypeptides of the invention are also useful for modulating the differentiation of normal and malignant cells, modulating the proliferation and/or differentiation of cancer and neoplastic cells, and modulating the immune response. Polynucleotides and polypeptides of the invention may represent a diagnostic marker for hematopoietic and immune diseases and/or disorders. The full- length protein should be a secreted protein, based upon homology to the CD33 family. Therefore, it is secreted into serum, uπne, or feces and thus the levels is assayable from patient samples. Assuming specific expression levels are reflective of the presence of immune disorders, this protein would provide a convenient diagnostic for early detection. In addition, expression of this gene product may also be linked to the progression of immune diseases, and therefore may itself actually represent a therapeutic or therapeutic target for the treatment of cancel

Polynucleotides and polypeptides of the invention may play an important role in the pathogenesis of human cancers and cellular transformation, particularly those of the immune and hematopoietic systems Polynucleotides and polypeptides of the invention may also be involved m the pathogenesis of developmental abnormalities based upon its potential effects on proliferation and differentiation of cells and tissue cell types. Due to the potential proliferating and differentiating activity of said polynucleotides and polypeptides, the invention is useful as a therapeutic agent inducing tissue regeneration, for treating inflammatory conditions (e.g., inflammatory bowel syndrome, diverticulitis, etc.). Moreover, the invention is useful in modulating the immune response to aberrant polypeptides, as may exist in rapidly proliferating cells and tissue cell types, particularly in adenocarcinoma cells, and other cancers This gene is expressed predominantly on NK cells, and to a lesser extent on T- cells. Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tιssue(s) or cell type(s) present in a biological sample and for diagnosis of the following diseases and conditions which include, but are not limited to, immune disorders and cancer, as well as the immunodiagnosis of acute leukemias. Similarly, polypeptides and antibodies directed to these polypeptides are useful to provide immunological probes for differential identification of the tιssue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, and breast tissue, expression of this gene at significantly higher or lower levels is detected in certain tissues or cell types (e.g. immune, cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue from an individual not having the disorder.

Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 34 as residues: Gly-12 to Tyr-26, Val-52 to Asp-59, Gln-88 to Asp-93, Arg-124 to Asn-129, His-193 to Arg-198, Gln-207 to Thr-213, Gln-338 to Arg- 346, Ser-378 to Ala-384. Ser-413 to Arg-420, Ser-428 to Glu-434, His-443 to Ser-451 , Glu-454 to Ser-461. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution in NK cells, in combination with the homology to siglec family of proteins indicates the protein product of this gene is useful for the diagnosis and treatment of a variety of immune system disorders. NK cells are bone-marrow derived granular lymphocytes that play an important role in natural immunity to infectious diseases and have the capacity to kill certain virally-infected cells and tumor cells that have down-regulated MHC Class-I antigen expression. The killing and proinflammatory activities of NK cells are regulated through a variety of cell surface receptors that can mediate either activity or inhibitory signals. The best understood receptors are those that recognize MHC Class I molecules at the cell surface and deliver a negative signal, thereby protecting normal host cells from cytotoxicity. These receptors can belong either to the C-type lectin superfamily or the Ig superfamily, although in humans the majority are members of the Ig superfamily known as killer cell Ig-like receptors (KIRs). Representative uses are described in the "Immune Activity" and "infectious disease" sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. Involvement in the regulation of cytokine production, antigen presentation, or other processes indicates a usefulness for treatment of cancer (e.g. by boosting immune responses). Expression cells of lymphoid ongm, indicates the natural gene product is involved in immune functions. Therefore it would also be useful as an agent for immunological disorders including arthntis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous Disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophi a, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-veisus-host diseases, or autoimmunity disordeis, such as autoimmune infertility, lense tissue injury, demyehnation. systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogien's Disease, and scleroderma. Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of vaπous blood lineages, and in the differentiation and/or proliferation of vaπous cell types Based upon the tissue distnbution of this protein, antagonists directed against this protein is useful in blocking the activity of this protein. Accordingly, preferred are antibodies which specifically bind a portion of the translation product of this gene. Also provided is a kit for detecting tumors in which expression of this protein occurs. Such a kit comprises in one embodiment an antibody specific for the translation product of this gene bound to a solid support. Also provided is a method of detecting these tumors in an individual which compπses a step of contacting an antibody specific for the translation product of this gene to a bodily fluid from the individual, preferably serum, and ascertaining whether antibody binds to an antigen found in the bodily fluid. Preferably the antibody is bound to a solid support and the bodily fluid is serum. The above embodiments, as well as other treatments and diagnostic tests (kits and methods), are more particularly descπbed elsewhere herein Furthermore, the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, addition to its use as a nutπtional supplement Protein, as well as, antibodies directed against the protein may show utility as a tumor markei and/or immunotheiapy targets for the above listed tissues

Many polynucleotide sequences, such as EST sequences, aie publicly available and accessible through sequence databases Some of these sequences aie related to SEQ ID NO 16 and may have been publicly available pπor to conception of the present invention Pieferably, such related polynucleotides aie specifically excluded fiom the scope of the present invention To list eveiy l elated sequence is cumbersome Accordingly, preferably excluded from the piesent invention are one or more polynucleotides comprising a nucleotide sequence described by the geneial formula of a- b, where a is any integer between 1 to 1734 of SEQ ID NO 16, b is an integer of 15 to 1748, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO 16, and where b is greater than or equal to a + 14

FEATURES OF PROTEIN ENCODED BY GENE NO: 7

This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides The polypeptide of the present invention has been putatively identified as a human integπn alpha 11 homolog derived from a human osteoblast II cDNA library More particularly, the polypeptide of the present invention has been putatively identified as a human integπn alpha 11-subunιt homolog, sometimes hereafter referred to as "integnn alpha 11", "integrin alpha 11- subunit", "al l", "Al l-subunit", and/or "Integπn al l-subunit". The invention also relates to inhibiting the action of such polypeptides.

The integnns are a large family of cell adhesion molecules consisting of noncovalently associated ab heterodimers. We have cloned and sequenced a novel human tegnn a -subunit cDNA, designated al 1 The al 1 cDNA encodes a protein with a 22 amino acid signal peptide, a large 1120 residue extracellulai domain that contains an I-domain of 207 lesidues and is linked by a transmembrane domain to a short cytoplasmic domain of 24 ammo acids The deduced al l piotein shows the typical structuial features of tegnn a-subunits and is similai to a distinct gioup of a-subunits fiom collagen-b dmg integnns However, it differs from most integrin a-chams by an incompletetely preserved cytoplasmic GFFKR motif

The human ITGA11 gene was located to bands q22 3-23 on chromosome 15, and its transcripts were found predominantly in bone, cartilage as well as in cardiac and skeletal muscle. Expression of the 5.5 kilobase al l mRNA was also detectable in ovary and small intestine.

All vertebrate cells express members of the integπn family of cell adhesion molecules, which mediate cellular adhesion to other cells and extracellular subtratum, cell migration and participate in important physiologic processes from signal transduction to cell proliferation and differentiation {Hynes, 92; Springer, 92 }.

Integrins are structurally homologous heterodimeπc type-I membrane glycoproteins formed by the noncovalent association of one of eight b -subunits with one of the 17 different a-subunits descπbed to date, resulting in at least 22 different ab complexes Their binding specificities for cellular and extracellular hgands are determined by both subunits and are dynamically regulated in a cell-type-specific mode by the cellular environment as well as by the developmental and activation state of the cell {Diamond and Spπnger, 94} In integπn a -subunits, the aminoterminal region of the large extracellular domain consists of a seven-fold repeated structure which is predicted to fold into a b -propeller domain {Corbi et al., 1987; Springer, 1997 }. The three or four C-terminal repeats contain putative divalent cation binding motifs that are thought to be important for ligand binding and subunit association {Diamond and Springer, 94}. The al, a2, a 10, aD, aE, aL, aM and aX-subunits contain an approximately 200 amino acid I- domain inserted between the second and third repeat that is not present in other a-chains {Larson et al., 1989}. Several isolated I-domains have been shown to independently bind the ligands of the parent integrin heterodimer {Kamata and Takada, 1994; Randi and Hogg, 1994}. The a3, a5-8, allb and aV-subunits are proteolytically processed at a conserved site into disulphide-linked heavy and light chains, while the a4-subunit is cleaved at a more aminoterminal site into two fragments that remain noncovalently associated {Hemler et al., 90}. Additional a-subunit variants are generated by alternative splicing of primary transcripts {Ziober et al., 93; Delwel et al., 95; Leung et al., 98 }. The extracellular domains of a-integrin subunits are connected by a single spanning transmembrane domain to short, diverse cytoplasmic domains whose only conserved feature is a membrane-proximal KXGFF(K/R)R motif {Sastry and Horwitz, 1993 }. The cytoplasmic domains have been implicated in the cell-type-specific modulation of integrin affinity states {Williams et al., 1994}.

The polypeptide of the present invention has been putatively identified as a member of the integrin family and has been termed integrin alpha 11 subunit ("al 1 "). This identification has been made as a result of amino acid sequence homology to the human integrin alpha 1 subunit (See Genbank Accession No. gi|346210).

Figures 19A-F show the nucleotide (SEQ ID NO: 17) and deduced amino acid sequence (SEQ ID NO:35) of al 1. Predicted amino acids from about 1 to about 22 constitute the predicted signal peptide (amino acid residues from about 1 to about 22 in SEQ ID NO:35) and are represented by the underlined amino acid regions; amino acids from about 666 to about 682, and/or amino acids from about 1145 to about 1161 constitute the predicted transmembrane domains (amino acids from about 666 to about 682, and/or ammo acids from about 1145 to about 1161 in SEQ ID NO:35) and are represented by the double underlined ammo acids; and amino acids from about 64 to about 96 constitute the predicted immunoglobulin and major histocompatibility complex protein domain (amino acids from about 64 to about 96 in SEQ ID NO:35) and are represented by the bold amino acids

Figure 20 shows the regions of similarity between the ammo acid sequences of the mtegnn alpha 11 subunit (al l) protein (SEQ ID NO:35) and the human integrin alpha 1 subunit (SEQ ID NO: 103) Figure 21 shows an analysis of the integrin alpha 1 1 subunit (al l) ammo acid sequence Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity, amphipathic regions; flexible regions; antigenic index and surface probability are shown

A polynucleotide encoding a polypeptide of the present invention is obtained from human ovary ,small intestine, fetal heart, fetal brain, large intestine, osteoblasts, human trabelcular bone cells, messangial cells, adipocytes, osteosarcoma, chondrosarcoma, breast cancer cells, and bone marrow tissues and cells. The polynucleotide of this invention was discovered in a human osteoblast II cDNA library Its translation product has homology to the characteristic immunoglobulin and major histocompatibility complex protein domain of integrin family members. As shown in Figures 19A-F, al 1 has transmembrane domains (the transmembrane domains comprise amino acids 666 - 682 and/or 1145 - 1161 of SEQ ID NO:35; which correspond to amino acids 666 - 682 and/or 1145 - 1161 of Figures 19A-F) with strong conservation between other members of the integπn family. The polynucleotide contains an open reading frame encoding the al 1 polypeptide of 1189 amino acids. The present invention exhibits a high degree of homology at the ammo acid level to the human integπn alpha 1 subunit (as shown in Figure 20)

Preferred polypeptides of the invention compπse the following amino acid sequence: TNGYQKTGDVYKCPVIHGNCTKLNLGRVTLSNV (SEQ ID NO: 102). Polynucleotides encoding these polypeptides are also provided.

The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the al 1 polypeptide having the ammo acid sequence shown m Figures 19A-F (SEQ ID NO:35) The nucleotide sequence shown in Figures 19A-F (SEQ ID NO 35) was obtained by sequencing a cloned cDNA (HOHBY69), which was deposited on Novembei 17 at the American Type Culture Collection, and given Accession Numbei 203484

The present invention is further directed to fragments of the isolated nucleic acid molecules descnbed herein By a fiagment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ ID NO: 17 is intended DNA fragments at least about 15nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and pπmers as discussed herein. Of course, larger fragments 50-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown SEQ ID NO 17. By a fragment at least 20 nt m length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NO: 17. In this context "about" includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Representative examples of al 1 polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, from about 501 to about 550, from about 551 to about 600, from about 601 to about 650, from about 651 to about 700, from about 701 to about 750, from about 751 to about 800, from about 801 to about 850, from about 851 to about 900, from about 901 to about 950, from about 951 to about 1000, from about 1001 to about 1050, from about 1051 to about 1100, from about 1101 to about 1150, from about 1151 to about 1200, from about 1201 to about 1250, from about 1251 to about 1300, from about 1301 to about 1350, from about 1351 to about 1400, from about 1401 to about 1450, from about 1451 to about 1500, from about 1501 to about 1550, from about 1551 to about 1600. from about 1601 to about 1650, from about 1651 to about 1700, from about 1701 to about 1750, from about 1751 to about 1800, from about 1801 to about 1850, from about 1851 to about 1900, from about 1901 to about 1950, from about 1951 to about 2000, from about 2001 to about 2050, from about 2051 to about 2100, from about 2101 to about 2150, from about 2151 to about 2200, from about 2201 to about 2250, from about 2251 to about 2300, from about 2301 to about 2350, from about 2351 to about 2400, from about 2401 to about 2450, from about 2451 to about 2500, from about 2501 to about 2550, from about 2551 to about 2600, from about 2601 to about 2650, from about 2651 to about 2700, from about 2701 to about 2750, from about 2751 to about 2800, from about 2801 to about 2850, from about 2851 to about 2900, from about 2901 to about 2950, from about 2951 to about 3000, from about 3001 to about 3050, from about 3051 to about 3100, from about 3101 to about 3150, from about 3151 to about 3200, from about 3201 to about 3250, from about 3251 to about 3300, from about 3301 to about 3350, from about 3351 to about 3400, from about 3401 to about 3450, from about 3451 to about 3500, from about 3501 to about 3550, from about 3551 to about 3600, from about 3601 to about 3650, from about 3651 to about 3700, from about 3701 to about 3750, from about 3751 to about 3800, from about 3801 to about 3850, from about 3851 to about 3900, from about 3901 to about 3950, from about 3951 to about 4000, from about 4001 to about 4050, from about 4051 to about 4100, from about 4101 to about 4150, from about 4151 to about 4200, from about 4201 to about 4250, from about 4251 to about 4300, from about 4301 to about 4350, from about 4351 to about 4400, from about 4401 to about 4450, from about 4451 to about 4500, from about 4501 to about 4550, from about 4551 to about 4600, from about 4601 to about 4650, from about 4651 to about 4700, from about 4701 to about 4750, from about 4751 to about 4800, from about 4801 to about 4850, from about 4851 to about 4900, from about 4901 to about 4950, from about 4951 to about 4995, from about, from about 1 to about 236, from about 144 to about 188, from about 231 to about 276 of SEQ ID NO.J7. or the complementary strand thereto, or the cDNA contained in the deposited gene. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.

Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding a member selected from the group: a polypeptide comprising or alternatively, consisting of, any one of the transmembrane domains (amino acid residues from about 666 to about 682 and/or 1145 to about 1161 in Figures 19A-F (amino acids from about 666 to about 682 and/or 1145 to about 1161 in SEQ ID NO:35), in addition to the immunoglobulin and major histocompatibility complex protein domain (amino acid residues from about 64 to about 96 in Figures 19A-F (amino acids from about 64 to about 96 in SEQ ID NO:35). Since the location of these domains have been predicted by computer analysis, one of ordinary skill would appreciate that the amino acid residues constituting these domains may vary slightly (e.g., by about 1 to 15 amino acid residues) depending on the criteria used to define each domain. In additional embodiments, the polynucleotides of the invention encode functional attributes of al l.

Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions"), turn and turn-forming regions ("turn- regions"), coil and coil-forming regions ("coil-regions"), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions of the present invention.

The data representing the structural or functional attπbutes of al 1 set forth in Figure 21 and oi Table VII, as descπbed above, was generated using the vaπous modules and algonthms of the DNA^STAR set on default parameters In a preferred embodiment, the data piesented in columns VIII, IX, XIII, and XIV of Table VII can be used to determine iegions of al 1 which exhibit a high degiee of potential for antigenicity Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/oi XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occui in the process of initiation of an immune response

Certain preferred regions in these legards aie set out in Figure 21, but may, as shown in Table VII, be represented or identified by using tabular representations of the data presented in Figure 21 The DNA STAR computei algorithm used to generate

Figuie 21 (set on the onginal default parameters) was used to present the data in Figure 21 in a tabular format (See Table VII). The tabular format of the data in Figure 21 is used to easily determine specific boundaπes of a preferred region The above-mentioned preferred regions set out in Figure 21 and in Table VII include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figures 19A-F. As set out in Figure 21 and in Table VII, such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions and Hopp-Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenic index and Emini surface-forming regions. Even if deletion of one or more amino acids from the N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimeπze, etc.) may still be retained. For example, the ability of shortened al 1 mutems to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majonty of the residues of the complete or mature polypeptide are removed from the N-terminus.

Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that an all mutein with a large number of deleted N-terminal ammo acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six al 1 ammo acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides having one oi more residues deleted from the amino terminus of the al 1 ammo acid sequence shown in Figures 19A-F, up to the threonine residue at position number 1184 and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides compnsmg the ammo acid sequence of residues n 1-1189 of Figures 19A-F, where nl is an integer from 2 to 1184 corresponding to the position of the amino acid residue in Figures 19A-F (which is identical to the sequence shown as SEQ ID NO:35). In anothei embodiment, N-terminal deletions of the al l polypeptide can be descπbed by the general formula n2-l 189, where n2 is a number from 2 to 1184, corresponding to the position of amino acid identified in Figure 19. N-termmal deletions of the al 1 polypeptide of the invention shown as SEQ ID NO:35 include polypeptides compπsing the amino acid sequence of residues: N-terminal deletions of the al 1 polypeptide of the invention shown as SEQ ID NO:35 include polypeptides compπsmg the amino acid sequence of residues: D-2 to E-l 189; L-3 to E-l 189; P-4 to E-l 189; R-5 toE-1189; G-6 to E-l 189; L-7 to fil l 89; V-8 to E-l 189; V-9 to E-l 189;A- 10 to E-l 189; W-l l to E-l 189; A- 12 to E-l 189; L-13 to E-1189;S-14 to E-1189; L-15 to E-1189; W-16 to E-1189; P-17 to E-1 189;G-18 to E-1189; F-19 to E-l 189; T-20 to E-l 189; D-21 to E-l 189;T-22 to E-l 189; F-23 to E- 1189; N-24 to E-1189; M-25 to E-1189;D-26 to E-1189; T-27 to E-1189; R-28 to E- 1189; K-29 to E-1189;P-30 to E-1189; R-31 to E-1189; V-32 to E-1189; 1-33 to E- 1189;P-34toE-1189;G-35toE-1189;S-36toE-1189;R-37toE-1189;T-38toE-1189; A-39 to E-1189; F-40 to E-1189; F-41 to E-l 189;G-42 to E-1189; Y-43 to E-l 189; T-44 to E-1189; V-45 to E-l 189;Q-46 to E-l 189; Q-47 to E-l 189; H-48 to E-l 189; D-49 to E- 1189J-50 to E- 1189; S-51 to E- 1189; G-52 to E- 1189; N-53 to E- 1189;K-54 to E- 1189; W-55 to E-1189; L-56 to E-l 189; V-57 to E-1189;V-58 to E-1189; G-59 to E-l 189; A- 60 to E-1189; P-61 to E-l 189;L-62 to E-1189; E-63 to E-l 189; T-64 to E-l 189; N-65 to E-1189;G-66toE-1189; Y-67 to E-1189; Q-68 to E-1189; K-69 to E-l 189;T-70 to E- 1189; G-71 to E-l 189; D-72 to E-1189; V-73 to E-l 189;Y-74 to E-l 189; K-75 to E- 1189; C-76 to E-l 189; P-77 to E-l 189;V-78 to E-l 189; 1-79 to E-l 189; H-80 to E-l 189; G-81 to E-l 189;N-82 to E-l 189; C-83 to E-1189; T-84 to E-l 189; K-85 to E-l 189;L-86 to E-1189; N-87 to E-l 189; L-88 to E-1189; G-89 to E-l 189;R-90 to E-1189; V-91 to E- 1189; T-92 to E-l 189; L-93 to E-l 189;S-94 to E-l 189; N-95 to E-l 189; V-96 to E-l 189; S-97 to E-1189;E-98 to E-1189; R-99 to E-1189; K-100 to E-1189; D-101 to E-l 189;N- 102 to E-1189; M-103 to E-l 189; R-104 to E-1189; L-105 toE-1189; G-106 to E-l 189; L-107toE-1189;S-108toE-1189;L-109toE-1189; A-l 10 to E-1189; T-l 11 to E-l 189; N-112 to E-l 189;P-113 to E-l 189; K-114 to E-1189; D-l 15 to E-l 189; N-116 toE-1189; S-117toE-1189;F-118toE-1189;L-119toE-1189;A-120toE-1189;C-121 to E-l 189; S-122 to E-l 189; P-123 to E-l 189;L-124 to E-l 189; W-125 to E-1189; S-126 to E-l 189; H-127toE-1189;E-128toE-1189;C-129toE-1189;G-130toE-1189;S-131toE-1189; S-132 to E-l 189; Y-133 to E-l 189; Y-134 to E-1189;T-135 to E-l 189; T-136 to E-l 189; G-137 to E-l 189; M-138 toE-1189; C-139 to E-l 189; S-140 to E-l 189; R-141 to E- 1189; V-142toE-1189; N-143 to E-1189; S-144 toE-1189; N-145 to E-1189;F-146 toE- 1189; R-147 toE-1189; F-148 to E-1189; S-149 toE-1189; K-150 to E-1189; T-151 toE- 1189; V-152 to E-1189; A-153to E-1189; P-154 to E-1189; A-155 to E-1189; L-156 to E-1189;Q-157toE-1189;R-158toE-1189;C-159toE-1189;Q-160toE-1189;T-161 to E-1189;Y-162toE-1189;M-163toE-1189;D-164toE-1189;I-165toE-1189;V-166to E-1189;I-167toE-1189;V-168toE-1189;L-169toE-1189;D-170toE-1189;G-171to E-1189;S-172toE-1189;N-173toE-1189;S-174toE-1189;I-175toE-1189; Y-176to E-1189;P-177toE-1189;W-178toE-1189;V-179toE-1189;E-180toE-1189; V-181 to E-1189; Q-182 to E-1189;H-183 to E-1189; F-184 to E-1189; L-185 to E-1189; 1-186 toE-1189;N-187toE-1189;I-188toE-1189;L-189toE-1189;K-190toE-1189;K-191 to E-1189; F-192 to E-1189; Y-193 to E-l 189:1-194 to E-1189; G-195 to E-1189; P-196 to E-1189; G-197 toE-1189; Q-198 to E-1189; 1-199 to E-1189; Q-200 to E-1189; V- 201 to E-l 189; G-202 to E-l 189; V-203 to E-l 189; V-204 to E-l 189;Q-205 to E-l 189; Y-206 to E-l 189; G-207 to E-l 189; E-208 toE-1189; D-209 to E-l 189; V-210 to E- 1189; V-211 to E-1189; H-212to E-1189; E-213 to E-l 189; F-214 to E-1189; H-215 to E-1189;L-216toE-1189;N-217toE-1189;D-218toE-1189; Y-219 toE-1189; R-220 to E-l 189; S-221 to E-l 189; V-222 to E-l 189; K-223to E-l 189; D-224 to E-l 189; V-225 to E-1189; V-226 to E-l 189;E-227 to E-l 189; A-228 to E-l 189; A-229 to E-l 189; S-230 toE-1189; H-231 to E-l 189; 1-232 to E-1189; E-233 to E-1189; Q-234to E-1189; R-235 to E-l 189; G-236 to E-l 189; G-237 to E-l 189;T-238 to E-l 189; E-239 to E-l 189; T-240 to E-1189; R-241 toE-1189; T-242 to E-1189; A-243 to E-1189; F-244 to E-1189; G- 245to E-l 189; 1-246 to E-l 189; E-247 to E-l 189; F-248 to E-l 189;A-249 to E-l 189; R- 250 to E-l 189; S-251 to E-l 189; E-252 toE-1189; A-253 to E-l 189; F-254 to E-l 189; Q- 255 to E-l 189; K-256to E-l 189; G-257 to E-l 189; G-258 to E-l 189; R-259 to E- 1189;K-260 to E-1189; G-261 to E-1189; A-262 to E-1189; K-263 toE-1189; K-264 to E-1189; V-265 to E-1189; M-266 to E-1189; I-267to E-1189; V-268 to E-1189; 1-269 to E-1189; T-270 to E-1189;D-271 to E-1189; G-272 to E-1189; E-273 to E-l 189; S-274 toE-1189; H-275 to E-l 189; D-276 to E-l 189; S-277 to E-l 189; P-278to E-l 189; D-279 to E-l 189; L-280 to E-l 189; E-281 to E-l 189;K-282 to E-l 189; V-283 to E-l 189; 1-284 to EJ189; Q-285 toEJ189; Q-286 to EJ189; S-287 to EJ189; E-288 to EJ 189; R- 289to E-l 189; D-290 to E-l 189; N-291 to E-l 189; V-292 to E-1189;T-293 to E-l 189; R-294 to E-1189; Y-295 to E-1189; A-296 toE-1189; V-297 to E-1189; A-298 to E- 1189; V-299 to E-l 189; L-300to E-l 189; G-301 to E-l 189; Y-302 to E-l 189; Y-303 to E-1189;N-304 to E-l 189; R-305 to E-1189; R-306 to E-1189; G-307 toE-1189; 1-308 to E-1189; N-309 to E-l 189; P-310 to E-l 189; E-31 lto E-l 189; T-312 to E-l 189; F-313 to E-l 189; L-314 to E-l 189;N-315 to E-1189; E-316 to E-1189; 1-317 to E-l 189; K-318 toE-1189; Y-319 to E-l 189; 1-320 to E-1189; A-321 to E-1189; S-322to E-1189; D-323 to E-1189; P-324 to E-l 189; D-325 to E-1189;D-326 to E-1189; K-327 to E-l 189; H- 328 to E-l 189; F-329 toE-1189; F-330 to E-l 189: N-331 to E-1189; V-332 to E-l 189; T- 333to E-l 189; D-334 to E-l 189; E-335 to E-l 189; A-336 to E-l 189;A-337 to E-l 189; L- 338 to E-1189; K-339 to E-l 189; D-340 toE-1189; I-34I to E-1189; V-342 to E-l 189; D-343 to E-l 189; A-344to E-l 189; L-345 to E-l 189; G-346 to E-l 189; D-347 to E- 1189;R-348 to E- 1189; 1-349 to E- 1189; F-350 to E- 1189; S-351 toE- 1189; L-352 to E- 1189; E-353 to E-l 189: G-354 to E- 1189; T-355to E-1189; N-356 to E- 1189; K-357 to E-l 189; N-358 to E-l 189;E-359 to E-l 189; T-360 to E-l 189; S-361 to E-l 189; F-362 toE-1189; G-363 to E-l 189; L-364 to E-l 189; E-365 to E-l 189; M-366to E-l 189; S-367 to E-1189; Q-368 to E-1189; T-369 to E-1189;G-370 to E-1189; F-371 to E-1189; S-372 to E-l 189; S-373 toE-1189; H-374 to E-l 189; V-375 to E-l 189; V-376 to E-l 189; E- 377to E-1189; D-378 to E-1189; G-379 to E-1189; V-380 to E-1189;L-381 to E-l 189; L- 382 to E-1189; G-383 to E-1189; A-384 toE-1189; V-385 to E-1189; G-386 to E-l 189; A-387 to E-1189; Y-388to E-l 189; D-389 to E-l 189; W-390 to E-l 189; N-391 to fil l 89;G-392 to E-l 189; A-393 to E-l 189; V-394 to E-l 189; L-395 toE-1189; K-396 to E-1189; E-397 to E-1189; T-398 to E-1189; S-399to E-1189; A-400 to E-1189; G-401 to E-1189; K-402 to E-l 189;V-403 to E-l 189; 1-404 to E-l 189; P-405 to E-l 189; L-406 toE-1189; R-407 to E-l 189; E-408 to E-1189; S-409 to E-1189; Y-410to E-1189; L-411 to E-l 189; K-412 to E-l 189; E-413 to E-l 189;F-414 to E-1189; P-415 to E-l 189; E-416 to E-l 189; E-417 toE-1189; L-418 to E-l 189; K-419 to E-l 189; N-420 to E-l 189; H- 421 to E-1189; G-422 to E-1189; A-423 to E-1189; Y-424 to E-1189;L-425 to E-1189; G-426 to E-1189; Y-427 to E-1189; T-428 toE-1189; V-429 to E-1189; T-430 to E-1189; S-431 to E-1189; V-432to E-1189; V-433 to E-1189; S-434 to E-1189; S-435 to E- 1189;R-436 to E-1189; Q-437 to E-1189; G-438 to E-1189; R-439 toE-1189; V-440 to E-l 189; Y-441 to E-1189; V-442 to E-l 189; A-443to E-1189; G-444 to E-l 189; A-445 to E- 1189; P-446 to E- 1189;R-447 to E- 1189; F-448 to E- 1189; N-449 to E- 1189; H-450 toE-1 189; T-451 to E-l 189; G-452 to E-l 189; K-453 to E-l 189; V-454to E-1189; 1-455 to E-1189; L-456 to E-l 189; F-457 to E-1189; T-458to E-1189; M-459 to E-1189; H-460 to E-1189; N-461 to E-1189;N-462 to E-1189; R-463 to E-1189; S-464 to E-1189; L-465 toE-1 189; T-466 to E-l 189; 1-467 to E-l 189; H-468 to E-l 189; Q-469to E-1189; A-470 to E-l 189; M-471 to E-l 189; R-472 to E-l 189;G-473 to E-l 189; Q-474 to E-l 189; Q- 475 to E-1189; 1-476 toE-1189; G-477 to E-1189; S-478 to E-l 189; Y-479 to E-1189; F- 480to E-l 189; G-481 to E-1189; S-482 to E-1189; E-483 to E-l 189J-484 to E-l 189; T- 485 to E-l 189; S-486 to E-l 189; V-487 toE-1189; D-488 to E-l 189; 1-489 to E-l 189; D- 490 to E-l 189; G-491to E-l 189; D-492 to E-l 189; G-493 to E-l 189; V-494 to fil l 89;T-495 to E-1189; D-496 to E-1189; V-497 to E-1189; L-498 toE-1189; L-499 to E- 1189; V-500 to E-l 189; G-501 to E-l 189; A-502to E-l 189; P-503 to E-1189; M-504 to E-1189; Y-505 to E-1189;F-506 to E-1189; N-507 to E-1189; E-508 to E-1189; G-509 toE-1189; R-510 to E-1189; E-511 to E-1189; R-512 to E-1189; G-513to E-1189; K-514 to E-1189; V-515 to E-1189; Y-516 to E-1189;V-517 to E-1189; Y-518 to E-1189; E- 519 to E-l 189; L-520 toE-1189; R-521 to E-l 189; Q-522 to E-l 189; N-523 to E-l 189; R-524to E-l 189; F-525 to E-l 189; V-526 to E-l 189; Y-527 to E-l 189;N-528 to E-l 189; G-529 to E-l 189; T-530 to E-l 189; L-531 toE-1189; K-532 to E-l 189; D-533 to E-l 189; S-534 to E-l 189; H-535to E-l 189; S-536 to E-l 189; Y-537 to E-l 189; Q-538 to E- 1189;N-539 to E-l 189; A-540 to E-l 189; R-541 to E-l 189; F-542 toE-1189; G-543 to E- 1189; S-544 to E-l 189; S-545 to E-1189; I-546to E-1189; A-547 to E-1189; S-548 to fil l 89; V-549 to E-l 189;R-550 to E-l 189; D-551 to E-l 189; L-552 to E-1189; N-553 toE-1189; Q-554 to E-l 189; D-555 to E-l 189; S-556 to E-l 189; Y-557to E-l 189; N-558 to E-l 189; D-559 to E-l 189; V-560 to E-l 189;V-561 to E-l 189; V-562 to E-l 189; G- 563 to E-l 189; A-564 toE-1189; P-565 to E-l 189; L-566 to E-l 189; E-567 to E-l 189; D-568to E-l 189; N-569 to E-l 189; H-570 to E-l 189; A-571 to E-l 189;G-572 to E-l 189; A-573 to E-1189; 1-574 to E-1189; Y-575 toE-1189; 1-576 to E-l 189; F-577 to E-l 189; H-578 to E-l 189; G-579to E-l 189; F-580 to E-1189; R-581 to E-l 189; G-582 to E- 1189;S-583 to E-l 189; 1-584 to E-1189; L-585 to E-l 189; K-586 toE-1189; T-587 to E- 1189; P-588 to E-1189; K-589 to E-l 189; Q-590to E-1189; R-591 to E-1189; 1-592 to E- 1189; T-593 to E-1189;A-594 to E-1189; S-595 to E-1189; E-596 to E-1189; L-597 toE- 1189; A-598 to E-1189; T-599 to E-1189; G-600 to E-1189; L-601to E-1189; Q-602 to E-1189; Y-603 to E-l 189; F-604 to E-1189;G-605 to E-l 189; C-606 to E-1189; S-607 to E-l 189; 1-608 toE-1189; H-609 to E-l 189; G-610 to E-l 189; Q-611 to E-l 189; L-612to E-1189; D-613 to E-l 189; L-614 to E-l 189; N-615 to E-1189;E-616 to E-1189; D-617 to E-1189; G-618 to E-1189; L-619 toE-1189; I-620 to E-1189; D-621 to E-1189; L-622 to E-1189; A-623to E-1189; V-624 to E-1189; G-625 to E-1189; A-626 to E-1189;L-627 to E-1189; G-628 to E-l 189; N-629 to E-1189; A-630 toE-1189; V-631 to E-1189; 1-632 to E-l 189; L-633 to E-l 189; W-634to E-l 189; S-635 to E-l 189; R-636 to E-l 189; P-637 to E-1189;V-638 to E-l 189; V-639 to E-1189; Q-640 to E-l 189; 1-641 toE-1189; N-642 to E-l 189; A-643 to E-l 189; S-644 to E-l 189; L-645to E-1189; H-646 to E-l 189; F-647 to E-1189; E-648 to E-l 189;P-649 to E-l 189; S-650 to E-1189; K-651 to E-l 189; 1-652 toE-1189; N-653 to E-l 189; 1-654 to E-l 189; F-655 to E-l 189; H-656to E-l 189; R-657 to E-1189; D-658 to E-1189; C-659 to E-1189;K-660 to E-1189; R-661 to E-1189; S-662 to E-1189; G-663 toE-1189; R-664 to E-1189; D-665 to E-1189; A-666 to E-1189; T- 667to E-1189; C-668 to E-1189; L-669 to E-1189; A-670 to E-1189;A-671 to E-1189; F- 672 to E-1189; L-673 to E-1189; C-674 toE-1189; F-675 to E-1189; T-676 to E-1189; P- 677 to E-l 189; 1-678 toE-1189; F-679 to E-l 189; L-680 to E-1189; A-681 to E-l 189; P- 682to E-1189; H-683 to E-1189; F-684 to E-1189; Q-685 to E-1189;T-686 to E-1189; T- 687 to E-l 189; T-688 to E-l 189; V-689 toE-1189; G-690 to E-l 189; 1-691 to E-l 189; R- 692 to E- 1189; Y-693to E-1189; N-694 to E- 1189; A-695 to E- 1189; T-696 to E- 1189;M-697 to E-1189; D-698 to E-1189; E-699 to E-1189; R-700 toE-1189; R-701 to E-1189; Y-702 to E-l 189; T-703 to E-l 189; P-704to E-1189; R-705 to E-1189; A-706 to E-1189; H-707 to E-1189;L-708 to E-1189; D-709 to E-1189; E-710 to E-1189; G-711 toE-1189; G-712 to E-l 189; D-713 to E-l 189; R-714 to E-l 189; F-715to E-l 189; T-716 to E-1189; N-717 to E-l 189; R-718 to E-1189;A-719 to E-1189; V-720 to E-1189; L- 721 to E-l 189; L-722 toE-1189; S-723 to E-1189; S-724 to E-l 189; G-725 to E-1189; Q- 726to E-1189; E-727 to E-1189; L-728 to E-1189; C-729 to E-1189;E-730 to E-1189; R- 731 to E-l 189; 1-732 to E-l 189; N-733 toE-1189; F-734 to E-l 189; H-735 to E-l 189; V- 736 to E-l 189; L-737to E-1189; D-738 to E-1189; T-739 to E-1189; A-740 to E-l 189;D- 741 to E-l 189; Y-742 to E-l 189; V-743 to E-l 189; K-744 toE-1189; P-745 to E-l 189; V-746 to E-l 189; T-747 to E-l 189; F-748to E-l 189; S-749 to E-l 189; V-750 to E-l 189; E-751 to E-l 189;Y-752 to E-l 189; S-753 to E-l 189; L-754 to E-l 189; E-755 toE-1189; D-756 to E-1189; P-757 to E-1189; D-758 to E-1189; H-759to E-1189; G-760 to E-

1189; P-761 to E-1189; M-762 to E-1189;L-763 to E-1189; D-764 to E-1189; D-765 to E-1189; G-766 toE-1189; W-767 to E-1189; P-768 to E-1189; T-769 to E-1189; T-770to E-l 189; L-771 to E-l 189; R-772 to E-l 189; V-773 to E-l 189;S-774 to E-l 189; V-775 to E-1189; P-776 to E-l 189; F-777 toE-1189; W-778 to E-l 189; N-779 to E-1189; G-780 to E-1189; C-781to E-1189; N-782 to E-1189; E-783 to E-1189; D-784 to E-1189;E-785 to E- 1189; H-786 to E- 1189; C-787 to E- 1189; V-788 toE- 1189; P-789 to E- 1189; D-790 to E-1189; L-791 to E-1189; V-792to E-l 189; L-793 to E-1189; D-794 to E-1189; A-795 to E-1189;R-796 to E-1189; S-797 to E-1189; D-798 to E-1189; L-799 toE-1189; P-800 to E-1189; T-801 to E-1189; A-802 to E-1189; M-803to E-1189; E-804 to E-l 189; Y- 805 to E-1189; C-806 to E-1189;Q-807 to E-1189; R-808 to E-1189; V-809 to E-1189; L-810 toE-1189; R-811 to E-1189; K-812 to E-1189; P-813 to E-1189; A-814to E-1189; Q-815 to E-1189; D-816 to E-1189; C-817 to E-1189;S-818 to E-1189; A-819 to E-1189; Y-820 to E-1189; T-821 toE-1189; L-822 to E-1189; S-823 to E-1189; F-824 to E-1189; D-825to E-l 189; T-826 to E-1189; T-827 to E-l 189; V-828 to E-l 189;F-829 to E-1189; 1-830 to E-l 189; 1-831 to E-1189; E-832 to E-1189;S-833 to E-l 189; T-834 to E-1189; R-835 to E-l 189; Q-836 toE-1189; R-837 to E-l 189; V-838 to E-l 189; A-839 to E- 1189; V-840to E-l 189; E-841 to E-l 189; A-842 to E-l 189; T-843 to E-l 189;L-844 to E- 1189; E-845 to E-l 189; N-846 to E-l 189; R-847 toE-1189; G-848 to E-l 189; E-849 to E-l 189; N-850 to E-l 189; A-851to E-l 189; Y-852 to E-1189; S-853 to E-l 189; T-854 to E-1189;V-855 to E-l 189; L-856 to E-1189; N-857 to E-1189; 1-858 toE-1189; S-859 to E-1189; Q-860 to E-1189; S-861 to E-1189; A-862to E-1189; N-863 to E-1189; L-864 to E-l 189; Q-865 to E-l 189;F-866 to E-l 189; A-867 to E-l 189; S-868 to E-l 189; L-869 toE-1189; 1-870 to E-1189; Q-871 to E-l 189; K-872 to E-l 189; E-873to E-l 189; D-874 to E- 1189; S-875 to E- 1189; D-876 to E- 1 189;G-877 to E- 1189; S-878 to E- 1189; 1-879 to E-l 189; E-880 toE-1189; C-881 to E-l 189; V-882 to E-l 189; N-883 to E-l 189; E- 884to E-l 189; E-885 to E-l 189; R-886 to E-l 189; R-887 to E-l 1891-888 to E-l 189; Q- 889 to E-1189; K-890 to E-1189; Q-891 toE-1189; V-892 to E-1189; C-893 to E-1189; N-894 to E-l 189; V-895to E-l 189; S-896 to E-l 189; Y-897 to E-l 189; P-898 to E- 1189;F-899 to E-1189; F-900 to E-1189; R-901 to E-1189; A-902 toE-1189; K-903 to fil l 89; A-904 to E-1189; K-905 to E-1189; V-906to E-1189; A-907 to E-1189; F-908 to E-1189; R-909 to E-l 189;L-910 to E-1189; D-911 to E-l 189; F-912 to E-l 189; E-913 toE-1189; F-914 to E-1189; S-915 to E-1189; K-916 to E-1189; S-917to E-1189; I-918 to E-l 189; F-919 to E-l 189; L-920 to E-l 189;H-921 to E-l 189; H-922 to E-l 189; L-923 to E-1189; E-924 toE-1189; 1-925 to E-1189; E-926 to E-1189; L-927 to E-1189; A- 928to E-l 189; A-929 to E-l 189; G-930 to E-l 189; S-931 to E-l 189;D-932 to E-l 189; S- 933 to E-1189; N-934 to E-1189; E-935 toE-1189; R-936 to E-1189; D-937 to E-1189; S-938 to E-l 189; T-939to E-l 189; K-940 to E-l 189; E-941 to E-l 189; D-942 to fil l 89;N-943 to E-l 189; V-944 to E-l 189; A-945 to E-l 189; P-946 toE-1189; L-947 to fil l 89; R-948 to E-1189; F-949 to E-1189; H-950to E-1189; L-951 to E-1189; K-952 to E-l 189; Y-953 to E-l 189;E-954 to E-l 189; A-955 to E-l 189; D-956 to E-l 189; V-957 toE-1189; L-958 to E-1189; F-959 to E-1189; T-960 to E-1189; R-961to E-1189; S-962 to E-l 189; S-963 to E-l 189; S-964 to E-l 189;L-965 to E-l 189; S-966 to E-l 189; H-967 to E-1189; Y-968 toE-1189; E-969 to E-1189; V-970 to E-1189; K-971 to E-l 189; L- 972to E-l 189; N-973 to E-l 189; S-974 to E-l 189; S-975 to E-l 189;L-976 to E-l 189; E- 977 to E-l 189; R-978 to E-l 189; Y-979 toE-1189; D-980 to E-l 189; G-981 to E-l 189; I- 982 to E- 1189; G-983to E-1189; P-984 to E- 1189; P-985 to E- 1 189; F-986 to E- 1189;S- 987 to E-l 189; C-988 to E-l 189; 1-989 to E-l 189; F-990 toE-1189; R-991 to E-l 189; I- 992 to E-l 189; Q-993 to E-l 189; N-994to E-1189; L-995 to E-1189; G-996 to E-l 189; L-997 to E-1189;F-998 to E-l 189; P-999 to E-l 189; 1-1000 to E-l 189; H-1001 toE- 1189; G-1002 to E-l 189; 1-1003 to E-l 189; M-1004 to E-l 189;M-1005 to E-l 189; K- 1006 to E-l 189; 1-1007 to E-l 189; T-1008 toE-1189; 1-1009 to E-l 189; P-1010 to fil l 89; I- 1011 to E-l 189;A- 1012 to E-1189; T-l 013 to E-1189; R-l 014 to E-1189; S- 1015 toE-1189; G-1016 to E-1189; N-1017 to E-1189; R-1018 to E-1 189;L-1019 to E-1189; L-1020 to E-1189; K-1021 to E-1189; L-1022 toE-1189; R-1023 to E-1189; D-1024 to E-l 189; F-1025 to E-l 189;L-1026 to E-1189; T-1027 to E-l 189; D-1028 to E-l 189; E- 1029 toE-1189; V-1030 to E-1189; A-1031 to E-1189; N-1032 to E-1189;T-1033 to E- 1189; S-1034 to E-l 189; C-1035 to E-1189; N-1036 toE-1189; 1-1037 to E-1189; W- 1038 to E-l 189; G-1039 to E-l 189;N-1040 to E-l 189; S-1041 to E-l 189; T-1042 to E- 1189; E-1043 toE-1189; Y-1044 to E-1189; R-1045 to E-1189; P-1046 to E-l 189;T- 1047 to E-l 189; P-1048 to E-1189; V-1049 to E-l 189; E-1050 toE-1189; E-1051 to E- 1189; D-1052 to E-1189; L-1053 to E-1189;R-1054 to E-1189; R-1055 to E-1189; A- 1056 to E-l 189; P-1057 toE-1189; Q-1058 to E-1189; L-1059 to E-l 189; N-1060 to E- 1189;H-1061 to E-1189; S-1062 to E-1189; N-1063 to E-l 189; S-1064 toE-1189; D- 1065 to E-l 189; V-1066 to E-l 189; V-1067 to E-l 189;S-1068 to E-l 189; 1-1069 to E- 1189; N-1070 to E-l 189; C-1071 toE-1189; N-1072 to E-1189; 1-1073 to E-1189; R- 1074 to E-l 189;L-1075 to E-l 189; V-1076 to E-l 189; P-1077 to E-l 189; N-1078 toE- 1189;Q-1079toE-1189;E-1080toE-1189;I-1081toE-1189;N-1082toE-1189;F- 1083 to E-1189; H-1084 to E-1189; L-1085 toE-1189; L-1086 to E-l 189; G-1087 to E- 1189; N-1088 to E-1189;L-1089 to E-1189; W-1090 to E-1189; L-1091 to E-1189; R- 1092 toE-1189; S-1093 to E-l 189; L-1094 to E-l 189; K-1095 to E-l 189;A-1096 to E- 1189;L-1097toE-1189;K-1098toE-1189; Y-1099 toE-1189; K-1100 to E-1189: S- 1101 to E-l 189; M-1102 to E-l 189;K-1103 to E-l 189; 1-1104 to E-l 189; M-1105 to E- 1189; VJ106toEJ189;NJ107toEJ189; A-l 108 to E-1189; A-1109 to E-1189;L- 1110 to E-l 189; Q-l 111 to E-l 189; R-l 112 to E-l 189; Q-l 113 toE-1189; F-1114 to fill 89; H-l 115 to E-1189; S-l 116 to E-l 189.PJ 117 to E-1189; F-1118 to E-1189; 1-1119 to E-l 189; F-1120 toE-1189; R-l 121 to E-l 189; E-l 122 to E-l 189; E-l 123 to E-l 189;D- 1124 to E-l 189; P-l 125 to E-l 189; S-l 126 to E-l 189; R-l 127 toE-1189; Q-l 128 to E- 1189; 1-1129 to E-l 189; V-l 130 to E-l 189;F-1131 to E-1189; E-l 132 to E-l 189; 1-1133 to E-l 189; S-l 134 toE-1189; K-1135 to E-l 189; Q-l 136 to E-l 189; E-l 137 to E- 1189;D-1138 to E-l 189; W-l 139 to E-l 189; Q-l 140 to E-l 189; V-l 141 toE-1189;P- 1142 to E-l 189; 1-1143 to E-l 189; W-l 144 to E-l 189J-1145 to E-l 189; 1-1146 to E- 1189; VJWtoEJ GJMStoE-HSΘ÷SJ^toEJlSg÷T-HSOtoEJ^L- llSl toEJ189;GJ152toE-H89;GJ153toEJ189;LJ154toE-n89;LJ155toE- 1189; L-l 156 to E-l 189; L-l 157 to E-l 189; A-l 158 to E-l 189;L-1159 to E-l 189; L- 1160 to E-l 189; V-l 161 to E-l 189; L-l 162 toE-1189; A-l 163 to E-l 189; L-l 164 to E- 1189; W-l 165 to E-l 189;K-1166 to E-l 189; L-l 167 to E-l 189; G-l 168 to E-l 189; F- 1169 toE-1189; F-1170 to E-l 189; R-l 171 to E-1189; S-l 172 to E-l 189;A-1173 to E- 1189; R-l 174 to E-l 189; R-l 175 to E-l 189; R-l 176 toE-1189; R-l 177 to E-l 189; E- 1178 to E-l 189; P-l 179 to E-l 189;G-1180 to E-1189; L-l 181 to E-1189; D-l 182 to fill 89; P-l 183 toE-1189; T-l 184 to E-1189; of SEQ ID NO:35. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification or loss of one or more biological functions of the protein, other functional activities (e.g., biological activities (e.g., ability to illicit mitogenic activity, induce differentiation of normal or malignant cells, ability to multimerize, etc.) may still be retained. For example the ability of the shortened al 1 mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that an al 1 mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six al 1 amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the al 1 polypeptide shown in Figures 19A-F, up to the glycine residue at position number 6, and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising the amino acid sequence of residues 1-ml of Figures 19A-F, where ml is an integer from 6 to 1189 corresponding to the position of the amino acid residue in Figures 19A-F. Moreover, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of C-terminal deletions of the al 1 polypeptide of the invention shown as SEQ ID NO:35 include polypeptides comprising the amino acid sequence of residues: M-1 to L-l 188; M-1 to V-l 187; M-1 to K-1186;M-1 to P-l 185; M-1 to T-l 184; M-1 to P-l 183; M-1 to D-1182; M-l to L-1181; M-l to G-1180; M-l toP-1179; M-l to E-1178; M-l to R-l 177; M-1 to R-l 176; M-1 to R-l 175; M-1 to R-l 174; M-1 to A-l 173; M-lto S-l 172; M-1 to R-l 171; M-1 to F-1170; M-1 to F-1169; M-1 to G-l 168; M-1 to L-l 167; M-1 to K-1166;M-1 to W-l 165; M-1 to L-l 164; M-1 to A-l 163; M-1 to L-l 162; M-1 to V- 1161; M-1 to L-l 160; M-1 toL-1159; M-1 to A-l 158; M-1 to L-l 157; M-1 to L-l 156; M-1 to L-l 155; M-1 to L-l 154; M-1 to G-l 153; M-lto G-l 152; M-1 to L-l 151; M-1 to T-l 150; M-1 to S-l 149; M-1 to G-l 148; M-1 to V-l 147; M-1 to 1-1146;M-1 to 1-1145; M-1 to W-l 144; M-1 to 1-1143; M-1 to P-l 142; M-1 to V-l 141; M-1 to Q-l 140; M-1 toW-1139; M-1 to D-l 138; M-1 to E-l 137; M-1 to Q-l 136; M-1 to K-1135; M-1 to S- 1134; M-1 to 1-1133; M-lto E-l 132; M-1 to F-1131; M-l to V-1130; M-l to 1-1129; M- 1 to Q-l 128; M-1 to R-l 127; M-1 to SJ 126;MJ to P-l 125; M-1 to D-l 124; M-1 to E- 1123; M-1 to E-1122; M-l to R-1121; M-l to F-1120; M-l toI-1119; M-l to F-1118; M- 1 to P-1117: M-l to S-1116; M-l to H-1115; M-l to F-1114; M-1 to Q-l 113; M-lto R- 1112; M-1 to Q-l l l l; M-l to L-1110; M-l to A-1109; M-1 to A-l 108; M-1 to N-1107; M-1 to V-l 106;M-1 to M-1105; M-1 to 1-1104; M-1 to K-1103; M-1 to M-1102; M-1 to S-l 101 ; M-1 to K-1100; M-1 toY-1099; M-1 to KJ098; MJ to LJ097; MJ to A- 1096; M-1 to K-1095; M-1 to L-1094; M-1 to S-1093; M-lto R-1092; M-1 to L-1091; M-1 to W-1090; M-1 to L-1089; M-1 to N-1088; M-1 to G-1087; M-1 to L-1086;M-1 to L- 1085; M-1 to H-1084; M-1 to F-1083; M-1 to N-1082; M-1 to 1-1081; M-1 to E-1080; M-1 toQ-1079; M-1 to N-1078; M-1 to P-1077; M-1 to V-1076; M-1 to L-1075; M-1 to R-1074; M-1 to 1-1073; M-lto N-1072; M-1 to C-1071; M-1 to N-1070; M-1 to 1-1069; M-1 to S-1068; M-1 to V-1067; M-1 to V-1066;M-1 to D-1065; M-1 to S-1064; M-1 to N-1063; M-1 to S-1062; M-1 to H-1061 ; M-1 to N-1060; M-1 toL-1059; M-1 to Q-1058; M-1 to P-1057; M-1 to A-1056; M-1 to R-1055; M-1 to R-1054; M-1 to L-1053; M-lto D-1052; M-1 to E-1051; M-1 to E-1050; M-1 to V-1049; M-1 to P-1048; M-1 to T-1047; M-1 to P-1046;M-1 to R-1045; M-1 to Y-1044; M-1 to E-1043; M-1 to T-1042; M-1 to S-1041; M-1 to N-1040; M-1 toG-1039; M-1 to W-1038; M-1 to 1-1037; M-1 to N-1036; M-1 to C-1035; M-1 to S-1034; M-1 to T-1033; M-lto N-1032; M-1 to A-1031; M-1 to V-1030; M-1 to E-1029; M-1 to D-1028; M-1 to T-1027; M-1 to L-1026;M-1 to F-1025; M-1 to D-1024; M-1 to R-1023; M-1 to L-1022; M-1 to K-1021; M-1 to L-1020; M-1 toL-1019; M-1 to R-1018; M-1 to N-1017; M-1 to G-1016; M-1 to S-1015; M-1 to R- 1014; M-1 to T-1013; M-lto A-1012; M-1 to 1-1011; M-1 to P-1010; M-1 to 1-1009; M- 1 to T-1008; M-1 to 1-1007; M-1 to K-1006; M-lto M-1005; M-1 to M-1004; M-1 to I- 1003; M-1 to G-1002; M-1 to H-1001; M-1 to 1-1000; M-1 to P-999;M-1 to F-998; M-1 to L-997; M-1 to G-996; M-1 to L-995; M-1 to N-994; M-1 to Q-993; M-1 to 1-992; M-1 toR-991; M-1 to F-990; M-1 to 1-989; M-1 to C-988; M-1 to S-987; M-1 to F-986; M-1 to P-985; M-1 to P-984;M-1 to G-983; M-1 to 1-982; M-1 to G-981 ; M-1 to D-980; M- 1 to Y-979; M-1 to R-978; M-1 to E-977; M-1 toL-976; M-1 to S-975; M-1 to S-974; M-1 to N-973; M-1 to L-972; M-1 to K-971; M-1 to V-970; M-1 toE-969; M-1 to Y-968; M-1 to H-967; M-1 to S-966; M-1 to L-965; M-1 to S-964; M-1 to S-963; M-1 to S-962;M-1 to R-961; M-1 to T-960; M-1 to F-959; M-1 to L-958: M-1 to V-957; M-1 to D-956; M-1 to A-955; M-1 toE-954; M-1 to Y-953; M-1 to K-952; M-1 to L-951; M-1 to H-950; M-1 to F-949; M-1 to R-948; M-1 toL-947; M-1 to P-946; M-1 to A-945; M-1 to V-944; M-1 to N-943; M-1 to D-942; M-1 to E-941; M-1 toK-940; M-1 to T-939; M-1 to S-938; M-1 to D-937; M-1 to R-936; M-1 to E-935; M-1 to N-934; M-1 toS-933; M-1 to D-932; M-1 to S-931; M-1 to G-930; M-1 to A-929; M-1 to A-928; M-1 to L-927; M-1 toE-926; M-1 to 1-925; M-1 to E-924; M-1 to L-923; M-1 to H-922; M-1 to H-921; M-1 to L-920; M-1 to F-919;M-1 to 1-918; M-1 to S-917; M-1 to K-916; M-1 to S-915; M-1 to F-914; M-1 to E-913; M-1 to F-912; M-1 toD-911; M-1 to L-910; M-1 to R-909; M-1 to F-908; M-1 to A-907; M-1 to V-906; M-1 to K-905; M-1 toA-904; M-1 to K-903; M-1 to A-902; M- 1 to R-901; M-1 to F-900; M-1 to F-899; M-1 to P-898; M-1 toY-897; M-1 to S-896; M- 1 to V-895; M-1 to N-894; M-1 to C-893; M-1 to V-892; M-1 to Q-891; M-1 toK-890; M-1 to Q-889; M-1 to L-888; M-1 to R-887; M-1 to R-886; M-1 to E-885; M-1 to E-884; M-1 toN-883; M-1 to V-882; M-1 to C-881; M-1 to E-880; M-1 to 1-879; M-1 to S-878; M-1 to G-877; M-1 to D-876;M-1 to S-875; M-1 to D-874; M-1 to E-873; M-1 to K-872; M-1 to Q-871; M-1 to 1-870; M-1 to L-869; M-1 toS-868; M-1 to A-867; M-1 to F-866; M-1 to Q-865; M-1 to L-864; M-1 to N-863; M-1 to A-862; M-1 toS-861; M-1 to Q-860; M-1 to S-859; M-1 to 1-858; M-1 to N-857; M-1 to L-856; M-1 to V-855; M-1 to T- 854;M-1 to S-853; M-1 to Y-852; M-1 to A-851; M-1 to N-850; M-1 to E-849; M-1 to G-848; M-1 to R-847; M-lto N-846; M-1 to E-845; M-1 to L-844; M-1 to T-843; M-1 to A-842; M-1 to E-841; M-1 to V-840; M-1 toA-839; M-1 to V-838; M-1 to R-837; M-1 to Q-836; M-1 to R-835; M-1 to T-834; M-1 to S-833; M-1 toE-832; M-1 to 1-831; M-1 to 1-830; M-1 to F-829; M-1 to V-828; M-1 to T-827; M-1 to T-826; M-1 to D-825;M-1 to F-824; M-1 to S-823; M-1 to L-822; M-1 to T-821; M-1 to Y-820; M-1 to A-819; M-1 to S-818; M-1 toC-817; M-1 to D-816; M-1 to Q-815; M-1 to A-814; M-1 to P-813; M-1 to K-812; M-1 to R-811; M-l toL-810; M-1 to V-809; M-1 to R-808; M-1 to Q-807; MJ to C-806; M-1 to Y-805; M-1 to E-804; M-1 toM-803; M-1 to A-802; M-1 to T-801; M-1 to P-800; M-1 to L-799; M-1 to D-798; M-1 to S-797; M-1 toR-796; M-1 to A-795; M-1 to D-794; M-1 to L-793; M-1 to V-792; M-1 to L-791; M-1 to D-790; M-1 toP-789; M-1 to V-788; M-1 to C-787; M-1 to H-786; M-1 to E-785; M-1 to D-784; M-1 to E-783; M- 1 toN-782; M-1 to C-781; M-1 to G-780; M-1 to N-779; M-1 to W-778; M-1 to F-777; M-1 to P-776; M-1 toV-775; M-1 to S-774; M-1 to V-773; M-1 to R-772; M-1 to L-771; M-1 to T-770; M-1 to T-769; M-1 to P-768;M-1 to W-767; M-1 to G-766; M-1 to D-765; M-1 to D-764; M-1 to L-763; M-1 to M-762; M-1 to P-761; M-lto G-760; M-1 to H- 759; M-1 to D-758; M-1 to P-757; M-1 to D-756; M-1 to E-755; M-1 to L-754; M-1 toS- 753; M-1 to Y-752; M-1 to E-751; M-1 to V-750; M-1 to S-749; M-1 to F-748; M-1 to T-747; M-1 to V-746;M-1 to P-745; M-1 to K-744; M-1 to V-743; M-1 to Y-742; M-1 to D-741; M-1 to A-740; M-1 to T-739; M-lto D-738; M-1 to L-737; M-1 to V-736; M-1 to H-735; M-1 to F-734; M-1 to N-733; M-1 to 1-732; M-1 toR-731 ; M-1 to E-730; M-1 to C-729; M-1 to L-728; M-1 to E-727; M-1 to Q-726; M-1 to G-725; M-1 toS-724; M-1 to S-723; M-1 to L-722; M-1 to L-721; M-1 to V-720; M-1 to A-719; M-1 to R-718; M-1 toN-717; M-1 to T-716; M-1 to F-715; M-1 to R-714; M-1 to D-713; M-1 to G-712; M-1 to G-711; M-1 toE-710; M-1 to D-709; M-1 to L-708; M-1 to H-707; M-1 to A-706; M-1 to R-705; M-1 to P-704; M-1 toT-703; M-1 to Y-702; M-1 to R-701; M-1 to R-700; M-1 to E-699; M-1 to D-698; M-1 to M-697; M-1 toT-696; M-1 to A-695; M-1 to N-694; M- 1 to Y-693; M-1 to R-692; M-1 to 1-691; M-1 to G-690; M-1 toV-689; M-1 to T-688; M- 1 to T-687; M-1 to T-686; M-1 to Q-685; M-1 to F-684; M-1 to H-683; M-1 to P-682;M- 1 to A-681; M-1 to L-680; M-1 to F-679; M-1 to 1-678; M-1 to P-677; M-1 to T-676; M- 1 to F-675; M-1 toC-674; M-1 to L-673; M-1 to F-672; M-1 to A-671; M-1 to A-670; M- 1 to L-669; M-1 to C-668; M-1 to T-667;M-1 to A-666; M-1 to D-665; M-1 to R-664; M-1 to G-663; M-1 to S-662; M-1 to R-661; M-1 to K-660; M-lto C-659; M-1 to D-658; M-1 to R-657; M-1 to H-656; M-1 to F-655; M-1 to 1-654; M-1 to N-653; M-1 toI-652; M-1 to K-651; M-1 to S-650; M-1 to P-649; M-1 to E-648; M-1 to F-647; M-1 to H-646; M-1 to L-645;M-1 to S-644; M-1 to A-643; M-1 to N-642; M-1 to 1-641; M-1 to Q-640; M-1 to V-639; M-1 to V-638; M-1 toP-637; M-1 to R-636; M-1 to S-635; M-1 to W-634; M-1 to L-633; M-1 to 1-632; M-1 to V-631; MJ to A-630;M-1 to N-629; M-1 to G-628; M-1 to L-627; M-1 to A-626; M-1 to G-625; M-1 to V-624; M-1 to A-623; M-lto L-622; M-1 to D-621 ; M-l to 1-620; M-1 to L-619; M-l to G-618; M-l to D-617; M-1 to E-616; M-1 toN-615; M-1 to L-614; M-1 to D-613; M-1 to L-612; M-1 to Q-611; M-1 to G-610; M-1 to H-609; M-1 tol-608; M-1 to S-607; M-1 to C-606; M-1 to G-605; M-1 to F-604; M-1 to Y-603; M-1 to Q-602; M-1 to L-601;M-1 to G-600; M-1 to T-599; M-1 to A-598; M-1 to L-597; M-1 to E-596; M-1 to S-595; M-1 to A-594; M-1 toT-593; M-1 to 1-592; M-1 to R-591; M-1 to Q-590; M-1 to K-589; M-1 to P-588; M-1 to T-587; M-1 to K- 586;M-1 to L-585; M-1 to 1-584; M-1 to S-583; M-1 to G-582; M-1 to R-581; M-1 to F- 580; M-1 to G-579; M-1 toH-578; M-1 to F-577; M-1 to 1-576; M-1 to Y-575; M-1 to I- 574; M-1 to A-573; M-1 to G-572; M-1 to A-571_.M-1 to H-570; M-1 to N-569; M-1 to D-568; M-1 to E-567; M-1 to L-566; M-1 to P-565; M-1 to A-564; M-lto G-563; M-1 to V-562; M-1 to V-561; M-1 to V-560; M-1 to D-559; M-1 to N-558; M-1 to Y-557; M-1 toS-556; M-1 to D-555; M-1 to Q-554; M-1 to N-553; M-1 to L-552; M-1 to D-551; M-1 to R-550; M-1 toV-549; M-1 to S-548; M-1 to A-547; M-1 to 1-546; M-1 to S-545; M-1 to S-544; M-1 to G-543; M-1 to F-542;M-1 to R-541; M-1 to A-540; M-1 to N-539; M-1 to Q-538; M-1 to Y-537; M-1 to S-536; M-1 to H-535; M-lto S-534; M-1 to D-533; M-1 to K-532; M-1 to L-531; M-1 to T-530; M-1 to G-529; M-1 to N-528; M-1 toY-527; M-1 to V-526; M-1 to F-525; M-1 to R-524; M-1 to N-523; M-1 to Q-522; M-1 to R-521; M- 1 toL-520; M-1 to E-519; M-1 to Y-518; M-1 to V-517; M-1 to Y-516; M-1 to V-515; M-1 to K-514; M-1 toG-513; M-1 to R-512; M-1 to E-511; M-1 to R-510; M-1 to G-509; M-1 to E-508; M-1 to N-507; M-1 toF-506; M-1 to Y-505; M-1 to M-504; M-1 to P-503; M-1 to A-502; M-1 to G-501; M-1 to V-500; M-1 toL-499; M-1 to L-498; M-1 to V-497; M-1 to D-496; M-1 to T-495; M-1 to V-494; M-1 to G-493; M-1 toD-492; M-1 to G- 491 ; M-1 to D-490; M-1 to 1-489; M-1 to D-488; M-1 to V-487; M-1 to S-486; M-1 to T- 485;MJ to 1-484; M-1 to E-483; M-1 to S-482; M-1 to G-481 ; M-1 to F-480; M-1 to Y- 479; M-1 to S-478; M-1 toG-477; M-1 to 1-476; M-1 to Q-475; M-1 to Q-474; M-1 to G- 473; M-1 to R-472; M-1 to M-471 ; M-1 toA-470; M-1 to Q-469; M-1 to H-468; M-1 to 1-467; M-1 to T-466; M-1 to L-465; M-1 to S-464; M-1 to R-463;M-1 to N-462; M-1 to N-461; M-1 to H-460; M-1 to M-459; M-1 to T-458; M-1 to F-457; M-1 to L-456; M-lto 1-455; M-1 to V-454; M-1 to K-453; M-1 to G-452; M-1 to T-451; M-1 to H-450; M-1 to N-449; M-1 toF-448; M-1 to R-447; M-1 to P-446; M-1 to A-445; M-1 to G-444; M-1 to A-443; M-1 to V-442; M-1 toY-441; M-1 to V-440; M-1 to R-439; M-1 to G-438; M-1 to Q-437; M-1 to R-436; M-1 to S-435; M-1 toS-434; M-1 to V-433; M-1 to V-432; M-1 to S-431; M-1 to T-430; M-1 to V-429; M-1 to T-428; M-1 to Y-427;M-1 to G-426; M-1 to L-425; M-1 to Y-424; M-1 to A-423; M-1 to G-422; M-1 to H-421; M-1 to N-420; M- lto K-419; M-1 to L-418; M-1 to E-417; M-1 to E-416; M-1 to P-415; M-1 to F-414; M- 1 to E-413; M-1 toK-412; M-1 to L-411 ; M-1 to Y-410; M-1 to S-409; M-1 to E-408; M- 1 to R-407; M-1 to L-406; M-1 to P-405;M-1 to 1-404; M-1 to V-403; M-1 to K-402; M- 1 to G-401; M-1 to A-400; M-1 to S-399; M-1 to T-398; M-1 toE-397; M-1 to K-396; M-1 to L-395; M-1 to V-394; M-1 to A-393; M-1 to G-392; M-1 to N-391; M-1 toW- 390; M-1 to D-389; M-1 to Y-388; M-1 to A-387; M-1 to G-386; M-1 to V-385; M-1 to A-384; M-1 toG-383; M-1 to L-382; M-1 to L-381; M-1 to V-380; M-1 to G-379; M-1 to D-378; M-1 to E-377; M-1 toV-376; M-1 to V-375; M-1 to H-374; M-1 to S-373; M-1 to S-372; M-1 to F-371; M-1 to G-370; M-1 toT-369; M-1 to Q-368; M-1 to S-367; M-1 to M-366; M-1 to E-365; M-1 to L-364; M-1 to G-363; M-1 toF-362; M-1 to S-361; M-1 to T-360; M-1 to E-359; M-1 to N-358; M-1 to K-357; M-1 to N-356; M-1 toT-355; M-1 to G-354; M-1 to E-353; M-1 to L-352; M-1 to S-351; M-1 to F-350; M-1 to 1-349; M-1 to R-348;M-1 to D-347; M-1 to G-346; M-1 to L-345; M-1 to A-344; M-1 to D-343; M-1 to V-342; M-1 to 1-341; M-1 toD-340; M-1 to K-339; M-1 to L-338; M-1 to A-337; M-1 to A-336; M-1 to E-335; M-1 to D-334; M-1 toT-333; M-1 to V-332; M-1 to N-331; M-1 to F-330; M-1 to F-329; M-1 to H-328; M-1 to K-327; M-1 toD-326; M-1 to D-325; M-1 to P-324; M-1 to D-323; M-1 to S-322; M-1 to A-321; M-1 to 1-320; M-1 to Y-319.MJ to K-318; M-1 to 1-317; M-1 to E-316; M-1 to N-315; M-1 to L-314; M-1 to F-313: M-1 to T-312;M-1 toE-311;M-l toP-310;M-l toN-309;M-l to 1-308; M-1 toG-307;M-l to R-306; M-1 to R-305; M-1 toN-304; M-1 to Y-303; M-1 to Y-302; M-1 to G-301; M-1 to L-300; M-1 to V-299; M-1 to A-298; M-1 toV-297; M-1 to A-296; M-1 to Y-295; M- 1 to R-294; M-1 to T-293; M-1 to V-292; M-1 to N-291; M-1 toD-290; M-1 to R-289; M-1 to E-288; M-1 to S-287; M-1 to Q-286; M-1 to Q-285; M-1 to 1-284; M-1 to V-

283;M-1 to K-282; M-1 to E-281; M-1 to L-280; M-1 to D-279; M-1 to P-278; M-1 to S- 277; M-1 to D-276; M-1 toH-275; M-1 to S-274; M-1 to E-273; M-1 to G-272; M-1 to D-271; M-1 to T-270; M-1 to 1-269; M-1 to V-268;M-1 to 1-267; M-1 to M-266; M-1 to V-265; M-1 to K-264; M-1 to K-263; M-1 to A-262; M-1 to G-261; M-lto K-260; M-1 to R-259; M-1 to G-258; M-1 to G-257; M-1 to K-256; M-1 to Q-255; M-1 to F-254; M- 1 toA-253; M-1 to E-252; M-1 to S-251; M-1 to R-250; M-1 to A-249; M-1 to F-248; M- 1 to E-247; M-1 to I-246;M-1 to G-245; M-1 to F-244; M-1 to A-243; M-1 to T-242; M- 1 to R-241; M-1 to T-240; M-1 to E-239; M-1 toT-238; M-1 to G-237; M-1 to G-236; M-1 to R-235; M-1 to Q-234; M-1 to E-233; M-1 to 1-232; M-1 toH-231; M-1 to S-230; M-1 to A-229; M-1 to A-228; M-1 to E-227; M-1 to V-226; M-1 to V-225; M-1 toD- 224; M-1 to K-223; M-1 to V-222; M-1 to S-221; M-1 to R-220; M-1 to Y-219; M-1 to D-218; M-1 toN-217; M-1 to L-216; M-1 to H-215; M-1 to F-214; M-1 to E-213; M-1 to H-212; M-1 to V-211; M-1 toV-210; M-1 to D-209; M-1 to E-208; M-1 to G-207; M-1 to Y-206; M-1 to Q-205; M-1 to V-204; M-1 toV-203; M-1 to G-202; M-1 to V-201; M- 1 to Q-200; M-1 to 1-199; M-1 to Q-198; M-1 to G-197; M-1 toP-196; M-1 to G-195; M- 1 to 1-194; M-1 to Y-193; M-1 to F-192; M-1 to K-191; M-1 to K-190; M-1 to L-189;M- 1 to 1-188; M-1 to N-187; M-1 to 1-186; M-1 to L-185; M-1 to F-184; M-1 to H-183; M- 1 to Q-182; M-1 toV-181; M- 1 to E-180; M-1 to V-179; M-1 to W-178; M-1 to P-177; M-1 to Y-176; M-l to 1-175; M-1 to S-174;M-l to N-173; M-1 to S-172; M-1 to G-171 ; M-1 to D-170; M-1 to L-169; M-1 to V-168; M-1 to 1-167; M-1 toV-166; M-1 to 1-165; M-1 to D-164; M-1 to M-163; M-1 to Y-162; M-1 to T-161; M-1 to Q-160; M-1 toC- 159; M-1 to R-158; M-1 to Q-157; M-1 to L-156; M-1 to A-155; M-1 to P-154; M-1 to A-153; M-1 toV-152; M-1 to T-151; M-l to KJ50; MJ to S-149; M-1 to F-148; M-l to R-147; M-1 to F-146; M-1 toN-145; M-1 to S-144; M-1 to N-143; M-1 to V-142; M-1 to R-141; M-1 to S-140; M-1 to C-139; M-1 toM-138; M-1 to G-137; M-1 to T-136; M-1 to T-135; M-1 to Y-134; M-1 to Y-133; M-1 to S-132; M-1 toS-131; M-1 to G-130; M-1 to C-129; M-1 to E-128; M-1 to H-127; M-1 to S-126; M-1 to W-125; M-1 toL-124; M-1 to P-123; M-1 to S-122; M-1 to C-121; M-1 to A-120; M-l to L-l 19; M-1 to F-118; M-1 to S-l 17;M-1 to N-116; M-1 to D-l 15; M-1 to K-114; M-1 to P-l 13; M-1 to N-112; M-1 to T-l 11; M-1 to A-l 10; M-lto L-109; M-1 to S-108; M-1 to LJ07; MJ to G-106; M-l to L-105; M-1 to R-104; M-1 to M-103; M-1 toN-102; M-1 to D-101 ; M-1 to K-100: M-1 to R-99; M-1 to E-98; M-1 to S-97; M-1 to V-96; M-1 to N-95; M-lto S-94; M-1 to L- 93; M-1 to T-92; M-1 to V-91; M-1 to R-90; M-1 to G-89; M-1 to L-88; M-1 to N-87; M-lto L-86; M-1 to K-85; M-1 to T-84; M-1 to C-83; M-1 to N-82; M-1 to G-81; M-1 to H-80; M-1 to 1-79; M-lto V-78; M-1 to P-77; M-1 to C-76; M-1 to K-75; M-1 to Y-74; M-1 to V-73; M-1 to D-72; M-1 to G-71; M-lto T-70; M-1 to K-69; M-1 to Q-68; M-1 to Y-67; M-1 to G-66; M-1 to N-65; M-1 to T-64; M-1 to E-63; M-lto L-62; M-1 to P- 61; M-1 to A-60; M-1 to G-59; M-1 to V-58; M-1 to V-57; M-1 to L-56; M-1 to W-55; M-lto K-54; M-1 to N-53; M-1 to G-52; M-1 to S-51; M-1 to 1-50; M-1 to D-49; M-1 to H-48; M-1 to Q-47; M-1 to Q-46; M-1 to V-45; M-1 to T-44; M-1 to Y-43; M-1 to G-42; M-1 to F-41; M-1 to F-40; M-1 to A-39; M-lto T-38; M-1 to R-37; M-1 to S-36; M-1 to G-35; M-1 to P-34; M-1 to 1-33; M-1 to V-32; M-1 to R-31; M-1 toP-30; M-1 to K-29; M-1 to R-28; M-1 to T-27; M-1 to D-26; M-1 to M-25; M-1 to N-24; M-1 to F-23; M-1 toT-22; M-l to D-21; M-l to T-20; M-l to FJ9; MJ to G-18; M-1 to P-17; M-l to W- 16; M-1 to L-15; M-1 toS-14; M-l to L-13; M-l to A-12; M-l to W-l 1; M-1 to AJO; M- 1 to V-9; M-1 to V-8; M-1 to L-7; M-1 to G-6; of SEQ ID NO:35. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

In addition, the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NOJ 7 which have been determined from the following related cDNA genes: HEEAB54R (SEQ ID NOJ04), HRDAF83R (SEQ ID NO: 105), HOUBC62R (SEQ ID NO: 106), HCDBI19R (SEQ ID NO: 107), HOHCU94R (SEQ ID NOJ08), HOACC13R (SEQ ID NO: 109), HCDAP21R (SEQ ID NO: 110), HNHHA34R (SEQ ID NO: 11 1), HOHEA75R (SEQ ID NO: 112) and HNGEL59R (SEQ ID NOJ 13).

Based on the sequence similarity to the human integrin alpha 1 subunit, translation product of this gene is expected to share at least some biological activities with integrin proteins, and specifically the integrin alpha 1 protein. Such activities are known in the art, some of which are described elsewhere herein. Specifically, polynucleotides and polypeptides of the invention are also useful for modulating the differentiation of normal and malignant cells, modulating the proliferation and/or differentiation of cancer and neoplastic cells, and modulating the immune response. Polynucleotides and polypeptides of the invention may represent a diagnostic marker for hematopoietic and immune diseases and/or disorders. The full- length protein should be a secreted protein, based upon homology to the integrin family. Therefore, it is secreted into serum, urine, or feces and thus the levels is assayable from patient samples. Assuming specific expression levels are reflective of the presence of immune disorders, this protein would provide a convenient diagnostic for early detection. In addition, expression of this gene product may also be linked to the progression of immune diseases, and therefore may itself actually represent a therapeutic or therapeutic target for the treatment of cancer. Polynucleotides and polypeptides of the invention may play an important role in the pathogenesis of human cancers and cellular transformation, particularly those of the immune and hematopoietic systems. Polynucleotides and polypeptides of the invention may also be involved in the pathogenesis of developmental abnormalities based upon its potential effects on proliferation and differentiation of cells and tissue cell types. Due to the potential proliferating and differentiating activity of said polynucleotides and polypeptides, the invention is useful as a therapeutic agent in inducing tissue regeneration, for treating inflammatory conditions (e.g., inflammatory bowel syndrome, diverticuhtis, etc.). Moreover, the invention is useful in modulating the immune response to aberrant polypeptides, as may exist in rapidly proliferating cells and tissue cell types, particularly in adenocarc oma cells, and other cancers.

Alternatively, the expression within cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis, treatment, and/or prevention of developmental diseases and disorders, including cancer, and other proliferative conditions. Representative uses are descπbed in the "Hyperpro ferative Disorders" and "Regeneration" sections below and elsewhere herein. Briefly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation.

Dysregulation of apoptosis can result in mappropπate suppression of cell death, as occurs in the development of some cancers, or m failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain neurodegenerative disorders, such as spinal muscular atrophy (SMA). Alternatively, this gene product is involved in the pattern of cellular proliferation that accompanies early embryogenesis. Thus, aberrant expression of this gene product in tissues - particularly adult tissues - may correlate with patterns of abnormal cellular proliferation, such as found in various cancers. Because of potential roles in proliferation and differentiation, this gene product may have applications in the adult for tissue regeneration and the treatment of cancers. It may also act as a morphogen to control cell and tissue type specification. Therefore, the polynucleotides and polypeptides of the present invention are useful in treating, detecting, and/or preventing said disorders and conditions, in addition to other types of degenerative conditions. Thus this protein may modulate apoptosis or tissue differentiation and is useful in the detection, treatment, and/or prevention of degenerative or proliferative conditions and diseases. The protein is useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues. The protein can also be used to gain new insight into the regulation of cellular growth and proliferation. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues. This gene is expressed almost exclusively in osteoblasts, human trabelcular bone cells, messangial cells, adipocytes, and to a lesser extent in osteosarcoma, chondrosarcoma, breast cancer cells, and bone marrow.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of the following diseases and conditions which include, but are not limited to, disorders of the skeletal system, connective tissues, and immune and hematpoietic diseases and/or disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful to provide immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the connective tissue and skeletal system, expression of this gene at significantly higher or lower levels is detected in certain tissues or cell types (e.g. immune, hematopoietic, skeletal, bone, cartilage, develpomental, reproductive, secretory, and cancerous and wounded tissues) or bodily fluids or cell types (e.g., lymph, serum, plasma, urine, synovial fluid or spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue from an individual not having the disorder.

Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 35 as residues: Phe-23 to Arg-31, Leu-62 to Asp-72, Val-96 to Asp-101, Thr-111 to Asn-116, Glu-128 to Thr-135, Val-142 to Ser-149, Asn-217 to Val- 222, GIu-233 to Arg-241, Gly-272 to Leu-280, Gln-286 to Thr-293, Tyr-303 to Ue-308, Gly-354 to Thr-360, Glu-408 to Lys-419, Glu-508 to Lys-514, Arg-521 to Val-526, Gly- 529 to Phe-542, Asp-551 to Tyr-557, Thr-587 to Thr-593, His-656 to Asp-665, Met-697 to Arg-705, Asp-709 to Thr-716, Glu-755 to Gly-760, Asn-779 to His-786, Leu-810 to Asp-816, Leu-844 to Ala-851, Gln-871 to Gly-877, Glu-884 to Gln-889, Ser-931 to Asn- 943, Ser-974 to Ile-982, Gly-1039 to Gln-1058, Arg-1121 to Arg-1127, Ser-1134 to Trp- 1139, Ser- 1172 to Pro-1183. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution in osteoblasts and homology to integrin alpha subunit 10 indicates that the protein products of this gene are useful for the treatment of disorders and conditions affecting the skeletal system, in particular osteoporosis as well as disorders afflicting connective tissues (e.g. arthritis, trauma, tendonitis, chrondomalacia and inflammation), such as in the diagnosis and treatment of various autoimmune disorders such as rheumatoid arthritis, lupus, scleroderma, and dermatomyositis as well as dwarfism, spmal deformation, and specific joint abnormalities as well as chondrodysplasias (ie. spondyloepiphyseal dysplasia congenita, familial osteoarthπtis, Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid). polynucleotides and polypeptides corresponding to this gene are useful for the treatment and diagnosis of hematopoietic related disorders such as anemia, pancytopenia, leukope a, thrombocytopenia oi leukemia since stromal cells aie important in the production of cells of hematopoietic lineages Such a use is consistent with the observed homology to integπn family members, in conjunction with The tissue distnbution m bone marrow cells Integnns play pivotal roles in cell migration, inflammation, proliferation, and cellulai mfiltiation Thus, the present invention is expected to share at least some of these activities Representative uses are descπbed in the "Immune Activity" and "infectious disease" sections below, in Example 1 1, 13, 14, 16, 18, 19, 20, and 27, and elsewhere heiein Briefly, the uses include bone marrow cell ex-vivo culture, bone marrow tiansplantation, bone marrow reconstitution, radiotherapy or chemotherapy of neoplasm The gene product may also be involved in lymphopoiesis, therefore, it can be used m immune disorders such as infection, inflammation, allergy, immunodeficiency etc. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of vanous blood lineages, and in the differentiation and or proliferation of vaπous cell types. Based upon the tissue distπbution of this protein, antagonists directed against this protein is useful blocking the activity of this protein. Accordingly, preferred are antibodies which specifically bind a portion of the translation product of this gene.

Also provided is a kit for detecting tumors in which expression of this protein occurs. Such a kit comprises in one embodiment an antibody specific for the translation product of this gene bound to a solid support. Also provided is a method of detecting these tumors in an individual which compπses a step of contacting an antibody specific for the translation product of this gene to a bodily fluid from the individual, preferably serum, and ascertaining whether antibody binds to an antigen found in the bodily fluid. Preferably the antibody is bound to a solid support and the bodily fluid is serum. The above embodiments, as well as other treatments and diagnostic tests (kits and methods), are more particularly described elsewhere herein. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues. Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 17 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 4981 of SEQ ID NO: 17, b is an integer of 15 to 4995, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 17, and where b is greater than or equal to a + 14.

FEATURES OF PROTEIN ENCODED BY GENE NO: 8

The present invention relates to three novel peptidoglycan recognition binding proteins expressed by keratinocytes, wound-healing tissues and chondrosarcoma tissue. More specifically, isolated nucleic acid molecules are provided encoding a human peptidoglycan recognition protein-related protein, sometimes referred to herein as "human tag7" or "tag7" or "htag7". Further provided are vectors, host cells and recombinant methods for producing the same. The invention also relates to both the inhibition and enhancement of activities of the tag7 protein, polypeptides and diagnostic methods for detecting tag7 gene expression.

Peptidoglycan, as well as Lipopolysacchande (LPS), is a surface component of many bactena which illicit a wide range of physiological and immune responses in humans. Specifically, peptidoglycan has been shown to manifest itself clinically by reproducing most of the symptoms of bacterial infection, including fevei , acute-phase response, inflammation, septic shock, leukocytosis, sleepiness, malaise, abcess formation, and arthritis (see Dziarski et al., JBC, 273 (15): 8680 (1998)). Furthermore, the type of peptidoglycan (i.e.- the specific stereoisomers or analogs of muramyl dipeptide, N-acetylglucosammyl-beta(l-4)-N-acteylmuramyl tetrapeptides, etc.), were shown to elicit a broad range of activities, including exhibiting greater pyrogenicity, inducing acute joint inflammation, stimulating macrophages, and causing hemorrhagic necrosis at a primed site (See Kotani et al., Fed Proc, 45(11): 2534 (1986)). It has been demonstrated in humans that a lipopolysacchande binding protein exists that was discovered as a trace plasma protein (See Schumann et al., Science, 249(4975): 1429 (1990)). It is thought that one of the modes of action by which this lipopolysacchande binding protein functions is by forming high-affinity complexes with lipopolysacchande, that then bind to macrophages and monocytes, inducing the secretion of tumor necrosis factor. Dziarski and Gupta (See Dziarski et al., JBC, 269(3): 2100 (1994)) demonstrated that a 70kDa receptor protein present on the surface of mouse lymphocytes served to bind hepaπn, hepaπnoids, bactenal poteichoic acids, peptidoglycan, and popolysacchaπdes. Recently, Dziarski et al. demonstrated that the CD14, a glycosylphosphatidyl ositol-linked protein present on the surface of macrophage and polymorphonuclear leukocytes, bound peptidoglycan and lipopolysacchande. Furthermore, the binding affinity of CD 14 for lipopolysacchande was significantly increased in the presence of a LPS-binding protein present in plasma. It is thought that the LPS-binding protein functions as a transfer molecule, whereby it binds LPS and presents it to the CD14 receptor (See Dziarski et al., JBC, 273(15): 8680 (1998)). Yoshida et al. isolated a peptidoglycan binding protein from the hemolymph of the Silkworm, Bombyx moπ, using column chromatography This protein was found to have a very specific affinity for peptidoglycan (See Yoshida et al., JBC, 271(23) 13854 (1996))

Additionally, Kang et al. recently cloned a peptidoglycan binding protein from the moth Trichoplusia ni. The peptidoglycan binding protein was shown to bind strongly to insoluble peptidoglycan (See Kang etal., PNAS, 95(17). 10078 (1998)). In this study the peptidoglycan binding protein was upiegulated by a bacterial infection m T. ni. The insect immune system is regarded as a model foi innate immunity. Thus, Kang et al weie able to gene both mouse and human homologs of the T ni peptidoglycan binding protein. All of these peptidoglycan binding proteins shared regions of homology, as well as foui conserved cysteine residues which may function in the tertiary structure of the protein, possibly in helping to form binding domains. Given that peptidoglycan is an integral component of bactenal cell walls, and that it induces many physiological responses from cytokine secretion to inflammation and macrophage activation, it appears as if this family of proteins is a ubiquitous group involved in the binding and recognition of peptidoglycan, the presentation of antigens (e.g., cell wall components, etc.), and the activation of the immune system, such as the secretion of cytokines, such as TNF. TNF is noted for its pro-mflammatory actions which result in tissue injury, such as induction of procoagulant activity on vascular endothelial cells (Pober, J.S. et al., J. Immunol. 136:1680 (1986)), increased adherence of neutrophils and lymphocytes (Pober, J.S. et al., J. Immunol. 138:3319 (1987)), and stimulation of the release of platelet activating factor from macrophages, neutrophils and vascular endothelial cells (Camussi, G. et al., J. Exp. Med. 166: 1390 (1987)). Recent evidence implicates TNF in the pathogenesis of many infections (Cerami, A. et al., Immunol. Today 9:28 (1988)), immune disorders, neoplastic pathology, e.g., in cachexia accompanying some malignancies (Oliff, A. et al., Cell 50:555 (1987)), and in autoimmune pathologies and graft-versus host pathology (Piguet, P.-F. et al., J. Exp. Med. 166: 1280 (1987)). The association of TNF with cancer and infectious pathologies is often related to the host's catabohc state. A major pioblem in cancer patients is weight loss, usually associated with anorexia. The extensive wasting which results is known as "cachexia" (Kern, K. A. et al. J. Parent. Enter Nutr. 12.286-298 (1988)). Cachexia includes progressive weight loss, anorexia, and persistent erosion of body mass in response to a malignant growth The cachectic state is thus associated with significant morbidity and is responsible for the majority of cancer mortality.

A number of studies have suggested that TNF is an important mediator of the cachexia in cancer, infectious pathology, and in other catabohc states. TNF is thought to play a cential role in the pathophysiological consequences of Gram-negative sepsis and endotoxic shock (Michie, H.R. et al., Br. J. Surg. 76:670-671 (1989); Debets, J. M. H. et al., Second Vienna Shock Forum, p.463-466 (1989); Simpson, S. Q. et al., Cnt. Care Clin. 5:27-47 (1989)), including fevei , malaise, anorexia, and cachexia. Endotoxin is a potent monocyte/macrophage activator which stimulates production and secretion of TNF (Kombluth, S.K. et al., J. Immunol. 137:2585-2591 (1986)) and other cytokines. Because TNF could mimic many biological effects of endotoxin, it was concluded to be a central mediator responsible for the clinical manifestations of endotoxin-related illness. TNF and other monocyte-denved cytokines mediate the metabolic and neurohormonal responses to endotoxin (Michie, H.R. et al., N. Eng. J. Med. 318:1481-1486 (1988)). Endotoxin administration to human volunteers produces acute illness with flu-like symptoms including fever, tachycardia, increased metabolic rate and stress hormone release (Revhaug, A. et al., Arch. Surg. 123: 162-170 (1988)). Elevated levels of circulating TNF have also been found in patients suffering from Gram-negative sepsis (Waage, A. et al., Lancet 1 :355-357 (1987); Hammerle, A.F. et al., Second Vienna Shock Forum p. 715-718 (1989); Debets, J. M. H. et al., Cnt. Care Med. 17:489-497 (1989); Calandra, T. et al., J. Infec. Dis. 161:982-987 (1990)). Passive immunotherapy directed at neutralizing TNF may have a beneficial effect in Gram-negative sepsis and endotoxemia, based on the increased TNF production and elevated TNF levels in these pathology states, as discussed above.

Antibodies to a "modulator" matenal which was characterized as cachectin (later found to be identical to TNF) were disclosed by Cerami et al. (EPO Patent Publication 0,212,489, March 4, 1987) Such antibodies were said to be useful in diagnostic immunoassays and in therapy of shock in bacterial infections. Rubin et al. (EPO Patent Publication 0,218,868, April 22, 1987) disclosed monoclonal antibodies to human TNF, the hybridomas secreting such antibodies, methods of producing such antibodies, and the use of such antibodies in immunoassay of TNF. Yone et al. (EPO Patent Publication 0,288,088, October 26, 1988) disclosed anti-TNF antibodies, including mAbs, and their utility in immunoassay diagnosis of pathologies, in particular Kawasaki's pathology and bactenal infection. The body fluids of patients with Kawasaki's pathology (infantile acute febrile mucocutaneous lymph node syndrome; Kawasaki, T., Allergy 16:178 (1967), Kawasaki, T., Shonica (Pediatπcs) 26:935 (1985)) were said to contain elevated TNF levels which were related to progress of the pathology (Yone et al., supra). Accordingly, there is a need to provide molecules that are involved in pathological conditions. Such novel proteins could be useful in augmenting the immune system m such areas as immune recognition, antigen presentation, and immune system activation. Antibodies or antagonists directed against these proteins is useful in reducing or eliminating disorders associated with TNF and TNF- ke cytokines, such as endotoxic shock and auto-immune disorders, for example.

The polypeptide of the present invention has been putatively identified as a member of the novel peptidoglycan recognition binding protein family and has been termed human tag7. This identification has been made as a result of amino acid sequence homology to the mouse tag7 (See Genbank Accession No. emb|CAA60133).

Figure 34 shows the nucleotide (SEQ ID NO: 18) and deduced ammo acid sequence (SEQ ID NO:36) of htag7. Predicted amino acids from about 1 to about 21 constitute the predicted signal peptide (amino acid residues from about 1 to about 21 in SEQ ID NO 36) and are represented by the underlined amino acid regions; and amino acids from about 34 to about 117 constitute the piedicted PGRP-hke domain (ammo acids from about 34 to about 117 in SEQ ID NO:36) and are repiesented by the double underlined amino acids Figuie 35 shows the regions of similarity between the amino acid sequences of the htag7 piotein (SEQ ID NO 36) and the mouse tag7 protein (SEQ ID NOJ 14)

Figure 36 shows an analysis of the htag7 amino acid sequence. Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown A polynucleotide encoding a polypeptide of the present invention is obtained from human chondrosarcoma cells, bone marrow, and neutrophils. The polynucleotide of this invention was discovered in a human chondrosarcoma cDNA library.

As shown in Figure 34, htag7 has a PGRP domain (the PGRP domain comprise amino acids from about 34 to about 117 of SEQ ID NO:36; which correspond to amino acids from about 34 to about 117 of Figure 34). The polynucleotide contains an open reading frame encoding the htag7 polypeptide of 198 amino acids. htag7 exhibits a high degree of homology at the amino acid level to the mouse tag7 (as shown in Figure 35). The present invention provides isolated nucleic acid molecules compnsmg a polynucleotide encoding the htag7 polypeptide having the ammo acid sequence shown in Figure 34 (SEQ ID NO:36). The nucleotide sequence shown in Figure 34 (SEQ ID

NO: 18) was obtained by sequencing a cloned cDNA (HCDDP40), which was deposited on November 17 at the American Type Culture Collection, and given Accession Number 203484.

The present invention is further directed to fragments of the isolated nucleic acid molecules described herein. By a fragment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ ID NO: 18 is intended DNA fragments at least about 15nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments 50-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in SEQ ID NOJ 8. By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NO: 18. In this context "about" includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Representative examples of htag7 polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, from about 501 to about 550, from about 551 to about 600, from about 601 to about 650, from about 651 to about 700, from about 701 to about 726, and from about 130 to about 379 of SEQ ID NO: 18, or the complementary strand thereto, or the cDNA contained in the deposited gene. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding a member selected from the group: a polypeptide compnsmg or alternatively, consisting of, the PGRP-hke domain (amino acid residues from about 34 to about 117 m Figure 34 (amino acids from about 34 to about 117 in SEQ ID NO:36) Since the location of these domains have been predicted by computer analysis, one of oidinaiy skill would appieciate that the ammo acid lesidues constituting these domains may vary slightly (e g , by about 1 to 15 amino acid lesidues) depending on the cπteiia used to define each domain As indicated, nucleic acid molecules of the present invention which encode a htag7 polypeptide may include, but aie not limited to those encoding the am o acid sequence of the PGRP-hke domain of the polypeptide, by itself, and the coding sequence for the PGRP-hke domain of the polypeptide and additional sequences, such as a pie-, or pro or prepro- piotein sequence In additional embodiments, the polynucleotides of the invention encode functional attributes of htag7.

Pieferred embodiments of the invention in this regard include fragments that compnse alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions"), turn and turn-forming regions ("turn- regions"), coil and coil-formmg regions ("coil-iegions"), hydiophihc regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions of htag7. The data representing the structural or functional attπbutes of htag7 set forth in Figure 36 and/or Table XII, as descnbed above, was generated using the vanous modules and algonthms of the DNA^STAR set on default parameters. In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table XII can be used to determine regions of htag7 which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.

Certain preferred regions in these regards are set out in Figure 36, but may, as shown in Table XII, be represented or identified by using tabular representations of the data presented in Figure 36. The DNA*STAR computer algorithm used to generate Figure 36 (set on the original default parameters) was used to present the data in Figure 36 in a tabular format (See Table XII). The tabular format of the data in Figure 36 is used to easily determine specific boundaries of a prefen-ed region. The above-mentioned preferred regions set out in Figure 36 and in Table XII include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figure 34. As set out in Figure 36 and in Table XII, such preferred regions include Garni er-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou- Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions and Hopp-Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenic index and

Emini surface-forming regions. Even if deletion of one or more amino acids from the N- terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, modulate cellular interaction, or signalling pathways, etc.) may still be retained. For example, the ability of shortened htag7 muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that an htag7 mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six htag7 ammo acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the htag7 ammo acid sequence shown in Figure 34, up to the proline residue at position number 191 and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides compnsmg the amino acid sequence of residues nl- 196 of Figure 34 , where nl is an integer from 2 to 191 coιτespondιng to the position of the amino acid residue in Figure 34 (which is identical to the sequence shown as SEQ ID NO:36). In another embodiment, N-terminal deletions of the htag7 polypeptide can be descnbed by the general formula n2-196, where n2 is a number from 2 to 191, corresponding to the position of ammo acid identified in Figure 34. N-terminal deletions of the htag7 polypeptide of the invention shown as SEQ ID NO:36 include polypeptides comprising the amino acid sequence of residues: N-termmal deletions of the htag7 polypeptide of the invention shown as SEQ ID NO:36 include polypeptides comprising the ammo acid sequence of residues: S-2 to P-196; R-3 to P-196; R-4 to P-196; S-5 to P-196; M-6 to P-196; L-7 to P-196; L-8 to P- 196; A-9 to P-196; W-10 to P-196, A-U to P-196; L-12 to P-196; P-13 to P-196; S-14 to P-196; L-15 to P-196; L-16 to P-196; R-17 to P-196; L-18 to P-196; G-19 to P-196; A-20 to P-196; A-21 to P-196; Q-22 to P-196; E-23 to P-196; T-24 to P-196; E-25 to P-196; D-26 to P-196; P-27 to P-196; A-28 to P-196; C-29 to P-196; C-30 to P-196; S-31 to P- 196; P-32 to P-196; 1-33 to P-196; V-34 to P-196; P-35 to P-196; R-36 to P-196; N-37 to P-196; E-38 to P-196; W-39 to P-196; K-40 to P-196; A-41 to P-196; L-42 to P-196; A- 43 to P-196; S-44 to P-196; E-45 to P-196; C-46 to P-196; A-47 to P-196; Q-48 to P- 196; H-49 to P-196; L-50 to P-196; S-51 to P-196; L-52 to P-196; P-53 to P-196; L-54 to P-196; R-55 to P-196; Y-56 to P-196; V-57 to P-196; V-58 to P-196; V-59 to P-196; S- 60 to P-196; H-61 to P-196; T-62 to P-196; A-63 to P-196; G-64 to P-196; S-65 to P- 196; S-66 to P-196; C-67 to P-196; N-68 to P-196; T-69 to P-196; P-70 to P-196; A-71 to P-196; S-72 to P-196; C-73 to P-196; Q-74 to P-196; Q-75 to P-196; Q-76 to P-196; A-77 to P-196; R-78 to P-196; N-79 to P-196; V-80 to P-196; Q-81 to P-196; H-82 to P- 196; Y-83 to P-196; H-84 to P-196; M-85 to P-196; K-86 to P-196; T-87 to P-196; L-88 to P-196; G-89 to P-196; W-90 to P-196; C-91 to P-196; D-92 to P-196; V-93 to P-196; G-94 to P-196; Y-95 to P-196; N-96 to P-196; F-97 to P-196; L-98 to P-196; 1-99 to P- 196; G- 100 to P-196; E-101 to P-196; D-l 02 to P-196; G-103 to P-196; L-l 04 to P-196; V-105 to P-196; Y-106 to P-196; E-l 07 to P- 196; G-108 to P-196; R-109 to P-196; G- 110 to P- 196; W-l 11 to P-196; N-112 to P-196; F-113 to P-196; T-l 14 to P-196; G-l 15 to P-196; A-l 16 to P-196; H-l 17 to P-196; S-l 18 to P-196; G-l 19 to P-196; H-120 to P- 196: L-121 to P-196; W-122 to P-196; N-123 to P-196; P-124 to P-196; M-125 to P-196; S-126 to P-196; 1-127 to P-196; G-128 to P-196; 1-129 to P-196; S-130 to P-196; F-131 to P-196; M-132 to P-196; G-133 to P-196; N-134 to P-196; Y-135 to P-196; M-136 to P-196; D-137 to P-196; R-138 to P-196; V-139 to P-196; P-l 40 to P-196; T-141 to P- 196; P-l 42 to P-196; Q-143 to P-196; A-144 to P-196; 1-145 to P-196; R-146 to P-196; A-147 to P-196; A-148 to P-196; Q-149 to P-196; G-150 to P-196; L-151 to P-196; L- 152 to P-196; A-153 to P-196; C-154 to P-196; G-155 to P-196; V-156 to P-196; A-157 to P-196; Q-158 to P-196; G-159 to P-196; A-160 to P-196; L-161 to P-196; R-162 to P- 196; S-163 to P-196; N-164 to P-196; Y-165 to P-196; V-166 to P-196; L-167 to P-196; K-168 to P-196; G-169 to P-196; H-170 to P-196; R-171 to P-196; D-172 to P-196; V- 173 to P-196; Q-174 to P-196; R-175 to P-196; T-176 to P-196; L-177 to P-196; S-178 to P-196; P-179 to P-196; G-l 80 to P-196; N-181 to P-196; Q-182 to P-196; L-l 83 to P- 196; Y-184 to P-196; H-l 85 to P-196; L-l 86 to P-196; 1-187 to P-196; Q-188 to P-196; N-189 to P-196; W-190 to P-196; P-191 to P-196; of SEQ ID NO:36. Polypeptides encoded by these polynucleotides are also encompassed by the invention. Also as mentioned above, even if deletion of one or more amino acids from the

C-terminus of a protein results in modification or loss of one or more biological functions of the protein, other functional activities (e.g., biological activities ) may still be retained. For example the ability of the shortened htag7 mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majoπty of the residues of the complete or mature polypeptide are removed from the C-termmus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described heiein and otherwise known in the art It is not unlikely that a htag7 mutein with a large numbei of deleted C-terminal ammo acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six htag7 amino acid residues may often evoke an immune response

Accordingly, the present invention furthei provides polypeptides having one oi more residues deleted from the carboxy terminus of the amino acid sequence of the htag7 polypeptide shown in Figure 34 , up to the methionine residue at position number 6, and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising the amino acid sequence of residues 1-ml of Figure 1, where ml is an integer from 6 to 196 corresponding to the position of the ammo acid residue in Figure 34 . Moreover, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the ammo acid sequence of C-terminal deletions of the htag7 polypeptide of the invention shown as SEQ ID NO:36 include polypeptides comprising the amino acid sequence of residues: M-1 to S-195; M-1 to R- 194; M-1 to Y-193; M-1 to H-192; M-1 to P-191; M-1 to W-190; M-1 to N-189; M-1 to Q-188; M-1 to 1-187; M-1 to L-186; M-1 to HI 85; M-1 to Y-184; M-1 to L-183; M-1 to Q-182; M-1 to N-181; M-1 to G-180; M-1 to P-179; M-1 to S-178; M-1 to L-177; M-1 to T-176; M-1 to R-175; M-1 to Q-174; M-1 to V-173; M-1 to D-172; M-1 to R-171; M-1 to H-170; M-1 to G-169; M-1 to K-168; M-1 to L-167; M-1 to V-166; M-1 to Y-165; M- 1 to N-164; M-1 to S-163, M-1 to R-162; M-1 to L-161; M-1 to A-160; M-1 to G-159; M-1 to Q-158; M-1 to A-157; M-1 to V-156; M-1 to G-155; M-1 to C-154; M-1 to A- 153; M-1 to L-152; M-1 to L-151; M-1 to G-150; M-1 to Q-149; M-1 to A-148; M-1 to A-147; M-1 to R-146; M-1 to 1-145; M-1 to A-144; M-1 to Q-143; M-1 to P-142; M-1 to T-141; M-1 to P-140; M-1 to V-139; M-1 to R-138; M-1 to D-137; M-1 to M-136; M-1 to Y-135; M-1 to N-134; M-1 to G-133; M-1 to M-132; M-1 to F-131; M-1 to S-130; M- 1 to 1-129; M-1 to G-128; M-1 to 1-127; M-1 to S-126; M-1 to M-125; M-1 to P-124; M- 1 to N-123; M-1 to W-122; M-1 to L-121 ; M-1 to H-120; M-l to G-l 19; M-1 to S-l 18; M-1 to H-l 17; M- 1 to A-l 16; M-1 to G-l 15; M-1 to T-l 14; M-1 to F-1 13; M-1 to N- 112; M-1 to W-111 ; M-1 to G-1 10; M-l to R-109; M-l to G-108; M-l to E-107; M-l to Y-106; M-1 to VJ05; MJ to LJ04; MJ to G-103; M-1 to D-102; M-l to E-101 ; M-l to G-100; M-1 to 1-99; M-1 to L-98; M-1 to F-97; M-1 to N-96; M-1 to Y-95; M-1 to G- 94; M-1 to V-93; M-1 to D-92; M-l to C-91; M-l to W-90; M-l to G-89; M-l to L-88; M-1 to T-87; M-1 to K-86: M-1 to M-85; M-1 to H-84; M-1 to Y-83; M-1 to H-82; M- 1 to Q-81 ; M-1 to V-80; M-1 to N-79; M-1 to R-78; M-1 to A-77; M-1 to Q-76; M-1 to Q- 75; M-1 to Q-74; M-l to C-73; M-1 to S-72; M-1 to A-71 ; M-1 to P-70; M-l to T-69; M- 1 to N-68; M-1 to C-67; M-1 to S-66; M-1 to S-65; M-1 to G-64; M-1 to A-63; M-1 to T- 62; M-1 to H-61; M-1 to S-60; M-1 to V-59; M-1 to V-58; M-1 to V-57; M-1 to Y-56; M-1 to R-55; M-1 to L-54; M-1 to P-53; M-1 to L-52; M-1 to S-51; M-1 to L-50; M-1 to H-49; M-1 to Q-48; M-1 to A-47; M-1 to C-46; M-1 to E-45; M-1 to S-44; M-1 to A-43; M-1 to L-42; M-1 to A-41 ; M-1 to K-40; M-1 to W-39; M-1 to E-38; M-1 to N-37; M-1 to R-36; M-1 to P-35; M-1 to V-34; M-1 to 1-33; M-1 to P-32; M-1 to S-31; M-1 to C- 30; M-1 to C-29; M-1 to A-28; M-1 to P-27; M-1 to D-26; M-1 to E-25; M-1 to T-24; M- 1 to E-23; M-1 to Q-22; M-1 to A-21; M-1 to A-20; M-1 to G-19; M-1 to L-18; M-1 to R-17; M-1 to L-16; M-1 to L-15; M-1 to S-14; M-1 to P-13; M-1 to L-12; M-1 to A-l l; M-1 to W-10; M-1 to A-9; M-1 to L-8; M-1 to L-7; M-1 to M-6; of SEQ ID NO:36. Polypeptides encoded by these polynucleotides are also encompassed by the invention. In addition, the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO:36 which have been determined from the following related cDNA genes: HBMTB79R (SEQ ID NOJ 15) and HCDDP40R (SEQ ID NOJ 16).

Based on the sequence similarity to the mouse tag7 and the PGRP-like domain, translation product of this gene is expected to share at least some biological activities with tag7 proteins, and specifically cytokine modulatory proteins. Such activities are known in the art, some of which are described elsewhere herein. Specifically, polynucleotides and polypeptides of the invention are also useful for modulating the differentiation of normal and malignant cells, modulating the proliferation and/or differentiation of cancer and neoplastic cells, and modulating the immune response. Polynucleotides and polypeptides of the invention may represent a diagnostic marker for hematopoietic and immune diseases and/or disorders. The full-length protein should be a secreted protein, based upon homology to the tag7 protein. Therefore, it is secreted into serum, urine, or feces and thus the levels is assayable from patient samples. Assuming specific expression levels are reflective of the presence of immune disorders, this protein would provide a convenient diagnostic for early detection. In addition, expression of this gene product may also be linked to the progression of immune diseases, and therefore may itself actually represent a therapeutic or therapeutic target for the treatment of cancer.

Polynucleotides and polypeptides of the invention may play an important role in the pathogenesis of human cancers and cellular transformation, particularly those of the immune and hematopoietic systems. Polynucleotides and polypeptides of the invention may also be involved in the pathogenesis of developmental abnormalities based upon its potential effects on proliferation and differentiation of cells and tissue cell types. Due to the potential proliferating and differentiating activity of said polynucleotides and polypeptides, the invention is useful as a therapeutic agent in inducing tissue regeneration, for treating inflammatory conditions (e.g., inflammatory bowel syndrome, diverticulitis, etc.). Moreover, the invention is useful in modulating the immune response to aberrant polypeptides, as may exist in rapidly proliferating cells and tissue cell types, particularly in adenocarcinoma cells, and other cancers. The translation product of this gene shares sequence homology with Tag7, which is a mouse cytokine that, m soluble form, triggers apoptosis in mouse L929 cells in vitro

The translation product of this gene also shaies sequence homology with antimicrobial BGP-A, a bovine antimiciobial peptide from bovine neutrophils. Preferred polypeptides of this invention comprise residues 184 to 196 shown in SEQ ID NO: 36 This polypeptide is believed to be the active mature form of the translation product of

This gene is expressed primarily in bone marrow and to a lesser extent in human chondrosarcoma and neutrophils.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tιssue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, infections, cancer, and disordeis of the immune system. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tιssue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of infected tissues and the immune system, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g. immune, hematopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 36 as residues: Ala-63 to Asn-68, Ala-71 to Gln-81, Tyr-135 to Thr- 141, Leu- 167 to Gin- 174, Pro-191 to Pro- 196. Polynucleotides encoding said polypeptides are also provided.

FEATURES OF PROTEIN ENCODED BY GENE NO: 9

This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. The polypeptide of the present invention has been putatively identified as a human butyrophilin homolog derived from a human testes tumor cDNA library. The polypeptide of the present invention is sometimes hereafter referred to as "Butyrophlin and B7-like IgG superfamily receptor", and/or "BBIR II". The invention also relates to inhibiting the action of such polypeptides.

Butyrophilin is a glycoprotein of the immunoglobulin superfamily that is secreted in association with the milk-fat-globule membrane from mammary epithelial cells. The butyrophilin gene appears to have evolved from a subset of genes in the immunoglobulin superfamily and genes encoding the B30.2 domain, which is conserved in a family of zinc-finger proteins. Furthermore, expression analysis of butyrophilin genes has shown that butyrophilin expression increases during lactation in conjunction with an increase in milk fat content. These results suggest that the stage-specific expression of milk fat globule membrane glycoproteins in mammary epithelial cells is regulated in a similar but not necessarily identical mechanism to that of a major milk protein, beta-casein.

The polypeptide of the present invention has been putatively identified as a member of the milk fat globule membrane glycoprotein family, and more particularly the butyrophilin family, and has been termed Butyrophlin and B7-like IgG superfamily receptor ("BBIR II"). This identification has been made as a result of amino acid sequence homology to the bovine butyrophilin precursor (See Genbank Accession No. gi|162773).

Preferred polypeptides of the invention comprise the following nucleic acid sequence: ACATCCATGGCTCTAATGCTCAGTTTGGTTCTGAGTCTCCTCAAGCTGGGATC AGGGCAGTGGCAGGTGTTTGGGCCAGACAAGCCTGTCCAGGCCTTGGTGGGG GAGGACGCAGCATTCTCCTGTTTCCTGTCTCCTAAGACCAATGCAGAGGCCA TGGAAGTGCGGTTCTTCAGGGGCCAGTTCTCTAGCGTGGTCCACCTCTACAG GGACGGGAAGGACCAGCCATTTATGCAGATGCCACAGTATCAAGGCAGGAC AAA ACTGGTGAAGGATTCTATTGCGGAGGGGCGCATCTCTCTGAGGCTGGA A AACATTACTGTGTTGGATGCTGGCCTCTATGGGTGCAGGATTAGTTCCCAGTC TTACTACCAGAAGGCCATCTGGGAGCTACAGGTGTCAGCACTGGGCTCAGTT CCTCTCATTTCCATCACGGGATATGTTGATAGAGACATCCAGCTACTCTGTCA GTCCTCGGGCTGGTTCCCCCGGCCCACAGCGAAGTGGAAAGGTCCACAAGGA C AGG ATTTGTCCACAGACTCC AGG AC AAAC AG AG AC ATGCATGGCCTGTTTG ATGTGGAGATCTCTCTGACCGTCCAAGAGAACGCCGGGAGCATATCCTGTTC CATGCGGCATGCTCATCTGAGCCGAGAGGTGGAATCCAGGGTACAGATAGG AGATACCTTTTTCGAGCCTATATCGTGGCACCTGGCTACCAAAGTACTGGGA ATACTCTGCTGTGGCCTATTTTTTGGCATTGTTGGACTGAAGATTTTCTTCTCC AAATTCCAGTGGAAAATCCAGGCGGAACTGGACTGGAGAAGAAAGCACGGA CAGGCAGAATTGAGAGACGCCCGGAAACACGCAGTGGAGGTGACTCTGGAT CCAGAGACGGCTCACCCGAAGCTCTGCGTTTCTGATCTGAAAACTGTAACCC ATAGAAAAGCTCCCCAGGAGGTGCCTCACTCTGAGAAGAGATTTACAAGGA AGAGTGTGGTGGCTTCTCAGAGTTTCCAAGCAGGGAAACATTACTGGGAGGT GGACGGAGGACACAATAAAAGGTGGCGCGTGGGAGTGTGCCGGGATGATGT GGACAGGAGGAAGGAGTACGTGACTTTGTCTCCCGATCATGGGTACTGGGTC CTCAGACTGAATGGAGAACATTTGTATTTCACATTAAATCCCCGTTTTATCAG CGTCTTCCCCAGGACCCCACCTACAAAAATAGGGGTCTTCCTGGACTATGAG TGTGGGACCATCTCCTTCTTCAACATAAATGACCAGTCCCTTATTTATACCCT GACATGTCGGTTTGAAGGCTTATTGAGGCCCTACATTGAGTATCCGTCCTATA ATGAGCAAAATGGAACTCCCAGAGACAAGCAACAGTGAGTCCTCCTCACAG GCAACCACGCCCTTCCTCCCCAGGGGTGAAATGTAGGATGAATCACATCCCA CATTCTTCTTTAGGGATATTAAGGTCTCTCTCCCAGATCCAAAGTCCCGCAGC AGCCGGCCAAGGTGGCTTCCAGATGAAGGGGGACTGGCCTGTCCACATGGG AGTCAGGTGTCATGGCTGCCCTGAGCTGGGAGGGAAGAAGGCTGACATTAC ATTTAGTTTGCTCTCACTCCATCTGGCTAAGTGATCTTGAAATACCACCTCTC AGGTG AAG A ACCGTCAGGAATTCCC ATCTCACAGGCTGTGGTGTAGATTAAG TAGACAAGGAATGTGAATAATGCTTAGATCTTATTGATGACAGAGTGTATCC TAATGGTTTGTTCATTATATTACACTTTCAGTAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA (SEQ ID NOJ 17), and/or ATGGCTCTAATGCTCAGTTTGGTTCTGAGTCTCCTCAAGCTGGGATCAGGGCA GTGGC AGGTGTTTGGGCC AG ACA AGCCTGTCCAGGCCTTGGTGGGGG AGG AC GCAGCATTCTCCTGTTTCCTGTCTCCTAAGACCAATGCAGAGGCCATGGAAG TGCGGTTCTTCAGGGGCCAGTTCTCTAGCGTGGTCCACCTCTACAGGGACGG GAAGGACCAGCCATTTATGCAGATGCCACAGTATCAAGGCAGGACAAAACT GGTGAAGGATTCTATTGCGGAGGGGCGCATCTCTCTGAGGCTGGAAAACATT ACTGTGTTGGATGCTGGCCTCTATGGGTGCAGGATTAGTTCCCAGTCTTACTA CCAGAAGGCCATCTGGGAGCTACAGGTGTCAGCACTGGGCTCAGTTCCTCTC ATTTCCATCACGGGATATGTTGATAGAGACATCCAGCTACTCTGTCAGTCCTC GGGCTGGTTCCCCCGGCCCACAGCGAAGTGGAAAGGTCCACAAGGACAGGA TTTGTCCACAGACTCCAGGACAAACAGAGACATGCATGGCCTGTTTGATGTG GAGATCTCTCTGACCGTCCAAGAGAACGCCGGGAGCATATCCTGTTCCATGC GGCATGCTCATCTGAGCCGAGAGGTGGAATCCAGGGTACAGATAGGAGATA CCTTTTTCGAGCCTATATCGTGGCACCTGGCTACCAAAGTACTGGGAATACTC TGCTGTGGCCTATTTTTTGGCATTGTTGGACTGAAGATTTTCTTCTCCAAATTC CAGTGGAAAATCCAGGCGGAACTGGACTGGAGAAGAAAGCACGGACAGGCA GAATTGAGAGACGCCCGGAAACACGCAGTGGAGGTGACTCTGGATCCAGAG ACGGCTCACCCGAAGCTCTGCGTTTCTGATCTGAAAACTGTAACCCATAGAA AAGCTCCCCAGGAGGTGCCTCACTCTGAGAAGAGATTTACAAGGAAGAGTGT GGTGGCTTCTCAGAGTTTCCAAGCAGGGAAACATTACTGGGAGGTGGACGGA GGACACAATAAAAGGTGGCGCGTGGGAGTGTGCCGGGATGATGTGGACAGG AGGAAGGAGTACGTGACTTTGTCTCCCGATCATGGGTACTGGGTCCTCAGAC TGAATGGAGAACATTTGTATTTCACATTAAATCCCCGTTTTATCAGCGTCTTC CCCAGGACCCCACCTACAAAAATAGGGGTCTTCCTGGACTATGAGTGTGGGA CCATCTCCTTCTTCAACATAAATGACCAGTCCCTTATTTATACCCTGACATGT CGGTTTGAAGGCTTATTGAGGCCCTACATTGAGTATCCGTCCTATAATGAGC AAAATGGAACTCCCAGAGACAAGCAACAGTGA (SEQ ID NO: 118). Polypeptide encoded by these polynucleotides are also provided. Preferred polypeptides of the invention comprise the following amino acid sequence:

MALMLSLVLSLLKLGSGQWQVFGPDKPVQALVGEDAAFSCFLSPKTNAEAMEV RFFRGQFSSVVHLYRDGKDQPFMQMPQYQGRTKLVKDSIAEGRISLRLENITVL DAGLYGCRISSQSYYQKAIWELQVSALGSVPLISITGYVDRDIQLLCQSSGWFPRP TAKWKGPQGQDLSTDSRTNRDMHGLFDVEISLTVQENAGSISCSMRHAHLSREV ESRVQIGDTFFEPISWHLATKVLGILCCGLFFGIVGLKIFFSKFQWKIQAELDWRR KHGQAELRDARKHAVEVTLDPETAHPKLCVSDLKTVTHRKAPQEVPHSEKRFT RKSVVASQSFQAGKHYWEVDGGHNKRWRVGVCRDDVDRRKEYVTLSPDHGY WVLRLNGEHLYFTLNPRFISVFPRTPPTKIGVFLDYECGTISFFNINDQSLIYTLTC RFEGLLRPYIEYPSYNEQNGTPRDKQQ (SEQ ID NO: 119). Polynucleotides encoding these polypeptides are also provided. A preferred polynucleotide splice variant of the invention comprises the following nucleic acid sequence:

ACCTTTTTCGAGCCTATATCGTGGCACCTGGCTACCAAAGTACTGGGAATACT CTGCTGTGGCCTATTTTTTGGCATTGTTGGACTGAAGATTTTCTTCTCCAAATT CCAGTGGAAAATCCAGGCGGAACTGGACTGGAGAAGAAAGCACGGACAGGC AGAATTGAGAGACGCCCGGAAACACGCAGTGGAGGTGACTCTGGATCCAGA GACGGCTCACCCGAAGCTCTGCGTTTCTGATCTGAAAACTGTAACCCATAGA AAAGCTCCCCAGGAGGTGCCTCACTCTGAGAAGAGATTTACAAGGAAGAGT GTGGTGGCTTCTCAGAGTTTCCAAGCAGGGAAACATTACTGGGAGGTGGACG GAGG AC ACAATAAAAGGTGGCGCGTGGG AGTGTGCCGGGATG ATGTGGAC A GGAGGAAGGAGTACGTGACTTTGTCTCCCGATCATGGGTACTGGGTCCTCAG ACTGAATGGAGAACATTTGTATTTCACATTAAATCCCCGTTTTATCAGCGTCT TCCCCAGGACCCCACCTACAAAAATAGGGGTCTTCCTGGACTATGAGTGTGG GACCATCTCCTTCTTCAACATAAATGACCAGTCCCTTATTTATACCCTGACAT GTCGGTTTGAAGGCTTATTGAGGCCCTACATTGAGTATCCGTCCTATAATGAG CAAAATGGAACTCCCAGAGACAAGCAACAGTGAGTCCTCCTCACAGGCAAC CACGCCCTTCCTCCCCAGGGGTGAAATGTAGGATGAATCACATCCCACATTC TTCTTTAGGGATATTAAGGTCTCTCTCCCAGATCCAAAGTCCCGCAGCAGCCG GCCAAGGTGGCTTCCAGATGAAGGGGGACTGGCCTGTCCACATGGGAGTCA GGTGTCATGGCTGCCCTGAGCTGGGAGGGAAGAAGGCTGACATTACATTTAG TTTGCTCTCACTCCATCTGGCTAAGTGATCTTGAAATACCACCTCTCAGGTGA AGAACCGTCAGGAATTCCCATCTCACAGGCTGTGGTGTAGATTAAGTAGACA AGGAATGTGAATAATGCTTAGATCTTATTGATGACAGAGTGTATCCTAATGG TTTGTTCATTATATTACACTTTCAGTAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAA (SEQ ID NO: 120). Polypeptides encoded by these polynucleotides are also provided. Figures 22A-D show the nucleotide (SEQ ID NO: 19) and deduced amino acid sequence (SEQ ID NO:37) of BBIR II. Predicted amino acids from about 1 to about 17 constitute the predicted signal peptide (amino acid residues from about 1 to about 17 in SEQ ID NO:37) and are represented by the underlined amino acid regions. Figure 23 shows the regions of similarity between the amino acid sequences of the Butyrophlin and B7-like IgG superfamily receptor (BBIR II) protein (SEQ ID NO:37) and the bovine butyrophilin precursor (SEQ ID NO: 121)

Figure 24 shows an analysis of the integrin alpha 11 subunit (BBIR II) amino acid sequence. Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.

A polynucleotide encoding a polypeptide of the present invention is obtained from human small intestine, colon tumor, and human testes tumor cells and tissues. The polynucleotide of this invention was discovered in a human testes tumor cDNA library. Its translation product has homology to the B30.2-like domain which is characteristic of proteins containing zinc-binding B-box motifs, and particularly for butyrophilin family members. The polynucleotide contains an open reading frame encoding the BBIR II polypeptide of 318 amino acids. BBIR II exhibits a high degree of homology at the amino acid level to the bovine butyrophilin precursor (as shown in Figure 23). The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the BBIR II polypeptide having the amino acid sequence shown in Figures 22A-D (SEQ ID NO: 37). The nucleotide sequence shown in Figures 22A-D (SEQ ID NO: 19) was obtained by sequencing a cloned cDNA (HTTDB46), which was deposited on November 17 at the American Type Culture Collection, and given Accession Number 203484.

The present invention is further directed to fragments of the isolated nucleic acid molecules described herein. By a fragment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ ID NO: 19 is intended DNA fragments at least about 15nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments 50-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in SEQ ID NOJ 9. By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NO: 19. In this context "about" includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Representative examples of BBIR II polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200. from about 201 to about 250, from about 251 to about 300, from about 301 to about 350. from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, from about 501 to about 550, from about 551 to about 600, from about 601 to about 650, from about 651 to about 700, from about 701 to about 750, from about 751 to about 800, from about 801 to about 850, from about 851 to about 900, from about 901 to about 950, from about 951 to about 1000, from about 1001 to about 1050, from about 1051 to about 1100, from about 1101 to about 1150, from about 1151 to about 1200, from about 1201 to about 1250, from about 1251 to about 1300, from about 1301 to about 1350, from about 1351 to about 1400, from about 1401 to about 1450, from about 1451 to about 1500, from about 1501 to about 1550, from about 1551 to about 1600, from about 1601 to about 1650, from about 1651 to about

1700, from about 1701 to about 1750, from about 1751 to about 1800, from about 1801 to about 1850, from about 1851 to about 1900, from about 1901 to about 1950, from about 1951 to about 2000. from about 2001 to about 2050, from about 2051 to about 2100, from about 2101 to about 2150, from about 2151 to about 2200, from about 2201 to about 2250, from about 2251 to about 2300, from about 2301 to about 2350, from about 2351 to about 2400, from about 2401 to about 2450, from about 2451 to about 2500, from about 2501 to about 2550, from about 2551 to about 2600, from about 2601 to about 2650, from about 2651 to about 2700, from about 2701 to about 2750, from about 2751 to about 2800, from about 2801 to about 2850, from about 2851 to about 2900, from about 2901 to about 2950, from about 2951 to about 3000, from about 3001 to about 3050, from about 3051 to about 3059 of SEQ ID NO: 19, or the complementary strand thereto, or the cDNA contained in the deposited gene. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1 ) nucleotides, at either terminus or at both termini.

Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding a member selected from the group: a polypeptide comprising or alternatively, consisting of, the mature BBIR II protein (amino acid residues from about 18 to about 318 in Figures 22A-D (amino acids from about 18 to about 318 in SEQ ID NO:37). Since the location of this form of the protein has been predicted by computer analysis, one of ordinary skill would appreciate that the amino acid residues constituting these domains may vary slightly (e.g., by about 1 to 15 amino acid residues) depending on the criteria used to define this location. In additional embodiments, the polynucleotides of the invention encode functional attributes of BBIR II.

Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions"), turn and turn-forming regions ("turn- regions"), coil and coil-forming regions ("coil-regions"), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions of BBIR II. The data representing the structural or functional attributes of BBIR II set forth in Figure 24 and/or Table VIII, as described above, was generated using the various modules and algorithms of the DNA*STAR set on default parameters. In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table VIII can be used to determine regions of BBIR II which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response. Certain preferred regions in these regards are set out in Figure 24, but may. as shown in Table VIII, be represented or identified by using tabular representations of the data presented in Figure 24. The DNA*STAR computer algorithm used to generate Figure 24 (set on the original default parameters) was used to present the data in Figure 24 in a tabular format (See Table VIII). The tabular format of the data in Figure 24 is used to easily determine specific boundaries of a preferred region. The above-mentioned preferred regions set out in Figure 24 and in Table VIII include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figures 22A-D. As set out in Figure 24 and in Table VIII, such preferred regions include Garni er-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions and Hopp-Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenic index and Emini surface-forming regions. Even if deletion of one or more amino acids from the N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, etc.) may still be retained. For example, the ability of shortened BBIR II muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that an BBIR II mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six BBIR II amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the BBIR II amino acid sequence shown in Figures 22A-D, up to the cystein residue at position number 313 and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising the amino acid sequence of residues n 1-318 of Figures 22A-D, where nl is an integer from 2 to 313 corresponding to the position of the amino acid residue in Figures 22A-D (which is identical to the sequence shown as SEQ ID NO:37). In another embodiment, N-terminal deletions of the BBIR II polypeptide can be described by the general formula n2-318, where n2 is a number from 2 to 313, corresponding to the position of amino acid identified in Figures 22A-D. N-terminal deletions of the BBIR II polypeptide of the invention shown as SEQ ID NO:37 include polypeptides comprising the amino acid sequence of residues: N-terminal deletions of the BBIR II polypeptide of the invention shown as SEQ ID NO:37 include polypeptides comprising the amino acid sequence of residues: A-2 to T-318; L-3 to T-318; M-4 to T- 318; L-5 to T-318; S-6to T-318; L-7 to T-318; V-8 to T-318; L-9 to T-318; S-10 to T- 318; L-l 1 to T-318; L-12 to T-318; K-13 toT-318; L-14 to T-318; G-15 to T-318; S-16 to T-318; G-17 to T-318; Q-18 to T-318; W-19 to T-318; Q-20 toT-318; V-21 to T-318; F-22 to T-318; G-23 to T-318; P-24 to T-318; D-25 to T-318; K-26 to T-318; P-27 toT- 318; V-28 to T-318; Q-29 to T-318; A-30 to T-318; L-31 to T-318; V-32 to T-318; G-33 to T-318; E-34 toT-318; D-35 to T-318; A-36 to T-318; A-37 to T-318; F-38 to T-318; S-39 to T-318; C-40 to T-318; F-41 toT-318; L-42 to T-318; S-43 to T-318; P-44 to T- 318; K-45 to T-318; T-46 to T-318; N-47 to T-318; A-48 toT-318; E-49 to T-318; A-50 to T-318; M-51 to T-318; E-52 to T-318; V-53 to T-318; R-54 to T-318; F-55 toT-318; F-56 to T-318; R-57 to T-318; G-58 to T-318; Q-59 to T-318; F-60 to T-318; S-61 to T- 318; S-62 toT-318; V-63 to T-318; V-64 to T-318; H-65 to T-318; L-66 to T-318; Y-67 to T-318; R-68 to T-318; D-69 toT-318; G-70 to T-318; K-71 to T-318; D-72 to T-318; Q-73 to T-318; P-74 to T-318; F-75 to T-318; M-76 toT-318; Q-77 to T-318; M-78 to T- 318; P-79 to T-318; Q-80 to T-318; Y-81 to T-318; Q-82 to T-318; G-83 toT-318; R-84 to T-318; T-85 to T-318; K-86 to T-318; L-87 to T-318; V-88 to T-318; K-89 to T-318; D-90 toT-318; S-91 to T-318; 1-92 to T-318; A-93 to T-318; E-94 to T-318; G-95 to T- 318; R-96 to T-318; 1-97 toT-318; S-98 to T-318; L-99 to T-318; R-100 to T-318; L-101 to T-318; E-l 02 to T-318; N-103 to T-318;I-104 to T-318; T-l 05 to T-318; V-106 to T- 318; L-107 to T-318; D-108 to T-318; A-109 to T-318; G-l 10 toT-318; L-l 11 to T-318; Y-112 to T-318; G-l 13 to T-318; C-114 to T-318; R-l 15 to T-318; 1-116 to T-318;S-1 17 to T-318; S-l 18 to T-318; Q-119 to T-318; S-120 to T-318; Y-121 to T-318; Y-122 to T- 318; Q-123 toT-318; K-124 to T-318; A- 125 to T-318; 1-126 to T-318; W-127 to T-318; E-128 to T-318; L-l 29 to T-318;Q-130 to T-318; V-131 to T-318; S-132 to T-318; A- 133 to T-318; L-l 34 to T-318; G-135 to T-318; S-136 toT-318; V-137 to T-318; P-138 to T-318; L-139 to T-318; 1-140 to T-318; S-141 to T-318; 1-142 to T-318;A-143 to T- 318; G-144 to T-318; Y-145 to T-318; V-146 to T-318; D-147 to T-318; R-148 to T-318; D-149to T-318; 1-150 to T-318; Q-151 to T-318; L-152 to T-318; L-153 to T-318; C-154 to T-318; Q-155 to T-318;S-156 to T-318; S-157 to T-318; G-158 to T-318; W-159 to T- 318; F-160 to T-318; P-161 to T-318; R-162to T-318; P-163 to T-318; T-l 64 to T-318; A- 165 to T-318; K-166 to T-318; W-167 to T-318; K-168 toT-318; G-169 to T-318; P- 170 to T-318; Q-171 to T-318; G-172 to T-318; Q-173 to T-318; D-174 to T-318;L-175 to T-318; S-176 to T-318; T-177 to T-318; D-178 to T-318; S-179 to T-318; R-180 to T- 318; T-181 toT-318; N-182 to T-318; R-l 83 to T-318; D-184 to T-318; M-185 to T-318; H-l 86 to T-318; G-l 87 to T-318;L-188 to T-318; F-189 to T-318; D-190 to T-318; V- 191 to T-318; E-192 to T-318; 1-193 to T-318; S-194 toT-318; L-195 to T-318; T-l 96 to T-318; V-197 to T-318; Q-198 to T-318; E-199 to T-318; N-200 to T-318;A-201 to T- 318; G-202 to T-318 ; S-203 to T-318; 1-204 to T-318; S-205 to T-318; C-206 to T-318; S-207 toT-318; M-208 to T-318; R-209 to T-318; H-210 to T-318; A-211 to T-318; H- 212 to T-318; L-213 to T-318;S-214 to T-318; R-215 to T-318; E-216 to T-318; V-217 to T-318; E-218 to T-318; S-219 to T-318; R-220 toT-318; V-221 to T-318; Q-222 to T- 318; 1-223 to T-318; G-224 to T-318; D-225 to T-318; W-226 to T-318;R-227 to T-318; R-228 to T-318; K-229 to T-318; H-230 to T-318; G-231 to T-318; Q-232 to T-318; A- 233to T-318; G-234 to T-318; K-235 to T-318; R-236 to T-318; K-237 to T-318; Y-238 to T-318; S-239 toT-318; S-240 to T-318; S-241 to T-318; H-242 to T-318; 1-243 to T- 318; Y-244 to T-318; D-245 to T-318;S-246 to T-318; F-247 to T-318; P-248 to T-318; S-249 to T-318; L-250 to T-318; S-251 to T-318; F-252 toT-318; M-253 to T-318; D- 254 to T-318; F-255 to T-318; Y-256 to T-318; 1-257 to T-318; L-258 to T-318;R-259 to T-318; P-260 to T-318; V-261 to T-318; G-262 to T-318; P-263 to T-318; C-264 to T- 318; R-265to T-318; A-266 to T-318; K-267 to T-318; L-268 to T-318; V-269 to T-318; M-270 to T-318 ; G-271 toT-318 ; T-272 to T-318 ; L-273 to T-318 ; K-274 to T-318 ; L- 275 to T-318; Q-276 to T-318; 1-277 to T-318;L-278 to T-318; G-279 to T-318; E-280 to T-318; V-281 to T-318; H-282 to T-318; F-283 to T-318; V-284to T-318; E-285 to T- 318; K-286 to T-318; P-287 to T-318; H-288 to T-318; S-289 to T-318; L-290 toT-318; L-291 to T-318; Q-292 to T-318; 1-293 to T-318; S-294 to T-318; G-295 to T-318; G- 296 to T-318;S-297 to T-318; T-298 to T-318; T-299 to T-318; L-300 to T-318; K-301 to T-318; K-302 to T-318; G-303to T-318; P-304 to T-318; N-305 to T-318; P-306 to T- 318; W-307 to T-318; S-308 to T-318; F-309 toT-318; P-310 to T-318; S-311 to T-318; P-312 to T-318; C-313 to T-318; of SEQ ID NO:37. Polypeptides encoded by these polynucleotides are also encompassed by the invention. Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results m modification or loss of one or more biological functions of the protein, other functional activities (e.g., biological activities (e.g., ability to illicit mitogenic activity, induce differentiation of normal or malignant cells, ability to multimerize, etc.) may still be retained. For example the ability of the shortened BBIR II mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majoπty of the residues of the complete or mature polypeptide aie removed from the C-termmus. Whether a particulai polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by loutine methods descπbed herein and otherwise known in the art. It is not unlikely that an BBIR II mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six BBIR II amino acid residues may often evoke an immune response. Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the ammo acid sequence of the BBIR II polypeptide shown in Figures 22A-D, up to the serine residue at position number 6, and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising the ammo acid sequence of residues 1-ml of Figure 1, where ml is an integer from 6 to 318 corresponding to the position of the amino acid residue in Figures 22A-D. Moreover, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the ammo acid sequence of C-termmal deletions of the BBIR II polypeptide of the invention shown as SEQ ID NO:37 include polypeptides comprising the amino acid sequence of residues M- 1 to P-317; M-1 to F-316; M-1 to L-315;M-1 to A-314; M-1 to C-313; M-1 to P-312; M- 1 to S-311; M-l to P-310; M-l to F-309; MJ to S-308; M-lto W-307; M-1 to P-306; M- 1 to N-305; M-1 to P-304; M-1 to G-303; M-1 to K-302; M-1 to K-301; M-1 toL-300; M-1 to T-299; M-1 to T-298; M-1 to S-297; M-1 to G-296; M-1 to G-295; M-1 to S-294;

M-1 toI-293; M-1 to Q-292; M-1 to L-291; M-1 to L-290; M-1 to S-289; M-1 to H-288;

M-1 to P-287; M-1 toK-286; M-1 to E-285; M-1 to V-284; M-1 to F-283; M-1 to H-282;

M-1 to V-281; M-1 to E-280; M-1 toG-279; M-1 to L-278; M-1 to 1-277; M-1 to Q-276; M-1 to L-275; M-1 to K-274; M-1 to L-273; M-1 toT-272; M-1 to G-271; M-1 to M-270;

M-1 to V-269; M-1 to L-268; M-1 to K-267; M-1 to A-266; M-1 toR-265; M-1 to C-264;

M-1 to P-263; M-1 to G-262; M-1 to V-261 ; M-1 to P-260; M-1 to R-259; M-1 toL-258;

M-1 to 1-257; M-1 to Y-256; M-1 to F-255; M-1 to D-254; M-1 to M-253; M-1 to F-252;

M-1 toS-251; M-1 to L-250; M-1 to S-249; M-1 to P-248; M-1 to F-247; M-1 to S-246; M-1 to D-245; M-1 toY-244; M-1 to 1-243; M-1 to H-242; M-1 to S-241 ; M-1 to S-240;

M-1 to S-239; M-1 to Y-238; M-1 toK-237; M-1 to R-236; M-1 to K-235; M-1 to G-234;

M-1 to A-233; M-1 to Q-232; M-1 to G-231; M-1 toH-230; M-1 to K-229; M-1 to R-

228; M-1 to R-227; M-1 to W-226; M-1 to D-225; M-1 to G-224; M-1 toI-223; M-1 to

Q-222; M-1 to V-221; M-1 to R-220; M-1 to S-219; M-1 to E-218; M-1 to V-217; M-1 toE-216; M-1 to R-215; M-1 to S-214; M-1 to L-213; M-1 to H-212; M-1 to A-211 ; M-1 to H-210; M-1 toR-209; M-1 to M-208; M-1 to S-207; M-1 to C-206; M-1 to S-205; M-1 to 1-204; M-1 to S-203; M-1 toG-202; M-1 to A-201; M-1 to N-200; M-1 to E-199; M-1 to Q-198; M-1 to V-197; M-1 to T-196; M-1 toL-195; M-1 to S-194; M-1 to 1-193; M-1 to E-192; M-1 to V-191; M-1 to D-190; M-1 to F-189; M-1 toL-188; M-1 to G-187; M-1 to H-186; M-1 to M-185; M-1 to D-184; M-1 to R-183; M-1 to N-182; M-1 toT-181; M-

1 to R-180; M-1 to S-179; M-1 to D-178; M-1 to T-177; M-1 to S-176; M-1 to L-175;

M-1 toD-174; M-1 to Q-173; M-1 to G-172; M-1 to Q-171; M-1 to P-170; M-1 to G-169;

M-1 to K-168; M-1 toW-167; M-1 to K-166; M-1 to A-165; M-1 to T-164; M-1 to P-

163; M-1 to R-162; M-1 to P-161; M-1 toF-160; M-1 to W-159; M-1 to G-158; M-1 to S-157; M-1 to S-156; M-1 to Q-155; M-1 to C-154; M-1 toL-153; M-1 to L-152; M-1 to

Q-151; M-1 to 1-150; M-1 to D-149; M-1 to R-148; M-1 to D-147; M-1 toV-146; M-1 to

Y-145; M-1 to G-144; M-1 to A-143; M-1 to 1-142; M-1 to S-141; M-1 to 1-140; M-1 toL-139; M-1 to P-138; M-1 to V-137; M-1 to S-136; M-1 to G-135; M-1 to L-134; M-1 to A-133; M-1 toS-132; M-1 to V-131; M-1 to Q-130; M-1 to L-129; M-1 to E-128; M-1 to W-127; M-1 to 1-126; M-1 toA-125; M-1 to K-124; M-1 to Q-123; M-1 to Y-122; M-1 to Y-121; M-1 to S-120; M-1 to Q-119; M-1 toS-118; M-1 to S-l 17; M-1 to 1-116; M-1 to R-l 15; M-1 to C-114; M-1 to G-l 13; M-1 to Y-l 12; M-1 toL-l l l; M-l to G-l 10; M-1 to A-109; M-1 to D-108; M-1 to L-107; M-1 to V-106; M-1 to T-105; M-1 tol-104; M-1 to N-103; M-1 to E-102; M-1 to L-101; M-1 to R-100; M-1 to L-99; M-1 to S-98; M-1 to I-97;M-1 to R-96; M-1 to G-95; M-1 to E-94; M-1 to A-93; M-1 to 1-92; M-1 to S-91; M-1 to D-90; M-1 to K-89;MJ to V-88; M-1 to L-87; M-1 to K-86; M-1 to T-85; M-1 to R-84; M-1 to G-83; M-1 to Q-82; M-1 to Y-81;M-1 to Q-80; M-1 to P-79; M-1 to M-78; M-1 to Q-77; M-1 to M-76; M-1 to F-75; M-1 to P-74; M-1 to Q-73;M-1 to D-72; M-1 to K-71; M-1 to G-70; M-1 to D-69; M-1 to R-68; M-1 to Y-67; M-1 to L-66; M-1 to H- 65;M-1 to V-64; M-l to V-63; M-1 to S-62; MJ to S-61 ; M-1 to F-60; M-1 to Q-59; M- 1 to G-58; M-1 to R-57;M-1 to F-56; M-1 to F-55; M-1 to R-54; M-1 to V-53; M-1 to E- 52; M-1 to M-51; M-1 to A-50; M-1 to E-49;M-1 to A-48; M-1 to N-47; M-1 to T-46; M-1 to K-45; M-1 to P-44; M-1 to S-43; M-1 to L-42; M-1 to F-41;M-1 to C-40; M-1 to S-39; M-1 to F-38; M-1 to A-37; M-1 to A-36; M-1 to D-35; M-1 to E-34; M-1 to G- 33;M-1 to V-32; M-1 to L-31; M-1 to A-30; M-1 to Q-29; M-1 to V-28; M-1 to P-27; M- 1 to K-26; M-1 to D-25;M-1 to P-24; M-1 to G-23; M-1 to F-22; M-1 to V-21; M-1 to Q- 20; M-1 to W-19; M-1 to Q-18; M-1 to G-17;M-1 to S-16; M-1 to G-15; M-1 to L-14; M-1 to K-13; M-1 to L-12; M-1 to L-l 1; M-1 to S-10; M-1 to L-9;M-1 to V-8; M-1 to L- 7; M-1 to S-6; of SEQ ID NO:37. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

In addition, the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO: 19 which have been determined from the following related cDNA genes: HTTDB46R (SEQ ID NO: 122), and HSIEA44R- (SEQ ID NO: 123). Based on the sequence similaπty to the bovin butyrophilin precursor, translation product of this gene is expected to share at least some biological activities with B30.2- like domain containing proteins, and specifically butyrophilin proteins. Such activities are known in the art, some of which are described elsewhere herein Specifically, polynucleotides and polypeptides of the invention are also useful for modulating the diffei en nation of normal and malignant cells, modulating the proliferation and/oi differentiation of cancer and neoplastic cells, and regulation of cell giowth and differentiation Polynucleotides and polypeptides of the invention may represent a diagnostic marker for breast diseases and/or disoideis, in addition to disorders of secretory organs and tissues (which include, testicular and gastrointestinal disordeis, particularly those cells which serve secretory functions for seminal fluid oi gastrointestinal hormones, and disorders of the mucosal membranes of such cells and tissues, etc.).

The full-length protein should be a secreted protein, based upon homology to the butyrophilin family of proteins. Therefore, it is secreted into milk, serum, unne, seminal fluid, or feces and thus the levels is assayable from patient samples. Assuming specific expression levels are reflective of the presence of breast disorders (i.e , breast cancer, breast dysfunction, etc.) this protein would provide a convenient diagnostic for eaily detection of such disorders In addition, expression of this gene product may also be linked to the progression of breast diseases, and therefore may itself actually represent a therapeutic or therapeutic target for the treatment of breast cancer. Polynucleotides and polypeptides of the invention may play an important role in the pathogenesis of human cancers and cellular transformation, particularly those of secretory cells and tissues. Polynucleotides and polypeptides of the invention may also be involved in the pathogenesis of developmental abnormalities based upon its potential effects on proliferation and differentiation of cells and tissue cell types Due to the potential proliferating and differentiating activity of said polynucleotides and polypeptides, the invention is useful as a therapeutic agent in inducing tissue regeneration, for treating inflammatory conditions. Moreover, the invention is useful in modulating the immune response to aberrant polypeptides, as may exist in rapidly proliferating cells and tissue cell types, particularly in cancers. The invention, including agonists and/or antagonists thereof, is useful in modulating the nutritional value of milk, its caloric content, its fat content, and may conceivably be useful in mediating the adaption of breast secretory function as a delivery vehicle for therapeutics (i.e., transgenic breast secretory tissue for transferring therapeutically active proteins to infants).

Alternatively, this gene product is involved in the pattern of cellular proliferation that accompanies early embryogenesis. Thus, aberrant expression of this gene product in tissues - particularly adult tissues - may correlate with patterns of abnormal cellular proliferation, such as found in various cancers. Because of potential roles in proliferation and differentiation, this gene product may have applications in the adult for tissue regeneration and the treatment of cancers. It may also act as a morphogen to control cell and tissue type specification. Therefore, the polynucleotides and polypeptides of the present invention are useful in treating, detecting, and/or preventing said disorders and conditions, in addition to other types of degenerative conditions. Thus this protein may modulate apoptosis or tissue differentiation and is useful in the detection, treatment, and/or prevention of degenerative or proliferative conditions and diseases. The protein is useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues. The protein can also be used to gain new insight into the regulation of cellular growth and proliferation. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

This gene is expressed primarily in small intestine, colon tumor, and to a lesser extent in human testes tumor cells.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, gastrointestinal diseases and/or disorders, in addition to lactation disorders, and tumors of the testes. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune and reproductive systems, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g. immune, testicular, gastrointestinal, and cancerous and wounded tissues) or bodily fluids (e.g. lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 37 as residues: Tyr-67 to Pro-74, Ser-117 to Gln-123, Pro-161 to Met-185, Gly-224 to His-242, Thr-299 to Trp-307. Polynucleotides encoding said polypeptides are also provided.

FEATURES OF PROTEIN ENCODED BY GENE NO: 10

The translation product of this gene contains a serine protease motif and accordingly is believed to possess serine protease activity. Assays for determining such activity are well known in the art. Preferred polypeptides of this invention possess such activity. Included in this invention as preferred domains are serine protease histidine active site domains, which were identified using the ProSite analysis tool (Swiss Institute of Bioinformatics). The catalytic activity of the serine proteases from the trypsin family is provided by a charge relay system involving an aspartic acid residue hydrogen- bonded to a histidine, which itself is hydrogen-bonded to a serine. The sequences in the vicinity of the active site serine and histidine residues are well conserved in this family of proteases [1]. Consensus pattern: [LIVM]-[ST]-A-[STAG]-H-C , H is the active site residue.

Preferred polypeptides of the invention comprise the following amino acid sequence: GTLVAEKHVLTAAHCIHDGKTYVKGTQ (SEQ ID NO: 124). Polynucleotides encoding these polypeptides are also provided.

Further preferred are polypeptides comprising the serine protease histidine active site domain of the sequence referenced in Table XIII for this gene, and at least 5, 10, 15, 20, 25, 30, 50, or 75 additional contiguous amino acid residues of this referenced sequence. The additional contiguous amino acid residues is N-terminal or C- terminal to the serine protease histidine active site domain.

Alternatively, the additional contiguous amino acid residues is both N-terminal and C-terminal to the serine protease histidine active site domain, wherein the total N- and C-terminal contiguous amino acid residues equal the specified number. The above preferred polypeptide domain is characteristic of a signature specific to serine protease proteins. Based on the sequence similarity, the translation product of this gene is expected to share at least some biological activities with serine proteases. Such activities are known in the art, some of which are described elsewhere herein.

In another embodiment, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention comprise the following amino acid sequence: GTRGQ AWEPRALSRRPHLSERRSEPRPGRAARRGTVLGMAGIPGLLFLLFF

LLCAVGQVSPYSAPWKPTWPAYRLPVVLPQSTLNLAKPDFGAEAKLEVSSSCGP QCHKGTPLPTYEEAKQYLSYETLYANGSRTETQVGIYILSSSGDGAQHRDSGSSG KSRRKRQIYGYDSRFSIFGKDFLLNYPFSTSVKLSTGCTGTLVAEKHVLTAAHCI HDGKTYVKGTQKLRVGFLKPKFKDGGRGANDSTSAMPEQMKFQWIRVKRTHV PKGWIKGNANDIGMDYDYALLELKKPHKRKFMKIGVSPPAKQLPGGRIHFSGYD NDRPGNLVYRFCDVKDETYDLLYQQCDSQPGASGSGVYVRMWKRQHQKWER KIIGMISGHQWVDMDGSPQEFTRGCSEITPLQYIPDISIGV (SEQ ID NO: 125). Polynucleotides encoding these polypeptides are also provided.

A preferred polypeptide variant of the invention comprises the following amino acid sequence:

MAGIPGLLFLLFFLLCAVGQVSPYSAPWKPTWPAYRLPVVLPQSTLNLAKPD FGAEAKLEVSSSCGPQCHKGTPLPTYEEAKQYLSYETLYANGSRTETQVGIYILS SSGDGAQHRDSGSSGKSRRKRQIYGYDSRFSIFGKDFLLNYPFSTSVKLSTGCTG TLVAEKHVLT

AAHCIHDGKTYVKGTQKLRVGFLKPKFKDGGRGANDSTSAMPEQMKFQWIRV KRTHVPKGWIKGNANDIGMDYDYALLELKKPHKRKFMKIGVSPPAKQLPGGRI HFSGYDNDRPGNLVYRFCDVKDETYDLLYQQCDAQPGASGSGVYVRMWKRQ QQKWERKIIGIFSGHQWVDMNGSPQDFNVAVRITPLKYAQICYWIKGNYLDCRE G (SEQ ID NO: 126). Polynucleotides encoding these polypeptides are also provided. Figures 25 A-B show the nucleotide (SEQ ID NO:20) and deduced amino acid sequence (SEQ ID NO:38) of the present invention. Predicted amino acids from about 1 to about 19 constitute the predicted signal peptide (amino acid residues from about 1 to about 19 in SEQ ID NO:38) and are represented by the underlined amino acid regions; amino acids from about 162 to about 188 constitutes the predicted serine protease histidine active site domain (amino acids residues from about 162 to about 188 in SEQ ID NO:38) and are represented by the double underlined amino acid regions; and amino acid residue 175 (amino acid residue 175 in SEQ ID NO:38) constitutes the predicted histidine active site residue and is represented by the bold amino acid.

Figure 26 shows the regions of similarity between the amino acid sequences of the present invention SEQ ID NO:38, and the Human Pancreatic Elastase 2 protein (gi|219620)(SEQ ID NO: 127).

Figure 27 shows an analysis of the amino acid sequence of SEQ ID NO:38. Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.

Northern analysis indicates that this gene is expressed highest in HUVEC, HUVEC +LPS, smooth muscle, fibroblasts, present in heart, brain, placenta, lung, liver, muscle, kidney, pancreas, spleen, thymus, prostate, testes, ovary, small intestine, colon and weakly in PBLs. The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the polypeptide having the amino acid sequence shown in Figures 25 A-B (SEQ ID NO:38), which was determined by sequencing a cloned cDNA. The nucleotide sequence shown in Figures 25 A-B (SEQ ID NO:20) was obtained by sequencing a cloned cDNA (HUSAQ05), which was deposited on Nov. 17, 1998 at the American Type Culture Collection, and given Accession Number 203484. The deposited gene is inserted in the pSport plasmid (Life Technologies, Rockville, MD) using the Sall/Notl restriction endonuclease cleavage sites.

The present invention is further directed to fragments of the isolated nucleic acid molecules described herein. By a fragment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ ID NO:20 is intended DNA fragments at least about 15nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments 50-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in SEQ ID NO:20. By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NO:20. In this context "about" includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Representative examples of polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, and from about 501 to about 550, and from about 551 to about 600, from about 601 to about 650, from about 651 to about 700, from about 701 to about 750, from about 751 to about 800, from about 801 to about 850, from about 851 to about 900, from about 901 to about 950, from about 951 to about 1000, from about 1001 to about 1050, from about 1051 to about 1100, from about 1101 to about 1150, from about 1151 to about 1200, from about 1201 to about 1250, from about 1251 to about 1300, from about 1301 to about 1350, from about 1351 to about 1400, from about 1401 to about 1450, from about 1451 to about 1500, from about 1501 to about 1550, from about 1551 to about 1600, from about 1601 to about 1650, from about 1651 to about 1699 of SEQ ID NO:20, or the complementary strand thereto, or the cDNA contained in the deposited gene. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. In additional embodiments, the polynucleotides of the invention encode functional attributes of the corresponding protein.

Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions"), turn and turn-forming regions ("turn- regions"), coil and coil-forming regions ("coil-regions"), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions. The data representing the structural or functional attributes of the protein set forth in Figure 27 and/or Table IX, as described above, was generated using the various modules and algorithms of the DNA*STAR set on default parameters. In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table IX can be used to determine regions of the protein which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.

Certain preferred regions in these regards are set out in Figure 27, but may, as shown in Table IX, be represented or identified by using tabular representations of the data presented in Figure 27. The DNA*STAR computer algorithm used to generate Figure 27 (set on the original default parameters) was used to present the data in Figure 27 in a tabular format (See Table IX). The tabular format of the data in Figure 27 is used to easily determine specific boundaries of a preferred region. The above-mentioned preferred regions set out in Figure 27 and in Table IX include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figure 1. As set out in Figure 27 and in Table IX, such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou- Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions and Hopp-Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenic index and

Emini surface-forming regions. Even if deletion of one or more amino acids from the N- terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, etc.) may still be retained. For example, the ability of shortened muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence shown in Figures 25A-B, up to the aspartic acid residue at position number 370 and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising the amino acid sequence of residues nl-375 of Figures 25A-B, where nl is an integer from 2 to 370 corresponding to the position of the amino acid residue in Figures 25A-B (which is identical to the sequence shown as SEQ ID NO:38). N-terminal deletions of the polypeptide of the invention shown as SEQ ID NO:38 include polypeptides comprising the amino acid sequence of residues: A-2 to V-375; G-3 to V-375; 1-4 to V-375; P-5 to V-375; G-6 to V-375; L-7 to V-375; L-8 to V-375; F-9 to V-375; L-10 to V-375; L-l 1 to V-375; F-12 to V-375; F-13 to V-375; L-14 to V-375; L- 15 to V-375; C-16 to V-375; A- 17 to V-375; V-l 8 to V-375; G-19 to V-375; Q-20 to V- 375; V-21 to V-375; S-22 to V-375; P-23 to V-375; Y-24 to V-375; S-25 to V-375; A-26 to V-375; P-27 to V-375; W-28 to V-375; K-29 to V-375; P-30 to V-375; T-31 to V-375; W-32 to V-375; P-33 to V-375; A-34 to V-375; Y-35 to V-375; R-36 to V-375; L-37 to V-375; P-38 to V-375; V-39 to V-375; V-40 to V-375; L-41 to V-375; P-42 to V-375; Q- 43 to V-375; S-44 to V-375; T-45 to V-375; L-46 to V-375; N-47 to V-375; L-48 to V- 375; A-49 to V-375; K-50 to V-375; P-51 to V-375; D-52 to V-375; F-53 to V-375; G-54 to V-375; A-55 to V-375; E-56 to V-375; A-57 to V-375; K-58 to V-375; L-59 to V-375; E-60 to V-375; V-61 to V-375; S-62 to V-375; S-63 to V-375; S-64 to V-375; C-65 to V- 375; G-66 to V-375; P-67 to V-375; Q-68 to V-375; C-69 to V-375; H-70 to V-375; K- 71 to V-375; G-72 to V-375; T-73 to V-375; P-74 to V-375; L-75 to V-375; P-76 to V- 375; T-77 to V-375; Y-78 to V-375; E-79 to V-375; E-80 to V-375; A-81 to V-375; K-82 to V-375; Q-83 to V-375; Y-84 to V-375; L-85 to V-375; S-86 to V-375; Y-87 to V-375; E-88 to V-375; T-89 to V-375; L-90 to V-375; Y-91 to V-375; A-92 to V-375; N-93 to V-375; G-94 to V-375; S-95 to V-375; R-96 to V-375; T-97 to V-375; E-98 to V-375; T- 99 to V-375; Q-100 to V-375; V-101 to V-375; G-102 to V-375; 1-103 to V-375; Y-104 to V-375; 1-105 to V-375; L-106 to V-375; S-107 to V-375; S-108 to V-375; S-109 to V- 375; G-l 10 to V-375; D-l 11 to V-375; G-l 12 to V-375; A-l 13 to V-375; Q-l 14 to V- 375; H-l 15 to V-375; R-l 16 to V-375; D-l 17 to V-375; S-l 18 to V-375; G-l 19 to V- 375; S-120 to V-375; S-121 to V-375; G-122 to V-375; K-123 to V-375; S-124 to V- 375; R- 125 to V-375; R-126 to V-375; K-127 to V-375; R-128 to V-375; Q-129 to V- 375; 1-130 to V-375; Y-131 to V-375; G-132 to V-375; Y-133 to V-375; D-l 34 to V- 375; S-135 to V-375; R-136 to V-375; F-137 to V-375; S-138 to V-375; 1-139 to V-375; F-140 to V-375; G-141 to V-375; K-142 to V-375; D-143 to V-375; F-144 to V-375; L- 145 to V-375; L-146 to V-375; N-147 to V-375; Y-148 to V-375; P-149 to V-375; F-150 to V-375; S-151 to V-375; T-152 to V-375; S-153 to V-375; V-l 54 to V-375; K-155 to V-375; L-156 to V-375; S-157 to V-375; T-158 to V-375; G-159 to V-375; C-160 to V- 375; T-161 to V-375; G-162 to V-375; T-163 to V-375; L-l 64 to V-375; V-165 to V- 375; A- 166 to V-375; E-l 67 to V-375; K-168 to V-375; H-l 69 to V-375; V-170 to V- 375; L-171 to V-375; T-172 to V-375; A-173 to V-375; A- 174 to V-375; H-175 to V- 375; C-176 to V-375; 1-177 to V-375; H-178 to V-375; D-179 to V-375; G-l 80 to V- 375; K-181 to V-375; T-l 82 to V-375; Y-183 to V-375; V-184 to V-375; K-185 to V- 375; G-186 to V-375; T-l 87 to V-375; Q-188 to V-375; K-189 to V-375; L-190 to V- 375; R-191 to V-375; V-192 to V-375; G-193 to V-375; F-194 to V-375; L-195 to V- 375; K-196 to V-375; P-197 to V-375; K-198 to V-375; F-199 to V-375; K-200 to V- 375; D-201 to V-375; G-202 to V-375; G-203 to V-375; R-204 to V-375; G-205 to V- 375; A-206 to V-375; N-207 to V-375; D-208 to V-375; S-209 to V-375; T-210 to V- 375; S-211 to V-375; A-212 to V-375; M-213 to V-375; P-214 to V-375; E-215 to V- 375; Q-216 to V-375; M-217 to V-375; K-218 to V-375; F-219 to V-375; Q-220 to V- 375; W-221 to V-375; 1-222 to V-375; R-223 to V-375; V-224 to V-375; K-225 to V- 375; R-226 to V-375; T-227 to V-375; H-228 to V-375; V-229 to V-375; P-230 to V- 375; K-231 to V-375; G-232 to V-375; W-233 to V-375; 1-234 to V-375; K-235 to V- 375; G-236 to V-375; N-237 to V-375; A-238 to V-375; N-239 to V-375; D-240 to V- 375; 1-241 to V-375; G-242 to V-375; M-243 to V-375; D-244 to V-375; Y-245 to V- 375; D-246 to V-375; Y-247 to V-375; A-248 to V-375; L-249 to V-375; L-250 to V- 375; E-251 to V-375; L-252 to V-375; K-253 to V-375; K-254 to V-375; P-255 to V- 375; H-256 to V-375; K-257 to V-375; R-258 to V-375; K-259 to V-375; F-260 to V- 375; M-261 to V-375; K-262 to V-375; 1-263 to V-375; G-264 to V-375; V-265 to V- 375; S-266 to V-375; P-267 to V-375; P-268 to V-375; A-269 to V-375; K-270 to V- 375; Q-271 to V-375; L-272 to V-375; P-273 to V-375; G-274 to V-375; G-275 to V- 375; R-276 to V-375; 1-277 to V-375; H-278 to V-375; F-279 to V-375; S-280 to V-375; G-281 to V-375; Y-282 to V-375; D-283 to V-375; N-284 to V-375; D-285 to V-375; R- 286 to V-375; P-287 to V-375; G-288 to V-375; N-289 to V-375; L-290 to V-375; V-291 to V-375; Y-292 to V-375; R-293 to V-375; F-294 to V-375; C-295 to V-375; D-296 to V-375; V-297 to V-375; K-298 to V-375; D-299 to V-375; E-300 to V-375; T-301 to V- 375; Y-302 to V-375; D-303 to V-375; L-304 to V-375; L-305 to V-375; Y-306 to V- 375; Q-307 to V-375; Q-308 to V-375; C-309 to V-375; D-310 to V-375; S-311 to V- 375; Q-312 to V-375; P-313 to V-375; G-314 to V-375; A-315 to V-375; S-316 to V- 375; G-317 to V-375; S-318 to V-375; G-319 to V-375; V-320 to V-375; Y-321 to V- 375; V-322 to V-375; R-323 to V-375; M-324 to V-375; W-325 to V-375; K-326 to V- 375; R-327 to V-375; Q-328 to V-375; H-329 to V-375; Q-330 to V-375; K-331 to V- 375; W-332 to V-375; E-333 to V-375; R-334 to V-375; K-335 to V-375; 1-336 to V- 375; 1-337 to V-375; G-338 to V-375; M-339 to V-375; 1-340 to V-375; S-341 to V-375; G-342 to V-375; H-343 to V-375; Q-344 to V-375; W-345 to V-375; V-346 to V-375; D-347 to V-375; M-348 to V-375; D-349 to V-375; G-350 to V-375; S-351 to V-375; P- 352 to V-375; Q-353 to V-375; E-354 to V-375; F-355 to V-375; T-356 to V-375; R-357 to V-375; G-358 to V-375; C-359 to V-375; S-360 to V-375; E-361 to V-375; 1-362 to V-375; T-363 to V-375; P-364 to V-375; L-365 to V-375; Q-366 to V-375; Y-367 to V- 375; 1-368 to V-375; P-369 to V-375; D-370 to V-375; of SEQ ID NO:38. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification or loss of one or more biological functions of the protein, other functional activities (e.g., biological activities such as ability to modulate the extracellular matrix, etc.) may still be retained. For example the ability to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the polypeptide shown in Figures 25A-B, up to the glycine residue at position number 6, and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising the amino acid sequence of residues 1-ml of Figures 25A-B, where ml is an integer from 6 to 375 corresponding to the position of the amino acid residue in Figures 25A-B. Moreover, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of C- terminal deletions of the polypeptide of the invention shown as SEQ ID NO:38 include polypeptides comprising the amino acid sequence of residues: M-1 to G-374; M-1 to I- 373; M-1 to S-372; M-1 to 1-371; M-1 to D-370; M-1 to P-369; M-1 to 1-368; M-1 to Y- 367; M-1 to Q-366; M-1 to L-365; M-1 to P-364; M-1 to T-363; M-1 to 1-362; M-1 to E- 361; M-1 to S-360; M-1 to C-359; M-1 to G-358; M-1 to R-357; M-1 to T-356; M-1 to F-355; M-1 to E-354; M-1 to Q-353; M-1 to P-352; M-1 to S-351; M-1 to G-350; M-1 to D-349; M-1 to M-348; M-1 to D-347; M-1 to V-346; M-1 to W-345; M-1 to Q-344; M-1 to H-343; M-1 to G-342; M-1 to S-341; M-1 to 1-340; M-1 to M-339; M-1 to G-338; M- 1 to 1-337; M-1 to 1-336; M-1 to K-335; M-1 to R-334; M-1 to E-333; M-1 to W-332; M- 1 to K-331; M-1 to Q-330; M-1 to H-329; M-1 to Q-328; M-1 to R-327; M-1 to K-326; M-1 to W-325; M-1 to M-324; M-1 to R-323; M-1 to V-322; M-1 to Y-321; M-1 to V- 320; M-1 to G-319; M-1 to S-318; M-1 to G-317; M-1 to S-316; M-1 to A-315; M-1 to G-314; M-1 to P-313; M-1 to Q-312; M-1 to S-311; M-1 to D-310; M-1 to C-309; M-1 to Q-308; M-1 to Q-307; M-1 to Y-306; M-1 to L-305; M-1 to L-304; M-1 to D-303; M- 1 to Y-302; M-1 to T-301; M-1 to E-300; M-1 to D-299; M-1 to K-298; M-1 to V-297; M-1 to D-296; M-1 to C-295; M-1 to F-294; M-1 to R-293; M-1 to Y-292; M-1 to V- 291; M-1 to L-290; M-1 to N-289; M-1 to G-288; M-1 to P-287; M-1 to R-286; M-1 to D-285; M-1 to N-284; M-1 to D-283; M-1 to Y-282; M-1 to G-281; M-1 to S-280; M-1 to F-279; M-1 to H-278; M-1 to 1-277; M-1 to R-276; M-1 to G-275; M-1 to G-274; M-1 to P-273; M-1 to L-272; M-1 to Q-271; M-1 to K-270; M-1 to A-269; M-1 to P-268; M-1 to P-267; M-1 to S-266; M-1 to V-265; M-1 to G-264; M-1 to 1-263; M-1 to K-262; M-1 to M-261; M-1 to F-260; M-1 to K-259; M-1 to R-258; M-1 to K-257; M-1 to H-256; M- 1 to P-255; M-1 to K-254; M-1 to K-253; M-1 to L-252; M-1 to E-251; M-1 to L-250; M-1 to L-249; M-1 to A-248; M-1 to Y-247; M-1 to D-246; M-1 to Y-245; M-1 to D- 244; M-1 to M-243; M-1 to G-242; M-1 to 1-241; M-1 to D-240; M-1 to N-239; M-1 to A-238; M-1 to N-237; M-1 to G-236; M-1 to K-235; M-1 to 1-234; M-1 to W-233; M-1 to G-232; M-1 to K-231; M-1 to P-230; M-1 to V-229; M-1 to H-228; M-1 to T-227; M- 1 to R-226; M-1 to K-225; M-1 to V-224; M-1 to R-223; M-1 to 1-222; M-1 to W-221; M-1 to Q-220; M-1 to F-219; M-1 to K-218; M-1 to M-217; M-1 to Q-216; M-1 to E- 215; M-1 to P-214; M-1 to M-213; M-1 to A-212; M-1 to S-211; M-1 to T-210; M-1 to S-209; M-1 to D-208; M-1 to N-207; M-1 to A-206; M-1 to G-205; M-1 to R-204; M-1 to G-203; M-1 to G-202; M-1 to D-201; M-1 to K-200; M-1 to F-199; M-1 to K-198; M- 1 to P-197; M-1 to K-196; M-1 to L-195; M-1 to F-194; M-1 to G-193; M-1 to V-192; M-1 to R-191; M-1 to L-190; M-1 to K-189; M-1 to Q-188; M-1 to T-187; M-1 to G- 186; M-1 to K-185; M-1 to V-184; M-1 to Y-183; M-1 to T-182; M-1 to K-181; M-1 to G-180; M-1 to D-179; M-1 to H-178; M-1 to 1-177; M-1 to C-176; M-1 to H-175; M-1 to A-174; M-1 to A-173; M-1 to T-172; M-1 to L-171; M-1 to V-170; M-1 to H-169; M-1 to K-168; M-1 toE-167;M-l to A-166; M-1 to V-165; M-1 toL-164;M-l toT-163;M- 1 to G-162; M-1 to T-161; M-1 to C-160; M-1 to G-159; M-1 to T-158; M-1 to S-157; M-1 to L-156; M-1 toK-155;M-l to V-154; M-1 to S-153; M-1 toT-152;M-l to S-151; M-1 to F-150; M-1 to P-149; M-1 to Y-148; M-1 to N-147; M-1 to L-146; M-1 to L-145; M-1 to F-144; M-1 to D-143; M-1 to K-142; M-1 to G-141; M-1 to F-140; M-1 to 1-139; M-1 to S-138; M-1 to F-137; M-1 to R-136; M-1 to S-135; M-1 to D-134; M-1 to Y-133; M-1 to G-132; M-1 to Y-131; M-1 to 1-130; M-1 to Q-129; M-1 to R-128; M-1 to K-127; M-1 to R-126; M-1 to R-125; M-1 to S-124; M-1 to K-123; M-1 to G-122; M-1 to S-121; M-1 to S-120; M-1 to G-l 19; M-1 to S-l 18; M-1 to D-117; M-1 to R-116; M-1 to H- 115; M-1 to Q-l14; M-1 to A-113;M-1 toG-112;M-l to D-l 11; M-1 to GJ 10; MJ to S-109; M-1 to S-108; M-1 to S-107; M-1 to L-106; M-1 to 1-105; M-1 to Y-104; M-1 to 1-103; M-1 to G-102; M-1 to V-101; M-1 to Q-100; M-1 to T-99; M-1 to E-98; M-1 to T- 97; M-1 to R-96; M-1 to S-95; M-1 to G-94; M-1 to N-93; M-1 to A-92; M-1 to Y-91; M-1 to L-90; M-1 to T-89; M-1 to E-88; M-1 to Y-87; M-1 to S-86; M-1 to L-85; M-1 to Y-84; M-1 to Q-83; M-1 to K-82; M-1 to A-81; M-1 to E-80; M-1 to E-79; M-1 to Y-78; M-1 to T-77; M-1 to P-76; M-1 to L-75; M-1 to P-74; M-1 to T-73; M-1 to G-72; M-1 to K-71; M-1 to H-70; M-1 to C-69; M-1 to Q-68; M-1 to P-67; M-1 to G-66; M-1 to C-65; M-1 to S-64; M-1 to S-63; M-1 to S-62; M-1 to V-61; M-1 to E-60; M-1 to L-59; M-1 to K-58; M-1 to A-57; M-1 to E-56; M-1 to A-55; M-1 to G-54; M-1 to F-53; M-1 to D-52; M-1 to P-51; M-1 to K-50; M-1 to A-49; M-1 to L-48; M-1 to N-47; M-1 to L-46; M-1 to T-45; M-1 to S-44; M-1 to Q-43; M-1 to P-42; M-1 to L-41; M-1 to V-40; M-1 to V- 39; M-1 to P-38; M-1 to L-37; M-1 to R-36; M-1 to Y-35; M-1 to A-34; M-1 to P-33; M-

1 to W-32; M-1 to T-31; M-1 to P-30; M-1 to K-29; M-1 to W-28; M-1 to P-27; M-1 to

A-26; M-1 to S-25; M-1 to Y-24; M-1 to P-23; M-1 to S-22; M-1 to V-21; M-1 to Q-20;

M-1 to G-19; M-1 to V-18; M-1 to A-17; M-1 to C-16; M-1 to L-15; M-1 to L-14; M-1 to F-13; M-l to F-12; M-l to L-l 1; M-1 to L-10; M-l to F-9; M-l to L-8; M-l to L-7;

M-1 to G-6; of SEQ ID NO:38. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

In addition, the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO:20 which have been determined from the following related cDNA genes: HFKCF40F (SEQ ID NO.128), HSRDF26R

(SEQ ID NOJ29), HTEBE07R (SEQ ID NOJ30), HFTBP82R (SEQ ID NOJ31),

HAQBJl lR (SEQ ID NO: 132), HAFBB 1 IR (SEQ ID NO: 133), HOEFO85R (SEQ ID

NO: 134), and HUVGY95R (SEQ ID NO: 135).

The gene encoding the disclosed cDNA is believed to reside on chromosome 12. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 12.

This gene is expressed primarily in endothelial cells, fibroblasts, smooth muscle, and osteoblasts, and to a lesser extent in brain, heart, placental tissues, lung, and many other tissues. Moreover, the transcript is present in HUVEC, HUVEC +LPS, smooth muscle, fibroblasts; present in heart, brain, placenta, lung, liver, muscle, kidney, pancreas, spleen, thymus, prostate, testes, ovary, small intestine, colon and weakly in

PBLs.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, disorders of vascularized tissues. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the vascular tissues, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g. vascular, skeletal, developmental, neural, cardiovascular, pulmonary, renal, immune, hematopoietic, reproductive, gastrointestinal, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, seminal, fluid, amniotic fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 38 as residues: Pro-67 to Thr-73, Pro-76 to Gln-83, Asn-93 to Thr-99, His-115 to Arg-128, His-178 to Lys-189, Pro-197 to Ala-212, Val-224 to Trp- 233, Lys-253 to Lys-259, Ser-280 to Asn-289, Asp-296 to Tyr-302, Gln-308 to Ala-315, Arg-327 to Lys-335, Asp-349 to Gly-358. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution in the vascularized endothelial cells indicates that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of diseases of vascularized tissues, such as atherosclerosis, ataxia malabsortion, and hyperlipidemia. These and other factors often result in other cardiovascular disease. Furthermore, translation product of this gene is useful for the treatment of wounds, and may facilitate the wound healing process. Moreover, the protein is useful in the detection, treatment, and/or prevention of a variety of vascular disorders and conditions, which include, but are not limited to miscrovascular disease, vascular leak syndrome, aneurysm, stroke, embolism, thrombosis, coronary artery disease, arteriosclerosis, and/or atherosclerosis. Based upon the tissue distribution of this protein, antagonists directed against this protein is useful in blocking the activity of this protein. Accordingly, preferred are antibodies which specifically bind a portion of the translation product of this gene.

Also provided is a kit for detecting tumors in which expression of this protein occurs. Such a kit comprises in one embodiment an antibody specific for the translation product of this gene bound to a solid support. Also provided is a method of detecting these tumors in an individual which comprises a step of contacting an antibody specific for the translation product of this gene to a bodily fluid from the individual, preferably serum, and ascertaining whether antibody binds to an antigen found in the bodily fluid. Preferably the antibody is bound to a solid support and the bodily fluid is serum. The above embodiments, as well as other treatments and diagnostic tests (kits and methods), are more particularly described elsewhere herein. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:20 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a- b, where a is any integer between 1 to 1685 of SEQ ID NO:20, b is an integer of 15 to 1699, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:20, and where b is greater than or equal to a + 14. FEATURES OF PROTEIN ENCODED BY GENE NO: 11

The translation product of this gene shares sequence homology with Cytotoxic- Regulatory T-Cell Associated Molecule (CRTAM) protein, which is thought to be important in the regulation of celluar physiology, development, differentiation or function of various cell types, including haematopoietic cells and various T-cell progenitors. See for example, PCT publication WO 96/34102 incorporated herein by reference in its entirety. Moreover, the protein product of this gene also shares homology with the thymocyte activation and developmental protein and the class-I MHC-restricted T cell associated molecule (See Genbank Accession Nos. gi|2665790, gb|AAB88491J, gb|AAC80267J, and gi|3930163; all information and references contained within these accessions are hereby incorporated herein by reference). Based on the sequence similarity, the translation product of this gene is expected to share at least some biological activities with T-cell modulatory proteins. Such activities are known in the art, some of which are described elsewhere herein.

Preferred polypeptides of the invention comprise the following amino acid sequence: MASVVLPSGSQCAAAAAAAAPPGLRLRLLLLLFSAAALIPTGDGQNLFTKDVTVI EGEVATISCQVNKSDDSVIQLLNPNRQTIYFRDFRPLKDSRFQLLNFSSSELKVSL TNVSISDEGRYFCQLYTDPPQESYTTITVLVPPRNLMIDIQKDTAVEGEEIEVNCT AMASKPATTIRWFKGNTELKGKSEVEEWSDMYTVTSQLMLKVHKEDDGVPVIC QVEHPAVTGNLQTQRYLEVQYKPQVHIQMTYPLQGLTREGDALELTCEAIGKPQ PVMVTWVRVDDEMPQHAVLSGPNLFINNLNKTDNGTYRCEASNIVGKAHSDY MLYVYDPPTTIPPPTTTTTTTTTTTTTILTIITDSRAGEEGSIRAVDHAVIGGVVAV VVFAMLCLLIILGRYFARHKGTYFTHEAKGADDAADADTAIINAEGGQNNSEEK KEYFI (SEQ ID NO: 136). Polynucleotides encoding these polypeptides are also provided.

The polypeptide of this latter embodiment has been determined to have a transmembrane domain at about amino acid position 379 - 395 of the amino acid sequence referenced in Table XIII for this gene. Moreover, a cytoplasmic tail encompassing amino acids 396 to 442 of this protein has also been determined. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type la membrane proteins.

Preferred polynucleotides comprise the following sequence: ATGGCGAGTGTAGTGC

TGCCGAGCGGATCCCAGTGTGCGGCGGCAGCGGCGGCGGCGGCGCCTCCCG GGCTCCGGCTCCGGCTTCTGCTGTTGCTCTTCTCCGCCGCGGCACTGATCCCC ACAGGTGATGGGCAGAATCTGTTTACGAAAGACGTGACAGTGATCGAGGGA GAGGTTGCGACCATCAGTTGCCAAGTCAATAAGAGTGACGACTCTGTGATTC AGCTACTG AATCCCAACAGGC AG ACCATTTATTTCAGGG ACTTCAGGCCTTT GAAGGACAGCAGGTTTCAGTTGCTGAATTTTTCTAGCAGTGAACTCAAAGTA TCATTGACAAACGTCTCAATTTCTGATGAAGGAAGATACTTTTGCCAGCTCTA TACCGATCCCCCACAGGAAAGTTACACCACCATCACAGTCCTGGTCCCACCA CGTAATCTGATGATCGATATCCAGAAAGACACTGCGGTGGAAGGTGAGGAG ATTGAAGTCAACTGCACTGCTATGGCCAGCAAGCCAGCCACGACTATCAGGT GGTTCAAAGGGAACACAGAGCTAAAAGGCAAATCGGAGGTGGAAGAGTGGT CAGACATGTACACTGTGACCAGTCAGCTGATGCTGAAGGTGCACAAGGAGG ACGATGGGGTCCCAGTGATCTGCCAGGTGGAGCACCCTGCGGTCACTGGAAA CCTGCAGACCCAGCGGTATCTAGAAGTACAGTATAAGCCTCAAGTGCACATT CAGATGACTTATCCTCTACAAGGCTTAACCCGGGAAGGGGACGCGCTTGAGT TAACATGTGAAGCCATCGGGAAGCCCCAGCCTGTGATGGTAACTTGGGTGAG AGTCGATGATGAAATGCCTCAACACGCCGTACTGTCTGGGCCCAACCTGTTC ATCAATAACCTAAACAAAACAGATAATGGTACATACCGCTGTGAAGCTTCAA ACATAGTGGGGAAAGCTCACTCGGATTATATGCTGTATGTATACGATCCCCC CACAACTATCCCTCCTCCCACAACAACCACCACCACCACCACCACCACCACC ACCACCATCCTTACCATCATCACAGATTCCCGAGCAGGTGAAGAAGGCTCGA TCAGGGCAGTGGATCATGCCGTGATCGGTGGCGTCGTGGCGGTGGTGGTGTT CGCCATGCTGTGCTTGCTCATCATTCTGGGGCGCTATTTTGCCAGACATAAAG GTACATACTTCACTCATGAAGCCAAAGGAGCCGATGACGCAGCAGACGCAG ACACAGCTATAATCAATGCAGAAGGAGGACAGAACAACTCCGAAGAAAAGA AAGAGTACTTCATCTAG (SEQ ID NOJ 37). Also preferred are the polypeptides encoded by these polynucleotides.

Figures 28A-B shows the nucleotide (SEQ ID NO:21) and deduced amino acid sequence (SEQ ID NO:39) of the present invention. Predicted amino acids from about 1 to about 44 constitute the predicted signal peptide (amino acid residues from about 1 to about 44 in SEQ ID NO:39) and are represented by the underlined amino acid regions. Figure 29 shows the regions of similarity between the amino acid sequences of the present invention SEQ ID NO:39, the human poliovirus receptor protein (gi| 1524088) (SEQ ID NO: 138), the human class-I MHC-restricted T cell associated molecule (WO9634102) (SEQ ID NO: 144), and the Gallus gallus thymocyte activation and developmental protein (gb|AAB88491J) (SEQ ID NO: 145). Figure 30 shows an analysis of the amino acid sequence of SEQ ID NO:39.

The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the polypeptide having the amino acid sequence shown in Figures 28A-B (SEQ ID NO:39), which was determined by sequencing a cloned cDNA. The nucleotide sequence shown in Figures 28A-B (SEQ ID NO:21) was obtained by sequencing a cloned cDNA (HOUDJ81), which was deposited on Nov. 17, 1998 at the American Type Culture Collection, and given Accession Number 203484. The deposited gene is inserted in the pSport plasmid (Life Technologies, Rockville, MD) using the Sall/Notl restriction endonuclease cleavage sites.

The present invention is further directed to fragments of the isolated nucleic acid molecules described herein. By a fragment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ ID NO:21 is intended DNA fragments at least about 15nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein- Of course, larger fragments 50-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in SEQ ID NO:2l. By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NO:2L In this context "about" includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Representative examples of polynucleotide fragments of the invention include, for example, fragments that comprise, or altematively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 1 1 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, and from about 501 to about 550, and from about 551 to about 600, from about 601 to about 650, from about 651 to about 700, from about 701 to about 750, from about 751 to about 800, from about 801 to about 850, from about 851 to about 900, from about 901 to about 950, from about 951 to about 1000, from about 1001 to about 1050, from about 1051 to about 1100, from about 1101 to about 1150, from about 1151 to about 1200, from about 1201 to about 1250, from about 1251 to about 1300, from about 1301 to about 1350, from about 1351 to about 1400, from about 1401 to about 1450, from about 1451 to about 1500, from about 1501 to about 1520 of SEQ ID NO:21, or the complementary strand thereto, or the cDNA contained in the deposited gene. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. In additional embodiments, the polynucleotides of the invention encode functional attributes of the corresponding protein.

Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions"), turn and turn-forming regions ("turn- regions"), coil and coil-forming regions ("coil-regions"), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions. The data representing the structural or functional attributes of the protein set forth in Figure 30 and/or Table X, as described above, was generated using the various modules and algorithms of the

DNA*STAR set on default parameters. In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table X can be used to determine regions of the protein which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.

Certain preferred regions in these regards are set out in Figure 30, but may, as shown in Table X, be represented or identified by using tabular representations of the data presented in Figure 30. The DNA*STAR computer algorithm used to generate

Figure 30 (set on the original default parameters) was used to present the data in Figure 30 in a tabular format (See Table X). The tabular format of the data in Figure 30 is used to easily determine specific boundaries of a preferred region. The above-mentioned preferred regions set out in Figure 30 and in Table X include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figures 28A-B. As set out in Figure 30 and in Table X, such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions and Hopp-Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions. Jameson-Wolf regions of high antigenic index and Emini surface-forming regions. Even if deletion of one or more amino acids from the N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, etc.) may still be retained. For example, the ability of shortened muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence shown in Figures 28A-B, up to the threonine residue at position number 359 and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising the amino acid sequence of residues n 1-364 of Figures 28A-B, where nl is an integer from 2 to 359 corresponding to the position of the amino acid residue in Figures 28A-B (which is identical to the sequence shown as SEQ ID NO:39). N-terminal deletions of the polypeptide of the invention shown as SEQ ID NO:39 include polypeptides comprising the amino acid sequence of residues: A-2 to R-364; S-3 to R- 364; V-4 to R-364; V-5 to R-364; L-6 to R-364; P-7 to R-364; S-8 to R-364; G-9 to R- 364; S-10 to R-364; Q-l 1 to R-364; C-12 to R-364; A-13 to R-364; A-14 to R-364; A-15 to R-364; A- 16 to R-364; A- 17 to R-364; A- 18 to R-364; A- 19 to R-364; A-20 to R-364; P-21 to R-364; P-22 to R-364; G-23 to R-364; L-24 to R-364; R-25 to R-364; L-26 to R- 364; R-27 to R-364; L-28 to R-364; L-29 to R-364; L-30 to R-364; L-31 to R-364; L-32 to R-364; F-33 to R-364; S-34 to R-364; A-35 to R-364; A-36 to R-364; A-37 to R-364; L-38 to R-364; 1-39 to R-364; P-40 to R-364; T-41 to R-364; G-42 to R-364; D-43 to R- 364; G-44 to R-364; Q-45 to R-364; N-46 to R-364; L-47 to R-364; F-48 to R-364; T-49 to R-364; K-50 to R-364; D-51 to R-364; V-52 to R-364; T-53 to R-364; V-54 to R-364; 1-55 to R-364; E-56 to R-364; G-57 to R-364; E-58 to R-364; V-59 to R-364; A-60 to R- 364; T-61 to R-364; 1-62 to R-364; S-63 to R-364; C-64 to R-364; Q-65 to R-364; V-66 to R-364; N-67 to R-364; K-68 to R-364; S-69 to R-364; D-70 to R-364; D-71 to R-364; S-72 to R-364; V-73 to R-364; 1-74 to R-364; Q-75 to R-364; L-76 to R-364; L-77 to R- 364; N-78 to R-364; P-79 to R-364; N-80 to R-364; R-81 to R-364; Q-82 to R-364; T-83 to R-364; 1-84 to R-364; Y-85 to R-364; F-86 to R-364; R-87 to R-364; D-88 to R-364; F-89 to R-364; R-90 to R-364; P-91 to R-364; L-92 to R-364; K-93 to R-364; D-94 to R- 364; S-95 to R-364; R-96 to R-364; F-97 to R-364; Q-98 to R-364; L-99 to R-364; L-100 to R-364; N-101 to R-364; F-102 to R-364; S-103 to R-364; S-104 to R-364; S-105 to R- 364; E-106 to R-364; L-107 to R-364; K-108 to R-364; V-109 to R-364; S-l 10 to R-364; L-l 11 to R-364; T-l 12 to R-364; N-113 to R-364; V-l 14 to R-364; S-l 15 to R-364; I- 116 to R-364; S-l 17 to R-364; D-l 18 to R-364; E-l 19 to R-364; G-120 to R-364; R-121 to R-364; Y-122 to R-364; F-123 to R-364; C-124 to R-364; Q-125 to R-364; L-126 to R-364; Y-127 to R-364; T-128 to R-364; D-129 to R-364; P-130 to R-364; P-131 to R- 364; Q-132 to R-364; E-133 to R-364; S-134 to R-364; Y-135 to R-364; T-136 to R-364; T-137 to R-364; 1-138 to R-364; T-139 to R-364; V-140 to R-364; L-141 to R-364; V- 142 to R-364; P-143 to R-364; P-144 to R-364; R-145 to R-364; N-146 to R-364; L-147 to R-364; M-148 to R-364; 1-149 to R-364; D-150 to R-364; 1-151 to R-364; Q-152 to R- 364; K-153 to R-364; D-154 to R-364; T-155 to R-364; A-156 to R-364; V-157 to R- 364; E-158 to R-364; G-159 to R-364; E-160 to R-364; E-161 to R-364; 1-162 to R-364; E-163 to R-364; V-164 to R-364; N-165 to R-364; C-166 to R-364; T-167 to R-364; A- 168 to R-364; M-169 to R-364; A-170 to R-364; S-171 to R-364; K-172 to R-364; P-173 to R-364; A-174 to R-364; T-175 to R-364; T-176 to R-364; 1-177 to R-364; R-178 to R- 364; W-l 79 to R-364; F-180 to R-364; K-181 to R-364; G-l 82 to R-364; N-183 to R- 364; T-l 84 to R-364; E-185 to R-364; L-l 86 to R-364; K-187 to R-364; G-188 to R-364; K-189 to R-364; S-190 to R-364; E-191 to R-364; V-192 to R-364; E-l 93 to R-364; E- 194 to R-364; W-l 95 to R-364; S-l 96 to R-364; D-l 97 to R-364; M-198 to R-364; Y- 199 to R-364; T-200 to R-364; V-201 to R-364; T-202 to R-364; S-203 to R-364; Q-204 to R-364; L-205 to R-364; M-206 to R-364; L-207 to R-364; K-208 to R-364; V-209 to R-364; H-210 to R-364; K-211 to R-364; E-212 to R-364; D-213 to R-364; D-214 to R- 364; G-215 to R-364; V-216 to R-364; P-217 to R-364; V-218 to R-364; 1-219 to R-364; C-220 to R-364; Q-221 to R-364; V-222 to R-364; E-223 to R-364; H-224 to R-364; P- 225 to R-364; A-226 to R-364; V-227 to R-364; T-228 to R-364; G-229 to R-364; N-230 to R-364; L-231 to R-364; Q-232 to R-364; T-233 to R-364; Q-234 to R-364; R-235 to R-364; Y-236 to R-364; L-237 to R-364; E-238 to R-364; V-239 to R-364; Q-240 to R- 364; Y-241 to R-364; K-242 to R-364; P-243 to R-364; Q-244 to R-364; V-245 to R- 364; H-246 to R-364; 1-247 to R-364; Q-248 to R-364; M-249 to R-364; T-250 to R-364; Y-251 to R-364; P-252 to R-364; L-253 to R-364; Q-254 to R-364; G-255 to R-364; L- 256 to R-364; T-257 to R-364; R-258 to R-364; E-259 to R-364; G-260 to R-364; D-261 to R-364; A-262 to R-364; L-263 to R-364; E-264 to R-364; L-265 to R-364; T-266 to R-364; C-267 to R-364; E-268 to R-364; A-269 to R-364; 1-270 to R-364; G-271 to R- 364; K-272 to R-364; P-273 to R-364; Q-274 to R-364; P-275 to R-364; V-276 to R-364; M-277 to R-364; V-278 to R-364; T-279 to R-364; W-280 to R-364; V-281 to R-364; R- 282 to R-364; V-283 to R-364; D-284 to R-364; D-285 to R-364; E-286 to R-364; M- 287 to R-364; P-288 to R-364; Q-289 to R-364; H-290 to R-364; A-291 to R-364; V-292 to R-364; L-293 to R-364; S-294 to R-364; G-295 to R-364; P-296 to R-364; N-297 to R-364; L-298 to R-364; F-299 to R-364; 1-300 to R-364; N-301 to R-364; N-302 to R- 364; L-303 to R-364; N-304 to R-364; K-305 to R-364; T-306 to R-364; D-307 to R- 364; N-308 to R-364; G-309 to R-364; T-310 to R-364; Y-311 to R-364; R-312 to R- 364; C-313 to R-364; E-314 to R-364; A-315 to R-364; S-316 to R-364; N-317 to R-364; 1-318 to R-364; V-319 to R-364; G-320 to R-364; K-321 to R-364; A-322 to R-364; H- 323 to R-364; S-324 to R-364; D-325 to R-364; Y-326 to R-364; M-327 to R-364; L-328 to R-364; Y-329 to R-364; V-330 to R-364; Y-331 to R-364; D-332 to R-364; P-333 to R-364; P-334 to R-364; T-335 to R-364; T-336 to R-364; 1-337 to R-364; P-338 to R- 364; P-339 to R-364; P-340 to R-364; T-341 to R-364; T-342 to R-364; T-343 to R-364; T-344 to R-364; T-345 to R-364; T-346 to R-364; T-347 to R-364; T-348 to R-364; T- 349 to R-364; T-350 to R-364; T-351 to R-364; T-352 to R-364; T-353 to R-364; 1-354 to R-364; L-355 to R-364; T-356 to R-364; 1-357 to R-364; 1-358 to R-364; T-359 to R- 364; of SEQ ID NO:39. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification or loss of one or more biological functions of the protein, other functional activities (e.g., biological activities such as ability to modulate the extracellular matrix, etc.) may still be retained. For example the ability to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response. Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the polypeptide shown in Figures 28A-B, up to the leucine residue at position number 6, and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising the amino acid sequence of residues 1-ml of Figures 28A-B, where ml is an integer from 6 to 364 con-esponding to the position of the amino acid residue in Figures 28 A-B. Moreover, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of C- terminal deletions of the polypeptide of the invention shown as SEQ ID NO:39 include polypeptides comprising the amino acid sequence of residues: M-1 to A-363; M-1 to R- 362; M-1 to S-361; M-1 to D-360; M-1 to T-359; M-1 to 1-358; M-1 to 1-357; M-1 to T- 356; M-1 to L-355; M-1 to 1-354; M-1 to T-353; M-1 to T-352; M-1 to T-351; M-1 to T- 350; M-1 to T-349; M-1 to T-348; M-1 to T-347; M-1 to T-346; M-1 to T-345; M-1 to T- 344; M-1 to T-343; M-1 to T-342; M-1 to T-341; M-1 to P-340; M-1 to P-339; M-1 to P- 338; M-1 to 1-337; M-1 to T-336; M-1 to T-335; M-1 to P-334; M-1 to P-333; M-1 to D- 332; M-1 to Y-331; M-1 to V-330; M-1 to Y-329; M-1 to L-328; M-1 to M-327; M-1 to Y-326; M-1 to D-325; M-1 to S-324; M-1 to H-323; M-1 to A-322; M-1 to K-321; M-1 to G-320; M-1 to V-319; M-1 to 1-318; M-1 to N-317; M-1 to S-316; M-1 to A-315; M-1 to E-314; M-1 to C-313; M-1 to R-312; M-1 to Y-311; M-1 to T-310; M-1 to G-309; M- 1 to N-308; M-1 to D-307; M-1 to T-306; M-1 to K-305; M-1 to N-304; M-1 to L-303; M-1 to N-302; M-1 to N-301 ; M-1 to 1-300; M-1 to F-299; M-1 to L-298; M-1 to N-297; M-1 to P-296; M-1 to G-295; M-1 to S-294; M-1 to L-293; M-1 to V-292; M-1 to A-291 ; M-1 to H-290; M-1 to Q-289; M-1 to P-288; M-1 to M-287; M-1 to E-286; M-1 to D- 285; M-1 to D-284; M-1 to V-283; M-1 to R-282; M-1 to V-281; M-1 to W-280; M-1 to T-279; M-1 to V-278; M-1 to M-277; M-1 to V-276; M-1 to P-275; M-1 to Q-274; M-1 to P-273; M-1 to K-272; M-1 to G-271; M-1 to 1-270; M-1 to A-269; M-1 to E-268; M-1 to C-267; M-1 to T-266; M-1 to L-265; M-1 to E-264; M-1 to L-263; M-1 to A-262; M-1 to D-261; M-1 to G-260; M-1 to E-259; M-1 to R-258; M-1 to T-257; M-1 to L-256; M- 1 to G-255; M-1 to Q-254; M-1 to L-253; M-1 to P-252; M-1 to Y-251; M-1 to T-250; M-1 to M-249; M-1 to Q-248; M-1 to 1-247; M-1 to H-246; M-1 to V-245; M-1 to Q- 244; M-1 to P-243; M-1 to K-242; M-1 to Y-241; M-1 to Q-240; M-1 to V-239; M-1 to E-238; M-1 to L-237; M-1 to Y-236; M-1 to R-235; M-1 to Q-234; M-1 to T-233; M-1 to Q-232; M-1 to L-231; M-1 to N-230; M-1 to G-229; M-1 to T-228; M-1 to V-227; M- 1 to A-226; M-1 to P-225; M-1 to H-224; M-1 to E-223; M-1 to V-222; M-1 to Q-221; M-1 to C-220; M-1 to 1-219; M-1 to V-218; M-1 to P-217; M-1 to V-216; M-1 to G-215; M-1 toD-214;M-ltoD-213;M-l to E-212; M-1 to K-211; M-1 to H-210; M-1 to V- 209; M-1 to K-208; M-1 to L-207; M-1 to M-206; M-1 to L-205; M-1 to Q-204; M-1 to S-203; M-1 to T-202; M-1 to V-201; M-1 to T-200; M-1 to Y-199; M-1 to M-198; M-1 to D-197; M-1 to S-196; M-1 to W-195; M-1 to E-194; M-1 to E-193; M-1 to V-192; M- 1 to E-191; M-1 to S-190; M-1 to K-189; M-1 to G-188; M-1 to K-187; M-1 to L-186; M-1 to E-185; M-1 to T-184; M-1 to N-183; M-1 to G-182; M-1 to K-181; M-1 to F- 180; M-1 to W-179; M-1 to R-178; M-1 to 1-177; M-1 to T-176; M-1 to T-175; M-1 to A-174; M-1 to P-173; M-1 to K-172; M-1 to S-171; M-1 to A-170; M-1 to M-169; M-1 to A-168; M-1 to T-167; M-1 to C-166; M-1 to N-165; M-1 to V-164; M-1 to E-163; M- 1 to 1-162; M-1 to E-161; M-1 to E-160; M-1 to G-159; M-1 to E-158; M-1 to V-157; M- 1 to A-156; M-1 to T-155; M-1 to D-154; M-1 to K-153; M-1 to Q-152; M-1 to 1-151; M-1 to D-150; M-1 to 1-149; M-1 to M-148; M-1 to L-147; M-1 to N-146; M-1 to R- 145; M-1 to P-144; M-1 to P-143; M-1 to V-142; M-1 to L-141; M-1 to V-140; M-1 to T-139; M-1 to 1-138; M-1 to T-137; M-1 to T-136; M-1 to Y-135; M-1 to S-134; M-1 to E-133; M-1 to Q-132; M-1 to P-131; M-1 to P-130; M-1 to D-129; M-1 to T-128; M-1 to Y-127; M-1 to L-126; M-1 to Q-125; M-1 to C-124; M-1 to F-123; M-1 to Y-122; M-1 to R-121; M-1 to G-120; M-1 to E-l 19; M-1 to D-l 18; M-1 to S-l 17; M-1 to 1-116; M-1 to S-l 15; M-1 to V-l 14; M-1 to N-113; M-1 to T-l 12; M-1 to L-l 11; M-1 to S-l 10; M-1 to V-109; M-1 to K-108; M-1 to L-107; M-1 to E-106; M-1 to S-105; M-1 to S-104; M-1 to S-103; M-1 to F-102; M-1 to N-101; M-1 to L-100; M-1 to L-99; M-1 to Q-98; M-1 to F-97; M-1 to R-96; M-1 to S-95; M-1 to D-94; MJ to K-93; M-l to L-92; MJ to P-91 ; M-1 to R-90; M-1 to F-89; M-1 to D-88; M-1 to R-87; M-1 to F-86; M-1 to Y-85; M-1 to 1-84; M-1 to T-83; M-1 to Q-82; M-1 to R-81; M-1 to N-80; M-1 to P-79; M-1 to N-78; M-1 to L-77; M-1 to L-76; M-1 to Q-75; M-1 to 1-74; M-1 to V-73; M-1 to S-72; M-1 to D-71; M-1 to D-70; M-1 to S-69; M-1 to K-68; M-1 to N-67; M-1 to V-66; M-1 to Q-65; M-1 to C-64; M-1 to S-63; M-1 to 1-62; M-1 to T-61; M-1 to A-60; M-1 to V-59; M-1 to E-58; M-1 to G-57; M-1 to E-56; M-1 to 1-55; M-1 to V-54; M-1 to T-53; M-1 to V-52; M-1 to D-51; M-1 to K-50; M-1 to T-49; M-1 to F-48; M-1 to L-47; M-1 to N-46; M-1 to Q-45; M-1 to G-44; M-1 to D-43; M-1 to G-42; M-1 to T-41; M-1 to P-40; M-1 to I- 39; M-1 to L-38; M-1 to A-37; M-1 to A-36; M-1 to A-35; M-1 to S-34; M-1 to F-33; M- 1 to L-32; M-1 to L-31; M-1 to L-30; M-1 to L-29; M-1 to L-28; M-1 to R-27; M-1 to L- 26; M-1 to R-25; M-1 to L-24; M-1 to G-23; M-1 to P-22; M-1 to P-21; M-1 to A-20; M- 1 to A-19; M-1 to A-18; M-1 to A-17; M-1 to A-16; M-1 to A-15; M-1 to A-14; M-1 to A-13; M-1 to C-12; M-1 to Q-11 ; M-1 to S-10; M-1 to G-9; M-1 to S-8; M-1 to P-7; M-1 to L-6; of SEQ ID NO:39. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

In addition, the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO:21 which have been determined from the following related cDNA genes: HSQFJ92R (SEQ ID NO: 139), HFLAB18F (SEQ ID NO: 140), HAQBH82R (SEQ ID NO: 141), HLHTMIOR (SEQ ID NO: 142), and HLHAL65R (SEQ ID NO: 143). This gene is expressed pnmaπly in immune system related tissues such as ulcerative colitis, rejected kidney tissues, and to a lesser extent in thymus and bone marrow. Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tιssue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune and hematopoietic diseases and/or disordeis, particulaily ulcerative colitis and rejected organs. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tιssue(s) or cell type(s) Foi a number of disorders of the above tissues or cells, particularly of the immune system, expiession of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g transplanted kidney, immune, hematopoeitic, renal, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder

Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 39 as residues: Gly-42 to Phe-48, Val-66 to Asp-71, Asn-78 to Thr-83, Asp-88 to Arg-96, Tyr- 127 to Tyr-135, Lys-181 to Trp- 195, Hιs-210 to Gly-215, Leu-303 to Thr-310, Thr-341 to Thr-350. Polynucleotides encoding said polypeptides are also provided.

The tissue distπbution pπmaπly in immune cells and tissues, combined with the homology to the CRT AM, thymocyte activation and developmental protein, the class-I MHC-restπcted T cell associated molecule protein, and the pohvirus receptor, indicates that the protein products of this gene are useful for the regulation of celluar physiology, development, differentiation or function of vaπous cell types, including haematopoietic cells and particularly T-cell progenitors. Representative uses are descnbed in the "Immune Activity" and "infectious disease" sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. The proteins can be used to develop products for the diagnosis and treatment of conditions associated with abnormal physiology or development, including abnormal proliferation, e.g. cancers, or degenerative conditions. The physiology or development of a cell can be modulated by contacting the cell with an agonist or antagonist (i.e. an anti- CRTAM-like peptide antibody). Further the CRTAM- like polypeptides of the present invention include treatment of ulcerative colitis, organ rejection and other immune system related disorders. Agonists or antagonists may treat or prevent such disorders as ulcerative colitis and rejected organs, such as kidney. Based upon the tissue distribution of this protein, antagonists directed against this protein is useful in blocking the activity of this protein. Accordingly, preferred are antibodies which specifically bind a portion of the translation product of this gene.

Also provided is a kit for detecting tumors in which expression of this protein occurs. Such a kit comprises in one embodiment an antibody specific for the translation product of this gene bound to a solid support. Also provided is a method of detecting these tumors in an individual which comprises a step of contacting an antibody specific for the translation product of this gene to a bodily fluid from the individual, preferably serum, and ascertaining whether antibody binds to an antigen found in the bodily fluid. Preferably the antibody is bound to a solid support and the bodily fluid is serum. The above embodiments, as well as other treatments and diagnostic tests (kits and methods), are more particularly described elsewhere herein. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues. Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:21 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a- b, where a is any integer between 1 to 1506 of SEQ ID NO:21, b is an integer of 15 to 1520, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:21, and where b is greater than or equal to a + 14.

FEATURES OFPROTEINENCODEDBY GENENO: 12

Figure 31 shows the nucleotide (SEQ ID NO:22) and deduced amino acid sequence (SEQ ID NO:40) of the present invention. Predicted amino acids from about 1 to about 23 constitute the predicted signal peptide (amino acid residues from about 1 to about 23 in SEQ ID NO:40) and are represented by the underlined amino acid regions. Figure 32 shows the regions of similarity between the amino acid sequences of the present invention SEQ ID NO:40 and the human FAP protein (gi| 1890647) (SEQ ID NO: 146).

Figure 33 shows an analysis of the amino acid sequence of SEQ ID NO:40. Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown. The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the polypeptide having the amino acid sequence shown in Figure 31 (SEQ ID NO:40), which was determined by sequencing a cloned cDNA. The nucleotide sequence shown in Figure 31 (SEQ ID NO:22) was obtained by sequencing a cloned cDNA (HPWCM76), which was deposited on Nov. 17, 1998 at the American Type Culture Collection, and given Accession Number 203484. The deposited gene is inserted in the pSport plasmid (Life Technologies, Rockville, MD) using the Sall/Notl restriction endonuclease cleavage sites.

The present invention is further directed to fragments of the isolated nucleic acid molecules described herein. By a fragment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ ID NO:22 is intended DNA fragments at least about 15nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments 50-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in SEQ ID NO:22. By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NO:22. In this context "about" includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Representative examples of polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, and from about 501 to about 550, and from about 551 to about 600, from about 601 to about 650, from about 651 to about 700, from about 701 to about 750, from about 751 to about 800, from about 801 to about 807 of SEQ ID NO:22, or the complementary strand thereto, or the cDNA contained in the deposited gene. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. In additional embodiments, the polynucleotides of the invention encode functional attributes of the corresponding protein.

Prefen-ed embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions"), turn and turn-forming regions ("turn- regions"), coil and coil-forming regions ("coil-regions"), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions. The data representing the structural or functional attributes of the protein set forth in Figure 33 and/or Table XI, as described above, was generated using the various modules and algorithms of the DNA*STAR set on default parameters. In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table XI can be used to determine regions of the protein which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.

Certain preferred regions in these regards are set out in Figure 33, but may, as shown in Table XI, be represented or identified by using tabular representations of the data presented in Figure 33. The DNA*STAR computer algorithm used to generate Figure 33 (set on the original default parameters) was used to present the data in Figure 33 in a tabular format (See Table XI). The tabular format of the data in Figure 33 is used to easily determine specific boundaries of a preferred region. The above-mentioned preferred regions set out in Figure 33 and in Table XI include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figure 31. As set out in Figure 33 and in Table XI, such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou- Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions and Hopp-Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenic index and Emini surface-forming regions. Even if deletion of one or more amino acids from the N- terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, etc.) may still be retained. For example, the ability of shortened muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response. Accordingly, the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence shown in Figure 31, up to the arginine residue at position number 61 and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising the amino acid sequence of residues nl-66 of Figure 31, where nl is an integer from 2 to 61 corresponding to the position of the amino acid residue in Figure 31 (which is identical to the sequence shown as SEQ ID NO:40). N-terminal deletions of the polypeptide of the invention shown as SEQ ID NO:40 include polypeptides comprising the amino acid sequence of residues: S-2 to N-66; S-3 to N-66; S-4 to N-66; S-5 to N-66; L-6 to N-66; K-7 to N-66; H-8 to N-66; L-9 to N-66; L-10 to N-66; C-11 to N-66; M-12 to N-66; A-13 to N-66; L-14 to N-66; S-15 to N-66; W-16 to N-66; F-17 to N-66; S-18 to N-66; S-l 9 to N-66; F-20 to N-66; 1-21 to N-66; S-22 to N-66; G-23 to N-66; E-24 to N-66; T-25 to N-66; S-26 to N-66; F-27 to N-66; S-28 to N-66; L-29 to N-66; L-30 to N- 66; N-31 to N-66; S-32 to N-66; F-33 to N-66; F-34 to N-66; L-35 to N-66; P-36 to N- 66; Y-37 to N-66; P-38 to N-66; S-39 to N-66; S-40 to N-66; R-41 to N-66; C-42 to N- 66; C-43 to N-66; C-44 to N-66; F-45 to N-66; S-46 to N-66; V-47 to N-66; Q-48 to N- 66; C-49 to N-66; S-50 to N-66; 1-51 to N-66; L-52 to N-66; D-53 to N-66; P-54 to N- 66; F-55 to N-66; S-56 to N-66; C-57 to N-66; N-58 to N-66; S-59 to N-66; M-60 to N- 66; R-61 to N-66; of SEQ ID NO:40. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the polypeptide shown in Figure 31, up to the leucine residue at position number 6, and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising the amino acid sequence of residues 1-ml of Figure 31, where ml is an integer from 6 to 66 corresponding to the position of the amino acid residue in Figure 31. Moreover, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of C-terminal deletions of the polypeptide of the invention shown as SEQ ID NO:40 include polypeptides comprising the amino acid sequence of residues: M-1 to E-65; M-1 to W- 64; M-1 to P-63; M-1 to F-62; M-1 to R-61; M-1 to M-60; M-1 to S-59; M-1 to N-58; M-1 to C-57; M-1 to S-56; M-1 to F-55; M-1 to P-54; M-1 to D-53; M-1 to L-52; M-1 to 1-51; M-1 to S-50: M-1 to C-49; M-1 to Q-48; M-1 to V-47; M-1 to S-46; M-1 to F-45; M-1 to C-44; M-1 to C-43; M-1 to C-42; M-1 to R-41 ; M-1 to S-40; M-1 to S-39; M-1 to P-38; M-1 to Y-37; M-1 to P-36; M-1 to L-35; M-1 to F-34; M-1 to F-33; M-1 to S-32; M-1 to N-31; M-1 to L-30; M-1 to L-29; M-1 to S-28; M-1 to F-27; M-1 to S-26; M-1 to T-25; M-1 to E-24; M-1 to G-23; M-1 to S-22; M-1 to 1-21; M-1 to F-20; M-1 to S-19; M-1 to S-18; M-1 to F-17; M-1 to W-16; M-1 to S-15; M-1 to L-14; M-1 to A-13; M-1 to M-12; M-l to C-11; M-1 to L-10; M-l to L-9; M-1 to H-8; M-1 to K-7; MJ to L-6; of SEQ ID NO:40. Polypeptides encoded by these polynucleotides are also encompassed by the invention. In addition, the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO:22 which have been determined from the following related cDNA genes: HPWCM76R (SEQ ID NO: 147).

This gene is expressed primarily in prostate BPH (benign prostatic hyperplasia) tissue. Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, inflammation of the prostate, or related tissues. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tιssue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the prostate, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g. prostate, cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disordei . The tissue distπbution in prostate BPH tissue indicates that polynucleotides and polypeptides corresponding to this gene are useful for the treatment of inflammatory conditions which result in an enlargement of the prostate, or related tissues. Polynucleotides and polypeptides corresponding to this gene are useful for the treatment and diagnosis of conditions concerning proper testicular function (e.g. endocπne function, sperm maturation), as well as cancer. Therefore, this gene product is useful in the treatment of male infertility and/or impotence. This gene product is also useful assays designed to identify binding agents, as such agents (antagonists) are useful as male contraceptive agents. Similarly, the protein is believed to be useful in the treatment and/or diagnosis of testicular cancer. The testes are also a site of active gene expression of transcnpts that is expressed, particularly at low levels, m other tissues of the body.

Therefore, this gene product is expressed in other specific tissues or organs where it may play related functional roles in other processes, such as hematopoiesis, inflammation, bone formation, and kidney function, to name a few possible target indications. Based upon the tissue distπbution of this protein, antagonists directed against this protein is useful in blocking the activity of this protein. Accordingly, preferred are antibodies which specifically bind a portion of the translation product of this gene. Also provided is a kit for detecting tumors in which expression of this protein occurs. Such a kit comprises in one embodiment an antibody specific for the translation product of this gene bound to a solid support Also provided is a method of detecting these tumors in an individual which composes a step of contacting an antibody specific for the translation product of this gene to a bodily fluid from the individual, preferably serum, and ascertaining whether antibody binds to an antigen found in the bodily fluid Preferably the antibody is bound to a solid support and the bodily fluid is serum The above embodiments, as well as othei treatments and diagnostic tests (kits and methods), are moie particularly descπbed elsewhere herein The homology to the FAP protein indicates that the protein product of this gene is useful in treating, detecting, and/or preventing iron metabolism disorders, particularly those resulting in high oxidative states, tissue damage, athersclerosis, free radical damage, vascular disordeis, iron binding protein dysfunction, nitnc oxide synthase dysfunction or aberration, vasodilation disorders, and tissue edema Based on the sequence similarity, the translation product of this gene is expected to share at least some biological activities with iron metabolism modulatory proteins Such activities are known in the art, some of which are descπbed elsewhere herein Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutπtional supplement Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO 22 and may have been publicly available pπor to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a- b, where a is any integer between 1 to 793 of SEQ ID NO:22, b is an integer of 15 to 807, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:22, and where b is greater than or equal to a + 14.

Table I

Res Position I I III V V VI II VIII IX X XI . XII XIII XIV

Met 1 B -0.10 0.44 -0.40 0.48

Val 2 B 0.08 0.01 0.15 0.63

Ala 3 B 0.47 0.01 0.40 0.76

Gin 4 B 0.51 -0.01 1.40 1.33

Asp 5 T Z 0.23 -0.20 . F 2.20 1.77

Pro 6 T T 0.02 -0.27 * * ] F 2.50 0.94

Gin 7 T T 0.88 -0.09 * * ] F 2.25 0.45

Gly 8 T T 0.66 -0.09 * F 2.00 0.47

Cys 9 \ B -0.01 0.60 * -0.10 0.25

Leu 10 \ B -0.82 0.74 * -0.35 0.08

Gin 11 A B -0.91 1.03 * -0.60 0.06

Leu 12 <\ B -0.91 0.99 * -0.60 0.16

Cys 13 \ B -1.42 0.41 * -0.60 0.34

Leu 14 A B -1.34 0.37 * -0.30 0.14

Ser 15 A B -0.53 0.47 * * -0.60 0.18

Glu 16 A B -0.88 0.19 * -0.30 0.53

Val 17 B T -0.88 0.04 » * 0.10 0.63

Ala 18 A T -0.10 0.04 * 0.10 0.39

Asn 19 T T 0.71 -0.34 * 1.31 0.44

Gly 20 T T 0.80 0.06 * * F 1.07 0.96

Leu 21 . C -0.06 -0.16 * * F 1.63 1.47

Arg 22 . C 0.50 -0.01 * * F 1.69 0.68

Asn 23 T C 0.49 -0.03 * F 2.10 0.92

Pro 24 B T -0.37 0.16 * F 1.24 1.10

Val 25 B T -0.06 0.11 • * 0.73 0.42

Ser 26 B T 0.17 0.61 * * 0.22 0.35

Met 27 B B -0.29 0.71 * -0.39 0.23

Val 28 B B -0.29 0.71 -0.60 0.31

His 29 B B -0.42 0.07 0.00 0.38

Ala 30 A B 0.12 0.11 0.50 0.38

Gly 31 T T . 0.39 -0.01 * F 2.15 0.74

Asp 32 T ^" T 1.10 -0.16 * * F 2.45 0.74

Gly 33 T C 1.26 -0.66 * * F 3.00 1.44

Thr 34 T C 0.59 -0.37 * * F 2.40 1.26

His 35 B B 0.32 -0.01 * * 1.20 0.65

Arg 36 B B 0.08 0.63 * 0.00 0.49

Phe 37 B B 0.08 0.70 * -0.30 0.34

Phe 38 B B 0.42 0.21 * -0.30 0.44 Val 39 \ . B -0.12 0.11 . * -0.30 0.39

Ala 40 \ . B -0.43 0.76 . * -0.60 0.33

Glu 41 \ . B -1.40 0.40 . * -0.60 0.38

Gin 42 \ . B -1.56 0.26 . . -0.30 0.38

Val 43 \ . B -1.14 0.26 . . -0.30 0.28

Gly 44 B B -1.14 0.67 . . -0.60 0.17

Val 45 B B -0.80 1.31 . . -0.60 0.07

Val 46 B B -1.61 1.67 . . -0.60 0.15

Trp 47 B B -1.82 1.71 . . -0.60 0.13

Val 48 B B -0.97 1.71 . . -0.60 0.27

Tyr 49 B -0.97 1.07 . . -0.16 0.60

Leu 50 B T -0.41 0.86 * . 0.28 0.56

Pro 51 T T 0.56 0.33 * * F 1.52 1.01

Asp 52 T T 0.03 -0.31 . * F 2.36 1.27

Gly 53 T C 0.89 -0.39 * * F 2.40 1.27

Ser 54 A C 1.13 -1.07 * . F 2.06 1.42

Arg 55 A B 1.73 -1.10 * * F 1.62 1.47

Leu 56 A B 1.24 -0.67 * . F 1.38 2.30

Glu 57 A B 0.43 -0.31 * . F 0.84 1.49

Gin 58 A B 0.78 -0.01 * * F 0.45 0.63

Pro 59 A A 0.27 -0.01 * * F 0.60 1.27

Phe 60 A A 0.20 -0.01 * * 0.30 0.60

Leu 61 A A 1.01 -0.01 * . 0.30 0.70

Asp 62 A A 0.12 -0.01 * . 0.30 0.73

Leu 63 A B -0.73 0.24 * . -0.30 0.59

Lys 64 A B -1.33 0.10 . . -0.30 0.53

Asn 65 B B -0.94 0.10 . . -0.30 0.26

He 66 B B -0.44 0.59 . * -0.60 0.46

Val 67 B B -0.66 0.39 . . -0.30 0.33

Leu 68 B B -0.13 0.81 * . -0.60 0.32

Thr 69 B B -1.07 1.33 . . F -0.45 0.48

Thr 70 B B -1.41 1.33 . . F -0.45 0.45

Pro 71 B B -0.52 1.11 . . F -0.45 0.54

Trp 72 B T 0.33 0.43 . . -0.20 0.62

He 73 B B 1.26 -0.06 . * 0.61 0.75

Gly 74 B B 1.22 -0.54 . . F 1.37 0.95

Asp 75 T C 0.83 -0.54 . . F 2.28 0.89

Glu 76 B T 0.23 -0.67 . . F 2.54 1.10

Arg 77 T T 0.18 -0.67 . * F 3.10 0.92

Gly 78 T T 0.26 -0.67 . . F 2.79 0.54

Phe 79 A 0.01 0.01 . * 0.83 0.26 Leu 80 A -0.69 0.51 * 0.22 0.13

Gly 81 A -0.72 1.30 * -0.09 0.12

Leu 82 A -1.04 1.37 * * -0.40 0.18

Ala 83 A -0.66 1.01 * -0.40 0.34

Phe 84 A -0.66 0.33 * -0.10 0.70

His 85 A r . 0.27 0.69 -0.20 0.73

Pro 86 A r 0.58 0.00 * 0.25 1.42

Lys 87 A T 1.39 0.00 * 0.56 2.23

Phe 88 A r 2.09 -0.39 * * 1.472.63

Arg 89 A 2.83 -0.89 * * 1.88 3.33

His 90 A T r . 2.17 -1.31 2.79 3.33

Asn 91 T T . 2.13 -0.53 * 3.10 3.33

Arg 92 T r ι.2θ -0.56 * 2.79 2.67

Lys 93 T r 1.66 0.13 1.58 1.37

Phe 94 B T 1.30 0.39 * # 0.87 1.34

Tyr 95 B B 1.03 0.74 -0.14 1.07

He 96 B B 0.37 1.13 -0.60 0.72

Tyr 97 B r -0.56 1.70 fc -0.20 0.44

Tyr 98 B r . -0.60 1.60 * -0.20 0.23

Ser 99 B r 0.14 0.84 -0.20 0.56

Cys 100 A r 0.43 0.16 0.100.71

Leu 101 A A 1.37 -0.60 0.600.91

Asp 102 A A 0.76 -1.36 F 0.90 1.35

Lys 103 A A 1.00 -1.10 F 0.90 1.87

Lys 104 A A 1.34 -1.67 F 0.90 3.94

Lys 105 A A 1.12 -2.36 * * F 0.90 4.71

Val 106 A A 2.04 -1.67 * F 0.90 1.65

Glu 107 A A 1.16 -1.67 * F 0.90 1.62

Lys 108 A A 0.81 -0.99 * F 0.75 0.57

He 109 A A 0.77 -0.60 * F 0.90 1.02

Arg 1 10 A A 0.12 -1.24 0.75 1.02

He 111 A A 1.02 -0.63 * 0.60 0.51 .

Ser 1 12 A A 0.17 -0.63 * F 0.90 1.45

Glu 1 13 A A -0.18 -0.67 * * F 0.75 0.55

Met 1 14 A A 0.82 -0.29 * * F 0.60 1.05

Lys 1 15 A A 0.12 -0.97 * F 0.90 1.53

Val 1 16 A B 1.01 -0.86 F 0.75 0.89

Ser 1 17 A A 1.10 -0.86 * F 0.90 1.51

Arg 1 18 A A 1.10 -1.04 F 0.90 1.17

Ala 1 19 A A 1.74 -0.64 * F 0.90 2.52

Asp 120 A T 1.1 1 - 1.29 •* F 1.30 3.77 Pro 121 A T 1.97 -1.17 ' " . 1 F 1.30 1.94

Asn 122 A T 1.46 -1.17 . * F 1.30 3.21

Lys 123 A T 1.39 -0.99 . * 1 F 1.30 1.59

Ala 124 A A 1.68 -0.99 * F 0.90 2.05

Asp 125 A A 1.68 -1.03 " * . F 0.90 1.71

Leu 126 A A 2.00 -1.43 ^« * * p 0.90 1.48

Lys 127 A A 1.14 -1.43 * * ] . 0.90 2.87

Ser 128 A A 0.21 -1.29 * * 1 F 0.90 1.28

Glu 129 A A -0.01 -0.60 * * 1 F 0.90 1.08

Arg 130 A A -0.01 -0.60 * * F 0.75 0.45

Val 131 A A -0.09 -0.60 * * 0.60 0.58

He 132 A A -0.13 -0.30 * * 0.30 0.23

Leu 133 A A 0.17 -0.30 * * 0.30 0.21

Glu 134 A A -0.04 -0.30 * * 0.30 0.48

He 135 \ A -0.74 -0.51 * * 0.75 1.06

Glu 136 \ \ -0.19 -0.70 ' * * p 0.90 1.30

Glu 137 K \ 0.70 -1.00 ' ' . F 0.90 1.01

Pro 138 \ T 1.48 -0.60 * ] F 1.302.31

Ala 139 \ T 1.48 -0.79 . 1 F 1.58 1.82

Ser 140 \ T 2.02 -0.39 . 1 F 1.56 1.69

Asn 141 T Z 1.68 0.04 . F 1.44 1.08

His 142 T C 1.68 0.04 . F 1.72 1.06

Asn 143 r T 1.08 -0.06 . F 2.80 1.37

Gly 144 r T 0.86 0.24 . F 1.77 0.70

Gly 145 Γ T 0.46 0.53 . F 1.19 0.43

Gin 146 A B B 0.11 0.81 . F 0.11 0.23

Leu 147 A B B -0.67 0.84 * -0.32 0.23

Leu 148 A B B -0.67 1.10 * -0.60 0.19

Phe 149 A B B -0.67 0.67 * -0.60 0.18

Gly 150 B B -0.57 0.70 * -0.60 0.22

Leu 151 B T -1.17 0.77 * -0.20 0.42

Asp 152 T T -0.60 0.70 * 0.20 0.48

Gly 153 T T -0.68 0.67 0.20 0.76

Tyr 154 B T -0.68 0.93 * -0.20 0.65

Met 155 B B -0.64 1.03 -0.60 0.33

Tyr 156 B B -0.18 1.51 * -0.60 0.49

He 157 B B -0.18 1.51 -0.60 0.31

Phe 158 B B -0.18 0.76 -0.60 0.52

Thr 159 B B -0.28 0.57 F -0.45 0.33

Gly 160 T T 0.32 0.24 F 0.88 0.46

Asp 161 T T -0.02 -0.04 F 1.71 0.93 Gly 162 r c 0.52 -0.33 F 1.74 0.65

Gly 163 T c 1.22 -0.39 F 1.97 0.65

Gin 164 . " 1.32 -0.81 * F 2.30 0.65

Ala 165 . ~* 0.97 -0.39 * F 1.92 1.01

Gly 166 B 0.62 -0.03 F 1.34 0.89

Asp 167 B T 0.16 -0.03 F 1.31 0.51

Pro 168 B T -0.20 0.26 F 0.48 0.41

Phe 169 B T -0.54 0.54 * -0.20 0.36

Gly 170 B T 0.04 0.54 * -0.20 0.21

Leu 171 B -0.20 0.94 -0.40 0.22

Phe 172 B -0.20 1.01 -0.40 0.26

Gly 173 B 0.01 0.63 * -0.10 0.46

Asn 174 0.76 0.60 * F 0.55 0.89

Ala 175 " 0.80 -0.09 F 1.902.05

Gin 176 = 1.31 -0.49 * F 2.20 2.78

Asn 177 T = 1.20 -0.53 F 3.00 2.32

Lys 178 r T 0.73 -0.24 * F 2.60 1.89

Ser 179 B T 0.39 -0.06 * F 1.75 0.90

Ser 180 B T 1.02 -0.03 * * F 1.45 0.55

Leu 181 B 0.17 -0.43 * * F 0.95 0.55

Leu 182 B B -0.64 0.21 * F -0.15 0.31

Gly 183 B B -0.58 0.51 * * F -0.45 0.19

Lys 184 B B -1.17 0.13 * * -0.30 0.45

Val 185 B B -0.87 0.13 * * -0.30 0.38

Leu 186 B B -0.91 -0.56 * * 0.60 0.64

Arg . 187 B B -0.10 -0.34 * 0.30 0.24

He 188 B B 0.36 0.06 * -0.30 0.52

Asp 189 B T -0.28 -0.59 * * 1.15 1.23

Val 190 B T 0.23 -0.77 * * 1.00 0.63

Asn 191 B T 0.74 -0.34 * * F 0.85 0.89

Arg 192 B T 0.60 -0.64 * F 1.49 0.72

Ala 193 T 1.14 -0.14 * * F 1.88 1.32

Gly 194 T T 1.19 -0.36 * F 2.27 0.81

Ser 195 T c 2.16 -0.76 * F 2.71 0.83

His 196 T T 1.91 -0.76 * * F 3.40 1.60

Gly 197 T T 1.91 -0.50 * * F 3.06 2.54

Lys 198 B 1.64 -0.93 * F 2.12 3.71

Arg 199 B B 1.78 -0.67 F 1.58 2.02

Tyr 200 B B 1.78 -0.74 * 1.43 3.16

Arg 201 B B 1.81 -0.79 * 1.43 2.12

Val 202 B B 2.16 -0.79 F 1.92 1.81 Pro 203 B T 1.90 -0.39 * 1 2.36 1.85

Ser 204 T T 1.09 -0.71 * F 3.40 1.46

Asp 205 T C 0.48 0.07 * F 1.96 1.71

Asn 206 T C 0.07 0.07 * F 1.47 0.82

Pro 207 C 0.92 0.03 F 0.93 0.82

Phe 208 B 0.92 -0.36 F 0.99 0.85

Val 209 B 0.88 0.07 F 0.33 0.82

Ser 210 B 0.29 0.10 1 0.61 0.52

Glu 211 B T 0.26 ^' 0.17 F 1.09 0.61

Pro 212 T T 0.26 -0.11 F 2.52 1.12

Gly 213 T T 0.37 -0.33 F 2.80 1.29

Ala 214 T ( Z 0.33 -0.21 2.02 0.75

His 215 Z 0.39 0.47 0.64 0.34

Pro 216 B B -0.20 0.80 -0.04 0.54

Ala 217 B B -0.23 0.87 -0.32 0.54

He 218 B B -0.23 1.13 -0.60 0.62

Tyr 219 B T -0.53 1.06 * -0.20 0.40

Ala 220 B T -0.39 1.31 -0.20 0.28

Tyr 221 B T -0.18 0.81 * -0.20 0.77

Gly 222 B T -0.19 0.53 * -0.20 0.79 lie 223 B B 0.41 0.39 * * -0.30 0.78

Arg 224 B B 0.77 0.80 * * -0.60 0.52

Asn 225 B T 0.69 0.04 * * 0.25 1.03

Met 226 B T 0.34 0.19 * * 0.100.79

Trp 227 B B -0.17 0.00 * * 0.30 0.41

Arg 228 B 0.72 0.64 * * -0.40 0.19

Cys 229 B 0.72 0.24 * * 0.24 0.32

Ala 230 B 0.38 -0.37 * 1.18 0.59

Val 231 B 0.98 -0.86 * 1.82 0.30

Asp 232 T T 1.06 -0.86 * . F 2.91 0.93

Arg 233 T T 0.06 -1.00 * . F 3.40 1.42

Gly 234 T T 0.41 -0.81 * * F 3.06 1.34

Asp 235 T C 1.11 -0.97 * . F 2.52 1.16

Pro 236 B 1.97 -0.97 * F 1.78 1.16

He 237 B 1.62 -0.57 * * F 1.78 2.03

Thr 238 B 1.62 -0.57 * * F 1.78 1.20

Arg 239 B 1.62 -0.57 * * F 2.12 1.52

Gin 240 B 1.73 -0.57 * * F 2.462.15

Gly 241 T T 1.06 -1.26 * F 3.40 2.92

Arg 242 T T 1.24 -1.06 * F 3.06 1.05

Gly 243 T T 0.89 -0.27 * F 2.27 0.52 Arg 244 18 T 0.43 -0.10 * F 1.530.28

He 245 B 0.43 -0.10 * 0.840.14

Phe 246 1 B -0.08 -0.10 * 0.500.24

Cys 247 B T -0.53 0.11 * 0.400.09

Gly 248 B T -0.19 0.54 0.400.13

Asp 249 r T -0.30 0.26 F 1.550.26

Val 250 T ( Z 0.70 -0.13 F 2.250.77

Gly 251 T ( Z 0.70 -0.70 * F 3.001.53

Gin 252 T < : 1.37 -0.34 * F 2.250.80

Asn 253 T ( Z 1.71 -0.34 * F 2.101.86

Arg 254 B T 0.86 -0.99 * F 1.903.25

Phe 255 A A 1.71 -0.77 F 1.201.39

Glu 256 A A 1.24 -1.17 * F 0.901.45

Glu 257 A A 0.36 -0.89 * 0.600.61

Val 258 A A B -0.46 -0.20 * 0.300.49

Asp 259 A A B -0.52 -0.30 * 0.300.23

Leu 260 A A B -0.17 -0.30 * * 0.300.27

He 261 A A B -0.51 0.13 * * -0.30 0.36

Leu 262 A T -0.51 -0.09 * * 0.700.21

Lys 263 r T 0.10 0.31 * * F 0.650.42

Gly 264 Γ T -0.24 0.39 * F 0.650.93

Gly 265 Γ T 0.28 0.13 * * F 0.801.12

Asn 266 T C 1.28 0.36 * * F 0.450.59

Tyr 267 T C 1.50 0.36 * * 0.451.17

Gly 268 T C 1.50 0.43 * * 0.151.19

Trp 269 B T 1.84 0.00 * * 0.851.48

Arg 270 A B 1.84 -0.40 * * 0.451.63

Ala 271 A B 1.14 -0.73 * * F 0.901.63

Lys 272 A A 0.80 -0.37 * * F 0.601.35

Glu 273 A A 0.48 -0.79 * * F 0.750.69

Gly 274 A A 0.52 -0.21 * F 0.450.37

Phe 275 A A 0.41 0.04 * * -0.30 0.29

Ala 276 A A 1.04 0.04 * -0.30 0.28

Cys 277 A T 1.04 0.04 * 0.100.56

Tyr 278 A T 0.23 -0.39 * 0.851.30

Asp 279 A T -0.09 -0.49 * F 1.001.06

Lys 280 A T 0.58 -0.41 * F 1.001.06

Lys 281 A A 1.17 -0.49 F 0.450.92

Leu 282 A A 1.24 -0.84 0.600.89

Cys 283 A A 1.19 -0.34 * 0.300.45

His 284 A B 0.38 0.04 * -0.30 0.30 Asn 285 B T 0.33 0.73 . * -0.20 0.30

Ala 286 A T 0.29 0.04 * 0.10 0.94

Ser 287 A T 0.24 -0.53 . . 1.15 1.15

Leu 288 A T 0.10 -0.39 . . 1 ' 0.85 0.53

Asp 289 B ] 3 -0.08 -0.10 . . 1 ' 0.45 0.43

Asp 290 B 1 3 -0.97 -0.17 . * 1 ³ 0.45 0.50

Val 291 B ] 3 -0.62 0.13 . . -0.30 0.42

Leu 292 B ] B -0.91 0.20 . . -0.30 0.40

Pro 293 B 1 B -0.34 0.70 * . -0.60 0.24

He 294 B B -0.69 1.46 . . -0.60 0.51

Tyr 295 B T -0.72 1.24 . . -0.20 0.61

Ala 296 B T -0.46 1.06 . . -0.20 0.54

Tyr 297 B T -0.50 1.13 . * -0.20 0.77

Gly 298 B T -0.63 1.09 * . -0.20 0.37

His 299 B B 0.30 0.76 * . -0.60 0.36

Ala 300 B B 0.24 0.26 * . -0.30 0.46

Val 301 B B -0.02 -0.11 * . 0.30 0.62

Gly 302 B T -0.09 0.10 * . 1 » 0.25 0.34

Lys 303 B T -0.09 0.09 * . 1 ^π 0.25 0.48

Ser 304 B T -0.40 0.01 * . 1 - 0.25 0.64

Val 305 B T -0.06 -0.20 . . 1 F 0.85 0.64

Thr 306 B T -0.06 0.13 . . 1 F 0.25 0.50

Gly 307 B T 0.04 0.77 . . 1 F -0.05 0.28

Gly 308 B T 0.11 1.14 . . ] F -0.05 0.59

Tyr 309 B T 0.07 0.50 . . -0.20 0.80

Val 310 B 0.26 0.44 * . -0.40 0.80

Tyr 311 B T 0.57 0.59 * . -0.20 0.43

Arg 312 B T 0.61 0.16 * . 0.10 0.48

Gly 313 B T 0.74 -0.21 * . F 1.13 0.87

Cys 314 B T 0.99 -0.43 * * F 1.41 0.85

Glu 315 B 1.03 -0.79 * * F 1.79 0.70

Ser 316 T C 1.28 -0.10 . * F 2.17 0.58

Pro 317 T T 0.82 -0.13 . * F 2.80 1.75

Asn 318 T T 0.36 -0.27 . . F 2.52 1.00

Leu 319 T T 0.78 0.41 . . F 1.19 0.62

Asn 320 B T -0.11 0.79 . . 0.360.63

Gly 321 B B -0.51 1.04 . . -0.32 0.27

Leu 322 B B -0.64 1.43 . . -0.60 0.29

Tyr 323 B B -0.64 1.17 . * -0.60 0.18

He 324 B B -0.53 0.77 * . -0.60 0.30

Phe 325 B B -1.13 1.13 * . -0.60 0.31 Gly 326 B B -1.09 1.06 * -0.60 0.20

Asp 327 B -0.62 0.69 ϋ -0.40 0.38

Phe 328 B -0.27 0.43 ie -0.40 0.43

Met 329 A T -0.19 -0.36 K 0.70 0.85

Ser 330 T ( Z -0.09 -0.10 . * F 1.05 0.42

Gly 331 A T -0.33 0.51 F -0.05 0.48

Arg 332 A T -1.14 0.23 0.10 0.49

Leu 333 A i \ -0.44 0.30 -0.30 0.30

Met 334 A A 0.16 0.31 . ^• * -0.30 0.53

Ala 335 A A 0.46 -0.11 * 0.30 0.47

Leu 336 A A 0.91 -0.11 . ' * 0.30 0.95

Gin . 337 A A 0.84 -0.80 0.75 1.87

Glu 338 A A 1.66 -1.41 . . F 0.90 3.71

Asp 339 A A 2.30 -1.51 * . F 0.90 7.23

Arg 340 A A 2.93 -2.20 - * . F 0.90 8.35

Lys 341 A A 3.46 -2.60 . ^> ^► F 0.90 9.64

Asn 342 A T 3.50 -1.69 * F 1.30 6.07

Lys 343 A T 3.54 -1.69 * F 1.30 6.20

Lys 344 A T 3.54 -1.69 * F 1.30 6.20

Trp 345 A T 3.43 -1.29 * F 1.30 6.68

Lys 346 A A 2.58 -1.69 F 0.90 5.58

Lys 347 \ A 1.91 -1.00 . F 0.90 2.30

Gin 348 A B 1.06 -0.43 * F 0.60 1.17

Asp 349 A B 0.67 -0.66 F 0.75 0.48

Leu 350 A B 0.66 -0.23 . F 0.45 0.24

Cys 351 A B 0.30 0.16 F -0.15 0.19

Leu 352 A B -0.06 0.24 F -0.15 0.16

Gly 353 A T -0.36 0.73 F -0.05 0.28

Ser 354 T T -1.02 0.43 F 0.35 0.70

Thr 355 T T -0.80 0.43 F 0.35 0.45

Thr 356 B T -0.83 0.24 F 0.25 0.46

Ser 357 B T -0.23 0.60 F -0.05 0.30

Cys 358 B -0.23 0.64 -0.40 0.32

Ala 359 B -0.74 0.59 -0.40 0.22

Phe 360 B T -1.32 0.79 -0.20 0.14

Pro 361 B T -1.31 1.09 -0.20 0.18

Gly 362 T T -1.32 0.90 0.20 0.24

Leu 363 B T -0.69 0.89 -0.20 0.39

He 364 B -0.40 0.60 * -0.40 0.35

Ser 365 A T 0.34 0.56 * * -0.20 0.47

Thr 366 A T -0.14 0.13 * F 0.40 1.13 His 367 i A r -0.69 0.23 * F 0.40 1.40

Ser 368 B T -0.77 0.23 " F 0.25 0.73

Lys 369 B 1 B -0.18 0.53 * -0.60 0.36

Phe 370 B 1 B -0.58 0.43 * > k -0.60 0.35

He 371 B B -0.86 0.71 * > fc -0.60 0.23

He 372 B B -0.82 0.83 fc -0.60 0.11

Ser 373 t \ B -0.52 0.83 fc -0.60 0.23

Phe 374 i A A -0.57 0.04 fc > ι< -0.30 0.54

Ala 375 . A A -0.46 -0.64 0.75 1.35

Glu 376 i A i A 0.09 -0.83 fc 0.75 1.01

Asp 377 . A . A 0.98 -0.79 fc F 0.90 1.16

Glu 378 A A 0.47 -1.57 F 0.90 1.99

Ala 379 A A 0.92 -1.39 F 0.75 0.95

Gly 380 A A 0.81 -0.63 F 0.75 0.89

Glu 381 A A . B 0.00 0.16 -0.30 0.44

Leu 382 A A . B -0.59 0.84 -0.60 0.36

Tyr 383 A A . B -0.90 0.84 -0.60 0.37

Phe 384 A A . B -0.61 0.90 -0.60 0.31

Leu 385 A B B -0.51 1.29 -0.60 0.50

Ala 386 A B B -0.72 1.36 -0.60 0.50

Thr 387 A B B -0.21 1.03 -0.60 0.89

Ser 388 A C -0.56 0.63 F -0.10 1.45

Tyr 389 T C -0.10 0.44 F 0.30 1.45

Pro 390 r T 0.12 0.70 F 0.50 1.58

Ser 391 T T 0.50 0.71 0.35 1.19

Ala 392 B T 0.92 0.76 0.08 1.17

Tyr 393 B 0.88 0.00 0.91 1.49

Ala 394 B T 0.82 0.00 * 1.24 1.10

Pro 395 B T 0.14 0.00 * p 1.52 1.46

Arg 396 T T . 0.20 0.19 F 1.30 0.65

Gly 397 B T 0.83 0.19 F 0.92 1.01

Ser 398 B B 0.38 -0.31 F 0.99 1.31

He 399 B B . 0.11 0.04 * -0.04 0.58

Tyr 400 B B 0.32 0.69 * * -0.47 0.43

Lys 401 B B 0.00 0.26 * * -0.30 0.54

Phe 402 B B 0.04 0.30 * 0.19 1.19

Val 403 B B 0.46 0.00 * F 1.28 1.02

Asp 404 B T 1.46 -0.76 * F 2.17 1.00

Pro 405 B T 1.11 -0.76 * F 2.66 2.26

Ser 406 T T 0.86 -1.04 * F 3.40 3.07

Arg 407 T T 1.34 -1.26 * F 3.06 2.85 Arg 408 T 1.86 -0.83 F 2.86 2.85

Ala 409 < Z 1.90 -0.83 F 2.66 2.10

Pro 410 T C 1.44 -1.21 * F 2.86 2.15

Pro 411 T T 1.79 -0.64 * = fc p 2.91 0.59

Gly 412 T T 1.43 -0.64 a fc p 3.40 1.16

Lys 413 T T 1.37 -0.39 * p 2.76 1.18

Cys 414 T T .1.74 -0.81 * F 2.72 1.52

Lys 415 B T 1.10 -0.81 fc p 1.98 2.38

Tyr 416 B T 1.10 -0.60 * p 1.49 0.88

Lys 417 B T 0.59 -0.17 * p 1.00 2.55

Pro 418 B B 0.66 -0.10 * p 0.45 0.95

Val 419 1 B B 1.01 -0.10 * F 0.60 1.18

Pro 420 1 B B 1.01 -0.37 * F 0.79 0.85

Val 421 B B 0.96 -0.37 * p 1.28 1.10

Arg 422 ] B B 0.96 -0.41 •> ^► F 1.62 1.99

Thr 423 ] B T 1.28 -1.06 * * p 2.66 2.58

Lys 424 T T 1.24 -1.49 * * p 3.40 6.80

Ser 425 T T 1.24 -1.44 * * p 3.06 2.43

Lys 426 T T 1.40 -1.01 * > ^► F 2.72 2.61

Arg 427 B 1.40 -0.71 > ► p 1.78 1.13

He 428 B 1.50 -0.71 * . fc p 1.44 1.65

Pro 429 B 0.64 -0.67 t fc 0.95 1.28

Phe 430 B 0.36 0.01 * fc -0.10 0.54

Arg 431 A B 0.36 0.51 * ι> -0.60 . 0.77

Pro 432 A B -0.07 -0.17 * * F 0.60 1.00

Leu 433 A A -0.03 -0.11 * i. p 0.60 1.67

Ala 434 A A -0.63 -0.26 * * F 0.45 0.63

Lys 435 A A 0.07 0.43 * .. F -0.45 0.34

Thr 436 A A -0.86 0.00 * » 0.30 0.68

Val 437 A A -1.46 0.00 * 0.30 0.56

Leu 438 A A -0.60 0.19 * -0.30 0.23

Asp 439 A A -0.01 0.19 * -0.30 0.32

Leu 440 A A -0.06 -0.30 * 0.30 0.74

Leu 441 A A -0.04 -0.54 * F 0.90 1.56

Lys 442 A A 0.81 -0.84 * F 0.90 1.25

Glu 443 A A 1.67 -0.84 * F 0.90 2.63

Gin 444 A A 1.08 -1.53 * F 0.90 6.38

Ser 445 A A 1.30 -1.71 * F 0.90 3.22

Glu 446 A A 2.22 -1.21 * F 0.90 1.88

Lys 447 A A 2.22 -1.21 * F 0.90 2.13

Ala 448 A A 1.92 -1.61 * F 0.90 3.17 Ala 449 A A 1.62 -1.61 * F 0.902.46

Arg 450 A . A 1.62 -1.23 * F 0.901.65

Lys 451 A A 1.03 -0.84 * F 0.902.18

Ser 452 A T 0.68 -0.84 * F 1.302.18

Ser 453 A T 0.46 -0.86 F 1.301.61

Ser 454 B T 0.46 -0.17 F 0.850.66

Ala 455 B T 0.04 0.33 F 0.250.50

Thr 456 B -0.34 0.33 -0.10 0.50

Leu 457 B -0.26 0.37 -0.10 0.37

Ala 458 B T -0.54 0.41 F -0.05 0.57

Ser 459 B T -0.24 0.41 F -0.05 0.40

Gly 460 T Z 0.00 0.33 F 0.450.83

Pro 461 T < Z -0.50 0.07 * F 0.450.81

Ala 462 . ^* . < Z 0.01 0.26 * F 0.250.50

Gin 463 A 0.60 0.26 F 0.050.68

Gly 464 B 0.94 -0.17 . F 0.650.76

Leu 465 B 0.94 -0.60 F 1.101.50

Ser 466 A 0.86 -0.67 * F 0.950.86

Glu 467 A 1.14 -0.69 . * F 1.101.16

Lys 468 A 1.19 -0.73 . F 1.101.89

Gly 469 A T T 1.58 -1.41 F 1.702.82

Ser 470 A T 1.58 -1.80 . F 1.303.26

Ser 471 A T 1.29 -1.11 . F 1.301.34

Lys 472 A T 0.99 -0.61 F 1.301.37

Lys 473 B 0.73 -0.66 F 1.101.37

Leu 474 B 0.77 -0.61 . F 1.101.58

Ala 475 B 0.77 -0.51 * F 1.401.14

Ser 476 B T 0.77 -0.13 . F 1.450.77

Pro 477 B T 0.77 0.26 F 1.301.24

Thr 478 T T 0.72 -0.43 F 2.602.46

Ser 479 T C 1.22 -0.53 . F 3.002.95

Ser 480 T T 1.00 -0.43 * * F 2.602.76

Lys 481 B T 1.41 -0.17 * * p 1.901.58

Asn 482 B T 1.28 -0.66 * * F 1.902.30

Thr 483 B T 1.38 -0.61 * * p 1.941.70

Leu 484 B 1.33 -0.57 * _{* F} 1.781.32

Arg 485 B 1.32 -0.14 * * p 1.670.81

Gly 486 B T 1.32 -0.06 * F 2.210.81

Pro 487 T T 1.37 -0.54 * p 3.401.96

Gly 488 T T 1.72 -1.23 . * p 3.062.00

Thr 489 T C 1.94 -1.23 . * p 2.524.05 Lys 490 A B 1.94 -1.16 . * F 1.58 2.65

Lys 491 A B 1.43 -1.59 * a F 1.24 5.24

Lys 492 A B 1.30 -1.37 * * p 0.90 2.69

Ala 493 A B 1.43 -1.43 * . F 0.90 1.33

Arg 494 A B 1.71 -1.00 * . F 0.90 1.03

Val 495 A B 0.81 -0.50 * * p 0.75 0.70

Gly 496 B T 0.88 0.14 * * p 0.25 0.52

Pro 497 B T 0.83 -0.36 * * p 0.85 0.52

His 498 B T 1.08 0.04 F 0.74 1.20

Val 499 B T 1.01 -0.17 * . F 1.68 1.20

Arg 500 B T 1.98 -0.60 * . F 2.32 1.55

Gin 501 B T 2.43 -1.03 * F 2.66 2.24

Gly 502 r T 2.69 -1.53 * F 3.40 5.90

Lys 503 . A , . T . 2.42 -2.17 F 2.66 6.03

Arg 504 - A 2.47 -1.79 * F 2.12 4.66

Arg 505 B 2.40 -1.50 F 1.78 3.89

Lys 506 B 2.10 -1.93 * F 1.44 3.89

Ser 507 B 2.41 -1.54 F 1.44 2.66

Leu 508 B 2.07 -1.04 F 1.78 1.85

Lys 509 B 1.61 -0.66 F 2.12 1.24

Ser 510 c 1.61 -0.23 * > fc p 2.21 0.91

His 511 T T 0.97 -0.61 * > ^► F 3.40 2.17

Ser 512 T c 1.38 -0.69 fc p 2.86 1.07

Gly 513 T T 1.98 -0.69 fc F 3.02 1.57

Arg 514 T T 1.63 -0.64 > " F 2.98 1.78

Met 515 c 1.34 -0.76 * F 2.54 1.78

Arg 516 T c 1.38 -0.64 * p 2.70 1.82

Pro 517 T c 1.68 -1.07 k F 3.00 1.61

Ser 518 A T 2.07 -0.67 * F 2.50 2.82

Ala 519 A T 2.07 -1.29 • * F 2.20 2.88

Glu 520 A A 2.08 -1.29 * * F 1.50 3.65

Gin 521 A A 1.62 -1.21 * * F 1.51 2.75

Lys 522 A A 1.94 -1.17 * F 1.52 2.69

Arg 523 A A 1.94 -1.67 * F 1.83 3.04

Ala 524 A T 1.72 -1.29 * F 2.542.36

Gly 525 T T 1.51 -1.00 * F 3.10 0.97

Arg 526 T T 1.12 -0.57 * F 2.79 0.77

Ser 527 T c 0.69 -0.14 * F 1.98 0.97

Leu 528 T c 0.19 -0.21 * 1.67 1.25

Pro 529 B 0.39 -0.21 * 0.81 0.82

Ter 530 T 0.34 0.21 * 0.30 0.78 PAGE INTENTIONALLY LEFT BLANK

Table II

Res Position I II III IV V VI VII VIII IX X XI . XII XIII XIV

Met 1 . . c 0.69 -0.24 * 1.19 1.74

Arg 2 . . c 0.38 -0.24 * 1.53 1.34

Pro 3 . . r c 0.88 0.11 1.32 0.91

Pro 4 . . r r 1.27 -0.31 2.61 1.80

Gly 5 . Γ r 0.96 -0.53 . . 1 F 3.40 1.48

Phe 6 . . Γ Γ 0.74 0.26 . . ] F 2.01 0.83

Arg 7 . A ] B -0.18 0.51 0.42 0.44

Asn 8 . t A ] B -0.78 0.77 * 0.08 0.37

Phe 9 . A ] B -1.16 1.03 * -0.26 0.35

Leu 10 - A ] B -1.11 0.74 * -0.60 0.18

Leu 11 . . A c -0.71 1.13 * -0.40 0.15

Leu 12 i A c -1.63 1.11 * -0.40 0.23

Ala 13 A c -2.44 1.01 -0.40 0.23

Ser 14 A c -2.44 1.01 -0.40 0.23

Ser 15 A c -2.22 1.11 -0.40 0.24

Leu 16 j A B -1.76 0.93 -0.60 0.24

Leu 17 A B -1.76 0.86 -0.60 0.18

Phe 18 A B -1.47 1.16 -0.60 0.11

Ala 19 A c -1.76 1.16 -0.40 0.18

Gly 20 A c -2.31 0.97 -0.40 0.22

Leu 21 A c -1.71 0.93 * -0.40 0.19

Ser 22 A c -0.90 0.57 * -0.40 0.29

Ala 23 A c -0.50 0.47 * -0.40 0.51

Val 24 A c -0.61 0.43 * -0.40 0.83

Pro 25 T c -0.57 0.53 * F 0.15 0.53

Gin 26 T T 0.03 0.53 * F 0.35 0.71

Ser 27 T T 0.03 0.46 * F 0.50 1.48

Phe 28 T c -0.19 0.20 * * F 0.60 1.28

Ser 29 T c 0.78 0.46 * * F 0.15 0.61

Pro 30 T c 0.69 0.06 * * F 0.450.89

Ser 31 T T 0.40 0.06 * * F 0.80 1.38

Leu 32 T T 0.49 0.19 * * F 0.80 1.08

Arg 33 T 0.84 0.23 * * F 0.60 1.08

Ser 34 T 0.56 0.23 * * F 0.45 0.80

Trp 35 T c 0.18 0.34 * * F 0.45 0.98

Pro 36 T c -0.19 0.16 * * F 0.45 0.50

Gly 37 T T 0.73 0.73 * * 0.20 0.20 Ala 38 T T -0.19 0.34 * * 0.50 0.38

Ala 39 c -0.19 0.11 * 0.10 0.20

Cys 40 1 B T 0.21 0.07 * 0.30 0.27

Arg 41 . A B -0.17 -0.36 * 0.30 0.53

Leu 42 . , A c 0.18 -0.36 * 0.50 0.53

Ser 43 . i A c 0.47 -0.86 * * 0.95 1.70

Arg 44 . . A c 1.06 -1.04 * F 1.10 1.16

Ala 45 A c 1.83 -1.04 * F 1.10 2.44

Glu 46 . / \ T 1.83 -1.73 * F 1.30 3.57

Ser 47 . . \ T 1.98 -2.11 * F 1.30 3.57

Glu 48 . . \ T 2.39 -1.54 * F 1.30 1.89

Arg 49 . . A T 1.69 -2.04 * F 1.30 2.14

Arg 50 . i A T 2.07 -1.54 * * F 1.58 1.62

Cys 51 . . A T 1.72 -1.50 * * 1.71 1.44

Arg 52 . i A T 2.02 -1.07 * * F 1.99 0.73

Ala 53 . r c 1.81 -0.67 * * F 2.47 0.64

Pro 54 T Γ 1.49 -0.24 * * F 2.80 1.86

Gly 55 T Γ 1.03 -0.39 * F 2.52 1.47

Gin 56 . Γ c 1.11 0.04 * * F 1.44 1.44

Pro 57 . Γ c 0.41 0.04 * * F 1.01 0.94

Pro 58 T Γ 0.19 0.11 F 0.93 0.96

Gly 59 T r -0.27 0.37 F 0.65 0.46

Ala 60 B . r 0.04 0.54 -0.20 0.16

Ala 61 B -0.30 0.61 * -0.40 0.14

Leu 62 B 0.02 0.61 * -0.40 0.14

Cys 63 T -0.11 0.19 * 0.61 0.27

His 64 T Γ 0.34 0.11 * 1.12 0.26

Gly 65 T Γ 0.27 -0.39 * F 2.18 0.63

Arg 66 T - T 0.86 -0.50 * F 2.49 0.63

Gly 67 T T 1.00 -1.07 F 3.10 0.77

Arg 68 T 1.32 -1.00 * F 2.59 0.42

Cys 69 T T 0.50 -1.00 * 2.33 0.21

Asp 70 T T 0.18 -0.36 * 1.72 0.16

Cys 71 T T -0.82 -0.21 * 1.41 0.04

Gly 72 T T -1.14 0.47 * 0.20 0.06

Val 73 B T -1.29 0.47 * -0.20 0.02

Cys 74 B B -1.48 0.97 -0.60 0.05

He 75 B B -1.79 1.04 * -0.60 0.03

Cys 76 B B -1.12 1.10 -0.60 0.07

His 77 B B -0.99 0.46 -0.60 0.22

Val 78 B T -0.48 . 0.31 0.10 0.48 Thr 79 B C -0.41 0.06 F 0.05 0.89

Glu 80 r C -0.22 0.10 * F 0.45 0.64

Pro 81 T r -0.26 0.39 F 0.65 0.75

Gly 82 T r -0.57 0.53 0.20 0.45

Met 83 T Γ 0.08 0.47 * 0.20 0.26

Phe 84 T -0.42 0.90 0.00 0.26

Phe 85 T -1.09 1.16 . 0.00 0.21

Gly 86 Γ C -0.88 1.30 0.00 0.12

Pro 87 r C -1.20 0.69 0.00 0.23

Leu 88 T r -0.63 0.47 * 0.20 0.14

Cys 89 T Γ 0.07 0.19 * 0.50 0.20

Glu 90 A T 0.48 -0.24 * 0.70 0.22

Cys 91 A T -0.03 0.24 0.10 0.28

His 92 A T -0.49 0.20 * 0.10 0.39

Glu 93 A T 0.32 0.20 * 0.10 0.12

Trp 94 A T 0.68 0.20 * 0.10 0.39

Val 95 A T 0.43 0.11 * 0.10 0.42

Cys 96 A T 1.10 0.37 * 0.38 0.38

Glu 97 A T 0.79 0.37 * 0.66 0.60

Thr 98 A T 0.49 -0.11 * F 1.69 0.80

Tyr 99 T T 0.47 -0.37 . F 2.52 1.99

Asp 100 T T 0.66 -0.46 F 2.80 1.66

Gly 101 T T 0.73 0.11 F 1.77 0.62

Ser 102 T T 0.39 0.13 F 1.49 0.40

Thr 103 T 0.67 -0.20 F 1.61 0.24

Cys 104 T T 0.57 0.30 * 0.78 0.32

Ala 105 T T 0.61 0.30 * 0.500.24

Gly 106 T T 0.29 -0.09 * 1.100.33

His 107 T. T 0.59 0.00 * 0.500.33

Gly 108 T 0.23 -0.57 * p 1.35 0.55

Lys 109 T 0.56 -0.50 * p 1.360.30

Cys 1 10 T T 1.19 -0.50 1.72 0.22

Asp 111 T T 0.87 -1.00 * 2.33 0.44

Cys 112 T T 0.94 -0.86 * 2.64 0.12

Gly 113 T T 0.62 -0.86 * F 3.10 0.44

Lys 114 T 0.58 -0.86 * p 2.59 0.14

Cys 115 T 1.24 -0.86 . * F 2.28 0.44

Lys 116 T 0.90 -1.03 * F 1.97 0.76

Cys 117 T 1.28 -1.03 * p 1.94 0.38

Asp 1 18 T T 1.38 -0.1 1 * p 1.81 0.74

Gin 119 T T 0.99 0.07 F 1.49 0.58 Gly 120 T T 1.66 0.50 . * F 1.62 1.07

Trp 121 T T 1.02 -0.07 . * F 2.80 1.07

Tyr 122 T 1.02 0.43 . . 1.12 0.62

Gly 123 T 1.02 0.60 . . 0.84 0.34

Asp 124 T 0.78 0.57 . . 0.56 0.56

Ala 125 T 0.91 0.41 . . 0.28 0.56

Cys 126 T 0.89 0.09 * . 0.30 0.87

Gin 127 T 1.13 0.14 * . 0.30 0.75

Tyr 128 ( : o.8i 0.54 -0.05 1.20

Pro 129 T T 0.81 0.61 . * F 0.50 1.20

Thr 130 T T 0.59 0.04 . ' * F 0.80 1.15

Asn 131 T T 0.94 0.33 . ^• * F 0.65 0.61

Cys 132 T T 0.99 0.06 fc 0.500.57

Asp 133 T 1.28 -0.37 . 0.90 0.79

Leu 134 T 1.53 -0.86 F 1.69 0.98

Thr 135 T 1.54 -1.26 . F 2.18 3.64

Lys 136 T 1.54 -1.44 F 2.52 2.92

Lys 137 T 2.21 -1.04 * F 2.86 5.70

Lys 138 T T 1.61 -1.33 * F 3.40 6.84

Ser 139 T T 1.76 -1.20 * F 3.06 3.39

Asn 140 T T 2.1 1 -0.63 * F 2.57 0.91

Gin 141 T T 2.07 -0.63 * F 2.57 0.91

Met 142 T 1.72 -0.23 * 2.07 1.09

Cys 143 T ^' T 1.68 -0.23 * 2.12 0.91

Lys 144 T T 1.98 -0.23 * F 2.61 0.91

Asn 145 T T 1.09 -0.63 * F 3.40 1.53

Ser 146 T T 0.20 -0.56 * F 3.06 2.00

Gin 147 B T 0.13 -0.44 * F 1.87 0.70

Asp 148 B T 0.50 0.13 * F 0.93 0.23

He 149 B B 0.46 0.11 * 0.040.23

He 150 B B -0.13 0.13 -0.30 0.22

Cys 151 B T -0.18 0.23 0.10 0.13

Ser 152 T T -0.49 0.66 0.20 0.19

Asn 153 T T -1.16 0.46 F 0.35 0.38

Ala 154 T T -0.30 0.34 F 0.65 0.38

Gly 155 T -0.08 0.27 F 0.45 0.39

Thr 156 T 0.24 0.46 0.00 0.13

Cys 157 T T 0.66 0.49 0.20 0.13

His 158 T T -0.01 -0.01 . * 1.10 0.25

Cys 159 T T 0.62 0.13 * 0.50 0.09

Gly 160 T T 0.30 -0.36 * 1.10 0.35 Arg 161 T 0.61 -0.36 . * 1.24 0.14

Cys 162 T T 1.28 -0.86 * F 2.23 0.43

Lys 163 T T 1.01 -1.03 . * F 2.57 0.69

Cys 164 T T 1.68 -1.07 . * F 2.91 0.47

Asp 165 T T 1.68 -1.07 . * F 3.40 1.48

Asn 166 T T 1.27 -1.21 . * F 2.91 0.73

Ser 167 T T 1.59 -0.83 . . F 2.72 1.83

Asp 168 T T 0.73 -0.97 . . F 2.38 1.08

Gly 169 T T 0.54 -0.29 . . F 1.59 0.56

Ser 170 B T 0.30 -0.04 . . F 0.85 0.31

Gly 171 B T -0.04 0.33 . * F 0.25 0.29

Leu 172 B B 0.30 0.76 .• * F -0.45 0.29

Val 173 B B -0.40 0.33 . * -0.30 0.43

Tyr 174 B T -0.72 0.73 . * -0.20 0.38

Gly 175 T T -0.42 0.87 . . 0.20 0.24

Lys 176 T T -0.74 0.19 . . 0.50 0.57

Phe 177 T T 0.07 0.11 . . 0.84 0.20

Cys 178 T T 0.92 -0.64 * * 2.08 0.33

Glu 179 T 1.28 -1.07 * . 2.22 0.28

Cys 180 T T 1.62 -1.07 * . 2.76 0.62

Asp 181 T T 0.91 -1.86 * * F 3.40 2.01

Asp 182 T T 0.72 -1.86 * * F 2.91 0.62

Arg 183 T T 1.39 -1.17 . * F 2.88 0.81

Glu 184 T 1.39 -1.74 . * 2.50 0.81

Cys 185 T 2.06 -1.74 . * 2.47 0.81

He 186 T 1.74 -1.74 * * 2.44 0.72

Asp 187 T T 1.74 -1.26 . * F 3.10 0.60

Asp 188 T C 1.63 -1.26 * * F 2.74 1.94

Glu 189 A T 0.74 -1.83 * . F 2.23 4.79

Thr 190 A T 0.74 -1.83 * . F 1.92 2.01

Glu 191 A 1.29 -1.26 * . F 1.26 0.65

Glu 192 A 0.94 -0.83 . . F 0.95 0.37

He 193 T 0.91 -0.40 . . 1.15 0.25

Cys 194 T T 0.57 -0.39 . . 1.60 0.20

Gly 195 T T 0.92 0.04 . . 1.25 0.1 1

Gly 196 T T 0.26 0.04 * . F 1.65 0.32

His 197 T T 0.01 -0.07 . . F 2.50 0.32

Gly 198 T T 0.23 0.11 . . F 1.65 0.51

Lys 199 T T 0.56 0.26 . . 1.25 0.28

Cys 200 T T 0.90 0.26 . . 1.00 0.20

Tyr 201 . T T 0.58 0.16 . . 0.75 0.33 Cys 202 T T 0.37 0.30 . 0.50 0.09

Gly 203 T T 0.04 1.06 * 0.20 0.26

Asn 204 T T 0.04 1.06 * 0.20 0.09

Cys 205 T T 0.12 0.30 * * 0.50 0.33

Tyr 206 T 0.02 0.23 * 0.30 0.34

Cys 207 T T 0.40 0.23 fc * 0.50 0.21

Lys 208 T T 0.71 0.74 fc * 0.20 0.40

Ala 209 T T 0.37 0.67 ι< * 0.20 0.35

Gly 210 T T 1.03 0.34 * * 0.81 0.65

Trp 211 T 1.32 -0.23 * * 1.52 0.54

His 212 T ( Z 1.32 -0.23 * * 1.98 1.07

Gly 213 T T 1.28 -0.16 * F 2.49 0.58

Asp 214 T T 1.17 -0.59 * F 3.10 0.96

Lys 215 T T 1.51 -0.71 * F 2.79 0.61

Cys 216 A T 1.13 -0.81 * 2.08 1.07

Glu 217 A ' T 1.17 -0.67 * 1.62 0.34

Phe 218 A T 0.62 -0.67 * 1.31 0.29

Gin 219 A T 0.31 0.01 * 0.10 0.37

Cys 220 A T 0.06 -0.07 * 0.70 0.31

Asp 221 A T 0.43 0.36 * 0.10 0.56

He 222 A . C 0.43 0.49 * -0.06 0.34

Thr 223 T C 0.83 0.09 * F 1.28 1.09

Pro 224 T T 0.88 -0.10 F 2.27 0.88

Trp 225 T T 1.66 -0.10 * F 2.76 2.50

Glu 226 T T 1.77 -0.79 * F 3.40 3.39

Ser 227 T T 1.99 -1.27 F 3.06 4.29

Lys 228 T T 1.99 -1.13 * * F 2.72 2.19

Arg 229 T T 1.90 -1.56 * * F 2.38 1.82

Arg 230 T T 1.98 -1.17 * F 2.38 1.82

Cys 231 T 1.98 -1.13 F 2.18 1.41

Thr 232 T 1.93 -1.13 * F 2.52 1.20

Ser 233 T C 1.93 -0.70 * * F 2.71 0.61

Pro 234 T T 0.93 -0.70 * * F 3.40 2.27

Asp 235 T T 0.16 -0.59 * * F 3.06 1.10

Gly 236 T T 0.52 -0.50 * F 2.58 0.44

Lys 237 T 0.83 -0.50 * F 2.35 0.38

He 238 T 1.24 -0.53 * 2.47 0.37

Cys 239 T T 1.11 -0.53 * 2.64 0.73

Ser 240 T T 0.80 -0.53 * F 3.10 0.36

Asn 241 T T 0.48 -0.04 F 2.49 0.74

Arg 242 T T -0.42 -0.16 F 2.18 0.74 Gly 243 1 B T -0.20 -0.09 . . 1 F 1.47 0.41

Thr 244 . B T 0.12 0.10 * . 1 F 0.56 0.14

Cys 245 B T 0.42 0.13 * . 0.10 0.07

Val 246 . B T -0.24 0.13 * . 0.10 0.12

Cys 247 T T -0.67 0.27 * * 0.50 0.04

Gly 248 T T -0.99 0.27 . . 0.50 0.12

Glu 249 T T -0.71 0.27 . . 0.50 0.09

Cys 250 T T -0.04 0.13 . . 0.50 0.22

Thr 251 T -0.04 -0.44 * 0.90 0.37

Cys 252 T 0.62 -0.23 . . 0.90 0.16

His 253 T 0.76 -0.23 . . 1.24 0.50

Asp 254 T 0.44 -0.37 . * 1.58 0.54

Val 255 T 0.77 -0.37 . * 2.07 1.44

Asp 256 T ( Z 1.08 -0.51 * F 2.86 1.05

Pro 257 T T 1.46 -1.01 * * F 3.40 1.05

Thr 258 T T 1.14 -0.10 * * F 2.76 1 48

Gly 259 T T 1.14 -0.31 . * 1 2.27 0.88

Asp 260 T 1.11 -0.31 . * F 1.73 0.95

Trp 261 T 1.08 -0.06 * F 1.39 0.46

Gly 262 . Z 0.94 -0.04 . * 1 F 0.85 0.63

Asp 263 T 1.26 -0.04 . * F 1.05 0.38

He 264 T 1.29 -0.04 . * 0.90 0.60

His 265 T T 0.62 -0.47 * . 1.10 0.87

Gly 266 T T 0.91 -0.33 * * F 1.25 0.28

Asp 267 T T 0.59 -0.33 . * F 1.56 0.69

Thr 268 T T 0.59 -0.44 . * F 1.87 0.27

Cys 269 T T 1.48 -0.94 * * 2.33 0.46

Glu 270 T T 1.62 -1.37 . . 2.64 0.48

Cys 271 T T 1.97 -1.37 * . F 3.10 0.65

Asp 272 T T 1.30 -1.86 * . F 2.94 2.01

Glu 273 T 1.72 -1.86 * * F 2.28 0.62

Arg 274 T T 1.80 -1.86 * . F 2.32 2.28

Asp 275 T T 0.94 -1.93 * . F 2.01 1.38

Cys 276 T T 1.37 -1.29 * . 1.40 0.59

Arg 277 T T 1.37 -0.53 * . 1.40 0.47

Ala 278 B B 1.48 -0.53 * . 0.60 0.47

Val 279 B B 1.12 -0.53 * . 1.09 1.73

Tyr 280 B T 0.82 -0.34 * * 1.53 1.38

Asp 281 T T 1.49 0.04 * . 1.67 1.83

Arg 282 T T 1.38 -0.46 * * F 2.76 4.12

Tyr 283 T T 1.27 -1.10 * * F 3.40 4.39 Ser 284 T T 1.46 -1.07 * > F 3.06 2.28

Asp 285 T 1.40 -0.50 * F 2.07 0.62

Asp 286 T 1.06 -0.11 * F 1.73 0.53

Phe 287 T 0.91 -0.44 * ' » 1.24 0.39

Cys 288 T T 0.81 -0.33 . 1.10 0.32

Ser 289 T T 1.11 0.10 0.50 0.19

Gly 290 T T 0.44 0.50 . ^■ fc p 0.35 0.38

His 291 T T 0.44 0.29 . ^■ fc p 0.87 0.38

Gly 292 T 0.48 0.11 . ^> ^► F 0.89 0.46

Gin 293 T 0.80 0.30 * ^■ 0.96 0.25

Cys 294 T T 1.21 0.30 * * 1.38 0.18

Asn 295 T T 0.89 -0.20 . ' fc 2.20 0.36

Cys 296 T T 0.92 -0.06 . ' fc 1.98 0.11

Gly 297 T T 0.60 -0.46 * ' 2.04 0.34

Arg 298 T 0.64 -0.46 * ^• 1.90 0.11

Cys 299 T T 0.72 -0.86 * fc 2.46 0.43

Asp 300 T T 0.38 -0.93 * fc 2.52 0.44

Cys 301 T T 0.76 -0.93 * fc 2.80 0.22

Lys 302 T T 0.86 -0.01 * fc 2.22 0.43

Ala 303 T 0.40 0.17 * fc 1.14 0.40

Gly 304 T 1.11 0.60 fc 0.56 0.75

Trp 305 T 1.16 0.03 fc 0.92 0.75

Tyr 306 T 1.16 0.03 fc 1.13 1.48

Gly 307 T T 1.11 0.10 * F 1.67 0.80

Lys 308 T T 1.67 -0.33 * F 2.76 1.32

Lys 309 T T 1.80 -0.74 F 3.40 1.15

Cys 310 T T 2.09 -1.07 F 3.06 1.79

Glu 311 T 2.03 -1.10 F 2.52 1.55

His 312 T C 1.71 -0.71 . F 2.18 1.04

Pro 313 T T 1.36 -0.14 . F 1.74 1.04

Gin 314 T T 0.50 -0.23 . F 1.25 0.87

Ser 315 T Γ 0.87 0.46 F 0.35 0.52

Cys 316 B T 0.28 0.34 F 0.25 0.45

Thr 317 B C 0.31 0.41 -0.40 0.27

Leu 318 B C 0.52 0.01 -0.10 0.34

Ser 319 B C 0.22 -0.37 * 0.65 1.11

Ala 320 A A -0.37 -0.56 * * F 0.90 1.03

Glu 321 A A 0.41 -0.36 * * p 0.45 0.87

Glu 322 A A 0.77 -1.04 * * p 0.90 1.28

Ser 323 . A T 0.91 -1.43 * * F 1.642.53

He 324 A . r 1.21 -1.36 * F 1.83 0.78 Arg 325 A T 1.46 -0.96 * F 2.17 0.78

Lys 326 A T 1.16 -0.53 * F 2.51 0.58

Cys 327 T T 0.86 -0.53 * F 3.40 1.11

Gin 328 T T 1.16 -0.83 * * p 2.91 0.76

Gly 329 T T 1.23 -0.83 * * F 2.57 0.63

Ser 330 T T 0.91 -0.14 * * p 1.93 0.97

Ser 331 T 0.20 -0.29 _* F 1.39 0.87

Asp 332 T 0.57 -0.11 * * 1 c 1.05 0.47

Leu 333 ( : 0.22 -0.16 * * p 1.16 0.47

Pro 334 T 0.68 -0.11 * * p 1.67 0.35

Cys 335 T T 0.63 -0.50 * p 2.18 0.41

Ser 336 T T 0.98 -0.07 * p 2.49 0.49

Gly 337 T T 0.31 -0.76 * 1 FT 3.10 0.63

Arg 338 T T 1.12 -0.61 * 1 F 2.79 0.63

Gly 339 T 0.67 -1.19 * F 2.56 0.82

Lys 340 T 0.99 -1.00 * * 1 F 2.53 0.44

Cys 341 T T 1.33 -1.00 * * F 2.70 0.22

Glu 342 T T 1.01 -1.00 * * 2.52 0.45

Cys 343 T T 0.59 -0.86 * * 2.80 0.12

Gly 344 T T 0.27 -0.37 2.22 0.33

Lys 345 T -0.02 -0.37 1.74 0.10

Cys 346 T 0.43 0.39 0.86 0.29

Thr 347 T 0.22 0.24 0.58 0.46

Cys 348 T 0.54 0.24 * 0.64 0.36

Tyr 349 0.89 0.67 * 0.28 0.66

Pro 350 T C 0.96 0.10 . F 1.47 0.76

Pro 351 T T 1.73 -0.39 * . F 2.76 2.78

Gly 352 T T 1.19 -0.96 * . F 3.40 3.47

Asp 353 T T 1.61 -1.07 * . F 3.06 1.67

Arg 354 T 1.51 -0.74 * * F 2.52 1.69

Arg 355 T 1.77 -0.74 * * F 2.18 1.69

Val 356 T 1.67 -1.17 * * 1.69 2.02

Tyr 357 T 1.34 -0.69 * * 1.35 1.49

Gly 358 T T 1.34 -0.11 * * F 1.25 0.41

Lys 359 T T 0.57 -0.11 * F 1.25 0.95

Thr 360 T T 0.46 -0.19 * * F 1.59 0.33

Cys 361 T T 1.31 -0.94 * * F 2.23 0.55

Glu 362 T 1.67 -1.37 * 2.22 0.46

Cys 363 T T 2.12 -1.37 * 2.76 0.62

Asp 364 T T 1.41 -1.86 * F 3.40 2.28

Asp 365 T T 1.72 -1.86 * F 2.91 0.70 Arg 366 T T 2.39 -1.86 * . F 3.03 2.28

Arg 367 T 1.58 -2.43 * . F 2.80 2.28

Cys 368 T 2.24 -1.74 . . F 2.77 1.12

Glu 369 T 1.90 -1.74 . . F 2.59 0.96

Asp 370 T T 1.04 -1.31 * . F 3.10 0.48

Leu 371 T T 0.08 -0.67 * . F 2.79 0.67

Asp 372 T T -0.70 -0.60 . . F 2.48 0.29

Gly 373 T T -0.38 -0.03 * 1.72 0.09

Val 374 B -0.72 0.40 * 0.21 0.11

Val 375 B -0.76 0.14 * -0.10 0.07

Cys 376 T T -0.29 0.64 0.20 0.09

Gly 377 T T -0.60 0.64 0.20 0.12

Gly 378 T T -0.92 0.49 . . F 0.35 0.23

His 379 T T -0.37 0.41 . . F 0.35 0.23

Gly 380 T -0.18 0.23 . . F 0.45 0.32

Thr 381 T 0.14 0.37 * . F 0.45 0.17

Cys 382 T T 0.60 0.37 * 0.50 0.12

Ser 383 T T 0.28 -0.13 1.28 0.25

Cys 384 T T -0.54 0.01 * 0.86 0.09

Gly 385 T T -0.87 0.17 * 1.04 0.13

Arg 386 T -0.56 0.17 * 1.02 0.05

Cys 387 B T 0.22 -0.21 * 1.80 0.16

Val 388 B 0.18 -0.79 * 1.52 0.32

Cys 389 B 0.56 -0.79 * * 1.34 0.16

Glu 390 T T 0.20 0.13 * 0.86 0.32

Arg 391 T T -0.26 0.34 0.68 0.38

Gly 392 T T 0.46 0.13 0.50 0.69

Trp 393 T T 0.50 -0.44 1.10 0.80

Phe 394 T 0.50 0.24 * 0.30 0.34

Gly 395 T 0.50 0.81 * 0.00 0.18

Lys 396 T 0.36 0.79 * 0.00 0.30

Leu 397 T 0.49 0.37 * * 0.64 0.47

Cys 398 T 0.89 0.01 * * 0.98 0.74

Gin 399 T 1.63 -0.41 * 1.92 0.72

His 400 T C 1.31 -0.41 * * 2.41 1.75

Pro 401 T T 1.27 -0.53 * * F 3.40 1.75

Arg 402 T T 1.48 -0.70 * F 3.06 1.63

Lys 403 T T 1.83 -0.49 F 2.42 1.18

Cys 404 T 1.83 -0.50 * 2.03 1.1 1

Asn 405 A _ C 1.87 -0.93 1.14 0.98

Met 406 A C 2.08 -0.93 * F 0.95 0.85 Thr 407 A Z 1.67 -0.53 * . F 1.44 2.73

Glu 408 A A 1.67 -0.71 * p 1.58 2.28

Glu 409 A A 2.33 -1.11 * . F 1.92 4.61

Gin 410 A T 1.52 -1.33 * . F 2.66 5.13

Ser 411 T r 1.46 -1.13 * . F 3.40 2.44

Lys 412 T Γ 1.77 -0.56 * . F 2.91 0.76

Asn 413 . Γ Z 1.47 -0.56 * . F 2.62 0.76

Leu 414 ' r C 0.88 -0.57 2.38 0.76

Cys 415 T 0.88 -0.46 ^• fc 1.99 0.38

Glu 416 T 0.83 -0.46 ' " . F 2.05 0.40

Ser 417 T r -0.10 -0.43 ^■ * . F 2.50 0.48

Ala 418 T T -0.91 -0.43 ^• " . F 2.25 0.62

Asp 419 T T -0.77 -0.31 ' * . F 2.00 0.30

Gly 420 T T -0.40 0.26 * . 1.00 0.12

He 421 B -0.74 0.26 fc 0.15 0.16

Leu 422 B -0.40 0.19 * * 0.15 0.09

Cys 423 T Γ -0.16 0.19 1.00 0.19

Ser 424 T Γ -0.46 0.19 fc * F 1.40 0.27

Gly 425 T Γ -0.78 -0.11 * F 2.25 0.43

Lys 426 T Γ 0.08 -0.23 _* F 2.50 0.43

Gly 427 T 0.22 -0.30 * F 2.05 0.44

Ser 428 T 0.54 -0.11 ' k * F 1.80 0.24

Cys 429 T 0.89 -0.11 fc * 1.40 0.12

His 430 T Γ 0.57 -0.11 1.35 0.24

Cys 431 T Γ -0.37 0.03 fc 0.50 0.10

Gly 432 T Γ -0.69 0.33 fc 0.50 0.12

Lys 433 T Γ -0.69 0.33 fc 0.50 0.05

Cys 434 T -0.61 0.21 fc 0.300.12

He 435 A T -0.58 0.14 fc 0.10 0.13

Cys 436 A B 0.09 -0.29 fc 0.30 0.11

Ser 437 A C 0.14 -0.29 fc 0.50 0.35

Ala 438 A C -0.14 0.06 * -0.10 0.52

Glu 439 A T -0.37 0.13 0.25 1.53

Glu 440 A B T 0.22 0.24 0.10 0.80

Trp 441 A B T 0.54 0.24 * 0.25 1.06

Tyr 442 A B T 0.84 0.17 * 0.10 0.61

He 443 B T 0.73 0.17 * 0.10 0.61

Ser 444 B T 0.07 0.96 * -0.20 0.50

Gly 445 T 0.07 0.61 * p 0.15 0.17

Glu 446 T -0.31 -0.14 * 0.90 0.41

Phe 447 T -0.07 -0.26 * 1.24 0.16 Cys 448 T T 0.82 -0.64 * K 2.08 0.28

Asp 449 T T 1.23 -1.07 * 2.420.27

Cys 450 T T 1.58 -1.07 * 2.760.60

Asp 451 T T 0.91 -1.86 * F 3.401.87

Asp 452 T T 1.61 -1.86 * F 2.910.60

Arg 453 T T 2.32 -1.86 * * F 2.721.87

Asp 454 T T 2.29 -2.43 * * F 2.722.24

Cys 455 T T 2.96 -1.93 . . F 2.721.83

Asp 456 T 2.61 -1.93 F 2.521.56

Lys 457 T 1.80 -1.50 * F 2.710.92

His 458 T T 0.80 -0.81 * F 3.401.42

Asp 459 T T 0.13 -0.70 F 2.910.60

Gly 460 T T 0.49 -0.13 . 2.120.16

Leu 461 B T 0.14 0.36 0.780.17

He 462 B 0.10 0.29 0.240.10

Cys 463 T T -0.21 0.69 * 0.200.16

Thr 464 T T -1.10 0.69 * F 0.350.20

Gly 465 T T -1.42 0.69 * F 0.350.20

Asn 466 T T -0.91 0.57 * F 0.350.20

Gly 467 T -0.69 0.39 F 0.450.18

He 468 T -0.37 0.47 * 0.000.10 -

Cys 469 T T -0.06 0.47 * 0.420.06

Ser 470 T T -0.38 0.47 * 0.640.10

Cys 471 T T -0.38 0.61 0.860.08

Gly 472 T T -0.70 -0.07 1.980.24

Asn 473 T T -0.10 -0.07 2.200.10

Cys 474 T T 0.57 0.46 1.080.19

Glu 475 T T 0.52 -0.11 1.760.32

Cys 476 T T 0.90 -0.11 1.540.20

Trp 477 T T 1.24 0.40 0.420.39

Asp 478 T T 0.90 0.23 0.500.36

Gly 479 T T 1.57 0.66 F 0.350.67

Trp 480 T T . 0.98 0.49 F 0.501.02

Asn 481 T C 0.98 0.07 • F 0.450.62

Gly 482 T C 1.27 0.64 * . F 0.150.33

Asn 483 T C 0.38 0.21 * 0.300.55

Ala 484 T C 0.43 -0.01 0.900.24

Cys 485 A T -0.09 0.50 -0.20 0.25

Glu 486 A B -0.43 0.76 -0.60 0.13

He 487 A T -0.39 0.79 -0.20 0.13

Trp 488 A T -0.39 0.67 -0.20 0.32 Leu 489 A C -0.04 0.10 -0.10 0.32

Gly 490 T T 0.41 0.86 * p 0.560.72

Ser 491 T C 0.02 0.60 F 0.721.05

Glu 492 T C 0.52 0.11 F 1.231.63

Tyr 493 T C 0.42 -0.14 . 1.892.11

Pro 494 T 0.84 -0.14 2.102.01

Ter 495 T 0.80 -0.10 1.891.48

Table III

Res Position II III IV V VI VII VIII IX X XI XII XIII XIV

Met 1 A A 0.10 -0.19 0.30 0.92

Glu 2 A A -0.32 -0.11 * * 0.300.72

Thr 3 A A 0.18 0.14 * -0.30 0.47

Gly 4 A A 0.68 -0.29 * 0.30 0.93

Ala 5 A A 0.86 -0.90 * . F 0.90 1.05

Leu 6 A A 1.46 -0.47 . F 0.60 1.12

Arg 7 A B 0.64 -0.56 . F 0.90 1.96

Arg 8 B 0.14 -0.30 * . F 0.80 1.60

Pro 9 A B 0.28 -0.11 . F 0.60 1.60

Gin 10 A B 0.06 -0.37 . F 0.60 1.26

Leu 11 A B 0.06 0.31 * -0.30 0.53

Leu 12 A B -0.87 1.00 -0.60 0.28

Pro 13 A B -1.79 1.26 * -0.60 0.14

Leu 14 A B -2.39 1.54 -0.60 0.14

Leu 15 A B -3.06 1.54 -0.60 0.14

Leu 16 A B -2.59 1.43 -0.60 0.05

Leu 17 A B -2.12 1.43 -0.60 0.06

Leu 18 A B -2.58 1.17 -0.60 0.07

Cys 19 B T -1.98 1.06 * * -0.20 0.04

Gly 20 T T -1.06 0.80 * * 0.200.08

Gly 21 T T -0.83 0.11 * F 0.65 0.20

Cys 22 B T -0.37 -0.07 * F 0.85 0.37

Pro 23 B 0.10 -0.21 * * F 0.96 0.37

Arg 24 T - T 0.10 -0.21 * F 1.87 0.37

Ala 25 T T 0.44 -0.07 * F 2.18 0.37

Gly 26 T T 0.79 -0.24 * F 2.49 0.38

Gly 27 T T 1.14 -0.67 * F 3.100.34 .

Cys 28 T 1.01 -0.19 * F 2.29 0.48

Asn 29 T C 0.30 -0.26 * F 1.98 0.48

Glu 30 B T 0.08 -0.07 F 1.47 0.48

Thr 31 B T 0.42 0.19 * F 0.56 0.74

Gly 32 B T 0.88 -0.39 * F 0.85 0.80

Met 33 A A 0.73 -0.79 * 0.60 0.91

Leu 34 A A 0.52 -0.10 * * 0.30 0.52

Glu 35 A A -0.29 -0.16 * 0.30 0.81

Arg 36 A A -0.64 0.10 * * -0.30 0.67 Leu 37 A A -0.64 0.06 * * -0.30 0.44

Pro 38 A A 0.00 -0.20 * * . 0.30 0.25

Leu 39 A ^' A 0.22 -0.20 * * . 0.30 0.26

Cys 40 A A -0.48 0.30 * * . -0.30 0.31

Gly 41 A A -1.18 0.40 * * . -0.30 0.18

Lys 42 A A -0.37 0.47 * * . -0.60 0.21

Ala 43 A A -0.76 -0.21 * * 0.30 0.67

Phe 44 A A -0.54 -0.17 * * 0.30 0.67

Ala 45 A A -0.22 0.01 * * . -0.30 0.33

Asp 46 A A 0.17 0.44 * * . -0.60 0.32

Met 47 A A -0.73 -0.06 * * . 0.30 0.75

Met 48 A A -0.14 -0.20 * * 0.30 0.55

Gly 49 A A -0.30 -0.70 * * 0.60 0.55

Lys 50 A . B 0.00 -0.06 " . 0.30 0.41

Val 51 A . B 0.04 0.24 * . -0.30 0.44

Asp 52 A . B 0.36 -0.37 * 0.30 0.89

Val 53 A . B 0.29 0.1 1 * -0.30 0.47

Trp 54 A . B 0.63 0.69 * -0.60 0.34

Lys 55 A . B -0.22 0.44 * -0.60 0.32

Trp 56 A . B 0.33 1.13 -0.60 0.36

Cys 57 A . B 0.33 0.87 -0.60 0.46

Asn 58 . B C 0.49 -0.04 * 0.500.40

Leu 59 . B C -0.11 0.74 * -0.40 0.33

Ser 60 . B C -1.01 0.51 * -0.40 0.43

Glu 61 B B -0.97 0.59 * -0.60 0.20

Phe 62 B B -0.54 0.94 -0.60 0.38

He 63 B B -0.54 1.01 * -0.60 0.44

Val 64 B B -0.03 0.63 * -0.60 0.44

Tyr 65 B B - -0.43 1.01 -0.60 0.68

Tyr 66 B B -0.74 1.01 * -0.60 0.84

Glu 67 B T -0.04 0.81 * -0.05 1.63

Ser 68 B T 0.18 0.57 -0.05 1.68

Phe 69 T T 0.72 0.39 * 0.50 0.57

Thr 70 T T 0.97 0.11 * F 0.65 0.48

Asn 71 T C 0.61 0.11 F 0.45 0.62

Cys 72 A T 0.61 0.34 * F 0.25 0.71

Thr 73 A A 0.32 -0.44 * F 0.45 0.85

Glu 74 A A 1.02 -0.43 * * 0.30 0.53

Met 75 A A 0.48 -0.43 * . 0.45 1.60

Glu 76 A A -0.38 -0.36 * . 0.300.82

Ala 77 A A -0.06 -0.20 * . 0.30 0.35 Asn 78 A A -0.41 0.23 * -0.30 0.35

Val 79 A B -0.66 0.19 * -0.30 0.11

Val 80 A B -0.34 0.94 * -0.60 0.17

Gly 81 A B -0.56 1.36 * -0.60 0.1 1

Cys 82 T 0.03 1.39 * 0.00 0.23

Tyr 83 T -0.18 1.14 * 0.00 0.50

Trp 84 B r -0.13 0.93 -0.20 0.78

Pro 85 Γ c 0.13 1.19 o . F 0.30 1.20

Asn 86 Γ c 0.48 1.11 . F 0.15 0.77

Pro 87 Γ 0.80 0.76 . F 0.30 1.27

Leu 88 0.34 0.27 fc . F 0.25 0.81

Ala 89 -0.26 0.63 fc . F -0.05 0.44

Gin 90 1 3 1 B -0.36 0.91 fc -0.60 0.20

Gly 91 B B -0.70 0.97 fc -0.60 0.35

Phe 92 B B -1.38 0.71 fc -0.60 0.34

He 93 B B -0.60 0.90 -0.60 0.14

Thr 94 B B 0.10 1.00 fc -0.60 0.19

Gly 95 B B 0.10 0.57 fc -0.60 0.43

He 96 B B -0.26 0.19 fc -0.15 1.06

His 97 B B -0.26 0.29 fc -0.30 0.64

Arg 98 B T 0.33 0.59 fc -0.20 0.56

Gin 99 B T 0.64 0.54 fc -0.05 1.06

Phe 100 B T 0.32 0.26 fc 0.25 1.26

Phe 101 T Γ 0.90 0.33 fc 0.50 0.34

Ser 102 T Γ 0.08 0.81 * 0.20 0.29

Asn 103 T Γ -0.03 1.06 0.20 0.25

Cys 104 T Γ 0.08 0.27 * 0.50 0.47

Thr 105 T -0.08 -0.51 1.20 0.69

Val 106 A T 0.59 -0.26 * 0.70 0.32

Asp 107 A B 0.08 -0.16 * 0.30 0.81

Arg 108 A B 0.08 -0.04 * 0.30 0.46

Val 109 A B 0.74 -0.53 * 0.75 1.08

His 110 A B 0.84 -1.17 * 0.75 1.08

Leu 111 A C 1.49 -0.74 * 0.80 0.85

Glu 112 A C 1.49 -0.31 * F 0.80 1.78

Asp 113 A C 1.38 -0.96 * * F 1.10 2.18

Pro 114 T C 1.38 -1.46 * F 1.50 4.58

Pro 115 T T 0.60 -1.50 * F 1.70 1.96

Asp 116 A T 0.52 -0.81 * F 1.15 0.97

Glu 117 A T 0.31 -0.13 * * 0.70 0.44

Val 118 A B -0.50 -0.13 * 0.30 0.44 Leu 1 19 B B -1.18 0.13 -0.30 0.22

He 120 B B -1.82 0.81 -0.60 0.09

Pro 121 B B -2.71 1.46 -0.60 0.09

Leu 122 B B -2.92 1.50 -0.60 0.07

He 123 B B -2.92 1.24 -0.60 0.16

Val 124 B B -2.97 1.20 -0.60 0.08

He 125 B B -2.89 1.41 -0.60 0.07

Pro 126 B B -2.99 1.41 -0.60 0.08

Val 127 B B -3.03 1.21 -0.60 0.16

Val 128 B B -2.73 1.21 -0.60 0.17

Leu 129 B B -2.48 1.03 -0.60 0.11

Thr 130 B B -2.18 1.21 -0.60 0.15

Val 131 B B -2.31 1.07 -0.60 0.20

Ala 132 A B -2.27 0.86 -0.60 0.25

Met 133 A B -2.27 0.86 -0.60 0.14

Ala 134 A B -2.31 1.01 -0.60 0.14

Gly 135 A ] B -2.29 1.01 * -0.60 0.10

Leu 136 A . B -1.32 1.43 < -0.60 0.11

Val 137 A . B -1.03 0.81 -0.60 0.21

Val 138 A 1 B -0.39 0.70 * -0.26 0.29

Trp 139 B B 0.31 0.27 < 0.38 0.70

Arg 140 B B 0.34 -0.41 F 1.62 1.84

Ser 141 B T 1.16 -0.57 . F 2.66 3.58

Lys 142 T T 1.70 -1.21 . F 3.40 5.68

Arg 143 T T 1.74 -1.64 ' F 3.064.19

Thr 144 T T 1.22 -0.96 F 2.72 2.58

Asp 145 A T 0.72 -0.66 F 1.98 1.06

Thr 146 A B 0.63 -0.23 ^• F 0.79 0.69

Leu 147 A B 0.20 0.20 -0.30 0.61

Leu 148 A B -0.30 0.14 -0.30 0.47

Ter 149 A B -0.38 0.57 -0.60 0.42

Table IV

Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV

Met 1 A A -0.76 0.27 . * -0.30 0.49

Arg 2 A A -1.07 0.34 * * -0.30 0.39

Leu 3 A A -1.49 0.70 * * -0.60 0.26

Leu 4 A A -1.40 0.96 . * -0.60 0.22

Ala 5 A A -1.82 0.73 * -0.60 0.15

Phe 6 A A -2.03 1.41 * * -0.60 0.15

Leu 7 A A -2.73 1.41 . * -0.60 0.15

Ser 8 A A -2.73 1.23 . . -0.60 0.15

Leu 9 A A -2.78 1.41 . . -0.60 0.14

Leu 10 A A -3.00 1.27 * . -0.60 0.13

Ala 11 A A -2.30 1.27 * . -0.60 0.08

Leu 12 A A -1.49 1.29 . . -0.60 0.17

Val 13 A A -1.50 0.60 . . -0.60 0.35

Leu 14 A A -1.03 0.40 . . -0.02 0.50

Gin 15 A A B -0.53 0.33 . . F 0.41 0.60

Glu 16 A T -0.53 0.13 . . F 1.24 1.17

Thr 17 A T -0.02 -0.01 * F 2.12 1.43

Gly 18 r T 0.02 -0.31 . . F 2.80 1.11

Thr 19 A T 0.62 -0.03 * * F 1.97 0.53

Ala 20 A 0.73 0.40 . . F 0.890.57

Ser 21 C 0.78 -0.09 . . F 1.56 1.12

Leu 22 A C 1.09 -0.51 * . F 1.38 1.55

Pro 23 A A 1.54 -1.00 * . F 0.902.66

Arg 24 A A 1.90 -1.50 * * F 0.90 3.88

Lys 25 A A 2.60 -1.89 . . F 0.90 9.42

Glu 26 A A 3.01 -2.57 . . F 0.90 11.93

Arg 27 A A 3.82 -3.00 . . F 0.90 11.93

Lys 28 A A 4.03 -3.00 . . F 0.90 10.33

Arg 29 A A 3.92 -3.00 . . F 0.90 10.33

Arg 30 A A 3.28 -2.60 . * F 0.90 9.13

Glu 31 A A 3.07 -1.99 * . F 0.90 4.52

Glu 32 A A 3.07 -1.56 . . F 1.24 3.57

Gin 33 A A 3.02 -1.56 * . F 1.58 3.57

Met 34 A C 2.57 -1.56 * . F 2.12 3.57

Pro 35 A T 2.46 -1.13 * . F 2.66 2.04

Are 36 T T 2.16 -1.13 * . F 3.40 1.97 Glu 37 A T 1.46 -1.14 * F 2.66 2.66

Gly 38 T T 1.46 -0.97 * * F 2.72 1.49

Asp 39 A 1.20 -1.40 * F 1.78 1.32

Ser 40 A 0.60 -0.76 * F 1.29 0.57

Phe 41 B 0.28 -0.07 0.50 0.47

Glu 42 B -0.53 -0.07 0.50 0.44

Val 43 B -0.08 0.61 -0.40 0.27

Leu 44 B -0.08 0.23 * -0.10 0.61

Pro 45 A 0.22 -0.16 * 0.50 0.56

Leu 46 A T 0.07 -0.16 * 0.85 1.27

Arg 47 A T -0.74 -0.16 * F 1.00 1.14

Asn 48 B T T 0.11 -0.16 * F 1.25 0.61

Asp 49 B T 0.71 -0.19 * F 1.00 1.19

Val 50 B 0.92 -0.44 * F 0.65 0.94

Leu 51 B 1.73 -0.44 * F 0.95 0.97

Asn 52 B T 1.38 -0.44 F 1.45 0.94

Pro 53 T C 1.03 0.31 F 1.50 1.98

Asp 54 T T 1.03 0.10 * F 2.00 2.37

Asn 55 T C 1.03 -0.59 F 3.00 2.56

Tyr 56 B B 0.96 -0.34 * F 1.80 1.23

Gly 57 B B 0.96 -0.09 1.20 0.52

Glu 58 B B 0.36 -0.09 * 0.90 0.53

Val 59 B B 0.06 0.20 * 0.00 0.28

He 60 B B 0.06 -0.17 0.30 0.38

Asp 61 B B 0.06 -0.20 0.30 0.35

Leu 62 B T 0.40 0.56 -0.20 0.75

Ser 63 T C 0.40 -0.09 1.05 1.85

Asn 64 T C 0.44 -0.77 F 1.50 1.91

Tyr 65 A T 1.02 -0.09 * F 1.34 1.91

Glu 66 A 1.02 -0.29 F 1.48 2.06

Glu 67 A 1.59 -0.67 * F 2.12 2.14

Leu 68 B 1.54 -0.31 * F 2.16 2.14

Thr 69 T T 1.54 -0.64 * F 3.40 1.22

Asp 70 T T 1.79 -0.64 * F 3.06 1.18

Tyr 71 T T 0.98 -0.24 * F 2.42 2.48

Gly 72 T T 0.77 -0.24 * F 2.08 1.42

Asp 73 A 1.58 -0.30 * F 1.23 1.31

Gin 74 A 1.03 -0.30 * * F 0.98 1.45

Leu 75 B 1.08 -0.41 * F 1.07 1.09

Pro 76 B 0.47 -0.84 * F 1.46 1.30

Glu 77 B B 0.50 -0.20 * F 0.90 0.56 Val 78 B B 0.20 -0.11 * p 0.81 0.98

Lys 79 B B -0.61 -0.41 * F 0.72 0.85

Val 80 B B -0.39 -0.16 . * F 0.63 0.40

Thr 81 B B -0.39 0.34 * F -0.06 0.55

Ser 82 B -0.98 0.13 * F 0.05 0.42

Leu 83 B -0.43 0.63 « -0.40 0.58

Ala 84 B -0.78 0.47 * -0.40 0.58

Pro 85 A -0.81 0.37 -0.10 0.58

Ala 86 B B -0.80 0.67 F -0.45 0.49

Thr 87 B B -0.71 0.37 F -0.15 0.65

Ser ^■ 88 B B -0.49 0.30 F 0.13 0.65

He 89 B B 0.14 0.37 F 0.41 0.65

Ser 90 B T 0.06 -0.13 . F 1.69 0.90

Pro 91 T C 0.33 -0.23 . F 2.17 0.90

Ala 92 T T 0.33 -0.13 . F 2.80 1.86

Lys 93 B T 0.04 -0.33 . F 2.12 2.00

Ser 94 B 0.72 -0.21 . F 1.64 1.31

Thr 95 B 0.68 -0.21 F 1.36 2.00

Thr 96 B 0.58 -0.29 . F 0.93 0.99

Ala 97 B 0.96 0.20 F 0.20 1.07

Pro 98 B 0.61 0.24 F 0.48 1.14

Gly 99 T 0.61 0.14 F 1.16 1.06

Thr 100 T C 0.92 0.04 F 1.44 1.41

Pro 101 T C 1.02 -0.06 F 2.32 1.46

Ser 102 T T 1.30 -0.06 F 2.80 2.28

Ser 103 T C 0.91 0.00 F 2.32 2.28

Asn 104 T C 0.94 0.13 F 1.44 1.46

Pro 105 T C 1.37 0.19 F 1.36 1.57

Thr 106 T . T 1.37 -0.20 F 2.08 2.30

Met 107 B T 1.36 -0.16 F 1.60 2.21

Thr 108 B 1.34 -0.07 . F 1.60 2.07

Arg 109 B T 0.76 -0.01 . F 2.00 2.07

Pro 110 B T 0.62 0.00 * F 1.80 2.11

Thr 111 B T 0.12 -0.19 F 1.60 1.45

Thr 112 B T -0.09 0.01 F 0.65 0.61

Ala 113 A B -0.59 0.70 F -0.25 0.32

Gly 114 A B -1.00 0.96 . -0.60 0.19

Leu 115 A B -1.09 0.86 -0.60 0.17

Leu 1 16 A B -0.78 0.76 -0.60 0.23

Leu 117 A B -0.68 0.66 * p -0.45 0.40

Ser 1 18 A B -0.09 0.66 F -0.29 0.75 Ser 119 B 0.22 0.37 . . F 0.52 1.46

Gin 120 B T 0.69 0.19 . . F 0.88 2.41

Pro 121 r T 0.69 -0.07 . . F 2.04 1.78

Asn 122 T T 1.29 0.23 . * F 1.60 1.10

His 123 T T 1.28 0.27 . . F 1.29 0.98

Gly 124 r 0.91 0.36 . . 0.78 0.91

Leu 125 T C 0.10 0.50 . . 0.32 0.30

Pro 126 r T -0.54 0.79 . . 0.36 0.18

Thr 127 Γ T -1.21 0.93 . . 0.20 0.14

Cys 128 B T -2.03 1.07 . . -0.20 0.09

Leu 129 B B -2.36 1.03 . . -0.60" 0.04

Val 130 B B -2.36 1.17 . . -0.60 0.02

Cys 131 B B -2.49 1.37 . . -0.60 0.02

Val 132 B B -2.48 1.23 . . -0.60 0.03

Cys 133 B B -2.11 0.93 . . -0.60 0.05

Leu 134 B B -2.16 0.67 . . -0.60 0.13

Gly 135 Γ T -1.54 0.74 . * F 0.35 0.13

Ser 136 Γ T -1.54 0.86 . * F 0.35 0.39

Ser 137 B T -0.69 0.86 . . F -0.05 0.25

Val 138 B T -0.02 0.17 . . 0.10 0.43

Tyr 139 B -0.10 -0.26 . . 0.50 0.53

Cys 140 B T 0.24 0.04 . * 0.10 0.28

Asp 141 B T -0.27 -0.34 . * 0.70 0.63

Asp 142 B T 0.03 -0.30 . * F 0.85 0.33

He 143 B T 0.89 -1.06 . . F 1.30 1.07

Asp 144 A B 0.24 -1.63 . . F 0.90 1.07

Leu 145 A B 0.70 -0.94 . . F 0.75 0.45

Glu 146 A B 0.49 -0.51 . * F 0.75 0.99

Asp 147 A B -0.32 -0.77 . * F 0.99 0.92

He 148 A B 0.36 -0.09 * * F 0.93 0.92

Pro 149 . C 0.47 -0.34 * . F 1.57 0.82

Pro 150 . C 1.39 -0.34 * . F 1.81 0.96

Leu 151 T C 1.08 -0.34 * . F 2.40 2.68

Pro 152 B T 0.49 -0.54 * . F 2.26 2.50

Arg 153 T T 1.13 -0.47 * . F 2.12 1.63

Arg 154 B T 0.53 -0.14 * . F 1.48 3.11

Thr 155 B B 0.50 -0.14 * . 0.69 1.66

Ala 156 B B 0.72 0.19 * * -0.15 1.33

Tyr 157 B B 1.04 0.69 * * -0.60 0.68

Leu 158 B B 0.23 0.69 * * -0.60 0.93

Tyr 159 B B 0.12 0.99 * * -0.60 0.80 Ala 160 B B 0.54 0.89 * * -0.60 0.82

Arg 161 B B 0.24 0.13 * * -0.15 1.94

Phe 162 B B 0.19 0.13 * * -0.30 0.87

Asn 163 B B 1.1 1 -0.24 * * 0.45 1.15

Arg 164 B B 0.47 -0.74 * F 1.08 1.15

He 165 B B 1.17 -0.06 * F 0.81 0.93

Ser 166 B Z 0.47 -0.84 * * F 1.64 1.13

Arg 167 B B 1.17 -0.74 * * F 1.47 0.59

He 168 B B 1.17 -0.74 * * F 1.80 1.45

Arg 169 B B 0.36 -1.43 * 1.47 1.80

Ala 170 A A 1.29 -1.03 * * F 1.29 0.80

Glu 171 A A 1.24 -1.03 * F 1.26 2.27

Asp 172 A A 0.32 -1.29 * F 1.08 1.15

Phe 173 A A 0.90 -0.60 * F 0.75 0.94

Lys 174 A A 0.83 -0.61 * F 0.75 0.78

Gly 175 A A 0.61 -0.61 * * F 0.75 0.94

Leu 176 A A 0.66 0.07 * * F -0.15 0.89

Thr 177 A A 0.77 -0.71 * * F 0.75 0.89

Lys 178 A A 0.58 -0.71 * * F 0.90 1.76

Leu 179 A A 0.53 -0.46 * * F 0.60 1.50

Lys 180 A B 0.07 -1.14 * * F 0.90 1.73

Arg 181 A B 0.58 -0.94 * * F 0.75 0.71

He 182 A B 0.89 -0.56 * F 0.90 1.16

Asp 183 A B 0.84 -0.84 * 0.60 0.93

Leu 184 B T 0.84 -0.44 * * F 0.85 0.77

Ser 185 B T -0.09 0.24 * * F 0.25 0.90

Asn 186 T C -0.50 0.24 * F 0.45 0.38

Asn 187 T C 0.09 0.63 * * F 0.15 0.62

Leu 188 B -0.80 0.33 * * -0.10 0.62

He 189 B 0.01 0.63 * -0.40 0.27

Ser 190 B 0.31 0.23 * F 0.05 0.28

Ser 191 B 0.31 0.23 * * F 0.05 0.54

He 192 B -0.28 -0.46 * F 0.80 1.29

Asp 193 B T -0.17 -0.64 * F 1.15 0.98

Asn 194 A T 0.83 -0.24 * * F 0.85 0.63

Asp 195 A T 0.32 -0.63 * F 1.30 1.76

Ala 196 A T -0.19 -0.63 * 1.00 0.87

Phe 197 A A 0.67 0.06 * -0.30 0.45

Arg 198 A A 0.08 0.16 * -0.30 0.36

Leu 199 A A -0.73 0.66 * -0.60 0.36

Leu 200 A A -0.73 0.84 * -0.60 0.35 His 201 A A -0.14 0.46 * * -0.60 0.31

Ala 202 A A -0.26 0.46 * * -0.60 0.62

Leu 203 A A -1.26 0.46 * * -0.60 0.62

Gin 204 A A -1.26 0.46 * -0.60 0.32

Asp 205 A B -0.66 0.64 -0.60 0.26

Leu 206 A 1 B -0.62 0.57 -0.60 0.49

He 207 A B -0.03 -0.11 0.300.49

Leu 208 . B T 0.78 -0.11 * . F 0.850.47

Pro 209 A T -0.03 0.29 F 0.250.99

Glu 210 A T -0.03 0.29 * p 0.401.16

Asn 211 A T 0.19 -0.40 F 1.002.44

Gin 212 A A 0.27 -0.59 * F 0.901.60

Leu 213 A A 0.87 -0.33 0.300.76

Glu 214 A A 0.22 0.10 -0.30 0.73

Ala 215 A ] B -0.59 0.34 -0.30 0.31

Leu 216 A B -0.80 0.63 -0.60 0.31

Pro 217 . B -1.10 0.37 -0.10 0.28

Val 218 . B -0.63 0.76 * -0.40 0.37

Leu 219 . B T -1.52 0.69 * . F -0.05 0.44

Pro 220 T Z -0.93 0.69 * * p 0.150.20

Ser 221 T Z -0.82 0.26 * . F 0.450.47

Gly 222 . B T -1.42 0.40 * . F 0.250.49

He 223 A B -0.57 0.40 * . F -0.15 0.26

Glu 224 A B -0.61 -0.03 * 0.300.33

Phe 225 A B -0.29 0.23 * -0.30 0.25

Leu 226 A B -0.80 -0.20 * 0.300.69

Asp 227 A A -0.46 -0.20 * * 0.300.33

Val 228 A A 0.54 0.20 * -0.30 0.61

Arg 229 A A -0.27 -0.59 * 0.751.45

Leu 230 A A 0.43 -0.59 * 0.880.71

Asn 231 A A 0.94 -0.19 * * 1.011.67

Arg 232 A T 0.64 -0.44 _{* * F} 1.841.14

Leu 233 A T 1.16 -0.06 * F 2.121.85

Gin 234 T T 0.16 -0.31 * F 2.801.14

Ser 235 T T 0.97 -0.03 * . F 2.370.41

Ser 236 T C 0.76 0.37 * * p 1.290.86

Gly 237 T T 0.06 0.11 * * p 1.210.76

He 238 A B 0.28 0.21 . . F 0.130.58

Gin 239 A B -0.42 0.33 . . F -0.15 0.43

Pro 240 A B -0.01 0.73 * * p -0.45 0.38

Ala 241 A A -0.30 0.30 * * -0.15 1.06 Ala 242 A . A -0.56 0.11 * -0.30 0.62

Phe 243 A t A 0.33 0.33 * * -0.30 0.40

Arg 244 A ^' A 0.38 -0.10 * t 0.30 0.68

Ala 245 A A -0.22 -0.60 * * 0.75 1.35

Met 246 A A 0.37 -0.41 * * 0.45 1.28

Glu 247 A A 0.26 -0.80 * * 0.75 1.13

Lys 248 A A 0.14 -0.01 * *> 0.30 0.97

Leu 249 A A -0.21 0.17 * fc -0.30 0.81

Gin 250 . \ . A -0.43 0.31 -0.30 0.73

Phe 251 A A -0.13 1.00 -0.60 0.30

Leu 252 A B -0.13 1.39 * -0.60 0.49

Tyr 253 B -0.18 0.70 * -0.40 0.47

Leu 254 B -0.18 0.70 * -0.23 0.88

Ser 255 B T -0.99 0.60 * 0.14 0.88

Asp 256 r T -0.29 0.60 * 0.71 0.46

Asn 257 Γ T 0.22 -0.16 * F 1.93 0.94

Leu 258 B T -0.42 -0.46 * F 1.70 0.94

Leu 259 B 0.18 -0.16 * F 1.33 0.39

Asp 260 B 0.13 0.27 * F 0.560.38

Ser 261 B -0.08 0.30 * F 0.39 0.45

He 262 B T -0.89 0.04 * F 0.42 0.85

Pro 263 B T -0.29 0.04 * F 0.25 0.42

Gly 264 T c 0.31 0.47 * F 0.15 0.48

Pro 265 T c 0.01 0.51 * F 0.30 1.07

Leu 266 . C -0.50 0.21 * * F 0.42 0.93

Pro 267 T C 0.50 0.47 * * F 0.49 0.77

Pro 268 T T 0.41 0.04 * * F 1.16 0.98

Ser 269 B T -0.10 0.00 * * F 1.68 1.59

Leu 270 B T 0.08 -0.04 * * F 1.700.76

Arg 271 B 0.08 0.03 * * F 0.73 0.67

Ser 272 B 0.29 0.29 0.41 0.41

Val 273 B 0.50 0.30 * 0.24 0.87

His 274 B 0.80 0.01 * 0.07 0.71

Leu 275 B T 0.80 0.41 * -0.20 0.86

Gin 276 T c -0.20 0.71 * F 0.15 0.95

Asn 277 T c 0.10 0.76 * F 0.15 0.49

Asn 278 T c 0.64 0.26 * * F 0.60 1.03

Leu 279 A A 0.08 0.06 * -0.30 0.86

He 280 A A 0.89 0.27 * * -0.30 0.53

Glu 281 A B 1.00 0.27 * -0.30 0.57

Thr 282 A A 1.00 -0.13 * 0.45 1.35 Met 283 A B 0.14 -0.81 * . F 0.90 3.21

Gin 284 A B 0.26 -0.86 * . F 0.90 1.38

Arg 285 A B 0.48 -0.07 * . F 0.45 0.83

Asp 286 A T 0.48 0.01 * . 0.10 0.45

Val 287 A B 0.58 -0.60 * . 0.60 0.43

Phe 288 A A 1.18 -0.57 * . . 0.60 0.34

Cys 289 . A A 1.18 -0.57 * . . 0.600.35

Asp 290 A A 1.03 -0.57 * . F 0.75 0.82

Pro 291 A A 1.08 -0.71 F 0.90 1.30

Glu 292 . A A 1.90 -1.50 F 0.904.83

Glu 293 A A 2.29 -1.57 F 0.90 3.94

His 294 A A 3.07 -1.09 * . F 0.90 3.67

Lys 295 A A 3.18 -1.51 F 0.904.16

His 296 A A 3.39 -1.51 * . F 0.904.70

Thr 297 A A 2.58 -1.11 * . F 0.90 5.98

Arg 298 A A 2.58 -0.93 " . F 0.90 2.47

Arg 299 A A 2.61 -0.93 * . F 0.90 3.14

Gin 300 A A 1.68 -1.43 * * F 0.90 3.63

Leu 301 A A 1.82 -1.23 * * F 0.90 1.30

Glu 302 A B 1.32 -1.23 * * F 0.90 1.30

Asp 303 A B 1.21 -0.54 * * F 0.75 0.62

He 304 A B 0.76 -0.94 * * F 1.11 1.25

Arg 305 A B 0.76 -1.20 * F 1.17 0.72

Leu 306 A B 1.36 -0.80 * * F 1.38 0.69

Asp 307 T T 0.47 -0.37 * * F 2.24 1.52

Gly 308 T C 0.47 -0.37 * F 2.10 0.54

Asn 309 T C 0.54 0.03 * * F 1.44 1.06

Pro 310 T C 0.13 0.03 * * F 1.08 0.52

He 311 B 0.13 0.41 F 0.17 0.71

Asn 312 B -0.57 0.67 -0.19 0.36

Leu 313 B -0.43 1.06 * -0.40 0.20

Ser 314 B -0.73 1.06 * -0.40 0.45

Leu 315 B -1.11 0.76 * -0.40 0.37

Phe 316 B T -0.47 0.86 * -0.20 0.46

Pro 317 T T -1.17 0.93 * 0.20 0.54

Ser 318 T T -1.02 1.33 * . 0.20 0.56

Ala 319 B T -1.53 1.21 -0.20 0.35

Tyr 320 B -0.93 1.11 * . -0.40 0.19

Phe 321 B -0.12 1.11 * . -0.40 0.21

Cys 322 B -0.72 0.73 * . -0.40 0.42

Leu 323 B -0.63 0.91 * -0.40 0.22 Pro 324 B -0.93 0.59 . * . -0.40 0.39

Arg 325 B B -1.03 0.49 . . -0.60 0.51

Leu 326 B B -0.22 0.34 . . -0.30 0.61

Pro 327 B T -0.26 -0.34 . ' * 0.700.78

He 328 B T 0.24 0.01 . . 0.100.34

Gly 329 B B 0.07 0.50 . ^• * F -0.60 0.60

Arg 330 B B -0.43 0.24 ' F -0.15 0.50

Phe 331 B B -0.01 0.24 * -0.30 0.91

Thr 332 B B -0.19 -0.01 * * . 0.451.17

Ter 333 B B 0.31 -0.01 * * 0.300.76

Table V

Res Position I I III IV V VI VII VIII IX X XI XII XIII XIV

Met 1 A B -1.30 0.70 -0.60 0.39

Leu 2 A B -1.72 0.96 -0.60 0.25

Leu 3 A B -2.14 1.21 -0.60 0.16

Pro 4 A B -2.06 1.47 -0.60 0.14

Leu 5 A B -1.97 1.24 * -0.60 0.22

Leu 6 A B -2.18 0.94 -0.60 0.36

Leu 7 A B -2.18 0.94 -0.60 0.19

Ser 8 A B -1.71 1.20 * -0.60 0.19

Ser 9 B B -1.84 0.94 F -0.45 0.23

Leu 10 B B -1.33 0.69 F -0.45 0.27

Leu 11 B Z -0.52 0.39 * F 0.05 0.27

Gly 12 r Z -0.30 0.40 F 0.45 0.35

Gly 13 Γ Z -0.60 0.51 F 0.15 0.43

Ser 14 B Γ -0.30 0.44 F 0.12 0.52

Gin 15 B Γ 0.17 -0.24 * F 1.19 0.88

Ala 16 B 1.09 -0.24 * F 1.16 0.88

Met 17 B Γ 0.73 -0.67 * p 1.98 1.28

Asp 18 B T 0.79 -0.27 . * p 1.70 0.64

Gly 19 T r 0.20 0.24 * * F 1.33 0.67

Arg 20 T T 0.31 0.43 * * 0.71 0.47

Phe 21 B B 0.04 -0.19 * * 0.64 0.56

Trp 22 B B 0.64 0.46 * * -0.43 0.42

He 23 B B 0.64 0.43 * * -0.60 0.37

Arg 24 B B 0.69 0.43 * * -0.60 0.74

Val 25 B B -0.28 0.03 * * -0.30 0.94

Gin 26 B C -0.18 -0.24 * * F 0.65 0.99

Glu 27 B C -0.74 -0.31 * * F 0.65 0.50

Ser 28 B T -0.07 0.33 * 0.10 0.50

Val 29 B B -0.18 0.11 * -0.30 0.45

Met 30 B B 0.09 -0.29 . 0.30 0.45

Val 31 B B -0.58 0.21 -0.30 0.34

Pro 32 B B -0.58 0.40 -0.30 0.24

Glu 33 B -1.17 -0.24 . 0.50 041

Ala 34 T -0.61 -0.17 . 0.90 0.39

Cys 35 B -0.87 -0.43 * 0.50 0.34

Asp 36 B T -0.22 -0.21 * 0.70 0.14 He 37 B B -0.68 0.21 -0.30 0.22

Ser 38 B B -0.98 0.29 * -0.30 0.22

Val 39 B T -1.09 0.10 * 0.100.18

Pro 40 B T -0.72 0.89 * -0.20 0.22

Cys 41 T T -0.97 0.59 * 0.20 0.22

Ser 42 T T -0.29 0.96 * 0.20 0.46

Phe 43 T 0.12 0.74 » 0.28 0.46

Ser 44 B 0.98 0.31 0.61 1.69

Tyr 45 B T 1.19 0.14 1.09 2.19

Pro 46 T T 1.57 -0.24 . F 2.52 4.22

Arg 47 T T 1.56 -0.11 . F 2.80 3.31

Gin 48 ^' r T 1.91 -0.01 . F 2.52 3.05

Asp 49 T 1.91 -0.34 * ] F 2.04 1.95

Trp 50 T T 1.84 -0.39 * F 1.96 1.33

Thr 51 T -* 1.84 0.10 * F 0.88 1.11

Gly 52 T T 1.14 0.13 * 1 F 0.80 1.03

Ser 53 T c 0.90 0.63 . F 0.15 0.99

Thr 54 . n 0.56 0.47 . F 0.10 1.07

Pro 55 T π 0.60 0.41 . F 0.30 1.07

Ala 56 ^' Γ T 0.62 0.74 0.35 1.26

Tyr 57 ^' Γ T 0.27 1.27 0.20 0.91

Gly 58 r T 0.61 1.57 0.200.51

Tyr 59 B B 0.33 1.14 * -0.45 1.01

Trp 60 B B -0.31 1.14 * -0.60 0.65

Phe 61 B B -0.03 1.03 * -0.60 0.49

Lys 62 B B 0.21 1.09 * -0.60 0.45

Ala 63 B B 0.24 0.33 * -0.30 0.74

Val 64 B B 0.18 -0.10 * 0.79 1.24

Thr 65 B B 0.51 -0.40 * F 1.13 0.89

Glu 66 B B 0.87 -0.40 * F 1.62 1.77

Thr 67 B r 0.23 -0.47 * F 2.362.36

Thr 68 Γ T 0.61 -0.61 * F 3.40 1.65

Lys 69 Γ T 0.61 -0.67 * F 3.06 1.48

Gly 70 T C 0.33 -0.03 F 2.07 0.76

Ala 71 T C 0.02 -0.01 * F 1.73 0.53

Pro 72 B 0.33 -0.01 * 0.84 0.38

Val 73 B 0.61 0.39 -0.10 0.62

Ala 74 B 0.57 0.46 -0.10 0.84

Thr 75 B 0.61 0.36 * * 0.50 0.94

Asn 76 C 1.31 0.31 F 1.30 1.70

His 77 T c 1.52 -0.33 * F 2.40 3.29 Gin 78 T c 1.52 -0.83 * . 3.00 3.95

Ser 79 T c 2.11 -0.67 * 1 F 2.70 1.82

Arg 80 B T 1.82 -1.07 * . F 2.20 2.32

Glu 81 A B 1.52 -0.96 * F 1.50 1.33

Val 82 A B 1.24 -0.97 * * F 1.54 1.33

Glu 83 A B 1.36 -0.87 * * 1.28 0.98

Met 84 A B 1.31 -0.87 * * 1.77 1.10

Ser 85 T c 1.31 -0.44 * * 1 : 2.56 1.47

Thr 86 T T 0.61 -1.09 * F 3.40 1.67

Arg 87 T T 1.47 -0.30 * 1 2.76 1.46

Gly 88 ' r T 0.66 -0.51 * 1 F 2.72 1.88

Arg 89 B 1 B 0.94 -0.21 * F 1.28 1.08

Phe 90 B B 0.90 -0.21 * 0.64 0.79

Gin 91 B B 1.21 0.21 * -0.30 0.79

Leu 92 B B 0.89 -0.21 * 0.30 0.68

Thr 93 B B 0.64 0.21 * F 0.34 1.21

Gly 94 . B c 0.58 -0.07 * 1 1.33 0.70

Asp 95 T c 0.93 -0.47 * * 1 2.22 1.71

Pro 96 T c 0.93 -0.73 * 1 F 2.86 1.17

Ala 97 Γ T 1.08 -0.81 . 3.40 1.90

Lys 98 Γ T 1.09 -0.67 * 1 F 2.91 0.61

Gly 99 Γ T 0.62 -0.29 * . F 2.27 0.53

Asn 100 B Γ T -0.23 -0.03 * F 1.93 0.43

Cys 101 B T -0.91 0.11 * * 0.44 0.16

Ser 102 B T -0.21 0.80 * * -0.20 0.11

Leu 103 A B B -0.26 0.37 * * -0.30 0.14

Val 104 A B B -0.50 -0.03 * * 0.30 0.43

He 105 A B B -0.50 -0.10 * 0.30 0.33

Arg 106 A B B -0.43 -0.09 * 0.30 0.68

Asp 107 A B B -0.13 -0.16 * 0.30 0.91

Ala 108 A B 0.68 -0.40 * 0.45 2.25

Gin 109 A B 1.53 -1.09 * 1.03 1.92 _,

Met 110 A c 2.12 -1.09 1.51 1.99

Gin 111 A B 2.01 -0.70 . F 1.74 2.64

Asp 112 • T T 1.77 -0.80 * F 2.82 2.64

Glu 113 . T T 1.66 -0.44 * . F 2.804.18

Ser 114 . T T 0.96 -0.27 * * F 2.52 2.09

Gin 115 . T T 1.67 0.11 * * F 1.64 1.08

Tyr 116 B B 0.81 0.11 * * 0.41 1.22

Phe 117 B B 0.81 0.76 * -0.32 0.68

Phe 1 18 B B 0.92 0.37 * * 0.04 0.68 Arg 119 B B 0.88 -0.03 * 0.98 0.85

Val 120 B B 0.58 -0.36 * 1.32 0.97

Glu 121 r T 0.58 -0.76 * . F 3.06 1.50

Arg 122 Γ T 0.42 -0.79 * . F 3.40 1.20

Gly 123 Γ T 1.23 -0.14 * * F 2.76 1.20

Ser 124 Γ T 0.88 -0.79 * * F 2.72 1.36

Tyr 125 B 1.73 -0.03 * 1.33 1.09

Val 126 B 1.03 0.37 * 0.39 1.76

Arg 127 B 0.32 0.73 -0.25 1.14

Tyr 128 B 0.67 0.96 * -0.40 0.72

Asn 129 B 0.97 0.60 * -0.25 1.56

Phe 130 B 0.87 -0.04 * 0.65 1.33

Met 131 B 1.02 0.39 * -0.10 0.84

Asn 132 Γ T 0.21 0.41 * 0.20 0.45

Asp 133 Γ T -0.36 0.80 0.20 0.45

Gly 134 Γ T -0.31 0.70 * 0.20 0.38

Phe 135 B T -0.47 0.09 0.10 0.47

Phe 136 B B -0.18 0.33 * -0.30 0.21

Leu 137 B B -1.03 0.81 -0.60 0.30

Lys 138 B B -1.84 1.03 -0.60 0.26

Val 139 B B -1.80 0.93 -0.60 0.25

Thr 140 B B -1.80 0.53 * -0.60 0.40

Val 141 B B -1.41 0.63 -0.60 0.17

Leu 142 B B -0.81 1.11 * -0.60 0.34

Ser 143 B B -0.74 0.90 * -0.60 0.36

Phe 144 B B -0.10 0.41 * * -0.26 0.96

Thr 145 T C 0.21 0.20 * * F 1.28 1.80

Pro 146 T C 1.07 -0.09 * * F 2.22 2.32

Arg 147 T C 1.84 -0.47 * F 2.564.48

Pro 148 T T 2.14 -0.76 * F 3.40 4.22

Gin 149 T 2.53 -0.84 * * F 2.86 4.39

Asp 150 T 2.84 -0.79 * F 2.52 3.24

His 151 T 2.24 -0.79 * F 2.18 3.50

Asn 152 T T 1.82 -0.53 * * F 2.04 1.67

Thr 153 T T 1.37 -0.44 F 1.40 1.44

Asp 154 T T 1.33 0.13 * F 0.65 0.57

Leu 155 B T 0.48 0.13 * * 0.10 0.48

Thr 156 B B 0.51 0.37 * -0.30 0.25

Cys 157 B B -0.19 -0.1 1 * 0.49 0.25

His 158 B B -0.18 0.67 * * -0.22 0.26

Val 159 B B -0.07 0.37 * * 0.27 0.24 Asp 160 B B 0.79 -0.11 * fc 1.06 0.88

Phe 161 B 0.76 -0.69 * 1.90 1.29

Ser 162 r T 0.57 -0.76 * F 2.46 1.72

Arg 163 Γ T 0.30 -0.76 * F 2.12 0.77

Lys 164 Γ T 0.57 -0.37 * F 1.78 1.18

Gly 165 Γ T 0.57 -0.66 * F 1.74 0.89

Val 166 1 B . c 1.38 -0.64 * F 0.95 0.79

Ser 167 ] B . •—. 1.37 -0.64 * F 0.95 0.77

Ala 168 B 1 B 0.40 -0.16 * F 0.60 1.13

Gin 169 B ] B 0.47 0.06 * k p 0.00 1.13

Arg 170 B B 0.00 -0.59 ^► F 0.90 1.65

Thr 171 B B 0.97 -0.29 * F 0.60 1.35

Val 172 B B 0.41 -0.79 * > 0.75 1.52

Arg 173 B 1 B 0.41 -0.54 0.60 0.58

Leu 174 B ] B 0.17 -0.04 * 0.30 0.40

Arg 175 B B -0.53 0.23 * > fc -0.30 0.85

Val 176 B B -0.43 0.09 * fc -0.30 0.44

Ala 177 B B 0.53 0.51 fc -0.60 0.82

Tyr 178 B B 0.42 -0.17 * fc 0.30 0.82 .

Ala 179 B T 0.42 -0.17 * fc 0.85 1.85

Pro 180 B T -0.54 -0.13 * fc 0.85 1.51

Arg 181 B T -0.58 0.01 F 0.25 0.72

Asp 182 B T -0.29 -0.06 * F 0.85 0.50

Leu 183 B B -0.93 -0.17 * 0.30 0.43

Val 184 B B -0.64 0.09 * -0.30 0.15

He 185 B B -0.32 0.47 * -0.26 0.12

Ser 186 B B -0.43 0.47 * 0.08 0.29

He 187 B B -0.43 -0.21 * 1.32 0.66

Ser 188 B T 0.07 -0.46 * F 2.36 1.52

Arg 189 T T 0.71 -0.66 * F 3.40 1.63

Asp 190 T T 1.01 -0.61 * F 3.06 3.61

Asn 191 T c 0.50 -0.80 * F 2.52 2.72

Thr 192 c 1.39 -0.50 * F 1.98 1.14

Pro 193 c 1.48 -0.50 F 1.64 1.19

Ala 194 T 1.37 -0.07 * F 1.20 1.14

Leu 195 B 1.16 -0.07 F 1.14 1.37

Glu 196 B 1.16 -0.13 * F 1.48 1.37

Pro 197 B 1.12 -0.16 * F 1.82 2.35

Gin 198 c 1.33 -0.23 * F 2.36 2.82

Pro 199 T T 1.07 -0.51 * p 3.40 2.62

Gin 200 T T 1.67 0.13 * p 2.16 1.26 Gly 201 T T 1.42 0.13 * p 1.82 1.12

Asn 202 T Z 0.82 0.49 * F 0.98 1.14

Val 203 B 0.82 0.74 * p 0.09 0.54

Pro 204 A B 0.44 0.34 -0.30 0.95

Tyr 205 A B 0.44 0.41 -0.60 0.59

Leu 206 A B 0.83 0.41 -0.17 1.39

Glu 207 A B 0.49 -0.23 . 1.01 1.79

Ala 208 A 1 B 1.34 -0.23 F 1.44 1.13

Gin 209 B T 0.86 -0.59 . F 2.42 2.38

Lys 210 T T 0.29 -0.49 * F 2.80 1.19

Gly 211 T T 1.21 0.20 * F 1.77 0.97

Gin 212 B T 0.40 -0.30 * F 1.84 1.10

Phe 213 A ] B 0.18 -0.01 * 0.86 0.45

Leu 214 A B -0.49 0.67 * -0.32 0.38

Arg 215 A B -1.12 0.81 * -0.60 0.12

Leu 216 A B -1.37 0.91 * -0.60 0.14

Leu 217 A B -1.37 0.63 * -0.60 0.17

Cys 218 A B -0.97 -0.06 * * 0.54 0.14

Ala 219 A T -0.16 0.33 * * 0.58 0.23

Ala 220 T T -0.48 0.04 * * F 1.37 0.49

Asp 221 T T 0.12 -0.21 . F 2.36 1.40

Ser 222 T C 0.34 -0.36 . F 2.40 2.15

Gin 223 T C 0.70 -0.36 F 2.16 2.15

Pro 224 T C 0.48 -0.37 . F 1.92 1.85

Pro 225 T T 0.77 0.31 * F 1.28 1.14

Ala 226 T T 0.48 0.31 F 0.89 0.88

Thr 227 B T -0.08 0.83 -0.20 0.60

Leu 228 B B -0.89 1.04 * * -0.60 0.29

Ser 229 B B -0.68 1.30 -0.60 0.24

Trp 230 B B -0.47 1.20 * -0.60 0.28

Val 231 B B 0.23 1.11 * -0.60 0.55

Leu 232 B B -0.31 0.43 * -0.60 0.80

Gin 233 B B -0.31 0.69 * F -0.45 0.57

Asn 234 B B -0.31 0.46 * F -0.45 0.63

Arg 235 B B -0.32 0.20 F 0.00 1.03

Val 236 B B 0.23 -0.10 F 0.45 0.79

Leu 237 B T 1.01 -0.11 * _* F 0.85 0.66

Ser 238 B T 0.80 -0.01 * F 0.85 0.46

Ser 239 T T 0.51 0.41 * F 0.35 0.96

Ser 240 T C 0.06 0.69 * F 0.30 1.22

His 241 T C 0.70 0.43 * F 0.15 0.90 Pro 242 r T 1.62 0.47 * 0.35 1.04

Trp 243 r T 1.71 0.09 * 0.65 1.52

Gly 244 T ( Z 1.20 0.13 * F 0.60 1.73

Pro 245 ( Z 1.16 0.31 F 0.25 0.92

Arg 246 T ( Z 0.38 0.31 F 0.45 0.87

Pro 247 T ( Z 0.59 0.09 * F 0.45 0.72

Leu 248 B T 0.07 -0.34 * 0.70 0.81

Gly 249 B T 0.20 -0.09 * 0.70 0.34

Leu 250 B 0.07 0.34 * -0.10 0.34

Glu 251 B -0.90 0.34 * -0.10 0.41

Leu 252 B -0.64 0.30 -0.10 0.31

Pro 253 B -0.42 -0.13 F 0.65 0.74

Gly 254 B -0.42 -0.31 F 0.65 0.43

Val 255 B 0.39 0.11 * F 0.05 0.52

Lys 256 B 0.09 -0.57 F 1.29 0.56

Ala 257 B 0.56 -0.61 * * p 1.63 0.76

Gly 258 r 0.88 -0.61 * * F 2.52 1.02

Asp 259 Γ r 0.98 -1.26 * * p 2.91 0.99

Ser 260 Γ Γ 1.52 -0.50 * * p 3.40 1.54

Gly 261 Γ Γ 0.81 -0.51 * * p 3.06 2.25

Arg 262 B ' Γ 1.51 -0.37 * sk p 1.87 0.72

Tyr 263 B B 1.27 -0.37 =fc p 1.28 1.05

Thr 264 B B 1.27 -0.26 0.79 1.08

Cys 265 B B 1.57 -0.69 0.94 0.95

Arg 266 B B 2.02 -0.29 0.98 0.98

Ala 267 B 1.10 -1.04 fc F 2.12 1.33

Glu 268 B 1.00 -0.84 * F 2.462.04

Asn 269 T T 1.01 -0.99 * * F 3.40 1.03

Arg 270 T r 1.68 -0.60 * F 3.06 1.37

Leu 271 T Γ 1.57 -0.70 * F 2.72 1.37

Gly 272 T T 2.27 -0.30 * F 2.08 1.47

Ser 273 C 1.68 -0.70 * fc F 1.64 1.47

Gin 274 A B 0.87 -0.20 * F 0.60 1.80

Gin 275 A B 0.76 -0.20 * F 0.60 1.50

Arg 276 A B 0.76 -0.63 F 0.90 1.87

Ala 277 A B 0.80 -0.33 F 0.45 0.89

Leu 278 A B 0.24 -0.34 0.30 0.69

Asp 279 A B 0.24 -0.10 * 0.30 0.26

Leu 280 A B 0.00 0.30 -0.30 0.45

Ser 281 B -0.32 0.56 -0.40 0.85

Val 282 B 0.06 0.30 * -0.10 0.79 Gin 283 B 0.87 0.73 -0.25 1.48

Tyr 284 B 0.87 0.04 0.25 1.91

Pro 285 r C 0.87 0.06 « Sfc 1.00 4.14

Pro 286 Γ C 1.28 0.10 fc sfc p 1.20 1.97

Glu 287 T Γ 1.28 -0.30 fc * p 2.20 2.47

Asn 288 B Γ 0.68 -0.41 fc sfc 2.00 1.18

Leu 289 B B 0.07 -0.23 1.10 0.76

Arg 290 B ] B -0.02 -0.01 0.90 0.32

Val 291 B B 0.19 0.37 0.10 0.27

Met 292 B B -0.40 0.37 fc * -0.10 0.57

Val 293 B B -0.40 0.19 -0.30 0.29

Ser 294 B 0.52 0.59 -0.40 0.63

Gin 295 B 0.10 -0.06 ' fc sfc 0.80 1.26

Ala 296 B 0.10 -0.19 - * . F 0.80 2.44

Asn 297 B -0.11 -0.19 * . F 0.80 1.35

Arg 298 B 0.74 0.11 * . F 0.05 0.64

Thr 299 B 1.04 -0.29 * . F 0.80 1.10

Val 300 B 0.23 -0.39 fc 0.65 1.10

Leu 301 B 0.48 -0.10 0.50 0.47

Glu 302 B 0.48 0.33 0.03 0.32

Asn 303 B 0.02 0.24 * . F 0.31 0.69

Leu 304 T T 0.02 0.03 * . F 1.04 0.83

Gly 305 T r 0.58 -0.17 * . F 1.77 0.69

Asn 306 T T 0.58 0.21 * . F 1.30 0.58

Gly 307 r C 0.37 0.50 * . F 0.67 0.58

Thr 308 C -0.49 0.24 F 0.64 0.90

Ser 309 B -0.49 0.46 F 0.01 0.42

Leu 310 B -0.14 0.74 -0.27 0.35

Pro 311 B -0.49 0.31 -0.10 0.42

Val 312 B -0.14 0.26 -0.10 0.31

Leu 313 B -0.13 0.27 F 0.05 0.64

Glu 314 B -0.64 -0.03 F 0.65 0.56

Gly 315 T T -0.50 0.23 F 0.65 0.62

Gin 316 T T -1.10 0.16 F 0.65 0.40

Ser 317 T T -1.10 0.16 F 0.65 0.19

Leu 318 B T -0.96 0.80 -0.20 0.14

Cys 319 B B -1.81 0.94 -0.60 0.04

Leu 320 B B -1.78 1.19 -0.60 0.02

Val 321 B B -1.81 1.29 -0.60 0.04

Cys 322 B B -1.81 1.10 -0.60 0.11

Val 323 B B -1.30 0.91 -0.60 0.18 Thr 324 B 13 -0.84 0.61 -0.60 0.32

His 325 T T -0.24 0.40 F 0.89 0.93

Ser 326 T C 0.02 0.26 * * F 1.08 1.93

Ser 327 T ( Z 0.80 0.11 * * F 1.32 1.35

Pro 328 T C 0.84 -0.37 F 2.16 1.95

Pro 329 T 0.86 -0.19 . F 2.40 1.20

Ala 330 T 0.60 -0.19 F 2.16 1.20

Arg 331 B 1 B 0.59 0.34 0.42 0.82

Leu 332 B B 0.89 0.40 0.18 0.76

Ser 333 B B 1.21 0.37 0.09 1.30

Trp 334 B B 1.08 -0.13 . 0.45 1.30

Thr 335 B B 1.67 0.30 . F 0.00 1.56

Gin 336 B T 0.70 0.01 F 0.40 2.02

Arg 337 B T 0.70 0.27 F 0.40 1.43

Gly 338 T T 0.70 0.04 F 0.65 0.82

Gin 339 B T 0.78 -0.06 F 0.85 0.63

Val 340 B 0.79 -0.03 . F 0.65 0.50

Leu 341 B 0.79 0.36 . F 0.05 0.67

Ser 342 B T 0.47 0.33 F 0.55 0.67

Pro 343 T r 0.51 0.36 F 1.40 1.41

Ser 344 T r 0.51 0.10 F 1.70 2.28

Gin 345 . Γ C 1.16 -0.59 F 2.702.85

Pro 346 T 1.62 -0.54 F 3.00 2.85

Ser 347 C 1.07 -0.54 F 2.50 2.10

Asp 348 . Γ C 0.47 -0.29 F 1.95 0.90

Pro 349 B . T 0.77 0.00 F 1.45 0.48

Gly 350 B . T -0.04 -0.43 F 1.15 0.62

Val 351 B . T -0.04 -0.13 fc 0.70 0.31

Leu 352 B 0.37 0.30 * -0.10 0.31

Glu 353 B -0.49 -0.13 * 0.50 0.61

Leu 354 B B -0.28 0.09 -0.30 0.61

Pro 355 B B -0.79 -0.16 F 0.60 1.27

Arg 356 A B B 0.07 -0.20 0.30 0.55

Val 357 A B B 0.84 -0.20 0.45 1.15

Gin 358 A B B 0.84 -0.39 * 0.45 1.01

Val 359 A B B 1.31 -0.81 0.60 0.89

Glu 360 A B B 1.52 -0.39 0.45 1.19

His 361 A C 0.71 -1.03 F 1.10 1.19

Glu 362 A T 1.26 -0.64 F 1.30 1.39

Gly 363 A T 0.59 -0.80 •fc F 1.30 1.16

Glu 364 A T 1.41 -0.23 F 0.85 0.46 Phe 365 A r 0.82 -0.23 0.70 0.36

Thr 366 A A 0.97 0.27 -0.30 0.37

Cys 367 A r 0.93 -0.16 0.70 0.41

His 368 A B 1.07 0.34 -0.30 0.65

Ala 369 A Γ 0.26 -0.01 0.95 0.70

Arg 370 A ( z 0.61 0.19 fc * 0.55 1.07

His 371 T ( z 0.62 0.04 * 1.05 0.78

Pro 372 Γ T 1.29 -0.07 2.25 1.03

Leu 373 r T 1.29 -0.17 F 2.50 0.91

Gly 374 r T 1.02 0.33 F 1.65 0.91

Ser 375 B ] 3 0.61 0.47 * F 0.30 0.44

Gin 376 B B -0.17 0.43 F 0.05 0.71

His 377 B B -0.26 0.43 -0.35 0.59

Val 378 B B -0.26 0.39 -0.30 0.59

Ser 379 B B -0.21 0.69 -0.60 0.28

Leu 380 B B -0.77 0.67 -0.60 0.28

Ser 381 B B -0.80 0.81 -0.60 0.28

Leu 382 B B -1.01 0.67 -0.60 0.28

Ser 383 B B -0.46 1.04 -0.60 0.54

Val 384 B B -0.37 0.74 -0.60 0.54

His 385 B B 0.49 0.79 -0.45 1.01

Tyr 386 B B -0.02 0.10 -0.15 1.50

Ser 387 B T -0.02 0.40 0.25 1.67

Pro 388 B T -0.07 0.44 F 0.10 1.01

Lys 389 T T 0.58 0.37 F 0.65 0.64

Leu 390 T T 0.31 0.04 * F 0.65 0.74

Leu 391 B -0.11 0.04 F 0.05 0.64

Gly 392 T C -0.11 0.19 F 0.45 0.17

Pro 393 T c -0.19 0.57 F 0.15 0.28

Ser 394 T c -0.23 0.80 F 0.15 0.36

Cys 395 T c -0.01 0.11 0.30 0.62

Ser 396 A _# c 0.80 0.19 -0.10 0.41

Tip 397 A B 0.80 -0.24 0.30 0.52

Glu 398 A B 0.20 -0.20 0.30 0.97

Ala 399 A T 0.47 -0.09 0.70 0.60

Glu 400 A T 0.47 0.03 F 0.10 0.77

Gly 401 A T 0.47 -0.31 0.70 0.24

Leu 402 A T 0.09 0.07 0.10 0.32

His 403 A T -0.21 0.14 0.10 0.10

Cys 404 T T 0.08 0.53 0.20 0.13

Ser 405 T T 0.08 0.49 0.20 0.22 Cys 406 T T -0.17 0.20 . . 0.500.27

Ser 407 T T 0.34 0.20 . * F 0.650.52

Ser 408 T 0.17 0.01 . . F 0.450.52

Gin 409 T 0.24 0.06 . . F 0.601.49

Ala 410 c 0.33 -0.01 . . F 1.241.13

Ser 411 c 0.70 0.03 . . F 0.881.30

Pro 412 c 0.19 0.03 . * F 1.121.00

Ala 413 T c 0.60 0.31 . * F 1.410.82

Pro 414 T c 0.31 -0.19 * * F 2.401.20

Ser 415 T c 0.61 0.34 * * F 1.410.82

Leu 416 B T 0.10 0.83 * * 0.520.85

Arg 417 B B -0.03 1.01 * * -0.12 0.45

Trp 418 B B 0.56 1.01 . * -0.36 0.33

Trp 419 A B c 0.77 0.63 . * -0.40 0.70

Leu 420 A B c 0.26 -0.06 . * 0.500.62

Gly 421 A B c 0.26 0.63 . * -0.40 0.49

Glu 422 A B 0.14 0.40 . * F -0.15 0.38

Glu 423 A B 0.09 -0.51 . . F 0.750.80

Leu 424 A c 0.38 -0.77 * . F 0.950.80

Leu 425 A c 0.89 -0.80 * . F 1.250.74

Glu 426 A T 0.93 -0.41 . . F 1.450.58

Gly 427 A T 0.93 -0.03 . . F 1.750.94

Asn 428 T T 0.93 -0.31 . . F 2.601.97

Ser 429 T c 1.44 -1.00 . . F 3.001.89

Ser 430 T c 1.56 -0.61 . . F 2.702.57

Gin 431 T c 1.56 -0.26 . . F 2.101.38

Asp 432 A c 1.04 -0.66 * . F 1.701.79

Ser 433 A B 0.73 -0.40 * . F 0.750.99

Phe 434 A B 0.82 -0.30 . * 0.300.82

Glu 435 A B 0.82 -0.27 . . 0.430.76

Val 436 A B 0.52 0.11 * . F 0.110.76

Thr 437 B T -0.07 0.11 . . F 0.791.18

Pro 438 T c -0.11 -0.17 . . F 1.570.69

Ser 439 T T 0.38 0.26 . . F 1.300.92

Ser 440 T c 0.09 0.04 . . F 0.970.98

Ala 441 c 0.36 0.47 . . F 0.340.67

Gly 442 T c 0.67 0.54 . . F 0.410.50

Pro 443 T T 0.58 0.56 . . F 0.480.61

Trp 444 T c 0.58 0.56 * . F 0.150.80

Ala 445 B T 0.07 0.44 * . F 0.101.09

Asn 446 B T 0.36 0.70 . . F -0.05 0.58 Ser 447 B T -0.11 0.66 * F -0.05 0.74

Ser 448 B T 0.07 0.43 sfc p -0.05 0.60

Leu 449 B T 0.01 0.43 -0.20 0.51

Ser 450 B 0.26 0.46 -0.40 0.38

Leu 451 B T -0.56 0.50 -0.20 0.28

His 452 B T -0.56 0.80 -0.20 0.28

Gly 453 T T -0.56 0.50 0.20 0.28

Gly 454 T Z -0.09 0.50 s_fc 0.15 0.45

Leu 455 . C -0.60 0.24 * * F 0.39 0.33

Ser 456 T C 0.32 0.43 * * F 0.43 0.27

Ser 457 T T -0.46 0.00 . * F 1.67 0.54

Gly 458 B T 0.00 0.26 . * F 0.81 0.54

Leu 459 B T -0.32 -0.43 * * 1.40 0.79

Arg 460 A B 0.49 -0.24 * * 0.86 0.32

Leu 461 A B 0.20 -0.63 * * 1.02 0.56

Arg 462 A B 0.21 -0.56 * * 0.88 0.68

Cys 463 A B 0.56 -0.33 * * 0.44 0.37

Glu 464 A B 0.51 0.07 . * -0.30 0.71

Ala 465 A T 0.37 0.03 * * 0.10 0.27

Trp 466 A T 0.83 0.53 * * -0.20 0.68

Asn 467 B T 0.13 0.39 . . 0.10 0.39

Val 468 B T 0.80 0.89 -0.20 0.39

His 469 T C 0.50 0.79 0.00 0.64

Gly 470 T C 0.74 0.26 0.30 0.54

Ala 471 . C 0.73 0.29 F 0.25 0.72

Gin 472 T C -0.16 0.03 F 0.45 0.71

Ser 473 T C -0.11 0.21 F 0.45 0.50

Gly 474 B T -0.08 0.47 F -0.05 0.41

Ser 475 B T -0.54 0.37 F 0.25 0.41

He 476 B -0.17 0.66 -0.40 0.25

Leu 477 B -0.17 0.70 * -0.06 0.39

Gin 478 B 0.18 0.27 * 0.58 0.49

Leu 479 B T 0.57 -0.11 * 1.87 1.39

Pro 480 B T 0.52 -0.80 * F 2.66 3.38

Asp 481 T T 0.60 -1.06 . F 3.40 1.93

Lys 482 T T 0.52 -0.77 F 3.06 1.93

Lys 483 B T 0.22 -0.77 F 2.17 0.88

Gly 484 B B 0.72 -0.81 F 1.43 0.70

Leu 485 B B 0.34 -0.33 F 0.79 0.51

He 486 B B -0.36 0.17 -0.30 0.26

Ser 487 B B -0.70 0.96 -0.60 0.22 Thr 488 B B -0.74 0.91 * . -0.60 0.36

Ala 489 B B -0.74 0.63 * -0.60 0.84

Phe 490 B T -0.52 0.37 . . F 0.25 0.62

Ser 491 T C -0.33 0.49 * . F 0.15 0.43

Asn 492 T C -0.84 0.79 F 0.15 0.37

Gly 493 T C -0.88 0.97 F 0.15 0.35

Ala 494 B C -1.18 0.61 -0.40 0.26

Phe 495 B B -0.82 0.91 -0.60 0.11

Leu 496 B B -1.41 0.94 -0.60 0.11

Gly 497 B B -1.72 1.20 -0.60 0.08

He 498 B B -1.97 1.19 -0.60 0.13

Gly 499 B B -2.19 0.90 -0.60 0.16

He 500 A B . . -2.30 0.90 -0.60 0.13

Thr 501 A B -2.19 1.16 -0.60 0.16

Ala 502 A B -2.66 1.26 -0.60 0.14

Leu 503 A B -2.43 1.51 -0.60 0.16

Leu 504 A B -2.90 1.40 -0.60 0.06

Phe 505 A B -2.60 1.60 -0.60 0.05

Leu 506 A B -3.10 1.60 -0.60 0.06

Cys 507 A A -3.40 1.60 -0.60 0.06

Leu 508 A A -3.48 1.60 -0.60 0.05

Ala 509 A A -3.27 1.50 * -0.60 0.04

Leu 510 A A -2.52 1.43 * -0.60 0.08

He 511 A B -2.60 0.86 * -0.60 0.19

He 512 A B -2.74 0.86 * -0.60 0.13

Met 513 A B -2.14 1.04 * -0.60 0.13

Lys 514 A B -1.51 0.79 * -0.26 0.28

He 515 A B -0.59 0.10 0.380.81

Leu 516 A B 0.41 -0.59 1.77 1.61

Pro 517 T C 0.99 -1.20 . . F 2.86 1.57

Lys 518 T T 1.59 -0.71 * F 3.40 3.24

Arg 519 T T 1.23 -1.00 sfc p 3.06 6.80 .

Arg 520 T T 2.12 -1.20 . . F 2.72 6.34

Thr 521 T 2.62 -1.63 . . F 2.18 5.49

Gin 522 B 2.62 -1.14 * . F 1.44 4.05

Thr 523 B . 2.69 -0.71 * . F 1.10 3.20

Glu 524 B 2.37 -0.71 * * F 1.104.34

Thr 525 B T 2.37 -0.77 . * F 1.30 3.87

Pro 526 T C 1.98 -1.17 . * F 1.50 5.26

Arg 527 T C 1.68 -0.87 * * F 1.84 2.63

Pro 528 T T 2.10 -0.49 * * F 2.08 2.44 Arg 529 r 2.07 -0.97 F 2.52 3.09

Phe 530 r 2.08 -0.90 F 2.86 2.15

Ser 531 r r 1.98 -0.51 * F 3.40 1.86

Arg 532 B Γ 0.98 -0.46 * * F 2.36 1.37

His 533 B Γ 0.38 0.23 F 1.42 1.11

Ser 534 B Γ 0.27 0.13 F 0.93 0.68

Thr 535 B 1 B 0.72 -0.26 0.64 0.58

Ile 536 B 1 B 0.13 0.50 -0.60 0.67

Leu 537 B ] B 0.02 0.69 -0.60 0.35

Asp 538 B 1 B -0.80 0.70 -0.60 0.39

Tyr 539 B 1 B -1.36 0.86 -0.60 0.41

Ile 540 B B -1.26 0.81 * -0.60 0.37

Asn 541 B 1 B -0.68 0.56 -0.60 0.34

Val 542 B 1 B ¹ -0.46 1.04 -0.60 0.32

Val 543 B B -0.80 0.79 -0.60 0.46

Pro 544 B -0.77 0.53 * . F -0.25 0.28

Thr 545 B Γ -0.69 0.56 * . F -0.05 0.59

Ala 546 B Γ -1.28 0.60 * . F -0.05 0.65

Gly 547 B Γ -0.42 0.46 * . F -0.05 0.43

Pro 548 B Γ 0.48 0.43 * . F 0.21 0.51

Leu 549 B 0.80 -0.06 F 1.32 1.01

Ala 550 B 1.11 -0.56 F 1.88 2.00

Gin 551 B 1.70 -0.59 F 2.14 2.08

Lys 552 B Γ 2.09 -0.61 * F 2.60 4.38

Arg 553 B Γ 1.71 -1.30 F 2.34 8.66

Asn 554 B Γ 2.21 -1.30 F 2.08 5.05

Gin 555 B T 2.59 -1.21 F 2.10 3.65

Lys 556 B 2.59 -0.79 F 1.92 2.88

Ala 557 C 2.24 -0.39 F 1.84 2.88

Thr 558 T C 1.92 -0.40 F 2.32 2.23

Pro 559 T T 2.03 -0.37 F 2.80 1.72

Asn 560 T T 1.72 -0.37 F 2.52 3.34

Ser 561 T C 1.47 -0.39 F 2.04 3.34

Pro 562 T 1.24 -0.44 F 1.76 3.34

Arg 563 T 1.34 -0.19 F 1.48 1.71

Thr 564 B 1.34 -0.16 F 0.80 1.98

Pro 565 B 1.00 -0.11 F 0.80 1.98

Leu 566 B 0.71 -0.11 F 0.65 1.00

Pro 567 B T 0.71 0.39 F 0.25 0.70

Pro 568 T T 0.30 0.33 F 0.65 0.70

Gly 569 T C 0.40 0.29 F 0.60 1.14 Ala 570 T C 0.61 0.03 F 0.94 1.14

Pro 571 C 1.12 -0.40 . F 1.68 1.27

Ser 572 T C 1.38 -0.44 F 2.22 1.72

Pro 573 T C 1.63 -0.87 . F 2.86 3.41

Glu 574 T T 1.98 -1.37 . F 3.40 4.41

Ser 575 T T 2.57 -1.40 . F 3.06 5.29

Lys 576 T T 2.82 -1.39 . F 2.94 5.93

Lys 577 T T 3.17 -1.81 F 2.82 6.84

Asn 578 T T 3.38 -1.81 F 2.70 10.21

Gin 579 T T 3.13 -1.80 * F 2.58 8.84

Lys 580 1 B 3.43 -1.04 * F 2.20 6.93

Lys 581 1 B 2.58 -0.64 * F 1.98 7.46

Gin 582 B 2.32 -0.36 * F 1.46 3.55

Tyr 583 B 2.02 -0.33 . 1.09 2.75

Gin 584 B 1.32 0.06 0.27 1.84

Leu 585 B T 1.07 0.84 -0.20 0.92

Pro 586 B T 1.02 0.87 F -0.05 0.91

Ser 587 B T 0.81 0.11 F 0.59 0.91

Phe 588 B T 1.10 0.14 F 1.08 1.70

Pro 589 ( : 0.80 -0.54 . F 2.32 2.20

Glu 590 ( 1.31 -0.59 . F 2.66 2.20

Pro 591 T T 1.21 -0.59 F 3.40 3.41

Lys 592 T T 1.51 -0.89 . F 3.06 3.18

Ser 593 T 1.62 -0.91 . F 2.52 3.18

Ser 594 T c 1.62 -0.41 * F 1.88 2.08

Thr 595 . c 1.62 -0.41 * F 1.64 1.61

Gin 596 . c 1.53 -0.41 * F 1.602.08

Ala 597 T c 1.49 -0.41 * F 2.102.08

Pro 598 T c 1.79 -0.40 . F 2.402.49

Glu 599 T c 1.79 -0.89 . F 3.00 2.49

Ser 600 T c 2.10 -0.90 . F 2.70 3.31

Gin 601 A . c 2.10 -1.00 . F 2.00 3.70

Glu 602 A C 2.69 -1.43 F 1.70 3.70

Ser 603 A C 2.09 -1.43 . F 1.40 4.79

Gin 604 A A 2.06 -1.13 F 0.902.28

Glu 605 A A 2.11 -1.03 * F 0.90 1.79

Glu 606 A A 1.52 -0.27 * F 0.602.09

Leu 607 A B 1.21 -0.16 . 0.45 1.22

His 608 A B 0.70 -0.07 0.45 1.02

Tyr 609 A B 0.70 0.61 -0.60 0.48

Ala 610 A B 0.00 1.01 * -0.60 0.95 Thr 611 A B -0.21 1.11 -0.60 0.60

Leu 612 A T 0.26 1.04 -0.20 0.59

Asn 613 A B -0.57 0.71 * -0.60 0.58

Phe 614 B B -0.21 0.86 -0.60 0.30

Pro 615 B T 0.17 0.37 * F 0.25 0.71

Gly 616 B T 0.59 0.11 * F 0.25 0.68

Val 617 B Z 1.19 -0.29 * F 0.80 1.54

Arg 618 T Z 1.19 -0.64 * F 1.50 1.54

Pro 619 T Z 1.30 -1.07 F 1.502.70

Arg 620 T " 1.62 -1.00 * 1 F 1.50 3.68

Pro 621 B T 1.37 -1.64 * . ] F 1.30 3.68

Glu 622 T 2.01 -1.03 F 1.84 2.35

Ala 623 B 1.94 -1.03 F 1.78 1.86

Arg 624 B 1.81 -1.03 1.97 2.40

Met 625 B T 1.39 -1.03 * * F 2.66 1.37

Pro 626 T T 1.60 -0.54 F 3.40 1.96

Lys 627 T T 1.01 -0.64 * F 3.06 1.74

Gly 628 T Z 1.60 -0.14 * . F 2.22 1.77

Thr 629 A . C 1.24 -0.76 * . F 1.78 1.91

Gln 630 A B 1.26 -0.43 . F 0.94 1.50

Ala 631 A B 1.47 0.07 . F 0.00 1.53

Asp 632 A B 0.57 -0.36 0.45 1.84

Tyr 633 A B 0.96 -0.20 * 0.30 0.79

Ala 634 A B 0.57 -0.60 0.75 1.56

Glu 635 A B 0.57 -0.31 0.30 0.81

Val 636 A B 0.77 0.09 -0.30 0.89

Lys 637 A B 0.38 -0.24 * 0.45 1.13

Phe 638 A B 0.23 -0.31 0.30 0.83

Gin 639 A B 0.43 0.11 -0.15 1.44

Ter 640 A B 0.04 -0.10 0.30 0.92

Table VI

Res Position I I II V V VI II VIII IX X XI XII XIII XIV

Met 1 A 3 -1.90 0.96 -0.60 0.21

Leu 2 A B -2.32 1.21 -0.60 0.13

Leu 3 A B -2.74 1.47 -0.60 0.09

Leu 4 A 1 B -2.57 1.73 -0.60 0.07

Leu 5 A ] B -2.99 1.54 -0.60 0.14

Leu 6 A ] B -3.20 1.54 -0.60 0.14

Leu 7 A B -2.68 1.54 -0.60 0.14

Pro 8 A B -2.21 1.77 -0.60 0.17

Leu 9 A B -1.29 1.51 -0.60 0.21

Leu 10 A B -0.48 0.83 -0.60 0.49

Trp 11 A z 0.44 0.14 * -0.10 0.55

Gly 12 A z 0.40 -0.29 . * F 0.80 1.31

Arg 13 A 0.61 -0.33 F 0.80 1.18

Glu 14 A B 1.08 -1.01 . * F 0.90 1.94

Arg 15 A B 1.89 -1.50 F 1.24 1.94

Val 16 A B 2.22 -1.53 F 1.58 1.71

Glu 17 A r 2.27 -1.53 F 2.32 1.98

Gly 18 r 2.16 -1.14 F 2.86 1.35

Gin 19 Γ T 2.27 -0.74 F 3.40 2.93

Lys 20 T c 2.20 -1.39 * F 2.86 3.32

Ser 21 T c 3.06 -1.39 F 2.86 6.70

Asn 22 Γ T 2.81 -1.81 F 3.06 6.46

Arg 23 Γ T 2.86 -1.46 F 3.06 5.06

Lys 24 Γ T 2.04 -1.07 F 3.06 5.06

Asp 25 Γ T 1.69 -0.77 F 3.40 2.60

Tyr 26 B T 1.39 -0.69 2.51 1.91

Ser 27 B B 1.39 -0.07 1.32 0.95 .

Leu 28 B B 0.98 0.33 0.38 0.98

Thr 29 B B 0.63 0.71 -0.26 0.84

Met 30 B T -0.22 0.34 0.10 0.84

Gin 31 B T -0.29 0.60 * F -0.05 0.76

Ser 32 B T -0.84 0.40 F 0.25 0.76

Ser 33 B T -0.03 0.56 F -0.05 0.57

Val 34 B B 0.28 0.34 F -0.15 0.57

Thr 35 B B 0.53 -0.06 F 0.45 0.73

Val 36 B B -0.07 -0.01 F 0.45 0.54 Gin 37 B B -0.43 0.21 F -0.15 0.72

Glu 38 B B -0.99 0.14 F -0.15 0.27

Gly 39 B T -0.17 0.30 * 0.10 0.27

Met 40 B B -0.71 0.16 * -0.30 0.21

Cys 41 B B 0.26 0.40 * * -0.30 0.09

Val 42 B B -0.41 0.40 * -0.30 0.18

His 43 B B -0.71 0.54 * * -0.60 0.10

Val 44 B B -1.07 0.31 * -0.30 0.24

Arg 45 B B -0.77 0.53 * -0.60 0.28

Cys 46 B r -0.34 0.27 * 0.100.28

Ser 47 T Γ 0.30 0.53 * 0.20 0.59

Phe 48 T r -0.52 0.31 * fc 0.500.46

Ser 49 B ' r 0.33 0.96 * * -0.20 0.64

Tyr 50 B -0.08 0.39 fc -0.10 0.80

Pro 51 B T 0.59 0.39 fc 0.25 1.24

Val 52 T r 0.58 0.00 * fc p 1.40 1.60

Asp 53 B T 1.28 0.10 * fc p 0.74 1.47

Ser 54 B . Γ 1.28 -0.66 . * F 1.98 1.59

Gin 55 B 1.52 -0.70 * p 2.12 2.87

Thr 56 T 1.52 -1.34 . * F 2.862.87

Asp 57 T Γ 1.52 -0.91 . * F 3.40 3.31

Ser 58 . Γ Z 1.49 -0.66 . F 2.86 1.42

Asp 59 B . Γ 1.44 -0.56 . F 2.32 1.34

Pro 60 B . Γ 1.20 -0.61 . F 1.83 0.79

Val 61 T 1.22 0.14 0.640.93

His 62 B . Γ 0.52 0.67 * -0.20 0.58

Gly 63 B . Γ 0.93 1.46 * -0.20 0.33

Tyr 64 B . Γ 0.34 1.03 * -0.20 0.86

Trp 65 B . Γ 0.21 0.89 * -0.20 0.64

Phe 66 B 1.07 0.81 * -0.09 0.64

Arg 67 B . Γ 1.10 0.79 * * 0.42 0.66

Ala 68 T Γ 0.56 0.03 * * 1.58 1.04

Gly 69 T T 0.50 -0.20 * * F 2.49 0.85

Asn 70 T r 0.50 -0.60 * * F 3.10 0.58

Asp 71 C 1.24 0.31 * * F 1.49 0.60

He 72 C 0.54 -0.19 * * F 1.93 1.22

Ser 73 A T 0.92 -0.11 1.32 0.76

Trp 74 A B 0.41 -0.09 0.61 0.71

Lys 75 A B -0.18 0.56 -0.60 0.75

Ala 76 A B -0.49 0.37 * -0.30 0.56

Pro 77 B 0.40 0.47 -0.40 0.77 Val 78 B 0.70 -0.04 0.50 0.62

Ala 79 B 0.78 0.36 -0.10 0.99

Thr 80 c 0.14 0.29 F 0.25 0.99

Asn 81 c 0.44 0.36 F 0.40 1.35

Asn 82 T c 0.07 0.63 F 0.30 1.41

Pro 83 T c 0.07 0.63 F 0.15 0.98

Ala 84 r r 0.66 0.79 0.20 0.45

Tip 85 Γ c 0.97 0.79 0.000.49

Ala 86 A B 0.97 0.39 -0.30 0.55

Val 87 A B 0.66 -0.04 0.30 0.94

Gin 88 A B 0.98 -0.06 F 0.94 1.29

Glu 89 A B 1.57 -0.97 F 1.58 2.50

Glu 90 A B 1.97 -1.47 F 1.92 5.62

Thr 91 T Γ 1.86 -2.11 F 3.06 6.36

Arg 92 T Γ 2.68 -1.73 F 3.40 3.18

Asp 93 T Γ 1.87 -1.23 F 3.06 2.50

Arg 94 B Γ 1.06 -0.54 2.17 1.43

Phe 95 A B 0.71 -0.34 0.98 0.60

His 96 A B 1.02 0.09 * 0.04 0.36

Leu 97 A c 0.70 0.09 -0.10 0.30

Leu 98 A T 0.70 0.51 -0.20 0.54

Gly 99 A T 0.28 0.13 F 0.59 0.69

Asp 100 c 1.02 0.11 * F 1.08 1.21

Pro 101 T 1.06 -0.57 F 2.52 2.93

Gin 102 T 1.20 -0.86 F 2.864.76

Thr 103 T Γ 1.70 -0.71 F 3.40 1.53

Lys 104 B Γ 1.23 -0.23 F 2.36 1.43

Asn 105 B Γ 0.93 0.03 F 1.27 0.68

Cys 106 B Γ 0.26 0.01 0.78 0.63

Thr 107 B B 0.37 0.21 0.04 0.22

Leu 108 B B 0.68 0.21 -0.30 0.27

Ser 109 B B 0.04 -0.19 0.30 0.84

He 110 B B 0.16 -0.26 0.30 0.59

Arg 111 B B 0.22 -0.74 F 0.90 1.39

Asp 112 A B 0.23 -0.81 F 0.90 1.03

Ala 113 A B 1.04 -0.81 F 0.90 1.97

Arg 114 A B 0.76 -1.50 1.03 1.68

Met 115 . A B 1.30 -1.00 F 1.46 1.01

Ser 116 A c 1.30 -0.57 F 1.79 0.99

Asp 117 T r 1.06 -1.07 F 2.67 0.99

Ala 1 18 Γ T 0.94 -0.31 F 2.80 1.57 Gly 1 19 T r 0.13 -0.14 * F 2.52 1.02

Arg 120 B T 0.84 0.26 0.94 0.53

Tyr 121 B B 0.54 0.26 0.41 1.02

Phe 122 B B 0.54 0.37 0.13 1.02

Phe 123 B B 1.18 -0.06 * 0.300.90

Arg 124 B B 1.18 -0.06 0.79 1.15

Met 125 B 1.07 -0.39 1.33 1.32

Glu 126 T r 0.42 -0.77 F 2.72 2.45

Lys 127 T Γ 1.17 -0.87 * F 2.91 0.88

Gly 128 T Γ 1.58 -0.87 * * F 3.40 1.77

Asn 129 T Γ 1.47 -0.57 * F 3.06 1.08

He 130 Z 1.82 -0.17 * F 1.87 0.86

Lys 131 T 1.87 0.59 0.83 1.37

Trp 132 T 1.58 0.16 * 0.79 1.70

Asn 133 B Γ 1.92 0.51 0.19 3.81

Tyr 134 B Γ 1.92 -0.17 * 1.33 3.18

Lys 135 B Γ 2.00 0.23 0.97 5.23

Tyr 136 T Γ 1.66 0.00 * F 2.362.68

Asp 137 T 1.09 -0.01 * F 2.40 2.30

Gin 138 B B 1.09 -0.13 * F 1.41 0.85

Leu 139 B B 0.48 0.27 0.42 0.87

Ser 140 B B 0.12 0.16 0.180.39

Val 141 B B -0.22 0.64 -0.36 0.32

Asn 142 B B -1.03 0.74 * -0.60 0.40

Val 143 B B -1.34 0.74 -0.60 0.24

Thr 144 B B -0.57 0.84 fc -0.60 0.47

Ala 145 B B -0.16 0.70 -0.60 0.40

Leu 146 B B 0.49 0.30 -0.15 1.06

Thr 147 B B 0.49 0.09 -0.15 1.14

His 148 B B 0.46 0.00 * 0.45 1.81

Arg 149 B Γ -0.04 0.19 0.25 1.54

Pro 150 B Γ -0.34 0.19 0.10 0.88

Asn 151 T T 0.26 0.39 0.500.45

He 152 B T 0.22 0.31 0.10 0.36

Leu 153 B -0.06 0.74 * -0.40 0.23

He 154 B T -0.98 0.80 -0.20 0.21

Pro 155 B r -0.77 1.09 * F -0.05 0.24

Gly 156 B T -1.07 0.40 F 0.25 0.51

Thr 157 B T -0.52 0.10 F 0.25 0.97

Leu 158 B -0.38 -0.16 F 0.65 0.62

Glu 159 B T -0.19 -0.01 F 0.85 0.34 Ser 160 T T 0.02 0.34 F 0.65 0.20

Gly 161 T T 0.37 0.26 F 0.61 0.42

Cys 162 T r -0.13 -0.03 1.02 0.39

Phe 163 B B 0.37 0.66 -0.72 0.24

Gin 164 B B -0.30 0.76 -0.76 0.35

Asn 165 . B T -0.30 0.90 * -0.40 0.35

Leu 166 B B -0.81 0.71 -0.76 0.55

Thr 167 . B T -0.36 0.57 -0.32 0.23

Cys 168 . B T 0.06 0.60 -0.28 0.22

Ser 169 . B T -0.53 1.11 -0.24 0.29

Val 170 B C -1.20 0.93 -0.40 0.20

Pro 171 T -0.39 1.01 0.00 0.20

Trp 172 T -0.08 0.44 0.00 0.26

Ala 173 B 0.24 0.46 -0.12 0.61

Cys 174 B 0.23 0.24 0.46 0.39

Glu 175 T Γ 0.88 0.30 . . F 1.49 0.53

Gin 176 T Γ 0.88 -0.19 . . F 2.37 0.81

Gly 177 T Γ 0.57 -0.26 . . F 2.80 2.35

Thr 178 Γ c 0.27 -0.21 . . F 2.32 1.34

Pro 179 c 0.63 0.47 * . F 0.79 0.54

Pro 180 . B c 0.34 0.46 * . F 0.31 0.74

Met 181 B B -0.26 0.94 -0.32 0.54

He 182 B B -0.26 1.07 -0.60 0.34

Ser 183 B B -0.26 1.07 -0.60 0.22

Trp 184 B B -0.34 1.13 -0.60 0.32

Met 185 B B -0.99 0.90 * -0.60 0.61

Gly 186 B T -0.69 0.86 -0.20 0.34

Thr 187 B c -0.01 0.86 * . F -0.25 0.43

Ser 188 . B c -0.52 0.37 * . F 0.05 0.67

Val 189 B B -0.27 0.44 F -0.45 0.56

Ser 190 c 0.12 0.51 F -0.05 0.53

Pro 191 c 0.17 0.46 . . F -0.05 0.61

Leu 192 c 0.17 0.46 _ F 0.34 1.10

His 193 B T 0.16 0.30 F 0.88 1.19

Pro 194 B T 1.12 0.40 F 0.82 1.11

Ser 195 T r 1.12 -0.03 F 2.36 2.64

Thr 196 r c 1.03 -0.33 F 2.402.60

Thr 197 B T 0.99 -0.44 F 1.96 2.25

Arg 198 B T 0.21 -0.23 F 1.72 1.25

Ser 199 B r 0.11 0.07 F 0.73 0.71

Ser 200 B T -0.40 0.07 F 0.49 0.71 Val 201 B B -0.98 0.27 -0.30 0.30

Leu 202 B B -0.88 0.96 -0.60 0.16

Thr 203 B B -0.99 1.00 -0.60 0.18

Leu 204 B B -0.90 1.01 -0.60 0.42

He 205 B B -0.60 0.80 * -0.60 0.79

Pro 206 B B 0.22 0.51 F -0.45 0.95

Gin 207 B 1.00 0.53 F -0.10 1.57

Pro 208 B 0.97 0.34 F 0.20 3.04

Gin 209 r 1.47 0.09 * F 0.60 1.95

His 210 Γ r 2.06 0.14 F 0.80 1.62

His 211 Γ T 1.46 0.13 F 0.80 1.41

Gly 212 r r 1.14 0.39 F 0.65 0.67

Thr 213 r Γ 0.69 0.47 F 0.35 0.71

Ser 214 B Γ 0.69 0.54 F -0.05 0.28

Leu 215 B B -0.13 0.44 -0.60 0.49

Thr 216 B B -0.41 0.66 -0.60 0.25

Cys 217 B B -0.88 0.66 » -0.60 0.27

Gin 218 B B -0.78 0.96 * -0.60 0.27

Val 219 B B -0.82 0.70 K -0.60 0.29

Thr 220 B B -0.60 0.64 » -0.60 0.54

Leu 221 B Γ -0.63 0.57 -0.20 0.31

Pro 222 B Γ -0.82 0.60 F -0.05 0.42

Gly 223 Γ T -1.13 0.60 F 0.35 0.21

Ala 224 r C -0.59 0.60 F 0.35 0.38

Gly 225 B -0.28 0.40 F 0.15 0.35

Val 226 B 0.64 0.37 F 0.65 0.57

Thr 227 B T 0.54 -0.06 F 1.80 1.10

Thr 228 B r 0.00 -0.07 F 2.00 1.61

Asn 229 B T 0.59 0.19 F 1.20 1.52

Arg 230 B T 0.12 -0.06 F 1.60 1.83

Thr 231 B B 0.98 0.14 * p 0.40 1.04

Ile 232 B B 0.43 0.06 * 0.05 1.04

Gin 233 B B 0.44 0.30 * -0.30 0.40

Leu 234 B B 0.20 0.69 * -0.60 0.37

Asn 235 B T -0.12 0.96 * -0.20 0.82

Val 236 B T -0.02 0.70 * -0.20 0.73

Ser 237 T C 0.87 0.73 * 0.15 1.37

Tyr 238 T C 0.87 0.44 * p 0.30 1.48

Pro 239 C 0.87 0.44 * F 0.10 3.21

Pro 240 T T 0.56 0.49 F 0.50 1.97

Gin 241 T T 0.56 0.59 F 0.50 1.82 Asn 242 B • r 0.54 0.47 F -0.05 0.87

Leu 243 B T -0.07 0.53 -0.20 0.81

Thr 244 B B -0.56 0.74 -0.60 0.35

Val 245 B B -0.34 1.13 * -0.60 0.19

Thr 246 B 1 B -0.69 1.13 -0.60 0.39

Val 247 B B -0.69 0.87 -0.60 0.27

Phe 248 B B -0.22 0.39 -0.30 0.63

Gin 249 B B -0.22 0.17 F 0.09 0.43

Gly 250 T 2 0.04 0.17 F 0.93 0.84

Glu 251 T ( Z 0.06 0.03 F 1.17 0.98

Gly 252 T ( z 0.60 -0.37 F 2.01 0.76

Thr 253 T ( 0.71 -0.29 F 2.40 1.11

Ala 254 B -0.10 -0.21 F 1.61 0.65

Ser 255 B -0.10 0.47 F 0.47 0.54

Thr 256 B -0.10 0.47 F 0.23 0.37

Ala 257 B -0.06 0.39 F 0.29 0.59

Leu 258 B -0.04 0.27 F 0.05 0.59

Gly 259 T 0.24 0.27 F 0.45 0.55

Asn 260 T Z -0.27 0.17 F 0.45 0.72

Ser 261 T z -0.26 0.36 F 0.45 0.72

Ser 262 T -0.52 0.06 F 0.45 0.98

Ser 263 T "^, -0.52 0.27 F 0.45 0.45

Leu 264 A B -0.18 0.56 F -0.45 0.28

Ser 265 A B -0.52 0.17 -0.30 0.36

Val 266 A B -0.22 0.21 -0.30 0.27

Leu 267 A B -0.22 0.23 -0.13 0.56

Glu 268 A B -0.73 -0.07 * F 0.79 0.56

Gly 269 T T 0.19 0.23 F 1.16 0.62

Gin 270 T T -0.32 -0.41 F 2.08 1.47

Ser 271 B T -0.32 -0.41 F 1.70 0.70

Leu 272 B T -0.18 0.23 F 0.93 0.53

Arg 273 B B -0.77 0.37 0.21 0.16

Leu 274 B B -1.28 0.47 -0.26 0.12

Val 275 B B -1.28 0.73 -0.43 0.11

Cys 276 B B -1.28 0.04 -0.30 0.09

Ala 277 B B -0.47 0.43 -0.60 0.15

Val 278 B B -0.79 0.14 0.00 0.33

Asp 279 T T -0.19 -0.07 F 1.85 0.96

Ser 280 T c 0.08 -0.21 F 2.10 1.46

Asn 281 T c 0.86 -0.21 F 2.40 1.99

Pro 282 . T c 0.63 -0.86 F 3.00 2.34 Pro 283 r 1.19 -0.17 F 2.40 1.44

Ala 284 Γ 0.90 -0.17 F 2.10 1.20

Arg 285 B B 0.89 0.34 0.300.82

Leu 286 B B 0.60 0.40 -0.30 0.76

Ser 287 B B 0.92 0.89 -0.60 0.79

Trp 288 B B 0.83 0.39 -0.30 0.79

Thr 289 B B 0.61 0.77 -0.45 1.29

Trp 290 B B 0.19 0.77 * -0.60 0.79

Arg 291 B B 0.19 0.87 -0.45 1.09

Ser 292 B B 0.24 0.64 -0.60 0.62

Leu 293 . B Γ 0.32 0.91 -0.20 0.93

Thr 294 . B . Γ 0.33 0.43 -0.20 0.73

Leu 295 . B < z 0.62 0.81 -0.40 0.73

Tyr 296 B r 0.30 0.83 F 0.10 1.53

Pro 297 Γ Γ 0.30 0.57 F 0.62 1.64

Ser 298 Γ Γ 1.11 0.47 F 0.74 2.67

Gin 299 Γ z 1.21 0.19 F 0.96 2.74

Pro 300 Γ Γ 1.21 _,. -0.14 F 1.88 2.74

Ser 301 Γ z 0.60 0.11 F 1.20 1.69

Asn 302 Γ z 0.00 0.37 F 0.93 0.72

Pro 303 B Γ 0.30 0.66 F 0.31 0.39

Leu 304 A B -0.51 0.23 * F 0.09 0.50

Val 305 A B -0.30 0.53 -0.48 0.26

Leu 306 A B -0.86 0.53 * -0.60 0.29

Glu 307 A B -0.89 0.74 * -0.60 0.26

Leu 308 A B -1.49 0.56 -0.60 0.47

Gin 309 A B -1.02 0.60 -0.60 0.47

Val 310 A B -0.17 0.34 -0.30 0.27

His 311 A B 0.64 0.34 -0.30 0.55

Leu 312 A z 0.30 -0.34 * 0.84 0.55

Gly 313 Γ c 1.11 -0.31 F 1.73 0.73

Asp 314 Γ Γ 0.41 -0.96 F 2.57 0.93

Glu 315 T T 0.96 -0.67 F 2.91 0.97

Gly 316 T T 0.32 -0.87 * F 3.40 1.42

Glu 317 A T 1.24 -0.73 F 2.51 0.46

Phe 318 A B 1.00 -0.73 1.62 0.52

Thr 319 A B 1.00 -0.23 0.98 0.53

Cys 320 A B 1.00 -0.26 0.89 0.53

Arg 321 A T 1.04 0.14 0.60 0.98

Ala 322 A T 0.23 -0.26 * F 1.60 0.91

Gin 323 A r 0.59 -0.06 F 2.00 1.40 Asn 324 T T 0.60 -0.20 * " F 2.50 0.71

Ser 325 . r C 1.27 0.19 * * F 1.45 0.94

Leu 326 . r c ι.i2 0.09 * * F 1.20 0.94

Gly 327 T r 0.86 0.19 . . F 1.15 0.79

Ser 328 B B 0.56 0.43 . . F -0.20 0.44

Gin 329 B B . . -0.26 0.43 . . F -0.45 0.71

His 330 B B 0.04 0.43 . * -0.60 0.59

Val 331 B B 0.04 0.40 . * -0.60 0.71

Ser 332 . - \ B 0.09 0.70 . * -0.60 0.34

Leu 333 . . \ B -0.42 0.69 . * -0.60 0.33

Asn 334 . - A B -0.42 0.87 . * -0.60 0.37

Leu 335 A C -0.39 0.63 . « -0.40 0.48

Ser 336 A C 0.47 0.64 . « -0.25 1.01

Leu 337 A B 0.52 -0.04 . * 0.45 1.09

Gin 338 A B 1.02 0.31 . * F 0.00 2.06

Gin 339 A B 0.68 0.11 . * F 0.34 2.22

Glu 340 A B 1.53 0.16 . * F 0.68 2.66

Tyr 341 B r 1.23 -0.53 * * F 2.32 3.08

Thr 342 T Γ 2.16 -0.31 * ι F 2.76 1.76

Gly 343 T Γ 1.94 -0.71 * * F 3.40 1.99

Lys 344 T Γ 1.09 -0.29 * * F 2.76 1.96

Met 345 B 0.79 -0.40 . * F 1.82 1.01

Arg 346 B 0.69 -0.50 * * F 1.48 1.37

Pro 347 B T 0.14 -0.50 * * F 1.19 0.68

Val 348 B T -0.32 0.14 . ^> * F 0.25 0.51

Ser 349 B T -1.18 0.21 * ^• k p 0.25 0.21

Gly 350 B T -0.92 0.90 * -0.20 0.11

Val 351 B B -1.62 0.90 * -0.60 0.15

Leu 352 B B -2.27 0.76 -0.60 0.1 1

Leu 353 B B -1.76 1.01 -0.60 0.09

Gly 354 B B -1.80 1.01 -0.60 0.11

Ala 355 B B -2.04 0.80 -0.60 0.14

Val 356 B B -1.53 0.61 -0.60 0.17

Gly 357 B -1.31 0.36 -0.10 0.17

Gly 358 T C -0.81 0.43 F 0.15 0.17

Ala 359 B T -1.06 0.41 F -0.05 0.33

Gly 360 B T -1.28 0.27 0.10 0.33

Ala 361 B T -1.28 0.53 -0.20 0.28

Thr 362 B B -1.63 0.74 -0.60 0.20

Ala 363 B B -2.10 1.03 -0.60 0.18

Leu 364 B B -1.81 1.29 -0.60 0.15 Val 365 B 1 B -2.17 1.17 -0.60 0.14

Phe 366 B 1 B -2.24 1.47 -0.60 0.12

Leu 367 B 1 B -2.79 1.54 -0.60 0.08

Ser 368 B 1 B -3.09 1.50 -0.60 0.08

Phe 369 B 1 B -2.98 1.54 -0.60 0.06

Cys 370 B B -3.01 1.54 -0.60 0.06

Val 371 B 1 B -3.17 1.54 -0.60 0.03

He 372 B B -3.21 1.80 -0.60 0.03

Phe 373 B ] B -2.80 1.66 -0.60 0.04

He 374 B B -2.40 1.09 -0.60 0.11

Val 375 B 1 B -2.40 0.83 -0.26 0.20

Val 376 B B -1.43 0.71 0.08 0.12

Arg 377 B r -0.50 -0.07 1.72 0.35

Ser 378 T Γ 0.24 -0.76 * . F 2.91 0.94

Cys 379 T Γ 0.83 -1.40 * . F 3.40 2.53

Arg 380 T Γ 1.10 -1.66 * . F 3.06 1.73

Lys 381 A T 2.07 -1.16 * . F 2.32 1.30

Lys 382 A T 1.74 -1.54 * . F 1.98 4.76

Ser 383 A C 1.46 -1.69 * . F 1.44 3.76

Ala 384 A B 1.53 -1.19 F 0.90 1.90

Arg 385 A B 1.42 -0.69 F 0.75 0.96

Pro 386 A B 0.52 -0.69 F 0.90 1.20

Ala 387 A B 0.13 -0.43 0.30 0.88

Ala 388 A B 0.43 -0.50 0.30 0.44

Asp 389 B T 0.13 -0.50 0.70 0.48

Val 390 B T -0.32 -0.24 0.70 0.33

Gly 391 B T -0.71 -0.31 * . F 0.85 0.33

Asp 392 B T -0.08 -0.20 * . F 0.85 0.19

He 393 A B 0.51 -0.20 F 0.45 0.52

Gly 394 A B -0.08 -0.84 * . F 0.75 0.88

Met 395 A B 0.78 -0.77 * . F 1.01 0.53

Lys 396 A B 0.81 -0.37 F 1.12 1.22

Asp 397 B T -0.08 -0.57 F 2.08 1.78

Ala 398 B T 0.92 -0.31 F 2.04 1.26

Asn 399 B T 0.92 -0.93 F 2.60 1.23

Thr 400 B T 1.22 -0.50 F 1.89 0.73

He 401 B T 0.59 -0.11 F 1.63 0.97

Arg 402 B T 0.29 -0.11 F 1.61 0.61

Gly 403 B T 0.88 -0.13 F 1.59 0.57

Ser 404 T C 0.53 -0.21 F 1.92 1.40

Ala 405 C 0.84 -0.47 F 1.81 0.71 Ser 406 r c 0.92 -0.07 * * F 2.40 1.15

Gin 407 Γ c 0.50 0.19 * . F 1.41 0.71

Gly 408 r c 0.84 0.29 F 1.32 1.01

Asn 409 r c 0.84 -0.21 . F 1.68 1.30

Leu 410 c 1.14 -0.21 . F 1.58 1.01

Thr 411 B r 0.86 0.30 * F 1.08 1.07

Glu 412 B Γ 0.86 0.37 * F 1.27 0.67

Ser 413 B Γ 1.20 -0.03 * F 2.36 1.36

Trp 414 T Γ 1.20 -0.71 * F 3.40 1.58

Ala 415 c 1.80 -0.80 * * F 2.66 1.46

Asp 416 T 2.22 -0.37 * . F 2.56 1.69

Asp 417 c 2.19 -0.76 * * F 2.66 3.15

Asn 418 Γ c 2.46 -1.17 * * F 2.86 4.24

Pro 419 Γ c 2.40 -1.17 . . F 2.86 3.45

Arg 420 T Γ 2.18 -0.74 * F 3.40 2.05

His 421 Γ c 1.59 -0.06 2.41 1.05

His 422 A c 1.00 0.04 0.92 0.69

Gly 423 A B 0.97 0.11 0.38 0.35

Leu 424 A B 0.88 0.61 -0.26 0.35

Ala 425 A B 0.47 0.50 -0.30 0.35

Ala 426 A c 0.16 0.39 0.500.47

His 427 Γ c 0.19 0.39 1.20 0.57

Ser 428 Γ c 0.53 -0.30 _* F 2.25 0.97

Ser 429 Γ c 1.46 -0.80 F 3.00 1.66

Gly 430 Γ c 2.04 -1.30 F 2.70 2.39

Glu 431 A c 1.74 -1.80 F 2.00 3.09

Glu 432 A c 1.78 -1.50 * F 1.70 1.62

Arg 433 A B 1.83 -1.49 * F 1.20 2.83

Glu 434 A B 1.54 -1.16 * F 0.90 2.56

He 435 A B 1.68 -0.66 0.75 1.49

Gin 436 A B 0.87 -0.23 * 0.45 1.18

Tyr 437 A B 0.57 0.46 * -0.60 0.5.6

Ala 438 c -0.24 0.84 * -0.05 1.07

Pro 439 B -0.28 0.94 * -0.40 0.54

Leu 440 B 0.66 1.04 * -0.40 0.47

Ser 441 c 0.31 0.29 * 0.10 0.92

Phe 442 c 0.56 0.21 0.10 0.59

His 443 T 0.93 -0.21 1.05 1.24

Lys 444 T 1.14 -0.47 F 1.50 1.43

Gly 445 c 1.96 -0.46 F 1.60 2.86

Glu 446 c 1.44 -1.24 F 2.20 3.51 Pro 447 c 1.84 -1.06 * F 2.50 1.45

Gin 448 r 1.53 -0.67 . F 3.00 1.96

Asp 449 c 1.49 -0.67 . F 2.50 1.12

Leu 450 T C 1.83 -0.27 . F 2.10 1.26

Ser 451 T C 1.24 -0.70 . F 2.10 1.26

Gly 452 B T 1.14 -0.60 . F 1.45 0.76

Gin 453 T C 1.14 -0.11 F 1.20 1.33

Glu 454 c 1.14 -0.40 F 1.00 1.60

Ala 455 c 1.96 -0.39 . F 1.30 2.59

Thr 456 c 2.01 -0.81 . F 1.90 2.59

Asn 457 ' r ( 2.06 -0.46 . F 2.10 2.35

Asn 458 ' r I z 2.06 -0.07 * F 2.40 3.11

Glu 459 ' r ( 1.17 -0.57 * F 3.00 3.73

Tyr 460 T r 1.80 -0.37 . _* F 2.60 1.63

Ser 461 T 1.22 -0.77 _* F 2.40 2.02

Glu 462 B 1.01 -0.49 F 1.25 0.82

He 463 B 1.06 -0.06 . F 1.22 0.81

Lys 464 B 0.67 -0.81 F 1.64 1.21

He 465 B 0.52 -0.77 * F 1.760.89

Pro 466 B 0.43 -0.34 . 1.73 1.62

Lys 467 T 0.04 -0.60 2.70 1.04

Ter 468 B 0.54 -0.17 * 1.73 1.89

Table VII

Res Position III 1 V V VI vn VIII IX X . KI XII XIII XIV

Met 1 B 0.43 0.01 -0.10 0.79

Asp 2 B 0.48 -0.41 0.65 1.21

Leu 3 B T 0.06 -0.41 0.70 0.94

Pro 4 T -0.41 -0.16 0.70 0.78

Arg 5 B T -0.88 -0.13 F 0.85 0.35

Gly 6 B T -0.87 0.51 F -0.05 0.31

Leu 7 B B -1.16 0.33 -0.30 0.20

Val 8 B B -0.93 0.81 -0.60 0.11

Val 9 B B -1.53 1.31 -0.60 0.11

Ala 10 B B -1.94 1.57 * -0.60 0.11

Trp 11 B B -2.41 1.27 -0.60 0.20

Ala 12 B B -1.89 1.31 -0.60 0.22

Leu 13 B B -1.24 1.59 -0.60 0.23

Ser 14 -0.73 1.51 -0.20 0.34

Leu 15 -0.84 1.03 -0.20 0.34

Trp 16 T z -0.87 1.31 0.00 0.35

Pro 17 T -0.28 1.11 0.00 0.38

Gly 18 T T 0.22 0.73 F 0.35 0.77

Phe 19 T T -0.18 0.53 F 0.50 1.06

Thr 20 . c 0.63 0.40 F 0.25 0.59

Asp 21 . c 0.32 0.37 F 0.25 0.96

Thr 22 . c 0.53 0.56 0.29 1.10

Phe 23 B 0.57 -0.23 1.33 1.27

Asn 24 B T 1.38 -0.23 1.87 1.10

Met 25 T T 1.73 -0.23 2.61 1.49

Asp 26 T T 1.52 -0.71 * F 3.40 3.44

Thr 27 T T 1.94 -1.07 F 3.06 3.31

Arg 28 T 1.79 -1.47 F 2.52 6.55

Lys 29 B 0.90 -1.44 F 1.78 2.91

Pro 30 B B 1.29 -0.76 F 1.24 1.41

Arg 31 B B 0.94 -0.81 F 0.90 1.12

Val 32 B B 0.96 -0.39 0.30 0.55

He 33 B T 0.96 0.00 F 0.22 0.48

Pro .34 B T 0.60 -0.43 F 0.79 0.48

Gly 35 T T 0.22 0.06 F 0.56 0.93

Ser 36 B T -0.59 -0.09 F 0.88 1.34 Arg 37 B B -0.43 0.01 F -0.30 0.75

Thr 38 B B 0.11 0.37 F -0.27 0.66

Ala 39 B B 0.08 0.37 -0.39 0.49

Phe 40 B B 0.11 0.74 -0.66 0.39

Phe 41 B B -0.44 1.23 -0.63 0.39

Gly 42 B B -0.56 1.39 -0.60 0.29

Tyr 43 B B -0.24 1.29 -0.60 0.57

Thr 44 B B 0.31 0.90 -0.45 1.14

Val 45 B B 1.01 0.61 -0.45 1.57

Gin 46 B B 0.82 0.19 -0.15 1.67

Gin 47 B B 0.87 0.11 -0.30 0.81

His 48 B B 0.77 0.01 0.10 1.47

Asp 49 B B 1.08 -0.20 F 0.95 0.84

He 50 . B r 1.98 -0.20 F 1.60 0.78

Ser 51 Γ Γ 1.69 -0.60 F 2.70 1.14

Gly 52 Γ Γ 0.88 -0.19 F 2.50 0.72

Asn 53 Γ Γ 0.06 0.50 F 1.35 0.85

Lys 54 Γ Γ -0.80 0.46 F 1.100.47

Trp 55 B B -0.26 0.71 -0.10 0.35

Leu 56 B B -0.54 0.71 -0.35 0.22

Val 57 B B -0.41 0.81 -0.60 0.11

Val 58 B B -1.22 1.24 -0.60 0.16

Gly 59 B B -1.27 1.01 -0.60 0.16

Ala 60 B -1.29 0.33 -0.10 0.38

Pro 61 C -0.48 0.17 0.10 0.73

Leu 62 C 0.03 -0.07 F 1.34 1.19

Glu 63 Γ 0.64 -0.07 * F 1.68 1.16

Thr 64 B Γ 0.99 0.19 fc F 1.42 1.18

Asn 65 Γ T 1.62 0.16 fc F 2.16 2.47

Gly 66 Γ r 1.52 -0.53 F 3.402.86

Tyr 67 Γ 1.99 -0.04 F 2.56 2.86

Gin 68 Γ 1.99 -0.10 F 2.48 1.76 .

Lys 69 B Γ T 1.44 -0.50 * F 2.602.97

Thr 70 B T 1.20 -0.29 * F 2.12 1.41

Gly 71 B T 1.59 -0.29 * F 2.04 1.27

Asp 72 B T 1.17 -0.69 * F 2.60 1.27

Val 73 B 0.96 -0.11 * F 1.69 0.47

Tyr 74 B T 0.06 -0.17 . 1.48 0.74

Lys 75 B T -0.52 0.04 0.62 0.33

Cys 76 B T -0.21 0.73 0.06 0.31

Pro 77 B T -0.56 0.59 -0.20 0.27 Val 78 B 0.30 0.26 -0.10 0.13

He 79 B -0.12 0.66 -0.40 0.40

His 80 B T -0.48 0.66 -0.20 0.14

Gly 81 B T 0.23 0.71 -0.20 0.27

Asn 82 r T -0.37 0.07 0.50 0.77

Cys 83 B T 0.49 0.07 0.25 0.47

Thr 84 B 0.57 -0.03 * F 0.65 0.76

Lys 85 B 0.26 0.23 F 0.05 0.39

Leu 86 B 0.71 0.26 -0.10 0.72

Asn 87 B -0.14 -0.31 0.50 0.97

Leu 88 B B 0.21 -0.16 0.30 0.36

Gly 89 B B -0.29 0.33 -0.30 0.63

Arg 90 B B -0.63 0.33 -0.30 0.32

Val 91 B B 0.18 0.31 fc -0.30 0.53

Thr 92 B B -0.68 0.03 fc -0.30 0.85

Leu 93 B B -0.17 0.24 -0.30 0.32

Ser 94 B B . 0.18 0.63 -0.60 0.58

Asn 95 B B 0.18 -0.01 _* F 0.45 0.70

Val 96 B B 1.08 -0.50 F 0.60 1.67

Ser 97 A 1.39 -1.19 * . F 1.102.49

Glu 98 A 2.20 -1.57 * . F 1.10 2.58

Arg 99 A T 1.90 -1.57 _* F 1.30 5.60

Lys 100 A T 2.01 -1.60 ^sk 1.304.13

Asp 101 A T 2.06 -1.99 _* F 1.30 4.67

Asn 102 A T 2.01 -1.30 1.15 1.97

Met 103 A 1.20 -0.87 * 0.80 0.97

Arg 104 A 0.79 -0.19 * 0.50 0.48

Leu 105 B -0.07 0.20 * * -0.10 0.40

Gly 106 B -0.66 0.49 -0.40 0.33

Leu 107 B -0.97 0.37 -0.10 0.17

Ser 108 B -0.37 0.86 -0.40 0.30

Leu 109 B -0.69 0.57 -0.06 0.49

Ala 110 B 0.17 0.57 0.28 0.92

Thr 111 B 0.51 -0.11 F 1.82 1.37

Asn 112 T C 1.32 -0.50 2.562.78

Pro 113 T T 1.32 -0.79 F 3.40 4.42

Lys 1 14 T T 1.43 -0.90 F 3.06 4.10

Asp 115 T T 1.21 -0.60 . . F 2.72 2.21

Asn 116 T T 0.93 -0.31 F 2.08 1.18

Ser 117 B T 0.27 -0.24 F 1.19 0.60

Phe 1 18 B T 0.18 0.33 0.10 0.19 Leu 119 B T -0.08 0.71 -0.20 0.16

Ala 120 B -0.89 0.74 -0.40 0.18

Cys 121 B -1.18 1.04 -0.40 0.17

Ser 122 T ( Z -1.18 1.17 0.00 0.22

Pro 123 T T -0.51 0.87 0.20 0.30

Leu 124 T T 0.30 0.87 0.20 0.75

Trp 125 T T 0.22 0.30 0.50 0.97

Ser 126 B 0.54 0.49 -0.27 0.34

His 127 B T 0.54 0.49 0.060.40

Glu 128 T T 0.46 0.19 0.89 0.51

Cys 129 T T 1.02 -0.34 F 1.77 0.51

Gly 130 T T 1.07 0.03 F 1.300.59

Ser 131 T T 1.06 0.29 F 1.17 0.54

Ser 132 T T 0.78 0.77 F 0.89 1.44

Tyr 133 T T 0.43 0.69 F 0.76 2.11

Tyr 134 B T 0.50 0.69 F 0.23 1.55

Thr 135 B 0.18 0.91 F -0.10 1.15

Thr 136 B 0.18 1.10 -0.40 0.39

Gly 137 B T 0.59 0.73 -0.20 0.34

Met 138 B T -0.02 -0.03 * 0.70 0.46

Cys 139 B T 0.22 0.13 0.100.23

Ser 140 B T 0.23 0.04 * 0.100.38

Arg 141 B 0.54 0.00 * * p 0.05 0.52

Val 142 B 0.19 -0.21 * F 0.80 1.55

Asn 143 B T 0.90 0.00 * F 0.61 1.00

Ser 144 T C 0.87 -0.39 * F 1.62 1.00

Asn 145 T r 0.87 0.40 * F 1.13 1.17

Phe 146 T r 0.80 0.14 1.340.97

Arg 147 T 1.34 -0.26 2.10 1.45

Phe 148 B T 0.49 -0.16 * 1.69 1.30

Ser 149 B B 0.20 0.09 * F 0.63 1.12

Lys 150 B C -0.01 -0.20 sfc F 1.07 0.58

Thr 151 B T 0.10 0.23 * F 0.61 1.03

Val 152 B C -0.82 -0.06 0.50 0.77

Ala 153 B B C -0.12 0.24 -0.10 0.32

Pro 154 A A 0.29 0.64 -0.60 0.38

Ala 155 A A -0.42 0.16 -0.15 1.01

Leu 156 A A B -0.1 1 0.09 -0.30 0.54

Gin 157 A A B 0.43 -0.01 0.30 0.60

Arg 158 A B B 0.78 0.04 * F -0.15 0.86

Cys 159 A B B 0.39 0.30 * F 0.00 1.63 Gin 160 . B B 0.98 0.23 -0.30 0.93

Thr 161 . B B 0.90 -0.17 0.30 0.80

Tyr 162 . B B 0.04 0.51 -0.45 1.04

Met 163 . B B -0.96 0.59 -0.60 0.45

Asp 164 . B B -1.14 0.87 -0.60 0.22

He 165 . B B -1.96 1.03 -0.60 0.10

Val 166 . B B -1.64 0.96 -0.60 0.09

He 167 . B B -1.74 0.34 -0.30 0.09

Val 168 . B B -1.44 0.77 -0.35 0.12

Leu 169 . B B -1.44 •0.47 -0.10 0.22

Asp 170 B T -0.86 0.23 F 1.00 0.50

Gly 171 T T -0.89 -0.07 1 F 2.25 0.90

Ser 172 T T -0.24 -0.03 F 2.50 0.77

Asn 173 T C 0.40 0.04 ' . 1 F 1.45 0.72

Ser 174 T C 0.92 0.47 * . 1 F 1.05 1.12

He 175 B C 0.07 0.96 fc 0.10 0.88

Tyr 176 B C 0.41 1.21 fc -0.15 0.41

Pro 177 . B B -0.14 0.81 -0.60 0.53

Trp 178 B B -0.14 1.07 -0.60 0.56

Val 179 B B 0.12 0.79 -0.60 0.62

Glu 180 B B 0.31 0.53 -0.60 0.54

Val 181 A B -0.26 0.89 -0.60 0.45

Gin 182 A B -0.93 0.66 -0.60 0.50

His 183 A B -0.64 0.70 -0.60 0.20

Phe 184 A B -0.68 1.10 fc -0.60 0.43

Leu 185 A B -1.49 1.14 * -0.60 0.18

He 186 A B -0.59 1.43 fc -0.60 0.11

Asn 187 A B -0.54 0.93 fc -0.60 0.25

He 188 A B -1.21 0.14 -0.30 0.60

Leu 189 A B -0.76 0.24 -0.30 0.74

Lys 190 B B -0.83 0.31 -0.30 0.72

Lys 191 B B -0.29 0.60 -0.60 0.72

Phe 192 B B -0.50 0.34 -0.30 0.86

Tyr 193 B B 0.04 0.09 * -0.30 0.67

He 194 B B 0.86 0.51 -0.60 0.33

Gly 195 T C -0.08 0.91 0.00 0.66

Pro 196 T T -0.12 0.81 F 0.35 0.30

Gly 197 T T -0.28 0.46 F 0.35 0.73

Gin 198 B T -0.38 0.41 F -0.05 0.55

He 199 B B -0.34 0.41 -0.60 0.35

Gin 200 B B -0.86 0.63 -0.60 0.26 Val 201 B B -0.64 0.84 -0.60 0.11

Gly 202 B B -0.54 0.84 -0.60 0.28

Val 203 B B -0.89 0.91 sk -0.60 0.25

Val 204 B B 0.00 0.94 -0.60 0.34

Gin 205 B B 0.00 0.30 -0.30 0.59

Tyr 206 1 B B 0.00 -0.13 0.45 1.32

Gly 207 A 1 B B -0.51 -0.13 F 0.60 1.32

Glu 208 A A B 0.31 -0.13 F 0.45 0.57

Asp 209 A A B 1.17 -0.03 F 0.45 0.49

Val 210 A A B 0.47 -0.79 0.60 0.86

Val 211 A A B 0.68 -0.43 0.30 0.43

His 212 A A B 0.21 0.07 -0.30 0.35

Glu 213 A A 0.21 0.76 -0.60 0.39

Phe 214 A A 0.21 0.51 -0.60 0.84

His 215 A A 0.82 -0.13 0.45 1.04

Leu 216 A A 1.79 0.13 0.04 0.94

Asn 217 A T 1.52 0.13 0.93 2.12

Asp 218 A T 0.67 -0.27 F 2.02 2.09

Tyr 219 r T 1.41 -0.13 F 2.76 1.88

Arg 220 Γ T 1.44 -0.81 F 3.40 2.34

Ser 221 A 1.40 -1.21 F 2.46 2.34

Val 222 A B 0.54 -0.57 F 1.92 1.11

Lys 223 A B 0.54 -0.69 F 1.43 0.42

Asp 224 A B 0.20 -0.69 F 1.09 0.54

Val 225 A A -0.50 -0.57 0.60 0.74

Val 226 A A -0.50 -0.71 0.60 0.37

Glu 227 A A 0.32 -0.33 0.30 0.30

Ala 228 A A -0.61 0.17 -0.30 0.55

Ala 229 A A -0.61 0.21 -0.30 0.52

Ser 230 A A 0.24 -0.43 0.30 0.52

His 231 A A 1.21 -0.03 0.64 0.89

He 232 A A 0.87 -0.53 1.43 1.72

Glu 233 A A 1.11 -0.60 F 1.92 1.27

Gin 234 T T 1.39 -0.56 F 2.91 0.92

Arg 235 T T 1.69 -0.57 F 3.40 1.90

Gly 236 T T 1.41 -1.26 F 3.06 1.90

Gly 237 T C 2.41 -0.77 F 2.68 1.59

Thr 238 C 2.10 -1.17 F 2.30 1.59

Glu 239 C 1.51 -0.69 F 2.12 2.31

Thr 240 . B 0.70 -0.61 F 1.74 2.36

Arg 241 . B 0.70 -0.26 F 1.60 1.42 Thr 242 B 0.16 -0.31 =fc F 1.29 0.81

Ala 243 A A 0.47 0.37 0.18 0.39

Phe 244 A A -0.23 -0.11 0.62 0.35

Gly 245 A A -0.51 0.67 -0.44 0.21

He 246 A A -0.51 0.69 -0.60 0.21

Glu 247 A A -0.50 0.19 -0.30 0.47

Phe 248 A A 0.09 -0.21 0.30 0.64

Ala 249 A A 0.20 -0.64 0.75 1.58

Arg 250 A A -0.16 -0.83 F 0.75 0.92

Ser 251 A A 0.73 -0.04 F 0.45 0.92

Glu 252 A A 0.78 -0.43 F 0.60 1.58

Ala 253 A A 1.13 -0.93 F 0.90 1.61

Phe 254 A A 1.38 -0.50 * sfc p 0.60 1.19

Gin 255 A T 1.38 -0.46 * . F 0.85 0.68

Lys 256 A T 1.72 -0.46 * . F 1.00 1.32

Gly 257 A T 1.38 -0.96 * . F 1.30 3.05

Gly 258 A T 1.38 -1.31 * . F 1.30 1.74

Arg 259 A A 2.12 -1.21 * . F 0.75 0.88

Lys 260 A A 2.17 -1.21 * . F 0.90 1.78

Gly 261 A A 1.27 -1.64 * . F 0.90 3.59

Ala 262 A A 1.01 -1.43 * . F 0.90 1.36

Lys 263 A B 0.47 -0.81 * . F 0.75 0.67

Lys 264 B B -0.50 -0.13 fc 0.30 0.48

Val 265 B B -1.43 0.09 fc -0.30 0.35

Met 266 B B -1.40 0.27 * -0.30 0.12

He 267 B B -0.81 0.76 * -0.60 0.09

Val 268 B B -1.20 0.76 * -0.60 0.20

He 269 B T -1.24 0.54 -0.20 0.20

Thr 270 B T -0.69 -0.07 F 0.85 0.49

Asp 271 B T -0.12 -0.37 F 0.85 0.89

Gly 272 T C 0.77 -0.51 F 1.50 1.73

Glu 273 . C 1.32 -1.20 F 1.60 2.00

Ser 274 . C 2.00 -1.30 F 1.90 1.60

His 275 . C 2.31 -0.87 F 2.20 2.51

Asp 276 . C 1.50 -1.30 * F 2.502.42

Ser 277 T C 1.84 -0.61 F 3.00 1.49

Pro 278 A T 1.89 -1.00 F 2.50 1.89

Asp 279 A T 1.33 -1.50 * . F 2.20 2.27

Leu 280 A T 0.48 -0.86 * . F 1.90 1.25

Glu 281 A A 0.48 -0.56 1.05 0.57

Lys 282 A A 0.78 -0.59 sfc p 0.75 0.59 Val 283 A A 0.69 -0.19 0.45 1.24

He 284 A A 0.69 -0.49 F 0.45 0.96

Gin 285 A B 1.61 -0.49 F 0.79 0.83

Gin 286 A A 1.61 -0.49 F 1.28 2.19

Ser 287 A A 1.57 -1.13 fc F 1.92 5.22

Glu 288 • r < Z 1.57 -1.41 F 2.86 4.85

Arg 289 T r 2.14 -1.17 F 3.40 2.08

Asp 290 T Γ 2.26 -1.09 F 3.06 2.24

Asn 291 T T 2.01 -1.47 F 2.72 2.53

Val 292 B B 1.72 -0.71 F 1.58 2.02

Thr 293 B B 0.87 -0.21 F 0.94 1.22

Arg 294 B 1 B 0.17 0.43 -0.60 0.57

Tyr 295 B ] B -0.69 0.53 -0.60 0.77

Ala 296 B ] B -1.50 0.53 -0.60 0.40

Val 297 B ] B -0.99 0.73 -0.60 0.17

Ala 298 B B -0.92 1.16 -0.60 0.11

Val 299 B B -1.28 1.16 -0.60 0.16

Leu 300 B B -1.03 1.41 -0.60 0.34

Gly 301 B B -0.33 1.17 -0.60 0.55

Tyr 302 B B 0.63 0.67 -0.45 1.45

Tyr 303 B 0.88 0.03 0.39 3.44

Asn 304 B Γ 0.84 -0.23 1.53 3.44

Arg 305 T Γ 1.66 0.03 F 1.82 1.54

Arg 306 T Γ 1.79 -0.33 F 2.76 1.58

Gly 307 T T 2.03 -0.66 F 3.40 1.52

He 308 C 1.97 -1.06 F 2.66 1.34

Asn 309 T C 1.27 -0.57 * F 2.37 0.99

Pro 310 T C 0.34 0.21 F 1.13 0.86

Glu 311 B T 0.23 0.47 F 0.44 1.02

Thr 312 B T 0.58 0.19 F 0.40 1.02

Phe 313 A A 0.58 -0.21 * 0.45 1.14

Leu 314 A A 0.62 0.04 -0.30 0.46

Asn 315 A A 0.59 0.04 -0.30 0.64

Glu 316 A A -0.30 0.31 F 000 1.16

He 317 A A . -0.58 0.21 -0.30 0.98

Lys 318 A A -0.18 0.03 -0.30 0.62

Tyr 319 A B 0.63 0.01 -0.30 0.48

He 320 A B 0.42 0.01 -0.15 1.14

Ala 321 A B 0.42 -0.24 0.64 0.88

Ser 322 A B 1.31 -0.24 0.98 0.94

Asp 323 T C 1.31 -1.00 F 2.52 2.23 Pro 324 A T 1.52 -1.69 . . F 2.664.42

Asp 325 T T 1.71 -1.69 . . F 3.404.49

Asp 326 A T 1.60 -1.29 . . F 2.662.33

Lys 327 A A 1.90 -0.50 . . F 1.621.30

His 328 A A 1.04 -0.53 . * 1.431.26

Phe 329 A B 0.94 0.11 * . 0.040.56

Phe 330 A B 0.94 0.60 * . -0.60 0.40

Asn 331 A A 0.94 0.60 * . -0.60 0.49

Val 332 A A 0.31 0.10 * . -0.30 0.99

Thr 333 A A -0.24 -0.19 * . F 0.601.15

Asp 334 A A -0.36 -0.47 * * F 0.450.72

Glu 335 A A 0.39 -0.19 . * 0.300.81

Ala 336 A A 0.39 -0.83 . . 0.751.12

Ala 337 A A 0.36 -1.31 . . 0.751.12

Leu 338 A A -0.19 -0.63 * . 0.600.45

Lys 339 A A -0.19 0.01 * . -0.30 0.33

Asp 340 A A -0.78 -0.49 * . 0.300.55

He 341 A A -1.00 -0.49 * . 0.300.67

Val 342 A A -0.76 -0.49 * . 0.300.28

Asp 343 A A 0.06 -0.06 * . 0.300.16

Ala 344 A A 0.12 -0.06 * . 0.300.39

Leu 345 A T -0.77 -0.74 * . 1.151.03

Gly 346 A T -0.58 -0.70 * . 1.000.43

Asp 347 A T -0.02 0.09 * . 0.100.37

Arg 348 B T -0.83 -0.03 * . 0.700.60

He 349 B -0.24 -0.03 * . 0.500.50

Phe 350 B 0.22 -0.46 * . 0.500.52

Ser 351 B 0.26 -0.03 * . 0.500.26

Leu 352 B 0.26 0.46 . . -0.10 0.54

Glu 353 C 0.19 0.17 . . F 1.001.01

Gly 354 C 1.08 -0.61 . . F 2.201.50

Thr 355 C 1.78 -0.60 . * F 2.502.93.

Asn 356 T C 1.77 -1.29 . . F 3.002.93

Lys 357 T C 2.28 -0.80 . * F 2.704.28

Asn 358 T C 1.58 -0.84 . . F 2.403.97

Glu 359 A T 1.58 -0.54 . . F 1.902.14

Thr 360 A T 1.08 -0.51 . * F 1.601.06

Ser 361 A T 1.08 0.17 . * F 0.250.54

Phe 362 A T 0.43 -0.23 . * 0.700.54

Gly 363 A T 0.13 0.39 . . 0.100.37

Leu 364 A 0.13 0.29 * -0.10 0.37 Glu 365 A 0.13 0.30 -0.10 0.74

Met 366 A 0.09 0.00 0.05 1.09

Ser 367 A T 0.09 0.00 F 0.40 1.30

Gin 368 A T 0.13 0.10 F 0.25 0.65

Thr 369 T C 0.64 0.49 F 0.15 0.88

Gly 370 T C 0.61 0.26 F 0.45 0.88

Phe 371 C 0.36 0.37 F 0.25 0.69

Ser 372 B C -0.20 0.61 F -0.25 0.36

Ser 373 B C -0.20 0.77 -0.40 0.27

His 374 1 B B 0.11 0.34 -0.30 0.53

Val 375 B B 0.11 -0.44 0.30 0.67

Val 376 B T -0.04 -0.40 0.70 0.49

Glu 377 B T -0.56 -0.14 0.70 0.27

Asp 378 A T -1.07 0.04 0.10 0.30

Gly 379 A T -1.38 0.09 0.10 0.33

Val 380 A A -1.11 -0.13 0.30 0.19

Leu 381 A A -1.11 0.37 -0.30 0.11

Leu 382 A B -1.46 1.01 -0.60 0.09

Gly 383 A B -2.04 1.01 -0.60 0.11

Ala 384 A B -1.94 0.87 -0.60 0.14

Val 385 A B -1.09 0.94 -0.60 0.27

Gly 386 B -0.57 0.26 -0.10 0.45

Ala 387 B 0.24 0.74 -0.40 0.47

Tyr 388 B 0.24 0.64 -0.25 1.01

Asp 389 r 0.24 0.43 0.15 1.01

Trp 390 A 0.24 0.50 -0.25 1.01

Asn 391 A -0.22 0.64 * -0.40 0.48

Gly 392 A A 0.41 0.57 * -0.60 0.24

Ala 393 A A 0.66 0.57 -0.60 0.45

Val 394 A A 0.34 -0.34 0.300.49

Leu 395 A A 0.33 -0.26 F 0.45 0.71

Lys 396 A A -0.26 -0.30 F 0.45 0.94

Glu 397 A A -0.26 -0.30 F 0.60 1.28

Thr 398 A A . 0.38 -0.51 F 0.90 1.54

Ser 399 A T . 0.38 -1.20 F 1.30 1.54

Ala 400 A T 0.30 -0.56 F 1.15 0.66

Gly 401 A T 0.04 0.13 F 0.25 0.32

Lys 402 B T -0.77 0.07 F 0.25 0.37

Val 403 B -0.34 0.37 -0.10 0.30

He 404 B -0.04 -0.13 0.50 0.60

Pro 405 B 0.24 -0.56 0.80 0.52 Leu 406 B 0.34 -0.17 . . 0.50 0.93

Arg 407 A -0.51 -0.06 . . 1 ³ 0.80 2.08

Glu 408 A 0.39 -0.06 . . I ^σ 0.80 1.11

Ser 409 A 1.28 -0.49 . * 1 ³ 0.80 2.69

Tyr 410 A A 0.79 -1.17 . . F 0.90 2.38

Leu 411 A A 1.39 -0.39 . . 1 ~ 0.60 1.19

Lys 412 A A 1.28 0.04 . . F 0.00 1.37

Glu 413 A A 1.28 -0.34 * . F 0.60 1.52

Phe 414 A A 0.77 -1.10 * . F 0.90 3.19

Pro 415 A A 1.06 -1.10 * . F 0.90 1.31

Glu 416 A A 1.87 -1.10 * . F 0.90 1.52

Glu 417 A A 1.79 -0.70 * * F 0.90 2.82

Leu 418 A A 1.44 -0.99 * * F 0.90 2.48

Lys 419 A A 1.56 -0.99 * * F 0.90 1.42

Asn 420 A T 1.52 -0.49 * . 0.70 0.83

His 421 . A T 0.71 0.27 * . 0.25 1.57

Gly 422 t A T 0.37 0.27 . . 0.10 0.65

Ala 423 B . T 0.93 0.70 . . -0.20 0.40

Tyr 424 B B 0.58 1.06 . . -0.60 0.46

Leu 425 B B -0.28 1.04 . . -0.60 0.67

Gly 426 B B -0.56 1.26 . . -0.60 0.49

Tyr 427 B B -0.51 1.24 . . -0.60 0.45

Thr 428 B B -0.78 0.87 . . -0.60 0.74

Val 429 B B -1.39 0.83 * . -0.60 0.55

Thr 430 B B -0.88 1.04 * . -0.60 0.26

Ser 431 B B -0.83 0.67 . . -0.60 0.24

Val 432 B B -0.48 0.57 . . -0.60 0.44

Val 433 B B -0.17 -0.07 . . F 0.79 0.60

Ser 434 B T 0.34 -0.16 . . F 1.53 0.77

Ser 435 B T 0.77 -0.11 . . F 2.02 1.03

Arg 436 B T 0.21 -0.76 . . F 2.66 2.71

Gin 437 T T 0.82 -0.76 . * F 3.40 1.50

Gly 438 B B 0.82 -0.39 * * F 1.96 1.75

Arg 439 B B 0.53 -0.13 * * F 1.47 0.66

Val 440 B B 0.49 0.37 * * 0.38 0.39

Tyr 441 B B -0.21 0.40 * * -0.26 0.39

Val 442 B B -0.42 0.47 * * -0.60 0.20

Ala 443 B B 0.03 0.90 * * -0.60 0.42

Gly 444 B B -0.78 0.26 * * -0.30 0.52

Ala 445 B . 0.08 0.29 . * -0.10 0.61

Pro 446 C 0.29 0.04 * * F 0.25 0.97 Arg 447 B 0.83 0.04 * ■» p 0.20 1.33

Phe 448 B 1.08 0.10 0.18 1.90

Asn 449 r 1.47 0.03 0.71 1.22

His 450 Γ T 1.20 -0.40 * p 1.79 1.24

Thr 451 T Z 0.52 0.24 * p 1.12 1.06

Gly 452 r T -0.40 0.14 * * F 1.30 0.46

Lys 453 B T -0.40 0.43 * * F 0.47 0.28

Val 454 B B -0.71 0.71 -0.21 0.17

He 455 B B -1.28 0.71 -0.34 0.25

Leu 456 B B -1.00 0.90 -0.47 0.12

Phe 457 B B -0.66 1.40 -0.60 0.22

Thr 458 B B -0.70 1.16 -0.60 0.51

Met 459 B B 0.27 0.87 -0.32 1.00

His 460 T " 0.86 0.19 1.01 2.26

Asn 461 T Z 0.86 -0.21 F 2.04 2.10

Asn 462 T Z 1.24 -0.01 F 2.32 1.75

Arg 463 r T 0.67 -0.14 F 2.80 1.85

Ser 464 . Z 1.23 0.04 F 1.37 0.81

Leu 465 A A 1.27 0.14 0.54 0.68

Thr 466 A B 0.68 0.14 0.26 0.60

He 467 A B 0.08 0.64 fc -0.32 0.46

His 468 A B 0.08 0.87 fc -0.60 0.55

Gin 469 A B 0.03 0.19 * * -0.30 0.74

Ala 470 A B 0.84 0.13 fc 0.02 1.05

Met 471 B T 1.16 -0.16 1.19 1.33

Arg 472 B T 1.16 -0.26 * * F 1.51 1.33

Gly 473 B T 0.84 0.03 * F 0.93 0.92

Gin 474 B T 0.54 -0.04 F 1.70 0.92

Gin 475 B 0.89 -0.27 F 1.33 0.63

He 476 B 0.79 0.49 F 0.41 1.00

Gly 477 B T 0.33 0.84 F 0.29 0.50

Ser 478 B T 0.38 0.87 F 0.12 0.29

Tyr 479 T C 0.38 0.86 0.00 0.55

Phe 480 B T -0.51 0.17 F 0.25 0.96

Gly 481 B . C 0.07 0.43 F -0.25 0.50

Ser 482 B . C 0.11 0.53 F -0.25 0.46

Glu 483 B B -0.44 0.16 F -0.15 0.71

He 484 B B -0.20 0.01 F -0.15 0.54

Thr 485 B B -0.39 -0.41 =1= p 0.45 0.67

Ser 486 B B -0.04 -0.1 1 * F 0.45 0.27

Val 487 B B -0.09 -0.1 1 * F 0.76 0.64 Asp 488 B B -0.09 -0.37 . * F 1.07 0.44

He 489 B 0.46 -0.86 * F 1.88 0.55

Asp 490 T T -0.09 -0.81 * F 2.79 0.73

Gly 491 T T -0.10 -0.81 * F 3.10 0.33

Asp 492 T T 0.76 -0.33 * * F 2.49 0.67

Gly 493 B T -0.10 -1.01 * * F 2.08 0.67

Val 494 B B -0.02 -0.37 . * ] F 1.07 0.50

Thr 495 B B -0.83 -0.11 . . ] F 0.76 0.25

Asp 496 B B -1.34 0.57 * . ] F -0.45 0.21

Val 497 B 1 3 -1.69 0.79 * . -0.60 0.21

Leu 498 B 1 B -1.93 0.57 . . -0.60 0.14

Leu 499 B 1 B -1.29 0.59 . . -0.60 0.09

Val 500 B 1 3 -1.58 1.01 . . -0.60 0.18

Gly 501 B 1 B -1.82 0.99 . . -0.60 0.21

Ala 502 B -1.67 1.06 . . -0.40 0.41

Pro 503 B -0.86 1.16 . . -0.40 0.48

Met 504 B -0.04 0.91 . . -0.40 0.77

Tyr 505 B 0.47 0.49 . . -0.25 1.33

Phe 506 B 0.92 0.41 * . -0.40 0.85

Asn 507 A T 1.51 -0.01 . * 0.85 1.68

Glu 508 A T 1.83 -0.63 . * F 1.30 1.86

Gly 509 A T 2.09 -1.39 . * F 1.30 4.20

Arg 510 A T 2.38 -1.74 . * F 1.30 2.58

Glu 511 A 2.22 -2.14 . * F 1.10 2.98

Arg 512 . B T 1.98 -1.50 . * F 1.30 2.24

Gly 513 . B T 1.12 -1.17 . * F 1.30 1.79

Lys 514 B B 1.22 -0.53 . * F 0.75 0.77

Val 515 B B 1.11 0.23 . * -0.30 0.61

Tyr 516 B B 0.30 0.23 . * -0.15 1.07

Val 517 B B 0.30 0.49 . * -0.60 0.44

Tyr 518 B 0.64 0.49 * * 0.01 1.17

Glu 519 B 0.60 0.24 * . 0.57 1.29 .

Leu 520 B 1.57 -0.11 . . 1.43 2.80

Arg 521 A T 1.11 -0.76 . . F 2.34 3.50

Gin 522 B T 1.11 -0.73 . . F 2.60 1.75

Asn 523 B T 1.11 -0.09 * . F 2.04 1.57

Arg 524 B T 1.11 -0.01 * * F 1.78 1.26

Phe 525 B 1.58 0.39 * . 0.57 1.17

Val 526 B T 1.16 0.41 * * 0.06 0.72

Tyr 527 B T 0.34 0.50 . * -0.20 0.53

Asn 528 B T 0.39 1.19 * F 0.29 0.50 Gly 529 T T 0.28 0.40 * * F 1.18 1.36

Thr 530 C 0.68 -0.24 . . F 2.02 1.45

Leu 531 r C 1.50 -0.61 . . F 2.86 1.21

Lys 532 T r . 1.44 -0.51 . * F 3.40 1.66

Asp 533 B ^' r . i.2o -0.56 . * F 2.66 1.54

Ser 534 B • r . 1.54 -0.29 . . F 2.022.93

His 535 B • r . 1.86 -0.57 . . 1.83 2.54

Ser 536 B . r . 2.08 -0.17 . * 1.19 2.44

Tyr 537 B . r . 2.14 0.33 . * 0.25 1.84

Gin 538 B . I" . 1.44 -0.06 . * 0.85 2.65

Asn 539 B 1.40 0.23 . * 0.05 1.71

Ala 540 B 1.13 0.27 . * 0.05 1.08

Arg 541 B 1.13 -0.10 * " 0.500.84

Phe 542 B 0.49 -0.11 * * F 0.65 0.70

Gly 543 . T r . -o.io 0.17 * ^• fc p 0.65 0.48

Ser 544 . r C -0.40 0.17 * ' * F 0.45 0.25

Ser 545 B . r -0.67 0.56 fc p -0.05 0.39

He 546 B . r -0.67 0.41 * ^> fc -0.20 0.29

Ala 547 B 1 3 0.03 -0.01 * 0.30 0.42

Ser 548 B ] B . -0.43 -0.40 0.30 0.53

Val 549 B B -0.13 -0.10 * 0.64 0.62

Arg 550 B B 0.17 -0.39 * F 1.13 0.99

Asp 551 B 1.06 -0.49 * F 1.82 1.28

Leu 552 B 1.34 -0.87 * F 2.46 2.88

Asn 553 T r 1.40 -1.13 * F 3.40 1.97

Gin 554 T r . 2.26 -0.37 * F 2.76 1.85

Asp 555 T r . 2.14 0.03 * F 1.82 3.60

Ser 556 T T . 1.29 -0.66 * F 2.38 3.74

Tyr 557 B T 1.24 -0.41 F 1.34 1.60

Asn 558 B B 0.39 -0.17 F 0.45 0.71

Asp 559 B B . 0.04 0.47 -0.60 0.39

Val 560 B B -0.54 0.51 * -0.60 0.25

Val 561 B B -0.46 0.26 -0.30 0.16

Val 562 B B -1.02 0.29 -0.30 0.14

Gly 563 B B -1.02 0.97 -0.60 0.16

Ala 564 A B -1.02 0.33 -0.30 0.38

Pro 565 A A -0.17 -0.31 . 0.30 0.85

Leu 566 A A 0.66 -0.56 0.75 1.37

Glu 567 A A 0.92 -0.49 F 0.60 1.85

Asp 568 A A 0.92 -0.49 0.45 1.21

Asn 569 A A 0.92 -0.49 . 0.45 1.45 His 570 A A 0.24 -0.67 0.60 0.85

Ala 571 A B 0.81 0.01 -0.30 0.36

Gly 572 A B -0.08 0.77 -0.60 0.35

Ala 573 A B -0.78 1.06 -0.60 0.18

He 574 B B -0.81 1.34 -0.60 0.15

Tyr 575 B B -1.12 1.34 -0.60 0.21

He 576 B B -1.23 1.34 -0.60 0.21

Phe 577 B B -0.78 1.63 -0.60 0.25

His 578 B B -0.53 0.94 fc * -0.60 0.32

Gly 579 B T 0.06 0.61 -0.20 0.45

Phe 580 T r -0.59 0.31 0.500.69

Arg 581 T Γ -0.51 0.21 " * F 0.65 0.36

Gly 582 T Γ 0.23 0.40 1 F 0.35 0.30

Ser 583 T Γ -0.04 -0.03 F 1.25 0.69

He 584 Z 0.09 -0.33 * * F 1.15 0.51

Leu 585 Z 0.83 0.10 F 0.85 0.79

Lys 586 Z 0.72 -0.33 * 1 F 1.90 1.18

Thr 587 Γ Z 1.18 -0.31 F 2.402.92

Pro 588 Γ Z 0.59 -1.00 F 3.00 6.94

Lys 589 B Γ 1.17 -1.00 F 2.50 2.43

Gin 590 B Γ 1.39 -0.51 F 2.20 2.43

Arg 591 B B 1.04 -0.50 * F 1.20 1.59

He 592 B B 1.36 -0.54 * F 1.20 1.06

Thr 593 A B B 0.76 -0.54 * F 0.90 1.06

Ala 594 A B B 0.12 -0.26 * F 0.45 0.45

Ser 595 A B -0.19 0.24 . F -0.15 0.65

Glu 596 A B -0.64 0.04 F -0.15 0.65

Leu 597 A A B -0.57 -0.01 . F 0.45 0.63

Ala 598 A A B -0.26 0.17 * -0.30 0.39

Thr 599 A A B 0.09 0.19 -0.30 0.39

Gly 600 A A B -0.31 0.94 -0.60 0.74

Leu 601 B B -0.66 1.04 * -0.60 0.63

Gin 602 B B -0.51 0.97 -0.60 0.44

Tyr 603 T T -0.22 1.06 0.20 0.24

Phe 604 B T -0.80 1.01 -0.20 0.38

Gly 605 B T -0.49 1.01 -0.20 0.15

Cys 606 B T -0.02 1.11 -0.20 0.13

Ser 607 B B -0.02 0.79 -0.60 0.15

He 608 B B -0.59 0.40 -0.60 0.27

His 609 B B 0.11 0.66 -0.60 0.41

Gly 610 B B -0.36 0.09 -0.30 0.52 Gin 611 B 0.31 0.39 -0.10 0.61

Leu 612 B 0.61 0.10 -0.10 0.72

Asp 613 B 1.50 -0.40 * 0.65 1.26

Leu 614 B 1.19 -0.83 0.95 1.21

Asn 615 A r 0.72 -0.80 . sk 1.30 1.45

Glu 616 A Γ -0.17 -0.80 fc F 1.15 0.72

Asp 617 A Γ 0.64 -0.11 * F 0.85 0.61

Gly 618 A Γ -0.17 -0.80 sk 1.15 0.63

Leu 619 A B . 0.06 -0.51 * 0.60 0.30

He 620 A B -0.80 -0.01 0.30 0.18

Asp 621 B B -1.14 0.63 -0.60 0.14

Leu 622 B B -1.73 0.63 -0.60 0.16

Ala 623 B B . . -2.20 0.44 -0.60 0.24

Val 624 A B -1.73 0.44 fc -0.60 0.12

Gly 625 A A -0.84 0.87 -0.60 0.14

Ala 626 A A -1.43 0.59 -0.60 0.22

Leu 627 A A -1.48 0.59 -0.60 0.30

Gly 628 A A -1.78 0.59 -0.60 0.23

Asn 629 A B 1 B -1.73 0.84 -0.60 0.16

Ala 630 A B ] B -1.68 1.03 -0.60 0.16

Val 631 A B B -1.39 1.26 fc -0.60 0.17 lie 632 A B B -0.47 1.21 -0.60 0.14

Leu 633 A B B -0.33 0.81 * -0.60 0.27

Trp 634 A B B -1.19 0.74 -0.60 0.57

Ser 635 A B B -1.46 0.74 -0.60 0.60

Arg 636 B B -0.60 0.70 F -0.45 0.54

Pro 637 B B -0.60 0.41 -0.60 0.89

Val 638 B B 0.21 0.19 -0.30 0.46

Val 639 B B -0.09 0.20 -0.30 0.38

Gin 640 B B -0.09 0.70 -0.60 0.25

He 641 B B -1.01 0.66 -0.60 0.45

Asn 642 B T -0.83 0.70 -0.20 0.50

Ala 643 B T -0.68 0.56 -0.20 0.39

Ser 644 B T 0.18 0.94 -0.20 0.48

Leu 645 A T . -0.03 0.26 0.10 0.52

His 646 A 0.56 0.29 0.18 0.80

Phe 647 A 0.60 0.17 * 0.46 0.80

Glu 648 A T . 0.30 -0.21 F 1.84 1.94

Pro 649 A T 0.60 -0.21 F 1.97 1.00

Ser 650 T T 0.52 -0.31 F 2.80 1.85

Lys 651 A T -0.14 -0.41 F 1.97 0.75 He 652 i V B 0.52 0.37 * * F 0.69 0.42

Asn 653 i A B 0.63 0.44 * * -0.04 0.43

He 654 i \ B 0.84 0.06 * * -0.02 0.42

Phe 655 B B 0.48 0.06 * * 0.04 1.00

His 656 B 1 r . 0.48 -0.06 * * 1.38 0.33

Arg 657 B 1 r . 1.48 -0.46 * * 1.72 0.95

Asp 658 T 1 r . ι.i8 -1.14 * . F 3.06 2.14

Cys 659 T 1 r 1.72 -1.54 * . F 3.40 2.11

Lys 660 T 2.53 -1.61 * . F 2.86 1.07

Arg 661 T 1 r 2.57 -1.61 * . F 3.03 1.25

Ser 662 T r . 1.87 -1.61 * . F 3.00 3.90

Gly 663 T 1 r 1.56 -1.69 * . F 2.97 1.97

Arg 664 T r 1.56 -1.20 * . F 2.94 1.45

Asp 665 T r o.7θ -0.63 * . F 3.10 0.58

Ala 666 B r o.oo -0.33 ^• F 2.090.48

Thr 667 B r . -0.29 -0.26 1.63 0.25

Cys 668 B I" -0.64 0.24 0.72 0.15

Leu 669 A A -1.57 1.03 -0.29 0.13

Ala 670 A A -2.23 1.21 -0.60 0.07

Ala 671 A A -2.34 1.30 . -0.60 0.07

Phe 672 A A -2.34 1.51 -0.60 0.08

Leu 673 A A -1.89 1.31 -0.60 0.11

Cys 674 A B -1.97 1.24 -0.60 0.17

Phe 675 B B -2.08 1.43 -0.60 0.14

Thr 676 B B -2.30 1.43 -0.60 0.14

Pro 677 B B -2.19 1.43 -0.60 0.22

He 678 A . B T -1.59 1.36 -0.20 0.26

Phe 679 A B B -0.96 1.00 -0.60 0.28

Leu 680 A B B -0.96 1.01 -0.60 0.24

Ala 681 A B C -0.64 1.37 -0.40 0.30

Pro 682 A B C -0.74 1.09 -0.40 0.60

His 683 A . B T -0.17 0.79 -0.05 1.05

Phe 684 A B T 0.22 0.59 -0.05 1.51

Gin 685 A B B 0.18 0.57 F -0.30 1.41

Thr 686 A B B 0.42 0.79 F -0.45 0.77

Thr 687 B B -0.26 0.71 * F -0.45 0.88

Thr 688 B B -0.11 0.61 * F -0.45 0.35

Val 689 B B 0.34 0.21 * -0.30 0.48

Gly 690 B B 0.34 0.49 * -0.60 0.52

He 691 B B 0.07 0.40 * -0.60 0.58

Arg 692 B B 0.07 0.41 * -0.60 0.79 Tyr 693 B B -0.22 0.26 -0.15 1.16

Asn 694 A 1 B B 0.63 0.44 -0.45 1.63

Ala 695 A I B B 0.98 -0.24 0.45 1.39

Thr 696 A B B 1.98 -0.24 0.45 1.54

Met 697 A B B 1.98 -1.00 F 0.90 1.87

Asp 698 A B 1.98 -1.40 F 0.90 3.63

Glu 699 A B 1.67 -1.14 F 1.20 3.94

Arg 700 A B 2.04 -1.14 F 1.50 5.75

Arg 701 A T 2.47 -1.33 * * F 2.20 5.33

Tyr 702 A T 2.48 -1.33 F 2.50 6.02

Thr 703 T C 2.44 -0.83 F 3.00 3.11

Pro 704 T ( Z 1.63 -0.33 F 2.40 2.16

Arg 705 B T 1.52 0.36 1.15 1.14

Ala 706 B T 1.41 -0.40 1.45 1.31

His 707 1 3 1.31 -0.89 1.59 1.47

Leu 708 B 1.28 -0.89 F 1.63 0.74

Asp 709 1 B T 1.49 -0.46 F 1.87 0.73

Glu 710 T T 1.49 -0.96 F 2.91 0.89

Gly 711 T T 1.38 -1.46 F 3.40 2.12

Gly 712 T T 1.10 -1.36 F 3.06 1.10

Asp 713 T 1.91 -0.87 F 2.37 0.92

Arg 714 A 2.02 -0.47 F 1.48 1.49

Phe 715 A 1.43 -0.90 F 1.44 2.95

Thr 716 B 0.92 -0.83 F 1.10 1.79

Asn 717 A B 0.46 -0.19 F 0.45 0.68

Arg 718 A B -0.36 0.50 F -0.45 0.64

Ala 719 A B -0.77 0.40 -0.60 0.37

Val 720 A B -0.37 0.30 -0.06 0.31

Leu 721 A B -0.40 0.29 0.18 0.21

Leu 722 A B -0.40 0.71 F 0.27 0.21

Ser 723 T C -0.51 0.61 F 1.11 0.48

Ser 724 T C -0.73 -0.03 F 2.40 1.01

Gly 725 T C -0.54 -0.03 F 2.16 1.01

Gin 726 A T 0.27 -0.14 * F 1.57 0.40

Glu 727 A A 1.19 -0.53 F 1.23 0.52

Leu 728 A A 0.60 -0.91 F 1.14 1.03

Cys 729 A A 0.90 -0.66 0.60 0.42

Glu 730 A A 0.54 -0.66 0.60 0.39

Arg 731 A A 0.51 0.13 -0.30 0.41

He 732 A A -0.34 -0.06 0.45 1.03

Asn 733 A A -0.34 0.01 -0.30 0.44 Phe 734 A A 0.32 0.70 fc -0.60 0.19

His 735 A B 0.01 0.70 fc -0.60 0.44

Val 736 A B -0.69 0.50 fc -0.60 0.40

Leu 737 A B 0.20 0.60 * -0.60 0.46

Asp 738 A A -0.04 -0.19 F 0.62 0.57

Thr 739 A T -0.20 0.07 F 0.74 1.20

Ala 740 A T -0.12 0.07 F 0.91 1.08

Asp 741 A T 0.52 -0.61 1.83 1.30

Tyr 742 B T 0.48 -0.19 1.70 1.39

Val 743 B B 0.17 -0.03 1.13 1.02

Lys 744 B B -0.22 -0.04 . F 0.96 0.88

Pro 745 B B 0.07 0.74 F -0.11 0.49

Val 746 B B -0.79 0.37 -0.13 0.88

Thr 747 B B . -0.54 0.37 fc -0.30 0.33

Phe 748 B B 0.07 0.37 fc -0.30 0.37

Ser 749 B B -0.28 0.70 fc -0.60 0.77

Val 750 B B -0.88 0.44 -0.60 0.72

Glu 751 B B -0.02 0.64 fc -0.60 0.68

Tyr 752 B 0.29 -0.14 fc 0.50 0.88

Ser 753 C 0.78 -0.53 * 1.49 1.99

Leu 754 T 1.08 -0.74 2.03 1.78

Glu 755 A 1.90 -0.74 F 2.12 1.89

Asp 756 A T 1.56 -1.00 F 2.66 1.92

Pro 757 T T 1.59 -0.96 F 3.40 2.31

Asp 758 T T 1.29 -1.21 F 3.06 2.06

His 759 T C 1.29 -0.60 F 2.52 1.22

Gly 760 C 1.29 0.09 F 0.93 0.65

Pro 761 B 1.29 -0.34 F 0.99 0.65

Met 762 B 1.16 -0.34 0.50 0.80

Leu 763 B 0.87 -0.41 F 0.89 0.80

Asp 764 T T 0.69 0.07 F 1.13 0.54

Asp 765 T T 0.72 0.07 F 1.37 0.85

Gly 766 T T 0.62 -0.06 F 2.36 1.48

Trp 767 T C 0.41 -0.26 _* F 2.40 1.28

Pro 768 B C 1.33 0.43 * F 0.71 0.63

Thr 769 B B 0.48 0.43 * F 0.42 1.25

Thr 770 B B 0.18 0.64 * F 0.03 0.89

Leu 771 B B -0.33 0.11 -0.06 0.77

Arg 772 B B -0.26 0.33 -0.30 0.39

Val 773 B B -0.74 0.27 -0.30 0.42

Ser 774 B B -0.72 0.57 -0.60 0.44 Val 775 B B -0.41 0.80 -0.60 0.24

Pro 776 B B 0.06 1.20 -0.60 0.52

Phe 777 B 1 [^■ . . -0.72 0.99 * -0.20 0.38

Trp 778 1 r T . o.i3 1.17 0.20 0.28

Asn 779 T C 0.43 0.93 0.00 0.29

Gly 780 1 r T 1.29 0.50 0.20 0.57

Cys 781 r T 1.50 -0.29 F 1.25 0.91

Asn 782 A r 2.17 -1.20 F 1.15 0.98

Glu 783 A 1 r . . 1.79 -1.10 F 1.30 1.35

Asp 784 A _' r . . 0.93 -0.96 F 1.30 1.35

Glu 785 A r ι.07 -0.89 F 1.15 0.62

His 786 A r . . 1.73 -0.86 1.00 0.56

Cys 787 A A 0.92 -0.86 0.60 0.56

Val 788 A 0.07 -0.17 0.50 0.26

Pro 789 A -0.74 0.47 F -0.25 0.14

Asp 790 A A -0.74 0.66 F -0.45 0.22

Leu 791 A A -1.30 0.09 -0.30 0.50

Val 792 A A -0.52 -0.06 0.30 0.33

Leu 793 A A 0.03 -0.49 0.30 0.38

Asp 794 A B 0.24 -0.10 0.30 0.62

Ala 795 A A -0.57 -0.79 F 0.90 1.40

Arg 796 A T . 0.03 -0.74 * F 1.30 1.40

Ser 797 A T . 0.58 -1.00 * F 1.30 1.29

Asp 798 A T C 0.80 -0.51 F 1.50 1.85

Leu 799 T C 0.20 -0.51 F 1.35 0.95

Pro 800 . A C 0.79 0.10 F 0.05 0.70

Thr 801 A A 0.43 -0.29 0.30 0.73

Ala 802 A A . 0.07 0.47 -0.45 1.39

Met 803 A A 0.07 0.36 -0.30 0.48

Glu 804 A A 0.99 0.33 -0.30 0.58

Tyr 805 A B 0.34 -0.16 0.45 1.12

Cys 806 A B -0.16 -0.01 0.30 0.84 .

Gin 807 A B 0.54 0.06 -0.30 0.40

Arg 808 A B 1.19 0.06 -0.30 0.50

Val 809 A B 0.98 -0.70 0.75 1.86

Leu 810 B B 0.63 -0.84 sk F 1.20 1.66

Arg 811 B B 1.30 -0.74 F 1.35 0.86

Lys 812 B B 1.30 -0.34 F 1.50 2.00

Pro 813 T 0.52 -0.99 F 2.70 4.06

Ala 814 T . 1.08 -1.10 F 3.00 1.1 1

Gin 815 B T 1.30 -0.71 F 2.35 0.74 Asp 816 . B r 0.94 -0.21 F 1.75 0.49

Cys 817 . B Γ 0.59 0.11 0.70 0.75

Ser 818 . B Γ -0.01 0.10 0.40 0.63

Ala 819 . B B 0.28 0.39 -0.30 0.31

Tyr 820 . B B -0.42 0.77 -0.60 0.78

Thr 821 . B B -0.42 0.99 -0.60 0.50

Leu 822 . B B -0.07 0.60 -0.60 0.83

Ser 823 . B B -0.08 0.59 -0.60 0.76

Phe 824 . B B -0.34 0.31 -0.30 0.76

Asp 825 . B B -0.80 0.47 F -0.45 0.69

Thr 826 . B B -1.38 0.57 F -0.45 0.44

Thr 827 . B B -1.46 0.87 * F -0.45 0.36

Val 828 . B B -1.16 0.77 -0.60 0.15

Phe 829 . B B -0.76 0.77 -0.60 0.18

He 830 . B B -1.07 0.67 -0.60 0.17

He 831 . B B -0.64 0.67 -0.60 0.33

Glu 832 A B -0.33 0.03 F -0.15 0.74

Ser 833 A Γ 0.63 -0.36 F 1.00 1.83

Thr 834 A T 0.48 -1.04 F 1.30 5.10

Arg 835 A r 0.78 -1.09 F 1.30 2.19

Gin 836 A Γ 0.81 -0.59 F 1.30 1.65

Arg 837 A A 0.81 -0.33 F 0.45 0.85

Val 838 A B 0.52 -0.81 0.600.75

Ala 839 A B 0.52 -0.31 0.30 0.44

Val 840 A B -0.40 -0.23 0.30 0.32

Glu 841 A A -0.40 0.46 -0.60 0.36

Ala 842 A A -0.51 -0.19 0.30 0.61

Thr 843 A A 0.46 -0.29 0.45 1.33

Leu 844 A A 0.70 -0.93 F 0.90 1.50

Glu 845 A A 1.56 -0.50 F 0.60 1.47

Asn 846 A Γ 1.56 -1.00 F 1.60 1.77

Arg 847 A Γ 1.56 -1.09 F 1.90 3.45

Gly 848 A T 1.62 -1.27 F 2.20 2.01

Glu 849 A T 2.13 -0.51 F 2.50 1.96

Asn 850 T C 1.82 -0.53 F 3.00 1.34

Ala 851 A T 0.97 -0.04 F 2.20 1.95

Tyr 852 B T 0.04 0.17 1.00 0.84

Ser 853 B T 0.39 0.86 0.40 0.43

Thr 854 B B -0.50 0.86 -0.30 0.68

Val 855 B B -0.80 1.04 + -0.60 0.31

Leu 856 B B -0.21 0.67 -0.60 0.31 Asn 857 . B B -0.27 0.69 -0.60 0.37

He 858 . B B -0.56 0.59 * F -0.45 0.66

Ser 859 . B -0.24 0.44 * F -0.25 0.81

Gin 860 . B ( Z -0.20 0.16 * F 0.25 0.81

Ser 861 T Z 0.61 0.44 * F 0.15 0.96

Ala 862 . B T -0.09 0.16 * F 0.40 1.23

Asn 863 . B T 0.21 0.56 * -0.20 0.62

Leu 864 A T 0.21 0.66 -0.20 0.47

Gin 865 A A -0.60 0.66 fc -0.60 0.62

Phe 866 A A -1.19 0.84 * -0.60 0.32

Ala 867 A A -0.60 1.13 fc -0.60 0.27

Ser 868 A A -0.56 0.84 fc -0.60 0.27

Leu 869 A A 0.26 0.44 -0.60 0.62

He 870 A A 0.26 -0.34 0.45 1.07

Gin 871 A A 0.66 -0.84 F 1.24 1.33

Lys 872 A A 1.24 -0.84 F 1.58 2.16

Glu 873 A A 1.20 -1.53 F 1.92 5.14

Asp 874 T T 1.71 -1.79 * F 3.06 2.94

Ser 875 T T 1.71 -1.80 * F 3.40 1.97

Asp 876 T T 1.71 -1.11 * F 2.91 0.80

Gly 877 T T 1.00 -1.11 * F 2.57 0.83

Ser 878 A A 0.14 -0.54 F 1.43 0.33

He 879 A A 0.14 -0.29 * 0.64 0.15

Glu 880 A A 0.44 0.11 -0.30 0.24

Cys 881 A B 0.44 -0.31 0.30 0.31

Val 882 A A 0.90 -0.70 0.60 0.76

Asn 883 A A 1.31 -1.39 * F 0.75 0.86

Glu 884 A A 1.39 -1.39 s_fc p 0.90 3.15

Glu 885 A A 1.39 -1.27 F 0.90 3.50

Arg 886 A A 2.10 -1.51 F 0.90 3.77

Arg 887 A A 2.96 -1.91 * F 0.904.35

Leu 888 A A 2.10 -1.51 F 0.90 4.35

Gin 889 A A 1.43 -0.87 F 0.90 1.65

Lys 890 A A 1.43 -0.30 F 0.45 0.45

Gin 891 A B 0.47 0.10 -0.30 0.88

Val 892 A B 0.06 0.06 * -0.30 0.38

Cys 893 A B 0.62 0.04 -0.30 0.25

Asn 894 B T 0.41 0.80 -0.20 0.23

Val 895 B T -0.33 0.83 -0.20 0.48

Ser 896 B . T -1.03 0.97 -0.20 0.77

Tyr 897 B T -0.07 1.19 -0.20 0.41 Pro 898 B B 0.01 0.79 * -0.45 1.09

Phe 899 A A B 0.06 0.64 " -0.60 0.82

Phe 900 A A B 0.32 0.26 * * -0.15 1.05

Arg 901 A A B 0.67 0.00 * -0.30 0.69

Ala 902 A A 0.06 -0.43 " 0.45 1.59

Lys 903 A A -0.32 -0.57 " * 1 F 0.90 1.36

Ala 904 A A -0.32 -0.86 " F 0.75 0.70

Lys 905 A A B 0.49 -0.07 " 0.30 0.60

Val 906 A A B -0.43 -0.57 * 0.60 0.59

Ala 907 A A B 0.16 0.11 -0.30 0.48

Phe 908 A A B -0.59 -0.39 . 0.30 0.40

Arg 909 A A B 0.00 0.40 « -0.60 0.47

Leu 910 A A -0.74 -0.24 " 0.30 0.80

Asp 911 A A -0.19 0.04 ι -0.30 0.80

Phe 912 A A 0.44 -0.36 * * 0.30 0.55

Glu 913 A A 0.84 -0.36 * 0.45 1.33

Phe 914 A A -0.16 -0.66 * * 0.75 1.07

Ser 915 A T -0.04 0.03 * F 0.25 0.87

Lys 916 A T -0.86 0.03 * F 0.25 0.43

Ser 917 A T -0.19 0.71 -0.20 0.41

He 918 A T -0.22 0.43 -0.20 0.42

Phe 919 A A -0.33 0.54 -0.60 0.28

Leu 920 A A -0.03 1.23 -0.60 0.18

His 921 A A -0.97 0.84 -0.60 0.43

His 922 A A -0.67 0.84 -0.60 0.35

Leu 923 A A -0.59 0.06 -0.30 0.74

Glu 924 A A -0.48 0.06 -0.30 0.45

He 925 A A -0.26 0.06 -0.30 0.33

Glu 926 A A -0.57 0.06 -0.30 0.41

Leu 927 A A -0.83 -0.20 0.300.23

Ala 928 A A -0.02 0.19 -0.30 0.44

Ala 929 A A -0.32 -0.50 0.30 0.43

Gly 930 A T 0.57 -0.11 F 0.85 0.69

Ser 931 T C 0.57 -0.40 F 1.20 1.11

Asp 932 T C 1.49 -0.90 F 1.50 1.89

Ser 933 T C 2.08 -1.40 F 1.84 3.75

Asn 934 C 2.37 -1.83 F 1.98 4.67

Glu 935 A 2.40 -1.83 F 2.12 3.75

Arg 936 A 2.74 -1.34 F 2.46 4.04

Asp 937 T T 2.74 -1.73 F 3.40 5.02

Ser 938 T C 3.04 -2.13 F 2.86 5.02 Thr 939 A r 3.04 -2.13 . F 2.32 4.28

Lys 940 A T 2.19 -1.73 * F 1.98 4.12

Glu 941 A A 1.49 -1.09 * F 1.24 2.28

Asp 942 A A 1.28 -0.97 . F 0.90 1.60

Asn 943 A A 0.77 -1.03 . * F 0.90 1.24

Val 944 A A 1.19 -0.34 . * 0.30 0.59

Ala 945 A A 0.44 -0.34 . * 0.30 0.69

Pro 946 A A 0.41 0.44 * -0.60 0.37

Leu 947 A A -0.40 0.54 fc -0.60 0.68

Arg 948 A A -0.36 0.59 -0.60 0.56

Phe 949 A A 0.26 0.09 -0.30 0.72

His 950 A A 0.84 0.41 -0.45 1.37

Leu 951 A A 0.47 -0.27 * * 0.45 1.21

Lys 952 A A 1.28 0.23 * -0.15 1.41

Tyr 953 A A 0.31 -0.56 * * 0.75 1.73

Glu 954 A A 0.20 -0.41 * 0.45 1.56

Ala 955 A B -0.47 -0.41 * 0.30 0.64

Asp 956 A B 0.03 0.37 * -0.30 0.35

Val 957 A B 0.10 0.10 fc -0.30 0.30

Leu 958 A B 0.04 0.10 -0.30 0.57

Phe 959 A B -0.26 -0.01 0.30 0.46

Thr 960 A B 0.03 0.37 F 0.06 0.83

Arg 961 A B -0.78 0.11 F 0.42 1.35

Ser 962 r T -0.22 0.11 F 1.43 1.29

Ser 963 T C 0.56 -0.29 F 2.04 1.19

Ser 964 T C 1.01 -0.27 . F 2.10 0.83

Leu 965 T C 1.32 0.49 F 0.99 0.97

Ser 966 . C 0.36 0.10 0.88 1.25

His 967 B 0.70 0.36 0.32 0.69

Tyr 968 B 0.19 -0.03 0.86 1.68

Glu 969 B 0.49 -0.03 . 0.65 1.04

Val 970 A 1.00 -0.01 * 0.65 1.22

Lys 971 A 1.00 -0.13 . F 0.80 1.05

Leu 972 A 0.22 -0.50 . F 0.65 0.81

Asn 973 T C 0.47 0.19 * F 0.45 0.90

Ser 974 A T 0.58 -0.46 * F 0.85 0.78

Ser 975 A T 1.19 -0.46 * F 1.00 1.85

Leu 976 B T 1.14 -0.39 * F 1.31 1.80

Glu 977 B 1.61 -0.79 * F 1.72 2.25

Arg 978 B T 0.72 -0.74 * F 2.23 1.66

Tyr 979 B T 0.68 -0.44 F 2.24 1.41 Asp 980 T T 0.77 -0.70 F 3.10 0.81

Gly 981 T T 1.37 -0.27 F 2.49 0.64

He 982 . T 0.67 0.16 F 1.38 0.63

Gly 983 C 0.26 0.19 * F 0.87 0.33

Pro 984 T C -0.17 0.57 F 0.46 0.44

Pro 985 T T -1.06 0.71 F 0.35 0.34

Phe 986 T T -1.41 0.71 0.20 0.24

Ser 987 B T -0.41 1.07 -0.20 0.13

Cys 988 B B -0.96 0.64 * -0.60 0.17

He 989 B B -0.74 0.90 -0.60 0.14

Phe 990 B B -0.53 0.51 -0.60 0.18

Arg 991 B B -0.64 0.53 -0.60 0.53

He 992 B B -0.69 0.64 -0.60 0.63

Gin 993 B B -0.83 0.39 -0.30 0.72

Asn 994 B r -0.64 0.29 0.10 0.30

Leu 995 B Γ -0.16 1.07 -0.20 0.37

Gly 996 B r -1.16 0.81 -0.20 0.33

Leu 997 ( : -0.30 1.10 -0.20 0.14

Phe 998 B -0.64 1.20 -0.40 0.24

Pro 999 B -1.53 0.94 -0.40 0.24

He 1000 A B -1.32 1.20 -0.60 0.20

His 1001 A B -1.58 1.13 -0.60 0.23

Gly 1002 A B -0.72 0.96 -0.60 0.15

He 1003 A B -0.91 0.53 -0.60 0.42

Met 1004 A B -1.01 0.53 -0.60 0.22

Met 1005 B B -1.01 0.51 -0.60 0.32

Lys 1006 B B -1.19 0.77 -0.60 0.32

He 1007 B B -1.73 0.51 -0.60 0.50

Thr 1008 B B -1.43 0.59 -0.60 0.35

Ile 1009 B B -1.14 0.47 -0.60 0.18

Pro 1010 B B -0.43 0.96 -0.60 0.37

He 1011 B B -0.78 0.27 0.04 0.50

Ala 1012 B B -0.23 0.17 0.38 0.95

Thr 1013 B T 0.08 -0.09 * F 1.87 0.61

Arg 1014 T T 1.08 -0.11 * F 2.76 1.40

Ser 1015 T T 0.48 -0.80 F 3.40 2.71

Gly 1016 T T 0.56 -0.61 * * F 3.06 1.55

Asn 1017 A T 1.19 -0.41 F 1.87 0.65

Arg 1018 A B 0.69 -0.41 F 1.13 0.97

Leu 1019 A B 0.69 -0.11 * F 0.79 0.81

Leu 1020 A B 0.99 -0.54 F 0.75 0.99 Lys 1021 A B 0.63 -0.94 * . F 0.75 0.84

Leu 1022 A B -0.18 -0.16 ι F 0.45 0.88

Arg 1023 A B -0.60 -0.16 * . F 0.45 0.88

Asp 1024 A B 0.21 -0.36 * * F 0.45 0.64

Phe 1025 A B 1.02 -0.36 * * 0.45 1.29

Leu 1026 A A 0.12 -1.04 ^"* * F 0.90 1.14

Thr 1027 A A 0.34 -0.40 * . F 0.45 0.51

Asp 1028 A A 0.23 0.10 F -0.15 0.59

Glu 1029 A A -0.08 -0.29 * . 0.45 1.15

Val 1030 A A 0.32 -0.49 0.45 1.15

Ala 1031 A A 0.47 -0.59 * . 0.60 0.93

Asn 1032 T T 0.78 -0.01 F 1.25 0.29

Thr 1033 T T -0.11 0.39 * . F 0.65 0.62

Ser 1034 3 T -0.40 0.43 * . F -0.05 0.43

Cys 1035 3 T 0.11 0.84 -0.20 0.28

Asn 1036 r 0.70 0.87 0.16 0.19

He 1037 ' Γ 0.40 0.79 0.320.23

Trp 1038 r T 0.40 0.79 0.68 0.58

Gly 1039 T <"! 0.70 0.70 F 0.790.52

Asn 1040 r T 1.12 0.30 F 1.60 1.28

Ser 1041 T 1.23 0.37 F 1.24 1.91

Thr 1042 . z 1.91 -0.54 F 1.78 3.79

Glu 1043 T 1.89 -0.54 F 1.82 3.64

Tyr 1044 T 2.02 -0.46 F 1.663.92

Arg 1045 T c 1.17 -0.41 F 1.804.20

Pro 1046 T c 1.47 -0.26 F 2.10 1.80

Thr 1047 T c 1.78 -0.26 F 2.40 1.99

Pro 1048 T C 1.78 -1.01 F 3.00 1.76

Val 1049 A A 1.21 -1.01 F 2.10 1.90

Glu 1050 A A 1.21 -0.76 F 1.80 1.09

Glu 1051 A A 1.53 -1.24 * F 1.50 1.38

Asp 1052 A A 1.26 -1.67 F 1.20 3.63

Leu 1053 A A 1.26 -1.81 F 0.902.12

Arg 1054 A A 2.11 -1.39 F 0.90 1.89

Arg 1055 A A 1.30 -0.99 F 0.90 1.96

Ala 1056 A A 1.30 -0.30 F 0.60 1.96

Pro 1057 A A 1.27 -0.59 F 0.90 1.61

Gin 1058 A A 1.78 -0.09 F 0.60 1.12

Leu 1059 A B 1.67 0.30 0.13 1.48

Asn 1060 A c 1.26 0.20 0.61 1.54

His 1061 T c 1.84 0.16 F 1.44 1.19 Ser 1062 T C 1.20 -0.24 . F 2.32 2.42

Asn 1063 T T 0.34 -0.29 . F 2.80 1.12

Ser 1064 T T . 0.86 -0.04 . F 2.37 0.61

Asp 1065 B B . . -0.03 -0.16 . F 1.29 0.61

Val 1066 B B 0.00 0.14 . F 0.41 0.27

Val 1067 B B . . -0.37 0.14 -0.02 0.32

Ser 1068 B T -0.37 0.33 0.10 0.10

He 1069 B T -0.96 0.73 -0.20 0.22

Asn 1070 B T -0.84 0.77 -0.20 0.21

Cys 1071 B T -0.80 0.13 0.10 0.31

Asn 1072 B B -0.80 0.43 -0.60 0.36

He 1073 B B -0.71 0.39 -0.30 0.17

Arg 1074 B B . . 0.18 0.41 -0.60 0.48

Leu 1075 B B . . 0.18 0.24 -0.30 0.48

Val 1076 B T 0.84 0.24 * F 0.40 1.18

Pro 1077 T C -0.04 -0.44 * F 1.20 1.05

Asn 1078 T T 0.84 0.24 F 0.65 0.89

Gin 1079 A T 0.03 -0.04 * F 1.00 1.93

Glu 1080 A B 0.81 0.10 * F 0.00 1.08

He 1081 A B 0.86 0.17 * F -0.15 0.91

Asn 1082 A B 0.26 0.46 -0.60 0.43

Phe 1083 A B -0.09 0.74 -0.60 0.21

His 1084 A 1 B -0.09 1.17 -0.60 0.29

Leu 1085 A 1 B -0.90 0.89 * -0.60 0.29

Leu 1086 A C -0.30 1.17 -0.40 0.28

Gly 1087 A T -1.11 1.30 -0.20 0.22

Asn 1088 A A -0.30 1.49 -0.60 0.22

Leu 1089 A A -0.57 0.80 -0.60 0.51

Trp 1090 A A -0.57 0.50 -0.60 0.69

Leu 1091 A A 0.29 0.76 -0.60 0.35

Arg 1092 A A 0.04 0.36 -0.30 0.86

Ser 1093 A A -0.77 0.17 -0.30 0.83

Leu 1094 A A 0.09 -0.06 * F 0.45 0.83

Lys 1095 A A 0.13 -0.74 F 0.75 0.84

Ala 1096 A A 0.99 0.01 -0.30 0.99

Leu 1097 A A . 0.58 -0.37 0.45 2.39

Lys 1098 A A 0.28 -0.67 F 0.90 1.60

Tyr 1099 A A 1.13 -0.06 F 0.60 1.57

Lys 1100 A A 0.20 -0.56 F 0.90 3.81

Ser 1101 A A 0.19 -0.56 F 0.90 1.34

Met 1102 A A 0.14 0.06 -0.30 0.84 Lys 1103 A A 0.10 -0.06 * 0.30 0.31

He 1104 A B -0.24 0.34 * -0.30 0.38

Met 1105 A A -0.88 0.46 * * -0.60 0.38

Val 1106 A A -1.39 0.34 * * -0.30 0.19

Asn 1107 A A -0.79 1.03 -0.60 0.23

Ala 1108 A A -0.72 0.74 -0.60 0.40

Ala 1109 A A 0.17 0.13 -0.15 1.05

Leu 1110 A A 0.07 -0.11 < 0.45 1.13

Gin 1111 A A 0.89 0.27 * . -0.30 0.97

Arg 1112 A A 0.59 0.27 * . -0.15 1.31

Gin 1113 A B 0.97 0.16 * . -0.15 2.13

Phe 1114 A T 0.86 -0.10 * . 0.85 1.90

His 1115 A C 0.78 0.29 * . -0.10 0.84

Ser 1116 A C 0.08 0.97 -0.40 0.34

Pro 1117 B < Z 0.08 1.36 -0.40 0.34

Phe 1118 A B ( Z 0.08 0.57 -0.40 0.49

He 1119 A B B 0.78 0.07 -0.30 0.63

Phe 1120 A B B 0.81 -0.31 . 0.30 0.71

Arg 1121 A A B 0.90 -0.74 . F 1.24 1.37

Glu 1122 . A T 0.81 -1.10 ^■ F 1.98 3.01

Glu 1123 . A Z 1.62 -1.40 F 2.124.66

Asp 1124 r C 2.51 -2.19 F 2.864.66

Pro 1125 T Γ 2.32 -1.79 F 3.404.66

Ser 1126 T T 1.36 -1.10 k F 3.06 1.89

Arg 1127 A T 0.66 -0.46 F 1.87 0.84

Gin 1128 A B 0.66 0.33 fc F 0.53 0.47

He 1129 A B -0.23 -0.10 * 0.64 0.61

Val 1130 A B -0.32 0.20 -0.30 0.22

Phe 1131 B B 0.02 0.59 -0.60 0.17

Glu 1132 B B -0.09 0.19 . -0.30 0.48

He 1133 B B -0.09 -0.10 F 0.60 1.12

Ser 1134 . A . C 0.80 -0.74 F 1.10 2.24

Lys 1135 . A T 1.37 -1.53 F 1.30 2.16

Gin 1136 . A T 2.07 -0.61 F 1.30 3.24

Glu 1137 A A 1.21 -0.90 F 0.90 4.18

Asp 1138 A B T 1.89 -0.64 F 1.30 1.55

Trp 1139 A B T 1.30 -0.21 0.85 1.39

Gin 1140 A B B 0.97 0.07 -0.30 0.56

Val 1141 A B B 0.08 0.99 * -0.60 0.35

Pro 1142 A B B -0.81 1.67 -0.60 0.24

He 1143 B B -1.67 1.44 -0.60 0.10 Trp 1144 B B -1.72 1.69 . * -0.60 0.10

He 1145 B B -2.02 1.47 . . -0.60 0.06

He 1146 B B -1.48 1.43 . . -0.60 0.12

Val 1147 B B -2.08 1.23 . . -0.60 0.16

Gly 1148 B B -1.53 1.00 . . 1 -0.45 0.19

Ser 1149 B c -1.59 0.74 . . I -0.25 0.27

Thr 1150 B c -1.51 0.49 . . 1 -0.25 0.35

Leu 1151 B c -1.43 0.53 . . 1 -0.25 0.30

Gly 1152 B r -1.39 0.79 . . 1 " -0.05 0.18

Gly 1153 A B B -1.86 1.09 . . -0.60 0.10

Leu 1154 A B B -2.14 1.29 . . -0.60 0.10

Leu 1155 B B -2.64 1.10 . . -0.60 0.11

Leu 1156 A A B -2.64 1.36 . . -0.60 0.09

Leu 1157 A A B -3.16 1.61 . . -0.60 0.09

Ala 1158 A A B -3.62 1.57 . . -0.60 0.08

Leu 1159 A A B -3.40 1.57 . . -0.60 0.08

Leu 1160 A A B -3.40 1.39 . . -0.60 0.10

Val 1161 A A B -2.88 1.39 . . -0.60 0.08

Leu 1162 A A B -2.02 1.80 * . -0.60 0.10

Ala 1163 A A B -2.24 1.11 . . -0.60 0.25

Leu 1164 A A B -1.78 1.11 . * -0.60 0.27

Trp 1165 A A B -1.67 0.90 . . -0.60 0.33

Lys 1166 A A B -1.51 1.00 . . -0.60 0.28

Leu 1167 A A B -0.59 1.29 . . -0.60 0.29

Gly 1168 A B -0.30 0.60 . . -0.60 0.55

Phe 1169 B B -0.08 0.07 * . -0.30 0.37

Phe 1170 . B B 0.32 0.57 * . -0.60 0.45

Arg 1171 . B B 0.39 -0.11 * . 0.30 0.89

Ser 1172 B c 1.31 -0.54 * . F 1.10 2.02

Ala 1173 c 1.77 -1.33 * . F 1.30 4.57

Arg 1174 c 2.47 -2.11 * . F 1.30 4.57

Arg 1175 T 2.96 -2.11 * . F 1.84 5.90

Arg 1176 T 2.50 -2.07 * . F 2.189.03

Arg 1177 T 1.99 -2.14 . . F 2.52 4.56

Glu 1178 T C 2.58 -1.46 . . F 2.86 1.92

Pro 1179 T T 2.26 -1.46 . . F 3.40 1.64

Gly 1180 T T 1.83 -1.03 . . F 3.06 1.29

Leu 1181 T C 1.51 -0.54 . * F 2.69 1.08

Asp 1182 T C 1.44 -0.11 . * F 2.22 1.08

Pro 1183 T C 0.59 -0.54 * * F 2.35 2.18

Thr 1184 T c -0.01 -0.33 * . F 1.88 1.96 Pro 1185 B T 0.33 -0.33 F 1.70 0.97

Lys 1186 A B 0.76 -0.33 F 1.28 1.08

Val 1187 A B 0.37 -0.33 F 0.96 0.96

Leu 1188 A A 0.19 -0.39 0.64 0.79

Glu 1189 A A 0.11 -0.39 0.47 0.51

Ter 1190 A A -0.07 0.04 -0.30 0.87

Table VIII

Res Position 1 I [II [V V VI VII VIII IX X XI XII XIII XIV

Met 1 A A -1.47 0.70 -0.60 0.31

Ala 2 A A -1.38 0.96 -0.60 0.20

Leu 3 A A -1.80 0.91 -0.60 0.21

Met 4 A A -2.27 1.17 -0.60 0.17

Leu 5 A i A -2.69 1.20 -0.60 0.13

Ser 6 A A -2.39 1.39 -0.60 0.13

Leu 7 A A -2.61 1.09 -0.60 0.17

Val 8 A A -2.61 1.16 -0.60 0.17

Leu 9 A A -1.97 1.16 -0.60 0.11

Ser 10 A A -1.97 0.77 -0.60 0.26

Leu 11 A B -2.01 0.77 -0.60 0.28

Leu 12 A B -1.50 0.56 -0.60 0.34

Lys 13 A B -0.99 0.26 F -0.15 0.34

Leu 14 A -0.18 0.30 F 0.05 0.41

Gly 15 T r -0.17 0.01 * F 0.65 0.86

Ser 16 Γ 0.64 0.24 F 0.45 0.45

Gly 17 Γ 0.60 0.64 * F 0.15 0.95

Gin 18 B Γ -0.14 0.60 F -0.05 0.71

Trp 19 B B 0.32 0.96 -0.60 0.46

Gin 20 B B 0.46 1.00 * -0.60 0.46

Val 21 B B 0.76 1.00 -0.60 0.41

Phe 22 B B 1.14 0.60 -0.30 0.65

Gly 23 T C 0.93 -0.31 1.50 0.75

Pro 24 T T 0.37 -0.29 F 2.30 1.57

Asp 25 T C 0.37 -0.29 F 2.40 1.34

Lys 26 T C 0.63 -0.67 F 3.00 2.35

Pro 27 B C 0.52 -0.60 F 2.30 1.54

Val 28 B B 0.01 -0.34 1.200.76

Gin 29 B B -0.12 0.30 0.30 0.28

Ala 30 B B -0.12 0.73 -0.30 0.18

Leu 31 B B -0.17 0.30 -0.30 0.42

Val 32 B B -0.54 -0.34 0.30 0.41

Gly 33 A B -0.28 -0.24 F 0.45 0.41

Glu 34 A A -0.98 -0.24 F 0.45 0.50

Asp 35 A A -0.69 -0.14 F 0.45 0.58

Ala 36 A A -0.54 -0.40 0.30 0.79 Ala 37 A A B -0.39 -0.26 0.30 0.24

Phe 38 A A 1 B -0.86 0.53 -0.60 0.13

Ser 39 A A B -1.16 1.21 -0.60 0.10

Cys 40 A A B -1.37 1.10 -0.60 0.14

Phe 41 A A B -0.73 1.03 -0.60 0.24

Leu 42 A B c -0.46 0.24 -0.10 0.36

Ser 43 T ( 0.24 0.34 * F 0.45 0.98

Pro 44 T c -0.04 0.17 * F 0.60 1.82

Lys 45 T c 0.62 -0.11 . si. F 1.20 2.23

Thr 46 A T 0.73 -0.80 _* F 1.30 2.88

Asn 47 A A 0.94 -0.69 sfc p 0.90 1.88

Ala 48 A A 1.24 -0.50 . 0.300.93

Glu 49 A A 0.60 -0.50 . 0.45 1.12

Ala 50 A A 0.67 -0.34 0.30 0.52

Met 51 A A 0.28 -0.74 * * 0.60 1.00

Glu 52 A > A -0.42 -0.46 ^■ 0.300.50

Val 53 A A 0.28 0.33 -0.30 0.43

Arg 54 A . A -0.07 -0.17 ' 0.300.85

Phe 55 A A 0.52 -0.36 0.30 0.48

Phe 56 A T 0.42 0.04 0.25 1.13

Arg 57 A T 0.12 0.19 0.100.50

Gly 58 r T 0.68 0.57 * . F 0.35 0.77

Gin 59 Γ T -0.29 0.17 fc * p 0.80 1.20

Phe 60 B . c -0.44 0.03 it sit 0.05 0.45

Ser 61 B . c 0.22 0.67 fc s* F -0.25 0.34

Ser 62 B B -0.70 0.74 * * -0.60 0.27

Val 63 B B -0.60 1.03 * -0.60 0.25

Val 64 B B -0.49 1.00 * -0.60 0.30

His 65 B B 0.21 0.61 * -0.26 0.43

Leu 66 B B 0.17 0.23 * 0.38 0.98

Tyr 67 B T 0.51 0.01 * 1.27 1.30

Arg 68 T T 1.37 -0.63 sk F 3.06 1.92

Asp 69 T T 2.22 -1.13 * F 3.40 3.88

Gly 70 T T 2.04 -1.41 * . F 3.06 4.29

Lys 71 T 2.16 -1.74 * . F 2.52 3.39

Asp 72 c 1.80 -0.96 . . F 1.98 1.76

Gin 73 c 1.69 -0.34 . . F 1.34 1.76

Pro 74 B 1.09 -0.37 . . F 0.80 1.52

Phe 75 B 1.22 0.24 -0.10 0.90

Met 76 B 1.18 0.67 -0.40 0.80

Gin 77 B 0.93 0.67 -0.40 0.90 Met 78 B 0.93 1.00 * -0.25 1.63

Pro 79 B 0.80 0.61 0.09 2.85

Gin 80 T 1.61 0.43 * * F 0.98 1.63

Tyr 81 T T 1.90 0.03 * F 1.82 3.23

Gin 82 i \ T 1.94 -0.10 * F 2.36 3.01

Gly 83 T T 1.73 -0.53 * F 3.40 3.48

Arg 84 B T 1.09 -0.24 * F 2.36 1.83

Thr 85 B 1.13 -0.36 * F 1.900.78

Lys 86 B 1.38 -0.76 . * F 2.24 1.59

Leu 87 B 1.08 -1.19 < ' * F 2.13 1.35

Val 88 B T 0.53 -0.80 . * F 2.22 1.26

Lys 89 B T -0.17 -0.60 . F 2.30 0.44

Asp 90 B T 0.14 -0.10 * . F 1.77 0.54

Ser 91 B T -0.24 -0.79 * t 1.84 1.26

He 92 A A 0.68 -1.00 * * 1.060.62

Ala 93 A A 0.64 -1.00 * F 0.98 0.73

Glu 94 A A 0.30 -0.31 * F 0.45 0.38

Gly 95 A A -0.51 -0.31 ' > * F 0.45 0.73

Arg 96 A A -0.10 -0.31 * * F 0.45 0.60

He 97 A A -0.02 -0.81 * F 0.75 0.67

Ser 98 A A 0.57 -0.13 0.30 0.56

Leu 99 A A 0.57 -0.56 0.60 0.50

Arg 100 A A 0.02 -0.16 0.45 1.14

Leu 101 A A -0.40 -0.16 fc * 0.30 0.60

Glu 102 A B -0.37 -0.06 * 0.45 1.04

Asn 103 A B -0.88 -0.10 0.30 0.40

He 104 A B -0.07 0.59 * -0.60 0.40

Thr 105 A B -0.77 -0.10 0.30 0.38

Val 106 A B -0.30 0.40 -0.60 0.24

Leu 107 A B -1.11 0.43 -0.60 0.34

Asp 108 A B -1.36 0.43 -0.60 0.19

Ala 109 A B -0.81 0.70 -0.60 0.41

Gly 110 . T -1.17 0.49 0.00 0.49

Leu 111 B T -0.20 0.37 0.10 0.16

Tyr 112 B T -0.28 0.37 0.10 0.30

Gly 113 B T -0.58 0.56 -0.20 0.22

Cys 114 B T -0.29 0.51 -0.20 0.35

Arg 115 B B 0.06 0.21 -0.30 0.30

He 116 B B 0.57 -0.14 F 0.45 0.52

Ser 117 B B 0.57 -0.19 F 0.76 1.31

Ser 118 B T 0.67 0.00 F 1.32 1.05 Gin 119 B T 1.33 0.76 . * F 0.582.35

Ser 120 T T 1.27 0.47 . * F 1.143.03

Tyr 121 T T 1.57 0.09 . . F 1.604.52

Tyr 122 . i \ T 0.98 0.20 . . 0.892.64

Gin 123 . / \ B 0.99 0.49 . . 0.031.38

Lys 124 . i \ B 0.99 1.01 * . -0.28 0.93

Ala 125 . . \ B 0.48 0.26 * . 0.011.02

He 126 . . \ B 0.72 0.19 . * -0.30 0.49

Trp 127 . i \ B 0.11 0.19 . * -0.30 0.42

Glu 128 A

-0.19 0.83 * * -0.60 0.31

Leu 129 A i A -0.82 0.71 * * -0.60 0.59

Gin 130 . . A B -1.04 0.53 . * -0.60 0.57

Val 131 . . \ B -0.50 0.30 . * -0.30 0.27

Ser 132 . . A ( Z -0.51 0.73 . * -0.40 0.33

Ala 133 . - A Z -1.37 0.43 . * -0.40 0.25

Leu 134 A B -0.77 0.67 . * -0.60 0.25

Gly 135 A 'r -1.58 0.46 -0.20 0.29

Ser 136 B B -1.61 0.76 . . -0.60 0.24

Val 137 B B -1.61 0.94 . . -0.60 0.20

Pro 138 B B -1.91 0.64 . . -0.60 0.27

Leu 139 B B -1.69 0.90 . . -0.60 0.14

He 140 B B -1.69 1.01 -0.60 0.19

Ser 141 B B -1.63 0.80 . . -0.60 0.12

He 142 B B -1.63 1.13 . . -0.60 0.24

Ala 143 B B -1.42 1.09 * . -0.60 0.25

Gly 144 B B -0.50 0.40 * . -0.34 0.31

Tyr 145 B B 0.39 0.01 * * 0.220.87

Val 146 B B -0.20 -0.67 * . 1.531.44

Asp 147 B T 0.69 -0.49 * * F 2.041.02

Arg 148 B T 0.47 -0.51 * . F 2.601.13

Asp 149 B T 0.00 -0.59 * . F 2.341.25

He 150 B T -0.42 -0.54 * . 1.780.62

Gin 151 A B 0.43 0.03 * . 0.220.17

Leu 152 A B 0.13 0.43 * . -0.34 0.18

Leu 153 A B -0.28 0.81 * . -0.60 0.34

Cys 154 A B -0.62 0.51 . * -0.60 0.26

Gin 155 A T -0.02 0.54 * * F -0.05 0.31

Ser 156 T T -0.72 0.77 * . F 0.350.40

Ser 157 T T -0.12 0.87 * . F 0.350.64

Gly 158 T T 0.80 0.73 * . F 0.350.57

Trp 159 T T 1.26 0.33 * . F 0.650.84 Phe 160 T C 0.94 0.37 * . F 0.45 0.97

Pro 161 T C 0.66 0.47 * * F 0.30 1.41

Arg 162 T C 1.00 0.54 * * F 0.30 1.36

Pro 163 T T 1.06 -0.37 * * F 1.40 3.13

Thr 164 T 1.39 -0.24 * * F 1.20 2.13

Ala 165 T 1.74 -0.67 * * F 1.50 2.17

Lys 166 T 1.74 -0.24 . * F 1.20 1.39

Trp 167 T 1.63 -0.24 * F 1.54 1.49

Lys 168 C 1.50 -0.33 . * F 1.68 2.55

Gly 169 C 1.81 -0.40 . * F 2.02 1.26

Pro 170 T C 2.40 0.00 * F 1.96 2.08

Gin 171 T T 1.54 -0.91 . * F 3.40 1.74

Gly 172 T ( Z 1.53 -0.23 . F 2.56 1.45

Gin 173 1 B • r 1.18 -0.27 F 2.02 1.26

Asp 174 1 B 1.52 -0.21 . F 1.82 1.05

Leu 175 B 1.43 -0.61 ^• * * F 2.12 1.77

Ser 176 B . Γ 1.54 -0.66 ' * * F 2.32 1.37

Thr 177 B . Γ 1.58 -1.06 ' * * F 2.66 1.60

Asp 178 T Γ 1.58 -0.57 ' " * F 3.40 2.80

Ser 179 . Γ C 1.69 -0.86 * * F 2.86 3.37

Arg 180 T Γ 2.50 -1.24 * * F 3.064.57

Thr 181 T Γ 2.20 -1.73 * . F 3.06 4.57

Asn 182 T Γ 2.48 -1.11 * . F 3.06 3.37

Arg 183 B . Γ 2.13 -1.00 * . F 2.66 2.34

Asp 184 T Γ 1.62 -0.57 * . F 3.40 1.61

Met 185 B . T 0.81 -0.37 « 2.06 0.82

His 186 B . T 1.12 0.01 fc 1.12 0.36

Gly 187 B . T 0.27 0.01 0.78 0.36

Leu 188 B B 0.16 0.66 -0.26 0.27

Phe 189 A B -0.73 0.04 -0.30 0.35

Asp 190 A B -0.43 0.23 -0.30 0.25

Val 191 A B -1.21 0.19 -0.30 0.40

Glu 192 A B -1.18 0.19 -0.30 0.38

He 193 A B -1.22 -0.11 0.30 0.33

Ser 194 A B -0.52 0.53 -0.60 0.33

Leu 195 A A B -0.52 0.29 -0.30 0.33

Thr 196 A A B 0.33 0.29 -0.30 0.81

Val 197 A A B -0.26 0.00 0.55 0.98

Gin 198 A A B 0.29 0.11 F 0.50 1.20

Glu 199 A A B 0.29 -0.14 F 1.20 0.82

Asn 200 T T 0.21 -0.24 F 2.40 1.48 Ala 201 T T 0.22 -0.20 F 2.50 0.60

Gly 202 T r 0.41 -0.21 F 2.25 0.46

Ser 203 T Γ 0.11 0.36 F 1.40 0.15

He 204 A -0.49 0.34 fc 0.40 0.21

Ser 205 A -0.38 0.46 fc -0.15 0.21

Cys 206 B 0.18 0.03 fc -0.10 0.30

Ser 207 A B -0.07 0.14 fc -0.30 0.58

Met 208 A A 0.20 -0.04 0.30 0.44

Arg 209 A A 0.28 0.07 -0.15 1.11

His 210 A A 0.28 0.19 -0.30 0.69

Ala 211 A A 1.06 0.19 -0.30 0.93

His 212 A A 1.36 -0.43 0.30 0.93

Leu 213 A A 1.10 -0.43 0.45 1.18

Ser 214 A A 0.99 -0.29 0.30 0.87

Arg 215 A A 0.72 -0.79 fc F 0.90 1.10

Glu 216 A A 1.42 -0.90 k F 0.90 1.79

Val 217 A . B 0.60 -1.59 k F 0.90 2.62

Glu 218 A . B 1.41 -1.33 fc F 0.75 0.99

Ser 219 A . B 0.82 -0.93 * fc F 0.75 0.99

Arg 220 B B 0.37 -0.24 * F 0.45 0.94

Val 221 B B 0.37 -0.46 * F 0.45 0.54

Gin 222 A B 0.93 -0.46 fc 0.64 0.67

He 223 A . T 1.04 0.07 * 0.78 0.36

Gly 224 A . T 1.46 0.07 * * F 1.27 0.95

Asp 225 T T 1.39 -0.57 * F 3.06 1.07

Trp 226 T T 2.21 -0.97 F 3.40 3.06

Arg 227 B 1.87 -1.16 F 2.464.20

Arg 228 T T 2.76 -1.16 F 2.722.49

Lys 229 ' T T 2.51 -0.76 F 2.38 4.10

His 230 T T 2.17 -1.17 F 2.382.12

Gly 231 . T C 2.50 -0.74 F 2.18 1.07

Gin 232 T 2.50 -0.74 F 2.52 1.07

Ala 233 C 2.43 -0.74 F 2.66 1.54

Gly 234 T T 2.14 -1.24 F 3.40 3.11

Lys 235 B T 1.88 -0.91 F 2.66 2.81

Arg 236 T T 1.92 -0.93 F 2.77 3.73

Lys 237 T T . 1.62 -1.04 * F 2.48 5.05

Tyr 238 B T 2.18 -1.09 F 1.79 3.39

Ser 239 B T 1.63 -0.59 F 1.50 2.35

Ser 240 B T 1.34 0.10 F 0.50 0.82

Ser 241 B T 1.23 0.86 F 0.15 0.82 His 242 B 0.89 0.10 * 0.20 1.03

He 243 B 0.43 0.10 * 0.15 1.03

Tyr 244 B 0.52 0.50 * -0.35 0.66

Asp 245 B 0.52 0.54 * -0.40 0.75

Ser 246 B 0.01 0.43 * F -0.10 1.44

Phe 247 B T -0.26 0.43 * F -0.05 0.76

Pro 248 T C -0.07 0.06 * F 0.45 0.61

Ser 249 T C -0.42 0.84 F 0.15 0.39

Leu 250 T C -0.42 1.07 0.00 0.45

Ser 251 B -0.82 0.29 -0.10 0.49

Phe 252 B B -0.37 0.64 -0.60 0.31

Met 253 B ] B -1.04 1.01 -0.60 0.60

Asp 254 B 1 B -1.56 1.01 -0.60 0.31

Phe 255 B 3 -0.63 1.31 -0.60 0.30

Tyr 256 B B -0.54 0.53 -0.60 0.59

He 257 B B -0.70 0.34 -0.30 0.54

Leu 258 B 1 B -0.44 0.99 * -0.60 0.47

Arg 259 B B -0.66 0.63 * -0.35 0.29

Pro 260 . B T -0.62 0.30 * F 0.75 0.65

Val 261 . B T -0.27 0.19 * * F 1.00 0.42

Gly 262 T C 0.03 -0.50 * * 1 F 2.35 0.42

Pro 263 T T 0.89 0.00 * * F 2.50 0.28

Cys 264 B T -0.03 -0.43 * * F 1.85 0.74

Arg 265 B T -0.68 -0.39 . 1.45 0.62

Ala 266 A B -0.42 -0.17 * 0.80 0.30

Lys 267 A B -0.42 0.01 -0.05 0.55

Leu 268 A B -0.52 -0.13 . 0.30 0.28

Val 269 A B -0.67 0.36 -0.30 0.40

Met 270 A A -0.73 0.54 -0.60 0.16

Gly 271 A A -0.96 0.54 * -0.60 0.40

Thr 272 A A -1.00 0.54 -0.60 0.44

Leu 273 A A -1.08 0.30 -0.30 0.77

Lys 274 A A -1.03 0.37 -0.30 0.55

Leu 275 A A -0.78 0.63 -0.60 0.31

Gin 276 A A -0.43 0.57 -0.60 0.37

He 277 A B -0.98 -0.11 0.30 0.32

Leu 278 A A -0.20 0.53 -0.60 0.29

Gly 279 A A -0.94 0.34 -0.30 0.23

Glu 280 A A -0.99 0.73 -0.60 0.28

Val 281 A A -0.99 0.69 * -0.60 0.26

His 282 A A -0.06 0.00 * 0.30 0.45 Phe 283 A A 0.54 -0.43 . 0.30 0.52

Val 284 A A 0.86 0.00 0.45 1.07

Glu 285 A A 0.56 -0.14 . . F 0.60 1.07

Lys 286 A T 0.60 -0.26 * F 1.00 1.66

Pro 287 A T -0.18 -0.36 * F 1.00 1.85

His 288 A T 0.52 -0.31 * . F 0.85 0.88

Ser 289 A T 0.49 0.09 * 0.10 0.76

Leu 290 A B 0.19 0.77 . ^• -0.60 0.35

Leu 291 B B -0.20 0.73 -0.60 0.34

Gin 292 B B -0.33 0.66 . . -0.60 0.25

He 293 B B ' -0.60 0.70 F -0.45 0.30

Ser 294 B T -0.61 0.40 F 0.25 0.49

Gly 295 r T -0.11 0.20 F 0.65 0.41

Gly 296 r T -0.11 0.29 * F 0.65 0.84

Ser 297 T Z -0.07 0.29 * F 0.45 0.52

Thr 298 B B 0.87 -0.10 * F 0.90 1.04

Thr 299 B B 0.82 -0.53 * F 1.50 2.11

Leu 300 B B 0.96 -0.53 * F 1.80 1.56

Lys 301 B T 1.30 -0.49 * F 2.20 1.67

Lys 302 T 1.39 -0.57 * F 3.00 1.86

Gly 303 T C 1.41 -0.63 * F 2.70 3.49

Pro 304 T C 1.42 -0.40 * F 2.10 1.83

Asn 305 T C 1.53 -0.01 * F 1.80 1.23

Pro 306 T T 1.28 0.77 * F 0.80 1.08

Trp 307 T 0.93 0.77 0.15 1.08

Ser 308 _m C 1.07 0.73 -0.20 0.90

Phe 309 B 0.61 0.76 F -0.25 0.90

Pro 310 B 0.02 0.90 F -0.25 0.46

Ser 311 T C -0.58 0.49 F 0.15 0.34

Pro 312 T T -0.99 0.79 F 0.35 0.33

Cys 313 T T -0.90 0.79 0.20 0.18

Ala 314 B T T -0.51 0.79 0.200.21

Leu 315 B -0.69 0.89 -0.40 0.20

Phe 316 B -0.78 0.89 -0.40 0.47

Pro 317 B -0.96 0.74 -0.40 0.60

Thr 318 B -0.68 0.67 -0.40 0.93

Ter 319 B -0.48 0.41 -0.25 1.37 Table IX

Res Position III IV V VI VII VIII IX X XI XII XIII XIV

Met 1 B -0.69 0.51 -0.40 0.26

Ala 2 B -0.64 0.51 -0.40 0.31

Gly 3 B -1.07 0.51 -0.40 0.24

He 4 B -1.49 0.77 -0.40 0.20

Pro 5 B -1.80 0.84 F -0.25 0.17

Gly 6 B B -2.01 1.13 -0.60 0.14

Leu 7 B B -2.23 1.39 -0.60 0.17

Leu 8 B I B -2.59 1.39 -0.60 0.09

Phe 9 B B -2.40 1.74 -0.60 0.08

Leu 10 B 1 B -3.00 2.10 -0.60 0.08

Leu 11 B B -3.47 2.10 -0.60 0.08

Phe 12 B B -3.32 2.10 -0.60 0.08

Phe 13 B B -3.10 1.89 -0.60 0.05

Leu 14 B B -3.26 1.70 -0.60 0.06

Leu 15 B B -2.79 1.66 -0.60 0.05

Cys 16 B B -1.98 1.30 -0.60 0.06

Ala 17 B B T -2.13 0.91 -0.20 0.13

Val 18 B B -1.73 0.87 -0.60 0.12

Gly 19 B B -1.13 0.57 -0.60 0.29

Gin 20 B B -0.57 0.43 F -0.45 0.45

Val 21 B B -0.20 0.69 F -0.45 0.95

Ser 22 B . T -0.20 0.43 F 0.10 1.28

Pro 23 B . T 0.44 0.50 F -0.05 0.75

Tyr 24 T T 0.50 0.53 * 0.35 1.56

Ser 25 T c 0.54 0.80 0.15 1.22

Ala 26 c 1.19 0.41 -0.05 1.58

Pro 27 T 1.18 0.41 0.15 1.56

Trp 28 T 1.10 0.14 0.45 1.68

Lys 29 T c 1.13 0.67 * F 0.30 1.75

Pro 30 T T 0.84 0.60 F 0.50 1.75

Thr 31 T T 1.19 0.67 F 0.50 1.68

Trp 32 B T 1.51 0.51 -0.05 1.32

Pro 33 B T 0.99 0.51 -0.05 1.67

Ala 34 T T 0.73 0.77 0.20 0.95

Tyr 35 T T 0.09 0.71 0.35 1.40

Arg 36 B T -0.46 0.44 -0.20 0.67 Leu 37 B B -0.98 0.66 . * -0.60 0.49

Pro 38 B B -0.98 0.84 * * -0.60 0.26

Val 39 B B -0.39 0.51 * * -0.60 0.21

Val 40 B B -0.44 0.91 * * -0.60 0.43

Leu 41 B B -0.87 0.61 * * F -0.45 0.37

Pro 42 B T -0.87 0.67 . . F -0.05 0.73

Gin 43 B T -0.66 0.71 . * F -0.05 0.81

Ser 44 B T -0.61 0.47 . . F 0.10 1.57

Thr 45 B T -0.34 0.47 * . F -0.05 0.84

Leu 46 B 0.51 0.54 * . F -0.25 0.49

Asn 47 B 0.51 0.14 . . 0.14 0.73

Leu 48 B 0.51 0.19 * * 0.38 0.78

Ala 49 B 0.11 -0.30 . . 1.37 1.59

Lys 50 T Z 0.08 -0.20 . . F 2.01 0.85

Pro 51 T Z 0.30 -0.17 . * F 2.40 1.02

Asp 52 T Z 0.30 -0.36 . * F 2.16 1.02

Phe 53 B T 0.52 -0.86 . =• 1.72 0.89

Gly 54 A A 1.16 -0.36 . « 0.78 0.58

Ala 55 A A 0.30 -0.79 . " 0.84 0.69

Glu 56 A A 0.51 -0.10 . " 0.30 0.66

Ala 57 A A -0.34 -0.89 . " F 0.90 1.16

Lys 58 A A 0.06 -0.67 . ^« * F 0.75 0.85

Leu 59 A B 0.10 -0.79 ι 0.60 0.66

Glu 60 A B 0.39 -0.40 . ^» fc 0.30 0.87

Val 61 A B -0.28 -0.51 . ^a k F 1.00 0.58

Ser 62 A r -0.03 0.06 . ^> " F 0.75 0.38

Ser 63 Γ T -0.29 -0.20 . ^> * F 2.00 0.22

Ser 64 Γ T 0.52 0.23 k F 1.65 0.45

Cys 65 Γ T -0.14 -0.01 F 2.50 0.58

Gly 66 T C 0.68 0.17 F 1.45 0.23

Pro 67 Γ 1.02 0.29 F 1.45 0.24

Gin 68 Γ 0.98 -0.10 1.90 0.89

Cys 69 B 0.97 -0.24 1.50 0.89

His 70 T T 1.42 -0.19 F 2.25 0.83

Lys 71 T T 0.96 -0.19 F 2.500.74

Gly 72 T T 0.96 0.10 F 1.80 1.14

Thr 73 T C 0.64 -0.04 F 1.95 1.29

Pro 74 C 1.07 -0.06 . F 1.35 0.93

Leu 75 C 1.10 0.70 F 0.61 1.48

Pro 76 T C 1.06 0.27 F 1.12 1.77

Thr 77 B T 0.81 -0.21 F 1.78 1.98 Tyr 78 B r 1.17 -0.14 F 2.04 2.43

Glu 79 B T 1.38 -0.83 F 2.60 3.14

Glu 80 A B 1.94 -0.86 F 1.94 3.77

Ala 81 A A 1.34 -0.59 F 1.68 3.77

Lys 82 A B 1.36 -0.66 F 1.42 1.79

Gin 83 A B 1.36 -0.27 F 0.86 1.39

Tyr 84 A B 1.36 0.49 -0.45 2.15

Leu 85 B B 1.04 -0.01 0.45 1.87

Ser 86 B B 0.82 0.47 -0.45 1.55

Tyr 87 A B B 0.53 0.76 -0.60 0.82

Glu 88 A B B -0.06 0.76 -0.45 1.55

Thr 89 A B B 0.19 0.57 -0.45 1.17

Leu 90 A B B 0.66 0.59 -0.45 1.20

Tyr 91 B T 0.66 0.26 0.36 0.69

Ala 92 T = 1.01 0.64 0.52 0.64

Asn 93 T 0.70 0.16 F 1.38 1.52

Gly 94 T z 1.01 -0.04 F 2.24 1.40

Ser 95 . z 1.51 -0.80 F 2.60 2.39

Arg 96 . B z 1.76 -0.81 F 2.14 2.15

Thr 97 B B 1.49 -0.81 F 1.68 3.76

Glu 98 B B 1.14 -0.60 F 1.42 2.08

Thr 99 B B 0.60 -0.56 F 1.16 1.05

Gin 100 B B 0.66 0.13 F -0.15 0.51

Val 101 B B -0.34 0.40 -0.60 0.46

Gly 102 B B -0.84 1.09 -0.60 0.22

He 103 B B -1.14 1.29 -0.60 0.11

Tyr 104 B B -1.13 1.27 -0.60 0.19

He 105 B B -1.43 1.01 -0.60 0.26

Leu 106 B B -0.92 0.97 -0.35 0.50

Ser 107 B T -0.58 0.71 F 0.45 0.32

Ser 108 T C -0.03 -0.04 * F 1.80 0.75

Ser 109 T C -0.38 -0.30 F 2.05 0.90

Gly 110 T T 0.51 -0.49 F 2.50 0.68

Asp 111 A T 1.29 -0.47 F 1.85 0.88

Gly 112 A c 1.70 -0.36 F 1.40 0.89

Ala 113 A B 2.00 -0.74 F 1.40 1.77

Gin 114 A B 2.00 -1.17 1.34 1.77

His 115 A B 2.00 -0.79 F 1.58 2.39

Arg 1 16 A B 1.70 -0.79 F 1.92 2.34

Asp 1 17 T T 1.74 -0.90 F 3.06 1.81

Ser 118 T T 1.99 -0.91 F 3.40 1.79 Gly 119 T T 2.03 -0.99 ^> F 2.91 0.90

Ser 120 T T 1.77 -0.99 * F 3.02 1.08

Ser 121 C 1.77 -0.60 . F 2.58 1.08

Gly 122 T C 1.88 -0.99 F 2.74 2.14

Lys 123 T T 2.22 -1.41 . F 2.90 3.13

Ser 124 T C 2.68 -1.80 F 3.00 4.66

Arg 125 B T 2.98 -2.19 . F 2.50 9.23

Arg 126 B 2.39 -2.21 F 2.00 7.99

Lys 127 B 3 2.49 -1.53 F 1.50 4.18

Arg 128 B 1 B 2.10 -1.16 F 1.20 3.35

Gin 129 B B 2.16 -0.73 0.75 1.69

He 130 B 1 B 2.04 0.03 -0.15 1.32

Tyr 131 B B 1.63 0.03 -0.15 1.13

Gly 132 B 1.70 0.41 -0.40 0.87

Tyr 133 B 0.89 0.01 0.05 2.44

Asp 134 B T 0.59 0.11 F 0.40 1.35

Ser 135 B T 0.59 -0.26 F 1.00 1.83

Arg 136 B T 0.13 0.00 F 0.25 0.82

Phe 137 B T 0.13 0.03 0.10 0.42

Ser 138 B B 0.42 0.46 -0.47 0.31

He 139 B B 0.42 0.07 -0.04 0.32

Phe 140 B B 0.02 0.07 0.09 0.62

Gly 141 T T -0.90 0.07 1.02 0.40

Lys 142 T T -1.01 0.37 F 1.300.47

Asp 143 T T -0.71 0.37 F 1.17 0.45

Phe 144 B T -0.07 -0.01 1.09 0.73

Leu 145 B 0.42 0.31 0.160.57

Leu 146 B 0.07 0.74 -0.27 0.53

Asn 147 B -0.28 1.53 -0.40 0.53

Tyr 148 T C -0.59 1.13 0.000.85

Pro 149 T T -0.19 0.93 0.35 1.50

Phe 150 T T -0.23 0.63 0.35 1.25

Ser 151 B T 0.62 0.87 F -0.05 0.59

Thr 152 B B -0.19 0.11 F -0.15 0.76

Ser 153 B B -0.24 0.37 * F -0.15 0.73

Val 154 B B -0.34 -0.03 F 0.45 0.73

Lys 155 B B 0.01 0.07 F -0.15 0.73

Leu 156 B B -0.36 0.01 * F -0.15 0.54

Ser 157 B T -0.36 0.20 F 0.25 0.39

Thr 158 B T -0.40 0.04 F 0.25 0.28

Gly 159 T T 0.14 0.47 F 0.35 0.34 Cys 160 T T -0.71 0.27 1 F 0.65 0.36

Thr 161 . . B -0.76 0.57 F -0.25 0.21

Gly 162 B -1.04 0.73 F -0.25 0.16

Thr 163 . A B -0.73 0.80 F -0.45 0.29

Leu 164 . A ] B -0.34 0.23 -0.30 0.35

Val 165 . A B 0.29 -0.26 0.30 0.71

Ala 166 . A B -0.26 -0.19 0.30 0.67

Glu 167 A A -0.72 -0.03 0.30 0.60

Lys 168 A A -0.72 -0.03 . 0.30 0.67

His 169 A A -0.50 -0.19 0.30 0.95

Val 170 A A -0.23 -0.19 0.30 0.56

Leu 171 A A 0.32 0.31 -0.30 0.28

Thr 172 A A -0.34 0.81 fc -0.60 0.28

Ala 173 A A -1.28 0.89 fc -0.60 0.20

Ala 174 A A -1.28 0.93 -0.60 0.17

His 175 i A B -0.42 0.74 -0.32 0.16

Cys 176 . A B 0.04 0.26 0.260.27

He 177 i A B 0.40 0.19 fc 0.54 0.26

His 178 T T 0.68 -0.31 fc 2.22 0.39

Asp 179 T T 1.02 -0.33 F 2.80 1.04

Gly 180 T T 0.20 -0.14 * si p 2.522.33

Lys 181 T T 0.91 -0.19 fc sfc p 2.24 1.27

Thr 182 B B 1.46 -0.69 * sfc 1.46 1.52

Tyr 183 B B 1.18 -0.26 * sfc 0.88 1.52

Val 184 B B 1.18 -0.20 * sfc 0.60 1.10

Lys 185 B B 1.57 0.20 * sfc p 0.12 1.32

Gly 186 B 0.71 -0.29 * sfc p 1.04 1.68

Thr 187 B 1.13 -0.36 sfc p 1.16 1.87

Gin 188 B 0.52 -1.00 k sfc p 1.58 1.83

Lys 189 B B 1.03 -0.36 1.20 1.37

Leu 190 B B 0.29 -0.36 * sfc 0.93 0.94

Arg 191 B B -0.18 -0.06 0.66 0.47

Val 192 B B 0.18 0.23 -0.06 0.19

Gly 193 B B -0.03 0.23 -0.18 0.47

Phe 194 B B -0.03 -0.03 0.64 0.37

Leu 195 B B 0.08 -0.03 1.13 1.00

Lys 196 B T 0.01 0.11 . * F 1.27 0.88

Pro 197 B T 0.87 -0.31 sfc sfc p 2.362.02

Lys 198 T T 0.87 -1.10 sfc sfc p 3.40 4.10

Phe 199 B T 1.22 -1.36 sfc sfc p 2.66 2.03

Lys 200 B 2.14 -0.93 fc * F 2.12 1.30 Asp 201 T T 1.76 -1.36 fc F 2.38 1.27

Gly 202 T T 1.38 -0.93 fc F 2.04 1.45

Gly 203 T T 1.33 -1.21 * * F 1.85 0.73

Arg 204 T C 2.03 -0.81 * fc F 1.95 0.71

Gly 205 c 1.69 -0.81 F 2.20 1.19

Ala 206 c 1.38 -0.86 F 2.50 1.62

Asn 207 T c 1.42 -0.80 F 3.00 1.19

Asp 208 T c 1.18 -0.41 F 2.40 1.61

Ser 209 T c 0.47 -0.34 F 2.10 1.61

Thr 210 T c 0.60 -0.23 F 1.65 0.99

Ser 211 c 1.19 -0.20 F 1.15 0.92

Ala 212 A c 1.19 -0.20 F 0.80 1.19

Met 213 A B 0.59 -0.19 0.45 1.42

Pro 214 A c 0.93 -0.06 * F 0.80 1.05

Glu 215 A A 0.54 -0.44 F 0.602.08

Gin 216 A A 0.84 -0.16 F 0.60 1.82

Met 217 A A 1.14 -0.37 * F 0.60 2.04

Lys 218 A 1 B 0.86 0.11 -0.15 1.24

Phe 219 A . B 1.18 0.80 -0.60 0.50

Gin 220 B B 0.32 0.40 -0.60 0.99

Trp 221 B B 0.37 0.43 * -0.60 0.37

He 222 B B 1.08 0.43 -0.37 0.85

Arg 223 B B 0.72 -0.36 0.76 0.96

Val 224 B B 1.39 -0.27 1.14 1.32

Lys 225 B r 0.53 -0.69 F 2.22 2.56

Arg 226 B Γ 0.61 -0.73 F 2.30 0.97

Thr 227 B Γ 1.54 -0.30 * F 1.92 2.02

His 228 B c 1.09 -0.94 F 1.79 2.02

Val 229 B . c 1.66 -0.51 F 1.56 1.02

Pro 230 B T 0.72 0.40 F 0.18 0.74

Lys 231 . T T 0.66 0.60 F 0.35 0.38

Gly 232 . T T 0.62 0.10 F 0.80 1.03

Trp 233 B T 0.66 -0.11 F 0.85 0.66

He 234 B 0.92 -0.14 F 0.92 0.53

Lys 235 B 1.13 0.36 F 0.59 0.54

Gly 236 c 1.09 0.33 F 1.060.83

Asn 237 T c 0.54 -0.59 F 2.58 1.98

Ala 238 T c 0.49 -0.59 F 2.70 0.69

Asn 239 T c 0.78 -0.16 F 2.13 0.69

Asp 240 T T 0.73 0.03 F 1.46 0.43

He 241 B 0.83 -0.37 1.04 0.71 Gly 242 . B 0.83 -0.11 * 0.77 0.69

Met 243 . B 1.18 -0.51 0.80 0.69

Asp 244 . B T 0.59 0.24 * 0.25 1.54

Tyr 245 . B T -0.22 0.06 * 0.25 1.57

Asp 246 . B T -0.14 0.31 * 0.25 1.31

Tyr 247 . B T 0.20 0.39 0.10 0.65

Ala 248 A A -0.01 0.39 fc -0.30 0.71

Leu 249 A A 0.03 0.31 -0.30 0.35

Leu 250 A A 0.32 0.31 -0.30 0.45

Glu 251 A A 0.11 -0.44 0.30 0.89

Leu 252 A A 0.32 -0.51 F 0.90 1.67

Lys 253 A A 0.96 -0.70 F 0.902.76

Lys 254 A T 1.88 -1.39 F 1.30 3.18

Pro 255 A T 2.73 -1.39 F 1.30 7.56

His 256 A T 2.03 -2.07 F 1.30 7.56

Lys 257 A T 2.24 -1.29 F 1.30 3.27

Arg 258 A 2.24 -0.67 F 1.10 2.09

Lys 259 B 1.31 -1.10 F 1.10 3.08

Phe 260 B B 1.18 -0.91 0.75 1.08

Met 261 B B 0.36 -0.49 fc 0.30 0.55

Lys 262 B B 0.01 0.16 fc -0.30 0.20

He 263 B B -0.31 0.54 * -0.60 0.31

Gly 264 B B -0.57 0.19 * -0.02 0.49

Val 265 B . C -0.46 0.00 F 0.61 0.38

Ser 266 B . C 0.19 0.50 * F 0.59 0.55

Pro 267 T C 0.14 -0.19 F 2.32 1.10

Pro 268 r T 0.22 -0.21 F 2.80 2.57

Ala 269 T T 0.36 -0.17 F 2.52 1.58

Lys 270 B T 0.87 -0.13 F 1.84 1.58

Gin 271 B 0.82 -0.13 F 1.36 1.01

Leu 272 B T 1.14 -0.13 F 1.13 0.99

Pro 273 B T 0.47 -0.63 F 1.15 0.97 .

Gly 274 T T 1.02 0.06 F 0.65 0.39

Gly 275 B T 0.28 0.16 F 0.25 0.65

Arg 276 B B -0.02 0.26 F -0.15 0.36

He 277 B B 0.44 0.21 -0.14 0.49

His 278 B B 0.41 0.21 0.02 0.49

Phe 279 B T 0.76 0.54 0.28 0.39

Ser 280 B T 1.10 0.54 0.44 0.94

Gly 281 T T 0.99 0.26 F 1.60 1.11

Tyr 282 T T 1.99 -0.24 F 2.04 2.14 Asp 283 T 1.81 -1.03 F 2.32 3.12

Asn 284 T 2.17 -0.99 F 2.50 4.88

Asp 285 . T 2.47 -0.99 F 2.68 3.08

Arg 286 r < : 2.oo -1.34 ' k F 2.86 2.97

Pro 287 T r 1.39 -0.66 fc F 3.40 1.52

Gly 288 T r 1.14 -0.41 fc F 2.61 0.68

Asn 289 B Γ 1.26 0.34 fc * F 1.27 0.54

Leu 290 B 8 0.56 0.34 * * 0.38 0.69

Val 291 B B -0.22 0.70 * . -0.26 0.60

Tyr 292 B B -0.01 0.84 * * -0.60 0.20

Arg 293 A B B -0.52 0.44 * * -0.60 0.40

Phe 294 A B B -0.48 0.40 * . -0.60 0.40

Cys 295 A B B 0.33 -0.24 ' fc 0.30 0.52

Asp 296 A B B 1.19 -1.00 ' fc 0.600.44

Val 297 A B 1.12 -1.00 ' fc 0.60 0.88

Lys 298 A T 0.77 -1.30 ' fc F 1.30 2.37

Asp 299 A T 1.47 -1.11 ' fc F 1.30 2.23

Glu 300 A B T 1.32 -1.11 ' k F 1.30 5.01

Thr 301 A . B T 0.51 -1.07 ' k F 1.30 2.06

Tyr 302 A . B T 1.12 -0.39 ' fc 0.85 1.02

Asp 303 A B B 1.08 0.37 -0.30 0.92

Leu 304 A B B 1.08 0.77 fc -0.45 1.11

Leu 305 A B B 0.41 0.69 fc -0.45 1.22

Tyr 306 A B B 0.72 0.50 fc -0.60 0.39

Gin 307 A B B 0.67 0.50 -0.60 0.80

Gin 308 A B B 0.67 0.20 F 0.00 1.29

Cys 309 A B B 1.27 -0.09 F 0.60 1.43

Asp 310 B T 1.73 -0.41 F 1.25 1.28

Ser 311 B 1.39 -0.39 F 1.15 0.73

Gin 312 B T 1.09 -0.29 F 1.75 1.37

Pro ^■313 T C 0.74 -0.47 F 2.20 1.10

Gly 314 T T 1.11 -0.04 F 2.50 0.81

Ala 315 T T C 0.77 -0.04 F 2.25 0.63

Ser 316 T C 0.21 -0.01 F 1.80 0.40

Gly 317 T C -0.03 0.20 F 0.95 0.30

Ser 318 B τ -0.68 0.53 sfc F 0.20 0.47

Gly 319 B T -0.22 0.67 F -0.05 0.26

Val 320 B B -0.23 0.29 -0.30 0.51

Tyr 321 B B -0.22 0.47 -0.60 0.38

Val 322 B B 0.17 1.00 -0.60 0.40

Arg 323 B B 0.58 0.57 -0.45 1.09 Met 324 B B 0.92 -0.07 * 0.45 1.36

Trp 325 B B 1.74 -0.43 » 0.45 3.17

Lys 326 B B 1.99 -0.57 fc * 0.75 2.20

Arg 327 A r 2.89 -0.17 F 1.00 3.85

Gin 328 A C 2.49 -0.79 F 1.10 7.33

His 329 A c 3.09 -0.79 F 1.10 3.85

Gin 330 A c 3.49 -0.79 F 1.10 3.41

Lys 331 A Γ 3.49 -0.79 fc F 1.30 3.85

Trp 332 A Γ 2.49 -1.19 fc * F 1.30 5.66

Glu 333 . . A c 1.60 -1.00 fc . F 1.10 2.29

Arg 334 A B B 1.29 -0.71 fc . F 0.75 0.80

Lys 335 A B B 0.69 -0.29 0.30 0.76

He 336 A B B -0.24 -0.59 fc 0.60 0.43

He 337 A B B -0.26 0.10 fc -0.30 0.15

Gly 338 A B B -0.60 0.49 -0.60 0.10

Met 339 B B -0.74 0.91 fc -0.60 0.15

He 340 B B -0.79 0.73 -0.60 0.28

Ser 341 r c -0.19 0.44 0.00 0.50

Gly 342 Γ c -0.16 0.93 0.00 0.53

His 343 Γ c 0.19 0.96 0.00 0.56

Gin 344 B Γ 0.19 0.27 0.10 0.70

Trp 345 B 1.08 0.50 -0.40 0.70

Val 346 B 1.03 0.07 0.20 0.86

Asp 347 B Γ 1.08 0.00 0.70 0.49

Met 348 T T 0.90 -0.01 F 2.15 0.62

Asp 349 T T 0.90 -0.50 F 2.60 1.30

Gly 350 T c 1.19 -0.74 F 3.00 1.35

Ser 351 T c 1.34 -0.74 F 2.70 2.35

Pro 352 T c 1.03 -0.57 F 2.40 1.22

Gin 353 T T 1.74 -0.09 F 2.25 1.78

Glu 354 B T 1.40 -0.51 F 2.10 2.60

Phe 355 B 1.08 -0.47 F 1.55 1.67

Thr 356 T T 1.08 -0.33 F 2.25 0.52

Arg 357 T T 1.29 -0.34 F 2.50 0.40

Gly 358 T T 0.40 -0.34 F 2.25 0.80

Cys 359 T T 0.09 -0.44 F 2.00 0.39

Ser 360 B c 0.58 -0.44 * F 1.15 0.29

Glu 361 B T 0.08 -0.01 F 1.10 0.45

He 362 B B -0.03 0.24 F -0.15 0.69

Thr 363 B B 0.07 0.07 F -0.15 0.89

Pro 364 B B -0.16 0.44 F -0.45 0.80 Leu 365 B B -0.07 1.13 -0.60 0.80

Gin 366 B B -0.07 0.87 * -0.60 0.86

Tyr 367 B B -0.07 0.39 -0.30 0.93

He 368 B B -0.06 0.64 -0.60 0.79

Pro 369 B B -0.73 0.34 -0.30 0.61

Asp 370 B B -0.27 0.63 F -0.45 0.27

He 371 B B -1.12 0.30 F -0.15 0.39

Ser 372 B B -1.27 0.26 -0.30 0.19

He 373 B B -0.77 0.26 -0.30 0.14

Gly 374 B B -0.94 0.69 -0.60 0.26

Val 375 B B -1.33 0.43 -0.60 0.25

Ter 376 B B -0.83 0.47 -0.60 0.45

Table X

Res Position ii in iv v vi VII vm ix : K XI XII XIII XIV

Met 1 B B . . . -1.26 0.43 . . -0.60 0.37

Ala 2 B B -1.68 0.64 -0.60 0.22

Ser 3 B B -1.50 0.90 -0.60 0.14

Val 4 B B -1.41 0.90 -0.60 0.22

Val 5 B B -1.37 0.67 -0.60 0.29

Leu 6 B T -1.07 0.60 F -0.05 0.21

Pro 7 T T -0.48 0.60 F 0.35 0.39

Ser 8 T T -0.84 0.36 F 0.65 0.90

Gly 9 T T -0.58 0.29 F 0.65 0.58

Ser 10 . T < : -0.31 0.10 F 0.45 0.38

Gin 11 A T -0.09 0.17 F 0.25 0.29

Cys 12 A T -0.47 0.29 0.10 0.29

Ala 13 A T -0.76 0.36 0.10 0.22

Ala 14 A A -1.00 0.47 -0.60 0.13

Ala 15 A A -1.29 0.57 -0.60 0.24

Ala 16 A A -1.88 0.50 -0.60 0.24

Ala 17 A A -1.42 0.50 -0.60 0.24

Ala 18 A A -1.04 0.43 -0.60 0.37

Ala 19 A A -0.80 0.36 -0.30 0.57

Ala 20 A A -1.02 0.29 * * -0.30 0.56

Pro 21 A T -0.32 0.47 * * F -0.05 0.46

Pro 22 A T -0.54 -0.03 . * F 0.85 0.89

Gly 23 A T 0.16 0.16 * F 0.25 0.72

Leu 24 A T -0.07 -0.34 0.70 0.92

Arg 25 A B -0.29 -0.09 * 0.30 0.49

Leu 26 A B -0.89 0.17 -0.30 0.41

Arg 27 A B -1.49 0.43 -0.60 0.41

Leu 28 A B . -1.96 0.43 -0.60 0.17

Leu 29 A B -1.84 1.11 -0.60 0.17

Leu 30 A B -2.26 1.21 -0.60 0.08

Leu 31 A A . -2.03 1.60 * * -0.60 0.12

Leu 32 A A -2.73 1.41 * * -0.60 0.15

Phe 33 A A -2.51 1.23 . . -0.60 0.18

Ser 34 A A -2.51 1.04 . . -0.60 0.23

Ala 35 A A -2.59 1.04 . . -0.60 0.23

Ala 36 A A -1.99 1.04 -0.60 0.18 Ala 37 A A . -1.49 0.69 -0.60 0.21

Leu 38 A B -1.13 0.79 -0.60 0.30

He 39 A B -0.83 0.71 -0.32 0.30

Pro 40 A B -0.59 0.21 F 0.41 0.49

Thr 41 T 0.00 0.14 F 1.29 0.59

Gly 42 T T 0.59 -0.14 F 2.52 1.45

Asp 43 T T 0.59 -0.43 F 2.80 1.51

Gly 44 T ( 0.78 -0.17 F 2.17 0.86

Gin 45 1 3 T 0.68 0.13 F 1.09 0.75

Asn 46 1 3 B 1.03 0.19 F 0.41 0.65

Leu 47 1 3 B 1.38 0.19 F 0.28 1.32

Phe 48 1 B B 0.52 -0.24 F 0.60 1.27

Thr 49 1 B B 0.56 0.00 F -0.15 0.59

Lys 50 1 3 B -0.30 0.09 F 0.00 1.02

Asp 51 . B B -1.19 0.04 F -0.15 0.88

Val 52 1 B B -0.38 -0.06 0.30 0.43

Thr 53 . B B -0.02 -0.54 0.60 0.37

Val 54 . B B 0.29 -0.1 1 0.30 0.22

He 55 ^' A B -0.61 -0.11 0.30 0.51

Glu 56 A B -1.20 -0.11 F 0.45 0.26

Gly 57 A -0.66 -0.10 F 0.65 0.36

Glu 58 A B -1.23 -0.26 F 0.45 0.74

Val 59 A B -0.68 -0.26 0.300.30

Ala 60 A B -0.46 0.13 fc -0.30 0.40

Thr 61 A B -0.46 0.27 * -0.30 0.12

Ile 62 A B -0.97 0.67 * -0.60 0.29

Ser 63 A B -0.97 0.67 -0.60 0.21

Cys 64 . B B -0.07 0.57 * -0.60 0.24

Gin 65 . B B 0.22 0.09 0.040.68

Val 66 . B B 0.53 -0.21 0.98 0.68

Asn 67 B T 1.42 -0.60 * F 2.32 2.12

Lys 68 B T 1.42 -1.17 * F 2.662.05

Ser 69 T T 1.23 -1.19 F 3.40 3.69

Asp 70 T T 0.34 -1.19 F 3.06 1.70

Asp 71 B T 1.20 -0.90 * F 2.17 0.60

Ser 72 B T 0.39 -0.50 * 1.38 0.77

Val 73 B B -0.47 -0.20 0.64 0.38

He 74 B B -0.17 0.49 -0.60 0.19

Gin 75 B B -0.38 0.89 -0.60 0.23

Leu 76 B B -0.38 0.93 * -0.60 0.47

Leu 77 B B 0.03 0.69 -0.17 1.08 Asn 78 T ( 2 0.89 0.00 . F 1.16 1.22

Pro 79 T < Z 1.47 0.00 F 1.44 2.56

Asn 80 T T 0.58 -0.20 F 2.52 4.49

Arg 81 T T 1.14 -0.20 * F 2.80 1.96

Gin 82 B 1 B 1.26 0.16 F 1.12 1.98

Thr 83 B B 1.37 0.51 F 0.54 1.07

He 84 B 1 3 1.58 0.11 0.41 1.07

Tyr 85 B ] B 0.88 0.11 0.13 1.03

Phe 86 B 1 3 0.88 0.50 * -0.60 0.62

Arg 87 B ] B 0.67 0.01 -0.15 1.73

Asp 88 B 1 B 0.17 -0.24 F 0.94 1.70

Phe 89 B 1.10 -0.31 F 1.48 1.62

Arg 90 ( Z 1.34 -1.10 F 2.32 1.66

Pro 91 ( Z 1.74 -1.10 F 2.66 1.66

Leu 92 T T 1.74 -0.71 F 3.40 2.56

Lys 93 T r 1.04 -1.50 F 3.062.56

Asp 94 T Γ 1.74 -0.71 F 2.72 1.44

Ser 95 A . Γ 0.82 -0.74 * F 1.98 3.01

Arg 96 A B 0.22 -0.74 F 1.24 1.24

Phe 97 A B 1.03 -0.06 . 0.30 0.61

Gin 98 A B 0.29 0.34 -0.30 0.74

Leu 99 A B -0.01 0.74 -0.60 0.33

Leu 100 A B -0.01 1.13 -0.60 0.50

Asn 101 A C -0.42 0.73 -0.40 0.39

Phe 102 A C 0.28 0.71 F -0.25 0.63

Ser 103 . T C -0.53 0.03 F 0.60 1.33

Ser 104 A . T 0.32 0.03 F 0.25 0.68

Ser 105 A . T 0.28 -0.37 F 1.00 1.58

Glu 106 A . T -0.02 -0.51 F 1.15 0.87

Leu 107 A B -0.13 -0.51 F 0.75 0.87

Lys 108 A B -0.14 -0.21 F 0.45 0.54

Val 109 A B 0.16 -0.11 0.300.45

Ser 110 B B -0.40 0.29 -0.30 0.87

Leu 111 B B -0.70 0.24 -0.30 0.32

Thr 112 B B . -0.78 0.63 -0.60 0.58

Asn 113 B B -1.12 0.67 -0.60 0.31

Val 114 B B -0.27 0.67 -0.60 0.50

Ser 115 B B 0.03 -0.01 0.64 0.58

He 116 B B 0.50 -0.50 F 1.13 0.62

Ser 117 B T 0.92 -0.47 F 1.87 0.83

Asp 118 T T 0.68 -1.11 F 3.06 1.21 Glu 119 T T 0.83 -0.74 * F 3.40 2.70

Gly 120 T T 0.47 -0.64 * F 3.06 1.74

Arg 121 1 B T 1.36 -0.46 * F 1.87 0.56

Tyr 122 1 B T 0.84 -0.06 * 1.38 0.56

Phe 123 B 1 3 0.60 0.63 * -0.26 0.47

Cys 124 B B 0.29 0.96 * -0.60 0.37

Gin 125 B 1 3 0.63 1.44 * -0.60 0.34

Leu 126 B ) B 0.31 0.69 * -0.60 0.66

Tyr 127 T 0.34 0.33 0.79 1.91

Thr 128 T 1.04 0.19 F 1.28 1.71

Asp 129 T ( Z 1.71 0.19 F 1.62 3.58

Pro 130 T < Z 1.41 -0.50 * F 2.86 3.96

Pro 131 T T 1.98 -0.87 F 3.40 3.68

Gin 132 T T 1.91 -0.60 F 3.06 3.45

Glu 133 B ] B 1.91 -0.11 F 1.62 3.22

Ser 134 B 1 3 1.02 -0.06 F 1.28 3.01

Tyr 135 B B 0.92 0.20 F 0.34 1.22

Thr 136 B B 0.28 0.29 F 0.00 1.01

Thr 137 B B -0.53 0.93 F -0.45 0.56

He 138 B B -1.39 1.23 -0.60 0.30

Thr 139 B B -1.30 1.11 -0.60 0.15

Val 140 B B -1.27 1.06 * -0.60 0.16

Leu 141 B B -0.84 1.00 * -0.60 0.36

Val 142 B B -0.53 0.31 * -0.30 0.49

Pro 143 B T -0.46 0.23 * F 0.40 1.06

Pro 144 r T -0.74 0.27 F 0.80 1.06

Arg 145 Γ T -0.78 0.20 F 0.80 1.41

Asn 146 A T 0.03 0.24 0.10 0.64

Leu 147 A B 0.00 -0.19 0.30 0.69

Met 148 A B 0.21 0.07 -0.30 0.25

He 149 A B 0.47 0.47 -0.60 0.27

Asp 150 A B 0.36 0.07 -0.30 0.64

He 151 A A 0.04 -0.61 0.75 1.09

Gin 152 A T 0.27 -0.74 F 1.302.24

Lys 153 A T 0.01 -0.93 F 1.30 1.36

Asp 154 A T 0.90 -0.29 F 1.00 1.43

Thr 155 A T 0.56 -0.97 F 1.30 1.43

Ala 156 A A 1.44 -0.94 F 0.75 0.71

Val 157 A A 1.44 -0.94 F 0.75 0.74

Glu 158 A A 0.51 -0.94 F 0.75 0.88

Gly 159 A A 0.51 -0.74 * F 0.75 0.61 Glu 160 A A -0.03 -1.24 F 0.90 1.43

Glu 161 A A . 0.56 -1.24 F O.75 0.61

He 162 A A 0.74 -0.84 F 0.75 1.00

Glu 163 A A 0.43 -0.70 * 0.60 0.31

Val 164 A A 0.19 -0.21 * 0.30 0.26

Asn 165 A A -0.41 0.29 -0.30 0.37

Cys 166 A A -1.00 0.21 -0.30 0.21

Thr 167 A A -0.41 0.71 * -0.60 0.29

Ala 168 A A -0.37 0.46 -0.60 0.24

Met 169 A 0.28 0.06 -0.10 0.90

Ala 170 A -0.31 -0.09 fc 0.50 0.96

Ser 171 A 0.04 -0.07 F 0.65 0.96

Lys 172 A 0.04 -0.09 fc F 0.80 1.40

Pro 173 A B -0.26 -0.21 F 0.60 2.00

Ala 174 A B 0.46 -0.03 F 0.60 1.05

Thr 175 B B 0.76 -0.41 F 0.60 1.02

Thr 176 B B 0.36 0.50 F -0.45 0.70

He 177 B B 0.36 0.86 -0.60 0.60

Arg 178 B B 0.22 0.36 -0.30 0.83

Trp 179 B B 0.81 0.30 * -0.30 0.57

Phe 180 T " 0.81 0.21 * * 0.45 1.30

Lys 181 T C 1.12 0.01 F 0.45 0.96

Gly 182 T C 1.20 0.01 F 0.60 1.58

Asn 183 T C 1.13 -0.21 F 1.20 1.50

Thr 184 A . C 1.08 -1.00 F 1.40 1.50

Glu 185 A A 1.82 -0.57 F 1.50 1.50

Leu 186 A A 1.48 -1.00 F 1.80 1.87

Lys 187 A A 1.82 -1.01 F 2.10 1.74

Gly 188 T C 0.97 -1.50 F 3.00 1.74

Lys 189 T C 1.28 -0.86 F 2.70 1.56

Ser 190 A T 1.28 -1.54 F 2.20 1.35

Glu 191 A T 1.80 -1.54 F 1.90 2.37

Val 192 A 1.46 -1.06 F 1.40 1.25

Glu 193 A 1.80 -0.67 F 1.10 1.25

Glu 194 A 1.16 -1.06 F 1.10 1.20

Trp 195 A T 1.21 -0.44 F 1.00 1.60

Ser 196 A T 0.90 -0.33 0.85 1.45

Asp 197 A T 0.90 0.16 0.25 1.21

Met 198 A T 0.59 0.80 -0.20 0.85

Tyr 199 A B . 0.29 0.37 -0.30 0.92

Thr 200 A B 0.58 0.37 -0.30 0.74 Val 201 A B 0.07 0.77 -0.45 1.29

Thr 202 A B -0.53 0.84 F -0.45 0.68

Ser 203 A A B -0.74 0.70 * F -0.45 0.47

Gin 204 A A B -0.46 0.90 * F -0.45 0.52

Leu 205 A A B -1.00 0.26 -0.30 0.72

Met 206 A A B -0.18 0.41 * -0.60 0.40

Leu 207 A A B 0.18 0.53 -0.60 0.31

Lys 208 A A B 0.48 0.13 -0.30 0.76

Val 209 A A 0.48 -0.56 1.09 1.32

His 210 A A 1.29 -1.17 * F 1.58 2.68

Lys 211 A A 1.54 -1.86 F 1.922.24

Glu 212 A T 1.50 -1.43 * F 2.66 2.98

Asp 213 T T 1.24 -1.43 F 3.40 1.63

Asp 214 T T 1.24 -1.50 * F 3.06 1.26

Gly 215 T T 0.39 -0.86 F 2.57 0.54

Val 216 B B -0.32 -0.17 0.98 0.23

Pro 217 B B -0.32 0.40 -0.26 0.07

Val 218 B B -1.18 0.80 -0.60 0.13

He 219 B B -1.18 1.01 -0.60 0.13

Cys 220 B B -0.87 0.37 -0.30 0.14

Gin 221 B B -0.22 0.44 -0.60 0.26

Val 222 B B -0.60 0.23 -0.30 0.58

Glu 223 B B -0.60 0.04 -0.15 1.08

His 224 B -0.02 0.11 -0.10 0.46

Pro 225 B 0.30 0.20 -0.10 0.90

Ala 226 T 0.30 -0.01 0.90 0.52

Val 227 T T C 0.34 0.39 0.50 0.61

Thr 228 T C 0.34 0.57 * F 0.15 0.33

Gly 229 T C 0.07 0.54 F 0.15 0.56

Asn 230 T C 0.28 0.53 * F 0.30 1.09

Leu 231 B B 0.98 0.29 . F 0.00 1.30

Gin 232 B B 1.59 -0.20 F 0.60 2.58

Thr 233 B B 1.09 0.13 . F 0.00 2.51

Gin 234 B B 1.43 0.41 . F -0.30 2.51

Arg 235 B B 0.58 -0.27 F 0.60 2.51

Tyr 236 B B 1.39 -0.03 • 0.45 1.29

Leu 237 B B 1.14 -0.11 0.45 1.29

Glu 238 B B 1.50 0.24 -0.15 1.03

Val 239 B B 1.29 0.24 -0.15 1.32

Gin 240 B T 1.18 -0.09 0.85 2.47

Tyr 241 B T 0.57 -0.37 0.85 2.47 Lys 242 B C 1.34 0.27 * F 0.20 2.47

Pro 243 A B 0.46 0.13 * F 0.00 1.94

Gin 244 B B 1.31 0.41 -0.60 0.87

Val 245 B B 0.71 0.06 -0.30 0.75

His 246 B B 0.64 0.67 -0.60 0.48

He 247 B B 0.36 0.73 -0.60 0.40

Gin 248 B B 0.36 1.09 -0.60 0.85

Met 249 B B -0.46 0.87 -0.60 0.96

Thr 250 B B 0.40 1.06 -0.45 1.13

Tyr 251 B T 0.09 0.77 -0.05 1.13

Pro 252 B T 0.17 0.80 -0.05 1.13

Leu 253 r T -0.14 0.87 0.20 0.65

Gin 254 B T 0.57 0.87 0.06 0.60

Gly 255 B 0.88 0.11 * . F 0.57 0.76

Leu 256 B 0.78 -0.31 * . F 1.58 1.59

Thr 257 B T 0.99 -0.57 * . F 2.19 0.91

Arg 258 B T 1.21 -0.97 * . 1 ~ 2.60 1.53

Glu 259 A T 0.40 -0.90 * . F 2.34 1.87

Gly 260 A T 0.74 -0.90 * . F 2.08 1.07

Asp 261 A A 0.74 -1.39 * . 1 F 1.27 0.95

Ala 262 A A 0.74 -0.70 * . F 1.01 0.45

Leu 263 A A -0.03 -0.21 0.30 0.66

Glu 264 A A -0.03 -0.07 0.30 0.21

Leu 265 A A -0.28 -0.07 0.300.36

Thr 266 A A -1.17 -0.07 0.30 0.44

Cys 267 A A -0.92 -0.07 0.30 0.18

Glu 268 A A -0.07 0.36 -0.10 0.22

Ala 269 A A -0.28 -0.33 0.70 0.30

He 270 A A 0.53 -0.39 0.900.86

Gly 271 A T 0.63 -0.56 . . F 1.95 0.86

Lys 272 C 0.44 -0.13 F 2.00 1.32

Pro 273 C -0.16 0.01 F 1.20 1.39 .

Gin 274 B C -0.42 -0.06 F 1.40 1.39

Pro 275 B B 0.16 0.16 F 0.25 0.52

Val 276 B B 0.21 0.64 -0.40 0.48

Met 277 . B B -0.69 1.13 -0.60 0.29

Val 278 B B . -0.37 1.37 -0.60 0.14

Thr 279 B B -1.22 0.94 -0.60 0.37

Trp 280 B B -1.01 0.94 -0.60 0.28

Val 281 B B -0.16 0.33 -0.30 0.63

Arg 282 B B 0.44 -0.31 0.30 0.73 Val 283 A I 3 0.70 -0.80 * 0.75 1.19

Asp 284 A 1 3 0.80 -1.10 * F 0.90 1.59

Asp 285 . A T 1.09 -1.31 * F 1.30 1.26

Glu 286 A A 1.91 -0.91 * F 0.90 2.93

Met 287 A A 1.21 -1.06 * F 0.90 2.39

Pro 288 A A 1.21 -0.56 F 0.90 1.45

Gin 289 A A 1 B 0.40 0.09 -0.30 0.62

His 290 A A 1 3 0.10 0.77 -0.60 0.52

Ala 291 A B 1 3 -0.24 0.54 -0.60 0.45

Val 292 A B I 3 0.14 0.54 -0.60 0.26

Leu 293 A B 1 3 0.36 0.57 -0.60 0.29

Ser 294 A 1 B C -0.46 0.47 F -0.25 0.46

Gly 295 T < Z -1.12 0.66 F 0.15 0.51

Pro 296 T < Z -1.42 0.80 F 0.15 0.54

Asn 297 T < Z -0.57 0.80 F 0.15 0.28

Leu 298 B T 0.24 0.81 -0.20 0.46

Phe 299 B -0.27 0.79 -0.40 0.48

He 300 B 0.08 1.04 -0.40 0.24

Asn 301 B 0.33 1.04 -0.40 0.48

Asn 302 B 0.02 0.36 0.39 1.10

Leu 303 . Z 0.83 0.06 F 1.08 2.27

Asn 304 T 1.53 -0.63 F 2.52 2.36

Lys 305 T 2.08 -0.63 F 2.86 2.36

Thr 306 T T 1.77 -0.60 * F 3.40 2.83

Asp 307 T T 1.52 -0.80 * F 3.06 2.54

Asn 308 T T 2.44 -0.44 F 2.42 1.99

Gly 309 T T 1.78 -0.44 * F 2.08 2.70

Thr 310 B 1.73 -0.36 F 0.99 0.87

Tyr 311 B 1.46 -0.36 * 0.50 0.93

Arg 312 B 1.16 -0.26 0.50 0.95

Cys 313 B 1.16 -0.30 0.50 0.88

Glu 314 B 0.61 -0.39 0.50 0.91

Ala 315 A T 0.07 -0.46 0.70 0.32

Ser 316 A T -0.03 0.19 0.10 0.45

Asn 317 A T -0.10 0.04 0.10 0.26 lie 318 A T -0.02 0.04 0.10 0.51

Val 319 A -0.06 0.04 0.10 0.38

Gly 320 A 0.23 0.16 0.30 0.32

Lys 321 A 0.53 0.14 0.50 0.62

Ala 322 A 0.29 -0.54 F 1.90 1.40

His 323 B T 0.58 -0.43 F 2.00 2.21 Ser 324 B T 0.62 -0.24 . * 1.65 1.09

Asp 325 B r 0.72 0.44 * 0.40 0.89

Tyr 326 B r -0.18 0.70 0.35 1.03

Met 327 B B 0.17 0.84 -0.40 0.57

Leu 328 B B 0.20 1.21 -0.60 0.53

Tyr 329 B B 0.29 1.21 -0.60 0.57

Val 330 B ] B 0.08 0.89 -0.60 0.89

Tyr 331 B 0.01 0.70 -0.25 1.67

Asp 332 B 0.30 0.50 * F -0.10 1.54

Pro 333 B Γ 0.22 0.23 F 0.40 2.99

Pro 334 T Γ 0.26 0.27 * F 0.80 1.34

Thr 335 T Γ 0.90 -0.06 * F 1.40 1.24

Thr 336 B Γ 0.93 0.37 F 0.40 1.24

He 337 B 0.62 0.37 F 0.32 1.24

Pro 338 B 0.52 0.43 F 0.14 1.24

Pro 339 Γ Z 0.42 0.43 F 0.66 1.24

Pro 340 Γ Z 0.42 0.43 F 0.78 2.55

Thr 341 Γ Z 0.42 0.23 F 1.202.38

Thr 342 B Γ 1.00 0.29 F 0.882.22

Thr 343 B B 0.90 0.34 F 0.362.07

Thr 344 B B 0.80 0.40 F -0.06 2.07

Thr 345 B B 0.70 0.40 F -0.18 2.07

Thr 346 B B 0.70 0.40 F -0.30 2.07

Thr 347 B B 0.70 0.40 F -0.30 2.07

Thr 348 B B 0.70 0.40 F -0.30 2.07

Thr 349 B B 0.70 0.40 F -0.30 2.07

Thr 350 B B 0.12 0.40 F -0.30 2.07

Thr 351 B B -0.38 0.60 F -0.30 1.01

Thr 352 B B . - -0.38 0.80 F -0.45 0.57

Thr 353 B B -0.96 0.80 * F -0.45 0.57

He 354 B B -1.53 1.00 * -0.60 0.28

Leu 355 B B -1.53 1.20 * -0.60 0.14

Thr 356 B B -1.22 1.20 * -0.60 0.14

He 357 B B -1.21 0.71 * -0.60 0.32

He 358 B B -0.79 0.41 -0.26 0.53

Thr 359 B B -0.49 -0.27 . * p 1.13 0.71

Asp 360 B T 0.43 -0.26 . s_fc p 2.02 1.03

Ser 361 T C 0.36 -0.94 * p 2.86 2.87

Arg 362 T T 0.86 -1.20 * F 3.402.54

Ala 363 T T 1.36 -1.26 . * 2.91 1.95

Arg 364 T 1.28 -0.83 . 2.37 1.86 Ter 365 . . . . T 0.89 -0.79 . * . 2.031.21

Table XI

Res Position I I III IV V VI II VIII IX X XI XII XIII XIV

Met 1 A 0.14 0.06 -0.10 0.89

Ser 2 A T -0.28 0.01 0.10 0.94

Ser 3 A T 0.16 0.27 0.10 0.60

Ser 4 A T 0.51 -0.16 0.85 1.22

Ser 5 A T 0.09 -0.27 F 1.00 1.24

Leu 6 A A -0.12 0.03 F -0.15 0.76

Lys 7 A A -0.49 0.33 F -0.15 0.47

His 8 A A -0.79 0.51 -0.60 0..9

Leu 9 A A -1.08 0.74 -0.60 0.23

Leu 10 A A -1.59 0.56 fc -0.60 0.11

Cys 11 A A -1.08 1.24 fc -0.60 0.07

Met 12 A A -1.41 1.13 -0.60 0.11

Ala 13 . A A -2.08 1.36 -0.60 0.14

Leu 14 t A A -1.57 1.46 -0.60 0.23

Ser 15 A . B -1.06 1.27 -0.60 0.31

Trp 16 A . B -1.09 1.04 -0.60 0.41

Phe 17 A . B -1.38 1.33 -0.60 0.43

Ser 18 . B c -1.09 1.33 -0.40 0.23

Ser 19 . B C -0.62 1.33 -0.40 0.29

Phe 20 . B c -0.32 0.84 -0.40 0.33

He 21 B c -0.34 0.06 -0.10 0.43

Ser 22 . B c 0.06 0.16 * F 0.05 0.46

Gly 23 c -0.34 0.16 . * F 0.25 0.71

Glu 24 c -0.34 0.16 . * F 0.25 0.88

Thr 25 c -0.46 -0.14 . * F 0.85 0.88

Ser 26 c -0.38 0.16 . F 0.25 0.73

Phe 27 A -0.08 0.41 -0.40 0.35

Ser 28 A -0.03 0.81 -0.40 0.39

Leu 29 c -0.73 0.71 -0.20 0.39

Leu 30 c -1.12 1.11 -0.20 0.39

Asn 31 T T -1.63 1.11 0.20 0.25

Ser 32 T T -1.14 1.41 0.20 0.25

Phe 33 T T -1.09 1.16 0.20 0.47

Phe 34 B T -0.49 1.23 -0.20 0.46

Leu 35 c 0.02 1.26 -0.20 0.53

Pro 36 T -0.28 1.26 0.00 0.82 Tyr 37 T T 0.13 0.86 * F 0.67 1.27

Pro 38 T T 0.17 0.07 F 1.14 3.02

Ser 39 T T 0.20 -0.04 * F 1.91 1.05

Ser 40 T T 0.34 0.10 F 1.33 0.36

Arg 41 B T -0.14 -0.09 F 1.70 0.12

Cys 42 B T -0.20 0.27 0.78 0.08

Cys 43 B T -0.84 0.27 0.61 0.08

Cys 44 B T -0.54 0.53 0.14 0.03

Phe 45 B T -0.91 0.93 -0.03 0.10

Ser 46 B T -1.32 0.93 -0.20 0.10

Val 47 B T -1.54 0.74 -0.20 0.25

Gin 48 B T -1.69 0.86 -0.20 0.20

Cys 49 B T -1.02 0.76 -0.20 0.12

Ser 50 B T -0.53 0.37 0.10 0.28

He 51 B T -0.93 0.16 0.10 0.25

Leu 52 B T -0.38 0.54 -0.20 0.40

Asp 53 . r Z -1.04 0.36 0.30 0.40

Pro 54 T Γ -0.38 0.54 . F 0.35 0.30

Phe 55 T Γ -0.38 0.26 0.50 0.59

Ser 56 T Γ -0.09 -0.04 1.10 0.48

Cys 57 T T 0.83 0.57 0.20 0.30

Asn 58 T T 0.13 0.14 0.50 0.69

Ser 59 T T 0.13 0.14 0.50 0.44

Met 60 T T 0.54 0.19 0.86 1.28

Arg 61 C 0.84 0.53 0.22 0.84

Phe 62 C 1.51 0.13 0.88 1.08

Pro 63 T 1.12 0.14 1.29 1.76

Trp 64 T 1.03 -0.04 2.10 1.15

Glu 65 1.24 0.39 0.89 1.70

Asn 66 T 0.74 0.03 1.08 1.40

Ter 67 T 1.06 0.03 0.87 1.70 Table XII

Res Position I II m iv v vi vii VIII IX X XI XII xm xiv

Met i B 0.97 -0.71 * . 1.64 1.91

Ser 2 B T 0.76 -0.76 . . 2.07 2.00

Arg 3 B T 0.33 -0.57 2.30 1.55

Arg 4 B T -0.09 -0.31 1.77 1.29

Ser 5 B T -0.29 -0.24 . 1.39 0.79

Met 6 i A B 0.02 -0.13 . 0.76 0.41

Leu 1 . . A B -0.27 0.79 -0.37 0.22

Leu 8 . . A B -1.19 1.29 -0.60 0.17

Ala 9 . i A B -1.51 1.59 . -0.60 0.14

Trp 10 . - A B -1.51 1.40 -0.60 0.26

Ala 11 . . A B -1.72 1.10 -0.60 0.42

Leu 12 B T -1.72 1.10 * * -0.20 0.34

Pro 13 B T -0.80 1.29 * * -0.20 0.27

Ser 14 B T -1.02 0.37 * * 0.10 0.52

Leu 15 B T -1.08 0.56 * * -0.20 0.52

Leu 16 A B -1.08 0.30 * * -0.30 0.33

Arg 17 A B -0.86 0.37 * * -0.30 0.25

Leu 18 A B -0.64 0.49 . * -0.60 0.31

Gly 19 A C -0.34 0.20 -0.10 0.65

Ala 20 A C 0.16 -0.49 0.50 0.57

Ala 21 A C 0.97 0.00 0.65 1.00

Gin 22 A B 0.86 -0.69 * F 1.21 1.75

Glu 23 A B 1.46 -1.11 F 1.52 2.90

Thr 24 A . r 1.21 -1.19 F 2.23 4.44

Glu 25 A . Γ 1.13 -1.19 F 2.54 2.59

Asp 26 . T T 1.06 -1.01 F 3.10 0.80

Pro 27 . T T 0.76 -0.44 F 2.49 0.30

Ala 28 . T T 0.54 -0.54 2.33 0.23

Cys 29 . T T -0.03 -0.11 1.72 0.21

Cys 30 B B -0.89 0.57 -0.29 0.10

Ser 31 B B . -1.10 0.79 -0.60 0.07

Pro 32 B B -0.78 0.71 -0.60 0.20

He 33 B B -0.19 0.14 0.00 0.75

Val 34 B T 0.48 -0.03 s_fc 1.45 0.90

Pro 35 B T 0.86 -0.41 _fc 1.90 1.01

Arg 36 T T 1.20 0.07 F 2.00 1.51 Asn 37 r C 0.82 -0.61 * * F 3.00 4.07

Glu 38 A r 0.90 -0.76 * * F 2.50 2.66

TΦ 39 A r 1.17 -0.50 * * F 2.20 1.12

Lys 40 A C 1.08 0.00 * * 1.10 0.70

Ala 41 A C 0.97 -0.01 * * 0.80 0.54

Leu 42 A C 0.30 -0.01 * 0.50 0.90

Ala 43 A A -0.29 -0.36 * k 0.30 0.24

Ser 44 A A 0.00 0.14 * -0.30 0.24

Glu 45 A A -0.08 0.04 * -0.30 0.50

Cys 46 A A -0.30 -0.14 * 0.30 0.68

Ala 47 A A 0.21 0.04 -0.30 0.42

Gin 48 A ] B -0.01 0.04 -0.30 0.32

His 49 A ] B 0.08 0.73 * » -0.60 0.50

Leu 50 A B -0.73 0.59 * -0.60 0.76

Ser 51 A B 0.04 0.77 * -0.60 0.36

Leu 52 B 0.39 0.37 * -0.10 0.52

Pro 53 B B -0.47 0.63 * fc -0.60 0.99

Leu 54 B B -1.29 0.59 fc -0.60 0.55

Arg 55 B B -1.33 0.84 fc -0.60 0.49

Tyr 56 B B -1.33 0.80 * -0.60 0.24

Val 57 B B -0.56 0.76 fc -0.60 0.39

Val 58 B B -0.66 0.57 -0.60 0.27

Val 59 B B -0.43 1.06 -0.60 0.25

Ser 60 B -0.89 0.80 -0.40 0.34

His 61 B -0.94 0.59 -0.40 0.45

Thr 62 B -0.39 0.33 -0.10 0.81

Ala 63 r -0.20 0.07 F 0.45 0.81

Gly 64 T T 0.66 0.26 F 0.65 0.32

Ser 65 T T 0.64 0.16 F 0.65 0.35

Ser 66 T T 0.47 0.16 F 0.65 0.51

Cys 67 T T 0.19 0.09 F 0.65 0.79

Asn 68 T 0.48 0.16 F 0.45 0.60

Thr 69 . C 0.16 0.16 F 0.25 0.60

Pro 70 T T 0.46 0.34 F 0.65 0.60

Ala 71 T T 0.76 0.17 * F 0.65 0.64

Ser 72 B T 1.42 0.17 sk p 0.25 0.77

Cys 73 B T 0.83 0.09 * sfc p 0.25 0.86

Gin 74 A B 1.26 0.16 * F -0.15 0.86

Gin 75 A B 1.47 -0.34 * sfc p 0.60 1.26

Gin 76 A B 1.20 -0.33 * sfc p 0.60 3.79

Ala 77 A B 1.50 -0.26 * F 0.60 1.62 Arg 78 . / A B 2.13 -0.26 * . F 0.60 1.62

Asn 79 . i A B 1.89 -0.16 * . 0.45 1.28

Val 80 t A B 1.86 0.20 * . -0.15 1.98

Gin 81 . i A B 1.26 0.20 * * -0.15 1.37

His 82 . / A B 1.89 0.81 * * -0.60 0.85

Tyr 83 B 1.47 0.41 * . -0.25 2.28

His 84 B 0.66 0.26 . . 0.05 1.90

Met 85 B 1 B 1.17 0.54 . . -0.45 1.15

Lys 86 B 1 B 0.88 0.47 . . -0.60 0.73

Thr 87 . 3 T . . 0.24 0.63 . . -0.20 0.56

Leu 88 . B T 0.49 0.70 * -0.20 0.30

Gly 89 . B T -0.33 0.09 . . 0.10 0.25

Trp 90 B 1 B -0.08 0.73 . . -0.60 0.13

Cys 91 B 1 B -0.37 0.67 . . -0.60 0.16

Asp 92 B T -0.06 0.74 . * -0.20 0.25

Val 93 B T 0.06 0.71 * . -0.20 0.38

Gly 94 B T -0.41 0.59 . * -0.20 0.61

Tyr 95 B T -1.01 0.70 . * -0.20 0.30

Asn 96 B B -0.69 1.39 . * -0.60 0.29

Phe 97 B B -0.69 1.17 . * -0.60 0.29

Leu 98 B B 0.17 0.74 . * -0.60 0.32

He 99 B B 0.17 -0.01 . . 0.30 0.33

Gly 100 B T -0.40 0.01 . . 0.10 0.38

Glu 101 B T -1.26 -0.09 . . ] F 0.85 0.38

Asp 102 T T -0.80 -0.13 . . F 1.25 0.40

Gly 103 T C 0.01 -0.06 . . F 1.05 0.63

Leu 104 B 0.56 -0.49 * * 0.50 0.63

Val 105 B 1.01 -0.06 * * 0.78 0.37

Tyr 106 B 0.67 -0.06 * * 1.06 0.74

Glu 107 B 0.38 -0.06 * . F 1.49 0.89

Gly 108 T T 0.72 0.17 . . F 1.92 1.25

Arg 109 T T 0.83 -0.07 . * F 2.80 1.29

Gly 110 T T 1.38 -0.04 . . F 2.37 0.64

Trp 111 T T 1.28 0.44 . * 1.04 0.94

Asn 112 C 0.69 0.44 . . 0.36 0.47

Phe 113 B 1.00 0.94 . . -0.12 0.48

Thr 114 C 0.59 1.01 . . -0.20 0.63

Gly 115 C 0.59 0.49 . * -0.20 0.52

Ala 116 C 0.84 0.51 . * -0.20 0.60

His 117 T C 0.03 0.23 . . 0.30 0.56

Ser 118 T C 0.44 0.43 . . 0.00 0.47 Gly 119 T C 0.76 0.91 0.00 0.49

His 120 T C 0.89 0.81 0.00 0.58

Leu 121 T 0.88 0.74 0.00 0.67

Trp 122 C 0.61 0.97 -0.20 0.67

Asn 123 C 0.02 0.93 -0.20 0.66

Pro 124 B B . . 0.02 1.11 -0.60 0.56

Met 125 B T -0.83 0.86 -0.20 0.52

Ser 126 B B . . -0.32 0.63 -0.60 0.23

He 127 B B . . -0.73 0.61 -0.60 0.20

Gly 128 B B . . -1.33 0.97 -0.60 0.17

He 129 B B . . -1.47 0.97 -0.60 0.13

Ser 130 B B . . -0.87 1.01 -0.60 0.18

Phe 131 B B -0.81 0.73 -0.60 0.29

Met 132 B r . -0.52 1.06 -0.20 0.66

Gly 133 T r -0.18 0.99 0.20 0.48

Asn 134 T r . 0.82 0.60 0.20 0.93

Tyr 135 T r 0.27 -0.19 1.25 1.85

Met 136 T 0.76 -0.16 1.31 1.39

Asp 137 T 1.04 -0.16 1.57 1.33

Arg 138 B 1.18 -0.07 F 1.58 1.23.

Val 139 B r . ι.i8 -0.40 * . F 2.04 1.92

Pro 140 B r . 0.83 -0.61 . F 2.60 1.99

Thr 141 . r C 0.54 -0.11 * F 2.24 1.03

Pro 142 B r 0.66 0.57 F 0.73 0.97

Gin 143 A B -0.04 -0.07 F 1.12 1.23

Ala 144 A B 0.22 0.00 0.56 0.86

He 145 A B 0.43 0.01 -0.30 0.56

Arg 146 A B 0.40 -0.01 0.30 0.56

Ala 147 A B -0.20 0.01 -0.30 0.55

Ala 148 A B -1.01 0.20 -0.30 0.65

Gin 149 A B -1.01 0.20 -0.30 0.27

Gly 150 A B -0.79 0.70 -0.60 0.27

Leu 151 A B . -1.24 0.77 -0.60 0.14

Leu 152 A B . -1.51 0.70 -0.60 0.08

Ala 153 A B -1.51 0.94 -0.60 0.06

Cys 154 A B -1.51 1.01 -0.60 0.08

Gly 155 A B . -1.51 0.73 -0.60 0.16

Val 156 A B -1.29 0.47 -0.60 0.16

Ala 157 A B -1.29 0.47 -0.60 0.29

Gin 158 A B -0.59 0.59 -0.60 0.25

Gly 159 A B -0.22 0.16 -0.30 0.65 Ala 160 . . A B 0.12 -0.10 * * F 0.45 0.86

Leu 161 . . A B 0.73 -0.20 * * F 0.45 0.80

Arg 162 B r 0.47 0.16 * * F 0.40 1.26

Ser 163 B T -0.34 0.37 * * F 0.25 0.93

Asn 164 B T 0.04 0.56 * F -0.05 0.93

Tyr 165 B T 0.29 -0.13 * 0.70 0.95

Val 166 B B 1.07 0.30 * -0.12 0.70

Leu 167 B B 1.07 0.41 * * . -0.24 0.59

Lys 168 B B 1.37 0.01 * . F 0.39 0.74

Gly 169 B 0.51 -0.74 * * F 1.82 1.67

His 170 B 1 B 0.76 -0.74 * * . F 1.80 1.50

Arg 171 B B 1.72 -1.03 * * . F 1.62 1.30

Asp 172 B B 2.22 -1.03 < ^k . F 1.44 2.57

Val 173 B 1 B 1.37 -0.97 « * . F 1.26 2.72

Gin 174 B ] B 1.41 -0.79 * * . F 1.08 1.15

Arg 175 B ] B 1.23 -0.40 * * . F 0.57 0.92

Thr 176 B B 0.78 0.03 ι ^k . F 0.24 1.92

Leu 177 . B c 0.78 -0.19 < * . F 1.16 1.10

Ser 178 . Γ c 1.63 -0.19 < ^k . F 1.53 0.90

Pro 179 . Γ c 0.82 0.21 ι ^k . F 1.20 1.08

Gly 180 T Γ 0.47 0.41 * " . F 0.98 1.08

Asn 181 T Γ 0.74 0.49 F 0.86 1.26

Gin 182 A B 0.74 0.60 ^► . F -0.06 1.11

Leu 183 A B 0.16 0.86 ^k . . -0.48 0.93

Tyr 184 A B 0.37 1.11 ^k . -0.60 0.40

His 185 A B 0.71 1.11 * . -0.60 0.40

Leu 186 A B 0.42 1.11 * . -0.60 0.79 lie 187 A B 0.21 1.34 * . . -0.60 0.53

Gin 188 A B 0.99 1.01 * . -0.60 0.60

Asn 189 A T 0.99 1.01 * . -0.20 0.99

Trp 190 . T c 1.13 1.09 * . 0.15 2.21

Pro 191 . T c 1.64 0.40 * . 0.45 2.50

His 192 T T 2.32 0.39 * 0.86 2.09

Tyr 193 T T 1.93 0.41 0.77 3.07

Arg 194 T 1.54 -0.07 1.68 2.54

Ser 195 c 1.44 -0.07 1.69 2.38

Pro 196 T 1.27 -0.14 * 2.10 1.94

Table XIII summarizes the information corresponding to each "Gene No." described above. The nucleotide sequence identified as "NT SEQ ID NO:X" was assembled from partially homologous ("overlapping") sequences obtained from the "cDNA clone ID" identified in Table XIII and, in some cases, from additional related DNA clones. The overlapping sequences were assembled into a single contiguous sequence of high redundancy (usually three to five overlapping sequences at each nucleotide position), resulting in a final sequence identified as SEQ ID NO:X.

The cDNA Clone ID was deposited on the date and given the corresponding deposit number listed in "ATCC Deposit No:Z and Date." Some of the deposits contain multiple different clones corresponding to the same gene. "Vector" refers to the type of vector contained in the cDNA Clone ID.

"Total NT Seq." refers to the total number of nucleotides in the contig identified by "Gene No." The deposited clone may contain all or most of these sequences, reflected by the nucleotide position indicated as "5' NT of Clone Seq." and the "3' NT of Clone Seq." of SEQ ID NO:X. The nucleotide position of SEQ ID NO:X of the putative start codon (methionine) is identified as "5' NT of Start Codon." Similarly , the nucleotide position of SEQ ID NO:X of the predicted signal sequence is identified as "5' NT of First AA of Signal Pep." The translated amino acid sequence, beginning with the methionine, is identified as

"AA SEQ ID NO:Y," although other reading frames can also be easily translated using known molecular biology techniques. The polypeptides produced by these alternative open reading frames are specifically contemplated by the present invention.

The first and last amino acid position of SEQ ID NO: Y of the predicted signal peptide is identified as "First AA of Sig Pep" and "Last AA of Sig Pep." The predicted first amino acid position of SEQ ID NO:Y of the secreted portion is identified as "Predicted First AA of Secreted Portion." Finally, the amino acid position of SEQ ID NO:Y of the last amino acid in the open reading frame is identified as "Last AA of ORF." SEQ ID NO:X (where X may be any of the polynucleotide sequences disclosed in the sequence listing) and the translated SEQ ID NO:Y (where Y may be any of the polypeptide sequences disclosed in the sequence listing) are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below. For instance, SEQ ID NO:X is useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO:X or the cDNA contained in the deposited clone. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from SEQ ID NO:Y may be used, for example, to generate antibodies which bind specifically to proteins containing the polypeptides and the secreted proteins encoded by the cDNA clones identified in Table XIII.

Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).

Accordingly, for those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO:X and the predicted translated amino acid sequence identified as SEQ ID NO:Y, but also a sample of plasmid DNA containing a human cDNA of the invention deposited with the ATCC, as set forth in Table XIII. The nucleotide sequence of each deposited clone can readily be determined by sequencing the deposited clone in accordance with known methods. The predicted amino acid sequence can then be verified from such deposits. Moreover, the amino acid sequence of the protein encoded by a particular clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited human cDNA, collecting the protein, and determining its sequence.

The present invention also relates to the genes corresponding to SEQ ID NO:X, SEQ ID NO:Y, or the deposited clone. The corresponding gene can be isolated in accordance with known methods using the sequence infomiation disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.

Also provided in the present invention are allelic variants, orthologs, and/or species homologs. Procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs, and/or species homologs of genes corresponding to SEQ ID NO:X, SEQ ID NO:Y, or a deposited clone, using information from the sequences disclosed herein or the clones deposited with the ATCC. For example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.

The polypeptides of the invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.

The polypeptides may be in the form of the secreted protein, including the mature form, or may be a part of a larger protein, such as a fusion protein (see below). It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification , such as multiple histidine residues, or an additional sequence for stability during recombinant production.

The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of a polypeptide, including the secreted polypeptide, can be substantially purified using techniques described herein or otherwise known in the art, such as, for example, by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988). Polypeptides of the invention also can be purified from natural, synthetic or recombinant sources using techniques described herein or otherwise known in the art, such as, for example, antibodies of the invention raised against the secreted protein.

The present invention provides a polynucleotide comprising, or alternatively consisting of, the nucleic acid sequence of SEQ ID NO:X, and/or a cDNA contained in

ATCC deposit Z. The present invention also provides a polypeptide comprising, or alternatively, consisting of, the polypeptide sequence of SEQ ID NO: Y and/or a polypeptide encoded by the cDNA contained in ATCC deposit Z. Polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO: Y and/or a polypeptide sequence encoded by the cDNA contained in ATCC deposit Z are also encompassed by the invention. Signal Sequences

The present invention also encompasses mature forms of the polypeptide having the polypeptide sequence of SEQ ID NO:Y and/or the polypeptide sequence encoded by the cDNA in a deposited clone. Polynucleotides encoding the mature forms (such as, for example, the polynucleotide sequence in SEQ ID NO:X and/or the polynucleotide sequence contained in the cDNA of a deposited clone) are also encompassed by the invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal or secretary leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Most mammalian cells and even insect cells cleave secreted proteins with the same specificity. However, in some cases, cleavage of a secreted protein is not entirely uniform, which results in two or more mature species of the protein. Further, it has long been known that cleavage specificity of a secreted protein is ultimately determined by the primary structure of the complete protein, that is, it is inherent in the amino acid sequence of the polypeptide.

Methods for predicting whether a protein has a signal sequence, as well as the cleavage point for that sequence, are available. For instance, the method of McGeoch, Virus Res. 3:271-286 (1985), uses the information from a short N-terminal charged region and a subsequent uncharged region of the complete (uncleaved) protein. The method of von Heinje, Nucleic Acids Res. 14:4683-4690 (1986) uses the information from the residues surrounding the cleavage site, typically residues -13 to +2, where +1 indicates the amino terminus of the secreted protein. The accuracy of predicting the cleavage points of known mammalian secretory proteins for each of these methods is in the range of 75-80%. (von Heinje, supra.) However, the two methods do not always produce the same predicted cleavage point(s) for a given protein.

In the present case, the deduced amino acid sequence of the secreted polypeptide was analyzed by a computer program called SignalP (Henrik Nielsen et al., Protein Engineering 10:1-6 (1997)), which predicts the cellular location of a protein based on the amino acid sequence. As part of this computational prediction of localization, the methods of McGeoch and von Heinje are incorporated. The analysis of the amino acid sequences of the secreted proteins described herein by this program provided the results shown in Table XIII. As one of ordinary skill would appreciate, however, cleavage sites sometimes vary from organism to organism and cannot be predicted with absolute certainty. Accordingly, the present invention provides secreted polypeptides having a sequence shown in SEQ ID NO:Y which have an N-terminus beginning within 5 residues (i.e., + or - 5 residues) of the predicted cleavage point. Similarly, it is also recognized that in some cases, cleavage of the signal sequence from a secreted protein is not entirely uniform, resulting in more than one secreted species. These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention. Moreover, the signal sequence identified by the above analysis may not necessarily predict the naturally occurring signal sequence. For example, the naturally occurring signal sequence may be further upstream from the predicted signal sequence. However, it is likely that the predicted signal sequence will be capable of directing the secreted protein to the ER. Nonetheless, the present invention provides the mature protein produced by expression of the polynucleotide sequence of SEQ ID NO:X and/or the polynucleotide sequence contained in the cDNA of a deposited clone, in a mammalian cell (e.g., COS cells, as desribed below). These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.

Polynucleotide and Polypeptide Variants

The present invention is directed to variants of the polynucleotide sequence disclosed in SEQ ID NO:X, the complementary strand thereto, and/or the cDNA sequence contained in a deposited clone. The present invention also encompasses variants of the polypeptide sequence disclosed in SEQ ID NO:Y and/or encoded by a deposited clone.

"Variant" refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the polynucleotide or polypeptide of the present invention.

The present invention is also directed to nucleic acid molecules which comprise, or alternatively consist of, a nucleotide sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for example, the nucleotide coding sequence in SEQ ID NO:X or the complementary strand thereto, the nucleotide coding sequence contained in a deposited cDNA clone or the complementary strand thereto, a nucleotide sequence encoding the polypeptide of SEQ ID NO: Y, a nucleotide sequence encoding the polypeptide encoded by the cDNA contained in a deposited clone, and/or polynucleotide fragments of any of these nucleic acid molecules (e.g., those fragments described herein). Polynucleotides which hybridize to these nucleic acid molecules under stringent hybridization conditions or lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.

The present invention is also directed to polypeptides which comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%;, 95%,

96%, 97%, 98%, 99%; identical to, for example, the polypeptide sequence shown in SEQ

ID NO:Y, the polypeptide sequence encoded by the cDNA contained in a deposited clone, and/or polypeptide fragments of any of these polypeptides (e.g., those fragments described herein). By a nucleic acid having a nucleotide sequence at least, for example, 95%

"identical" to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the polypeptide. In other words, to obtain a nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be an entire sequence shown in Table XIII, the ORF (open reading frame), or any fragment specified as described herein.

As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the presence invention can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245(1990)). In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identiy are: Matrix=Unitary, k-tuple=4,

Mismatch Penalty=l, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=l, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the lenght of the subject nucleotide sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5' or 3' deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for 5' and 3' truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5' or 3' ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of the present invention. Only bases outside the 5' and 3' bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.

For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5' end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5' end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5' and 3' ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5' or 3' of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5' and 3' of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to made for the purposes of the present invention.

By a polypeptide having an amino acid sequence at least, for example, 95%

"identical" to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, (indels) or substituted with another amino acid. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence. As a practical matter, whether any particular polypeptide is at least 80%, 85%,

90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, an amino acid sequences shown in Table XIII (SEQ ID NO:Y) or to the amino acid sequence encoded by cDNA contained in a deposited clone can be determined conventionally using known computer programs. A preferred method for determing the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245(1990)). In a sequence alignment the query and subject sequences are either both nucleotide sequences or both ammo acid sequences The result of said global sequence alignment is in percent identity Preferred parameters used in a FASTDB amino acid alignment are.

Matπx=PAM 0, k-tuple=2, Mismatch Penalty=l, Joining Penalty=20, Randomization

Group Length=0, Cutoff Score=l, Window Sιze=sequence length, Gap Penalty=5, Gap Size Penalty=0 05, Window Sιze=500 or the length of the subject amino acid sequence, whichevei is shorter

If the subject sequence is shortei than the queiy sequence due to N- or C-teiminal deletions, not because of internal deletions, a manual correction must be made to the results This is because the FASTDB program does not account foi N- and C-termmal truncations of the subject sequence when calculating global pet cent identity For subject sequences tiuncated at the N- and C- termini, relative to the queiy sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score This final percent identity score is what is used foi the purposes of the present invention Only residues to the N- and C-termmi of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence

For example, a 90 ammo acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N- terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequnce are manually corrected for. No other manual corrections are to made for the purposes of the present invention. The variants may contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Moreover, variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coli).

Naturally occurring variants are called "allelic variants," and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism.

(Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985).) These allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present invention. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis. Using known methods of protein engineering and recombinant DNA technology, variants may be generated to improve or alter the characteristics of the polypeptides of the present invention. For instance, one or more amino acids can be deleted from the N- terminus or C-terminus of the secreted protein without substantial loss of biological function. The authors of Ron et al., J. Biol. Chem. 268: 2984-2988 (1993), reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 aminoterminal amino acid residues. Similarly, Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216 (1988).)

Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein. For example, Gayle and coworkers (J. Biol. Chem 268:22105-22111 (1993)) conducted extensive mutational analysis of human cytokine IL-la. They used random mutagenesis to generate over 3,500 individual IL-la mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The investigators found that "[m]ost of the molecule could be altered with little effect on either [binding or biological activity]." (See, Abstract.) In fact, only 23 unique amino acid sequences, out of more than 3,500 nucleotide sequences examined, produced a protein that significantly differed in activity from wild-type.

Furthermore, even if deleting one or more amino acids from the N-terminus or C- terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce and/or to bind antibodies which recognize the secreted form will likely be retained when less than the majority of the residues of the secreted form are removed from the N-terminus or C-terminus. Whether a particular polypeptide lacking N- or C-terminal residues of a protein retains such immunogenic activities can readily be determined by routine methods described herein and otherwise known in the art.

Thus, the invention further includes polypeptide variants which show substantial biological activity. Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity. For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., Science 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.

The first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By compaπng amino acid sequences different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating ammo acid substitution could be modified while still maintaining biological activity ot the protein. The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identity regions cπtical for protein function. For example, site directed mutagenesis or alanme-scanning mutagenesis (introduction of single alanine mutations at every residue m the molecule) can be used. (Cunningham and Wells, Science 244: 1081-1085 (1989).) The resulting mutant molecules can then be tested for biological activity.

As the authors state, these two strategies have levealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which ammo acid changes are likely to be permissive at certain amino acid positions in the protein. For example, most buried (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Moreover, tolerated conservative ammo acid substitutions involve replacement of the aliphatic or hydrophobic ammo acids Ala, Val, Leu and He; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gin, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.

Besides conservative ammo acid substitution, variants of the present invention include (l) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or

(ii) substitution with one or more of amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), or (iv) fusion of the polypeptide with additional amino acids, such as, for example, an IgG Fc fusion region peptide, or leader or secretory sequence, or a sequence facilitating purification. Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.

For example, polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).)

A further embodiment of the invention relates to a polypeptide which comprises the amino acid sequence of the present invention having an amino acid sequence which contains at least one amino acid substitution, but not more than 50 amino acid substitutions, even more preferably, not more than 40 amino acid substitutions, still more preferably, not more than 30 amino acid substitutions, and still even more preferably, not more than 20 amino acid substitutions. Of course, in order of ever-increasing preference, it is highly preferable for a peptide or polypeptide to have an amino acid sequence which comprises the amino acid sequence of the present invention, which contains at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions. In specific embodiments, the number of additions, substitutions, and/or deletions in the amino acid sequence of the present invention or fragments thereof (e.g., the mature form and/or other fragments described herein), is 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, conservative amino acid substitutions are preferable. Polynucleotide and Polypeptide Fragments

The present invention is also directed to polynucleotide fragments of the polynucleotides of the invention. In the present invention, a "polynucleotide fragment" refers to a short polynucleotide having a nucleic acid sequence which: is a portion of that contained in a deposited clone, or encoding the polypeptide encoded by the cDNA in a deposited clone; is a portion of that shown in SEQ ID NO:X or the complementary strand thereto, or is a portion of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:Y. The nucleotide fragments of the invention are preferably at least about 15 nt. and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt, at least about 50 nt, at least about 75 nt, or at least about 150 nt in length. A fragment "at least 20 nt in length," for example, is intended to include 20 or more contiguous bases from the cDNA sequence contained in a deposited clone or the nucleotide sequence shown in SEQ ID NO:X. In this context "about" includes the particularly recited value, a value larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. These nucleotide fragments have uses that include, but are not limited to, as diagnostic probes and primers as discussed herein. Of course, larger fragments (e.g., 50, 150, 500, 600, 2000 nucleotides) are preferred. Moreover, representative examples of polynucleotide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851- 900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601- 1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, or 2001 to the end of SEQ ID NO:X, or the complementary strand thereto, or the cDNA contained in a deposited clone. In this context "about" includes the particularly recited ranges, and ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has biological activity. More preferably, these polynucleotides can be used as probes or primers as discussed herein. Polynucleotides which hybridize to these nucleic acid molecules under stringent hybridization conditions or lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.

In the present invention, a "polypeptide fragment" refers to an amino acid sequence which is a portion of that contained in SEQ ID NO:Y or encoded by the cDNA contained in a deposited clone. Protein (polypeptide) fragments may be "free-standing." or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, or 161 to the end of the coding region. Moreover, polypeptide fragments can be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids in length. In this context "about" includes the particularly recited ranges or values, and ranges or values larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes. Polynucleotides encoding these polypeptides are also encompassed by the invention. Preferred polypeptide fragments include the secreted protein as well as the mature form. Further preferred polypeptide fragments include the secreted protein or the mature form having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1-60, can be deleted from the amino terminus of either the secreted polypeptide or the mature form. Similarly, any number of amino acids, ranging from 1-30, can be deleted from the carboxy terminus of the secreted protein or mature form. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotides encoding these polypeptide fragments are also preferred. Also preferred are polypeptide and polynucleotide fragments characterized by structural or functional domains, such as fragments that compnse alpha-helix and alpha- helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions. Polypeptide fragments of SEQ ID NO:Y falling within conserved domains aie specifically contemplated by the present invention Moreover, polynucleotides encoding these domains are also contemplated Other preferred polypeptide fragments are biologically active fragments. Biologically active fragments are those exhibiting activity similar, but not necessanly identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity Polynucleotides encoding these polypeptide fragments are also encompassed by the invention. Preferably, the polynucleotide fragments of the invention encode a polypeptide which demonstrates a functional activity. By a polypeptide demonstrating a "functional activity" is meant, a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) polypeptide of invention protein. Such functional activities include, but are not limited to, biological activity, antigenicity [ability to bind (or compete with a polypeptide of the invention for binding) to an antibody to the polypeptide of the invention], immunogenicity (ability to generate antibody which binds to a polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide of the invention.

The functional activity of polypeptides of the invention, and fragments, variants deπvatives, and analogs thereof, can be assayed by various methods.

For example, in one embodiment where one is assaying for the ability to bind or compete with full-length polypeptide of the invention for binding to an antibody of the polypeptide of the invention, various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay),

"sandwich" immunoassays, lmmunoradiometπc assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutmation assays), complement fixation assays, immunofluorescence assays, protein A assays, and lmmunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondaiy antibody is labeled Many means aie known in the art for detecting binding in an immunoassay and are within the scope of the present invention.

In another embodiment, where a ligand for a polypeptide of the invention identified, or the ability of a polypeptide fragment, variant or derivative of the invention to multimeπze is being evaluated, binding can be assayed, e.g., by means well-known the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky, E., et al., 1995, Microbiol. Rev. 59:94-123. In another embodiment, physiological correlates of binding of a polypeptide of the invention to its substrates (signal transduction) can be assayed. In addition, assays described herein (see Examples) and otherwise known in the art may routinely be applied to measure the ability of polypeptides of the invention and fragments, variants denvatives and analogs thereof to elicit related biological activity related to that of the polypeptide of the invention (either in vitro or in vivo). Other methods will be known to the skilled artisan and are within the scope of the invention.

Epitopes & Antibodies

The present invention is also directed to polypeptide fragments compπsing, or alternatively consisting of, an epitope of the polypeptide sequence shown in SEQ ID NO:Y, or the polypeptide sequence encoded by the cDNA contained in a deposited clone.

Polynucleotides encoding these epitopes (such as, for example, the sequence disclosed in

SEQ ID NO:X) are also encompassed by the invention, as is the nucleotide sequences of the complementary strand of the polynucleotides encoding these epitopes. And polynucleotides which hybridize to the complementary strand under stringent hybridization conditions or lower stringency conditions.

In the present invention, "epitopes" refer to polypeptide fragments having antigenic or immunogenic activity in an animal, especially in a human. A preferred embodiment of the present invention relates to a polypeptide fragment comprising an epitope, as well as the polynucleotide encoding this fragment. A region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope." In contrast, an "immunogenic epitope" is defined as a part of a protein that elicits an antibody response. (See, for instance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998- 4002 (1983).)

Fragments which function as epitopes may be produced by any conventional means. (See, e.g.. Houghten, R. A., Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985) further described in U.S. Patent No. 4,631,211.)

In the present invention, antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, and most preferably between about 15 to about 30 amino acids. Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983).) Similarly, immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. (See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985).) A preferred immunogenic epitope includes the secreted protein. The immunogenic epitopes may be presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse) or, if it is long enough (at least about 25 amino acids), without a earner. However, immunogenic epitopes compnsmg as few as 8 to 10 ammo acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e g , in Western blotting )

Epitope-beanng polypeptides ot the ptesent invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e g , Sutcliffe et al., supra. Wilson et al.. supra, and Bittle et al , J Gen Virol., 66.2347-2354 (1985). If in vivo immunization is used, animals may be immunized with fiee peptide, however, anti-peptide antibody titer may be boosted by coupling of the peptide to a macromolecular earner, such as keyhole limpet hemacyamn (KLH) or tetanus toxoid For instance, peptides containing cysteine residues may be coupled to a carrier using a linker such as -maleimidobenzoyl- N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intrapentoneal and/or intradermal injection of emulsions containing about 100 μgs of peptide or carrier protein and Freund's adjuvant. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known m the art.

As one of skill in the art will appreciate, and discussed above, the polypeptides of the present invention comprising an immunogenic or antigenic epitope can be fused to heterologous polypeptide sequences. For example, the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG,

IgM), or portions thereof (CHI, CH2, CH3, any combination thereof including both entire domains and portions thereof) resulting in chimeric polypeptides. These fusion proteins facilitate purification, and show an increased half-life in vivo. This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, e.g., EPA 0,394,827; Traunecker et al., Nature, 331 :84-86 (1988).

Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion can also be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag to aid in detection and purification of the expressed polypeptide.

Additional fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as "DNA shuffling"). DNA shuffling may be employed to modulate the activities of polypeptides corresponding to SEQ ID NO:Y thereby effectively generating agonists and antagonists of the polypeptides. See,generally, U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten, P.A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, S., Trends Biotechnol. 16(2):76-82 (1998); Hansson, L.O., et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R., Biotechniques 24(2):308-13 (1998) (each of these patents and publications are hereby incorporated by reference). In one embodiment, alteration of polynucleotides corresponding to SEQ ID NO:X and corresponding polypeptides may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments into a desired molecule corresponding to SEQ ID NO:X polynucleotides of the invention by homologous, or site-specific, recombination. In another embodiment, polynucleotides corresponding to SEQ ID NO:X and corresponding polypeptides may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of coding polynucleotide corresponding to

SEQ ID NO:X, or the polypeptide encoded thereby may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

Antibodies

The present invention further relates to antibodies and T-cell antigen receptors (TCR) which specifically bind the polypeptides of the present invention. The antibodies of the present invention include IgG (including IgGi, IgG2, IgG3, and IgG4), IgA (including IgAl and IgA2), IgD, IgE, or IgM, and IgY. As used herein, the term "antibody" (Ab) is meant to include whole antibodies, including single-chain whole antibodies, and antigen- binding fragments thereof. Most preferably the antibodies are human antigen binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a V, or V_H domain. The antibodies may be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine, rabbit, goat, guinea pig, camel, horse, or chicken. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entire or partial of the following: hinge region, CHI, CH2, and CH3 domains. Also included in the invention are any combinations of variable region(s) and hinge region, CHI, CH2, and CH3 domains. The present invention further includes monoclonal, polyclonal, chimeric, humanized, and human monoclonal and human polyclonal antibodies which specifically bind the polypeptides of the present invention. The present invention further includes antibodies which are anti-idiotypic to the antibodies of the present invention.

The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater mul specificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for heterologous compositions, such as a heterologous polypeptide or solid support material. See, e.g., WO 93/17715; WO 92/08802; WO 91/00360, WO 92/05793, Tutt, et al., J. Immunol 147:60-69 (1991); US Patents

5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648, Kostelny et al., J Immunol

148.1547-1553 (1992)

Antibodies of the present invention may be described or specified in terms of the epιtope(s) or portιon(s) of a polypeptide of the present invention which are recognized or specifically bound by the antibody The epιtope(s) or polypeptide portιon(s) may be specified as descπbed herein, e g , by N-teimmal and C-terminal positions, by size in contiguous amino acid residues, or listed in the Tables and Figures. Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefoie, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of the polypeptides of the present invention are included. Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and descnbed herein) to a polypeptide of the present invention are also included in the present invention. Further included m the present invention are antibodies which only bind polypeptides encoded by polynucleotides which hybndize to a polynucleotide of the present invention under stnngent hybπdization conditions (as described herein). Antibodies of the present invention may also be descπbed or specified in terms of their binding affinity. Preferred binding affinities include those with a dissociation constant or Kd less than 5X10⁶M, 10 ⁶M, 5X10⁷M, 10⁷M, 5X10⁸M, 10⁸M, 5X10⁹M, 10⁹M, 5X10 ¹⁰M, 10 ¹⁰M, 5X10 "M, 10 "M, 5X10^'12M, 10 ¹²M, 5X10^"I M, 10^'13M, 5X10^"14M, 10 ¹⁴M, 5X10^"I5M, and 10 ¹⁵M.

Antibodies of the present invention have uses that include, but are not limited to, methods known in the art to purify, detect, and target the polypeptides of the present invention including both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference in the entirety).

The antibodies of the present invention may be used either alone or in combination with other compositions. The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions. For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, or toxins. See, e.g., WO 92/08495; WO 91/14438; WO 89/12624; US Patent 5,314,995; and EP 0 396 387.

The antibodies of the present invention may be prepared by any suitable method known in the art. For example, a polypeptide of the present invention or an antigenic fragment thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies. The term "monoclonal antibody" is not a limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technology.

Hybridoma techniques include those known in the art and taught in Harlow et al., ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties). Fab and F(ab')2 fragments may be produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce

F(ab')2 fragments). Alternatively, antibodies of the present invention can be produced through the application of recombinant DNA and phage display technology or through synthetic chemistry using methods known in the art. For example, the antibodies of the present invention can be prepared using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of a phage particle which carries polynucleotide sequences encoding them. Phage with a desired binding property are selected from a repertoire or combinatorial antibody library (e.g. human or murine) by selecting directly with antigen, typically antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and US Patents 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743 (said references incorporated by reference in their entireties). As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host including mammalian cells, insect cells, plant cells, yeast, and bacteria. For example, techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in WO 92/22324;

Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34

(1995); and Better et al., Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties).

Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Patents 4,946,778 and 5,258,498; Huston et al.,

Methods in Enzymology 203:46-88 (1991); Shu, L. et al., PNAS 90:7995-7999 (1993); and Skeπ-a et al., Science 240:1038-1040 (1988). For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric. humanized, or human antibodies. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; and US Patent 5,807,715. Antibodies can be humanized using a variety of techniques including CDR-grafting (EP 0 239 400; WO 91/09967; US Patent 5,530,101; and 5,585,089), veneering or resurfacing (EP 0 592 106; EP 0 519 596; Padlan E.A., Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (US Patent 5,565,332). Human antibodies can be made by a variety of methods known in the art including phage display methods described above. See also, US Patents 4,444,887, 4,716,111, 5,545,806, and 5,814,318; and WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741 (said references incorporated by reference in their entireties).

Further included in the present invention are antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide of the present invention. The antibodies may be specific for antigens other than polypeptides of the present invention. For example, antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors. Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and punfication methods using methods known m the art. See e.g., Harbor et al. supra and WO 93/21232; EP 0 439

095; Naramura et al., Immunol. Lett. 39-91-99 (1994); US Patent 5,474,981 ; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al , J Immunol. 146:2446-2452 (1991) (said references incorpoiated by reference in their entireties)

The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions For example, the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof The antibody portion fused to a polypeptide of the piesent invention may comprise the hinge region, CHI domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof. The polypeptides of the present invention may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art. The polypeptides may also be fused or conjugated to the above antibody portions to form multimers. For example, Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions. Higher multimenc forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See e.g., US Patents 5,336,603,

5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,112,946; EP 0 307 434, EP 0 367 166; WO 96/04388, WO 91/06570; Ashkenazi et al., PNAS 88:10535-10539 (1991); Zheng et al., J. Immunol. 154:5590-5600 (1995); and Vii et al., PNAS 89:11337-11341 (1992) (said references incorporated by reference in their entireties). The invention further relates to antibodies which act as agonists or antagonists of the polypeptides of the present invention. For example, the present invention includes antibodies which disrupt the receptor/hgand interactions with the polypeptides of the invention either partially or fully. Included are both receptor-specific antibodies and ligand-specific antibodies. Included are receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. Also included are receptor-specific antibodies which both prevent ligand binding and receptor activation. Likewise, included are neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor.

Further included are antibodies which activate the receptor. These antibodies may act as agonists for either all or less than all of the biological activities affected by ligand- mediated receptor activation. The antibodies may be specified as agonists or antagonists for biological activities comprising specific activities disclosed herein. The above antibody agonists can be made using methods known in the art. See e.g., WO 96/40281; US Patent 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen, et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4): 1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon, et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. l l l(Pt2):237-247 (1998); Pitard et al., J. Immunol. Methods 205(2):177-190 (1997); Liautard et al., Cytokinde 9(4):233-241 (1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(l):14-20 (1996) (said references incorporated by reference in their entireties).

As discussed above, antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to ligand can be used to generate anti-idiotypes that "mimic" the polypeptide mutimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand. For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligands/receptors, and thereby block its biological activity. The invention further relates to a diagnostic kit for use in screening serum containing antibodies specific against proliferative and/or cancerous polynucleotides and polypeptides. Such a kit may include a substantially isolated polypeptide antigen comprising an epitope which is specifically immunoreactive with at least one anti- polypeptide antigen antibody. Such a kit also includes means for detecting the binding of said antibody to the antigen. In specific embodiments, the kit may include a recombinantly produced or chemically synthesized polypeptide antigen. The polypeptide antigen of the kit may also be attached to a solid support.

In a more specific embodiment the detecting means of the above-described kit includes a solid support to which said polypeptide antigen is attached. Such a kit may also include a non-attached reporter-labelled anti-human antibody. In this embodiment, binding of the antibody to the polypeptide antigen can be detected by binding of the said reporter- labelled antibody.

The invention further includes a method of detecting proliferative and/or cancerous disorders and conditions in a test subject. This detection method includes reacting serum from a test subject (e.g. one in which proliferative and/or cancerous cells or tissues may be present) with a substantially isolated polypeptide and/or polynucleotide antigen, and examining the antigen for the presence of bound antibody. In a specific embodiment, the method includes a polypeptide antigen attached to a solid support, and the serum is reacted with the support. Subsequently, the support is reacted with a reporter labelled anti-human antibody. The solid support is then examined for the presence of reporter- label led antibody.

Additionally, the invention includes a proliferative condition vaccine composition. The composition includes a substantially isolated polypeptide and/or polynucleotide antigen, where the antigen includes an epitope which is specifically immunoreactive with at least antibody specific for the epitope. The peptide and/or polynucleotide antigen may be produced according to methods known in the art, including recombinant expression or chemical synthesis. The peptide antigen is preferably present in a pharmacologically effective dose in a pharmaceutically acceptable carrier.

Further, the invention includes a monoclonal antibody that is specifically immunoreactive with polypeptide and/or polynucleotide epitopes. The invention includes a substantially isolated preparation of polyclonal antibodies specifically immunoreactive with polynucleotides and/or polypeptides of the present invention. In a more specific embodiment, such polyclonal antibodies are prepared by affinity chromatography, in addition to, other methods known in the art.

In another emodiment, the invention includes a method for producing antibodies to polypeptide and/or polynucleotide antigens. The method includes administering to a test subject a substantially isolated polypeptide and/or polynucleotide antigen, where the antigen includes an epitope which is specifically immunoreactive with at least one anti- polypeptide and/or polynucleotide antibody. The antigen is administered in an amount sufficient to produce an immune response in the subject.

In an additional embodiment, the invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the invention. The diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody is attached to a solid support. In a specific embodiment, the antibody may be a monoclonal antibody. The detecting means of the kit may include a second, labelled monoclonal antibody. Alternatively, or in addition, the detecting means may include a labelled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention. After binding with specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labelled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support. The reagent is again washed to remove unbound labelled antibody, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric or colorimetric substrate (Sigma, St. Louis, MO).

The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s). Thus, the invention provieds an assay system or kit for carrying out this diagnostic method. The kit generally includes a support with surface-bound recombinant antigens, and a reporter-labelled anti-human antibody for detecting surface-bound anti-antigen antibody.

Fusion Proteins

Any polypeptide of the present invention can be used to generate fusion proteins. For example, the polypeptide of the present invention, when fused to a second protein, can be used as an antigenic tag. Antibodies raised against the polypeptide of the present invention can be used to indirectly detect the second protein by binding to the polypeptide. Moreover, because secreted proteins target cellular locations based on trafficking signals, the polypeptides of the present invention can be used as targeting molecules once fused to other proteins. Examples of domains that can be fused to polypeptides of the present invention include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but may occur through linker sequences. Moreover, fusion proteins may also be engineered to improve charactenstics of the polypeptide of the present invention. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence during puπfication from the host cell or subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate puπfication Such regions may be removed prior to final preparation of the polypeptide The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.

Moreover, polypeptides of the present invention, including fragments, and specifically epitopes, can be combined with parts of the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CHI, CH2, CH3, and any combination thereof, including both entire domains and portions thereof), resulting in chimeric polypeptides. These fusion proteins facilitate punfication and show an increased half-life in vivo. One reported example descπbes chimeπc proteins consisting of the first two domains of the human CD4-poly peptide and vaπous domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP A 394,827; Traunecker et al., Nature 331:84-86 (1988).) Fusion proteins having disulfide-linked dimenc structures (due to the IgG) can also be more efficient in binding and neutralizing other molecules, than the monomenc secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995).) Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising vanous portions of constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K.

Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).)

Moreover, the polypeptides of the present invention can be fused to marker sequences, such as a peptide which facilitates purification of the fused polypeptide. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the "HA" tag, corresponds to an epitope derived from the influenza hemagglutinin protein. (Wilson et al., Cell 37:767 (1984).)

Thus, any of these above fusions can be engineered using the polynucleotides or the polypeptides of the present invention.

Vectors. Host Cells, and Protein Production

The present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by recombinant techniques. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells. The polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged hpid. If the vector is a virus, it may be packaged in vitro using an appropπate packaging cell line and then transduced into host cells.

The polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, tip, phoA and tac promoters, the

SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Othet suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcπption initiation, termination, and, in the tianscπbed region, a πbosome binding site for translation. The coding portion of the transcnpts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated. As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for cultunng in E. coli and other bacteπa. Representative examples of appropriate hosts include, but are not limited to, bactenal cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-descnbed host cells are known in the art.

Among vectors preferred for use m bactena include pQE70, pQE60 and ρQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescπpt vectors, pNH8A, pNHlόa, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.

Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular

Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.

A polypeptide of this invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification. Polypeptides of the present invention, and preferably the secreted form, can also be recovered from: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked. In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with the polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination (see, e.g., U.S. Patent No. 5,641,670, issued June 24, 1997; International Publication No. WO 96/29411, published September 26, 1996; International Publication No. WO 94/12650, published August 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties). In addition, polypeptides of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller et al., Nature, 310:105-111 (1984)). For example, a polypeptide corresponding to a fragment of a polypeptide sequence of the invention can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, omithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

The invention encompasses polypeptides which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH₄; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc. Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression. The polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.

Also provided by the invention are chemically modified derivatives of the polypeptides of the invention which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Patent NO: 4,179,337). The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about" indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).

The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of the protein. There are a number of attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group. One may specifically desire proteins chemically modified at the N-terminus. Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the

N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.

The polypeptides of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers). Accordingly, the present invention relates to monomers and multimers of the polypeptides of the invention, their preparation, and compositions (preferably, Therapeutics) containing them. In specific embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In additional embodiments, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.

Multimers encompassed by the invention may be homomers or heteromers. As used herein, the term homomer, refers to a multimer containing only polypeptides corresponding to the amino acid sequence of SEQ ID NO:Y or encoded by the cDNA contained in a deposited clone (including fragments, variants, splice variants, and fusion proteins, corresponding to these polypeptides as described herein). These homomers may contain polypeptides having identical or different amino acid sequences. In a specific embodiment, a homomer of the invention is a multimer containing only polypeptides having an identical amino acid sequence. In another specific embodiment, a homomer of the invention is a multimer containing polypeptides having different amino acid sequences. In specific embodiments, the multimer of the invention is a homodimer (e.g., containing polypeptides having identical or different amino acid sequences) or a homotrimer (e.g., containing polypeptides having identical and/or different amino acid sequences). In additional embodiments, the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., polypeptides of different proteins) in addition to the polypeptides of the invention. In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer. Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation. Thus, in one embodiment, multimers of the invention, such as, for example, homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution. In another embodiment, heteromultimers of the invention, such as, for example, heterotrimers or heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution. In other embodiments, multimers of the invention are formed by covalent associations with and/or between the polypeptides of the invention. Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence ( e.g., that recited in the sequence listing, or contained in the polypeptide encoded by a deposited clone). In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occurring) polypeptide. In another instance, the covalent associations are the consequence of chemical or recombinant manipulation. Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a fusion protein of the invention.

In one example, covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., US Patent Number 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in an Fc fusion protein of the invention (as described herein). In another specific example, covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another protein that is capable of forming covalently associated multimers, such as for example, oseteoprotegeπn (see, e.g.,

International Publication NO: WO 98/49305, the contents of which are herein incorporated by reference in its entirety). In another embodiment, two or more polypeptides of the invention are joined through peptide linkers. Examples include those peptide linkers descnbed in U.S. Pat. No. 5,073,627 (hereby incorporated by reference). Proteins comprising multiple polypeptides of the invention separated by peptide linkers may be pioduced using conventional recombinant DNA technology

Another method for preparing multimer polypeptides of the invention involves use of polypeptides of the invention fused to a leucine zipper or isoleucme zipper polypeptide sequence Leucine zipper and isoleucme zipper domains are polypeptides that promote multimeπzation of the proteins in which they are found Leucine zippers were originally identified in several DNA-bind g proteins (Landschulz et al., Science 240:1759, (1988)), and have since been found in a variety of different proteins Among the known leucine zippers are naturally occurring peptides and denvatives thereof that dimeπze or tnmeπze Examples of leucine zipper domains suitable for producing soluble multimenc proteins of the invention are those descnbed in PCT application WO 94/10308, hereby incorporated by reference. Recombinant fusion proteins compnsmg a polypeptide of the invention fused to a polypeptide sequence that dimenzes or tπmenzes in solution are expressed suitable host cells, and the resulting soluble multimenc fusion protein is recovered from the culture supernatant using techniques known in the art.

Tπmenc polypeptides of the invention may offer the advantage of enhanced biological activity. Preferred leucine zipper moieties and isoleucme moieties are those that preferentially form tnmers. One example is a leucme zipper deπved from lung surfactant protein D (SPD), as descπbed m Hoppe et al. (FEBS Letters 344:191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby incorporated by reference. Other peptides deπved from naturally occurnng tnmeπc proteins may be employed in prepanng tπmenc polypeptides of the invention. In another example, proteins of the invention are associated by interactions between Flag® polypeptide sequence contained in fusion proteins of the invention containing Flag® polypeptide seuqence. In a further embodiment, associations proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in Flag® fusion proteins of the invention and anti-Flag® antibody.

The multimers of the invention may be generated using chemical techniques known in the art. For example, polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety). Additionally, multimers of the invention may be generated using techniques known in the art to form one or more inter- molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., US Patent Number 5,478,925, which is herein incoiporated by reference in its entirety). Further, polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., US Patent Number 5,478,925, which is herein incoiporated by reference in its entirety). Additionally, techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using genetic engineering techniques known in the art. In one embodiment, polypeptides contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety). In a specific embodiment, polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N- terminus (lacking the leader sequence) (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety). In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain (or hyrophobic or signal peptide) and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).

Uses of the Polynucleotides

Each of the polynucleotides identified herein can be used in numerous ways as reagents. The following description should be considered exemplary and utilizes known techniques.

The polynucleotides of the present invention are useful for chromosome identification. There exists an ongoing need to identify new chromosome markers, since few chromosome marking reagents, based on actual sequence data (repeat polymorphisms), are presently available. Each polynucleotide of the present invention can be used as a chromosome marker.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the sequences shown in SEQ ID NO:X. Primers can be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the SEQ ID NO:X will yield an amplified fragment.

Similarly, somatic hybrids provide a rapid method of PCR mapping the polynucleotides to particular chromosomes. Three or more clones can be assigned per day using a single thermal cycler. Moreover, sublocalization of the polynucleotides can be achieved with panels of specific chromosome fragments. Other gene mapping strategies that can be used include in situ hybridization, prescreening with labeled flow-sorted chromosomes, and preselection by hybridization to construct chromosome specific-cDNA libraries.

Precise chromosomal location of the polynucleotides can also be achieved using fluorescence in situ hybridization (FISH) of a metaphase chromosomal spread. This technique uses polynucleotides as short as 500 or 600 bases; however, polynucleotides

2,000-4,000 bp are preferred. For a review of this technique, see Verma et al., "Human Chromosomes: a Manual of Basic Techniques," Pergamon Press, New York (1988).

For chromosome mapping, the polynucleotides can be used individually (to mark a single chromosome or a single site on that chromosome) or in panels (for marking multiple sites and/or multiple chromosomes). Preferred polynucleotides correspond to the noncoding regions of the cDNAs because the coding sequences are more likely conserved within gene families, thus increasing the chance of cross hybridization during chromosomal mapping.

Once a polynucleotide has been mapped to a precise chromosomal location, the physical position of the polynucleotide can be used in linkage analysis. Linkage analysis establishes coinheritance between a chromosomal location and presentation of a particular disease. (Disease mapping data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library) .) Assuming 1 megabase mapping resolution and one gene per 20 kb, a cDNA precisely localized to a chromosomal region associated with the disease could be one of 50-500 potential causative genes. Thus, once coinheritance is established, differences in the polynucleotide and the corresponding gene between affected and unaffected individuals can be examined. First, visible structural alterations in the chromosomes, such as deletions or translocations, are examined in chromosome spreads or by PCR. If no structural alterations exist, the presence of point mutations are ascertained. Mutations observed in some or all affected individuals, but not in normal individuals, indicates that the mutation may cause the disease. However, complete sequencing of the polypeptide and the corresponding gene from several normal individuals is required to distinguish the mutation from a polymorphism. If a new polymoφhism is identified, this polymoφhic polypeptide can be used for further linkage analysis.

Furthermore, increased or decreased expression of the gene in affected individuals as compared to unaffected individuals can be assessed using polynucleotides of the present invention. Any of these alterations (altered expression, chromosomal rearrangement, or mutation) can be used as a diagnostic or prognostic marker.

Thus, the invention also provides a diagnostic method useful during diagnosis of a disorder, involving measuring the expression level of polynucleotides of the present invention in cells or body fluid from an individual and comparing the measured gene expression level with a standard level of polynucleotide expression level, whereby an increase or decrease in the gene expression level compared to the standard is indicative of a disorder.

In still another embodiment, the invention includes a kit for analyzing samples for the presence of proliferative and/or cancerous polynucleotides derived from a test subject. In a general embodiment, the kit includes at least one polynucleotide probe containing a nucleotide sequence that will specifically hybridize with a polynucleotide of the present invention and a suitable container. In a specific embodiment, the kit includes two polynucleotide probes defining an internal region of the polynucleotide of the present invention, where each probe has one strand containing a 31'mer-end internal to the region. In a further embodiment, the probes may be useful as primers for polymerase chain reaction amplification.

Where a diagnosis of a disorder, has already been made according to conventional methods, the present invention is useful as a prognostic indicator, whereby patients exhibiting enhanced or depressed polynucleotide of the present invention expression will experience a worse clinical outcome relative to patients expressing the gene at a level nearer the standard level.

By "measuring the expression level of polynucleotide of the present invention" is intended qualitatively or quantitatively measuring or estimating the level of the polypeptide of the present invention or the level of the mRNA encoding the polypeptide in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the polypeptide level or mRNA level in a second biological sample). Preferably, the polypeptide level or mRNA level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having a disorder. As will be appreciated in the art, once a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison. By "biological sample" is intended any biological sample obtained from an individual, body fluid, cell line, tissue culture, or other source which contains the polypeptide of the present invention or mRNA. As indicated, biological samples include body fluids (such as semen, lymph, sera, plasma, urine, synovial fluid and spinal fluid) which contain the polypeptide of the present invention, and other tissue sources found to express the polypeptide of the present invention. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.

The method(s) provided above may preferrably be applied in a diagnostic method and/or kits in which polynucleotides and/or polypeptides are attached to a solid support. In one exemplary method, the support may be a "gene chip" or a "biological chip" as described in US Patents 5,837,832, 5,874,219, and 5,856,174. Further, such a gene chip with polynucleotides of the present invention attached may be used to identify polymoφhisms between the polynucleotide sequences, with polynucleotides isolated from a test subject. The knowledge of such polymoφhisms (i.e. their location, as well as, their existence) would be beneficial in identifying disease loci for many disorders, including cancerous diseases and conditions. Such a method is described in US Patents 5,858,659 and 5,856,104. The US Patents referenced supra are hereby incoφorated by reference in their entirety herein.

The present invention encompasses polynucleotides of the present invention that are chemically synthesized, or reproduced as peptide nucleic acids (PNA), or according to other methods known in the art. The use of PNAs would serve as the preferred form if the polynucleotides are incoφorated onto a solid support, or gene chip. For the puφoses of the present invention, a peptide nucleic acid (PNA) is a polyamide type of DNA analog and the monomeric units for adenine, guanine, thymine and cytosine are available commercially (Perceptive Biosystems). Certain components of DNA, such as phosphorus, phosphorus oxides, or deoxyribose derivatives, are not present in PNAs. As disclosed by P. E. Nielsen, M. Egholm, R. H. Berg and O. Buchardt, Science 254, 1497 (1991); and M. Egholm, O. Buchardt, L.Christensen, C. Behrens, S. M. Freier, D. A. Driver, R. H. Berg, S. K. Kim, B. Norden, and P. E. Nielsen, Nature 365, 666 (1993), PNAs bind specifically and tightly to complementary DNA strands and are not degraded by nucleases. In fact, PNA binds more strongly to DNA than DNA itself does. This is probably because there is no electrostatic repulsion between the two strands, and also the polyamide backbone is more flexible. Because of this, PNA/DNA duplexes bind under a wider range of stringency conditions than DNA/DNA duplexes, making it easier to perform multiplex hybridization. Smaller probes can be used than with DNA due to the strong binding. In addition, it is more likely that single base mismatches can be determined with PNA DNA hybridization because a single mismatch in a PNA/DNA 15-mer lowers the melting point (T.sub.m) by 8°-20° C, vs. 4°-16° C for the DNA/DNA 15-mer duplex. Also, the absence of charge groups in PNA means that hybridization can be done at low ionic strengths and reduce possible interference by salt during the analysis. The present invention is useful for detecting cancer in mammals. In particular the invention is useful during diagnosis of pathological cell proliferative neoplasias which include, but are not limited to: acute myelogenous leukemias including acute monocytic leukemia, acute _.myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute erythroleukemia, acute megakaryocytic leukemia, and acute undifferentiated leukemia, etc.; and chronic myelogenous leukemias including chronic myelomonocytic leukemia, chronic granulocytic leukemia, etc. Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans.

Particularly preferred are humans. Pathological cell proliferative disorders are often associated with inappropriate activation of proto-oncogenes. (Gelmann, E. P. et al., "The Etiology of Acute Leukemia: Molecular Genetics and Viral Oncology," in Neoplastic Diseases of the Blood, Vol 1., Wiemik, P. H. et al. eds., 161-182 (1985)). Neoplasias are now believed to result from the qualitative alteration of a normal cellular gene product, or from the quantitative modification of gene expression by insertion into the chromosome of a viral sequence, by chromosomal translocation of a gene to a more actively transcribed region, or by some other mechanism. (Gelmann et al., supra) It is likely that mutated or altered expression of specific genes is involved in the pathogenesis of some leukemias, among other tissues and cell types. (Gelmann et al., supra) Indeed, the human counteφarts of the oncogenes involved in some animal neoplasias have been amplified or translocated in some cases of human leukemia and carcinoma. (Gelmann et al., supra)

For example, c-myc expression is highly amplified in the non-lymphocytic leukemia cell line HL-60. When HL-60 cells are chemically induced to stop proliferation, the level of c- myc is found to be downregulated. (International Publication Number WO 91/15580) However, it has been shown that exposure of HL-60 cells to a DNA construct that is complementary to the 5' end of c-myc or c-myb blocks translation of the corresponding mRNAs which downregulates expression of the c-myc or c-myb proteins and causes arrest of cell proliferation and differentiation of the treated cells. (International Publication Number WO 91/15580; Wickstrom et al., Proc. Natl. Acad. Sci. 85:1028 (1988); Anfossi et al., Proc. Natl. Acad. Sci. 86:3379 (1989)). However, the skilled artisan would appreciate the present invention's usefulness would not be limited to treatment of proliferative disorders of hematopoietic cells and tissues, in light of the numerous cells and cell types of varying origins which are known to exhibit proliferative phenotypes.

In addition to the foregoing, a polynucleotide can be used to control gene expression through triple helix formation or antisense DNA or RNA. Antisense techniques are discussed, for example, in Okano, J. Neurochem. 56: 560 (1991);

"Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression,CRCPress, Boca Raton, FL (1988). Triple helix formation is discussed in, for instance Lee et al, Nucleic Acids Research 6: 3073 (1979); Cooney et al., Science 241 : 456 (1988); and Dervan et al., Science 251 : 1360 (1991). Both methods rely on binding of the polynucleotide to a complementary DNA or RNA. For these techniques, preferred polynucleotides are usually oligonucleotides 20 to 40 bases in length and complementary to either the region of the gene involved in transcription (triple helix - see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991) ) or to the mRNA itself (antisense - Okano, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988).) Triple helix formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques are effective in model systems, and the information disclosed herein can be used to design antisense or triple helix polynucleotides in an effort to treat disease.

Polynucleotides of the present invention are also useful in gene therapy. One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the genetic defect. The polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner. Another goal is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell.

The polynucleotides are also useful for identifying individuals from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymoφhism (RFLP) for identification of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identifying personnel.

This method does not suffer from the cmrent limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult. The polynucleotides of the present invention can be used as additional DNA markers for RFLP.

The polynucleotides of the present invention can also be used as an alternative to RFLP, by determining the actual base-by-base DNA sequence of selected portions of an individual's genome. These sequences can be used to prepare PCR primers for amplifying and isolating such selected DNA, which can then be sequenced. Using this technique, individuals can be identified because each individual will have a unique set of DNA sequences. Once an unique ID database is established for an individual, positive identification of that individual, living or dead, can be made from extremely small tissue samples.

Forensic biology also benefits from using DNA-based identification techniques as disclosed herein. DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, semen, synovial fluid, amniotic fluid, breast milk, lymph, pulmonary sputum or surfactant,urine,fecal matter, etc., can be amplified using PCR. In one prior art technique, gene sequences amplified from polymoφhic loci, such as DQa class II HLA gene, are used in forensic biology to identify individuals. (Erlich, H., PCR Technology, Freeman and Co. (1992).) Once these specific polymoφhic loci are amplified, they are digested with one or more restriction enzymes, yielding an identifying set of bands on a Southern blot probed with DNA corresponding to the DQa class II HLA gene. Similarly, polynucleotides of the present invention can be used as polymoφhic markers for forensic puφoses.

There is also a need for reagents capable of identifying the source of a particular tissue. Such need arises, for example, in forensics when presented with tissue of unknown origin. Appropriate reagents can comprise, for example, DNA probes or primers specific to particular tissue prepared from the sequences of the present invention. Panels of such reagents can identify tissue by species and/or by organ type. In a similar fashion, these reagents can be used to screen tissue cultures for contamination.

In the very least, the polynucleotides of the present invention can be used as molecular weight markers on Southern gels, as diagnostic probes for the presence of a specific mRNA in a particular cell type, as a probe to "subtract-out" known sequences in the process of discovering novel polynucleotides, for selecting and making oligomers for attachment to a "gene chip" or other support, to raise anti-DNA antibodies using DNA immunization techniques, and as an antigen to elicit an immune response.

Uses of the Polypeptides

Each of the polypeptides identified herein can be used in numerous ways. The following description should be considered exemplary and utilizes known techniques. A polypeptide of the present invention can be used to assay protein levels in a biological sample using antibody-based techniques. For example, protein expression in tissues can be studied with classical immunohistological methods. (Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell . Biol. 105:3087-3096 (1987).) Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine (1251, 1211), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin. In addition to assaying secreted protein levels in a biological sample, proteins can also be detected in vivo by imaging. Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, NMR or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incoφorated into the antibody by labeling of nutrients for the relevant hybridoma.

A protein-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example, 1311, 112In, 99mTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously, or intraperitoneally) into the mammal. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S.W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments." (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).)

Thus, the invention provides a diagnostic method of a disorder, which involves (a) assaying the expression of a polypeptide of the present invention in cells or body fluid of an individual; (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.

Moreover, polypeptides of the present invention can be used to treat disease. For example, patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., insulin), to supplement absent oi decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B,

SOD, catalase, DNA repair proteins), to inhibit the activity of a polypeptide (e.g., an oncogene or tumor supressor), to activate the activity of a polypeptide (e.g., by binding to a receptor), to reduce the activity of a membrane bound receptor by competing with it tot- free ligand (e.g., soluble TNF receptors used in reducing inflammation), oi to bπng about a desired response (e.g., blood vessel growth inhibition, enhancement of the immune response to proliferative cells or tissues)

Similarly, antibodies directed to a polypeptide of the present invention can also be used to treat disease. For example, administration of an antibody directed to a polypeptide of the present invention can bind and reduce oveφroduction of the polypeptide. Similarly, administration of an antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor)

At the very least, the polypeptides of the present invention can be used as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art. Polypeptides can also be used to raise antibodies, which in turn are used to measure protein expression from a recombinant cell, as a way of assessing transformation of the host cell. Moreover, the polypeptides of the present invention can be used to test the following biological activities. Gene Therapy Methods

Another aspect of the present invention is to gene therapy methods for treating disorders, diseases and conditions. The gene therapy methods relate to the introduction of nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into an animal to achieve expression of a polypeptide of the present invention. This method requires a polynucleotide which codes for a polypeptide of the invention that operatively linked to a promoter and any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques are known in the art, see, for example, WO90/11092, which is herein incoφorated by reference. Thus, for example, cells from a patient may be engineered with a polynucleotide

(DNA or RNA) comprising a promoter operably linked to a polynucleotide of the invention ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide. Such methods are well-known in the art. For example, see Belldegrun et al., J. Natl. Cancer Inst., 85:207-216 (1993); Ferrantini et al., Cancer Research. 53:107-1112 (1993); Ferrantini et al., J. Immunology 153: 4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995); Ogura et al., Cancer Research 50: 5102-5106 (1990); Santodonato, et al., Human Gene Therapy 7:1-10 (1996); Santodonato, et al., Gene Therapy 4:1246-1255 (1997); and Zhang, et al., Cancer Gene Therapy 3: 31-38 (1996)), which are herein incoφorated by reference. In one embodiment, the cells which are engineered are arterial cells. The arterial cells may be reintroduced into the patient through direct injection to the artery, the tissues surrounding the artery, or through catheter injection.

As discussed in more detail below, the polynucleotide constructs can be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, and the like). The polynucleotide constructs may be delivered in a pharmaceutically acceptable liquid or aqueous carrier. In one embodiment, the polynucleotide of the invention is delivered as a naked polynucleotide. The term "naked" polynucleotide, DNA or RNA refers to sequences that are free from any delivery vehicle that acts to assist, promote or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, the polynucleotides of the invention can also be delivered in liposome formulations and lipofectin formulations and the like can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Patent Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein incoφorated by reference. The polynucleotide vector constructs of the invention used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL available from Pharmacia; and pEFl/V5, pcDNA3J, and pRc/CMV2 available from Invitrogen. Other suitable vectors will be readily apparent to the skilled artisan.

Any strong promoter known to those skilled in the art can be used for driving the expression of polynucleotide sequence of the invention. Suitable promoters include adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Heφes Simplex thymidine kinase promoter; retroviral LTRs; the b-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter for the polynucleotides of the invention.

Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.

The polynucleotide construct of the invention can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular, fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.

For the naked/TMc/e/c acid sequence injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 mg/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration.

The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked DNA constructs can be delivered to artenes duπng angioplasty by the catheter used in the procedure.

The naked polynucleotides are delivered by any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, cathetei infusion, and so-called "gene guns" These delivery methods aie known in the ait.

The constructs may also be dehveied with dehveiy vehicles such as viral sequences, vnal particles, liposome formulations, lipofectin, precipitating agents, etc Such methods of delivery are known in the art

In certain embodiments, the polynucleotide consttucts of the invention are complexed in a liposome preparation. Liposomal preparations for use in the instant invention include cationic (positively charged), anionic (negatively charged) and neutral preparations However, cationic liposomes are particularly preferred because a tight charge complex can be formed between the cationic liposome and the polyanionic nucleic acid Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Feigner et al., Proc. Natl. Acad. Sci. USA , 84 7413-7416 (1987), which is herein incoφorated by reference); mRNA (Malone et al , Proc. Natl. Acad. Sci. USA , 86:6077-6081 (1989), which is herein incoφorated by reference); and punfied transcnption factors (Debs et al., J. Biol. Chem., 265:10189-10192 (1990), which is herein incoφorated by reference), in functional form.

Cationic liposomes are readily available. For example, N[l-2,3-dιoleyloxy)propyl]-N,N,N-tnethylammonιum (DOTMA) liposomes are particularly useful and are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y (See, also, Feigner et al , Proc Natl Acad. Sci. USA , 84:7413-7416 (1987), which is herein incoφorated by reference). Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boehnnger). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g. PCT Publication NO: WO 90/11092 (which is herein incoφorated by reference) for a description of the synthesis of DOTAP (1,2- bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparation of DOTMA liposomes is explained in the literature, see, e.g., Feigner et al., Proc. Natl. Acad. Sci.

USA, 84:7413-7417, which is herein incoiporated by reference. Similar methods can be used to prepare liposomes from other cationic lipid materials.

Similarly, anionic and neutral liposomes are readily available, such as from Avanti

Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials. Such materials include phosphatidyl, choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art. For example, commercially dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine (DOPE) can be used in various combinations to make conventional liposomes, with or without the addition of cholesterol. Thus, for example, DOPG DOPC vesicles can be prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas into a sonication vial. The sample is placed under a vacuum pump overnight and is hydrated the following day with deionized water. The sample is then sonicated for 2 hours in a capped vial, using a Heat Systems model 350 sonicator equipped with an inverted cup (bath type) probe at the maximum setting while the bath is circulated at 15EC. Alternatively, negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles or by extrusion through nucleopore membranes to produce unilamellar vesicles of discrete size. Other methods are known and available to those of skill in the art.

The liposomes can comprise multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs being preferred. The various liposome-nucleic acid complexes are prepared using methods well known in the art. See, e.g., Straubinger et al., Methods of Immunology , 101:512-527 (1983), which is herein incoφorated by reference. For example, MLVs containing nucleic acid can be prepared by depositing a thin film of phospholipid on the walls of a glass tube and subsequently hydrating with a solution of the material to be encapsulated. SUVs are prepared by extended sonication of MLVs to produce a homogeneous population of unilamellar liposomes. The material to be entrapped is added to a suspension of preformed

MLVs and then sonicated. When using liposomes containing cationic lipids, the dried lipid film is resuspended in an appropriate solution such as sterile water or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated, and then the preformed liposomes are mixed directly with the DNA. The liposome and DNA form a very stable complex due to binding of the positively charged liposomes to the cationic DNA. SUVs find use with small nucleic acid fragments. LUVs are prepared by a number of methods, well known in the art. Commonly used methods include Ca²⁺-EDTA chelation (Papahadjopoulos et al., Biochim. Biophys. Acta, 394:483 (1975); Wilson et al., Cell , 17:77 (1979)); ether injection (Deamer et al., Biochim. Biophys. Acta, 443:629 (1976); Ostro et al., Biochem. Biophys. Res. Commun., 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA, 76:3348 (1979)); detergent dialysis (Enoch et al., Proc. Natl. Acad. Sci. USA , 76:145 (1979)); and reverse-phase evaporation (REV) (Fraley et al., J. Biol. Chem., 255:10431 (1980); Szoka et al., Proc. Natl. Acad. Sci. USA , 75:145 (1978); Schaefer-Ridder et al., Science, 215:166 (1982)), which are herein incoφorated by reference.

Generally, the ratio of DNA to liposomes will be from about 10:1 to about 1:10. Preferably, the ration will be from about 5:1 to about 1:5. More preferably, the ration will be about 3:1 to about 1:3. Still more preferably, the ratio will be about 1:1. U.S. Patent NO: 5,676,954 (which is herein incoφorated by reference) reports on the injection of genetic material, complexed with cationic liposomes carriers, into mice. U.S. Patent Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication NO: WO 94/9469 (which are herein incoφorated by reference) provide cationic lipids for use in transfecting DNA into cells and mammals. U.S. Patent Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication NO: WO 94/9469 (which are herein incoφorated by reference) provide methods for delivering DNA-cationic lipid complexes to mammals. In certain embodiments, cells are engineered, ex vivo or in vivo, using a retroviral particle containing RNA which comprises a sequence encoding polypeptides of the invention. Retroviruses from which the retroviral plasmid vectors may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.

The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86. GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy , 1:5-14 (1990), which is incoφorated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO₄ precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particles which include polynucleotide encoding polypeptides of the invention. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express polypeptides of the invention. In certain other embodiments, cells are engineered, ex vivo or in vivo, with polynucleotides of the invention contained in an adenovirus vector. Adenovirus can be manipulated such that it encodes and expresses polypeptides of the invention, and at the same time is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. Adenovirus expression is achieved without integration of the viral DNA into the host cell chromosome, thereby alleviating concerns about insertional mutagenesis. Furthermore, adenoviruses have been used as live enteric vaccines for many years with an excellent safety profile (Schwartzet al., Am. Rev. Respir. Dis., 109:233-238 (1974)). Finally, adenovirus mediated gene transfer has been demonstrated in a number of instances including transfer of alpha- 1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld et al., Science , 252:431-434 (1991); Rosenfeld et al., Cell, 68: 143-155 (1992)). Furthermore, extensive studies to attempt to establish adenovirus as a causative agent in human cancer were uniformly negative (Green et al. Proc. Natl. Acad. Sci. USA , 76:6606 (1979)). Suitable adenoviral vectors useful in the present invention are described, for example, in Kozarsky and Wilson,

Opin. Genet. Devel., 3:499-503 (1993); Rosenfeld et al., Cell , 68:143-155 (1992); Engelhardt et al., Human Genet. Ther., 4:759-769 (1993); Yang et al., Nature Genet., 7:362-369 (1994); Wilson et al., Nature , 365:691-692 ( 1993); and U.S. Patent NO: 5,652,224, which are herein incoφorated by reference. For example, the adenovirus vector Ad2 is useful and can be grown in human 293 cells. These cells contain the El region of adenovirus and constitutively express Ela and Elb, which complement the defective adenoviruses by providing the products of the genes deleted from the vector. In addition to Ad2, other varieties of adenovirus (e.g., Ad3, Ad5, and Ad7) are also useful in the present invention. Preferably, the adenoviruses used in the present invention are replication deficient.

Replication deficient adenoviruses require the aid of a helper virus and/or packaging cell line to form infectious particles. The resulting virus is capable of infecting cells and can express a polynucleotide of interest which is operably linked to a promoter, but cannot replicate in most cells. Replication deficient adenoviruses may be deleted in one or more of all or a portion of the following genes: Ela, Elb, E3, E4, E2a, or LI through L5.

In certain other embodiments, the cells are engineered, ex vivo or in vivo, using an adeno-associated virus (AAV). AAVs are naturally occurring defective viruses that require helper viruses to produce infectious particles (Muzyczka, Curr. Topics in Microbiol. Immunol., 158:97 (1992)). It is also one of the few viruses that may integrate its DNA into non-dividing cells. Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate, but space for exogenous DNA is limited to about 4.5 kb.

Methods for producing and using such AAVs are known in the art. See, for example, U.S. Patent Nos. 5,139,941, 5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and

5,589,377.

For example, an appropriate AAV vector for use in the present invention will include all the sequences necessary for DNA replication, encapsidation, and host-cell integration. The polynucleotide construct containing polynucleotides of the invention is inserted into the AAV vector using standard cloning methods, such as those found in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989). The recombinant AAV vector is then transfected into packaging cells which are infected with a helper virus, using any standard technique, including lipofection. electroporation, calcium phosphate precipitation, etc. Appropriate helper viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or heφes viruses. Once the packaging cells are transfected and infected, they will produce infectious AAV viral particles which contain the polynucleotide construct of the invention. These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo. The transduced cells will contain the polynucleotide construct integrated into its genome, and will express the desired gene product.

Another method of gene therapy involves operably associating heterologous control regions and endogenous polynucleotide sequences (e.g. encoding the polypeptide sequence of interest) via homologous recombination (see, e.g., U.S. Patent NO: 5,641,670, issued June 24, 1997; International Publication NO: WO 96/29411, published September 26, 1996; International Publication NO: WO 94/12650, published August 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); and Zijlstra et al., Nature, 342:435-438 (1989). This method involves the activation of a gene which is present in the target cells, but which is not normally expressed in the cells, or is expressed at a lower level than desired.

Polynucleotide constructs are made, using standard techniques known in the art, which contain the promoter with targeting sequences flanking the promoter. Suitable promoters are described herein. The targeting sequence is sufficiently complementary to an endogenous sequence to permit homologous recombination of the promoter-targeting sequence with the endogenous sequence. The targeting sequence will be sufficiently near the 5' end of the desired endogenous polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination. The promoter and the targeting sequences can be amplified using PCR. Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5' and 3' ends. Preferably, the 3' end of the first targeting sequence contains the same restriction enzyme site as the 5' end of the amplified promoter and the 5' end of the second targeting sequence contains the same restriction site as the 3' end of the amplified promoter. The amplified promoter and targeting sequences are digested and ligated together.

The promoter-targeting sequence construct is delivered to the cells, either as naked polynucleotide, or in conjunction with transfection-facilitating agents, such as liposomes, viral sequences, viral particles, whole viruses, lipofection, precipitating agents, etc., described in more detail above. The P promoter-targeting sequence can be delivered by any method, included direct needle injection, intravenous injection, topical administration, catheter infusion, particle accelerators, etc. The methods are described in more detail below.

The promoter-targeting sequence construct is taken up by cells. Homologous recombination between the construct and the endogenous sequence takes place, such that an endogenous sequence is placed under the control of the promoter. The promoter then drives the expression of the endogenous sequence.

The polynucleotides encoding polypeptides of the present invention may be administered along with other polynucleotides encoding other angiongenic proteins. Angiogenic proteins include, but are not limited to, acidic and basic fibroblast growth factors, VEGF-1, VEGF-2 (VEGF-C), VEGF-3 (VEGF-B), epidermal growth factor alpha and beta, platelet-derived endothelial cell growth factor, platelet-derived growth factor, tumor necrosis factor alpha, hepatocyte growth factor, insulin like growth factor, colony stimulating factor, macrophage colony stimulating factor, granulocyte/macrophage colony stimulating factor, and nitric oxide synthase.

Preferably, the polynucleotide encoding a polypeptide of the invention contains a secretory signal sequence that facilitates secretion of the protein. Typically, the signal sequence is positioned in the coding region of the polynucleotide to be expressed towards or at the 5' end of the coding region. The signal sequence may be homologous or heterologous to the polynucleotide of interest and may be homologous or heterologous to the cells to be transfected. Additionally, the signal sequence may be chemically synthesized using methods known in the art.

Any mode of administration of any of the above-described polynucleotides constructs can be used so long as the mode results in the expression of one or more molecules in an amount sufficient to provide a therapeutic effect. This includes direct needle injection, systemic injection, catheter infusion, biolistic injectors, particle accelerators (i.e., "gene guns"), gelfoam sponge depots, other commercially available depot materials, osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid (tablet or pill) pharmaceutical formulations, and decanting or topical applications during surgery. For example, direct injection of naked calcium phosphate-precipitated plasmid into rat liver and rat spleen or a protein-coated plasmid into the portal vein has resulted in gene expression of the foreign gene in the rat livers. (Kaneda et al., Science, 243:375 (1989)). A preferred method of local administration is by direct injection. Preferably, a recombinant molecule of the present invention complexed with a delivery vehicle is administered by direct injection into or locally within the area of arteries. Administration of a composition locally within the area of arteries refers to injecting the composition centimeters and preferably, millimeters within arteries. Another method of local administration is to contact a polynucleotide construct of the present invention in or around a surgical wound. For example, a patient can undergo surgery and the polynucleotide construct can be coated on the surface of tissue inside the wound or the construct can be injected into areas of tissue inside the wound. Therapeutic compositions useful in systemic administration, include recombinant molecules of the present invention complexed to a targeted delivery vehicle of the present invention. Suitable delivery vehicles for use with systemic administration comprise liposomes comprising ligands for targeting the vehicle to a particular site.

Preferred methods of systemic administration, include intravenous injection, aerosol, oral and percutaneous (topical) delivery. Intravenous injections can be performed using methods standard in the art. Aerosol delivery can also be performed using methods standard in the art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA , 189:11277-11281 (1992), which is incoφorated herein by reference). Oral delivery can be performed by complexing a polynucleotide construct of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal.

Examples of such carriers, include plastic capsules or tablets, such as those known in the art. Topical delivery can be performed by mixing a polynucleotide construct of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into the skin. Determining an effective amount of substance to be delivered can depend upon a number of factors including, for example, the chemical structure and biological activity of the substance, the age and weight of the animal, the precise condition requiring treatment and its severity, and the route of administration. The frequency of treatments depends upon a number of factors, such as the amount of polynucleotide constructs administered per dose, as well as the health and history of the subject. The precise amount, number of doses, and timing of doses will be determined by the attending physician or veterinarian. Therapeutic compositions of the present invention can be administered to any animal, preferably to mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs, with humans being particularly Biological Activities

The polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used in assays to test for one or more biological activities If these polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity Thus, the polynucleotides or polypeptides, or agonists or antagonists could be used to treat the associated disease.

Immune Activity

The polynucleotides or polypeptides, or agonists or antagonists of the present invention may be useful in treating deficiencies or disorders of the immune system, by activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of immune cells Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluπpotent stem cells The etiology of these immune deficiencies or disorders may be genetic, somatic, such as cancer or some autoimmune disorders, acquired (e.g., by chemotherapy or toxins), or infectious. Moreover, a polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used as a marker or detector of a particular immune system disease or disorder.

A polynucleotides or polypeptides, or agonists or antagonists of the present invention may be useful in treating or detecting deficiencies or disorders of hematopoietic cells. A polynucleotides or polypeptides, or agonists or antagonists of the present invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluπpotent stem cells, in an effort to treat those disorders associated with a decrease in certain (or many) types hematopoietic cells. Examples of immunologic deficiency syndromes include, but are not limited to blood protein disorders (e.g. agammaglobul emia, dysgammaglobuhnemia), ataxia telangiectasia, common vanable immunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction, severe combined immunodeficiency (SCIDs), Wiskott-Aldnch Disorder, anemia, thrombocytopenia, or hemoglobinuna Moreover, a polynucleotides or polypeptides, or agonists or antagonists of the present invention could also be used to modulate hemostatic (the stopping of bleeding) oi thrombolytic activity (clot formation). For example, by inci easing hemostatic or thiombolytic activity, a polynucleotides or polypeptides, or agonists or antagonists of the present invention could be used to treat blood coagulation disorders (e.g , afibπnogenemia, factoi deficiencies), blood platelet disorders (e.g thrombocytopenia), or wounds resulting from tiauma, surgery, or other causes Alternatively, a polynucleotides or polypeptides, ot agonists ot antagonists of the present invention that can decrease hemostatic or thrombolytic activity could be used to inhibit or dissolve clotting These molecules could be important in the treatment of heart attacks (infarction), strokes, or scarπng. A polynucleotides or polypeptides, or agonists or antagonists of the present invention may also be useful m treating or detecting autoimmune disorders. Many autoimmune disorders result from inappropπate recognition of self as foreign matenal by immune cells. This inappropπate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of a polynucleotides or polypeptides, or agonists or antagonists of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing autoimmune disorders.

Examples of autoimmune disorders that can be treated or detected by the present invention include, but are not limited to- Addison's Disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephπtis, Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, Myasthenia Gravis, Neuntis, Ophthalmia, Bullous Pemphigoid, Pemphigus, Polyendocπnopathies, Puφura, Reiter's Disease, Stiff -Man Syndrome, Autoimmune Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonaiy Inflammation,

Guillain-Barre Syndrome, insulin dependent diabetes melhtis, and autoimmune inflammatory eye disease.

Similarly, allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated by a polynucleotides or polypeptides, or agonists or antagonists of the present invention Moieover, these molecules can be used to treat anaphylaxts, hypei sensitivity to an antigenic molecule, oi blood group incompatibility

A polynucleotides or polypeptides, ot agonists or antagonists of the present invention may also be used to tieat and/oi prevent organ rejection or graft-versus-host disease (GVHD) Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response Similarly, an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues The administration of a polynucleotides oi polypeptides, or agonists oi antagonists of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing organ rejection or GVHD

Similarly, a polynucleotides or polypeptides, or agonists or antagonists of the present invention may also be used to modulate inflammation For example, the polypeptide or polynucleotide oi agonists or antagonist may inhibit the proliferation and differentiation of cells involved m an inflammatory response These molecules can be used to treat inflammatory conditions, both chronic and acute conditions, including inflammation associated with infection (e.g , septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephntis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, or resulting from over production of cytokines (e.g., TNF or IL-1 ) Hyperproliferative Disorders

A polynucleotides or polypeptides, or agonists or antagonists of the invention can be used to treat or detect hypeφroliferative disorders, including neoplasms. A polynucleotides or polypeptides, or agonists or antagonists of the present invention may inhibit the proliferation of the disorder through direct or indirect interactions.

Alternatively, a polynucleotides or polypeptides, or agonists or antagonists of the present invention may proliferate other cells which can inhibit the hypeφroliferative disorder.

For example, by increasing an immune response, particularly increasing antigenic qualities of the hypeφroliferative disorder or by proliferating, differentiating, or mobilizing T-cells, hypeφroliferative disorders can be treated. This immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, decreasing an immune response may also be a method of treating hypeφroliferative disorders, such as a chemotherapeutic agent.

Examples of hypeφroliferative disorders that can be treated or detected by a polynucleotides or polypeptides, or agonists or antagonists of the present invention include, but are not limited to neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital. Similarly, other hypeφroliferative disorders can also be treated or detected by a polynucleotides or polypeptides, or agonists or antagonists of the present invention. Examples of such hypeφroliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, puφura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hypeφroliferative disease, besides neoplasia, located in an organ system listed above. One pieferred embodiment utilizes polynucleotides of the present invention to inhibit aberrant cellular division, by gene therapy using the present invention, and/or protein fusions or fragments thereof.

Thus, the present invention provides a method for treating cell proliferative disorders by inserting into an abnormally proliferating cell a polynucleotide of the present invention, wherein said polynucleotide represses said expression.

Another embodiment of the piesent invention provides a method of treating cell- prohferative disorders in individuals comprising administration of one or more active gene copies of the present invention to an abnormally proliferating cell or cells. In a preferred embodiment, polynucleotides of the piesent invention is a DNA construct comprising a recombinant expression vector effective in expressing a DNA sequence encoding said polynucleotides. In another prefeπ-ed embodiment of the present invention, the DNA construct encoding the poynucleottdes of the piesent invention is inserted into cells to be treated utilizing a retrovirus, or more prefeπably an adenoviral vector (See G J. Nabel, et al., PNAS 1999 96: 324-326, which is hereby incorporated by reference). In a most preferred embodiment, the viral vector is defective and will not transform non-prohferatmg cells, only proliferating cells. Moreover, in a preferred embodiment, the polynucleotides of the present invention inserted into proliferating cells either alone, or in combination with or fused to other polynucleotides, can then be modulated via an external stimulus (i.e. magnetic, specific small molecule, chemical, or drug administration, etc.), which acts upon the promoter upstream of said polynucleotides to induce expression of the encoded protein product. As such the beneficial therapeutic affect of the present invention may be expressly modulated (i.e. to increase, decrease, or inhibit expression of the present invention) based upon said external stimulus. Polynucleotides of the present invention may be useful in repressing expression of oncogenic genes or antigens. By "repressing expression of the oncogenic genes " is intended the suppression of the transcription of the gene, the degradation of the gene transcπpt (pre-message RNA), the inhibition of splicing, the destruction of the messenger RNA, the prevention of the post-translational modifications of the protein, the destruction of the protein, or the inhibition of the normal function of the protein.

For local administration to abnormally proliferating cells, polynucleotides of the present invention may be administered by any method known to those of skill in the art including, but not limited to transfection, electroporation, microinjection of cells, or in vehicles such as liposomes, lipofectin, or as naked polynucleotides, or any other method described throughout the specification. The polynucleotide of the present invention may be delivered by known gene delivery systems such as, but not limited to, retroviral vectors

(Gilboa, J. Virology 44:845 (1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad. Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol. Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yates et al., Nature 313:812 (1985)) known to those skilled in the art. These references are exemplary only and are hereby incoφorated by reference. In order to specifically deliver or transfect cells which are abnormally proliferating and spare non-dividing cells, it is preferable to utilize a retrovirus, or adenoviral (as described in the art and elsewhere herein) delivery system known to those of skill in the art. Since host DNA replication is required for retroviral DNA to integrate and the retrovirus will be unable to self replicate due to the lack of the retrovirus genes needed for its life cycle. Utilizing such a retroviral delivery system for polynucleotides of the present invention will target said gene and constructs to abnormally proliferating cells and will spare the non-dividing normal cells.

The polynucleotides of the present invention may be delivered directly to cell proliferative disorder/disease sites in internal organs, body cavities and the like by use of imaging devices used to guide an injecting needle directly to the disease site. The polynucleotides of the present invention may also be administered to disease sites at the time of surgical intervention.

By "cell proliferative disease" is meant any human or animal disease or disorder, affecting any one or any combination of organs, cavities, or body parts, which is characterized by single or multiple local abnormal proliferations of cells, groups of cells, or tissues, whether benign or malignant.

Any amount of the polynucleotides of the present invention may be administered as long as it has a biologically inhibiting effect on the proliferation of the treated cells. Moreover, it is possible to administer more than one of the polynucleotide of the present invention simultaneously to the same site. By "biologically inhibiting" is meant partial oi total growth inhibition as well as decreases in the rate of proliferation or growth of the cells The biologically inhibitory dose may be determined by assessing the effects of the polynucleotides of the present invention on target malignant or abnormally proliferating cell growth m tissue culture, tumor growth in animals and cell cultures, or any othei method known to one of ordinary skill in the art.

The present invention is further directed to antibody-based therapies which involve administering of anti-polypeptides and anti-polynucleotide antibodies to a mammalian, preferably human, patient for treating one or more of the descπbed disorders. Methods for producing anti-polypeptides and anti-polynucleotide antibodies polyclonal and monoclonal antibodies are described in detail elsewhere herein. Such antibodies may be provided in pharmaceutically acceptable compositions as known in the art or as descπbed herein.

A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are descπbed in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, momtoπng or therapeutic puφoses without undue expenmentation. In particular, the antibodies, fragments and deπvatives of the present invention are useful for treating a subject having or developing cell proliferative and/or differentiation disorders as descnbed herein. Such treatment compnses administering a single or multiple doses of the antibody, or a fragment, denvative, or a conjugate thereof. The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokmes or hematopoietic growth factors, for example, which serve to increase the number or activity of effector cells which interact with the antibodies. It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, tragments oi iegions theieof, for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fiagements thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity foi polynucleotides or polypeptides, including fragements thereof. Preferred binding affinities include those with a dissociation constant or Kd less than 5X10⁶M, 10⁶M, 5X10 ⁷M, 10 ⁷M, 5X10 ⁸M, 10 ⁸M, 5X10⁹M, 10⁹M, 5X10 ^l0M, 10 ¹⁰M, 5X10 "M, 10 "M, 5X10 ^l2M, 10 ¹²M, 5X10 ^πM, 10 ^r,M, 5X10 ^l4M, 10 ^l4M, 5X10 ¹⁵M, and 10 ¹⁵M.

Moreover, polypeptides of the present invention are useful in inhibiting the angiogenesis of proliferative cells or tissues, either alone, as a protein fusion, or in combination with other polypeptides directly or indirectly, as descπbed elsewhere herein. In a most preferred embodiment, said anti-angiogenesis effect may be achieved indirectly, for example, through the inhibition of hematopoietic, tumor-specific cells, such as tumor- associated macrophages (See Joseph IB, et al. J Natl Cancer Inst, 90(21): 1648-53 (1998), which is hereby incorporated by reference). Antibodies directed to polypeptides or polynucleotides of the present invention may also result in inhibition of angiogenesis directly, or indirectly (See Witte L, et al., Cancer Metastasis Rev. 17(2):155-61 (1998), which is hereby incoφorated by reference)).

Polypeptides, including protein fusions, of the present invention, or fragments thereof may be useful in inhibiting proliferative cells or tissues through the induction of apoptosis. Said polypeptides may act either directly, or indirectly to induce apoptosis of proliferative cells and tissues, for example in the activation of a death-domain receptor, such as tumor necrosis factor (TNF) receptor- 1, CD95 (Fas/APO-1), TNF-receptor-related apoptosis-mediated protein (TRAMP) and TNF-related apoptosts-inducmg ligand (TRAIL) receptor-1 and -2 (See Schulze-Osthoff K, etal., Eur J Biochem 254(3):439-59 (1998), which is hereby incoφorated by reference) Moreover, in another preferred embodiment of the present invention, said polypeptides may induce apoptosis through other mechanisms, such as in the activation of other proteins which will activate apoptosis, or through stimulating the expiession of said proteins, eithei alone or in combination with small molecule drugs ot adjuviants, such as apoptonin, galectms, thioredoxtns, antnnflammatory proteins (See foi example, Mutat Res 400(1-2) 447-55 (1998), Med

Hypotheses 50(5) 423-33 (1998), Chem Btol Interact Apr 24,111-112.23-34 (1998), J Mol Med 76(6) 402-12 (1998). Int J Tissue React,20(l):3-15 (1998), which are all hereby incoφoi ated by reference)

Polypeptides, including piotein fusions to, or fragments thereof, of the present invention are useful in inhibiting the metastasis of proliferative cells or tissues Inhibition may occur as a direct result of admintsteiing polypeptides, or antibodies directed to said polypeptides as described elsewere herein, or indirectly, such as activating the expression of proteins known to inhibit metastasis, for example alpha 4 integnns, (See, e.g , Curr Top Microbiol Immunol 1998,231.125-41, which is hereby incoφorated by reference). Such thereapeutic affects of the piesent invention may be achieved either alone, or in combination with small molecule drugs or adjuvants. In another embodiment, the invention provides a method of delivering compositions containing the polypeptides of the invention (e.g., compositions containing polypeptides or polypeptide antibodes associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs) to targeted cells expressing the polypeptide of the present invention Polypeptides or polypeptide antibodes of the invention may be associated with with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions Polypeptides, protein fusions to, or fragments thereof, of the present invention are useful in enhancing the immunogenicity and/or antigenicity of proliferating cells or tissues, either directly, such as would occur if the polypeptides of the present invention 'vaccinated' the immune response to respond to proliferative antigens and immunogens, or indirectly, such as in activating the expression of proteins known to enhance the immune response (e.g. chemokines), to said antigens and immunogens.

Cardiovascular Disorders

Polynucleotides or polypeptides, ot agonists or antagonists of the invention may be used to treat cardiovascular disorders, including peripheral artery disease, such as limb ischemia. Cardiovascular disorders include cardiovascular abnormalities, such as arteπo- artenal fistula, artenovenous fistula, ceiebral atteπovenous malformations, congenital heart defects, pulmonary atresta, and Scimitar Syndrome. Congenital heart defects include aortic coarctatton, cor tπatπatum, coronary vessel anomalies, crisscross heart, dextrocardia, patent ductus arteπosus, Ebstem's anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of fallot, transposition of great vessels, double outlet right ventricle, tπcuspid atresia, persistent truncus arteπosus, and heart septal defects, such as aortopulmonary septal defect, endocardial cushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventπcular heart septal defects.

Cardiovascular disorders also include heart disease, such as arrhythmias, carc oid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bactenal), heart aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy, congestive cardiomyopathy, left ventncular hypertrophy, nght ventncular hypertrophy, post-infarction heart rupture, ventricular septal rupture, heart valve diseases, myocardial diseases, myocardial ischemia, pericardial effusion, pericarditis (including constrictive and tuberculous), pneumopeπcardium, postpencardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemta, cardiovascular pregnancy complications, Scimitar Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.

Arrhythmias include sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block, sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, and ventricular fibrillation. Tachycardias include paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

Heart valve disease include aortic valve insufficiency, aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency, and tricuspid valve stenosis.

Myocardial diseases include alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Keams Syndrome, myocardial reperfusion injury, and myocarditis.

Myocardial ischemias include coronary disease, such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning.

Cardiovascular diseases also include vascular diseases such as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease, Klippel-

Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabetic angiopathies, diabetic retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids, hepatic veno- occlusive disease, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal vein occlusion, Scimitar syndrome, superior vena cava syndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagic telangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis, and venous insufficiency.

Aneurysms include dissecting aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart aneurysms, and iliac aneurysms. Arterial occlusive diseases include arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya disease, renal artery obstruction, retinal artery occlusion, and thromboangiitis obliterans.

Cerebrovascular disorders include carotid artery diseases, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery diseases, cerebral embolism and thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient), subclavian steal syndrome, periventricular leukomalacia, vascular headache, cluster headache, migraine, and vertebrobasilar insufficiency.

Embolisms include air embolisms, amniotic fluid embolisms, cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and thromoboembolisms. Thrombosis include coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, and thrombophlebitis.

Ischemia includes cerebral ischemia, ischemic colitis, compartment syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion injuries, and peripheral limb ischemia. Vasculitis includes aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph node syndrome, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-Henoch puφura, allergic cutaneous vasculitis, and

Wegener's granulomatosis.

Polynucleotides or polypeptides, or agonists or antagonists of the invention, are especially effective for the treatment of critical limb ischemia and coronary disease

Polypeptides may be administered using any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, biohstic injectors, particle accelerators, gelfoam sponge depots, other commercially available depot matenals, osmotic pumps, oral or suppositoπal solid pharmaceutical formulations, decanting oi topical applications during surgery, aerosol delivery. Such methods are known in the art Polypeptides of the invention may be administered as part of a Therapeutic, described in more detail below. Methods of dehvenng polynucleotides of the invention are described in more detail herein.

Anti-Angiogenesis Activity

The naturally occurring balance between endogenous stimulators and inhibitors of angiogenesis is one in which inhibitory influences predominate. Rastmejad et al, Cell 56:345-355 (1989). In those rare instances in which neovasculanzation occurs under normal physiological conditions, such as wound healing, organ regeneration, embryonic development, and female reproductive processes, angiogenesis is stnngently regulated and spatially and temporally delimited. Under conditions of pathological angiogenesis such as that characterizing solid tumor growth, these regulatory controls fail. Unregulated angiogenesis becomes pathologic and sustains progression of many neoplastic and non- neoplastic diseases. A number of serious diseases are dominated by abnormal neovasculanzation including solid tumor growth and metastases, arthritis, some types of eye disorders, and psoriasis. See, e.g., reviews by Moses et al., Biotech. 9:630-634 (1991); Folkman et ah, N. Engl. J. Med., 333: 1151-1163 (1995); Auerbach et al, J. Microvasc. Res. 29:401-411 (1985); Folkman, Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985); Patz, Am. J. Opthalmol.

94:115-143 (1982); and Folkman et al., Science 227 :719-725 (1983). In a number of pathological conditions, the process of angiogenesis contributes to the disease state. For example, significant data have accumulated which suggest that the growth of solid tumors is dependent on angiogenesis. Folkman and Klagsbrun, Science 235:442-441 (1987).

The present invention provides for treatment of diseases or disorders associated with neovasculanzation by administration of the polynucleotides and/or polypeptides of the invention, as well as agonists or antagonists of the present invention. Malignant and metastatic conditions which can be treated with the polynucleotides and polypeptides, or agonists or antagonists of the invention include, but are not limited to, malignancies, solid tumors, and cancers described herein and otherwise known in the art (for a review of such disorders, see Fishman et al, Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)).Thus, the present invention provides a method of treating an angiogenesis-related disease and/or disorder, comprising administering to an individual in need thereof a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist of the invention. For example, polynucleotides, polypeptides, antagonists and/or agonists may be utilized in a variety of additional methods in order to therapeutically treat a cancer or tumor. Cancers which may be treated with polynucleotides, polypeptides, antagonists and/or agonists include, but are not limited to solid tumors, including prostate, lung, breast, ovarian, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, thyroid cancer; primary tumors and metastases; melanomas; glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non- small cell lung cancer; colorectal cancer; advanced malignancies; and blood bom tumors such as leukemias. For example, polynucleotides, polypeptides, antagonists and/or agonists may be delivered topically, in order to treat cancers such as skin cancer, head and neck tumors, breast tumors, and Kaposi's sarcoma.

Within yet other aspects, polynucleotides, polypeptides, antagonists and/or agonists may be utilized to treat superficial forms of bladder cancer by, for example, intravesical administration. Polynucleotides, polypeptides, antagonists and/or agonists may be delivered directly into the tumor, or near the tumor site, via injection or a catheter. Of course, as the artisan of ordinary skill will appreciate, the appropriate mode of administration will vary according to the cancer to be treated. Other modes of delivery are discussed herein.

Polynucleotides, polypeptides, antagonists and/or agonists may be useful in treating other disorders, besides cancers, which involve angiogenesis. These disorders include, but are not limited to: benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; artheroscleric plaques; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth) of the eye; rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis; vasculogenesis; granulations; hypertrophic scars (keloids); nonunion fractures; scleroderma; trachoma; vascular adhesions; myocardial angiogenesis; coronary collaterals; cerebral collaterals; arteriovenous malformations; ischemic limb angiogenesis; Osier-Webber Syndrome; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; fibromuscular dysplasia; wound granulation; Crohn's disease; and atherosclerosis.

For example, within one aspect of the present invention methods are provided for treating hypertrophic scars and keloids, comprising the step of administering a polynucleotide, polypeptide, antagonist and/or agonist of the invention to a hypertrophic scar or keloid.

Within one embodiment of the present invention polynucleotides, polypeptides, antagonists and/or agonists are directly injected into a hypertrophic scar or keloid, in order to prevent the progression of these lesions. This therapy is of particular value in the prophylactic treatment of conditions which are known to result in the development of hypertrophic scars and keloids (e.g., burns), and is preferably initiated after the proliferative phase has had time to progress (approximately 14 days after the initial injury), but before hypertrophic scar or keloid development. As noted above, the present invention also provides methods for treating neovascular diseases of the eye, including for example, co eal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, retrolental fibroplasia and macular degeneration. Moreover, Ocular disorders associated with neovascularization which can be treated with the polynucleotides and polypeptides of the present invention (including agonists and/or antagonists) include, but are not limited to: neovascular glaucoma, diabetic retinopathy. retinoblastoma, retrolental fibroplasia, uveitis, retinopathy of prematurity macular degeneration, comeal graft neovascularization, as well as other eye inflammatory diseases, ocular tumors and diseases associated with choroidal or iris neovascularization. See, e.g., reviews by Waltman et al., Am. J. Ophthal. 55:704-710 (1978) and Gartner et al, Surv. Ophthal. 22:291-312 (1978).

Thus, within one aspect of the present invention methods are provided for treating neovascular diseases of the eye such as comeal neovascularization (including comeal graft neovascularization), comprising the step of administering to a patient a therapeutically effective amount of a compound (as described above) to the cornea, such that the formation of blood vessels is inhibited. Briefly, the cornea is a tissue which normally lacks blood vessels. In certain pathological conditions however, capillaries may extend into the cornea from the pericomeal vascular plexus of the limbus. When the cornea becomes vascularized, it also becomes clouded, resulting in a decline in the patient's visual acuity. Visual loss may become complete if the cornea completely opacitates. A wide variety of disorders can result in corneal neovascularization, including for example, corneal infections (e.g., trachoma, heφes simplex keratitis, leishmaniasis and onchocerciasis), immunological processes (e.g., graft rejection and Stevens-Johnson's syndrome), alkali bums, trauma, inflammation (of any cause), toxic and nutritional deficiency states, and as a complication of wearing contact lenses.

Within particularly preferred embodiments of the invention, may be prepared for topical administration in saline (combined with any of the preservatives and antimicrobial agents commonly used in ocular preparations), and administered in eyedrop form. The solution or suspension may be prepared in its pure form and administered several times daily. Alternatively, anti-angiogenic compositions, prepared as described above, may also be administered directly to the comea. Within preferred embodiments, the anti-angiogenic composition is prepared with a muco-adhesive polymer which binds to comea. Within further embodiments, the anti-angiogenic factors or anti-angiogenic compositions may be utilized as an adjunct to conventional steroid therapy. Topical therapy may also be useful prophylactically in comeal lesions which are known to have a high probability of inducing an angiogenic response (such as chemical bums). In these instances the treatment, likely in combination with steroids, may be instituted immediately to help prevent subsequent complications.

Within other embodiments, the compounds described above may be injected directly into the comeal stroma by an ophthalmologist under microscopic guidance. The preferred site of injection may vary with the moφhology of the individual lesion, but the goal of the administration would be to place the composition at the advancing front of the vasculature (i.e., interspersed between the blood vessels and the normal comea). In most cases this would involve perilimbic comeal injection to "protect" the comea from the advancing blood vessels. This method may also be utilized shortly after a comeal insult in order to prophylactically prevent comeal neovascularization. In this situation the material could be injected in the perilimbic comea interspersed between the comeal lesion and its undesired potential limbic blood supply. Such methods may also be utilized in a similar fashion to prevent capillary invasion of transplanted corneas. In a sustained-release form injections might only be required 2-3 times per year. A steroid could also be added to the injection solution to reduce inflammation resulting from the injection itself. Within another aspect of the present invention, methods are provided for treating neovascular glaucoma, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eye, such that the formation of blood vessels is inhibited. In one embodiment, the compound may be administered topically to the eye in order to treat early forms of neovascular glaucoma. Within other embodiments, the compound may be implanted by injection into the region of the anterior chamber angle. Withm other embodiments, the compound may also be placed in any location such that the compound is continuously released into the aqueous humor. Withm another aspect of the present invention, methods are provided for treating proliferative diabetic retinopathy, compπsing the step of admimstenng to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/oi agonist to the eyes, such that the formation of blood vessels is inhibited.

Within particularly preferred embodiments of the invention, proliferative diabetic retinopathy may be treated by injection into the aqueous humor or the vitreous, in order to increase the local concentration of the polynucleotide, polypeptide, antagonist and/oi agonist in the retina. Preferably, this treatment should be initiated prior to the acquisition of severe disease requtnng photocoagulation.

Within another aspect of the present invention, methods are provided for treating retrolental fibroplasia, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eye, such that the formation of blood vessels is inhibited. The compound may be administered topically, via intravitreous injection and/or via intraocular implants.

Additionally, disorders which can be treated with the polynucleotides, polypeptides, agonists and/or agonists include, but are not limited to, hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing, granulations, hemophihc joints, hypertrophic scars, nonunion fractures, Osier-Weber syndrome, pyogemc granuloma, scleroderma, trachoma, and vascular adhesions.

Moreover, disorders and/or states, which can be treated with be treated with the the polynucleotides, polypeptides, agonists and/or agonists include, but are not limited to, solid tumors, blood bo tumors such as leukemias, tumor metastasis, Kaposi's sarcoma, benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyoge c granulomas, rheumatoid arthritis, psoriasis, ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, co eal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, and uvietis, delayed wound healing, endometriosis, vascluogenesis, granulations, hypertrophic scars (keloids), nonunion fractures, scleroderma, trachoma, vascular adhesions, myocardial angiogenesis, coronary collaterals, cerebral collaterals, arteriovenous malformations, ischemic limb angiogenesis, Osier-Webber Syndrome, plaque neovascularization, telangiectasia, hemophiliac joints, angiofibroma fibromuscular dysplasia, wound granulation, Crohn's disease, atherosclerosis, birth control agent by preventing vascularization required for embryo implantation controlling menstruation, diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa), ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.

In one aspect of the birth control method, an amount of the compound sufficient to block embryo implantation is administered before or after intercourse and fertilization have occuπ'ed, thus providing an effective method of birth control, possibly a "morning after" method. Polynucleotides, polypeptides, agonists and/or agonists may also be used in controlling menstruation or administered as either a peritoneal lavage fluid or for peritoneal implantation in the treatment of endometriosis.

Polynucleotides, polypeptides, agonists and/or agonists of the present invention may be incoφorated into surgical sutures in order to prevent stitch granulomas. Polynucleotides, polypeptides, agonists and/or agonists may be utilized in a wide variety of surgical procedures. For example, within one aspect of the present invention a compositions (in the form of, for example, a spray or film) may be utilized to coat or spray an area prior to removal of a tumor, in order to isolate normal surrounding tissues from malignant tissue, and/or to prevent the spread of disease to surrounding tissues. Within other aspects of the present invention, compositions (e.g., in the form of a spray) may be delivered via endoscopic procedures in order to coat tumors, or inhibit angiogenesis in a desired locale. Within yet other aspects of the present invention, surgical meshes which have been coated with anti- angiogenic compositions of the present invention may be utilized in any procedure wherein a surgical mesh might be utilized. For example, within one embodiment of the invention a surgical mesh laden with an anti-angiogenic composition may be utilized during abdominal cancer resection surgery (e.g., subsequent to colon resection) in order to provide support to the structure, and to release an amount of the anti-angiogenic factor.

Within further aspects of the present invention, methods are piovided for treating tumoi excision sites, comprising administering a polynucleotide, polypeptide, agonist and/or agonist to the resection margins of a tumor subsequent to excision, such that the local recurrence of cancer and the formation of new blood vessels at the site is inhibited Within one embodiment of the invention, the anti-angiogemc compound is administered directly to the tumor excision site (e g., applied by swabbing, brushing or otheiwise coating the resection margins of the tumor with the anti-angiogenic compound). Alternatively, the anti -angiogenic compounds may be incoφorated into known surgical pastes prior to administration. Withm particularly preferred embodiments of the invention, the anti-angiogenic compounds are applied after hepatic resections for malignancy, and after neurosurgical operations.

Within one aspect of the present invention, polynucleotides, polypeptides, agonists and/or agonists may be administered to the resection margin of a wide variety of tumors, including for example, breast, colon, brain and hepatic tumors. For example, within one embodiment of the invention, anti-angiogenic compounds may be administered to the site of a neurological tumor subsequent to excision, such that the formation of new blood vessels at the site are inhibited.

The polynucleotides, polypeptides, agonists and/or agonists of the present invention may also be administered along with other anti-angiogenic factors. Representative examples of other anti-angiogenic factors include: Anti-Invasive Factor, retinoic acid and derivatives thereof, pachtaxel, Suramin, Tissue Inhibitor of Metalloproteιnase-1, Tissue Inhibitor of Metalloproteιnase-2, Plasminogen Activator Inhibitor- 1 , Plasminogen Activator Inhιbιtor-2, and vanous forms of the lighter "d group" transition metals Lighter "d group" transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition metal species may form transition metal complexes. Suitable complexes of the above-mentioned transition metal species include oxo transition metal complexes Representative examples of vanadium complexes include oxo vanadium complexes such as vanadate and vanadyl complexes Suitable vanadate complexes include metavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate hydrates such as vanadyl sulfate mono- and tπhydrates.

Representative examples of tungsten and molybdenum complexes also include oxo complexes Suitable oxo tungsten complexes include tungstate and tungsten oxide complexes Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl complexes. Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic acid Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include hydroxo derivatives derived from, for example, glycerol, tartaπc acid, and sugars.

A wide variety of other anti-angiogenic factors may also be utilized within the context of the present invention. Representative examples include platelet factor 4; protamine sulphate; sulphated chitin derivatives (prepared from queen crab shells), (Murata et al., Cancer Res. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex (SP- PG) (the function of this compound may be enhanced by the presence of steroids such as estrogen, and tamoxifen citrate), Staurospoπne; modulators of matrix metabolism, including for example, proline analogs, cishydroxyproline, d,L-3,4- dehydroproline, Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate; 4-propyl-

5-(4-pyridinyI)-2(3H)-oxazolone; Methotrexate; Mitoxantrone; Heparin; Interferons; 2

Macroglobulin-seru ; ChIMP-3 (Pavloff et al., J. Bio. Chem. 267:17321-17326, 1992); Chymostatin (Tomkinson et al., Biochem J. 286:475-480, 1992); Cyclodextrin

Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-

557, 1990); Gold Sodium Thiomalate ("GST"; Matsubara and Ziff, J. Clin. Invest.

79: 1440-1446, 1987); anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol.

Chem. 262(4): 1659-1664, 1987); Bisantrene (National Cancer Institute); Lobenzarit disodium (N-(2)-carboxyphenyl-4- chloroanthronilic acid disodium or "CCA"; Takeuchi et al., Agents Actions 36:312-316, 1992); Thalidomide; Angostatic steroid; AGM-1470; carboxynaminolmidazole; and metalloproteinase inhibitors such as BB94.

Diseases at the Cellular Level Diseases associated with increased cell survival or the inhibition of apoptosis that could be treated or detected by the polynucleotides or polypeptides and/or antagonists or agonists of the invention, include cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) and viral infections (such as heφes viruses, pox viruses and adenoviruses), inflammation, graft v. host disease, acute graft rejection, and chronic graft rejection. In preferred embodiments, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention are used to inhibit growth, progression, and/or metasis of cancers, in particular those listed above.

Additional diseases or conditions associated with increased cell survival that could be treated or detected by the polynucleotides or polypeptides, or agonists or antagonists of the invention, include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias

(e.g., chronic myelocytic (granulocyttc) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g . Hodgkm's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothehosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarc oma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcmoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, chonocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, ghoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, p ealoma, hemangioblastoma, acoustic neuroma, oligodendroghoma, menangioma, melanoma, neuroblastoma, and retinoblastoma. Diseases associated with increased apoptosis that could be treated or detected by the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, include AIDS; neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumor or pπor associated disease); autoimmune disorders (such as, multiple sclerosis,

Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephπtis and rheumatoid arthntis) myelodysplastic syndromes (such as aplastic anemia), graft v. host disease, ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury (e.g., hepatitis lelated liver injury, ischemia/reperfusion injury, cholestosis (bile duct injury) and hvei cancer); toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia

Wound Healing and Epithelial Cell Proliferation

In accordance with yet a further aspect of the present invention, there is provided a process for utilizing the polynucleotides oi polypeptides, and/or agonists or antagonists of the invention, for therapeutic purposes, for example, to stimulate epithelial cell proliferation and basal keratinocytes for the puφose of wound healing, and to stimulate hair follicle production and healing of dermal wounds. Polynucleotides or polypeptides, as well as agonists or antagonists of the invention, may be clinically useful in stimulating wound healing including surgical wounds, excisional wounds, deep wounds involving damage of the dermis and epidermis, eye tissue wounds, dental tissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers, venous stasis ulcers, bu s resulting from heat exposure or chemicals, and other abnormal wound healing conditions such as uremia, malnutπtion, vitamin deficiencies and complications associted with systemic treatment with steroids, radiation therapy and antineoplastic drugs and antimetabolites. Polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to promote dermal reestabhshment subsequent to dermal loss The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to increase the adherence of skin grafts to a wound bed and to stimulate re-epitheliahzation from the wound bed. The following are a non-exhaustive list of grafts that polynucleotides or polypeptides, agonists or antagonists of the invention, could be used to increase adherence to a wound bed: autografts, artificial skin, allografts, autodermic graft, autoepdermic grafts, avacular grafts, Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft, delayed graft, dermic graft, epidermic graft, fascia graft, full thickness graft, heterologous graft, xenograft, homologous graft, hypeφlastic graft, lamellar graft, mesh graft, mucosal graft, Ollier-Thiersch graft, omenpal graft, patch graft, pedicle graft, penetrating graft, split skin graft, thick split graft. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, can be used to promote skin strength and to improve the appearance of aged skin.

It is believed that the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, will also produce changes in hepatocyte proliferation, and epithelial cell proliferation in the lung, breast, pancreas, stomach, small intesting, and large intestine. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could promote proliferation of epithelial cells such as sebocytes, hair follicles, hepatocytes, type II pneumocytes, mucin-producing goblet cells, and other epithelial cells and their progenitors contained within the skin, lung, liver, and gastrointestinal tract. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, may promote proliferation of endothelial cells, keratinocytes, and basal keratinocytes.

The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could also be used to reduce the side effects of gut toxicity that result from radiation, chemotherapy treatments or viral infections. The polynucleotides or polypeptides, and or agonists or antagonists of the invention, may have a cytoprotective effect on the small intestine mucosa. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, may also stimulate healing of mucositis (mouth ulcers) that result from chemotherapy and viral infections. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could further be used in full regeneration of skin in full and partial thickness skin defects, including bu s, (i.e., repopulation of hair follicles, sweat glands, and sebaceous glands), treatment of other skin defects such as psoriasis. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to treat epidermolysis bullosa, a defect in adherence of the epidermis to the underlying dermis which results in frequent, open and painful blisters by accelerating reepithelialization of these lesions. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could also be used to treat gastric and doudenal ulcers and help heal by scar formation of the mucosal lining and regeneration of glandular mucosa and duodenal mucosal lining more rapidly. Inflamamatory bowel diseases, such as Crohn's disease and ulcerative colitis, are diseases which result in destruction of the mucosal surface of the small or large intestine, respectively. Thus, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to promote the resurfacing of the mucosal surface to aid more rapid healing and to prevent progression of inflammatory bowel disease. Treatment with the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, is expected to have a significant effect on the production of mucus throughout the gastrointestinal tract and could be used to protect the intestinal mucosa from injurious substances that are ingested or following surgery. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to treat diseases associate with the under expression of the polynucleotides of the invention.

Moreover, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to prevent and heal damage to the lungs due to various pathological states. A growth factor such as the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, which could stimulate proliferation and differentiation and promote the repair of alveoli and brochiolar epithelium to prevent or treat acute or chronic lung damage. For example, emphysema, which results in the progressive loss of aveoli, and inhalation injuries, i.e., resulting from smoke inhalation and bums, that cause necrosis of the bronchiolar epithelium and alveoli could be effectively treated using the polynucleotides or polypeptides, and/or agonists or antagonists of the invention. Also, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to stimulate the proliferation of and differentiation of type II pneumocytes, which may help treat or prevent disease such as hyaline membrane diseases, such as infant respiratory distress syndrome and bronchopulmonary displasia, in premature infants. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could stimulate the proliferation and differentiation of hepatocytes and, thus, could be used to alleviate or treat liver diseases and pathologies such as fulminant liver failure caused by cirrhosis, liver damage caused by viral hepatitis and toxic substances

(i.e., acetaminophen, carbon tetraholoride and other hepatotoxins known in the art). In addition, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used treat or prevent the onset of diabetes mellitus. In patients with newly diagnosed Types I and II diabetes, where some islet cell function remains, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to maintain the islet function so as to alleviate, delay or prevent permanent manifestation of the disease. Also, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used as an auxiliary in islet cell transplantation to improve or promote islet cell function.

Infectious Disease A polypeptide or polynucleotide and/or agonist or antagonist of the present invention can be used to treat or detect infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases may be treated. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, polypeptide or polynucleotide and/or agonist or antagonist of the present invention may also directly inhibit the infectious agent, without necessarily eliciting an immune response. Viruses are one example of an infectious agent that can cause disease or symptoms that can be treated or detected by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention. Examples of viruses, include, but are not limited to

Examples of viruses, include, but are not limited to the following DNA and RNA viruses and viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Bimaviridae,

Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue, EBV, HIV,

Flaviviridae, Hepadnaviridae (Hepatitis), Heφes viridae (such as, Cytomegalovirus, Heφes

Simplex, Heφes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus,

Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A, Influenza B, and parainfluenza), Papiloma virus, Papovaviridae, Parvoviridae, Picomaviridae, Poxviridae (such as

Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lenti virus), and Togaviridae (e.g., Rubi virus). Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to: arthritis, bronchiollitis, respiratory syncytial virus, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta),

Japanese B encephalitis, Junin, Chikungunya, Rift Valley fever, yellow fever, meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia. polynucleotides or polypeptides, or agonists or antagonists of the invention, can be used to treat or detect any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat: meningitis, Dengue, EBV, and/or hepatitis (e.g., hepatitis B). In an additional specific embodiment polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat patients nonresponsive to one or more other commercially available hepatitis vaccines. In a further specific embodiment polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat AIDS. Similarly, bacterial or fungal agents that can cause disease or symptoms and that can be treated or detected by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention include, but not limited to, include, but not limited to, the following Gram-Negative and Gram-positive bacteria and bacterial families and fungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia), Cryptococcus neoformans, Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae,

Blastomycosis, Bordetella, Borrelia (e.g., Borrelia burgdorferi, Brucellosis, Candidiasis,

Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, E. coli (e.g.,

Enterotoxigenic E. coli and Enterohemorrhagic E. coli), Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, and Salmonella paratyphi), Se atia, Yersinia),

Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales, Mycobacterium leprae, Vibrio cholerae, Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Menigococcal), Meisseria meningitidis, Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus (e.g., Heamophilus influenza type B), Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp., Staphylococcal, Meningiococcal, Pneumococcal and Streptococcal (e.g., Streptococcus pneumoniae and Group B Streptococcus). These bacterial or fungal families can cause the following diseases or symptoms, including, but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (e.g., AIDS related infections), paronychia, prosthesis-related infections, Reiter's Disease, respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis (e.g., mengitis types A and B), Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections, wound infections. Polynucleotides or polypeptides, agonists or antagonists of the invention, can be used to treat or detect any of these symptoms or diseases. In specific embodiments, Ppolynucleotides, polypeptides, agonists or antagonists of the invention are used to treat: tetanus, Diptheria, botulism, and/or meningitis type B.

Moreover, parasitic agents causing disease or symptoms that can be treated or detected by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention include, but not limited to, the following families or class: Amebiasis,

Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic,

Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax, Plasmodium falciparium,

Plasmodium malariae and Plasmodium ovale). These parasites can cause a variety of diseases or symptoms, including, but not limited to: Scabies, Trombiculiasis, eye infections, intestinal disease (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS related), malaria, pregnancy complications, and toxoplasmosis. polynucleotides or polypeptides, or agonists or antagonists of the invention, can be used to treat or detect any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat malaria.

Preferably, treatment using a polypeptide or polynucleotide and/or agonist or antagonist of the present invention could either be by administering an effective amount of a polypeptide to the patient, or by removing cells from the patient, supplying the cells with a polynucleotide of the present invention, and returning the engineered cells to the patient (ex vivo therapy). Moreover, the polypeptide or polynucleotide of the present invention can be used as an antigen in a vaccine to raise an immune response against infectious disease.

Regeneration

A polynucleotide or polypeptide and/or agonist or antagonist of the present invention can be used to differentiate, proliferate, and attract cells, leading to the regeneration of tissues. (See, Science 276:59-87 (1997).) The regeneration of tissues could be used to repair, replace, or protect tissue damaged by congenital defects, trauma

(wounds, bu s, incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarthritis, periodontal disease, liver failure), surgery, including cosmetic plastic surgery, fibrosis, reperfusion injury, or systemic cytokine damage. Tissues that could be regenerated using the present invention include organs (e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac), vasculature (including vascular and lymphatics), nervous, hematopoietic, and skeletal

(bone, cartilage, tendon, and ligament) tissue. Preferably, regeneration occurs without or decreased scarring. Regeneration also may include angiogenesis. Moreover, a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase regeneration of tissues difficult to heal. For example, increased tendon/ligament regeneration would quicken recovery time after damage. A polynucleotide or polypeptide and/or agonist or antagonist of the present invention could also be used prophylactically in an effort to avoid damage. Specific diseases that could be treated include of tendinitis, caφal tunnel syndrome, and other tendon or ligament defects. A further example of tissue regeneration of non-healing wounds includes pressure ulcers, ulcers associated with vascular insufficiency, surgical, and traumatic wounds. Similarly, nerve and brain tissue could also be regenerated by using a polynucleotide or polypeptide and/or agonist or antagonist of the present invention to proliferate and differentiate nerve cells. Diseases that could be treated using this method include central and peripheral nervous system diseases, neuropathies, or mechanical and traumatic disorders (e.g., spinal cord disorders, head trauma, cerebrovascular disease, and stoke). Specifically, diseases associated with peripheral nerve injuries, peripheral neuropathy (e.g., resulting from chemotherapy or other medical therapies), localized neuropathies, and central nervous system diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome), could all be treated using the polynucleotide or polypeptide and/or agonist or antagonist of the present invention. Chemotaxis

A polynucleotide or polypeptide and/or agonist or antagonist of the present invention may have chemotaxis activity. A chemotaxic molecule attracts or mobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and or endothelial cells) to a particular site in the body, such as inflammation, infection, or site of hypeφroliferation. The mobilized cells can then fight off and/or heal the particular trauma or abnormality.

A polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase chemotaxic activity of particular cells. These chemotactic molecules can then be used to treat inflammation, infection, hypeφroliferative disorders, or any immune system disorder by increasing the number of cells targeted to a particular location in the body. For example, chemotaxic molecules can be used to treat wounds and other trauma to tissues by attracting immune cells to the injured location. Chemotactic molecules of the present invention can also attract fibroblasts, which can be used to treat wounds.

It is also contemplated that a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may inhibit chemotactic activity. These molecules could also be used to treat disorders. Thus, a polynucleotide or polypeptide and/or agonist or antagonist of the present invention could be used as an inhibitor of chemotaxis.

Binding Activity

A polypeptide of the present invention may be used to screen for molecules that bind to the polypeptide or for molecules to which the polypeptide binds. The binding of the polypeptide and the molecule may activate (agonist), increase, inhibit (antagonist), or decrease activity of the polypeptide or the molecule bound. Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors),or small molecules. Preferably, the molecule is closely related to the natural ligand of the polypeptide, e.g., a fragment of the ligand, or a natural substrate, a ligand, a structural or functional mimetic. (See, Coligan et al., Current Protocols in Immunology l(2):Chapter 5 (1991).)

Similarly, the molecule can be closely related to the natural receptor to which the polypeptide binds, or at least, a fragment of the receptor capable of being bound by the polypeptide (e.g., active site). In either case, the molecule can be rationally designed using known techniques.

Preferably, the screening for these molecules involves producing appropriate cells which express the polypeptide, either as a secreted protein or on the cell membrane. Prefeired cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide (or cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound potentially containing the molecule to observe binding, stimulation, or inhibition of activity of either the polypeptide or the molecule.

The assay may simply test binding of a candidate compound to the polypeptide, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the candidate compound results in a signal generated by binding to the polypeptide.

Alternatively, the assay can be carried out using cell-free preparations, polypeptide/molecule affixed to a solid support, chemical libraries, or natural product mixtures. The assay may also simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide, measuring polypeptide/molecule activity or binding, and comparing the polypeptide/molecule activity or binding to a standard.

Preferably, an ΕLISA assay can measure polypeptide level or activity in a sample (e.g., biological sample) using a monoclonal or polyclonal antibody. The antibody can measure polypeptide level or activity by either binding, directly or indirectly, to the polypeptide or by competing with the polypeptide for a substrate.

Additionally, the receptor to which a polypeptide of the invention binds can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting (Cohgan, et al., Current Protocols m Immun., 1(2), Chapter 5,

(1991)). For example, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the polypeptides, for example, NIH3T3 cells which are known to contain multiple receptors for the FGF family proteins, and SC-3 cells, and a cDNA library created from this RNA is divided into pools and used to transtect COS cells or other cells that are not responsive to the polypeptides. Transfected cells which are grown on glass slides are exposed to the polypeptide of the present invention, after they have been labelled. The polypeptides can be labeled by a variety of means including lodination or inclusion of a recognition site for a site-specific protein kinase Following fixation and incubation, the slides are subjected to auto-radiographic analysis Positive pools are identified and sub-pools are prepared and re-transfected using an iterative sub-pooling and re-screening process, eventually yielding a single clones that encodes the putative receptor.

As an alternative approach for receptor identification, the labeled polypeptides can be photoaffmity linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked matenal is resolved by PAGE analysis and exposed to X- ray film. The labeled complex containing the receptors of the polypeptides can be excised, resolved into peptide fragments, and subjected to protein microsequenc g The amino acid sequence obtained from microsequencmg would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the genes encoding the putative receptors.

Moreover, the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as "DNA shuffling") may be employed to modulate the activities of polypeptides of the invention thereby effectively generating agonists and antagonists of polypeptides of the invention. See generally, U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten, P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, S. Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O., et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R. Biotechniques 24(2):308-13 (1998) (each of these patents and publications are hereby incoφorated by reference). In one embodiment, alteration of polynucleotides and corresponding polypeptides of the invention may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments into a desired polynucleotide sequence of the invention molecule by homologous, or site-specific, recombination. In another embodiment, polynucleotides and corresponding polypeptides of the invention may be alterred by being subjected to random mutagenesis by enτ>r-prone

PCR, random nucleotide insertion oi othei methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of the polypeptides of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc of one or more heterologous molecules. In preferred embodiments, the heterologous molecules are family members. In furthei prefeπ-ed embodiments, the heterologous molecule is a growth factoi such as, foi example, platelet-derived growth factor (PDGF), insulin-like growth tactor (IGF-I), transtorming growth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblast growth factor (FGF), TGF-beta, bone moφhogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7, activ s A and B, decapentaplegιc(dpp), 60A, OP-2, dorsahn, growth differentiation factors (GDFs), nodal, MIS, inhibin-alpha, TGF-betal, TGF-beta2, TGF-beta3, TGF- beta5, and glial-deπved neurotrophic factor (GDNF). Other preferred fragments are biologically active fragments of the polypeptides of the invention. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.

Additionally, this invention provides a method of screening compounds to identify those which modulate the action of the polypeptide of the present invention. An example of such an assay comprises combining a mammalian fibroblast cell, a the polypeptide of the present invention, the compound to be screened and 3[H] thymidine under cell culture conditions where the fibroblast cell would normally proliferate. A control assay may be performed in the absence of the compound to be screened and compared to the amount of fibroblast proliferation in the presence of the compound to determine if the compound stimulates proliferation by determining the uptake of 3[H] thymidine in each case. The amount of fibroblast cell proliferation is measured by liquid scintillation chromatography which measures the incorporation of 3[H] thymidine. Both agonist and antagonist compounds may be identified by this procedute

In another method, a mammalian cell ot membtane prepat ation expressing a leceptor for a polypeptide of the present invention is incubated with a labeled polypeptide of the present invention in the presence of the compound The ability ot the compound to enhance or block this interaction could then be measured Alternatively, the response of a known second messenger system following interaction of a compound to be screened and the receptor is measured and the ability of the compound to bind to the receptor and elicit a second messenger response is measured to determine if the compound is a potential agonist or antagonist Such second messenger systems include but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.

All of these above assays can be used as diagnostic or prognostic markers. The molecules discovered using these assays can be used to treat disease or to bring about a particular result in a patient (e.g., blood vessel growth) by activating or inhibiting the polypeptide/molecule. Moreover, the assays can discover agents which may inhibit or enhance the production of the polypeptides of the invention from suitably manipulated cells or tissues. Therefore, the invention includes a method of identifying compounds which bind to the polypeptides of the invention compnsmg the steps of: (a) incubating a candidate binding compound with the polypeptide; and (b) determining if binding has occurred. Moreover, the invention includes a method of identifying agonists/antagonists compnsmg the steps of: (a) incubating a candidate compound with the polypeptide, (b) assaying a biological activity , and (b) determining if a biological activity of the polypeptide has been altered.

Also, one could identify molecules bind a polypeptide of the invention experimentally by using the beta-pleated sheet regions contained in the polypeptide sequence of the protein. Accordingly, specific embodiments of the invention are directed to polynucleotides encoding polypeptides which compnse, or alternatively consist of, the amino acid sequence of each beta pleated sheet regions m a disclosed polypeptide sequence Additional embodiments of the invention are directed to polynucleotides encoding polypeptides which comprise, or alternatively consist of, any combination oi all of contained in the polypeptide sequences of the invention Additional piefeπed embodiments of the invention are dnected to polypeptides which comprise, or alternatively consist of, the amino acid sequence of each of the beta pleated sheet regions in one of the polypeptide sequences of the invention Additional embodiments ot the invention are directed to polypeptides which compnse, ot alternatively consist ot, any combination or all of the beta pleated sheet regions in one of the polypeptide sequences of the invention

Targeted Delivery In another embodiment, the invention provides a method of delivering compositions to targeted cells expressing a receptor for a polypeptide of the invention, or cells expressing a cell bound form of a polypeptide of the invention

As discussed herein, polypeptides or antibodies of the invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxms, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions

In one embodiment, the invention provides a method for the specific delivery of compositions of the invention to cells by administenng polypeptides of the invention (including antibodies) that are associated with heterologous polypeptides or nucleic acids. In one example, the invention provides a method for delivenng a therapeutic protein into the targeted cell In another example, the invention provides a method for delivenng a single stranded nucleic acid (e.g., antisense or πbozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcnbed) into the targeted cell In another embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention (e.g., polypeptides of the invention or antibodies of the invention) in association with toxins or cytotoxic prodrugs. By "toxin" is meant compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death. Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. By "cytotoxic prodrug" is meant a non-toxic compound that is converted by an enzyme, normally present in the cell, into a cytotoxic compound. Cytotoxic prodrugs that may be used according to the methods of the invention include, but are not limited to, glutamyl derivatives of benzoic acid mustard alkylating agent, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubisin, and phenoxyacetamide derivatives of doxorubicin.

Drug Screening

Further contemplated is the use of the polypeptides of the present invention, or the polynucleotides encoding these polypeptides, to screen for molecules which modify the activities of the polypeptides of the present invention. Such a method would include contacting the polypeptide of the present invention with a selected compound(s) suspected of having antagonist or agonist activity, and assaying the activity of these polypeptides following binding.

This invention is particularly useful for screening therapeutic compounds by using the polypeptides of the present invention, or binding fragments thereof, m any of a vanety of drug screening techniques The polypeptide or fragment employed in such a test may be affixed to a solid support, expressed on a cell surface, free solution, or located mtracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays

One may measure, for example, the formulation ot complexes between the agent being tested and a polypeptide of the piesent invention

Thus, the present invention piovides methods of screening for drugs or any other agents which affect activities mediated by the polypeptides of the present invention These methods comprise contacting such an agent with a polypeptide of the present invention oi a fragment thereof and assaying for the presence of a complex between the agent and the polypeptide or a fragment thereof, by methods well known in the art In such a competitive binding assay, the agents to screen are typically labeled Following incubation, tree agent is separated from that present in bound form, and the amount of free or uncomplexed label is a measuie of the ability of a particular agent to bind to the polypeptides of the present invention.

Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the polypeptides of the present invention, and is descnbed in great detail in European Patent Application 84/03564, published on

September 13, 1984, which is incoφorated herein by reference herein. Bnefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic p s or some other surface. The peptide test compounds are reacted with polypeptides of the present invention and washed. Bound polypeptides are then detected by methods well known in the art. Purified polypeptides are coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies may be used to capture the peptide and immobilize it on the solid support. This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding polypeptides of the present invention specifically compete with a test compound for binding to the polypeptides or fragments thereof. In this manner, the antibodies are used to detect the presence of any peptide which shares one or more antigenic epitopes with a polypeptide of the invention

Antisense And Ribozyme (Antagonists)

In specific embodiments, antagonists according to the present invention ate nucleic acids corresponding to the sequences contained in SEQ ID NO X, oi the complementai y strand thereof, and/or to nucleotide sequences contained a deposited clone In one embodiment, antisense sequence is generated internally by the organism, in another embodiment, the antisense sequence is sepaiately administered (see, foi example, O'Connor, Neurochem , 56:560 (1991) Ohgodeoxynucleotides as Anitsense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988) Antisense technology can be used to control gene expression through antisense DNA or RNA, or through tnple-he x formation. Antisense techniques are discussed for example, in Okano, Neurochem., 56:560 (1991); Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988). Tπple helix formation is discussed in, for instance, Lee et al., Nucleic Acids Research, 6:3073 (1979), Cooney et al, Science, 24L456 (1988); and Dervan et al., Science, 251:1300 (1991). The methods are based on binding of a polynucleotide to a complementary DNA or RNA.

For example, the use of c-myc and c-myb antisense RNA constructs to inhibit the growth of the non-lymphocytic leukemia cell line HL-60 and other cell lines was previously described. (Wickstrom et al. (1988); Anfossi et al. (1989)). These expeπments were performed in vitro by incubating cells with the oligoribonucleotide. A similar procedure for in vivo use is descnbed in WO 91/15580. Bnefly, a pair of oligonucleotides for a given antisense RNA is produced as follows- A sequence complimentary to the first 15 bases of the open reading frame is flanked by an EcoRI site on the 5 end and a Hindlll site on the 3 end. Next, the pair of oligonucleotides is heated at 90°C for one mmute and then annealed in 2X ligation buffer (20mM TRIS HCl pH 7.5, lOmM MgC12, 10MM dithiothreitol (DTT) and 0.2 mM ATP) and then ligated to the EcoRl/Hmd III site of the retroviral vector PMV7 (WO 91/15580). For example, the 5' coding portion of a polynucleotide that encodes the mature polypeptide of the present invention may be used to design an antisense RNA o gonucleotide of from about 10 to 40 base pairs in length A DNA o gonucleotide is designed to be complementary to a region of the gene involved m tianscπption thereby preventing transcription and the production of the receptoi . The antisense RNA ohgonucleotide hybridizes to the mRNA m vivo and blocks translation of the mRNA molecule into receptor polypeptide

In one embodiment, the antisense nucleic acid of the invention is produced mtracellularly by transcription from an exogenous sequence. For example, a vector or a portion thereof, is transcribed, producing an antisense nucleic acid (RNA) of the invention Such a vector would contain a sequence encoding the antisense nucleic acid of the invention. Such a vector can remain episomal or become chtomosomally integrated, as long as it can be transcnbed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in vertebrate cells. Expression of the sequence encoding a polypeptide of the invention, or fragments thereof, can be by any promoter known in the art to act in vertebrate, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include, but are not limited to, the SV40 early promoter region (Bemoist and Chambon, Nature, 29:304- 310 (1981), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell, 22:787-797 (1980), the heφes thymidme promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A., 78:1441-1445 (1981), the regulatory sequences of the metallothionein gene (Bnnster et al., Nature, 296:39-42 (1982)), etc. The antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a gene of interest. However, absolute complementarity, although preferred, is not required. A sequence "complementary to at least a portion of an RNA," referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double stranded antisense nucleic acids of the invention, a single strand of the duplex

DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid Generally, the larger the hybridizing nucleic acid, the more base mismatches with a RNA sequence of the invention it may contain and still form a stable duplex (or triplex as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5' end of the message, e.g., the 5' untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3' untranslated sequences of mRNAs have been shown to be effective at inhibiting translation of mRNAs as well. See generally, Wagner, R., Nature, 372:333-335 (1994). Thus, oligonucleotides complementary to either the 5' - or 3' - non- translated, non-coding regions of a polynucleotide sequence of the invention could be used in an antisense approach to inhibit translation of endogenous mRNA. Oligonucleotides complementary to the 5' untranslated region of the mRNA should include the complement of the AUG start codon. Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention. Whether designed to hybridize to the 5' -, 3' - or coding region of mRNA, antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides. The polynucleotides of the invention can be DNA or RNA or chimenc mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The o gonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybndization, etc. The ohgonucleotide may include other appended gioups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al.,

Proc. Natl. Acad. Set U.S.A 86:6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad Sci ,

84 648-652 (1987); PCT Publication NO. WO88/09810, published Decembei 15, 1988) or the blood-bra barrier (see, e.g., PCT Publication NO: WO89/10134, published Apπl 25, 1988), hybridization-tπggered cleavage agents. (See, e.g., Krol et al., BioTechniques, 6:958-976 (1988)) or intercalating agents. (See, e.g., Zon, Pharm. Res., 5:539-549 (1988)). To this end, the ohgonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cioss-hnking agent, transport agent, hybπdization- tπggered cleavage agent, etc. The antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracιl, 5-bromouracιl, 5-chlorouracιl, 5-ιodouracιl, hypoxanthme, xantine, 4-acetylcytosme, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylamιnomethyl-2-thιoundιne, 5-carboxymethylamιnomethyluracιl, dihydrouractl, beta-D-galactosylqueosme, inosine, N6-ιsopentenyladenιne, 1-methylguanιne, 1-methyhnosιne, 2,2-dιmethylguanme,

2-methyladenιne, 2-methylguanιne, 3-methylcytosιne, 5-methylcytosιne, N6-adenme, 7-methylguanιne, 5-methylamιnomethyluracιl, 5-methoxyamιnomethyl-2-thιouracιl, beta- D-mannosylqueosme, 5 '-methoxycarboxymethyluracil, 5-methoxyuracιl, 2-methylthιo-N6-ιsopentenyladenιne, uracil-5-oxyacetιc acid (v), wybutoxos e, pseudouracil, queosine, 2-thιocytosme, 5-methyl-2-thιouracιl, 2-thιouracιl, 4-thιouracιl, 5-methyluracιl, uracιl-5-oxyacetιc acid methylester, uracιl-5-oxyacetιc acid (v), 5-methyl- 2-thιouracιl, 3-(3-amιno-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-dιamιnopuπne. The antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group including, but not limited to, a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.

In yet another embodiment, the antisense oligonucleotide is an a-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gautier et al.. Nucl. Acids Res., 15:6625-6641 (1987)). The oligonucleotide is a 2-0-methylribonucleotide (Inoue et al., Nucl. Acids Res., 15:6131-6148 (1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett. 215:327-330 (1987)). Polynucleotides of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (Nucl. Acids Res., 16:3209 (1988)), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A., 85:7448-7451 (1988)), etc.

While antisense nucleotides complementary to the coding region sequence of the invention could be used, those complementary to the transcribed untranslated region are most preferred. Potential antagonists according to the invention also include catalytic RNA, or a ribozyme (See, e.g., PCT International Publication WO 90/11364, published October 4, 1990; Sarver et al, Science, 247: 1222-1225 (1990). While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy mRNAs corresponding to the polynucleotides of the invention, the use of hammerhead nbozymes is preferred

Hammerhead nbozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5' -UG-3' . The construction and production of hammerhead nbozymes is well known in the art and is descnbed more fully in Haseloff and Gerlach, Natuie, 334:585-591 (1988) There aie numerous potential hammerhead ii bozy me cleavage sites within each nucleotide sequence disclosed in the sequence listing Preferably, the ribozyme is engmeeied so that the cleavage recognition site is located near the 5^' end of the mRNA corresponding to the polynucleotides of the invention, l e Jo increase efficiency and minimize the intracellular accumulation of nonfunctional mRNA transcripts

As in the antisense appioach, the ribozymes ot the invention can be composed of modified oligonucleotides (e g for improved stability, targeting, etc.) and should be delivered to cells which exptess the polynucleotides of the invention in vivo. DNA constructs encoding the ribozyme may be introduced into the cell m the same manner as described above for the introduction of antisense encoding DNA. A preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive promoter, such as, for example, pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the nbozyme to destroy endogenous messages and inhibit translation. Since nbozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.

Antagonist/agonist compounds may be employed to inhibit the cell growth and proliferation effects of the polypeptides of the present invention on neoplastic cells and tissues, i.e. stimulation of angiogenesis of tumors, and, therefore, retard or prevent abnormal cellular growth and proliferation, for example, in tumor formation or growth

The antagonist/agonist may also be employed to prevent hyper-vascular diseases, and prevent the proliferation of epithelial lens cells after extracapsular cataract surgery. Prevention of the mitogenic activity of the polypeptides of the present invention may also be desirous in cases such as restenosis after balloon angioplasty.

The antagonist/agonist may also be employed to prevent the growth of scar tissue dunng wound healing. The antagonist/agonist may also be employed to treat the diseases described herein.

Thus, the invention provides a method of treating disorders or diseases, including but not limited to the disorders or diseases listed throughout this application, associated with overexpression of a polynucleotide of the present invention by administering to a patient

(a) an antisense molecule directed to the polynucleotide of the present invention, and/or (b) a ribozyme directed to the polynucleotide of the present invention

Other Activities

The polypeptide of the present invention, as a result of the ability to stimulate vascular endothelial cell growth, may be employed in treatment foi stimulating re- vasculaπzation of ischemic tissues due to various disease conditions such as thrombosis, arteriosclerosis, and other cardiovascular conditions. These polypeptide may also be employed to stimulate angiogenesis and limb regeneration, as discussed above.

The polypeptide may also be employed for treating wounds due to mjunes, bums, post-operative tissue repair, and ulcers since they are mitogenic to vaπous cells of different origins, such as fibroblast cells and skeletal muscle cells, and therefore, facilitate the repair or replacement of damaged or diseased tissue.

The polypeptide of the present invention may also be employed stimulate neuronal growth and to treat and prevent neuronal damage which occurs in certain neuronal disorders or neuro-degenerative conditions such as Alzheimer's disease, Parkinson's disease, and AIDS-related complex. The polypeptide of the invention may have the ability to stimulate chondrocyte growth, therefore, they may be employed to enhance bone and periodontal regeneration and aid tissue transplants or bone grafts.

The polypeptide of the present invention may be also be employed to prevent sk aging due to sunburn by stimulating keratmocyte growth. The polypeptide of the invention may also be employed for preventing hair loss, since FGF family members activate hair-forming cells and promotes melanocyte growth.

Along the same lines, the polypeptides of the present invention may be employed to stimulate growth and differentiation of hematopoietic cells and bone marrow cells when used in combination with other cytokines.

The polypeptide of the invention may also be employed to maintain organs before transplantation or for supporting cell culture of primary tissues.

The polypeptide of the present invention may also be employed for inducing tissue of mesodermal origin to differentiate in early embryos. The polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also increase or decrease the differentiation or proliferation of embryonic stem cells, besides, as discussed above, hematopoietic lineage.

The polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also be used to modulate mammalian characteristics, such as body height, weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation, size, and shape (e.g., cosmetic surgery). Similarly, polypeptides or polynucleotides and/or agonist or antagonists of the present invention may be used to modulate mammalian metabolism affecting catabolism, anabolism, processing, utilization, and storage of energy.

Polypeptide or polynucleotides and/or agonist or antagonists of the present invention may be used to change a mammal's mental state or physical state by influencing biorhythms, caricadic rhythms, depression (including depressive disorders), tendency for violence, tolerance for pain, reproductive capabilities (preferably by Activin or Inhibin-like activity), hormonal or endocrine levels, appetite, libido, memory, stress, or other cognitive qualities. Polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also be used as a food additive or preservative, such as to increase or decrease storage capabilities, fat content, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional components. Other Preferred Embodiments

Other preferred embodiments of the claimed invention include an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 50 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table XIII.

Also preferred is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO:X in the range of positions beginning with the nucleotide at about the position of the 5' Nucleotide of the Clone Sequence and ending with the nucleotide at about the position of the 3' Nucleotide of the Clone Sequence as defined for SEQ ID NO:X in Table XIII.

Also preferred is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO:X in the range of positions beginning with the nucleotide at about the position of the 5' Nucleotide of the Start Codon and ending with the nucleotide at about the position of the 3' Nucleotide of the Clone Sequence as defined for SEQ ID NO:X in Table XIII.

Similarly preferred is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO:X in the range of positions beginning with the nucleotide at about the position of the 5' Nucleotide of the First Amino Acid of the Signal Peptide and ending with the nucleotide at about the position of the ' Nucleotide of the Clone Sequence as defined for SEQ ID NO:X in Table XIII.

Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 150 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X.

Further preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 500 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X. A further preferred embodiment is a nucleic acid molecule compnsmg a nucleotide sequence which is at least 95% identical to the nucleotide sequence of SEQ ID NO:X beginning with the nucleotide at about the position of the 5' Nucleotide of the First Amino

Acid of the Signal Peptide and ending with the nucleotide at about the position of the 3' Nucleotide of the Clone Sequence as defined foi SEQ ID NO:X in Table XIII

A further prefeπed embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the complete nucleotide sequence of SEQ ID NO X.

Also preferred is an isolated nucleic acid molecule which hybridizes under stπngent hybndizatton conditions to a nucleic acid molecule, wherein said nucleic acid molecule which hybridizes does not hybndize under stπngent hybπdization conditions to a nucleic acid molecule having a nucleotide sequence consisting of only A residues or of only T residues

Also preferred is a composition of matter comprising a DNA molecule which compπses a human cDNA clone identified by a cDNA Clone Identifier in Table XIII, which DNA molecule is contained in the material deposited with the American Type Culture Collection and given the ATCC Deposit Number shown in Table XIII for said cDNA Clone Identifier.

Also preferred is an isolated nucleic acid molecule compπsing a nucleotide sequence which is at least 95% identical to a sequence of at least 50 contiguous nucleotides in the nucleotide sequence of a human cDNA clone identified by a cDNA Clone Identifier in Table XIII, which DNA molecule is contained in the deposit given the ATCC Deposit Number shown in Table XIII.

Also preferred is an isolated nucleic acid molecule, wherein said sequence of at least 50 contiguous nucleotides is included in the nucleotide sequence of the complete open reading frame sequence encoded by said human cDNA clone. Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to sequence of at least 150 contiguous nucleotides in the nucleotide sequence encoded by said human cDNA clone.

A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to sequence of at least 500 contiguous nucleotides in the nucleotide sequence encoded by said human cDNA clone.

A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the complete nucleotide sequence encoded by said human cDNA clone. A further prefeired embodiment is a method for detecting in a biological sample a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table XIII; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table XIII and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table XIII; which method comprises a step of comparing a nucleotide sequence of at least one nucleic acid molecule in said sample with a sequence selected from said group and determining whether the sequence of said nucleic acid molecule in said sample is at least 95% identical to said selected sequence. Also preferred is the above method wherein said step of comparing sequences comprises determining the extent of nucleic acid hybridization between nucleic acid molecules in said sample and a nucleic acid molecule comprising said sequence selected from said group. Similarly, also preferred is the above method wherein said step of comparing sequences is performed by comparing the nucleotide sequence determined from a nucleic acid molecule in said sample with said sequence selected from said group. The nucleic acid molecules can comprise DNA molecules or RNA molecules.

A further preferred embodiment is a method for identifying the species, tissue or cell type of a biological sample which method comprises a step of detecting nucleic acid molecules in said sample, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table XIII; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table XIII and contained in the deposit with the

ATCC Deposit Numbei shown foi said cDNA clone in Table XIII.

The method for identifying the species, tissue or cell type of a biological sample can comprise a step of detecting nucleic acid molecules compπsing a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group

Also preferred is a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a gene encoding a secreted protein identified in Table XIII, which method comprises a step ot detecting in a biological sample obtained from said subject nucleic acid molecules, if any, compnsmg a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table XIII; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table XIII and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table XIII.

The method for diagnosing a pathological condition can comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group.

Also preferred is a composition of matter compπsing isolated nucleic acid molecules wherein the nucleotide sequences of said nucleic acid molecules compnse a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table XIII; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table XIII and contained in the deposit with the ATCC Deposit Number shown foi said cDNA clone in Table XIII The nucleic acid molecules can compnse DNA molecules oi RNA molecules

Also preferred is an isolated polypeptide comprising an ammo acid sequence at least 90% identical to a sequence of at least about 10 contiguous ammo acids in the ammo acid sequence of SEQ ID NO Y wherein Y is any integei as defined in Table XIII

Also preferred is a polypeptide, wherein said sequence of contiguous amino acids is included in the ammo acid sequence of SEQ ID NO Y in the lange of positions beginning with the residue at about the position of the First Amino Acid of the Secreted Portion and ending with the residue at about the Last Amino Acid of the Open Reading Frame as set forth for SEQ ID NO:Y in Table XIII

Also preferred is an isolated polypeptide compnsmg an amino acid sequence at least 95% identical to a sequence of at least about 30 contiguous amino acids in the ammo acid sequence of SEQ ID NO.Y

Further preferred is an isolated polypeptide comprising an ammo acid sequence at least 95% identical to a sequence of at least about 100 contiguous amino acids the amino acid sequence of SEQ ID NO Y

Further preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to the complete am o acid sequence of SEQ ID NO:Y.

Further preferred is an isolated polypeptide comprising an amino acid sequence at least 90% identical to a sequence of at least about 10 contiguous amino acids in the complete ammo acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table XIII and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone m Table XIII. Also preferred is a polypeptide wherein said sequence of contiguous amino acids is included in the amino acid sequence of a secreted portion of the secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table XIII and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table XIII

Also preferred is an isolated polypeptide comprising an ammo acid sequence at least 95% identical to a sequence of at least about 30 contiguous amino acids in the amino acid sequence of the secreted portion of the piotein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table XIII and contained in the deposit with the ATCC Deposit Number shown foi said cDNA clone in Table XIII

Also preferred is an isolated polypeptide compπsing an ammo acid sequence at least 95%> identical to a sequence of at least about 100 contiguous amino acids in the amino acid sequence of the secreted portion of the protein encoded by a human cDNA clone identified by a cDNA Clone Identifier Table XIII and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table XIII.

Also preferred is an isolated polypeptide compnsmg an ammo acid sequence at least 95% identical to the ammo acid sequence of the secreted portion of the protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table XIII and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table XIII.

Further preferred is an isolated antibody which binds specifically to a polypeptide compnsmg an ammo acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table XIII; and a complete ammo acid sequence of a protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table XIII and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table XIII Further preferred is a method for detecting in a biological sample a polypeptide compnsmg an ammo acid sequence which is at least 90% identical to a sequence of at least

10 contiguous ammo acids in a sequence selected from the group consisting of an amino acid sequence of SEQ ID NO Y wherein Y is any integer as defined in Table XIII; and a complete amino acid sequence of a protein encoded by a human cDNA clone identified by a cDNA Clone Identifiei in Table XIII and contained in the deposit with the ATCC

Deposit Numbei shown foi said cDNA clone in Table XIII, which method compπses a step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group and determining whether the sequence of said polypeptide molecule said sample is at least 90%; identical to said sequence of at least 10 contiguous ammo acids

Also preferred is the above method wheiein said step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group comprises determining the extent of specific binding of polypeptides in said sample to an antibody which binds specifically to a polypeptide compπsing an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous ammo acids in a sequence selected from the group consisting of an amino acid sequence of SEQ ID NO Y wherein Y is any integer as defined in Table XIII, and a complete ammo acid sequence of a protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table XIII and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone Table XIII

Also preferred is the above method wherein said step of companng sequences is performed by companng the amino acid sequence determined from a polypeptide molecule in said sample with said sequence selected from said group. Also preferred is a method for identifying the species, tissue or cell type of a biological sample which method compnses a step of detecting polypeptide molecules in said sample, if any, compnsmg an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous ammo acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table XIII; and a complete ammo acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table XIII and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table XIII.

Also preferred is the above method for identifying the species, tissue or cell type of a biological sample, which method comprises a step of detecting polypeptide molecules compπsing an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the above group

Also preferred is a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a gene encoding a secreted protein identified in Table XIII, which method comprises a step of detecting in a biological sample obtained from said subject polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence m said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table XIII; and a complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table XIII and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table XIII.

In any of these methods, the step of detecting said polypeptide molecules includes using an antibody.

Also preferred is an isolated nucleic acid molecule compnsmg a nucleotide sequence which is at least 95% identical to a nucleotide sequence encoding a polypeptide wherein said polypeptide comprises an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table XIII; and a complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table XIII and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in

Table XIII. Also preferred is an isolated nucleic acid molecule, wherein said nucleotide sequence encoding a polypeptide has been optimized for expression of said polypeptide in a prokaryotic host.

Also preferred is an isolated nucleic acid molecule, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table XIII; and a complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table XIII and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table XIII.

Further preferred is a method of making a recombinant vector comprising inserting any of the above isolated nucleic acid molecule into a vector. Also preferred is the recombinant vector produced by this method. Also preferred is a method of making a recombinant host cell comprising introducing the vector into a host cell, as well as the recombinant host cell produced by this method.

Also preferred is a method of making an isolated polypeptide comprising culturing this recombinant host cell under conditions such that said polypeptide is expressed and recovering said polypeptide. Also preferred is this method of making an isolated polypeptide, wherein said recombinant host cell is a eukaryotic cell and said polypeptide is a secreted portion of a human secreted protein comprising an amino acid sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y beginning with the residue at the position of the First Amino Acid of the Secreted Portion of SEQ ID NO:Y wherein Y is an integer set forth in Table XIII and said position of the First Amino Acid of the Secreted Portion of SEQ ID NO:Y is defined in Table XIII; and an amino acid sequence of a secreted portion of a protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table XIII and contained in the deposit with the ATCC

Deposit Number shown for said cDNA clone in Table XIII. The isolated polypeptide produced by this method is also preferred.

Also preferred is a method of treatment of an individual in need of an increased level of a secreted protein activity, which method comprises administering to such an individual a pharmaceutical composition comprising an amount of an isolated polypeptide, polynucleotide, or antibody of the claimed invention effective to increase the level of said protein activity in said individual.

The above-recited applications have uses in a wide variety of hosts. Such hosts include, but are not limited to, human, murine, rabbit, goat, guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig, chicken, goat, cow, sheep, dog, cat, non-human primate, and human. In specific embodiments, the host is a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat. In preferred embodiments, the host is a mammal. In most preferred embodiments, the host is a human.

Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.

Examples

Example 1; Isolation of a Selected cDNA Clone From the Deposited Sample

Each cDNA clone in a cited ATCC deposit is contained in a plasmid vector. Table XIII identifies the vectors used to construct the cDNA library from which each clone was isolated. In many cases, the vector used to construct the library is a phage vector from which a plasmid has been excised. The table immediately below correlates the related plasmid for each phage vector used in constructing the cDNA library. For example, where a particular clone is identified in Table XIII as being isolated in the vector "Lambda Zap," the corresponding deposited clone is in "pBluescript." Vector Used to Construct Library Corresponding Deposited Plasmid

Lambda Zap pBluescript (pBS)

Uni-Zap XR pBluescript (pBS) Zap Express pBK lafmid BA plafmid BA pSportl pSportl pCMVSport 2.0 pCMVSport 2.0 pCMVSport 3.0 pCMVSport 3.0 pCR^®2J pCR^®2J

Vectors Lambda Zap (U.S. Patent Nos. 5.128,256 and 5,286,636), Uni-Zap XR (U.S. Patent Nos. 5,128, 256 and 5,286.636), Zap Express (U.S. Patent Nos. 5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. et al., Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. and Short, j. M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees, M. A. et al.. Strategies 5:58-61 (1992)) are commercially available from

Stratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, CA, 92037. pBS contains an ampicillin resistance gene and pBK contains a neomycin resistance gene. Both can be transformed into E. coli strain XL-1 Blue, also available from Stratagene. pBS comes in 4 forms SK+, SK-, KS+ and KS. The S and K refers to the orientation of the polylinker to the T7 and T3 primer sequences which flank the polylinker region ("S" is for Sad and "K" is for Kpnl which are the first sites on each respective end of the linker). "+" or "-" refer to the orientation of the fl origin of replication ("on"), such that in one orientation, single stranded rescue initiated from the f 1 ori generates sense strand DNA and in the other, antisense. Vectors pSportl, pCMVSport 2.0 and pCMVSport 3.0, were obtained from Life

Technologies, Inc., P. O. Box 6009, Gaithersburg, MD 20897. All Sport vectors contain an ampicillin resistance gene and may be transformed into E. coli strain DH10B, also available from Life Technologies. (See, for instance, Gruber, C. E., et al., Focus 15:59 (1993).) Vector lafmid BA (Bento Soares, Columbia University, NY) contains an ampicillin resistance gene and can be transformed into E. coli strain XL-1 Blue. Vector pCR^®2.1, which is available from Invitrogen, 1600 Faraday Avenue, Carlsbad, CA 92008, contains an ampicillin resistance gene and may be transformed into E. coli strain DH10B, available from Life Technologies. (See, for instance, Clark, J. M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al., Bio/Technology 9: (1991).) Preferably, a polynucleotide of the present invention does not compnse the phage vector sequences identified for the particular clone in Table XIII, as well as the corresponding plasmid vector sequences designated above. The deposited material in the sample assigned the ATCC Deposit Numbei cited in

Table XIII for any given cDNA clone also may contain one oi more additional plasmids, each comprising a cDNA clone different from that given clone Thus, deposits sharing the same ATCC Deposit Numbei contain at least a plasmid for each cDNA clone identified in

Table XIII Typically, each ATCC deposit sample cited in Table XIII comprises a mixture of appioximately equal amounts (by weight) of about 50 plasmid DNAs, each containing a different cDNA clone; but such a deposit sample may include plasmids for oie or less than 50 cDNA clones, up to about 500 cDNA clones.

Two approaches can be used to isolate a particular clone from the deposited sample of plasmid DNAs cited for that clone in Table XIII. Fust, a plasmid is directly isolated by screening the clones using a polynucleotide probe corresponding to SEQ ID NO:X.

Particularly, a specific polynucleotide with 30-40 nucleotides is synthesized using an Applied Biosystems DNA synthesizer according to the sequence reported. The o gonucleotide is labeled, for instance, with ³²P-γ-ATP using T4 polynucleotide kinase and purified according to routine methods (E.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spnng Harbor Press, Cold Spnng, NY (1982).) The plasmid mixture is transformed into a suitable host, as indicated above (such as XL-1 Blue (Stratagene)) using techniques known to those of skill in the art, such as those provided by the vector supplier or in related publications or patents cited above. The transformants are plated on 1.5% agar plates (containing the appropriate selection agent, e.g., ampicillin) to a density of about 150 transformants (colonies) per plate. These plates are screened using Nylon membranes according to routine methods for bactenal colony screening (e.g., Sambrook et al, Molecular Cloning. A Laboratory Manual, 2nd Edit., (1989), Cold Spnng Harbor Laboratory Press, pages 1.93 to 1J04), or other techniques known to those of skill in the art.

Alternatively, two primers of 17-20 nucleotides derived from both ends of the SEQ

ID NO:X (i.e., within the region of SEQ ID NO:X bounded by the 5^' NT and the 3' NT of the clone defined in Table XIII) are synthesized and used to amplify the desired cDNA using the deposited cDNA plasmid as a template. The polymerase chain reaction is canied out under routine conditions, for instance, in 25 ul of reaction mixture with 0.5 ug of the above cDNA template. A convenient reaction mixture is 1.5-5 mM MgCl₂, 0.01% (w/v) gelatin, 20 uM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase. Thirty five cycles of PCR (denaturation at 94 degree C for 1 min; annealing at 55 degree C for 1 min; elongation at 72 degree C for 1 min) are petformed with a Perkin-Elmer Cetus automated thermal cycler. The amplified product is analyzed by agarose gel electrophoresis and the DNA band with expected molecular weight is excised and purified. The PCR product is verified to be the selected sequence by subcloning and sequencing the DNA product.

Several methods are available for the identification of the 5' or 3" non-coding portions of a gene which may not be present in the deposited clone. These methods include but are not limited to, filter probing, clone enrichment using specific probes, and protocols similar or identical to 5' and 3" "RACE" protocols which are well known in the art. For instance, a method similar to 5' RACE is available for generating the missing 5' end of a desired full-length transcript. (Fromont-Racine et al., Nucleic Acids Res. 21(7)J683J684 (1993).)

Briefly, a specific RNA oligonucleotide is ligated to the 5' ends of a population of RNA presumably containing full-length gene RNA transcripts. A primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specific to a known sequence of the gene of interest is used to PCR amplify the 5' portion of the desired full- length gene. This amplified product may then be sequenced and used to generate the full length gene. This above method starts with total RNA isolated from the desired source, although poly-A+ RNA can be used The RNA preparation can then be treated with phosphatase if necessary to eliminate 5^' phosphate groups on degraded or damaged RNA which may interfere with the later RNA ligase step. The phosphatase should then be inactivated and the RNA treated with tobacco acid pyrophosphatase in oider to remove the cap structuie present at the 5' ends of messengei RNAs. This reaction leaves a 5' phosphate group at the 5' end of the cap cleaved RNA which can then be ligated to an RNA o gonucleotide using T4 RNA ligase

This modified RNA prepaiatton is used as a template for first strand cDNA synthesis using a gene specific o gonucleotide The fust strand synthesis reaction is used as a template for PCR amplification of the desired 5' end using a pnmer specific to the ligated RNA ohgonucleotide and a pnmer specific to the known sequence of the gene of interest. The resultant product is then sequenced and analyzed to confirm that the 5' end sequence belongs to the desired gene

Example 2; Isolation of Genomic Clones Corresponding to a Polynucleotide

A human genomic PI library (Genomic Systems, Inc.) is screened by PCR using pnmers selected for the cDNA sequence corresponding to SEQ ID NO.X., according to the method described in Example 1. (See also, Sambrook.)

Example 3: Tissue Distribution of Polypeptide

Tissue distπbution of mRNA expression of polynucleotides of the present invention is determined using protocols for Northern blot analysis, descnbed by, among others, Sambrook et al. For example, a cDNA probe produced by the method descnbed Example 1 is labeled with P³² using the redipπme™ DNA labeling system (Amersham Life Science), according to manufacturer's instructions. After labeling, the probe is punfted using CHROMA SPIN- 100™ column (Clontech Laboratoπes, Inc.), according to manufacturer's protocol number PT 1200-1. The purified labeled probe is then used to examine various human tissues for mRNA expression.

Multiple Tissue Northern (MTN) blots containing various human tissues (H) or human immune system tissues (IM) (Clontech) are examined with the labeled probe using ExpressHyb™ hybridization solution (Clontech) according to manufacturer's protocol number PT 1190-1. Following hybridization and washing, the blots are mounted and exposed to film at -70 degree C overnight, and the films developed according to standard procedures.

Example 4: Chromosomal Mapping of the Polynucleotides

An oligonucleotide primer set is designed according to the sequence at the 5' end of SEQ ID NO:X. This primer preferably spans about 100 nucleotides. This primer set is then used in a polymerase chain reaction under the following set of conditions : 30 seconds,95 degree C; 1 minute, 56 degree C; 1 minute, 70 degree C. This cycle is repeated 32 times followed by one 5 minute cycle at 70 degree C. Human, mouse, and hamster DNA is used as template in addition to a somatic cell hybrid panel containing individual chromosomes or chromosome fragments (Bios, Ine). The reactions is analyzed on either 8% polyacrylamide gels or 3.5 % agarose gels. Chromosome mapping is determined by the presence of an approximately 100 bp PCR fragment in the particular somatic cell hybrid.

Example 5: Bacterial Expression of a Polypeptide

A polynucleotide encoding a polypeptide of the present invention is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' ends of the DNA sequence, as outlined in Example 1, to synthesize insertion fragments. The primers used to amplify the cDNA insert should preferably contain restriction sites, such as BamHI and Xbal, at the 5' end of the primers in order to clone the amplified product into the expression vector. For example, BamHI and Xbal correspond to the restriction enzyme sites on the bactenal expiession vector pQE-9 (Qiagen, Ine , Chatsworth, CA) This plasmid vector encodes antibiotic resistance (Amp^1*), a bactenal ongm of replication (on), an IPTG-regulatable promoter/operator (P/O), a πbosome binding site (RBS), a 6-hιstιdιne tag (6-Hιs), and restπction enzyme cloning sites The pQE-9 vector is digested with BamHI and Xbal and the amplified fragment is ligated into the pQE-9 vectoi maintaining the reading ft me initiated at the bacterial RBS The ligation mixtuie is then used to transform the E coli strain M15/rep4 (Qiagen, Ine ) which contains multiple copies of the plasmid pREP4, which expresses the lad lepressor and also confers kanamycin resistance (Kan^r) Ttansformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected Plasmid DNA is isolated and confirmed by restπction analysis

Clones containing the desned constructs aie grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml) The O/N culture is used to inoculate a large culture at a ratio of 1 100 to 1 250 The cells are grown to an optical density 600 (O D ⁶⁰⁰) of between 0 4 and 0 6 IPTG (Isopropyl-B-D- thiogalacto pyranoside) is then added to a final concentration of 1 mM IPTG induces by inactivating the lad repressor, cleaπng the P/O leading to increased gene expression

Cells are grown for an extra 3 to 4 hours Cells are then harvested by centnfugation (20 mins at 6000Xg) The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl by stirπng foi 3-4 hours at 4 degree C The cell debπs is removed by centnfugation, and the supernatant containing the polypeptide is loaded onto a nickel- mtnlo-tπ-acetic acid ("Ni-NTA") affinity resin column (available from QIAGEN, Inc., supra) Proteins with a 6 x His tag bind to the Ni-NTA resin with high affinity and can be punfied m a simple one-step procedure (for details see The QIAexpressionist (1995) QIAGEN, Ine , supra).

Briefly, the supernatant is loaded onto the column 6 M guanidine-HCl, pH 8, the column is first washed with 10 volumes of 6 M guanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finally the polypeptide is eluted with 6 M guanidine-HCl, pH 5.

The purified protein is then renatured by dialyzing it against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaCI. Alternatively, the protein can be successfully refolded while immobilized on the Ni-NTA column. The recommended conditions are as follows: renature using a linear 6M-1M urea gradient in

500 mM NaCI, 20% glycerol, 20 mM Tris/HCI pH 7.4, containing protease inhibitors. The renaturation should be performed over a period of 1.5 hours or more. After renaturation the proteins are eluted by the addition of 250 mM immidazole. Immidazole is removed by a final dialyzing step against PBS or 50 mM sodium acetate pH 6 buffer plus 200 mM NaCI. The purified protein is stored at 4 degree C or frozen at -80 degree C.

In addition to the above expression vector, the present invention further includes an expression vector comprising phage operator and promoter elements operatively linked to a polynucleotide of the present invention, called pHE4a. (ATCC Accession Number 209645, deposited on February 25, 1998.) This vector contains: 1) a neomycinphosphotransferase gene as a selection marker, 2) an E. coli origin of replication, 3) a T5 phage promoter sequence, 4) two lac operator sequences, 5) a Shine-Delgarno sequence, and 6) the lactose operon repressor gene (laclq). The origin of replication (oriC) is derived from pUC19 (LTI, Gaithersburg, MD). The promoter sequence and operator sequences are made synthetically.

DNA can be inserted into the pHEa by restricting the vector with Ndel and Xbal, BamHI, Xhol, or Asp718, running the restricted product on a gel, and isolating the larger fragment (the stuffer fragment should be about 310 base pairs). The DNA insert is generated according to the PCR protocol described in Example 1, using PCR primers having restriction sites for Ndel (5' primer) and Xbal, BamHI, Xhol, or Asp718 (3' primer). The PCR insert is gel purified and restricted with compatible enzymes. The insert and vector are ligated according to standard protocols. The engineered vector could easily be substituted in the above protocol to express protein in a bacterial system.

Example 6: Purification of a Polypeptide from an Inclusion Body The following alternative method can be used to purify a polypeptide expressed in

E coli when it is present in the form of inclusion bodies. Unless otherwise specified, all of the following steps are conducted at 4-10 degree C.

Upon completion of the production phase of the E. coli fermentation, the cell culture is cooled to 4-10 degree C and the cells harvested by continuous centrifugation at 15,000 φm (Heraeus Sepatech). On the basis of the expected yield of protein per unit weight of cell paste and the amount of purified protein required, an appropriate amount of cell paste, by weight, is suspended in a buffer solution containing 100 mM Tris, 50 mM EDTA, pH 1.4. The cells are dispersed to a homogeneous suspension using a high shear mixer. The cells are then lysed by passing the solution through a microfluidizer

(Microfuidics, Coφ. or APV Gaulin, Inc.) twice at 4000-6000 psi. The homogenate is then mixed with NaCI solution to a final concentration of 0.5 M NaCI, followed by centrifugation at 7000 xg for 15 min. The resultant pellet is washed again using 0.5M NaCI, 100 mM Tris, 50 mM EDTA, pH 1.4. The resulting washed inclusion bodies are solubilized with 1.5 M guanidine hydrochloride (GuHCl) for 2-4 hours. After 7000 xg centrifugation for 15 min., the pellet is discarded and the polypeptide containing supernatant is incubated at 4 degree C overnight to allow further GuHCl extraction.

Following high speed centrifugation (30,000 xg) to remove insoluble particles, the GuHCl solubilized protein is refolded by quickly mixing the GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH 4.5, 150 mM NaCI, 2 mM EDTA by vigorous stirring. The refolded diluted protein solution is kept at 4 degree C without mixing for 12 hours prior to further purification steps. To clarify the refolded polypeptide solution, a previously prepared tangential filtration unit equipped with 0J6 um membrane filter with appropriate surface area (e.g.,

Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loaded onto a cation exchange resin (e.g., Poros HS-50, Perseptive Biosystems). The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM, 1000 mM, and 1500 mM NaCI in the same buffer, in a stepwise manner. The absorbance at 280 nm of the effluent is continuously monitored. Fractions are collected and further analyzed by SDS-PAGE.

Fractions containing the polypeptide are then pooled and mixed with 4 volumes of water. The diluted sample is then loaded onto a previously prepared set of tandem columns of strong anion (Poros HQ-50, Perseptive Biosystems) and weak anion (Poros CM-20, Perseptive Biosystems) exchange resins. The columns are equilibrated with 40 mM sodium acetate, pH 6.0. Both columns are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCI. The CM-20 column is then eluted using a 10 column volume linear gradient ranging from 0.2 M NaCI. 50 mM sodium acetate, pH 6.0 to 1.0 M NaCI, 50 mM sodium acetate, pH 6.5. Fractions are collected under constant A₂₈₀ monitoring of the effluent. Fractions containing the polypeptide (determined, for instance, by 16% SDS- PAGE) are then pooled.

The resultant polypeptide should exhibit greater than 95% purity after the above refolding and purification steps. No major contaminant bands should be observed from

Commassie blue stained 16% SDS-PAGE gel when 5 ug of purified protein is loaded. The purified protein can also be tested for endotoxin/LPS contamination, and typically the LPS content is less than 0J ng/ml according to LAL assays.

Example 7: Cloning and Expression of a Polypeptide in a Baculovirus Expression System

In this example, the plasmid shuttle vector pA2 is used to insert a polynucleotide into a baculovirus to express a polypeptide. This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites such as BamHI, Xba I and Asp718. The polyadenylation site of the simian virus 40 ("SV40") is used for efficient polyadenylation.

For easy selection of recombinant virus, the plasmid contains the beta-galactosidase gene from E. coli under control of a weak Drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhedrin gene. The inserted genes are flanked on both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate a viable virus that express the cloned polynucleotide.

Many other baculovirus vectors can be used in place of the vector above, such as pAc373, pVL941, and pAcIMl, as one skilled in the art would readily appreciate, as long as the construct provides appropriately located signals for transcription, translation, secretion and the like, including a signal peptide and an in-frame AUG as required. Such vectors are described, for instance, in Luckow et al., Virology 170:31-39 (1989).

Specifically, the cDNA sequence contained in the deposited clone, including the AUG initiation codon and the naturally associated leader sequence identified in Table XIII, is amplified using the PCR protocol described in Example 1. If the naturally occurring signal sequence is used to produce the secreted protein, the pA2 vector does not need a second signal peptide. Alternatively, the vector can be modified (pA2 GP) to include a baculovirus leader sequence, using the standard methods described in Summers et al., "A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures," Texas Agricultural Experimental Station Bulletin No. 1555 (1987).

The amplified fragment is isolated from a 1% agarose gel using a commercially available kit ("Geneclean," BIO 101 Inc., La Jolla, Ca.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel. The plasmid is digested with the corresponding restriction enzymes and optionally, can be dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art. The DNA is then isolated from a 1% agarose gel using a commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.). The fragment and the dephosphorylated plasmid are ligated together with T4 DNA ligase. E. coli HB101 or other suitable E. coli hosts such as XL-1 Blue (Stratagene

Cloning Systems, La Jolla, CA) cells are transformed with the ligation mixture and spread on culture plates. Bacteria containing the plasmid are identified by digesting DNA from individual colonies and analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing.

Five ug of a plasmid containing the polynucleotide is co-transfected with 1.0 ug of a commercially available linearized baculovirus DNA ("BaculoGold™ baculovirus DNA",

Pharmingen, San Diego, CA), using the lipofection method described by Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). One ug of BaculoGold™ virus DNA and 5 ug of the plasmid are mixed in a sterile well of a microtiter plate containing 50 ul of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, MD). Afterwards, 10 ul Lipofectin plus 90 ul Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added drop-wise to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is then incubated for 5 hours at 27 degrees C. The transfection solution is then removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. Cultivation is then continued at 27 degrees C for four days. After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, supra. An agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of gal- expressing clones, which produce blue-stained plaques. (A detailed description of a "plaque assay" of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10.) After appropriate incubation, blue stained plaques are picked with the tip of a micropipettor (e.g., Εppendorf). The agar containing the recombinant viruses is then resuspended in a microcentrifuge tube containing 200 ul of Grace's medium and the suspension containing the recombinant baculovirus is used to mfect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then they are stored at 4 degree C.

To verify the expression of the polypeptide, Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells are infected with the recombinant baculovirus containing the polynucleotide at a multiplicity of infection ("MOI") of about 2.

If radiolabeled proteins are desired, 6 hours later the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies

Inc., Rockville, MD). After 42 hours, 5 uCi of ^,5S-methιonme and 5 uCi ¹⁵S-cysteιne (available from Amersham) are added. The cells are further incubated for 16 hours and then are harvested by centnfugation The proteins in the supernatant as well as the intracellular proteins are analyzed by SDS-PAGE followed by autoradiography (if radiolabeled).

Microsequencmg of the amino acid sequence of the amino terminus of punfied protein may be used to determine the amino terminal sequence of the produced protein.

Example 8: Expression of a Polypeptide in Mammalian Cells

The polypeptide of the present invention can be expressed in a mammalian cell. A typical mammalian expression vector contains a promoter element, which mediates the initiation of transcription of mRNA, a protein coding sequence, and signals required for the termination of transcnption and polyadenylation of the transcnpt. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcπption is achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter). Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat

(ATCC 37152), pSV2d-.fr (ATCC 37146), pBC12MI (ATCC 67109), pCMVSport 2.0, and pCMVSport 3.0. Mammalian host cells that could be used include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CVI, quail QCl-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.

Alternatively, the polypeptide can be expressed in stable cell lines containing the polynucleotide integrated into a chromosome. The co-transfection with a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) marker is useful in developing cell lines that carry several hundred or even several thousand copies of the gene of interest. (See, e.g., Alt, F. W., et al., J. Biol. Chem. 253:1357-1370 (1978); Ha lin, J. L. and Ma, C, Biochem. et Biophys. Acta, 1097: 107-143 (1990); Page, M. J. and Sydenham, M. A.,

Biotechnology 9:64-68 (1991).) Another useful selection marker is the enzyme glutamine synthase (GS) (Muφhy et al., Biochem J. 227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175 (1992). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the production of proteins.

Derivatives of the plasmid pSV2-dhfr (ATCC Accession No. 37146), the expression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCC Accession No.209647) contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447 (March, 1985)) plus a fragment of the CMV- enhancer (Boshart et al., Cell 41:521-530 (1985).) Multiple cloning sites, e.g., with the restriction enzyme cleavage sites BamHI, Xbal and Asp718, facilitate the cloning of the gene of interest. The vectors also contain the 3' intron, the polyadenylation and termination signal of the rat preproinsulin gene, and the mouse DHFR gene under control of the SV40 early promoter.

Specifically, the plasmid pC6, for example, is digested with appropriate restriction enzymes and then dephosphorylated using calf intestinal phosphates by procedures known in the art. The vector is then isolated from a 1% agarose gel.

A polynucleotide of the present invention is amplified according to the protocol outlined in Example 1. If the naturally occurring signal sequence is used to produce the secreted protein, the vector does not need a second signal peptide. Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, e.g., WO 96/34891.)

The amplified fragment is isolated from a 1% agarose gel using a commercially available kit ("Geneclean," BIO 101 Inc., La Jolla, Ca.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.

The amplified fragment is then digested with the same restriction enzyme and purified on a 1% agarose gel. The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. E. cσ/-^' HB101 or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC6 using, for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene is used for transfection. Five μg of the expression plasmid pC6 a pC4 is cotransfected with 0.5 ug of the plasmid pSVneo using lipofectin (Feigner et al., supra). The plasmid pSV2-neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418. The cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/ml G418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 uM, 2 uM, 5 uM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained which grow at a concentration of 100 - 200 uM. Expression of the desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reversed phase

HPLC analysis.

Example 9: Protein Fusions

The polypeptides of the present invention are preferably fused to other proteins. These fusion proteins can be used for a variety of applications. For example, fusion of the present polypeptides to His-tag, HA-tag, protein A, IgG domains, and maltose binding protein facilitates purification. (See Example 5; see also EP A 394.827; Traunecker, et al., Nature 331:84-86 (1988).) Similarly, fusion to IgG-1, IgG-3, and albumin increases the halflife time in vivo. Nuclear localization signals fused to the polypeptides of the present invention can target the protein to a specific subcellular localization, while covalent heterodimer or homodimers can increase or decrease the activity of a fusion protein. Fusion proteins can also create chimeric molecules having more than one function. Finally, fusion proteins can increase solubility and/or stability of the fused protein compared to the non-fused protein. All of the types of fusion proteins described above can be made by modifying the following protocol, which outlines the fusion of a polypeptide to an IgG molecule, or the protocol described in Example 5.

Briefly, the human Fc portion of the IgG molecule can be PCR amplified, using primers that span the 5' and 3' ends of the sequence described below. These primers also should have convenient restriction enzyme sites that will facilitate cloning into an expression vector, preferably a mammalian expression vector.

For example, if pC4 (Accession No. 209646) is used, the human Fc portion can be ligated into the BamHI cloning site. Note that the 3' BamHI site should be destroyed. Next, the vector containing the human Fc portion is re-restricted with BamHI, linearizing the vector, and a polynucleotide of the present invention, isolated by the PCR protocol described in Example 1, is ligated into this BamHI site. Note that the polynucleotide is cloned without a stop codon, otherwise a fusion protein will not be produced.

If the naturally occurring signal sequence is used to produce the secreted protein, pC4 does not need a second signal peptide. Altematively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence.

(See, e.g., WO 96/34891.)

Human IgG Fc region: GGG ATCCGGAGCCCAAATCTTCTGAC AA A ACTCACAC ATGCCCACCGTGCCC A GCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAA GGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACG TAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGA GGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC CGTGTGGTCAGCGTCCTCACCGTCCTGC ACCAGGACTGGCTG AATGGCAAGGA GTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCA TCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCA TCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG CTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC TACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCT CATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT (SEQ ID NOJ)

Example 10: Production of an Antibody from a Polypeptide

The antibodies of the present invention can be prepared by a variety of methods. (See, Current Protocols, Chapter 2.) As one example of such methods, cells expressing a polypeptide of the present invention is administered to an animal to induce the production of sera containing polyclonal antibodies. In a preferred method, a preparation of the secreted protein is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.

In the most preferred method, the antibodies of the present invention are monoclonal antibodies (or protein binding fragments thereof). Such monoclonal antibodies can be prepared using hybridoma technology. (Kδhler et al., Nature 256:495

(1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981).) In general, such procedures involve immunizing an animal (preferably a mouse) with polypeptide or, more preferably, with a secreted polypeptide- expressing cell. Such cells may be cultured in any suitable tissue culture medium; however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56 degrees C), and supplemented with about 10 g/1 of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 ug/ml of streptomycin.

The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP2O), available from the ATCC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80:225-232 (1981).) The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the polypeptide.

Alternatively, additional antibodies capable of binding to the polypeptide can be produced in a two-step procedure using anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, and therefore, it is possible to obtain an antibody which binds to a second antibody. In accordance with this method, protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the protein- specific antibody can be blocked by the polypeptide. Such antibodies comprise anti- idiotypic antibodies to the protein-specific antibody and can be used to immunize an animal to induce formation of further protein-specific antibodies.

It will be appreciated that Fab and F(ab')2 and other fragments of the antibodies of the present invention may be used according to the methods disclosed herein. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). Alternatively, secreted protein-binding fragments can be produced through the application of recombinant DNA technology or through synthetic chemistry.

For in vivo use of antibodies in humans, it may be preferable to use "humanized" chimeric monoclonal antibodies. Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric antibodies are known in the art. (See, for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Patent No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671 ; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985).)

Example 11; Production Of Secreted Protein For High-Throughput Screening Assays The following protocol produces a supernatant containing a polypeptide to be tested. This supernatant can then be used in the Screening Assays described in Examples 13-20. First, dilute Poly-D-Lysine (644 587 Boehringer-Mannheim) stock solution

(lmg/ml in PBS) 1:20 in PBS (w/o calcium or magnesium 17-516F Biowhittaker) for a working solution of 50ug/ml. Add 200 ul of this solution to each well (24 well plates) and incubate at RT for 20 minutes. Be sure to distribute the solution over each well (note: a 12-channel pipetter may be used with tips on every other channel). Aspirate off the Poly- D-Lysine solution and rinse with 1ml PBS (Phosphate Buffered Saline). The PBS should remain in the well until just prior to plating the cells and plates may be poly-lysine coated in advance for up to two weeks.

Plate 293T cells (do not carry cells past P+20) at 2 x 10⁵ cells/well in .5ml DMEM(Dulbecco's Modified Eagle Medium)(with 4.5 G/L glucose and L-glutamine (12- 604F Biowhittaker))/10% heat inactivated FBS(14-503F Biowhittaker)/lx Penstrep(17- 602E Biowhittaker). Let the cells grow overnight.

The next day, mix together in a sterile solution basin: 300 ul Lipofectamine (18324-012 Gibco/BRL) and 5ml Optimem I (31985070 Gibco/BRL)/96-well plate. With a small volume multi-channel pipetter, aliquot approximately 2ug of an expression vector containing a polynucleotide insert, produced by the methods described in Examples 8 or 9, into an appropriately labeled 96-well round bottom plate. With a multi-channel pipetter, add 50ul of the Lipofectamine/Optimem I mixture to each well. Pipette up and down gently to mix. Incubate at RT 15-45 minutes. After about 20 minutes, use a multi-channel pipetter to add 150ul Optimem I to each well. As a control, one plate of vector DNA lacking an insert should be transfected with each set of transfections.

Preferably, the transfection should be performed by tag-teaming the following tasks. By tag-teaming, hands on time is cut in half, and the cells do not spend too much time on PBS. First, person A aspirates off the media from four 24-well plates of cells, and then person B rinses each well with .5-lml PBS. Person A then aspirates off PBS rinse, and person B, using al2-channel pipetter with tips on every other channel, adds the 200ul of DNA Lipofectamine/Optimem I complex to the odd wells first, then to the even wells, to each row on the 24-well plates. Incubate at 37 degrees C for 6 hours. While cells are incubating, prepare appropriate media, either 1%BSA in DMEM with lx penstrep, or CHO-5 media (116.6 mg/L of CaC12 (anhyd); 0.00130 mg L CuSO₄-

5H₂O; 0.050 mg/L of Fe(NO₃)₃-9H₂O; 0.417 mg/L of FeSO₄-7H₂O; 311.80 mg L of Kcl;

28.64 mg/L of MgCl₂; 48.84 mg/L of MgSO₄; 6995.50 mg/L of NaCI; 2400.0 mg/L of NaHCO₃; 62.50 mg L of NaH₂PO₄-H₂0; 71.02 mg L of Na₂HPO4; .4320 mg/L of ZnSO₄-

7H₂O; .002 mg/L of Arachidonic Acid ; 1.022 mg/L of Cholesterol; .070 mg/L of DL- alpha-Tocopherol-Acetate; 0.0520 mg/L of Linoleic Acid; 0.010 mg/L of Linolenic Acid;

0.010 mg/L of Myristic Acid; 0.010 mg L of Oleic Acid; 0.010 mg/L of Palmitric Acid;

0.010 mg/L of Palmitic Acid; 100 mg/L of Pluronic F-68; 0.010 mg/L of Stearic Acid; 2.20 mg/L of Tween 80; 4551 mg/L of D-Glucose; 130.85 mg/ml of L- Alanine; 147.50 mg/ml of L-Arginine-HCL; 7.50 mg/ml of L-Asparagine-H₂0; 6.65 mg/ml of L-Aspartic Acid; 29.56 mg/ml of L-Cystine-2HCL-H₂0; 31.29 mg/ml of L-Cystine-2HCL; 7.35 mg/ml of L-Glutamic Acid; 365.0 mg/ml of L-Glutamine; 18.75 mg/ml of Glycine; 52.48 mg/ml of L-Histidine-HCL-H₂0; 106.97 mg/ml of L-Isoleucine; 111.45 mg/ml of L-Leucine; 163.75 mg/ml of L-Lysine HCL; 32.34 mg/ml of L-Methionine; 68.48 mg/ml of L-

Phenylalainine; 40.0 mg/ml of L-Proline; 26.25 mg/ml of L-Serine; 101.05 mg/ml of L- Threonine; 19.22 mg/ml of L-Tryptophan; 91.79 mg/ml of L-Tryrosine-2Na-2H₂0; 99.65 mg/ml of L- Valine; 0.0035 mg/L of Biotin; 3.24 mg/L of D-Ca Pantothenate; 11.78 mg/L of Choline Chloride; 4.65 mg/L of Folic Acid; 15.60 mg/L of i-Inositol; 3.02 mg/L of Niacinamide; 3.00 mg L of Pyridoxal HCL; 0.031 mg/L of Pyridoxine HCL; 0.319 mg/L of Riboflavin; 3J7 mg L of Thiamine HCL; 0.365 mg/L of Thymidine; and 0.680 mg/L of Vitamin B₁₂; 25 mM of HEPES Buffer; 2.39 mg/L of Na Hypoxanthine; 0J05 mg/L of Lipoic Acid; 0.081 mg/L of Sodium Putrescine-2HCL; 55.0 mg/L of Sodium Pyruvate; 0.0067 mg L of Sodium Selenite; 20uM of Ethanolamine; 0J22 mg/L of Ferric Citrate; 41.70 mg/L of Methyl-B-Cyclodextrin complexed with Linoleic Acid; 33.33 mg L of Methyl-B-Cyclodextrin complexed with Oleic Acid; and 10 mg/L of Methyl-B- Cyclodextrin complexed with Retinal) with 2mm glutamine and lx penstrep. (BSA (81- 068-3 Bayer) lOOgm dissolved m IL DMEM for a 10% BSA stock solution) Filter the media and collect 50 ul for endotoxin assay in 15ml polystyrene conical.

The transfection reaction is terminated, preferably by tag-teaming, at the end of the incubation penod. Person A aspirates off the transfection media, while person B adds 1.5ml appropriate media to each well. Incubate at 37 degrees C for 45 or 72 hours depending on the media used. 1%BSA foi 45 houis ot CHO-5 foi 72 hours

On day four, using a 300ul multichannel pipettei , aliquot 600ul in one 1ml deep well plate and the remaining supernatant into a 2ml deep well The supernatants from each well can then be used in the assays descπbed in Examples 13-20. It is specifically understood that when activity is obtained in any of the assays described below using a supernatant, the activity originates from eithei the polypeptide directly (e.g., as a secreted protein) or by the polypeptide inducing expression of other proteins, which aie then secreted into the supernatant. Thus, the invention further provides a method of identifying the protein in the supernatant charactenzed by an activity in a particular assay.

Example 12: Construction of GAS Reporter Construct

One signal transduction pathway involved in the differentiation and proliferation of cells is called the Jaks-STATs pathway. Activated proteins in the Jaks-STATs pathway bind to gamma activation site "GAS" elements or interferon-sensitive responsive element ("ISRE"), located in the promoter of many genes. The binding of a protein to these elements alter the expression of the associated gene.

GAS and ISRE elements are recognized by a class of transcnption factors called Signal Transducers and Activators of Transcnption, or "STATs." There are six members of the STATs family. Statl and Stat3 are present in many cell types, as is Stat2 (as response to IFN-alpha is widespread). Stat4 is more restricted and is not in many cell types though it has been found in T helper class I, cells after treatment with IL-12. Stat5 was oπgmally called mammary growth factor, but has been found at higher concentrations in other cells including myeloid cells. It can be activated in tissue culture cells by many cytokines.

The STATs are activated to translocate from the cytoplasm to the nucleus upon tyrosine phosphorylation by a set of kinases known as the Janus Kinase ("Jaks") family. Jaks represent a distinct family of soluble tyrosine kinases and include Tyk2, Jakl, Jak2, and Jak3. These kinases display significant sequence similarity and are generally catalyticaliy inactive in resting cells.

The Jaks are activated by a wide range of receptors summarized in the Table below. (Adapted from review by Schidler and Darnell, Ann. Rev. Biochem. 64:621-51 (1995).) A cytokine receptor family, capable of activating Jaks, is divided into two groups: (a) Class 1 includes receptors for IL-2, IL-3, IL-4, IL-6, IL-7. IL-9, IL-11, IL-12, IL-15, Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; and (b) Class 2 includes IFN-a, IFN-g, and IL-10. The Class 1 receptors share a conserved cysteine motif (a set of four conserved cysteines and one tryptophan) and a WSXWS motif (a membrane proximal region encoding Tφ-Ser-Xxx-Tφ-Ser (SEQ ID NO:2)).

Thus, on binding of a ligand to a receptor, Jaks are activated, which in turn activate STATs, which then translocate and bind to GAS elements. This entire process is encompassed in the Jaks-STATs signal transduction pathway.

Therefore, activation of the Jaks-STATs pathway, reflected by the binding of the GAS or the ISRE element, can be used to indicate proteins involved in the proliferation and differentiation of cells. For example, growth factors and cytokines are known to activate the Jaks-STATs pathway. (See Table below.) Thus, by using GAS elements linked to reporter molecules, activators of the Jaks-STATs pathway can be identified. JAKs STATS GAS, elements) or ISRE

Ligand tyk- Jakl Jak2 Jak3

IFN familv

IFN-a/B + + - - 1.2.3 ISRE

IFN-g + + - 1 GAS (IRFl>Lys6>IFP)

11-10 + 9 ? - 13 gpl30 familv

IL-6 (Pleiotrophic) + + + 9 1.3 GAS (IRFl>Lys6>IFP)

Il-l l(Pleiotrophic) ? + 9 9 1.3

OnM(Pleiotrophic) ') + + 9 1.3

LIF(Pleiotrophic) 9 + + 9 1.3

CNTF(Pleiotrophic) -/+ + + 9 1.3

G-CSF(Pleiotrophic) 9 + 9 9 1.3

IL-12(Pleiotrophic) + - + + 1.3

2-C famiiv

IL-2 (lymphocytes) - + - + 1.3.5 GAS

IL-4 (lymph/myeloid) - + - + 6 GAS (IRF1 = IFP »Lv6)(leH)

IL-7 (lymphocytes) - + - + 5 GAS

IL-9 (lymphocytes) - + - + 5 GAS

IL-13 (lymphocyte) - + 9 9 6 GAS

IL-15 ? + 9 + 5 GAS gpl40 familv

IL-3 (myeloid) - - + - 5 GAS (IRFl >IFP»Ly6)

IL-5 (myeloid) - - + - 5 GAS

GM-CSF (myeloid) - - + - 5 GAS

Growth hormone familv /

GH 9 - + - 5

PRL 9 +/- + - 1.3.5

EPO 9 - + - 5 GAS(B-CAS>IRFl=IFP»Ly6)

Receptor Tvrosine Kinases

EGF 9 + + - 1.3 GAS (IRFl )

PDGF 9 + + - 13

CSF-1 9 + + - 1.3 GAS (not ΙRFl )

To construct a synthetic GAS containing promoter element, which is used in the

Biological Assays described in Examples 13-14, a PCR based strategy is employed to generate a GAS-SV40 promoter sequence. The 5' primer contains four tandem copies of the GAS binding site found in the IRF1 promoter and previously demonstrated to bind STATs upon induction with a range of cytokines (Rothman et al., Immunity 1 :457-468

(1994).), although other GAS or ISRE elements can be used instead. The 5' primer also contains 18bp of sequence complementary to the SV40 early promoter sequence and is flanked with an Xhol site. The sequence of the 5' primer is:

5':GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAA ATGATTTCCCCGAAATATCTGCCATCTCAATTAG:3' (SEQ ID NO:3)

The downstream primer is complementary to the SV40 promoter and is flanked with a Hind III site: 5':GCGGCAAGCTTTTTGCAAAGCCTAGGC:3' (SEQ ID NOJ) PCR amplification is performed using the SV40 promoter template present in the B-gal:promoter plasmid obtained from Clontech. The resulting PCR fragment is digested with Xhol/Hind III and subcloned into BLSK2-. (Stratagene.) Sequencing with forward and reverse primers confirms that the insert contains the following sequence: 5 ' : CTCGAG ATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGA AATGA TTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTA ACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCAT GGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAG CTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAA GCTT:3' (SEQ ID NO:5)

With this GAS promoter element linked to the SV40 promoter, a GAS:SEAP2 reporter construct is next engineered. Here, the reporter molecule is a secreted alkaline phosphatase, or "SEAP." Clearly, however, any reporter molecule can be instead of

SEAP, in this or in any of the other Examples. Well known reporter molecules that can be used instead of SEAP include chloramphenicol acetyltransferase (CAT), luciferase, alkaline phosphatase, B-galactosidase, green fluorescent protein (GFP), or any protein detectable by an antibody.

The above sequence confirmed synthetic GAS-SV40 promoter element is subcloned into the pSEAP-Promoter vector obtained from Clontech using Hindlll and Xhol, effectively replacing the SV40 promoter with the amplified GAS:SV40 promoter element, to create the GAS-SEAP vector. However, this vector does not contain a neomycin resistance gene, and therefore, is not prefeired for mammalian expression systems.

Thus, in order to generate mammalian stable cell lines expressing the GAS-SEAP reporter, the GAS-SEAP cassette is removed from the GAS-SEAP vector using Sail and Notl, and inserted into a backbone vector containing the neomycin resistance gene, such as pGFP-1 (Clontech), using these restriction sites in the multiple cloning site, to create the GAS-SEAP/Neo vector. Once this vector is transfected into mammalian cells, this vector can then be used as a reporter molecule for GAS binding as described in Examples 13-14. Other constructs can be made using the above description and replacing GAS with a different promoter sequence. For example, construction of reporter molecules containing NFK-B and EGR promoter sequences are described in Examples 15 and 16. However, many other promoters can be substituted using the protocols described in these Examples. For instance, SRE, IL-2, NFAT, or Osteocalcin promoters can be substituted, alone or in combination (e.g., GAS/NF-KB/EGR, GAS NF-KB, I1-2/NFAT, or NF-KB/GAS).

Similarly, other cell lines can be used to test reporter construct activity, such as HELA (epithelial), HUVEC (endothelial), Reh (B-cell), Saos-2 (osteoblast), HUVAC (aortic), or Cardiomyocyte.

Example 13: High-Throughput Screening Assay for T-cell Activity.

The following protocol is used to assess T-cell activity by identifying factors, and determining whether supemate containing a polypeptide of the invention proliferates and or differentiates T-cells. T-cell activity is assessed using the GAS/SEAP/Neo construct produced in Example 12 Thus, factors that increase SEAP activity indicate the ability to activate the Jaks-STATS signal transduction pathway. The T-cell used in this assay is Jurkat T-cells (ATCC Accession No. TIB- 152), although Molt-3 cells (ATCC

Accession No. CRL-1552) and Molt-4 cells (ATCC Accession No. CRL-1582) cells can also be used.

Jurkat T-cells are lymphoblasttc CD4+ Thl helpei cells In order to genetate stable cell lines, approximately 2 million Jurkat cells are ttansfected with the GAS-SEAP/neo vectoi using DMRIE-C (Life Technologιes)(transfectιon procedure described below) The transfected cells are seeded to a density of approximately 20,000 cells per well and transfectants resistant to 1 mg/ml genticin selected Resistant colonies are expanded and then tested for their response to increasing concentrations of interferon gamma The dose response of a selected clone is demonstrated

Specifically, the following protocol will yield sufficient cells for 75 wells containing 200 ul of cells Thus, it is either scaled up, or performed in multiple to generate sufficient cells for multiple 96 well plates. Jurkat cells are maintained in RPMI + 10% serum with l%Pen-Strep Combine 2.5 mis of OPTI-MEM (Life Technologies) with 10 ug of plasmid DNA in a T25 flask. Add 2.5 ml OPTI-MEM containing 50 ul of DMRIE-C and incubate at room temperature for 15-45 mins.

Duπng the incubation peπod, count cell concentration, spin down the required number of cells (10⁷ per transfection), and resuspend in OPTI-MEM to a final concentration of 10⁷ cells/ml Then add 1ml of 1 x 10⁷ cells m OPTI-MEM to T25 flask and incubate at 37 degrees C for 6 hrs. After the incubation, add 10 ml of RPMI + 15% serum.

The Jurkat:GAS-SEAP stable reporter lines are maintained in RPMI + 10% serum, 1 mg/ml Genticin, and 1% Pen-Strep. These cells are treated with supernatants containing polypeptides of the invention and or induced polypeptides of the invention as produced by the protocol descnbed in Example 11. On the day of treatment with the supernatant, the cells should be washed and resuspended in fresh RPMI + 10% serum to a density of 500,000 cells per ml. The exact number of cells required will depend on the number of supernatants being screened. For one 96 well plate, approximately 10 million cells (for 10 plates, 100 million cells) are required.

Transfer the cells to a triangular reservoir boat, in order to dispense the cells into a 96 well dish, using a 12 channel pipette. Using a 12 channel pipette, transfer 200 ul of cells into each well (therefore adding 100, 000 cells per well).

After all the plates have been seeded, 50 ul of the supernatants are transferred directly from the 96 well plate containing the supernatants into each well using a 12 channel pipette. In addition, a dose of exogenous interferon gamma (0J, 1.0, 10 ng) is added to wells H9, H10, and HI 1 to serve as additional positive controls for the assay.

The 96 well dishes containing Jurkat cells treated with supernatants are placed in an incubator for 48 hrs (note: this time is variable between 48-72 hrs). 35 ul samples from each well are then transferred to an opaque 96 well plate using a 12 channel pipette. The opaque plates should be covered (using sellophene covers) and stored at -20 degrees C until SEAP assays are performed according to Example 17. The plates containing the remaining treated cells are placed at 4 degrees C and serve as a source of material for repeating the assay on a specific well if desired. As a positive control, 100 Unit/ml interferon gamma can be used which is known to activate Jurkat T cells. Over 30 fold induction is typically observed in the positive control wells.

The above protocol may be used in the generation of both transient, as well as, stable transfected cells, which would be apparent to those of skill in the art.

Example 14: High-Throughput Screening Assay Identifying Myeloid Activity

The following protocol is used to assess myeloid activity by determining whether polypeptides of the invention proliferates and/or differentiates myeloid cells. Myeloid cell activity is assessed using the GAS/SEAP/Neo construct produced in Example 12. Thus, factors that increase SEAP activity indicate the ability to activate the Jaks-STATS signal transduction pathway. The myeloid cell used in this assay is U937, a pre-monocyte cell line, although TF-1, HL60, or KG1 can be used. To transiently transfect U937 cells with the GAS/SEAP/Neo construct produced in

Example 12, a DEAE-Dextran method (Kharbanda et. al., 1994, Cell Growth &

Differentiation, 5:259-265) is used. First, harvest 2 l0e⁷ U937 cells and wash with PBS. The U937 cells are usually grown in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/ml penicillin and 100 mg/ml streptomycin.

Next, suspend the cells in 1 ml of 20 mM Tris-HCI (pH 1.4) buffer containing 0.5 mg/ml DEAE-Dextran, 8 ug GAS-SEAP2 plasmid DNA, 140 mM NaCI, 5 mM KCl, 375 uM Na HPO₄.7H₂O, 1 mM MgCl₂, and 675 uM CaCl₂. Incubate at 37 degrees C for 45 min. Wash the cells with RPMI 1640 medium containing 10% FBS and then resuspend in 10 ml complete medium and incubate at 37 degrees C for 36 hr.

The GAS-SEAP/U937 stable cells are obtained by growing the cells in 400 ug/ml G418. The G418-free medium is used for routine growth but every one to two months, the cells should be re-grown in 400 ug/ml G418 for couple of passages. These cells are tested by harvesting 1x10 cells (this is enough for ten 96-well plates assay) and wash with PBS. Suspend the cells in 200 ml above described growth medium, with a final density of 5xl0⁵ cells/ml. Plate 200 ul cells per well in the 96-well plate (or lxlO⁵ cells/well).

Add 50 ul of the supernatant prepared by the protocol described in Example 11. Incubate at 37 degrees C for 48 to 72 hr. As a positive control, 100 Unit/ml interferon gamma can be used which is known to activate U937 cells. Over 30 fold induction is typically observed in the positive control wells. SEAP assay the supernatant according to the protocol described in Example 17. Example 15: High-Throughput Screening Assay Identifying Neuronal Activity.

When cells undergo differentiation and proliferation, a group of genes are activated through many different signal transduction pathways. One of these genes, EGR1 (early growth response gene 1), is induced in various tissues and cell types upon activation. The promoter of EGR1 is responsible for such induction. Using the EGR1 promoter linked to reporter molecules, activation of cells can be assessed.

Particularly, the following protocol is used to assess neuronal activity in PC 12 cell lines. PC12 cells (rat phenochromocytoma cells) are known to proliferate and/or differentiate by activation with a number of mitogens, such as TPA (tetradecanoyl phorbol acetate), NGF (nerve growth factor), and EGF (epidermal growth factor). The EGR1 gene expression is activated during this treatment. Thus, by stably transfecting PC12 cells with a construct containing an EGR promoter linked to SEAP reporter, activation of PC12 cells can be assessed. The EGR/SEAP reporter construct can be assembled by the following protocol.

The EGR-1 promoter sequence (-633 to +l)(Sakamoto K et al., Oncogene 6:867-871 (1991)) can be PCR amplified from human genomic DNA using the following primers:

5' GCGCTCGAGGGATGACAGCGATAGAACCCCGG -3' (SEQ ID NO:6)

5' GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3' (SEQ ID NO:7) Using the GAS:SEAP/Neo vector produced in Example 12, EGR1 amplified product can then be inserted into this vector. Linearize the GAS:SEAP/Neo vector using restriction enzymes Xhol/Hindlll, removing the GAS/SV40 stuffer. Restrict the EGR1 amplified product with these same enzymes. Ligate the vector and the EGR1 promoter.

To prepare 96 well-plates for cell culture, two mis of a coating solution (1:30 dilution of collagen type I (Upstate Biotech Inc. Cat#08-115) in 30% ethanol (filter sterilized)) is added per one 10 cm plate or 50 ml per well of the 96-well plate, and allowed to air dry for 2 hr. PC 12 cells are routinely grown in RPMI- 1640 medium (Bio Whittaker) containing

10% horse serum (JRH BIOSCIENCES, Cat. # 12449-78P), 5% heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/ml penicillin and 100 ug/ml streptomycin on a precoated 10 cm tissue culture dish. One to four split is done every three to four days. Cells are removed from the plates by scraping and resuspended with pipetting up and down for more than 15 times.

Transfect the EGR/SEAP/Neo construct into PC 12 using the Lipofectamine protocol described in Example 11. EGR-SEAP/PC12 stable cells are obtained by growing the cells in 300 ug/ml G418. The G418-free medium is used for routine growth but every one to two months, the cells should be re-grown in 300 ug/ml G418 for couple of passages. To assay for neuronal activity, a 10 cm plate with cells around 70 to 80% confluent is screened by removing the old medium. Wash the cells once with PBS (Phosphate buffered saline). Then starve the cells in low serum medium (RPMI-1640 containing 1% horse serum and 0.5% FBS with antibiotics) overnight. The next morning, remove the medium and wash the cells with PBS. Scrape off the cells from the plate, suspend the cells well in 2 ml low serum medium. Count the cell number and add more low serum medium to reach final cell density as 5x10^ cells/ml.

Add 200 ul of the cell suspension to each well of 96-well plate (equivalent to 1x10-^ cells/well). Add 50 ul supernatant produced by Example 11, 37°C for 48 to 72 hr. As a positive control, a growth factor known to activate PC 12 cells through EGR can be used, such as 50 ng/ul of Neuronal Growth Factor (NGF). Over fifty-fold induction of SEAP is typically seen in the positive control wells. SEAP assay the supernatant according to Example 17.

Example 16: High-Throughput Screening Assay for T-cell Activity

NF-KB (Nuclear Factor KB) is a transcription factor activated by a wide variety of agents including the inflammatory cytokines IL-1 and TNF, CD30 and CD40, lymphotoxin-alpha and lymphotoxin-beta, by exposure to LPS or thrombin, and by expression of certain viral gene products. As a transcription factor, NF-KB regulates the expression of genes involved in immune cell activation, control of apoptosis (NF- KB appears to shield cells from apoptosis), B and T-cell development, anti-viral and antimicrobial responses, and multiple stress responses. In non-stimulated conditions, NF- KB is retained in the cytoplasm with I-KB

(Inhibitor KB). However, upon stimulation, I- KB is phosphorylated and degraded, causing NF- KB to shuttle to the nucleus, thereby activating transcription of target genes.

Target genes activated by NF- KB include IL-2, IL-6, GM-CSF. ICAM-1 and class 1

MHC. Due to its central role and ability to respond to a range of stimuli, reporter constructs utilizing the NF-KB promoter element are used to screen the supernatants produced in Example 11. Activators or inhibitors of NF-KB would be useful in treating diseases. For example, inhibitors of NF-KB could be used to treat those diseases related to the acute or chronic activation of NF-KB, such as rheumatoid arthritis. To construct a vector containing the NF-KB promoter element, a PCR based strategy is employed. The upstream primer contains four tandem copies of the NF-KB binding site (GGGGACTTTCCC) (SEQ ID NO:8), 18 bp of sequence complementary to the 5' end of the SV40 early promoter sequence, and is flanked with an Xhol site: 5':GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTT CCATCCTGCCATCTCAATTAG:3' (SEQ ID NO:9)

The downstream primer is complementary to the 3' end of the SV40 promoter and is flanked with a Hind III site: 5':GCGGCAAGCTTTTTGCAAAGCCTAGGC:3' (SEQ ID NOJ)

PCR amplification is performed using the SV40 promoter template present in the pB-gal:promoter plasmid obtained from Clontech. The resulting PCR fragment is digested with Xhol and Hind III and subcloned into BLSK2-. (Stratagene) Sequencing with the T7 and T3 primers confirms the insert contains the following sequence: 5':CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCCATC

TGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCG

CCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTT

TTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTA GTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTT:3' (SEQ ID

NO: 10)

Next, replace the SV40 minimal promoter element present in the pSEAP2-promoter plasmid (Clontech) with this NF-KB/SV40 fragment using Xhol and Hindlll. However, this vector does not contain a neomycin resistance gene, and therefore, is not prefeired for mammalian expression systems.

In order to generate stable mammalian cell lines, the NF-KB /SV40/SEAP cassette is removed from the above NF-KB/SEAP vector using restriction enzymes Sail and Notl, and inserted into a vector containing neomycin resistance. Particularly, the NF- KB/SV40/SEAP cassette was inserted into pGFP-1 (Clontech), replacing the GFP gene, after restricting pGFP-1 with Sail and Notl.

Once NF-KB/SV40/SEAP/Neo vector is created, stable Jurkat T-cells are created and maintained according to the protocol described in Example 13. Similarly, the method for assaying supernatants with these stable Jurkat T-cells is also described in Example 13. As a positive control, exogenous TNF alpha (0.1,1, 10 ng) is added to wells H9, H10, and HI 1, with a 5-10 fold activation typically observed.

Example 17: Assay for SEAP Activity

As a reporter molecule for the assays described in Examples 13-16, SEAP activity is assayed using the Tropix Phospho-light Kit (Cat. BP-400) according to the following general procedure. The Tropix Phospho-light Kit supplies the Dilution, Assay, and Reaction Buffers used below.

Prime a dispenser with the 2.5x Dilution Buffer and dispense 15 ul of 2.5x dilution buffer into Optiplates containing 35 ul of a supernatant. Seal the plates with a plastic sealer and incubate at 65 degree C for 30 min. Separate the Optiplates to avoid uneven heating.

Cool the samples to room temperature for 15 minutes. Empty the dispenser and prime with the Assay Buffer. Add 50 ml Assay Buffer and incubate at room temperature 5 min. Empty the dispenser and prime with the Reaction Buffer (see the table below). Add

50 ul Reaction Buffer and incubate at room temperature for 20 minutes. Since the intensity of the chemiluminescent signal is time dependent, and it takes about 10 minutes to read 5 plates on luminometer, one should treat 5 plates at each time and start the second set 10 minutes later. Read the relative light unit in the luminometer. Set H12 as blank, and print the results. An increase in chemiluminescence indicates reporter activity.

Reaction Buffer Formulation:

# of plates Rxn buffer diluent (ml) CSPD (ml) 10 60 3

11 65 3.25

12 70 3.5

13 75 3.75

14 80 4

15 85 4.25

16 90 4.5

17 95 4.75

18 100 5

19 105 5.25

20 110 5.5

21 115 5.75

22 120 6

23 125 6.25

24 130 6.5

25 135 6.75

26 140 7

27 145 7.25

28 150 7.5

29 155 7.75

30 160 8

31 165 8.25

32 170 8.5

33 175 8.75

34 180 9

35 185 9.25

36 190 9.5

37 195 9.75

38 200 10

39 205 10.25 40 210 10.5

41 215 10.75

42 220 11

43 225 11.25

44 230 1 1.5

45 235 11.75

46 240 12

47 245 12.25

48 250 12.5

49 255 12.75

50 260 13

Example 18: High-Throughput Screening Assay Identifying Changes in Small Molecule Concentration and Membrane Permeability

Binding of a ligand to a receptor is known to alter intracellular levels of small molecules, such as calcium, potassium, sodium, and pH, as well as alter membrane potential. These alterations can be measured in an assay to identify supernatants which bind to receptors of a particular cell. Although the following protocol describes an assay for calcium, this protocol can easily be modified to detect changes in potassium, sodium, pH, membrane potential, or any other small molecule which is detectable by a fluorescent probe.

The following assay uses Fluorometric Imaging Plate Reader ("FLIPR") to measure changes in fluorescent molecules (Molecular Probes) that bind small molecules. Clearly, any fluorescent molecule detecting a small molecule can be used instead of the calcium fluorescent molecule, fluo-4 (Molecular Probes, Inc.; catalog no. F-14202), used here.

For adherent cells, seed the cells at 10,000 -20,000 cells/well in a Co-star black 96- well plate with clear bottom. The plate is incubated in a CO, incubator for 20 hours. The adherent cells are washed two times in Biotek washer with 200 ul of HBSS (Hank's Balanced Salt Solution) leaving 100 ul of buffer after the final wash. A stock solution of 1 mg/ml fluo-4 is made in 10% pluronic acid DMSO. To load the cells with fluo-4 , 50 ul of 12 ug/ml fluo-4 is added to each well. The plate is incubated at 37 degrees C in a CO₂ incubator for 60 min. The plate is washed four times in the Biotek washer with HBSS leaving 100 ul of buffer. For non-adherent cells, the cells are spun down from culture media. Cells are resuspended to 2-5xl0⁶ cells/ml with HBSS in a 50-ml conical tube. 4 ul of 1 mg/ml fluo-4 solution in 10% pluronic acid DMSO is added to each ml of cell suspension. The tube is then placed in a 37 degrees C water bath for 30-60 min. The cells are washed twice with HBSS, resuspended to lxlO⁶ cells/ml, and dispensed into a microplate, 100 ul/well. The plate is centrifuged at 1000 rpm for 5 min. The plate is then washed once in Denley CellWash with 200 ul, followed by an aspiration step to 100 ul final volume.

For a non-cell based assay, each well contains a fluorescent molecule, such as fluo- 4 . The supernatant is added to the well, and a change in fluorescence is detected. To measure the fluorescence of intracellular calcium, the FLIPR is set for the following parameters: (1) System gain is 300-800 mW; (2) Exposure time is 0J second; (3) Camera F/stop is F/2; (4) Excitation is 488 nm; (5) Emission is 530 nm; and (6) Sample addition is 50 ul. Increased emission at 530 nm indicates an extracellular signaling event

which has resulted in an increase in the intracellular Ca⁺⁺ concentration.

Example 19: High-Throughput Screening Assay Identifying Tyrosine Kinase Activity

The Protein Tyrosine Kinases (PTK) represent a diverse group of transmembrane and cytoplasmic kinases. Within the Receptor Protein Tyrosine Kinase RPTK) group are receptors for a range of mitogenic and metabolic growth factors including the PDGF, FGF, EGF, NGF, HGF and Insulin receptor subfamilies. In addition there are a large family of RPTKs for which the corresponding ligand is unknown. Ligands for RPTKs include mainly secreted small proteins, but also membrane-bound and extracellular matrix proteins.

Activation of RPTK by ligands involves ligand-mediated receptor dimerization, resulting in transphosphorylation of the receptor subunits and activation of the cytoplasmic tyrosine kinases. The cytoplasmic tyrosine kinases include receptor associated tyrosine kinases of the src-family (e.g., src, yes, lck, lyn, fyn) and non-receptor linked and cytosolic protein tyrosine kinases, such as the Jak family, members of which mediate signal transduction triggered by the cytokine superfamily of receptors (e.g., the Interleukins,

Interferons, GM-CSF, and Leptin).

Because of the wide range of known factors capable of stimulating tyrosine kinase activity, the identification of novel human secreted proteins capable of activating tyrosine kinase signal transduction pathways are of interest. Therefore, the following protocol is designed to identify those novel human secreted proteins capable of activating the tyrosine kinase signal transduction pathways.

Seed target cells (e.g., primary keratinocytes) at a density of approximately 25,000 cells per well in a 96 well Loprodyne Silent Screen Plates purchased from Nalge Nunc (Naperville, IL). The plates are sterilized with two 30 minute rinses with 100%) ethanol, rinsed with water and dried overnight. Some plates are coated for 2 hr with 100 ml of cell culture grade type I collagen (50 mg/ml), gelatin (2%) or polylysine (50 mg/ml), all of which can be purchased from Sigma Chemicals (St. Louis, MO) or 10%; Matrigel purchased from Becton Dickinson (Bedford,MA), or calf serum, rinsed with PBS and stored at 4 degree C. Cell growth on these plates is assayed by seeding 5,000 cells/well in growth medium and indirect quantitation of cell number through use of alamarBlue as described by the manufacturer Alamar Biosciences, Inc. (Sacramento, CA) after 48 hr. Falcon plate covers #3071 from Becton Dickinson (Bedford,MA) are used to cover the Loprodyne Silent Screen Plates. Falcon Microtest III cell culture plates can also be used in some proliferation experiments.

To prepare extracts, A431 cells are seeded onto the nylon membranes of Loprodyne plates (20,000/200ml/well) and cultured overnight in complete medium. Cells are quiesced by incubation in serum-free basal medium for 24 hr. After 5-20 minutes treatment with EGF (60ng/ml) or 50 ul of the supernatant produced in Example 11, the medium was removed and 100 ml of extraction buffer ((20 mM HEPES pH 7.5, 0J5 M NaCI, 1% Triton X-100, 0.1% SDS, 2 mM Na3VO4, 2 mM Na4P2O7 and a cocktail of protease inhibitors (# 1836170) obtained from Boeheringer Mannheim (Indianapolis, IN) is added to each well and the plate is shaken on a rotating shaker for 5 minutes at 4 degrees C. The plate is then placed in a vacuum transfer manifold and the extract filtered through the 0.45 mm membrane bottoms of each well using house vacuum. Extracts are collected in a 96-well catch/assay plate in the bottom of the vacuum manifold and immediately placed on ice. To obtain extracts clarified by centrifugation, the content of each well, after detergent solubilization for 5 minutes, is removed and centrifuged for 15 minutes at 4 degrees C at 16,000 x g.

Test the filtered extracts for levels of tyrosine kinase activity. Although many methods of detecting tyrosine kinase activity are known, one method is described here.

Generally, the tyrosine kinase activity of a supernatant is evaluated by determining its ability to phosphorylate a tyrosine residue on a specific substrate (a biotinylated peptide). Biotinylated peptides that can be used for this putpose include PSK1 (cotresponding to amino acids 6-20 of the cell division kinase cdc2-p34) and PSK2 (con-esponding to amino acids 1-17 of gastrin). Both peptides are substrates for a range of tyrosine kinases and are available from Boehringer Mannheim. The tyrosine kinase reaction is set up by adding the following components in order.

First, add lOul of 5uM Biotinylated Peptide, then lOul ATP/Mg ₊ (5mM ATP/50mM

MgCl ), then lOul of 5x Assay Buffer (40mM imidazole hydrochloride, pH7.3, 40 mM beta-glycerophosphate, ImM EGTA, lOOmM MgCl , 5 mM MnCl_2> 0.5 mg/ml BSA), then 5ul of Sodium Vanadate(lmM), and then 5ul of water. Mix the components gently and preincubate the reaction mix at 30 degrees C for 2 min. Initial the reaction by adding lOul of the control enzyme or the filtered supernatant.

The tyrosine kinase assay reaction is then terminated by adding 10 ul of 120mm EDTA and place the reactions on ice.

Tyrosine kinase activity is determined by transferring 50 ul aliquot of reaction mixture to a microtiter plate (MTP) module and incubating at 37 degrees C for 20 min.

This allows the streptavadin coated 96 well plate to associate with the biotinylated peptide. Wash the MTP module with 300ul/well of PBS four times. Next add 75 ul of anti- phospotyrosme antibody conjugated to horse radish peroxιdase(antι-P-Tyr-POD(0.5u/ml)) to each well and incubate at 37 degrees C for one hour. Wash the well as above.

Next add lOOul of peroxidase substrate solution (Boehrmger Mannheim) and incubate at room temperature for at least 5 mins (up to 30 min). Measure the absorbance of the sample at 405 nm by using ELISA reader. The level of bound peroxidase activity is quantttated using an ELISA reader and teflects the level of tyiostne kinase activity

Example 20: High-Throughput Screening Assay Identifying Phosphorylation Activity

As a potential alternative and/or compliment to the assay of protein tyrosine kinase activity described in Example 19. an assay which detects activation (phosphorylation) of major intracellular signal transduction intermediates can also be used. For example, as described below one particular assay can detect tyiostne phosphorylation of the Erk-1 and Etk-2 kinases However, phosphorylation of other molecules, such as Raf, JNK, p38 MAP, Map kinase kinase (MEK), MEK kinase, Src, Muscle specific kinase (MuSK), IRAK, Tec, and Janus, as well as any other phosphoseπne, phosphotyrosine, or phosphothreontne molecule, can be detected by substituting these molecules for Erk-1 or Erk-2 m the following assay.

Specifically, assay plates are made by coating the wells of a 96-well ELISA plate with 0.1ml of protein G (lug/ml) for 2 hr at room temp, (RT). The plates are then nnsed with PBS and blocked with 3% BSA/PBS for 1 hr at RT. The protein G plates are then treated with 2 commercial monoclonal antibodies (lOOng/well) against Erk-land Erk-2 (1 hr at RT) (Santa Cruz Biotechnology). (To detect other molecules, this step can easily be modified by substituting a monoclonal antibody detecting any of the above descπbed molecules.) After 3-5 πnses with PBS, the plates are stored at 4 degrees C until use. A431 cells are seeded at 20,000/well in a 96-well Loprodyne ftlterplate and cultured overnight in growth medium. The cells are then starved for 48 hr in basal medium (DMEM) and then treated with EGF (6ng/well) or 50 ul of the supernatants obtained in Example 11 for 5-20 minutes. The cells are then solubilized and extracts filtered directly into the assay plate.

After incubation with the extract for 1 hr at RT, the wells are again rinsed. As a positive control, a commercial preparation of MAP kinase (lOng/well) is used in place of A431 extract. Plates are then treated with a commercial polyclonal (rabbit) antibody

(lug/ml) which specifically recognizes the phosphorylated epitope of the Erk-1 and Erk-2 kinases (1 hr at RT). This antibody is biotinylated by standard procedures. The bound polyclonal antibody is then quantitated by successive incubations with Europium- streptavidin and Europium fluorescence enhancing reagent in the Wallac DELFIA instrument (time-resolved fluorescence). An increased fluorescent signal over background indicates a phosphorylation.

Example 21: Method of Determining Alterations in a Gene Corresponding to a Polynucleotide RNA isolated from entire families or individual patients presenting with a phenotype of interest (such as a disease) is be isolated. cDNA is then generated from these RNA samples using protocols known in the art. (See, Sambrook.) The cDNA is then used as a template for PCR, employing primers surrounding regions of interest in SEQ ID NO:X. Suggested PCR conditions consist of 35 cycles at 95 degrees C for 30 seconds; 60- 120 seconds at 52-58 degrees C; and 60-120 seconds at 70 degrees C, using buffer solutions described in Sidransky et al., Science 252:706 (1991).

PCR products are then sequenced using primers labeled at their 5' end with T4 polynucleotide kinase, employing SequiTherm Polymerase. (Epicentre Technologies). The intron-exon borders of selected exons is also determined and genomic PCR products analyzed to confirm the results. PCR products harboring suspected mutations is then cloned and sequenced to validate the results of the direct sequencing.

PCR products is cloned into T-tailed vectors as described in Holton et al., Nucleic Acids Research, 19:1156 (1991) and sequenced with T7 polymerase (United States Biochemical). Affected individuals are identified by mutations not present in unaffected individuals.

Genomic rearrangements are also observed as a method of determining alterations m a gene corresponding to a polynucleotide Genomic clones isolated according to Example 2 are nick-translated with digoxigenindeoxy-uπdme 5'-tπphosphate (Boehπnger

Manheim), and FISH performed as described in Johnson et al., Methods Cell Biol. 35:73-

99 (1991) Hybridization with the labeled probe is carried out using a vast excess of human cot-1 DNA for specific hybridization to the coπ-espondmg genomic locus.

Chromosomes are counterstamed with 4,6-dιamιno-2-phenyhdole and propidium iodide, producing a combination of C- and R-bands. Aligned images for precise mapping are obtained using a triple-band filter set (Chioma Technology, Brattleboro, VT) in combination with a cooled charge-coupled device camera (Photometries, Tucson, AZ) and variable excitation wavelength filters. (Johnson et al., Genet. Anal. Tech. Appl., 8:75 (1991).) Image collection, analysis and chromosomal fractional length measurements are performed using the ISee Graphical Program System. (Inovision Corporation, Durham, NC.) Chromosome alterations of the genomic region hybπdized by the probe are identified as insertions, deletions, and trans locations. These alterations are used as a diagnostic marker for an associated disease.

Example 22: Method of Detecting Abnormal Levels of a Polypeptide in a Biological Sample

A polypeptide of the present invention can be detected in a biological sample, and if an increased or decreased level of the polypeptide is detected, this polypeptide is a marker for a particular phenotype. Methods of detection are numerous, and thus, it is understood that one skilled m the art can modify the following assay to fit their particular needs.

For example, antibody-sandwich ELISAs are used to detect polypeptides in a sample, preferably a biological sample. Wells of a microtiter plate are coated with specific antibodies, at a final concentration of 0.2 to 10 ug/ml. The antibodies are either monoclonal or polyclonal and are produced by the method described in Example 10 The wells are blocked so that non-specific binding of the polypeptide to the well is reduced.

The coated wells are then incubated for > 2 hours at RT with a sample containing the polypeptide. Preferably, senal dilutions of the sample should be used to validate results. The plates are then washed three times with deionized or distilled watei to remove unbounded polypeptide.

Next, 50 ul of specific antibody-alkaline phosphatase conjugate, at a concentration of 25-400 ng, is added and incubated for 2 houis at room temperature. The plates aie again washed three times with deionized or distilled water to remove unbounded conjugate

Add 75 ul of 4-methylumbelhferyl phosphate (MUP) oi p-nitrophenyl phosphate (NPP) substrate solution to each well and incubate 1 hour at room temperature Measure the reaction by a microtiter plate reader. Prepare a standard curve, using serial dilutions of a control sample, and plot polypeptide concentration on the X-axis (log scale) and fluorescence or absorbance of the Y-axis (linear scale). Interpolate the concentration of the polypeptide m the sample using the standard curve.

Example 23: Formulation

The invention also provides methods of treatment and/or prevention of diseases or disorders (such as, for example, any one or more of the diseases or disorders disclosed herein) by administration to a subject of an effective amount of a Therapeutic. By therapeutic is meant a polynucleotides or polypeptides of the invention (including fragments and variants), agonists or antagonists thereof, and/or antibodies thereto, in combination with a pharmaceutically acceptable earner type (e.g., a stenle earner). The Therapeutic will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment with the Therapeutic alone), the site of delivery, the method of administration, the scheduling of administration, and other factors known to practitioners. The "effective amount" for purposes herein is thus determined by such considerations.

As a general proposition, the total pharmaceutically effective amount of the

Therapeutic administered parenterally per dose will be in the range of about lug/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably foi humans between about 0.01 and 1 mg/kg/day for the hormone If given continuously, the Therapeutic is typically administered at a dose rate of about 1 ug/kg/houi to about 50 ug/kg/houi, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump An intravenous bag solution may also be employed The length of treatment needed to observe changes and the interval following treatment foi responses to occur appears to vary depending on the desired effect.

Therapeutics can be are administered orally, lectally, parenterally, lntracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oial or nasal spray. "Pharmaceutically acceptable earner" refers to a non-toxic solid, semisohd or liquid filler, diluent, encapsulating material or formulation auxiliary of any The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, lntrapeπtoneal, lntrastemal, subcutaneous and mtraarticular injection and infusion. Therapeutics of the invention are also suitably administered by sustained-release systems. Suitable examples of sustained-release Therapeutics are administered orally, rectally, parenterally, lntracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray. "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semisohd or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intrapentoneal, mtrasternal, subcutaneous and mtraarticular injection and infusion. Therapeutics of the invention are also suitably administered by sustained-release systems. Suitable examples of sustained-release Therapeutics include suitable polymeric materials (such as, for example, semi-permeable polymer matrices in the form of shaped articles, e.g., films, or mirocapsules), suitable hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, and sparingly soluble derivatives

(such as, for example, a sparingly soluble salt).

Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP

58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sid an et al.,

Biopolymers 22:547-556 (1983)), poly (2- hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res. 15: 167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., Id.) or poly-D- (-)-3-hydroxybutyric acid (EP 133,988).

Sustained-release Therapeutics also include liposomally entrapped Therapeutics of the invention (see generally. Langer, Science 249: 1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 317 -327 and 353-365 (1989)). Liposomes containing the Therapeutic are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci.(USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-1 18008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal Therapeutic.

In yet an additional embodiment, the Therapeutics of the invention are delivered by way of a pump (Λee Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)).

Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)). For parenteral administration, in one embodiment, the Therapeutic is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form

(solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to the Therapeutic.

Generally, the formulations are prepared by contacting the Therapeutic uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such cairier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes. The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.

The Therapeutic is typically formulated in such vehicles at a concentration of about 0J mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be understood that the use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of polypeptide salts.

Any pharmaceutical used for therapeutic administration can be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes) Therapeutics generally are placed into a container having a sterile access port, for example, an intravenous solution bag oi vial having a stoppei pieiceable by a hypodermic injection needle.

Therapeutics ordinarily will be stoied in unit or multi-dose containers, for example, sealed ampoules oi vials, as an aqueous solution or as a lyophilized formulation foi reconstitution As an example of a lyophilized formulation, 10-ml vials are filled with 5 ml of steπle-filteied 1% (w/v) aqueous Theiapeutic solution, and the resulting mixture is lyophilized The infusion solution is prepaied by reconstituting the lyophilized Therapeutic using bacteπostatic Watei-for-Injection

The invention also provides a pharmaceutical pack or kit compnsmg one oi more containers filled with one or moie of the ingredients of the Therapeutics of the invention. Associated with such contamer(s) can be a notice in the form prescnbed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the Therapeutics may be employed in conjunction with other therapeutic compounds.

The Therapeutics of the invention may be administered alone or in combination with adjuvants. Adjuvants that may be administered with the Therapeutics of the invention include, but are not limited to, alum, alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21 (Genentech, Inc.), BCG, and MPL. In a specific embodiment, Therapeutics of the invention are administered in combination with alum. In another specific embodiment, Therapeutics of the invention are administered in combination with QS-21. Further adjuvants that may be administered with the Therapeutics of the invention include, but are not limited to, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology.

Vaccines that may be administered with the Therapeutics of the invention include, but are not limited to, vaccines directed toward protection against MMR (measles, mumps, rubella), polio, varicella, tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae B, whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus, cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies, typhoid fever, and pertussis.

Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration "in combination" further includes the separate administration of one of the compounds or agents given first, followed by the second.

The Therapeutics of the invention may be administered alone or in combination with other therapeutic agents. Therapeutic agents that may be administered in combination with the Therapeutics of the invention, include but not limited to, other members of the TNF family, chemotherapeutic agents, antibiotics, steroidal and non-steroidal anti- inflammatories, conventional immunotherapeutic agents, cytokines and/or growth factors. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration "in combination" further includes the separate administration of one of the compounds or agents given first, followed by the second.

In one embodiment, the Therapeutics of the invention are administered in combination with members of the TNF family. TNF, TNF-related or TNF-like molecules that may be administered with the Therapeutics of the invention include, but are not limited to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta

(found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L,

4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO 96/14328),

AIM-I (International Publication No. WO 97/33899), endokine-alpha (International Publication No. WO 98/07880), TR6 (International Publication No. WO 98/30694), OPG, and neutrokine-alpha (International Publication No. WO 98/18921 , OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2

(International Publication No. WO 96/34095), DR3 (International Publication No WO

97/33904), DR4 (International Publication No. WO 98/32856), TR5 (International Publication No. WO 98/30693), TR6 (International Publication No WO 98/30694), TR7 (International Publication No. WO 98/41629), TRANK, TR9 (International Publication No. WO 98/56892),TR10 (International Publication No. WO 98/54202), 312C2 (International Publication No. WO 98/06842), and TR12, and soluble forms CD154, CD70, and CD 153. In certain embodiments, Therapeutics of the invention are administered in combination with antiretroviral agents, nucleoside reverse transcriptase inhibitors, non- nucleoside reverse transcπptase inhibitors, and/or protease inhibitors. Nucleoside reverse transcnptase inhibitors that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, RETROVIR™ (zidovudine/AZT), VIDEX™ (didanosine/ddl), HIVID™ (zalcitabine/ddC), ZERIT™ (stavudιne/d4T), EPIVIR™ (lamιvudme/3TC), and COMBIVIR™ (zidovudine/lamivudme). Non-nucleoside reverse transcπptase inhibitors that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, VIRAMUNE™ (nevirapme), RESCRIPTOR™ (delavirdine), and SUSTIVA™ (efavirenz). Protease inhibitors that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, CRIXIVAN™ (indinavir), NORVIR™ (ntonavir), INVIRASE™ (saquinavir), and VIRACEPT™ (nelfmavir). In a specific embodiment, antiretroviral agents, nucleoside reverse transcnptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors may be used in any combination with Therapeutics of the invention to treat AIDS and/or to prevent or treat HIV infection.

In other embodiments, Therapeutics of the invention may be administered in combination with anti-opportunistic infection agents. Anti-opportunistic agents that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLE™ , DAPSONE™,

PENTAMIDINE™ , ATOVAQUONE™ . ISONIAZID™ , RIFAMPIN™,

PYRAZINAMIDE™ , ETHAMBUTOL™ , RIFABUTIN™, CLARITHROMYCIN™,

AZITHROMYCIN™ , GANCICLOVIR™ , FOSCARNET™ , CIDOFOVIR™ , FLUCONAZOLE™, ITRACONAZOLE™ , KETOCONAZOLE™ , ACYCLOVIR™, FAMCICOLVIR™, PYRIMETHAMINE™ , LEUCOVORIN™ , NEUPOGEN™ (filgrastim/G-CSF), and LEUKINE™ (sargramostim/GM-CSF). In a specific embodiment, Therapeutics of the invention are used in any combination with TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™ , PENTAMIDINE™, and/or ATOVAQUONE™ to prophylactically treat or prevent an opportunistic Pneumocystis carinii pneumonia infection. In another specific embodiment, Therapeutics of the invention are used in any combination with ISONIAZID™, RIFAMPIN™ , PYRAZINAMIDE™, and/or ETHAMBUTOL™ to prophylactically treat or prevent an opportunistic Mycobacterium avium complex infection. In another specific embodiment, Therapeutics of the invention are used in any combination with RIFABUTIN™ , CLARITHROMYCIN™, and/or AZITHROMYCIN™ to prophylactically treat or prevent an opportunistic Mycobacterium tuberculosis infection. In another specific embodiment, Therapeutics of the invention are used in any combination with GANCICLOVIR™, FOSCARNET™ , and/or CIDOFOVIR™ to prophylactically treat or prevent an opportunistic cytomegalovirus infection. In another specific embodiment, Therapeutics of the invention are used in any combination with FLUCONAZOLE™ ,

ITRACONAZOLE™, and/or KETOCONAZOLE™ to prophylactically treat or prevent an opportunistic fungal infection. In another specific embodiment, Therapeutics of the invention are used in any combination with ACYCLOVIR™ and/or FAMCICOLVIR™ to prophylactically treat or prevent an opportunistic herpes simplex virus type I and/or type II infection. In another specific embodiment, Therapeutics of the invention are used in any combination with PYRIMETHAMINE™ and/or LEUCOVORIN™ to prophylactically treat or prevent an opportunistic Toxoplasma gondii infection. In another specific embodiment, Therapeutics of the invention are used in any combination with

LEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat oi prevent an opportunistic bacterial infection.

In a further embodiment, the Therapeutics of the invention are administered in combination with an antiviral agent Antiviral agents that may be administered with the Therapeutics of the invention include, but are not limited to, acyclovir, ribavinn, amantadine, and remantidme.

In a further embodiment, the Theiapeutics of the invention are administered in combination with an antibiotic agent Antibiotic agents that may be administered with the Therapeutics of the invention include, but are not limited to, amoxicillm, beta-lactamases, am oglycosides, beta-lactam (glycopeptide), beta-lactamases, Clindamycm, chloramphenicol, cephalospoπns, ciprofloxacm, ciprofloxacin, erythromycin, fluoroquinolones, macrohdes, metronidazole, penicillins, qumolones, πfampm, streptomycin, sulfonamide, tetracycl es, tπmethopπm, tπmethopπm-sulfamthoxazole, and vancomycm.

Conventional nonspecific immunosuppressive agents, that may be administered in combination with the Therapeutics of the invention include, but are not limited to, steroids, cyclospoπne, cyclosponne analogs, cyclophosphamide methylprednisone, prednisone, azathiopnne, FK-506, 15-deoxyspergualιn, and other immunosuppressive agents that act by suppressing the function of responding T cells.

In specific embodiments, Therapeutics of the invention are administered in combination with immunosuppressants. Immunosuppressants preparations that may be administered with the Therapeutics of the invention include, but are not limited to, ORTHOCLONE™ (OKT3), SANDIMMUNE™/NEORAL™/SANGDYA™ (cyclosporin),

PROGRAF™ (tacrolimus), CELLCEPT™ (mycophenolate), Azathioprine, glucorticosteroids, and RAPAMUNE™ (sirolimus). In a specific embodiment, immunosuppressants may be used to prevent rejection of organ or bone marrow transplantation.

In an additional embodiment, Therapeutics of the invention are administered alone or in combination with one or more intravenous immune globulin preparations.

Intravenous immune globulin preparations that may be administered with the Therapeutics of the invention include, but not limited to, GAMMAR™ , IVEEGAM™, SANDOGLOBULIN™ . GAMMAGARD S/D™, and GAMIMUNE™ . In a specific embodiment, Therapeutics of the invention are administered in combination with intravenous immune globulin preparations in transplantation therapy (e.g., bone marrow transplant).

In an additional embodiment, the Therapeutics of the invention are administered alone or in combination with an anti -inflammatory agent. Anti-inflammatory agents that may be administered with the Therapeutics of the invention include, but are not limited to, glucocorticoids and the nonsteroidal anti-inflammatories, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4- hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, and tenidap.

In another embodiment, compostions of the invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered with the Therapeutics of the invention include, but are not limited to, antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, floxundine, interferon alpha-2b, glutamic acid, phcamycin, mercaptopuπne, and 6- thioguanme), cytotoxic agents (e.g , carmustme, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustme, hydroxyurea, procarbazine, mitomycm, busulfan, cis-platm, and vincnstme sulfate); hormones (e.g., medroxyprogesterone, estramustme phosphate sodium, ethmyl estradiol, esti adiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chloiotnanisene, and testolactone), nitrogen mustaid derivatives (e.g , mephalen, chorambucil, mechlorethamine ( tiogen mustard) and thiotepa), steroids and combinations (e g., bethamethasone sodium phosphate), and others (e g , dicarbaz e, asparagmase, mitotane, v cnstine sulfate, v blastine sulfate, and etoposide)

In a specific embodiment, Thei apeutics of the invention aie admi steied in combination with CHOP (cyclophosphamide, doxorubicin, vincnstme, and prednisone) or any combination of the components of CHOP In anothei embodiment, Therapeutics of the invention are administered in combination with Rituximab. In a furthei embodiment, Therapeutics of the invention are administered with Rituxmab and CHOP, or Rituxmab and any combination of the components of CHOP.

In an additional embodiment, the Therapeutics of the invention are administered in combination with cytokines. Cytokines that may be administered with the Therapeutics of the invention include, but are not limited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, antι-CD40, CD40L, IFN-gamma and TNF-alpha. In another embodiment, Therapeutics of the invention may be administered with any mterleukin, including, but not limited to, IL-lalpha, IL-lbeta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, and IL-21

In an additional embodiment, the Therapeutics of the invention are administered m combination with angiogenic proteins. Angiogenic proteins that may be administered with the Therapeutics of the invention include, but are not limited to, Ghoma Derived Growth Factoi (GDGF), as disclosed in European Patent Number EP-399816; Platelet Derived Growth Factor-A (PDGF-A), as disclosed in European Patent Number EP-682110; Platelet Derived Growth Factor-B (PDGF-B), as disclosed in European Patent Number EP-282317;

Placental Growth Factor (P1GF), as disclosed in International Publication Number WO

92/06194; Placental Growth Factor-2 (P1GF-2), as disclosed in Hauser et al., Gorwth

Factors, 4:259-268 (1993); Vascular Endothelial Growth Factor (VEGF), as disclosed in International Publication Number WO 90/13649; Vascular Endothelial Growth Factor-A

(VEGF-A), as disclosed in European Patent Number EP-506477; Vascular Endothelial

Growth Factor-2 (VEGF-2), as disclosed in International Publication Number WO

96/39515; Vascular Endothelial Growth Factor B (VEGF-3); Vascular Endothelial Growth

Factor B-186 (VEGF-B 186), as disclosed in International Publication Number WO 96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number WO 98/02543; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E), as disclosed in German Patent Number DE19639601. The above mentioned references are incoφorated herein by reference herein. In an additional embodiment, the Therapeutics of the invention are administered in combination with hematopoietic growth factors. Hematopoietic growth factors that may be administered with the Therapeutics of the invention include, but are not limited to, LEUKINE™ (SARGRAMOSTIM™) and NEUPOGEN™ (FILGRASTIM™).

In an additional embodiment, the Therapeutics of the invention are administered in combination with Fibroblast Growth Factors. Fibroblast Growth Factors that may be administered with the Therapeutics of the invention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15. In additional embodiments, the Therapeutics of the invention are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.

Example 24: Method of Treating Decreased Levels of the Polypeptide The present invention relates to a method for treating an individual in need of an increased level of a polypeptide of the invention in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an agonist of the invention (including polypeptides of the invention). Moreover, it will be appreciated that conditions caused by a decrease in the standard or normal expression level of a secreted protein in an individual can be treated by administering the polypeptide of the present invention, preferably in the secreted form. Thus, the invention also provides a method of treatment of an individual in need of an increased level of the polypeptide comprising administering to such an individual a Therapeutic comprising an amount of the polypeptide to increase the activity level of the polypeptide in such an individual.

For example, a patient with decreased levels of a polypeptide receives a daily dose OJ-100 ug/kg of the polypeptide for six consecutive days. Preferably, the polypeptide is in the secreted form. The exact details of the dosing scheme, based on administration and formulation, are provided in Example 23.

Example 25: Method of Treating Increased Levels of the Polypeptide

The present invention also relates to a method of treating an individual in need of a decreased level of a polypeptide of the invention in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an antagonist of the invention (including polypeptides and antibodies of the invention).

In one example, antisense technology is used to inhibit production of a polypeptide of the present invention. This technology is one example of a method of decreasing levels of a polypeptide, preferably a secreted form, due to a variety of etiologies, such as cancer. For example, a patient diagnosed with abnormally increased levels of a polypeptide is administered intravenously antisense polynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment is repeated after a 7-day rest period if the treatment was well tolerated. The formulation of the antisense polynucleotide is provided in Example 23. Example 26: Method of Treatment Using Gene Therapy-Ex Vivo

One method of gene therapy transplants fibroblasts, which are capable of expressing a polypeptide, onto a patient. Generally, fibroblasts are obtained from a subject by skm biopsy. The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperatuie, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin) is added. The flasks are then incubated at 37 degree C for approximately one week.

At this time, fresh media is added and subsequently changed every several days After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks. pMV-7 (Kirschmeier, P.T. et al., DNA, 7:219-25 (1988)), flanked by the long terminal repeats of the Moloney munne sarcoma virus, is digested with EcoRI and Hindlll and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and punfied, using glass beads.

The cDNA encoding a polypeptide of the present invention can be amplified using PCR pnmers which correspond to the 5' and 3' end sequences respectively as set forth in Example 1 using pnmers and having appropnate restnction sites and mitiation/stop codons, if necessary. Preferably, the 5' pnmer contains an EcoRI site and the 3' pnmer includes a Hindlll site. Equal quantities of the Moloney munne sarcoma virus linear backbone and the amplified EcoRI and Hindlll fragment are added together, m the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropnate for ligation of the two fragments. The ligation mixture is then used to transform bactena HB101, which are then plated onto agar containing kanamycin for the purpose of confirming that the vector has the gene of interest properly inserted. The amphotropic pA317 or GP+aml2 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum

(CS), penicillin and streptomycin. The MSV vector containing the gene is then added to the media and the packaging cells transduced with the vector. The packaging cells now produce infectious viral particles containing the gene (the packaging cells are now referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his. Once the fibroblasts have been efficiently infected, the fibroblasts are analyzed to determine whether protein is produced.

The engineered fibroblasts are then transplanted onto the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads.

Example 27: Gene Therapy Using Endogenous Genes Corresponding To Polynucleotides of the Invention

Another method of gene therapy according to the present invention involves operably associating the endogenous polynucleotide sequence of the invention with a promoter via homologous recombination as described, for example, in U.S. Patent NO: 5,641,670, issued June 24, 1997; International Publication NO: WO 96/29411, published September 26, 1996; International Publication NO: WO 94/12650, published August 4, 1994; Koller et al., Proc. Natl Acad. Sci. USA, 86:8932-8935 (1989); and Zijlstra et al., Nature, 342:435-438 (1989). This method involves the activation of a gene which is present in the target cells, but which is not expressed m the cells, or is expressed at a lower level than desired.

Polynucleotide constructs are made which contain a promoter and targeting sequences, which are homologous to the 5' non-coding sequence of endogenous polynucleotide sequence, flanking the promoter. The targeting sequence will be sufficiently near the 5' end of the polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination The promoter and the targeting sequences can be amplified using PCR. Preferably, the amplified promoter contains distinct restπction enzyme sites on the 5' and 3' ends. Preferably, the 3' end of the first targeting sequence contains the same restriction enzyme site as the 5' end of the amplified promoter and the 5' end of the second targeting sequence contains the same restriction site as the 3' end of the amplified promoter.

The amplified promoter and the amplified targeting sequences are digested with the appropriate restnction enzymes and subsequently treated with calf intestinal phosphatase. The digested promoter and digested targeting sequences are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropπate for ligation of the two fragments. The construct is size fractionated on an agarose gel then puπfied by phenol extraction and ethanol precipitation.

In this Example, the polynucleotide constructs are administered as naked polynucleotides via electroporation. However, the polynucleotide constructs may also be administered with transfection-faci tatmg agents, such as liposomes, viral sequences, viral particles, precipitating agents, etc. Such methods of delivery are known in the art.

Once the cells are transfected, homologous recombination will take place which results in the promoter being operably linked to the endogenous polynucleotide sequence. This results m the expression of polynucleotide corresponding to the polynucleotide m the cell. Expression may be detected by immunological staining, or any other method known m the art. Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in DMEM + 10% fetal calf serum. Exponentially growing or early stationary phase fibroblasts are trypsinized and rinsed from the plastic surface with nutrient medium. An aliquot of the cell suspension is removed for counting, and the remaining cells are subjected to centrifugation. The supernatant is aspirated and the pellet is resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 1.3, 137 mM NaCI, 5 mM KCl, 0.7 mM

Na₂ HPO , 6 mM dextrose). The cells are recentrifuged, the supernatant aspirated, and the cells resuspended in electroporation buffer containing 1 mg/ml acetylated bovine serum albumin. The final cell suspension contains approximately 3X10⁶ cells/ml. Electroporation should be performed immediately following resuspension.

Plasmid DNA is prepared according to standard techniques. For example, to construct a plasmid for targeting to the locus corresponding to the polynucleotide of the invention, plasmid pUC18 (MBI Fermentas, Amherst, NY) is digested with Hindlll. The CMV promoter is amplified by PCR with an Xbal site on the 5' end and a BamHI site on the 3'end. Two non-coding sequences are amplified via PCR: one non-coding sequence (fragment 1) is amplified with a Hindlll site at the 5' end and an Xba site at the 3'end; the other non-coding sequence (fragment 2) is amplified with a BamHI site at the 5'end and a Hindlll site at the 3'end. The CMV promoter and the fragments (1 and 2) are digested with the appropriate enzymes (CMV promoter - Xbal and BamHI; fragment 1 - Xbal; fragment 2 - BamHI) and ligated together. The resulting ligation product is digested with Hindlll, and ligated with the Hindlll-digested pUC18 plasmid.

Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap (Bio-Rad). The final DNA concentration is generally at least 120 μg/ml. 0.5 ml of the cell suspension (containing approximately 1.5.X10⁶ cells) is then added to the cuvette, and the cell suspension and DNA solutions are gently mixed. Electroporation is performed with a

Gene-Pulser apparatus (Bio-Rad). Capacitance and voltage are set at 960 μ¥ and 250-300 V, respectively. As voltage increases, cell survival decreases, but the percentage of surviving cells that stably incorporate the introduced DNA into their genome increases dramatically. Given these parameters, a pulse time of approximately 14-20 mSec should be observed.

Electroporated cells are maintained at room temperature for approximately 5 min, and the contents of the cuvette are then gently removed with a stenle transfer pipette. The cells are added directly to 10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cm dish and incubated at 37 degree C. The following day, the media is aspirated and replaced with 10 ml of fresh media and incubated for a furthei 16-24 hours.

The engineered fibroblasts are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcarner beads The fibroblasts now produce the protein product. The fibroblasts can then be introduced into a patient as descnbed above

Example 28: Method of Treatment Using Gene Therapy - In Vivo

Another aspect of the present invention is using in vivo gene therapy methods to treat disorders, diseases and conditions. The gene therapy method relates to the introduction of naked nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences into an animal to increase or decrease the expression of the polypeptide. The polynucleotide of the present invention may be operatively linked to a promoter or any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques and methods are known in the art, see, for example, WO90/11092, WO98/11779; U.S. Patent NO. 5693622, 5705151, 5580859;

Tabata et al., Cardiovasc. Res. 35(3):470-479 (1997); Chao et al., Pharmacol. Res.

35(6):517-522 (1997); Wolff, Neuromuscul. Disord. 7(5):314-318 (1997); Schwartz et al.,

Gene Ther. 3(5):405-411 (1996); Tsurumi et al., Circulation 94(12):3281-3290 (1996) (incorporated herein by reference).

The polynucleotide constructs may be delivered by any method that delivers mjectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, sk , lung, liver, intestine and the like). The polynucleotide constructs can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

The term "naked" polynucleotide, DNA or RNA, refers to sequences that are free from any delivery vehicle that acts to assist, promote, or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, the polynucleotides of the present invention may also be delivered in liposome formulations (such as those taught in Feigner P.L. et al. (1995) Ann. NY Acad. Sci. 772:126-139 and Abdallah B. et al. (1995) Biol. Cell 85(l): l-7) which can be prepared by methods well known to those skilled in the art.

The polynucleotide vector constructs used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Any strong promoter known to those skilled in the art can be used for driving the expression of DNA. Unlike other gene therapies techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non- replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.

The polynucleotide construct can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides. For the naked polynucleotide injection, an effective dosage amount of DNA or

RNA will be in the range of fiom about 0.05 g/kg body weight to about 50 mg/kg body weight Pieferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and moie preferably from about 0 05 mg/kg to about 5 mg/kg Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being tieated and the route of administration The preferred route of administration is by the paienteral route ot injection into the interstitial space of tissues Howevei, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particulai ly foi delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked polynucleotide constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.

The dose response effects of injected polynucleotide in muscle in vivo is determined as follows. Suitable template DNA for production of mRNA coding for polypeptide of the present invention is prepared in accordance with a standard recombinant DNA methodology The template DNA, which may be either circular or linear, is either used as naked DNA or complexed with liposomes. The quadriceps muscles of mice are then injected with vanous amounts of the template DNA.

Five to six week old female and male Balb/C mice are anesthetized by intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incision is made on the anterior thigh, and the quadriceps muscle is directly visualized. The template DNA is injected in 0J ml of earner m a 1 cc synnge through a 27 gauge needle over one minute, approximately 0.5 cm from the distal insertion site of the muscle into the knee and about 0.2 cm deep. A suture is placed over the injection site for future localization, and the skin is closed with stainless steel clips.

After an appropriate incubation time (e.g., 7 days) muscle extracts are prepared by excising the entire quadriceps. Every fifth 15 um cross-section of the individual quadriceps muscles is histochemically stained for protein expression. A time course foi protein expression may be done in a similar fashion except that quadriceps from different mice aie harvested at diffeient times. Persistence of DNA in muscle following injection may be determined by Southern blot analysis after preparing total cellulai DNA and HIRT supernatants fiom injected and control mice. The results of the above experimentation in mice can be use to extrapolate proper dosages and other treatment parameters m humans and other animals using naked DNA

Example 29: Transgenic Animals.

The polypeptides of the invention can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate transgenic animals. In a specific embodiment, techniques described herein or otherwise known in the art, are used to express polypeptides of the invention in humans, as part of a gene therapy protocol. Any technique known in the art may be used to introduce the transgene (i.e., polynucleotides of the invention) into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to, pronuclear microinjection (Paterson et al., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology (NY) 11: 1263-1270 (1993); Wnght et al., Biotechnology (NY) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; gene targeting in embryonic stem cells (Thompson et al., Cell 56:313-321 (1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol. 3: 1803-1814 (1983)); introduction of the polynucleotides of the invention using a gene gun

(see, e.g., Ulmer et al., Science 259:1745 (1993); introducing nucleic acid constructs into embryonic pleuripotent stem cells and transferring the stem cells back into the blastocyst; and sperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989); etc. For a review of such techniques, see Gordon, "Transgenic Animals/' Intl. Rev. Cytol. 115: 171-

229 (1989), which is incoφorated by reference herein in its entirety.

Any technique known in the art may be used to produce transgenic clones containing polynucleotides of the invention, for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (Campell et al., Nature 380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).

The present invention provides for transgenic animals that carry the transgene in all their cells, as well as animals which can-y the transgene in some, but not all their cells, i.e., mosaic animals or chimeric. The transgene may be integrated as a single transgene or as multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236 (1992)). The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. When it is desired that the polynucleotide transgene be integrated into the chromosomal site of the endogenous gene, gene targeting is prefeired. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the puφose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene. The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type, by following, for example, the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.

Once transgenic animals have been generated, the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the transgene has taken place The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcπptase-PCR (rt-PCR). Samples of transgenic gene-expressing tissue may also be evaluated immunocytochemically oi immunohistochemically using antibodies specific for the transgene product

Once the foundei animals are produced, they may be bred, inbred, outbred, oi crossbred to produce colonies of the particular animal. Examples of such breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the transgene on a distinct background that is appropnate for an expenmental model of interest.

Transgenic animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying conditions and/or disorders associated with aberrant expression, and in screening for compounds effective in ameliorating such conditions and/or disorders. Example 30: Knock-Out Animals.

Endogenous gene expression can also be reduced by inactivating or "knocking out" the gene and/or its promoter using targeted homologous recombination. (E.g., see Smithies et al., Nature 317:230-234 (1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell 5:313-321 (1989); each of which is incoφorated by reference herein in its entirety). For example, a mutant, non-functional polynucleotide of the invention (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous polynucleotide sequence (either the coding regions or regulatory regions of the gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express polypeptides of the invention in vivo. In another embodiment, techniques known in the art are used to generate knockouts in cells that contain, but do not express the gene of interest. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the targeted gene. Such approaches are particularly suited in research and agricultural fields where modifications to embryonic stem cells can be used to generate animal offspring with an inactive targeted gene (e.g., see Thomas & Capecchi 1987 and Thompson 1989, supra). However this approach can be routinely adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors that will be apparent to those of skill in the art.

In further embodiments of the invention, cells that are genetically engineered to express the polypeptides of the invention, or alternatively, that are genetically engineered not to express the polypeptides of the invention (e.g., knockouts) are administered to a patient in vivo. Such cells may be obtained from the patient (i.e., animal, including human) or an MHC compatible donor and can include, but are not limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cells are genetically engineered in vitro using recombinant DNA techniques to introduce the coding sequence of polypeptides of the invention into the cells, or alternatively, to disrupt the coding sequence and/or endogenous regulatory sequence associated with the polypeptides of the invention, e.g., by transduction (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. The coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention. The engineered cells which express and preferably secrete the polypeptides of the invention can be introduced into the patient systemically, e.g., in the circulation, or intraperitoneally.

Alternatively, the cells can be incoiporated into a matrix and implanted in the body, e.g., genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft. (See, for example, Anderson et al. U.S. Patent No. 5,399,349; and Mulligan & Wilson, U.S. Patent No. 5,460,959 each of which is incorporated by reference herein in its entirety).

When the cells to be administered are non-autologous or non-MHC compatible cells, they can be administered using well known techniques which prevent the development of a host immune response against the introduced cells. For example, the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.

Transgenic and "knock-out" animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying conditions and/or disorders associated with aberrant expression, and in screening for compounds effective in ameliorating such conditions and/or disorders. Example 31. Isolation of antibody fragments directed against polypeptides of the invention from a library of scFvs.

Naturally occurring V-genes isolated from human PBLs are constructed into a large library of antibody fragments which contain reactivities against a polypeptide having the amino acid sequence of SEQ ID NO:Y to which the donor may or may not have been exposed (see e.g., U.S. Patent 5,885,793 incoiporated herein in its entirety by reference).

Rescue of the library.

A library of scFvs is constructed from the RNA of human PBLs as described in WO92/01047. To rescue phage displaying antibody fragments, approximately 10⁹ E. coli harboring the phagemid are used to inoculate 50 ml of 2x TY containing 1% glucose and 100 micrograms/ml of ampicillin (2xTY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Five ml of this culture is used to inoculate 50 ml of 2xTY-AMP- GLU, 2 x 108 TU of delta gene 3 helper (M13 delta gene III, see WO92/01047) are added and the culture incubated at 37°C for 45 minutes without shaking and then at

37°C for 45 minutes with shaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and the pellet resuspended in 2 liters of of 2x TY containing 100 micrograms/ml ampicillin and 50 micrograms/ml kanamycin and grown overnight. Phage are prepared as described in WO92/01047. M13 delta gene III is prepared as follows: M13 delta gene III helper phage does not encode gene III protein, hence the phage(mid) displaying antibody fragments have a greater avidity of binding to antigen. Infectious M13 delta gene III particles are made by growing the helper phage in cells harboring a pUC19 derivative supplying the wild type gene III protein during phage moφhogenesis. The culture is incubated for 1 hour at 37°C without shaking and then for a further hour at 37°C with shaking. Cells were spun down (IEC-Centra 8, 4000 revs/min for 10 min), resuspended in 300 ml 2x TY broth containing 100 micrograms ampicillin/ml and 25 micrograms kanamycin/ml (2x TY- AMP-KAN) and grown overnight, shaking at 37°C. Phage particles are purified and concentrated from the culture medium by two PEG-precipitations (Sambrook et al.,

1990), resuspended in 2 ml PBS and passed through a 0.45 micrometer filter (Minisart

NML; Sartonus) to give a final concentration of approximately 10ⁿ transducing units/ml (ampicilhn-resistant clones). Panning the Library.

Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100 micrograms/ml or 10 micrograms/ml of a polypeptide of the present invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at 37°C and then washed 3 times in PBS.

Approximately 10^π TU of phage is applied to the tube and incubated foi 30 minutes at room temperature tumbling on an over and undei turntable and then left to stand foi another 1.5 hours. Tubes are washed 10 times with PBS 0.1%; Tween-20 and 10 times with PBS. Phage are eluted by adding 1 ml of 100 mM tπethylamine and rotating 15 minutes on an under and over turntable after which the solution is immediately neutralized with 0.5 ml of 1.0M Tπs-HCl, pH 7.4 Phage are then used to infect 10 ml of mid-log E. coli TGI by mcubatmg eluted phage with bacteria for 30 minutes at 37°C. The E. coli are then plated on TYE plates containing 1% glucose and 100 micrograms/ml ampicillin. The resulting bactenal library is then rescued with delta gene 3 helper phage as descnbed above to prepare phage for a subsequent round of selection. This process is then repeated for a total of 4 rounds of affinity punfication with tube-washing increased to 20 times with PBS, 0.1 % Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders.

Eluted phage from the third and fourth rounds of selection are used to infect E. coli HB 2151 and soluble scFv is produced (Marks, et al., 1991) from single colonies for assay. ELISAs are performed with microtiter plates coated with either 10 picograms/ml of the polypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clones positive in ELISA are further characteπzed by PCR ftngeφπnting (see e.g., WO92/01047) and then by sequencing. Example 32: Assays Detecting Stimulation or Inhibition of B cell Proliferation and Differentiation

Generation of functional humoral immune responses requires both soluble and cognate signaling between B-lmeage cells and their microenvironment Signals may impart a positive stimulus that allows a B-hneage cell to continue its progi ammed development, oi a negative stimulus that instructs the cell to aπest its cunent developmental pathway To date, numerous stimulatory and mhibitoiy signals have been found to influence B cell responsiveness including IL-2. IL-4. IL-5, IL-6, IL-7, ILK), IL-13, IL-14 and IL-15 Inteiestingly, these signals are by themselves weak effectors but can. in combination with vaπous co-stimulatory proteins, induce activation, proliferation, diffeientiation. homing, tolerance and death among B cell populations

One of the best studied classes of B-cell co-stimulatoiy proteins is the TNF- superfamily Withm this family CD40, CD27, and CD30 along with then lespective ligands

CD 154, CD70, and CD 153 have been found to legulate a variety of immune responses Assays which allow for the detection and/or observation of the piohferation and differentiation of these B-cell populations and their precursors are valuable tools in determining the effects vaπous proteins may have on these B-cell populations in terms of proliferation and differentiation Listed below are two assays designed to allow for the detection of the differentiation, proliferation, or inhibition of B-cell populations and their precursors. In Vitro Assay- Purified polypeptides of the invention, or truncated forms thereof, is assessed for its ability to induce activation, proliferation, differentiation or inhibition and/or death m B-cell populations and their precursors. The activity of the polypeptides of the invention on purified human tonsillar B cells, measured qualitatively over the dose range from OJ to 10,000 ng/mL, is assessed in a standard B-lymphocyte co-stimulation assay in which purified tonsillar B cells are cultured in the presence of either formahn- fixed Staphylococcus aureus Cowan I (SAC) or immobilized anti-human IgM antibody as the pnming agent. Second signals such as IL-2 and IL-15 synergize with SAC and IgM crosshnking to elicit B cell proliferation as measured by tπtiated-thymidine incoφoration Novel synergizmg agents can be readily identified using this assay The assay involves isolating human tonsillar B cells by magnetic bead (MACS) depletion of CD3-posιtιve cells. The resulting cell population is greater than 95% B cells as assessed by expression of CD45R(B220). Vaπous dilutions of each sample are placed into individual wells of a 96-well plate to which aie added 10⁵ B-cells suspended in cultuie medium (RPMI 1640 containing 10%

FBS, 5 X 10⁵M 2ME, lOOU/ml penicillin, lOug/ml streptomycin, and 10⁵ dilution of

SAC) in a total volume of 150ul Proliferation oi inhibition is quantitated by a 20h pulse

(luCi/well) with 3H-thymιdιne (6.7 Ci/mM) beginning 72h post factor addition. The positive and negative controls are IL2 and medium respectively

In Vivo Assay- BALB/c mice are injected (l p ) twice per day with buffer only, oi 2 mg/Kg of a polypeptide of the invention, oi truncated forms thereof. Mice receive this treatment foi 4 consecutive days, at which time they are sacrificed and various tissues and serum collected for analyses. Comparison of H&E sections from normal spleens and spleens treated with polypeptides of the invention identify the results of the activity of the polypeptides on spleen cells, such as the diffusion of pen-arterial lymphatic sheaths, and/or significant increases in the nucleated cellulaπty of the red pulp regions, which may indicate the activation of the differentiation and proliferation of B-cell populations Immunohistochemical studies using a B cell marker, antι-CD45R(B220), are used to determine whether any physiological changes to splenic cells, such as splenic disorganization, are due to increased B-cell representation within loosely defined B-cell zones that infiltrate established T-cell regions.

Flow cytometnc analyses of the spleens from mice treated with polypeptide is used to indicate whether the polypeptide specifically increases the proportion of ThB+, CD45R(B220)dull B cells over that which is observed in control mice.

Likewise, a predicted consequence of increased mature B-cell representation m vivo is a relative increase in serum Ig titers. Accordingly, serum IgM and IgA levels are compared between buffer and polypeptide-treated mice. The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.

Example 33: T Cell Proliferation Assay

A CD3-induced proliferation assay is performed on PBMCs and is measured by the uptake of ³H-thymidine. The assay is performed as follows. Ninety-six well plates are coated with 100 μl/well of mAb to CD3 (HIT3a, Pharmingen) or isotype-matched control mAb (B33J) overnight at 4 degrees C (1 μg/ml in .05M bicarbonate buffer, pH 9.5), then washed three times with PBS. PBMC are isolated by F/H gradient centrifugation from human peripheral blood and added to quadruplicate wells (5 x 10 /well) of mAb coated plates in RPMI containing 10% FCS and P/S in the presence of varying concentrations of polypeptides of the invention (total volume 200 ul). Relevant protein buffer and medium alone are controls. After 48 hr. culture at 37 degrees C, plates are spun for 2 min. at 1000 φm and 100 μl of supernatant is removed and stored -20 degrees C for measurement of IL-2 (or other cytokines) if effect on proliferation is observed. Wells are supplemented with 100 ul of medium containing 0.5 uCi of ³H-thymidine and cultured at 37 degrees C for 18-24 hr. Wells are harvested and incoφoration of ³H-thymidine used as a measure of proliferation. Anti-CD3 alone is the positive control for proliferation. IL-2 (100 U/ml) is also used as a control which enhances proliferation. Control antibody which does not induce proliferation of T cells is used as the negative controls for the effects of polypeptides of the invention.

The studies described in this example tested activity of polypeptides of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention. Example 34: Effect of Polypeptides of the Invention on the Expression of MHC Class

II. Costimulatory and Adhesion Molecules and Cell Differentiation of Monocytes and

Monocyte-Derived Human Dendritic Cells

Dendritic cells are generated by the expansion of proliferating precursors found in the peripheral blood: adherent PBMC or elutriated monocytic fractions are cultured for 7- 10 days with GM-CSF (50 ng/ml) and IL-4 (20 ng/ml). These dendritic cells have the characteristic phenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHC class II antigens). Treatment with activating factors, such as TNF-α, causes a rapid change in surface phenotype (increased expression of MHC class I and II, costimulatory and adhesion molecules, downregulation of FCγRII, upregulation of CD83). These changes correlate with increased antigen-presenting capacity and with functional maturation of the dendritic cells.

FACS analysis of surface antigens is performed as follows. Cells are treated 1-3 days with increasing concentrations of polypeptides of the invention or LPS (positive control), washed with PBS containing 1% BSA and 0.02 mM sodium azide, and then incubated with 1:20 dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at 4 degrees C. After an additional wash, the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).

Effect on the production of cytokines. Cytokines generated by dendritic cells, in particular IL-12, are important in the initiation of T-cell dependent immune responses. IL- 12 strongly influences the development of Thl helper T-cell immune response, and induces cytotoxic T and NK cell function. An ELISA is used to measure the IL-12 release as follows. Dendritic cells (10⁶/ml) are treated with increasing concentrations of polypeptides of the invention for 24 hours. LPS (100 ng/ml) is added to the cell culture as positive control. Supernatants from the cell cultures are then collected and analyzed for IL-12 content using commercial ELISA kit (e..g, R & D Systems (Minneapolis, MN)). The standard protocols provided with the kits are used. Effect on the expression of MHC Class II, costimulatory and adhesion molecules. Three major families of cell surface antigens can be identified on monocytes: adhesion molecules, molecules involved in antigen presentation, and Fc receptor. Modulation of the expression of MHC class II antigens and other costimulatory molecules, such as B7 and ICAM-1, may result in changes in the antigen presenting capacity of monocytes and ability to induce T cell activation. Increase expression of Fc receptors may correlate with improved monocyte cytotoxic activity, cytokine release and phagocytosis.

FACS analysis is used to examine the surface antigens as follows. Monocytes are treated 1-5 days with increasing concentrations of polypeptides of the invention or LPS (positive control), washed with PBS containing 1% BSA and 0.02 mM sodium azide, and then incubated with 1 :20 dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at 4 degreesC. After an additional wash, the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).

Monocyte activation and/or increased survival. Assays for molecules that activate (or alternatively, inactivate) monocytes and/or increase monocyte survival (or alternatively, decrease monocyte survival) are known in the art and may routinely be applied to determine whether a molecule of the invention functions as an inhibitor or activator of monocytes. Polypeptides, agonists, or antagonists of the invention can be screened using the three assays described below. For each of these assays, Peripheral blood mononuclear cells (PBMC) are purified from single donor leukopacks (American Red Cross, Baltimore, MD) by centrifugation through a Histopaque gradient (Sigma). Monocytes are isolated from PBMC by counterflow centrifugal elutriation.

Monocyte Survival Assay. Human peripheral blood monocytes progressively lose viability when cultured in absence of serum or other stimuli. Their death results from internally regulated process (apoptosis). Addition to the culture of activating factors, such as TNF-alpha dramatically improves cell survival and prevents DNA fragmentation.

Propidium iodide (PI) staining is used to measure apoptosis as follows. Monocytes are cultured for 48 hours in polypropylene tubes m serum-free medium (positive control), in the presence of 100 ng/ml TNF-alpha (negative control), and in the presence of varying concentrations of the compound to be tested. Cells are suspended at a concentration of 2 x

10⁶/ml in PBS containing PI at a final concentration of 5 μg/ml, and then incubaed at room temperature foi 5 minutes before FACScan analysis. PI uptake has been demonstrated to correlate with DNA fragmentation in this experimental paiadigm

Effect on cytokine release. An important function of monocytes/macrophages is their regulatory activity on other cellulai populations of the immune system through the release of cytokines after stimulation. An ELISA to measure cytokine release is performed as follows. Human monocytes are incubated at a density of 5x10^ cells/ml with increasing concentrations of the a polypeptide of the invention and under the same conditions, but in the absence of the polypeptide. For IL-12 production, the cells are primed overnight with IFN (100 U/ml) in presence of a polypeptide of the invention. LPS (10 ng/ml) is then added Conditioned media are collected after 24h and kept frozen until use. Measurement of TNF-alpha, IL-10, MCP-1 and IL-8 is then performed using a commercially available ELISA kit (e..g, R & D Systems (Minneapolis, MN)) and applying the standard protocols provided with the kit.

Oxidative burst. Punfied monocytes are plated in 96-w plate at 2-lxlO⁵ cell/well. Increasing concentrations of polypeptides of the invention are added to the wells in a total volume of 0.2 ml culture medium (RPMI 1640 + 10% FCS, glutamine and antibiotics). After 3 days incubation, the plates are centrifuged and the medium is removed from the wells. To the macrophage monolayers, 0.2 ml per well of phenol red solution (140 mM NaCI, 10 mM potassium phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol red and 19 U/ml of HRPO) is added, together with the stimulant (200 nM PMA). The plates are incubated at 37°C for 2 hours and the reaction is stopped by adding 20 μl IN NaOH per well. The absorbance is read at 610 nm. To calculate the amount of H₂O₂ produced by the macrophages, a standard curve of a H₂O, solution of known molarity is performed for each experiment. The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polypeptides, polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 35: Biological Effects of Polypeptides of the Invention

Astrocyte and Neuronal Assays-

Recombinant polypeptides of the invention, expressed in Escherichia coli and purified as described above, can be tested for activity in promoting the survival, neurite outgrowth, or phenotypic differentiation of cortical neuronal cells and for inducing the proliferation of glial fibrillary acidic protein immunoposifive cells, astrocytes. The selection of cortical cells for the bioassay is based on the prevalent expression of FGF-1 and FGF-2 in cortical structures and on the previously reported enhancement of cortical neuronal survival resulting from FGF-2 treatment. A thymidine incoφoration assay, for example, can be used to elucidate a polypeptide of the invention's activity on these cells. Moreover, previous reports describing the biological effects of FGF-2 (basic FGF) on cortical or hippocampal neurons in vitro have demonstrated increases in both neuron survival and neurite outgrowth (Walicke et al., "Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension." Proc. Natl. Acad. Sci. USA 53:3012-3016. (1986), assay herein incorporated by reference in its entirety). However, reports from experiments done on PC- 12 cells suggest that these two responses are not necessarily synonymous and may depend on not only which FGF is being tested but also on which receptor(s) are expressed on the target cells. Using the primary cortical neuronal culture paradigm, the ability of a polypeptide of the invention to induce neurite outgrowth can be compared to the response achieved with FGF-2 using, for example, a thymidine incoφoration assay.

5 Fibroblast and endothelial cell assays-

Human lung fibroblasts are obtained from Clonetics (San Diego, CA) and maintained in growth media from Clonetics. Dermal microvascular endothelial cells are obtained from Cell Applications (San Diego, CA). For proliferation assays, the human lung fibroblasts and dermal microvascular endothelial cells can be cultured at 5,000 _Q cells/well in a 96-well plate for one day in growth medium. The cells are then incubated for one day in 0.1% BSA basal medium. After replacing the medium with fresh 0.1% BSA medium, the cells are incubated with the test proteins for 3 days. Alamar Blue (Alamar Biosciences, Sacramento, CA) is added to each well to a final concentration of 10%. The cells are incubated for 4 hr. Cell viability is measured by reading in a CytoFluor 5 fluorescence reader. For the PGE, assays, the human lung fibroblasts are cultured at 5,000 cells/well in a 96-well plate for one day. After a medium change to 0.1% BSA basal medium, the cells are incubated with FGF-2 or polypeptides of the invention with or without IL-lα for 24 hours. The supernatants are collected and assayed for PGE₂ by EIA kit (Cayman, Ann Arbor, MI). For the IL-6 assays, the human lung fibroblasts are cultured Q at 5,000 cells/well in a 96-well plate for one day. After a medium change to 0.1% BSA basal medium, the cells are incubated with FGF-2 or with or without polypeptides of the invention IL-lα for 24 hours. The supernatants are collected and assayed for IL-6 by ELISA kit (Endogen, Cambridge, MA).

Human lung fibroblasts are cultured with FGF-2 or polypeptides of the invention _ζ for 3 days in basal medium before the addition of Alamar Blue to assess effects on growth of the fibroblasts. FGF-2 should show a stimulation at 10 - 2500 ng/ml which can be used to compare stimulation with polypeptides of the invention. Parkinson Models.

The loss of motor function in Parkinson's disease is attributed to a deficiency of striatal dopamine resulting from the degeneration of the nigrostriatal dopaminergic projection neurons. An animal model for Parkinson's that has been extensively characterized involves the systemic administration of l-methyl-4 phenyl 1,2,3,6- tetrahydropyridine (MPTP). In the CNS, MPTP is taken-up by astrocytes and catabolized by monoamine oxidase B to l-methyl-4-phenyl pyridine (MPP⁺) and released. Subsequently, MPP⁺ is actively accumulated in dopaminergic neurons by the high-affinity reuptake transporter for dopamine. MPP⁺ is then concentrated in mitochondria by the electrochemical gradient and selectively inhibits nicotidamide adenine disphosphate: ubiquinone oxidoreductionase (complex I), thereby interfering with electron transport and eventually generating oxygen radicals.

It has been demonstrated in tissue culture paradigms that FGF-2 (basic FGF) has trophic activity towards nigral dopaminergic neurons (Ferrari et al., Dev. Biol. 1989). Recently, Dr. Unsicker's group has demonstrated that administering FGF-2 in gel foam implants in the striatum results in the near complete protection of nigral dopaminergic neurons from the toxicity associated with MPTP exposure (Otto and Unsicker, J. Neuroscience, 1990). Based on the data with FGF-2, polypeptides of the invention can be evaluated to determine whether it has an action similar to that of FGF-2 in enhancing dopaminergic neuronal survival in vitro and it can also be tested in vivo for protection of dopaminergic neurons in the striatum from the damage associated with MPTP treatment. The potential effect of a polypeptide of the invention is first examined in vitro in a dopaminergic neuronal cell culture paradigm. The cultures are prepared by dissecting the midbrain floor plate from gestation day 14 Wistar rat embryos. The tissue is dissociated with trypsin and seeded at a density of 200,000 cells/cm² on polyorthinine-laminin coated glass coverslips. The cells are maintained in Dulbecco's Modified Eagle's medium and F12 medium containing hormonal supplements (Nl) The cultures are fixed with paraformaldehyde after 8 days in vitro and are processed for tyrosine hydroxylase, a specific marker for dopminergic neurons, immunohistochemical staining. Dissociated cell cultures are prepared from embryonic rats. The culture medium is changed every third day and the factoi s are also added at that time.

Since the dopamineigic neurons are isolated from animals at gestation day 14, a developmental time which is past the stage when the dopaminergic precursor cells are prohfei ating, an increase in the number of tyrosine hydroxylase immunopositive neurons would represent an increase in the numbei of dopaminergic neurons surviving in vitro Therefoie, if a polypeptide of the invention acts to prolong the survival of dopaminergic neurons, it would suggest that the polypeptide may be involved in Parkinson's Disease.

The studies described in this example tested activity of a polypeptide of the invention However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 36: The Effect of Polypeptides of the Invention on the Growth of Vascular Endothelial Cells

On day 1, human umbilical vein endothelial cells (HUVEC) are seeded at 2-5x10⁴ cells/35 mm dish density in M199 medium containing 4% fetal bovme serum (FBS), 16 units/ml heparin, and 50 units/ml endothelial cell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the medium is replaced with Ml 99 containing 10% FBS, 8 units/ml heparin. A polypeptide having the amino acid sequence of SEQ ID NO:Y, and positive controls, such as VEGF and basic FGF (bFGF) are added, at varying concentrations. On days 4 and 6, the medium is replaced. On day 8, cell number is determined with a Coulter Counter.

An increase in the number of HUVEC cells indicates that the polypeptide of the invention may proliferate vascular endothelial cells. The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 37: Stimulatory Effect of Polypeptides of the Invention on the Proliferation of Vascular Endothelial Cells

For evaluation of mitogenic activity of growth factors, the colorimetric MTS

(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H- tetrazolium) assay with the electron coupling reagent PMS (phenazine methosulfate) was performed (CellTiter 96 AQ, Promega). Cells are seeded in a 96-well plate (5,000 cells/well) in 0J mL serum-supplemented medium and are allowed to attach overnight. After serum-starvation for 12 hours in 0.5% FBS, conditions (bFGF, VEGF,₆₅ or a polypeptide of the invention in 0.5% FBS) with or without Heparin (8 U/ml) are added to wells for 48 hours. 20 mg of MTS/PMS mixture (1:0.05) are added per well and allowed to incubate for 1 hour at 37°C before measuring the absorbance at 490 nm in an ELISA plate reader. Background absorbance from control wells (some media, no cells) is subtracted, and seven wells are performed in parallel for each condition. See, Leak et al. In Vitro Cell. Dev. Biol. 504:512-518 (1994). The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 38: Inhibition of PDGF-induced Vascular Smooth Muscle Cell Proliferation Stimulatory Effect

HAoSMC proliferation can be measured, for example, by BrdUrd incoφoration. Briefly, subconfluent, quiescent cells grown on the 4-chamber slides are transfected with CRP or FITC-labeled AT2-3LP. Then, the cells are pulsed with 10% calf serum and 6 mg/ml BrdUrd. After 24 h, immunocytochemistry is performed by using BrdUrd Staining

Kit (Zymed Laboratories). In brief, the cells are incubated with the biotinylated mouse anti-BrdUrd antibody at 4 degrees C for 2 h after being exposed to denatuπng solution and then incubated with the stieptavidin-peroxidase and diaminobenzidine Aftei counterstainmg with hematoxyhn, the cells are mounted for microscopic examination, and the BrdUrd-positive cells are counted. The BrdUid index is calculated as a percent of the

BrdUrd-positive cells to the total cell number In addition, the simultaneous detection of the BrdUrd staining (nucleus) and the FITC uptake (cytoplasm) is performed for individual cells by the concomitant use of bright field illumination and dark field-UV fluorescent illumination. See, Hayashida et al . J. Biol. Chem. 6:271(36).21985-21992 (1996)

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 39: Stimulation of Endothelial Migration

This example will be used to explore the possibility that a polypeptide of the invention may stimulate lymphatic endothelial cell migration. Endothelial cell migration assays are performed using a 48 well microchemotaxis chamber (Neuroprobe Inc., Cabin John, MD; Falk, W., et al., J. Immunological Methods 1980;33:239-247). Polyvmylpyrrohdone-free polycarbonate filters with a pore size of 8 u (Nucleopore Coφ. Cambndge, MA) are coated with 0.1% gelatin for at least 6 hours at room temperature and dried under sterile air. Test substances are diluted to appropriate concentrations in M199 supplemented with 0.25% bovine serum albumin (BSA), and 25 ul of the final dilution is placed in the lower chamber of the modified Boyden apparatus. Subconfluent, early passage (2-6) HUVEC or BMEC cultures are washed and trypsinized for the minimum time required to achieve cell detachment. After placing the filter between lower and upper chamber, 2.5 x 10⁵ cells suspended in 50 ul M199 containing 1% FBS are seeded in the upper compartment. The apparatus is then incubated for 5 hours at 37°C in a humidified chamber with 5% CO2 to allow cell migration. After the incubation period, the filter is removed and the upper side of the filter with the non-migrated cells is scraped with a rubber policeman. The filters are fixed with methanol and stained with a Giemsa solution (Diff-Quick, Baxter, McGraw Park, IL). Migration is quantified by counting cells of three random high-power fields (40x) in each well, and all groups are performed in quadruplicate.

Example 40: Stimulation of Nitric Oxide Production by Endothelial Cells Nitric oxide released by the vascular endothelium is believed to be a mediator of vascular endothelium relaxation. Thus, activity of a polypeptide of the invention can be assayed by determining nitric oxide production by endothelial cells in response to the polypeptide.

Nitric oxide is measured in 96-well plates of confluent microvascular endothelial cells after 24 hours starvation and a subsequent 4 hr exposure to various levels of a positive control (such as VEGF-1) and the polypeptide of the invention. Nitric oxide in the medium is determined by use of the Griess reagent to measure total nitrite after reduction of nitric oxide-derived nitrate by nitrate reductase. The effect of the polypeptide of the invention on nitric oxide release is examined on HUVEC. Briefly, NO release from cultured HUVEC monolayer is measured with a NO- specific polarographic electrode connected to a NO meter (Iso-NO, World Precision Instruments Inc.) (1049). Calibration of the NO elements is performed according to the following equation: 2 KNO₂ + 2 KI + 2 H₂SO₄ 6 2 NO + I₂ + 2 H₂O + 2 K₂SO₄

The standard calibration curve is obtained by adding graded concentrations of

KNO₂ (0, 5, 10, 25, 50, 100, 250, and 500 nmol/L) into the calibration solution containing

KI and H₂SO₄. The specificity of the Iso-NO electrode to NO is previously determined by measurement of NO from authentic NO gas (1050). The culture medium is removed and

HUVECs are washed twice with Dulbecco's phosphate buffered saline. The cells are then bathed in 5 ml of filtered Krebs-Henseleit solution in 6-well plates, and the cell plates are kept on a slide warmer (Lab Line Instruments Inc.) To maintain the temperature at 37°C.

The NO sensor probe is inserted vertically into the wells, keeping the tip of the electrode 2 mm under the surface of the solution, before addition of the different conditions. S-nitroso acetyl penicillamin (SNAP) is used as a positive control. The amount of released NO is expressed as picomoles per lxlO⁶ endothelial cells. All values reported are means of four to six measurements in each group (number of cell culture wells). See, Leak et al. Biochem. and Biophys. Res. Comm. 277:96-105 (1995). The studies described in this example tested activity of polypeptides of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and or antagonists of the invention.

Example 41: Effect of Polypepides of the Invention on Cord Formation in Angiogenesis

Another step in angiogenesis is cord formation, marked by differentiation of endothelial cells. This bioassay measures the ability of microvascular endothelial cells to form capillary-like structures (hollow structures) when cultured in vitro. CADMEC (microvascular endothelial cells) are purchased from Cell Applications,

Inc. as proliferating (passage 2) cells and are cultured in Cell Applications' CADMEC Growth Medium and used at passage 5. For the in vitro angiogenesis assay, the wells of a 48-well cell culture plate are coated with Cell Applications' Attachment Factor Medium (200 ml/well) for 30 min. at 37°C. CADMEC are seeded onto the coated wells at 7,500 cells/well and cultured overnight in Growth Medium. The Growth Medium is then replaced with 300 mg Cell Applications' Chord Formation Medium containing control buffer or a polypeptide of the invention (0J to 100 ng/ml) and the cells are cultured for an additional 48 hr. The numbers and lengths of the capillary-like chords are quanfitated through use of the Boeckeler VIA- 170 video image analyzer. All assays are done in triplicate.

Commercial (R&D) VEGF (50 ng/ml) is used as a positive control, b-esteradiol (1 ng/ml) is used as a negative control. The appropriate buffer (without protein) is also utilized as a control.

Example 42: Angiogenic Effect on Chick Chorioallantoic Membrane

Chick chorioallantoic membrane (CAM) is a well-established system to examine angiogenesis. Blood vessel formation on CAM is easily visible and quantifiable. The ability of polypeptides of the invention to stimulate angiogenesis in CAM can be examined.

Fertilized eggs of the White Leghorn chick (Gallus gallus) and the Japanese qual (Coturnix cotumix) are incubated at 37.8°C and 80% humidity. Differentiated CAM of 16-day-old chick and 13-day-old qual embryos is studied with the following methods.

On Day 4 of development, a window is made into the egg shell of chick eggs. The embryos are checked for normal development and the eggs sealed with cellotape. They are further incubated until Day 13. Thermanox coverslips (Nunc, Naperville, IL) are cut into disks of about 5 mm in diameter. Sterile and salt-free growth factors are dissolved in distilled water and about 3.3 mg/ 5 ml are pipetted on the disks. After air-drying, the inverted disks are applied on CAM. After 3 days, the specimens are fixed in 3% glutaraldehyde and 2% formaldehyde and rinsed in 0J2 M sodium cacodylate buffer.

They are photographed with a stereo microscope [Wild M8] and embedded for semi- and ultrathin sectioning as described above. Controls are performed with carrier disks alone. The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 43: Angiogenesis Assay Using a Matrigel Implant in Mouse

In vivo angiogenesis assay of a polypeptide of the invention measures the ability of an existing capillary network to form new vessels in an implanted capsule of murine extracellular matrix material (Matrigel). The protein is mixed with the liquid Matrigel at 4 degree C and the mixture is then injected subcutaneously in mice where it solidifies. After 7 days, the solid "plug" of Matrigel is removed and examined for the presence of new blood vessels. Matrigel is purchased from Becton Dickinson Labware/Collaborative Biomedical Products.

When thawed at 4 degree C the Matrigel material is a liquid. The Matrigel is mixed with a polypeptide of the invention at 150 ng/ml at 4 degrees C and drawn into cold 3 ml syringes. Female C57B1/6 mice approximately 8 weeks old are injected with the mixture of Matrigel and experimental protein at 2 sites at the midventral aspect of the abdomen (0.5 ml/site). After 7 days, the mice are sacrificed by cervical dislocation, the Matrigel plugs are removed and cleaned (i.e., all clinging membranes and fibrous tissue is removed). Replicate whole plugs are fixed in neutral buffered 10% formaldehyde, embedded in paraffin and used to produce sections for histological examination after staining with Masson's Trichrome. Cross sections from 3 different regions of each plug are processed. Selected sections are stained for the presence of vWF. The positive control for this assay is bovme basic FGF (150 ng/ml). Matngel alone is used to determine basal levels of angiogenesis.

Example 44: Rescue of Ischemia in Rabbit Lower Limb Model

To study the in vivo effects of polynucleotides and polypeptides of the invention on ischemia, a rabbit hmdlimb ischemia model is cieated by surgical removal of one femoral arteries as described previously (Takeshita et al , Am J. Pathol 747: 1649-1660 (1995)) The excision of the femoral artery results in retrograde propagation of thrombus and occlusion of the external iliac artery Consequently, blood flow to the ischemic limb is dependent upon collateral vessels originating from the internal iliac artery (Takeshitaet al. Am J. Pathol 147:1649-1660 (1995)). An interval of 10 days is allowed for post-operative recovery of rabbits and development of endogenous collateral vessels. At 10 day post- operatively (day 0), after performing a baseline angiogram, the internal iliac artery of the ischemic limb is transfected with 500 mg naked expression plasmid containing a polynucleotide of the invention by arterial gene transfer technology using a hydrogel- coated balloon catheter as described (Riessen et al. Hum Gene Ther. 4:749-758 (1993); Leclerc et al. J. Clin. Invest. 90: 936-944 (1992)). When a polypeptide of the invention is used in the treatment, a single bolus of 500 mg polypeptide of the invention or control is delivered into the internal iliac artery of the ischemic limb over a penod of 1 min. through an infusion catheter. On day 30, vaπous parameters are measured in these rabbits: (a) BP ratio - The blood pressure ratio of systolic pressure of the ischemic limb to that of normal limb; (b) Blood Flow and Flow Reserve - Resting FL: the blood flow dunng undilated condition and Max FL: the blood flow during fully dilated condition (also an indirect measure of the blood vessel amount) and Flow Reserve is reflected by the ratio of max FL: resting FL; (c) Angiographic Score - This is measured by the angiogram of collateral vessels. A score is determined by the percentage of circles in an overlaying grid that with crossing opacifted arteries divided by the total number m the rabbit thigh; (d) Capillary density - The number of collateral capillaries determined in light microscopic sections taken from hindhmbs

The studies descnbed in this example tested activity of polynucleotides and polypeptides of the invention Howevei , one skilled in the art could easily modify the exemplified studies to test the agonists, and/or antagonists of the invention

Example 45: Effect of Polypeptides of the Invention on Vasodilation

Since dilation of vascular endothelium is important in reducing blood pressuie, the ability of polypeptides of the invention to affect the blood pressure in spontaneously hypertensive rats (SHR) is examined Increasing doses (0, 10, 30, 100, 300, and 900 mg/kg) of the polypeptides of the invention aie administeied to 13- 14 week old spontaneously hypertensive rats (SHR) Data are expressed as the mean +/- SEM. Statistical analysis are performed with a paired t-test and statistical significance is defined as p<0.05 vs. the response to buffer alone

The studies described in this example tested activity of a polypeptide of the invention However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g , gene therapy), agonists, and/or antagonists of the invention.

Example 46: Rat Ischemic Skin Flap Model

The evaluation parameters include skin blood flow, skm temperature, and factor VIII immunohistochemistry or endothelial alkaline phosphatase reaction. Expression of polypeptides of the invention, during the skin ischemia, is studied using in situ hybndization.

The study in this model is divided into three parts as follows: a) Ischemic skin b) Ischemic skin wounds c) Normal wounds

The experimental protocol includes: a) Raising a 3x4 cm, single pedicle full-thickness random skin flap (myocutaneous flap over the lower back of the animal). b) An excisional wounding (4-6 mm in diameter) in the ischemic skin (skin-flap). c) Topical treatment with a polypeptide of the invention of the excisional wounds (day 0, 1, 2, 3, 4 post-wounding) at the following various dosage ranges: lmg to 100 mg. d) Harvesting the wound tissues at day 3, 5, 7, 10, 14 and 21 post-wounding for histological, immunohistochemical, and in situ studies.

Example 47: Peripheral Arterial Disease Model

Angiogenic therapy using a polypeptide of the invention is a novel therapeutic strategy to obtain restoration of blood flow around the ischemia in case of peripheral arterial diseases. The experimental protocol includes: a) One side of the femoral artery is ligated to create ischemic muscle of the hindlimb, the other side of hindlimb serves as a control. b) a polypeptide of the invention, in a dosage range of 20 mg - 500 mg, is delivered intravenously and/or intramuscularly 3 times (perhaps more) per week for 2-3 weeks. c) The ischemic muscle tissue is collected after ligation of the femoral artery at 1, 2, and 3 weeks for the analysis of expression of a polypeptide of the invention and histology. Biopsy is also performed on the other side of normal muscle of the contralateral hindlimb. The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 48: Ischemic Myocardial Disease Model

A polypeptide of the invention is evaluated as a potent mitogen capable of stimulating the development ot collateral vessels, and restructuring new vessels aftei coronary artery occlusion. Alteration of expression of the polypeptide is investigated in situ. The experimental protocol includes. a) The heart is exposed through a left-side thoracotomy in the rat. Immediately, the left coronary artery is occluded with a thin suture (6-0) and the thorax is closed b) a polypeptide of the invention, in a dosage range of 20 mg - 500 mg, is delivered intravenously and/or intramuscularly 3 times (perhaps more) per week foi 2-4 weeks. c) Thirty days after the surgery, the heart is removed and cross-sectioned for moφhometπc and situ analyzes.

Example 49: Rat Corneal Wound Healing Model

This animal model shows the effect of a polypeptide of the invention on neovasculanzation. The expenmental protocol includes: a) Making a 1-1.5 mm long incision from the center of comea into the stromal layer. b) Inserting a spatula below the lip of the incision facing the outer comer of the eye. c) Making a pocket (its base is 1-1.5 mm form the edge of the eye). d) Positioning a pellet, containing 50ng- 5ug of a polypeptide of the invention, within the pocket. e) Treatment with a polypeptide of the invention can also be applied topically to the coineal wounds in a dosage range of 20mg - 500mg (daily treatment for five days).

The studies descπbed in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention

Example 50: Diabetic Mouse and Glucocorticoid-Impaired Wound Healing Models A. Diabetic db+/db+ Mouse Model. To demonstrate that a polypeptide of the invention accelerates the healing process, the genetically diabetic mouse model of wound healing is used. The full thickness wound healing model in the db+/db+ mouse is a well characterized, clinically relevant and reproducible model of impaired wound healing. Healing of the diabetic wound is dependent on formation of granulation tissue and re-epitheliahzation rather than contraction (Gartner, M.H. et al, J. Surg. Res. 52:389 (1992); Greenhalgh, D.G. et al, Am. J. Pathol. 136:1235 (1990)).

The diabetic animals have many of the characteristic features observed in Type II diabetes melhtus. Homozygous (db+/db+) mice are obese in comparison to their normal heterozygous (db+/+m) httermates. Mutant diabetic (db+/db+) mice have a single autosomal recessive mutation on chromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci. USA 77:283-293 (1982)). Animals show polyphagia, polydipsia and polyuna. Mutant diabetic mice (db+/db+) have elevated blood glucose, increased or normal insulin levels, and suppressed cell-mediated immunity (Mandel et al, J. Immunol. 120: 1315 (1978); Debray-Sachs, M. et al, Clin. Exp. Immunol. 51(1):1-1 (1983); Leiter et al, Am. J. of

Pathol. 114:46-55 (1985)). Peripheral neuropathy, myocardial complications, and microvascular lesions, basement membrane thickening and glomerular filtration abnormalities have been described in these animals (Norido, F. et al., Exp. Neurol. S5(2):221-232 (1984); Robertson et al, Diabetes 29(1):60-61 (1980); Giacomelli et al,

Lab Invest. 40(4):460-413 (1979); Coleman, D.L., Diabetes 31 (Suppl): l-6 (1982)). These homozygous diabetic mice develop hyperglycemia that is resistant to insulin analogous to human type II diabetes (Mandel et al, J. Immunol. 120:1315-1311 (1978)).

The characteristics observed in these animals suggests that healing in this model may be similar to the healing observed in human diabetes (Greenhalgh, et al. Am. J. of Pathol. 736: 1235-1246 (1990)).

Genetically diabetic female C57BL/KsJ (db+/db+) mice and their non-diabetic (db+/+m) heterozygous littermates are used in this study (Jackson Laboratories). The animals are purchased at 6 weeks of age and are 8 weeks old at the beginning of the study. Animals are individually housed and received food and water ad libitum. All manipulations are performed using aseptic techniques. The experiments are conducted according to the rules and guidelines of Human Genome Sciences, Inc. Institutional Animal Care and Use Committee and the Guidelines for the Care and Use of Laboratory Animals. Wounding protocol is performed according to previously reported methods

(Tsuboi, R. and Rifkin, D.B., J. Exp. Med. 172:245-251 (1990)). Briefly, on the day of wounding, animals are anesthetized with an intraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanol and 2-methyl-2-butanol dissolved in deionized water. The dorsal region of the animal is shaved and the skin washed with 70% ethanol solution and iodine. The surgical area is dried with sterile gauze prior to wounding. An 8 mm full- thickness wound is then created using a Keyes tissue punch. Immediately following wounding, the surrounding skin is gently stretched to eliminate wound expansion. The wounds are left open for the duration of the experiment. Application of the treatment is given topically for 5 consecutive days commencing on the day of wounding. Prior to treatment, wounds are gently cleansed with stenle saline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at the day of surgery and at two day intervals thereafter. Wound closure is determined by daily measurement on days 1-5 and on day 8. Wounds are measured horizontally and vertically using a calibrated Jameson cahper. Wounds are considered healed if granulation tissue is no longer visible and the wound is covered by a continuous epithelium.

A polypeptide of the invention is administered using at a range different doses, from 4mg to 500mg per wound per day for 8 days in vehicle Vehicle control groups received 50mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300mg/kg). The wounds and surrounding skin are then harvested for histology and immunohistochemistry. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing. Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls) are evaluated: 1) Vehicle placebo control, 2) untreated group, and 3) treated group.

Wound closure is analyzed by measuring the area in the vertical and hoπzontal axis and obtaining the total square area of the wound. Contraction is then estimated by establishing the differences between the initial wound area (day 0) and that of post treatment (day 8). The wound area on day 1 is 64mm², the corresponding size of the dermal punch. Calculations are made using the following formula:

[Open area on day 8] - [Open area on day 1] / [Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are sectioned peφendicular to the wound surface (5mm) and cut using a Reichert-Jung microtome. Routine hematoxyhn-eosin (H&E) staining is performed on cross-sections of bisected wounds. Histologic examination of the wounds are used to assess whether the healing process and the morphologic appearance of the repaired skin is altered by treatment with a polypeptide of the invention. This assessment included veπfication of the presence of cell accumulation, inflammatory cells, capillaries, fibroblasts, re- epitheliahzation and epidermal matunty (Greenhalgh, D.G. et al, Am. J. Pathol. 136:1235 (1990)). A calibrated lens micrometer is used by a blinded observer.

Tissue sections are also stained lmmunohistochemically with a polyclonal labbit anti-human keratin antibody using ABC Elite detection system Human skin is used as a positive tissue control while non-immune IgG is used as a negative control Keratinocyte growth is determined by evaluating the extent of reepithehahzation of the wound using a calibrated lens micrometer.

Proliferating cell nuclear antigen/cyclm (PCNA) in skm specimens is demonstrated by using anti-PCNA antibody (1 :50) with an ABC Elite detection system. Human colon cancer can serve as a positive tissue control and human bram tissue can be used as a negative tissue control. Each specimen includes a section with omission of the primary antibody and substitution with non-immune mouse IgG. Ranking of these sections is based on the extent of proliferation on a scale of 0-8, the lower side of the scale reflecting slight proliferation to the higher side reflecting intense proliferation.

Experimental data are analyzed using an unpaired t test. A p value of < 0.05 is considered significant.

B. Steroid Impaired Rat Model

The inhibition of wound healing by steroids has been well documented in various in vitro and in vivo systems (Wahl, Glucocorticoids and Wound healing. In: Anti- Inflammatory Steroid Action: Basic and Clinical Aspects. 280-302 (1989); Wahlet α/., J. Immunol. 115: 476-481 (1975); Werb et al., J. Exp. Med. 747: 1684-1694 (1978)). Glucocorticoids retard wound healing by inhibiting angiogenesis, decreasing vascular permeability (Ebert et al., An. Intern. Med. 37:101-105 (1952)), fibroblast proliferation, and collagen synthesis (Beck et al, Growth Factors. 5: 295-304 (1991); Haynes et al, J. Clin. Invest. 61: 703-797 (1978)) and producing a transient reduction of circulating monocytes (Haynes et al., J. Clin. Invest. 61. 703-797 (1978); Wahl, "Glucocorticoids and wound healing", In: Antnnflammatory Steroid Action: Basic and Clinical Aspects,

Academic Press, New York, pp. 280-302 (1989)) The systemic administration of steroids to impaired wound healing is a well establish phenomenon in rats (Beck et al., Growth

Factors. 5. 295-304 (1991), Haynes et al, J Cl n. Invest. 61: 703-797 (1978), Wahl,

"Glucocorticoids and wound healing", In. Antnnflammatory Steroid Action: Basic and

Clinical Aspects, Academic Press, New Yoik, pp 280-302 (1989); Pierce et al, Proc.

Natl. Acad. Sci. USA 86: 2229-2233 (1989)). To demonstrate that a polypeptide of the invention can accelerate the healing process, the effects of multiple topical applications of the polypeptide on full thickness excisional skin wounds in rats in which healing has been impaired by the systemic administration of methylprednisolone is assessed.

Young adult male Sprague Dawley rats weighing 250-300 g (Charles Rivei Laboratones) are used in this example. The animals are purchased at 8 weeks of age and are 9 weeks old at the beginning of the study. The healing response of rats is impaired by the systemic administration of methylprednisolone (17mg/kg/rat intramuscularly) at the time of wounding. Animals are individually housed and received food and water ad libitum. All manipulations are performed using aseptic techniques. This study is conducted according to the rules and guidelines of Human Genome Sciences, Inc. Institutional Animal Care and Use Committee and the Guidelines for the Care and Use of Laboratory Animals.

The wounding protocol is followed according to section A, above. On the day of wounding, animals are anesthetized with an intramuscular injection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsal region of the animal is shaved and the skin washed with 70% ethanol and iodine solutions. The surgical area is dried with sterile gauze prior to wounding. An 8 mm full-thickness wound is created using a Keyes tissue punch. The wounds are left open for the duration of the expeπment. Applications of the testing materials are given topically once a day for 7 consecutive days commencing on the day of wounding and subsequent to methylprednisolone administration. Prior to treatment, wounds are gently cleansed with sterile saline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at the day of wounding and at the end of treatment. Wound closure is determined by daily measurement on days 1 -5 and on day 8. Wounds are measured horizontally and vertically using a calibrated Jameson caliper. Wounds are considered healed if granulation tissue is no longer visible and the wound is covered by a continuous epithelium.

The polypeptide of the invention is administered using at a range different doses, from 4mg to 500mg per wound per day for 8 days in vehicle. Vehicle control groups received 50mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300mg/kg). The wounds and surrounding skin are then harvested for histology. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.

Four groups of 10 animals each (5 with methylprednisolone and 5 without glucocorticoid) are evaluated: 1) Untreated group 2) Vehicle placebo control 3) treated groups.

Wound closure is analyzed by measuring the area in the vertical and horizontal axis and obtaining the total area of the wound. Closure is then estimated by establishing the differences between the initial wound area (day 0) and that of post treatment (day 8). The wound area on day 1 is 64mm², the corresponding size of the dermal punch. Calculations are made using the following formula:

[Open area on day 8] - [Open area on day 1] / [Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are sectioned perpendicular to the wound surface (5mm) and cut using an Olympus microtome. Routine hematoxylin-eosin (H&E) staining is performed on cross-sections of bisected wounds. Histologic examination of the wounds allows assessment of whether the healing process and the morphologic appearance of the repaired skin is improved by treatment with a polypeptide of the invention. A calibrated lens micrometer is used by a blinded observer to determine the distance of the wound gap.

Example 51: Lymphadema Animal Model or The purpose of this experimental approach is to create an appropriate and consistent lymphedema model for testing the therapeutic effects of a polypeptide of the invention in lymphangiogenesis and re-establishment of the lymphatic circulatory system in the rat hind limb. Effectiveness is measured by swelling volume of the affected limb, quantification of the amount of lymphatic vasculature, total blood plasma protein, and histopathology. Acute lymphedema is observed for 7-10 days. Perhaps more importantly, the chronic progress of the edema is followed for up to 3-4 weeks.

Prior to beginning surgery, blood sample is drawn for protein concentration analysis. Male rats weighing approximately ~350g are dosed with Pentobarbital. Subsequently, the right legs are shaved from knee to hip. The shaved area is swabbed with gauze soaked in 70% EtOH. Blood is drawn for serum total protein testing. Circumference and volumetric measurements are made prior to injecting dye into paws after marking 2 measurement levels (0.5 cm above heel, at mid-pt of dorsal paw). The intradermal dorsum of both right and left paws are injected with 0.05 ml of 1 % Evan's Blue. Circumference and volumetric measurements are then made following injection of dye into paws.

Using the knee joint as a landmark, a mid-leg inguinal incision is made circumferentially allowing the femoral vessels to be located. Forceps and hemostats are used to dissect and separate the skin flaps. After locating the femoral vessels, the lymphatic vessel that runs along side and underneath the vessel(s) is located. The main lymphatic vessels in this area are then electrically coagulated suture ligated.

Using a microscope, muscles in back of the leg (near the semitendinosis and adductors) are bluntly dissected The popliteal lymph node is then located. The 2 proximal and 2 distal lymphatic vessels and distal blood supply of the popliteal node aie then and ligated by suturing. The popliteal lymph node, and any accompanying adipose tissue, is then removed by cutting connective tissues

Care is taken to control any mild bleeding resulting from this procedure After lymphatics aie occluded, the sk flaps are sealed by using liquid skin (Vetbond) (AJ Buck). The separated skm edges are sealed to the underlying muscle tissue while leaving a gap of -0.5 cm around the leg. Skm also may be anchored by suturing to underlying muscle when necessary.

To avoid infection, animals are housed individually with mesh (no bedding). Recovering animals are checked daily through the optimal edematous peak, which typically occurred by day 5-7. The plateau edematous peak are then observed. To evaluate the intensity of the lymphedema, the circumference and volumes of 2 designated places on each paw before operation and daily for 7 days are measured. The effect plasma proteins on lymphedema is determined and whether protein analysis is a useful testing perimeter is also investigated. The weights of both control and edematous limbs are evaluated at 2 places. Analysis is performed m a blind manner.

Circumference Measurements: Under brief gas anesthetic to prevent limb movement, a cloth tape is used to measure limb circumference. Measurements are done at the ankle bone and dorsal paw by 2 different people then those 2 readings are averaged

Readings are taken from both control and edematous limbs

Volumetric Measurements On the day of surgery, animals are anesthetized with

Pentobarbital and are tested pnor to surgery For daily volumetrics animals are under brief halothane anesthetic (rapid immobilization and quick recovery), both legs aie shaved and equally maiked using waterproof marker on legs Legs aie fust dipped in watei , then dipped into insti ument to each marked level then measuied by Buxco edema software(Chen/Vιctoι) Data is lecoided by one person, while the othei is dipping the limb to maiked area Blood-plasma protein measurements Blood is drawn, spun, and serum separated pnor to suigery and then at conclusion foi total piotein and Ca2+ compaiison

Limb Weight Compaiison Aftei drawing blood, the animal is prepared foi tissue collection The limbs are amputated using a quil tine, then both expeπmental and contiol legs are cut at the ligature and weighed A second weighing is done as the tibio-cacaneal joint is disarticulated and the foot is weighed

Histological Preparations The transveise muscle located behind the knee (popliteal) area is dissected and arranged in a metal mold, filled with freezeGel, dipped into cold methylbutane, placed into labeled sample bags at - 80EC until sectioning Upon sectioning, the muscle is observed under fluorescent microscopy for lymphatics The studies described in this example tested activity of a polypeptide of the invention However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e g., gene therapy), agonists, and/or antagonists of the invention

Example 52: Suppression of TNF alpha-induced adhesion molecule expression by a Polypeptide of the Invention

The recruitment of lymphocytes to areas of inflammation and angiogenesis involves specific receptor-hgand interactions between cell surface adhesion molecules (CAMs) on lymphocytes and the vascular endothelium. The adhesion process, in both normal and pathological settings, follows a multi-step cascade that involves intercellular adhesion molecule- 1 (ICAM-1), vascular cell adhesion molecule- 1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin) expression on endothelial cells (EC). The expression of these molecules and others on the vascular endothelium determines the efficiency with which leukocytes may adhere to the local vasculature and extravasate into the local tissue during the development of an inflammatory response. The local concentration of cytokines and growth factor participate in the modulation of the expression of these CAMs. Tumor necrosis factor alpha (TNF-a), a potent proinflammatory cytokine, is a stimulator of all three CAMs on endothelial cells and may be involved in a wide variety of inflammatory responses, often resulting in a pathological outcome.

The potential of a polypeptide of the invention to mediate a suppression of TNF-a induced CAM expression can be examined. A modified ELISA assay which uses ECs as a solid phase absorbent is employed to measure the amount of CAM expression on TNF-a treated ECs when co-stimulated with a member of the FGF family of proteins.

To perform the experiment, human umbilical vein endothelial cell (HUVEC) cultures are obtained from pooled cord harvests and maintained in growth medium (EGM- 2; Clonetics, San Diego, CA) supplemented with 10% FCS and 1% penicillin/streptomycin in a 37 degree C humidified incubator containing 5% CO?. HUVECs are seeded in 96- well plates at concentrations of 1 x 10⁴ cells/well in EGM medium at 37 degree C for 18- 24 hrs or until confluent. The monolayers are subsequently washed 3 times with a serum- free solution of RPMI-1640 supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin, and treated with a given cytokine and/or growth factor(s) for 24 h at 37 degree C. Following incubation, the cells are then evaluated for CAM expression.

Human Umbilical Vein Endothelial cells (HUVECs) are grown in a standard 96 well plate to confluence. Growth medium is removed from the cells and replaced with 90 ul of 199 Medium (10% FBS). Samples for testing and positive or negative controls are added to the plate m tπphcate (in 10 ul volumes). Plates are incubated at 37 degree C for either 5 h (selectin and integrin expression) or 24 h (integrin expression only). Plates are aspirated to remove medium and 100 μl of 0.1% paraformaldehyde-PBS(wιth Ca++ and

Mg++) is added to each well. Plates are held at 4°C for 30 min. Fixative is then removed from the wells and wells are washed IX with

PBS(+Ca,Mg)+0.5% BSA and drained Do not allow the wells to dry. Add 10 μl of diluted primary antibody to the test and control wells. Antι-ICAM-1-Bιotm, Anti-VCAM-

1-Bιotm and Anti-E-selectin-Biotm are used at a concentration of 10 μg/ml (1 J0 dilution of 0J mg/ml stock antibody). Cells are incubated at 37°C for 30 mm. in a humidified environment. Wells are washed X3 with PBS(+Ca,Mg)+0.5% BSA.

Then add 20 μl of diluted ExtiAvidm-Alkal e Phosphotase (1:5,000 dilution) to each well and incubated at 37°C for 30 min. Wells are washed X3 with PBS(+Ca,Mg)+0.5% BSA. 1 tablet of p-Nitrophenol Phosphate pNPP is dissolved in 5 ml of glycine buffer (pH 10.4). 100 μl of pNPP substrate in glycine buffer is added to each test well. Standard wells in triplicate are prepared from the working dilution of the ExtrAvidm-Alkahne Phosphotase in glycine buffer: 1:5,000 (10°) > 10⁰⁵ > 10 ' > 10 ^{1 5}. 5 μl of each dilution is added to tnphcate wells and the resulting AP content m each well is 5.50 ng, 1.74 ng, 0.55 ng, 0J8 ng. 100 μl of pNNP reagent must then be added to each of the standard wells. The plate must be incubated at 37°C for 4h. A volume of 50 μl of 3M NaOH is added to all wells. The results are quantified on a plate reader at 405 nm. The background subtraction option is used on blank wells filled with glycme buffer only. The template is set up to indicate the concentration of AP-conjugate in each standard well [ 5.50 ng; 1.74 ng; 0.55 ng; 0J8 ng]. Results are indicated as amount of bound AP- conjugate in each sample. The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention. It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.

The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background of the Invention, Detailed Description, and Examples is hereby incoiporated herein by reference. Further, the hard copy of the sequence listing submitted herewith and the corresponding computer readable form are both incoφorated herein by reference in their entireties.

Example 53: Cloning, sequence analysis and chromosomal localization of the novel human integrin alpha 11 subunit. Abstract The integrins are a large family of cell adhesion molecules consisting of noncovalently associated αβ heterodimers. We have cloned and sequenced a novel human integrin α - subunit cDNA, designated al l. The all cDNA encodes a protein with a 22 amino acid signal peptide, a large 1120 residue extracellular domain that contains an I-domain of 207 residues and is linked by a transmembrane domain to a short cytoplasmic domain of 24 amino acids. The deduced al l protein shows the typical structural features of integrin α- subunits and is similar to a distinct group of α-subunits from collagen-binding integrins. However, it differs from most integrin α-chains by an incompletetely preserved cytoplasmic GFFKR motif. The human ITGA11 gene was located to bands q22.3-23 on chromosome 15, and its transcripts were found predominantly in bone, cartilage as well as in cardiac and skeletal muscle. Expression of the 5.5 kilobase al l mRNA was also detectable in ovary and small intestine. Introduction

All vertebrate cells express members of the integrin family of cell adhesion molecules, which mediate cellular adhesion to other cells and extracellular subtratum, cell migration and participate in important physiologic processes from signal transduction to cell proliferation and differentiation {Hynes, 92; Springer, 92 } . Integrins are structurally homologous heterodimeric type-I membrane glycoproteins formed by the noncovalent association of one of eight β -subunits with one of the 17 different α-subunits described to date, resulting in at least 22 different αβ complexes. Their binding specificities for cellular and extracellular ligands are determined by both subunits and are dynamically regulated in a cell-type-specific mode by the cellular environment as well as by the developmental and activation state of the cell {Diamond and Springer, 94 } . In integrin α -subunits, the aminoterminal region of the large extracellular domain consists of a seven-fold repeated structure which is predicted to fold into a β -propeller domain { Corbi et al, 1987; Springer, 1997 } . The three or four C-terminal repeats contain putative divalent cation binding motifs that are thought to be important for ligand binding and subunit association {Diamond and Springer, 94}. The α¹, α², α ¹⁰, α^D, α^E, α , α^M and α^x-subunits contain an approximately 200 amino acid I-domain inserted between the second and third repeat that is not present in other α-chains {Larson et al., 1989}. Several isolated I-domains have been shown to independently bind the ligands of the parent integrin heterodimer {Kamata and Takada, 1994; Randi and Hogg, 1994 } . The α\ α ^5"8, α "^b and α ^v-subunits are proteolytically processed at a conserved site into disulphide-linked heavy and light chains, while the α⁴-subunit is cleaved at a more aminoterminal site into two fragments that remain noncovalently associated {Hemler et al, 90}. Additional α-subunit variants are generated by alternative splicing of primary transcripts {Ziober et al., 93; Delwel et al., 95; Leung et al, 98 }. The extracellular domains of α-integrin subunits are connected by a single spanning transmembrane domain to short, diverse cytoplasmic domains whose only conserved feature is a membrane-proximal KXGFF(K/R)R motif { Sastry and Horwitz, 1993 }. The cytoplasmic domains have been implicated in the cell-type-specific modulation of integrin affinity states {Williams et al, 1994 }.

Here we report the cDNA cloning, sequence analysis, expression and chromosomal localization of the human α-integrin subunit.

Materials and Methods

Library screening and DNA sequencing.

A human fetal heart cDNA library in λgtlO (Clontech Laboratories, Inc., Palo Alto, CA, USA) was screened with ^,2P-labelled (redipτi^'me, Amersham New Zealand Ltd., Auckland, New Zealand) probes corresponding to the regions 473 to 749 and 2394 to 3189 of the αl 1 cDNA using standard procedures. Inserts were subcloned from λgtlO into pUC21 and sequenced on both strands according to a successive specific primer strategy on an automated sequencer (Applied Biosystems 373A, The Centre for Gene Technology. School of Biological Sciences, The University of Auckland).

Northern Blot Analysis and Tissue distribution.

A 1341bp PCR fragment corresponding to the region 351-1692 of the α cDNA was ³²P- labelled (red/prime) and hybridized with human multiple tissue Northern blots (MTN I and MTN II, Clontech) for 16h at 60∞C in ExpressHyb solution (Clontech). Filters were washed twice with OJxSSC/1 % SDS at 50∞C for 30min, and autoradiographed. Human DNA from 63 tissue-specific cDNA libraries (Express-Check™, American Type Culture Collection, Manassas, VA, USA) was amplified using primers KL120 (5'- GCAGGGATGCCACCTGCC) and KL119 (5'-GATGAAGACTGTGGTGTCGAAGG) according to the manufacturers instructions. PCR-products were resolved by agarose gel electrophoresis and transferred to Hybond C+ (Amersham). Filters were hybridized by standard procedures { Ausubel et al, 98 } with a 502bp ³²P-labelled (rediprime) probe fragment obtained from the cloned "" cDNA with the same oligonucleotides. Chromosomal assignment.

500ng genomic DNA prepared from a panel of 21 human-rodent somatic cell hybrids or from human, mouse and hamster cells { Kelsell et al., 95 } was amplified with oligonuleotides KL175 (5'-GGTGCCAGACCTACATGGAC) and KL189 (5'- CGTGCAAATTCAATGCCAAATGCC) in a standard PCR reaction of 30 cycles (94∞C for lmin, 55∞C for lmin, 72∞C for 2min). All PCR reactions were resolved in a 2% agarose gel. Southern hybridization was performed as detailed above, except that the probe fragment was obtained from clone HOHBY69 with oligonucleotides KL175 and KL189. For fluorescent in situ hybridization, metaphase spreads were prepared from phytohemagglutinin-stimulated peripheral blood lymphocytes of a 46,XY male donor using standard cytogenetic procedures. A purified 3.7kB fragment representing the entire coding region of clone HOHBY69 was labelled with biotin-16-dUTP using the High Prime labelling kit (Roche Molecular Biochemicals, Auckland, NZ). Conditions for hybridization and immunofluorescent detection were essentially as described {Morris et al, 93 }, except that C₀t-1 suppression was not required, slides were washed to a stringency of 0JxSSC/60∞C after hybridization, and an additional amplification step was needed because of the small size of the probe. For precise chromosome band localization, DAPI and FITC images were captured using a Photometries KAF1400 CCD camera and QUIPS Smartcapture FISH software version 1.3 (Vysis Inc., Downers Grove, IL, USA). QUIPS CGH/Karyotyping software (version 3.0.2) assisted karyotype analysis.

Results

Cloning of a novel human -integrin subunit cDNA: A protein homology search {Altschul et al., 90} of the human expressed sequence tag (EST) databases of Human Genome Sciences, Inc. {Ni et al, 97 } and The Institute for Genomic Research { Kirkness and Kerlavage, 97 } identified the clones HRDAF83 and HOEAM34 as candidate novel integrin α -subunit cDNAs. Clone HRDAF83 was isolated from a human rhabdomyosarcoma cDNA library and sequenced on both strands. The

1223bp insert contains largely incompletely processed hnRNA and a 277bp region that showed homology to the aminoterminal half of the αl-integrin I-domain . The 2517bp insert of clone HOEAM34 was derived from a human osteoblast cDNA library. It is homologous to the C-terminal part of the human αl-subunit and contains 1324 nucleotides of 3'-untranslated region . In order to isolate the full-length cDNAs for these integrin α- subunits, a cDNA library prepared from human fetal heart in λgtlO was screened with the

277bp fragment from clone HRDAF83 homologous to the αl-I-domain. Two clones, λ831 and λ832, were isolated and both strands of their inserts sequenced. Clone λ832 contains the entire 5' half of a novel α-subunit cDNA, while clone λ831 covers the same region, but is 358bp and 173bp shorter than λ831 at its 5'- and 3'-ends, respectively. A screening of the same library with a 795bp fragment from the extreme 5'-terminus of clone HOEAM34 identified clone λ342, which contained essentially the same region as clone HOEAM34 but has a 317bp shorter 3'-untranslated region . Rescreening the EST databases with the sequences derived from the human fetal heart library led to the identification of clone HOHBY69, which was isolated from a osteoblast cDNA libray. Both strands of the 468 lbp insert of clone HOHBY69 were sequenced. The 5'-region of HOHBY69 was identical to the HRDAF83/λ832/λ831 -group, while the 3'-region of HOHBY69 was largely identical to HOEAM34 and λ342, thereby demonstrating that the two groups of partial cDNAs represent the 5'- and 3'-portions of the same cDNA . One major difference between the HOHBY69 and HOEAM34/λ342 is the presence of an additional GTA-triplet at position 3088 in HOHBY69. From the overlapping clones, a total of 4986bp of cDNA was assembled to the composite sequence shown in Figure 2 and has been submitted to GenBank™ with accession number AF109681. This cDNA encodes a previously unidentifed human integrin α-subunit that was designated all. Structure of the human al l-subunit.

The al l cDNA contains a 5'-untranslated region of 72 nucleotides and a single open reading frame extending from a predicted translation initiation codon at position +1 to a

TGA termination codon at position 3570 . This is followed by 1324 nucleotides of 3' untranslated region which contains an AATTAAA polyadenylation signal {Wahle and

Keller, 1996 } 12 nucleotides upstream of a poly(A) stretch. The deduced amino acid sequence contains a 22 residue N-terminal region with the characteristics of a cleaved signal peptide { von Heijne, 83; Nielsen et al, 97 }, a large extracellular domain of 1120 amino acids followed by a 23 amino acid hydrophobic stretch that resembles a transmembrane domain, and a short 24 residue cytoplasmic domain. The molecular weight of the mature 1167 amino acid αl 1-subunit is predicted to be 131 kDa, but the addition of carbohydrate side chains to any of the 15 potential N-glycosylation sequons [NX(S/T)] within the extracellular domain is likely to increase the molecular weight of the native protein. An I-domain of 207 amino acids is inserted between the second and third repeat . Consistent with the structure of an typical I-domain-containing integrin α-subunit, it lacks a potential dibasic protease cleavage site in the C-terminal region of the extracellular domain.

The αl 1-subunit is most closely related to the recently discovered αlO-subunit (Camper et al, 98, Lehnert et al, in preparation} and the αl- and α2-subunits. Overall, the mature αl l-protein is 45% identical to the αlO chain, while the homologies to the αl- and α2- subunits are 41% and 39%, respectively. Even greater homology exists between the I- domains of the αlO- and αl l-subunits which are 60% identical to each other. The high degree of homology seen in the extracellular domains of the subunits is in contrast to the low similarity of their cytoplasmic domains. Interestingly, the KXGFF(K/R)R motif that is absolutely conserved in all other α-subunit cytoplasmic domains is only partially preserved in both subunits. The sequence in al l is KLGFFRS, while the alO-subunit contains a KLGFFAH motif. A graphical comparison of the similarity between all integrin α-subunits is shown in Fig. 3. Together with the α-subunits from the collagen-binding integrins αlβl, α2βl and αlOβl, the αll-subunit forms a group distinct from the other I-domain- containing integπn subunits.

Tissue distribution and expression of the integrin al l-subunit. The tissue distnbution of the all mRNA was assessed by screening multiple human tissue Northern blots with a probe corresponding to the region 351-1692 of the al l cDNA. A single transcript of approximately 5.5kb was found weakly expressed only in ovary and small intestine . Integπn αl l-subunit expression was further analyzed by amplification and Southern hybndization of a 502bp fragment coπ-esponding to the region 1988-2490 in the al l cDNA from tissue-specific human cDNA libraries, al l cDNA was detected in five diffeient cDNA libraries piepared from fetal heart (day 57-75), in two fetal brain libraries, and in a cDNA library from large intestine (not shown). An analysis of the Human Genome Sciences Database revealed eight different al l -related ESTs in human osteoblast hbranes, three EST in a human chondrosarcoma cDNA library and two EST in a human stromal osteoclastoma library

Chromosomal localization of the integrin al l-subunit.

Genomic DNA from a collection af 21 human-rodent somatic cell hybrids {Kelsell et al.,

95 } was amplified by PCR using ohgonucleotide primers directed the the region 473 to 749 of the human all cDNA. In Southern hybndization, a signal corresponding to a l,4kb fragment was detectable only with DNA from a hybrid cell line that contains human chromosome 15. A fragment of the same size was also amplified from human genomic DNA, but not from mouse or hamster DNA (Fig. 5C). Cloning and sequence of the PCR product from chromosome 15 revealed the presence of a 1154bp intron inserted after cDNA-position 600, thus resulting in a PCR-product of 143 lbp. The ITGAll gene was also localized by fluorescent in situ hybndization of metaphase chromosomes with the entire coding region from clone HOHBY69. All of 20 metaphase cells analyzed showed fluorescent signal on both chromosomes 15, specifically across bands q22.3-q23. No additional signals were detected on any other chromosome (Fig. 5A).

Discussion: We have cloned and sequenced a novel cDNA encoding a protein that shares extensive structural homology with integrin α-chains. The aminoterminal 22 amino acids of the deduced protein sequence show the characteristic features of a hydrophobic leader peptide, including a signal peptidase recognition motif at positions -3 and -1 { von Heijne 83 } . Proteolytic cleavage of the precursor protein at this position would result in an aminoterminal sequence for the mature al l-chain of FNMD, which is similar to the consensus sequence[(F/Y)N(L/V)D] of all other integrin αl l-subunits {Tuckwell et al., 94 }. The N-terminal half of the large extracellular region of al l is composed of seven repeats that each contain FG— GAP— GxxY consensus motifs (FG-GAP repeats). These repeats can be found in all integrin αl-subunits and are predicted to fold into a β-propeller domain { Springer, 97 }. Inserted between the second and third FG-GAP repeats is a 207 amino acid I-domain spanning from glutamine¹³⁸ to methionine³⁴⁴. It contains a divalent cation coordination motif that has been shown to directly bind Mg²⁺ ions in the α^M subunit { Michishita et al, 96 } . The noncontiguous amino acid side chains involved in the coordination of magnesium or manganese ions have been identified by mutagenesis analysis and from crystal structures of the isolated α², α and α^M-subunit I-domains {Emsley et al, 97; Qu and Leahy, 95; Lee et al, 95; }. All residues required for the coordination of the divalent cations in these subunits are preserved in the αll-I-domain. These are the asparagines at positions 148 and 249, the serine residues at position 150 and 152, and the threonine at position 218. The crystal sctructure of the α2-subunit has revealed a small "-helix that is not present in the I-domains of the β2-associated α-subunits. Together with the MIDAS sphere, amino acid residues from this C-helix and the adjacent turn region have been proposed to make physical contacts to a collagen triple helix {Emsley et al., 97 }. Interestingly, the small C- helix is structurally conserved in the α-subunits of the collagen-binding integrins αlβl, α2βl and αlOβl, and is also present in the αl l l-domain (G²⁷⁹YYNR²⁸³). In addition, asparagine¹⁵⁴ and histidine²⁵⁸ of the α2-I-domain were predicted to contact the collagen triple helix, and both are preserved in the α l, αlO and αll-I-domains, but not in other integrin αl l-subunits. The conservation of structural motifs required for collagen binding suggests that collagen may be a ligand for the αl 1 integrin. Each of the repeats 5-7 of the αl l-subunit accomodates the sequence Dx(D/N)xDxxxD. Three or four copies of these putative divalent cation binding sites are conserved in all integrin α-subunits and their presence is consistent with the divalent cation requirement for the adhesive function of integrins {Larson et al, 89; Fujimura and Phillips, 83; Hynes, 92 } . The extracellular domain of the integrin αl l-subunit contains 20 cysteine residues. Only the intramolecular disulfide bonds in the "^llb subunit have been biochemically characterized {Calvete et al, 89 }, but the location of many cysteines is conserved in integrin α-subunits. In the αl l- subunit, the cysteine residues 637 and 646, 652 and 707, 759 and 765, and 859 and 871 are homologous to the residues that form the four carboxyterminal disulfide bonds in the heavy chain of "^lib {Calvete et al, 89}. Based on the proposed structure of the integrin α- subunit propeller domain {Springer et al, 97 }, additional disulfide bonds within the al l subunit can be predicted between cysteine residues 54 and 61, 99 and 117, and between 107 and 137. Two additional cysteine residues are found within a short segment (residues 783 to 798) that is unique to the αl 1-subunit.

The integrin cytoplasmic domains play central roles in integrin affinity modulation and in cellular signal transmission. The membrane-proximal sequence KxGFF(K/R)R is strictly conserved among integrin α-subunit cytoplasmic domains {Williams et al, 94}. Within this motif, both phenylalanine residues and the last arginine have been implicated in maintaining the default low affinity state of integrins α^Lβ₂ and α^Ilbβ₃, as their substitution or deletion resulted in constitutively activated ligand binding {O'Toole et al, 94, Lu and Spriner 97}. Interestingly, the last arginine residue is replaced by a serine in the al l cytoplasmic domain and with a histidine in the αlO subunit, suggesting that both integrins might be in a default "high" affinity state. It will be interesting to analyze whether substitution of these residues with a conserved arginine will affect their affinity status.

We have isolated all cDNAs from osteoclast, osteoblast, myosarcoma and fetal heart libraries. Amongst the HGS EST databases, integrin al l transcripts were predominantly found in libraries prepared from osteoblast, osteoclast and chondrosarcoma cells. A search for further ""-related sequences in the EST division of the GenBank database revealed two clones (accession numbers Z50157 and Z50167) from primary human myoblasts { Genini et al., 96}, two clones from human trabecular bone cells (AA852614 and AA852615), as well as clones from fibroblast cells (W45078), pancreatic tumor (U53091) and breast tissue (H16112). In contrast, Northern blot analysis detected al l -expression only in ovary and small intestine, only fetal heart, fetal brain and large intestine. Of the tissues represented in the tissue-specific cDNA-library panel, only fetal heart, fetal brain and large intestine showed detectable al l-expression. However, bone- and muscle-derived tissues were not included in the Northern blot, and cDNA libraries prepared from these tissues were also not represented in the tissue-specific cDNA panel.

The ITGAllgenc was localized to chromosome 15, bands q22.3-23, by FISH and PCR analysis of human-rodent somatic cell hybrids. This segment is overrepresented in squamous cell carcinomas {Wolff et al., 1998 }, but appears to only infrequently affected in other cancers. Genes at this region encode neogenin, a protein expressed ubiquitously expressed in human tissues {Meyerhardt et al, 91 } ; tropomyosin 1, expressed in cardiac and skeletal muscle tissues {Tiso et al, 97 } ; and the human homologue of the metalloprotease-disintegrin kuzbanian, which is overexpressed in tumors of sympathoadrenal origin { Yavari et al, 98}. In addition, the region 15q22.3-q23 is linked to Bardet-Biedl syndrome 4, a heterogeneous autosomal disorder characterized by obesity and associated with cardiovascular anomalities {Carmi et al., 95 }.

In conclusion, we have cloned and sequenced the cDNA for the novel integrin αl 1-subunit which is closely related to the "-subunits of the collagen-binding integrins αlβl, α2βl and αlOβl. The high degree of homology of al l to these subunits suggests that it associates with the integrin βl-subunit, and may function as an additional collagen receptor.

All references referred to above and presented below are hereby incoφorated herein by reference:

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Camper L., Hellman, U., and Lundgren-Akerlund, E. (1998). Isolation, cloning and sequence analysis of the integrin subunit αlO, a βl-associated collagen binding integrin expressed on chondrocytes. Journal of Biological Chemistry 273: 20383-20389.

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Corbi A. L., Miller, L. J., O'Connor, K., Larson, R. S., and Springer, T. A. (1987). cDNA cloning and complete primary structure of the alpha subunit of a leukocyte adhesion glycoprotein, pl50,95. EMBO Journal 6: 4023-4028. De Melker A. A., Kramer, D., Kuikman, I., and Sonnenberg, A. (1997). The two phenylalanines in the GFFKR motif of the integrin alphaόA subunit are essential for heterodimerization. Biochemical Journal 328: 529-537.

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Emsley J., King, S. L., Bergelson, J. M., and Liddington, R. C. (1997). Crystal structure of the I domain from integrin alpha2betal . Journal of Biological Chemistry 272: 28512- 28517.

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Spurr, N. K. (1995). Development of a panel of monochromosomal somatic cell hybrids for rapid gene mapping. Annals of Human Genetics 59: 233-241.

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Leung E., Lim, S. P., Berg, R., Yang, Y., Ni, J., Wang, S. X., and Krissansen, G. W. (1998). A novel extracellular domain variant of the human integrin alpha 7 subunit generated by alternative intron splicing. Biochemical & Biophysical Research Communications 243: 317-325.

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Michishita M., Videm, V., and Amaout, M. A. (1993). A novel divalent cation-binding site in the A domain of the beta 2 integrin CR3 (CDl lb/CD18) is essential for ligand binding. Cell 72: 857-867. Meyerhardt J. A., Look, A. T., Bigner, S. H., and Fearon, E. R. (1997). Identification and characterization of neogenin, a DCC-related gene. Oncogene 14: 1129-1136.

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Sastry S. K. and Horwitz, A. F. (1993). Integrin cytoplasmic domains: mediators of cytoskeletal linkages and extra- and intracellular initiated transmembrane signaling. Current Opinion in Cell Biology 5: 819-831. Springer T. A. (1997). Folding of the N-terminal, ligand-binding region of integrin alpha- subunits into a beta-propeller domain. Proceedings of the National Academy of Sciences of the United States of America 94: 65-72.

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Tiso N., Rampoldi, L., Pallavicini, A., Zimbello, R., Pandolfo, Valle, G., Lanfranchi, G., and Danieli, G. A. (1997). Fine mapping of five human skeletal muscle genes: alpha- tropomyosin, beta-tropomyosin, troponin-I slow-twitch, troponin-I fast-twitch, and troponin-C fast. Biochemical & Biophysical Research Communications 230: 347-350.

Tuckwell D. S., Humphries, M. J., and Brass, A. (1994). A secondary structure model of the integrin alpha subunit N-terminal domain based on analysis of multiple alignments. Cell Adhesion & Communication 2: 385-402.

von Heijne G. (1983). Patterns of amino acids near signal-sequence cleavage sites. European Journal of Biochemistry 133: 17-21.

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Williams M. J., Hughes, P. E., O'Toole, T. E., and Ginsberg, M. H. (1998). The inner world of cell adhesion: Integrin cytoplasmic domains. Trends in Cell Biology 4: 109-112.

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Ziober B. L., Vu, M. P., Waleh, N., Crawford, J., Lin, C. S., Kramer, and RH (1993). Alternative extracellular and cytoplasmic domains of the integrin alpha 7 subunit are differentially expressed during development. Journal of Biological Chemistry 268: _--Z-L

Applicant's nr agent's file reference number PF489PCT International appiica < r ■•, wass^ηgd--,..

RO/US 1 5 FEE 2000

I DICAΗONS RELAΗNG TO A DEPOSITED MICROORGANISM

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A. The indications made beiow relate to the microorganism referred to in the description on page 8 , ii„e 30 n. IDENTIΠCATIONOFDEPOSΓΓ Further deposits are identified on an additional sheet | |

Name of depositary institution American Type Culture Collection

Address of depositary institution t including postal code and country) 10801 University Boulevard Maπassas, Virginia 20110-2209 United States of Ameπca

Dateof deposit Accession Number

November 17, 1998 203484

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Claims

What Is Claimed Is:

1. An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of:

(a) a polynucleotide fragment of SEQ ID NO:X or a polynucleotide fragment of the cDNA sequence included in ATCC Deposit No:Z, which is hybridizable to SEQ ID NO:X;

(b) a polynucleotide encoding a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA sequence included in ATCC Deposit No:Z, which is hybridizable to SEQ ID NO:X;

(c) a polynucleotide encoding a polypeptide domain of SEQ ID NO:Y or a polypeptide domain encoded by the cDNA sequence included in ATCC Deposit No:Z, which is hybridizable to SEQ ID NO:X;

(d) a polynucleotide encoding a polypeptide epitope of SEQ ID NO:Y or a polypeptide epitope encoded by the cDNA sequence included in ATCC Deposit No:Z, which is hybridizable to SEQ ID NO:X;

(e) a polynucleotide encoding a polypeptide of SEQ ID NO:Y or the cDNA sequence included in ATCC Deposit No:Z, which is hybridizable to SEQ ID NO:X, having biological activity;

(f) a polynucleotide which is a variant of SEQ ID NO:X;

(g) a polynucleotide which is an allelic variant of SEQ ID NO:X;

(h) a polynucleotide which encodes a species homologue of the SEQ ID NO:Y;

(i) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(h), wherein said polynucleotide does not hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence of only A residues or of only T residues.

2. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide fragment comprises a nucleotide sequence encoding a secreted protein.

3. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide fragment comprises a nucleotide sequence encoding the sequence identified as SEQ ID NO:Y or the polypeptide encoded by the cDNA sequence included in ATCC Deposit No:Z, which is hybridizable to SEQ ID NO:X.

4. The isolated nucleic acid molecule of claim 1 , wherein the polynucleotide fragment comprises the entire nucleotide sequence of SEQ ID NO:X or the cDNA sequence included in ATCC Deposit No:Z, which is hybridizable to SEQ ID NO:X.

5. The isolated nucleic acid molecule of claim 2, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the C-terminus or the N- terminus.

6. The isolated nucleic acid molecule of claim 3, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the C-terminus or the N- terminus.

7. A recombinant vector comprising the isolated nucleic acid molecule of claim 1.

8. A method of making a recombinant host cell comprising the isolated nucleic acid molecule of claim 1.

9. A recombinant host cell produced by the method of claim 8.

10. The recombinant host cell of claim 9 comprising vector sequences.

11. An isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence selected from the group consisting of:

(a) a polypeptide fragment of SEQ ID NO:Y or the encoded sequence included in ATCC Deposit No:Z;

(b) a polypeptide fragment of SEQ ID NO:Y or the encoded sequence included in ATCC Deposit No:Z, having biological activity;

(c) a polypeptide domain of SEQ ID NO:Y or the encoded sequence included in ATCC Deposit No:Z; (d) a polypeptide epitope of SEQ ID NO:Y or the encoded sequence included in ATCC Deposit No:Z;

(e) a secreted form of SEQ ID NO:Y or the encoded sequence included in ATCC Deposit No:Z;

(f) a full length protein of SEQ ID NO:Y or the encoded sequence included in ATCC Deposit No:Z;

(g) a variant of SEQ ID NO:Y;

(h) an allelic variant of SEQ ID NO:Y; or (i) a species homologue of the SEQ ID NO:Y.

12. The isolated polypeptide of claim 1 1 , wherein the secreted form or the full length protein comprises sequential amino acid deletions from either the C-terminus or the N-terminus.

13. An isolated antibody that binds specifically to the isolated polypeptide of claim 1 1.

14. A recombinant host cell that expresses the isolated polypeptide of claim 11.

15. A method of making an isolated polypeptide comprising:

(a) culturing the recombinant host cell of claim 14 under conditions such that said polypeptide is expressed; and

(b) recovering said polypeptide.

16. The polypeptide produced by claim 15.

17. A method for preventing, treating, or ameliorating a medical condition, comprising administering to a mammalian subject a therapeutically effective amount of the polypeptide of claim 11 or the polynucleotide of claim 1.

18. A method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising:

(a) determining the presence or absence of a mutation in the polynucleotide of claim 1 ; and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or absence of said mutation.

19. A method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising:

(a) determining the presence or amount of expression of the polypeptide of claim 11 in a biological sample; and

(b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or amount of expression of the polypeptide.

20. A method for identifying a binding partner to the polypeptide of claim 1 1 comprising:

(a) contacting the polypeptide of claim 1 1 with a binding partner; and

(b) determining whether the binding partner effects an activity of the polypeptide.

21. The gene corresponding to the cDNA sequence of SEQ ID NO:Y.

22. A method of identifying an activity in a biological assay, wherein the method comprises:

(a) expressing SEQ ID NO:X in a cell;

(b) isolating the supernatant;

(c) detecting an activity in a biological assay; and

(d) identifying the protein in the supernatant having the activity.

23. The product produced by the method of claim 20.