SECRETED AND TRANSMEMBRANE POLYPEPTIDES AND NUCLEIC ACIDS ENCODING THE SAME

POLYPEPTIDES SECRETES ET TRANSMEMBRANAIRES ET ACIDES NUCLEIQUES CODANT POUR CEUX-CI

English Abstract

The present invention is directed to novel polypeptides and to nucleic acid
molecules encoding those polypeptides. Also provided herein are vectors and
host cells comprising those nucleic acid sequences, chimeric polypeptide
molecules comprising the polypeptides of the present invention fused to
heterologous polypeptide sequences, antibodies which bind to the polypeptides
of the present invention and to methods for producing the polypeptides of the
present invention.

French Abstract

L'invention concerne des nouveaux polypeptides et des molécules d'acides nucléiques codant pour lesdits polypeptides. L'invention porte également sur des vecteurs et des cellules hôtes comprenant lesdites séquences nucléotidiques, sur des molécules de polypeptides chimères comprenant les polypeptides de l'invention fusionnés à des séquences polypeptidiques hétérologues, sur des anticorps se liant aux polypeptides de l'invention et sur des procédés de production des polypeptides de l'invention.

Claims

WHAT IS CLAIMED IS:

1. Isolated nucleic acid having at least 80% nucleic acid sequence identity to
a nucleotide sequence
that encodes an amino acid sequence selected from the group consisting of the
amino acid sequence shown in
Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:4), Figure 6 (SEQ ID NO:6), Figure
8 (SEQ ID NO:8), Figure
(SEQ ID NO:10), Figure 12 (SEQ ID NO:12), Figure 14 (SEQ ID NO:14), Figure 16
(SEQ ID NO:16),
Figure 18 (SEQ ID NO:18), Figure 20 (SEQ ID NO:20), Figure 22 (SEQ ID NO:22),
Figure 24 (SEQ ID
NO:24), Figure 26 (SEQ ID NO:26), Figure 28 (SEQ ID NO:28), Figure 30 (SEQ ID
NO:30), Figure 32 (SEQ
ID NO:32), Figure 34 (SEQ ID NO:34), Figure 36 (SEQ ID NO:36), Figure 38 (SEQ
ID NO:38), Figure 40
(SEQ ID NO:40), Figure 42 (SEQ ID NO:42), Figure 44 (SEQ ID NO:44), Figure 46
(SEQ ID NO:46), Figure
48 (SEQ ID NO:48), Figure 50 (SEQ ID NO:50), Figure 52 (SEQ ID NO:52), Figure
54 (SEQ ID NO:54),
Figure 56 (SEQ ID NO:56), Figure 58 (SEQ ID NO:58), Figure 60 (SEQ ID NO:60),
Figure 62 (SEQ ID
NO:62), Figure 64 (SEQ ID NO:64), Figure 66 (SEQ ID NO:66), Figure 68 (SEQ ID
NO:68), Figure 70 (SEQ
ID NO:70), Figure 72 (SEQ ID NO:72), Figure 74 (SEQ ID NO:74), Figure 76 (SEQ
ID NO:76), Figure 78
(SEQ ID NO:78), Figure 80 (SEQ ID NO:80), Figure 82 (SEQ ID NO:82), Figure 84
(SEQ ID NO:84), Figure
86 (SEQ ID NO:86), Figure 88 (SEQ ID NO:88), Figure 90 (SEQ ID NO:90), Figure
92 (SEQ ID NO:92),
Figure 94 (SEQ ID NO:94), Figure 96 (SEQ ID NO:96), Figure 98 (SEQ ID NO:98),
Figure 100 (SEQ ID
NO:100), Figure 102 (SEQ ID NO:102), Figure 104 (SEQ ID NO:104), Figure 106
(SEQ ID NO:106), Figure
108 (SEQ ID NO:108), Figure 110 (SEQ ID NO:110), Figure 112 (SEQ ID NO:112),
Figure 114 (SEQ ID
NO:114), Figure 116 (SEQ ID NO:116), Figure 118 (SEQ ID NO:118), Figure 120
(SEQ ID NO:120), Figure
122 (SEQ ID NO:122), Figure 124 (SEQ ID NO:124), Figure 126 (SEQ ID NO:126),
Figure 128 (SEQ ID
NO:128), Figure 130 (SEQ ID NO:130), Figure 132 (SEQ ID NO:132), Figure 134
(SEQ ID NO:134), Figure
136 (SEQ ID NO:136), Figure 138 (SEQ ID NO:138), Figure 140 (SEQ ID NO:140),
Figure 142 (SEQ ID
NO:142), Figure 144 (SEQ ID NO:144), Figure 146 (SEQ ID NO:146), Figure 148
(SEQ ID NO:148), Figure
150 (SEQ ID NO:150), Figure 152 (SEQ ID NO:152), Figure 154 (SEQ ID NO:154),
Figure 156 (SEQ ID
NO:156), Figure 158 (SEQ ID NO:158), Figure 160 (SEQ ID NO:160), Figure 162
(SEQ ID NO:162), Figure
164 (SEQ ID NO:164), Figure 166 (SEQ ID NO:166) and Figure 168 (SEQ ID
NO:168).

2.Isolated nucleic acid having at least 80% nucleic acid sequence identity to
a nucleotide sequence
selected from the group consisting of the nucleotide sequence shown in Figure
1 (SEQ ID NO:1 ), Figure 3 (SEQ
ID NO:3), Figure 5 (SEQ ID NO:5), Figure 7 (SEQ ID NO:7), Figure 9 (SEQ ID
NO:9), Figure 11 (SEQ ID
NO:11), Figure 13 (SEQ ID NO:13), Figure 15 (SEQ ID NO:15), Figure 17 (SEQ ID
NO:17), Figure 19 (SEQ
ID NO:19), Figure 21 (SEQ ID NO:21), Figure 23 (SEQ ID NO:23), Figure 25 (SEQ
ID NO:25), Figure 27
(SEQ ID NO:27), Figure 29 (SEQ ID NO:29), Figure 31 (SEQ ID NO:31), Figure 33
(SEQ ID NO:33), Figure
35 (SEQ ID NO:35), Figure 37 (SEQ ID NO:37), Figure 39 (SEQ ID NO:39), Figure
41 (SEQ ID NO:41),
Figure 43 (SEQ ID NO:43), Figure 45 (SEQ ID NO:45), Figure 47 (SEQ ID NO:47),
Figure 49 (SEQ ID
NO:49), Figure 51 (SEQ ID NO:51), Figure 53 (SEQ ID NO:53), Figure 55 (SEQ ID
NO:55), Figure 57 (SEQ
ID NO:57), Figure 59 (SEQ ID NO:59), Figure 61 (SEQ ID NO:61), Figure 63 (SEQ
ID NO:63), Figure 65
(SEQ ID NO:65), Figure 67 (SEQ ID NO:67), Figure 69 (SEQ ID NO:69), Figure 71
(SEQ ID NO:71), Figure

99



73 (SEQ ID NO:73), Figure 75 (SEQ ID NO:75), Figure 77 (SEQ ID NO:77), Figure
79 (SEQ ID NO:79),
Figure 81 (SEQ ID NO:81), Figure 83 (SEQ ID NO:83), Figure 85 (SEQ ID NO:85),
Figure 87 (SEQ ID
NO:87), Figure 89 (SEQ ID NO:89), Figure 91 (SEQ ID NO:91), Figure 93 (SEQ ID
NO:93), Figure 95 (SEQ
ID NO:95), Figure 97 (SEQ ID NO:97), Figure 99 (SEQ ID NO:99), Figure 101 (SEQ
ID NO:101), Figure
103 (SEQ ID NO:103), Figure 105 (SEQ ID NO:105), Figure 107 (SEQ ID NO:107),
Figure 109 (SEQ ID
NO:109), Figure 111 (SEQ ID NO:111), Figure 113 (SEQ ID NO:113), Figure 115
(SEQ ID NO:115), Figure
117 (SEQ ID NO:117), Figure 119 (SEQ ID NO:119), Figure 121 (SEQ ID NO:121),
Figure 123 (SEQ ID
NO:123), Figure 125 (SEQ ID NO:125), Figure 127 (SEQ ID NO:127), Figure 129
(SEQ ID NO:129), Figure
131 (SEQ ID NO:131), Figure 133 (SEQ ID NO:133), Figure 135 (SEQ ID NO:135),
Figure 137 (SEQ ID
NO:137), Figure 139 (SEQ ID NO:1390), Figure 141 (SEQ ID NO:141), Figure 143
(SEQ ID NO:143), Figure
145 (SEQ ID NO:145), Figure 147 (SEQ ID NO:147), Figure 149 (SEQ ID NO:149),
Figure 151 (SEQ ID
NO:151), Figure 153 (SEQ ID NO:153), Figure 155 (SEQ ID NO:155), Figure 157
(SEQ ID NO:157), Figure
159 (SEQ ID NO:159), Figure 161 (SEQ ID NO:161), Figure 163 (SEQ ID NO:163),
Figure 165 (SEQ ID
NO:165) and Figure 167 (SEQ ID NO:167).

3. Isolated nucleic acid having at least 80% nucleic acid sequence identity to
a nucleotide sequence
selected from the group consisting of the full-length coding sequence of the
nucleotide sequence shown in Figure
1 (SEQ ID NO:1), Figure 3 (SEQ ID NO:3), Figure 5 (SEQ ID NO:5), Figure 7 (SEQ
ID NO:7), Figure 9
(SEQ ID NO:9), Figure 11 (SEQ ID NO:11), Figure 13 (SEQ ID NO:13), Figure 15
(SEQ ID NO:15), Figure
17 (SEQ ID NO:17), Figure 19 (SEQ ID NO:19), Figure 21 (SEQ ID NO:21), Figure
23 (SEQ ID NO:23),
Figure 25 (SEQ ID NO:25), Figure 27 (SEQ ID NO:27), Figure 29 (SEQ ID NO:29),
Figure 31 (SEQ ID
NO:31), Figure 33 (SEQ ID NO:33), Figure 35 (SEQ ID NO:35), Figure 37 (SEQ ID
NO:37), Figure 39 (SEQ
ID NO:39), Figure 41 (SEQ ID NO:41), Figure 43 (SEQ ID NO:43), Figure 45 (SEQ
ID NO:45), Figure 47
(SEQ ID NO:47), Figure 49 (SEQ ID NO:49), Figure 51 (SEQ ID NO:51), Figure 53
(SEQ ID NO:53), Figure
55 (SEQ ID NO:55), Figure 57 (SEQ ID NO:57), Figure 59 (SEQ ID NO:59), Figure
61 (SEQ ID NO:61),
Figure 63 (SEQ ID NO:63), Figure 65 (SEQ ID NO:65), Figure 67 (SEQ ID NO:67),
Figure 69 (SEQ ID
NO:69), Figure 71 (SEQ ID NO:71), Figure 73 (SEQ ID NO:73), Figure 75 (SEQ ID
NO:75), Figure 77 (SEQ
ID NO:77), Figure 79 (SEQ ID NO:79), Figure 81 (SEQ ID NO:81), Figure 83 (SEQ
ID NO:83), Figure 85
(SEQ ID NO:85), Figure 87 (SEQ ID NO:87), Figure 89 (SEQ ID NO:89), Figure 91
(SEQ ID NO:91), Figure
93 (SEQ ID NO:93), Figure 95 (SEQ ID NO:95), Figure 97 (SEQ ID NO:97), Figure
99 (SEQ ID NO:99),
Figure 101 (SEQ ID NO:101), Figure 103 (SEQ ID NO:103), Figure 105 (SEQ ID
NO:105), Figure 107 (SEQ
ID NO:107), Figure 109 (SEQ ID NO:109), Figure 111 (SEQ ID NO:111), Figure 113
(SEQ ID NO:113),
Figure 115 (SEQ ID NO:115), Figure 117 (SEQ ID NO:117), Figure 119 (SEQ ID
NO:119), Figure 121 (SEQ
ID NO:121), Figure 123 (SEQ ID NO:123), Figure 125 (SEQ ID NO:125), Figure 127
(SEQ ID NO:127),
Figure 129 (SEQ ID NO:129), Figure 131 (SEQ ID NO:131), Figure 133 (SEQ ID
NO:133), Figure 135 (SEQ
ID NO:135), Figure 137 (SEQ ID NO:137), Figure 139 (SEQ ID NO:1390), Figure
141 (SEQ ID NO:141),
Figure 143 (SEQ ID NO:143), Figure 145 (SEQ ID NO:145), Figure 147 (SEQ ID
NO:147), Figure 149 (SEQ
ID NO:149), Figure 151 (SEQ ID NO:151), Figure 153 (SEQ ID NO:153), Figure 155
(SEQ ID NO:155),

100



Figure 157 (SEQ ID NO:157), Figure 159 (SEQ ID NO:159), Figure 161 (SEQ ID
NO:161), Figure 163 (SEQ
ID NO:163), Figure 165 (SEQ ID NO:165) and Figure 167 (SEQ ID NO:167).

4. Isolated nucleic acid having at least 80 % nucleic acid sequence identity
to the full-length coding
sequence of the DNA deposited under any ATCC accession number shown in Table
7.

5. A vector comprising the nucleic acid of Claim 1.

6. The vector of Claim 5 operably linked to control sequences recognized by a
host cell
transformed with the vector.

7. A host cell comprising the vector of Claim 5.

8. The host cell of Claim 7, wherein said cell is a CHO cell.

9. The host cell of Claim 7, wherein said cell is an E. coli.

10. The host cell of Claim 7, wherein said cell is a yeast cell.

11. A process for producing a PRO polypeptides comprising culturing the host
cell of Claim 7
under conditions suitable for expression of said PRO polypeptide and
recovering said PRO polypeptide from the
cell culture.

12. An isolated polypeptide having at least 80 % amino acid sequence identity
to an amino acid
sequence selected from the group consisting of the amino acid sequence shown
in Figure 2 (SEQ ID NO:2),
Figure 4 (SEQ ID NO:4), Figure 6 (SEQ ID NO:6), Figure 8 (SEQ ID NO:8), Figure
10 (SEQ ID NO:10),
Figure 12 (SEQ ID NO:12), Figure 14 (SEQ ID NO:14), Figure 16 (SEQ ID NO:16),
Figure 18 (SEQ ID
NO:18), Figure 20 (SEQ ID NO:20), Figure 22 (SEQ ID NO:22), Figure 24 (SEQ ID
NO:24), Figure 26 (SEQ
ID NO:26), Figure 28 (SEQ ID NO:28), Figure 30 (SEQ ID NO:30), Figure 32 (SEQ
ID NO:32), Figure 34
(SEQ ID NO:34), Figure 36 (SEQ ID NO:36), Figure 38 (SEQ ID NO:38), Figure 40
(SEQ ID NO:40), Figure
42 (SEQ ID NO:42), Figure 44 (SEQ ID NO:44), Figure 46 (SEQ ID NO:46), Figure
48 (SEQ ID NO:48),
Figure 50 (SEQ ID NO:50), Figure 52 (SEQ ID NO:52), Figure 54 (SEQ ID NO:54),
Figure 56 (SEQ ID
NO:56), Figure 58 (SEQ ID NO:58), Figure 60 (SEQ ID NO:60), Figure 62 (SEQ ID
NO:62), Figure 64 (SEQ
ID NO:64), Figure 66 (SEQ ID NO:66), Figure 68 (SEQ ID NO:68), Figure 70 (SEQ
ID NO:70), Figure 72
(SEQ ID NO:72), Figure 74 (SEQ ID NO:74), Figure 76 (SEQ ID NO:76), Figure 78
(SEQ ID NO:78), Figure
80 (SEQ ID NO:80), Figure 82 (SEQ ID NO:82), Figure 84 (SEQ ID NO:84), Figure
86 (SEQ ID NO:86),
Figure 88 (SEQ ID NO:88), Figure 90 (SEQ ID NO:90), Figure 92 (SEQ ID NO:92),
Figure 94 (SEQ ID
NO:94), Figure 96 (SEQ ID NO:96), Figure 98 (SEQ ID NO:98), Figure 100 (SEQ ID
NO:100), Figure 102

101



(SEQ ID NO:102), Figure 104 (SEQ ID NO:104), Figure 106 (SEQ ID NO:106),
Figure 108 (SEQ ID
NO:108), Figure 110 (SEQ ID NO: 110), Figure 112 (SEQ ID NO:112), Figure 114
(SEQ ID NO:114), Figure
116 (SEQ ID NO:116), Figure 118 (SEQ ID NO:118), Figure 120 (SEQ ID NO:120),
Figure 122 (SEQ ID
NO:122), Figure 124 (SEQ ID NO:124), Figure 126 (SEQ ID NO:126), Figure 128
(SEQ ID NO:128), Figure
130 (SEQ ID NO:130), Figure 132 (SEQ ID NO:132), Figure 134 (SEQ ID NO:134),
Figure 136 (SEQ ID
NO:136), Figure 138 (SEQ ID NO:138), Figure 140 (SEQ ID NO:140), Figure 142
(SEQ ID NO:142), Figure
144 (SEQ ID NO:144), Figure 146 (SEQ ID NO:146), Figure 148 (SEQ ID NO:148),
Figure 150 (SEQ ID
NO:150), Figure 152 (SEQ ID NO:152), Figure 154 (SEQ ID NO:154), Figure 156
(SEQ ID NO:156), Figure
158 (SEQ ID NO:158), Figure 160 (SEQ ID NO:160), Figure 162 (SEQ ID NO:162),
Figure 164 (SEQ ID
NO:164), Figure 166 (SEQ ID NO:166) and Figure 168 (SEQ ID NO:168).

13. An isolated polypeptide having at least 80 % amino acid sequence identity
to an amino acid
sequence encoded by the full-length coding sequence of the DNA deposited under
any ATCC accession number
shown in Table 7.

14. A chimeric molecule comprising a polypeptide according to Claim 12 fused
to a heterologous
amino acid sequence.

15. The chimeric molecule of Claim 14, wherein said heterologous amino acid
sequence is an
epitope tag sequence.

16. The chimeric molecule of Claim 14, wherein said heterologous amino acid
sequence is a Fc
region of an immunoglobulin.

17. An antibody which specifically binds to a polypeptide according to Claim
12.

18. The antibody of Claim 17, wherein said antibody is a monoclonal antibody,
a humanized
antibody or a single-chain antibody.

19. Isolated nucleic acid having at least 80 % nucleic acid sequence identity
to:
(a) a nucleotide sequence encoding the polypeptide shown in Figure 2 (SEQ ID
NO:2), Figure 4
(SEQ ID NO:4), Figure 6 (SEQ ID NO:6), Figure 8 (SEQ ID NO:8), Figure 10 (SEQ
ID NO:10), Figure 12
(SEQ ID NO:12), Figure 14 (SEQ ID NO:14), Figure 16 (SEQ ID NO:16), Figure 18
(SEQ ID NO:18), Figure
20 (SEQ ID NO:20), Figure 22 (SEQ ID NO:22), Figure 24 (SEQ ID NO:24), Figure
26 (SEQ ID NO:26),
Figure 28 (SEQ ID NO:28), Figure 30 (SEQ ID NO:30), Figure 32 (SEQ ID NO:32),
Figure 34 (SEQ ID
NO:34), Figure 36 (SEQ ID NO:36), Figure 38 (SEQ ID NO:38), Figure 40 (SEQ ID
NO:40), Figure 42 (SEQ
ID NO:42), Figure 44 (SEQ ID NO:44), Figure 46 (SEQ ID NO:46), Figure 48 (SEQ
ID NO:48), Figure 50
(SEQ ID NO:50), Figure 52 (SEQ ID NO:52), Figure 54 (SEQ ID NO:54), Figure 56
(SEQ ID NO:56), Figure

102



58 (SEQ ID NO:58), Figure 60 (SEQ ID NO:60), Figure 62 (SEQ ID NO:62), Figure
64 (SEQ ID NO:64),
Figure 66 (SEQ ID NO:66), Figure 68 (SEQ ID NO:68), Figure 70 (SEQ ID NO:70),
Figure 72 (SEQ ID
NO:72), Figure 74 (SEQ ID NO:74), Figure 76 (SEQ ID NO:76), Figure 78 (SEQ ID
NO:78), Figure 80 (SEQ
ID NO:80), Figure 82 (SEQ ID NO:82), Figure 84 (SEQ ID NO:84), Figure 86 (SEQ
ID NO:86), Figure 88
(SEQ ID NO:88), Figure 90 (SEQ ID NO:90), Figure 92 (SEQ ID NO:92), Figure 94
(SEQ ID NO:94), Figure
96 (SEQ ID NO:96), Figure 98 (SEQ ID NO:98), Figure 100 (SEQ ID NO:100),
Figure 102 (SEQ ID NO:102),
Figure 104 (SEQ ID NO:104), Figure 106 (SEQ ID NO:106), Figure 108 (SEQ ID
NO:108), Figure 110 (SEQ
ID NO:110), Figure 112 (SEQ ID NO:112), Figure 114 (SEQ ID NO:114), Figure 116
(SEQ ID NO:116),
Figure 118 (SEQ ID NO:118), Figure 120 (SEQ ID NO:120), Figure 122 (SEQ ID
NO:122), Figure 124 (SEQ
ID NO:124), Figure 126 (SEQ ID NO:126), Figure 128 (SEQ ID NO:128), Figure 130
(SEQ ID NO:130),
Figure 132 (SEQ ID NO:132), Figure 134 (SEQ ID NO:134), Figure 136 (SEQ ID
NO:136), Figure 138 (SEQ
ID NO:138), Figure 140 (SEQ ID NO:140), Figure 142 (SEQ ID NO:142), Figure 144
(SEQ ID NO:144),
Figure 146 (SEQ ID NO:146), Figure 148 (SEQ ID NO:148), Figure 150 (SEQ ID
NO:150), Figure 152 (SEQ
ID NO:152), Figure 154 (SEQ ID NO:154), Figure 156 (SEQ ID NO:156), Figure 158
(SEQ ID NO:158),
Figure 160 (SEQ ID NO:160), Figure 162 (SEQ ID NO:162), Figure 164 (SEQ ID
NO:164), Figure 166 (SEQ
ID NO:166) or Figure 168 (SEQ ID NO:168), lacking its associated signal
peptide;
(b) a nucleotide sequence encoding an extracellular domain of the polypeptide
shown in Figure 2
(SEQ ID NO:2), Figure 4 (SEQ ID NO:4), Figure 6 (SEQ ID NO:6), Figure 8 (SEQ
ID NO:8), Figure 10 (SEQ
ID NO:10), Figure 12 (SEQ ID NO:12), Figure 14 (SEQ ID NO:14), Figure 16 (SEQ
ID NO:16), Figure 18
(SEQ ID NO:18), Figure 20 (SEQ ID NO:20), Figure 22 (SEQ ID NO:22), Figure 24
(SEQ ID NO:24), Figure
26 (SEQ ID NO:26), Figure 28 (SEQ ID NO:28), Figure 30 (SEQ ID NO:30), Figure
32 (SEQ ID NO:32),
Figure 34 (SEQ ID NO:34), Figure 36 (SEQ ID NO:36), Figure 38 (SEQ ID NO:38),
Figure 40 (SEQ ID
NO:40), Figure 42 (SEQ ID NO:42), Figure 44 (SEQ ID NO:44), Figure 46 (SEQ ID
NO:46), Figure 48 (SEQ
ID NO:48), Figure 50 (SEQ ID NO:50), Figure 52 (SEQ ID NO:52), Figure 54 (SEQ
ID NO:54), Figure 56
(SEQ ID NO:56), Figure 58 (SEQ ID NO:58), Figure 60 (SEQ ID NO:60), Figure 62
(SEQ ID NO:62), Figure
64 (SEQ ID NO:64), Figure 66 (SEQ ID NO:66), Figure 68 (SEQ ID NO:68), Figure
70 (SEQ ID NO:70),
Figure 72 (SEQ ID NO:72), Figure 74 (SEQ ID NO:74), Figure 76 (SEQ ID NO:76),
Figure 78 (SEQ ID
NO:78), Figure 80 (SEQ ID NO:80), Figure 82 (SEQ ID NO:82), Figure 84 (SEQ ID
NO:84), Figure 86 (SEQ
ID NO:86), Figure 88 (SEQ ID NO:88), Figure 90 (SEQ ID NO:90), Figure 92 (SEQ
ID NO:92), Figure 94
(SEQ ID NO:94), Figure 96 (SEQ ID NO:96), Figure 98 (SEQ ID NO:98), Figure 100
(SEQ ID NO:100),
Figure 102 (SEQ ID NO:102), Figure 104 (SEQ ID NO:104), Figure 106 (SEQ ID
NO:106), Figure 108 (SEQ
ID NO:108), Figure 110 (SEQ ID NO:110), Figure 112 (SEQ ID NO:112), Figure 114
(SEQ ID NO:114),
Figure 116 (SEQ ID NO:116), Figure 118 (SEQ ID NO:118), Figure 120 (SEQ ID
NO:120), Figure 122 (SEQ
ID NO:122), Figure 124 (SEQ ID NO:124), Figure 126 (SEQ ID NO:126), Figure 128
(SEQ ID NO:128),
Figure 130 (SEQ ID NO:130), Figure 132 (SEQ ID NO:132), Figure 134 (SEQ ID
NO:134), Figure 136 (SEQ
ID NO:136), Figure 138 (SEQ ID NO:138), Figure 140 (SEQ ID NO:140), Figure 142
(SEQ ID NO:142),
Figure 144 (SEQ ID NO:144), Figure 146 (SEQ ID NO:146), Figure 148 (SEQ ID
NO:148), Figure 150 (SEQ
ID NO:150), Figure 152 (SEQ ID NO:152), Figure 154 (SEQ ID NO:154), Figure 156
(SEQ ID NO:156),

103



Figure 158 (SEQ ID NO:158), Figure 160 (SEQ ID NO:160), Figure 162 (SEQ ID
NO:162), Figure 164 (SEQ
ID NO:164), Figure 166 (SEQ ID NO:166) or Figure 168 (SEQ ID NO:168), with its
associated signal peptide;
or
(c) a nucleotide sequence encoding an extracellular domain of the polypeptide
shown in Figure 2
(SEQ ID NO:2), Figure 4 (SEQ ID NO:4), Figure 6 (SEQ ID NO:6), Figure 8 (SEQ
ID NO:8), Figure 10 (SEQ
ID NO:10), Figure 12 (SEQ ID NO:12), Figure 14 (SEQ ID NO:14), Figure 16 (SEQ
ID NO:16), Figure 18
(SEQ ID NO:18), Figure 20 (SEQ ID NO:20), Figure 22 (SEQ ID NO:22), Figure 24
(SEQ ID NO:24), Figure
26 (SEQ ID NO:26), Figure 28 (SEQ ID NO:28), Figure 30 (SEQ ID NO:30), Figure
32 (SEQ ID NO:32),
Figure 34 (SEQ ID NO:34), Figure 36 (SEQ ID NO:36), Figure 38 (SEQ ID NO:38),
Figure 40 (SEQ ID
NO:40), Figure 42 (SEQ ID NO:42), Figure 44 (SEQ ID NO:44), Figure 46 (SEQ ID
NO:46), Figure 48 (SEQ
ID NO:48), Figure 50 (SEQ ID NO:50), Figure 52 (SEQ ID NO:52), Figure 54 (SEQ
ID NO:54), Figure 56
(SEQ ID NO:56), Figure 58 (SEQ ID NO:58), Figure 60 (SEQ ID NO:60), Figure 62
(SEQ ID NO:62), Figure
64 (SEQ ID NO:64), Figure 66 (SEQ ID NO:66), Figure 68 (SEQ ID NO:68), Figure
70 (SEQ ID NO:70),
Figure 72 (SEQ ID NO:72), Figure 74 (SEQ ID NO:74), Figure 76 (SEQ ID NO:76),
Figure 78 (SEQ ID
NO:78), Figure 80 (SEQ ID NO:80), Figure 82 (SEQ ID NO:82), Figure 84 (SEQ ID
NO:84), Figure 86 (SEQ
ID NO:86), Figure 88 (SEQ ID NO:88), Figure 90 (SEQ ID NO:90), Figure 92 (SEQ
ID NO:92), Figure 94
(SEQ ID NO:94), Figure 96 (SEQ ID NO:96), Figure 98 (SEQ ID NO:98), Figure 100
(SEQ ID NO:100),
Figure 102 (SEQ ID NO:102), Figure 104 (SEQ ID NO:104), Figure 106 (SEQ ID
NO:106), Figure 108 (SEQ
ID NO:108), Figure 110 (SEQ ID NO:110), Figure 112 (SEQ ID NO:112), Figure 114
(SEQ ID NO:114),
Figure 116 (SEQ ID NO:116), Figure 118 (SEQ ID NO:118), Figure 120 (SEQ ID
NO:120), Figure 122 (SEQ
ID NO:122), Figure 124 (SEQ ID NO:124), Figure 126 (SEQ ID NO:126), Figure 128
(SEQ ID NO:128),
Figure 130 (SEQ ID NO:130), Figure 132 (SEQ ID NO:132), Figure 134 (SEQ ID
NO:134), Figure 136 (SEQ
ID NO:136), Figure 138 (SEQ ID NO:138), Figure 140 (SEQ ID NO:140), Figure 142
(SEQ ID NO:142),
Figure 144 (SEQ ID NO:144), Figure 146 (SEQ ID NO:146), Figure 148 (SEQ ID
NO:148), Figure 150 (SEQ
ID NO:150), Figure 152 (SEQ ID NO:152), Figure 154 (SEQ ID NO:154), Figure 156
(SEQ ID NO:156),
Figure 158 (SEQ ID NO:158), Figure 160 (SEQ ID NO:160), Figure 162 (SEQ ID
NO:162), Figure 164 (SEQ
ID NO:164), Figure 166 (SEQ ID NO:166) or Figure 168 (SEQ ID NO:168), lacking
its associated signal
peptide.

20. An isolated polypeptide having at least 80% amino acid sequence identity
to:
(a) an amino acid sequence of the polypeptide shown in Figure 2 (SEQ ID NO:2),
Figure 4 (SEQ
ID NO:4), Figure 6 (SEQ ID NO:6), Figure 8 (SEQ ID NO:8), Figure 10 (SEQ ID
NO:10), Figure 12 (SEQ
ID NO:12), Figure 14 (SEQ ID NO:14), Figure 16 (SEQ ID NO:16), Figure 18 (SEQ
ID NO:18), Figure 20
(SEQ ID NO:20), Figure 22 (SEQ ID NO:22), Figure 24 (SEQ ID NO:24), Figure 26
(SEQ ID NO:26), Figure
28 (SEQ ID NO:28), Figure 30 (SEQ ID NO:30), Figure 32 (SEQ ID NO:32), Figure
34 (SEQ ID NO:34),
Figure 36 (SEQ ID NO:36), Figure 38 (SEQ ID NO:38), Figure 40 (SEQ ID NO:40),
Figure 42 (SEQ ID
NO:42), Figure 44 (SEQ ID NO:44), Figure 46 (SEQ ID NO:46), Figure 48 (SEQ ID
NO:48), Figure 50 (SEQ
ID NO:50), Figure 52 (SEQ ID NO:52), Figure 54 (SEQ ID NO:54), Figure 56 (SEQ
ID NO:56), Figure 58

104




(SEQ ID NO:58), Figure 60 (SEQ ID NO:60), Figure 62 (SEQ ID NO:62), Figure 64
(SEQ ID NO:64), Figure
66 (SEQ ID NO:66), Figure 68 (SEQ ID NO:68), Figure 70 (SEQ ID NO:70), Figure
72 (SEQ ID NO:72),
Figure 74 (SEQ ID NO:74), Figure 76 (SEQ ID NO:76), Figure 78 (SEQ ID NO:78),
Figure 80 (SEQ ID
NO:80), Figure 82 (SEQ ID NO:82), Figure 84 (SEQ ID NO:84), Figure 86 (SEQ ID
NO:86), Figure 88 (SEQ
ID NO:88), Figure 90 (SEQ ID NO:90), Figure 92 (SEQ ID NO:92), Figure 94 (SEQ
ID NO:94), Figure 96
(SEQ ID NO:96), Figure 98 (SEQ ID NO:98), Figure 100 (SEQ ID NO:100), Figure
102 (SEQ ID NO:102),
Figure 104 (SEQ ID NO:104), Figure 106 (SEQ ID NO:106), Figure 108 (SEQ ID
NO:108), Figure 110 (SEQ
ID NO:110), Figure 112 (SEQ ID NO:112), Figure 114 (SEQ ID NO:114), Figure 116
(SEQ ID NO:116),
Figure 118 (SEQ ID NO:118), Figure 120 (SEQ ID NO:120), Figure 122 (SEQ ID
NO:122), Figure 124 (SEQ
ID NO:124), Figure 126 (SEQ ID NO:126), Figure 128 (SEQ ID NO:128), Figure 130
(SEQ ID NO:130),
Figure 132 (SEQ ID NO:132), Figure 134 (SEQ ID NO:134), Figure 136 (SEQ ID
NO:136), Figure 138 (SEQ
ID NO:138), Figure 140 (SEQ ID NO:140), Figure 142 (SEQ ID NO:142), Figure 144
(SEQ ID NO:144),
Figure 146 (SEQ ID NO:146), Figure 148 (SEQ ID NO:148), Figure 150 (SEQ ID
NO:150), Figure 152 (SEQ
ID NO:152), Figure 154 (SEQ ID NO:154), Figure 156 (SEQ ID NO:156), Figure 158
(SEQ ID NO:158),
Figure 160 (SEQ ID NO:160), Figure 162 (SEQ ID NO:162), Figure 164 (SEQ ID
NO:164), Figure 166 (SEQ
ID NO:166) or Figure 168 (SEQ ID NO:168), lacking its associated signal
peptide;

(b) an amino acid sequence of an extracellular domain of the polypeptide shown
in Figure 2 (SEQ
ID NO:2), Figure 4 (SEQ ID NO:4), Figure 6 (SEQ ID NO:6), Figure 8 (SEQ ID
NO:8), Figure 10 (SEQ ID
NO:10), Figure 12 (SEQ ID NO:12), Figure 14 (SEQ ID NO:14), Figure 16 (SEQ ID
NO:16), Figure 18 (SEQ
ID NO:18), Figure 20 (SEQ ID NO:20), Figure 22 (SEQ ID NO:22), Figure 24 (SEQ
ID NO:24), Figure 26
(SEQ ID NO:26), Figure 28 (SEQ ID NO:28), Figure 30 (SEQ ID NO:30), Figure 32
(SEQ ID NO:32), Figure
34 (SEQ ID NO:34), Figure 36 (SEQ ID NO:36), Figure 38 (SEQ ID NO:38), Figure
40 (SEQ ID NO:40),
Figure 42 (SEQ ID NO:42), Figure 44 (SEQ ID NO:44), Figure 46 (SEQ ID NO:46),
Figure 48 (SEQ ID
NO:48), Figure 50 (SEQ ID NO:50), Figure 52 (SEQ ID NO:52), Figure 54 (SEQ ID
NO:54), Figure 56 (SEQ
ID NO:56), Figure 58 (SEQ ID NO:58), Figure 60 (SEQ ID NO:60), Figure 62 (SEQ
ID NO:62), Figure 64
(SEQ ID NO:64), Figure 66 (SEQ ID NO:66), Figure 68 (SEQ ID NO:68), Figure 70
(SEQ ID NO:70), Figure
72 (SEQ ID NO:72), Figure 74 (SEQ ID NO:74), Figure 76 (SEQ ID NO:76), Figure
78 (SEQ ID NO:78),
Figure 80 (SEQ ID NO:80), Figure 82 (SEQ ID NO:82), Figure 84 (SEQ ID NO:84),
Figure 86 (SEQ ID
NO:86), Figure 88 (SEQ ID NO:88), Figure 90 (SEQ ID NO:90), Figure 92 (SEQ ID
NO:92), Figure 94 (SEQ
ID NO:94), Figure 96 (SEQ ID NO:96), Figure 98 (SEQ ID NO:98), Figure 100 (SEQ
ID NO:100), Figure
102 (SEQ ID NO:102), Figure 104 (SEQ ID NO:104), Figure 106 (SEQ ID NO:106),
Figure 108 (SEQ ID
NO:108), Figure 110 (SEQ ID NO:110), Figure 112 (SEQ ID NO:112), Figure 114
(SEQ ID NO:114), Figure
116 (SEQ ID NO:116), Figure 118 (SEQ ID NO:118), Figure 120 (SEQ ID NO:120),
Figure 122 (SEQ ID
NO:122), Figure 124 (SEQ ID NO:124), Figure 126 (SEQ ID NO:126), Figure 128
(SEQ ID NO:128), Figure
130 (SEQ ID NO:130), Figure 132 (SEQ ID NO:132), Figure 134 (SEQ ID NO:134),
Figure 136 (SEQ ID
NO:136), Figure 138 (SEQ ID NO:138), Figure 140 (SEQ ID NO:140), Figure 142
(SEQ ID NO:142), Figure
144 (SEQ ID NO:144), Figure 146 (SEQ ID NO:146), Figure 148 (SEQ ID NO:148),
Figure 150 (SEQ ID
NO:150), Figure 152 (SEQ ID NO:152), Figure 154 (SEQ ID NO:154), Figure 156
(SEQ ID NO:156), Figure

105



158 (SEQ ID NO:158), Figure 160 (SEQ ID NO:160), Figure 162 (SEQ ID NO:162),
Figure 164 (SEQ ID
NO:164), Figure 166 (SEQ ID NO:166) or Figure 168 (SEQ ID NO:168), with its
associated signal peptide;
or
(c) an amino acid sequence of an extracellular domain of the polypeptide shown
in Figure 2 (SEQ
ID NO:2), Figure 4 (SEQ ID NO:4), Figure 6 (SEQ ID NO:6), Figure 8 (SEQ ID
NO:8), Figure 10 (SEQ ID
NO:10), Figure 12 (SEQ ID NO:12), Figure 14 (SEQ ID NO:14), Figure 16 (SEQ ID
NO:16), Figure 18 (SEQ
ID NO:18), Figure 20 (SEQ ID NO:20), Figure 22 (SEQ ID NO:22), Figure 24 (SEQ
ID NO:24), Figure 26
(SEQ ID NO:26), Figure 28 (SEQ ID NO:28), Figure 30 (SEQ ID NO:30), Figure 32
(SEQ ID NO:32), Figure
34 (SEQ ID NO:34), Figure 36 (SEQ ID NO:36), Figure 38 (SEQ ID NO:38), Figure
40 (SEQ ID NO:40),
Figure 42 (SEQ ID NO:42), Figure 44 (SEQ ID NO:44), Figure 46 (SEQ ID NO:46),
Figure 48 (SEQ ID
NO:48), Figure 50 (SEQ ID NO:50), Figure 52 (SEQ ID NO:52), Figure 54 (SEQ ID
NO:54), Figure 56 (SEQ
ID NO:56), Figure 58 (SEQ ID NO:58), Figure 60 (SEQ ID NO:60), Figure 62 (SEQ
ID NO:62), Figure 64
(SEQ ID NO:64), Figure 66 (SEQ ID NO:66), Figure 68 (SEQ ID NO:68), Figure 70
(SEQ ID NO:70), Figure
72 (SEQ ID NO:72), Figure 74 (SEQ ID NO:74), Figure 76 (SEQ ID NO:76), Figure
78 (SEQ ID NO:78),
Figure 80 (SEQ ID NO:80), Figure 82 (SEQ ID NO:82), Figure 84 (SEQ ID NO:84),
Figure 86 (SEQ ID
NO:86), Figure 88 (SEQ ID NO:88), Figure 90 (SEQ ID NO:90), Figure 92 (SEQ ID
NO:92), Figure 94 (SEQ
ID NO:94), Figure 96 (SEQ ID NO:96), Figure 98 (SEQ ID NO:98), Figure 100 (SEQ
ID NO:100), Figure
102 (SEQ ID NO:102), Figure 104 (SEQ ID NO:104), Figure 106 (SEQ ID NO:106),
Figure 108 (SEQ ID
NO:108), Figure 110 (SEQ ID NO:110), Figure 112 (SEQ ID NO:112), Figure 114
(SEQ ID NO:114), Figure
116 (SEQ ID NO:116), Figure 118 (SEQ ID NO:118), Figure 120 (SEQ ID NO:120),
Figure 122 (SEQ ID
NO:122), Figure 124 (SEQ ID NO:124), Figure 126 (SEQ ID NO:126), Figure 128
(SEQ ID NO:128), Figure
130 (SEQ ID NO:130), Figure 132 (SEQ ID NO:132), Figure 134 (SEQ ID NO:134),
Figure 136 (SEQ ID
NO:136), Figure 138 (SEQ ID NO:138), Figure 140 (SEQ ID NO:140), Figure 142
(SEQ ID NO:142), Figure
144 (SEQ ID NO:144), Figure 146 (SEQ ID NO:146), Figure 148 (SEQ ID NO:148),
Figure 150 (SEQ ID
NO:150), Figure 152 (SEQ ID NO:152), Figure 154 (SEQ ID NO:154), Figure 156
(SEQ ID NO:156), Figure
158 (SEQ ID NO:158), Figure 160 (SEQ ID NO:160), Figure 162 (SEQ ID NO:162),
Figure 164 (SEQ ID
NO:164), Figure 166 (SEQ ID NO:166) or Figure 168 (SEQ ID NO:168), lacking its
associated signal peptide.

21. A method of detecting a polypeptide designated as A, B, C or D in a sample
suspected of
containing an A, B, C or D polypeptide, said method comprising contacting said
sample with a polypeptide
designated herein as E, F, G, H or I and determining the formation of a A/E,
B/F, B/G, C/H or D/I polypeptide
conjugate in said sample, wherein the formation of said conjugate is
indicative of the presence of an A, B, C or
D polypeptide in said sample and wherein A is a PRO10272 polypeptide, B is a
PRO20110 polypeptide, C is
a PRO10096 polypeptide, D is a PRO19670 polypeptide, E is a PRO5801
polypeptide, F is a PRO1 polypeptide,
G is a PRO20040 polypeptide, H is a PRO20233 polypeptide and I is a PRO1890
polypeptide.

22. The method according to Claim 21, wherein said sample comprises cells
suspected of
expressing said A, B, C or D polypeptide.

106



23. The method according to Claim 21, wherein said E, F, G, H or I polypeptide
is labeled with
a detectable label.

24. The method according to Claim 21, wherein said E, F, G, H or I polypeptide
is attached to
a solid support.

25. A method of detecting a polypeptide designated as E, F, G, H or I in a
sample suspected of
containing an E, F, G, H or I polypeptide, said method comprising contacting
said sample with a polypeptide
designated herein as A, B, C or D and determining the formation of a A/E, B/F,
B/G, C/H or D/I polypeptide
conjugate in said sample, wherein the formation of said conjugate is
indicative of the presence of an A, B, C or
D polypeptide in said sample and wherein A is a PRO10272 polypeptide, B is a
PRO20110 polypeptide, C is
a PRO10096 polypeptide, D is a PRO19670 polypeptide, E is a PRO5801
polypeptide, F is a PRO1 polypeptide,
G is a PRO20040 polypeptide, H is a PRO20233 polypeptide and I is a PRO1890
polypeptide.

26. The method according to Claim 25, wherein said sample comprises cells
suspected of
expressing said E, F, G, H or I polypeptide.

27. The method according to Claim 25, wherein said A, B, C or D polypeptide is
labeled with a
detectable label.

28. The method according to Claim 25, wherein said A, B, C or D polypeptide is
attached to a
solid support.

29. A method of linking a bioactive molecule to a cell expressing a
polypeptide designated as A,
B, C or D, said method comprising contacting said cell with a polypeptide
designated as E, F, G, H or I that
is bound to said bioactive molecule and allowing said A, B, C or D and said E,
F, G, H or I polypeptides to bind
to one another, thereby linking said bioactive molecules to said cell, wherein
A is a PRO10272 polypeptide, B
is a PRO20110 polypeptide, C is a PRO10096 polypeptide, D is a PRO19670
polypeptide, E is a PRO5801
polypeptide, F is a PRO1 polypeptide, G is a PRO20040 polypeptide, H is a
PRO20233 polypeptide and I is a
PRO1890 polypeptide.

30. The method according to Claim 29, wherein said bioactive molecule is a
toxin, a radiolabel
or an antibody.

31. The method according to Claim 29, wherein said bioactive molecule causes
the death of said
cell.


107



32. A method of linking a bioactive molecule to a cell expressing a
polypeptide designated as E,
F, G, H or I, said method comprising contacting said cell with a polypeptide
designated as A, B, C or D that
is bound to said bioactive molecule and allowing said A, B, C or D and said E,
F, G, H or I polypeptides to bind
to one another, thereby linking said bioactive molecules to said cell, wherein
A is a PRO10272 polypeptide, B
is a PRO20110 polypeptide, C is a PRO10096 polypeptide, D is a PRO19670
polypeptide, E is a PRO5801
polypeptide, F is a PRO1 polypeptide, G is a PRO20040 polypeptide, H is a
PR020233 polypeptide and I is a
PRO1890 polypeptide.
33. The method according to Claim 32, wherein said bioactive molecule is a
toxin, a radiolabel
or an antibody.
34. The method according to Claim 32, wherein said bioactive molecule causes
the death of said
cell.
35. A method of modulating at least one biological activity of a cell
expressing a polypeptide
designated as A, B, C or D, said method comprising contacting said cell with a
polypeptide designated as E, F,
G, H or I or an anti-A, B, C or D polypeptide antibody, whereby said E, F, G,
H or I polypeptide or anti-A,
B, C or D polypeptide antibody binds to said A, B, C or D polypeptide, thereby
modulating at least one
biological activity of said cell, wherein A is a PRO10272 polypeptide, B is a
PRO20110 polypeptide, C is a
PRO10096 polypeptide, D is a PRO19670 polypeptide, E is a PRO5801 polypeptide,
F is a PRO1 polypeptide,
G is a PRO20040 polypeptide, H is a PRO20233 polypeptide and I is a PR01890
polypeptide.
36. The method according to Claim 35, wherein said cell is killed.
37. A method of modulating at least one biological activity of a cell
expressing a polypeptide
designated as E, F, G, H or I, said method comprising contacting said cell
with a polypeptide designated as A,
B, C or D or an anti-E, F, G, H or I polypeptide antibody, whereby said A, B,
C or D polypeptide or anti-E,
F, G, H or I polypeptide antibody binds to said E, F, G, H or I polypeptide,
thereby modulating at least one
biological activity of said cell, wherein A is a PRO10272 polypeptide, B is a
PRO20110 polypeptide, C is a
PRO10096 polypeptide, D is a PRO19670 polypeptide, E is a PRO5801 polypeptide,
F is a PRO1 polypeptide,
G is a PRO20040 polypeptide, H is a PRO20233 polypeptide and I is a PRO1890
polypeptide.
38. The method according to Claim 37, wherein said cell is killed.
108

Description

CA 02380355 2002-03-O1
WO 01/16318 PCT/US00/23328
SECRETED AND TRANSMEMBRANE POLYPEPTIDES AND NUCLEIC ACIDS ENCODING THE
SAME
FIELD OF THE INVENTION
The present invention relates generally to the identification and isolation of
novel DNA and to the
recombinant production of novel polypeptides.
BACKGROUND OF THE INVENTION
Extracellular proteins play important roles in, among other things, the
formation, differentiation and
maintenance of multicellular organisms. The fate of many individual cells,
e.g., proliferation, migration,
differentiation, or interaction with other cells, is typically governed by
information received from other cells
and/or the immediate environment. This information is often transmitted by
secreted polypeptides (for instance,
mitogenic factors, survival factors, cytotoxic factors, differentiation
factors, neuropeptides, and hormones) which
are, in turn, received and interpreted by diverse cell receptors or membrane-
bound proteins. These secreted
polypeptides or signaling molecules normally pass through the cellular
secretory pathway to reach their site of
action in the extracellular environment.
Secreted proteins have various industrial applications, including as
pharmaceuticals, diagnostics,
biosensors and bioreactors. Most protein drugs available at present, such as
thrombolytic agents, interferons,
interleukins, erythropoietins, colony stimulating factors, and various other
cytokines, are secretory proteins.
Their receptors, which are membrane proteins, also have potential as
therapeutic or diagnostic agents. Efforts
are being undertaken by both industry and academia to identify new, native
secreted proteins. Many efforts are
focused on the screening of mammalian recombinant DNA libraries to identify
the coding sequences for novel
secreted proteins. Examples of screening methods and techniques are described
in the literature [see, for
example, Klein et al., Proc. Natl. Acad. Sci. 93:7108-7113 (1996); U.S. Patent
No. 5,536,637)].
Membrane-bound proteins and receptors can play important roles in, among other
things, the formation,
differentiation and maintenance of multicellular organisms. The fate of many
individual cells, e. g. , proliferation,
migration, differentiation, or interaction with other cells, is typically
governed by information received from
other cells and/or the immediate environment. This information is often
transmitted by secreted polypeptides
(for instance, mitogenic factors, survival factors, cytotoxic factors,
differentiation factors, neuropeptides, and
hormones) which are, in turn, received and interpreted by diverse cell
receptors or membrane-bound proteins.
Such membrane-bound proteins and cell receptors include, but are not limited
to, cytokine receptors, receptor
kinases, receptor phosphatases, receptors involved in cell-cell interactions,
and cellular adhesin molecules like
selectins and integrins. For instance, transduction of signals that regulate
cell growth and differentiation is
regulated in part by phosphorylation of various cellular proteins. Protein
tyrosine kinases, enzymes that catalyze
that process, can also act as growth factor receptors. Examples include
fibroblast growth factor receptor and
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nerve growth factor receptor.
Membrane-bound proteins and receptor molecules have various industrial
applications, including as
pharmaceutical and diagnostic agents. Receptor immunoadhesins, for instance,
can be employed as therapeutic
agents to block receptor-ligand interactions. The membrane-bound proteins can
also be employed for screening
of potential peptide or small molecule inhibitors of the relevant
receptor/ligand interaction.
Efforts are being undertaken by both industry and academia to identify new,
native receptor or
membrane-bound proteins. Many efforts are focused on the screening of
mammalian recombinant DNA libraries
to identify the coding sequences for novel receptor or membrane-bound
proteins.
SUMMARY OF THE INVENTION
In one embodiment, the invention provides an isolated nucleic acid molecule
comprising a nucleotide
sequence that encodes a PRO polypeptide.
In one aspect, the isolated nucleic acid molecule comprises a nucleotide
sequence having at least about
80 % nucleic acid sequence identity, alternatively at least about 81 % nucleic
acid sequence identity, alternatively
at least about 82 % nucleic acid sequence identity, alternatively at least
about 83 % nucleic acid sequence identity,
alternatively at least about 84 % nucleic acid sequence identity,
alternatively at least about 85 % nucleic acid
sequence identity, alternatively at least about 86 % nucleic acid sequence
identity, alternatively at least about 87
nucleic acid sequence identity, alternatively at least about 88 % nucleic acid
sequence identity, alternatively at
least about 89% nucleic acid sequence identity, alternatively at least about
90% nucleic acid sequence identity,
alternatively at least about 91 % nucleic acid sequence identity,
alternatively at least about 92 % nucleic acid
sequence identity, alternatively at least about 93 % nucleic acid sequence
identity, alternatively at least about 94 %
nucleic acid sequence identity, alternatively at least about 95 % nucleic acid
sequence identity, alternatively at
least about 96% nucleic acid sequence identity, alternatively at least about
97% nucleic acid sequence identity,
alternatively at least about 98% nucleic acid sequence identity and
alternatively at least about 99% nucleic acid
sequence identity to (a) a DNA molecule encoding a PRO polypeptide having a
full-length amino acid sequence
as disclosed herein, an amino acid sequence lacking the signal peptide as
disclosed herein, an extracellular
domain of a transmembrane protein, with or without the signal peptide, as
disclosed herein or any other
specifically defined fragment of the full-length amino acid sequence as
disclosed herein, or (b) the complement
of the DNA molecule of (a).
In other aspects, the isolated nucleic acid molecule comprises a nucleotide
sequence having at least about
80% nucleic acid sequence identity, alternatively at least about 81 % nucleic
acid sequence identity, alternatively
at least about 82 % nucleic acid sequence identity, alternatively at least
about 83 % nucleic acid sequence identity,
alternatively at least about 84 % nucleic acid sequence identity,
alternatively at least about 85 % nucleic acid
sequence identity, alternatively at least about 86 % nucleic acid sequence
identity, alternatively at least about 87
nucleic acid sequence identity, alternatively at least about 88% nucleic acid
sequence identity, alternatively at
least about 89% nucleic acid sequence identity, alternatively at least about
90% nucleic acid sequence identity,
alternatively at least about 91% nucleic acid sequence identity, alternatively
at least about 92% nucleic acid
sequence identity, alternatively at least about 93 % nucleic acid sequence
identity, alternatively at least about 94
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nucleic acid sequence identity, alternatively at least about 95 % nucleic acid
sequence identity, alternatively at
least about 96 % nucleic acid sequence identity, alternatively at least about
97 % nucleic acid sequence identity,
alternatively at least about 98 % nucleic acid sequence identity and
alternatively at least about 99 % nucleic acid
sequence identity to (a) a DNA molecule comprising the coding sequence of a
full-length PRO polypeptide cDNA
as disclosed herein, the coding sequence of a PRO polypeptide lacking the
signal peptide as disclosed herein,
the coding sequence of an extracellular domain of a transmembrane PRO
polypeptide, with or without the signal
peptide, as disclosed herein or the coding sequence of any other specifically
defined fragment of the full-length
amino acid sequence as disclosed herein, or (b) the complement of the DNA
molecule of (a).
In a further aspect, the invention concerns an isolated nucleic acid molecule
comprising a nucleotide
sequence having at least about 80% nucleic acid sequence identity,
alternatively at least about 81 % nucleic acid
sequence identity, alternatively at least about 82 % nucleic acid sequence
identity, alternatively at least about 83 %
nucleic acid sequence identity, alternatively at least about 84 % nucleic acid
sequence identity, alternatively at
least about 85 % nucleic acid sequence identity, alternatively at least about
86 % nucleic acid sequence identity,
alternatively at least about 87 % nucleic acid sequence identity,
alternatively at least about 88 % nucleic acid
sequence identity, alternatively at least about 89 % nucleic acid sequence
identity, alternatively at least about 90
nucleic acid sequence identity, alternatively at least about 91 % nucleic acid
sequence identity, alternatively at
least about 92 % nucleic acid sequence identity, alternatively at least about
93 % nucleic acid sequence identity,
alternatively at least about 94 % nucleic acid sequence identity,
alternatively at least about 95 % nucleic acid
sequence identity, alternatively at least about 96 % nucleic acid sequence
identity, alternatively at least about 97
nucleic acid sequence identity, alternatively at least about 98 % nucleic acid
sequence identity and alternatively
at least about 99% nucleic acid sequence identity to (a) a DNA molecule that
encodes the same mature
polypeptide encoded by any of the human protein cDNAs deposited with the ATCC
as disclosed herein, or (b)
the complement of the DNA molecule of (a).
Another aspect the invention provides an isolated nucleic acid molecule
comprising a nucleotide
sequence encoding a PRO polypeptide which is either transmembrane domain-
deleted or transmembrane domain-
inactivated, or is complementary to such encoding nucleotide sequence, wherein
the transmembrane domains)
of such polypeptide are disclosed herein. Therefore, soluble extracellular
domains of the herein described PRO
polypeptides are contemplated.
Another embodiment is directed to fragments of a PRO polypeptide coding
sequence, or the complement
thereof, that may fmd use as, for example, hybridization probes, for encoding
fragments of a PRO polypeptide
that may optionally encode a polypeptide comprising a binding site for an anti-
PRO antibody or as antisense
oligonucleotide probes. Such nucleic acid fragments are usually at least about
20 nucleotides in length,
alternatively at least about 30 nucleotides in length, alternatively at least
about 40 nucleotides in length,
alternatively at least about 50 nucleotides in length, alternatively at least
about 60 nucleotides in length,
alternatively at least about 70 nucleotides in length, alternatively at least
about 80 nucleotides in length,
alternatively at least about 90 nucleotides in length, alternatively at least
about 100 nucleotides in length,
alternatively at least about 110 nucleotides in length, alternatively at least
about 120 nucleotides in length,
alternatively at least about 130 nucleotides in length, alternatively at least
about 140 nucleotides in length,
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alternatively at least about 150 nucleotides in length, alternatively at least
about 160 nucleotides in length,
alternatively at least about 170 nucleotides in length, alternatively at least
about 180 nucleotides in length,
alternatively at least about 190 nucleotides in length, alternatively at least
about 200 nucleotides in length,
alternatively at least about 250 nucleotides in length, alternatively at least
about 300 nucleotides in length,
alternatively at least about 350 nucleotides in length, alternatively at least
about 400 nucleotides in length,
alternatively at least about 450 nucleotides in length, alternatively at least
about 500 nucleotides in length,
alternatively at least about 600 nucleotides in length, alternatively at least
about 700 nucleotides in length,
alternatively at least about 800 nucleotides in length, alternatively at least
about 900 nucleotides in length and
alternatively at least about 1000 nucleotides in length, wherein in this
context the term "about" means the
referenced nucleotide sequence length plus or minus 10% of that referenced
length. It is noted that novel
fragments of a PRO polypeptide-encoding nucleotide sequence may be determined
in a routine manner by
aligning the PRO polypeptide-encoding nucleotide sequence with other known
nucleotide sequences using any
of a number of well known sequence alignment programs and determining which
PRO polypeptide-encoding
nucleotide sequence fragments) are novel. All of such PRO polypeptide-encoding
nucleotide sequences are
contemplated herein. Also contemplated are the PRO polypeptide fragments
encoded by these nucleotide
molecule fragments, preferably those PRO polypeptide fragments that comprise a
binding site for an anti-PRO
antibody.
In another embodiment, the invention provides isolated PRO polypeptide encoded
by any of the isolated
nucleic acid sequences hereinabove identified.
In a certain aspect, the invention concerns an isolated PRO polypeptide,
comprising an amino acid
sequence having at least about 80 % amino acid sequence identity,
alternatively at least about 81 % amino acid
sequence identity, alternatively at least about 82 % amino acid sequence
identity, alternatively at least about 83
amino acid sequence identity, alternatively at least about 84 % amino acid
sequence identity, alternatively at least
about 85 % amino acid sequence identity, alternatively at least about 86 %
amino acid sequence identity,
alternatively at least about 87 % amino acid sequence identity, alternatively
at least about 88 % amino acid
sequence identity, alternatively at least about 89 % amino acid sequence
identity, alternatively at least about 90
amino acid sequence identity, alternatively at least about 91 % amino acid
sequence identity, alternatively at least
about 92 % amino acid sequence identity, alternatively at least about 93 %
amino acid sequence identity,
alternatively at least about 94 % amino acid sequence identity, alternatively
at least about 95 % amino acid
sequence identity, alternatively at least about 96 % amino acid sequence
identity, alternatively at least about 97
amino acid sequence identity, alternatively at least about 98% amino acid
sequence identity and alternatively at
least about 99 % amino acid sequence identity to a PRO polypeptide having a
full-length amino acid sequence
as disclosed herein, an amino acid sequence lacking the signal peptide as
disclosed herein, an extracellular
domain of a transmembrane protein, with or without the signal peptide, as
disclosed herein or any other
specifically defined fragment of the full-length amino acid sequence as
disclosed herein.
In a further aspect, the invention concerns an isolated PRO polypeptide
comprising an amino acid
sequence having at least about 80% amino acid sequence identity, alternatively
at least about 81 % amino acid
sequence identity, alternatively at least about 82 % amino acid sequence
identity, alternatively at least about 83
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amino acid sequence identity, alternatively at least about 84 % amino acid
sequence identity, alternatively at least
about 85 % amino acid sequence identity, alternatively at least about 86 %
amino acid sequence identity,
alternatively at least about 87 % amino acid sequence identity, alternatively
at least about 88 % amino acid
sequence identity, alternatively at least about 89 % amino acid sequence
identity, alternatively at least about 90
amino acid sequence identity, alternatively at least about 91 % amino acid
sequence identity, alternatively at least
about 92 % amino acid sequence identity, alternatively at least about 93 %
amino acid sequence identity,
alternatively at least about 94 % amino acid sequence identity, alternatively
at least about 95 % amino acid
sequence identity, alternatively at least about 96 % amino acid sequence
identity, alternatively at least about 97
amino acid sequence identity, alternatively at least about 98 % amino acid
sequence identity and alternatively at
least about 99% amino acid sequence identity to an amino acid sequence encoded
by any of the human protein
cDNAs deposited with the ATCC as disclosed herein.
In a specific aspect, the invention provides an isolated PRO polypeptide
without the N-terminal signal
sequence and/or the initiating methionine and is encoded by a nucleotide
sequence that encodes such an amino
acid sequence as hereinbefore described. Processes for producing the same are
also herein described, wherein
those processes comprise culturing a host cell comprising a vector which
comprises the appropriate encoding
nucleic acid molecule under conditions suitable for expression of the PRO
polypeptide and recovering the PRO
polypeptide from the cell culture.
Another aspect the invention provides an isolated PRO polypeptide which is
either transmembrane
domain-deleted or transmembrane domain-inactivated. Processes for producing
the same are also herein
described, wherein those processes comprise culturing a host cell comprising a
vector which comprises the
appropriate encoding nucleic acid molecule under conditions suitable for
expression of the PRO polypeptide and
recovering the PRO polypeptide from the cell culture.
In yet another embodiment, the invention concerns agonists and antagonists of
a native PRO polypeptide
as defined herein. In a particular embodiment, the agonist or antagonist is an
anti-PRO antibody or a small
molecule.
In a further embodiment, the invention concerns a method of identifying
agonists or antagonists to a
PRO polypeptide which comprise contacting the PRO polypeptide with a candidate
molecule and monitoring a
biological activity mediated by said PRO polypeptide. Preferably, the PRO
polypeptide is a native PRO
polypeptide.
In a still further embodiment, the invention concerns a composition of matter
comprising a PRO
polypeptide, or an agonist or antagonist of a PRO polypeptide as herein
described, or an anti-PRO antibody, in
combination with a carrier. Optionally, the carrier is a pharmaceutically
acceptable carrier.
Another embodiment of the present invention is directed to the use of a PRO
polypeptide, or an agonist
or antagonist thereof as hereinbefore described, or an anti-PRO antibody, for
the preparation of a medicament
useful in the treatment of a condition which is responsive to the PRO
polypeptide, an agonist or antagonist
thereof or an anti-PRO antibody.
In other embodiments of the present invention, the invention provides vectors
comprising DNA
encoding any of the herein described polypeptides. Host cell comprising any
such vector are also provided. By
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way of example, the host cells may be CHO cells, E. coli, or yeast. A process
for producing any of the herein
described polypeptides is further provided and comprises culturing host cells
under conditions suitable for
expression of the desired polypeptide and recovering the desired polypeptide
from the cell culture.
In other embodiments, the invention provides chimeric molecules comprising any
of the herein described
polypeptides fused to a heterologous polypeptide or amino acid sequence.
Example of such chimeric molecules
comprise any of the herein described polypeptides fused to an epitope tag
sequence or a Fc region of an
immunoglobulin.
In another embodiment, the invention provides an antibody which binds,
preferably specifically, to any
of the above or below described polypeptides. Optionally, the antibody is a
monoclonal antibody, humanized
antibody, antibody fragment or single-chain antibody.
In yet other embodiments, the invention provides oligonucleotide probes useful
for isolating genomic
and cDNA nucleotide sequences or as antisense probes, wherein those probes may
be derived from any of the
above or below described nucleotide sequences.
In yet other embodiments, the present invention is directed to methods of
using the PRO polypeptides
of the present invention for a variety of uses based upon the functional
biological assay data presented in the
Examples below.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a nucleotide sequence (SEQ ID NO:1) of a native sequence PR0180
cDNA, wherein
SEQ ID NO:1 is a clone designated herein as "DNA26843-1389".
Figure 2 shows the amino acid sequence (SEQ ID N0:2) derived from the coding
sequence of SEQ ID
NO:1 shown in Figure 1.
Figure 3 shows a nucleotide sequence (SEQ ID N0:3) of a native sequence PR0218
cDNA, wherein
SEQ ID N0:3 is a clone designated herein as "DNA30867-1335".
Figure 4 shows the amino acid sequence (SEQ ID N0:4) derived from the coding
sequence of SEQ ID
N0:3 shown in Figure 3.
Figure 5 shows a nucleotide sequence (SEQ ID NO:S) of a native sequence PR0263
cDNA, wherein
SEQ ID NO:S is a clone designated herein as "DNA34431-1177".
Figure 6 shows the amino acid sequence (SEQ ID N0:6) derived from the coding
sequence of SEQ ID
NO:S shown in Figure 5.
Figure 7 shows a nucleotide sequence (SEQ ID N0:7) of a native sequence PR0295
cDNA, wherein
SEQ ID N0:7 is a clone designated herein as "DNA38268-1188".
Figure 8 shows the amino acid sequence (SEQ ID N0:8) derived from the coding
sequence of SEQ ID
N0:7 shown in Figure 7.
Figure 9 shows a nucleotide sequence (SEQ ID N0:9) of a native sequence PR0874
cDNA, wherein
SEQ ID N0:9 is a clone designated herein as "DNA40621-1440".
Figure 10 shows the amino acid sequence (SEQ ID NO:10) derived from the coding
sequence of SEQ
ID N0:9 shown in Figure 9.
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Figure 11 shows a nucleotide sequence (SEQ ID NO:11) of a native sequence
PR0300 cDNA, wherein
SEQ ID NO:11 is a clone designated herein as "DNA40625-1189".
Figure 12 shows the amino acid sequence (SEQ ID N0:12) derived from the coding
sequence of SEQ
ID NO:11 shown in Figure 11.
Figure 13 shows a nucleotide sequence (SEQ ID N0:13) of a native sequence
PR01864 cDNA, wherein
SEQ ID N0:13 is a clone designated herein as "DNA45409-2511".
Figure 14 shows the amino acid sequence (SEQ ID N0:14) derived from the coding
sequence of SEQ
ID N0:13 shown in Figure 13.
Figure 15 shows a nucleotide sequence (SEQ ID N0:15) of a native sequence
PR01282 cDNA, wherein
SEQ ID N0:15 is a clone designated herein as "DNA45495-1550".
Figure 16 shows the amino acid sequence (SEQ ID N0:16) derived from the coding
sequence of SEQ
ID N0:15 shown in Figure 15.
Figure 17 shows a nucleotide sequence (SEQ ID N0:17) of a native sequence
PR01063 cDNA, wherein
SEQ ID N0:17 is a clone designated herein as "DNA49820-1427".
Figure 18 shows the amino acid sequence (SEQ ID N0:18) derived from the coding
sequence of SEQ
ID N0:17 shown in Figure 17.
Figure 19 shows a nucleotide sequence (SEQ ID N0:19) of a native sequence
PR01773 cDNA, wherein
SEQ ID N0:19 is a clone designated herein as "DNA56406-1704".
Figure 20 shows the amino acid sequence (SEQ ID N0:20) derived from the coding
sequence of SEQ
ID N0:19 shown in Figure 19.
Figure 21 shows a nucleotide sequence (SEQ ID N0:21 ) of a native sequence
PR01013 cDNA, wherein
SEQ ID N0:21 is a clone designated herein as "DNA56410-1414".
Figure 22 shows the amino acid sequence (SEQ ID N0:22) derived from the coding
sequence of SEQ
ID N0:21 shown in Figure 21.
Figure 23 shows a nucleotide sequence (SEQ ID N0:23) of a native sequence
PR0937 cDNA, wherein
SEQ ID N0:23 is a clone designated herein as "DNA56436-1448".
Figure 24 shows the amino acid sequence (SEQ ID N0:24) derived from the coding
sequence of SEQ
ID N0:23 shown in Figure 23.
Figure 25 shows a nucleotide sequence (SEQ ID N0:25) of a native sequence
PR0842 cDNA, wherein
SEQ ID N0:25 is a clone designated herein as "DNA56855-1447".
Figure 26 shows the amino acid sequence (SEQ ID N0:26) derived from the coding
sequence of SEQ
ID N0:25 shown in Figure 25.
Figure 27 shows a nucleotide sequence (SEQ ID N0:27) of a native sequence
PR01180 cDNA, wherein
SEQ ID N0:27 is a clone designated herein as "DNA56860-1510".
Figure 28 shows the amino acid sequence (SEQ ID N0:28) derived from the coding
sequence of SEQ
ID N0:27 shown in Figure 27.
Figure 29 shows a nucleotide sequence (SEQ ID N0:29) of a native sequence
PR0831 cDNA, wherein
SEQ ID N0:29 is a clone designated herein as "DNA56862-1343".
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Figure 30 shows the amino acid sequence (SEQ ID N0:30) derived from the coding
sequence of SEQ
ID N0:29 shown in Figure 29.
Figure 31 shows a nucleotide sequence (SEQ ID N0:31 ) of a native sequence
PRO1115 cDNA, wherein
SEQ ID N0:31 is a clone designated herein as "DNA56868-1478".
Figure 32 shows the amino acid sequence (SEQ ID N0:32) derived from the coding
sequence of SEQ
ID N0:31 shown in Figure 31.
Figure 33 shows a nucleotide sequence (SEQ ID N0:33) of a native sequence PRO
1277 cDNA, wherein
SEQ ID N0:33 is a clone designated herein as "DNA56869-1545".
Figure 34 shows the amino acid sequence (SEQ ID N0:34) derived from the coding
sequence of SEQ
ID N0:33 shown in Figure 33.
Figure 35 shows a nucleotide sequence (SEQ ID N0:35) of a native sequence
PR01074 cDNA, wherein
SEQ ID N0:35 is a clone designated herein as "DNA57704-1452".
Figure 36 shows the amino acid sequence (SEQ ID N0:36) derived from the coding
sequence of SEQ
ID N0:35 shown in Figure 35.
Figure 37 shows a nucleotide sequence (SEQ ID N0:37) of a native sequence PRO
1344 cDNA, wherein
SEQ ID N0:37 is a clone designated herein as "DNA58723-1588".
Figure 38 shows the amino acid sequence (SEQ ID N0:38) derived from the coding
sequence of SEQ
ID N0:37 shown in Figure 37.
Figure 39 shows a nucleotide sequence (SEQ ID N0:39) of a native sequence PRO
1136 cDNA, wherein
SEQ ID N0:39 is a clone designated herein as "DNA57827-1493".
Figure 40 shows the amino acid sequence (SEQ ID N0:40) derived from the coding
sequence of SEQ
ID N0:39 shown in Figure 39.
Figure 41 shows a nucleotide sequence (SEQ ID N0:41 ) of a native sequence PRO
1109 cDNA, wherein
SEQ ID N0:41 is a clone designated herein as "DNA58737-1473".
Figure 42 shows the amino acid sequence (SEQ ID N0:42) derived from the coding
sequence of SEQ
ID N0:41 shown in Figure 41.
Figure 43 shows a nucleotide sequence (SEQ ID N0:43) of a native sequence PRO
1003 cDNA, wherein
SEQ ID N0:43 is a clone designated herein as "DNA58846-1409".
Figure 44 shows the amino acid sequence (SEQ ID N0:44) derived from the coding
sequence of SEQ
ID N0:43 shown in Figure 43.
Figure 45 shows a nucleotide sequence (SEQ ID N0:45) of a native sequence
PR01138 cDNA, wherein
SEQ ID N0:45 is a clone designated herein as "DNA58850-1495".
Figure 46 shows the amino acid sequence (SEQ ID N0:46) derived from the coding
sequence of SEQ
ID N0:45 shown in Figure 45.
Figure 47 shows a nucleotide sequence (SEQ ID N0:47) of a native sequence
PR0994 cDNA, wherein
SEQ ID N0:47 is a clone designated herein as "DNA58855-1422".
Figure 48 shows the amino acid sequence (SEQ ID N0:48) derived from the coding
sequence of SEQ
ID N0:47 shown in Figure 47.
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Figure 49 shows a nucleotide sequence (SEQ ID N0:49) of a native sequence PRO
1069 cDNA, wherein
SEQ ID N0:49 is a clone designated herein as "DNA59211-1450".
Figure 50 shows the amino acid sequence (SEQ ID N0:50) derived from the coding
sequence of SEQ
ID N0:49 shown in Figure 49.
Figure 51 shows a nucleotide sequence (SEQ ID N0:51 ) of a native sequence PRO
1411 cDNA, wherein
SEQ ID N0:51 is a clone designated herein as "DNA59212-1627".
Figure 52 shows the amino acid sequence (SEQ ID N0:52) derived from the coding
sequence of SEQ
ID N0:51 shown in Figure 51.
Figure 53 shows a nucleotide sequence (SEQ ID N0:53) of a native sequence PRO
1129 cDNA, wherein
SEQ ID N0:53 is a clone designated herein as "DNA59213-1487".
Figure 54 shows the amino acid sequence (SEQ ID N0:54) derived from the coding
sequence of SEQ
ID N0:53 shown in Figure 53.
Figure 55 shows a nucleotide sequence (SEQ ID N0:55) of a native sequence
PR01027 cDNA, wherein
SEQ ID N0:55 is a clone designated herein as "DNA59605-1418".
Figure 56 shows the amino acid sequence (SEQ ID N0:56) derived from the coding
sequence of SEQ
ID N0:55 shown in Figure 55.
Figure 57 shows a nucleotide sequence (SEQ ID N0:57) of a native sequence
PR01106 cDNA, wherein
SEQ ID N0:57 is a clone designated herein as "DNA59609-1470" .
Figure 58 shows the amino acid sequence (SEQ ID N0:58) derived from the coding
sequence of SEQ
ID N0:57 shown in Figure 57.
Figure 59 shows a nucleotide sequence (SEQ ID N0:59) of a native sequence
PR01291 cDNA, wherein
SEQ ID N0:59 is a clone designated herein as "DNA59610-1556".
Figure 60 shows the amino acid sequence (SEQ ID N0:60) derived from the coding
sequence of SEQ
ID N0:59 shown in Figure 59.
Figure 61 shows a nucleotide sequence (SEQ ID N0:61 ) of a native sequence
PR03573 cDNA, wherein
SEQ ID N0:61 is a clone designated herein as "DNA59837-2545".
Figure 62 shows the amino acid sequence (SEQ ID N0:62) derived from the coding
sequence of SEQ
ID N0:61 shown in Figure 61.
Figure 63 shows a nucleotide sequence (SEQ ID N0:63) of a native sequence
PR03566 cDNA, wherein
SEQ ID N0:63 is a clone designated herein as "DNA59844-2542".
Figure 64 shows the amino acid sequence (SEQ ID N0:64) derived from the coding
sequence of SEQ
ID N0:63 shown in Figure 63.
Figure 65 shows a nucleotide sequence (SEQ ID N0:65) of a native sequence PRO
1098 cDNA, wherein
SEQ ID N0:65 is a clone designated herein as "DNA59854-1459".
Figure 66 shows the amino acid sequence (SEQ ID N0:66) derived from the coding
sequence of SEQ
ID N0:65 shown in Figure 65.
Figure 67 shows a nucleotide sequence (SEQ ID N0:67) of a native sequence PRO
1158 cDNA, wherein
SEQ ID N0:67 is a clone designated herein as "DNA60625-1507".
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Figure 68 shows the amino acid sequence (SEQ ID N0:68) derived from the coding
sequence of SEQ
ID N0:67 shown in Figure 67.
Figure 69 shows a nucleotide sequence (SEQ ID N0:69) of a native sequence
PR01124 cDNA, wherein
SEQ ID N0:69 is a clone designated herein as "DNA60629-1481".
Figure 70 shows the amino acid sequence (SEQ ID N0:70) derived from the coding
sequence of SEQ
ID N0:69 shown in Figure 69.
Figure 71 shows a nucleotide sequence (SEQ ID N0:71 ) of a native sequence
PR01287 cDNA, wherein
SEQ ID N0:71 is a clone designated herein as "DNA61755-1554°'.
Figure 72 shows the amino acid sequence (SEQ ID N0:72) derived from the coding
sequence of SEQ
ID N0:71 shown in Figure 71.
Figure 73 shows a nucleotide sequence (SEQ ID N0:73) of a native sequence
PR01335 cDNA, wherein
SEQ ID N0:73 is a clone designated herein as "DNA62812-1594".
Figure 74 shows the amino acid sequence (SEQ ID N0:74) derived from the coding
sequence of SEQ
ID N0:73 shown in Figure 73.
Figure 75 shows a nucleotide sequence (SEQ ID N0:75) of a native sequence
PR01315 cDNA, wherein
SEQ ID N0:75 is a clone designated herein as "DNA62815-1576".
Figure 76 shows the amino acid sequence (SEQ ID N0:76) derived from the coding
sequence of SEQ
ID N0:75 shown in Figure 75.
Figure 77 shows a nucleotide sequence (SEQ ID N0:77) of a native sequence PRO
1357 cDNA, wherein
SEQ ID N0:77 is a clone designated herein as "DNA64881-1602".
Figure 78 shows the amino acid sequence (SEQ ID N0:78) derived from the coding
sequence of SEQ
ID N0:77 shown in Figure 77.
Figure 79 shows a nucleotide sequence (SEQ ID N0:79) of a native sequence PRO
1356 cDNA, wherein
SEQ ID N0:79 is a clone designated herein as "DNA64886-1601".
Figure 80 shows the amino acid sequence (SEQ ID N0:80) derived from the coding
sequence of SEQ
ID N0:79 shown in Figure 79.
Figure 81 shows a nucleotide sequence (SEQ ID N0:81) of a native sequence
PR01557 cDNA, wherein
SEQ ID N0:81 is a clone designated herein as "DNA64902-1667".
Figure 82 shows the amino acid sequence (SEQ ID N0:82) derived from the coding
sequence of SEQ
ID N0:81 shown in Figure 81.
Figure 83 shows a nucleotide sequence (SEQ ID N0:83) of a native sequence
PR01347 cDNA, wherein
SEQ ID N0:83 is a clone designated herein as "DNA64950-1590".
Figure 84 shows the amino acid sequence (SEQ ID N0:84) derived from the coding
sequence of SEQ
ID N0:83 shown in Figure 83.
Figure 85 shows a nucleotide sequence (SEQ ID N0:85) of a native sequence
PR01302 cDNA, wherein
SEQ ID N0:85 is a clone designated herein as "DNA65403-1565".
Figure 86 shows the amino acid sequence (SEQ ID N0:86) derived from the coding
sequence of SEQ
ID N0:85 shown in Figure 85.


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Figure 87 shows a nucleotide sequence (SEQ ID N0:87) of a native sequence PRO
1270 cDNA, wherein
SEQ ID N0:87 is a clone designated herein as "DNA66308-1537".
Figure 88 shows the amino acid sequence (SEQ ID N0:88) derived from the coding
sequence of SEQ
ID N0:87 shown in Figure 87.
Figure 89 shows a nucleotide sequence (SEQ ID N0:89) of a native sequence PRO
1268 cDNA, wherein
SEQ ID N0:89 is a clone designated herein as "DNA66519-1535".
Figure 90 shows the amino acid sequence (SEQ ID N0:90) derived from the coding
sequence of SEQ
ID N0:89 shown in Figure 89.
Figure 91 shows a nucleotide sequence (SEQ ID N0:91 ) of a native sequence PRO
1327 cDNA, wherein
SEQ ID N0:91 is a clone designated herein as "DNA66521-1583".
Figure 92 shows the amino acid sequence (SEQ ID N0:92) derived from the coding
sequence of SEQ
ID N0:91 shown in Figure 91.
Figure 93 shows a nucleotide sequence (SEQ ID N0:93) of a native sequence PRO
1328 cDNA, wherein
SEQ ID N0:93 is a clone designated herein as "DNA66658-1584".
Figure 94 shows the amino acid sequence (SEQ ID N0:94) derived from the coding
sequence of SEQ
ID N0:93 shown in Figure 93.
Figure 95 shows a nucleotide sequence (SEQ ID N0:95) of a native sequence PRO
1329 cDNA, wherein
SEQ ID N0:95 is a clone designated herein as "DNA66660-1585".
Figure 96 shows the amino acid sequence (SEQ ID N0:96) derived from the coding
sequence of SEQ
ID N0:95 shown in Figure 95.
Figure 97 shows a nucleotide sequence (SEQ ID N0:97) of a native sequence PRO
1340 cDNA, wherein
SEQ ID N0:97 is a clone designated herein as "DNA66663-1598".
Figure 98 shows the amino acid sequence (SEQ ID N0:98) derived from the coding
sequence of SEQ
ID N0:97 shown in Figure 97.
Figure 99 shows a nucleotide sequence (SEQ ID N0:99) of a native sequence PRO
1342 cDNA, wherein
SEQ ID N0:99 is a clone designated herein as "DNA66674-1599".
Figure 100 shows the amino acid sequence (SEQ ID NO:100) derived from the
coding sequence of SEQ
ID N0:99 shown in Figure 99.
Figure 101 shows a nucleotide sequence (SEQ ID NO:101) of a native sequence
PR03579 cDNA,
wherein SEQ ID NO:101 is a clone designated herein as "DNA68862-2546".
Figure 102 shows the amino acid sequence (SEQ ID N0:102) derived from the
coding sequence of SEQ
ID NO:101 shown in Figure 101.
Figure 103 shows a nucleotide sequence (SEQ ID N0:103) of a native sequence
PR01472 cDNA,
wherein SEQ ID N0:103 is a clone designated herein as "DNA68866-1644".
Figure 104 shows the amino acid sequence (SEQ ID N0:104) derived from the
coding sequence of SEQ
ID N0:103 shown in Figure 103.
Figure 105 shows a nucleotide sequence (SEQ ID NO:105) of a native sequence
PR01461 cDNA,
wherein SEQ ID NO:105 is a clone designated herein as "DNA68871-1638".
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Figure 106 shows the amino acid sequence (SEQ ID N0:106) derived from the
coding sequence of SEQ
ID NO:105 shown in Figure 105.
Figure 107 shows a nucleotide sequence (SEQ ID N0:107) of a native sequence
PR01568 cDNA,
wherein SEQ ID N0:107 is a clone designated herein as "DNA68880-1676".
Figure 108 shows the amino acid sequence (SEQ ID N0:108) derived from the
coding sequence of SEQ
ID N0:107 shown in Figure 107.
Figure 109 shows a nucleotide sequence (SEQ ID N0:109) of a native sequence
PR01753 cDNA,
wherein SEQ ID N0:109 is a clone designated herein as "DNA68883-1691".
Figure 110 shows the amino acid sequence (SEQ ID NO:110) derived from the
coding sequence of SEQ
ID N0:109 shown in Figure 109.
Figure 111 shows a nucleotide sequence (SEQ ID NO:111) of a native sequence
PR01570 cDNA,
wherein SEQ ID NO:111 is a clone designated herein as "DNA68885-1678".
Figure 112 shows the amino acid sequence (SEQ ID N0:112) derived from the
coding sequence of SEQ
ID NO:111 shown in Figure 111.
Figure 113 shows a nucleotide sequence (SEQ ID N0:113) of a native sequence
PR01446 cDNA,
wherein SEQ ID N0:113 is a clone designated herein as "DNA71277-1636".
Figure 114 shows the amino acid sequence (SEQ ID N0:114) derived from the
coding sequence of SEQ
ID N0:113 shown in Figure 113.
Figure 115 shows a nucleotide sequence (SEQ ID NO:115) of a native sequence
PR01565 cDNA,
wherein SEQ ID NO:115 is a clone designated herein as "DNA73727-1673".
Figure 116 shows the amino acid sequence (SEQ ID N0:116) derived from the
coding sequence of SEQ
ID NO:115 shown in Figure 115.
Figure 117 shows a nucleotide sequence (SEQ ID N0:117) of a native sequence
PR01572 cDNA,
wherein SEQ ID N0:117 is a clone designated herein as "DNA73734-1680".
Figure 118 shows the amino acid sequence (SEQ ID N0:118) derived from the
coding sequence of SEQ
ID N0:117 shown in Figure 117.
Figure 119 shows a nucleotide sequence (SEQ ID NO:119) of a native sequence
PR01573 cDNA,
wherein SEQ ID N0:119 is a clone designated herein as "DNA73735-1681 ".
Figure 120 shows the amino acid sequence (SEQ ID N0:120) derived from the
coding sequence of SEQ
ID N0:119 shown in Figure 119.
Figure 121 shows a nucleotide sequence (SEQ ID N0:121) of a native sequence
PRO1550 cDNA,
wherein SEQ ID N0:121 is a clone designated herein as "DNA76393-1664".
Figure 122 shows the amino acid sequence (SEQ ID N0:122) derived from the
coding sequence of SEQ
ID N0:121 shown in Figure 121.
Figure 123 shows a nucleotide sequence (SEQ ID N0:123) of a native sequence
PR01693 cDNA,
wherein SEQ ID N0:123 is a clone designated herein as "DNA77301-1708".
Figure 124 shows the amino acid sequence (SEQ ID N0:124) derived from the
coding sequence of SEQ
ID N0:123 shown in Figure 123.
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Figure 125 shows a nucleotide sequence (SEQ ID N0:125) of a native sequence
PR01566 cDNA,
wherein SEQ ID N0:125 is a clone designated herein as "DNA77568-1626".
Figure 126 shows the amino acid sequence (SEQ ID N0:126) derived from the
coding sequence of SEQ
ID N0:125 shown in Figure 125.
Figure 127 shows a nucleotide sequence (SEQ ID N0:127) of a native sequence
PR01774 cDNA,
wherein SEQ ID N0:127 is a clone designated herein as "DNA77626-1705".
Figure 128 shows the amino acid sequence (SEQ ID N0:128) derived from the
coding sequence of SEQ
ID N0:127 shown in Figure 127.
Figure 129 shows a nucleotide sequence (SEQ ID N0:129) of a native sequence
PR01928 cDNA,
wherein SEQ ID N0:129 is a clone designated herein as "DNA81754-2532".
Figure 130 shows the amino acid sequence (SEQ ID N0:130) derived from the
coding sequence of SEQ
ID N0:129 shown in Figure 129.
Figure 131 shows a nucleotide sequence (SEQ ID N0:131) of a native sequence
PR01865 cDNA,
wherein SEQ ID N0:131 is a clone designated herein as "DNA81757-2512".
Figure 132 shows the amino acid sequence (SEQ ID N0:132) derived from the
coding sequence of SEQ
ID N0:131 shown in Figure 131.
Figure 133 shows a nucleotide sequence (SEQ ID N0:133) of a native sequence
PR01925 cDNA,
wherein SEQ ID N0:133 is a clone designated herein as "DNA82302-2529".
Figure 134 shows the amino acid sequence (SEQ ID N0:134) derived from the
coding sequence of SEQ
ID N0:133 shown in Figure 133.
Figure 135 shows a nucleotide sequence (SEQ ID N0:135) of a native sequence
PR01926 cDNA,
wherein SEQ ID N0:135 is a clone designated herein as "DNA82340-2530".
Figure 136 shows the amino acid sequence (SEQ ID N0:136) derived from the
coding sequence of SEQ
ID N0:135 shown in Figure 135.
Figure 137 shows a nucleotide sequence (SEQ ID N0:137) of a native sequence
PR01801 cDNA,
wherein SEQ ID N0:137 is a clone designated herein as "DNA83500-2506" .
Figure 138 shows the amino acid sequence (SEQ ID N0:138) derived from the
coding sequence of SEQ
ID N0:137 shown in Figure 137.
Figure 139 shows a nucleotide sequence (SEQ ID N0:139) of a native sequence
PR04405 cDNA,
wherein SEQ ID N0:139 is a clone designated herein as "DNA84920-2614".
Figure 140 shows the amino acid sequence (SEQ ID N0:140) derived from the
coding sequence of SEQ
ID N0:139 shown in Figure 139.
Figure 141 shows a nucleotide sequence (SEQ ID N0:141) of a native sequence
PR03435 cDNA,
wherein SEQ ID N0:141 is a clone designated herein as "DNA85066-2534".
Figure 142 shows the amino acid sequence (SEQ ID N0:142) derived from the
coding sequence of SEQ
ID N0:141 shown in Figure 141.
Figure 143 shows a nucleotide sequence (SEQ ID N0:143) of a native sequence
PR03543 cDNA,
wherein SEQ ID N0:143 is a clone designated herein as "DNA86571-2551".
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Figure 144 shows the amino acid sequence (SEQ ID N0:144) derived from the
coding sequence of SEQ
ID N0:143 shown in Figure 143.
Figure 145 shows a nucleotide sequence (SEQ ID N0:145) of a native sequence
PR03443 cDNA,
wherein SEQ ID N0:145 is a clone designated herein as "DNA87991-2540".
Figure 146 shows the amino acid sequence (SEQ ID N0:146) derived from the
coding sequence of SEQ
ID N0:145 shown in Figure 145.
Figure 147 shows a nucleotide sequence (SEQ ID N0:147) of a native sequence
PR03442 cDNA,
wherein SEQ ID N0:147 is a clone designated herein as "DNA92238-2539".
Figure 148 shows the amino acid sequence (SEQ ID N0:148) derived from the
coding sequence of SEQ
ID N0:147 shown in Figure 147.
Figure 149 shows a nucleotide sequence (SEQ ID N0:149) of a native sequence
PR05990 cDNA,
wherein SEQ ID N0:149 is a clone designated herein as "DNA96042-2682".
Figure 150 shows the amino acid sequence (SEQ ID NO:150) derived from the
coding sequence of SEQ
ID N0:149 shown in Figure 149.
Figure 151 shows a nucleotide sequence (SEQ ID NO:151) of a native sequence
PR04342 cDNA,
wherein SEQ ID NO:151 is a clone designated herein as "DNA96787-2534".
Figure 152 shows the amino acid sequence (SEQ ID N0:152) derived from the
coding sequence of SEQ
ID NO:151 shown in Figure 151.
Figure 153 shows a nucleotide sequence (SEQ ID N0:153) of a native sequence
PR010096 cDNA,
wherein SEQ ID N0:153 is a clone designated herein as "DNA125185-2806".
Figure 154 shows the amino acid sequence (SEQ ID N0:154) derived from the
coding sequence of SEQ
ID N0:153 shown in Figure 153.
Figure 155 shows a nucleotide sequence (SEQ ID NO:155) of a native sequence
PR010272 cDNA,
wherein SEQ ID NO:155 is a clone designated herein as "DNA147531-2821".
Figure 156 shows the amino acid sequence (SEQ ID N0:156) derived from the
coding sequence of SEQ
ID NO:155 shown in Figure 155.
Figure 157 shows a nucleotide sequence (SEQ ID N0:157) of a native sequence
PR05801 cDNA,
wherein SEQ ID N0:157 is a clone designated herein as "DNA115291-2681".
Figure 158 shows the amino acid sequence (SEQ ID N0:158) derived from the
coding sequence of SEQ
ID N0:157 shown in Figure 157.
Figure 159 shows a nucleotide sequence (SEQ ID N0:159) of a native sequence
PR020110 cDNA,
wherein SEQ ID N0:159 is a clone designated herein as "DNA166819".
Figure 160 shows the amino acid sequence (SEQ ID N0:160) derived from the
coding sequence of SEQ
ID N0:159 shown in Figure 159.
Figure 161 shows a nucleotide sequence (SEQ ID N0:161) of a native sequence
PR020040 cDNA,
wherein SEQ ID N0:161 is a clone designated herein as "DNA164625-2890".
Figure 162 shows the amino acid sequence (SEQ ID N0:162) derived from the
coding sequence of SEQ
ID N0:161 shown in Figure 161.
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Figure 163 shows a nucleotide sequence (SEQ ID N0:163) of a native sequence
PR020233 cDNA,
wherein SEQ ID N0:163 is a clone designated herein as "DNA165608".
Figure 164 shows the amino acid sequence (SEQ ID N0:164) derived from the
coding sequence of SEQ
ID N0:163 shown in Figure 163.
Figure 165 shows a nucleotide sequence (SEQ ID N0:165) of a native sequence
PR019670 cDNA,
wherein SEQ ID N0:165 is a clone designated herein as "DNA131639-2874".
Figure 166 shows the amino acid sequence (SEQ ID N0:166) derived from the
coding sequence of SEQ
ID N0:165 shown in Figure 165.
Figure 167 shows a nucleotide sequence (SEQ ID N0:167) of a native sequence
PR01890 cDNA,
wherein SEQ ID N0:167 is a clone designated herein as "DNA79230-2525".
Figure 168 shows the amino acid sequence (SEQ ID N0:168) derived from the
coding sequence of SEQ
ID N0:167 shown in Figure 167.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions
The terms "PRO polypeptide" and "PRO" as used herein and when immediately
followed by a
numerical designation refer to various polypeptides, wherein the complete
designation (i.e., PRO/number) refers
to specific polypeptide sequences as described herein. The terms "PRO/number
polypeptide" and
"PRO/number" wherein the term "number" is provided as an actual numerical
designation as used herein
encompass native sequence polypeptides and polypeptide variants (which are
further defined herein). The PRO
polypeptides described herein may be isolated from a variety of sources, such
as from human tissue types or
from another source, or prepared by recombinant or synthetic methods. The term
"PRO polypeptide" refers to
each individual PRO/number polypeptide disclosed herein. All disclosures in
this specification which refer to
the "PRO polypeptide" refer to each of the polypeptides individually as well
as jointly. For example,
descriptions of the preparation of, purification of, derivation of, formation
of antibodies to or against,
administration of, compositions containing, treatment of a disease with, etc.,
pertain to each polypeptide of the
invention individually. The term "PRO polypeptide" also includes variants of
the PRO/number polypeptides
disclosed herein.
A "native sequence PRO polypeptide" comprises a polypeptide having the same
amino acid sequence
as the corresponding PRO polypeptide derived from nature. Such native sequence
PRO polypeptides can be
isolated from nature or can be produced by recombinant or synthetic means. The
term "native sequence PRO
polypeptide" specifically encompasses naturally-occurring truncated or
secreted forms of the specific PRO
polypeptide (e.g., an extracellular domain sequence), naturally-occurring
variant forms (e.g., alternatively
spliced forms) and naturally-occurring allelic variants of the polypeptide. In
various embodiments of the
invention, the native sequence PRO polypeptides disclosed herein are mature or
full-length native sequence
polypeptides comprising the full-length amino acids sequences shown in the
accompanying figures. Start and
stop codons are shown in bold font and underlined in the figures. However,
while the PRO polypeptide
disclosed in the accompanying figures are shown to begin with methionine
residues designated herein as amino


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acid position 1 in the figures, it is conceivable and possible that other
methionine residues located either upstream
or downstream from the amino acid position 1 in the figures may be employed as
the starting amino acid residue
for the PRO polypeptides.
The PRO polypeptide "extracellular domain" or "ECD" refers to a form of the
PRO polypeptide which
is essentially free of the transmembrane and cytoplasmic domains. Ordinarily,
a PRO polypeptide ECD will have
less than 1 % of such transmembrane and/or cytoplasmic domains and preferably,
will have less than 0.5 % of
such domains. It will be understood that any transmembrane domains identified
for the PRO polypeptides of
the present invention are identified pursuant to criteria routinely employed
in the art for identifying that type of
hydrophobic domain. The exact boundaries of a transmembrane domain may vary
but most likely by no more
than about 5 amino acids at either end of the domain as initially identified
herein. Optionally, therefore, an
extracellular domain of a PRO polypeptide may contain from about 5 or fewer
amino acids on either side of the
transmembrane domain/extracellular domain boundary as identified in the
Examples or specification and such
polypeptides, with or without the associated signal peptide, and nucleic acid
encoding them, are comtemplated
by the present invention.
The approximate location of the "signal peptides" of the various PRO
polypeptides disclosed herein are
shown in the present specification and/or the accompanying figures. It is
noted, however, that the C-terminal
boundary of a signal peptide may vary, but most likely by no more than about 5
amino acids on either side of
the signal peptide C-terminal boundary as initially identified herein, wherein
the C-terminal boundary of the
signal peptide may be identified pursuant to criteria routinely employed in
the art for identifying that type of
amino acid sequence element (e.g., Nielsen et al., Prot. En~. 10:1-6 (1997)
and von Heinje et al., Nucl. Acids.
Res. 14:4683-4690 ( 1986)). Moreover, it is also recognized that, in some
cases, cleavage of a signal sequence
from a secreted polypeptide is not entirely uniform, resulting in more than
one secreted species. These mature
polypeptides, where the signal peptide is cleaved within no more than about 5
amino acids on either side of the
C-terminal boundary of the signal peptide as identified herein, and the
polynucleotides encoding them, are
contemplated by the present invention.
"PRO polypeptide variant" means an active PRO polypeptide as defined above or
below having at least
about 80 % amino acid sequence identity with a full-length native sequence PRO
polypeptide sequence as
disclosed herein, a PRO polypeptide sequence lacking the signal peptide as
disclosed herein, an extracellular
domain of a PRO polypeptide, with or without the signal peptide, as disclosed
herein or any other fragment of
a full-length PRO polypeptide sequence as disclosed herein. Such PRO
polypeptide variants include, for
instance, PRO polypeptides wherein one or more amino acid residues are added,
or deleted, at the N- or C-
terminus of the full-length native amino acid sequence. Ordinarily, a PRO
polypeptide variant will have at least
about 80 % amino acid sequence identity, alternatively at least about 81 %
amino acid sequence identity,
alternatively at least about 82 % amino acid sequence identity, alternatively
at least about 83 % amino acid
sequence identity, alternatively at least about 84 % amino acid sequence
identity, alternatively at least about 85
amino acid sequence identity, alternatively at least about 86 % amino acid
sequence identity, alternatively at least
about 87 % amino acid sequence identity, alternatively at least about 88 %
amino acid sequence identity,
alternatively at least about 89 % amino acid sequence identity, alternatively
at least about 90 % amino acid
16


CA 02380355 2002-03-O1
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sequence identity, alternatively at least about 91 % amino acid sequence
identity, alternatively at least about 92
amino acid sequence identity, alternatively at least about 93 % amino acid
sequence identity, alternatively at least
about 94 % amino acid sequence identity, alternatively at least about 95 %
amino acid sequence identity,
alternatively at least about 96 % amino acid sequence identity, alternatively
at least about 97 % amino acid
sequence identity, alternatively at least about 98 % amino acid sequence
identity and alternatively at least about
99 % amino acid sequence identity to a full-length native sequence PRO
polypeptide sequence as disclosed herein,
a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an
extracellular domain of a PRO
polypeptide, with or without the signal peptide, as disclosed herein or any
other specifically defined fragment
of a full-length PRO polypeptide sequence as disclosed herein. Ordinarily, PRO
variant polypeptides are at least
about 10 amino acids in length, alternatively at least about 20 amino acids in
length, alternatively at least about
30 amino acids in length, alternatively at least about 40 amino acids in
length, alternatively at least about 50
amino acids in length, alternatively at least about 60 amino acids in length,
alternatively at least about 70 amino
acids in length, alternatively at least about 80 amino acids in length,
alternatively at least about 90 amino acids
in length, alternatively at least about 100 amino acids in length,
alternatively at least about 150 amino acids in
length, alternatively at least about 200 amino acids in length, alternatively
at least about 300 amino acids in
length, or more.
"Percent (%) amino acid sequence identity" with respect to the PRO polypeptide
sequences identified
herein is defined as the percentage of amino acid residues in a candidate
sequence that are identical with the
amino acid residues in the specific PRO polypeptide sequence, after aligning
the sequences and introducing gaps,
if necessary, to achieve the maximum percent sequence identity, and not
considering any conservative
substitutions as part of the sequence identity. Alignment for purposes of
determining percent amino acid
sequence identity can be achieved in various ways that are within the skill in
the art, for instance, using publicly
available computer software such as BLAST, BLAST-2, ALIGN or Megalign
(DNASTAR) software. Those
skilled in the art can determine appropriate parameters for measuring
alignment, including any algorithms needed
to achieve maximal alignment over the full length of the sequences being
compared. For purposes herein,
however, % amino acid sequence identity values are generated using the
sequence comparison computer program
ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided
in Table 1 below. The
ALIGN-2 sequence comparison computer program was authored by Genentech, Inc.
and the source code shown
in Table 1 below has been filed with user documentation in the U. S. Copyright
Office, Washington D. C. , 20559,
where it is registered under U.S. Copyright Registration No. TXU510087. The
ALIGN-2 program is publicly
available through Genentech, Inc., South San Francisco, California or may be
compiled from the source code
provided in Table 1 below. The ALIGN-2 program should be compiled for use on a
UNIX operating system,
preferably digital UNIX V4.OD. All sequence comparison parameters are set by
the ALIGN-2 program and
do not vary.
In situations where ALIGN-2 is employed for amino acid sequence comparisons,
the % amino acid
sequence identity of a given amino acid sequence A to, with, or against a
given amino acid sequence B (which
can alternatively be phrased as a given amino acid sequence A that has or
comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence B) is
calculated as follows:
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100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by
the sequence alignment program
ALIGN-2 in that program's alignment of A and B, and where Y is the total
number of amino acid residues in
B. It will be appreciated that where the length of amino acid sequence A is
not equal to the length of amino acid
sequence B, the % amino acid sequence identity of A to B will not equal the %
amino acid sequence identity of
B to A. As examples of % amino acid sequence identity calculations using this
method, Tables 2 and 3
demonstrate how to calculate the % amino acid sequence identity of the amino
acid sequence designated
"Comparison Protein" to the amino acid sequence designated "PRO", wherein
"PRO" represents the amino acid
sequence of a hypothetical PRO polypeptide of interest, "Comparison Protein"
represents the amino acid
sequence of a polypeptide against which the "PRO" polypeptide of interest is
being compared, and "X, "Y" and
"Z" each represent different hypothetical amino acid residues.
Unless specifically stated otherwise, all % amino acid sequence identity
values used herein are obtained
as described in the immediately preceding paragraph using the ALIGN-2 computer
program. However, % amino
acid sequence identity values may also be obtained as described below by using
the WU-BLAST-2 computer
program (Altschul et al., Methods in Enzvmolo~v 266:460-480 (1996)). Most of
the WU-BLAST-2 search
parameters are set to the default values. Those not set to default values,
i.e., the adjustable parameters, are set
with the following values: overlap span = 1, overlap fraction = 0.125, word
threshold (T) = 11, and scoring
matrix = BLOSUM62. When WU-BLAST-2 is employed, a % amino acid sequence
identity value is
determined by dividing (a) the number of matching identical amino acid
residues between the amino acid
sequence of the PRO polypeptide of interest having a sequence derived from the
native PRO polypeptide and
the comparison amino acid sequence of interest (i.e., the sequence against
which the PRO polypeptide of interest
is being compared which may be a PRO variant polypeptide) as determined by WU-
BLAST-2 by (b) the total
number of amino acid residues of the PRO polypeptide of interest. For example,
in the statement "a polypeptide
comprising an the amino acid sequence A which has or having at least 80 %
amino acid sequence identity to the
amino acid sequence B", the amino acid sequence A is the comparison amino acid
sequence of interest and the
amino acid sequence B is the amino acid sequence of the PRO polypeptide of
interest.
Percent amino acid sequence identity may also be determined using the sequence
comparison program
NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The
NCBI-BLAST2 sequence
comparison program may be downloaded from http://www.ncbi.nlm.nih.gov or
otherwise obtained from the
National Institute of Health, Bethesda, MD. NCBI-BLAST2 uses several search
parameters, wherein all of those
search parameters are set to default values including, for example, unmask =
yes, strand = all, expected
occurrences = 10, minimum low complexity length = 15/5, mufti-pass e-value =
0.01, constant for mufti-pass
= 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62.
In situations where NCBI-BLAST2 is employed for amino acid sequence
comparisons, the % amino
acid sequence identity of a given amino acid sequence A to, with, or against a
given amino acid sequence B
(which can alternatively be phrased as a given amino acid sequence A that has
or comprises a certain % amino
acid sequence identity to, with, or against a given amino acid sequence B) is
calculated as follows:
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100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by
the sequence aligrunent program
NCBI-BLAST2 in that program's alignment of A and B, and where Y is the total
number of amino acid residues
in B. It will be appreciated that where the length of amino acid sequence A is
not equal to the length of amino
acid sequence B, the % amino acid sequence identity of A to B will not equal
the % amino acid sequence identity
ofBtoA.
"PRO variant polynucleotide" or "PRO variant nucleic acid sequence" means a
nucleic acid molecule
which encodes an active PRO polypeptide as defined below and which has at
least about 80 % nucleic acid
sequence identity with a nucleotide acid sequence encoding a full-length
native sequence PRO polypeptide
sequence as disclosed herein, a full-length native sequence PRO polypeptide
sequence lacking the signal peptide
as disclosed herein, an extracellular domain of a PRO polypeptide, with or
without the signal peptide, as
disclosed herein or any other fragment of a full-length PRO polypeptide
sequence as disclosed herein.
Ordinarily, a PRO variant polynucleotide will have at least about 80% nucleic
acid sequence identity,
alternatively at least about 81 % nucleic acid sequence identity,
alternatively at least about 82 % nucleic acid
sequence identity, alternatively at least about 83 % nucleic acid sequence
identity, alternatively at least about 84
nucleic acid sequence identity, alternatively at least about 85 % nucleic acid
sequence identity, alternatively at
least about 86% nucleic acid sequence identity, alternatively at least about
87% nucleic acid sequence identity,
alternatively at least about 88 % nucleic acid sequence identity,
alternatively at least about 89 % nucleic acid
sequence identity, alternatively at least about 90 % nucleic acid sequence
identity, alternatively at least about 91
nucleic acid sequence identity, alternatively at least about 92% nucleic acid
sequence identity, alternatively at
least about 93 % nucleic acid sequence identity, alternatively at least about
94 % nucleic acid sequence identity,
alternatively at least about 95% nucleic acid sequence identity, alternatively
at least about 96% nucleic acid
sequence identity, alternatively at least about 97 % nucleic acid sequence
identity, alternatively at least about 98 %
nucleic acid sequence identity and alternatively at least about 99% nucleic
acid sequence identity with a nucleic
acid sequence encoding a full-length native sequence PRO polypeptide sequence
as disclosed herein, a full-length
native sequence PRO polypeptide sequence lacking the signal peptide as
disclosed herein, an extracellular domain
of a PRO polypeptide, with or without the signal sequence, as disclosed herein
or any other fragment of a full-
length PRO polypeptide sequence as disclosed herein. Variants do not encompass
the native nucleotide
sequence.
Ordinarily, PRO variant polynucleotides are at least about 30 nucleotides in
length, alternatively at least
about 60 nucleotides in length, alternatively at least about 90 nucleotides in
length, alternatively at least about
120 nucleotides in length, alternatively at least about 150 nucleotides in
length, alternatively at least about 180
nucleotides in length, alternatively at least about 210 nucleotides in length,
alternatively at least about 240
nucleotides in length, alternatively at least about 270 nucleotides in length,
alternatively at least about 300
nucleotides in length, alternatively at least about 450 nucleotides in length,
alternatively at least about 600
nucleotides in length, alternatively at least about 900 nucleotides in length,
or more.
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"Percent (%) nucleic acid sequence identity" with respect to PRO-encoding
nucleic acid sequences
identified herein is defined as the percentage of nucleotides in a candidate
sequence that are identical with the
nucleotides in the PRO nucleic acid sequence of interest, after aligning the
sequences and introducing gaps, if
necessary, to achieve the maximum percent sequence identity. Alignment for
purposes of determining percent
nucleic acid sequence identity can be achieved in various ways that are within
the skill in the art, for instance,
using publicly available computer software such as BLAST, BLAST-2, ALIGN or
Megalign (DNASTAR)
software. For purposes herein, however, % nucleic acid sequence identity
values are generated using the
sequence comparison computer program ALIGN-2, wherein the complete source code
for the ALIGN-2 program
is provided in Table 1 below. The ALIGN-2 sequence comparison computer program
was authored by
Genentech, Inc. and the source code shown in Table 1 below has been filed with
user documentation in the U. S.
Copyright Office, Washington D.C., 20559, where it is registered under U.S.
Copyright Registration No.
TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc.,
South San Francisco,
California or may be compiled from the source code provided in Table 1 below.
The ALIGN-2 program should
be compiled for use on a UNIX operating system, preferably digital UNIX V4.OD.
All sequence comparison
parameters are set by the ALIGN-2 program and do not vary.
In situations where ALIGN-2 is employed for nucleic acid sequence comparisons,
the % nucleic acid
sequence identity of a given nucleic acid sequence C to, with, or against a
given nucleic acid sequence D (which
can alternatively be phrased as a given nucleic acid sequence C that has or
comprises a certain % nucleic acid
sequence identity to, with, or against a given nucleic acid sequence D) is
calculated as follows:
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the
sequence alignment program ALIGN-2
in that program's alignment of C and D, and where Z is the total number of
nucleotides in D. It will be
appreciated that where the length of nucleic acid sequence C is not equal to
the length of nucleic acid sequence
D, the % nucleic acid sequence identity of C to D will not equal the % nucleic
acid sequence identity of D to
C. As examples of % nucleic acid sequence identity calculations, Tables 4 and
5, demonstrate how to calculate
the % nucleic acid sequence identity of the nucleic acid sequence designated
"Comparison DNA" to the nucleic
acid sequence designated "PRO-DNA", wherein "PRO-DNA" represents a
hypothetical PRO-encoding nucleic
acid sequence of interest, "Comparison DNA" represents the nucleotide sequence
of a nucleic acid molecule
against which the "PRO-DNA" nucleic acid molecule of interest is being
compared, and "N", "L" and "V" each
represent different hypothetical nucleotides.
Unless specifically stated otherwise, all % nucleic acid sequence identity
values used herein are obtained
as described in the immediately preceding paragraph using the ALIGN-2 computer
program. However,
nucleic acid sequence identity values may also be obtained as described below
by using the WU-BLAST-2
computer program (Altschul et al., Methods in Enzymology 266:460-480 (1996)).
Most of the WU-BLAST-2
search parameters are set to the default values. Those not set to default
values, i.e., the adjustable parameters,
are set with the following values: overlap span = 1, overlap fraction = 0.125,
word threshold (T) = 11, and


CA 02380355 2002-03-O1
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scoring matrix = BLOSUM62. When WU-BLAST-2 is employed, a % nucleic acid
sequence identity value
is determined by dividing (a) the number of matching identical nucleotides
between the nucleic acid sequence
of the PRO polypeptide-encoding nucleic acid molecule of interest having a
sequence derived from the native
sequence PRO polypeptide-encoding nucleic acid and the comparison nucleic acid
molecule of interest (i.e., the
sequence against which the PRO polypeptide-encoding nucleic acid molecule of
interest is being compared which
may be a variant PRO polynucleotide) as determined by WU-BLAST-2 by (b) the
total number of nucleotides
of the PRO polypeptide-encoding nucleic acid molecule of interest. For
example, in the statement "an isolated
nucleic acid molecule comprising a nucleic acid sequence A which has or having
at least 80 % nucleic acid
sequence identity to the nucleic acid sequence B", the nucleic acid sequence A
is the comparison nucleic acid
molecule of interest and the nucleic acid sequence B is the nucleic acid
sequence of the PRO polypeptide
encoding nucleic acid molecule of interest.
Percent nucleic acid sequence identity may also be determined using the
sequence comparison program
NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The
NCBI-BLAST2 sequence
comparison program may be downloaded from http://www.ncbi.nlm.nih.gov or
otherwise obtained from the
National Institute of Health, Bethesda, MD. NCBI-BLAST2 uses several search
parameters, wherein all of those
search parameters are set to default values including, for example, unmask =
yes, strand = all, expected
occurrences = 10, minimum low complexity length = 15/5, multi-pass e-value =
0.01, constant for multi-pass
= 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62.
In situations where NCBI-BLAST2 is employed for sequence comparisons, the %
nucleic acid sequence
identity of a given nucleic acid sequence C to, with, or against a given
nucleic acid sequence D (which can
alternatively be phrased as a given nucleic acid sequence C that has or
comprises a certain % nucleic acid
sequence identity to, with, or against a given nucleic acid sequence D) is
calculated as follows:
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the
sequence alignment program NCBI-
BLAST2 in that program's alignment of C and D, and where Z is the total number
of nucleotides in D. It will
be appreciated that where the length of nucleic acid sequence C is not equal
to the length of nucleic acid sequence
D, the % nucleic acid sequence identity of C to D will not equal the % nucleic
acid sequence identity of D to
C.
In other embodiments, PRO variant polynucleotides are nucleic acid molecules
that encode an active
PRO polypeptide and which are capable of hybridizing, preferably under
stringent hybridization and wash
conditions, to nucleotide sequences encoding a full-length PRO polypeptide as
disclosed herein. PRO variant
polypeptides may be those that are encoded by a PRO variant polynucleotide.
"Isolated," when used to describe the various polypeptides disclosed herein,
means polypeptide that has
been identified and separated and/or recovered from a component of its natural
environment. Contaminant
components of its natural environment are materials that would typically
interfere with diagnostic or therapeutic
uses for the polypeptide, and may include enzymes, hormones, and other
proteinaceous or non-proteinaceous
21


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solutes. In preferred embodiments, the polypeptide will be purified (1) to a
degree sufficient to obtain at least
15 residues of N-terminal or internal amino acid sequence by use of a spinning
cup sequenator, or (2) to
homogeneity by SDS-PAGE under non-reducing or reducing conditions using
Coomassie blue or, preferably,
silver stain. Isolated polypeptide includes polypeptide in situ within
recombinant cells, since at least one
component of the PRO polypeptide natural environment will not be present.
Ordinarily, however, isolated
polypeptide will be prepared by at least one purification step.
An "isolated" PRO polypeptide-encoding nucleic acid or other polypeptide-
encoding nucleic acid is a
nucleic acid molecule that is identified and separated from at least one
contaminant nucleic acid molecule with
which it is ordinarily associated in the natural source of the polypeptide-
encoding nucleic acid. An isolated
polypeptide-encoding nucleic acid molecule is other than in the form or
setting in which it is found in nature.
Isolated polypeptide-encoding nucleic acid molecules therefore are
distinguished from the specific polypeptide-
encoding nucleic acid molecule as it exists in natural cells. However, an
isolated polypeptide-encoding nucleic
acid molecule includes polypeptide-encoding nucleic acid molecules contained
in cells that ordinarily express the
polypeptide where, for example, the nucleic acid molecule is in a chromosomal
location different from that of
natural cells.
The term "control sequences" refers to DNA sequences necessary for the
expression of an operably
linked coding sequence in a particular host organism. The control sequences
that are suitable for prokaryotes,
for example, include a promoter, optionally an operator sequence, and a
ribosome binding site. Eukaryotic cells
are known to utilize promoters, polyadenylation signals, and enhancers.
Nucleic acid is "operably linked" when it is placed into a functional
relationship with another nucleic
acid sequence. For example, DNA for a presequence or secretory leader is
operably linked to DNA for a
polypeptide if it is expressed as a preprotein that participates in the
secretion of the polypeptide; a promoter or
enhancer is operably linked to a coding sequence if it affects the
transcription of the sequence; or a ribosome
binding site is operably linked to a coding sequence if it is positioned so as
to facilitate translation. Generally,
"operably linked" means that the DNA sequences being linked are contiguous,
and, in the case of a secretory
leader, contiguous and in reading phase. However, enhancers do not have to be
contiguous. Linking is
accomplished by ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide
adaptors or linkers are used in accordance with conventional practice.
The term "antibody" is used in the broadest sense and specifically covers, for
example, single anti-PRO
monoclonal antibodies (including agonist, antagonist, and neutralizing
antibodies), anti-PRO antibody
compositions with polyepitopic specificity, single chain anti-PRO antibodies,
and fragments of anti-PRO
antibodies (see below). The term "monoclonal antibody" as used herein refers
to an antibody obtained from a
population of substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population are
identical except for possible naturally-occurring mutations that may be
present in minor amounts.
"Stringency" of hybridization reactions is readily determinable by one of
ordinary skill in the art, and
generally is an empirical calculation dependent upon probe length, washing
temperature, and salt concentration.
In general, longer probes require higher temperatures for proper annealing,
while shorter probes need lower
temperatures. Hybridization generally depends on the ability of denatured DNA
to reanneal when
22


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complementary strands are present in an environment below their melting
temperature. The higher the degree
of desired homology between the probe and hybridizable sequence, the higher
the relative temperature which
can be used. As a result, it follows that higher relative temperatures would
tend to make the reaction conditions
more stringent, while lower temperatures less so. For additional details and
explanation of stringency of
hybridization reactions, see Ausubel et al., Current Protocols in Molecular
Biology, Wiley Interscience
Publishers, (1995).
"Stringent conditions" or "high stringency conditions", as defined herein, may
be identified by those
that: (1) employ low ionic strength and high temperature for washing, for
example 0.015 M sodium
chloride/0.0015 M sodium citrate/0.1 % sodium dodecyl sulfate at 50°C;
(2) employ during hybridization a
denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1
% bovine serum
albumin/0.1 % Ficoll/0.1 % polyvinylpyrrolidone/SOmM sodium phosphate buffer
at pH 6.5 with 750 mM sodium
chloride, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5
x SSC (0.75 M NaCI, 0.075 M
sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1 % sodium pyrophosphate,
5 x Denhardt's solution,
sonicated salmon sperm DNA (50 ~cg/ml), 0.1 % SDS, and 10% dextran sulfate at
42°C, with washes at 42°C
in 0.2 x SSC (sodium chloride/sodium citrate) and 50 % formamide at 55
°C, followed by a high-stringency wash
consisting of 0.1 x SSC containing EDTA at 55°C.
"Moderately stringent conditions" may be identified as described by Sambrook
et al., Molecular
Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and
include the use of washing
solution and hybridization conditions (e.g., temperature, ionic strength and %
SDS) less stringent that those
described above. An example of moderately stringent conditions is overnight
incubation at 37°C in a solution
comprising: 20% formamide, 5 x SSC (150 mM NaCI, 15 mM trisodium citrate), 50
mM sodium phosphate (pH
7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured
sheared salmon sperm DNA,
followed by washing the filters in 1 x SSC at about 37-50°C. The
skilled artisan will recognize how to adjust
the temperature, ionic strength, etc. as necessary to accommodate factors such
as probe length and the like.
The term "epitope tagged" when used herein refers to a chimeric polypeptide
comprising a PRO
polypeptide fused to a "tag polypeptide" . The tag polypeptide has enough
residues to provide an epitope against
which an antibody can be made, yet is short enough such that it does not
interfere with activity of the polypeptide
to which it is fused. The tag polypeptide preferably also is fairly unique so
that the antibody does not
substantially cross-react with other epitopes. Suitable tag polypeptides
generally have at least six amino acid
residues and usually between about 8 and 50 amino acid residues (preferably,
between about 10 and 20 amino
acid residues).
As used herein, the term "immunoadhesin" designates antibody-like molecules
which combine the
binding specificity of a heterologous protein (an "adhesin") with the effector
functions of immunoglobulin
constant domains. Structurally, the irnmunoadhesins comprise a fusion of an
amino acid sequence with the
desired binding specificity which is other than the antigen recognition and
binding site of an antibody (i.e., is
"heterologous"), and an immunoglobulin constant domain sequence. The adhesin
part of an immunoadhesin
molecule typically is a contiguous amino acid sequence comprising at least the
binding site of a receptor or a
ligand. The immunoglobulin constant domain sequence in the immunoadhesin may
be obtained from any
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immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including
IgA-1 and IgA-2), IgE, IgD
or IgM.
"Active" or "activity" for the purposes herein refers to forms) of a PRO
polypeptide which retain a
biological and/or an immunological activity of native or naturally-occurring
PRO, wherein "biological" activity
refers to a biological function (either inhibitory or stimulatory) caused by a
native or naturally-occurring PRO
other than the ability to induce the production of an antibody against an
antigenic epitope possessed by a native
or naturally-occurring PRO and an "immunological" activity refers to the
ability to induce the production of an
antibody against an antigenic epitope possessed by a native or naturally-
occurring PRO.
The term "antagonist" is used in the broadest sense, and includes any molecule
that partially or fully
blocks, inhibits, or neutralizes a biological activity of a native PRO
polypeptide disclosed herein. In a similar
manner, the term "agonist" is used in the broadest sense and includes any
molecule that mimics a biological
activity of a native PRO polypeptide disclosed herein. Suitable agonist or
antagonist molecules specifically
include agonist or antagonist antibodies or antibody fragments, fragments or
amino acid sequence variants of
native PRO polypeptides, peptides, antisense oligonucleotides, small organic
molecules, etc. Methods for
identifying agonists or antagonists of a PRO polypeptide may comprise
contacting a PRO polypeptide with a
candidate agonist or antagonist molecule and measuring a detectable change in
one or more biological activities
normally associated with the PRO polypeptide.
"Treatment" refers to both therapeutic treatment and prophylactic or
preventative measures, wherein
the object is to prevent or slow down (lessen) the targeted pathologic
condition or disorder. Those in need of
treatment include those already with the disorder as well as those prone to
have the disorder or those in whom
the disorder is to be prevented.
"Chronic" administration refers to administration of the agents) in a
continuous mode as opposed to
an acute mode, so as to maintain the initial therapeutic effect (activity) for
an extended period of time.
"Intermittent" administration is treatment that is not consecutively done
without interruption, but rather is cyclic
m nature.
"Mammal" for purposes of treatment refers to any animal classified as a
mammal, including humans,
domestic and farm animals, and zoo, sports, or pet animals, such as dogs,
cats, cattle, horses, sheep, pigs, goats,
rabbits, etc. Preferably, the mammal is human.
Administration "in combination with" one or more further therapeutic agents
includes simultaneous
(concurrent) and consecutive administration in any order.
"Carriers" as used herein include pharmaceutically acceptable carriers,
excipients, or stabilizers which
are nontoxic to the cell or mammal being exposed thereto at the dosages and
concentrations employed. Often
the physiologically acceptable carrier is an aqueous pH buffered solution.
Examples of physiologically
acceptable carriers include buffers such as phosphate, citrate, and other
organic acids; antioxidants including
ascorbic acid; low molecular weight (less than about 10 residues) polypeptide;
proteins, such as serum albumin,
gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine,
glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and
other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols
such as mannitol or sorbitol; salt-
24


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forming counterions such as sodium; and/or nonionic surfactants such as
TWEENT"', polyethylene glycol (PEG),
and PLURONICS~".
"Antibody fragments" comprise a portion of an intact antibody, preferably the
antigen binding or
variable region of the intact antibody. Examples of antibody fragments include
Fab, Fab', F(ab')2, and Fv
fragments; diabodies; linear antibodies (Zapata et al., Protein Ene. 8(10):
1057-1062 [1995]); single-chain
antibody molecules; and multispecific antibodies formed from antibody
fragments.
Papain digestion of antibodies produces two identical antigen-binding
fragments, called "Fab"
fragments, each with a single antigen-binding site, and a residual "Fc"
fragment, a designation reflecting the
ability to crystallize readily. Pepsin treatment yields an F(ab'), fragment
that has two antigen-combining sites
and is still capable of cross-linking antigen.
"Fv" is the minimum antibody fragment which contains a complete antigen-
recognition and -binding
site. This region consists of a dimer of one heavy- and one light-chain
variable domain in tight, non-covalent
association. It is in this configuration that the three CDRs of each variable
domain interact to define an antigen-
binding site on the surface of the VH-VL dimer. Collectively, the six CDRs
confer antigen-binding specificity
to the antibody. However, even a single variable domain (or half of an Fv
comprising only three CDRs specific
for an antigen) has the ability to recognize and bind antigen, although at a
lower affinity than the entire binding
site.
The Fab fragment also contains the constant domain of the light chain and the
first constant domain
(CH 1 ) of the heavy chain. Fab fragments differ from Fab' fragments by the
addition of a few residues at the
carboxy terminus of the heavy chain CH1 domain including one or more cysteines
from the antibody hinge
region. Fab'-SH is the designation herein for Fab' in which the cysteine
residues) of the constant domains bear
a free thiol group. F(ab')z antibody fragments originally were produced as
pairs of Fab' fragments which have
hinge cysteines between them. Other chemical couplings of antibody fragments
are also known.
The "light chains" of antibodies (immunoglobulins) from any vertebrate species
can be assigned to one
of two clearly distinct types, called kappa and lambda, based on the amino
acid sequences of their constant
domains.
Depending on the amino acid sequence of the constant domain of their heavy
chains, immunoglobulins
can be assigned to different classes. There are five major classes of
immunoglobulins: IgA, IgD, IgE, IgG, and
IgM, and several of these may be further divided into subclasses (isotypes),
e.g., IgGl, IgG2, IgG3, IgG4, IgA,
and IgA2.
"Single-chain Fv" or "sFv" antibody fragments comprise the VH and V~ domains
of antibody, wherein
these domains are present in a single polypeptide chain. Preferably, the Fv
polypeptide further comprises a
polypeptide linker between the VH and V~ domains which enables the sFv to form
the desired structure for
antigen binding. For a review of sFv, see Pluckthun in The PharmacoloQV of
Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
The term "diabodies" refers to small antibody fragments with two antigen-
binding sites, which
fragments comprise a heavy-chain variable domain (VH) connected to a light-
chain variable domain (V~) in the
same polypeptide chain (VH-VL). By using a linker that is too short to allow
pairing between the two domains


CA 02380355 2002-03-O1
WO 01/16318 PCT/US00/23328
on the same chain, the domains are forced to pair with the complementary
domains of another chain and create
two antigen-binding sites. Diabodies are described more fully in, for example,
EP 404,097; WO 93/11161; and
Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
An "isolated" antibody is one which has been identified and separated and/or
recovered from a
component of its natural environment. Contaminant components of its natural
environment are materials which
would interfere with diagnostic or therapeutic uses for the antibody, and may
include enzymes, hormones, and
other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the
antibody will be purified (1)
to greater than 95 % by weight of antibody as determined by the Lowry method,
and most preferably more than
99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-
terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-
PAGE under reducing or
nonreducing conditions using Coomassie blue or, preferably, silver stain.
Isolated antibody includes the antibody
in situ within recombinant cells since at least one component of the
antibody's natural environment will not be
present. Ordinarily, however, isolated antibody will be prepared by at least
one purification step.
An antibody that "specifically binds to" or is "specific for" a particular
polypeptide or an epitope on
a particular polypeptide is one that binds to that particular polypeptide or
epitope on a particular polypeptide
without substantially binding to any other polypeptide or polypeptide epitope.
The word "label" when used herein refers to a detectable compound or
composition which is conjugated
directly or indirectly to the antibody so as to generate a "labeled" antibody.
The label may be detectable by itself
(e.g. radioisotope labels or fluorescent labels) or, in the case of an
enzymatic label, may catalyze chemical
alteration of a substrate compound or composition which is detectable.
By "solid phase" is meant a non-aqueous matrix to which the antibody of the
present invention can
adhere. Examples of solid phases encompassed herein include those formed
partially or entirely of glass (e.g.,
controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides,
polystyrene, polyvinyl alcohol and
silicones. In certain embodiments, depending on the context, the solid phase
can comprise the well of an assay
plate; in others it is a purification column (e.g., an affinity chromatography
column). This term also includes
a discontinuous solid phase of discrete particles, such as those described in
U.S. Patent No. 4,275,149.
A "liposome" is a small vesicle composed of various types of lipids,
phospholipids and/or surfactant
which is useful for delivery of a drug (such as a PRO polypeptide or antibody
thereto) to a mammal. The
components of the liposome are commonly arranged in a bilayer formation,
similar to the lipid arrangement of
biological membranes.
A "small molecule" is defined herein to have a molecular weight below about
500 Daltons.
26


CA 02380355 2002-03-O1
WO 01/16318 PCT/US00/23328
Table 1
/*
* C-C
increased
from
12
to
15


* Z
is
average
of
EQ


$ * B
is
average
of
ND


* matchwith stop is M; stop-stop = 0; J (joker) match = 0


* ~


#/defineM -8 /* value of a match with a stop */


int _day[26][26] _ {


/* A B C D E F G H I J K L M N O P Q R S T U V W X Y Z */


/* A { 2, 0,-2, 0, 0,-4, 1,-1,-1, 0,-1,-2,-1, O, M, 1, 0,-2,
*! 1, 1, 0, 0,-6, 0,-3, 0},


/* B { 0, 3,-4, 3, 2,-5, 0, 1,-2, 0, 0,-3,-2, 2, M,-1, 1, 0,
*/ 0, 0, 0,-2,-5, 0,-3, 1},


/* C {-2,-4,15,-5,-5,-4,-3,-3,-2, 0,-5,-6,-5,-4, M,-3,-5,-4,
*/ 0,-2, 0,-2,-8, 0, 0,-5},


/* D { 0, 3,-5, 4, 3,-6, 1, 1,-2, 0, 0,-4,-3, 2, M,-1, 2,-1,
*/ 0, 0, 0,-2,-7, 0,-4, 2},


/* E { 0, 2,-5, 3, 4,-5, 0, 1,-2, 0, 0,-3,-2, 1, M,-1, 2,-1,
*/ 0, 0, 0,-2,-7, 0,-4, 3},


/* F {-4,-5,-4,-6,-5, 9,-5,-2, 1, 0,-5, 2, 0,-4, M,-5,-5,-4,-3,-3,
*/ 0,-1, 0, 0, 7,-5},


/* G { 1, 0,-3, 1, 0,-5, 5,-2,-3, 0,-2,-4,-3, O, M,-1,-1,-3,
*/ 1, 0, 0,-1,-7, 0,-5, 0},


/* H {-1, 1,-3, 1, 1,-2,-2, 6,-2, 0, 0,-2,-2, 2, M, 0, 3, 2,-1,-1,
*/ 0,-2,-3, 0, 0, 2},


/* I {-1,-2,-2,-2,-2, 1,-3,-2, 5, 0,-2, 2, 2,-2, M,-2,-2,-2,-1,
*/ 0, 0, 4,-5, 0,-1,-2},


/* J { 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, O, M, 0, 0, 0,
*/ 0, 0, 0, 0, 0, 0, 0, 0},


/* K {-1, 0,-5, 0, 0,-S,-2, 0,-2, 0, 5,-3, 0, 1, M,-1, 1, 3,
*/ 0, 0, 0,-2,-3, 0,-4, 0},


/* L {-2,-3,-6,-4,-3, 2,-4,-2, 2, 0,-3, 6, 4,-3, M,-3,-2,-3,-3,-1,
*/ 0, 2,-2, 0,-1,-2},


/* M {-1,-2,-5,-3,-2, 0,-3,-2, 2, 0, 0, 4, 6,-2, M,-2,-1, 0,-2,-1,
*/ 0, 2,-4, 0,-2,-1},


/* N { 0, 2,-4, 2, 1,-4, 0, 2,-2, 0, 1,-3,-2, 2, M,-1, 1, 0,
*/ 1, 0, 0,-2,-4, 0,-2, 1},


/* O { M,_M,
*/ M,_M, M, M. M, M, M, M, M, M, M, M, O, M, M, M, M, M, M,
M, M, M, M, M},


/* P _
*/ { 1,-1,-3 1,-1,-5,-1, 0,-2, 0,-1,-3,-2,-1, M, 6, 0, 0,
1, 0, 0,-1,-6, 0,-5, 0},


/* Q { 0, 1,-5, 2, 2,-5,-1, 3,-2, 0, 1,-2,-1, 1,_M, 0, 4, 1,-1,-1,
*/ 0,-2,-5, 0,-4, 3},


/* R {-2, 0,-4,-1,-1,-4,-3, 2,-2, 0, 3,-3, 0, O, M, 0, 1, 6,
*/ 0,-1, 0,-2, 2, 0,-4, 0},


/* S { 1, 0, 0, 0, 0,-3, 1,-1,-1, 0, 0,-3,-2, 1, M, 1,-1, 0,
*/ 2, 1, 0,-1,-2, 0,-3, 0},


/* T { 1, 0,-2, 0, 0,-3, 0,-1, 0, 0, 0,-1,-1, O, M, 0,-1,-1,
*/ 1, 3, 0, 0,-5, 0,-3, 0},


/* U { 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, O, M, 0, 0, 0,
*/ 0, 0, 0, 0, 0, 0, 0, 0},


/* V { 0,-2,-2,-2,-2,-1,-1,-2, 4, 0,-2, 2, 2,-2, M,-1,-2,-2,-1,
*/ 0, 0, 4,-6, 0,-2,-2},


/* W {-6,-5,-8,-7,-7, 0,-7,-3,-5, 0,-3,-2,-4,-4, M,-6,-5, 2,-2,-5,
*/ 0,-6,17, 0, 0,-6},


/* x { o, o, o, o, o, o, o, o, o, o, o, o, o, o,
*/ M, o, o, o, o, o, o, o, o, o, o, o},


/* Y _
*/ {-3,-3, 0,-4,-4, 7,-5, 0,-1, 0,-4,-1,-2,-2, M,-5,-4,-4,-3,-3,
0,-2, 0, 0,10,-4},


/* Z { 0, 1,-5, 2, 3,-5, 0, 2,-2, 0, 0,-2,-1, 1, M, 0, 3, 0,
*/ 0, 0, 0,-2,-6, 0,-4, 4}


};


45
55
27


CA 02380355 2002-03-O1
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Table 1 ~cont')
/*


*/


#include<
stdio.
h
>


#include<
ctype.h
>



#defineMAXJMP16 /* max jumps in a diag */


#def'meMAXGAP /* don't continue to penalize
24 gaps larger than this */


#defineJMPS 1024 /* max jmps in an path */


#defineMX 4 /* save if there's at least
MX-1 bases since last jmp
*/



#defineDMAT 3 /* value of matching bases
*/


#defineDMIS 0 /* penalty for mismatched
bases */


#defineDINSO8 /* penalty for a gap */


#defineDINSI1 /* penalty per base */


IS #definePINSO8 /* penalty for a gap */


#definePINS14 /* penalty per residue *!


struct
jmp
{


shortn[MAXJMP];
/* size
of jmp
(neg for
dely)
*/


unsignedshort x[MAXJMP];
/* base
no. of
jmp in
seq x
*!


}; /* limits seq to 2"16 -1 */


struct
diag
{


int score; /* score at lastjmp */


long offset; /* offset of prev block */


shortijmp; /* current jmp index */


struct /* list of jmps */
jmp
jp;


};


struct
path
{


int spc; /* number of leading spaces
*/


shortn[JMPS];
/* size
of jmp
(gap)
*/


int x[JMPS]; jmp (last elem before gap)
/* loc */
of


};



char *ofile; /* output file name */


char *namex[2];/* seq names: getseqsQ */


char *prog; /* prog name for err msgs
*/


char *seqx[2]; /* seqs: getseqs0 */


int dmax; /* best diag: nwQ */


int dmax0; /* final diag */


int dna; /* set if dna: main() */


int endgaps; /* set if penalizing end gaps
*/


int gapx, gapy;/* total gaps in seqs */


int len0, lenl;/* seq lens */


int ngapx, /* total size of gaps */
ngapy;


int smax; /* max score: nw() */


int *xbm; l* bitmap for matching */


long offset; /* current offset in jmp file
*/


structdiag *dx; /* holds diagonals */


structpath pp(2]; /* holds path for seqs */


char *callocQ, Q, *indexQ, *strcpy0;
*malloc


char *getseqQ,
*g callocQ;


5 5


28


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Table 1 (cony)
/* Needleman-Wunsch alignment program
* usage: progs filet filet
* where filet and filet are two dna or two protein sequences.
* The sequences can be in upper- or lower-case an may contain ambiguity
* Any lines beginning with '; ' > ' or ' < ' are ignored
* Max file length is 65535 (limited by unsigned short x in the jmp struct)
* A sequence with 1/3 or more of its elements ACGTU is assumed to be DNA
* Output is in the file "align.out"
* The program may create a tmp file in /tmp to hold info about traceback.
* Original version developed under BSD 4.3 on a vax 8650
*/
#include "nw.h"
IS #include "day.h"
static _dbval[26] _ {
1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0
static ~bval[26] _ {
1, 2~(1< <('D'-'A'))~(1 < <('N'-'A')), 4, 8, 16, 32, 64,
128, 256, OxFFFFFFF, I < < 10, 1 < < I I, 1 < < 12, 1 < < 13, 1 < < 14,
1«15, 1«16, 1«17, I«18, 1«19, 1«20, I«21, 1«22,
1«23, 1«24, 1«25(1«('E'-'A'))~(1«('Q'-'A'))
main(ac, av) main
int ac;
char *av( ];
{
prog = av(0];


if(ac!=3){


fprintf(stderr,"usage: %s filet filet\n", prog);


fprintf(stderr,"where filet and filet are two dna
or two protein sequences.\n");


fprintf(stderr,"The sequences can be in upper-
or lower-case\n");


fprintf(stderr, "Any lines beginning with ';' or
' < ' are ignored\n");


fprintf(stderr,"Output is in the file \"align.out\"\n");


exit( 1 );


)


namex[0] = av[1];


namex(1] = av[2];


seqx[0] = getseq(namex[0], &len0);


seqx[I] = getseq(namex[1], &lenl);


xbm = (dna)? dbval : .pbval;


endgaps = 0; /* 1 to penalize endgaps */


ofile = "align.out"; /* output file */


nw(); /* fill in the matrix, get the possible jmps
*/


readjmpsQ; /* get the actual jmps *!


print(); /* print slats, alignment */


cleanup(0); /* unlink any tmp files */
)
29


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Table 1 (cony)
/* do the alignment, return best score: main()
* dna: values in Fitch and Smith, PNAS, 80, 1382-1386, 1983
* pro: PAM 250 values
* When scores are equal, we prefer mismatches to any gap, prefer
* a new gap to extending an ongoing gap, and prefer a gap in seqx
* to a gap in seq y.
*/
nw
nwQ
{
char *px, *py; /* seqs and ptrs */
int *ndely, *dely; /* keep track of dely */
int ndelx, delx; /* keep track of delx */
int *tmp; /* for swapping row0, rowl */
int mis; /* score for each type */
IS int ins0, insl; /* insertion penalties */
register id; /* diagonalindex */
register ij; /* jmp index */
register *col0, *coll; /* score for curr, last row */
register xx, yy; /* index into seqs */
dx = (struct diag *)g calloc("to get diags", len0+lenl+1, sizeof(struct
diag));
ndely = (int *)g calloc("to get ndely", lent+1, sizeof(int));
dely = (int *)g calloc("to get dely", lent+1, sizeof(int));
col0 = (int *)g calloc("to get col0", lenl + 1, sizeof(int));
coil = (int *)g calloc("to get coil", lenl+1, sizeof(int));
ins0 = (dna)? DINSO : PINSO;
ins 1 = (dna)? DINS 1 : PINS 1;
smax = -10000;
if (endgaps)
for (col0[0] = dely[0] _ -ins0, yy = 1; yy < = lenl; yy++) {
col0[yy] = dely[yy] = col0[yy-1] - insl;
ndely[yy] = yy;
)
col0[0] = 0; /* Waterman Bull Math Biol 84 */
{
else
for (yy = 1; yy < = lenl; yy++)
dely[yy] _ -ins0;
/* fill in match matrix
*1
for (px = seqx[0], xx = 1; xx < = len0; px++, xx++)
/* initialize first entry in col
*/
if (endgaps) {
if (xx == 1)
coil[0] = delx = -(ins0+insl);
else
colt[0] = delx = col0[0) - insl;
ndelx = xx;
{
else {
col l [0] = 0;
delx = -ins0;
ndelx = 0;
30


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Table 1 (cony)
for (py = seqx[1], yy = 1; yy < = lenl; py++,
yy++) {


mis = col0[yy-1];


if (dna)


mis +_ (xbm[*px-'A']&xbm[*py-'A'])? DMAT : DMIS;


else


mis += day[*px-'A'][*py-'A'];


/* update penalty for del in x seq;


* favor new del over ongong del


* ignore MAXGAP if weighting endgaps


*/


if (endgaps ~ ~ ndely[yy] < MAXGAP) {


if (col0[yy] - ins0 > = dely[yy]) {


dely[yy] = col0[yy] - (ins0+insl);


ndely(yy] = 1;


} else {


dely[yy] -= insl;


ndely[yy] + +;


}


} else {


if (col0[yy] - (ins0+insl) > = dely[yy]) {


dely[yy] = col0[yy] - (ins0+insl);


ndely[yy] = 1;


} else


ndely[yy] + +;


}


/* update penalty for del in y seq;


* favor new del over ongong del


*/


if (endgaps ~ ~ ndelx < MAXGAP) {


if (coll[yy-1] - ins0 > = delx) {


delx = colt[yy-1] - (ins0+insl);


ndelx = 1;


else


deli -= insl;


ndelx+ +;


}
} else {
if (coll[yy-1] - (ins0+insl) > = delx) {
delx = colt[yy-1] - (ins0+insl);
ndelx = 1;
} else
ndelx+ +;
}
/* pick the maximum score; we're favoring
* mis over any del and delx over dely
*/
60
...nw
31


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Table 1 (cont'1
id = xx - yy + lent - 1;
if (mis > = delx && mis > = dely(yy])
col l [yy] = mis;
else if (delx > = dely[yy]) {
col l [yy] = delx;
ij = dx[id].ijmp;
if (dx[id].jp.n[0] && (!dna ~ ~ (ndelx > = MAXJMP
&& xx > dx[id].jp.x[ij]+MX) ~ ~ mis > dx[id].score+DINSO)) {
dx[id].ijmp++;
if (++ij > = MAXJMP) {
writejmps(id);
ij = dx[id].ijmp = 0;
dx[id].offset = offset;
1$ offset += sizeof(struct jmp) + sizeof(offset);
}
}
dx[id].jp.n[ij] = ndelx;
dx[id].jp.x[ij] = xx;
dx[id].score = delx;
}
else {
coll(yy] = dely[yy];
ij = dx[id].ijmp;
if (dx[id].jp.n[0] && (!dna ~ ~ (ndely[yy] > = MAXJMP
&& xx > dx[id].jp.x[ij]+MX) ~ ~ mis > dx(id].score+DINSO)) {
dx[id].ijmp++;
if (++ij > = MAXJMP) {
writejmps(id);
ij = dx[id].ijmp = 0;
dx[id].offset = offset;
offset + = sizeof(struct jmp) + sizeof(offset);
}
dx[id].jp.n[ij] _ -ndely(yy];
dx[id].jp.x[ij] = xx;
dx[id].score = dely[yy];
}
if (xx == len0 && yy < lent) {
/* last col
*/
if (endgaps)
coll(yy] -= ins0+insl*(lenl-yy);
if (col l [yy] > smax)
smax = coil[yy];
dmax = id;
}
}
}
if (endgaps && xx < len0)
coil[yy-1] -= ins0+insl*(len0-xx);
if (toll[yy-1] > smax) {
smax = coil[yy-1];
dmax = id;
tmp = col0; col0 = toll; coil = tmp;
}
(void) free((char *)ndely);
(void) free((char *)dely);
(void) free((char *)col0);
(void) free((char *)coll); }
...nw
32


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Table 1 (cony)
/*
* print() -- only routine visible outside this module
* static:
* getmatQ -- trace back best path, count matches: print()
* pr align() -- print alignment of described in array p[ ]: print()
* dumpblockQ -- dump a block of lines with numbers, stars: pr align()
* numsQ -- put out a number line: dumpblockQ
* putlineQ -- put out a line (name, [num], seq, [num]): dumpblockQ
* stars() - -put a line of stars: dumpblockQ
* stripnameQ -- strip any path and prefix from a seqname
*/
#include "nw.h"
#defme SPC 3
#define P_LINE 256 /* maximum output line */
#defme P_SPC 3 /* space between name or num and seq */
extern _day[26][26];
int olen; /* set output line length */
FILE *fx; /* output file */
print()
print
{
int lx, 1y, firstgap, lastgap; /* overlap */
if ((fx = fopen(ofile, "w")) _ = 0) {
fprintf(stderr, " % s: can't write % s\n", prog, ofile);
cleanup(1);
fprintf(fx, " < first sequence: % s (length = % d)\n", namex[0], len0);
fprintf(fx, "<second sequence: %s (length = %d)\n", namex[1], lenl);
olen = 60;
lx = IenO;
1y = lent;
firstgap = lastgap = 0;
if (dmax < lenl - 1) { /* leading gap in x */
pp[0].spc = firstgap = lent - dmax - 1;
1y -= pp[0].spc;
else if (dmax > lenl - 1) { /* leading gap in y */
pp[1].spc = firstgap = dmax - (lenl - 1);
4$ lx -= pp[1].spc;
if (dmax0 < len0 - 1) { /* trailing gap in x */
lastgap = len0 - dmax0 -l;
lx -= lastgap;
else if (dmax0 > IenO - 1) { /* trailing gap in y */
lastgap = dmax0 - (len0 - 1);
1y - = lastgap;
SS getmat(Ix, 1y, firstgap, lastgap);
pr align();
33


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Table 1 (cony)
/*
* trace back the best path, count matches
*/
static
getmat(lx, 1y, firstgap, lastgap) getIriat
int lx, 1y; /* "core" (minus endgaps) */
int firstgap, lastgap; ' /* leading trailing overlap */
{
int nm, i0, i1, siz0, sizl;
char outx(32];
double pct;
register n0, n1;
register char *p0, *pl;
/* get total matches, score
*/
i0 = i1 = siz0 = sizl = 0;
p0 = seqx[0] + pp[1].spc;
p1 = seqx[1] + pp[0].spc;
n0 = pp[1].spc + 1;
n1 = pp[0].spc + 1;
nm=0;
while ( *p0 && *pl ) {
if (siz0) {
p1++;
n1++;
siz0--;
else if (sizl) {
p0++;
n0++;
sizl--;
else {
if (xbm[*p0-'A']&xbm[*pl-'A'])
nm++;
if (n0++ _= pp[0].x[i0])
siz0 = pp[0].n[i0++];
if (n1++ _= pp[1].x[il])
sizl = pp[1].n[il++];
p0++;
p1++;
)
)
/* pct homology:
* if penalizing endgaps, base is the shorter seq
* else, knock off overhangs and take shorter core
*/
if (endgaps)
lx = (IenO < lenl)? len0 : lenl;
else
lx = (Lx < 1y)? lx : 1y;
pct = 100.*(double)nm/(double)lx;
fprintf(fx, "\n");
fprintf(fx, " < %d match% s in an overlap of %d: %.2f percent similarity\n",
ntn, (nm == 1)? "' . "es", lx, pct);
34


CA 02380355 2002-03-O1
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Table 1 (cony)
fprintf(fx, "<gaps in first sequence: %d", gapx); ...getillat
if (gapx)
(void) sprintf(outx, " ( % d % s % s)",
S ngapx, (dna)? "base":"residue", (ngapx == 1)? "':"s");
fprintf(fx," % s", outx);
fprintf(fx, ", gaps in second sequence: %d", gapy);
if (gaPY) {
(void) sprintf(outx, " ( % d % s % s)",
ngapy, (dna)? "base":"residue", (ngapy == 1)? "':"s");
fprintf(fx,"%s", outx);
}
if (dna)
IS fprintf(fx,
"\n < score: % d (match = % d, mismatch = % d, gap penalty = % d + %d per
base)\n",
smax, DMAT, DMIS, DINSO, DINS1);
else
fprintf(fx,
"\n < score: %d (Dayhoff PAM 250 matrix, gap penalty = % d + % d per
residue)\n",
smax, PINSO, PINS1);
if (endgaps)
fprintf(fx,
"<endgaps penalized. left endgap: %d %s%s, right endgap: %d %s%s\n",
2$ firstgap, (dna)? "base" : "residue", (firstgap == 1)? "' . "s",
lastgap, (dna)? "base" : "residue", (lastgap == 1)? "' . "s");
else
fprintf(fx, " < endgaps not penalized\n" );
}
static nm; /* matches in core -- for checking */
static lmax; /* lengths of stripped file names */
static ij[2]; /* jmp index for a path */
static nc[2]; /* number at start of current line */
static ni[2]; /* current elem number -- for gapping */
static siz[2];
static char *ps[2]; /* ptr to current element */
static char *po[2]; /* ptr to next output char slot */
static char out[2][P LINE]; /* output line */
static char star[P LINE]; /* set by stars() */
/*
* print alignment of described in struct path pp [ ]
*/
static
pr align() pr ahgri
int nn; /* char count */
int more;
register i;
for (i = 0, lmax = 0; i < 2; i++) {
nn = stripname(namex[i]);
if (nn > lmax)
lmax = nn;
nc[i] = 1;
ni[i] = l;
' siz[i] = ij[i] = 0;
ps[i] = seqx[i];
po[i] = out[i]; }


CA 02380355 2002-03-O1
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Table 1 (cony)
for (nn = nm = 0, more = 1; more; ) { ... pr align
for (i = more = 0; i < 2; i++) {
/*
* do we have more of this sequence?
*/
if (!*ps[i])
continue;
more++;
if (pp[i].spc) { /* leading space */
*po[i]++ _ ' ,
pp[i] . spc--;
IS )
else if (siz[i]) { /* in a gap */
*po[i]++ _ ,
siz[i]--;
else { l* we're putting a seq element
*/
*Po[i] _ *Ps[i];
if (islower(*ps[i]))
*ps[i] = toupper(*ps[i]);
po[i]++;
ps[i] + +;
/*
* are we at next gap for this seq?
*/
if (ni[i] _= pp[i].x[ij[i]]) {
/*
* we need to merge all gaps
* at this location
*/
siz[i] = pp[i].n[ij[i]++];
while (ni[i] _= pp[i].x[ij[i]])
siz[i] += pp[i].n[ij[i]++];
ni[i] + +;
)
)
if (++nn == olen ~ ( !more && nn) {
dumpblockQ;
for (i = 0; i < 2; i++)
po[i] = out[i];
nn = 0;
/*
* dump a block of lines, including numbers, stars: pr align()
*%
static
dumpblockQ dumpblock
{
register i;
for (i = 0; i < 2; i++)
*po[i]__ _ '\0';
36


CA 02380355 2002-03-O1
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Table 1 (cony)
(void) putc(' \n' , fx);
for (i = 0; i < 2; i++)
if (*out[i] && (*out[i] ! _ ' ' I I *(po[i]) ! _ ' ')) {
if (i == 0)
nums(i);
if (i == 0 && *out[1])
starsQ;
putline(i);
if (i == 0 && *out[1])
fprintf(fx, star);
if (i == 1)
nums(i);
1$
... dumpblock
/*
* put out a number line: dumpblockQ
*/
static
nums(ix) nums
int ix; /* index in out[] holding seq line */
{
char mine[P LINE];
register i, j;
register char *pn, *px, *py;
for (pn = nline> i = 0; i < lmax+P SPC; i++, pn++)
*Pn = >
for (i = nc[ix], py = out[ix]; *py; py++, pn++)
if (*py =- ' I I *PY =_ -')
*Pn = >
else {
if (i% 10 == 0 I I (i == 1 && nc[ix] != 1)) {
j = (i < 0)? -i : i;
for (px = pn; j; j /= 10, px--)
*px=j%10+'0';
if (i < o)
*px = >
{
else
*Pn = >
i++;
)
)
*Pn = ~ \0' ;
nc[ix] = i;
for (pn = mine; *pn; pn++)
(void) putc(*pn, fx);
(void) putc('\n', fx);
/*
* put out a line (name, [num], seq, [num]): dumpblock()
*/
static
putline(ix) puthrie
f)0 int ix; {
37


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Table 1 (cony)
...putline
int i;


register char *px;



for (px = namex[ix], i = 0; *px && *px ! _ ':'; px++, i++)


(void) putt(*px, fX);


for (; i < lmax+P SPC; i++)


(void) putt(' ', fx);



/* these count from 1:


* ni[] is current element (from 1)


* nc[] is number at start of current line


*/


IS for (px = out[ix]; *px; px++)


(void) putt(*px&Ox7F, fx);


(void) putt('\n', fx);



/*


* line of stars (seqs always in out[0], out[1]): dumpblockQ
put
a


*/


static


stars()
stars


{


int i;


register char *p0, *p 1, cx, *px;


if (!*out[0] ~ ~ (*out[0] _- ' ' && *(po(0]) _- ' ') ~ ~


!*out[1] ~ ~ (*out[1] _- ' && *(po[1]) _- ' '))


return;


px = star;


for (i = lmax+P SPC; i; i--)


*px++ _ ,


for (p0 = out[0], p1 = out[1]; *p0 && *pl; p0++, p1++) {


if (isalpha(*p0) && isalpha(*pl)) {


if (xbm[*p0-'A')&xbm[*pl-'A']) {


cx = '*';


nm++;



else if (!dna && day[*p0-'A'][*pl-'A'] > 0)


cx = . ,


else


cx = ,



else


cx = ,


*px++ = cx;



*px++ _ '\n';


*px = '\0';


i


38


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Table 1 (cony)
/*
* strip path or prefix from pn, return len: pr align()
*%
static
stripname(pn) stripname
char *pn; /* file name (may be path) */
f
register char *px, *py;
py=0;
for (px = pn; *px; px++)
if (*px =- '/')
py=px+1;
if (pY)
IS (void) strcpy(pn, py);
return(strlen(pn));
25
35
45
55
39


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Table 1 (cony)
/*
* cleanup() -- cleanup any tmp file
* getseqQ -- read in seq, set dna, len, maxlen
* g calloc() -- callocQ with error checkin
* readjmpsQ -- get the good jmps, from tmp file if necessary
* writejmpsQ -- write a filled array of jmps to a tmp file: nwQ
*/
#include "nw.h"
#include < syslfile.h >
char *jname = "/tmp/homgXXXXXX"; /* tmp file for jmps */
FILE *fj;
int cleanupQ; /* cleanup tmp file */
IS long lseek0;
/*
* remove any tmp file if we blow
*/
cleanup(i) Cleanup
int i;
{
if (fj)
(void) unlink(jname);
exit(i);
/*
* read, return ptr to seq, set dna, len, maxlen
* skip lines starting with ' ; , ' < ' , or ' > '
* seq in upper or lower case
*/
char
getseq(file, len) getseq
char *file; /* file name */
int *len; /* seq len */
{
char line[1024], *pseq;
register char *px, *py;
int natgc, tlen;
FILE *fp;
if ((fp = fopen(file,"r")) _ = 0) {
fprintf(stderr,"%s: can't read %s\n", prog, file);
exit( 1 );
I
tlen = natgc = 0;
while (fgets(line, 1024, fp)) {
if (*line =- ' ~ ~ *line =- ' <' ~ ~ *line =- ' >')
continue;
for (px = line; *px !_ '\n'; px++)
if (isupper(*px) ~ ~ islower(*px))
tlen++;
if ((pseq = malloc((unsigned)(tlen+6))) _ = 0) {
fprintf(stderr,"%s: mallocQ failed to get %d bytes for %s\n", prog, tlen+6,
file);
exit( 1 );
pseq[0] = pseq[1] = pseq[2] = pseq[3] _ '\0';
40


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Table 1 (cony)
...getseq
py = pseq + 4;
*len = tlen;
rewind(fp);
while (fgets(line, 1024, fp)) {
if (*line =- ' ~ C *line =- ' <' ~ ~ *line =- ' >')
continue;
for (px = line; *px ! _ '\n'; px++) {
if (isupper(*px))
*py + + _ *px;
else if (islower(*px))
*py++ = toupper(*px);
if (index("ATGCU",*(py-1)))
natgc+ +;
1
*py++ _ '\0';
*PY = ~\0~;
(void) fclose(fp);
dna = natgc > (tlen/3);
return(pseq+4);
char
g calloc(msg, nx, sz) g-CaI~OC
char *msg; /* program, calling routine */
int nx, sz; /* number and size of elements */
{
char *px, *calloc0;
if ((px = calloc((unsigned)nx, (unsigned)sz)) _ = 0) {
if (*msg) {
fprintf(stderr, "%s: g callocQ failed %s (n=%d, sz=%d)\n", prog, msg, nx, sz);
exit(1);
return(px);
/*
* get final jmps from dx[] or tmp file, set pp[], reset dmax: main()
*/
readjmps0 readjmps
{
int fd = -1;
int siz, i0, i1;
register i> j, xx;
if (tj) {
(void) fclose(fj);
if ((fd = open(jname, O_RDONLY, 0)) < 0) {
fprintf(stderr, "%s: can't open() %s\n", prog, jname);
cleanup( 1 );
1
)
for (i = i0 = i1 = 0, dmax0 = dmax, xx = len0; ; i++)
while (1) {
for (j = dx[dmax].ijmp; j > = 0 && dx[dmax].jp.x[j] > = xx; j--)
,
41


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Table 1 (cony)
...readjmps
if (j < 0 && dx(dmax].offset && fj) {
(void) lseek(fd, dx[dmax].offset, 0);
(void) read(fd, (char *)&dx[dmax].jp, sizeof(struct jmp));
(void) read(fd, (char *)&dx[dmax].offset, sizeof(dx[dmax].offset));
dx[dmax].ijmp = MAXJMP-1;
else
break;
if (i > = JMPS) {
fprintf(stderr, " % s: too many gaps in alignment\n", prog);
cleanup(1);
if (j >=o){
siz = dx[dmax].jp.n[j];
xx = dx[dmax].jp.x[j];
dmax + = siz;
if (siz < 0) { /* gap in second seq */
pp[1].n[il] _ -siz;
xx + = siz;
/*id=xx-yy+lenl-1
*/
pp(1].x(il] = xx - dmax + lenl - 1;
gapy+ +;
ngapy -= siz;
/* ignore MAXGAP when doing endgaps */
siz = (-siz < MAXGAP ~ ~ endgaps)? -siz : MAXGAP;
i1++;
1
else if (siz > 0) { /* gap in first seq */
pp[0].n[i0] = siz;
pp[0].x[i0] = xx;
gapx+ +;
ngapx + = siz;
/* ignore MAXGAP when doing endgaps */
siz = (siz < MAXGAP ~ ~ endgaps)? siz : MAXGAP;
i0++;
1
else
break;
/* reverse the order of jmps
*/
for (j = 0, i0--; j < i0; j + + , i0--) {
i = pp[0].n[j]; pp[0].n[j] = pp[0].n[i0]; pp[0].n[i0] = i;
i = pp[0].x[j]; pp[0].x[j] = pp[0].x[i0]; pp[0].x[i0] = i;
for (j = 0, i1--; j < i1; j++, i1--) {
i = pp[1].n[j]; pp[1].n[j] = pp[1].n[il]; pp[1].n[il] = i;
i = pp[1].x[j]; pp[1].x[j] = pp[1].x[il]; pp[1].x[il] = i;
if (fd > = 0)
(void) close(fd);
if (fj) {
(void) unlink(jname);
fj=0;
offset = 0;
42


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Table 1 (cony)
/*
* write a filled jmp struct offset of the prev one (if any): nw()
*/
writejmps(ix) Writejmps
int ix;
char *mktempQ;
if (!fj) {
if (mktemp(jname) < 0) {
fprintf(stderr, "%s: can't mktempQ %s\n", prog, jname);
cleanup(1);
IS if ((fj = fopen(jname, "w")) _= 0) {
fprintf(stderr, "%s: can't write %s\n", prog, jname);
exit( 1 );
(void) fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1, fj);
(void) fwrite((char *)&dx[ix].offset, sizeof(dx[ix].offset), l, fj);
30
40
50
60
43


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Table 2
PRO XXXXXXXXXXXXXXX (Length = 15 amino acids)
Comparison Protein XXXXXYYYYYYY (Length = 12 amino acids)
% amino acid sequence identity =
(the number of identically matching amino acid residues between the two
polypeptide sequences as determined
by ALIGN-2) divided by (the total number of amino acid residues of the PRO
polypeptide) _
5 divided by 15 = 33.3
44


CA 02380355 2002-03-O1
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Table 3
PRO XXXXXXXXXX (Length = 10 amino acids)
Comparison Protein XXXXXYYYYYYZZYZ (Length = 15 amino acids)
% amino acid sequence identity =
(the number of identically matching amino acid residues between the two
polypeptide sequences as determined
by ALIGN-2) divided by (the total number of amino acid residues of the PRO
polypeptide) _
5 divided by 10 = 50%


CA 02380355 2002-03-O1
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Table 4
PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides)
Comparison DNA NNNNNNLLLLLLLLLL (Length = 16 nucleotides)
% nucleic acid sequence identity =
(the number of identically matching nucleotides between the two nucleic acid
sequences as determined by
ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic
acid sequence) _
6 divided by 14 = 42.9
46


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Table 5
PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides)
Comparison DNA NNNNLLLVV (Length = 9 nucleotides)
% nucleic acid sequence identity =
(the number of identically matching nucleotides between the two nucleic acid
sequences as determined by
ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic
acid sequence) _
4 divided by 12 = 33.3
47


CA 02380355 2002-03-O1
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II. Compositions and Methods of the Invention
A. Full-Length PRO Polypeptides
The present invention provides newly identified and isolated nucleotide
sequences encoding polypeptides
referred to in the present application as PRO polypeptides. In particular,
cDNAs encoding various PRO
polypeptides have been identified and isolated, as disclosed in further detail
in the Examples below. It is noted
that proteins produced in separate expression rounds may be given different
PRO numbers but the UNQ number
is unique for any given DNA and the encoded protein, and will not be changed.
However, for sake of
simplicity, in the present specification the protein encoded by the full
length native nucleic acid molecules
disclosed herein as well as all further native homologues and variants
included in the foregoing definition of
PRO, will be referred to as "PRO/number", regardless of their origin or mode
of preparation.
As disclosed in the Examples below, various cDNA clones have been deposited
with the ATCC. The
actual nucleotide sequences of those clones can readily be determined by the
skilled artisan by sequencing of the
deposited clone using routine methods in the art. The predicted amino acid
sequence can be determined from
the nucleotide sequence using routine skill. For the PRO polypeptides and
encoding nucleic acids described
herein, Applicants have identified what is believed to be the reading frame
best identifiable with the sequence
information available at the time.
B. PRO Polypeptide Variants
In addition to the full-length native sequence PRO polypeptides described
herein, it is contemplated that
PRO variants can be prepared. PRO variants can be prepared by introducing
appropriate nucleotide changes into
the PRO DNA, and/or by synthesis of the desired PRO polypeptide. Those skilled
in the art will appreciate that
amino acid changes may alter post-translational processes of the PRO, such as
changing the number or position
of glycosylation sites or altering the membrane anchoring characteristics.
Variations in the native full-length sequence PRO or in various domains of the
PRO described herein,
can be made, for example, using any of the techniques and guidelines for
conservative and non-conservative
mutations set forth, for instance, in U.S. Patent No. 5,364,934. Variations
may be a substitution, deletion or
insertion of one or more codons encoding the PRO that results in a change in
the amino acid sequence of the
PRO as compared with the native sequence PRO. Optionally the variation is by
substitution of at least one amino
acid with any other amino acid in one or more of the domains of the PRO.
Guidance in determining which
amino acid residue may be inserted, substituted or deleted without adversely
affecting the desired activity may
be found by comparing the sequence of the PRO with that of homologous known
protein molecules and
minimizing the number of amino acid sequence changes made in regions of high
homology. Amino acid
substitutions can be the result of replacing one amino acid with another amino
acid having similar structural
and/or chemical properties, such as the replacement of a leucine with a
serine, i.e., conservative amino acid
replacements. Insertions or deletions may optionally be in the range of about
1 to 5 amino acids. The variation
allowed may be determined by systematically making insertions, deletions or
substitutions of amino acids in the
sequence and testing the resulting variants for activity exhibited by the full-
length or mature native sequence.
48


CA 02380355 2002-03-O1
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PRO polypeptide fragments are provided herein. Such fragments may be truncated
at the N-terminus
or C-terminus, or may lack internal residues, for example, when compared with
a full length native protein.
Certain fragments lack amino acid residues that are not essential for a
desired biological activity of the PRO
polypeptide.
PRO fragments may be prepared by any of a number of conventional techniques.
Desired peptide
fragments may be chemically synthesized. An alternative approach involves
generating PRO fragments by
enzymatic digestion, e.g., by treating the protein with an enzyme known to
cleave proteins at sites defined by
particular amino acid residues, or by digesting the DNA with suitable
restriction enzymes and isolating the
desired fragment. Yet another suitable technique involves isolating and
amplifying a DNA fragment encoding
a desired polypeptide fragment, by polymerase chain reaction (PCR).
Oligonucleotides that define the desired
termini of the DNA fragment are employed at the 5' and 3' primers in the PCR.
Preferably, PRO polypeptide
fragments share at least one biological and/or immunological activity with the
native PRO polypeptide disclosed
herein.
In particular embodiments, conservative substitutions of interest are shown in
Table 6 under the heading
of preferred substitutions. If such substitutions result in a change in
biological activity, then more substantial
changes, denominated exemplary substitutions in Table 6, or as further
described below in reference to amino
acid classes, are introduced and the products screened.
49


CA 02380355 2002-03-O1
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Table 6
Original Exemplary Preferred


Residue Substitutions Substitutions


Ala (A) val; leu; ile val


Arg (R) lys; gln; asn lys


Asn (N) gln; his; lys; arg gln


Asp (D) glu glu


Cys (C) ser ser


Gln (Q) asn asn


Glu (E) asp asp


Gly (G) pro; ala ala


His (H) asn; gln; lys; arg arg


Ile (I) leu; val; met; ala; phe;


norleucine leu


Leu (L) norleucine; ile; val;


met; ala; phe ile


Lys (K) arg; gln; asn arg


Met (M) leu; phe; ile leu


Phe (F) leu; val; ile; ala; tyr leu


Pro (P) ala ala


Ser (S) thr thr


Thr (T) ser ser


Trp (W) tyr; phe tyr


Tyr (Y) trp; phe; thr; ser phe


Val (V) ile; leu; met; phe;


ala; norleucine leu


Substantial modifications in function or immunological identity of the PRO
polypeptide are accomplished
by selecting substitutions that differ significantly in their effect on
maintaining (a) the structure of the polypeptide
backbone in the area of the substitution, for example, as a sheet or helical
conformation, (b) the charge or
hydrophobicity of the molecule at the target site, or (c) the bulk of the side
chain. Naturally occurring residues
are divided into groups based on common side-chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;
(4) basic: asn, gln, his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and
(6) aromatic: trp, tyr, phe.
Non-conservative substitutions will entail exchanging a member of one of these
classes for another class.
Such substituted residues also may be introduced into the conservative
substitution sites or, more preferably, into
the remaining (non-conserved) sites.
The variations can be made using methods known in the art such as
oligonucleotide-mediated (site-
directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed
mutagenesis [Carter et al., Nucl.
Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)],
cassette mutagenesis [Wells et
al., Gene, 34:315 (1985)], restriction selection mutagenesis [Wells et al.,
Philos. Trans. R. Soc. London SerA,


CA 02380355 2002-03-O1
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317:415 (1986)] or other known techniques can be performed on the cloned DNA
to produce the PRO variant
DNA.
Scanning amino acid analysis can also be employed to identify one or more
amino acids along a
contiguous sequence. Among the preferred scanning amino acids are relatively
small, neutral amino acids. Such
amino acids include alanine, glycine, serine, and cysteine. Alanine is
typically a preferred scanning amino acid
among this group because it eliminates the side-chain beyond the beta-carbon
and is less likely to alter the main
chain conformation of the variant [Cunningham and Wells, Science, 244: 1081-
1085 (1989)]. Alanine is also
typically preferred because it is the most common amino acid. Further, it is
frequently found in both buried and
exposed positions [Creighton, The Proteins, (W.H. Freeman & Co., N.Y.);
Chothia, J. Mol. Biol., 150:1
(1976)]. If alanine substitution does not yield adequate amounts of variant,
an isoteric amino acid can be used.
C. Modifications of PRO
Covalent modifications of PRO are included within the scope of this invention.
One type of covalent
modification includes reacting targeted amino acid residues of a PRO
polypeptide with an organic derivatizing
agent that is capable of reacting with selected side chains or the N- or C-
terminal residues of the PRO.
Derivatization with bifunctional agents is useful, for instance, for
crosslinking PRO to a water-insoluble support
matrix or surface for use in the method for purifying anti-PRO antibodies, and
vice-versa. Commonly used
crosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,
glutaraldehyde, N-hydroxysuccinimide
esters, for example, esters with 4-azidosalicylic acid, homobifunctional
imidoesters, including disuccinimidyl
esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides
such as bis-N-maleimido-1,8
octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.
Other modifications include deamidation of glutaminyl and asparaginyl residues
to the corresponding
glutamyl and aspartyl residues, respectively, hydroxylation of proline and
lysine, phosphorylation of hydroxyl
groups of seryl or threonyl residues, methylation of the a-amino groups of
lysine, arginine, and histidine side
chains [T.E. Creighton, Proteins: Structure and Molecular Properties, W.H.
Freeman & Co., San Francisco,
pp. 79-86 (1983)], acetylation of the N-terminal amine, and amidation of any C-
terminal carboxyl group.
Another type of covalent modification of the PRO polypeptide included within
the scope of this
invention comprises altering the native glycosylation pattern of the
polypeptide. "Altering the native
glycosylation pattern" is intended for purposes herein to mean deleting one or
more carbohydrate moieties found
in native sequence PRO (either by removing the underlying glycosylation site
or by deleting the glycosylation
by chemical and/or enzymatic means), and/or adding one or more glycosylation
sites that are not present in the
native sequence PRO. In addition, the phrase includes qualitative changes in
the glycosylation of the native
proteins, involving a change in the nature and proportions of the various
carbohydrate moieties present.
Addition of glycosylation sites to the PRO polypeptide may be accomplished by
altering the amino acid
sequence. The alteration may be made, for example, by the addition of, or
substitution by, one or more serine
or threonine residues to the native sequence PRO (for O-linked glycosylation
sites). The PRO amino acid
sequence may optionally be altered through changes at the DNA level,
particularly by mutating the DNA
encoding the PRO polypeptide at preselected bases such that codons are
generated that will translate into the
51


CA 02380355 2002-03-O1
WO 01/16318 . PCT/US00/23328
desired amino acids.
Another means of increasing the number of carbohydrate moieties on the PRO
polypeptide is by
chemical or enzymatic coupling of glycosides to the polypeptide. Such methods
are described in the art, e.g.,
in WO 87/05330 published 11 September 1987, and in Aplin and Wriston, CRC
Crit. Rev. Biochem., pp. 259-
306 (1981).
Removal of carbohydrate moieties present on the PRO polypeptide may be
accomplished chemically
or enzymatically or by mutational substitution of codons encoding for amino
acid residues that serve as targets
for glycosylation. Chemical deglycosylation techniques are known in the art
and described, for instance, by
Hakimuddin, et al., Arch. Biochem. Bi~hys., 259:52 (1987) and by Edge et al.,
Anal. Biochem., 118:131
(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be
achieved by the use of a variety
of endo- and exo-glycosidases as described by Thotakura et al., Meth.
Enzymol., 138:350 (1987).
Another type of covalent modification of PRO comprises linking the PRO
polypeptide to one of a variety
of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene
glycol, or polyoxyalkylenes, in
the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144;
4,670,417; 4,791,192 or 4,179,337.
The PRO of the present invention may also be modified in a way to form a
chimeric molecule
comprising PRO fused to another, heterologous polypeptide or amino acid
sequence.
In one embodiment, such a chimeric molecule comprises a fusion of the PRO with
a tag polypeptide
which provides an epitope to which an anti-tag antibody can selectively bind.
The epitope tag is generally placed
at the amino- or carboxyl- terminus of the PRO. The presence of such epitope-
tagged forms of the PRO can be
detected using an antibody against the tag polypeptide. Also, provision of the
epitope tag enables the PRO to
be readily purified by affinity purification using an anti-tag antibody or
another type of affinity matrix that binds
to the epitope tag. Various tag polypeptides and their respective antibodies
are well known in the art. Examples
include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly)
tags; the flu HA tag polypeptide and its
antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc
tag and the 8F9, 3C7, 6E10,
G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and Cellular
BioloQV, 5:3610-3616 (1985)]; and the
Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et
al., Protein Engineering, 3(6):547-
553 (1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,
BioTechnolo~v, 6:1204-1210
(1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194 (1992)];
an a-tubulin epitope peptide
[Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10
protein peptide tag [Lutz-
Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)].
In an alternative embodiment, the chimeric molecule may comprise a fusion of
the PRO with an
immunoglobulin or a particular region of an immunoglobulin. For a bivalent
form of the chimeric molecule (also
referred to as an "immunoadhesin" ), such a fusion could be to the Fc region
of an IgG molecule. The Ig fusions
preferably include the substitution of a soluble (transmembrane domain deleted
or inactivated) form of a PRO
polypeptide in place of at least one variable region within an Ig molecule. In
a particularly preferred
embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the
hinge, CH1, CH2 and CH3
regions of an IgGl molecule. For the production of immunoglobulin fusions see
also US Patent No. 5,428,130
issued June 27, 1995.
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CA 02380355 2002-03-O1
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D. Preparation of PRO
The description below relates primarily to production of PRO by culturing
cells transformed or
transfected with a vector containing PRO nucleic acid. It is, of course,
contemplated that alternative methods,
which are well known in the art, may be employed to prepare PRO. For instance,
the PRO sequence, or
portions thereof, may be produced by direct peptide synthesis using solid-
phase techniques [see, e.g., Stewart
et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, CA
(1969); Merrifield, J. Am. Chem.
Soc., 85:2149-2154 (1963)]. In vitro protein synthesis may be performed using
manual techniques or by
automation. Automated synthesis may be accomplished, for instance, using an
Applied Biosystems Peptide
Synthesizer (Foster City, CA) using manufacturer's instructions. Various
portions of the PRO may be
chemically synthesized separately and combined using chemical or enzymatic
methods to produce the full-length
PRO.
1. Isolation of DNA Encoding PRO
DNA encoding PRO may be obtained from a cDNA library prepared from tissue
believed to possess
the PRO mRNA and to express it at a detectable level. Accordingly, human PRO
DNA can be conveniently
obtained from a cDNA library prepared from human tissue, such as described in
the Examples. The PRO-
encoding gene may also be obtained from a genomic library or by known
synthetic procedures (e.g., automated
nucleic acid synthesis).
Libraries can be screened with probes (such as antibodies to the PRO or
oligonucleotides of at least
about 20-80 bases) designed to identify the gene of interest or the protein
encoded by it. Screening the cDNA
or genomic library with the selected probe may be conducted using standard
procedures, such as described in
Sambrook et al. , Molecular Cloning: A Laboratory Manual (New York: Cold
Spring Harbor Laboratory Press,
1989). An alternative means to isolate the gene encoding PRO is to use PCR
methodology [Sambrook et al.,
supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor
Laboratory Press, 1995)].
The Examples below describe techniques for screening a cDNA library. The
oligonucleotide sequences
selected as probes should be of sufficient length and sufficiently unambiguous
that false positives are minimized.
The oligonucleotide is preferably labeled such that it can be detected upon
hybridization to DNA in the library
being screened. Methods of labeling are well known in the art, and include the
use of radiolabels like 3ZP-labeled
ATP, biotinylation or enzyme labeling. Hybridization conditions, including
moderate stringency and high
stringency, are provided in Sambrook et al., supra.
Sequences identified in such library screening methods can be compared and
aligned to other known
sequences deposited and available in public databases such as GenBank or other
private sequence databases.
Sequence identity (at either the amino acid or nucleotide level) within
defined regions of the molecule or across
the full-length sequence can be determined using methods known in the art and
as described herein.
Nucleic acid having protein coding sequence may be obtained by screening
selected cDNA or genomic
libraries using the deduced amino acid sequence disclosed herein for the first
time, and, if necessary, using
conventional primer extension procedures as described in Sambrook et al.,
supra, to detect precursors and
processing intermediates of mRNA that may not have been reverse-transcribed
into cDNA.
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CA 02380355 2002-03-O1
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2. Selection and Transformation of Host Cells
Host cells are transfected or transformed with expression or cloning vectors
described herein for PRO
production and cultured in conventional nutrient media modified as appropriate
for inducing promoters, selecting
transformants, or amplifying the genes encoding the desired sequences. The
culture conditions, such as media,
temperature, pH and the like, can be selected by the skilled artisan without
undue experimentation. In general,
principles, protocols, and practical techniques for maximizing the
productivity of cell cultures can be found in
Mammalian Cell Biotechnolog~,y: a Practical Approach, M. Butler, ed. (IRL
Press, 1991) and Sambrooket al.,
su ra.
Methods of eukaryotic cell transfection and prokaryotic cell transformation
are known to the ordinarily
skilled artisan, for example, CaCl2, CaP04, liposome-mediated and
electroporation. Depending on the host cell
used, transformation is performed using standard techniques appropriate to
such cells. The calcium treatment
employing calcium chloride, as described in Sambrook et al., supra, or
electroporation is generally used for
prokaryotes. Infection with Agrobacterium tumefaciens is used for
transformation of certain plant cells, as
described by Shaw et al. , Gene, 23:315 ( 1983) and WO 89/05859 published 29
June 1989. For mammalian cells
without such cell walls, the calcium phosphate precipitation method of Graham
and van der Eb, ViroloQV,
52:456-457 (1978) can be employed. General aspects of mammalian cell host
system transfections have been
described in U.S. Patent No. 4,399,216. Transformations into yeast are
typically carried out according to the
method of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao et al.,
Proc. Natl. Acad. Sci. (USA), 76:3829
(1979). However, other methods for introducing DNA into cells, such as by
nuclear microinjection,
electroporation, bacterial protoplast fusion with intact cells, or
polycations, e.g., polybrene, polyornithine, may
also be used. For various techniques for transforming mammalian cells, see
Keown et al., Methods in
Enzymolo~y, 185:527-537 (1990) and Mansour et al., Nature, 336:348-352 (1988).
Suitable host cells for cloning or expressing the DNA in the vectors herein
include prokaryote, yeast,
or higher eukaryote cells. Suitable prokaryotes include but are not limited to
eubacteria, such as Gram-negative
or Gram-positive organisms, for example, Enterobacteriaceae such as E. coli.
Various E. coli strains are
publicly available, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli
X1776 (ATCC 31,537); E, coli
strain W3110 (ATCC 27,325) and KS 772 (ATCC 53,635). Other suitable
prokaryotic host cells include
Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia,
Klebsiella, Proteus, Salmonella,
e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B.
subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed in DD
266,710 published 12 April 1989),
Pseudomonas such as P. aeruginosa, and Streptomyces. These examples are
illustrative rather than limiting.
Strain W3110 is one particularly preferred host or parent host because it is a
common host strain for recombinant
DNA product fermentations. Preferably, the host cell secretes minimal amounts
of proteolytic enzymes. For
example, strain W3110 may be modified to effect a genetic mutation in the
genes encoding proteins endogenous
to the host, with examples of such hosts including E. coli W3110 strain 1A2,
which has the complete genotype
tonA ; E. coli W3110 strain 9E4, which has the complete genotype tonA ptr3; E.
coli W3110 strain 27C7
(ATCC 55,244), which has the complete genotype tonA ptr3 phoA EI S (argF-
lac)169 degP ompT kan'; E. coli
W3110 strain 37D6, which has the complete genotype tonA ptr3 phoA El5 (argF-
lac)169 degP ompT rbs7
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CA 02380355 2002-03-O1
WO 01/16318 PCT/US00/23328
ilvG kan'; E. coli W3110 strain 40B4, which is strain 37D6 with a non-
kanamycin resistant degP deletion
mutation; and an E. coli strain having mutant periplasmic protease disclosed
in U. S. Patent No. 4,946,783 issued
7 August 1990. Alternatively, in vitro methods of cloning, e.g., PCR or other
nucleic acid polymerase
reactions, are suitable.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or
yeast are suitable cloning
or expression hosts for PRO-encoding vectors. Saccharomyces cerevisiae is a
commonly used lower eukaryotic
host microorganism. Others include Schizosaccharomyces pombe (Beach and Nurse,
Nature, 290: 140 [1981];
EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Patent No.
4,943,529; Fleer et al.,
Bio/Technoloev, 9:968-975 (1991)) such as, e.g., K. lactis (MW98-8C, CBS683,
CBS4574; Louvencourt et
al., J. Bacteriol., 154(2):737-742 [1983]), K. fragilis (ATCC 12,424), K.
bulgaricus (ATCC 16,045), K.
wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC
36,906; Van den Berg et al.,
Bio/Technoloey, 8:135 ( 1990)), K. thermotolerans, and K. marxianus; yarrowia
(EP 402,226); Pichia pastoris
(EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278 [1988]);
Candida; Trichoderma reesia (EP
244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-
5263 [1979]); Schwanniomyces
such as Schwanniomyces occidentalis (EP 394,538 published 31 October 1990);
and filamentous fungi such as,
e.g., Neurospora, Penicillium, Tolypocladium(W091/00357published lOJanuary
1991), andAspergillushosts
such as A. nidulans (Ballance et al., Biochem. Biophvs. Res. Commun., 112:284-
289 [1983]; Tilburn et al.,
Gene, 26:205-221 [1983]; Yeltonet al., Proc. Natl. Acad. Sci. USA, 81: 1470-
1474 [1984]) andA. niger(Kelly
and Hynes, EMBO J., 4:475-479 [1985]). Methylotropic yeasts are suitable
herein and include, but are not
limited to, yeast capable of growth on methanol selected from the genera
consisting of Hansenula, Candida,
Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of
specific species that are exemplary
of this class of yeasts may be found in C. Anthony, The Biochemistrv of
Methylotrophs, 269 (1982).
Suitable host cells for the expression of glycosylated PRO are derived from
multicellular organisms.
Examples of invertebrate cells include insect cells such as Drosophila S2 and
Spodoptera Sf9, as well as plant
cells. Examples of useful mammalian host cell lines include Chinese hamster
ovary (CHO) and COS cells.
More specific examples include monkey kidney CV1 line transformed by SV40 (COS-
7, ATCC CRL 1651);
human embryonic kidney line (293 or 293 cells subcloned for growth in
suspension culture, Graham et al., J.
Gen Virol., 36:59 (1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and
Chasin, Proc. Natl. Acad.
Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.,
23:243-251 (1980)); human lung
cells (W 138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse
mammary tumor (MMT
060562, ATCC CCL51). The selection of the appropriate host cell is deemed to
be within the skill in the art.
3. Selection and Use of a Replicable Vector
The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO may be inserted into
a replicable vector
for cloning (amplification of the DNA) or for expression. Various vectors are
publicly available. The vector
may, for example, be in the form of a plasmid, cosmid, viral particle, or
phage. The appropriate nucleic acid
sequence may be inserted into the vector by a variety of procedures. In
general, DNA is inserted into an
appropriate restriction endonuclease sites) using techniques known in the art.
Vector components generally


CA 02380355 2002-03-O1
WO 01/16318 PCT/US00/23328
include, but are not limited to, one or more of a signal sequence, an origin
of replication, one or more marker
genes, an enhancer element, a promoter, and a transcription termination
sequence. Construction of suitable
vectors containing one or more of these components employs standard ligation
techniques which are known to
the skilled artisan.
The PRO may be produced recombinantly not only directly, but also as a fusion
polypeptide with a
heterologous polypeptide, which may be a signal sequence or other polypeptide
having a specific cleavage site
at the N-terminus of the mature protein or polypeptide. In general, the signal
sequence may be a component of
the vector, or it may be a part of the PRO-encoding DNA that is inserted into
the vector. The signal sequence
may be a prokaryotic signal sequence selected, for example, from the group of
the alkaline phosphatase,
penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion
the signal sequence may be, e.g.,
the yeast invertase leader, alpha factor leader (including Saccharomyces and
Kluyveromyces a-factor leaders,
the latter described in U.S. Patent No. 5,010,182), or acid phosphatase
leader, the C. albicans glucoamylase
leader (EP 362,179 published 4 April 1990), or the signal described in WO
90/13646 published 15 November
1990. In mammalian cell expression, mammalian signal sequences may be used to
direct secretion of the
protein, such as signal sequences from secreted polypeptides of the same or
related species, as well as viral
secretory leaders.
Both expression and cloning vectors contain a nucleic acid sequence that
enables the vector to replicate
in one or more selected host cells. Such sequences are well known for a
variety of bacteria, yeast, and viruses.
The origin of replication from the plasmid pBR322 is suitable for most Gram-
negative bacteria, the 2~ plasmid
origin is suitable for yeast, and various viral origins (SV40, polyoma,
adenovirus, VSV or BPV) are useful for
cloning vectors in mammalian cells.
Expression and cloning vectors will typically contain a selection gene, also
termed a selectable marker.
Typical selection genes encode proteins that (a) confer resistance to
antibiotics or other toxins, e.g., ampicillin,
neomycin, methotrexate, or tetracycline, (b) complement auxotrophic
deficiencies, or (c) supply critical nutrients
not available from complex media, e.g., the gene encoding D-alanine racemase
for Bacilli.
An example of suitable selectable markers for mammalian cells are those that
enable the identification
of cells competent to take up the PRO-encoding nucleic acid, such as DHFR or
thymidine kinase. An
appropriate host cell when wild-type DHFR is employed is the CHO cell line
deficient in DHFR activity,
prepared and propagated as described by Urlaub et al., Proc. Natl. Acad. Sci.
USA, 77:4216 (1980). A suitable
selection gene for use in yeast is the trpl gene present in the yeast plasmid
YRp7 [Stinchcomb et al., Nature,
282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene,
10:157 (1980)]. The trpl gene
provides a selection marker for a mutant strain of yeast lacking the ability
to grow in tryptophan, for example,
ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].
Expression and cloning vectors usually contain a promoter operably linked to
the PRO-encoding nucleic
acid sequence to direct mRNA synthesis. Promoters recognized by a variety of
potential host cells are well
known. Promoters suitable for use with prokaryotic hosts include the (3-
lactamase and lactose promoter systems
[Chang et al . , Nature, 275:615 ( 1978); Goeddel et al . , Nature, 281:544 (
1979)] , alkaline phosphatase, a
tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980);
EP 36,776), and hybrid
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CA 02380355 2002-03-O1
WO 01/16318 , PCT/US00/23328
promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA,
80:21-25 (1983)]. Promoters
for use in bacterial systems also will contain a Shine-Dalgarno (S.D.)
sequence operably linked to the DNA
encoding PRO.
Examples of suitable promoting sequences for use with yeast hosts include the
promoters for 3-
phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem., 255:2073 (1980)] or
other glycolytic enzymes [Hess
et al., J. Adv. Enzyme Red, 7:149 (1968); Holland, Biochemistry, 17:4900
(1978)], such as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase,
phosphofructokinase, glucose-
6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase,
triosephosphate isomerase, phosphoglucose
isomerase, and glucokinase.
Other yeast promoters, which are inducible promoters having the additional
advantage of transcription
controlled by growth conditions, are the promoter regions for alcohol
dehydrogenase 2, isocytochrome C, acid
phosphatase, degradative enzymes associated with nitrogen metabolism,
metallothionein, glyceraldehyde-3-
phosphate dehydrogenase, and enzymes responsible for maltose and galactose
utilization. Suitable vectors and
promoters for use in yeast expression are further described in EP 73,657.
PRO transcription from vectors in mammalian host cells is controlled, for
example, by promoters
obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK
2,211,504 published 5 July
1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian
sarcoma virus, cytomegalovirus, a
retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous
mammalian promoters, e.g., the
actin promoter or an immunoglobulin promoter, and from heat-shock promoters,
provided such promoters are
compatible with the host cell systems.
Transcription of a DNA encoding the PRO by higher eukaryotes may be increased
by inserting an
enhancer sequence into the vector. Enhancers are cis-acting elements of DNA,
usually about from 10 to 300
bp, that act on a promoter to increase its transcription. Many enhancer
sequences are now known from
mammalian genes (globin, elastase, albtunin, a-fetoprotein, and insulin).
Typically, however, one will use an
enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on
the late side of the replication
origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma
enhancer on the late side of the
replication origin, and adenovirus enhancers. The enhancer may be spliced into
the vector at a position 5' or
3' to the PRO coding sequence, but is preferably located at a site 5' from the
promoter.
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant,
animal, human, or nucleated
cells from other multicellular organisms) will also contain sequences
necessary for the termination of
transcription and for stabilizing the mRNA. Such sequences are commonly
available from the 5' and,
occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs.
These regions contain nucleotide
segments transcribed as polyadenylated fragments in the untranslated portion
of the mRNA encoding PRO.
Still other methods, vectors, and host cells suitable for adaptation to the
synthesis of PRO in
recombinant vertebrate cell culture are described in Gething et al. , Nature,
293:620-625 ( 1981 ); Mantei et al. ,
Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.
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4. Detecting Gene Am~lification/Ex rep ssion
Gene amplification and/or expression may be measured in a sample directly, for
example, by
conventional Southern blotting, Northern blotting to quantitate the
transcription of mRNA [Thomas, Proc. Natl.
Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ
hybridization, using an
appropriately labeled probe, based on the sequences provided herein.
Alternatively, antibodies may be employed
that can recognize specific duplexes, including DNA duplexes, RNA duplexes,
and DNA-RNA hybrid duplexes
or DNA-protein duplexes. The antibodies in turn may be labeled and the assay
may be carried out where the
duplex is bound to a surface, so that upon the formation of duplex on the
surface, the presence of antibody bound
to the duplex can be detected.
Gene expression, alternatively, may be measured by immunological methods, such
as
immunohistochemical staining of cells or tissue sections and assay of cell
culture or body fluids, to quantitate
directly the expression of gene product. Antibodies useful for
immunohistochemical staining and/or assay of
sample fluids may be either monoclonal or polyclonal, and may be prepared in
any mammal. Conveniently, the
antibodies may be prepared against a native sequence PRO polypeptide or
against a synthetic peptide based on
the DNA sequences provided herein or against exogenous sequence fused to PRO
DNA and encoding a specific
antibody epitope.
5. Purification of Polypeptide
Forms of PRO may be recovered from culture medium or from host cell lysates.
If membrane-bound,
it can be released from the membrane using a suitable detergent solution (e.g.
Triton-X 100) or by enzymatic
cleavage. Cells employed in expression of PRO can be disrupted by various
physical or chemical means, such
as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing
agents.
It may be desired to purify PRO from recombinant cell proteins or
polypeptides. The following
procedures are exemplary of suitable purification procedures: by fractionation
on an ion-exchange column;
ethanol precipitation; reverse phase HPLC; chromatography on silica or on a
canon-exchange resin such as
DEAF; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel
filtration using, for example,
Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgG;
and metal chelating columns
to bind epitope-tagged forms of the PRO. Various methods of protein
purification may be employed and such
methods are known in the art and described for example in Deutscher, Methods
in Enzymolo~v, 182 (1990);
Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New
York (1982). The purification
steps) selected will depend, for example, on the nature of the production
process used and the particular PRO
produced.
E. Uses for PRO
Nucleotide sequences (or their complement) encoding PRO have various
applications in the art of
molecular biology, including uses as hybridization probes, in chromosome and
gene mapping and in the
generation of anti-sense RNA and DNA. PRO nucleic acid will also be useful for
the preparation of PRO
polypeptides by the recombinant techniques described herein.
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CA 02380355 2002-03-O1
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The full-length native sequence PRO gene, or portions thereof, may be used as
hybridization probes
for a cDNA library to isolate the full-length PRO cDNA or to isolate still
other cDNAs (for instance, those
encoding naturally-occurring variants of PRO or PRO from other species) which
have a desired sequence identity
to the native PRO sequence disclosed herein. Optionally, the length of the
probes will be about 20 to about 50
bases. The hybridization probes may be derived from at least partially novel
regions of the full length native
nucleotide sequence wherein those regions may be determined without undue
experimentation or from genomic
sequences including promoters, enhancer elements and introns of native
sequence PRO. By way of example,
a screening method will comprise isolating the coding region of the PRO gene
using the known DNA sequence
to synthesize a selected probe of about 40 bases. Hybridization probes may be
labeled by a variety of labels,
including radionucleotides such as 3'P or 35S, or enzymatic labels such as
alkaline phosphatase coupled to the
probe via avidin/biotin coupling systems. Labeled probes having a sequence
complementary to that of the PRO
gene of the present invention can be used to screen libraries of human cDNA,
genomic DNA or mRNA to
determine which members of such libraries the probe hybridizes to.
Hybridization techniques are described in
further detail in the Examples below.
Any EST sequences disclosed in the present application may similarly be
employed as probes, using
the methods disclosed herein.
Other useful fragments of the PRO nucleic acids include antisense or sense
oligonucleotides comprising
a singe-stranded nucleic acid sequence (either RNA or DNA) capable of binding
to target PRO mRNA (sense)
or PRO DNA (antisense) sequences. Antisense or sense oligonucleotides,
according to the present invention,
comprise a fragment of the coding region of PRO DNA. Such a fragment generally
comprises at least about 14
nucleotides, preferably from about 14 to 30 nucleotides. The ability to derive
an antisense or a sense
oligonucleotide, based upon a cDNA sequence encoding a given protein is
described in, for example, Stein and
Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al. (BioTechniqnes
6:958, 1988).
Binding of antisense or sense oligonucleotides to target nucleic acid
sequences results in the formation
of duplexes that block transcription or translation of the target sequence by
one of several means, including
enhanced degradation of the duplexes, premature termination of transcription
or translation, or by other means.
The antisense oligonucleotides thus may be used to block expression of PRO
proteins. Antisense or sense
oligonucleotides further comprise oligonucleotides having modified sugar-
phosphodiester backbones (or other
sugar linkages, such as those described in WO 91/06629) and wherein such sugar
linkages are resistant to
endogenous nucleases. Such oligonucleotides with resistant sugar linkages are
stable in vivo (i.e., capable of
resisting enzymatic degradation) but retain sequence specificity to be able to
bind to target nucleotide sequences.
Other examples of sense or antisense oligonucleotides include those
oligonucleotides which are
covalently linked to organic moieties, such as those described in WO 90/10048,
and other moieties that increases
affinity of the oligonucleotide for a target nucleic acid sequence, such as
poly-(L-lysine). Further still,
intercalating agents, such as ellipticine, and alkylating agents or metal
complexes may be attached to sense or
antisense oligonucleotides to modify binding specificities of the antisense or
sense oligonucleotide for the target
nucleotide sequence.
59


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Antisense or sense oligonucleotides may be introduced into a cell containing
the target nucleic acid
sequence by any gene transfer method, including, for example, CaP04-mediated
DNA transfection,
electroporation, or by using gene transfer vectors such as Epstein-Barr virus.
In a preferred procedure, an
antisense or sense oligonucleotide is inserted into a suitable retroviral
vector. A cell containing the target nucleic
acid sequence is contacted with the recombinant retroviral vector, either in
vivo or ex vivo. Suitable retroviral
vectors include, but are not limited to, those derived from the murine
retrovirus M-MuLV, N2 (a retrovirus
derived from M-MuLV), or the double copy vectors designated DCTSA, DCTSB and
DCTSC (see WO
90/13641).
Sense or antisense oligonucleotides also may be introduced into a cell
containing the target nucleotide
sequence by formation of a conjugate with a ligand binding molecule, as
described in WO 91/04753. Suitable
ligand binding molecules include, but are not limited to, cell surface
receptors, growth factors, other cytokines,
or other ligands that bind to cell surface receptors. Preferably, conjugation
of the ligand binding molecule does
not substantially interfere with the ability of the ligand binding molecule to
bind to its corresponding molecule
or receptor, or block entry of the sense or antisense oligonucleotide or its
conjugated version into the cell.
Alternatively, a sense or an antisense oligonucleotide may be introduced into
a cell containing the target
nucleic acid sequence by formation of an oligonucleotide-lipid complex, as
described in WO 90/10448. The
sense or antisense oligonucleotide-lipid complex is preferably dissociated
within the cell by an endogenous lipase.
Antisense or sense RNA or DNA molecules are generally at least about 5 bases
in length, about 10
bases in length, about 15 bases in length, about 20 bases in length, about 25
bases in length, about 30 bases in
length, about 35 bases in length, about 40 bases in length, about 45 bases in
length, about 50 bases in length,
about 55 bases in length, about 60 bases in length, about 65 bases in length,
about 70 bases in length, about 75
bases in length, about 80 bases in length, about 85 bases in length, about 90
bases in length, about 95 bases in
length, about 100 bases in length, or more.
The probes may also be employed in PCR techniques to generate a pool of
sequences for identification
of closely related PRO coding sequences.
Nucleotide sequences encoding a PRO can also be used to construct
hybridization probes for mapping
the gene which encodes that PRO and for the genetic analysis of individuals
with genetic disorders. The
nucleotide sequences provided herein may be mapped to a chromosome and
specific regions of a chromosome
using known techniques, such as in situ hybridization, linkage analysis
against known chromosomal markers,
and hybridization screening with libraries.
When the coding sequences for PRO encode a protein which binds to another
protein (example, where
the PRO is a receptor), the PRO can be used in assays to identify the other
proteins or molecules involved in
the binding interaction. By such methods, inhibitors of the receptor/ligand
binding interaction can be identified.
Proteins involved in such binding interactions can also be used to screen for
peptide or small molecule inhibitors
or agonists of the binding interaction. Also, the receptor PRO can be used to
isolate correlative ligand(s).
Screening assays can be designed to find lead compounds that mimic the
biological activity of a native PRO or
a receptor for PRO. Such screening assays will include assays amenable to high-
throughput screening of
chemical libraries, making them particularly suitable for identifying small
molecule drug candidates. Small


CA 02380355 2002-03-O1
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molecules contemplated include synthetic organic or inorganic compounds. The
assays can be performed in a
variety of formats, including protein-protein binding assays, biochemical
screening assays, immunoassays and
cell based assays, which are well characterized in the art.
Nucleic acids which encode PRO or its modified forms can also be used to
generate either transgenic
animals or "knock out" animals which, in turn, are useful in the development
and screening of therapeutically
useful reagents. A transgenic animal (e.g., a mouse or rat) is an animal
having cells that contain a transgene,
which transgene was introduced into the animal or an ancestor of the animal at
a prenatal, e.g., an embryonic
stage. A transgene is a DNA which is integrated into the genome of a cell from
which a transgenic animal
develops. In one embodiment, cDNA encoding PRO can be used to clone genomic
DNA encoding PRO in
accordance with established techniques and the genomic sequences used to
generate transgenic animals that
contain cells which express DNA encoding PRO. Methods for generating
transgenic animals, particularly
animals such as mice or rats, have become conventional in the art and are
described, for example, in U. S. Patent
Nos. 4,736,866 and 4,870,009. Typically, particular cells would be targeted
for PRO transgene incorporation
with tissue-specific enhancers. Transgenic animals that include a copy of a
transgene encoding PRO introduced
into the germ line of the animal at an embryonic stage can be used to examine
the effect of increased expression
of DNA encoding PRO. Such animals can be used as tester animals for reagents
thought to confer protection
from, for example, pathological conditions associated with its overexpression.
In accordance with this facet of
the invention, an animal is treated with the reagent and a reduced incidence
of the pathological condition,
compared to untreated animals bearing the transgene, would indicate a
potential therapeutic intervention for the
pathological condition.
Alternatively, non-human homologues of PRO can be used to construct a PRO
"knock out" animal
which has a defective or altered gene encoding PRO as a result of homologous
recombination between the
endogenous gene encoding PRO and altered genomic DNA encoding PRO introduced
into an embryonic stem
cell of the animal. For example, cDNA encoding PRO can be used to clone
genomic DNA encoding PRO in
accordance with established techniques. A portion of the genomic DNA encoding
PRO can be deleted or
replaced with another gene, such as a gene encoding a selectable marker which
can be used to monitor
integration. Typically, several kilobases of unaltered flanking DNA (both at
the 5' and 3' ends) are included
in the vector [see e.g., Thomas and Capecchi, Cell, 51:503 (1987) for a
description of homologous
recombination vectors]. The vector is introduced into an embryonic stem cell
line (e.g., by electroporation) and
cells in which the introduced DNA has homologously recombined with the
endogenous DNA are selected [see
e.g., Li et al., Cell, 69:915 (1992)]. The selected cells are then injected
into a blastocyst of an animal (e.g.,
a mouse or rat) to form aggregation chimeras [see e.g., Bradley, in
Teratocarcinomas and Embryonic Stem
Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-
152]. A chimeric embryo can
then be implanted into a suitable pseudopregnant female foster animal and the
embryo brought to term to create
a "knock out" animal. Progeny harboring the homologously recombined DNA in
their germ cells can be
identified by standard techniques and used to breed animals in which all cells
of the animal contain the
homologously recombined DNA. Knockout animals can be characterized for
instance, for their ability to defend
against certain pathological conditions and for their development of
pathological conditions due to absence of
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the PRO polypeptide.
Nucleic acid encoding the PRO polypeptides may also be used in gene therapy.
In gene therapy
applications, genes are introduced into cells in order to achieve in vivo
synthesis of a therapeutically effective
genetic product, for example for replacement of a defective gene. "Gene
therapy" includes both conventional
gene therapy where a lasting effect is achieved by a single treatment, and the
administration of gene therapeutic
agents, which involves the one time or repeated administration of a
therapeutically effective DNA or mRNA.
Antisense RNAs and DNAs can be used as therapeutic agents for blocking the
expression of certain genes in
vivo. It has already been shown that short antisense oligonucleotides can be
imported into cells where they act
as inhibitors, despite their low intracellular concentrations caused by their
restricted uptake by the cell
membrane. (Zamecnik et al., Proc. Natl. Acad. Sci. USA 83:4143-4146 [1986]).
The oligonucleotides can be
modified to enhance their uptake, e.g. by substituting their negatively
charged phosphodiester groups by
uncharged groups.
There are a variety of techniques available for introducing nucleic acids into
viable cells. The
techniques vary depending upon whether the nucleic acid is transferred into
cultured cells in vitro, or in vivo in
the cells of the intended host. Techniques suitable for the transfer of
nucleic acid into mammalian cells in vitro
include the use of liposomes, electroporation, microinjection, cell fusion,
DEAE-dextran, the calcium phosphate
precipitation method, etc. The currently preferred in vivo gene transfer
techniques include transfection with viral
(typically retroviral) vectors and viral coat protein-liposome mediated
transfection (Dzau et al., Trends in
Biotechnolo~y 11, 205-210 [1993]). In some situations it is desirable to
provide the nucleic acid source with
an agent that targets the target cells, such as an antibody specific for a
cell surface membrane protein or the
target cell, a ligand for a receptor on the target cell, etc. Where liposomes
are employed, proteins which bind
to a cell surface membrane protein associated with endocytosis may be used for
targeting and/or to facilitate
uptake, e.g. capsid proteins or fragments thereof tropic for a particular cell
type, antibodies for proteins which
undergo internalization in cycling, proteins that target intracellular
localization and enhance intracellular half-life.
The technique of receptor-mediated endocytosis is described, for example, by
Wu et al., J. Biol. Chem. 262,
4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414
(1990). For review of gene
marking and gene therapy protocols see Anderson et al., Science 256, 808-813
(1992).
The PRO polypeptides described herein may also be employed as molecular weight
markers for protein
electrophoresis purposes and the isolated nucleic acid sequences may be used
for recombinantly expressing those
markers.
The nucleic acid molecules encoding the PRO polypeptides or fragments thereof
described herein are
useful for chromosome identification. In this regard, there exists an ongoing
need to identify new chromosome
markers, since relatively few chromosome marking reagents, based upon actual
sequence data are presently
available. Each PRO nucleic acid molecule of the present invention can be used
as a chromosome marker.
The PRO polypeptides and nucleic acid molecules of the present invention may
also be used
diagnostically for tissue typing, wherein the PRO polypeptides of the present
invention may be differentially
expressed in one tissue as compared to another, preferably in a diseased
tissue as compared to a normal tissue
of the same tissue type. PRO nucleic acid molecules will fmd use for
generating probes for PCR, Northern
62


CA 02380355 2002-03-O1
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analysis, Southern analysis and Western analysis.
The PRO polypeptides described herein may also be employed as therapeutic
agents. The PRO
polypeptides of the present invention can be formulated according to known
methods to prepare pharmaceutically
useful compositions, whereby the PRO product hereof is combined in admixture
with a pharmaceutically
acceptable carrier vehicle. Therapeutic formulations are prepared for storage
by mixing the active ingredient
having the desired degree of purity with optional physiologically acceptable
carriers, excipients or stabilizers
(Remineton's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in
the form of lyophilized
formulations or aqueous solutions. Acceptable carriers, excipients or
stabilizers are nontoxic to recipients at the
dosages and concentrations employed, and include buffers such as phosphate,
citrate and other organic acids;
antioxidants including ascorbic acid; low molecular weight (less than about 10
residues) polypeptides; proteins,
such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such
as polyvinylpyrrolidone, amino
acids such as glycine, glutamine, asparagine, arginine or lysine;
monosaccharides, disaccharides and other
carbohydrates including glucose, mannose, or dextrins; chelating agents such
as EDTA; sugar alcohols such as
mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic
surfactants such as TWEENT"'',
PLURONICST"' or PEG.
The formulations to be used for in vivo administration must be sterile. This
is readily accomplished by
filtration through sterile filtration membranes, prior to or following
lyophilization and reconstitution.
Therapeutic compositions herein generally are placed into a container having a
sterile access port, for
example, an intravenous solution bag or vial having a stopper pierceable by a
hypodermic injection needle.
The route of administration is in accord with lrnown methods, e.g. injection
or infusion by intravenous,
intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial or
intralesional routes, topical
administration, or by sustained release systems.
Dosages and desired drug concentrations of pharmaceutical compositions of the
present invention may
vary depending on the particular use envisioned. The determination of the
appropriate dosage or route of
administration is well within the skill of an ordinary physician. Animal
experiments provide reliable guidance
for the determination of effective doses for human therapy. Interspecies
scaling of effective doses can be
performed following the principles laid down by Mordenti, J. and Chappell, W.
"The use of interspecies scaling
in toxicokinetics" In Toxicokinetics and New Drug Development, Yacobi et al.,
Eds., Pergamon Press, New
York 1989, pp. 42-96.
When in vivo administration of a PRO polypeptide or agonist or antagonist
thereof is employed, normal
dosage amounts may vary from about 10 ng/kg to up to 100 mg/kg of mammal body
weight or more per day,
preferably about 1 ~,g/kg/day to 10 mg/kg/day, depending upon the route of
administration. Guidance as to
particular dosages and methods of delivery is provided in the literature; see,
for example, U.S. Pat. Nos.
4,657,760; 5,206,344; or 5,225,212. It is anticipated that different
formulations will be effective for different
treatment compounds and different disorders, that administration targeting one
organ or tissue, for example, may
necessitate delivery in a manner different from that to another organ or
tissue.
Where sustained-release administration of a PRO polypeptide is desired in a
formulation with release
characteristics suitable for the treatment of any disease or disorder
requiring administration of the PRO
63


CA 02380355 2002-03-O1
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polypeptide, microencapsulation of the PRO polypeptide is contemplated.
Microencapsulation of recombinant
proteins for sustained release has been successfully performed with human
growth hormone (rhGH), interferon-
(rhIFN- ), interleukin-2, and MN rgp120. Johnson et al., Nat. Med., 2:795-799
(1996); Yasuda, Biomed.
Ther., 27:1221-1223 (1993); Hora et al., Bio/Technolo~v, 8:755-758 (1990);
Cleland, "Design and Production
of Single Immunization Vaccines Using Polylactide Polyglycolide Microsphere
Systems," in Vaccine Design:
The Subunit and Adiuvant Approach, Powell and Newman, eds, (Plenum Press: New
York, 1995), pp. 439-462;
WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat. No. 5,654,010.
The sustained-release formulations of these proteins were developed using poly-
lactic-coglycolic acid
(PLGA) polymer due to its biocompatibility and wide range of biodegradable
properties. The degradation
products of PLGA, lactic and glycolic acids, can be cleared quickly within the
human body. Moreover, the
degradability of this polymer can be adjusted from months to years depending
on its molecular weight and
composition. Lewis, "Controlled release of bioactive agents from
lactide/glycolide polymer," in: M. Chasm
and R. Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems (Marcel
Dekker: New York, 1990),
pp. 1-41.
This invention encompasses methods of screening compounds to identify those
that mimic the PRO
polypeptide (agonists) or prevent the effect of the PRO polypeptide
(antagonists). Screening assays for
antagonist drug candidates are designed to identify compounds that bind or
complex with the PRO polypeptides
encoded by the genes identified herein, or otherwise interfere with the
interaction of the encoded polypeptides
with other cellular proteins. Such screening assays will include assays
amenable to high-throughput screening
of chemical libraries, making them particularly suitable for identifying small
molecule drug candidates.
The assays can be performed in a variety of formats, including protein-protein
binding assays,
biochemical screening assays, immunoassays, and cell-based assays, which are
well characterized in the art.
All assays for antagonists are common in that they call for contacting the
drug candidate with a PRO
polypeptide encoded by a nucleic acid identified herein under conditions and
for a time sufficient to allow these
two components to interact.
In binding assays, the interaction is binding and the complex formed can be
isolated or detected in the
reaction mixture. In a particular embodiment, the PRO polypeptide encoded by
the gene identified herein or the
drug candidate is immobilized on a solid phase, e.g., on a microtiter plate,
by covalent or non-covalent
attachments. Non-covalent attachment generally is accomplished by coating the
solid surface with a solution of
the PRO polypeptide and drying. Alternatively, an immobilized antibody, e.g.,
a monoclonal antibody, specific
for the PRO polypeptide to be immobilized can be used to anchor it to a solid
surface. The assay is performed
by adding the non-immobilized component, which may be labeled by a detectable
label, to the immobilized
component, e.g., the coated surface containing the anchored component. When
the reaction is complete, the
non-reacted components are removed, e.g., by washing, and complexes anchored
on the solid surface are
detected. When the originally non-immobilized component carries a detectable
label, the detection of label
immobilized on the surface indicates that complexing occurred. Where the
originally non-immobilized
component does not carry a label, complexing can be detected, for example, by
using a labeled antibody
specifically binding the immobilized complex.
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If the candidate compound interacts with but does not bind to a particular PRO
polypeptide encoded by
a gene identified herein, its interaction with that polypeptide can be assayed
by methods well known for detecting
protein-protein interactions. Such assays include traditional approaches, such
as, e.g., cross-linking, co-
immunoprecipitation, and co-purification through gradients or chromatographic
columns. In addition, protein-
protein interactions can be monitored by using a yeast-based genetic system
described by Fields and co-workers
(Fields and Song, Nature (London), 340:245-246 (1989); Chien et al., Proc.
Natl. Acad. Sci. USA, 88:9578-
9582 (1991)) as disclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA,
89: 5789-5793 (1991). Many
transcriptional activators, such as yeast GAL4, consist of two physically
discrete modular domains, one acting
as the DNA-binding domain, the other one functioning as the transcription-
activation domain. The yeast
expression system described in the foregoing publications (generally referred
to as the "two-hybrid system")
takes advantage of this property, and employs two hybrid proteins, one in
which the target protein is fused to
the DNA-binding domain of GAL4, and another, in which candidate activating
proteins are fused to the
activation domain. The expression of a GAL1-ZacZ reporter gene under control
of a GAL4-activated promoter
depends on reconstitution of GAL4 activity via protein-protein interaction.
Colonies containing interacting
polypeptides are detected with a chromogenic substrate for (3-galactosidase. A
complete kit
(MATCHMAKERT"') for identifying protein-protein interactions between two
specific proteins using the two-
hybrid technique is commercially available from Clontech. This system can also
be extended to map protein
domains involved in specific protein interactions as well as to pinpoint amino
acid residues that are crucial for
these interactions.
Compounds that interfere with the interaction of a gene encoding a PRO
polypeptide identified herein
and other infra- or extracellular components can be tested as follows: usually
a reaction mixture is prepared
containing the product of the gene and the infra- or extracellular component
under conditions and for a time
allowing for the interaction and binding of the two products. To test the
ability of a candidate compound to
inhibit binding, the reaction is run in the absence and in the presence of the
test compound. In addition, a
placebo may be added to a third reaction mixture, to serve as positive
control. The binding (complex formation)
between the test compound and the infra- or extracellular component present in
the mixture is monitored as
described hereinabove. The formation of a complex in the control reactions)
but not in the reaction mixture
containing the test compound indicates that the test compound interferes with
the interaction of the test compound
and its reaction partner.
To assay for antagonists, the PRO polypeptide may be added to a cell along
with the compound to be
screened for a particular activity and the ability of the compound to inhibit
the activity of interest in the presence
of the PRO polypeptide indicates that the compound is an antagonist to the PRO
polypeptide. Alternatively,
antagonists may be detected by combining the PRO polypeptide and a potential
antagonist with membrane-bound
PRO polypeptide receptors or recombinant receptors under appropriate
conditions for a competitive inhibition
assay. The PRO polypeptide can be labeled, such as by radioactivity, such that
the number of PRO polypeptide
molecules bound to the receptor can be used to determine the effectiveness of
the potential antagonist. The gene
encoding the receptor can be identified by numerous methods known to those of
skill in the art, for example,
ligand panning and FACS sorting. Coligan et al., Current Protocols in Immun.,
1(2): Chapter 5 (1991).


CA 02380355 2002-03-O1
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Preferably, expression cloning is employed wherein polyadenylated RNA is
prepared from a cell responsive to
the PRO polypeptide and a cDNA library created from this RNA is divided into
pools and used to transfect COS
cells or other cells that are not responsive to the PRO polypeptide.
Transfected cells that are grown on glass
slides are exposed to labeled PRO polypeptide. The PRO polypeptide can be
labeled by a variety of means
including iodination or inclusion of a recognition site for a site-specific
protein kinase. Following fixation and
incubation, the slides are subjected to autoradiographic analysis. Positive
pools are identified and sub-pools are
prepared and re-transfected using an interactive sub-pooling and re-screening
process, eventually yielding a
single clone that encodes the putative receptor.
As an alternative approach for receptor identification, labeled PRO
polypeptide can be photoaffmity-
linked with cell membrane or extract preparations that express the receptor
molecule. Cross-linked material is
resolved by PAGE and exposed to X-ray film. The labeled complex containing the
receptor can be excised,
resolved into peptide fragments, and subjected to protein micro-sequencing.
The amino acid sequence obtained
from micro- sequencing would be used to design a set of degenerate
oligonucleotide probes to screen a cDNA
library to identify the gene encoding the putative receptor.
In another assay for antagonists, mammalian cells or a membrane preparation
expressing the receptor
would be incubated with labeled PRO polypeptide in the presence of the
candidate compound. The ability of
the compound to enhance or block this interaction could then be measured.
More specific examples of potential antagonists include an oligonucleotide
that binds to the fusions of
immunoglobulin with PRO polypeptide, and, in particular, antibodies including,
without limitation, poly- and
monoclonal antibodies and antibody fragments, single-chain antibodies, anti-
idiotypic antibodies, and chimeric
or humanized versions of such antibodies or fragments, as well as human
antibodies and antibody fragments.
Alternatively, a potential antagonist may be a closely related protein, for
example, a mutated form of the PRO
polypeptide that recognizes the receptor but imparts no effect, thereby
competitively inhibiting the action of the
PRO polypeptide.
Another potential PRO polypeptide antagonist is an antisense RNA or DNA
construct prepared using
antisense technology, where, e.g., an antisense RNA or DNA molecule acts to
block directly the translation of
mRNA by hybridizing to targeted mRNA and preventing protein translation.
Antisense technology can be used
to control gene expression through triple-helix formation or antisense DNA or
RNA, both of which methods are
based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding
portion of the polynucleotide
sequence, which encodes the mature PRO polypeptides herein, is used to design
an antisense RNA
oligonucleotide of from about 10 to 40 base pairs in length. A DNA
oligonucleotide is designed to be
complementary to a 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); Dervan et al. ,
Science, 251:1360 ( 1991 )), thereby
preventing transcription and the production of the PRO polypeptide. The
antisense RNA oligonucleotide
hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule
into the PRO polypeptide
(antisense - Okano, Neurochem., 56:560 (1991); Oli~odeoxynucleotides as
Antisense Inhibitors of Gene
Ex ression (CRC Press: Boca Raton, FL, 1988). The oligonucleotides described
above can also be delivered
to cells such that the antisense RNA or DNA may be expressed in vivo to
inhibit production of the PRO
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polypeptide. When antisense DNA is used, oligodeoxyribonucleotides derived
from the translation-initiation site,
e.g., between about -10 and +10 positions of the target gene nucleotide
sequence, are preferred.
Potential antagonists include small molecules that bind to the active site,
the receptor binding site, or
growth factor or other relevant binding site of the PRO polypeptide, thereby
blocking the normal biological
activity of the PRO polypeptide. Examples of small molecules include, but are
not limited to, small peptides
or peptide-like molecules, preferably soluble peptides, and synthetic non-
peptidyl organic or inorganic
compounds.
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific
cleavage of RNA.
Ribozymes act by sequence-specific hybridization to the complementary target
RNA, followed by
endonucleolytic cleavage. Specific ribozyme cleavage sites within a potential
RNA target can be identified by
known techniques. For further details see, e.g. , Rossi, Current Biolo~y,
4:469-471 ( 1994), and PCT publication
No. WO 97/33551 (published September 18, 1997).
Nucleic acid molecules in triple-helix formation used to inhibit transcription
should be single-stranded
and composed of deoxynucleotides. The base composition of these
oligonucleotides is designed such that it
promotes triple-helix formation via Hoogsteen base-pairing rules, which
generally require sizeable stretches of
purines or pyrimidines on one strand of a duplex. For further details see,
e.g., PCT publication No. WO
97/33551, supra.
These small molecules can be identified by any one or more of the screening
assays discussed
hereinabove and/or by any other screening techniques well known for those
skilled in the art.
Diagnostic and therapeutic uses of the herein disclosed molecules may also be
based upon the positive
functional assay hits disclosed and described below.
F. Anti-PRO Antibodies
The present invention further provides anti-PRO antibodies. Exemplary
antibodies include polyclonal,
monoclonal, humanized, bispecific, and heteroconjugate antibodies.
1. Polvclonal Antibodies
The anti-PRO antibodies may comprise polyclonal antibodies. Methods of
preparing polyclonal
antibodies are known to the skilled artisan. Polyclonal antibodies can be
raised in a mammal, for example, by
one or more injections of an immunizing agent and, if desired, an adjuvant.
Typically, the immunizing agent
and/or adjuvant will be injected in the mammal by multiple subcutaneous or
intraperitoneal injections. The
immunizing agent may include the PRO polypeptide or a fusion protein thereof.
It may be useful to conjugate
the immunizing agent to a protein known to be immunogenic in the mammal being
immunized. Examples of
such immunogenic proteins include but are not limited to keyhole limpet
hemocyanin, serum albumin, bovine
thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may
be employed include Freund's
complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic
trehalose dicorynomycolate).
The immunization protocol may be selected by one skilled in the art without
undue experimentation.
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2. Monoclonal Antibodies
The anti-PRO antibodies may, alternatively, be monoclonal antibodies.
Monoclonal antibodies may be
prepared using hybridoma methods, such as those described by Kohler and
Milstein, Nature, 256:495 (1975).
In a hybridoma method, a mouse, hamster, or other appropriate host animal, is
typically immunized with an
immunizing agent to elicit lymphocytes that produce or are capable of
producing antibodies that will specifically
bind to the immunizing agent. Alternatively, the lymphocytes may be immunized
in vitro.
The immunizing agent will typically include the PRO polypeptide or a fusion
protein thereof.
Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of
human origin are desired, or spleen
cells or lymph node cells are used if non-human mammalian sources are desired.
The lymphocytes are then
fused with an immortalized cell line using a suitable fusing agent, such as
polyethylene glycol, to form a
hybridoma cell [coding, Monoclonal Antibodies: Principles and Practice,
Academic Press, (1986) pp. 59-103].
Immortalized cell lines are usually transformed mammalian cells, particularly
myeloma cells of rodent, bovine
and human origin. Usually, rat or mouse myeloma cell lines are employed. The
hybridoma cells may be
cultured in a suitable culture medium that preferably contains one or more
substances that inhibit the growth or
survival of the unfused, immortalized cells. For example, if the parental
cells lack the enzyme hypoxanthine
guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the
hybridomas typically will
include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which
substances prevent the growth of
HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support
stable high level expression of
antibody by the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More
preferred immortalized cell lines are murine myeloma lines, which can be
obtained, for instance, from the Salk
Institute Cell Distribution Center, San Diego, California and the American
Type Culture Collection, Manassas,
Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have
been described for the
production of human monoclonal antibodies [Kozbor, J. Immunol . , 133:3001 (
1984); Brodeur et al. , Monoclonal
Antibody Production Techniques and Applications, Marcel Dekker, Inc., New
York, (1987) pp. 51-63].
The culture medium in which the hybridoma cells are cultured can then be
assayed for the presence of
monoclonal antibodies directed against PRO. Preferably, the binding
specificity of monoclonal antibodies
produced by the hybridoma cells is determined by immunoprecipitation or by an
in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such
techniques and assays are
known in the art. The binding affinity of the monoclonal antibody can, for
example, be determined by the
Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
After the desired hybridoma cells are identified, the clones may be subcloned
by limiting dilution
procedures and grown by standard methods [coding, su ra]. Suitable culture
media for this purpose include,
for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.
Alternatively, the hybridoma cells
may be grown in vivo as ascites in a mammal.
The monoclonal antibodies secreted by the subclones may be isolated or
purified from the culture
medium or ascites fluid by conventional immunoglobulin purification procedures
such as, for example, protein
A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or
affinity chromatography.
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The monoclonal antibodies may also be made by recombinant DNA methods, such as
those described
in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the
invention can be readily isolated
and sequenced using conventional procedures (e.g., by using oligonucleotide
probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). The hybridoma cells of the
invention serve as a preferred source of such DNA. Once isolated, the DNA may
be placed into expression
vectors, which are then transfected into host cells such as simian COS cells,
Chinese hamster ovary (CHO) cells,
or myeloma cells that do not otherwise produce immunoglobulin protein, to
obtain the synthesis of monoclonal
antibodies in the recombinant host cells. The DNA also may be modified, for
example, by substituting the
coding sequence for human heavy and light chain constant domains in place of
the homologous murine sequences
[U.S. Patent No. 4,816,567; Morrison et al., supra] or by covalently joining
to the immunoglobulin coding
sequence all or part of the coding sequence for a non-immunoglobulin
polypeptide. Such a non-immunoglobulin
polypeptide can be substituted for the constant domains of an antibody of the
invention, or can be substituted for
the variable domains of one antigen-combining site of an antibody of the
invention to create a chimeric bivalent
antibody.
The antibodies may be monovalent antibodies. Methods for preparing monovalent
antibodies are well
known in the art. For example, one method involves recombinant expression of
immunoglobulin light chain and
modified heavy chain. The heavy chain is truncated generally at any point in
the Fc region so as to prevent
heavy chain crosslinking. Alternatively, the relevant cysteine residues are
substituted with another amino acid
residue or are deleted so as to prevent crosslinking.
In vitro methods are also suitable for preparing monovalent antibodies.
Digestion of antibodies to
produce fragments thereof, particularly, Fab fragments, can be accomplished
using routine techniques known
in the art.
3. Human and Humanized Antibodies
The anti-PRO antibodies of the invention may further comprise humanized
antibodies or human
antibodies. Humanized forms of non-human (e.g., murine) antibodies are
chimeric immunoglobulins,
immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')Z or
other antigen-binding
subsequences of antibodies) which contain minimal sequence derived from non-
human immunoglobulin.
Humanized antibodies include human immunoglobulins (recipient antibody) in
which residues from a
complementary determining region (CDR) of the recipient are replaced by
residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the desired
specificity, affinity and capacity. In
some instances, Fv framework residues of the human immunoglobulin are replaced
by corresponding non-human
residues. Humanized antibodies may also comprise residues which are found
neither in the recipient antibody
nor in the imported CDR or framework sequences. In general, the humanized
antibody will comprise
substantially all of at least one, and typically two, variable domains, in
which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all or
substantially all of the FR regions are
those of a human immunoglobulin consensus sequence. The humanized antibody
optimally also will comprise
at least a portion of an immunoglobulin constant region (Fc), typically that
of a human immunoglobulin [Jones
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et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329
(1988); and Presta, Curr. On.
Struct. Biol., 2:593-596 (1992)].
Methods for humanizing non-human antibodies are well known in the art.
Generally, a humanized
antibody has one or more amino acid residues introduced into it from a source
which is non-human. These non-
human amino acid residues are often referred to as "import" residues, which
are typically taken from an "import"
variable domain. Humanization can be essentially performed following the
method of Winter and co-workers
[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-
327 (1988); Verhoeyen et al.,
Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences
for the corresponding
sequences of a human antibody. Accordingly, such "humanized" antibodies are
chimeric antibodies (U.S. Patent
No. 4,816,567), wherein substantially less than an intact human variable
domain has been substituted by the
corresponding sequence from a non-human species. In practice, humanized
antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues are
substituted by residues from
analogous sites in rodent antibodies.
Human antibodies can also be produced using various techniques known in the
art, including phage
display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks
et al., J. Mol. Biol., 222:581
(1991)]. The techniques of Cole et al. and Boerner et al. are also available
for the preparation of human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Theranv,
Alan R. Liss, p. 77 (1985) and
Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies
can be made by introducing
of human immunoglobulin loci into transgenic animals, e.g., mice in which the
endogenous immunoglobulin
genes have been partially or completely inactivated. Upon challenge, human
antibody production is observed,
which closely resembles that seen in humans in all respects, including gene
rearrangement, assembly, and
antibody repertoire. This approach is described, for example, in U.S. Patent
Nos. 5,545,807; 5,545,806;
5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific
publications: Marks et al.,
Bio/Technolo~y 10, 779-783 ( 1992); Lonberg et al., Nature 368 856-859 (1994);
Morrison, Nature 368, 812-13
(1994); Fishwild et al., Nature Biotechnoloey 14, 845-51 (1996); Neuberger,
Nature Biotechnolo~y 14, 826
(1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).
The antibodies may also be affinity matured using known selection and/or
mutagenesis methods as
described above. Preferred affinity matured antibodies have an affinity which
is five times, more preferably 10
times, even more preferably 20 or 30 times greater than the starting antibody
(generally murine, humanized or
human) from which the matured antibody is prepared.
4. Bispecific Antibodies
Bispecific antibodies are monoclonal, preferably human or humanized,
antibodies that have binding
specificities for at least two different antigens. In the present case, one of
the binding specificities is for the
PRO, the other one is for any other antigen, and preferably for a cell-surface
protein or receptor or receptor
subunit.
Methods for making bispecific antibodies are known in the art. Traditionally,
the recombinant
production of bispecific antibodies is based on the co-expression of two
immunoglobulin heavy-chain/light-chain


CA 02380355 2002-03-O1
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pairs, where the two heavy chains have different specificities [Milstein and
Cuello, Nature, 305:537-539 ( 1983)] .
Because of the random assortment of immunoglobulin heavy and light chains,
these hybridomas (quadromas)
produce a potential mixture of ten different antibody molecules, of which only
one has the correct bispecific
structure. The purification of the correct molecule is usually accomplished by
affinity chromatography steps.
Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in
Traunecker et al., EMBO
J., 10:3655-3659 (1991).
Antibody variable domains with the desired binding specificities (antibody-
antigen combining sites) can
be fused to immunoglobulin constant domain sequences. The fusion preferably is
with an immunoglobulin
heavy-chain constant domain, comprising at least part of the hinge, CH2, and
CH3 regions. It is preferred to
have the first heavy-chain constant region (CH1) containing the site necessary
for light-chain binding present in
at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain
fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression vectors, and
are co-transfected into a suitable
host organism. For further details of generating bispecific antibodies see,
for example, Suresh et al., Methods
in Enzymology, 121:210 (1986).
According to another approach described in WO 96/27011, the interface between
a pair of antibody
molecules can be engineered to maximize the percentage of heterodimers which
are recovered from recombinant
cell culture. The preferred interface comprises at least a part of the CH3
region of an antibody constant domain.
In this method, one or more small amino acid side chains from the interface of
the first antibody molecule are
replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory
"cavities" of identical or similar
size to the large side chains) are created on the interface of the second
antibody molecule by replacing large
amino acid side chains with smaller ones (e.g. alanine or threonine). This
provides a mechanism for increasing
the yield of the heterodimer over other unwanted end-products such as
homodimers.
Bispecific antibodies can be prepared as full length antibodies or antibody
fragments (e.g. F(ab')z
bispecific antibodies). Techniques for generating bispecific antibodies from
antibody fragments have been
described in the literature. For example, bispecific antibodies can be
prepared can be prepared using chemical
linkage. Brennan et al. , Science 229:81 ( 1985) describe a procedure wherein
intact antibodies are proteolytically
cleaved to generate F(ab')z fragments. These fragments are reduced in the
presence of the dithiol complexing
agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular
disulfide formation. The Fab'
fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an
equimolar amount of the other Fab'-TNB derivative to form the bispecific
antibody. The bispecific antibodies
produced can be used as agents for the selective immobilization of enzymes.
Fab' fragments may be directly recovered from E. coli and chemically coupled
to form bispecific
antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the
production of a fully humanized
bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately
secreted from E. coli and subjected
to directed chemical coupling in vitro to form the bispecific antibody. The
bispecific antibody thus formed was
able to bind to cells overexpressing the ErbB2 receptor and normal human T
cells, as well as trigger the lytic
activity of human cytotoxic lymphocytes against human breast tumor targets.
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Various technique for making and isolating bispecific antibody fragments
directly from recombinant cell
culture have also been described. For example, bispecific antibodies have been
produced using leucine zippers.
Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipper
peptides from the Fos and Jun
proteins were linked to the Fab' portions of two different antibodies by gene
fusion. The antibody homodimers
were reduced at the hinge region to form monomers and then re-oxidized to form
the antibody heterodimers.
This method can also be utilized for the production of antibody homodimers.
The "diabody" technology
described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)
has provided an alternative
mechanism for making bispecific antibody fragments. The fragments comprise a
heavy-chain variable domain
(V") connected to a light-chain variable domain (V~) by a linker which is too
short to allow pairing between the
two domains on the same chain. Accordingly, the V f, and V~ domains of one
fragment are forced to pair with
the complementary VL and VH domains of another fragment, thereby forming two
antigen-binding sites. Another
strategy for making bispecific antibody fragments by the use of single-chain
Fv (sFv) dimers has also been
reported. See, Gruber et al., J. Immunol. 152:5368 (1994).
Antibodies with more than two valencies are contemplated. For example,
trispecific antibodies can be prepared.
Tutt et al., J. Immunol. 147:60 (1991).
Exemplary bispecific antibodies may bind to two different epitopes on a given
PRO polypeptide herein.
Alternatively, an anti-PRO polypeptide arm may be combined with an arm which
binds to a triggering molecule
on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or
B7), or Fc receptors for IgG
(FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD 16) so as to focus
cellular defense mechanisms
to the cell expressing the particular PRO polypeptide. Bispecific antibodies
may also be used to localize
cytotoxic agents to cells which express a particular PRO polypeptide. These
antibodies possess a PRO-binding
arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such
as EOTUBE, DPTA, DOTA,
or TETA. Another bispecific antibody of interest binds the PRO polypeptide and
further binds tissue factor
(TF).
5. Heteroconiu~ate Antibodies
Heteroconjugate antibodies are also within the scope of the present invention.
Heteroconjugate
antibodies are composed of two covalently joined antibodies. Such antibodies
have, for example, been proposed
to target immune system cells to unwanted cells [U. S. Patent No. 4,676,980],
and for treatment of HIV infection
[WO 91/00360; WO 92/200373; EP 03089]. It is contemplated that the antibodies
may be prepared in vitro
using known methods in synthetic protein chemistry, including those involving
crosslinking agents. For
example, immunotoxins may be constructed using a disulfide exchange reaction
or by forming a thioether bond.
Examples of suitable reagents for this purpose include iminothiolate and
methyl-4-mercaptobutyrimidate and
those disclosed, for example, in U.S. Patent No. 4,676,980.
6. Effector Function En ineering
It may be desirable to modify the antibody of the invention with respect to
effector function, so as to
enhance, e.g., the effectiveness of the antibody in treating cancer. For
example, cysteine residues) may be
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introduced into the Fc region, thereby allowing interchain disulfide bond
formation in this region. The
homodimeric antibody thus generated may have improved internalization
capability and/or increased complement-
mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See
Caron et al. , J. Exp Med. , 176:
1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric
antibodies with enhanced
anti-tumor activity may also be prepared using heterobifunctional cross-
linkers as described in Wolff et al.
Cancer Research, 53: 2560-2565 ( 1993). Alternatively, an antibody can be
engineered that has dual Fc regions
and may thereby have enhanced complement lysis and ADCC capabilities. See
Stevenson et al., Anti-Cancer
Drug Desisn, 3: 219-230 (1989).
7. Immunoconiugates
The invention also pertains to immunoconjugates comprising an antibody
conjugated to a cytotoxic agent
such as a chemotherapeutic agent, toxin (e.g. , an enzymatically active toxin
of bacterial, fungal, plant, or animal
origin, or fragments thereof), or a radioactive isotope (i.e., a
radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have
been described above.
Enzymatically active toxins and fragments thereof that can be used include
diphtheria A chain, nonbinding active
fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins,
Phytolaca americana proteins
(PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, croon,
sapaonaria officinalis inhibitor,
gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the
tricothecenes. A variety of radionuclides are
available for the production of radioconjugated antibodies. Examples include
2''-Bi, '3'I, '3'In, ~°Y, and '86Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of
bifunctional protein-coupling
agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT), bifunctional
derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters
(such as disuccinimidyl suberate),
aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-
azidobenzoyl) hexanediamine), bis-
diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-
diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2,4-
dinitrobenzene). For example, a ricin
immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098
(1987). Carbon-14-labeled 1-
isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is
an exemplary chelating agent
for conjugation of radionucleotide to the antibody. See W094/11026.
In another embodiment, the antibody may be conjugated to a "receptor" (such
streptavidin) for
utilization in tumor pretargeting wherein the antibody-receptor conjugate is
administered to the patient, followed
by removal of unbound conjugate from the circulation using a clearing agent
and then administration of a
"ligand" (e.g., avidin) that is conjugated to a cytotoxic agent (e.g., a
radionucleotide).
8. Immunoliposomes
The antibodies disclosed herein may also be formulated as immunoliposomes.
Liposomes containing
the antibody are prepared by methods known in the art, such as described in
Epstein et al., Proc. Natl. Acad.
Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030
(1980); and U.S. Pat. Nos.
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4,485,045 and 4,544,545. Liposomes with enhanced circulation time are
disclosed in U.S. Patent No.
5,013,556.
Particularly useful liposomes can be generated by the reverse-phase
evaporation method with a lipid
composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized
phosphatidylethanolamine (PEG-
PE). Liposomes are extruded through filters of defined pore size to yield
liposomes with the desired diameter.
Fab' fragments of the antibody of the present invention can be conjugated to
the liposomes as described in Martin
et al ., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange
reaction. A chemotherapeutic agent
(such as Doxorubicin) is optionally contained within the liposome. See Gabizon
et al. , J. National Cancer Inst. ,
81(19): 1484 (1989).
9. Pharmaceutical Compositions of Antibodies
Antibodies specifically binding a PRO polypeptide identified herein, as well
as other molecules
identified by the screening assays disclosed hereinbefore, can be administered
for the treatment of various
disorders in the form of pharmaceutical compositions.
If the PRO polypeptide is intracellular and whole antibodies are used as
inhibitors, internalizing
antibodies are preferred. However, lipofections or liposomes can also be used
to deliver the antibody, or an
antibody fragment, into cells. Where antibody fragments are used, the smallest
inhibitory fragment that
specifically binds to the binding domain of the target protein is preferred.
For example, based upon the variable-
region sequences of an antibody, peptide molecules can be designed that retain
the ability to bind the target
protein sequence. Such peptides can be synthesized chemically and/or produced
by recombinant DNA
technology. See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-
7893 (1993). The fomn>)ation
herein may also contain more than one active compound as necessary for the
particular indication being treated,
preferably those with complementary activities that do not adversely affect
each other. Alternatively, or in
addition, the composition may comprise an agent that enhances its function,
such as, for example, a cytotoxic
agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such
molecules are suitably present in
combination in amounts that are effective for the purpose intended.
The active ingredients may also be entrapped in microcapsules prepared, for
example, by coacervation
techniques or by interfacial polymerization, for example,
hydroxymethylcellulose or gelatin-microcapsules and
poly-(methylmethacylate) microcapsules, respectively, in colloidal drug
delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles, and
nanocapsules) or in macroemulsions.
Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.
The formulations to be used for in vivo administration must be sterile. This
is readily accomplished by
filtration through sterile filtration membranes.
Sustained-release preparations may be prepared. Suitable examples of sustained-
release preparations
include semipermeable matrices of solid hydrophobic polymers containing the
antibody, which matrices are in
the form of shaped articles, e.g., films, or microcapsules. Examples of
sustained-release matrices include
polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate),
orpoly(vinylalcohol)), polylactides (U.S.
Pat. No. 3,773,919), copolymers of L-glutamic acid and y ethyl-L-glutamate,
non-degradable ethylene-vinyl
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acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON
DEPOT T"' (injectable
microspheres composed of lactic acid-glycolic acid copolymer and leuprolide
acetate), and poly-D-(-)-3-
hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic
acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins for shorter
time periods. When encapsulated
antibodies remain in the body for a long time, they may denature or aggregate
as a result of exposure to moisture
at 37°C, resulting in a loss of biological activity and possible
changes in immunogenicity. Rational strategies
can be devised for stabilization depending on the mechanism involved. For
example, if the aggregation
mechanism is discovered to be intermolecular S-S bond formation through thio-
disulfide interchange, stabilization
may be achieved by modifying sulfhydryl residues, lyophilizing from acidic
solutions, controlling moisture
content, using appropriate additives, and developing specific polymer matrix
compositions.
G. Uses for anti-PRO Antibodies
The anti-PRO antibodies of the invention have various utilities. For example,
anti-PRO antibodies may
be used in diagnostic assays for PRO, e.g. , detecting its expression (and in
some cases, differential expression)
in specific cells, tissues, or serum. Various diagnostic assay techniques
known in the art may be used, such as
competitive binding assays, direct or indirect sandwich assays and
immunoprecipitation assays conducted in
either heterogeneous or homogeneous phases [Zola, Monoclonal Antibodies: A
Manual of Techniq-ues, CRC
Press, Inc. (1987) pp. 147-158]. The antibodies used in the diagnostic assays
can be labeled with a detectable
moiety. The detectable moiety should be capable of producing, either directly
or indirectly, a detectable signal.
For example, the detectable moiety may be a radioisotope, such as 3H, '4C, 3zp
'sS, or 'zSI, a fluorescent or
chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or
luciferin, or an enzyme, such
as alkaline phosphatase, beta-galactosidase or horseradish peroxidase. Any
method known in the art for
conjugating the antibody to the detectable moiety may be employed, including
those methods described by Hunter
et al., Nature, 144:945 (1962); David et al., Biochemistry, 13:1014 (1974);
Pain et al., J. Immunol. Meth.,
40:219 (1981); and Nygren, J. Histochem. and Cvtochem., 30:407 (1982).
Anti-PRO antibodies also are useful for the affinity purification of PRO from
recombinant cell culture
or natural sources. In this process, the antibodies against PRO are
immobilized on a suitable support, such a
Sephadex resin or filter paper, using methods well known in the art. The
immobilized antibody then is contacted
with a sample containing the PRO to be purified, and thereafter the support is
washed with a suitable solvent that
will remove substantially all the material in the sample except the PRO, which
is bound to the immobilized
antibody. Finally, the support is washed with another suitable solvent that
will release the PRO from the
antibody.
The following examples are offered for illustrative purposes only, and are not
intended to limit the scope
of the present invention in any way.
All patent and literature references cited in the present specification are
hereby incorporated by reference
in their entirety.


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EXAMPLES
Commercially available reagents referred to in the examples were used
according to manufacturer's
instructions unless otherwise indicated. The source of those cells identified
in the following examples, and
throughout the specification, by ATCC accession numbers is the American Type
Culture Collection, Manassas,
VA.
EXAMPLE 1: Extracellular Domain Homoloey Screening to Identify Novel
Po~peptides and cDNA Encoding
Therefor
The extracellular domain (ECD) sequences (including the secretion signal
sequence, if any) from about
950 known secreted proteins from the Swiss-Prot public database were used to
search EST databases. The EST
databases included public databases (e.g., Dayhoff, GenBank), and proprietary
databases (e.g. LIFESEQT"',
Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the
computer program BLAST or
BLAST-2 (Altschul et al., Methods in EnzymologY 266:460-480 (1996)) as a
comparison of the ECD protein
sequences to a 6 frame translation of the EST sequences. Those comparisons
with a BLAST score of 70 (or in
some cases 90) or greater that did not encode known proteins were clustered
and assembled into consensus DNA
sequences with the program "phrap" (Phil Green, University of Washington,
Seattle, WA).
Using this extracellular domain homology screen, consensus DNA sequences were
assembled relative
to the other identified EST sequences using phrap. In addition, the consensus
DNA sequences obtained were
often (but not always) extended using repeated cycles of BLAST or BLAST-2 and
phrap to extend the consensus
sequence as far as possible using the sources of EST sequences discussed
above.
Based upon the consensus sequences obtained as described above,
oligonucleotides were then
synthesized and used to identify by PCR a cDNA library that contained the
sequence of interest and for use as
probes to isolate a clone of the full-length coding sequence for a PRO
polypeptide. Forward and reverse PCR
primers generally range from 20 to 30 nucleotides and are often designed to
give a PCR product of about 100-
1000 by in length. The probe sequences are typically 40-55 by in length. In
some cases, additional
oligonucleotides are synthesized when the consensus sequence is greater than
about 1-1.Skbp. In order to screen
several libraries for a full-length clone, DNA from the libraries was screened
by PCR amplification, as per
Ausubel et al., Current Protocols in Molecular Biolo~y, with the PCR primer
pair. A positive library was then
used to isolate clones encoding the gene of interest using the probe
oligonucleotide and one of the primer pairs.
The cDNA libraries used to isolate the cDNA clones were constructed by
standard methods using
commercially available reagents such as those from Invitrogen, San Diego, CA.
The cDNA was primed with
oligo dT containing a NotI site, linked with blunt to SaII hemikinased
adaptors, cleaved with NotI, sized
appropriately by gel electrophoresis, and cloned in a defined orientation into
a suitable cloning vector (such as
pRKB or pRKD; pRKSB is a precursor of pRKSD that does not contain the SfiI
site; see, Holmes et al., Science,
253:1278-1280 (1991)) in the unique XhoI and NotI sites.
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EXAMPLE 2: Isolation of cDNA clones by Amy lase Screening
Preparation of oli_.go dT primed cDNA library
mRNA was isolated from a human tissue of interest using reagents and protocols
from Invitrogen, San
Diego, CA (Fast Track 2). This RNA was used to generate an oligo dT primed
cDNA library in the vector
pRKSD using reagents and protocols from Life Technologies, Gaithersburg, MD
(Super Script Plasmid System).
In this procedure, the double stranded cDNA was sized to greater than 1000 by
and the SaII/NotI linkered cDNA
was cloned into XhoI/NotI cleaved vector. pRKSD is a cloning vector that has
an sp6 transcription initiation
site followed by an SfiI restriction enzyme site preceding the XhoI/NotI cDNA
cloning sites.
2. Preparation of random primed cDNA library
A secondary cDNA library was generated in order to preferentially represent
the 5' ends of the primary
cDNA clones. Sp6 RNA was generated from the primary library (described above),
and this RNA was used
to generate a random primed cDNA library in the vector pSST-AMY.O using
reagents and protocols from Life
Technologies (Super Script Plasmid System, referenced above). In this
procedure the double stranded cDNA
was sized to 500-1000 bp, tinkered with blunt to NotI adaptors, cleaved with
SfiI, and cloned into SfiI/NotI
cleaved vector. pSST-AMY.O is a cloning vector that has a yeast alcohol
dehydrogenase promoter preceding
the cDNA cloning sites and the mouse amylase sequence (the mature sequence
without the secretion signal)
followed by the yeast alcohol dehydrogenase terminator, after the cloning
sites. Thus, cDNAs cloned into this
vector that are fused in frame with amylase sequence will lead to the
secretion of amylase from appropriately
transfected yeast colonies.
3. Transformation and Detection
DNA from the library described in paragraph 2 above was chilled on ice to
which was added
electrocompetent DH10B bacteria (Life Technologies, 20 ml). The bacteria and
vector mixture was then
electroporated as recommended by the manufacturer. Subsequently, SOC media
(Life Technologies, 1 ml) was
added and the mixture was incubated at 37°C for 30 minutes. The
transformants were then plated onto 20
standard 150 mm LB plates containing ampicillin and incubated for 16 hours
(37°C). Positive colonies were
scraped off the plates and the DNA was isolated from the bacterial pellet
using standard protocols, e.g. CsCI-
gradient. The purified DNA was then carried on to the yeast protocols below.
The yeast methods were divided into three categories: (1) Transformation of
yeast with the
plasmid/cDNA combined vector; (2) Detection and isolation of yeast clones
secreting amylase; and (3) PCR
amplification of the insert directly from the yeast colony and purification of
the DNA for sequencing and further
analysis.
The yeast strain used was HD56-SA (ATCC-90785). This strain has the following
genotype: MAT
alpha, ura3-52, leu2-3, leu2-112, his3-11, his3-15, MAL+, SUC+, GAL+.
Preferably, yeast mutants can be
employed that have deficient post-translational pathways. Such mutants may
have translocation deficient alleles
in sec7l, sec72, sec62, with truncated sec71 being most preferred.
Alternatively, antagonists (including
antisense nucleotides and/or ligands) which interfere with the normal
operation of these genes, other proteins
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implicated in this post translation pathway (e.g., SEC6lp, SEC72p, SEC62p,
SEC63p, TDJIp or SSAlp-4p)
or the complex formation of these proteins may also be preferably employed in
combination with the amylase-
expressing yeast.
Transformation was performed based on the protocol outlined by Gietz et al. ,
Nucl. Acid. Res. ,
20:1425 (1992). Transformed cells were then inoculated from agar into YEPD
complex media broth (100 ml)
and grown overnight at 30°C. The YEPD broth was prepared as described
in Kaiser et al., Methods in Yeast
Genetics, Cold Spring Harbor Press, Cold Spring Harbor, NY, p. 207 (1994). The
overnight culture was then
diluted to about 2 x 106 cells/ml (approx. ODD=0.1) into fresh YEPD broth (500
ml) and regrown to 1 x 10'
cells/ml (approx. ODD=0.4-0.5).
The cells were then harvested and prepared for transformation by transfer into
GS3 rotor bottles in a
Sorval GS3 rotor at 5,000 rpm for 5 minutes, the supernatant discarded, and
then resuspended into sterile water,
and centrifuged again in 50 ml falcon tubes at 3,500 rpm in a Beckman GS-6KR
centrifuge. The supernatant
was discarded and the cells were subsequently washed with LiAc/TE (10 ml, 10
mM Tris-HCI, 1 mM EDTA
pH 7.5, 100 mM LiZOOCCH3), and resuspended into LiAc/TE (2.5 ml).
Transformation took place by mixing the prepared cells ( 100 ~.1) with freshly
denatured single stranded
salmon testes DNA (Lofstrand Labs, Gaithersburg, MD) and transforming DNA (1
~,g, vol. < 10 ~,1) in
microfuge tubes. The mixture was mixed briefly by vortexing, then 40% PEG/TE
(600 ~.1, 40% polyethylene
glycol-4000, 10 mM Tris-HCI, 1 mM EDTA, 100 mM Li,00CCH3, pH 7.5) was added.
This mixture was
gently mixed and incubated at 30°C while agitating for 30 minutes. The
cells were then heat shocked at 42°C
for 15 minutes, and the reaction vessel centrifuged in a microfuge at 12,000
rpm for 5-10 seconds, decanted and
resuspended into TE (500 ~,1, 10 mM Tris-HCI, 1 mM EDTA pH 7.5) followed by
recentrifugation. The cells
were then diluted into TE ( 1 ml) and aliquots (200 ~,1) were spread onto the
selective media previously prepared
in 150 mm growth plates (VWR).
Alternatively, instead of multiple small reactions, the transformation was
performed using a single, large
scale reaction, wherein reagent amounts were scaled up accordingly.
The selective media used was a synthetic complete dextrose agar lacking uracil
(SCD-Ura) prepared as
described in Kaiser et al., Methods in Yeast Genetics, Cold Spring Harbor
Press, Cold Spring Harbor, NY, p.
208-210 (1994). Transformants were grown at 30°C for 2-3 days.
The detection of colonies secreting amylase was performed by including red
starch in the selective
growth media. Starch was coupled to the red dye (Reactive Red-120, Sigma) as
per the procedure described by
Biely et al., Anal. Biochem., 172:176-179 (1988). The coupled starch was
incorporated into the SCD-Ura agar
plates at a final concentration of 0.15 % (w/v), and was buffered with
potassium phosphate to a pH of 7.0 (50-
100 mM final concentration).
The positive colonies were picked and streaked across fresh selective media
(onto 150 mm plates) in
order to obtain well isolated and identifiable single colonies. Well isolated
single colonies positive for amylase
secretion were detected by direct incorporation of red starch into buffered
SCD-Ura agar. Positive colonies were
determined by their ability to break down starch resulting in a clear halo
around the positive colony visualized
directly.
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4. Isolation of DNA by PCR Amplification
When a positive colony was isolated, a portion of it was picked by a toothpick
and diluted into sterile
water (30 ~,1) in a 96 well plate. At this time, the positive colonies were
either frozen and stored for subsequent
analysis or immediately amplified. An aliquot of cells (5 ~.1) was used as a
template for the PCR reaction in a
25 ~1 volume containing: 0.5 ~,1 Klentaq (Clontech, Palo Alto, CA); 4.0 ~,l 10
mM dNTP's (Perkin Elmer-
Cetus); 2.5 ~1 Kentaq buffer (Clontech); 0.25 ~1 forward oligo 1; 0.25 ~,1
reverse oligo 2; 12.5 ~,1 distilled water.
The sequence of the forward oligonucleotide 1 was:
5'-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3' (SEQ ID N0:169)
The sequence of reverse oligonucleotide 2 was:
5'-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3' (SEQ ID N0:170)
PCR was then performed as follows:
a. Denature 92°C, 5 minutes
b. 3 cycles of: Denature 92°C, 30 seconds
Anneal 59°C, 30 seconds
Extend 72°C, 60 seconds
c. 3 cycles of: Denature 92°C, 30 seconds
Anneal 57°C, 30 seconds
Extend 72°C, 60 seconds
d. 25 cycles of: Denature 92°C, 30 seconds
Anneal 55°C, 30 seconds
Extend 72°C, 60 seconds
e. Hold 4°C
The underlined regions of the oligonucleotides annealed to the ADH promoter
region and the amylase
region, respectively, and amplified a 307 by region from vector pSST-AMY.O
when no insert was present.
Typically, the first 18 nucleotides of the 5' end of these oligonucleotides
contained annealing sites for the
sequencing primers. Thus, the total product of the PCR reaction from an empty
vector was 343 bp. However,
signal sequence-fused cDNA resulted in considerably longer nucleotide
sequences.
Following the PCR, an aliquot of the reaction (5 ~.l) was examined by agarose
gel electrophoresis in
a 1 % agarose gel using a Tris-Borate-EDTA (TBE) buffering system as described
by Sambrook et al. , supra.
Clones resulting in a single strong PCR product larger than 400 by were
further analyzed by DNA sequencing
after purification with a 96 Qiaquick PCR clean-up column (Qiagen Inc.,
Chatsworth, CA).
EXAMPLE 3: Isolation of cDNA Clones Using Si na~orithm Analysis
Various polypeptide-encoding nucleic acid sequences were identified by
applying a proprietary signal
sequence finding algorithm developed by Genentech, Inc. (South San Francisco,
CA) upon ESTs as well as
clustered and assembled EST fragments from public (e.g., GenBank) and/or
private (LIFESEQ~, Incyte
Pharmaceuticals, Inc., Palo Alto, CA) databases. The signal sequence algorithm
computes a secretion signal
score based on the character of the DNA nucleotides surrounding the first and
optionally the second methionine
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CA 02380355 2002-03-O1
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codon(s) (ATG) at the 5'-end of the sequence or sequence fragment under
consideration. The nucleotides
following the first ATG must code for at least 35 unambiguous amino acids
without any stop codons. If the first
ATG has the required amino acids, the second is not examined. If neither meets
the requirement, the candidate
sequence is not scored. In order to determine whether the EST sequence
contains an authentic signal sequence,
the DNA and corresponding amino acid sequences surrounding the ATG codon are
scored using a set of seven
sensors (evaluation parameters) known to be associated with secretion signals.
Use of this algorithm resulted
in the identification of numerous polypeptide-encoding nucleic acid sequences.
EXAMPLE 4: Isolation of cDNA Clones Encodin~Human PRO Polypeptides
Using the techniques described in Examples 1 to 3 above, numerous full-length
cDNA clones were
identified as encoding PRO polypeptides as disclosed herein. These cDNAs were
then deposited under the terms
of the Budapest Treaty with the American Type Culture Collection, 10801
University Blvd., Manassas, VA
20110-2209, USA (ATCC) as shown in Table 7 below.
Table 7
Material ATCC Dep. No. Deposit Date


DNA26843-1389 203099 August 4, 1998


DNA30867-1335 209807 April 28, 1998


DNA34431-1177 209399 October 17, 1997


DNA38268-1188 209421 October 28, 1997


DNA40621-1440 209922 June 2, 1998


DNA40625-1189 209788 April 21, 1998


DNA45409-2511 203579 January 12, 1999


DNA45495-1550 203156 August 25, 1998


DNA49820-1427 209932 June 2, 1998


DNA56406-1704 203478 November 17, 1998


DNA56410-1414 209923 June 2, 1998


DNA56436-1448 209902 May 27, 1998


DNA56855-1447 203004 June 23, 1998


DNA56860-1510 209952 June 9, 1998


DNA56862-1343 203174 September 1, 1998


DNA56868-1478 203024 June 23, 1998


DNA56869-1545 203161 August 25, 1998


DNA57704-1452 209953 June 9, 1998


DNA58723-1588 203133 August 18, 1998


DNA57827-1493 203045 July 1, 1998


DNA58737-1473 203136 August 18, 1998


DNA58846-1409 209957 June 9, 1998


DNA58850-1495 209956 June 9, 1998


DNA58855-1422 203018 June 23, 1998


DNA59211-1450 209960 June 9, 1998


DNA59212-1627 203245 September 9, 1998


DNA59213-1487 209959 June 9, 1998


DNA59605-1418 203005 June 23, 1998


DNA59609-1470 209963 June 9, 1998


DNA59610-1556 209990 June 16, 1998


DNA59837-2545 203658 February 9, 1999


DNA59844-2542 203650 February 9, 1999


DNA59854-1459 209974 June 16, 1998




CA 02380355 2002-03-O1
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Table 7 (cony)
DNA60625-1507 209975 June 16, 1998


DNA60629-1481 209979 June 16, 1998


DNA61755-1554 203112 August 11, 1998


DNA62812-1594 203248 September 9, 1998


DNA62815-1576 203247 September 9, 1998


DNA64881-1602 203240 September 9, 1998


DNA64886-1601 203241 September 9, 1998


DNA64902-1667 203317 October 6, 1998


DNA64950-1590 203224 September 15, 1998


DNA65403-1565 203230 September 15, 1998


DNA66308-1537 203159 August 25, 1998


DNA66519-1535 203236 September 15, 1998


DNA66521-1583 203225 September 15, 1998


DNA66658-1584 203229 September 15, 1998


DNA66660-1585 203279 September 22, 1998


DNA66663-1598 203268 September 22, 1998


DNA66674-1599 203281 September 22, 1998


DNA68862-2546 203652 February 9, 1999


DNA68866-1644 203283 September 22, 1998


DNA68871-1638 203280 September 22, 1998


DNA68880-1676 203319 October 6, 1998


DNA68883-1691 203535 December 15, 1998


DNA68885-1678 203311 October 6, 1998


DNA71277-1636 203285 September 22, 1998


DNA73727-1673 203459 November 3, 1998


DNA73734-1680 203363 October 20, 1998


DNA73735-1681 203356 October 20, 1998


DNA76393-1664 203323 October 6, 1998


DNA77301-1708 203407 October 27, 1998


DNA77568-1626 203134 August 18, 1998


DNA77626-1705 203536 December 15, 1998


DNA81754-2532 203542 December 15, 1998


DNA81757-2512 203543 December 15, 1998


DNA82302-2529 203534 December 15, 1998


DNA82340-2530 203547 December 22, 1998


DNA83500-2506 203391 October 29, 1998


DNA84920-2614 203966 April 27, 1999


DNA85066-2534 203588 January 12, 1999


DNA86571-2551 203660 February 9, 1999


DNA87991-2540 203656 February 9, 1999


DNA92238-2539 203602 January 20, 1999


DNA96042-2682 PTA-382 July 20, 1999


DNA96787-2534 203589 January 12, 1999


DNA125185-2806 PTA-1031 December 7, 1999


DNA147531-2821 PTA-1185 January 11, 2000


DNA115291-2681 PTA-202 June 8, 1999


DNA 164625-28890 PTA-1535 March 21, 2000


DNA131639-2874 PTA-1784 April 25, 2000


DNA79230-2525 203549 December 22, 1998


These deposits were made under the provisions of the Budapest Treaty on the
International Recognition
of the Deposit of Microorganisms for the Purpose of Patent Procedure and the
Regulations thereunder (Budapest
Treaty). This assures maintenance of a viable culture of the deposit for 30
years from the date of deposit. The
81


CA 02380355 2002-03-O1
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deposits will be made available by ATCC under the terms of the Budapest
Treaty, and subject to an agreement
between Genentech, Inc. and ATCC, which assures permanent and unrestricted
availability of the progeny of
the culture of the deposit to the public upon issuance of the pertinent U.S.
patent or upon laying open to the
public of any U.S. or foreign patent application, whichever comes first, and
assures availability of the progeny
to one determined by the U.S. Commissioner of Patents and Trademarks to be
entitled thereto according to 35
USC ~ 122 and the Commissioner's rules pursuant thereto (including 37 CFR ~
1.14 with particular reference
to 886 OG 638).
The assignee of the present application has agreed that if a culture of the
materials on deposit should
die or be lost or destroyed when cultivated under suitable conditions, the
materials will be promptly replaced on
notification with another of the same. Availability of the deposited material
is not to be construed as a license
to practice the invention in contravention of the rights granted under the
authority of any government in
accordance with its patent laws.
EXAMPLE 5: Use of PRO as a hybridization probe
The following method describes use of a nucleotide sequence encoding PRO as a
hybridization probe.
DNA comprising the coding sequence of full-length or mature PRO as disclosed
herein is employed as
a probe to screen for homologous DNAs (such as those encoding naturally-
occurring variants of PRO) in human
tissue cDNA libraries or human tissue genomic libraries.
Hybridization and washing of filters containing either library DNAs is
performed under the following
high stringency conditions. Hybridization of radiolabeled PRO-derived probe to
the filters is performed in a
solution of 50 % formamide, Sx SSC, 0.1 %o SDS, 0.1 % sodium pyrophosphate, 50
mM sodium phosphate, pH
6.8, 2x Denhardt's solution, and 10% dextran sulfate at 42°C for 20
hours. Washing of the filters is performed
in an aqueous solution of 0. lx SSC and 0.1 % SDS at 42°C.
DNAs having a desired sequence identity with the DNA encoding full-length
native sequence PRO can
then be identified using standard techniques known in the art.
EXAMPLE 6: Expression of PRO in E. coli
This example illustrates preparation of an unglycosylated form of PRO by
recombinant expression in
E. coli.
The DNA sequence encoding PRO is initially amplified using selected PCR
primers. The primers
should contain restriction enzyme sites which correspond to the restriction
enzyme sites on the selected
expression vector. A variety of expression vectors may be employed. An example
of a suitable vector is
pBR322 (derived from E. coli; see Bolivar et al., Gene, 2:95 (1977)) which
contains genes for ampicillin and
tetracycline resistance. The vector is digested with restriction enzyme and
dephosphorylated. The PCR
amplified sequences are then ligated into the vector. The vector will
preferably include sequences which encode
for an antibiotic resistance gene, a trp promoter, a polyhis leader (including
the first six STII codons, polyhis
sequence, and enterokinase cleavage site), the PRO coding region, lambda
transcriptional terminator, and an
argU gene.
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The ligation mixture is then used to transform a selected E. coli strain using
the methods described in
Sambrook et al. , supra. Transformants are identified by their ability to grow
on LB plates and antibiotic resistant
colonies are then selected. Plasmid DNA can be isolated and confirmed by
restriction analysis and DNA
sequencing .
Selected clones can be grown overnight in liquid culture medium such as LB
broth supplemented with
antibiotics. The overnight culture may subsequently be used to inoculate a
larger scale culture. The cells are
then grown to a desired optical density, during which the expression promoter
is turned on.
After culturing the cells for several more hours, the cells can be harvested
by centrifugation. The cell
pellet obtained by the centrifugation can be solubilized using various agents
known in the art, and the solubilized
PRO protein can then be purified using a metal chelating column under
conditions that allow tight binding of the
protein.
PRO may be expressed in E, coli in a poly-His tagged form, using the following
procedure. The DNA
encoding PRO is initially amplified using selected PCR primers. The primers
will contain restriction enzyme
sites which correspond to the restriction enzyme sites on the selected
expression vector, and other useful
sequences providing for efficient and reliable translation initiation, rapid
purification on a metal chelation
column, and proteolytic removal with enterokinase. The PCR-amplified, poly-His
tagged sequences are then
ligated into an expression vector, which is used to transform an E. coli host
based on strain 52 (W3110
fuhA(tonA) Ion galE rpoHts(htpRts) clpP(lacIq). Transformants are first grown
in LB containing 50 mg/ml'
carbenicillin at 30°C with shaking until an O.D.600 of 3-5 is reached.
Cultures are then diluted 50-100 fold into
CRAP media (prepared by mixing 3.57 g (NH4)ZS04, 0.71 g sodium citrate~2H20,
1.07 g KCI, 5.36 g Difco
yeast extract, 5.36 g Sheffield hycase SF in 500 mL water, as well as 110 mM
MPOS, pH 7.3, 0.55% (w/v)
glucose and 7 mM MgS04) and grown for approximately 20-30 hours at 30°C
with shaking. Samples are
removed to verify expression by SDS-PAGE analysis, and the bulk culture is
centrifuged to pellet the cells. Cell
pellets are frozen until purification and refolding.
E. colt paste from 0.5 to 1 L fermentations (6-10 g pellets) is resuspended in
10 volumes (w/v) in 7 M
guanidine, 20 mM Tris, pH 8 buffer. Solid sodium sulfite and sodium
tetrathionate is added to make final
concentrations of O.1M and 0.02 M, respectively, and the solution is stirred
overnight at 4°C. This step results
in a denatured protein with all cysteine residues blocked by sulfitolization.
The solution is centrifuged at 40,000
rpm in a Beckman Ultracentifuge for 30 min. The supernatant is diluted with 3-
5 volumes of metal chelate
column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22
micron filters to clarify. The
clarified extract is loaded onto a 5 ml Qiagen Ni-NTA metal chelate column
equilibrated in the metal chelate
column buffer. The column is washed with additional buffer containing 50 mM
imidazole (Calbiochem, Utrol
grade), pH 7.4. The protein is eluted with buffer containing 250 mM imidazole.
Fractions containing the
desired protein are pooled and stored at 4°C. Protein concentration is
estimated by its absorbance at 280 nm
using the calculated extinction coefficient based on its amino acid sequence.
The proteins are refolded by diluting the sample slowly into freshly prepared
refolding buffer consisting
of: 20 mM Tris, pH 8.6, 0.3 M NaCI, 2.5 M urea, 5 mM cysteine, 20 mM glycine
and 1 mM EDTA.
Refolding volumes are chosen so that the final protein concentration is
between 50 to 100 micrograms/ml. The
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refolding solution is stirred gently at 4°C for 12-36 hours. The
refolding reaction is quenched by the addition
of TFA to a final concentration of 0.4% (pH of approximately 3). Before
further purification of the protein, the
solution is filtered through a 0.22 micron filter and acetonitrile is added to
2-10% final concentration. The
refolded protein is chromatographed on a Poros R1/H reversed phase column
using a mobile buffer of 0.1
TFA with elution with a gradient of acetonitrile from 10 to 80% . Aliquots of
fractions with A280 absorbance
are analyzed on SDS polyacrylamide gels and fractions containing homogeneous
refolded protein are pooled.
Generally, the properly refolded species of most proteins are eluted at the
lowest concentrations of acetonitrile
since those species are the most compact with their hydrophobic interiors
shielded from interaction with the
reversed phase resin. Aggregated species are usually eluted at higher
acetonitrile concentrations. In addition
to resolving misfolded forms of proteins from the desired form, the reversed
phase step also removes endotoxin
1~ from the samples.
Fractions containing the desired folded PRO polypeptide are pooled and the
acetonitrile removed using
a gentle stream of nitrogen directed at the solution. Proteins are formulated
into 20 mM Hepes, pH 6.8 with
0.14 M sodium chloride and 4 % mannitol by dialysis or by gel filtration using
G25 Superfine (Pharmacia) resins
equilibrated in the formulation buffer and sterile filtered.
Many of the PRO polypeptides disclosed herein were successfully expressed as
described above.
EXAMPLE 7: Expression of PRO in mammalian cells
This example illustrates preparation of a potentially glycosylated form of PRO
by recombinant
expression in mammalian cells.
The vector, pRKS (see EP 307,247, published March 15, 1989), is employed as
the expression vector.
Optionally, the PRO DNA is ligated into pRKS with selected restriction enzymes
to allow insertion of the PRO
DNA using ligation methods such as described in Sambrook et al. , supra. The
resulting vector is called pRKS-
PRO.
In one embodiment, the selected host cells may be 293 cells. Human 293 cells
(ATCC CCL 1573) are
grown to confluence in tissue culture plates in medium such as DMEM
supplemented with fetal calf serum and
optionally, nutrient components and/or antibiotics. About 10 ~.g pRKS-PRO DNA
is mixed with about 1 ~g
DNA encoding the VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)] and
dissolved in 500 ~l of 1 mM
Tris-HCI, 0.1 mM EDTA, 0.227 M CaCIZ. To this mixture is added, dropwise, 500
~,I of 50 mM HEPES (pH
7.35), 280 mM NaCI, 1.5 mM NaP04, and a precipitate is allowed to form for 10
minutes at 25°C. The
precipitate is suspended and added to the 293 cells and allowed to settle for
about four hours at 37°C. The
culture medium is aspirated off and 2 ml of 20% glycerol in PBS is added for
30 seconds. The 293 cells are
then washed with serum free medium, fresh medium is added and the cells are
incubated for about 5 days.
Approximately 24 hours after the transfections, the culture medium is removed
and replaced with culture
medium (alone) or culture medium containing 200 ~Ci/ml 35S-cysteine and 200
P,Ci/ml 35S-methionine. After
a 12 hour incubation, the conditioned medium is collected, concentrated on a
spin filter, and loaded onto a 15
SDS gel. The processed gel may be dried and exposed to film for a selected
period of time to reveal the
presence of PRO polypeptide. The cultures containing transfected cells may
undergo further incubation (in
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serum free medium) and the medium is tested in selected bioassays.
In an alternative technique, PRO may be introduced into 293 cells transiently
using the dextran sulfate
method described by Somparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981).
293 cells are grown to
maximal density in a spinner flask and 700 ~,g pRKS-PRO DNA is added. The
cells are first concentrated from
the spinner flask by centrifugation and washed with PBS. The DNA-dextran
precipitate is incubated on the cell
pellet for four hours. The cells are treated with 20% glycerol for 90 seconds,
washed with tissue culture
medium, and re-introduced into the spinner flask containing tissue culture
medium, 5 ~.g/ml bovine insulin and
0.1 ~g/ml bovine transferrin. After about four days, the conditioned media is
centrifuged and filtered to remove
cells and debris. The sample containing expressed PRO can then be concentrated
and purified by any selected
method, such as dialysis and/or column chromatography.
In another embodiment, PRO can be expressed in CHO cells. The pRKS-PRO can be
transfected into
CHO cells using known reagents such as CaP04 or DEAE-dextran. As described
above, the cell cultures can
be incubated, and the medium replaced with culture medium (alone) or medium
containing a radiolabel such as
ssS-methionine. After determining the presence of PRO polypeptide, the culture
medium may be replaced with
serum free medium. Preferably, the cultures are incubated for about 6 days,
and then the conditioned medium
is harvested. The medium containing the expressed PRO can then be concentrated
and purified by any selected
method.
Epitope-tagged PRO may also be expressed in host CHO cells. The PRO may be
subcloned out of the
pRKS vector. The subclone insert can undergo PCR to fuse in frame with a
selected epitope tag such as a poly-
his tag into a Baculovirus expression vector. The poly-his tagged PRO insert
can then be subcloned into a SV40
driven vector containing a selection marker such as DHFR for selection of
stable clones. Finally, the CHO cells
can be transfected (as described above) with the SV40 driven vector. Labeling
may be performed, as described
above, to verify expression. The culture medium containing the expressed poly-
His tagged PRO can then be
concentrated and purified by any selected method, such as by Ni2+-chelate
affinity chromatography.
PRO may also be expressed in CHO and/or COS cells by a transient expression
procedure or in CHO
cells by another stable expression procedure.
Stable expression in CHO cells is performed using the following procedure. The
proteins are expressed
as an IgG construct (immunoadhesin), in which the coding sequences for the
soluble forms (e.g. extracellular
domains) of the respective proteins are fused to an IgGI constant region
sequence containing the hinge, CH2
and CH2 domains and/or is a poly-His tagged form.
Following PCR amplification, the respective DNAs are subcloned in a CHO
expression vector using
standard techniques as described in Ausubel et al., Current Protocols of
Molecular Biology, Unit 3.16, John
Wiley and Sons (1997). CHO expression vectors are constructed to have
compatible restriction sites 5' and 3'
of the DNA of interest to allow the convenient shuttling of cDNA's. The vector
used expression in CHO cells
is as described in Lucas et al., Nucl. Acids Res. 24:9 (1774-1779 (1996), and
uses the SV40 early
promoter/enhancer to drive expression of the cDNA of interest and
dihydrofolate reductase (DHFR). DHFR
expression permits selection for stable maintenance of the plasmid following
transfection.


CA 02380355 2002-03-O1
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Twelve micrograms of the desired plasmid DNA is introduced into approximately
10 million CHO cells
using commercially available transfection reagents Superfect~ (Quiagen),
Dospei or Fugene~ (Boehringer
Mannheim). The cells are grown as described in Lucas et al., supra.
Approximately 3 x 10-' cells are frozen
in an ampule for further growth and production as described below.
The ampules containing the plasmid DNA are thawed by placement into water bath
and mixed by
vortexing. The contents are pipetted into a centrifuge tube containing 10 mLs
of media and centrifuged at 1000
rpm for 5 minutes. The supernatant is aspirated and the cells are resuspended
in 10 mL of selective media (0.2
~m filtered PS20 with 5 % 0.2 ~cm diafiltered fetal bovine serum). The cells
are then aliquoted into a 100 mL
spinner containing 90 mL of selective media. After 1-2 days, the cells are
transferred into a 250 mL spinner
filled with 150 mL selective growth medium and incubated at 37°C. After
another 2-3 days, 250 mL, 500 mL
and 2000 mL spinners are seeded with 3 x 105 cells/mL. The cell media is
exchanged with fresh media by
centrifugation and resuspension in production medium. Although any suitable
CHO media may be employed,
a production medium described in U.S. Patent No. 5,122,469, issued June 16,
1992 may actually be used. A
3L production spinner is seeded at 1.2 x 106 cells/mL. On day 0, the cell
number pH ie determined. On day
1, the spinner is sampled and sparging with filtered air is commenced. On day
2, the spinner is sampled, the
temperature shifted to 33°C, and 30 mL of 500 g/L glucose and 0.6 mL of
10% antifoam (e.g., 35%
polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion) taken.
Throughout the production,
the pH is adjusted as necessary to keep it at around 7.2. After 10 days, or
until the viability dropped below
70 % , the cell culture is harvested by centrifugation and filtering through a
0.22 ~m filter. The filtrate was either
stored at 4°C or immediately loaded onto columns for purification.
For the poly-His tagged constructs, the proteins are purified using a Ni-NTA
column (Qiagen). Before
purification, imidazole is added to the conditioned media to a concentration
of 5 mM. The conditioned media
is pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4,
buffer containing 0.3 M NaCI
and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4°C. After loading,
the column is washed with additional
equilibration buffer and the protein eluted with equilibration buffer
containing 0.25 M imidazole. The highly
purified protein is subsequently desalted into a storage buffer containing 10
mM Hepes, 0.14 M NaCI and 4
mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -
80°C.
Immunoadhesin (Fc-containing) constructs are purified from the conditioned
media as follows. The
conditioned medium is pumped onto a 5 ml Protein A column (Pharmacia) which
had been equilibrated in 20
mM Na phosphate buffer, pH 6.8. After loading, the column is washed
extensively with equilibration buffer
before elution with 100 mM citric acid, pH 3.5. The eluted protein is
immediately neutralized by collecting 1
ml fractions into tubes containing 275 ~L of 1 M Tris buffer, pH 9. The highly
purified protein is subsequently
desalted into storage buffer as described above for the poly-His tagged
proteins. The homogeneity is assessed
by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman
degradation.
Many of the PRO polypeptides disclosed herein were successfully expressed as
described above.
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EXAMPLE 8: Expression of PRO in Yeast
The following method describes recombinant expression of PRO in yeast.
First, yeast expression vectors are constructed for intracellular production
or secretion of PRO from
the ADH2/GAPDH promoter. DNA encoding PRO and the promoter is inserted into
suitable restriction enzyme
sites in the selected plasmid to direct intracellular expression of PRO. For
secretion, DNA encoding PRO can
be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH
promoter, a native PRO
signal peptide or other mammalian signal peptide, or, for example, a yeast
alpha-factor or invertase secretory
signal/leader sequence, and linker sequences (if needed) for expression of
PRO.
Yeast cells, such as yeast strain AB 110, can then be transformed with the
expression plasmids described
above and cultured in selected fermentation media. The transformed yeast
supernatants can be analyzed by
precipitation with 10 % trichloroacetic acid and separation by SDS-PAGE,
followed by staining of the gels with
Coomassie Blue stain.
Recombinant PRO can subsequently be isolated and purified by removing the
yeast cells from the
fermentation medium by centrifugation and then concentrating the medium using
selected cartridge filters. The
concentrate containing PRO may further be purified using selected column
chromatography resins.
Many of the PRO polypeptides disclosed herein were successfully expressed as
described above.
EXAMPLE 9: E~ression of PRO in Baculovirus-Infected Insect Cells
The following method describes recombinant expression of PRO in Baculovirus-
infected insect cells.
The sequence coding for PRO is fused upstream of an epitope tag contained
within a baculovirus
expression vector. Such epitope tags include poly-his tags and immunoglobulin
tags (like Fc regions of IgG).
A variety of plasmids may be employed, including plasmids derived from
commercially available plasmids such
as pVL1393 (Novagen). Briefly, the sequence encoding PRO or the desired
portion of the coding sequence of
PRO such as the sequence encoding the extracellular domain of a transmembrane
protein or the sequence
encoding the mature protein if the protein is extracellular is amplified by
PCR with primers complementary to
the 5' and 3' regions. The 5' primer may incorporate flanking (selected)
restriction enzyme sites. The product
is then digested with those selected restriction enzymes and subcloned into
the expression vector.
Recombinant baculovirus is generated by co-transfecting the above plasmid and
BaculoGoldT"' virus
DNA (Pharmingen) into Spodoptera frugiperda ("Sf~") cells (ATCC CRL 1711 )
using lipofectin (commercially
available from GIBCO-BRL). After 4 - 5 days of incubation at 28°C, the
released viruses are harvested and used
for further amplifications. Viral infection and protein expression are
performed as described by O'Reilley et
al., Baculovirus e~ression vectors: A Laboratory Manual, Oxford: Oxford
University Press (1994).
Expressed poly-his tagged PRO can then be purified, for example, by Ni2+-
chelate affinity
chromatography as follows. Extracts are prepared from recombinant virus-
infected Sf9 cells as described by
Rupert et al., Nature, 362:175-179 (1993). Briefly, Sf9 cells are washed,
resuspended in sonication buffer (25
mL Hepes, pH 7.9; 12.5 mM MgCIZ; 0.1 mM EDTA; 10% glycerol; 0.1 % NP-40; 0.4 M
KCl), and sonicated
twice for 20 seconds on ice. The sonicates are cleared by centrifugation, and
the supernatant is diluted 50-fold
in loading buffer (SO mM phosphate, 300 mM NaCI, 10% glycerol, pH 7.8) and
filtered through a 0.45 ,um
87


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filter. A Ni2+-NTA agarose column (commercially available from Qiagen) is
prepared with a bed volume of 5
mL, washed with 25 mL of water and equilibrated with 25 mL of loading buffer.
The filtered cell extract is
loaded onto the column at 0.5 mL per minute. The column is washed to baseline
AZBO with loading buffer, at
which point fraction collection is started. Next, the column is washed with a
secondary wash buffer (50 mM
phosphate; 300 mM NaCI, 10% glycerol, pH 6.0), which elutes nonspecifically
bound protein. After reaching
AZ$o baseline again, the column is developed with a 0 to 500 mM Imidazole
gradient in the secondary wash
buffer. One mL fractions are collected and analyzed by SDS-PAGE and silver
staining or Western blot with
Niz+-NTA-conjugated to alkaline phosphatase (Qiagen). Fractions containing the
eluted His,o tagged PRO are
pooled and dialyzed against loading buffer.
Alternatively, purification of the IgG tagged (or Fc tagged) PRO can be
performed using known
chromatography techniques, including for instance, Protein A or protein G
column chromatography.
Many of the PRO polypeptides disclosed herein were successfully expressed as
described above.
EXAMPLE 10: Preparation of Antibodies that Bind PRO
This example illustrates preparation of monoclonal antibodies which can
specifically bind PRO.
Techniques for producing the monoclonal antibodies are known in the art and
are described, for
instance, in Goding, supra. Immunogens that may be employed include purified
PRO, fusion proteins containing
PRO, and cells expressing recombinant PRO on the cell surface. Selection of
the immunogen can be made by
the skilled artisan without undue experimentation.
Mice, such as Balb/c, are immunized with the PRO immunogen emulsified in
complete Freund's
adjuvant and injected subcutaneously or intraperitoneally in an amount from 1-
100 micrograms. Alternatively,
the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research,
Hamilton, MT) and
injected into the animal's hind foot pads. The immunized mice are then boosted
10 to 12 days later with
additional immunogen emulsified in the selected adjuvant. Thereafter, for
several weeks, the mice may also be
boosted with additional immunization injections. Serum samples may be
periodically obtained from the mice
by retro-orbital bleeding for testing in ELISA assays to detect anti-PRO
antibodies.
After a suitable antibody titer has been detected, the animals "positive" for
antibodies can be injected
with a final intravenous injection of PRO. Three to four days later, the mice
are sacrificed and the spleen cells
are harvested. The spleen cells are then fused (using 35 % polyethylene
glycol) to a selected murine myeloma
cell line such as P3X63AgU.l, available from ATCC, No. CRL 1597. The fusions
generate hybridoma cells
which can then be plated in 96 well tissue culture plates containing HAT
(hypoxanthine, aminopterin, and
thymidine) medium to inhibit proliferation of non-fused cells, myeloma
hybrids, and spleen cell hybrids.
The hybridoma cells will be screened in an ELISA for reactivity against PRO.
Determination of
"positive" hybridoma cells secreting the desired monoclonal antibodies against
PRO is within the skill in the art.
The positive hybridoma cells can be injected intraperitoneally into syngeneic
Balb/c mice to produce
ascites containing the anti-PRO monoclonal antibodies. Alternatively, the
hybridoma cells can be grown in tissue
culture flasks or roller bottles. Purification of the monoclonal antibodies
produced in the ascites can be
accomplished using ammonium sulfate precipitation, followed by gel exclusion
chromatography. Alternatively,
88


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affinity chromatography based upon binding of antibody to protein A or protein
G can be employed.
EXAMPLE 11: Purification of PRO Polypentides Using Specific Antibodies
Native or recombinant PRO polypeptides may be purified by a variety of
standard techniques in the art
of protein purification. For example, pro-PRO polypeptide, mature PRO
polypeptide, or pre-PRO polypeptide
is purified by immunoaffinity chromatography using antibodies specific for the
PRO polypeptide of interest. In
general, an immunoaffmity column is constructed by covalently coupling the
anti-PRO polypeptide antibody to
an activated chromatographic resin.
Polyclonal immunoglobulins are prepared from immune sera either by
precipitation with ammonium
sulfate or by purification on immobilized Protein A (Pharmacia LKB
Biotechnology, Piscataway, N.J.).
Likewise, monoclonal antibodies are prepared from mouse ascites fluid by
ammonium sulfate precipitation or
chromatography on immobilized Protein A. Partially purified immunoglobulin is
covalently attached to a
chromatographic resin such as CnBr-activated SEPHAROSET"' (Pharmacia LKB
Biotechnology). The antibody
is coupled to the resin, the resin is blocked, and the derivative resin is
washed according to the manufacturer's
instructions.
Such an immunoaffinity column is utilized in the purification of PRO
polypeptide by preparing a fraction
from cells containing PRO polypeptide in a soluble form. This preparation is
derived by solubilization of the
whole cell or of a subcellular fraction obtained via differential
centrifugation by the addition of detergent or by
other methods well known in the art. Alternatively, soluble PRO polypeptide
containing a signal sequence may
be secreted in useful quantity into the medium in which the cells are grown.
A soluble PRO polypeptide-containing preparation is passed over the
immunoaffinity column, and the
column is washed under conditions that allow the preferential absorbance of
PRO polypeptide (e.g. , high ionic
strength buffers in the presence of detergent). Then, the column is eluted
under conditions that disrupt
antibody/PRO polypeptide binding (e.g. , a low pH buffer such as approximately
pH 2-3, or a high concentration
of a chaotrope such as urea or thiocyanate ion), and PRO polypeptide is
collected.
EXAMPLE 12: Drug Screening
This invention is particularly useful for screening compounds by using PRO
polypeptides or binding
fragment thereof in any of a variety of drug screening techniques. The PRO
polypeptide or fragment employed
in such a test may either be free in solution, affixed to a solid support,
borne on a cell surface, or located
intracellularly. One method of drug screening utilizes eukaryotic or
prokaryotic host cells which are stably
transformed with recombinant nucleic acids expressing the PRO polypeptide or
fragment. Drugs are screened
against such transformed cells in competitive binding assays. Such cells,
either in viable or fixed form, can be
used for standard binding assays. One may measure, for example, the formation
of complexes between PRO
polypeptide or a fragment and the agent being tested. Alternatively, one can
examine the diminution in complex
formation between the PRO polypeptide and its target cell or target receptors
caused by the agent being tested.
Thus, the present invention provides methods of screening for drugs or any
other agents which can
affect a PRO polypeptide-associated disease or disorder. These methods
comprise contacting such an agent with
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an PRO polypeptide or fragment thereof and assaying (I) for the presence of a
complex between the agent and
the PRO polypeptide or fragment, or (ii) for the presence of a complex between
the PRO polypeptide or fragment
and the cell, by methods well known in the art. In such competitive binding
assays, the PRO polypeptide or
fragment is typically labeled. After suitable incubation, free PRO polypeptide
or fragment is separated from that
present in bound form, and the amount of free or uncomplexed label is a
measure of the ability of the particular
agent to bind to PRO polypeptide or to interfere with the PRO polypeptide/cell
complex.
Another technique for drug screening provides high throughput screening for
compounds having suitable
binding affinity to a polypeptide and is described in detail in WO 84/03564,
published on September 13, 1984.
Briefly stated, large numbers of different small peptide test compounds are
synthesized on a solid substrate, such
as plastic pins or some other surface. As applied to a PRO polypeptide, the
peptide test compounds are reacted
with PRO polypeptide and washed. Bound PRO polypeptide is detected by methods
well known in the art.
Purified PRO polypeptide can also be coated directly onto plates for use in
the aforementioned drug screening
techniques. In addition, non-neutralizing antibodies can 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 PRO polypeptide specifically compete with a test
compound for binding to PRO
polypeptide or fragments thereof. In this manner, the antibodies can be used
to detect the presence of any
peptide which shares one or more antigenic determinants with PRO polypeptide.
EXAMPLE 13: Rational Drug Design
The goal of rational drug design is to produce structural analogs of
biologically active polypeptide of
interest (i. e. , a PRO polypeptide) or of small molecules with which they
interact, e. g. , agonists, antagonists, or
inhibitors. Any of these examples can be used to fashion drugs which are more
active or stable forms of the
PRO polypeptide or which enhance or interfere with the function of the PRO
polypeptide in vivo (c.f., Hodgson,
Bio/TechnoloQV, 9: 19-21 (1991)).
In one approach, the three-dimensional structure of the PRO polypeptide, or of
an PRO
polypeptide-inhibitor complex, is determined by x-ray crystallography, by
computer modeling or, most typically,
by a combination of the two approaches. Both the shape and charges of the PRO
polypeptide must be ascertained
to elucidate the structure and to determine active sites) of the molecule.
Less often, useful information regarding
the structure of the PRO polypeptide may be gained by modeling based on the
structure of homologous proteins.
In both cases, relevant structural information is used to design analogous PRO
polypeptide-like molecules or to
identify efficient inhibitors. Useful examples of rational drug design may
include molecules which have improved
activity or stability as shown by Braxton and Wells, Biochemistry. 31:7796-
7801 (1992) or which act as
inhibitors, agonists, or antagonists of native peptides as shown by Athauda et
al., J. Biochem., 113:742-746
(1993).
It is also possible to isolate a target-specific antibody, selected by
functional assay, as described above,
and then to solve its crystal structure. This approach, in principle, yields a
pharmacore upon which subsequent
drug design can be based. It is possible to bypass protein crystallography
altogether by generating anti-idiotypic


CA 02380355 2002-03-O1
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antibodies (anti-ids) to a functional, pharmacologically active antibody. As a
mirror image of a mirror image,
the binding site of the anti-ids would be expected to be an analog of the
original receptor. The anti-id could then
be used to identify and isolate peptides from banks of chemically or
biologically produced peptides. The isolated
peptides would then act as the pharmacore.
By virtue of the present invention, sufficient amounts of the PRO polypeptide
may be made available
to perform such analytical studies as X-ray crystallography. In addition,
knowledge of the PRO polypeptide
amino acid sequence provided herein will provide guidance to those employing
computer modeling techniques
in place of or in addition to x-ray crystallography.
EXAMPLE 14: Pericyte c-Fos Induction (Assay 93)
This assay shows that certain polypeptides of the invention act to induce the
expression of c-fos in
pericyte cells and, therefore, are useful not only as diagnostic markers for
particular types of pericyte-associated
tumors but also for giving rise to antagonists which would be expected to be
useful for the therapeutic treatment
of pericyte-associated tumors. Induction of c-fos expression in pericytes is
also indicative of the induction of
angiogenesis and, as such, PRO polypeptides capable of inducing the expression
of c-fos would be expected to
be useful for the treatment of conditions where induced angiogenesis would be
beneficial including, for example,
wound healing, and the like. Specifically, on day 1, pericytes are received
from VEC Technologies and all but
5 ml of media is removed from flask. On day 2, the pericytes are trypsinized,
washed, spun and then plated onto
96 well plates. On day 7, the media is removed and the pericytes are treated
with 100 ~,1 of PRO polypeptide
test samples and controls (positive control = DME+5% serum +/- PDGF at 500
ng/ml; negative control =
protein 32). Replicates are averaged and SD/CV are determined. Fold increase
over Protein 32 (buffer control)
value indicated by chemiluminescence units (RLU) luminometer reading verses
frequency is plotted on a
histogram. Two-fold above Protein 32 value is considered positive for the
assay. ASY Matrix: Growth media
= low glucose DMEM = 20% FBS + 1X pen strep + 1X fungizone. Assay Media = low
glucose DMEM
+5 % FBS.
The following polypeptides tested positive in this assay: PR01347 and PR01340.
EXAMPLE 15: Abilit~of PRO Polypeptides to Stimulate the Release of
ProteoQlycans from Cartilage (Assay
The ability of various PRO polypeptides to stimulate the release of
proteoglycans from cartilage tissue
was tested as follows.
The metacarphophalangeal joint of 4-6 month old pigs was aseptically
dissected, and articular cartilage
was removed by free hand slicing being careful to avoid the underlying bone.
The cartilage was minced and
cultured in bulk for 24 hours in a humidified atmosphere of 95 % air, 5 % COZ
in serum free (SF) media
(DME/F12 1:1) woth 0.1 % BSA and 100U/ml penicillin and 100~.g/ml
streptomycin. After washing three
times, approximately 100 mg of articular cartilage was aliquoted into
micronics tubes and incubated for an
additional 24 hours in the above SF media. PRO polypeptides were then added at
1 % either alone or in
combination with 18 ng/ml interleukin-la, a known stimulator of proteoglycan
release from cartilage tissue.
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The supernatant was then harvested and assayed for the amount of proteoglycans
using the 1,9-dimethyl-
methylene blue (DMB) colorimetric assay (Farndale and Buttle, Biochem.
Biophys. Acta 883:173-177 (1985)).
A positive result in this assay indicates that the test polypeptide will find
use, for example, in the treatment of
sports-related joint problems, articular cartilage defects, osteoarthritis or
rheumatoid arthritis.
When various PRO polypeptides were tested in the above assay, the polypeptides
demonstrated a marked
ability to stimulate release of proteoglycans from cartilage tissue both
basally and after stimulation with
interleukin-1 a and at 24 and 72 hours after treatment, thereby indicating
that these PRO polypeptides are useful
for stimulating proteoglycan release from cartilage tissue. As such, these PRO
polypeptides are useful for the
treatment of sports-related joint problems, articular cartilage defects,
osteoarthritis or rheumatoid arthritis. The
polypeptides testing positive in this assay are: PR01565, PR01693, PR01801 and
PR010096.
EXAMPLE 16: Detection of Polypentides That Affect Glucose or FFA Uptake in
Skeletal Muscle (Assay 106)
This assay is designed to determine whether PRO polypeptides show the ability
to affect glucose or FFA
uptake by skeletal muscle cells. PRO polypeptides testing positive in this
assay would be expected to be useful
for the therapeutic treatment of disorders where either the stimulation or
inhibition of glucose uptake by skeletal
muscle would be beneficial including, for example, diabetes or hyper- or hypo-
insulinemia.
In a 96 well format, PRO polypeptides to be assayed are added to primary rat
differentiated skeletal
muscle, and allowed to incubate overnight. Then fresh media with the PRO
polypeptide and +/- insulin are
added to the wells. The sample media is then monitored to determine glucose
and FFA uptake by the skeletal
muscle cells. The insulin will stimulate glucose and FFA uptake by the
skeletal muscle, and insulin in media
without the PRO polypeptide is used as a positive control, and a limit for
scoring. As the PRO polypeptide being
tested may either stimulate or inhibit glucose and FFA uptake, results are
scored as positive in the assay if
greater than 1.5 times or less than 0.5 times the insulin control.
The following PRO polypeptides tested positive as either stimulators or
inhibitors of glucose and/or FFA
uptake in this assay: PR04405.
EXAMPLE 17: Identification of PRO Polypentides That Stimulate TNF-a Release In
Human Blood (Assay 128)
This assay shows that certain PRO polypeptides of the present invention act to
stimulate the release of
TNF-a in human blood. PRO polypeptides testing positive in this assay are
useful for, among other things,
research purposes where stimulation of the release of TNF-a would be desired
and for the therapeutic treatment
of conditions wherein enhanced TNF-a release would be beneficial.
Specifically, 200 ~1 of human blood
supplemented with SOmM Hepes buffer (pH 7.2) is aliquotted per well in a 96
well test plate. To each well is
then added 300,1 of either the test PRO polypeptide in 50 mM Hepes buffer (at
various concentrations) or 50
mM Hepes buffer alone (negative control) and the plates are incubated at
37°C for 6 hours. The samples are
then centrifuged and SOp,I of plasma is collected from each well and tested
for the presence of TNF-a by ELISA
assay. A positive in the assay is a higher amount of TNF-a in the PRO
polypeptide treated samples as compared
to the negative control samples.
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The following PRO polypeptides tested positive in this assay: PR0263. PR0295,
PR01282, PRO 1063,
PR01356, PR03543, and PR05990.
EXAMPLE 18: Tumor Versus Normal Differential Tissue Expression Distribution
Oligonucleotide probes were constructed from some of the PRO polypeptide-
encoding nucleotide
sequences shown in the accompanying figures for use in quantitative PCR
amplification reactions. The
oligonucleotide probes were chosen so as to give an approximately 200-600 base
pair amplified fragment from
the 3' end of its associated template in a standard PCR reaction. The
oligonucleotide probes were employed in
standard quantitative PCR amplification reactions with cDNA libraries isolated
from different human tumor and
normal human tissue samples and analyzed by agarose gel electrophoresis so as
to obtain a quantitative
determination of the level of expression of the PRO polypeptide-encoding
nucleic acid in the various tumor and
normal tissues tested. (3-actin was used as a control to assure that
equivalent amounts of nucleic acid was used
in each reaction. Identification of the differential expression of the PRO
polypeptide-encoding nucleic acid in
one or more tumor tissues as compared to one or more normal tissues of the
same tissue type renders the
molecule useful diagnostically for the determination of the presence or
absence of tumor in a subject suspected
of possessing a tumor as well as therapeutically as a target for the treatment
of a tumor in a subject possessing
such a tumor. These assays provided the following results.
Molecule is more hi hg >y expressed in: as compared to:
DNA26843-1389 normal lung lung tumor
rectum tumor normal rectum
DNA30867-1335 normal kidney kidney tumor
DNA40621-1440 normal lung lung tumor
DNA40625-1189 normal lung lung tumor
DNA45409-2511 melanoma tumor normal skin
DNA56406-1704 kidney tumor normal kidney
normal skin melanoma tumor
DNA56410-1414 normal stomach stomach tumor
DNA56436-1448 normal skin melanoma tumor
DNA56855-1447 normal esophagus esophageal tumor
rectum tumor normal rectum
DNA56860-1510 normal kidney kidney tumor
rectum tumor normal rectum
DNA56862-1343 kidney tumor normal kidney
normal lung lung tumor
93


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Molecule is more h~hlv expressedas compared to:
in:


DNA56868-1478 normal stomach stomach tumor


normal lung lung tumor


DNA56869-1545 normal esophagus esophageal tumor


normal skin melanoma tumor


DNA57704-1452 normal stomach stomach tumor


rectum tumor normal rectum


DNA58723-1588 normal stomach stomach tumor


kidney tumor normal kidney


normal skin melanoma tumor


DNA57827-1493 normal stomach stomach tumor


normal skin melanoma tumor


DNA58737-1473 esophageal tumor normal esophagus


normal stomach stomach tumor


DNA58846-1409 lung tumor normal lung


DNA58850-1495 esophageal tumor normal esophagus


kidney tumor normal kidney


DNA58855-1422 normal stomach stomach tumor


rectum tumor normal rectum


DNA59211-1450 normal kidney kidney tumor


DNA59212-1627 normal skin melanoma tumor


DNA59213-1487 normal stomach stomach tumor


normal skin melanoma tumor


DNA59605-1418 melanoma tumor normal skin


DNA59609-1470 esophageal tumor normal esophagus


DNA59610-1556 esophageal tumor normal esophagus


lung tumor normal lung


normal skin melanoma tumor


DNA59837-2545 normal skin melanoma tumor


DNA59844-2542 normal skin melanoma tumor


esophageal tumor normal esophagus


DNA59854-1459 normal esophagus esophageal tumor


stomach tumor normal stomach


normal lung lung tumor


DNA60625-1507 normal lung lung tumor


DNA60629-1481 normal esophagus esophageal tumor


normal rectum rectum tumor


94


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Molecule is more highly expressedas compared to:
in:


DNA61755-1554 normal stomach stomach tumor


kidney tumor normal kidney


DNA62812-1594 normal stomach stomach tumor


normal lung lung tumor


normal rectum rectum tumor


normal skin melanoma tumor


DNA62815-1576 esophageal tumor normal esophagus



DNA64881-1602 normal stomach stomach tumor


normal lung lung tumor


DNA64902-1667 esophageal tumor normal esophagus


kidney tumor normal kidney


DNA65403-1565 normal esophagus esophageal tumor


DNA66308-1537 normal lung lung tumor



DNA66519-1535 kidney tumor normal kidney


DNA66521-1583 normal esophagus esophageal tumor


normal stomach stomach tumor


normal lung lung tumor


normal rectum rectum tumor


normal skin melanoma tumor


DNA66658-1584 normal lung lung tumor


melanoma tumor normal skin


DNA66660-1585 lung tumor normal lung


DNA66674-1599 kidney tumor normal kidney


normal lung lung tumor


DNA68862-2546 melanoma tumor normal skin


DNA68866-1644 normal stomach stomach tumor



DNA68871-1638 lung tumor normal lung


normal skin melanoma tumor


DNA68880-1676 normal lung lung tumor


normal skin melanoma tumor


DNA68883-1691 esophageal tumor normal esophagus


DNA68885-1678 lung tumor normal lung



DNA71277-1636 normal stomach stomach tumor


DNA73734-1680 normal lung lung tumor


95


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WO 01/16318 PCT/US00/23328
Molecule is more hi hg 1y expressed in: as compared to:
DNA73735-1681 esophageal tumor normal esophagus
normal kidney kidney tumor
lung tumor normal lung
normal skin melanoma tumor
DNA76393-1664 esophageal tumor normal esophagus
stomach tumor normal stomach
lung tumor normal lung
rectum tumor normal rectum
DNA77568-1626 normal stomach stomach tumor
lung tumor normal lung
DNA77626-1705 normal rectum rectum tumor
DNA81754-2532 normal skin melanoma tumor


DNA81757-2512 esophageal tumor normal esophagus


normal stomach stomach tumor


melanoma tumor normal skin


DNA82302-2529 normal stomach stomach tumor


normal lung lung tumor


DNA82340-2530 normal esophagus esophageal tumor


DNA85066-2534 lung tumor normal lung


normal skin melanoma tumor


DNA87991-2540 esophageal tumor normal esophagus


DNA92238-2539 normal skin melanoma tumor


DNA96787-2534 normal kidney kidney tumor
EXAMPLE 19: Identification of Receptor/LiQand Interactions
In this assay, various PRO polypeptides are tested for ability to bind to a
panel of potential receptor or
ligand molecules for the purpose of identifying receptor/ligand interactions.
The identification of a ligand for
a known receptor, a receptor for a known ligand or a novel receptor/ligand
pair is useful for a variety of
indications including, for example, targeting bioactive molecules (linked to
the ligand or receptor) to a cell
known to express the receptor or ligand, use of the receptor or ligand as a
reagent to detect the presence of the
ligand or receptor in a composition suspected of containing the same, wherein
the composition may comprise
cells suspected of expressing the ligand or receptor, modulating the growth of
or another biological or
immunological activity of a cell known to express or respond to the receptor
or ligand, modulating the immune
response of cells or toward cells that express the receptor or ligand,
allowing the preparaion of agonists,
antagonists and/or antibodies directed against the receptor or ligand which
will modulate the growth of or a
biological or immunological activity of a cell expressing the receptor or
ligand, and various other indications
which will be readily apparent to the ordinarily skilled artisan.
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The assay is performed as follows. A PRO polypeptide of the present invention
suspected of being a
ligand for a receptor is expressed as a fusion protein containing the Fc
domain of human IgG (an
immunoadhesin). Receptor-ligand binding is detected by allowing interaction of
the immunoadhesin polypeptide
with cells (e.g. Cos cells) expressing candidate PRO polypeptide receptors and
visualization of bound
immunoadhesin with fluorescent reagents directed toward the Fc fusion domain
and examination by microscope.
Cells expressing candidate receptors are produced by transient transfection,
in parallel, of defined subsets of a
library of cDNA expression vectors encoding PRO polypeptides that may function
as receptor molecules. Cells
are then incubated for 1 hour in the presence of the PRO polypeptide
immunoadhesin being tested for possible
receptor binding. The cells are then washed and fixed with paraformaldehyde.
The cells are then incubated with
fluorescent conjugated antibody directed against the Fc portion of the PRO
polypeptide immunoadhesin (e.g.
FITC conjugated goat anti-human-Fc antibody). The cells are then washed again
and examined by microscope.
A positive interaction is judged by the presence of fluorescent labeling of
cells transfected with cDNA encoding
a particular PRO polypeptide receptor or pool of receptors and an absence of
similar fluorescent labeling of
similarly prepared cells that have been transfected with other cDNA or pools
of cDNA. If a defined pool of
cDNA expression vectors is judged to be positive for interaction with a PRO
polypeptide immunoadhesin, the
individual cDNA species that comprise the pool are tested individually (the
pool is "broken down") to determine
the specific cDNA that encodes a receptor able to interact with the PRO
polypeptide immunoadhesin.
In another embodiment of this assay, an epitope-tagged potential ligand PRO
polypeptide (e.g. 8
histidine "His" tag) is allowed to interact with a panel of potential receptor
PRO polypeptide molecules that have
been expressed as fusions with the Fc domain of human IgG (immunoadhesins).
Following a 1 hour
co-incubation with the epitope tagged PRO polypeptide, the candidate receptors
are each immunoprecipitated
with protein A beads and the beads are washed. Potential ligand interaction is
determined by western blot
analysis of the immunoprecipitated complexes with antibody directed towards
the epitope tag. An interaction
is judged to occur if a band of the anticipated molecular weight of the
epitope tagged protein is observed in the
western blot analysis with a candidate receptor, but is not observed to occur
with the other members of the panel
of potential receptors.
Using these assays, the following receptor/ligand interactions have been
herein identified:
(1) PR010272 binds to PR05801.
(2) PR020110 binds to the human IL-17 receptor (Yao et al., Cytokine 9(11):794-
800 (1997); also herein
designated as PROD and to PR020040.
(3) PR010096 binds to PR020233.
(4) PR019670 binds to PR01890.
The foregoing written specification is considered to be sufficient to enable
one skilled in the art to
practice the invention. The present invention is not to be limited in scope by
the construct deposited, since the
deposited embodiment is intended as a single illustration of certain aspects
of the invention and any constructs
that are functionally equivalent are within the scope of this invention. The
deposit of material herein does not
constitute an admission that the written description herein contained is
inadequate to enable the practice of any
aspect of the invention, including the best mode thereof, nor is it to be
construed as limiting the scope of the
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claims to the specific illustrations that it represents. Indeed, various
modifications of the invention in addition
to those shown and described herein will become apparent to those skilled in
the art from the foregoing
description and fall within the scope of the appended claims.
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Representative Drawing