USOO5766866A United States Patent (19) 11 Patent Number: 5,766,866 Center et al. 45) Date of Patent: Jun. 16, 1998 54 LYMPHOCYTE CHEMOATTRACTANT Center, et al. The Journal of Immunology. 128:2563-2568 FACTOR AND USES THEREOF (1982). 75 Inventors: David M. Center. Wellesley Hills; Cruikshank, et al. The Journal of Immunology, William W. Cruikshank. Westford; 138:3817-3823 (1987). Hardy Kornfeld, Brighton, all of Mass. Cruikshank, et al., The Journal of Immunology, 73) Assignee: Research Corporation Technologies, 146:2928-2934 (1991). Inc., Tucson, Ariz. Cruikshank, et al., EMBL Database. Accession No. M90301 (21) Appl. No.: 580,680 (1992). 22 Filed: Dec. 29, 1995 Cruikshank. et al. The Journal of Immunology, 128:2569-2574 (1982). Related U.S. Application Data Rand, et al., J. Exp. Med., 173:1521-1528 (1991). 60 Division of Ser. No. 480,156, Jun. 7, 1995, which is a continuation-in-part of Ser. No. 354,961, Dec. 13, 1994, Harlow (1988) Antibodies, a laboratory manual. Cold Spring which is a continuation of Ser. No. 68,949, May 21, 1993, Harbor Laboratory, 285, 287. abandoned. Waldman (1991) Science. vol. 252, 1657-1662. (51) Int. Cl. ....................... G01N 33/53; CO7K 1700; CO7K 16/00: A61K 45/05 Hams et al. (1993) TIBTECH Feb. 1993 vol. 111, 42-44. 52 U.S. Cl. ......................... 424/7.24; 435/7.1; 435/7.92: 435/975; 530/350:530/351; 530/387.1: Center, et al. (Feb. 1995) "The Lymphocyte Chemoattractant 530/388.23: 530/389.2: 424/85.1: 424/130.1 Factor”. J. Lab. Clin. Med. 125(2):167-172. 58) Field of Search ..................................... 435/7.24, 7.1. Cruikshank, et al. (May 24, 1994) "Molecular and Func 435/7.92.975; 530/350, 351,387.1388.23. tional Analysis of a Lymphocyte Chemoattractant Factor: 389.2: 424/130.1. 85.1 Association of Biologic Function with CD4 Expression", 56) References Cited Proc. Natl. Acad. Sci. USA 91(11):5109-5113. U.S. PATENT DOCUMENTS (May 1996) "Terminology Note: Interleukin 16 (IL-16)". Eur: J. Inmunol. 26(5)1196. 4486,530 2A1984 David et al. 4.745,051 5/1988 Smith et al. FOREIGN PATENT DOCUMENTS Primary Examiner-Anthony C. Caputa Assistant Examiner-Khalid Masood WO 94/28134 12/1994 WIPO Attorney; Agent, or Firm-Scully. Scott, Murphy & Presser OTHER PUBLICATIONS 57 ABSTRACT Berman, et al., Cellular Immunology, 95:105-112 (1985). Berman, et al., AM Rev. Respir. Dis... 142:238-257 (1990). Disclosed is a substantially pure antibody which specifically Cambell, "Monoclonal Antibody Technology, The Produc binds a LCF polypeptide and methods of using such anti tion and Characterization of Rodent and Human Hybrido bodies. mas." Elsevier Science Publishers (Amsterdam), pp. 1-4 and 29 (1984). 5 Claims, 19 Drawing Sheets U.S. Patent Jun. 16, 1998 Sheet 1 of 19 5,766,866 28S 18S FIG. U.S. Patent Jun. 16, 1998 Sheet 2 of 19 5,766,866 1. TTCCTCGAGAGCTGTCAACACAGGCTGAGGAATCTCAAGGCCCAGTGCTCAAGATGCCT 6O AGCCAGCGAGCACGGAGCTTCCCCCTGACCAGGTCCCAGTCCTGTGAGACGAAGCTACT 19 TGACGAAAAGACCAGCAAACTCTATCTATCACCAGCCAGTGTCATCGGCTGTCATGAA 178 ATCCTTGCTGGCCTTCCATCTTCTATCTCCTGTGCCCAGACTCCCTGCATCCCCAAGG 237 CAGGGGCATCTCCAACATCATCATCCAACGAAGACTCAGCTGCAAATGGTTCTGCTGAA 296 ACATCTGCCTTGGACACGGGGTTCTCGCTCAACC TTCAGAGCTGAGAGAATATACAGA 355 GGGTCTCACGGAAGCCAAGGAAGACGATGATGGGGACCACAGTTCCTTCAGTCTGGTCA 41.4 GTCCGT TATCTCCCTGCTGAGCTCAGAAGAATTAAAAAAACTCATCGAGGAGGTGAAGG 473 TTCTGGATGAAGCAACATAAAGCAATTAGACGGCATCCAGTCACCATCTTACACAAG 532 GAGGAAGGTCGTGGTCTGGGTTCAGCTTGGCAGGAGGAGCAGATCTAGAAAACAAGGT 59. GATTACGG CACAGAGTGTCCAAATGGGCTGGCCTCCCAGGAAGGGACTATTCAGA 6SO AGGGCAATGAGGTTCTTCCATCAACGGCAAGTCTCCAAGGGGACCACGCACCATGAT 709 GCCTTGGCCATCCTCCGCCAAGCTCGAGAGCCCAGGCAAGCTGTGATTGTCACAAGGAA 768 GCTGACTCCAGAGCC ATG CCC GAC CTC AAC TCC TCC ACT GAC CT GCA Met Pro Asp Leu Asn Ser Ser Thr Asp Ser Ala 816 GCC TCA GCC TCT GCA GCC AGT GAT GTT TCT GTA GAA TCT ACA GCA 12 Ala Ser Ala Ser Ala Ala Ser Asp val Ser wall Glu Ser Thr Ala 861 GAG GCC ACA GTC TGC ACG GTG ACA CTG GAG AAG ATG TCG GCA GGG 27 Gu Ala Thr Val Cys Thr Val Thr Leu Gu Lys Met Ser Ala Gly 906 CTG GGC TTC AGC CTG GAA GGA GGG AAG GGC. TCC CTA CAC GGA GAC 42 Leu Gly Phe Ser Leu Glu Gly Gly Lys Gly Ser Leu His Gly Asp 951 AAG CCT CTC ACC ATT. AAC AGG ATT TTC AAA GGA GCA GCC TCA GAA 57 lys Pro Leu Thir Ile Asn Arg Ile Phe Lys Gly Ala Ala Ser Glu 996 CAA AGT GAG ACA GTC CAG CCT GGA GAT GAA ATC TTG CAG CTG GGT 72 Gln Ser Gu Thr Val Gln Pro Gly Asp Glu Ile Leu Gln Leu Gly 1041 GGC ACT GCC ATG CAG GGC CTC ACA CGG TTG GAA GCC TGG AAC ATC 87 Gly Thr Ala Met Gln Gly Leu Thr Arg Phe Glu Ala Trp Asn Ile 1086 ATC AAG GCA CTG CCT GAT GGA CCT GTC ACG ATT GTC ATC AGG AGA 102 Ile lys Ala Leu Pro Asp Gly Pro Val Thr Ile vali Ile Arg Arg 1131 AAA AGC CTC CAG TCC AAG GAA ACC ACA GCT GCT GGA GAC TCC TAG 117 Lys Ser Leu Gln Ser Lys Glu Thr Thr Ala Ala Gly Asp Ser - 1176 GCAGGACATGCTGAAGCCAAAGCCAATAACACACAGCTAACACACAGCTCCCATAACC 1235 CGATTCTCAGGGTCTCTGCTGCCGCCCCACCCAGATGGGGGAAAGCACAGGTGGGCTT 1294 CCCAGTGGCTGCTGCCCAGGCCCAGACCTTCTAGGACGCCACCCAGCAAAAGGTTGTTC 1353 CTAAAATAAGGGCAGAGTCACACTGGGGCAGCTGATACAAATTGCAGACTGTGTAAAAA 1412 GAGAGCTTAATGATAATATTGTGGTGCCACAAATAAAATGGATTTATTAGAATTCCATA 1471. TGACA TCATGCCTGGCTTCGCAAAATGT CAAG ACTGAACTGTGTCATGATTCAC 1530 CCCCAAACAGTGACATTTATTTTTCTCATGAATCTGCAATGTGGGCAGAGATTGGAATG 1589 GGCAGCTCATCTCTGTCCCACTTGGCATCAGCTGGCGTCATGCAAAGTCATGCAAAGGC 1648 TGGGACCACCTGAGATCATTCACTCATACATCTGGCCGTTGATGTTGGCTGGGAACTCA 1707 CCTGGGGCTGCTGGCCTGAATGCTTATAGGTGGCCTCTCCTTGTTGCCTGGGCTCCTCA 1755 CAACATGGTGTCTGGATTCCCAGGATGAGCATCCCAGGATCGCAAGAGCCACGTAGAAG 1825 CTGCATCTTGT TATACCTTTGCCTTGGAAGTTGCATGGCATCACCTCCACCATACTCC 1884 ATCAGTTAGAGCTGACACAAACCTGCCTGGGTTTAAGGGGAGAGGAAATATTGCTGGGG 1943 TCATT TATGAAAAATACAGTTTGTCACATGAAACATTTGCAAAATTGTTTTTGGTTGGA 2002 TTGGAGAAGTAATCCTAGGGAAGGGTGGTGGAGCCAGTAAATAGAGGAGTACAGTGTAA 2061 GCACCAAGCTCAAAGCGTGGACAGGTGTGCCGACAGAAGGAACCAGCGTGTATATGAGG 2120 GTATCAAATAAAATTGCTACTACTTACCACC FG.2 U.S. Patent Jun. 16, 1998 Sheet 3 of 19 5,766,866 FG3A U.S. Patent Jun. 16, 1998 Sheet 4 of 19 5,766,866 FIG.3B U.S. Patent Jun. 16, 1998 Sheet 5 of 19 5,766,866 FG4 U.S. Patent Jun. 16, 1998 Sheet 6 of 19 5,766,866 N N N N HNNN Nip O O : N 3 Migration (%. Control) U.S. Patent Jun. 16, 1998 Sheet 7 of 19 5,766,866 -K o * - o i : ck S. 8 8 s & 8 8 Cell Migration (/. Control) U.S. Patent Jun. 16, 1998 Sheet 8 of 19 5,766,866 Control an r O O t Nu CD O Os 24 hr O CD h O 2 - O CD 48 hr Green Fluorescence (IL-2R) FG.7 U.S. Patent Jun. 16, 1998 Sheet 9. Of 19 5,766,866 5OOOOO 2OO 4OOOOO 8O s E O 3OOOOO 16O O 5 2OOOOO l4O s O IOOOOO 2O OO 3O Froction (minutes) FIG.8A U.S. Patent Jun. 16, 1998 Sheet 10 of 19 5,766,866 kDa A B 31 U.S. Patent Jun. 16, 1998 Sheet 11 of 19 5,766,866 sap??dad O O£ O6O8O/O9OGOt?O2O| La––––--------~---------------------—] O 2"|-- U.S. Patent Jun. 16, 1998 Sheet 12 of 19 5,766,866 Ceil Migration (%. Control) OO 8O 4O N N 2 N 3 N 4 N 5 N 6 N 7 N 8 N 9 BAL Somples From Normal Subjects FIGO U.S. Patent Jun. 16, 1998 Sheet 13 of 19 5,766,866 Cell Migration (%. Control) 2OO 8O 6O 4 O 2O OO 8O L 2 3 4 5 L 6 L 7 BAL Somples Post Cho lenge FG U.S. Patent Jun. 16, 1998 Sheet 14 of 19 5,766,866 Cell Migrotion (A Control ) 22O 2OO 8O - b -2 b -3b L4b L5b L6 b L7 a BAL. Somples Post Antigen-Challenge FIG2O Cell Migration (%. Control ) 22O 2OO 8O 4 O 2O OO L2 L3 b L 4b LS b L. 6 L 7 BAL Somples Post Antigen - Challenge FG.2b U.S. Patent Jun. 16, 1998 Sheet 15 of 19 5,766,866 Cel. Migration (%. Contro) 22O 2OO 8 O 6 O 4 O k r 2 O wis A ps s st a OO 3 E h L b 2 | 3 b - 4 b 5 b l S | L 7 O BAL. Somples Post Antigen - Cho lenge FIG 3 U.S. Patent Jun. 16, 1998 Sheet 16 of 19 5,766,866 : . Total cells ( millions) U.S. Patent Jun. 16, 1998 Sheet 17 of 19 5,766,866 s i CX s s | i Tolo cells (millions) U.S. Patent Jun. 16, 1998 Sheet 18 of 19 5,766,866 4uno0ºC]O OO9 O62 Og O9 O9 Toto number cells (milions) U.S. Patent Jun. 16, 1998 Sheet 19 of 19 5,766,866 7O O. 6OO T SOO G.E 4oo T 9. S.N 3OO I 2OO T OO ahme 1. O O 2 3 4 5 6 7 8 9 O Week FIG 7 5,766.866 1 2 LYMPHOCYTE CHEMOATTRACTANT capable of promoting or stimulating the migration of unac FACTOR AND USES THEREOF tivated or activated CD4“lymphocytes, eosinophils, monocytes, and the like. By "polypeptide" is meant any This is a divisional of application Ser. No. 08/480.156. chain of amino acids, regardless of length or post filed on Jun. 7, 1995; which is a continuation-in-part of U.S. 5 translational modification (e.g. glycosylation). By a "sub Ser. No. 08/354,961, filed Dec. 13, 1994; which is a con stantially identical" amino acid sequence is meant an amino tinuation of U.S. Ser. No. 08/068.949, filed May 21, 1993 acid sequence which differs only by conservative amino acid now abandoned. substitutions. for example, substitution of one amino acid for This work was supported in part by a grant from the another of the same class (e.g., valine for glycine, arginine O for lysine and the like) or by one or more non-conservative Federal Government and the Government therefore has amino acid substitutions. deletions, or insertions located at certain rights in the invention. positions of the amino acid sequence which do not destroy BACKGROUND OF THE INVENTION the biological activity of the polypeptide. Such equivalent polypeptides can be isolated by extraction from tissues or This invention relates to recombinant LCF, DNA, and cells of any animal which naturally produce such a polypep uses thereof. 15 tide or which can be induced to do so.