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United States Patent (19) 11 Patent Number: 6,114,143 Eda Et Al

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United States Patent (19) 11 Patent Number: 6,114,143 Eda Et Al USOO611.4143A United States Patent (19) 11 Patent Number: 6,114,143 Eda et al. (45) Date of Patent: *Sep. 5, 2000 54). ANTI-HIV MONOCLONAL ANTIBODY 90O3984 4/1990 WIPO. 9015078 12/1990 WIPO. 75 Inventors: Yasuyuki Eda; Hiroaki Maeda; 9304090 3/1993 WIPO. Keiichi Makizumi, Kouichi Shiosaki, all of Kumamoto; Kiyoshi Osatomi, OTHER PUBLICATIONS Nagasaki, Kazuhiko Kimachi, Kumamoto; Hirofumi Higuchi, Kumamoto; Sachio Tokiyoshi, Matsushita, et al. : Characterization of a Kumamoto, all of Japan mouse/human . : AIDS Res. and Hum. Retro. v. 8, No. 6: pp. 1107–1115, 1992. 73 Assignee: Juridical Foundation The Chemo-Sero-Therapeutic Research Ohno, et al. : A broadly neutalizing monoclonal . Proc. Institute, Kumamoto, Japan Natl. Acad. Sci. : V. 88: pp. 10726–10729, 1991. * Notice: This patent issued on a continued pros Scott, et al. : Human monoclonal antibody that ecution application filed under 37 CFR recognizes ... : Proc. Natl. Acad. Sci. : V. 8: pp. 8597-8601, 1.53(d), and is subject to the twenty year 1990. patent term provisions of 35 U.S.C. 154(a)(2). Primary Examiner Jeffrey Stucker 21 Appl. No.: 08/513,968 ASSistant Examiner Brett Nelson 22 PCT Filed: Mar. 9, 1994 Attorney, Agent, or Firm-Browdy and Neimark 86 PCT No.: PCT/JP94/00371 57 ABSTRACT S371 Date: Sep. 11, 1995 A monoclonal antibody useful for clinical application which S 102(e) Date: Sep. 11, 1995 recognizes the conserved region of V3-PND region of 87 PCT Pub. No.: WO9420632 glycoprotein antigen having a molecular weight of about PCT Pub. Date: Sep. 15, 1994 1.2x10 daltons (gp120) on a coating membrane of human 30 Foreign Application Priority Data immunodeficiency virus (HIV) and which has an ability to neutralize a broad range of various HIV variants, or a Mar. 11, 1993 JP Japan .................................... 5-O78913 fragment thereof, and the chimeric and humanized antibod 51) Int. Cl." ............................ C12N 15/09; C12P 21/04; ies derived therefrom are provided. By using as an immu C12P 11/00; A61K 39/395 nogen a plurality of peptides having PND-Tip region con 52 U.S. Cl. ....................... 435/69.3; 435/69.1; 435/69.6; taining the highly conserved GPGR sequence within PND of 424/130.1; 424/137.1; 424/139.1; 424/141.1; HIV gp120, a monoclonal antibody having a neutralizing 424/147.1; 424/160.1 activity to many HIV variants can be prepared. By trans 58 Field of Search .................................. 435/69.6, 69.1, planting the gene fragment coding for the variable region of 435/69.3; 424/130.1, 137.1, 139.1, 141.1, Said monoclonal antibody or complementarity determining 147.1, 160.1 region (CDR) of Said region to a human antibody gene, a chimeric antibody or a reshaped antibody having an anti 56) References Cited HIV neutralizing activity which are effective for clinical FOREIGN PATENT DOCUMENTS application can be obtained. 0339504 11/1989 European Pat. Off.. 8809181 12/1988 WIPO. 18 Claims, 26 Drawing Sheets U.S. Patent Sep. 5, 2000 Sheet 1 of 26 6,114,143 SERUM D LUTION (X) - D - SP-1 ( MN) - - - - A ---- SP -6 (NI61) ----- - - - - SP - 9 (NI23) - - - a as O --- SP - 11 (N 54-2) - O - SP - 12 (NI53 ) - - - - - A ----- SP - 14 (RF) - A - SP - 17 (NI 18) - - - - - A ----- SP-2O (NI 63-2) - - SP-30 (GPGR) 5 SYNTHETIC PEPT DES (CLN CALLY SOLATED STRAIN) U.S. Patent Sep. 5, 2000 Sheet 2 of 26 6,114,143 3. e C S. 2 u O 2. C C Or O 1 AV (f) ON g ASSSSC-SN------- S-SèSisiesA-. SA a A - - - - S&s O 52 5 3 5 4. 5 5 5 6 5 7 5 8 5 9 5 1 O SERUM D LUT ON (X) - O - SP-1 (MN) O - A - - - - - SP-6 (NI61) ---- I -, - ... SP-9 (N123) ---, - O - - - - - SP-11 (NI 54-2) - O - SP-12 (NI53) - - - - - A ---- SP - 14 (RF) - A - SP-17 (NI 18) ----- A ----- SP-2O (NI63-2) - - SP-3 O(GPGR) 5 SYNTHETC PEPT DES (CLIN CALLY SOLATED STRAN) U.S. Patent Sep. 5, 2000 Sheet 4 of 26 6,114,143 A/G. 4 OO1 O1 1 1 1 O DLUTED CONCENTRATION OF ANTIBODY (ug / m ) - O - SP-1 (MN) -- - - A ---- SP-6 (NIS) - - - - - - SP-9 (NI 23) ---. - A - - - SP-11 (NI54-2) 1- a a- O -...-- SP-12 (NI53) - O - SP-2O(N 63-2) ----- A ----- SP-3O(GPGR) 5 SYNTHEf C PEPT DES (CLIN CALLY SOLATED STRAIN) U.S. Patent Sep. 5, 2000 Sheet 5 of 26 6,114,143 A/G 5 D LUTED CONCENTRATION OF ANTBOOY ( ug / ml ) - O - SP-1 ( MN) - - - - - - A ----- SP-6 (NI61) - - - - --- SP-9 (N 23) ----- A --- SP-11 (NI54-2) ----- O --- SP-12 (N53 ) - O - SP-2O (NI 63-2) - - - - A - - - - SP-3O (GPGR) 5 SYNTHETIC PEPT DES (CLIN CALLY SOLATED STRAIN) U.S. Patent Sep. 5, 2000 Sheet 6 of 26 6,114,143 A/G. 64 A/G. 6A A/G. 66 U.S. Patent Sep. 5, 2000 Sheet 7 of 26 6,114,143 A/G 7 2 C CD es l E CD C aws R s L 1 CD 2 g O C?) C O 3 4. 5 6 7 8 9 1 O 2O NO. OF AMNO AC DS U.S. Patent Sep. 5, 2000 Sheet 8 of 26 6,114,143 A/G 3 SUBSTITUTION OF THE 1 St AMNO ACD (I) WITH OTHER AM NO ACDS Li O 2 On 1 CC OC | | | A C D E F G H K L M N P Q R S T W W Y 2 SUBSTITUTION OF THE 2nd AMNO ACD (H) WITH OTHER AMNO AC DS U. O 2 1 O CCO | | | A C D E F G H K L M N P Q R S T W W Y U.S. Patent Sep. 5, 2000 Sheet 9 of 26 6,114,143 A/G. f.O SUBSTITUTION OF THE 3rd AMNO ACID ( I.) WITH OTHER AMNO AC DS SUBSTITUTION OF THE 4th AMNO ACD (G) WITH OTHER AM NO ACDS U.S. Patent Sep. 5, 2000 Sheet 10 of 26 6,114,143 A/ G. /2 h AMNO ACD (G) O U.S. Patent Sep. 5, 2000 Sheet 11 of 26 6,114,143 A/G. 74 SUBSTITUTION OF THE 7th AMNO AC D (R) WITH OTHER AM NO ACDS SUBSTITUTION OF THE 8th AM NO ACID (A) WITH OTHER AM NO. A CDS U.S. Patent Sep. 5, 2000 Sheet 12 of 26 6,114,143 A/6. f6 SUBSTITUTION OF THE 9th AMNO ACID (F) WITH OTHER AM NO AC DS O1 SUBSTITUTION OF THE 1 Oth AM NO. A CD (Y) WITH OTHER AM NO. A CDS A C D E F G H K LM, N P Q R U.S. Patent Sep. 5, 2000 Sheet 13 of 26 6,114,143 A/G 16 Isolated HIV strains Sequence of PND u5. 5 O. 64 C25 HV-MN CTRPNYNKRKRIHI GPGRAFYTTKNIIGTIRQAHC - -- NIOC - 4 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - -- KM-Ol - - - -SN-T--S- - - - - - - - - - - - GD- - -D- - - - - - -- - -- N29 - - - - -N-T--S- - - - - - - - W - - - GE---N- - - - - - -- - N 60-3 - - - - -N-T -- G -- M - - - G- - - - - GE - - -D- - - - - - -- - - TM2 - - - - - NKA-G-LSV - - - -S----RQ-T-D- - - - - - - -- NI23 - - - - -N-T--S-P- - - - - - - - - -GE---N- - - - - - - -- NI85-16 - - - - -N-T--S-N- - - - - - - - - - GD- - - D - - - - - - - -- NI42 - - - - -N-T--S-P- - - - - - - - - - GD - - -D--K--- o - -- NI26 - - - - -N-T--S-P- - - - - - - - - - GD - - - D - - - - - - - -- PAS - - - - -N-T--S-R-Z - - - - -L-A-GG- - -D- - - - - - au- - -- TMO 4. - - - - -N-T--G-R- - - - - -W-A-EK--- D - - - - - - -- -- N85-9 - - - - -N-T--G-R- - - - - -W--AEK- - -D- - - - - - -- DBA3 - - - - -N-T--G-R- - - - - -V-A-EK---D- - - - - - -- -- NI61-1 - - - - -N-T--G-R- - - - - -W-A-GK---N- - - - - - -- -- N6-2 - - - - -N-T --G-R- - - - - -W-A-GK- - - D - - - - - - -- NI5 4-2 - - - - -N-T - - G - RW - - - - - -A-EK--- D - - - - - - - + -- NI36-2 - - - - - N-TK-S-RMX-W- - -W-A-GK-M-D- - - - - - - NI 63-2 - - - - -N-T-RG-R- - - - - - - -A-DK - - -D- - - - - - -- -- N53 - - - - - N-TK-A-RV - - - -TL-A-RR- - -D- - - - - - -- -- SF2 - - - - -N-T--S-Y- - - - - - - H --GR---D--K--- -- - -- NIll - - --SMKT--G--L -WK-TM-A-GE-K-D- - - - - - - - NI55 - Ol - - - - -N-T--S- - - - - - - - - -A-GD- - -D- - - - - - -- - -- N55 - 04 - - - - - - - - - - - - - - A- - - - - - - - - - - - - N- - - - - - - KMO-01 - - - -N-T--S- - - - - - - - - -A-GE---N- - - - - - -- --- -- KMO-O2 - - - - -N-T--S-N- - - - - - - -A-GE---D--K- - - - - -- TI-O1 -----N-T--S-N- - - - - - - - --GQ---N------ - - -- TI-O - - - - - N-T--S- - - - - - - - - - - - GQ---------- -- - -- YHI - O - - - - - NYTG--VS- - - - - - - R--GA- - -D--K--- au- - -- HHA- O - - - - SN-T- - S - - - - - - - - - - - - GD - - -D- - - - - - -- - -- HTU-O1 - - - - -N-T--S- - - - - - S - - - - - GE---D- - - - - - -- - Binding rate (%) 333 26% 83% (10/30) (8/30) (25/30) U.S. Patent Sep. 5, 2000 Sheet 14 of 26 6,114,143 A/G fe (MHL4ver. 4) AAGGGCCGCCACC Leader FRI 60 ATGGAATGGAGCTGGGTCTTTATCTTTCTCCTGTCAGTAACTGCAGGTGTCCACTCCCAG MetGluTrpSerTrpVal PhellePheleuILeuServal ThrAlaGlyVal His SerGln FR1 120 GTCCAGCTGCAGCAGTCTGGAGCTGAGCTGGTAAGGCCTGGGACTTCAGTGAAGATGTTC ValGln LeuglinGlnSerGlyAlaGluLeuValArg ProGlyThrSerVallysMetPhe FR1 CDR1 FR2 18O TGCAAGGCTGCTGGATACACCTTCACTAACTCCTGGATAGGTTGGTTTAGGCAGAGGCCT Cys Lys Ala AlaGlyTyrThrPheThrAsnSerTrpIleGlyTrp PheArgGln ArgPro CDR2 240 GGACATGGCCTTGAGTGGATTGGAGATATTTACCCTGGAGGTGGTTATACTAACTACAAT GlyHis GlyLeugluTrpIleGlyAspIleTyr ProGlyGlyGlyTyrThrAsnTyrASn FR3 3 O O. GAGATCTTCAAGGGCAAGGCCACACTGACTGCAGACACATCCTCCAGCACAGCCTATATG GlullePhelysGlyLys AlaThrLeu ThrAlaAspThrSer SerSerThrAlaTyrMet FR3 CDR3 360 CAGCTCAGCAGCCTGACATCTGAGGACTCTGCCATCTATTACTGTTCAAGGGGGATACCG GlnLeuSerSerLeuThrSerGluAspSerAlalleTyrTyrCys SerArgGlyIlePro FR4 | CY 2a 420 GGATATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACA GluTyrAlaMetAspTyrTrpGlyGlnGlyThrSerValThrValSerSerAlaLys Thr JH4 (MHCver . 1) ACAGCCCCATCGGTCTATGCACTCCCGGGATCC ThrAlaProServalTyrProLeu ProGly Ser : Primer U.S. Patent Sep. 5, 2000 Sheet 15 of 26 6,114,143 A/G 2O ACTAGTCGAC Leader (MKL7ver.2) ATGGGCATCAAGATGGAGTCACAGATTCTGGTCCTCATGTCCCTGCTGTTCTGGGTATCT 60 MetGlyIleLysMetGluSerGlnIleLeuValleuMet SerLeuLeuPheTrpVal Ser FR1 GGTACCTGTGGGGACATTGTGATGACACAGTCTCCATCCTCCCTGACTGTGACAGCAGGA 120 GlyThrCysGlyAspIleValMetThrClnSerPro Ser SerLeuThrValThrAlaGly CDR1 GAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAACAGTGGAGATCAAAAG 180 Glullys ValThrMet SerCys Lys SerSerGln SerLeuLeuAsnSerGlyAspGln Lys FR2 CDR2 AACTACTTGACCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAACTGTTGATCTATTGG 24 O AsnTyrLeuThrTrpTyrglin Glin Lys ProGlyGln ProProLys LeuLeuIleTyrTrp CDR2 FR3 GCATCCACTGGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGAAACAGAT 300 Alaser Thr(GlyGluSerGlyVal ProAspArg PheThrgly SerGlySerGlu ThrAsp FR3 CDR3 TTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGAAT 360 PheThrLeu ThrIle Ser SerVal Glin AlaGluAspLeuhalaValTyr TyrCysGlnAsn FR4 C - GATTATAGTTATCCGTGGACGTTCGGTGGAGGCACCAAACTGGAAATCAAACGGGCTGAT 420 AspTyr SerTyr ProTrpThrPheGlyGlyGlyThrLys Leuglu IleLys ArgAlaAsp Jk1 (MKCver . 1) GCTGCACCAACTGTATCCACTCCCACCACCACCCGGGAC Ala Ala ProThr Val SerIle Phe Pro : Primer U.S.
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