Sites on antibodies having affinity for photoaffinity compounds, in particular purine or azidopurine containing compounds are taught. These sites provide for the site-specific attachment of nucleotide photoaffinity compounds to antibodies, e.g., ATP- or GTP-analog photoaffinity compounds by photochemical reaction. These nucleotide photoaffinity compounds may additionally be attached to molecules having a desired therapeutic or diagnostic activity, and the resultant conjugates used as diagnostics or therapeutics

Haley, Boyd E.

Köhler, Heinz

Pavlinkova, Gabriela

Rajagopalan, Krishnan

English

Sites on antibodies having affinity for photoaffinity compounds, in particular purine or azidopurine containing compounds are taught. Such photoaffinity sites are contained on a conserved region of different antibody molecules. These sites provide for the site-specific attachment of nucleotide photoaffinity compounds to antibodies, e.g., ATP- or GTP-analog photoaffinity compounds by photochemical means. These nucleotide photoaffinity compounds may additionally be attached to molecules having a desired therapeutic or diagnostic activity, and the resultant conjugates used as diagnostics or therapeutics

University of Kentucky

University of KentuckyUKnowledgeChemistry Faculty Patents Chemistry9-1-1998Nucleotide or Nucleoside PhotoaffinityCompound Modified Antibodies, Methods forTheir Manufacture and Use Thereof as Diagnosticsand TherapeuticsBoyd E. HaleyUniversity of KentuckyHeinz KöhlerUniversity of Kentucky, heinz.kohler@uky.eduKrishnan RajagopalanUniversity of KentuckyGabriela PavlinkovaUniversity of KentuckyRight click to open a feedback form in a new tab to let us know how this document benefits you.Follow this and additional works at: https://uknowledge.uky.edu/chemistry_patentsPart of the Chemistry CommonsThis Patent is brought to you for free and open access by the Chemistry at UKnowledge. It has been accepted for inclusion in Chemistry Faculty Patentsby an authorized administrator of UKnowledge. For more information, please contact UKnowledge@lsv.uky.edu.Recommended CitationHaley, Boyd E.; Köhler, Heinz; Rajagopalan, Krishnan; and Pavlinkova, Gabriela, "Nucleotide or Nucleoside Photoaffinity CompoundModified Antibodies, Methods for Their Manufacture and Use Thereof as Diagnostics and Therapeutics" (1998). Chemistry FacultyPatents. 29.https://uknowledge.uky.edu/chemistry_patents/29‘ 800991A USOOS UIllted States Patent [19] [11] Patent Number: 5,800,991 Haley et al. {45] Date of Patent: Sep. 1, 1998 [54] NUCLEOTIDE OR NUCLEOSIDE 5.106.951 4/1992 Morgan ct a1. .................... .. 530/3919 PHOTOAFFINITY COMPOUND MODIFIED 5.272.055 12/1993 Haley ........................................ .. 435/6 ANTIBODIES, METHODS FOR THEIR MANUFACTURE AND USE THEREOF AS OTHER PUBLICATIONS DIAGNOSTICS AND THERAPEUTICS Canevari. et a_l.. Annals of Oncology, vol. 5. pp. 698-701. 1994. [751 Inventors: Boyd E- Halpy. Nicholjtsvillc: Heinz Stein. et 31.. Science, vol. 261. pp. 1004-1012. 1993. K01ller. Lexington‘; Krlshl’l?ll _ Borrebaeck. J. Immunol. Methods, vol. 123. pp. 157-165. RaJagopalan. Lexington; Gabriela 1939 Pavimkm- l?xmgton- 1*“ 0f KY- Spalding. Bio/Technology, vol. 11. pp. 423-429. 1993. [73] Assignee: University of Kentucky Research 3:933‘ Immunology Today’ vol’ 11‘ NO' 6‘ pp‘ F‘mndau‘m‘ Lexmgwn‘ Ky‘ Harris et al.. 72mph, vol. 11. pp. 42-44. 1993. Bach et al.. Immunolo yToda . vol. 14. No.9. .421-425. [21] Appl. No.: 681,432 1993‘ g y pp [22] Filed; Jul, 23, 1996 Waldmann. Science. vol. 252. pp. 1657-1662. 1991. Related Us. Application Data Primary Examiner—T0ni R. Scheiner Attorney, Agent, or Fim:—Bu.rns. Doane. Swecker & [63] Continuation-impart of $61.’. No. 208,822, Mar. 11, 1994, Pat Mathis L-L-P N .5 96,081. 1 ° 5 [57] ABSTRACT [51] Int. Cl.6 ........................ .. G01N 33/53; C07K 16/00; ClZP 21/00; C12Q 1/68 Sites on antibodies having af?nity for photoa?inity [52] US. Cl. ............................... .. 435/6; 435/79; 435/75; 9091991949 in particular purin9 9r azidopurine containing 530/3911; 530/3913; 530/3915; 530/391]; compounds are taught. Such photoa?‘inity 51165 are contained 530/3919 on a conserved region of different antibody molecules. [53] Field of Search ................................... .. 435/79. 7.92. “3'?” ms PM!“ for ‘116 sltc'specl?f‘ amfchmcm 0f “cle 435/6. 7.5; 530/3911. 391.3. 391.5. 391.7. 0"“ Phmm?mty comm!!!“ ‘0 an'lmdlcs- °-g-~ ATP- 0’ 391.9 GTP-analog photoa?inity compounds by photochemical means. These nucleotide photoa?inity compounds may [56] References Cited additionally be attached to molecules having a desired therapeutic or diagnostic activity. and the resultant conju U-S- PATENT DOCUMENTS gates used as diagnostics or therapeutics. 4,861,579 8/1989 Meyer et a]. . 5,045,451 9/1991 Uhr et a1. ............................. .. 435/723 15 Claims, 21 Drawing Sheets 5,800,991 US. Patent Sep. 1, 1998 Sheet 1 0f 21 com :23 nEqmzéE S08 %com 0000? oooom oooom 0000.“. SEO U.S. Patent Sep. 1, 1998 Sheet 2 0f 21 5,800,991 00w oom SEO w5E<US. Patent Sep. 1, 1998 Sheet 3 0f 21 5,800,991 mm 82620258895 593mm om wm m .OEor ON on ow om cm on ow om coEnEE “xvUS. Patent Sep. 1, 1998 Sheet 4 0f 21 5,800,991 33 262:6@2825 05m ~ 0@NQO8.5mg_  .. 82 E3 28mooom U.S. Patent Sep. 1, 1998 Sheet 5 0f 21 5,800,991 63 Q< @289500 Fom ow ow ow cow coEnEE o\oUS. Patent Sep. 1, 1998 Sheet 6 0f 21 5,800,991 5% ? ?aws-AT? S g pm: we"; ma 1% 2:1‘ 12 a? a a @amiam? US. Patent Sep. 1, 1998 Sheet 7 of 21 5,800,991 U.S. Patent Sep. 1, 1998 Sheet 8 0f 21 5,800,991 US. Patent Sep. 1, 1998 Sheet 9 0f 21 5,800,991 FIG. 9A 40000 30000 - CPM 20000 - 10000 - O I I I I 0 100 200 300 400 500 [V32 P]-8-N 3 ATP [UM] US. Patent Sep. 1, 1998 Sheet 10 0f 21 5,800,991 FIG. 9B 350 300 250 200 CPM 150 100 50 0 l | I r l 1 O 25 5O 75 100 125 150 175 [732 P]-8-N3 ATP [UM] US. Patent Sep. 1, 1998 Sheet 11 0f 21 5,800,991 FIG. 9C 9.622 Q0 1000 800 600 400 200 ATP, FM US. Patent Sep. 1, 1998 Sheet 12 0f 21 5,800,991 FIG. 9D 1000 800 600 400 200 ATP, PM US. Patent Sep. 1,1998 Sheet 13 0f 21 ‘ 5,800,991 FIG. 10A 120 100 80 % labeling J>~ o)o N O l Tryptophan 0 T I I l 0 200 400 600 800 1000 Amino acid, PM US. Patent Sep. 1, 1998 Sheet 14 0f 21 5,800,991 FIG. 10B 120 100 80 Tyrosine % labeling A c»o l |N O l Tryptophan O I I l I 0 200 400 600 800 1000 Amino acid, PM US. Patent Sep. 1, 1998 Sheet 15 0f 21 5,800,991 FIG. 11A ‘I .. \\ .... :L. \l\|\|.|lJin lllll 1|. llllhlrlllllmalllhh' lalluranlllnMlll If: ‘If! I SSH-0.1 J x A \ uh. _ O 0O 54| m=m0 *010000 1000 100 10 Fluorescence Intensity [log] Sep. 1, 1998 Sheet 16 0f 21 5,800,991 US. Patent FIG. 11B Fluorescence Intensity [log] US. Patent Sep. 1, 1998 Sheet 17 of 21 5,800,991 FIG. 12A 0.16 TmTvEc 3m00 2.0 40 5. O04| 1OTQIVQE x550 Fraction Number US. Patent Sep. 1, 1998 Sheet 18 0f 21 5,800,991 FIG. 12B 05050 21100 0.0.00.0. 00000 ___. h _u my. a u c J an PH 9 n 2 T ___ 5050505. 33221.10 TQLQE x2% O. O 90 80 6O 7O Fraction Number 50 US. Patent Sep. 1, 1998 Sheet 19 0f 21 5,800,991 om? on zzziwwoo 50:55amw>wwm<5§ $592?:385 0 8 ; HI> mamHI> movwnI> mOPwHx> 20m