Method And Molecules

Abstract

The present invention provides a bioconjugation method and compounds for use therein. The bioconjugation method comprises the step of conjugating a biological molecule containing a first unsaturated functional group with a payload comprising a second unsaturated functional group, wherein the first and second unsaturated functional groups are complementary to each other such that conjugation is a reaction of said functional groups via a Diels-Alder reaction which forms a cyclohexene ring.

1 . A bioconjugation method, comprising:
 conjugating an antibody molecule containing a first unsaturated functional group with a payload comprising a second unsaturated functional group, wherein the first and second unsaturated functional groups are complementary to each other such that conjugation is a reaction of said functional groups via a Diels-Alder reaction which forms a cyclohexene ring.   
     
     
         2 . The bioconjugation method according to  claim 1 , wherein the first functional group is a diene. 
     
     
         3 . The bioconjugation method according to  claim 2 , wherein the second functional groups is a dieneophile, esters of maleic acid, esters of fumaric acid, esters of acrylic acid, methacrylic acid, acrylonitrile, acrylamide, methacrylamide, methyl vinyl ketone, amides, and esters of but-2ynedioic acid, quinone, acetylenes. 
     
     
         4 . The bioconjugation method according to  claim 1 , wherein the second functional group is a diene. 
     
     
         5 . The bioconjugation method according to  claim 4 , wherein the first functional group is a dieneophile selected from the group consisting of esters of maleic acid, maleimide, esters of fumaric acid, esters of acrylic acid, methoacrylic acid, acrylonitrile, acrylamide, methacrylamide, methyl vinyl ketone, amides, and esters of but-2ynedioic acid, quinone, acetylenes. 
     
     
         6 . (canceled) 
     
     
         7 . The bioconjugation method according to  claim 2 , wherein the diene is a linear dienes, carbocyclic diene, or heterocyclic diene. 
     
     
         8 . The bioconjugation method according to  claim 7 , wherein the diene comprises a butadiene, a cyclopentadiene, a 1, 3-cyclohexadiene, a furan, or anthracene. 
     
     
         9 . The bioconjugation method according to  claim 2 , wherein the diene is contained in a non-natural amino acid. 
     
     
         10 . The bioconjugation method according to  claim 9 , wherein the diene is a non-natural amino acid derived from lysine, cysteine, selenocysteine, aspartic acid, glutamic acid, serine, threonine, and tyrosine. 
     
     
         11 . The bioconjugation method according to  claim 8 , wherein the diene is in a side chain of the amino acid. 
     
     
         12 - 22 . (canceled) 
     
     
         23 . The bioconjugation method according to  claim 9 , wherein the non-natural amino acid is selected from the group comprising: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         24 . The bioconjugation method according to  claim 3 , wherein the dienophile is the side chain of a non-natural amino acid. 
     
     
         25 . The bioconjugation method according to  claim 1 , wherein before conjugation of the antibody molecule with the payload, the diene or dienophile is conjugated via a linker to an amino acid residue in the antibody molecule wherein the amino acid is a cysteine or lysine. 
     
     
         26 - 27 . (canceled) 
     
     
         28 . The bioconjugation method according to  claim 25 , wherein the diene has a structure: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         29 - 36 . (canceled) 
     
     
         37 . An antibody molecule conjugated to a payload, wherein a conjugation reaction linking the antibody molecule to the payload was via a Diels-Alder reaction between a diene and a dieneophile to form a cyclohexene ring. 
     
     
         38 - 41 . (canceled) 
     
     
         42 . The antibody molecule conjugated to a payload according to  claim 1 , wherein the payload is selected from:
 a. an auristatin;   b. comprising a maytansinoid,   c. is a toxin,   d. a polymer.   
     
     
         43 . A pharmaceutical composition according comprising the antibody molecule conjugated to a payload as defined in  claim 37  and diluent, carrier, and/or excipient. 
     
     
         44 - 49 . (canceled) 
     
     
         50 . A non-natural amino comprising a diene or dienophile, wherein the non-natural amino acid is derived from lysine asparagine, glutamine, cysteine, aspartic acid, glutamic acid. 
     
     
         51 . (canceled) 
     
     
         52 . The non-natural amino acid according to  claim 50 , wherein the non-natural amino acid has a formula (II): 
       
         
           
           
               
               
           
         
         or a salt thereof wherein
 X 2  represents —C—, —CH 2  or O; 
 R a  represents
 i) a saturated or an unsaturated branched or unbranched C 1-8  alkylene chain, wherein at least one carbon is replaced by a heteroatom selected from O, N, S(O) 0-3 , wherein said chain is optionally, substituted by one or more groups independently selected from oxo, halogen, amino; or 
 ii) together with a carbon from the 5 membered ring represents a cyclopropane ring linked to a saturated or unsaturated branched or unbranched C 1-6  alkylene chain, wherein at least one carbon is replaced by a heteroatom selected from O, N, S(O) p , wherein said chain is optionally, substituted by one or more groups independently selected from oxo, halogen, sulfo, sulfhydryl, amino; 
 
 R b  represents H, —OC 1-3 alkyl, C 1-6 alkyl optionally bearing a hydroxyl substituent, —C 1-3 alkyleneN 3 , or —C 2-5 alkynyl; 
 R c  represents H, —OC 1-3 alkyl, C 1-6 alkyl optionally bearing a hydroxyl substituent, —C 1-3 alkyleneN 3 , or —C 2-5 alkynyl; 
 R d  represents H, —OC 1-3 alkyl, C 1-6 alkyl optionally bearing a hydroxyl substituent, —C 1-3 alkyleneN 3 , or —C 2-5 alkynyl; 
 R e  represents H, saturated or unsaturated branched or unbranched C 1-8  alkylene chain, wherein one or more carbons are optionally replaced by —O— and the chain is optionally substituted by one or more halogen atoms N 3 , or —C 2-5 alkynyl. 
 
       
     
     
         53 . The non-natural amino acid according to  claim 52 , wherein R a  is —(CH 2 )mC(O)—, —CH 2 (CH 3 )C(O)—, —(CH 2 )mCH 2 OC(O)—, —CHCHCH 2 OC(O)—, —OCH 2 CH 2 COC(O)—; and
 m represents 0 or 1. 
 
     
     
         54 . The non-natural amino acid according to  claim 52 , wherein R b  is H, —OC 1-3 alkyl, —CH 3 , —CH(CH 3 ) 2 , CH 2 OH, —CH 2 N 3 , or —CCH. 
     
     
         55 . The non-natural amino acid according to  claim 52 , wherein R c  is H, —OC 1-3 alkyl, —CH 3 , —CH(CH 3 ) 2 , CH 2 OH, —CH 2 N 3 , or —CCH. 
     
     
         56 . The non-natural amino acid according to  claim 52 , wherein R d  is H, —OC 1-3 alkyl, —CH 3 , —CH(CH 3 ) 2 , CH 2 OH, —CH 2 N 3 , or —CCH. 
     
     
         57 . The non-natural amino acid according to  claim 52 , wherein R e  represents H or —CH 2 OCH 2 CH 2 N 3 . 
     
     
         58 . The non-natural amino acid according to  claim 52 , wherein the non-natural amino acid is a residue of the structure of formula (IIa): 
       
         
           
           
               
               
           
         
         or a salt thereof wherein R a , R b , R c , R d , R e  and X 2  are defined above compounds of formula (II). 
       
     
     
         59 . The non-natural amino acid according to  claim 52 , wherein the non-natural amino acid has the structure of formula (IIb): 
       
         
           
           
               
               
           
         
         or a salt thereof wherein R a , R b , R c , R d , R e  and X 2  are defined above compounds of formula (II). 
       
     
     
         60 . The non-natural amino acid according to  claim 52 , wherein the non-natural amino acid has the structure of formula (IIc): 
       
         
           
           
               
               
           
         
         or a salt thereof wherein R a , R b , R c , R d , R e  are defined above and X 2 ′ is —C— or —CR′ as defined above. 
       
     
     
         61 . The non-natural amino acid according to  claim 60 , wherein the non-natural amino acid is selected from the group comprising: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         or a salt of any one of the same. 
       
     
     
         62 . (canceled)