Method of synthesising adcs using affinity resins

Abstract

This invention relates to a solid phase method of synthesising biomolecule-drug-conjugates. In particular, this invention relates to a solid phase method of synthesising antibody-drug-conjugates (ADCs). This invention also relates to intermediate methods of producing immobilised, chemically modified biomolecules, e.g. antibodies. The invention also relates to various uses of capture resins and to biomolecule-drug-conjugates, intermediate products and compositions of the methods of the invention.

1 - 31 . (canceled) 
     
     
         32 . A method of synthesising an antibody-DM1-conjugate, the method comprising:
 (i) contacting an antibody with FAbsorbent F1P HF resin, MAbsorbent A1P resin, MAbsorbent A2P resin or a capture resin comprising a capture moiety for the antibody, wherein the capture moiety is:
 a branched ligand scaffold of formula: 
   
       
         
           
           
               
               
           
         
         
           wherein R 1  and R 2  are the same or different and are each optionally substituted alkyl or aryl ligands, and R 3  is a solid support optionally attached by a spacer motif; or 
           a branched triazyl scaffold of formula: 
         
       
       
         
           
           
               
               
           
         
         
           wherein Q represents the attachment point to a solid support matrix, optionally with a spacer motif and Groups A and B are phenyl or naphthyl groups substituted with one or more substituents capable of hydrogen bonding; 
           under conditions suitable to immobilise the antibody and therefore provide immobilised antibody; 
         
         (ii) reacting the antibody with a crosslinker moiety to provide an activated, immobilised antibody; 
         (iii) contacting the activated, immobilised antibody with DM1 to form an immobilised antibody-DM1-conjugate; and 
         (iv) releasing the antibody-DM1-conjugate from the capture resin. 
       
     
     
         33 . The method of  claim 32 , wherein step (i) comprises incubating antibody with the capture resin. 
     
     
         34 . The method of  claim 33 , wherein the incubation is carried out at temperature from 10 to 40° C. 
     
     
         35 . The method of  claim 33 , wherein the incubation is carried out at temperature from 15 to 37° C. 
     
     
         36 . The method of  claim 33 , wherein the incubation is performed for a period of time of 10 minutes to 18 hours. 
     
     
         37 . The method of  claim 33 , wherein the incubation is carried out in a buffer solution. 
     
     
         38 . The method of  claim 37 , wherein the buffer solution is phosphate buffered saline (PBS). 
     
     
         39 . The method of  claim 33 , wherein the incubation is carried out at a pH range of from 5 to 8. 
     
     
         40 . The method of  claim 32 , wherein after step (i) the immobilised antibody is washed to remove any antibody that has not been immobilised on the capture resin. 
     
     
         41 . The method of claim  31 , wherein the crosslinker moiety is succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC). 
     
     
         42 . The method of  claim 41 , wherein step (ii) is carried out in a buffer solution. 
     
     
         43 . The method of  claim 42 , wherein the buffer solution is phosphate buffered saline (PBS). 
     
     
         44 . The method of  claim 41 , wherein step (ii) is carried out at a pH of from 7 to 8. 
     
     
         45 . The method of  claim 41 , wherein step (ii) is carried out in the presence of a chelating agent. 
     
     
         46 . The method of  claim 45 , wherein the chelating agent is EDTA. 
     
     
         47 . The method of  claim 32 , wherein after step (ii) the activated, immobilised antibody is washed to remove any crosslinker moiety. 
     
     
         48 . The method of  claim 32 , wherein step (iii) comprises contacting the immobilised antibody with DM1 in a buffer solution. 
     
     
         49 . The method of  claim 32 , wherein step (iii) comprises contacting the immobilised antibody with DM1 at a pH of from 7 to 8, preferably 7.4. 
     
     
         50 . The method of  claim 32 , wherein step (iii) is carried out in the presence of a chelating agent. 
     
     
         51 . The method of  claim 50 , wherein the chelating agent is EDTA. 
     
     
         52 . The method of  claim 32 , wherein step (iii) comprises incubating the immobilised antibody with DM1 for a period of time from 6 hours to 18 hours. 
     
     
         53 . The method of  claim 32 , wherein after step (iii) immobilised antibody-DM1-conjugate is washed to remove any unreacted DM1. 
     
     
         54 . The method of  claim 32 , wherein step (iv) comprises altering the pH to break the support-antibody bond. 
     
     
         55 . The method of  claim 54 , wherein the pH is decreased to less than pH 5. 
     
     
         56 . The method of  claim 55 , wherein the pH is decreased to pH 3. 
     
     
         57 . The method of  claim 55 , wherein the eluted antibody-DM1-conjugate is neutralised after the step of releasing the conjugate from the capture resin. 
     
     
         58 . The method of  claim 57 , wherein the antibody-DM1-conjugate is captured into 2% v/v of 1 M tris(hydroxymethyl)aminoethane (TRIS). 
     
     
         59 . The method of  claim 32 , wherein the ligand of the capture resin has a structure: 
       
         
           
           
               
               
           
         
       
     
     
         60 . The method of  claim 32 , wherein the antibody is a monoclonal antibody. 
     
     
         61 . The method of  claim 32 , wherein the antibody is trastuzumab.