Chemically-defined non-polymeric valency platform molecules and conjugates thereof

Abstract

Chemically-defined, non-polymeric valency platform molecules and conjugates comprising chemically-defined valency platform molecules and biological or chemical molecules including polynucleotide duplexes of at least 20 base pairs that have significant binding activity for human lupus anti-dsDNA autoantibodies.

1 . A conjugate comprising (a) biological or chemical molecules reacted with (b) a chemically-defined, non-polymeric valency platform molecule of the formula:
   G [1] {T [1] } n[1]   Formula 1     or     G [2] {L [2] −J [2] −Z [2] (T [2] ) n[2] } n[2]   Formula 2   
     wherein
 each of G [1]  and G [2] , if present, is independently a linear, branched or multiply-branched chain comprising 1-2000 chain atoms selected from the group C, N, O, Si, P and S; 
 each of the n [1]  moieties shown as T [1]  and each of the p [ 2]×n [2]  moieties shown as T [2]  is independently chosen from the group NHR SUB  (amine), C(═O)NHNHR SUB  (hydrazide), NHNHR SUB  (hydrazine), C(═O)OH (carboxylic acid), C(═O)OR ESTER  (activated ester), C(═O)OC(═O)R B  (anhydride), C(═O)X (acid halide), S(═O) 2 X (sulfonyl halide), C(═NR SUB )OR SUB  (imidate ester), NCO (isocyanate), NCS (isothiocyanate), OC(═O)X (haloformate), C(═O)OC(═NR SUB )NHR SUB  (carbodiimide adduct), C(═O)H (aldehyde), C(═O)R B  (ketone), SH (sulfhydryl or thiol), OH (alcohol), C(═O)CH 2 X (haloacetyl), R ALK X (alkyl halide), S(═O) 2 OR ALK X (alkyl sulfonate), NR 1 R 2  wherein R 1 R 2  is —C(═O)CH═CHC(═O)— (maleimide), C(═O)CRB═CRB2 (α,β-unsaturated carbonyl), R ALK —Hg—X (alkyl mercurial), and S(═O)CR B ═CR B   2  (α,β-unsaturated sulfone); 
 
     wherein
 each X is independently a halogen of atomic number greater than 16 and less than 54 or other good leaving group; 
 each R ALK  is independently a linear, branched, or cyclic alkyl (1-20C) group; 
 each R SUB  is independently H, linear, branched, or cyclic alkyl (1-20C), aryl (6-20C), or alkaryl (7-30C); 
 each R ESTER  is independently N-hydroxysuccinimidyl, p-nitrophenoxy, pentafluorophenoxy, or other activating group; 
 each R B  is independently a radical comprising 1-50 atoms selected from the group C, H, N, O, Si, P and S; 
 each of the n [2]  moieties shown as L [2] , if present, is independently chosen from the group O, NR SUB  and S; 
 each of the n [2]  moieties shown as J [2] , if present, is independently chosen from the group C(═O) and C(═S); 
 n [1] =1 to 32; 
 n [2] =1 to 32; 
 p [2] =1 to 8; 
 with the proviso that the product n [ 2]×p [ 2] be greater than 1 and less than 33; 
 each of the n [ 2] moieties shown as Z [2]  is independently a radical comprising 1-200 atoms selected from the group C, H, N, O, Si, P and S, containing attachment sites for at least p [ 2] functional groups on alkyl, alkenyl, or aromatic carbon atoms. 
 
   
   
       2 . A conjugate according to  claim 1 , wherein the biological molecules comprise polynucleotide duplexes of at least about 20 base pairs each bound to the valency platform molecule, the duplexes each having a significant binding activity for human systemic lupus erythematosus anti-dsDNA autoantibodies. 
   
   
       3 . A conjugate according to  claim 1 , wherein the biological or chemical molecules are selected from the group consisting of carbohydrates, lipid, lipopolysaccharides, peptides, proteins, glycoproteins, single-stranded or double-stranded oligonucleotides, haptens, or chemical analogs thereof such as mimotopes, aptamers. 
   
   
       4 . A conjugate according to  claim 1 , wherein the biological or chemical molecules are analogs of immunogens wherein (a) the analog binds specifically to B cells to which the immunogen binds specifically and (b) the conjugate lacks a T cell epitope. 
   
   
       5 . The conjugate of  claim 1 , wherein the valency platform molecule is derivatized by a reagent selected from the group consisting of DABA, BAHA, BAHA ox , and AHAB. 
   
   
       6 . The conjugate of  claim 2 , wherein a linker molecule couples the duplexes to the valency platform molecule. 
   
   
       7 . The conjugate of  claim 6 , wherein the linker molecule is selected from the group consisting of HAD and HADPS. 
   
   
       8 . The conjugate of  claim 2 , wherein the duplexes are substantially homogeneous in length. 
   
   
       9 . The conjugate of  claim 2 , wherein the duplexes are substantially homogeneous in nucleotide composition. 
   
   
       10 . The conjugate of  claim 2 , wherein the duplexes are 20 to 50-bp in length. 
   
   
       11 . The conjugate of  claim 2 , wherein the duplexes are bound to the valency platform molecule at or proximate one of their ends. 
   
   
       12 . The conjugate of  claim 2 , wherein the conjugate is a tolerogen for human systemic lupus erythematosus. 
   
   
       13 . A conjugate according to  claim 2 , wherein the polynucleotide duplexes have a B-DNA type helical structure and a significant binding activity for human systemic lupus erythematosus anti-dsDNA autoantibodies. 
   
   
       14 . A pharmaceutical composition for treating lupus comprising the conjugate of  claim 2  formulated with a pharmaceutically acceptable injectable vehicle. 
   
   
       15 . A method for treating an individual for lupus comprising administering a therapeutically effective amount of the composition  claim 14  to an individual in need of such treatment. 
   
   
       16 . A method for making the conjugate of  claim 2 , comprising:
 (a) bonding a multiplicity of single-stranded polynucleotides of at least about 20 base pairs each on the valency platform molecule; and   (b) annealing complementary single-stranded polynucleotides to the single-stranded polynucleotides conjugated to the valency platform molecule to form said duplexes.   
   
   
       17 . A pharmaceutical composition for treating an antibody-mediated pathology comprising a therapeutically effective amount of the conjugate of  claim 2 , combined with a pharmaceutically acceptable carrier. 
   
   
       18 . A method of inducing specific B cell anergy to an immunogen in an individual comprising administering to the individual an effective amount of the conjugate of  claim 17 . 
   
   
       19 . A method of treating an individual for an antibody-mediated pathology in which undesired antibodies are produced in response to an immunogen comprising administering a therapeutically effective amount of the conjugate of  claim 17  to the individual. 
   
   
       20 . A method for making a conjugate according to  claim 2 , comprising
 (a) covalently bonding the analog of the immunogen lacking T cell epitopes to the chemically-defined valency platform molecule to form a conjugate; and   (b) recovering the conjugate from the reaction mixture.   
   
   
       21 . A chemically-defined, non-polymeric valency platform molecule of the formula:
   G [6] {O—C(═O)—NR SUB -Q [6] (T [6] ) p[6] } n[6]   Formula 6     or     G [7] {O—C(═O)—N[Q [7] (T [7] ) p[7]/2 ] 2 } n[7]   Formula 7   
     wherein
 each of G [6]  and G [7 ], if present, is independently a linear, branched or multiply-branched chain comprising 1-2000 chain atoms selected from the group C, N, O, Si, P and S; 
 each of the n [6] ×p [6]  moieties shown as T [6]  and each of the n [7] ×p [7]  moieties shown as T [7]  is independently chosen from the group 
 
     NHR SUB  (amine), C(═O)NHNHR SUB  (hydrazide), NHNHR SUB  (hydrazine), C(═O)OH (carboxylic acid), C(═O)OR ESTER  (activated ester), C(═O)OC(═O)R B  (anhydride), C(═O)X (acid halide), S(═O) 2 X (sulfonyl halide), C(═NR SUB )OR SUB  (imidate ester), NCO (isocyanate), NCS (isothiocyanate), OC(═O)X (haloformate), C(═O)OC(═NR SUB )NHR SUB  (carbodiimide adduct), C(═O)H (aldehyde), C(═O)R B  (ketone), SH (sulfhydryl or thiol), OH (alcohol), C(═O)CH 2 X (haloacetyl), R ALK X (alkyl halide), S(═O) 2 OR ALK X (alkyl sulfonate), NR 1 R 2  wherein R 1 R 2  is —C(═O)CH═CHC(═O)— (maleimide), C(═O)CR B ═CR B   2  (α,β-unsaturated carbonyl), R ALK —Hg—X (alkyl mercurial), and S(═O)CR B ═CR B   2  (α,β-unsaturated sulfone); 
     wherein
 each X is independently a halogen of atomic number greater than 16 and less than 54 or other good leaving group; 
 each R ALK  is independently a linear, branched, or cyclic alkyl (1-20C) group; 
 each R SUB  is independently H, linear, branched, or cyclic alkyl (1-20C), aryl (1-20C), or alkaryl (1-30C); 
 each R ESTER  is independently N-hydroxysuccinimidyl, p-nitrophenoxy, pentafluorophenoxy, or other activating group; 
 each R B  is independently a radical comprising 1-50 atoms selected from the group C, H, N, O, Si, P and S; 
 n [6] =1 to 32; 
 P [6] =1 to 8; 
 with the proviso that the product n [6] ×p [6]  be greater than 1 and less than 33; 
 n [7] =1 to 32; 
 p [7] =2, 4, 6 or 8; 
 with the proviso that the product n [7] ×p [7]  be greater than 1 and less than 33; 
 each of the n [6]  moieties shown as Q [6]  and each of the 2×n [7]  moieties shown as Q [7]  is independently a radical comprising 1-100 atoms selected from the group C, H, N, O, Si, P and S, containing attachment sites for at least p [6]  (for Q [6] ) or p [7] /2 (for Q [7] , where p [7] /2 is an integer) functional groups on alkyl, alkenyl, or aromatic carbon atoms.