Multi-specific human albumin nanoparticles decorated with antibody fragments and loaded with cytotoxics

Serum albumin nanoparticles (Alb-NP) decorated with at least one decoration chain comprising:

• a linker, bound to the nanoparticles by means of an —S— thioether bond, and
• at least one biological molecule,
• wherein the at least one biological molecule is bound to the linker through an amide bond formed by means of a transamidation (or transglutamination) reaction, mediated by the enzyme transglutaminase,
• said nanoparticles having the following formula (I):
• wherein:
• Alb-NP is a serum albumin nanoparticle containing, on the surface, thiol functionalities (—SH) reacted with an electrophilic group of Z to form the —S— thioether bond;
• Z-spacer-X—NH— is a linker wherein:
• Z derives from a functional group containing an electrophilic group capable of reacting with the —SH group of the albumin, thereby forming the —S— thioether bond, wherein said functional group is selected from:
• 2-bromoacetic acid, 3-bromopropanoic acid, 3-chloropropanoic acid, 4-bromobutyric acid, 5-chlorobutyric acid, 5-bromopentanoic acid, 5-chloropentanoic acid, 4-bromomethyl benzoic acid, 4-chloromethyl benzoic acid, 2-maleimidoacetic acid, 3-maleimidopropionic acid, 4-maleimidobutyric acid, 5-maleimidopentanoic acid, 6-maleimidohexanoic acid, 3-maleimidobenzoic acid, 4-maleimidobenzoic acid, 4-(2-N-Maleimido)methyl benzoic acid, 1-bromoacetic acid and 1-chloroacetic acid;
• the spacer is selected from:
• —NH—(CH2—O)n—CH2—CO—, with n from 2 to 10, preferably 2, 3, 4 and 5;
• —NH—(CH2—CH2—O)n—CH2—CO—, with n from 2 to 10, preferably 2, 3, 4 and 5;
• or the spacer is a Ym group,
• wherein Y is selected from:
• an —NH—(CH2)n—CO— amino acid, with n from 3 to 10, preferably between 3 and 5, more preferably with n=3, 4 or 5;
• glycine, alanine;
• and combinations thereof;
• m is a number between 1 and 5;
• wherein the —NH— group of the spacer forms an amide bond with the carboxylic group of the precursor of Z and the —CO— group of the spacer forms an amide bond with the —NH— group of the subsequent unit X;
• the —X—NH— group is selected from:
• —NH—(CH2)n—NH— with n ranging between 3 and 10, preferably 3, 4 and 5;
• —NH—(O—CH2)n—NH— with n ranging between 2 and 10, preferably 2, 3, 4 and 5;
• —NH—(O—CH2—CH2)n—NH— with n ranging between 2 and 10, preferably 2, 3, 4 and 5;
• L-lysine amino acid;
• L-ornithine amino acid;
• C-terminal amidated L-lysine amino acid;
• C-terminal amidated L-ornithine amino acid;
• wherein the two —NH— groups of the —X—NH— group form an amide bond, respectively, with the —CO— group of the spacer and with the glutamine of a consensus sequence with the formula (VI):
• AA1-AA2-Q-AA3-AA4 Formula (VI)
• wherein:
• AA1 is leucine (L;
• Leu) or is absent;
• AA2 is leucine (L;
• Leu) or is threonine (T;
• Thr);
• Q is glutamine with the formula —CO—(CH2)2—CH—(NH)—CO—;
• AA3 is serine (S;
• Ser) or is glycine (G;
• Gly);
• AA4 is proline (P;
• Pro) or alanine (A;
• Ala) or is absent;
• preferably, AA1 and AA4 are not simultaneously absent;
• R1 and R2, different from or identical to each other, are selected from:
• a Fab, an scFv, a nanobody (NB), an antibody and combinations thereof,
• or
• said albumin nanoparticles having the formula (II):
• wherein,
• Z-Spacer-AA1-AA2-Q-AA3-AA4 is a linker;
• Alb-NP, Z, spacer, R1 and R2 are as defined for the formula (I);
• AA1-AA2-Q-AA3-AA4, with Q equal to glutamine with the formula —CO—(CH2)2—CH—(NH)—CO—, is as defined for the formula (I) and is bound to the spacer by means of an amide bond between the —CO— terminal of the spacer and an —NH— group of the amino acid AA1, if present, or AA2;
• the peptide sequence containing a lysine (K;
• Lys) has the formula (IX):
• (AA)w-K-(AA)p Formula (IX)
• wherein
• w and p are whole numbers comprised between 0 and 8, preferably between 1 and 5, with the condition that w and p are never simultaneously equal to 0;
• AA indicates an amino acid selected from:
• alanine (A;
• Ala), tyrosine (Y;
• Tyr), phenylalanine (F;
• Phe), glycine (G;
• Gly), tryptophan (W;
• Trp) and serine (S;
• Ser);
• K is a lysine (Lys).