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JP-7856631-B2 - Method for preparing bioconjugates

JP7856631B2JP 7856631 B2JP7856631 B2JP 7856631B2JP-7856631-B2

Inventors

  • ヴァイデベン， マリア アントニア
  • ファン ゲール， レモン
  • デ ビーバー， ローリーン
  • ファン バーケル， サンダー セバスチャーン
  • ファン デルフト， フロリス ルイス

Assignees

  • シンアフィックス ビー．ブイ．

Dates

Publication Date
20260511
Application Date
20210902
Priority Date
20200902

Claims (15)

  1. A method for preparing a bioconjugate of structure B-(Z-L-D) x (1), (i) Alkyne or alkene compounds of structure Q-L-D(2) [In the structure, Q is a click probe that includes a cyclic alkyne portion or a cyclic alkene portion. L is Linker, D is the payload] and (ii) the molecule of structure B-(F) x (3) [in the structure, B is a biomolecule functionalized with x click probes F, F is a click probe that can react to Q, A method comprising the step of reacting x, an integer in the range of 1 to 10, with a surfactant containing a negatively charged portion to form a bioconjugate in which the payload is covalently bonded to a biomolecule via a connecting group Z formed by a click reaction between Q and F.
  2. The method according to claim 1, wherein the surfactant is an anionic surfactant.
  3. The method according to claim 1 or 2, wherein the surfactant is selected from the group consisting of sodium decanoate, sodium dodecanoate, sodium lauryl sulfate (SDS), and sodium deoxycholate, and preferably the surfactant is sodium decanoate or sodium deoxycholate.
  4. The method according to any one of claims 1 to 3, wherein the reaction is carried out in a solvent system containing water and an organic solvent in a ratio of 50/50 to 100/0, preferably in the range of 75/25 to 95/5.
  5. The method according to any one of claims 1 to 4, wherein the concentration of the molecule of structure (3) is in the range of 1 to 100 mg/mL, preferably in the range of 5 to 50 mg/mL, and more preferably in the range of 10 to 20 mg/mL.
  6. The method according to any one of claims 1 to 5, wherein the click probe Q comprises a cyclic alkyne moiety, and the click probe F is selected from the group consisting of azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, orthoquinones, dioxothiophenes, and cydonones, preferably with the click probe F being an azide moiety.
  7. Click probe Q is selected from the group consisting of (Q22) to (Q36), Or, the (hetero)cycloalkynyl moiety Q matches structure (Q37), [In the structure, R15 is independently selected from the group consisting of hydrogen, halogen, -OR16, -NO2, -CN, -S(O)2R16 , -S ( O) 3 (-) , C1 - C24 alkyl groups, C6 - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups, and C7 - C24 (hetero)arylalkyl groups, wherein the alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups, and (hetero)arylalkyl groups are optionally substituted, and the two substituents R15 may be linked to form optionally substituted condensed cycloalkyl or optionally substituted condensed (hetero)arene substituents, and R16 is independently selected from the group consisting of hydrogen, halogen, C1 - C24 alkyl groups, C6 - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups, and C7 -C 24 Independently selected from the group consisting of (hetero)arylalkyl groups, Y2 is C( R31 ) 2 , O, S, or NR31 , and R31 is R15 or -LD individually. u is 0, 1, 2, 3, 4, or 5. u' is 0, 1, 2, 3, 4, or 5, and u + u' = 4, 5, 6, 7, or 8. v is an integer in the range of 8 to 16. Preferably, the cyclooctinyl moiety Q corresponds to structure (Q38), [In the structure, R15 is independently selected from the group consisting of hydrogen, halogen, -OR16, -NO2, -CN, -S(O)2R16 , -S ( O) 3 (-) , C1 - C24 alkyl groups, C5 - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups, and C7 - C24 (hetero)arylalkyl groups, wherein the alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups, and (hetero)arylalkyl groups are optionally substituted, and the two substituents R15 may be linked to form optionally substituted condensed cycloalkyl or optionally substituted condensed (hetero)arene substituents, and R16 is independently selected from the group consisting of hydrogen, halogen, C1 - C24 alkyl groups, C6 - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups, and C7 -C 24 Independently selected from the group consisting of (hetero)arylalkyl groups, R 18 is independently selected from the group consisting of hydrogen, halogen, C1 - C24 alkyl groups, C6 - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups, and C7 - C24 (hetero)arylalkyl groups. R 19 is selected from the group consisting of hydrogen, -LD, halogen, C1 - C24 alkyl groups, C6 - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups, and C7 - C24 (hetero)arylalkyl groups, wherein the alkyl group is optionally interrupted by one or more heteroatoms selected from the group consisting of O, N, and S, and the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are independently optionally substituted. l is an integer in the range of 0 to 10. Or, the (hetero)cyclooctinyl moiety Q matches structure (Q39) [In the structure, R15 is independently selected from the group consisting of hydrogen, halogen, -OR16, -NO2, -CN, -S(O)2R16 , -S ( O) 3 (-) , C1 - C24 alkyl groups, C5 - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups, and C7 - C24 (hetero)arylalkyl groups, wherein the alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups, and (hetero)arylalkyl groups are optionally substituted, and the two substituents R15 may be linked to form optionally substituted condensed cycloalkyl or optionally substituted condensed (hetero)arene substituents, and R16 is independently selected from the group consisting of hydrogen, halogen, C1 - C24 alkyl groups, C6 - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups, and C7 -C 24 Independently selected from the group consisting of (hetero)arylalkyl groups, Y is N or CR 15 . The method according to any one of claims 1 to 6.
  8. Click probe Q is selected from the group consisting of optionally substituted (hetero)cyclopropenyl groups, (hetero)cyclobutenyl groups, trans-(hetero)cycloheptenyl groups, trans-(hetero)cyclooctenyl groups, trans-(hetero)cyclononenyl groups, or trans-(hetero)cyclodecynyl groups, preferably click probe Q is selected from the group consisting of (Q40) to (Q50). The method according to any one of claims 1 to 6, wherein in (Q44) and (Q45), the R group(s) of Si are alkyl or aryl.
  9. The method according to any one of claims 1 to 8, wherein payload D is a cytotoxin, preferably colchicine, vinca alkaloid, anthracycline, camptothecin, doxorubicin, daunorubicin, taxane, calicheamicin, tubulicin, irinotecan, inhibitory peptide, amanitin, de-bouganin, duocalmycin, meitansine, auristatin, engine, pyrrolobenzodiazepine (PBD) or indolinobenzodiazepine dimer (IGN), or a cytotoxin selected from PNU-159, 682 and their derivatives, more preferably calicheamicin, PBD dimer, SN-38, MMAE, or exatecan.
  10. The method according to any one of claims 1 to 9, wherein the biomolecule is selected from the group consisting of proteins (including glycoproteins such as antibodies), polypeptides, peptides, glycans, lipids, nucleic acids, oligonucleotides, polysaccharides, oligosaccharides, enzymes, hormones, amino acids, and monosaccharides, more preferably from the group consisting of proteins, polypeptides, peptides, and glycans, and most preferably the biomolecule is a protein.
  11. The method according to claim 10, wherein the biomolecule is selected from the group consisting of mAb, Fab, VHH, scFv, diabody, minibody, afibody, affinin, affimer, atrimer, finomer, Cys-not, DARPin, adnectin/ centinin , notchin, anticarin®, FN3, Knitz domain , bicyclic peptide, and tricyclic peptide.
  12. The method according to any one of claims 1 to 11, wherein the click probe F is attached to a monosaccharide portion, preferably to the terminal monosaccharide portion of a glycoprotein glycan, and most preferably to the terminal monosaccharide portion of an antibody glycan.
  13. The use of a surfactant containing a negatively charged moiety in a bioconjugation reaction for preparing a bioconjugate of structure B-(Z-L-D) x (1), wherein x payloads D are covalently bonded to biomolecule B via connecting groups Z formed by a click reaction between click probe Q and click probe F, and the reaction proceeds as follows: (i) Alkyne or alkene compounds of structure Q-L-D(2) [In the structure, Q is a click probe that includes a cyclic alkyne portion or a cyclic alkene portion. L is Linker, D is the payload] and (ii) the molecule of structure B-(F) x (3) [in the structure, B is a biomolecule functionalized with x click probes F, F is a click probe that can react to Q, x is an integer in the range of 1 to 10.
  14. (i) To increase the conversion rate of the bioconjugation reaction, (ii) To increase the yield of the bioconjugation reaction, (iii) Reduce the amount of organic co-solvent in the solvent system in which the bioconjugation reaction takes place. (iv) To provide flexibility in the concentration of biomolecules during bioconjugation reactions. (v) Reduce the excess amount of alkyne or alkene functionalized payload used during the bioconjugation reaction. (vi) Reduce the degree of aggregate formation during the bioconjugation reaction. (vii) The use according to claim 13 for one or more of the purposes of simplifying the downstream processing of the bioconjugate.
  15. The use according to claim 13 or 14 for improving the drug-antibody ratio (DAR) of a bioconjugate.

Description

Field of Invention [0001] The present invention relates to the field of bioconjugation, and more particularly to a method for preparing bioconjugates in the presence of a surfactant. Background of the Invention [0002] Antibody-drug conjugates (ADCs), considered miracle drugs in treatment, consist of antibodies bound to a drug. Antibodies (also called ligands) can be in small protein formats (scFv, Fab fragments, DARPin, aphibodies, etc.), but are generally monoclonal antibodies (mAbs) selected based on high selectivity and affinity for a given antigen, a long circulating half-life, and little to no immunogenicity. Thus, mAbs as protein ligands for carefully selected biological receptors provide an ideal delivery platform for the selective targeting of drugs. For example, monoclonal antibodies known to selectively bind to specific cancer-associated antigens can be used for the delivery of chemically conjugated cytotoxic substances to tumors via binding, internalization, intracellular processing, and finally release of active catabolites. Cytotoxic substances can be in other formats such as small molecule toxins, protein toxins, or oligonucleotides. As a result, tumor cells can be selectively eradicated while preserving normal cells that were not targeted by the antibody. Similarly, while chemical conjugation of antibacterial agents (antibiotics) to antibodies can be applied to the treatment of bacterial infections, the conjugation of anti-inflammatory drugs is under investigation for the treatment of autoimmune diseases. For example, the attachment of oligonucleotides to antibodies is a potentially promising technique for the treatment of neuromuscular diseases. Therefore, the concept of targeted delivery of active drugs to selected specific cellular sites is a powerful technique for the treatment of a wide range of diseases, offering many advantages over systemic delivery of the same drug. [0003] An alternative strategy for targeting and delivering specific protein substances using monoclonal antibodies involves gene fusion of the latter protein to one (or more) of the antibody's terminals, which may be the N-terminus or C-terminus of the light chain or heavy chain (or both). In this case, the bioactive protein of interest, such as a protein toxin like Pseudomonas exotoxin A (PE38) or an anti-CD3 single-chain variable fragment (scFv), is gene-encoded as a fusion to the antibody, though not necessarily, and likely via a peptide spacer, and the antibody is expressed as a fusion protein. The peptide spacer may or may not contain a protease-sensitive cleavage site. [0004] In the field of ADCs, chemical linkers are typically employed to attach pharmaceuticals to antibodies. These linkers must possess several important attributes, including the need for plasma stability after prolonged drug administration. A stable linker allows for the localization of the ADC to a planned site or cell in the body and prevents premature release of the payload in circulation. Premature release indiscriminately induces all kinds of undesirable biological responses, thereby reducing the therapeutic index of the ADC. Upon internalization, the ADC should be processed so that the payload is effectively released and thus able to bind to its target. [0005] Linkers belong to two families: non-cleavable linkers and cleavable linkers. Non-cleavable linkers consist of a chain of atoms between the antibody and the payload and are sufficiently stable under physiological conditions regardless of the organ or biological compartment in which the antibody-drug conjugate resides. As a result, the release of the payload from an ADC containing a non-cleavable linker depends on the complete (lysosomal) degradation of the antibody after the ADC has been moved into the cell. As a result of this degradation, the payload (still containing the linker), as well as peptide fragments and/or amino acids, are released from the antibody to which the linker was originally attached. Cleavable linkers utilize the intrinsic properties of the cell or cellular compartment for the selective release of the payload from the ADC, thereby generally leaving no trace of linker after metabolic processing. There are three commonly used mechanisms for cleavable linkers: 1) sensitivity to specific enzymes, 2) pH sensitivity, and 3) sensitivity to the cellular redox state (or its microenvironment). The cleavable linker may also include, for example, self-destructive units based on para-aminobenzyl alcohol groups and their derivatives. The linker may also include additional non-functional elements, often called spacer or stretcher units, for connecting the linker to reactive groups for reactions with biomolecules. [0006] Currently, payloads used in ADCs mainly include microtubule disruptors [e.g., auristatins such as monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF), maytansinoids such as DM1 and DM4, tubulinin], DNA damaging agents [e.g., calicheamycin, pyrroloben