Linkers for site-specific antibody conjugation

Abstract

The present specification reports a polypeptide-linker-nucleic acid conjugate, wherein the linker comprises a 3-aminopropanamide unit, a 2,6-diaminohexanoic acid amide unit, and a 1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d]-1,2,3-triazole unit, the polypeptide comprises a C-terminal lysine residue, the nucleic acid comprises an oxygen atom at the 5' or 3' end linked to a phosphorus atom in oxidation state V, and the 3-amino group of the 3-aminopropanamide unit and the carboxy functional group of the lysine residue of the polypeptide are linked by / form an amide bond, and the 3-aminopropanamide unit is linked to the carboxy functional group of the lysine residue of the polypeptide. The carboxyl functional group at position 1 and the alpha amino group of the 2,6-diaminohexanoic acid amide unit are linked by / form an amide bond, the 6-amino group of the 2,6-diaminohexanoic acid amide unit is the nitrogen of the 1,2,3-triazole element of the 1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d]-1,2,3-triazole unit, and the oxygen linked to the phosphorus atom of the nucleic acid is covalently linked to the cyclopropane element of the 1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d]-1,2,3-triazole unit.

Description

[Technical Field]

[0001] The present invention is in the field of protein-drug conjugates, more particularly in the field of covalent conjugates of targeting antibodies to therapeutic nucleic acids. [Background technology]

[0002] Background of the Invention A drug is a chemical substance used to treat, cure, or prevent disease. Drugs can be administered via several routes, and many drugs can be administered by more than one route. Typical administration routes include, but are not limited to, injection of a solution, suspension, or emulsion (e.g., intramuscular, intravenous, intraperitoneal, intraocular, intraosseous, subcutaneous, or intrathecal), oral, rectal, sublingual, or topical. Drugs are usually distributed systemically within a patient's body, and it is clear that they may have adverse effects, for example, due to their activity in non-target tissues. Other tissues may be difficult to reach. Targeted therapy aims to overcome this drawback by using drugs directed to the cells, tissues, or organs in the body that are intended to act. Targeted therapy is expected to be more effective and have fewer side effects than traditional non-targeted forms of treatment.

[0003] One method of targeting a therapeutic entity to its intended site of action is by conjugating it to an antibody that specifically binds to the target cell or tissue. One of the challenges associated with antibody-drug conjugates (ADCs), such as antibody-oligonucleotide conjugates (AOCs), is their manufacture. Most drug-antibody conjugates utilize either partially reduced interchain disulfide bonds that allow thiol-maleimide chemistry or lysine functionalization using activated esters. These methods result in a statistical mixture of conjugated antibody species with different numbers of drugs attached to different sites. For ADCs with a monomethyl auristatin E payload, ADC species with drug-to-antibody ratios (DARs) of 4, 6, or 8 were demonstrated to be increasingly hydrophobic and much more prone to aggregation than species with a DAR of 2 (Adem et al., Bioconj. Chem. 25 (2014) 656-664).

[0004] Therefore, it is desirable to provide ADC for targeted therapy, such as AOC, by more precisely controlling the number and site of attachment of therapeutic entity.This should improve the uniformity of ADC.Improved site-specific conjugation technology continues to be the interest of many pharmaceutical companies, because of its potential use in the preparation of therapeutic ADC and diagnostic antibody-label conjugate or antibody-enzyme conjugate.

[0005] Microbial transglutaminase from Streptomyces mobaraensis has emerged as an inexpensive and easy-to-use enzyme for protein cross-linking as well as site-specific protein labeling (Ando et al., 2014; Strop et al., 2013).

[0006] WO 2015 / 162563 disclosed antibody-drug conjugates with high drug loading.

[0007] Magdalena Dorywalska et al. disclosed the effect of the attachment site on the stability of cleavable antibody-drug conjugates (Bioconj. Chem. 26 (2015) 650-659).

[0008] US Patent Application Publication No. 2020 / 0249231 disclosed microbial transglutaminase, its substrate and methods of use thereof.

[0009] Ian Huggins et al. disclosed site-selective antibody-oligonucleotide conjugation via microbial transglutaminase (Mol. 24 (2019) 3287). The discovery of a novel transglutaminase from Kutzneria albida and the identification of its respective peptide substrate were described by Steffen et al. (2017). KalbTG catalyzes the formation of an isopeptide bond between a glutamine (Gln, Q) and a lysine (Lys, K) side chain. YRYRQ (SEQ ID NO: 17) was identified as the KalbTG Gln-containing motif (Q-amino acid sequence, Q-tag), and RYESK (SEQ ID NO: 16) was identified as the Lys-containing acceptor motif (K-amino acid sequence, K-tag). KalbTG exhibits similar efficiency but improved specificity and developability compared to previously described microbial transglutaminase (mTG). Summary of the Invention

[0010] The present invention is based, at least in part, on the discovery that for conjugation of antibodies to therapeutic nucleic acids using transglutaminase from Kutschnaria albida (KalbTG) or a functionally active variant thereof, the linker attached to the K-amino acid (K-tag) affects the conjugation efficiency.

[0011] The present invention is further based, at least in part, on the discovery that the linker that covalently conjugates the antibody and the therapeutic nucleic acid affects the in vivo stability of the conjugate.

[0012] The present invention is further based, at least in part, on the discovery that it is not possible to incorporate a Q-tag into every site within an IgG1 antibody that is compatible with conjugation to a payload. Antibody constant domain regions were screened for KalbTG-mediated conjugation. In this regard, KalbTG Q-tags were inserted into surface-exposed inter- and intra-domain flexible loops within the human IgG1 heavy and light chain constant regions, as well as into the C-termini of each heavy and light chain. A total of nine different sites within the IgG1 heavy and light chains were tested, each containing a KalbTG Q-tag motif inserted within two flexible linkers (GGGSYRYRQGGGS) (SEQ ID NO: 25). Furthermore, three antibodies (mAb1 to mAb3) with different binding specificities were tested. These molecules with single-site insertions were evaluated for their expression rates. The results are shown in the Examples (see Table 1).

[0013] The present invention includes at least the following independent aspects and dependent embodiments.

[0014] 1. A polypeptide-linker-nucleic acid conjugate comprising: said linker is a 3-aminopropanamide unit (i.e., a 3-amino derivative of propanamide); 2,6-diaminohexanoic acid amide units, 1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d]-1,2,3-triazole units and Including, said polypeptide is C-terminal lysine residue Including, said nucleic acid Oxygen linked at the 5' or 3' end to a fluorophore in oxidation state V (i.e., a phosphorus atom in oxidation state V) Including, the 3-amino group of the 3-aminopropanamide unit and the carboxy functional group of the lysine residue of the polypeptide are linked by / form an amide bond; the carboxy functional group of the 3-aminopropanamide unit and the alpha amino group of the 2,6-diaminohexanoic acid amide unit are linked by / form an amide bond; the 6-amino group of the 2,6-diaminohexanoic acid amide unit is the nitrogen of the 1,2,3-triazole element of the 1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d]-1,2,3-triazole unit; The oxygen linked to the fluorophore (i.e., phosphorus atom) of the nucleic acid is covalently linked to the cyclopropane element of the 1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d]-1,2,3-triazole unit. A polypeptide-linker-nucleic acid conjugate, characterized in that:

[0015] 2. A polypeptide-linker-nucleic acid conjugate, comprising: said linker is 1,2-diaminoethyl units, 3-aminopropionic acid units, 1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d]-1,2,3-triazole units and Including, said polypeptide is C-terminal lysine residue Including, said nucleic acid Oxygen linked at the 5' or 3' end to a fluorophore in oxidation state V (i.e., a phosphorus atom in oxidation state V) Including, the 2-amino group of the 1,2-diaminoethyl unit and the carboxyl functional group of the lysine residue of the polypeptide are linked by / form an amide bond; the 1-amino group of the 1,2-diaminoethyl unit and the carboxy group of the 3-aminopropionic acid unit are linked by / form an amide bond; the 3-amino group of the 3-aminopropionic acid unit is the nitrogen of the 1,2,3-triazole element of the 1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d]-1,2,3-triazole unit; The oxygen linked to the fluorophore (i.e., phosphorus atom) of the nucleic acid is covalently linked to the cyclopropane element of the 1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d]-1,2,3-triazole unit. A polypeptide-linker-nucleic acid conjugate, characterized in that:

[0016] 3. The polypeptide-linker-nucleic acid conjugate of embodiment 1 or 2, wherein the 6-amino group of the 2,6-diaminohexanoic acid amide unit or the 3-amino group of the 3-aminopropionic acid unit is the nitrogen at position 1 (numbering according to Figure 3) of the 1,2,3-triazole element of the 1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d]-1,2,3-triazole unit.

[0017] 4. The polypeptide-linker-nucleic acid conjugate of any one of embodiments 1 to 3, wherein the amide group of the 2,6-diaminohexanoic acid amide unit is an NHR(1) or NR(1)R(2) group, and R(1) and R(2) are independently selected from the group consisting of lower alkyl and oxyalkyl, including at least methyl, ethyl, propyl, butyl, pentyl, hexyl, oxymethyl, oxyethyl, oxypropyl, oxybutyl, oxypentyl, and oxyhexyl.

[0018] 5. The polypeptide-linker-nucleic acid conjugate of any one of embodiments 1 to 4, wherein the oxygen linked to the fluorophore (i.e., phosphorus atom) of the nucleic acid is covalently linked to the carbon at position 6 (numbering according to Figure 3) of the cyclopropane element of the 1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d]-1,2,3-triazole unit.

[0019] 6. The polypeptide-linker-nucleic acid conjugate of any one of embodiments 1 to 5, wherein the oxygen linked to the fluorophore (i.e., phosphorus atom) of the nucleic acid is covalently linked by a methyl or ethyl unit to the carbon at position 6 (numbering according to Figure 3) of the cyclopropane element of the 1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d]-1,2,3-triazole unit.

[0020] 7. The polypeptide-linker-nucleic acid conjugate of any one of embodiments 1 to 6, wherein the oxygen is linked to a fluorophore in oxidation state V (i.e., a phosphorus atom in oxidation state V) at the 5' end of the nucleic acid.

[0021] 8. The polypeptide-linker-nucleic acid conjugate of any one of embodiments 1 to 7, wherein the oxygen linked to the fluorophore in the oxidation state V (i.e., the phosphorus atom in the oxidation state V) is a phosphate or a phosphorothioate.

[0022] 9. The polypeptide-linker-nucleic acid conjugate according to any one of embodiments 1 to 8, wherein the polypeptide comprises the K-amino acid sequence RYESK, in which the 3-amino group of the 3-aminopropanamide unit or the 2-amino group of the 1,2-diaminoethyl unit in the sequence is linked by / forms an amide bond with the carboxyl functional group of the lysine residue.

[0023] 10. The polypeptide-linker-nucleic acid conjugate of any one of embodiments 1 to 9, wherein the polypeptide comprises the K-amino acid sequence RYESK, in which the 3-amino group of the 3-aminopropanamide unit or the 2-amino group of the 1,2-diaminoethyl unit and the carboxyl functional group of a lysine residue are linked by / form an amide bond, and the epsilon-amino group of the lysine is linked to the side chain of a glutamine residue by an isopeptide bond.

[0024] 11. The polypeptide-linker-nucleic acid conjugate of any one of embodiments 1 to 10, wherein the polypeptide comprises the K-amino acid sequence RYESK, in which the 3-amino group of a 3-aminopropanamide unit or the 2-amino group of a 1,2-diaminoethyl unit and the carboxyl functional group of a lysine residue are linked by / form an amide bond, and the epsilon-amino group of the lysine is linked to the side chain of a glutamine residue by an isopeptide bond, and the glutamine residue is within a Q-amino acid sequence of at least 5 amino acid residues.

[0025] 12. The polypeptide-linker-nucleic acid conjugate according to any one of embodiments 1 to 11, wherein the polypeptide comprises the K-amino acid sequence RYESK, wherein the 3-amino group of the 3-aminopropanamide unit or the 2-amino group of the 1,2-diaminoethyl unit in the K-amino acid sequence and the carboxy functional group of the lysine residue in the K-amino acid sequence are linked by / form an amide bond, and the epsilon-amino group of the lysine in the K-amino acid sequence is linked to the side chain of a glutamine residue by an isopeptide bond, and wherein the glutamine residue is within the Q-amino acid sequence RYGQR (SEQ ID NO: 11), RWRQR (SEQ ID NO: 12), YRQRT (SEQ ID NO: 13), IRQRQ (SEQ ID NO: 14), FRYRQ (SEQ ID NO: 15) or YRYRQ (SEQ ID NO: 17), and in one preferred embodiment YRYRQ (SEQ ID NO: 17).

[0026] 13. The polypeptide-linker-nucleic acid conjugate according to any one of embodiments 1 to 12, wherein the polypeptide comprises the K-amino acid sequence RYESK, wherein the 3-amino group of the 3-aminopropanamide unit or the 2-amino group of the 1,2-diaminoethyl unit in the K-amino acid sequence and the carboxy functional group of the lysine residue in the K-amino acid sequence are linked by / form an amide bond, and the epsilon-amino group of the lysine in the K-amino acid sequence is linked to the side chain of a glutamine residue by an isopeptide bond, and the glutamine residue is within a Q-amino acid sequence of RYGQR (SEQ ID NO: 11), RWRQR (SEQ ID NO: 12), YRQRT (SEQ ID NO: 13), IRQRQ (SEQ ID NO: 14), FRYRQ (SEQ ID NO: 15) or YRYRQ (SEQ ID NO: 17), in one preferred embodiment YRYRQ (SEQ ID NO: 17), and wherein the Q-amino acid sequence comprises one or two spacers at its direct termini.

[0027] 14. The polypeptide-linker-nucleic acid conjugate according to any one of embodiments 1 to 13, wherein the polypeptide comprises the K-amino acid sequence RYESK, wherein the 3-amino group of the 3-aminopropanamide unit or the 2-amino group of the 1,2-diaminoethyl unit in the K-amino acid sequence and the carboxy functional group of the lysine residue in the K-amino acid sequence are linked by / form an amide bond, and the epsilon-amino group of the lysine in the K-amino acid sequence is linked to the side chain of a glutamine residue by an isopeptide bond, and the glutamine residue is within a Q-amino acid sequence of RYGQR (SEQ ID NO: 11), RWRQR (SEQ ID NO: 12), YRQRT (SEQ ID NO: 13), IRQRQ (SEQ ID NO: 14), FRYRQ (SEQ ID NO: 15) or YRYRQ (SEQ ID NO: 17), in one preferred embodiment YRYRQ (SEQ ID NO: 17), and wherein the Q-amino acid sequence comprises one or two spacers consisting mainly or entirely of Gly and Ser directly at its termini.

[0028] 15. The polypeptide comprises the K-amino acid sequence RYESK, wherein the 3-amino group of the 3-aminopropanamide unit or the 2-amino group of the 1,2-diaminoethyl unit in the K-amino acid sequence and the carboxy functional group of the lysine residue in the K-amino acid sequence are linked by / form an amide bond, the epsilon-amino group of the lysine in the K-amino acid sequence is linked to the side chain of a glutamine residue by an isopeptide bond, and the glutamine residue is located within a Q-amino acid sequence of RYGQR (SEQ ID NO: 11), RWRQR (SEQ ID NO: 12), YRQRT (SEQ ID NO: 13), IRQRQ (SEQ ID NO: 14), FRYRQ (SEQ ID NO: 15) or YRYRQ (SEQ ID NO: 17), and in one preferred embodiment, YRYRQ (SEQ ID NO: 17), and the Q-amino acid sequences are, independently of each other, directly terminated at their termini with (Gly-Gly-Gly-Ser). n 15. The polypeptide-linker-nucleic acid conjugate of any one of embodiments 1 to 14, comprising one or two spacers consisting of (SEQ ID NO: 19), wherein n=1, 2, 3, 4 or 5, preferably n=1.

[0029] 16. The polypeptide comprises the K-amino acid sequence RYESK, wherein the 3-amino group of the 3-aminopropanamide unit or the 2-amino group of the 1,2-diaminoethyl unit in the K-amino acid sequence and the carboxy functional group of the lysine residue in the K-amino acid sequence are linked by / form an amide bond, the epsilon-amino group of the lysine in the K-amino acid sequence is linked to a glutamine residue side chain by an isopeptide bond, and the glutamine residue is selected from the group consisting of RYGQR (SEQ ID NO: 11), RWRQR (SEQ ID NO: 12), YRQRT (SEQ ID NO: 13), IRQRQ (SEQ ID NO: 14), and the like. 16. The polypeptide-linker-nucleic acid conjugate according to any one of embodiments 1 to 15, wherein the Q-amino acid sequence is within the Q-amino acid sequence of FRYRQ (SEQ ID NO: 15) or YRYRQ (SEQ ID NO: 17), and in one preferred embodiment YRYRQ (SEQ ID NO: 17), wherein the Q-amino acid sequence is at a position selected from positions 110 (LC110), 143 (LC143) and 214 (LC214) of the antibody light chain and positions 118 (HC118), 177 (HC177), 297 (HC297), 341 (HC341), 401 (HC401) and 446 (HC446) of the antibody heavy chain (numbering according to Kabat).

[0030] 17. The polypeptide comprises the K-amino acid sequence RYESK, wherein the 3-amino group of the 3-aminopropanamide unit or the 2-amino group of the 1,2-diaminoethyl unit in the K-amino acid sequence and the carboxy functional group of the lysine residue in the K-amino acid sequence are linked by / form an amide bond, and the epsilon-amino group of the lysine in the K-amino acid sequence is linked to a glutamine residue side chain by an isopeptide bond, and the glutamine residue is selected from the group consisting of RYGQR (SEQ ID NO: 11), RWRQ 17. The polypeptide-linker-nucleic acid conjugate according to any one of embodiments 1 to 16, wherein the Q-amino acid sequence is within the Q-amino acid sequence of R (SEQ ID NO: 12), YRQRT (SEQ ID NO: 13), IRQRQ (SEQ ID NO: 14), FRYRQ (SEQ ID NO: 15) or YRYRQ (SEQ ID NO: 17), in one preferred embodiment YRYRQ (SEQ ID NO: 17), and wherein said Q-amino acid sequence is at a position selected from position 214 of the antibody light chain (LC214), position 177 of the antibody heavy chain (HC177) and position 297 of the antibody heavy chain (HC297) (numbering according to Kabat).

[0031] 17a. A polypeptide-linker-nucleic acid conjugate according to any one of embodiments 1 to 17, wherein the Q-amino acid sequence is in a chain of an antibody comprising at least one light chain and at least one heavy chain.

[0032] 18. A polypeptide-linker-nucleic acid conjugate according to any one of embodiments 1 to 17a, wherein the light chain constant domain not comprising the Q amino acid sequence comprises an amino acid sequence that is at least 96%, 97%, 98% or 99%, in particular 100%, identical to the amino acid sequence of any of SEQ ID NOs: 6 or 7, or wherein the heavy chain constant region not comprising the Q amino acid sequence comprises an amino acid sequence that is at least 96%, 97%, 98% or 99%, in one preferred embodiment 100% identical to the amino acid sequence of SEQ ID NOs: 1 to 5.

[0033] 19. A polypeptide-linker-nucleic acid conjugate according to any one of embodiments 1 to 18, wherein the light chain constant domain comprises or consists of an amino acid sequence that is at least 96%, 97%, 98% or 99%, in particular 100%, identical to the amino acid sequence of SEQ ID NO: 10, and / or the heavy chain constant region comprises or consists of an amino acid sequence that is at least 96%, 97%, 98% or 99%, in one preferred embodiment 100% identical to the amino acid sequence of SEQ ID NO: 8 or 9 or 34.

[0034] 20. The polypeptide-linker-nucleic acid conjugate of any one of embodiments 1 to 19, wherein the 3-aminopropanamide unit is 3-[2-aminoethoxy]propanamide.

[0035] 21. The polypeptide-linker-nucleic acid conjugate of any one of embodiments 1 to 20, wherein the 3-aminopropanamide unit is 3-[2-(2-aminoethoxy)ethoxy]propanamide.

[0036] 22. The polypeptide-linker-nucleic acid conjugate of any one of embodiments 1 to 21, wherein the 3-aminopropanamide unit is 3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propanamide.

[0037] 23. The polypeptide-linker-nucleic acid conjugate of any one of embodiments 1 to 22, wherein the 3-aminopropanamide unit is 3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]propanamide.

[0038] 24. The polypeptide-linker-nucleic acid conjugate of any one of embodiments 1 to 23, wherein the 3-aminopropanamide unit is 3-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]propanamide.

[0039] 25. The polypeptide-linker-nucleic acid conjugate of any one of embodiments 1 to 24, wherein the 3-aminopropanamide unit is 3-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanamide.

[0040] 26. The polypeptide-linker-nucleic acid conjugate of any one of embodiments 2 to 25, wherein the 3-aminopropionic acid unit is a 4-aminobutanoic acid unit.

[0041] 27. The polypeptide-linker-nucleic acid conjugate of any one of embodiments 2 to 26, wherein the 3-aminopropionic acid unit is a 4-aminopentanoic acid unit.

[0042] 28. The polypeptide-linker-nucleic acid conjugate of any one of embodiments 2 to 27, wherein the 3-aminopropionic acid unit is a 4-aminoethanoic acid unit.

[0043] 29. The polypeptide-linker-nucleic acid conjugate of any one of embodiments 1, 3-25, wherein the 2,6-diaminohexanoic acid amide unit is 2,5-diaminopentanoic acid.

[0044] 30. The polypeptide-linker-nucleic acid conjugate of any one of embodiments 1, 3-25, and 29, wherein the 2,6-diaminohexanoic acid amide unit is a 2,4-diaminobutanoic acid unit.

[0045] 31. The polypeptide-linker-nucleic acid conjugate of any one of embodiments 1, 3-25, and 29-30, wherein the 2,6-diaminohexanoic acid amide unit is a 2,3-diaminopropionic acid unit.

[0046] 32. The polypeptide-linker-nucleic acid conjugate of any one of embodiments 1, 3 to 25, and 29 to 31, wherein the 2,6-diaminohexanoic acid amide unit is a 2,2-diaminoethanoic acid unit.

[0047] 33. The polypeptide-linker-nucleic acid conjugate according to any one of embodiments 1 to 32, wherein the conjugate is a covalent conjugate.

[0048] 34. The linker has the following structure (the point of attachment to the nucleic acid (-OP(V)) and the K-amino acid sequence (-NH-C(=O)) are indicated by wavy lines): A polypeptide-linker-nucleic acid conjugate described in any one of embodiments 1 to 33, having TIFF2025525560000001.tif79128.

[0049] 35. The linker has the following structure (the point of attachment to the nucleic acid (-OP(V)) and the K-amino acid sequence (-NH-C(=O)) are indicated by wavy lines): A polypeptide-linker-nucleic acid conjugate described in any one of embodiments 1 to 34, having TIFF2025525560000002.tif77128.

[0050] 36. The linker has the following structure (the point of attachment to the nucleic acid (-OP(V)) and the K-amino acid sequence (-NH-C(=O)) are indicated by wavy lines): A polypeptide-linker-nucleic acid conjugate described in any one of embodiments 1 to 35, having TIFF2025525560000003.tif84128.

[0051] 37. The linker has the following structure (the point of attachment to the nucleic acid (-OP(V)) and the K-amino acid sequence (-NH-C(=O)) are indicated by wavy lines): A polypeptide-linker-nucleic acid conjugate described in any one of embodiments 1 to 36, having TIFF2025525560000004.tif90128.

[0052] 38. A polypeptide-linker-nucleic acid conjugate according to any one of embodiments 1 to 37, wherein the antibody recognizes and binds to a target at its complementarity-determining region (CDR), in particular the target is a biological molecule present on a cell.

[0053] 39. The polypeptide-linker-nucleic acid conjugate of any one of embodiments 1 to 38, wherein the nucleic acid is selected from the group consisting of RNA, siRNA, antisense oligonucleotides (ASO), LNA, and ASO comprising LNA nucleotides.

[0054] 40. a) the antibody recognizes one target, which is a receptor that induces receptor-mediated endocytosis, such as transferrin receptor protein 1 (TfR1), insulin-like growth factor 1 receptor (IGF-1R), low-density lipoprotein receptor-related protein 1 (LRP1) or low-density lipoprotein receptor-related protein 8 (LRP8), in particular TfR1; and / or b) the antibody recognizes one or two targets, and the one or two targets are specific to a particular cell type, for example, a tumor marker specific to tumor cells, such as breast cancer cells; A polypeptide-linker-nucleic acid conjugate according to any one of embodiments 1 to 39.

[0055] 41. a) providing an antibody comprising the Q-amino acid sequence RYGQR (SEQ ID NO: 11), RWRQR (SEQ ID NO: 12), YRQRT (SEQ ID NO: 13), IRQRQ (SEQ ID NO: 14), FRYRQ (SEQ ID NO: 15), or YRYRQ (SEQ ID NO: 17), in one preferred embodiment YRYRQ (SEQ ID NO: 17), at one or more positions selected from positions 110 (LC110), 143 (LC143), and 214 (LC214) of the antibody light chain and positions 118 (HC118), 177 (HC177), 297 (HC297), 341 (HC341), 401 (HC401), and 446 (HC446) of the antibody heavy chain (numbering according to Kabat); b) providing a polypeptide comprising the amino acid sequence RYESK (SEQ ID NO: 16) conjugated to the terminal amino group of (3-[2-[2-(2-aminoethoxy)ethoxy]propanamido)-6-azidohexanamide via an amide bond formed between the carboxy group of the C-terminal lysine residue and the terminal amino group of (3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propanamido)-6-azidohexanamide; c) reacting the antibody of a) with the polypeptide of b) in the presence of KalbTG or a functionally active variant thereof under conditions that promote the activity of KalbTG, thereby forming an isopeptide bond between the Q-amino acid sequence of the antibody and the polypeptide comprising the amino acid sequence of RYESK to produce (3-[2-[2-(2-antibody aminoethoxy)ethoxy]ethoxy]propanamido)-6-azidohexanamide (antibody azide); d) reacting the antibody azide with O-2-(bicyclo[6.1.0]non-4-yn-9-yl)-ethyl-O'-nucleic acid thiophosphonate, thereby producing the polypeptide-linker-nucleic acid conjugate of any one of embodiments 1 to 40. 41. A method for producing a polypeptide-linker-nucleic acid conjugate according to any one of embodiments 1 and 3 to 40, comprising:

[0056] 42. The reaction product of step c) has the following structure: The method of embodiment 41, having TIFF2025525560000005.tif58133.

[0057] 43. The reaction product of step d) has the following structure: The method of embodiment 41 or 42, having TIFF2025525560000006.tif118128.

[0058] 44. a) providing an antibody comprising the Q-amino acid sequence RYGQR (SEQ ID NO: 11), RWRQR (SEQ ID NO: 12), YRQRT (SEQ ID NO: 13), IRQRQ (SEQ ID NO: 14), FRYRQ (SEQ ID NO: 15), or YRYRQ (SEQ ID NO: 17), in one preferred embodiment YRYRQ (SEQ ID NO: 17), at one or more positions selected from positions 110 (LC110), 143 (LC143), and 214 (LC214) of the antibody light chain and positions 118 (HC118), 177 (HC177), 297 (HC297), 341 (HC341), 401 (HC401), and 446 (HC446) of the antibody heavy chain (numbering according to Kabat); b) providing a polypeptide comprising the amino acid sequence RYESK (SEQ ID NO: 16) conjugated to the terminal amino group of [2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethyl]propanamido)-6-azidopropanamide via an amide bond formed between the carboxy group of the C-terminal lysine residue and the terminal amino group of [2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethyl]propanamido)-6-azidopropanamide; c) reacting the antibody of a) with the polypeptide of b) in the presence of KalbTG or a functionally active variant thereof under conditions that promote the activity of KalbTG, thereby forming an isopeptide bond between the Q-amino acid sequence of the antibody and the polypeptide comprising the amino acid sequence of RYESK to produce (3-[2-[2-(2-antibody aminoethoxy)ethoxy]ethoxy]propanamido)-6-azidopropanamide (antibody azide); d) reacting the antibody azide with O-2-(bicyclo[6.1.0]non-4-yn-9-yl)-ethyl-O'-nucleic acid thiophosphonate, thereby producing the polypeptide-linker-nucleic acid conjugate of any one of embodiments 2 to 40. 41. A method for producing a polypeptide-linker-nucleic acid conjugate according to any one of embodiments 2 to 40, comprising:

[0059] 45. The reaction product of step c) has the following structure: The method of embodiment 44, having TIFF2025525560000007.tif58133.

[0060] 46. The reaction product of step d) has the following structure: The method of any one of embodiments 44 to 45, having TIFF2025525560000008.tif71128.

[0061] 47. A polypeptide-linker-nucleic acid conjugate according to any one of embodiments 1 to 40, or a polypeptide-linker-nucleic acid conjugate produced according to the method according to any one of embodiments 41 to 46, for use as a medicament.

[0062] 48. A polypeptide-linker-nucleic acid conjugate according to any one of embodiments 1 to 40, or a polypeptide-linker-nucleic acid conjugate produced according to the method of any one of embodiments 41 to 46, for use in treating a neurological or brain disease, such as Alzheimer's disease or Parkinson's disease.

[0063] 49. A polypeptide-linker-nucleic acid conjugate according to any one of embodiments 1 to 40, or a polypeptide-linker-nucleic acid conjugate produced according to the method of any one of embodiments 41 to 46, for use in treating cancer, such as breast cancer.

[0064] In addition to the various embodiments depicted and claimed, the presently disclosed subject matter is directed to other embodiments having other combinations of the features disclosed and claimed herein. Thus, particular features presented herein may be combined with each other in other manners within the scope of the presently disclosed subject matter, such that the presently disclosed subject matter includes any suitable combination of features disclosed herein. The foregoing descriptions of specific embodiments of the presently disclosed subject matter have been presented for purposes of illustration and description and are not intended to be exhaustive or to limit the presently disclosed subject matter to the disclosed embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0065] Detailed Description of Embodiments of the Invention The present invention relates to modified antibodies comprising a heavy chain and a light chain, wherein the heavy chain and / or light chain comprise one or more first recognition site(s) for transglutaminase from Kutschneria albida (KalbTG) or a functionally active variant thereof. The one or more first recognition site(s) are introduced at one or more selected position(s) within the heavy and / or light chain of the antibody. The present invention further relates to one or more nucleic acids encoding modified antibodies according to the present invention, as well as covalent conjugates comprising (i) a modified antibody according to the present invention and (ii) one or more non-antibody moieties (payload(s)) covalently conjugated to the one or more first recognition site(s) either directly or via a first linker. In certain embodiments, the non-antibody moiety comprises a therapeutic entity and, optionally, a second linker. The present invention further relates to methods of covalently conjugating modified antibodies according to the present invention to non-antibody moieties. If the non-antibody moiety comprises a therapeutic entity, the present invention further relates to conjugates of the modified antibody according to the invention and the therapeutic entity for use as a medicament and as pharmaceutical compositions for use in the treatment of diseases.

[0066] The C-terminus of the IgG heavy chain has been described as a suitable Q-tag insertion site for mTG for antibody labeling (see, for example, WO 2021 / 174091). The inventors have observed, inter alia, that conjugating a payload to the moiety increases aggregation and hydrophobicity. To be able to use KalbTG to prepare medicament-useful antibody-drug conjugates, the KalbTG Q-tag must be introduced into a defined site within the IgG backbone.

[0067] To avoid the above drawbacks, the objective of the present invention was to identify sites within IgG molecules for the incorporation of Q-tag motifs, which would result in improved properties of modified antibodies for KalbTG-mediated conjugation. The incorporation of Q-tags should provide the necessary accessibility for conjugation and therapeutic activity of therapeutic entities without impairing antibody folding or function or reducing expression yields. Successful identification of such sites would allow the incorporation of one or more therapeutic moieties per IgG molecule in a defined stoichiometry and controlled, site-specific manner. This could provide more uniform conjugate products, for example, reducing the required purification and separation efforts and resulting in molecules with more favorable drug-like properties.

[0068] It can be seen that expression titers changed, ie decreased or even improved, depending on the insertion site, which was completely unexpected.

[0069] The modified antibodies were further tested for Q-tag conjugation and accessibility to KalbTG. Small molecules (fluorescent dyes) and small single-stranded nucleic acids were tested as payloads. When the antibody was symmetric, i.e., containing two identical heavy and light chain pairs, there were two Q-tags per molecule. When the antibody was asymmetric, i.e., containing two different heavy and light chain pairs, there was a single Q-tag per molecule. Therefore, a drug-to-antibody ratio (DAR, i.e., the number of payload molecules per antibody molecule) of 2 was expected for symmetric antibodies with 100% conjugation efficiency, and a DAR of 1 for asymmetric antibodies. The results for fluorescent dyes in the case of mAb5 are shown in Table 2. The results for single-stranded nucleic acids of 15 nucleotides in length for mAb-1 and 2 and 20 nucleotides in length for mAb-4 are shown in Table 3 (determined by HIC and UV-vis). MAbs 1 and 2 were conjugated in a one-step method, mAb-4 was conjugated in a two-step method (click chemistry was used to first conjugate the K-tag and then the K-tag to the nucleic acid).

[0070] As an example, for mAb-2, five different Q-tags, RYGQR (SEQ ID NO: 11), RWRQR (SEQ ID NO: 12), YRQRT (SEQ ID NO: 13), IRQRQ (both Qs can be modified; SEQ ID NO: 14), and FRYRQ (SEQ ID NO: 15), were introduced into three different positions, namely, HC297, HC446, and LC143, respectively. The expression yield (1 L, μg / mL) and conjugation (reverse; HIC / UV-vis) results are shown in Table 4.

[0071] Further biophysical characterization of the modified antibodies conjugated to single-stranded nucleic acids of 15 and 20 nucleotides, respectively, was performed by hydrophobic interaction chromatography (HIC), as exemplarily shown in Table 5 and Figure 1 for mAb-2 (15 nucleotides). The retention time of the hydrophilic marker was 9.25 min, and the relative retention time of the hydrophobic marker was 25.9 min (mAb2).

[0072] Thus, we have successfully identified several KalbTG Q-tag insertion sites spanning the length of the IgG backbone that do not adversely affect expression yield and provide enzymatic accessibility.

[0073] Suitable insertion sites for Q-tags are at positions 110 (LC110), 143 (LC143), and 214 (LC214) in the light chain and at positions 118 (HC118), 177 (HC177), 297 (HC297), 341 (HC341), 401 (HC401), and 446 (HC446) in the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat).

[0074] Thus, in a first aspect, the present invention relates to a modified antibody comprising a heavy chain and a light chain, wherein the heavy and / or light chain comprises one or more (first) recognition site(s) for transglutaminase from Kutneria albida (KalbTG) at one or more positions selected from positions 110 (LC110), 143 (LC143), and 214 (LC214) of the light chain and positions 118 (HC118), 177 (HC177), 297 (HC297), 341 (HC341), 401 (HC401), and 446 (HC446) of the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat). Thus, in the modified antibody of the present invention, the recognition site(s) for KalbTG are located within the constant region of the Ig heavy chain polypeptide, i.e., not at either end and / or within or at the C-terminus of the Ig light chain constant domain. Furthermore, the modified antibodies of the present invention may contain an additional first recognition site for KalbTG at position 446 (HC446) of the heavy chain, i.e., the C-terminus. By inserting a recognition site for KalbTG at a position, it is meant that the amino acid present at that position in the unmodified sequence is replaced by the recognition site, or that a recognition site is preferably further inserted after that position.

[0075] In certain embodiments, the one or more positions are selected from the group of positions comprising position 214 of the light chain (LC214), and positions 118 (HC118), 177 (HC177), 297 (HC297), and 341 (HC341) of the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat).

[0076] In a preferred embodiment, the one or more positions are selected from the group of positions comprising position 214 of the light chain (LC214), and positions 341 (HC341), 297 (HC297), and 177 (HC177) of the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat).

[0077] In certain embodiments, modified antibodies according to the present invention comprise two identical heavy chains or heavy chain Fc regions. Such modified antibodies may comprise two, four, or more (primary) recognition sites for transglutaminase from Kutneria albida (KalbTG) at one or more positions selected from positions 110 (LC110), 143 (LC143), and 214 (LC214) in the light chain and positions 118 (HC118), 177 (HC177), 297 (HC297), 341 (HC341), 401 (HC401), and 446 (HC446) in the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat).

[0078] In certain embodiments, modified antibodies according to the present invention comprise two different heavy chains, whereby the differences result from mutations in each to induce heterodimerization. Such modified antibodies may comprise two or more (primary) recognition sites for transglutaminase from Kutneria albida (KalbTG) at one or more identical or different positions selected from positions 110 (LC110), 143 (LC143), and 214 (LC214) in the light chain and positions 118 (HC118), 177 (HC177), 297 (HC297), 341 (HC341), 401 (HC401), and 446 (HC446) in the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat).

[0079] Similarly, the present invention relates to modified antibody Fc regions comprising a heavy chain Fc region, the heavy chain Fc region comprising one or more (primary) recognition site(s) for transglutaminase from Kutneria albida (KalbTG) at one or more positions selected from the group consisting of positions 118 (HC118), 177 (HC177), 297 (HC297), 341 (HC341), 401 (HC401), and 446 (HC446) of the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat). Thus, in the modified antibody Fc regions of the present invention, the recognition site for KalbTG is located within the constant region of the Ig heavy chain Fc region polypeptide, i.e., not at any of the termini. Furthermore, the modified antibody Fc regions of the present invention may comprise an additional primary recognition site for KalbTG at position 446 (HC446) of the heavy chain, i.e., at the C-terminus. In reference to inserting a recognition site for KalbTG at a position, it means that the amino acid present at that position in the unmodified sequence is replaced by the recognition site, or that the recognition site is preferably further inserted after that position.

[0080] In certain embodiments, the one or more positions are selected from the group of positions comprising positions 118 (HC118), 177 (HC177), 297 (HC297), and 341 (HC341) of the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat).

[0081] In a preferred embodiment, the one or more positions are selected from the group of positions comprising positions 341 (HC341), 297 (HC297) and 177 (HC177) of the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat).

[0082] The terms "antibody" and "Ig" are used interchangeably herein. They are used in the broadest sense and include, for example, monoclonal antibodies regardless of binding specificity (including agonist, antagonist, neutralizing, full-length or intact monoclonal antibodies), monovalent antibodies (e.g., full-length antibodies lacking one Fab), multivalent antibodies, i.e., antibodies that are bivalent or tetravalent multispecific antibodies, and fragments of full-length antibodies, so long as they contain at least one of the modifications outlined above.

[0083] Naturally occurring antibodies are produced by the assembly of heavy chains alone or heavy and light chains. Each heavy chain consists of four domains: a variable domain (VH) and three constant domains (CH1, CH2, and CH3). Light chains consist of a variable domain (VL) and a constant domain (CL). When heavy and light chains are present, the light chain pairs with a cognate heavy chain Fab fragment, which contains the VH and CH1 domains. The Fab fragments of related light and heavy chains are collectively referred to as a Fab fragment. The heavy chain CH2 and CH3 domains, together referred to as the heavy chain Fc region, dimerize with additional heavy chain CH2 and CH3 domains from a second chain to form an Fc region. The Fc region is connected to the Fab fragment via a flexible hinge region. The hinge region contains several disulfide bridges that covalently link the two heavy chain Fc regions to each other. In the Fab fragment, the light chain and heavy chain Fab fragment are also connected by a single disulfide bridge. However, the connectivity varies between IgG subclasses. The overall structure of full-length IgG resembles a Y-shape, with the Fc region forming the base and the two Fab fragments forming the arms, available for binding to antigen.

[0084] Within the variable domain are loops called complementarity-determining regions (CDRs). These are primarily responsible for the direct interaction of the antibody with its antigen. Due to the significant variation in the number of amino acids in these CDRs, there are multiple numbering schemes for variable regions. As used herein, the amino acid positions of all heavy and light chain constant regions and domains are numbered according to the Kabat numbering system described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), and are referred to herein as "numbering according to Kabat." Specifically, the Kabat numbering system of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see pages 647-660) is used for the light chain constant domains CL of kappa and lambda isotypes, and the Kabat EU index numbering system (see pages 661-723) is used for the heavy chain constant domains (CH1, hinge, CH2, and CH3, which is further clarified herein by saying "numbering is according to the Kabat EU index" in this case).

[0085] As used herein, the term "modified antibody" refers to an antibody or antibody Fc region according to the present invention that contains at least one (artificial) internal Q-tag (at a desired site). Modified antibodies include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinant antibodies, multispecific antibodies (including bispecific antibodies), humanized antibodies, camelized antibodies, chimeric antibodies, intrabodies, anti-idiotypic (anti-id) antibodies, and functional fragments thereof. The term "functional fragment" refers to a portion of an intact antibody that retains some or all of the binding activity of the antibody from which the fragment is derived. Non-limiting examples of functional antibody fragments include Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, etc. In particular, modified antibodies according to the present invention include antibody molecules and immunologically active portions of antibody molecules, such as molecules containing antigen-binding domains or antigen-binding sites (e.g., one or more complementarity-determining regions (CDRs)), so long as modifications according to the present invention are present. The modified antibodies provided herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY, and in certain embodiments, IgG), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, and in one preferred embodiment, IgG1), or any subclass (e.g., IgG2a and IgG2b). Antibodies can be humanized, chimeric, and / or affinity matured, as well as antibodies from other species, such as mouse, rabbit, and sheep.

[0086] According to the present invention, in certain embodiments, the modified antibody comprises at least one heavy chain and at least one light chain. Thus, in certain embodiments, the modified antibody may comprise one, two, three, or four Fab fragments. In certain embodiments, the modified antibody is a monovalent, monospecific antibody comprising one (full-length) light chain and one (full-length) heavy chain forming a cognate light chain-heavy chain pair (comprising one binding site), and one heavy chain Fc region fragment comprising the hinge region associated with the Fc region of the (full-length) heavy chain.

[0087] According to the present invention, in certain embodiments, the modified antibody may be based on an IgG1, IgG2, IgG3, or IgG4 antibody, in particular a humanized, mouse, rabbit, or sheep antibody. Exemplary and suitable sequences are given below, with X ↓ indicates the insertion site of the Q-tag motif.

[0088] Human heavy chain constant region of IgG1 subclass (G1m1, 17 Caucasian allotypes): (SEQ ID NO: 1) TIFF2025525560000009.tif43142

[0089] Human heavy chain constant region of IgG1 subclass (G1m17-African American allotype): (SEQ ID NO: 2) TIFF2025525560000010.tif43142

[0090] Human heavy chain constant region of the IgG2 subclass: (SEQ ID NO: 3) TIFF2025525560000011.tif44142

[0091] Human heavy chain constant region of the IgG3 subclass: (SEQ ID NO: 4) TIFF2025525560000012.tif50142

[0092] Human heavy chain constant region of IgG4 subclass: (SEQ ID NO: 5) TIFF2025525560000013.tif43142

[0093] Thus, in some embodiments, the heavy chain constant region is based on a human Ig heavy chain constant region, such as a human IgG1, human IgG2, human IgG3, or human IgG4 heavy chain constant region. In certain embodiments, the heavy chain constant region comprises at least one Q-tag according to the invention.

[0094] In certain embodiments, human IgG1 heavy chain polypeptides on which modified antibodies according to the present invention may be based comprise a constant region amino acid sequence that is 75% or more, for example, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, and up to 100% identical to the amino acid sequence set forth in SEQ ID NO: 1 or 2. In certain embodiments, human IgG2 heavy chain polypeptides on which modified antibodies according to the present invention may be based comprise a constant region amino acid sequence that is 75% or more, for example, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, and up to 100% identical to the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, human IgG3 heavy chain polypeptides on which modified antibodies according to the present invention may be based comprise a constant region amino acid sequence that is 75% or more, for example, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, and up to 100% identical to the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, human IgG4 heavy chain polypeptides on which modified antibodies according to the invention may be based comprise a constant region amino acid sequence that is 75% or more, such as 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, and up to 100% identical to the amino acid sequence set forth in SEQ ID NO:5.

[0095] In certain embodiments, the modified antibody according to the invention comprises the following additional mutations (numbering according to Kabat): a) L234A, L235A in both Fc region polypeptides; b) P329G in both Fc region polypeptides; c) T366W in one Fc region polypeptide and T366S, L368A, Y407V in the other Fc region polypeptide; d) S354C in one Fc region polypeptide and Y349C in the other Fc region polypeptide; e) a) and b); f) a) and b) and c); or g) a) and b) and c) and d).

[0096] In a preferred embodiment, a modified antibody according to the invention comprises the mutations L234A, L235A, P329G, T366W in a first Fc-region polypeptide and the mutations L234A, L235A, P329G, T366S, L368A, Y407V in a second Fc-region polypeptide. In a specific embodiment, the modified antibody further comprises one of the mutations S354C and Y349C in the first Fc-region polypeptide and the other in the second Fc-region polypeptide.

[0097] In a preferred embodiment, the human heavy chain polypeptide on which the modified antibodies according to the present invention can be based comprises a constant region amino acid sequence that is 75% or more, for example 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, and up to 100% identical to the amino acid sequence set forth in SEQ ID NO:8 or SEQ ID NO:9 or SEQ ID NO:34.

[0098] HC177( SEQ ID NO: 34) TIFF2025525560000014.tif42142 or HC297( SEQ ID NO: 8) TIFF2025525560000015.tif42142 or HC341( SEQ ID NO: 9) TIFF2025525560000016.tif42142

[0099] According to the present invention, in certain embodiments, the modified antibody may be based on an IgG1, IgG2, IgG3 or IgG4 antibody, in particular a humanized antibody further comprising a light chain constant domain. Exemplary suitable sequences are given below, with X ↓ indicates the insertion site of the Q-tag motif.

[0100] Human kappa light chain constant domain ( SEQ ID NO: 6) TIFF2025525560000017.tif18142 Human lambda light chain constant domain ( SEQ ID NO: 7) TIFF2025525560000018.tif18140

[0101] In certain embodiments, the human light chain polypeptide on which the modified antibodies according to the present invention can be based comprises a constant region amino acid sequence that is 75% or more, e.g., 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, and up to 100% identical to the amino acid sequence set forth in SEQ ID NO: 6 or 7.

[0102] In a preferred embodiment, the human light chain polypeptide on which the modified antibody according to the present invention can be based comprises a constant region amino acid sequence that is 75% or more, for example 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, and up to 100% identical to the amino acid sequence set forth in SEQ ID NO: 10.

[0103] LC214( SEQ ID NO: 10) TIFF2025525560000019.tif17141

[0104] Preferably, the unmodified light chain constant domain comprises an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100% identical to the amino acid sequence of any of SEQ ID NOs: 6 or 7, and / or the unmodified heavy chain constant region comprises an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100% identical to the amino acid sequence of SEQ ID NOs: 1 to 5. Also preferably, the light chain constant domain comprises or consists of an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100% identical to the amino acid sequence of SEQ ID NO: 10, and / or the heavy chain constant region comprises or consists of an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100% identical to the amino acid sequence of SEQ ID NO: 8 or 9 or 34.

[0105] As detailed above, modified antibodies according to the present invention may be bispecific or multispecific antibodies. Exemplary embodiments include the following: -Full length antibodies with domain swapping: A multispecific IgG antibody comprising a first Fab fragment and a second Fab fragment, wherein in the first Fab fragment: a) only the CH1 and CL domains are replaced by each other (i.e., the light chain of the first Fab fragment comprises the VL and CH1 domains, and the heavy chain of the first Fab fragment comprises the VH and CL domains); b) only the VH and VL domains are replaced by one another (i.e. the light chain of the first Fab fragment comprises the VH and CL domains and the heavy chain of the first Fab fragment comprises the VL and CH1 domains); or c) the CH1 and CL domains are replaced with each other and the VH and VL domains are replaced with each other (i.e., the light chain of the first Fab fragment comprises the VH and CH1 domains, and the heavy chain of the first Fab fragment comprises the VL and CL domains); and the second Fab fragment comprises a light chain comprising a VL and CL domain and a heavy chain comprising a VH and CH1 domain, and the full-length antibody with domain swapping may comprise a first heavy chain comprising a CH3 domain and a second heavy chain comprising a CH3 domain, both CH3 domains being complementarily engineered by respective amino acid substitutions to support heterodimerization of the first heavy chain and the modified second heavy chain; -Full-length antibody with domain swapping and an additional heavy chain C-terminal binding site: 1. A multispecific IgG antibody, comprising: a) a full-length antibody comprising two pairs of full-length antibody light chains and two pairs of full-length antibody heavy chains, wherein the binding site formed by each pair of full-length heavy chains and full-length light chains specifically binds to a first antigen; b) an additional Fab fragment fused to the C-terminus of one heavy chain of the full-length antibody, the binding site of the additional Fab fragment specifically binding to a second antigen; Including, a multispecific IgG antibody, wherein the additional Fab fragment that specifically binds to a second antigen i) comprises a domain crossover such that a) the light chain variable domain (VL) and the heavy chain variable domain (VH) are substituted for each other, or b) the light chain constant domain (CL) and the heavy chain constant domain (CH1) are substituted for each other, or ii) is a single-chain Fab fragment; -1-arm single chain format (=1-arm single chain antibody): An antibody comprising a first binding site that specifically binds to a first epitope or antigen and a second binding site that specifically binds to a second epitope or antigen, wherein the individual chains are as follows: -Light chain (variable light domain + constant light kappa domain) -Light / heavy chain combination (variable light domain + light constant domain + peptide linker + variable heavy domain + CH1 + hinge + CH2 + CH3 knob mutations) -Heavy chain (variable heavy domain + CH1 + hinge + CH2 + CH3 hole mutations); -2-arm single chain format (=2-arm single chain antibody): An antibody comprising a first binding site that specifically binds to a first epitope or antigen and a second binding site that specifically binds to a second epitope or antigen, wherein the individual chains are as follows: -Light chain / heavy chain 1 combination (variable light chain domain + light chain constant domain + peptide linker + variable heavy chain domain + CH1 + hinge + CH2 + CH3 hole mutation) - light chain / heavy chain 2 combination (variable light chain domain + light chain constant domain + peptide linker + variable heavy chain domain + CH1 + hinge + CH2 + CH3 knob mutations); -Common light chain bispecific formats (=common light chain bispecific antibodies): An antibody comprising a first binding site that specifically binds to a first epitope or antigen and a second binding site that specifically binds to a second epitope or antigen, wherein the individual chains are as follows: -Light chain (variable light domain + constant light domain) -Heavy chain 1 (variable heavy chain domain + CH1 + hinge + CH2 + CH3 hole mutation) -Heavy chain 2 (variable heavy domain + CH1 + hinge + CH2 + CH3 with knob mutation).

[0106] As used herein, the term "replaced with each other" refers to the above-mentioned domain crossover with respect to corresponding heavy chain domains and light chain domains. Thus, when CH1 and CL domains are "replaced with each other", this term refers to the domain crossover described under item (i) and the resulting heavy chain and light chain domain sequences. Thus, when VH and VL are "replaced with each other", this term refers to the domain crossover described under item (ii); and when CH1 and CL domains are "replaced with each other" and VH and VL domains are "replaced with each other", this term refers to the domain crossover described under item (iii).

[0107] In certain embodiments, a multispecific antibody also comprises at least one Fab fragment comprising either the domain crossover of the CH1 and CL domains described in item (i) above, or the domain crossover of the VH and VL domains described in item (ii) above, or the domain crossover of the VH-CH1 and VL-VL domains described in item (iii) above. In the case of a multispecific antibody with domain crossover, Fabs that specifically bind to the same antigen(s) are constructed to have the same domain sequence. Therefore, when more than one Fab with domain crossover is included in a multispecific antibody, the Fab(s) specifically bind to the same antigen.

[0108] The term "antigen" refers to a predetermined target to which an antibody can selectively bind. An antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten or fragment thereof, or other naturally occurring or synthetic compound. In certain embodiments, the antigen is a polypeptide. In other embodiments, the antigen is therapeutically relevant. In yet other embodiments, the antigen is specific to or allows delivery to a particular region in the body, such as a particular organ or cell type or diseased region. It can be a receptor or a specific structure on the surface of a cell, such as a tumor marker. In a preferred embodiment, the antigen is the human transferrin receptor.

[0109] Preferably, (i) the antibody comprises two pairs of heavy and / or light chains, each of which comprises one or more first recognition sites, or (ii) the antibody comprises two pairs of heavy and / or light chains, each of which comprises one or more first recognition sites, or (iii) the antibody comprises one pair of heavy and light chains and one additional heavy chain Fc region, each of which comprises one or more first recognition sites.

[0110] According to the present invention, modified antibodies contain one or more recognition sites for KalbTG in the heavy and / or light chain polypeptides. The recognition sites include a KalbTG motif, which can catalyze the formation of an isopeptide bond between the modified antibody and a compound (payload) coupled to the modified antibody. Typically, the isopeptide bond is formed between a glutamine (Gln) side chain and a lysine (Lys) side chain. Preferably, the modification introduced into the antibody to obtain the modified antibody of the present invention includes the creation of an (artificial) Q-tag in the antibody heavy and / or light chain polypeptide, i.e., a Gln-containing motif recognized by KalbTG. Suitable Q-tags are disclosed in WO 2017 / 102759 A1, which is expressly incorporated herein by reference. The one or more first recognition sites, independently of one another, comprise or have a Gln-containing motif, particularly the sequence RYGQR (SEQ ID NO: 11), RWRQR (SEQ ID NO: 12), YRQRT (SEQ ID NO: 13), IRQRQ (SEQ ID NO: 14), FRYRQ (SEQ ID NO: 15), or YRYRQ (SEQ ID NO: 17), in particular YRYRQ (SEQ ID NO: 17). Q-tags can be generated by one or more amino acid modifications, such as substitutions or insertions, preferably insertions. More preferably, Q-tags are generated by insertion and / or substitution of an amino acid sequence comprising YRYRQ (SEQ ID NO: 17) or RVRQR (SEQ ID NO: 18), in particular YRYRQ (SEQ ID NO: 17). Preferably, the inserted amino acid sequence has a length of 5 to 20 amino acids and comprises YRYRQ (SEQ ID NO: 17) or RVRQR (SEQ ID NO: 18), in particular YRYRQ (SEQ ID NO: 17). In certain embodiments, the insertion is a Q-tag motif without a spacer, i.e., the insertion consists of YRYRQ (SEQ ID NO: 17) or RVRQR (SEQ ID NO: 18), particularly YRYRQ (SEQ ID NO: 17). The introduction of one or more recognition sites for KalbTG may be the only modification in the light and / or heavy chain constant regions. Alternatively, additional modifications, such as a tag for purification (e.g., a His tag) or other modifications, such as those for increasing stability or heterodimerization or modifying effector function, may be present alone or in any combination.

[0111] As described above, one or more first recognition site(s) for KalbTG are inserted into specific sites in the antibody, namely, at one or more positions selected from positions 110 (LC110), 143 (LC143), and 214 (LC214) of the light chain and positions 118 (HC118), 177 (HC177), 297 (HC297), 341 (HC341), 401 (HC401), and 446 (HC446) of the heavy chain, in particular HC177 and / or HC297 and / or LC214. In certain embodiments, the modified antibodies of the invention may comprise an additional recognition site for KalbTG at position 446 (HC446) of the heavy chain, i.e., at the C-terminus, in particular for coupling to a domain different from that coupled by the first recognition site. Preferably, one or more of the recognition sites are independent of each other at positions 177 (HC177) or 297 (HC297) of the heavy chain or at position 214 (LC214) of the light chain.

[0112] In certain embodiments, the Q-tag is inserted into the antibody light / heavy chain amino acid sequence via one or two spacers. The spacers may increase flexibility or allow conjugation with larger payloads. In certain embodiments, each spacer sequence, independently of the others, contains 1 to 20 amino acids, preferably 1 to 10 amino acids, and more preferably 1 to 5 amino acids. More preferably, the spacers do not substantially interfere with the function of the Q-tag, KalbTG, antibody folding, or the payload attached to the modified antibody. The spacers may be attached to the N-terminus and / or C-terminus of the Q-tag. In certain embodiments, the spacer amino acid is a small amino acid such as glycine or serine. Amino acid linkers and their compositions are known in the art (see, for example, Chichili et al., Prot. Sci. 22 (2013) 153-167). The amino acids glycine, serine, alanine, threonine, and glutamic acid typically constitute the amino acids of flexible linkers. Thus, the linker(s) may consist mainly or entirely of Gly and / or Ser and / or Ala and / or Thr and / or Glu, e.g., GGGP (SEQ ID NO: 20), ESGS (SEQ ID NO: 21) or APAP (SEQ ID NO: 22). Also, spacers may be present which comprise or consist of KESGSVSSEQLAQFRSLD (SEQ ID NO: 23) or EGKSSGSGSESKST (SEQ ID NO: 24). In a preferred embodiment, the spacers, independently of each other, consist mainly or entirely of Gly and Ser, e.g., (Gly m Ser) n wherein m=1, 2, 3, or 4 and n=1, 2, 3, 4, or 5, and m and n are independently preferably m=3 and n=1. Preferably, one or more first recognition sites are inserted into the heavy chain and / or light chain amino acid sequence via one or two spacers at their ends, and in particular, the spacers are mainly or entirely composed of Gly and Ser, for example (Gly-Gly-Gly-Ser). n (SEQ ID NO: 19) where n=1, 2, 3, 4 or 5, preferably n=1.

[0113] When a spacer is present, it is preferred to insert the amino acid sequence X1-YRYRQ-X2 (SEQ ID NO: 17) or X1-RVRQR-X2 (SEQ ID NO: 18) into the antibody. X1 and X2 are, independently of each other, absent or a spacer, particularly a spacer amino acid. In a specific embodiment, the insertion is a Q-tag motif with two flexible spacers, particularly GGGSYRYRQGGGS (SEQ ID NO: 25) or GGGSRVRQRGGGS (SEQ ID NO: 26), particularly GGGSYRYRQGGGS (SEQ ID NO: 25).

[0114] Exemplary Fc regions comprising portions of the hinge region with (referred to as SEQ ID NO: 32, 113) and without (referred to as SEQ ID NO: 33, 110) the first recognition site (bold) have the following sequences: TIFF2025525560000020.tif87128

[0115] In one aspect, the invention relates to one or more nucleic acids encoding a chain of a modified antibody according to the invention.

[0116] Nucleic acids encoding modified antibodies of the invention can be isolated or generated in vitro for recombinant production of the antibody. The nucleic acid can be inserted into a replicable vector for further cloning (amplification of the DNA) or for further expression.

[0117] The term "nucleic acid" encompasses DNA (gDNA and cDNA) and RNA molecules, and nucleotides, the basic structural units of nucleic acids, include not only naturally occurring nucleotides but also analogs with modified sugars or base moieties. The nucleic acid sequences encoding the heavy chain variable regions and light chain variable regions of the present invention may be modified. Such modifications include the addition, deletion, or non-conservative or conservative substitution of nucleotides, as long as the encoded sequence remains unchanged.

[0118] DNA encoding modified antibodies according to the invention can be isolated or synthesized using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to DNA encoding the heavy and light chains of the antibody).

[0119] Many transfer and expression vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0120] As used herein, the term "vector" refers to a means for expressing a gene of interest in a host cell, such as a plasmid vector, a cosmid vector, or a viral vector, for example, a bacteriophage vector, an adenovirus vector, a retrovirus vector, or an adeno-associated virus vector. The nucleic acid encoding the modified antibody in the vector is operably linked to a promoter and a polyadenylation signal sequence.

[0121] "Operably linked" refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence, whereby the control sequence controls the transcription and / or translation of the other nucleic acid.

[0122] When a eukaryotic cell is used as the host, a promoter derived from the genome of a mammalian cell (e.g., metallotinein promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) may be used, or a mammalian promoter derived from an animal virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and murine sarcoma virus (RSV) promoter) may be used. In addition, a polyadenylation signal sequence is present after the coding nucleic acid as a transcription termination sequence.

[0123] Cells can be transformed with the aforementioned vectors. The cells used to produce the antibodies of the present invention can be, but are not limited to, prokaryotic cells, yeast cells, or higher eukaryotic cells.

[0124] However, of most interest are animal cells, and examples of useful host cell lines include, but are not limited to, COS-7, BHK, CHO, CHO-S, CHO-K1, GS-CHO, CHO DXB-11, CHO DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080. In a preferred embodiment, the host cells are CHO cells.

[0125] In one aspect, the invention relates to a covalent conjugate comprising (i) a modified antibody according to the invention and (ii) one or more non-antibody (payload) domain(s) covalently conjugated to one or more (first) recognition site(s) for KalbTG or a functionally active variant thereof, wherein the non-antibody domain(s) comprise a second recognition site for KalbTG. In certain embodiments, the non-antibody domain is a therapeutic moiety comprising a therapeutic entity and, optionally, a second linker.

[0126] As described above, the modified antibodies of the present invention are provided with the aid of KalbTG to specifically conjugate one or more payloads, such as therapeutic moieties, to one or more internal sites of the antibody. This results in an ADC that can be used for targeted therapy. The conjugate can bind to the target of interest, thereby transporting the payload, such as therapeutic moiety, to the intended tissue or organ in the body. In a preferred embodiment, the modified antibody recognizes and binds to the target through its complementarity-determining region (CDR), and the target is particularly a biomolecule present on a cell.

[0127] As detailed above, it may be desirable to target a therapeutic moiety to a specific tissue or organ in a patient's body. This can improve in vivo distribution and reduce adverse side effects. Clearly, it may be intended to deliver a therapeutic moiety to tissues or organs that are otherwise difficult to reach. As an example, it may be envisioned to guide a therapeutic moiety into the brain. Due to the blood-brain barrier, it is difficult to deliver a "naked" therapeutic moiety to the brain unless it is administered directly, especially if the therapeutic moiety exceeds a certain size limit. A therapeutic approach that overcomes the blood-brain barrier and helps transport a therapeutic moiety into the brain would clearly be advantageous. Notably, the same approach can be used to guide a therapeutic agent to another region in the body.

[0128] In the present invention, the molecular recognition units of antibodies that specifically bind to structures within the body are used to target therapeutic moieties. In view of the above, it is clear that a therapeutic entity can be any compound useful in the treatment or prevention of a disease of interest, particularly a compound that is delivered to a specific tissue within the body, such as a specific organ or cell type or diseased area.

[0129] The terms "treat," "treating," and "treatment" are meant to include alleviating or arresting a condition, disorder, or disease, or one or more symptoms associated with a condition, disorder, or disease, or alleviating or eradicating the cause(s) of the condition, disorder, or disease itself. The terms "prevent," "preventing," and "prevention" are meant to include methods of delaying and / or eliminating the onset of a condition, disorder, or disease, and / or its attendant symptoms; methods of barring a subject from acquiring a condition, disorder, or disease; or methods of reducing a subject's risk of acquiring a condition, disorder, or disease.

[0130] For this purpose, a non-antibody payload containing a therapeutic entity and optionally a second linker is covalently conjugated to the modified antibody of the present invention. The therapeutic moiety includes an active therapeutic entity or a prodrug. Optionally, a second linker may be present. The second linker may be, for example, a chemical linker containing an alkyl group or a polyethylene group, or a peptide linker.

[0131] In a preferred embodiment, the therapeutic entity is a nucleic acid such as an RNA, siRNA or ASO (antisense oligonucleotide), in particular an ASO comprising LNA nucleotides, and / or the therapeutic entity is a toxin or a small organic molecule or an immunomodulator.

[0132] In a preferred embodiment, the therapeutic entity is a nucleic acid. The nucleic acid can be, for example, DNA or RNA, or a mixture thereof. The term RNA also includes antisense RNA as well as small interfering RNA (siRNA), a class of double-stranded, non-coding RNA molecules, typically 20-24 base pairs long, that are similar to miRNAs and operate within the RNA interference (RNAi) pathway. They disrupt the expression of specific genes with complementary nucleotide sequences by post-transcriptionally degrading mRNA, thereby preventing translation. The nucleic acid may also contain one or more locked nucleic acids (LNAs), which are modified RNA nucleotides in which the ribose moiety is modified with an extra bridge connecting the 2' oxygen and 4' carbon. The bridge "locks" the ribose in the 3'-endo (north) conformation. This structure may be due to increased stability against enzymatic degradation, and furthermore, the LNA structure has improved specificity and affinity as a monomer or building block of an oligonucleotide. LNA nucleotides can be mixed with DNA or RNA residues in an oligonucleotide or nucleic acid.

[0133] Additionally or alternatively, the therapeutic entity can be a small molecule. In the field of pharmacology, a small molecule has a low molecular weight (<2,500 daltons, particularly <1,000 daltons). Many small molecule therapeutic entities are small organic molecules. Small organic molecules typically bind to specific biological macromolecules and act as effectors, altering the activity or function of the target. These compounds can be natural (such as primary and secondary metabolites) or artificial (i.e., not naturally occurring), and they have beneficial effects against diseases.

[0134] In the present invention, the therapeutic entity is contained in a non-antibody domain, which is covalently coupled to the modified antibody of the present invention, optionally via a second linker. The length, rigidity, and chemical composition of the linker can affect the conjugation kinetics and the stability of the resulting conjugate, so the linker may depend on the intended target and therapeutic entity. In a preferred embodiment, the linker is an alkyl linker, a polyethylene linker, a peptide linker, or a mixture thereof. In certain embodiments, the linker comprises ethylene glycol (PEG) units, for example, about 2 to 50 ethylene glycol units. Exemplary linkers include a polyethylene glycol linker ((-NH-C(=O)-PEG n In another embodiment, the linker is an aliphatic carbon chain. The linker may be an unsubstituted or substituted C 1~6 alkyl, and may include unsubstituted or substituted alkyl, such as C 1~6 The alkyl may be substituted with one or more substituents selected from the group consisting of alkoxy, acyl, acyloxy, alkoxycarbonyl, carbonylalkoxy, acylamino, amino, aminoacyl, aminocarbonylamino, aminocarbonyloxy, cycloalkyl, cycloalkenyl, cyano, azido, halo, hydroxyl, nitro, carboxyl, thiol, thioalkyl, alkyl, alkenyl, alkynyl, heterocyclyl, aminosulfonyl, sulfonylamino, sulfonyl, and oxo. In yet another embodiment, the linker is a peptide linker, i.e., a linker composed of amino acids.

[0135] In a preferred embodiment, the conjugate comprises a modified antibody according to the invention and one or more therapeutic nucleic acids, such as ASOs, each of which is linked to a single Q-tag via an amide bond to the terminal residue of the Q-tag via a second linker, in particular a PEG linker, as defined above.

[0136] In certain embodiments, the conjugate has a DAR ranging from about 1 to about 8, from about 1 to about 4, or from about 1 to about 2. In other embodiments, the conjugate has a DAR of about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8.

[0137] Once the antibody binds to the target, the conjugate is transported into the respective cell by endocytosis and the therapeutic entity is released and can act in the intended manner (e.g., treat a disease).

[0138] In a preferred embodiment, the modified antibody recognizes one target, which is a receptor that induces receptor-mediated endocytosis, such as human transferrin receptor 1 (TfR1), human insulin-like growth factor 1 receptor (IGF-1R), human low-density lipoprotein receptor-related protein 1 (LRP1) or human low-density lipoprotein receptor-related protein 8 (LRP8), in particular TfR1.

[0139] For example, in the case of specific receptors such as TfR1 or IGF-1R, when the antibody binds to the target, the conjugate is transported into the respective cell by endocytosis, released from the endosome, and exocytosed again from the cell. If the cell is part of a barrier such as the blood-brain barrier, transport across the respective barrier is thereby achieved. The therapeutic entity is thereby transported to body compartments that could not be reached by the therapeutic entity not conjugated to the modified antibody according to the present invention.

[0140] In one highly preferred conjugate, the modified antibody can be conjugated to a structure that allows it to cross the blood-brain barrier, such as the transferrin receptor.

[0141] In a preferred embodiment, the modified antibody recognizes one or two targets, which are specific to a particular cell type, such as a tumor marker specific to tumor cells (e.g., breast cancer cells).

[0142] In a preferred embodiment, the modified antibody according to the invention is conjugated to a therapeutic entity comprising an RNA or LNA or ASO or siRNA, and optionally a non-antibody domain comprising a PEG linker, for treating or preventing a brain disease, such as Parkinson's disease or Alzheimer's disease.

[0143] In one aspect, the present invention relates to a method of covalently conjugating a modified antibody according to the present invention to a therapeutic entity, the method comprising: a) providing a modified antibody according to the invention; b) providing a non-antibody domain comprising: (i) a therapeutic entity; (ii) a second recognition site for KalbTG, in particular the second recognition site comprising or having a Lys-containing motif, in particular the sequence RYESK (SEQ ID NO: 16); and (iii) optionally a second linker between the therapeutic entity and the second recognition site; c) reacting the modified antibody of a) and the non-antibody domain of b) in the presence of KalbTG or a functionally active variant thereof and under conditions that promote the activity of KalbTG, thereby forming an isopeptide bond between the first recognition site and the second recognition site, and thus conjugating the modified antibody to a therapeutic entity; Includes.

[0144] In the first step of the method, a modified antibody and non-antibody domain of the present invention are provided. The non-antibody domain comprises (i) a therapeutic entity; (ii) a second recognition site for KalbTG, particularly a second recognition site comprising or having a Lys-containing motif, particularly the sequence RYESK (SEQ ID NO: 16) (K-tag); and (iii) optionally, a second linker between the therapeutic entity and the second recognition site. The domains may be as defined above. The modified antibody and non-antibody domain are reacted in the presence of KalbTG or a functionally active variant thereof under conditions that promote the activity of KalbTG, thereby forming an isopeptide bond between the first and second recognition sites, thus conjugating the modified antibody to the therapeutic entity. In certain embodiments, in this method according to the present invention, the antibody comprises one or more Q-tags that are conjugated using the activity of KalbTG. The non-antibody domain also comprises a second recognition site for KalbTG. In certain embodiments, the non-antibody domain comprises a K-tag having at least 80% sequence identity to the peptide sequence RYESK (SEQ ID NO: 16).

[0145] Microbial transglutaminases (mTG), including KalbTG, catalyze the formation of Gln-Lys isopeptide bonds and are widely used for cross-linking proteins and peptides in food and biotechnology applications (e.g., to improve the texture of protein-rich foods or to generate antibody-drug conjugates). KalbTG exhibits essentially no cross-reactivity with known mTG substrates or commonly used target proteins, such as antibodies, thus enabling specific conjugation at predetermined sites. Thus, essentially any payload containing a second recognition site for KalbTG (K-tag), particularly one in which the second recognition site contains or has a Lys-containing motif, particularly the sequence RYESK (SEQ ID NO: 16), can be coupled to a modified antibody at one or more first recognition sites. KalbTG or a functionally active variant thereof can be as defined in Steffen et al. (2017) or WO 2016 / 100735 A1 (both of which are expressly incorporated herein by reference). The functionally active variant can be a transglutaminase having at least 80%, 90%, 95%, or 99% sequence identity to KalbTG of WO 2016 / 100735 A1 (see SEQ ID NO: 6). Alternatively, KalbTG or a functionally active variant thereof can be part of a fusion protein that additionally includes a label, such as a tag, for example, for purification purposes.

[0146] In certain embodiments, KalbTG has the amino acid sequence Includes TIFF2025525560000021.tif125142.

[0147] In certain embodiments, in the methods of the present invention, the coupling is controlled, e.g., achieved at a stoichiometric ratio of non-antibody domain to modified antibody, e.g., about 1: 1. Multiple conjugation can also be achieved by using two or more first recognition sites on one modified antibody to attach two or even multiple payloads to the modified antibody.

[0148] The following table provides an overview of the linkers (1376, 1018, 1185, and 1181) tested in a two-step process of first enzymatic conjugation of a K-amino acid sequence-linker-conjugate (7456) to an antibody with a Q-amino acid sequence, followed by conjugation of an ASO to the antibody-K-amino acid sequence-linker conjugate to yield a polypeptide-linker-nucleic acid conjugate (R3 = oligonucleotide). The resulting conjugated linker moieties are designated as 7267 (based on 1018), 7268 (based on 1376), and 7371 (based on 1181). TIFF2025525560000022.tif220141

[0149] The results of the two-step process are shown in the table below. The target DAR is 1 because the antibody contains only a single Q-amino acid sequence. TIFF2025525560000023.tif50128 * High amounts of unwanted DAR3 species (three ASO linkers attached per mAb)

[0150] Therefore, linkers 1018 and 1376 are the best linkers in terms of conjugation efficiency and conjugate quality.

[0151] Additionally, the serum stability of the conjugate was tested and the results are shown in the table below. TIFF2025525560000024.tif41142n.dect.=Not detected

[0152] The conjugate with linker 1185 shows a higher degree of endonuclease cleavage, while the conjugate with linker 1018 is found to be the most stable.

[0153] One aspect of the present invention is a polypeptide-linker-nucleic acid conjugate comprising: -The linker 3-aminopropanamide units, 2,6-diaminohexanoic acid amide units, 1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d]-1,2,3-triazole units and Including, The polypeptide is Lysine residue (K-amino acid sequence) Including, - the nucleic acid is an oxygen atom linked at the 5' or 3' end to a fluorophore in oxidation state V (this term is synonymous with a phosphorus atom in oxidation state V) Including, the 3-amino group of the 3-aminopropanamide unit and the carboxyl functional group of the lysine residue of the polypeptide are linked by / form an amide bond; the carboxy functional group of the 3-aminopropanamide unit and the alpha amino group of the 2,6-diaminohexanoic acid amide unit are linked by / form an amide bond; the 6-amino group of the 2,6-diaminohexanoic acid amide unit is the nitrogen of the 1,2,3-triazole element of the 1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d]-1,2,3-triazole unit; The oxygen linked to the nucleic acid fluorophore (the term is synonymous with the phosphorus atom) is covalently linked to the cyclopropane element of the 1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d]-1,2,3-triazole unit A polypeptide-linker-nucleic acid conjugate, characterized in that:

[0154] One aspect of the present invention is a polypeptide-linker-nucleic acid conjugate comprising: -The linker 1,2-diaminoethyl units, 3-aminopropionic acid units, 1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d]-1,2,3-triazole units and Including, The polypeptide is Lysine residue (K-amino acid sequence) Including, - the nucleic acid is Oxygen linked at the 5' or 3' end to a fluorophore in oxidation state V (phosphorus atom in oxidation state V) Including, the 2-amino group of the 1,2-diaminoethyl unit and the carboxyl functional group of the lysine residue of the polypeptide are linked by / form an amide bond, The 1-amino group of the 1,2-diaminoethyl unit and the carboxy group of the 3-aminopropionic acid unit are linked by / form an amide bond, the 3-amino group of the 3-aminopropionic acid unit is the nitrogen of the 1,2,3-triazole element of the 1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d]-1,2,3-triazole unit; The oxygen linked to the nucleic acid fluorophore (phosphorus atom) is covalently linked to the cyclopropane element of the 1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d]-1,2,3-triazole unit. The present invention relates to a polypeptide-linker-nucleic acid conjugate, characterized in that

[0155] In a particular embodiment of all aspects and embodiments of the present invention, the 6-amino group of the 2,6-diaminohexanoic acid amide unit or the 3-amino group of the 3-aminopropionic acid unit is the nitrogen in position 1 (numbering according to Figure 3) of the 1,2,3-triazole element of the 1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d]-1,2,3-triazole unit.

[0156] In certain embodiments of all aspects and embodiments of the present invention, the amide group of the 2,6-diaminohexanoic acid amide unit is an NHR(1) or NR(1)R(2) group, where R(1) and R(2) are independently selected from the group consisting of lower alkyl and oxyalkyl, including at least methyl, ethyl, propyl, butyl, pentyl, hexyl, oxymethyl, oxyethyl, oxypropyl, oxybutyl, oxypentyl, and oxyhexyl.

[0157] In certain embodiments of all aspects and embodiments of the present invention, the oxygen linked to the fluorophore (i.e., the phosphorus atom) of the nucleic acid is covalently linked to the carbon at the 6-position (numbering according to Figure 3) of the cyclopropane element of the 1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d]-1,2,3-triazole unit.

[0158] In certain embodiments of all aspects and embodiments of the present invention, the oxygen linked to the fluorophore (i.e., the phosphorus atom) of the nucleic acid is covalently linked by a methyl or ethyl unit to the carbon at the 6-position (numbering according to Figure 3) of the cyclopropane element of the 1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d]-1,2,3-triazole unit.

[0159] In certain embodiments of all aspects and embodiments of the present invention, oxygen is linked to a fluorophore in oxidation state V (phosphorus atom in oxidation state V) at the 5' end of the nucleic acid.

[0160] In certain embodiments of all aspects and embodiments of the present invention, the oxygen linked to the fluorophore in oxidation state V (phosphorus atom in oxidation state V) is phosphate or phosphorothioate.

[0161] In certain embodiments of all aspects and embodiments of the invention, the polypeptide comprises a K-amino acid sequence RYESK in which the 3-amino group of the 3-aminopropanamide unit or the 2-amino group of the 1,2-diaminoethyl unit is linked by / forms an amide bond to the carboxy function of the lysine residue.

[0162] In certain embodiments of all aspects and embodiments of the invention, the polypeptide comprises a K-amino acid sequence RYESK in which the 3-amino group of the 3-aminopropanamide unit or the 2-amino group of the 1,2-diaminoethyl unit is linked by / forms an amide bond to the carboxyl functional group of a lysine residue, and the epsilon-amino group of the lysine is linked to the side chain of a glutamine residue by an isopeptide bond.

[0163] In certain embodiments of all aspects and embodiments of the invention, the polypeptide comprises a K-amino acid sequence RYESK in which the 3-amino group of the 3-aminopropanamide unit or the 2-amino group of the 1,2-diaminoethyl unit is linked by / forms an amide bond to the carboxyl functional group of a lysine residue, and the epsilon-amino group of the lysine is linked by an isopeptide bond to the side chain of a glutamine residue, the glutamine residue being within a Q-amino acid sequence of at least 5 amino acid residues.

[0164] In particular embodiments of all aspects and embodiments of the invention, the polypeptide comprises a K-amino acid sequence RYESK in which the 3-amino group of the 3-aminopropanamide unit or the 2-amino group of the 1,2-diaminoethyl unit is linked by / forms an amide bond to the carboxyl functional group of a lysine residue, and the epsilon-amino group of the lysine is linked by an isopeptide bond to the side chain of a glutamine residue, the glutamine residue being within the Q-amino acid sequence RYGQR (SEQ ID NO:11), RWRQR (SEQ ID NO:12), YRQRT (SEQ ID NO:13), IRQRQ (SEQ ID NO:14), FRYRQ (SEQ ID NO:15), or YRYRQ (SEQ ID NO:17), in particular YRYRQ (SEQ ID NO:17).

[0165] In particular embodiments of all aspects and embodiments of the invention, the polypeptide comprises a K-amino acid sequence RYESK in which the 3-amino group of the 3-aminopropanamide unit or the 2-amino group of the 1,2-diaminoethyl unit is linked by / forms an amide bond to the carboxyl functional group of a lysine residue, and the epsilon-amino group of the lysine is linked to the side chain of a glutamine residue by an isopeptide bond, and the glutamine residue is within a Q-amino acid sequence of RYGQR (SEQ ID NO:11), RWRQR (SEQ ID NO:12), YRQRT (SEQ ID NO:13), IRQRQ (SEQ ID NO:14), FRYRQ (SEQ ID NO:15), or YRYRQ (SEQ ID NO:17), in particular YRYRQ (SEQ ID NO:17), and the Q-amino acid sequence comprises one or two spacers at its termini.

[0166] In certain embodiments of all aspects and embodiments of the invention, the polypeptide comprises a K-amino acid sequence RYESK in which the 3-amino group of the 3-aminopropanamide unit or the 2-amino group of the 1,2-diaminoethyl unit is linked by / forms an amide bond to the carboxyl functional group of a lysine residue, and the epsilon-amino group of the lysine is linked to the side chain of a glutamine residue by an isopeptide bond, and the glutamine residue is located within a Q-amino acid sequence of RYGQR (SEQ ID NO:11), RWRQR (SEQ ID NO:12), YRQRT (SEQ ID NO:13), IRQRQ (SEQ ID NO:14), FRYRQ (SEQ ID NO:15), or YRYRQ (SEQ ID NO:17), in particular YRYRQ (SEQ ID NO:17), and the Q-amino acid sequence comprises one or two spacers at its termini, consisting primarily of Gly and Ser.

[0167] In certain preferred embodiments of all aspects and embodiments of the invention, the polypeptide comprises a K-amino acid sequence RYESK in which the 3-amino group of the 3-aminopropanamide unit or the 2-amino group of the 1,2-diaminoethyl unit is linked by / forms an amide bond to the carboxyl functional group of a lysine residue, and the epsilon-amino group of the lysine is linked by an isopeptide bond to the side chain of a glutamine residue, and the glutamine residue is within a Q-amino acid sequence of RYGQR (SEQ ID NO:11), RWRQR (SEQ ID NO:12), YRQRT (SEQ ID NO:13), IRQRQ (SEQ ID NO:14), FRYRQ (SEQ ID NO:15), or YRYRQ (SEQ ID NO:17), in particular YRYRQ (SEQ ID NO:17), and the Q-amino acid sequences are each independently terminated by (Gly-Gly-Gly-Ser) at their termini. n (SEQ ID NO: 19), where n=1, 2, 3, 4 or 5.

[0168] In certain embodiments of all aspects and embodiments of the invention, the polypeptide comprises a K-amino acid sequence RYESK, wherein the 3-amino group of the 3-aminopropanamide unit or the 2-amino group of the 1,2-diaminoethyl unit in the K-amino acid sequence and the carboxy functional group of the lysine residue in the sequence are linked by / form an amide bond, and the epsilon amino group of the lysine is linked to the glutamine residue side chain by an isopeptide bond, and the glutamine residue is selected from the group consisting of RYGQR (SEQ ID NO: 11), RWRQR (SEQ ID NO: 12), YRQRT (SEQ ID NO: 13), and the like. No. 13), IRQRQ (SEQ ID NO: 14), FRYRQ (SEQ ID NO: 15) or YRYRQ (SEQ ID NO: 17), in particular within the Q-amino acid sequence of YRYRQ (SEQ ID NO: 17), wherein the Q-amino acid sequence is at a position selected from positions 110 (LC110), 143 (LC143) and 214 (LC214) of the antibody light chain and positions 118 (HC118), 177 (HC177), 297 (HC297), 341 (HC341), 401 (HC401) and 446 (HC446) of the antibody heavy chain (numbering according to Kabat).

[0169] In certain preferred embodiments of all aspects and embodiments of the invention, the polypeptide comprises a K-amino acid sequence RYESK, wherein the 3-amino group of the 3-aminopropanamide unit or the 2-amino group of the 1,2-diaminoethyl unit in the K-amino acid sequence and the carboxy functional group of the lysine residue in the sequence are linked by / form an amide bond, and the epsilon-amino group of the lysine is linked to the side chain of a glutamine residue by an isopeptide bond, and the glutamine residue is located within the Q-amino acid sequence of RYGQR (SEQ ID NO:11), RWRQR (SEQ ID NO:12), YRQRT (SEQ ID NO:13), IRQRQ (SEQ ID NO:14), FRYRQ (SEQ ID NO:15) or YRYRQ (SEQ ID NO:17), in particular YRYRQ (SEQ ID NO:17), and wherein the Q-amino acid sequence is located at a position selected from position 214 of the antibody light chain (LC214), position 177 of the antibody heavy chain (HC177), and position 297 of the antibody heavy chain (HC297) (numbering according to Kabat).

[0170] In particular embodiments of all aspects and embodiments of the invention, the light chain constant domain that does not comprise the Q amino acid sequence comprises an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100%, identical to the amino acid sequence of any of SEQ ID NOs: 6 or 7, or the heavy chain constant region that does not comprise the Q amino acid sequence comprises an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100% identical to the amino acid sequence of SEQ ID NOs: 1 to 5.

[0171] In particular embodiments of all aspects and embodiments of the invention, the light chain constant domain comprises or consists of an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100% identical to the amino acid sequence of SEQ ID NO: 10, and / or the heavy chain constant region comprises or consists of an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100% identical to the amino acid sequence of SEQ ID NO: 8 or 9 or 34.

[0172] In certain embodiments of all aspects and embodiments of the present invention, the 3-aminopropanamide unit is 3-[2-aminoethoxy]propanamide.

[0173] In certain embodiments of all aspects and embodiments of the present invention, the 3-aminopropanamide unit is 3-[2-(2-aminoethoxy)ethoxy]propanamide.

[0174] In certain preferred embodiments of all aspects and embodiments of the present invention, the 3-aminopropanamide unit is 3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propanamide.

[0175] In certain embodiments of all aspects and embodiments of the present invention, the 3-aminopropanamide unit is 3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]propanamide.

[0176] In certain embodiments of all aspects and embodiments of the present invention, the 3-aminopropanamide unit is 3-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]propanamide.

[0177] In certain embodiments of all aspects and embodiments of the present invention, the 3-aminopropanamide unit is 3-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanamide.

[0178] In certain embodiments of all aspects and embodiments of the present invention, the 3-aminopropionic acid unit is a 4-aminobutanoic acid unit.

[0179] In certain embodiments of all aspects and embodiments of the present invention, the 3-aminopropionic acid unit is a 4-aminopentanoic acid unit.

[0180] In certain embodiments of all aspects and embodiments of the present invention, the 3-aminopropionic acid unit is a 2-aminoethanoic acid unit.

[0181] In certain embodiments of all aspects and embodiments of the present invention, the 2,6-diaminohexanoic acid amide units are 2,5-diaminopentanoic acid units.

[0182] In certain embodiments of all aspects and embodiments of the present invention, the 2,6-diaminohexanoic acid amide unit is a 2,4-diaminobutanoic acid unit.

[0183] In certain embodiments of all aspects and embodiments of the present invention, the 2,6-diaminohexanoic acid amide units are 2,3-diaminopropionic acid units.

[0184] In certain embodiments of all aspects and embodiments of the present invention, the 2,6-diaminohexanoic acid amide units are 2,2-diaminoethanoic acid units.

[0185] In certain embodiments of all aspects and embodiments of the present invention, the conjugate is a covalent conjugate.

[0186] In certain embodiments of all aspects and embodiments of the invention, the linker has the following structure (points of attachment to the nucleic acid and K-amino acid polypeptide are shown with wavy lines): I have TIFF2025525560000025.tif66128.

[0187] In certain preferred embodiments of all aspects and embodiments of the present invention, the linker has the following structure (with points of attachment to the nucleic acid and K-amino acid polypeptide shown as wavy lines): I have TIFF2025525560000026.tif89128.

[0188] In certain embodiments of all aspects and embodiments of the invention, the linker has the following structure (points of attachment to the nucleic acid and K-amino acid polypeptide are shown with wavy lines): I have TIFF2025525560000027.tif71128.

[0189] In certain embodiments of all aspects and embodiments of the invention, the linker has the following structure (points of attachment to the nucleic acid and K-amino acid polypeptide are shown with wavy lines): I have TIFF2025525560000028.tif82128.

[0190] In certain embodiments of all aspects and embodiments of the present invention, the antibody recognizes and binds to a target through its complementarity determining regions (CDRs), and in particular the target is a biomolecule present on a cell.

[0191] In certain embodiments of all aspects and embodiments of the present invention, the nucleic acid is selected from the group consisting of RNA, siRNA, antisense oligonucleotides (ASO), LNA, and ASO comprising LNA nucleotides.

[0192] In particular embodiments of all aspects and embodiments of the present invention: a) the antibody recognizes a target, which is a receptor that induces receptor-mediated endocytosis, such as transferrin receptor protein 1 (TfR1), insulin-like growth factor 1 receptor (IGF-1R), low-density lipoprotein receptor-related protein 1 (LRP1) or low-density lipoprotein receptor-related protein 8 (LRP8), in particular TfR1; and / or b) The antibody recognizes one or two targets, which are specific for a particular cell type, for example, a tumor marker specific for tumor cells, such as breast cancer cells.

[0193] In a preferred embodiment of all aspects and embodiments of the present invention, the polypeptide-linker-nucleic acid conjugate comprises: K - the amino acid sequence RYESK (SEQ ID NO: 16); a Q-amino acid sequence selected from the group of Q-amino acid sequences consisting of RYGQR (SEQ ID NO: 11), RWRQR (SEQ ID NO: 12), YRQRT (SEQ ID NO: 13), IRQRQ (SEQ ID NO: 14), FRYRQ (SEQ ID NO: 15), and YRYRQ (SEQ ID NO: 17), preferably YRYRQ (SEQ ID NO: 17); a nucleic acid having a phosphorothioate at the 5' end; A linker having the following structure: TIFF2025525560000029.tif131128 The oxygen atom (shown as -O in the structure) of the 2-oxyethyl group attached to the cyclopropane element of the 1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d]-1,2,3-triazole unit of the linker is the oxygen atom of the phosphorothioate of the nucleic acid (i.e., directly bonded to the phosphorothioate fluorophore), The 3-amino group of 3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propanamide (shown as -N in the structure) and the carboxyl functional group of the lysine residue in the K-amino acid sequence form an amide bond; the epsilon amino group of a lysine in the K-amino acid sequence is linked by an isopeptide bond to the side chain of a glutamine residue in the Q-amino acid sequence; a linker in which a Q-amino acid sequence is inserted at a position selected from antibody light chain position 214 (LC214), antibody heavy chain position 177 (HC177) and antibody heavy chain position 297 (HC297) (numbering according to Kabat) of each antibody chain, whereby the antibody comprises a binding site that specifically binds to a target, preferably the human transferrin receptor; The present invention is characterized by comprising:

[0194] One aspect of the present invention is a method for producing a polypeptide-linker-nucleic acid conjugate according to the present invention, comprising the steps of: a) providing an antibody comprising a Q-amino acid sequence of RYGQR (SEQ ID NO: 11), RWRQR (SEQ ID NO: 12), YRQRT (SEQ ID NO: 13), IRQRQ (SEQ ID NO: 14), FRYRQ (SEQ ID NO: 15), or YRYRQ (SEQ ID NO: 17), in particular YRYRQ (SEQ ID NO: 17), at one or more positions selected from positions 110 (LC110), 143 (LC143), and 214 (LC214) of the antibody light chain and positions 118 (HC118), 177 (HC177), 297 (HC297), 341 (HC341), 401 (HC401), and 446 (HC446) of the antibody heavy chain (numbering according to Kabat); b) providing a polypeptide comprising the amino acid sequence RYESK (SEQ ID NO: 16) conjugated to the terminal amino group of (3-[2-[2-(2-aminoethoxy)ethoxy]propanamido)-6-azidohexanamide via an amide bond formed between the carboxy group of the C-terminal lysine residue and the terminal amino group of (3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propanamido)-6-azidohexanamide; c) reacting the antibody of a) with the polypeptide of b) in the presence of KalbTG or a functionally active variant thereof under conditions that promote the activity of KalbTG, thereby forming an isopeptide bond between the Q-amino acid sequence of the antibody and the polypeptide containing the amino acid sequence of RYESK to produce (3-[2-[2-(2-antibody aminoethoxy)ethoxy]ethoxy]propanamido)-6-azidohexanamide (antibody azide); d) reacting the antibody azide with O-2-(bicyclo[6.1.0]non-4-yn-9-yl)-ethyl-O'-nucleic acid thiophosphonate, thereby producing a polypeptide-linker-nucleic acid conjugate according to the invention; The method includes:

[0195] In certain embodiments of all aspects and embodiments of the present invention, the reaction product of step c) has the following structure: I have TIFF2025525560000030.tif58133.

[0196] In certain embodiments of all aspects and embodiments of the present invention, the reaction product of step d) has the following structure: I have TIFF2025525560000031.tif76136.

[0197] One aspect of the present invention is a method for producing a polypeptide-linker-nucleic acid conjugate according to the present invention, comprising the steps of: a) providing an antibody comprising a Q-amino acid sequence of RYGQR (SEQ ID NO: 11), RWRQR (SEQ ID NO: 12), YRQRT (SEQ ID NO: 13), IRQRQ (SEQ ID NO: 14), FRYRQ (SEQ ID NO: 15), or YRYRQ (SEQ ID NO: 17), in particular YRYRQ (SEQ ID NO: 17), at one or more positions selected from positions 110 (LC110), 143 (LC143), and 214 (LC214) of the antibody light chain and positions 118 (HC118), 177 (HC177), 297 (HC297), 341 (HC341), 401 (HC401), and 446 (HC446) of the antibody heavy chain (numbering according to Kabat); b) providing a polypeptide comprising the amino acid sequence RYESK (SEQ ID NO: 16) conjugated to the terminal amino group of [2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethyl]propanamido)-6-azidopropanamide via an amide bond formed between the carboxy group of a C-terminal lysine residue and the terminal amino group of [2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethyl]propanamido)-6-azidopropanamide; c) reacting the antibody of a) with the polypeptide of b) in the presence of KalbTG or a functionally active variant thereof under conditions that promote the activity of KalbTG, thereby forming an isopeptide bond between the Q-amino acid sequence of the antibody and the polypeptide comprising the amino acid sequence of RYESK to produce (3-[2-[2-(2-antibody aminoethoxy)ethoxy]ethoxy]propanamido)-6-azidopropanamide (antibody azide); d) reacting the antibody azide with O-2-(bicyclo[6.1.0]non-4-yn-9-yl)-ethyl-O'-nucleic acid thiophosphonate, thereby producing a polypeptide-linker-nucleic acid conjugate according to the invention; The method includes:

[0198] In certain embodiments of all aspects and embodiments of the present invention, the reaction product of step c) has the following structure: I have TIFF2025525560000032.tif57133.

[0199] In certain embodiments of all aspects and embodiments of the present invention, the reaction product of step d) has the following structure: I have TIFF2025525560000033.tif111128.

[0200] One aspect of the present invention is a polypeptide-linker-nucleic acid conjugate according to the present invention, or a polypeptide-linker-nucleic acid conjugate produced according to the method according to the present invention, for use as a medicament.

[0201] One aspect of the present invention is a polypeptide-linker-nucleic acid conjugate according to the present invention, or a polypeptide-linker-nucleic acid conjugate produced according to a method according to the present invention, for use in the treatment of a neurological or brain disease, such as Alzheimer's disease or Parkinson's disease.

[0202] One aspect of the invention is a polypeptide-linker-nucleic acid conjugate according to the invention, or a polypeptide-linker-nucleic acid conjugate produced according to a method according to the invention, for use in the treatment of cancer, such as breast cancer.

[0203] In one aspect, the covalent conjugate comprising the modified antibody of the invention or comprising the linker of the invention or produced according to the method of the invention is for use as a pharmaceutical, particularly for use in the treatment of a neurological or brain disease such as Alzheimer's disease or Parkinson's disease, or for use in the treatment of cancer, such as breast cancer.

[0204] In certain embodiments, the disease is a neurological disease. In certain embodiments, the neurological disease is selected from the group consisting of neuropathy, neurodegenerative disease, cancer, eye disease, seizure disorder, lysosomal storage disease, amyloidosis, viral or microbial disease, ischemia, behavioral disorder, CNS inflammation, Alzheimer's disease, Parkinson's disease, multiple sclerosis, CD20-positive cancer with brain metastasis, and HER2-positive cancer with brain metastasis.

[0205] In certain embodiments, the neurological disease is selected from the group consisting of a neuropathic disorder, a neurodegenerative disease, a cancer, an ocular disorder, a seizure disorder, a lysosomal storage disease, an amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, and a CNS inflammation.

[0206] As detailed above, the modified antibodies according to the present invention or conjugates comprising a linker according to the present invention may be used as medicaments, in particular for use in the treatment of neurological or brain diseases such as Alzheimer's disease or Parkinson's disease, or in the treatment of cancers such as breast cancer.

[0207] Conjugate can be included in composition.This composition, also called pharmaceutical composition, refers to a composition intended for use in the pharmaceutical field or as a medicine, and is in a form that allows the biological activity of the active ingredient contained therein to be effective, and does not contain additional ingredients that are unacceptably toxic to the subject to which the pharmaceutical composition is administered.It can optionally contain pharmaceutically acceptable excipients, diluents or carriers, such as buffer substances, stabilizers or preservatives, and optionally further active ingredients, particularly those known in relation to pharmaceutical compositions.

[0208] Generally, the nature of the additional ingredients depends on the specific form of the pharmaceutical composition and the mode of administration used. Pharmaceutically acceptable carriers can be used to enhance or stabilize the composition or to facilitate its preparation. Such carriers include, but are not limited to, physiologically compatible saline, buffered saline, dextrose, water, glycerol, solvents, dispersion media, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, as well as combinations thereof. The formulation must be appropriate for the mode of administration. For example, parenteral formulations typically contain injectable fluids containing pharmaceutically and physiologically acceptable fluids as vehicles, such as water, saline, balanced salt solutions, aqueous dextrose, glycerol, and the like. In addition to biologically neutral carriers, pharmaceutical compositions to be administered may contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents.

[0209] The pharmaceutical composition may contain a stabilizer. The term "stabilizer" refers to a substance that protects the composition from harmful conditions, such as those encountered during heating or freezing, and / or extends the stability or shelf life of the conjugate of the present invention under certain conditions or conditions. Examples of stabilizers include, but are not limited to, sugars such as sucrose, lactose, and mannose; sugar alcohols such as mannitol; amino acids such as glycine or glutamic acid; and proteins such as human serum albumin or gelatin.

[0210] Typically, a therapeutically effective dose or effective dose of the conjugate is used in the pharmaceutical composition of the present invention. The amount of the conjugate to be administered can be initially determined based on the dosage and / or administration regimen guidance of an equivalent unlinked therapeutic agent. Generally, the conjugate can provide targeted delivery, and thus can provide at least one of a reduced dose or reduced administration in the administration regimen. Thus, the conjugate can provide a reduced dose and / or reduced administration in the administration regimen compared to the previous therapeutic agent present in the conjugate of the present invention. As described above, the conjugate can provide a controlled stoichiometry of drug delivery, so the dosage of the conjugate can be calculated based on the number of drug molecules provided per antibody-therapeutic entity conjugate.

[0211] The pharmaceutical compositions of the present invention can be administered once or several times, or multiple times. The frequency of administration of the conjugate can vary depending on various factors, such as the severity of symptoms. For example, in some embodiments, the conjugate is administered once a month, twice a month, three times a month, every other week (qow), once a week (qw), twice a week (biw), three times a week (tiw), four times a week, five times a week, six times a week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid).

[0212] In certain embodiments, a conjugate or pharmaceutical composition of the invention is administered simultaneously with one or more additional compounds, hi certain embodiments, a conjugate or pharmaceutical composition of the invention is administered before or after the additional compound(s).

[0213] The pharmaceutical composition of the present invention can be used as a medicine for treating an individual.The individual is a mammal.Mammals include, but are not limited to, livestock animals (e.g., cows, sheep, cats, dogs and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits and rodents (e.g., mice and rats).Preferably, the individual is a human.

[0214] Pharmaceutical compositions containing the conjugates described herein can be delivered to cells, cell groups, tumors, tissues, or subjects using delivery techniques known in the art. Generally, any suitable art-recognized method for delivering conjugates can be adapted for use with the compositions described herein. For example, delivery can be by local administration (e.g., direct injection, implantation, or topical administration), systemic administration, or parenteral routes, including subcutaneous, intravenous, intraocular, intraperitoneal, or intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), or intramuscular administration. The covalent conjugates of the present invention are preferably administered intravenously, intramuscularly, or intraarterially, more preferably intravenously. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate the above-described pharmaceutical compositions in unit dosage form. As used herein, unit dosage form refers to physically discrete units suitable as unit dosages, each containing a predetermined amount of active ingredient calculated to produce the desired therapeutic effect in combination with the necessary pharmaceutical carrier. Examples of such unit dosage forms include injectable solutions or suspensions.

[0215] As detailed above, the conjugates / pharmaceutical compositions of the present invention are particularly useful for treating neurological or brain diseases, such as Alzheimer's disease or Parkinson's disease. The term "neurological disease" encompasses, inter alia, neurodegenerative diseases, neuroinflammatory diseases, or seizure disorders, particularly those of the brain. Neurodegenerative diseases are characterized by the progressive loss of neuronal structure or function, including neuronal death. Many neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, and multiple sclerosis, occur as a result of neurodegenerative processes. There are many similarities between different neurodegenerative disorders, including atypical protein assembly and induced cell death. Neurodegeneration can also be observed at many different levels of neural circuitry, ranging from molecular to systemic. The terms "neurodegenerative disease" and "neuroinflammatory disease" have overlapping scopes. Inflammatory responses are a hallmark of neurodegenerative diseases and are involved in or contribute to neuronal cell death through different mechanisms. Tryptophan catabolism along the kynurenine pathway (KP) represents one of these mechanisms. Seizure disorders are brain disorders characterized by abnormal signaling between brain cells. Seizure disorders can affect parts of the brain (partial seizures) or the entire brain (generalized seizures). The most prominent seizure disorder is epilepsy. Receptor-mediated or receptor-induced endocytosis can be used as a target for conjugates to cross the blood-brain barrier. Examples include transferrin receptor 1 (TfR1), insulin-like growth factor 1 receptor (IGF-1R), low-density lipoprotein receptor-related protein 1 (LRP1), or low-density lipoprotein receptor-related protein 8 (LRP8), particularly TfR1.

[0216] Unless otherwise defined, all technical and scientific terms and any acronyms used herein have the same meaning as commonly understood by one of ordinary skill in the art of this disclosure. Although any methods and materials similar or equivalent to those described herein can be used in the practice presented herein, particular methods and materials are described herein.

[0217] The present invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. Any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, and exemplary methods and materials are described herein. Furthermore, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

[0218] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the words "comprise," "contain," and "encompass" are to be interpreted inclusively rather than exclusively. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. The term "plurality" refers to two or more.

[0219] The following figures, sequences, and examples are intended to illustrate various embodiments of the present invention. Therefore, the specific changes described should not be construed as limitations on the scope of the present invention. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the present invention, and therefore, it should be understood that such equivalent embodiments are included herein. [Brief explanation of the drawings]

[0220] [Figure 1A] Hydrophobic interaction chromatography of mAb2 (HER2) with nine different Q-tag insertion sites conjugated to single-stranded nucleic acids with 15 nucleotides / residues. A: Simplified; B: Fully annotated. [Figure 1B] See legend to Figure 1A. [Figure 2A]1 shows exemplary reactions for KalbTG-mediated conjugation of a mAb with a nucleic acid payload: A: one-step conjugation; B: two-step conjugation. [Figure 2B] See legend to Figure 2A. [Figure 3] The numbering of 1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d]-1,2,3-triazole units used herein. [Example]

[0221] Example 1: Recombinant production of modified antibodies according to the invention Gene synthesis The desired gene segments were prepared by chemical synthesis, and the synthesized gene fragments were cloned by Twist Bioscience (San Francisco, USA) into vectors suitable for expression in HEK293 and Expi293 cells.

[0222] Expression of modified antibodies in mammalian cells Antibody production was performed by transient cotransfection of single expression cassette plasmids in HEK293 cells cultured in F17 medium (Invitrogen, Carlsbad, CA, USA) or Expi293 cells in Expi293 expression medium (Thermo Scientific, Waltham, MA, USA). Transfections were performed as specified in the manufacturer's instructions, using a 1:1 ratio of HC:LC expression plasmids for the symmetric standard IgG1 format or a 1:1:1 ratio of HC1:HC2:LC expression plasmids for the asymmetric knob-into-hole format. Cell culture supernatants were harvested 7 days after transfection and stored at low temperatures (e.g., -20°C).

[0223] Protein titer quantification Protein titers of supernatant samples were determined by affinity chromatography using a POROS A 20 μm column, 2.1 × 30 mm (Life Technologies, Carlsbad, CA, USA) on a high-performance liquid chromatography system (Ultimate 3000 HPLC system, Thermo Scientific, Waltham, MA, USA). The supernatant was loaded onto a column equilibrated with 0.2 M NaHPO, pH 7.4, followed by elution with 0.1 M citric acid, 0.2 M NaCl, pH 2.5. Titers were quantified by measuring absorbance at 280 nm, and protein concentrations were subsequently calculated by comparing the analyte elution peak area (under the curve) with a reference standard curve.

[0224] Purification of modified antibodies from mammalian culture supernatants Antibodies in the culture supernatant were captured by Protein A affinity chromatography using a Mab Select SuRe column (GE Healthcare, Chicago, IL, USA) equilibrated with PBS buffer, pH 7.4. Unbound proteins were removed by washing with the equilibration buffer. The modified antibodies were eluted with 50 mM citrate (pH 3.0), and the pH of the eluate was immediately adjusted to pH 7.5 by adding 2 M Tris (pH 9.0). A second purification step was performed by size exclusion chromatography using a Superdex 200™ column (GE Healthcare, Chicago, IL, USA) in 20 mM histidine, 140 mM NaCl, pH 6.0. The purified modified antibodies were stored at -80°C.

[0225] Antibody purification using medium-scale automation (MilAn) Antibodies were purified in one step using Protein A affinity chromatography as described above using MabSelectSure-Sepharose (Cytiva, Marlborough, MA, USA) on a liquid handling system (Tecan, Männedorf, Switzerland) equipped with a column from Repligen (Waltham, MA, USA). Equilibration, sample loading, and washing steps were performed as described, and antibodies were eluted from the column using 25 mM citrate, pH 3.0. Eluted antibody fractions were neutralized with 1.5 M Tris (pH 7.5), and concentrations were determined by measuring the optical density (OD) at 280 nm.

[0226] Exemplary Antibody Overview Antibody 110: explanation: Anti-HER2 antibody based on IgG1 subclass with P329G / L234A / L235A mutations; Q-tag insertion into HC after amino acid position 177 (EU numbering); Q-tag and spacer sequence: GGGSYRYRQGGGS (SEQ ID NO: 25) Heavy chain constant region sequence TIFF2025525560000034.tif49129Light chain constant domain sequence TIFF2025525560000035.tif17129

[0227] Antibody 113: explanation: Anti-HER2 antibody based on IgG1 subclass with P329G / L234A / L235A mutations; Q-tag insertion into HC after amino acid position 401 (EU numbering); Q-tag and spacer sequence: GGGSYRYRQGGGS (SEQ ID NO: 25) Heavy chain constant region sequence TIFF2025525560000036.tif49130Light chain constant domain sequence TIFF2025525560000037.tif17129

[0228] Table 1. Expression yields of various antibodies with Q-tags inserted at various positions TIFF2025525560000038.tif114138wt=wild type; LC=light chain; HC=heavy chain

[0229] Expression of modified antibodies was affected by the position of the Q-tag introduced: highest yields were obtained when it was introduced after LC110, LC214, HC118, HC177, HC297, HC341, and HC401.

[0230] Example 2: KalbTG conjugation of modified antibodies according to the present invention to fluorescent dyes Conjugation with KalbTG The purified antibody containing the Q-tag was transferred via dialysis into conjugation buffer (histidine buffer containing NaCl, pH 8.5). For the KalbTG reaction, the antibody was mixed with a K-tag small molecule (a fluorescent dye, 10x molar excess) and KalbTG was added (molar ratio mAb:KalbTG>100:1). The reaction mixture was incubated at 37°C with shaking, and the reaction was then quenched by adding 10 mM ammonium sulfate to the solution. To remove unconjugated payload and residual enzyme, the conjugated modified antibody was purified by size exclusion chromatography using a Superdex200™ column (GE Healthcare, Chicago, IL, USA) in PBS pH 7.5. The purified conjugate was stored at -80°C.

[0231] Analysis of conjugates Protein quantification was performed using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Additionally, qualitative DAR measurements were performed by hydrophobic interaction chromatography as described in Example 4 below. Conjugate purity was analyzed by CE-SDS under denaturing and reducing conditions using a Caliper LabChip® GXII Touch™ Protein Characterization System according to the manufacturer's instructions (Perkin Elmer, Waltham, MA, USA).

[0232] Aggregate content was determined by SEC using a TSKgel UP-SW 3000 analytical size exclusion column (Tosoh Bioscience, Griesheim, Germany) equilibrated with 0.2 M K2HPO4 / KH2PO4, 0.25 M KCl, pH 6.2 on a high-performance liquid chromatography system (Ultimate 3000 HPLC system, Thermo Fisher Scientific, Waltham, MA, USA).

[0233] The identity of the conjugate was confirmed by ESI-Q-ToF-MS (Bruker maXis 433, Bruker, Billerica, MA, USA). For MS analysis, samples were deglycosylated using N-glycosidase F (Roche, Basel, Switzerland) and subsequently desalted into 2% formic acid, 40% acetonitrile.

[0234] Table 2. Conjugation efficiency of fluorescent dyes to antibody mAb-5 with Q-tags at various positions TIFF2025525560000039.tif93128

[0235] Example 3: KalbTG conjugation of modified antibodies according to the invention to nucleic acids Synthesis of antisense oligonucleotides Single-stranded LNA oligonucleotides were synthesized using standard phosphoramidite chemistry. DNA and LNA phosphoramidites and all standard reagents were purchased from Merck KGaA (Darmstadt, Germany). K-tag peptides were custom synthesized by Schafer-N Ap (Copenhagen, Denmark) and Biosyntan (Berlin, Germany).

[0236] Oligonucleotides were synthesized on a 130 mmol scale on a NittoPhase HL UnyLinker 350 support (Kinovate, Oceanside, CA) on an AKTA Oligopilot (GE Healthcare, Brondby, Denmark). After synthesis, the oligonucleotides were cleaved from the support overnight. The oligonucleotides were purified by ion exchange chromatography and desalted using Millipore membranes. After lyophilization, the compounds were finally characterized by liquid chromatography-mass spectrometry (reverse phase and electrospray ionization-mass spectrometry).

[0237] The oligonucleotides were conjugated to either the respective linker for conjugation to the K-tag (one-step reaction) or to an acceptor moiety for click chemistry conjugation (two-step reaction), and the conjugates were then directly subjected to purification by reverse-phase HPLC as described below.

[0238] After precipitation of the linker oligonucleotide with 2% lithium perchlorate in acetone, the resulting precipitate was washed with acetone, dried under vacuum, and redissolved in PBS. 1.5 equivalents of K-tag peptide dissolved in PBS was added. After 1 h at room temperature, the reaction mixture was directly subjected to purification by reverse-phase HPLC.

[0239] Both reactions were purified by reverse-phase HPLC on a Waters XBridge Peptide BEH C 18 OBD Prep Column, 300 Å, 10 μm, 10 mm × 150 mm, using 0.1 M ammonium acetate and acetonitrile as eluents. Pooled fractions were lyophilized, redissolved in water, and the pH adjusted to pH 7.0 with aqueous NaOH. After final lyophilization, the compounds were finally characterized by liquid chromatography-mass spectrometry (reverse-phase and electrospray ionization-mass spectrometry).

[0240] Enzymatic conjugation of LNA-ASO to Q-tagged antibodies One-step conjugation using KalbTG Purified antibodies containing Q-tags were transferred via dialysis into conjugation buffer (histidine buffer containing approximately 150 mM chloride ions, pH 8.5). For the KalbTG reaction, the antibodies were mixed with an excess of K-tagged oligonucleotides, and KalbTG (Roche Diagnostics, Mannheim, Germany) was added. The reaction mixture was incubated at 37°C with shaking, and the reaction was then quenched by adding 10 mM ammonium sulfate to the solution. To remove unconjugated payload and residual enzyme, the conjugated modified antibodies were purified by size exclusion chromatography using a Superdex200™ column (GE Healthcare, Chicago, IL, USA) in PBS, 250 mM arginine, pH 7.5. The purified conjugate was stored at -80°C.

[0241] Two-step conjugation using KalbTG and click chemistry The purified antibody containing the Q-tag was transferred to conjugation buffer (histidine buffer containing NaCl, pH 8.5) via dialysis. For the KalbTG reaction, the antibody was mixed with an excess of a K-tagged linker (10-fold molar excess) containing the first part of the click conjugation, and KTG was added. The reaction mixture was incubated at 37°C with shaking, and then the reaction was quenched by adding 10 mM ammonium sulfate to the solution. To remove unconjugated linker and residual enzyme, the conjugated modified antibody was purified by size exclusion chromatography using a Superdex200™ column (GE Healthcare, Chicago, IL, USA) in PBS, 250 mM arginine, pH 7.5. The purified antibody-linker conjugate was added to an excess amount of oligonucleotides conjugated to each of the other parts of the click conjugation in PBS, 250 mM arginine, pH 7.5, and the reaction mixture was incubated overnight at room temperature with shaking. The antibody-oligonucleotide conjugates were purified by size exclusion chromatography as described above, and the purified conjugates were stored at -80°C.

[0242] Analysis of conjugates Quantitation of oligonucleotide conjugates was performed by UV / Vis spectroscopy at 260, 280, and 350 nm using a SoloVPE system (C Technologies, Bridgewater, NJ, USA). The Beer-Lambert equation was used to calculate conjugate concentrations and quantitative drug-antibody ratios (DARs). Additionally, qualitative DAR measurements were performed by hydrophobic interaction chromatography as described in Example 4 below. Conjugate purity was analyzed by CE-SDS under denaturing and reducing conditions using a Caliper LabChip® GXII Touch™ Protein Characterization System (Perkin Elmer, Waltham, MA, USA). Aggregate content was determined by SEC using a TSKgel UP-SW 3000 analytical size-exclusion column (Tosoh Bioscience, Griesheim, Germany) equilibrated with 0.2 M KHPO / KHPO, 0.25 M KCl, pH 6.2 on a high-performance liquid chromatography system (Ultimate 3000 HPLC system, Thermo Fisher Scientific, Waltham, MA, USA). The identity of the conjugate was confirmed by ESI-Q-ToF-MS (Bruker maXis 433, Bruker, Billerica, MA, USA). For MS analysis, samples were deglycosylated using N-glycosidase F (Roche, Basel, Switzerland) and subsequently desalted into 2% formic acid, 40% acetonitrile.

[0243] Table 3. Conjugation efficiency of nucleic acids consisting of 15 or 20 nucleotides to different antibodies with Q-tags at various positions. TIFF2025525560000040.tif135136n.d.=Undecided

[0244] Conjugation of modified antibodies is affected by the position of the introduced Q-tag: introduction after positions LC110, LC143, LC214, HC118, HC177, HC297, and HC341 resulted in the best conjugation efficiency.

[0245] Table 4. Expression yield and conjugation efficiency of modified antibodies with different Q-tags TIFF2025525560000041.tif95161n.d.=Undecided

[0246] Example 4: Hydrophobic interaction chromatography of KalbTG conjugate-modified antibodies according to the present invention Hydrophobic interaction chromatography (HIC) was performed on a high-performance liquid chromatography system (Ultimate 3000 HPLC system, Thermo Fisher Scientific, Waltham, MA, USA) using a TSKgel Butyl-NPR column (2.5 μm, 4.6 × 35 mm, TOSOH Bioscience, Tokyo, Japan) at a flow rate of 1 mL / min. The column was equilibrated with eluent A (20 mM NaHPO dihydrate, 1.5 M (NH)SO, pH 7.0), and 60 μg of each sample was loaded onto the column. Subsequently, a gradient between eluent A and eluent B (20 mM NaHPO dihydrate, 25% (v / v) isopropanol, pH 7.0) was applied.

[0247] gradient: 0 minutes 5%B 0~30 minutes 5%B→80%B 30~34 minutes 80%B→100%B 34~44 minutes 100%B 45~55 minutes 0%B

[0248] Elution profiles were obtained by continuously measuring absorbance at 280 nm. Drug-to-antibody ratios (DARs) were determined by peak integration using Chromeleon 7.2 (Thermo Fisher Scientific, Waltham, MA, USA).

[0249] Exemplary results are shown in FIG.

[0250] Table 5. Retention times of modified antibodies conjugated to nucleic acids consisting of 15 nucleotides (mAb-2) and 20 nucleotides (mAb4). TIFF2025525560000042.tif133145

[0251] The hydrophilic marker had a retention time of 9.25 min and the hydrophobic markers had relative retention times of 25.9 min (mAb2) or 9.00 min and 24.7 min (mAb4), respectively.

[0252] The hydrophobicity of the conjugates is affected by the position of the introduced Q-tag: later introduction of LC110, LC214, and HC297 resulted in a maximum two-fold change in the relative retention time of the conjugated modified antibody relative to the unconjugated antibody, while HC177 had the lowest relative retention time of all internal insertion sites tested.

[0253] Example 5: Synthesis of antisense oligonucleotides with K-amino acid sequence linker variants Single-stranded LNA oligonucleotides were synthesized using standard phosphoramidite chemistry. DNA and LNA phosphoramidites and all standard reagents were purchased from Merck KGaA (Darmstadt, Germany). Amino linker C6 was purchased from Link Technologies (Bellshill, Scotland). N-succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker was purchased from Biosynth Carbosynth (Newbury, UK). Endo-BCN-PEG3-NHS ester was purchased from BroadPharm (San Diego, CA). K-amino acids were custom synthesized by Schafer-N Ap (Copenhagen, Denmark) and Biosyntan (Berlin, Germany).

[0254] 5'-amino linker C6 oligonucleotides were synthesized on a 130 mmol scale on a NittoPhase HL UnyLinker 350 support (Kinovate, Oceanside, CA) on an AKTA Oligopilot (GE Healthcare, Brondby, Denmark). After synthesis, the oligonucleotides were cleaved from the support overnight at 65°C using aqueous ammonia. The oligonucleotides were purified by ion exchange on a SuperQ-5PW gel (Tosoh Bioscience, Griesheim, Germany) using a gradient of 10 mM NaOH buffer and 2 M NaCl, and desalted using a Millipore membrane. After lyophilization, the compounds were finally characterized by liquid chromatography-mass spectrometry (reverse phase and electrospray ionization-mass spectrometry).

[0255] Linker variants were prepared from the appropriate 5'-amino linker C6 precursor. The precursor was dissolved to 5 mM in 20 mM aqueous sodium bicarbonate, pH 8, and 1 volume of DMF was added. Five equivalents of SMCC linker in 0.1 volume of DMF was added, and the mixture was incubated at 40°C for 2 hours. After precipitation of the SMCC linker oligonucleotide with 2% lithium perchlorate in acetone, the resulting precipitate was washed with acetone, dried under vacuum, and redissolved in PBS. 1.5 equivalents of the K-amino acid sequence peptide were added, dissolved in PBS. After 1 hour at room temperature, the reaction mixture was directly subjected to purification by reverse-phase HPLC.

[0256] The reaction was purified by reverse-phase HPLC on a Waters XBridge Peptide BEH C 18 OBD Prep Column, 300 Å, 10 μm, 10 mm × 150 mm, using 0.1 M ammonium acetate and acetonitrile as eluents. Pooled fractions were lyophilized, redissolved in water, and the pH was adjusted to 7.0 with aqueous NaOH. After final lyophilization, the compound was finally characterized by liquid chromatography-mass spectrometry (reverse-phase and electrospray ionization-mass spectrometry).

[0257] Table 6. Summary of the synthesized linkers TIFF2025525560000043.tif172143TIFF2025525560000044.tif216143TIFF2025525560000045.tif232143TIFF2025525560000046.tif231143

[0258] Example 6: Enzymatic conjugation of ASO-K-amino acid sequences to antibodies bearing Q-amino acid sequences Purified antibodies containing the Q-amino acid sequence were transferred via dialysis into conjugation buffer (His, approximately 150 mM NaCl, pH 8.5). For the KTG reaction, the antibody (c = 15 mg / mL) was mixed with the K-amino acid sequence-conjugated oligonucleotide (10-fold molar excess), and KTG (Roche Diagnostics, Mannheim, Germany) was added (molar ratio antibody:KTG = 300:1). The reaction mixture was incubated at 37 °C and 350 rpm for 3 hours, and the reaction was then quenched by adding 10 mM ammonium sulfate to the solution. To remove unconjugated free material and residual enzyme, the conjugated antibody was purified by size exclusion chromatography using a Superdex200™ column (GE Healthcare, Chicago, IL, USA) in PBS, 250 mM arginine, pH 7.5 for oligonucleotide conjugates. The purified conjugates were stored at -80 °C.

[0259] The conjugates were analyzed according to the method of Example 4.

[0260] Table 7. Results obtained with mAb1 (CD163) TIFF2025525560000047.tif148142n.d.=Undecided

[0261] Example 7: Synthesis of linkers with improved in vivo stability and improved synthesis Single-stranded LNA oligonucleotides were synthesized using standard phosphoramidite chemistry. DNA and LNA phosphoramidites and all standard reagents were purchased from Merck KGaA (Darmstadt, Germany). Amino linker C6 was purchased from Link Technologies (Bellshill, Scotland). Endo-BCN-PEG3-NHS ester was purchased from BroadPharm (San Diego, CA). HEG spacer phosphoramidite (Spacer CE-phosphoramidite 18) and BCN CEP I phosphoramidite (CAS number: 1393528-83-8) were purchased from Biosearch Technologies. BCN-succinimidyl ester (CAS number: 1516551-46-4) was purchased from Merck (Darmstadt).

[0262] 5'-amino linker C6 oligonucleotides were synthesized on a 20 μmol scale on a NittoPhase HL UnyLinker 350 support (Kinovate, Oceanside, CA) on a BioAutomation MerMade 12 (LGC Biosearch). After synthesis, the oligonucleotides were cleaved from the support using aqueous ammonia at 65°C overnight. The deprotected 5'-amino linker C6 oligonucleotides were precipitated with 2% lithium perchlorate in acetone, and the resulting precipitate was washed with acetone and dried under vacuum.

[0263] Linker variants 1376 and 1185 were prepared from the appropriate 5'-amino linker C6 precursor. The precursor was dissolved to 5 mM in 20 mM aqueous sodium bicarbonate, pH 8, and 1 volume of DMF was added. Five equivalents of endo-BCN-X-NHS (X represents the respective moiety of the linker variant; see the table below for details) in 0.1 volume of DMF were added, and the mixture was incubated at room temperature (40°C) for 2 hours.

[0264] The linker variants were purified by reverse-phase HPLC on a Waters XBridge Peptide BEH C 18 OBD Prep Column, 300Å, 10 μm, 10 mm × 150 mm, using 0.1 M ammonium acetate and acetonitrile as eluents. Pooled fractions were lyophilized, redissolved in water, and the pH was adjusted to 7.0 with aqueous NaOH. After final lyophilization, the compounds were finally characterized by liquid chromatography-mass spectrometry (reverse-phase and electrospray ionization-mass spectrometry).

[0265] Table 8. Summary of the linkers synthesized (R3 = oligonucleotide) TIFF2025525560000048.tif220141

[0266] Example 8: Enzymatic conjugation of a K-amino acid sequence to an antibody bearing a Q-amino acid sequence and conjugation of an ASO to the antibody-K-amino acid sequence conjugate Purified antibody containing the Q-amino acid sequence was transferred to conjugation buffer (His, approximately 150 mM NaCl, pH 8.5) via dialysis. For the KTG reaction, the antibody (c = 15 mg / mL) was mixed with a linker-containing K-amino acid sequence (10-fold molar excess of linker ID 7456) and KTG was added (molar ratio antibody:KTG = 300:1). The reaction mixture was incubated at 37 °C and 350 rpm for 3 hours, and the reaction was then quenched by adding 10 mM ammonium sulfate to the solution. To remove unconjugated linker and residual enzyme, the conjugated antibody was purified by size exclusion chromatography using a Superdex 200™ column (GE Healthcare, Chicago, IL, USA) in PBS, 250 mM arginine, pH 7.5.

[0267] The purified antibody-K-amino acid sequence conjugate was added to the BCN-containing oligonucleotide (10-fold molar excess) in PBS, 250 mM arginine, pH 7.5, and the reaction mixture was incubated O / N at 25° C. and 350 rpm. The antibody-oligonucleotide conjugate was purified by size exclusion chromatography as described above, and the purified conjugate was stored at −80° C.

[0268] The conjugates were analyzed as described in Examples 3 and 4 above.

[0269] Further endotoxin levels were measured using an EndogenNexgen MCS'' machine (Charles River Laboratories, Wilmington, Massachusetts, USA).

[0270] Table 9: Conjugation results (target DAR=1 since the antibody contains only one Q-amino acid sequence) TIFF2025525560000049.tif50128 * High amounts of unwanted DAR3 species (three ASO linkers attached per mAb)

[0271] Linkers 1018 and 1376 are the best linkers in terms of conjugation efficiency and conjugate quality.

[0272] Example 9: Conjugate stability Serum incubation: Three of the antibody oligonucleotides generated in Example 8 were incubated in human serum and oligonucleotide cleavage determined. The greater the number of degradation fragments, the more nucleotides have been cleaved.

[0273] Table 10: Incubation results TIFF2025525560000050.tif41142n.dect.=Not detected

[0274] The conjugate with linker 1185 shows a higher degree of endonuclease cleavage, while the conjugate with linker 1018 is found to be the most stable.

[0275] In mice: Antibody-oligonucleotide conjugates with linkers 1376 and 1018 were administered to mice. Seven hours after administration, blood samples were collected and oligonucleotide cleavage was determined. Results from serum incubation were confirmed; antibody-oligonucleotide conjugates with linker 1018 showed lower degradation than antibody-oligonucleotide conjugates with linker 1376.

[0276] References TIFF2025525560000051.tif89133

Claims

1. A polypeptide-linker-nucleic acid conjugate comprising: - said linker is 3-aminopropanamide units, 2,6-diaminohexanoic acid amide units; 1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d]-1,2,3-triazole units Including, - the polypeptide is C-terminal lysine residue Including, - the nucleic acid is Oxygen linked to a phosphorus atom in oxidation state V at the 5' or 3' end Including, the 3-amino group of the 3-aminopropanamide unit and the carboxy functional group of the lysine residue of the polypeptide form an amide bond; the carboxy functional group of the 3-aminopropanamide unit and the alpha amino group of the 2,6-diaminohexanoic acid amide unit form an amide bond; the 6-amino group of the 2,6-diaminohexanoic acid amide unit is the nitrogen of the 1,2,3-triazole element of the 1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d]-1,2,3-triazole unit; the oxygen linked to the phosphorus atom of the nucleic acid is covalently linked to the cyclopropane element of the 1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d]-1,2,3-triazole unit; A polypeptide-linker-nucleic acid conjugate, characterized in that:

2. 2. The polypeptide-linker-nucleic acid conjugate of claim 1, wherein the 6-amino group of the 2,6-diaminohexanoic acid amide unit is the nitrogen at position 1 (numbering according to Figure 3) of the 1,2,3-triazole element of the 1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d]-1,2,3-triazole unit.

3. 3. The polypeptide-linker-nucleic acid conjugate of claim 1 or 2, wherein the oxygen linked to the phosphorus atom of the nucleic acid is covalently linked by a methyl or ethyl unit to the carbon at the 6-position (numbering according to Figure 3) of the cyclopropane element of the 1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d]-1,2,3-triazole unit.

4. The polypeptide-linker-nucleic acid conjugate of any one of claims 1 to 3, wherein the oxygen linked to the phosphorus atom in the oxidation state V is phosphate or phosphorothioate.

5. The polypeptide comprises a K-amino acid sequence RYESK, wherein the 3-amino group of the 3-aminopropanamide unit and the carboxyl functional group of a lysine residue in the K-amino acid sequence form an amide bond, and the epsilon-amino group of the lysine in the K-amino acid sequence is linked by an isopeptide bond to the side chain of a glutamine residue in a Q-amino acid sequence of RYGQR (SEQ ID NO:11), RWRQR (SEQ ID NO:12), YRQRT (SEQ ID NO:13), IRQRQ (SEQ ID NO:14), FRYRQ (SEQ ID NO:15), or YRYRQ (SEQ ID NO:17), in particular YRYRQ (SEQ ID NO:17), and wherein, at the direct N-terminus or the direct C-terminus or the direct N-terminus and the direct C-terminus of the Q-amino acid sequence, independently of each other, n=1, 2, 3, 4, or 5, preferably n=1 (Gly-Gly-Gly-Ser) ｎ 5. The polypeptide-linker-nucleic acid conjugate of claim 1, wherein there is a spacer(s) consisting of (SEQ ID NO: 19).

6. 6. The polypeptide-linker-nucleic acid conjugate of claim 1, wherein the polypeptide comprises a K-amino acid sequence RYESK, wherein the 3-amino group of the 3-aminopropanamide unit and the carboxy functional group of a lysine residue in the K-amino acid sequence form an amide bond, and the epsilon-amino group of the lysine in the K-amino acid sequence is linked by an isopeptide bond to the side chain of a glutamine residue in a Q-amino acid sequence of RYGQR (SEQ ID NO:11), RWRQR (SEQ ID NO:12), YRQRT (SEQ ID NO:13), IRQRQ (SEQ ID NO:14), FRYRQ (SEQ ID NO:15), or YRYRQ (SEQ ID NO:17), in particular YRYRQ (SEQ ID NO:17), and wherein the Q-amino acid sequence is located at a position selected from position 214 of the antibody light chain (LC214), position 177 of the antibody heavy chain (HC177), and position 297 of the antibody heavy chain (HC297) (numbering according to Kabat).

7. 7. The polypeptide-linker-nucleic acid conjugate of claim 5 or 6, wherein the Q-amino acid sequence is in a chain of an antibody comprising at least one light chain and at least one heavy chain, and wherein the light chain constant domain not comprising the Q-amino acid sequence comprises an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100% identical to the amino acid sequence of any of SEQ ID NOs: 6 or 7, or the heavy chain constant region not comprising the Q-amino acid sequence comprises an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100% identical to the amino acid sequence of SEQ ID NOs: 1 to 5.

8. The polypeptide-linker-nucleic acid conjugate of any one of claims 1 to 7, wherein the 3-aminopropanamide unit is 3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propanamide.

9. The linker has the following structure: The polypeptide-linker-nucleic acid conjugate of any one of claims 1 to 8, having:

10. The polypeptide-linker-nucleic acid conjugate of any one of claims 1 to 9, wherein the nucleic acid is selected from the group consisting of RNA, siRNA, antisense oligonucleotides (ASO), LNA, and ASO comprising LNA nucleotides.

11. a) the antibody recognizes a target, which is a receptor that induces receptor-mediated endocytosis, such as transferrin receptor protein 1 (TfR1), insulin-like growth factor 1 receptor (IGF-1R), low-density lipoprotein receptor-related protein 1 (LRP1) or low-density lipoprotein receptor-related protein 8 (LRP8), in particular TfR1; and / or b) the antibody recognizes one or two targets, and the one or two targets are specific to a particular cell type, for example, a tumor marker specific to tumor cells, such as breast cancer cells; The polypeptide-linker-nucleic acid conjugate according to any one of claims 7 to 10.

12. a) providing an antibody comprising a Q-amino acid sequence of RYGQR (SEQ ID NO:11), RWRQR (SEQ ID NO:12), YRQRT (SEQ ID NO:13), IRQRQ (SEQ ID NO:14), FRYRQ (SEQ ID NO:15), or YRYRQ (SEQ ID NO:17), in particular YRYRQ (SEQ ID NO:17), at one or more positions selected from positions 110 (LC110), 143 (LC143), and 214 (LC214) of the antibody light chain, and positions 118 (HC118), 177 (HC177), 297 (HC297), 341 (HC341), 401 (HC401), and 446 (HC446) of the antibody heavy chain (numbering according to Kabat); b) providing a polypeptide comprising the amino acid sequence RYESK (SEQ ID NO: 16) conjugated to the terminal amino group of said (3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propanamido)-6-azidohexanamide via an amide bond formed between the carboxy group of said C-terminal lysine residue and the terminal amino group of said (3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propanamido)-6-azidohexanamide; c) reacting the antibody of a) with the polypeptide of b) in the presence of KalbTG or a functionally active variant thereof, thereby forming an isopeptide bond between the Q-amino acid sequence of the antibody and the polypeptide comprising the amino acid sequence of RYESK to produce (3-[2-[2-(2-antibody aminoethoxy)ethoxy]ethoxy]propanamido)-6-azidohexanamide (antibody azide); d) reacting the antibody azide with O-2-(bicyclo[6.1.0]non-4-yn-9-yl)-ethyl-O'-nucleic acid thiophosphonate, thereby producing the polypeptide-linker-nucleic acid conjugate of any one of claims 1 to 11; 12. A method for producing a polypeptide-linker-nucleic acid conjugate according to any one of claims 1 to 11, comprising:

13. The reaction product of step c) has the following structure:

13. The method of claim 12, comprising:

14. 5. The reaction product of step d) has the following structure:

14. The method of claim 12 or 13, comprising:

15. A polypeptide-linker-nucleic acid conjugate according to any one of claims 1 to 12, or a polypeptide-linker-nucleic acid conjugate produced according to the method of any one of claims 12 to 14, for use as a medicament.

Images