Linkers for site-specific protein conjugation

Abstract

The present invention relates to the field of polypeptide conjugates, more in particular to conjugates comprising a polypeptide, a nucleic acid and a linker, wherein the conjugation involves a click chemistry between an organic azide and dibenzocyclooctine (DBCO) derivatives. The invention relates as well to methods to obtain such conjugates, as well as to their use in the treatment of a neurological disease, a brain disease, or cancer.

Description

[0001] Linkers for site-specific protein conjugation

[0002] The current invention is in the field of protein-drug conjugates, more specifically in the field of covalent conjugates of targeting antibodies to therapeutic nucleic acids.

[0003] Background of the invention

[0004] A pharmaceutical is a chemical substance used to treat, cure or prevent a disease. It can be administered via a number of routes, and many pharmaceuticals can be administered by more than one route. Typical administration routes include without limitation injection as a solution, suspension or emulsion (e.g. intramuscular, intravenous, intraperitoneal, intraocular, intraosseous, subcutaneous or intrathecal), orally, rectally, sublingually or topically. A pharmaceutical is usually systemically distributed in the body of the patient and may have adverse effects due to its activity at, e.g., non-targeted tissues. Other tissues may be difficult to reach. Targeted therapy aims to overcome this disadvantage by using pharmaceuticals that are directed to cells, tissues or organs in the body, where they are intended to act. Targeted therapies are expected to be more effective than conventional non-targeted forms of treatments and have less side effects.

[0005] One way of directing a therapeutic entity to the intended place of action is by conjugating it to an antibody specifically binding at the target cells or tissues. One of the challenges associated with antibody drug conjugates (ADCs), such as antibody-oligonucleotide conjugates (AOCs), is their manufacturing. Most drug-to- antibody conjugates make use either of partially reduced interchain disulfide bonds enabling thiol-maleimide chemistry or of lysine functionalization using activated esters. These methods result in statistical mixtures of conjugated antibody species with different numbers of drugs attached at different sites. It has been shown that in an ADC with an monomethyl auristatin E payload, the ADC species with a drug-to- antibody ratio (DAR) of 4, 6, or 8, which were demonstrated to be increasingly hydrophobic, were much more prone to aggregation than the DAR2 species (Adem et al., Bioconj. Chem. 25 (2014) 656-664).

[0006] Therefore, it is desirable to provide ADCs, such as AOCs, for targeted therapy with more precise control over the number and site of the attachment of the therapeutic entities. Thereby homogeneity of the ADC should be increased. Improved sitespecific conjugation technologies remain the target of interest of many pharmaceutical companies for their potential use in the preparation of therapeutic ADCs as well as diagnostic antibody-label or antibody-enzyme conjugates.

[0007] Microbial transglutaminase from Streptomyces mobaraensis has emerged as an inexpensive and easy to use enzyme for protein crosslinking as well as site-specific protein labelling (Ando et al., 2014; Strop et al., 2013).

[0008] WO 2015 / 162563 disclosed antibody-drug conjugates with high drug loading.

[0009] Magdalena Dorywalska et al. disclosed the effect of attachment site on stability of cleavable antibody drug conjugates (Bioconj. Chem. 26 (2015) 650-659).

[0010] US 2020 / 0249231 disclosed microbial transglutaminases, substrates therefor and methods for the use thereof.

[0011] 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 the respective peptide substrates have been described by Steffen et al. (2017). KalbTG catalyzes the formation of an isopeptide bond between a glutamine side (Gin, Q) chain and a lysine (Lys, K) side chain. YRYRQ (SEQ ID NO: 17) was identified as a KalbTG Gln-containing-motif (Q-amino acid sequence, Q-tag), while RYESK (SEQ ID NO: 16) was identified as Lys-containing-acceptor-motif (K-amino acid sequence, K- tag). KalbTG shows similar efficiency, but improved specificity and developability compared to previously described microbial transglutaminases (mTGs).

[0012] WO 2023 / 118398 disclosed site-specific antibody conjugation and its use.

[0013] Summary of the invention

[0014] The present application is directed to the conjugation of a polypeptide (preferably an antibody) to a nucleic acid via DBCO-based linkers, namely DBCO-A3 (short A3), DBCO-A6 (short A6) and DBCO-AmTEG (short Am TEG), see Example 1.

[0015] The current invention is further based, at least in part, on the finding that the linker covalently conjugating a polypeptide, preferably an antibody, and a therapeutic nucleic acid influences the in vivo stability of the conjugate. See Example 6, showing the stability properties of the Brainshuttle™ (BrS) antibody-DBCO-based linker- ASO conjugates according to the invention. The current invention is further based, at least in part, on the finding that the linker covalently conjugating a polypeptide, preferably an antibody, and a therapeutic nucleic acid influences the in vivo activity of the conjugate. See Example 4, showing improved activity properties for an exemplary BrS antibody-DBCO-based linker- ASO conjugate according to the invention compared to a conjugate comprising a BCN-based linker.

[0016] It has been previously shown that a Q-tag for enzymatic conjugation with a transglutaminase cannot be incorporated at all sites within an IgGl antibody while maintaining suitability for conjugation to a payload. See WO 2023 / 118398, the contents of which are incorporated herein. In total, nine suitable and distinct sites within the IgGl heavy and light chains were identified.

[0017] The present application is directed to the KalbTG-mediated conjugation of a polypeptide (preferably an antibody) to a nucleic acid via DBCO-based linkers, namely DBCO-A3 (short A3), DBCO-A6 (short A6) and DBCO-AmTEG (short Am TEG), see Example 1.

[0018] The conjugates of the invention comprise a linker which is a dibenzocyclooctine (DBCO)-derived linker, which is generated by a click chemistry reaction between an organic azide and dibenzocyclooctine (DBCO) derivatives. In the click chemistry, the azide may approach the alkyne in different ways as depicted below, generating two regio-isomers. Although chemical structures may be provided in the application for one isomer only, the present invention covers the conjugates deriving from both isomers: The current invention is based, at least in part, on the finding that for conjugation of an antibody to a therapeutic nucleic acid by the KalbTG, or a functionally active variant thereof, the linker attached to the K-amino acid sequence (K-Tag) influences the conjugation efficiency. See Example 2.

[0019] The current invention is further based, at least in part, on the finding that the linker covalently conjugating an antibody and a therapeutic nucleic acid influences the in vivo stability of the conjugate. See Example 6, showing the stability properties of the Brainshuttle™ (BrS) antibody-DBCO-based linker-ASO conjugates according to the invention.

[0020] The current invention is further based, at least in part, on the finding that the linker covalently conjugating an antibody and a therapeutic nucleic acid influences the in vivo activity of the conjugate. See Example 4, showing improved activity properties for the BrS antibody-DBCO-based linker-ASO conjugates according to the invention compared to a conjugate comprising a BCN-based linker.

[0021] The current invention comprises at least the following independent aspects and dependent embodiments:

[0022] 1. A conjugate comprising a polypeptide, a nucleic acid and a linker, wherein:

[0023] The linker comprises the following chemical structure (A): The linker connects the polypeptide and the nucleic acid, for the avoidance of doubt, the polypeptide and the nucleic acid are not connected directly to each other by a covalent bond,

[0024] The linker is conjugated to the C-terminal amino acid of the polypeptide at the position noted as 1 in structure (A), either directly by a covalent bond or via an additional spacer,

[0025] The linker is conjugated to an oxygen linked to a phosphor of the oxidation state V of the nucleic acid at the position noted as 2 in structure (A), either directly by a covalent bond or via an additional spacer, a ranges from 0 to 20, wherein in a preferred embodiment a is 2, b ranges from 0 to 20, wherein in a preferred embodiment b is 4, each R1 is independently selected from hydrogen, halogen, hydroxy, alcoxy, nitrate, nitrite, and Cl -CIO organic group, wherein in one embodiment up to three R1 are not hydrogen, wherein in a preferred embodiment all R1 are hydrogen, and

[0026] - R2 is selected from NH2, OH, a monosubstituted amine residue (such as NH- Alkyl), a disubstituted amine residue (such as N(Alkyl)l(Alkyl)2), wherein in a preferred embodiment R2 is NH2.

[0027] 2. A conjugate according to the previous embodiment wherein:

[0028] The linker comprises the following chemical structure (1):

[0029]

[0030] The linker connects the polypeptide and the nucleic acid,

[0031] The linker is conjugated to the C-terminal amino acid of the polypeptide at the position noted as 1 in structure (1), either directly by a covalent bond or via an additional spacer,

[0032] The linker is conjugated to an oxygen linked to a phosphor of the oxidation state V of the nucleic acid at the position noted as 2 in structure (1), either directly by a covalent bond or via an additional spacer. 3. The conjugate according to the previous embodiment, wherein the oxygen linked to the phosphor of the oxidation state V (i.e. a phosphorous atom of the oxidation state V) is at the 5’ terminus of the nucleic acid.

[0033] 4. The conjugate according to any of the previous embodiments, wherein the oxygen is linked to the phosphor of the oxidation state V (i.e. a phosphorous atom of the oxidation state V) in a phosphate or phosphorothioate.

[0034] 5. The conjugate according to any of the previous embodiments, wherein the linker is conjugated to the nucleic acid via an amide bond. 6. The conjugate according to any of the previous embodiments, wherein the linker is conjugated to the nucleic acid via an unbranched spacer of formula (CH2)n interspaced between the amide bond and the oxygen linked to the phosphor of the nucleic acid, i.e. the carbonyl group of (1) is conjugated to an N(H)(CH2)n group spacer.

[0035] 7. The conjugate according to any of the previous embodiments, wherein the amino group in the amide bond between the linker and the nucleic acid is provided by a compound selected from the group consisting of metanamide, etanamide, propanamide, butanamide, pentanamide, hexanamide, heptanamide and octanamide nonanamide and decanamide.

[0036] 8. The conjugate according to any of the previous embodiments, wherein the amino group in the amide bond between the linker and the nucleic acid is provided by a compound selected from propanamide and hexanamide.

[0037] 9. The conjugate according to any of the previous embodiments, wherein the linker is conjugated to the 5’end of the nucleic acid via a spacer selected from a group consisting of -CH2- group, a CH2-CH2- group, a -CH2-CH2-CH2- group, a -CH2-CH2-CH2-CH2- group, a -CH2-CH2-CH2-CH2-CH2- group, a -CH2-CH2-CH2-CH2-CH2-CH2- group, a -CH2-CH2-CH2-CH2-CH2- CH2- group, and a -CH2-CH2-CH2-CH2-CH2-CH2-CH2- group.

[0038] 10. The conjugate according to the previous embodiment, wherein the linker is conjugated to the nucleic acid via a spacer selected from a -CH2-CH2-CH2- group and a -CH2-CH2-CH2-CH2-CH2-CH2- group.

[0039] 11. The conjugate according to the previous embodiment, wherein the linker is conjugated to the nucleic acid via a spacer selected from a -NH-CH2-CH2- CH2- group and a -NH-CH2-CH2-CH2-CH2-CH2-CH2- group.

[0040] 12. The conjugate according to any of the previous embodiments, wherein the conjugate comprises the following structure: The conjugate according to any of embodiments 1-11, wherein the conjugate comprises the following structure: The conjugate according to any of embodiments 1-5, wherein the linker is conjugated to the nucleic acid via n units of ethylene glycol (PEG). The conjugate according to the previous embodiment, wherein n=3 or 4. The conjugate according to any of embodiments 1-5 and 14-15, wherein the conjugate comprises the following structure:

[0041] 17. The conjugate according to any of embodiments 1-4, wherein the linker is conjugated directly at the position noted as 2 in structure (1) to the oxygen linked to the phosphor of the oxidation state V of the nucleic acid. 18. The conjugate according to any of the previous embodiments, wherein the linker is conjugated to the C-terminal amino acid of the polypeptide by direct covalent binding between the C-terminal amino acid of the polypeptide and the position noted as 1 in structure (1) via an -NH- group.

[0042] 19. The conjugate according to any of the previous embodiments, wherein the linker is conjugated to the C-terminal amino acid of the polypeptide by direct covalent binding between the C-terminal amino acid of the polypeptide and the position noted as 1 in structure (1) via an amide bond. 0. The conjugate according to any of the previous embodiments, wherein the linker is conjugated to the carboxy group at the C terminus of the polypeptide via -NH-(PEG)m-propionate-NH-. 1. The conjugate according to the previous embodiment, wherein m=3 or 4. 2. The conjugate according to any of embodiments 1-17, wherein the linker is conjugated to the C-terminal amino acid of the polypeptide by direct covalent binding between the C-terminal amino acid of the polypeptide and the position noted as 1 in structure (1). The conjugate according to any of embodiments 1 to 21, wherein the conjugate comprises the following structure: The conjugate according to any of the previous embodiments, wherein the polypeptide comprises a lysine residue. The conjugate according to any of the previous embodiments, wherein the polypeptide comprises a lysine residue, wherein said lysine residue belongs to a tag sequence RYESK (SEQ ID NO: 16) comprised at the C-terminal end of the polypeptide, wherein the carboxy group of said lysine is conjugated to the linker via an amide bond. The conjugate according to the previous embodiment, wherein the polypeptide comprises the K-amino acid sequence RYESK (SEQ ID NO: 16), wherein the conjugate comprises the following structure:

[0043]

[0044] 27. The conjugate according to any of embodiments 23-26, wherein the conjugate comprises the following structure: 28. The conjugate according to any of embodiments 23-26, wherein the conjugate comprises the following structure:

[0045]

[0046] 29. The conjugate according to any of embodiments 23-26, wherein the conjugate comprises the following structure: Nucleic acid 30. The conjugate according to any of the previous embodiments, wherein the polypeptide comprises the K-amino acid sequence (K-tag) RYESK (SEQ ID NO: 16), wherein the epsilon amino group of the lysine (K) is linked via an isopeptide bond to a glutamine (Q) residue side chain of a second polypeptide, wherein the glutamine residue is within a Q-amino acid sequence (Q-tag) of at least 5 amino acid residues.

[0047] 31. The conjugate according to any of the previous embodiments, wherein the polypeptide comprises the K-amino acid sequence RYESK (SEQ ID NO: 16), wherein the epsilon amino group of the lysine in the K-amino acid sequence is linked by an isopeptide bond to a glutamine residue side chain of a second polypeptide, wherein the glutamine residue is within a Q-amino acid sequence (Q-tag) selected from the group 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).

[0048] 32. The conjugate according to any one of the previous embodiments, wherein the polypeptide comprises the K-amino acid sequence RYESK, wherein the epsilon amino group of the lysine in the K-amino acid sequence is linked by an isopeptide bond to a glutamine residue side chain of a second polypeptide, wherein 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), wherein the Q-amino acid sequence comprises one or two spacer(s) directly at its termini.

[0049] 33. The conjugate according to any of the previous embodiments, wherein the polypeptide comprises the K-amino acid sequence RYESK (SEQ ID NO: 16), wherein the epsilon amino group of the lysine in the K-amino acid sequence is linked by an isopeptide bond to a glutamine residue side chain of a second polypeptide, wherein 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), wherein the Q-amino acid sequence comprises one or two spacer(s) consisting primarily or fully of Gly and Ser directly at its termini.

[0050] 34. The conjugate according to any of the previous embodiments, wherein the polypeptide comprises the K-amino acid sequence RYESK (SEQ ID NO: 16), wherein the epsilon amino group of the lysine in the K-amino acid sequence is linked by an isopeptide bond to a glutamine residue side chain of a second polypeptide, wherein 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), wherein the Q-amino acid sequence comprises one or two spacer(s) consisting independently of each other of (Gly-Gly-Gly-Ser)p (SEQ ID NO: 19) with p = 1, 2, 3, 4 or 5, preferably p=l, directly at its termini.

[0051] 35. The conjugate according to any of the previous embodiments, wherein the polypeptide comprises the K-amino acid sequence RYESK (SEQ ID NO: 16), wherein the epsilon amino group of the lysine in the K-amino acid sequence is linked by an isopeptide bond to a glutamine residue side chain of a second polypeptide, wherein 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), wherein the Q-amino acid sequence is at a position selected from position 110 (LC110), position 143 (LC143) and position 214 (LC214) of an antibody light chain and position 118 (HC118), position 177 (HC177), position 297 (HC297), position 341 (HC341), position 401 (HC401) and position 446 (HC446) of an antibody heavy chain (numbering according to Kabat).

[0052] 36. The conjugate according to any of the previous embodiments, wherein the polypeptide comprises the K-amino acid sequence RYESK (SEQ ID NO: 16), wherein the epsilon amino group of the lysine in the K-amino acid sequence is linked by an isopeptide bond to a glutamine residue side chain of a second polypeptide, wherein 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), wherein the Q-amino acid sequence is at a position selected from position 214 (LC214) of an antibody light chain, position 177 (HC177) of an antibody heavy chain, and position 297 (HC297) of an antibody heavy chain (numbering according to Kabat). 37. The conjugate according to any of the previous embodiments, wherein the Q- amino acid sequence is in a chain of an antibody that comprises at least one light chain, preferably one complete light chain, and at least one heavy chain, preferably one complete heavy chain.

[0053] 38. The conjugate according to the previous embodiment, wherein the Q-amino acid sequence is in one chain of an antibody that comprises two light chains, preferably two complete light chains, and two heavy chains, preferably two complete heavy chains.

[0054] 39. The conjugate according to any of the embodiments 37 and 38, wherein the Q-amino acid sequence is in two chains of an antibody that comprises two light chains, preferably two complete light chains, and two heavy chains, preferably two complete heavy chains.

[0055] 40. The conjugate according to any of embodiments 37-39, wherein the light chain constant domain not comprising a 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 NO: 6 or 7; and / or wherein the heavy chain constant region not comprising a 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 NO: 1 to 5.

[0056] 41. The conjugate according to any of embodiments 37-40, wherein 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 wherein 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 31.

[0057] 42. The conjugate according to any of the embodiments 35-41, wherein the antibody recognizes a target and binds thereto with its complementarity determining regions (CDRs), particularly wherein the target is a biomolecule present on a cell. The conjugate according to any of the previous embodiments, wherein the nucleic acid is selected from the group consisting of a RNA, siRNA, antisense oligonucleotide (ASO), LNA, and an ASO comprising LNA nucleotides. The conjugate according to any of the embodiments 35-43, wherein a) the antibody recognizes one target and said target is a receptor inducing receptor-mediated endocytosis, such as transferrin receptor 1 (TfRl), insulin-like growth factor 1 receptor (IGF-1R), low density lipoprotein receptor-related protein 1 (LRP1), low density lipoprotein receptor-related protein 8 (LRP8), CD98 (SLC3A2), or podocalyxin (PODXL), particularly TfRl; and / or b) wherein the antibody recognizes one or further target(s) and said one or further targets is / are specific for a specific cell type, and / or said one of further target(s) is / are therapeutic targets. The conjugate according to the previous embodiment, wherein the therapeutic target is a tumor target, a neurological target or a metabolic target. A compound of the formula (2): The compound according to the previous claim for forming a conjugate according to any of the embodiments 1-12, 18-27 and 30-45. A compound of the formula (3): The compound according to the previous claim for forming a conjugate according to any of the embodiments 1-11, 13, 18-26, 28 and 30-45. A compound of the formula (4): The compound according to the previous claim for forming a conjugate according to any of the embodiments 1-11, 14-26 and 29-45. An azide of the formula (5) for forming a conjugate according to any of the previous embodiments. A method for producing a polypeptide-linker-nucleic acid conjugate according to any of embodiments 1-16, 18-21 and 23-45 comprising the following steps: 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:

[0058] 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 position 110 (LC110), position 143 (LC143) and position 214 (LC214) of an antibody light chain and position 118 (HC118), position 177 (HC177), position 297 (HC297), position 341 (HC341), position 401 (HC401) and position 446 (HC446) of an antibody heavy chain (numbering according to Kabat), b) providing a polypeptide comprising the amino acid sequence RYESK

[0059] (SEQ ID NO: 16), which is conjugated to the terminal amino group of (3-[2-[2-(2-aminoethoxy)ethoxy]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 the (3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propanamido)-6- azidohexanami de, c) reacting the antibody of a) and the polypeptide of b) in the presence of

[0060] KalbTG or a functionally active variant thereof and under conditions conducive to the activity of KalbTG, thereby forming a isopeptide bond between the Q-amino acid sequence of the antibody and the polypeptide comprising the amino acid sequence of RYESK, thus producing an (3-[2-[2-(2-antibody aminoethoxy)ethoxy]ethoxy] propanamido)-6-azidohexanamide (antibody azide), d) reacting the antibody azide with the compound according to any of embodiments 46, 48 and 50, and thereby producing the polypeptide- linker-nucleic acid conjugate according to any one of embodiments 1- 16, 18-21 and 23-45. 54. The method according to the previous embodiment, wherein the reaction product of step c) comprises the following structure isopeptide bond to glutamine

[0061] 55. The method according to any one of embodiments 53 to 54, wherein the reaction product of step d) comprises the following structure:

[0062] 56. The conjugate according to any one of embodiments 1 to 45, or the polypeptide-linker-nucleic acid conjugate produced according to the method of any one of embodiments 53 to 55 for use as a medicament. 57. The conjugate according to any one of embodiments 1 to 45, or the polypeptide-linker-nucleic acid conjugate produced according to the method of any one of embodiment 53 to 55 for its use in the manufacture of a medicament.

[0063] 58. Use of the conjugate according to any one of embodiments 1 to 45, or the polypeptide-linker-nucleic acid conjugate produced according to the method of any one of embodiment 53 to 55 in the manufacture of a medicament. 59. The conjugate according to any one of embodiments 1 to 45, or the polypeptide-linker-nucleic acid conjugate produced according to the method of any one of embodiment 53 to 55 for use in treating a neurological disease or a brain disease.

[0064] 60. The conjugate according to the previous embodiment, wherein the neurological disease or the brain disease is selected from Alzheimer’s disease and Parkinson’s disease.

[0065] 61. The conjugate according to any one of embodiments 1 to 45, or the polypeptide-linker-nucleic acid conjugate produced according to the method of any one of embodiment 53 to 55 for use in treating cancer

[0066] 62. The conjugate according to the previous embodiment, wherein the cancer is breast cancer.

[0067] 63. Method for the treatment of a neurological disease or a brain disease in a subject in need thereof comprising the administration to said subject of a therapeutically effective amount of the conjugate according to any one of embodiments 1 to 45, or the polypeptide-linker-nucleic acid conjugate produced according to the method of any one of embodiment 53 to 55.

[0068] 64. The method according to the previous embodiment, wherein the neurological disease or the brain disease is selected from Alzheimer’s disease and Parkinson’s disease.

[0069] 65. Method for the treatment of cancer in a subject in need thereof comprising the administration to said subject of a therapeutically effective amount of the conjugate according to any one of embodiments 1 to 45, or the polypeptide- linker-nucleic acid conjugate produced according to the method of any one of embodiment 53 to 55.

[0070] 66. The method according to the previous claim, wherein the cancer is breast cancer. 67. The conjugate according to any one of embodiments 1 to 45, or the polypeptide-linker-nucleic acid conjugate produced according to the method of any one of embodiment 53 to 55 for its use in the manufacture of a medicament for treating a neurological disease or a brain disease.

[0071] 68. The conjugate for use according to the previous embodiment, wherein the neurological disease or the brain disease is selected from Alzheimer’s disease and Parkinson’s disease.

[0072] 69. The conjugate according to any one of embodiments 1 to 45, or the polypeptide-linker-nucleic acid conjugate produced according to the method of any one of embodiment 53 to 55, for its use in the manufacture of a medicament for treating cancer.

[0073] 70. The conjugate for use according to the previous embodiment, wherein the cancer is breast cancer.

[0074] 71. Use of the conjugate according to any one of embodiments 1 to 43, or the polypeptide-linker-nucleic acid conjugate produced according to the method of any one of embodiment 53 to 55 in the manufacture of a medicament for treating a neurological disease or a brain disease.

[0075] 72. The use of the conjugate according to the previous embodiment, wherein the neurological disease or the brain disease is selected from Alzheimer’s disease and Parkinson’s disease.

[0076] 73. Use of the conjugate according to any one of embodiments 1 to 45, or the polypeptide-linker-nucleic acid conjugate produced according to the method of any one of embodiment 53 to 55, in the manufacture of a medicament for treating cancer.

[0077] 74. The use of the conjugate according to the previous embodiment, wherein the cancer is breast cancer. Detail Description of Embodiments of the Invention

[0078] A modified antibody comprising a heavy chain and a light chain, wherein the heavy chain or / and the light chain comprises one or more first recognition site(s) for the transglutaminase from Kutzneria albida (KalbTG) or a functionally active variant thereof, has been previously described. See WO 2023 / 118398, the contents of which are incorporated herein. The one or more first recognition site(s) are introduced at one or more selected position(s) within the antibody’s heavy chain and / or light chain. The one or more nucleic acids encoding said modified antibody, as well as a covalent conjugate comprising (i) the modified antibody 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, have also been described.

[0079] According to the present invention, the non-antibody moiety comprises a therapeutic entity and a second linker, wherein this second linker comprises a dibenzocyclooctyne (DBCO) derivative. The present invention further relates to a method of covalently conjugating an antibody to non-antibody moieties using a linker according to the invention. In case the non-antibody moiety comprises a therapeutic entity, the present invention further relates to the conjugate according to the invention of the modified antibody and the therapeutic entity as pharmaceutical composition, for use as a medicament as well as for use in treating a disease.

[0080] The IgG heavy chain C-terminus has been described as a suitable Q-tag insertion site for mTGs for antibody labeling (see, e.g., WO 2021 / 174091). However, increased aggregation and hydrophobicity when conjugating payloads to that site, have been observed. In order to be able to use KalbTG to prepare antibody-drug conjugates useful as pharmaceuticals, the KalbTG Q-tag has to be introduced at a defined site within the IgG backbone.

[0081] The incorporation of the Q-tag should not impair the antibody folding or function or diminish expression yields and should provide the required accessibility for the conjugation as well as therapeutic activity of the therapeutic entity. The successful identification of such sites allows the incorporation of one or more therapeutic moieties per IgG molecule in a defined stoichiometry as well as a controlled sitespecific manner. Thereby more homogeneous conjugate products can be provided, e.g., reducing the required purification and separation efforts and yield molecules with more favorable drug-like properties. In case the antibody is symmetric, i.e. comprises two identical heavy and light chain pairs, two Q-tags are present per molecule. In case the antibody is asymmetric, i.e. comprises two different heavy and light chain pairs, a single Q-tag is present per molecule. Therefore, a drug-to-antibody ratio (DAR, i.e. number of payload molecules per antibody molecule) of 2 was expected at 100 % conjugation efficiency for the symmetric antibody and a DAR of 1 for the asymmetric antibody.

[0082] A number of KalbTG Q-tag insertion sites spanning the length of the IgG backbone, which do not negatively impact on expression yield and provide enzyme accessibility, were identified. See WO 2023 / 118398.

[0083] The suitable insertion sites for the Q-tag are at positions 110 (LC110), 143 (LC143) and 214 (LC214) of the light chain and position 118 (HC118), 177 (HC177), 297 (HC297), 341 (HC341), 401 (HC401), and 446 (HC446) of the heavy chain (the amino acid numbering follows Rabat’s EU-numbering scheme).

[0084] Accordingly, in a first aspect the present invention relates to a conjugate comprising a linker according to the current invention, comprising a modified antibody comprising a heavy chain and a light chain, wherein the heavy chain or / and the light chain comprises one or more (first) recognition site(s) for the transglutaminase from Kutzneria albida (KalbTG) at one or more of the positions selected from position 110 (LC110), position 143 (LC143), and position 214 (LC214) of the light chain and position 118 (HC118), position 177 (HC177), position 297 (HC297), position 341 (HC341), position 401 (HC401) and position 446 (HC446) of the heavy chain (the amino acid numbering follows Rabat’s EU-numbering scheme). Accordingly, in the antibody part of the conjugate of the present invention, the recognition site for KalbTG is positioned internally in the constant regions of the Ig heavy chain polypeptide, i.e. not at any terminus, or / and internally or at the C-terminus of the Ig light chain constant domain. In addition, the modified antibody in the conjugate of the present invention may include a further first recognition site for KalbTG at position 446 (HC446) of the heavy chain, i.e. at the C-terminal end. In the context of the current invention the insertion of a recognition site for KalbTG at a position means that the amino acid present at that position in the unmodified sequence is either replaced by the recognition site or the recognition site is preferably inserted after that position in addition.

[0085] In certain embodiments, the one or more positions are selected from the group of positions comprising position 214 (LC214) of the light chain and position 118 (HC118), position 177 (HC177), position 297 (HC297) and position 341 (HC341) of the heavy chain (the amino acid numbering follows Kabat’s EU-numbering scheme)

[0086] In one preferred embodiment, the one or more positions are selected from the group of positions comprising position 214 (LC214) of the light chain and position 341 (HC341), position 297 (HC297) and position 177 (HC177) of the heavy chain (the amino acid numbering follows Kabat’s EU-numbering scheme).

[0087] In the most preferred embodiment, the position is position 297 (HC297) of the heavy chain (the amino acid numbering follows Kabat’s EU-numbering scheme).

[0088] In certain embodiments, the antibody in the conjugate according to the invention comprises two identical heavy chains or heavy chain Fc-regions. Such an antibody may comprise two, four or more (first) recognition site(s) for the transglutaminase from Kutzneria albida (KalbTG) at one or more of the positions selected from position 110 (LC110), position 143 (LC143) and position 214 (LC214) of the light chain and position 118 (HC118), position 177 (HC177), position 297 (HC297), position 341 (HC341), position 401 (HC401) and position 446 (HC446) of the heavy chain (the amino acid numbering follows Kabat’s EU-numbering scheme).

[0089] In certain embodiments, the antibody in the conjugate according to the invention comprises two different heavy chains whereby the difference arises from the respective mutations for inducing heterodimerization. Such a modified antibody may comprise one, two or more (first) recognition site(s) for the transglutaminase from Kutzneria albida (KalbTG) at one or more identical or different positions selected from position 110 (LC110), position 143 (LC143) and position 214 (LC214) of the light chain and position 118 (HC118), position 177 (HC177), position 297 (HC297), position 341 (HC341), position 401 (HC401) and position 446 (HC446) of the heavy chain (the amino acid numbering follows Kabat’s EU-numbering scheme).

[0090] Likewise, the current invention relates to a conjugate comprising an antibody, wherein the antibody Fc-region comprises a heavy chain Fc-region, wherein the heavy chain Fc-region comprises one or more (first) recognition site(s) for the transglutaminase from Kutzneria albida (KalbTG) at one or more of the positions selected from the group of positions comprising position 118 (HC118), position 177 (HC177), position 297 (HC297), position 341 (HC341), position 401 (HC401) and position 446 (HC446) of the heavy chain (the amino acid numbering follows Kabat’s EU-numbering scheme). Accordingly, in the antibody Fc-region in the conjugate of the present invention, the recognition site for KalbTG is positioned internally in the constant regions of the Ig heavy chain Fc-region polypeptide, i.e. not at any terminus. In addition, the antibody Fc-region of the conjugate of the present invention may include a further first recognition site for KalbTG at position 446 (HC446) of the heavy chain, i.e. at the C-terminal end. In the context of the insertion of a recognition site for KalbTG at a position means that the amino acid present at that position in the unmodified sequence is either replaced by the recognition site or the recognition site is preferably inserted after that position in addition.

[0091] In certain embodiments, the one or more positions are selected from the group of positions comprising position 118 (HC118), position 177 (HC177), position 297 (HC297) and position 341 (HC341) of the heavy chain (the amino acid numbering follows Kabat’s EU-numbering scheme).

[0092] In one preferred embodiment, the one or more positions are selected from the group of positions comprising position 341 (HC341), position 297 (HC297) and position 177 (HC177) of the heavy chain (the amino acid numbering follows Kabat’s EU- numbering scheme).

[0093] In the most preferred embodiment, the position is position 297 (HC297) of the heavy chain (the amino acid numbering follows Kabat’s EU-numbering scheme).

[0094] The terms “antibody” and “Ig” are used interchangeably herein. They are used in the broadest sense and include, for example, monoclonal antibodies independent of the binding specificity (including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies), monovalent antibodies, for example full-length antibodies lacking one Fab), multivalent antibodies, i.e. antibodies that are two- or tetravalent, multispecific antibodies and fragments of full-length antibodies so long as they comprise at least one of the modifications as outlined above.

[0095] Naturally occurring antibodies are generated by the assembly of heavy chains only or heavy and light chains. Each heavy chain is composed of four domains: the variable domain (VH) and three constant domains (CHI, CH2, and CH3). The light chain is composed of variable domain (VL) and a constant domain (CL). In case heavy and light chains being present, the light chain pairs with a cognate heavy chain Fab-fragment comprising the VH and CHI domains. The associated light chain and heavy chain Fab fragment together are denoted as Fab fragment. The heavy chain CH2 and CH3 domains, together denoted as heavy chain Fc-region, dimerize with additional heavy chain CH2 and CH3 domains from a second chain to form the Fc- region. The Fc-region is connected to the Fab-fragment(s) via a flexible hinge region. The hinge region comprises several disulfide bridges that covalently link two heavy chain Fc-regions together. In the Fab-fragment, the light chain and the heavy chain Fab fragment are also connected by one disulfide bridge. However, the connectivity differs among the IgG subclasses. The overall structure of full-length IgGs resembles a Y-shape, with the Fc-region forming the base while the two Fab-fragments form the arms and are available for binding to the antigen.

[0096] Within the variable domains reside loops called complementarity determining regions (CDRs). These are mainly responsible for the direct interaction of the antibody with its antigen. Because of the significant variability in the number of amino acids in these CDRs, there are multiple numbering schemes for the variable regions. As used herein, the amino acid positions of all constant regions and domains of the heavy and light chain 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 is referred to as “numbering according to Kabat” herein. Specifically, the Kabat numbering system (see pages 647-660) of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) is used for the light chain constant domain CL of kappa and lambda isotype, and the Kabat EU index numbering system (see pages 661-723) is used for the constant heavy chain domains (CHI, Hinge, CH2 and CH3, which is herein further clarified by referring to “numbering according to Kabat EU index” in this case).

[0097] The term “modified antibody” as used herein, denotes antibodies or antibody Fc- regions comprising 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 fragments of an antibody include Fab- fragments, F(ab’)-fragments, F(ab)2-fragments, F(ab’)2-fragments, etc. In particular, modified antibodies include antibody molecules and immunologically active portions of antibody molecules, for example, antigen-binding domains or molecules that contain an antigen-binding site that binds to the antigen (e.g., one or more complementarity determining regions (CDRs)) as long as the modification is present. The modified antibodies, in certain embodiments, can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY, in a preferred embodiments IgG), any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2, in one preferred embodiment IgGl), or any subclass (e.g. IgG2a and IgG2b). An antibody can be humanized, chimeric and / or affinity matured as well as an antibody from other species, for example, mouse, rabbit and sheep.

[0098] In accordance with the present invention, in certain embodiments, the antibody in the conjugate of the invention comprises at least one heavy chain and at least one light chain. Accordingly, the antibody in certain embodiments may comprise one or two or three or four Fab-fragments. In certain embodiments, the 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 (including one binding site) and one heavy chain Fc-region fragment including a hinge region associated with the Fc-region of the (full-length) heavy chain.

[0099] In accordance with the present invention, in certain embodiments, the antibody in the conjugate of the invention may be based on an IgGl, IgG2, IgG3, or IgG4 antibody, particularly a humanized, mouse, rabbit, or sheep antibody. Exemplary and suitable sequences are given in the following, wherein X4indicates the possible insertion sites of Q-tag motifs:

[0100] Human-Heavy chain-constant region of IgGl subclass (GlmE17 - Caucasian allotype): (SEQ ID NO. 1)

[0101] AiSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFP AVLQ SSiGL YSLS S VVT VPS S SLGTQT YICNVNHKP SNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNiSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGiQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDiGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGi

[0102] Human-Heavy chain-constant region of IgGl subclass (Glml7 - Afro-American allotype): (SEQ ID NO. 2) AiSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFP AVLQ SSiGL YSLS S VVT VPS S SLGTQT YICNVNHKP SNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNiSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGiQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDiGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGi

[0103] Human-Heavy chain-constant region of IgG2 subclass: (SEQ ID NO. 3)

[0104] AiSTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSiGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERK CCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQ FNWYVDGVEVHNAKTKPREEQFNiSTFRVVSVLTVVHQDWLNGKEYKCK VSNKGLPAPIEKTISKTKGiQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPMLDSDiGSFFLYSKLTVDKSRWQQGNV F SC S VMHEALHNHYTQKSLSLSPGi

[0105] Human-Heavy chain-constant region of IgG3 subclass: (SEQ ID NO. 4)

[0106] AiSTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSiGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVEL KTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTP PPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQF KWYVDGVEVHNAKTKPREEQYNiSTFRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKTKGiQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESSGQPENNYNTTPPMLDSDiGSFFLYSKLTVDKSRWQQGNIF

[0107] SCSVMHEALHNRFTQKSLSLSPGi

[0108] Human-Heavy chain-constant region of IgG4 subclass: (SEQ ID NO. 5)

[0109] AiSTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFP AVLQ SSiGL YSLS S VVT VPS S SLGTKT YTCNVDHKP SNTKVDKRVSPN MVPHAHHAQAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREEQFNiSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGiQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDiGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGi

[0110] Thus, in some embodiments, the heavy chain constant region is based on a human Ig heavy chain constant region, e.g., human IgGl, 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.

[0111] In certain embodiments, the human IgGl heavy chain polypeptide, on which the antibody of the conjugate according to the invention may be based, includes a constant region amino acid sequence 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: 1 or 2.

[0112] In certain embodiments, the human IgG2 heavy chain polypeptide, on which the modified antibody in the conjugate according to the invention may be based, includes a constant region amino acid sequence 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: 3.

[0113] In certain embodiments, the human IgG3 heavy chain polypeptide, on which the modified antibody in the conjugate according to the current invention may be based, includes a constant region amino acid sequence 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: 4.

[0114] In certain embodiments, the human IgG4 heavy chain polypeptide, on which the modified antibody in the conjugate according to the current invention may be based, includes a constant region amino acid sequence 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: 5. In certain embodiments, the antibody of the conjugate according to the invention comprises the following further 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).

[0115] In one preferred embodiment, the antibody of the conjugate according to the invention comprises in the first Fc-region polypeptide the mutations L234A, L235A, P329G, T366W and in the second Fc-region polypeptide the mutations L234A, L235A, P329G, T366S, L368A, Y407V and the amino acid sequence YRYRQ (SEQ ID NO: 17) inserted at position 297 (HC297) of the heavy chain (the amino acid numbering follows Kabat’ s EU-numbering scheme), optionally with one or two spacer(s) consisting independently of each other of (Gly-Gly-Gly-Ser)p (SEQ ID NO: 19) with p = 1, 2, 3, 4 or 5, preferably p=l, directly at it C- and N-terminus. In certain embodiments, the antibody further comprises one of the mutation S354C and Y349C in the first Fc-region polypeptide and the other in the second Fc-region polypeptide.

[0116] In one preferred embodiment, the human heavy chain polypeptide, on which the antibody of the conjugate according to the invention may be based, includes a constant region amino acid sequence 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: 8 or SEQ ID NO: 9 or SEQ ID NO: 30, preferably SEQ ID NO: 8:

[0117] HC177 (SEQ ID NO: 30) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFP AVLQ SSGGGS YRYRQGGGSGL YSLS S VVTVP S S SLGTQT YICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG or

[0118] HC297 (SEQ ID NO: 8)

[0119] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFP AVLQS SGL YSLS SVVTVPS S SLGTQT YICNVNHKP SNTKVDKKVEPK SC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNGGGSYRYRQGGGSSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG or

[0120] HC341 (SEQ ID NO: 9)

[0121] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFP AVLQS SGL YSLS SVVTVPS S SLGTQT YICNVNHKP SNTKVDKKVEPK SC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI< CT<VSNI<ALPAPIEI<TISI<AI<GGGGSYRYRQGGGSQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0122] In accordance with the present invention, in certain embodiments, the antibody in the conjugate of the invention may be based on an IgGl, IgG2, IgG3, or IgG4 antibody, particularly a humanized antibody, further comprising a light chain constant domain. Exemplary and suitable sequences for the constant region amino acid sequence of the light chain are given in the following, wherein X4indicates the insertion sites of Q-tag motifs: Human-kappa light chain-constant domain (SEQ ID NO: 6)

[0123] RTYiAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREiAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGECl

[0124] Human-lambda light chain-constant domain (SEQ ID NO: 7)

[0125] QPKiAAPSVTLFPPSSEELQANKATLVCLISDFYPGAiVTVAWKADSSPVK AGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTV APTECiS

[0126] In certain embodiments, the human light chain polypeptide, on which the modified antibody in the conjugate according to the invention may be based, includes a constant region amino acid sequence 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.

[0127] In one preferred embodiment, the human light chain polypeptide, on which the modified antibody in the conjugate according to the invention may be based, includes a constant region amino acid sequence 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: 10:

[0128] LC214 (SEQ ID NO: 10)

[0129] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGECGGGSYRYRQGGGS

[0130] 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 NO: 6 or 7; and / or wherein 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 NO: 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 wherein 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 30, preferably SEQ ID NO: 8.

[0131] As detailed above, the antibody in the conjugate according to the invention may be a bi- or multispecific antibody. Exemplary embodiments include: full-length antibody with domain exchange: a multispecific IgG antibody comprising a first Fab fragment and a second Fab fragment, wherein in the first Fab fragment a) only the CHI and CL domains are replaced by each other (i.e. the light chain of the first Fab fragment comprises a VL and a CHI domain and the heavy chain of the first Fab fragment comprises a VH and a CL domain); b) only the VH and VL domains are replaced by each other (i.e. the light chain of the first Fab fragment comprises a VH and a CL domain and the heavy chain of the first Fab fragment comprises a VL and a CHI domain); or c) the CHI and CL domains are replaced by each other and the VH and VL domains are replaced by each other (i.e. the light chain of the first Fab fragment comprises a VH and a CHI domain and the heavy chain of the first Fab fragment comprises a VL and a CL domain); and wherein the second Fab fragment comprises a light chain comprising a VL and a CL domain, and a heavy chain comprising a VH and a CHI domain; the full length antibody with domain exchange may comprises a first heavy chain including a CH3 domain and a second heavy chain including a CH3 domain, wherein both CH3 domains are engineered in a complementary manner by respective amino acid substitutions, in order to support heterodimerization of the first heavy chain and the modified second heavy chain;

[0132] - full-length antibody with domain exchange and additional heavy chain C-terminal binding site: a multispecific IgG antibody comprising a) one full length antibody comprising two pairs each of a full length antibody light chain and a full length antibody heavy chain, wherein the binding sites formed by each of the pairs of the full length heavy chain and the full length light chain specifically bind to a first antigen, and b) one additional Fab fragment, wherein the additional Fab fragment is fused to the C-terminus of one heavy chain of the full length antibody, wherein the binding site of the additional Fab fragment specifically binds to a second antigen, wherein the additional Fab fragment specifically binding to the second antigen i) comprises a domain crossover such that a) the light chain variable domain (VL) and the heavy chain variable domain (VH) are replaced by each other, or b) the light chain constant domain (CL) and the heavy chain constant domain (CHI) are replaced by each other, or ii) is a single chain Fab fragment;

[0133] - the one-armed single chain format (= one-armed single chain antibody): 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, whereby the individual chains are as follows

[0134] - light chain (variable light chain domain + light chain kappa constant domain)

[0135] - combined light / heavy chain (variable light chain domain + light chain constant domain + peptidic linker + variable heavy chain domain + CHI + Hinge + CH2 + CH3 with knob mutation)

[0136] - heavy chain (variable heavy chain domain + CHI + Hinge + CH2 + CH3 with hole mutation);

[0137] - the two-armed single chain format (= two-armed single chain antibody): 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, whereby the individual chains are as follows

[0138] - combined light / heavy chain 1 (variable light chain domain + light chain constant domain + peptidic linker + variable heavy chain domain + CHI + Hinge + CH2 + CH3 with hole mutation) - combined light / heavy chain 2 (variable light chain domain + light chain constant domain + peptidic linker + variable heavy chain domain + CHI + Hinge + CH2 + CH3 with knob mutation);

[0139] - the common light chain bispecific format (= common light chain bispecific antibody): 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, whereby the individual chains are as follows

[0140] - light chain (variable light chain domain + light chain constant domain)

[0141] - heavy chain 1 (variable heavy chain domain + CHI + Hinge + CH2 + CH3 with hole mutation)

[0142] - heavy chain 2 (variable heavy chain domain + CHI + Hinge + CH2 + CH3 with knob mutation).

[0143] As used herein the term “replaced by each other” with respect to corresponding heavy and light chain domains refers to the aforementioned domain crossovers. As such, when CHI and CL domains are “replaced by each other” it is referred to the domain crossover mentioned under item (i) and the resulting heavy and light chain domain sequence. Accordingly, when VH and VL are “replaced by each other” it is referred to the domain crossover mentioned under item (ii); and when the CHI and CL domains are “replaced by each other” and the VH and VL domains are “replaced by each other” it is referred to the domain crossover mentioned under item (iii).

[0144] Multispecific antibodies also comprise in certain embodiments at least one Fab fragment including either a domain crossover of the CHI and the CL domains as mentioned under item (i) above, or a domain crossover of the VH and the VL domains as mentioned under item (ii) above, or a domain crossover of the VH-CH1 and the VL-VL domains as mentioned under item (iii) above. In case of multi specific antibodies with domain crossover, the Fabs specifically binding to the same antigen(s) are constructed to be of the same domain sequence. Hence, in case more than one Fab with a domain crossover is contained in the multispecific antibody, said Fab(s) specifically bind to the same antigen.

[0145] 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 another embodiment, the antigen is of therapeutic relevance. In still another embodiment, the antigen is specific for or allows delivery to a specific area in the body, such as to a particular organ or cell type or diseased area. This may be a specific structure on the surface of a cell such as a receptor or a tumor marker. In one preferred embodiment, the antigen is human transferrin receptor.

[0146] Preferably, (i) the antibody in the conjugate of the invention comprises two pairs each of a heavy chain and a light chain, wherein each of the heavy chains or / and the light chains comprises the one or more first recognition sites, or (ii) the antibody comprises two pairs each of a heavy chain and a light chain, wherein one of the heavy chains or / and the light chains comprises the one or more first recognition sites, or (iii) the antibody comprises one pair of a heavy chain and a light chain and one additional heavy chain Fc-region, wherein the heavy chain or / and the heavy chain Fc-region fragment or the light chain comprises the one or more first recognition sites.

[0147] According to the present invention, the antibody in the conjugate of the invention includes one or more recognition sites for KalbTG in the heavy chain polypeptide and / or the light chain polypeptide. The recognition site includes a motif for KalbTG, wherein KalbTG can catalyze the formation of an isopeptide bond between the modified antibody and a compound (payload) to be coupled to the modified antibody. Typically, the isopeptide bond is formed between a glutamine (Gin) side chain and a lysine (Lys) side chain. Preferably, the modification introduced into an antibody to obtain the modified antibody of the present invention includes the generation of a (artificial) Q-tag, i.e. a Gin containing motif recognized by the KalbTG, within the antibody heavy and / or light chain polypeptide. Suitable Q-tags are disclosed in WO 2017 / 102759 Al, expressly incorporated by reference herein. The one or more first recognition sites independently of each other comprise or have a Gin-containing motif, especially 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), particularly YRYRQ (SEQ ID NO: 17). The Q-tag may be generated by one or more amino acid modifications, such as substitutions or insertions, preferably insertions. More preferably, the Q-tag is generated by insertion of and / or replacement by an amino acid sequence comprising YRYRQ (SEQ ID NO: 17) or RVRQR (SEQ ID NO: 18), especially 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), especially YRYRQ (SEQ ID NO: 17). In a specific embodiment, the insertion is a Q-tag motif without spacers, i.e. the insertion is consisting of YRYRQ (SEQ ID NO: 17) or RVRQR (SEQ ID NO: 18), particularly YRYRQ (SEQ ID NO: 17). The introduction of the one or more recognition sites for KalbTG may be the only modifications in the constant regions of the light and / or heavy chains. Alternatively further modifications may be present, such as a tag for purification (e.g. a His-tag) or other modifications, e.g. for increasing stability or heterodimerization or effector function modification, either alone or in any combination.

[0148] As stated above, the one or more first recognition site(s) for KalbTG are inserted into the antibody at specific sites, namely at one or more of the positions selected from position 110 (LC110), 143 (LC143) and 214 (LC214) of the light chain and position 118 (HC118), 177 (HC177), 297 (HC297), 341 (HC341), 401 (HC401), and 446 (HC446) of the heavy chain, particularly HC177 and / or HC297 and / or LC214. In certain embodiments, the antibody of the conjugate of the present invention may include a further recognition site for KalbTG at position 446 (HC446) of the heavy chain, i.e. at the C-terminal end, particularly for coupling to a domain different from that coupled at the first recognition site. Preferably, the one or more first recognition sites are independently of each other at position 177 (HC177) or 297 (HC297) of the heavy chain or at position 214 (LC214) of the light chain.

[0149] In certain embodiments, the Q-tag is inserted into the antibody light / heavy chain amino acid sequence via one or two spacer(s). The spacers may increase flexibility or allow for conjugation to larger payloads. In certain embodiments, each spacer sequence comprises independently of each other between 1 to 20 amino acids, preferably 1 to 10 amino acids, more preferred 1 to 5 amino acids, and more preferred said spacer do not interfere essentially with the function of the Q-tag, the KalbTG, the folding of the antibody and the payload to be attached to the modified antibody. The spacer may be N- and / or C-terminally attached to the Q-tag. In certain embodiments, the spacer amino acids are small amino acids, like glycine or serine. Amino acid linkers and their compositions are known in the art (see, e.g. Chichili et al., Prot. Sci. 22 (2013) 153-167). Amino acids glycine, serine, alanine, threonine and glutamate usually constitute amino acids of flexible linkers. Accordingly, the linker(s) may consist primarily or fully of Gly and / or Ser and / or Ala and / or Thr and / or Glu, such as GGGP (SEQ ID NO: 20), ESGS (SEQ ID NO: 21) or APAP (SEQ ID NO: 22). Also, a spacer comprising or consisting of KESGSVSSEQLAQFRSLD (SEQ ID NO: 23) or EGKSSGSGSESKST (SEQ ID NO: 24) may be present. In one preferred embodiment, the spacers are consisting independently of each other primarily or fully of Gly and Ser, such as (GlymSer)nwith m = 1, 2, 3 or 4 and n = 1, 2, 3, 4 or 5, m and n being independently of each other, preferably m=3 and n=l. Preferably, the one or more first recognition sites are inserted into the heavy or / and light chain amino acid sequence via one or two spacer(s) at its termini, especially wherein the spacer consists primarily or fully of Gly and Ser, such as (Gly-Gly-Gly-Ser)n (SEQ ID NO: 19) with n = 1, 2, 3, 4 or 5, preferably n=l.

[0150] If a spacer is present, the insertion of the amino acid sequence X1-YRYRQ-X2 (SEQ ID NO: 17) or X1-RVRQR-X2 (SEQ ID NO: 18) into the antibody is preferred. XI and X2 are independently from each other absent or a spacer, particularly spacer amino acids. 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), especially GGGSYRYRQGGGS (SEQ ID NO: 25).

[0151] An exemplary Fc-region including part of the hinge region with (SEQ ID NO: 28, referred to as 113) and without (SEQ ID NO: 29, referred to as 110) a first recognition site (in bold) has the following sequences:

[0152] 113 : DKTHTCPPCP APEAAGGPSV FLFPPKPKDT LMISRTPEVT

[0153] 110 : DKTHTCPPCP APEAAGGPSV FLFPPKPKDT LMISRTPEVT

[0154] 113 : CVWDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY

[0155] 110 : CVWDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY

[0156] 113 : RWSVLTVLH QDWLNGKEYK CKVSNKALGA PIEKTISKAK

[0157] 110 : RWSVLTVLH QDWLNGKEYK CKVSNKALGA PIEKTISKAK

[0158] 113 : GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE

[0159] 110 : GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE

[0160] 113 : WESNGQPENN YKTTPPVLDS DGGGSYRYRQ GGGSGSFFLY

[0161] 110 : WESNGQPENN YKTTPPVLDS D GSFFLY

[0162] 113 : SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK

[0163] 110 : SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK The nucleic acid encoding the antibody in the conjugate of the present invention can be isolated or generated in vitro to produce the antibody recombinantly. The nucleic acid may be inserted into a replicable vector for further cloning (amplification of DNA) or for further expression.

[0164] “Nucleic acid” has the meaning of comprehensively including DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are basic structural units in nucleic acids, include not only natural nucleotides, but also analogs with modified sugar or base sites. The sequence of the nucleic acid encoding the heavy and light chain variable regions of the present invention can be modified. Such modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides as long as the encoded sequence is not changed.

[0165] The DNA encoding the modified antibody in the conjugate according to the invention can be isolated or synthesized using conventional procedures (e.g., by using an oligonucleotide probe capable of specifically binding to DNA encoding the heavy and light chains of the antibody).

[0166] Many transfer and expression vectors are available. Vector components generally include, but are not limited to, one or more of the following: signal sequence, origin of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences.

[0167] The term "vector" as used herein refers to a plasmid vector as a means for expressing a gene of interest in a host cell; Cozmid vector; viral vectors such as bacteriophage vectors, adenovirus vectors, retroviral vectors and adeno-associated viral vectors, and the like. The nucleic acid encoding the modified antibody in the vector is operably linked to a promoter and a polyadenylation signal sequence.

[0168] “Operatively 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 regulatory factor binding sites) and another nucleic acid sequence, whereby the control sequence controls the other nucleic acid’s transcription and / or translation.

[0169] In the case of using eukaryotic cells as a host, promoters derived from the genome of mammalian cells (e.g., metallothioneine promoter, P-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or mammalian promoters derived from animal viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, Moloney virus promoter, Epstein-Bar virus (EB V) promoter and Rous Sarcoma virus (RSV) promoter) may be used. In addition, a polyadenylation signal sequence is present after the encoding nucleic acid as a transcription termination sequence.

[0170] Cells may be transformed with the aforementioned vectors. The cells used to generate the antibodies used in the conjugate of the present invention may be prokaryotic, yeast or higher eukaryotic cells, but are not limited thereto.

[0171] However, the greatest interest is in animal cells and examples of useful host cell lines are 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, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6 , SP2 / 0, NS-0, U20S, or HT1080, but is not limited thereto. In one preferred embodiment, the host cell is a CHO cell.

[0172] In one aspect, the present invention relates to a conjugate comprising (i) a polypeptide, preferably an antibody as previously described (ii) one or more nonantibody (payload) domain(s) covalently conjugated to the one or more (first) recognition site(s) for KalbTG or a functionally active variant thereof, wherein the non-antibody domain comprises 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.

[0173] As detailed above, the antibody in the conjugate of the present invention is provided for conjugating one or more payloads, such as, e.g., therapeutic moieties, specifically to one or more internal site(s) of the antibody with the aid of KalbTG. Thereby, an ADC is obtained, which may be used in the targeted therapy. The conjugate is capable of binding to a target of interest and thereby transports the payload, e.g., the therapeutic moiety, to the intended tissue or organ in the body. In one preferred embodiment, the modified antibody recognizes a target and binds thereto with its complementarity determining regions (CDRs), particularly wherein the target is a therapeutic target such as, e.g., a biomolecule present on a cell.

[0174] As detailed above, it may be desirable to target a therapeutic moiety to a specific tissue or organ in a patient’s body. This may improve in vivo distribution and reduce adverse side effects. Evidently, it may be intended to deliver a therapeutic moiety to a tissue or organ which is otherwise hard to reach. As an example, it may be envisioned to direct a therapeutic moiety into the brain. Due to the blood-brain- barrier it is difficult to deliver a “naked” therapeutic moiety thereto, if not administered directly into the brain, especially in case the therapeutic moiety exceeds certain size limitations. Therapeutic approaches aiding in overcoming the bloodbrain-barrier and transporting the therapeutic moiety into the brain are evidently of advantage. Markedly, the same approach may be used to direct a therapeutic to another area in the body.

[0175] In the present invention, the molecular recognition units of antibodies specifically binding to structures in the body are employed in the targeting of the therapeutic moiety. In view of the above, it is evident that the therapeutic entity may be any compound useful in the treatment or prevention of the disease of interest, especially a compound, which is to be delivered to a specific tissue in the body, such as to a particular organ or cell type or diseased area.

[0176] The terms “treat”, “treating” and “treatment” are meant to include alleviating or abrogating a condition, disorder, or disease, or one or more of the symptoms associated with the 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 a method of delaying and / or precluding the onset of a condition, disorder, or disease, and / or its attendant symptoms; barring a subject from acquiring a condition, disorder, or disease; or reducing a subject’s risk of acquiring a condition, disorder, or disease.

[0177] For this, a non-antibody payload comprising a therapeutic entity and optionally a second linker is covalently conjugated to the modified antibody in the conjugate of the invention. The therapeutic moiety comprises the active therapeutic entity or a prodrug. Optionally, a second linker may be present.

[0178] In one preferred embodiment, the therapeutic entity is a nucleic acid, such as RNA, siRNA or ASO (anti-sense oligonucleotide), particularly an ASO comprising LNA nucleotides; and / or the therapeutic entity is a toxin or a small organic molecule or an immune modulator.

[0179] In one preferred embodiment, the therapeutic entity is a nucleic acid. The nucleic acid may be for example DNA or RNA or a mixture thereof. The term RNA also includes anti-sense RNAs as well as small interfering RNAs (siRNAs) which is a class of double-stranded, non-coding RNA molecules, typically 20-24 base pairs in length, similar to miRNA, and operating within the RNA interference (RNAi) pathway. It interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, thereby preventing translation. The nucleic acid may also comprise one or more locked nucleic acid (LNA), which is a modified RNA nucleotide 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 can be attributed to the increased stability against enzymatic degradation; moreover, the structure of LNA has improved specificity and affinity as a monomer or a constituent of an oligonucleotide. The LNA nucleotide can be mixed with DNA or RNA residues in the oligonucleotide or nucleic acid.

[0180] Additionally or alternatively, the therapeutic entity may be a small molecule. In the field of pharmacology, a small molecule is of low molecular weight (< 2,500 Daltons, particularly < 1,000 Daltons). Many small molecule therapeutic entities are small organic molecules. Small organic molecules typically bind specific biological macromolecules and act as an effector, 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); they have a beneficial effect against a disease.

[0181] In the present invention, the therapeutic entity is comprised in a non-antibody domain, which is covalently coupled to the modified antibody of conjugate of the invention, optionally via a second linker. The linker may depend on the intended target and therapeutic entity, as the length, rigidity and chemical composition of the linker may impact the conjugation reaction rates and the stability of the resulting conjugatesv

[0182] In one preferred embodiment, the conjugate comprises an antibody and one or more therapeutic nucleic acids, such as ASOs, wherein each therapeutic nucleic acid is linked to a single Q-tag via an amide bond to the terminal residue of the Q-tag via a second linker.

[0183] 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 another embodiment, the conjugate has a DAR of about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8. Upon binding of the antibody to the target, the conjugate is transported into the respective cell by endocytosis, the therapeutic entity is released and can act in the intended manner (e.g., treat a disease).

[0184] In one preferred embodiment, the modified antibody recognizes one target and said target is a receptor inducing receptor-mediated endocytosis, such as, e.g., the human transferrin receptor 1 (TfRl), the human insulin-like growth factor 1 receptor (IGF-1R), the human low density lipoprotein receptor-related protein 1 (LRP1), or the human low density lipoprotein receptor-related protein 8 (LRP8), CD98, or PODXL, particularly TfRl.

[0185] The "transferrin receptor" ("TfR") is a transmembrane glycoprotein (with a molecular weight of about 180,000 Da) composed of two disulphide-bonded subunits (each of apparent molecular weight of about 90,000 Da) involved in iron uptake in vertebrates. In one embodiment, the TfR herein is human TfR comprising the amino acid sequence as in Schneider et al. Nature 311 : 675 - 678 (1984), for example.

[0186] CD98 (also referred to as CD98 heavy chain; 42F heavy chain; SLC3A2) is a type II transmembrane glycoprotein. The human CD98hc sequence is set forth inUNTPROT Accession No. P08195. The protein comprises a 75 amino acid N-terminal intracellular cytoplasmic domain, a single transmembrane domain, and a 426 amino acid C-terminal extracellular domain (Parmacek et al., Nucleic Acids Res. 17: 1915- 1931, 1989). CD98 covalently links via a disulfide bond to one of several light chains (SLC7A5, 6, 7, 8, 10, or 1 1), which are L-type amino acid transporters. This interaction is required for the cell surface expression and amino acid transport function of the light chains. CD98 also associates with integrin P3 subunits, thereby regulating integrin signaling that controls cell proliferation, survival, migration, and epithelial adhesion / polarity (Cai et al., J. Cell Sci. 1 18: 889-899, 2005).

[0187] PODXL, also known as podocalyxin-like protein- 1, PCLP1 or PCX, is a type I transmembrane protein of the sialomucin family of CD34. The human PODXL sequence is set forth in UNIPOROT Accession No. 000592. Preserving the basic structure of family members CD34 and endoglycan, PODXL consists of a highly conserved cytoplasmic domain with a C-terminal PDZ binding region (DTHL motif), a single-pass transmembrane domain, and an extensively O-glycosylated and sialylated extracellular domain A "neurological disorder" as used herein refers to a disease or disorder which affects the CNS and / or which has an etiology in the CNS. Exemplary CNS diseases or disorders include, but are not limited to, neuropathy, amyloidosis, cancer, an ocular disease or disorder, viral or microbial infection, inflammation, ischemia, neurodegenerative disease, seizure, behavioral disorders, and a lysosomal storage disease. For the purposes of this application, the CNS will be understood to include the eye, which is normally sequestered from the rest of the body by the blood-retina barrier. Specific examples of neurological disorders include, but are not limited to, neurodegenerative diseases (including, but not limited to, Lewy body disease, postpoliomyelitis syndrome, Shy-Draeger syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, tauopathies (including, but not limited to, Alzheimer disease and supranuclear palsy), prion diseases (including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutzfeldt- Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsy, motor neuron disease, and nervous system heterodegenerative disorders (including, but not limited to, Canavan disease, Huntington's disease, neuronal ceroid- lipofuscinosis, Alexander's disease, Tourette's syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Halervorden- Spatz syndrome, lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome), dementia (including, but not limited to, Pick's disease, and spinocerebellar ataxia), cancer (e.g. of the CNS and / or brain, including brain metastases resulting from cancer elsewhere in the body).

[0188] For specific receptors, such as, e.g., the TfRl or IGF-1R, upon binding of the antibody 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, thereby transport across the respective barrier is achieved. Thereby, the therapeutic entity is transported to a compartment of the body, which could not have been reached by the therapeutic entity not conjugated to the antibody of the conjugate according to the invention.

[0189] In one highly preferred conjugate, the antibody is capable of binding to a structure allowing crossing the blood-brain barrier, such as the transferrin receptor.

[0190] In a preferred embodiment, the antibody of the conjugate according to the invention recognizes one or further targets, wherein said target(s) is / are specific for a specific cell type, and / or wherein said target(s) is / are therapeutic target(s). In one preferred embodiment, the modified antibody recognizes one or two target(s) and said one or two targets is / are specific for a specific cell type, such as a tumor marker being specific for a tumor cell, such a breast cancer cell. In one preferred embodiment, the therapeutic target is selected from the group comprising a tumor target, a neurological target and a metabolic target. Tumor targets, neurological targets and metabolic targets have been described in the prior art and known for the skilled person.

[0191] In one preferred embodiment, the modified antibody in the conjugate according to the invention is conjugated to a non-antibody domain comprising a therapeutic entity comprising RNA or LNA or an ASO or an siRNA for treating or preventing a brain disease, such a Parkinson’s disease or Alzheimer’s disease.

[0192] In one aspect, the present invention relates to a method of conjugating an antibody to a therapeutic entity, the method comprising a) providing an antibody, b) providing a non-antibody domain, wherein the non-antibody domain comprises (i) a therapeutic entity, (ii) a second recognition site for KalbTG, especially wherein the second recognition site comprises or has a Lys- containing motif, especially the sequence RYESK (SEQ ID NO: 16) , and (iii) optionally a second linker between the therapeutic entity and the second recognition site; and c) reacting the antibody of a) and the non-antibody domain of b) in the presence of KalbTG or a functionally active variant thereof and under conditions conducive to the activity of KalbTG, thereby forming an isopeptide bond between the first and the second recognition site, thus conjugating the antibody to the therapeutic entity.

[0193] In the first steps of the method, the antibody and the non-antibody domain are provided. The non-antibody domain comprises (i) a therapeutic entity, (ii) a second recognition site for KalbTG, especially wherein the second recognition site comprises or has a Lys-containing motif, especially 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 antibody and the non-antibody domain are reacted in the presence of KalbTG or a functionally active variant thereof and under conditions conducive to the activity of KalbTG, thereby an isopeptide bond between the first and the second recognition site is formed, thus conjugating the antibody to the therapeutic entity. In certain embodiments, in this method according to the present invention, the antibody comprises one or more Q-tag(s), which is / 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).

[0194] Microbial transglutaminases (mTGs), including KalbTG catalyze the formation of Gln-Lys isopeptide bonds and are widely used for the cross-linking of proteins and peptides in food and biotechnological applications (e.g. to improve the texture of protein-rich foods or in generating antibody-drug conjugates). KalbTG exhibits essentially no cross-reactivity with known mTG substrates or commonly used target proteins, such as antibodies and therefore allows for the specific conjugation at predetermined sites. Accordingly, essentially any payload comprising a second recognition site (K-tag) for KalbTG, especially wherein the second recognition site comprises or has a Lys-containing motif, especially the sequence RYESK (SEQ ID NO: 16), may be coupled to the modified antibody at the one or more first recognition site(s). KalbTG or a functionally active variant thereof may be as defined in Steffen at al. (2017) or WO 2016 / 100735 Al, both expressly incorporated by reference herein. The functionally active variant may be a transglutaminase having at least 80%, 90%, 95% or 99% sequence identity to the KalbTG of WO 2016 / 100735 Al (see SEQ ID NO: 6 therein). Alternatively, the KalbTG or a functionally active variant thereof may be part of a fusion protein additionally comprising a label such as a tag, e.g. for purification purposes.

[0195] In certain embodiments, the KalbTG comprises the amino acid sequence

[0196] Met Hi s Lys Trp Phe Leu Arg Ala Ala Vai Vai Ala Ala Vai Gly Phe Gly Leu Pro Thr Leu l ie Ala Thr Thr Ala Gin Ala Ala Ala Vai Ala Ala Pro Thr Pro Arg Ala Pro Leu Ala Pro Pro Leu Ala Glu Asp Arg Ser Tyr Arg Thr Trp Arg Vai Glu Asp Tyr Vai Glu Ala Trp Glu Arg Tyr Hi s Gly Arg Glu Met Thr Glu Asp Glu Arg Glu Asn Leu Ala Arg Gly Cys T ie Gly Vai Thr Vai Vai Asn Leu Asn Arg Glu Asp Leu Ser Asn Pro Pro Leu Asn Leu Ser Phe Gly Ser Leu Arg Thr Ala Glu Ala Vai Gin Ala Ala Leu Asn Lys T ie Vai Asp Thr His Pro Ser Pro Ala Gin Tyr Glu Ala Ala Vai Ala Lys Asp Pro T ie Leu Lys Arg Leu Lys Asn Vai Vai Lys Ala Leu Pro Ser Trp T ie Asp Ser Ala Lys Leu Lys Ala Ser T ie Phe Ser Lys Arg Phe Tyr Ser Trp Gin Asn Pro Asp Trp Ser Glu Glu Arg Ala His Thr Thr Tyr Arg Pro Asp Arg Glu Thr Asp Gin Vai Asp Met Ser Thr Tyr Arg Tyr Arg Ala Arg Pro Gly Tyr Vai Asn Phe Asp Tyr Gly Trp Phe Asp Gin Asp Thr Asn Thr Trp Trp Hi s Ala Asn Hi s Glu Glu Pro Arg Met Vai Vai Tyr Gin Ser Thr Leu Arg Hi s Tyr Ser Arg Pro Leu Gin Asp Phe Asp Glu Gin Vai Phe Thr Vai Ala Phe Ala Lys Lys Asp

[0197] (SEQ ID NO: 27).

[0198] In certain embodiments, in the method according to the present invention said coupling is controlled and, for example, achieved in a stoichiometric ratio of nonantibody domain and modified antibody, for example at about 1 : 1. Multiple conjugation can also be achieved by using more than one first recognition sites on one antibody in order to attach two or even multiple payloads to the modified antibody.

[0199] All the conjugates tested, including the antibody-BCN linker-nucleic acid conjugate, and the three antibody-DBCO linker (A3, A6, AmTEG)-nucleic acid conjugates comprised the same Brainshuttle™ (BrS) antibody and the same nucleic acid, in particular the same antisense oligonucleotide (ASO).

[0200] The following Table provides an overview of the dibenzocyclooctyne (DBCO)- based linkers within the scope of the present invention that have been tested in a two- step process of a first enzymatic conjugation of a K-amino acid sequence-linker- conjugate (7456, including RYESK, SEQ ID NO: 16) to antibodies with Q-amino acid sequences followed by the conjugation of a nucleic acid (in this case an ASO) to the antibody-K-amino acid sequence-linker conjugate to obtain a polypeptide(antibody)-linker-nucleic acid conjugate by click chemistry. The resulting conjugated linker parts as shown as A3 conjugate (based on DBCO-A3 linker), A6 conjugate (based on DBCO-A6 linker) and Am TEG conjugate (based on DBCO-AmTEG conjugate). A BCN-based linker was also tested for comparative purposes, as shown in the following Table. The results of the two-step process are shown in the following Table. As the antibody comprises only a single Q-amino acid sequence the target DAR is one.

[0201] *corrected for the amount of non-acidified antibody

[0202] All DBCO-based linkers tested (A3, A6, Am TEG) showed conjugation efficiency and conjugate quality that are improved, better than those obtained with the BCN- based linker, especially at reduced excess.

[0203] Additionally serum stability of the conjugates has been tested. The results are shown in the following Table.

[0204] It can be seen that the conjugates are stable, the conjugate comprising the linker A6 being the most stable after an incubation period of 48h.

[0205] The relative accumulation rate (or relative LUCA rate) of the conjugates of the invention has been tested. The relative accumulation rate measures the cell nonspecific uptake, so the lower the relative accumulation value, the more specific the cellular uptake is. The results are shown in the following Table: The lowest cellular accumulation rate was shown for the conjugate with linker A3, followed by the conjugate with the linker Am TEG.

[0206] In vitro potency and efficacy of different antibody-DBCO linker-ASO conjugates according to the invention was evaluated. Figure 1 shows the target expression decrease for different antibody-DBCO / BCN-linker-ASO conjugates (A) and for DBCO / BCN-linker-ASO alone (B). The results showed a strong knock-down of the antibody-DBCO linker-ASO conjugates according to the invention (noted as BrS- A6-ASO, BrS-AmTEG-ASO and BrS-A3-ASO conjugates) compared to the conjugate with the BCN linker (A, noted as BrS-BCN ASO conjugate, diamond symbol).

[0207] Figure 2 shows the in vitro potency and efficacy of different antibody-DBCO-linker- ASO conjugates according to the invention. Results showed a strong knockdown by the antibody-DBCO-linker-ASO conjugates (Figure 2 A, B, C) compared to the antibody-BCN-linker-ASO conjugates (Figure 2, D). The highest knockdown and most efficient dose-response is shown for the A3 variant conjugate (B, black line with triangle symbols), superior to the ASO alone variant (B, grey line with circle symbols).

[0208] While the application of the BCN-linker-conjugate (Figure 2, D) led to a 40% knockdown of the target gene expression, the conjugates variants with DBCO- linkers (Figure 2, A-C) showed a higher knockdown efficiency of 60%. In comparison to the BCN-ASO (KD 60%), the BrS-BCN-ASO conjugate has a lower efficiency (40%) (Figure 2, D). However, for the conjugates with the DBCO linker, the conjugates reach a similar level of knockdown comparable to their ASO alone counterparts (Figure 2, A, B, C). Of interest, within the 3 variants of the DBCO linker conjugates, the DBCO-A3 linker conjugate (Figure 2, B) showed the best doseresponse curve to their ASO-alone counterparts. IC50 values, maximal knockdown (KD) and area-under-curve values of DBCO-conjugates and ASO-linker alone counterparts are shown in the following Table.

[0209] Figure 3 shows the cellular accumulation rate of BCN-linker- ASO Brainshuttle™ (BrS) conjugate versus different DBCO-linker-Brainshuttle™ (BrS)-ASO conjugates. The lowest uptake corresponds to the BrS-DBCO-A3-ASO conjugate. The BrS-DBCO linker- ASO conjugates decrease cellular accumulation rate (A3, Am TEG) or show the same level on cellular accumulation rate (A6) compared to the conjugate with the BCN linker. The lowest cellular accumulation rate is shown in the BrS-DBCO-A3-ASO conjugate (A3 variant conjugate).

[0210] Overall, linker A3 shows the best performance of the DBCO linkers. One aspect of the present invention is a conjugate comprising a polypeptide, a nucleic acid and a linker, characterized in that:

[0211] The linker comprises the following chemical structure (A): The linker connects the polypeptide and the nucleic acid, ie the polypeptide and the nucleic acid are not connected directly to each other by a covalent bond,

[0212] The linker is conjugated to the C-terminal amino acid of the polypeptide at the position noted as 1 in structure (A), either directly by a covalent bond or via an additional spacer,

[0213] The linker is conjugated to an oxygen linked to a phosphor of the oxidation state V of the nucleic acid at the position noted as 2 in structure (A), either directly by a covalent bond or via an additional spacer, a ranges from 0 to 20, b ranges from 0 to 20, each R1 is independently selected from hydrogen, halogen, hydroxy, alcoxy, nitrate, nitrite, and Cl -C10 organic group, and

[0214] - R2 is selected from NH2, OH, a monosubstituted amine residue (such as NH- Alkyl), a disubstituted amine residue (such as N(Alkyl)l(Alkyl)2), wherein in a preferred embodiment R2 is NH2.

[0215] In an aspect of the conjugate of the invention, the linker comprising the chemical structure (A), which connects the polypeptide and the nucleic acid, is conjugated to the C-terminal amino acid of the polypeptide at the position noted as 1 in structure (A) directly by a covalent bond. In an alternative embodiment, the linker comprising the chemical structure (A), which connects the polypeptide and the nucleic acid, is conjugated to the C-terminal amino acid of the polypeptide at the position noted as 1 in structure (A) via an additional spacer.

[0216] In an aspect of the conjugate of the invention, the linker comprising the chemical structure (A), which connects the polypeptide and the nucleic acid, is conjugated to an oxygen linked to a phosphor of the oxidation state V of the nucleic acid at the position noted as 2 in structure (A) directly by a covalent bond. In an alternative aspect of the conjugate of the invention, the linker comprising the chemical structure (A), which connects the polypeptide and the nucleic acid, is conjugated to an oxygen linked to a phosphor of the oxidation state V of the nucleic acid at the position noted as 2 in structure (A) via an additional spacer.

[0217] In an aspect of the conjugate of the invention, the a value according to the chemical structure (A) ranges from 0 to 20, i.e. the number of -CH2- groups ranges from 0 (no -CH2- group) to 20. In a particular aspect, the a value ranges from 1 to 15. In a more particular aspect, the a value ranges from 1 to 12, more particularly the a value ranges from 1 to 10, more particularly the a value ranges from 1 to 8. In a more preferred aspect, the a value ranges from 1 to 5. In a more preferred aspect, the a value is 2.

[0218] In an aspect of the conjugate of the invention, the b value according to the chemical structure (A) ranges from 0 to 20, i.e. the number of -CH2- groups ranges from 0 (no -CH2- group) to 20. In a particular aspect, the b value ranges from 0 to 15. In a more particular aspect, the b value ranges from 1 to 12, more particularly from 1 to 10, more particularly from 1 to 8, more particularly from 1 to 6. In a more preferred aspect, the b value is 4.

[0219] In an aspect of the conjugate of the invention, R1 according to the chemical structure (A) is independently selected from hydrogen, halogen, hydroxy, alcoxy, nitrate, nitrite, and Cl -CIO organic group, more particularly a C1-C8 organic group, more particularly a C1-C6 organic group, more particularly a C1-C4 organic group. In one aspect of the conjugate of the invention, up to three R1 according to the chemical structure (A) are not hydrogen (H). In a preferred aspect of the conjugate of the invention, all R1 according to the chemical structure (A) are hydrogen (H).

[0220] The term "organic group" is used for the purpose of this invention to mean a hydrocarbon group that is classified as an aliphatic group, cyclic group, or combination of aliphatic and cyclic groups (e.g., alkaryl and aralkyl groups). In the context of the present invention, suitable organic groups for compounds of this invention are those that do not interfere with the reaction of an alkyne with a 1,3- dipole-fimctional compound to form a heterocyclic compound. In the context of the present invention, the term "aliphatic group" means a saturated or unsaturated linear or branched hydrocarbon group. This term is used to encompass alkyl, alkenyl, and alkynyl groups, for example. The term "alkyl group" means a saturated linear or branched monovalent hydrocarbon group including, for example, methyl, ethyl, n- propyl, isopropyl, tert-butyl, amyl, heptyl, and the like. The term "alkenyl group" means an unsaturated, linear or branched monovalent hydrocarbon group with one or more olefinically unsaturated groups (i.e., carbon-carbon double bonds), such as a vinyl group. The term "alkynyl group" means an unsaturated, linear or branched monovalent hydrocarbon group with one or more carbon-carbon triple bonds. The term "cyclic group" means a closed ring hydrocarbon group that is classified as an alicyclic group, aromatic group, or heterocyclic group. The term "alicyclic group" means a cyclic hydrocarbon group having properties resembling those of aliphatic groups. The term "aromatic group" or "aryl group" means a mono- or polynuclear aromatic hydrocarbon group. The term "heterocyclic group" means a closed ring hydrocarbon in which one or more of the atoms in the ring is an element other than carbon (e.g., nitrogen, oxygen, sulfur, etc.).

[0221] In an aspect of the conjugate of the invention, R2 according to the chemical structure (A) is selected from NH2, OH, a monosubstituted amine residue (such as NH-alkyl), and a disubstituted amine residue (such as N(alkyl)l(alkyl)2). In a preferred aspect of the conjugate of the invention, R2 is NH2.

[0222] In an aspect of the invention, the conjugate of the invention comprises a polypeptide, a nucleic acid and a linker, wherein: the linker comprises the following chemical structure (A)

[0223]

[0224] The linker connects the polypeptide and the nucleic acid,

[0225] The linker is conjugated to the C-terminal amino acid of the polypeptide at the position noted as 1 in structure (A), either directly by a covalent bond or via an additional spacer,

[0226] The linker is conjugated to an oxygen linked to a phosphor of the oxidation state V of the nucleic acid at the position noted as 2 in structure (A), either directly by a covalent bond or via an additional spacer, a value is 2

[0227] - b value is 4, all R1 are hydrogen (H), and

[0228] - R2 is NH2.

[0229] Thus, one aspect of the present invention is a conjugate comprising a polypeptide, a nucleic acid and a linker, characterized in that:

[0230] The linker comprises the following chemical structure (1):

[0231]

[0232] The linker connects the polypeptide and the nucleic acid,

[0233] The linker is conjugated to the C-terminal amino acid of the polypeptide by the position noted as 1 in structure (1), either directly by a covalent bond or via an additional spacer,

[0234] The linker is conjugated to an oxygen linked to a phosphor of the oxidation state V of the nucleic acid by the position noted as 2 in structure (1), either directly by a covalent bond or via an additional spacer.

[0235] In an aspect of the conjugate of the invention, the linker comprising the chemical structure (1), which connects the polypeptide and the nucleic acid, is conjugated to the C-terminal amino acid of the polypeptide at the position noted as 1 in structure (1) directly by a covalent bond. In an alternative embodiment, the linker comprising the chemical structure (1), which connects the polypeptide and the nucleic acid, is conjugated to the C-terminal amino acid of the polypeptide at the position noted as 1 in structure (1) via an additional spacer.

[0236] In an aspect of the conjugate of the invention, the linker comprising the chemical structure (1), which connects the polypeptide and the nucleic acid, is conjugated to an oxygen linked to a phosphor of the oxidation state V of the nucleic acid at the position noted as 2 in structure (1) directly by a covalent bond. In an alternative aspect of the conjugate of the invention, the linker comprising the chemical structure (1), which connects the polypeptide and the nucleic acid, is conjugated to an oxygen linked to a phosphor of the oxidation state V of the nucleic acid at the position noted as 2 in structure (1) via an additional spacer.

[0237] In an aspect of the conjugate of the invention, the oxygen linked to the phosphor of the oxidation state V (i.e. a phosphorous atom of the oxidation state V) is at the 5’ terminus of the nucleic acid.

[0238] In an aspect of the conjugate of the invention, the oxygen is linked to the phosphor of the oxidation state V (i.e. a phosphorous atom of the oxidation state V) in a phosphate or phosphorothioate.

[0239] In an aspect of the conjugate of the invention, the linker is conjugated to the nucleic acid via an amide bond.

[0240] In an aspect of the conjugate of the invention, the linker is conjugated to the nucleic acid via an unbranched spacer of formula (CH2)n interspaced between the amide bond and the oxygen linked to the phosphor of the nucleic acid, i.e. the carbonyl group of (1) is conjugated to an N(H)(CH2)n group spacer.

[0241] In an aspect of the conjugate of the invention, the linker is conjugated to the nucleic acid via an amide bond, wherein the amino group in the amide bond between the linker and the nucleic acid is provided by a compound selected from the group consisting of metanamide, etanamide, propanamide, butanamide, pentanamide, hexanamide, heptanamide and octanamide nonanamide and decanamide.

[0242] In a preferred aspect of the conjugate of then invention, the linker is conjugated to the nucleic acid via an amide bond, wherein the amino group in the amide bond is provided by a compound selected from the group consisting of metanamide, etanamide, propanamide, butanamide, pentanamide, hexanamide, heptanamide and octanamide nonanamide and decanamide; more preferably wherein the amino group in the amide bond is provided by a compound selected from propanamide and hexanamide. In an aspect of the conjugate of the invention, the linker is conjugated to 5’end of the nucleic acid via a spacer selected from a group consisting of -CH2- group, a CH2- CH2- group, a -CH2-CH2-CH2- group, a -CH2-CH2-CH2-CH2- group, a -CH2- CH2-CH2-CH2-CH2- group, a -CH2-CH2-CH2-CH2-CH2-CH2- group, a -CH2- CH2-CH2-CH2-CH2-CH2- group, and a -CH2-CH2-CH2-CH2-CH2-CH2-CH2- group. In a preferred aspect of the conjugate of the invention, the linker is conjugated to 5’end of the nucleic acid via a spacer selected from a -CH2-CH2-CH2- group and a -CH2-CH2-CH2-CH2-CH2-CH2- group.

[0243] In an aspect of the conjugate of the invention, the linker is conjugated to the nucleic acid via an amide bond. In a preferred aspect, the linker is conjugated to 5’end of the nucleic acid via a spacer selected from a group consisting of -CH2- group, a CH2- CH2- group, a -CH2-CH2-CH2- group, a -CH2-CH2-CH2-CH2- group, a -CH2- CH2-CH2-CH2-CH2- group, a -CH2-CH2-CH2-CH2-CH2-CH2- group, a -CH2- CH2-CH2-CH2-CH2-CH2- group, and a -CH2-CH2-CH2-CH2-CH2-CH2-CH2- group. In a preferred aspect of the conjugate of the invention, the linker is conjugated to 5’end of the nucleic acid via a spacer selected from a -CH2-CH2-CH2- group and a -CH2-CH2-CH2-CH2-CH2-CH2- group. In a more preferred aspect, the linker is conjugated to the nucleic acid via a spacer selected from a -NH-CH2-CH2-CH2- group and a -NH-CH2-CH2-CH2-CH2-CH2-CH2- group.

[0244] In an aspect of the conjugate of the invention, the conjugate comprises the following structure: The conjugate of the invention comprising the structure above comprises a DBCO- based linker referred to as A3 or DBC0-A3 or DBCO-based A3 linker.

[0245] In an aspect of the conjugate of the invention, the conjugate comprises the following structure:

[0246] The conjugate of the invention comprising the structure above comprises a DBCO- based linker referred to as A6 or DBCO-A6 or DBCO-based A6 linker.

[0247] In an aspect of the conjugate of the invention, the linker is conjugated to the nucleic acid via n units of ethylene glycol (PEG). In a preferred aspect, the n value is n=3 or n=4.

[0248] In an aspect of the conjugate of the invention, the conjugate comprises the following structure:

[0249]

[0250] The conjugate of the invention comprising the structure above comprises a DBCO- based linker referred to as AmTEG or DBCO-AmTEG or DBCO-based AmTEG linker. In an aspect of the conjugate of the invention, the linker is conjugated directly at the position noted as 2 in (1) to the oxygen linked to the phosphor of the oxidation state V of the nucleic acid.

[0251] In an aspect of the conjugate of the invention, the linker is conjugated to the C- terminal amino acid of the polypeptide by direct covalent binding between the C- terminal amino acid of the polypeptide and the position noted as 1 in (1) via an -NH- group.

[0252] In an aspect of the conjugate of the invention, the linker is conjugated to the C- terminal amino acid of the polypeptide by direct covalent binding between the C- terminal amino acid of the polypeptide and the position noted as 1 in (1) via an amide bond.

[0253] In an aspect of the conjugate of the invention, the linker is conjugated to the carboxy group at the C terminus of the polypeptide via -NH-(PEG)m-propionate-NH-. In a particular aspect of the invention, m=3 or m=4. In an aspect of the conjugate of the invention, the linker is conjugated to the C- terminal amino acid of the polypeptide by direct covalent binding between the C- terminal amino acid of the polypeptide and the position noted as 1 in (1).

[0254] In an aspect of the conjugate of the invention, the conjugate comprises the following structure:

[0255] In an aspect of the conjugate of the invention, the polypeptide comprises a lysine (K, Lys) residue.

[0256] In an aspect of the conjugate of the invention, the polypeptide comprises a lysine residue, wherein said lysine residue belongs to a tag sequence RYESK.

[0257] In an aspect of the conjugate of the invention, the polypeptide comprises a lysine residue, wherein said lysine residue belongs to a tag sequence RYESK in N- to C- terminal direction comprised at the C-terminal end of the polypeptide, wherein the carboxy group of said lysine is conjugated to the linker via an amide bond. In an aspect of the conjugate of the invention, the polypeptide comprises the K-amino acid sequence RYESK, wherein the conjugate comprises the following structure:

[0258]

[0259] In an aspect of the conjugate of the invention, the conjugate comprises the following structure: In an aspect of the conjugate of the invention, the conjugate comprises the following structure:

[0260]

[0261] In an aspect of the conjugate of the invention, the conjugate comprises the following structure: In an aspect of the conjugate of the invention, the polypeptide comprises the K-amino acid sequence RYESK, wherein the epsilon amino group of the lysine (K) is linked via an isopeptide bond to a glutamine (Q) residue side chain of a second polypeptide, wherein the glutamine residue is within a Q-amino acid sequence (Q-tag) of at least 5 amino acid residues. In a particular aspect of the invention, the second polypeptide is an antibody, preferably an antibody as described previously in the application.

[0262] In an aspect of the conjugate of the invention, the polypeptide comprises the K-amino acid sequence RYESK, wherein the epsilon amino group of the lysine in the K-amino acid sequence is linked by an isopeptide bond to a glutamine residue side chain of a second polypeptide, wherein 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). In a particular aspect of the invention, the second polypeptide is an antibody, preferably an antibody as described previously in the application.

[0263] In an aspect of the conjugate of the invention, the polypeptide comprises the K-amino acid sequence RYESK, wherein the epsilon amino group of the lysine in the K-amino acid sequence is linked by an isopeptide bond to a glutamine residue side chain of a second polypeptide, wherein 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), wherein the Q-amino acid sequence comprises one or two spacer(s) directly at its termini. In a particular aspect of the invention, the second polypeptide is an antibody, preferably an antibody as described previously in the application.

[0264] In an aspect of the conjugate of the invention, the polypeptide comprises the K-amino acid sequence RYESK, wherein the epsilon amino group of the lysine in the K-amino acid sequence is linked by an isopeptide bond to a glutamine residue side chain of a second polypeptide, wherein 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), wherein the Q-amino acid sequence comprises one or two spacer(s) consisting primarily or fully of Gly and Ser directly at its termini. In a particular aspect of the invention, the second polypeptide is an antibody, preferably an antibody as described previously in the application.

[0265] In an aspect of the conjugate of the invention, the polypeptide comprises the K-amino acid sequence RYESK, wherein the epsilon amino group of the lysine in the K-amino acid sequence is linked by an isopeptide bond to a glutamine residue side chain of a second polypeptide, wherein 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 a particular aspect, the glutamine residue is within the Q-amino acid sequence YRYRQ (SEQ ID NO: 17), wherein the Q-amino acid sequence comprises one or two spacer(s) consisting independently of each other of (Gly-Gly-Gly-Ser)p (SEQ ID NO: 19) with p = 1, 2, 3, 4 or 5, preferably p=l, directly at its termini. In a particular aspect of the invention, the second polypeptide is an antibody, preferably an antibody as described previously in the application.

[0266] In an aspect of the conjugate of the invention, the polypeptide comprises the K-amino acid sequence RYESK, wherein the epsilon amino group of the lysine in the K-amino acid sequence is linked by an isopeptide bond to a glutamine residue side chain of a second polypeptide, wherein 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), wherein the Q-amino acid sequence is at a position selected from position 110 (LC110), position 143 (LC143) and position 214 (LC214) of an antibody light chain and position 118 (HC118), position 177 (HC177), position 297 (HC297), position 341 (HC341), position 401 (HC401) and position 446 (HC446) of an antibody heavy chain (numbering according to Kabat). In a particular aspect of the invention, the second polypeptide is an antibody, preferably an antibody as described previously in the application.

[0267] In an aspect of the conjugate of the invention, the polypeptide comprises the K-amino acid sequence RYESK, wherein the epsilon amino group of the lysine in the K-amino acid sequence is linked by an isopeptide bond to a glutamine residue side chain of a second polypeptide, wherein 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), wherein the Q-amino acid sequence is at a position selected from position 214 (LC214) of an antibody light chain, position 177 (HC177) of an antibody heavy chain, and position 297 (HC297) of an antibody heavy chain (numbering according to Kabat). ). In a particular aspect of the invention, the second polypeptide is an antibody, preferably an antibody as described previously in the application.

[0268] In an aspect of the conjugate of the invention, the polypeptide comprises the K-amino acid sequence RYESK, wherein the epsilon amino group of the lysine (K) is linked via an isopeptide bond to a glutamine (Q) residue side chain of a second polypeptide, wherein the glutamine residue is within a Q-amino acid sequence (Q-tag) of at least 5 amino acid residues.

[0269] In a particular aspect of the conjugate of the invention, the Q-amino acid sequence is in a chain of an antibody that comprises at least one light chain, preferably complete light chain, and at least one heavy chain, preferably complete heavy chain.

[0270] In a particular aspect of the conjugate of the invention, the Q-amino acid sequence is in one chain of an antibody that comprises two light chains, preferably two complete light chains, and two heavy chains, preferably two complete heavy chains.

[0271] In a particular aspect of the conjugate of the invention, the Q-amino acid sequence is in two chains of an antibody that comprises two light chains, preferably two complete light chains, and two heavy chains, preferably two complete heavy chains.

[0272] In a particular aspect of the conjugate of the invention, the light chain constant domain not comprising a 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 NO: 6 or 7; and / or wherein the heavy chain constant region not comprising a 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 NO: 1 to 5.

[0273] In a particular aspect of the conjugate 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 wherein 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 30.

[0274] In a particular aspect of the conjugate of the invention, the antibody recognizes a target and binds thereto with its complementarity determining regions (CDRs), particularly wherein the target is a biomolecule present on a cell. In a particular aspect of the invention, the biomolecule present on a cell is a cell receptor. In a more particular aspect of the invention, the cell receptor is located in the surface of the cell.

[0275] In a particular aspect of the conjugate of the invention, the nucleic acid is selected from the group consisting of a RNA, siRNA, anti-sense oligonucleotide (ASO), LNA, and an ASO comprising LNA nucleotides. In a more particular aspect of the conjugate of the invention, the nucleic acid is an ASO. In an alternative particular aspect of the invention, the nucleic acid is a siRNA.

[0276] In a particular aspect of the conjugate of the invention: a) the antibody recognizes one target and said target is a receptor inducing receptor-mediated endocytosis, such as transferrin receptor 1 (TfRl), insulin-like growth factor 1 receptor (IGF-1R), low density lipoprotein receptor-related protein 1 (LRP1), low density lipoprotein receptor-related protein 8 (LRP8), CD98, or PODXL, particularly TfRl; and / or b) the antibody recognizes one or further target(s) and said one or further targets is / are specific for a specific cell type, and / or said one of further target(s) is / are therapeutic targets.

[0277] In a particular aspect of the conjugate of the invention, the antibody recognizes one or further therapeutic target(s). In a more particula aspect, the therapeutic target is selected from the group comprising tumor targets, neurological targets and metabolic targets.

[0278] In an aspect of the invention, it relates to a compound of the following formula:

[0279] In a particular aspect, the invention relates to a compound of the formula as above for forming a conjugate according to the invention.

[0280] In an aspect of the invention, it relates to a compound of the following formula:

[0281] In a particular aspect, the invention relates to a compound of the formula as above for forming a conjugate according to the invention.

[0282] In an aspect of the invention, it relates to a compound of the following formula: In a particular aspect, the invention relates to a compound of the formula as above for forming a conjugate according to the invention.

[0283] In an aspect of the invention, it relates an azide of the following formula:

[0284]

[0285] In a particular aspect, the invention relates to an azide of the formula as above for forming a conjugate according to the invention.

[0286] In a particular aspect, the invention relates to a method for producing a polypeptide- linker-nucleic acid conjugate according to the invention comprising the following steps: a) providing an antibody comprising a Q-amino acid sequence of RYGQR

[0287] (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 position 110 (LC110), position 143 (LC143) and position 214 (LC214) of an antibody light chain and position 118 (HC118), position 177 (HC177), position 297 (HC297), position 341 (HC341), position 401 (HC401) and position 446 (HC446) of an antibody heavy chain (numbering according to Kabat), b) providing a polypeptide comprising the amino acid sequence RYESK

[0288] (SEQ ID NO: 16), which is conjugated to the terminal amino group of (3-[2-[2-(2-aminoethoxy)ethoxy]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 the (3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propanamido)-6- azidohexanami de, c) reacting the antibody of a) and the polypeptide of b) in the presence of KalbTG or a functionally active variant thereof and under conditions conducive to the activity of KalbTG, thereby forming a isopeptide bond between the Q-amino acid sequence of the antibody and the polypeptide comprising the amino acid sequence of RYESK, thus producing an (3-[2-[2-(2-antibody aminoethoxy)ethoxy]ethoxy]propanamido)-6-azidohexanamide (antibody azide), d) reacting the antibody azide with the conjugate as previously described, and thereby producing the polypeptide-linker-nucleic acid conjugate of the invention as previously described.

[0289] In a particular aspect of the method of the invention, the reaction product of step c) as described above comprises the following structure: isopeptide bond to glutamine In a particular aspect of the method of the invention, the reaction product of step d) as described above comprises the following structure: Medical uses of the conjugates of the invention

[0290] In another embodiment, the invention relates to the conjugate as previously described, or the polypeptide-linker-nucleic acid conjugate produced according to the method as previously described, for its use as a medicament.

[0291] In an alternative embodiment, the invention relates to the conjugate as previously described, or the polypeptide-linker-nucleic acid conjugate produced according to the method previously described, for its use in the manufacture of a medicament.

[0292] In an alternative embodiment, the invention relates to the use of the conjugate as previously described, or the polypeptide-linker-nucleic acid conjugate produced according to the method as previoulse described, in the manufacture of a medicament.

[0293] In another embodiment, the invention relates to the conjugate as previously described, or the polypeptide-linker-nucleic acid conjugate as previously described for use in treating a neurological disease or a brain disease. In a preferred embodiment, said neurological disease or brain disease is Alzheimer’s disease. In an alternative preferred embodiment, said neurological disease or brain disease or Parkinson’s disease.

[0294] In an alternative embodiment, the invention relates to the conjugate as previously described, or the polypeptide-linker-nucleic acid conjugate produced according to the method as previously described for use in treating cancer. In a preferred embodiment, the cancer is breast cancer.

[0295] In an alternative embodiment, the invention relates to a method for the treatment of a neurological disease or a brain disease in a subject in need thereof comprising the administration to said subject of a therapeutically effective amount of the conjugate according to the invention as previously described, or the polypeptide-linker-nucleic acid conjugate produced according to the method of the invention as previously described. In a preferred embodiment, said neurological disease or brain disease is Alzheimer’s disease. In an alternative preferred embodiment, said neurological disease or brain disease or Parkinson’s disease.

[0296] In an alternative embodiment, the invention relates to a method for the treatment of cancer in a subject in need thereof comprising the administration to said subject of a therapeutically effective amount of the conjugate according to the invention as previously described, or the polypeptide-linker-nucleic acid conjugate produced according to the method of the invention as previously described. In a preferred embodiment, the cancer is breast cancer.

[0297] In an alternative embodiment, the invention relates to the conjugate as previously described, or the polypeptide-linker-nucleic acid conjugate produced according to the method as previously described for its use in the manufacture of a medicament for treating a neurological disease or a brain disease. In a preferred embodiment, said neurological disease or brain disease is Alzheimer’s disease. In an alternative preferred embodiment, said neurological disease or brain disease or Parkinson’s disease.

[0298] In an alternative embodiment, the invention relates to the conjugate as previously described, or the polypeptide-linker-nucleic acid conjugate produced according to the method as previously described, for its use in the manufacture of a medicament for treating cancer. In a preferred embodiment, the cancer is breast cancer.

[0299] In an alternative embodiment, the invention relates to the use of the conjugate as previously described, or the polypeptide-linker-nucleic acid conjugate produced according to the method as previously described in the manufacture of a medicament for treating a neurological disease or a brain disease. In a preferred embodiment, said neurological disease or brain disease is Alzheimer’s disease. In an alternative preferred embodiment, said neurological disease or brain disease or Parkinson’s disease.

[0300] In an alternative embodiment, the invention relates to the use of the conjugate as previously described, or the polypeptide-linker-nucleic acid conjugate produced according to the method as previously described, in the manufacture of a medicament for treating cancer. In a preferred embodiment, the cancer is breast cancer.

[0301] In one aspect, the covalent conjugate comprising the modified antibody of the present invention or the linker according to the invention or produced according to the method of the present invention is for use as a medicament, particularly for use in treating a neurological disease or a brain disease, such as Alzheimer’s disease or Parkinson’s disease or for use in treating cancer, such as breast cancer.

[0302] In certain embodiments, the disease is a neurological disease. In certain embodiments, the neurological disease is selected from the group consisting of a neuropathy disorder, a neurodegenerative disease, cancer, an ocular disease disorder, a seizure disorder, a lysosomal storage disease, amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, CNS inflammation, Alzheimer’s Disease, Parkinson’s Disease, multiple sclerosis, CD20 positive cancer with brain metastases, and HER2 positive cancer with brain metastases.

[0303] In certain embodiments, the neurological disease is selected from the group consisting of a neuropathy disorder, a neurodegenerative disease, cancer, an ocular disease disorder, a seizure disorder, a lysosomal storage disease, amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, and CNS inflammation.

[0304] As detailed above, the conjugates comprising the modified antibody in the conjugate according to the invention or the linker according to the invention may be used as a medicament, particularly in the treatment of a neurological disease or a brain disease, such as Alzheimer’s disease or Parkinson’s disease, or for use in treating cancer, such as breast cancer.

[0305] The conjugate may be encompassed in a composition. Such a composition also referred to as pharmaceutical composition, is a composition intended for use in the pharmaceutical field or as pharmaceutic and refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the pharmaceutical composition would be administered. It may optionally contain a pharmaceutically acceptable excipient, diluent or carrier, such as buffer substances, stabilizers or preservative and optionally further active ingredients, especially ingredients known in connection with pharmaceutical compositions.

[0306] In general, the nature of the additional ingredient will depend on the particular form of pharmaceutical composition and the mode of administration being employed. Pharmaceutically acceptable carriers enhance or stabilize the composition, or can be used to facilitate preparation of the composition. Such carriers may include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, solvents, dispersion media, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible as well as combinations thereof. The formulation should suit the mode of administration. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like.

[0307] The pharmaceutical composition may comprise a stabilizer. The term "stabilizer" refers to a substance, which protects the composition from adverse conditions, such as those which occur during heating or freezing, and / or prolongs the stability or shelf-life of the conjugate of the invention in a condition or state. 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.

[0308] Typically, a therapeutically effective dose or efficacious dose of the conjugate is employed in the pharmaceutical compositions of the invention. The amount of conjugate administered can be initially determined based on guidance of a dose and / or dosage regimen of a comparable uncoupled therapeutic. In general, the conjugates can provide for targeted delivery, thus providing for at least one of reduced dose or reduced administrations in a dosage regimen. Thus, the conjugates can provide for reduced dose and / or reduced administration in a dosage regimen relative to the therapeutic prior to being in a conjugate of the present invention. As noted above, because the conjugates can provide for controlled stoichiometry of drug delivery, dosages of conjugates can be calculated based on the number of drug molecules provided on a per antibody-therapeutic entity conjugate basis.

[0309] A pharmaceutical composition of the invention may be administered once or several times or on multiple occasions. The frequency of administration of a conjugate can vary depending on any of a variety of factors, e.g., severity of the symptoms, etc. For example, in some embodiments, the conjugate is administered once per month, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid).

[0310] In certain embodiments, the conjugate or pharmaceutical composition of the invention is administered simultaneously with one or more additional compounds. In certain embodiments, the conjugate or pharmaceutical composition of the invention is administered before or after the additional compound(s).

[0311] The pharmaceutical composition of the invention may be used as a medicament for treating an individual. The individual is a mammal. Mammals include, but are not limited to, domesticated 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.

[0312] The pharmaceutical compositions including a conjugate described herein can be delivered to a cell, group of cells, tumor, tissue, or subject using delivery technologies known in the art. In general, any suitable method recognized in the art for delivering the conjugate can be adapted for use with the herein described compositions. For example, delivery can be by local administration, (e.g., direct injection, implantation, or topical administering), systemic administration, or subcutaneous, intravenous, intraocular, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), or intramuscular administration. The covalent conjugate of the present invention is preferably to be administered intravenously, intramuscularly, or intraarterially, more preferably intravenously. It is especially advantageous to formulate the aforementioned pharmaceutical compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Examples of such unit dosage forms are injectable solutions or suspensions and the like.

[0313] As detailed above, the conjugate / pharmaceutical composition of the invention is particularly useful in treating a neurological disease or a brain disease, such as Alzheimer’s disease or Parkinson’s disease. The term “neurological diseases” encompasses, among others, neurodegenerative diseases, neuroinflammatory diseases or seizure disorders, particularly of the brain. Neurodegenerative diseases are characterized by progressive loss of structure or function of neurons, including death of neurons. Many neurodegenerative diseases including Parkinson's, Alzheimer's, Huntington's, Amyotrophic lateral sclerosis and Multiple Sclerosis occur as a result of neurodegenerative processes. There are many parallels between different neurodegenerative disorders including atypical protein assemblies as well as induced cell death. Neurodegeneration can further be found in many different levels of neuronal circuitry ranging from molecular to systemic. The terms “Neurodegenerative diseases” and “Neuroinflammatory diseases” have a partially overlapping scope. Inflammatory responses are a hallmark of neurodegenerative disease and participate, or contribute, through different mechanisms in the neuronal cell death. The tryptophan catabolism along the Kynurenine pathway (KP) represents one of these mechanisms. Seizure disorders are brain disorders which are characterized by abnormal signaling between brain cells. Seizure disorders can affect part of the brain (partial seizures) or the entire brain (generalized seizures). The most prominent seizure disorder is epilepsy. In order to pass the blood-brain barrier a receptor mediating receptor-induced endocytosis may be used as a target for the conjugate. Examples thereof include transferrin receptor 1 (TfRl), 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 TfR.1.

[0314] Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of the disclosure. Although any methods and materials similar or equivalent to those described herein can be used in the practice as presented herein, the specific methods, and materials are described herein.

[0315] The invention is not limited to the particular methodology, protocols, and reagents described herein because they may vary. Any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the exemplary methods, and materials are described herein. Further, 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.

[0316] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference 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 indicates otherwise. The term "plurality" refers to two or more.

[0317] The following Figures, Sequences and Examples are intended to illustrate various embodiments of the invention. As such, the specific modifications discussed are not to be construed as limitations on the scope of the invention. It will be apparent to the person skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the invention, and it is thus to be understood that such equivalent embodiments are to be included herein.

[0318] FIGURES

[0319] Figure 1. Dose-response curve showing target expression decrease in iPSC neurons for different antibody-DBCO / BCN-linker-ASO Brainshuttle (BrS) conjugates (A) and for DBCO / BCN-linker-ASO alone (B). All conjugates comprised the same Brainshutte antibody and the same ASO sequence, and differentiated in their linker (DBCO-based, namely A3, A6 and Am TEG, or BCN-based).

[0320] Figure 2. Dose-response curve showing target expression decrease in iPSC neurons for different antibody-DBCO / BCN-linker-ASO Brainshuttle (BrS) conjugates vs. the corresponding DBCO / BCN-linker-ASO alone. For DBCO linker: antibody-A6 linker-ASO conjugate vs A6 linker-ASO alone (A), antibody-A3 linker-ASO conjugate vs A3 linker-ASO alone (B) and antibody- Am TEG linker-ASO conjugate vs AmTEG linker-ASO alone (C). For BCN linker: antibody-BCN linker-ASO conjugate vs BCN linker-ASO alone (D). All conjugates comprised the same Brainshutte antibody and the same ASO sequence, and differentiated in their linker (DBCO-based, namely A3, A6 and AmTEG, or BCN-based).

[0321] Figure 3. Cellular accumulation rate of BCN-linker-ASO Brainshuttle (BrS) conjugates and different DBCO-linker-brain shuttle (BrS)-ASO conjugates. In vitro human PK estimation of BrS-linker-ASO conjugates with different linker variants (A6, A3, Cl, BCN) was evaluated in human LSECs, showing an impact of cellular accumulation rate. All conjugates comprised the same Brainshutte antibody and the same ASO sequence, and differentiated in their linker (DBCO-based, namely A3, A6 and AmTEG, or BCN-based).

[0322] Figure 4. Conjugate of the invention based on the DBCO-A3 linker. In a preferred embodiment, the nucleic acid is an ASO.

[0323] Figure 5. Conjugate of the invention based on the DBCO-A6 linker. In a preferred embodiment, the nucleic acid is an ASO.

[0324] Figure 6. Conjugate of the invention based on the DBCO-AmTEG linker. In a preferred embodiment, the nucleic acid is an ASO.

[0325] Example 1: KalbTG conjugation of an antibody to a nucleic acid according to the invention

[0326] Synthesis of DBCO-linked ASOs according to the invention

[0327] 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). Aminolinker C6 and TEG were purchased from Link Technologies (Bellshill, Scotland) and Aminolinker C3 from Boc Sciences (NY, USA).

[0328] Oligonucleotides were synthesized on NittoPhase HL UnyLinker 400 support (Kinovate, Oceanside, CA) on an AKTA Oligopilot (GE Healthcare, Brondby, Denmark) at 275mmol scale. After synthesis, the oligonucleotides were cleaved from the support using aqueous ammonia / EtOH 3 / 1 at 55 °C for 3h. The crude oligonucleotides were diafiltrated on an AKTA Crossflow (GE Healthcare, Brondby, Denmark) first with 0.1M NaHCO3 and then with water. After lyophilization, the compounds were finally characterized by liquid chromatography-mass spectrometry (reverse phase and electrospray ionization-mass spectrometry).

[0329] Synthesis of DBCO-active ester l l,12-Didehydro-y-oxodibenz[b,f]azocine-5(6H)-butanoic acid was purchased from Synthonix (Wake Forest, USA).

[0330] To a solution of 1 l,12-Didehydro-y-oxodibenz[b,f]azocine-5(6H)-butanoic acid (655umol, leq) in DCM / THF 1 / 1 (2ml) bis(pentafluorophenyl) carbonate (786umol, 1 ,2eq) and DMAP (655umol, leq) were added. The reaction mixture was stirred for 2h at RT. The solution was diluted with DCM (10ml) and washed with water (10ml). The org. layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude active ester was directly used for the coupling with amino-modified ASOs. MS ES+ [M+H] 472.1

[0331] Coupling of amino-modified ASOs to DBCO-carboxylate derivatives

[0332] The crude amino-modified oligonucleotides were prepared from the appropriate aminolinker precursors as described above. The DBCO active ester was prepared as described above. The DBCO derivative ASOs were coupled according to the following procedure: To a solution of amino-modified ASOs (63umol, leq) in 0.5M NaHC03 (800ul) was added to a solution of DBCO-active ester (102umol, 3.5eq) in DMSO / THF 3 / 2 (4ml). The reaction was stirred for 45min at 45°C. A mixture of EtOH / Et2O 1 / 1 (10ml) was added to the reaction mixture and the suspension was stored overnight at -20°C. The cold suspension was centrifuged for 20min. The supernatant was removed and the solid dried under vacuum. The crude material was purified by reversed phase HPLC on a YMC-Triat C18-S Prep Column, 120nm, 10μm, 250x 4.6mm using 0.1 M triethylamine acetate and acetonitrile as eluent. Pooled fractions were desalted by Centrifugal filtration (amicon filter 3kD). After a final lyophilization, the compounds were finally characterized by liquid chromatographymass spectrometry (reverse phase and electrospray ionization-mass spectrometry).

[0333] Table 1 provides an overview of the synthesized DBCO-linked ASOs.

[0334] Table 1. DBCO-linked ASOs according to the invention. A6, A3 and Am-TEG. Enzymatic conjugation of LNA-ASOs to antibodies with Q-tags

[0335] Antibodies with Q-tag Gene segments of human transferrin-targeting antibodies with Q-tag insertions were prepared by chemical synthesis and the synthesized gene fragments were cloned into a suitable vector for expression in HEK-293F and Expi293F cells by Twist Bioscience (San Francisco, CA, USA). Antibody production was performed by transient transfection of HEK-293F cells cultivated in F17 Medium (Invitrogen, Carlsbad, CA, USA) or Expi293F cells in Expi293 Expression medium (Thermo Scientific, Waltham, MA, USA), respectively. Transfection was carried out as specified in the manufacturer’s instructions. Cell culture supernatants were harvested seven days after transfection.

[0336] 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. Unbound protein was removed by washing with an equilibration buffer. The antibody was eluted with 50 mM Citrate pH 3.0 and the pH of the eluate was immediately adjusted to 7.5 with 2 M Tris pH 9.0. Size exclusion chromatography using a Superdex 200TM column (GE Healthcare, Chicago, IL, USA) in 20 mM Histidine, 140 mM NaCl pH 6.0 was performed as a second purification step. The purified antibody was stored at -80 °C.

[0337] Protein quantification was performed with a Nanodrop spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). Purity of the antibody variants 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-SW3000 analytical size-exclusion column (Tosoh Bioscience, Griesheim, Germany), equilibrated with 0.2 M K2HPO4 / KH2PO4, 0.25M KC1 pH 6.2, on a High Performance Liquid Chromatography system (Ultimate 3000 HPLC system, Thermo Fisher Scientific, Waltham, MA, USA). The identity of the antibodies was confirmed by ESLQ-ToF-MS (Bruker maXis 433, Bruker, Billerica, MA, USA). For MS analysis, the samples were deglycosylated using N-glycosidase F (Roche, Basel, Switzerland) and subsequently desalted into 2% formic acid, 40% acetonitrile. Endotoxin levels were determined with an EndogenNexgen MCS" machine (Charles River Laboratories, Wilmington, MA, USA). Conjugation using KalbTG

[0338] Purified antibodies containing a Q-amino acid sequence were transferred into a conjugation buffer (20 mM histidine 140 mM NaCl pH 8.5) via dialysis. For the KTG reaction, the antibody (c = 15 mg / mL) was mixed with K-amino acid sequence comprising linker (10 x molar excess, linker ID 7456) and KTG was added (molar ratio antibody:KTG = 300:1). The reaction mixture was incubated O / N at 25 °C, 350 rpm. To remove unconjugated linker and residual enzyme, the conjugated antibody was purified by size exclusion chromatography using a Superdex 200TM column (GE Healthcare, Chicago, IL, USA) in PBS, 250 mM arginine pH 7.5.

[0339] The purified antib ody-K-amino acid sequence conjugate was added to the BCN- or DBCO-containing oligonucletiode in PBS, 250 mM arginine pH 7.5 and the reaction mixture was incubated O / N at 25 °C, 350 rpm. The antibody-oligonucleotide conjugate was purified by size exclusion chromatography as described above and purified conjugates were stored at -80 °C.

[0340] Example 2. Characterization of the antibody-nucleic acid conjugates of the invention: Conjugation efficiency

[0341] The purified antib ody-K-amino acid sequence conjugate was added to the BCN- or DBCO-containing oligonucletiode (at 5x, 2x or 1.5x molar excess) in PBS, 250 mM arginine pH 7.5 and the reaction mixture was incubated O / N at 25 °C, 350 rpm. The antibody-oligonucleotide conjugate was purified as described above.

[0342] Conjugation efficiency for BCN and DBCO-based linkers when 5x, 2x and 1.5x concentrations over the antibody concentration were used is shown in Table 2.

[0343] Table 2. Conjugation efficiency of various linkers at different excess concentrations

[0344] *corrected for the amount of non acidified antibody

[0345] A lower conjugation efficiency was observed for the BCN-based linker compared to DBCO-based linkers when a 1.5x concentration over antibody concentration was used.

[0346] Example 3. Characterization of the antibody-nucleic acid conjugates of the invention: Hidrophobicity

[0347] Hydrophobic Interaction Chromatography (HIC)

[0348] 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 x 35 mm, TOSOH Bioscience, Tokyo, Japan) with a flow rate of 1 mL / min. The column was equilibrated with Eluent A (20 mM Na2HPO4 dihydrate, 1.5 M (NH4)2SO4, pH 7.0) and 60 pg of each sample was loaded onto the column. Subsequently, a gradient between Eluent A and Eluent B (20 mM Na2HPO4 dihydrate, 25 % (v / v) isopropanol, pH 7.0) was applied.

[0349] Gradient:

[0350] 0 min 5 % B

[0351] 0-21 min 5 % B -> 100 % B

[0352] 21 - 22.2 min 100 % B

[0353] 22.5 min -> 5 % B

[0354] 25 min 5 % B

[0355] The elution profile was obtained by continuous measurement of the absorbance at 214 nm. Drug to antibody ratios (DAR) were determined by peak integration using Chromeleon 7.2 (Thermo Fisher Scientific, Waltham, MA, USA). The hydrophilic marker had a retention time of 6.1 min. and the hydrophobic marker had a relative retention time of 13.9 min. Retention times of the mAb-ASO conjugates including DBCO linkers or BCN linker are shown in Table 3. The data show that the hydrophobicity of the conjugate is not influenced by the chemical nature of the linker.

[0356] Table 3. Retention times of BrS-linker-ASO conjugates containing different DBCO-linkers and containing BCN-linker.

[0357] Example 4. Characterization of the antibody-nucleic acid conjugates of the invention: Activity

[0358] To assess the activity of the conjugates according to the invention in a physiological in vitro system, a neuronal target gene knock-down assay was performed. Therefore, induced pluripotent stem cells (iPSC)-derived neurons were treated with three antibody-DBCO linker- ASO conjugates according to the invention (A3, A6, Am TEG) or with an antibody-BCN linker- ASO, and with the corresponding linker- ASO alone, in a dose-dependent manner.

[0359] Cell cultures

[0360] Icell Glutaneurons (FUJIFILM Cellular Dynamics, Inc. (FCDI), Madison, WI, USA, #R1061) were thawed according to the manufacturer's instructions. One to two days later, the neurons were treated with different concentrations of ASO or BrS-ASO conjugates (0.16 nM - 1000 nM). Neuronal medium was changed twice per week and neurons were harvested after 14 days. qPCR RNA for qPCR was extracted from the cells using the Qiagen RNeasy 96 Kit according to the manufacturer’s protocol (74192, Qiagen, Strasse 1, 40724 Hilden, Germany). Expression of the target gene and reference gene was quantified in a duplex reaction using qScript™ XLT One-Step RT-qPCR ToughMix®, Low ROX™ (Quanta Bioscience, 700 Great Seneca Hwy Rockville, MD 20850, USA) and measured on the Applied Biosystem ViiA 7 Real-Time PCR System. The gene expression assays were from Thermo Fischer (Thermo Fisher Scientific, Waltham, MA). Relative expression levels of the target gene were calculated using the deltadelta Ct method. The dose-response curves were fitted from the relative expression values for each compound using a 4-Parameter Sigmoidal Dose-Response Model in Graphpad Prism (Boston, MA, USA).

[0361] In vitro potency and efficacy of different antibody-DBCO linker-ASO conjugates according to the invention was evaluated. Figure 1 shows the target expression decrease for different antibody-DBCO / BCN-linker-ASO conjugates (A) and for DBCO / BCN-linker-ASO alone (B). The results showed a strong knock-down of the optimised antibody-DBCO linker-ASO conjugates according to the invention (noted as BrS-A6-ASO, BrS-AmTEG-ASO and BrS-A3-ASO conjugates) compared to the conjugate with the BCN linker (A, noted as BrS-BCN ASO conjugate, diamond symbol).

[0362] Figure 2 shows the in vitro potency and efficacy of different antibody-DBCO-linker- ASO conjugates according to the invention. Results showed a strong knockdown of the antibody-DBCO-linker-ASO conjugates (Figure 2 A, B, C) compared to the antibody-BCN-linker-ASO conjugates (Figure 2, D). The highest knockdown and most efficient dose-response is shown for the A3 variant conjugate (B, black line with triangle symbols), superior to the ASO alone variant (B, grey line with circle symbols).

[0363] While the treatment with the BCN-linker-conjugate (Figure 2, D) led to a 40% knockdown of the target gene expression, the conjugates variants with DBCO- linkers (Figure 2, A-C) showed a higher knockdown efficiency of 60%. In comparison to the BCN-ASO (KD 60%), the BrS-BCN-ASO conjugate has a 20% lower efficiency (40%) (Figure X, D). However, for the conjugates with the DBCO linker, the conjugates reach a similar level of knockdown comparable to their ASO alone counterparts (Figure 2, A, B, C). Of interest, within the 3 variants of the DBCO linker conjugates, the DBCO-A3 linker conjugate (Figure 2, B) showed a superior dose-response curve to their ASO-alone counterparts.

[0364] Table 4: IC50 values, maximal knockdown (KD) and area-under-curve values of DBCO-conjugates and ASO-linker alone counterparts.

[0365] Example 5. PK characterization of the conjugates of the invention

[0366] Antibody labelling

[0367] Antibodies and mAb-linker-ASO conjugates according to the invention were labelled using the SiteClick™ Antibody Azido Modification Kit (S20026, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. A pH-sensitive amine-reactive dye (Amax = 523 nm, G9841, Promega, Madison, WI, USA) was coupled to a sulfo DBCO PEG4 amine (# 1228-100, Click Chemistry Tools, Newark, CA, USA). Antibodies and conjugates were labelled with a molar dye excess of 2. Excess dye was removed using the Amicon® Ultra-2 Centrifugal Filter with a MWCO of 50 kD (# UFC200324, EMD Millipore, Burlington, MA, USA) and the labelled products were re-buffered in 20 mM histidine buffer (pH 5.5). The concentration of the labelled antibodies or labelled mAb-ASO conjugates [a] as well as the degree of labelling (DOL) [b] was determined with a Nanodrop spectrometer at 280 nm and 532 nm, as follows:

[0368] Antibody concentration = [A280nm- [A280nm* CF(Dye)]] / s(AB) [a]

[0369] DOL = [A532nm* MW(AB)] / [c(AB) * e(Dye)] [b] wherein: a (Dye) = 47225, CF(Dye) = 0.36, A = absorbance, a = extinction coefficient, and CF = correction factor.

[0370] To confirm the absence of unbound dye and to verify the concentration measured at the spectrometer, a size exclusion chromatography of the labelled antibodies / conjugates was performed. Samples were separated using a BioSuite Diol (OH) column (186002165, Waters, Milford, MA, USA186002165) with a potassium dihydrogen phosphate buffer (pH 6.2) as the mobile phase at a flow rate of 0.5ml / min and 15 pg of each sample was loaded onto the column. Absorption at 280 and 532 nm was detected and detectors at 280 nm and 532 nm were used to quantify and analyze the labelled antibodies / conjugates. The area under the curve (AUCs) at 280 nm and 532 nm was extracted to calculate the concentration of the labelled antibodies / conjugates. The geo-mean of the AUCs from all injected antibodies was computed and the calculated deviation from each injected antibody to this geo-mean was identified. For an antibody to be reliable within the PK assay, the difference from the geo-mean was expected to be below 15%.

[0371] Cell cultures and assays

[0372] Human liver-derived endothelial cells (#HLECP2,CL1AA1266, Lonza, Basel, Switzerland) were maintained in EBMTM-2 Endothelial Cell Growth Basal Medium-2 (#CC-3156, Lonza, Basel, Switzerland) supplemented with EGMTM-2 SingleQuotsTM Supplements (#CC-4176, Lonza, Basel, Switzerland).

[0373] For biological replicates the assay ran on two different days in triplicates. Five respectively six days prior antibody treatment, cells were plated onto coated 100 mm collagen I dishes (#354450, Coming, New York, USA). Two days prior treatment cells were sub-cultured into coated 96-well plates at a cell density of 4xl04cells / well to allow adherence for 48 hours. For the second measurement-day three days prior treatment cells were sub-cultured with a cell density of 2 xl04 cells / well. Medium was changed after 24 hours and cells were kept at 37 °C and 5 % CO2. The day of the experiment, samples were prepared in the form of a dosing solution as a mastermix. Samples were measured in triplicates at two time points. For normalization, the dosing solution was diluted 1 :4 with 0.2 M citric acid buffer pH 4.5 (25 pl dosing solution + 75 pl acid). The mixtures were measured in the plate reader (bottom Reading kex = 532 nm, kern = 560 nm). Cells were washed twice with 200 pl pre-warmed medium and subsequently incubated with 100 pl of 400 nM labeled antibodies / conjugates or 20 mM histidine buffer (pH 5.5) as negative control in medium. After 2 and 4 hours, the antib ody(conjugate solution was removed and cells were washed once with 200 pl ice-cold DPBS (w / o Mg, w / o Ca) and detached by applying 100 pl Trypsin (#P 10-027100, PAN-Biotech GmbH, Aidenbach, Germany) for 2.5 minutes at 37 °C. Trypsin was inactivated by the addition of 100 pl FACS Buffer (20 % FBS (#FBS-HI-12A, Capricorn Scientific GmbH, Ebsdorfergrund, Germany), 1 mM EDTA (#15575020, Life Technologies GmbH, Darmstadt, Germany) in DPBS (#P04-36500, PAN-Biotech GmbH, Aidenbach, Germany).

[0374] The mean fluorescent intensity (MFI, more specifically the geometric mean) of the internalized antibodies / conjugates was acquired using the LSRFortessa™ X-20 (BD Biosciences, San Jose, CA, USA) equipped with a laser to excite at 532 nm and a filter to collect emitted light at 572 / 35nm. Identical conditions, gains and gates were used for both time points (2 and 4 hours). The experiment was carried out on two consecutive days (biological replicates) to improve accuracy and robustness of the assay. Data extraction was performed using the FloJo VIO software (BD Biosciences, San Jose, CA, USA). Value of the negative control (background cellular fluorescence) was subtracted from all geo-mean values for that particular day, followed by dividing / normalizing each value by the corresponding fluorescence emission readout of the dosing solution. The “dose normalized” value of control antibody 1 was subtracted from those of the samples for that individual day. The values were further divided by the linear slope of control antibody 2, obtained by linear regression analysis of 2 & 4 hour values. The now “dose and control” normalized geo-mean values from each antib ody / conjugate from both days were plotted together and a linear regression analysis was carried out. The resultant slope of the linear fit is the calculated relative cellular accumulation rate or the so- called “relative LUCA rate” for that particular antibody / conjugate.

[0375] The LUCA assay was performed to estimate the human PK. Primary human liver endothelial cells were incubated with labelled BrS-ASO conjugates containing either different forms of the DBCO linker (A3, A6, Am TEG) or the BCN linker. After incubation the cell uptake was measured. In comparison to the BrS-BCN-ASO conjugates, BrS-DBCO-linker-ASO conjugates A3 and AmTEG reduced the cellular accumulation rate. The BrS-DBCO-linker-ASO conjugate A6 showed similar cellular accumulation as BrS-BCN-ASO conjugate. BrS-A3 linker-ASO conjugate showed the lowest accumulation rate and BrS-AmTEG-ASO conjugate showed a decreased cellular accumulation rate in comparison to BrS-BCN-ASO.

[0376] Figure 3 shows the cellular accumulation rate of BCN-linker-ASO Brainshuttle (BrS) conjugate versus different DBCO-linker-brain shuttle (BrS)-ASO conjugates. The lowest uptake corresponds to the BrS-DBCO-A3-ASO conjugate. The optimised BrS-DBCO linker-ASO conjugates decrease cellular accumulation rate (A3, AmTEG) or show the same level on cellular accumulation rate (A6) compared to the conjugate with the BCN linker. The lowest cellular accumulation rate is shown in the BrS-DBCO- A3-ASO conjugate (A3 variant conjugate).

[0377] Example 6. Characterization of the antibody-nucleic acid conjugates of the invention: Stability

[0378] LC-MS Instrumentation

[0379] An Ultimate 3000 HPLC system was used in combination with an Orbitrap Exploris 480 Mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The isolated antibody conjugates were analyzed on a reversed-phase 4um macroporous polymer particles column MAbPac (RP) from Thermo Fisher Scientific, 1.0 mm i.d. * 150 mm, at a flow of 50 ul / min. The heating of the system was circumvented by using an off-line column heater HotDog XL5090 from ProLab (Basel, Switzerland) to maintain the MAbPac column temperature at 80°C. The system’ s autosampler was used to directly inject aliquots of the sample onto the MAbPAC using Eluents at low pH. Detection was performed on a Thermo Scientific orbitrap Exploris 480 Mass Spectrometer equipped with the BioPharma option. Acquisition was performed in the intact Protein application mode at High Pressure. The orbitrap mass range was set up from 600 to 4.500 Da at a Resolution of 30.000. Positive ionization mode was used for the intact mass analysis of the antibody oligonucleotide conjugates.

[0380] Chromatographic Separation

[0381] A 50μL aliquot of the eluate, following immunoaffmity extraction, was directly injected onto a polymeric reversed phase HPLC column MAbPac RP, 2.1 mm i.d. x 100 mm (Thermo Fisher Scientific, Waltham, MA, USA), kept at 80°C, using high- pressure gradient elution at a flow rate of 50 pL / min. The elution was performed with a mixture of 0.1% formic acid (El) and acetonitrile containing 0.1% formic acid (E2). Both Eluents contain in addition 0.005% Trifluoroacetic acid. After injection, eluent E2 was kept at 2.5% for 1.0 min, and then increased to 20% within 1.0 min, and further raised to 95% in 18 min. Thereafter, E2 was maintened at 95% for an additional 6.0 min. While E2 was raised, El was reduced complementarity. After 24.0 min following injection, E2 was decreased to 2.5%. The overall run time of analysis was 37 min.

[0382] Data Analysis

[0383] Raw data files were processed with BioPharmaFinder 4.0 software (Thermo Fisher Scientific, Waltham, MA, USA). For the isotopically unresolved mass spectra, the ReSpect Algorithm (Thermo Fisher Scientific, Waltham, MA, USA) was applied.

[0384] Immunoaffmity extraction

[0385] Streptavidin Mag SepharoseTM Beads in solution obtained from GE Healthcare (Chicago, IL, USA) were homogenized to a slurry at ambient temperature. A 100 pL aliquot of the bead slurry was added to 300 μL of 0.05% Tween-20 detergent in buffered saline (1* DPBS; pH 6.8-7.8) in 1.5 mL Lobind microtubes (Eppendorf, Hamburg, Germany) and homogenized at ambient temperature in a thermomixer for 2.0 min. The beads were separated by magnet and the solvent removed. Resuspending of beads and further washing with 0.05% Tween-20 in DPBS was repeated three times.

[0386] Magnetic beads were resuspended in 500 0.05%μTLween in DPBS and 200 pmol of monoclonal capture antibody (CMAB Biopharma, Suzhou, China) were added to beads slurry. Beads were incubated at ambient temperature for at least 2 h using gentle mixing with the Thermomixer (Eppendorf, Hamburg, Germany). Thereafter, beads were washed three times in 0.3 mL of 0.05% Tween20 in DPBS. Beads were homogenized in a wash solution for about 2.0 min before separating the magnetic beads and removing the solvent.

[0387] For immunocapture from plasma, separated beads were re-suspended in 250 pL 0.05% Tween in DPBS and a 50 plaμsLma aliquot was added to the beads solution and incubated at ambient temperature for 2 h applying gentle mixing in the Thermomixer. Thereafter beads were washed twice in 300 0.05%TweeμnL in DPBS using gentle homogenization for about 2.0 min. Finally, the beads were washed with 300 ul DPBS without Tween.

[0388] For elution of substrate from monoclonal capture antibody, 120 of 10 rμnML glycine-HCl buffer (pH 2.0) were added to the beads, followed by incubation at ambient temperature for ca. 2 h applying gentle mixing in the thermomixer. Beads were separated magnetically and the supernatant containing the protein of interest was saved. 100 μL of the supernatant containing the intact mAb oligonucleotide conjugates and possible degradation products were diluted with 100ul 50mM Tris- HC1 at pH8 buffer and 50ul were used for direct analysis and the remainder was used for deglycosylation or digestion with trypsin or other enzymes.

[0389] This setup depleted the mAb-ASO conjugate and all possible degradation products from the linker ASO part.

[0390] Deglycosylation After the injection of 50 ul for intact mass analysis the left over, 2 ul of N- Glycosidase (PNGase F, Cat No V483A, Promega, Madison, WI, USA) was added and incubated for 15 min at 50°C to cleave off N-linked oligosaccharides. 25-150ul of the deglycosylated antibodies were then injected to the LC-HRMS for intact protein analysis.

[0391] In vitro stability in serum

[0392] The antibody-ASO conjugate, at a concentration of 200 pg / ml, was incubated in human serum for durations of 24 and 48 hours. This incubation was performed in a thermomixer with gentle agitation at 1200 rpm at a temperature of 37°C.

[0393] The antibody-ASO conjugate and its possible degradation products were immunoextracted from plasma using a biotinylated monoclonal capture antibody (mcAb) coupled to streptavidin Mag SepharoseTM coated magnetic beads. After incubation of the beads in 50pl of plasma, the captured analytes were released from the isolated beads. The immuno-extraction eluate was analyzed on a polymeric reversed phase MAbPac column (1.0 mm i.d. x 150 mm) heated at 80°C to isolate the antibody conjugate. The intact antibody-ASO conjugate was analyzed by LC-MS on an orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific. The eluents were 0.1% formic acid, 0.005%Trifluoroacetic acid and acetonitrile. Acetonitrile raise to 95% in 20 min at a flow rate of 50 pl / min. The effluent of the analytical was directed to a heated electrospray ion source of the Exploris480 in the positive ionization source mode. The orbitrap full MS spectra were acquired in the mass range m / z 600 to 4500 using the intact protein mode at high pressure.

[0394] Stability assessment revealed that no new mass peaks emerged at either 24 or 48 hours for any of the three DBCO-based linkers tested. The initial deconvoluted mass spectra at 0 hours were consistent with those observed at subsequent time points for each conjugate, indicating that all linkers maintained complete stability for up to 48 hours.

[0395] Table 5. Percentage of intact BrS antibody-DBCO linker-ASO conjugates after incubation in human serum at 37°C

Claims

Claims1. A conjugate comprising a polypeptide, a nucleic acid and a linker, wherein:The linker comprises the following chemical structure (1):The linker connects the polypeptide and the nucleic acid,The linker is conjugated to the C-terminal amino acid of the polypeptide at the position noted as 1 in structure (1),The linker is conjugated to an oxygen linked to a phosphor of the oxidation state V of the nucleic acid at the position noted as 2 in structure (1).

2. The conjugate according to the previous claim, wherein the oxygen linked to the phosphor of the oxidation state V is at the 5’ terminus of the nucleic acid, and / or wherein the oxygen is linked to the phosphor of the oxidation state V (i.e. a phosphorous atom of the oxidation state V) in a phosphate or phosphorothi oate .

3. The conjugate according to any of the previous claims, wherein the linker is conjugated to the nucleic acid via an amide bond.The conjugate according to any of the previous claims, wherein the conjugate comprises the following structure:or the conjugate comprises the following structure:

5. The conjugate according to any of the previous claims, wherein the linker is conjugated to the C-terminal amino acid of the polypeptide by direct covalent binding between the C-terminal amino acid of the polypeptide and the position noted as 1 in (1) via an amide bond.

6. The conjugate according to any of the previous claims, wherein the linker is conjugated to the carboxy group at the C terminus of the polypeptide via -NH-(PEG)m-propionate-NH-, wherein m=3 or 4.

7. The conjugate according to any of the previous claims, wherein the conjugate comprises the following structure:

8. The conjugate according to any of the previous claims, wherein the polypeptide comprises a lysine residue, wherein said lysine residue belongs to a tag sequence RYESK in N- to C-terminal direction comprised at the C- terminal end of the polypeptide, wherein the carboxy group of said lysine is conjugated to the linker via an amide bond, and wherein the conjugate comprises the following structure9. The conjugate according to any of the previous claims, wherein the conjugate comprises the following structure: idor the conjugate comprises the following structureor the conjugate comprises the following structure acid10. The conjugate according to any of the previous claims, wherein the polypeptide comprises the K-amino acid sequence RYESK, wherein the epsilon amino group of the lysine (K) is linked via an isopeptide bond to a glutamine (Q) residue side chain of a second polypeptide, wherein the glutamine residue is within a Q-amino acid sequence (Q-tag) of at least 5 amino acid residues.

11. The conjugate according to any of the previous claims, wherein the polypeptide comprises the K-amino acid sequence RYESK, wherein the epsilon amino group of the lysine in the K-amino acid sequence is linked by an isopeptide bond to a glutamine residue side chain of a second polypeptide, wherein 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 IDNO: 17), preferably YRYRQ (SEQ ID NO: 17), wherein the Q-amino acid sequence is at a position selected from position 110 (LC110), position 143 (LC143) and position 214 (LC214) of an antibody light chain and position 118 (HC118), position 177 (HC177), position 297 (HC297), position 341 (HC341), position 401 (HC401) and position 446 (HC446) of an antibody heavy chain (numbering according to Kabat).

12. The conjugate according to any of claims 10-11, wherein the Q-amino acid sequence is in a chain of an antibody that comprises at least one light chain and at least one heavy chain.

13. The conjugate according to the previous claim, wherein the light chain constant domain not comprising a 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 NO: 6 or 7; and / or wherein the heavy chain constant region not comprising a Q-amino acid sequence comprises an amino acid sequence that is at least 96 %, 97 %, 98 %, or 99 %, preferably 100 % identical to the amino acid sequence of SEQ ID NO: 1 to 5.

14. The conjugate according to any of the previous claims, wherein the nucleic acid is selected from the group consisting of a RNA, siRNA, antisense oligonucleotide (ASO), LNA, and an ASO comprising LNA nucleotides.

15. The conjugate according to any of the previous claims, wherein a) the antibody recognizes one target and said target is a receptor inducing receptor-mediated endocytosis, such as transferrin receptor 1 (TfRl), insulin-like growth factor 1 receptor (IGF-1R), low density lipoprotein receptor-related protein 1 (LRP1), low density lipoprotein receptor-related protein 8 (LRP8), CD98, or podocalyxin (PODXL), particularly TfRl; and / or b) wherein the antibody recognizes one or further target(s) and said one or further targets is / are specific for a specific cell type, and / or said oneof further target(s) is / are therapeutic targets, wherein the therapeutic target is a tumor target, a neurological target or a metabolic target.

16. A compound of the formula:(i)or of the formulaor of the formula17. The compound according to the previous claim for forming a conjugate according to any of claims 1-15.

18. A method for producing a polypeptide-linker-nucleic acid conjugate according to any of claims 1-15 comprising the following steps: 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), preferably YRYRQ (SEQ ID NO: 17) at one or more positions selected from position 110 (LC110), position 143 (LC143) and position 214 (LC214) of an antibody light chain and position 118 (HC118), position 177 (HC177), position 297 (HC297), position 341 (HC341), position 401 (HC401) and position 446 (HC446) of an antibody heavy chain (numbering according to Kabat), b) providing a polypeptide comprising the amino acid sequence RYESK(SEQ ID NO: 16), which is conjugated to the terminal amino group of (3-[2-[2-(2-aminoethoxy)ethoxy]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 the (3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propanamido)-6- azidohexanami de, c) reacting the antibody of a) and the polypeptide of b) in the presence of KalbTG or a functionally active variant thereof and under conditions conducive to the activity of KalbTG, thereby forming a isopeptidebond between the Q-amino acid sequence of the antibody and the polypeptide comprising the amino acid sequence of RYESK, thus producing an (3-[2-[2-(2-antibody aminoethoxy)ethoxy]ethoxy]propanamido)-6-azidohexanamide (antibody azide), d) reacting the antibody azide with the compound according to any of claim16 (i) to (iii), and thereby producing the polypeptide-linker-nucleic acid conjugate according to any one of claims 1-15.

19. The method according to the previous claim, wherein the reaction product of step c) comprises the following structureisopeptide bond to glutamine and / or wherein the reaction product of step d) comprises the following structure:

20. The conjugate according to any one of claims 1 to 15, or the polypeptide- linker-nucleic acid conjugate produced according to the method of any one of claims 18-19 for use as a medicament.

21. The conjugate according to any one of claims 1 to 15, or the polypeptide- linker-nucleic acid conjugate produced according to the method of any one of claims 18-19 for use in treating a neurological disease or a brain disease or cancer.

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