Site-specific antibody conjugation and uses thereof

Abstract

To provide modified antibodies useful as pharmaceutical compositions.SOLUTION: Provided are modified antibodies comprising at least an antibody heavy chain, the antibody heavy chain comprising one or two or three or four or more first recognition sites for transglutaminase (KalbTG) derived from Kutzneria albida inserted at or after one or more positions independently selected from the group of positions comprising: position 118 (HC118), position 177 (HC177), position 297 (HC297), position 341 (HC341), and position 401 (HC401) of the antibody heavy chain (numbering according to Kabat).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention is in the field of antibody technology, particularly in the field of antibody-drug conjugates. In particular, the present invention relates to modified antibodies containing one or more artificial recognition sites for transglutaminase derived from Kutzneria albida, and antibody-drug conjugates comprising the modified antibodies. [Background technology]

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

[0003] One way to target a therapeutic entity to its intended site of action is by conjugating it to an antibody that specifically binds to it in target cells or tissues. One of the challenges associated with antibody-drug conjugates (ADCs), such as antibody-oligonucleotide conjugates (AOCs), is their manufacture. Most drug-antibody linkages utilize either partially reduced interchain disulfide bonds, which allow for thiol-maleimide chemistry, or lysine functionalization using activated esters or artificially introduced cysteine ​​residues. This method results in a statistical mixture of conjugated antibody species with different numbers of drugs attached to different sites. In ADCs with a monomethyl auristatin E payload, ADC species with drug-to-antibody ratios (DARs) of 4, 6, or 8 were demonstrated to be increasingly hydrophobic and much more prone to aggregation than species with a DAR of 2 (Adem et al.).

[0004] Therefore, it is desirable to provide ADCs, such as AOCs, for targeted therapy with more precise control over the number and sites of attachment of therapeutic entities. This should increase the uniformity of ADCs. Improved site-specific conjugation techniques remain of interest to many pharmaceutical companies for their potential use in the preparation of therapeutic ADCs as well as diagnostic antibody labels or antibody-enzyme conjugates.

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

[0006] The discovery of a novel transglutaminase (KalbTG) from Kutzneria albida and the identification of its respective peptide substrate were described by Steffen et al. (2017). KalbTG exhibits similar efficiency but improved specificity and developability compared to previously described microbial transglutaminases (mTG).

[0007] US Patent Application Publication No. 2020 / 0249231 reported a system and method for the identification and characterization of transglutaminase species.

[0008] The IgG heavy chain C-terminus has been described as a suitable Q-tag insertion site for mTG for antibody labeling (see, e.g., WO 2021 / 174091). Summary of the Invention

[0009] To avoid the above drawbacks, the objective of the present invention was to identify sites within IgG molecules for incorporation of Q-tag motifs that would result in improved properties of modified antibodies with respect to KalbTG-mediated conjugation. Incorporation of a Q-tag should provide the necessary accessibility for conjugation and therapeutic activity of the therapeutic entity without impairing antibody folding or function or reducing expression yield. Successful identification of such sites by the present invention allows for the incorporation of one or more therapeutic moieties per IgG molecule in a defined stoichiometry and controlled, site-specific manner. This can provide a more uniform conjugate product, for example, reducing the required purification and separation effort and resulting in molecules with more favorable drug-like properties. Furthermore, the risk of compromising the conjugation process and / or the binding of the antibody to its antigen by the conjugated payload is reduced.

[0010] The present inventors have observed, inter alia, that attachment of a payload to the C-terminus of an IgG heavy chain increases aggregation and hydrophobicity. To be able to use KalbTG to prepare pharmaceutically useful antibody-drug conjugates, the KalbTG Q-tag must be introduced at a defined site within the IgG backbone according to the present invention.

[0011] The present invention relates to modified antibodies comprising heavy and light chains, wherein the heavy and / or light chains comprise one or more first recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) or a functionally active variant thereof, wherein the one or more first recognition site(s) are introduced at one or more selected position(s) within the heavy and / or light chains of the antibody.

[0012] One aspect of the invention is a modified antibody comprising a heavy chain and a light chain, wherein the heavy and / or light chains comprise one or more (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) at or inserted after one or more positions (numbering according to Kabat) selected from position 110 (LC110), position 143 (LC143), and position 214 (LC214) in the light chain and position 118 (HC118), position 177 (HC177), position 297 (HC297), position 341 (HC341), and position 401 (HC401) in the heavy chain. Thus, in the modified antibodies of the invention, the recognition site(s) for KalbTG are located within the constant region of the antibody heavy chain polypeptide, i.e., not at either the N- or C-terminus of the antibody heavy chain or / and not within or at the C-terminus of the antibody light chain constant domain.

[0013] In certain embodiments of all aspects and embodiments of the invention, the modified antibodies of the invention further comprise a recognition site for KalbTG at or fused to the C-terminus of the heavy chain, i.e., at position 446 (HC446) or position 447 (HC447) (numbering according to Kabat).

[0014] In the context of the present invention, the term "insertion of a recognition site for KalbTG at a position" means that the amino acid residue present at that position in the unmodified amino acid sequence of each chain of the antibody is replaced by the recognition site, i.e., the recognition site is inserted in place of the amino acid residue, or the recognition site is inserted, in a preferred embodiment, either directly after that position in the amino acid sequence, spaced between two short flexible linker peptides, i.e., the amino acid residue is maintained.

[0015] In particular embodiments of all aspects and embodiments of the invention, 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 (numbering according to Kabat).

[0016] In a preferred embodiment of all aspects and embodiments of the invention, 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 (numbering according to Kabat).

[0017] In certain embodiments of all aspects and embodiments of the invention, the modified antibody comprises two identical heavy chains or heavy chain Fc regions.

[0018] In certain embodiments of all aspects and embodiments of the invention, the modified antibody comprises two, four, or more (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) inserted at or after one or more 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), and position 401 (HC401) of the heavy chain (numbering according to Kabat).

[0019] In particular embodiments of all aspects and embodiments of the invention, the modified antibodies according to the invention comprise two different heavy chains or antibody Fc regions, whereby the differences arise at least from the respective mutations to induce heterodimerization.

[0020] In certain embodiments, the modified antibody comprises one, two, three or more (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) inserted independently of one another at or after one or more 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), and position 401 (HC401) of the heavy chain (numbering according to Kabat).

[0021] Similarly, the present invention relates to a modified antibody Fc region comprising one or more (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) inserted after one or more positions selected from the group of positions including position 118 (HC118), position 177 (HC177), position 297 (HC297), position 341 (HC341), and position 401 (HC401) of the heavy chain (numbering according to Kabat).

[0022] Thus, in the modified antibody Fc regions of the present invention, the recognition site for KalbTG is located within the constant region of the antibody heavy chain Fc region polypeptide, i.e., not at either end. Furthermore, the modified antibody Fc regions of the present invention may comprise an additional first recognition site for KalbTG at position 446 (HC446) or position 447 (HC447) of the heavy chain, i.e., at the C-terminus (numbering according to Kabat). In the context of the present invention, the term "insertion of a recognition site for KalbTG at a certain position" means that the amino acid present at that position in the unmodified sequence is replaced by the recognition site, or, in a preferred embodiment, a recognition site is further inserted after that position, either directly or spaced apart between two flexible short linker peptides.

[0023] In particular embodiments of all aspects and embodiments of the invention, the one or more positions are selected from the group of positions comprising heavy chain position 118 (HC118), position 177 (HC177), position 297 (HC297), and position 341 (HC341) (numbering according to Kabat).

[0024] In a preferred embodiment of all aspects and embodiments of the invention, 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 (numbering according to Kabat).

[0025] In a preferred embodiment of all aspects and embodiments of the present invention, the modified antibody comprises at least one (cognate) pair of antibody heavy and light chains, wherein the heavy chain comprises one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) inserted at or after position 297 (HC297) of the heavy chain (numbering according to Kabat).

[0026] In a preferred embodiment of all aspects and embodiments of the present invention, the modified antibody comprises at least one (cognate) pair of antibody heavy and light chains, wherein the heavy chain comprises one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) inserted at or after position 177 (HC177) of the heavy chain (numbering according to Kabat).

[0027] In a preferred embodiment of all aspects and embodiments of the present invention, the modified antibody comprises at least one (cognate) pair of antibody heavy chain and antibody light chain, wherein the light chain comprises one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) inserted at or after position 214 (LC214) of the light chain (numbering according to Kabat).

[0028] In a preferred embodiment of all aspects and embodiments of the present invention, the modified antibody comprises at least one (cognate) pair of antibody heavy and light chains, wherein the heavy chain comprises two (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) inserted at or after positions 297 (HC297) and 177 (HC177) of the heavy chain (numbering according to Kabat).

[0029] In a preferred embodiment of all aspects and embodiments of the present invention, the modified antibody comprises at least one (cognate) pair of antibody heavy and light chains, wherein the heavy chain comprises one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) inserted at or after position 297 (HC297) of the heavy chain and the light chain comprises one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) inserted at or after position 214 (LC214) of the light chain (numbering according to Kabat).

[0030] In a preferred embodiment of all aspects and embodiments of the present invention, the modified antibody comprises at least one (cognate) pair of antibody heavy and light chains, wherein the heavy chain comprises one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) inserted at or after position 177 (HC177) of the heavy chain and the light chain comprises one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) inserted at or after position 214 (LC214) of the light chain (numbering according to Kabat).

[0031] In a preferred embodiment of all aspects and embodiments of the invention, the modified antibody comprises at least one (cognate) pair of antibody heavy and light chains, wherein the heavy chain comprises two (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) inserted at or after position 297 (HC297) and position 177 (HC177) of the heavy chain, and the light chain comprises one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) inserted at or after position 214 (LC214) of the light chain (numbering according to Kabat).

[0032] In a preferred embodiment of all aspects and embodiments of the invention, the modified antibody comprises two (cognate) pairs of antibody heavy and light chains, wherein the heavy chains of the first pair comprise one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) inserted at or after position 297 (HC297) of the heavy chain, and the light chains of the second pair comprise one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) inserted at or after position 214 (LC214) of the light chain (numbering according to Kabat).

[0033] In a preferred embodiment of all aspects and embodiments of the invention, the modified antibody comprises two (cognate) pairs of antibody heavy and light chains, wherein the heavy chains of the first pair comprise one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) inserted at or after position 177 (HC177) of the heavy chain, and the light chains of the second pair comprise one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) inserted at or after position 214 (LC214) of the light chain (numbering according to Kabat).

[0034] In a preferred embodiment of all aspects and embodiments of the present invention, the modified antibody comprises two (cognate) pairs of antibody heavy and light chains, wherein the heavy chains of the first pair comprise one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) inserted at or after position 297 (HC297) of the heavy chain, the heavy chains of the second pair comprise one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) inserted at or after position 177 (HC177) of the heavy chain, and the light chains of the second pair comprise one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) inserted at or after position 214 (LC214) of the light chain. It contains one (first) recognition site(s) for transglutaminase from Kluyveromyces albida (KalbTG) (numbering according to Kabat).

[0035] In a preferred embodiment of all aspects and embodiments of the present invention, the modified antibody comprises two (cognate) pairs of antibody heavy and light chains, wherein the heavy chains of the first pair comprise one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) inserted at or after position 177 (HC177) of the heavy chain, the heavy chains of the second pair comprise one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) inserted at or after position 297 (HC297) of the heavy chain, and the light chains of the second pair comprise one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) inserted at or after position 214 (LC214) of the light chain. It contains one (first) recognition site(s) for transglutaminase from Kluyveromyces albida (KalbTG) (numbering according to Kabat).

[0036] In a preferred embodiment of all aspects and embodiments of the invention, the modified antibody comprises two (cognate) pairs of antibody heavy and light chains, wherein the heavy chains of the first pair comprise two (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) inserted at or after position 297 (HC297) and position 177 (HC177) of the heavy chain, and the light chains of the second pair comprise one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) inserted at or after position 214 (LC214) of the light chain (numbering according to Kabat).

[0037] In certain embodiments of all aspects and embodiments of the present invention, a recognition site(s) for Kutzneria albida transglutaminase (KalbTG) is inserted immediately after that position. In certain embodiments, the recognition site(s) for Kutzneria albida transglutaminase (KalbTG) is inserted between two flexible peptide linkers. In certain embodiments, the recognition site(s) for Kutzneria albida transglutaminase (KalbTG) has the amino acid sequence GGGSYRYRQGGGS (SEQ ID NO: 25).

[0038] The present invention further relates to one or more nucleic acids encoding modified antibodies according to the invention, as well as covalent conjugates comprising (i) a modified antibody according to the invention, and (ii) one or more non-antibody moieties (payload(s)) covalently conjugated to one or more first recognition site(s) either directly or via a first linker. In certain embodiments, the non-antibody moiety comprises a therapeutic entity and, optionally, a second linker.

[0039] The present invention further relates to a method for covalently conjugating a modified antibody according to the invention to a non-antibody moiety, and when the non-antibody moiety comprises a therapeutic entity, the present invention further relates to a conjugate of the modified antibody according to the invention with the therapeutic entity as a pharmaceutical composition for use as a medicament and for use in the treatment of disease.

[0040] KalbTG catalyzes the formation of an isopeptide bond between a glutamine (Gln, Q) side chain and a lysine (Lys, K) side chain. The isopeptide bond formed is an ε-(γ-glutamyl)lysine bond / crosslink. The enzymatic bond formation is covalent and therefore largely irreversible. YRYRQ (SEQ ID NO: 17) was identified as the KalbTG Gln-containing motif (Q tag), and RYESK (SEQ ID NO: 16) was identified as the Lys-containing acceptor motif (K tag).

[0041] Therefore, the present invention encompasses at least the following embodiments.

[0042] 1. A modified antibody comprising at least an antibody heavy chain, wherein the antibody heavy chain comprises one or two or three or four or more first recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) inserted at or after one or more positions independently selected from the group of positions comprising: position 118 (HC118), position 177 (HC177), position 297 (HC297), position 341 (HC341) and position 401 (HC401) of the antibody heavy chain (numbering according to Kabat).

[0043] 2. A modified antibody comprising at least an antibody light chain, wherein the antibody light chain comprises one or two or three or four or more first recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) inserted at or after one or more positions independently selected from the group of positions comprising: position 110 (LC110), position 143 (LC143) and position 214 (LC214) of the antibody light chain (numbering according to Kabat).

[0044] 3. A modified antibody comprising a heavy chain and a light chain, wherein the heavy chain and / or light chain comprise one or two or three or four or more first recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) inserted at or after one or more positions independently selected from the group of positions comprising: position 110 (LC110), position 143 (LC143) and position 214 (LC214) of the antibody light chain, and position 118 (HC118), position 177 (HC177), position 297 (HC297), position 341 (HC341) and position 401 (HC401) of the antibody heavy chain (numbering according to Kabat).

[0045] 4. The modified antibody according to any one of embodiments 1 to 3, wherein a first recognition site for KalbTG is present within the amino acid sequence of each antibody chain.

[0046] 5. The modified antibody of any one of embodiments 1 to 4, wherein the modified antibody further comprises a first recognition site for KalbTG at or fused to the C-terminus of the antibody heavy chain.

[0047] 6. The modified antibody of any one of embodiments 1 to 5, wherein the modified antibody further comprises a first recognition site for KalbTG at position 446 (HC446) or position 447 (HC447) of the antibody heavy chain (numbering according to Kabat).

[0048] 7. The modified antibody of any one of embodiments 1 to 6, wherein one or two or three or four or more positions are independently selected from the group of positions comprising: position 110 (LC110), position 143 (LC143) and position 214 (LC214) of the antibody light chain, and position 118 (HC118), position 177 (HC177), position 297 (HC297), position 341 (HC341) and position 401 (HC401) of the antibody heavy chain (numbering according to Kabat).

[0049] 8. The modified antibody of any one of embodiments 1 to 7, wherein one or two or three or four or more positions are independently selected from the group of positions comprising position 214 (LC214) of the antibody light chain, and position 118 (HC118), position 177 (HC177), position 297 (HC297) and position 341 (HC341) of the antibody heavy chain (numbering according to Kabat).

[0050] 9. The modified antibody of any one of embodiments 1 to 8, wherein one or two or three or four or more positions are independently selected from the group of positions comprising position 214 (LC214) of the antibody light chain, and position 341 (HC341), position 297 (HC297) and position 177 (HC177) of the antibody heavy chain (numbering according to Kabat).

[0051] 10. The modified antibody of any one of embodiments 1 to 9, wherein one or two or three or four or more positions are independently selected from the group of positions comprising position 214 (LC214) of the antibody light chain, and position 297 (HC297) and position 177 (HC177) of the antibody heavy chain (numbering according to Kabat).

[0052] 11. The modified antibody of any one of embodiments 1 to 10, wherein the modified antibody comprises an even number of first recognition site(s) for KalbTG at or inserted after one or more positions independently selected from the group consisting of position 110 (LC110), position 143 (LC143), and position 214 (LC214) of the antibody light chain, and position 118 (HC118), position 177 (HC177), position 297 (HC297), position 341 (HC341), and position 401 (HC401) of the antibody heavy chain (numbering according to Kabat).

[0053] 12. The modified antibody of embodiment 11, wherein the even number is 2 or 4 or 6 or 8.

[0054] 13. The modified antibody of any one of embodiments 11-12, wherein the even number is 2 or 4.

[0055] 14. The modified antibody of any one of embodiments 1 to 13, wherein the modified antibody comprises an even number of first recognition site(s) for KalbTG at or inserted after one or more positions independently selected from the group consisting of position 110 (LC110), position 143 (LC143), and position 214 (LC214) of the antibody light chain, and position 118 (HC118), position 177 (HC177), position 297 (HC297), position 341 (HC341), and position 401 (HC401) of the antibody heavy chain (numbering according to Kabat).

[0056] 15. The modified antibody of embodiment 14, wherein the odd number is 1 or 3 or 5 or 7.

[0057] 16. The modified antibody of any one of embodiments 14 to 15, wherein the odd number is 1 or 3.

[0058] 17. The modified antibody of any one of embodiments 1 to 16, wherein the modified antibody comprises at least one cognate pair of an antibody heavy chain and an antibody light chain.

[0059] 18. The modified antibody of any one of embodiments 1 to 17, wherein the antibody heavy chain comprises one first recognition site for KalbTG inserted at or after position 297 (HC297) of the antibody heavy chain (numbering according to Kabat).

[0060] 19. The modified antibody of any one of embodiments 1 to 18, wherein the antibody heavy chain comprises one first recognition site for KalbTG inserted at or after position 177 (HC177) of the antibody heavy chain (numbering according to Kabat).

[0061] 20. The modified antibody of any one of embodiments 1 to 19, wherein the antibody light chain comprises one primary recognition site for KalbTG inserted at or after position 214 (LC214) of the antibody light chain (numbering according to Kabat).

[0062] 21. The modified antibody of any one of embodiments 1 to 20, wherein the antibody heavy chain comprises two first recognition sites for KalbTG inserted at or after position 297 (HC297) and position 177 (HC177) of the antibody heavy chain (numbering according to Kabat).

[0063] 22. The modified antibody of any one of embodiments 1 to 20, wherein the antibody heavy chain comprises one first recognition site for KalbTG inserted at or after position 297 (HC297) of the antibody heavy chain, and the antibody light chain comprises one first recognition site for KalbTG inserted at or after position 214 (LC214) of the antibody light chain (numbering according to Kabat).

[0064] 23. The modified antibody of any one of embodiments 1 to 20, wherein the antibody heavy chain comprises one first recognition site for KalbTG inserted at or after position 177 (HC177) of the antibody heavy chain, and the antibody light chain comprises one first recognition site for KalbTG inserted at or after position 214 (LC214) of the antibody light chain (numbering according to Kabat).

[0065] 24. The modified antibody of any one of embodiments 1 to 20, wherein the antibody heavy chain comprises one first recognition site for KalbTG inserted at or after position 297 (HC297) and position 177 (HC177) of the antibody heavy chain, and the antibody light chain comprises two first recognition sites for KalbTG inserted at or after position 214 (LC214) of the antibody light chain (numbering according to Kabat).

[0066] 25. The modified antibody of any one of embodiments 1 to 24, wherein the modified antibody comprises two pairs of antibody heavy chains and cognate antibody light chains.

[0067] 26. (i) the modified antibody comprises two pairs of antibody heavy chains and two pairs of antibody light chains, each of which comprises one or more first recognition sites for KalbTG; or (ii) the modified antibody comprises two pairs each of antibody heavy chains and antibody light chains, and one of the antibody heavy chains or / and antibody light chains comprises one or more first recognition sites for KalbTG; or (iii) The modified antibody of any one of embodiments 1 to 25, wherein the modified antibody comprises one pair of antibody heavy chains and one additional antibody heavy chain Fc region polypeptide, and the antibody heavy chain and / or antibody heavy chain Fc region fragment or antibody light chain comprises one or more first recognition sites for KalbTG.

[0068] 27. The modified antibody of embodiment 25, wherein the antibody heavy chain of a first pair comprises one first recognition site for KalbTG inserted at or after position 297 (HC297) of the antibody heavy chain, and the antibody light chain of a second pair comprises one first recognition site for KalbTG inserted at or after position 214 (LC214) of the antibody light chain (numbering according to Kabat).

[0069] 28. The modified antibody of embodiment 25, wherein the antibody heavy chain of a first pair comprises one first recognition site for KalbTG inserted at or after position 177 (HC177) of the antibody heavy chain, and the antibody light chain of a second pair comprises one first recognition site for KalbTG inserted at or after position 214 (LC214) of the antibody light chain (numbering according to Kabat).

[0070] 29. The modified antibody of embodiment 25, wherein a first pair of antibody heavy chains comprises one first recognition site for KalbTG inserted at or after position 297 (HC297) of the antibody heavy chain, a second pair of antibody heavy chains comprises one first recognition site for KalbTG inserted at or after position 177 (HC177) of the antibody heavy chain, and a second pair of antibody light chains comprises one first recognition site for KalbTG inserted at or after position 214 (LC214) of the antibody light chain (numbering according to Kabat).

[0071] 30. The modified antibody of embodiment 25, wherein a first pair of antibody heavy chains comprises one first recognition site for KalbTG inserted at or after position 177 (HC177) of the antibody heavy chain, a second pair of antibody heavy chains comprises one first recognition site for KalbTG inserted at or after position 297 (HC297) of the antibody heavy chain, and a second pair of antibody light chains comprises one first recognition site for KalbTG inserted at or after position 214 (LC214) of the antibody light chain (numbering according to Kabat).

[0072] 31. The modified antibody of embodiment 25, wherein the antibody heavy chain of a first pair comprises two first recognition sites for KalbTG inserted at or after position 297 (HC297) and position 177 (HC177) of the antibody heavy chain, and the antibody light chain of a second pair comprises one first recognition site for KalbTG inserted at or after position 214 (LC214) of the antibody light chain (numbering according to Kabat).

[0073] 32. The modified antibody of any one of embodiments 1 to 25, wherein both antibody heavy chains comprise one first recognition site for KalbTG inserted at or after position 297 (HC297) of the antibody heavy chain (numbering according to Kabat).

[0074] 33. The modified antibody of any one of embodiments 1 to 25, wherein both antibody heavy chains comprise one first recognition site for KalbTG inserted at or after position 177 (HC177) of the antibody heavy chain (numbering according to Kabat).

[0075] 34. The modified antibody of any one of embodiments 1 to 25, wherein both antibody light chains comprise one primary recognition site for KalbTG inserted at or after position 214 (LC214) of the antibody light chain (numbering according to Kabat).

[0076] 35. The modified antibody of any one of embodiments 1 to 25, wherein both antibody heavy chains comprise two first recognition sites for KalbTG inserted at or after position 297 (HC297) and position 177 (HC177) of the antibody heavy chain (numbering according to Kabat).

[0077] 36. The modified antibody of any one of embodiments 1 to 25, wherein both antibody heavy chains comprise one primary recognition site for KalbTG inserted at or after position 297 (HC297) of the antibody heavy chain, and both antibody light chains comprise one primary recognition site for KalbTG inserted at or after position 214 (LC214) of the antibody light chain (numbering according to Kabat).

[0078] 37. The modified antibody of any one of embodiments 1 to 25, wherein both antibody heavy chains comprise one primary recognition site for KalbTG inserted at or after position 177 (HC177) of the antibody heavy chain, and both antibody light chains comprise one primary recognition site for KalbTG inserted at or after position 214 (LC214) of the antibody light chain (numbering according to Kabat).

[0079] 38. The modified antibody of any one of embodiments 1 to 25, wherein both antibody heavy chains comprise two first recognition sites for KalbTG inserted at or after position 297 (HC297) and position 177 (HC177) of the antibody heavy chain, and both antibody light chains comprise one first recognition site for KalbTG inserted at or after position 214 (LC214) of the antibody light chain (numbering according to Kabat).

[0080] 39. The modified antibody of any one of embodiments 1-38, wherein the modified antibody comprises two identical antibody heavy chains.

[0081] 40. The modified antibody of any one of embodiments 1-38, wherein the modified antibody comprises two different antibody heavy chains.

[0082] 41. The modified antibody of embodiment 40, wherein the differences result from at least one mutation to induce heterodimerization.

[0083] 42. The modified antibody of any one of embodiments 1 to 41, wherein the modified antibody comprises, in addition to the recognition site(s) for KalbTG, the following 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).

[0084] 43. A modified antibody Fc region comprising at least one modified antibody heavy chain Fc region polypeptide, wherein the modified antibody heavy chain Fc region polypeptide comprises one or two or three or four or more first recognition site(s) for transglutaminase from Kutneria albida (KalbTG) at or after one or more positions independently selected from the group of positions comprising: position 118 (HC118), position 177 (HC177), position 297 (HC297), position 341 (HC341), and position 401 (HC401) of the antibody heavy chain (numbering according to Kabat).

[0085] 44. The modified antibody Fc region of embodiment 43, wherein a first recognition site for KalbTG is present within the amino acid sequence of each antibody chain.

[0086] 45. The modified antibody Fc region of any one of embodiments 43-44, wherein the modified antibody heavy chain Fc region polypeptide further comprises a first recognition site for KalbTG fused at or to its C-terminus.

[0087] 46. ​​The modified antibody Fc region of any one of embodiments 43 to 45, wherein the antibody heavy chain Fc region polypeptide further comprises a first recognition site for KalbTG at position 446 (HC446) or position 447 (HC447) of the antibody heavy chain (numbering according to Kabat).

[0088] 47. The modified antibody Fc region of any one of embodiments 43 to 46, wherein one or two or three or four or more positions are independently selected from the group of positions comprising position 118 (HC118), position 177 (HC177), position 297 (HC297) and position 341 (HC341) of the antibody heavy chain (numbering according to Kabat).

[0089] 48. The modified antibody Fc region of any one of embodiments 43 to 47, wherein one or two or three or four or more positions are independently selected from the group of positions comprising position 341 (HC341), position 297 (HC297) and position 177 (HC177) of the antibody heavy chain (numbering according to Kabat).

[0090] 49. The modified antibody of any one of embodiments 43 to 48, wherein one or two or three or four or more positions are independently selected from the group of positions comprising position 297 (HC297) and position 177 (HC177) of the antibody heavy chain (numbering according to Kabat).

[0091] 50. The modified antibody Fc region of any one of embodiments 43 to 49, wherein the modified antibody Fc region comprises an even number of first recognition site(s) for KalbTG inserted at or after one or more positions independently selected from the group of positions comprising position 118 (HC118), position 177 (HC177), position 297 (HC297), position 341 (HC341), and position 401 (HC401) of the antibody heavy chain (numbering according to Kabat).

[0092] 51. The modified antibody Fc region of embodiment 50, wherein the even number is 2 or 4 or 6 or 8.

[0093] 52. The modified antibody Fc region of any one of embodiments 49-50, wherein the even number is 2 or 4.

[0094] 53. The modified antibody Fc region of any one of embodiments 43 to 49, wherein the modified antibody Fc region comprises an odd number of first recognition site(s) for KalbTG inserted at or after one or more positions independently selected from the group of positions comprising position 118 (HC118), position 177 (HC177), position 297 (HC297), position 341 (HC341), and position 401 (HC401) of the antibody heavy chain (numbering according to Kabat).

[0095] 54. The modified antibody Fc region of embodiment 53, wherein the odd number is 1 or 3 or 5 or 7.

[0096] 55. The modified antibody Fc region of any one of embodiments 53-54, wherein the odd number is 1 or 3.

[0097] 56. The modified antibody Fc region of any one of embodiments 43 to 55, wherein the modified antibody heavy chain Fc region polypeptide comprises one first recognition site for KalbTG inserted at or after position 297 (HC297) of the antibody heavy chain (numbering according to Kabat).

[0098] 57. The modified antibody Fc region of any one of embodiments 43 to 56, wherein the modified antibody heavy chain Fc region polypeptide comprises one first recognition site for KalbTG inserted at or after position 177 (HC177) of the antibody heavy chain (numbering according to Kabat).

[0099] 58. The modified antibody Fc region of any one of embodiments 43 to 57, wherein the modified antibody heavy chain Fc region polypeptide comprises two first recognition sites for KalbTG inserted at or after position 297 (HC297) and position 177 (HC177) of the antibody heavy chain (numbering according to Kabat).

[0100] 59. The modified antibody Fc region of any one of embodiments 43-58, wherein the modified antibody Fc region comprises two modified antibody heavy chain Fc region polypeptides of any one of embodiments 43-58.

[0101] 60. The modified antibody Fc region of any one of embodiments 43 to 58, wherein the modified antibody Fc region comprises two modified antibody heavy chain Fc region polypeptides, one modified antibody heavy chain Fc region polypeptide comprising one first recognition site for KalbTG inserted at or after position 297 (HC297) of the antibody heavy chain, and the other modified antibody heavy chain Fc region polypeptide comprising one first recognition site for KalbTG inserted at or after position 177 (HC177) of the antibody heavy chain (numbering according to Kabat).

[0102] 61. The modified antibody Fc region of any one of embodiments 43-58, wherein the modified antibody Fc region comprises two modified antibody heavy chain Fc region polypeptides, both of which comprise a single first recognition site for KalbTG inserted at or after position 297 (HC297) of the antibody heavy chain (numbering according to Kabat).

[0103] 62. The modified antibody Fc region of any one of embodiments 43 to 58, wherein the modified antibody Fc region comprises two modified antibody heavy chain Fc region polypeptides, both of which comprise a first recognition site for KalbTG inserted at or after position 177 (HC177) of the antibody heavy chain (numbering according to Kabat).

[0104] 63. The modified antibody Fc region of any one of embodiments 43 to 58, wherein the modified antibody Fc region comprises two modified antibody heavy chain Fc region polypeptides, both of which have two first recognition sites for KalbTG inserted at or after position 297 (HC297) and position 177 (HC177) of the antibody heavy chain (numbering according to Kabat).

[0105] 64. The modified antibody Fc region of any one of embodiments 43-63, wherein the modified antibody Fc region comprises two identical antibody heavy chain Fc region polypeptides.

[0106] 65. The modified antibody Fc region of any one of embodiments 43-63, wherein the modified antibody Fc region comprises two different antibody heavy chain Fc region polypeptides.

[0107] 66. The modified antibody Fc region of embodiment 65, wherein the differences result from at least each mutation to induce heterodimerization.

[0108] 67. The modified antibody Fc region of any one of embodiments 43-66, wherein the modified antibody comprises, in addition to the recognition site(s) for KalbTG, the following 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).

[0109] 68. A modified antibody comprising a modified antibody Fc region according to any one of embodiments 43 to 67.

[0110] 69. The modified antibody or modified antibody Fc region of any one of embodiments 1 to 68, wherein the first recognition site(s) for KalbTG comprise or have a Gln-containing five amino acid motif.

[0111] 70. The modified antibody or modified antibody Fc region of any one of embodiments 1 to 69, wherein the first recognition site(s) for KalbTG are independently selected from the group of first recognition sites comprising the amino acid sequences 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), YRYRQ (SEQ ID NO: 17) and RVRQR (SEQ ID NO: 18).

[0112] 71. The modified antibody or modified antibody Fc region of any one of embodiments 1 to 70, wherein the first recognition site(s) for KalbTG are, independently of each other, selected from the amino acid sequences YRYRQ (SEQ ID NO: 17) or RVRQR (SEQ ID NO: 18).

[0113] 72. The modified antibody or modified antibody Fc region of any one of embodiments 1-71, wherein the first recognition site(s) of KalbTG is YRYRQ (SEQ ID NO: 17).

[0114] 73. The modified antibody or modified antibody Fc region of any one of embodiments 1 to 72, wherein recognition site(s) for KalbTG are inserted after position:

[0115] 74. The modified antibody or modified antibody Fc region of any one of embodiments 1 to 73, wherein the recognition site(s) for KalbTG are interposed between two flexible peptide linkers.

[0116] 75. The modified antibody or modified antibody Fc region of embodiment 74, wherein each peptide linker independently comprises 1 to 20 amino acids.

[0117] 76. The modified antibody or modified antibody Fc region of any one of embodiments 74-75, wherein each peptide linker independently comprises 1 to 10 amino acids.

[0118] 77. The modified antibody or modified antibody Fc region of any one of embodiments 74-76, wherein each peptide linker independently comprises 1 to 5 amino acids.

[0119] 78. The modified antibody or modified antibody Fc region of any one of embodiments 74 to 77, wherein the peptide linker does not essentially interfere with the function of the Q tag, KalbTG, antibody folding, and payload attached to the modified antibody.

[0120] 79. The modified antibody or modified antibody Fc region of any one of embodiments 74 to 79, wherein the peptide linker comprises predominantly or exclusively Gly and / or Ser and / or Ala and / or Thr and / or Glu amino acid residues.

[0121] 80. The modified antibody or modified antibody Fc region of any one of embodiments 74 to 80, wherein the peptide linkers are independently selected from the group of peptide linkers comprising GGGP (SEQ ID NO: 20), ESGS (SEQ ID NO: 21), APAP (SEQ ID NO: 22), KESGSVSSEQLAQFRSLD (SEQ ID NO: 23), and EGKSSGSGSESKST (SEQ ID NO: 24).

[0122] 81. The modified antibody or modified antibody Fc region of any one of embodiments 74 to 80, wherein the peptide linker consists primarily or entirely of Gly and Ser, independently of each other.

[0123] 82. The peptide linker is (Gly m Ser) n 82. The modified antibody or modified antibody Fc region of any one of embodiments 74 to 81, wherein m=1, 2, 3 or 4 and n=1, 2, 3, 4 or 5, and m and n are selected independently of each other.

[0124] 83. The modified antibody or modified antibody Fc region of any one of embodiments 82, wherein m=3 and n=1.

[0125] 84. The modified antibody or modified antibody Fc region of any one of embodiments 74 to 83, wherein the peptide linker comprises one or two or three or four or five repeating units, each having the amino acid sequence Gly-Gly-Gly-Ser (n=1, 2, 3, 4, 5; SEQ ID NO: 19).

[0126] 85. The modified antibody or modified antibody Fc region of embodiment 84, wherein the peptide linker comprises one repeating unit having the amino acid sequence Gly-Gly-Gly-Ser (n=1; SEQ ID NO: 19).

[0127] 86. The modified antibody of any one of embodiments 1-85, wherein the modified antibody comprises one or two or three or four Fab fragments.

[0128] 87. The modified antibody of any one of embodiments 1 to 86, wherein the modified antibody is a monovalent, monospecific antibody comprising one full-length antibody light chain and one full-length antibody heavy chain forming a cognate antibody light chain-heavy chain pair, and one antibody heavy chain Fc region polypeptide fragment comprising a hinge region associated with the Fc region of the full-length antibody heavy chain.

[0129] 88. The modified antibody of any one of embodiments 1-87, wherein the modified antibody is based on a human Ig heavy chain constant region.

[0130] 89. The modified antibody of any one of embodiments 1-88, wherein the modified antibody is based on the heavy chain constant region of human IgG1, human IgG2, human IgG3, or human IgG4.

[0131] 90. The modified antibody of any one of embodiments 1 to 89, wherein the modified antibody has a constant region amino acid sequence that is 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 99% or more, or up to 100% identical to the amino acid sequence set forth in SEQ ID NO: 1 or 2, based on a human IgG1 heavy chain polypeptide.

[0132] 91. The modified antibody of any one of embodiments 1 to 89, wherein the modified antibody has a constant region amino acid sequence that is 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 99% or more, or up to 100% identical to the amino acid sequence set forth in SEQ ID NO: 3, based on a human IgG2 heavy chain polypeptide.

[0133] 92. The modified antibody of any one of embodiments 1 to 89, wherein the modified antibody has a constant region amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or up to 100% identical to the amino acid sequence set forth in SEQ ID NO:4, based on a human IgG3 heavy chain polypeptide.

[0134] 93. The modified antibody of any one of embodiments 1 to 89, wherein the modified antibody has a constant region amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or up to 100% identical to the amino acid sequence set forth in SEQ ID NO: 5, based on a human IgG4 heavy chain polypeptide.

[0135] 94. The modified antibody comprises the sequence of SEQ ID NO: 01 [Table 1] [Table 2] a first recognition site for KalbTG is inserted at or after one or more positions identified by [Table 3] Only if an additional first recognition site for KalbTG is inserted at one of the positions identified by [Table 4] and a first recognition site for KalbTG inserted after the position identified by

[0136] 95. The modified antibody comprises the sequence of SEQ ID NO: 02 [Table 5] [Table 6] a first recognition site for KalbTG is inserted at or after one or more positions identified by [Table 7] Only if an additional first recognition site for KalbTG is inserted at one of the positions identified by [Table 8] and a first recognition site for KalbTG inserted after the position identified by

[0137] 96. The modified antibody is SEQ ID NO: 03 [Table 9] [Table 10] a first recognition site for KalbTG is inserted at or after one or more positions identified by [Table 11] Only if an additional first recognition site for KalbTG is inserted at one of the positions identified by [Table 12] and a first recognition site for KalbTG inserted after the position identified by

[0138] 97. The modified antibody comprises SEQ ID NO: 04 [Table 13] [Table 14] a first recognition site for KalbTG is inserted at or after one or more positions identified by [Table 15] Only if an additional first recognition site for KalbTG is inserted at one of the positions identified by [Table 16] and a first recognition site for KalbTG inserted after the position identified by

[0139] 98. The modified antibody comprises the sequence of SEQ ID NO: 05 [Table 17] [Table 18] a first recognition site for KalbTG is inserted at or after one or more positions identified by [Table 19] Only if an additional first recognition site for KalbTG is inserted at one of the positions identified by [Table 20] and a first recognition site for KalbTG inserted after the position identified by

[0140] 99. The modified antibody of any one of embodiments 1 to 98, wherein the modified antibody comprises an antibody heavy chain constant region amino acid sequence that is 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 99% or more, or up to 100% identical to the amino acid sequence set forth in SEQ ID NO:8 or SEQ ID NO:9 or SEQ ID NO:34.

[0141] 100. The modified antibody of any one of embodiments 1 to 99, wherein the modified antibody comprises an antibody heavy chain constant region having the amino acid sequence of SEQ ID NO:8.

[0142] 101. The modified antibody of any one of embodiments 1 to 99, wherein the modified antibody comprises an antibody heavy chain constant region having the amino acid sequence of SEQ ID NO:9.

[0143] 102. The modified antibody of any one of embodiments 1-99, wherein the modified antibody comprises an antibody heavy chain constant region having the amino acid sequence of SEQ ID NO: 34.

[0144] 103. The modified antibody of any one of embodiments 1 to 102, wherein the modified antibody has an antibody light chain constant region amino acid sequence that is 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 99% or more, or up to 100% identical to the amino acid sequence set forth in SEQ ID NO: 6 or 7, based on the light chain of a human kappa or lambda antibody.

[0145] 104. The modified antibody of any one of embodiments 1 to 89, wherein the modified antibody has a constant region amino acid sequence that is 75% or more, such as 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 99% or more, or up to 100% identical to the amino acid sequence set forth in SEQ ID NO: 10.

[0146] 105. The modified antibody comprises SEQ ID NO: 06 [Table 21] [Table 22] and a first recognition site for KalbTG inserted at or after one or more positions identified by:

[0147] 106. The modified antibody comprises SEQ ID NO: 07 [Table 23] [Table 24] and a first recognition site for KalbTG inserted at or after one or more positions identified by:

[0148] 107. The modified antibody of any one of embodiments 1-106, wherein the modified antibody comprises an antibody light chain constant region having the amino acid sequence set forth in SEQ ID NO:10.

[0149] 108. The modified antibody of any one of embodiments 1 to 107, wherein the modified antibody comprises (i) an unmodified antibody light chain constant domain having an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100% identical to the amino acid sequence of any of SEQ ID NOs: 6 or 7, or (ii) an unmodified antibody heavy chain constant region having an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100% identical to the amino acid sequence of SEQ ID NOs: 1 to 5.

[0150] 109. The modified antibody is a) an unmodified antibody light chain constant domain comprising an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100%, identical to the amino acid sequence of any of SEQ ID NOs: 6 or 7; and b) A modified antibody according to any one of embodiments 1 to 107, comprising a modified antibody heavy chain constant region comprising or consisting 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 NOs: 1 to 5, and into which at least one recognition site for KalbTG has been inserted.

[0151] 110. The modified antibody is a) a modified antibody light chain constant domain comprising an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100%, identical to the amino acid sequence of any of SEQ ID NOs: 6 or 7, wherein at least one recognition site for KalbTG has been inserted; and b) A modified antibody according to any one of embodiments 1 to 107, comprising an unmodified antibody heavy chain constant region comprising or consisting 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 NOs: 1 to 5.

[0152] 111. The modified antibody is a) an antibody light chain constant domain comprising or consisting of an amino acid sequence that is at least 96%, 97%, 98% or 99%, in particular 100%, identical to the amino acid sequence of SEQ ID NO: 6 or 7; and b) A modified antibody according to any one of embodiments 1 to 107, comprising a modified antibody heavy chain constant region comprising or consisting 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.

[0153] 112. The modified antibody is a) an antibody light chain constant domain comprising or consisting of an amino acid sequence that is at least 96%, 97%, 98% or 99%, in particular 100%, identical to the amino acid sequence of SEQ ID NO: 6 or 7; and b) A modified antibody according to any one of embodiments 1 to 107, comprising a modified antibody heavy chain constant region comprising or consisting 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: 9.

[0154] 113. The modified antibody is a) an antibody light chain constant domain comprising or consisting of an amino acid sequence that is at least 96%, 97%, 98% or 99%, in particular 100%, identical to the amino acid sequence of SEQ ID NO: 6 or 7; and b) A modified antibody according to any one of embodiments 1 to 107, comprising a modified antibody heavy chain constant region comprising or consisting 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: 34.

[0155] 114. The modified antibody is a) a modified antibody light chain constant domain comprising or consisting 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 b) A modified antibody according to any one of embodiments 1 to 107, comprising an antibody heavy chain constant region comprising or consisting 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 NOs: 1 to 5.

[0156] 115. The modified antibody is a) a modified antibody light chain constant domain comprising or consisting 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 b) A modified antibody according to any one of embodiments 1 to 107, comprising an antibody heavy chain constant region comprising or consisting 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 NOs: 1 to 5.

[0157] 116. The modified antibody is a) a modified antibody light chain constant domain comprising or consisting 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 b) A modified antibody according to any one of embodiments 1 to 107, comprising an antibody heavy chain constant region comprising or consisting 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 NOs: 1 to 5.

[0158] 117. The modified antibody is a) a modified antibody light chain constant domain comprising or consisting 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 b) A modified antibody according to any one of embodiments 1 to 107, comprising a modified antibody heavy chain constant region comprising or consisting 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.

[0159] 118. The modified antibody is a) a modified antibody light chain constant domain comprising or consisting 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 b) A modified antibody according to any one of embodiments 1 to 107, comprising a modified antibody heavy chain constant region comprising or consisting 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: 9.

[0160] 119. The modified antibody is a) a modified antibody light chain constant domain comprising or consisting 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 b) A modified antibody according to any one of embodiments 1 to 107, comprising a modified antibody heavy chain constant region comprising or consisting 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: 34.

[0161] 120. The modified antibody is a) a modified antibody light chain constant domain comprising the amino acid sequence of SEQ ID NO: 10; and b) A modified antibody according to any one of embodiments 1 to 107, comprising a modified antibody heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 8.

[0162] 121. The modified antibody is a) a modified antibody light chain constant domain comprising the amino acid sequence of SEQ ID NO: 10; and b) A modified antibody according to any one of embodiments 1 to 107, comprising a modified antibody heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 9.

[0163] 122. The modified antibody is a) a modified antibody light chain constant domain comprising the amino acid sequence of SEQ ID NO: 10; and b) A modified antibody according to any one of embodiments 1 to 107, comprising a modified antibody heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 34.

[0164] 123. The modified antibody of any one of embodiments 1-122, wherein the modified antibody binds to an antigen.

[0165] 124. The modified antibody of any one of embodiments 1-123, wherein the modified antibody specifically binds to a structure that allows it to cross the blood-brain barrier.

[0166] 125. The modified antibody of any one of embodiments 1-124, wherein the modified antibody specifically binds to a receptor that induces receptor-mediated endocytosis.

[0167] 126. The modified antibody of any one of embodiments 1 to 125, wherein the modified antibody specifically binds to an antigen selected from the group of antigens consisting of human transferrin receptor 1 (TfR1), human insulin-like growth factor 1 receptor (IGF-1R), human low-density lipoprotein receptor-related protein 1 (LRP1), and human low-density lipoprotein receptor-related protein 8 (LRP8).

[0168] 127. The modified antibody of any one of embodiments 1-126, wherein the modified antibody specifically binds to human transferrin receptor 1.

[0169] 128. The modified antibody of any one of embodiments 1-127, wherein the modified antibody specifically binds to one or two antigens, and wherein the one or two antigens are specific for a particular cell type.

[0170] 129. The modified antibody of embodiment 128, wherein one or both of the antigens is a tumor marker specific for tumor cells.

[0171] 130. The modified antibody of any one of embodiments 128-129, wherein the cell type is a breast cancer cell.

[0172] 131. A covalent conjugate comprising: (i) a modified antibody according to any one of embodiments 1 to 130; and (ii) one or more non-antibody domain(s) covalently conjugated to the one or more first recognition site(s) for KalbTG of the modified antibody of (i), A covalent conjugate wherein the non-antibody domain contains a second recognition site for KalbTG.

[0173] 132. The covalent conjugate according to embodiment 131, wherein the non-antibody domain is a therapeutic moiety comprising a therapeutic entity and, optionally, a second linker.

[0174] 133. The non-antibody domain is (i) the treatment entity; (ii) a second recognition site for KalbTG that is complementary to the first recognition site present in the modified antibody, i.e., if the modified antibody comprises a Q tag, the non-antibody domain comprises a K tag, or vice versa, and in particular, the second recognition site comprises or has a Lys-containing motif, in particular the sequence RYESK (SEQ ID NO: 16) (K tag); (iii) The covalent conjugate of embodiment 131 or 132, optionally comprising a second linker between the therapeutic entity and the second recognition site.

[0175] 134. The covalent conjugate according to any one of embodiments 131 to 133, wherein the second recognition site for KalbTG is a K tag having at least 80% sequence identity with the peptide sequence RYESK (SEQ ID NO: 16).

[0176] 135. The covalent conjugate according to any one of embodiments 131-134, wherein the therapeutic entity is a nucleic acid.

[0177] 136. The covalent conjugate according to any one of embodiments 131-135, wherein the therapeutic entity is DNA or RNA, or a mixture thereof.

[0178] 137. The covalent conjugate according to any one of embodiments 131-136, wherein the therapeutic entity is a non-coding RNA molecule.

[0179] 138. The covalent conjugate according to any one of embodiments 131-137, wherein the therapeutic entity is a non-coding RNA molecule of 15 to 24 base pairs in length.

[0180] 139. The covalent conjugate according to any one of embodiments 131-138, wherein the therapeutic entity comprises one or more locked nucleic acid (LNA) residues.

[0181] 140. The covalent conjugate according to any one of embodiments 131-139, wherein the therapeutic entity is an LNA oligonucleotide mixed with DNA or RNA residues.

[0182] 141. The covalent conjugate according to any one of embodiments 131 to 140, wherein the therapeutic entity is an siRNA or an antisense oligonucleotide.

[0183] 142. The covalent conjugate according to any one of embodiments 131 to 141, wherein the therapeutic entity is selected from the group comprising antisense oligonucleotides, including LNA nucleotides, toxins, small organic molecules and immunomodulators.

[0184] 143. The covalent conjugate of embodiment 142, wherein the small organic molecule has a molecular weight of 2,500 daltons or less.

[0185] 144. The covalent conjugate of any one of embodiments 142-143, wherein the small organic molecule has a molecular weight of 1,000 daltons or less.

[0186] 145. The covalent conjugate according to any one of embodiments 131-144, wherein the second linker is an alkyl linker or a polyethylene linker or a peptide linker or a mixture thereof.

[0187] 146. The covalent conjugate according to any one of embodiments 131-145, wherein the second linker comprises an ethylene glycol unit.

[0188] 147. The covalent conjugate according to any one of embodiments 131-146, wherein the second linker comprises about 2 to 50 ethylene glycol units.

[0189] 148. The covalent conjugate according to any one of embodiments 131-147, wherein the second linker comprises an aliphatic carbon chain.

[0190] 149. The second linker is an unsubstituted or substituted C 1~6 may contain alkyl, substituted C 1~6 149. The covalent conjugate of any one of embodiments 131-148, wherein, in the case of alkyl, the substituents are selected from the group consisting of alkoxy, acyl, acyloxy, alkoxycarbonyl, carbonylalkoxy, acylamino, amino, aminoacyl, aminocarbonylamino, aminocarbonyloxy, cycloalkyl, cycloalkenyl, cyano, azido, halo, hydroxyl, nitro, carboxyl, thiol, thioalkyl, alkyl, alkenyl, alkynyl, heterocyclyl, aminosulfonyl, sulfonylamino, sulfonyl, and oxo.

[0191] 150. The covalent conjugate according to any one of embodiments 131-145, wherein the second linker is a peptide linker.

[0192] 151. The covalent conjugate of any one of embodiments 1 to 150, wherein the conjugate comprises a modified antibody of any one of embodiments 131 to 130 and one or more therapeutic nucleic acids, each therapeutic nucleic acid linked to a single Q tag via an isopeptide bond and, optionally, amide-linked to the terminal residue of a K tag via a second linker.

[0193] 152. The covalent conjugate of embodiment 151, wherein the one or more therapeutic nucleic acid(s) is(are) antisense oligonucleotide(s).

[0194] 153. The covalent conjugate according to any one of embodiments 151-152, wherein the second linker comprises one or more ethylene glycol units.

[0195] 154. The covalent conjugate according to any one of embodiments 131 to 153, wherein the non-antibody domain comprises a therapeutic entity comprising RNA or LNA, and optionally a PEG linker, for treating or preventing a brain disease.

[0196] 155. The covalent conjugate according to embodiment 154, wherein the brain disease is Parkinson's disease or Alzheimer's disease.

[0197] 156. The method is a) providing a modified antibody according to any one of embodiments 1 to 130; b) providing a non-antibody domain, the non-antibody domain comprising (i) a therapeutic entity, (ii) a second recognition site for KalbTG that is complementary to the first recognition site present in the modified antibody provided under (i), and (iii) optionally, a second linker between the therapeutic entity and the second recognition site for KalbTG; c) incubating the modified antibody of a) and the non-antibody domain of b) in the presence of KalbTG or a functionally active variant or fragment thereof, thereby forming an isopeptide bond between the first and second recognition sites for KalbTG; A method of covalently conjugating a modified antibody according to any one of embodiments 1 to 130 to a therapeutic entity, comprising incubating the modified antibody with the therapeutic entity, thereby conjugating the modified antibody to the therapeutic entity.

[0198] 157. The method of embodiment 156, wherein step c) is carried out under conditions that promote the activity of KalbTG.

[0199] 158. The method according to any one of embodiments 156 to 157, wherein the first recognition site for KalbTG has the amino acid sequence YRYRQ (SEQ ID NO: 17).

[0200] 159. The method according to any one of embodiments 156 to 158, wherein the amino acid sequence of the second recognition site for KalbTG is RYESK (SEQ ID NO: 16).

[0201] 160. The modified antibody, modified Fc region, covalent conjugate and method of any one of embodiments 1-159, wherein KalbTG comprises the amino acid sequence (three letter code). [Table 25]

[0202] 161. A pharmaceutical composition comprising a covalent conjugate according to any one of embodiments 131-155 and 160.

[0203] 162. A covalent conjugate according to any one of embodiments 131-155 and 160, or a covalent conjugate prepared according to the method of any one of embodiments 156-160, for use as a medicament.

[0204] 163. The use according to embodiment 162, wherein the use is for treating a neurological or brain disease or cancer.

[0205] 164. The use according to any one of embodiments 162-163, wherein the neurological disease is selected from the group consisting of neuropathy, neurodegenerative disease, cancer, eye disease, seizure disorder, lysosomal storage disease, amyloidosis, viral or microbial disease, ischemia, behavioral disorder, CNS inflammation, Alzheimer's disease, Parkinson's disease, multiple sclerosis, CD20-positive cancer with brain metastasis, and HER2-positive cancer with brain metastasis.

[0206] 165. The use according to any one of embodiments 162 to 164, wherein the neurological disorder is selected from a neuropathic disorder, a neurodegenerative disorder, a cancer, an ophthalmic disorder, a seizure disorder, a lysosomal storage disease, an amyloidosis, a viral or microbial disease, an ischemia, a behavioral disorder, and a CNS inflammation.

[0207] 166. The use according to any one of embodiments 162 to 165, wherein the use is for treating Alzheimer's disease or Parkinson's disease or breast cancer.

[0208] 167. A nucleic acid or nucleic acid composition encoding a modified antibody according to any one of embodiments 1 to 130.

[0209] 168. A cell comprising a nucleic acid or nucleic acid composition according to embodiment 167.

[0210] 169. A method for producing a modified antibody according to any one of embodiments 1 to 130, comprising: - culturing the cells according to embodiment 168, - recovering the modified antibody from the cells and / or the culture medium, thereby producing a modified antibody according to any one of embodiments 1 to 130.

[0211] 170. The method of embodiment 169, wherein the cell is a mammalian cell.

[0212] 171. The method of any one of embodiments 169-170, wherein the cells are CHO cells. DETAILED DESCRIPTION OF THE INVENTION

[0213] The present invention is based, at least in part, on the discovery that Q-tags cannot be incorporated into every site within antibodies, such as IgG1 antibodies, with the attendant compatibility for conjugation to a payload. Antibody constant domain regions were screened for KalbTG-mediated conjugation. In this context, KalbTG Q-tags were inserted into surface-exposed inter- and intra-domain flexible loops within the human IgG1 heavy and light chain constant regions, as well as into the C-termini of each heavy and light chain. In total, nine different sites within the IgG1 heavy and light chains were tested. The KalbTG Q-tag motif was inserted spaced apart between / within two flexible linkers as the amino acid sequence GGGS-YRYRQ-GGGS (SEQ ID NO: 25). To demonstrate the general applicability of the findings, five antibodies (mAb-1 to mAb-5) with different binding specificities were further tested. These molecules with a single KalbTG recognition site insertion were evaluated for their expression rates. The results are shown in the Examples and Table 1. [Table 26]

[0214] It can be seen that expression titers changed, ie decreased or even improved, depending on the insertion site, which was completely unexpected.

[0215] The modified antibodies were further tested for Q-tag conjugation and accessibility for enzymatic modification with KalbTG. Two different payloads were tested: a small molecule (a fluorescent dye) and a small single-stranded nucleic acid. When the antibody was symmetric, i.e., containing two identical heavy and light chain pairs, there were two Q-tags per molecule. When the antibody was asymmetric, i.e., containing two different heavy and light chain pairs, there was a single Q-tag per molecule. Therefore, for symmetric antibodies, a drug-to-antibody ratio (DAR, i.e., the number of payload molecules per antibody molecule) of 2 was expected with 100% conjugation efficiency, and for asymmetric antibodies, a DAR of 1 was expected with 100% conjugation efficiency. The results for fluorescent dyes in the case of mAb-5 are shown in Table 2. [Table 27]

[0216] The results for single-stranded nucleic acids of 15 nucleotides in length for mAb-1 and 2 and 20 nucleotides in length for mAb-4 are shown in Table 3 (determined by HIC and UV-vis). [Table 28]

[0217] Mab-1 and mAb-2 were conjugated in a one-step method, while mAb-4 was conjugated in a two-step method (using click chemistry to first conjugate the K tag and then the K tag to the nucleic acid), thereby demonstrating the general applicability of the method according to the invention using antibodies according to the invention, i.e., the method is independent of the binding specificity of the antibody and the technique used to conjugate the payload to the K tag, i.e., either directly or sequentially.

[0218] As an example, for mAb-2, five different Q tags, RYGQR (SEQ ID NO: 11), RWRQR (SEQ ID NO: 12), YRQRT (SEQ ID NO: 13), IRQRQ (both Qs can be modified; SEQ ID NO: 14), and FRYRQ (SEQ ID NO: 15), were introduced into three different positions, namely, HC297, HC446, and LC143. The expression yield (1 L, μg / mL) and conjugation (reverse; HIC / UV-vis) results are shown in Table 4. [Table 29]

[0219] Further biophysical characterization of the modified antibodies conjugated to single-stranded nucleic acids consisting of 15 and 20 nucleotides, respectively, was performed by hydrophobic interaction chromatography (HIC), as exemplarily shown for mAb-2 (15 nucleotides) in Table 5 and Figure 1. The retention time of the hydrophilic marker was 9.25 min, and the relative retention time of the hydrophobic marker was 25.9 min (mAb-2). [Table 30]

[0220] Thus, we have successfully identified several KalbTG Q-tag insertion sites spanning the length of the IgG backbone that do not adversely affect expression yield and allow / provide enzymatic accessibility.

[0221] Therefore, suitable insertion sites for Q-tag expression yield and enzymatic accessibility are positions 110 (LC110), 143 (LC143), and 214 (LC214) of the light chain and positions 118 (HC118), 177 (HC177), 297 (HC297), 341 (HC341), and 401 (HC401) of the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat).

[0222] Furthermore, in vivo analysis using human FcRn transgenic mice was performed to determine the pharmacokinetic parameters of the modified antibodies conjugated to one or two single-stranded nucleic acids, respectively. The results are shown in Table 6. [Table 31]

[0223] Therefore, preferred insertion sites for the PK properties of the Q-tag are at position 214 of the light chain (LC214) and positions 177 (HC177) and 297 (HC297) of the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat).

[0224] Thus, in a first aspect, the present invention relates to a modified antibody comprising a heavy chain and a light chain, wherein the heavy and / or light chain comprises one or more (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) at or inserted after one or more positions (amino acid numbering according to the EU numbering scheme of Kabat) selected from position 110 (LC110), position 143 (LC143), and position 214 (LC214) in the light chain, and position 118 (HC118), position 177 (HC177), position 297 (HC297), position 341 (HC341), and position 401 (HC401) in the heavy chain. Thus, in the modified antibody of the present invention, the recognition site(s) for KalbTG are located within the constant region of the Ig heavy chain polypeptide, i.e., not at either end and / or not within or at the C-terminus of the Ig light chain constant domain. Furthermore, the modified antibodies of the invention may comprise an additional first recognition site for KalbTG at position 446 (HC446) of the heavy chain, i.e., at the C-terminus. By inserting a recognition site for KalbTG at a position, it is meant that the amino acid present at that position in the unmodified sequence is replaced by the recognition site, or that a recognition site is preferably further inserted after that position.

[0225] In particular embodiments of all aspects and embodiments of the invention, 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 (amino acid numbering according to the EU numbering scheme of Kabat).

[0226] In certain embodiments of all aspects and embodiments of the invention, 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 (amino acid numbering according to the EU numbering scheme of Kabat).

[0227] In a preferred embodiment of all aspects and embodiments of the invention, the one or more positions are selected from the group of positions comprising position 214 (LC214) of the light chain, and position 297 (HC297) and position 177 (HC177) of the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat).

[0228] In certain embodiments of all aspects and embodiments of the invention, modified antibodies according to the invention comprise two identical heavy chains or heavy chain Fc regions. In further embodiments, such modified antibodies comprise two, four, or more (primary) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) at one or more positions selected from position 110 (LC110), position 143 (LC143), and position 214 (LC214) in the light chain and position 118 (HC118), position 177 (HC177), position 297 (HC297), position 341 (HC341), and position 401 (HC401) in the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat).

[0229] In certain embodiments of all aspects and embodiments of the invention, modified antibodies according to the invention comprise two different heavy chains, whereby the differences arise from respective mutations to induce heterodimerization. In further embodiments, such modified antibodies comprise one, two, or more (first) recognition site(s) for 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), and position 401 (HC401) of the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat).

[0230] Similarly, the present invention relates to modified antibody Fc regions comprising heavy chain Fc regions, wherein the heavy chain Fc regions comprise one or more (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) at or inserted after one or more positions selected from the group consisting of positions 118 (HC118), 177 (HC177), 297 (HC297), 341 (HC341), and 401 (HC401) of the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat). Thus, in the modified antibody Fc regions of the present invention, the recognition site(s) for KalbTG are located within the constant region of the Ig heavy chain Fc region polypeptide, i.e., not at any of the termini. Furthermore, the modified antibody Fc regions of the present invention may comprise an additional first recognition site for KalbTG at position 446 (HC446) of the heavy chain, i.e., at the C-terminus. In reference to inserting a recognition site for KalbTG at a position, it means that the amino acid present at that position in the unmodified sequence is replaced by the recognition site, or that a recognition site is preferably further inserted after that position.

[0231] In particular embodiments of all aspects and embodiments of the invention, the one or more positions are selected from the group of positions comprising heavy chain position 118 (HC118), position 177 (HC177), position 297 (HC297), and position 341 (HC341) (amino acid numbering according to the EU numbering scheme of Kabat).

[0232] In particular embodiments of all aspects and embodiments of the invention, 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 (amino acid numbering according to the EU numbering scheme of Kabat).

[0233] In a preferred embodiment of all aspects and embodiments of the invention, the one or more positions are selected from the group of positions comprising position 297 (HC297) and position 177 (HC177) of the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat).

[0234] In a preferred embodiment of all aspects and embodiments of the present invention, the modified antibody comprises at least one (cognate) pair of antibody heavy chain and antibody light chain, wherein the heavy chain comprises one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) at position 297 (HC297) of the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat).

[0235] In a preferred embodiment of all aspects and embodiments of the present invention, the modified antibody comprises at least one (cognate) pair of antibody heavy chain and antibody light chain, wherein the heavy chain comprises one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) at position 177 (HC177) of the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat).

[0236] In a preferred embodiment of all aspects and embodiments of the present invention, the modified antibody comprises at least one (cognate) pair of antibody heavy chain and antibody light chain, wherein the light chain comprises one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) at position 214 (LC214) of the light chain (amino acid numbering according to the Kabat numbering scheme).

[0237] In a preferred embodiment of all aspects and embodiments of the present invention, the modified antibody comprises at least one (cognate) pair of antibody heavy and light chains, wherein the heavy chain comprises two (first) recognition sites for transglutaminase from Kutzneria albida (KalbTG) at positions 297 (HC297) and 177 (HC177) of the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat).

[0238] In a preferred embodiment of all aspects and embodiments of the present invention, the modified antibody comprises at least one (cognate) pair of antibody heavy and light chains, wherein the heavy chain comprises one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) at position 297 (HC297) of the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat) and the light chain comprises one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) at position 214 (LC214) of the light chain (amino acid numbering according to the Kabat numbering scheme).

[0239] In a preferred embodiment of all aspects and embodiments of the present invention, the modified antibody comprises at least one (cognate) pair of antibody heavy and light chains, wherein the heavy chain comprises one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) at position 177 (HC177) of the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat) and the light chain comprises one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) at position 214 (LC214) of the light chain (amino acid numbering according to the Kabat numbering scheme).

[0240] In a preferred embodiment of all aspects and embodiments of the present invention, the modified antibody comprises at least one (cognate) pair of antibody heavy and light chains, wherein the heavy chain comprises two (primary) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) at positions 177 (HC177) and 297 (HC297) of the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat) and the light chain comprises one (primary) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) at position 214 (LC214) of the light chain (amino acid numbering according to the Kabat numbering scheme).

[0241] In a preferred embodiment of all aspects and embodiments of the present invention, the modified antibody comprises two (cognate) pairs of antibody heavy and light chains, wherein the heavy chain of the first pair comprises one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) at position 297 (HC297) of the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat) and the light chain of the second pair comprises one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) at position 214 (LC214) of the light chain (amino acid numbering according to the Kabat numbering scheme).

[0242] In a preferred embodiment of all aspects and embodiments of the present invention, the modified antibody comprises two (cognate) pairs of antibody heavy and light chains, wherein the heavy chain of the first pair comprises one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) at position 177 (HC177) of the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat) and the light chain of the second pair comprises one (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) at position 214 (LC214) of the light chain (amino acid numbering according to the Kabat numbering scheme).

[0243] In a preferred embodiment of all aspects and embodiments of the present invention, the modified antibody comprises two (cognate) pairs of antibody heavy and light chains, wherein the heavy chains of the first pair comprise a (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) at position 297 (HC297) of the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat), the heavy chains of the second pair comprise a (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) at position 177 (HC177) of the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat), and the light chains of the second pair comprise a (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) at position 214 (LC214) of the light chain. The nucleotide sequence of the ...

[0244] In a preferred embodiment of all aspects and embodiments of the present invention, the modified antibody comprises two (cognate) pairs of antibody heavy and light chains, wherein the heavy chains of the first pair comprise a (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) at position 177 (HC177) of the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat), the heavy chains of the second pair comprise a (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) at position 297 (HC297) of the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat), and the light chains of the second pair comprise a (first) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) at position 214 (LC214) of the light chain. The nucleotide sequence of the ...

[0245] In a preferred embodiment of all aspects and embodiments of the present invention, the modified antibody comprises two (cognate) pairs of antibody heavy and light chains, wherein the heavy chains of the first pair comprise two (primary) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) at positions 297 (HC297) and 177 (HC177) of the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat), and the light chains of the second pair comprise one (primary) recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) at position 214 (LC214) of the light chain (amino acid numbering according to the Kabat numbering scheme).

[0246] In certain embodiments of all aspects and embodiments of the present invention, a recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) is inserted immediately after that position. In certain embodiments, the recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) is inserted between two flexible linkers. In certain embodiments, the recognition site(s) for transglutaminase from Kutzneria albida (KalbTG) has the amino acid sequence GGGSYRYRQGGGS (SEQ ID NO: 25).

[0247] The terms "antibody" and "Ig" are used interchangeably herein. They are used in the broadest sense and include, for example, monoclonal antibodies regardless of binding specificity (including agonist, antagonist, neutralizing, full-length or intact monoclonal antibodies), monovalent antibodies (e.g., full-length antibodies lacking one Fab), multivalent antibodies, i.e., antibodies that are bivalent or tetravalent multispecific antibodies, and fragments of full-length antibodies, so long as they contain at least one of the modifications outlined above.

[0248] Naturally occurring antibodies are generated by the assembly of heavy chains alone (e.g., heavy-chain-only VHH antibodies) or heavy and light chains (e.g., wild-type IgG antibodies). Each heavy chain is composed of four domains: a variable domain (VH) and three constant domains (CH1, CH2, and CH3), which are connected by a flexible hinge region. A light chain is composed of a variable domain (VL) and a constant domain (CL). When heavy and light chains are present, the light chain pairs with a cognate heavy chain, i.e., a Fab fragment of the heavy chain containing the VH and CH1 domains, to form the antibody binding site. The associated light and heavy chain Fab fragments are collectively referred to as Fab fragments. The heavy chain CH2 and CH3 domains are collectively referred to as an antibody heavy chain Fc region polypeptide. One antibody heavy chain Fc region polypeptide dimerizes with an additional antibody heavy chain Fc region polypeptide, for example, from a second antibody heavy chain, to form an Fc region. The Fc region is connected to the Fab fragment(s) via a flexible hinge region. Thus, in certain embodiments, an antibody heavy chain Fc region polypeptide further comprises all or part of a hinge region in addition to the CH2 and CH3 domains. The hinge region contains several disulfide bridges that covalently link two antibody heavy chain Fc region polypeptides to each other. In Fab fragments, the light and heavy chain Fab fragments are also connected by a single disulfide bridge. However, the connectivity differs between IgG subclasses. The overall structure of a full-length IgG resembles a Y-shape, with the Fc region forming the base and the two Fab fragments forming the arms available for antigen binding.

[0249] Within the variable domains are loops called complementarity-determining regions (CDRs), which are primarily responsible for the direct interaction of the antibody with its antigen. Due to the significant variation in the number of amino acids in these CDRs, multiple numbering schemes exist for variable regions. As used herein, the amino acid positions of all heavy and light chain constant regions and domains are numbered according to the Kabat numbering system as described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), and are referred to herein as "numbering according to Kabat." Specifically, the Kabat numbering system of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see pages 647-660) is used for the light chain constant domains CL of kappa and lambda isotypes, and the Kabat EU index numbering system (see pages 661-723) is used for the heavy chain constant domains (CH1, hinge, CH2, and CH3, which is further clarified in this specification by saying "numbering is according to the Kabat EU index" in this case).

[0250] As used herein, the term "modified antibody" refers to an antibody or antibody Fc region according to the present invention 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, heavy chain-only antibodies, and functional fragments thereof. The term "functional fragment" refers to a portion of an intact antibody that retains some or all of the antigen-binding activity of the antibody from which the fragment is derived. Non-limiting examples of functional antibody fragments include Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, etc. In particular, modified antibodies according to the present invention include antibody molecules and immunologically active portions of antibody molecules, such as molecules comprising an antigen-binding domain or antigen-binding site (e.g., one or more complementarity-determining regions (CDRs)), so long as modifications according to the present invention are present. The modified antibodies provided herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY, and in certain embodiments, IgG), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, and in one preferred embodiment, IgG1), or any subclass (e.g., IgG2a and IgG2b). Antibodies can be humanized, chimeric, and / or affinity matured, and antibodies from other species, such as mouse, rabbit, and sheep.

[0251] According to the present invention, in certain embodiments, the modified antibody comprises at least one heavy chain and at least one light chain. Thus, in certain embodiments, the modified antibody comprises one, two, three, or four Fab fragments. In certain embodiments, the modified antibody is a monovalent, monospecific antibody comprising one (full-length) light chain and one (full-length) heavy chain forming a cognate light chain-heavy chain pair (comprising one binding site), and one heavy chain Fc region fragment comprising the hinge region associated with the Fc region of the (full-length) heavy chain.

[0252] According to the present invention, in certain embodiments, the modified antibody may be based on an IgG1, IgG2, IgG3, or IgG4 antibody, in particular a humanized, mouse, rabbit, or sheep antibody. Exemplary suitable sequences are shown below: [Table 32] In the annotation, "X" indicates an amino acid residue / position, [Table 33] indicates the insertion site of the Q-tag motif (with or without a linker sequence): [Table 34] [Table 35] [Table 36] [Table 37] [Table 38]

[0253] Thus, in some embodiments, the heavy chain constant region of a modified antibody according to the invention is based on a human Ig heavy chain constant region, such as a human IgG1, human IgG2, human IgG3, or human IgG4 heavy chain constant region. In certain embodiments, the heavy chain constant region comprises at least one Q tag according to the invention.

[0254] In certain embodiments, human IgG1 heavy chain polypeptides on which modified antibodies according to the invention may be based comprise a constant region amino acid sequence that is 75% or more, for example 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 99% or more, and up to 100% identical to the amino acid sequence set forth in SEQ ID NO: 1 or 2. In a preferred embodiment of all aspects and embodiments of the invention, modified antibodies according to the invention are based on an IgG1 antibody and are therefore modified / variant IgG1 antibodies. In certain embodiments, human IgG2 heavy chain polypeptides on which modified antibodies according to the invention may be based comprise a constant region amino acid sequence that is 75% or more, for example 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 99% or more, and up to 100% identical to the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, human IgG3 heavy chain polypeptides on which modified antibodies according to the present invention can be based comprise a constant region amino acid sequence that is 75% or more, for example 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 99% or more, and up to 100% identical to the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, human IgG4 heavy chain polypeptides on which modified antibodies according to the present invention can be based comprise a constant region amino acid sequence that is 75% or more, for example 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 99% or more, and up to 100% identical to the amino acid sequence set forth in SEQ ID NO:5.

[0255] In particular embodiments of all aspects and embodiments of the invention, the modified antibody according to the invention comprises, in addition to the inserted KalbTG recognition site according to the invention, 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).

[0256] In a preferred embodiment of all aspects and embodiments of the invention, a modified antibody according to the invention comprises the mutations L234A, L235A, P329G, T366W in a first Fc-region polypeptide and the mutations L234A, L235A, P329G, T366S, L368A, Y407V in a second Fc-region polypeptide. In a particular embodiment of all aspects and embodiments of the invention, the modified antibody further comprises one of the mutations S354C and Y349C in the first Fc-region polypeptide and the other in the second Fc-region polypeptide.

[0257] In a preferred embodiment of all aspects and embodiments of the invention, the modified antibody according to the invention is based on a human heavy chain polypeptide and has a constant region amino acid sequence that is 75% or more, such as 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 99% or more and up to 100% identical to the amino acid sequence set forth in SEQ ID NO:8 or SEQ ID NO:9 or SEQ ID NO:34.

[0258] [Table 39]

[0259] According to the present invention, in certain embodiments of all aspects and embodiments of the present invention, the modified antibody may be based on an IgG1, IgG2, IgG3 or IgG4 antibody, in particular a humanized antibody further comprising a light chain constant domain. Exemplary suitable sequences of modified light chain constant domains according to the present invention are shown below: [Table 40] In the annotation, "X" indicates an amino acid residue / position, [Table 41] indicates the insertion site of the Q tag motif (with or without linker sequence): [Table 42]

[0260] In particular embodiments of all aspects and embodiments of the invention, the human light chain polypeptide on which the modified antibodies according to the invention may be based comprises a constant region amino acid sequence that is 75% or more, such as 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 99% or more and up to 100% identical to the amino acid sequence set forth in SEQ ID NO: 6 or 7.

[0261] In a preferred embodiment of all aspects and embodiments of the invention, the human light chain polypeptide of the modified antibody according to the invention is SEQ ID NO: 10: [Table 43] The constant region amino acid sequence is 75% or more, for example, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 99% or more, and up to 100% identical to the amino acid sequence shown in

[0262] Preferably, the unmodified light chain constant domain comprises an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100%, identical to the amino acid sequence of any of SEQ ID NOs: 6 or 7, or the unmodified heavy chain constant region comprises an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100% identical to the amino acid sequence of any of SEQ ID NOs: 1 to 5.

[0263] In a preferred embodiment of all aspects and embodiments of the invention, the modified antibody according to the invention comprises: a) an unmodified light chain constant domain comprising an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100%, identical to the amino acid sequence of any of SEQ ID NOs: 6 or 7; and b) A modified heavy chain constant region comprising or consisting 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 NOs: 1 to 5, and into which at least one recognition site for KalbTG has been inserted.

[0264] In a preferred embodiment of all aspects and embodiments of the invention, the modified antibody according to the invention comprises: a) a modified light chain constant domain comprising an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100%, identical to the amino acid sequence of any of SEQ ID NOs: 6 or 7, wherein at least one KalbTG recognition site has been inserted; and b) comprises an unmodified heavy chain constant region comprising or consisting 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 NOs: 1 to 5.

[0265] 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, or the heavy chain constant region comprises or consists of an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100% identical to the amino acid sequence of SEQ ID NO: 8 or 9 or 34.

[0266] In one preferred embodiment of all aspects and embodiments of the invention, the modified antibody according to the invention comprises a) a light chain constant domain comprising or consisting 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 b) a modified heavy chain constant region comprising or consisting of an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100% identical to the amino acid sequence of SEQ ID NO: 8 or 9 or 34.

[0267] As detailed above, modified antibodies according to the present invention may be bispecific or multispecific antibodies. Exemplary bispecific or multispecific antibodies include: -Full length antibodies with domain swapping: A multispecific IgG antibody comprising a first Fab fragment and a second Fab fragment, wherein in the first Fab fragment: a) only the CH1 and CL domains are replaced by each other (i.e., the light chain of the first Fab fragment comprises the VL and CH1 domains, and the heavy chain of the first Fab fragment comprises the VH and CL domains); b) only the VH and VL domains are replaced by one another (i.e. the light chain of the first Fab fragment comprises the VH and CL domains and the heavy chain of the first Fab fragment comprises the VL and CH1 domains); or c) the CH1 and CL domains are replaced with each other and the VH and VL domains are replaced with each other (i.e., the light chain of the first Fab fragment comprises the VH and CH1 domains, and the heavy chain of the first Fab fragment comprises the VL and CL domains); and the second Fab fragment comprises a light chain comprising a VL and a CL domain and a heavy chain comprising a VH and a CH1 domain; A full-length antibody with domain swapping may comprise a first heavy chain comprising a CH3 domain and a second heavy chain comprising a CH3 domain, both CH3 domains being complementarily engineered by respective amino acid substitutions to support heterodimerization of the first heavy chain and the modified second heavy chain; -Full-length antibody with domain swapping and an additional heavy chain C-terminal binding site: 1. A multispecific IgG antibody, comprising: a) a full-length antibody comprising two pairs of full-length antibody light chains and two pairs of full-length antibody heavy chains, wherein the binding site formed by each pair of full-length heavy chains and full-length light chains specifically binds to a first antigen; b) an additional Fab fragment fused to the C-terminus of one heavy chain of the full-length antibody, wherein the binding site of the additional Fab fragment specifically binds to a second antigen; a multispecific IgG antibody, wherein the additional Fab fragment that specifically binds to a second antigen i) comprises a domain crossover such that a) the light chain variable domain (VL) and the heavy chain variable domain (VH) are substituted for each other, or b) the light chain constant domain (CL) and the heavy chain constant domain (CH1) are substituted for each other, or ii) is a single-chain Fab fragment; -1-arm single chain format (=1-arm single chain antibody): An antibody comprising a first binding site that specifically binds to a first epitope or antigen and a second binding site that specifically binds to a second epitope or antigen, wherein the individual chains are as follows: -Light chain (variable light domain + constant light kappa domain) -Light / heavy chain combination (variable light domain + light constant domain + peptide linker + variable heavy domain + CH1 + hinge + CH2 + CH3 knob mutations) -Heavy chain (variable heavy domain + CH1 + hinge + CH2 + CH3 with hole mutation); -2-arm single chain format (=2-arm single chain antibody): An antibody comprising a first binding site that specifically binds to a first epitope or antigen and a second binding site that specifically binds to a second epitope or antigen, wherein the individual chains are as follows: -Light chain / heavy chain 1 combination (variable light chain domain + light chain constant domain + peptide linker + variable heavy chain domain + CH1 + hinge + CH2 + CH3 hole mutation) - light chain / heavy chain 2 combination (variable light chain domain + light chain constant domain + peptide linker + variable heavy chain domain + CH1 + hinge + CH2 + CH3 knob mutations); -Common light chain bispecific formats (=common light chain bispecific antibodies): An antibody comprising a first binding site that specifically binds to a first epitope or antigen and a second binding site that specifically binds to a second epitope or antigen, wherein the individual chains are as follows: -Light chain (variable light domain + constant light domain) -Heavy chain 1 (variable heavy chain domain + CH1 + hinge + CH2 + CH3 hole mutation) -Heavy chain 2 (variable heavy domain + CH1 + hinge + CH2 + CH3 knob mutations).

[0268] The term "specifically binds" means that an antibody binds to at least 10 -8 Dissociation constant in mol / l (=K Diss) interacts with its cognate antigen. Thus, the term "specifically binds" refers to a -8 Dissociation constant (=K Diss ) to its cognate antigen. The terms "not specifically binding" or "does not specifically bind" are used to refer to binding of an antibody having a specific binding affinity of up to 10 -7 mol / l or more, i.e., for example, 10 -5 Dissociation constant in mol / l (=K Diss ) indicates the binding of an antibody to an unrelated antigen. At the same time, the property of "not specifically binding" is -7 K in mol / l or less Diss In certain embodiments, an antibody that specifically binds to an antigen has a K of at least 100-fold between its reactivity to its cognate antigen and its reactivity to an unrelated antigen. Diss -There will be gaps.

[0269] Antibodies, especially K Diss The binding properties of can be assessed by a BIAcore® instrument. In this method, the binding properties are assessed by changes in surface plasmon resonance (SPR). It is convenient to attach the antibody under investigation to a solid phase (called a chip) and assess its binding by applying the antigen of interest to this coated chip.

[0270] As used herein, the term "replaced by one another" with respect to corresponding heavy and light chain domains refers to the domain crossover described above. Thus, when the CH1 and CL domains are "replaced by one another," the term refers to the domain crossover described under item (i) and the resulting heavy and light chain domain sequences. Thus, when the VH and VL are "replaced by one another," the term refers to the domain crossover described under item (ii), and when the CH1 and CL domains are "replaced by one another" and the VH and VL domains are "replaced by one another," the term refers to the domain crossover described under item (iii).

[0271] In certain embodiments, multispecific antibodies also comprise at least one Fab fragment comprising either the domain crossover of the CH1 and CL domains described in item (i) above, or the domain crossover of the VH and VL domains described in item (ii) above, or the domain crossover of the VH-CH1 and VL-VL domains described in item (iii) above. In the case of multispecific antibodies with domain crossovers, Fabs that specifically bind to the same antigen(s) are constructed to have the same domain sequence. Therefore, when more than one Fab with domain crossovers is included in a multispecific antibody, the Fab(s) specifically bind to the same antigen(s).

[0272] The term "antigen" refers to a predetermined target to which an antibody can selectively bind. An antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten or fragment thereof, or other naturally occurring or synthetic compound. In certain embodiments, the antigen is a polypeptide. In other embodiments, the antigen is therapeutically relevant. In yet other embodiments, the antigen is specific to or allows delivery to a particular area in the body, such as a particular organ or cell type or diseased area. It can be a receptor or a specific structure on the surface of a cell, such as a tumor marker. In one preferred embodiment of all aspects and embodiments of the present invention, the antigen is the human transferrin receptor.

[0273] Preferably, (i) the antibody comprises two pairs of heavy and / or light chains, each of which comprises one or more first recognition sites, or (ii) the antibody comprises two pairs of heavy and light chains, each of which comprises one or more first recognition sites, or (iii) the antibody comprises one pair of heavy and light chains and one additional heavy chain Fc region, each of which comprises one or more first recognition sites.

[0274] According to the present invention, modified antibodies contain one or more recognition sites for KalbTG in the heavy and / or light chain polypeptides. The recognition sites include a KalbTG motif, which can catalyze the formation of an isopeptide bond between the modified antibody and a compound (payload) that binds to the modified antibody. Typically, the isopeptide bond is formed between a glutamine (Gln, Q) side chain and a lysine (Lys, K) side chain. Preferably, the modification introduced into the antibody to obtain the modified antibody of the present invention includes the creation of an (artificial) Q tag in the antibody heavy and / or light chain polypeptide, i.e., a Gln-containing motif recognized by KalbTG. Suitable Q tags are disclosed in WO 2017 / 102759, which is expressly incorporated herein by reference. In certain embodiments, one or more first KalbTG recognition sites, independent of one another, comprise or have a Gln-containing motif, particularly the sequence RYGQR (SEQ ID NO:11), RWRQR (SEQ ID NO:12), YRQRT (SEQ ID NO:13), IRQRQ (SEQ ID NO:14), FRYRQ (SEQ ID NO:15), or YRYRQ (SEQ ID NO:17), in particular YRYRQ (SEQ ID NO:17). Q-tags can be generated by one or more amino acid modifications, such as substitutions or insertions, preferably insertions. In certain embodiments, Q-tags are generated by insertion and / or substitution of an amino acid sequence comprising YRYRQ (SEQ ID NO:17) or RVRQR (SEQ ID NO:18), in particular YRYRQ (SEQ ID NO:17). Preferably, the inserted amino acid sequence has a length of 5 to 20 amino acids and comprises YRYRQ (SEQ ID NO:17) or RVRQR (SEQ ID NO:18), in particular YRYRQ (SEQ ID NO:17). In certain embodiments, the insertion is a Q-tag motif without a linker, i.e., the insertion consists of YRYRQ (SEQ ID NO: 17) or RVRQR (SEQ ID NO: 18), particularly YRYRQ (SEQ ID NO: 17). Introduction of one or more recognition sites for KalbTG may be the only modification in the light and / or heavy chain constant regions. Alternatively, further modifications, such as tags for purification (e.g., His tags) or other modifications, such as increased stability or heterodimerization or modified effector function, may be present alone or in any combination.

[0275] As described above, one or more first recognition site(s) for KalbTG are inserted into specific sites in the antibody, namely at one or more positions selected from positions 110 (LC110), 143 (LC143), and 214 (LC214) of the light chain and positions 118 (HC118), 177 (HC177), 297 (HC297), 341 (HC341), and 401 (HC401) of the heavy chain, in particular HC177 and / or HC297 and / or LC214. In certain embodiments, the modified antibodies of the invention may comprise an additional (second) recognition site for KalbTG at position 446 (HC446) of the heavy chain, i.e., at the C-terminus, in particular for linking to a domain different from the domain bound by the first recognition site. Preferably, one or more of the recognition sites are independent of each other at positions 177 (HC177) or 297 (HC297) of the heavy chain or position 214 (LC214) of the light chain.

[0276] In certain embodiments, the Q tag is inserted into the antibody light / heavy chain amino acid sequence, spaced apart between / via one or two linker(s). The linker may provide increased flexibility or allow conjugation with larger payloads. In certain embodiments, each linker sequence, independently of the others, comprises 1 to 20 amino acids, in certain embodiments 1 to 10 amino acids, and in further embodiments 1 to 5 amino acids, which linkers do not essentially interfere with the function of the Q tag, KalbTG, antibody folding, or payloads attached to the modified antibody. The linker may be attached N-terminally and / or C-terminally to the Q tag. In certain embodiments, the linker amino acid is a small amino acid such as glycine or serine. Amino acid linkers and their compositions are known in the art (see, e.g., Chichili et al.). The amino acids glycine, serine, alanine, threonine, and glutamic acid typically constitute the amino acids of flexible linkers. Thus, the linker(s) may consist mainly or entirely of Gly and / or Ser and / or Ala and / or Thr and / or Glu, e.g., GGGP (SEQ ID NO: 20), ESGS (SEQ ID NO: 21) or APAP (SEQ ID NO: 22). There may also be linkers present which comprise or consist of KESGSVSSSEQLAQFRSLD (SEQ ID NO: 23) or EGKSSGSGSESKST (SEQ ID NO: 24). In a preferred embodiment of all aspects and embodiments of the invention, the linkers consist mainly or entirely of Gly and Ser, independently of each other, e.g., (Gly m Ser) n wherein m=1, 2, 3 or 4 and n=1, 2, 3, 4 or 5, and m and n are independently of each other, preferably m=3 and n=1. In one preferred embodiment of all aspects and embodiments of the present invention, the one or more first recognition sites are inserted at their ends into the heavy chain and / or light chain amino acid sequence via one or two linker(s), in particular the linkers consist mainly or entirely of Gly and Ser, for example (Gly-Gly-Gly-Ser). n (SEQ ID NO: 19) where n=1, 2, 3, 4 or 5, preferably n=1.

[0277] If a linker is present, it is an insertion of the amino acid sequence X1-YRYRQ-X2 (SEQ ID NO: 17) or X1-RVRQR-X2 (SEQ ID NO: 18) into the antibody. X1 and X2 are, independently of each other, absent or a linker, particularly a linker amino acid. In a preferred embodiment of all aspects and embodiments of the invention, the insertion is of a Q tag motif with two flexible linkers, particularly GGGSYRYRQGGGS (SEQ ID NO: 25) or GGGSRVRQRGGGS (SEQ ID NO: 26), in particular GGGSYRYRQGGGS (SEQ ID NO: 25).

[0278] Exemplary Fc regions comprising portions of the hinge region with (referred to as SEQ ID NO: 32, 113) and without (referred to as SEQ ID NO: 33, 110) the first recognition site (bold) have the following sequences: [Table 44]

[0279] In a second aspect, the present invention relates to one or more nucleic acids encoding the chains of a modified antibody according to the invention.

[0280] Nucleic acids encoding modified antibodies of the invention can be isolated or produced in vitro for recombinant production of the antibody. The nucleic acid can be inserted into a replicable vector for further cloning (amplification of the DNA) or for further expression.

[0281] The term "nucleic acid" encompasses DNA (gDNA and cDNA) and RNA molecules, and nucleotides, the basic structural units of nucleic acids, include not only naturally occurring nucleotides but also analogs with modified sugars or base moieties. The nucleic acid sequences encoding the heavy chain variable regions and light chain variable regions of the present invention may be modified. Such modifications include addition, deletion, or non-conservative or conservative substitution of nucleotides, as long as the encoded sequence remains unchanged.

[0282] DNA encoding modified antibodies according to the invention can be isolated or synthesized using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to DNA encoding the heavy and light chains of the antibody).

[0283] Many transfer and expression vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0284] As used herein, the term "vector" refers to a means for expressing a gene of interest in a host cell, such as a plasmid vector, a cosmid vector, or a viral vector, for example, a bacteriophage vector, an adenovirus vector, a retrovirus vector, or an adeno-associated virus vector. The nucleic acid encoding the modified antibody in the vector is operably linked to a promoter and a polyadenylation signal sequence.

[0285] "Operably linked" refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence, whereby the control sequence controls the transcription and / or translation of the other nucleic acid.

[0286] When a eukaryotic cell is used as the host, a promoter derived from the genome of a mammalian cell (e.g., metallothionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) may be used, or a mammalian promoter derived from an animal virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and murine sarcoma virus (RSV) promoter) may be used. In addition, a polyadenylation signal sequence is present after the coding nucleic acid as a transcription termination sequence.

[0287] Cells can be transformed with the aforementioned vectors. The cells used to produce the antibodies of the present invention can be, but are not limited to, prokaryotic cells, yeast cells, or higher eukaryotic cells.

[0288] However, of most interest are animal cells, and examples of useful host cell lines include, but are not limited to, COS-7, BHK, CHO, CHO-S, CHO-K1, GS-CHO, CHO DXB-11, CHO DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL3A, W138, HepG2, SK-Hep, MMT, TRI, MRC5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080. In a preferred embodiment, the host cells are CHO cells.

[0289] In a third aspect, the present invention relates to a covalent conjugate comprising (i) a modified antibody according to the invention, and (ii) one or more non-antibody (payload) domain(s) covalently conjugated to one or more (first) recognition site(s) for KalbTG, wherein the non-antibody domain(s) comprise a complementary second recognition site(s) for KalbTG. In certain embodiments of all aspects and embodiments of the invention, the non-antibody domain is a therapeutic moiety comprising a therapeutic entity and optionally a second linker.

[0290] As described above, the modified antibodies of the present invention are provided with the aid of KalbTG to specifically conjugate one or more payloads, e.g., therapeutic moieties, to one or more internal sites of the antibody. This results in an ADC that can be used for targeted therapy. The conjugate can bind to a target of interest, thereby transporting the payload, e.g., therapeutic moiety, to the intended area in the body. In certain embodiments, the modified antibodies of the present invention recognize and bind to a target through its complementarity-determining region (CDR), and in particular, the target is a biomolecule present on a cell.

[0291] As detailed above, it may be desirable to target a therapeutic moiety to a specific region in a patient's body. This can improve in vivo distribution and reduce adverse side effects. Clearly, it may be intended to deliver a therapeutic moiety to an area that is otherwise difficult to reach. As an example, it may be envisioned to guide a therapeutic moiety into the brain. Due to the blood-brain barrier, it is difficult to deliver a "naked" therapeutic moiety to the brain unless it is administered directly, especially if the therapeutic moiety exceeds a certain size limit. A therapeutic approach that overcomes the blood-brain barrier and helps transport a therapeutic moiety into the brain would clearly be advantageous. Notably, the same approach can be used to guide a therapeutic agent to another region in the body.

[0292] In the present invention, the molecular recognition unit of an antibody that specifically binds to a structure within the body is used to target a therapeutic moiety. In view of the above, it is clear that the therapeutic entity can be any compound useful in the treatment or prevention of a disease of interest, particularly a compound that is delivered to a specific region within the body, such as a specific organ or cell type or diseased area.

[0293] The terms "treat," "treating," and "treatment" are meant to include alleviating or arresting a condition, disorder, or disease, or one or more symptoms associated with a condition, disorder, or disease, or alleviating or eradicating the cause(s) of the condition, disorder, or disease itself. The terms "prevent," "preventing," and "prevention" are meant to include methods of delaying and / or eliminating the onset of a condition, disorder, or disease and / or its attendant symptoms; methods of barring a subject from acquiring a condition, disorder, or disease; or methods of reducing a subject's risk of acquiring a condition, disorder, or disease.

[0294] For this purpose, a non-antibody payload comprising a therapeutic entity and, optionally, a second linker, is covalently conjugated to the modified antibody of the present invention. The therapeutic moiety comprises an active therapeutic entity or a prodrug. Optionally, a second linker may be present. The second linker may be, for example, a chemical linker comprising an alkyl group or a polyethylene group, or a peptide linker.

[0295] In a preferred embodiment of all aspects and embodiments of the present invention, the therapeutic entity is a nucleic acid such as an RNA, siRNA or ASO (antisense oligonucleotide), in particular an ASO comprising LNA nucleotides, and / or the therapeutic entity is a toxin or a small organic molecule or an immunomodulator.

[0296] In a preferred embodiment of all aspects and embodiments of the present invention, the therapeutic entity is a nucleic acid. In certain embodiments, the nucleic acid is DNA or RNA, or a mixture thereof. The term RNA also includes antisense RNA and small interfering RNA (siRNA), a class of double-stranded, non-coding RNA molecules, typically 20-24 base pairs in length, that are similar to miRNAs and operate within the RNA interference (RNAi) pathway. They disrupt the expression of specific genes with complementary nucleotide sequences by post-transcriptionally degrading mRNA, thereby preventing translation. In certain embodiments, the nucleic acid comprises one or more locked nucleic acids (LNAs). LNAs are modified RNA nucleotides in which the ribose moiety is modified with an extra bridge connecting the 2' oxygen and the 4' carbon. The bridge "locks" the ribose in the 3'-endo (north) conformation. This structure may be due to increased stability against enzymatic degradation, and furthermore, the LNA structure has improved specificity and affinity as a monomer or building block of an oligonucleotide. In certain embodiments, LNA nucleotides are mixed with DNA or RNA residues in an oligonucleotide or nucleic acid.

[0297] In certain embodiments, the therapeutic entity may additionally or alternatively be a small molecule. In the field of pharmacology, a small molecule has a low molecular weight (<2,500 daltons, particularly <1,000 daltons). Many small molecule therapeutic entities are small organic molecules. Small organic molecules typically bind to specific biological macromolecules and act as effectors, altering the activity or function of the target. These compounds may be natural (such as primary and secondary metabolites) or artificial (i.e., not naturally occurring), and they have beneficial effects against diseases.

[0298] In the present invention, the therapeutic entity is a non-antibody domain that is covalently attached to the modified antibody 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 affect the conjugation kinetics and the stability of the resulting conjugate. In one preferred embodiment of all aspects and embodiments of the present invention, the (second) linker is an alkyl linker, a polyethylene linker, or a peptide linker, or a mixture thereof. In certain embodiments, the (second) linker comprises ethylene glycol (PEG) units, for example, about 2 to 50 ethylene glycol units. Exemplary linkers include polyethylene glycol linkers ((-NH-C(=O)-PEG n In certain embodiments, the (second) linker is an aliphatic carbon chain. The linker may be an unsubstituted or substituted C 1~6 alkyl, which may include unsubstituted or substituted alkyl, such as C 1~6 The alkyl may be substituted with one or more substituents selected from the group consisting of alkoxy, acyl, acyloxy, alkoxycarbonyl, carbonylalkoxy, acylamino, amino, aminoacyl, aminocarbonylamino, aminocarbonyloxy, cycloalkyl, cycloalkenyl, cyano, azido, halo, hydroxyl, nitro, carboxyl, thiol, thioalkyl, alkyl, alkenyl, alkynyl, heterocyclyl, aminosulfonyl, sulfonylamino, sulfonyl, and oxo. In certain embodiments, the (second) linker is a peptide linker, i.e., a linker composed of amino acids.

[0299] In a preferred embodiment of all aspects and embodiments of the present invention, the conjugate comprises a modified antibody according to the present invention and one or more therapeutic nucleic acids, e.g., ASOs, each therapeutic nucleic acid linked via an isopeptide bond to a single Q-tag and, optionally, amide-linked to the terminal residue of a K-tag via a second linker, in particular a PEG linker, as defined above.

[0300] In certain embodiments of all aspects and embodiments of the invention, the conjugate has a DAR ranging from about 1 to about 8, from about 1 to about 4, or from about 1 to about 2. In other embodiments, the conjugate has a DAR of about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8.

[0301] Once the antibody binds to the target, the conjugate is transported into the respective cell by endocytosis and the therapeutic entity is released and can act in the intended manner (e.g., treat a disease).

[0302] In a preferred embodiment of all aspects and embodiments of the present invention, the modified antibody recognizes one target, which is a receptor that induces receptor-mediated endocytosis, such as human transferrin receptor 1 (TfR1), human insulin-like growth factor 1 receptor (IGF-1R), human low-density lipoprotein receptor-related protein 1 (LRP1) or human low-density lipoprotein receptor-related protein 8 (LRP8), in particular TfR1.

[0303] For example, in the case of specific receptors such as TfR1 or IGF-1R, when the antibody binds to the target, the conjugate is transported into the respective cell by endocytosis, released from the endosome, and exocytosed again from the cell. If the cell is part of a barrier such as the blood-brain barrier, transport across the respective barrier is thereby achieved. The therapeutic entity is thereby transported to body compartments that could not be reached by a therapeutic entity not conjugated to a modified antibody according to the present invention.

[0304] In a preferred embodiment of all aspects and embodiments of the present invention, the modified antibody specifically binds to a structure that allows for passage across the blood-brain barrier, hi a particular embodiment, the structure that allows for passage across the blood-brain barrier is the human transferrin receptor.

[0305] In certain embodiments, the modified antibody recognizes one or two targets(es), wherein the one or two targets are specific to a particular cell type, such as a tumor marker specific to tumor cells (e.g., breast cancer cells).

[0306] In a preferred embodiment of all aspects and embodiments of the present invention, the modified antibody according to the present invention is conjugated to a therapeutic entity comprising RNA or LNA, and optionally a non-antibody domain comprising a PEG linker, for treating or preventing a brain disease, such as Parkinson's disease or Alzheimer's disease.

[0307] In a fourth aspect, the present invention relates to a method for covalently conjugating a modified antibody according to the present invention to a therapeutic entity, said method comprising: a) providing a modified antibody according to the invention; b) providing a non-antibody domain, the non-antibody domain comprising: (i) Therapeutic entity; (ii) a second recognition site for KalbTG that is complementary to the first recognition site present in the modified antibody provided under (i), in a preferred embodiment, the second recognition site comprises or has a Lys-containing motif, in particular the sequence RYESK (SEQ ID NO: 16); and (iii) optionally providing a non-antibody domain comprising a second linker between the therapeutic entity and a second recognition site; c) incubating or reacting the modified antibody of a) and the non-antibody domain of b) in the presence of KalbTG or a functionally active variant or fragment thereof under conditions that promote the activity of KalbTG, thereby forming an isopeptide bond between the first recognition site and the second recognition site, and thus conjugating the modified antibody to the therapeutic entity.

[0308] In the first step of the method, modified antibodies and non-antibody domains of the invention are provided.

[0309] The non-antibody domain is (i) the treatment entity; (ii) a second recognition site for KalbTG that is complementary to the first recognition site present in the modified antibody, i.e., if the modified antibody comprises a Q tag, the non-antibody domain comprises a K tag, or vice versa, and in particular, the second recognition site comprises or has a Lys-containing motif, in particular the sequence RYESK (SEQ ID NO: 16) (K tag); (iii) optionally, a second linker between the therapeutic entity and the second recognition site.

[0310] The domain may be as defined above.

[0311] The modified antibody and non-antibody domain are incubated, i.e., reacted, in the presence of KalbTG or a functionally active variant or fragment thereof (meaning a variant or fragment of KalbTG having enzymatic activity equivalent to that of wild-type KalbTG) and under conditions that promote the activity of KalbTG, thereby forming an isopeptide bond between the first and second recognition sites and thus conjugating the modified antibody to the therapeutic entity.

[0312] In particular embodiments of all aspects and embodiments of this method according to the invention, the antibody comprises one or more Q tags(s) that are conjugated to the respective one or more K tags using the enzymatic activity of KalbTG.

[0313] The non-antibody domain comprises a second recognition site for KalbTG. In certain embodiments of all aspects and embodiments of this method according to the invention, the non-antibody domain comprises a K tag having at least 80% sequence identity to the peptide sequence RYESK (SEQ ID NO: 16).

[0314] Microbial transglutaminases (mTGs), including KalbTG, catalyze the formation of Gln-Lys isopeptide bonds and are widely used for cross-linking proteins and peptides in food and biotechnology applications (e.g., to improve the texture of protein-rich foods or in generating antibody-drug conjugates).

[0315] However, KalbTG exhibits essentially no cross-reactivity with known mTG substrates or commonly used target proteins, such as antibodies, thus allowing for specific conjugation at a predetermined site.

[0316] Thus, essentially any payload comprising a second recognition site for KalbTG (K tag), in particular where the second recognition site comprises or has a Lys-containing motif, in particular the sequence RYESK (SEQ ID NO: 16), can be conjugated / bound to an antibody modified with one or more first recognition site(s), i.e., Q tags.

[0317] KalbTG or a functionally active variant or fragment thereof may be as defined in Steffen at al. (2017) or WO 2016 / 100735 A1 (both of which are expressly incorporated herein by reference).

[0318] The functionally active variant or fragment may be a transglutaminase having at least 80%, 90%, 95%, or 99% sequence identity to KalbTG of WO 2016 / 100735 A1 (see SEQ ID NO: 6 therein). Alternatively, KalbTG or a functionally active variant thereof may be part of a fusion protein that additionally comprises a label, such as a tag, for example for purification purposes.

[0319] In certain embodiments, KalbTG comprises the amino acid sequence (three letter code). [Table 45]

[0320] In certain embodiments of all aspects and embodiments of the methods according to the invention, the linkage is controlled, e.g., achieved at a stoichiometric ratio of non-antibody domain to modified antibody, e.g., about 1:1 per Q-tag / K-tag pair. Multiple conjugation can also be achieved by using two or more first recognition sites on one modified antibody to attach two or even multiple payloads to the modified antibody.

[0321] In a fifth aspect, the covalent conjugate comprising the modified antibody of the invention or produced according to the method of the invention is for use as a medicament, in particular for use in the treatment of a neurological or brain disease such as Alzheimer's disease or Parkinson's disease, or for use in the treatment of cancer, such as breast cancer.

[0322] In certain embodiments of all aspects and embodiments of the invention, the disease is a neurological disease, hi certain embodiments, the neurological disease is selected from the group consisting of a neuropathy, a neurodegenerative disease, cancer, an eye disorder, a seizure disorder, a lysosomal storage disease, an amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, CNS inflammation, Alzheimer's disease, Parkinson's disease, multiple sclerosis, a CD20-positive cancer with brain metastasis, and a HER2-positive cancer with brain metastasis.

[0323] In certain embodiments of all aspects and embodiments of the invention, the neurological disorder is selected from the group consisting of a neuropathic disorder, a neurodegenerative disorder, a cancer, an ocular disorder, a seizure disorder, a lysosomal storage disease, an amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, and CNS inflammation.

[0324] As detailed above, conjugates comprising modified antibodies according to the invention can be used as medicines, in particular in the treatment of neurological or brain diseases (in a preferred embodiment Alzheimer's disease or Parkinson's disease) or for use in the treatment of cancers such as breast cancer.

[0325] The conjugate can be included in a composition. Such a composition, also called a pharmaceutical composition, refers to a composition intended for use in the pharmaceutical field or as a medicine, and is in a form that allows the biological activity of the active ingredient contained therein to be effective, and is a preparation that does not contain additional ingredients that are unacceptably toxic to the subject to which the pharmaceutical composition is administered. The composition can optionally contain pharmaceutically acceptable excipients, diluents or carriers, such as buffer substances, stabilizers or preservatives, and optionally further active ingredients, particularly ingredients known in the context of pharmaceutical compositions.

[0326] Generally, the nature of the additional ingredients will depend on the particular form of the pharmaceutical composition and the mode of administration used. Pharmaceutically acceptable carriers can be used to enhance or stabilize the composition or to facilitate preparation of the composition. Such carriers include, but are not limited to, physiologically compatible saline, buffered saline, dextrose, water, glycerol, solvents, dispersion media, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, as well as combinations thereof. The formulation should be appropriate for the mode of administration. For example, parenteral formulations usually comprise injectable fluids that contain pharmaceutically and physiologically acceptable fluids such as water, saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a vehicle. In addition to biologically neutral carriers, pharmaceutical compositions to be administered may contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents.

[0327] The pharmaceutical composition may contain a stabilizer. The term "stabilizer" refers to a substance that protects the composition from harmful conditions, such as those encountered during heating or freezing, and / or extends the stability or shelf life of the conjugates of the present invention under certain conditions or conditions. Examples of stabilizers include, but are not limited to, sugars such as sucrose, lactose, and mannose; sugar alcohols such as mannitol; amino acids such as glycine or glutamic acid; and proteins such as human serum albumin or gelatin.

[0328] Typically, a therapeutically effective dose or effective dose of the conjugate is used in the pharmaceutical composition of the present invention. The amount of the conjugate to be administered can be initially determined based on the dosage and / or administration regimen guidance of an equivalent unlinked therapeutic agent. Generally, the conjugate can provide targeted delivery and thus can provide at least one of a reduced dose or reduced administration in the administration regimen. Thus, the conjugate can provide a reduced dose and / or reduced administration in the administration regimen compared to the previous therapeutic agent present in the conjugate of the present invention. As described above, the conjugate can provide a controlled stoichiometry of drug delivery, so the dosage of the conjugate can be calculated based on the number of drug molecules provided per antibody-therapeutic entity conjugate.

[0329] The pharmaceutical compositions of the present invention may be administered once or several times, or multiple times. The frequency of administration of the conjugate may vary depending on various factors, such as the severity of symptoms. For example, in some embodiments, the conjugate is administered once every 6 months, once every 5 months, once every 4 months, once every 3 months, once every 2 months, once every month, twice a month, three times a month, every other week (qow), once a week (qw), twice a week (biw), three times a week (tiw), four times a week, five times a week, six times a week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid).

[0330] In certain embodiments, a conjugate or pharmaceutical composition of the invention is administered simultaneously with one or more additional compounds, hi certain embodiments, a conjugate or pharmaceutical composition of the invention is administered before or after the additional compound(s).

[0331] The pharmaceutical composition of the present invention can be used as a medicine to treat an individual. The individual is a mammal. Mammals include, but are not limited to, livestock animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the individual is a human.

[0332] Pharmaceutical compositions containing the conjugates described herein can be delivered to cells, groups of cells, tumors, tissues, or subjects using delivery techniques known in the art. Generally, any suitable art-recognized method for delivering conjugates can be adapted for use with the compositions described herein. For example, delivery can be by local administration (e.g., direct injection, implantation, or topical administration), systemic administration, or parenteral routes, including subcutaneous, intravenous, intraocular, intraperitoneal, or intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), or intramuscular administration. The covalent conjugates of the present invention are preferably administered intravenously, intramuscularly, or intraarterially, more preferably intravenously. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate the above-described pharmaceutical compositions in unit dosage form. As used herein, unit dosage form refers to physically discrete units suitable as unit dosages, each unit containing a predetermined amount of active ingredient calculated to produce the desired therapeutic effect in combination with the necessary pharmaceutical carrier. Examples of such unit dosage forms include injectable solutions or suspensions.

[0333] As detailed above, the conjugates / pharmaceutical compositions of the present invention are particularly useful for treating neurological or brain diseases, such as Alzheimer's disease or Parkinson's disease. The term "neurological disease" encompasses, inter alia, neurodegenerative diseases, neuroinflammatory diseases, or seizure disorders, particularly those of the brain. Neurodegenerative diseases are characterized by the progressive loss of neuronal structure or function, including neuronal death. Many neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, and multiple sclerosis, occur as a result of neurodegenerative processes. There are many similarities between different neurodegenerative disorders, including atypical protein assembly and induced cell death. Neurodegeneration can also be observed at many different levels of neural circuitry, ranging from molecular to systemic. The terms "neurodegenerative disease" and "neuroinflammatory disease" have partially overlapping scopes. Inflammatory responses are a hallmark of neurodegenerative diseases and are involved in or contribute to neuronal cell death through different mechanisms. Tryptophan catabolism along the kynurenine pathway (KP) represents one of these mechanisms. Seizure disorders are brain disorders characterized by abnormal signal transduction between brain cells. Seizure disorders can affect parts of the brain (partial seizures) or the entire brain (generalized seizures). The most prominent seizure disorder is epilepsy. Receptor-mediated or receptor-induced endocytosis can be used as a target for conjugates to cross the blood-brain barrier. Examples include transferrin receptor 1 (TfR1), insulin-like growth factor 1 receptor (IGF-1R), low-density lipoprotein receptor-related protein 1 (LRP1), or low-density lipoprotein receptor-related protein 8 (LRP8), particularly TfR1.

[0334] Unless otherwise defined, all technical and scientific terms and any acronyms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice presented herein, particular methods and materials are described herein.

[0335] The present invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. Any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, and exemplary methods and materials are described herein. Furthermore, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

[0336] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the words "comprise," "contain," and "encompass" are to be interpreted inclusively rather than exclusively. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. The term "plurality" refers to two or more.

[0337] The following figures, sequences, and examples are intended to illustrate various embodiments of the present invention. Therefore, the specific changes described should not be construed as limitations on the scope of the invention. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the invention, and therefore, such equivalent embodiments should be understood to be included herein. [Brief explanation of the drawings]

[0338] [Figure 1] 1 shows hydrophobic interaction chromatography of mAb-2 (HER 2) with nine different Q-tag insertion sites conjugated to a single-stranded nucleic acid with 15 residues. [Figure 2] 1 shows an exemplary reaction of a single-step KalbTG-mediated conjugation of a mAb with a nucleic acid payload. [Example]

[0339] Example 1 Recombinant production of modified antibodies according to the invention Gene synthesis The desired gene segments were prepared by chemical synthesis, and the synthesized gene fragments were cloned by Twist Bioscience (San Francisco, USA) into vectors suitable for expression in HEK293 and Expi293 cells.

[0340] Expression of modified antibodies in mammalian cells Antibody production was performed by transient cotransfection of single expression cassette plasmids into HEK293 cells cultured in F17 medium (Invitrogen, Carlsbad, CA, USA) or Expi293 cells in Expi293 expression medium (Thermo Scientific, Waltham, MA, USA). Transfections were performed as specified in the manufacturer's instructions, using a 1:1 ratio of HC:LC expression plasmids for the symmetric standard IgG1 format or a 1:1:1 ratio of HC1:HC2:LC expression plasmids for the asymmetric knob-into-hole format. Cell culture supernatants were harvested 7 days after transfection and stored at low temperature (e.g., -20°C).

[0341] Protein titer quantification Protein titers of supernatant samples were determined by affinity chromatography using a POROS A 20 μm column, 2.1 × 30 mm (Life Technologies, Carlsbad, CA, USA) on a high-performance liquid chromatography system (Ultimate 3000 HPLC system, Thermo Scientific, Waltham, MA, USA). The supernatant was loaded onto a column equilibrated with 0.2 M NaHPO, pH 7.4, followed by elution with 0.1 M citric acid, 0.2 M NaCl, pH 2.5. Titers were quantified by measuring absorbance at 280 nm, and protein concentrations were subsequently calculated by comparing the analyte elution peak area (under the curve) with a reference standard curve.

[0342] Purification of modified antibodies from mammalian culture supernatants Antibodies in the culture supernatant were captured by Protein A affinity chromatography using a Mab Select SuRe column (GE Healthcare, Chicago, IL, USA) equilibrated with PBS buffer, pH 7.4. Unbound proteins were removed by washing with the equilibration buffer. The modified antibodies were eluted with 50 mM citrate (pH 3.0), and the pH of the eluate was immediately adjusted to pH 7.5 by adding 2 M Tris (pH 9.0). A second purification step was performed by size exclusion chromatography using a Superdex 200™ column (GE Healthcare, Chicago, IL, USA) in 20 mM histidine, 140 mM NaCl, pH 6.0. The purified modified antibodies were stored at -80°C.

[0343] Antibody purification using mid-scale automation (Milan) Antibodies were purified in one step using Protein A affinity chromatography as described above, using MabSelect SuRe-Sepharose (Cytiva, Marlborough, MA, USA) on a liquid handling system (Tecan, Männedorf, Switzerland) equipped with a column from Repligen (Waltham, MA, USA). Equilibration, sample loading, and washing steps were performed as described, and antibodies were eluted from the column using 25 mM citrate, pH 3.0. Eluted antibody fractions were neutralized with 1.5 M Tris (pH 7.5), and concentrations were determined by measuring the optical density (OD) at 280 nm.

[0344] Exemplary Antibody Overview Antibody 110: explanation: Anti-HER2 antibody based on IgG1 subclass with P329G / L234A / L235A mutations; Q-tag inserted into HC after amino acid residue 177 (HC177) (EU numbering); Q-tag and spacer sequence: GGGSYRYRQGGGS (SEQ ID NO: 25) [Table 46]

[0345] Antibody 113: explanation: Anti-HER2 antibody based on IgG1 subclass with P329G / L234A / L235A mutations; Q-tag inserted into the HC after amino acid residue 401 (HC401) (EU numbering); Q-tag and spacer sequence: GGGSYRYRQGGGS (SEQ ID NO: 25) [Table 47] [Table 48]

[0346] The expression of modified antibodies is affected by the position of the introduced Q-tag: the highest yields were obtained when introduced after LC110, LC214, HC118, HC177, HC297, HC341, and HC401.

[0347] Example 2 KalbTG conjugation of modified antibodies according to the present invention to fluorescent dyes Conjugation with KalbTG The purified antibody containing the Q tag was transferred via dialysis into conjugation buffer (histidine buffer containing NaCl, pH 8.5). For the KalbTG reaction, the antibody was mixed with a K tag small molecule (a fluorescent dye, 10x molar excess) and KalbTG was added (molar ratio mAb:KalbTG > 100:1). The reaction mixture was incubated at 37°C with shaking, and the reaction was subsequently quenched by adding 10 mM ammonium sulfate to the solution. To remove unconjugated payload and residual enzyme, the conjugated modified antibody was purified by size exclusion chromatography using a Superdex 200™ column (GE Healthcare, Chicago, IL, USA) in PBS pH 7.5. The purified conjugate was stored at -80°C.

[0348] Analysis of conjugates Protein quantification was performed using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Additionally, qualitative DAR measurements were performed by hydrophobic interaction chromatography as described in Example 4 below. Conjugate purity was analyzed by CE-SDS under denaturing and reducing conditions using a Caliper LabChip® GXII Touch™ Protein Characterization System according to the manufacturer's instructions (Perkin Elmer, Waltham, MA, USA).

[0349] Aggregate content was determined by SEC using a TSKgel UP-SW 3000 analytical size exclusion column (Tosoh Bioscience, Griesheim, Germany) equilibrated with 0.2 M K2HPO4 / KH2PO4, 0.25 M KCl, pH 6.2 on a high-performance liquid chromatography system (Ultimate 3000 HPLC system, Thermo Fisher Scientific, Waltham, MA, USA).

[0350] The identity of the conjugate was confirmed by ESI-Q-ToF-MS (Bruker maXis 433, Bruker, Billerica, MA, USA). For MS analysis, samples were deglycosylated using N-glycosidase F (Roche, Basel, Switzerland) and subsequently desalted into 2% formic acid, 40% acetonitrile. [Table 49]

[0351] Example 3 KalbTG conjugation of modified antibodies according to the invention to nucleic acids Synthesis of antisense oligonucleotides Single-stranded LNA oligonucleotides were synthesized using standard phosphoramidite chemistry. DNA and LNA phosphoramidites and all standard reagents were purchased from Merck KGaA (Darmstadt, Germany). K-tag peptides were custom synthesized by Schafer-N Ap (Copenhagen, Denmark) and Biosyntan (Berlin, Germany).

[0352] Oligonucleotides were synthesized on a 130 mmol scale on a NittoPhase HL UnyLinker 350 support (Kinovate, Oceanside, CA) on an AKTA Oligopilot (GE Healthcare, Brondby, Denmark). After synthesis, the oligonucleotides were cleaved from the support overnight. The oligonucleotides were purified by ion exchange chromatography and desalted using Millipore membranes. After lyophilization, the compounds were finally characterized by liquid chromatography-mass spectrometry (reverse phase and electrospray ionization-mass spectrometry).

[0353] The oligonucleotides were conjugated either to the respective linker for conjugation to the K tag (one-step reaction) or to an acceptor moiety for click chemistry conjugation (two-step reaction), and the conjugates were then directly subjected to purification by reverse-phase HPLC as described below.

[0354] After precipitation of the linker oligonucleotide with 2% lithium perchlorate in acetone, the resulting precipitate was washed with acetone, dried under vacuum, and redissolved in PBS. 1.5 equivalents of K-tag peptide dissolved in PBS was added. After 1 h at room temperature, the reaction mixture was directly subjected to purification by reverse-phase HPLC.

[0355] Both reactions were purified by reverse-phase HPLC on a Waters XBridge Peptide BEH C 18 OBD Prep Column, 300 Å, 10 μm, 10 mm × 150 mm, using 0.1 M ammonium acetate and acetonitrile as eluents. Pooled fractions were lyophilized, redissolved in water, and the pH adjusted to pH 7.0 with aqueous NaOH. After final lyophilization, the compounds were finally characterized by liquid chromatography-mass spectrometry (reverse-phase and electrospray ionization-mass spectrometry).

[0356] Enzymatic conjugation of LNA-ASO to Q-tagged antibodies One-step conjugation using KalbTG Purified antibodies containing Q tags were transferred via dialysis into conjugation buffer (histidine buffer containing approximately 150 mM chloride ions, pH 7.5). For the KalbTG reaction, the antibodies were mixed with an excess of K-tagged oligonucleotides, and KalbTG (Roche Diagnostics, Mannheim, Germany) was added. The reaction mixture was incubated at 37°C with shaking, and the reaction was subsequently quenched by adding 10 mM ammonium sulfate to the solution. To remove unconjugated payload and residual enzyme, the conjugated modified antibodies were purified by size exclusion chromatography using a Superdex 200™ column (GE Healthcare, Chicago, IL, USA) in PBS, 250 mM arginine, pH 7.5. The purified conjugate was stored at -80°C.

[0357] Two-step conjugation using KalbTG and click chemistry The purified antibody containing the Q tag was transferred via dialysis into conjugation buffer (histidine buffer containing NaCl, pH 8.5). For the KalbTG reaction, the antibody was mixed with an excess of a K-tagged linker (10-fold molar excess) containing the first part of the click conjugation, and KalbTG was added. The reaction mixture was incubated at 37°C with shaking, and the reaction was then quenched by adding 10 mM ammonium sulfate to the solution. To remove unconjugated linker and residual enzyme, the conjugated modified antibody was purified by size exclusion chromatography using a Superdex 200™ column (GE Healthcare, Chicago, IL, USA) in PBS, 250 mM arginine, pH 7.5. The purified antibody-linker conjugate was added to an excess of oligonucleotides conjugated to each of the other parts of the click conjugation in PBS, 250 mM arginine, pH 7.5, and the reaction mixture was incubated overnight at room temperature with shaking. The antibody-oligonucleotide conjugates were purified by size exclusion chromatography as described above, and the purified conjugates were stored at -80°C.

[0358] Analysis of conjugates Quantitation of oligonucleotide conjugates was performed by UV / Vis spectroscopy at 260, 280, and 350 nm using a SoloVPE system (C Technologies, Bridgewater, NJ, USA). The Beer-Lambert equation was used to calculate conjugate concentrations and quantitative drug-antibody ratios (DARs). Additionally, qualitative DAR measurements were performed by hydrophobic interaction chromatography as described in Example 4 below. Conjugate purity was analyzed by CE-SDS under denaturing and reducing conditions using a Caliper LabChip® GXII Touch™ Protein Characterization System (Perkin Elmer, Waltham, MA, USA). Aggregate content was determined by SEC using a TSKgel UP-SW 3000 analytical size-exclusion column (Tosoh Bioscience, Griesheim, Germany) equilibrated with 0.2 M KHPO / KHPO, 0.25 M KCl, pH 6.2 on a high-performance liquid chromatography system (Ultimate 3000 HPLC system, Thermo Fisher Scientific, Waltham, MA, USA). The identity of the conjugate was confirmed by ESI-Q-ToF-MS (Bruker maXis 433, Bruker, Billerica, MA, USA). For MS analysis, samples were deglycosylated using N-glycosidase F (Roche, Basel, Switzerland) and subsequently desalted into 2% formic acid, 40% acetonitrile. [Table 50]

[0359] Conjugation of modified antibodies is affected by the position of the introduced Q-tag: introduction after positions LC110, LC143, LC214, HC118, HC177, HC297, and HC341 resulted in the best conjugation efficiency. [Table 51]

[0360] Example 4 Hydrophobic interaction chromatography of KalbTG conjugate-modified antibodies according to the present invention Hydrophobic interaction chromatography (HIC) was performed on a high-performance liquid chromatography system (Ultimate 3000 HPLC system, Thermo Fisher Scientific, Waltham, MA, USA) using a TSKgel Butyl-NPR column (2.5 μm, 4.6 × 35 mm, TOSOH Bioscience, Tokyo, Japan) at a flow rate of 1 mL / min. The column was equilibrated with eluent A (20 mM NaHPO dihydrate, 1.5 M (NH)SO, pH 7.0), and 60 μg of each sample was loaded onto the column. Subsequently, a gradient between eluent A and eluent B (20 mM NaHPO dihydrate, 25% (v / v) isopropanol, pH 7.0) was applied. gradient: 0 minutes 5%B 0~30 minutes 5%B→80%B 30~34 minutes 80%B->100%B 34~44 minutes 100%B 45~55 minutes 0%B

[0361] Elution profiles were obtained by continuously measuring absorbance at 280 nm. Drug-to-antibody ratios (DARs) were determined by peak integration using Chromeleon 7.2 (Thermo Fisher Scientific, Waltham, MA, USA).

[0362] Exemplary results are shown in FIG. [Table 52]

[0363] The hydrophilic marker had a retention time of 9.25 min and the hydrophobic markers had relative retention times of 25.9 min (mAb-2) or 9.00 min and 24.7 min (mAb-4), respectively.

[0364] The hydrophobicity of the conjugates is affected by the position of the introduced Q-tag: later introduction of LC110, LC214, and HC297 resulted in up to a two-fold change in the relative retention time of the conjugated modified antibody relative to the unconjugated antibody, while HC177 had the lowest relative retention time of all internal insertion sites tested.

[0365] Example 5 in vivo analysis Test Plan Human FcRn transgenic mice (hFcRn Tg32+ / + mice) were randomly assigned to 12 cohorts (n=3 / cohort) according to the 12 different compounds to be tested.

[0366] All compounds contain a cognate pair of full-length antibody light chains and an antibody Fc region fragment. [Table 53] The antibody is based on a one-arm anti-transferrin receptor antibody comprising a full-length antibody heavy chain pair having the amino acid sequence of

[0367] The Q-tags had the amino acid sequence GGGSYRYRQGGGS (SEQ ID NO: 25) inserted after each position.

[0368] Compound 1: Reference without conjugated nucleic acid Compound 2: One nucleic acid conjugated to a Q tag inserted after position HC118 of the full-length heavy chain Compound 3: One nucleic acid conjugated to a Q tag inserted after position HC177 of the full-length heavy chain Compound 4: One nucleic acid conjugated to a Q-tag inserted after position HC295 of the full-length heavy chain Compound 5: One nucleic acid conjugated to a Q tag inserted after position HC297 of the full-length heavy chain Compound 6: One nucleic acid conjugated to a Q tag inserted after position HC341 of the full-length heavy chain Compound 7: One nucleic acid conjugated to a Q tag inserted after position HC401 of the full-length heavy chain Compound 8: One nucleic acid conjugated to a Q tag inserted after position HC446 of the full-length heavy chain Compound 9: One nucleic acid conjugated to a Q-tag inserted after position LC214 Compound 10: Two nucleic acids conjugated to Q tags inserted after position HC297 of the full-length heavy chain and the Fc region fragment Compound 11: Two nucleic acids conjugated to Q tags inserted after position HC341 of the full-length heavy chain and the Fc region fragment Compound 12: Two nucleic acids conjugated to Q tags inserted after position HC446 of the full-length heavy chain and the Fc region fragment

[0369] The compounds were formulated as solutions in 20 mM histidine (pH 6) and administered intravenously at a single nominal dose of 20 mg / kg body weight and a volume of 2 mL / kg (except for Compound 1, which was administered at a volume of 3.3 mL / kg and a dose level of 16.7 mg / kg). Each formulation was injected as a slow bolus into one of two lateral tail veins. Serial microsampling of blood (20 μL / time point / mouse) was performed by tail vein puncture. Blood samples were collected into K3-EDTA-coated Minivette® POCT tubes 5 minutes, 1 hour, 7 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, and 336 hours after administration. Once the Minivette® was filled, the blood was immediately transferred to a 0.2 ml PCR Eppendorf tube and centrifuged at approximately 10,000 × g for approximately 5 minutes at 4 °C. For terminal sampling, blood was collected by cardiac puncture and transferred to K3-EDTA coated polypropylene tubes. Plasma samples were stored at -20°C for further processing and analysis.

[0370] bioanalysis The pharmacokinetics (PK) of the compounds were determined by two separate methods: one method quantifying the antibody component and a second method quantifying the locked nucleic acid component of the conjugate.

[0371] To determine PK in plasma samples, a generic electrochemiluminescence immunoassay (ECLIA) method specific for the human CH2 domain was used on a cobas® e411 (Roche Diagnostics GmbH, Mannheim, Germany) device under non-GLP conditions based on Stubenrauch et al. Briefly, test samples were added stepwise to the detection vessel along with a first detection antibody (biotinylated), a second detection antibody (ruthenium-conjugated), and SA beads. The calibration range of the specific standard curve for each compound was 0.69 ng / mL to 1,500 ng / mL assay concentration in 1% C57BL / 6 mouse plasma. The analytical sensitivity was 69 ng / mL in 100% plasma. Standard curves, quality controls, and sample dilutions were prepared in assay buffer containing C57BL / 6 mouse plasma, resulting in a 1% matrix concentration. Plasma samples were analyzed at two different dilutions (1:100 to 1:800).

[0372] The same plasma samples were analyzed using a hybridization enzyme-linked immunosorbent assay (hELISA) under non-GLP conditions.

[0373] For the hELISA method, standards, quality control, and prediluted samples were prepared using unconjugated nucleic acid as a reference. Biotinylated capture oligonucleotide probes and digoxigenylated detection oligonucleotide probes were added for hybridization and transferred to a streptavidin-coated microtiter plate. Polyclonal anti-digoxigenin-POD Fab fragments and TMB solution were used for detection. Color intensity was analyzed photometrically at 450 nm (reference wavelength 690 nm). Color intensity was proportional to the analyte concentration in the test sample. The calibration range of the standard curve was 0.8 pM to 111 pM assay concentration in 1% C57BL / 6 mouse plasma. For plasma samples, standard curves, and quality controls, all dilutions were prepared in assay buffer containing C57BL / 6 mouse plasma, resulting in a 1% matrix concentration. Plasma samples were analyzed at two different dilutions (1:100 to 1:400,000). The analytical sensitivity was 90 pM in 100% plasma.

[0374] result Table 54

[0375] Table 55

Claims

1. 1. A modified antibody comprising at least an antibody heavy chain, immediately after position 297 (HC297) of the antibody heavy chain (numbering according to Kabat), one first recognition site for transglutaminase from Kutzneria albida (KalbTG), or One first recognition site for transglutaminase (KalbTG) from Kutzneria albida, interposed between two flexible linker peptides. Either of the following is inserted, wherein the first recognition site for KalbTG is independently selected from the group of first recognition sites having the amino acid sequences 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), YRYRQ (SEQ ID NO: 17), and RVRQR (SEQ ID NO: 18); the linker peptide has one or two or three or four or five repeating units each having the amino acid sequence Gly-Gly-Gly-Ser (n=1, 2, 3, 4, 5; SEQ ID NO: 19); Modified antibodies.

2. 1. A modified antibody comprising a heavy chain and a light chain, immediately after position 143 (LC143) of the antibody light chain (numbering according to Kabat), one first recognition site for transglutaminase from Kutzneria albida (KalbTG), or One first recognition site for transglutaminase (KalbTG) from Kutzneria albida, interposed between two flexible linker peptides. inserted; and immediately after position 297 (HC297) of the antibody heavy chain (numbering according to Kabat), one first recognition site for transglutaminase from Kutzneria albida (KalbTG), or One first recognition site for transglutaminase (KalbTG) from Kutzneria albida, interposed between two flexible linker peptides. Either of the following is inserted, wherein the first recognition site for KalbTG is independently selected from the group of first recognition sites having the amino acid sequences 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), YRYRQ (SEQ ID NO: 17), and RVRQR (SEQ ID NO: 18); the linker peptide has one or two or three or four or five repeating units each having the amino acid sequence Gly-Gly-Gly-Ser (n=1, 2, 3, 4, 5; SEQ ID NO: 19); Modified antibodies.

3. The modified antibody of claim 1 or 2, wherein the modified antibody comprises an additional first recognition site for KalbTG fused to or at the C-terminus of the antibody heavy chain.

4. 3. The modified antibody of claim 1 or 2, wherein the modified antibody further comprises a first recognition site for KalbTG at position 401 (HC401), position 446 (HC446), or position 447 (HC447) of the antibody heavy chain (numbering according to Kabat).

5. The modified antibody of claim 1 or 2, wherein the modified antibody comprises two identical antibody heavy chains.

6. The modified antibody of claim 1 or 2, wherein the modified antibody comprises two different antibody heavy chains.

7. The modified antibody comprises, in addition to the recognition site(s) for KalbTG, the following 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) The modified antibody of claim 1 or 2, comprising:

8. 1. A modified antibody Fc region comprising at least one modified antibody heavy chain Fc region polypeptide, immediately after position 297 (HC297) of the antibody heavy chain (numbering according to Kabat), one first recognition site for transglutaminase from Kutzneria albida (KalbTG), or One first recognition site for transglutaminase (KalbTG) from Kutzneria albida, interposed between two flexible linker peptides. Either of the following is inserted, wherein the first recognition site for KalbTG is independently selected from the group of first recognition sites having the amino acid sequences 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), YRYRQ (SEQ ID NO: 17), and RVRQR (SEQ ID NO: 18); the linker peptide has one or two or three or four or five repeating units each having the amino acid sequence Gly-Gly-Gly-Ser (n=1, 2, 3, 4, 5; SEQ ID NO: 19); Modified antibody Fc region.

9. The modified antibody has the sequence of SEQ ID NO: 01: Table 1 (where Table 2 a first recognition site for KalbTG is inserted immediately after the position identified by Table 3 an additional first recognition site for KalbTG is inserted immediately after the one or more positions identified by Table 4 if and only if an additional first recognition site for KalbTG is inserted at one of the positions identified by Table 5 The first recognition site for KalbTG is inserted immediately after the position identified by 9. The modified antibody of claim 1, comprising an antibody heavy chain constant region having the amino acid sequence:

10. The modified antibody has the sequence of SEQ ID NO: 02: Table 6 (where Table 7 a first recognition site for KalbTG is inserted immediately after the position identified by Table 8 an additional first recognition site for KalbTG is inserted immediately after the one or more positions identified by Table 9 if and only if an additional first recognition site for KalbTG is inserted at one of the positions identified by Table 10 The first recognition site for KalbTG is inserted immediately after the position identified by 9. The modified antibody of claim 1, comprising an antibody heavy chain constant region having the amino acid sequence:

11. The modified antibody has the sequence of SEQ ID NO: 03: Table 11 (where Table 12 a first recognition site for KalbTG is inserted immediately after the position identified by Table 13 an additional first recognition site for KalbTG is inserted immediately after the one or more positions identified by Table 14 if and only if an additional first recognition site for KalbTG is inserted at one of the positions identified by Table 15 The first recognition site for KalbTG is inserted immediately after the position identified by 9. The modified antibody of claim 1, comprising an antibody heavy chain constant region having the amino acid sequence:

12. The modified antibody has the sequence of SEQ ID NO: 04: Table 16 (where Table 17 a first recognition site for KalbTG is inserted immediately after the position identified by Table 18 an additional first recognition site for KalbTG is inserted immediately after the one or more positions identified by Table 19 if and only if an additional first recognition site for KalbTG is inserted at one of the positions identified by Table 20 The first recognition site for KalbTG is inserted immediately after the position identified by 9. The modified antibody of claim 1, comprising an antibody heavy chain constant region having the amino acid sequence:

13. The modified antibody has the sequence of SEQ ID NO: 05: Table 21 (where Table 22 a first recognition site for KalbTG is inserted immediately after the position identified by Table 23 an additional first recognition site for KalbTG is inserted immediately after the one or more positions identified by Table 24 if and only if an additional first recognition site for KalbTG is inserted at one of the positions identified by Table 25 The first recognition site for KalbTG is inserted immediately after the position identified by 9. The modified antibody of claim 1, comprising an antibody heavy chain constant region having the amino acid sequence:

14. 9. The modified antibody of any one of claims 1, 2 and 8, comprising an antibody heavy chain constant region having the amino acid sequence of SEQ ID NO:

8.

15. The modified antibody has the sequence of SEQ ID NO: 06: Table 26 (where Table 27 a first recognition site for KalbTG is inserted immediately after the position identified by Table 28 an additional first recognition site for KalbTG is inserted immediately after the one or more positions identified by 9. The modified antibody of claim 1, comprising an antibody light chain constant region having the amino acid sequence:

16. The modified antibody has the sequence of SEQ ID NO: 07: Table 29 (where Table 30 a first recognition site for KalbTG is inserted immediately after the position identified by Table 31 an additional first recognition site for KalbTG is inserted immediately after the one or more positions identified by 9. The modified antibody of claim 1, comprising an antibody light chain constant region having the amino acid sequence:

17. The modified antibody of any one of claims 1, 2 and 8, wherein the modified antibody specifically binds to a receptor that induces receptor-mediated endocytosis.

18. The modified antibody of any one of claims 1, 2 and 8, wherein the modified antibody specifically binds to an antigen selected from the group of antigens consisting of human transferrin receptor 1 (TfR1), human insulin-like growth factor 1 receptor (IGF-1R), human low-density lipoprotein receptor-related protein 1 (LRP1) and human low-density lipoprotein receptor-related protein 8 (LRP8).

19. A covalent conjugate comprising: (i) A modified antibody according to any one of claims 1, 2 and 8; (ii) one or more non-antibody domain(s) covalently conjugated to the one or more first recognition site(s) for KalbTG of the modified antibody of (i); and Including, the non-antibody domain comprises a second recognition site for KalbTG; and The second recognition site for KalbTG is a K tag having the sequence RYESK (SEQ ID NO: 16); Covalent conjugates.

20. the non-antibody domain is (i) the therapeutic entity; and (ii) a second recognition site for KalbTG; and 20. The covalent conjugate of claim 19, comprising:

21. the non-antibody domain is (iii) a second linker between the therapeutic entity and the second recognition site; 21. The covalent conjugate of claim 20, further comprising:

22. 22. The covalent conjugate of claim 21, wherein the second linker is an alkyl linker, a polyethylene linker, a peptide linker, or a mixture thereof.

23. The KalbTG has the amino acid sequence (three letter code): Table 32 20. The covalent conjugate of claim 19, comprising:

24. A method of covalently conjugating a modified antibody according to any one of claims 1, 2 and 8 to a therapeutic entity, said method comprising: a) providing a modified antibody according to any one of claims 1, 2 and 8; b) providing a non-antibody domain, said non-antibody domain comprising: (i) the therapeutic entity; and (ii) a second recognition site for KalbTG that is complementary to the first recognition site present in the modified antibody provided under (i); providing a non-antibody domain comprising: and c) incubating the modified antibody of a) and the non-antibody domain of b) in the presence of KalbTG or a functionally active variant or fragment thereof, thereby forming an isopeptide bond between the first recognition site and the second recognition site for KalbTG; thereby conjugating the modified antibody to the therapeutic entity. Including, The second recognition site for KalbTG is a K tag having the sequence RYESK (SEQ ID NO: 16); method.

25. b) the non-antibody domain is (iii) a second linker between the therapeutic entity and the second recognition site for KalbTG.

25. The method of claim 24, further comprising:

26. The KalbTG has the amino acid sequence (three letter code): Table 33 25. The method of claim 24, comprising:

27. A pharmaceutical composition comprising the covalent conjugate of claim 19.

28. 20. The covalent conjugate of claim 19 for use as a medicament.

29. 20. Use of the covalent conjugate of claim 19 in the preparation of a medicament for the treatment or prevention of a neurological disorder selected from the group consisting of neurological disorders, neurodegenerative diseases, cancer, eye disorders, seizure disorders, lysosomal storage diseases, amyloidosis, viral or microbial diseases, ischemia, behavioral disorders, CNS inflammation, Alzheimer's disease, Parkinson's disease, multiple sclerosis, CD20 positive cancer with brain metastases, and HER2 positive cancer with brain metastases.

30. A method comprising the covalent conjugate of claim 19. A pharmaceutical composition for the treatment or prevention of a neurological disorder selected from the group consisting of neurological disorders, neurodegenerative diseases, cancer, eye disorders, seizure disorders, lysosomal storage diseases, amyloidosis, viral or microbial diseases, ischemia, behavioral disorders, CNS inflammation, Alzheimer's disease, Parkinson's disease, multiple sclerosis, CD20-positive cancer with brain metastasis, and HER2-positive cancer with brain metastasis.

31. A nucleic acid or composition of nucleic acids encoding a modified antibody described in any one of claims 1, 2 and 8.

32. A cell comprising the nucleic acid or nucleic acid composition of claim 31.

33. A method for producing a modified antibody according to any one of claims 1, 2 and 8, comprising: - culturing the cells according to claim 32, - recovering the modified antibody from the cells and / or culture medium; A method comprising the step of thereby producing a modified antibody according to any one of claims 1, 2 and 8.

34. The KalbTG has the amino acid sequence (three letter code): Table 34 The modified antibody or modified Fc region of any one of claims 1, 2 and 8, comprising:

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