Site-Specific Antibody Conjugation and Its Use

Abstract

To provide a modified antibody comprising heavy chain and light chain for use as medicine and as a pharmaceutical composition for use in the treatment of diseases, as well as a conjugate of modified antibody and a therapeutic entity.SOLUTION: Disclosed is a modified antibody comprising at least antibody heavy chain, where the antibody heavy chain comprises one or two or three or four first recognition site(s) for the transglutaminase from Kutzneria albida (KalbTG) at or inserted after one or more of the positions independently selected from the group consisting of 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 relates to the field of antibody technology, particularly to the field of antibody-drug conjugates. In particular, the present invention relates to modified antibodies comprising one or more artificial recognition sites for transglutaminase derived from Kutzneria albida, and to antibody-drug conjugates comprising said modified antibodies. [Background technology]

[0002] Pharmaceuticals are chemical substances used to treat, cure, or prevent diseases. Pharmaceuticals can be administered via several routes, and many pharmaceuticals can be administered via more than one route. Typical routes of administration include, but are not limited to, oral, rectal, sublingual, or topical injection of solutions, suspensions, or emulsions (e.g., intramuscular, intravenous, intraperitoneal, intraocular, intraosseous, subcutaneous, or intrathecal). Clearly, pharmaceuticals are usually distributed systemically throughout a patient's body and may have adverse effects due to their activity in undesirable areas. Reaching other areas can be difficult. Targeted therapy aims to overcome this drawback by using pharmaceuticals directed towards areas of the body where they are intended to act. Targeted therapy is expected to be more effective and have fewer side effects than conventional untargeted forms of treatment.

[0003] One way to direct a therapeutic entity to its intended site of action is by conjugating it to an antibody that specifically binds to 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 conjugates utilize either partially reduced interchain disulfide bonds that enable thiol-maleimide chemistry, or lysine functionalization using activated esters or artificially introduced cysteine ​​residues. This method results in a statistical mixture of conjugate antibody species having different numbers of drugs bound to different sites. In ADCs with a monomethyl auristatin E payload, it has been demonstrated that ADC species with drug-to-antibody ratios (DAR) of 4, 6, or 8 become progressively more hydrophobic and are shown to aggregate much more readily than DAR2 species (Adem et al.).

[0004] Therefore, it is desirable to provide ADCs, such as AOCs, for targeted therapy by more precisely controlling the number and site of binding of therapeutic entities. This should improve the uniformity of ADCs. Improved site-directed conjugation technology continues to be of interest to many pharmaceutical companies for its potential use in the preparation of therapeutic ADCs and diagnostic antibody labels or antibody-enzyme conjugates.

[0005] Microbial transglutaminase derived from Streptomyces mobaraensis is emerging as an inexpensive and easy-to-use enzyme for protein crosslinking and 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 substrates were described by Steffen et al. (2017). KalbTG exhibits similar efficiency to previously described microbial transglutaminases (mTGs), but with improved specificity and developmental potential.

[0007] U.S. Patent Application Publication No. 2020 / 0249231 reports 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, for example, International Publication No. 2021 / 174091). [Overview of the Initiative]

[0009] To avoid the aforementioned drawbacks, the object of the present invention was to identify sites within the IgG molecule for the incorporation of Q-tag motifs, which result in improved properties of modified antibodies with respect to KalbTG-mediated conjugation. The incorporation of the 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 according to the present invention enables the incorporation of one or more therapeutic moieties per IgG molecule in a defined stoichiometric and controlled site-specific manner. This makes it possible to provide a more homogeneous conjugate product, for example, reducing the necessary purification and separation efforts and obtaining molecules with more desirable drug-like properties. Furthermore, the risk of impairing the binding of the antibody to its antigen by the conjugation process and / or conjugate payload is reduced.

[0010] The inventors observed, among other things, that aggregation and hydrophobicity increased when the payload was attached to the C-terminus of the IgG heavy chain. In order to prepare pharmaceutically useful antibody-drug conjugates using KalbTG, the KalbTG Q-tag must be introduced at a specified site within the IgG backbone according to the present invention.

[0011] The present invention relates to a modified antibody comprising a heavy chain and a light chain, wherein the heavy chain and / or the light chain comprises one or more first recognition sites for transglutaminase (KalbTG) derived from Kutzneria albida or a functionally active variant thereof. The one or more first recognition sites are introduced at one or more selected positions within the heavy chain and / or light chain of the antibody.

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

[0013] In all aspects of the present invention and in certain embodiments of the embodiments, the modified antibody of the present invention further includes 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 relation to the present invention, the term "insertion of a KalbTG recognition site at a certain position" means that an 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 either directly immediately after that position in the amino acid sequence, spaced between two short flexible linker peptides, i.e., the amino acid residue is maintained, in one preferred embodiment.

[0015] In all aspects of the present invention and in certain embodiments of the embodiments, one or more positions are selected from the group of positions including the light chain position 214 (LC214) and the heavy chain positions 118 (HC118), 177 (HC177), 297 (HC297), and 341 (HC341) (numbering follows Kabat).

[0016] In all aspects and one preferred embodiment of the present invention, one or more positions are selected from the group of positions including the light chain position 214 (LC214) and the heavy chain positions 341 (HC341), 297 (HC297), and 177 (HC177) (numbering follows Kabat).

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

[0018] In all aspects of the present invention and in certain embodiments of the embodiments, the modified antibody comprises two, four, or more (first) recognition sites for transglutaminase (KalbTG) derived from Kutzneria albida, 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 (numbering follows Kabat), or inserted thereafter.

[0019] In certain embodiments of all aspects and embodiments of the present invention, the modified antibody according to the present invention comprises two different heavy chains or antibody Fc regions, whereby the differences result from respective mutations that at least 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 each other at or after 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 (numbering according to Kabat).

[0021] Similarly, the present invention relates to a modified antibody Fc region, the 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 comprising positions 118 (HC118), 177 (HC177), 297 (HC297), 341 (HC341) and 401 (HC401) of the heavy chain (numbering according to Kabat).

[0022] Thus, in the modified antibody Fc region of the present invention, the recognition site of KalbTG is located within the constant region of the antibody heavy chain Fc region polypeptide, i.e., not at any terminus. Furthermore, the modified antibody Fc region of the present invention may include a further first recognition site for KalbTG at position 446 (HC446) or position 447 (HC447) of the heavy chain, i.e., at the C-terminus (numbering follows 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, the recognition site is inserted either directly or with an intervening spacer after that position, either directly or between two flexible short linker peptides.

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

[0024] In a preferred embodiment of all aspects and embodiments of the present invention, one or more positions are selected from the group of positions including position 341 (HC341), position 297 (HC297), and position 177 (HC177) of the heavy chain (numbering follows Kabat).

[0025] In a preferred embodiment of all aspects and embodiments of the present invention, the modified antibody comprises at least one (homologous) pair of an antibody heavy chain and an antibody light chain, and 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 follows Kabat).

[0026] In all aspects and one preferred embodiment of the present invention, the modified antibody comprises at least one (homogeneous) pair of antibody heavy chain and antibody light chain, the heavy chain comprising one (first) recognition site (possibly multiple) for transglutaminase (KalbTG) derived from Kutzneria albida inserted at or after position 177 (HC177) of the heavy chain (numbering follows Kabat).

[0027] In all aspects and one preferred embodiment of the present invention, the modified antibody comprises at least one (homogeneous) pair of antibody heavy chain and antibody light chain, the light chain comprising one (or more) recognition sites for Kutzneria albida-derived transglutaminase (KalbTG) inserted at or after position 214 (LC214) of the light chain (numbering follows Kabat).

[0028] In all aspects and one preferred embodiment of the present invention, the modified antibody comprises at least one (homogeneous) pair of antibody heavy chain and antibody light chain, the heavy chain comprising two (first) recognition sites (possibly multiple) for transglutaminase (KalbTG) derived from Kutzneria albida, inserted at or after positions 297 (HC297) and 177 (HC177) of the heavy chain (numbering follows Kabat).

[0029] In all aspects and one preferred embodiment of the present invention, the modified antibody comprises at least one (homogeneous) pair of an antibody heavy chain and an antibody light chain, wherein the heavy chain comprises one (or more) recognition sites for Kutzneria albida transglutaminase (KalbTG) inserted at or after position 297 (HC297) of the heavy chain, and the light chain comprises one (or more) recognition sites for Kutzneria albida transglutaminase (KalbTG) inserted at or after position 214 (LC214) of the light chain (numbering follows Kabat).

[0030] In all aspects and one preferred embodiment of the present invention, the modified antibody comprises at least one (homogeneous) pair of an antibody heavy chain and an antibody light chain, wherein the heavy chain comprises one (or more) recognition sites for Kutzneria albida transglutaminase (KalbTG) at or after position 177 (HC177), and the light chain comprises one (or more) recognition sites for Kutzneria albida transglutaminase (KalbTG) at or after position 214 (LC214) (numbering follows Kabat).

[0031] In all aspects and one preferred embodiment of the present invention, the modified antibody comprises at least one (homogeneous) pair of an antibody heavy chain and an antibody light chain, wherein the heavy chain comprises two (first) recognition sites for Kutzneria albida transglutaminase (KalbTG) inserted at or after positions 297 (HC297) and 177 (HC177), and the light chain comprises one (first) recognition site for Kutzneria albida transglutaminase (KalbTG) inserted at or after position 214 (LC214) (numbering follows Kabat).

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

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

[0034] In all aspects and one preferred embodiment of the present invention, the modified antibody comprises two (homogeneous) pairs of antibody heavy chains and antibody light chains, wherein the heavy chain of the first pair comprises one (preferably more) recognition site for Kutzneria albida transglutaminase (KalbTG) inserted at or after position 297 (HC297) of the heavy chain, the heavy chain of the second pair comprises one (preferably more) recognition site for Kutzneria albida (KalbTG) transglutaminase inserted at or after position 177 (HC177) of the heavy chain, and the light chain of the second pair comprises one (preferably more) recognition site for Kutzneria albida (KalbTG) transglutaminase inserted at or after position 214 (LC214) of the light chain It contains one (or more) recognition sites for transglutaminase (KalbTG) derived from Albida (numbering follows Kabat).

[0035] In all aspects and one preferred embodiment of the present invention, the modified antibody comprises two (homogeneous) pairs of antibody heavy chains and antibody light chains, wherein the heavy chain of the first pair comprises one (preferably more) recognition site for Kutzneria albida transglutaminase (KalbTG) inserted at or after position 177 (HC177) of the heavy chain, the heavy chain of the second pair comprises one (preferably more) recognition site for Kutzneria albida (KalbTG) transglutaminase inserted at or after position 297 (HC297) of the heavy chain, and the light chain of the second pair comprises one (preferably more) recognition site for Kutzneria albida (KalbTG) transglutaminase inserted at or after position 214 (LC214) of the light chain It contains one (or more) recognition sites for transglutaminase (KalbTG) derived from Albida (numbering follows Kabat).

[0036] In all aspects and one preferred embodiment of the present invention, the modified antibody comprises two (homogeneous) pairs of antibody heavy chains and antibody light chains, wherein the heavy chain of the first pair comprises two (first) recognition sites for Kutzneria albida transglutaminase (KalbTG) inserted at or after positions 297 (HC297) and 177 (HC177) of the heavy chain, and the light chain of the second pair comprises one (first) recognition site for Kutzneria albida transglutaminase (KalbTG) inserted at or after position 214 (LC214) of the light chain (numbering follows Kabat).

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

[0038] The present invention further relates to one or more nucleic acids encoding a modified antibody according to the present invention, and a covalent conjugate comprising (i) a modified antibody according to the present invention, and (ii) one or more non-antibody portions (payloads) covalently conjugated to one or more first recognition sites, either directly or via a first linker. In certain embodiments, the non-antibody portion 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 present invention to a non-antibody portion. Where the non-antibody portion includes a therapeutic entity, the present invention further relates to a conjugate of a modified antibody according to the present invention and a therapeutic entity as a pharmaceutical composition for use as a pharmacopoeia and for use in the treatment of a disease.

[0040] KalbTG catalyzes the formation of isopeptide bonds between the glutamine side (Gln, Q) chain and the lysine side (Lys, K) chain. The formed isopeptide bond is an ε-(γ-glutamyl)lysine bond / crosslink. Enzymatic bond formation is covalent and therefore nearly 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, two, 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 including positions 118 (HC118), 177 (HC177), 297 (HC297), 341 (HC341), and 401 (HC401) of the antibody heavy chain (numbering follows Kabat).

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

[0044] 3. A modified antibody comprising a heavy chain and a light chain, wherein the heavy chain and / or light chain comprises one or more first recognition sites (or multiple sites) for transglutaminase (KalbTG) derived from Kutzneria albida, independently selected from the group of positions including positions 110 (LC110), 143 (LC143), and 214 (LC214) of the antibody light chain, and positions 118 (HC118), 177 (HC177), 297 (HC297), 341 (HC341), and 401 (HC401) of the antibody heavy chain, or inserted thereafter (numbering follows Kabat).

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

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

[0047] 6. The modified antibody according to 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 follows Kabat).

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

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

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

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

[0052] 11. The modified antibody according to any one of Embodiments 1 to 10, wherein the modified antibody includes an even number of first recognition sites for KalbTG, which are contained in or inserted after the positions of 110 (LC110), 143 (LC143), and 214 (LC214) of the antibody light chain, and 118 (HC118), 177 (HC177), 297 (HC297), 341 (HC341), and 401 (HC401) of the antibody heavy chain, independently selected from each other.

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

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

[0055] 14. The modified antibody according to any one of Embodiments 1 to 13, wherein the modified antibody includes an even number of first recognition sites for KalbTG, which are contained in or inserted after the positions of 110 (LC110), 143 (LC143), and 214 (LC214) of the antibody light chain, and 118 (HC118), 177 (HC177), 297 (HC297), 341 (HC341), and 401 (HC401) of the antibody heavy chain, independently selected from each other.

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

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

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

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

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

[0061] 20. A modified antibody according to any one of Embodiments 1 to 19, wherein the antibody light chain includes a first recognition site for KalbTG inserted at or after position 214 (LC214) of the antibody light chain (numbering follows Kabat).

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

[0063] 22. A modified antibody according to 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 follows Kabat).

[0064] 23. A modified antibody according to 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 follows Kabat).

[0065] 24. A modified antibody according to any one of Embodiments 1 to 20, wherein the antibody heavy chain comprises one first recognition site for KalbTG inserted at or after positions 297 (HC297) and 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 follows Kabat).

[0066] 25. The modified antibody according to any one of Embodiments 1 to 24, wherein the modified antibody comprises two pairs: an antibody heavy chain and a congener antibody light chain.

[0067] 26. (i) The modified antibody comprises two pairs each of an antibody heavy chain and an antibody light chain, and each of the antibody heavy chain and / or an antibody light chain contains 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 and / or antibody light chains contains one or more first recognition sites for KalbTG, or (iii) The modified antibody according to any one of Embodiments 1 to 25, wherein the modified antibody comprises an antibody heavy chain and 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 according to Embodiment 25, wherein the 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, and the 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 follows Kabat).

[0069] 28. The modified antibody according to Embodiment 25, wherein the 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, and the 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 follows Kabat).

[0070] 29. The modified antibody according to Embodiment 25, wherein the 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, the 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 the 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 follows Kabat).

[0071] 30. The modified antibody according to Embodiment 25, wherein the 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, the 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 the 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 follows Kabat).

[0072] 31. The modified antibody according to Embodiment 25, wherein the first pair of antibody heavy chains comprises two first recognition sites for KalbTG inserted at or after positions 297 (HC297) and 177 (HC177) of the antibody heavy chain, and the 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 follows Kabat).

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

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

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

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

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

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

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

[0080] 39. A modified antibody according to any one of Embodiments 1 to 38, wherein the modified antibody comprises two identical antibody heavy chains.

[0081] 40. A modified antibody according to any one of Embodiments 1 to 38, wherein the modified antibody comprises two different antibody heavy chains.

[0082] 41. The modified antibody according to Embodiment 40, wherein the difference arises from at least each mutation for inducing heterodimerization.

[0083] 42. The modified antibody according to any one of Embodiments 1 to 41, wherein the modified antibody includes the following mutations (numbering follows Kabat) in addition to the recognition site(s) for KalbTG: a) L234A and 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 more first recognition sites (or more) for transglutaminase (KalbTG) derived from Cutnelia alvida at one or more positions independently selected from the group of positions including positions 118 (HC118), 177 (HC177), 297 (HC297), 341 (HC341), and 401 (HC401) of the antibody heavy chain, or after them (numbering follows Kabat).

[0085] 44. The modified antibody Fc region according to Embodiment 43, wherein the first recognition site for KalbTG is located within the amino acid sequence of each antibody chain.

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

[0087] 46. ​​The modified antibody Fc region according to 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 follows Kabat).

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

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

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

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

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

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

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

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

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

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

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

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

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

[0101] 60. A modified antibody Fc region according to any one of Embodiments 43 to 58, wherein the modified antibody Fc region comprises two modified antibody heavy chain Fc region polypeptides, one of which comprises a 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 comprises a first recognition site for KalbTG inserted at or after position 177 (HC177) of the antibody heavy chain (numbering follows Kabat).

[0102] 61. A modified antibody Fc region according to 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 include a first recognition site for KalbTG inserted at or after position 297 (HC297) of the antibody heavy chain (numbering follows Kabat).

[0103] 62. A modified antibody Fc region according to 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 include a first recognition site for KalbTG inserted at or after position 177 (HC177) of the antibody heavy chain (numbering follows Kabat).

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

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

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

[0107] 66. The modified antibody Fc region according to Embodiment 65, wherein the difference arises from at least each mutation to induce heterodimerization.

[0108] 67. The modified antibody Fc region according to any one of Embodiments 43 to 66, comprising the following mutations (numbering follows Kabat) in addition to the recognition site(s) for KalbTG: a) L234A and 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 the modified antibody Fc region described in any one of embodiments 43 to 67.

[0110] 69. A modified antibody or modified antibody Fc region according to any one of Embodiments 1 to 68, wherein the first recognition site (multiple sites are possible) for KalbTG contains or has a Gln-containing 5-amino acid long motif.

[0111] 70. A modified antibody or modified antibody Fc region according to 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 including 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. A modified antibody or modified antibody Fc region according to any one of Embodiments 1 to 70, wherein the first(or more) recognition sites for KalbTG are independently selected from the amino acid sequence YRYRQ (SEQ ID NO: 17) or RVRQR (SEQ ID NO: 18).

[0113] 72. A modified antibody or modified antibody Fc region according to any one of Embodiments 1 to 71, wherein the first recognition site (or multiple sites) of KalbTG is YRYRQ (SEQ ID NO: 17).

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

[0115] 74. A modified antibody or modified antibody Fc region according to 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 according to Embodiment 74, wherein each peptide linker independently contains 1 to 20 amino acids.

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

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

[0119] 78. A modified antibody or modified antibody Fc region according to 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 the payload that binds to the modified antibody.

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

[0121] 80. A modified antibody or modified antibody Fc region according to any one of embodiments 74 to 80, wherein the peptide linker is independently selected from the group of peptide linkers including 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. A modified antibody or modified antibody Fc region according to any one of embodiments 74 to 80, wherein the peptide linker consists of Gly and Ser independently of each other, primarily or entirely.

[0123] 82. Peptide linker (Gly m Ser) n A modified antibody or modified antibody Fc region according to 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 independently selected from each other.

[0124] 83. A modified antibody or modified antibody Fc region according to any one of Embodiments 82, wherein m=3 and n=1.

[0125] 84. A modified antibody or modified antibody Fc region according to any one of embodiments 74 to 83, wherein the peptide linker comprises one, two, three, 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 according to Embodiment 84, wherein the peptide linker includes one repeating unit having the amino acid sequence Gly-Gly-Gly-Ser(n=1; SEQ ID NO: 19).

[0127] 86. A modified antibody according to any one of Embodiments 1 to 85, wherein the modified antibody comprises one, two, three, or four Fab fragments.

[0128] 87. The modified antibody according to 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 homologous antibody light-heavy chain pair, and one antibody heavy chain Fc region polypeptide fragment including a hinge region that associates with the Fc region of the full-length antibody heavy chain.

[0129] 88. A modified antibody according to any one of Embodiments 1 to 87, wherein the modified antibody is based on the human Ig heavy chain constant region.

[0130] 89. A modified antibody according to any one of Embodiments 1 to 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 according to any one of Embodiments 1 to 89, wherein the modified antibody is based on a human IgG1 heavy chain polypeptide and 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 shown in SEQ ID NO: 1 or 2.

[0132] 91. The modified antibody according to any one of Embodiments 1 to 89, wherein the modified antibody is based on a human IgG double-chain polypeptide and 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 shown in SEQ ID NO: 3.

[0133] 92. The modified antibody according to any one of Embodiments 1 to 89, wherein the modified antibody is based on a human IgG triple-chain polypeptide and 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 shown in SEQ ID NO: 4.

[0134] 93. The modified antibody according to any one of Embodiments 1 to 89, wherein the modified antibody is based on a human IgG quadrilateral polypeptide and 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 shown in SEQ ID NO: 5.

[0135] 94. Modified antibody, SEQ ID NO: 01 [Table 1] [Table 2] A first recognition site for KalbTG is inserted at or after one or more locations identified by, [Table 3] Only if a further first recognition site for KalbTG is inserted into one of the locations identified by, [Table 4] A modified antibody according to any one of Embodiments 1 to 89, comprising an antibody heavy chain constant region having an amino acid sequence of (where a first recognition site for KalbTG is inserted after the position identified by ).

[0136] 95. Modified antibody, SEQ ID NO: 02 [Table 5] [Table 6] A first recognition site for KalbTG is inserted at or after one or more locations identified by, [Table 7] Only if a further first recognition site for KalbTG is inserted into one of the locations identified by, [Table 8] A modified antibody according to any one of Embodiments 1 to 89, comprising an antibody heavy chain constant region having an amino acid sequence of (where a first recognition site for KalbTG is inserted after the position identified by ).

[0137] 96. Modified antibody, SEQ ID NO: 03 [Table 9] [Table 10] A first recognition site for KalbTG is inserted at or after one or more locations identified by, [Table 11] Only if a further first recognition site for KalbTG is inserted into one of the locations identified by, [Table 12] A modified antibody according to any one of Embodiments 1 to 89, comprising an antibody heavy chain constant region having an amino acid sequence of (where a first recognition site for KalbTG is inserted after the position identified by ).

[0138] 97. Modified antibody, SEQ ID NO: 04 [Table 13] [Table 14] A first recognition site for KalbTG is inserted at or after one or more locations identified by, [Table 15] Only if a further first recognition site for KalbTG is inserted into one of the locations identified by, [Table 16] A modified antibody according to any one of Embodiments 1 to 89, comprising an antibody heavy chain constant region having an amino acid sequence of (where a first recognition site for KalbTG is inserted after the position identified by ).

[0139] 98. Modified antibody, SEQ ID NO: 05 [Table 17] [Table 18] A first recognition site for KalbTG is inserted at or after one or more locations identified by, [Table 19] Only if a further first recognition site for KalbTG is inserted into one of the locations identified by, [Table 20] A modified antibody according to any one of Embodiments 1 to 89, comprising an antibody heavy chain constant region having an amino acid sequence of (where a first recognition site for KalbTG is inserted after the position identified by ).

[0140] 99. The modified antibody according to any one of Embodiments 1 to 98, wherein the modified antibody contains 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 shown in SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 34.

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

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

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

[0144] 103. A modified antibody according to any one of Embodiments 1 to 102, wherein the modified antibody is based on the light chain of a human kappa antibody or a lambda antibody and 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 shown in SEQ ID NO: 6 or 7.

[0145] 104. The modified antibody according to any one of Embodiments 1 to 89, wherein the modified antibody has 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, or up to 100% identical to the amino acid sequence shown in SEQ ID NO: 10.

[0146] 105. Modified antibody, SEQ ID NO: 06 [Table 21] [Table 22] A modified antibody according to any one of embodiments 1 to 104, comprising an antibody light chain constant region having an amino acid sequence (where a first recognition site for KalbTG is inserted at or after one or more positions identified by ).

[0147] 106. Modified antibody, SEQ ID NO: 07 [Table 23] [Table 24] A modified antibody according to any one of embodiments 1 to 104, comprising an antibody light chain constant region having an amino acid sequence (where a first recognition site for KalbTG is inserted at or after one or more positions identified by ).

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

[0149] 108. The modified antibody according to 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 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 sequences of SEQ ID NOs. 1 to 5.

[0150] 109. Modified antibodies, 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 either 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 which includes or comprises an amino acid sequence that is at least 96%, 97%, 98%, or 99%, particularly 100%, identical to the amino acid sequences of SEQ ID NOs: 1 to 5, wherein at least one recognition site for KalbTG is inserted.

[0151] 110. Modified antibodies, 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 either SEQ ID NO: 6 or 7, wherein a recognition site for at least one KalbTG is 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 sequences of SEQ ID NOs. 1 to 5.

[0152] 111. Modified antibodies, a) an 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: 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. Modified antibodies, a) an 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: 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. Modified antibodies, a) an 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: 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. Modified antibodies, a) Modified antibody light chain constant domains 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 sequences of SEQ ID NOs. 1 to 5.

[0156] 115. Modified antibodies, a) Modified antibody light chain constant domains 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 sequences of SEQ ID NOs. 1 to 5.

[0157] 116. Modified antibodies, a) Modified antibody light chain constant domains 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 sequences of SEQ ID NOs. 1 to 5.

[0158] 117. Modified antibodies, a) Modified antibody light chain constant domains 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. Modified antibodies, a) Modified antibody light chain constant domains 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. Modified antibodies, a) Modified antibody light chain constant domains 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. Modified antibodies, a) Modified antibody light chain constant domain containing 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 containing the amino acid sequence of SEQ ID NO: 8.

[0162] 121. Modified antibodies, a) Modified antibody light chain constant domain containing 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 containing the amino acid sequence of SEQ ID NO: 9.

[0163] 122. Modified antibodies, a) Modified antibody light chain constant domain containing 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 containing the amino acid sequence of SEQ ID NO: 34.

[0164] 123. A modified antibody according to any one of Embodiments 1 to 122, wherein the modified antibody binds to an antigen.

[0165] 124. A modified antibody according to any one of Embodiments 1 to 123, wherein the modified antibody specifically binds to a structure that enables passage through the blood-brain barrier.

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

[0167] 126. The modified antibody according to 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. A modified antibody according to any one of Embodiments 1 to 126, wherein the modified antibody specifically binds to human transferrin receptor 1.

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

[0170] 129. The modified antibody according to Embodiment 128, wherein one or both of the antigens are tumor markers specific to tumor cells.

[0171] 130. A modified antibody according to any one of Embodiments 128 to 129, wherein the cell type is breast cancer cells.

[0172] 131. A covalent conjugate, (i) A modified antibody according to any one of Embodiments 1 to 130, (ii) comprising one or more non-antibody domains covalently conjugated to one or more first recognition sites (or more) of the modified antibody of (i) for KalbTG, A covalent conjugate in which 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 entity and a therapeutic portion optionally comprising a second linker.

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

[0175] 134. A 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. A covalent conjugate according to any one of embodiments 131 to 134, wherein the therapeutic entity is nucleic acid.

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

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

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

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

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

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

[0183] 142. A 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 immunomodulatory factors.

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

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

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

[0187] 146. A covalent conjugate according to any one of embodiments 131 to 145, wherein the second linker comprises ethylene glycol units.

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

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

[0190] 149. The second linker is non-substituted or substituted C 1~6 May contain alkyl, substituted C 1~6 In the case of alkyl groups, the substituent is selected from the group consisting of alkoxy, acyl, acyloxy, alkoxycarbonyl, carbonylalkoxy, acylamino, amino, aminoacyl, aminocarbonylamino, aminocarbonyloxy, cycloalkyl, cycloalkenyl, cyano, azide, halo, hydroxyl, nitro, carboxyl, thiol, thioalkyl, alkyl, alkenyl, alkynyl, heterocyclyl, aminosulfonyl, sulfonylamino, sulfonyl, and oxo, as described in any one of Embodiments 131 to 148, covalently bonded conjugate

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

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

[0193] 152. The covalent conjugate according to Embodiment 151, wherein one or more therapeutic nucleic acids are antisense oligonucleotides.

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

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

[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) To provide a modified antibody according to any one of Embodiments 1 to 130, b) To provide a non-antibody domain, wherein the non-antibody domain comprises (i) a therapeutic entity, (ii) a second recognition site for KalbTG complementary to a 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 recognition site and the second recognition site for KalbTG. A method for covalently conjugating a modified antibody to a therapeutic entity according to any one of embodiments 1 to 130, comprising conjugating the modified antibody to a therapeutic entity and incubating it.

[0198] 157. The method according to 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] 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. KalbTG comprises an amino acid sequence (3-letter notation) and a modified antibody, modified Fc region, covalent conjugate, and method according to any one of Embodiments 1 to 159. [Table 25]

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

[0203] 162. A covalent conjugate for use as a pharmaceutical, as described in any one of embodiments 131 to 155 and 160, or a covalent conjugate manufactured according to the method described in any one of embodiments 156 to 160.

[0204] 163. The use described in Embodiment 162, wherein the use is for treating neurological or brain diseases or cancer.

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

[0206] 165. Use according to any one of Embodiments 162 to 164, wherein the neurological disorder is selected from neuropathic disorders, neurodegenerative diseases, cancer, ocular disorders, seizure disorders, lysosomal storage disorders, amyloidosis, viral or microbial diseases, ischemia, behavioral disorders, and CNS inflammation.

[0207] 166. Use according to any one of Embodiments 162 to 165, wherein the use is for treating Alzheimer's disease, 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 the nucleic acid or nucleic acid composition described in Embodiment 167.

[0210] 169. A method for producing a modified antibody according to any one of Embodiments 1 to 130, - The step of culturing cells as described in Embodiment 168, A method comprising the step of recovering a modified antibody from cells and / or culture medium, thereby producing a modified antibody according to any one of Embodiments 1 to 130.

[0211] 170. The method according to Embodiment 169, wherein the cells are mammalian cells.

[0212] 171. The method according to any one of embodiments 169 to 170, wherein the cells are CHO cells. [Modes for carrying out the invention]

[0213] This invention is based, at least in part, on the finding that Q tags cannot be incorporated into all sites within antibodies such as IgG1 antibodies, and that compatibility is associated with conjugation to a payload. Antibody constant domain regions were screened for conjugation via KalbTG. In this context, KalbTG Q tags were inserted between surface-exposed domains and within flexible loops within the constant regions of human IgG1 heavy and light chains, as well as at the C-terminus of the respective heavy and light chains. In total, nine different sites within the IgG1 heavy and light chains were tested. The KalbTG Q-tag motif was inserted spaced 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 with different binding specificities (mAb-1 to mAb-5) 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 was found that the expression titer changed, or decreased or even improved, depending on the insertion site, which was completely unexpected.

[0215] Modified antibodies were further tested for the conjugation and accessibility of Q tags for enzymatic modification by KalbTG. Two different payloads, small molecules (fluorescent dyes) and small single-stranded nucleic acids, were tested. When the antibody was symmetric, i.e., contained two identical heavy and light chain pairs, two Q tags were present per molecule. When the antibody was asymmetric, i.e., contained two different heavy and light chain pairs, a single Q tag was present per molecule. Therefore, a drug-to-antibody ratio (DAR, i.e., number of payload molecules per antibody molecule) of 2 was expected for symmetric antibodies with 100% conjugation efficiency, and a DAR of 1 was expected for asymmetric antibodies 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 of single-stranded nucleic acids with a length of 15 nucleotides for mAb-1 and mAb-2 and a length of 20 nucleotides for mAb-4 are shown in Table 3 (determined by HIC and UV-vis).

Table 28

[0217] Mab-1 and mAb-2 were conjugated by a one-step method, and mAb-4 was conjugated by a two-step method (using click chemistry, first a K-tag and then conjugating the K-tag to the nucleic acid). Thereby, the general applicability of the method according to the invention using the antibody according to the invention is shown, 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 at three different positions, namely HC297, HC446 and LC143, respectively. The results of expression yield (1 L, μg / mL) and conjugation (backward; HIC / UV-vis) are shown in Table 4.

Table 29

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

Table 30

[0220] Therefore, the inventors successfully identified several KalbTG Q-tag insertion sites spanning the length of the IgG backbone that enable / provide enzyme accessibility without adversely affecting expression yields.

[0221] Therefore, suitable insertion sites for Q-tag expression yield and enzyme 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 follows the Kabat EU numbering scheme).

[0222] Furthermore, pharmacokinetic parameters of modified antibodies conjugated to one or two single-stranded nucleic acids, respectively, were generated by in vivo analysis using human FcRn transgenic mice. These 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 (LC214) of the light chain and positions 177 (HC177) and 297 (HC297) of the heavy chain (amino acid numbering follows the Kabat EU numbering scheme).

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

[0225] In all aspects of the present invention and in certain embodiments of the embodiments, one or more positions are selected from the group of positions including light chain position 214 (LC214) and heavy chain positions 118 (HC118), 177 (HC177), 297 (HC297), and 341 (HC341) (amino acid numbering follows Kabat's EU numbering scheme).

[0226] In all aspects of the present invention and in certain embodiments of the embodiments, one or more positions are selected from the group of positions including light chain position 214 (LC214) and heavy chain positions 341 (HC341), 297 (HC297), and 177 (HC177) (amino acid numbering follows Kabat's EU numbering scheme).

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

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

[0229] In all aspects and in certain embodiments of the present invention, the modified antibody according to the present invention comprises two different heavy chains, thereby the difference arising from each mutation to induce heterodimerization. In further embodiments, such a modified antibody comprises one, two or more (first) recognition sites for transglutaminase derived from Kutzneria albida (KalbTG) at one or more identical or different 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 (amino acid numbering follows Kabat's EU numbering scheme).

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

[0231] In all aspects of the present invention and in certain embodiments of the embodiments, one or more positions are selected from the group of positions including heavy chain positions 118 (HC118), 177 (HC177), 297 (HC297), and 341 (HC341) (amino acid numbering follows Kabat's EU numbering scheme).

[0232] In all aspects of the present invention and in certain embodiments of the embodiments, one or more positions are selected from the group of positions including heavy chain positions 341 (HC341), 297 (HC297), and 177 (HC177) (amino acid numbering follows Kabat's EU numbering scheme).

[0233] In all aspects and one preferred embodiment of the present invention, one or more positions are selected from the group of positions including heavy chain positions 297 (HC297) and 177 (HC177) (amino acid numbering follows Kabat's EU numbering scheme).

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

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

[0236] In all aspects and one preferred embodiment of the present invention, the modified antibody comprises at least one (homogeneous) pair of antibody heavy chain and antibody light chain, the light chain comprising one (or more) recognition sites for Kutzneria albida-derived transglutaminase (KalbTG) at position 214 (LC214) of the light chain (amino acid numbering follows Kabat's 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 an antibody heavy chain and an antibody light chain, and the heavy chain comprises two (first) recognition sites (s) for transglutaminase (KalbTG) derived from Kutzneria albida at positions 297 (HC297) and 177 (HC177) of the heavy chain (amino acid numbering follows the Kabat EU numbering scheme).

[0238] In a preferred embodiment of all aspects and embodiments of the present invention, the modified antibody comprises at least one (cognate) pair of an antibody heavy chain and an antibody light chain, the heavy chain comprises one (first) recognition site (s) for transglutaminase (KalbTG) derived from Kutzneria albida at position 297 (HC297) of the heavy chain (amino acid numbering follows the Kabat EU numbering scheme), and the light chain comprises one (first) recognition site (s) for transglutaminase (KalbTG) derived from Kutzneria albida at position 214 (LC214) of the light chain (amino acid numbering follows 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 an antibody heavy chain and an antibody light chain, the heavy chain comprises one (first) recognition site (s) for transglutaminase (KalbTG) derived from Kutzneria albida at position 177 (HC177) of the heavy chain (amino acid numbering follows the Kabat EU numbering scheme), and the light chain comprises one (first) recognition site (s) for transglutaminase (KalbTG) derived from Kutzneria albida at position 214 (LC214) of the light chain (amino acid numbering follows the Kabat numbering scheme).

[0240] In all aspects and one preferred embodiment of the present invention, the modified antibody comprises at least one (homogeneous) pair of an antibody heavy chain and an antibody light chain, wherein the heavy chain comprises two (first) recognition sites for transglutaminase (KalbTG) derived from Kutzneria albida at positions 177 (HC177) and 297 (HC297) (amino acid numbering follows Kabat's EU numbering scheme), and the light chain comprises one (first) recognition site for transglutaminase (KalbTG) derived from Kutzneria albida at position 214 (LC214) (amino acid numbering follows Kabat's numbering scheme).

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

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

[0243] In all aspects and one preferred embodiment of the present invention, the modified antibody comprises two (homogeneous) pairs of antibody heavy chains and antibody light chains, wherein the heavy chain of the first pair contains a (first) recognition site(s) for transglutaminase derived from Kutzneria albida (KalbTG) at position 297 (HC297) (amino acid numbering follows the Kabat EU numbering scheme), the heavy chain of the second pair contains a (first) recognition site(s) for transglutaminase derived from Kutzneria albida (KalbTG) at position 177 (HC177) (amino acid numbering follows the Kabat EU numbering scheme), and the light chain of the second pair contains a recognition site(s) for Kutzneria albida (KalbTG) at position 214 (LC214) It contains (a)(

[0244] In all aspects and one preferred embodiment of the present invention, the modified antibody comprises two (homogeneous) pairs of antibody heavy chains and antibody light chains, wherein the heavy chain of the first pair contains a (first) recognition site(s) for transglutaminase derived from Kutzneria albida (KalbTG) at position 177 (HC177) (amino acid numbering follows the Kabat EU numbering scheme), the heavy chain of the second pair contains a (first) recognition site(s) for transglutaminase derived from Kutzneria albida (KalbTG) at position 297 (HC297) (amino acid numbering follows the Kabat EU numbering scheme), and the light chain of the second pair contains a recognition site(s) for Kutzneria albida (KalbTG) at position 214 (LC214) It contains (a)(

[0245] In all aspects and one preferred embodiment of the present invention, the modified antibody comprises two (homogeneous) pairs of antibody heavy chains and antibody light chains, wherein the heavy chain of the first pair comprises two (first) recognition sites (or more) for transglutaminase (KalbTG) derived from Kutzneria albida at positions 297 (HC297) and 177 (HC177) (amino acid numbering follows Kabat's EU numbering scheme), and the light chain of the second pair comprises one (first) recognition site (or more) for transglutaminase (KalbTG) derived from Kutzneria albida at position 214 (LC214) (amino acid numbering follows Kabat's numbering scheme).

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

[0247] The terms “antibody” and “Ig” are used interchangeably herein. They are used in their broadest sense and include, for example, monoclonal antibodies unrelated to binding specificity (including agonists, antagonists, neutralizing antibodies, full-length or intact monoclonal antibodies), monovalent antibodies (e.g., full-length antibodies lacking one Fab), multivalent antibodies, i.e., bivalent or tetravalent multispecific antibodies, and fragments of full-length antibodies insofar as they contain at least one of the modifications outlined above.

[0248] Naturally occurring antibodies are produced either as heavy chains only (e.g., VHH antibodies consisting only of heavy chains) or as assemblies of heavy and light chains (e.g., wild-type IgG antibodies). Each heavy chain consists of four domains: a variable domain (VH) and three constant domains (CH1, CH2, CH3). These are connected by a flexible hinge region. The light chain consists of a variable domain (VL) and a constant domain (CL). When both heavy and light chains are present, the light chain pairs with a Fab fragment of a congeneral heavy chain, i.e., a 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 the Fab fragment. The heavy chain CH2 and CH3 domains are collectively referred to as the 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. Therefore, in certain embodiments, the 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 crosslinks that covalently link the two antibody heavy chain Fc region polypeptides to each other. In the Fab fragment, the Fab fragments of the light chain and heavy chain are also connected by a single disulfide crosslink. However, the connectivity differs among IgG subclasses. The overall structure of full-length IgG resembles a Y shape, with the Fc region forming the base and the two Fab fragments forming the arms, which are available for binding to the antigen.

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

[0250] As used herein, the term “modified antibody” refers to an antibody or antibody Fc region according to the present invention that includes 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-idiotype (anti-id) antibodies, heavy-chain-only antibodies, and their functional fragments. 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 originates. Non-limiting examples of functional fragments of an antibody include Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, etc. In particular, modified antibodies according to the present invention include antibody molecules and molecules containing immunologically active portions of antibody molecules, such as antigen-binding domains or antigen-binding sites that bind to antigens (e.g., one or more cross-complementarity-determining regions (CDRs)), insofar as the modifications according to the present invention are present. The modified antibodies provided herein may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY; IgG in a particular embodiment), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2; IgG1 in a preferred embodiment), or any subclass (e.g., IgG2a and IgG2b). The antibodies may be humanized, chimeric, and / or affinity-matured, and may be derived 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. Therefore, 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 homogeneous light-heavy chain pair (containing one binding site), and one heavy chain Fc region fragment containing a hinge region related to 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, particularly a humanized mouse, rabbit, or sheep antibody. Exemplary and suitable sequences are shown below. [Table 32] In the annotation, "X" indicates an amino acid residue / position. [Table 33] This indicates the insertion site of the Q tag motif (with or without linker array): [Table 34] [Table 35] [Table 36] [Table 37] [Table 38]

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

[0254] In certain embodiments, the human IgG1 heavy chain polypeptide that can serve as the basis for the modified antibody according to the present invention includes a constant region amino acid sequence that is 75% or more identical, 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 SEQ ID NO: 1 or 2. In all aspects and embodiments of the present invention, in one preferred embodiment, the modified antibody according to the present invention is based on an IgG1 antibody and is therefore a modified / variant IgG1 antibody. In certain embodiments, the human IgG2 heavy chain polypeptide that can serve as the basis for the modified antibody according to the present invention includes a constant region amino acid sequence that is 75% or more identical, 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 SEQ ID NO: 3. In certain embodiments, the human IgG3 heavy chain polypeptide that can serve as the basis for the modified antibody according to the present invention includes a constant region amino acid sequence that is 75% or more identical, 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 SEQ ID NO: 4. In certain embodiments, the human IgG4 heavy chain polypeptide that can serve as the basis for the modified antibody according to the present invention includes a constant region amino acid sequence that is 75% or more identical, 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 SEQ ID NO: 5.

[0255] In all aspects of the present invention and in specific embodiments of its embodiments, the modified antibody according to the present invention comprises the following further mutations (numbering follows Kabat) in addition to the inserted KalbTG recognition site according to the present invention: a) L234A and 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 all aspects and one preferred embodiment of the present invention, the modified antibody according to the present invention comprises mutations L234A, L235A, P329G, and T366W in the first Fc region polypeptide and mutations L234A, L235A, P329G, T366S, L368A, and Y407V in the second Fc region polypeptide. In all aspects and a particular embodiment of the present invention, the modified antibody further comprises one of mutations S354C and Y349C in the first Fc region polypeptide and the other in the second Fc region polypeptide.

[0257] In all aspects and one preferred embodiment of the present invention, the modified antibody according to the present invention is based on a human heavy chain polypeptide and has 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 shown in SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 34.

[0258] [Table 39]

[0259] According to the present invention, in all aspects and in specific 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 and 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] The symbol indicates the insertion site of the Q tag motif (with or without linker array): [Table 42]

[0260] In all aspects and in specific embodiments of the present invention, the human light chain polypeptide that can serve as the basis for the modified antibody according to the present invention includes 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 shown in SEQ ID NO: 6 or 7.

[0261] In all aspects and one preferred embodiment of the present invention, the human light chain polypeptide of the modified antibody according to the present invention is SEQ ID NO: 10: [Table 43] The amino acid sequence shown is identical to that of the constant region amino acid sequence by 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%.

[0262] Preferably, the unmodified light chain constant domain includes an amino acid sequence that is at least 96%, 97%, 98%, or 99%, particularly 100%, identical to the amino acid sequence of either SEQ ID NO: 6 or 7, or the unmodified heavy chain constant region includes an amino acid sequence that is at least 96%, 97%, 98%, or 99%, particularly 100%, identical to the amino acid sequences of SEQ ID NOs: 1 to 5.

[0263] In all aspects and one preferred embodiment of the present invention, the modified antibody according to the present invention is 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 either SEQ ID NO: 6 or 7; and b) A modified heavy chain constant region comprising or having an amino acid sequence that is at least 96%, 97%, 98%, or 99%, particularly 100%, identical to the amino acid sequences of SEQ ID NOs. 1 to 5, wherein at least one recognition site for KalbTG is inserted.

[0264] In all aspects and one preferred embodiment of the present invention, the modified antibody according to the present invention is 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 either SEQ ID NO: 6 or 7, wherein at least one KalbTG recognition site is inserted; and b) A non-modified heavy chain constant region comprising an amino acid sequence that is at least 96%, 97%, 98%, or 99%, and especially 100%, identical to the amino acid sequences of SEQ ID NOs. 1-5.

[0265] Preferably, the light chain constant domain contains or comprises an amino acid sequence that is at least 96%, 97%, 98%, or 99%, particularly 100%, identical to the amino acid sequence of SEQ ID NO: 10, or the heavy chain constant region contains or comprises an amino acid sequence that is at least 96%, 97%, 98%, or 99%, particularly 100%, identical to the amino acid sequence of SEQ ID NO: 8, 9, or 34.

[0266] In all aspects and one preferred embodiment of the present invention, the modified antibody according to the present invention comprises a) a light chain constant domain comprising or 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 NO: 10, and b) a modified heavy chain constant region comprising or 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 NO: 8, 9, or 34.

[0267] As detailed above, the modified antibody according to the present invention may be a bispecific antibody or a multispecific antibody. Examples of bispecific or multispecific antibodies include the following: - Full-length antibodies with domain exchange: A multispecific IgG antibody comprising a first Fab fragment and a second Fab fragment, wherein in the first Fab fragment, a) Only the CH1 and CL domains are substituted for each other (i.e., the light chain of the first Fab fragment contains the VL and CH1 domains, and the heavy chain of the first Fab fragment contains the VH and CL domains); b) Only the VH and VL domains are replaced by each other (i.e., the light chain of the first Fab fragment contains the VH and CL domains, and the heavy chain of the first Fab fragment contains the VL and CH1 domains); or c) The CH1 and CL domains are substituted for each other, and the VH and VL domains are substituted for each other (i.e., the light chain of the first Fab fragment contains the VH and CH1 domains, and the heavy chain of the first Fab fragment contains the VL and CL domains); and The second Fab fragment comprises a light chain containing VL and CL domains and a heavy chain containing VH and CH1 domains. Full-length antibodies involving domain exchange may contain a first heavy chain containing a CH3 domain and a second heavy chain also containing a CH3 domain, and both CH3 domains are complementarily manipulated by their respective amino acid substitutions to support heterodimerization of the first and modified second heavy chains; - Full-length antibodies with domain exchange and additional heavy chain C-terminal binding sites: A multispecific IgG antibody, a) A full-length antibody comprising two pairs each of full-length antibody light chains and full-length antibody heavy chains, wherein the binding sites formed by each pair of full-length heavy chains and full-length light chains specifically bind to a first antigen, b) an additional Fab fragment fused to the C-terminus of one heavy chain of a full-length antibody, wherein the binding site of the additional Fab fragment specifically binds to a second antigen, A multispecific IgG antibody in which an additional Fab fragment that specifically binds to a second antigen includes a domain crossover such as i) a) a light chain variable domain (VL) and heavy chain variable domain (VH) are substituted for each other, or b) a light chain constant domain (CL) and heavy chain constant domain (CH1) are substituted for each other, or ii) 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 chain domain + light chain kappa constant domain) - Light chain / heavy chain combination (variable light chain domain + constant light chain domain + peptide linker + variable heavy chain domain + CH1 + hinge + CH2 + CH3 knob mutations) - Heavy chain (variable heavy chain domain + CH1 + hinge + CH2 + CH3 hole mutation present); - 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 combination (variable light chain domain + constant light chain domain + peptide linker + variable heavy chain domain + CH1 + hinge + CH2 + CH3 hole mutation present) - Light chain / heavy chain 2 combination (variable light chain domain + constant light chain domain + peptide linker + variable heavy chain domain + CH1 + hinge + CH2 + CH3 knob mutations); - Common light chain bispecificity format (= common light chain bispecificity 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 chain domain + constant light chain domain) - Heavy chain 1 (variable heavy chain domain + CH1 + hinge + CH2 + CH3 hole mutation present) - Heavy chain 2 (variable heavy chain domain + CH1 + hinge + CH2 + CH3 knob mutation present).

[0268] The term "specifically binds" means that the antibody binds at least 10 times -8 The dissociation constant (=K) of mol / l Diss) shows interaction with its cognate antigen. Thus, the term "specifically binds" means the binding of an antibody having a dissociation constant (=K -8 ) of 10 Diss mol / l or less to its cognate antigen. The terms "does not specifically bind" or "not specifically binding" mean the binding of an antibody to an unrelated antigen at a dissociation constant (=K -7 ) of 10 -5 mol / l or more, i.e., for example, 10 Diss mol / l. At the same time, the property of "not specifically binding" is ensured by a K -7 of 10 Diss mol / l or less for each unrelated antigen. In certain embodiments, an antibody that specifically binds an antigen will have at least a 100-fold K Diss -gap between its reactivity to its cognate antigen and its reactivity to an unrelated antigen.

[0269] The binding properties of antibodies, particularly K Diss , can be evaluated by a BIAcore® instrument. In this method, the binding properties are evaluated by changes in surface plasmon resonance (SPR). It is convenient to evaluate the binding by binding the antibody under investigation to a solid phase (referred to as a chip) and applying the antigen in question to this coated chip.

[0270] As used herein, the term “substituting for each other” with respect to the corresponding heavy and light chain domains refers to the domain crossover described above. Thus, when the CH1 and CL domains are “substituting for each other,” the term refers to the domain crossover mentioned under item (i), and the resulting heavy and light chain domain sequences. Accordingly, when the VH and VL domains are “substituting for each other,” the term refers to the domain crossover mentioned under item (ii), and when the CH1 and CL domains are “substituting for each other” and the VH and VL domains are “substituting for each other,” the term refers to the domain crossover mentioned under item (iii).

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

[0272] The term "antigen" refers to a predetermined target to which an antibody can selectively bind. An antigen may 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 therapeutic. In yet another embodiment, the antigen is specific to or enables delivery to a specific area in the body, such as a particular organ or cell type or disease area. This may be a specific structure on the surface of a cell, such as a receptor or tumor marker. In one preferred embodiment of all aspects and embodiments of the present invention, the antigen is a human transferrin receptor.

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

[0274] According to the present invention, a modified antibody comprises a heavy chain polypeptide and / or a light chain polypeptide containing one or more recognition sites for KalbTG. The recognition site comprises a motif of KalbTG, which can catalyze the formation of an isopeptide bond between the modified antibody and a compound (payload) bound 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 involves the generation of an (artificial) Q tag in the antibody heavy chain and / or light chain polypeptide, i.e., a Gln-containing motif recognized by KalbTG. Suitable Q tags are disclosed in International Publication No. 2017 / 102759, expressly incorporated herein by reference. In certain embodiments, one or more independent first KalbTG recognition sites include 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), particularly YRYRQ (SEQ ID NO: 17). The Q tag can be generated by one or more amino acid modifications, preferably substitution or insertion. In certain embodiments, the Q tag is generated by insertion and / or substitution of an amino acid sequence including YRYRQ (SEQ ID NO: 17) or RVRQR (SEQ ID NO: 18), particularly YRYRQ (SEQ ID NO: 17). Preferably, the inserted amino acid sequence has a length of 5 to 20 amino acids and includes YRYRQ (SEQ ID NO: 17) or RVRQR (SEQ ID NO: 18), particularly 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). The introduction of one or more recognition sites to KalbTG may be the sole modification in the constant region of the light chain and / or heavy chain. Alternatively, further modifications such as a purification tag (e.g., a His tag) or other modifications, e.g., increased stability or heterodimerization or modification of effector function, may be present alone or in any combination.

[0275] As described above, one or more first recognition sites of KalbTG are inserted into specific sites on the antibody, namely positions 110 (LC110), 143 (LC143), and 214 (LC214) on the light chain, and positions 118 (HC118), 177 (HC177), 297 (HC297), 341 (HC341), and 401 (HC401) on the heavy chain, particularly one or more selected from HC177 and / or HC297 and / or LC214. In certain embodiments, the modified antibody of the present invention may include a further (second) recognition site of KalbTG at position 446 (HC446) on the heavy chain, i.e., the C-terminus, in order to link to a domain different from the domain bound at the first recognition site. Preferably, one or more recognition sites are located independently of each other at positions 177 (HC177) or 297 (HC297) on the heavy chain or at position 214 (LC214) on the light chain.

[0276] In certain embodiments, the Q tag is inserted into the antibody light / heavy chain amino acid sequence, spaced between / through one or more linkers. The linkers can increase flexibility or enable conjugation with larger payloads. In certain embodiments, each linker sequence independently comprises 1 to 20 amino acids, 1 to 10 amino acids in certain embodiments, and 1 to 5 amino acids in further embodiments, and the linkers do not inherently interfere with the function of the Q tag, KalbTG, antibody folding, and the payload conjugated to the modified antibody. The linkers may be N-terminal and / or C-terminal to the Q tag. In certain embodiments, the linker amino acids are small amino acids such as glycine or serine. Amino acid linkers and their compositions are known in the art (see, for example, Chichili et al.). The amino acids glycine, serine, alanine, threonine, and glutamic acid typically constitute the amino acids of flexible linkers. Therefore, linkers may consist mainly or entirely of Gly and / or Ser and / or Ala and / or Thr and / or Glu, for example GGGP (SEQ ID NO: 20), ESGS (SEQ ID NO: 21), or APAP (SEQ ID NO: 22). There may also be linkers that include or consist of KESGSVSSEQLAQFRSLD (SEQ ID NO: 23) or EGKSSGSGSESKST (SEQ ID NO: 24). In one preferred embodiment of all aspects and embodiments of the present invention, the linkers consist mainly or entirely of Gly and Ser, independently of each other, for example (Gly m Ser) n The m=1, 2, 3 or 4 and n=1, 2, 3, 4 or 5, where m and n are independent of each other, preferably m=3 and n=1. In all aspects and one preferred embodiment of the present invention, one or more first recognition sites are inserted into the heavy chain and / or light chain amino acid sequence via one or more linkers at their terminals, the linkers being primarily or entirely from Gly and Ser, for example (Gly-Gly-Gly-Ser). n (Sequence ID 19), where n=1, 2, 3, 4, or 5, preferably n=1.

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

[0278] An exemplary Fc region, which includes parts of a hinge region having (referred to as sequence numbers 32 and 113) and not having (referred to as sequence numbers 33 and 110) a first recognition region (bold), has the following sequence: [Table 44]

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

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

[0281] The term "nucleic acid" comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and the basic structural unit of nucleic acids, nucleotides, includes not only natural nucleotides but also analogs having modified sugar or base sites. The nucleic acid sequences encoding the heavy-chain variable region and light-chain variable region of the present invention can be modified. Such modifications include the addition, deletion, or non-conservative or conserved substitution of nucleotides, as long as the encoded sequence does not change.

[0282] The DNA encoding the modified antibody according to the present invention can be isolated or synthesized using conventional procedures (for example, by using oligonucleotide probes that can specifically bind to the DNA encoding the heavy and light chains of the antibody).

[0283] Many transfer vectors and expression vectors are available. Vector components generally include, but are not limited to, one or more of the following: signal sequences, origins of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences.

[0284] As used herein, the term “vector” refers to plasmid vectors; cosmid vectors; viral vectors, such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors, as means for expressing a target gene in a host cell. The nucleic acid encoding the modified antibody in the vector is operably ligated to a promoter and a polyadenylation signal sequence.

[0285] "Operationally linked" refers to a functional linkage between a nucleic acid expression regulatory sequence (e.g., a promoter, signal sequence, or array of transcription factor binding sites) and another nucleic acid sequence, thereby allowing the regulatory sequence to control the transcription and / or translation of the other nucleic acid.

[0286] When eukaryotic cells are used as the host, promoters derived from the genome of mammalian cells (e.g., metallothione promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) may be used, or mammalian promoters derived from animal viruses (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 mouse sarcoma virus (RSV) promoter) may be used. Furthermore, 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 may be, but are not limited to, prokaryotic cells, yeast cells, or higher eukaryotic cells.

[0288] However, the most interesting 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 one preferred embodiment, the host cell is a CHO cell.

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

[0290] As detailed above, the modified antibody of the present invention is provided for the purpose of specifically conjugating one or more payloads, such as a therapeutic moiety, to one or more internal sites of the antibody with the help of KalbTG. 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, such as the therapeutic moiety, to an intended area in the body. In certain embodiments, the modified antibody according to the present invention recognizes a target and binds to it at its complementarity-determining region (CDR), and in particular the target is a biomolecule present on a cell.

[0291] As detailed above, it is sometimes desirable to target the therapeutic portion to a specific area within the patient's body. This can improve in vivo distribution and reduce harmful side effects. Clearly, the therapeutic portion may be intended to be delivered to areas that are otherwise difficult to reach. As an example, it may be conceivable to guide the therapeutic portion into the brain. Due to the blood-brain barrier, it is difficult to deliver a "naked" therapeutic portion there, especially if it exceeds certain size limitations, unless it is administered directly to the brain. A therapeutic approach that overcomes the blood-brain barrier and helps transport the therapeutic portion into the brain is clearly advantageous. Notably, the same approach may be used to guide therapeutic drugs to other areas within the body.

[0292] In this invention, the molecular recognition unit of an antibody that specifically binds to a structure in the body is used for targeting the therapeutic area. Considering the above, it is clear that the therapeutic entity may be any compound useful for treating or preventing the disease of interest, in particular a compound delivered to a specific area in the body, such as a specific organ or cell type or disease area.

[0293] The terms “treat,” “treating,” and “treatment” include alleviating or suppressing a symptom, disorder, or disease, or one or more symptoms associated with a symptom, disorder, or disease, or alleviating or eradicating the cause(s) of the symptom, disorder, or disease itself. The terms “prevent,” “preventing,” and “prevention” include methods of delaying and / or eliminating the onset of a symptom, disorder, or disease and / or its associated symptoms; methods of preventing a subject from acquiring a symptom, disorder, or disease; or methods of reducing the risk of a subject acquiring a symptom, disorder, or disease.

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

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

[0296] In all aspects and one preferred embodiment 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, as well as small interfering RNAs (siRNAs), which are a class of double-stranded non-coding RNA molecules, typically 20-24 base pairs long, that are similar to miRNAs and operate within the RNA interference (RNAi) pathway. They interfere with the expression of certain genes having complementary nucleotide sequences by degrading mRNA after transcription, thereby interfering with translation. In certain embodiments, the nucleic acid comprises one or more locked nucleic acids (LNAs). An LNA is a modified RNA nucleotide in which the ribose portion is modified with an extra crosslink connecting the 2' oxygen and 4' carbon. The crosslink "locks" the ribose in the 3'-end (north) conformation. This structure may result in increased stability against enzymatic degradation, and furthermore, the structure of the LNA has improved specificity and affinity as a component of monomers or oligonucleotides. In certain embodiments, the LNA nucleotide is mixed with DNA or RNA residues in the oligonucleotide or nucleic acid.

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

[0298] In the present invention, the therapeutic entity is a non-antibody domain covalently bound to the modified antibody of the present invention via a second linker, optionally. The linker may depend on the intended target and therapeutic entity, as the length, stiffness, and chemical composition of the linker may affect the conjugation reaction rate and the stability of the resulting conjugate. In all aspects and one preferred embodiment of the present invention, the (second) linker is an alkyl linker, a polyethylene linker, a peptide linker, or a mixture thereof. In certain embodiments, the (second) linker contains ethylene glycol (PEG) units, e.g., about 2 to 50 ethylene glycol units. An exemplary linker is the polyethylene glycol linker ((-NH-C(=O)-PEG n Examples include -NH2 (n=2~25). In certain embodiments, the (second) linker is an aliphatic carbon chain. The linker is unsubstituted or substituted C 1~6 It may also contain unsubstituted or substituted alkyl groups such as alkyl groups, C 1~6 The alkyl group 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, azide, 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 all aspects and one preferred embodiment of the present invention, the conjugate comprises a modified antibody according to the present invention and one or more therapeutic nucleic acids, such as ASOs, each therapeutic nucleic acid linked to a single Q-tag via an isopeptide bond and optionally amide-bonded to a terminal residue of the K-tag via a second linker, particularly a PEG linker, as defined above.

[0300] In all aspects and certain embodiments of the present invention, the conjugate has a DAR in the range of about 1 to about 8, about 1 to about 4, or about 1 to about 2. In another embodiment, the conjugate has a DAR of about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8.

[0301] When an antibody binds to a target, the conjugate is transported to the respective cell by endocytosis, releasing the therapeutic entity and allowing it to act in the intended manner (e.g., to treat a disease).

[0302] In all aspects and one preferred embodiment of the present invention, the modified antibody recognizes a target, the target being 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), particularly 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 to the respective cell by endocytosis, released from the endosome, and then exocytated again from the cell. If the cell is part of a barrier such as the blood-brain barrier, this enables transport across the respective barrier. As a result, the therapeutic entity is transported to compartments of the body that could not be reached by the therapeutic entity not conjugated by the modified antibody according to the present invention.

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

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

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

[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, wherein the method is: a) To provide a modified antibody according to the present invention, b) To provide a non-antibody domain, wherein the non-antibody domain is (i) The actual treatment, (ii) A second recognition site for KalbTG that is complementary to the first recognition site present in the modified antibody provided under (i), preferably a second recognition site comprising or having a Lys-containing motif, particularly the sequence RYESK (SEQ ID NO: 16), and (iii) optionally, to provide a non-antibody domain including a second linker between the therapeutic entity and the second recognition site, c) The modified antibody of a) and the non-antibody domain of b) are incubated or reacted in the presence of KalbTG or a functionally active variant or fragment thereof, under conditions that enhance the activity of KalbTG, thereby forming an isopeptide bond between the first recognition site and the second recognition site, and thus conjugating the modified antibody to a therapeutic entity.

[0308] In the first step of this method, the modified antibody and non-antibody domain of the present invention are provided.

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

[0310] The domain can be as defined above.

[0311] The modified antibody and the 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 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 the modified antibody is conjugated to the therapeutic entity.

[0312] In all aspects of this method according to the present invention and in specific embodiments of the embodiments, the antibody comprises one or more Q tags, each conjugated to one or more K tags using the enzymatic activity of KalbTG.

[0313] The non-antibody domain includes a second recognition site for KalbTG. In all aspects of this method according to the present invention and in certain embodiments of the embodiments, the non-antibody domain includes a K tag having at least 80% sequence identity with respect 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 crosslinking proteins and peptides in food and biotechnology applications (e.g., to improve the texture of protein-rich foods or to generate antibody-drug conjugates).

[0315] However, KalbTG does not inherently exhibit cross-reactivity with known mTG substrates or commonly used target proteins, such as antibodies, and therefore allows for specific conjugation at a given site.

[0316] Therefore, any payload containing a second recognition site (K tag) for KalbTG, particularly any payload in which the second recognition site contains 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 sites, i.e., Q tags.

[0317] KalbTG or its functionally active variants or fragments may be as defined in Steffen et al. (2017) or International Publication No. 2016 / 100735A1 (both expressly incorporated herein by reference).

[0318] A functionally active variant or fragment may be a transglutaminase having at least 80%, 90%, 95%, or 99% sequence identity with KalbTG of International Publication 2016 / 100735A1 (see Sequence ID No. 6 therein). Alternatively, KalbTG or its functionally active variant may be part of a fusion protein that additionally includes a label, such as a tag, for purification purposes.

[0319] In certain embodiments, KalbTG includes an amino acid sequence (a three-letter code). [Table 45]

[0320] In all aspects of the method according to the present invention and in specific embodiments of the embodiments, the conjugation is controlled and achieved, for example, at a stoichiometric ratio of non-antibody domains to modified antibodies, e.g., about 1:1 per Q-tag / K-tag pair. Multiple conjugations can also be achieved by using two or more first recognition sites on a single modified antibody to conjugate two or more payloads to the modified antibody.

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

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

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

[0324] As detailed above, the conjugate containing the modified antibody according to the present invention can be used as a pharmaceutical, particularly in the treatment of neurological or brain diseases (in a preferred embodiment of Alzheimer's disease or Parkinson's disease), or in the treatment of cancers such as breast cancer.

[0325] Conjugates may be incorporated into a composition. Such compositions, also called pharmaceutical compositions, are compositions intended for use in the pharmaceutical field or as pharmaceuticals, in a form that allows the biological activity of the active ingredient contained therein to be effective, and do not contain additional ingredients that are unacceptably toxic to the subject to which the pharmaceutical composition is administered. The composition may optionally contain pharmaceutically acceptable excipients, diluents or carriers, such as buffers, stabilizers or preservatives, and optionally further active ingredients, in particular ingredients known in relation to pharmaceutical compositions.

[0326] Generally, the properties of additional components depend on the specific form of the pharmaceutical composition and the mode of administration used. Pharmaceutically acceptable carriers can be used to enhance or stabilize the composition or to facilitate its preparation. Such carriers include, but are not limited to, physiologically compatible salines, buffered salines, dextrose, water, glycerol, solvents, dispersions, antimicrobial and antifungal agents, isotonic and absorption retardants, and combinations thereof. The formulation should be suitable for the mode of administration. For example, parenteral formulations typically contain an injectable fluid that includes a pharmaceutical and physiologically acceptable fluid as a vehicle, such as water, saline, equilibrium salt solutions, aqueous dextrose, or glycerol. In addition to a biologically neutral carrier, the administered pharmaceutical composition may contain small amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffers.

[0327] The pharmaceutical composition may contain stabilizers. The term “stabilizer” refers to a substance that protects the composition from harmful conditions that occur during heating or freezing, and / or extends the stability or shelf life of the conjugate of the present invention under certain conditions or states. 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 conjugate administered can first be determined based on the guidance of the dose and / or administration regimen of an equivalent unconjugated therapeutic agent. Generally, the conjugate can provide targeted delivery and therefore can provide at least one of dose reduction or dose reduction in the administration regimen. Thus, the conjugate can provide dose reduction and / or dose reduction in the administration regimen compared to the previous therapeutic agent present in the conjugate of the present invention. As described above, since the conjugate can provide a controlled stoichiometry of drug delivery, the dose of the conjugate can be calculated based on the number of drug molecules delivered per antibody-therapeutic entity conjugate.

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

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

[0331] The pharmaceutical compositions of the present invention can be used as pharmaceuticals for treating individuals. The individuals are mammals. Examples of mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, e.g., monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the individual is a human.

[0332] Pharmaceutical compositions comprising the conjugates described herein can be delivered to cells, cell populations, tumors, tissues, or subjects using delivery techniques known in the art. Generally, any suitable method recognized in the art for delivering conjugates can be adapted for use with the compositions described herein. For example, delivery may be by local administration (e.g., direct injection, implantation, or topical administration), systemic administration, 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, and more preferably, administered intravenously, intramuscularly, or intraarterially. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate the above-described pharmaceutical compositions into unit dosage forms. As used herein, a unit dosage form refers to a physically distinct unit suitable as a unit dose, each unit containing a predetermined amount of the active ingredient calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier. Examples of such unit dosage forms are injectable solutions or suspensions, etc.

[0333] As detailed above, the conjugate / pharmaceutical composition of the present invention is particularly useful for the treatment of neurological or cerebrovascular diseases such as Alzheimer's disease or Parkinson's disease. The term “neurological disease” includes, among other things, 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 be seen in many different levels of neural circuits, from molecular to systemic. The terms “neurodegenerative disease” and “neuroinflammatory disease” have a partially overlapping scope. Inflammatory responses are a prominent feature of neurodegenerative diseases and are involved in or contribute to neuronal cell death through different mechanisms. Tryptophan catabolism along the kynurenine pathway (KP) represents one of these mechanisms. Seizure disorders are brain disorders characterized by abnormal signaling between brain cells. Seizure disorders can affect a part of the brain (partial seizures) or the entire brain (generalized seizures). The most prominent seizure disorder is epilepsy. To cross the blood-brain barrier, receptor-mediated receptor-induced endocytosis can be used as a conjugate target. 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 those skilled in the art of this disclosure. Any methods and materials similar to or equivalent to those described herein may be used in the practices presented herein, but certain methods and materials are described herein.

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

[0336] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple objects unless the context explicitly indicates otherwise. Similarly, the words “comprise,” “contain,” and “encompass” are interpreted inclusively, not exclusively. Likewise, the word “or” is intended to include “and” unless the context explicitly indicates otherwise. The term “plural” 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 modifications described should not be construed as limiting 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 it should be understood that such equivalent embodiments are included herein. [Brief explanation of the drawing]

[0338] [Figure 1] This shows hydrophobic interaction chromatography of mAb-2(HER2) with nine different Q-tag insertion sites conjugated to a single-stranded nucleic acid having 15 residues. [Figure 2] This shows an exemplary reaction of single-step KalbTG-mediated conjugation between an mAb and a nucleic acid payload. [Examples]

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

[0340] Expression of modified antibodies in mammalian cells Antibody production was induced by transient co-transfection of a single-expression cassette plasmid in HEK293 cells cultured in F17 medium (Invitrogen, Carlsbad, California, USA) or Expi293 cells in Expi293 expression medium (Thermo Scientific, Waltham, Massachusetts, USA). Transfection was performed as specified in the manufacturer's instructions, with a plasmid ratio of HC:LC expression plasmid = 1:1 for the symmetric standard IgG1 format, or with a plasmid ratio of HC1:HC2:LC expression plasmid = 1:1:1 for the asymmetric knob-into-hole format. Cell culture supernatant was collected 7 days after transfection. The supernatant was stored at a low temperature (e.g., -20°C).

[0341] Quantitative determination of protein titer The protein titer of the supernatant sample was determined by affinity chromatography using a high-performance liquid chromatography system (Ultimate 3000 HPLC system, Thermo Scientific, Waltham, Massachusetts, USA) with a POROS A 20 μm column, 2.1 × 30 mm (Life Technologies, Carlsbad, California, USA). The supernatant was loaded onto a column equilibrated with 0.2 M Na2HPO4, pH 7.4, and subsequently eluted with 0.1 M citrate, 0.2 M NaCl, pH 2.5. The titer was quantified by measuring the absorbance at 280 nm, and the protein concentration was then calculated by comparing the elution peak area (below the curve) of the analyte with a reference standard curve.

[0342] Purification of modified antibodies from mammalian culture supernatant Antibodies in the culture supernatant were captured by protein A affinity chromatography using a Mab Select SuRe column (GE Healthcare, Chicago, Illinois, USA) equilibrated with PBS buffer, pH 7.4. Unbound proteins were removed by washing with the equilibration buffer. 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 size exclusion chromatography procedure using a Superdex 200® column (GE Healthcare, Chicago, Illinois, USA) in 20 mM histidine, 140 mM NaCl, pH 6.0 was performed as a second purification step. The purified modified antibodies were stored at -80°C.

[0343] Antibody purification using medium-scale automation (Milan) The antibody was purified in one step using protein A affinity chromatography as described above, with a MabSelectSuRe-Sepharose (Cytiva, Marlborough, Massachusetts, USA) in a liquid processing system (Tecan, Mönnendorf, Switzerland) equipped with a column from Repligen (Waltham, Massachusetts, USA). The equilibration, sample loading, and washing steps were performed as described, and the antibody was eluted from the column using 25 mM citrate, pH 3.0. The eluted antibody fraction was neutralized with 1.5 M Tris (pH 7.5), and the concentration was determined by measuring the optical density (OD) at 280 nm.

[0344] Overview of Exemplary Antibodies Antibody 110: explanation: Anti-HER2 antibody based on IgG1 subclass with P329G / L234A / L235A mutation; Q-tag insertion 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 mutation; Q-tag insertion into 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 influenced by the position of the introduced Q tag. The best yields were obtained when introduced after LC110, LC214, HC118, HC177, HC297, HC341, and HC401.

[0347] Example 2 KalbTG conjugation of modified antibodies to fluorescent dyes according to the present invention Conjugation using KalbTG The purified antibody containing the Q tag was transferred to a conjugation buffer (histidine buffer containing NaCl, pH 8.5) via dialysis. For the KalbTG reaction, the antibody was mixed with a K-tag small molecule (fluorescent dye, 10 × 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 the unconjugated payload and residual enzymes, the conjugated modified antibody was purified by size exclusion chromatography using a Superdex 200™ column (GE Healthcare, Chicago, Illinois, USA) in PBS pH 7.5. The purified conjugate was stored at -80°C.

[0348] Conjugate Analysis Protein quantification was performed using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Furthermore, qualitative DAR measurements were performed by hydrophobic interaction chromatography as described in Example 4 below. The purity of the conjugate was analyzed by CE-SDS under denaturation and reduction conditions using a Caliper LabChip® GXII Touch® protein characterization system, in accordance with the manufacturer's instructions (Perkin Elmer, Waltham, Massachusetts, USA).

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

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

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

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

[0353] Oligonucleotides were conjugated to either a linker (one-step reaction) for conjugation to a K tag or an acceptor (two-step reaction) for click chemical conjugation. The conjugates were then directly purified 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-tagged peptide were dissolved in PBS and added. After 1 hour at room temperature, the reaction mixture was directly purified by reverse-phase HPLC.

[0355] Both of the above reaction products were purified by reverse-phase HPLC using 0.1 M ammonium acetate and acetonitrile as eluents on a Waters XBridge Peptide BEH C 18 OBD Prep Column, 300 Å, 10 μm, 10 mm × 150 mm. The pooled fractions were freeze-dried, redissolved in water, and the pH was adjusted to pH 7.0 with aqueous NaOH solution. After final freeze-drying, the compounds were 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 The purified antibody containing the Q tag was transferred via dialysis to a conjugation buffer (histidine buffer containing approximately 150 mM chloride ions, pH 7.5). For the KalbTG reaction, the antibody was mixed with an excess of K-tagged oligonucleotide, 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 the unconjugated payload and residual enzymes, the conjugated modified antibody was purified by size exclusion chromatography using a Superdex 200™ column (GE Healthcare, Chicago, Illinois, USA) in PBS, 250 mM arginine, pH 7.5. The purified conjugate was stored at -80°C.

[0357] Two-stage conjugation using KalbTG and click chemistry The purified antibody containing the Q tag was transferred via dialysis to a conjugation buffer (histidine buffer containing NaCl, pH 8.5). For the KalbTG reaction, the antibody was mixed with an excess of K-tagged linker (10x molar excess) containing the first portion of the click conjugation, and KalbTG 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 the unconjugated linker and residual enzymes, the conjugated modified antibody was purified by size exclusion chromatography using a Superdex 200™ column (GE Healthcare, Chicago, Illinois, USA) in PBS, 250 mM arginine, pH 7.5. The purified antibody-linker conjugate was added to an excess amount of oligonucleotides conjugated to the other portions 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 conjugate was purified by size exclusion chromatography as described above, and the purified conjugate was stored at -80°C.

[0358] Conjugate Analysis The oligonucleotide conjugate was quantified by UV / Vis spectroscopy at 260, 280, and 350 nm using the SoloVPE system (C Technologies, Bridgewater, New Jersey, USA). The conjugate concentration and quantitative drug-antibody ratio (DAR) were calculated using the Lambert-Beer equation. Furthermore, qualitative DAR measurements were performed by hydrophobic interaction chromatography as described in Example 4 below. The purity of the conjugate was analyzed by CE-SDS under denaturation and reduction conditions using the Caliper LabChip® GXII Touch® protein characterization system (Perkin Elmer, Waltham, Massachusetts, USA). Aggregate content was determined by SEC using a TSKgel UP-SW 3000 analytical size exclusion column (Tosoh Bioscience, Griesheim, Germany) equilibrated with 0.2M K2HPO4 / KH2PO4, 0.25M KCl, and pH 6.2 on a high-performance liquid chromatography system (Ultimate 3000 HPLC system, Thermo Fisher Scientific, Waltham, Massachusetts, USA). Conjugate identity was confirmed by ESI-Q-ToF-MS (Bruker maXis 433, Bruker, Billerica, Massachusetts, USA). For MS analysis, the sample was deglycosylated using N-glycosidase F (Roche, Basel, Switzerland), followed by desalting with 2% formic acid and 40% acetonitrile. [Table 50]

[0359] The conjugation of modified antibodies is influenced by the position of the introduced Q tag. Introduction after the positions of 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 using a high-performance liquid chromatography system (Ultimate 3000 HPLC system, Thermo Fisher Scientific, Waltham, Massachusetts, USA) with 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 Na₂HPO₄ dihydrate, 1.5 M (NH₄)₂SO₄, 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 Na₂HPO₄ 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] The elution profile was obtained by continuously measuring the absorbance at 280 nm. The drug-antibody ratio (DAR) was determined by peak integration using Chromeleon 7.2 (Thermo Fisher Scientific, Waltham, Massachusetts, USA).

[0362] An example of the results is shown in Figure 1. [Table 52]

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

[0364] The hydrophobicity of the conjugate is influenced by the position of the introduced Q-tag. Introduction after the positions of LC110, LC214, and HC297 resulted in up to a twofold change in the unconjugated antibody compared to the conjugated modified antibody, while HC177 showed the lowest relative retention time for 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 12 different compounds to be tested.

[0366] All compounds consist of a homologous pair of full-length antibody light chains and an antibody Fc region fragment. [Table 53] Based on a one-arm anti-transferrin receptor antibody containing a full-length antibody heavy chain pair.

[0367] The Q tags had the amino acid sequence GGGSYRYRQGGGS (sequence number 25) inserted after each position.

[0368] Compound 1: Reference compound that does not contain conjugated nucleic acids Compound 2: A single nucleic acid conjugated to a Q tag inserted after the HC118 position in the full-length heavy chain. Compound 3: A single nucleic acid conjugated to a Q tag inserted after the HC177 position in the full-length heavy chain. Compound 4: A single nucleic acid conjugated to a Q tag inserted after the HC295 position in the full-length heavy chain. Compound 5: A single nucleic acid conjugated to a Q tag inserted after the HC297 position in the full-length heavy chain. Compound 6: A single nucleic acid conjugated to a Q tag inserted after the full-length heavy chain at position HC341. Compound 7: A single nucleic acid conjugated to a Q tag inserted after the HC401 position in the full-length heavy chain. Compound 8: A single nucleic acid conjugated to a Q tag inserted after position HC446 in the full-length heavy chain. Compound 9: A single nucleic acid conjugated to a Q tag inserted after position LC214. Compound 10: Two nucleic acids conjugated to a Q tag inserted after the HC297 and Fc region fragments of the full-length heavy chain. Compound 11: Two nucleic acids conjugated to a Q tag inserted after the HC341 and Fc region fragments of the full-length heavy chain. Compound 12: Two nucleic acids conjugated to a Q tag inserted after the HC446 and Fc region fragments of the full-length heavy chain.

[0369] The compounds were formulated as a solution in 20 mM histidine (pH 6) and administered intravenously at a single nominal dose of 20 mg / kg body weight and a dose of 2 mL / kg (exceptionally, compound 1 was administered at dose levels of 3.3 mL / kg and 16.7 mg / kg). Each formulation was slowly injected as a bolus into one of the two lateral tail veins. Continuous microsampling of blood (20 μL / time point / mouse) was performed by tail vein puncture. Blood samples were collected in K3-EDTA coated Minivette® POCT at 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 4°C at approximately 10,000 × g for approximately 5 minutes. For final sampling, blood was collected by cardiac puncture and transferred to a K3-EDTA coated polypropylene tube. The plasma sample was stored at -20°C for further processing and analysis.

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

[0371] To determine the pharmacokinetics (PK) in plasma samples, a general electrochemiluminescence immunoassay (ECLIA) specific to the human CH2 domain was used on a cobas®e411 (Roche Diagnostics GmbH, Mannheim, Germany) instrument under non-GLP conditions based on Stubenrauch et al. Briefly, the test sample was added stepwise to the detection vessel with the first detection antibody (biotinylated), the second detection antibody (rutheniumized), and SA beads. The calibration range for specific standard curves for each compound was 0.69 ng / mL to 1,500 ng / mL assay concentrations 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 yielding a 1% matrix concentration. Plasma samples were analyzed in two different dilutions (1:100 to 1:800).

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

[0373] In the hELISA method, standards, quality control, and pre-diluted samples were prepared using non-conjugated nucleic acids as a reference. Biotinylated capture oligonucleotide probes and digoxigenylated detection oligonucleotide probes were added for hybridization and transferred to streptavidin-coated microtiter plates. Polyclonal anti-digoxigenin-POD Fab fragments and TMB solution were used for detection. Color intensity was analyzed photometrically at 450 nm (690 nm reference wavelength). Color intensity was proportional to the analyte concentration in the test sample. Calibration range of the standard curve for 0.8 pM to 111 pM assay concentrations in 1% C57BL / 6 mouse plasma. For plasma samples, standard curves, and quality control, all dilutions were prepared in assay buffer containing C57BL / 6 mouse plasma yielding a 1% matrix concentration. Plasma samples were analyzed in 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, wherein said 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 said antibody heavy chain (numbering according to Kabat).

2. 1. 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 Kutschneria 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).

3. 1. A modified antibody comprising a heavy chain and a light chain, wherein the heavy chain and / or the light chain comprise one or two or three or four or more first recognition site(s) for transglutaminase from Kutschneria 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).

4. The modified antibody of any one of claims 1 to 3, wherein the first recognition site for KalbTG is present within the amino acid sequence of each of the antibody chains.

5. The modified antibody of any one of claims 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.

6. 6. The modified antibody of any one of claims 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).

7. The modified antibody of any one of claims 1 to 6, wherein the modified antibody comprises two identical antibody heavy chains.

8. The modified antibody of any one of claims 1 to 6, wherein the modified antibody comprises two different antibody heavy chains.

9. 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 any one of claims 1 to 8, comprising:

10. 1. 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, two, 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).

11. 11. The modified antibody or modified antibody Fc region of any one of claims 1 to 10, 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).

12. The modified antibody or modified antibody Fc region of any one of claims 1 to 11, wherein the recognition site(s) for KalbTG are inserted after said position.

13. The modified antibody or modified antibody Fc region of any one of claims 1 to 12, wherein the recognition site(s) for KalbTG are interposed between two flexible peptide linkers.

14. The modified antibody or modified antibody Fc region of claim 13, 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).

15. The modified antibody has the sequence of SEQ ID NO: 01: Table 56 Table 57 a first recognition site for KalbTG is inserted after one or more positions identified by Table 58 if and only if an additional first recognition site for KalbTG is inserted at one of the positions identified by Table 59 A first recognition site for KalbTG is inserted after the position identified by 15. The modified antibody of any one of claims 1 to 14, comprising an antibody heavy chain constant region having the amino acid sequence:

16. The modified antibody has the sequence of SEQ ID NO: 02: Table 60 Table 61 a first recognition site for KalbTG is inserted after one or more positions identified by Table 62 if and only if an additional first recognition site for KalbTG is inserted at one of the positions identified by Table 63 A first recognition site for KalbTG is inserted after the position identified by 15. The modified antibody of any one of claims 1 to 14, comprising an antibody heavy chain constant region having the amino acid sequence:

17. The modified antibody has the sequence of SEQ ID NO: 03: Table 64 Table 65 a first recognition site for KalbTG is inserted after one or more positions identified by Table 66 if and only if an additional first recognition site for KalbTG is inserted at one of the positions identified by Table 67 A first recognition site for KalbTG is inserted after the position identified by 15. The modified antibody of any one of claims 1 to 14, comprising an antibody heavy chain constant region having the amino acid sequence:

18. The modified antibody has the sequence of SEQ ID NO: 04: Table 68 Table 69 a first recognition site for KalbTG is inserted after one or more positions identified by Table 70 if and only if an additional first recognition site for KalbTG is inserted at one of the positions identified by Table 71 A first recognition site for KalbTG is inserted after the position identified by 15. The modified antibody of any one of claims 1 to 14, comprising an antibody heavy chain constant region having the amino acid sequence:

19. The modified antibody has the sequence of SEQ ID NO: 05: Table 72 Table 73 a first recognition site for KalbTG is inserted after one or more positions identified by Table 74 if and only if an additional first recognition site for KalbTG is inserted at one of the positions identified by Table 75 A first recognition site for KalbTG is inserted after the position identified by 15. The modified antibody of any one of claims 1 to 14, comprising an antibody heavy chain constant region having the amino acid sequence:

20. The modified antibody of any one of claims 1 to 14, wherein the modified antibody comprises an antibody heavy chain constant region having the amino acid sequence of SEQ ID NO:

8.

21. The modified antibody of any one of claims 1 to 14, wherein the modified antibody comprises an antibody heavy chain constant region having the amino acid sequence of SEQ ID NO:

9.

22. The modified antibody of any one of claims 1 to 14, wherein the modified antibody comprises an antibody heavy chain constant region having the amino acid sequence of SEQ ID NO:

34.

23. The modified antibody has the sequence of SEQ ID NO: 06: Table 76 Table 77 a first recognition site for KalbTG is inserted after one or more positions identified by 23. The modified antibody of any one of claims 1 to 22, comprising an antibody light chain constant region having the amino acid sequence:

24. The modified antibody has the sequence of SEQ ID NO: 07: Table 78 Table 79 a first recognition site for KalbTG is inserted after one or more positions identified by 23. The modified antibody of any one of claims 1 to 22, comprising an antibody light chain constant region having the amino acid sequence:

25. The modified antibody of any one of claims 1 to 22, wherein the modified antibody comprises an antibody light chain constant region having the amino acid sequence shown in SEQ ID NO:

10.

26. The modified antibody of any one of claims 1 to 25, wherein the modified antibody specifically binds to a receptor that induces receptor-mediated endocytosis.

27. The modified antibody of any one of claims 1 to 26, 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).

28. A covalent conjugate comprising: (i) a modified antibody according to any one of claims 1 to 27; (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, A covalent conjugate wherein the non-antibody domain comprises a second recognition site for KalbTG.

29. the non-antibody domain is (i) a therapeutic entity; and (ii) a second recognition site for KalbTG that is complementary to the first recognition site present on the modified antibody; and (iii) optionally, a second linker between the therapeutic entity and the second recognition site; 29. The covalent conjugate of claim 28, comprising:

30. 30. The covalent conjugate of any one of claims 28 to 29, 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).

31. The covalent conjugate of any one of claims 29 to 30, wherein the second linker is an alkyl linker or a polyethylene linker or a peptide linker or a mixture thereof.

32. 28. A method for covalently conjugating a modified antibody according to any one of claims 1 to 27 to a therapeutic entity, said method comprising: a) providing a modified antibody according to any one of claims 1 to 27; b) providing a non-antibody domain, said non-antibody domain comprising: (i) a 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); (iii) optionally, a second linker between the therapeutic entity and the second recognition site for KalbTG; providing a non-antibody domain comprising: 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. A method comprising:

33. 33. The method of claim 32, wherein the second recognition site for KalbTG has the amino acid sequence of RYESK (SEQ ID NO: 16).

34. The KalbTG has the amino acid sequence (three letter code): Table 80 The modified antibody, modified Fc region, covalent conjugate and method of any one of claims 1 to 33, comprising:

35. A pharmaceutical composition comprising the covalent conjugate of any one of claims 29 to 31 and 34.

36. A covalent conjugate according to any one of claims 29 to 31 and 34, or a covalent conjugate produced according to the method of any one of claims 32 to 34, for use as a medicament.

37. 37. The use of claim 36, 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.

38. A nucleic acid or composition of nucleic acids encoding the modified antibody of any one of claims 1 to 27.

39. 39. A cell comprising the nucleic acid or nucleic acid composition of claim 38.

40. A method for producing a modified antibody according to any one of claims 1 to 27, comprising the steps of: - culturing the cells according to claim 39, - 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 to 27.

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