Modified antibodies for site-specific conjugation and diagnostic uses thereof

Abstract

The present invention relates to modified antibodies comprising a heavy chain and a light chain, one or more of whose immunoglobulin polypeptide chains have been modified to comprise one or more first recognition sites for transglutaminase from Kutsneria albida (KalbTG) or a functionally active variant thereof. The one or more first recognition sites are introduced at one or more selected positions within the antibody heavy chain and / or the antibody light chain. The present invention further relates to one or more nucleic acids encoding the immunoglobulin polypeptide chains comprising one or more recognition sites, site-specifically conjugated antibodies comprising the modified antibody and one or more label domains covalently linked to the one or more first recognition sites, kits for producing the conjugated antibodies, methods for specifically labeling the modified antibodies by site-specific conjugation, the use of the modified antibodies for producing the site-specifically conjugated antibodies, methods for detecting a target in a sample, and the use of the site-specifically conjugated antibodies in the detection and / or diagnosis of a target.

Description

[Technical field]

[0001] The present invention relates to modified antibodies comprising a heavy chain and a light chain, one or more of whose immunoglobulin polypeptide chains have been modified to contain one or more first recognition sites for transglutaminase from Kutsneria albida (KalbTG) or a functionally active variant thereof. The one or more first recognition sites are introduced at one or more selected positions within the antibody heavy chain and / or the antibody light chain. The present invention further relates to one or more nucleic acids encoding the immunoglobulin polypeptide chains comprising one or more recognition sites, site-specifically conjugated antibodies comprising the modified antibody and one or more labeling domains covalently linked to one or more first recognition sites, kits for producing the conjugated antibodies, methods for specifically labeling the modified antibodies by site-specific conjugation, in particular the use of the modified antibodies for producing the site-specifically conjugated antibodies, methods for detecting a target in a sample, and the use of the site-specifically conjugated antibodies in the detection and / or diagnosis of a target. [Background technology]

[0002] A diagnostic test is a procedure that is performed to confirm or determine the presence of a disease in an individual suspected of having the disease, usually after reporting symptoms or based on other medical test results. Quantification of a target substance, cell type, or another specific entity is a common output of, for example, most blood tests. This can answer not only whether the target entity is present or absent, but also how much is present. In blood tests, quantification is relatively well specified, such as given by mass concentration. Targets of interest are often detected by the use of target-specific labeled markers.

[0003] For this reason, target-specific antibody labels or antibody-enzyme conjugates are widely used in diagnostic tests. Many of the commercially available tests, such as the Roche Elecsys® assay, use conjugated or polymerized antibodies with multiple ruthenium labels per antibody as detection molecules.

[0004] Typically, these antibody-ruthenium conjugates are currently generated by random targeting of surface lysine residues using succinimide-based chemistry (non-site-specific conjugation). Over the past decade, many techniques for site-specific conjugation have emerged. Taken together, they result in products with more precise control over the number and insertion site of labels as well as improved homogeneity. These techniques include numerous chemical and enzymatic methods targeting engineered cysteines (e.g. ThioMab), ribosomally introduced unnatural amino acids, antibody-related carbohydrate chains or various engineered enzyme-recognition peptide tags (e.g. sortase, formylglycine generating enzyme or transglutaminase tags) (Agarwal and Bertozzi, 2015; Zhou and Kim, 2015). Different methods present different sets of advantages, disadvantages and limitations.

[0005] However, improved site-specific conjugation techniques remain the target of interest for many medical companies because of their potential use in the preparation of therapeutic antibody-drug conjugates as well as diagnostic antibody labels or antibody-enzyme conjugates.

[0006] Microbial transglutaminase from Streptomyces mobaraensis has emerged as an inexpensive and easy-to-use enzyme for protein cross-linking as well as site-specific protein labeling (Ando et al., 2014). It has also been used to generate IgG with drug loading at multiple different sites and show that the site of drug loading on IgG greatly influences the performance profile of antibody-drug conjugates (Strop et al., 2013).

[0007] The discovery of a novel transglutaminase (Kutschneria albida transglutaminase; KalbTG) and the identification of its respective peptide substrates have been described (Steffen et al., 2017). KalbTG catalyzes the formation of an isopeptide bond between a glutamine (Gln, Q) and a lysine (Lys, K) side chain. YRYRQ (SEQ ID NO: 14) was identified as the KalbTG Gln-containing motif (Q-tag) and RYESK (SEQ ID NO: 21) was identified as the KalbTG Lys-containing acceptor motif (K-tag). KalbTG shows similar efficiency but improved specificity and developability compared to other previously described microbial transglutaminases (mTG).

[0008] So far, only the IgG heavy chain C-terminus has been described as a suitable Q-tag insertion site for mTG for antibody labeling (WO 2021 / 174091). This limits the application of the method to two labeling rates per antibody and prevents investigating the label / drug performance dependency on the binding site. In order to be able to use KalbTG to label Roche Elecsys® detection antibodies with ruthenium / iridium, or to prepare antibody-drug / -enzyme conjugates in general, the KalbTG Q-tag needs to be engineered into the IgG backbone at one or more sites according to the desired labeling rate and the preferred labeling site. Internal labeling of the antibody may also reduce the mobility of the attached label and increase the compatibility of the labeled antibody. Summary of the Invention

[0009] To avoid the above mentioned drawbacks, the aim was to identify potential sites within IgG molecules for the incorporation of recognition sites for transglutaminase from Kutneria albida, and in particular for the incorporation of Q-tag motifs. The incorporation should not impair the folding or function of the antibody and should provide the necessary accessibility for the action of the enzyme. The successful identification of such sites allows the incorporation of not only two but multiple labeling molecules per IgG molecule, as the application requires in a controlled site-specific manner providing a more uniform conjugate product with better lot-to-lot consistency. Furthermore, molecules with single insertions at various sites allow the dependence of the labeling performance (e.g. signal or blank of ruthenium / iridium labeling, or pharmacodynamic / kinetic properties of the drug) on ​​the position within the IgG to be investigated, as well as preselected combinations that provide the desired properties (e.g. less ruthenium / iridium labeling quenching).

[0010] Antibody constant domain regions were screened for conjugation via KalbTG. In this context, KalbTG Q-tags were inserted into surface-exposed inter- and intra-domain flexible loops in the human IgG1 constant region as well as in the chain C-terminus. A total of 24 different sites were tested in the heavy and light chains of IgG. Each had the insertion of the KalbTG Q-tag motif within two flexible linkers (GGGSYRYRQGGGS) (SEQ ID NO: 28) and, in some cases, without flexible linker flanking (YRYRQ) (SEQ ID NO: 14). These molecules with single-site insertions were evaluated for their expression rate, folding success, stability and conjugation efficiency. Insertion sites that caused dramatic loss of yield or aggregation were excluded. To eliminate further inappropriately modified antibodies, the remaining modified antibodies were tested for conjugation and accessibility of the KalbTG Q-tag. The inventors have successfully identified several KalbTG Q-tag insertion sites spanning the length of the IgG backbone that provide enzymatic accessibility and do not adversely affect IgG cellular expression or folding. The 12 successful insertion sites are at positions 108 (LC108), 110 (LC110) and 214 (LC214) in the light chain and at positions 118 (HC118), 177 (HC177), 207 (HC207), 235 (HC235), 269 (HC269), 297 (HC297), 328 (HC328), 341 (HC341) and 401 (HC401) in the heavy chain (amino acid numbering according to the EU numbering scheme). Additionally, successful combinations of insertion sites have been identified as well.

[0011] Thus, in a first aspect, the present invention relates to a modified antibody comprising a heavy chain and a light chain, modified to comprise one or more first recognition sites for transglutaminase from Kutneria albida (KalbTG) by insertions, each insertion being located after one or more positions independently selected from positions 108 (LC108), 110 (LC110) and 214 (LC214) in the light chain and positions 118 (HC118), 177 (HC177), 207 (HC207), 235 (HC235), 269 (HC269), 297 (HC297), 328 (HC328), 341 (HC341) and 401 (HC401) in the heavy chain. Amino acid numbering is according to the EU numbering scheme of Kabat. Thus, in the modified antibodies of the present invention, the recognition site for KalbTG is located within the constant region of the Ig heavy chain polypeptide and / or the Ig light chain domain. In addition, the modified antibodies of the present invention may contain an additional first recognition site for KalbTG at position 446 (HC446) of the heavy chain, i.e., at the C-terminus. In the context of inserting a recognition site for KalbTG at a position, it means that the amino acid present at that position in the unmodified sequence is replaced by the recognition site or, preferably, is further inserted after that position.

[0012] In one embodiment, the modified antibody comprises at least one (cognate) pair of antibody heavy chain and antibody light chain, wherein the heavy chain comprises one or more recognition sites for transglutaminase from Kutneria albida (KalbTG), and the one or more recognition sites are at a position selected from the group consisting of position 118 (HC118), position 177 (HC177), position 207 (HC207), position 235 (HC235), position 269 (HC269), position 297 (HC297), position 328 (HC328), position 341 (HC341) and position 401 (HC401) of the heavy chain (amino acid numbering according to the EU numbering scheme of Kabat). In one embodiment, each position of the recognition site is independently selected.

[0013] In one embodiment, the modified antibody comprises at least one (cognate) pair of antibody heavy chains and antibody light chains, wherein the light chain comprises one or more recognition sites for transglutaminase from Kutneria albida (KalbTG), and the one or more recognition sites are at a position selected from the group consisting of position 108 (LC108), position 110 (LC110) and position 214 (LC214) of the light chain (amino acid numbering according to the EU numbering scheme of Kabat). In one embodiment, each position of the recognition site is independently selected.

[0014] The terms "antibody" and "Ig" are used interchangeably herein and are used in the broadest sense to specifically include, for example, individual monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full-length or intact monoclonal antibodies), monovalent antibodies, polyvalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific so long as they exhibit the desired binding activity and contain the recognition sites defined above), single chain antibodies, and fragments of antibodies.

[0015] Naturally occurring antibodies are generated by the assembly of heavy chains alone or heavy and light chains. Each heavy chain is composed of four domains: variable domain (VH), CH1, CH2, and CH3. The light chain is composed of a variable domain (VL) and a constant domain (CL). When heavy and light chains are present, the light chain is paired with a cognate heavy chain Fab fragment, which contains the VH and CH1 domains. The Fab fragments of related light and heavy chains are collectively referred to as a Fab fragment. The heavy chain CH2 and CH3 domains, together referred to as the heavy chain Fc region, dimerize with additional heavy chain CH2 and CH3 domains from a second chain to form an Fc region. The Fc region is connected to the Fab fragment through a flexible hinge region. The hinge region contains several disulfide bridges that covalently link the two heavy chain Fc regions to each other. In the Fab fragment, the light and heavy chains are also connected by one disulfide bridge. However, the connectivity differs between IgG subclasses. The overall structure of full-length IgG resembles a Y shape, with the Fc region forming the base and the two Fab fragments forming the arms available for binding to antigen.

[0016] Within the variable domains are loops called complementarity determining regions (CDRs). These are primarily responsible for the direct interaction of the antibody with its antigen. Due to the significant variation in the number of amino acids in these CDRs, there are multiple numbering schemes for the variable regions. 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 as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), and are referred to herein as "Kabat numbering." Specifically, the Kabat numbering system of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see pages 647-660) is used for the light chain constant domains CL of the kappa and lambda isotypes, and the Kabat EU index numbering system (see pages 661-723) is used for the heavy chain constant domains (CH1, hinge, CH2 and CH3, which is further clarified herein by reference to "numbering according to the Kabat EU index").

[0017] The term "modified antibody" as used herein refers to an antibody or antibody Fc region according to the present invention that contains at least one (artificial) internal Q-tag at a desired site. A modified antibody is capable of binding to a specific antigen and is composed of two pairs of polypeptide chains, each pair having one heavy chain and one light chain, each amino-terminal portion of each chain containing a variable region of about 100 to about 130 or more amino acids, and each carboxyl-terminal portion of each chain containing a constant region. Modified antibodies also include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinant antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, intrabodies, anti-idiotypic (anti-id) antibodies, and functional fragments thereof. The term "functional fragment" refers to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the modified antibody from which the fragment is derived, the functional fragment containing at least one internal Q-tag. Non-limiting examples of functional fragments of antibodies include single chain Fvs (scFvs) (e.g., including monospecific or bispecific), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, antibody / Fc fusion proteins, disulfide-linked Fvs (sdFvs), and the like. In particular, the modified antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as antigen-binding domains or molecules that contain an antigen-binding site that binds to an antigen (e.g., one or more complementarity determining regions (CDRs)). The antibodies provided herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY, preferably IgG), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecule. Antibodies can be human, humanized, chimeric and / or affinity matured, as well as antibodies from other species, such as goat, mouse, rat, rabbit or sheep.

[0018] According to the invention, the modified antibody comprises at least one heavy chain and at least one light chain. Thus, in certain embodiments, the modified antibody comprises one or two or three or four or more Fab domains. In one embodiment, the modified antibody is a monovalent monospecific antibody comprising one (full-length) light chain and one (full-length) heavy chain forming a cognate light-heavy chain pair (comprising one binding site) and one heavy chain Fc region fragment associated with the Fc region of the heavy chain. In another embodiment, the antibody comprises two Fab domains. In such an embodiment, the modified antibody is a bivalent monospecific antibody comprising two (full-length) light chains and two (full-length) heavy chains forming a cognate light-heavy chain pair (comprising an antigen binding site) and a heavy chain Fc region with each pair of heavy chain polypeptide domains. Additionally, modified multivalent antibodies having one or more first recognition sites for transglutaminase from Kutneria albida (KalbTG) are contemplated, wherein the unmodified multivalent antibody is any of the recombinantly obtainable antibodies disclosed in WO2019057816.

[0019] The invention provides modified antibodies comprising a heavy chain and a light chain modified to include one or more first recognition sites for transglutaminase from Kutneria albida (KalbTG) by insertions, each insertion being located after one or more positions independently selected from positions 108 (LC108), 110 (LC110) and 214 (LC214) of the light chain and positions 118 (HC118), 177 (HC177), 207 (HC207), 235 (HC235), 269 (HC269), 297 (HC297), 328 (HC328), 341 (HC341) and 401 (HC401) of the heavy chain. In one embodiment, the modified antibody includes an additional first recognition site for KalbTG at position 446 (HC446) of the heavy chain.

[0020] According to the invention, the antibody to be modified may be an IgG1, IgG2, IgG3 or IgG4 antibody, in particular a human, mouse, rabbit or sheep antibody. Exemplary suitable sequences are shown below, in which in the "X" annotation, "X" indicates an amino acid residue / position and " ↓ " indicates the site of insertion of a Q tag motif (with or without additional moieties such as, but not limited to, a label and a linker).

[0021] Human Heavy Chain Constant (IgG1) (SEQ ID NO:1) [ka] Human Heavy Chain Constant (IgG2) (SEQ ID NO:2) [ka] Human heavy chain constant (IgG3) (SEQ ID NO:3) [ka] Human heavy chain constant (IgG4) (SEQ ID NO: 4) [ka] Mouse Heavy Chain Constant (IgG1) (SEQ ID NO:5) [ka] Mouse Heavy Chain Constant (IgG2a) (SEQ ID NO:6) [ka] Mouse Heavy Chain Constant (IgG2b) (SEQ ID NO: 7) [ka] Rabbit Heavy Chain Constant (SEQ ID NO:8) [ka] Sheep heavy chain constant (SEQ ID NO:9) [ka] Human-Light-Chain-Constant (SEQ ID NO: 10) [ka] Mouse-Light-Constant (SEQ ID NO:11) [ka] Rabbit-Light-Constant (SEQ ID NO:12) [ka] Sheath-Light-Constant (SEQ ID NO: 13) [ka]

[0022] Thus, optionally, the Ig heavy chain constant region is a human Ig heavy chain constant region, such as a human IgG1, human IgG2, human IgG3 or human IgG4 heavy chain constant region. Optionally, the heavy chain constant region comprises at least one Q tag according to the invention.

[0023] Optionally, the Ig heavy chain constant region is a mouse Ig heavy chain constant region, such as a mouse IgG1, mouse IgG2a, or mouse IgG2b heavy chain constant region. Optionally, the Ig heavy chain constant region is a rabbit Ig heavy chain constant region or a sheep Ig heavy chain constant region. Optionally, the Ig light chain constant region is a human, mouse, rabbit, or sheep Ig light chain constant region.

[0024] In certain embodiments, the modified antibody comprises a heavy chain represented by any of the foregoing SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 9. The heavy chain of the antibody has been modified to contain one or more first recognition sites for transglutaminase from Kutneria albida (KalbTG) by insertions, each insertion being located after one or more positions indicated by an "X" annotation, where "X" indicates an amino acid residue / position, and " ↓ " indicates the site of insertion of the Q tag motif (not including binding moieties such as, but not limited to, linkers and labels).

[0025] Optionally, the human IgG1 heavy chain polypeptide on which the modified antibody may be based comprises a constant region amino acid sequence that is 75% or more, e.g., 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, up to 100% identical to the amino acid sequence shown in SEQ ID NO: 1. Optionally, the human IgG2 heavy chain polypeptide on which the modified antibody may be based comprises a constant region amino acid sequence that is 75% or more, e.g., 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, up to 100% identical to the amino acid sequence shown in SEQ ID NO: 2. Optionally, the human IgG3 heavy chain polypeptide on which the modified antibody may be based comprises a constant region amino acid sequence that is 75% or more, e.g., 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, up to 100% identical to the amino acid sequence shown in SEQ ID NO: 3. Optionally, the human IgG4 heavy chain polypeptide on which the modified antibody may be based comprises a constant region amino acid sequence that is 75% or more, for example 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, up to 100% identical to the amino acid sequence shown in SEQ ID NO: 4. Optionally, the mouse IgG1 heavy chain polypeptide on which the modified antibody may be based comprises a constant region amino acid sequence that is 75% or more, for example 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, up to 100% identical to the amino acid sequence shown in SEQ ID NO: 5. Optionally, the mouse IgG2a heavy chain polypeptide on which the modified antibody may be based comprises a constant region amino acid sequence that is 75% or more, for example 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, up to 100% identical to the amino acid sequence shown in SEQ ID NO: 6. Optionally, a mouse IgG2b heavy chain polypeptide on which the modified antibody may be based comprises a constant region amino acid sequence that is 75% or more, e.g., 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, up to 100% identical to the amino acid sequence set forth in SEQ ID NO: 7. Optionally, a rabbit heavy chain polypeptide on which the modified antibody may be based comprises a constant region amino acid sequence that is 75% or more, e.g., 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, up to 100% identical to the amino acid sequence set forth in SEQ ID NO:8.Optionally, the sheep heavy chain polypeptide on which the modified antibody may be based comprises a constant region amino acid sequence that is 75% or more, e.g., 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, up to 100% identical to the amino acid sequence set forth in SEQ ID NO: 9. Optionally, the human light chain polypeptide on which the modified antibody may be based comprises a constant region amino acid sequence that is 75% or more, e.g., 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, up to 100% identical to the amino acid sequence set forth in SEQ ID NO: 10. Optionally, the mouse light chain polypeptide on which the modified antibody may be based comprises a constant region amino acid sequence that is 75% or more, e.g., 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, up to 100% identical to the amino acid sequence set forth in SEQ ID NO: 11. Optionally, the rabbit light chain polypeptide on which the modified antibody can be based comprises a constant region amino acid sequence that is 75% or more, for example 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, up to 100% identical to the amino acid sequence shown in SEQ ID NO: 12. Optionally, the sheep light chain polypeptide on which the modified antibody can be based comprises a constant region amino acid sequence that is 75% or more, for example 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, up to 100% identical to the amino acid sequence shown in SEQ ID NO: 13. Even more preferably, the unmodified light chain comprises an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100% identical to the amino acid sequence of any of SEQ ID NOs: 10 to 13, and / or the unmodified heavy chain comprises an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100% identical to the amino acid sequence of any of SEQ ID NOs: 1 to 9.

[0026] In certain embodiments, the modified antibody comprises a light chain represented by any of SEQ ID NOs: 10, 11, 12, and 13, as described above. The light chain of the antibody has been modified to contain one or more first recognition sites for transglutaminase from Kutneria albida (KalbTG) by insertions, each insertion being located after one or more positions indicated by an "X" annotation, where "X" indicates an amino acid residue / position, and " ↓ " indicates the site for insertion of a Q tag motif (with or without binding moieties such as, but not limited to, a linker and label).

[0027] In a preferred embodiment, (i) the unmodified light chain constant domain (CL) comprises an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100% identical to the amino acid sequence of any one of SEQ ID NOs: 10 to 13, and / or (ii) any one of the unmodified heavy chain constant domains CH3, CH2 and CH1 comprises an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100% identical to the amino acid sequence of the respective domain in the amino acid sequence of SEQ ID NOs: 1 to 9, and / or (iii) the unmodified heavy chain constant domain comprises an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100% identical to the amino acid sequence of each of SEQ ID NOs: 1 to 9. and / or (vi) the unmodified light chain constant domain (CL) consists of an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100% identical to the amino acid sequence of any one of SEQ ID NOs: 10 to 13, and / or (v) any one of the unmodified heavy chain constant domains CH3, CH2 and CH1 consists of an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100% identical to the amino acid sequence of the respective domain in the amino acid sequence of SEQ ID NOs: 1 to 9, and / or (vi) the unmodified heavy chain constant domain consists of an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100% identical to the amino acid sequence of each of SEQ ID NOs: 1 to 9.

[0028] As detailed above, the antibody may be a bispecific or multispecific antibody. Exemplary bispecific or multispecific antibodies include: -Full length antibodies with domain swapping: A multispecific IgG antibody comprising a first Fab fragment and a second Fab fragment, wherein in the first Fab fragment: a) only the CH1 and CL domains are replaced with each other (i.e. the light chain of the first Fab fragment comprises the VL and CH1 domains and the heavy chain of the first Fab fragment comprises the VH and CL domains); b) only the VH and VL domains are replaced with each other (i.e. the light chain of the first Fab fragment comprises the VH and CL domains and the heavy chain of the first Fab fragment comprises the VL and CH1 domains); or c) the CH1 and CL domains are replaced with each other and the VH and VL domains are replaced with each other (i.e. the light chain of the first Fab fragment comprises the VH and CH1 domains and the heavy chain of the first Fab fragment comprises the VL and CL domains); and the second Fab fragment comprises a light chain comprising a VL domain and a CL domain, and a heavy chain comprising a VH domain and a CH1 domain; A full-length antibody with domain swapping may comprise a first heavy chain comprising a CH3 domain and a second heavy chain comprising a CH3 domain, both CH3 domains being complementarily engineered by respective amino acid substitutions to support heterodimerization of the first heavy chain and the modified second heavy chain; -Full-length antibody with domain swapping and additional heavy chain C-terminal binding site: A multispecific IgG antibody, comprising: a) a full-length antibody comprising two pairs of full-length antibody light chains and full-length antibody heavy chains, wherein a binding site formed by each pair of full-length heavy chains and full-length light chains specifically binds to a first antigen; b) one further Fab fragment, fused to the C-terminus of one heavy chain of the full-length antibody, wherein the binding site of the further Fab fragment specifically binds to a second antigen; and Including, a multispecific IgG antibody, wherein the further Fab fragment which specifically binds to the second antigen i) comprises a domain crossover such that a) the light chain variable domain (VL) and the heavy chain variable domain (VH) are replaced by each other, or b) the light chain constant domain (CL) and the heavy chain constant domain (CH1) are replaced by each other, or ii) is a single chain Fab fragment; -1-arm single chain format (=1-arm single chain antibody): An antibody comprising a first binding site that specifically binds to a first epitope or antigen and a second binding site that specifically binds to a second epitope or antigen, wherein the individual chains are as follows: -Light chain (variable light domain + constant light kappa domain) -Light / heavy chain combination (variable light domain + light constant domain + peptide linker + variable heavy domain + CH1 + hinge + CH2 + CH3 with knob mutation) -heavy chain (variable heavy domain + CH1 + hinge + CH2 + CH3 with hole mutation); -2-arm single chain format (=2-arm single chain antibody): An antibody comprising a first binding site that specifically binds to a first epitope or antigen and a second binding site that specifically binds to a second epitope or antigen, wherein the individual chains are as follows: - Light / heavy chain combination 1 (variable light domain + light chain constant domain + peptide linker + variable heavy domain + CH1 + hinge + CH2 + CH3 with hole mutation); - light / heavy chain combination 2 (variable light domain + light chain constant domain + peptide linker + variable heavy domain + CH1 + hinge + CH2 + CH3 with knob mutation); -Common light chain bispecific formats (=common light chain bispecific antibodies): An antibody comprising a first binding site that specifically binds to a first epitope or antigen and a second binding site that specifically binds to a second epitope or antigen, wherein the individual chains are as follows: -Light chain (variable light domain + constant light domain) -Heavy Chain 1 (VH domain + CH1 + hinge + CH2 + CH3 with hole mutation) - Heavy chain 2 (variable heavy domain + CH1 + hinge + CH2 + CH3 with knob mutation).

[0029] As used herein, the term "replaced by each other" with respect to corresponding heavy and light chain domains refers to the aforementioned domain crossover. Thus, when CH1 and CL domains are "replaced by each other", this term refers to the domain crossover referred to under item (i) and the resulting heavy and light chain domain sequences. Thus, when VH and VL are "replaced by each other", this term refers to the domain crossover referred to under item (ii), and when CH1 and CL domains are "replaced by each other" and VH and VL domains are "replaced by each other", this term refers to the domain crossover referred to under item (iii).

[0030] In one embodiment, the multispecific antibody also comprises at least one Fab fragment comprising either the domain crossover of CH1 and CL domains described in the above item (i), or the domain crossover of VH and VL domains described in the above item (ii), or the domain crossover of VH-CH1 and VL-VL domains described in the above item (iii). In the case of a multispecific antibody with domain crossover, Fabs that specifically bind to the same antigen are constructed to have the same domain sequence. Thus, when more than one Fab with domain crossover is included in a multispecific antibody, the Fabs specifically bind to the same antigen.

[0031] Multivalent IgG derivatives such as those disclosed in WO2019057816 can be designed with CH1-VH domains fused either to the C-terminus or N-terminus of the IgG heavy chain, which together with the associated light chain (CL-VL) form additional binding sites, thus turning the bivalent IgG into a tetravalent or hexavalent antibody. Similarly, further C- or N-terminal additions result in longer heavy chain fusion constructs, thus allowing even greater multivalency. Importantly, since the domains in these constructs correspond to those with the Q-tag insertion sites shown herein, a multivalent IgG derivative with an additional C-terminally or N-terminally added CH1-VH domain is a further embodiment of a modified antibody comprising a heavy chain and a light chain, which has been modified to comprise one or more first recognition sites for transglutaminase from Kutneria albida (KalbTG) by insertions, each insertion being located after one or more positions independently selected from position 108 (LC108), position 110 (LC110) and position 214 (LC214) in the light chain and position 118 (HC118), position 177 (HC177), position 207 (HC207), position 235 (HC235), position 269 (HC269), position 297 (HC297), position 328 (HC328), position 341 (HC341) and position 401 (HC401) in the heavy chain.

[0032] In one embodiment, the antibody comprises two pairs of heavy and light chains, each of which comprises one or more first recognition sites. In another embodiment, the antibody comprises three or more pairs of heavy and light chains, each of which comprises one or more additional Fab domains VH and CH1 heavy chain fragments fused to either the VH N-terminus or the Fc C-terminus of the heavy chain, and each of the fusion polypeptides or / and light chains comprises one or more first recognition sites. In yet another embodiment, the antibody comprises three or more pairs of heavy and light chains, each of which comprises one or more additional Fab domains VL and CL light chain fragments fused to either the VH N-terminus or the Fc C-terminus of the heavy chain, and each of the fusion polypeptides or / and light chains or / and Fab domains VH and CH1 heavy chain fragments comprises one or more first recognition sites.

[0033] The term "antigen" refers to a predetermined target to which an antibody can selectively bind. An antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten or fragment thereof, or other naturally occurring or synthetic compound. In one embodiment, the target antigen is a polypeptide. In another embodiment, the target antigen is related to diagnosis.

[0034] According to the present invention, the modified antibody comprises one or more recognition sites for KalbTG in the heavy and / or light chain polypeptides. The recognition site comprises a motif of KalbTG, which can catalyze the formation of an isopeptide bond between the modified antibody and a compound that binds to the antibody. Typically, it is formed between a glutamine (Gln) side chain and a lysine (Lys) side chain. In certain embodiments, the Lys side chain can be replaced with a primary amine compound that can act as a functional analog of the peptidic KalbTG K-tag. Preferably, the modification of the antibody to generate the modified antibody of the present invention comprises the generation of a Q-tag in the antibody, i.e., a Gln-containing motif recognized by KalbTG. Suitable Q-tags are disclosed in WO 2017 / 102759 et seq. The Q-tag can be generated by one or more amino acid modifications, such as substitutions or insertions, preferably insertions. In certain embodiments, the one or more first recognition sites, independently of each other, comprise or have a Gln-containing motif. More preferably, the Q tag is generated by insertion and / or substitution of an amino acid sequence comprising any of the sequences of SEQ ID NOs: 14-20, in particular YRYRQ (SEQ ID NO: 17) or RVRQR (SEQ ID NO: 18), in particular YRYRQ (SEQ ID NO: 17). The introduction of one or more recognition sites for KalbTG may be the only modification in the constant regions of the light and / or heavy chains. Alternatively, further modifications such as tags for purification (e.g. His tags) or other modifications, e.g. increased stability or heterodimerization or modifications of effector function, may be present alone or in any combination.

[0035] More preferably, the Q-tag is generated by insertion of an amino acid sequence comprising YRYRQ (SEQ ID NO: 14) or RVRQR (SEQ ID NO: 15), in particular YRYRQ (SEQ ID NO: 14). Preferably, the inserted amino acid sequence has a length of 5 to 20 amino acids and comprises YRYRQ (SEQ ID NO: 14) or RVRQR (SEQ ID NO: 15), in particular YRYRQ (SEQ ID NO: 14). In a particular embodiment, the insertion is a Q-tag motif without a linker, i.e., the insertion is YRYRQ (SEQ ID NO: 14) or RVRQR (SEQ ID NO: 15), in particular (YRYRQ) (SEQ ID NO: 14). The introduction of one or more recognition sites for KalbTG may be the only modification in the constant regions of the light and / or heavy chains. Alternatively, further modifications such as a tag for purification (e.g. His-tag) or other modifications, such as increasing stability or heterodimerization or modifying effector functions, may be present alone or in any combination.

[0036] As mentioned above, one or more first recognition sites for KalbTG are inserted at specific sites of the antibody, i.e. at one or more positions selected from position 108 (LC108), position 110 (LC110) and position 214 (LC214) of the light chain and position 118 (HC118), position 177 (HC177), position 207 (HC207), position 235 (HC235), position 269 (HC269), position 297 (HC297), position 328 (HC328), position 341 (HC341) and position 401 (HC401) of the heavy chain. Furthermore, the modified antibody of the invention may contain an additional first recognition site for KalbTG at position 446 (HC446) of the heavy chain, i.e. at the C-terminus. Amino acid numbering is according to the established EU numbering scheme (Kabat, 1991). When a recognition site is inserted into the amino acid sequence of the light or heavy chain, it is inserted immediately after the position specified above. The one or more first recognition sites inserted can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more recognition sites inserted. The recognition sites can be inserted into one, two, three or four chains of the antibody. For example, the one or more insertions can be - Only one heavy chain, or - Only one light chain, or - both heavy chains, or - both light chains, or - one heavy chain and one light chain, or - one heavy chain and two light chains, or - two heavy chains and one light chain, or - 2 heavy chains and 2 light chains, may exist in

[0037] When two light chains or two heavy chains are modified, the inserted recognition sites can be at different or identical positions for the two chains. For example, both heavy chains can have a first recognition site inserted at position 328 (HC328) (same position), or one heavy chain has a first recognition site inserted at position 328 (HC328) and the other heavy chain has a first recognition site inserted at position 341 (HC341) (different positions). As detailed above, it may be preferable for multiple first recognition sites to be present in one antibody to increase the number of potential labeling sites in the antibody.

[0038] In a preferred embodiment of the invention, the modified antibody according to the first aspect of the invention comprises: a) two first recognition sites for KalbTG, in particular -HC118 / HC177; or -HC118 / HC328; or -HC118 / HC341; or -HC118 / HC401; or -HC177 / HC207; or -HC177 / HC328; or -HC177 / HC341; or -HC177 / HC401; or -HC207 / HC328; or -HC207 / HC341; or -HC207 / HC401; or -HC328 / HC446; or -HC341 / HC446; or -HC118 / LC214; or -HC177 / LC108; or -HC177 / LC214; or -HC207 / LC214; or -HC446 / LC214 At the position especially -HC118 / HC177; or -HC118 / HC328; or -HC118 / HC341; or -HC177 / HC341; or -HC207 / HC341; or -HC328 / HC446; or -HC341 / HC446; or -HC118 / LC214; or -HC177 / LC108; or -HC177 / LC214; or -HC207 / LC214; or -HC446 / LC214 At the position or b) three primary recognition sites for KalbTG, in particular -HC118 / HC235 / HC341; or -HC207 / HC235 / HC341; or -HC118 / HC341 / HC446; or -HC207 / HC341 / HC446; or -HC118 / HC235 / HC328; or -HC177 / HC235 / HC328; or -HC207 / HC235 / HC328 At the position or c) four primary recognition sites for KalbTG, in particular -HC118 / HC235 / HC341 / HC446; or -HC207 / HC235 / HC341 / HC446 Includes at the position.

[0039] As detailed above, the modified antibody comprises one or more recognition sites for KalbTG in an immunoglobulin (Ig) heavy chain polypeptide and / or an immunoglobulin (Ig) heavy chain polypeptide. The recognition site comprises a motif of KalbTG, which can catalyze the formation of an isopeptide bond between the modified antibody and a compound that binds to the antibody. Typically, the isopeptide bond is formed between a glutamine (Gln) side chain and a lysine (Lys) side chain. However, the K tag is not entirely limited to the presence of a Lys moiety, since it has been found that some primary amines can functionally replace the Lys acceptor site. Preferably, the modification of the antibody to generate the modified antibody of the invention comprises the generation of a Q tag in the antibody, i.e., a Gln-containing motif recognized by KalbTG. Suitable Q tags are disclosed in WO 2017 / 102759. Examples include YRYRQ (SEQ ID NO: 14), YRQRT (SEQ ID NO: 16), RYGQR (SEQ ID NO: 17), RWRQR (SEQ ID NO: 18), RVRQR (SEQ ID NO: 15), IRQRQ (SEQ ID NO: 19) and FRYRQ (SEQ ID NO: 20), in particular YRYRQ (SEQ ID NO: 14). Q-tags can be generated by one or more amino acid modifications, such as substitutions or insertions, preferably insertions. The one or more first recognition sites, independently of each other, comprise or have a Gln-containing motif. More preferably, Q-tags are generated by insertion and / or substitution of an amino acid sequence comprising any of the sequences of SEQ ID NOs: 14-20, in particular YRYRQ (SEQ ID NO: 14) or RVRQR (SEQ ID NO: 15), in particular YRYRQ (SEQ ID NO: 14). Preferably, the inserted amino acid sequence has a length of 5 to 20 amino acids and comprises YRYRQ (SEQ ID NO: 14) or RVRQR (SEQ ID NO: 15), in particular YRYRQ (SEQ ID NO: 14). In certain embodiments, the insertion is a Q tag motif without a linker, ie, the insertion is YRYRQ (SEQ ID NO: 14) or RVRQR (SEQ ID NO: 15), in particular (YRYRQ) (SEQ ID NO: 14).

[0040] In a preferred embodiment, the Q-tag is inserted into the antibody light / heavy chain amino acid sequence via one or two linkers. The linker may increase flexibility or allow attachment of larger labels. Preferably, the linker sequence comprises 1 to 20 amino acids, preferably 1 to 10 amino acids, more preferably 1 to 5 amino acids, more preferably said amino acids do not essentially interfere with the function of the Q-tag, KalbTG, antibody folding, and the label attached to the modified antibody. The linker may be attached N-terminally and / or C-terminally to the Q-tag. Preferred linker amino acids are small amino acids such as glycine or alanine. Amino acid linkers and their compositions are known in the art (see, for example, Chichili et al. Linkers in the structural biology of protein-protein interactions Protein Sci. 2013 February;22(2):153-167). The amino acids glycine, serine, alanine, threonine and glutamic acid usually constitute the amino acids of flexible linkers. Thus, the linker may consist mainly or completely of Gly and / or Ser and / or Ala and / or Thr and / or Glu, e.g., GGGP, ESGS or APAP. There may also be linkers that comprise or consist of KESGSVSSEQLAQFRSLD (SEQ ID NO: 26) and EGKSSGSGSESKST (SEQ ID NO: 27). Linkers consisting mainly or completely of Gly and Ser, e.g., where m=1, 2, 3 or 4 and n=1, 2, 3, 4 or 5 (Gly m Ser) n is particularly preferred, where m and n are independent of each other, preferably m = 3 and n = 1. Preferably, the first recognition site or any one of the two or more first recognition sites is linked to the antibody at one or both ends of each first recognition site via one or two linkers, in particular the linkers are Gly and Ser, e.g. (Gly-Gly-Gly-Ser) where n = 1, 2, 3, 4 or 5, preferably n = 1. n(SEQ ID NO:22), or in particular the linker comprises or consists of GGGP (SEQ ID NO:23), ESGS (SEQ ID NO:24) or APAP (SEQ ID NO:25). In embodiments having multiple linkers, the amino acid sequence of each linker is independently selected.

[0041] If a linker is present, it is preferred to insert the amino acid sequence X1-YRYRQ-X2 (SEQ ID NO: 14) or X1-RVRQR-X2 (SEQ ID NO: 15) into the antibody. X1 and X2, independently of each other, are absent or a linker, in particular a linker amino acid. In a particular embodiment, the insertion is a Q-tag motif with two flexible linkers, in particular (GGGSYRYRQGGGS) (SEQ ID NO: 28) or GGGSRVRQRGGGS (SEQ ID NO: 29), in particular GGGSYRYRQGGGS) (SEQ ID NO: 28).

[0042] In a highly preferred embodiment, the light chain constant domain comprises or consists of an amino acid sequence that is at least 96%, 97%, 98% or 99%, in particular 100% identical to an amino acid sequence of any of SEQ ID NOs: 10 to 13, and / or the heavy chain constant domain comprises or consists of an amino acid sequence that is at least 96%, 97%, 98% or 99%, in particular 100% identical to an amino acid sequence of SEQ ID NOs: 1 to 9.

[0043] In a second aspect, the present invention relates to a nucleic acid encoding the polypeptide chains of a modified antibody of the present invention.

[0044] Nucleic acids encoding modified antibodies of the invention can be isolated or generated in vitro for recombinant production of the antibodies. The nucleic acids can be isolated and inserted into replicable vectors for further cloning (amplification of the DNA) or for further expression.

[0045] The term "nucleic acid" encompasses DNA (gDNA and cDNA) and RNA molecules, and the basic structural unit of nucleic acid, nucleotide, includes not only natural nucleotides but also analogs having modified sugars or base moieties. The sequences of the nucleic acids encoding the heavy chain variable region and light chain variable region of the present invention may be modified. Such modifications include addition, deletion, or non-conservative or conservative substitution of nucleotides, as long as the encoded sequence is not changed.

[0046] DNA encoding the antibody is readily isolated or synthesized using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to DNA encoding the heavy and light chains of the antibody). Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0047] As used herein, the term "vector" refers to a plasmid vector, a cosmid vector, a viral vector, such as a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector, as a means for expressing a gene of interest in a host cell. The nucleic acid encoding the modified antibody in the vector is operably linked to a promoter.

[0048] "Operably linked" refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence, where the control sequence is another nucleic acid and controls the transcription and / or translation of the sequence.

[0049] When a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, etc.), a ribosome binding site for conversion initiation, and a transcription / translation termination sequence are used. In addition, for example, when the host is a eukaryotic cell, a promoter derived from the genome of a mammalian cell (e.g., metallothionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter), a mammalian promoter derived from an animal virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter), Moloney virus promoter, Epstein-Barr virus (EBV) promoter, Rous sarcoma virus (RSV) promoter can be used, and these promoters generally have a polyadenylation sequence as a transcription termination sequence.

[0050] In some cases, the vector may be fused to other sequences to facilitate purification of the antibodies expressed therefrom, such as glutathione S-transferase (USP), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6xHis (hexahistidine; Qiagen, USA).

[0051] The vectors contain antibiotic resistance genes commonly used in the art as selection markers, for example, for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline. Resistance genes are present.

[0052] The cells may be transformed with the above-mentioned 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.

[0053] Prokaryotic host cells such as Escherichia coli, Bacillus strains, such as Bacillus subtilis, Bacillus thuringensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, and Staphylococcus (e.g., Staphylococcus carnosus) can be used.

[0054] However, of greatest interest are animal cells and examples of useful host cell lines include, but are not limited to, COS-7, BHK, CHO, CHO-S, CHOK1, GS-CHO, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S or HT1080.

[0055] In a third aspect, the present invention relates to a site-specifically conjugated antibody, which comprises a conjugated antibody of the present invention (as defined above) and (i) one or more labeling domains, each of which comprises a second recognition site for KalbTG or a functionally active variant thereof, wherein the second recognition site is covalently linked to a first recognition site for KalbTG or a functionally active variant thereof present in the modified antibody, and the labeling domain comprises a detectable label; or (ii) one or more chemical linker moieties, each of the one or more covalently attached chemical linker moieties comprising a second recognition site for KalbTG or a functionally active variant thereof, the second recognition site being covalently attached to the first recognition site of the antibody, the chemical linker moieties comprising a functional group capable of forming a covalent bond in a chemical (non-enzymatic) coupling reaction, in particular a functional group with a reaction partner of the carbodiimide or N-hydroxysuccinimide coupling chemistry or with a reaction partner of the so-called "click" chemistry, in particular a functional group with an azide or alkyne moiety; Includes.

[0056] In this regard, "click" chemistry refers to a group of reactions that are fast, simple to use, easy to purify, versatile, regiospecific, and give high product yields. Although there are many reactions that meet the criteria, the Huisgen 1,3-dipolar cycloaddition of azides and terminal alkynes, especially cyclic alkynes, is widely used. "Click" chemistry applications exist in a wide variety of research fields, including materials science, polymer chemistry, and especially biochemistry and pharmaceutical science. In certain embodiments, the chemical linker moiety comprises an alkyne or azide group that can be used as a reactive partner in the "click" reaction. Among the various known "click" chemistries, the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition of azides and terminal alkynes to form 1,2,3-triazoles is one of the most frequently reported examples. This reaction forms exclusively 1,4-substituted products, making it regiospecific. The reaction typically does not require temperature elevation, but can be carried out over a wide range of temperatures (0-160°C), in a variety of solvents (including water), and over a wide range of pH values ​​(5-12). The reaction proceeds up to 107 times faster than the uncatalyzed version, and purification is usually easy to carry out. Furthermore, the reaction does not appear to be affected by steric factors, i.e., the substituents attached to the reactive partners in the "click" reaction. All of these features make this cycloaddition particularly popular among the other click reactions mentioned above.

[0057] In one embodiment, the functional group with a so-called "click" chemistry reaction partner is an azide group that can undergo a "click" reaction using an alkyne or a phosphine as a reactant. In particular, cyclooctyne derivatives are known to those skilled in the art for the modification of biomolecules under mild conditions (WO 2006 / 050262). In a particular embodiment. Thus, the site-specifically conjugated antibody comprises a chemical linker moiety covalently attached (via a K tag or a functional equivalent thereof) to (i) an alkyne or (ii) an azide reaction partner that can undergo a "click" reaction with the respective "click" reaction partner.

[0058] As detailed above, the modified antibodies of the present invention are provided by using KalbTG to specifically link one or more label domains to one or more internal sites of the antibody, thus producing specifically labeled antibodies that can be used to detect a target of interest. The modified antibodies can bind to a target antigen of interest and be detected via one or more labels attached thereto. This allows the target of interest to be detected and / or quantified.

[0059] Label detection methods are well known and frequently used in the field of in vitro diagnostics. Exemplary methods include fluorescent, isotopic, chemiluminescent, chemiluminescent and nanoparticle labels. Fluorescent label detection methods are the most common and convenient technique for transferring information from molecular events. Fluorescent probes are stable, easy to operate and provide good sensitivity and resolution. In antibody sandwich assay systems, fluorophores are used as secondary antibody labels. In this way, direct non-native effects on the molecule of interest can be omitted. Fluorescent labels are now available in many forms, from quantum dots and small organic molecules to fluorescent proteins with a range of brightness and specificity that can be selected as needed. The advent of advanced fluorescence techniques such as fluorescence resonance energy transfer (FRET), bimolecular fluorescence complementation (BiFC), and fluorescence correlation spectroscopy (FCS) has allowed the use of smaller samples with lower detection limits. Exemplary labels include fluorescein, rhodamine and cyanine derivatives. One of the earliest methods for molecular detection involved the use of radioisotopes, which allowed for precise quantification of the target. Although radioactivity is suitable due to its sensitivity and specificity and the possibility of fluorescent detection, the use of isotopically labeled molecules raises several safety concerns. However, the labels used include: 3 H, 32 P, 35 S and 125I. Chemiluminescence is another phenomenon useful for converting molecular interactions into analyzable color information. When a molecule obtains its energy from an exothermic reaction, different wavelengths are emitted during its ground state during molecular relaxation depending on the amount of energy obtained. Heterogeneous methods are the more widely used chemiluminescence assays. Chemiluminescence methods can be direct (using a luminophore marker) or indirect (using an enzyme marker). In direct chemiluminescence methods, the luminophore labels used are typically acridinium and ruthenium esters, while enzyme labels used in indirect methods include alkaline phosphatase with adamantyl 1,2-dioxetane aryl phosphate (AMPPD) substrate and horseradish peroxidase with luminol or its derivatives as substrate. Detectable labels can also be indirect labels, the visualization of which requires a secondary reporter molecule. Those skilled in the art are further familiar with binding pairs consisting of a first and a second binding partner, which can be formed by specific non-covalent interactions between the first and second binding partners. Well-known exemplary binding pairs comprising such first and second binding partners include: - antigens and antigen-specific antibodies; - haptens and hapten-specific antibodies; -Ligands and specific ligand binding domains; - an oligo- or polysaccharide and a lectin, where the lectin is capable of specifically binding to the oligo- or polysaccharide; -Histidine tag and Zn 2+ , Ni 2+ , Co 2+ , and Cu 2+ a metal chelate complex comprising a metal ion selected from the group consisting of a histidine tag and a metal chelate complex, wherein the metal chelate complex is capable of binding to the histidine tag; - indium chelate complex and CHA255 antibody; - host residues of Cucurbitaceae[n]uril and guest residues capable of binding to host residues; - a first and a second protein dimerization domain, optionally in the presence of a dimerization inducer or enhancer; and - a first and a second oligonucleotide spiegelmer, each consisting of an L-ribose or L-2'-deoxyribose containing nucleoside monomer, wherein the first oligonucleotide spiegelmer is capable of hybridizing with the second oligonucleotide spiegelmer; - a first and a second oligomer consisting of a beta-L-LNA nucleoside monomer, the first oligomer being capable of hybridizing to the second oligomer; The label can therefore generally be a single-stranded oligonucleotide or an analogue thereof. However, single-stranded all-L-LNA oligomers as described in WO 2019 / 243391 and WO 2020 / 245377 are particularly advantageous as labels. The conjugated single-stranded L-LNA oligomer as a label can be bound by a further single-stranded L-LNA oligomer that has a complementary sequence and can hybridize with the label oligomer. The further single-stranded L-LNA oligomer in an exemplary case is coupled to a moiety that can generate a detectable signal.

[0060] Biotin, also known as vitamin H, is often used as a label. Biotin can be detected and visualized using the highly efficient biotin binder streptavidin. Thus, biotinylated compounds are easily detected via streptavidin carrying either a reporter enzyme or a fluorescent dye. However, endogenous biotin from biological samples often interferes with biotin detection, so the use of digoxigenin, a steroid present only in digitalis plants, may be preferred. Visualization of incorporated digoxigenin is achieved in a similar manner by digoxigenin antibody conjugates with reporter enzymes or fluorescent dyes.

[0061] The choice of label and detection method is generally determined by the particular downstream application. While speed of detection and ease of quantification are the main advantages of fluorescent labels, some indirect methods using secondary reporter molecules allow signal amplification with a resulting increase in sensitivity. The above methods can be competitive or non-competitive and include sandwich immunoassays and competitive immunoassays.

[0062] In a highly preferred method, the site-specifically conjugated antibody is labeled with a label that allows electrochemiluminescence detection, such as luminol, acridinium ester, ruthenium ester, iridium ester, or especially ruthenium or iridium ester. In particular, a label selected from any of a biotin moiety, a fluorescent dye, an iridium label, a ruthenium label, a radioactive label, a single-stranded oligonucleotide or analogue thereof, and a chemiluminescent label is covalently attached to a chemical linker.

[0063] In a preferred embodiment, a) the label or label domain is suitable for detection by an enzyme-mediated reaction, such as an enzyme-mediated chromogenic, fluorogenic or metallographic reaction, or by direct or indirect fluorimetry, or by radiometry, or by electrochemiluminescence; and / or b) the label or label domain is or comprises an enzyme, an enzyme substrate, a chromophore, a fluorophore, a quencher, a radiolabel, biotin, a metal, or an electrochemiluminescent moiety, in particular an electrochemiluminescent moiety containing iridium or ruthenium; and / or c) Each site-specifically conjugated antibody is labeled with at least one, at least two, at least three, at least four, at least five, at least six, at least seven or at least eight label domains, in particular at least two, at least four, at least six or at least eight labels or label domains.

[0064] These labels and detection methods are well known in the art.

[0065] In one embodiment of the site-specifically conjugated antibody of the present invention, the linker is a monofunctional or bifunctional chemical linker moiety that allows the covalent attachment of one or two additional reactive compounds, respectively. A conjugate refers to a compound in which two or more compounds are covalently linked. Two or more compounds can be covalently linked by a bifunctional linker, where a first covalent bond is formed between a first reactive group of the linker and the first compound, and a second covalent bond is formed between a second reactive group of the linker and the second compound.

[0066] In a fourth aspect, the present invention provides a kit for producing a site-specifically conjugated antibody, the kit comprising a modified antibody of the invention (as defined above); (i) KalbTG or a functionally active variant thereof, (ii) a labeling domain capable of binding to one or more first recognition sites by KalbTG or a functionally active variant thereof, the labeling domain comprising a detectable label; and (iii) a chemical linker moiety capable of being attached to one or more first recognition sites by KalbTG or a functionally active variant thereof, which comprises a functional group capable of forming a covalent bond in a chemical (non-enzymatic) coupling reaction, in particular a functional group with a reaction partner of the carbodiimide or N-hydroxysuccinimide coupling chemistry or with a reaction partner of the so-called "click" chemistry, in particular an azide or alkyne moiety; Includes.

[0067] Microbial transglutaminase (MTG), including KalbTG, catalyzes the formation of Gln-Lys isopeptide bonds and is widely used for cross-linking of proteins and peptides in food and biotechnology applications (e.g., to improve the texture of protein-rich foods or in generating antibody-drug conjugates). KalbTG shows essentially no cross-reactivity with known MTG substrates or commonly used target proteins, such as antibodies, thus allowing specific labeling at a predefined site. Thus, essentially any label domain comprising a detectable label and a second recognition site for KalbTG, particularly where the second recognition site comprises or has a Lys-containing motif (K-tag), in particular the sequence RYESK (SEQ ID NO: 21), can be conjugated to a modified antibody at one or more first recognition sites. KalbTG or its functionally active variants can be described in Steffen et al., 2017 or WO 2016 / 100735. The functionally active variant may be a transglutaminase having at least 80%, 90%, 95% or 100% sequence identity to KalbTG of WO 2016 / 100735 (see SEQ ID NO: 6). Alternatively, KalbTG or a functionally active variant thereof may be part of a fusion protein that additionally comprises a label, such as a tag, for example for purification purposes.

[0068] The label domain comprises a detectable label and a second recognition site for KalbTG. The detectable label can be any label as defined above, for example in the context of the third aspect of the invention. Suitable labels include biotin moieties, fluorescent dyes, iridium labels, ruthenium labels, radioactive labels, single-stranded oligonucleotides or analogs thereof, and chemiluminescent labels. For the purposes of this disclosure, it is understood that the detectable label forming part of the label domain does not block the second recognition site of KalbTG from accessing the active center of KalbTG, but also allows the second recognition site to contact the first recognition site. Thus, the second recognition site can be optionally linked to the detectable label via a linker that covalently links the detectable label to the second recognition site.

[0069] In a fifth aspect, the present invention provides a method for site-specific conjugation of a modified antibody, the method comprising the steps of: a) providing a modified antibody of the invention (as defined above); b) providing a labeling domain, the labeling domain comprising (i) a detectable label, (ii) a second recognition site for KalbTG, in particular a motif having a primary amine group, more particularly a Lys-containing motif (K tag), in particular the second recognition site comprising or having the sequence RYESK (SEQ ID NO: 21), and (iii) optionally a linker between the label and the second recognition site; c) reacting the modified antibody of a) with the label domain of b) in the presence of KalbTG or a functionally active variant thereof and under conditions that promote the activity of KalbTG to form an isopeptide bond between the first recognition site and the second recognition site, thereby site-specifically conjugating the modified antibody; Including, or a) providing a modified antibody of the invention (as defined above); b) providing a chemical linker moiety, which comprises (i) a functional group capable of forming a covalent bond in a chemical (non-enzymatic) coupling reaction, in particular a functional group with a reaction partner of the carbodiimide or N-hydroxysuccinimide coupling chemistry or with a reaction partner of the so-called "click" chemistry, in particular a functional group with an azide or alkyne moiety, and (ii) a second recognition site for KalbTG, in particular a second recognition site which comprises or has a motif bearing a primary amine group, more particularly a Lys-containing motif, in particular the sequence RYESK (SEQ ID NO: 21); c) reacting the modified antibody of a) with the chemical linker moiety of b) in the presence of KalbTG or a functionally active variant thereof and under conditions that promote the activity of KalbTG to form an isopeptide bond between the first recognition site and the second recognition site, thereby site-specifically conjugating the modified antibody; Includes.

[0070] In this method, a modified antibody and a labeling domain of the invention are provided. In a first alternative, the labeling domain comprises (i) a detectable label, (ii) a second recognition site for KalbTG, in particular the second recognition site comprises or has a Lys-containing motif (K tag), in particular the sequence RYESK (SEQ ID NO: 21). The labeling domain may be as defined above. The modified antibody and the labeling domain react in the presence of KalbTG or a functionally active variant thereof and under conditions that promote the activity of KalbTG, thereby forming an isopeptide bond between the first and second recognition sites, thus specifically labeling the modified antibody. Preferably, in this method according to the invention, the antibody comprises one or more Q tags that are labeled with the activity of KalbTG. For example, the attached label is selected from an enzyme, biotin, a radioactive group, a dye such as a fluorescent dye, an isotope and a metal. The labeling domain also comprises a second recognition site for KalbTG. Preferably, the label domain comprises a K-tag with at least 80% sequence identity to the peptide sequence RYESK (SEQ ID NO: 21). In a second alternative, the chemical linker moiety comprises (i) a functional group capable of forming a covalent bond in a chemical (non-enzymatic) coupling reaction, in particular with a reaction partner of carbodiimide or N-hydroxysuccinimide coupling chemistry, or with a reaction partner of the so-called "click" chemistry, in particular with an azide or alkyne moiety, and (ii) a second recognition site of KalbTG, in particular comprising or having a motif with a primary amine group, more particularly a Lys-containing motif, in particular the sequence RYESK (SEQ ID NO: 21). The chemical linker moiety may be as defined above. The modified antibody and the chemical linker moiety react in the presence of KalbTG or a functionally active variant thereof and under conditions that promote the activity of KalbTG, thereby forming an isopeptide bond between the first and second recognition sites, thus site-specifically conjugating the modified antibody. Preferably, in this method according to the invention, the antibody comprises one or more Q tags which are labelled with the activity of KalbTG, the labelling domain also comprising a second recognition site for KalbTG.Preferably, the chemical linker moiety comprises a K tag having at least 80% sequence identity to the peptide sequence RYESK (SEQ ID NO:21).

[0071] The method according to the present invention is preferred, in which the labeling is controlled, for example achieved at a stoichiometric ratio of label to antibody, for example about 1:1.In another embodiment, the controlled reaction takes into account the number of first recognition sites per antibody and the stoichiometric ratio of label and antibody.Thus, multiple labeling can be achieved, and it is preferred to use two or more first recognition sites on one antibody to attach two or even multiple labels to the antibody.The present invention is particularly useful for immunological reactions and assays in which quantification is desired.

[0072] In a sixth aspect, the present invention relates to the use of a modified antibody of the present invention (as defined above), KalbTG or a functionally active variant thereof, and (i) a second recognition site for KalbTG linked to a detectable label or (ii) a second recognition site for KalbTG linked to a chemical linker moiety comprising a functional group capable of forming a covalent bond in a chemical coupling reaction, for the preparation of a site-specifically conjugated antibody. The definitions and comments regarding the fifth aspect apply.

[0073] In a seventh aspect, the present invention relates to a method for detecting a target in a sample, the method comprising the steps of: a) contacting a sample suspected of containing a target with a site-specifically conjugated antibody of the invention (as defined above), where the site-specifically conjugated antibody is capable of specifically binding to the target under conditions favoring binding of the antibody to the target; b) optionally removing unbound site-specifically conjugated antibody; c) detecting the target by detecting the label of the site-specifically conjugated antibody that specifically binds to the target; Includes.

[0074] In the first step, a sample is provided and contacted with the site-specifically conjugated antibody of the present invention. The sample can be any sample suspected of containing the target of interest, including a sample from a subject. A sample is a limited amount of material that is intended to be identical to and represents a larger amount of that material. The act of obtaining a sample can be performed by a human or automatically. The sample can be taken or provided for testing, analysis, examination, investigation, verification, or experimental use. Sampling can be ongoing continuously. The sample can include or consist of a solid, liquid, or gas. It can be a gel or a material of some intermediate character, such as sputum, tissue, organism, or a combination of these. Preferably, the sample is a liquid or suspension that allows easy distribution.

[0075] A sample in this context is a quantity of a substance suspected of containing one or more targets to be detected or measured and quantified. As used herein, the term includes, but is not limited to, specimens (e.g., biopsies or medical specimens), cultures (e.g., microbial cultures) or environmental samples such as water or soil. Samples can be from subjects such as animals or humans and can be fluids, solids (e.g., stool), suspensions or tissues. The term "sample from a subject" includes all biological fluids, excretions and tissues isolated from any given subject. Preferably, the subject is an animal, more preferably a mammal, or even more preferably a human. Samples can be obtained from domestic animals, as well as from all of the various families of wild animals or wild animals, including, but not limited to, animals such as ungulates, bears, fish, rodents, etc.

[0076] Examples of samples include, but are not limited to, cell or tissue cultures, blood, serum, plasma, needle aspirates, urine, semen, sperm, seminal fluid, seminal plasma, prostatic fluid, stool, tears, saliva, sweat, biopsy fluid, ascites, cerebrospinal fluid, pleural fluid, amniotic fluid, peritoneal fluid, interstitial fluid, sputum, milk, lymph, bronchial and other lavage fluid samples, or tissue extract samples. The source of the sample may be a fresh, frozen and / or preserved organ or tissue sample or a solid tissue such as a biopsy or aspirate, or may be cells from any point in the subject's pregnancy or development.

[0077] Samples may contain compounds that are not naturally mixed with the source of the sample in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0078] The target of interest may represent a microorganism (such as a pathogen) and may be useful in diagnosing a disease such as an infection. Infections may be caused by bacteria, viruses, fungi, and parasites, or other nucleic acid containing subjects. Pathogens may be exogenous (obtained from environmental or animal sources or other humans) or endogenous (obtained from normal flora). Samples may be selected based on signs and symptoms, should be indicative of a disease process, and should be collected prior to administration of an antimicrobial agent. The amount of nucleic acid in an untreated sample may indicate the severity of the disease.

[0079] Alternatively, the target may indicate a disease or disorder. The disorder relates to the disorder or incorrect functioning of an organ, part, structure, or system of the body due to genetic or developmental errors, infection, poisoning, nutritional deficiency or imbalance, toxicity, or the influence of unfavorable environmental factors. This may be a human, animal, or plant disease caused by infection or health disorder. Thus, many markers (such as blood markers) indicate a disease state.

[0080] A sample suspected of containing the target is contacted with a site-specifically conjugated antibody of the invention (as defined above), which is a site-specifically conjugated antibody capable of specifically binding to the target, under conditions that favor binding of the antibody to the target. The antibody recognizes and binds to the target with a complementarity determining region (CDR) formed by one or more variable regions of the antibody. "Binding" as used in reference to an antibody means binding to the target with a specific binding site. -6 M or less, e.g. 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 Affinity (K D It may refer to a physical interaction between an antibody and a target (e.g., an antigen) that is characterized by a lower K D A value of 10 corresponds to a higher binding affinity (i.e., stronger binding) and thus -7 K of M D The value is 10 -6 K of M D The binding affinity of the target is higher than the value. After contact and binding, the unbound site-specifically conjugated antibody can be removed, for example, by a washing step. The target is then detected by detecting the label of the site-specifically conjugated antibody that is specifically bound to the target. The detection method obviously depends on the nature of the detectable label. In the method of the present invention, the presence, amount or concentration of the target can be determined. Detection of the presence or altered amount relative to a control indicates, for example, the presence of a disease, a microorganism, or a condition.

[0081] In electrochemiluminescence methods (e.g., immunodetection using Roche Elecsys® analyzers), antibodies are coupled to ruthenium esters (e.g., [Ru(bpy)3] 2+ , tris(2,2'-bipyridine)ruthenium(II) (Ru 2+In the presence of the target, the site-specifically conjugated antibody bearing the label is immobilized on the working electrode (e.g., via magnetic beads on a second antibody that binds to the target; sandwich assay). A co-reactant (e.g., oxalate ion (CO4 2- ), peroxydisulfuric acid (persulfate, SO), tri-n-propylamine (TPrA) and other amine-related derivatives, and hydrogen peroxide (HO) are oxidized at the electrode to generate radical cations, which rapidly deprotonate to form radicals. The radical cations and radical cations react with luminophores (e.g., ruthenium esters) to emit photons that are detected.

[0082] In preferred embodiments, the target is a microorganism, a cell, particularly a tumor cell, a virus, a bacterium, a fungus, a mammalian species, a genetic condition, a physiological condition or disease, or a protein, such as a protein that exhibits another antibody.

[0083] In another preferred embodiment, the sample is obtained from a source, cell culture or subject suspected of contamination, in particular the subject is selected from the group consisting of humans, animals and plants, in particular a human.

[0084] In yet another embodiment, the sample is selected from the group consisting of a body fluid, blood, plasma, serum, urine, bile, cerebrospinal fluid, synovial fluid, a swab, a clinical specimen, an organ sample, and a tissue sample.

[0085] The method of the present invention is of particular interest in the medical field, such as in diagnosis or treatment monitoring, and can be used to detect and / or quantify targets of interest indicative of specific microorganisms, cells, viruses, bacteria, fungi, mammalian species, genetic conditions or diseases. Accordingly, the method can be used to detect pathogens. Pathogens can cause disease. Typically, pathogens are used to describe infectious agents such as viruses, bacteria, prions, fungi, or even other microorganisms. Of course, the method of the present invention can also be used to detect non-pathogenic microorganisms.

[0086] Exemplary pathogens include, but are not limited to, the following: - Bacteria: Streptococcus, Staphylococcus, Pseudomonas aeruginosa, Burkholderia, Mycobacterium, Chlamydophila, Ehrlichia, Rickettsia, Salmonella, Neisseria, Brucella, Mycobacterium, Nocardia, Listeria, Francisella, Legionella, Yersinia -Viruses: Adenovirus, Herpes Simplex Virus, Varicella Zoster Virus, Cytomegalovirus Papillomavirus, Hepatitis B Virus, Hepatitis C Virus, Hepatitis E Virus, Poliovirus, Yellow Fever Virus, Dengue Virus, West Nile Virus, TBE Virus, HIV, Influenza Virus, Lassa Virus, Rotavirus and Ebola Virus -Fungi: Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis and Stachybotrys - Parasites: protozoan parasites, helminthic parasites and arthropod parasites

[0087] It is clear that reliable detection and optionally quantification of pathogens can be of high relevance to diagnosing the presence and severity of disease.

[0088] The method of the present invention can be used to detect and quantify specific cells, for example subpopulations of cells. Examples of such cells include cancer cells, such as circulating tumor cells or circulating tumor microemboli, specific blood cells, such as B cells, T cells, eosinophils, etc. The cells can be rare cells, in particular in a population where the ratio of rare cells to total cells is at most 5%, preferably at most 1%, in particular at most 0.1%, for example at most 0.01%. The rare cells can in particular be circulating tumor cells (CTCs) and circulating tumor microemboli (CTMs) in the blood of a patient. The ability to find and quantify "rare" tumor cells (only a few CTCs mixed with approximately 10 million white blood cells and 5 billion red blood cells in 1 ml of blood) and distinguish them from other cells, in particular epithelial non-tumor cells and white blood cells, is particularly relevant for the early detection of cancer. These cells can be detected long before the tumor itself becomes detectable, which is obviously a great advantage in the treatment of cancerous diseases.

[0089] Cancer cells are characterized by certain markers (tumor markers), which can be detected or quantified in the present method. Examples that may be mentioned are in particular oncogenes and tumor suppressor genes such as p53, genes such as erb-B2 of the ras family, c-myc, mdm2, c-fos, DPC4, FAP, nm23, RET, WT1, such as LOH for genes such as p53, DCC, APC, Rb, and microsatellite instability such as BRCA1 and BRCA2, MSH2, MLH1, WT1 in hereditary tumors, tumor RNAs such as CEA, cytokeratins, such as CK20, BCL-2, MUC1, in particular its tumor-specific splice variants, MAGE3, Muc18, tyrosinase, PSA, PSM, BA46, Mage-1, etc., or morphogenic RNAs such as maspin, hCG, GIP, motilin, hTG, SCCA. -1, AR, ER, PR, various hormones, etc. - as well as RNAs and proteins that specifically influence the metastatic profile, i.e. molecules involved in angiogenesis, motility, adhesion and matrix degradation, e.g. bFGF, bFGF-R, VEGF, VEGF-R, e.g. VEGF-R1 or VEGF-R2, E-cadherin, integrins, selectins, MMPs, TIMPs, SF, SF-R, etc., cell cycle profile or proliferation profile, e.g. cyclins (e.g. expression ratio of cyclins D, E and B), Ki67, p120, p21, PCNA, etc., or apoptosis profile, e.g. expression of FAS(L+R), TNF(L+R), perforin, granzyme B, BAX, bcl-2, caspase 3, etc.

[0090] Alternative cells that may be determined by the method of the invention include cardiovascular or vascular cells released by inflammatory processes or fetal cells, such as fetal cells in maternal blood, stem cells (e.g. cancer stem cells), cells showing minimal residual disease, cancer cells (e.g. leukemia cells). In this regard, the method may be used for genotyping, diagnosis, prognosis, treatment monitoring, etc.

[0091] The methods may also be used to detect and quantitate cellular content (eg, food control), genetic conditions (eg, when detecting or monitoring genetic disorders) or diseases of mammalian species.

[0092] In an eighth aspect, the present invention relates to the use of a site-specifically conjugated antibody of the present invention (as defined above) in the detection of a target and / or in diagnosis, in particular for the detection of a disease or pathogen, or in patient monitoring, in particular in disease treatment monitoring or determining treatment efficiency. The definitions and comments regarding the other aspects of the invention apply. The use according to this eighth aspect is preferably in vitro. In a particular embodiment, the use according to this eighth aspect is not carried out in the human or animal body. In a preferred embodiment, the target is detected in an ex vivo biological sample.

[0093] 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 the present invention. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, 1994 (ISBN 0-19-854287-9), published by Oxford University Press, Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, 1994 (ISBN 0-632-02182-9), published by Blackwell Science, and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, 1995 (ISBN 1-56081-569-8), published by VCH Publishers, Inc.

[0094] The present invention is not limited to the specific methodology, protocols, and reagents described herein, because these may vary. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred methods and materials are described herein. Furthermore, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

[0095] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the words "comprise," "contain," and "encompass" are to be interpreted inclusively rather than exclusively. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. The term "plurality" refers to two or more.

[0096] The following examples are intended to illustrate various embodiments of the present invention.Therefore, the specific modifications described should not be interpreted as limitations on the scope of the present invention.It is clear to those skilled in the art that various equivalents, changes and modifications can be made without departing from the scope of the present invention, and therefore, such equivalent embodiments should be understood to be included in this specification. [Brief description of the drawings]

[0097] [Figure 1] Schematic diagram of KalbTG enzyme labeling reaction. [Diagram 2] A schematic diagram of an IgG molecule is shown indicating sites for Q-tag insertion with likely candidates indicated by an asterisk. [Diagram 3] FIG. 1 shows a decision flow chart for evaluation of single-site Q-tagged IgG. [Figure 4] 1 shows a decision flow chart for evaluation of multi-site Q-tagged IgG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0098] Example 1: Identification of suitable sites for labeling The KalbTG Q-tag was inserted into surface exposed inter- and intradomain flexible loops within the human IgG1 constant region as well as the C-terminus of the chain, see schematic Figure 2.

[0099] A total of 24 different sites within the IgG heavy and light chains were selected for testing, each with an insertion of a KalbTG Q-tag motif within two flexible linkers (GGGSYRYRQGGGS) (SEQ ID NO:28) and optionally without flexible linker flanking (YRYRQ) (SEQ ID NO:14).

[0100] The above sequence set (SEQ ID NOs: 1-13) includes markings for insertion sites within the human IgG1 heavy chain and human light chain sequences, as well as homologous sites within the IgG heavy and light chain sequences of other species. The nucleic acid sequences encoding the aforementioned modified IgGs were cloned into a standard mammalian cell expression vector with a major extracellular secretion signal sequence. The vectors were transfected into HEK293 cells, and the modified IgG constructs were expressed following standard transient expression protocols. The modified IgG molecules were then purified from cell culture supernatants using Protein A affinity chromatography.

[0101] Purified IgG molecules with insertions at a single site in either the heavy or light chain were evaluated for their expression rate (mg IgG per mL of culture supernatant). Furthermore, successful folding and aggregation tendency were evaluated by size-exclusion chromatography. Furthermore, the stability of the modified IgG was tested using dynamic light scattering (DLS) thermal scanning. Finally, the conjugation efficiency of IgG molecules with single-site insertions (i.e., the success of the enzymatic conjugation reaction targeting these sites) was evaluated by enzymatically labeling the IgG with two different labels in independent labeling experiments under standard reaction conditions. In the standard experiment, two labels were addressed: biotin (Bi) and sulfo-ruthenium (sBPRu), both of which were linked to a K-tag (RYESK) (SEQ ID NO: 21) via a PEG-based linker. For a detailed overview of the results, see Table 1 and the respective legends. In a first evaluation step, sites were selected based on favorable expression yields and low aggregation tendency. Sites without dramatic loss of expression yield (50-100%) and with less than 15% aggregates were selected for follow-up. Furthermore, several sites that at least partially fulfilled the set criteria were also selected for follow-up (checkpoint 1). In a second evaluation step, sites were shortlisted based on conjugation efficiency. Sites that allowed quantitative conjugation of targets exhibiting accessibility of the Q-tag to the enzyme were selected (checkpoint 2). See Figure 3 for an overview of the decision flow chart. Twelve successful internal and one C-terminal insertion / conjugation binding sites were identified.

[0102] Table 1 (attached below) provides a summary of the molecule (IgG with single site insertion) evaluation data, with a more detailed explanation included in the table legend. [Table 1] TIFF2025501578000016.tif131165

[0103] As shown in Table 1, we successfully identified several KalbTG Q-tag insertion sites spanning the length of the IgG backbone that provided enzymatic accessibility and did not adversely affect IgG cellular expression or folding.

[0104] Example 2: Identification of combinations of sites that allow multiple labeling The single sites identified in Example 1 serve as building blocks for the construction of IgG scaffolds with multiple insertion motifs (Q-tags) for multi-site specific conjugation via KalbTG. Furthermore, they allow us to further investigate the attachment site(s) effect on labeling performance. Therefore, IgG molecules containing combinations of 2, 3, 4, 5 or 6 of the single insertion sites of Example 1 were tested.

[0105] The nucleic acid sequence encoding the modified IgG was cloned into a standard mammalian cell expression vector with a major extracellular secretion signal sequence. The vector was transfected into HEK293 cells, and the modified IgG construct was expressed according to a standard transient expression protocol. The modified IgG molecule was then purified from the cell culture supernatant using Protein A affinity chromatography.

[0106] Purified IgG molecules with insertions at a single site in either the heavy or light chain were evaluated for their expression rate (mg IgG per ml culture supernatant). Successful folding and aggregation tendency were also evaluated by size exclusion chromatography. Finally, the conjugation efficiency of IgG with multiple site insertions (i.e., successful enzymatic conjugation reactions targeting these sites) was evaluated by enzymatic labeling of IgG with K-tag azide label under standard reaction conditions. K-tag-azide is a heterobifunctional linker, in which the K-tag (RYESK) (SEQ ID NO: 21) is linked to the azide moiety via a PEG-like linker. The azide group serves as a handle for a subsequent "click" reaction to attach any label / protein with a complementary reactive group (e.g., an alkyne group). For a detailed overview of the results, see Table 2 and its legend.

[0107] At checkpoint 1 (CP1), sites were selected that did not result in dramatic loss of yield and did not show significant aggregation. Other sites were excluded. At checkpoint 2 (CP2), sites were selected that allowed additional conjugation at 4 or more inserted KalbTG recognition sites in the target, and the Q-tag was accessible to the enzyme. See Figure 4 for an outline of the decision flow chart for this. In this process, 21 surprising successful combinations were identified.

[0108] Table 2 (appended below) provides a summary of the molecule (IgG with multiple insertion sites) evaluation data, with a more detailed explanation in the table legend. [Table 2] TIFF2025501578000018.tif236165 TIFF2025501578000019.tif83165

[0109] As shown in Table 2, we have successfully identified several IgG molecules with combinations of 2, 3 and even 4 multiple sites that allow site-specific insertion of 4, 6 and 8 labels, further enhancing the ability to scan the site-dependence of labeling performance.

[0110] References -Agarwal, P. & Bertozzi, CRSite-specific antibody-drug conjugates: the nexus of bioorthogonal chemistry, protein engineering, and drug development.Bioconjug Chem 26,176-192,doi:10.1021 / bc5004982(2015). -Ando, ​​H. et al.Purification and Characteristics of a Novel Transglutaminase Derived from Microorganisms.Agricultural and Biological Chemistry 53,2613-2617,doi:10.1080 / 00021369.1989.10869735(2014). -Chichili et al.Linkers in the structural biology of protein-protein interactions Protein Sci.2013 Feb;22(2):153-167 -Steffen,W.et al.Discovery of a microbial transglutaminase enabling highly site-specific labeling of proteins.J Biol Chem,doi:10.1074 / jbc.M117.797811(2017). -Strop,P.et al.Location matters:site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates.Chem Biol 20,161-167,doi:10.1016 / j.chembiol.2013.01.010(2013). - International Publication No. 2016 / 100735 - International Publication No. 2017 / 102759 -International Publication No. 2021 / 174091 -Zhou,Q.&Kim,J.Advances in the Development of Site-Specific Antibody-Drug Conjugation.(2015).

Claims

1. 1. A modified antibody comprising a heavy chain and a light chain, the antibody being modified to include one or more first recognition sites for transglutaminase from Kutneria albida (KalbTG) by insertions, each insertion being located after one or more positions independently selected from position 108 (LC108), position 110 (LC110), and position 214 (LC214) of the light chain and position 118 (HC118), position 177 (HC177), position 207 (HC207), position 235 (HC235), position 269 (HC269), position 297 (HC297), position 328 (HC328), position 341 (HC341), and position 401 (HC401) of the heavy chain.

2. 2. The modified antibody of claim 1, comprising an additional first recognition site for KalbTG at position 446 (HC446) of the heavy chain.

3. (i) the antibody comprises two pairs each of heavy and light chains, and each of the heavy chains or / and the light chains comprises the one or more first recognition sites; or (ii) the antibody comprises three or more pairs of heavy and light chains, each heavy chain being a fusion polypeptide comprising one or more additional Fab domains, VH and CH1 heavy chain fragments, fused to either the VH N-terminus or Fc C-terminus of the heavy chain, and each of the fusion polypeptides and / or the light chains comprising the one or more first recognition sites; or (iii) the antibody comprises three or more pairs of heavy and light chains, each heavy chain being a fusion polypeptide comprising one or more additional Fab domains VL and CL light chain fragments fused to either the VH N-terminus or the Fc C-terminus of the heavy chain, each fused Fab domain VL and CL light chain fragment being paired with a Fab domain VH and CH1 heavy chain fragment, and each of the fusion polypeptides and / or the light chains and / or the Fab domain VH and CH1 heavy chain fragments comprising the one or more first recognition sites; The modified antibody of claim 1.

4. a) the antibody recognizes two first recognition sites for KalbTG, in particular: - HC118 / HC177; or - HC118 / HC328; or - HC118 / HC341; or - HC118 / HC401; or - HC177 / HC207; or - HC177 / HC328; or - HC177 / HC341; or - HC177 / HC401; or - HC207 / HC328; or - HC207 / HC341; or - HC207 / HC401; or - HC328 / HC446; or - HC341 / HC446; or - HC118 / LC214; or -HC177 / LC108; or -HC177 / LC214; or - HC207 / LC214; or -HC446 / LC214 At the position especially, - HC118 / HC177; or - HC118 / HC328; or - HC118 / HC341; or - HC177 / HC341; or - HC207 / HC341; or - HC328 / HC446; or - HC341 / HC446; or - HC118 / LC214; or -HC177 / LC108; or -HC177 / LC214; or - HC207 / LC214; or -HC446 / LC214 or or b) the antibody recognizes the three first recognition sites for KalbTG, in particular: - HC118 / HC235 / HC341; or - HC207 / HC235 / HC341; or - HC118 / HC341 / HC446; or - HC207 / HC341 / HC446; or - HC118 / HC235 / HC328; or - HC177 / HC235 / HC328; or -HC207 / HC235 / HC328 or or c) the antibody recognizes the four first recognition sites for KalbTG, in particular: - HC118 / HC235 / HC341 / HC446; or -HC207 / HC235 / HC341 / HC446 Including at the position The modified antibody of claim 1.

5. The modified antibody of claim 1, wherein the one or more first recognition sites comprise or have Gln-containing motifs independently selected from the group of sequences consisting of YRYRQ (SEQ ID NO: 14), YRQRT (SEQ ID NO: 16), RYGQR (SEQ ID NO: 17), RWRQR (SEQ ID NO: 18), RVRQR (SEQ ID NO: 15), IRQRQ (SEQ ID NO: 19) and FRYRQ (SEQ ID NO: 20), particularly YRYRQ (SEQ ID NO: 17) or RVRQR (SEQ ID NO: 18), particularly YRYRQ (SEQ ID NO: 17).

6. The first recognition site or any one of the two or more first recognition sites is linked to the antibody via one or two linkers at one or both ends of each of the first recognition sites, and in particular, the linker is mainly or completely composed of Gly and Ser, for example, (Gly-Gly-Gly-Gly-Ser). ｎ (SEQ ID NO: 22), where n=1, 2, 3, 4 or 5, preferably n=1, or in particular, the linker comprises or consists of GGGP (SEQ ID NO: 23), ESGS (SEQ ID NO: 24) or APAP (SEQ ID NO: 25), and each of the two linkers at both ends is independently selected.

7. the unmodified light chain constant domain (CL) comprises an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100% identical to the amino acid sequence of any of SEQ ID NOs: 10 to 13, and / or any of the unmodified heavy chain constant domains CH3, CH2 and CH1 comprise an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100% identical to the amino acid sequence of the respective domain in the amino acid sequence of SEQ ID NOs: 1 to 9, and / or the unmodified heavy chain constant domain comprises an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100% identical to the amino acid sequence of any of SEQ ID NOs: 1 to 9, and / or the unmodified light chain constant domain (CL) consists of an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100% identical to the amino acid sequence of any of SEQ ID NOs: 10 to 13, and / or any of the unmodified heavy chain constant domains CH3, CH2 and CH1 consists of an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100% identical to the amino acid sequence of each of the domains in the amino acid sequences of SEQ ID NOs: 1 to 9, and / or the unmodified heavy chain constant domain consists of an amino acid sequence that is at least 96%, 97%, 98% or 99%, particularly 100% identical to the amino acid sequence of each of SEQ ID NOs: 1 to 9, The modified antibody of claim 1.

8. The antibody a) a complete antibody or one or more functional fragments thereof, such as a single chain Fv (scFv), a Fab fragment, a F(ab') fragment, a F(ab)2 fragment, a F(ab')2 fragment, an antibody / Fc fusion protein, or a disulfide-linked Fvs (sdFv); and / or b) is of goat, mouse, rat, rabbit or sheep origin; and / or c) are human, humanized, chimeric and / or affinity matured; The modified antibody of claim 1.

9. A nucleic acid encoding a polypeptide chain of a modified antibody according to any one of claims 1 to 8.

10. A site-specifically conjugated antibody comprising the modified antibody of any one of claims 1 to 8 and (i) one or more label domains, each of which comprises a second recognition site for KalbTG or a functionally active variant thereof, wherein the second recognition site is covalently linked to the first recognition site of the modified antibody, and wherein the label domain comprises a detectable label; or (ii) one or more chemical linker moieties, each of the one or more covalently attached chemical linker moieties comprising a second recognition site for KalbTG or a functionally active variant thereof, the second recognition site being covalently attached to the first recognition site of the antibody, the chemical linker moieties comprising a functional group capable of forming a covalent bond in a chemical (non-enzymatic) coupling reaction, in particular a functional group with a reaction partner of carbodiimide or N-hydroxysuccinimide coupling chemistry, or a functional group with a reaction partner of so-called "click" chemistry, in particular an azide or alkyne moiety; A site-specifically conjugated antibody comprising:

11. 11. The site-specifically conjugated antibody of claim 10, wherein a label is covalently attached to the chemical linker moiety, the label being selected from the group consisting of a biotin moiety, a fluorescent dye, an iridium label, a ruthenium label, a radioactive label, a single-stranded oligonucleotide or analog thereof, and a chemiluminescent label.

12. a) the label or the label domain is suitable for detection by an enzyme-mediated reaction, such as an enzyme-mediated chromogenic, fluorogenic or metallurgical reaction, or by direct or indirect fluorometry, or by radiometry, or by electrochemiluminescence; and / or b) the label or the label domain is or comprises an enzyme, an enzyme substrate, a chromophore, a fluorophore, a quencher, a radiolabel, biotin, a metal, or an electrochemiluminescent moiety, in particular an electrochemiluminescent moiety containing iridium or ruthenium; and / or c) each site-specifically conjugated antibody is conjugated to at least two, at least four, at least six, or at least eight labels or label domains; The site-specifically conjugated antibody of claim 10.

13. 12. The site-specifically conjugated antibody of claim 11, wherein the linker is a monofunctional or bifunctional chemical linker moiety that allows for the covalent attachment of one or two reactive additional compounds, respectively.

14. A kit for producing a site-specifically conjugated antibody, comprising a modified antibody according to any one of claims 1 to 8 and (i) KalbTG or a functionally active variant thereof; (ii) a label domain capable of being bound to the one or more first recognition sites by KalbTG or a functionally active variant thereof, the label domain comprising a detectable label; and (iii) a chemical linker moiety that can be attached to the one or more first recognition sites by KalbTG or a functionally active variant thereof, the chemical linker moiety comprising a functional group that can form a covalent bond in a chemical (non-enzymatic) coupling reaction, in particular a functional group with a reaction partner of carbodiimide or N-hydroxysuccinimide coupling chemistry, or a functional group with a reaction partner of so-called "click" chemistry, in particular an azide or alkyne moiety. and at least one further component selected from the group consisting of: Includes a kit.

15. 1. A method for site-specific conjugation of a modified antibody, comprising: a) providing a modified antibody according to any one of claims 1 to 8; b) providing a label domain, said label domain comprising: (i) a detectable label; (ii) a second recognition site for KalbTG, in particular the second recognition site comprising or having a motif with a primary amine group, more particularly a Lys-containing motif, in particular the sequence RYESK (SEQ ID NO: 21); and (iii) optionally a linker between said label and said second recognition site; c) reacting the modified antibody of a) with the label domain of b) in the presence of KalbTG or a functionally active variant thereof under conditions that promote the activity of KalbTG to form an isopeptide bond between the first recognition site and the second recognition site, thereby site-specifically conjugating the modified antibody; contains, or a) providing a modified antibody according to any one of claims 1 to 8; b) providing a chemical linker moiety, which comprises (i) a functional group capable of forming a covalent bond in a chemical (non-enzymatic) coupling reaction, in particular a functional group with a reaction partner of carbodiimide or N-hydroxysuccinimide coupling chemistry, or a functional group with a reaction partner of so-called "click" chemistry, in particular a functional group with an azide or alkyne moiety, and (ii) a second recognition site for KalbTG, in particular a second recognition site which comprises or has a motif with a primary amine group, more in particular a Lys-containing motif, in particular the sequence RYESK (SEQ ID NO: 21); c) reacting the modified antibody of a) with the chemical linker moiety of b) in the presence of KalbTG or a functionally active variant thereof and under conditions that promote the activity of KalbTG to form an isopeptide bond between the first recognition site and the second recognition site, thereby site-specifically conjugating the modified antibody. A method comprising:

16. Use of a modified antibody according to any one of claims 1 to 8, KalbTG or a functionally active variant thereof, and (i) a second recognition site for KalbTG linked to a detectable label or (ii) a second recognition site for KalbTG linked to a chemical linker moiety comprising a functional group capable of forming a covalent bond in a chemical coupling reaction, for producing a site-specifically conjugated antibody.

17. 1. A method for detecting a target in a sample, comprising: a) contacting the sample suspected of containing the target with a site-specifically conjugated antibody according to claim 10, wherein the site-specifically conjugated antibody is capable of specifically binding to the target under conditions conducive to binding of the antibody to the target; b) optionally removing unbound site-specifically conjugated antibody; c) detecting the target by detecting the label of the site-specifically conjugated antibody that specifically binds to the target; A method comprising:

18. The method described in claim 17, wherein the site-specifically conjugated antibody is the site-specifically conjugated antibody described in claim 11.

19. The method described in claim 17, wherein the site-specifically conjugated antibody is the site-specifically conjugated antibody described in claim 12.

20. The method of claim 17, wherein the site-specifically conjugated antibody is the site-specifically conjugated antibody described in claim 13.

21. a) the target is a microorganism, a cell, in particular a tumor cell, a virus, a bacterium, a fungus, a mammalian species, a protein, such as a protein indicative of a genetic condition, a physiological condition or a disease, or another antibody; and / or b) the sample is obtained from a source, cell culture or subject suspected to be contaminated, in particular the subject is selected from the group consisting of humans, animals and plants, in particular a human; and / or c) the sample is selected from the group consisting of body fluids, blood, plasma, serum, urine, bile, cerebrospinal fluid, synovial fluid, swabs, clinical specimens, organ samples and tissue samples; 18. The method of claim 17.

22. 11. In vitro use of a site-specifically conjugated antibody according to claim 10 in the detection of a target and / or in diagnostics, in particular for the detection of a disease or pathogen, or in patient monitoring, in particular in the treatment monitoring of a disease or in determining the efficiency of a treatment.

23. The in vitro use described in claim 22, wherein the site-specifically conjugated antibody is the site-specifically conjugated antibody described in claim 11.

24. The in vitro use described in claim 22, wherein the site-specifically conjugated antibody is the site-specifically conjugated antibody described in claim 12.

25. The in vitro use described in claim 22, wherein the site-specifically conjugated antibody is the site-specifically conjugated antibody described in claim 13.

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