Transglutaminase conjugation method with glycine based linker

Abstract

To provide a method for generating an antibody-payload conjugate by means of a microbial transglutaminase (MTG).SOLUTION: The method comprises a step of conjugating a linker comprising or having the peptide structure (shown in the N→C direction) Gly-(Aax)m-B-(Aax)n via the N-terminal primary amine of the N-terminal glycine (Gly) residue to a glutamine (Gln) residue included in the heavy or light chain of an antibody. One object of the present invention is to provide a transglutaminase-based antibody conjugation approach which does not require prior deglycosylation of the antibody, in particular of N297.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a method for producing antibody-payload conjugates by microbial transglutaminase.The present invention further provides linkers, linker-payload constructs and / or antibody-payload constructs. [Background technology]

[0002] 2. Background of the Invention Conjugating highly potent payloads to antibodies is of increasing interest for the targeted treatment of cancer or inflammatory diseases, the resulting constructs being called antibody-payload conjugates, or antibody-drug conjugates (ADCs).

[0003] Currently, seven ADCs (Adcetris, Kadcyla, Besponsa, Mylotarg, Polivy, Padcev, Enhertu) are FDA approved, all of which have payloads chemically linked to antibodies in a non-site-specific manner. Thus, the resulting products are highly heterogeneous, both in terms of the stoichiometric relationship between antibody and payload (payload to antibody ratio or drug to antibody ratio, DAR) and the conjugation site on the antibody. Each of the resulting molecular species, although in the same drug product, has distinct properties and can potentially result in a wide range of different in vivo pharmacokinetic properties and activities.

[0004] Previous in vivo studies (Lhospice et al., 2015) demonstrated that site-specific drug conjugation resulted in significantly higher tumor uptake (approximately 2-fold) and reduced uptake in non-target tissues compared to FDA-approved ADCs, with a maximum tolerated dose that was at least 3-fold higher. These data suggest that stoichiometrically well-defined ADCs exhibit improved pharmacokinetics and superior therapeutic index compared to chemically modified ADCs.

[0005] As a site-specific technique, enzymatic conjugation has attracted great interest because these conjugation reactions are typically fast and can be carried out under physiological conditions. Among the available enzymes, microbial transglutaminase (MTG) from the species Streptomyces mobaraensis has attracted increasing interest as an attractive alternative to traditional chemical protein conjugation of functional moieties, including antibodies. MTG catalyzes, under physiological conditions, the transamidation reaction between a "reactive" glutamine of a protein or peptide and a "reactive" lysine residue of a protein or peptide, the latter of which may be a simple low molecular weight primary amine such as a 5-aminopentyl group (Jeger et al., 2010, Strop et al., 2014).

[0006] The bond that is formed is an isopeptide bond, which is an amide bond that does not form part of the peptide bond backbone of the respective polypeptide or protein, that is formed between the γ-carboxamide of a glutamyl residue of an acylglutamine-containing amino acid donor sequence and the primary (1°) amine of a substrate that includes an amino donor according to the invention.

[0007] Our experience, and that of others, indicates that only a few glutamines are typically targeted by MTG, making it an attractive tool for site-specific and stoichiometric protein modification.

[0008] Previously, glutamine 295 (Q295) was identified as the only reactive glutamine on the heavy chain of various IgG types that is specifically targeted by MTG with low molecular weight primary amine substrates (Jeger et al. 2010).

[0009] However, quantitative conjugation to Q295 was only possible when the glycan moiety at asparagine residue 297 (N297) was removed by PNGase F, and glycosylated antibodies could not be conjugated efficiently (conjugation efficiency <20%). This finding is also supported by the studies of Mindt et al. (2008) and Jeger et al. (2010) and Dickgiesser et al. 2020.

[0010] To obviate the need for deglycosylation, a point mutation can be inserted at residue N297, resulting in the loss of glycosylation, referred to as aglycosylation.

[0011] However, both approaches have significant drawbacks: the enzymatic deglycosylation step is undesirable from a GMP standpoint, as it must be ensured that both the deglycosylation enzyme (e.g., PNGase F) and the cleaved glycans are removed from the medium to ensure a high purity product.

[0012] Substitution of N297 for another amino acid also results in H This has undesirable effects, since it may affect the overall stability of the two domains and, as a result, the efficacy of the entire conjugate. Furthermore, the glycans present at N297 have important immunomodulatory effects, since they induce antibody-dependent cellular cytotoxicity (ADCC), etc. These immunomodulatory effects will be lost upon deglycosylation or substitution of N297 with another amino acid.

[0013] Furthermore, engineering antibodies for payload attachment can have drawbacks in that sequence insertions can increase immunogenicity and reduce the overall stability of the antibody. [Prior art documents] [Non-patent literature]

[0014] [Non-Patent Document 1] Lhospice et al., Site-Specific Conjugation of Monomethyl Auristatin E to Anti-Cd30 Antibodies Improves Their Pharmacokinetics and Therapeutic Index in Rodent Models, Mol Pharm 12 (6), 1863-1871. 2015 [Non-Patent Document 2] Jeger et al, Site-specific and stoichiometric modification of antibodies by bacterial transglutaminase. Angew Chem Int Ed Engl. 2010 Dec 17;49(51):9995-7 [Non-Patent Document 3] Strop, et al., Versatility of Microbial Transglutaminase. Bioconjugate Chemistry 2014, 25 (5), 855-862. [Non-Patent Document 4] Mindt, et al., Modification of different IgG1 antibodies via glutamine and lysine using bacterial and human tissue transglutaminase. Bioconjugate chemistry 2008, 19 (1), 271-8. [Non-Patent Document 5] Dickgiesser S. et al., Site-Specific Conjugation of Native Antibodies Using Engineered Microbial Transglutaminases. Bioconjug Chem. 2020 Mar 12. doi: 10.1021 / acs.bioconjchem.0c00061. Summary of the Invention [Problem to be solved by the invention]

[0015] Thus, one object of the present invention is to provide a transglutaminase-based antibody conjugation approach that does not require prior deglycosylation of the antibody, in particular of N297. That is the thing.

[0016] Another object of the present invention is to provide a method for the preparation of a H The objective of this invention is to provide a transglutaminase-based antibody conjugation approach that does not require replacement or modification of N297 of domain 2.

[0017] A further object of the present invention is to provide an antibody conjugation technique that allows for the production of highly uniform conjugation products, both with respect to stoichiometry as well as site specificity of conjugation.

[0018] These and further objects are met by the methods and means according to the independent claims. The dependent claims relate to specific embodiments. [Means for solving the problem]

[0019] Summary of the Invention The present invention relates to methods and linker structures for producing antibody-linker conjugates and / or antibody-payload conjugates by microbial transglutaminase (MTG). General advantages of the present invention and its features are discussed in detail below. [Brief description of the drawings]

[0020] [Figure 1]FIG. 1 shows a diagram of one embodiment of the present invention. MTG=microbial transglutaminase. The star symbol indicates the payload or linking moiety B. Gp is ​​a Gly residue that is at the N-terminus of the peptide and is a substrate for MTG. Note that this process allows glycosylation at N297 to be maintained. Note that if B / star is a linking moiety, the actual payload must still be conjugated to this moiety. As discussed elsewhere herein, B / star can be or include a linking moiety, such as a bio-orthogonal group (e.g., azide / N3 group), suitable for strain-promoted alkyne-azide cycloaddition (SPAAC) click chemistry reaction to, for example, a DBCO-containing payload (e.g., a toxin or fluorescent dye or metal chelator, such as DOTA or NODA-GA). This click chemistry-based "two-step chemoenzymatic" approach to attaching functional moieties to antibodies has a major advantage since it is possible to click at a low molecular excess compared to the antibody, typically at e.g. 5 or even less equivalents / conjugation site (Dennler et al. 2014). This allows for cost-effective production of ADCs. Moreover, virtually any probe ranging from fluorescent dyes to metal chelators can be clicked with this approach (see Spycher et al. 2017, Dennler et al. 2015). The B / star may also be the actual payload, e.g. a toxin. Such an embodiment allows for rapid manufacture of the resulting compound in one step, facilitating purification and production.

[0021] [Diagram 2]FIG. 2 shows an example of a linker peptide comprising an oligopeptide according to the invention. The sequence is GlyAlaArgLys(N3) (GARK1, K1=Lys(N3)). Lys(N3) is a Lys residue in which the primary amine is replaced by an azide group (-NN≡N or -N3). According to the nomenclature of the present invention, Lys(N3) or only N3 can be considered as the linking part B (in this example, N3 is suitable for click chemistry). The peptide efficiently conjugates to the native IgG1 antibody at position Q295 (about 77.3% estimated from LC-MS analysis under non-optimized conditions). It is important to understand that in some linker peptides shown herein, the C-terminal part is simply indicated as N3. However, this should be understood as an abbreviation for Lys(N3). For example, GAR(N3) corresponds to the peptide GlyAlaArgLys(N3) or GARK(N3). That is, 6-azido-L-lysine can be abbreviated as Lys(N3) in the three-letter notation, or K(N3) or (N3) in the one-letter notation. It should therefore be understood that K(N3), when part of a peptide, always refers to the single amino acid residue Lys(N3), but not to the dipeptide Lys-Lys(N3). On the other hand, the dipeptide Lys-Lys(N3) would be represented in the one-letter notation as KK(N3). It is further important to understand that in the various linker peptides shown herein, the primary amine on the C-terminus or side chain may or may not be protected, even if otherwise shown. Protection can be achieved, for example, by amidation of the C-terminus and / or acetylation of the primary amine on the side chain. In the context of the present invention, both protected and unprotected linker peptides are encompassed. For example, GARK(N3) actually encompasses two variants, with or without the C-terminus protected. The diagram below shows the C-terminal Lys(N3) residue, where the C-terminus is protected by amidation: [ka]

[0022] [Diagram 3] Figure 3 shows the results of screening a small given peptide library against a native IgG1 antibody. Various peptides containing MTG-reactive N-terminal amino acid residues or derivatives (beta-alanine) were screened. As can be seen, single or double N-terminal glycines work most efficiently. LC-MS was used for the analysis.

[0023] [Figure 4] Figures 4 and 5 show embodiments in which the linker comprises a Cys residue with a free sulfhydryl group suitable for conjugating a maleimide-containing toxin linker construct. Figure 4 shows the conjugation reaction and Figure 5 shows some potential linker constructs.

[0024] [Diagram 5] Figures 4 and 5 show embodiments in which the linker comprises a Cys residue with a free sulfhydryl group suitable for conjugating a maleimide-containing toxin linker construct. Figure 4 shows the conjugation reaction and Figure 5 shows some potential linker constructs.

[0025] [Figure 6] Figure 6 shows a two-step conjugation process (Figure 6A) in which a peptide is conjugated to a Gln of an antibody (e.g., Q295 of an IgG or molecularly engineered) and a one-step conjugation process according to the present invention (Figure 6B). Table 1 below clarifies the two terms used herein: [Table 1] In the two-step process, the linker peptide is Gly-(Aax). n A Gly residue is conjugated to a Gln residue of the antibody via microbial transglutaminase, and then the linking moiety, in this case a Cys residue with a free sulfhydryl group, is conjugated via a maleimide to the payload, in this case an MMAE toxin with a MC / VC / PABDC linker structure. In the one-step process, the linker peptide Gly-(Aax) m is already conjugated to the payload. A Gly residue is conjugated to a Gln residue of the antibody, and the payload consists of an MMAE toxin with a VC / PABC structure. A valine residue of the VC structure is conjugated to the last amino acid of the linker peptide by a peptide bond.

[0026] [Figure 7]Figure 7 shows two examples of linkers, including linkers suitable for dual payload attachment. Figure 7A shows a peptide with a first linking moiety that is an azide (N3) and a second linking moiety that is a tetrazine (both bioorthogonal). The structure of the oligopeptide is GlyAlaArgLys(N3)Lys(tetrazine) (GARK1K2, K1=Lys(N3), K2=Lys(tetrazine)). Figure 7B shows a peptide with a free sulfhydryl group from an azide (N3) and a Cys moiety. The structure of the oligopeptide is GlyAlaArgLys(N3)Cys (GARK1C, K1=Lys(N3)). Each of the linking moieties is a bioorthogonal compatible group that can be clicked simultaneously. Thus, these linkers allow two different payloads to be conjugated to Q295 of the CH2 domain of an antibody. The use of a second payload allows the development of a completely new class of antibody-payload conjugates that exceed current therapeutic approaches in terms of efficacy and potency. New fields of application are envisioned, such as dual imaging for imaging and therapy or intra- / post-operative surgery (see Azhdarinia A. et al., Molec Imaging and Biology, 2012). For example, dual-labeled antibodies that include molecular imaging agents for pre-operative positron emission tomography (PET) and near-infrared fluorescent (NIRF) dyes for guided delineation of surgical margins can greatly enhance cancer diagnosis, staging and resection (see Houghton JL. et al., PNAS 2015). PET and NIRF optical imaging provide complementary clinical applications, respectively allowing non-invasive whole-body imaging to localize disease and identification of tumor margins during surgery. However, to date, the creation of such dual-labeled probes has been difficult due to the lack of suitable site-specific methods; linking two different probes by chemical means results in nearly impossible analysis and reproducibility due to random conjugation of the probes.Furthermore, in a study by Levengood M. et al., (Angewandte Chemie, 2016), a dual-drug labeled antibody conjugated with two different auristatin toxins (having different physicochemical properties and exerting complementary anticancer activity) conferred activity in cell lines and xenograft models refractory to ADCs composed of the individual auristatin components. This suggests that dual-labeled ADCs may enable cancer heterogeneity and resistance to be addressed more effectively than a single conventional ADC alone. Since one resistance mechanism to ADCs involves active pumping of cytotoxic moieties from cancer cells, another dual-drug application may involve additional simultaneous delivery of a drug that specifically blocks the efflux mechanism of the cytotoxic drug. Thus, such dual-labeled ADCs may help overcome cancer resistance to ADCs more effectively than conventional ADCs. Similar structures in which alkynes or tetrazine / trans-cyclooctene are used as linkers are equally suitable and are within the scope and spirit of the present invention. It is important to understand that in some linker peptides shown herein, the C-terminal portion is simply indicated as N3. However, this should be understood as an abbreviation of Lys(N3). For example, GAR(N3) or GARK(N3) actually means GARK1, K1=Lys(N3) or GlyAlaArgLys(N3). It is further important to understand that in various linker peptides shown herein, the C-terminus may be protected or not, even if it is indicated otherwise. Protection may be achieved by amidation of the C-terminus. Since conjugation of the linker to the antibody is achieved through the primary amine of the N-terminal glycine residue of the linker, the N-terminus of the linker is preferably not protected. In the context of the present invention, both protected and unprotected linker peptides are encompassed. For example, GARK(N3) actually encompasses two variants: a) unprotected at both termini as discussed above, or b) only protected at the C-terminus as discussed above. The question of whether the C-terminus is amidated or not is a practical matter depending on the conjugation conditions (buffer, medium, reactivity of other reaction components, etc.).

[0027] [Figure 8] Figure 8A and Figure 8B show two possible linker structures, each with two azide linker moieties. Figure 8A shows GlyGlyAlaArgLys(N3)Lys(N3) (GGARK1K2, K1 and K2 = Lys(N3)). Figure 8B shows GlyGlyAlaArgLys(N3)ArgLys(N3) (GGARK1RK2; K1 and K2 = Lys(N3)). In this way, an antibody payload ratio of 4 can be obtained. The presence of a charged Arg residue helps to keep the hydrophobic payload in solution. It is important to understand that in some linker peptides shown herein, the C-terminal portion is simply shown as N3. However, this should be understood as an abbreviation for Lys(N3). For example, GAR(N3) or GARK(N3) actually means GARK1, K1=Lys(N3) or GlyAlaArgLys(N3).

[0028] [Figure 9-1] FIG. 9 shows further linkers suitable for MTG-mediated conjugation to native antibodies. [Figure 9-2] FIG. 9 shows further linkers suitable for MTG-mediated conjugation to native antibodies. [Figure 9-3] Figure 9 shows further linkers suitable for MTG-mediated conjugation to native antibodies. These linker structures contain a linking moiety (azide, N3) suitable for click chemistry-based attachment of a functional payload in a second step, or a Cys residue that provides a thiol group suitable for attachment to a maleimide. Because these structures are based on peptides whose chemistry is well understood and assembled from single amino acid building blocks, new linkers can be rapidly and easily synthesized and evaluated. Table 2 below provides an overview: [Table 2]

[0029] [Figure 10] Figure 10 shows that the light chain of the IgG1 antibody is not modified by conjugation. The deconvoluted LC-MS spectrum of the IgG1 light chain is shown.

[0030] [Figure 11-1] Figure 11A shows the deconvoluted LC-MS spectrum of the Trastuzumab native IgG1 heavy chain selectively modified with the N3-functional linker GGARK(N3). The spectrum shows that the heavy chain was selectively and quantitatively (>95%) modified with only one peptide linker, since the observed mass difference corresponds to the expected peptide mass shift (Mw unmodified heavy chain = 50595 Da, expected Mw = 51091 Da, measured Mw = 51092 Da). Figure 11B shows the deconvoluted LC-MS spectrum of the Trastuzumab native IgG1 heavy chain selectively clicked with DBCO-PEG4-Ahx-DM1 to the N3-functional linker GGARK(N3) pre-attached to the heavy chain. The spectrum shows that the heavy chain was selectively and quantitatively (>95%) clicked. Figure 11C shows the deconvoluted LC-MS of another IgG1 heavy chain modified with GGARK(N3) under non-optimized conjugation conditions. Conjugation ratio: 83%. [Figure 11-2]Figure 11A shows the deconvoluted LC-MS spectrum of the Trastuzumab native IgG1 heavy chain selectively modified with the N3-functional linker GGARK(N3). The spectrum shows that the heavy chain was selectively and quantitatively (>95%) modified with only one peptide linker, since the observed mass difference corresponds to the expected peptide mass shift (Mw unmodified heavy chain = 50595 Da, expected Mw = 51091 Da, measured Mw = 51092 Da). Figure 11B shows the deconvoluted LC-MS spectrum of the Trastuzumab native IgG1 heavy chain selectively clicked with DBCO-PEG4-Ahx-DM1 to the N3-functional linker GGARK(N3) pre-attached to the heavy chain. The spectrum shows that the heavy chain was selectively and quantitatively (>95%) clicked. Figure 11C shows the deconvoluted LC-MS of another IgG1 heavy chain modified with GGARK(N3) under non-optimized conjugation conditions. Conjugation ratio: 83%.

[0031] [Figure 12] Figure 12A shows the deconvoluted LC-MS of GARK(N3)-modified trastuzumab heavy chain. A conjugation efficiency of over 95% was achieved. Figure 12B shows the deconvoluted LC-MS of GARK(N3)-modified trastuzumab heavy chain clicked with DBCO-PEG4-Ahx-DM1. A click efficiency of over 95% was achieved to give an ADC with DAR2.

[0032] [Figure 13] Figure 13 shows an overview of the Ig CH2 domain according to various numbering schemes. For the purposes of the present invention, EU numbering is used.

[0033] [Figure 14] FIG. 14 shows a transglutaminase reaction to conjugate a linker with an N-terminal Gly residue with a free primary amine to the free primary amine of the Q295 residue of an antibody.

[0034] [Figure 15] Figure 15. Click chemistry reaction scheme to payload-labeled dibenzocyclooctyne (strain-promoted alkyne-azide cycloaddition (SPAAC) to conjugate linker GlyAlaArgLys(N3) (GARK1, K1 = Lys(N3)).

[0035] [Figure 16] FIG. 16 shows a variety of peptide linkers that can be used in the context of the present invention, each of which contains an unnatural amino acid.

[0036] [Figure 17-1]FIG. 17 shows various linker-toxin constructs that can be conjugated to antibodies by the methods described herein. In all cases, the Gly residue has a primary amine for transglutaminase conjugation. FIG. 17A. This figure shows a non-cleavable GGARR-Ahx-May peptide-toxin conjugate with two arginine groups that serve to increase the solubility of the hydrophobic payload maytansine (May). The primary amine of the N-terminal glycine residue serves for conjugation to the antibody via MTG. Because the linker is non-cleavable, the Ahx-spacer serves to cleave the positively charged arginine from May, helping May bind to its target more efficiently. FIG. 17B. This figure shows a non-cleavable GGARR-PEG4-May peptide-toxin conjugate with two arginine groups and a PEG4 spacer, with all three moieties serving to increase the solubility of the hydrophobic payload May. The primary amine of the N-terminal glycine residue serves for conjugation to the antibody via MTG. Since the linker is non-cleavable, PEG4 also helps to cleave the positively charged arginine from May, helping May bind to its target more efficiently. Figure 17C. This figure shows a cleavable GGARR-PEG4-VC-MMAE peptide-toxin conjugate with two arginine groups, a PEG4 spacer, a PABC group, and a val-cit sequence (VC). The primary amine of the N-terminal glycine residue serves for conjugation to the antibody via MTG, the arginine group and the PEG4 spacer serve to increase solubility, and the PABC group and val-cit sequence help to release the toxin. Figure 17D. This figure shows a cleavable GGARR-MMAE peptide-toxin conjugate with two arginine groups and a PABC group, but no PEG spacer or val-cit sequence.Because the GGARR group is inherently degradable by peptidases, a val-cit sequence is not required for toxin release through the self-immolative PABC moiety, and because the two arginine groups are extremely hydrophilic, a PEG spacer is not required, so the entire peptide-toxin conjugate can be kept as small as possible to minimize undesirable interactions with other molecules during blood circulation. [Figure 17-2]FIG. 17 shows various linker-toxin constructs that can be conjugated to antibodies by the methods described herein. In all cases, the Gly residue has a primary amine for transglutaminase conjugation. FIG. 17A. This figure shows a non-cleavable GGARR-Ahx-May peptide-toxin conjugate with two arginine groups that serve to increase the solubility of the hydrophobic payload maytansine (May). The primary amine of the N-terminal glycine residue serves for conjugation to the antibody via MTG. Because the linker is non-cleavable, the Ahx-spacer serves to cleave the positively charged arginine from May, helping May bind to its target more efficiently. FIG. 17B. This figure shows a non-cleavable GGARR-PEG4-May peptide-toxin conjugate with two arginine groups and a PEG4 spacer, with all three moieties serving to increase the solubility of the hydrophobic payload May. The primary amine of the N-terminal glycine residue serves for conjugation to the antibody via MTG. Since the linker is non-cleavable, PEG4 also helps to cleave the positively charged arginine from May, helping May bind to its target more efficiently. Figure 17C. This figure shows a cleavable GGARR-PEG4-VC-MMAE peptide-toxin conjugate with two arginine groups, a PEG4 spacer, a PABC group, and a val-cit sequence (VC). The primary amine of the N-terminal glycine residue serves for conjugation to the antibody via MTG, the arginine group and the PEG4 spacer serve to increase solubility, and the PABC group and val-cit sequence help to release the toxin. Figure 17D. This figure shows a cleavable GGARR-MMAE peptide-toxin conjugate with two arginine groups and a PABC group, but no PEG spacer or val-cit sequence.Because the GGARR group is inherently degradable by peptidases, a val-cit sequence is not required for toxin release through the self-immolative PABC moiety, and because the two arginine groups are extremely hydrophilic, a PEG spacer is not required, so the entire peptide-toxin conjugate can be kept as small as possible to minimize undesirable interactions with other molecules during blood circulation.

[0037] [Figure 18] Figure 18 shows the results of a cytotoxicity assay performed according to Example 3. Her-GARK(N3)(P684) and Her-GGARK(N3)(P579) N-terminal glycine ADCs made by methods according to the invention and containing a May moiety click-conjugated to each linker have potency against SK-BR3 cells similar to Kadcyla. Thus, the advantages offered by the novel linker technology (ease of manufacture, site specificity, stable stoichiometry, no need to deglycosylate the antibody) are not accompanied by disadvantages in terms of cytotoxicity.

[0038] [Figure 19] Figure 19: Structure of βAla-Gly-Ala-Arg-Lys (N3). βAla represents β-alanine, which is structurally similar to glycine. However, the linker has poor conjugation efficiency compared to GGARK (N3) (see Example 2) which has an N-terminal glycine. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] Detailed Description of the Invention Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific components or process steps of the method described, and such devices and methods may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting. It should be noted that, as used in the specification and the appended claims, the singular forms "a", "an" and "the" include singular and / or plural referents unless the context clearly dictates otherwise. It should be further understood that when a parameter range defined by numerical values ​​is given, the range is deemed to include these limits.

[0040] It should be further understood that the embodiments disclosed herein are not intended to be understood as separate embodiments unrelated to each other. Features discussed in one embodiment are intended to be disclosed in the context of other embodiments shown herein. In some cases, if a particular feature is not disclosed in one embodiment but is disclosed in another embodiment, a person skilled in the art will understand that this does not necessarily mean that the feature is not intended to be disclosed in the other embodiment. Although it is the intent of the present application to disclose the feature in other embodiments, merely for the sake of clarity, the following description may be omitted. It should be understood that in order to keep the specification manageable, this is not being done.

[0041] Furthermore, the contents of the documents mentioned herein are incorporated by reference, in particular with reference to documents that disclose standard or routine methods, in which case the incorporation by reference has the primary purpose of providing a fully enabling disclosure and avoiding redundant repetition.

[0042] According to a first aspect, there is provided a method for producing antibody-payload or antibody-linker conjugates by microbial transglutaminase (MTG), comprising the step of transglutaminase (MTG) binding to a peptide structure (shown in N→C orientation) via the N-terminal primary amine of an N-terminal glycine (Gly) residue: Gly-(Aax) m -B-(Aax) n (In the formula, m is an integer between 0 and 12, n is an integer between 0 and 12, m+n≧0, Aax is an amino acid or an amino acid derivative, B is the payload or linkage) to a glutamine (Gln) residue contained in the heavy or light chain of the antibody.

[0043] As used herein, the term "primary amine" refers to an amine substituted with two hydrogen atoms, of the general formula R-NH2.

[0044] In certain embodiments, the peptide linker may comprise two or more linking moieties and / or payloads. That is, the linker may comprise a peptide structure (shown in the N→C orientation): Gly-(Aax) m -B1-(Aax) n -B2-(Aax) o (In the formula, m, n, and o are integers between 0 and 12, m+n+o≧0, Aax is an amino acid or an amino acid derivative, B1 and B2 are payloads and / or linking moieties, and B1 and B2 may be the same or different from each other. may have:

[0045] In other embodiments, the peptide linker may comprise three linking moieties and / or payloads, i.e., the linker may have the peptide structure (shown in the N→C orientation): Gly-(Aax) m -B1-(Aax) n -B2-(Aax) o -B3-(Aax) p (In the formula, m, n, o, and p are integers between 0 and 12, m+n+o+p≧0, Aax is an amino acid or an amino acid derivative, B1, B2 and B3 are payloads and / or linking moieties, and B1, B2 and B3 may be the same or different from each other. may have:

[0046] It should be understood that the present invention also encompasses linkers that contain more than three linking moieties and / or payloads, for example, four, five or six linking moieties and / or payloads. In this case, the peptide structure of the linker may contain two or three linking moieties and / or follows the same pattern as described above for the linker containing the payload.

[0047] In certain embodiments, a method for making antibody-payload conjugates by microbial transglutaminase (MTG) is provided, comprising the step of: cleaving the peptide structure (shown in N→C orientation) via the N-terminal primary amine of the N-terminal glycine (Gly) residue: Gly-(Aax) m -B-(Aax) n (In the formula, m is an integer between 0 and 12, n is an integer between 0 and 12, m+n≧0, Aax is a naturally occurring or non-naturally occurring L- or D-amino acid, or an amino acid derivative or mimetic; B is the payload or linkage) to a glutamine (Gln) residue contained in the heavy or light chain of the antibody.

[0048] In certain embodiments, the present invention provides a method for making antibody-payload or antibody-linker conjugates by microbial transglutaminase (MTG), which comprises ligating the peptide structure (shown in the N→C orientation) Gly-(Aax) m -B-(Aax) n to a glutamine (Gln) residue in the heavy or light chain of the antibody. In this case, it should be understood that moiety B may comprise more than one payload and / or linking moiety. For example, B may be (B'-(Aax) o -B″), where B′ and B″ are payload and / or linking moieties, and o is an integer between 0 and 12. Alternatively, B may represent (B′-(Aax) o -B''-(Aax) p -B'''), where B', B'' and B''' are payload and / or linking moieties, and o and p are integers between 0 and 12, inclusive.

[0049] Thus, in a particular embodiment, the present invention relates to a method according to the present invention, wherein the linker comprises two or more payloads and / or linking moieties. In another embodiment, the present invention relates to a method according to the present invention, wherein the two or more payloads and / or linking moieties B are different from each other.

[0050] That is, the linker according to the present invention can comprise a single payload or a linking moiety. In certain embodiments, the linker comprises two linking moieties, and the two linking moieties are identical. In other embodiments, the linker comprises two linking moieties, and the two linking moieties are different. In yet another embodiment, the linker comprises two identical or different payloads. The present invention further encompasses the linker comprising one or more payloads and one or more linking moieties.

[0051] It should further be understood that not all payloads or linking moieties can function as intrachain payloads or linking moieties, for example because not all payloads or linking moieties have functional groups that form peptide or amide bonds with the C-terminal carboxyl group of a first Aax moiety and the N-terminal amine group of a second Aax moiety. In this case, such payloads or linking moieties are preferably located at the C-terminus of the linker and attached to the carboxyl group of the C-terminal Aax moiety of the linker. When a payload or linking moiety is in an intrachain position of the linker, it is preferred that the payload or linking moiety is an amino acid, an amino acid derivative, or is attached to a molecule having the general structure -NH-CHR-CO-. is preferred.

[0052] In preferred embodiments, m and / or n are 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 11 or more. In other preferred embodiments, m and / or n are 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less. In further preferred embodiments, m+n is 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 11 or more. In still further preferred embodiments, m+n is 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less.

[0053] Members of both ranges can be combined with one another to disclose preferred length ranges having lower and upper limits.

[0054] Thus, in a particular embodiment, the invention relates to a method according to the invention, wherein m+n, and optionally m+n+o and m+n+o+p, are 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less or 4 or less.

[0055] It is important to understand that in the various linker peptides shown herein, the C-terminus may or may not be protected, even if shown otherwise. Protection can be achieved by amidation of the C-terminus. In the context of the present invention, both protected and unprotected linker peptides are encompassed.

[0056] The inventors have shown that this process is suitable for extremely cost-effective and rapid production (24-36 h, or, if desired, 48 h) of site-specific antibody-payload conjugates, thus enabling the generation of large libraries of such molecules and their subsequent screening in high-throughput screening systems.

[0057] In contrast, the Cys engineering process to create antibody-payload conjugates, in which a payload is conjugated to an antibody via an engineered Cys residue, requires at least about 3-4 weeks.

[0058] In general, the method allows multiple payloads to be conjugated to the antibody. For each payload, a suitable peptide linker structure can be identified from a large linker pool to provide optimal clinical and non-clinical characteristics. This is not possible with other methods where the linker structure is fixed. Furthermore, the method according to the present invention allows the creation of antibody-payload conjugates that contain two or more different payloads, each payload being site-specifically conjugated to the antibody. Thus, the method according to the present invention can be used to create antibodies with novel and / or superior therapeutic or diagnostic capabilities.

[0059] The linker may comprise any amino acid, including, but not limited to, α-, β-, γ-, δ-, and ε-amino acids. In the case of α-amino acids, the linker may comprise any naturally occurring L- or D-amino acid. Naturally occurring L- or D-amino acids include any L- or D-amino acid found in nature. That is, the term "naturally occurring L- or D-amino acid" includes all standard or proteinogenic amino acids used as building blocks in naturally occurring proteins. In addition, the term "naturally occurring L- or D-amino acid" includes all non-standard amino acids found in nature, e.g., as metabolic intermediates or degradation products, or as building blocks of other, non-proteinogenic macromolecules. It includes L- or D-amino acids.

[0060] Additionally, the linker may include non-naturally occurring L- or D-amino acids, which include any molecule having the general structure H2N-CHR-COOH not previously found in nature.

[0061] Those skilled in the art are aware of the resources and databases to consult when determining whether an L- or D-amino acid is naturally occurring or non-naturally occurring. However, in case of doubt, it should be understood that the term "naturally occurring or non-naturally occurring L- or D-amino acid" encompasses the L- and D-isomers of any molecule having the general structure H2N-CHR-COOH, regardless of the origin of the molecule.

[0062] In certain embodiments, the linkers of the invention may also include naturally occurring or non-naturally occurring non-chiral amino acids having the general structure H2N-CR1R2-COOH.

[0063] Furthermore, the linker of the present invention may comprise an amino acid derivative. An amino acid derivative is a compound obtained from a naturally occurring or non-naturally occurring amino acid by one or more chemical reactions, for example, chemical reactions of the α-amino group, the α-carboxylic acid group and / or the amino acid side chain. That is, the term amino acid derivative encompasses any molecule having the structure -NH-CHR-CO- obtained from a naturally occurring or non-naturally occurring L- or D-amino acid. Since it is envisaged that the amino acid derivative of the present invention is part of a peptide-based linker, it is preferred that the amino acid derivative is obtained by one or more reactions of the amino acid side chain of a naturally occurring or non-naturally occurring L- or D-amino acid, or the alpha-carboxylic acid group of a naturally occurring or non-naturally occurring L- or D-amino acid, if the amino acid derivative is located at the C-terminus of the peptide. It should be noted that naturally occurring and non-naturally occurring amino acids may be amino acid derivatives, and vice versa.

[0064] Examples of non-standard, non-naturally occurring amino acids and amino acid derivatives that can be included in the linkers of the invention include, but are not limited to, α-aminobutyric acid, α-aminoisobutyric acid, ornithine, hydroxyproline, agmatine, (S)-2-amino-4-((2-amino)pyrimidinyl)butanoic acid, alpha-aminoisobutyric acid, p-benzoyl-L-phenylalanine, t-butylglycine, citruiline, cyclohexylalanine, desaminotyrosine, L-(4-guanidino)phenylalanine, homoarginine, homocysteine, homoserine, homolysine, n-formyltryptophan, norleucine, norvaline, phenylglycine, (S)-4-piperidyl-(N-amidino)glycine, parabenzoyl-L-phenylalanine, sarcosine and 2-thienylalanine.

[0065] In addition to the alpha-amino acids described above, the linker of the present invention may also include one or more β-, γ-, δ- or ε-amino acids. Thus, in certain embodiments, the linker may be a peptidomimetic. A peptidomimetic does not only contain a classical peptide bond formed between two α-amino acids, but may additionally or alternatively include one or more amide bonds formed between an alpha amino acid and a β-, γ-, δ- or ε-amino acid, or between two β-, γ-, δ- or ε-amino acids. An example of a linker that is a peptidomimetic and includes an amide bond between an α-amino acid and a β-amino acid is shown in FIG. 16 (Gly-β-Ala-Arg-Lys(N3)). Thus, in any example of the present invention where the linker is described as a peptide, the linker may also be a peptidomimetic, and thus not only composed of α-amino acids, but may instead include one or more β-, γ-, δ- or ε-amino acids or molecules that are not classified as amino acids. It should be understood that examples of β-, γ-, δ- or ε-amino acids that may be included in the linkers of the present invention include, but are not limited to, β-alanine, γ-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, 6-aminohexanoic acid and statins.

[0066] The term "D-amino acid" is understood to include the D-counterparts of both naturally occurring and non-naturally occurring amino acids.

[0067] Since the peptide linkers of the present invention are peptide-based, they may be hydrolyzed by host cell peptidases once the antibody-payload conjugate is internalized into the target cell. Thus, in certain embodiments, the linker does not necessarily need to include a cathepsin cleavage site. Thus, in one embodiment, the linker that includes or has a peptide structure is not cleavable by cathepsin. This includes, in particular, cathepsin B. In one further embodiment, the linker that includes or has a peptide structure does not include a valine-alanine motif or a valine-citrulline motif. However, it should be understood that the present invention also encompasses linkers that include a cathepsin cleavage site, such as valine-alanine or valine-citrulline. For example, linkers that include non-standard or D-amino acids cannot be efficiently cleaved by host cell peptidases. In this case, the cathepsin cleavage site in the linker may improve the release of the payload after internalization into the host cell. If necessary, the linker may further include other motifs or self-immolative groups that allow efficient release of the payload in the target cell.

[0068] One typical dipeptide structure used in ADC linkers, but lacking a Lys residue, is provided, for example, in brentuximab vedotin and is discussed in Dubowchik and Firestone 2002. The valine-citrulline motif provided by SGN-CD33A is a linker that can be cleaved by cathepsin B to release the toxin at the disease site. The same applies to the valine-alanine motif provided by SGN-CD33A, for example.

[0069] In a further embodiment, the linker does not comprise polyethylene glycol or a polyethylene glycol derivative.

[0070] Polyethylene glycol (PEG) is a polyether compound that has many applications from industrial manufacturing to medicine. Depending on its molecular weight, PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE). The structure of PEG is H-(O-CH2-CH2) n It is commonly represented as -OH. However, it should be understood that the linkers of the present invention can include PEG or PEG derivatives.

[0071] Thus, since B can be either a payload or a linking moiety, it is important to understand that the method according to the invention has two main embodiments, as shown in Table 3 below: [Table 3]

[0072] That is, in certain embodiments, the payload is attached to the linker by chemical synthesis. Thus, the linker has the structure Gly-(Aax) m -Payload or Gly-(Aax) m -Payload-(Aax) n For example, the payload can be attached to the C-terminus of the peptide by chemical synthesis. Thus, in certain embodiments, the linker can have the structure Gly-Ala-Arg-payload, Gly-Ala-Arg-Arg-payload, Gly-Gly-Ala-Arg-payload, Gly-Gly-Ala-Arg-Arg-payload, or Gly-Gly-Gly-payload.

[0073] According to a further embodiment of the invention, the antibody comprises IgG, IgE, IgM, IgD, IgA and IgY IgG1, IgG2, IgG3, IgG4, IgA1 and IgA, and / or · Retains target binding properties and C H 2 domain, fragments or recombinant variants thereof At least one selected from the group consisting of:

[0074] The antibody is preferably a monoclonal antibody.

[0075] The antibodies may be of human origin, but may also be from mouse, rat, goat, donkey, hamster or rabbit. If the conjugate is therapeutic, the mouse or rabbit antibodies may be chimeric or humanized as appropriate.

[0076] C H Fragments or recombinant variants of antibodies comprising two domains can be, for example: Antibody formats containing only the heavy chain domain (Shark Antibody / IgNAR(V H -C H 1-C H 2-C H 3-C H 4-C H 5)2 or camel antibody / hcIgG(V H -C H 2-C H 3)2) scFv-Fc(VH-VL-CH2-CH3)2 Fc fusion peptides, comprising an Fc domain and one or more receptor domains. It is.

[0077] Antibodies may also be bispecific (e.g., DVD-IgG, cross-Mab, adjunct IgG-HC fusions) or biparatopic. For an overview, see Brinkmann and Kontermann (2017).

[0078] Thus, in a particular embodiment, the invention relates to a method for the treatment of cancer, comprising administering to a patient a therapeutically effective amount of an antibody, the antibody being an IgG, IgE, IgM, IgD, IgA or IgY antibody, or a fragment or recombinant variant thereof, which fragment or recombinant variant retains target binding properties and is H The present invention relates to a method according to the present invention, comprising:

[0079] In a preferred embodiment, the antibody is an IgG antibody. That is, the antibody can be an IgG antibody, preferably glycosylated at residue N297. Alternatively, the antibody can be a deglycosylated antibody, preferably with glycan cleaved at residue N297 with enzyme PNGaseF. Furthermore, the antibody can be an aglycosylated antibody, preferably with residue N297 replaced with a non-asparagine residue. Methods for deglycosylating antibodies and for producing aglycosylated antibodies are known in the art.

[0080] As discussed herein, IgG antibodies glycosylated at residue N297 have several advantages over non-glycosylated antibodies. Moreover, it has been demonstrated that the linkers of the present invention can be conjugated with unexpectedly high efficiency to antibodies glycosylated at residue N297. Thus, in an even more preferred embodiment, the antibody is H It is an IgG antibody that is glycosylated at residue N297 (EU numbering) in domain 2.

[0081] In a particular embodiment, the invention relates to a method according to the invention, wherein (a) a linker comprising a payload or a linking moiety B is conjugated to a Gln residue introduced into the heavy or light chain of an antibody by molecular engineering, or (b) a linker comprising a payload or a linking moiety B is conjugated to a Gln residue in the Fc domain of an antibody.

[0082] According to a further embodiment of the invention, the payload or linking moiety is conjugated to a Gln residue that has been introduced into the heavy or light chain of the antibody by molecular engineering.

[0083] The term "molecular engineering" as used herein relates to the use of molecular biology methods to manipulate nucleic acid sequences. In the present invention, molecular engineering can be used to introduce Gln residues into the heavy or light chains of an antibody. In general, two different strategies for introducing Gln residues into the heavy or light chains of an antibody are envisioned in the present invention. First, a single residue of the heavy or light chain of an antibody can be replaced with a Gln residue. Second, a Gln-containing peptide tag consisting of two or more amino acid residues can be incorporated into the heavy or light chain of an antibody. To this end, the peptide tag can be incorporated into an internal position of the heavy or light chain, i.e., between two existing amino acid residues of the heavy or light chain, or the peptide tag can be fused (added) to the N-terminus or C-terminus of the heavy or light chain of an antibody.

[0084] In the first case, any amino acid residue in the heavy or light chain of the antibody can be replaced with a Gln residue, in which case the resulting antibody can be conjugated with a linker of the invention by microbial transglutaminase. In certain embodiments, the antibody has a C of an IgG antibody. HThe antibody has a substitution at amino acid residue N297 (EU numbering) of the 2 domain, in particular the substitution is N297Q substitution. The antibody comprising N297Q mutation can be conjugated to more than one linker per heavy chain of the antibody. For example, the antibody comprising N297Q mutation can be conjugated to four linkers, one linker is conjugated to residue Q295 of the first heavy chain of the antibody, one linker is conjugated to residue N297Q of the first heavy chain of the antibody, one linker is conjugated to residue Q295 of the second heavy chain of the antibody, and one linker is conjugated to residue N297Q of the second heavy chain of the antibody. Those skilled in the art know that by replacing residue N297 of an IgG antibody with a Gln residue, an aglycosylated antibody can be obtained.

[0085] In certain embodiments, the present invention relates to a method for the preparation of a polypeptide of the present invention, wherein the Gln residue introduced into the heavy or light chain of the antibody by molecular engineering is (a) incorporated into the heavy or light chain of the antibody, or (b) contained in a peptide fused to the N-terminus or C-terminus of the heavy or light chain of the antibody. This relates to a method according to the present invention.

[0086] Thus, instead of replacing a single amino acid residue of an antibody, a peptide tag containing a transglutaminase-accessible Gln residue can be introduced into the heavy or light chain of the antibody. Such a peptide tag can be fused to the N-terminus or C-terminus of the heavy or light chain of the antibody. Preferably, a peptide tag containing a transglutaminase-accessible Gln residue is fused to the C-terminus of the heavy chain of the antibody. Even more preferably, a peptide tag containing a transglutaminase-accessible Gln residue is fused to the C-terminus of the heavy chain of an IgG antibody. Several peptide tags that can be fused to the C-terminus of an antibody heavy chain and serve as substrates for microbial transglutaminase have been described in WO2012 / 059882, WO2016 / 144608, WO2016 / 100735, WO2016 / 096785, as well as by Steffen et al. (JBC, 2017) and Malesevic et al. (Chembiochem, 2015).

[0087] Exemplary peptide linkers that may be introduced into the heavy or light chain of an antibody, in particular fused to the C-terminus of the heavy chain of the antibody, are LLQGG, LLQG, LSLSQG, GGGLLQGG, GLLQG, LLQ, GSPLAQSHGG, GLLQGGG, GLLQGG, GLLQ, LLQLLQGA, LLQGA, LLQYQGA, LLQGSG, LLQYQG, LLQLLQG, SLLQG, LLQLQ, LLQLLQ, LLQGR, EEQYASTY, EEQYQSTY, EEQYNSTY, EEQYQS, EEQYQST, EQYQSTY, QYQS, QYQSTY, YRYRQ, DYALQ, FGLQRPY, EQKLISEEDL, LQR and YQR.

[0088] Those skilled in the art can easily substituted amino acid residues in an antibody by the method of molecular cloning described, for example, in Sambrook, Joseph. (2001). Molecular cloning: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. We know how to introduce peptide tags into antibodies.

[0089] According to a further embodiment of the invention, the payload or linking moiety is conjugated to a Gln in the Fc domain of the antibody.

[0090] That is, a linker of the invention can be conjugated to any Gln residue in the Fc domain of an antibody that can serve as a substrate for microbial transglutaminase.

[0091] Typically, the term Fc domain, as used herein, refers to the last two constant region immunoglobulin domains (Cc domains) of IgA, IgD and IgG. H 2 and C H 3), and the last three constant region domains of IgE, IgY and IgM (C H 2. C H 3 and C H 4) That is, the linker containing the payload or the linking moiety B is attached to the C of the antibody. H 2. C H 3, and, where applicable, C H It can be conjugated to the 4 domain.

[0092] According to a further embodiment of the invention, the payload or linking moiety is H In a particular embodiment, the present invention relates to an antibody conjugated to a Gln residue Q295 (EU numbering) in the Fc domain of an IgG. H The present invention relates to a method according to the present invention, in which the Gln residue Q295 (EU numbering) of domain 2 is

[0093] It is important to understand that Q295 is a highly conserved amino acid residue in IgG type antibodies. It is conserved in human IgG1, 2, 3, 4, and especially rabbit and rat antibodies. Thus, the ability to use Q295 is a valuable tool in the design of therapeutic antibody-payload conjugates or diagnostic conjugates, where the antibody is typically of non-human origin. This is a considerable advantage for creating gates. Thus, the method according to the invention provides a very versatile and widely applicable tool. Residue Q295 is highly conserved among IgG type antibodies, but some IgG type antibodies, such as mouse IgG2a or IgG2b, do not have this residue. Therefore, it is preferable that the antibody used in the method of the invention is preferably C H It should be understood that this is an IgG type antibody that contains residue Q295 (EU numbering) in domain 2.

[0094] Furthermore, engineered conjugates using Q295 for payload attachment demonstrated good pharmacokinetics and efficacy (Lhospice et al. 2015), and were not found to be unstable and prone to degradation. It has been shown that some bacteria can also have toxins (Dorywalska et al. 2015). It is expected that a similar effect will be seen with this site-specific method, since the same residues are modified, but in glycosylated antibodies. As glycosylation may further contribute to overall ADC stability, removal of glycan moieties, such as by the mentioned approach, has been shown to result in less stable antibodies (Zheng et al. 2011).

[0095] According to a further embodiment of the invention, the antibody to which the payload or linking moiety is conjugated is glycosylated.

[0096] Typical IgG antibodies are C H It is N-glycosylated at position N297 (Asp-X-Ser / Thr-motif) in the 2 domain.

[0097] Thus, in certain embodiments, the present invention provides an antibody comprising an Fc domain in which the Gln residue is C H C of an IgG antibody that is glycosylated at residue N297 (EU numbering) in the 2 domain H The present invention relates to a method according to the present invention, in which the Gln residue Q295 (EU numbering) of domain 2 is

[0098] C of the linker by transglutaminase H In the literature discussing conjugation to 2 Gln residues, the focus is on small, low molecular weight substrates. However, the prior art literature always describes the need for a deglycosylation step at N297 position or the use of an aglycosylated antibody to achieve such conjugation (WO2015 / 015448; WO2017 / 025179; WO2013 / 092998).

[0099] However, quite surprisingly and contrary to all expectations, site-specific conjugation of glycosylated antibodies to Q295 is indeed efficiently possible by using the oligopeptide structures discussed above.

[0100] Q295 is very close to N297, which is glycosylated in its native state, but the method according to the invention allows for the conjugation of a linker or payload thereto using a specified linker.

[0101] However, as shown, the method according to the invention does not require prior enzymatic deglycosylation of Q295, nor the use of an aglycosylation antibody, nor the substitution of N297 for another amino acid, nor the introduction of a T299A mutation to prevent glycosylation.

[0102] These two points provide significant advantages in terms of production: an enzymatic deglycosylation step is undesirable from a GMP standpoint, since it requires the removal of both the deglycosylation enzyme (e.g., PNGase F) and the cleaved glycans from the medium.

[0103] Furthermore, genetic engineering of antibodies for payload attachment is not required, resulting in improved immunity. Sequence insertions that may increase immunogenicity and decrease the overall stability of the antibody can be avoided.

[0104] Substitution of N297 for another amino acid can also result in increased antibody aggregation and reduced solubility, which is particularly important for hydrophobic payloads such as PBDs (Zheng et al. 2011), resulting in reduced overall stability of the entire Fc domain (Subedi et al, 2015) and reduced conjugation. This has undesirable effects since it may affect the efficacy of the entire gate. Furthermore, the glycans present on N297 have important immunomodulatory effects since they induce antibody-dependent cellular cytotoxicity (ADCC) and the like. These immunomodulatory effects would be lost with deglycosylation or any of the other approaches discussed above to obtain aglycosylated antibodies. Furthermore, any sequence modification of an established antibody may result in regulatory issues, which is problematic since often accepted, clinically validated antibodies are used as starting points for ADC conjugation.

[0105] Thus, the method according to the invention makes it possible to generate stoichiometrically well-defined ADCs with site-specific payload attachment easily and without drawbacks.

[0106] In view of the above, the method of the present invention preferably comprises the steps of: H It is stated that the antibody is used for conjugation at residue Q295 (EU numbering) of the C2 domain, where the antibody is H The antibody is glycosylated at residue N297 (EU numbering) of domain 2. However, it is explicitly stated that the method of the invention also encompasses the conjugation of deglycosylated or aglycosylated antibodies at residue Q295 or any other suitable Gln residue of the antibody, where the Gln residue may be an endogenous Gln residue or a Gln residue that has been introduced by molecular engineering.

[0107] The present invention also encompasses the conjugation of antibodies of isotypes other than IgG antibodies, such as IgA, IgE, IgM, IgD or IgY antibodies, which may be at endogenous Gln residues, such as endogenous Gln residues in the Fc domain of the antibody, or at Gln residues that have been introduced into the antibody by molecular engineering.

[0108] Generally, a person skilled in the art knows how to determine at which position of an antibody a linker is conjugated. For example, the conjugation site can be determined by proteolytic digestion of the antibody-payload conjugate and LC-MS / MS analysis of the resulting fragments. For example, the sample can be deglycosylated with GlycINATOR (Genovis) according to the respective instruction manual and then digested with Trypsin Gold (mass spectrometry grade, Promega). Thus, 1 μg of protein can be incubated with 50 ng of trypsin at 37° C. overnight. LC-MS / MS analysis can be performed using a nanoAcquity HPLC system connected to a Synapt-G2 mass spectrometer (Waters). For this, 100 ng of peptide solution can be loaded onto an Acquity UPLC Symmetry C18 trap column (Waters, part number 186006527) and trapped for 3 min with 1% buffer A (water, 0.1% formic acid) and 99% buffer B (acetonitrile, 0.1% formic acid) at a flow rate of 5 μL / min. Peptides can then be eluted with a linear gradient of 3% to 65% buffer B within 25 min. Data can be acquired in a mass range of 50-2000 m / z in positive polarity decomposition mode. Other instrument settings can be as follows: capillary voltage 3,2 kV, sampling cone 40 V, extraction cone 4.0 V, source temperature 130 °C, cone gas 35 L / h, nanoflow gas 0.1 bar and purge gas 150 L / h. The mass spectrometer can be calibrated with [Glu1]-fibrinopeptide.

[0109] Furthermore, those skilled in the art know how to determine the drug to antibody (DAR) ratio or payload to antibody ratio of an antibody-payload construct. For example, the DAR can be determined by hydrophobic interaction chromatography (HIC) or LC-MS.

[0110] For hydrophobic interaction chromatography (HIC), samples can be adjusted to 0.5 M ammonium sulfate and evaluated via a MAB PAK HIC Butyl column (5 μm, 4.6×100 mm, Thermo Scientific) using a full gradient from A (1.5 M ammonium sulfate, 25 mM Tris HCl, pH 7.5) to B (20% isopropanol, 25 mM Tris HCl, pH 7.5) over 20 minutes at 1 mL / min and 30° C. Typically, 40 μg of sample can be used and signals can be recorded at 280 nm. Relative HIC retention times (HIC-RRT) can be calculated by dividing the absolute retention times of the ADC DAR2 species by the retention times of the respective unconjugated mAbs.

[0111] For LC-MS DAR determination, the ADC can be diluted with NH4HCO3 to a final concentration of 0.025 mg / mL. 40 μL of this solution can then be reduced with 1 μL of TCEP (500 mM) for 5 min at room temperature and then alkylated by adding 10 μL of chloroacetamide (200 mM) followed by overnight incubation at 37 °C in the dark. For reversed-phase chromatography, a Dionex U3000 system in combination with the software Chromeleon can be used. The system can be equipped with a RP-1000 column (1000 Å, 5 μm, 1.0 × 100 mm, Sepax) heated to 70 °C and a UV detector set at a wavelength of 214 nm. Solvent A can consist of water + 0.1% formic acid, and solvent B can include 85% acetonitrile + 0.1% formic acid. The reduced and alkylated sample can be loaded onto the column and separated by a gradient of 30-50% solvent B over 14 min. The liquid chromatography system can be connected to a Synapt-G2 mass spectrometer to identify the DAR species. The mass spectrometer capillary voltage can be set to 3 kV, the sampling cone to 30 V, and the extraction cone to a total value of 5 V. The source temperature can be set to 150 °C, the desolvation temperature to 500 °C, the cone gas to 20 l / h, the desolvation gas to 600 l / h, and the acquisition can be performed in positive mode, with a 1 s scan time, and in the mass range of 600-5000 Da. The instrument can be calibrated with sodium iodide. Deconvolution of the spectra can be performed with the MaxEnt1 algorithm of MassLynx until convergence. After the assignment of the DAR species to the chromatographic peaks, the DAR can be calculated based on the integrated peak areas of the reversed-phase chromatograms.

[0112] According to a further embodiment of the invention, the net charge of the linker is neutral or positive.

[0113] The net charge of a peptide is usually calculated at neutral pH (7.0). In the simplest approach, the net charge is determined by adding the number of positively charged amino acid residues (Arg and Lys and, optionally, His) and the number of negatively charged amino acid residues (Asp and Glu) and calculating the difference between the two groups. If the linker contains non-standard amino acids, the skilled artisan knows how to determine the charge of the non-standard amino acid at neutral pH.

[0114] According to a further embodiment of the invention, the linker does not contain any negatively charged amino acid residues.

[0115] Preferably, the oligopeptide is also negatively charged at the negatively charged amino acid residues Glu and Asp. It also does not contain any charged non-standard amino acids.

[0116] According to a further embodiment of the invention, the linker comprises a positively charged amino acid residue.

[0117] According to one embodiment of the invention, the linker comprises: - ricin or a ricin derivative or a ricin mimetic, Arginine, and / or Histidine and wherein the amino acid residues are selected from the group consisting of:

[0118] In certain embodiments, the linker is - ricin or a ricin derivative or a ricin mimetic, Arginine, and Histidine and wherein the amino acid residue is selected from the group consisting of:

[0119] In certain embodiments, the linker is Lysine, Arginine, and Histidine and wherein the amino acid residue is selected from the group consisting of:

[0120] In certain embodiments, the linker is Arginine, and Histidine and wherein the amino acid residue is selected from the group consisting of:

[0121] In certain embodiments, the linker comprises at least one arginine residue.

[0122] Table 8 shows that linkers with negative, neutral and positive net charges can be conjugated to glycosylated antibodies using the methods of the present invention. In particular, linkers containing a positively charged arginine residue can be conjugated to glycosylated antibodies with high efficiency.

[0123] That is, in certain embodiments, the linker according to the present invention has a neutral or positive net charge. In certain embodiments, the linker according to the present invention has a neutral or positive net charge and comprises at least one arginine and / or histidine residue. In certain embodiments, the linker according to the present invention has a neutral or positive net charge and comprises at least one arginine residue. In certain embodiments, the linker according to the present invention does not comprise a lysine residue. In certain embodiments, the linker according to the present invention has a neutral or positive net charge and comprises no lysine residue.

[0124] Table 8 further shows that a linker having the amino acid sequence Gly-[Gly / Ala]-Arg-B can be efficiently conjugated to a glycosylated antibody. Thus, in certain embodiments, a linker according to the invention has the sequence Gly-[Gly / Ala]-Arg-B or Gly-[Gly / Ala]-Arg-B-(Aax) n has.

[0125] In certain embodiments, the linker comprising one or more linking moieties B is GDC, G RCD, GRDC, GGDC, GGCD, GGEC, GGK(N3)D, GGRCD, GGGDC, GC, GRC, GGRC, GRAC, GARC, GGHK(N3), GGK(N3)RC, GARK(N3) and GGARK(N3). In a preferred embodiment, the linker comprising one or more linking moieties B is selected from the group consisting of GGK(N3)D, GGRCD, GC, GRC, GGRC, GARC, GGK(N3)RC, GARK(N3) and GGARK(N3). In a more preferred embodiment, the linker comprising one or more linking moieties B is selected from the group consisting of GGRCD, GC, GGRC, GARC, GGK(N3)RC, GARK(N3) and GGARK(N3). In a most preferred embodiment, the linker comprising one or more linking moieties B is selected from the group consisting of GC, GGRC, GARC and GGARK(N3). In certain embodiments, the linker comprising one or more linking moieties B is GGGK(N3).

[0126] According to a further embodiment of the invention, the antibody is H 2 domain contains Asn residue N297 (EU numbering).

[0127] According to a further embodiment of the invention, the N297 residue is glycosylated.

[0128] According to a further embodiment of the invention, the linker comprising the payload or the linking moiety B is conjugated to the amide side chain of the Gln residue, i.e. the amide side chain of the Gln residue of the antibody is conjugated to the N-terminal amino group of the linker via an isopeptide bond.

[0129] According to one further embodiment of the present invention, the microbial transglutaminase is derived from a Streptomyces species, in particular Streptomyces mobaraensis, and preferentially has 80% sequence identity with the native enzyme. Thus, MTG may be the native enzyme or an engineered variant of the native enzyme. As shown in Figure 8, high conjugation efficiency was obtained with native MTG variants that are not optimized for conjugation of glycosylated antibodies.

[0130] One such microbial transglutaminase is commercially available from Zedira (Germany). It is recombinantly produced in E. coli. Streptomyces mobaraensis transglutaminase has the amino acid sequence disclosed in SEQ ID NO: 48. S. mobaraensis MTG variants with other amino acid sequences have been reported and are also encompassed by the present invention (SEQ ID NOs: 28 and 49).

[0131] In another embodiment, the microbial transglutaminase Streptomyces ladakanum (formerly known as Streptoverticillium ladakanum) is used. Streptomyces ladakanum transglutaminase (US Pat. No. 6,660,510 (B2)) has the amino acid sequence disclosed in SEQ ID NO:27.

[0132] Both of the above transglutaminases may be sequence modified. In some embodiments, transglutaminases having 80% or more sequence identity with SEQ ID NOs: 27, 28, 48 and 49 may be used.

[0133] Another suitable microbial transglutaminase is commercially available from Ajinomoto and is called ACTIVA TG. Compared to the transglutaminase from Zedira, ACTIVA TG lacks four N-terminal amino acids but has similar activity.

[0134] Further microbial transglutaminases that can be used in the context of the present invention are described in Kieliszek and Misiewicz 2014, the contents of which are incorporated herein by reference in their entirety. As disclosed in WO2015 / 191883A1, WO2008 / 102007A1 and U.S. Patent Application Publication No. 2010 / 0143970.

[0135] In certain embodiments, a mutant variant of microbial transglutaminase is used for conjugating the linker to the antibody. That is, the microbial transglutaminase used in the methods of the present invention can be a variant of S. mobaraensis transglutaminase as set forth in SEQ ID NO: 27 or 29. In certain embodiments, the recombinant S. morabaensis transglutaminase as set forth in SEQ ID NO: 29 is a variant of the mutant G25 In certain embodiments, the recombinant S. morabaensis transglutaminase set forth in SEQ ID NO: 29 comprises the mutations G250D and E300D. In the form of the recombinant S. morabaensis transglutaminase shown in SEQ ID NO:29, , D4E and G250D. In one particular embodiment, the recombinant S. morabaensis transglutaminase set forth in SEQ ID NO:29 comprises the mutations E120A and G250D. In one particular embodiment, the recombinant S. morabaensis is shown in SEQ ID NO:29. The transglutaminase comprises the mutations A212D and G250D. In certain embodiments, the recombinant S. morabaensis transglutaminase set forth in SEQ ID NO:29 comprises the mutations A212D and G250D. Contains the mutations G250D and K327T.

[0136] The microbial transglutaminase can be added to the conjugation reaction at any concentration that allows for efficient conjugation of the antibody and the linker. In certain embodiments, the microbial transglutaminase can be added to the conjugation reaction at a concentration of less than 100 U / mL, 90 U / mL, 80 U / mL, 70 U / mL, 60 U / mL, 50 U / mL, 40 U / mL, 30 U / mL, 20 U / mL, 10 U / mL, or 7 U / mL.

[0137] The method according to the present invention includes the use of microbial transglutaminase. However, it should be noted that the equivalent reaction can also be carried out by an enzyme containing transglutaminase activity of non-microbial origin. Thus, the antibody-payload conjugate according to the present invention can also be produced using an enzyme containing transglutaminase activity of non-microbial origin.

[0138] To obtain efficient conjugation, it is preferred to add the linker in molar excess to the antibody, i.e., in certain embodiments, the antibody is mixed with at least a 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 molar equivalent excess of peptide linker relative to the antibody.

[0139] The method according to the invention is preferably carried out at a pH in the range of 6 to 8.5. Examples 1 and 2 show that the conjugation efficiency is highest at pH 7.6. Thus, in a preferred embodiment, the invention relates to a method according to the invention, wherein the conjugation of the linker to the antibody is achieved at a pH in the range of 6 to 8.5, more preferably at a pH in the range of 7 to 8. In a most preferred embodiment, the invention relates to a method according to the invention, wherein the conjugation of the linker to the antibody is achieved at pH 7.6.

[0140] The method of the present invention can be carried out in any buffer suitable for conjugating the linker or linker-payload construct to an antibody according to the method of the present invention. Buffers suitable for the method of the present invention include, but are not limited to, Tris, MOPS, HEPES, PBS or Bis-Tris. Furthermore, the buffer can contain any salt concentration suitable for carrying out the method of the present invention. For example, the buffer used in the method of the present invention can be 150 mM or less, 140 mM or less, or 150 mM or less. The salt concentration may be 1 mM or less, 130 mM or less, 120 mM or less, 110 mM or less, 100 mM or less, 90 mM or less, 80 mM or less, 70 mM or less, 60 mM or less, 50 mM or less, 40 mM or less, 30 mM or less, 20 mM or less, 10 mM or less, or 1 mM. In certain embodiments, the buffer may be salt-free.

[0141] It should be noted that optimal reaction conditions (e.g., pH, buffer, salt concentration) may vary between payloads and may depend, to some extent, on the physicochemical properties of the linker and / or payload. However, undue experimentation is not required by one of ordinary skill in the art to identify suitable reaction conditions for carrying out the methods of the present invention.

[0142] According to a further embodiment of the invention, the linking moiety B is Bioorthogonal marker groups, or Other non-bio-orthogonal entities for crosslinking At least one selected from the group consisting of:

[0143] In certain embodiments of the invention, the linking moiety B is Bioorthogonal marker groups, or Non-bio-orthogonal entities for crosslinking Includes.

[0144] The term "bio-orthogonal marker group" was established by Sletten and Bertozzi (2011) to indicate a reactive group that can result in a chemical reaction occurring inside a biological system without interfering with the natural biochemical process. A "non-bio-orthogonal entity for crosslinking" can be any molecule that contains or consists of a first functional group, which can be crosslinked chemically or enzymatically to a payload that contains a compatible second functional group. Even if the crosslinking reaction is a non-bio-orthogonal reaction, it is preferred that the reaction does not introduce additional modifications to the antibody other than the crosslinking of the payload to the linker. In view of the above, the linking moiety B can consist of a "bio-orthogonal marker group" or a "non-bio-orthogonal entity" or can include a "bio-orthogonal marker group" or a "non-bio-orthogonal entity". For example, in the case of the linking moiety Lys(N3), both the entire Lys(N3) and the azide group alone can be considered as a bio-orthogonal marker group in the present invention.

[0145] According to a further embodiment of the invention, the bioorthogonal marker group or the non-bioorthogonal entity comprises: -NN≡N or -N3 Lys(N3) Tetrazine Alkynes DBCO ·BCN Norborene Trans-cyclooctene -RCOH (aldehyde) Acyl trifluoroborates -SH, and Cysteine At least one selected from the group consisting of:

[0146] These groups may, for example, participate in any of the coupling reactions shown in Table 4: [Table 4]

[0147] In Table 4 above, the linking moiety may be or may include the so-called "binding partner 1" or "binding partner 2" therein.

[0148] According to a further embodiment of the invention, linking moiety B is a Cys residue having a free sulfhydryl group.

[0149] The free sulfhydryl group of such a Cys residue (or derivative) can be used to conjugate a maleimide-containing linker toxin construct thereto. See Figure 5 for further details of some of the conjugation reactions and some potential linker constructs.

[0150] Toxins containing maleimide linkers, such as Adcetris, are frequently used and approved by medical authorities. Thus, drugs containing MMAE toxins are conjugated to linkers containing (i) a p-aminobenzyl spacer, (ii) a dipeptide, and (iii) a maleimidocaproyl linker, which allows the construct to be conjugated to the free sulfhydryl group of a Cys residue of an antibody.

[0151] Thus, providing a Cys residue in a linker according to the invention has the advantage that an antibody-payload construct can be made using a pre-made toxin-maleimide construct, or more generally, the advantages of Cys-maleimide conjugation chemistry can be fully utilized, while at the same time pre-made antibodies can be used that do not need to be deglycosylated.

[0152] In specific embodiments, the Cys residue is at the C-terminus of the peptide linker, or intrachain.

[0153] In another embodiment, the linking moiety B comprises an azide group. Those skilled in the art are aware of molecules that comprise an azide group, such as 6-azido-lysine (Lys(N3)) or 4-azido-homoalanine (Xaa(N3)), that can be incorporated into the linker according to the present invention. Linking moieties that comprise an azide group can be used as substrates in various bioorthogonal reactions, such as strain-promoted azide-alkyne cycloaddition (SPAAC), copper-catalyzed azide-alkyne cycloaddition (CuAAC) or Staudinger ligation. For example, in certain embodiments, payloads that comprise cyclooctene derivatives, such as DBCO, can be attached to linkers that comprise an azide group by SPAAC (see FIG. 15).

[0154] In yet another embodiment, the linking moiety B comprises a tetrazine. Those skilled in the art are aware of tetrazine-containing molecules, preferably amino acid derivatives containing a tetrazine group, that can be incorporated into the linker according to the present invention (see, for example, FIG. 7A). The linking moiety containing a tetrazine can be used as a substrate in bioorthogonal tetrazine ligation. For example, in certain embodiments, a payload containing a cyclopropene, norborene or cyclooctyne group, such as bicyclo[6.1.0]nonyne (BCN), can be attached to a linker containing a tetrazine group.

[0155] The present invention further encompasses linkers comprising two different bioorthogonal marker groups and / or non-bioorthogonal entities. For example, a linker according to the present invention may comprise a linking moiety comprising an azide, such as Lys(N3) or Xaa(N3), and a linking moiety comprising a sulfhydryl, such as cysteine. In certain embodiments, a linker according to the present invention may comprise a linking moiety comprising an azide, such as Lys(N3) or Xaa(N3), and a linking moiety comprising a tetrazine, such as a tetrazine-modified amino acid. In certain embodiments, a linker according to the present invention may comprise a linking moiety comprising a sulfhydryl, such as cysteine, and a linking moiety comprising a tetrazine, such as a tetrazine-modified amino acid. A linker comprising two different bioorthogonal marker groups and / or non-bioorthogonal entities has the advantage that it can accommodate two separate payloads, thus obtaining an antibody-payload conjugate comprising more than one payload.

[0156] According to one further embodiment of the invention it is provided that when B is a linking moiety, a further step of linking the actual payload to the linking moiety is performed.

[0157] between azides and cyclooctynes ​​(also called copper-free click chemistry, Baskin et al. (2007)) and between nitrones and cyclooctynes ​​(Ning et al. (2010)). ,3-Dipolar cycloaddition, oxime / hydrazone formation from aldehydes and ketones (Yarema, et al (1998)), tetrazine ligation (Blackman et al (2008)), isothiocyanate ligation (Ito et al (2009)), and cycloaddition of aldehydes and ketones (Yarema, et al (1998)). sononitrile-based click reactions (Stoeckmann et al. (2011)) and more recently quadricyclane ligation (Sletten & Bertozzi (JACS, 2011)), copper(I) Catalytic azide-alkyne cycloaddition (CuAAC, Kolb & Sharpless (2003)), strain-promoted azide-alkyne cycloaddition (SPAAC, Agard et al (2004)) or strain-promoted Biomolecular Biology, including alkyne-nitrone cycloaddition (SPANC, MacKenzie et al (2014)) Several chemical ligation strategies have been developed that meet the requirements of orthogonality.

[0158] All these documents are incorporated herein by reference in order to provide a sufficient enabling disclosure and to avoid redundant repetition.

[0159] It should be understood that preferably, the linker according to the invention is conjugated to the Gln residue of the antibody by microbial transglutaminase, followed by binding of the payload to the bioorthogonal marker group or non-bioorthogonal entity of said linker. However, the present invention also encompasses antibody-payload conjugates in which, in a first step, the payload is bound to a linker comprising a linking moiety, and in a second step, the resulting linker-payload construct is conjugated to the antibody by microbial transglutaminase.

[0160] In a particular embodiment, the present invention relates to a method according to the invention, wherein the payload is linked to the linking moiety B of the antibody-linker conjugate via a click reaction, such as any one of the click reactions described above. In a preferred embodiment, the click reaction is SPAAC.

[0161] According to a further embodiment of the invention, payload B is ·toxin Cytokines ·Growth factors Radionuclides ·hormone Antiviral agents Antibacterial Fluorescent dyes Immunoregulatory / immunostimulatory agents Half-life increase part Increased solubility Polymer-toxin conjugates ·Nucleic acid Biotin or streptavidin moiety ·vitamin a target binding moiety, and Anti-inflammatory At least one selected from the group consisting of:

[0162] The half-life increasing moiety is, for example, a PEG moiety (polyethylene glycol moiety; PEGylation), other polymer moieties, PAS moieties (oligopeptides containing proline, alanine and serine; PASylation), or serum albumin binders. The solubility increasing moiety is, for example, a PEG moiety (PEGylation) or a PAS moiety (PASylation).

[0163] A polymer-toxin conjugate is a polymer that can carry many payload molecules. Such conjugates are sometimes used, for example, in Mersana therapeutics. Also known as fleximer, sold by tics.

[0164] One example of a nucleic acid payload is MCT-485, an extremely small non-coding double-stranded RNA with oncolytic and immune activating properties, developed by MultiCell Technologies, Inc.

[0165] Anti-inflammatory agents are, for example, anti-inflammatory cytokines that, when conjugated to a target-specific antibody, can ameliorate inflammation caused by, for example, an autoimmune disease.

[0166] The term "fluorochrome" as used herein refers to a dye that absorbs light at a first wavelength and emits at a second wavelength that is longer than the first wavelength. In certain embodiments, the fluorescent dye is a near-infrared fluorescent dye that emits at wavelengths between 650-900 nm. In this region, tissue autofluorescence is low, so low fluorescence quenching enhances deep tissue penetration with minimal background interference. Thus, near-infrared fluorescence imaging can be used to make tissues to which the antibody-payload conjugates of the invention are bound visible during surgery. "Near-infrared fluorescent dyes" are known in the art and commercially available. In certain embodiments, the near-infrared fluorescent dye can be IRDye 800CW, Cy7, Cy7.5, NIR CF750 / 770 / 790, DyLight 800 or Alexa Fluor 750.

[0167] The term "radionuclide" as used herein refers to, e.g., 90 Y, 111 In, 67 Cu, 77 Lu, 99 Tc, 161 Tb, 225 The present invention relates to medically useful radionuclides, including positively charged ions of radiometals such as Y, In, Tb, Ac, Cu, Lu, Tc, Re, Co, Fe, etc., such as Ac. The radionuclides can be included in chelating agents. Furthermore, the radionuclides can be therapeutic radionuclides or radionuclides that can be used as contrast agents in the imaging techniques discussed below. Radionuclides or molecules that contain radionuclides are known in the art and are commercially available.

[0168] The term "toxin" as used herein relates to any compound that is toxic to a cell or organism. Thus, a toxin may be, for example, a small molecule, a nucleic acid, a peptide or a protein. Specific examples are neurotoxins, necrotizing toxins, hematotoxins and cytotoxins. According to one further embodiment of the invention, the toxin is Pyrrolobenzodiazepines (PBDs) Auristatins (e.g., MMAE, MMAF) Maytansinoids (maytansine, DM1, DM4, DM21) Duocarmycin Tubulysin Enediyenes (e.g., calicheamicin) PNU, doxorubicin Pyrrole-based kinesin spindle protein (KSP) inhibitors Calicheamicin Amanitin (e.g. α-amanitin), and / or Camptothecins (e.g., exatecan, deruxtecan) At least one selected from the group consisting of:

[0169] In certain embodiments, the payload is an auristatin. As used herein, the term "auristatin" refers to a phylogenetic tree of anti-mitotic agents. Auristatin derivatives are also included within the definition of the term "auristatin." Examples of auristatins include, but are not limited to, synthetic analogs of auristatin E (AE), monomethylauristatin E (MMAE), monomethylauristatin F(MMAF) and dolastatins.

[0170] In certain embodiments, the payload is a maytansinoid. In the context of the present invention, the term "maytansinoid" refers to a class of highly cytotoxic drugs originally isolated from the African shrub Maytenus ovatus, as well as additional maytansinol and C-3 esters of natural maytansinol (U.S. Pat. No. 4,151,042); synthetic C-3 ester analogs of maytansinol (Kupchan et al., J. Med. Chem. 21: 31-37, 1978; Higashide et al., Nature 270: 721-722, 1977; Kawai et al., Chem. Farm. Bull. 32: 3441-3451; and U.S. Pat. No. 5,416,064); C-3 esters of simple carboxylic acids (U.S. Pat. Nos. 4,248,870; 4,265,814; 4,308,268; 4,308,269; 4,309,428; 4,317,821; 4,322,348; and 4,331,598); and C-3 esters with derivatives of N-methyl-L-alanine (U.S. Pat. Nos. 4,137,230; 4,260,608; and Kawai et al., Chem. Pharm Bull. 12: 3441, 1984). Exemplary maytansinoids that may be used in the methods of the invention or that may be included in the antibody-payload conjugates of the invention are DM1, DM3, DM4 and / or DM21.

[0171] In certain embodiments, the toxic payload molecule is a duocarmycin. Suitable duocarmycins may be, for example, duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin CI, duocarmycin C2, duocarmycin D, duocarmycin SA, duocarmycin MA, and CC-1065. The term "duocarmycin" should also be understood to refer to synthetic analogs of duocarmycins, such as adozelesin, bizelesin, carzelesin, KW-2189, and CBI-TMI.

[0172] Toxins can also be inhibitors of drug efflux transporters in the sense of the present invention. Antibody-payload conjugates containing toxins and inhibitors of drug efflux transporters can have the advantage that, once internalized in a cell, the inhibitors of drug efflux transporters prevent the toxins from being effluxed out of the cell. Within the present invention, the drug efflux transporter can be P-glycoprotein. Some common pharmacological inhibitors of P-glycoprotein include amiodarone, clarithromycin, cyclosporine, colchicine, diltiazem, erythromycin, felodipine, ketoconazole, lansoprazole, omeprazole and other proton pump inhibitors, nifedipine, paroxetine, reserpine, saquinavir, sertraline, quinidine, tamoxifen, verapamil and duloxetine. Elacridar and CP100356 are other common P-gp inhibitors. Zosuquidar and tariquidar were also developed in this context. Finally, Valspodar and Reversan are other examples of such drugs.

[0173] The vitamins may be selected from the group consisting of folates, which include folic acid, folacin and vitamin B9.

[0174] The target binding moiety may be a protein or a small molecule capable of specifically binding to a protein or non-protein target. In one embodiment, such a target binding moiety is an antibody mimetic, including an scFv-type antibody, a Fab fragment, a F(ab)2 fragment, a nanobody, an affibody, a diabody, a VHH-type antibody, or a DARPIN.

[0175] It should be understood that the payload can be attached to the linking portion of the linker by any suitable reaction, such as a Click reaction, or can be attached to the linker by chemical synthesis.

[0176] According to a further embodiment of the invention, the linker has two or more linking moieties B.

[0177] In such an embodiment, for example, an antibody-payload conjugate can be made in which two payloads are conjugated to each Q295 residue, resulting in an antibody to payload ratio of 4.

[0178] According to a further embodiment of the invention, the two or more linking moieties B are different from each other.

[0179] In such an embodiment, the first linking moiety can be or include, for example, an azide (N3) and the second linking moiety can be or include a tetrazine. Thus, such an oligopeptide linker can link two different payloads to two Gln residues of an antibody, i.e., two C1 residues of an antibody. H This allows for conjugation to the Q295 residue of domain 2.

[0180] In this way, an antibody-payload ratio of 2+2 can be obtained. The use of a second payload makes it possible to develop an entirely new class of antibody-payload conjugates that exceed current therapeutic approaches in terms of efficacy and potency.

[0181] Such an embodiment allows, inter alia, targeting two different structures within a cell, such as DNA and microtubules. Some cancers may be resistant to certain drugs, such as microtubule poisons, so that DNA poisons can still kill cancer cells.

[0182] According to another embodiment, two drugs can be used that are sufficiently potent only if released simultaneously and in the same tissue, which may result in reduced off-target toxicity if the antibody is partially degraded in healthy tissue or one drug is lost prematurely.

[0183] Furthermore, the dual-labeled probe can be used for non-invasive imaging and treatment, or intra- / post-operative imaging / surgery.In such an embodiment, tumor patients can be selected by non-invasive imaging.The tumor can then be surgically removed using other imaging agents (e.g., fluorescent dyes) that help surgeons or robots identify all cancerous tissue.

[0184] According to another aspect of the invention there is provided an antibody-payload conjugate produced using a method according to any one of the above steps.

[0185] According to another aspect of the invention, the peptide structure (shown in N→C orientation) Gly-(Aax) m -B-(Aax) n wherein Gly contains an N-terminal primary amine; m is an integer between 0 and 12, n is an integer between 0 and 12, m+n≧0, Aax is an amino acid or an amino acid derivative, B is the payload or linkage) which can be conjugated to an antibody via the N-terminal primary amine of the N-terminal Gly of the linker by microbial transglutaminase.

[0186] The linker is suitable for conjugation via the N-terminal primary amine of an N-terminal glycine (Gly) residue to a glutamine (Gln) residue contained in an antibody heavy or light chain by transglutaminase enzyme.

[0187] In general, the advantages and embodiments discussed above of the method of the present invention also apply to this aspect, i.e., the linker as a composition of matter. Therefore, these embodiments shall be considered to be disclosed by the linker as a composition of matter as well.

[0188] It is important to understand that in the various linker peptides shown herein, the C-terminus may or may not be protected, even if shown otherwise. Protection can be achieved by amidation. In the context of the present invention, C-terminus protected and unprotected linker peptides are encompassed.

[0189] In a particular embodiment, the present invention relates to a linker according to the invention, which comprises two or more payloads and / or linking moieties B.

[0190] In certain embodiments, the linker may comprise two or more linking moieties and / or payloads. That is, the linker may comprise a peptide structure (shown in the N→C orientation): Gly-(Aax) m -B1-(Aax) n -B2-(Aax) o (In the formula, m, n, and o are integers between 0 and 12, m+n+o≧0, Aax is an amino acid or an amino acid derivative, B1 and B2 are payloads and / or linking moieties, and B1 and B2 may be the same or different from each other. and the linker can be conjugated to the antibody via the N-terminal primary amine of the N-terminal Gly of the linker by microbial transglutaminase.

[0191] In other embodiments, the linker may comprise three linking moieties and / or payloads, i.e., the linker may comprise a peptide structure (shown in the N→C orientation): Gly-(Aax) m -B1-(Aax) n -B2-(Aax) o-B3-(Aax) p (In the formula, m, n, o, and p are integers between 0 and 12, m+n+o+p≧0, Aax is an amino acid or an amino acid derivative, B1, B2 and B3 are payloads and / or linking moieties, and B1, B2 and B3 may be the same or different from each other. and the linker can be conjugated to the antibody via the N-terminal primary amine of the N-terminal Gly of the linker by microbial transglutaminase.

[0192] It should be understood that the present invention also encompasses linkers that contain more than three linking moieties and / or payloads, for example, four, five or six linking moieties and / or payloads, in which case the peptide structure of the linker follows the same pattern as described above for linkers that contain two or three linking moieties and / or payloads.

[0193] In certain embodiments, the present invention provides a peptide structure (shown in the N→C orientation): Gly-(Aax) m -B-(Aax) n (In the formula, m is an integer between 0 and 12, n is an integer between 0 and 12, m+n≧0, Aax is an amino acid or an amino acid derivative, B is the payload or linkage) which can be conjugated to an antibody via the N-terminal primary amine of the N-terminal Gly of the linker by microbial transglutaminase.

[0194] In this case, it should be understood that moiety B may include more than one payload and / or linking moiety. For example, B may be (B'-(Aax) o-B″), where B′ and B″ are payload and / or linking moieties, and o is an integer between 0 and 12. Alternatively, B may represent (B′-(Aax) o -B''-(Aax) p -B'''), where B', B'' and B''' are payload and / or linking moieties, and o and p are integers between 0 and 12, inclusive.

[0195] In preferred embodiments, m and / or n are 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 11 or more. In other preferred embodiments, m and / or n are 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less. In further preferred embodiments, m+n is 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 11 or more. In still further preferred embodiments, m+n is 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less.

[0196] Members of both ranges can be combined with one another to disclose preferred length ranges having lower and upper limits.

[0197] Thus, in a particular embodiment, the invention relates to a linker according to the invention, wherein m+n is 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, or 4 or less.

[0198] In further embodiments, the linker is not cleavable by cathepsin B, and / or the linker does not contain a valine-alanine or valine-citrulline motif, and / or the linker does not contain polyethylene glycol or a polyethylene glycol derivative.

[0199] According to one embodiment, the linking moiety B is Bioorthogonal markers Other non-bio-orthogonal entities for crosslinking At least one selected from the group consisting of:

[0200] In certain embodiments, at least one linking moiety B of the linker is bioorthogonal marker groups; or Non-bio-orthogonal entities for crosslinking It comprises or consists of.

[0201] According to one embodiment, the bioorthogonal marker group or the non-bioorthogonal entity is -NN≡N or -N3 Lys(N3) Tetrazine Alkynes DBCO ·BCN Norbolene Trans-cyclooctene -RCOH (aldehyde) Acyl trifluoroborates -SH, and Cysteine At least one selected from the group consisting of:

[0202] In further embodiments, the net charge of the linker is neutral or positive, and / or the linker does not contain a negatively charged amino acid residue, and / or the linker contains a positively charged amino acid residue, and / or the linker is Lysine Arginine, and / or Histidine and wherein the amino acid residues are selected from the group consisting of:

[0203] In certain embodiments, the linker is Lysine, Arginine, and Histidine and wherein the amino acid residue is selected from the group consisting of:

[0204] In certain embodiments, the linker is Arginine, and Histidine and wherein the amino acid residue is selected from the group consisting of:

[0205] That is, in certain embodiments, the linker according to the present invention has a neutral or positive net charge. In certain embodiments, the linker according to the present invention has a neutral or positive net charge and comprises at least one arginine and / or histidine residue. In certain embodiments, the linker according to the present invention does not comprise a lysine residue. In certain embodiments, the linker has a neutral or positive net charge and does not comprise a lysine residue.

[0206] According to one embodiment, the primary amine groups are suitable to act as substrates for microbial transglutaminase (MTG).

[0207] According to a further embodiment, the linker is suitable for generating antibody-payload conjugates by microbial transglutaminase (MTG). According to a further embodiment, the linker is a) the list shown in Table 5, and / or b) any one of SEQ ID NOs: 1 to 25 is selected from.

[0208] In a particular embodiment, the invention relates to a linker according to the invention, wherein the linker is selected from the list presented in Table 5.

[0209] According to yet another aspect of the present invention, at least a) a linker according to the above description, and b) one or more payloads wherein the linker and / or payload are optionally chemically modified during attachment to allow for covalent or non-covalent attachment to form the construct.

[0210] In certain embodiments, at least a) a linker according to the above description, and b) one or more payloads wherein one or more payloads are covalently or non-covalently attached to the linker.

[0211] In certain embodiments, the present invention relates to a linker-payload construct according to the present invention, in which one or more payloads in the construct are covalently attached to the linking moiety B of the linker in a click reaction, i.e., one or more payloads may be attached to the linking moiety B by any of the click reactions discussed above, such as, but not limited to, SPAAC, tetrazine ligation or thiol-maleimide conjugation.

[0212] In addition to the click reaction between the linking moiety in the linker and the payload, the payload can be covalently attached to the linker by any enzymatic or non-enzymatic reaction known in the art. For this purpose, the payload can be attached to the C-terminus of the linker or to an amino acid side chain of the linker.

[0213] In certain embodiments, the payload is attached to the linker by chemical synthesis. Those skilled in the art know how to attach the payload to the peptide linker by chemical reaction. For example, the payload containing amine, or the payload containing thiol (e.g., maytansine analog), or the payload containing hydroxyl (e.g., SN-38 analog) can be attached to the C-terminus of the peptide linker by chemical synthesis to obtain the linker shown in FIG. 17, for example. However, those skilled in the art know additional reactions and reactive groups that can be utilized to couple the payload to the C-terminus or side chain of amino acid or amino acid derivative by chemical synthesis. Typical reactions that can be used to attach the payload to the peptide linker by chemical synthesis include, but are not limited to, peptide coupling, activated ester coupling (NHS ester, PFP ester), click reaction (CuAAC, SPAAC), Michael addition (thiol maleimide conjugation). Coupling of payloads to peptides has been reported, for example, by Costoplus et al. (ACS Med Chem, 2019), Sonzini et al. (Bioconj Chem, 2019), Bodero et al. (Belstein, 2018), Nunes et al. (RSC Adv, 2017), Doronina et al. (Bioconj Chem, 2006), Nakada et al. (Bioorg Med Chem, 2016) and Dickgiesser et al. (Bioconj Chem, 2020) , have been described extensively in the prior art.

[0214] In a particular embodiment, the present invention relates to a linker-payload construct according to the invention, in which the linker and / or the payload are chemically modified, as required during attachment, to allow covalent or non-covalent attachment to form said construct.

[0215] When two or more payloads are used, the payloads may be the same or different from one another.

[0216] In one embodiment, the payload comprises: ·toxin Cytokines ·Growth factors Radionuclides ·hormone Antiviral agents Antibacterial Fluorescent dyes Immunomodulators / immunostimulants Half-life increase part Increased solubility Polymer-toxin conjugates ·Nucleic acid Biotin or streptavidin moiety ·vitamin a target binding moiety, and Anti-inflammatory Protein disaggregators (PROTACs) At least one selected from the group consisting of:

[0217] In another embodiment, the toxin is Pyrrolobenzodiazepines (PBDs) Auristatins (e.g., MMAE, MMAF) Maytansinoids (maytansine, DM1, DM4, DM21) Duocarmycin Tubulysin Enediynes (e.g., calicheamicin) PNU, doxorubicin Pyrrole-based kinesin spindle protein (KSP) inhibitors Calicheamicin Amanitin (e.g. α-amanitin), and / or Camptothecins (e.g., exatecan, deruxtecan) At least one selected from the group consisting of:

[0218] According to another aspect of the present invention, a) one or more linker-payload constructs according to the above description, and b) an antibody containing at least one Gln residue in the heavy or light chain wherein the linker-payload construct and / or the antibody are optionally chemically modified during conjugation to allow for covalent or non-covalent conjugation to form the conjugate.

[0219] In certain embodiments, the present invention provides a method for producing a pharmaceutical composition comprising: a) one or more linker-payload constructs according to the above description, and b) an antibody containing at least one Gln residue in the heavy or light chain wherein the linker-payload construct is conjugated to the amide side chain of a Gln residue in the heavy or light chain of the antibody via the N-terminal primary amine of an N-terminal glycine residue contained in the linker-payload construct. Concerning antibody-payload conjugates.

[0220] In a particular embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein conjugation is achieved by means of microbial transglutaminase (MTG).

[0221] In certain embodiments, the present invention relates to an antibody-payload conjugate according to the present invention, in which conjugation is achieved before or after the formation of the linker-payload construct. That is, the present invention encompasses antibody-payload conjugates in which in a first step, the linker is conjugated to the antibody, and then in a second step, one or more payloads are attached to the linking portion of the linker. However, the present invention also encompasses antibody-payload conjugates in which in a first step, one or more payloads are attached to the linking portion of the linker, and then in a second step, the resulting linker-payload construct is conjugated to the antibody. Furthermore, in a one-step reaction, one or more payloads can be attached to the peptide linker by chemical synthesis, and then the resulting linker-payload construct can be conjugated to the antibody.

[0222] In a particular embodiment, the invention relates to a method for the treatment of cancer, comprising administering to a patient a therapeutically effective amount of an antibody, the antibody being an IgG, IgE, IgM, IgD, IgA or IgY antibody, or a fragment or recombinant variant thereof, which fragment or recombinant variant retains target binding properties and is capable of inhibiting the binding of a target molecule. H The present invention relates to an antibody-payload conjugate according to the present invention which comprises two domains.

[0223] In a particular embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is an IgG antibody.

[0224] In a particular embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is a glycosylated antibody, a deglycosylated antibody or an aglycosylated antibody.

[0225] In certain embodiments, the present invention relates to a glycosylated antibody. H It is an IgG antibody that is glycosylated at residue N297 (EU numbering) in the C2 domain, or the glycosylated antibody is an IgG antibody. HThe present invention relates to an antibody-payload conjugate according to the invention, which is an antibody of a different isotype that is glycosylated at a residue homologous to residue N297 (EU numbering) of domain 2.

[0226] In a particular embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein (a) the linker-payload construct is conjugated to a Gln residue introduced into the heavy or light chain of the antibody by molecular engineering, or (b) the linker-payload construct is conjugated to a Gln residue in the Fc domain of the antibody.

[0227] In a particular embodiment, the present invention relates to a method for the preparation of a IgG antibody comprising administering to a patient ... H In one embodiment of the present invention, the Gln residue Q295 (EU numbering) in domain 2 or a homologous Gln residue in an antibody of a different isotype is an antibody-payload conjugate according to the present invention.

[0228] In a specific embodiment, the present invention provides an antibody comprising a glycine residue in the Fc domain of the antibody, the glycine residue being replaced by a glycine residue in the Fc domain of the antibody. H C of an IgG antibody that is glycosylated at residue N297 (EU numbering) in the 2 domain H The Gln residue Q295 (EU numbering) of domain 2 of the antibody-payload conjugate according to the invention.

[0229] The antibody of the method or antibody-payload conjugate of the present invention can be any antibody, preferably can be any IgG type antibody. For example, the antibody can be, but is not limited to, brentuximab, trastuzumab, gemtuzumab, inotuzumab, avelumab, cetuximab, rituximab, daratumumab, pertuzumab, vedolizumab, ocrelizumab, tocilizumab, ustekinumab, golimumab, obinutuzumab, polatuzumab, or enfortumab.

[0230] Thus, in certain embodiments, the present invention relates to an antibody-payload conjugate according to the present invention, wherein the antibody is brentuximab. In a further embodiment, the present invention relates to an antibody-payload conjugate according to the present invention, wherein the antibody is trastuzumab. In a further embodiment, the present invention relates to an antibody-payload conjugate according to the present invention, wherein the antibody is gemtuzumab. In a further embodiment, the present invention relates to an antibody-payload conjugate according to the present invention, wherein the antibody is inotuzumab. In a further embodiment, the present invention relates to an antibody-payload conjugate according to the present invention, wherein the antibody is avelumab. In a further embodiment, the present invention relates to an antibody-payload conjugate according to the present invention, wherein the antibody is cetuximab. In a further embodiment, the present invention relates to an antibody-payload conjugate according to the present invention, wherein the antibody is rituximab. In a further embodiment, the present invention relates to an antibody-payload conjugate according to the present invention, wherein the antibody is daratumumbab. In a further embodiment, the present invention relates to an antibody-payload conjugate according to the present invention, wherein the antibody is pertuzumab. In a further embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is vedolizumab. In a further embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is ocrelizumab. In a further embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is tocilizumab. In a further embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is ustekinumab. In a further embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is golimumab. In a further embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is obinutuzumab. In a further embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is polatuzumab. In a further embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is enfortumab.

[0231] In a particular embodiment, the present invention relates to a glycosylated IgG antibody in which a Gln residue has been introduced into the heavy or light chain of the antibody by molecular engineering. H The antibody-payload conjugate according to the invention is N297Q (EU numbering) of the 2 domain.

[0232] In a particular embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the Gln residue introduced into the antibody heavy or light chain by molecular engineering is comprised in a peptide that is (a) incorporated into the antibody heavy or light chain or (b) fused to the N-terminus or C-terminus of the antibody heavy or light chain.

[0233] In a particular embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein a peptide comprising a Gln residue is fused to the C-terminus of the heavy chain of the antibody.

[0234] In a particular embodiment, the present invention relates to a peptide comprising a Gln residue selected from the group consisting of LLQGG, LLQG, LSLSQG, GGGLLQGG, GLLQG, LLQ, GSPLAQSHGG, GLLQGGG, GLLQGG, GLLQ, LLQLLQGA, LLQGA, LLQYQGA, LLQGSG, LLQYQG, LLQLLQG, SLLQG, LLQLQ, LLQLLQ, LLQGR, EEQYASTY, EEQYQSTY, EEQYNSTY, EEQYQS, EEQYQST, EQYQSTY, QYQS, QYQSTY, YRYRQ, DYALQ, FGLQRPY, EQKLISEEDL, LQR, and YQR. The present invention relates to an antibody-payload conjugate according to the present invention.

[0235] In a particular embodiment, the present invention relates to an antibody-payload conjugate according to the invention, comprising at least one toxin.

[0236] That is, the antibody-payload conjugate of the present invention comprises an antibody that is conjugated to at least one linker that comprises at least one toxin.In certain embodiments, the antibody-payload conjugate comprises two linkers, and each heavy chain of the antibody is conjugated to one linker.In certain embodiments, the antibody-payload conjugate comprises four linkers, and each heavy chain of the antibody is conjugated to two linkers.In such cases, each linker can contain one or more payloads, such as toxins.

[0237] In certain embodiments, the antibody-payload conjugate according to the present invention comprises two linkers, each linker comprising one payload, for example a toxin. In other embodiments, the antibody-payload conjugate according to the present invention comprises two linkers, each linker comprising two payloads, for example one toxin and one other payload, or two identical or different toxins. In embodiments in which the antibody-payload conjugate comprises two linkers, it is preferred that the linkers are conjugated to residue Q295 of the two heavy chains of an IgG antibody. Even more preferably, the antibody is an IgG antibody that is glycosylated at residue N297.

[0238] In certain embodiments, the antibody-payload conjugate according to the present invention comprises four linkers, each linker comprising one payload, for example a toxin. In other embodiments, the antibody-payload conjugate according to the present invention comprises four linkers, each linker comprising two payloads, for example one toxin and one other payload, or two identical or different toxins. In the embodiment in which the antibody-payload conjugate comprises four linkers, it is preferred that the linkers are conjugated to residues Q295 and N297Q of the two heavy chains of an IgG antibody.

[0239] In a particular embodiment, the invention relates to an antibody-payload conjugate according to the invention, which comprises two different toxins.

[0240] In certain embodiments, the antibody-payload conjugate according to the present invention comprises two different toxins. That is, in certain embodiments, the antibody-payload conjugate may comprise two linkers, each linker comprising two different toxins. The antibody-payload conjugate comprising two different toxins has the advantage that it may have increased cytotoxic activity. Such increased cytotoxic activity may be achieved by combining two toxins that target two different cellular mechanisms. For example, the antibody-payload conjugate according to the present invention may comprise a first toxin that inhibits cell division and a second toxin that is a toxin that interferes with DNA replication and / or transcription.

[0241] Thus, in a particular embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the first toxin is a toxin that inhibits cell division and the second toxin is a toxin that interferes with DNA replication and / or transcription.

[0242] Toxins that inhibit cell division, such as mitotic inhibitors or spindle poisons, are agents that have the ability to inhibit or prevent mitosis of cells. Spindle poisons are poisons that disrupt cell division by affecting the protein threads that connect the centromeric regions of chromosomes, known as the mitotic spindle. Spindle poisons block the mitotic division of cell division at the spindle assembly checkpoint (SAC). By interrupting the mitotic phase, it effectively stops the production of new cells. The mitotic spindle is composed of microtubules (polymerized tubulin) along with regulatory proteins; which aid each other in their activities to properly separate the replicated chromosomes. Certain compounds that affect the mitotic spindle have proven highly effective against solid tumors and hematological malignancies.

[0243] Two specific families of mitotic inhibitors, the vinca alkaloids and the taxanes, disrupt cell division by perturbing microtubule dynamics. The vinca alkaloids act by causing inhibition of the polymerization of tubulin into microtubules, resulting in G2 / M arrest in the cell cycle, and ultimately cell death. In contrast, the taxanes arrest the mitotic cell cycle by stabilizing microtubules against depolymerization. Although there are numerous other spindle proteins that could be targets for novel chemotherapy, tubulin-binding agents are the only class in clinical use. Agents that affect the motor protein kinesin are beginning to enter clinical trials. Another class, paclitaxel, acts by binding to tubulin within existing microtubules. Preferred toxins that inhibit cell division in the present invention are auristatins, such as MMAE and MMAF, and maytansinoids, such as DM1, DM3, DM4, and / or DM21.

[0244] In a particular embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein at least one of the toxins is an auristatin or a maytansinoid.

[0245] Some drugs that prevent the correct replication and / or transcription of DNA molecules and have been shown to be suitable for cancer treatment are known to those skilled in the art.For example, antimetabolites such as nucleotide or nucleoside analogues that are misincorporated into newly formed DNA and / or RNA molecules are known in the art and are summarized by Tsesmetzis et al, Cancers (Basel), 2018, 10(7): 240.Another toxin known to interfere with DNA replication and / or transcription is duoromycin.

[0246] Thus, in certain embodiments, an antibody-payload conjugate according to the invention comprises two different toxins, where the first toxin is a duolomycin and the second payload is an auristatin or a maytansinoid.

[0247] In certain embodiments, the invention relates to an antibody-payload conjugate according to the invention, which comprises two different auristatins.

[0248] One major advantage of an antibody-payload conjugate containing two different toxins is that the antibody-payload conjugate may still act on target cells that escape the mechanism of action of one of the toxins and / or that the antibody-payload conjugate may have high efficacy against heterogeneous tumors.

[0249] In a particular embodiment, the invention relates to an antibody-payload conjugate according to the invention comprising a toxin and an inhibitor of a drug efflux transporter.

[0250] In a particular embodiment, the invention relates to an antibody-payload conjugate according to the invention comprising a toxin and a solubility-enhancing moiety.

[0251] That is, an antibody-payload conjugate may contain two payloads, the first payload being a toxin and the second payload being a solubility-increasing moiety. Structure 5 in FIG. 9 shows a peptide linker containing a solubility-increasing moiety attached to a lysine side chain. Thus, an antibody-payload conjugate containing a toxin and a solubility-increasing moiety may be obtained by clicking the toxin onto the azide group of the linker shown in Structure 5 in FIG. 9. Alternatively, Antibody-linker conjugates can be obtained by clicking a toxin onto an azide-containing linking moiety of the linker and a maleimide-containing solubility-enhancing moiety onto a cysteine ​​side chain of the same linker. Alternatively, the toxin and / or the solubility-enhancing moiety can be attached to the linker by chemical synthesis.

[0252] In a particular embodiment, the invention relates to an antibody-payload conjugate according to the invention comprising a toxin and an immunostimulant.

[0253] As used herein, depending on the context, the term "immunostimulant" includes compounds that increase a subject's immune response to an antigen. Examples of immune stimulants include immunostimulants and immune cell activating compounds. The antibody-payload conjugates of the present invention may contain immune stimulants that help program immune cells to recognize ligands and enhance antigen presentation. Immune cell activating compounds include Toll-like receptor (TLR) agonists. Such agonists include pathogen-associated molecular patterns (PAMPs), such as bacterially-derived immunomodulators (aka danger signals), infection-mimicking compositions, and damage-associated molecular patterns (DAMPs), such as compositions that mimic stress or damaged cells. TLR agonists include nucleic acids or lipid compositions (e.g., monophosphoryl lipid A (MPLA)). In one example, the TLR agonist includes a TLR9 agonist, such as a cytosine-guanosine oligonucleotide (CpG-ODN), a poly(ethyleneimine) (PEI) condensed oligonucleotide (ODN), such as PEI-CpG-ODN, or double-stranded deoxyribonucleic acid (DNA). In another example, the TLR agonist includes a TLR3 agonist, such as polyinosine-polycytidylic acid (poly(I:C)), PEI-poly(I:C), polyadenylic acid-polyuridylic acid (poly(A:U)), PEI-poly(A:U), or double-stranded ribonucleic acid (RNA). Other exemplary vaccine immunostimulatory compounds include lipopolysaccharide (LPS), chemokines / cytokines, fungal beta-glucans (such as lentinan), imiquimod, CRX-527, and OM-174.

[0254] In a particular embodiment, the invention relates to an antibody-payload conjugate according to the invention, which comprises two different immunostimulants.

[0255] In a particular embodiment, the present invention relates to an antibody-payload conjugate according to the invention, wherein at least one immunostimulant is a TLR agonist.

[0256] The term "TLR agonist" as used herein refers to a molecule that can trigger a signaling response through the TLR signaling pathway, either as a direct ligand or indirectly through endogenous or exogenous production. Agonistic ligands of TLR receptors are (i) the natural ligands of the actual TLR receptors, or functionally equivalent variants thereof that conserve the ability to bind to the TLR receptors and induce costimulatory signals thereon, or (ii) agonistic antibodies against the TLR receptors, or functionally equivalent variants thereof, that can specifically bind to the TLR receptors, more particularly the extracellular domains of said receptors, and induce some of the immune signals controlled by the receptors and associated proteins. The binding specificity can be for the human TLR receptors, or for TLR receptors of different species that are homologous to the human TLR receptors.

[0257] In a particular embodiment, the present invention relates to an antibody-payload conjugate according to the invention comprising a radionuclide and a fluorescent dye.

[0258] In a particular embodiment, the present invention relates to a method for the detection of radionuclides, the radionuclides being suitable for use in tomography, in particular single photon emission computed tomography (SPECT) or positron emission tomography (PET), and The present invention relates to an antibody-payload conjugate according to the present invention, wherein the photodye is a near infrared fluorescent dye.

[0259] The term "radionuclide" as used herein has the same meaning as radioactive nuclide, radioisotope or radioisotope.

[0260] The radionuclide is preferably detectable by nuclear medicine molecular imaging techniques such as positron emission tomography (PET), single photon emission computed tomography (SPECT), a hybrid of SPECT and / or PET, or a combination thereof.

[0261] Single photon emission computed tomography (SPECT) as used herein includes planar scintigraphy (PS).

[0262] Hybrids of SPECT and / or PET are for example SPECT / CT, PET / CT, PET / IRM or SPECT / IRM.

[0263] SPECT and / or PET obtain information about the concentration (or uptake) of radionuclides introduced into the subject's body. PET creates images by detecting gamma ray pairs emitted indirectly by positron-emitting radionuclides. A PET analysis results in a series of thin slice images of the body over the region of interest (e.g., brain, breast, liver, ...). These thin slice images can be assembled into a three-dimensional representation of the area being investigated. SPECT is similar to PET, but the radioactive materials used in SPECT have longer decay times than those used in PET, and emit single gamma rays instead of double gamma rays. SPECT images show lower sensitivity than PET images and are less detailed than PET images, but the SPECT technique offers the advantage of being much cheaper than PET and not requiring the proximity of a particle accelerator. Actual clinical PET offers higher sensitivity and better spatial resolution than SPECT, and offers the advantage of more accurate attenuation correction due to the higher energy of the photons; therefore, PET provides more accurate quantitative data than SPECT. Planar scintigraphy (PS) is similar to SPECT in that it uses the same radionuclides. However, PS only produces 2D information.

[0264] SPECT provides a computer-generated picture of localized radiotracer uptake, while CT provides a 3D anatomical image of X-ray density of the human body. Combined SPECT / CT imaging provides sequential functional information from SPECT and anatomical information from CT, obtained during a single examination. CT data are also used for rapid and optimal attenuation correction of single-photon emission data. By precisely localizing areas of abnormal and / or physiologic tracer uptake, SPECT / CT improves sensitivity and specificity, but can also help to achieve accurate dosimetry estimates and to guide interventional procedures or better define target volumes for external beam radiation therapy. Gamma camera imaging with single-photon emitting radiotracers has become the majority of procedures.

[0265] The radionuclide is technetium-99m( 99m Tc), Gallium-67( 67 Ga), Gallium-68( 68 Ga), Yttrium-90( 90 Y), Indium-111( 111 In), Rhenium-186( 186 Re), Fluorine-18( 18 F), copper-64( 64 Cu), Terbium-149( 149 Tb) or thallium-201( 201 The radionuclide may be selected from the group consisting of: TI (Trifluoromethyl) and TI (Trifluoromethyl). The radionuclide may be contained in a molecule or may be bound to a chelating agent.

[0266] According to another aspect of the present invention, a linker according to the above description, a linker-pay according to the above description Pharmaceutical compositions comprising the loaded construct and / or the antibody-payload conjugate according to the above description are provided.

[0267] According to another aspect of the present invention there is provided a pharmaceutical product comprising an antibody-payload conjugate according to the above description, or a pharmaceutical composition according to the above description, and at least one further pharma- ceutically acceptable carrier.

[0268] A pharma- ceutically acceptable carrier refers to an ingredient in a pharmaceutical formulation, other than the active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0269] Pharmaceutical formulations of the antibody-payload conjugates described herein are prepared by mixing such conjugates having the desired purity, in the form of a lyophilized formulation or an aqueous solution, with one or more optional pharma- ceutically acceptable carriers (Flemington's Pharmaceutical Sciences 16th edition, Oslo, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (such as 1 0 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt forming counterions such as sodium; metal complexes (e.g., Zn protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmacologic carriers herein include interstitial drug dispersing agents, such as soluble neutral active hyaluronidase glycoproteins. Further included are soluble PH-20 hyaluronidase glycoproteins (sHASEGPs), such as human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0270] In one embodiment the present invention relates to an antibody-payload conjugate according to the invention, a pharmaceutical composition according to the invention or a pharmaceutical product according to the invention for use in therapy and / or diagnosis.

[0271] That is, the antibody-payload conjugate of the present invention can be used in the treatment of a subject or in the diagnosis of a disease or condition of a subject.The individual or subject is a mammal.Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs and horses), primates (e.g., humans and non-human primates, such as macaques), rabbits, and rodents (e.g., mice and rats).In certain embodiments, the individual or subject is a human.

[0272] According to another aspect of the invention, the method comprises administering to the patient a therapeutically effective amount of the compound according to the invention. is an infectious disease · Suffering from are at risk of developing and / or Have been diagnosed with There is provided a pharmaceutical composition according to the above description or a product according to the above description for treating a patient or for preventing or preventing (for the manufacture of a medicament) such a condition.

[0273] Preferably, the present invention relates to an antibody-payload conjugate according to the invention, a pharmaceutical composition according to the invention or a pharmaceutical product according to the invention for use in the treatment of a patient suffering from a neoplastic disease.

[0274] The term "neoplastic disease" as used herein refers to a condition characterized by uncontrolled, abnormal growth of cells. Neoplastic diseases include cancer. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancer include breast cancer, prostate cancer, colon cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, ovarian cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, liver cancer, bladder cancer, hepatoma, colorectal cancer, cervical cancer, endometrial cancer, salivary gland cancer, renal cancer, vulvar cancer, thyroid cancer, liver cancer, skin cancer, melanoma, brain cancer, ovarian cancer, neuroblastoma, myeloma, various types of head and neck cancer, acute lymphoblastic leukemia, acute myeloid leukemia, Ewing's sarcoma, and peripheral neuroepithelioma. Preferred cancers include liver cancer, lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, Ewing's sarcoma, and peripheral neuroepithelioma.

[0275] That is, the antibody-payload conjugate of the present invention is preferably used for the treatment of cancer. Thus, in certain embodiments, the antibody-payload conjugate comprises an antibody that specifically binds to an antigen present on a tumor cell. In certain embodiments, the antigen is an antigen on the surface of a tumor cell. In certain embodiments, the antigen on the surface of a tumor cell is internalized into the cell together with the antibody-payload conjugate upon binding of the antibody-payload conjugate to the antigen.

[0276] When antibody-payload conjugate is used in the treatment of cancer, it is preferred that antibody-payload conjugate comprises at least one payload that has the ability to kill or inhibit the proliferation of tumor cells to which antibody-drug conjugate is bound.In certain embodiments, at least one payload exhibits cytotoxic activity after antibody-payload conjugate is internalized by tumor cells.In certain embodiments, at least one payload is a toxin.

[0277] According to another aspect of the present invention there is provided a method of treating or preventing a neoplastic disease, comprising the step of administering to a patient in need thereof an antibody-payload conjugate as described above, a pharmaceutical composition as described above or a product as described above.

[0278] The inflammatory disease may be an autoimmune disease. The infectious disease may be a bacterial infection or a viral infection.

[0279] In a particular embodiment, the present invention relates to an antibody-payload conjugate according to the invention, a pharmaceutical composition according to the invention or a pharmaceutical product according to the invention for use in pre-operative, intra-operative and / or post-operative imaging.

[0280] That is, the antibody-payload conjugates according to the invention can be used in imaging. For this purpose, the antibody-payload conjugates can be used to target specific molecules, cells or tissues. The antibody-payload conjugate can be visualized while bound to tissue. Various techniques for visualizing a particular payload are known in the art. For example, if the payload is a radionuclide, the molecule, cell or tissue to which the antibody-payload conjugate binds can be visualized by PET or SPECT. If the payload is a fluorescent dye, the molecule, cell or tissue to which the antibody-payload conjugate binds can be visualized by fluorescence imaging. In certain embodiments, the antibody-payload conjugate according to the present invention comprises two different payloads, for example a radionuclide and a fluorescent dye. In this case, the molecule, cell or tissue to which the antibody-payload conjugate binds can be visualized using two different and / or complementary imaging techniques, for example PET / SPECT and fluorescence imaging.

[0281] The antibody-payload conjugates may be used for pre-operative, intra-operative and / or post-operative imaging.

[0282] Preoperative imaging encompasses all imaging techniques that can be carried out before surgery to make certain target molecules, cells or tissues visible when diagnosing a certain disease or condition, and to provide surgical guidance if necessary.Preoperative imaging can include the step of making tumor visible by PET or SPECT before surgery is carried out, by using antibody-payload conjugates that comprise antibodies that specifically bind to antigens on tumors and are conjugated to payloads that comprise radionuclides.

[0283] Intraoperative imaging encompasses all imaging techniques that can be implemented during surgery to make specific target molecules, cells or tissues visible, thus providing surgical guidance.In certain embodiments, near-infrared fluorescent dye-containing antibody-payload conjugates can be used to visualize tumors during surgery by near-infrared fluorescent imaging.Intraoperative imaging can allow surgeons to identify specific tissues, such as tumor tissues, during surgery, thus allowing complete removal of tumor tissues.

[0284] Post-operative imaging encompasses all imaging techniques that can be performed after surgery to visualize specific target molecules, cells or tissues and evaluate the outcome of the surgery. Post-operative imaging can be performed similarly to pre-operative surgery.

[0285] In certain embodiments, the present invention relates to an antibody-payload conjugate comprising two or more different payloads. For example, the antibody-payload conjugate may comprise a radionuclide and a near-infrared fluorescent dye. Such an antibody-payload conjugate may be used for imaging by PET / SPECT and near-infrared fluorescent imaging. The advantage of such an antibody is that it can be used to visualize target tissues, such as tumors, before and after surgery by PET or SPECT. At the same time, the tumor can be visualized by near-infrared fluorescent imaging. It can be visualized during surgery.

[0286] In a particular embodiment, the present invention relates to an antibody-payload conjugate according to the invention, a pharmaceutical composition according to the invention or a pharmaceutical product according to the invention for use in intraoperative imaging guided cancer surgery.

[0287] As described above, the antibody-payload conjugates of the present invention can be used to visualize target molecules, cells or tissues to guide surgeons or robots during surgery, i.e., the antibody-payload conjugates can be used to visualize tumor tissue during surgery, for example by near-infrared imaging, to allow complete removal of the tumor tissue.

[0288] The conjugate or product is administered to a human or animal subject in an amount or dosage that effectively treats the disease, or a corresponding method of treatment is provided.

[0289] The antibody-payload conjugates of the present invention can be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and, if desired for localized treatment, intralesional, intrauterine or intravesical administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any suitable route, for example, injection, such as intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. Various administration schedules are contemplated herein, including, but not limited to, single administration or multiple administrations over various time periods, bolus administration, and pulse infusion.

[0290] The antibody-payload conjugates of the present invention are formulated, dosed and administered in a manner consistent with good medical practice. Factors to consider in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the administration schedule, and other factors known to the physician. The antibody-payload conjugates need not be formulated with one or more agents currently used to prevent or treat the disorder in question, but are formulated as needed. The effective amount of such other agents depends on the amount of antibody-payload conjugate present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and routes of administration as described herein, or about 1-99% of the dosages described herein, or any dosages and any routes that have been empirically / clinically determined to be appropriate.

[0291] The appropriate dosage of the antibody-payload conjugate of the present invention (when used alone or in combination with one or other additional therapeutic agents) to prevent or treat a disease depends on the type of disease being treated, the type of antibody-payload conjugate, the severity and course of the disease, whether the antibody-payload conjugate is administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody-payload conjugate, and the discretion of the attending physician. The antibody-payload conjugate is suitably administered to the patient at one time or over a series of treatments.

[0292] Table 5 below shows various linkers that may be used in the context of the present invention, and their SEQ ID NOs. For the avoidance of doubt, in case of any discrepancy with the electronic WIPO ST 25 sequence listing, the sequences in this table shall be considered accurate.

[0293] It is important to understand that in some linker peptides shown herein, the C-terminal portion is simply indicated as N3. However, this should be understood as an abbreviation for Lys(N3). For example, GARK(N3) or GlyAlaArgLys(N3) actually means GARK1 (K1=Lys(N3)).

[0294] It is further important to understand that in the various linker peptides shown herein, the C-terminus, even if shown otherwise, may or may not be protected.

[0295] Protection can be achieved by amidation of the C-terminus. In the context of the present invention, both protected and unprotected linker peptides are encompassed.

[0296] For example, GARK(N3) actually encompasses two variants, one with and one without a protected C-terminus. On the other hand, for example, GARK(N3)-COOH explicitly designates an unprotected peptide, i.e., a peptide with an unprotected C-terminus. do.

[0297] Table 5 below shows some linkers that are encompassed and suitable for use in the context of the present invention: [Table 5] EXAMPLES

[0298] The invention has been illustrated and described in detail in the drawings and foregoing description, and such illustrations and The following description and explanations should be considered as illustrative or representative, and not restrictive; the invention is not limited to the disclosed embodiments. Other variations of the disclosed embodiments can be understood and made by those skilled in the art in the practice of the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that combinations of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0299] All amino acid sequences disclosed herein are presented from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are presented from 5' to 3'.

[0300] Example 1 Conjugation efficiency Peptides were used as received, dissolved at appropriate stock concentrations (e.g., 25 mM), aliquots prepared and stored at -20°C according to the manufacturer's instructions. Two antibodies of the IgG subclass (Antibody 1: anti-Her2 IgG1, Antibody 2: anti-CD38 IgG1) were modified as follows: 1 mg / mL of non-deglycosylated antibody (~6.67 μM) was mixed with 80 molar equivalents of peptide linker (i.e., ~533 μM), 6 U / mL MTG and buffer. The reaction mixture was incubated at 37°C for 20 hours and then subjected to LC-MS analysis under reducing conditions.

[0301] Table 6 below shows the linker (( * ) versus other linkers: [Table 6]

[0302] It can be clearly seen that the peptide containing an N-terminal Gly residue, but with no further primary amines except for the N-terminal primary amine, has by far the best conjugation efficiency with the Q295 residue of the glycosylated antibody, even though other peptides also contain an N-terminal primary amine (although not included in the Gly residue).

[0303] Example 2 Conjugation efficiency Peptides were used as received, dissolved at appropriate stock concentrations (e.g., 25 mM), aliquots prepared and stored at −20° C. according to manufacturer's instructions. Two antibodies of the IgG subclass (Antibody 1: anti-Her2 IgG1, Antibody 2: anti-CD38 IgG1) were modified as follows: 1 mg / mL of non-deglycosylated antibody (approximately 6.67 μM) was mixed with 80 molar equivalents of peptide linker (i.e., approximately 533 μM), 6 U / mL MTG and buffer. The reaction mixture was incubated at 37° C. for 20 hours and then subjected to LC-MS analysis under reducing conditions.

[0304] Table 7 below shows the linker (( * 19 shows the conjugation efficiency of the linker βAGARK(N3) (marked with a ∇βA) versus another linker shown in FIG. 19 (note that βA stands for β-alanine).

[0305] [Table 7]

[0306] It can be clearly seen that a peptide containing an N-terminal Gly residue, but with no further primary amines except for the N-terminal primary amine, has a much better conjugation efficiency with the Q295 residue of the glycosylated antibody compared to a structurally similar linker with an N-terminal β-Ala residue.

[0307] Example 3 Cytotoxicity assay Cell lines and culture: MDA-MB-231 and SK-BR-3 were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI-1640 according to standard cell culture protocols.

[0308] SK-BR-3 is a breast cancer cell line isolated by Memorial Sloan-Kettering Cancer Center in 1970 that is used in therapeutic research, especially in the context of HER2 targeting. MDA-MB-231 cells are derived from a "basal" type human breast adenocarcinoma and are triple negative (ER, PR and HER2 negative). Adcetris (brentuximab vedotin) is a commercially available antibody-drug conjugate that targets CD30 and is therefore not expected to be active against cells that do not express CD30, such as MDA-MB-231 and SK-BR-3. Kadcyla (trastuzumab emtansine) is a commercially available antibody-drug conjugate that targets Her2 and is therefore not expected to be active against cells that express Her2 (e.g., SK-BR-3) but not against cells that do not express Her2 (e.g., MDA-MB-231). p684 and p579 are antibody drug conjugates made by the linker technology specified herein, where the linker has an N-terminal glycine (GARK(N3)(P684) and GGARK(N3)(P579)). To make the antibody-payload conjugates, a May (maytansine) molecule (see below) attached to a DBCO group was clicked onto the azide group of the linker. Both conjugates use a non-deglycosylated antibody, target Her2, and have a drug to antibody ratio of 2, thus two May (maytansine) molecules. Herceptin is a non-deglycosylated, non-conjugated antibody that targets Her2. [ka]

[0309] Cytotoxicity assay: Cells were seeded in 96-well plates (white-walled, clear flat-bottom plates) at a density of 10,000 cells / well and incubated overnight at 37°C and 5% CO2. Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) were serially diluted 1:4 in medium at a starting concentration of 10 μg / mL (66.7 mM). Medium was removed from the cells and mAb / ADC dilutions were added. Cells treated with medium alone served as a reference for 100% viability. Cells were incubated with antibodies for 3 days at 37°C and 5% CO2.

[0310] Cell viability was assessed by Cell Titer-Glo® (Promega) according to the manufacturer's instructions, as briefly outlined here. Plates were equilibrated to room temperature for 30 minutes. Cell Titer-Glo® reagent was prepared by adding Cell Titer-Glo buffer to substrate. 50 μL / well of Cell Titer-Glo® reagent was added and incubated at room temperature with shaking for 2 minutes, followed by an additional 30 minutes at room temperature. Luminescence was detected on a Perkin Elmer 2030 Multilabel Reader Victor™ X3 plate reader using an integration time of 1 second.

[0311] Data was processed as follows: luminescence values ​​of wells treated with medium alone were averaged and served as a reference for 100% viability. % viability of mAb / ADC-treated wells was calculated using the following equation:

number

[0312] Normalized % viability was plotted versus the logarithm of the mAb / ADC concentration and data was fitted using GraphPad Prism 7.00.

[0313] As can be seen in Figure 18, P684 and P579 have the same potency against SK-BR3 cells as Kadcyla. Thus, the advantages offered by the novel linker technology (ease of manufacture, site specificity, stable stoichiometry, no need to deglycosylate the antibody) are not accompanied by the disadvantages in terms of cytotoxicity. This is even more important since P684 and P579 have a DAR of 2, whereas Kadcyla has an average DAR of 3.53±0.05, thus allowing more toxin to be delivered to the target cells. The following table shows the potency (IC50): [Table 8]

[0314] Example 4 Conjugation efficiency Peptides were used as received, dissolved at appropriate stock concentrations (e.g., 25 mM), aliquots prepared and stored at -20°C according to manufacturer's instructions. Anti-Her2 IgG1 antibody (Trastuzumab) was modified as follows: 1 mg / mL of non-deglycosylated antibody (~6.67 μM) was mixed with 80 molar equivalents of peptide linker (i.e., ~533 μM), 6 U / mL MTG and buffer. The reaction mixture was incubated at 37°C for 20 hours and then subjected to LC-MS analysis under reducing conditions.

[0315] Table 8 below shows the conjugation efficiency (CE (%)) of various linkers falling within the scope of the present invention.

[0316] [Table 9]

[0317] References Dorywalska et al (2015), Site-Dependent Degradation of a Non-Cleavable Auristatin-Based Linker-Payload in Rodent Plasma and Its Effect on AD C Efficacy. PLoS ONE 10(7): e0132282 Sletten & Bertozzi, A Bioorthogonal Quadricyclane Ligation. J Am Chem Soc 2011, 133 (44), 17570-17573. Agard et al, J Am Chem Soc. 2004 Nov 24;126(46):15046-7. Stoeckmann et al (2011). "Exploring isonitrile-based click chemistry for ligation with biomolecules". Organic & Biomolecular Chemistry. 9 (21): 7303. Blackman et al (2008). "The Tetrazine Ligation: Fast Bioconjugation based on Inverse-electron-demand Diels-Alder Reactivity". Journal of the American Chemical Society. 130 (41): 13518-9. Yarema, et al (1998). "Metabolic Delivery of Ketone Groups to Sialic Acid Residues. Application To Cell Surface Glycoform Engineering". Journal of Biological Chemistry. 273 (47): 31168-79. Ning et al (2010). "Protein Modification by Strain-Promoted Alkyne-Nitrone Cycloaddition". Angewandte Chemie International Edition. 49 (17): 3065. Baskin et al (2007). "Copper-free click chemistry for dynamic in vivo imaging". Proceedings of the National Academy of Sciences. 104 (43): 16793-7. MacKenzie, DA; Sherratt, AR; Chigrinova, M; Cheung, LL; Pezacki, JP (Aug 2014). "Strain-promoted cycloadditions involving nitrones and alkynes-rapid tunable reactions for bioorthogonal labeling". Curr Opin Chem Biol. 21: 81-8. Agard, N. J.; Baskin, J. M.; Prescher, J. A.; Lo, A.; Bertozzi, C. R. (2006). "A Comparative Study of Bioorthogonal Reactions with Azides". ACS Chem. Biol. 1: 644-648 Kolb, H.C.; Sharpless, B.K. (2003). "The growing impact of click chemistry on drug discovery". Drug Discov Today. 8 (24): 1128-1137. Lhospice et al., Site-Specific Conjugation of Monomethyl Auristatin E to Anti-Cd30 Antibodies Improves Their Pharmacokinetics and Therapeutic Index in Rodent Models, Mol Pharm 12 (6), 1863-1871. 2015 Jeger et al, Site-specific and stoichiometric modification of antibodies by bacterial transglutaminase. Angew Chem Int Ed Engl. 2010 Dec 17;49(51):9995-7 Strop, et al., Versatility of Microbial Transglutaminase. Bioconjugate Chemistry 2014, 25 (5), 855-862. Spycher et al., Dual Site-Specifically Modified Antibodies With Solid-Phase Immobilized Microbial Transglutaminase. Chembiochem. 2017 Aug 03; 18(19): 1923-1927 Dennler et al., Transglutaminase-based chemo-enzymatic conjugation approach yields homogeneous antibody-drug conjugates. Bioconjug Chem. 2014 Mar 19;25(3):569-78 Dennler et al. Microbial transglutaminase and c-myc-tag: a strong couple for the functionalization of antibody-like protein scaffolds from discovery platforms. Chembiochem. 2015 Mar 23;16(5):861-7 Mindt, et al., Modification of different IgG1 antibodies via glutamine and lysine using bacterial and human tissue transglutaminase. Bioconjugate chemistry 2008, 19 (1), 271-8. Dubowchik et al., Cathepsin B-labile dipeptide linkers for lysosomal release of doxorubicin from internalizing immunoconjugates: model studies of enzymatic drug release and antigen-specific in vitro anticancer activity. Bioconjug Chem. 2002 Jul-Aug; 13(4):855-69. Zheng, et al., The impact of glycosylation on monoclonal antibody conformation and stability. Mabs-Austin 2011, 3 (6), 568-576. Subedi, et al., The Structural Role of Antibody N-Glycosylation in Receptor Interactions. Structure 2015, 23 (9), 1573-1583. Kieliszek and Misiewicz, Folia Microbiol (Praha). 2014; 59(3): 241-250 Brinkmann and Kontermann, The making of bispecific antibodies. MAbs. 2017 Feb-Mar; 9(2): 182-212. Azhdarinia A. et al., Dual-Labeling Strategies for Nuclear and Fluorescence Molecular Imaging: A Review and Analysis. Mol Imaging Biol. 2012 Jun; 14(3): 261-276. Houghton JL. et al., Site-specifically labeled CA19.9-targeted immunoconjugates for the PET, NIRF, and multimodal PET / NIRF imaging of pancreatic cancer. Proc Natl Acad Sci U S A. 2015 Dec 29;112(52):15850-5 Levengood M. et al., Orthogonal Cysteine Protection Enables Homogeneous Multi‐Drug Antibody-Drug Conjugates. Angewandte Chemie, Volume56, Issue3, January 16, 2017 Costoplus JA. et al., Peptide-Cleavable Self-immolative Maytansinoid Antibody-Drug Conjugates Designed To Provide Improved Bystander Killing. ACS Med Chem Lett. 2019 Sep 27;10(10):1393-1399. Sonzini S. et al., Improved Physical Stability of an Antibody-Drug Conjugate Using Host-Guest Chemistry. Bioconjug Chem. 2020 Jan 15;31(1):123-129. Bodero L. et al., Synthesis and biological evaluation of RGD and isoDGR peptidomimetic-α-amanitin conjugates for tumor-targeting. Beilstein J. Org. Chem. 2018, 14, 407-415. Nunes JPM. et al., Use of a next generation maleimide in combination with THIOMAB TM antibody technology delivers a highly stable, potent and near homogeneous THIOMAB TM antibody-drug conjugate (TDC). RSC Adv., 2017,7, 24828-24832. Doronina SO. et al., Enhanced activity of monomethylauristatin F through monoclonal antibody delivery: effects of linker technology on efficacy and toxicity. Bioconjug Chem. 2006 Jan-Feb;17(1):114-24. Nakada T. et al., Novel antibody drug conjugates containing exatecan derivative-based cytotoxic payloads. Bioorg Med Chem Lett. 2016 Mar 15;26(6):1542-1545. Dickgiesser S. et al., Site-Specific Conjugation of Native Antibodies Using Engineered Microbial Transglutaminase. Bioconjug Chem. 2020 Mar 12. doi: 10.1021 / acs.bioconjchem.0c00061. Steffen W. et al., Discovery of a microbial transglutaminase enabling highly site-specific labeling of proteins. The Journal of Biological Chemistry. July 27, 2017 doi: 10.1074 / jbc.M117.797811. Malesevic M. et al., A fluorescence-based array screen for transglutaminase substrates. Chembiochem. 2015 May 26;16(8):1169-74.

[0318] Disclaimer It is important to understand that in some of the linker peptides shown herein, the C-terminal portion is simply shown as N3. However, this should be understood as an abbreviation for Lys(N3). For example, GAR(N3) is actually GARK1, K1=Lys(N3 ) or GlyAlaArgLys(N3).

[0319] It is further important to understand that in the various linker peptides shown herein, the C-terminus may be protected or not, even if it is shown otherwise. Protection can be achieved by amidation. In the context of the present invention, both protected and unprotected linker peptides are included. For example, GARK(N3) actually includes two variants, with and without C-terminus protection.

Claims

1. A method for producing antibody-linker conjugates by microbial transglutaminase (MTG) is described, which comprises linking the peptide structure (shown in N→C orientation) via the N-terminal primary amine of the N-terminal glycine (Gly) residue: Gly-(｡ax) ｍ -B-(A+) ｎ (In the formula, m is an integer greater than 0 and less than or equal to 12, n is an integer between 0 and 12, m+n>0, Aax is an amino acid or amino acid derivative, ・B is the connecting part) to a glutamine (Gln) residue contained in the heavy chain or light chain of the antibody.

2. The method described in claim 1, wherein the linker comprises two or more payloads B.

3. The method described in claim 2, wherein the two or more payloads B are different from each other.

4. One or more payloads, ·toxin Cytokines ・Growth factors ・Radioactive nuclides ·hormone ・Antiviral agents ・Antibacterial agents ・Fluorescent dye - Immunomodulators / immunostimulants ・Half-life increase part - Increased solubility part Polymer-toxin conjugates ・Nucleic acid Biotin or streptavidin moiety ·vitamin a target binding moiety, and Anti-inflammatory agents 4. The method of claim 1, wherein the compound is selected from the group consisting of:

5. The toxin Pyrrolobenzodiazepines (PBDs) Auristatins (e.g., MMAE, MMAF) Maytansinoids (maytansine, DM1, DM4, DM21) Duocarmycin -Tubulysin Enediynes (e.g., calicheamicin) - PNU, doxorubicin ・Pyrrole-based kinesin spindle protein (KSP) inhibitors Calicheamicin amanitin (e.g., α-amanitin), and Camptothecins (e.g., exatecan, deruxtecan) The method of claim 4, wherein the at least one selected from the group consisting of:

6. A method according to any one of claims 1 to 5, wherein the antibody is an IgG, IgE, IgM, IgD, IgA or IgY antibody, or a fragment or recombinant variant thereof, wherein the fragment or recombinant variant retains target binding properties and comprises a C H 2 domain.

7. The method described in claim 6, wherein the antibody is a glycosylated antibody, a deglycosylated antibody or an aglycosylated antibody.

8. The method of claim 7, wherein the glycosylated antibody is an IgG antibody that is glycosylated at residue N297 (EU numbering) of the C H 2 domain.

9. The method of claim 1, wherein (a) the linker containing the payload is conjugated to a Gln residue introduced into the heavy or light chain of the antibody by molecular engineering, or (b) the linker containing the payload is conjugated to a Gln residue in the Fc domain of the antibody.

10. The antibody wherein the Gln residue in the Fc domain is Gln residue Q295 (EU numbering) in the C H 2 domain of an IgG antibody; or the Gln residue introduced by molecular engineering into the heavy or light chain of the antibody is N297Q (EU numbering) in the CH2 domain of an aglycosylated IgG antibody; or The Gln residue introduced into the heavy or light chain of the antibody by molecular engineering is (a) incorporated into the heavy or light chain of the antibody, or (b) contained in a peptide fused to the N-terminus or C-terminus of the heavy or light chain of the antibody.

10. The method of claim 9.

11. i) m + n ≦ 12, 11, 10, 9, 8, 7, 6, 5 or 4; ii) the net charge of the linker is neutral or positive; iii) the linker does not contain any negatively charged amino acid residues; iv) the linker comprises at least one positively charged amino acid residue; and / or v) the linker is Lysine, arginine, and Histidine and / or vi) the linker is not cleavable by cathepsins; and / or vii) the linker does not contain a valine-alanine or valine-citrulline motif 11. The method according to any one of claims 1 to 10.

12. A method described in any one of claims 1 to 11, wherein the linker comprising at least one payload is conjugated to the amide side chain of the Gln residue.

13. A method according to any one of claims 1 to 12, wherein the microbial transglutaminase is derived from a Streptomyces species, in particular Streptomyces mobaraensis.

14. An antibody comprising: a) an antibody containing at least one Gln residue in the heavy or light chain; and b) Peptide structure (shown in N→C orientation): Gly-(Aax) m -B-(Aax) n wherein Gly contains an N-terminal primary amine; m is an integer greater than 0 and less than or equal to 12, n is an integer between 0 and 12, m+n>0, Aax is an amino acid or amino acid derivative, ・B is the connecting part) 1. An antibody-payload conjugate comprising a linker comprising: An antibody-payload conjugate, wherein the linker is conjugated to the amide side chain of a Gln residue in the heavy or light chain of the antibody via the N-terminal primary amine of an N-terminal glycine residue contained in the linker-payload construct.

15. The antibody-payload conjugate of claim 14, wherein conjugation is achieved by microbial transglutaminase (MTG).

16. An antibody-payload conjugate described in claim 14 or 15, wherein the linker comprises two or more payloads.

17. The two or more payloads: ·toxin Cytokines ・Growth factors ・Radioactive nuclides ·hormone ・Antiviral agents ・Antibacterial agents ・Fluorescent dye - Immunomodulators / immunostimulants ・Half-life increase part - Increased solubility part Polymer-toxin conjugates ・Nucleic acid Biotin or streptavidin moiety ·vitamin a target binding moiety, and Anti-inflammatory agents The antibody-payload conjugate of any one of claims 14 to 16, selected from the group consisting of:

18. The toxin of claim 17, Pyrrolobenzodiazepines (PBDs) Auristatins (e.g., MMAE, MMAF) Maytansinoids (maytansine, DM1, DM4, DM21) Duocarmycin -Tubulysin Enediynes (e.g., calicheamicin) - PNU, doxorubicin ・Pyrrole-based kinesin spindle protein (KSP) inhibitors Calicheamicin amanitin (e.g., α-amanitin), and Camptothecins (e.g., exatecan, deruxtecan) 18. The antibody-payload conjugate of claim 17, which is at least one selected from the group consisting of:

19. i) the linker is not cleavable by cathepsins; and / or ii) the linker does not contain a valine-alanine or valine-citrulline motif; and / or iii) m+n≦12, 11, 10, 9, 8, 7, 6, 5, or 4; and / or vi) the net charge of the linker is neutral or positive; and / or v) the linker does not contain any negatively charged amino acid residues; and / or vi) the linker comprises at least one positively charged amino acid residue; and / or vii) the linker is Lysine, arginine, and Histidine and / or viii) the linker is selected from the list shown in Table 5 An antibody-payload conjugate according to any one of claims 14 to 18.

20. An antibody-payload conjugate described in any one of claims 14 to 19, wherein the one or more payloads are covalently or non-covalently bound to the linker.

21. An antibody-payload conjugate according to any one of claims 14 to 20, wherein the antibody is an IgG, IgE, IgM, IgD, IgA or IgY antibody, or a fragment or recombinant variant thereof, wherein the fragment or recombinant variant retains target binding properties and comprises a C H 2 domain.

22. The antibody-payload conjugate of claim 21, wherein the antibody is a glycosylated antibody, a deglycosylated antibody, or an aglycosylated antibody.

23. The antibody-payload conjugate of claim 22, wherein the glycosylated antibody is an IgG antibody that is glycosylated at residue N297 (EU numbering) of the C H 2 domain.

24. The antibody-payload conjugate of any one of claims 14 to 23, wherein (a) the linker-payload construct is conjugated to a Gln residue introduced into the heavy or light chain of the antibody by molecular engineering, or (b) the linker-payload construct is conjugated to a Gln residue in the Fc domain of the antibody.

25. The antibody wherein the Gln residue in the Fc domain is Gln residue Q295 (EU numbering) in the C H2 domain of an IgG antibody; or the Gln residue introduced by molecular engineering into the heavy or light chain of the antibody is N297Q (EU numbering) in the C H 2 domain of an aglycosylated antibody; or The Gln residue introduced into the heavy or light chain of the antibody by molecular engineering is (a) incorporated into the heavy or light chain of the antibody, or (b) contained in a peptide fused to the N-terminus or C-terminus of the heavy or light chain of the antibody. The antibody-payload conjugate of claim 24.

26. Comprising at least one toxin; or including inhibitors of toxins and drug efflux transporters; or Contains a toxin and a solubility-increasing moiety; or containing toxins and immunostimulants; or Contains two different toxins; or containing two immunostimulants; or Contains radionuclides and fluorescent dyes An antibody-payload conjugate according to any one of claims 14 to 25.

27. ​​The antibody-payload conjugate described in claim 26, wherein the antibody-payload conjugate comprises two different toxins, the first toxin being a toxin that inhibits cell division and the second toxin being a toxin that interferes with DNA replication and / or transcription.

28. An antibody-payload conjugate according to any one of claims 26 to 27, wherein at least one of the toxins is an auristatin or a maytansinoid.

29. The antibody-payload conjugate of claim 26, wherein at least one immunostimulant is a TLR agonist.

30. The antibody-payload conjugate of claim 26, comprising a radionuclide and a fluorescent dye, wherein the radionuclide is a radionuclide suitable for use in tomography, particularly single photon emission computed tomography (SPECT) or positron emission tomography (PET), and the fluorescent dye is a near-infrared fluorescent dye.

31. A pharmaceutical composition comprising an antibody-payload conjugate according to any one of claims 14 to 30.

32. A pharmaceutical product comprising an antibody-payload conjugate according to any one of claims 14 to 30 or a pharmaceutical composition according to claim 31, and at least one further pharmaceutically acceptable ingredient.

33. An antibody-payload conjugate according to any one of claims 14 to 30, a pharmaceutical composition according to claim 31 or a pharmaceutical product according to claim 32 for use in therapy and / or diagnosis.

34. A neoplastic disease, a neurological disease, an autoimmune disease, an inflammatory disease, or an infectious disease. - Suffering from are at risk of developing, and / or - Has been diagnosed with An antibody-payload conjugate according to any one of claims 14 to 30, a pharmaceutical composition according to claim 31 or a pharmaceutical product according to claim 32 for use in the treatment of a patient.

35. An antibody-payload conjugate according to any one of claims 14 to 30, a pharmaceutical composition according to claim 31 or a pharmaceutical product according to claim 32 for use in treating a patient suffering from a neoplastic disease.

36. A neoplastic disease, a neurological disease, an autoimmune disease, an inflammatory disease, or an infectious disease. - Suffering from are at risk of developing, and / or - Has been diagnosed with Use of an antibody-payload conjugate according to any one of claims 14 to 30, a pharmaceutical composition according to claim 31 or a pharmaceutical product according to claim 32 for the manufacture of a medicament for treating a patient.

37. A method for treating or preventing a neoplastic disease, comprising administering to a patient in need thereof an antibody-payload conjugate according to any one of claims 14 to 30, a pharmaceutical composition according to claim 31 or a pharmaceutical product according to claim 32.

38. An antibody-payload conjugate according to any one of claims 14 to 30, a pharmaceutical composition according to claim 31 or a pharmaceutical product according to claim 32 for use in pre-, intra- or post-operative imaging.

39. An antibody-payload conjugate according to any one of claims 14 to 30, a pharmaceutical composition according to claim 31 or a pharmaceutical product according to claim 32 for use in intraoperative imaging-guided cancer surgery.

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