Methods for Producing Antibody-Linker Conjugates

Abstract

The present invention relates to a method for making an antibody-payload conjugate using transglutaminase. The method comprises the step of conjugating a linker comprising the structure (shown in the N→C direction) (Sp1)-K-(Sp2)-B-(Sp3) or (Sp1)-B-(Sp2)-K-(Sp3) to a Gln residue contained in an antibody, where (Sp1) is a chemical spacer or absent; (Sp2) is a chemical spacer or absent; (Sp3) is a chemical spacer or absent; K is lysine or a lysine derivative or a lysine mimic; B is a linking moiety or a payload; the linker is conjugated to the Gln residue contained in the antibody via a primary amine contained in the side chain of the lysine residue, the lysine derivative, or the lysine mimic; and contacting the antibody with less than 80 molar equivalents of the linker. Furthermore, the present invention relates to antibody-linker conjugates, antibody-drug conjugates, and linker constructs comprising a lysine residue.

Description

[Technical field]

[0001] The present invention relates to methods for producing antibody-linker conjugates using transglutaminase.The present invention further provides antibody-linker conjugates, and pharmaceutical compositions comprising the antibody-linker conjugates of the invention, and uses thereof. [Background technology]

[0002] Antibody-drug conjugates (ADCs) are typically composed of an antibody and a small molecule drug conjugated to the antibody via a chemical linker. After decades of preclinical and clinical research, a series of ADCs have been approved for the treatment of specific tumor types, such as brentuximab vedotin (Adcetris®) for relapsed Hodgkin lymphoma and systemic anaplastic large cell lymphoma, gemtuzumab ozogamicin (Mylotarg®) for acute myeloid leukemia, adotrastuzumab emtansine (Kadcyla®) for HER2-positive metastatic breast cancer, inotuzumab ozogamicin (Besponsa®) for B-cell malignancies, and most recently polatuzumab vedotin-piiq (Polivy®). Most recently, enfortumab vedotin (Padcev®), trastuzumab deruxtecan (Enhertu®), sacituzumab govitecan (Trodelvy®), and belantamab mafadotin (Blenrep®) have received marketing approval. For a review on ADCs, see, for example, (Zhao P.et al., 2020, Acta Pharmaceutica Sinica B, 10, 1589-1600). While many ADCs have demonstrated impressive anticancer activity, many patients fail to respond to these treatments, experience severe side effects before signs of efficacy appear, or experience relapse after a period of time. Therefore, there remains a great medical need for novel ADC formats that have favorable drug-like properties, can be produced in sufficient quantities and quality to support drug development at a reasonable cost, and are suitable as therapeutic agents.

[0003] A key step in the preparation of ADCs is the covalent conjugation of the payload to the antibody. Most ADCs currently in clinical development are made by conjugation to endogenous lysine or cysteine ​​residues of the antibody, carefully controlling the average degree of modification to achieve an average drug-to-antibody ratio (DAR) in the range of 3.5 to 4.0. Historically, this ratio was chosen based on (a) minimizing the amount of unconjugated antibody and (b) avoiding very high DAR species in the mixture, which can be problematic in production and formulation due to their high hydrophobicity and low solubility (Lambert JM and Berkenbilt A., 2018, Annu. Rev. Med. 69, 191-207), typically resulting in poor pharmacokinetic properties (Lyon RP, et al., 2015, Nat Biotechnol, 33, 733-735). Recently, various genetic, chemical, and enzymatic methods for site-specific conjugation have been developed, allowing the DAR to be 2 (or 4) while avoiding undermodification or overmodification of the antibody. An overview of these methodologies has been reported by Yamada et al. (reviewed in Kei Yamada and Yuji Ito, 2019, ChemBioChem, 20, 2729-2739).

[0004] Enzymatic conjugation has attracted great interest because these conjugation reactions are usually rapid, site-specific, and can be performed under physiological conditions. Among the available enzymes, microbial transglutaminase (MTG) from the species Streptomyces mobaraensis has gained 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 S. et al., 2010, Angew. Chem. Int. Ed., 49, 9995-9997).

[0005] Jeger et al. stated that conjugation of antibodies using transglutaminase as an enzyme occurs at the Q295 residue, but conjugation is only possible when the glycan moiety at asparagine residue 297 (N297) is removed with PNGase F, and glycosylated antibodies could not be conjugated efficiently (conjugation efficiency less than 20%) (Jeger S. et al., 2010, Angew. Chem. Int. Ed., 49, 9995-9997; Mindt T. et al. 2008, Bioconj Chem, 9, 271-278).

[0006] Another approach to generate ADCs with MTG is based on the use of aglycosylated antibodies, where residue N297 is replaced with an amino acid residue that cannot undergo glycosylation. However, replacing N297 with another amino acid can have undesirable effects, as it can affect the overall stability of the entire Fc domain (Subedi GP and Barb AW., 2015, Structure, 23, 1573-1583) and the efficacy of the entire conjugate. This can result in increased aggregation of the antibody and reduced solubility, which is especially important for hydrophobic payloads. Furthermore, the glycan present on N297 has important immunomodulatory effects, as it induces effector functions such as antibody-dependent cellular cytotoxicity (ADCC). These immunomodulatory effects are lost by deglycosylation or any of the other approaches mentioned above to obtain aglycosylated antibodies. Furthermore, modifying any sequence of an established antibody can lead to regulatory issues, which is problematic since in many cases approved and clinically validated antibodies are used as the starting point for ADC conjugation.

[0007] Recently, Spycher et al. have disclosed a transglutaminase-based conjugation approach that does not require prior deglycosylation of the antibody in order to conjugate the payload (Spycher et al., WO 2019 / 057772). The ability to conjugate natively glycosylated antibodies offers a significant manufacturing advantage: an enzymatic deglycosylation step is undesirable from a Good Manufacturing Process (GMP) perspective, since it must be ensured that both the deglycosylation enzyme (e.g., PNGase F) and the cleaved glycan are removed from the reaction mixture. Furthermore, there is no need to genetically engineer the antibody in order to conjugate the payload, thus avoiding the insertion of sequences that may increase immunogenicity and decrease the overall stability of the antibody.

[0008] In view of the above, there remains a need in the art for improved methods for making ADCs with high conjugation efficiency. Summary of the Invention

[0009] The present invention is characterized in the embodiments and claims provided herein. In particular, the present invention relates inter alia to the following embodiments:

[0010] 1. A method for producing an antibody-linker conjugate using transglutaminase, comprising the following structure (shown in N→C orientation): (Sp 1 )-K-(Sp 2 )-B-(Sp 3 ) or (Sp 1 )-B-(Sp 2 )-K-(Sp 3 ) to a Gln residue contained in the antibody, (Sp 1 ) is a chemical spacer or is absent; (Sp 2 ) is a chemical spacer or is absent; (Sp 3 ) is a chemical spacer or is absent; K is lysine, or a lysine derivative or a lysine mimetic; B is a linking moiety or payload; the linker is conjugated to a Gln residue in the antibody via a primary amine in the side chain of the lysine residue, the lysine derivative, or the lysine mimetic; The method wherein the antibody is contacted with less than 80 molar equivalents of the linker.

[0011] 2. The method of embodiment 1, wherein the antibody is contacted with 20 molar equivalents or less than 20 molar equivalents of the linker.

[0012] 3. The method of embodiment 1 or 2, wherein the antibody is contacted with 2 to 20 molar equivalents of the linker.

[0013] 4. The method of any one of embodiments 1 to 3, wherein the antibody is added to the conjugation reaction at a concentration ranging from 1 to 50 mg / mL.

[0014] 5. The method according to any one of embodiments 1 to 4, wherein the transglutaminase is added to the conjugation reaction at a concentration ranging from 1 to 20 U / mg antibody.

[0015] 6. The method of any one of embodiments 1 to 5, wherein conjugation of the linker to the antibody is accomplished at a pH in the range of 6 to 8.5.

[0016] 7. The chemical spacer (Sp 1 ), (Sp 2 ), and (Sp 3 7. The method of any one of embodiments 1 to 6, wherein each of the following independently comprises 0 to 12 amino acid residues:

[0017] 8. The method of any one of embodiments 1-7, wherein the linker comprises no more than 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 amino acid residues.

[0018] 9. The method of any one of embodiments 1 to 8, wherein the net charge of the linker is neutral or positive.

[0019] 10. The method of any one of embodiments 1 to 9, wherein the linker does not contain a negatively charged amino acid residue.

[0020] 11. The method of any one of the preceding embodiments, wherein B is a linking moiety.

[0021] 12. The linking moiety B is Bioorthogonal marker groups, or Non-bio-orthogonal entities for crosslinking 12. The method of embodiment 11, comprising:

[0022] 13. The bioorthogonal marker group or the non-bioorthogonal entity for crosslinking is -NN≡N or -N 3 ; Lys(N 3 ); Tetrazine; ·Alkynes; ·Distorted cyclooctyne; · BCN; · Strained alkenes; Photoreactive groups; ·aldehyde; Acyltrifluoroborates; ·Proteolytic agents (“PROTACs”); ·Cyclopentadiene / spirolocyclopentadiene; ·Thioselective electrophiles; -SH; and Cysteine 13. The method of embodiment 12, consisting of or comprising at least one molecule or moiety selected from the group consisting of:

[0023] 14. The method of any one of embodiments 11 to 13, comprising the further step of conjugating one or more payloads to said linking moiety B.

[0024] 15. The method of embodiment 14, wherein the one or more payloads are conjugated to the linking moiety B via a Click reaction.

[0025] 16. The method of any one of embodiments 1 to 10, wherein B is a payload.

[0026] 17. The payload comprises: ·toxin; Cytokines; ·Growth factors; Radionuclides; ·hormone; Antiviral agents; Antibacterial agents; · Fluorescent dyes; ·Immunomodulators / immunostimulants; · Half-life extending moieties; ·Solubility enhancing moiety; Polymer-toxin conjugates; ·Nucleic acid; · a biotin or streptavidin moiety; ·vitamin; ·Proteolytic agents (“PROTACs”); a target-binding moiety; and / or Anti-inflammatory 17. The method according to any one of embodiments 14 to 16, comprising at least one of:

[0027] 18. The toxin is Pyrrolobenzodiazepines (e.g. PBD); Auristatins (e.g., MMAE, MMAF); Maytansinoids (e.g. maytansine, DM1, DM4, DM21); ·Duocarmycin; · Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors; ·Tubulysin; Enediynes (e.g., calicheamicin); · Anthracycline derivatives (PNU) (e.g. doxorubicin); · Pyrrole-based kinesin spindle protein (KSP) inhibitors; · Cryptophycin; Drug efflux pump inhibitors; Sandramycin; Amanitin (e.g., α-amanitin); and Camptothecins (e.g., exatecan, deruxtecan) 18. The method of embodiment 17, wherein the at least one selected from the group consisting of:

[0028] 19. The chemical spacer (Sp 2 19. The method of any one of the preceding claims, wherein said polymerizable compound comprises a self-immolative moiety.

[0029] 20. The method of embodiment 19, wherein the self-immolative moiety is directly attached to the payload B.

[0030] 21. The method of embodiment 19 or 20, wherein the self-immolative moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety or a self-immolative aminomethylene spacer.

[0031] 22. The method according to any one of the preceding embodiments, wherein the antibody is an IgG antibody, in particular an IgG1 antibody.

[0032] 23. The Gln residue to which the linker is conjugated is contained in the Fc domain of the antibody, and in particular, the Gln residue to which the linker is conjugated is the C domain of an IgG antibody. H 23. The method of embodiment 22, wherein the Gln residue Q295 (EU numbering) of domain 2 is Gln residue Q295 (EU numbering).

[0033] 24. The method according to embodiment 22, wherein the Gln residue to which the linker is conjugated has been introduced into the heavy or light chain of the antibody by molecular engineering.

[0034] 25. The Gln residue introduced into the heavy or light chain of an antibody by molecular engineering is C of an aglycosylated IgG antibody. H 25. The method of embodiment 24, wherein the 2 domain is N297Q (EU numbering).

[0035] 26. The method of embodiment 24, wherein the Gln residue introduced into the antibody heavy or light chain by molecular engineering is (a) incorporated into the antibody heavy or light chain or (b) contained in a peptide fused to the N-terminus or C-terminus of the antibody heavy or light chain.

[0036] 27. The method of embodiment 26, wherein the peptide containing the Gln residue is fused to the C-terminus of the heavy chain of the antibody.

[0037] 28. The IgG antibody is a glycosylated IgG antibody, particularly when the IgG antibody is C H28. The method of any one of embodiments 22-24 or 26-27, wherein the IL-2 domain is glycosylated at residue N297 (EU numbering).

[0038] 29. The method of any one of embodiments 1-28, wherein the antibody is selected from the group consisting of brentuximab, trastuzumab, gemtuzumab, inotuzumab, avelumab, cetuximab, rituximab, daratumumab, pertuzumab, vedolizumab, ocrelizumab, tocilizumab, ustekinumab, golimumab, obinutuzumab, sacituzumab, belantamab, polatuzumab, and enfortumab.

[0039] 30. The method according to any one of embodiments 1 to 29, wherein the linker is conjugated to the γ-carboxamide group of a Gln residue contained in the antibody.

[0040] 31. The method of any one of embodiments 1 to 30, wherein the linker is suitable for conjugation to a glycosylated antibody with a conjugation efficiency of at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0041] 32. The method according to any one of the preceding embodiments, wherein the transglutaminase is a microbial transglutaminase, preferably derived from a Streptomyces species, in particular Streptomyces mobaraensis.

[0042] 33. An antibody-linker conjugate produced by the method according to any one of embodiments 1 to 32.

[0043] 34. A pharmaceutical composition comprising the antibody-linker conjugate of embodiment 33.

[0044] 35. The pharmaceutical composition according to embodiment 34, comprising at least one additional therapeutically active agent.

[0045] 36. The antibody-linker conjugate according to embodiment 33 or the pharmaceutical composition according to embodiment 34 or 35, for use in therapy and / or diagnosis, in particular wherein the antibody-linker conjugate comprises at least one payload.

[0046] 37. Neoplastic disease, neurological disease, autoimmune disease, inflammatory disease, or infectious disease suffer from, are at risk of developing and / or Diagnosed with The antibody-linker conjugate of embodiment 33 or the pharmaceutical composition of embodiment 34 or 35, for use in the treatment of a patient, particularly wherein the antibody-linker conjugate comprises at least one toxin.

[0047] 38. The antibody-linker conjugate or pharmaceutical composition according to embodiment 37, wherein the antibody-linker conjugate comprises polatuzumab and the neoplastic disease is a B-cell-related cancer.

[0048] 39. The antibody-linker conjugate or pharmaceutical composition according to embodiment 38, wherein said B cell related cancer is non-Hodgkin's lymphoma, in particular said B cell related cancer is diffuse large B cell lymphoma.

[0049] 40. The antibody-linker conjugate or pharmaceutical composition according to embodiment 37, wherein the antibody-linker conjugate comprises trastuzumab and the neoplastic disease is a HER2-positive cancer, in particular a HER2-positive breast cancer, gastric cancer, ovarian cancer, or lung cancer.

[0050] 41. The antibody-linker conjugate or pharmaceutical composition according to embodiment 37, wherein the antibody-linker conjugate comprises enfortumab or an enfortumab variant and the neoplastic disease is a nectin-4 positive cancer, in particular a nectin-4 positive pancreatic cancer, lung cancer, bladder cancer, or breast cancer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] Thus, in one embodiment, the invention provides a method for producing an antibody-linker conjugate using transglutaminase, comprising the following structure (shown in the N→C orientation): (Sp 1 )-K-(Sp 2 )-B-(Sp 3 ) or (Sp 1 )-B-(Sp 2 )-K-(Sp 3 ) to a Gln residue contained in the antibody, (Sp 1 ) is a chemical spacer or is absent; (Sp 2 ) is a chemical spacer or is absent; (Sp 3 ) is a chemical spacer or is absent; K is lysine, or a lysine derivative or a lysine mimetic; B is a linking moiety or payload; the linker is conjugated to a Gln residue in the antibody via a primary amine in the side chain of the lysine residue, the lysine derivative, or the lysine mimetic; wherein said antibody is contacted with less than 80 molar equivalents of said linker.

[0052] Thus, the present invention is based, at least in part, on the surprising discovery that, under optimized reaction conditions, lysine-based linkers can be conjugated to glycosylated antibodies with extremely high efficiency. In particular, the inventors have shown that optimizing the linker to antibody ratio results in surprisingly high conjugation efficiency.

[0053] In WO 2019 / 057772, it was demonstrated for the first time that lysine-based peptide linkers can be conjugated to glycosylated antibodies. It was believed in the art that glycosylated antibodies must be contacted with a large excess of linkers to achieve efficient conjugation. As a result, in WO 2019 / 057772, glycosylated antibodies are contacted with 80 molar equivalents of lysine-based linkers to achieve conjugation. Contrary to previous belief, the inventors surprisingly found that lowering the linker-to-antibody ratio results in significantly more efficient conjugation of glycosylated antibodies. As a result, quantitative conjugation of various linkers, including bulky payloads, was achieved in a single step.

[0054] In particular, it has been demonstrated that contacting the antibody with less than 80 molar equivalents of the linker improves conjugation efficiency. In a preferred embodiment, the antibody is contacted with less than 70 molar equivalents of the linker. In a more preferred embodiment, the antibody is contacted with less than 60 molar equivalents of the linker. In a further preferred embodiment, the antibody is contacted with less than 50 molar equivalents of the linker. In a further preferred embodiment, the antibody is contacted with less than 40 molar equivalents of the linker. In a further preferred embodiment, the antibody is contacted with less than 30 molar equivalents of the linker. In a further preferred embodiment, the antibody is contacted with less than 20 molar equivalents of the linker. In a further preferred embodiment, the antibody is contacted with less than 15 molar equivalents of the linker. In a most preferred embodiment, the antibody is contacted with less than 10 molar equivalents of the linker.

[0055] Alternatively, the antibody may be contacted with 2 to 80 molar equivalents of the linker, preferably 2 to 70 molar equivalents of the linker, more preferably 2 to 60 molar equivalents of the linker, even more preferably 2 to 50 molar equivalents of the linker, even more preferably 2 to 40 molar equivalents of the linker, even more preferably 2 to 30 molar equivalents of the linker, even more preferably 2 to 25 molar equivalents of the linker, even more preferably 2 to 20 molar equivalents of the linker, even more preferably 2 to 15 molar equivalents of the linker, even more preferably 2 to 10 molar equivalents of the linker, and most preferably 2 to 8 molar equivalents of the linker.

[0056] Alternatively, the antibody may be contacted with 2.5 to 80 molar equivalents of the linker, preferably 2.5 to 70 molar equivalents of the linker, more preferably 2.5 to 60 molar equivalents of the linker, even more preferably 2.5 to 50 molar equivalents of the linker, even more preferably 2.5 to 40 molar equivalents of the linker, even more preferably 2.5 to 30 molar equivalents of the linker, even more preferably 2.5 to 25 molar equivalents of the linker, even more preferably 2.5 to 20 molar equivalents of the linker, even more preferably 2.5 to 15 molar equivalents of the linker, even more preferably 2.5 to 10 molar equivalents of the linker, and most preferably 2.5 to 8 molar equivalents of the linker.

[0057] The antibody may be added to the conjugation reaction at any concentration. However, it is preferred to add the antibody to the conjugation reaction at a concentration in the range of 0.1 to 50 mg / mL. That is, in a particular embodiment, the present invention relates to a method according to the present invention, in which the antibody is added to the conjugation reaction at a concentration in the range of 0.1 to 50 mg / mL, preferably 1 to 50 mg / mL, more preferably 1 to 25 mg / mL, more preferably 2.5 to 20 mg / mL, even more preferably 5 to 20 mg / mL, and most preferably 5 to 17 mg / mL.

[0058] The method according to the invention is preferably carried out at a pH in the range of 6 to 9. Thus, in a preferred embodiment, the invention relates to a method according to the invention, wherein 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 6.5 to 8, even 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 conjugation of the linker to the antibody is achieved at pH 7.6.

[0059] The method of the invention can be carried out in any buffer suitable for conjugation of the payload to the linker. Buffers suitable for the method of the invention include, but are not limited to, Tris, MOPS, HEPES, PBS, or BisTris buffers. The concentration of the buffer depends, inter alia, on the concentration of the antibody and / or linker and may range from 10 to 1000 mM, 10 to 500 mM, 10 to 400 mM, 10 to 250 mM, 10 to 150 mM, or 10 to 100 mM. Furthermore, the buffer may have any salt concentration suitable for carrying out the method of the invention. For example, the buffers used in the methods of the invention may have a salt concentration of <150 mM, <140 mM, <130 mM, <120 mM, <110 mM, <100 mM, <90 mM, <80 mM, <70 mM, <60 mM, <50 mM, <40 mM, <30 mM, <20 mM, or <10 mM, or may have no salt. In certain embodiments, the methods of the invention are carried out in 50 mM Tris, pH 7.6, preferably without salt.

[0060] It should be noted that optimal reaction conditions (e.g., pH, buffer, salt concentration) may vary from payload to payload and will depend in part on the physicochemical properties of the linker and / or payload, however, one of skill in the art would not need to carry out undue experimentation to identify suitable reaction conditions for carrying out the methods of the invention.

[0061] Transglutaminase may be added to the conjugation reaction at any concentration that allows the antibody and linker to be efficiently conjugated. In certain embodiments, the concentration of transglutaminase in the conjugation reaction may depend on the amount of antibody used in the same reaction. For example, transglutaminase may be added to the conjugation reaction at a concentration of less than 100 U / mg antibody, less than 90 U / mg antibody, less than 80 U / mg antibody, less than 70 U / mg antibody, less than 60 U / mg antibody, less than 50 U / mg antibody, less than 40 U / mg antibody, less than 30 U / mg antibody, less than 20 U / mg antibody, less than 10 U / mg antibody, or less than 6 U / mg antibody. In certain embodiments, transglutaminase may be added to the conjugation reaction at a concentration of 1 U / mg antibody, 3 U / mg antibody, 5 U / mg antibody, or 6 U / mg antibody.

[0062] That is, in certain embodiments, transglutaminase may be added to the conjugation reaction at a concentration in the range of 1 to 20 U / mg antibody, preferably 1 to 10 U / mg antibody, more preferably 1 to 7.5 U / mg antibody, even more preferably 2 to 6 U / mg antibody, even more preferably 2 to 4 U / mg antibody, and most preferably 3 U / mg antibody.

[0063] The method according to the invention is preferably catalyzed by a microbial transglutaminase. However, it should be noted that an equivalent reaction can be carried out by an enzyme having transglutaminase activity of non-microbial origin. Thus, the antibody-linker conjugate according to the invention can be produced using an enzyme having transglutaminase activity of non-microbial origin.

[0064] It is to be understood that the present application encompasses any combination of linkers, antibodies, transglutaminases, and / or buffer concentrations disclosed above.

[0065] In a preferred embodiment, the present invention provides a method for producing an antibody-linker conjugate using transglutaminase, comprising the following structure (shown in the N→C orientation): (Sp 1 )-K-(Sp 2 )-B-(Sp 3 ) or (Sp 1 )-B-(Sp 2 )-K-(Sp 3 ) to a Gln residue contained in the antibody, (Sp 1 ) is a chemical spacer or is absent; (Sp 2 ) is a chemical spacer or is absent; (Sp 3 ) is a chemical spacer or is absent; K is lysine, or a lysine derivative or a lysine mimetic; B is a linking moiety or payload; the linker is conjugated to a Gln residue in the antibody via a primary amine in the side chain of the lysine residue, the lysine derivative, or the lysine mimetic; contacting the antibody with 2-70 molar equivalents of the linker; and / or adding the antibody to the conjugation reaction at a concentration ranging from 0.1-50 mg / mL; and optionally adding the transglutaminase to the conjugation reaction at a concentration ranging from 1-20 U / mg antibody.

[0066] In a more preferred embodiment, the present invention provides a method for producing an antibody-linker conjugate using transglutaminase, comprising the following structure (shown in the N→C orientation): (Sp 1 )-K-(Sp 2 )-B-(Sp 3 ) or (Sp 1 )-B-(Sp 2 )-K-(Sp 3 ) to a Gln residue contained in the antibody, (Sp 1 ) is a chemical spacer or is absent; (Sp2 ) is a chemical spacer or is absent; (Sp 3 ) is a chemical spacer or is absent; K is lysine, or a lysine derivative or a lysine mimetic; B is a linking moiety or payload; the linker is conjugated to a Gln residue in the antibody via a primary amine in the side chain of the lysine residue, the lysine derivative, or the lysine mimetic; contacting the antibody with 2-50 molar equivalents of the linker; and / or adding the antibody to the conjugation reaction at a concentration ranging from 1-50 mg / mL; and optionally adding the transglutaminase to the conjugation reaction at a concentration ranging from 1-20 U / mg antibody.

[0067] In an even more preferred embodiment, the present invention relates to a method for producing an antibody-linker conjugate using transglutaminase, comprising the following structure (shown in the N→C orientation): (Sp 1 )-K-(Sp 2 )-B-(Sp 3 ) or (Sp 1 )-B-(Sp 2 )-K-(Sp 3 ) to a Gln residue contained in the antibody, (Sp 1 ) is a chemical spacer or is absent; (Sp 2 ) is a chemical spacer or is absent; (Sp 3 ) is a chemical spacer or is absent; K is lysine, or a lysine derivative or a lysine mimetic; B is a linking moiety or payload; the linker is conjugated to a Gln residue in the antibody via a primary amine in the side chain of the lysine residue, the lysine derivative, or the lysine mimetic; contacting the antibody with 2-25 molar equivalents of the linker; and / or adding the antibody to the conjugation reaction at a concentration in the range of 2.5-25 mg / mL; and optionally adding the transglutaminase to the conjugation reaction at a concentration in the range of 1-15 U / mg antibody.

[0068] In an even more preferred embodiment, the present invention relates to a method for producing an antibody-linker conjugate using transglutaminase, comprising the following structure (shown in the N→C orientation): (Sp 1 )-K-(Sp 2 )-B-(Sp 3 ) or (Sp 1 )-B-(Sp 2 )-K-(Sp 3 ) to a Gln residue contained in the antibody, (Sp 1 ) is a chemical spacer or is absent; (Sp 2 ) is a chemical spacer or is absent; (Sp 3 ) is a chemical spacer or is absent; K is lysine, or a lysine derivative or a lysine mimetic; B is a linking moiety or payload; the linker is conjugated to a Gln residue in the antibody via a primary amine in the side chain of the lysine residue, the lysine derivative, or the lysine mimetic; contacting the antibody with 2-20 molar equivalents of the linker; and / or adding the antibody to the conjugation reaction at a concentration in the range of 2.5-20 mg / mL; and optionally adding the transglutaminase to the conjugation reaction at a concentration in the range of 1-15 U / mg antibody.

[0069] In an even more preferred embodiment, the present invention relates to a method for producing an antibody-linker conjugate using transglutaminase, comprising the following structure (shown in the N→C orientation): (Sp 1 )-K-(Sp 2 )-B-(Sp 3 ) or (Sp 1 )-B-(Sp 2 )-K-(Sp 3 ) to a Gln residue contained in the antibody, (Sp 1 ) is a chemical spacer or is absent; (Sp 2 ) is a chemical spacer or is absent; (Sp 3 ) is a chemical spacer or is absent; K is lysine, or a lysine derivative or a lysine mimetic; B is a linking moiety or payload; the linker is conjugated to a Gln residue in the antibody via a primary amine in the side chain of the lysine residue, the lysine derivative, or the lysine mimetic; contacting the antibody with 2.5 to 15 molar equivalents of the linker; and / or adding the antibody to the conjugation reaction at a concentration in the range of 2.5 to 20 mg / mL; and optionally adding the transglutaminase to the conjugation reaction at a concentration in the range of 1 to 10 U / mg antibody.

[0070] In an even more preferred embodiment, the present invention relates to a method for producing an antibody-linker conjugate using transglutaminase, comprising the following structure (shown in the N→C orientation): (Sp 1 )-K-(Sp 2 )-B-(Sp 3 ) or (Sp 1 )-B-(Sp 2 )-K-(Sp 3 ) to a Gln residue contained in the antibody, (Sp 1 ) is a chemical spacer or is absent; (Sp 2 ) is a chemical spacer or is absent; (Sp 3 ) is a chemical spacer or is absent; K is lysine, or a lysine derivative or a lysine mimetic; B is a linking moiety or payload; the linker is conjugated to a Gln residue in the antibody via a primary amine in the side chain of the lysine residue, the lysine derivative, or the lysine mimetic; contacting the antibody with 2.5 to 10 molar equivalents of the linker; and / or adding the antibody to the conjugation reaction at a concentration in the range of 5 to 20 mg / mL; and optionally adding the transglutaminase to the conjugation reaction at a concentration in the range of 2 to 10 U / mg antibody.

[0071] In a most preferred embodiment, the present invention provides a method for producing an antibody-linker conjugate using transglutaminase, comprising the following structure (shown in the N→C orientation): (Sp 1 )-K-(Sp 2 )-B-(Sp 3 ) or (Sp 1 )-B-(Sp 2 )-K-(Sp 3 ) to a Gln residue contained in the antibody, (Sp 1 ) is a chemical spacer or is absent; (Sp 2 ) is a chemical spacer or is absent; (Sp 3 ) is a chemical spacer or is absent; K is lysine, or a lysine derivative or a lysine mimetic; B is a linking moiety or payload; the linker is conjugated to a Gln residue in the antibody via a primary amine in the side chain of the lysine residue, the lysine derivative, or the lysine mimetic; contacting the antibody with 2.5 to 8 molar equivalents of the linker; and / or adding the antibody to the conjugation reaction at a concentration in the range of 5 to 17 mg / mL; and optionally adding the transglutaminase to the conjugation reaction at a concentration in the range of 2 to 10 U / mg antibody.

[0072] In another embodiment, the invention provides a method of producing an antibody-linker conjugate using transglutaminase, comprising the following structure (shown in the N→C orientation): (Sp 1 )-K-(Sp 2 )-B-(Sp 3 ) or (Sp 1 )-B-(Sp 2 )-K-(Sp 3 ) to a Gln residue contained in the antibody, (Sp 1 ) is a chemical spacer or is absent; (Sp 2 ) is a chemical spacer or is absent; (Sp 3 ) is a chemical spacer or is absent; K is lysine, or a lysine derivative or a lysine mimetic; B is a linking moiety or payload; the linker is conjugated to a Gln residue in the antibody via a primary amine in the side chain of the lysine residue, the lysine derivative, or the lysine mimetic; contacting the antibody with 5-40 molar equivalents of the linker; and / or adding the antibody to the conjugation reaction at a concentration ranging from 5-17 mg / mL; and optionally adding the transglutaminase to the conjugation reaction at a concentration ranging from 2-10 U / mg antibody.

[0073] In another embodiment, the invention provides a method of producing an antibody-linker conjugate using transglutaminase, comprising the following structure (shown in the N→C orientation): (Sp 1 )-K-(Sp 2 )-B-(Sp 3 ) or (Sp 1 )-B-(Sp 2 )-K-(Sp 3 ) to a Gln residue contained in the antibody, (Sp 1 ) is a chemical spacer or is absent; (Sp 2 ) is a chemical spacer or is absent; (Sp 3 ) is a chemical spacer or is absent; K is lysine, or a lysine derivative or a lysine mimetic; B is a linking moiety or payload; the linker is conjugated to a Gln residue in the antibody via a primary amine in the side chain of the lysine residue, the lysine derivative, or the lysine mimetic; contacting the antibody with 5-20 molar equivalents of the linker; and / or adding the antibody to the conjugation reaction at a concentration in the range of 5-17 mg / mL; and optionally adding the transglutaminase to the conjugation reaction at a concentration in the range of 2-10 U / mg antibody.

[0074] In the present invention, it is preferred that the linker has the structure (Sp 1 )-K-(Sp 2 )-B-(Sp 3 ) or (Sp 1 )-B-(Sp 2 )-K-(Sp 3) wherein the linker is conjugated to a glutamine residue in the antibody via a primary amine contained in residue K of the linker. In certain embodiments, residue K is a lysine residue. However, in certain embodiments, residue K may be a lysine mimetic or a lysine derivative, provided that the lysine mimetic or the lysine derivative contains a primary amine in its amino acid side chain.

[0075] Thus, in certain embodiments, the residue K may be a lysine mimetic. The term "lysine mimetic" as used herein refers to a compound that has a different structure from lysine but similar properties to lysine and thus can be used to replace lysine in a peptide or protein without significantly changing the function and / or structure of the peptide or protein. In certain embodiments, a lysine mimetic may differ from lysine in the length or composition of the aliphatic chain connecting the primary amine to the α-carbon atom. Thus, in certain embodiments, a lysine mimetic may be ornithine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, 2,5-diaminopentanoic acid, 2,6-diaminohexanoic acid, or 2,7-diaminoheptanoic acid. In certain embodiments, a lysine mimetic may be a β-amino acid, such as β-homolysine.

[0076] In certain embodiments, the residue K may be a lysine derivative. The term "lysine derivative" as used herein refers to a lysine or lysine mimic in which one or more functional groups contained in the lysine or lysine mimic are modified or substituted. In the present invention, the amino group in the side chain of the lysine derivative is preferably not modified, so that it is available for conjugation to a glutamine residue in a protein. In embodiments, when the residue K is located at the C-terminal position of the linker, K may be a lysine derivative in which the α-carboxyl group is modified or substituted. In certain embodiments, the α-carboxyl group of the lysine mimic may be amidated.

[0077] In the present invention, the lysine-based linker has the structure (Sp1 )-K-(Sp 2 )-B-(Sp 3 ) or (Sp 1 )-B-(Sp 2 )-K-(Sp 3 ), i.e., the linker may comprise one or more chemical spacers (Sp). The term "chemical spacer", as used herein, refers to a chemical moiety that is covalently attached to a chemical residue of the linker and / or that is interposed between two chemical residues of the linker.

[0078] In certain embodiments, the present invention provides a chemical spacer (Sp 1 ), (Sp 2 ), and (Sp 3 ) each independently contain 0 to 12 amino acid residues.

[0079] That is, in certain embodiments, the chemical spacer (Sp 1 ), (Sp 2 ) and / or (Sp 3 ) may or may not be present. (Sp 1 ), (Sp 2 ), and / or (Sp 3 In embodiments where (Sp 1 ), (Sp 2 ), and / or (Sp 3 ) may contain one or more amino acid residues. In such embodiments, (Sp 1 ), (Sp 2 ), and / or (Sp 3 ) may each contain 0 to 12 amino acid residues. 1 ), (Sp 2 ), and / or (Sp 3 It should be noted that ) may contain non-amino acid residues, as disclosed in more detail below.

[0080] Chemical spacer (Sp 1 ), (Sp 2 ), and / or (Sp3 The "amino acid residue" contained in may be an amino acid, an amino acid mimetic, or an amino acid derivative. It should be understood that the term amino acid encompasses not only α-amino acids, but also other amino acids such as β-, γ-, or δ-amino acids. The α-amino acid residue may be separated from the chemical spacer (Sp 1 ), (Sp 2 ), and / or (Sp 3 ) in its L- or D-form. 1 ), (Sp 2 ), and / or (Sp 3 ) contains a chiral β-, γ-, or δ-amino acid, the chiral β-, γ-, or δ-amino acid may be present in its S- or R-form. Thus, in its broadest sense, the term "amino acid residue" as used herein refers to any residue that includes an amino group (-NH 2 ) and a carboxyl group (-COOH). Thus, whenever an "amino acid" or "amino acid residue" is referred to throughout this disclosure, it should be understood that the term amino acid residue can also encompass amino acid mimetics or derivatives.

[0081] Furthermore, the term amino acid residue is not limited to the known set of proteinogenic amino acids, i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, but also encompasses non-standard and unnatural amino acids. A "non-standard amino acid", as used herein, may be any amino acid that is not part of the set of proteinogenic amino acids but can be obtained from natural sources. However, it should be noted that some non-standard amino acids can be found in naturally occurring peptides and / or proteins.

[0082] "Unnatural amino acids" or "synthetic amino acids", as used herein, may be any molecule that fits the general definition of an amino acid, i.e., contains an amino group and a carboxyl group, but is not found in nature. Thus, unnatural amino acids are preferably obtained by chemical synthesis. It should be understood that the distinction between non-standard and unnatural amino acids may be uncertain in some cases. For example, an amino acid defined as an unnatural amino acid may later be identified in nature and reclassified as a non-standard amino acid.

[0083] Examples of non-standard or unnatural amino acids are D-amino acids (such as D-alanine, D-arginine, D-methionine), homoamino acids (such as homoserine, homoarginine, homocysteine, α-aminoadipic acid), N-methylated amino acids (such as sarcosine, N-Me-leucine), α-methyl amino acids (such as α-methyl-histidine, α-aminoisobutyric acid), β-amino acids (such as β-alanine, D-3-aminoisobutyric acid, L-β-homoalanine), γ-amino acids (such as γ-aminobutyric acid), and mimetics or derivatives of alanine. (β-cyclopropylalanine, phenylglycine, dehydro-alanine, β-cyanoalanine, β-(3-pyridyl)-alanine, β-(1,2,4-triazol-1-yl)-alanine, β-(1-piperazinyl)-alanine, etc.), mimics or derivatives of phenylalanine (4-iodophenylalanine, pentafluoro-phenylalanine, naphthyl-alanine, 4-aminophenylalanine, etc.), mimics or derivatives of arginine (β-ureidoalanine, ω-methylarginine, etc.), mimics of lysine mimetics or derivatives (e.g., (3-(3-methyl-3H-diazirin-3-yl)propamino)carbonyl-L-lysine, Nε,Nε,Nε-trimethyllysine), histidine mimetics or derivatives (e.g., 2,5-di-iodohistidine, 1-methylhistidine), tyrosine mimetics or derivatives (e.g., 3-aminotyrosine, tyronine, 3,5-dinitrotyrosine, 3-hydroxy-methyl-tyrosine, O-phospho-L-tyrosine), tryptophan mimetics or derivatives (e.g., 5-hydroxytryptophan, 1-methyl ... ptophan, etc.), serine mimetics or derivatives (β-(2-thienyl)-serine, β-(3,4-dihydroxyphenyl)-serine, O-phosphoserine, etc.), threonine mimetics or derivatives (allo-threonine, O-phosphothreonine, etc.), proline mimetics or derivatives (hydroxyproline, 3,4-dehydro-proline, pyroglutamic acid, thiaproline, cis-octahydroindole-2-carboxylic acid, etc.), leucine and isoleucine mimetics or derivatives (allo-isoleucine, norleucine, 4,5-dehydroleucine, (4S)-4-hydroxy-L-isoleucine, etc.), valine mimics or derivatives (norvaline, gamma-hydroxyvaline, etc.), citrulline mimics or derivatives (thiocitrulline, homocitrulline, etc.), cysteine ​​mimics or derivatives (penicillamine, selenocysteine, buthionine sulfoximine, etc.), methionine mimics or derivatives (S-methylmethionine, L-methionine sulfone, L-methionine sulfoxide, L -methionine sulfo-oximine, selenomethionine, etc.), aspartic acid mimetics or derivatives (DL-threo-β-hydroxyaspartic acid, L-aspartic acid β-methyl ester, etc.), glutamic acid mimetics or derivatives (γ-methylene glutamic acid, γ-carboxyglutamic acid, etc., γ-hydroxyglutamic acid, L-glutamic acid 5-methyl ester, L-2-aminoheptanedioic acid, etc.), asparagine mimetics or derivatives (L-threo-3 -hydroxyasparagine, N,N-dimethyl-L-asparagine, L-2-amino-2-carboxyethanesulfonamide, 5-diazo-4-oxo-L-norvaline, etc.), mimetics or derivatives of glutamine (4-F-(2S,4R)-fluoroglutamine, γ-glutamylmethylamide, theanine, L-glutamic acid γ-monohydroxamate, etc.), amino acids containing cyclic moieties (4-aminopiperidine-4-carboxylic acid, azetidine-2-carbo The amino acid may be, but is not limited to, an amino acid containing a bioorthogonal moiety (such as propargylglycine, α-allylglycine, L-azido-homoalanine, p-benzoyl-l-phenylalanine, p-2-fluoroacetyl-l-phenylalanine, (S)-2-amino-3-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)propanoic acid, etc.).

[0084] In addition to the α-amino acids mentioned above, chemical spacers (Sp 1 ), (Sp 2 ), and / or (Sp 3) may contain one or more β-, γ-, δ-, or ε-amino acids. Thus, in certain embodiments, the linker may be a peptidomimetic. A peptidomimetic may not only contain a classical peptide bond formed between two α-amino acids, but may additionally or alternatively contain one or more amide bonds formed between an α-amino acid and a β-, γ-, δ-, or ε-amino acid, or between two β-, γ-, δ-, or ε-amino acids, respectively. Thus, in any example of the present invention where the linker is described as being a peptide, it is understood that the linker may be a peptidomimetic and thus may not only consist of α-amino acids, but may instead contain one or more β-, γ-, δ-, or ε-amino acids or molecules that are not classified as amino acids. 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.

[0085] In addition, chemical spacers (Sp 1 ), (Sp 2 ), and / or (Sp 3 ) may include amino acid derivatives and / or amino acid mimetics. 1 ), (Sp 2 ), and / or (Sp 3 In embodiments where Sp comprises one or more amino acid derivatives, it is preferred that the amino acid derivatives have free amino and carboxyl groups such that they can undergo peptide or isopeptide bond formation. 1 ), (Sp 2 ), and / or (Sp 3In embodiments where the amino acid mimic comprises one or more amino acid mimetics, the amino acid mimetics may have free amino and carboxyl groups such that they can undergo peptide or isopeptide bond formation. However, in certain embodiments, the amino acid mimetics or derivatives may have substituted amino groups that do not prevent peptide bond formation. Examples of such amino acid mimetics or derivatives may be N-methylated amino acids, such as sarcosine or N-Me-leucine.

[0086] (Sp 1 ) or (Sp 3 In embodiments where the amino acid residue included in (a) is a terminal amino acid residue, the terminal amino acid residue may comprise a modified, protected, or substituted N-terminal amino group or a C-terminal carboxyl group.

[0087] Additionally, the amino acid mimetic or derivative may be an amino acid that contains a derivatized amino group, e.g., a mimetic or derivative of proline or other cyclic amino acids such as azetidine-2-carboxylic acid, pipecolic acid, or spinacin. Additionally, the amino acid mimetic may contain other functional groups that replace the amino and / or carboxyl groups of standard amino acids, allowing the amino acid mimetic to undergo additional bond formation with adjacent amino acids, amino acid derivatives, and / or amino acid mimetics to form peptidomimetics.

[0088] The term "amino acid mimetic" as used herein refers to a compound that has a different structure from a particular amino acid, but functions similarly to the particular amino acid, and can therefore be used to replace the particular amino acid. An amino acid mimetic is said to function similarly to a particular amino acid if it meets at least some similar structural and / or functional characteristics of the amino acid it mimics. The term "amino acid derivative" refers to an amino acid, as defined herein, in which one or more functional group(s) contained in the amino acid have been modified or replaced. The amino acid derivative may preferably be a derivative of a proteinogenic amino acid or a non-standard amino acid. Any functional group of the amino acid derivative may be replaced or modified.

[0089] In embodiments where the linker includes one or more terminal amino acid residue(s), the terminal amino acid residue(s) may be protected. For example, (Sp 1 In embodiments where the spacer (Sp) comprises an N-terminal amino acid residue, the N-terminal amino group may be protected. For example, in certain embodiments, the spacer (Sp 1 ) may be acetylated. In other embodiments, the lysine residue K may be the N-terminal amino acid of the linker. In such embodiments, the N-terminal amino group of the lysine, lysine mimetic, or lysine derivative may be protected, for example, by acetylation. In certain embodiments, the linking moiety B or payload B may be an amino acid or may be based on an amino acid. In such embodiments, the N-terminal amino group of the amino acid-based payload or linking moiety B may be protected, for example, by acetylation.

[0090] Similarly, (Sp 3 In embodiments where the spacer (Sp) comprises a C-terminal amino acid residue, the C-terminal carboxyl group may be protected. For example, in certain embodiments, the spacer (Sp 3) may be amidated. In other embodiments, the K residue may be the C-terminal amino acid of the linker. In such embodiments, the C-terminal carboxyl group of the lysine, lysine mimetic, or lysine derivative may be protected, e.g., by amidation. In certain embodiments, linking moiety B or payload B may be an amino acid or may be based on an amino acid. In such embodiments, the C-terminal carboxyl group of the amino acid-based payload or linking moiety B may be protected, e.g., by amidation.

[0091] In certain embodiments, the chemical spacer (Sp 1 ), (Sp 2 ), and / or (Sp 3 ) may each contain 0-12 amino acid residues, including amino acid derivatives and amino acid mimetics. 1 ) may contain 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues, 2 ) may contain 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues, 3 ) may contain 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues.

[0092] In a particular embodiment, the present invention relates to a method according to the invention, wherein the linker comprises 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or less amino acid residues.

[0093] That is, in certain embodiments, the linker may contain 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid residues, including amino acid mimetics and amino acid derivatives. The amino acid residues contained in the linker, including amino acid mimetics and amino acid derivatives, are preferably chemical spacers (Sp) in the K residues. 1 ), (Sp 2 ), and / or (Sp 3 ), and in certain embodiments, amino acid residues also contained in B when B is an amino acid-based linking moiety or payload.

[0094] In certain embodiments, the linker may comprise 2-25 amino acid residues, including amino acid mimetics and amino acid derivatives. In other embodiments, the linker may comprise 2-20 amino acid residues, including amino acid mimetics and amino acid derivatives. In other embodiments, the linker may comprise 2-15 amino acid residues, including amino acid mimetics and amino acid derivatives. In other embodiments, the linker may comprise 2-10 amino acid residues, including amino acid mimetics and amino acid derivatives. In other embodiments, the linker may comprise 3-10 amino acid residues, including amino acid mimetics and amino acid derivatives. In other embodiments, the linker may comprise 3-8 amino acid residues, including amino acid mimetics and amino acid derivatives. In other embodiments, the linker may comprise 3-6 amino acid residues, including amino acid mimetics and amino acid derivatives.

[0095] In a particular embodiment, the present invention relates to a method according to the invention, wherein the net charge of the linker is neutral or positive.

[0096] In certain embodiments, the linker is a peptide linker (or a peptidomimetic as disclosed herein). That is, a chemical spacer (Sp 1 ), (Sp 2 ), and / or (Sp3 ), when present, consist only of amino acids, amino acid mimetics, or amino acid derivatives. 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 or amino acid derivatives in which the charged functional group has been modified or substituted, the skilled artisan will know how to determine the charge of the non-standard amino acid or amino acid derivative at neutral pH.

[0097] In certain embodiments, the payload or linking moiety B, or (Sp 1 ), (Sp 2 ), and / or (Sp 3 Any non-amino acid moieties contained in the linker may also contribute to the net charge of the linker. However, the skilled artisan will know how to calculate the net charge of the entire linker, including any non-amino acid moieties, preferably at neutral pH (7.0).

[0098] In certain embodiments, the net charge of the linker is calculated based only on the amino acid residues contained in the linker, including amino acid mimetics and amino acid derivatives.Thus, in certain embodiments, the present invention relates to a method according to the present invention, wherein the net charge of the amino acid residues contained in the linker is neutral or positive.

[0099] In a particular embodiment, the invention relates to a method according to the invention, wherein the linker does not contain any negatively charged amino acid residues.

[0100] That is, the linker may not contain negatively charged amino acid residues, including amino acid mimetics and amino acid derivatives. A negatively charged amino acid residue is an amino acid, amino acid mimetics, or amino acid derivative that has a negative charge at neutral pH (7.0). Standard negatively charged amino acids are glutamic acid and aspartic acid. However, negatively charged non-standard amino acids, amino acid mimetics, and amino acid derivatives are known in the art.

[0101] In a particular embodiment, the present invention relates to a method according to the invention, wherein the linker comprises at least one positively charged amino acid residue other than the residue K, i.e. (Sp 1 ), (Sp 2 ), and / or (Sp 3 ) may contain at least one positively charged amino acid. In certain embodiments, (Sp 1 ), (Sp 2 ), and / or (Sp 3 ) comprises at least one histidine or arginine residue. However, it is preferred herein that the linker does not comprise the sequence motif RK, where R is arginine, an arginine mimetic, or an arginine derivative, and K is lysine, a lysine mimetic, or a lysine derivative. Thus, in a particularly preferred embodiment, the present invention relates to a method for producing an antibody-linker conjugate using transglutaminase, comprising the following structure (shown in the N→C direction): (Sp 1 )-K-(Sp 2 )-B-(Sp 3 ) or (Sp 1 )-B-(Sp 2 )-K-(Sp 3 ) to a Gln residue contained in the antibody, (Sp 1 ) is a chemical spacer or is absent; (Sp 2 ) is a chemical spacer or is absent; (Sp 3) is a chemical spacer or is absent; K is lysine, or a lysine derivative or a lysine mimetic; B is a linking moiety or payload; the linker is conjugated to a Gln residue in the antibody via a primary amine in the side chain of the lysine residue, the lysine derivative, or the lysine mimetic; contacting the antibody with less than 80 molar equivalents of the linker; wherein said residue K is not directly coupled to the N-terminal arginine, arginine mimetic, or arginine derivative.

[0102] In certain embodiments, (Sp 1 ), (Sp 2 ), and / or (Sp 3 ) comprises at least one histidine residue. In certain embodiments, the histidine residue is directly linked to a lysine residue, a lysine mimetic, or a lysine derivative. That is, in certain embodiments, a linker according to the invention comprises the motif HK.

[0103] In certain embodiments, the present invention provides a method for producing an antibody-linker conjugate using transglutaminase, comprising the following structure (shown in the N→C orientation): (Sp 1 )-K-(Sp 2 )-B-(Sp 3 ) or (Sp 1 )-B-(Sp 2 )-K-(Sp 3 ) to a Gln residue contained in the antibody, (Sp 1 ) is a chemical spacer or is absent; (Sp 2 ) is a chemical spacer or is absent; (Sp 3 ) is a chemical spacer or is absent; K is lysine, or a lysine derivative or a lysine mimetic; B is a linking moiety or payload; the linker is conjugated to a Gln residue in the antibody via a primary amine in the side chain of the lysine residue, the lysine derivative, or the lysine mimetic; the antibody is contacted with less than 80 molar equivalents of the linker; The method relates to a method in which the linker comprises at least one histidine residue, in particular wherein the at least one histidine residue is directly linked to residue K, in particular wherein the linker comprises the sequence motif HK.

[0104] In certain embodiments, the present invention provides a compound having the following structure (shown in the N→C orientation): (Sp 1 )-K-(Sp 2 )-B-(Sp 3 ) or (Sp 1 )-B-(Sp 2 )-K-(Sp 3 ) An antibody conjugate comprising a linker comprising or consisting of (Sp 1 ) is a chemical spacer or is absent; (Sp 2 ) is a chemical spacer or is absent; (Sp 3 ) is a chemical spacer or is absent; K is lysine, or a lysine derivative or a lysine mimetic; B is a linking moiety or payload; the linker is conjugated to a Gln residue in the antibody via a primary amine in the side chain of the lysine residue, the lysine derivative, or the lysine mimetic; It relates to conjugates wherein said linker comprises at least one histidine residue, in particular said at least one histidine residue is directly linked to residue K, in particular wherein said linker comprises the sequence motif HK.

[0105] In addition to or instead of amino acid residues, including amino acid mimetics and derivatives, chemical spacers (Sp 1 ), (Sp 2 ), and / or (Sp 3 ) may comprise or consist of non-amino acid moieties.

[0106] That is, in certain embodiments, the chemical spacer (Sp 1 ), (Sp 2 ), and / or (Sp 3 ) may not be composed solely of amino acids, amino acid mimetics, or amino acid derivatives. 1 ), (Sp 2 ), and / or (Sp 3 ) may contain or consist exclusively of non-amino acid components. In certain embodiments, the chemical spacer (Sp 1 ), (Sp 2 ), and / or (Sp 3 ) may contain amino acid components and non-amino acid components.

[0107] For example, but not limited to, chemical spacers (Sp 1 ), (Sp 2 ), and / or (Sp 3 ), each may comprise a carbon containing backbone of 1-200 atoms, optionally at least 10 atoms substituted at one or more atoms, e.g., 10-100 atoms or 20-100 atoms, optionally the carbon containing backbone is a linear hydrocarbon or may comprise a cyclic group, a symmetrically or asymmetrically branched hydrocarbon, a monosaccharide, a disaccharide, a linear or branched oligosaccharide (asymmetrically or symmetrically branched), other naturally occurring linear or branched oligomer (asymmetrically or symmetrically branched), or more generally, any dimer, trimer or higher oligomer (linear, asymmetrically or symmetrically branched) resulting from any chain-growth or step-growth polymerization process.

[0108] (Sp 1 ), (Sp2 ), and / or (Sp 3 ) is any linear, branched and / or cyclic C 2-30 Alkyl, C 2-30 Alkenyl, C 2-30 Alkynyl, C 2-30 Heteroalkyl, C 2-30 Heteroalkenyl, C 2-30 Heteroalkynyl (optionally containing one or more homocyclic aromatic or heterocyclic radicals); in particular any linear or branched C 2-5 Alkyl, C 5-10 Alkyl, C 11-20 Alkyl, -OC 1-5 Alkyl, -OC 5-10 Alkyl, -OC 11-20 Alkyl, or (CH 2 -CH 2 -O-) 1-24 , or (CH 2 ) x1 -(CH 2 -O-CH 2 ) 1-24 -(CH 2 ) x2 - group (wherein x1 and x2 are independently an integer selected from the range of 0 to 20), an amino acid, an oligopeptide, a sugar chain, a sulfate, a phosphate, or a carboxylate. 1 ), (Sp 2 ), and / or (Sp 3 ) is C 2-6 It may contain alkyl groups.

[0109] In certain embodiments, the chemical spacer (Sp 1 ), (Sp 2 ), and / or (Sp 3) may contain one or more polyethylene glycol (PEG) moieties or equivalent condensation polymers such as poly(carboxybetaine methacrylate) (pCBMA), polyoxazoline, polyglycerol, polyvinylpyrrolidone, or poly(hydroxyethyl methacrylate) (pHEMA). Polyethylene glycol (PEG) is a polyether compound with many applications ranging from industrial manufacturing to pharmaceuticals. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE) depending on its molecular weight. The structure of PEG is generally H-(O-CH 2 -CH 2 ) n Those skilled in the art will recognize how to functionalize condensation polymers so that they can be coupled to an amino acid residue or a payload.

[0110] Thus, in a particular embodiment, the present invention relates to a method according to the present invention, wherein the linker comprises one or more PEG moieties. In a particular embodiment, the PEG moiety is a chemical spacer (Sp 1 ), (Sp 2 ), and / or (Sp 3 In certain embodiments, each PEG moiety included in the linker can include 2 to 20 ethylene glycol monomers, 2 to 15 ethylene glycol monomers, 2 to 10 ethylene glycol monomers, or 2 to 5 ethylene glycol monomers. In certain embodiments, the PEG moiety can include a (Sp 2 In certain embodiments, the PEG moiety comprises a linking moiety or payload in the form of (Sp 2 ) to connect to amino acid residues in (Sp 2 In certain embodiments, the PEG moiety is included in (Sp 2 In certain embodiments, the PEG moiety is included in the formula (Sp 2 ) to attach to the self-immolative moiety, which is then attached to the payload (Sp 2) is included.

[0111] In certain embodiments, the chemical spacer (Sp 1 ), (Sp 2 ), and / or (Sp 3 ) may include dextran. The term "dextran" as used in the present invention refers to a complex, branched glucan composed of chains of various lengths, which may have a weight ranging from 3 to 2000 kDa. The linear chains typically consist of α-1,6 glycosidic bonds between glucose molecules, while the branches begin with α-1,3 bonds. Dextran may be synthesized from sucrose, for example, by lactic acid bacteria. In the context of the present invention, dextran used as a carrier may preferably have a molecular weight of about 15 to 1500 kDa.

[0112] In certain embodiments, the chemical spacer (Sp 1 ), (Sp 2 ), and / or (Sp 3 ) may include oligonucleotides. The term "oligonucleotide", as used herein, refers to an oligomer or polymer of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), as well as oligonucleotides that do not occur in nature. Due to their increased stability, oligonucleotides are preferably polymers of DNA.

[0113] In certain embodiments, the chemical spacer (Sp 1 ), (Sp 2 ), and / or (Sp 3 ), when present, consists solely of amino acid residues, including amino acid mimetics and derivatives, and PEG moieties. In certain embodiments, the chemical spacer (Sp 1 ), (Sp 2 ), and / or (Sp 3 ), when present, consists solely of amino acid residues, including amino acid mimetics and derivatives. In certain embodiments, the chemical spacer (Sp 1 ), (Sp 2 ), and / or (Sp 3) are α-L-amino acids. That is, in certain embodiments, the linker, excluding the payload or the linking moiety B, consists only of amino acid residues. In certain embodiments, the linker, excluding the payload or the linking moiety B, consists only of α-L-amino acid residues. Such peptide-based linkers may contain protecting groups at the N-terminus and / or C-terminus. That is, the N-terminal amino group may be acetylated and / or the C-terminal carboxyl group may be amidated.

[0114] Chemical spacer (Sp 1 ), (Sp 2 ), (Sp 3 It should be noted that the chemical spacers (Sp 1 ), (Sp 2 ), and / or (Sp 3 ) have different structures and / or chemical spacers (Sp 1 ), (Sp 2 ), and / or (Sp 3 ) are preferably not present at the same time. That is, in certain embodiments, the chemical spacers (Sp 1 ), (Sp 2 ), and / or (Sp 3 ) may be present in the linker.

[0115] In certain embodiments, the K residue may be directly connected to one or more small hydrophobic amino acid residues, for example, in certain embodiments, the K residue may be directly connected to one or more alanine residues.

[0116] In the present invention, the linker is preferably conjugated to the antibody via a primary amine contained in the side chain of residue K. Thus, the chemical spacer (Sp 1 ), (Sp 2 ), and / or (Sp 3) preferably does not contain additional lysine residues, lysine mimetics, or lysine derivatives that can function as additional amine donors in transglutaminase-based conjugation reactions. In other embodiments, any free N-terminal amino group contained in the linker may be substituted, e.g., acetylated, such that it cannot function as a substrate for transglutaminase.

[0117] The linker of the present invention further comprises at least one linking moiety or payload B. The linker of the present invention can be used to directly conjugate a payload to an antibody in a one-step conjugation process. In other embodiments, a linker comprising one or more linking moieties may be conjugated to an antibody in a first step, and then one or more payloads may be linked to the antibody-linker conjugate in a second step. Table 1 below clarifies the two terms as used herein.

[0118] [Table 1]

[0119] In certain embodiments, the linker may comprise one or more linking moieties B. Thus, in certain embodiments, the invention relates to a method according to the invention, wherein B is a linking moiety.

[0120] A "linking moiety", as used herein, generally refers to a molecule that is at least bifunctional. In the present invention, the linking moiety comprises a first functional group that couples the linking moiety to the linker of the present invention, and a second functional group that can be used to couple additional molecules to the linker before or after the linker is conjugated to the antibody. In certain embodiments, the linking moiety of the present invention is an amino acid, an amino acid mimetic, or an amino acid derivative. In such embodiments, the linking moiety is preferably connected to the linker through its amino group, but the functional group contained in the amino acid side chain can be used to couple additional molecules to the linker. Alternatively, the linking moiety may be connected to the linker through its carboxyl group, but the functional group contained in the amino acid side chain can be used to couple additional molecules to the linker.

[0121] In certain embodiments, the present invention provides a method for the preparation of a medicament for use in ... pharmaceutical composition comprising the steps of: Bioorthogonal marker groups, or Non-bio-orthogonal entities for crosslinking The present invention relates to a method according to the present invention, comprising:

[0122] The term "bioorthogonal marker group" was established by Sletten and Bertozzi (A Bioorthogonal Quadricyclane Ligation. J Am Chem Soc 2011,133(44),17570-17573) to name reactive groups that can undergo chemical reactions in biological systems without interfering with native biochemical processes. A "non-bio-orthogonal entity for crosslinking" may be any molecule that comprises or consists of a first functional group that can be crosslinked chemically or enzymatically to a payload that contains a compatible second functional group. Even if the crosslinking reaction is a non-bioorthogonal 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 may consist of a "bioorthogonal marker group" or a "non-bioorthogonal entity" or may include a "bioorthogonal marker group" or a "non-bioorthogonal entity". For example, the linking moiety Lys(N 3 ), Lys(N 3 Both the entire Lys(N) and the azide group alone can be considered as bioorthogonal marker groups in the present invention. 3 ) refers to 6-azido-L-lysine, and K(N 3 ) is sometimes abbreviated as

[0123] In certain embodiments, the present invention provides a method for the preparation of a bioorthogonal marker group or a non-bioorthogonal entity for crosslinking, comprising: -NN≡N or -N 3 ; Lys(N 3 ); Tetrazine; ·Alkynes; ·Distorted cyclooctyne; · BCN; · Strained alkenes; Photoreactive groups; ·aldehyde; Acyltrifluoroborates; ·Proteolytic agents (“PROTACs”); ·Cyclopentadiene / spirolocyclopentadiene; ·Thioselective electrophiles; -SH; and Cysteine The present invention relates to a method according to the present invention, which comprises or consists of at least one molecule or moiety selected from the group consisting of:

[0124] The bioorthogonal marker groups contained in the linker or the non-bioorthogonal entities for cross-linking can participate in any of the conjugation reactions shown in Table 2, for example.

[0125] [Table 2]

[0126] Linking moiety B may be or include what is referred to in Table 2 as "binding partner 1" or "binding partner 2."

[0127] In certain embodiments, linking moiety B can be a cysteine, a cysteine ​​mimetic, or a cysteine ​​derivative having a free sulfhydryl group.

[0128] The free sulfhydryl group of such Cys residue (or mimic or derivative) can be conjugated to a payload construct that contains a thioselective electrophile, such as maleimide. Toxin constructs that contain maleimide moieties are frequently used, such as Adcetris, and are approved by medical authorities. Thus, toxin constructs that contain MMAE toxins can be coupled to the free sulfhydryl group of Cys residue in the linker of the present invention.

[0129] It should be noted that other thioselective electrophiles such as 3-arylpropionitriles (APN) or phosphonamidates may be used in place of the maleimides in the process of the present invention.

[0130] Thus, providing a Cys residue in the linker according to the invention has the advantage that it allows the antibody-payload conjugate to be made using a pre-made toxin-maleimide construct or, more generally, allows the full exploitation of the advantages of Cys-maleimide coupling chemistry. At the same time, it allows the use of pre-made antibodies that do not need to be deglycosylated. In certain embodiments, the Cys residue may be present at the C-terminus or intrachain in the amino acid-based linker.

[0131] In another embodiment, the linking moiety B may comprise an azide group. One skilled in the art may 3 )) or 4-azido-homoalanine (Xaa(N 3 We are aware of molecules that contain an azide group that can be incorporated into the linkers of the present invention, such as azide-containing cycloaddition (SPAAC), copper-catalyzed azide-alkyne cycloaddition (CuAAC), or Staudinger ligation. Linking moieties that contain an azide group can be used as substrates in a variety of bioorthogonal reactions, such as, for example, strain-promoted azide-alkyne cycloaddition (SPAAC), copper-catalyzed azide-alkyne cycloaddition (CuAAC), or Staudinger ligation. For example, in certain embodiments, payloads that contain cyclooctyne derivatives, such as DBCO, DIBO, BCN, or BARAC, can be coupled to linkers that contain an azide group by SPAAC.

[0132] In yet another embodiment, the linking moiety B may comprise a tetrazine group. Those skilled in the art will be aware of tetrazine-containing molecules, preferably amino acid derivatives containing a tetrazine group, that can be incorporated into the linker of the present invention. The linking moiety containing tetrazine can be used as a substrate in bioorthogonal tetrazine ligation. For example, in certain embodiments, a payload containing a cyclooctyne group, such as cyclopropene, norborene, norborene derivative, or bicyclo[6.1.0]nonyne (BCN), can be coupled to a linker containing a tetrazine group.

[0133] In certain embodiments, the linking moiety B may comprise a cyclic diene, such as a cyclopentadiene derivative. Cyclopentadiene derivatives that can potentially be linked to maleimide-containing payload molecules are described by Amant et al., Tuning the Diels-Alder Reaction for Bioconjugation to Maleimide Drug-Linkers; Bioconjugate Chem. 2018, 29, 7, 2406-2414 and Amant et al., A Reactive Antibody Platform for One-Step Production of Antibody-Drug Conjugates through a Diels-Alder Reaction with Maleimide; Bioconjugate Chem. 2019, 30, 9, 2340-2348.

[0134] In certain embodiments, the linking moiety B may comprise a photoreactive group. The term "photoreactive group", as used herein, refers to a chemical group that responds to an applied external energy source to undergo active species generation, and thus covalently bonds to an adjacent chemical structure (e.g., abstractable hydrogen). Examples of photoreactive groups include, but are not limited to, aryl azides, such as phenyl azide, o-hydroxyphenyl azide, m-hydroxyphenyl azide, tetrafluorophenyl azide, o-nitrophenyl azide, m-nitrophenyl azide, or azido-methylcoumarin, diazirine, psoralen, or benzophenone.

[0135] In a particular embodiment, the present invention relates to a method according to the invention comprising the further step of conjugating one or more payloads to the linking moiety B.

[0136] Instead of directly conjugating a linker comprising one or more payloads to an antibody in a one-step process, the present invention refers in certain embodiments to a two-step process in which a linker comprising at least one linking moiety B can be conjugated to an antibody in a first step, followed by coupling of one or more payloads to the linking moiety B in a second step.

[0137] The term "payload" as used herein refers to any naturally occurring or synthetically produced molecule, including small molecules or chemical entities that can be chemically synthesized, and larger molecules or biological entities that must be produced by fermentation of a host cell or can also be chemically synthesized and that confer novel functionality to the antibody. The payload may be a linking moiety or a chemical spacer (Sp) contained in a linker. 1 ) and / or (Sp 3 It will be appreciated that other portions of the linker, such as the aryl, aryl, or K residues, may contain additional structures or functional groups that allow for coupling of a payload.

[0138] In the two-step conjugation process, the payload can be linked to the linking moiety by any suitable method known in the art.Preferably, the payload can be linked to any of the bioorthogonal marker groups disclosed herein or non-bioorthogonal entities for crosslinking.That is, the payload preferably comprises a functional group that is compatible with the bioorthogonal marker group or non-bioorthogonal entities for crosslinking contained in at least one linking moiety B.

[0139] Several bioorthogonal reactions that can be used to link a payload to a bioorthogonal marker group contained in linking moiety B are known in the art. For example, 1,3-dipolar cycloaddition between azides and cyclooctynes ​​(also called copper-free click chemistry, Baskin et al. (“Copper-free click chemistry for dynamic in vivo imaging”. Proceedings of the National Academy of Sciences. 104(43):16793-7)), between nitrones and cyclooctynes ​​(Ning et al. (“Protein Modification by Strain-Promoted Alkyne-Nitrone Cycloaddition”. Angewandte Chemie International Edition. 49(17):3065)), oxime / hydrazone formation from aldehydes and ketones (Yarema,et al. (“Metabolic Delivery of Ketone Groups to Sialic Acid Residues. Application To Cell Surface Glycoform Engineering”. Journal of Biological Chemistry. 273(47):31168-79)), tetrazine ligation (Blackman et al. (“The Tetrazine Ligation: Fast Bioconjugation based on Inverse-electron-demand Diels-Alder Reactivity”. Journal of the American Chemical Society. 130(41):13518-9)), isonitrile-based click reaction (Stockmann et al. ("Exploring isonitrile-based click chemistry for ligation with biomolecules". Organic & Biomolecular Chemistry.9(21):7303)), and most recently, quadricyclane ligation (Sletten & Bertozzi (JACS, A Bioorthogonal Quadricyclane Ligation. J Am Chem Soc 2011,133(44),17570-17573)), copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC, Kolb & Sharpless ("The growing impact of click chemistry on drug discovery". Drug Discov Today.8(24):1128-1137)), strain-promoted azide-alkyne cycloaddition (SPAAC, Agard et al ("A Comparative Study of Bioorthogonal Reactions with Azides". ACS Chem. Biol.1:644-648)), or strain-promoted alkyne-nitrone cycloaddition (SPANC, MacKenzie et al ("Strain-promoted cycloadditions A number of chemical ligation strategies have been developed that meet the requirements of bioorthogonal labeling, including "Curr Opin Chem Biol. 21:81-8) involving nitrones and alkynes-rapid tunable reactions for bioorthogonal labeling". All of these documents are incorporated herein by reference in order to provide a sufficient enabling disclosure and to avoid redundant repetition.

[0140] It should be understood that the payload is preferably coupled to a bioorthogonal marker group contained in the linker or a non-bioorthogonal entity for crosslinking after the linker of the present invention is conjugated to a Gln residue of the antibody using transglutaminase. However, the present invention also encompasses antibody-linker conjugates in which one or more payloads are coupled to a linker comprising at least one linking moiety B in a first step and the resulting linker-payload construct is conjugated to the antibody by transglutaminase in a second step.

[0141] In a particular embodiment, the present invention relates to a method according to the invention, wherein one or more payloads are conjugated to the linking moiety B via a Click reaction.

[0142] That is, one or more payloads may be linked to linking moiety B in a Click reaction, particularly any of the Click reactions disclosed herein.

[0143] In a particularly preferred embodiment, at least one payload may be conjugated to a linking moiety B contained in the linker via thiol-maleimide conjugation. That is, in certain embodiments, the payload may comprise a maleimide group and the linking moiety B may be a molecule containing a thiol group, such as, but not limited to, a cysteine ​​residue or a cysteine ​​mimic such as homocysteine. However, B may also be a non-amino acid molecule containing a free thiol group. In another embodiment, the payload may comprise a free thiol group and the linking moiety B may comprise a maleimide group.

[0144] In another particularly preferred embodiment, at least one payload may be conjugated to a linking moiety B contained in the linker via strain-promoted azide-alkyne cycloaddition (SPAAC). That is, in certain embodiments, the payload may comprise an alkyne group, such as, but not limited to, a cycloocytin group, and the linking moiety B may be a molecule comprising an azide group, such as, but not limited to, the lysine derivative Lys(N) disclosed herein. 3 ). However, B may also be a non-amino acid molecule containing a free azide group. In another embodiment, the payload may contain an alkyne group, such as a cyclooctyne group, and the linking moiety B may contain an azide group.

[0145] Besides the Click reaction between the linking moiety in the linker and a functional group in the payload, the payload can be covalently attached to the linking moiety by any enzymatic or non-enzymatic reaction known in the art.

[0146] The payload is preferably linked to the linking moiety via a covalent bond. However, in certain embodiments, the payload may be linked to the linking moiety via a strong non-covalent bond. That is, in certain embodiments, the linking moiety B may comprise a biotin moiety, such as, but not limited to, the lysine derivative biocytin. In such embodiments, a payload comprising a streptavidin moiety may be linked to a linker comprising a biotin moiety.

[0147] In a particular embodiment, the invention relates to a method according to the invention, wherein B is a payload.

[0148] In certain embodiments, the payload may already be part of the linker so that the payload can be conjugated to the antibody in a one-step process. In such embodiments, the linker is preferably coupled to the linker by chemical synthesis. The payload is preferably coupled to a chemical spacer contained in the linker or directly to the K residue. In embodiments in which the payload is coupled to an amino acid residue, including amino acid mimetics and derivatives, the payload may be coupled to the C-terminal carboxyl group or the N-terminal amino group of the amino acid residue. Alternatively, the payload may be coupled to a functional group contained in the side chain of the amino acid residue. Those skilled in the art are aware of how to functionalize the payload so that it can be coupled to a carboxyl group, an amino group, or an amino acid side chain.

[0149] Furthermore, those skilled in the art are aware of methods for coupling payloads to amino acid-based linkers by chemical synthesis. For example, payloads that contain amines, or payloads that contain thiols (e.g., in the case of maytansine analogs), or payloads that contain hydroxyls (e.g., in the case of SN-38 analogs) can be attached to the C-terminus of amino acid-based linkers by chemical synthesis. However, those skilled in the art are aware of additional reactions and reactive groups that can be utilized to couple payloads to the N-terminus, C-terminus, or side chain of amino acids or amino acid derivatives by chemical synthesis. Exemplary reactions that can be used to couple payloads to amino acid-based linkers by chemical synthesis include, but are not limited to, peptide coupling, activated ester coupling (NHS ester, PFP ester), click reaction (CuAAC, SPAAC), and Michael addition (thiol-maleimide conjugation).Regarding the coupling of a payload to a peptide, for example, Costoplus 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 et al. (Improved Physical Stability of an Antibody-Drug Conjugate Using Host-Guest Chemistry. Bioconjug Chem. 2020 Jan 15; 31(1): 123-129), Bodero 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 et al. (Use of a next generation maleimide in combination with THIOMAB. TM antibody technology delivers a highly stable, potent and near homogeneous THIOMAB TMantibody-drug conjugate(TDC).RSC Adv.,2017,7,24828-24832), Doronina 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 et al.(Novel antibody drug conjugates containing exatecan derivative-based cytotoxic payloads.Bioorg Med Chem Lett.2016 Mar 15;26(6):1542-1545), and Dickgiesser et al. (Site-Specific Conjugation of Native Antibodies Using Engineered Microbial Transglutaminases.Bioconjug Chem.2020 Mar 12.doi:10.1021 / acs.bioconjchem.0c00061) and has been described extensively in the prior art.

[0150] It is understood that the payload can be coupled to the N-terminus or C-terminus of a peptide-based or peptide-containing linker of the present invention. In certain embodiments, the payload can be directly coupled to the N-terminal amino group or C-terminal carboxyl group of a peptide or amino acid residue.

[0151] Those skilled in the art are aware of suitable reactive groups for coupling a payload to an amino acid residue.For example, a payload containing an amine can be coupled to the C-terminal carboxyl group of an amino acid residue via an amide bond.Alternatively, a payload containing a thiol group or and a hydroxyl group can be coupled to the C-terminal carboxyl group of an amino acid via a thioester or ester bond, respectively.A payload containing a carboxylic acid group can be coupled to the N-terminal amino group of an amino acid residue via an amide bond.

[0152] In certain embodiments, the payload can be indirectly coupled to the N-terminus or C-terminus of a peptide or amino acid residue contained in the linker of the present invention. Those skilled in the art are aware of linker molecules that can be used to couple a payload to the N-terminal amino group or C-terminal carboxyl group of an amino acid residue contained in the linker of the present invention.

[0153] In certain embodiments, a payload containing a hydroxyl group can be coupled to the N-terminus of an amino acid residue via a linker molecule, for example, a payload containing a hydroxyl group can be coupled to the N-terminal amino group via a carbamate linker molecule.

[0154] In certain embodiments, the payload containing a thiol group can be coupled to the N-terminus of an amino acid residue via a linker molecule. For example, the payload containing a thiol group can be coupled to the N-terminus amino group via a thiocarbamate linker molecule. Alternatively, the payload containing a thiol group can be coupled to the N-terminus amino group via an alkyl linker molecule containing a carboxyl group and a thiol group. In certain embodiments, the alkyl linker molecule can be a 3-mercaptopropionic acid linker molecule, in which the payload forms a disulfide bond with the thiol group contained in the 3-mercaptopropionic acid linker molecule.

[0155] In certain embodiments, a payload containing an amide group can be coupled to the N-terminus of an amino acid residue via a linker molecule. For example, a payload containing an amine group can be coupled to the N-terminal amino group via a dicarboxylic acid linker molecule that forms an amide bond with the payload and the amino group of the N-terminal amino acid residue. Examples of dicarboxylic acids that can be used as linker molecules in the present invention include, but are not limited to, succinic acid or pimelic acid.

[0156] Alternative linker molecules for indirectly coupling a payload to the N-terminus of an amino acid residue comprised in a linker of the invention or linker molecules suitable for indirectly coupling a payload to the C-terminus of an amino acid residue comprised in a linker of the invention have been described in the art and are encompassed by the present invention.

[0157] In certain embodiments, the present invention provides a method for the preparation of a peptide comprising: ·toxin; Cytokines; ·Growth factors; Radionuclides; ·hormone; Antiviral agents; Antibacterial agents; · Fluorescent dyes; ·Immunomodulators / immunostimulants; · Half-life extending moieties; ·Solubility enhancing moiety; Polymer-toxin conjugates; ·Nucleic acid; · a biotin or streptavidin moiety; ·vitamin; ·Proteolytic agents (“PROTACs”); a target-binding moiety; and / or Anti-inflammatory The present invention relates to a method comprising at least one of the following steps:

[0158] Any one of the payloads disclosed herein may be directly coupled to a linker for use in the one-step conjugation process disclosed herein, or may be linked to a linking moiety included in an antibody-linker conjugate made as part of the two-step process disclosed herein.

[0159] In certain embodiments, the payload may be a cytokine. The term "cytokine" as used herein means any secreted polypeptide that affects the function of other cells and regulates interactions between cells in immune or inflammatory responses. Cytokines include, but are not limited to, monokines, lymphokines, and chemokines, regardless of the cell that produces them. For example, monokines are commonly said to be produced and secreted by monocytes, although many other cells also produce monokines, such as natural killer cells, fibroblasts, basophils, neutrophils, endothelial cells, brain astrocytes, bone marrow stromal cells, epidermal keratinocytes, and B lymphocytes. Lymphokines are commonly said to be produced by lymphocyte cells. Examples of cytokines include, but are not limited to, interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor alpha (TNFα), and tumor necrosis factor beta (TNFβ).

[0160] In certain embodiments, the payload may be an anti-inflammatory agent. The term "anti-inflammatory agent" as used in the present invention refers to the class of agents whose main mechanism of action and use is in the area of ​​treating inflammation, as well as any other agent from another therapeutic class that has a useful anti-inflammatory effect. Such anti-inflammatory agents include, but are not limited to, nonsteroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs), macrolide antibiotics, and statins. Preferably, NSAIDs include, but are not limited to, salicylates (e.g., aspirin), arylpropionic acids (e.g., ibuprofen), anthranilic acids (e.g., mefenamic acid), pyrazoles (e.g., phenylbutazone), cyclic acetic acids (indomethicin), and oxicams (e.g., piroxicam). Preferably, anti-inflammatory agents for use in the methods of the present invention include sulindac, diclofenac, tenoxicam, ketorolac, naproxen, nabumetone, diflunisal, ketoprofen, arylpropionic acid, tenidap, hydroxychloroquine, sulfasalazine, celecoxib, rofecoxib, meloxicam, etoricoxib, valdecoxib, methotrexate, etanercept, infliximab, adalimumab, atorvastatin, fluvastatin, lovastatin, pravastatin, simvastatin, clarithromycin, azithromycin, roxithromycin, erythromycin, ibuprofen, dexibuprofen, flurbiprofen, fenoprofen, fenbufen, benoxaprofen, dexketoprofen, tolfenamic acid, nimesulide, and oxaprozin.

[0161] In certain embodiments, the anti-inflammatory agent may be an anti-inflammatory cytokine, which when conjugated to a target-specific antibody can ameliorate inflammation caused by, for example, an autoimmune disease. Cytokines with anti-inflammatory activity may be, but are not limited to, IL-1RA, IL-4, IL-6, IL-10, IL-11, IL-13, or TGF-β.

[0162] In certain embodiments, the payload may be a growth factor. The term "growth factor" as used herein refers to a naturally occurring substance that can stimulate cell growth, proliferation, cell differentiation, and / or cell maturation. Growth factors exist in the form of proteins or steroid hormones. Growth factors are important for controlling various cellular processes. Growth factors typically act as signaling molecules between cells. However, different growth factors have different abilities to promote cell growth, proliferation, cell differentiation, and cell maturation. A non-limiting list of examples of growth factors includes basic fibroblast growth factor, adrenomedullin, angiopoietin, autocrine motility stimulating factor, bone morphogenetic protein, brain-derived neurotrophic factor, epidermal growth factor, epidermal growth factor, fibroblast growth factor, glial cell line-derived neurotrophic factor, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, growth differentiation factor-9, hepatocyte growth factor, hepatocellular carcinoma-derived growth factor, insulin growth factor, insulin-like growth factor, migration stimulating factor, myostatin, nerve growth factor, and other neurotrophins, platelet-derived growth factor, transforming growth factor alpha, transforming growth factor beta, tumor necrosis factor alpha, vascular endothelial growth factor, placental growth factor, bovine fetal somatotrophin, and cytokines (e.g., IL-1-cofactor for IL-3 and IL-6, IL-2-t cell growth factor, IL-3, IL-4, IL-5, IL-6, and IL-7, etc.).

[0163] In certain embodiments, the payload may be a hormone. The term "hormone," as used herein, refers to a chemical released by cells or glands in one part of the body that sends out a message that affects cells in other parts of the organism.Examples of hormones useful in the present invention include, but are not limited to, melatonin (MT), serotonin (5-HT), thyroxine (T4), triiodothyronine (T3), epinephrine or adrenaline (EPI), norepinephrine or noradrenaline (NRE), dopamine (DPM or DA), anti-Mullerian hormone or Mullerian inhibitory hormone (AMH), adiponectin (Acrp30), adrenocorticotropic hormone or corticotropin (ACTH), angiotensinogen and angiotensin (AGT), antidiuretic hormone or These include vasopressin (ADH), atrial natriuretic peptide or atriopeptin (ANP), calcitonin (CT), cholecystokinin (CCK), corticotropin releasing hormone (CRH), erythropoietin (EPO), follicle stimulating hormone (FSH), gastrin (GRP), ghrelin, glucagon (GCG), gonadotrophin releasing hormone (GnRH), growth hormone releasing hormone (GHRH), human chorionic gonadotrophin (hCG), human placental lactogen (HPL), growth hormone (GH or hGH), inhibin, insulin Insulin (INS), insulin-like growth factor or somatomedin (IGF), leptin (LEP), luteinizing hormone (LH), melanocyte-stimulating hormone (MSH or α-MSH), orexin, oxytocin (OXT), parathyroid hormone (PTH), prolactin (PRL), relaxin (RLN), secretin (SCT), somatostatin (SRIF), thrombopoietin (TPO), thyroid-stimulating hormone or thyrotropin (TSH), thyrotropin-releasing hormone (TRH), cortisol, aldosterone, testosterone, dehydrogenase These are pyandrosterone (DHEA), androstenedione, dihydrotestosterone (DHT), estrone, estriol (E3), progesterone, calcitriol, calcidiol, prostaglandins (PG), leukotrienes (LT), prostacyclin (PGI2), thromboxane (TXA2), prolactin-releasing hormone (PRH), lipotropin (PRH), brain natriuretic peptide (BNP), neuropeptide Y (NPY), histamine, endothelin, pancreatic polypeptide, renin, and enkephalin.

[0164] In certain embodiments, the payload may be an antiviral agent. The term "antiviral agent" as used herein means an agent (compound or biological) effective to inhibit virus formation and / or replication in a mammal. This includes agents that interfere with either the host or viral mechanisms necessary for virus formation and / or replication in a mammal. Antiviral agents include, for example, ribavirin, amantadine, VX-497 (merimepodib, Vertex Pharmaceuticals), VX-498 (Vertex Pharmaceuticals), levovirin, viramidine, ceprene (maxamine), XTL-001 and XTL-002 (XTL Biopharmaceuticals).

[0165] In certain embodiments, the payload may be an antibacterial agent. The term "antibacterial agent" as used herein refers to any substance, compound, combination of substances, or combination of compounds that can (i) inhibit, reduce, or prevent bacterial growth, (ii) inhibit or reduce the ability of bacteria to cause an infection in a subject, or (iii) inhibit or reduce the ability of bacteria to grow or remain infectious in an environment. The term "antibacterial agent" also refers to a compound that can reduce the infectivity or pathogenicity of bacteria.

[0166] In certain embodiments, the payload may be an immunomodulator. The term "immunomodulator" as used herein for combination therapy refers to a substance that acts to suppress, mask, or enhance the host's immune system. Examples of immunomodulators include, but are not limited to, proteinaceous agents such as cytokines, peptidomimetics, and antibodies (e.g., human, humanized, chimeric, monoclonal, polyclonal, Fv, ScFv, Fab or F(ab)2 fragments, or epitope-binding fragments), nucleic acid molecules (e.g., antisense nucleic acid molecules, iRNA, and triple helices), small molecules, organic compounds, and inorganic compounds. Specifically, immunomodulatory agents include, but are not limited to, methotrexate, leflunomide, cyclophosphamide, cytoxan, imuran, cyclosporine A, minocycline, azathioprine, antibiotics (e.g., FK506 (tacrolimus)), methylprednisolone (MP), corticosteroids, steroids, mycophenolate mofetil, rapamycin (sirolimus), mizoribine, deoxyspergualin, brequinar, malononitriloamines (e.g., leflunamide), T cell receptor modulators, and cytokine receptor modulators.

[0167] In certain embodiments, the immunomodulator may be an immunostimulant. The term "immunostimulant" as used herein preferably refers to any substance or material that can elicit an immune response (e.g., an immune response against a particular pathogen). Immune cell activating compounds include Toll-like receptor (TLR) agonists. Such agonists include pathogen-associated molecular patterns (PAMPs), such as infection-mimicking compositions, such as bacterial-derived immunomodulators (also known as danger signals), and damage-associated molecular patterns (DAMPs), such as compositions that mimic stressed or damaged cells. TLR agonists include nucleic acid or lipid compositions, such as monophosphoryl lipid A (MPLA). In one example, the TLR agonist includes TLR9 agonists, such as cytosine-guanosine oligonucleotides (CpG-ODN), poly(ethyleneimine) (PEI)-degenerate oligonucleotides (ODN), such as PEI-CpG-ODN, or double-stranded deoxyribonucleic acid (DNA). In another example, the TLR agonist includes a TLR3 agonist such as polyinosinic-polycytidylic acid (poly(I:C)), PEI-poly(I:C), polyadenylic-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 β-glucans (such as lentinan), imiquimod, CRX-527, and OM-174.

[0168] In certain embodiments, the payload may be a half-life extending moiety or a solubility enhancing moiety. The half-life extending moiety is, for example, a PEG moiety (polyethylene glycol moiety; PEGylation), other polymer moieties, a PAS moiety (oligopeptide containing proline, alanine, and serine; PASylation), or a serum albumin binder. The solubility enhancing moiety is, for example, a PEG moiety (PEGylation) or a PAS moiety (PASylation).

[0169] In certain embodiments, the payload may be a polymer-toxin conjugate. A polymer-toxin conjugate is a polymer that can carry many payload molecules. Such conjugates may also be called fleximers, as sold, for example, by Mersana therapeutics. A polymer-toxin conjugate may include any of the toxins disclosed herein.

[0170] In certain embodiments, the payload may be a nucleotide. One example of a nucleic acid payload is MCT-485, a very small non-coding double-stranded RNA with oncolytic and immune activating properties developed by MultiCell Technologies, Inc.

[0171] In certain embodiments, the payload may be a fluorescent dye. The term "fluorochrome", as used herein, refers to a dye that absorbs light at a first wavelength and emits light at a second wavelength longer than the first wavelength. In certain embodiments, the fluorescent dye is a near-infrared fluorescent dye that emits light at a wavelength between 650 and 900 nm. In this region, tissue autofluorescence is low and fluorescence quenching is low, enhancing deep tissue penetration while minimizing background interference. Thus, near-infrared fluorescence imaging can be used to visualize tissues to which the antibody-payload conjugates of the invention are bound during surgery. "Near-infrared fluorescent dyes" are known in the art and are 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.

[0172] In certain embodiments, the payload may comprise a radionuclide. The term "radionuclide" as used herein refers to medically useful radionuclides including positively charged ions of radioactive metals such as Y, In, Tb, Ac, Cu, Lu, Tc, Re, Co, Fe, etc., e.g. 90 Y, 111 In,67 Cu, 77 Lu, 99 Tc, 161 Tb, 225 Ac and the like. The radionuclide may be contained in a chelating agent such as DOTA or NODA-GA. Furthermore, the radionuclide may be a therapeutic radionuclide or may be a radionuclide that can be used as a contrast agent in imaging techniques as described below. Radionuclides or molecules containing radionuclides are known in the art and are commercially available.

[0173] In certain embodiments, the payload may be a vitamin, which may be selected from the group consisting of folates, including folic acid, folacin, and vitamin B9.

[0174] In certain embodiments, the present invention relates to a toxin comprising Pyrrolobenzodiazepines (e.g. PBD); Auristatins (e.g., MMAE, MMAF); Maytansinoids (e.g. maytansine, DM1, DM4, DM21); ·Duocarmycin; · Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors; ·Tubulysin; Enediynes (e.g., calicheamicin); · Anthracycline derivatives (PNU) (e.g. doxorubicin); · Pyrrole-based kinesin spindle protein (KSP) inhibitors; · Cryptophycin; Drug efflux pump inhibitors; Sandramycin; Amanitin (e.g., α-amanitin); and Camptothecins (e.g., exatecan, deruxtecan) The present invention relates to a method according to the present invention, wherein the method is at least one selected from the group consisting of:

[0175] That is, the antibody-linker conjugates produced by the methods of the present invention preferably contain a toxin payload. The term "toxin" as used in the present invention relates to any compound produced by a living cell or organism and that is toxic to the cell or organism. Thus, the toxin may be, for example, a small molecule, a peptide, or a protein. Specific examples are neurotoxins, necrotic toxins, hematotoxins, and cytotoxins. In a particular embodiment, the toxin is a toxin used in the treatment of neoplastic diseases. That is, the toxin can be conjugated to an antibody in the methods of the present invention and delivered to malignant cells by the targeting specificity of the antibody.

[0176] In certain embodiments, the toxin may be an auristatin. The term "auristatin" as used herein refers to a family of antimitotic drugs. Auristatin derivatives are also included within the definition of the term "auristatin". Examples of auristatins include, but are not limited to, auristatin E (AE), monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), and synthetic analogs of dolastatins.

[0177] In certain embodiments, the toxin may be a maytansinoid. In the context of the present invention, the term "maytansinoid" refers to a group of highly cytotoxic drugs originally isolated from the African shrub Maytenus ovatus, as well as maytansinol and the C-3 ester 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 ... 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 maytansine, DM1, DM3, DM4, and / or DM21.

[0178] In certain embodiments, the toxin may be a duocarmycin. Suitable duocarmycins may be, for example, duocarmycin A, duocarmycin Bl, duocarmycin B2, duocarmycin CI, duocarmycin C2, duocarmycin D, duocarmycin SA, duocarmycin MA, and CC-1065. It should be understood that the term "duocarmycin" also refers to synthetic analogs of duocarmycins, such as adozelesin, bizeresin, carzelesin, KW-2189, and CBI-TMI.

[0179] In certain embodiments, the toxin may be a NAMPT inhibitor. The terms "NAMPT inhibitor" and "nicotinamide phosphoribosyltransferase inhibitor" as used herein refer to inhibitors that reduce the activity of NAMPT. The term "NAMPT inhibitor" may also include prodrugs of NAMPT inhibitors. Examples of NAMPT inhibitors include, but are not limited to, FK866 (also known as APO866), GPP78 hydrochloride, ST118804, STF31, pyridyl cyanoguanidine (also known as CH-828), GMX-1778, and P7C3. Additional NAMPT inhibitors are known in the art and may be suitable for use in the compositions and methods described herein. See, for example, WO 2015 / 054060, U.S. Pat. Nos. 8,211,912, and 9,676,721, which are incorporated herein by reference in their entireties. In some embodiments, the NAMPT inhibitor is FK866. In some embodiments, the NAMPT inhibitor is GMX-1778.

[0180] In certain embodiments, the toxin may be a tubulysin. Tubulysins are cytotoxic peptides and include nine members (A-I). Tubulysin A has potential applications as an anti-cancer drug. It arrests cells in the G2 / M phase. Tubulysin A inhibits polymerization and induces depolymerization of isolated microtubules more efficiently than vinblastine. Tubulysin A has a strong cytostatic effect on various tumor cell lines, with its IC50 in the picomolar range. Another tubulysin that can be used in the methods of the present invention may be tubulysin E.

[0181] In certain embodiments, the toxin may be an enediyne. The term "enediyne," as used herein, refers to a group of bacterial natural products characterized by either a 9- or 10-membered ring containing two triple bonds separated by a double bond (see, e.g., KC Nicolaou; A.L. Smith; E.W. Yue (1993). "Chemistry and biology of natural and designed enediynes". PNAS 90(13):5881-5888; the entire contents of which are incorporated herein by reference). Some enediynes can undergo Bergmann cyclization, and the resulting diradical, a 1,4-dehydrobenzene derivative, can abstract a hydrogen atom from the sugar backbone of DNA, resulting in DNA strand cleavage (see, e.g., S. Walker; R. Landovitz; W. D. Ding; G. A. Ellestad; D. Kahne (1992). "Cleavage behavior of calicheamicin gamma 1 and calicheamicin T". Proc Natl Acad Sci USA 89(10):4608-12, the entire contents of which are incorporated herein by reference). Reactivity with DNA endows many enediynes with antibiotic properties, and several enediynes are being clinically investigated as anticancer antibiotics. Non-limiting examples of enediynes are dynemicin, neocarzinostatin, calicheamicin, esperamicin (see, e.g., Adrian L. Smith and KC Bicolaou, "The Enediyne Antibiotics" J. Med. Chem., 1996, 39(11), pp 2103-2117; and Donald Borders, "Enediyne antibiotics as antitumor agents," Informa Healthcare; 1st edition (Nov. 23, 1994, ISBN-10:0824789385; the entire contents of which are incorporated herein by reference). In certain embodiments, the toxin may be a calicheamicin.

[0182] In certain embodiments, the toxin may be doxorubicin. "Doxorubicin," as used herein, refers to a member of the anthracycline family derived from the bacterium Streptomyces peucetius var. caesius, which is of the Streptomyces genus, and includes doxorubicin, daunorubicin, epirubicin, and idarubicin.

[0183] In certain embodiments, the toxin may be a kinesin spindle protein inhibitor. The term "kinesin spindle protein inhibitor" refers to a compound that inhibits kinesin spindle proteins, which are involved in the assembly of bipolar spindles during cell division. Kinesin spindle protein inhibitors are being investigated as cancer treatments. Examples of kinesin spindle protein inhibitors include ispinesib. Additionally, the term "kinesin spindle protein inhibitor" includes SB715992 or SB743921 from GlaxoSmithKline, and pentamidine / chlorpromarin from CombinatoRx.

[0184] In certain embodiments, the toxin may be a cryptophycin, as described in U.S. Patent Application Publication No. 20180078656A1, which is incorporated by reference.

[0185] In certain embodiments, the toxin may be sandramycin, a depsipeptide first isolated from Nocardioides species (ATCC 39419) that has been shown to have cytotoxic and antitumor activity.

[0186] In certain embodiments, the toxin may be an amatoxin. Amatoxins (including alpha-amanitin, beta-amanitin, and amanitin) are cyclic peptides composed of eight amino acids. They can be isolated from the mushroom Amanita phalloides or prepared synthetically from the building blocks. Amatoxins specifically inhibit DNA-dependent RNA polymerase II in mammalian cells, thereby affecting cellular transcription and protein biosynthesis. When transcription is inhibited in a cell, growth and proliferation ceases. Although not covalently bound, the complex between amanitin and RNA polymerase II is very tight (KD=3nM). Dissociation of amanitin from the enzyme is a very slow process, making it unlikely that affected cells will recover. If transcription inhibition continues too long in a cell, the cell will undergo programmed cell death (apoptosis). In a preferred embodiment, the term "amatoxin" as used herein refers to α-amanitin or a variant thereof, e.g. as described in WO 2010 / 115630, WO 2010 / 115629, WO 2012 / 119787, WO 2012 / 041504 and WO 2014 / 135282.

[0187] In certain embodiments, the toxin may be camptothecin. The term "camptothecin" as used herein is intended to mean camptothecin or a camptothecin derivative that functions as a topoisomerase I inhibitor. Exemplary camptothecins include, for example, topotecan, exatecan, deruxtecan, irinotecan, DX-8951f, SN38, BN80915, lurtotecan, 9-nitrocamptothecin, and aminocamptothecin. Various camptothecins, including camptothecin, have been reported for use in treating human cancer patients. Some camptothecins are described, for example, in Kehrer et al., Anticancer Drugs, 12(2):89-105, (2001) or Li et al., ACS Med.Chem.Lett.2019,10,10,1386-1392).

[0188] A toxin in the sense of the present invention may be an inhibitor of a drug efflux transporter. An antibody-payload conjugate comprising a toxin and an inhibitor of a drug efflux transporter may have the advantage that when internalized in a cell, the inhibitor of the drug efflux transporter prevents the toxin from being excreted outside the cell. In the present invention, the drug efflux transporter may 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 with this in mind. Finally, Valspodar and Libersan are other examples of such agents.

[0189] It should be understood that payload B as defined herein is not only to be understood as an actual payload, but rather as a payload molecule, which when used in the present invention may contain additional structures, for example to facilitate coupling of the payload to a linking moiety B or K residue or a chemical spacer via chemical synthesis.

[0190] That is, in certain embodiments, the actual payload may be contained in a payload molecule that is linked to a linker of the invention. The payload molecule may have the following structure: X-(spacer)-payload, (wherein payload represents the actual payload, e.g., one of the compounds disclosed herein, X represents a reactive group suitable for attaching the payload molecule to a compatible functional group in the linking moiety (two-step process) or the K residue of the chemical spacer or linker (one-step process), and (spacer) represents a chemical spacer that spatially separates the actual payload from the reactive group X). However, it should be understood that in certain embodiments, the reactive group X may be part of the spacer or the actual payload. For example, the spacer may comprise a peptide or amino acid residue, and the reactive group X may be the amino group of the N-terminal amino acid residue contained in the spacer. In other embodiments, the spacer may not be present. In embodiments where a spacer is not present, a functional group may be included in the actual payload. In certain embodiments, the spacer can be used to attach a functional group of interest, i.e., a functional group compatible with the functional group contained in the linking moiety, to the actual payload. In certain embodiments, the reactive group X may be a maleimide group, or a cyclooctyne group, such as, but not limited to, a DBCO group or a BCN group.

[0191] In certain embodiments, the present invention provides a chemical spacer (Sp 2 ) comprises a self-immolative moiety.

[0192] That is, the linker may contain a self-immolative moiety to facilitate release of the payload in the target cell or tissue. The self-immolative moiety may be contained in any part of the linker. However, the self-immolative moiety is preferably contained within a chemical spacer (Sp) that separates the payload from the K residue. 2 Alternatively, the self-immolative moiety may be comprised in a (spacer) comprised in the payload molecule, as defined above.

[0193] The term "self-immolative moiety", as used herein, refers to an at least bifunctional molecule that may be included in a linker and that spontaneously degrades after an initial reaction has occurred, thereby releasing the payload. The initial reaction may be hydrolysis of a covalent bond between the self-immolative moiety and an amino acid residue. In certain embodiments, the covalent bond between the self-immolative moiety and an amino acid residue may be an amide bond formed between the α-carboxyl group of the amino acid and an amine group included in the self-immolative moiety, and the initial reaction may be catalyzed by a peptidase or protease. However, other chemical entities are also encompassed by the present invention.

[0194] In a particular embodiment, the present invention relates to a method according to the invention, wherein the self-immolative moiety is directly attached to payload B.

[0195] More preferably, the self-immolative moiety is directly attached to the payload B, such that the payload is released upon degradation of the self-immolative moiety. In certain embodiments, the self-immolative moiety is located between the payload and a K residue contained in the linker. That is, the self-immolative moiety may be coupled to the N-terminus of the K residue or to the C-terminus of the K residue. Alternatively, the self-immolative moiety may be coupled to the payload via a chemical spacer (Sp 2 ) and preferably at the N-terminus or C-terminus of said amino acid residues. Furthermore, the self-immolative moiety may be fused to the payload and a chemical spacer (Sp) by any method known in the art. 2 ) may be located between a non-amino acid residue included in the

[0196] It will be appreciated that the choice of self-immolative moiety will depend, inter alia, on the functional groups available on the payload molecule.

[0197] In a particular embodiment, the invention relates to a method according to the invention, wherein the self-immolative moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety or an aminomethylene spacer.

[0198] That is, in certain embodiments, the linker may comprise a self-immolative moiety p-aminobenzylcarbamoyl (PABC). PABC comprises a free amine group suitable for coupling to the C-terminus of an amino acid residue or peptide and a carbamoyl group via which it can be coupled to a payload, in particular a payload comprising an amine. However, the skilled artisan will be aware of how to functionalize a payload to comprise an amine group. The self-immolative moiety PABC is preferably located between the payload and an amino acid residue comprised in the linker. The amino acid residue is preferably a residue K or a chemical spacer (Sp 2 In certain embodiments, the self-immolative moiety PABC is an amino acid contained in the payload and a chemical spacer (Sp 2 ) and an alanine residue contained in the payload. In certain embodiments, the self-immolative moiety may be located between the payload and a peptidase cleavage site. In certain embodiments, the self-immolative moiety may be located between the payload and a cathepsin cleavage site. That is, the self-immolative moiety may be located between the payload and a motif known to be cleavable by cathepsin.

[0199] The term "cathepsin" as used herein refers to a family of proteases. The term cathepsin includes cathepsin A, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin F, cathepsin G, cathepsin H, cathepsin K, cathepsin L1, cathepsin L2, cathepsin O, cathepsin S, cathepsin W, and cathepsin Z. In certain embodiments, the cleavable moiety may be a motif that is specifically hydrolyzed by cathepsin B, such as valine-alanine, valine-citrulline, or alanine-alanine. Further motifs that can be specifically hydrolyzed by peptidases are disclosed by Salomon et al., Optimizing Lysosomal Activation of Antibody-Drug Conjugates (ADCs) by Incorporation of Novel Cleavable Dipeptide Linkers, Mol Pharm. 2019, 16(12), p. 4817-4825.

[0200] One typical dipeptide structure used in ADC linkers is the valine-citrulline motif, as provided for example in brentuximab vedotin, discussed in Dubowchik and Firestone;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;13(4);p.855-69. This linker can be cleaved by cathepsin B to release the actual payload at the disease site. The same is true for the valine-alanine motif, as provided for example in SGN-CD33A.

[0201] Thus, in certain embodiments, the linker has the structure (Sp 1 )-K-(Sp 2 )-Val-Cit-(self-immolative moiety)-payload. In certain embodiments, the linker may comprise the structure (Sp 1 )-K-(Sp 2 )-Val-Cit-payload. In certain embodiments, the linker may comprise the structure (Sp 1 )-K-(Sp 2 )-Val-Cit-PABC-payload.

[0202] In certain embodiments, the linker may comprise or consist of the structure K-Val-Cit-(self-immolative moiety)-payload. In certain embodiments, the linker may comprise or consist of the structure K-Val-Cit-PABC-payload. In certain embodiments, the linker may comprise or consist of the structure K-Val-Cit-PABC-MMAE. In certain embodiments, the linker may comprise or consist of the structure K-Val-Cit-PABC-maytansine.

[0203] It should be noted that the peptide cleavage site may also be a motif cleavable by other peptidases such as caspase 3, legumain, or neutrophil elastase, or may be a motif as described in Dal Corso et al., Innovative Linker Strategies for Tumor-Targeted Drug Conjugates; Chemistry; 25(65); p. 14740-14757.

[0204] However, it should be noted that cells contain a wide range of cellular peptidases, and other, less conserved amino acid motifs may also be efficiently cleaved by peptidases. Thus, in certain embodiments, the linker may have the structure (Sp 1 )-K-(Sp 2)-PABC-payload, where (Sp 2 ) may contain absent or consisting of amino acid residues).

[0205] In certain embodiments, the linker has the structure (Sp 1 )-K-(Sp 2 )-PABC-payload, where (Sp 2 ) is the PABC part and (Sp 2 ) or a PEG moiety between the C-terminal most amino acid residue contained in the K residue.

[0206] In certain embodiments, a linker comprising the self-immolative moiety PABC is coupled to an amine-containing payload, in particular a payload comprising a primary or secondary amine. In certain embodiments, the amine-containing payload is an auslistatin, such as MMAE. In certain embodiments, the amine-containing payload is a maytansinoid, such as maytansine.

[0207] It should be noted that the payload can be coupled to the self-immolative PABC moiety via an additional linker molecule. For example, an amine-containing payload can be coupled to the PABC moiety via a p-nitrophenol (PNP) group. Further linker molecules that can couple payloads containing reactive groups other than amines to the PABC moiety are disclosed in Su et al., Bioconjugate Chem. 2018, 29, 4, 1155-1167; and Dokter et al., Mol Cancer Ther. 2014 Nov; 13(11): 2618-29. For example, a payload containing an alcohol or phenol group can be coupled to the PABC moiety via an ethylenediamine (EDA) linker.

[0208] In a particular embodiment, the present invention relates to a method according to the present invention, wherein the self-immolative moiety comprises a methylamine group, which has previously been demonstrated to be usable as a self-immolative moiety in peptide-based linkers of ADCs (Costoplus et al., ACS Med. Chem. Lett., 2019, 10, 10, 1393-1399 and Li et al., ACS Med. Chem. Lett. 2019, 10, 10, 1386-1392).

[0209] In particular, a self-immolative moiety containing a methylamine group can be coupled to the C-terminus of an amino acid residue via an amide bond formed between the α-carboxyl group of the amino acid residue and the amine contained in the methylamine group. 2 ) or residue K. The methyl group contained in the methylamine group may be coupled to the payload by an ether or thioether bond. Thus, when the payload contains a hydroxyl or thiol group, it is preferred to use a methylamine group as the self-immolative group. In certain embodiments, the hydroxyl-containing payload may be a camptothecin, such as the exatecan derivative Dxd, or an anthracycline, such as PNU-159682. In certain embodiments, the thiol-containing payload may be a maytansinoid, such as DM1, DM4, or DM21.

[0210] Linkers containing aminomethylene spacers have the molecular structure C-(NH)-(CH 3 )-OC or C-(NH)-(CH 3 )-SC.

[0211] It will be appreciated that for coupling a payload to the C-terminal carboxyl group of an amino acid residue, PABCs containing an aminomethylene spacer and self-immolative moieties are preferably used.

[0212] Other self-immolative moieties that can be used to couple a payload to the C-terminal carboxyl group of an amino acid residue include the p-aminobenzylethanol (PABE) linker for coupling a phenol-containing payload to the C-terminal carboxyl group of an amino acid residue (Zhang et al., Bioconjugate Chem. 2018, 29, 6, 1852-1858) or the paramethylaniline (PMA) linker for coupling a tertiary amine or heteroaryl moiety-containing payload to the C-terminal carboxyl group of an amino acid residue (Staben et al., Nature Chemistry volume 8, pages 1112-1119 (2016)). A non-limiting example of a payload containing a phenol group is duocarmycin GA or pyrrolobenzodiazepine PBD. A non-limiting example of a payload containing a tertiary amine is duocarmycin GA.

[0213] However, the payload can also be coupled to the N-terminal amino group via a self-immolative moiety. For example, the payload can be coupled to the N-terminal amino group of an amino acid residue via a self-immolative moiety comprising an ortho-hydroxy-protected aryl sulfate. For example, ortho-hydroxy-protected aryl sulfate (OHPAS) can be used to couple a phenolic payload, such as a PBD, to the N-terminal amino group of an amino acid residue. The OHPAS moiety preferably comprises a carboxyl group, through which it can be directly coupled to the N-terminal amino group of an amino acid residue. Alternatively, the OHPAS moiety can be coupled to a functionalized PEG linker, such as, but not limited to, a functionalized (PEG) 2 It may be coupled to the N-terminal amino group of the amino acid residue via a linker. Preferably, the PEG linker is functionalized with an amino group at one end to allow coupling to the carboxyl group contained in the OHPAS moiety, and functionalized with a carboxyl group at the other end to allow coupling to the N-terminal amino group of the amino acid residue (Park et al., Bioconjugate Chem. 2019, 30, 7, 1957-1968).

[0214] Alternatively or additionally, a linker molecule may be placed between the sulfate group of OHPAS and the payload to allow for coupling of non-phenolic payloads to OHPAS. For example, a para-hydroxybenzyl (PHB) linker molecule can be used to couple a payload containing a primary or secondary amine to the OHPAS moiety through the formation of a carbamate. A payload containing a tertiary amine can be coupled to an OHPAS containing linker through the formation of a quaternary ammonium. Furthermore, a para-hydroxybenzylethylenediamine (PHB-EDA) linker molecule can be used to couple a payload containing a hydroxyl group to the OHPAS moiety through the formation of a carbamate (Park et al., Bioconjugate Chem. 2019, 30, 7, 1957-1968).

[0215] In certain embodiments, the payload can be coupled to an amino acid residue contained in the linker via a cleavable moiety. A "cleavable moiety", as used herein, is a chemical unit that can be separated from the actual payload by enzymatic or non-enzymatic hydrolysis. In certain embodiments, the cleavable moiety can be an amino acid motif that is hydrolyzable by a peptidase or protease.

[0216] In other embodiments, the cleavable moiety included in the linker may be a carbohydrate moiety. In such embodiments, the cleavable moiety may be a moiety that is cleavable by a glucosidase. Thus, in certain embodiments, the cleavable moiety may be a moiety that is cleavable by β-glucuronidase or β-galactosidase.

[0217] In other embodiments, the cleavable moiety included in the linker may be a phosphate moiety. In such embodiments, the cleavable moiety may be a moiety that is cleavable by a phosphatase. Thus, in certain embodiments, the cleavable moiety may be a moiety that is cleavable by beta-lysosomal acid pyrophosphatase or an acid phosphatase.

[0218] Further examples of cleavable moieties that can be used to release the payload from the linker molecule are described by Bargh et al., Cleavable linkers in antibody-drug conjugates; Chem Soc Rev. 2019 Aug 12; 48(16):4361-4374. In certain embodiments, the linker can comprise the structure (cleavable moiety)-(self-immolative moiety)-payload. In such embodiments, the self-immolative moiety can degrade upon cleavage of the cleavable moiety, releasing the payload.

[0219] In certain embodiments, a linker may comprise a single linking moiety or payload.

[0220] In certain embodiments, the linker may comprise two or more linking moieties and / or payloads B. That is, in certain embodiments, the linker may comprise the following structure: a)(Sp 1 )-K-(Sp 2 )-B 1 -(Sp 3 )-B 2 -(Sp 4 ), b)(Sp 4 )-B 2 -(Sp 1 )-K-(Sp 2 )-B 1 -(Sp 3 ), c)(Sp 1 )-B 1 -(Sp 2 )-K-(Sp 3 )-B 2-(Sp 4 ), or d)(Sp 4 )-B 2 -(Sp 1 )-B 1 -(Sp 2 )-K-(Sp 3 ).

[0221] In such an embodiment, the chemical spacer (Sp 1 ), (Sp 2 ), (Sp 3 ), and the K residue may have the same characteristics as defined above. 1 and B. 2 may be any one of the linking moieties and / or payloads defined above. In addition, a chemical spacer (Sp 4 ) is a chemical spacer (Sp 1 ), (Sp 2 ), or (Sp 3 ) may or may not be present.

[0222] Thus, in certain embodiments, the present invention provides a method for the preparation of a medicament comprising the steps of: 2 Including, in particular, B 2 is a chemical spacer (Sp 1 ) or (Sp 3 The present invention relates to a method in which the compound is connected to a linker via

[0223] That is, the payload or connecting portion B 2 is a chemical spacer (Sp 1 ) or (Sp 3 ), and the payload or connecting portion B 1 The payload or connecting part B may be directly connected to the 2 is B 2 (Sp 1 ), (Sp 3 ), or B 1 The compound may include any functional group suitable for coupling to the functional group contained in

[0224] In certain embodiments, the payload or linking moiety B 2 B 2 (Sp 3 ) or B 1 That is, B may contain an amino group connected to 2 is reacted with the amino group (Sp 3 ) or B 1 In certain embodiments, the carboxyl group contained in (Sp 3 The carboxyl group in the chemical spacer (Sp 3 In certain embodiments, the amino acid residue of B 1 The carboxyl group contained in B may be the α-carboxyl group of an amino acid-based payload or a linking moiety. 2 is connected via a linker molecule (Sp 3 ) or B 1 In certain embodiments, the linker molecule may comprise a self-immolative moiety.

[0225] In certain embodiments, the payload or linking moiety B 2 B 2 (Sp 1 ) or B 1 That is, B may contain a carboxyl group connected to 2 is reacted with the carboxyl group (Sp 1 ) or B 1 In certain embodiments, the amine group contained in (Sp 1 The amine group in the chemical spacer (Sp 1 In certain embodiments, the amino acid residue of B 1 The amine group contained in B may be an α-amino group of an amino acid based payload or a linking moiety. 2 is connected via a linker molecule (Sp 1 ) or B 1In certain embodiments, the linker molecule may comprise a self-immolative moiety.

[0226] However, B 2 It should be noted that B may contain functional groups other than amine or carboxyl groups. In such embodiments, B 2 can be linked to (Sp 1 ), (Sp 3 ), or B 1 can be coupled to

[0227] In certain embodiments, the payload or linking moiety B 2 is (Sp 1 ) or (Sp 3 ) can be coupled to the amino acid side chains contained in B 2 is connected via a compatible functional group (Sp 1 ) or (Sp 3 ) can be attached to a functional group of an amino acid side chain contained in

[0228] In certain embodiments, (Sp 1 ), (Sp 2 ), (Sp 3 ), and K residues consist solely of amino acids, amino acid mimetics, and / or amino acid derivatives. 1 and / or B. 2 Also includes an amino acid backbone. In such embodiments, the linker may be a linear peptide or a peptidomimetic. 1 In embodiments where is an amino acid, an amino acid mimetic, or an amino acid derivative, the linker has the structure (Sp 1 )-K-(Sp 2 )-B 1 (Sp 1 )-K-(Sp 2 )-B 1 is a linear peptide or peptidomimetic. 1In embodiments where is an amino acid, an amino acid mimetic, or an amino acid derivative, the linker has the structure (Sp 1 )-K-(Sp 2 )-B 1 -(Sp 3 ), (Sp 1 )-K-(Sp 2 )-B 1 -(Sp 3 ) is a linear peptide or peptidomimetic. 1 In embodiments where is an amino acid, an amino acid mimetic, or an amino acid derivative, the linker has the structure K-(Sp 2 )-B 1 -(Sp 3 ), and K-(Sp 2 )-B 1 -(Sp 3 ) is a linear peptide or peptidomimetic. 1 In embodiments where is an amino acid, an amino acid mimetic, or an amino acid derivative, the linker has the structure K-(Sp 2 )-B 1 K-(Sp 2 )-B 1 is a linear peptide or peptidomimetic. 1 In embodiments where is an amino acid, amino acid mimetic, or amino acid derivative, the linker has the structure KB 1 -(Sp 3 ), KB 1 -(Sp 3 ) is a linear peptide or peptidomimetic. 1 In embodiments where is an amino acid, amino acid mimetic, or amino acid derivative, the linker has the structure KB 1 KB 1 is a linear peptide or a peptidomimetic.

[0229] B 1 and B. 2 In embodiments where is an amino acid, an amino acid mimetic, or an amino acid derivative, the linker has the structure (Sp 1 )-K-(Sp 2 )-B 1 -(Sp3 )-B 2 -(Sp 4 ), (Sp 1 )-K-(Sp 2 )-B 1 -(Sp 3 )-B 2 -(Sp 4 ) is a linear peptide or peptidomimetic. 1 and B. 2 In other embodiments where is an amino acid, amino acid mimetic, or amino acid derivative, the linker has the structure (Sp 4 )-B 2 -(Sp 1 )-K-(Sp 2 )-B 1 -(Sp 3 ), (Sp 4 )-B 2 -(Sp 1 )-K-(Sp 2 )-B 1 -(Sp 3 ) is a linear peptide or peptidomimetic. 1 and B. 2 In other embodiments where is an amino acid, amino acid mimetic, or amino acid derivative, the linker has the structure (Sp 4 )-B 2 -(Sp 1 )-B 1 -(Sp 2 )-K-(Sp 3 ), (Sp 4 )-B 2 -(Sp 1 )-B 1 -(Sp 2 )-K-(Sp 3 ) is a linear peptide or peptidomimetic.

[0230] B 1 In embodiments where the linker is not an amino acid, amino acid mimetic, or amino acid derivative, the linker has the structure (Sp 1 )-K-(Sp 2 )-B 1 -(Sp 3 ), (Sp 1 )-K-(Sp2 ) is a linear peptide or peptidomimetic, B 1 is (Sp 2 ) is connected to the C-terminal carboxyl group contained in B 1 In embodiments where the linker is not an amino acid, amino acid mimetic, or amino acid derivative, the linker has the structure (Sp 1 )-B 1 -(Sp 2 )-K-(Sp 3 ), (Sp 2 )-K-(Sp 3 ) is a linear peptide or peptidomimetic, B 1 is (Sp 2 ) is connected to the N-terminal amino group contained in B 1 It should be noted that B does not necessarily have to be directly coupled to the peptide or peptidomimetic. Instead, B 1 may be coupled to the peptide or peptidomimetic via a linker molecule and / or a self-immolative moiety.

[0231] B 1 is an amino acid, an amino acid mimetic, or an amino acid derivative; B 2 In embodiments where S is not an amino acid, amino acid mimetic, or amino acid derivative, the linker has the structure (Sp 1 )-K-(Sp 2 )-B 1 -(Sp 3 )-B 2 -(Sp 4 ), (Sp 4 )-B 2 -(Sp 1 )-K-(Sp 2 )-B 1 -(Sp 3 ), (Sp 1 )-B 1 -(Sp 2 )-K-(Sp 3 )-B 2 -(Sp 4 ), or (Sp 4 )-B 2 -(Sp 1 )-B 1 -(Sp2 )-K-(Sp 3 ), (Sp 1 )-K-(Sp 2 )-B 1 -(Sp 3 ) or (Sp 1 )-B 1 -(Sp 2 )-K-(Sp 3 ) is a linear peptide or peptidomimetic, B 2 is (Sp 3 ), B 1 , or the C-terminal carboxyl group contained in the K residue, or (Sp 1 ), B 1 , or is coupled to the N-terminal amino group of the K residue.

[0232] In such embodiments, for example, antibody-payload conjugates can be made with antibody to payload ratios of 2 or 4, for example with 1 or 2 payloads conjugated to each Q295 residue.

[0233] In a particular embodiment, the present invention provides a method for the preparation of a medicament for the treatment of a cancer, comprising: 1 and B. 2 are identical or different from each other.

[0234] That is, the payload or connecting portion B 1 and B. 2 may be identical, i.e., have the same chemical structure, or may be structurally different. 1 and B. 2 are both payloads or both linking moieties. 1 and B. 2 In an embodiment where both are payloads, payload B 1 and B. 2 may be the same or may have different payloads. 1 and B. 2 In embodiments where both are linking moieties, linking moiety B 1 and B. 2may be the same or different linking moieties. In certain embodiments, B 1 is the connecting part, and B 2 may be the payload, or vice versa.

[0235] All payloads or connecting parts are B 1 The reason is that the nucleotide sequence at one end (Sp 2 ) or K residue at the other end (Sp 3 ), (Sp 1 ), or B 2 It should be understood that this is because B does not have a functional group for forming a covalent bond with 1 In embodiments where B is the payload or linking moiety in a chain, 1 is preferably a divalent or polyvalent molecule. For example, B 1 may be an amino acid, an amino acid mimetic, or an amino acid derivative. In such embodiments, B 1 is linked to the amino group (Sp 2 ) or to the C-terminal carboxyl group of K, and via that carboxyl group (Sp 3 ) or B 2 Alternatively, B 1 is reacted with the carboxyl group (Sp 2 ) or to the N-terminal amino group of K, and via that amino group (Sp 1 ) or B 2 can be coupled to the C-terminal carboxyl group of

[0236] In certain embodiments, the linker comprises two linking moieties B 1 and B. 2 may include.

[0237] That is, in certain embodiments, the present invention encompasses a linker comprising two bioorthogonal marker groups and / or non-bioorthogonal entities. For example, a linker according to the present invention may comprise a linker having a structure similar to that of Lys(N 3 ) or Xaa(N 3) and sulfhydryl-containing linking moieties such as cysteine. In certain embodiments, the linkers of the present invention include a linking moiety that is 3 ) or Xaa(N 3 ) and a linking moiety comprising a tetrazine, such as a tetrazine modified amino acid. In certain embodiments, the linkers of the present invention may comprise a linking moiety comprising a sulfhydryl, such as a cysteine, and a linking moiety comprising a tetrazine, such as a tetrazine modified amino acid. Linkers comprising two different bioorthogonal marker groups and / or non-bioorthogonal entities have the advantage of being able to accept two different payloads, thus resulting in antibody-payload conjugates comprising more than one payload.

[0238] In this way, a 2+2 antibody-payload ratio can be achieved. The use of a second payload may enable the development of entirely new classes of antibody-payload conjugates that exceed current therapeutic approaches in terms of efficacy and potency.

[0239] Such an embodiment may in particular allow for targeting two different structures within a cell, such as DNA and microtubules, since some cancers are resistant to certain drugs, such as microtubule toxins, the DNA toxins can still kill the cancer cells.

[0240] According to another embodiment, two drugs can be used that are only fully effective if released at the same time in the same tissue, potentially reducing off-target toxicity if the antibody is partially degraded in healthy tissue or one drug is lost prematurely.

[0241] Furthermore, dual-labeled probes may be used for non-invasive imaging and therapy or intra- / post-operative imaging / surgery. In such an embodiment, tumor patients can be selected using non-invasive imaging. The tumor can then be surgically removed using the other contrast agent (e.g., fluorescent dye) that helps the surgeon or robot identify all cancerous tissue during surgery.

[0242] In certain embodiments, B 1 and B. 2 may be a linking moiety containing a thiol group, such as cysteine, and B 1 and B. 2 The other is Lys(N 3 ) may be a linking moiety that includes an azide moiety. In such embodiments, two different payloads may be coupled to the linker, one via thiol-maleimide conjugation and the other via a SPAAC reaction.

[0243] In certain embodiments, a linker may comprise two payloads. A linker that contains only a payload and no linking moiety can be conjugated to an antibody in a one-step process.

[0244] B 1 and B. 2 In an embodiment where both are payloads, B 1 and B. 2 It should be understood that the linker may be the same or may have different structures. In certain embodiments, the linker containing one or more payloads can be chemically synthesized. Alternatively, one or more payloads can be coupled to the linking moiety contained in the linker by any of the methods disclosed herein before the linker is conjugated to the antibody.

[0245] In certain embodiments, the linkers of the invention provide HIt may be possible to conjugate two different payloads to residue Q295 of the 2 domain. The use of a second payload allows the development of an entirely new class of antibody-payload conjugates that exceed current therapeutic approaches in terms of efficacy and potency. New application areas are also envisioned, such as dual-type imaging for imaging and therapy or intra- / post-operative surgery (see 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). For example, dual-labeled antibodies encompassing a molecular imaging agent for preoperative positron emission tomography (PET) and a near-infrared fluorescent (NIRF) dye for guided delineation of surgical margins can greatly improve cancer diagnosis, staging, and resection (see 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 US A.2015 Dec 29;112(52):15850-5). PET and NIRF optical imaging offer complementary clinical applications that enable non-invasive whole-body imaging to localize disease and identify tumor margins during surgery, respectively. However, to date, the creation of such dual-labeled probes has been difficult due to the lack of suitable site-specific methods. Conjugating two different probes by chemical means results in random conjugation of the probes, making analysis and reproducibility nearly impossible.

[0246] Furthermore, in a study by Levengood M. et al. (Orthogonal Cysteine ​​Protection Enables Homogeneous Multi-Drug Antibody-Drug Conjugates. Angewandte Chemie,Volume56,Issue3,January 16,2017), a dual-drug-conjugated antibody conjugated with two different auristatin toxins (which have different physicochemical properties and exert complementary anticancer activity) conferred activity in cell lines and xenograft models that were resistant to ADCs composed of the individual auristatin components. This suggests that dual-conjugated ADCs may enable cancer heterogeneity and resistance to be addressed more effectively than a single conventional ADC alone. Since one of the resistance mechanisms to ADCs involves active pumping out of the cytotoxic moiety from cancer cells, another dual-drug application may involve additionally and simultaneously delivering a drug that specifically blocks the efflux mechanism of the cytotoxic drug. Thus, such dual-conjugated ADCs may help overcome cancer resistance to ADCs more effectively than conventional ADCs.

[0247] The term "antibody" is used herein in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity. The terms "antibody" and "antibodies" broadly encompass naturally occurring forms of antibodies (e.g., IgG, IgA, IgM, IgE).

[0248] The antibody is preferably a monoclonal antibody. The antibody 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 antibody may optionally be chimerized or humanized.

[0249] C H Fragments or recombinant variants of antibodies comprising two domains can be, for example: Antibody formats that contain only heavy chain domains (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-C H 2-CH3)2 Fc fusion peptides containing an Fc domain and one or more receptor domains It may be.

[0250] Antibodies may also be bispecific (e.g. DVD-IgG, crossMab, additional IgG-HC fusions) or biparatopic. For an overview see Brinkmann and Kontermann; Bispecific antibodies; Drug Discov Today; 2015; 20(7); p. 838-47.

[0251] In a particular embodiment, the invention relates to a method according to the invention, wherein the antibody is an IgG antibody, in particular an IgG1 antibody.

[0252] "IgG" as used herein means a polypeptide belonging to the class of antibodies substantially encoded by recognized immunoglobulin gamma genes. In humans, IgG includes the subclasses or isotypes IgG1, IgG2, IgG3, and IgG4. In mice, IgG includes IgG1, IgG2a, IgG2b, IgG3. Full-length IgG consists of two identical pairs of two immunoglobulin chains, each pair having one light chain and one heavy chain, each light chain containing immunoglobulin domains VL and CL, and each heavy chain containing immunoglobulin domains VH, Cγ1 (also called CH1), Cγ2 (also called CH2), and Oγ3 (also called CH3). In the context of human IgG1, "CH1" refers to positions 118-215, the CH2 domain refers to positions 231-340, and the CH3 domain refers to positions 341-447, according to the EU index of Kabat. IgG1 also contains a hinge domain, which in the case of IgG1 corresponds to positions 216-230.

[0253] The antibody used in the methods of the invention or the antibody-payload conjugates of the invention may be or comprise any antibody, preferably any IgG type antibody. For example, the antibody may be, but is not limited to, brentuximab, trastuzumab, gemtuzumab, inotuzumab, avelumab, cetuximab, rituximab, daratumumab, pertuzumab, vedolizumab, ocrelizumab, tocilizumab, ustekinumab, golimumab, obinutuzumab, sacituzumab, belantamab, polatuzumab, and enfortumab.

[0254] Thus, in a particular embodiment, the invention relates to a method according to the invention, wherein the antibody is selected from the group consisting of brentuximab, trastuzumab, gemtuzumab, inotuzumab, avelumab, cetuximab, rituximab, daratumumab, pertuzumab, vedolizumab, ocrelizumab, tocilizumab, ustekinumab, golimumab, obinutuzumab, sacituzumab, belantamab, polatuzumab, and enfortumab.

[0255] In a preferred embodiment, the invention relates to a method according to the invention, wherein the antibody is selected from the group consisting of the antibodies brentuximab, gemtuzumab, trastuzumab, inotuzumab, polatuzumab, enfortumab, sacituzumab and belantamab.

[0256] In a more preferred embodiment the invention relates to a method according to the invention, wherein the antibody is polatuzumab or trastuzumab or enfortumab.

[0257] That is, in certain embodiments, the invention relates to an antibody-linker conjugate, wherein the antibody is polatuzumab and the linker is any one of the linkers disclosed herein.

[0258] In another embodiment, the invention relates to an antibody-linker conjugate, wherein the antibody is trastuzumab and the linker is any one of the linkers disclosed herein.

[0259] In another embodiment, the invention relates to an antibody-linker conjugate, wherein the antibody is enfortumab and the linker is any one of the linkers disclosed herein.

[0260] The antibody for use in the methods of the present invention may be a glycosylated antibody, a deglycosylated antibody, or an aglycosylated antibody.

[0261] That is, in certain embodiments, the antibody may be an IgG antibody, preferably glycosylated at residue N297. Thus, in certain embodiments, the invention provides that the IgG antibody is a glycosylated IgG antibody, in particular that the IgG antibody is C H The method according to the invention relates to a method according to the invention in which the polypeptide is glycosylated at residue N297 (EU numbering) of domain 2.

[0262] As discussed herein, IgG antibodies glycosylated at residue N297 have several advantages over non-glycosylated antibodies.

[0263] However, the antibody may preferably be a deglycosylated antibody, in which the carbohydrate chain at residue N297 has been cleaved with the enzyme PNGase F. Additionally, the antibody may preferably be an aglycosylated antibody, in which residue N297 has been substituted with a non-asparagine residue. Methods for deglycosylating antibodies and for making aglycosylated antibodies are known in the art.

[0264] In certain embodiments, a linker of the invention can be conjugated to an endogenous Gln residue in the Fc domain of an antibody or to a Gln residue introduced into the antibody by molecular engineering.

[0265] Thus, in a particular embodiment, the present invention provides a method for the preparation of ... 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

[0266] The linkers of the present invention can be conjugated to any Gln residue in the Fc domain of an antibody that can serve as a substrate for transglutaminase. Typically, the term Fc domain, as used herein, refers to the last two constant region immunoglobulin domains (C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C21, C31, C42, C53, C64, C75, C86, C97, C98, C99, C100, C111, C122, C133, C143, C154, C155, C166, C177, C187, C188, C190, C191, C192, C193, C194, C195, C 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 according to the present invention is a C H 2. C H 3, and C, if applicable H 4 domain.

[0267] In certain embodiments, the endogenous Gln residue is located at the C of an IgG antibody. HIn a particular embodiment, the present invention relates to an antibody in which the Gln residue in the Fc domain of an IgG antibody is a Gln residue Q295 (EU numbering) in the C2 domain of an IgG antibody. H In a particularly preferred embodiment, the Gln residue in the Fc domain of the antibody is the Gln residue Q295 (EU numbering) of the CH2 domain of a glycosylated IgG antibody, in particular a glycosylated IgG antibody with an unmodified constant region.

[0268] It is important to understand that Q295 is a highly conserved amino acid residue in IgG type antibodies. In particular, it is conserved in human IgG1, 2, 3, 4, as well as rabbit and rat antibodies. The availability of Q295 is therefore a great advantage in the creation of therapeutic antibody-payload conjugates or diagnostic conjugates, where the antibodies are often of non-human origin. Thus, the method according to the invention provides a highly versatile and widely applicable tool. Despite the fact that residue Q295 is highly conserved among IgG type antibodies, there are IgG type antibodies that do not have this residue, such as mouse and rat IgG2a antibodies. Thus, the antibodies used in the method according to the invention are preferably C295-Q295. H It is to be understood that this is an IgG type antibody that contains residue Q295 (EU numbering) in domain 2.

[0269] Furthermore, it has been shown that genetically engineered artificial conjugates using Q295 for payload attachment have shown good pharmacokinetics and efficacy (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; 2015; 12(6), p. 1863-1871) and can carry unstable toxins that are prone to degradation (Dorywalska et al.; Site-Dependent Degradation of a Non-Cleavable Auristatin-Based Linker-Payload in Rodent Plasma and Its Effect on ADC Efficacy. PLoS ONE; 2015; 10(7): e0132282). Therefore, it is expected that this site-specific approach will have similar effects since the same residues are modified except for the glycosylated antibody. Glycosylation may further contribute to the overall stability of the ADC, and removal of the glycan moiety, similar to the approach mentioned above, has been shown to result in less stable antibodies (Zheng et al.; The impact of glycosylation on monoclonal antibody conformation and stability. Mabs-Austin; 2011, 3(6), p. 568-576).

[0270] C of the linker using transglutaminase H The literature discussing conjugation to Gln residues of 2 has focused on small, low molecular weight substrates, but the prior art literature describes that a deglycosylation step at position N297 or the use of an aglycosylated antibody is always necessary to achieve such conjugation (WO 2015 / 015448, WO 2017 / 025179, WO 2013 / 092998).

[0271] However, quite surprisingly, and contrary to all expectations, the use of the above-mentioned linker structures indeed allows efficient site-specific conjugation of glycosylated antibodies to Q295: in particular, the coupling of linkers containing toxin molecules was achieved with a conjugation efficiency of over 80%.

[0272] Although Q295 is in close proximity to N297, and N297 is glycosylated in the native state, the methods of the present invention still allow for the conjugation of a linker or payload to it using the specified linker.

[0273] As shown, the method of the present invention does not require prior enzymatic deglycosylation of N297 to prevent glycosylation, nor the use of an aglycosylation antibody, nor the substitution of N297 with another amino acid, nor the introduction of a T299A mutation.

[0274] These two points are of great advantage in terms of production. Enzymatic deglycosylation is undesirable from the viewpoint of GMP, because it is necessary to reliably remove both the deglycosylation enzyme (e.g., PNGase F) and the cleaved glycan from the medium.

[0275] Furthermore, because there is no need to genetically engineer the antibody to attach a payload, the insertion of sequences that may increase immunogenicity and decrease the overall stability of the antibody can be avoided.

[0276] Substitution of N297 with another amino acid also has undesirable effects, since it may affect the overall stability of the entire Fc domain (Subedi et al, The Structural Role of Antibody N-Glycosylation in Receptor Interactions. Structure 2015, 23(9), 1573-1583) and the efficacy of the entire conjugate, which may result in increased antibody aggregation and reduced solubility (Zheng et al.; The impact of glycosylation on monoclonal antibody conformation and stability. Mabs-Austin 2011, 3(6), 568-576), which is particularly important for hydrophobic payloads such as PBDs. Furthermore, the glycans present on N297 have important immunomodulatory effects, such as initiating antibody-dependent cellular cytotoxicity (ADCC). These immunomodulatory effects are lost by deglycosylation or any of the other approaches mentioned above to obtain aglycosylated antibodies. Furthermore, any sequence modifications of established antibodies may lead to regulatory issues, which is problematic since approved and clinically validated antibodies are often used as the starting point for ADC conjugation.

[0277] Thus, the method of the present invention makes it possible to easily and without disadvantages generate ADCs with well-defined stoichiometry to which site-specific payloads are attached.

[0278] In view of the above, the method of the present invention preferably comprises the steps of: H The C of an antibody that is glycosylated at residue N297 (EU numbering) of the C2 domain HIt is described that the method is used for the conjugation of an IgG antibody at residue Q295 (EU numbering) of domain 2. However, it is specified 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, which may be an endogenous Gln residue or a Gln residue introduced by molecular engineering.

[0279] Thus, in a particular embodiment, the invention relates to a method according to the invention, wherein a linker-conjugating Gln residue has been introduced into the heavy or light chain of the antibody by molecular engineering.

[0280] The term "molecular engineering" as used herein refers 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 envisaged in the present invention. First, a single residue in 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 or by replacing 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 the antibody.

[0281] For example, amino acid residues in the heavy or light chain of the antibody may be replaced with Gln residues, so long as the resulting antibody can be conjugated to a linker of the invention by 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, and in particular, the substitution is a N297Q substitution. The antibody comprising the N297Q mutation can be conjugated to more than one linker per heavy chain of the antibody. For example, the antibody comprising the N297Q mutation can be conjugated to four linkers, one linker conjugated to residue Q295 of the first heavy chain of the antibody, one linker conjugated to residue N297Q of the first heavy chain of the antibody, one linker conjugated to residue Q295 of the second heavy chain of the antibody, and one linker conjugated to residue N297Q of the second heavy chain of the antibody. Those skilled in the art recognize that the substitution of residue N297 of an IgG antibody with a Gln residue results in an aglycosylated antibody.

[0282] Thus, in a particular embodiment, the present invention relates to a method for the preparation of a glycosylated IgG antibody in which a Gln residue is introduced into the heavy or light chain of the antibody by molecular engineering. H The present invention relates to a method according to the present invention, which is a 2 domain N297Q (EU numbering).

[0283] In a particular embodiment, the invention relates to a method 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.

[0284] Instead of replacing a single amino acid residue of an antibody, a peptide tag containing a transglutaminase-accessible Gln residue may be introduced into the heavy or light chain of the antibody. Such a peptide tag may be fused to the N-terminus or C-terminus of the heavy or light chain of the antibody. Alternatively, the peptide tag may be inserted into a suitable position 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 the heavy chain of an antibody and function as a substrate for transglutaminase are described in WO 2012 / 059882 and WO 2016 / 144608.

[0285] Thus, in a particular embodiment, the invention relates to a method according to the invention, wherein a peptide comprising a Gln residue is fused to the C-terminus of the heavy chain of the antibody.

[0286] Exemplary peptide tags 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 an antibody, are LLQGG (SEQ ID NO:1), LLQG (SEQ ID NO:2), LSLSQG (SEQ ID NO:3), GGGLLQGG (SEQ ID NO:4), GLLQG (SEQ ID NO:5), LLQ,GSPLAQSHGG (SEQ ID NO:6), GLLQGGG (SEQ ID NO:7), GLLQGG (SEQ ID NO:8), GLLQ (SEQ ID NO:9), LLQLLQGA (SEQ ID NO:10), LLQGA (SEQ ID NO:11), LLQYQGA (SEQ ID NO:12), LLQGSG (SEQ ID NO:13), LLQYQG (SEQ ID NO:14), LLQLLQG (SEQ ID NO:15), LLQLLQG (SEQ ID NO:16), LLQLLQG (SEQ ID NO:17), LLQLLQG (SEQ ID NO:18), LLQLLQG (SEQ ID NO:19), LLQLLQG (SEQ ID NO:20), LLQLLQG (SEQ ID NO:21), LLQLLQG (SEQ ID NO:22), LLQLLQG (SEQ ID NO:23), LLQLLQG (SEQ ID NO:24), LLQLLQG (SEQ ID NO:25), LLQLLQG (SEQ ID NO:26), LLQLLQG (SEQ ID NO:27), LLQLLQG (SEQ ID NO:28), LLQLLQG (SEQ ID NO:29), LLQLLQG (SEQ ID NO:30), LLQLLQG (SEQ ID NO:31), LLQLLQG (SEQ ID NO:32), LLQLLQG (SEQ ID NO:3 No. 15), SLLQG (SEQ ID NO: 16), LLQLQ (SEQ ID NO: 17), LLQLLQ (SEQ ID NO: 18), LLQGR (SEQ ID NO: 19), EEQYASTY (SEQ ID NO: 20), EEQYQSTY (SEQ ID NO: 21), EEQYNSTY (SEQ ID NO: 22), EEQYQS (SEQ ID NO: 23), EEQYQST (SEQ ID NO: 24), EQYQSTY (SEQ ID NO: 25), QYQS (SEQ ID NO: 26), QYQSTY (SEQ ID NO: 27), YRYRQ (SEQ ID NO: 28), DYALQ (SEQ ID NO: 29), FGLQRPY (SEQ ID NO: 30), EQKLISEEDL (SEQ ID NO: 31), LQR, and YQR.

[0287] The skilled artisan knows how to replace amino acid residues in an antibody or introduce peptide tags into an antibody by methods of molecular cloning, e.g. as described in Sambrook, Joseph. (2001). Molecular cloning: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

[0288] In general, a person skilled in the art knows how to determine at which position on the antibody the linker is conjugated. For example, the conjugation site can be determined by proteolytic digestion of the antibody-payload conjugate and LC-MS analysis of the resulting fragments. For example, samples 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 overnight at 37° C. LC-MS analysis can be performed using a nanoAcquity HPLC system coupled to a Synapt-G2 mass spectrometer (Waters). For this purpose, 100 ng of peptide solution can be loaded onto an Acquity UPLC Symmetry C18 trap column (Waters, part number 186006527) and trapped for 3 minutes 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 may then be eluted with a linear gradient from 3% to 65% buffer B within 25 min. Data may be acquired in positive polarity separation mode and a mass range of 50-2000 m / z. Other instrument settings may 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 may be calibrated with [Glu1]-fibrinopeptide.

[0289] Additionally, one of skill in the art is aware of methods to determine the drug-to-antibody (DAR) 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.

[0290] For hydrophobic interaction chromatography (HIC), samples may be adjusted to 0.5 M ammonium sulfate and evaluated through a MAB PAK HIC Butyl column (5 μm, 4.6×100 mm, Thermo Scientific) with 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 may be used and the signal at 280 nm may be recorded. Relative HIC retention times (HIC-RRT) may be calculated by dividing the absolute retention times of the two ADC DARs by the retention times of the respective unconjugated mAbs.

[0291] To calculate the LC-MS DAR, add ADC to NH to a final concentration of 0.025 mg / mL. 4 HCO 3The solution may then be diluted 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 incubation at 37° C. overnight in the dark. For reversed-phase chromatography, a Dionex U3000 system may be used in combination with the software Chromeleon. The system may 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 may consist of water with 0.1% formic acid, and solvent B may comprise 85% acetonitrile with 0.1% formic acid. The reduced and alkylated sample may be loaded onto the column and separated by a gradient of 30→55% solvent B over 14 min. To identify the DAR species, the liquid chromatography system may be coupled to a Synapt-G2 mass spectrometer. The mass spectrometer capillary voltage may be set at 3 kV, the sampling cone at 30 V, and the extraction cone at a total value of 5 V. The source temperature may be set at 150 °C, the desolvation temperature at 500 °C, the cone gas at 20 l / h, the desolvation gas at 600 l / h, and the acquisition may be performed in positive mode with a scan time of 1 second in the mass range of 600-5000 Da. The instrument may be calibrated with sodium iodide. Spectra may be deconvoluted until convergence with the MaxEnt1 algorithm in MassLynx. After assigning DAR species to the chromatographic peaks, the DAR can be calculated based on the integrated peak area of ​​the reversed-phase chromatogram.

[0292] In a particular embodiment, the present invention relates to a method according to the invention, wherein a linker is conjugated to the γ-carboxamide group of a Gln residue contained in the antibody.

[0293] That is, the linker of the present invention is preferably conjugated to the amide group of the side chain of a Gln residue contained in the antibody, preferably any one of the Gln residues disclosed herein, more preferably Gln residue Q295 (EU numbering).

[0294] In certain embodiments, the invention relates to a method according to the invention, wherein the linker is suitable for conjugation to a glycosylated antibody with a conjugation efficiency of at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0295] That is, in certain embodiments, the linker may be a linker that can be conjugated to a glycosylated antibody with at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% efficiency. In a preferred embodiment, the linker may be a linker that can be conjugated to a glycosylated antibody with at least 70% efficiency. In another preferred embodiment, the linker may be a linker that can be conjugated to a glycosylated antibody with at least 75% efficiency. In another preferred embodiment, the linker may be a linker that can be conjugated to a glycosylated antibody with at least 80% efficiency. In another preferred embodiment, the linker may be a linker that can be conjugated to a glycosylated antibody with at least 85% efficiency. In another preferred embodiment, the linker may be a linker that can be conjugated to a glycosylated antibody with at least 90% efficiency. In another preferred embodiment, the linker may be a linker capable of conjugating to a glycosylated antibody with at least 95% efficiency. Preferably, the glycosylated antibody is a glycosylated IgG antibody, more preferably an IgG antibody glycosylated at residue N297 (EU numbering).

[0296] Those skilled in the art are aware of how to determine the conjugation efficiency of an antibody with a particular linker. For example, the conjugation efficiency can be determined as described herein. That is, an antibody, particularly an IgG1 antibody, can be incubated under conditions defined herein. After the incubation period, the conjugation efficiency can be determined by LC-MS analysis under reducing conditions. The transglutaminase can be microbial transglutaminase (MTG) from Streptomyces mobaraensis available from Zedira (Germany). Suitable buffers can be Tris, MOPS, HEPES, PBS, or BisTris buffers. However, it should be understood that the choice of buffer system can be different and can be highly dependent on the chemical nature of the linker. However, those skilled in the art can identify optimal buffer conditions based on the disclosure of the present invention. Alternatively, conjugation efficiency may be determined as described by Spycher et al. (Dual, Site-Specific Modification of Antibodies by Using Solid-Phase Immobilized Microbial Transglutaminase, ChemBioChem 2019 18(19):1923-1927) and analyzed according to Benjamin et al. (Thiolation of Q295: Site-Specific Conjugation of Hydrophobic Payloads without the Need for Genetic Engineering, Mol. Pharmaceutics 2019,16:2795-2807).

[0297] In certain embodiments, antibodies may be conjugated as described in Example 1. Briefly, native glycosylated monoclonal antibodies at 5 mg / mL may be incubated in 50 mM Tris pH 7.6 with microbial transglutaminase (MTG, Zedira) at a concentration of 5 U / mg antibody and 5 molar equivalents of the indicated linker payload at 37° C. for 24 hours on a rotating thermomixer.

[0298] In a particular embodiment, the present invention relates to a method according to the invention, wherein the microbial transglutaminase is derived from a Streptomyces species, in particular from Streptomyces mobaraensis.

[0299] That is, the microbial transglutaminase used in the method of the present invention may be derived from a Streptomyces species, in particular Streptomyces mobaraensis, and preferentially has 80% sequence identity to the native enzyme, and thus MTG may be the native enzyme or a genetically engineered variant of the native enzyme.

[0300] 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: 32. S. mobaraensis MTG variants with other amino acid sequences have been reported and are also encompassed by the present invention (SEQ ID NOs: 33 and 34).

[0301] In another embodiment, a microbial transglutaminase from Streptomyces ladakanum (formerly known as Streptoverticillium ladakanum) may be used. Streptomyces ladakanum transglutaminase (U.S. Patent No. 6,660,510 B2) has the amino acid sequence disclosed in SEQ ID NO:35.

[0302] Any of the above transglutaminases may have a modified sequence. In some embodiments, a transglutaminase having 80%, 85%, 90%, or 95% or more sequence identity with any one of SEQ ID NOs: 32 to 35 can be used.

[0303] Another suitable microbial transglutaminase is commercially available from Ajinomoto, called ACTIVA TG. Compared to the Zedira transglutaminase, ACTIVA TG is missing four amino acids at the N-terminus, but has similar activity.

[0304] Further microbial transglutaminases that may be used in the context of the present invention are disclosed in Kieliszek and Misiewicz (Folia Microbiol (Praha). 2014; 59(3): 241-250), WO 2015 / 191883 A1, WO 2008 / 102007 A1, and U.S. Patent Application Publication No. 2010 / 0143970, the contents of which are hereby incorporated by reference in their entireties.

[0305] In certain embodiments, mutant variants of microbial transglutaminase can be used for conjugating the linker to the antibody. That is, the microbial transglutaminase used in the method of the present invention can be a variant of S. mobaraensis transglutaminase as set forth in SEQ ID NO: 32 or 33. In certain embodiments, the recombinant S. mobaraensis transglutaminase as set forth in SEQ ID NO: 32 can comprise the mutation G254D. In certain embodiments, the recombinant S. mobaraensis transglutaminase as set forth in SEQ ID NO: 32 can comprise the mutations G254D and E304D. In certain embodiments, the recombinant S. mobaraensis transglutaminase as set forth in SEQ ID NO: 32 can comprise the mutations D8E and G254D. In certain embodiments, the recombinant S. mobaraensis transglutaminase as set forth in SEQ ID NO: 32 can comprise the mutations E124A and G254D. In certain embodiments, the recombinant S. mobaraensis transglutaminase set forth in SEQ ID NO: 32 may comprise the mutations A216D and G254D. In certain embodiments, the recombinant S. mobaraensis transglutaminase set forth in SEQ ID NO: 32 may comprise the mutations G254D and K331T.

[0306] In a particular embodiment, the invention relates to an antibody-linker conjugate produced by the method according to the invention.

[0307] That is, the present invention relates to an antibody-linker conjugate produced via any of the aforementioned processes.

[0308] Furthermore, the present invention relates to a pharmaceutical composition comprising the antibody-linker conjugate according to the present invention.

[0309] Thus, in a particular embodiment, the present invention relates to a pharmaceutical composition comprising an antibody-linker conjugate according to the invention, in particular, said antibody-linker conjugate comprising at least one payload and at least one pharma- ceutically acceptable component.

[0310] It is to be understood that the pharmaceutical composition may comprise the antibody-payload conjugates produced in the one-step or two-step process disclosed herein.

[0311] The type of payload contained in the antibody-payload construct contained in the pharmaceutical composition depends on the use of the pharmaceutical composition. In an embodiment in which the pharmaceutical composition is used for the treatment of a disease, the payload is preferably a drug. If the disease is a tumor disease, the payload is preferably a toxin. In an embodiment in which the pharmaceutical composition is used for diagnosis, the payload is preferably an imaging agent.

[0312] In a particular embodiment, the present invention relates to a pharmaceutical composition according to the present invention, which comprises at least one additional therapeutically active agent.

[0313] The pharmaceutical compositions according to the present invention may comprise at least one pharma- ceutically acceptable ingredient.

[0314] A pharma- ceutically acceptable ingredient refers to an ingredient, other than an active ingredient, in a pharmaceutical formulation that is non-toxic to a subject. Pharmaceutically acceptable ingredients include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0315] Pharmaceutical formulations of the antibody-linker conjugates described herein in the form of lyophilized formulations or aqueous solutions are prepared by mixing such conjugates having the desired purity with one or more optional pharma- ceutically acceptable ingredients (Flemington's Pharmaceutical Sciences 16th edition, Oslo, A. Ed. (1980)). Pharmaceutically acceptable ingredients are generally non-toxic to recipients at the dosages and concentrations used, and may include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexahexamethonium 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 (less than about 10 residues) polypeptides; Examples of suitable pharmacokinetic or pharmacokinetically acceptable components include, but are not limited to, peptides; 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 dextrin; 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 pharmacokinetically acceptable components herein further include interstitial drug dispersing agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), such as human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use thereof are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968.For example, a sHASEGP may be combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0316] In a particular embodiment, the present invention relates to an antibody-linker conjugate according to the invention, in particular wherein the antibody-linker conjugate comprises at least one payload, or a pharmaceutical composition according to the invention for use in therapy and / or diagnosis.

[0317] That is, the antibody-linker conjugate or pharmaceutical composition of the present invention can be used in the treatment of a subject or in the diagnosis of a disease or condition in a subject. The individual or subject is preferably a mammal. Mammals include, but are not limited to, livestock animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as macaques), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human. When the antibody-linker conjugate or pharmaceutical composition comprising the antibody-linker conjugate of the present invention is used for treatment, it is preferred that the linker comprises a drug. When the antibody-linker conjugate or pharmaceutical composition comprising the antibody-linker conjugate of the present invention is used for diagnosis, it is preferred that the linker comprises at least one imaging agent.

[0318] In a particular embodiment, the present invention relates to an antibody-linker conjugate according to the present invention, in particular where the antibody-linker conjugate comprises at least one payload, or to a method for treating a neoplastic disease, a neurological disease, an autoimmune disease, an inflammatory disease, or an infectious disease. suffer from, are at risk of developing and / or Diagnosed with A pharmaceutical composition according to the invention for use in treating a patient.

[0319] In a particular embodiment, the present invention relates to an antibody-linker conjugate according to the invention, in particular wherein the antibody-linker conjugate comprises at least one payload, or a pharmaceutical composition according to the invention for use in the treatment of a patient suffering from a neoplastic disease.

[0320] The term "neoplastic disease" as used herein refers to a condition characterized by uncontrolled abnormal growth of cells. Neoplastic diseases also 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, hepatocellular carcinoma, colorectal cancer, cervical cancer, endometrial cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, liver cancer, skin cancer, melanoma, brain tumor, ovarian cancer, neuroblastoma, myeloma, various head and neck cancers, 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.

[0321] That is, the antibody-linker conjugate of the present invention is preferably used for the treatment of cancer. Thus, in certain embodiments, the antibody-linker conjugate of the present invention comprises an antibody that specifically binds to an antigen present on a tumor cell. In certain embodiments, the antigen may be an antigen on the surface of a tumor cell. In certain embodiments, the antigen on the surface of a tumor cell may be translocated to the inside of the cell together with the antibody-linker conjugate when the antibody-linker conjugate binds to the antigen.

[0322] When the antibody-linker conjugate of the present invention is used to treat cancer, the antibody-linker conjugate preferably comprises at least one payload capable of killing or inhibiting the proliferation of tumor cells to which the antibody-linker conjugate binds. In certain embodiments, the at least one payload exhibits cytotoxic activity after the antibody-linker conjugate is internalized in the tumor cell. In certain embodiments, the at least one payload is a toxin.

[0323] The inflammatory disease may be an autoimmune disease. The infectious disease may be a bacterial or viral infection.

[0324] In certain embodiments, the antibody-linker conjugates and / or pharmaceutical compositions of the invention may be used in the treatment of B-cell related cancers.

[0325] Thus, in certain embodiments, the invention relates to an antibody-linker conjugate or a pharmaceutical composition for use according to the invention, wherein the antibody-linker conjugate comprised in the pharmaceutical composition comprises polatuzumab, and the neoplastic disease is a B-cell related cancer.

[0326] To this end, the antibody-linker conjugate preferably comprises an anti-CD79b antibody as disclosed herein, which preferably is internalized in a target cell upon binding to CD79b. In a particular embodiment, the anti-CD79b antibody is polatuzumab, having a heavy chain as set forth in SEQ ID NO: 36 and a light chain as set forth in SEQ ID NO: 37. Additionally, it is preferred that the antibody-linker conjugate comprises at least one toxin.

[0327] B-cell related cancers include high-, intermediate-, and low-grade lymphomas (e.g., B-cell lymphomas such as mucosa-associated lymphoid tissue B-cell lymphoma and non-Hodgkin's lymphoma (NHL), mantle cell lymphoma, Burkitt's lymphoma, small lymphocytic lymphoma, marginal zone lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, and Hodgkin's lymphoma, and T-cell lymphoma), and chronic lymphomas such as leukemias (secondary leukemia, B-cell leukemia (CD5+ B lymphocytes)). The cancer may be any one selected from the group consisting of myeloid leukemia (CLL), myeloid leukemias such as acute myeloid leukemia, chronic myeloid leukemia, lymphocytic leukemia such as acute lymphoblastic leukemia (ALL), and myelodysplasia, as well as other hematological cancers including cancers of additional hematopoietic cells including polymorphonuclear leukocytes such as basophils, eosinophils, neutrophils, and monocytes, dendritic cells, platelets, erythrocytes, and natural killer cells, and / or B-cell or T-cell associated cancers. Also included are cancerous B-cell proliferative disorders selected from: lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, recurrent aggressive NHL, recurrent indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle cell lymphoma.

[0328] In a particular embodiment, the invention relates to an antibody-linker conjugate or a pharmaceutical composition for use according to the invention, wherein the B cell related cancer is non-Hodgkin's lymphoma, in particular wherein the B cell related cancer is diffuse large B cell lymphoma.

[0329] Furthermore, the anti-CD79b antibody-linker conjugate and / or pharmaceutical composition comprising an anti-CD79b antibody-linker conjugate may be used in combination with other therapies suitable for the treatment of B-cell related cancers.

[0330] Thus, in certain embodiments, the invention relates to an antibody-linker conjugate or pharmaceutical composition for use according to the invention, wherein the antibody-linker conjugate or pharmaceutical composition is administered in combination with bendamustine and / or rituximab.

[0331] It is understood that the antibody-linker conjugate or pharmaceutical composition does not necessarily have to be administered simultaneously with additional therapeutic agents such as bendamustine and / or rituximab. Instead, the antibody-linker conjugate or pharmaceutical composition may be administered on a different dosing schedule, and therefore on different days, than other therapeutic agents used to treat the same disease.

[0332] In certain embodiments, the antibody-linker conjugates and / or pharmaceutical compositions of the invention can be used in the treatment of HER2-positive cancer.

[0333] Thus, in a particular embodiment, the present invention relates to an antibody-linker conjugate or pharmaceutical composition for use according to the present invention, wherein the antibody-linker conjugate comprised in the pharmaceutical composition comprises trastuzumab, and the neoplastic disease is HER2-positive cancer, in particular HER2-positive breast cancer, gastric cancer, ovarian cancer, or lung cancer.

[0334] To this end, the antibody-linker conjugate preferably comprises an anti-HER2 / neu antibody as disclosed herein, which preferably is internalized in a target cell upon binding to HER2 / neu. In a particular embodiment, the anti-HER2 / neu antibody is trastuzumab, having a heavy chain set forth in SEQ ID NO: 38 and a light chain set forth in SEQ ID NO: 39. Additionally, it is preferred that the antibody-linker conjugate comprises at least one toxin.

[0335] HER2 positive cancer, as used herein, may be, but is not limited to, HER2 positive breast cancer, gastric cancer, ovarian cancer, or lung cancer. Those skilled in the art can determine whether a cancer is a HER2 positive cancer. For example, tumor cells can be isolated by biopsy, and the presence of HER2 / neu can be determined by any method known in the art.

[0336] Additionally, the anti-HER2 / neu antibody-linker conjugate and / or pharmaceutical compositions comprising the anti-HER2 / neu antibody-linker conjugate may be used in combination with other therapies suitable for the treatment of HER2 cell positive cancers.

[0337] Thus, in certain embodiments, the invention relates to an antibody-linker conjugate or pharmaceutical composition for use according to the invention, wherein the antibody-linker conjugate or pharmaceutical composition is administered in combination with lapatinib, capecitabine, and / or a taxane.

[0338] It is understood that the antibody-linker conjugate or pharmaceutical composition does not necessarily have to be administered simultaneously with additional therapeutic agents such as lapatinib, capecitabine, and / or taxanes. Instead, the antibody-linker conjugate or pharmaceutical composition may be administered on a different dosing schedule, and therefore on different days, than other therapeutic agents used to treat the same disease.

[0339] In certain embodiments, the antibody-linker conjugates and / or pharmaceutical compositions according to the present invention can be used in the treatment of Nectin-4 positive cancers.

[0340] Thus, in a particular embodiment, the invention relates to an antibody-linker conjugate or a pharmaceutical composition for use according to the invention, wherein the antibody-linker conjugate comprised in the pharmaceutical composition comprises enfortumab or an enfortumab variant, and the neoplastic disease is a Nectin-4 positive cancer, in particular a Nectin-4 positive pancreatic cancer, lung cancer, bladder cancer, or breast cancer.

[0341] For this purpose, it is preferred that the antibody-linker conjugate comprises an anti-Nectin-4 antibody disclosed herein, which is preferably internalized in target cells upon binding to Nectin-4. In a particular embodiment, the anti-Nectin-4 antibody is Enfortumab having a heavy chain set forth in SEQ ID NO: 40 or SEQ ID NO: 42 and a light chain set forth in SEQ ID NO: 41. It is further preferred that the antibody-linker conjugate comprises at least one toxin.

[0342] Nectin-4 positive cancer, as used herein, may be, but is not limited to, Nectin-4 positive pancreatic cancer, lung cancer, bladder cancer, or breast cancer. Those skilled in the art can determine whether a cancer is a Nectin-4 positive cancer. For example, tumor cells can be isolated by biopsy, and the presence of Nectin-4 can be determined by any method known in the art.

[0343] Furthermore, the anti-Nectin-4 antibody-linker conjugate and / or the pharmaceutical composition comprising the anti-Nectin-4 antibody-linker conjugate may be used in combination with other therapies suitable for treating Nectin-4 cell-positive cancers.

[0344] Thus, in certain embodiments, the invention relates to an antibody-linker conjugate or pharmaceutical composition for use according to the invention, wherein the antibody-linker conjugate or pharmaceutical composition is administered in combination with a cisplatin-based chemotherapeutic agent and / or pembrolizumab.

[0345] It is understood that the antibody-linker conjugate or pharmaceutical composition does not necessarily have to be administered simultaneously with additional therapeutic agents, such as cisplatin-based chemotherapy and / or pembrolizumab. Instead, the antibody-linker conjugate or pharmaceutical composition may be administered on a different schedule, and therefore on different days, than other therapeutic agents used to treat the same disease.

[0346] In a particular embodiment, the present invention relates to the use of an antibody-linker conjugate according to the invention, in particular where the antibody-linker conjugate comprises at least one payload, or to a method for treating a neoplastic, neurological, autoimmune, inflammatory or infectious disease. suffer from, are at risk of developing and / or Diagnosed with The use of a pharmaceutical composition according to the invention for the manufacture of a medicament for treating a patient.

[0347] In a particular embodiment, the present invention relates to a method for treating or preventing a neoplastic disease, comprising administering to a patient in need thereof an antibody-linker conjugate according to the invention, in particular an antibody-linker conjugate comprising at least one payload, or a pharmaceutical composition according to the invention.

[0348] In a particular embodiment, the present invention relates to an antibody-linker conjugate according to the invention or a pharmaceutical composition according to the invention for use in pre-operative, intra-operative or post-operative imaging, in particular wherein the antibody-linker conjugate comprises at least one payload.

[0349] That is, the antibody-linker conjugate of the present invention can be used in medical imaging. To this end, the antibody-linker conjugate can be visualized while bound to a specific target molecule, cell, or tissue. To visualize a specific payload, various techniques are known in the art. For example, if the payload is a radionuclide, the molecule, cell, or tissue to which the antibody-linker 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-linker conjugate binds can be visualized by fluorescence imaging. In certain embodiments, the antibody-linker conjugate of 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-linker conjugate binds can be visualized using two different and / or complementary imaging techniques, such as PET / SPECT and fluorescence imaging.

[0350] The antibody-linker conjugates can be used for pre-operative, intra-operative, and / or post-operative imaging.

[0351] Preoperative imaging encompasses all imaging techniques that may be performed prior to surgery to visualize specific target molecules, cells, or tissues in diagnosing a particular disease or condition, and optionally to provide surgical guidance. Preoperative imaging may include visualizing tumors by PET or SPECT prior to surgery, using an antibody-linker conjugate that includes an antibody that specifically binds to an antigen on the tumor and is conjugated to a payload that includes a radionuclide.

[0352] Intraoperative imaging encompasses all imaging techniques that can be performed during surgery to visualize specific target molecules, cells, or tissues and provide surgical guidance. In certain embodiments, antibody-linker conjugates containing near-infrared fluorescent dyes can be used to visualize tumors during surgery by near-infrared fluorescent imaging. Intraoperative imaging allows surgeons to identify specific tissues, such as tumor tissues, during surgery, so that tumor tissues can be completely removed.

[0353] Post-operative imaging encompasses all imaging techniques that can be performed after surgery to visualize specific target molecules, cells, or tissues and evaluate the results of surgery. Post-operative imaging can be performed in the same manner as pre-operative surgery.

[0354] In certain embodiments, the present invention relates to an antibody-linker conjugate that comprises two or more different payloads. For example, the antibody-linker conjugate can comprise a radionuclide and a near-infrared fluorescent dye. Such an antibody-payload conjugate can be used for imaging by PET / SPECT and near-infrared fluorescence imaging. The advantage of such an antibody is that it can be used to visualize target tissue, such as tumor, before and after surgery by PET or SPECT. At the same time, tumor can also be visualized during surgery by near-fluorescence infrared imaging.

[0355] In a particular embodiment, the present invention relates to an antibody-linker conjugate according to the invention or a pharmaceutical composition according to the invention, in particular wherein the antibody-linker conjugate comprises at least one payload, for use in intraoperative image-guided cancer surgery.

[0356] As mentioned above, the antibody-linker conjugates of the present invention can be used to visualize target molecules, cells, or tissues and guide surgeons or robots during surgery, i.e., the antibody-linker conjugates can be used to visualize tumor tissue during surgery, for example by near-infrared imaging, allowing complete removal of the tumor tissue.

[0357] The antibody-linker conjugates or pharmaceutical compositions according to the invention may be administered to a human or animal subject in an amount or dosage sufficient to effectively treat a disease or for diagnostic purposes.

[0358] The antibody-linker conjugate or pharmaceutical composition of the present invention can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, as well as intralesional, intrauterine, or intravesical administration if localized treatment is desired. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing may be by any suitable route, for example, injection, such as intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. A variety of dosing schedules are contemplated herein, including, but not limited to, single or multiple doses over various time periods, bolus administration, and pulse infusion.

[0359] The antibody-linker conjugate or pharmaceutical composition of the present invention can be formulated, dosed, and administered in a manner consistent with the present invention. Factors to consider in this context include the specific disorder being treated, the specific 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-linker conjugate or pharmaceutical composition of the present invention is optionally, but not necessarily, formulated with one or more agents currently used to prevent or treat the disorder of interest. The effective amount of such other agents depends on the amount of antibody-linker 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 at about 1-99% of the dosages described herein, or at any dosage and by any route determined to be empirically / clinically appropriate.

[0360] The appropriate dosage of the antibody-linker conjugate or pharmaceutical composition of the invention (when used alone or in combination with one or more 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-linker conjugate is administered for prophylactic or therapeutic purposes, the medical history, the patient's clinical history and response to the antibody-linker conjugate, and the discretion of the attending physician. The antibody-linker conjugate or pharmaceutical composition of the invention is suitably administered to the patient at one time or over a series of treatments. EXAMPLES

[0361] general law The antibody trastuzumab (Herceptin®, Roche, purchased from a pharmacy), as well as the peptide-linker and linker-payload (custom synthesised by LifeTein and Levena Biopharma, respectively) were all commercially available. Polatuzumab, with heavy and light chains consisting of the sequences of SEQ ID NOs: 36 and 37, was transiently transfected into suspension-adapted CHO-K1 cells and expressed in serum-free / animal component-free medium. Protein was purified from the supernatant by Protein A affinity chromatography (Mab Select Sure column; GE Healthcare).

[0362] Conjugation reactions were performed by mixing native glycosylated monoclonal antibodies, microbial transglutaminase (MTG, Zedira), and the indicated peptide-linker or linker-payload in buffer in a rotating thermomixer. Conjugation efficiency was assessed by LC-MS under DTT reducing conditions. Reduction of samples was achieved by incubating them for 15 min at 37 °C in 50 mM DTT (final) and 50 mM Tris buffer. Probes were analyzed on an Acquity UPLC H-Class System (Waters) and a Xevo G2-XS QTOF (Waters) coupled to an ACQUITY UPLC BEH C18 column. Conjugation efficiency (CE) was calculated from the deconvoluted spectra and presented in %. For the calculation according to the following formula, the intensities obtained from both glycoforms (G1F and G0F) were considered.

[0363]

number

[0364] Wherein cj = conjugated, ncj = unconjugated.

[0365] Example 1: Identification of optimized reaction conditions method Reactions were carried out using two different sets of reaction conditions: Condition 1: native glycosylated trastuzumab antibody at 1 mg / mL in Tris 50 mM pH 7.6, MTG at a concentration of 6 U / mg, and 80 molar equivalents of the indicated peptide-linker or linker-payload in a rotating thermomixer for 20 hours at 37° C., or Condition 2: native glycosylated trastuzumab antibody at 5 mg / mL in Tris 50 mM pH 7.6, MTG at a concentration of 5 U / mg, and 5 molar equivalents of the indicated peptide-linker or linker-payload in a rotating thermomixer for 24 hours at 37° C. Conjugation efficiency was assessed by LCMS as described in General Methods.

[0366] result Surprisingly, superior conjugation efficiency was obtained when substantially fewer equivalents of peptide linker and linker payload (80 vs. 5 molar equivalents) and lower MTG concentrations (5 U / mg vs. 6 U / mg) were used, as shown in Table 3. Even more strikingly, when functionalized peptides or linker payloads were used, conjugation efficiency was significantly improved under condition 2, i.e., using 5 molar equivalents of peptide-linker or linker-payload instead of 80. This finding was true not only for different linkers, but also for different payload classes (three cytotoxins: MMAE, maytansine, and exatecan, and one steroid: cortisol), which is quite surprising.

[0367] [Table 3]

[0368] Example 2: Identification of further reaction conditions To demonstrate that conjugation with lysine-linker-payload tolerates a wide variety of reaction conditions, conjugation of the linker-payload to polatuzumab was performed using a variety of reaction conditions with varying parameters.

[0369] method Standard conditions used the following parameters: native glycosylated polatuzumab antibody at 5 mg / mL in Tris 50 mM pH 7.6, MTG at a concentration of 5 U / mg, and 5 molar equivalents of RKAA-PABC-MMAE in a rotating thermomixer at 37° C. for 24 hours.

[0370] The variable parameters are shown in Table 4.

[0371] Conjugation efficiency was assessed by LCMS as follows: Conjugation efficiency (CE) was calculated from the deconvoluted spectra and presented in %. For the calculation according to the following formula, the intensities obtained from both glycoforms (G1F and G0F) were taken into account.

[0372]

number

[0373] Wherein cj = conjugated, ncj = unconjugated.

[0374] result The RKAA-PABC-MMAE linker-payload was conjugated with very high conjugation efficiency over a very wide range of reaction conditions: over 80% conjugation efficiency was obtained with antibody concentrations ranging from 5 to 17 mg / mL and MTG concentrations relative to antibody concentration (U / mg) ranging from 2 to 10 U / mg. Furthermore, high conjugation efficiency was obtained with linker-to-antibody molarities (2 to 8 equiv.) and a very wide range of pH (conjugation efficiency of 67% at pH 6.0 to 86% at pH 8).

[0375] Surprisingly, lowering the linker payload excess relative to antibody resulted in higher conjugation efficiency, i.e., 2-20 equivalents of linker payload resulted in higher conjugation efficiency than 80 equivalents of linker payload, which was contrary to expectations (Table 4).

[0376] [Table 4]

[0377] Example 3: Identification of further reaction conditions To demonstrate the importance of linker concentration, additional linkers with different sequences and payloads were conjugated to the antibodies polatuzumab and trantuzumab at different concentrations.

[0378] method The following parameters were used as standard conditions: native glycosylated polatuzumab or trastuzumab antibody at 3.5 mg / mL in Tris 50 mM pH 7.6 and MTG at a concentration of 5 U / mg in a rotating thermomixer for 24 hours at 37° C. Linkers RKAA-PABC-MMAE, KAR-PABC-MMAE, and AHK-PABC-Exa were added to the reaction mixture at various concentrations.

[0379] The variable parameters are shown in Table 5.

[0380] Conjugation efficiency was evaluated by RPLC as follows: After reduction, samples were analyzed by UHPLC Dionex UltiMate 3000 (Thermo Fisher) using a BioResolve RP mAb Polyphenyl column. Conjugation efficiency (CE) was calculated using the relative peak areas extracted from the RPLC chromatograms according to the formula and is presented in %.

[0381]

number

[0382] Wherein cj = conjugated, ncj = unconjugated.

[0383] result For all linkers tested, the addition of 5–20 equivalents of linker payload to the reaction resulted in exceptionally high conjugation efficiencies.

[0384]

Table 5

Claims

1. 1. A method of producing an antibody-linker conjugate using transglutaminase, comprising the following structure (shown in the N→C orientation): (Sp １ ) - K - (Sp ２ ) - B - (Sp ３ ) or (Sp １ ) - B - (Sp ２ ) - K - (Sp ３ ) to a Gln residue contained in the antibody, ・(Sp １ ) is a chemical spacer or is absent; ・(Sp ２ ) is a chemical spacer or is absent; ・(Sp ３ ) is a chemical spacer or is absent; K is lysine, or a lysine derivative or lysine mimetic; B is a linking moiety or payload; the linker is conjugated to a Gln residue in the antibody via a primary amine in the side chain of the lysine residue, lysine derivative, or lysine mimetic; the antibody is contacted with less than 80 molar equivalents of the linker; method.

2. The Gln residue to which the linker is conjugated is C of an IgG antibody. Ｈ The method of claim 1, wherein the Gln residue Q295 (EU numbering) in domain 2 is Gln residue Q295 (EU numbering).

3. The IgG antibody is a glycosylated IgG antibody, and in particular, the IgG antibody is Ｈ The method of claim 2, wherein the IL-2 domain is glycosylated at residue N297 (EU numbering).

4. The method of any one of claims 1 to 3, wherein the antibody is contacted with 20 molar equivalents or less than 20 molar equivalents of the linker.

5. The method of any one of claims 1 to 3, wherein the antibody is contacted with 2 to 20 molar equivalents of the linker.

6. The method of any one of claims 1 to 3, wherein the antibody is added to the conjugation reaction at a concentration ranging from 1 to 50 mg / mL.

7. 4. The method of claim 1, wherein the transglutaminase is added to the conjugation reaction at a concentration ranging from 1 to 20 U / mg antibody.

8. 4. The method of any one of claims 1 to 3, wherein conjugation of the linker to the antibody is achieved at a pH in the range of 6 to 8.

5.

9. The chemical spacer (Sp １ ), (Sp ２ ), and (Sp ３ 4. The method of claim 1, wherein each of the following independently contains 0 to 12 amino acid residues:

10. 4. The method of any one of claims 1 to 3, wherein the linker comprises no more than 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 amino acid residues.

11. The method of any one of claims 1 to 3, wherein the net charge of the linker is neutral or positive.

12. The method of any one of claims 1 to 3, wherein the linker does not contain any negatively charged amino acid residues.

13. The method of claim 1 , wherein B is a linking moiety.

14. The connecting portion B is a bioorthogonal marker group, or Non-bio-orthogonal entities for crosslinking 14. The method of claim 13, comprising:

15. the bioorthogonal marker group or the non-bioorthogonal entity for crosslinking is -N-N≡N or -N ３ ; ・Lys (N) ３ ); tetrazine; Alkynes; - strained cyclooctyne; - BCN; Strained alkenes; photoreactive groups; ·aldehyde; acyltrifluoroborates; - proteolytic agents ("PROTACs"); Cyclopentadiene / spirolocyclopentadiene; Thioselective electrophiles; -SH; and ・Cysteine 15. The method of claim 14, consisting of or comprising at least one molecule or moiety selected from the group consisting of:

16. The method of any one of claims 13 to 15, comprising the further step of conjugating one or more payloads to said linking moiety B.

17. 17. The method of claim 16, wherein the one or more payloads are conjugated to the linking moiety B via a Click reaction.

18. The method of any one of claims 1 to 3, wherein B is a payload.

19. The payload is ·toxin; Cytokines; ・Growth factors; - Radionuclides; ·hormone; - antiviral agents; - antibacterial agents; fluorescent dyes; - immunomodulatory / immunostimulatory agents; Half-life extending moieties; - solubility-enhancing moieties; Polymer-toxin conjugates; ・Nucleic acid; a biotin or streptavidin moiety; ·vitamin; - proteolytic agents ("PROTACs"); a target binding moiety; and / or Anti-inflammatory agents 17. The method of claim 16, comprising at least one of:

20. The payload is ·toxin; Cytokines; ・Growth factors; - Radionuclides; ·hormone; - antiviral agents; - antibacterial agents; fluorescent dyes; - immunomodulatory / immunostimulatory agents; Half-life extending moieties; - solubility-enhancing moieties; Polymer-toxin conjugates; ・Nucleic acid; a biotin or streptavidin moiety; ·vitamin; - proteolytic agents ("PROTACs"); a target binding moiety; and / or Anti-inflammatory agents 20. The method of claim 17, comprising at least one of:

21. The payload, ·toxin; Cytokines; ・Growth factors; - Radionuclides; ·hormone; - antiviral agents; - antibacterial agents; fluorescent dyes; - immunomodulatory / immunostimulatory agents; Half-life extending moieties; - solubility-enhancing moieties; Polymer-toxin conjugates; ・Nucleic acid; a biotin or streptavidin moiety; ·vitamin; - proteolytic agents ("PROTACs"); a target binding moiety; and / or Anti-inflammatory agents 20. The method of claim 18, comprising at least one of:

22. The toxin is - pyrrolobenzodiazepines (e.g. PBD); - auristatins (e.g., MMAE, MMAF); maytansinoids (e.g., maytansine, DM1, DM4, DM21); Duocarmycin; - nicotinamide phosphoribosyltransferase (NAMPT) inhibitors; - Tubulysin; enediynes (e.g., calicheamicin); - anthracycline derivatives (PNU) (e.g., doxorubicin); - Pyrrole-based kinesin spindle protein (KSP) inhibitors; Cryptophycin; - drug efflux pump inhibitors; Sandramycin; amanitin (e.g., α-amanitin); and Camptothecins (e.g., exatecan, deruxtecan) 20. The method of claim 19, wherein the at least one selected from the group consisting of:

23. The toxin of claim 1, - pyrrolobenzodiazepines (e.g. PBD); - auristatins (e.g., MMAE, MMAF); maytansinoids (e.g., maytansine, DM1, DM4, DM21); Duocarmycin; - nicotinamide phosphoribosyltransferase (NAMPT) inhibitors; - Tubulysin; enediynes (e.g., calicheamicin); - anthracycline derivatives (PNU) (e.g., doxorubicin); - Pyrrole-based kinesin spindle protein (KSP) inhibitors; Cryptophycin; - drug efflux pump inhibitors; Sandramycin; amanitin (e.g., α-amanitin); and Camptothecins (e.g., exatecan, deruxtecan) 21. The method of claim 20, wherein the at least one selected from the group consisting of:

24. The toxin of claim 1, - pyrrolobenzodiazepines (e.g. PBD); - auristatins (e.g., MMAE, MMAF); maytansinoids (e.g., maytansine, DM1, DM4, DM21); Duocarmycin; - nicotinamide phosphoribosyltransferase (NAMPT) inhibitors; - Tubulysin; enediynes (e.g., calicheamicin); - anthracycline derivatives (PNU) (e.g., doxorubicin); - Pyrrole-based kinesin spindle protein (KSP) inhibitors; Cryptophycin; - drug efflux pump inhibitors; Sandramycin; amanitin (e.g., α-amanitin); and Camptothecins (e.g., exatecan, deruxtecan) 22. The method of claim 21, wherein the at least one selected from the group consisting of:

25. The chemical spacer (Sp ２ 10. The method of claim 1, wherein said polymerizable compound comprises a self-immolative moiety.

26. 26. The method of claim 25, wherein the self-immolative moiety is directly attached to the payload B.

27. 27. The method of claim 25 or 26, wherein the self-immolative moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety or a self-immolative aminomethylene spacer.

28. The method according to any one of claims 1 to 3, wherein the antibody is an IgG antibody, in particular an IgG1 antibody.

29. 4. The method of any one of claims 1 to 3, wherein the antibody is selected from the group consisting of brentuximab, trastuzumab, gemtuzumab, inotuzumab, avelumab, cetuximab, rituximab, daratumumab, pertuzumab, vedolizumab, ocrelizumab, tocilizumab, ustekinumab, golimumab, obinutuzumab, sacituzumab, belantamab, polatuzumab, and enfortumab.

30. The method of any one of claims 1 to 3, wherein the linker is conjugated to the γ-carboxamide group of a Gln residue contained in the antibody.

31. 4. The method of claim 1, wherein the linker is suitable for conjugation to a glycosylated antibody with a conjugation efficiency of at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%.

32. 4. The method according to any one of claims 1 to 3, wherein the transglutaminase is a microbial transglutaminase, preferably derived from a Streptomyces species, in particular Streptomyces mobaraensis.

33. An antibody-linker conjugate produced by the method of claim 1.

34. A pharmaceutical composition comprising the antibody-linker conjugate of claim 33.

35. 35. The pharmaceutical composition of claim 34, comprising at least one additional therapeutically active agent.

36. 36. A composition comprising the antibody-linker conjugate of claim 33 or a pharmaceutical composition according to claim 34 or 35, in particular wherein said antibody-linker conjugate comprises at least one payload, for use in therapy and / or diagnosis.

37. Neoplastic disease, neurological disease, autoimmune disease, inflammatory disease, or infectious disease - Suffering from are at risk of developing, and / or -Diagnosed with 36. A composition comprising the antibody-linker conjugate of claim 33, or a pharmaceutical composition according to claim 34 or 35, in particular wherein said antibody-linker conjugate comprises at least one toxin, for use in the treatment of a patient.

38. 38. A composition or pharmaceutical composition comprising the antibody-linker conjugate of claim 37, wherein the antibody-linker conjugate comprises polatuzumab and the neoplastic disease is a B-cell-related cancer.

39. 39. A composition or pharmaceutical composition comprising the antibody-linker conjugate of claim 38, wherein the B cell related cancer is non-Hodgkin's lymphoma, particularly wherein the B cell related cancer is diffuse large B cell lymphoma.

40. 38. A composition or pharmaceutical composition comprising the antibody-linker conjugate of claim 37, wherein the antibody-linker conjugate comprises trastuzumab and the neoplastic disease is HER2-positive cancer, in particular HER2-positive breast cancer, gastric cancer, ovarian cancer, or lung cancer.

41. 38. A composition or pharmaceutical composition comprising the antibody-linker conjugate of claim 37, wherein the antibody-linker conjugate comprises enfortumab or an enfortumab variant, and the neoplastic disease is Nectin-4 positive cancer, particularly Nectin-4 positive pancreatic cancer, lung cancer, bladder cancer, or breast cancer.