Peptide linkers containing two or more payloads

Abstract

The present invention relates to a peptide linker comprising (a) an amino acid residue comprising a primary amine and (b) two or more payloads, each of which can be independently attached to (i) the N-terminus of the peptide linker, (ii) the C-terminus of the peptide linker, or (iii) a side chain of an amino acid residue contained in the peptide linker. The present invention further relates to an antibody-payload conjugate comprising the peptide linker of the present invention, a method for making the antibody-payload conjugate, and uses thereof.

Description

[Background technology]

[0001] The present invention relates to a method for producing an antibody-payload conjugate using transglutaminase. The present invention further provides a peptide linker comprising two or more payloads for producing the antibody-payload conjugate. Also included are pharmaceutical compositions comprising the antibody-payload conjugate of the present invention and uses thereof.

[0002] Antibody-based therapeutics play an important role in the targeted treatment of various diseases, such as cancer and immune disorders. In recent years, antibody-drug conjugates (ADCs) have been widely studied to effectively deliver drugs to target sites. While many ADCs have shown 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 at reasonable cost in sufficient quantity and quality to support drug development, 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 conjugating to endogenous lysine or cysteine ​​residues of antibodies, carefully controlling the average degree of modification to achieve an average drug-to-antibody ratio (DAR) in the range of 3.5-4.0. More recently, ADCs with DARs of 7-8 have shown significantly improved efficacy, due to the delivery of much more toxic payloads to tumor sites (Ogitani et al., 2016. Clin Cancer Res, 22(20):5097-5108).

[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, a 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). Thus, transglutaminase (TGase) transfers moieties bearing an amine donor group to an acceptor glutamine residue by transglutamination.

[0005] Full-length IgG antibodies of human isotype contain a conserved glutamine residue (Q295) at position 295 of the heavy chain. This glutamine 295 residue is close to the N-glycosylation site (N297), so it was generally believed that Q295 of full-length antibodies would be inaccessible to TGase when the antibody is N-glycosylated. To allow TGase to act on full-length antibodies, the Fc region of the antibody was deglycosylated or mutated to remove the N-glycosylation site before TGase-mediated conjugation. However, Jeger et al. described that conjugation of antibodies using transglutaminase as an enzyme occurred at the Q295 residue, and conjugation was only possible when the glycan moiety at asparagine residue 297 (N297) was removed with PNGase F, but 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] Alternatively, glutamine-containing sequence "tags" have been inserted into the light or heavy chains of antibodies to provide acceptor glutamine sites (see, e.g., WO 2012 / 059882).Historically, therefore, site-specific ADC technology has relied on artificial antibody mutants that can lead to immunogenicity and in vivo instability.

[0007] Hu and Allen found that conjugation could be achieved at Q295 of a natively glycosylated antibody using an artificial transglutaminase (WO 2015 / 191883). Indeed, the authors showed that conjugation efficiency was higher with the artificial transglutaminase compared to wild-type transglutaminase.

[0008] More recently, Spycher et al. disclosed a wild-type 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 and WO 2019 / 188061). Surprisingly, Spycher et al. were able to demonstrate high conjugation efficiency using lysine- or glycine-based linkers.

[0009] In summary, Hu and Allen (WO 2015 / 191883) and Spycher et al. (WO 2019 / 057772 and WO 2020 / 188061) describe two-step and one-step conjugation approaches. However, neither Hu and Allen nor Spycher et al. have experimentally demonstrated the conjugation of a linker containing two or more payloads to a native glycosylated antibody in one step. In contrast, it was reasoned that a two-step process was required to obtain a DAR4 ADC, in which a linker containing two functional groups is conjugated to the antibody in the first step, and then the payload is chemically coupled to the antibody-linker conjugate in the second step. Specifically, it was hypothesized that direct conjugation of a linker containing two or more payloads to an antibody in one step would be inefficient due to steric hindrance in the binding pocket of transglutaminase. Another concern was that linkers containing two or more payloads have been reported to be less soluble and more prone to aggregation.

[0010] More recently, a research group reported the preparation of DAR4 ADCs using wild-type transglutaminase (Yamazaki et al. 2021, Nat Comm). The synthesis of the ADC required both antibody reengineering (removing the glycan moiety at asparagine residue 297 by mutating N297 to alanine) and a two-step chemoenzymatic approach, further demonstrating the difficulty of obtaining DAR4 ADCs in a simple one-step process.

[0011] From the viewpoint of manufacturing, one-step process is obviously preferred.Unfortunately, the efficient conjugation of native glycosylated antibody with a linker that comprises two or more payloads has not been achieved so far.As a result, there is a need in the art for a linker that comprises two or more payloads that can be efficiently conjugated to two native glycosylated antibodies.

[0012] Thus, the technical problem addressed by the present invention can be to provide a linker containing two or more payloads for efficient conjugation to native glycosylated antibodies. Summary of the Invention

[0013] The present invention is characterized in the embodiments and claims provided herein. In particular, the present invention relates inter alia to the following embodiments: 1. a) an amino acid residue containing a primary amine; b) Two or more payloads A peptide linker comprising: each of the two or more payloads independently; i) the N-terminus of the peptide linker; ii) at the C-terminus of the peptide linker, or iii) Side chains of amino acid residues contained in the peptide linker A peptide linker that can be attached to

[0014] 2. The primary amine contained in the amino acid residue is a) a primary amine in the side chain of a lysine, lysine derivative, or lysine mimetic; or b) A primary amine contained in the N-terminal amino acid residue having the structure NH2-(Y)-COOH. 2. The peptide linker of embodiment 1,

[0015] 3. Y is (R2C)n and n is an integer ranging from 1 to 20, 1 to 15, 1 to 10.

[0016] 4. The peptide linker of embodiment 3, wherein at least one R moiety of each -(R2C)- monomer is hydrogen, or both R moieties of each -(R2C)- monomer are hydrogen.

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

[0018] 6. A peptide linker according to any one of embodiments 1 to 5, wherein the linker comprises at least one arginine and / or histidine residue.

[0019] 7. A peptide linker according to any one of embodiments 1 to 6, wherein the linker comprises the sequence motif RK.

[0020] 8. A peptide linker according to any one of embodiments 1 to 7, wherein the linker comprises any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 29.

[0021] 9. A peptide linker according to any one of embodiments 1 to 8, wherein the linker comprises two to four payloads.

[0022] 10. A peptide linker described in any one of embodiments 1 to 9, wherein at least one of the two or more payloads is attached to the peptide linker via a chemical linker.

[0023] 11. The peptide linker of embodiment 10, wherein the chemical linker is an enzymatically and / or chemically cleavable linker.

[0024] 12. A peptide linker according to embodiment 10 or 11, wherein the chemical linker is or comprises a self-immolative linker.

[0025] 13. The self-immolative linker comprises: a) a p-aminobenzyl alcohol moiety; or b) a 2,4-bis(hydroxymethyl)aniline moiety; or c) p-aminobenzyl quaternary ammonium; or d) an ethylenediamine-based moiety; or e) an (aminomethyl)pyrrolidine-based moiety; or f) Aminomethyl-based moieties 13. The peptide linker of embodiment 12, comprising:

[0026] 14. The peptide linker of embodiment 13, wherein the hydroxyl group contained in the p-aminobenzyl alcohol moiety forms a carbamate with the payload.

[0027] 15. The peptide linker of embodiment 13, wherein each of the hydroxyl groups contained in the 2,4-bis(hydroxymethyl)aniline moiety forms a carbamate with the payload.

[0028] 16. The peptide linker of embodiment 13, wherein the quaternary ammonium cation contained in the p-aminobenzyl quaternary ammonium originates from an amine contained in the payload.

[0029] 17. The peptide linker of embodiment 13, wherein an amino group contained in the ethylenediamine-based moiety or the (aminomethyl)pyrrolidine-based moiety forms a carbamate with a payload.

[0030] 18. The peptide linker of embodiment 13, wherein the amino group contained in the aminomethyl-based moiety forms a hemiaminal or thiohemiaminal with the payload.

[0031] 19. A peptide linker according to any one of embodiments 1 to 18, wherein at least one payload is attached to a side chain of an amino acid residue contained in the peptide linker.

[0032] 20. The peptide linker of embodiment 19, wherein at least one payload is attached to a side chain of a glutamic acid residue, an aspartic acid residue, a tryptophan residue, a cysteine ​​residue, a lysine residue, a tyrosine residue, a serine residue, or a threonine residue, or a derivative or mimetic of each thereof.

[0033] 21. A peptide linker according to any one of embodiments 1 to 20, wherein the peptide linker comprises two peptide moieties, the two peptide moieties being connected via their N-terminal amino acid residues with a dicarboxylic acid linker or an activated version thereof.

[0034] 22. The payload is: ·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; biotin or streptavidin moieties; ·vitamin; ·Proteolytic agents (“PROTACs”); Ligands or substrates for receptors; a target binding moiety; and / or Anti-inflammatory 22. The peptide linker according to any one of embodiments 1 to 21, wherein the peptide linker is at least one of:

[0035] 23. 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; Thymidylate synthase inhibitors; Amanitin (e.g., α-amanitin); and Camptothecins (e.g., exatecan, deruxtecan) 23. The peptide linker of embodiment 22, which is at least one selected from the group consisting of:

[0036] 24. A peptide linker described in any one of embodiments 1 to 23, wherein the two or more payloads are identical.

[0037] 25. A peptide linker described in any one of embodiments 1 to 23, wherein at least two of the two or more payloads are different from each other.

[0038] 26. A peptide linker according to any one of embodiments 1 to 25, wherein the linker is suitable to function as a substrate for transglutaminase.

[0039] 27. An antibody-payload conjugate comprising an antibody conjugated to a peptide linker described in any one of embodiments 1 to 26.

[0040] 28. The antibody-payload conjugate of embodiment 27, wherein the peptide linker is conjugated to the antibody via an isopeptide bond formed between the γ-carboxamide group of a glutamine residue contained in the antibody and the primary amine contained in an amino acid residue of the peptide linker.

[0041] 29. The antibody-payload conjugate of embodiment 27 or 28, wherein the antibody is an IgG antibody.

[0042] 30. The antibody-payload conjugate of embodiment 29, wherein the peptide linker is conjugated to a glutamine residue contained in the Fc domain of the antibody.

[0043] 31. The glutamine residue to which the peptide linker is conjugated is C of an IgG antibody. H 31. The antibody-payload conjugate of embodiment 30, wherein the glutamine residue Q295 (EU numbering) of domain 2 is

[0044] 32. An antibody-payload conjugate according to embodiment 29, wherein the glutamine residue to which the peptide linker is conjugated has been introduced into the heavy or light chain of the antibody by molecular engineering.

[0045] 33. The glutamine residue introduced into the heavy or light chain of the antibody by molecular engineering is C of an aglycosylated IgG antibody. H 33. The antibody-payload conjugate of embodiment 32, which is N297Q (EU numbering) of the 2 domains.

[0046] 34. The antibody-payload conjugate of embodiment 32, wherein the glutamine 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.

[0047] 35. The antibody-payload conjugate of embodiment 34, wherein the peptide comprising the Gln residue is fused to the C-terminus of the heavy chain of the antibody.

[0048] 36. The antibody-payload conjugate of any one of embodiments 29 to 32 or 34 to 35, wherein the IgG antibody is a glycosylated IgG antibody.

[0049] 37. The IgG antibody is H The antibody-payload conjugate of embodiment 36, which is glycosylated at residue N297 (EU numbering) of domain 2.

[0050] 38. 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, enfortumab, endrecolomab, gemtuzumab, loncastuximab, mecbotamab, adecatuzumab, D93, gatipotuzumab, and la. 38. The antibody-payload conjugate according to any one of embodiments 27 to 37, selected from the group consisting of betuzumab, tusamitamab, upifitamab, rifastuzumab, mirvetuximab, sofituzumab, anetumab, tisotumab, cofituzumab, pralzatamab, radriatuzumab, belantamab, patritumab, cetuximab, nimotuzumab, matuzumab, portuzumab, sitatuzumab, tucotuzumab, and endrecolomab.

[0051] 39. The antibody-payload conjugate of any one of embodiments 27 to 38, wherein the antibody is selected from the group consisting of brentuximab, gemtuzumab, trastuzumab, inotuzumab, polatuzumab, enfortumab, sacituzumab, and belantamab.

[0052] 40. The antibody-payload conjugate of any one of embodiments 27 to 39, wherein the antibody is polatuzumab, or trastuzumab, or enfortumab.

[0053] 41. A method for preparing an antibody-payload conjugate, comprising the step of conjugating a peptide linker described in any one of embodiments 1 to 26 to an antibody.

[0054] 42. A method for conjugating a peptide linker comprising two or more payloads to an antibody using transglutaminase (TG), comprising: a) mixing the antibody, the peptide linker, and the TG in a fluid, thereby conjugating the linker-payloads to the antibody in one step under the catalytic action of the TG; and b) extracting the conjugate obtained in step a) from the fluid.

[0055] 43. The method of embodiment 42, wherein the peptide linker is a peptide linker described in any one of embodiments 1 to 26.

[0056] 44. The method of embodiment 42 or 43, wherein the peptide linker is conjugated to a glutamine residue in the antibody via a primary amine in an amino acid residue of the peptide linker.

[0057] 45. The method of any one of embodiments 41 to 44, wherein the antibody is an antibody fragment.

[0058] 46. ​​The method of any one of embodiments 41 to 44, wherein the antibody is an IgA, IgD, IgE, IgG, or IgM antibody.

[0059] 47. A method according to any one of embodiments 41 to 46, wherein the peptide linker is conjugated to a glutamine residue contained in the Fc domain of the antibody.

[0060] 48. The glutamine residue to which the peptide linker is conjugated is C of an IgG antibody. H The method of any one of embodiments 41 to 47, wherein the glutamine residue Q295 (EU numbering) of the 2 domain is

[0061] 49. A method according to any one of embodiments 41 to 47, wherein the glutamine residue to which the peptide linker is conjugated has been introduced into the heavy or light chain of the antibody by molecular engineering.

[0062] 50. The glutamine residue introduced into the heavy or light chain of the antibody by molecular engineering is C of an aglycosylated IgG antibody. H 50. The method of embodiment 49, wherein the 2 domain is N297Q (EU numbering).

[0063] 51. The method of embodiment 50, wherein the glutamine residue introduced into the heavy or light chain of the antibody by molecular engineering is (a) incorporated into the heavy or light chain of the antibody or (b) contained in a peptide fused to the N-terminus or C-terminus of the heavy or light chain of the antibody.

[0064] 52. The method of embodiment 51, wherein the peptide containing the Gln residue is fused to the C-terminus of the heavy chain of the antibody.

[0065] 53. The method of any one of embodiments 41-49 or 51-52, wherein the antibody is a glycosylated IgG antibody.

[0066] 54. The IgG antibody is H 54. The method of embodiment 53, wherein the IL-2 domain is glycosylated at residue N297 (EU numbering).

[0067] 55. 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, enfortumab, endrecolomab, gemtuzumab, loncastuximab, mecbotamab, adecatuzumab, D93, and gatipo. The method according to any one of embodiments 41 to 54, wherein the agent is selected from the group consisting of tuzumab, labetuzumab, tusamitamab, upifitamab, rifastuzumab, mirvetuximab, sofituzumab, anetumab, tisotumab, cofituzumab, pralzatamab, radriatuzumab, belantamab, patritumab, cetuximab, nimotuzumab, matuzumab, portuzumab, sitatuzumab, tucotuzumab, and endrecolomab.

[0068] 56. The method of any one of embodiments 41 to 55, wherein the antibody is selected from the group consisting of brentuximab, gemtuzumab, trastuzumab, inotuzumab, polatuzumab, enfortumab, sacituzumab, and belantamab.

[0069] 57. The method of any one of embodiments 41 to 56, wherein the antibody is polatuzumab, or trastuzumab, or enfortumab.

[0070] 58. The method of any one of embodiments 41 to 57, wherein the peptide linker is conjugated to the γ-carboxamide group of a Gln residue contained in the antibody.

[0071] 59. The method of any one of embodiments 41 to 58, wherein the peptide 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%.

[0072] 60. The method of any one of embodiments 41 to 59, wherein the transglutaminase is microbial transglutaminase (MTG).

[0073] 61. The method of embodiment 60, wherein the microbial transglutaminase is derived from a Streptomyces species, in particular Streptomyces mobaraensis.

[0074] 62. The method of any one of embodiments 41 to 61, wherein the antibody is contacted with 2 to 100 molar equivalents of the linker.

[0075] 63. A method according to any one of embodiments 41 to 62, wherein the antibody is added to the conjugation reaction at a concentration of 0.1 to 50 mg / mL.

[0076] 64. The method of any one of embodiments 41 to 63, wherein the transglutaminase is added to the conjugation reaction at a concentration of less than 200 U / mg antibody.

[0077] 65. A method according to any one of embodiments 41 to 64, wherein the conjugation reaction is carried out in a buffered solution.

[0078] 66. The buffer solution is a) a pH in the range of 5 to 10; and / or b) a buffer concentration in the range of 10 to 1000 mM; and / or c) Salt concentrations in the range of less than 250 mM 66. The method of embodiment 65, comprising:

[0079] 67. An antibody-payload conjugate produced by a method according to any one of embodiments 41 to 66.

[0080] 68. A pharmaceutical composition comprising an antibody-payload conjugate according to any one of embodiments 27 to 40 or embodiment 67, and at least one pharma- ceutically acceptable ingredient.

[0081] 69. The pharmaceutical composition of embodiment 68, comprising at least one additional therapeutically active agent.

[0082] 70. An antibody-payload conjugate according to any one of embodiments 27 to 40 or embodiment 67 or a pharmaceutical composition according to embodiment 68 or 69, for use in therapy and / or diagnosis.

[0083] 71. Neoplastic, neurological, autoimmune, inflammatory, or infectious diseases suffer from, are at risk of developing and / or Diagnosed with An antibody-payload conjugate according to any one of embodiments 27 to 40 or embodiment 67, or a pharmaceutical composition according to embodiment 68 or 69, for use in the treatment of a patient.

[0084] 72. The antibody-payload conjugate or pharmaceutical composition for use according to embodiment 71, wherein the antibody-payload conjugate comprises polatuzumab and the neoplastic disease is a B-cell-related cancer.

[0085] 73. The antibody-payload conjugate or pharmaceutical composition for use according to embodiment 72, wherein the B cell-related cancer is non-Hodgkin's lymphoma, in particular, the B cell-related cancer is diffuse large B cell lymphoma.

[0086] 74. An antibody-payload conjugate or pharmaceutical composition for use according to embodiment 72 or 73, administered in combination with bendamustine and / or rituximab.

[0087] 75. The antibody-payload conjugate or pharmaceutical composition for use according to embodiment 71, wherein the antibody-payload 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.

[0088] 76. The antibody-payload conjugate or pharmaceutical composition for use according to embodiment 75, administered in combination with lapatinib, capecitabine, and / or a taxane.

[0089] 77. The antibody-payload conjugate or pharmaceutical composition for use according to embodiment 71, wherein the antibody-payload 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.

[0090] 78. The antibody-payload conjugate or pharmaceutical composition for use according to embodiment 77, administered in combination with a platinum-based chemotherapy agent and / or pembrolizumab.

[0091] 79. Neoplastic, neurological, autoimmune, inflammatory, or infectious diseases suffer from, are at risk of developing and / or Diagnosed with Use of an antibody-payload conjugate according to any one of embodiments 27 to 40 or embodiment 67 or a pharmaceutical composition according to embodiment 68 or 69 for the manufacture of a medicament for treating a patient.

[0092] 80. A method for treating or preventing a neoplastic disease, comprising administering to a patient in need thereof an antibody-payload conjugate described in any one of embodiments 27 to 40 or embodiment 67 or a pharmaceutical composition described in embodiment 68 or 69. [Brief description of the drawings]

[0093] [Figure 1] Chemical structure of the linker Ac-RKAA-PABC-(MMAE)2. [Diagram 2] Chemical structure of the linker Ac-RKAA-PABC-PABC-(MMAE)2. [Diagram 3] Chemical structure of the linker MMAE-PABC-AA-C2-RKAA-PABC-MMAE. [Figure 4] Chemical structure of the linker Ac-RKAA-PABC-(Exa)2. [Diagram 5] Chemical structure of the linker Ac-ARK-PABC-(Exa)2. [Figure 6] Chemical structure of the linker Ac-RKARA-PABC-(Exa)2. [Figure 7] Chemical structure of the linker Ac-RKAAAA-PABC-(Exa)2. [Figure 8] Chemical structure of the linker Ac-RKAAAAAA-PABC-(Exa)2. [Figure 9] Chemical structure of the linker Ac-RKAASGSG-PABC-(Exa)2. [Figure 10] Chemical structure of the linker Ac-RKHA-PABC-(Exa)2. [Figure 11] Chemical structure of the linker Ac-RKHAAA-PABC-(Exa)2. [Figure 12] Chemical structure of the linker Ac-HKA-PABC-(Exa)2. [Figure 13] Chemical structure of the linker Ac-RKAA-PABC-(G-Exa)2. [Figure 14] Chemical structure of the linker Exa-PABC-AA-C2-RKAA-PABC-Exa. [Figure 15] Chemical structure of the linker GGR-PABC-(Exa)2. [Figure 16] Chemical structure of the linker GGRG-PABC-(G-Exa)2. [Figure 17] Chemical structure of the linker Ac-RKAA-PABC-(G-Exa')2. [Figure 18] Chemical structure of the linker GGRG-PABC-(G-Exa')2. [Figure 19] Chemical structure of the linker Ac-E(A-PABC-MMAE)ARKAA-PABC-(MMAE)2. [Figure 20] Chemical structure of the linker (MMAE)2-PABC-AA-C2-RKAA-PABC-(MMAE)2. [Figure 21] Chemical structure of the linker Exa-PABC-AA-C2-RKAA-PABC-(MMAE)2. [Figure 22] Chemical structure of the linker May-C5-RKAE(A-PABC-MMAE)A-EDA-Cortisol. [Diagram 23] Chemical structure of the linker RhKAA-PABC-(MMAE)2. [Figure 24] Chemical structure of the linker NH2-C5-GRG-PABC-(MMAE)2. [Diagram 25] Chemical structure of the linker RKVCit-PABC-PABC-(MMAE)2. [Figure 26] Chemical structure of the linker Exa-PABC-RA-C3-RKAR-PABC-MMAE. [Figure 27] Cryptophycin-AA-C2-RKVA-Cyptophycin chemical structure. [Figure 28] Chemical structure of the linker KAR-PABC-EDA-BHMC-(MMAF)2. [Figure 29] Chemical structure of the linker RK-E(PEG12-FA)AA-PABC-MMAE. [Diagram 30] Chemical structure of the linker E(AA-AM-Dxd)RKAA-AM-Dxd. [Diagram 31] Chemical structure of the linker cRGD-PEG4-RKAH-PABC-EDA-PNU. [Diagram 32] Chemical structure of linker-biotin-RKAN-PABQ-Rifalog. [Diagram 33] Chemical structure of the linker May-RKGGFG-PABC-AMP-AE. [Diagram 34] Chemical structure of the linker resiquimod-CitV-C2-RKGP-STING. [Diagram 35] Chemical structure of the linker C(May)-RKAA-AM-May. [Diagram 36]Chemical structure of the linker K(SMCC-May)-RKAA-(ValCit-PABC-MMAE)2. [Figure 37] Chemical structure of the linker S(Glyco)-RKAA-(AA-PABC-MMAE)2. [Figure 38] Chemical structure of the linker Exa-gluc-C3-RK-C3-gluc-Exa. [Figure 39] Chemical structure of the linker D(AA-AM-Dxd)-D(AA-AM-Dxd)-RKAA-AM-Dxd. [Diagram 40] Chemical structure of the linker (E(AA-PABC-G-Dxd))2RKVCit-PABC-PABC-(G-Dxd)2. [Diagram 41] Chemical structure of the linker NH2-PEG2-PABC-(MMAE)2. [Diagram 42] Chemical structure of the linker MMAE-PABC-AA-C2-KAR-PABC-MMAE. [Diagram 43] Chemical structure of the linker RKN(PABC-MMAE)A-PABC-MMAE. [Diagram 44] Antitumor efficacy of two DAR4 linkers of the present invention compared to the benchmark antibody enfortumab vedotin in a Nectin-4 positive solid tumor model. [Diagram 45] Antitumor efficacy of two DAR4 linkers of the present invention in a CD79b-positive liquid tumor model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0094] That is, the present invention is based, at least in part, on the surprising finding that a peptide linker comprising two or more payloads can be efficiently conjugated to a natively glycosylated antibody. As can be seen from the accompanying examples, a peptide linker comprising two or more payloads can be conjugated to a natively glycosylated antibody in a single reaction step with a very high efficiency of at least 60%. Moreover, a peptide linker comprising two payloads can be conjugated to a natively glycosylated antibody in a single reaction step with an efficiency of 80-100%.

[0095] To the inventors' surprise, the peptide linkers according to the invention have been found to be particularly well suited for the one-step conjugation of ADCs with a DAR > 4, in contrast to non-peptide linkers (such as amino-PEG linkers known in the art; see Example 9), which achieved conjugation efficiencies of less than 30%.

[0096] Even more surprising, high conjugation efficiency was achieved for all peptide linkers. It is important to note that quantitative conjugation of linkers containing two or more payloads to native glycosylated antibodies in one step has never been reported. In this light, it must be considered even more surprising that peptide linkers containing two or more bulky payloads can be conjugated to native glycosylated antibodies with such high efficiency.

[0097] In addition to these extremely high conjugation efficiencies, the present inventors have found that antibodies conjugated with the peptide linkers of the present invention have in vivo activity (see Example 11). Furthermore, it has been surprisingly found that antibodies conjugated with peptide linkers containing payloads at their N-terminus and C-terminus have stronger antitumor activity than the benchmark antibody enfortumab vedotin.

[0098] In a specific embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: a) an amino acid residue containing a primary amine; b) Two or more payloads A peptide linker comprising: each of the two or more payloads independently; i) the N-terminus of the peptide linker; ii) at the C-terminus of the peptide linker, or iii) Side chains of amino acid residues contained in the peptide linker The present invention relates to a peptide linker that can be attached to

[0099] Thus, the present invention relates to a peptide linker comprising two or more payloads covalently attached to the peptide moiety. In the prior art, a two-step approach to generate antibody-payload conjugates with a drug-to-antibody (DAR) ratio >4 has been envisaged, in which in a first step a linker comprising two reactive groups is conjugated to a glycosylated antibody by microbial transglutaminase, and in a second step a payload molecule is attached to the reactive groups contained in the antibody-linker conjugate (WO 2019 / 057772 and WO 2015 / 191883). In particular, due to the limited accessibility to the endogenous conjugation site Q295 and the limited space of the substrate binding pocket of transglutaminase, it has been considered difficult to conjugate a linker comprising a bulky payload such as a toxin to a native glycosylated antibody in a single reaction step. Despite these known difficulties, the inventors have surprisingly found that peptide linkers of the invention comprising two or more payloads can be conjugated to glycosylated antibodies with extremely high conjugation efficiency.

[0100] A "peptide linker" in the sense of the present invention is a molecule comprising at least two amino acid residues, which are coupled via a peptide bond. It is assumed that the peptide linker is suitable as a substrate for microbial transglutaminase. In particular, it is assumed that the peptide linker is suitable for conjugation with glutamine residues contained in antibodies. Therefore, the peptide linker according to the present invention must contain at least one amino acid residue containing a primary amine.

[0101] Thus, in a specific embodiment, the present invention provides a method for preparing a cyclic amine comprising administering to a subject an amino acid residue comprising the steps of: a) a primary amine in the side chain of a lysine, lysine derivative, or lysine mimetic; or b) relates to a peptide linker according to the invention which is a primary amine contained in the N-terminal amino acid residue having the structure NH2-(Y)-COOH.

[0102] That is, in a preferred embodiment, the amino acid residue containing a primary amine is a lysine residue. In such an embodiment, the peptide linker comprises a peptide moiety that includes at least one lysine residue.

[0103] However, linkers according to the invention may also comprise lysine mimetics or derivatives, provided that the lysine mimetics or derivatives contain a free primary amine in the amino acid side chain.

[0104] In certain embodiments, the amino acid residue containing a primary amine 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 characteristics to lysine and can therefore 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, homolysine, or 2,7-diaminoheptanoic acid (exemplary linkers containing homolysine are shown in Figure 23). In certain embodiments, a lysine mimetic may be a β-amino acid, such as β-homolysine.

[0105] In certain embodiments, the amino acid residue containing primary amine 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 glutamine residues in proteins. Thus, the "lysine derivative" contained in the peptide linker of the present invention preferably contains a modified or substituted α-amino group and / or α-carboxyl group.

[0106] In certain embodiments, the primary amine contained in the amino acid residue may be a primary amine contained in the N-terminal amino acid residue having the structure NH2-(Y)-COOH.

[0107] In certain embodiments, the primary amine may be the α-amino group of an α-amino acid, which may be any proteinogenic α-amino acid, including alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine.

[0108] In particularly preferred embodiments, the primary amine may be the α-amino group of a glycine residue. In such embodiments, it is preferred that the glycine residue is the N-terminal amino acid residue of the peptide linker, such that the α-amino group is available for conjugation to the glycine residue via microbial transglutaminase.

[0109] The amino acid containing a primary amine may be a non-standard or synthetic amino acid. 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 may be found in naturally occurring peptides and / or proteins. A "synthetic amino acid", as used herein, may be any molecule that falls under the general definition of an amino acid (NH2-(Y)-COOH), i.e., that contains an amino group and a carboxyl group, but is not found in nature. Thus, non-natural amino acids are preferably obtained by chemical synthesis. It should be understood that the distinction between non-standard and synthetic amino acids may be uncertain in some cases. For example, an amino acid defined as a synthetic amino acid may be identified in nature at a later time and reclassified as a non-standard amino acid. A non-standard or synthetic amino acid may be an α-, β-, γ-, δ-, or ε-amino acid.

[0110] In certain embodiments, an amino acid containing a primary amine may have the structure NH2-(Y)-COOH.

[0111] In certain embodiments, the moiety Y may comprise a carbon comprising a 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 comprising the backbone is a linear hydrocarbon or comprises 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) obtained from any chain-growth or step-growth polymerization process.

[0112] Y may further include 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 CH2-CH2-O-) 1-24 , or (CH2) x1 -(CH2-O-CH2) 1-24 -(CH2) x2 - group, where x1 and x2 are independently an integer selected from the range of 0 to 20, an amino acid, an oligopeptide, a glycan, a sulfate, a phosphate, or a carboxylate. 2-6 It may contain alkyl groups.

[0113] In certain embodiments, the present invention provides a method for the preparation of a compound according to the present invention, wherein Y is -(RC)n -, and n is an integer ranging from 1 to 20, 1 to 15, or 1 to 10. That is, Y can have the structure:

[0114] [ka]

[0115] In certain embodiments, Y can be a substituted or unsubstituted alkyl or alkenyl chain. It is understood that when Y is a substituted or unsubstituted alkenyl chain, there must be at least two R moieties attached to consecutive carbon molecules.

[0116] The term "substituted alkyl," as used herein, generally refers to an alkyl group having an additional group or groups attached to any carbon of the alkyl group. That is, a substituted alkyl has the structure -(RC) n where each R can independently be hydrogen or a functional group such as alkyl, lower alkyl, aryl, acyl, halogen, alkylhalo, hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy, aryloxy, aryloxyalkyl, mercapto, both saturated and unsaturated cyclic hydrocarbons, heterocycles, and other organic groups.

[0117] In certain embodiments, an amino acid containing a primary amine has the structure NH2-(Y)-COOH, where Y is -(RC) n -, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In certain embodiments, nAt least one, two, three, four, or five of the moieties R in - may be a functional group such as alkyl, lower alkyl, aryl, acyl, halogen, alkylhalo, hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy, aryloxy, aryloxyalkyl, mercapto, both saturated and unsaturated cyclic hydrocarbons, heterocycles, and other organic groups.

[0118] In a specific embodiment, the invention relates to a peptide linker according to the invention, wherein at least one R moiety of each -(RC)- monomer is hydrogen.

[0119] That is, in certain embodiments, one R moiety of each -(R2C)-monomer may be hydrogen, while the other R moiety may be a functional group such as alkyl, lower alkyl, aryl, acyl, halogen, alkylhalo, hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy, aryloxyalkyl, mercapto, both saturated and unsaturated cyclic hydrocarbons, heterocycles, and other organic groups. Alternatively, one R moiety of each -(R2C)-monomer may be hydrogen, while the other R moiety may be absent (in the case of alkenes). In certain embodiments, some -(R2C)-monomers in the moiety Y may contain two hydrogen substituents, and some -(R2C)-monomers in the same moiety Y may contain one hydrogen substituent and one substituent R as defined herein.

[0120] In a specific embodiment, the invention relates to a peptide linker according to the invention, wherein both R moieties of each -(RC)- monomer are hydrogen.

[0121] In certain embodiments, the structure -(RC) n - is the structure - (R2C) n - may be an unsubstituted alkyl chain in which all moieties R are hydrogen atoms. That is, in certain embodiments, the structure -(RC) n- can be a methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, or octadecyl group.

[0122] That is, in certain embodiments, an amino acid containing a primary amine has the structure NH2-(Y)-COOH, where Y is -(CH2) n -, where n is an integer from 1 to 20. In certain embodiments, an amino acid containing a primary amine may have the structure NH2-(Y)-COOH, where Y is -(CH2) n -, where n is an integer from 1 to 15, and in certain embodiments, an amino acid containing a primary amine can have the structure NH2-(Y)-COOH, where Y is -(CH2) n -, where n is an integer from 1 to 10, and in certain embodiments, an amino acid containing a primary amine can have the structure NH2-(Y)-COOH, where Y is -(CH2) n -, where n is an integer from 1 to 9, and in certain embodiments, an amino acid containing a primary amine can have the structure NH2-(Y)-COOH, where Y is -(CH2) n -, where n is an integer from 1 to 8, and in certain embodiments, an amino acid containing a primary amine can have the structure NH2-(Y)-COOH, where Y is -(CH2) n -, where n is an integer from 1 to 7, and in certain embodiments, an amino acid containing a primary amine can have the structure NH2-(Y)-COOH, where Y is -(CH2) n -, where n is an integer from 1 to 6.

[0123] In certain embodiments, Y is the structure -(CH) n - where n is 1. That is, in certain embodiments, the amino acid containing a primary amine may be glycine.

[0124] In certain embodiments, Y is the structure -(CH)n - where n is 2. That is, in certain embodiments, the amino acid comprising a primary amine may be β-alanine.

[0125] In certain embodiments, Y is the structure -(CH) n - where n is 3. That is, in certain embodiments, the amino acid containing a primary amine may be 4-aminobutyric acid.

[0126] In certain embodiments, Y is the structure -(CH) n - (wherein n is 4). That is, in certain embodiments, the amino acid containing a primary amine may be 5-aminopentanoic acid. (An exemplary linker containing 5-aminopentanoic acid is shown in Figure 24.)

[0127] In certain embodiments, Y is the structure -(CH) n - where n is 5. That is, in certain embodiments, the amino acid containing a primary amine may be 6-aminohexanoic acid.

[0128] In certain embodiments, Y is the structure -(CH) n - where n is 6. That is, in certain embodiments, the amino acid containing a primary amine may be 7-aminoheptanoic acid.

[0129] In certain embodiments, Y is the structure -(CH) n - where n is 7. That is, in certain embodiments, the amino acid containing a primary amine may be 8-aminooctanoic acid.

[0130] In certain embodiments, Y is the structure -(CH) n - where n is 8. That is, in certain embodiments, the amino acid containing a primary amine may be 9-aminononanoic acid.

[0131] In certain embodiments, Y is the structure -(CH) n - where n is 9. That is, in certain embodiments, the amino acid containing a primary amine may be 10-aminodecanoic acid.

[0132] In certain embodiments, Y is the structure -(CH) n - where n is 10. That is, in certain embodiments, the amino acid containing a primary amine may be 11-aminoundecanoic acid.

[0133] In certain embodiments, the amino acid containing a primary amine has the structure NH2-(CH2) n -X-(CH2) n -COOH, where X is a substituted or unsubstituted alkyl or heteroalkyl chain, and n is an integer from 0 to 20, 0 to 10, or 0 to 6.

[0134] That is, in certain embodiments, an amino acid containing a primary amine has the structure NH2-(CH2) n -X-COOH, where X is a substituted or unsubstituted alkyl or heteroalkyl chain and n is an integer from 1 to 20, 1 to 10, or 1 to 6.

[0135] In certain embodiments, the amino acid containing a primary amine has the structure NH2-X-(CH2) n -COOH, where X is a substituted or unsubstituted alkyl or heteroalkyl chain, and n is an integer from 1 to 20, 1 to 10, or 1 to 6.

[0136] In a preferred embodiment, the amino acid comprising a primary amine comprises at least one methylene group (CH2). More preferably, at least one methylene group is directly coupled to the primary amine. That is, the amino acid comprising a primary amine preferably comprises the structure NH2-CH2-.

[0137] In a preferred embodiment, the present invention relates to a method for preparing a cyclic amine having a primary amine group comprising the steps of: a) a primary amine in the side chain of a lysine, lysine derivative, or lysine mimetic; or b) Structure NH2-(CH2) n The peptide linker according to the present invention is a primary amine contained in the N-terminal amino acid residue having -COOH (wherein n is an integer ranging from 1 to 10).

[0138] In certain embodiments, the payload is attached to the N-terminus of the peptide linker. In such embodiments, the primary amine contained in the amino acid residue is preferably a primary amine in the side chain of a lysine, lysine derivative, or lysine mimetic, more preferably a primary amine in the side chain of a lysine residue.

[0139] In a particular embodiment, the invention relates to a peptide linker according to the invention, wherein the linker comprises no more than 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 amino acid residues.

[0140] The peptide linker of the present invention preferably comprises at least two amino acid residues and not more than 25 amino acid residues. In a preferred embodiment, all amino acid residues contained in the peptide linker of the present invention form a single peptide. However, it should be understood that the peptide linker may comprise two or more peptide moieties. For example, in certain embodiments, the peptide linker may comprise two peptide moieties, which are covalently connected to each other, but not by a peptide bond. Examples of such peptide linkers are further provided below.

[0141] In a particular embodiment, the invention relates to a peptide linker according to the invention, wherein the net charge of the linker is neutral or positive.

[0142] 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, Lys, and 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 containing charged functional groups, one skilled in the art can calculate the net charge at neutral pH accordingly.

[0143] In certain embodiments, the payload may also contribute to the net charge of the linker, however, one of skill in the art will know how to calculate the net charge of the entire linker, including any payload, preferably at neutral pH (7.0).

[0144] In certain embodiments, the net charge of the peptide linker is calculated based only on the amino acid residues contained in the linker, including amino acid mimetics and amino acid derivatives. Thus, in a specific embodiment, the present invention relates to a peptide linker according to the present invention, in which the net charge of the amino acid residues contained in the peptide linker is neutral or positive.

[0145] In a particular embodiment, the invention relates to a peptide linker according to the invention, wherein the linker does not contain any negatively charged amino acid residues.

[0146] That is, the linker may not contain negatively charged amino acid residues, including negatively charged 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 carries a negative charge at neutral pH (7.0). Negatively charged standard 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.

[0147] It should be noted that the peptide linker of the present invention may contain one or more glutamic or aspartic acid residues, however, it is preferred that the carboxyl group contained in the side chain of the aspartic or glutamic acid is coupled to the payload.

[0148] In a specific embodiment, the invention relates to a peptide linker according to the invention, wherein the linker comprises at least one positively charged amino acid residue.

[0149] In certain embodiments, the peptide linker comprises a positively charged lysine residue, which provides a primary amine for conjugation to the antibody via transglutaminase. However, it is preferred herein that the peptide linker comprises at least one additional positively charged amino acid. The additional positively charged amino acid may be a standard amino acid residue, such as arginine or histidine. However, the additional positively charged amino acid may also be a non-standard amino acid.

[0150] In a specific embodiment, the invention relates to a peptide linker according to the invention, wherein the linker comprises at least one arginine residue.

[0151] It has been demonstrated herein that the linker containing arginine residue can be conjugated to glycosylated antibody with high efficiency.Therefore, it is preferred herein that the peptide linker according to the present invention contains at least one arginine residue.It should be noted that the arginine residue can also be replaced with arginine mimic or arginine derivative.

[0152] The arginine residue may be located at any position of the peptide linker. In certain embodiments, the arginine residue is adjacent to an amino acid residue that contains a primary amine. In certain embodiments, the arginine residue is coupled to the N-terminus of an amino acid that contains a primary amine, i.e., a lysine residue, a lysine mimic, or a lysine derivative (e.g., an RK motif). In certain embodiments, the arginine residue is coupled to the C-terminus of an amino acid that contains a primary amine, i.e., a lysine residue, a lysine mimic, or a lysine derivative (e.g., a KR motif). In certain embodiments, the arginine residue is coupled to an amino acid that contains a primary amine, i.e., a lysine residue, a lysine mimic, or a lysine derivative, via another amino acid residue, preferably an alanine residue (KAR or RAK motif). In certain embodiments, the peptide linker comprises an arginine residue and a histidine residue.

[0153] In a specific embodiment, the invention relates to a peptide linker according to the invention, wherein the linker comprises at least one histidine residue.

[0154] It has been demonstrated herein that a linker containing histidine residue can be conjugated to glycosylated antibody with high efficiency.Therefore, it is preferred herein that the peptide linker according to the present invention contains at least one histidine residue.It should be noted that the histidine residue can also be replaced with a histidine mimic or histidine derivative.

[0155] The histidine residue may be located at any position of the peptide linker. In certain embodiments, the histidine residue is adjacent to an amino acid residue that contains a primary amine. In certain embodiments, the histidine residue is coupled to the N-terminus of an amino acid that contains a primary amine, i.e., a lysine residue, a lysine mimic, or a lysine derivative (e.g., an HK motif). In certain embodiments, the histidine residue is coupled to the C-terminus of an amino acid that contains a primary amine, i.e., a lysine residue, a lysine mimic, or a lysine derivative (e.g., a KH motif). In certain embodiments, the histidine residue is coupled to an amino acid that contains a primary amine, i.e., a lysine residue, a lysine mimic, or a lysine derivative, via another amino acid residue, preferably an alanine residue (KAH or HAK motif). In certain embodiments, the peptide linker comprises a histidine residue and an arginine residue.

[0156] In a particular embodiment, the invention relates to a peptide linker according to the invention, wherein the linker comprises the sequence motif RK.

[0157] The present inventors have shown that peptide linkers containing the sequence motif RK (arginyl-lysyl) can be conjugated to glycosylated antibodies with extremely high efficiency, even when the linker contains two or more payloads. It should be understood that the lysine residue contained in the RK motif contains a primary amine through which the peptide linker is conjugated to the glutamine residue contained in the antibody. That is, the lysine residue contained in the RK motif is preferably an amino acid containing a primary amine.

[0158] As used herein, it is preferred that the motif RK consists of the amino acids arginine and lysine, however, it is understood that the arginine and / or lysine residues may be replaced with arginine mimetics / derivatives and / or lysine mimetics / derivatives.

[0159] That is, in certain embodiments, the motif RK may comprise an arginine mimetic. The term "arginine mimetic" as used herein refers to a compound that has a different structure from arginine but similar characteristics to arginine and can therefore be used to replace arginine in a peptide or protein without significantly changing the function and / or structure of the peptide or protein. An arginine mimetic may differ from arginine in the length or composition of the aliphatic chain connecting the guanidino group to the α-carbon atom. Alternatively or in addition, an arginine mimetic may differ from arginine in the guanidino group itself. That is, an arginine mimetic may include a functional group that has similar physicochemical properties to the guanidino group. In certain embodiments, an arginine mimetic may be homoarginine, 2-amino-3-guanidino-propionic acid, β-ureidoalanine, or citrulline.

[0160] In certain embodiments, the motif RK may comprise an arginine derivative. The term "arginine derivative" as used herein refers to an arginine or arginine mimetic in which one or more functional groups contained in the arginine or arginine mimetic are modified or substituted. The arginine derivative may be an arginine or arginine mimetic in which the guanidino group is substituted or modified. In certain embodiments, the arginine derivative may be ω-methylarginine. In embodiments, when the residue R is located at the N-terminal position of the linker, R may be an arginine derivative in which the α-amino group is modified or substituted. In certain embodiments, the α-amino group of the arginine or arginine mimetic may be acetylated.

[0161] In certain embodiments, motif RK may comprise a lysine mimetic or a lysine derivative as defined elsewhere herein.

[0162] In certain embodiments, motif RK may include lysine mimetics / derivatives and arginine mimetics / derivatives.

[0163] In certain embodiments, the lysine residue, or the lysine mimic or lysine derivative may be separated from the arginine residue, or the arginine mimic or arginine derivative by only one amino acid residue. That is, the peptide linker of the present invention may comprise the sequence motif RXK or KXR, where X may be any amino acid. In a preferred embodiment, the lysine residue, or the lysine mimic or lysine derivative may be separated from the arginine residue, or the arginine mimic or arginine derivative by an alanine residue. That is, the peptide linker of the present invention may comprise the sequence motif RAK or KAR. It was demonstrated in Example 10 that the linker containing the sequence motif KAR can be conjugated to a glycosylated antibody with extremely high conjugation efficiency.

[0164] In a particular embodiment, the invention relates to a peptide linker according to the invention, wherein the linker comprises the sequence motif HK.

[0165] The present inventors have shown that peptide linkers containing the sequence motif HK (histidyl-lysyl) can be conjugated to glycosylated antibodies with very high efficiency, even when the linker contains two or more payloads (e.g., Example 3). It should be understood that the lysine residue contained in the HK motif contains a primary amine through which the peptide linker is conjugated to the glutamine residue contained in the antibody. That is, the lysine residue contained in the HK motif is preferably an amino acid containing a primary amine.

[0166] It is preferred herein that the motif HK consists of the amino acids histidine and lysine, however it is understood that the histidine and / or lysine residues may be replaced by histidine mimetics / derivatives and / or lysine mimetics / derivatives.

[0167] That is, in certain embodiments, the motif HK may comprise a histidine mimetic. The term "histidine mimetic" as used herein refers to a compound that has a different structure from histidine but has similar characteristics to histidine and can therefore be used to replace histidine in a peptide or protein without significantly changing the function and / or structure of the peptide or protein. A histidine mimetic may differ from histidine in the length or composition of the aliphatic chain connecting the imidazole group to the α-carbon atom. Alternatively or in addition, a histidine mimetic may differ from histidine in the imidazole group itself. That is, a histidine mimetic may comprise a functional group that has similar physicochemical properties to an imidazole group. In certain embodiments, a histidine mimetic may be a homohistidine.

[0168] In certain embodiments, the motif HK may comprise a histidine derivative. The term "histidine derivative" as used herein refers to a histidine or histidine mimetic in which one or more functional groups contained in the histidine or histidine mimetic are modified or substituted. The histidine derivative may be a histidine or histidine mimetic in which the imidazole group is substituted or modified. In embodiments, when the residue H is located at the N-terminal position of the linker, H may be a histidine derivative in which the α-amino group is modified or substituted. In certain embodiments, the α-amino group of the histidine or histidine mimetic may be acetylated.

[0169] In certain embodiments, the motif HK may comprise a lysine mimetic or a lysine derivative as defined elsewhere herein.

[0170] In certain embodiments, the motif HK may include lysine mimetics / derivatives and histidine mimetics / derivatives.

[0171] In certain embodiments, the lysine residue, or the lysine mimic or lysine derivative may be separated from the arginine residue, or the arginine mimic or arginine derivative by only one amino acid residue. That is, the peptide linker of the present invention may comprise the sequence motif HXK or KXH, where X may be any amino acid. In a preferred embodiment, the lysine residue, or the lysine mimic or lysine derivative may be separated from the arginine residue, or the arginine mimic or arginine derivative by an alanine residue. That is, the peptide linker of the present invention may comprise the sequence motif HAK or KAH.

[0172] In a specific embodiment, the present invention relates to a peptide linker according to the present invention, wherein the linker comprises any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 29 or 82 to 93.

[0173] That is, the peptide linker according to the present invention may contain any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 29 or 82 to 93.

[0174] That is, in certain embodiments, the peptide linker may comprise the peptide sequence RKAA (SEQ ID NO: 1). Several linkers comprising the sequence RKAA are shown herein (see Figures 1, 2, 3, 4, 13, 14, 17, 20, 21, 30, 35, 36, 37, and 39). Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKAA.

[0175] In certain embodiments, the peptide linker may comprise the peptide sequence ARK (SEQ ID NO: 2). A linker comprising SEQ ID NO: ARK is illustrated in Figure 5. Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide ARK.

[0176] In certain embodiments, the peptide linker may comprise the peptide sequence RKARA (SEQ ID NO: 3). A linker comprising the sequence RKARA is illustrated in Figure 6. Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKARA.

[0177] In certain embodiments, the peptide linker may comprise the peptide sequence RAAAA (SEQ ID NO: 4). A linker comprising the sequence RAAAA is illustrated in Figure 7. Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RAAAA.

[0178] In certain embodiments, the peptide linker may comprise the peptide sequence RKAAAAAA (SEQ ID NO: 5). A linker comprising the sequence RKAAAAAA is illustrated in Figure 8. Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKAAAAAA.

[0179] In certain embodiments, the peptide linker may comprise the peptide sequence RKAASGSG (SEQ ID NO: 6). A linker comprising the sequence RKAASGSG is illustrated in Figure 9. Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKAASGSG.

[0180] In certain embodiments, the peptide linker may comprise the peptide sequence RKHA (SEQ ID NO: 7). A linker comprising the sequence RKHA is illustrated in Figure 10. Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKHA.

[0181] In certain embodiments, the peptide linker may comprise the peptide sequence RKHAAA (SEQ ID NO: 8). A linker comprising the sequence RKHAAA is illustrated in Figure 11. Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKHAAA.

[0182] In certain embodiments, the peptide linker may comprise the peptide sequence GGR (SEQ ID NO: 9). A linker comprising the sequence GGR is illustrated in Figure 15. Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide GGR.

[0183] In certain embodiments, the peptide linker may comprise the peptide sequence GGRG (SEQ ID NO: 10). Linkers comprising the sequence GGRG are illustrated in Figures 16 and 18. Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide GGRG.

[0184] In certain embodiments, the peptide linker may comprise the peptide sequence EARKAA (SEQ ID NO: 11). A linker comprising the sequence EARKAA is illustrated in FIG. 19. Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide EARKAA. Furthermore, it is preferred that one or more payloads are attached to the side chain of a glutamic acid residue. It should be understood that when an amine-containing payload is attached to the side chain of a glutamic acid residue, the peptide sequence of the linker can also be considered as QARKAA (SEQ ID NO: 84).

[0185] In certain embodiments, the peptide linker may comprise the peptide sequence RKAEA (SEQ ID NO: 12). A linker comprising the sequence RKAEA is illustrated in FIG. 22. Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKAEA. Furthermore, it is preferred that one or more payloads are attached to the side chain of a glutamic acid residue. It should be understood that when an amine-containing payload is attached to the side chain of a glutamic acid residue, the peptide sequence of the linker can also be considered as RKAQA (SEQ ID NO: 85).

[0186] In certain embodiments, the peptide linker may comprise the peptide sequence HKA (SEQ ID NO: 13). A linker comprising the sequence HKA is illustrated in Figure 12. Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide HKA.

[0187] In certain embodiments, the peptide linker may comprise the peptide sequence RhKAA (SEQ ID NO: 14). A linker comprising the sequence RhKAA is illustrated in Figure 23. Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RhKAA.

[0188] In certain embodiments, the peptide linker comprises the peptide sequence XGRG (SEQ ID NO: 15), where X is the structure NH2-(CH2) n -COOH, where n is an integer between 1 and 20, preferably between 1 and 10. A linker comprising the sequence XGRG is illustrated in Figure 24. Preferably, one or more payloads are attached to the C-terminus of the peptide XGRG.

[0189] In certain embodiments, the peptide linker may comprise the peptide sequence RKVCit (SEQ ID NO: 16). A linker comprising the sequence RKVCit is illustrated in Figure 25. Preferably, one or more payload peptides are attached to the N-terminus and / or C-terminus of the peptide sequence RKVCit.

[0190] In certain embodiments, the peptide linker may comprise the peptide sequence RKAR (SEQ ID NO: 17). A linker comprising the sequence RKAR is illustrated in Figure 26. Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKAR.

[0191] In certain embodiments, the peptide linker may comprise the peptide sequence RKVA (SEQ ID NO: 18). A linker comprising the sequence RKVA is illustrated in Figure 27. Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKVA.

[0192] In certain embodiments, the peptide linker may comprise the peptide sequence KAR (SEQ ID NO: 19). Linkers comprising SEQ ID NO: KAR are illustrated in Figures 28 and 42. Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide KAR.

[0193] In certain embodiments, the peptide linker may comprise the peptide sequence RKEAA (SEQ ID NO: 20). A linker comprising the sequence RKEAA is illustrated in FIG. 29. Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKEAA. Furthermore, it is preferred that one or more payloads are attached to the side chain of a glutamic acid residue. It is to be understood that when an amine-containing payload is attached to the side chain of a glutamic acid residue, the peptide sequence of the linker can also be considered as RKQAA (SEQ ID NO: 86).

[0194] In certain embodiments, the peptide linker may comprise the peptide sequence RKDA (SEQ ID NO: 82). A linker comprising the sequence RKDA is illustrated in FIG. 43. Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKDA. Furthermore, it is preferred that one or more payloads are attached to the side chain of an aspartic acid residue. It should be understood that when an amine-containing payload is attached to the side chain of an aspartic acid residue, the peptide sequence of the linker can also be considered as RKNA (SEQ ID NO: 83).

[0195] In certain embodiments, the peptide linker may comprise the peptide sequence ERKAA (SEQ ID NO: 21). A linker comprising the sequence ERKAA is illustrated in FIG. 30. Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide ERKAA. Furthermore, it is preferred that one or more payloads are attached to the side chain of a glutamic acid residue. It is to be understood that when an amine-containing payload is attached to the side chain of a glutamic acid residue, the peptide sequence of the linker can also be considered as QRKAA (SEQ ID NO: 87).

[0196] In certain embodiments, the peptide linker may comprise the peptide sequence RKAH (SEQ ID NO: 22). A linker comprising the sequence RKAH is illustrated in Figure 31. Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKAH.

[0197] In certain embodiments, the peptide linker may comprise the peptide sequence RKAN (SEQ ID NO: 23). A linker comprising the sequence RKAN is illustrated in Figure 32. Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKAN.

[0198] In certain embodiments, the peptide linker may comprise the peptide sequence RKGGFG (SEQ ID NO: 24). A linker comprising the sequence RKGGFG is illustrated in Figure 33. Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKGGFG.

[0199] In certain embodiments, the peptide linker may comprise the peptide sequence RKGP (SEQ ID NO: 25). A linker comprising the sequence RKGP is illustrated in Figure 34. Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide RKGP.

[0200] In certain embodiments, the peptide linker may comprise the peptide sequence KRKAA (SEQ ID NO: 26). A linker comprising the sequence KRKAA is illustrated in Figure 36. Preferably, the one or more payloads are attached to the N-terminus and / or C-terminus of the peptide KRKAA. Furthermore, it is preferred that the one or more payloads are attached to one of the lysine residues, preferably to the side chain of the N-terminal lysine residue.

[0201] In certain embodiments, the peptide linker may comprise the peptide sequence SRKAA (SEQ ID NO: 27). A linker comprising the sequence SRKAA is illustrated in Figure 37. Preferably, the one or more payloads are attached to the N-terminus and / or C-terminus of the peptide SRKAA. Furthermore, it is preferred that the one or more payloads are attached to the side chain of a serine residue.

[0202] In certain embodiments, the peptide linker may comprise the peptide sequence DDRKAA (SEQ ID NO: 28). A linker comprising the sequence DDRKAA is illustrated in FIG. 39. Preferably, one or more payloads are attached to the N-terminus and / or C-terminus of the peptide DDRKAA. Furthermore, it is preferred that one or more payloads are attached to the side chain of an aspartic acid residue. It is to be understood that when an amine-containing payload is attached to the side chain of an aspartic acid residue, the peptide sequence of the linker can also be considered as DNRKAA (SEQ ID NO: 88), NDRKAA (SEQ ID NO: 89), or NNRKAA (SEQ ID NO: 90).

[0203] In certain embodiments, the peptide linker may comprise the peptide sequence EERKValCit (SEQ ID NO: 29). The sequence is illustrated in FIG. 40 as a linker comprising the peptide sequence EERKValCit. Preferably, the one or more payloads are attached to the N-terminus and / or C-terminus of the peptide sequence EERKValCit. Furthermore, it is preferred that the one or more payloads are attached to the side chain of a glutamic acid residue. It should be understood that when an amine-containing payload is attached to the side chain of a glutamic acid residue, the peptide sequence of the linker can also be considered as EQRKValCit (SEQ ID NO: 91), QERKValCit (SEQ ID NO: 92), or QQRKValCit (SEQ ID NO: 93).

[0204] In certain embodiments, the peptide linker is any one of the linkers shown in Figures 1-40 or 42-43.

[0205] In a particular embodiment, the invention relates to a peptide linker according to the invention, wherein the linker comprises two to four payloads.

[0206] The peptide linker of the present invention can be used to generate antibody-payload conjugates with a payload to antibody ratio of 4 or more using microbial transglutaminase. Native glycosylated antibodies have a single conjugation site at glutamine residue 295 (Q295) of the heavy chain. Since an antibody comprises two heavy chains, conjugating a linker comprising two payloads to each glutamine residue results in an antibody-payload conjugate comprising four payloads. Similarly, conjugating a linker comprising three or four payloads to each glutamine residue results in an antibody-payload conjugate comprising six or eight payloads, respectively. Thus, in certain embodiments, the peptide linker of the present invention comprises two, three, or four payloads.

[0207] The inventors have identified various ways to couple two or more payloads to a peptide linker. In certain embodiments, two payloads can be coupled to the C-terminus of the peptide linker (see Figures 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 23, 24, 25, 28, 36, 37, and 40). In other embodiments, two payloads can be coupled to the N-terminus of the peptide linker (see Figure 20). In yet other embodiments, one or two payloads can be coupled to the N-terminus of the peptide linker and the C-terminus of the peptide linker, respectively (see Figures 3, 14, 20, 21, 22, 26, 27, 31, 32, 33, 34, and 38).

[0208] In embodiments in which no payload is attached to either the N-terminus or the C-terminus of the peptide linker, it is preferred that each terminus is modified, i.e., the N-terminus of the peptide linker is preferably acetylated and the C-terminus of the peptide linker is preferably amidated.

[0209] In addition to coupling a payload to the terminus of a peptide linker, one or more payloads can be coupled to an amino acid side chain (see Figures 19, 22, 29, 30, 35, 36, 37, 39, and 40). Those skilled in the art will recognize amino acid residues that have functional groups on their amino acid side chains that allow for attachment of a payload. Amino acids with functional groups on their side chains include, but are not limited to, those described by deGruiter et al. in Biochemistry 2017, 56, 30, 3863-3873. Additionally, payloads can be coupled to the side chains of non-standard amino acids, including, but not limited to, pAcF, CpK, pAMF, SCpHK, AzK, and Sec.

[0210] That is, in a particular embodiment, the present invention relates to a peptide linker according to the present invention, in which at least one payload is attached to the side chain of a glutamic acid residue, an aspartic acid residue, a tryptophan residue, a cysteine ​​residue, a lysine residue, a tyrosine residue, a serine residue, or a threonine residue contained in the peptide linker.

[0211] In specific embodiments, one or two payloads can be attached to the carboxylic acid side chains of glutamic acid or aspartic acid (see Figures 19, 22, 29, 30, 39, and 40).

[0212] In a specific embodiment, one or two payloads can be attached to the amines of the lysine side chains (see Figure 36).

[0213] In a specific embodiment, one or two payloads can be attached to the thiol of a cysteine ​​side chain (see Figure 35).

[0214] In a specific embodiment, one or two payloads can be attached to the side chain hydroxyls of serine, threonine, or tyrosine (see Figure 37).

[0215] The payload can be directly coupled to the peptide linker. For example, an amine-containing payload can be coupled to the C-terminus of the peptide linker via an isopeptide bond (see FIG. 27). Similarly, a carboxyl-containing payload can be coupled to the N-terminus of the peptide linker via an isopeptide bond (see FIG. 32), or a thiol-containing payload can be coupled to the side chain of a cysteine ​​residue contained in the peptide linker.

[0216] However, it is preferred herein to couple the payloads to the peptide linker via a chemical linker, particularly when the two payloads are attached to either the N-terminus or the C-terminus of the peptide linker, using a chemical linker between the two payloads and the N-terminus or the C-terminus.

[0217] Thus, in a particular embodiment, the present invention relates to a peptide linker according to the invention, wherein at least one of the two or more payloads is attached to the peptide linker via a chemical linker.

[0218] In the present invention, it is preferred that at least one of the two or more payloads is coupled to the peptide linker via a chemical linker, but even more preferably, all payloads are coupled to the peptide linker via a chemical linker.

[0219] The chemical linker may have various purposes. In certain embodiments, the chemical linker simply functions as an "adapter" to couple one payload to the peptide linker. For example, a chemical linker containing an amine group can be used to couple a payload to the C-terminus of the peptide linker via an amide bond. In such embodiments, it is preferred that the chemical linker contains one or more functional groups other than amines, so that the payload can be coupled to the chemical linker via these additional functional groups.

[0220] In certain embodiments, the chemical linker functions as an "amplifier moiety" to couple several payloads to the peptide linker. For example, a chemical linker containing a disubstituted amine can be used as a dendron to attach two payloads (exemplary linkers containing an amplifier are shown in Figures 36 and 37). Another example of an amplifier is the 2,6-bis-(hydroxymethyl)-p-cresol moiety (shown in Figure 28).

[0221] Similarly, chemical linkers containing a carboxyl group can be used to couple one or more payloads to the N-terminus of a peptide linker via an amide bond. For example, a dicarboxylic acid molecule can be used to couple an amine-containing payload to the N-terminus of a peptide (see Figures 22, 31, and 33).

[0222] Additionally, chemical linkers containing compatible functional groups can be used to couple the payload to amino acid side chains contained in the peptide linker.

[0223] In any of the embodiments disclosed above, one of skill in the art can identify a suitable chemical linker for coupling a payload to the peptide linker, whether that chemical acts as an "adapter" or an "amplifier moiety." That is, one of skill in the art can identify a linker that has the necessary functional groups to couple a payload of interest to a functional group contained in the peptide linker.

[0224] However, the chemical linker can not only function as an adapter between the payload(s) and the peptide linker, but can also serve other functions.

[0225] Thus, in a particular embodiment, the present invention relates to a peptide linker according to the invention, wherein the chemical linker is an enzymatically and / or chemically cleavable linker.

[0226] The cleavable linker may be any enzymatically and / or chemically cleavable linker known in the art, including, but not limited to, those described by Bargh et al. (Chem. Soc. Rev., 2019, 48, 4361), which is incorporated by reference in its entirety.

[0227] Cleavable linkers have the advantage that they can control and / or facilitate the release of the payload from the antibody. For example, one or more payloads can be coupled to a peptide linker via an enzymatically and / or chemically cleavable chemical linker.

[0228] In certain embodiments, the chemical linker is cleavable in vivo. The cleavable linker may include a chemically or enzymatically labile or degradable linkage. The cleavable linker generally relies on a biological process to release the payload, such as reduction in the cytoplasm, exposure to acidic conditions in the lysosome, or cleavage by specific proteases or other enzymes inside or outside the cell. The cleavable linker generally incorporates one or more chemical bonds that are chemically or enzymatically cleavable. In certain embodiments, the linker includes a chemically labile group, such as a hydrazone group and / or a disulfide group. Linkers that include chemically labile groups take advantage of the differential properties between plasma and some cytoplasmic compartments. The intracellular conditions that promote payload release of hydrazone-containing linkers are the acidic environment of endosomes and lysosomes, while disulfide-containing linkers are reduced in the cytoplasm, which contains high concentrations of thiols, such as glutathione. In certain embodiments, the plasma stability of linkers containing chemically labile groups can be increased by introducing steric hindrance with substituents in the vicinity of the chemically labile group.

[0229] Acid labile groups such as hydrazones or carbonates remain intact in the systemic circulation in the neutral pH environment of blood (pH 7.3-7.5) and undergo hydrolysis to release the payload when the ADC is internalized into the weakly acidic endosomal (pH 5.0-6.5) and lysosomal (pH 4.5-5.0) compartments of cells. This pH-dependent release mechanism is associated with non-specific release of the payload. To increase the stability of the hydrazone group of the linker, the linker may be altered by chemical modification, e.g., substitution, such that more efficient release can be achieved in the lysosomes while minimizing losses in the circulation. Linkers containing hydrazones or carbonates may contain additional cleavage sites, e.g., additional acid labile cleavage sites and / or enzyme labile cleavage sites. Exemplary linkers with carbonate acid labile groups are shown in FIG. 34.

[0230] Other acid-labile groups that can be included in chemical linkers include cis-aconityl-containing linkers. Cis-aconityl chemistry uses a carboxylic acid juxtaposed to the amide bond to accelerate hydrolysis of the amide under acidic conditions.

[0231] Cleavable chemical linkers may contain disulfide groups. Disulfides are thermodynamically stable at physiological pH and are designed to release the payload upon internalization into cells where the cytoplasm provides a significantly more reducing environment compared to the extracellular environment. Disulfide bond cleavage generally requires the presence of a cytoplasmic thiol cofactor such as (reduced) glutathione (GSH), so that disulfide-containing linkers are reasonably stable in circulation and selectively release the payload in the cytoplasm. The intracellular enzyme protein disulfide isomerase or similar enzymes capable of cleaving disulfide bonds may also contribute to preferential cleavage of disulfide bonds within cells. GSH is reported to be present within cells in a concentration range of 0.5-10 mM, compared to significantly lower concentrations of circulating GSH or cysteine ​​(the most abundant small thiol) at approximately 5 μM. Tumor cells, where hypoxia is caused by irregular blood flow, have higher glutathione concentrations due to increased activity of reductase enzymes. In certain embodiments, chemical modifications of the linker, such as the use of steric hindrance adjacent to the disulfide bond, can increase the stability of disulfide-containing linkers in vivo. Exemplary linkers with disulfide groups are shown in FIG.

[0232] Another type of cleavable linker that can be used is a chemical linker that is specifically cleaved by an enzyme. Such linkers are typically peptide-based or contain a peptide region that acts as a substrate for the enzyme. Peptide-based linkers tend to be more stable in plasma and in the extracellular environment than chemically labile linkers. Peptide bonds generally have good serum stability because lysosomal proteolytic enzymes are very low in activity in blood due to endogenous inhibitors and an unfavorably high pH value in blood compared to lysosomes. Release of the payload from the antibody occurs specifically by the action of lysosomal proteases, such as cathepsin, legumain, and plasmin. These lysosomal proteases can be present at high levels in certain tumor cells, but can also be found extracellularly in the tumor microenvironment. Peptide-based linkers can also be cleaved by non-lysosomal extracellular proteases, such as matrix metalloproteinases. Non-peptide-based linkers can also be specifically cleaved by glycosidases.

[0233] In an exemplary embodiment, the cleavable peptide is a tetrapeptide such as Gly-Phe-Leu-Gly (SEQ ID NO: 30), Ala-Leu-Ala-Leu (SEQ ID NO: 31), Gly-Gly-Phe-Gly (SEQ ID NO: 32), or Ala-Ala, Ala-Arg, Val-Cit, Val-Ala, Met-(D)Lys, Asn-(D)Lys, Val-(D)Asp, Phe-Lys, Ile- The dipeptide is selected from dipeptides such as Val, Asp-Val, His-Val, NorVal-(D)Asp, Ala-(D)Asp, Met-Lys, Asn-Lys, Ile-Pro, Me3Lys-Pro, phenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Met-(D)Lys, Asn-(D)Lys. In certain embodiments, dipeptides are preferred over longer polypeptides, since longer peptides are more hydrophobic. That is, the linker comprising the amino acids set forth in SEQ ID NOs: 1-29 or 82-93 may further comprise any of the dipeptide or tetrapeptide motifs described above. Preferably, the dipeptide or tetrapeptide motifs are coupled to the payload directly or via a self-immolative spacer.

[0234] The enzymatically cleavable linker may contain a self-immolative spacer to spatially separate the payload from the enzymatic cleavage site. Direct attachment of the payload to the peptide linker may result in proteolytic release of the amino acid adducts of the payload, thereby compromising activity. The use of a self-immolative spacer allows for release of a fully active and chemically unmodified payload upon hydrolysis of the amide or glycosidic bond.

[0235] In certain embodiments, the present invention provides a method for the preparation of a self-immolative linker comprising the steps of: a) a p-aminobenzyl alcohol moiety; or b) a 2,4-bis(hydroxymethyl)aniline moiety; or c) p-aminobenzyl quaternary ammonium; or d) an ethylenediamine-based moiety; or e) an (aminomethyl)pyrrolidine-based moiety; or f) Aminomethyl-based moieties The present invention relates to a peptide linker comprising:

[0236] One self-immolative spacer is the bifunctional para-aminobenzyl alcohol group, which links to peptides via the amino group to form an amide bond, while amine-containing drugs can be attached to the linker's benzylic hydroxyl group (PABC) via a carbamate functional group. The resulting prodrug is activated upon protease-mediated cleavage, resulting in a 1,6-elimination reaction that releases the unmodified drug, carbon dioxide, and a remnant of the linker group. Heterocyclic variants of this self-immolative group have also been described. See, for example, U.S. Pat. No. 7,989,434, which is incorporated herein by reference. The para-aminobenzyl alcohol moiety can also be used to link phenol- or hydroxyl-containing payloads through the formation of a carbonate (see FIG. 34). The para-aminobenzyl moiety can also be used to link tertiary or heteroaryl-amine-containing payloads through the formation of a quaternary ammonium (PABQ) (see FIG. 32). That is, in a particular embodiment, the present invention relates to a peptide linker according to the present invention, in which the quaternary ammonium cation contained in the p-aminobenzyl quaternary ammonium originates from an amine contained in the payload, preferably a tertiary amine or a heteroaryl amine.

[0237] Another self-immolative spacer is the 2,4-bis(hydroxymethyl)aniline group, which links to peptides through the amino group to form an amide bond, whereas amine-containing drugs can be attached through two benzylic hydroxyl groups of the linker via two carbamate functionalities. The resulting prodrug is activated upon protease-mediated cleavage and releases the payload via sequential 1,6- and 1,4-elimination processes.

[0238] For hydroxyl-containing drugs, suitable self-immolative spacers include, but are not limited to, ethylenediamine-based carbamates (EDA) (see Figure 22 31), (aminomethyl)pyrrolidine-based carbamates (AMP) (see Figure 33), or aminomethyl moieties (AM) (see Figures 30, 39, and 40). This latter release mechanism relies on the lability of the hemiaminal functional group, which readily undergoes 1,2-elimination to release the desired alcohol.

[0239] For thiol-containing drugs, suitable self-immolative spacers include, but are not limited to, aminomethyl moieties (AM) (see Figure 35). This latter release mechanism takes advantage of the lability of the thiohemiaminal functional group, which readily undergoes 1,2-elimination to release the desired thiol.

[0240] In some embodiments, the enzymatically cleavable linker is a β-glucuronic acid-based linker. Easy release of the payload can be achieved through cleavage of the β-glucuronide glycosidic bond by the lysosomal enzyme β-glucuronidase. This enzyme is abundant in lysosomes and is overexpressed in some tumor types, but the enzymatic activity outside the cell is low. β-glucuronic acid-based linkers can be used to avoid the tendency of antibody-payload conjugates to aggregate due to the hydrophilic nature of β-glucuronide (see FIG. 38).

[0241] As mentioned above, in a specific embodiment, the present invention relates to a peptide linker according to the invention, wherein the chemical spacer is or comprises a self-immolative linker.

[0242] It is preferred herein that the payload is attached to the peptide linker via a self-immolative linker to facilitate the release of the unmodified drug. Even more preferably, the self-immolative linker is coupled to a peptide sequence that is efficiently cleaved by a protease or peptidase. A cleavable peptide can be defined as part of the peptide linker or as part of the chemical linker that connects the peptide linker and the payload(s).

[0243] The self-immolative linker may be any self-immolative linker known in the art, however, it is preferred that the self-immolative linker comprises a p-aminobenzyl alcohol moiety or a 2,4-bis(hydroxymethyl)aniline moiety.

[0244] Thus, in a specific embodiment, the present invention relates to a peptide linker according to the invention, wherein the self-immolative linker comprises a p-aminobenzyl alcohol moiety or a 2,4-bis(hydroxymethyl)aniline moiety.

[0245] A self-immolative linker containing a p-aminobenzyl alcohol moiety can be used to couple a payload to the C-terminus of a peptide. That is, the amino group of the p-aminobenzyl alcohol moiety can be coupled to the C-terminal carboxyl group of a peptide linker via an amide bond (see, for example, FIG. 3). Alternatively or additionally, the amino group of the p-aminobenzyl alcohol moiety can be coupled to a carboxyl group in the side chain of an aspartic acid or glutamic acid residue in the peptide linker via an amide bond (see, for example, FIG. 19 or 22).

[0246] The payload can be attached to the hydroxyl group of the p-aminobenzyl alcohol moiety via a carbamate. In certain embodiments, the C-terminal amino acid of the peptide linker to which the p-aminobenzyl alcohol moiety can be attached can be composed of motifs that are efficiently cleaved by peptidases, such as, but not limited to, the sequence motif valine-citrulline.

[0247] It should be understood that the peptide linker of the present invention may contain more than one p-aminobenzyl alcohol moiety. For example, the peptide linker of the present invention may contain two peptide moieties, which are linked to each other via their N-terminus. In such an embodiment, the peptide linker has two C-termini, both of which can be conjugated to a payload via a p-aminobenzyl alcohol moiety. An exemplary linker with a payload attached to both termini (the C-terminus via a p-aminobenzyl alcohol moiety, and the N-terminus via a second peptide moiety and a p-aminobenzyl alcohol moiety) is shown in FIG. 3.

[0248] The p-aminobenzyl alcohol moiety can also be used to couple a payload to an amino acid side chain. For example, a payload can be coupled to a carboxyl group in the side chain of a glutamic acid or aspartic acid residue via the p-aminobenzyl alcohol moiety. The p-aminobenzyl alcohol moiety can be coupled to a carboxyl group in the side chain of a glutamic acid or aspartic acid residue directly or via one or more amino acid residues, as shown in Figures 19, 22, 30, 39, and 40. In certain embodiments, the p-aminobenzyl alcohol moiety can be coupled to a carboxyl group in the side chain of a glutamic acid or aspartic acid residue via a valine-citrulline sequence or an alanine-alanine sequence.

[0249] In certain embodiments, an amine-containing payload can be coupled to a carboxyl group in a peptide linker by two or more aminobenzyl alcohol moieties (see FIG. 3).

[0250] A self-immolative linker containing a 2,4-bis(hydroxymethyl)aniline moiety can be used to couple two payloads to a single functional group contained in a peptide linker. That is, the 2,4-bis(hydroxymethyl)aniline moiety can be coupled to a carboxyl group contained in the peptide linker through its amino group. A payload can then be coupled to each hydroxyl group through a carbamate. An exemplary peptide linker in which two payloads are coupled to the C-terminus of the peptide linker through a 2,4-bis(hydroxymethyl)aniline moiety is shown in FIG. 1.

[0251] By using a linker containing a 2,4-bis(hydroxymethyl)aniline moiety, a peptide linker containing more than two payloads can be obtained. For example, a linker containing four payloads can be obtained by coupling two payloads to the N-terminus of a peptide linker through a 2,4-bis(hydroxymethyl)aniline moiety (indirectly through a second peptide moiety) and coupling two payloads to the C-terminus of the peptide linker through another 2,4-bis(hydroxymethyl)aniline moiety (see FIG. 20). Similarly, a peptide linker containing three payloads can be obtained by coupling two payloads to the 2,4-bis(hydroxymethyl)aniline moiety and coupling a third payload to the peptide linker through a p-aminobenzyl alcohol moiety (see FIG. 19).

[0252] In a specific embodiment, the present invention relates to a peptide linker according to the invention, wherein a hydroxyl group contained in the p-aminobenzyl alcohol moiety forms a carbamate with the payload.

[0253] As mentioned above, the payload can be attached to the p-aminobenzyl alcohol moiety via a carbamate. That is, the payload preferably comprises a free amine group suitable for undergoing carbamate formation. Those skilled in the art will be aware of methods for forming a carbamate between the p-aminobenzyl alcohol moiety and an amine-containing payload.

[0254] In a specific embodiment, the present invention relates to a peptide linker according to the invention, wherein the hydroxyl group contained in the p-aminobenzyl alcohol moiety forms a carbonate with the payload.

[0255] The payload can be attached to the p-aminobenzyl alcohol moiety via a carbonate. That is, the payload preferably comprises a free hydroxyl group suitable for undergoing carbonate formation (see FIG. 34). Those skilled in the art will be aware of methods for forming a carbonate between the p-aminobenzyl alcohol moiety and a hydroxyl-containing payload.

[0256] In a specific embodiment, the present invention relates to a peptide linker according to the invention, wherein each hydroxyl group contained in the 2,4-bis(hydroxymethyl)aniline moiety forms a carbamate with the payload.

[0257] That is, the 2,4-bis(hydroxymethyl)aniline moiety contained in the peptide linker of the present invention can form two carbamates with a payload that contains two individual amines.

[0258] In a specific embodiment, the present invention relates to a peptide linker according to the invention, wherein the p-aminobenzyl moiety forms a quaternary ammonium with the payload.

[0259] As mentioned above, the payload can be attached to the p-aminobenzyl via a quaternary ammonium. That is, the payload preferably comprises a tertiary amine or a heteroarylamine suitable for undergoing the formation of a quaternary ammonium. Those skilled in the art will be aware of how to form a quaternary ammonium between a p-aminobenzyl moiety and a payload comprising a tertiary amine or a heteroarylamine.

[0260] In a specific embodiment, the invention relates to a peptide linker according to the invention, wherein the self-immolative linker comprises an ethylenediamine carbamate (EDA) moiety.

[0261] That is, a payload can be coupled to the peptide linker of the present invention via an ethylenediamine carbamate (EDA) moiety. The EDA moiety can be directly coupled to the C-terminus of the peptide or can be coupled to the side chain of aspartic acid or glutamic acid via an amide bond. The EDA moiety preferably undergoes carbamate formation with a payload containing a hydroxyl group. Examples of linkers containing an EDA moiety are shown in Figures 22 and 31. As shown in Figure 28, an amplifier linked to two payloads can also be connected using an EDA site.

[0262] In a specific embodiment, the present invention relates to a peptide linker according to the invention, wherein the self-immolative linker comprises an (aminomethyl)pyrrolidine-based carbamate (AMP) moiety.

[0263] That is, a payload can be coupled to a peptide linker of the present invention via an (aminomethyl)pyrrolidine-based carbamate (AMP) moiety. The AMP moiety can be directly coupled to the C-terminus of the peptide or can be coupled to the side chain of aspartic acid or glutamic acid via an amide bond. The AMP moiety preferably undergoes carbamate formation with a payload that contains a hydroxyl group. An example of a linker containing an AMP moiety is shown in FIG.

[0264] In a specific embodiment, the present invention relates to a peptide linker according to the invention, wherein the self-immolative linker comprises an aminomethyl (AM) moiety.

[0265] That is, the payload can be coupled to the peptide linker of the present invention via an aminomethyl (AM) moiety. The AM moiety can be directly coupled to the C-terminus of the peptide or can be coupled to the side chain of aspartic acid or glutamic acid via an amide bond. The AM moiety is preferably used to link a payload containing a hydroxyl group, thereby forming a hemiaminal. Examples of linkers containing an AM moiety are shown in Figures 30, 39, and 40. However, the AM moiety can also be used to link a payload containing a thiol group, thereby forming a thiohemiaminal. An example of a linker containing an AM moiety with a thiol-containing payload is shown in Figure 35.

[0266] In a specific embodiment, the present invention relates to a peptide linker according to the present invention, wherein at least one payload is attached to the side chain of a glutamic acid residue, an aspartic acid residue, a tryptophan residue, a cysteine ​​residue, a lysine residue, a tyrosine residue, a serine residue, or a threonine residue contained in the peptide linker.

[0267] As mentioned above, one or more payloads can be coupled to the amino acid side chains contained in the peptide linker. Those skilled in the art are aware of suitable chemical linkers for coupling payloads to amino acid side chains, i.e., carboxyl groups in the side chains of glutamic or aspartic acid residues, thiol groups in the side chains of cysteine ​​residues, amino groups in the side chains of lysine residues, or hydroxyl groups in the side chains of tyrosine, serine, or threonine residues. Examples of linkers containing payloads in amino acid side chains are shown in Figures 19, 22, 29, 30, 35, 36, 37, 39, and 40.

[0268] In a specific embodiment, the present invention relates to a peptide linker according to the invention, wherein the peptide linker comprises two peptide moieties, said two peptide moieties being connected via their N-terminal amino acid residues with a dicarboxylic acid linker (HO2C-R-CO2H).

[0269] Particular linkers within the scope of the present invention comprise two peptide moieties linked via their N-terminal amino acid residues (see, for example, FIG. 3). The N-terminal amino acids of the two peptide moieties may also be linked via a dicarboxylic acid, each of whose carboxylic acid groups forms an amide bond with the N-terminal amino group of the peptide moiety.

[0270] Any dicarboxylic acid can be used to link two peptide moieties via their N-terminal amino acid residues. In certain embodiments, the dicarboxylic acid can be an aliphatic dicarboxylic acid. That is, the dicarboxylic acid can be ethanedioic acid, propanedioic acid, butanedioic acid, pentanedioic acid, hexanedioic acid, heptanedioic acid, octanedioic acid, nonanedioic acid, or decanedioic acid. In certain embodiments, the two peptide moieties are linked to a butanedioic acid molecule via their N-terminal amino acids (see Figures 3, 14, 20, 21, 27, and 34). In certain embodiments, the two peptide moieties are linked to a pentanedioic acid molecule via their N-terminal amino acids (see Figure 26). The aliphatic dicarboxylic acid can include a substituted or unsubstituted alkyl or alkenyl chain.

[0271] In certain embodiments, the dicarboxylic acid may be an aromatic dicarboxylic acid, including, but not limited to, phthalic acid, isophthalic acid, or terephthalic acid.

[0272] It should be understood that linking two peptide moieties via the N-terminal amino acid results in a peptide construct that does not contain a free N-terminal amino group that may be suitable for conjugation to a glutamine residue contained in an antibody. Thus, a peptide linker that comprises two N-terminally linked peptide moieties preferably contains a lysine residue, a lysine mimetic, or a lysine derivative to enable conjugation of the peptide linker to a glutamine moiety contained in an antibody.

[0273] In certain embodiments, the first peptide portion included in the peptide linker may comprise any of the amino acid sequences set forth in SEQ ID NOs: 1-8, 11-14, 16-29, or 82-93. The second peptide portion may have any amino acid sequence. In certain embodiments, the second peptide portion may have a length of 2-100, preferably 2-50, more preferably 2-25, even more preferably 2-10, and most preferably 2-5 amino acid residues. In certain embodiments, the second peptide portion may be a dipeptide or a tripeptide. However, it should be noted that the second peptide portion may be a single amino acid or a longer peptide. To allow efficient release of the payload, the second peptide portion preferably comprises a peptide sequence that is efficiently cleaved by a peptidase. In certain embodiments, the second peptide portion may have the sequence Asn, Ala, Ala-Ala, Ala-Asn, Val-Ala, Val-Cit, Ala-Arg, Arg-Ala, Ala-Ala-Arg (SEQ ID NO: 34), Ala-Arg-Ala (SEQ ID NO: 35), Ala-Ala-Asn (SEQ ID NO: 36).

[0274] That is, in certain embodiments, the peptide linker has the structure: [payload 1]-[peptide 1]-[dicarboxylic acid]-[peptide 2]-[payload 2]; [Payload 1] and [Payload 2] are payloads, [Peptide 1] is a first peptide moiety, [Peptide 2] is a second peptide moiety, [Dicarboxylic acid] is a dicarboxylic acid. It may have At least one of said peptide moieties 1 and / or 2 comprises a free amine; the N-terminus of peptide 1 and the N-terminus of peptide 2 are connected via the dicarboxylic acid, Payload 1 is attached to the C-terminus of Peptide 1, preferably via a chemical linker; and Payload 2 is preferably attached to the C-terminus of peptide 2 via a chemical linker.

[0275] Preferably, the peptide moiety comprising a free amine group is a peptide moiety comprising a lysine residue, a lysine mimetic, or a lysine derivative as defined elsewhere herein, or a peptide linker comprising any one of the amino acid sequences set forth in SEQ ID NOs: 1-8, 11-14, 16-29, or 82-93.

[0276] In certain embodiments, the peptide linker can comprise a first peptide portion comprising a sequence set forth in SEQ ID NOs: 1-8, 11-14, 16-29, or 82-93 and a second portion comprising the sequence Ala-Ala, wherein the first and second peptide portions are linked to a butanedioic acid molecule via their N-terminal amino acids.

[0277] In certain embodiments, the peptide linker can comprise a first peptide portion comprising the sequence RKAA and a second portion comprising the sequence Ala-Ala, the first and second peptide portions being linked to a butanedioic acid molecule via their N-terminal amino acids.

[0278] Instead of coupling the two peptide moieties via their N-terminal amino acid residues, the second peptide moiety may be coupled to an amino acid side chain of the first peptide moiety. That is, the first peptide moiety contained in the peptide linker may comprise any of the amino acid sequences set forth in SEQ ID NOs: 6, 11-12, 20-21, 23, 26-29, or 82-93. The second peptide moiety, i.e., the one located at the amino acid side chain, may have any amino acid sequence. In certain embodiments, the second peptide moiety may be a dipeptide or a tripeptide. However, it should be noted that the second peptide moiety may be a single amino acid or a longer peptide. To allow efficient release of the payload, the second peptide moiety preferably comprises a peptide sequence that is efficiently cleaved by a peptidase. In certain embodiments, the second peptide portion may have the sequence Asn, Ala, Ala-Ala, Ala-Asn, Val-Ala, Val-Cit, Ala-Arg, Arg-Ala, Ala-Ala-Arg (SEQ ID NO: 34), Ala-Arg-Ala (SEQ ID NO: 35), Ala-Ala-Asn (SEQ ID NO: 36).

[0279] The peptide linker according to the present invention comprises two or more payloads. The peptide linker comprising two or more payloads is preferably obtained by chemical synthesis.

[0280] Those skilled in the art are aware of methods for coupling a payload to an amino acid-based linker by chemical synthesis. For example, a payload containing an amine (e.g., in the case of an auristatin analogue), or a payload containing a thiol (e.g., in the case of a maytansine analogue), or a payload containing a hydroxyl (e.g., in the case of an SN-38 analogue) can be attached to the C-terminus of an amino acid-based linker by chemical synthesis. However, those skilled in the art are aware of additional reactions and reactive groups that can be utilized to couple a payload to the N-terminus, C-terminus, or side chain of an amino acid or amino acid derivative by chemical synthesis. Exemplary reactions that can be used to couple a payload to an amino acid-based linker 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).

[0281] 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 extensively described in the prior art.

[0282] It should be understood that the payload can be coupled to the N-terminus and / or C-terminus of the 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 the C-terminal carboxyl group of the peptide or amino acid residue.

[0283] 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 bond or an ester bond, respectively (see FIG. 34). 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 (see FIG. 32).

[0284] In certain embodiments, the payload can be indirectly coupled to the N-terminus and / 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.

[0285] 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.

[0286] In certain embodiments, a payload containing a thiol group can be coupled to the N-terminus of an amino acid residue via a linker molecule. For example, a payload containing a thiol group can be coupled to the N-terminus amino group via a thiocarbamate linker. Alternatively, a 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 disulfur bond with the thiol group contained in the 3-mercaptopropionic acid linker molecule.

[0287] 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, with each of the carboxylic acid groups contained in the dicarboxylic acid linker forming 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.

[0288] Alternative linker molecules for indirectly coupling a payload to the N-terminus of an amino acid residue contained in a peptide linker of the present invention or linker molecules suitable for indirectly coupling a payload to the C-terminus of an amino acid residue contained in a peptide linker of the present invention have been described in the art and are encompassed by the present invention.

[0289] The present inventors surprisingly found that a peptide linker in which payloads are attached to the N-terminus and C-terminus of the peptide linker has stronger antitumor activity than a linker in which all payloads are attached to the C-terminus of the peptide linker (see Examples 11 and 12).

[0290] Thus, in a specific embodiment, the present invention relates to a peptide linker according to the present invention, wherein one or more payloads are attached to the N-terminus of the amine-containing peptide linker and one or more payloads are attached to the C-terminus of the amine-containing peptide linker.

[0291] In certain embodiments, one or two payloads may be attached to the N-terminus of an amine-containing peptide linker and one or two payloads may be attached to the C-terminus of the amine-containing peptide linker, i.e., the peptide linker may be a DAR4, DAR6, or DAR8 linker.

[0292] In certain embodiments, one payload may be attached to the N-terminus of an amine-containing peptide linker and one payload may be attached to the C-terminus of the amine-containing peptide linker. In such embodiments, the peptide linker may be a "linear" DAR4 linker.

[0293] The amine-containing peptide linker may be any one of the lysine-containing peptide linkers disclosed herein, including peptide linkers that contain a lysine mimetic or a lysine derivative as defined herein.

[0294] In certain embodiments, a "linear" DAR4 linker has the following structure (N→C orientation): [Payload 1] - [(Aa) m -(Lys)-(Aa) n ]-[Payload 2]; [Payload 1] and [Payload 2] are payloads, Aa can be any amino acid residue; m and n may be integers ranging from 0 to 10, preferably from 0 to 6, more preferably from 0 to 4; Lys is a lysine residue, a lysine mimetic, or a lysine derivative. It may have [Payload 1] is directly or indirectly attached to the N-terminus of residue (Aa) or (Lys), [Payload 2] is directly or indirectly attached to the C-terminus of residue (Aa) or (Lys).

[0295] In certain embodiments, a "linear" DAR4 linker has the following structure: [Payload 1] - [(Aa) m -(Arg / His)-(Aa) n -(Lys)-(Aa) o ]-[Payload 2]; [Payload 1] and [Payload 2] are payloads, Aa can be any amino acid residue; m, n, and o may be integers ranging from 0 to 10, preferably from 0 to 6, more preferably from 0 to 4; Arg can be an arginine residue, an arginine mimetic, or an arginine derivative; His can be a histidine residue, a histidine mimetic, or a histidine derivative Lys is a lysine residue, a lysine mimetic, or a lysine derivative. It may have [Payload 1] is directly or indirectly attached to the N-terminus of a residue of (Aa) or (Arg / His), [Payload 2] is directly or indirectly attached to the C-terminus of residue (Aa) or (Lys).

[0296] In certain embodiments, a "linear" DAR4 linker has the following structure: [Payload 1] - [(Aa) m -(Lys)-(Aa) n -(Arg / His)-(Aa) o ]-[Payload 2]; [Payload 1] and [Payload 2] are payloads, Aa can be any amino acid residue; m, n, and o may be integers ranging from 0 to 10, preferably from 0 to 6, more preferably from 0 to 4; Arg can be an arginine residue, an arginine mimetic, or an arginine derivative; His can be a histidine residue, a histidine mimetic, or a histidine derivative Lys is a lysine residue, a lysine mimetic, or a lysine derivative. It may have [Payload 1] is directly or indirectly attached to the N-terminus of residue (Aa) or (Lys), [Payload 2] is directly or indirectly attached to the C-terminus of the (Aa) or (Arg / His) residue.

[0297] It is understood that the payloads can be directly or indirectly attached to the N-terminus and C-terminus of the peptide linker, in one embodiment, a first payload can be directly attached to the N-terminal amino group of the peptide linker and a second payload can be directly attached to the C-terminal carboxyl group of the peptide linker.

[0298] However, it is preferred that the payload is indirectly attached to the N-terminus and C-terminus of the peptide linker, for example using any one of the chemical linkers described herein. In particular, the payload can be indirectly attached to the N-terminus of the peptide linker via a dicarboxylic acid and a second peptide moiety, as described in more detail elsewhere herein. Furthermore, it is preferred that all payloads are attached to the peptide or chemical linker via a self-immolative moiety, such as any one of the self-immolative moieties disclosed herein.

[0299] In certain embodiments, the present invention provides a method for the preparation of a medicament comprising administering to said patient a medicament the payload of which is ·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; biotin or streptavidin moieties; ·vitamin; ·Proteolytic agents (“PROTACs”); · Receptor ligands; a target binding moiety; and / or Anti-inflammatory The present invention relates to a peptide linker according to the present invention, which is at least one of the following:

[0300] 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 cell-cell interactions in immune or inflammatory responses. Cytokines include, but are not limited to, monokines, lymphokines, and chemokines, regardless of which cell produces them. For example, monokines are generally 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 generally 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).

[0301] 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 (indomethacin), 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.

[0302] 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.

[0303] 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, growth factors differ in their ability 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.).

[0304] 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 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 (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, dehydroepiandrosterone, Androsterone (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.In a specific embodiment, the hormone is cortisol.

[0305] 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).

[0306] 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.

[0307] Suitable antibiotics that can be used as payloads in the present invention include, but are not limited to, macrolides, penicillins, cephalosporins, quinolones, fluoroquinolones, sulfonamides, tetracyclines, monobactams, carbapenems, aminoglycosides, rifamycins, β-lactams, ansamycins, oxazolidinones, streptogramins, glycopeptides, polypeptides, and arsphenamines, or pharmaceutically acceptable salts thereof, more preferably the antibiotic is erythromycin, azithromycin, clarithromycin, dirithromycin, clindamycin, doxycycline, minocycline, tigecycline, trimethoprim, pyocyanin, vancomycin, streptomycin, dihydrostreptomycin, amikacin, apramycin, arbekacin, astromycin, bekanamycin, dibekacin, flavin ... The anti-inflammatory agent is selected from mycetin, gentamicin, hygromycin, isepamicin, kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin, ribostamycin, sisomicin, spectinomycin, tobramycin, verdamycin, polymyxin, glycylcycline, carbapenem, carbacephem, chloramphenicol, clindamycin, lincomycin, daptomycin, novobiocin, clindamycin, ethambutol, fosfomycin, fusidic acid, furazolidone, isoniazid, linezolid, metronidazole, mupirocin, nitrofurantoin, platensimycin, pyrazinamide, quinupristin, benzalkonium, dalfopristin, rifampin, rifampicin, rifabutin, rifaximin, rifalog, tinidazole, viomycin, and capreomycin, or a pharma- ceutically acceptable salt thereof.

[0308] In a particular embodiment, a peptide linker of the present invention comprises a rifalog as the payload (see FIG. 32).

[0309] 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, malononitriloamide (e.g., leflunamide), T cell receptor modulators, and cytokine receptor modulators.

[0310] In certain embodiments, the immunomodulator may be an immunostimulator. The term "immunostimulator" 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 STING agonists (e.g., STING agonist-3, extracted from Example 10 of WO2017175147A1), lipopolysaccharide (LPS), chemokines / cytokines, fungal β-glucans (such as lentinan), imiquimod, CRX-527, and OM-174.

[0311] In certain embodiments, the immunostimulant may be a toll-like receptor (TLR) 7 / 8 agonist, such as, but not limited to, imiquimod, resiquimod, 852-A, vesatolimod, AZD8848, motolimod, or sergantolimod.

[0312] In certain embodiments, the peptide linker of the invention may comprise two different immunostimulants. For example, the peptide linker of the invention may comprise STING agonist 3 and resiquimod (see FIG. 34).

[0313] 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).

[0314] In certain embodiments, the payload may be a polymer-toxin conjugate. A polymer-toxin conjugate is a polymer that can carry one or multiple payload molecules. Examples include Fleximer polymer toxins developed by Mersana therapeutics, PSAR polymer toxins developed by Mablink, and XTEN polymer toxins developed by Amunix. Alternatively, the polymer toxin conjugate may include any of the toxins disclosed herein.

[0315] 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.

[0316] 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 penetration into deep tissues while minimizing background interference. Thus, near-infrared fluorescence imaging can be used to visualize tissues to which the antibody-payload conjugates of the invention bind 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.

[0317] In certain embodiments, the payload may comprise a radionuclide. The term "radionuclide" as used herein relates to medically useful radionuclides comprising positively charged ions of radioactive metals such as Y, In, Tb, Ac, Cu, Lu, Tc, Re, Co, Fe, etc., such as 90Y, 111In, 67Cu, 77Lu, 99Tc, 161Tb, 225Ac, etc. The radionuclide may be comprised in a chelating agent such as DOTA or NODA-GA. Furthermore, the radionuclide may be a therapeutic radionuclide or a radionuclide that can be used as a contrast agent in imaging techniques as described below. Radionuclides or molecules comprising radionuclides are known in the art and are commercially available.

[0318] In certain embodiments, the payload may be a ligand of a receptor or a substrate of a receptor. In particular, the payload may be a ligand or substrate of a receptor known to be highly expressed in cancer cells. That is, by coupling such a ligand or substrate of a receptor to an antibody via a peptide linker of the present invention, the specificity of the antibody-payload conjugate may be improved, and the internalization of the antibody-payload conjugate into target cells, such as cancer cells, may be further improved. For example, to improve the targeting of cancer cells overexpressing the folate receptor FRα, the payload may be folic acid. However, the payload may also be a derivative or analog of folic acid that binds to FRα with high affinity. Furthermore, the payload may be any other ligand or substrate of FRα, in particular a ligand or substrate that binds to FRα with high affinity.

[0319] In other embodiments, the payload may be a ligand or substrate for the biotin receptor, i.e., the payload may be biotin, an analog or derivative of biotin, or any other molecule that binds with high affinity to the biotin receptor.

[0320] In other embodiments, the payload may be a ligand or substrate for the epidermal growth factor receptor (EGFR). That is, the payload may be epidermal growth factor (EGF) or any derivative, analog, or fragment thereof that binds with high affinity to EGFR. Additionally, the payload may be any molecule that binds with high affinity to EGFR.

[0321] Other examples of peptide / small molecule ligands that can be used as payloads to target receptors known to be highly expressed in cancer cells include, but are not limited to, tumor-homing peptides: RGD peptide and its derivatives (iRGD, cilengitide, SFITGv6, CNGRC, etc.), extracellular matrix homing peptides (DAG, ZD2, CSG, PlGF-2, BT1718), tumor-associated macrophage targeting agents (RP-182, M2pep, mUNO), EGFR targeting peptides (GE11), Angiopep-2, peptides targeting aberrant cell signaling pathways (LP4, NBD, H1), PSMA binders (urea-based or phosphoramidate-based binders).

[0322] Both "ligand" and "substrate" are defined herein as molecules that bind to a receptor with a particular affinity. However, it should be understood that a "ligand" is usually a small molecule, while a "substrate" is usually a macromolecule such as a peptide or protein. It should be understood that the ligand or substrate included in the peptide linker of the present invention may be a naturally occurring ligand or substrate, a derivative of a naturally occurring ligand or substrate, or a chemically modified version of a naturally occurring ligand or substrate.

[0323] In certain embodiments, the payload may be a vitamin. The vitamin may be selected from the group consisting of folates, including folic acid, folacin, and vitamin B9. The vitamin may be selected from the group consisting of biotin and vitamin B7.

[0324] In a specific embodiment, 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; Thymidylate synthase inhibitors; Amanitin (e.g., α-amanitin); and Camptothecins (e.g., exatecan, deruxtecan) The present invention relates to a peptide linker according to the present invention, which is at least one selected from the group consisting of:

[0325] That is, the peptide linker of the present invention preferably comprises a toxin payload. The term "toxin" as used in the present invention relates to any compound that is toxic to a cell or organism. Preferably, the toxin is produced by the cell or organism. However, the toxin may also be a chemical derivative or analogue of the toxin produced by the cell or organism. The toxin may be, but is not limited to, a small molecule, a peptide, or a protein. Specific examples are neurotoxins, necrotic toxins, hematotoxins, and cytotoxins. In certain embodiments, the toxin is a toxin used in the treatment of neoplastic diseases. That is, the toxin can be conjugated to an antibody in the method of the present invention and delivered to or into malignant cells by the targeting specificity of the antibody.

[0326] 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.

[0327] 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 can be used in the methods of the invention or that can be included in the antibody-payload conjugates of the invention are maytansine, DM1, DM3, DM4, and / or DM21.

[0328] 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.

[0329] 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, International Publication No. WO 2015 / 054060, U.S. Patent Nos. 8,211,912, and 9,676,721, which are incorporated herein by reference in their entirety. In some embodiments, the NAMPT inhibitor is FK866. In some embodiments, the NAMPT inhibitor is GMX-1778.

[0330] 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.

[0331] 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 confers antibiotic characteristics to many enediynes, 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 a specific embodiment, the toxin may be a calicheamicin.

[0332] 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.

[0333] 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 protein, which is involved in the assembly of bipolar spindles during cell division. Kinesin spindle protein inhibitors are being investigated for the treatment of cancer. 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.

[0334] In certain embodiments, the toxin may be cryptophycin or a derivative as described in U.S. Patent Application Publication Nos. 20180078656A1, 20210163458A1, and 20210228726A1, which are incorporated by reference.

[0335] In certain embodiments, the toxin may be sandramycin, a depsipeptide originally isolated from Nocardioides species (ATCC 39419) that has been shown to have cytotoxic and antitumor activity.

[0336] In certain embodiments, the toxin may be a thymidine synthase (or thymidylate synthase) inhibitor. Thymidylate synthase inhibitors are chemical agents that inhibit the enzyme thymidylate synthase and are promising anti-cancer chemotherapy. This inhibition prevents methylation of C5 of deoxyuridine monophosphate (dUMP), thereby inhibiting the synthesis of deoxythymidine monophosphate (dTMP). The lack of dTMP, a necessary precursor for dTTP, prevents cells from properly synthesizing DNA, which promotes cell death as a downstream effect. In the present invention, thymidylate synthase inhibitors may be, but are not limited to, raltitrexed, pemetrexed, nolatrexed, ZD9331, GS7904L, fluorouracil, BGC-945, and OSI-7904L.

[0337] In certain embodiments, the toxin may be an amatoxin. Amatoxins (including α-amanitin, β-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.

[0338] 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 to be used to treat 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). In certain embodiments, the camptothecin is an exatecan derivative shown as compound 10 in Li et al., ACS Med. Chem. Lett. 2019, 10, 10, 1386-1392. In certain embodiments, the camptothecin derivative is glycinated exatecan (G-Exa; see Figures 13 and 16).

[0339] 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.

[0340] The peptide linker of the present invention comprises at least two payloads, which may be identical or may have different structures.

[0341] Thus, in a specific embodiment, the present invention relates to a peptide linker according to the invention, wherein two or more payloads are identical.

[0342] By coupling two or more identical payloads to the peptide linker, the concentration of the payload in the target tissue or cell of the antibody-payload conjugate can be increased. For example, when the peptide linker of the antibody-payload conjugate contains two or more identical toxins (resulting in a DAR>4 ADC), the toxin concentration in the target tissue or cell is higher than that of a conventional DAR2 ADC. Using the peptide linker of the present invention, ADCs containing four, six, or eight identical payload molecules can be obtained.

[0343] In a particular embodiment, the present invention relates to a peptide linker according to the invention, wherein at least two of the two or more payloads are different from each other.

[0344] In certain embodiments, the peptide linker of the present invention allows two different payloads to be conjugated to the antibody. The use of a second payload allows the development of a completely new class of antibody-payload conjugates that exceed current therapeutic approaches in terms of efficacy and potency. New application areas are also envisioned, such as dual-type imaging for imaging and therapy or intra- / post-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, including molecular imaging agents for preoperative positron emission tomography (PET) and near-infrared fluorescent (NIRF) dyes 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. Attaching two different probes by chemical means results in random conjugation of the probes, making analysis and reproducibility nearly impossible.

[0345] In a specific embodiment, the present invention relates to peptide linkers according to the invention resulting in theranostic agents (combined therapeutic and diagnostic strategies), where one payload serves imaging / detection purposes (fluorescent molecules or radioligands) and one payload serves treatment purposes.

[0346] Furthermore, a study by Levengood M. et al. (Orthogonal Cysteine ​​Protection Enables Homogeneous Multi-Drug Antibody-Drug Conjugates. Angewandte Chemie, Volume 56, Issue 3, January 16, 2017) showed that a dual-drug conjugated antibody attached 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 refractory to ADCs composed of individual auristatin components. This suggests that dual-conjugated ADCs may be able to address cancer heterogeneity and resistance more effectively than a single conventional ADC alone.

[0347] Thus, in certain embodiments, the peptide linker of the present invention comprises at least two different toxins. The at least two different toxins may be any of the toxins known in the art and / or disclosed herein. In particular, the two or more toxins coupled to the peptide linker of the present invention may have different modes of action.

[0348] In certain embodiments, peptide linkers of the invention comprise one or more auristatins and one or more camptothecins. In certain embodiments, peptide linkers of the invention comprise one or more MMAE molecules and one or more exatecan or exatecan derivatives (see FIG. 21).

[0349] In certain embodiments, a peptide linker of the invention comprises two different auristatins, hi certain embodiments, a peptide linker of the invention comprises MMAE and MMAF.

[0350] In certain embodiments, peptide linkers of the present invention include a toxin and a hormone. In certain embodiments, peptide linkers of the present invention include a toxin and cortisol. In certain embodiments, peptide linkers of the present invention include an auristatin and cortisol. In certain embodiments, peptide linkers of the present invention include MMAE and cortisol (see FIG. 22). In certain embodiments, peptide linkers of the present invention include an auristatin, a maytansinoid, and cortisol.

[0351] Since one of the resistance mechanisms to ADCs involves the active efflux of the cytotoxic moiety from cancer cells, another dual drug application may involve the additional and simultaneous delivery of a drug that specifically blocks the efflux mechanism of the cytotoxic drug. Thus, such dual-labeled ADCs may help overcome cancer resistance to ADCs more effectively than conventional ADCs.

[0352] In certain embodiments, the peptide linker of the present invention may comprise at least one toxin and at least one ligand of a receptor, preferably a receptor expressed in a cancer cell. In certain embodiments, the peptide linker of the present invention may comprise at least one toxin and a folate molecule.

[0353] In certain embodiments, a peptide linker according to the invention comprises three different payloads.

[0354] In a particular embodiment, the invention relates to a peptide linker according to the invention, wherein the linker is suitable to function as a substrate for transglutaminase.

[0355] That is, the peptide linker of the present invention is designed to function as a substrate for transglutaminase, an enzyme that in nature primarily catalyzes the formation of an isopeptide bond between the γ-carboxamide group (-(C=O)NH2) of the side chain of a glutamine residue and the ε-amino group (-NH2) of a lysine residue, followed by the release of ammonia (NH3). However, it is known in the art that this enzyme is rather tolerant and will accept primary amines other than the ε-amino group of lysine.

[0356] Herein, it is preferred that the peptide linker is conjugated to the glutamine residue of the antibody by transglutaminase. Therefore, the peptide linker must contain a primary amine to function as a substrate for transglutaminase. The primary amine is preferably contained in the side chain of lysine, lysine mimic or lysine derivative, or an amino acid residue having the structure NH2-(Y)-COOH as defined herein above. The transglutaminase may be any transglutaminase as defined herein, preferably a microbial transglutaminase as defined herein.

[0357] In a particular embodiment, the present invention relates to an antibody-payload conjugate comprising an antibody conjugated to a peptide linker according to the invention.

[0358] Thus, the present invention further encompasses antibody-linker conjugates comprising any of the peptide linkers defined herein, where it is preferred that an amine-containing peptide linker according to the invention is conjugated to a glutamine residue in an antibody.

[0359] Thus, in a particular embodiment, the present invention relates to an antibody-payload conjugate according to the invention, in which the peptide linker is conjugated to the antibody via an isopeptide bond formed between a γ-carboxamide group of a glutamine residue in the antibody and a primary amine in an amino acid residue of the peptide linker.

[0360] 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).

[0361] 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 for therapeutic use, the mouse or rabbit antibody may optionally be chimerized or humanized.

[0362] 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.

[0363] The term "antibody" also includes antigen-binding fragments of antibodies. Preferably, the peptide linker according to the present invention is a C-type peptide linker of an IgG antibody. H The antibody or antibody fragment of the present invention is conjugated to glutamine residue 295 (Q295) in the C2 domain. H It is preferred that it comprises two domains.

[0364] CH 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-CH2-CH3)2 Fc fusion peptides containing an Fc domain and one or more receptor domains It may be.

[0365] In a particular embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is an IgG antibody.

[0366] "IgG" as used herein refers to a polypeptide belonging to the class of antibodies substantially encoded by the 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 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 (Cγ2), Cγ3 (Cγ4), Cγ5 (Cγ6), Cγ7 (Cγ8), Cγ9 (Cγ10), Cγ11 (Cγ12), Cγ13 (Cγ14), Cγ15 (Cγ16), Cγ17 (Cγ18), Cγ19 (Cγ19), Cγ20 (Cγ21), Cγ21 (Cγ22), Cγ22 (Cγ23), Cγ24 (Cγ25), Cγ25 (Cγ26), Cγ27 (Cγ28), Cγ29 (Cγ29), Cγ30 (Cγ31), Cγ32 (Cγ32), Cγ33 (Cγ34), Cγ35 (Cγ35), Cγ36 (Cγ37), Cγ38 (Cγ39), Cγ40 (Cγ41), Cγ42 (Cγ42), Cγ53 (Cγ43), Cγ54 (Cγ55), Cγ55 (Cγ56), Cγ67 (Cγ68), Cγ70 (Cγ71), Cγ81 (Cγ82), Cγ93 (Cγ94), Cγ10 (Cγ11), Cγ12 (Cγ13), Cγ14 (Cγ15), Cγ15 (Cγ16), Cγ16 (Cγ17), Cγ18 (Cγ19), H 1), Cγ2 (C H 2) and Oγ3 (C H In the context of human IgG1, the EU index of Kabat states that H "1" refers to 118th to 215th place, and C H The second domain is 231-340, and the second domain is C H The 3 domain refers to positions 341 to 447. IgG1 also includes the hinge domain, which for IgG1 refers to positions 216 to 230.

[0367] In a preferred embodiment, the antibody is an IgG1 antibody. In a particularly preferred embodiment, the antibody is a human IgG1 antibody.

[0368] In a specific embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the peptide linker is conjugated to a glutamine residue comprised in the Fc domain of the antibody.

[0369] That is, the peptide linker of the present invention is preferably conjugated to a glutamine residue contained in the Fc domain of an antibody. The linker of the present invention can be conjugated to any Gln residue in the Fc domain of an antibody that can function as a substrate for transglutaminase. Typically, the term Fc domain, as used in the present invention, 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, C20, C19, C21, C31, C42, C43, C54, C65, C76, C87, C98, C99, C100, C111, C122, C131, C142, C153, C164, C175, C185, C196, C197, C198, C199, C211, C222, C31, C32, C43, C44, C54, C65, C76, C77, C78, ​​C89, C99, C101, C112, C123, C124, C125, C132, C145, C153, C154, C165, C176, C186, C187, C188, C189, C211, C189, C212, C189, C221, C189, C231, C189, C241, C252, C261, C272, C31, C32, C43, C44, C45, C46, ​​C47, C48, C49, C54, C48, 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.

[0370] For example, a peptide linker according to the invention can be conjugated to an endogenous glutamine residue (eg, Q295 in an IgG1 antibody) or a glutamine residue introduced into the Fc domain of an antibody by genetic engineering.

[0371] That is, in certain antibodies, the present invention provides a method for determining whether a glutamine residue to which a peptide linker is conjugated is located at the C H In particular, the glutamine residue Q295 (EU numbering) of domain 2 of the antibody-payload conjugate according to the present invention.

[0372] 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, some IgG type antibodies, such as mouse and rat IgG2a antibodies, do not possess this residue. 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.

[0373] C using transglutaminase H Literature discussing the conjugation of linkers to the Gln residue of 2 has focused on small, low molecular weight substrates, however, prior art literature has described the need for deglycosylation of the asparagine residue at position N297 or the use of an aglycosylated antibody to achieve such conjugation (WO 2015 / 015448, WO 2017 / 025179, WO 2013 / 092998).

[0374] However, rather surprisingly and contrary to all expectations, the above peptide linker structures indeed allow efficient site-specific conjugation of glycosylated antibodies to Q295. In particular, couples of peptide linkers containing two or more payloads were achieved with conjugation efficiencies of over 90%, most of which were achieved.

[0375] Although Q295 is in close proximity to N297, which is glycosylated in the native state, the method of the present invention still allows efficient conjugation to Q295 using the designated peptide linker.

[0376] 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.

[0377] These two points are of great advantage in terms of production: an enzymatic deglycosylation step is undesirable from a GMP perspective, because it is necessary to ensure that both the deglycosylation enzyme (e.g., PNGase F) and the cleaved glycans are removed from the medium.

[0378] Furthermore, because there is no need to genetically engineer the antibody to attach a payload, one can avoid the insertion of sequences that may increase immunogenicity and decrease the overall stability of the antibody.

[0379] Substitution of N297 with another amino acid may also have 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). Furthermore, the glycans present at 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, clinically validated antibodies are very often used as the starting point for ADC conjugation.

[0380] Thus, the method of the present invention using the peptide linkers of the present invention allows for the easy and trouble-free generation of stoichiometrically well-defined ADCs to which a site-specific payload is bound.

[0381] In view of the above, the method of the present invention preferably comprises the steps of: H The C of an antibody in which residue N297 (EU numbering) in the C2 domain is glycosylated HThe method is used for the conjugation of IgG antibodies at residue Q295 (EU numbering) of domain 2. However, it is expressly stated that the method of the invention also encompasses the conjugation of deglycosylated or aglycosylated antibodies at residue Q295 or any other suitable Gln residue of the antibody, which may be an endogenous Gln residue or a Gln residue introduced by molecular engineering.

[0382] Thus, in a particular embodiment, the invention relates to an antibody-payload conjugate according to the invention, in which a glutamine residue to which a peptide linker is conjugated has been introduced into the heavy or light chain of the antibody by molecular engineering.

[0383] 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.

[0384] 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 microbial transglutaminase. In certain embodiments, the antibody has a C of an IgG antibody. HThe antibody has a substitution at amino acid residue N297 (EU numbering) of the 2 domain, 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, where one linker is conjugated to residue Q295 of the first heavy chain of the antibody, one linker is conjugated to residue N297Q of the first heavy chain of the antibody, one linker is conjugated to residue Q295 of the second heavy chain of the antibody, and one linker is conjugated to residue N297Q of the second heavy chain of the antibody. Those skilled in the art are aware that the substitution of residue N297 of an IgG antibody with a Gln residue results in an aglycosylated antibody.

[0385] Thus, in a particular embodiment, the present invention relates to an antibody-payload conjugate according to the invention, wherein the glutamine residue introduced by molecular engineering into the heavy or light chain of the antibody is N297Q (EU numbering) in the CH2 domain of an aglycosylated IgG antibody.

[0386] In a specific embodiment, the present invention relates to an antibody-payload conjugate according to the invention, wherein the glutamine residue introduced into the antibody heavy or light chain by molecular engineering is (a) incorporated into the antibody heavy or light chain or (b) comprised in a peptide fused to the N-terminus or C-terminus of the antibody heavy or light chain.

[0387] 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 microbial transglutaminase are described in WO 2012 / 059882 and WO 2016 / 144608.

[0388] Thus, in a specific embodiment, the invention relates to an antibody-payload conjugate according to the invention, in which a peptide comprising a Gln residue is fused to the C-terminus of the heavy chain of the antibody.

[0389] Exemplary peptide tags that can be introduced into the heavy or light chain of an antibody, and in particular fused to the C-terminus of the heavy chain of an antibody, are LLQGG (SEQ ID NO: 70), LLQG (SEQ ID NO: 37), LSLSQG (SEQ ID NO: 38), GGGLLQGG (SEQ ID NO: 39), GLLQG (SEQ ID NO: 40), LLQ (SEQ ID NO: 41), GSPLAQSHGG (SEQ ID NO: 42), GLLQGGG (SEQ ID NO: 43), GLLQGG (SEQ ID NO: 44), GLLQ (SEQ ID NO: 45), LLQLLQGA (SEQ ID NO: 46), LLQGA (SEQ ID NO: 47), LLQYQGA (SEQ ID NO: 48), LLQGSG (SEQ ID NO: 49), LLQYQG (SEQ ID NO: 50), LLQLL QG (SEQ ID NO:51), SLLQG (SEQ ID NO:52), LLQLQ (SEQ ID NO:53), LLQLLQ (SEQ ID NO:54), LLQGR (SEQ ID NO:55), EEQYASTY (SEQ ID NO:56), EEQYQSTY (SEQ ID NO:57), EEQYNSTY (SEQ ID NO:58), EEQYQS (SEQ ID NO:59), EEQYQST (SEQ ID NO:60), EQYQSTY (SEQ ID NO:61), QYQS (SEQ ID NO:62), QYQSTY (SEQ ID NO:63), YRYRQ (SEQ ID NO:64), DYALQ (SEQ ID NO:65), FGLQRPY (SEQ ID NO:66), EQKLISEEDL (SEQ ID NO:67), LQR (SEQ ID NO:68), and YQR (SEQ ID NO:69).

[0390] 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.

[0391] In general, those skilled in the art are aware of methods to determine at which position of an antibody a peptide 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) and then digested with Trypsin Gold (mass spectrometry grade, Promega) according to the respective instruction manual. Thus, 1 μg of protein can be incubated with 50 ng of trypsin at 37° C. overnight. LC-MS analysis can be performed using a nanoAcquity HPLC system coupled to a Synapt-G2 mass spectrometer (Waters). To that end, 100 ng of peptide solution may be loaded onto an Acquity UPLC Symmetry C18 trap column (Waters, part number 186006527) and trapped for 3 min with 1% buffer A (water, 0.1% formic acid) and 99% buffer B (acetonitrile, 0.1% formic acid) at a flow rate of 5 μL / min. Peptides 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.

[0392] 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.

[0393] 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) using a full gradient from A (1.5 M ammonium sulfate, 25 mM Tris HCl, pH 7.5) to B (20% isopropanol, 25 mM Tris HCl, pH 7.5) over 20 minutes at 1 mL / min and 30° C. Typically, 40 μg of sample 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 ADC DAR2 species by the retention times of the respective unconjugated mAbs.

[0394] For LC-MS DAR, ADC may be diluted with NH4HCO3 to a final concentration of 0.025 mg / mL. 40 μL of this solution may then be reduced with 1 μL of TCEP (500 mM) for 5 minutes at room temperature, 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 minutes. 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 the 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. The deconvolution of the spectra may be performed until convergence with the MaxEnt1 algorithm of MassLynx. After the assignment of the DAR species to the chromatographic peaks, the DAR can be calculated based on the integrated peak area of ​​the reversed-phase chromatogram.

[0395] In a particular embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the IgG antibody is a glycosylated IgG antibody.

[0396] That is, it is preferred herein that the peptide linker according to the present invention is conjugated to a glycosylated IgG antibody. It is particularly preferred that the peptide linker according to the present invention is conjugated to a native glycosylated IgG antibody. A native IgG antibody contains a single conjugation site at glutamine residue 295 (Q295). Therefore, it is particularly preferred herein that the peptide linker according to the present invention is conjugated to residue Q295 of a native glycosylated antibody. The only glycosylation site of a native IgG antibody is asparagine residue 297 (N297).

[0397] Thus, in a specific embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the IgG antibody is glycosylated at residue N297 (EU numbering) in the CH2 domain.

[0398] In a particularly preferred embodiment, the peptide linker according to the invention is conjugated to position Q295 of an IgG antibody which is glycosylated at position N297. More preferably, the antibody is an IgG1 antibody.

[0399] In a specific embodiment, the invention relates to an antibody that is selected from the group consisting of brentuximab (anti-CD30), trastuzumab (anti-Her2 / neu), gemtuzumab (anti-CD33), inotuzumab (anti-CD22), avelumab (anti-PD-L1), cetuximab (anti-EGFR), rituximab (anti-CD20), daratumumab (anti-CD38), pertuzumab (anti-HER2), vedolizumab (anti-integrin α4β7), ocrelizumab (anti-CD20), tocilizumab (anti-CD20), and rituximab (anti-CD22). mab (anti-IL-6-R), ustekinumab (anti-IL-12 / 23), golimumab (anti-TNFα), obinutuzumab (anti-CD20), sacituzumab (anti-Trop-2), belantamab (anti-BCMA), polatuzumab (anti-CD79b), enfortumab (anti-nectin-4), endrecolomab (anti-EpCAM), gemtuzumab (anti-CD33), loncastuximab (anti-CD19), mecbotamab (anti-AXL), adecatumumab (anti-EpCA M), D93 (anti-dn-collagen), gatipotuzumab (anti-TA-MUC1), labetuzumab (anti-carcinoembryonic cell adhesion molecule 5), tusamitamab (anti-CEACAM5), upifitamab (anti-NaPi2b), rifastuzumab (anti-NaPi2b), mirvetuximab (anti-FRα), sofituzumab (anti-MUC16), anetumab (anti-mesothelin), tisotumab (anti-TF), cofituzumab (anti-Trop-2), pralzatamab (anti-CD 166), radriatuzumab (anti-LIV-1), belantamab (anti-BCMA), patritumab (anti-ERBB3), cetuximab (anti-EGFR), nimotuzumab (anti-EGFR), matuzumab (anti-EGFR), portuzumab (anti-HER2), sitatuzumab (anti-TACSTD1), tucotuzumab (anti-EpCAM), and endrecolomab (anti-EpCAM).

[0400] In a specific embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is selected from the group consisting of brentuximab (anti-CD30), gemtuzumab (anti-CD30), trastuzumab (anti-Her2 / neu), inotuzumab (anti-CD22), polatuzumab (anti-CD79b), enfortumab (anti-nectin-4), sacituzumab (anti-Trop-2), and belantamab (anti-BCMA).

[0401] In a particular embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the antibody is polatuzumab (anti-CD79b) or trastuzumab (anti-Her2 / neu) or enfortumab (anti-nectin-4).

[0402] In specific embodiments, the invention relates to antibodies that are capable of binding to, or are capable of binding to, CD30, Her2 / neuCD33, CD22, PD-L1, EGFR, CD20, CD38, HER2, integrin gα4β7, CD20, IL-6-R, IL-12, IL-23, TNFα, CD20, Trop-2, BCMA, CD79b, Nectin-4, EpCAM, CD33, CD19, AXL, dn-collagen, TA-MUC1, carcinoembryonic cell adhesion molecule 5, CEACAM, or other antigen-specific antibodies. The present invention relates to an antibody-payload conjugate according to the present invention which specifically binds to an antigen selected from the group consisting of CD30, Her2 / neu, CD22, CD79b, Nectin-4, Trop-2, and BCMA, more preferably CD79b, Her2 / neu, and Nectin-4.

[0403] In a particular embodiment, the present invention relates to a method for preparing an antibody-payload conjugate comprising the step of conjugating a peptide linker according to the invention to an antibody.

[0404] That is, any of the peptide linkers comprising two or more payloads disclosed herein can be conjugated to an antibody. In particular, any of the peptide linkers comprising amines disclosed herein can be conjugated to the glutamine residue of an antibody via transglutaminase. As disclosed elsewhere herein, the glutamine residue to which the peptide linker is conjugated can be an endogenous glutamine residue (e.g., Q295 of an IgG antibody) or a glutamine residue introduced into the antibody by molecular engineering.

[0405] In a specific embodiment, the present invention relates to a method for conjugating a peptide linker comprising two or more payloads to an antibody using transglutaminase (TG), comprising: (a) mixing the antibody, the peptide linkers, and the TG in a fluid, thereby conjugating the linker-payloads to the antibody in one step under the catalytic action of the TG; and (b) extracting the conjugates obtained in step (a) from the fluid.

[0406] Thus, the present invention further encompasses a method for conjugating a peptide linker comprising two or more payloads to an antibody in a one-step reaction using transglutaminase. For this purpose, the antibody may be mixed with the peptide linker according to the invention and transglutaminase in a fluid. A "fluid" in the sense of the present invention is a liquid. Preferably, the liquid is an aqueous solution, even more preferably a buffered aqueous solution.

[0407] The peptide linker according to the present invention can be mixed with the antibody and transglutaminase by mixing a solution containing the peptide linker with a solution containing the antibody and a solution containing transglutaminase. Alternatively, solutions containing the peptide linker, the antibody and the transglutaminase separately can be added to an aqueous solution. In particular, each component can be added to the aqueous solution at a defined concentration. The peptide linker according to the present invention is conjugated to the antibody under the catalytic action of the transglutaminase. That is, the individual components can be mixed under conditions suitable for efficient conjugation of the peptide linker to the antibody. Such conditions are defined elsewhere herein.

[0408] In a second method step, the resulting antibody-payload conjugate must be removed from the liquid. The skilled artisan is aware of methods for isolating the antibody-payload conjugate from an aqueous solution. Furthermore, the skilled artisan is aware of methods for separating the antibody-payload conjugate from unconjugated antibodies or peptide linkers or from incompletely conjugated antibodies. For example, the antibody-payload conjugate according to the invention can be isolated from the mixture by HPLC.

[0409] It should be understood that "extracting the conjugate from a fluid" is synonymous with "isolating the conjugate from a mixture," i.e., the conjugate can also be extracted by removing transglutaminase and unconjugated antibody and peptide linker from the fluid.

[0410] The peptide linker used in the method according to the invention may be any one of the peptide linkers disclosed herein, in particular any peptide linker falling within the definition provided herein above or any peptide linker shown in the experimental examples.

[0411] Thus, in a particular embodiment, the present invention relates to a method according to the invention, wherein the peptide linker is a peptide linker according to the invention.

[0412] Furthermore, the antibody may be as defined in more detail elsewhere herein, i.e. for the antibody-payload conjugates according to the invention.

[0413] In particular, the peptide linker may comprise an amino acid sequence as set forth in SEQ ID NOs: 1 to 29 or 82 to 93. Furthermore, the linker may be any one of the linkers shown in Figures 1 to 40 or 42 to 43.

[0414] Thus, in a specific embodiment, the present invention relates to a method according to the present invention, wherein the peptide linker is conjugated to a glutamine residue in the antibody via a primary amine contained in an amino acid residue of the peptide linker.

[0415] In a particular embodiment, the invention relates to a method according to the invention, wherein the antibody is an antibody fragment.

[0416] In a specific embodiment, the invention relates to a method according to the invention, wherein the antibody is an IgA, IgD, IgE, IgG or IgM antibody.

[0417] In a specific embodiment, the invention relates to a method according to the invention, wherein the peptide linker is conjugated to a glutamine residue contained in the Fc domain of the antibody.

[0418] In a specific embodiment, the present invention relates to a method for the preparation of a peptide linker conjugated to a glutamine residue in an IgG antibody. H The glutamine residue Q295 (EU numbering) of domain 2 relates to a method according to the present invention.

[0419] In a particular embodiment, the method according to the invention relates to a method wherein a glutamine residue to which a peptide linker is conjugated has been introduced into the heavy or light chain of the antibody by molecular engineering.

[0420] In a specific embodiment, the present invention relates to a method for the preparation of a glycosylated IgG antibody in which a glutamine 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).

[0421] In a specific embodiment, the invention relates to a method according to the invention, wherein the glutamine 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.

[0422] 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.

[0423] In a particular embodiment, the invention relates to a method according to the invention, wherein the antibody is a glycosylated IgG antibody.

[0424] In a specific embodiment, the present invention relates to an IgG antibody. 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.

[0425] In a specific embodiment, the invention relates to an antibody that is selected from the group consisting of brentuximab (anti-CD30), trastuzumab (anti-Her2 / neu), gemtuzumab (anti-CD33), inotuzumab (anti-CD22), avelumab (anti-PD-L1), cetuximab (anti-EGFR), rituximab (anti-CD20), daratumumab (anti-CD38), pertuzumab (anti-HER2), vedolizumab (anti-integrin α4β7), ocrelizumab (anti-CD20), Tocilizumab (anti-IL-6-R), ustekinumab (anti-IL-12 / 23), golimumab (anti-TNFα), obinutuzumab (anti-CD20), sacituzumab (anti-Trop-2), belantamab (anti-BCMA), polatuzumab (anti-CD79b), enfortumab (anti-nectin-4), endrecolomab (anti-EpCAM), gemtuzumab (anti-CD33), loncastuximab (anti-CD19), mecbotamab (anti-AXL), adecatumab (anti-IL-6-R), sacituzumab (anti-Trop-2), belantamab (anti-BCMA), polatuzumab (anti-CD79b), enfortumab (anti-N ...IL-6-R), sacituzumab (anti-IL-6-R), sacituzumab (anti-IL-6-R), sacituzumab (anti-IL-6-R), sacituzumab (anti-IL-6-R), sacituzumab (anti-IL-6-R), sacituzumab (anti-IL-6-R), sacituzum (anti-EpCAM), D93 (anti-dn-collagen), gatipotuzumab (anti-TA-MUC1), labetuzumab (anti-carcinoembryonic cell adhesion molecule 5), tusamitamab (anti-CEACAM5), upifitamab (anti-NaPi2b), rifastuzumab (anti-NaPi2b), mirvetuximab (anti-FRα), sofituzumab (anti-MUC16), anetumab (anti-mesothelin), tisotumab (anti-TF), cofituzumab (anti-Trop-2), The present invention relates to a method according to the present invention, in which the antibody is selected from the group consisting of larzatamab (anti-CD166), radriatuzumab (anti-LIV-1), belantamab (anti-BCMA), patritumab (anti-ERBB3), cetuximab (anti-EGFR), nimotuzumab (anti-EGFR), matuzumab (anti-EGFR), portuzumab (anti-HER2), sitatuzumab (anti-TACSTD1), tucotuzumab (anti-EpCAM), and endrecolomab (anti-EpCAM).

[0426] In a specific embodiment, the invention relates to a method according to the invention, wherein the antibody is selected from the group consisting of brentuximab (anti-CD30), gemtuzumab (anti-CD30), trastuzumab (anti-Her2 / neu), inotuzumab (anti-CD22), polatuzumab (anti-CD79b), enfortumab (anti-nectin-4), sacituzumab (anti-Trop-2), and belantamab (anti-BCMA).

[0427] In a specific embodiment, the invention relates to a method according to the invention, wherein the antibody is polatuzumab (anti-CD79b) or trastuzumab (anti-Her2 / neu) or enfortumab (anti-Nectin-4).

[0428] In specific embodiments, the invention relates to antibodies that are capable of binding to, or are capable of binding to, CD30, Her2 / neuCD33, CD22, PD-L1, EGFR, CD20, CD38, HER2, integrin α4β7, CD20, IL-6-R, IL-12, IL-23, TNFα, CD20, Trop-2, BCMA, CD79b, Nectin-4, EpCAM, CD33, CD19, AXL, dn-collagen, TA-MUC1, carcinoembryonic cell adhesion molecule 5, The present invention relates to a method which specifically binds to an antigen selected from the group consisting of CEACAM5, NaPi2b, FRα, MUC16, mesothelin, TF, CD166, LIV-1, ERBB3, EGFR, and TACSTD1, preferably CD30, Her2 / neu, CD22, CD79b, nectin-4, Trop-2, and BCMA, more preferably CD79b, Her2 / neu, and nectin-4.

[0429] In a specific embodiment, the invention relates to a method according to the invention, wherein the peptide linker is conjugated to the γ-carboxamide group of a Gln residue contained in the antibody.

[0430] In specific embodiments, the invention relates to a method according to the invention, wherein the peptide 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%.

[0431] That is, in certain embodiments, the peptide linker of the present invention 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 peptide linker of the present invention can be conjugated to a glycosylated antibody with at least 70% efficiency. In another preferred embodiment, the peptide linker of the present invention can be conjugated to a glycosylated antibody with at least 75% efficiency. In another preferred embodiment, the peptide linker of the present invention can be conjugated to a glycosylated antibody with at least 80% efficiency. In another preferred embodiment, the peptide linker of the present invention can be conjugated to a glycosylated antibody with at least 85% efficiency. In another preferred embodiment, the peptide linker of the present invention can be conjugated to a glycosylated antibody with at least 90% efficiency. In another preferred embodiment, the peptide linker of the present invention can be conjugated 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).

[0432] The skilled artisan will be aware of how to determine the conjugation efficiency of an antibody with a particular peptide linker. For example, the conjugation efficiency can be determined as described herein. That is, an antibody, particularly an IgG1 antibody, may be incubated at a concentration of 1-5 mg / mL with 5-20 eq molar equivalents of linker per mg of antibody and 3-6 U of microbial transglutaminase in a suitable buffer at 37° C. for 20-48 hours or as described in Example 1. After the incubation period, the conjugation efficiency can be determined by LC-MS analysis under reducing conditions. The microbial transglutaminase may be, for example, MTG from Streptomyces mobaraensis available from Zedira (Germany). Suitable buffers may be Tris, MOPS, HEPES, PBS or BisTris buffers. However, it should be understood that the choice of buffer system may be different and may depend largely on the chemical nature of the linker. However, the skilled artisan will be able to 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).

[0433] In a particular embodiment, the antibody can be conjugated by incubating 5 mg / mL of native glycosylated monoclonal antibody in 50 mM Tris pH 7.6 with microbial transglutaminase (MTG, Zedira) at a concentration of 5-10 U / mg antibody and 5 molar equivalents of the indicated linker payload for 24 hours at 37° C. in a rotating thermomixer. However, it should be understood that the conditions, particularly the buffer conditions and peptide linker concentration, may be adjusted depending on the properties of the payload(s). However, the skilled artisan will be able to identify optimal reaction conditions based on the teachings provided in the present invention.

[0434] In a particular embodiment, the present invention relates to a method according to the invention, wherein the transglutaminase is a microbial transglutaminase (MTG).

[0435] The transglutaminase for use in the methods of the invention may be any transglutaminase suitable for conjugating the peptide linker of the invention to an antibody. The transglutaminase may be of any origin, for example, the transglutaminase may be of bacterial, archaeal or eukaryotic origin.

[0436] In certain embodiments, the transglutaminase may be a mammalian transglutaminase, including human transglutaminase, hi certain embodiments, the transglutaminase may be a microbial transglutaminase, including bacterial and fungal transglutaminase.

[0437] 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.

[0438] 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. Thus, MTG may be the native enzyme or a genetically engineered variant of the native enzyme.

[0439] 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: 78. S. mobaraensis MTG variants with other amino acid sequences have also been reported and are also encompassed by the present invention (SEQ ID NOs: 79 and 80).

[0440] One such microbial transglutaminase may be the MTG-TX variant from S. mobaraensis described in Jin et al. 2016, Journal of Molecular Catalysis B:Enzymatic, which exhibits high salt tolerance and wide range of pH and temperature stability.

[0441] 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:81.

[0442] Any of the above transglutaminases may have a modified sequence. In some embodiments, a transglutaminase having 80%, 85%, 90%, 95% or more sequence identity to any one of SEQ ID NOs: 78 to 81 may be used.

[0443] Another suitable microbial transglutaminase is commercially available from Ajinomoto, called ACTIVA TG. Compared to the transglutaminase from Zedira, ACTIVA TG is missing four amino acids at the N-terminus, but has similar activity.

[0444] Further microbial transglutaminases that can 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 entire contents of which are incorporated herein by reference.

[0445] 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: 78 or 79. In certain embodiments, the recombinant S. mobaraensis transglutaminase as set forth in SEQ ID NO: 78 can comprise the mutation G254D. In certain embodiments, the recombinant S. mobaraensis transglutaminase as set forth in SEQ ID NO: 78 can comprise the mutations G254D and E304D. In certain embodiments, the recombinant S. mobaraensis transglutaminase as set forth in SEQ ID NO: 78 can comprise the mutations D8E and G254D. In certain embodiments, the recombinant S. mobaraensis transglutaminase as set forth in SEQ ID NO: 78 can comprise the mutations E124A and G254D. In certain embodiments, the recombinant S. mobaraensis transglutaminase set forth in SEQ ID NO: 78 may comprise the mutations A216D and G254D. In certain embodiments, the recombinant S. mobaraensis transglutaminase set forth in SEQ ID NO: 78 may comprise the mutations G254D and K331T.

[0446] In a particular embodiment, the present invention relates to a method according to the invention, wherein transglutaminase is added to the conjugation reaction in a concentration of less than 200 U / mg antibody.

[0447] The microbial transglutaminase may be added to the conjugation reaction at any concentration that allows the antibody to be efficiently conjugated to the linker. In certain embodiments, the concentration of the microbial transglutaminase in the conjugation reaction may depend on the amount of antibody used in the same reaction. For example, the microbial transglutaminase may be added to the conjugation reaction at a concentration of less than 200 U / mg antibody, 150 U / mg antibody, 100 U / mg antibody, 90 U / mg antibody, 80 U / mg antibody, 70 U / mg antibody, 60 U / mg antibody, 50 U / mg antibody, 40 U / mg antibody, 30 U / mg antibody, 20 U / mg antibody, 10 U / mg antibody, or 6 U / mg antibody.

[0448] In certain embodiments, microbial transglutaminase may be added to the conjugation reaction at a concentration of 1 U / mg antibody. In certain embodiments, microbial transglutaminase may be added to the conjugation reaction at a concentration of 3 U / mg antibody. In certain embodiments, microbial transglutaminase may be added to the conjugation reaction at a concentration of 5 U / mg antibody. In certain embodiments, microbial transglutaminase may be added to the conjugation reaction at a concentration of 6 U / mg antibody. In certain embodiments, microbial transglutaminase may be added to the conjugation reaction at a concentration of 7.5 U / mg antibody. In certain embodiments, microbial transglutaminase may be added to the conjugation reaction at a concentration of 10 U / mg antibody.

[0449] In certain embodiments, microbial transglutaminase may be added to the conjugation reaction at a concentration of 1-100 U / mg antibody. In certain embodiments, microbial transglutaminase may be added to the conjugation reaction at a concentration of 3-50 U / mg antibody. In certain embodiments, microbial transglutaminase may be added to the conjugation reaction at a concentration of 5-25 U / mg antibody.

[0450] In certain embodiments, microbial transglutaminase may be added to the conjugation reaction at a concentration of 1-20 U / mg antibody, preferably 3-15 U / mg antibody, and more preferably 5-10 U / mg antibody.

[0451] Preferably, the transglutaminase for use in the method of the present invention is a microbial transglutaminase. However, it should be noted that an equivalent reaction can be carried out by an enzyme with transglutaminase activity of non-microbial origin. Thus, the antibody-payload conjugate of the present invention can also be produced using an enzyme with transglutaminase activity of non-microbial origin.

[0452] In a specific embodiment, the present invention relates to a method according to the invention, wherein the antibody is added to the conjugation reaction at a concentration of 0.1 to 50 mg / mL.

[0453] The antibody may be added to the conjugation reaction at any concentration suitable to obtain efficient conjugation of the antibody. 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 specific 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 of 0.1 to 50 mg / mL, preferably 0.25 to 25 mg / mL / mL, more preferably 0.5 to 12.5 mg / mL, even more preferably 1 to 10 mg / mL, even more preferably 2 to 7.5 mg / mL, and most preferably about 5 mg / mL.

[0454] Alternatively, the antibody may be added to the conjugation reaction at a concentration in the range of 1 to 20 mg / mL, preferably 2.5 to 20 mg / mL, more preferably 5 to 20 mg / mL, and most preferably 5 to 17 mg / mL.

[0455] In a specific embodiment, the invention relates to a method according to the invention, wherein the antibody is contacted with 2 to 100 molar equivalents of the peptide linker.

[0456] To obtain efficient conjugation, it is preferred to add the linker to the antibody in molar excess, i.e., in certain embodiments, the antibody is mixed with at least 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 molar equivalents of linker.

[0457] That is, in a specific embodiment, the present invention relates to a method according to the present invention, in which an antibody is contacted with 2 to 100 molar equivalents of a linker, preferably 2 to 80 molar equivalents of a linker, more preferably 2 to 70 molar equivalents of a linker, even more preferably 2 to 60 molar equivalents of a linker, even more preferably 2 to 50 molar equivalents of a linker, even more preferably 2 to 40 molar equivalents of a linker, even more preferably 2 to 30 molar equivalents of a linker, even more preferably 2 to 25 molar equivalents of a linker, even more preferably 2 to 20 molar equivalents of a linker, even more preferably 2 to 15 molar equivalents of a linker, and most preferably 2 to 10 molar equivalents of a linker.

[0458] Alternatively, the antibody may be contacted with 2.5 to 100 molar equivalents of the linker, preferably 2.5 to 80 molar equivalents of the linker, more preferably 2.5 to 70 molar equivalents of the linker, even 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 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.

[0459] Alternatively, the antibody may be contacted with 5 to 100 molar equivalents of the linker, preferably 5 to 80 molar equivalents of the linker, more preferably 5 to 70 molar equivalents of the linker, even more preferably 5 to 60 molar equivalents of the linker, even more preferably 5 to 50 molar equivalents of the linker, even more preferably 5 to 40 molar equivalents of the linker, even more preferably 5 to 30 molar equivalents of the linker, even more preferably 5 to 20 molar equivalents of the linker, even more preferably 5 to 15 molar equivalents of the linker, and most preferably 5 to 10 molar equivalents of the linker.

[0460] In a particular embodiment, the present invention relates to a method according to the invention, wherein the conjugation reaction is carried out in a buffered solution.

[0461] The method according to the invention is preferably carried out at a pH in the range of 5 to 10. 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 5 to 10, preferably at a pH in the range of 6 to 9, more preferably at a pH in the range of 6 to 8.5, even more preferably at a pH in the range of 6.5 to 8 and most preferably at a pH in the range of 6.6 to 7.6.

[0462] In a particular embodiment, the invention relates to a method according to the invention, wherein conjugation of the linker to the antibody is achieved at pH 6.6.

[0463] In a particular 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.

[0464] 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, buffers used in the methods of the invention may have a salt concentration of <250 mM, <200 mM, <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 contain no salt.

[0465] That is, in a specific embodiment, the present invention relates to a buffer solution comprising: a) a pH in the range of 5 to 10; and / or b) a buffer concentration in the range of 10 to 1000 mM; and / or c) Salt concentrations in the range of less than 250 mM The present invention relates to a method comprising the steps of:

[0466] In a preferred embodiment, the present invention relates to a buffer solution comprising: a) a pH in the range of 6 to 9; and / or b) a buffer concentration in the range of 10 to 1000 mM; and / or c) Salt concentrations in the range of less than 250 mM The present invention relates to a method comprising the steps of:

[0467] In a more preferred embodiment, the present invention relates to a buffer solution comprising: a) a pH in the range of 6 to 8; and / or b) a buffer concentration in the range of 10 to 500 mM; and / or c) Salt concentration less than 150 mM The present invention relates to a method comprising the steps of:

[0468] In an even more preferred embodiment, the present invention relates to a buffer solution comprising: a) a pH in the range of 6 to 8; and / or b) a buffer concentration in the range of 10 to 200 mM; and / or c) Salt concentration less than 50 mM The present invention relates to a method comprising the steps of:

[0469] In a preferred embodiment, the method of the invention is carried out in 50 mM Tris, pH 7.6, preferably without salt.

[0470] In another preferred embodiment, the method of the invention is carried out in 50 mM BisTris, pH 6.6, preferably without salt.

[0471] In another preferred embodiment, the method of the invention is carried out in 50 mM BisTris, pH 7.5, preferably without salt.

[0472] 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 undertake undue experimentation to identify suitable reaction conditions for carrying out the methods of the invention.

[0473] It is to be understood that the present application encompasses any combination of linkers, antibodies, MTG, and / or buffer concentrations disclosed above.

[0474] In certain embodiments, the invention relates to methods according to the invention, wherein the antibody is contacted with 2-80 molar equivalents of linker; and / or microbial transglutaminase is added to the conjugation reaction at a concentration ranging from 1-20 U / mg antibody, and optionally the antibody is added to the conjugation reaction at a concentration ranging from 0.1-20 mg / mL.

[0475] In a preferred embodiment, the invention relates to a method according to the invention, wherein the antibody is contacted with 2-50 molar equivalents of a linker; and / or microbial transglutaminase is added to the conjugation reaction at a concentration in the range of 1-15 U / mg antibody, and optionally the antibody is added to the conjugation reaction at a concentration in the range of 1-20 mg / mL.

[0476] In a more preferred embodiment, the invention relates to a method according to the invention, wherein the antibody is contacted with 2-30 molar equivalents of linker; and / or microbial transglutaminase is added to the conjugation reaction at a concentration in the range of 2-15 U / mg antibody, and optionally the antibody is added to the conjugation reaction at a concentration in the range of 2.5-20 mg / mL.

[0477] In an even more preferred embodiment, the invention relates to a method according to the invention, wherein the antibody is contacted with 2-20 molar equivalents of linker; and / or microbial transglutaminase is added to the conjugation reaction at a concentration in the range of 5-15 U / mg antibody, and optionally the antibody is added to the conjugation reaction at a concentration in the range of 2.5-20 mg / mL.

[0478] In an even more preferred embodiment, the invention relates to a method according to the invention, wherein the antibody is contacted with 2-15 molar equivalents of linker; and / or microbial transglutaminase is added to the conjugation reaction at a concentration in the range of 5-15 U / mg antibody, and optionally the antibody is added to the conjugation reaction at a concentration in the range of 5-20 mg / mL.

[0479] In a most preferred embodiment, the invention relates to a method according to the invention, wherein the antibody is contacted with 2.5 to 12.5 molar equivalents of linker; and / or microbial transglutaminase is added to the conjugation reaction at a concentration in the range of 5 to 15 U / mg antibody, and optionally the antibody is added to the conjugation reaction at a concentration in the range of 5 to 20 mg / mL.

[0480] In another preferred embodiment, the invention relates to a method according to the invention, wherein the antibody is contacted with 2-20 molar equivalents of linker; and / or microbial transglutaminase is added to the conjugation reaction at a concentration in the range of 5-15 U / mg antibody, and optionally the antibody is added to the conjugation reaction at a concentration in the range of 2.5-20 mg / mL.

[0481] It should be noted that the specific reaction mixtures disclosed above can be freely combined with any of the buffer conditions disclosed herein, however, the specific components defined above are preferably mixed at a pH in the range of 6-8.

[0482] In a particular embodiment, the present invention relates to an antibody-payload conjugate produced by the method according to the present invention.

[0483] Thus, the present invention further relates to antibody-linker conjugates produced using any of the aforementioned method steps.

[0484] In a particular embodiment, the present invention relates to a pharmaceutical composition comprising an antibody-payload conjugate according to the invention and at least one pharma- ceutically acceptable ingredient.

[0485] Thus, the present invention further relates to a pharmaceutical composition comprising an antibody-payload conjugate according to the present invention.

[0486] The term "pharmaceutical composition" as used herein refers to any composition containing a chemical or active ingredient, intended for use in the medical cure, treatment, or prevention of disease, and in a form that allows the active ingredient to be effective. In particular, a pharmaceutical composition does not contain excipients that are unacceptably toxic to the subject to which the composition is administered. A pharmaceutical composition is sterile, i.e., aseptic and free of all viable microorganisms and their spores. The pharmaceutical composition of the present invention is preferably liquid.

[0487] The type of payload included in the antibody-payload construct included in the pharmaceutical composition will vary depending on the intended use of the pharmaceutical composition. In embodiments where the pharmaceutical composition is used to treat a disease, the payload is preferably a drug. If the disease is a neoplastic disease, the payload is preferably a toxin. In embodiments where the pharmaceutical composition is used in diagnosis, the payload is preferably an imaging agent.

[0488] The pharmaceutical composition according to the present invention may comprise the antibody-drug conjugate disclosed herein. The pharmaceutical composition comprising the antibody-drug conjugate is preferably used for the treatment of a disease.

[0489] The pharmaceutical compositions according to the present invention may comprise at least one pharma- ceutically acceptable ingredient.

[0490] A pharma- ceutically acceptable ingredient refers to an ingredient in a pharmaceutical formulation other than an active ingredient that is non-toxic to a subject. Pharmaceutically acceptable ingredients include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0491] Pharmaceutical formulations of the antibody-payload 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 the recipient 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; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; Examples of suitable pharmacokinetic or pharmacokinetically acceptable components include, but are not limited to, glycines, 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 insterstitial drug dispersing agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., 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.

[0492] In a specific embodiment, the present invention relates to a pharmaceutical composition according to the invention, comprising at least one additional therapeutically active agent.

[0493] That is, the pharmaceutical composition comprising the antibody-payload conjugate may contain one or more additional therapeutically active agents. It should be understood that the antibody-payload conjugate can be used in various therapeutic areas. Thus, the additional therapeutically active agent in the pharmaceutical composition may vary depending on the use of the pharmaceutical composition.

[0494] In certain embodiments, pharmaceutical compositions comprising the antibody-payload conjugates of the present invention can be used to treat cancer. In such embodiments, the pharmaceutical compositions may contain one or more additional anti-cancer drugs. As used herein, the term "anti-cancer" drug refers to one or a combination of drugs conventionally used to treat cancer.

[0495] For example, a pharmaceutical composition comprising an antibody-payload conjugate according to the invention may further comprise one or more chemotherapeutic agents. As used herein, the term "chemotherapeutic agent" or "chemotherapeutic agent" or "chemotherapeutic drug" refers to an agent that can be used in a pharma- ceutical effective amount to reduce, prevent, reduce, limit, and / or slow the growth of metastases or neoplasms, or to directly kill neoplastic cells by neoplastic necrosis or apoptosis or any other mechanism, or to otherwise reduce, prevent, reduce, limit, and / or slow the growth of metastases or neoplasms in a subject with a neoplastic disease. Chemotherapeutic agents include, for example, fluoropyrimidines; pyrimidine nucleosides; purine nucleosides; antifolates, platinum agents; anthracyclines / anthracenediones; epipodophyllotoxins; camptothecins; hormones; hormone conjugates; antihormones; enzymes, proteins, peptides, and polyclonal and / or monoclonal antibodies; vinca alkaloids; taxanes; epothilones; antimicrotubule agents; alkylating agents; antimetabolites; topoisomerase inhibitors; antiviral agents; and various other cytotoxic and cytostatic agents.

[0496] In a particular embodiment, the present invention relates to an antibody-payload conjugate according to the invention or a pharmaceutical composition according to the invention for use in therapy and / or diagnosis.

[0497] That is, the antibody-payload conjugate or pharmaceutical composition according to 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, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as macaques), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human. When the antibody-payload conjugate or pharmaceutical composition comprising the antibody-payload conjugate according to the present invention is used for treatment, it is preferred that the payload is a drug. When the antibody-payload conjugate or pharmaceutical composition comprising the antibody-payload conjugate according to the present invention is used for diagnosis, it is preferred that the linker comprises at least one imaging agent as a payload.

[0498] In a specific embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: Neoplastic, neurological, autoimmune, inflammatory, or infectious diseases suffer from, are at risk of developing and / or Diagnosed with The present invention relates to an antibody-payload conjugate according to the invention or a pharmaceutical composition according to the invention for use in the treatment of a patient.

[0499] In a particular embodiment, the invention relates to an antibody-payload conjugate according to the invention or a pharmaceutical composition according to the invention for use in the treatment of a patient suffering from a neoplastic disease.

[0500] The patient suffering from cancer may be a patient who has not been previously treated with any anti-cancer therapy, however, the patient suffering from cancer may also be a patient who has been refractory to previous anti-cancer treatments.

[0501] 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 carcinoma, salivary gland carcinoma, kidney cancer, vulvar cancer, thyroid cancer, liver carcinoma, 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.

[0502] That is, the antibody-payload conjugate of the present invention is preferably used for the treatment of cancer. Thus, in certain embodiments, the antibody-payload conjugate 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 internalized together with the antibody-payload conjugate when the antibody-payload conjugate binds to the antigen.

[0503] When the antibody-payload conjugates of the present invention are used to treat cancer, the antibody-payload conjugates preferably contain at least one payload capable of killing or inhibiting the proliferation of tumor cells to which the antibody-payload conjugate binds. In certain embodiments, the at least one payload exhibits cytotoxic activity after the antibody-payload conjugate is internalized in tumor cells. In certain embodiments, the at least one payload is a toxin.

[0504] In a particular embodiment, the invention relates to an antibody-payload conjugate according to the invention or a pharmaceutical composition according to the invention for use in the treatment of a patient suffering from an autoimmune disease.

[0505] In a particular embodiment, the invention relates to an antibody-payload conjugate according to the invention or a pharmaceutical composition according to the invention for use in the treatment of a patient suffering from a bacterial or viral infection.

[0506] In a particular embodiment, the antibody-payload conjugates and / or pharmaceutical compositions according to the invention may be used in the treatment of B-cell related cancers.

[0507] Thus, in a specific embodiment, the invention relates to an antibody-payload conjugate or a pharmaceutical composition for use according to the invention, wherein the antibody-payload conjugate comprises polatuzumab and the neoplastic disease is a B-cell related cancer.

[0508] To that end, it is preferred that the antibody-payload conjugate comprises an anti-CD79b antibody as disclosed herein, which is preferably internalized into target cells 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: 71 and a light chain as set forth in SEQ ID NO: 72. It is further preferred that the antibody-payload conjugate comprises at least one toxin.

[0509] In certain embodiments, the anti-CD79b antibody comprised in the antibody-payload conjugate or pharmaceutical composition may be conjugated to any one of the linkers shown in Figures 1-40 or any one of the linkers disclosed herein.

[0510] 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.

[0511] In a particular embodiment, the invention relates to an antibody-payload 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.

[0512] Furthermore, the anti-CD79b antibody-payload conjugates and / or pharmaceutical compositions comprising anti-CD79b antibody-payload conjugates may be used in combination with other therapies suitable for the treatment of B-cell related cancers.

[0513] Thus, in a specific embodiment, the invention relates to an antibody-payload conjugate or a pharmaceutical composition for use according to the invention, which is administered in combination with bendamustine and / or rituximab.

[0514] It is understood that the antibody-payload 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-payload conjugate or pharmaceutical composition may be administered on a different dosing schedule, and consequently on different days, than other therapeutic agents used to treat the same disease.

[0515] In certain embodiments, the antibody-payload conjugates and / or pharmaceutical compositions according to the invention may be used in the treatment of HER2-positive cancers.

[0516] Thus, in a specific embodiment, the invention relates to an antibody-payload conjugate or a pharmaceutical composition for use according to the invention, wherein the antibody-payload 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.

[0517] To that end, the antibody-payload conjugate preferably comprises an anti-HER2 / neu antibody as disclosed herein, which preferably is internalized into target cells 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: 73 and a light chain set forth in SEQ ID NO: 74. Additionally, the antibody-payload conjugate preferably comprises at least one toxin.

[0518] In certain embodiments, the anti-HER2 / neu antibody comprised in the antibody-payload conjugate or pharmaceutical composition can be conjugated to any one of the linkers shown in Figures 1-40 or any one of the linkers disclosed herein.

[0519] 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.

[0520] Furthermore, the anti-HER2 / neu antibody-payload conjugates and / or pharmaceutical compositions comprising anti-HER2 / neu antibody-payload conjugates may be used in combination with other therapies suitable for the treatment of HER2 cell positive cancers.

[0521] Thus, in a specific embodiment, the invention relates to an antibody-payload conjugate or pharmaceutical composition for use according to the invention, wherein the antibody-payload conjugate or pharmaceutical composition is administered in combination with lapatinib, capecitabine, and / or a taxane.

[0522] It is understood that the antibody-payload 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-payload conjugate or pharmaceutical composition may be administered on a different dosing schedule, and consequently on different days, than other therapeutic agents used to treat the same disease.

[0523] In a particular embodiment, the antibody-payload conjugate and / or pharmaceutical composition according to the present invention can be used for the treatment of Nectin-4 positive cancers.

[0524] Thus, in a specific embodiment, the present invention relates to an antibody-payload conjugate or a pharmaceutical composition for use according to the present invention, wherein the antibody-payload 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.

[0525] To that end, it is preferred that the antibody-payload conjugate comprises an anti-Nectin-4 antibody disclosed herein, which is preferably internalized into 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: 75 and a light chain set forth in SEQ ID NO: 76 or 77. Furthermore, it is preferred that the antibody-payload conjugate comprises at least one toxin.

[0526] In certain embodiments, the anti-Nectin-4 antibody contained in the antibody-payload conjugate or pharmaceutical composition can be conjugated to any one of the linkers shown in Figures 1 to 40 or any one of the linkers disclosed herein.

[0527] 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.

[0528] The anti-Nectin-4 antibody-payload conjugate and / or pharmaceutical composition comprising the anti-Nectin-4 antibody-payload conjugate of the present invention may be administered alone to patients who have previously received a PD-1 or PD-L1 inhibitor in combination with a platinum-based chemotherapy agent before or after surgery.

[0529] Furthermore, the anti-Nectin-4 antibody-payload conjugate and / or the pharmaceutical composition comprising the anti-Nectin-4 antibody-payload conjugate may be used in combination with other therapies suitable for the treatment of Nectin-4 positive cancers.

[0530] Thus, in a specific embodiment, the invention relates to an antibody-payload conjugate or a pharmaceutical composition for use according to the invention, wherein the antibody-payload conjugate or the pharmaceutical composition is administered in combination with a cisplatin-based chemotherapeutic agent and / or pembrolizumab.

[0531] It is understood that the antibody-payload conjugate or pharmaceutical composition does not necessarily have to be administered simultaneously with additional therapeutic agents such as cisplatin-based chemotherapeutic agents and / or pembrolizumab. Instead, the antibody-payload conjugate or pharmaceutical composition may be administered on a different schedule, and consequently on different days, than other therapeutic agents used to treat the same disease.

[0532] In specific embodiments, the present invention relates 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 The present invention relates to the use of an antibody-payload conjugate according to the invention or a pharmaceutical composition according to the invention for the manufacture of a medicament for treating a patient.

[0533] In a particular embodiment, the present invention relates to a method for treating or preventing a neoplastic disease, comprising administering an antibody-payload conjugate according to the invention or a pharmaceutical composition according to the invention to a patient in need thereof.

[0534] In a particular embodiment, the present invention relates to an antibody-payload conjugate according to the invention or a pharmaceutical composition according to the invention for use in pre-operative, intra-operative or post-operative imaging.

[0535] That is, the antibody-payload conjugate of the present invention can be used in medical imaging. To this end, the antibody-payload 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-payload conjugate binds can be visualized by PET or SPECT. If the payload is a fluorescent dye, the molecule, cell, or tissue to which the antibody-payload conjugate binds can be visualized by fluorescence imaging. In a particular embodiment, the antibody-payload conjugate of the present invention comprises two different payloads, e.g., a radionuclide and a fluorescent dye. In this case, the molecule, cell, or tissue to which the antibody-payload conjugate binds can be visualized using two different and / or complementary imaging techniques, such as PET / SPECT and fluorescence imaging.

[0536] The antibody-payload conjugates can be used for pre-operative, intra-operative, and / or post-operative imaging.

[0537] 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 the tumor by PET or SPECT prior to performing surgery, by 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.

[0538] Intraoperative imaging encompasses all imaging techniques that can be performed during surgery to visualize specific target molecules, cells, or tissues and provide guidance to the surgeon. 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 the surgeon to identify specific tissues, such as tumor tissues, during surgery, so that the tumor tissue can be completely removed.

[0539] Postoperative imaging encompasses all imaging techniques that can be performed after surgery to visualize specific target molecules, cells, or tissues and evaluate the outcome of surgery. Postoperative imaging can be performed in the same manner as preoperative imaging.

[0540] In particular, the present invention relates to an antibody-payload conjugate that comprises two or more different payloads. For example, an 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.

[0541] In a particular embodiment, the present invention relates to an antibody-payload conjugate according to the invention or a pharmaceutical composition according to the invention, in particular an antibody-payload conjugate comprising two payloads for use in intraoperative image-guided cancer surgery.

[0542] As mentioned above, the antibody-payload 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-payload conjugates can be used to visualize tumor tissue during surgery, for example by near-infrared imaging, to allow complete removal of the tumor tissue.

[0543] The antibody-payload 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.

[0544] The antibody-payload 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.

[0545] The antibody-payload conjugates or pharmaceutical compositions of the present invention may be formulated, dosed, and administered in a manner consistent with good medical practice. 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-payload conjugates or pharmaceutical compositions of the present invention are 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-payload conjugate present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and routes of administration as described herein, or at about 1-99% of the dosages described herein, or at any dosage and by any route determined empirically / clinically appropriate.

[0546] The appropriate dosage of the antibody-payload conjugate or pharmaceutical composition of the invention (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-payload conjugate or pharmaceutical composition of the invention is suitably administered to the patient at one time or over a series of treatments. EXAMPLES

[0547] general law The antibody trastuzumab (Herceptin®, Roche, purchased from a pharmacy) and all peptide-linkers and linker-payloads (custom synthesis by LifeTein and Levena Biopharma, respectively) were commercially available. DNA constructs encoding polatuzumab with heavy and light chains consisting of the sequences of SEQ ID NOs: 71 and 72 and enfortumab with heavy and light chains consisting of the sequences of SEQ ID NOs: 75 and 76 were transiently transfected into suspension-adapted CHO-K1 cells and expressed in serum-free / animal component-free medium. Proteins were purified from the supernatant by Protein A affinity chromatography (Mab Select Sure columns; GE Healthcare).

[0548] Conjugation reactions were performed by mixing 5 mg / mL native glycosylated monoclonal antibody, microbial transglutaminase (MTG, Zedira) at a concentration of 5-10 U / mg, and 5-20 molar equivalents of the indicated linker-payload in a rotating thermomixer at 37°C for 24 hours in Tris 50 mM pH 7.6 or BisTris pH 6.0-6.8. Conjugation efficiency was assessed by LC-MS or RPLC under DTT reducing conditions. Reduction of samples was achieved by incubating samples in 50 mM DTT (final) and 50 mM Tris buffer at 37°C for 15 minutes.

[0549] LCMS: After reduction, samples 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 %. The intensities obtained from both glycoforms (G1F and G0F) were taken into account for the calculation according to the following formula:

[0550]

number

[0551] RPLC: 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 relative peak areas extracted from RPLC chromatograms according to the formula and is presented in %.

[0552]

number

[0553] Example 1: Conjugation of various MMAE linker-payload constructs to prepare trastuzumab DAR4 ADCs method Reaction conditions: 5 mg / mL native store-bought fully glycosylated trastuzumab antibody (store-bought Herceptin®), MTG at a concentration of 5 U / mg, and 5 molar equivalents of the indicated linker-payload in Tris 50 mM pH 7.6 at 37° C. for 24 hours in a rotating thermomixer. Conjugation efficiency was assessed by LCMS as described above.

[0554] result Surprisingly, using various MMAE-linker-payload constructs according to the invention to prepare DAR4 ADCs resulted in excellent conjugation efficiencies to native, fully glycosylated trastuzumab (Table 1).

[0555] [Table 1]

[0556] Example 2: Conjugation of various MMAE linker-payload constructs with two other antibodies to prepare DAR4 ADCs To demonstrate the versatility of the invention, DAR4 MMAE ADCs were generated using a variety of MMAE linker payloads and with two different parent antibodies: polatuzumab and enfortumab.

[0557] method Conjugation reactions were carried out by mixing 5 mg / mL of the indicated natively glycosylated antibody, MTG at a concentration of 6-7.5 U / mg, and 5 molar equivalents of the indicated linker-payload in Tris 50 mM pH 7.6 in a rotating thermomixer for 24 hours at 37° C. Conjugation efficiency was assessed by LCMS as described above.

[0558] result Surprisingly, using various MMAE-linker-payload constructs according to the invention to prepare DAR4 ADCs, excellent conjugation efficiencies were obtained with the native, fully glycosylated polatuzumab (Table 2a) and enfortumab (Table 2b) antibodies, respectively.

[0559] [Table 2a]

[0560] [Table 2b]

[0561] Example 3: Conjugation of various exatecan linker-payload constructs to prepare exatecan DAR4 ADCs To demonstrate the broad applicability of the present invention, a linker payload containing exatecan (a topoisomerase I inhibitor) for preparing a trastuzumab DAR4 exatecan ADC was designed and evaluated.

[0562] method Reaction conditions: native glycosylated trastuzumab antibody at 5 mg / mL, MTG at a concentration of 10 U / mg, and 7.5-12.5 molar equivalents of the indicated linker-payload in BisTris pH 6.6 at 37° C. for 24 hours in a rotating thermomixer. Conjugation efficiency was assessed by LCMS or RPLC as described above.

[0563] result Surprisingly, excellent conjugation efficiencies were obtained with native, fully glycosylated trastuzumab using various exatecan linker-payload constructs according to the invention to prepare DAR4 ADCs (Table 3).

[0564] [Table 3]

[0565] Example 4: Conjugation of various exatecan analogue linker-payload constructs to trastuzumab to prepare DAR4 ADCs To demonstrate high tolerability of the reaction, a linker-payload construct for preparing a DAR4 ADC was evaluated using an analog of exatecan (compound 10) described in Li et al. 2019. This analog is referred to as exatecan' (or Exa') in the context of Examples 4 and 5.

[0566] method Reaction conditions: 5 mg / mL native glycosylated trastuzumab antibody, MTG at a concentration of 10 U / mg, and 7.5 molar equivalents of the indicated linker-payload in BisTris pH 6.6 at 37° C. for 24 hours in a rotating thermomixer. Conjugation efficiency was assessed by LCMS as described above.

[0567] result Surprisingly, excellent conjugation efficiencies were obtained with native, fully glycosylated trastuzumab using various exatecan' linker-payload constructs according to the invention to prepare DAR4 ADCs (Table 4).

[0568] [Table 4]

[0569] Example 5: Conjugation of various exatecan (or exatecan analog) linker-payload constructs with two other antibodies to prepare DAR4 ADCs To demonstrate the versatility of the reaction, various exatecan (Exa) or exatecan analog (Exa') linker-payload constructs were conjugated to two additional antibodies: polatuzumab and enfortumab to generate polatuzumab DAR4 Exa or polatuzumab DAR4 Exa' and enfortumab DAR4 Exa or enfortumab DAR4 Exa' ADCs.

[0570] method Conjugation reactions were carried out by mixing 5 mg / mL of the indicated natively glycosylated antibody, MTG at a concentration of 7.5 U / mg, and 10 molar equivalents of the indicated linker-payload in Tris 50 mM pH 7.6 in a rotating thermomixer for 24 hours at 37° C. Conjugation efficiency was assessed by RPLC as described above.

[0571] result Surprisingly, using various exatecan (or analog) linker-payload constructs according to the invention to prepare DAR4 ADCs resulted in excellent conjugation efficiencies to the native, fully glycosylated polatuzumab (Table 5a) and enfortumab (Table 5b) antibodies, respectively.

[0572] [Table 5a]

[0573] [Table 5b]

[0574] Example 6: Conjugation of various MMAE linker-payload constructs to prepare trastuzumab DAR6 or DAR8 ADCs To demonstrate the broad applicability of the present invention, we sought to generate ADCs with a DAR > 4. Therefore, various MMAE linker-payload constructs were designed and evaluated to generate DAR6 or DAR8 ADCs.

[0575] method Reaction conditions: 5 mg / mL native glycosylated trastuzumab antibody, MTG at a concentration of 8 U / mg, and 5 molar equivalents of the indicated linker-payload in BisTris 50 mM pH 7.5 for 22 hours at 37° C. in a rotating thermomixer. Conjugation efficiency was assessed by LCMS as described above.

[0576] result Remarkably, structures according to the invention containing three or four MMAE moieties per linker-payload construct provided good or excellent conjugation efficiencies (Table 6) to native fully glycosylated trastuzumab, resulting in trastuzumab-DAR6 MMAE or DAR8 MMAE ADCs. The conjugation efficiency was surprisingly greater than 67%, significantly higher than that shown historically for lysine-based linkers resulting in DAR2 ADCs (see Example 5 of WO 2015 / 191883, where only 40% conjugation efficiency was achieved).

[0577] [Table 6]

[0578] Example 7: Conjugation of MMAE-Exatecan Linker-Payload Constructs to Prepare a DAR6 Trastuzumab Dual Payload ADC Containing Four MMAEs and Two Exatecans To demonstrate that the invention can also include linker payloads containing different types of drugs in one linker payload, a structure containing two different drugs (MMAE and exatecan) was designed and conjugated to trastuzumab, resulting in a DAR6 trastuzumab dual payload ADC containing four MMAEs and two exatecans.

[0579] method Reaction conditions: 5 mg / mL native glycosylated trastuzumab antibody, MTG at a concentration of 8 U / mg, and 5 molar equivalents of the indicated linker-payload in BisTris 50 mM pH 7.5 for 22 hours at 37° C. in a rotating thermomixer. Conjugation efficiency was assessed by LCMS as described above.

[0580] result Surprisingly, structures according to the invention containing two different types of drugs per linker-payload construct (i.e., 2× MMAE and 1× exatecan) resulted in good conjugation efficiency to native fully glycosylated trastuzumab (Table 7), resulting in a DAR6 trastuzumab dual payload ADC containing 4 MMAE and 2 exatecan.

[0581] [Table 7]

[0582] Example 8: Conjugation of MMAE-maytansine-cortisol linker-payload constructs to prepare DAR6 trastuzumab triple payload ADCs containing two MMAEs, two maytansines, and two cortisols To demonstrate that the invention can also include three different drug types and payload classes on one linker-payload construct, a construct containing three different payloads (MMAE, maytansine, and cortisol CS) was designed and conjugated to trastuzumab, resulting in a DAR6 trastuzumab triple payload ADC containing two MMAEs, two maytansines, and two cortisols.

[0583] method Reaction conditions: 5 mg / mL native glycosylated trastuzumab antibody, MTG at a concentration of 8 U / mg, and 5 molar equivalents of the indicated linker-payload in BisTris 50 mM pH 7.5 for 22 hours at 37° C. in a rotating thermomixer. Conjugation efficiency was assessed by LCMS as described above.

[0584] result Surprisingly, structures according to the invention containing three different types of payloads per linker-payload construct (i.e., 1×MMAE, 1×May, and 1×CS) resulted in excellent conjugation efficiencies to native, fully glycosylated trastuzumab (Table 8), resulting in a DAR6 trastuzumab triple payload ADC containing two MMAEs, two maytansines, and two cortisols.

[0585] [Table 8]

[0586] Example 9: Conjugation of peptide-containing (according to the invention) or peptide-free (not according to the invention) MMAE linker-payload constructs to prepare trastuzumab DAR4 ADCs

[0587] method Reaction conditions: 5 mg / mL native glycosylated trastuzumab antibody, MTG at a concentration of 8 U / mg, and 5 molar equivalents of the indicated linker-payload in BisTris 50 mM pH 7.5 for 22 hours at 37° C. in a rotating thermomixer. Conjugation efficiency was assessed by LCMS as described above.

[0588] result Linkers according to the invention comprising a peptide and two MMAEs provided excellent conjugation efficiency to native, fully glycosylated trastuzumab (Table 9), resulting in trastuzumab-DAR4 MMAE ADCs, whereas, in striking contrast, constructs with amino-PEG and two MMAEs (but not according to the invention, since they do not contain a peptide) conjugated very poorly to native, fully glycosylated trastuzumab.

[0589] [Table 6]

[0590] Example 10: Identification of optimal reaction conditions To demonstrate the importance of linker-payload concentration for reaching optimal conjugation efficiency, various linker sequences and linker-payload concentrations were tested for conjugation to trastuzumab.

[0591] method The following parameters were used as standard conjugation protocol: 3.5 mg / mL native glycosylated trastuzumab antibody and 5 U / mg MTG in Tris 50 mM pH 7.6 for 24 hours at 37 °C in a rotating thermomixer. Linker payloads MMAE-PABC-AA-C2-KAR-PABC-MMAE, RKN(PABC-MMAE)A-PABC-MMAE, and RKAA-PABC-(MMAE)2 were added to the reaction mixture at various concentrations ranging from 2 to 80 molar equivalents. The parameters are shown in Table 7. Conjugation efficiency was assessed by LCMS as described above.

[0592] result Surprisingly, extremely high conjugation efficiencies were obtained when 5-20 equivalents of linker-payload were added to the reaction, regardless of the sequence of the linker-payload tested.

[0593] [Table 7]

[0594] Example 11: Anti-Nectin-4 DAR4 ADCs show efficient tumor growth inhibition in vivo in Nectin-4 positive solid tumor models The anti-Nectin-4 ADCs of the present invention, ARA-04-MMAE-PABC-AA-C2-RKAA-PABC-MMAE (DAR 4.0) and ARA-04-RKAA-PABC-(MMAE)2 (DAR 3.8), were examined in vivo for tumor growth inhibition in a SUM190PT (Nectin-4 positive, solid tumor) xenograft model.

[0595] method For SUM190PT xenografts, 2 × 10 6 Cells were injected into the mammary fat pad of CB17 SCID mice (Janvier). Tumor dimensions and body weights were recorded three times a week. Volume = (width) 2 Tumor volume was calculated according to the formula: × length × 0.5. The average tumor size was approximately 200 mm 3Upon reaching 10 μg / kg of mouse body weight, mice were assigned to treatment groups containing 6 mice each using a non-random stratification protocol. ADC was injected intravenously once on the day of randomization. The ADC of the present invention was generated in-house as described in Example 2. Enfortumab vedotin (DAR4) is commercially available and was purchased from a pharmacy. ARA-04-MMAE-PABC-AA-C2-RKAA-PABC-MMAE (DAR4.0) and ARA-04-RKAA-PABC-(MMAE)2 (DAR3.8) were injected at an ADC dose equivalent to a payload dose of 10 μg / kg of mouse body weight (10 μg / kg). Enfortumab vedotin (DAR4) was injected at a payload dose of 15 μg / kg of mouse body weight (15 μg / kg). Mice in the control group were injected with PBS. All mouse experiments were performed according to Swiss guidelines and approved by the Veterinary Office of Zurich, Switzerland.

[0596] result The ADCs of the present invention, ARA-04-MMAE-PABC-AA-C2-RKAA-PABC-MMAE and ARA-04-RKAA-PABC-(MMAE)2, were compared to enfortumab vedotin in the SUM190PT xenograft model.

[0597] A single iv injection of 10 μg / kg body weight resulted in efficient antitumor responses in all mice with the ADCs of the present invention (FIG. 44). Enfortumab vedotin administered at 15 μg / kg resulted in transient tumor regression, but none were tumor-free at day 20 after treatment, and 6 / 6 animals showed significant regrowth after day 40. Most surprisingly, ARA-04-MMAE-PABC-AA-C2-RKAA-PABC-MMAE showed better efficacy in 4 / 6 mice, leading to complete long-term responses, whereas, in contrast, injection of ARA-04-RKAA-PABC-(MMAE)2 at the same dose resulted in tumor control for approximately 20 days in 6 / 6 mice, followed by tumor regrowth. Thus, very surprisingly, these data show that ARA-04-MMAE-PABC-AA-C2-RKAA-PABC-MMAE, in which a payload is coupled to each N-terminus and C-terminus of a peptide linker, exhibits more efficient antitumor activity compared to ARA-04-RKAA-PABC-(MMAE)2, in which a payload is coupled only to the C-terminus of the peptide linker.

[0598] The anti-Nectin-4 ADCs of the present invention, ARA-04-MMAE-PABC-AA-C2-RKAA-PABC-MMAE and ARA-04-RKAA-PABC-(MMAE)2, which consist of the same antibody and payload as the respective benchmark ADC (Enfortumab Vedotin), are summarized as active in vivo. Surprisingly, only ARA-04-MMAE-PABC-AA-C2-RKAA-PABC-MMAE, in which the payload is coupled to each N-terminus and C-terminus of the peptide linker, showed superior efficacy with survival benefit.

[0599] Example 12: Anti-CD79b DAR4 ADCs demonstrate efficient tumor growth inhibition in vivo in CD79b-positive liquid tumor models The anti-CD79b ADCs of the present invention, ARA-01-MMAE-PABC-AA-C2-RKAA-PABC-MMAE (DAR 4.0) and ARA-01-RKAA-PABC-(MMAE)2 (DAR 3.9), were examined for in vivo tumor growth inhibition in a Ramos (CD79b positive, liquid tumor) xenograft model.

[0600] method For Ramos xenografts, 20 × 10 6 Cells were injected subcutaneously into CB17 SCID mice (Janvier). Tumor dimensions and body weights were recorded three times a week. Volume = (width) 2 Tumor volume was calculated according to the formula: × length × 0.5. The average tumor size was approximately 200 mm 3 Upon reaching 100 mg / kg, mice were assigned to treatment groups containing 6 mice each using a non-random stratification protocol. ADC was injected intravenously once on the day of randomization. ADCs according to the invention were generated in-house as described in Example 2. ARA-01-MMAE-PABC-AA-C2-RKAA-PABC-MMAE (DAR4.0) and ARA-01-RKAA-PABC-(MMAE)2 (DAR3.9) were injected intravenously once at an ADC dose equivalent to a payload dose of 25 μg / kg mouse body weight (25 μg / kg). Mice in the control group were injected with PBS. All mouse experiments were performed according to Swiss guidelines and approved by the Veterinary Office of Zurich, Switzerland.

[0601] result The ADCs of the present invention, ARA-01-MMAE-PABC-AA-C2-RKAA-PABC-MMAE and ARA-01-RKAA-PABC-(MMAE)2, were compared to each other in the Ramos xenograft model.

[0602] With a single iv injection of 25 μg / kg, the ADCs according to the invention showed an efficient antitumor response in all mice compared to the PBS-treated control group (FIG. 45). Surprisingly, compared to ARA-01-RKAA-PABC-(MMAE)2 injected at the same dose, which showed a transient reduction in tumor volume in all animals followed by tumor regrowth, ARA-01-MMAE-PABC-AA-C2-RKAA-PABC-MMAE showed superior efficacy with complete disappearance of tumors in 5 out of 6 mice 21 days after treatment. These data support the results of Example 11 and surprisingly substantiate the finding that ADCs with payloads coupled to each N-terminus and C-terminus of the peptide linker show more efficient antitumor activity than comparable ADCs with payloads coupled only to the C-terminus.

[0603] It is summarized that the anti-CD79b ADCs of the present invention, ARA-01-MMAE-PABC-AA-C2-RKAA-PABC-MMAE and ARA-01-RKAA-PABC-(MMAE)2, are active in vivo. Surprisingly, ARA-01-MMAE-PABC-AA-C2-RKAA-PABC-MMAE, in which a payload is coupled to each N-terminus and C-terminus of a peptide linker, exhibited superior efficacy in providing survival benefits compared to ARA-01-RKAA-PABC-(MMAE)2, in which a payload is coupled only to the C-terminus of the peptide linker, supporting the data shown in FIG. 11 showing that the MMAE-PABC-AA-C2-RKAA-PABC-MMAE linker provided superior anti-tumor efficacy compared to RKAA-PABC-(MMAE)2.

Claims

1. a) an amino acid residue containing a primary amine; b) two or more payloads; A peptide linker comprising: each of the two or more payloads independently, i) the N-terminus of the peptide linker; ii) the C-terminus of the peptide linker; or iii) a side chain of an amino acid residue contained in the peptide linker A peptide linker that can be attached to

2. The primary amine contained in the amino acid residue is a) a primary amine in the side chain of a lysine, lysine derivative, or lysine mimetic; or b) Structure NH ２ A primary amine contained in the N-terminal amino acid residue having —(Y)—COOH The peptide linker of claim 1,

3. Y is (R ２ C) ｎ 3. The peptide linker of claim 2, wherein n is an integer ranging from 1 to 20, 1 to 15, or 1 to 10.

4. Each - (R ２ C) At least one R moiety of the -monomer is hydrogen, or each -(R ２ C) - The peptide linker of claim 3, wherein both R moieties of the monomer are hydrogen.

5. 5. The peptide linker of claim 1, wherein the linker comprises 25 or less, 20 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, or 4 or less amino acid residues.

6. The peptide linker according to any one of claims 1 to 4, wherein the linker comprises at least one arginine and / or histidine residue.

7. The peptide linker according to any one of claims 1 to 4, wherein the linker comprises the sequence motif RK.

8. The peptide linker according to any one of claims 1 to 4, wherein the linker comprises any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 29 or 82 to 93.

9. The peptide linker of any one of claims 1 to 4, wherein the linker comprises two to four payloads.

10. The linker has the following structure (in the N→C direction): [Payload 1]-[(Aa) ｍ -(Lys)-(Aa) ｎ -(Arg / His)-(Aa) ｏ ] - [Payload 2]; [Payload 1] and [Payload 2] are payloads, (Aa) can be any amino acid residue; m, n, and o may be integers ranging from 0 to 10, preferably from 0 to 6, more preferably from 0 to 4; (Arg) can be an arginine residue, an arginine mimetic, or an arginine derivative; (His) can be a histidine residue, a histidine mimetic, or a histidine derivative (Lys) is a lysine residue, a lysine mimetic, or a lysine derivative consisting of or comprising [Payload 1] is attached directly or indirectly to the N-terminus of the (Aa) or (Lys) residue; The peptide linker according to any one of claims 1 to 4, wherein [Payload 2] is directly or indirectly attached to the C-terminus of the (Aa) or (Arg / His) residue.

11. The peptide linker of any one of claims 1 to 4, wherein at least one of the two or more payloads is attached to the peptide linker via a chemical linker.

12. 12. The peptide linker of claim 11, wherein the chemical linker is an enzymatically and / or chemically cleavable linker.

13. 12. The peptide linker of claim 11, wherein the chemical linker is or comprises a self-immolative linker.

14. the self-immolative linker a) a p-aminobenzyl alcohol moiety; or b) a 2,4-bis(hydroxymethyl)aniline moiety; or c) p-aminobenzyl quaternary ammonium; or d) an ethylenediamine-based moiety; or e) an (aminomethyl)pyrrolidine-based moiety; or f) Aminomethyl-based moieties 14. The peptide linker of claim 13, comprising:

15. The peptide linker of claim 14, wherein the hydroxyl group contained in the p-aminobenzyl alcohol moiety forms a carbamate with the payload.

16. 15. The peptide linker of claim 14, wherein each of the hydroxyl groups contained in the 2,4-bis(hydroxymethyl)aniline moiety forms a carbamate with a payload.

17. The peptide linker of claim 14, wherein the quaternary ammonium cation contained in the p-aminobenzyl quaternary ammonium originates from an amine contained in the payload.

18. 15. The peptide linker of claim 14, wherein an amino group contained in the ethylenediamine-based moiety or the (aminomethyl)pyrrolidine-based moiety forms a carbamate with a payload.

19. 15. The peptide linker of claim 14, wherein the amino group contained in the aminomethyl-based moiety forms a hemiaminal or thiohemiaminal with the payload.

20. The peptide linker according to any one of claims 1 to 4, wherein at least one payload is attached to a side chain of an amino acid residue contained in the peptide linker.

21. 21. The peptide linker of claim 20, wherein at least one payload is attached to a side chain of a glutamic acid residue, an aspartic acid residue, a tryptophan residue, a cysteine ​​residue, a lysine residue, a tyrosine residue, a serine residue, or a threonine residue, or a derivative or mimetic of each thereof.

22. The peptide linker according to any one of claims 1 to 4, wherein the peptide linker comprises two peptide moieties connected via their N-terminal amino acid residues with a dicarboxylic acid linker or an activated version thereof.

23. The linker has the structure: [payload 1]-[peptide 1]-[dicarboxylic acid]-[peptide 2]-[payload 2]; [Payload 1] and [Payload 2] are payloads, [Peptide 1] is the first peptide moiety, [Peptide 2] is a second peptide moiety; [Dicarboxylic acid] is a dicarboxylic acid. consisting of or comprising at least one of [Peptide 1] and / or [Peptide 2] comprises a free amine, preferably the free amine is comprised in the side chain of a lysine residue, a lysine mimetic, or a lysine derivative; the N-terminus of [peptide 1] and the N-terminus of [peptide 2] are connected via the dicarboxylic acid; [Payload 1] is attached to the C-terminus of [Peptide 1], preferably via a chemical linker; 23. The peptide linker of claim 22, wherein [Payload 2] is attached to the C-terminus of [Peptide 2], preferably via a chemical linker.

24. 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; biotin or streptavidin moieties; ·vitamin; - proteolytic agents ("PROTACs"); - Ligands or substrates of receptors; a target binding moiety; and / or Anti-inflammatory agents The peptide linker according to any one of claims 1 to 4, which is at least one of:

25. 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; - thymidylate synthase inhibitors; amanitin (e.g., α-amanitin); and Camptothecins (e.g., exatecan, deruxtecan) 25. The peptide linker of claim 24, which is at least one selected from the group consisting of:

26. The peptide linker according to any one of claims 1 to 4, wherein the two or more payloads are identical.

27. The peptide linker according to any one of claims 1 to 4, wherein at least two of the two or more payloads are different from each other.

28. The peptide linker of any one of claims 1 to 4, wherein the linker is suitable to function as a substrate for transglutaminase.

29. An antibody-payload conjugate comprising an antibody conjugated to the peptide linker of claim 1.

30. 30. The antibody-payload conjugate of claim 29, wherein the peptide linker is conjugated to the antibody via an isopeptide bond formed between a γ-carboxamide group of a glutamine residue contained in the antibody and the primary amine contained in an amino acid residue of the peptide linker.

31. The antibody-payload conjugate of claim 29 or 30, wherein the antibody is an IgG antibody.

32. The antibody-payload conjugate of claim 31 , wherein the peptide linker is conjugated to a glutamine residue contained in the Fc domain of the antibody.

33. The glutamine residue to which the peptide linker is conjugated is C of an IgG antibody. Ｈ 33. The antibody-payload conjugate of claim 32, wherein the glutamine residue is Q295 (EU numbering) of domain 2.

34. The antibody-payload conjugate of claim 31 , wherein the glutamine residue to which the peptide linker is conjugated has been introduced into the heavy or light chain of the antibody by molecular engineering.

35. The glutamine residue introduced into the heavy or light chain of the antibody by molecular engineering is C of an aglycosylated IgG antibody. Ｈ 35. The antibody-payload conjugate of claim 34, wherein the two domains are N297Q (EU numbering).

36. The antibody-payload conjugate of claim 34, wherein the glutamine residue introduced into the antibody heavy or light chain by molecular engineering is contained in a peptide (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.

37. The antibody-payload conjugate of claim 36, wherein the peptide containing the Gln residue is fused to the C-terminus of the heavy chain of the antibody.

38. The antibody-payload conjugate of claim 31 , wherein the IgG antibody is a glycosylated IgG antibody.

39. The IgG antibody is Ｈ 39. The antibody-payload conjugate of claim 38, which is glycosylated at residue N297 (EU numbering) of domain 2.

40. 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, enfortumab, endrecolomab, gemtuzumab, loncastuximab, mecbotamab, adecatuzumab, D93, and gatipotuzumab.

31. The antibody-payload conjugate of claim 29 or 30, wherein the antibody-payload conjugate is selected from the group consisting of mab, labetuzumab, tusamitamab, upifitamab, rifastuzumab, mirvetuximab, sofituzumab, anetumab, tisotumab, cofituzumab, pralzatamab, radriatuzumab, belantamab, patritumab, cetuximab, nimotuzumab, matuzumab, portuzumab, sitatuzumab, tucotuzumab, and endrecolomab.

41. 31. The antibody-payload conjugate of claim 29 or 30, wherein the antibody is selected from the group consisting of brentuximab, gemtuzumab, trastuzumab, inotuzumab, polatuzumab, enfortumab, sacituzumab, and belantamab.

42. The antibody-payload conjugate of claim 29 or 30, wherein the antibody is polatuzumab, or trastuzumab, or enfortumab.

43. A method for preparing an antibody-payload conjugate, comprising the step of conjugating a peptide linker according to any one of claims 1 to 4 to an antibody.

44. A method for conjugating a peptide linker comprising two or more payloads to an antibody using transglutaminase (TG), the method comprising: a) mixing the antibody, the peptide linker, and the TG in a fluid, thereby conjugating the linker-payloads to the antibody in one step under the catalytic action of the TG; and b) extracting the conjugate obtained in step a) from the fluid.

45. The method of claim 44, wherein the peptide linker is a peptide linker according to any one of claims 1 to 4.

46. 45. The method of claim 44, wherein the peptide linker is conjugated to a glutamine residue in the antibody via a primary amine contained in an amino acid residue of the peptide linker.

47. 44. The method of claim 43, wherein the antibody is an antibody fragment.

48. 44. The method of claim 43, wherein the antibody is an IgA, IgD, IgE, IgG, or IgM antibody.

49. 44. The method of claim 43, wherein the peptide linker is conjugated to a glutamine residue contained in the Fc domain of the antibody.

50. The glutamine residue to which the peptide linker is conjugated is C of an IgG antibody. Ｈ The method of claim 43, wherein the glutamine residue is Q295 (EU numbering) of domain 2.

51. 44. The method of claim 43, wherein the glutamine residue to which the peptide linker is conjugated has been introduced into the heavy or light chain of the antibody by molecular engineering.

52. The glutamine residue introduced into the heavy or light chain of the antibody by molecular engineering is C of an aglycosylated IgG antibody. Ｈ 52. The method of claim 51, wherein the nucleotide sequence is N297Q (EU numbering) of the 2 domains.

53. The method of claim 52, wherein the glutamine residue introduced into the heavy or light chain of the antibody by molecular engineering is contained in a peptide that is (a) incorporated into the heavy or light chain of the antibody or (b) fused to the N-terminus or C-terminus of the heavy or light chain of the antibody.

54. 54. The method of claim 53, wherein the peptide comprising the Gln residue is fused to the C-terminus of the heavy chain of the antibody.

55. 44. The method of claim 43, wherein the antibody is a glycosylated IgG antibody.

56. The IgG antibody is Ｈ 56. The method of claim 55, wherein the 2 domain is glycosylated at residue N297 (EU numbering).

57. The antibody may be selected from the group consisting of brentuximab, trastuzumab, gemtuzumab, inotuzumab, avelumab, cetuximab, rituximab, daratumumab, pertuzumab, vedolizumab, ocrelizumab, tocilizumab, ustekinumab, golimumab, obinutuzumab, sacituzumab, belantamab, polatuzumab, enfortumab, endrecolomab, gemtuzumab, loncastuximab, mecbotamab, adecatuzumab, D 93, gatipotuzumab, labetuzumab, tusamitamab, upifitamab, rifastuzumab, mirvetuximab, sofituzumab, anetumab, tisotumab, cofituzumab, pralzatamab, radriatuzumab, belantamab, patritumab, cetuximab, nimotuzumab, matuzumab, portuzumab, sitatuzumab, tucotuzumab, and endrecolomab.

58. 44. The method of claim 43, wherein the antibody is selected from the group consisting of brentuximab, gemtuzumab, trastuzumab, inotuzumab, polatuzumab, enfortumab, sacituzumab, and belantamab.

59. The antibody is polatuzumab, or trastuzumab, or enfortumab.

43. The method according to claim 43.

60. The method of claim 43, wherein the peptide linker is conjugated to the gamma-carboxamide group of a Gln residue contained in the antibody.

61. 44. The method of claim 43, wherein the peptide 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%.

62. 44. The method of claim 43, wherein the transglutaminase is a microbial transglutaminase (MTG).

63. 63. The method of claim 62, wherein the microbial transglutaminase is derived from a Streptomyces species, in particular Streptomyces mobaraensis.

64. 44. The method of claim 43, wherein the antibody is contacted with 2 to 100 molar equivalents of the linker.

65. 44. The method of claim 43, wherein the antibody is added to the conjugation reaction at a concentration of 0.1 to 50 mg / mL.

66. 44. The method of claim 43, wherein the transglutaminase is added to the conjugation reaction at a concentration of less than 200 U / mg antibody.

67. 44. The method of claim 43, wherein the conjugation reaction is carried out in a buffered solution.

68. The buffer solution a) a pH in the range of 5 to 10; and / or b) a buffer concentration in the range of 10 to 1000 mM; and / or c) salt concentrations in the range of less than 250 mM 68. The method of claim 67, comprising:

69. 44. An antibody-payload conjugate produced by the method of claim 43.

70. 30. A pharmaceutical composition comprising the antibody-payload conjugate of claim 29 and at least one pharmaceutically acceptable ingredient.

71. 71. The pharmaceutical composition of claim 70, comprising at least one additional therapeutically active agent.

72. An antibody-payload conjugate according to claim 29 or 30 or a pharmaceutical composition according to claim 70 or 71 for use in therapy and / or diagnosis.

73. Neoplastic, neurological, autoimmune, inflammatory, or infectious diseases - Suffering from are at risk of developing, and / or -Diagnosed with 72. An antibody-payload conjugate according to claim 29 or 30 or a pharmaceutical composition according to claim 70 or 71 for use in the treatment of a patient.

74. 74. The antibody-payload conjugate or pharmaceutical composition for use according to claim 73, wherein said antibody-payload conjugate comprises polatuzumab and said neoplastic disease is a B-cell related cancer.

75. 75. The antibody-payload conjugate or pharmaceutical composition for use according to claim 74, wherein said B cell related cancer is non-Hodgkin's lymphoma, in particular said B cell related cancer is diffuse large B cell lymphoma.

76. 75. The antibody-payload conjugate or pharmaceutical composition for use according to claim 74, administered in combination with bendamustine and / or rituximab.

77. 74. The antibody-payload conjugate or pharmaceutical composition for use according to claim 73, wherein said antibody-payload conjugate comprises trastuzumab and said neoplastic disease is a HER2 positive cancer, in particular a HER2 positive breast cancer, gastric cancer, ovarian cancer or lung cancer.

78. 78. The antibody-payload conjugate or pharmaceutical composition for use according to claim 77, administered in combination with lapatinib, capecitabine, and / or a taxane.

79. 74. The antibody-payload conjugate or pharmaceutical composition for use according to claim 73, wherein said antibody-payload conjugate comprises enfortumab or an enfortumab variant and said neoplastic disease is a Nectin-4 positive cancer, in particular a Nectin-4 positive pancreatic cancer, lung cancer, bladder cancer or breast cancer.

80. 80. The antibody-payload conjugate or pharmaceutical composition for use according to claim 79, administered in combination with a platinum-based chemotherapeutic agent and / or pembrolizumab.

81. Neoplastic, neurological, autoimmune, inflammatory, or infectious diseases - Suffering from are at risk of developing, and / or -Diagnosed with Use of an antibody-payload conjugate according to claim 29 or 30 or a pharmaceutical composition according to claim 70 or 71 for the manufacture of a medicament for treating a patient.

82. 71. A method for treating or preventing a neoplastic disease, comprising administering to a patient in need thereof an antibody-payload conjugate of claim 29 or 30 or a pharmaceutical composition of claim 70.

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