Means and methods for producing antibody-linker conjugates

Abstract

The present invention relates to a method for producing antibody-payload conjugates by microbial transglutaminase (MTG), comprising conjugating a linker having the structure (shown in the N→C direction) (Sp1)-RK-(Sp2)-B-(Sp3) or (Sp1)-B-(Sp2)-RK-(Sp3) to a Gln residue contained in the antibody, where (Sp1) is a chemical spacer or absent; (Sp2) is a chemical spacer or absent; (Sp3) is a chemical spacer or absent; R is arginine or an arginine derivative or an arginine mimetic; K is lysine or a lysine derivative or a lysine mimetic; and B is a linking moiety or a payload, and the linker is conjugated to the Gln residue contained in the antibody via a primary amine contained in the side chain of the lysine residue, lysine derivative, or lysine mimetic.

Description

[Technical Field]

[0001] The present invention relates to a method for producing an antibody-linker conjugate by microbial transglutaminase. The present invention further provides an antibody-linker conjugate, an antibody-drug conjugate, a linker construct, and a pharmaceutical composition comprising the antibody-linker conjugate or antibody-drug conjugate of the present invention, and uses thereof. [Background technology]

[0002] Antibody-drug conjugates (ADCs) typically consist of an antibody and a small molecule drug conjugated to the antibody via a chemical linker. After decades of preclinical and clinical research, a series of ADCs have been approved to treat specific tumor types, such as brentuximab vedotin (Adcetris®) for recurrent Hodgkin's lymphoma and systemic anaplastic large cell lymphoma, gemtuzumab ozogamicin (Mylotarg®) for acute myeloid leukemia, ado-trastuzumab emtansine (Kadcyla®) for HER2-positive metastatic breast cancer, inotuzumab ozogamicin (Besponsa®) for B-cell malignancies, and most recently polatuzumab vedotin-piiq (Polivy®). Recently, enfortumab vedotin (Padcev®), trastuzumab deruxtecan (Enhertu®), sacituzumab govitecan (Trodelvy®), and belantamab mafodotin (Blenrep®) have received marketing approval. For a review of ADCs, see, for example, (Zhao P. et al., 2020, Acta Pharmaceutica Sinica B, 10, 1589-1600). While many ADCs have demonstrated excellent anticancer activity, many patients either do not respond to these treatments, experience significant side effects before efficacy is seen, or relapse after a period of time. Therefore, there remains a significant medical need for novel ADC formats that have favorable drug-like properties, can be produced in sufficient quantities and quality at reasonable cost 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 have been generated by conjugation to endogenous lysine or cysteine ​​residues of the antibody, with careful control of the average degree of modification to achieve an average drug-to-antibody ratio (DAR) in the range of 3.5–4.0. Historically, this ratio was chosen based on (a) minimizing the amount of unconjugated antibody and (b) avoiding species in the mixture with very high DARs, which can be problematic in manufacturing and formulation due to their higher hydrophobicity and lower solubility (Lambert JM and Berkenbilt A., 2018, Annu. Rev. Med. 69, 191–207) and typically result in poor pharmacokinetic properties (Lyon RP, et al., 2015, Nat. Biotechnol. 33, 733–735). In recent years, various genetic, chemical, and enzymatic methods have been developed for site-specific conjugation, which may enable a DAR of 2 (or 4) while avoiding over- or under-modification of the antibody. An overview of these methodologies is provided by Yamada et al. (reviewed in Kei Yamada and Yuji Ito, 2019, ChemBioChem, 20, 2729-2739).

[0004] Enzymatic conjugation has attracted considerable attention because these conjugation reactions are typically rapid, site-specific, and can be performed under physiological conditions. Among available enzymes, microbial transglutaminase (MTG) from the species Streptomyces mobaraensis has increasingly emerged as an attractive alternative to traditional chemical protein conjugation of functional moieties, including antibodies. Under physiological conditions, MTG catalyzes the transamidation reaction between a "reactive" glutamine and a "reactive" lysine residue in a protein or peptide, where the latter may be a simple, low-molecular-weight primary amine, such as a 5-aminopentyl group (Jeger S. et al., 2010, Angew. Chem. Int. Ed., 49, 9995-9997).

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

[0006] Another approach to generating ADCs using MTG is based on the use of aglycosylated antibodies in which residue N297 is replaced with an amino acid residue that cannot be subject to glycosylation. However, replacing N297 with another amino acid can have undesirable effects, as it can affect the overall stability of the entire Fc domain (Subedi GP and Barb AW., 2015, Structure, 23, 1573-1583) and the efficacy of the entire conjugate. Consequently, this can lead to increased antibody aggregation and reduced solubility, which is particularly important for hydrophobic payloads. Furthermore, the glycan present at N297 has important immunomodulatory effects, as it induces effector functions such as antibody-dependent cellular cytotoxicity (ADCC). These immunomodulatory effects are lost upon deglycosylation or one of the other approaches discussed above to obtain aglycosylated antibodies. Furthermore, modifying any sequence of an established antibody can also pose regulatory issues, which is problematic because in many cases, approved and clinically validated antibodies are used as the starting point for ADC conjugation. Recently, Spycher et al. disclosed a transglutaminase-based conjugation approach that does not require prior deglycosylation of antibodies for payload conjugation (Spycher et al., WO 2019 / 057772). The ability to conjugate natively glycosylated antibodies offers significant advantages in manufacturing: an enzymatic deglycosylation step is undesirable in good manufacturing practice (GMP) because it must ensure that not only the cleaved glycans but also the deglycosylation enzyme (e.g., PNGase F) are removed from the reaction mixture. Furthermore, genetic engineering of the antibody for payload attachment is not required, thereby avoiding the insertion of sequences that could increase immunogenicity and reduce the overall stability of the antibody. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Zhao P. et al., 2020, Acta Pharmaceutica Sinica B, 10, 1589-1600 [Non-patent document 2] Lambert JM and Berkenbilt A., 2018, Annu. Rev. Med. 69, 191-207 [Non-patent document 3] Lyon RP, et al., 2015, Nat Biotechnol, 33, 733-735 [Non-patent document 4] Yamada et al. (Kei Yamada and Yuji Ito, 2019, ChemBioChem, 20, 2729-2739 [Non-Patent Document 5] Jeger S. et al., 2010, Angew. Chem. Int. Ed., 49, 9995-9997 [Non-patent document 6] Mindt T. et al. 2008, Bioconj Chem, 9, 271-278 [Non-Patent Document 7] Subedi GP and Barb AW., 2015, Structure, 23, 1573-1583 Summary of the Invention [Means for solving the problem]

[0008] In view of the foregoing, there remains a need in the art for improved methods for generating ADCs with high conjugation efficiency.

[0009] Furthermore, there is a need in the art for novel ADCs with improved efficacy and / or pharmacokinetic properties, as well as highly defined drug-to-antibody ratios.

[0010] Summary of the Invention The present invention is characterized in the embodiments and claims provided herein. In particular, the present invention relates, inter alia, to the following embodiments:

[0011] 1. A method for producing antibody-linker conjugates by microbial transglutaminase (MTG), comprising the structure (shown in the N→C orientation): (Sp1)-RK-(Sp2)-B-(Sp3) or (Sp1)-B-(Sp2)-RK-(Sp3) to a Gln residue contained in the antibody, wherein - (Sp1) is a chemical spacer or is absent; - (Sp2) is a chemical spacer or is absent; - (Sp3) is a chemical spacer or is absent; R is arginine or an arginine derivative or arginine mimetic; K is lysine or a lysine derivative or a lysine mimetic; - B is a linking moiety or payload; and the linker is conjugated to a Gln residue in the antibody via a primary amine in the side chain of a lysine residue, lysine derivative, or lysine mimetic.

[0012] 2. 2. The method of embodiment 1, wherein the chemical spacers (Sp1), (Sp2) and (Sp3) each independently comprise 0 to 12 amino acid residues.

[0013] 3. 3. The method of embodiment 1 or 2, wherein the linker comprises no more than 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 amino acid residues.

[0014] 4. 4. The method of any one of embodiments 1 to 3, wherein the net charge of the linker is neutral or positive.

[0015] 5. 5. The method of any one of embodiments 1 to 4, wherein the linker does not contain any negatively charged amino acid residues.

[0016] 6. 6. The method of any one of embodiments 1 to 5, wherein the linker comprises the amino acid sequence RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3) or RKR (SEQ ID NO: 4).

[0017] 7. 7. The method of any one of embodiments 1 to 6, wherein the linker comprises the amino acid sequence RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2) or ARK (SEQ ID NO: 3).

[0018] 8. 8. The method of any one of embodiments 1 to 7, wherein the linker comprises the amino acid sequence RKAA (SEQ ID NO: 1).

[0019] 9. 9. The method of any one of embodiments 1 to 8, wherein B is a linking moiety.

[0020] 10. The connecting part B is - bioorthogonal marker groups, or - Non-bio-orthogonal entities for crosslinking 10. The method of embodiment 9, comprising:

[0021] 11. The bioorthogonal marker group or the non-bioorthogonal entity for crosslinking is - -NN≡N, or -N3; - Lys(N3); - tetrazine; - alkynes; - Distorted cyclooctyne; - BCN; - strained alkenes; - photoreactive groups; - aldehydes; - acyltrifluoroborates; - Protein degrading agents ("PROTACs"); - Cyclopentadiene / spirolocyclopentadiene; - Thioselective electrophiles; - -SH; and - Cysteine 11. The method of embodiment 10, consisting of or comprising at least one molecule or moiety selected from the group consisting of:

[0022] 12. 12. The method of any one of embodiments 9 to 11, comprising the further step of conjugating one or more payloads to linking moiety B.

[0023] 13. 13. The method of embodiment 12, wherein one or more payloads are conjugated to linking moiety B by a click reaction.

[0024] 14. 9. The method of any one of embodiments 1 to 8, wherein B is a payload.

[0025] 15. The payload is - toxin; - cytokines; - growth factors; - Radionuclides; - Hormones; - antiviral agents; - antibacterial agents; - Fluorescent dyes: - immunoregulatory / immunostimulant agents; - Half-life increasing part; - solubility increasing part; - polymer-toxin conjugates; - Nucleic acids; - a biotin or streptavidin moiety; - Vitamins; - Protein degrading agents ("PROTACs"); - a target binding moiety; and / or - Anti-inflammatory 15. The method of any one of embodiments 12 to 14, comprising at least one of:

[0026] 16. The toxin, - Pyrrolobenzodiazepines (e.g., PBD); - Auristatins (e.g., MMAE, MMAF); - Maytansinoids (e.g., maytansine, DM1, DM4, DM21); - Duocarmycin; - Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors; - Tubulyshyn; - Enzymes (e.g., calicheamicin); - anthracycline derivatives (PNU) (e.g., doxorubicin); - Pyrrole-based kinesin spindle protein (KSP) inhibitors; - Cryptophycin; - Drug efflux pump inhibitors; - Sandramycin; amanitin (e.g., α-amanitin); and - Camptothecins (e.g., exatecan, deruxtecan) 16. The method of embodiment 15, wherein the at least one selected from the group consisting of:

[0027] 17. 17. The method of any one of embodiments 14 to 16, wherein the chemical spacer (Sp2) comprises a self-immolative moiety.

[0028] 18. 18. The method of embodiment 17, wherein the self-immolative moiety is attached directly to payload B.

[0029] 19. 19. The method of embodiment 17 or 18, wherein the self-immolative moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.

[0030] 20. 20. The method of any one of embodiments 1 to 19, wherein the antibody is an IgG antibody, particularly an IgG1 antibody.

[0031] twenty one. The Gln residue to which the linker is conjugated is contained in the Fc domain of the antibody, and in particular, the Gln residue to which the linker is conjugated is located in the C H 21. The method of embodiment 20, wherein the Gln residue Q295 (EU numbering) of the 2 domain.

[0032] twenty two. 21. The method of embodiment 20, wherein the Gln residue to which the linker is conjugated has been introduced into the heavy or light chain of the antibody by molecular engineering.

[0033] twenty three. Gln residues introduced into the heavy or light chain of an antibody by molecular engineering are C H 23. The method of embodiment 22, wherein the nucleotide sequence is N297Q (EU numbering) of the 2 domain.

[0034] twenty four. 23. The method of embodiment 22, wherein the Gln residue introduced into the antibody heavy or light chain by molecular engineering is (a) incorporated into the antibody heavy or light chain, or (b) contained in a peptide fused to the N- or C-terminus of the antibody heavy or light chain.

[0035] twenty five. 25. The method of embodiment 24, wherein the peptide comprising a Gln residue is fused to the C-terminus of the heavy chain of the antibody.

[0036] 26. The IgG antibody is a glycosylated IgG antibody, especially when the IgG antibody is C H 26. The method of any one of embodiments 20-22 or 24-25, wherein the IgG2 domain is glycosylated at residue N297 (EU numbering).

[0037] 27. 27. The method of any one of embodiments 1 to 26, wherein the antibody is selected from the group consisting of brentuximab, trastuzumab, gemtuzumab, inotuzumab, avelumab, cetuximab, rituximab, daratumumab, pertuzumab, vedolizumab, ocrelizumab, tocilizumab, ustekinumab, golimumab, obinutuzumab, sacituzumab, belantamab, polatuzumab, and enfortumab.

[0038] 28. 28. The method of any one of embodiments 1 to 27, wherein the antibody is selected from the group consisting of brentuximab, gemtuzumab, trastuzumab, inotuzumab, polatuzumab, enfortumab, sacituzumab, and belantamab.

[0039] 29. 29. The method of any one of embodiments 1 to 28, wherein the antibody is polatuzumab or trastuzumab or enfortumab.

[0040] 30. 30. The method of any one of embodiments 1 to 29, wherein the linker is conjugated to the gamma-carboxamide group of a Gln residue contained in the antibody.

[0041] 31. 31. The method of any one of embodiments 1 to 30, wherein the linker is suitable for conjugation to a glycosylated antibody with a conjugation efficiency of at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95%.

[0042] 32. 32. The method according to any one of the preceding embodiments, wherein the microbial transglutaminase is derived from a Streptomyces species, in particular Streptomyces mobaraensis.

[0043] 33. 33. An antibody-linker conjugate produced by the method of any one of embodiments 1 to 32.

[0044] 34. a) an antibody; and b) Structure: (Sp1)-RK-(Sp2)-B-(Sp3) or (Sp1)-B-(Sp2)-RK-(Sp3) (In the formula, - (Sp1) is a chemical spacer or is absent; - (Sp2) is a chemical spacer or is absent; - (Sp3) is a chemical spacer or is absent; R is arginine or an arginine derivative or arginine mimetic; K is lysine or a lysine derivative or a lysine mimetic; - B is the linking portion or payload) a linker comprising 1. An antibody-linker conjugate comprising: An antibody-linker conjugate, wherein the linker is conjugated to the antibody by an isopeptide bond formed between the γ-carboxamide group of a glutamine residue contained in the antibody and a primary amine contained in the side chain of a lysine residue, lysine derivative, or lysine mimetic contained in an RK motif contained in the linker.

[0045] 35. 35. The antibody-linker conjugate of embodiment 34, wherein the chemical spacers (Sp1), (Sp2) and (Sp3) each independently comprise from 0 to 12 amino acid residues.

[0046] 36. 36. The antibody-linker conjugate of embodiment 34 or 35, wherein the linker comprises no more than 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 amino acid residues.

[0047] 37. 37. The antibody-linker conjugate of any one of embodiments 34 to 36, wherein the net charge of the linker is neutral or positive.

[0048] 38. 38. The antibody-linker conjugate of any one of embodiments 34 to 37, wherein the linker does not contain any negatively charged amino acid residues.

[0049] 39. 39. The antibody-linker conjugate of any one of embodiments 34 to 38, wherein the linker comprises an amino acid sequence selected from the group consisting of RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3) and RKR (SEQ ID NO: 4).

[0050] 40. 40. The antibody-linker conjugate of any one of embodiments 34 to 39, wherein the linker comprises an amino acid sequence selected from the group consisting of RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2) and ARK (SEQ ID NO: 3).

[0051] 41. 41. The antibody-linker conjugate of any one of embodiments 34 to 40, wherein the linker comprises the amino acid sequence RKAA (SEQ ID NO: 1).

[0052] 42. 42. The antibody-linker conjugate of any one of embodiments 34 to 41, wherein B is a linking moiety.

[0053] 43. The connecting part B is - bioorthogonal marker groups, or - Non-bio-orthogonal entities for crosslinking 43. The antibody-linker conjugate of embodiment 42, comprising:

[0054] 44. The bioorthogonal marker group or the non-bioorthogonal entity for crosslinking is - -NN≡N, or -N3; - Lys(N3); - tetrazine; - alkynes; - Distorted cyclooctyne; - BCN; - strained alkenes; - photoreactive groups; - aldehydes; - acyltrifluoroborates; - Protein degrading agents ("PROTACs"); - Cyclopentadiene / spirolocyclopentadiene; - Thioselective electrophiles; - -SH; and - Cysteine 44. The antibody-linker conjugate of embodiment 43, consisting of or comprising at least one molecule or moiety selected from the group consisting of:

[0055] 45. 45. The antibody-linker conjugate of any one of embodiments 42 to 44, wherein one or more payloads are conjugated to linking moiety B.

[0056] 46. 46. ​​The antibody-linker conjugate of embodiment 45, wherein one or more payloads are conjugated to linking moiety B by a click reaction.

[0057] 47. 42. The antibody-linker conjugate of any one of embodiments 34 to 41, wherein B is a payload.

[0058] 48. The payload is - toxin; - cytokines; - growth factors; - Radionuclides; - Hormones; - antiviral agents; - antibacterial agents; - Fluorescent dyes: - immunoregulatory / immunostimulant agents; - Half-life increasing part; - solubility increasing part; - polymer-toxin conjugates; - Nucleic acids; - a biotin or streptavidin moiety; - Vitamins; - Protein degrading agents ("PROTACs"); - a target binding moiety; and / or - Anti-inflammatory 48. The antibody-linker conjugate of any one of embodiments 45 to 47, comprising at least one of:

[0059] 49. The toxin, - Pyrrolobenzodiazepines (e.g., PBD); - Auristatins (e.g., MMAE, MMAF); - Maytansinoids (e.g., maytansine, DM1, DM4, DM21); - Duocarmycin; - Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors; - Tubulyshyn; - Enzymes (e.g., calicheamicin); - anthracycline derivatives (PNU) (e.g., doxorubicin); - Pyrrole-based kinesin spindle protein (KSP) inhibitors; - Cryptophycin; - Drug efflux pump inhibitors; - Sandramycin; amanitin (e.g., α-amanitin); and - Camptothecins (e.g., exatecan, deruxtecan) 49. The antibody-linker conjugate of embodiment 48, which is at least one selected from the group consisting of:

[0060] 50. 50. The antibody-linker conjugate of any one of embodiments 47 to 49, wherein the chemical spacer (Sp2) comprises a self-immolative moiety.

[0061] 51. 51. The antibody-linker conjugate of embodiment 50, wherein the self-immolative moiety is attached directly to payload B.

[0062] 52. 52. The antibody-linker conjugate of embodiment 50 or 51, wherein the self-immolative moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.

[0063] 53. The antibody-linker conjugate of any one of embodiments 34 to 52, wherein the antibody is an IgG antibody, in particular an IgG1 antibody.

[0064] 54. The Gln residue to which the linker is conjugated is contained in the Fc domain of the antibody, and in particular, the Gln residue to which the linker is conjugated is located in the C H 54. The antibody-linker conjugate of embodiment 53, wherein the Gln residue Q295 (EU numbering) of the 2 domain is Gln residue Q295 (EU numbering).

[0065] 55. 54. The antibody-linker conjugate of embodiment 53, wherein the Gln residue to which the linker is conjugated has been introduced into the heavy or light chain of the antibody by molecular engineering.

[0066] 56. Gln residues introduced into the heavy or light chain of an antibody by molecular engineering are C H 56. The antibody-linker conjugate of embodiment 55, which is 2 domain N297Q (EU numbering).

[0067] 57. 56. The antibody-linker conjugate of embodiment 55, wherein the Gln residue introduced into the antibody heavy or light chain by molecular engineering is either (a) incorporated into the antibody heavy or light chain, or (b) contained in a peptide fused to the N- or C-terminus of the antibody heavy or light chain.

[0068] 58. 58. The antibody-linker conjugate of embodiment 57, wherein the peptide comprising a Gln residue is fused to the C-terminus of the heavy chain of the antibody.

[0069] 59. The IgG antibody is a glycosylated IgG antibody, especially when the IgG antibody is C H 59. The antibody-linker conjugate of any one of embodiments 53 to 55 or 57 to 58, wherein the antibody-linker conjugate is glycosylated at residue N297 (EU numbering) of the 2 domain.

[0070] 60. 60. The antibody-linker conjugate of any one of embodiments 34 to 59, wherein the antibody is selected from the group consisting of brentuximab, trastuzumab, gemtuzumab, inotuzumab, avelumab, cetuximab, rituximab, daratumumab, pertuzumab, vedolizumab, ocrelizumab, tocilizumab, ustekinumab, golimumab, obinutuzumab, sacituzumab, belantamab, polatuzumab, and enfortumab.

[0071] 61. 61. The antibody-linker conjugate of any one of embodiments 34 to 60, wherein the antibody is selected from the group consisting of brentuximab, gemtuzumab, trastuzumab, inotuzumab, polatuzumab, enfortumab, sacituzumab, and belantamab.

[0072] 62. The antibody-linker conjugate of any one of embodiments 34 to 61, wherein the antibody is polatuzumab or trastuzumab or enfortumab.

[0073] 63. a) IgG antibodies; and b) a linker comprising a drug moiety B, wherein drug moiety B is covalently linked to an amino acid sequence selected from the group consisting of RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), or RKR (SEQ ID NO: 4). 1. An antibody-drug conjugate comprising: The linker is located at the C H An antibody-drug conjugate conjugated to an IgG antibody via an isopeptide bond formed between the γ-carboxamide group of glutamine residue Q295 (EU numbering) in domain 2 and the primary amine in the side chain of a lysine residue in the linker.

[0074] 64. 64. The antibody-drug conjugate of embodiment 63, wherein drug moiety B is linked to the N- or C-terminus of the amino acid sequence comprised in the linker by a self-immolative moiety.

[0075] 65. 65. The antibody-drug conjugate of embodiment 64, wherein the self-immolative moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.

[0076] 66. The IgG antibody is a glycosylated IgG antibody, especially when the IgG antibody is C H 66. The antibody-drug conjugate of any one of embodiments 63 to 65, wherein the antibody-drug conjugate is glycosylated at residue N297 (EU numbering) of the 2 domain.

[0077] 67. 67. The antibody-drug conjugate of any one of embodiments 63 to 66, wherein the IgG antibody is an IgG1 antibody.

[0078] 68. The antibody-drug conjugate of any one of embodiments 63 to 67, wherein the IgG antibody is polatuzumab or an antibody comprising a heavy chain set forth in SEQ ID NO:5 and a light chain set forth in SEQ ID NO:6.

[0079] 69. The antibody-drug conjugate of any one of embodiments 63 to 67, wherein the IgG antibody is trastuzumab or an antibody comprising a heavy chain set forth in SEQ ID NO: 7 and a light chain set forth in SEQ ID NO: 8.

[0080] 70. 68. The antibody-drug conjugate of any one of embodiments 63 to 67, wherein the IgG antibody is enfortumab or an antibody comprising a heavy chain set forth in SEQ ID NO: 9 and a light chain set forth in SEQ ID NO: 10 or 11.

[0081] 71. Drugs, - Pyrrolobenzodiazepines (e.g., PBD); - Auristatins (e.g., MMAE, MMAF); - Maytansinoids (e.g., maytansine, DM1, DM4, DM21); - Duocarmycin; - Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors; - Tubulyshyn; - Enzymes (e.g. calicheamicin); - anthracycline derivatives (PNU) (e.g., doxorubicin); - Pyrrole-based kinesin spindle protein (KSP) inhibitors; - Cryptophycin; - Drug efflux pump inhibitors; - Sandramycin; amanitin (e.g., α-amanitin); and - Camptothecins (e.g., exatecan, deruxtecan) 71. The antibody-drug conjugate of any one of embodiments 63 to 70, wherein the toxin is selected from the group consisting of:

[0082] 72. 72. The antibody-drug conjugate of any one of embodiments 63 to 71, wherein the linker has the structure RKAA-PABC-B (particularly, wherein B is an auristatin or a maytansinoid, particularly wherein the auristatin is MMAE and the maytansinoid is DM1 or maytansine).

[0083] 73. 72. The antibody-drug conjugate of any one of embodiments 63 to 71, wherein the linker has the structure RKA-PABC-B (particularly, wherein B is an auristatin or a maytansinoid, particularly wherein the auristatin is MMAE and the maytansinoid is DM1 or maytansine).

[0084] 74. 72. The antibody-drug conjugate of any one of embodiments 63 to 71, wherein the linker has the structure ARK-PABC-B (particularly, wherein B is an auristatin or a maytansinoid, particularly wherein the auristatin is MMAE and the maytansinoid is DM1 or maytansine).

[0085] 75. 72. The antibody-drug conjugate of any one of embodiments 63 to 71, wherein the linker has the structure RKR-PABC-B (particularly, wherein B is an auristatin or a maytansinoid, particularly wherein the auristatin is MMAE and the maytansinoid is DM1 or maytansine).

[0086] 76. structure: (Sp1)-RK-(Sp2)-B-(Sp3) or (Sp1)-B-(Sp2)-RK-(Sp3) (In the formula, - (Sp1) is a chemical spacer or is absent; - (Sp2) is a chemical spacer or is absent; - (Sp3) is a chemical spacer or is absent; R is arginine or an arginine derivative or arginine mimetic; K is lysine or a lysine derivative or a lysine mimetic; - B is the linking portion or payload) A linker construct comprising:

[0087] 77. 77. The linker construct of embodiment 76, wherein the chemical spacers (Sp1), (Sp2) and (Sp3) each independently comprise 0 to 12 amino acid residues.

[0088] 78. 78. The linker construct of embodiment 76 or 77, wherein the linker comprises no more than 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 amino acid residues.

[0089] 79. 79. A linker construct according to any one of embodiments 76 to 78, wherein the net charge of the linker is neutral or positive.

[0090] 80. 80. The linker construct of any one of embodiments 76 to 79, wherein the linker does not contain any negatively charged amino acid residues.

[0091] 81. 81. A linker construct according to any one of embodiments 76 to 80, wherein the linker comprises the amino acid sequence RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3) or RKR (SEQ ID NO: 4).

[0092] 82. 82. The linker construct of any one of embodiments 76 to 81, wherein B is a linking moiety.

[0093] 83. The connecting part B is - bioorthogonal marker groups, or - Non-bio-orthogonal entities for crosslinking 83. The linker construct of embodiment 82, comprising:

[0094] 84. The bioorthogonal marker group or the non-bioorthogonal entity for crosslinking is - -NN≡N, or -N3; - Lys(N3); - tetrazine; - alkynes; - Distorted cyclooctyne; - BCN; - strained alkenes; - photoreactive groups; - aldehydes; - acyltrifluoroborates; - Protein degrading agents ("PROTACs"); - Cyclopentadiene / spirolocyclopentadiene; - Thioselective electrophiles; - -SH; and - Cysteine 84. The linker construct of embodiment 83, consisting of or comprising at least one molecule or moiety selected from the group consisting of:

[0095] 85. 85. A linker construct according to any one of embodiments 76 to 84, consisting of or comprising the structure RKAA-B, in particular where B is Lys(N3) or cysteine.

[0096] 86. 85. A linker construct according to any one of embodiments 76 to 84, consisting of or comprising the structure RKA-B, in particular wherein B is Lys(N3) or cysteine.

[0097] 87. 85. A linker construct according to any one of embodiments 76 to 84, consisting of or comprising the structure ARK-B (in particular, where B is Lys(N3) or cysteine).

[0098] 88. 85. A linker construct according to any one of embodiments 76 to 84, consisting of or comprising the structure B-RKR, in particular where B is Lys(N3) or cysteine.

[0099] 89. 82. The linker construct of any one of embodiments 76 to 81, wherein B is a payload.

[0100] 90. The payload is - toxin; - cytokines; - growth factors; - Radionuclides; - Hormones; - antiviral agents; - antibacterial agents; - Fluorescent dyes: - immunoregulatory / immunostimulant agents; - Half-life increasing part; - solubility increasing part; - polymer-toxin conjugates; - Nucleic acids; - a biotin or streptavidin moiety; - Vitamins; - Protein degrading agents ("PROTACs"); - a target binding moiety; and / or - Anti-inflammatory 90. The linker construct of embodiment 89, comprising at least one of:

[0101] 91. The toxin, - Pyrrolobenzodiazepines (e.g., PBD); - Auristatins (e.g., MMAE, MMAF); - Maytansinoids (e.g., maytansine, DM1, DM4, DM21); - Duocarmycin; - Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors; - Tubulyshyn; - Enzymes (e.g. calicheamicin); - anthracycline derivatives (PNU) (e.g., doxorubicin); - Pyrrole-based kinesin spindle protein (KSP) inhibitors; - Cryptophycin; - Drug efflux pump inhibitors; - Sandramycin; amanitin (e.g., α-amanitin); and - Camptothecins (e.g., exatecan, deruxtecan) 91. The linker construct of embodiment 90, wherein the linker construct is at least one selected from the group consisting of:

[0102] 92. 92. The linker construct of any one of embodiments 89 to 91, wherein the chemical spacer (Sp2) comprises a self-immolative moiety.

[0103] 93. 93. The linker construct of embodiment 92, wherein the self-immolative moiety is directly attached to payload B.

[0104] 94. 94. The linker construct of embodiment 92 or 93, wherein the self-immolative moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.

[0105] 95. 95. A linker construct according to any one of embodiments 89 to 94, consisting of or comprising the structure RKAA-PABC-B (particularly, wherein B is an auristatin or a maytansinoid, in particular wherein the auristatin is MMAE and the maytansinoid is DM1 or maytansine).

[0106] 96. 95. A linker construct according to any one of embodiments 89 to 94, consisting of or comprising the structure RKA-PABC-B (particularly, wherein B is an auristatin or a maytansinoid, in particular wherein the auristatin is MMAE and the maytansinoid is DM1 or maytansine).

[0107] 97. 95. A linker construct according to any one of embodiments 89 to 94, consisting of or comprising the structure ARK-PABC-B (particularly, wherein B is an auristatin or a maytansinoid, in particular wherein the auristatin is MMAE and the maytansinoid is DM1 or maytansine).

[0108] 98. 95. A linker construct according to any one of embodiments 89 to 94, consisting of or comprising the structure B-PABC-RKR (particularly, wherein B is an auristatin or a maytansinoid, in particular wherein the auristatin is MMAE and the maytansinoid is DM1 or maytansine).

[0109] 99. 99. Use of a linker construct according to any one of embodiments 76 to 98 in the production of an antibody-linker conjugate by microbial transglutaminase.

[0110] 100. The use according to embodiment 99, wherein the antibody is an IgG antibody, in particular an IgG1 antibody.

[0111] 101. The use of embodiment 65 or 66, wherein the antibody is polatuzumab or trastuzumab or enfortumab.

[0112] 102. a) an antibody-linker conjugate according to any one of embodiments 33 to 62, in particular an antibody-linker conjugate comprising at least one payload; or b) An antibody drug-conjugate according to any one of embodiments 63 to 75. 1. A pharmaceutical composition comprising:

[0113] 103. The pharmaceutical composition according to embodiment 102, comprising at least one further therapeutically active agent.

[0114] 104. An antibody-linker conjugate according to any one of embodiments 33 to 62, comprising at least one payload, an antibody-drug conjugate according to any one of embodiments 63 to 75, or a pharmaceutical composition according to embodiment 102 or 103, particularly for use in therapy and / or diagnosis.

[0115] 105. In particular, neoplastic, neurological, autoimmune, inflammatory or infectious diseases - Are you suffering from - are at risk of developing it, and / or - diagnosed with it An antibody-linker conjugate according to any one of embodiments 33 to 62, comprising at least one payload, the antibody-drug conjugate according to any one of embodiments 63 to 75, or the pharmaceutical composition according to embodiment 102 or 103, for use in treating a patient.

[0116] 106. 106. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to embodiment 105, wherein the antibody-linker conjugate or antibody-drug conjugate comprised in the pharmaceutical composition comprises polatuzumab, and the neoplastic disease is a cancer associated with B cells.

[0117] 107. 107. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to embodiment 106, wherein the cancer associated with B cells is non-Hodgkin's lymphoma, in particular the cancer associated with B cells is diffuse large B-cell lymphoma.

[0118] 108. 108. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to embodiment 106 or 107, which is administered in combination with bendamustine and / or rituximab.

[0119] 109. 106. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to embodiment 105, wherein the antibody-linker conjugate or antibody-drug conjugate comprised in the pharmaceutical composition 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.

[0120] 110. 110. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to embodiment 109, wherein said antibody is administered in combination with lapatinib, capecitabine and / or a taxane.

[0121] 111. 106. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to embodiment 105, wherein the antibody-linker conjugate or antibody-drug conjugate comprised in the pharmaceutical composition comprises enfortumab or an enfortumab variant, and the neoplastic disease is Nectin-4 positive cancer, in particular Nectin-4 positive pancreatic cancer, lung cancer, bladder cancer or breast cancer.

[0122] 112. 112. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to embodiment 111, administered in combination with a cisplatin-based chemotherapy agent and / or pembrolizumab.

[0123] 113. In particular, the antibody-linker conjugate Neoplastic, neurological, autoimmune, inflammatory or infectious diseases - Are you suffering from - are at risk of developing it, and / or - diagnosed with it Use of the antibody-linker conjugate of any one of embodiments 33 to 62, comprising at least one payload, the antibody-drug conjugate of any one of embodiments 63 to 75, or the pharmaceutical composition of embodiment 102 or 103, for the manufacture of a medicament for the treatment of a patient.

[0124] 114. A method for treating or preventing a neoplastic disease, comprising administering to a patient in need thereof an antibody-linker conjugate according to any one of embodiments 33 to 62, in particular wherein the antibody-linker conjugate comprises at least one payload, an antibody-drug conjugate according to any one of embodiments 63 to 75, or a pharmaceutical composition according to embodiment 102 or 103.

[0125] Thus, in one embodiment, the present invention provides a method for producing antibody-linker conjugates by microbial transglutaminase (MTG), comprising the structure (shown in the N→C orientation): (Sp1)-RK-(Sp2)-B-(Sp3) or (Sp1)-B-(Sp2)-RK-(Sp3) to a Gln residue contained in the antibody, wherein - (Sp1) is a chemical spacer or is absent; - (Sp2) is a chemical spacer or is absent; - (Sp3) is a chemical spacer or is absent; R is arginine or an arginine derivative or arginine mimetic; K is lysine or a lysine derivative or a lysine mimetic; - B is a linking moiety or payload; and wherein the linker is conjugated to a Gln residue in the antibody through a primary amine in the side chain of a lysine residue, lysine derivative, or lysine mimetic.

[0126] That is, the present invention is based at least in part on the surprising finding that linkers containing the peptide motif RK (arginyl-lysyl) can be conjugated to glycosylated antibodies with high efficiency. Patent application WO2019 / 057772 demonstrated that peptide-based linkers can be efficiently conjugated to glutamine residues of glycosylated antibodies via lysine residues in the linker. However, it has now been surprisingly shown that the extended motif RK leads to even further improved conjugation efficiency.

[0127] The inventors have shown that lysine-containing linkers without an RK motif, when directly attached to a drug molecule, result in conjugation efficiencies ranging from 27 to 77% (see Table 4). Linkers containing an RK motif, as provided herein, have been conjugated to glycosylated antibodies with efficiencies of at least 82%, and in certain instances, up to 100% (see Tables 3 and 5). Thus, linkers containing an RK motif are particularly preferred over other lysine-based linkers for MTG-based conjugation to glycosylated antibodies, especially when the payload is directly conjugated to the glycosylated antibody in a one-step reaction.

[0128] Within the scope of the present invention, it is preferred that the linker comprises the structure (Sp1)-RK-(Sp2)-B-(Sp3) or (Sp1)-B-(Sp2)-RK-(Sp3), and that the linker is conjugated to a glutamine residue in the antibody via a primary amine contained in residue K in the RK motif of the linker. In certain embodiments, residue K is a lysine residue. However, in certain embodiments, residue K may be a lysine mimetic or a lysine derivative, so long as the lysine mimetic or lysine derivative contains a primary amine in its amino acid side chain.

[0129] Thus, in certain embodiments, residue K may be a lysine mimetic. The term "lysine mimetic," as used herein, refers to a compound that has a different structure from lysine but similar characteristics to lysine and can therefore be used to replace lysine in a peptide or protein without significantly altering the function and / or structure of the peptide or protein. In certain embodiments, the lysine mimetic may differ from lysine in the length or composition of the fatty chain attached to the primary amine and the α-carbon atom. Thus, in certain embodiments, the lysine mimetic may be ornithine or 2,7-diaminoheptanoic acid. In certain embodiments, the lysine mimetic may be a beta-amino acid, such as beta-homolisine.

[0130] In certain embodiments, residue K may be a lysine derivative. The term "lysine derivative," as used herein, refers to a lysine or lysine mimetic in which one or more functional groups contained therein have been modified or substituted. Within the scope of the present invention, it is preferred that the amino group of the side chain of the lysine derivative is not modified so that it is available for conjugation to a glutamine residue in a protein. In embodiments in which residue K is located at the C-terminal position of the linker, K may be a lysine derivative in which the α-carboxy group has been modified or substituted. In certain embodiments, the α-carboxy group of the lysine mimetic may be amidated.

[0131] The linker further comprises a residue R, which together with residue K forms the RK motif of the linker. In certain embodiments, residue R is an arginine residue. However, in certain embodiments, residue R may be an arginine mimetic or an arginine derivative.

[0132] Thus, in certain embodiments, residue R may be an arginine mimetic. As used herein, the term "arginine mimetic" refers to a compound that has a different structure from arginine but similar characteristics to arginine, and thus can be used to replace arginine in a peptide or protein without significantly altering the function and / or structure of the peptide or protein. An arginine mimetic may differ from arginine in the length or composition of the fatty chain attached to the guanidino group and 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 contain a functional group with 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.

[0133] In certain embodiments, residue R may be 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 have been modified or substituted. The arginine derivative may be an arginine or arginine mimetic in which the guanidino group has been substituted or modified. In certain embodiments, the arginine derivative may be ω-methylarginine. In embodiments in which residue R is located at the N-terminal position of the linker, R may be an arginine derivative in which the α-amino group has been modified or substituted. In certain embodiments, the α-amino group of the arginine mimetic may be acetylated.

[0134] It should be understood that the RK motif preferably consists of the amino acids arginine and lysine. However, the arginine or lysine residues, or both, may be replaced by mimetics or derivatives as disclosed above. In certain embodiments, the RK motif may consist of the amino acids arginine and ornithine. In certain embodiments, the RK motif may consist of the amino acids arginine and 2,7-diaminoheptanoic acid. In certain embodiments, the RK motif may consist of the amino acids homoarginine and lysine. In certain embodiments, the RK motif may consist of the amino acids 2-amino-3-guanidino-propionic acid and lysine. In certain embodiments, the RK motif may consist of the amino acids homoarginine and ornithine. In certain embodiments, the RK motif may consist of the amino acids homoarginine and 2,7-diaminoheptanoic acid. In certain embodiments, the RK motif may consist of the amino acids 2-amino-3-guanidino-propionic acid and ornithine. In certain embodiments, the RK motif may consist of the amino acids 2-amino-3-guanidino-propionic acid and 2,7-diaminoheptanoic acid.

[0135] Within the scope of the present invention, the RK motif is embedded in the structure (Sp1)-RK-(Sp2)-B-(Sp3) or (Sp1)-B-(Sp2)-RK-(Sp3). That is, the linker may contain one or more chemical spacers (Sp). The term "chemical spacer", as used herein, describes a chemical moiety that is covalently attached to a chemical residue of the linker and / or inserted between two chemical residues of the linker.

[0136] In a particular embodiment, the present invention relates to a method according to the present invention, wherein the chemical spacers (Sp1), (Sp2) and (Sp3) each independently comprise from 0 to 12 amino acid residues.

[0137] That is, in certain embodiments, chemical spacers (Sp1), (Sp2), and / or (Sp3) may be present or absent. In embodiments in which (Sp1), (Sp2), and / or (Sp3) are present, (Sp1), (Sp2), and / or (Sp3) may comprise one or more amino acid residues. In such embodiments, each of (Sp1), (Sp2), and / or (Sp3) may comprise 0 to 12 amino acid residues. It should be noted that chemical spacers (Sp1), (Sp2), and / or (Sp3) may also comprise non-amino acid residues, as disclosed in more detail below.

[0138] The "amino acid residue" contained in the chemical spacers (Sp1), (Sp2), and / or (Sp3) may be an amino acid, an amino acid mimetic, or an amino acid derivative. It should be understood that the term amino acid not only encompasses α-amino acids, but also other amino acids such as β-, γ-, or δ-amino acids. An α-amino acid residue may be present in the chemical spacers (Sp1), (Sp2), and / or (Sp3) in its L- or D-form. In embodiments in which (Sp1), (Sp2), and / or (Sp3) contain chiral β-, γ-, or δ-amino acids, the chiral β-, γ-, or δ-amino acids may be present in their S- or R-form. Thus, in its broadest sense, the term "amino acid residue," as used herein, may refer to any organic compound containing an amino group (—NH2) and a carboxy group (—COOH). Thus, whenever "amino acid" or "amino acid residue" is referred to throughout this disclosure, it should be understood that the term amino acid residue may also encompass an amino acid mimetic or derivative.

[0139] Furthermore, the term amino acid residue is not limited to the known set of proteinogenic amino acids, i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, but should be understood to encompass non-canonical amino acids and unnatural amino acids. A "non-canonical 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-canonical amino acids may also be found in naturally occurring peptides and / or proteins.

[0140] As used herein, a "non-natural amino acid" or "synthetic amino acid" may be any molecule that falls under the general definition of an amino acid, i.e., any molecule that contains an amino group and a carboxy 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-canonical amino acids and non-natural amino acids may be unclear in some instances. For example, an amino acid defined as a non-natural amino acid may later be identified in nature and thus reclassified as a non-canonical amino acid.

[0141] Examples of non-canonical or unnatural amino acids include, but are not limited to, D-amino acids (e.g., D-alanine, D-arginine, D-methionine), homo-amino acids (e.g., homoserine, homoarginine, homocysteine, α-aminoadipic acid), N-methylated amino acids (e.g., sarcosine, N-Me-leucine), α-methyl amino acids (e.g., α-methyl-histidine, α-aminoisobutyric acid), β-amino acids (e.g., β-alanine, D-3-aminoisobutyric acid, L-β-homoalanine), γ-amino acids (e.g., γ-aminobutyric acid). , alanine mimics or derivatives (e.g., β-cyclopropylalanine, phenylglycine, dehydroalanine, β-cyanoalanine, β-(3-pyridyl)-alanine, β-(1,2,4-triazol-1-yl)-alanine, β-(1-piperazinyl)-alanine), phenylalanine mimics or derivatives (e.g., 4-iodophenylalanine, pentafluorophenylalanine, naphthyl-alanine, 4-aminophenylalanine), arginine mimics or derivatives (e.g., β-ureidoalanine, ω-methylarginine), , lysine mimics or derivatives (e.g., (3-(3-methyl-3H-diazirin-3-yl)propamino)carbonyl-1-lysine, Nε,Nε,Nε-trimethyllysine), histidine mimics or derivatives (e.g., 2,5-di-iodohistidine, 1-methylhistidine), tyrosine mimics or derivatives (e.g., 3-aminotyrosine, tyronine, 3,5-dinitrotyrosine, 3-hydroxy-methyl-tyrosine, O-phospho-L-tyrosine), tryptophan mimics or derivatives (e.g., 5-hydroxy-tryptophan, 1-methyl tryptophan), serine mimics or derivatives (e.g., β-(2-thienyl)-serine, β-(3,4-dihydroxyphenyl)-serine, O-phosphoserine), threonine mimics or derivatives (e.g., allo-threonine, O-phosphothreonine), proline mimics or derivatives (e.g., hydroxyproline, 3,4-dehydro-proline, pyroglutamic acid, thiaproline, cis-octahydroindole-2-carboxylic acid), leucine and isoleucine mimics or derivatives (e.g., allo-isoleucine, norleucine, 4,5-dehydroleucine, (4S)-4-hydroxy-L-isoleucine), valine mimics or derivatives (e.g., norvaline, γ-hydroxyvaline), citrulline mimics or derivatives (e.g., thiocitrulline, homocitrulline), cysteine ​​mimics or derivatives (e.g., penicillamine, selenocysteine, buthionine sulfoximine), methionine mimics or derivatives (e.g., S-methylmethionine, L-methionine sulfone, L-methionine sulfone, sulfoxide, L-methionine sulf-oximine, selenomethionine), aspartic acid mimics or derivatives (e.g., DL-threo-β-hydroxyaspartic acid, L-aspartic acid β-methyl ester), glutamic acid mimics or derivatives (e.g., γ-methyleneglutamic acid, γ-carboxyglutamic acid, γ-hydroxyglutamic acid, L-glutamic acid 5-methyl ester, L-2-aminoheptanedioic acid), asparagine mimics or derivatives glutamine mimetics or derivatives (e.g., 4-F-(2S,4R)-fluoroglutamine, γ-glutamylmethylamide, theanine, L-glutamic acid γ-monohydroxamate), amino acids containing cyclic moieties (e.g., 4-aminopiperidine-4-carboxylate), The amino acid may be an amino acid containing a bioorthogonal moiety (e.g., propargylglycine, α-allylglycine, L-azido-homoalanine, p-benzoyl-l-phenylalanine, p-2-fluoroacetyl-l-phenylalanine, (S)-2-amino-3-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)propanoic acid).

[0142] In addition to the alpha-amino acids described above, the chemical spacers (Sp1), (Sp2), and / or (Sp3) may contain one or more β-, γ-, δ-, or ε-amino acids. Thus, in certain embodiments, the linker may be a peptidomimetic. A peptidomimetic may not exclusively contain a classical peptide bond formed between two α-amino acids, but may additionally or alternatively include one or more amide bonds formed between an alpha amino acid and a β-, γ-, δ-, or ε-amino acid, or between two β-, γ-, δ-, or ε-amino acids, respectively. Thus, in any example of the present invention in which the linker is described as a peptide, it should be understood that the linker may be a peptidomimetic and thus may not be exclusively composed of α-amino acids, but may instead include one or more β-, γ-, δ-, or ε-amino acids or molecules not classified as amino acids. Examples of β-, γ-, δ-, or ε-amino acids that may be included in the linkers of the invention include, but are not limited to, β-alanine, γ-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, 6-aminohexanoic acid, and statins.

[0143] Additionally, chemical spacers (Sp1), (Sp2), and / or (Sp3) may comprise amino acid derivatives and / or amino acid mimetics. In embodiments in which (Sp1), (Sp2), and / or (Sp3) comprise one or more amino acid derivatives, it is preferred that the amino acid derivatives have free amino and carboxy groups so that they can undergo peptide or isopeptide bond formation. In embodiments in which (Sp1), (Sp2), and / or (Sp3) comprise one or more amino acid mimetics, the amino acid mimetics may have free amino and carboxy groups so that they can undergo peptide or isopeptide bond formation. However, in certain embodiments, the amino acid mimetics or derivatives may have substituted amino groups that do not prevent peptide bond formation. Examples of such amino acid mimetics or derivatives may be N-methylated amino acids, such as sarcosine or N-Me-leucine.

[0144] In embodiments in which the amino acid residue included in (Sp1) or (Sp3) is a terminal amino acid residue, the terminal amino acid residue may comprise a modified, protected or substituted N-terminal amino group or C-terminal carboxy group.

[0145] Additionally, the amino acid mimetic or derivative may be a mimetic or derivative of an amino acid containing a derivatized amino group, such as proline or other cyclic amino acids, such as azetidine-2-carboxylic acid, pipecolic acid, or spinacin. Additionally, the amino acid mimetic may contain other functional groups that replace the amino and / or carboxy groups of standard amino acids and allow the amino acid mimetic to undergo alternative bond formation with adjacent amino acids, amino acid derivatives, and / or amino acid mimetics, as well as to form peptidomimetics.

[0146] The term "amino acid mimetic" as used herein refers to a compound that has a different structure from a specific amino acid but functions similarly to the specific amino acid, and can therefore be used to replace the specific amino acid. An amino acid mimetic is said to function similarly to a specific amino acid if it has at least some structural and / or functional characteristics similar to the amino acid it mimics. The term "amino acid derivative" refers to an amino acid as defined herein, in which one or more functional groups contained in the amino acid have been modified or substituted. The amino acid derivative may preferably be a derivative of a proteinogenic or non-canonical amino acid. Any functional group of the amino acid derivative may be substituted or modified.

[0147] In embodiments where the linker comprises one or more terminal amino acid residues, the terminal amino acid residues may be protected. For example, in embodiments where (Sp1) comprises an N-terminal amino acid residue, the N-terminal amino group may be protected. For example, in certain embodiments, the N-terminal amino acid residue contained in the spacer (Sp1) may be acetylated. In other embodiments, the R residue contained in the RK motif may be the N-terminal amino acid of the linker. In such embodiments, the N-terminal amino group of the arginine, arginine mimetic, or arginine derivative may be protected, for example, by acetylation. In certain embodiments, linking moiety B or payload B may be an amino acid or be based on an amino acid. In such embodiments, the N-terminal amino group of the amino acid-based payload or linking moiety B may be protected, for example, by acetylation.

[0148] Similarly, in embodiments in which (Sp3) comprises a C-terminal amino acid residue, the C-terminal carboxy group may be protected. For example, in certain embodiments, the C-terminal amino acid residue of the spacer (Sp3) may be amidated. In other embodiments, the K residue contained in the RK motif may be the C-terminal amino acid of the linker. In such embodiments, the C-terminal carboxy group of the lysine, lysine mimetic, or lysine derivative may be protected, e.g., by amidation. In certain embodiments, linking moiety B or payload B may be an amino acid or be based on an amino acid. In such embodiments, the C-terminal carboxy group of the amino acid-based payload or linking moiety B may be protected, e.g., by amidation.

[0149] In certain embodiments, each of the chemical spacers (Sp1), (Sp2), and / or (Sp3) may contain 0 to 12 amino acid residues, including amino acid derivatives and amino acid mimetics. That is, in certain embodiments, (Sp1) may contain 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues, (Sp2) may contain 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues, and (Sp3) may contain 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues.

[0150] In certain embodiments, the invention relates to a method 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.

[0151] That is, in certain embodiments, the linker may contain 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid residues, including amino acid mimetics and amino acid derivatives. It should be understood that the amino acid residues contained in the linker containing amino acid mimetics and amino acid derivatives are preferably amino acid residues contained in the RK motif, chemical spacer (Sp1), (Sp2), and / or (Sp3), and in certain embodiments, B when B is an amino acid-based linking moiety or payload. In embodiments where the linker contains only two amino acid residues, the two amino acid residues are contained in the RK motif. In such embodiments, (Sp1), (Sp2), and / or (Sp3) are absent or do not contain any amino acid, amino acid mimetic, or amino acid derivative.

[0152] In certain embodiments, the linker may comprise 2 to 25 amino acid residues, including amino acid mimetics and amino acid derivatives. In other embodiments, the linker may comprise 2 to 20 amino acid residues, including amino acid mimetics and amino acid derivatives. In other embodiments, the linker may comprise 2 to 15 amino acid residues, including amino acid mimetics and amino acid derivatives. In other embodiments, the linker may comprise 2 to 10 amino acid residues, including amino acid mimetics and amino acid derivatives. In other embodiments, the linker may comprise 3 to 10 amino acid residues, including amino acid mimetics and amino acid derivatives. In other embodiments, the linker may comprise 3 to 8 amino acid residues, including amino acid mimetics and amino acid derivatives. In other embodiments, the linker may comprise 3 to 6 amino acid residues, including amino acid mimetics and amino acid derivatives.

[0153] In certain embodiments, the present invention relates to a method according to the present invention, wherein the net charge of the linker is neutral or positive.

[0154] In certain embodiments, the linker is a peptide linker (or a peptidomimetic as disclosed herein). That is, the chemical spacers (Sp1), (Sp2), and / or (Sp3), when present, are exclusively composed of amino acids, amino acid mimetics, or amino acid derivatives. The net charge of a peptide is typically calculated at neutral pH (7.0). In the simplest approach, the net charge is determined by adding the number of positively charged amino acid residues (Arg and Lys, and optionally His) and the number of negatively charged amino acid residues (Asp and Glu) and calculating the difference between the two groups. If the linker contains a non-canonical amino acid or amino acid derivative in which the charged functional group has been modified or substituted, those skilled in the art will know how to determine the charge of the non-canonical amino acid or amino acid derivative at neutral pH.

[0155] In certain embodiments, the payload or linking moiety B or any non-amino acid moieties contained in (Sp1), (Sp2) and / or (Sp3) may also contribute to the net charge of the linker. However, those skilled in the art know how to calculate the net charge of the entire linker, including any non-amino acid moieties, preferably at neutral pH (7.0).

[0156] In certain embodiments, the net charge of the linker is calculated solely based on the amino acid residues contained in the linker, including amino acid mimetics and amino acid derivatives. Thus, in certain embodiments, the present invention relates to a method according to the present invention, wherein the net charge of the amino acid residues contained in the linker is neutral or positive.

[0157] In a particular embodiment, the present invention relates to a method according to the invention, wherein the linker does not comprise a negatively charged amino acid residue.

[0158] That is, the linker may not contain negatively charged amino acid residues, including amino acid mimetics and amino acid derivatives. Negatively charged amino acid residues are amino acids, amino acid mimetics, or amino acid derivatives that have a negative charge at neutral pH (7.0). Negatively charged canonical amino acids are glutamic acid and aspartic acid. However, negatively charged non-canonical amino acids, amino acid mimetics, and amino acid derivatives are known in the art.

[0159] In certain embodiments, the present invention relates to a method according to the present invention, wherein the linker comprises at least one positively charged amino acid residue outside the RK motif, i.e., (Sp1), (Sp2) and / or (Sp3) comprise at least one positively charged amino acid. In certain embodiments, (Sp1), (Sp2) and / or (Sp3) comprise at least one histidine residue.

[0160] In addition to or instead of amino acid residues, including amino acid mimetics and derivatives, the chemical spacers (Sp1), (Sp2) and / or (Sp3) may comprise or consist of non-amino acid moieties.

[0161] That is, in certain embodiments, chemical spacers (Sp1), (Sp2), and / or (Sp3) may not consist exclusively of amino acids, amino acid mimetics, or amino acid derivatives. That is, chemical spacers (Sp1), (Sp2), and / or (Sp3) may include or consist exclusively of non-amino acid components. In certain embodiments, chemical spacers (Sp1), (Sp2), and / or (Sp3) may include amino acids and non-amino acid components.

[0162] For example, without limitation, each of chemical spacers (Sp1), (Sp2) and / or (Sp3) may comprise a framework comprising 1 to 200 carbon atoms, optionally substituted with one or more atoms, and optionally comprising at least 10 atoms, e.g., 10 to 100 atoms or 20 to 100 carbon atoms, where the carbon-containing framework is linear hydrocarbon or comprises cyclic groups, symmetrically or asymmetrically branched hydrocarbons, monosaccharides, disaccharides, linear or branched oligosaccharides (asymmetrically branched or symmetrically branched), other naturally occurring linear or branched oligomers (asymmetrically branched or symmetrically branched), or more generally, any dimer, trimer, or higher oligomer (linear, asymmetrically branched or symmetrically branched) resulting from any chain-growth or step-growth polymerization process.

[0163] (Sp1), (Sp2) and / or (Sp3) are any linear, branched and / or cyclic C alkyl radicals optionally interrupted by one or more homocyclic aromatic or heterocyclic radicals. 2~30 Alkyl, C 2~30 Alkenyl, C 2~30 Alkynyl, C 2~30 Heteroalkyl, C 2~30 Heteroalkenyl, C 2~30 Heteroalkynyl; in particular, any linear or branched C alkyl group, wherein x1 and x2 are independently integers selected from the range of 0 to 20. 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 In some embodiments, (Sp1), (Sp2) and / or (Sp3) may be a C2~6 It may contain an alkyl group.

[0164] In certain embodiments, the chemical spacers (Sp1), (Sp2), and / or (Sp3) may comprise one or more polyethylene glycol (PEG) moieties or equivalent condensation polymers, such as poly(carboxybetaine methacrylate) (pCBMA), polyoxazoline, polyglycerol, polyvinylpyrrolidone, or poly(hydroxyethyl methacrylate) (pHEMA). Polyethylene glycol (PEG) is a polyether compound with many applications, from industrial manufacturing to medicine. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular weight. The structure of PEG is typically H—(O—CH—CH) n Those skilled in the art know how to functionalize condensation polymers so that they can be linked to amino acid residues or payloads.

[0165] The present inventors have shown that linkers containing a PEG moiety can be conjugated to glycosylated antibodies as efficiently as equivalent linkers without a PEG moiety. For example, the linkers ARK-PEG2-PABC-MMAE (Figure 14) and ARK-PEG2-(NH)-(CH3)-S-C4-maytansine (Figure 15) were conjugated to glycosylated polatuzumab with 92 and 90% efficiency, respectively (compared to 94% for ARK-PABC-MMAE). In another example, the linkers ARK-PEG2-PABC-MMAE (Figure 14) and ARK-PEG2-(NH)-(CH3)-S-C4-maytansine (Figure 15) were conjugated to glycosylated trastuzumab with 99% efficiency (compared to 100% for ARK-PABC-MMAE).

[0166] Thus, in certain embodiments, the present invention relates to a method according to the present invention, wherein the linker comprises one or more PEG moieties. In certain embodiments, the PEG moieties may be included in the chemical spacers (Sp1), (Sp2), and / or (Sp3). In certain embodiments, each PEG moiety included in the linker may comprise 2 to 20 ethylene glycol monomers, 2 to 15 ethylene glycol monomers, 2 to 10 ethylene glycol monomers, or 2 to 5 ethylene glycol monomers. In certain embodiments, the PEG moiety is included in (Sp2) so as to directly connect the linking moiety to the RK motif or the payload. In certain embodiments, the PEG moiety is included in (Sp2) so as to connect the linking moiety to the amino acid residue included in (Sp2) or the payload. In certain embodiments, the PEG moiety is included in (Sp2) so as to connect the RK motif to the self-immolative moiety, which in turn is connected to the payload. In certain embodiments, the PEG moiety is included in (Sp2) so as to connect the amino acid residue included in (Sp2) to the self-immolative moiety, which in turn is connected to the payload.

[0167] In certain embodiments, the chemical spacers (Sp1), (Sp2), and / or (Sp3) may comprise dextran. The term "dextran," as used herein, refers to a complex, branched glucan composed of chains of various lengths, which may have weights ranging from 3 to 2,000 kDa. The linear chains typically consist of alpha-1,6 glycosidic linkages between glucose molecules, while the branches begin with alpha-1,3 linkages. Dextran can be synthesized from sucrose, for example, by lactic acid bacteria. In the context of the present invention, dextran used as a carrier may preferably have a molecular weight of approximately 15 to 1,500 kDa.

[0168] In certain embodiments, the chemical spacers (Sp1), (Sp2), and / or (Sp3) may comprise an oligonucleotide. The term "oligonucleotide" as used herein refers to an oligomer or polymer of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), as well as non-naturally occurring oligonucleotides. Due to their greater stability, the oligonucleotide is preferably a polymer of DNA.

[0169] In certain embodiments, chemical spacers (Sp1), (Sp2), and / or (Sp3), when present, are exclusively comprised of amino acid residues, including amino acid mimetics and derivatives, and PEG moieties. In certain embodiments, chemical spacers (Sp1), (Sp2), and / or (Sp3), when present, are exclusively comprised of amino acid residues, including amino acid mimetics and derivatives. In certain embodiments, all amino acid residues contained in chemical spacers (Sp1), (Sp2), and / or (Sp3) are α-L-amino acids. That is, in certain embodiments, the linker, excluding the payload or linking moiety B, is exclusively comprised of amino acid residues. In certain embodiments, the linker, excluding the payload or linking moiety B, is exclusively comprised of α-L-amino acid residues. Such peptide-based linkers may contain protecting groups at the N- and / or C-termini. That is, the N-terminal amino group may be acetylated and / or the C-terminal carboxy group may be amidated.

[0170] It should be noted that the chemical spacers (Sp1), (Sp2), and / or (Sp3) may have the same structure. However, it is preferred that each of the chemical spacers (Sp1), (Sp2), and / or (Sp3) have a different structure, and / or that not all of the chemical spacers (Sp1), (Sp2), and / or (Sp3) are present at the same time. That is, in certain embodiments, only one or two of the chemical spacers (Sp1), (Sp2), and / or (Sp3) may be present in the linker.

[0171] In certain embodiments, the RK motif may be directly connected to one or more small, hydrophobic amino acid residues, for example, in certain embodiments, the RK motif may be directly connected to one or more alanine residues.

[0172] That is, in certain embodiments, the present invention relates to a method according to the present invention, wherein the linker comprises the amino acid sequence RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4) or RK-Val-Cit (SEQ ID NO: 54). It should be understood that the RK motif used to conjugate the linker to a glutamine residue of the antibody may be comprised in the amino acid sequence RKAA, RKA, ARK, RKR or RK-Val-Cit.

[0173] In a particular embodiment, the present invention relates to a method according to the invention, wherein the linker comprises the amino acid sequence RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2) or ARK (SEQ ID NO: 3).

[0174] In a particular embodiment, the present invention relates to a method according to the invention, wherein the linker comprises the amino acid sequence RKAA (SEQ ID NO: 1).

[0175] In a particular embodiment, the present invention relates to a method according to the present invention, wherein the linker comprises the amino acid sequence RK-Val-Cit (SEQ ID NO: 54).

[0176] Within the scope of the present invention, it is preferred that the linker be conjugated to the antibody via the primary amine contained in the side chain of residue K contained in the RK motif. Therefore, it is preferred that the chemical spacers (Sp1), (Sp2), and / or (Sp3) do not contain additional lysine residues, lysine mimetics, or lysine derivatives that could serve as additional amine donors in transglutaminase-based conjugation reactions. In other embodiments, any free N-terminal amino group contained in the linker may be substituted, e.g., acetylated, so that it cannot serve as a substrate for microbial transglutaminase.

[0177] The linker according to the present invention further comprises at least one linking moiety or payload B. The linker according to the present invention can be used to directly conjugate a payload to an antibody in a one-step conjugation process. In other embodiments, a linker comprising one or more linking moieties may be conjugated to an antibody in a first step, and then one or more payloads may be linked to the antibody-linker conjugate in a second step. Table 1 below clarifies the two terms used herein. Table 1: One- and two-step conjugation [Table 1]

[0178] In certain embodiments, the linker may comprise one or more linking moieties B. Thus, in certain embodiments, the present invention relates to a method according to the invention, wherein B is a linking moiety.

[0179] As used herein, the term "linking moiety" generally refers to a molecule with at least two functionalities. Within the scope of the present invention, the linking moiety comprises a first functional group that connects the linking moiety to the linker of the present invention and a second functional group that can be used to connect additional molecules to the linker before or after the linker is conjugated to an antibody. In certain embodiments, the linking moiety of the present invention is an amino acid, an amino acid mimic, or an amino acid derivative. In such embodiments, the linking moiety is preferably connected to the linker via its amino group, while the functional group contained in the amino acid side chain can be used to connect additional molecules to the linker. Alternatively, the linking moiety can be connected to the linker via its carboxy group, while the functional group contained in the amino acid side chain can be used to connect additional molecules to the linker.

[0180] In a particular embodiment, the present invention relates to a method according to the present invention, wherein linking moiety B is - bioorthogonal marker groups, or - Non-bio-orthogonal entities for crosslinking The present invention relates to a method, comprising:

[0181] The term "bioorthogonal marker group" was established by Sletten and Bertozzi (A Bioorthogonal Quadricyclane Ligation. J Am Chem Soc 2011, 133 (44), 17570-17573) to refer to a reactive group that can cause a chemical reaction to occur inside a biological system without interfering with native biochemical processes. A "non-bioorthogonal entity for crosslinking" can be any molecule containing or consisting of a first functional group, where the first functional group can be chemically or enzymatically crosslinked to a payload containing a compatible second functional group. Even if the crosslinking reaction is non-bioorthogonal, it is preferable that the reaction does not introduce any further modifications to the antibody other than crosslinking the payload to the linker. In view of the above, linking moiety B can consist of a "bioorthogonal marker group" or a "non-bioorthogonal entity," or can include a "bioorthogonal marker group" or a "non-bioorthogonal entity." For example, in the case of the linking moiety Lys(N3), both the entire Lys(N3) and the azido group alone may be considered bioorthogonal marker groups within the scope of the present invention. Lys(N3) refers to 6-azido-L-lysine, which may be abbreviated as K(N3).

[0182] In certain embodiments, the present invention relates to a method according to the present invention, wherein the bioorthogonal marker group or the non-bioorthogonal entity for crosslinking is: - -NN≡N, or -N3; - Lys(N3); - tetrazine; - alkynes; - Distorted cyclooctyne; - BCN; - strained alkenes; - photoreactive groups; - aldehydes; - acyltrifluoroborates; - Protein degrading agents ("PROTACs"); - Cyclopentadiene / spirolocyclopentadiene; - Thioselective electrophiles; - -SH; and - Cysteine The present invention relates to a method comprising or consisting of at least one molecule or moiety selected from the group consisting of:

[0183] The bioorthogonal marker group contained in the linker or the non-bioorthogonal entity for cross-linking can participate in any of the conjugation reactions shown in Table 2, for example. Table 2 [Table 2]

[0184] Linking moiety B may be or include what is referred to as "binding partner 1" or "binding partner 2" in Table 2.

[0185] In certain embodiments, linking moiety B may be a cysteine, a cysteine ​​mimetic, or a cysteine ​​derivative having a free sulfhydryl group.

[0186] The free sulfhydryl group of such a Cys residue (or a mimetic or derivative) may be conjugated to a payload construct containing a thio-selective electrophile, such as maleimide. Toxin constructs containing maleimide moieties are frequently used and approved by medical authorities, such as Adcetris. Thus, toxin constructs containing MMAE toxins can be attached to the free sulfhydryl group of a Cys residue of the linker of the present invention.

[0187] It should be noted that other thio-selective electrophiles, such as 3-arylpropionitriles (APN) or phosphonamidates, can also be used in place of maleimides in the methods of the present invention.

[0188] Thus, providing a Cys residue in the linker according to the present invention has the advantage that it allows antibody-payload conjugates to be produced using pre-made toxin-maleimide constructs, and more generally, it allows the full utilization of Cys-maleimide conjugation chemistry. At the same time, pre-made antibodies that do not need to be deglycosylated can be used. In certain embodiments, the Cys residue can be at the C-terminus of the amino acid-based linker or within the chain.

[0189] In another embodiment, the linking moiety B may contain an azide group. Those skilled in the art are familiar with molecules containing an azide group that can be incorporated into the linker of the present invention, such as 6-azido-lysine (Lys(N3)) or 4-azido-homoalanine (Xaa(N3)). Linking moieties containing an azide group may be used as substrates in various bioorthogonal reactions, such as strain-promoted azide-alkyne cycloaddition (SPAAC), copper-catalyzed azide-alkyne cycloaddition (CuAAC), or Staudinger ligation. For example, in certain embodiments, payloads containing cyclooctyne derivatives such as DBCO, DIBO, BCN, or BARAC may be attached to linkers containing an azide group via SPAAC.

[0190] In another embodiment, the linking moiety B may contain a tetrazine group. Those skilled in the art are aware of tetrazine-containing molecules, preferably amino acid derivatives containing a tetrazine group, that can be incorporated into the linker of the present invention. A linking moiety containing a tetrazine can be used as a substrate in bioorthogonal tetrazine ligation. For example, in certain embodiments, a payload containing a cyclopropene, norborene, norborene derivative, or cyclooctyne group, such as bicyclo[6.1.0]nonyne (BCN), can be attached to a linker containing a tetrazine group.

[0191] In certain embodiments, linking moiety B may comprise a cyclic diene, such as a cyclopentadiene derivative. Potential cyclopentadiene derivatives that can be linked to maleimide-containing payload molecules are described in Amant et al., "Tuning the Diels-Alder Reaction for Bioconjugation to Maleimide Drug-Linkers; Bioconjugate Chem. 2018, 29, 7, 2406-2414" and "A Reactive Antibody Platform for One-Step Production of Antibody-Drug Conjugates through a Diels-Alder Reaction with Maleimide; Bioconjugate Chem. 2019, 30, 9, 2340-2348."

[0192] In certain embodiments, the linking moiety B may comprise a photoreactive group. The term "photoreactive group," as used herein, refers to a chemical group that responds to an applied external energy source to undergo active species generation and forms a covalent bond with an adjacent chemical structure (e.g., an abstractable hydrogen). Examples of photoreactive groups include, but are not limited to, aryl azides, such as phenyl azide, o-hydroxyphenyl azide, m-hydroxyphenyl azide, tetrafluorophenyl azide, o-nitrophenyl azide, m-nitrophenyl azide, or azidomethylcoumarin, diazirine, psoralen, or benzophenone.

[0193] In certain embodiments, the present invention relates to a method according to the present invention, which comprises the further step of conjugating one or more payloads to linking moiety B.

[0194] Instead of directly conjugating a linker comprising one or more payloads to an antibody in a one-step process, the present invention, in certain embodiments, refers to a two-step process, in which a linker comprising at least one linking moiety B is conjugated to an antibody in a first step, and then one or more payloads can be attached to linking moiety B in a second step.

[0195] The term "payload," as used herein, refers to any naturally occurring or synthetically produced molecule, including low molecular weight molecules or chemical entities that can be chemically synthesized, and larger molecules or biological entities that must be produced by fermentation of host cells or can also be chemically synthesized and that confer novel functionality to the antibody. It should be understood that the payload may include additional structures or functional groups that allow for attachment to the linking moiety included in the linker of the payload or to other parts of the linker, such as the chemical spacers (Sp1) and / or (Sp3) or the RK motif.

[0196] In the two-step conjugation process, the payload can be linked to the linking moiety by any suitable method known in the art.Preferably, the payload can be linked to any of the bioorthogonal marker groups disclosed herein or non-bioorthogonal entities for crosslinking.That is, the payload preferably comprises a functional group that is compatible with the bioorthogonal marker group or non-bioorthogonal entities for crosslinking contained in at least one linking moiety B.

[0197] Several bioorthogonal reactions that can be used to link a payload to a bioorthogonal marker group contained in linking moiety B are known in the art. For example, 1,3-dipolar cycloaddition between azides and cyclooctynes ​​(also called copper-free click chemistry, Baskin et al. ("Copper-free click chemistry for dynamic in vivo imaging", Proceedings of the National Academy of Sciences. 104 (43): 16793-7)), between nitrones and cyclooctynes ​​(Ning et al. ("Protein Modification by Strain-Promoted Alkyne-Nitrone Cycloaddition", Angewandte Chemie International Edition. 49 (17): 3065)), oxime / hydrazone formation from aldehydes and ketones (Yarema, et al. ("Metabolic Delivery of Ketone Groups to Sialic Acid Residues. Application To Cell Surface Glycoform Engineering", Journal of Biological Chemistry. 273 (47): 31168-79)), and tetrazine ligation (Blackman et al. ("The Tetrazine Ligation: Fast Bioconjugation based on Inverse-electron-demand Diels-Alder Reactivity. Journal of the American Chemical Society. 130 (41): 13518-9), isonitrile-based click reaction (Stockmann et al. ("Exploring isonitrile-based click chemistry for ligation with biomolecules". Organic & Biomolecular Chemistry.9 (21): 7303), and more recently, quadricyclane ligation (Sletten & Bertozzi (JACS, A Bioorthogonal Quadricyclane Ligation. J Am Chem Soc 2011, 133 (44), 17570-17573)), copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC, Kolb & Sharpless ("The growing impact of click chemistry on drug discovery". Drug Discov Today. 8 (24): 1128-1137)), strain-promoted azide-alkyne cycloaddition (SPAAC, Agard et al ("A Comparative Study of Bioorthogonal Reactions with Azides". ACS Chem. Biol. 1: 644-648)), or strain-promoted alkyne-nitrone cycloaddition (SPANC, MacKenzie et al Several chemical ligation strategies that meet the requirements of bioorthogonal labeling have been developed, including "Strain-promoted cycloadditions involving nitrones and alkynes—rapid tunable reactions for bioorthogonal labeling". Curr Opin Chem Biol. 21: 81-8). All of these documents are incorporated herein by reference to provide sufficient enabling disclosure and to avoid redundant repetition.

[0198] It should be understood that it is preferable to conjugate the payload to a bioorthogonal marker group or a non-bioorthogonal entity for cross-linking contained in a linker according to the present invention after the linker has been conjugated to a Gln residue of the antibody by microbial transglutaminase. However, the present invention also encompasses antibody-linker conjugates in which one or more payloads are attached to a linker comprising at least one linking moiety B in a first step, and the resulting linker-payload construct is conjugated to an antibody in a second step by microbial transglutaminase.

[0199] In certain embodiments, the present invention relates to a method according to the present invention, wherein one or more payloads are conjugated to linking moiety B by a Click reaction.

[0200] That is, one or more payloads can be linked to linking moiety B in a Click reaction, particularly any of the Click reactions disclosed herein.

[0201] In certain preferred embodiments, at least one payload can be conjugated to a linking moiety B included in the linker by thiol-maleimide conjugation. That is, in certain embodiments, the payload can include a maleimide group, and linking moiety B can be a molecule containing a thiol group, for example, but not limited to, a cysteine ​​residue or a cysteine ​​mimetic such as homocysteine. However, B can also be a non-amino acid molecule containing a free thiol group. In another embodiment, the payload can include a free thiol group, and linking moiety B can include a maleimide group.

[0202] In another particularly preferred embodiment, at least one payload can be conjugated to a linking moiety B included in the linker by strain-promoted azide-alkyne cycloaddition (SPAAC). That is, in certain embodiments, the payload can include an alkyne group, such as, but not limited to, a cyclooctyne group, and the linking moiety B can be a molecule containing an azide group, such as, but not limited to, the lysine derivative Lys(N3) disclosed herein. However, B can also be a non-amino acid molecule containing a free azide group. In another embodiment, the payload can include an alkyne group, such as, for example, a cyclooctyne group, and the linking moiety B can include an azide group.

[0203] Besides the Click reaction between the linking moiety of the linker and the functional group of the payload, the payload may be covalently attached to the linking moiety by any enzymatic or non-enzymatic reaction known in the art.

[0204] Preferably, the payload is linked to the linking moiety by a covalent bond. However, in certain embodiments, the payload may be linked to the linking moiety by a strong non-covalent bond. That is, in certain embodiments, the linking moiety B may comprise a biotin moiety, such as, but not limited to, the lysine derivative biocytin. In such embodiments, a payload comprising a streptavidin moiety may be linked to a linker comprising a biotin moiety.

[0205] In a particular embodiment, the present invention relates to a method according to the invention, wherein B is a payload.

[0206] In certain embodiments, the payload may already be part of the linker so that it can be conjugated to the antibody in a one-step process. In such embodiments, the linker is preferably attached to the linker by chemical synthesis. The payload is preferably attached to a chemical spacer contained in the linker or directly attached to the RK motif. In embodiments in which the payload is attached to an amino acid residue, including amino acid mimetics and derivatives, the payload may be attached to the C-terminal carboxy group or N-terminal amino group of the amino acid residue. Alternatively, the payload may be attached to a functional group contained in the side chain of the amino acid residue. Those skilled in the art are familiar with methods for functionalizing the payload so that it can be attached to the carboxy group, amino group, or amino acid side chain.

[0207] Furthermore, those skilled in the art know how to connect payloads to amino acid-based linkers by chemical synthesis.For example, amine-containing payloads, or thiol-containing payloads (for example, maytansine analogs), or hydroxyl-containing payloads (for example, SN-38 analogs) can be attached to the C-terminus of amino acid-based linkers by chemical synthesis.However, those skilled in the art know that other reactions and reactive groups can be used to connect payloads to the N-terminus, C-terminus, or side chain of amino acids or amino acid derivatives by chemical synthesis.Typical reactions that can be used to connect payloads to amino acid-based linkers by chemical synthesis include, but are not limited to, peptide bond, activated ester bond (NHS ester, PFP ester), click reaction (CuAAC, SPAAC), and Michael addition (thiol-maleimide conjugation).Conjugation of a payload to a peptide can be performed using techniques described in the prior art, e.g., 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. (Site-Specific Conjugation of Native Antibodies Using Engineered Microbial Transglutaminases. Bioconjug Chem. 2020 Mar 12. doi: 10.1021 / acs.bioconjchem.0c00061).

[0208] It should be understood that the payload can be attached to the N-terminus or C-terminus of a peptide-based or peptide-containing linker according to the present invention. In certain embodiments, the payload can be directly attached to the N-terminal amino group or C-terminal carboxy group of a peptide or amino acid residue (see, e.g., Figure 22).

[0209] Those skilled in the art are familiar with reactive groups suitable for binding a payload to an amino acid residue. For example, an amine-containing payload can be bound to the C-terminal carboxyl group of an amino acid residue via an amide bond (Figure 22). Alternatively, a thiol or hydroxyl group-containing payload can be bound to the C-terminal carboxyl group of an amino acid via a thioester or ester bond, respectively. A carboxylic acid group-containing payload can be bound to the N-terminal amino group of an amino acid residue via an amide bond.

[0210] In certain embodiments, the payload can be indirectly linked to the N- or C-terminus of a peptide or amino acid residue contained in the linker according to the present invention. Those skilled in the art are aware of linker molecules that can be used to link a payload to the N-terminal amino group or C-terminal carboxy group of an amino acid residue contained in the linker according to the present invention.

[0211] In certain embodiments, a payload containing a hydroxy group can be attached to the N-terminus of an amino acid residue by a linker molecule, for example, a payload containing a hydroxy group can be attached to the N-terminal amino group by a carbamate linker molecule (Figure 24).

[0212] In certain embodiments, a payload containing a thiol group can be attached to the N-terminus of an amino acid residue via a linker molecule. For example, a payload containing a thiol group can be attached to the N-terminal amino group via a thiocarbamate linker molecule (Figure 28). Alternatively, a payload containing a thiol group can be attached to the N-terminal 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, and the payload forms a di-sulfur bond with the thiol group contained in the 3-mercaptopropionic acid linker molecule (Figure 29).

[0213] In certain embodiments, the payload that comprises an amide group can be linked to the N-terminus of an amino acid residue through a linker molecule.For example, the payload that comprises an amine group can be linked to the N-terminus amino acid group through a dicarboxylic acid linker molecule, where the dicarboxylic acid linker forms an amide bond with the payload and the amino group of the N-terminus 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 (see Figures 9 and 30).

[0214] Alternative linker molecules for indirectly attaching a payload to the N-terminus of an amino acid residue comprised in a linker according to the invention or linker molecules that are suitable for indirectly attaching a payload to the C-terminus of an amino acid residue comprised in a linker according to the invention have been described in the art and are encompassed by the present invention.

[0215] In a particular embodiment, the present invention relates to a method according to the present invention, wherein the payload comprises: - toxin; - cytokines; - growth factors; - Radionuclides; - Hormones; - antiviral agents; - antibacterial agents; - Fluorescent dyes: - immunoregulatory / immunostimulant agents; - Half-life increasing part; - solubility increasing part; - polymer-toxin conjugates; - Nucleic acids; - a biotin or streptavidin moiety; - Vitamins; - Protein degrading agents ("PROTACs"); - a target binding moiety; and / or - Anti-inflammatory The present invention relates to a method comprising at least one of the following:

[0216] Any one of the payloads disclosed herein can be attached directly to a linker for use in the one-step conjugation process disclosed herein, or can be attached to a linking moiety included in an antibody-linker conjugate produced as part of the two-step process disclosed herein.

[0217] In certain embodiments, the payload may be a cytokine. As used herein, the term "cytokine" refers to any secreted polypeptide that affects the function of other cells and modulates cell-cell interactions in immune or inflammatory responses. Cytokines include, but are not limited to, monokines, lymphokines, and chemokines, regardless of the cell that produces them. For example, monokines are generally said to be produced and secreted by monocytes; however, many other cells, such as natural killer cells, fibroblasts, basophils, neutrophils, endothelial cells, brain astrocytes, bone marrow stromal cells, epidermal keratinocytes, and B lymphocytes, also produce monokines. Lymphokines are generally said to be produced by lymphocytes. 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).

[0218] In certain embodiments, the payload may be an anti-inflammatory agent. As used herein, the term "anti-inflammatory agent" refers to a class of drugs whose primary mode of action and use is in the area of ​​treating inflammation, as well as any other drug from another therapeutic class that has a useful anti-inflammatory effect. Such anti-inflammatory agents include, but are not limited to, nonsteroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs), macrolide antibiotics, and statins. Preferably, NSAIDs include, but are not limited to, salicylates (e.g., aspirin), arylpropionic acids (e.g., ibuprofen), anthranilic acids (e.g., mefenamic acid), pyrazoles (e.g., phenylbutazone), cyclic acetic acids (indomethicin), and oxicams (e.g., piroxicam). Preferably, anti-inflammatory agents for use in the methods of the present invention include sulindac, diclofenac, tenoxicam, ketorolac, naproxen, nabumetone, diflunasal, ketoprofen, arlypropionic 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.

[0219] In certain embodiments, the anti-inflammatory agent may be an anti-inflammatory cytokine that, 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.

[0220] In certain embodiments, the payload may be a growth factor. The term "growth factor," as used herein, refers to a naturally occurring substance capable of stimulating cell growth, proliferation, cell differentiation, and / or cell maturation. Growth factors exist in the form of either proteins or steroid hormones. Growth factors are important for regulating various cellular processes. Growth factors typically act as signaling molecules between cells. However, their ability to promote cell growth, proliferation, cell differentiation, and cell maturation varies among growth factors. 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, endothelial 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, hepatoma-derived growth factor, insulin growth factor, insulin-like growth factor, migration-stimulating factor, myostatin, nerve growth factor, and other neutrophils, platelet-derived growth factor, transforming growth factor alpha, transforming growth factor beta, tumor necrosis factor-alpha, vascular endothelial growth factor, placental growth factor, fetal bovine somatotropin, 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).

[0221] 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 that are 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 (ADH), and the like. atrial natriuretic peptide or atriopeptin (ANP), calcitonin (CT), cholecystokinin (CCK), corticotrophin-releasing hormone (CRH), erythropoietin (EPO), follicle-stimulating hormone (FSH), gastrin (GRP), ghrelin, glucagon (GCG), gonadotropin-releasing hormone (GnRH), growth hormone-releasing hormone (GHRH), human chorionic gonadotropin (hCG), human placental lactogen (HPL), growth hormone (GH or hGH), inhibin, 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, dehydrogenase Epiandrosterone (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.

[0222] In certain embodiments, the payload may be an antiviral agent. The term "antiviral agent," as used herein, refers to an agent (compound or biological agent) that is effective in inhibiting viral formation and / or replication in a mammal. This includes agents that interfere with host or viral mechanisms necessary for viral 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, Ceplene (Maxamine), XTL-001, and XTL-002 (XTL Biopharmaceuticals).

[0223] 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 infection in a subject; or (iii) inhibit or reduce the ability of bacteria to increase or persist infection in an environment. The term "antibacterial agent" also refers to a compound that can reduce the infectivity or virulence of bacteria.

[0224] In certain embodiments, the payload may be an immunomodulatory agent. The term "immunomodulatory agent," as used herein in reference to combination therapy, refers to a substance that acts to suppress, mask, or enhance the host's immune system. Examples of immunomodulatory agents include, but are not limited to, proteinaceous agents such as cytokines, peptidomimetics, and antibodies (e.g., human, humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs, 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. In particular, immunomodulatory agents include, but are not limited to, methotrexate, leflunomide, cyclophosphamide, cytoxan, Immuran, cyclosporine A, minocycline, azathioprine, antibiotics (e.g., FK506 (tacrolimus)), methylprednisolone (MP), corticosteroids, steriods, mycophenolate mofetil, rapamycin (sirolimus), mizoribine, deoxyspergualin, brequinar, malononitriloamindes (e.g., leflunamide), T cell receptor modulators, and cytokine receptor modulators.

[0225] In certain embodiments, the immunoregulatory agent may be an immunostimulatory agent. As used herein, the term "immunostimulatory agent" preferably refers to any substance or substance capable of inducing an immune response (e.g., an immune response against a specific pathogen). Compounds that activate immune cells include Toll-like receptor (TLR) agonists. Such agonists include pathogen-associated molecular patterns (PAMPs), such as bacterial immunomodulators (also known as danger signals) that mimic infection, and damage-associated molecular patterns (DAMPs), such as stressed or damaged cells. TLR agonists include nucleic acids or lipid compositions (e.g., monophosphoryl lipid A (MPLA)). In one example, the TLR agonist includes a TLR9 agonist, such as a cytosine-guanosine oligonucleotide (CpG-ODN), a poly(ethyleneimine) (PEI)-condensed oligonucleotide (ODN), such as PEI-CpG-ODN, or double-stranded deoxyribonucleic acid (DNA). In another example, the TLR agonist includes a TLR3 agonist, such as polyinosinic-polycytidylic acid (poly(I:C)), PEI-poly(I:C), polyadenylic-polyuridylic acid (poly(A:U)), PEI-poly(A:U), or double-stranded ribonucleic acid (RNA). Other exemplary vaccine immunostimulatory compounds include lipopolysaccharide (LPS), chemokines / cytokines, fungal beta-glucans (e.g., lentinan), imiquimod, CRX-527, and OM-174.

[0226] In certain embodiments, the payload may be a half-life increasing moiety or a solubility increasing moiety.The half-life increasing 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 increasing moiety is, for example, a PEG moiety (PEGylation) or a PAS moiety (PASylation).

[0227] In certain embodiments, the payload may be a polymer-toxin conjugate. A polymer-toxin conjugate is a polymer that can carry multiple payload molecules. Such a conjugate is also called a fleximer, for example, commercially available from Mersana Therapeutics. The polymer-toxin conjugate may include any of the toxins disclosed herein.

[0228] 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 immunostimulatory properties developed by MultiCell Technologies, Inc.

[0229] 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 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 wavelengths between 650 and 900 nm. This range exhibits less tissue autofluorescence and enhanced deep tissue penetration while minimizing background interference due to low fluorescence quenching. Therefore, near-infrared fluorescent imaging can be used to visualize tissues to which the antibody-payload conjugates of the present invention are bound during surgery. "Near-infrared fluorescent dyes" are known in the art and commercially available. In certain embodiments, the near-infrared fluorescent dye may be IRDye 800CW, Cy7, Cy7.5, NIR CF750 / 770 / 790, DyLight 800, or Alexa Fluor 750.

[0230] In certain embodiments, the payload may comprise a radionuclide. The term "radionuclide," as used herein, refers to a medically useful radionuclide comprising a positively charged ion of a radioactive metal such as, for example, Y, In, Tb, Ac, Cu, Lu, Tc, Re, Co, Fe, etc., e.g., 90Y, 111 In, 67 Cu, 77 Lu, 99 Tc, 161 Tb, 225 Ac and the like. The radionuclide may be contained in a chelating agent such as DOTA or NODA-GA. Furthermore, the radionuclide may be a therapeutic radionuclide or a radionuclide that can be used as a contrast agent in the imaging techniques discussed below. Radionuclides or molecules containing radionuclides are known in the art and are commercially available.

[0231] In certain embodiments, the payload may be a vitamin, which may be selected from the group consisting of folates, including folic acid, folacin, and vitamin B9.

[0232] In a particular embodiment, the present invention relates to a method according to the present invention, wherein the toxin is - Pyrrolobenzodiazepines (e.g., PBD); - Auristatins (e.g., MMAE, MMAF); - Maytansinoids (e.g., maytansine, DM1, DM4, DM21); - Duocarmycin; - Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors; - Tubulyshyn; - Enzymes (e.g., calicheamicin); - anthracycline derivatives (PNU) (e.g., doxorubicin); - Pyrrole-based kinesin spindle protein (KSP) inhibitors; - Cryptophycin; - Drug efflux pump inhibitors; - Sandramycin; amanitin (e.g., α-amanitin); and - Camptothecins (e.g., exatecan, deruxtecan) The method is at least one selected from the group consisting of:

[0233] That is, the antibody-linker conjugates produced using the methods of the present invention preferably contain a toxin payload. The term "toxin," as used herein, refers to any compound produced by living cells and organisms that is toxic to the cells or organisms. Thus, a toxin can be, for example, 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, a toxin can be conjugated to an antibody using the methods of the present invention and delivered to or into malignant cells via the targeting specificity of the antibody.

[0234] In certain embodiments, the toxin may be an auristatin. As used herein, the term "auristatin" refers to a family of antimitotic agents. Auristatin derivatives are also included within the definition of the term "auristatin." Examples of auristatins include, but are not limited to, synthetic analogs of auristatin E (AE), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and dolastatins.

[0235] In certain embodiments, the toxin may be a maytansinoid. In the context of the present invention, the term "maytansinoid" refers to a class of highly cytotoxic drugs originally isolated from the African shrub Maytenus ovatus and further includes maytansinol and natural C-3 esters of maytansinol (U.S. Pat. No. 4,151,042); synthetic C-3 ester analogs of maytansinol (Kupchan et al., J. Med. Chem. 21: 31-37, 1978; Higashide et al., Nature 270: 721-722, 1977; Kawai et al., Chem. Farm. Bull. 32: 3441-3451; and U.S. Pat. No. 5,416,064); C-3 esters of simple carboxylic acids (U.S. Pat. Nos. 4,248,870; 4,265,814; 4,308,268; 4,308,269; 4,309,428; 4,317,821; 4,322,348; and 4,331,598); and C-3 esters with derivatives of N-methyl-L-alanine (U.S. Pat. Nos. 4,137,230; 4,260,608; and Kawai et al., Chem. Pharm Bull. 12: 3441, 1984). Exemplary maytansinoids that 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.

[0236] In certain embodiments, the toxin may be a duocarmycin. Suitable duocarmycins may be, for example, duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, duocarmycin MA, and CC-1065. The term "duocarmycin" should also be understood to refer to synthetic analogs of duocarmycins, for example, adozelesin, bizelesin, carzelesin, KW-2189, and CBI-TMI.

[0237] In certain embodiments, the toxin may be a NAMPT inhibitor. As used herein, the terms "NAMPT inhibitor" and "nicotinamide phosphoribosyltransferase inhibitor" 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), GPP 78 hydrochloride, ST 118804, 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, PCT Publication WO2015 / 054060, U.S. Patent Nos. 8,211,912, and 9,676,721, which are incorporated herein by reference in their entireties. In some embodiments, the NAMPT inhibitor is FK866. In some embodiments, the NAMPT inhibitor is GMX-1778.

[0238] In certain embodiments, the toxin may be a tubulysin. Tubulysins are cytotoxic peptides containing nine members (A-I). Tubulysin A has potential use as an anticancer drug. Tubulysin A arrests cells in the G2 / M phase. Tubulysin A inhibits polymerization more efficiently than vinblastine and induces depolymerization of isolated microtubules. Tubulysin A has potent cytostatic activity against various tumor cell lines with an IC50 in the picomolar range. Another tubulysin that can be used in the methods of the present invention may be tubulysin E.

[0239] In certain embodiments, the toxin may be an enediyne. The term "enediyne," as used herein, refers to a class of bacterial natural products characterized by a 9- and 10-membered ring containing two triple bonds separated by a double bond (see, e.g., KC Nicolaou; AL Smith; EW 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). Their reactivity with DNA endows many enediynes with antibiotic properties, and several enediynes are under clinical investigation as anticancer antibiotics. Non-limiting examples of enediynes are dynemicin, neocarzinostatin, calicheamicin, and esperamicin (see, e.g., Adrian L. Smith and KC Bicolaou, "The Enediyne Antibiotics," J. Med.Chem., 1996, 39 (11), pp. 2103-2117; and Donald Borders, "Enediyne antibiotics as antitumor agents," Informa Healthcare; 1st edition (Nov. 23, 1994, ISBN-10:0824789385), the entire contents of which are incorporated herein by reference).In certain embodiments, the toxin may be a calicheamicin.

[0240] 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, of the Streptomyces genus, and includes doxorubicin, daunorubicin, epirubicin, and idarubicin.

[0241] In certain embodiments, the toxin may be a kinesin spindle protein inhibitor. The term "kinesin spindle protein inhibitor" refers to a compound that inhibits kinesin spindle proteins, which are involved in the assembly of bipolar spindles during cell division. Kinesin spindle protein inhibitors are being investigated 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.

[0242] In certain embodiments, the toxin may be a cryptophycin, as described in US20180078656A1, which is incorporated by reference.

[0243] In certain embodiments, the toxin may be sandramycin, a depsipeptide first isolated from Nocardioides sp. (ATCC 39419) and shown to have cytotoxic and antitumor activity.

[0244] In certain embodiments, the toxin may be amatoxin. Amatoxins (including alpha-amanitin, beta-amanitin, and amanitin) are cyclic peptides composed of eight amino acids. They are isolated from the Amanita phalloides mushroom and can be synthetically prepared from linking blocks. Amatoxins specifically inhibit DNA-dependent RNA polymerase II in mammalian cells, thereby inhibiting transcription and protein biosynthesis in affected cells. Inhibition of transcription in cells halts growth and proliferation. Although not covalently bound, the complex between amanitin and RNA polymerase II is very tight (KD = 3 nM). Dissociation of amanitin from the enzyme is a very slow process, making affected cells unlikely to recover. If transcription inhibition persists in a cell for too long, the cell undergoes programmed cell death (apoptosis). In a preferred embodiment, the term "amatoxin" as used herein refers to alpha-amanitin or a variant thereof as described, for example, in WO2010 / 115630, WO2010 / 115629, WO2012 / 119787, WO2012 / 041504, and WO2014 / 135282.

[0245] In certain embodiments, the toxin may be camptothecin. As used herein, the term "camptothecin" 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, BN 80915, raltotecan, 9-nitrocamptothecin, and aminocamptothecin. Various camptothecins, including camptothecin, have been described for use in treating human cancer patients. Some camptothecins have been 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).

[0246] Toxins may also be inhibitors of drug efflux transporters within the meaning of the present invention. Antibody-payload conjugates containing a toxin and a drug efflux transporter inhibitor may have the advantage that, when translocated into cells, the drug efflux transporter inhibitor prevents the toxin from being excreted from the cell. Within the scope of 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 CP 100356 are other common P-gp inhibitors. Zosuquidar and tariquidar have also been developed based on this idea. Finally, valspodar and reversan are other examples of such drugs.

[0247] It should be understood that the payload B defined herein should not be understood exclusively as the actual payload itself, but rather as a payload molecule, which as used herein may include additional structures that facilitate the attachment of the payload to the linking moiety B or RK motif or chemical spacer, for example, by chemical synthesis.

[0248] That is, in certain embodiments, the actual payload may be contained in a payload molecule that is linked to a linker of the invention. The payload molecule may have the structure: X-(spacer)-payload, (wherein payload represents the actual payload, e.g., one of the compounds disclosed herein; X represents a reactive group suitable for attaching the payload molecule to a compatible functional group of the linking moiety (two-step process) or the chemical spacer of the linker or the RK motif (one-step process), where (spacer) represents a chemical spacer that spatially separates the actual payload from the reactive group X). However, it should be understood that in certain embodiments, the reactive group X may be part of the spacer or the actual payload. For example, the spacer may comprise a peptide or amino acid residue, and the reactive group X may be the amino group of the N-terminal amino acid residue contained in the spacer. In other embodiments, the spacer may be absent. In embodiments where a spacer is absent, a functional group may be contained in the actual payload. In certain embodiments, the spacer can be used to attach a functional group of interest, i.e., a functional group that is compatible with the functional group contained in the linking moiety, to the actual payload. In certain embodiments, the reactive group X may be a maleimide group or a cyclooctyne group, such as, but not limited to, a DBCO or BCN group.

[0249] In certain embodiments, the present invention relates to a method according to the present invention, wherein the chemical spacer (Sp2) comprises a self-immolative moiety.

[0250] That is, the linker may include a self-immolative moiety that facilitates release of the payload from the target cell or tissue. The self-immolative moiety may be included in any part of the linker. However, the self-immolative moiety is preferably included in the chemical spacer (Sp2) that separates the payload from the RK motif. Alternatively, the self-immolative moiety may be included in the payload molecule (spacer) as defined above.

[0251] As used herein, the term "self-immolative moiety" refers to an at least bifunctional molecule that may be included in a linker and that spontaneously decomposes after an initial reaction, thereby releasing a payload. The initial reaction may be hydrolysis of a covalent bond between the self-immolative moiety and an amino acid residue. In certain embodiments, the covalent bond between the self-immolative moiety and the amino acid residue may be an amide bond formed between the α-carboxy group of the amino acid and an amine group included in the self-immolative moiety, and the initial reaction may be catalyzed by a peptidase or protease. However, other chemical structures are encompassed by the present invention.

[0252] In a particular embodiment, the present invention relates to a method according to the invention, wherein the self-immolative moiety is attached directly to payload B.

[0253] More preferably, the self-immolative moiety is attached directly to payload B such that the payload is released upon degradation of the self-immolative moiety. In certain embodiments, the self-immolative moiety is located between the payload and the RK motif contained in the linker. That is, the self-immolative moiety may be attached to the N-terminus of residue R or the C-terminus of residue K. Alternatively, the self-immolative moiety may be located between the payload and an amino acid residue contained in the chemical spacer (Sp2), preferably at the N- or C-terminus of said amino acid residue. Furthermore, the self-immolative moiety may be located between the payload and a non-amino acid residue contained in the chemical spacer (Sp2) by any method known in the art.

[0254] It should be understood that the selection of the self-immolative moiety will depend, among other things, on the functional groups that are available on the payload molecule.

[0255] In certain embodiments, the present invention relates to a method according to the present invention, wherein the self-immolative moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.

[0256] That is, in certain embodiments, the linker may comprise a self-immolative moiety p-aminobenzylcarbamoyl (PABC). PABC comprises a free amine group suitable for attachment to the C-terminus of an amino acid residue or peptide and a carbamoyl group that can be used to attach to a payload, particularly an amine-containing payload. However, those skilled in the art will know how to functionalize a payload so that it contains an amine group. The self-immolative moiety PABC is preferably located between the payload and an amino acid residue contained in the linker. The amino acid residue is preferably residue K contained in an RK motif or an amino acid contained in the chemical spacer (Sp2). In certain embodiments, the self-immolative moiety PABC is located between the payload and an alanine residue contained in the chemical spacer (Sp2). In certain embodiments, the self-immolative moiety may be located between the payload and a peptidase cleavage site. In certain embodiments, the self-immolative moiety may be located between the payload and a cathepsin cleavage site. That is, the self-immolative moiety may be located between the payload and a motif known to be cleavable by cathepsin.

[0257] The term "cathepsin," as used herein, refers to a family of proteases. The term cathepsin includes cathepsin A, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin F, cathepsin G, cathepsin H, cathepsin K, cathepsin L1, cathepsin L2, cathepsin O, cathepsin S, cathepsin W, and cathepsin Z. In certain embodiments, the cleavable moiety may be a motif that is specifically hydrolyzed by cathepsin B, such as valine-alanine, valine-citrulline, or alanine-alanine. Further motifs that can be specifically hydrolyzed by peptidases are disclosed in Salomon et al., Optimizing Lysosomal Activation of Antibody-Drug Conjugates (ADCs) by Incorporation of Novel Cleavable Dipeptide Linkers, Mol Pharm. 2019, 16(12), pp. 4817-4825.

[0258] A typical dipeptide structure used in ADC linkers is, for example, the valine-citrulline motif found in brentuximab vedotin, as discussed in Dubowchik and Firestone; Cathepsin B-labile dipeptide linkers for lysosomal release of doxorubicin from internalizing immunoconjugates: model studies of enzymatic drug release and antigen-specific in vitro anticancer activity; Bioconjug Chem; 2002; 13(4); pp. 855-69. This linker can be cleaved by cathepsin B to release the actual payload at the disease site. The same applies to the valine-alanine motif found in SGN-CD33A, for example.

[0259] Thus, in certain embodiments, a linker may comprise the structure (Sp1)-RK-(Sp2)-Val-Cit-(self-immolative moiety)-payload. In certain embodiments, a linker may comprise the structure (Sp1)-RK-(Sp2)-Val-Cit-payload. In certain embodiments, a linker may comprise the structure (Sp1)-RK-(Sp2)-Val-Cit-PABC-payload.

[0260] In certain embodiments, the linker may comprise the structure RK-Val-Cit (SEQ ID NO: 54). In certain embodiments, the linker may comprise or consist of the structure RK-Val-Cit-(self-immolative moiety)-payload. In certain embodiments, the linker may comprise or consist of the structure RK-Val-Cit-PABC-payload. In certain embodiments, the linker may comprise or consist of the structure RK-Val-Cit-PABC-MMAE. In certain embodiments, the linker may comprise or consist of the structure RK-Val-Cit-PABC-maytansine.

[0261] It should be noted that the peptide cleavage site may be cleavable by other peptidases, such as caspase 3, legumain, or neutrophil elastase, or may be a motif described in Dal Corso et al., Innovative Linker Strategies for Tumor-Targeted Drug Conjugates; Chemistry; 25(65); p. 14740-14757.

[0262] However, it should be noted that cells contain a wide range of cellular peptidases, and other less conserved amino acid motifs may also be efficiently cleaved by peptidases. Thus, in certain embodiments, the linker may comprise the structure (Sp1)-RK-(Sp2)-PABC-payload, where (Sp2) is absent or consists of an amino acid residue.

[0263] In certain embodiments, the linker may comprise the structure (Sp1)-RK-(Sp2)-PABC-payload, where (Sp2) comprises a PEG moiety between the PABC moiety and the most C-terminal amino acid residue contained in the (Sp2) or RK motif.

[0264] In certain embodiments, a linker comprising the self-immolative moiety PABC is linked to an amine-containing payload, particularly a payload comprising a primary or secondary amine. In certain embodiments, the amine-containing payload is an auristatin, e.g., MMAE. In certain embodiments, the amine-containing payload is a maytansinoid, e.g., maytansine.

[0265] It should be noted that the payload can be linked to the self-immolative PABC moiety via an additional linker molecule. For example, an amine-containing payload can be linked to the PABC moiety via a p-nitrophenol (PNP) group. Additional linker molecules that enable the binding of payloads containing reactive groups other than amines to the PABC moiety are disclosed in Su et al., Bioconjugate Chem. 2018, 29, 4, 1155-1167; and Dokter et al., Mol Cancer Ther. 2014 Nov;13(11):2618-29. For example, a payload containing an alcohol or phenol group can be linked to the PABC moiety via an ethylenediamine (EDA) linker (see Figures 18 and 19).

[0266] In certain embodiments, the present invention relates to a method according to the present invention, wherein the self-immolative moiety comprises a methylamine group. It has previously been demonstrated that methylamine groups can be used as self-immolative moieties in peptide-based linkers of ADCs (Costoplus et al., ACS Med. Chem. Lett., 2019, 10, 10, 1393-1399 and Li et al., ACS Med. Chem. Lett. 2019, 10, 10, 1386-1392).

[0267] In particular, a self-immolative moiety containing a methylamine group may be attached to the C-terminus of an amino acid residue via an amide bond formed between the α-carboxy group of the amino acid residue and the amine contained in the methylamine group. The amino acid residue may be an amino acid residue contained in (Sp2) or residue K contained in an RK motif. The methyl group contained in the methylamine group may be attached to the payload via an ether or thioether bond. Thus, a methylamine group may be preferably used as a self-immolative group when the payload contains a hydroxyl group or a thiol group. In certain embodiments, the hydroxyl-containing payload may be a camptothecin, e.g., an anthracycline such as the exatecan derivative Dxd or PNU-159682. In certain embodiments, the thiol-containing payload may be a maytansinoid, e.g., DM1, DM4, or DM21.

[0268] A linker containing a methylamine group may have the molecular structure C—(NH)—(CH)—OC or C—(NH)—(CH)—SC. Exemplary linkers containing a methylamine group are shown in Figures 15, 17, and 21.

[0269] It should be understood that PABC and self-immolative moieties containing a methylamine group are preferably used to attach a payload to the C-terminal carboxy group of an amino acid residue.

[0270] Other self-immolative moieties that can be used to attach a payload to the C-terminal carboxy group of an amino acid residue include a p-aminobenzylethanol (PABE) linker (Zhang et al., Bioconjugate Chem. 2018, 29, 6, 1852-1858) for attaching a phenol-containing payload to the C-terminal carboxy group of an amino acid residue, or a para-methylaniline (PMA) linker (Staben et al., Nature Chemistry volume 8, pages 1112-1119 (2016)) for attaching a tertiary amine or heteroaryl moiety-containing payload to the C-terminal carboxy group of an amino acid residue (see Figures 20 and 23, respectively). Non-limiting examples of payloads containing a phenol group include duocarmycin GA or pyrrolobenzodiazepine PBD. Non-limiting examples of payloads containing a tertiary amine include duocarmycin GA.

[0271] However, the payload may also be attached to the N-terminal amino group via a self-immolative moiety. For example, the payload may be attached to the N-terminal amino group of the amino acid residue via a self-immolative moiety comprising an ortho-hydroxy-protected aryl sulfate. For example, ortho-hydroxy-protected aryl sulfate (OHPAS) can be used to attach a phenol payload such as a PBD to the N-terminal amino group of the amino acid residue (see Figure 27). The OHPAS moiety preferably includes a carboxy group, by which the OHPAS can be directly attached to the N-terminal amino group of the amino acid residue. Alternatively, the OHPAS moiety may be attached to the N-terminal amino group of the amino acid residue via a functionalized PEG linker, for example, but not limited to, a functionalized (PEG)2 linker. Preferably, the PEG linker is functionalized at one end with an amino group to allow attachment to a carboxy group contained in the OHPAS moiety and at the other end with a carboxy group to allow attachment to the N-terminal amino group of an amino acid residue (Park et al., Bioconjugate Chem. 2019, 30, 7, 1957-1968) (see Figure 26).

[0272] Alternatively or additionally, a linker molecule may be positioned between the sulfate group of the OHPAS and the payload to enable the attachment of non-phenolic payloads to the OHPAS. For example, a para-hydroxybenzyl (PHB) linker molecule may be used to enable the attachment of a payload containing a primary or secondary amine to the OHPAS via the formation of a carbamate (see Figures 31 and 32). A payload containing a tertiary amine may be attached to the OHPAS containing linker via the formation of a quaternary ammonium (see Figures 33 and 34). Furthermore, a para-hydroxybenzylethylenediamine (PHB-EDA) linker molecule may be used to attach a hydroxyl-containing payload to the OHPAS moiety via the formation of a carbamate (Park et al., Bioconjugate Chem. 2019, 30, 7, 1957-1968) (see Figure 25).

[0273] In certain embodiments, the payload can be linked to an amino acid residue contained in the linker via a cleavable moiety. As used herein, a "cleavable moiety" is a chemical unit that can be separated from the actual payload by enzymatic or non-enzymatic hydrolysis. In certain embodiments, the cleavable moiety can be an amino acid motif that is hydrolyzable by peptidases or proteases.

[0274] In other embodiments, the cleavable moiety included in the linker may be a carbohydrate moiety. In such embodiments, the cleavable moiety may be a moiety that is cleavable by a glucosidase. Thus, in certain embodiments, the cleavable moiety may be a moiety that is cleavable by beta-glucuronidase or beta-galactosidase.

[0275] In other embodiments, the cleavable moiety included in the linker may be a phosphate moiety. In such embodiments, the cleavable moiety may be a moiety that is cleavable by a phosphatase. Thus, in certain embodiments, the cleavable moiety may be a moiety that is cleavable by beta-lysosomal acid pyrophosphatase or acid phosphatase.

[0276] Additional examples of cleavable moieties that can be used to release a payload from a linker molecule are described in Bargh et al., Cleavable linkers in antibody-drug conjugates; Chem Soc Rev. 2019 Aug 12;48(16):4361-4374. In certain embodiments, a linker may comprise the structure (cleavable moiety)-(self-immolative moiety)-payload. In such embodiments, the self-immolative moiety can decompose upon cleavage of the cleavable moiety, releasing the payload.

[0277] In certain embodiments, the present invention relates to a method according to the present invention, wherein the linker is any one of the linkers shown in Figure 1, Figure 2, Figure 3, Figure 8, Figure 9, Figure 14, Figure 15, Figure 17, Figure 18, Figure 19, Figure 20, Figure 21, Figure 22, Figure 23, Figure 24, Figure 25, Figure 26, Figure 27, Figure 28, Figure 29, Figure 30, Figure 31, Figure 32, Figure 33 or Figure 34.

[0278] In certain embodiments, the linker may comprise two or more linking moieties and / or payloads B. That is, in certain embodiments, the linker has the structure a)(Sp1)-RK-(Sp2)-B1-(Sp3)-B2-(Sp4), b)(Sp4)-B2-(Sp1)-RK-(Sp2)-B1-(Sp3), c) (Sp1)-B1-(Sp2)-RK-(Sp3)-B2-(Sp4), or d)(Sp4)-B2-(Sp1)-B1-(Sp2)-RK-(Sp3) may include:

[0279] In such embodiments, the chemical spacers (Sp1), (Sp2), (Sp3) and the RK motif may have the same characteristics as defined above. Furthermore, moieties B1 and B2 may be any one of the linking moieties and / or payloads defined above. Furthermore, chemical spacer (Sp4) may have the same characteristics as chemical spacers (Sp1), (Sp2), or (Sp3), or may be absent.

[0280] Thus, in a particular embodiment, the present invention relates to a method according to the invention, wherein the linker comprises a second linking moiety or payload B2, in particular B2 being connected to the linker by a chemical spacer (Sp1) or (Sp3).

[0281] That is, payload or linking moiety B2 may be connected to chemical spacer (Sp1) or (Sp3) or directly to payload or linking moiety B1. Payload or linking moiety B2 may contain any functional group that is suitable for attaching B2 to a functional group contained in (Sp1), (Sp3) or B1.

[0282] In certain embodiments, the payload or linking moiety B2 may comprise an amino group through which B2 is connected to (Sp3) or B1. That is, B2 may be connected to a carboxy group contained in (Sp3) or B1 through said amino group. In certain embodiments, the carboxy group contained in (Sp3) may be a carboxy group contained in the C-terminal amino acid residue of the chemical spacer (Sp3). In certain embodiments, the carboxy group contained in B1 may be the α-carboxy group of an amino acid-based payload or linking moiety. In certain embodiments, B2 may be attached to a carboxy group contained in (Sp3) or B1 by a linker molecule. In certain embodiments, the linker molecule may comprise a self-immolative moiety.

[0283] In certain embodiments, the payload or linking moiety B2 may comprise a carboxy group through which B2 is connected to (Sp1) or B1. That is, B2 may be connected to an amine group contained in (Sp1) or B1 via said carboxy group. In certain embodiments, the amine group contained in (Sp1) may be an amine group contained in the N-terminal amino acid residue of the chemical spacer (Sp1). In certain embodiments, the amine group contained in B1 may be an α-amino group of an amino acid-based payload or linking moiety. In certain embodiments, B2 may be attached to an amine group contained in (Sp1) or B1 by a linker molecule. In certain embodiments, the linker molecule may comprise a self-immolative moiety.

[0284] However, it should be noted that B2 may contain functional groups other than amine or carboxy groups. In such embodiments, B2 may be attached to (Sp1), (Sp3), or B1 by any method known in the art, either directly or through a linker or self-immolative group.

[0285] In certain embodiments, the payload or linking moiety B2 may be attached to an amino acid side chain contained in (Sp1) or (Sp3), i.e., B2 may be connected to a functional group of an amino acid side chain contained in (Sp1) or (Sp3) by a compatible functional group.

[0286] In certain embodiments, (Sp1), (Sp2), (Sp3), and the RK motif are exclusively amino acids, amino acid mimetics, and / or amino acid derivatives. In certain embodiments, B1 and / or B2 also comprise an amino acid backbone. In such embodiments, the linker may be a linear peptide or peptidomimetic. In embodiments where B1 is an amino acid, amino acid mimetic, or amino acid derivative, the linker may have the structure (Sp1)-RK-(Sp2)-B1, where (Sp1)-RK-(Sp2)-B1 is a linear peptide or peptidomimetic. In embodiments where B1 is an amino acid, amino acid mimetic, or amino acid derivative, the linker may have the structure (Sp1)-RK-(Sp2)-B1-(Sp3), where (Sp1)-RK-(Sp2)-B1-(Sp3) is a linear peptide or peptidomimetic. In embodiments where B1 is an amino acid, amino acid mimetic, or amino acid derivative, the linker may have the structure RK-(Sp2)-B1-(Sp3), where RK-(Sp2)-B1-(Sp3) is a linear peptide or peptidomimetic. In embodiments where B1 is an amino acid, amino acid mimetic, or amino acid derivative, the linker may have the structure RK-(Sp2)-B1, where RK-(Sp2)-B1 is a linear peptide or peptidomimetic. In embodiments where B1 is an amino acid, amino acid mimetic, or amino acid derivative, the linker may have the structure RK-B1-(Sp3), where RK-B1-(Sp3) is a linear peptide or peptidomimetic. In embodiments where B1 is an amino acid, amino acid mimetic, or amino acid derivative, the linker may have the structure RK-B1, where RK-B1 is a linear peptide or peptidomimetic.

[0287] In embodiments where B1 and B2 are amino acids, amino acid mimetics, or amino acid derivatives, the linker may have the structure (Sp1)-RK-(Sp2)-B1-(Sp3)-B2-(Sp4), where (Sp1)-RK-(Sp2)-B1-(Sp3)-B2-(Sp4) is a linear peptide or peptidomimetic. In other embodiments where B1 and B2 are amino acids, amino acid mimetics, or amino acid derivatives, the linker may have the structure (Sp4)-B2-(Sp1)-RK-(Sp2)-B1-(Sp3), where (Sp4)-B2-(Sp1)-RK-(Sp2)-B1-(Sp3) is a linear peptide or peptidomimetic. In other embodiments where B1 and B2 are amino acids, amino acid mimetics or amino acid derivatives, the linker may have the structure (Sp4)-B2-(Sp1)-B1-(Sp2)-RK-(Sp3), where (Sp4)-B2-(Sp1)-B1-(Sp2)-RK-(Sp3) is a linear peptide or peptidomimetic.

[0288] In embodiments in which B1 is not an amino acid, amino acid mimetic, or amino acid derivative, the linker may have the structure (Sp1)-RK-(Sp2)-B1-(Sp3), where (Sp1)-RK-(Sp2) is a linear peptide or peptidomimetic and B1 is attached to the C-terminal carboxy group of (Sp2). In embodiments in which B1 is not an amino acid, amino acid mimetic, or amino acid derivative, the linker may have the structure (Sp1)-B1-(Sp2)-RK-(Sp3), where (Sp2)-RK-(Sp3) is a linear peptide or peptidomimetic and B1 is attached to the N-terminal amino group of (Sp2). However, it should be noted that B1 does not necessarily have to be directly attached to the peptide or peptidomimetic. Instead, B1 may be attached to the peptide or peptidomimetic by a linker molecule and / or a self-immolative moiety.

[0289] In embodiments where B1 is an amino acid, amino acid mimetic or amino acid derivative and B2 is not an amino acid, amino acid mimetic or amino acid derivative, the linker may have the structure (Sp1)-RK-(Sp2)-B1-(Sp3)-B2-(Sp4), (Sp4)-B2-(Sp1)-RK-(Sp2)-B1-(Sp3), (Sp1)-B1-(Sp2)-RK-(Sp3)-B2-(Sp4) or It can have the formula (Sp4)-B2-(Sp1)-B1-(Sp2)-RK-(Sp3), where (Sp1)-RK-(Sp2)-B1-(Sp3) or (Sp1)-B1-(Sp2)-RK-(Sp3) is a linear peptide or peptidomimetic, and B2 is attached to the C-terminal carboxy group contained in (Sp3), B1, or RK or to the N-terminal amino group of (Sp1), B1, or RK.

[0290] In such embodiments, antibody-payload conjugates can be produced at antibody to payload ratios of, for example, 2 or 4, with, for example, 1 or 2 payloads conjugated to each Q295 residue.

[0291] In a particular embodiment, the present invention relates to a method according to the present invention, wherein B1 and B2 are the same or different from each other.

[0292] That is, the payloads or linking moieties B1 and B2 may be the same, i.e., have the same chemical structure, or may be structurally different. In certain embodiments, B1 and B2 are both payloads or both linking moieties. In embodiments where B1 and B2 are both payloads, the payloads B1 and B2 may be the same or different payloads. In embodiments where B1 and B2 are both linking moieties, the linking moieties B1 and B2 may be the same or different linking moieties. In certain embodiments, B1 may be a linking moiety and B2 may be a payload, or vice versa.

[0293] It should be understood that not all payloads or linking moieties can function as intra-chain payloads or linking moieties at position B1, for example, because not all payloads or linking moieties have functional groups that form covalent bonds with (Sp2) or RK on the one hand and with (Sp3), (Sp1), or B2 on the other. Thus, in embodiments in which B1 is an intra-chain payload or linking moiety, B1 is preferably a bivalent or multivalent molecule. For example, B1 may be an amino acid, amino acid mimetic, or amino acid derivative. In such embodiments, B1 may be attached to the C-terminal carboxy group of (Sp2) or RK through its amino group and to the N-terminal amino group of (Sp3) or B2 through its carboxy group. Alternatively, B1 may be attached to the N-terminal amino group of (Sp2) or RK through its carboxy group and to the C-terminal carboxy group of (Sp1) or B2 through its amino group.

[0294] In certain embodiments, the linker may comprise two linking moieties B1 and B2.

[0295] That is, in certain embodiments, the present invention encompasses linkers comprising two bioorthogonal marker groups and / or non-bioorthogonal entities. For example, a linker according to the present invention may comprise an azide-containing linking moiety, such as Lys(N3) or Xaa(N3), and a sulfhydryl-containing linking moiety, such as cysteine. In certain embodiments, a linker according to the present invention may comprise an azide-containing linking moiety, such as Lys(N3) or Xaa(N3), and a tetrazine-containing linking moiety, such as a tetrazine-modified amino acid. In certain embodiments, a linker according to the present invention may comprise a sulfhydryl-containing linking moiety, such as cysteine, and a tetrazine-containing linking moiety, such as a tetrazine-modified amino acid. Linkers comprising two different bioorthogonal marker groups and / or non-bioorthogonal entities have the advantage that they can accept two separate payloads, thereby resulting in antibody-payload conjugates comprising two or more payloads.

[0296] In this way, a 2+2 antibody-payload ratio can be achieved. The use of a second payload may enable the development of an entirely new class of antibody-payload conjugates that surpass current therapeutic approaches in terms of efficacy and potency.

[0297] Such embodiments may, among other things, allow for targeting two different structures within a cell, such as DNA and microtubules. Some cancers may be resistant to one drug, such as a microtubule toxin, so the DNA toxin may still kill the cancer cells.

[0298] According to another embodiment, two drugs can be used that are only fully effective if released simultaneously and in the same tissue, which may result in reduced off-target toxicity if the antibody is partially degraded in healthy tissue or one drug is lost prematurely.

[0299] Furthermore, the dual-labeled probe can be used for non-invasive imaging and treatment or intraoperative / postoperative imaging / surgery. In such an embodiment, tumor patients can be selected by non-invasive imaging. Then, the tumor can be surgically removed using other imaging agents (e.g., fluorescent dyes) that help the surgeon or robot identify all cancerous tissue during surgery.

[0300] In certain embodiments, one of B1 and B2 can be a linking moiety that includes a thiol group, e.g., cysteine, and the other of B1 and B2 can be a linking moiety that includes an azide moiety, e.g., Lys(N3). In such embodiments, two separate payloads can be attached to the linker, one via thiol-maleimide conjugation and the other via a SPAAC reaction.

[0301] In certain embodiments, a linker may comprise two payloads. A linker comprising only a payload but no linking moiety may be conjugated to an antibody in a one-step process.

[0302] In embodiments where B1 and B2 are both payloads, it should be understood that B1 and B2 may have the same or different structures.In certain embodiments, the linker comprising one or more payloads may be chemically synthesized.Alternatively, one or more payloads may be bound to the linking moiety comprised in the linker by any of the methods disclosed herein before the linker is conjugated to the antibody.

[0303] In certain embodiments, the linkers of the invention allow two different payloads to be attached to the C of an antibody. HThe second payload can be conjugated to the Q295 residue of the second domain.The use of the second payload allows the development of a completely new class of antibody-payload conjugates that surpass current therapeutic approaches in terms of efficacy and potency.In addition, new application fields are envisioned, such as imaging and therapy or intraoperative / postoperative dual-type imaging (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 incorporating a molecular imaging agent for preoperative positron emission tomography (PET) and a near-infrared fluorescent (NIRF) dye to guide surgical margin delineation can greatly enhance 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 U S A. 2015 Dec 29;112(52):15850-5). PET and NIRF optical imaging offer complementary clinical applications, enabling noninvasive whole-body imaging to identify disease location and intraoperative tumor margin identification, respectively. However, to date, the generation of such dual-labeled probes has been challenging due to the lack of suitable site-specific methods, and attaching two distinct probes by chemical means often results in random probe conjugation, making analysis and reproducibility nearly impossible.

[0304] 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) found that a dual-drug-conjugated antibody with two different auristatin toxins attached conferred activity in cell lines and xenograft models that were resistant to ADCs composed of individual auristatin components (which have different physiochemical properties and exert complementary anticancer activities). This suggests that dual-conjugated ADCs may address cancer heterogeneity and resistance more effectively than single conventional ADCs alone. Because one resistance mechanism to ADCs involves the active pumping of cytotoxic moieties out of 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-conjugated ADCs may help overcome cancer resistance to ADCs more effectively than conventional ADCs.

[0305] 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 of a length exhibiting a desired biological activity. The terms "antibody(s)" broadly encompass naturally occurring forms of antibodies (e.g., IgG, IgA, IgM, IgE).

[0306] The antibody is preferably a monoclonal antibody. The antibody may be of human origin, but may also be derived from mouse, rat, goat, donkey, hamster, or rabbit. If the conjugate is for therapeutic use, the mouse or rabbit antibody may be chimeric or humanized as needed.

[0307] C H Fragments or recombinant variants of antibodies comprising two domains can be e.g. - Just the heavy chain domain (Shark antibody / IgNAR(V H -C H 1-C H 2-C H 3-C H 4-C H 5)2 or camelized antibody / hcIgG(V H -C H 2-C H 3) Antibody formats including 2) - scFv-Fc(VH-VL-CH2-CH3)2 - an Fc fusion peptide containing an Fc domain and one or more receptor domains; may be.

[0308] Antibodies may be bispecific (e.g., DVD-IgG, crossMab, additional IgG-HC fusions) or biparatopic. For an overview, see Brinkmann and Kontermann; Bispecific antibodies; Drug Discovery Today; 2015; 20(7); pp. 838-47.

[0309] In a particular embodiment, the invention relates to a method according to the invention, wherein the antibody is an IgG antibody, in particular an IgG1 antibody.

[0310] By "IgG," as used herein, is meant a polypeptide belonging to the class of antibodies substantially encoded by recognized immunoglobulin gamma genes. In humans, IgG includes the subclasses or isotypes IgG1, IgG2, IgG3, and IgG4. In mice, IgG includes IgG1, IgG2a, IgG2b, and 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 (also called CH1), Cγ2 (also called CH2), and Cγ3 (also called CH3). In the context of human IgG1, "CH1" refers to positions 118-215, the CH2 domain refers to positions 231-340, and the CH3 domain refers to positions 341-447, according to the EU index of Kabat. IgG1 also contains a hinge domain, which in the case of IgG1 refers to positions 216-230.

[0311] The antibody or antibody-payload conjugate of the present invention used in the method of the present invention can be or comprise any antibody, preferably any IgG type antibody.For example, the antibody can be, but is not limited to, brentuximab, trastuzumab, gemtuzumab, inotuzumab, avelumab, cetuximab, rituximab, daratumumab, pertuzumab, vedolizumab, ocrelizumab, tocilizumab, ustekinumab, golimumab, obinutuzumab, sacituzumab, belantamab, polatuzumab and enfortumab.

[0312] Thus, in a particular embodiment, the invention relates to a method according to the invention, wherein the antibody is selected from the group consisting of brentuximab, trastuzumab, gemtuzumab, inotuzumab, avelumab, cetuximab, rituximab, daratumumab, pertuzumab, vedolizumab, ocrelizumab, tocilizumab, ustekinumab, golimumab, obinutuzumab, sacituzumab, belantamab, polatuzumab and enfortumab.

[0313] In a preferred embodiment, the invention relates to a method according to the invention, wherein the antibody is selected from the group consisting of brentuximab, gemtuzumab, trastuzumab, inotuzumab, polatuzumab, enfortumab, sacituzumab and belantamab.

[0314] In a more preferred embodiment, the invention relates to a method according to the invention, wherein the antibody is polatuzumab or trastuzumab or enfortumab.

[0315] That is, in certain embodiments, the invention relates to an antibody-linker conjugate wherein the antibody is polatuzumab and the linker is any one of the linkers disclosed herein.

[0316] In another embodiment, the invention relates to an antibody-linker conjugate wherein the antibody is trastuzumab and the linker is any one of the linkers disclosed herein.

[0317] In another embodiment, the invention relates to an antibody-linker conjugate wherein the antibody is enfortumab and the linker is any one of the linkers disclosed herein.

[0318] Antibodies for use in the methods according to the invention may be glycosylated, deglycosylated or aglycosylated antibodies.

[0319] That is, in certain embodiments, the antibody may be an IgG antibody which is preferably glycosylated at residue N297. Thus, in certain embodiments, the invention relates to a method according to the invention, wherein the IgG antibody is a glycosylated IgG antibody, in particular an IgG antibody C H The method relates to a method in which the polypeptide is glycosylated at residue N297 (EU numbering) of domain 2.

[0320] As discussed herein, IgG antibodies glycosylated at residue N297 have several advantages over non-glycosylated antibodies.

[0321] However, the antibody may also be a deglycosylated antibody, preferably in which the glycan at residue N297 has been cleaved with the enzyme PNGase F. Additionally, the antibody may be an aglycosylated antibody, preferably in which residue N297 has been replaced with a non-asparagine residue. Methods for deglycosylating antibodies and for producing aglycosylated antibodies are known in the art.

[0322] In certain embodiments, the linkers of the invention may be conjugated to an endogenous Gln residue in the Fc domain of an antibody or to a Gln residue introduced into the antibody by molecular engineering.

[0323] Thus, in a particular embodiment, the present invention relates to a method according to the present invention, wherein the Gln residue to which the linker is conjugated is comprised in the Fc domain of the antibody, in particular the Gln residue to which the linker is conjugated is comprised in the C domain of an IgG antibody. H The method relates to a method in which the Gln residue Q295 (EU numbering) of the 2 domain is

[0324] The linkers of the present invention may be conjugated to any Gln residue in the Fc domain of an antibody that can serve as a substrate for microbial transglutaminase. Typically, the term Fc domain, as used herein, refers to the last two constant region immunoglobulin domains (C) of IgA, IgD and IgG. H 2 and C H 3) and the last three constant region domains of IgE, IgY, and IgM (C H 2. C H 3 and C H 4). That is, the linker according to the present invention is a C H 2. C H 3, and, if applicable, C H4 domain.

[0325] In certain embodiments, the endogenous Gln residue may be Gln residue Q295 (EU numbering) in the CH2 domain of an IgG antibody. Thus, in certain embodiments, the invention relates to a method according to the invention, wherein the Gln residue in the Fc domain of the antibody is Gln residue Q295 (EU numbering) in the CH2 domain of an IgG antibody.

[0326] It is important to understand that Q295 is a highly conserved amino acid residue in IgG antibodies. Q295 is particularly conserved in human IgG1, 2, 3, and 4, as well as in rabbit and rat antibodies. Therefore, the availability of Q295 is a significant advantage for generating therapeutic antibody-payload conjugates or diagnostic conjugates, where the antibodies are often of non-human origin. The method according to the present invention therefore provides a highly versatile and widely applicable tool. Although residue Q295 is extremely conserved among IgG antibodies, some IgG antibodies, such as mouse and rat IgG2a antibodies, do not possess this residue. Therefore, the antibodies used in the method of the present invention may be C H It should be understood that it is preferred that the antibody is an IgG type antibody comprising residue Q295 (EU numbering) of the 2 domain.

[0327] Furthermore, engineered conjugates using Q295 for payload attachment have been shown to exhibit favorable pharmacokinetics and efficacy (Lhospice et al., Site-Specific Conjugation of Monomethyl Auristatin E to Anti-Cd30 Antibodies Improves Their Pharmacokinetics and Therapeutic Index in Rodent Models, Mol Pharm; 2015; 12(6), pp. 1863-1871), and are even capable of carrying unstable toxins prone to degradation (Dorywalska et al., Site-Dependent Degradation of a Non-Cleavable Auristatin-Based Linker-Payload in Rodent Plasma and Its Effect on ADC Efficacy. PLoS ONE; 2015; 10(7): e0132282). Therefore, since the same residues are modified, except for those of glycosylated antibodies, we expect similar efficacy for this site-specific approach. Glycosylation can further contribute to overall ADC stability, and removal of glycan moieties, similar to the mentioned approach, has been shown to result in less stable antibodies (Zheng et al.; The impact of glycosylation on monoclonal antibody conformation and stability. Mabs-Austin; 2011, 3(6), pp.568-576).

[0328] C of the linker by transglutaminase H In the literature discussing conjugation to two Gln residues, attention has been focused on small, low-molecular-weight substrates, but the prior art always describes a deglycosylation step at position N297 or the use of an aglycosylated antibody to accomplish such conjugation, if necessary (WO2015 / 015448; WO2017 / 025179; WO2013 / 092998).

[0329] However, quite surprisingly and contrary to all expectations, site-specific conjugation of glycosylated antibodies to Q295 was quite efficient using the linker structures discussed above. In particular, attachment of linkers containing toxin molecules was achieved with conjugation efficiencies of over 80%.

[0330] Although Q295 is in close proximity to N297, which is glycosylated in its native state, the method according to the invention still allows the conjugation of a linker or payload to it using specific linkers.

[0331] As shown, the method according to the invention does not require prior enzymatic deglycosylation of N297, nor the use of an aglycosylation antibody, nor the substitution of N297 with another amino acid, nor the introduction of a T299A mutation to prevent glycosylation.

[0332] These two points provide considerable advantages in manufacturing aspects: an enzymatic deglycosylation step is undesirable in GMP aspects, as it must be ensured that not only the cleaved glycans but also the deglycosylation enzyme (e.g., PNGase F) are removed from the medium.

[0333] Furthermore, it is necessary not to genetically engineer the antibody for the attachment of the payload, thereby avoiding the insertion of sequences that may increase immunogenicity and decrease the overall stability of the antibody.

[0334] Substituting N297 for another amino acid can also have undesirable effects, as it can 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, consequently, the efficacy of the entire conjugate, which can lead to increased antibody aggregation and reduced solubility, which is particularly important for hydrophobic payloads such as PBDs (Zheng et al.; The impact of glycosylation on monoclonal antibody conformation and stability. Mabs-Austin 2011, 3 (6), 568-576). Furthermore, the glycan present at N297 has important immunomodulatory effects, such as eliciting antibody-dependent cellular cytotoxicity (ADCC). These immunomodulatory effects would be lost upon deglycosylation or any of the other approaches discussed above to obtain aglycosylated antibodies. Furthermore, modifying any sequence of an established antibody can also pose regulatory issues, which is problematic because in many cases, approved and clinically validated antibodies are used as the starting point for ADC conjugation.

[0335] Thus, the method according to the invention allows for the preparation of stoichiometrically well-defined ADCs with site-specific payload attachment easily and without disadvantages.

[0336] In view of the above, the method of the present invention is H Preferably, the antibody is used for conjugation of an IgG antibody at residue Q295 (EU numbering) of the C2 domain, where the antibody is HIt can be stated that the antibody is glycosylated at residue N297 (EU numbering) of domain 2. However, it can be clearly stated that the methods of the present invention also encompass the conjugation of antibodies that are deglycosylated or aglycosylated at residue Q295 or any other suitable Gln residue of the antibody, where the Gln residue may be an endogenous Gln residue or a Gln residue introduced by molecular engineering.

[0337] Thus, in a particular embodiment, the present invention relates to a method according to the invention, wherein a Gln residue to which a linker is conjugated has been introduced into the heavy or light chain of the antibody by molecular engineering.

[0338] The term "molecular engineering" as used herein refers to the use of molecular biology methods to manipulate nucleic acid sequences. Within the scope of the present invention, molecular engineering can be used to introduce Gln residues into the heavy or light chains of antibodies. Generally, two different strategies for introducing Gln residues into the heavy or light chains of antibodies are envisioned within the scope of 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 in the heavy or light chain, or the peptide tag can be fused (attached) to the N- or C-terminus of the heavy or light chain of an antibody.

[0339] For example, amino acid residues in the heavy or light chain of the antibody may be substituted 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 the C of an IgG antibody. HThe antibody has a substitution at amino acid residue N297 (EU numbering) in the 2 domain, particularly the substitution is an N297Q substitution.An antibody comprising an N297Q mutation may be conjugated to two or more linkers per heavy chain of the antibody.For example, an antibody comprising an N297Q mutation may be conjugated to four linkers, one linker conjugated to residue Q295 of the first heavy chain of the antibody, one linker conjugated to residue N297Q of the first heavy chain of the antibody, one linker conjugated to residue Q295 of the second heavy chain of the antibody, and one linker conjugated to residue N297Q of the second heavy chain of the antibody.Those skilled in the art are aware that residue N297 of an IgG antibody can be replaced with a Gln residue to obtain an aglycosylated antibody.

[0340] Thus, in a particular embodiment, the present invention provides a method according to the present invention, wherein the Gln residue introduced into the heavy or light chain of the antibody by molecular engineering is in the C of an aglycosylated IgG antibody. H 2 domain N297Q (EU numbering).

[0341] In a particular embodiment, the present invention relates to a method according to the present invention, wherein the Gln residue introduced into the antibody heavy or light chain by molecular engineering is (a) incorporated into the antibody heavy or light chain, or (b) comprised in a peptide fused to the N- or C-terminus of the antibody heavy or light chain.

[0342] Instead of substituting a single amino acid residue in 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- or C-terminus of the heavy or light chain of the antibody. Alternatively, the peptide tag may be inserted into the heavy or light chain of the antibody at a suitable position. Preferably, the peptide tag containing a transglutaminase-accessible Gln residue is fused to the C-terminus of the heavy chain of the antibody. Even more preferably, the 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 serve as a substrate for microbial transglutaminase are described in WO2012 / 059882 and WO2016 / 144608.

[0343] Thus, in a particular embodiment, the invention relates to a method according to the invention, wherein a peptide comprising a Gln residue is fused to the C-terminus of the heavy chain of the antibody.

[0344] Exemplary peptide tags that can be introduced into the heavy or light chain of an antibody, particularly fused to the C-terminus of the heavy chain of an antibody, are LLQGG (SEQ ID NO: 16), LLQG (SEQ ID NO: 17), LSLSQG (SEQ ID NO: 18), GGGLLQGG (SEQ ID NO: 19), GLLQG (SEQ ID NO: 20), LLQ (SEQ ID NO: 21), GSPLAQSHGG (SEQ ID NO: 22), GLLQGGG (SEQ ID NO: 23), GLLQGG (SEQ ID NO: 24), GLLQ (SEQ ID NO: 25), LLQLLQGA (SEQ ID NO: 26), LLQGA (SEQ ID NO: 27), LLQYQGA (SEQ ID NO: 28), LLQGSG (SEQ ID NO: 29), LLQYQG (SEQ ID NO: 30), LLQLLQG (SEQ ID NO: 31). Sequence number 31), SLLQG (SEQ ID NO: 32), LLQLQ (SEQ ID NO: 33), LLQLLQ (SEQ ID NO: 34), LLQGR (SEQ ID NO: 35), EEQYASTY (SEQ ID NO: 36), EEQYQSTY (SEQ ID NO: 37), EEQYNSTY (SEQ ID NO: 38), EEQYQS (SEQ ID NO: 39), EEQYQST (SEQ ID NO: 40), EQYQSTY (SEQ ID NO: 41), QYQS (SEQ ID NO: 42), QYQSTY (SEQ ID NO: 43), YRYRQ (SEQ ID NO: 44), DYALQ (SEQ ID NO: 45), FGLQRPY (SEQ ID NO: 46), EQKLISEEDL (SEQ ID NO: 47), LQR (SEQ ID NO: 48) and YQR (SEQ ID NO: 49).

[0345] Those skilled in the art know how to substitute amino acid residues in an antibody or how to introduce peptide tags into an antibody by molecular cloning methods described, for example, in Sambrook, Joseph. (2001). Molecular cloning: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

[0346] Generally, those skilled in the art know how to determine the position of the antibody to which the linker is conjugated. For example, the conjugation site can be determined by proteolytic digestion of the antibody-payload conjugate and LC-MS analysis of the resulting fragments. For example, the sample can be deglycosylated with GlyciNATOR (Genovis) according to the instruction manual, and then digested with Trypsin Gold (mass spectrometry grade, Promega). Thus, 1 μg of protein can be incubated with 50 ng of trypsin overnight at 37 ° C. LC-MS analysis can be performed using a nanoAcquity HPLC system connected to a Synapt-G2 mass spectrometer (Waters). To do so, 100 ng of peptide solution is loaded onto an Acquity UPLC Symmetry C18 capture column (Waters, part number 186006527) and captured for 3 min at a flow rate of 5 µL / min with 1% Buffer A (Water, 0.1% formic acid) and 99% Buffer B (acetonitrile, 0.1% formic acid). Peptides are then eluted with a linear gradient of 3% to 65% Buffer B over 25 min. Data are acquired in positive polarity resolution mode and a mass range of 50 to 2000 m / z. Other instrument settings are as follows: capillary voltage 3.2 kV, sampling cone 40 V, extraction cone 4.0 V, source temperature 130 °C, cone gas 35 L / h, nanoflow gas 0.1 bar, and purge gas 150 L / h. The mass spectrometer can be calibrated with [Glu1]-fibrinopeptide.

[0347] Furthermore, those skilled in the art know how to determine the drug-to-antibody (DAR) ratio or payload-to-antibody ratio of an antibody-payload construct. For example, the DAR can be determined by hydrophobic interaction chromatography (HIC) or LC-MS.

[0348] For hydrophobic interaction chromatography (HIC), samples can be adjusted to 0.5 M ammonium sulfate and evaluated on 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) at 1 mL / min and 30°C over 20 minutes. Typically, 40 μg of sample can be used, and the signal can be recorded at 280 nm. Relative HIC retention times (HIC-RRT) can be calculated by dividing the absolute retention times of the two ADC DARs by the retention times of the respective unconjugated mAbs.

[0349] To determine the LC-MS DAR, the ADC can be diluted with NH4HCO3 to a final concentration of 0.025 mg / mL. Next, 40 μL of this solution can 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 overnight at 37 °C in the dark. For reverse-phase chromatography, a Dionex U3000 system can be used in combination with Chromeleon software. This system can be equipped with a RP-1000 column (1000 Å, 5 μm, 1.0 × 100 mm, Sepax) heated to 70 °C and a UV detector set at a wavelength of 214 nm. Solvent A can consist of water with 0.1% formic acid, and solvent B can contain 85% acetonitrile with 0.1% formic acid. The reduced and alkylated sample was loaded onto the column and separated over 14 minutes using a 30-55% gradient of solvent B. The liquid chromatography system was coupled to a Synapt-G2 mass spectrometer for identification of DAR species. The mass spectrometer capillary voltage was set to 3 kV, the sampling cone to 30 V, and a maximum voltage of 5 V was applied to the extraction cone. The source temperature was set to 150 °C, the desolvation temperature to 500 °C, the cone gas to 20 L / h, and the desolvation gas to 600 L / h. Acquisition was performed in positive mode over a mass range of 600-5000 Da with a 1-second scan time. The instrument was calibrated with sodium iodide. Spectral deconvolution was performed using the MassLynx MaxEnt1 algorithm until convergence. After assigning DAR species to chromatographic peaks, calculations were performed based on the integrals of the peak areas in the reversed-phase chromatogram.

[0350] In a particular embodiment, the present invention relates to a method according to the present invention, wherein the linker is conjugated to the γ-carboxamide group of a Gln residue comprised in the antibody.

[0351] That is, the linker according to the present invention is preferably conjugated to the amide group of the side chain of a Gln residue contained in the antibody, preferably any one of the Gln residues disclosed herein, more preferably Gln residue Q295 (EU numbering).

[0352] In certain embodiments, the invention relates to a method according to the invention, wherein the linker is suitable for conjugation to a glycosylated antibody with a conjugation efficiency of at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95%.

[0353] That is, in certain embodiments, the linker can be a linker that can be conjugated to a glycosylated antibody with at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% efficiency. In a preferred embodiment, the linker can be a linker that can be conjugated to a glycosylated antibody with at least 70% efficiency. In another preferred embodiment, the linker can be a linker that can be conjugated to a glycosylated antibody with at least 75% efficiency. In another preferred embodiment, the linker can be a linker that can be conjugated to a glycosylated antibody with at least 80% efficiency. In another preferred embodiment, the linker can be a linker that can be conjugated to a glycosylated antibody with at least 85% efficiency. In another preferred embodiment, the linker can be a linker that can be conjugated to a glycosylated antibody with at least 90% efficiency. In another preferred embodiment, the linker can be a linker that 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).

[0354] Those skilled in the art are aware of methods for determining the conjugation efficiency of an antibody with a particular linker. For example, conjugation efficiency can be determined as described herein. That is, an antibody, particularly an IgG1 antibody, can be incubated in a suitable buffer with 5-20 eq molar equivalents of linker per mg of antibody and 3-6 U of microbial transglutaminase at a concentration of 1-5 mg / mL at 37°C for 20-48 hours, or as described in Example 1. After the incubation period, conjugation efficiency can be determined by LC-MS analysis under reducing conditions. The microbial transglutaminase may be 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 vary and may depend greatly on the chemical properties of the linker. However, those skilled in the art will be able to identify optimal buffer conditions based on the present disclosure. Alternatively, conjugation efficiency can be determined as described in Spycher et al. (Dual, Site-Specific Modification of Antibodies by Using Solid-Phase Immobilized Microbial Transglutaminase, ChemBioChem 2019 18(19):1923-1927) and analyzed as in Benjamin et al. (Thiolation of Q295: Site-Specific Conjugation of Hydrophobic Payloads without the Need for Genetic Engineering, Mol. Pharmaceutics 2019, 16: 2795-2807).

[0355] In certain embodiments, antibodies may be conjugated as described in Example 1. That is, 5 mg / ml of native glycosylated monoclonal antibody may be incubated in a rotary thermomixer at 37° C. for 24 hours in 50 mM Tris pH 7.6 containing microbial transglutaminase (MTG, Zedira) at a concentration of 5 U per mg of antibody and 5 molar equivalents of the specified linker-payload.

[0356] 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 Streptomyces mobaraensis.

[0357] That is, the microbial transglutaminase used in the method of the present invention is derived from a Streptomyces species, in particular Streptomyces mobaraensis, and may preferentially have 80% sequence identity with the naturally occurring enzyme. Thus, MTG may be the naturally occurring enzyme or an engineered variant of the naturally occurring enzyme.

[0358] 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: 12. MTG variants of S. mobaraensis with other amino acid sequences have been reported and are also encompassed by the present invention (SEQ ID NOs: 13 and 14).

[0359] In another embodiment, a microbial transglutaminase from Streptomyces ladakanum (formerly known as Streptoverticillium ladakanum) may be used. Streptomyces ladakanum transglutaminase (U.S. Pat. No. 6,660,510 B2) has the amino acid sequence disclosed in SEQ ID NO: 15.

[0360] Both of the above transglutaminases may be sequence modified. In some embodiments, a transglutaminase having 80%, 85%, 90%, or 95% or more sequence identity with any one of SEQ ID NOs: 12-15 may be used.

[0361] Another suitable microbial transglutaminase is commercially available from Ajinomoto Co., Inc. and is called ACTIVA TG. Compared to the transglutaminase from Zedira, ACTIVA TG lacks four N-terminal amino acids but has similar activity.

[0362] Further microbial transglutaminases that may be used in the context of the present invention are disclosed in Kieliszek and Misiewicz (Folia Microbiol (Praha). 2014; 59(3): 241-250), WO2015 / 191883 A1, WO2008 / 102007 A1 and US2010 / 0143970, the contents of which are incorporated herein by reference in their entirety.

[0363] In certain embodiments, mutant variants of microbial transglutaminase may be used for conjugating linkers to antibodies. That is, the microbial transglutaminase used in the methods of the present invention may be a variant of the S. mobaraensis transglutaminase set forth in SEQ ID NO: 12 or 13. In certain embodiments, the recombinant S. mobaraensis transglutaminase set forth in SEQ ID NO: 12 may comprise the mutation G254D. In certain embodiments, the recombinant S. mobaraensis transglutaminase set forth in SEQ ID NO: 12 may comprise the mutations G254D and E304D. In certain embodiments, the recombinant S. mobaraensis transglutaminase set forth in SEQ ID NO: 12 may comprise the mutations D8E and G254D. In certain embodiments, the recombinant S. mobaraensis transglutaminase set forth in SEQ ID NO: 12 may comprise the mutations E124A and G254D. In certain embodiments, the recombinant S. morabaensis transglutaminase set forth in SEQ ID NO: 12 may include the mutations A216D and G254D. In certain embodiments, the recombinant S. morabaensis transglutaminase set forth in SEQ ID NO: 12 may include the mutations G254D and K331T.

[0364] Microbial transglutaminase may be added to the conjugation reaction at any concentration that allows efficient conjugation of the antibody with the linker. In certain embodiments, the concentration of microbial transglutaminase in the conjugation reaction may depend on the amount of antibody used in the same reaction. For example, microbial transglutaminase may be added to the conjugation reaction at a concentration of 100 U per mg of antibody, 90 U per mg of antibody, 80 U per mg of antibody, 70 U per mg of antibody, 60 U per mg of antibody, 50 U per mg of antibody, 40 U per mg of antibody, 30 U per mg of antibody, 20 U per mg of antibody, 10 U per mg of antibody, or less than 6 U per mg of antibody. In certain embodiments, microbial transglutaminase may be added to the conjugation reaction at a concentration of 1, 3, 5, or 6 U per mg of antibody.

[0365] That is, in certain embodiments, microbial transglutaminase may be added to the conjugation reaction at a concentration ranging from 1 to 20 U per mg of antibody, preferably 1 to 10 U per mg of antibody, more preferably 1 to 7.5 U per mg of antibody, even more preferably 2 to 6 U per mg of antibody, even more preferably 2 to 4 U per mg of antibody, and most preferably 3 U per mg of antibody.

[0366] The method according to the present invention involves the use of a microbial transglutaminase. However, it should be noted that an equivalent reaction may be carried out by an enzyme containing transglutaminase activity that is not of microbial origin. Thus, the antibody-linker conjugate according to the present invention may also be produced by an enzyme containing transglutaminase activity that is not of microbial origin.

[0367] The antibody may be added to the conjugation reaction at any concentration. However, it is preferred that the antibody be added to the conjugation reaction at a concentration in the range of 0.1 to 20 mg / mL. That is, in a specific embodiment, the present invention relates to a method according to the present invention, wherein the antibody is added to the conjugation reaction at a concentration of 0.1 to 20 mg / mL, preferably 0.25 to 15 mg / 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.

[0368] Alternatively, the antibody may be added to the conjugation reaction at a concentration ranging from 1 to 20 mg / ml, preferably from 2.5 to 20 mg / mL, more preferably from 5 to 20 mg / mL, and most preferably from 5 to 17 mg / mL.

[0369] To obtain efficient conjugation, the linker is preferably added in molar excess to the antibody, 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 the linker.

[0370] Thus, in a particular embodiment, the invention relates to a method according to the invention, wherein the antibody is contacted with 2 to 100 molar equivalents of the linker, preferably 2 to 80 molar equivalents of the linker, more preferably 2 to 70 molar equivalents of the linker, even more preferably 2 to 60 molar equivalents of the linker, even more preferably 2 to 50 molar equivalents of the linker, even more preferably 2 to 40 molar equivalents of the linker, even more preferably 2 to 30 molar equivalents of the linker, even more preferably 2 to 25 molar equivalents of the linker, even more preferably 2 to 20 molar equivalents of the linker, even more preferably 2 to 15 molar equivalents of the linker, and most preferably 2 to 10 molar equivalents of the linker.

[0371] Alternatively, the antibody can 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.

[0372] 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.

[0373] The method according to the invention is preferably carried out at a pH in the range of 6 to 9. Thus, in a preferred embodiment, the invention relates to a method according to the invention, wherein conjugation of the linker to the antibody is achieved at a pH in the range of 6 to 8.5, more preferably at a pH in the range of 6.5 to 8, even more preferably at a pH in the range of 7 to 8. In a most preferred embodiment, the invention relates to a method according to the invention, wherein conjugation of the linker to the antibody is achieved at pH 7.6.

[0374] The methods of the present invention can be carried out in any buffer suitable for conjugating a payload to a linker. Suitable buffers for the methods of the present invention include, but are not limited to, Tris, MOPS, HEPES, PBS, or BisTris buffers. The buffer concentration 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 can contain any salt concentration suitable for carrying out the methods of the present invention. For example, the buffer used in the methods of the invention may have a salt concentration of 150 mM or less, 140 mM or less, 130 mM or less, 120 mM or less, 110 mM or less, 100 mM or less, 90 mM or less, 80 mM or less, 70 mM or less, 60 mM or less, 50 mM or less, 40 mM or less, 30 mM or less, 20 mM or less, or 10 mM or less, or may have no salt. In certain embodiments, the methods of the invention are carried out in 50 mM Tris (pH 7.6), preferably without salt.

[0375] It should be noted that optimal reaction conditions (e.g., pH, buffer, salt concentration) may vary between payloads and, to some extent, depend on the physicochemical properties of the linker and / or payload. However, one of ordinary skill in the art would not require undue experimentation to identify reaction conditions that are suitable for carrying out the methods of the present invention.

[0376] It should be understood that the present application encompasses any combination of linker, antibody MTG and / or buffer concentrations disclosed above.

[0377] In a preferred embodiment, the present invention provides a method for producing antibody-linker conjugates by microbial transglutaminase (MTG), comprising the structure (shown in the N→C orientation): (Sp1)-RK-(Sp2)-B-(Sp3) or (Sp1)-B-(Sp2)-RK-(Sp3) to a Gln residue contained in the antibody, wherein - (Sp1) is a chemical spacer or is absent; - (Sp2) is a chemical spacer or is absent; - (Sp3) is a chemical spacer or is absent; R is arginine or an arginine derivative or arginine mimetic, K is lysine or a lysine derivative or a lysine mimetic; B is a linking moiety or payload, the linker is conjugated to a Gln residue in the antibody through a primary amine in the side chain of a lysine residue, lysine derivative, or lysine mimetic; and the antibody is contacted with 2 to 80 molar equivalents of the linker; and / or The method relates to a method in which the microbial transglutaminase is added to the conjugation reaction at a concentration ranging from 1 to 20 U / mg of antibody, and optionally the antibody is added to the conjugation reaction at a concentration ranging from 0.1 to 20 mg / mL.

[0378] In a more preferred embodiment, the present invention provides a method for producing antibody-linker conjugates by microbial transglutaminase (MTG), comprising the structure (shown in the N→C orientation): (Sp1)-RK-(Sp2)-B-(Sp3) or (Sp1)-B-(Sp2)-RK-(Sp3) to a Gln residue contained in the antibody, wherein - (Sp1) is a chemical spacer or is absent; - (Sp2) is a chemical spacer or is absent; - (Sp3) is a chemical spacer or is absent; R is arginine or an arginine derivative or arginine mimetic, K is lysine or a lysine derivative or a lysine mimetic; B is a linking moiety or payload, the linker is conjugated to a Gln residue in the antibody through a primary amine in the side chain of a lysine residue, lysine derivative, or lysine mimetic; and the antibody is contacted with 2 to 50 molar equivalents of the linker; and / or The method relates to a method in which the microbial transglutaminase is added to the conjugation reaction at a concentration ranging from 1 to 10 U / mg of antibody, and optionally the antibody is added to the conjugation reaction at a concentration ranging from 1 to 20 mg / mL.

[0379] In an even more preferred embodiment, the present invention provides a method for producing antibody-linker conjugates by microbial transglutaminase (MTG), comprising the steps of: (Sp1)-RK-(Sp2)-B-(Sp3) or (Sp1)-B-(Sp2)-RK-(Sp3) to a Gln residue contained in the antibody, wherein - (Sp1) is a chemical spacer or is absent; - (Sp2) is a chemical spacer or is absent; - (Sp3) is a chemical spacer or is absent; R is arginine or an arginine derivative or arginine mimetic, K is lysine or a lysine derivative or a lysine mimetic; B is a linking moiety or payload, the linker is conjugated to a Gln residue in the antibody through a primary amine in the side chain of a lysine residue, lysine derivative, or lysine mimetic; and the antibody is contacted with 2 to 30 molar equivalents of the linker; and / or The method relates to a method in which microbial transglutaminase is added to the conjugation reaction at a concentration ranging from 2 to 10 U / mg of antibody, and optionally, the antibody is added to the conjugation reaction at a concentration ranging from 5 to 20 mg / mL.

[0380] In an even more preferred embodiment, the present invention provides a method for producing antibody-linker conjugates by microbial transglutaminase (MTG), comprising the steps of: (Sp1)-RK-(Sp2)-B-(Sp3) or (Sp1)-B-(Sp2)-RK-(Sp3) to a Gln residue contained in the antibody, wherein - (Sp1) is a chemical spacer or is absent; - (Sp2) is a chemical spacer or is absent; - (Sp3) is a chemical spacer or is absent; R is arginine or an arginine derivative or arginine mimetic, K is lysine or a lysine derivative or a lysine mimetic; B is a linking moiety or payload, the linker is conjugated to a Gln residue in the antibody through a primary amine in the side chain of a lysine residue, lysine derivative, or lysine mimetic; and and / or microbial transglutaminase is added to the conjugation reaction at a concentration ranging from 2 to 10 U / mg of antibody, and optionally, the antibody is added to the conjugation reaction at a concentration ranging from 5 to 20 mg / mL.

[0381] In an even more preferred embodiment, the present invention provides a method for producing antibody-linker conjugates by microbial transglutaminase (MTG), comprising the steps of: (Sp1)-RK-(Sp2)-B-(Sp3) or (Sp1)-B-(Sp2)-RK-(Sp3) to a Gln residue contained in the antibody, wherein - (Sp1) is a chemical spacer or is absent; - (Sp2) is a chemical spacer or is absent; - (Sp3) is a chemical spacer or is absent; R is arginine or an arginine derivative or arginine mimetic, K is lysine or a lysine derivative or a lysine mimetic; B is a linking moiety or payload, the linker is conjugated to a Gln residue in the antibody through a primary amine in the side chain of a lysine residue, lysine derivative, or lysine mimetic; and the antibody is contacted with about 2.5-15 molar equivalents of linker; and / or the microbial transglutaminase is added to the conjugation reaction at a concentration ranging from 2-10 U / mg of antibody, and optionally the antibody is added to the conjugation reaction at a concentration ranging from 5-20 mg / mL.

[0382] In a most preferred embodiment, the present invention provides a method for producing antibody-linker conjugates by microbial transglutaminase (MTG), comprising the structure (shown in the N→C orientation): (Sp1)-RK-(Sp2)-B-(Sp3) or (Sp1)-B-(Sp2)-RK-(Sp3) to a Gln residue contained in the antibody, wherein - (Sp1) is a chemical spacer or is absent; - (Sp2) is a chemical spacer or is absent; - (Sp3) is a chemical spacer or is absent; R is arginine or an arginine derivative or arginine mimetic, K is lysine or a lysine derivative or a lysine mimetic; B is a linking moiety or payload, the linker is conjugated to a Gln residue in the antibody through a primary amine in the side chain of a lysine residue, lysine derivative, or lysine mimetic; and the antibody is contacted with about 2.5-10 molar equivalents of linker; and / or the microbial transglutaminase is added to the conjugation reaction at a concentration ranging from 2-10 U / mg of antibody, and optionally the antibody is added to the conjugation reaction at a concentration ranging from 5-20 mg / mL.

[0383] In certain embodiments, the present invention relates to antibody-linker conjugates produced using the methods according to the present invention.

[0384] That is, the present invention relates to antibody-linker conjugates produced using any of the aforementioned steps.

[0385] In certain embodiments, the present invention provides a method for producing a pharmaceutical composition comprising: a) an antibody; and b) Structure: (Sp1)-RK-(Sp2)-B-(Sp3) or (Sp1)-B-(Sp2)-RK-(Sp3); (In the formula, - (Sp1) is a chemical spacer or is absent; - (Sp2) is a chemical spacer or is absent; - (Sp3) is a chemical spacer or is absent; R is arginine or an arginine derivative or arginine mimetic, K is lysine or a lysine derivative or a lysine mimetic; - B is the linking portion or payload) an antibody-linker conjugate comprising a linker comprising The present invention relates to an antibody-linker conjugate in which the linker is conjugated to the antibody by an isopeptide bond formed between the γ-carboxamide group of a glutamine residue contained in the antibody and a primary amine contained in the side chain of a lysine residue, lysine derivative, or lysine mimetic contained in an RK motif contained in the linker.

[0386] That is, the present invention further relates to antibody-linker conjugates produced using the methods of the present invention. In particular, the present invention refers to antibodies conjugated at a glutamine residue contained in the heavy or light chain of the antibody with any one of the linkers disclosed herein in connection with the methods of the present invention. That is, all linkers disclosed above in connection with the methods of the present invention can be included in the antibody-linker constructs of the present invention. Preferably, the linkers of the present invention are conjugated to a glutamine residue of the antibody through an amide bond formed between the amide side chain of the glutamine residue contained in the antibody and the primary amine contained in residue K in the RK motif of the linker. In certain embodiments, the primary amine contained in residue K is an amine group contained in the side chain of a lysine residue, lysine mimetic, or lysine derivative disclosed herein. In certain embodiments, K is a lysine residue, and the primary amine (by which the linker is conjugated to the antibody) is the ε-amino group contained in the lysine residue.

[0387] The chemical spacer included in the antibody-linker constructs disclosed herein can be any one of the RK-containing linkers disclosed herein, i.e., the linker can be a linker containing a single linking moiety or payload B, or a linker containing two or more linking moieties and / or payloads B1, B2, etc.

[0388] In certain embodiments, the present invention relates to an antibody-linker conjugate according to the present invention, wherein the chemical spacers (Sp1), (Sp2) and (Sp3) each independently comprise from 0 to 12 amino acid residues.

[0389] In certain embodiments, the invention relates to an antibody-linker conjugate 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.

[0390] In certain embodiments, the present invention relates to an antibody-linker conjugate according to the present invention, wherein the net charge of the linker is neutral or positive.

[0391] In a particular embodiment, the present invention relates to an antibody-linker conjugate according to the invention, wherein the linker does not comprise any negatively charged amino acid residues.

[0392] In certain embodiments, the present invention relates to an antibody-linker conjugate according to the present invention, wherein the linker comprises an amino acid sequence selected from the group consisting of RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3) and RKR (SEQ ID NO: 4).

[0393] In certain embodiments, the present invention relates to an antibody-linker conjugate according to the present invention, wherein the linker comprises an amino acid sequence selected from the group consisting of RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2) and ARK (SEQ ID NO: 3).

[0394] In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the linker comprises the amino acid sequence RKAA (SEQ ID NO: 1).

[0395] In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the linker comprises the amino acid sequence RK-Val-Cit (SEQ ID NO: 54).

[0396] In certain embodiments, the invention relates to an antibody-linker conjugate according to the invention, wherein B is a linking moiety.

[0397] In a particular embodiment, the present invention relates to an antibody-linker conjugate according to the present invention, wherein linking moiety B is - bioorthogonal marker groups, or - Non-bio-orthogonal entities for crosslinking The present invention relates to an antibody-linker conjugate comprising:

[0398] In certain embodiments, the present invention provides an antibody-linker conjugate according to the present invention, wherein the bioorthogonal marker group or the non-bioorthogonal entity for crosslinking is: - -NN≡N, or -N3; - Lys(N3); - tetrazine; - alkynes; - Distorted cyclooctyne; - BCN; - strained alkenes; - photoreactive groups; - aldehydes; - acyltrifluoroborates; - Protein degrading agents ("PROTACs"); - Cyclopentadiene / spirolocyclopentadiene; - Thioselective electrophiles; - -SH; and - Cysteine The present invention relates to an antibody-linker conjugate consisting of or comprising at least one molecule or moiety selected from the group consisting of:

[0399] That is, an antibody-linker conjugate according to the present invention may be an antibody conjugated to a linker comprising one or more linking moieties. Such an antibody-linker conjugate can then be customized with one or more payloads, particularly payloads that may be suitable for binding to one or more linking moieties.

[0400] In certain embodiments, the present invention relates to an antibody-linker conjugate according to the present invention, wherein one or more payloads are conjugated to linking moiety B.

[0401] In certain embodiments, the present invention relates to an antibody-linker conjugate according to the present invention, wherein one or more payloads are conjugated to linking moiety B by a Click reaction.

[0402] That is, the antibody-linker conjugate according to the present invention may be an antibody-payload conjugate produced in the two-step process disclosed herein.

[0403] In certain embodiments, the invention relates to an antibody-linker conjugate according to the invention, wherein B is a payload.

[0404] In certain embodiments, the present invention provides an antibody-linker conjugate according to the present invention, wherein the payload is - toxin; - cytokines; - growth factors; - Radionuclides; - Hormones; - antiviral agents; - antibacterial agents; - Fluorescent dyes: - immunoregulatory / immunostimulant agents; - Half-life increasing part; - solubility increasing part; - polymer-toxin conjugates; - Nucleic acids; - a biotin or streptavidin moiety; - Vitamins; - Protein degrading agents ("PROTACs"); - a target binding moiety; and / or - Anti-inflammatory The present invention relates to an antibody-linker conjugate comprising at least one of:

[0405] In certain embodiments, the present invention provides an antibody-linker conjugate according to the present invention, wherein the toxin is - Pyrrolobenzodiazepines (e.g., PBD); - Auristatins (e.g., MMAE, MMAF); - Maytansinoids (e.g., maytansine, DM1, DM4, DM21); - Duocarmycin; - Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors; - Tubulyshyn; - Enzymes (e.g., calicheamicin); - anthracycline derivatives (PNU) (e.g., doxorubicin); - Pyrrole-based kinesin spindle protein (KSP) inhibitors; - Cryptophycin; - Drug efflux pump inhibitors; - Sandramycin; amanitin (e.g., α-amanitin); and - Camptothecins (e.g., exatecan, deruxtecan) The present invention relates to an antibody-linker conjugate, which is at least one selected from the group consisting of:

[0406] In a particular embodiment, the present invention relates to an antibody-linker conjugate according to the present invention, wherein the chemical spacer (Sp2) comprises a self-immolative moiety.

[0407] In a particular embodiment, the present invention relates to an antibody-linker conjugate according to the present invention, wherein the self-immolative moiety is attached directly to payload B.

[0408] In certain embodiments, the invention relates to an antibody-linker conjugate according to the invention, wherein the self-immolative moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.

[0409] That is, the antibody-linker conjugate according to the present invention may be an antibody-payload conjugate produced in the one-step process disclosed herein.

[0410] The antibody comprised in the antibody-linker conjugate according to the invention may be any one of the antibodies disclosed herein in relation to the methods according to the invention, in particular any one of the IgG type antibodies, i.e., the antibody comprised in the antibody-linker conjugate according to the invention may comprise the same glycosylation pattern, mutations and / or modifications as the antibodies disclosed herein in relation to the methods according to the invention.

[0411] In a particular embodiment, the present invention relates to an antibody-payload conjugate according to the present invention, wherein the linker is any one of the linkers shown in Figure 1, Figure 2, Figure 3, Figure 8, Figure 9, Figure 14, Figure 15, Figure 17, Figure 18, Figure 19, Figure 20, Figure 21, Figure 22, Figure 23, Figure 24, Figure 25, Figure 26, Figure 27, Figure 28, Figure 29, Figure 30, Figure 31, Figure 32, Figure 33 or Figure 34.

[0412] In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the antibody is an IgG antibody, in particular an IgG1 antibody.

[0413] In a particular embodiment, the present invention relates to an antibody-linker conjugate according to the present invention, wherein the Gln residue to which the linker is conjugated is comprised in the Fc domain of the antibody, in particular the Gln residue to which the linker is conjugated is comprised in the C domain of an IgG antibody. H Regarding the antibody-linker conjugate, the Gln residue Q295 (EU numbering) of the 2 domain is

[0414] In a particular embodiment, the present invention relates to an antibody-linker conjugate according to the present invention, wherein the Gln residue to which the linker is conjugated has been introduced into the heavy or light chain of the antibody by molecular engineering.

[0415] In a particular embodiment, the present invention relates to an antibody-linker conjugate according to the present invention, wherein the Gln residue introduced into the heavy or light chain of the antibody by molecular engineering is C of an aglycosylated IgG antibody. H The antibody-linker conjugate is N297Q (EU numbering) of the two domains.

[0416] In a specific embodiment, the present invention relates to an antibody-linker conjugate according to the present invention, wherein the Gln residue introduced into the antibody heavy or light chain by molecular engineering is (a) incorporated into the antibody heavy or light chain, or (b) contained in a peptide fused to the N- or C-terminus of the antibody heavy or light chain.

[0417] In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein a peptide comprising a Gln residue is fused to the C-terminus of the heavy chain of the antibody.

[0418] In a particular embodiment, the present invention relates to an antibody-linker conjugate according to the present invention, wherein the IgG antibody is a glycosylated IgG antibody, in particular an IgG antibody. H The antibody-linker conjugate is glycosylated at residue N297 (EU numbering) of the 2 domain.

[0419] In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the antibody is selected from the group consisting of brentuximab, trastuzumab, gemtuzumab, inotuzumab, avelumab, cetuximab, rituximab, daratumumab, pertuzumab, vedolizumab, ocrelizumab, tocilizumab, ustekinumab, golimumab, obinutuzumab, sacituzumab, belantamab, polatuzumab, and enfortumab.

[0420] In certain embodiments, the invention relates to an antibody-linker conjugate according to the invention, wherein the antibody is selected from the group consisting of brentuximab, gemtuzumab, trastuzumab, inotuzumab, polatuzumab, enfortumab, sacituzumab, and belantamab.

[0421] In a particular embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the antibody is polatuzumab or trastuzumab or enfortumab.

[0422] In certain embodiments, the present invention relates to antibody-drug conjugates, i.e., the antibody may be conjugated to a linker according to the present invention, wherein the linker comprises one or more toxins.

[0423] Thus, in certain embodiments, the present invention provides a) IgG antibodies; and b) a linker comprising a drug moiety B, wherein drug moiety B is covalently linked to an amino acid sequence selected from the group consisting of RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), or RKR (SEQ ID NO: 4). 1. An antibody-drug conjugate comprising: The linker is located at the C HThe present invention relates to an antibody-drug conjugate conjugate that is conjugated to an IgG antibody via an isopeptide bond formed between the γ-carboxamide group of glutamine residue Q295 (EU numbering) in domain 2 and the primary amine contained in the side chain of a lysine residue contained in the linker.

[0424] In certain embodiments, the present invention provides a) IgG antibodies; and b) a linker comprising a drug moiety B, wherein drug moiety B is covalently linked to an amino acid sequence comprising or consisting of the sequence RK-Val-Cit (SEQ ID NO: 54). 1. An antibody-drug conjugate comprising: The linker is located at the C H The present invention relates to an antibody-drug conjugate conjugate that is conjugated to an IgG antibody via an isopeptide bond formed between the γ-carboxamide group of glutamine residue Q295 (EU numbering) in domain 2 and the primary amine contained in the side chain of a lysine residue contained in the linker.

[0425] That is, in certain embodiments, the linker may comprise any one of the sequences RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4), or RK-Val-Cit (SEQ ID NO: 54), and the linker is conjugated to a glutamine residue of the antibody through a primary amine contained in residue K. It should be understood that drug moiety B need not be directly linked to the structure RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4), or RK-Val-Cit (SEQ ID NO: 54). Instead, drug moiety B may be indirectly linked to the structure RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4), or RK-Val-Cit (SEQ ID NO: 54). For example, the linker may further comprise a chemical structure positioned between drug moiety B and the structure RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4), or RK-Val-Cit (SEQ ID NO: 54). Such chemical structures may be any of the structures disclosed herein for chemical spacers (Sp1), (Sp2), or (Sp3). In certain embodiments, the linker may comprise one or more amino acid residues positioned between drug moiety B and the structure RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4), or RK-Val-Cit (SEQ ID NO: 54). In certain embodiments, the linker may comprise one or more PEG moieties positioned between drug moiety B and the structure RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4), or RK-Val-Cit (SEQ ID NO: 54). In certain embodiments, the linker may comprise a cleavable and / or self-immolative moiety positioned between the drug moiety B and the structure RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4) or RK-Val-Cit (SEQ ID NO: 54).

[0426] That is, in certain embodiments, the present invention relates to an antibody-drug conjugate according to the present invention, wherein drug moiety B is linked to the N- or C-terminus of an amino acid sequence comprised in the linker by a self-immolative moiety.

[0427] In certain embodiments, the invention relates to an antibody-drug conjugate according to the invention, wherein the self-immolative moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.

[0428] That is, the self-immolative moiety included in a linker according to the invention can be any one of the self-immolative moieties disclosed herein, hi certain embodiments, the self-immolative moiety can be a PABC or methylamine group disclosed herein.

[0429] In a particular embodiment, the present invention relates to an antibody-drug conjugate according to the present invention, wherein the IgG antibody is a glycosylated IgG antibody, particularly an IgG antibody. H The antibody-drug conjugate is glycosylated at residue N297 (EU numbering) of the 2 domain.

[0430] In a particular embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the IgG antibody is an IgG1 antibody.

[0431] That is, the antibody is preferably an IgG antibody, in particular an IgG1 antibody, and in particular the IgG or IgG1 antibody is glycosylated at residue N297 (EU numbering).

[0432] The antibody-drug conjugate may comprise one or more of the toxins disclosed herein. Thus, in certain embodiments, the invention provides an antibody-drug conjugate according to the invention, wherein the drug is: - Pyrrolobenzodiazepines (e.g., PBD); - Auristatins (e.g., MMAE, MMAF); - Maytansinoids (e.g., maytansine, DM1, DM4, DM21); - Duocarmycin; - Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors; - Tubulyshyn; - Enzymes (e.g. calicheamicin); - anthracycline derivatives (PNU) (e.g., doxorubicin); - Pyrrole-based kinesin spindle protein (KSP) inhibitors; - Cryptophycin; - Drug efflux pump inhibitors; - Sandramycin; amanitin (e.g., α-amanitin); and - Camptothecins (e.g., exatecan, deruxtecan) The present invention relates to an antibody-drug conjugate, wherein the toxin is selected from the group consisting of:

[0433] It should be understood that the toxin may be attached directly to the linker by chemical synthesis, however, in other embodiments, the toxin may be attached to a linking moiety included in the linker in a two-step process.

[0434] In certain embodiments, the present invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure RKAA-B or RKAA-(linker molecule)-B.

[0435] That is, payload B may be directly attached to the C-terminus of the alanine residue, or may be attached to the C-terminus of the alanine residue via a linker molecule. It should be understood that the choice of linker molecule largely depends on the functional group available on payload B. Linker molecules suitable for attaching payloads having different functional groups to peptides are disclosed herein. The linker molecule may be a cleavable or non-cleavable linker molecule. In particular, the linker molecule may comprise a self-immolative moiety, particularly any one of the self-immolative moieties disclosed herein. Thus, in certain embodiments, the present invention relates to an antibody-drug conjugate according to the present invention, wherein the linker comprises or consists of the structure RKAA-(self-immolative moiety)-B.

[0436] Alternatively, the payload may be attached to the N-terminus of the arginine residue directly or via a linker molecule, for example, via any one of the linker molecules disclosed herein. That is, in certain embodiments, the present invention relates to an antibody-drug conjugate according to the present invention, wherein the linker comprises or consists of the structure B-RKAA or B-(linker molecule)-RKAA. In certain embodiments, the present invention relates to an antibody-drug conjugate according to the present invention, wherein the linker comprises or consists of the structure B-(self-immolative moiety)-RKAA. In certain embodiments, the self-immolative moiety comprised in the structure B-(self-immolative moiety)-RKAA may be a self-immolative moiety comprising an ortho-hydroxy-protected aryl sulfate (OHPAS) moiety disclosed herein.

[0437] In a particular embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker has the structure RKAA-PABC-B (particularly, wherein B is an auristatin or a maytansinoid, particularly wherein the auristatin is MMAE and the maytansinoid is DM1 or maytansine).

[0438] In certain embodiments, the linker may have the structure RKAA-PABC-B. That is, the linker may comprise the linear peptide RKAA, in which the carboxy group of the C-terminal alanine residue is linked to the amino group contained in PABC by an amide bond. Toxin B may be attached to PABC via carbamate formation. It should be understood that not all toxins contain functional groups that allow for carbamate formation with PABC. Thus, the toxin may be connected to PABC by a linker.

[0439] In certain embodiments, the toxin may be a toxin containing a primary or secondary amine, hi certain embodiments, the toxin may be MMAE or maytansine.

[0440] In certain embodiments, the linker may have a protected N-terminus. In certain embodiments, the N-terminus may be acetylated. In certain embodiments, the linker is a linker shown in Figure 1 or Figure 8.

[0441] In certain embodiments, the linker may have the structure RKAA-PABC-MMAE. In certain embodiments, the linker may have the structure RKAA-(PEG) n -PABC-MMAE, where n is an integer between 2 and 20. In certain embodiments, the linker may have the structure RKAA-(PEG)2-PABC-MMAE. In certain embodiments, the linker may have the structure RKAA-MMAE. In certain embodiments, the linker may have the structure RKAA-Val-Cit-PABC-MMAE. In certain embodiments, the linker may include an additional linker between the PABC moiety and the MMAE. In certain embodiments, the additional linker may be a p-nitrophenol (PNP) group.

[0442] It should be noted that the linker may include a self-immolative moiety other than PABC. That is, the linker may have the structure RKAA-(self-immolative moiety)-toxin. Those skilled in the art will know of other self-immolative moieties that can be used within the scope of the present invention. Furthermore, those skilled in the art will know of toxins that can be attached to a self-immolative moiety, if necessary, by an additional linker.

[0443] In certain embodiments, the toxin may be a toxin containing a hydroxy group, and the linker may include a self-immolative methylamine group. That is, the linker may have the structure RKAA-(NH)-(CH)-O-toxin. In certain embodiments, the hydroxy-containing toxin may be a camptothecin, e.g., an anthracycline such as exatecan or an exatecan derivative, particularly the exatecan derivative Dxd, or PNU-159682.

[0444] In certain embodiments, the toxin may be a toxin containing a thiol group, and the linker may include a self-immolative methylamine group. That is, the linker may have the structure RKAA-(NH)-(CH)-S-toxin. In certain embodiments, the thiol-containing toxin may be a maytansinoid, such as DM1, or a thiol-containing derivative thereof.

[0445] In certain embodiments, the present invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure RKA-B or RKA-(linker molecule)-B.

[0446] That is, payload B may be directly attached to the C-terminus of the alanine residue, or may be attached to the C-terminus of the alanine residue via a linker molecule. It should be understood that the choice of linker molecule largely depends on the functional group available on payload B. Linker molecules suitable for attaching payloads having different functional groups to peptides are disclosed herein. The linker molecule may be a cleavable or non-cleavable linker molecule. In particular, the linker molecule may comprise a self-immolative moiety, particularly any one of the self-immolative moieties disclosed herein. Thus, in certain embodiments, the present invention relates to an antibody-drug conjugate according to the present invention, wherein the linker comprises or consists of the structure RKA-(self-immolative moiety)-B.

[0447] Alternatively, the payload may be attached to the N-terminus of the arginine residue directly or via a linker molecule, for example, via any one of the linker molecules disclosed herein. That is, in certain embodiments, the present invention relates to an antibody-drug conjugate according to the present invention, wherein the linker comprises or consists of the structure B-RKA or B-(linker molecule)-RKA. In certain embodiments, the present invention relates to an antibody-drug conjugate according to the present invention, wherein the linker comprises or consists of the structure B-(self-immolative moiety)-RKA. In certain embodiments, the self-immolative moiety comprised in the structure B-(self-immolative moiety)-RKA may be a self-immolative moiety comprising an ortho-hydroxy-protected aryl sulfate (OHPAS) moiety disclosed herein.

[0448] In certain embodiments, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker has the structure RKA-PABC-B (particularly, wherein B is an auristatin or a maytansinoid, particularly wherein the auristatin is MMAE and the maytansinoid is DM1 or maytansine).

[0449] In certain embodiments, the linker may have the structure RKA-PABC-B. That is, the linker may include a linear peptide RKA, in which the carboxy group of the C-terminal alanine residue is linked to an amino group contained in PABC by an amide bond. Toxin B may be attached to PABC via carbamate formation. It should be understood that not all toxins contain functional groups that allow for carbamate formation with PABC. Thus, the toxin may be connected to PABC by a linker.

[0450] In certain embodiments, the toxin may be a toxin containing a primary or secondary amine, hi certain embodiments, the toxin may be MMAE or maytansine.

[0451] In certain embodiments, the linker may have a protected N-terminus. In certain embodiments, the N-terminus may be acetylated. In certain embodiments, the linker is the linker shown in Figure 2.

[0452] In certain embodiments, the linker may have the structure RKA-PABC-MMAE. In certain embodiments, the linker may have the structure RKA-(PEG) n -PABC-MMAE, where n is an integer between 2 and 20. In certain embodiments, the linker may have the structure RKA-(PEG)2-PABC-MMAE. In certain embodiments, the linker may have the structure RKA-MMAE. In certain embodiments, the linker may have the structure RKA-Val-Cit-PABC-MMAE. In certain embodiments, the linker may include an additional linker between the PABC moiety and the MMAE. In certain embodiments, the additional linker may be a p-nitrophenol (PNP) group.

[0453] It should be noted that the linker may include a self-immolative moiety other than PABC. That is, the linker may have the structure RKA-(self-immolative moiety)-toxin. Those skilled in the art will know other self-immolative moieties that can be used within the scope of the present invention. Furthermore, those skilled in the art will know toxins that can be attached to a self-immolative moiety, if necessary, by an additional linker.

[0454] In certain embodiments, the toxin may be a toxin containing a hydroxy group, and the linker may include a self-immolative methylamine group. That is, the linker may have the structure RKA-(NH)-(CH)-O-toxin. In certain embodiments, the hydroxy-containing toxin may be a camptothecin, e.g., an anthracycline such as exatecan or an exatecan derivative, particularly the exatecan derivative Dxd, or PNU-159682.

[0455] In certain embodiments, the toxin may be a toxin containing a thiol group, and the linker may include a self-immolative methylamine group. That is, the linker may have the structure RKA-(NH)-(CH)-S-toxin. In certain embodiments, the thiol-containing toxin may be a maytansinoid, such as DM1, or a thiol-containing derivative thereof.

[0456] In a particular embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure ARK-B or ARK-(linker molecule)-B.

[0457] That is, payload B may be directly attached to the C-terminus of the lysine residue, or may be attached to the C-terminus of the lysine residue via a linker molecule. It should be understood that the choice of linker molecule largely depends on the functional group available on payload B. Linker molecules suitable for attaching payloads having different functional groups to peptides are disclosed herein. The linker molecule may be a cleavable or non-cleavable linker molecule. In particular, the linker molecule may comprise a self-immolative moiety, in particular any one of the self-immolative moieties disclosed herein. Thus, in certain embodiments, the present invention relates to an antibody-drug conjugate according to the present invention, wherein the linker comprises or consists of the structure ARK-(self-immolative moiety)-B.

[0458] Alternatively, the payload may be attached to the N-terminus of the alanine residue directly or via a linker molecule, for example, via any one of the linker molecules disclosed herein. That is, in certain embodiments, the present invention relates to an antibody-drug conjugate according to the present invention, wherein the linker comprises or consists of the structure B-ARK or B-(linker molecule)-ARK. In certain embodiments, the present invention relates to an antibody-drug conjugate according to the present invention, wherein the linker comprises or consists of the structure B-(self-immolative moiety)-ARK. In certain embodiments, the self-immolative moiety comprised in the structure B-(self-immolative moiety)-ARK may be a self-immolative moiety comprising an ortho-hydroxy-protected aryl sulfate (OHPAS) moiety disclosed herein.

[0459] In a particular embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker has the structure ARK-PABC-B (particularly, wherein B is an auristatin or a maytansinoid, particularly wherein the auristatin is MMAE and the maytansinoid is DM1 or maytansine).

[0460] In certain embodiments, the linker may have the structure ARK-PABC-B. That is, the linker may comprise a linear peptide ARK, in which the carboxy group of the C-terminal lysine residue is linked to an amino group contained in PABC by an amide bond. Toxin B may be attached to PABC via carbamate formation. It should be understood that not all toxins contain functional groups that allow for carbamate formation with PABC. Thus, the toxin may be connected to PABC by a linker.

[0461] In certain embodiments, the toxin may be a toxin containing a primary or secondary amine, hi certain embodiments, the toxin may be MMAE or maytansine.

[0462] In certain embodiments, the linker may have a protected N-terminus. In certain embodiments, the N-terminus may be acetylated. In certain embodiments, the linker is the linker shown in Figure 3.

[0463] In certain embodiments, the linker may have the structure ARK-PABC-MMAE. In certain embodiments, the linker may have the structure ARK-(PEG) n -PABC-MMAE, where n is an integer between 2 and 20. In certain embodiments, the linker may have the structure ARK-(PEG)2-PABC-MMAE (see Figure 14). In certain embodiments, the linker may have the structure ARK-MMAE. In certain embodiments, the linker may have the structure ARK-Val-Cit-PABC-MMAE. In certain embodiments, the linker may include an additional linker between the PABC moiety and the MMAE. In certain embodiments, the additional linker may be a p-nitrophenol (PNP) group.

[0464] It should be noted that the linker may contain a self-immolative moiety other than PABC. That is, the linker may have the structure ARK-(self-immolative moiety)-toxin. Those skilled in the art will know other self-immolative moieties that can be used within the scope of the present invention. Furthermore, those skilled in the art will know toxins that can be attached to a self-immolative moiety, if necessary, by an additional linker.

[0465] In certain embodiments, the toxin may be a toxin containing a hydroxy group, and the linker may include a self-immolative methylamine group. That is, the linker may have the structure ARK-(NH)-(CH)-O-toxin. In certain embodiments, the hydroxy-containing toxin may be a camptothecin, e.g., an anthracycline such as exatecan or an exatecan derivative, particularly the exatecan derivative Dxd, or PNU-159682.

[0466] In certain embodiments, the toxin may be a thiol-containing toxin and the linker may include a self-immolative methylamine group. That is, the linker may have the structure ARK-(NH)-(CH)-S-toxin (similar to Figure 15). In certain embodiments, the thiol-containing toxin may be a maytansinoid, e.g., DM1, or a thiol-containing derivative thereof. In certain embodiments, the linker is a linker shown in FIG. 14 or FIG.

[0467] In a particular embodiment, the present invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure RKR-B or RKR-(linker molecule)-B.

[0468] That is, payload B may be directly attached to the C-terminus of the arginine residue, or may be attached to the C-terminus of the arginine residue via a linker molecule. It should be understood that the choice of linker molecule largely depends on the functional group available on payload B. Linker molecules suitable for attaching payloads having different functional groups to peptides are disclosed herein. The linker molecule may be a cleavable or non-cleavable linker molecule. In particular, the linker molecule may comprise a self-immolative moiety, particularly any one of the self-immolative moieties disclosed herein. Thus, in certain embodiments, the present invention relates to an antibody-drug conjugate according to the present invention, wherein the linker comprises or consists of the structure RKR-(self-immolative moiety)-B.

[0469] Alternatively, the payload may be attached to the N-terminus of the arginine residue directly or via a linker molecule, for example, via any one of the linker molecules disclosed herein. That is, in certain embodiments, the present invention relates to an antibody-drug conjugate according to the present invention, wherein the linker comprises or consists of the structure B-RKR or B-(linker molecule)-RKR. In certain embodiments, the linker may be a dicarboxylic acid linker (see Figure 9). In certain embodiments, the present invention relates to an antibody-drug conjugate according to the present invention, wherein the linker comprises or consists of the structure B-(self-immolative moiety)-RKR. In certain embodiments, the self-immolative moiety comprised in the structure B-(self-immolative moiety)-RKR may be a self-immolative moiety comprising an ortho-hydroxy-protected aryl sulfate (OHPAS) moiety disclosed herein.

[0470] In a particular embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker has the structure RKR-PABC-B (particularly, wherein B is an auristatin or a maytansinoid, particularly wherein the auristatin is MMAE and the maytansinoid is DM1 or maytansine).

[0471] In certain embodiments, the linker may have the structure RKR-PABC-B. That is, the linker may comprise the linear peptide RKR, in which the carboxy group of the C-terminal arginine residue is linked to the amino group contained in the PABC by an amide bond. Toxin B may be attached to the PABC via carbamate formation. It should be understood that not all toxins contain functional groups that allow for carbamate formation with the PABC. Thus, the toxin may be connected to the PABC by the linker.

[0472] In certain embodiments, the toxin may be a toxin containing a primary or secondary amine, hi certain embodiments, the toxin may be MMAE or maytansine.

[0473] In certain embodiments, the linker may have a protected N-terminus, hi certain embodiments, the N-terminus may be acetylated.

[0474] In certain embodiments, the linker may have the structure RKR-PABC-MMAE. In certain embodiments, the linker may have the structure RKR-(PEG) n-PABC-MMAE, where n is an integer between 2 and 20. In certain embodiments, the linker may have the structure RKR-(PEG)2-PABC-MMAE. In certain embodiments, the linker may have the structure RKR-MMAE. In certain embodiments, the linker may have the structure RKR-Val-Cit-PABC-MMAE. In certain embodiments, the linker may include an additional linker between the PABC moiety and the MMAE. In certain embodiments, the additional linker may be a p-nitrophenol (PNP) group.

[0475] It should be noted that the linker may contain a self-immolative moiety other than PABC. That is, the linker may have the structure RKR-(self-immolative moiety)-toxin. Those skilled in the art will know of other self-immolative moieties that can be used within the scope of the present invention. Furthermore, those skilled in the art will know of toxins that can be attached to a self-immolative moiety, if necessary, by an additional linker.

[0476] In certain embodiments, the toxin may be a toxin containing a hydroxy group, and the linker may include a self-immolative methylamine group. That is, the linker may have the structure RKR-(NH)-(CH)-O-toxin. In certain embodiments, the hydroxy-containing toxin may be a camptothecin, e.g., an anthracycline such as exatecan or an exatecan derivative, particularly the exatecan derivative Dxd, or PNU-159682.

[0477] In certain embodiments, the toxin may be a thiol-containing toxin and the linker may include a self-immolative methylamine group. That is, the linker may have the structure RKR-(NH)-(CH)-S-toxin (similar to Figure 15). In certain embodiments, the thiol-containing toxin may be a maytansinoid, e.g., DM1, or a thiol-containing derivative thereof.

[0478] In a particular embodiment, the present invention relates to an antibody-drug conjugate according to the present invention, wherein the linker comprises or consists of the structure RK-Val-Cit-B or RK-Val-Cit-(linker molecule)-B.

[0479] That is, payload B may be directly linked to the C-terminus of the citrulline residue, or may be linked to the C-terminus of the citrulline residue via a linker molecule. It should be understood that the choice of linker molecule largely depends on the functional group available on payload B. Linker molecules suitable for linking payloads having different functional groups to peptides are disclosed herein. The linker molecule may be a cleavable or non-cleavable linker molecule. In particular, the linker molecule may comprise a self-immolative moiety, particularly any one of the self-immolative moieties disclosed herein. Thus, in certain embodiments, the present invention relates to an antibody-drug conjugate according to the present invention, wherein the linker comprises or consists of the structure RK-Val-Cit-(self-immolative moiety)-B.

[0480] In a particular embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker has the structure RK-Val-Cit-PABC-B (particularly, wherein B is an auristatin or a maytansinoid, particularly wherein the auristatin is MMAE and the maytansinoid is DM1 or maytansine).

[0481] In certain embodiments, the linker may have the structure RK-Val-Cit-PABC-B. That is, the linker may comprise the linear peptide RK-Val-Cit, in which the carboxy group of the C-terminal citrulline residue is linked to the amino group contained in the PABC by an amide bond. Toxin B may be attached to the PABC via carbamate formation. It should be understood that not all toxins contain functional groups that allow for carbamate formation with the PABC. Thus, the toxin may be connected to the PABC by a linker.

[0482] In certain embodiments, the toxin may be a toxin containing a primary or secondary amine, hi certain embodiments, the toxin may be MMAE or maytansine.

[0483] In certain embodiments, the linker may have a protected N-terminus, hi certain embodiments, the N-terminus may be acetylated.

[0484] In certain embodiments, the linker may have the structure RK-Val-Cit-PABC-MMAE. In certain embodiments, the linker may have the structure RK-(PEG) n -Val-Cit-PABC-MMAE, where n is an integer between 2 and 20. In certain embodiments, the linker may have the structure RK-(PEG)2-Val-Cit-PABC-MMAE. In certain embodiments, the linker may have the structure RK-Val-Cit-MMAE. In certain embodiments, the linker may include an additional linker between the PABC moiety and the MMAE. In certain embodiments, the additional linker may be a p-nitrophenol (PNP) group.

[0485] It should be noted that the linker may include a self-immolative moiety other than PABC. That is, the linker may have the structure RK-Val-Cit-(self-immolative moiety)-toxin. Those skilled in the art will know of other self-immolative moieties that can be used within the scope of the present invention. Furthermore, those skilled in the art will know of toxins that can be attached to a self-immolative moiety, if necessary, by an additional linker.

[0486] In certain embodiments, the toxin may be a toxin containing a hydroxy group, and the linker may include a self-immolative methylamine group. That is, the linker may have the structure RK-Val-Cit-(NH)-(CH)-O-toxin. In certain embodiments, the hydroxy-containing toxin may be a camptothecin, e.g., an anthracycline such as exatecan or an exatecan derivative, particularly the exatecan derivative Dxd, or PNU-159682.

[0487] In certain embodiments, the toxin may be a thiol-containing toxin and the linker may include a self-immolative methylamine group. That is, the linker may have the structure RK-Val-Cit-(NH)-(CH)-S-toxin. In certain embodiments, the thiol-containing toxin may be a maytansinoid, such as DM1, or a thiol-containing derivative thereof.

[0488] In certain embodiments, the invention relates to antibody-linker conjugates or antibody drug conjugates comprising the antibody polatuzumab, or alternatively, an anti-CD79b antibody.

[0489] That is, in certain embodiments, the present invention provides a) polatuzumab or anti-CD79b antibody; and b) Structure: (Sp1)-RK-(Sp2)-B-(Sp3) or (Sp1)-B-(Sp2)-RK-(Sp3); (In the formula, - (Sp1) is a chemical spacer or is absent; - (Sp2) is a chemical spacer or is absent; - (Sp3) is a chemical spacer or is absent; R is arginine or an arginine derivative or arginine mimetic; K is lysine or a lysine derivative or a lysine mimetic; - B is the linking portion or payload) a linker comprising 1. An antibody-linker conjugate comprising: The present invention relates to an antibody-linker conjugate in which the linker is conjugated to the polatuzumab or anti-CD79b antibody by an isopeptide bond formed between the γ-carboxamide group of a glutamine residue contained in the polatuzumab or anti-CD79b antibody and a primary amine contained in the side chain of a lysine residue, lysine derivative, or lysine mimetic contained in the RK motif contained in the linker.

[0490] In certain embodiments, the present invention relates to an antibody-payload conjugate comprising polatuzumab or an anti-CD79b antibody, wherein the linker is any one of the linkers shown in Figure 1, Figure 2, Figure 3, Figure 8, Figure 9, Figure 14, Figure 15, Figure 17, Figure 18, Figure 19, Figure 20, Figure 21, Figure 22, Figure 23, Figure 24, Figure 25, Figure 26, Figure 27, Figure 28, Figure 29, Figure 30, Figure 31, Figure 32, Figure 33 or Figure 34.

[0491] Polatuzumab is commercially available as the antibody-drug conjugate polatuzumab vedotin, sold under the name Polivy. Polatuzumab vedotin contains the anti-CD79b antibody polatuzumab and the linker maleimidocaproyl-L-valine-L-citrulline-PABC-MMAE (mc-vc-PABC-MMAE), commonly known as vedotin. The mc-vc-PABC-MMAE linker of polatuzumab vedotin is conjugated to a free cysteine ​​residue contained in the antibody. The antibody polatuzumab is disclosed in WO2009 / 012268, the entire contents of which are incorporated herein by reference. Furthermore, cysteine-engineered variants of polatuzumab are disclosed in WO2009 / 099728, the entire contents of which are incorporated herein by reference.

[0492] The present inventors have shown that polatuzumab conjugates containing the linker according to the present invention have a longer half-life in plasma compared to the commercially available conjugate polatuzumab vedotin. Thus, antibody-linker conjugates conjugated to the linker according to the present invention using microbial transglutaminase are surprisingly more stable than antibodies produced by other techniques, for example, by conjugating a maleimide-containing linker to a cysteine ​​residue of an antibody. Thus, the antibody-linker conjugates according to the present invention are more likely to reach their target cells or tissues without losing their payload too quickly.

[0493] In certain embodiments, the antibody is polatuzumab comprising a heavy chain set forth in SEQ ID NO: 5 and a light chain set forth in SEQ ID NO: 6. However, the invention also encompasses variants of polatuzumab in which the heavy and / or light chains comprise at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 5 and / or SEQ ID NO: 6, respectively. In particular, the antibody may comprise any of the sequence variations disclosed in WO2009 / 012268 or WO2009 / 099728.

[0494] It is preferred herein that polatuzumab, or an anti-CD79b antibody, is present in the antibody-linker conjugate in a glycosylated form. That is, polatuzumab, or an anti-CD79b antibody, is preferably glycosylated at residue N297 (EU numbering). However, polatuzumab, or an anti-CD79b antibody, may also be deglycosylated as described herein.

[0495] Polatuzumab, or an anti-CD79b antibody, may be conjugated to any one of the linkers disclosed herein, particularly for the methods according to the invention. Preferably, the linker is conjugated to the antibody glutamine residue Q295 (EU numbering) via MTG catalysis. However, the linker may also be conjugated to an engineered glutamine residue such as N297Q (EU numbering) and / or any one of the glutamine-containing tags disclosed herein.

[0496] The linker conjugated to polatuzumab, or the anti-CD79b antibody, may contain a single linking moiety or payload B, or may contain multiple linking moieties and / or payloads B1, B2, etc.

[0497] That is, in certain embodiments, the linker included in a polatuzumab-linker conjugate may include one or more linking moieties B. Such polatuzumab-linker conjugates may then be functionalized with a suitable payload in a two-step process disclosed herein.

[0498] In certain embodiments, the linker included in the polatuzumab-payload conjugate may comprise one or more payloads B. Such polatuzumab-payload conjugates may be obtained by a two-step process, in which a linker comprising a linking moiety is conjugated to polatuzumab in a first step, and a payload is linked to the linking moiety in a second step. Alternatively, the polatuzumab-payload conjugates may be obtained by a one-step process, in which a linker comprising a payload is directly conjugated to polatuzumab.

[0499] In certain embodiments, the present invention relates to antibody-drug conjugates comprising polatuzumab or an anti-CD79B antibody, i.e., the linker included in the antibody-drug conjugate may comprise one or more toxins described herein.

[0500] In certain embodiments, an antibody-drug conjugate comprising polatuzumab or an anti-CD79b antibody may comprise a linker comprising the amino acid sequence RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4), or RK-Val-Cit (SEQ ID NO: 54).

[0501] That is, in certain embodiments, the present invention provides a) polatuzumab or anti-CD79b antibody; and b) a linker comprising a drug moiety B, wherein drug moiety B is covalently linked to an amino acid sequence selected from the group consisting of RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4), or RK-Val-Cit (SEQ ID NO: 54). 1. An antibody-drug conjugate comprising: The linker is located at the C Hand an antibody-drug conjugate conjugated to polatuzumab or an anti-CD79b antibody via an isopeptide bond formed between the γ-carboxamide group of glutamine residue Q295 (EU numbering) in domain 2 and a primary amine contained in the side chain of a lysine residue contained in the linker.

[0502] Preferably, the antibody is polatuzumab comprising a heavy chain set forth in SEQ ID NO: 5 and a light chain set forth in SEQ ID NO: 6. Thus, in a particular embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the IgG antibody is polatuzumab or an antibody comprising a heavy chain set forth in SEQ ID NO: 5 and a light chain set forth in SEQ ID NO: 6.

[0503] In certain embodiments, a linker conjugated to polatuzumab, or an anti-CD79b antibody, may comprise the structure RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4), or RK-Val-Cit (SEQ ID NO: 54). In certain embodiments, a linker comprising the structure RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4), or RK-Val-Cit (SEQ ID NO: 54) may be conjugated to residue Q295 of polatuzumab, or an anti-CD79b antibody, through the primary amine contained in residue K.

[0504] In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to a linker RKAA-B (wherein B is preferably a toxin) disclosed herein. In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to a linker RKAA-(self-immolative moiety)-B (wherein B is preferably a toxin) disclosed herein. The self-immolative moiety may be any self-immolative moiety known in the art and / or disclosed herein that is suitable for attaching a payload to the C-terminus of a peptide. In certain embodiments, the self-immolative moiety may be a group comprising PABC or a methyl-amine.

[0505] In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to the linker RKAA-PABC-B. In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to the linker RKAA-PABC-MMAE (see Figure 1). In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to the linker RKAA-PABC-maytansine (see Figure 8). In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to the linker RKAA-MMAE. In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to the linker RKAA-PABC-PNP-MMAE. In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker RKAA-(NH)-(CH)-OB (where O is the oxygen atom of an ether bond and B is a hydroxyl-containing toxin). In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker RKAA-(NH)-(CH)-O-camptothecin. In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker RKAA-(NH)-(CH)-SB (where S is the sulfur atom of a thioether bond and B is a thiol-containing toxin). In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker RKAA-(NH)-(CH)-S-DM1.

[0506] In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to a linker B-RKAA (wherein B is preferably a toxin) disclosed herein. In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to a linker B-(self-immolative moiety)-RKAA (wherein B is preferably a toxin) disclosed herein. The self-immolative moiety may be any self-immolative moiety known in the art and / or disclosed herein that is suitable for attaching a payload to the N-terminus of a peptide. In certain embodiments, the self-immolative moiety may be a self-immolative moiety comprising an ortho-hydroxy-protected aryl sulfate (OHPAS) moiety. In certain embodiments, the amine comprising payload B may be attached to the N-terminal arginine moiety by a dicarboxylic acid linker, similar to Figure 9.

[0507] In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to a linker RKA-B (wherein B is preferably a toxin) disclosed herein. In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to a linker RKA-(self-immolative moiety)-B (wherein B is preferably a toxin) disclosed herein. The self-immolative moiety may be any self-immolative moiety known in the art and / or disclosed herein that is suitable for attaching a payload to the C-terminus of a peptide. In certain embodiments, the self-immolative moiety may be a group comprising PABC or a methyl-amine.

[0508] In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to the linker RKA-PABC-B. In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to the linker RKA-PABC-MMAE (see Figure 2). In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to the linker RKA-PABC-maytansine. In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to the linker RKA-MMAE. In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to the linker RKA-PABC-PNP-MMAE. In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker RKA-(NH)-(CH)-OB (where O is the oxygen atom of an ether bond and B is a hydroxyl-containing toxin). In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker RKA-(NH)-(CH)-O-camptothecin. In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker RKA-(NH)-(CH)-SB (where S is the sulfur atom of a thioether bond and B is a thiol-containing toxin). In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker RKA-(NH)-(CH)-S-DM1.

[0509] In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to a linker B-RKA (wherein B is preferably a toxin) disclosed herein. In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to a linker B-(self-immolative moiety)-RKA (wherein B is preferably a toxin) disclosed herein. The self-immolative moiety may be any self-immolative moiety known in the art and / or disclosed herein that is suitable for attaching a payload to the N-terminus of a peptide. In certain embodiments, the self-immolative moiety may be a self-immolative moiety comprising an ortho-hydroxy-protected aryl sulfate (OHPAS) moiety. In certain embodiments, the amine comprising payload B may be attached to the N-terminal arginine moiety by a dicarboxylic acid linker, similar to Figure 9.

[0510] In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to the linker ARK-B (wherein B is preferably a toxin) disclosed herein. In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to the linker ARK-(self-immolative moiety)-B (wherein B is preferably a toxin) disclosed herein. The self-immolative moiety may be any self-immolative moiety known in the art and / or disclosed herein that is suitable for attaching a payload to the C-terminus of a peptide. In certain embodiments, the self-immolative moiety may be a group comprising PABC or a methyl-amine.

[0511] In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to the linker ARK-PABC-B. In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to the linker ARK-PABC-MMAE (see Figure 3). In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to the linker ARK-PABC-maytansine. In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to the linker ARK-MMAE. In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to the linker ARK-PABC-PNP-MMAE. In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker ARK-(NH)-(CH)-OB (where O is the oxygen atom of an ether bond and B is a hydroxyl-containing toxin). In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker ARK-(NH)-(CH)-O-camptothecin. In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker ARK-(NH)-(CH)-SB (where S is the sulfur atom of a thioether bond and S is a thiol-containing toxin). In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker ARK-(NH)-(CH)-S-DM1.

[0512] In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to a linker B-ARK (wherein B is preferably a toxin) disclosed herein. In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to a linker B-(self-immolative moiety)-ARK (wherein B is preferably a toxin) disclosed herein. The self-immolative moiety may be any self-immolative moiety known in the art and / or disclosed herein that is suitable for attaching a payload to the N-terminus of a peptide. In certain embodiments, the self-immolative moiety may be a self-immolative moiety comprising an ortho-hydroxy-protected aryl sulfate (OHPAS) moiety.

[0513] In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to the linker RKR-B (wherein B is preferably a toxin) disclosed herein. In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to the linker RKR-(self-immolative moiety)-B (wherein B is preferably a toxin) disclosed herein. The self-immolative moiety may be any self-immolative moiety known in the art and / or disclosed herein that is suitable for attaching a payload to the C-terminus of a peptide. In certain embodiments, the self-immolative moiety may be a group comprising PABC or a methyl-amine.

[0514] In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker RKR-PABC-B. In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker RKR-PABC-MMAE. In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker RKR-PABC-maytansine. In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker RKR-MMAE. In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker RKR-PABC-PNP-MMAE. In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker RKR-(NH)-(CH)-OB (where O is the oxygen atom of an ether bond and B is a hydroxy-containing toxin). In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker RKR-(NH)-(CH)-O-camptothecin. In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker RKR-(NH)-(CH)-SB (where S is the sulfur atom of a thioether bond and S is a thiol-containing toxin). In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker RKR-(NH)-(CH)-S-DM1.

[0515] In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to a linker B-RKR (wherein B is preferably a toxin) disclosed herein. In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to a linker B-(self-immolative moiety)-RKR (wherein B is preferably a toxin) disclosed herein. The self-immolative moiety may be any self-immolative moiety known in the art and / or disclosed herein that is suitable for attaching a payload to the N-terminus of a peptide. In certain embodiments, the self-immolative moiety may be a self-immolative moiety comprising an ortho-hydroxy-protected aryl sulfate (OHPAS) moiety. In certain embodiments, the amine comprising payload B may be attached to the N-terminal arginine moiety by a dicarboxylic acid linker, as illustrated in Figure 9.

[0516] In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to the linker RK-Val-Cit-B, where B is preferably a toxin, as disclosed herein. In certain embodiments, polatuzumab, or an anti-CD79b antibody, may be conjugated to the linker RK-Val-Cit-(self-immolative moiety)-B, where B is preferably a toxin, as disclosed herein. The self-immolative moiety may be any self-immolative moiety known in the art and / or disclosed herein that is suitable for attaching a payload to the C-terminus of a peptide. In certain embodiments, the self-immolative moiety may be a group comprising PABC or a methyl-amine.

[0517] In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker RK-Val-Cit-PABC-B. In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker RK-Val-Cit-PABC-MMAE. In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker RK-Val-Cit-PABC-maytansine. In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker RK-Val-Cit-MMAE. In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker RK-Val-Cit-PABC-PNP-MMAE. In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker RK-Val-Cit-(NH)-(CH)-OB (where O is the oxygen atom of an ether bond and B is a hydroxyl-containing toxin). In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker RK-Val-Cit-(NH)-(CH)-O-camptothecin. In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker RK-Val-Cit-(NH)-(CH)-SB (where S is the sulfur atom of a thioether bond and B is a thiol-containing toxin). In certain embodiments, polatuzumab or an anti-CD79b antibody may be conjugated to the linker RK-Val-Cit-(NH)-(CH)-S-DM1.

[0518] In certain embodiments, the invention relates to antibody-linker conjugates or antibody drug-conjugates comprising the antibody trastuzumab, or alternatively, an anti-HER2 / neu antibody.

[0519] That is, in certain embodiments, the present invention provides a) trastuzumab or anti-HER2 / neu antibodies; and b) Structure: (Sp1)-RK-(Sp2)-B-(Sp3) or (Sp1)-B-(Sp2)-RK-(Sp3); (In the formula, - (Sp1) is a chemical spacer or is absent; - (Sp2) is a chemical spacer or is absent; - (Sp3) is a chemical spacer or is absent; R is arginine or an arginine derivative or arginine mimetic; K is lysine or a lysine derivative or a lysine mimetic; - B is the linking portion or payload) a linker comprising 1. An antibody-linker conjugate comprising: The present invention relates to an antibody-linker conjugate in which the linker is conjugated to the trastuzumab or anti-HER2 / neu antibody by an isopeptide bond formed between the gamma-carboxamide group of a glutamine residue contained in the trastuzumab or anti-HER2 / neu antibody and a primary amine contained in the side chain of a lysine residue, lysine derivative, or lysine mimetic contained in the RK motif contained in the linker.

[0520] In certain embodiments, the present invention relates to an antibody-payload conjugate comprising trastuzumab or an anti-HER2 / neu antibody, wherein the linker is any one of the linkers shown in Figure 1, Figure 2, Figure 3, Figure 8, Figure 9, Figure 14, Figure 15, Figure 17, Figure 18, Figure 19, Figure 20, Figure 21, Figure 22, Figure 23, Figure 24, Figure 25, Figure 26, Figure 27, Figure 28, Figure 29, Figure 30, Figure 31, Figure 32, Figure 33 or Figure 34.

[0521] Trastuzumab is commercially available as the antibody-drug conjugate trastuzumab emtacin, sold under the name Kadcyla. Trastuzumab emtacin comprises the anti-HER2 / neu antibody trastuzumab and the toxin DM1, linked to trastuzumab via an N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) linker. Linker-DM1 constructs can be conjugated to up to eight different lysine residues in the antibody, resulting in antibodies with various drug-to-antibody ratios. Preferably, trastuzumab comprises a heavy chain set forth in SEQ ID NO:7 and a light chain set forth in SEQ ID NO:8. However, the present invention also encompasses variants of trastuzumab in which the heavy and / or light chains comprise at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO:7 and / or SEQ ID NO:8, respectively. In certain embodiments, the antibody may be an anti-HER2 / neu antibody, for example, but not limited to, those disclosed in WO1998 / 006692, WO1999 / 905536, WO2003 / 087131, which are incorporated by reference in their entireties.

[0522] It is preferred herein that trastuzumab, or an anti-HER2 / neu antibody, is present in the antibody-linker conjugate in a glycosylated form. That is, trastuzumab, or an anti-HER2 / neu antibody, is preferably glycosylated at residue N297 (EU numbering). However, trastuzumab, or an anti-HER2 / neu antibody, may also be deglycosylated as described herein.

[0523] Trastuzumab or an anti-HER2 / neu antibody may be conjugated to any one of the linkers disclosed herein, particularly for the method according to the present invention. Preferably, the linker is conjugated to the glutamine residue Q295 (EU numbering) of the antibody by MTG catalysis. However, the linker may also be conjugated to an engineered glutamine residue such as N297Q (EU numbering) and / or any one of the glutamine-containing tags disclosed herein.

[0524] The linker conjugated to trastuzumab, or an anti-HER2 / neu antibody, may contain a single linking moiety or payload B, or may contain multiple linking moieties and / or payloads B1, B2, etc.

[0525] That is, in certain embodiments, the linker included in a trastuzumab-linker conjugate may comprise one or more linking moieties B. Such trastuzumab-linker conjugates may then be functionalized with a suitable payload in a two-step process disclosed herein.

[0526] In certain embodiments, the linker included in the trastuzumab-payload conjugate may comprise one or more payloads B. Such trastuzumab-payload conjugates may be obtained by a two-step process, in which a linker comprising a linking moiety is conjugated to trastuzumab in a first step, and a payload is linked to the linking moiety in a second step. Alternatively, the trastuzumab-payload conjugates may be obtained by a one-step process, in which a linker comprising a payload is directly conjugated to trastuzumab.

[0527] In certain embodiments, the present invention relates to antibody-drug conjugates comprising trastuzumab or an anti-HER2 / neu antibody, i.e., the linker included in the antibody-drug conjugate may comprise one or more toxins described herein.

[0528] In certain embodiments, an antibody-drug conjugate comprising trastuzumab or an anti-HER2 / neu antibody may comprise a linker comprising the amino acid sequence RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4), or RK-Val-Cit (SEQ ID NO: 54).

[0529] That is, in certain embodiments, the present invention provides a) trastuzumab or anti-HER2 / neu antibodies; and b) an antibody-drug conjugate comprising a linker comprising a drug moiety B, wherein drug moiety B is covalently linked to an amino acid sequence selected from the group consisting of RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4) or RK-Val-Cit (SEQ ID NO: 54); The linker is located at the C H and an antibody-drug conjugate conjugated to trastuzumab or an anti-HER2 / neu antibody via an isopeptide bond formed between the γ-carboxamide group of glutamine residue Q295 (EU numbering) in domain 2 and a primary amine contained in the side chain of a lysine residue contained in the linker.

[0530] Preferably, the antibody is trastuzumab comprising a heavy chain set forth in SEQ ID NO: 7 and a light chain set forth in SEQ ID NO: 8. Thus, in a particular embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the IgG antibody is trastuzumab or an antibody comprising a heavy chain set forth in SEQ ID NO: 7 and a light chain set forth in SEQ ID NO: 8.

[0531] In certain embodiments, a linker conjugated to trastuzumab or an anti-HER2 / neu antibody may comprise the structure RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4), or RK-Val-Cit (SEQ ID NO: 54). In certain embodiments, a linker comprising the structure RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4), or RK-Val-Cit (SEQ ID NO: 54) may be conjugated to residue Q295 of trastuzumab or an anti-HER2 / neu antibody through the primary amine contained in residue K.

[0532] In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, may be conjugated to a linker RKAA-B, where B is preferably a toxin, as disclosed herein. In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, may be conjugated to a linker RKAA-(self-immolative moiety)-B, where B is preferably a toxin, as disclosed herein. The self-immolative moiety may be any self-immolative moiety known in the art and / or disclosed herein that is suitable for attaching a payload to the C-terminus of a peptide. In certain embodiments, the self-immolative moiety may be a group comprising PABC or a methyl-amine.

[0533] In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RKAA-PABC-B. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RKAA-PABC-MMAE (see Figure 1). In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RKAA-PABC-maytansine (see Figure 8). In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RKAA-MMAE. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RKAA-PABC-PNP-MMAE. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RKAA-(NH)-(CH)-OB, where O is the oxygen atom of an ether bond and B is a hydroxyl-containing toxin. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RKAA-(NH)-(CH)-O-camptothecin. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RKAA-(NH)-(CH)-SB, where S is the sulfur atom of a thioether bond and B is a thiol-containing toxin. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RKAA-(NH)-(CH)-S-DM1.

[0534] In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, may be conjugated to the linker B-RKAA (wherein B is preferably a toxin) disclosed herein. In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, may be conjugated to the linker B-(self-immolative moiety)-RKAA (wherein B is preferably a toxin) disclosed herein. The self-immolative moiety may be any self-immolative moiety known in the art and / or disclosed herein that is suitable for attaching a payload to the N-terminus of a peptide. In certain embodiments, the self-immolative moiety may be a self-immolative moiety comprising an ortho-hydroxy-protected aryl sulfate (OHPAS) moiety. In certain embodiments, the amine comprising payload B may be attached to the N-terminal arginine moiety by a dicarboxylic acid linker, similar to Figure 9.

[0535] In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, may be conjugated to the linker RKA-B, where B is preferably a toxin, as disclosed herein. In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, may be conjugated to the linker RKA-(self-immolative moiety)-B, where B is preferably a toxin, as disclosed herein. The self-immolative moiety may be any self-immolative moiety known in the art and / or disclosed herein that is suitable for attaching a payload to the C-terminus of a peptide. In certain embodiments, the self-immolative moiety may be a group comprising PABC or a methyl-amine.

[0536] In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RKA-PABC-B. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RKA-PABC-MMAE (see Figure 2). In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RKA-PABC-maytansine. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RKA-MMAE. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RKA-PABC-PNP-MMAE. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RKA-(NH)-(CH)-OB, where O is the oxygen atom of an ether bond and B is a hydroxyl-containing toxin. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RKA-(NH)-(CH)-O-camptothecin. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RKA-(NH)-(CH)-SB, where S is the sulfur atom of a thioether bond and B is a thiol-containing toxin. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RKA-(NH)-(CH)-S-DM1.

[0537] In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, may be conjugated to the linker B-RKA (wherein B is preferably a toxin) disclosed herein. In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, may be conjugated to the linker B-(self-immolative moiety)-RKA (wherein B is preferably a toxin) disclosed herein. The self-immolative moiety may be any self-immolative moiety known in the art and / or disclosed herein that is suitable for attaching a payload to the N-terminus of a peptide. In certain embodiments, the self-immolative moiety may be a self-immolative moiety comprising an ortho-hydroxy-protected aryl sulfate (OHPAS) moiety. In certain embodiments, the amine comprising payload B may be attached to the N-terminal arginine moiety by a dicarboxylic acid linker, similar to Figure 9.

[0538] In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, may be conjugated to the linker ARK-B (wherein B is preferably a toxin) disclosed herein. In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, may be conjugated to the linker ARK-(self-immolative moiety)-B (wherein B is preferably a toxin) disclosed herein. The self-immolative moiety may be any self-immolative moiety known in the art and / or disclosed herein that is suitable for attaching a payload to the C-terminus of a peptide. In certain embodiments, the self-immolative moiety may be a group comprising PABC or a methyl-amine.

[0539] In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker ARK-PABC-B. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker ARK-PABC-MMAE (see Figure 3). In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker ARK-PABC-maytansine. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker ARK-MMAE. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker ARK-PABC-PNP-MMAE. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker ARK-(NH)-(CH)-OB, where O is the oxygen atom of an ether bond and B is a hydroxy-containing toxin. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker ARK-(NH)-(CH)-O-camptothecin. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker ARK-(NH)-(CH)-SB, where S is the sulfur atom of a thioether bond and S is a hydroxy-containing toxin. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker ARK-(NH)-(CH)-S-DM1.

[0540] In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, may be conjugated to the linker B-ARK (wherein B is preferably a toxin) disclosed herein. In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, may be conjugated to the linker B-(self-immolative moiety)-ARK (wherein B is preferably a toxin) disclosed herein. The self-immolative moiety may be any self-immolative moiety known in the art and / or disclosed herein that is suitable for attaching a payload to the N-terminus of a peptide. In certain embodiments, the self-immolative moiety may be a self-immolative moiety comprising an ortho-hydroxy-protected aryl sulfate (OHPAS) moiety.

[0541] In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, may be conjugated to the linker RKR-B (wherein B is preferably a toxin) disclosed herein. In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, may be conjugated to the linker RKR-(self-immolative moiety)-B (wherein B is preferably a toxin) disclosed herein. The self-immolative moiety may be any self-immolative moiety known in the art and / or disclosed herein that is suitable for attaching a payload to the C-terminus of a peptide. In certain embodiments, the self-immolative moiety may be a group comprising PABC or a methyl-amine.

[0542] In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RKR-PABC-B. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RKR-PABC-MMAE. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RKR-PABC-maytansine. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RKR-MMAE. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RKR-PABC-PNP-MMAE. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RKR-(NH)-(CH)-OB (where O is the oxygen atom of an ether bond and B is a hydroxy-containing toxin). In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RKR-(NH)-(CH)-O-camptothecin. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RKR-(NH)-(CH)-SB (where S is the sulfur atom of a thioether bond and S is a thiol-containing toxin). In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RKR-(NH)-(CH)-S-DM1.

[0543] In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, may be conjugated to a linker B-RKR (wherein B is preferably a toxin) disclosed herein. In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, may be conjugated to a linker B-(self-immolative moiety)-RKR (wherein B is preferably a toxin) disclosed herein. The self-immolative moiety may be any self-immolative moiety known in the art and / or disclosed herein that is suitable for attaching a payload to the N-terminus of a peptide. In certain embodiments, the self-immolative moiety may be a self-immolative moiety comprising an ortho-hydroxy-protected aryl sulfate (OHPAS) moiety. In certain embodiments, the amine comprising payload B may be attached to the N-terminal arginine moiety by a dicarboxylic acid linker, as illustrated in Figure 9.

[0544] In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, may be conjugated to the linker RK-Val-Cit-B, where B is preferably a toxin, as disclosed herein. In certain embodiments, trastuzumab, or an anti-HER2 / neu antibody, may be conjugated to the linker RK-Val-Cit-(self-immolative moiety)-B, where B is preferably a toxin, as disclosed herein. The self-immolative moiety may be any self-immolative moiety known in the art and / or disclosed herein that is suitable for attaching a payload to the C-terminus of a peptide. In certain embodiments, the self-immolative moiety may be a group containing PABC or a methyl-amine.

[0545] In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RK-Val-Cit-PABC-B. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RK-Val-Cit-PABC-MMAE. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RK-Val-Cit-PABC-maytansine. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RK-Val-Cit-MMAE. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RK-Val-Cit-PABC-PNP-MMAE. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RK-Val-Cit-(NH)-(CH)-OB, where O is the oxygen atom of an ether bond and B is a hydroxyl-containing toxin. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RK-Val-Cit-(NH)-(CH)-OB camptothecin. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RK-Val-Cit-(NH)-(CH)-SB, where S is the sulfur atom of a thioether bond and B is a thiol-containing toxin. In certain embodiments, trastuzumab or an anti-HER2 / neu antibody may be conjugated to the linker RK-Val-Cit-(NH)-(CH)-S-DM1.

[0546] In certain embodiments, the present invention relates to antibody-linker conjugates or antibody drug-conjugates comprising the antibody enfortumab, or alternatively, anti-Nectin-4 antibodies.

[0547] That is, in certain embodiments, the present invention provides a) enfortumab or anti-Nectin-4 antibody; and b) Structure: (Sp1)-RK-(Sp2)-B-(Sp3) or (Sp1)-B-(Sp2)-RK-(Sp3); (In the formula, - (Sp1) is a chemical spacer or is absent; - (Sp2) is a chemical spacer or is absent; - (Sp3) is a chemical spacer or is absent; R is arginine or an arginine derivative or arginine mimetic; K is lysine or a lysine derivative or a lysine mimetic; - B is the linking portion or payload) a linker comprising 1. An antibody-linker conjugate comprising: The present invention relates to an antibody-linker conjugate in which the linker is conjugated to enfortumab or the anti-Nectin-4 antibody by an isopeptide bond formed between the gamma-carboxamide group of a glutamine residue contained in enfortumab or the anti-Nectin-4 antibody and a primary amine contained in the side chain of a lysine residue, lysine derivative, or lysine mimetic contained in the RK motif contained in the linker.

[0548] In certain embodiments, the present invention relates to an antibody-payload conjugate comprising enfortumab or an anti-Nectin-4 antibody, wherein the linker is any one of the linkers shown in Figure 1, Figure 2, Figure 3, Figure 8, Figure 9, Figure 14, Figure 15, Figure 17, Figure 18, Figure 19, Figure 20, Figure 21, Figure 22, Figure 23, Figure 24, Figure 25, Figure 26, Figure 27, Figure 28, Figure 29, Figure 30, Figure 31, Figure 32, Figure 33 or Figure 34.

[0549] Enfortumab is commercially available as an antibody-drug conjugate called enfortumab vedotin, sold under the name Padcev. Enfortumab vedotin contains the anti-Nectin-4 antibody enfortumab and the linker maleimidocaproyl-L-valine-L-citrulline-PABC-MMAE (mc-vc-PABC-MMAE), commonly known as vedotin. The mc-vc-PABC-MMAE linker of enfortumab vedotin is conjugated to a free cysteine ​​residue contained in the antibody. The antibody enfortumab is disclosed in WO2012 / 047724, the entire contents of which are incorporated herein by reference.

[0550] In certain embodiments, the antibody is enfortumab, comprising a heavy chain set forth in SEQ ID NO:9 and a light chain set forth in SEQ ID NO:10. However, the present invention also encompasses variants of enfortumab in which the heavy and / or light chains comprise at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO:9 and / or SEQ ID NO:10, respectively. In particular, the antibody may comprise any of the sequence variations disclosed in WO2012 / 047724. In certain embodiments, the light chain of enfortumab may comprise a mutation at residue Q55 of SEQ ID NO:10. In particular, the mutation is Q55N (SEQ ID NO:11).

[0551] It is preferred herein that enfortumab or anti-Nectin-4 antibody is present in the antibody-linker conjugate in glycosylated form.That is, enfortumab or anti-Nectin-4 antibody is preferably glycosylated at residue N297 (EU numbering).However, enfortumab or anti-Nectin-4 antibody may also be deglycosylated as described herein.

[0552] Enfortumab or anti-Nectin-4 antibody may be conjugated to any one of the linkers disclosed herein, particularly for the method according to the present invention.Preferably, the linker is conjugated to the glutamine residue Q295 (EU numbering) of the antibody by MTG catalysis.However, the linker may also be conjugated to an engineered glutamine residue such as N297Q (EU numbering) and / or any one of the glutamine-containing tags disclosed herein.

[0553] The linker conjugated to enfortumab, or the anti-Nectin-4 antibody, may contain a single linking moiety or payload B, or may contain multiple linking moieties and / or payloads B1, B2, etc.

[0554] That is, in certain embodiments, the linker included in an enfortumab-linker conjugate may include one or more linking moieties B. Such enfortumab-linker conjugates may then be functionalized with a suitable payload in a two-step process disclosed herein.

[0555] In certain embodiments, the linker included in the enfortumab-payload conjugate may comprise one or more payloads B. Such enfortumab-payload conjugates may be obtained by a two-step process, in which a linker containing a linking moiety is conjugated to enfortumab in a first step, and a payload is linked to the linking moiety in a second step. Alternatively, the enfortumab-payload conjugates may be obtained by a one-step process, in which a linker containing a payload is directly conjugated to enfortumab.

[0556] In certain embodiments, the present invention relates to an antibody-drug conjugate comprising enfortumab or an anti-Nectin-4 antibody, i.e., the linker included in the antibody-drug conjugate may comprise one or more toxins described herein.

[0557] In certain embodiments, an antibody-drug conjugate comprising enfortumab or an anti-Nectin-4 antibody may comprise a linker comprising the amino acid sequence RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4), or RK-Val-Cit (SEQ ID NO: 54).

[0558] That is, in certain embodiments, the present invention provides a) Enfortumab or anti-Nectin-4 antibody: and b) an antibody-drug conjugate comprising a linker comprising a drug moiety B, wherein drug moiety B is covalently linked to an amino acid sequence selected from the group consisting of RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4) or RK-Val-Cit (SEQ ID NO: 54); The linker is located at the C H The present invention relates to an antibody-drug conjugate conjugated to enfortumab or an anti-Nectin-4 antibody via an isopeptide bond formed between the γ-carboxamide group of glutamine residue Q295 (EU numbering) in Domain 2 and the primary amine contained in the side chain of a lysine residue contained in the linker.

[0559] Preferably, the antibody is enfortumab comprising a heavy chain set forth in SEQ ID NO: 9 and a light chain set forth in SEQ ID NO: 10. Thus, in a particular embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the IgG antibody is enfortumab or an antibody comprising a heavy chain set forth in SEQ ID NO: 9 and a light chain set forth in SEQ ID NO: 10.

[0560] In certain embodiments, a linker conjugated to enfortumab or an anti-Nectin-4 antibody may comprise the structure RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4), or RK-Val-Cit (SEQ ID NO: 54). In certain embodiments, a linker comprising the structure RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4), or RK-Val-Cit (SEQ ID NO: 54) may be conjugated to residue Q295 of enfortumab or an anti-Nectin-4 antibody via the primary amine contained in residue K.

[0561] In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker RKAA-B (wherein B is preferably a toxin) disclosed herein. In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker RKAA-(self-immolative moiety)-B (wherein B is preferably a toxin) disclosed herein. The self-immolative moiety may be any self-immolative moiety known in the art and / or disclosed herein that is suitable for attaching a payload to the C-terminus of a peptide. In certain embodiments, the self-immolative moiety may be a group containing PABC or methyl-amine.

[0562] In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker RKAA-PABC-B. In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker RKAA-PABC-MMAE (see Figure 1). In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker RKAA-PABC-maytansine (see Figure 8). In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker RKAA-MMAE. In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker RKAA-PABC-PNP-MMAE. In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker RKAA-(NH)-(CH)-OB (where O is the oxygen atom of an ether bond and B is a hydroxyl-containing toxin). In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker RKAA-(NH)-(CH)-O-camptothecin. In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker RKAA-(NH)-(CH)-SB (where S is the sulfur atom of a thioether bond and B is a thiol-containing toxin). In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker RKAA-(NH)-(CH)-S-DM1.

[0563] In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker B-RKAA (wherein B is preferably a toxin) disclosed herein. In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker B-(self-immolative moiety)-RKAA (wherein B is preferably a toxin) disclosed herein. The self-immolative moiety may be any self-immolative moiety known in the art and / or disclosed herein that is suitable for attaching a payload to the N-terminus of a peptide. In certain embodiments, the self-immolative moiety may be a self-immolative moiety comprising an ortho-hydroxy-protected aryl sulfate (OHPAS) moiety. In certain embodiments, the amine comprising payload B may be attached to the N-terminal arginine moiety by a dicarboxylic acid linker, similar to Figure 9.

[0564] In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker RKA-B (wherein B is preferably a toxin) disclosed herein. In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker RKA-(self-immolative moiety)-B (wherein B is preferably a toxin) disclosed herein. The self-immolative moiety may be any self-immolative moiety known in the art and / or disclosed herein that is suitable for attaching a payload to the C-terminus of a peptide. In certain embodiments, the self-immolative moiety may be a group containing PABC or methyl-amine.

[0565] In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker RKA-PABC-B. In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker RKA-PABC-MMAE (see Figure 2). In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker RKA-PABC-maytansine. In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker RKA-MMAE. In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker RKA-PABC-PNP-MMAE. In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker RKA-(NH)-(CH)-OB (where O is the oxygen atom of an ether bond and B is a hydroxyl-containing toxin). In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker RKA-(NH)-(CH)-O-camptothecin. In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker RKA-(NH)-(CH)-SB (where S is the sulfur atom of a thioether bond and B is a thiol-containing toxin). In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker RKA-(NH)-(CH)-S-DM1.

[0566] In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker B-RKA (wherein B is preferably a toxin) disclosed herein. In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker B-(self-immolative moiety)-RKA (wherein B is preferably a toxin) disclosed herein. The self-immolative moiety may be any self-immolative moiety known in the art and / or disclosed herein that is suitable for attaching a payload to the N-terminus of a peptide. In certain embodiments, the self-immolative moiety may be a self-immolative moiety comprising an ortho-hydroxy-protected aryl sulfate (OHPAS) moiety. In certain embodiments, the amine comprising payload B may be attached to the N-terminal arginine moiety by a dicarboxylic acid linker, similar to Figure 9.

[0567] In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker ARK-B (wherein B is preferably a toxin) disclosed herein. In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker ARK-(self-immolative moiety)-B (wherein B is preferably a toxin) disclosed herein. The self-immolative moiety may be any self-immolative moiety known in the art and / or disclosed herein that is suitable for attaching a payload to the C-terminus of a peptide. In certain embodiments, the self-immolative moiety may be a group containing PABC or methyl-amine.

[0568] In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker ARK-PABC-B. In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker ARK-PABC-MMAE (see Figure 3). In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker ARK-PABC-maytansine. In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker ARK-MMAE. In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker ARK-PABC-PNP-MMAE. In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker ARK-(NH)-(CH)-OB (where O is the oxygen atom of an ether bond and B is a hydroxyl-containing toxin). In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker ARK-(NH)-(CH)-O-camptothecin. In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker ARK-(NH)-(CH)-SB (where S is the sulfur atom of a thioether bond and B is a thiol-containing toxin). In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker ARK-(NH)-(CH)-S-DM1.

[0569] In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker B-ARK (wherein B is preferably a toxin) disclosed herein. In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker B-(self-immolative moiety)-ARK (wherein B is preferably a toxin) disclosed herein. The self-immolative moiety may be any self-immolative moiety known in the art and / or disclosed herein that is suitable for attaching a payload to the N-terminus of a peptide. In certain embodiments, the self-immolative moiety may be a self-immolative moiety comprising an ortho-hydroxy-protected aryl sulfate (OHPAS) moiety.

[0570] In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker RKR-B (wherein B is preferably a toxin) disclosed herein. In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker RKR-(self-immolative moiety)-B (wherein B is preferably a toxin) disclosed herein. The self-immolative moiety may be any self-immolative moiety known in the art and / or disclosed herein that is suitable for attaching a payload to the C-terminus of a peptide. In certain embodiments, the self-immolative moiety may be a group containing PABC or methyl-amine.

[0571] In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker RKR-PABC-B. In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker RKR-PABC-MMAE. In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker RKR-PABC-maytansine. In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker RKR-MMAE. In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker RKR-PABC-PNP-MMAE. In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker RKR-(NH)-(CH)-OB (where O is the oxygen atom of an ether bond and B is a hydroxyl-containing toxin). In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker RKR-(NH)-(CH)-O-camptothecin. In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker RKR-(NH)-(CH)-SB (where S is the sulfur atom of a thioether bond and S is a thiol-containing toxin). In certain embodiments, enfortumab or anti-Nectin-4 antibodies may be conjugated to the linker RKR-(NH)-(CH)-S-DM1.

[0572] In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker B-RKR (wherein B is preferably a toxin) disclosed herein. In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker B-(self-immolative moiety)-RKR (wherein B is preferably a toxin) disclosed herein. The self-immolative moiety may be any self-immolative moiety known in the art and / or disclosed herein that is suitable for attaching a payload to the N-terminus of a peptide. In certain embodiments, the self-immolative moiety may be a self-immolative moiety comprising an ortho-hydroxy-protected aryl sulfate (OHPAS) moiety. In certain embodiments, the amine comprising payload B may be linked to the N-terminal arginine moiety by a dicarboxylic acid linker, as illustrated in Figure 9.

[0573] In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker RK-Val-Cit-B (wherein B is preferably a toxin) disclosed herein. In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker RK-Val-Cit-(self-immolative moiety)-B (wherein B is preferably a toxin) disclosed herein. The self-immolative moiety may be any self-immolative moiety known in the art and / or disclosed herein that is suitable for attaching a payload to the C-terminus of a peptide. In certain embodiments, the self-immolative moiety may be a group containing PABC or methyl-amine.

[0574] In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker RK-Val-Cit-PABC-B. In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker RK-Val-Cit-PABC-MMAE. In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker RK-Val-Cit-PABC-maytansine. In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker RK-Val-Cit-MMAE. In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker RK-Val-Cit-PABC-PNP-MMAE. In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker RK-Val-Cit-(NH)-(CH)-OB (where O is an oxygen atom of an ether bond and B is a hydroxyl-containing toxin). In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker RK-Val-Cit-(NH)-(CH)-OB camptothecin. In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker RK-Val-Cit-(NH)-(CH)-SB (where S is a sulfur atom of a thioether bond and B is a thiol-containing toxin). In certain embodiments, enfortumab or an anti-Nectin-4 antibody may be conjugated to the linker RK-Val-Cit-(NH)-(CH)-S-DM1.

[0575] Furthermore, the present invention relates to a linker construct comprising the motif RK. The linker construct according to the present invention can be used for the conjugation of a wide range of antibodies. Due to the highly conserved conjugation site Q295, the linker conjugate according to the present invention can be used "off the shelf" to generate antibody-payload conjugates of essentially any IgG-type antibody. Compared to linkers known from the prior art, the RK linker of the present invention can be used for the highly efficient conjugation of glycosylated antibodies, and can achieve high conjugation efficiency even when bulky payloads such as toxins are included.

[0576] Thus, in certain embodiments, the present invention provides a compound having the structure: (Sp1)-RK-(Sp2)-B-(Sp3) or (Sp1)-B-(Sp2)-RK-(Sp3); (In the formula, - (Sp1) is a chemical spacer or is absent; - (Sp2) is a chemical spacer or is absent; - (Sp3) is a chemical spacer or is absent; R is arginine or an arginine derivative or arginine mimetic, K is lysine or a lysine derivative or a lysine mimetic; - B is the linking portion or payload) The present invention relates to a linker construct comprising:

[0577] It should be understood that the linker construct may have the same structure and / or characteristics as the linkers disclosed above with respect to the methods according to the invention, the antibody-linker conjugates according to the invention and / or the antibody-drug conjugates according to the invention.

[0578] In certain embodiments, the present invention relates to a linker construct according to the present invention, wherein the chemical spacers (Sp1), (Sp2) and (Sp3) each independently comprise from 0 to 12 amino acid residues.

[0579] In a particular embodiment, the present invention relates to a linker construct 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.

[0580] In certain embodiments, the present invention relates to a linker construct according to the present invention, wherein the net charge of the linker is neutral or positive.

[0581] In a particular embodiment, the present invention relates to a linker construct according to the present invention, wherein the linker does not comprise any negatively charged amino acid residues.

[0582] In a particular embodiment, the present invention relates to a linker construct according to the present invention, wherein the linker comprises the amino acid sequence RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4) or RK-Val-Cit (SEQ ID NO: 54).

[0583] In a particular embodiment, the present invention relates to a linker construct according to the invention, wherein B is a linking moiety.

[0584] In a particular embodiment, the present invention relates to a linker construct according to the present invention, wherein linking moiety B is - bioorthogonal marker groups, or - Non-bio-orthogonal entities for crosslinking The present invention relates to a linker construct comprising:

[0585] In certain embodiments, the present invention provides a linker construct according to the present invention, wherein the bioorthogonal marker group or the non-bioorthogonal entity for crosslinking is: - -NN≡N, or -N3; - Lys(N3); - tetrazine; - alkynes; - Distorted cyclooctyne; - BCN; - strained alkenes; - photoreactive groups; - aldehydes; - acyltrifluoroborates; - Protein degrading agents ("PROTACs"); - Cyclopentadiene / spirolocyclopentadiene; - Thioselective electrophiles; - -SH; and - Cysteine The present invention relates to a linker construct consisting of or comprising at least one molecule or moiety selected from the group consisting of:

[0586] In a particular embodiment, the present invention relates to a linker construct according to the invention, which consists of or comprises the structure RKAA-B or B-RKAA, in particular wherein B is Lys(N3) or cysteine.

[0587] That is, in certain embodiments, the present invention relates to a linker comprising the structure RKAA-B or B-RKAA (wherein B is a linking moiety). It should be understood that B may be any linking moiety known in the art and / or disclosed herein. In certain embodiments, B may be a thiol-containing linking moiety, such as cysteine, an azide-containing linking moiety, such as Lys(N3), or a tetrazine-containing linking moiety. It should be understood that a linker comprising the structure RKAA-B or B-RKAA may comprise additional amino acid residues, linking moieties, payloads, and / or other chemical groups, such as, but not limited to, a PEG moiety. In certain embodiments, the linker construct consists of the structure RKAA-B or B-RKAA.

[0588] In a particular embodiment, the present invention relates to a linker construct according to the invention, which consists of or comprises the structure RKA-B or B-RKA, in particular wherein B is Lys(N3) or cysteine.

[0589] That is, in certain embodiments, the present invention relates to a linker comprising the structure RKA-B or B-RKA (wherein B is a linking moiety). It should be understood that B may be any linking moiety known in the art and / or disclosed herein. In certain embodiments, B may be a thiol-containing linking moiety, such as cysteine, an azide-containing linking moiety, such as Lys(N3), or a tetrazine-containing linking moiety. It should be understood that a linker comprising the structure RKA-B or B-RKA may comprise additional amino acid residues, linking moieties, payloads, and / or other chemical groups, such as, but not limited to, a PEG moiety. In certain embodiments, the linker construct consists of the structure RKA-B or B-RKA.

[0590] In a particular embodiment, the present invention relates to a linker construct according to the invention, which consists of or comprises the structure ARK-B or B-ARK, in particular wherein B is Lys(N3) or cysteine.

[0591] That is, in certain embodiments, the present invention relates to a linker comprising the structure ARK-B or B-ARK (wherein B is a linking moiety). It should be understood that B may be any linking moiety known in the art and / or disclosed herein. In certain embodiments, B may be a thiol-containing linking moiety, such as cysteine, an azide-containing linking moiety, such as Lys(N3), or a tetrazine-containing linking moiety. It should be understood that a linker comprising the structure ARK-B or B-ARK may comprise additional amino acid residues, linking moieties, payloads, and / or other chemical groups, such as, but not limited to, a PEG moiety. In certain embodiments, the linker construct consists of the structure ARK-B or B-ARK.

[0592] In a particular embodiment, the present invention relates to a linker construct according to the invention, which consists of or comprises the structure RKR-B or B-RKR, in particular wherein B is Lys(N3) or cysteine.

[0593] That is, in certain embodiments, the present invention relates to a linker comprising the structure RKR-B or B-RKR, where B is a linking moiety. It should be understood that B may be any linking moiety known in the art and / or disclosed herein. In certain embodiments, B may be a thiol-containing linking moiety, such as cysteine, an azide-containing linking moiety, such as Lys(N3), or a tetrazine-containing linking moiety. It should be understood that a linker comprising the structure RKR-B or B-RKR may comprise additional amino acid residues, linking moieties, payloads, and / or other chemical groups, such as, but not limited to, a PEG moiety. In certain embodiments, the linker construct consi...

Claims

1. a) an IgG antibody; and b) A linker comprising a drug moiety B, wherein the drug moiety B is a toxin covalently linked to an amino acid sequence selected from the group consisting of RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), or RK-Val-Cit (SEQ ID NO: 54), wherein the drug moiety B is linked to the N- or C-terminus of the amino acid sequence contained in the linker by a p-aminobenzylcarbamoyl (PABC) moiety.

1. An antibody-drug conjugate comprising: The linker is a C Ｈ an antibody-drug conjugate conjugated to the IgG antibody via an isopeptide bond formed between the γ-carboxamide group of glutamine residue Q295 (EU numbering) of domain 2 and a primary amine contained in the side chain of a lysine residue contained in the linker.

2. The antibody-drug conjugate of claim 1, wherein the IgG antibody is a glycosylated IgG antibody.

3. The IgG antibody is Ｈ 3. The antibody-drug conjugate of claim 2, wherein the antibody-drug conjugate is glycosylated at residue N297 (EU numbering) of domain 2.

4. The antibody-drug conjugate of any one of claims 1 to 3, wherein the IgG antibody is an IgG1 antibody.

5. The antibody-drug conjugate of any one of claims 1 to 4, wherein the IgG antibody is polatuzumab or an antibody comprising a heavy chain shown in SEQ ID NO: 5 and a light chain shown in SEQ ID NO:

6.

6. The antibody-drug conjugate of any one of claims 1 to 4, wherein the IgG antibody is trastuzumab or an antibody comprising a heavy chain shown in SEQ ID NO: 7 and a light chain shown in SEQ ID NO:

8.

7. The antibody-drug conjugate of any one of claims 1 to 4, wherein the IgG antibody is enfortumab or an antibody comprising a heavy chain shown in SEQ ID NO: 9 and a light chain shown in SEQ ID NO: 10 or 11.

8. the drug moiety - pyrrolobenzodiazepines; - auristatins; - maytansinoids; - duocarmycin; - nicotinamide phosphoribosyltransferase (NAMPT) inhibitors; - Tubulysin; - Enzyme; - anthracycline derivatives (PNU); - pyrrole-based kinesin spindle protein (KSP) inhibitors; - cryptophycin; - drug efflux pump inhibitors; - Sandramycin; - Amanitin; and - Camptothecin The antibody-drug conjugate of any one of claims 1 to 7, selected from the group consisting of:

9. - said pyrrolobenzodiazepine is PBD; - the auristatin is MMAE or MMAF; - the maytansinoid is maytansine, DM1, DM4 or DM21; - the enediyne is calicheamicin; - said anthracycline derivative (PNU) is doxorubicin; - said amanitin is α-amanitin; and - the camptothecin is exatecan or deruxtecan The antibody-drug conjugate of claim 8, wherein

10. 10. The antibody-drug conjugate of any one of claims 1 to 9, wherein the linker has the structure RKAA-PABC-B, RKA-PABC-B, ARK-PABC-B, or RK-Val-Cit-PABC-B.

11. The antibody-drug conjugate of claim 10, wherein B is an auristatin or a maytansinoid.

12. The antibody-drug conjugate of claim 11, wherein the auristatin is MMAE and the maytansinoid is DM1 or maytansine.

13. The antibody-drug conjugate of any one of claims 1 to 12, wherein the linker has the following structure: 【Chemistry 1】

14. a) an IgG antibody, wherein the IgG antibody is polatuzumab or an antibody comprising a heavy chain set forth in SEQ ID NO: 5 and a light chain set forth in SEQ ID NO: 6; and b) a linker having the following structure: 【Chemistry 2】 1. An antibody-drug conjugate comprising: The linker is a C Ｈ an antibody-drug conjugate conjugated to the IgG antibody via an isopeptide bond formed between the γ-carboxamide group of glutamine residue Q295 (EU numbering) of domain 2 and a primary amine contained in the side chain of a lysine residue contained in the linker.

15. a) an IgG antibody, wherein the IgG antibody is trastuzumab or an antibody comprising a heavy chain set forth in SEQ ID NO: 7 and a light chain set forth in SEQ ID NO: 8; and b) a linker having the following structure: 【Transformation 3】 1. An antibody-drug conjugate comprising: The linker is a C Ｈ an antibody-drug conjugate conjugated to the IgG antibody via an isopeptide bond formed between the γ-carboxamide group of glutamine residue Q295 (EU numbering) of domain 2 and a primary amine contained in the side chain of a lysine residue contained in the linker.

16. a) an IgG antibody, wherein the IgG antibody is enfortumab or an antibody comprising a heavy chain set forth in SEQ ID NO: 9 and a light chain set forth in SEQ ID NO: 10 or 11; and b) a linker having the following structure: 【Chemistry 4】 1. An antibody-drug conjugate comprising: The linker is a C Ｈ an antibody-drug conjugate conjugated to the IgG antibody via an isopeptide bond formed between the γ-carboxamide group of glutamine residue Q295 (EU numbering) of domain 2 and a primary amine contained in the side chain of a lysine residue contained in the linker.

17. A linker construct comprising the structure: RKAA-PABC-B, RKA-PABC-B, ARK-PABC-B or RK-Val-Cit-PABC-B, PABC is a p-aminobenzylcarbamoyl moiety, and B is a toxin Linker constructs.

18. The B is - pyrrolobenzodiazepines; - auristatins; - maytansinoids; - duocarmycin; - nicotinamide phosphoribosyltransferase (NAMPT) inhibitors; - Tubulysin; - Enzymes (e.g. calicheamicin); - anthracycline derivatives (PNU); - pyrrole-based kinesin spindle protein (KSP) inhibitors; - cryptophycin; - drug efflux pump inhibitors; - Sandramycin; - Amanitin; and - Camptothecin 18. The linker construct of claim 17, wherein the linker construct is at least one selected from the group consisting of:

19. - said pyrrolobenzodiazepine is PBD; - the auristatin is MMAE or MMAF; - the maytansinoid is maytansine, DM1, DM4 or DM21; - the enediyne is calicheamicin; - said anthracycline derivative (PNU) is doxorubicin; - said amanitin is α-amanitin; and - the camptothecin is exatecan or deruxtecan 19. The linker construct of claim 18, wherein:

20. 20. The linker construct of any one of claims 17 to 19, wherein B is an auristatin or a maytansinoid.

21. 21. The linker construct of claim 20, wherein the auristatin is MMAE and the maytansinoid is DM1 or maytansine.

22. The linker has the structure: 【Transformation 5】 22. The linker construct of any one of claims 17 to 21, having:

23. Use of a linker construct according to any one of claims 17 to 22 in the production of antibody-drug conjugates by microbial transglutaminase.

24. 24. The use according to claim 23, wherein the antibody is an IgG antibody.

25. 25. The use according to claim 23 or 24, wherein the antibody is polatuzumab or trastuzumab or enfortumab.

26. A pharmaceutical composition comprising the antibody drug-conjugate of any one of claims 1 to 16, wherein the pharmaceutical composition comprises at least one pharmaceutically acceptable ingredient.

27. 27. The pharmaceutical composition of claim 26, comprising at least one additional therapeutically active agent.

28. A composition comprising the antibody-drug conjugate of any one of claims 1 to 16, or the pharmaceutical composition of claim 26 or 27, for use in therapy.

29. Neoplastic, neurological, autoimmune, inflammatory or infectious diseases - Are you suffering from - be at risk of developing it, and / or - diagnosed with A composition comprising the antibody-drug conjugate of any one of claims 1 to 16, or the pharmaceutical composition of claim 26 or 27, for use in treating a patient.

30. 30. The composition for use of claim 29, wherein the antibody-drug conjugate comprises polatuzumab or an antibody comprising a heavy chain set forth in SEQ ID NO:5 and a light chain set forth in SEQ ID NO:6, and the neoplastic disease is a cancer associated with B cells.

31. 31. The composition for use of claim 30, wherein the cancer associated with B cells is non-Hodgkin's lymphoma or diffuse large B-cell lymphoma.

32. The composition for use according to claim 30 or 31, wherein the antibody-drug conjugate or the pharmaceutical composition is administered in combination with bendamustine and / or rituximab.

33. 30. The composition for use of claim 29, wherein the antibody-drug conjugate comprises trastuzumab or an antibody comprising a heavy chain set forth in SEQ ID NO: 7 and a light chain set forth in SEQ ID NO: 8, and the neoplastic disease is a HER2-positive cancer.

34. 34. The composition for use of claim 33, wherein the HER2-positive cancer is HER2-positive breast cancer, gastric cancer, ovarian cancer or lung cancer.

35. The composition for use according to claim 33 or 34, wherein the antibody-drug conjugate or the pharmaceutical composition is administered in combination with lapatinib, capecitabine and / or a taxane.

36. 30. The composition for use of claim 29, wherein the antibody-drug conjugate comprises enfortumab or an enfortumab variant or an antibody comprising a heavy chain set forth in SEQ ID NO: 9 and a light chain set forth in SEQ ID NO: 10 or 11, and the neoplastic disease is a Nectin-4 positive cancer.

37. The composition for use according to claim 36, wherein the Nectin-4 positive cancer is Nectin-4 positive pancreatic cancer, lung cancer, bladder cancer or breast cancer.

38. The composition for use according to claim 36 or 37, wherein the antibody-drug conjugate or the pharmaceutical composition is administered in combination with a cisplatin-based chemotherapy agent and / or pembrolizumab.

39. Neoplastic, neurological, autoimmune, inflammatory or infectious diseases - Are you suffering from - be at risk of developing it, and / or - diagnosed with 28. Use of an antibody-drug conjugate according to any one of claims 1 to 16, or a pharmaceutical composition according to claim 26 or 27, for the manufacture of a medicament for the treatment of a patient.

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